Targeted chemical drug and preparation method therefor, pharmaceutical composition, and use of targeted chemical drug

Abstract

Provided are a targeted chemical drug and a preparation method therefor, a pharmaceutical composition, and the use of the targeted chemical drug. The targeted chemical drug comprises a targeting nucleic acid vector and a chemical small molecule drug loaded on the targeting nucleic acid vector; and the targeting nucleic acid vector comprises a nucleic acid vector and a targeting molecule connected to the nucleic acid vector, wherein the nucleic acid vector is a DNA vector or an RNA vector, and the nucleic acid vector comprises sequence a, sequence, b and sequence c that form a self-assembled structure. The targeting chemical drug can more stably and reliably deliver the chemical small molecule drug, and enables the chemical small molecule drug to produce a combination therapeutic effect with other drugs carried by the vector.

Description

Targeted chemical drug and preparation method thereof, pharmaceutical composition, and application of targeted chemical drug

[0001] This application is based on the Chinese application with CN application number 202310871629.2 and application date July 14, 2023, and claims its priority. The disclosed content of the CN application is again introduced as a whole into this application. Technical Field

[0002] The present invention relates to the field of medicine, and in particular to a targeted chemical drug and a preparation method thereof, a pharmaceutical composition, and applications of the targeted chemical drug. Background Art

[0003] Traditional cancer chemotherapy relies on the principle that tumor cells, due to their rapid proliferation, are more susceptible to anticancer drug extermination. However, representative cytotoxic chemotherapeutics such as doxorubicin, paclitaxel, and cisplatin cannot distinguish cancer cells from normal cells. This lack of selectivity can lead to a phenomenon of "killing one thousand enemies while injuring eight hundred of one's own," or even worse side effects. In particular, the high doses of cytotoxic agents required to eradicate tumors put patients at risk of systemic cytotoxicity. Therefore, specific delivery systems that recognize the inherent differences between tumor and normal cells are urgently needed for effective cancer treatment. The development and technological advancement of multi-target-mediated nucleic acid nanocarriers offer the potential for specific delivery. However, the reliability of existing nucleic acid nanocarriers for delivering these small molecule drugs needs to be further improved.

[0004] Summary of the Invention

[0005] The main purpose of the present invention is to provide a targeted chemical drug and its preparation method, pharmaceutical composition, and application of the targeted chemical drug to solve the following problems in the prior art: targeted delivery of small molecule chemical drugs, toxicity reduction and efficacy enhancement, and expansion of the therapeutic window.

[0006] To achieve the above-mentioned object, according to one aspect of the present invention, a targeted chemical drug is provided, which includes a targeted nucleic acid vector and a chemical small molecule drug mounted on the targeted nucleic acid vector, and the targeted nucleic acid vector includes a nucleic acid vector and a targeting molecule connected to the nucleic acid vector; wherein the nucleic acid vector is a DNA vector or an RNA vector, and the nucleic acid vector comprises an a sequence, a b sequence and a c sequence that form a self-assembly structure, wherein the a sequence is SEQ ID NO:1: 5'-ACGAGCGTTCCG-3'; the b sequence is SEQ ID NO:2: 5'-CGGTTCGCCG-3'; the c sequence is SEQ ID NO:3: 5'-CGGCCATAGCCGT-3'; or, the a sequence is SEQ ID NO:4: 5'-ACGAGCGUUCCG-3'; the b sequence is SEQ ID NO:5: 5'-CGGUUCGCCG-3'; and the c sequence is SEQ ID NO:6: 5'-CGGCCAUAGCCGU-3'; optionally, at least one base is substituted, inserted or deleted in the a sequence, the b sequence and the c sequence.

[0007] According to a second aspect of the present invention, a pharmaceutical composition is provided, which comprises the above-mentioned targeted chemical drug and a pharmaceutically acceptable carrier and / or excipient.

[0008] According to a third aspect of the present invention, there is provided a use of the above-mentioned targeted chemical drug in the preparation of a drug for treating tumors.

[0009] Furthermore, the cancer tumor is gastric cancer and / or lung cancer; preferably, the drug is administered into the tumor, intravenously or intraperitoneally during the application process.

[0010] According to the fourth aspect of the present invention, there is also provided a second type of method for preventing and / or treating cancer, comprising: providing the above-mentioned targeted chemical drug or pharmaceutical composition; and administering an effective amount of the targeted chemical drug or pharmaceutical composition to a tumor patient.

[0011] Furthermore, the tumor is a male tumor, a gynecological tumor, a respiratory system tumor, a digestive system tumor, a hematological tumor, a urinary system tumor, a bone marrow tumor, a neurological tumor, a dermatological tumor, a general surgical tumor or an ENT tumor; preferably, the male tumor is prostate cancer, penile cancer, testicular tumor or male urethral cancer, the gynecological tumor is ovarian cancer, cervical cancer, endometrial cancer, uterine fibroids, vulvar cancer or malignant hydatidiform mole, the respiratory tumor is lung cancer, non-small cell lung cancer, small cell lung cancer, nasopharyngeal cancer, tracheal tumor, lung cancer metastasis, pulmonary pseudotumor or radiation lung cancer, the digestive system tumor is liver cancer, gastric cancer, colorectal cancer, gallbladder cancer, esophageal cancer, rectal cancer, pancreatic cancer or colon cancer, the hematological tumor is Leukemia, lymphoma, lymphosarcoma or multiple myeloma, renal cancer, bladder cancer or urinary cancer, bone marrow tumors are giant cell tumors of bone, osteochondroma or osteosarcoma, neurological tumors are brain tumors, meningiomas, brain tuberculomas, pituitary tumors, neuroblastomas, glioblastomas or brain gliomas, dermatological tumors are skin cancer or melanoma, general surgical tumors are breast cancer, lipomas, thyroid cancer or thyroid tumors, ENT tumors are oral cancer, tongue cancer, laryngeal cancer, middle ear cancer, gingival cancer or orbital tumors; preferably, the drug is administered by tumor administration, intravenous administration or intraperitoneal administration during the application; preferably, the daily dosage of the drug during the application is 0.1 μg / Kg to 100 mg / Kg.

[0012] According to the fifth aspect of the present invention, a method for preparing the above-mentioned targeted chemical drug is also provided, which comprises the following steps: forming a targeted nucleic acid vector with an optional oligonucleotide effector molecule and an optional immunostimulant by sequence self-assembly; connecting a small molecule chemical drug to the targeted nucleic acid vector to obtain a targeted chemical drug.

[0013] The targeted chemical drug provided by the present invention includes a targeted nucleic acid vector and a small chemical molecule drug mounted on the targeted nucleic acid vector, and the targeted nucleic acid vector includes a nucleic acid vector and a targeting molecule connected to the nucleic acid vector; wherein the nucleic acid vector is a DNA vector or an RNA vector, and the nucleic acid vector contains a sequence a, a sequence b, and a sequence c that form a self-assembly structure. On the one hand, due to the coating effect of the above-mentioned DNA vector, the small chemical molecule drug can be wrapped in the vector so that it will not fall off during the delivery process; on the other hand, the vector has better targeting than other nucleic acid vectors after being modified with a targeting aptamer; on the other hand, the small chemical molecule drug and other drugs carried by the vector produce a combined therapeutic effect. In short, the targeted chemical drug can deliver the small chemical molecule drug more stably and reliably, and can produce a combined therapeutic effect with the small chemical molecule drug and other drugs carried by the vector. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The accompanying drawings, which constitute part of this application, are intended to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are intended to explain the present invention and do not constitute an undue limitation of the present invention. In the accompanying drawings:

[0015] FIG1 shows the HPLC chromatography (SEC) detection results of the crude product of the targeting nucleic acid vector after sequence assembly in Example 6 of the present invention;

[0016] FIG2 shows the HPLC chromatography (SEC) spectrum detection results of the targeted nucleic acid vector after HPLC purification in Example 6 of the present invention;

[0017] FIG3 shows the HPLC chromatography (reverse phase) spectrum detection results of the crude targeted chemical drug after loading epirubicin in Example 6 of the present invention;

[0018] FIG4 shows the HPLC chromatographic (reverse phase) spectrum detection results of the crude targeted chemical drug after HPLC purification in Example 6 of the present invention;

[0019] FIG5 shows a HPLC chromatographic (reverse phase) spectrum detection diagram of the targeted chemical drug obtained by changing the molar ratio of epirubicin to carrier to 10:1 in Example 6 of the present invention;

[0020] FIG6 shows a HPLC chromatographic (reverse phase) spectrum detection diagram of the targeted chemical drug obtained by changing the molar ratio of epirubicin to carrier to 20:1 in Example 6 of the present invention;

[0021] FIG7 shows a HPLC chromatographic (reverse phase) spectrum detection diagram of the targeted chemical drug obtained by changing the molar ratio of epirubicin to carrier to 30:1 in Example 6 of the present invention;

[0022] FIG8 shows the relative weight growth curves of mice in each group in an in vivo efficacy test of the epirubicin targeted drug Apt AS1411-Apt EGFR-Apt A15-2*Bio-DNA-epirubicin prepared in Example 5 of the present invention on an N87 mouse gastric cancer model;

[0023] FIG9 shows the tumor growth curves of each group of mice in an in vivo efficacy test of the epirubicin targeted drug Apt AS1411-Apt EGFR-Apt A15-2*Bio-DNA-epirubicin prepared in Example 5 of the present invention on the N87 mouse gastric cancer model;

[0024] FIG10 shows the changes in tumor volume of each group of mice in an in vivo efficacy test of the epirubicin targeted drug Apt AS1411-Apt EGFR-Apt A15-2*Bio-DNA-epirubicin prepared in Example 5 of the present invention on an N87 mouse gastric cancer model;

[0025] Figure 11 shows the mouse body weight change curves in an in vivo efficacy test on the N87 mouse gastric cancer model comparing the epirubicin targeted drug Apt AS1411-Apt EGFR-Apt A15-2*Bio-DNA-epirubicin prepared in Example 5 of the present invention and the epirubicin targeted drug Apt AS1411-Apt EGFR-Apt A15-2*Bio-AmiR-21-DNA-epirubicin prepared in Example 7;

[0026] Figure 12 shows the curves of mouse tumor volume changes in an in vivo efficacy test on an N87 mouse gastric cancer model comparing the epirubicin targeted drug Apt AS1411-Apt EGFR-Apt A15-2*Bio-DNA-epirubicin prepared in Example 5 of the present invention and the epirubicin targeted drug Apt AS1411-Apt EGFR-Apt A15-2*Bio-AmiR-21-DNA-epirubicin prepared in Example 7;

[0027] Figure 13 shows the curves of changes in relative tumor volume in mice in an in vivo efficacy test on the N87 mouse gastric cancer model of the epirubicin targeted drug Apt AS1411-Apt EGFR-Apt A15-2*Bio-DNA-epirubicin prepared in Example 5 of the present invention and the epirubicin targeted drug Apt AS1411-Apt EGFR-Apt A15-2*Bio-AmiR-21-DNA-epirubicin prepared in Example 7;

[0028] FIG14 shows the curve of mouse tumor volume change in an in vivo efficacy test of the epirubicin targeted drug Apt AS1411-4*Bio-DNA-epirubicin prepared in Example 3 of the present invention on an A549 mouse lung cancer model;

[0029] FIG15 shows the curve of the relative tumor volume change in mice in an in vivo efficacy test of the epirubicin targeted drug Apt AS1411-4*Bio-DNA-epirubicin prepared in Example 3 of the present invention on an A549 mouse lung cancer model;

[0030] Figure 16 shows the curves of mouse tumor volume changes in an in vivo efficacy test of the epirubicin targeted drug Apt AS1411-Apt EGFR-Apt A15-DNA-epirubicin prepared in Example 6 of the present invention and the epirubicin targeted drug Apt AS1411-Apt EGFR-Apt A15-AmiR-21-DNA-epirubicin prepared in Example 20 on an A549 mouse lung cancer model;

[0031] Figure 17 shows the curves of changes in relative tumor volume in mice in an in vivo efficacy test of the epirubicin targeted drug Apt AS1411-Apt EGFR-Apt A15-DNA-epirubicin prepared in Example 6 of the present invention and the epirubicin targeted drug Apt AS1411-Apt EGFR-Apt A15-AmiR-21-DNA-epirubicin prepared in Example 20 on an A549 mouse lung cancer model;

[0032] FIG18 shows the curve of mouse tumor volume change in an in vivo efficacy test of the epirubicin targeted drug Apt AS1411-Apt EGFR-AptA15-DNA-epirubicin prepared in Example 6 of the present invention on a HepG2 mouse liver cancer model;

[0033] FIG19 shows the curve of the change in relative tumor volume of mice in an in vivo efficacy test of the epirubicin targeted drug Apt AS1411-Apt EGFR-AptA15-DNA-epirubicin prepared in Example 6 of the present invention on a HepG2 mouse liver cancer model;

[0034] FIG20 shows tumor photos of Groups 1, 2, and 4 in an in vivo efficacy test of the epirubicin targeted drug Apt TfRA3-Apt AS1411-Apt A15-DNA-epirubicin prepared in Example 9 of the present invention on a U251 glioma mouse model;

[0035] Figure 21 shows the weight change curves of mice in each group in the in vivo efficacy test of the epirubicin targeted drug Apt TfRA4-Apt AS1411-Apt A15-DNA-epirubicin (1) prepared in Example 8 of the present invention on the GL261-Luc brain glioma mouse model;

[0036] FIG22 shows the percentage change in body weight of mice in each group in an in vivo efficacy test of the epirubicin targeted drug Apt TfRA4-Apt AS1411-Apt A15-DNA-epirubicin (1) prepared in Example 8 of the present invention on a GL261-Luc brain glioma mouse model;

[0037] FIG23 shows an in vivo imaging image of the dorsal side of mice in an in vivo efficacy test of the epirubicin targeted drug Apt TfRA4-Apt AS1411-Apt A15-DNA-epirubicin (1) prepared in Example 8 of the present invention on a GL261-Luc brain glioma mouse model;

[0038] FIG24 shows an in vivo imaging image of the left side of a mouse in the in vivo efficacy test of the epirubicin targeted drug Apt TfRA4-Apt AS1411-Apt A15-DNA-epirubicin (1) prepared in Example 8 of the present invention on a GL261-Luc brain glioma mouse model;

[0039] FIG25 shows an in vivo imaging image of the right side of a mouse in the in vivo efficacy test of the epirubicin targeted drug Apt TfRA4-Apt AS1411-Apt A15-DNA-epirubicin (1) prepared in Example 8 of the present invention on a GL261-Luc brain glioma mouse model;

[0040] FIG26 shows an in vivo imaging image of the ventral side of a mouse in an in vivo efficacy test of the epirubicin targeted drug Apt TfRA4-Apt AS1411-Apt A15-DNA-epirubicin (1) prepared in Example 8 of the present invention on a GL261-Luc brain glioma mouse model;

[0041] Figure 27 shows the BLI value change curve of mouse in vivo imaging in the in vivo efficacy test of the epirubicin targeted drug Apt TfRA4-Apt AS1411-Apt A15-DNA-epirubicin (1) prepared in Example 8 of the present invention on the GL261-Luc brain glioma mouse model; wherein a is the BLI value change curve of the dorsal in vivo imaging of the mouse, b is the BLI value change curve of the left in vivo imaging of the mouse, c is the BLI value change curve of the right in vivo imaging of the mouse, and d is the BLI value change curve of the ventral in vivo imaging of the mouse;

[0042] FIG28 shows the survival curves of mice in each group in the in vivo efficacy test of the epirubicin targeted drug Apt TfRA4-Apt AS1411-Apt A15-DNA-epirubicin (1) prepared in Example 8 of the present invention on the GL261-Luc brain glioma mouse model. DETAILED DESCRIPTION

[0043] It should be noted that, in the absence of conflict, the embodiments and features of the embodiments in this application can be combined with each other. The present invention will be described in detail below with reference to the accompanying drawings and in combination with the embodiments.

[0044] Explanation of terms:

[0045] Oligonucleotide refers to an oligonucleotide molecule including small interfering nucleic acid (siRNA), antisense nucleic acid (ASO), microRNA (miRNA) and nucleic acid aptamer (Aptamer). Oligonucleotide drugs are composed of nucleotides and are a completely new drug category that is completely different from small molecule drugs and antibody drugs. Compared with traditional chemical drug molecules, oligonucleotide drugs have the advantages of strong target specificity, high efficiency, long-lasting drug effect, simple drug design, and rich candidate targets. The main oligonucleotide drugs are siRNA drugs and antisense nucleic acid drugs, which mainly act on cytoplasmic mRNA, recognize and inhibit target mRNA through base complementarity, realize the regulation of protein expression, and achieve the purpose of treating diseases. The nucleic acid or small nucleic acid of this application refers to small nucleic acids.

[0046] Antisense oligonucleotides (ASOs): ASOs, or antisense oligonucleotides, are typically short (16-53 nucleotides), synthetic oligonucleotides designed to block the function of RNA (including mRNA or miRNA). These nucleotides are typically highly complementary to small RNAs and may contain chemically modified nucleotides or have specific sequences added to their ends to increase their binding affinity and resist intracellular nucleic acid degradation.

[0047] When targeting mRNA, ASOs are typically designed to form complementary pairs with specific regions of the target mRNA to prevent reading by the translation machinery, cause mRNA degradation, or modify mRNA splicing.

[0048] When targeting miRNA, ASOs (also called anti-miRNA molecules based on their function) are typically designed to form a complementary pair with a specific miRNA, preventing the miRNA from binding to its target mRNA, thereby inhibiting the function of the miRNA (miRNA is a class of short non-coding RNAs that can form a complementary pair with the 3' untranslated region of the target mRNA, thereby affecting the stability and translation of the mRNA and further regulating protein expression). ASOs that act as anti-miRNA molecules in this application include, but are not limited to, A-miR21, A-miR-10a, A-miR-30c, AmiR1306, and the like.

[0049] MicroRNA (miRNA): A short, non-coding RNA approximately 22 nucleotides in length. Its mechanism of action is to form complementary pairs with the 3' untranslated region (3'UTR) of target mRNAs, thereby regulating the stability or translation of target mRNAs, thereby affecting protein expression. MiRNAs have been shown to play a key role in many biological processes, including development, differentiation, proliferation, and apoptosis.

[0050] MicroRNAs exist in various forms, the most primitive of which is pri-miRNA. Pri-miRNA undergoes a single process to become pre-miRNA, or a microRNA precursor. Pre-miRNA is then cleaved by the Dicer enzyme into mature miRNA. In actual research, pre-miRNAs are the earliest and most widely used, with many commercial microRNA libraries being in pre-miRNA form. Recent studies have also revealed that the arms of microRNAs play a crucial role in the formation of mature miRNAs, leading to an increasing use of the natural pri-miRNA form.

[0051] In addition, as part of a therapeutic strategy, in order to increase the level of specific miRNAs (such as miR-34a or miR-122) in the body, existing studies have proposed the concept of miRNA supplements. Such supplements are usually miRNA analogs or mimics, which can be in RNA form or DNA form. When in DNA form, as long as they can be transcribed into RNA with the same sequence, they can perform the same function as normal miRNAs in cells.

[0052] Using DNA sequences as miRNA supplements has several advantages. First, DNA is more stable and resistant to degrading enzymes in the body. Second, DNA can be read by the cell's transcription machinery and transcribed into the corresponding RNA, thereby increasing miRNA levels within the cell. Finally, DNA synthesis is relatively easy and inexpensive, making it easier to design and manufacture, especially for large-scale production and application.

[0053] Taking miR-34a as an example, miR-34a has been shown to play an important anti-tumor role in many types of cancer. Therefore, increasing miR-34a levels in the body may be an effective cancer treatment strategy. By providing miR-34a supplements in the form of DNA sequences (i.e., miRNA mimics), miR-34a levels in the body can be increased, thereby inhibiting the growth and survival of cancer cells.

[0054] Therefore, in the application, the meaning of miRNA is broad, including various forms, such as the above-mentioned pri-miRNA, pre-miRNA, mature miRNA and miRNA analogs (DNA form, which can form oligonucleotides with the same sequence and the same function as mature miRNA through transcription). In this application, miRNA analogs include but are not limited to one or more of miR-34, miR-542, miR-126 and miR-122.

[0055] Although there are many carriers in the prior art that improve the efficiency of drug delivery, including nucleic acid carriers, it is still difficult to solve the problem of limited clinical application of drugs; at the same time, drug combination is a conventional means of clinical treatment, but it usually refers to the combination of single drugs, and the technical difficulty of the drug itself containing multiple effector molecules is extremely great; in order to solve the above problems, the present invention provides a chemical small molecule drug, which includes a targeted nucleic acid carrier and a chemical small molecule drug mounted on the targeted nucleic acid carrier, and the targeted nucleic acid carrier includes a nucleic acid carrier and a targeting molecule connected to the nucleic acid carrier; wherein the nucleic acid carrier is a DNA vector or an RNA vector, and the nucleic acid vector comprises an a sequence (sequence a), a b sequence (sequence b) and a c sequence (sequence c) that form a self-assembly structure, the a sequence is SEQ ID NO: 1: 5'-ACGAGCGTTCCG-3'; the b sequence is SEQ ID NO: 2: 5'-CGGTTCGCCG-3'; the c sequence is SEQ ID NO: 3: 5'-CGGCCATAGCCGT-3'; or, the a sequence is SEQ ID NO:7: 5'-ACGAGCGUUCCG-3'; the b sequence is SEQ ID NO:8: 5'-CGGUUCGCCG-3'; the c sequence is SEQ ID NO:9: 5'-CGGCCAUAGCCGU-3'; optionally, at least one base is substituted, inserted or deleted in the a sequence, b sequence and c sequence.

[0056] The present invention utilizes a unique nucleic acid vector. This DNA vector maintains the inherent resistance of DNA molecules to nuclease degradation and overcomes the previous problem of DNA's rigid structure hindering its self-assembly. The vector is self-assembled from three pure DNA strands. RNA vectors also exhibit improved self-assembly properties. The inventors also discovered that after the nucleic acid vector is loaded with chemical small molecule drugs and connected to the targeting molecule, it can be covalently inserted into the vector CG structure, or connected to the vector sequence in the form of base substitution or extension, or covalently connected to the vector with a linker, thereby temporarily losing its activity. During the delivery process, it mainly exists in the form of hanging on the vector, will not fall off or rarely exists in a free form, thus greatly reducing its toxicity; on the other hand, the vector can better target the target tissue, target tissue microenvironment and target cell after being mediated by targeting molecules, especially composite targeting adaptors, enter the target cell through endocytosis, cytotoxicity, etc., and release the chemical small molecule drug under the combined action of the low pH environment in the cell, glutathione reduction and enzymatic cleavage, thereby achieving efficient targeted mediation for chemical small molecule drugs and greatly improving the bioavailability of chemical small molecule drugs. At the same time, when the carrier is simultaneously loaded with other drugs (such as gene regulation, immune regulation) and chemical small molecule drugs, the chemical small molecule drugs can very well produce a synergistic effect with other drug effector molecules to achieve combined efficacy, greatly improving the efficiency of single-drug combination therapy and reducing toxicity.

[0057] In a preferred embodiment, the 5' end and 3' end of the sequence a, the sequence b and the sequence c are each independently connected to an extended base segment, and the extended base segment includes 0 to 14 bases; preferably, the nucleic acid vector comprises the following sequence that forms a self-assembly structure:

[0058] The 1st set of sequences:

[0059] a-sequence: SEQ ID NO:7: 5'-GCGGCGCCCACGAGCGTTCCGGGAGC-3';

[0060] b-sequence: SEQ ID NO: 8: 5'-GCTCCCGGTTCGCCGCCAGCCGCC-3';

[0061] c-sequence: SEQ ID NO:9: 5'-GGCGGCAGGCGGCCATAGCCGTGGGCGCCGC-3';

[0062] or

[0063] The 2nd set of sequences:

[0064] a Sequence: SEQ ID NO: 10: 5'-GCGGCGCCCACGAGCGTTCCGGGAGAGGAGC-3';

[0065] b sequence: SEQ ID NO: 11: 5'-GCTCCTCTCCCGGTTCGCCGCGAGCCGCG-3';

[0066] c sequence: SEQ ID NO: 12: 5'-CGCGGCTCGCGGCCATAGCCGTGGGCGCCGC-3';

[0067] or

[0068] The 3rd set of sequences:

[0069] a Sequence: SEQ ID NO: 10: 5'-GCGGCGCCCACGAGCGTTCCGGGAGAGGAGC-3';

[0070] b sequence: SEQ ID NO: 13: 5'-GCTCCTCTCCCGGTTCGCCAGCCAGCCGCGG-3';

[0071] c sequence: SEQ ID NO: 14: 5'-GGCGGCTGGCGGCCATAGCCGTGGGCGCCGC-3';

[0072] or

[0073] The 4th set of sequences:

[0074] a Sequence: SEQ ID NO: 15: 5'-GCGGCGCCCACGAGCGTTCCGGGAGAGGCC-3';

[0075] b sequence: SEQ ID NO: 16: 5'-GGCCTCTCCCGGTTCGCCGCCAGCCGCC-3';

[0076] c sequence: SEQ ID NO: 14: 5'-GGCGGCTGGCGGCCATAGCCGTGGGCGCCGC-3';

[0077] or

[0078] The 5th set of sequences:

[0079] a Sequence: SEQ ID NO: 17: 5'-GCGGCGCCCACGAGCGUUCCGGGAGAGGCC-3';

[0080] b sequence: SEQ ID NO: 18: 5'-GGCCUCUCCCGGUUCGCCGCCAGCCGCC-3';

[0081] c sequence: SEQ ID NO: 19: 5'-GGCGGCUGGCGGCCAUAGCCGUGGGCGCCGC-3';

[0082] or

[0083] The 6th set of sequences:

[0084] a Sequence: SEQ ID NO: 20: 5'-GCGGCGCCCACGAGCGTTCCGGGAGAGGAGGCC-3';

[0085] b sequence: SEQ ID NO: 21: 5'-GGCCTCCTCTCCCGGTTCGCCGCCAGCCGCC-3';

[0086] c sequence: SEQ ID NO: 14: 5'-GGCGGCTGGCGGCCATAGCCGTGGGCGCCGC-3';

[0087] or

[0088] The 7th set of sequences:

[0089] a Sequence: SEQ ID NO: 10: 5'-GCGGCGCCCACGAGCGTTCCGGGAGAGGAGC-3';

[0090] b sequence: SEQ ID NO: 22: 5'-GCTCCTCTCCCGGTTCGCCGCCAGCCGCC-3';

[0091] c sequence: SEQ ID NO: 14: 5'-GGCGGCTGGCGGCCATAGCCGTGGGCGCCGC-3';

[0092] or

[0093] The 8th set of sequences:

[0094] a Sequence: SEQ ID NO: 23: 5'-GCGACGCCCACGAGCGTTCCGGGAGAGGAG-3';

[0095] b sequence: SEQ ID NO: 24: 5'-CTCCTCTCCCGGTTCGCCGCGAGCCGCG-3';

[0096] c sequence: SEQ ID NO: 25: 5'-CGCGGCACGCGGCCATAGCCGTGGGCGTCGC-3';

[0097] or

[0098] The 9th set of sequences:

[0099] a Sequence: SEQ ID NO: 26: 5'-GCGACGCCCACGAGCGTTCCGGGAGAGGAGC-3';

[0100] b sequence: SEQ ID NO: 11: 5'-GCTCCTCTCCCGGTTCGCCGCGAGCCGCG-3';

[0101] c sequence: SEQ ID NO: 27: 5'-CGCGGCTCGCGGCCATAGCCGTGGGCGTCGC-3';

[0102] or

[0103] The 10th set of sequences:

[0104] a Sequence: SEQ ID NO: 28: 5'-GACGCCCACGAGCGTTCCGGGAGAGG-3';

[0105] b sequence: SEQ ID NO: 29: 5′-CCTCTCCCGGTTCGCCGCGAGCCT-3′;

[0106] c sequence: SEQ ID NO: 30: 5'-GGCTCGCGGCCATAGCCGTGGGCGTCTGCTGCTGCTGCTG-3';

[0107] or

[0108] The 11th set of sequences:

[0109] a Sequence: SEQ ID NO: 31: 5'-GCCCACGAGCGTTCGGAGA-3';

[0110] b sequence: SEQ ID NO: 32: 5'-TCTCCCGGTTCGCCGCCAGCCGCC-3';

[0111] c sequence: SEQ ID NO: 33: 5'-GGCGGCTGGCGGCCATAGCCGTGGGC-3'.

[0112] The nucleic acid vector formed by self-assembly of the above-mentioned groups of sequences can more fully exert the above-mentioned effects.

[0113] As mentioned above, using the above-mentioned nucleic acid vector provided by the present invention, when the vector is simultaneously loaded with other drugs (such as gene regulation, immune regulation) and chemical small molecule drugs, the chemical small molecule drugs can very well produce a synergistic effect of combined efficacy with other drug effector molecules, greatly improving the efficiency of single drug combination and reducing toxicity. Specifically, the targeted chemical drug further comprises an oligonucleotide effector molecule and / or an immunostimulant mounted on a targeting nucleic acid carrier; preferably, the oligonucleotide effector molecule comprises one or more of ASO, siRNA, miRNA, and nucleic acid aptamer; more preferably, the siRNA comprises one or more of ASAP1, ATAD2, CD24, CD47, EGFR, HBV, HSP, HS70, PD-L1, PAPP-1, Survivin, TAP, TIM-3, TGF-β1 (TGF-beta1), and VEGF-C; further preferably, the siRNA comprises one or more of ASAP1, CD47, PD-L1, and TGF-β1; more preferably, the miRNA comprises one or more of A-miR21, A-miR-10a, A-miR-30c, miR-34, miR-542, miR-126-3p, and miR-122; further preferably, the miRNA is A-miR21;More preferably, the nucleic acid aptamer includes a nucleic acid aptamer in the form of DNA and / or a nucleic acid aptamer in the form of RNA, and preferably the nucleic acid aptamer includes A1, A15, AS1411, AFP, ATP, Act-12c, A18, BAF7-1, C-Met-SL1, CH6, CA2, C12, CRAC Orail, CEA, CEA-18, CEA-T84, CSC1, CSC13, CD40, CD16a, CD19, CD3-4, CD44, CD12 (HDLBP), CD20, CD24, CD33, CD38, CD105, CD117, CD63, CD123, EGFR, EpCAM, EcR, FAP, anti-FAP, GPC-1, GSK836, GPC3 (APS63-1), Her2, Her3, HMGA2 , H2, HbsAg, IFN-y(B4), IL-4Ra, IL-17, LZH8, MUC1, M5, M7, M1, N5, NG-Dua, NKG2D(20-N-15), NSE, Np-A15, Np-A 48. Np-A58, Np-A61, OX40, PSMA, PDGFRβ (PDGFRbeta), PDGF, PD-L1, PD-1, PTK-7, ProGRP-48, SF, TBA15, TBA29, TfRA4, TfRA3, TTA1, TLS9a, TGF-betaII(S58)(TGF-betaII(S58)), TNF-a, TNF, T1, VEGF, VCAM-1, VCAM-12d, CH6, PL-45, EP66, AGC, Karpas299, SW620, MDA-MB-231, MCF-7, PC-3, BCMA, CTLA-4, CCL1, CD4-3, CD28, FGF2(F2), FGF2, FGF5, LAG-3, MRP1, TIM3, TIMC-1 1, VEGF165, 4-1BB; further preferably, the nucleic acid aptamer includes one or more of A1, A15, AS1411, C12, CD40L, EGFR, GPC-1, IL-4Ra, MUC1, OX40, PD-L1, TTA1, TfRA3, and TfRA4; preferably, the immunostimulant includes one or more of CPG2006, CPG1826, CPG2216, CPG2395, CPG-ODNT7, and CPG-ODN-PCIF1; more preferably, the immunostimulant is CPG2006.

[0114] Specifically, the sequence structure of the sense strand and antisense strand of each siRNA from the 5' end to the 3' end is as follows:

[0115] ASAP1: the sense strand thereof is SEQ ID NO: 34: 5'-UGAUAUUAUGGAAGCAAAUUU-3', and the antisense strand thereof is SEQ ID NO: 35: 5'-AUUUGCUUCCAUAAUAUCAUU-3'; or the sense strand thereof is SEQ ID NO: 36: 5'-UUAGGUUUGGGGUUGGAUCUU-3', and the antisense strand thereof is SEQ ID NO: 37: 5'-GAUCCAACCCCAAACCUAAUU-3';

[0116] ATAD2: its sense strand is SEQ ID NO: 38: 5'-AAUCCUACAACUUCGACGCUU-3', and its antisense strand is SEQ ID NO: 39: 5'-GCGUCGAAGUUGUAGGAUUUU-3';

[0117] CD24: its sense strand is SEQ ID NO:40: 5'-UGUUUACAUUGUUGAGGUAUU-3', and its antisense strand is SEQ ID NO:41: 5'-UACCUCAACAAUGUAAACUUU-3';

[0118] CD47: its sense strand is SEQ ID NO:42: 5'-GGUGAUUACCCAGAGAUAUTT-3', and its antisense strand is SEQ ID NO:43: 5'-AUAUCUCUGGGUAAUCACCTT-3'; or, its sense strand is SEQ ID NO:44: 5'-UGGUGAAAGAGGUCAUUCCUU-3', and its antisense strand is SEQ ID NO:45: 5'-GGAAUGACCUCUUUCACCAUU-3'; or, its sense strand is SEQ ID NO:46: 5'-GGAAUGACCUCUUUCACCATT-3', and its antisense strand is SEQ ID NO:47: 5'-UGGUGAAAGAGGUCAUUCCTT-3'; or, its sense strand is SEQ ID NO:48: 5'-GGUGAUUACCCAGAGAUAUUU-3', and its antisense strand is SEQ ID NO:49: 5'-AUAUCUCUGGGUAAUCACCUU-3'; preferably, the base modifications in the CD47 are as follows: a thiolation modification is performed between two adjacent bases among the 2nd to 4th bases from the 5' end of the sense chain, a thiolation modification is performed between two adjacent bases among the 1st to 3rd bases from the 3' end of the sense chain, a thiolation modification is performed between two adjacent bases among the 1st to 2nd bases from the 5' end of the antisense chain, and a thiolation modification is performed between two adjacent bases among the 1st to 3rd bases from the 3' end of the antisense chain; it should be noted that the purpose of the above-mentioned base modifications is to further improve the structural stability of the drug, and the base modifications appearing below are all for this purpose and will not be repeated here.

[0119] EGFR: its sense strand is SEQ ID NO:50: 5'-GGCUGGUUAUGUCCUCAUUUU-3', and its antisense strand is SEQ ID NO:51: 5'-AAUGAGGACAUAACCAGCCUU-3'; or, its sense strand is SEQ ID NO:52: 5'-UUAGAUAAGACUGCUAAGGUU-3', and its antisense strand is SEQ ID NO:53: 5'-UUAGAUAAGACUGCUAAGGUU-3'; or, its sense strand is SEQ ID NO:54: 5'-UGCCUUAGCAGUCUUAUCUAAUU-3', and its antisense strand is SEQ ID NO:55: 5'-UUAGAUAAGACUGCUAAGGCAUU-3'; or, its sense strand is SEQ ID NO:78: 5'-UGCCUUAGCAGUCUUAUCUAAUUUU-3', and its antisense strand is SEQ ID NO:79:5'-AAUUAGAUAAAGACUGCUAAGGCAUU-3';

[0120] HBV: The sense strand is SEQ ID NO: 56: 5'-GGACUUCUCUCAAUUUUCUUU-3', and the antisense strand is SEQ ID NO: 57: 5'-AGAAAAUUGAGAGAAGUCCUU-3'; preferably, C and U in the HBV are modified to 2'-F;

[0121] HSP: The sense strand is SEQ ID NO: 58: 5'-CGCAGAACACCGUGUUCGAUU-3', and the antisense strand is SEQ ID NO: 59: 5'-UCGAACACGGUGUUCUGCGUU-3'; preferably, the thiocyanate modification is present between two adjacent bases in the first to third bases from the 5' end of the sense strand, and the thiocyanate modification is present between two adjacent bases in the first to third bases from the 3' end of the antisense strand;

[0122] HS70: Its sense strand is SEQ ID NO:60: 5'-GGCCAACAAGAUCACCAUC-3', and its antisense strand is SEQ ID NO:61: 5'-GAUGGUGAUCUUGUUGGCCUU-3'; preferably, in the antisense strand of HS70, the thiocyanate modification is between two adjacent bases in the first to third bases from the 5' end, and the thiocyanate modification is between two adjacent bases in the first to third bases from the 3' end;

[0123] PD-L1: the sense strand is SEQ ID NO:62: 5'-CCAGCACACUGAGAAUCAAUU-3', and the antisense strand is SEQ ID NO:63: 5'-UUGAUUCUCAGUGUGCUGGUU-3'; or the sense strand is SEQ ID NO:64: 5'-AGACGUAAGCAGUGUUGAATT-3', and the antisense strand is SEQ ID NO:65: 5'-UUCAACACUGCUUACGUCUTT-3';

[0124] PAPP-1: its sense strand is SEQ ID NO: 66: 5'-GAGGAAGGUAUCAACAAAUTT-3', and its antisense strand is SEQ ID NO: 67: 5'-AUUUGUUGAUACCUUCCUCTT-3';

[0125] Survivin:

[0126] The sense strand is SEQ ID NO: 70: 5'-GCAGGUUCCUUAUCUGUCACAUU-3', and the antisense strand is SEQ ID NO: 71: 5'-UGUGACAGAUAAGGAACCUGCAGUU-3'; or,

[0127] The sense strand is SEQ ID NO: 72: 5'-GGAAUUGGAAGGCUGGGAACCUU-3', and the antisense strand is SEQ ID NO: 73: 5'-GGUUCCCAGCCUUCCAAUUCCUU-3'; preferably, the sense strand is thiolated between two adjacent bases from the 1st to 3rd bases from the 5' end, and the antisense strand is thiolated between two adjacent bases from the 1st to 3rd bases from the 5' end, and the antisense strand is thiolated between the 1st to 2nd bases from the 3' end; or,

[0128] The sense strand is: SEQ ID NO: 74: 5'-UGCAGGUUCCUUAUCUGUCATT-3', and the antisense strand is: SEQ ID NO: 75: 5'-UGACAGAUAAGGAACCUGCTT-3'; or,

[0129] The sense strand is SEQ ID NO: 76: 5'-CUGCAGGUUCCUUAUCUGUCACAUU-3', and the antisense strand is SEQ ID NO: 77: 5'-UGUGACAGAUAAGGAACCUGCAGUU-3'; preferably, the sense strand has a thiolation modification between two adjacent bases from the 1st to 3rd bases from the 5' end and a thiolation modification between two adjacent bases from the 1st to 3rd bases from the 3' end, and the antisense strand has a thiolation modification between two adjacent bases from the 1st to 3rd bases from the 5' end and a thiolation modification between the 1st to 3rd bases from the 3' end;

[0130] TAP:

[0131] The sense strand is SEQ ID NO:80: 5'-GCUGCACACGGUUCAGAAUUU-3', and the antisense strand is SEQ ID NO:81: 5'-AUUCUGAACCGUGUGCAGCUU-3'; preferably, the sense strand is thiolated between two adjacent bases from the 1st to 3rd bases from the 5' end, and the antisense strand is thiolated between two adjacent bases from the 1st to 3rd bases from the 5' end, and is thiolated between two adjacent bases from the 1st to 3rd bases from the 3' end; or,

[0132] The sense strand is SEQ ID NO:82: 5'-CAGGAUGAGUUACUUGAAAUU-3', and the antisense strand is SEQ ID NO:83: 5'-UUUCAAGUAACUCAUCCUGUU-3';

[0133] TIM-3: its sense strand is SEQ ID NO: 86: 5'-GUGCUCAGGACUGAUGAAATT-3', and its antisense strand is SEQ ID NO: 87: 5'-UUUCAUCAGUCCUGAGCACTT-3';

[0134] TGF-β1: the sense strand thereof is SEQ ID NO:88: 5'-GUCAACUGUGGAGCAACACUU-3', and the antisense strand thereof is SEQ ID NO:89: 5'-GUGUUGCUCCACAGUUGACUU-3'; or the sense strand thereof is SEQ ID NO:90: ​​5'-GCAACAACGCCAUCUAUGATT-3', and the antisense strand thereof is SEQ ID NO:91: 5'-UCAUAGAUGGCGUUGUUGCTT-3';

[0135] VEGF-C:

[0136] The sense strand is SEQ ID NO:92: 5'-GCAAGACGUUGUUUGAAAUUAUU-3', and the antisense strand is SEQ ID NO:93: 5'-UAAUUUCAAACAACGUCUUGCUU-3'; preferably, the sense strand is thiolated between two adjacent bases from the 1st to 3rd bases from the 5' end, and the antisense strand is thiolated between two adjacent bases from the 1st to 3rd bases from the 5' end, and is thiolated between two adjacent bases from the 1st to 3rd bases from the 3' end; or,

[0137] The sense strand is SEQ ID NO:84:5'-CAGCAAGACGUUGUUUGAAAUUAUU-3', and the antisense strand is SEQ ID NO:85:5'-UAAUUUCAAACAACGUCUUGCUGUU-3'; or,

[0138] The sense strand is SEQ ID NO:94: 5'-CAGGAUGGUAAAGACUACAUU-3', and the antisense strand is SEQ ID NO:95: 5'-UGUAGUCUUUACCAUCCUGUU-3'; preferably, the sense strand is thiolated between two adjacent bases from the 1st to 3rd bases from the 5' end, and the antisense strand is thiolated between two adjacent bases from the 1st to 3rd bases from the 5' end, and is thiolated between two adjacent bases from the 1st to 3rd bases from the 3' end;

[0139] Preferably, the miRNA includes one or more of miR-34, miR-542, miR-126-3p and miR-122;

[0140] Preferably, the ASO includes one or more of A-miR21, A-miR-10a, A-miR-30c, and A-miR-1306;

[0141] The sequence structure of each miRNA and ASO from 5' to 3' end is as follows:

[0142] A-miR21:

[0143] 5'-GATAAGCT-3'; preferably, each base thereof is modified with a locked nucleic acid; or,

[0144] 5'-GAUAAGCU-3'; preferably, each base thereof is modified with a locked nucleic acid; or,

[0145] SEQ ID NO:96:5'-GTCAACATCAGTCTGATAAGCTA-3'; or,

[0146] SEQ ID NO:97:5'-GUCAACAUCAGUCUGAUAAGCUA-3'; or,

[0147] SEQ ID NO:98:5'-TCAACATCAGTCTGATAAGCTA; or,

[0148] 5'-GATAAGCT-3'; preferably, thiolation modification between two adjacent bases;

[0149] A-miR-10a: 5'-ACAGGGTA-3'; preferably, each base thereof is modified with a locked nucleic acid;

[0150] A-miR-30c: SEQ ID NO:99: 5'-GCTGAGAGTGTAGGATGTTTACA-3';

[0151] miR-34: 5′-TGTGACAG-3′;

[0152] miR-542: 5′-TGGCAGTGT-3′;

[0153] miR-126-3p: 5'-UCGUACC-3'; preferably, the adjacent bases are thiolated;

[0154] miR-122: 5′-GGAAGTGT-3;

[0155] AmiR-1306: SEQ ID NO: 100: 5'-CATCACCACCAGAGCCAACGTC-3'; preferably, the 5' end of the 5' base sequence is thiolated between two adjacent bases;

[0156] The sequence structure of each nucleic acid aptamer from the 5' end to the 3' end is as follows:

[0157] A1: SEQ ID NO: 101: 5'-GGTTGCATGCCGTGGGGAGGGGGGTGGGTTTTATAGCGTACTCAG-3';

[0158] A15: SEQ ID NO: 102: 5'-CCCTCCTACATAGGG-3'; or, SEQ ID NO: 227: 5'-CCCUCCUACAUAGGG-3';

[0159] AS1411: SEQ ID NO: 103: 5'-GGTGGTGGTGGTTGTGGTGGTGGTGG-3'; or, SEQ ID NO: 228: 5'-GGUGGUGGUGGUUGUGGUGGUGGUGG-3'; or, SEQ ID NO: 229: AUUCUGAACCGUGUGCAGCACCACGCUGCACACGGUUCAGAAUACACACGAGGCTAT CTAGAATGTAC-3';

[0160] AFP: SEQ ID NO: 104: 5'-GGCAGGAAGACAAACAAGCTTGGCGGCGGGAAGGTGTTTAAATTCCCGGGTCTGCGT GGTCTGTGGTGCTGT-3'; or, SEQ ID NO: 105: 5'-ACCTGGGGAGTATTGGGGAGGAAGG-3';

[0161] ATP: SEQ ID NO: 106: 5'-ACCTGGGGAGTATTGGGGAGGAAGG-3'; or, SEQ ID NO: 107: 5'-GGGAGGACGATGCGGAGGAAGGGTAGG-3', preferably a thiolation modification between two adjacent bases in the first to fourth bases from the 5' end;

[0162] Act-12c: SEQ ID NO: 108: 5'-CGGGGAAAGTCACGGGGGGTTTCAGATGTTCTGATCGGTGTGGAG-3';

[0163] A18: SEQ ID NO:109: 5'-CCAGATAGTCCCTGG-3';

[0164] BAF7-1: SEQ ID NO: 110: 5'-GATAACGGGCACGAATTCGGAGTG-3';

[0165] C-Met-SL1: SEQ ID NO: 111: 5'-ATCAGGCTGGATGGTAGCTCGGTCGGGGTGGGTGGGTTGGCAAGTCTGAT-3';

[0166] CH6: SEQ ID NO: 112: 5'-AGTCTGTTGGACCGAATCCCGTGGACGCACCCTTTGGACG-3';

[0167] CA2: SEQ ID NO: 113: 5'-CCCACCGTCTGCGCTTAGCTCCTGGGCCTGGATGGGC-3';

[0168] C12: SEQ ID NO: 114: 5'-GTGGATTGTTGTGTTCTGTTGGTTTTTGTGTTGTC-3'; preferably, the first to third bases from the 5' end are thiolated between two adjacent bases;

[0169] CRAC Orail: SEQ ID NO: 115: 5'-CCAGTAGCCATACCGGTTTGTGGATGGGGTGTATGCGAGT-3';

[0170] CEA: SEQ ID NO: 116: 5'-CTAGGATCCCCACTCACCATCTCTCAGCTTGCTTCCTAGC-3'; or, SEQ ID NO: 234: 5'-CUAGGAUCCCCACUCACCAUCUCAGCUUGCUUCCUAGC-3'; CEA-18: SEQ ID NO: 117: 5'-TTAACTTATTCGACCATA-3';

[0171] CEA-T84: SEQ ID NO: 118: 5'-TCGCGCGAGTCGTCTGGGGAACCATCGAGTTACACCGACCTTCTATGTGCGGCCCCCC GCATCGTCCTCCC-3';

[0172] CSC1: SEQ ID NO:119: 5’-ACCTTGGCTGTCGTGTTGTAGGTGGTTTGCTGCGGTGGGCTCAAGAAGAAAGCGCAA AGAGGTCAGTGGTCAGAGCGT-3’;

[0173] CSC13: SEQ ID NO:120: 5’-ACCTTGGCTGTCGTGTTGTGGGGTGTCGTATCTTTCGTGTCTTATTATTTTCTAGGTGGA GGTCAGTGGTCAGAGCGT-3’;

[0174] CD40: SEQ ID NO:121: 5’-CCAACGAGTAGGCGATAGCGCGTGG-3’; or, SEQ ID NO:122: 5’-AGAGACGATGCGGCCAACGAGTAGGCGATAGCGCGTGGCAGAGCGTCGCT-3’;

[0175] CD16a: SEQ ID NO:123: 5’-CCACTGCGGGGGTCTATACGTGAGGAAGAAGTGG-3’;

[0176] CD19: SEQ ID NO:124: 5’-TGCGTGTGTAGTGTGTGTCGTTTCTCCTTTTTTTGGTTGCTGCTCTTAGGGATTTGGGC GG-3’; or, SEQ ID NO:125: 5’-TGCGTGTGTAGTGTGTCTGTTCTCCTTTTTTTGGTTGCTGCTCTTAGGGATTTGGGCGG-3’;

[0177] CD3-4: SEQ ID NO:126: 5’-TCTCGGACGCGTGTGGTCGGCCGAGTGGCCCACGGTAGAAGGGTTAGAACTGCTGGT TGGTGAATCTCGCTGCCTGGCCCTAGAGTG-3’;

[0178] CD44:

[0179] SEQ ID NO:127: 5’-CCAAGGCCTGCAAGGGAACCAAGGACACAG-3’; or,

[0180] SEQ ID NO: 128: 5'-CCAAGGCCTGCAAGGGAACCAAGGACACAG-3'; preferably, the thiolation modification is between two adjacent bases from the 3rd to 5th bases from the 5' end, the thiolation modification is between two adjacent bases from the 12th to 14th bases from the 5' end, the thiolation modification is between the 1st to 2nd bases from the 3' end, the thiolation modification is between the 3rd to 4th bases from the 3' end, the thiolation modification is between the 5th to 6th bases from the 3' end, and the thiolation modification is between two adjacent bases from the 12th to 14th bases from the 3' end; or,

[0181] SEQ ID NO:237: 5'-GGGAUGGAUCCAAGCUUACUGGCAUCUGGAUUUGCGGCGUGCCAGAAUAAAGAGUAUAACGUGUGAAUGGGAAGCUUCGAUAGGAAUUCGG-3';

[0182] CD12 (HDLBP): SEQ ID NO: 129: 5'-GTGGATTGTTGTGTTCTGTTGGTTTTTGTGTTGTC-3';

[0183] CD20: SEQ ID NO: 130: 5'-TGCGTGTGTAGTGTGTCTGTTTTTTATCTTCTTTTATCTACTCTTAGGGATTTGGGCGG-3';

[0184] CD24: SEQ ID NO: 131: 5'-TATGTGGGTGGGTGGGCGGTTATGCTGAGTCAGCCTTGCT-3'; or, SEQ ID NO: 132: 5'-ATCCAGAGTGACGCAGCATATGTGGGTGGGTGGGCGGTTATGCTGAGTCAGCCTTGCT TGGACACGGTGGCTTAGT-3';

[0185] CD33: SEQ ID NO: 133: 5'-TACCAGTGCGATGCTCAGCACGCTTATAGGGGCTGGACAAAATTCTACCCAGCCTTT-3';

[0186] CD38: SEQ ID NO: 134: 5'-TACGTGAATCTCGTACGATACTCTGTAAGCGT-3';

[0187] CD105: SEQ ID NO:135: 5’-GATCAGTTTTCCATGCCAGTTGGTATTCCGCGACAGTTTGATCTC-3’;

[0188] CD117: SEQ ID NO:136: 5’-GGGGCCGGGGCAAGGGGGGGGTACCGTGGTAGGAC-3’;

[0189] CD63: SEQ ID NO:137: 5’-CACCCCACCTCGCTCCCGTGACACTAATGCTA-3’;

[0190] CD123: SEQ ID NO:138: 5’-TGCGTGTGTAGTGTGTCTGGGCTACATCGATGAGCTGCCTAGGGTCCCTCTTAGGGATTTGGGCGG-3’;

[0191] EGFR: SEQ ID NO:139: 5’-GCCTTAGTAACGTGCTTTGATGTCGATTCGACAGGAGGC-3’; or, SEQ ID NO:239: 5’-GCCUUAGUAACGUGCUUUGAUGUCGAUUCGACAGGAGGC-3’; or, SEQ ID NO:240: 5’-UGCCGCUAUAAUGCACGGAUUUAAUCGCCGUAGAAAAGCAUGUCAAAGCCGUU-3’;

[0192] EpCAM: SEQ ID NO:140: 5’-CACTACAGAGGTTGCGTCTGTCCCACGTTGTCATGGGGGGTTGGCCTG-3’; or, SEQ ID NO:141: 5’-GACAAACGGGGGAAGATTTGACGTCGACGAC-3’; or, SEQ ID NO:241: 5’-GCGACUGGUUACCCGGUCG-3’;

[0193] EcR: SEQ ID NO:142: 5’-GCAGGTCCACTGCGGGGGTCTATACGTGAGGAAGAAGTGGGCAGGTC-3’;

[0194] FAP: SEQ ID NO:143: 5’-TGGGGGTTGAGGCTAAGCCGA-3’;

[0195] Anti-FAP: SEQ ID NO: 144: 5'-CCGCTCGAGCTAGTCTGACAAAGAGAAACAC-3';

[0196] GPC-1: SEQ ID NO: 145: 5'-AACGGAGTGTGGCTAACTCGA-3';

[0197] GSK836: SEQ ID NO: 147: 5'-GCAGAGGTGAAGCGAAGTCG-3'; preferably, the 5' end of the nucleotide sequence of bases 1 to 6 is thiolated between two adjacent bases;

[0198] GPC3 (APS63-1): SEQ ID NO: 148: 5'-TAACGCTGACCTTAGCTGCATGGCTTTACATGTTCCA-3'; preferably, the 5' end and the 1st to 4th bases from the 3' end are thiolated between two adjacent bases;

[0199] Her2: SEQ ID NO: 149: 5'-AGCCGCGAGGGGAGGGATAGGGTAGGGCGCGGCT-3'; or, SEQ ID NO: 150: 5'-GGGAGCTCAGAATAAACGCTCAAAGGGTCAAGCTGATTACACTTTGTCCACTATTGGG TCCTTCGACATGAGGCCCGGATC-3'; or, SEQ ID NO: 246: 5'-AGCCGCGAGGGGAGGGAUAGGGUAGGGCGCGGCU-3';

[0200] Her3: SEQ ID NO: 151: 5'-GGGAGCTCAGAATAAACGCTCAAGGCTAACAGCACGCAACGGGGGGGAGTAATCGTG TCTGTTCGACATGAGGCCCGGATC-3'; or, SEQ ID NO: 247: 5'-CAGCGAAAGUUGCGUAUGGGUCACAUCGCAGGCACAUGUCAUCUGGGCG-3'; or, SEQ ID NO:248: 5'-GAAUUCCGCGUGUGCCAGCGAAAGUUGCGUAUGGGCCACAUCGCAGGCACAUGUCAUCUGGGCGGUCCGUUCGGGAUCC-3';

[0201] HMGA2: SEQ ID NO: 152: 5'-GGAAAAAATTTTTTAAAAAACCC-3'; preferably, a thiolation modification is performed between two adjacent bases;

[0202] H2: SEQ ID NO: 146: 5'-GGGCCGTCGAACACGAGCATGGTGCGTGGACCTAGGATGACCTGAGTACTGTCC-3';

[0203] HBsAg: SEQ ID NO: 153: 5'-CACAGCGAACAGCGGCGGACATAATAGTGCTTACTACGAC-3'; preferably, the bases 1 to 4 from the 5' end are thiolated between two adjacent bases;

[0204] IFN-y (B4): SEQ ID NO: 154: 5'-CCGCCCAAATCCCTAAGAGAAGACTGTAATGACATCAAACCAGACACACACACTACACACGCA-3';

[0205] IL-4Ra: SEQ ID NO: 155: 5'-GGAGGACGAUGCGGAAAAAGCAACAGGGUGCUCCAUGCGCAUGGAACCUGCGCGCAGACGACUCGCUGAGGAUCCGAGA-3'; or, SEQ ID NO: 156: 5'-AAAAAGCACAGGGUGCUCCAUGCGCAUGGAACCUGCGCG-3';

[0206] IL-17: SEQ ID NO: 157: 5'-CTTGGATCACCATAGTCGCTAGTCGAGGCT-3'; or, SEQ ID NO: 158: 5'-GCGGCATCCTATCACGCATTGACC-3';

[0207] LZH8: SEQ ID NO: 159: 5'-ATCCAGAGTGACGCAGCATATTAGTACGGCTTAACCCCATGGTGGACACGGTGGCTTAGT-3';

[0208] MUC1: SEQ ID NO:160: 5’-GCAGTTGATCCTTTGGATACCCTGG-3’; or, SEQ ID NO:161: 5’-GAAGTGAAAATGACAGAACACAACA-3’; or, SEQ ID NO:162: 5’-AACCGCCCAAATCTCTAAGAGTCGGACTGCAACCTATGCTATCGTTGATGTCTGTCCAAGCAACACAGACACACTACACACACGCACA-3’; or, SEQ ID NO:163: 5’-AATGACAGAACACAACATT-3’; or, SEQ ID NO:250: 5’-GCAGUUGAUCCUUUGGAUACCCUGG-3’;

[0209] M5: SEQ ID NO:164: 5’-AGCAGCACAGAGGTCAGATGCTTGGTTCCACCGTACTGACTGTAGTAAAATCTGATCACTCCTATGCGTGCTACCGTGAA-3’;

[0210] M7: SEQ ID NO:165: 5’-AGCAGCACAGAGGTCAGATGTAGTCGGTCTTCTTGTTTGAAACTGCTAATTTTGAAAAAACCTATGCGTGCTACCGTGAA-3’;

[0211] M1: SEQ ID NO:166: 5’-AGCAGCACAGAGGTCAGATGATATAACCTTAATAAATAAAATATAAATTATTTAATCTTACCTATGCGTGCTACCGTGAA-3’;

[0212] N5: SEQ ID NO:167: 5’-GATTGAGTAGATAGTGGTTCTGTACGTAGTGAAAGAGTGG-3’;

[0213] N-G-Dua: SEQ ID NO:168: 5’-CAAGTTGCTCGTCGCGATACGTTTGGTTGGTGTGGTTGGCAGTATCGCAGGTCCAAGTTGCTCGTCGCGATACAACGGAGTGTGGCTAACTCGA-3’;

[0214] NKG2D(20-N-15):SEQ ID NO:169:5’-CAAGTTGCTCGTCGCGATACGTTTGGTTGGTGTGGTTGGCAGTATC-3’;

[0215] NSE:SEQ ID NO:170:5’-TCACACGGACCTCTCTCTACATTAATTGCGCATTTCGTT-3’;

[0216] Np-A15:SEQ ID NO:171:5’-GCTGGATGTTCATGCTGGCAAAATTCCTTAGGGGCACCGTTACTTTGACACATCCAGC-3’;

[0217] Np-A48:SEQ ID NO:172:5’-GCTGGATGTCGCTTACGACAATATTCCTTAGGGGCACCGCTACATTGACACATCCAGC-3’;

[0218] Np-A58:SEQ ID NO:173:5’-GCTGGATGTCACCGGATTGTCGGACATCGGATTGTCTGAGTCATATGACACATCCAGC-3’;

[0219] Np-A61:SEQ ID NO:174:5’-GCTGGATGTTGACCTTTACAGATCGGATTCTGTGGGGCGTTAAACTGACACATCCAGC-3’;

[0220] OX40: SEQ ID NO:175: 5’-GGGAGGACGATGCGGCAGTCTGCATCGTAGGAATCGCCACCGTATACTTTCCCACCAGACGACTCGCTGAGGATCCGAGA-3’; or, SEQ ID NO:176: 5’-CAGTCTGCATCGTAGGATTAGCCACCGUATCTTTCCCAC-3’; or, SEQ ID NO:177: 5’-CCAACGAGTAGGCGATAGCGCGTGG-3’; or, SEQ ID NO:252: 5’-GGGAGGACGAUGCGGCAGUCUGCAUCGUAGGAAUCGCCACCGUAUACUUUCCCACCAGACGACUCGCUGAGGAUCCGAGA-3’; or, SEQ ID NO:253: 5’-GGGAGGACGAUGCGGCAGUCUGCAUCGUAGGAAUCGCCACCGUAUACUUUCCCACCAGACGACUCGCUG-3’; or, SEQ ID NO:254: 5’-CAGUCUGCAUCGUAGGAAUCGCCACCGUAUACUUUCCCAC-3’; or, SEQ ID NO:255: 5’-GGGAUGCGGAAAAAAGAACACUUCCGAUUAGGGCCCACCCUAACGGCCGCAGAC-3’;

[0221] PSMA: SEQ ID NO:179: 5’-GGGAGGACGATGCGGATCAGCCATGTTTACGTCACTCCT-3’; or, SEQ ID NO:180: 5’-GCGTTTTCGCTTTTGCGTTTTGGGTCATCTGCTTACGATAGCAATGCT-3’; or, SEQ ID NO:256: 5’-GGGACCGAAAAAGACCUGACUUCUAUACUAAGUCUACGUUCCC-3’; or, SEQ ID NO:257: 5’-GGGAGGACGAUGCGGAUCAGCCAUGUUUACGUCACUCCU-3’;

[0222] PDGFRβ: SEQ ID NO:181: 5’-TGTCGTGGGGCATCGAGTAAATGCAATTCGACA-3’; or, SEQ ID NO:258: 5’-UGUCGUGGGGCAUCGAGUAAAUGCAAUUCGACA-3’;

[0223] PDGF: SEQ ID NO:182: 5’-CAGGCTACGGCACGTAGAGCATCACCATGATCCTG-3’;

[0224] PD-L1: SEQ ID NO:183: 5’-AACAACGTATAACAATGCCCACGTCACCAGAGTACTATGG-3’; or, SEQ ID NO:184: 5’-GCCCCAGTTATGCTTTCCCCCTCTGTCTCTTTG-3’; or, SEQ ID NO:185: 5’-ATCGCCCGCAGCACCCATTTGTTTTTTTTTG-3’; or, SEQ ID NO:69: ACGGGCCACATCAACTCATTGATAGACAATGCGTCCACTGCCCGT; or, SEQ ID NO:186: 5’-TGCCCGCACATCAACTCATTGATAGACAATGCGTCCACTCGGGCA-3’; or, SEQ ID NO:187: 5’-TGCCCGCACATCAACTCATTGATAGACAATGCGTCCACTACGGGC-3’; or, SEQ ID NO:188: 5’-CGGGCACACATCAACTCATTGATAGACAATGCGTCCACTGCCCGT-3’; or, SEQ ID NO:189: 5’-GTTGGTCACATCAACTCATTGATAGACAATGCGTCCACTACCAAC-3’; or, SEQ ID NO:190: 5’-GGGCCACATCAACTCATTGATAGACAATGCGTCCACTGCCC-3’; or, SEQ ID NO:191: 5’-TGGTTGCACATCAACTCATTGATAGACAATGCGTCCACTCAACCA-3’; or, SEQ ID NO:200: 5’-TACAGGTTCTGGGGGGTGGGTGGGGAACCTGTT-3’; or, SEQ ID NO:238: 5’-CTACGAGACGAACTTATGCGTAATAATGACTGTCGTAG-3’;

[0225] PD-1: SEQ ID NO:192: 5’-GACGATAGCGGTGACGGCACAGACGGTACAGTTCCCGTCCCTGCACTACACGTATGCC GCTTCCGTCCGTCGCTC-3’; or, SEQ ID NO:193: 5’-GAGCGACGGACGGAAGCGGCATACGTGTAGTGCAGGGACGGGAACTGTACCGTCTGT GCCGTCACCGCTATCGTC-3’; or, SEQ ID NO:194: 5’-GGATCCTAGACGCATTGACCCGCTGCCTCTACTGAGGCTGTGTCAGTGTGCGGCTCGG ACTGTTGAATTC-3’; or, SEQ ID NO:195: 5’-AGCGGTGACGGCACAGACGGTACAGTTCCCGTCCCTGCACTACACGTATGCCGCTGA GAGAGAGGGAGGC-3’; or, SEQ ID NO:196: 5’-ACCGACAGTGAAGGACTCAGCGAACTCTCAGACTCGGTTC-3’;

[0226] PTK-7: SEQ ID NO:197: 5’-ATCTAACTGCTGCGCCGCCGGGAAAATACTGTACGGTTAGA-3’;

[0227] ProGRP-48: SEQ ID NO:198: 5’-CATGCGGAGTAGAGCGAGCCCAGATAGTCCCTGGTTATTTCCTTAGG-3’;

[0228] SF: SEQ ID NO:199: 5’-GATCTCTCTCTGCCCTAAGTCCGCACCCGTGCTTCCCTGT-3’;

[0229] TBA15: SEQ ID NO:201: 5’-GGTTGGTGTGGTTGG-3’;

[0230] TBA29: SEQ ID NO:202: 5’-AGTCCGTGGTAGGGCAGGTTGGGGTGACT-3’;

[0231] TfRA4: SEQ ID NO:203: 5’-GCGTGGTACCACGC-3’; or, SEQ ID NO:259: 5’-GCGUGGUACCACGC-3’;

[0232] TfRA3: SEQ ID NO:204: 5'-GCGTGGTCACACGC-3'; or, SEQ ID NO:205: 5'-GCGGCGCCCACGAGCGTTCGCGTGGTCACACGCGTTCCGCCCTCCTACATAGGGCGCATAGCCGTGGGCGCCGC-3'; or, SEQ ID NO:260: 5'-GCGUGGUCACACGC-3';

[0233] TTA1: SEQ ID NO:206: 5'-CCTGCACTTGGCTTGGATTTCAGAAGGGAGACCC-3'; or, SEQ ID NO:286: 5'-CTGCACTTGGCTTGGATTTCAGAAGGGAGACCC-3'; or, SEQ ID NO:261: 5'-CCUGCACUUGGCTTGGAUUUCAGAAGGGAGACCC-3';

[0234] TLS9a: SEQ ID NO:207: 5'-AGTCCATTTTATTCCTGAATATTTGTTAACCTCATGGAC-3';

[0235] TGF-βII(S58): SEQ ID NO:208: 5'-ACATTGCTGCGTGATCGCCTCACATGGGTTTGTCTGGTCGATTTGGAGGTGGTGGGTGGC-3';

[0236] TNF-a: SEQ ID NO:209: 5'-GCGGCCGATAAGGTCTTTCCAAGCGAACGAAAA-3';

[0237] TNF: SEQ ID NO:210: 5'-GCGCCACTACAGGGGAGCTGCCATTCGAATAGGTGGGCCGC-3';

[0238] T1: SEQ ID NO:211: 5'-CGCTCGATAGATCGAGCTTCGCTCGATGTGGTGTTGTGGGGGCTTGTATTGGTCGATCACGCTCTAGAGCACTG-3';

[0239] VEGF: SEQ ID NO:212: 5’-TGTGGGGGTGGACTGGGTGGGTACC-3’; or, SEQ ID NO:213: 5’-TGTGGGGGTGGACGGGCCGGGTAGA-3’; or, SEQ ID NO:214: 5’-GGTGGGGGTGGACGGGCCGGGTAGA-3’; or, SEQ ID NO:266: 5’-AUGCAGUUUGAGAAGUCGCGCAU-3’; preferably with phosphorothioate modification between the 6th and 7th bases from the 5’ end.

[0240] VCAM-1: SEQ ID NO:215: 5’-ATACCAGCTTATTCAATTGGACACGGCAAAGGGGTATAGCCTACCGGACCGTGAACATGGAATGGTGTGCTGCGTGGAGATAGTAAGTGCAATCT-3’; or, SEQ ID NO:216: 5’-GGACACGGCAAAGGGGTATAGCCTACCGGACCGTGAACATGGAATGGTGTGCTGCGTGG-3’;

[0241] VCAM-12d: SEQ ID NO:217: 5’-AGGGAATCTTGCCTAGGGAGGGAGTAGCGAAAGGGCTCA-3’;

[0242] CH6: SEQ ID NO:218: 5’-AGTCTGTTGGACCGAATCCCGTGGACGCACCCTTTGGACG-3’;

[0243] PL-45: SEQ ID NO:219: 5’-ACTCATAGGGTTAGGGGCTGCTGGCCAGATACTCAGATGGTAGGGTTACTATGAGC-3’;

[0244] EP66: SEQ ID NO:220: 5’-AACAGAGGGACAAACGGGGGAAGATTTGACGTCGACGACA-3’;

[0245] AGC: SEQ ID NO:221: 5’-CGACCCGGCACAAACCCAGAACCATATACACGATCATTCGTCTCCTGGGCCG-3’;

[0246] Karpas299:SEQ ID NO:222:5'-ATCCAGATGACGCAGCACCACCACCGTACAATTTTTTCATTACCTACTCGGC-3';

[0247] SW620:SEQ ID NO:223:5'-CCCATCAATGTTACGACCCGCTAGGGCTGCTGTGCCATCGGGTAA-3';

[0248] MDA-MB-231:SEQ ID NO:224:5'-AGAATTCAGTCGGACAGCGAAGTAGTTTTCCTTCTAACCTAAGAACCCGCGGCAGTTTAATGTAGA-3':

[0249] MCF-7:SEQ ID NO:225:5'-GCATGGGGTTTCGGCGTTTCGTCTATCTTGTTTCTGTTAGCGTCT-3':

[0250] PC-3:SEQ ID NO:226:5'-TGCCACTACAGCTGGTTCGGTTTGGTGACTTCGTTCTTCGTTGTGGTGCTTAGTGGC-3';

[0251] BCMA:SEQ ID NO:230:5'-AGUGCAAGACGUUCGCAGAUUAGCGAAAAGGGUCAUUGACUAGUAC-3';

[0252] CTLA-4:SEQ ID NO:231:5'-GGUGGAAGAGGGAUGGGCCGACGUGCCGCAU-3';

[0253] CCL1:SEQ ID NO:233:5'-UGACUCCCUCUGACAGCCUAAUUUCUCCCGAUUACCCUG-3':

[0254] CD4-3:SEQ ID NO:235:5'-GGGAGGACGAUGCGGUUUGGGGUUUUCCCGUGCCCCAGACGACUCGCCCGA-3';

[0255] CD28: SEQ ID NO:236: 5’-GGGAGAGAGGAAGAGGGAUGGGGAUUAGACCAUAGGCUCCCAACCCCCCCCGGGAGAGAGGAAGAGGGAUGGGGAUUAGACCAUAGGCUCCCAACCCCCGGG-3’;

[0256] FGF2(F2): SEQ ID NO:242: 5’-GGGAUACUAGGGCAUUAAUGUUACCAGUGUAGUCCC-3’;

[0257] FGF2: SEQ ID NO:243: 5’-GGGAAACUAGGGCGUUAACGUGACCAGUGUUUCUCGA-3’; or, SEQ ID NO:244: 5’-GGGAAACUAGGGCGUUAACGUGACCAGUGUUUCCC-3’;

[0258] FGF5: SEQ ID NO:245: 5’-GGGCGACCUCUCCGUACUGACCUACAGAGCGACAUACUAGUGUAUCCAGAUCGCCC-3’;

[0259] LAG-3: SEQ ID NO:249: 5’-GGGAGAGAGAUAUAAGGGCCUCCUGAUACCCGCUGCUAUCUGGACCGAUCCCAUUACCAAAUUCUCUCCC-3’;

[0260] MRP1: SEQ ID NO:251: �-GGGAGAAUAGUCAACAAAUCGUUUGGGGCGACUUCUCCUUCCUUUCUCCC-3’;

[0261] TIM3: SEQ ID NO:262: 5’-GGGAGAGGACCAGUA-GCCACUAUGGUGUUGGAGCUAGCGG-CAGAGCGUCGCGGUCCCUCCC-3’; or, SEQ ID NO:263: 5’-GGGAGAGGACCAGUA-CUGGUAGUUCUCUGUGCGACUCCUA-CAGAGCGUCGCGGUCCCUCCC-3’;

[0262] TIMC-11: SEQ ID NO: 264: 5'-AAGCAACACUUAGUCGCGAUUGAUACGUGCGCAGUCAU-3'; or, SEQ ID NO: 265: 5'-GGAGGACGAUGCGGGGAAGCAACACUUAGUCGCGAUUGAUACGUGCGCAGUCAUCAGACGACUCGCUGAGGAUCCGAGA-3';

[0263] VEGF165: SEQ ID NO:267: 5'-CGGAAUCAGUGAAUGCUUA UACAUCCG-3';

[0264] 4-1BB: SEQ ID NO:268: 5'-GGGAGAGAGGAAGAGGGAUGGGCGACCGAACGUGCCCUUCAAAGCCGUUCACUAACCAGUGGCAUAACCCAGAGGUCGAUAGUACUGGAUCCCCCC-3';

[0265] The sequence structure of each of the immunostimulants from the 5' end to the 3' end is as follows:

[0266] CPG2006:

[0267] SEQ ID NO: 269: 5'-TCGTCGTTTTGTCGTTTTGTCGTT-3'; preferably, a thiolation modification between two adjacent bases; or,

[0268] SEQ ID NO:270: 5'-UCGUCGUUUUGUCGUUUUGUCGUU-3';

[0269] CPG1826: SEQ ID NO: 271: TCCATGACGTTCCTGACG-3'; preferably, a thiolation modification is performed between two adjacent bases;

[0270] CPG2216: SEQ ID NO:272: 5'-GGGGGACGATCGTCGGGGGG-3';

[0271] CPG2395: SEQ ID NO:273:TCGTCGTTTTCGGCGCGCGCCG-3';

[0272] CPG-ODNT7: SEQ ID NO:274: 5'-TCGTCGTCGTCGTCGTCGTCG-3'; or, SEQ ID NO:275: 5'-TCGTCGTCGTCGTCGTCGTCGTCG-3';

[0273] CPG-ODN-PCIF1:5'-AGCGAA-3'.

[0274] Preferably, when the targeted chemical drug includes a nucleic acid aptamer, the targeting molecule includes a nucleic acid aptamer and / or a small molecule target head; when the targeted chemical drug does not include a nucleic acid aptamer, the targeting molecule is a small molecule target head, wherein the small molecule target head is selected from one or more of folic acid, biotin, vitamin B12 and mannose.

[0275] Selecting the above-mentioned oligonucleotide effector molecules, immunostimulants, and small molecule targets, along with chemical small molecule drugs, and attaching them to specific nucleic acid vectors can further enhance synergistic effects and achieve even more beneficial outcomes. For example, the nucleolin aptamer AS1411 can bind specifically and with high affinity to nucleolin on the surface of cancer cells, while having no affinity for normal cells. Binding to nucleolin on the tumor cell membrane induces macropinocytosis, allowing rapid cell entry. This is further facilitated by inducing the activity of EGFR, Akt, P38, and Racl. Nucleolin, as a multifunctional shuttle protein, acts as a molecular chaperone, shuttling between the cell membrane, cytoplasm, and nucleus during this process. EGFR-targeted aptamers, as auxiliary target molecules for combined targeting combinations, are highly expressed in tumor cells. Aptamers targeting EGFR can bind specifically and with high affinity to the EGFR receptor protein on the surface of cancer cells, while having no or low affinity for normal cells. CD133+ tumor cells have stronger self-renewal, differentiation and tumorigenic capabilities. CD133 is a common marker similar to tumor stem cells and is highly expressed in a variety of solid tumors (e.g., glioma, breast cancer, gastric cancer, liver cancer, lung cancer, pancreatic cancer, prostate cancer, colorectal cancer, etc.). A15, as an aptamer of CD133, has a targeting effect of tracking and specifically binding to CD133+ tumor cells in vivo. The present invention modifies the A15 aptamer at one end of the above-mentioned DNA vector trident structure in a sequence extension manner, and more preferably assembles it with the AS1411 and EGFR aptamers modified at the other two ends of the nanonucleic acid vector to form a composite targeted and epirubicin-loaded improved chemical drug. The targeted improved epirubicin targeted drug, when administered intravenously, can multiple-anchor tumor cells overexpressing nucleolin, EGFR, and CD133 in tumor tissues to synergistically enhance the chance and efficiency of targeted binding between the targeted drug and the tumor cells. After internalization into the cell, the nanonucleic acid aggregate undergoes reduction and enzymatic cleavage under the action of low pH, high glutathione, and nuclease, releasing the epirubicin original drug. Epirubicin enters the cell nucleus, and its acene four-ring structure specifically inserts into the GC / CG site of the target gene DNA, inhibiting DNA transcription, thereby inhibiting the proliferation and development of the tumor. The A15 aptamer mediates the nanonucleic acid aggregate to further track CD133+ tumor stem cells, inhibiting the proliferation of key tumor "seed" cells, thereby achieving control or regression of tumor development. MiR-21 is a gene that confers drug resistance. Using its resistance sequence can inhibit the development of drug resistance, enhancing the drug's toxicity and thus more effectively killing tumor cells. The TfRA4 target head mediates nanoparticles that cross the blood-brain barrier, carrying epirubicin and more effectively killing tumor cells in the brain.In addition to AS1411, A15, EGFR, and TfRA4 aptamers, other targeting aptamers provided by the present invention can be connected to the above-mentioned DNA vector and can also play a corresponding role to achieve a better delivery effect of epirubicin.

[0276] The chemical small molecule drugs used in the present invention can be of the common types in the field, including but not limited to anthracycline drugs, pyrimidine drugs, platinum drugs, glutamic acid derivative drugs, flavonoid drugs, botanical drugs and their derivatives, folic acid drugs, salicylic acid drugs or acridine drugs. Considering the better binding stability with the nucleic acid carrier, in a preferred embodiment, the anthracycline drug is selected from doxorubicin, epirubicin, pirarubicin, daunorubicin, idarubicin, mitoxantrone, valrubicin, or the free base or hydrochloride of the above drugs; preferably, the pyrimidine drug is selected from gemcitabine, 5-fluorouracil, cytarabine or capecitabine; preferably, the platinum drug is selected from cisplatin, oxaliplatin, carboplatin, nedaplatin or lobaplatin; preferably, The glutamate derivative drug is selected from lenalidomide, thalidomide or pomalidomide; preferably, the flavonoid drug is selected from flavonoids, flavonols, dihydroflavonoids, isoflavones or chalcones; preferably, the botanical drug and its derivatives are selected from vincristine, dihydroartemisinin, paclitaxel, maytansine, docetaxel or 10-hydroxycamptothecin; preferably, the folic acid drug is selected from methotrexate; preferably, the salicylic acid drug is selected from aspirin or sodium salicylate; preferably, the acridine drug is selected from tacrine or amsacrine; more preferably, the small molecule chemical drug is selected from one or more of epirubicin or its free base or its hydrochloride, gemcitabine, 5-fluorouracil, paclitaxel, and maytansine.

[0277] Preferably, when the small molecule target head, oligonucleotide effector molecule and immunostimulatory agent are connected to the nucleic acid vector, they are directly connected to the terminal bases of each sequence of the nucleic acid vector, or connected to the terminal bases of each sequence of the nucleic acid vector through a base connection bridge comprising 1 to 10 bases; preferably, when the chemical small molecule drug is an anthracycline drug or an acridine drug, the chemical small molecule drug is inserted into the GC adjacent base pairs of the targeting nucleic acid vector in the form of a specific insertion; when the chemical small molecule drug is a pyrimidine drug, the small molecule target head, oligonucleotide effector molecule and immunostimulatory agent are connected to the terminal bases of each sequence of the nucleic acid vector through a base connection bridge comprising 1 to 10 bases. The small molecule chemical drug is connected to the nucleic acid vector in a manner of at least partially replacing the base in the base connection bridge and / or as an extension of the base and / or replacing the base of the vector backbone; when the small molecule chemical drug is a platinum-based chemical drug, a glutamic acid derivative chemical drug, a flavonoid chemical drug, a botanical drug and its derivatives, a folic acid chemical drug or a salicylic acid chemical drug, the small molecule chemical drug is covalently connected to the nucleic acid vector through a linker; preferably, the linker is selected from a long-chain primary amine compound, more preferably a long-chain primary amine compound containing C2 to C14, and further preferably 5-bromomethyl-6-bromo-1-hexylamine (C7). After condensation with two molecules of phosphate, a base phosphoramidite monomer containing a linker is further prepared (such as N4-benzoyl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxycytidine nucleoside-3'-(6-aminohexyl-2-hydroxymethyl)-N,N'-diisopropyl phosphoramidite, or N2-benzoyl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxyguanosine nucleoside-3'-(6-aminohexyl-2-hydroxymethyl)-N,N'-diisopropyl phosphoramidite). The loading amount of each of the above-mentioned small molecule drugs can vary between 1 and the theoretical loading amount. Here, the theoretical loading amount refers to the maximum value that can be loaded based on the theoretical structure of the nucleic acid vector sequence. For example, if the small molecule drug is specifically inserted between adjacent GC base pairs of the targeting nucleic acid vector, the number of adjacent GC base pairs in the sequence is the theoretical loading amount, which should be understood by those skilled in the art. In the actual loading process, the molar ratio of epirubicin to the nucleic acid carrier is 2 to 300:1, preferably 2 to 290:1, more preferably 2 to 29:1, further preferably 10 to 50:1, and most preferably 15 to 25:1.

[0278] In order to further improve the drug size and / or stability during delivery, as well as to optimize the efficacy of drug combinations and reduce toxicity and increase efficacy, in a preferred embodiment, the present invention provides the following more reliable targeted chemical drugs:

[0279] (1) The targeted chemical drug includes a DNA vector, a chemical small molecule drug, and a nucleic acid aptamer EGFR and optional biotin connected to the DNA vector, preferably the chemical small molecule drug is epirubicin or its free base or its hydrochloride; preferably, the DNA vector contains a first group of sequences, the nucleic acid aptamer EGFR is connected to the 5' end of the b sequence, and the molar ratio of the nucleic acid aptamer EGFR to the DNA vector is 1:1; preferably, the nucleic acid aptamer EGFR is connected to the 5' end of the b sequence through a base bridge TTTTT; when the targeted chemical drug includes a DNA vector, a chemical small molecule drug, and a nucleic acid aptamer EGFR and biotin connected to the DNA vector, each DNA vector is connected to 2 to 4 biotins, and each biotin is respectively connected to the 5' end or 3' end of the a sequence or the c sequence. Specifically, the targeted chemical drug includes a targeted nucleic acid vector formed by self-assembly of the following sequences and epirubicin mounted on the targeted nucleic acid vector:

[0280] Drug 1: Apt EGFR-DNA-epirubicin

[0281] Note: The bold part is the a sequence, b sequence or c sequence in the nucleic acid vector, the same below.

[0282] In the aforementioned drug 1, the nanonucleic acid carrier is modified with an EGFR aptamer in a sequence-extending manner to act as an active targeting mediator for specific binding to the EGFR protein, which is highly expressed in tumor tissue cells. Approximately 30 epirubicin-mounting sites are designed on the three double-stranded DNA arms formed by self-assembly of the three core backbone sequences. During preparation, a 40:1 excess (epirubicin: nanonucleic acid carrier) is preferably used, and 4% paraformaldehyde is used as a coupling agent. The benzotetracyclic ring structure of the epirubicin molecule specifically intercalates into the GC / CG sites of the nanonucleic acid carrier. The amino group of the daunose structure interacts with the amino group of a guanosine ring in the carrier's GC / CG binding site, forming a Schiff base covalent bonding reaction catalyzed by paraformaldehyde, creating a stable mounting mechanism of physical intercalation and covalent attachment. In addition to the nanoparticle's natural passive "funnel" aggregation effect on tumor tissue, the drug exhibits a strong affinity for tumor tissues and cells that highly express the EGFR protein. These two targeting effects enable high concentrations to accumulate at tumor sites. After the EGFR aptamer binds to the highly expressed EGFR protein on the surface of tumor cells, it enters the cytoplasm through mechanisms such as endocytosis. Under the catalysis of the low pH value and high concentration of glutathione reducing agent in the tumor cell, epirubicin dissociates and is released from the nanocarrier. The released free epirubicin enters the cell nucleus and specifically binds to the GC / CG site embedded in the double-stranded DNA in the nucleus, blocking DNA transcription, thereby inhibiting the differentiation and proliferation of tumor cells and producing an anti-tumor effect that inhibits the occurrence and development of tumors. Specifically: The delivery, transport, release, sustained release and inhibitory mechanisms of this drug are as follows:

[0283] 1. Delivery: EGFR-DNA + epirubicin nanoparticles enter the body through targeted delivery, which has a natural passive targeted "funnel" aggregation effect.

[0284] 2. Transport: After the EGFR aptamer binds to the highly expressed EGFR protein on the surface of tumor cells, it enters the cell through mechanisms such as endocytosis. This binding causes the nanoparticles to accumulate at high concentrations at the tumor site, enhancing the drug's efficacy.

[0285] 3. Release: When the drug enters the tumor cells, epirubicin is dissociated and released from the nanonucleic acid carrier under the catalysis of low pH and high concentration of glutathione reducing agent.

[0286] 4. Sustained release: Epirubicin molecules are combined with nano-nucleic acid carriers through a stable mounting mode of physical chimerism and covalent linkage, which can achieve slow release, thereby reducing the toxic side effects of the drug in the body.

[0287] 5. Inhibitory Mechanism: Released epirubicin enters the cell nucleus and specifically binds to the GC / CG sites of double-stranded DNA embedded in the nucleus. This binding blocks DNA transcription, thereby inhibiting the differentiation and proliferation of tumor cells and producing an anti-tumor effect.

[0288] In summary, the delivery, transport, release, sustained-release, and inhibitory mechanisms of drug 1 make it a promising targeted anti-tumor drug. By specifically targeting tumor tissues and cells that overexpress EGFR protein, leveraging the passive targeting and aggregation effect of nanoparticles, effectively releasing the drug and achieving sustained release, and specifically blocking tumor cell DNA transcription, this drug offers a new research direction for cancer treatment.

[0289] Drug 2: Apt EGFR-4*Bio-DNA-epirubicin

[0290] In the aforementioned drug 2, the nanonucleic acid carrier modifies the EGFR aptamer by sequence extension, and the ends of the other two arms are modified with four biotins via PEG to act as active targeting mediators for specific binding to the EGFR protein and biotin receptor protein highly expressed in tumor tissue cells. Approximately 30 GC / CG epirubicin-mounting sites are designed on the three double-stranded DNA arms formed by self-assembly of the three core backbone sequences, forming a physical chimera + covalently linked stable mounting mode. The drug's delivery, transport, release, sustained-release, and inhibitory mechanisms are as follows:

[0291] 1. Delivery: Nanoparticle nucleic acid carriers achieve active targeting by modifying EGFR aptamers and linking biotin. This structure enables the drug to specifically bind to EGFR proteins and biotin receptor proteins that are highly expressed in tumor tissue cells.

[0292] 2. Transport: After the EGFR aptamer and biotin bind to the target protein on the surface of tumor cells, they enter the cell through mechanisms such as endocytosis. This binding causes the nanoparticles to accumulate at high concentrations at the tumor site, enhancing the drug's efficacy.

[0293] 3. Release: When the drug enters the tumor cells, epirubicin is dissociated and released from the nanonucleic acid carrier under the catalysis of low pH and high concentration of glutathione reducing agent.

[0294] 4. Sustained release: Epirubicin molecules are combined with nano-nucleic acid carriers through a stable mounting mode of physical chimerism and covalent linkage, which can achieve slow release, thereby reducing the toxic side effects of the drug in the body.

[0295] 5. Inhibitory Mechanism: Released epirubicin enters the cell nucleus and specifically binds to the GC / CG sites of double-stranded DNA embedded in the nucleus. This binding blocks DNA transcription, thereby inhibiting the differentiation and proliferation of tumor cells and producing an anti-tumor effect.

[0296] In summary, the drug's delivery, transport, release, sustained-release, and inhibitory mechanisms make it a promising targeted anti-tumor drug. By specifically targeting tumor tissues and cells that overexpress EGFR and biotin receptor proteins, leveraging the passive targeting and aggregation effects of nanoparticles, effectively releasing the drug and achieving sustained release, and specifically blocking tumor cell DNA transcription, this drug offers a new research direction for cancer treatment.

[0297] (2) Targeted chemical drugs include DNA vectors, chemical small molecule drugs, and nucleic acid aptamers AS1411 in DNA form, nucleic acid aptamers EGFR, nucleic acid aptamers A15 in DNA form, and optional biotin connected to the DNA vector, preferably the chemical small molecule drug is epirubicin or its free base or its hydrochloride; preferably, the DNA vector comprises a first group of sequences, the molar ratio of nucleic acid aptamers AS1411 in DNA form to the DNA vector is 1:1, and the aptamers are connected to the 5' end of the a sequence; preferably, the nucleic acid aptamers AS1411 are connected to the 5' end of the a sequence via a base bridge TTTTT; the molar ratio of nucleic acid aptamers EGFR to the DNA vector is 1:1, and the aptamers are connected to the b sequence. The 5' end of the sequence; preferably, the nucleic acid aptamer EGFR is connected to the 5' end of the b sequence through the base bridge TTTTT; the molar ratio of the DNA-formed nucleic acid aptamer A15 to the DNA vector is 1:1, and is connected to the 5' end of the c sequence; preferably, the nucleic acid aptamer A15 is connected to the 5' end of the c sequence through the base bridge TTTTT; when the targeted chemical drug includes a DNA vector, a chemical small molecule drug, and a DNA-formed nucleic acid aptamer AS1411, a nucleic acid aptamer EGFR, a DNA-formed nucleic acid aptamer A15, and biotin connected to the DNA vector, each DNA vector is connected to 2 biotins, and each biotin is respectively connected to the 3' end of the a sequence and the c sequence. Specifically, the targeted chemical drug includes a targeted nucleic acid vector formed by self-assembly of the following sequences and epirubicin mounted on the targeted nucleic acid vector:

[0298] Drug 3: Apt AS1411-Apt EGFR-Apt A15-2*Bio-DNA-Epirubicin:

[0299] Drug 4: Apt AS1411-Apt EGFR-Apt A15-DNA-epirubicin:

[0300] In the above-mentioned drugs 3 and 4, the nanonucleic acid carrier is modified with AS1411 aptamer, EGFR aptamer, and A15 aptamer at the 5' end of the three oligonucleotide sequences constituting the nucleic acid carrier backbone by sequence extension, and biotin Bio is optionally modified at the 3' end of the a chain and c chain by PEG connection. A composite targeted nanonucleic acid carrier is formed that specifically binds to nucleolin, EGFR protein, stem cell-like CD133 receptor, and biotin receptor protein, and covalently mounts epirubicin drug. This drug is a nanonucleic acid carrier that achieves composite targeting by modifying AS1411 aptamer, EGFR aptamer, A15 aptamer, and biotin Bio. The drug delivery, transport, release, sustained release, and inhibitory mechanisms are as follows:

[0301] 1. Delivery: The nanoparticle nucleic acid carrier is modified with AS1411 aptamer, EGFR aptamer, A15 aptamer, and optionally biotin to achieve complex targeting. This enables the drug to specifically bind to different types of tumor cells, leveraging the affinity of the aptamer with the corresponding tumor cell surface receptor to achieve specific binding.

[0302] 2. Transport: After the aptamer binds to the target receptor on the surface of the tumor cell, it enters the cell through mechanisms such as the endoplasmic reticulum pathway. This binding causes the nanoparticles to accumulate at high concentrations at the tumor site, improving the drug's efficacy.

[0303] 3. Release: When the drug enters the tumor cells, epirubicin is dissociated and released from the nanonucleic acid carrier under the catalysis of low pH and high concentration of glutathione reducing agent.

[0304] 4. Sustained release: Epirubicin molecules are combined with nano-nucleic acid carriers through a stable mounting mode of physical chimerism and covalent linkage, which can achieve slow release, thereby reducing the toxic side effects of the drug in the body.

[0305] 5. Inhibitory Mechanism: Released epirubicin enters the cell nucleus and specifically binds to the GC / CG sites of double-stranded DNA embedded in the nucleus. This binding blocks DNA transcription, thereby inhibiting the differentiation and proliferation of tumor cells and producing an anti-tumor effect.

[0306] In summary, the delivery, transport, release, sustained-release, and inhibitory mechanisms of drug 3 make it a promising, complex, targeted anti-tumor agent. By specifically targeting different receptors in tumor tissues and cells, leveraging the passive targeting and aggregation effects of nanoparticles, effectively releasing the drug and achieving sustained release, and specifically blocking tumor cell DNA transcription, this drug offers a new research direction for cancer treatment.

[0307] (3) Targeted chemical drugs include DNA vectors, chemical small molecule drugs, and DNA-formed nucleic acid aptamers AS1411 and biotin connected to the DNA vector, preferably the chemical small molecule drug is epirubicin or its free base or its hydrochloride; preferably, the DNA vector contains a first group of sequences, the DNA-formed nucleic acid aptamer AS1411 is connected to the 5' end of the b sequence, and the molar ratio of the DNA-formed nucleic acid aptamer AS1411 to the DNA vector is 1:1; preferably, the nucleic acid aptamer AS1411 is connected to the 5' end of the b sequence through a base bridge TTTTT; 2 to 4 biotins are connected to each DNA vector, and each biotin is connected to the 5' end or 3' end of the a sequence or the c sequence, respectively. Specifically, the targeted chemical drug includes a targeted nucleic acid vector formed by self-assembly of the following sequences and epirubicin mounted on the targeted nucleic acid vector:

[0308] Drug 5: Apt AS1411-4*Bio-DNA-epirubicin:

[0309] In the aforementioned drug 5, the nanonucleic acid carrier achieves active targeting specific binding by modifying the AS1411 aptamer and optionally linking biotin. The drug's delivery, transport, release, sustained release, and inhibitory mechanisms are as follows:

[0310] 1. Delivery: The nanonucleic acid carrier achieves active targeting by modifying the AS1411 aptamer and optionally linking biotin. This structure enables the drug to specifically bind to ribonucleic acid (NCL) and biotin receptor proteins that are highly expressed in tumor tissue cells.

[0311] 2. Transport: After the AS1411 aptamer and biotin bind to the target protein on the surface of tumor cells, they enter the cell through mechanisms such as endocytosis. This binding allows the nanoparticles to accumulate at high concentrations at the tumor site, enhancing the drug's efficacy.

[0312] 3. Release: When the drug enters the tumor cells, epirubicin is dissociated and released from the nanonucleic acid carrier under the catalysis of low pH and high concentration of glutathione reducing agent.

[0313] 4. Sustained release: Epirubicin molecules are combined with nano-nucleic acid carriers through a stable mounting mode of physical chimerism and covalent linkage, which can achieve slow release, thereby reducing the toxic side effects of the drug in the body.

[0314] 5. Inhibitory Mechanism: Released epirubicin enters the cell nucleus and specifically binds to the GC / CG sites of double-stranded DNA embedded in the nucleus. This binding blocks DNA transcription, thereby inhibiting the differentiation and proliferation of tumor cells and producing an anti-tumor effect.

[0315] In summary, the delivery, transport, release, sustained-release, and inhibitory mechanisms of drug 5 make it a promising targeted anti-tumor drug. By specifically targeting tumor tissues and cells that highly express ribonucleic acid (NCL) and biotin receptor proteins, leveraging the passive targeting and aggregation effects of nanoparticles, effectively releasing the drug and achieving sustained release, and specifically blocking tumor cell DNA transcription, this drug offers a new research direction for cancer treatment.

[0316] (4) Targeted chemical drugs include DNA vectors, chemical small molecule drugs, and nucleic acid aptamers A1 and DNA-formed nucleic acid aptamers A15 connected to the DNA vector, preferably the chemical small molecule drug is epirubicin or its free base or its hydrochloride; preferably, the DNA vector contains a first group of sequences, the molar ratio of nucleic acid aptamer A1 to the DNA vector is 1:1, and it is connected to the 5' end of the b sequence; preferably, the nucleic acid aptamer A1 is connected to the 5' end of the b sequence via a base bridge TTTTT; the molar ratio of nucleic acid aptamer A15 to the DNA vector is 1:1, and it is connected to the 5' end of the c sequence; preferably, the nucleic acid aptamer A15 is connected to the 5' end of the b sequence via a base bridge TTTTT. Specifically, the targeted chemical drug includes a targeted nucleic acid vector formed by self-assembly of the following sequences and epirubicin mounted on the targeted nucleic acid vector:

[0317] Drug 6: Apt A1-Apt A15-DNA-epirubicin:

[0318] In the aforementioned drug 6, the nanonucleic acid carrier achieves active targeting specific binding by modifying the A1 aptamer and the A15 aptamer. The drug's delivery, transport, release, sustained release, and inhibitory mechanisms are as follows:

[0319] 1. Delivery: The nanonucleic acid carrier achieves active targeting by modifying the A1 and A15 aptamers. This structure enables the drug to specifically bind to tumor tissue cells, leveraging the affinity of the A1 and A15 aptamers for the corresponding CD133 receptor on the surface of tumor cells.

[0320] 2. Transport: After binding to target receptors on the surface of tumor cells, the A1 and A15 aptamers enter the cells through mechanisms such as endocytosis. This binding allows the nanoparticles to accumulate at high concentrations at the tumor site, enhancing the drug's efficacy.

[0321] 3. Release: When the drug enters the tumor cells, epirubicin is dissociated and released from the nanonucleic acid carrier under the catalysis of low pH and high concentration of glutathione reducing agent.

[0322] 4. Sustained release: Epirubicin molecules are combined with nano-nucleic acid carriers through a stable mounting mode of physical chimerism and covalent linkage, which can achieve slow release, thereby reducing the toxic side effects of the drug in the body.

[0323] 5. Inhibitory Mechanism: Released epirubicin enters the cell nucleus and specifically binds to the GC / CG sites of double-stranded DNA embedded in the nucleus. This binding blocks DNA transcription, thereby inhibiting the differentiation and proliferation of tumor cells and producing an anti-tumor effect.

[0324] In summary, the delivery, transport, release, sustained-release, and inhibitory mechanisms of drug 6 make it a promising targeted anti-tumor agent. By specifically targeting tumor tissues and cells, leveraging the passive targeting and aggregation effects of nanoparticles, effectively releasing the drug and achieving sustained release, and specifically blocking tumor cell DNA transcription, this drug offers a new research direction for cancer treatment.

[0325] (5) Targeted chemical drugs include DNA vectors, chemical small molecule drugs, and nucleic acid aptamers AS1411 in DNA form, nucleic acid aptamers EGFR, nucleic acid aptamers A15 in DNA form, optional biotin and oligonucleotide A-miR-21 connected to the DNA vector, preferably the chemical small molecule drug is epirubicin or its free base or its hydrochloride; preferably, the DNA vector comprises a first group of sequences, the molar ratio of nucleic acid aptamers AS1411 in DNA form to the DNA vector is 1:1, and the DNA vector is connected to the 5' end of the a sequence; preferably, the nucleic acid aptamer AS1411 is connected to the 5' end of the a sequence via a base bridge TTTTT; the nucleic acid aptamer EGFR is connected to the DNA vector; The molar ratio of the A vector is 1:1, and it is connected to the 5' end of the b sequence; preferably, the nucleic acid aptamer EGFR is connected to the 5' end of the b sequence through the base bridge TTTTT; the molar ratio of the DNA-formed nucleic acid aptamer A15 to the DNA vector is 1:1, and it is connected to the 5' end of the c sequence; preferably, the nucleic acid aptamer A15 is connected to the 5' end of the c sequence through the base bridge TTTTT; the molar ratio of the oligonucleotide A-miR-21 to the DNA vector is 1:1, and it is connected to the 3' end of the c sequence; optionally, each DNA vector is connected to 2 biotins, and each biotin is respectively connected to the 3' end of the a sequence and the end of the oligonucleotide A-miR-21 away from the c sequence. Specifically, the targeted chemical drug includes a targeted nucleic acid vector formed by self-assembly of the following sequences and epirubicin mounted on the targeted nucleic acid vector:

[0326] Drug 7: Apt AS1411-Apt EGFR-Apt A15-2*Bio-AmiR-21-DNA-Epirubicin

[0327] Drug 8: Apt AS1411-Apt EGFR-Apt A15-AmiR-21-DNA-epirubicin

[0328] In the aforementioned drugs 7 and 8, the nanonucleic acid carrier achieves a tertiary targeting effect through AS1411, EGFR, A15 aptamers, and optionally biotin. The following is a detailed analysis of the molecular functions and tertiary targeting effects:

[0329] 1. AS1411 aptamer: AS1411 is an oligonucleotide that specifically binds to nucleolin receptors on the surface of tumor cells. This binding promotes drug uptake by tumor cells, achieving targeted anti-tumor effects.

[0330] 2. EGFR aptamers: EGFR is an epidermal growth factor receptor that is overexpressed in a variety of malignant tumor cells. Through EGFR aptamers, drugs can specifically bind to tumor cells that overexpress EGFR, increasing drug concentration in tumor tissue.

[0331] 3. A15 aptamer: A15 is an aptamer that specifically binds to the CD133 receptor on cancer stem cells (CSFs). CSCs are a key factor in tumor recurrence and drug resistance, and the A15 aptamer enables drug targeting of CSCs.

[0332] 4. A-miR-21: A-miR-21 is an anti-tumor ASO that targets microRNA-21. MicroRNA-21 is overexpressed in various tumors and promotes tumor development. A-miR-21 can inhibit the function of microRNA-21, thereby suppressing tumor development.

[0333] 5. Epirubicin: Inhibits the differentiation and proliferation of tumor cells by blocking DNA transcription.

[0334] Through a three-level targeting-mediated effect, these drugs achieve effective delivery, transport, release, sustained release, and inhibitory effects. The focus is on targeting cancer stem cells to inhibit tumor initiation and progression. This multi-targeting strategy is expected to improve drug efficacy, reduce toxic side effects, and provide a new therapeutic approach for the treatment of various malignancies.

[0335] (6) Targeted chemical drugs include DNA vectors, chemical small molecule drugs, and DNA-based nucleic acid aptamers TfRA4, AS1411, and A15 connected to the DNA vector, preferably the chemical small molecule drug is epirubicin or its free base or its hydrochloride; preferably, the DNA vector comprises a first group of sequences, the molar ratio of the DNA-based nucleic acid aptamer TfRA4 to the DNA vector is 1:1, and the DNA vector is connected to the 5' end of the a sequence; preferably, the nucleic acid aptamer TfRA4 ... A4 is connected to the 5' end of sequence a via a base bridge TTTTT; the molar ratio of the DNA-based nucleic acid aptamer AS1411 to the DNA vector is 1:1, and it is connected to the 5' end of sequence b; preferably, the nucleic acid aptamer AS1411 is connected to the 5' end of sequence b via a base bridge TTTTT; the molar ratio of the DNA-based nucleic acid aptamer A15 to the DNA vector is 1:1, and it is connected to the 5' end of sequence c; preferably, the nucleic acid aptamer A15 is connected to the 5' end of sequence c via a base bridge TTTTT. Specifically, the targeted chemical drug includes a targeted nucleic acid vector formed by self-assembly of the following sequences and epirubicin mounted on the targeted nucleic acid vector:

[0336] Drug 9: Apt TfRA4-Apt AS1411-Apt A15-DNA-epirubicin (1):

[0337] In the above-mentioned drug 9, the AS1411 aptamer, TfRA4 aptamer, and A15 aptamer are modified at the 5' end of the three oligonucleotide sequences that constitute the nucleic acid vector backbone, forming a composite targeted nanonucleic acid carrier that specifically binds to nucleolin, BBB transferrin, stem cell-like CD133 receptor, and biotin receptor protein, and covalently mounts epirubicin to form a targeted epirubicin drug. This nanonucleic acid targeted drug can specifically bind to transferrin on the BBB and cross the BBB accompanied by the AS1411 chaperone, reaching the brain parenchyma and binding to the nucleolin receptor highly expressed on the surface of glial tumors. In particular, it can also specifically bind to tumor stem cell-like proteins that highly express CD133 receptor proteins and enter the cell, inhibiting the occurrence and development of tumors. Specifically, the drug achieves its delivery, transport, release, sustained release, and inhibitory mechanisms through a three-level targeting-mediated effect. The following is a detailed analysis of the functions of each molecule and the three-level targeting-mediated effect:

[0338] 1. Delivery: The nanoparticle nucleic acid carrier is combined with the TfRA4 aptamer, enabling the drug to specifically bind to the transferrin receptor on the blood-brain barrier (BBB). This helps the drug cross the BBB and enter the brain.

[0339] 2. Transport: After the drug crosses the BBB, the AS1411 aptamer helps the drug specifically bind to the nucleolin receptors highly expressed on the surface of glial tumors in the brain, thereby targeting glial tumor cells.

[0340] 3. Release and sustained release: After the epirubicin covalently attached to the nanonucleic acid carrier enters the tumor cells, it is gradually released under the action of low pH and high concentrations of glutathione reducing agents. This release method can achieve a sustained release effect, reduce the drug's toxic side effects, and improve efficacy.

[0341] 4. Inhibitory Mechanism: The A15 aptamer enables the drug to specifically bind to tumor stem cells that highly express the CD133 receptor protein, allowing it to enter the cell and exert an inhibitory effect. This is crucial for killing and inhibiting brain gliomas, as tumor stem cells are a key factor in tumor recurrence and drug resistance.

[0342] In summary, drug 9, through a three-level targeting-mediated effect, specifically targets transferrin, nucleolin receptors, and CD133 receptors on the BBB, achieving effective drug delivery, transport, release, sustained release, and inhibitory effects. The focus is on targeting cancer stem cells to inhibit the occurrence and progression of gliomas. This multi-targeting strategy is expected to improve drug efficacy, reduce toxic side effects, and provide a new therapeutic approach for the treatment of gliomas and other malignancies.

[0343] (7) The targeted chemical drug includes a DNA vector, a chemical small molecule drug, and a DNA-formed nucleic acid aptamer TfRA4, a DNA-formed nucleic acid aptamer AS1411, and a DNA-formed nucleic acid aptamer A15 connected to the DNA vector, wherein the chemical small molecule drug is epirubicin or its free base or its hydrochloride; the DNA vector comprises an eleventh group of sequences, the molar ratio of the DNA-formed nucleic acid aptamer TfRA4 to the DNA vector is 1:1, and the nucleic acid aptamer TfRA4 is connected to the 5' end of the a sequence; preferably, the nucleic acid aptamer TfRA4 is connected to the 5' end of the a sequence via a transition base segment SEQ ID NO: 276: 5'-TTGCGGCGAGCGGCGA-3', and the targeted chemical drug further comprises a complementary sequence SEQ ID NO: 277: 5'-TCGCCGCTCGCCGCTT-3', which is aligned with the transition base segment SEQ ID NO:276: 5'-TTGCGGCGAGCGGCGA-3' is complementary and connected, and its 3' end is connected to the 5' segment of the nucleic acid aptamer TfRA4; the molar ratio of the nucleic acid aptamer AS1411 in DNA form to the DNA vector is 1:1, and it is connected to the 5' end of the b sequence; preferably, the nucleic acid aptamer AS1411 is connected to the 5' end of the b sequence through the base bridge TTTTT; the molar ratio of the nucleic acid aptamer A15 in DNA form to the DNA vector is 1:1, and it is connected to the 5' end of the c sequence; preferably, the nucleic acid aptamer A15 is connected to the 5' end of the c sequence through the base bridge TTTTT. Specifically, the targeted chemical drug includes a targeted nucleic acid vector formed by self-assembly of the following sequences and epirubicin mounted on the targeted nucleic acid vector:

[0344] Drug 10: Apt TfRA4-Apt AS1411-Apt A15-DNA+Epirubicin (2):

[0345] The above-mentioned drug 10 is a variation of drug 9. The difference from drug 8 is that the 14 base sequences before and after the TfRA4 aptamer (the transition base segment TTGCGGCGAGCGGCGA and the complementary sequence TCGCCGCTCGCCGCTT described above) are completely complementary. After self-assembly, a neck structure is formed through a pair of TT / TT spacers and then extended. This further improves the stability of the drug structure and solves the problem of multimers that are easily caused by self-assembly of TFRA4.

[0346] (8) Targeted chemical drugs include DNA vectors, chemical small molecule drugs, and DNA-based nucleic acid aptamers AS1411, TfRA3, and A15 connected to the DNA vector, preferably the chemical small molecule drug is epirubicin or its free base or its hydrochloride; preferably, the DNA vector comprises a first group of sequences, the molar ratio of the DNA-based nucleic acid aptamer AS1411 to the DNA vector is 1:1, and the nucleic acid aptamer AS1411 is connected to the 5' end of the a sequence; preferably, the nucleic acid aptamer AS 1411 is connected to the 5' end of sequence a via a base bridge TTTTT; the molar ratio of the DNA-based nucleic acid aptamer TfRA3 to the DNA vector is 1:1, and it is connected to the 5' end of sequence b; preferably, the nucleic acid aptamer TfRA3 is connected to the 5' end of sequence b via a base bridge TTTTT; the molar ratio of the DNA-based nucleic acid aptamer A15 to the DNA vector is 1:1, and it is connected to the 5' end of sequence c; preferably, the nucleic acid aptamer A15 is connected to the 5' end of sequence b via a base bridge TTTTT. Specifically, the targeted chemical drug includes a targeted nucleic acid vector formed by self-assembly of the following sequences and epirubicin mounted on the targeted nucleic acid vector:

[0347] Drug 11: Apt TfRA3-Apt A1S1411-Apt A15-DNA-epirubicin

[0348] In the above-mentioned drug 11, the nanonucleic acid carrier modifies the AS1411 aptamer, TfRA3 aptamer, and A15 aptamer at the 5' end of the three oligonucleotide sequences that constitute the nucleic acid carrier backbone by sequence extension, forming a composite targeted nanonucleic acid carrier that specifically binds to nucleolin, BBB transferrin, stem cell-like CD133 receptor, and biotin receptor protein, and covalently mounts epirubicin to form a targeted epirubicin drug. This nanonucleic acid targeted drug can specifically bind to transferrin on the BBB and cross the BBB accompanied by the AS1411 chaperone, reaching the brain parenchyma and binding to the nucleolin receptor highly expressed on the surface of glial tumors. In particular, it can also specifically bind to tumor stem cell-like proteins that highly express CD133 receptor proteins and enter the cell, inhibiting the occurrence and development of tumors. Specifically, this drug 11 is a nanonucleic acid carrier that achieves composite targeting by modifying the AS1411 aptamer, TfRA3 aptamer, and A15 aptamer. The drug delivery, transport, release, sustained release, and inhibitory mechanisms are as follows:

[0349] 1. Delivery: Nanoparticle nucleic acid carriers are modified with AS1411, TfRA3, and A15 aptamers to specifically bind to nucleolin, BBB transferrin, and stem cell-like CD133 receptors. This enables the drug to specifically bind to transferrin on the BBB, cross the BBB, and reach the brain parenchyma.

[0350] 2. Tertiary Targeting: a. Primary Targeting: The drug binds to transferrin on the BBB via the TfRA3 aptamer and crosses the BBB accompanied by the AS1411 chaperone. b. Secondary Targeting: After reaching the brain parenchyma, the drug binds to the nucleolin receptor, which is highly expressed on the surface of glial tumors, via the AS1411 aptamer. c. Tertiary Targeting: The drug specifically binds to cancer stem cells that highly express the CD133 receptor protein via the A15 aptamer.

[0351] 3. Transport: After the aptamer binds to the target receptor on the surface of the tumor cell, it enters the cell through mechanisms such as the endoplasmic reticulum pathway.

[0352] 4. Release: When the drug enters the tumor cells, epirubicin is dissociated and released from the nanonucleic acid carrier under the catalysis of low pH and high concentration of glutathione reducing agent.

[0353] 5. Sustained release: Epirubicin molecules are combined with nano-nucleic acid carriers through a stable mounting mode of physical chimerism and covalent linkage, which can achieve slow release, thereby reducing the toxic side effects of the drug in the body.

[0354] 6. Inhibitory Mechanism: Released epirubicin enters the cell nucleus and specifically binds to GC / CG sites embedded in double-stranded DNA within the nucleus. This binding blocks DNA transcription, thereby inhibiting tumor cell differentiation and proliferation, resulting in an anti-tumor effect. In particular, the A15 aptamer binds to tumor stem cells that highly express the CD133 receptor protein, enabling the drug to enter and inhibit tumor stem cells. This is crucial for its mechanism of action in killing and inhibiting gliomas.

[0355] Cancer stem cells are cells with the potential for self-renewal and multidirectional differentiation, and they play a key role in tumor growth, recurrence, and drug resistance. By targeting the CD133 receptor, which is highly expressed on cancer stem cells, the drug 11 can more effectively target these cells, thereby eliminating the source of the tumor and inhibiting tumor recurrence.

[0356] In summary, the drug 11's delivery, transport, release, sustained-release, and inhibitory mechanisms are achieved through a three-level targeting-mediated effect, with a focus on targeting cancer stem cells to inhibit the occurrence and progression of gliomas. This composite targeting strategy is expected to improve drug efficacy, reduce toxic side effects, and provide a new therapeutic approach for the treatment of gliomas and other malignant tumors.

[0357] (9) Targeted chemical drugs include DNA vectors, chemical small molecule drugs, and immunostimulants CPG2006, nucleic acid aptamers CD40, and nucleic acid aptamers PD-L1 in DNA form connected to the DNA vector, preferably the chemical small molecule drug is epirubicin or its free base or its hydrochloride; preferably, the DNA vector contains the ninth group of sequences, the molar ratio of the immunostimulant CPG2006 to the DNA vector is 1:1, and is connected to the 5' end of the a sequence; preferably, the immunostimulant CPG2006 is connected to the 5' end of the a sequence via a base bridge TTTTT; the molar ratio of the nucleic acid aptamer CD40 to the DNA vector is 1:1, and is connected to the 5' end of the b sequence; preferably, the nucleic acid aptamer CD40 is connected to the 5' end of the b sequence via a base bridge TTTTT; the molar ratio of the nucleic acid aptamer PD-L1 in DNA form to the DNA vector is 1:1, and is connected to the 5' end of the c sequence; preferably, the nucleic acid aptamer PD-L1 is connected to the 5' end of the c sequence via a base bridge TTTTTT. Specifically, the targeted chemical drug includes a targeted nucleic acid vector formed by self-assembly of the following sequence and epirubicin mounted on the targeted nucleic acid vector:

[0358] Drug 12: CPG2006 (DNA)-Apt CD40-Apt PD-L1-DNA-Epirubicin

[0359] Note: The adjacent bases at positions 1-24 at the 5' end of sequence A are all thiolated, and the adjacent bases at positions 1-3 at the 3' end are all thiolated; the adjacent bases at positions 1-3 at the 5' end and 3' end of sequences B and C are all thiolated.

[0360] In the aforementioned drug 12, the nanoparticle nucleic acid carrier integrates three aptamers (CPG2006, CD40, and PD-L1) to achieve active combined immunotherapy and chemotherapy targeting the tumor microenvironment. The following is a detailed analysis of the functions of each effector molecule and the synergistic tumor suppression effect generated by the integrated targeting of the tumor microenvironment:

[0361] 1. CPG2006 aptamer: CPG2006 is a CpG oligonucleotide that acts as an immune activator, activating the immune system by recognizing the TLR9 receptor. In the tumor microenvironment, CPG2006 activates dendritic cells (DCs) and B cells, which in turn stimulates the activation and expansion of T cells, enhancing anti-tumor immune responses.

[0362] 2. CD40 aptamer: CD40 is a membrane-bound protein that binds to the CD40 receptor, thereby activating dendritic cells and B cells. By activating immune cells, CD40L helps improve the tumor microenvironment and enhance anti-tumor immune responses.

[0363] 3. PD-L1 aptamers: PD-L1 is an immunosuppressive molecule. In the tumor microenvironment, tumor cells express PD-L1 to suppress T cell activity and evade immune surveillance. By designing PD-L1 aptamers, drugs can block the interaction between PD-L1 and the PD-1 receptor, restore T cell activity, and enhance anti-tumor immune responses.

[0364] 4. Epirubicin: Epirubicin is an anti-tumor drug that inhibits the differentiation and proliferation of tumor cells by blocking DNA transcription.

[0365] By integrating three aptamers into an active immunotherapy agent, epirubicin, this drug achieves combined targeting of immunotherapy and classic chemotherapy drugs to the tumor microenvironment. Within the tumor microenvironment, the CPG2006 and CD40L aptamers activate dendritic cells and B cells, respectively, promoting T cell activation and expansion. The PD-L1 aptamer restores T cell activity by blocking the interaction between PD-L1 and the PD-1 receptor. These three aptamers work together to enhance the anti-tumor immune response. Meanwhile, epirubicin, a classic chemotherapy drug, inhibits the differentiation and proliferation of tumor cells by blocking DNA transcription. This combined chemotherapy and immunotherapy approach makes tumor cells more susceptible to immune cell recognition and elimination under the action of chemotherapy drugs.

[0366] The tumor microenvironment is a complex network of cells and molecules, including tumor cells, immune cells, supporting cells, and extracellular matrix. The drug achieves a synergistic tumor-suppressing effect of immunotherapy and chemotherapy by integratedly targeting the tumor microenvironment. First, the drug regulates the tumor microenvironment by activating immune cells (such as dendritic cells, B cells, and T cells), making it more conducive to anti-tumor immune responses. Secondly, the drug enhances the ability of T cells to attack tumor cells by blocking the interaction between PD-L1 and PD-1. Finally, epirubicin has a direct cytotoxic effect on tumor cells, further enhancing the clearance of tumor cells by immune cells.

[0367] In summary, this drug achieves a synergistic tumor-suppressing effect by integrating immunotherapy and chemotherapy drugs, targeting the tumor microenvironment. This integrated strategy is expected to improve the efficacy of tumor treatment and provide patients with better quality of life and prognosis.

[0368] (10) Targeted chemical drugs include DNA vectors, small molecule chemical drugs, and immunostimulants CPG2006, nucleic acid aptamers CD40, nucleic acid aptamers PD-L1 in DNA form, and nucleic acid aptamers C12 connected to the DNA vector; wherein the DNA vector also includes a d sequence and an e sequence as complementary sequences, the d sequence is SEQ ID NO: 278: 5'-GCCACCGTGCTACA-3', and the e sequence is SEQ ID NO: 279: 5'-CAGCAGCAGCAGCA-3'; the 3' end of the b sequence in the DNA vector is connected to a transition base segment SEQ ID NO: 280: 5'-GTAGCACGGTGGC-3', and the d sequence is linked to the transition base segment SEQ ID NO: 281 through chain complementarity. NO:280: 5'-GTAGCACGGTGGC-3' complementary connection, the e sequence is complementary to the c sequence of the DNA vector in a chain complementary manner; preferably, the chemical small molecule drug is epirubicin or its free base or its hydrochloride; preferably, the DNA vector comprises the tenth group of sequences, the molar ratio of the immunostimulant CPG2006 to the DNA vector is 2:1, and they are connected to the 5' end of the a sequence and the 5' end of the c sequence respectively; preferably, the immunostimulant CPG2006 is connected to the 5' end of the a sequence through a base connection bridge TTTTTT, and is connected to the 5' end of the c sequence through a base connection bridge TTTTT; the nucleic acid adaptor The molar ratio of the ligand CD40 to the DNA vector is 1:1, and it is connected to the 5' end of the b sequence; preferably, the nucleic acid aptamer CD40 is connected to the 5' end of the b sequence via a base bridge TTTTT; the molar ratio of the nucleic acid aptamer PD-L1 to the DNA vector is 1:1, and it is connected to the 5' end of the d sequence; preferably, the nucleic acid aptamer PD-L1 is connected to the 5' end of the d sequence via a base bridge TTTTTT; the molar ratio of the nucleic acid aptamer C12 to the DNA vector is 1:1, and it is connected to the 5' end of the e sequence; preferably, the nucleic acid aptamer C12 is connected to the 5' end of the e sequence via a base bridge TTTTT. Specifically, the targeted chemical drug includes a targeted nucleic acid vector formed by self-assembly of the following sequences and epirubicin mounted on the targeted nucleic acid vector:

[0369] Drug 13: 2*CPG2006(DNA)-Apt CD40-Apt PD-L1-Apt C12-DNA-Epirubicin

[0370] Note: The adjacent bases at positions 1-24 on the 5' end of sequences A and C are all thiolated; the adjacent bases at positions 1-3 on the 5' end of sequences B, D, and E are all thiolated.

[0371] In drug 13, the nanoparticle nucleic acid vector binds to three aptamers (CPG2006, CD40, and PD-L1) and targets the C12 receptor protein, which is highly expressed on the surface of small cell lung cancer cells. This allows for combined immunotherapy and traditional chemotherapy drugs to target the tumor microenvironment. This drug is highly effective in inhibiting recurrent, resistant, and small cell lung cancer cells.

[0372] The CPG2006 aptamer is a CpG oligonucleotide mimetic that activates the Toll-like receptor 9 (TLR9) pathway, thereby stimulating the activation and proliferation of dendritic cells and B cells. In this way, the CPG2006 aptamer helps enhance anti-tumor immune responses. The CD40 aptamer activates dendritic cells, B cells, and other anti-tumor immune cells by binding to the CD40 receptor, promoting anti-tumor immune responses. The PD-L1 aptamer blocks the interaction between PD-L1 and the PD-1 receptor, restoring T cell activity. This approach helps enhance T cell attack capabilities against tumor cells. The C12 aptamer is an aptamer that specifically targets a receptor protein highly expressed on the surface of small cell lung cancer cells. Through the action of the C12 aptamer, drugs can accurately identify and target tumor cells, improving the precision of treatment. Epirubicin, a classic chemotherapy drug, inhibits the growth and spread of tumor cells by blocking DNA replication and transcription.

[0373] In this integrated nanonucleic acid carrier, the aptamers and epirubicin work together to achieve combined immunotherapy and chemotherapy targeting the tumor microenvironment. The CPG2006-CD40-PD-L1 aptamer activates immune cells and enhances the anti-tumor immune response; the C12 aptamer achieves specific targeting of small cell lung cancer cells; and epirubicin directly produces cytotoxic effects on tumor cells. This synergistic tumor-suppressing effect can significantly improve the effectiveness of tumor treatment. Specifically, the following points explain the mechanism of action of the integrated nanonucleic acid carrier in the treatment of small cell lung cancer:

[0374] 1. Improvement of the tumor microenvironment: The CPG2006-CD40-PD-L1 aptamer acts on the tumor microenvironment, changing the immunosuppressive state. This helps to enhance the anti-tumor immune response and improve the effectiveness of tumor cell clearance.

[0375] 2. Targeted delivery: The C12 aptamer can specifically bind to receptor proteins highly expressed on the surface of small cell lung cancer cells, enabling precise drug delivery. This targeted delivery strategy helps reduce drug distribution in normal tissues and minimize side effects.

[0376] 3. Tumor cell apoptosis: Epirubicin inhibits the growth and spread of tumor cells by blocking DNA replication and transcription, thereby inducing tumor cell apoptosis.

[0377] 4. Immune surveillance restoration: PD-L1 aptamers restore T cell activity by blocking the interaction between PD-L1 and the PD-1 receptor. This helps improve the immune system's ability to monitor tumor cells and prevent tumor recurrence and metastasis.

[0378] 5. Immune memory formation: CD40 aptamers activate dendritic cells and B cells, helping the immune system to remember tumor antigens. This helps prolong the effects of immunotherapy and prevent tumor recurrence.

[0379] In summary, drug 13 exhibits a synergistic tumor-suppressing effect in the treatment of small cell lung cancer. The aptamers and epirubicin work together to improve the tumor microenvironment, enhance anti-tumor immune responses, achieve precise delivery, induce tumor cell apoptosis, restore immune surveillance, and establish immune memory. This integrated nanoparticle nucleic acid delivery system holds promise for the treatment of recurrent, resistant, and small cell lung cancer.

[0380] (11) Targeted chemical drugs include DNA vectors, chemical small molecule drugs, and immunostimulants CPG2006, nucleic acid aptamers C12, siRNA oligonucleotides CD47, nucleic acid aptamers PD-L1 in DNA form, and siRNA oligonucleotides PD-L1 connected to the DNA vector; preferably, the chemical small molecule drug is epirubicin or its free base or its hydrochloride; preferably, the DNA vector contains the ninth group of sequences, the molar ratio of siRNA oligonucleotides PD-L1 to the DNA vector is 1:1, and its sense chain is connected to the 5' end of the c sequence in the DNA vector as a transition base segment, and the antisense chain is connected to the sense chain as a complementary sequence, and the nucleic acid aptamer PD-L1 in DNA form is connected to the 5' end of the antisense chain of the siRNA oligonucleotides PD-L1; preferably, the nucleic acid aptamer PD -L1 is connected to the 5' end of the antisense strand of the siRNA oligonucleotide PD-L1 through a base bridge TTTTT; the molar ratio of the immunostimulator CPG2006 to the DNA vector is 1:1, and it is connected to the 5' end of the a sequence; preferably, the immunostimulator CPG2006 is connected to the 5' end of the a sequence through a base bridge TTTTT; the molar ratio of the siRNA oligonucleotide CD47 to the DNA vector is 1:1, and its sense strand is connected to the 5' end of the b sequence as a transition base segment, and the antisense strand is complementary to the sense strand as a complementary sequence; the molar ratio of the nucleic acid aptamer C12 to the DNA vector is 1:1, and it is connected to the 5' end of the antisense strand of the siRNA oligonucleotide CD47; preferably, the nucleic acid aptamer C12 is connected to the 5' end of the antisense strand of the siRNA oligonucleotide CD47 through a base bridge TTTTT. Specifically, the targeted chemical drug includes a targeted nucleic acid vector formed by self-assembly of the following sequences and epirubicin mounted on the targeted nucleic acid vector:

[0381] Drug 14: CPG2006 (DNA)-Apt C12-CD47 siRNA-Apt PD-L1-PD-L1 siRNA-DNA-epirubicin

[0382] Note: The adjacent bases at positions 1-24 of the 5' end of sequence A are all thiolated, and the adjacent bases at positions 1-3 of the 3' end are all thiolated; the adjacent bases at positions 1-3 of the 5' end and 3' end of sequence B are all thiolated, and the adjacent bases at positions 19-21 of the 5' end of sequence B are all thiolated; the adjacent bases at positions 1-3 of the 5' end and 3' end of sequence C are all thiolated, and the adjacent bases at positions 19-21 of the 5' end of sequence C are all thiolated. The 5'-end of sequence D contains thiosulfate modifications between adjacent bases 19-21; the 5'-end and 3'-end of sequence D contain thiosulfate modifications between adjacent bases 19-21; the 5'-end and 3'-end of sequence E contain thiosulfate modifications between adjacent bases 1-3, and the 3'-end of sequence E contain thiosulfate modifications between adjacent bases 19-21. The C / U2'F modifications are underlined in the RNA sequence.

[0383] In drug 14, the nanonucleic acid carrier is modified with CpG2006 and C12 / PD-L1 aptamers in a sequence-extended manner to actively target and mediate specific binding to HDLBP (high-density lipoprotein binding protein) and PD-L1 proteins, both highly expressed on cancer cell surfaces, as well as CD47 siRNA and PD-L1 siRNA gene regulators. Approximately 30 epirubicin-mounting sites are designed on the three double-stranded DNA arms formed by self-assembly of the three core backbone sequences. During preparation, a 40:1 excess (epirubicin: nanonucleic acid carrier) is preferably used, along with 4% paraformaldehyde as a coupling agent. The benzotetracycline ring structure of the epirubicin molecule is specifically inserted into the GC / CG sites of the nanonucleic acid carrier. The amino group of the daunose structure interacts with the amino group of a guanosine ring in the carrier's GC / CG binding site, forming a Schiff base covalent bonding reaction catalyzed by paraformaldehyde, resulting in a stable mounting mechanism characterized by physical intercalation and covalent attachment. In addition to the nanoparticle's natural passive targeting "funnel" aggregation effect on tumor tissue, this nanoparticle also exhibits a targeted affinity for tumor tissues and cells that overexpress HDLBP and PD-L1 proteins. These two targeting effects mediate high concentrations of the drug at the tumor site. After binding to the highly expressed HDLBP protein on the surface of tumor cells, the C12 aptamer enters the cytoplasm through mechanisms such as endocytosis. Under the catalytic influence of the low pH and high concentration of glutathione reducing agents within the tumor cell, epirubicin dissociates from the nanocarrier. The released epirubicin then enters the cell nucleus and specifically binds to the GC / CG sites of double-stranded DNA embedded in the nucleus, blocking DNA transcription. This inhibits tumor cell differentiation and proliferation, producing an anti-tumor effect that suppresses tumor development and progression.

[0384] The above-mentioned drug 14 combines multiple treatment approaches, including immunotherapy, gene knockdown, and chemotherapy, to jointly inhibit advanced, recurrent, and metastatic refractory small cell lung cancer. The following is the mechanism of action of each component:

[0385] 1. CpG 2006: CpG 2006 is a CpG oligonucleotide that modulates immune responses by activating Toll-like receptor 9 (TLR9). Activating TLR9 can enhance anti-tumor immune responses, such as stimulating dendritic cell and B cell activation, promoting Th1 cytokine production, and enhancing the activity of cytotoxic T cells and natural killer cells.

[0386] 2. C12: C12 is an aptamer that targets HDLBP (high-density lipoprotein binding protein), which is highly expressed on the surface of small cell lung cancer cells. It helps to specifically target nanoparticles to tumor cells, improving therapeutic efficacy and reducing toxicity to normal tissues.

[0387] 3. CD47 siRNA: CD47 siRNA is a small interfering RNA that inhibits tumor cell immune escape by knocking down CD47 protein expression. CD47 normally acts as a "don't eat me" signal, and by binding to SIRPα, it inhibits macrophage phagocytosis of tumor cells. Reducing CD47 expression helps restore macrophage phagocytosis, thereby enhancing anti-tumor immunity.

[0388] 4. PD-L1: PD-L1 aptamers are aptamers that target PD-L1. By binding to PD-L1, they prevent its interaction with the PD-1 receptor, thereby activating an anti-tumor immune response. The PD-1 / PD-L1 pathway plays a key role in tumor immune escape, inhibiting T cell activity. By blocking this pathway, immune cells can enhance their attack on tumor cells.

[0389] 5. PD-L1 siRNA: PD-L1 siRNA is a small interfering RNA that targets the PD-L1 gene. It enhances immune cell attacks against tumor cells by reducing PD-L1 protein expression. Combined with the Apt PD-L1 aptamer, PD-L1 siRNA can more effectively block the PD-1 / PD-L1 pathway and activate the immune response.

[0390] 6. Nanonucleic acid vectors are formed by self-assembly of three partially continuous complementary DNA oligonucleotides. They connect the aforementioned multiple effective nucleic acid therapeutic molecules through sequence extension, improving their stability and targeting, and enabling synergistic therapy.

[0391] 7. Epirubicin, a DNA topoisomerase II inhibitor, can block DNA replication and transcription, thereby inhibiting tumor cell growth. Nanoparticles deliver epirubicin to tumor tissues and cells, where it then produces a sustained-release effect under the catalytic action of low pH and high glutathione reduction, increasing drug concentration in tumor tissue and reducing systemic toxicity.

[0392] In summary, the mechanism of action of drug 14 includes immune response activation via CpG 2006, tumor targeting via the C12 aptamer, combined immune evasion blockade by CD47 and PD-L1 siRNAs, and tumor cell growth inhibition via epirubicin. This nanoparticle therapy integrates multiple therapeutic approaches to jointly inhibit advanced, recurrent, and metastatic, refractory cancers, such as small cell lung cancer, from different pathways.

[0393] (12) Targeted chemical drugs include DNA vectors, chemical small molecule drugs, and miRNA oligonucleotide A-miR-21, siRNA oligonucleotide TGF-β1, nucleic acid aptamer TfRA4, and nucleic acid aptamer PD-L1 connected to the DNA vector; preferably, the chemical small molecule drug is epirubicin or its free base or its hydrochloride; preferably, the DNA vector contains a second group of sequences, The molar ratio of the siRNA oligonucleotide TGF-β1 to the DNA vector is 1:1, and its antisense strand is connected to the 3' end of the c sequence as a transition base segment, and the sense strand is complementary to the antisense strand as a complementary sequence; the molar ratio of the nucleic acid aptamer TfRA4 to the DNA vector is 1:1, and it is connected to the 5' end of the a sequence; preferably, the nucleic acid aptamer TfRA4 is connected to the 5' end of the a sequence through a base bridge TTTTT; the molar ratio of the nucleic acid aptamer PD-L1 to the DNA vector is 1:1, and it is connected to the 5' end of the b sequence; preferably, the nucleic acid aptamer PD-L1 is connected to the 5' end of the b sequence through a base bridge TTTTT; the molar ratio of the miRNA oligonucleotide A-miR-21 to the DNA vector is 2:1, and it is sequentially connected to the 5' end of the sense strand of the siRNA oligonucleotide TGF-B. Specifically, the targeted chemical drug includes a targeted nucleic acid vector formed by self-assembly of the following sequences and epirubicin mounted on the targeted nucleic acid vector:

[0394] Drug 15: A-miR-21-TGF-β1siRNA-Apt TfRA4-Apt PD-L1-DNA-epirubicin

[0395] Note: The 5' end of sequence B contains thiolation between bases 1-3; the 5' end of sequence C contains thiolation between bases 29-31 and between bases 1-3; the 3' end of sequence D contains thiolation between bases 1-3 and between bases 19-21. C / U2'F modifications are underlined in the RNA sequence.

[0396] Drug 15 is a nano-nucleic acid integrated aptamer active gene combined immunotherapy agent that can achieve combined immunotherapy and classical chemotherapy drugs to target the tumor microenvironment, effectively inhibiting recurrent, resistant, brain glioma or brain metastatic tumor cells. The following are the functions of each effector molecule of this drug and their synergistic effects in tumor suppression:

[0397] 1. A-miR-21: AmiR-21 is an ASO that targets microRNA-21 (miR-21). MiR-21 is a ubiquitously expressed microRNA that is associated with the development, progression, invasion, and metastasis of various tumors. A-miR-21 binds to miR-21 and inhibits the regulation of its target genes, thereby reducing the proliferation, invasion, and metastasis of tumor cells.

[0398] 2. TGF-β1 siRNA: TGF-β1 siRNA is a small interfering RNA (siRNA) that specifically knocks down TGF-β1. TGF-β1 is a transforming growth factor that is often overexpressed in the tumor microenvironment, promoting tumor cell growth, invasion, and metastasis. By reducing TGF-β1 expression, TGF-β1 siRNA can inhibit tumor cell growth and metastasis.

[0399] 3. Apt TfRA4: TfRA4 is a nucleic acid aptamer targeting the transferrin receptor (TfR). TfR is highly expressed on tumor cells and the blood-brain barrier (BBB). Apt TfRA4 actively targets the drug to tumor cells and the BBB by binding to TfR. This helps increase drug concentration in tumor tissue and reduce toxicity to normal tissues.

[0400] 4. Apt PD-L1: PD-L1 aptamers are nucleic acid aptamers that target programmed death ligand 1 (PD-L1) on the surface of tumor cells. PD-L1 binds to PD-1 on immune cells, inhibiting their activity and allowing tumor cells to evade immune surveillance. Apt PD-L1 binds to PD-L1, preventing its interaction with PD-1, restoring immune cell activity and enhancing the effectiveness of tumor immunotherapy.

[0401] 5. The drug's nanonucleic acid carrier backbone combines molecules such as AmiR-21, TGF-β1 siRNA, TfRA4 aptamer, and PD-L1 aptamer to form an integrated targeted drug. The nanonucleic acid carrier backbone also stabilizes the entire structure, improving the drug's biostability and duration of action.

[0402] 6. Epirubicin: Epirubicin is a widely used anti-tumor drug that inhibits tumor growth by primarily suppressing DNA synthesis and cell division in tumor cells. Covalently attaching epirubicin to a nanonucleic acid carrier backbone enables combined therapy with immunotherapeutics, enhancing anti-tumor efficacy.

[0403] Specifically, the above-mentioned drug 15 achieves highly effective treatment through the following synergistic tumor-suppressing effects:

[0404] 1. Combined targeting: AmiR-21, TGF-β1 siRNA, TfRA4 aptamer, and PD-L1 aptamer target tumor cells' miR-21, TGF-β1, TfR, and PD-L1, respectively, achieving multi-pathway tumor suppression. This combined targeting strategy helps improve drug efficacy and reduce side effects.

[0405] 2. Combination therapy: Epirubicin, as a classic chemotherapy drug, can be combined with gene therapy agents such as AmiR-21 and TGF-β1 siRNA and PD-L1 aptamer immunotherapy to achieve comprehensive treatment and enhance anti-tumor effects.

[0406] 3. Synergy of immunotherapy: PD-L1 aptamers can activate immune cells in the tumor microenvironment by relieving immunosuppression, and produce synergistic anti-tumor effects with chemotherapy drugs such as epirubicin.

[0407] 4. Knockdown of key signaling pathways: AmiR-21 and TGF-β1 siRNAs inhibit the growth, invasion and metastasis of tumor cells by knocking down key signaling pathways in tumor cells, thereby enhancing the anti-tumor effect of drugs.

[0408] In summary, drug 15 utilizes multiple targeting strategies and combined treatment approaches to exert a synergistic effect in suppressing tumors. The following are the specific manifestations of the synergistic tumor suppression effect:

[0409] 5. Improved drug permeability: By binding to TfR, the TfRA4 aptamer enables the entire nanodrug to more easily cross the blood-brain barrier (BBB), increasing the drug concentration in tumor tissue. This is of great significance for the treatment of gliomas and brain metastases.

[0410] 6. Reduced toxicity and side effects: Multi-pathway targeting and combined treatment strategies can help reduce drug dosage, thereby reducing toxicity to normal cells and tissues during treatment. In addition, integrating drugs into nanonucleic acid carriers can increase the drug's biostability and duration of action, further reducing side effects.

[0411] 7. Combating drug resistance: Combination therapy strategies can overcome drug resistance that may arise from single-drug treatments. By targeting multiple signaling pathways and mechanisms in tumor cells, they can reduce tumor cell tolerance to drugs, thereby improving efficacy.

[0412] 8. Prolonging survival and improving quality of life: Drug 15, through multi-pathway synergistic tumor suppression, can effectively control tumor growth, invasion, and metastasis, thereby prolonging patient survival. At the same time, reducing toxicity and side effects helps improve patients' quality of life.

[0413] In summary, the above-mentioned drug 15 is a nano-nucleic acid integrated aptamer active gene combined immunotherapy agent, whose effector molecules exert synergistic anti-tumor effects, achieving combined targeting of the tumor microenvironment by immunotherapy and classical chemotherapy drugs. This innovative therapy has important value in effectively inhibiting recurrent, resistant, brain glioma or brain metastatic tumor cells.

[0414] (13) Targeted chemical drugs include DNA vectors, chemical small molecule drugs, and nucleic acid aptamers IL-4Ra, siRNA oligonucleotide CD47, DNA-formed nucleic acid aptamers TfRA4, siRNA oligonucleotide PD-L1, and nucleic acid aptamers GPC-1 connected to the DNA vector; preferably, the chemical small molecule drug is epirubicin or its free base or its hydrochloride; the DNA vector contains a third group of sequences, the molar ratio of the nucleic acid aptamer TfRA4 to the DNA vector is 1:1, and the nucleic acid aptamer is connected to the 5' end of the a sequence; preferably, the nucleic acid aptamer TfRA4 is connected to the 5' end of the a sequence via a base bridge TTTTT; the molar ratio of the nucleic acid aptamer GPC-1 to the DNA vector is 1:1, and the nucleic acid aptamer GPC-1 is connected to the 5' end of the c sequence ; The molar ratio of siRNA oligonucleotide PD-L1 to DNA vector is 1:1, and its sense chain is connected to the 5' end of sequence b as a transition base segment, and the antisense chain is complementary to the sense chain as a complementary sequence and is connected to the 3' end of sequence a; The molar ratio of siRNA oligonucleotide CD47 to DNA vector is 1:1, and its sense chain is connected to the 3' end of sequence c as a transition base segment, and the antisense chain is complementary to the sense chain as a complementary sequence; The molar ratio of nucleic acid adaptor IL-4Ra to DNA vector is 1:1, and is connected to the 5' end of the antisense chain of siRNA oligonucleotide CD47; Preferably, nucleic acid adaptor IL-4Ra is connected to the 5' end of the antisense chain of siRNA oligonucleotide CD47 through a base bridge AAAA. Specifically, the targeted chemical drug includes a targeted nucleic acid vector formed by self-assembly of the following sequences and epirubicin mounted on the targeted nucleic acid vector:

[0415] Drug 16: Apt IL-4Ra-CD47 siRNA-Apt TfRA4-PD-L1 siRNA-Apt GPC-1-DNA-epirubicin

[0416] Note: The adjacent bases at positions 1-3 of the 3' end of sequence A are all thiolated, and the adjacent bases at positions 19-21 of the 3' end of sequence A are all thiolated; the adjacent bases at positions 1-3 of the 5' end of sequence B are all thiolated, and the adjacent bases at positions 19-21 of the 5' end of sequence B are all thiolated; the adjacent bases at positions 1-3 of the 3' end of sequence C are all thiolated, and the adjacent bases at positions 19-21 are all thiolated; the adjacent bases at positions 1-4 of the 5' end of sequence D are all thiolated, and the adjacent bases at positions 1-3 of the 3' end of sequence D are all thiolated, and the adjacent bases at positions 19-21 of the 3' end of sequence D are all thiolated. In sequences A, B, and C, the underlined RNA sequences are C / U2'F modified; in sequence D, the first 44 bases are C / U2'F and A / G2'-OMe modified, and bases 45-64 are C / U2'F modified.

[0417] Drug 16, described above, is a nano-nucleic acid integrated aptamer active gene combined immunotherapy agent that combines multiple therapeutic strategies, including immunotherapy, targeted therapy, gene therapy, and classical chemotherapy, to achieve combined targeting of the tumor microenvironment. This integrated treatment approach is highly effective in inhibiting recurrent, resistant, and brain glioma or brain metastatic tumor cells. The following are the functions of each effector molecule and their role in the synergistic tumor suppression effect:

[0418] 1. IL-4Ra: IL-4Ra is a subunit of the interleukin-4 receptor. As an immunotherapy target, it can regulate the polarization of tumor-associated macrophages (TAMs), thereby affecting the tumor microenvironment. By regulating the polarization of TAMs, tumor growth and metastasis can be inhibited.

[0419] 2. CD47 siRNA: CD47 is an immune checkpoint protein that inhibits macrophage phagocytosis of tumor cells by exerting a "don't eat me" signal. CD47 siRNA specifically knocks down CD47 expression, eliminating the "don't eat me" effect and enhancing macrophage phagocytosis of tumor cells.

[0420] 3. TfRA4 aptamer: TfRA4 aptamer can specifically bind to the transferrin receptor on the blood-brain barrier (BBB), prompting nanomedicines to cross the BBB and enter brain tumor tissue, thereby increasing the concentration of drugs at the tumor site.

[0421] 4. PD-L1 siRNA: PD-L1 is an immune checkpoint protein that, by binding to PD-1, inhibits T cell activity and reduces the immune system's ability to clear tumors. PD-L1 siRNA can specifically knock down PD-L1 expression, relieving its inhibitory effect on T cells and enhancing the immune system's ability to clear tumors.

[0422] 5. GPC-1 aptamer: GPC-1 aptamer can specifically bind to and inhibit the highly expressed receptor proteins on the surface of glioma or brain metastasis cells, thereby exerting a targeted inhibitory effect on tumor cells and slowing tumor growth and metastasis.

[0423] 6. Nano-nucleic acid carriers: They can combine aptamers and siRNA to achieve highly integrated nano-drug design, improving the targeting and biological activity of drugs.

[0424] 7. Epirubicin is a classic anti-tumor drug that inhibits DNA replication and cell division in tumor cells. Combining epirubicin with other immune and gene therapy agents via nanocarriers offers the advantages of combined therapy and enhances anti-tumor effects.

[0425] Through the synergistic effects of these effector molecules, this drug, based on a nano-nucleic acid integrated aptamer active gene combined with immunotherapy, has significant advantages in inhibiting recurrent, resistant, and brain glioma or brain metastatic tumor cells. This combined treatment strategy can achieve the synergistic effects of multiple therapeutic approaches, by regulating the tumor microenvironment, enhancing the immune system's ability to clear tumors, promoting drug cross-border blood-brain barrier, and specifically inhibiting tumor cells, thereby achieving highly effective inhibition of recurrent, resistant, and brain glioma or brain metastatic tumor cells.

[0426] (14) Targeted chemical drugs include DNA vectors, chemical small molecule drugs, and DNA-based nucleic acid aptamers AS1411, DNA-based nucleic acid aptamers EGFR, and DNA-based nucleic acid aptamers A15 connected to the DNA vector, preferably the chemical small molecule drug is gemcitabine; preferably, the DNA vector contains the fourth group of sequences, the molar ratio of the DNA-based nucleic acid aptamer AS1411 to the DNA vector is 1:1, and the DNA vector is connected to the 5' end of the a sequence; the molar ratio of the DNA-based nucleic acid aptamer EGFR to the DNA vector is 1:1. The ratio is 1:1, and it is connected to the 5' end of the b sequence; the molar ratio of the DNA aptamer A15 to the DNA vector is 1:1, and it is connected to the 5' end of the c sequence; preferably, gemcitabine is connected in the form of at least one base insertion at one or more of the following positions: between the DNA aptamer AS1411 and the a sequence, at the 3' end of the a sequence, between the DNA aptamer EGFR and the b sequence, at the 3' end of the b sequence, between the DNA aptamer A15 and the c sequence, and at the 3' end of the c sequence. Specifically, the targeted chemical drug is a gemcitabine targeted chemical drug formed by self-assembly of the following sequence:

[0427] Drug 17: Apt AS1411-Apt EGFR-Apt A15-DNA + gemcitabine

[0428] Note: The J above represents gemcitabine, which is directly synthesized in the sequence in the form of base substitution and base extension during sequence synthesis, which is equivalent to 36 gemcitabine molecules connected to a single DNA targeting vector.

[0429] SEQ ID NO:103:5'-GGTGGTGGTGGTTGTGGTGGTGGTGG-3';

[0430] SEQ ID NO:15:5'-GCGGCGCCCACGAGCGTTCCGGGAGAGGCC-3';

[0431] SEQ ID NO:139:5'-GCCTTAGTAACGTGCTTTGATGTCGATTCGACAGGAGGC-3';

[0432] SEQ ID NO:16:5'-GGCCTCTCCCGGTTCGCCGCCAGCCGCC-3';

[0433] SEQ ID NO:102:5'-CCCTCCTACATAGGG-3';

[0434] SEQ ID NO: 14: 5'-GGCGGCTGGCGGCCATAGCCGTGGGCGCCGC-3'.

[0435] In the aforementioned drug 17, highly specific targeting of tumor cells is achieved by integrating different aptamers into a nanonucleic acid carrier. Among them, gemcitabine can be directly incorporated into the carrier aptamer sequence during synthesis using an oligonucleotide synthesizer. The functions of each aptamer are as follows:

[0436] 1. AS1411: AS1411 is a nucleolin aptamer that can specifically bind to nucleolin receptors on the surface of tumor cells, thereby achieving tumor cell targeting.

[0437] EGFR: EGFR aptamers have the ability to specifically bind to the epidermal growth factor receptor (EGFR). EGFR is overexpressed in many tumor cells, so EGFR aptamers can enhance the targeting of nanonucleic acid vectors to tumor cells.

[0438] 3. A15: The A15 aptamer can specifically bind to the stem cell marker CD133 receptor protein. This enables the nanonucleic acid carrier to better target cancer stem cells, thereby combating tumor recurrence and drug resistance.

[0439] 4. Nano-nucleic acid carriers serve as the basic framework to integrate aptamers with drugs to improve the biological activity and targeting of drugs.

[0440] 5. Gemcitabine is a widely used anti-tumor drug that inhibits DNA synthesis and cell division. By combining gemcitabine with other aptamers via nanoparticles, it achieves highly specific targeting of tumor cells.

[0441] In summary, drug 17 combines multiple aptamers to achieve highly specific targeting of tumor cells. By targeting nucleolin, EGFR, and CD133 receptors, this nanonucleic acid carrier can effectively inhibit tumor cells, especially cancer stem cells. The introduction of gemcitabine further enhances the anti-tumor effect, making this nanonucleic acid carrier have great potential for clinical application.

[0442] (15) Targeted chemical drugs include RNA vectors, chemical small molecule drugs, and DNA-based nucleic acid aptamers AS1411, DNA-based nucleic acid aptamers EGFR, and DNA-based nucleic acid aptamers A15 connected to the RNA vector, preferably the chemical small molecule drug is 5' fluorouracil; preferably, the RNA vector contains the fifth group of sequences, the molar ratio of the DNA-based nucleic acid aptamer AS1411 to the RNA vector is 1:1, and the nucleic acid aptamer AS1411 is connected to the 5' end of the a sequence; the molar ratio of the DNA-based nucleic acid aptamer EGFR to the RNA vector is 1:1. The molar ratio of DNA aptamer A15 to RNA vector is 1:1, and it is connected to the 5' end of sequence b; the molar ratio of DNA aptamer A15 to RNA vector is 1:1, and it is connected to the 5' end of sequence c; preferably, 5-fluorouracil is connected in the form of at least one base insertion at one or more of the following positions: between DNA aptamer AS1411 and sequence a, at the 3' end of sequence a, between DNA aptamer EGFR and sequence b, at the 3' end of sequence b, between DNA aptamer A15 and sequence c, and at the 3' end of sequence c. Specifically, the targeted chemical drug is a 5'-fluorouracil targeted chemical drug formed by self-assembly of the following sequence:

[0443] Drug 18: Apt AS1411-Apt EGFR-Apt A15-RNA-5'-fluorouracil

[0444] Note: The N above represents 5'-fluorouracil (5-FU), which is directly synthesized into the sequence during sequence synthesis via base substitution and base extension. Each base between two adjacent 5'-fluorouracil residues is thiolated, equivalent to 36 5-fluorouracil residues attached to a single DNA targeting vector. Sequence A is thiolated between the first two bases at the 5' end; sequence B is thiolated between the 5' and 3' ends; and sequence C is thiolated between the 5' end and the 2' end. RNA C / U2F and A / G2'_OMe are thiolated between the 5' and 3' ends. C / U2'F and A / G2'_OMe modifications are underlined in the RNA sequence.

[0445] Drug 18, described above, achieves highly specific targeting of tumor cells through the integration of multiple aptamers via a nanonucleic acid carrier. 5'-fluorouridine can be directly incorporated into the carrier aptamer sequence during synthesis using an oligonucleotide synthesizer. The functions of the various components of this nanonucleic acid carrier are as follows:

[0446] 1. 5'-Fluorouracil is a widely used anti-tumor drug that inhibits DNA and RNA synthesis. 5'-Fluorouracil is a nucleoside analog of 5-FU and is more effective in entering cells and releasing its therapeutic effects.

[0447] 2. AS1411: AS1411 is a nucleolin aptamer that can specifically bind to nucleolin receptors on the surface of tumor cells to achieve tumor cell targeting.

[0448] EGFR: EGFR aptamers have the ability to specifically bind to the epidermal growth factor receptor (EGFR). EGFR is overexpressed in many tumor cells, so EGFR aptamers can enhance the targeting of nanonucleic acid vectors to tumor cells.

[0449] 4. A15: The A15 aptamer can specifically bind to the stem cell marker CD133 receptor protein. This enables the nanonucleic acid carrier to better target cancer stem cells, thereby combating tumor recurrence and drug resistance.

[0450] 5. Nano-nucleic acid carriers serve as the basic framework to integrate aptamers with drugs to improve the biological activity and targeting of drugs.

[0451] In summary, drug 18 achieves highly specific targeting of tumor cells by integrating multiple aptamers. This nanonucleic acid carrier utilizes a strategy of targeting nucleolin, EGFR, and CD133 receptors, resulting in potent inhibitory effects on tumor cells, particularly cancer stem cells. Furthermore, the incorporation of 5'-fluorouridine further enhances the anti-tumor effect, giving this nanonucleic acid carrier promising clinical application potential.

[0452] (16) Targeted chemical drugs include DNA vectors, chemical small molecule drugs, and DNA-formed nucleic acid aptamers AS1411, DNA-formed nucleic acid aptamers EGFR, and DNA-formed nucleic acid aptamers A15 connected to the DNA vector, preferably the chemical small molecule drug is paclitaxel; preferably, the DNA vector contains the sixth group of sequences, the molar ratio of the DNA-formed nucleic acid aptamer AS1411 to the DNA vector is 1:1, and the nucleic acid aptamer is connected to the 5' end of the a sequence; preferably, the nucleic acid aptamer AS1411 is connected to the 5' end of the a sequence via a base bridge TTTTT; the molar ratio of the DNA-formed nucleic acid aptamer EGFR to the DNA vector is 1:1, and the nucleic acid aptamer EGFR is connected to the 5' end of the b sequence; preferably, the nucleic acid aptamer EGFR is connected to the 5' end of the b sequence via a base bridge TTTTT; the molar ratio of the DNA-formed nucleic acid aptamer A15 to the DNA vector is 1:1, and the nucleic acid aptamer is connected to the 5' end of the c sequence; preferably, the nucleic acid aptamer A15 is connected to the 5' end of the c sequence via a base bridge TTTTT. Specifically, the targeted chemical drug includes the following sequences that self-assemble to form a targeted nucleic acid carrier and paclitaxel mounted thereon:

[0453] Drug 19: Apt AS1411-Apt EGFR-Apt A15-DNA-paclitaxel

[0454] Note: The 5' and 3' ends of the A, B, and C sequences are all thiolated between bases 1-2. The 35th, 39th, 54th, and 59th bases from the 5' end of the A sequence are amino- (-NH2) modified. The 50th, 55th, 66th, and 70th bases from the 5' end of the B sequence are amino- (-NH2) modified. The 24th and 28th bases from the 5' end of the C sequence are amino- (-NH2) modified.

[0455] In the aforementioned drug 19, during the synthesis of the three DNA oligonucleotide / aptamer base sequences, the nanonucleic acid carrier synthesizes multiple amino linkers at the sequence design site, which can be covalently linked to a linker with a disulfide bond modified on the 2'HO of paclitaxel through a linker reaction. 8-12 paclitaxel molecules are attached to the nanoparticle, which efficiently aggregates in the tumor microenvironment and enters the target cells under the guidance of the three-aptamer composite targeting, releasing the paclitaxel molecules. This targeted paclitaxel nanoparticle can effectively inhibit a variety of paclitaxel-sensitive tumors and has many advantages such as high targeting, controlled release, and low toxicity, and is expected to improve the efficacy of paclitaxel treatment and reduce side effects. Specifically, the drug has the following properties:

[0456] 1. Highly Targeted: The combination of AS1411, EGFR, and A15 aptamers enables the nanoparticles to achieve high targeting. The AS1411 aptamer primarily targets tumor cell nucleolar proteins, the EGFR aptamer primarily targets the epidermal growth factor receptor on tumor cells, and the A15 aptamer specifically recognizes and binds to cancer stem cell-like proteins. These three aptamers work synergistically, enabling the nanoparticles to efficiently accumulate in the tumor microenvironment and enter target cells.

[0457] 2. Controlled Release: Paclitaxel molecules are covalently linked to nanoparticles via a disulfide linker. Once the nanoparticles enter tumor cells, the intracellular reducing environment (e.g., high glutathione concentrations) cleaves the disulfide bonds, releasing the paclitaxel molecules. This controlled release mechanism helps minimize nonspecific accumulation of paclitaxel in normal tissues and reduces toxicity.

[0458] 3. Low toxicity: Due to the highly targeted and controlled release characteristics of nanoparticles, the nonspecific accumulation of paclitaxel in normal tissues is reduced, thereby reducing toxicity to normal tissues. This is conducive to improving therapeutic efficacy and reducing side effects.

[0459] 4. Anti-tumor Effect: Upon release, the paclitaxel molecules loaded into the nanoparticles can inhibit the mitotic process of tumor cells and induce apoptosis by blocking the dynamic equilibrium between microtubule polymerization and depolymerization. Furthermore, paclitaxel can further inhibit tumor development and spread by inhibiting tumor cell migration, invasion, and angiogenesis.

[0460] 5. Addressing drug resistance: The three aptamers in the nanonucleic acid carrier target multiple sites, which helps reduce the likelihood of tumor cells developing resistance to paclitaxel. Furthermore, the nanoparticles' highly targeted and controlled release properties result in higher concentrations of paclitaxel in the tumor microenvironment, further enhancing the killing effect on drug-resistant tumor cells.

[0461] 6. Combination Therapy: The AS1411-EGFR-A15-DNA-paclitaxel nanoparticle can be used in combination with other anti-tumor drugs, radiotherapy, and immunotherapy to produce synergistic tumor-suppressing effects. For example, when used in combination with immunotherapy, it can enhance the anti-tumor effects of immune cells by improving the tumor microenvironment; when used in combination with radiotherapy, it can increase the sensitivity of tumor cells to radiotherapy through radiosensitization.

[0462] 7. Personalized treatment: Based on the expression of targets on the surface of tumor cells of different patients, the aptamer combination can be adjusted and the nano-nucleic acid carrier can be customized to achieve personalized treatment.

[0463] In summary, drug 19 demonstrates excellent anti-tumor activity through its highly targeted, controlled release, and low toxicity. By combining it with other treatments, addressing drug resistance, and enabling personalized therapy, this nanonucleic acid carrier is expected to become an effective treatment for a variety of tumors.

[0464] (17) The targeted chemical drug includes a DNA vector, a chemical small molecule drug, and a DNA-formed nucleic acid aptamer AS1411, a DNA-formed nucleic acid aptamer EGFR, and a DNA-formed nucleic acid aptamer A15 connected to the DNA vector, preferably the chemical small molecule drug is metansine; preferably, the DNA vector contains the seventh group of sequences, the molar ratio of the DNA-formed nucleic acid aptamer AS1411 to the DNA vector is 1:1, and is connected to the 5' end of the a sequence; preferably, the nucleic acid aptamer AS1411 is connected to the 5' end of the a sequence via a base bridge TTTTT; the molar ratio of the DNA-formed nucleic acid aptamer EGFR to the DNA vector is 1:1, and is connected to the 5' end of the b sequence; preferably, the nucleic acid aptamer EGFR is connected to the 5' end of the b sequence via a base bridge TTTTT; the molar ratio of the DNA-formed nucleic acid aptamer A15 to the DNA vector is 1:1, and is connected to the 5' end of the c sequence; preferably, the nucleic acid aptamer A15 is connected to the 5' end of the c sequence via a base bridge TTTTT. Specifically, the targeted chemical drug includes the following sequences that self-assemble to form a targeted nucleic acid carrier and maytansine mounted thereon:

[0465] Drug 20: Apt AS1411-Apt EGFR-AptA15-DNA + Maytansine

[0466] Note: The 5' and 3' ends of the A, B, and C sequences are all thiolated between bases 1-2. The 43rd and 61st bases from the 5' end of the A sequence are amino- (-NH2) modified, the 48th and 63rd bases from the 5' end of the B sequence are amino- (-NH2) modified, and the 24th and 43rd bases from the 5' end of the C sequence are amino- (-NH2) modified.

[0467] In the aforementioned drug 20, during the synthesis of three DNA oligonucleotide / aptamer base sequences, multiple amino linkers are synthesized at the designed sequence sites. These amino linkers can then covalently bind to the modified linker on the 2' HO residue of maytansine. Six maytansine molecules are attached to the nanoparticle, and under the guidance of the three-aptamer complex targeting mechanism, they efficiently accumulate in the tumor microenvironment and enter target cells, releasing the highly toxic maytansine molecules. This targeted paclitaxel nanoparticle is highly effective in inhibiting a variety of maytansine-sensitive tumors.

[0468] The drug can efficiently accumulate in the tumor microenvironment and enter target cells under the guidance of a triple-aptamer composite targeting agent, releasing highly toxic maytansine molecules. This targeted nanoparticle can effectively inhibit a variety of tumors sensitive to maytansine. The following is a detailed analysis of the nanoparticle's mechanism of action:

[0469] 1. Targeting: The AS1411-EGFR-A15-DNA-maytansine nanoparticle carrier achieves high targeting of tumor cells through three aptamers (AS1411, EGFR, and A15). The AS1411 aptamer specifically binds to the nucleolin receptor, the EGFR aptamer specifically binds to the epidermal growth factor receptor, and the A15 aptamer binds to the CD133 receptor protein. Through these three aptamers, the nanoparticle carrier can accurately identify and target tumor cells.

[0470] 2. Drug Loading Performance: In the design of the nanonucleic acid carrier, the maytansine molecule is covalently linked to the DNAh oligonucleotide / aptamer base sequence via an amino linker. Each nanoparticle can carry six maytansine molecules, thereby increasing the drug loading capacity.

[0471] 3. Drug Release: After the nanonucleic acid vector successfully enters the tumor cell, the maytansine molecule is released from the linker under appropriate conditions, thereby exerting a toxic effect on the tumor cell. Maytansine is a DNA synthesis inhibitor that can prevent the synthesis of tumor cell DNA, thereby causing tumor cell apoptosis.

[0472] 4. Tumor Suppression: Maytansine nanoparticles achieve highly effective tumor cell inhibition through their targeting, drug-carrying properties, and controlled release. Furthermore, Maytansine can further inhibit tumor development and spread by inhibiting tumor cell migration, invasion, and angiogenesis.

[0473] 5. Addressing drug resistance: The three aptamers in the nanonucleic acid carrier target multiple sites, which helps reduce the likelihood of tumor cells developing resistance to maytansine. Furthermore, the nanoparticles' highly targeted and controlled release properties also help reduce maytansine's toxicity to normal cells, thereby improving therapeutic efficacy and patient tolerance.

[0474] 6. Biodistribution and Pharmacokinetics: The design of the nanoparticle carrier optimizes the in vivo distribution of maytansine for tumor treatment. The nanoparticles' high stability in the circulatory system effectively prevents rapid clearance from non-target organs such as the liver and kidneys. Furthermore, the nanoparticle carrier can achieve high accumulation in tumor tissues through the enhanced permeation and retention effect (EPR effect).

[0475] 7. Safety and Biocompatibility: The materials and synthesis process of these drugs exhibit excellent biocompatibility, reducing the likelihood of adverse reactions in patients during treatment. Furthermore, their highly targeted and controlled release properties reduce their toxicity to normal cells, enhancing the safety of treatment.

[0476] In summary, drug 19, through the targeting mediation of a triple aptamer complex, achieves efficient accumulation in the tumor microenvironment and entry into target cells, releasing the highly toxic maytansine molecule. This targeted nanoparticle leverages advantages in targeting, drug loading, controlled release, tumor suppression, resistance mitigation, biodistribution and pharmacokinetics, safety, and biocompatibility, effectively inhibiting a variety of maytansine-sensitive tumors.

[0477] (18) Targeted chemical drugs include DNA vectors, chemical small molecule drugs, and nucleic acid aptamers MUC1, nucleic acid aptamers AS1411, and nucleic acid aptamers GPC-1 connected to the DNA vector, preferably the chemical small molecule drug is gemcitabine; preferably, the DNA vector contains the ninth group of sequences, the molar ratio of nucleic acid aptamer MUC1 to the DNA vector is 1:1, and is connected to the 5' end of sequence a; the molar ratio of nucleic acid aptamer AS1411 to the DNA vector is 1:1, and is connected to the 5' end of sequence b; the molar ratio of nucleic acid aptamer GPC-1 to the DNA vector is 1:1, and is connected to the 5' end of sequence c. Specifically, the targeted chemical drug includes the following sequences self-assembled to form a targeted nucleic acid vector and epirubicin mounted thereon:

[0478] Drug 21: Apt MUC1-Apt AS1411-Apt GPC1 + gemcitabine-DNA-epirubicin

[0479] Note: The J above represents gemcitabine, which was directly synthesized into the sequence during sequence synthesis through base substitution and base extension. Each adjacent base of gemcitabine is thiolated, equivalent to 36 gemcitabine residues linked to a single DNA targeting vector. Sequences A, B, and C above are all thiolated between bases 1 and 2 at the 5' end.

[0480] SEQ ID NO: 160: 5'-GCAGTTGATCCTTTGGATACCCTGG-3';

[0481] SEQ ID NO: 26: 5'-GCGACGCCCACGAGCGTTCCGGGAGAGGAGC-3';

[0482] SEQ ID NO: 103: 5'-GGTGGTGGTGGTTGTGGTGGTGGTGG-3';

[0483] SEQ ID NO: 11: 5'-GCTCCTCTCCCGGTTCGCCGCGAGCCGCG-3';

[0484] SEQ ID NO: 145: 5'-AACGGAGTGTGGCTAACTCGA-3';

[0485] SEQ ID NO: 27: 5'-CGCGGCTCGCGGCCATAGCCGTGGGCGTCGC-3'.

[0486] In the above-mentioned drug 21, the nanonucleic acid vector is composed of three DNA vector core skeleton oligonucleotides arranged in a sequence extension manner according to the designed base arrangement. The three aptamer sequences and the continuously arranged gemcitabine molecules are synthesized at one time by a DNA synthesizer into three single strands with complementary pairing relationships in some sequences, and then combined based on the principle of base complementary self-assembly to form a MUC1 / AS1411 / GPC1-gemcitabine-DNAh vector. Then, epirubicin is covalently modified on the pre-designed nanonucleic acid vector GC / CG mounting sites (30) by physical insertion and amino cross-linking to form a MUC1 / AS1411 / GPC1-gemcitabine-DNAh-epirubicin targeted nanoparticle drug. Its combined targeting function ensures that the nanoparticles specifically bind and accumulate in the tumor microenvironment and tumor cells, where MUC1 / nucleolin / GPC1 proteins are highly expressed. Epirubicin is released by reductive dissociation in the low pH and high glutathione concentrations of tumor cells. Dissociated epirubicin and enzymatically cleaved gemcitabine then enter the cell nucleus and specifically bind to the GC / CG sites of double-stranded DNA chromosomes in the nucleus, blocking DNA transcription and alternatively blocking DNA synthesis, thereby inhibiting tumor cell division and proliferation and suppressing tumor progression. The MUC1 / AS1411 / GPC1-gemcitabine-DNAh3.3-epirubicin nanoparticle triple aptamer + gemcitabine + epirubicin integrated targeted chemotherapy drug possesses unique mechanisms of action and advantages. The following analysis will detail its mechanism of action, synergistic combined targeted delivery, epirubicin raw material, sustained-release gemcitabine, and comparative advantages.

[0487] 1. Mechanism of Action: This nanoparticle drug combines three aptamers: MUC1, AS1411, and GPC1, each targeting a tumor-associated biomarker. MUC1 is a membrane-associated glycoprotein overexpressed in various cancers; AS1411 is an oligonucleotide aptamer that binds to the nucleolin receptor; and GPC1 is a cell surface glycoprotein associated with the growth and invasion of pancreatic and other cancers. These three aptamers work together to aggregate the drug within target cells, thereby enhancing therapeutic efficacy.

[0488] 2. Synergistic composite targeted delivery effect: The three aptamers work synergistically, giving the nanoparticle drug a high degree of specificity and selectivity, enabling it to specifically bind and aggregate within the tumor microenvironment and tumor cells. This synergistic effect significantly increases the drug concentration in tumor tissue, thereby enhancing therapeutic efficacy and reducing damage to normal tissue.

[0489] 3. Sustained-release effect of epirubicin and gemcitabine: Epirubicin is released by reductive dissociation in the low pH and high glutathione concentrations of tumor cells. It enters the cell nucleus and specifically binds to the GC / CG target site on double-stranded DNA chromosomes, blocking DNA transcription and thereby inhibiting tumor cell division and proliferation. Gemcitabine, in turn, is dissociated through enzymatic cleavage, alternatively blocking DNA synthesis. This sustained-release effect allows for sustained drug release in tumor tissue, enhancing therapeutic efficacy and minimizing side effects.

[0490] 4. Comparative advantages: (1) High specificity: The synergistic effect of the three aptamers makes the nanoparticle drug highly specific, effectively reducing damage to normal tissues. (2) Enhanced therapeutic effect: The synergistic composite targeted delivery effect greatly increases the concentration of the drug in tumor tissue, thereby improving the therapeutic effect. The combined action of epirubicin and gemcitabine inhibits tumor cell division and proliferation by blocking DNA transcription and alternatively blocking DNA synthesis, thereby enhancing the anti-tumor effect. (3) Reduced side effects: The sustained release effect of epirubicin and gemcitabine allows the drug to be continuously released in tumor tissue, reducing the systemic toxicity of the drug and alleviating side effects. (4) Reduced drug resistance: Gemcitabine and epirubicin have different mechanisms of action. Their combined use helps reduce the resistance of tumor cells to a single drug. (5) Widely applicable: MUC1, AS1411, and GPC1 aptamers target biomarkers overexpressed in multiple cancers, making the nanoparticle drug suitable for a variety of tumor types.

[0491] In summary, drug 21 has significant advantages. Its highly specific targeting, synergistic composite targeting delivery effect, sustained-release effect of epirubicin original drug and gemcitabine, and wide applicability make this drug have high potential and application value in anti-tumor treatment.

[0492] The above-mentioned targeted chemical drug structures help further improve the reliability of targeted delivery of small molecule drugs, giving them superior specificity. At the same time, the use of the above-mentioned targeted chemical drug structures also further promotes the loading stability and loading quantity of small molecule drugs. Through the above-mentioned connection method, the drug size is more appropriate, allowing it to enter the cell membrane through cell surface receptor-mediated phagocytosis while avoiding nonspecific cell penetration and being filtered out by the kidneys. Therefore, the favorable particle size helps improve the pharmacokinetics, pharmacodynamics, biodistribution, and toxicological distribution.

[0493] According to a second aspect of the present invention, a pharmaceutical composition is provided, which comprises the above-mentioned targeted chemical drug and a pharmaceutically acceptable carrier and / or excipient.

[0494] The aforementioned pharmaceutically acceptable excipients may vary depending on the desired dosage form and / or mode and route of administration of the drug, and may be appropriately selected from existing pharmaceutical excipients. These include, but are not limited to, pharmaceutically acceptable carriers, excipients, adjuvants, and other excipients. Furthermore, the targeted chemical drug can be formulated into various dosage forms to accommodate diverse routes of administration, including oral, parenteral, and transdermal administration. The dosage forms of the drug include capsules, tablets, oral solutions, injections, and transdermal formulations.

[0495] Specifically, the targeted chemical drugs of the present application can be made into capsules, tablets, oral liquids, injections or transdermal absorbents according to methods known in the pharmaceutical industry. Preferably, the injection is an intravenous injection. When preparing capsules, tablets, and oral liquids suitable for oral administration, sucrose, lactose, galactose, corn starch, gelatin, microcrystalline cellulose, carboxymethyl cellulose, etc. can be used as carriers or excipients. In addition, the drugs of the present application can also be made into solutions and suspensions suitable for oral administration using methods and auxiliary ingredients known in the pharmaceutical industry. If solutions and suspensions suitable for parenteral administration are prepared, distilled water, water for injection, isotonic sodium chloride or glucose solution, or low concentration (e.g., 1-100 mm) phosphate buffered saline (PBS) can be used as carriers or diluents. One or more other auxiliary ingredients or additives can be added to these parenteral preparations, such as ascorbic acid can be used as an antioxidant, and sodium benzoate, etc. can be used as a preservative. Other solubilizers, disintegrants, colorants, dispersants, or surfactants can also be contained in the preparations of these dosage forms.

[0496] According to the third aspect of the present invention, there is provided a use of the above-mentioned targeted chemical drug in the preparation of a drug for treating tumors. Furthermore, the tumor is a male tumor, a gynecological tumor, a respiratory system tumor, a digestive system tumor, a hematological tumor, a urinary system tumor, a bone marrow tumor, a neurological tumor, a dermatological tumor, a general surgical tumor or an ENT tumor; preferably, the male tumor is prostate cancer, penile cancer, testicular cancer or male urethra cancer, the gynecological tumor is ovarian cancer, cervical cancer, endometrial cancer, uterine fibroids, vulvar cancer or malignant hydatidiform mole, the respiratory system tumor is lung cancer, non-small cell lung cancer, small cell lung cancer, nasopharyngeal cancer, tracheal tumor, lung cancer metastasis, inflammatory pseudotumor or radiation lung cancer, the digestive system tumor is liver cancer, gastric cancer, colorectal cancer, gallbladder cancer, esophageal cancer, rectal cancer, pancreatic cancer or colon cancer, the hematological tumor is Leukemia, lymphoma, lymphosarcoma or multiple myeloma, renal cancer, bladder cancer or urinary cancer, bone marrow tumors are giant cell tumors of bone, osteochondroma or osteosarcoma, neurological tumors are brain tumors, meningiomas, brain tuberculomas, pituitary tumors, neuroblastomas, glioblastomas or brain gliomas, dermatological tumors are skin cancer or melanoma, general surgical tumors are breast cancer, lipomas, thyroid cancer or thyroid tumors, ENT tumors are oral cancer, tongue cancer, laryngeal cancer, middle ear cancer, gingival cancer or orbital tumors; preferably, the drug is administered by tumor administration, intravenous administration or intraperitoneal administration during the application; preferably, the daily dosage of the drug during the application is 0.1 μg / Kg to 100 mg / Kg.

[0497] According to the fourth aspect of the present invention, there is also provided a method for preventing and / or treating cancer, which comprises: providing the above-mentioned targeted chemical drug or pharmaceutical composition; administering an effective amount of the targeted chemical drug or pharmaceutical composition to a tumor patient. Preferably, the tumor is a male tumor, a gynecological tumor, a respiratory system tumor, a digestive system tumor, a hematological tumor, a urinary system tumor, a bone marrow tumor, a neurological tumor, a dermatological tumor, a general surgical tumor or an ENT tumor; preferably, the male tumor is prostate cancer, penile cancer, testicular cancer or male urethra cancer, the gynecological tumor is ovarian cancer, cervical cancer, endometrial cancer, uterine fibroids, vulvar cancer or malignant hydatidiform mole, the respiratory system tumor is lung cancer, non-small cell lung cancer, small cell lung cancer, nasopharyngeal cancer, tracheal tumor, lung cancer metastasis, inflammatory pseudotumor or radiation lung cancer, the digestive system tumor is liver cancer, gastric cancer, colorectal cancer, gallbladder cancer, esophageal cancer, rectal cancer, etc. Intestinal cancer, pancreatic cancer or colon cancer, hematological tumors are leukemia, lymphoma, lymphosarcoma or multiple myeloma, kidney cancer, bladder cancer or urinary cancer, bone marrow tumors are giant cell tumor of bone, osteochondroma or osteosarcoma, neurological tumors are brain tumors, meningioma, brain tuberculoma, pituitary tumor, neuroblastoma, glioblastoma or glioma, dermatological tumors are skin cancer or melanoma, general surgical tumors are breast cancer, lipoma, thyroid cancer or thyroid tumor, ENT tumors are oral cancer, tongue cancer, laryngeal cancer, middle ear cancer, gingival cancer or orbital tumor; preferably, the drug is administered into the tumor, intravenously or intraperitoneally.

[0498] The effective amount herein includes a prophylactic effective amount and / or a therapeutically effective amount. A therapeutically effective amount refers to an amount that effectively achieves the desired therapeutic outcome, such as relief of gastric cancer or lung cancer, within the necessary dosage and timeframe. In a specific embodiment, the dosage can be adjusted to provide the optimal therapeutic response dose. The therapeutically effective amount can vary based on factors such as the individual's disease state, age, sex, weight, and the ability of the formulation to elicit the desired response in the individual. The meaning of a therapeutically effective amount also includes an amount in which the beneficial effects of the treatment outweigh its toxic or deleterious effects. A prophylactic effective amount refers to an amount that effectively achieves the desired prophylactic outcome, such as preventing or inhibiting the occurrence of acute leukemia, breast cancer, and / or ovarian cancer, within the necessary dosage and timeframe. The prophylactic effective amount can be determined based on the above description of the therapeutically effective amount. For any particular subject, the specific dosage can be adjusted over time based on individual needs and the professional judgment of the person administering the dose.

[0499] According to the fifth aspect of the present invention, a method for preparing a targeted chemical drug is also provided, characterized in that it includes the following steps: forming a targeted nucleic acid vector with an optional oligonucleotide effector molecule and an optional immunostimulant by sequence self-assembly; and connecting a small chemical molecule drug to the targeted nucleic acid vector to obtain a targeted chemical drug. As described above, the targeted chemical drug includes a targeted nucleic acid vector and a small chemical molecule drug mounted on the targeted nucleic acid vector, and the targeted nucleic acid vector includes a nucleic acid vector and a targeting molecule connected to the nucleic acid vector; optionally, it also includes an oligonucleotide effector molecule and / or an immunostimulant mounted on the targeted nucleic acid vector. During sequence self-assembly, the targeted nucleic acid vector can directly form a single chain during sequence design and synthesis, and then be formed in a self-assembly manner. And the small chemical molecule drug can be bound to the targeted nucleic acid vector during sequence design and synthesis or by being mounted on the targeted nucleic acid vector, which should be understood by those skilled in the art.

[0500] In the actual process of self-assembly of the synthetic sequence, it can be set as multiple single sequences according to the base distribution of the target nucleic acid vector, and then self-assembled to obtain a targeted nucleic acid vector. In a preferred embodiment, the step of forming a targeted nucleic acid vector with an optional oligonucleotide effector molecule and an optional immunostimulant by sequence self-assembly includes: dissolving at least three sequences with an optional oligonucleotide effector molecule, an optional immunostimulant and a targeting molecule in an assembly solution and performing a denaturation reaction to form a self-assembled crude product; the self-assembled crude product is sequentially purified, eluted and evaporated to obtain the targeted nucleic acid vector with an optional oligonucleotide effector molecule and an optional immunostimulant. Self-assembly with at least three sequences reduces the difficulty of assembly and the length of the single sequence is more appropriate.

[0501] To make self-assembly more efficient, preferably, the at least three sequences include at least the following three sequences: sequence A: sequence a, directly linked at its end or linked via a base bridge to an optional oligonucleotide effector molecule, an optional immunostimulator, and an optional targeting molecule; sequence B: sequence b, directly linked at its end or linked via a base bridge to an optional oligonucleotide effector molecule, an optional immunostimulator, and an optional targeting molecule; sequence C: sequence c, directly linked at its end or linked via a base bridge to an optional oligonucleotide effector molecule, an optional immunostimulator, and an optional targeting molecule; the molar ratio of sequence A, sequence B, and sequence C is 0.90-1.10:0.90-1.10:0.90-1.10, more preferably 1:1:1; when the at least three sequences include sequences other than sequence a, sequence b, and sequence c, the molar ratio of each sequence to sequence A is 0.90-1.10:0.90-1.10, more preferably 1:1. Controlling the sequence molar ratio within the above range improves assembly efficiency.

[0502] Preferably, when at least three sequences contain sequences other than sequence A, sequence B, and sequence C, they are recorded as complementary sequences. At this time, any 5' end or 3' end of sequence a, sequence b, and sequence c is independently connected to a transition base segment (at least one of the 5' end or 3' end of sequence a, the 5' end or 3' end of sequence b, and the 5' end or 3' end of sequence c is connected to the transition base segment, and one transition base segment corresponds to one complementary sequence), and the complementary sequence and the transition base segment present a base complementary structure so that the complementary sequence and the transition base segment are complementary connected during the self-assembly process; more preferably, the complementary sequence itself is the sense chain or antisense chain of the siRNA oligonucleotide effector molecule and the miRNA oligonucleotide effector molecule (the sense chain or antisense chain of the siRNA oligonucleotide effector molecule, the sense chain or antisense chain of the miRNA oligonucleotide effector molecule), and / or, the complementary sequence is used to provide a mounting position for a small molecule target head, a targeting aptamer, or an immunostimulant. The use of the above complementary sequences is conducive to shortening the length of the single sequence during the self-assembly process, making the assembly more efficient and the final drug stability higher.

[0503] Further preferably, when at least three sequences contain complementary sequences, complementary sequences carrying optional small molecule target heads, optional targeting aptamers, and optional immunostimulants are added to the assembly solution at the same time to carry out a denaturation reaction; at this time, the complementary sequences can also serve as carrier sequences for small molecule target heads, targeting aptamers, and immunostimulants, and are mounted in the final targeted chemical drug during the self-assembly process.

[0504] In a preferred embodiment, the assembly liquid is a TMS aqueous solution, a sodium chloride aqueous solution, a magnesium chloride aqueous solution or purified water; preferably, the temperature of the denaturation reaction is 80-99°C, more preferably 85-99°C, and further pre-selected as 90-99°C; preferably, after the denaturation reaction is completed, the reaction system is cooled to a holding temperature for a holding reaction, and finally cooled to obtain an assembly product; wherein the holding temperature is 70-50°C, more preferably 65-55°C, and further preferably 63-57°C; the holding reaction time is 3-15 min, more preferably 3-10 min, and further preferably 3-5 min; preferably, in the process of cooling the reaction system to the holding temperature, the cooling rate is 2-10°C / min, more preferably 2-6°C / min, and further preferably 2-3°C / min; preferably, the cooling end temperature is 0-25°C, more preferably 0-15°C, and further preferably 0-4°C; preferably, the pH value of the denaturation reaction is 5.4-8.8. Carrying out the self-assembly deformation reaction of each sequence under the above process conditions has a higher yield and a more stable reaction.

[0505] Depending on the structural type of chemical small molecule drugs, the following methods can be used for drug loading:

[0506] When the chemical small molecule drug is an anthracycline drug or an acridine drug, the step of mounting the chemical small molecule drug on the targeted nucleic acid carrier includes: dissolving the chemical small molecule, a coupling agent, and a targeted nucleic acid carrier with an optional oligonucleotide effector molecule and an optional immunostimulant in a first solvent and reacting under light-proof conditions so that the chemical small molecule is specifically embedded in the GC adjacent base pairs of the targeted nucleic acid carrier to obtain a crude reaction product; the crude reaction product is crystallized, washed, and evaporated to dryness to obtain the targeted chemical drug; preferably, the coupling agent is formaldehyde and / or paraformaldehyde; preferably, the reaction temperature is 0 to 37°C, more preferably 4 to 20°C, and further preferably 4 to 8°C; preferably, the reaction pH is is 6 to 8; preferably, the reaction time is 24 to 96 hours, more preferably 48 to 72 hours, and further preferably 72 hours; preferably, the first solvent is purified water and / or PBS buffer; preferably, the detergent used in the washing process is a halogenated alkane solvent, an ether solvent or a benzene solvent, more preferably chloroform, dichloromethane, ether, toluene or xylene; preferably, the solvent used in the crystallization process is a lower alcohol solvent, more preferably methanol, ethanol or isopropanol; preferably, the average number of small molecule drugs mounted on each targeted nucleic acid carrier with an optional oligonucleotide effector molecule and an optional immunostimulant is 1 to 50, more preferably 10 to 40, and further preferably 20 to 35;

[0507] When the chemical small molecule drug is a pyrimidine drug, the small molecule target head, the oligonucleotide effector molecule and the immunostimulatory agent are connected to the terminal bases of each sequence of the nucleic acid vector through a base connection bridge comprising 1 to 10 bases. The step of connecting the chemical small molecule drug to the targeted nucleic acid vector with the optional oligonucleotide effector molecule and the optional immunostimulatory agent comprises: in the step of forming the targeted nucleic acid vector with the optional oligonucleotide effector molecule and the optional immunostimulatory agent by sequence self-assembly, the chemical small molecule drug is connected to at least one sequence of the nucleic acid vector in the form of at least partial replacement of the base in the base connection bridge and / or as an extended base and / or in the form of a carrier backbone base replacement, and then the connection of the chemical small molecule drug is completed by sequence self-assembly; preferably, the average number of chemical small molecule drugs mounted on each targeted nucleic acid vector molecule is 1 to 30, more preferably 10 to 20, and even more preferably 12 to 18;

[0508] When the chemical small molecule drug is a platinum drug, a glutamic acid derivative drug, a flavonoid drug, a botanical drug and its derivatives, a folic acid drug, or a salicylic acid drug, the step of linking the chemical small molecule drug to a targeted nucleic acid vector with an optional oligonucleotide effector molecule and an optional immunostimulant comprises: during the synthesis of each sequence of the nucleic acid vector linked to the targeting molecule, binding a linker to at least one sequence of the nucleic acid vector with the optional oligonucleotide effector molecule, the optional immunostimulant, and the targeting molecule to obtain a targeted nucleic acid vector linked to the linker; dissolving the targeted nucleic acid vector linked to the linker and the chemical small molecule drug in a second solvent for reaction, and then cooling, purifying, washing, and evaporating to dryness to obtain the targeted chemical drug;

[0509] Preferably, when the small molecule drug itself carries a succinimide lipid group, the targeted nucleic acid vector connected to the linker and the small molecule drug are dissolved in a second solvent for reaction. When the small molecule drug itself does not carry a succinimide lipid group, before dissolving the small molecule drug in the second solvent for reaction, the method further includes: reacting the small molecule drug with bis(2-hydroxyethyl) disulfide and N,N-diisopropylethylamine in sequence to modify the succinimide lipid group on the small molecule drug. Preferably, the second solvent is purified water and / or PBS buffer.

[0510] In order to enable the targeting aptamer to stretch freely, maintain its three-dimensional conformation unaffected, and maintain its specificity and affinity, these targeting aptamers can also be connected to one end of the single-stranded sequence by using a fragment sequence containing several bases, such as 3 to 6 bases, as a base bridge. For example, the EGFR aptamer is synthesized and connected to the 5' end of the b chain using a base bridge "TTTTT" (see the sequence structure of each drug described above for details). This method should be understood by those skilled in the art and will not be repeated here. If the targeting aptamer is another small molecule structure, it can also be directly synthesized at one end of the single chain by chemical modification, which is also a conventional method in the art.

[0511] The present application is further described in detail below with reference to specific embodiments. These embodiments should not be construed as limiting the scope of protection claimed in this application.

[0512] Example 1

[0513] In this example, the epirubicin targeting drug Apt EGFR-DNA-epirubicin was synthesized.

[0514] Step 1: Synthesize the Apt EGFR-DNA targeting vector, the sequence of which is shown in Table 1 below:

[0515] Table 1

[0516] Note: The bold part is the a sequence, b sequence or c sequence in the nucleic acid vector, the same below.

[0517] 1. Experimental materials and reagents:

[0518] (1)A sequence;

[0519] (2) B sequence;

[0520] (3) C sequence;

[0521] (4) TMS buffer (Tris hydrochloride, sodium chloride, magnesium chloride);

[0522] (5) TBM (Tris-borate magnesium) buffer

[0523] 2. Experimental methods:

[0524] Sequence A, sequence B, and sequence C were dissolved in TMS buffer at a molar ratio of 1:1:1, respectively; after mixing, the pH of the reaction solution was controlled to 6, the mixture was heated to 80°C, maintained for 5 minutes, then decreased to 70°C at a rate of 10°C / min and kept for reaction for 15 minutes, and then decreased to 4°C at a rate of 10°C / min to obtain a crude product.

[0525] The product was loaded onto an 8% (m / v) non-denaturing PAGE gel and the vector was purified by electrophoresis at 100 V at 4°C in TBM buffer; the target band was cut out and eluted in DNA elution buffer at 37°C, followed by ethanol precipitation overnight and evaporation at low temperature under reduced pressure to obtain the self-assembled product - EGFR-DNA targeting vector.

[0526] Step 2: Mount Epirubicin

[0527] 1. Experimental Materials and Reagents

[0528] (1) the Apt EGFR-DNA targeting vector prepared in step 1;

[0529] (2)DEPC water.

[0530] (3) PBS buffer (pH 7.4).

[0531] (4) 4% formaldehyde

[0532] (5) Epirubicin hydrochloride.

[0533] (6)Chloroform.

[0534] (7) Anhydrous ethanol.

[0535] 2. Experimental methods:

[0536] (1) Accurately weigh epirubicin hydrochloride (0.79 mg, 1.354 μmoL) and dissolve it in DEPC water (1.0 mL) and PBS buffer (1.25 mL). Add formaldehyde aqueous solution (0.25 mL) under ice-water bath cooling and mix well. Mix the mixture with EGFR-DNA targeting vector (1 mg) and react at 25°C for 24 hours under dark conditions. Control the reaction pH value at 6.

[0537] (2) The reaction solution was washed with chloroform (10 mL × 3), and 25 mL of anhydrous ethanol was added to the aqueous phase. After mixing, the mixture was placed in the dark at 4°C to allow the product to precipitate completely (4 hours). Centrifugation was performed (4000 rpm), and the supernatant was transferred. The solid product was washed again with ethanol (50 mL). The supernatant was removed, and the residue was evaporated to dryness under reduced pressure at low temperature to obtain a dark red solid product, which is the epirubicin targeted drug Apt EGFR-DNA-epirubicin.

[0538] (4) Calculation of mount rate:

[0539] 1. Prepare 100 μL of epirubicin-PBS standard solutions of known concentrations: 2 μM, 4 μM, 6 μM, 8 μM, and 10 μM;

[0540] 2. Dissolve Apt EGFR-DNA-epirubicin in 100ul PBS;

[0541] 3. Place the standard solution and Apt EGFR-DNA-epirubicin in a PCR plate, heat at 85°C for 5 minutes, and then cool to room temperature;

[0542] 4. Use a microplate reader to measure the absorbance of epirubicin at 492 nm, draw a standard curve, and calculate the molar concentration of epirubicin in the mounted product;

[0543] 5. Use a spectrophotometer to measure the absorbance of DNA at 260 nm to obtain the mass concentration of DNA vector in each sample;

[0544] 6. Calculate the loading efficiency based on the measured epirubicin molar concentration and the mass concentration of the DNA vector. The theoretical maximum loading efficiency of epirubicin for the aforementioned epirubicin-targeted drug, Apt EGFR-DNA-epirubicin, is approximately 23. Testing has shown an actual average loading efficiency of 20.

[0545] Example 2

[0546] In this embodiment, the epirubicin targeting drug Apt EGFR-4*Bio-DNA-epirubicin was synthesized.

[0547] Step 1: Synthesize the Apt EGFR-4*Bio-DNA targeting vector, the single-stranded sequence of which is shown in Table 2 below:

[0548] Table 2

[0549] 1. Experimental materials and reagents:

[0550] (1)A sequence;

[0551] (2) B sequence;

[0552] (3) C sequence;

[0553] (4) sodium chloride aqueous solution;

[0554] (5) TBM (Tris-borate magnesium) buffer

[0555] 2. Experimental methods:

[0556] Sequence A, sequence B, and sequence C were dissolved in a sodium chloride aqueous solution at a molar ratio of 1.05:1:1.05; after mixing, the pH of the reaction solution was controlled to 7. The mixture was heated to 85°C, maintained for 5 minutes, then cooled to 50°C at a rate of 4°C / min, incubated for 10 minutes, and then cooled to 10°C at a rate of 4°C / min to obtain a crude product.

[0557] The product was loaded onto an 8% (m / v) non-denaturing PAGE gel and purified by electrophoresis at 100 V in TBM buffer at 4°C. The target band was excised and eluted in DNA elution buffer at 37°C, followed by ethanol precipitation overnight and evaporation at low temperature under reduced pressure to obtain the self-assembled product—Apt EGFR-4*Bio-DNA targeting vector.

[0558] Step 2: Mount Epirubicin

[0559] 1. Experimental materials and reagents:

[0560] (1) Apt EGFR-4*Bio-DNA targeting vector prepared in step 1;

[0561] (2)DEPC water.

[0562] (3) PBS buffer (5.8).

[0563] (4) 4% paraformaldehyde

[0564] (5) Epirubicin hydrochloride.

[0565] (6)Chloroform.

[0566] (7) Anhydrous ethanol.

[0567] 2. Experimental methods:

[0568] (1) Accurately weigh epirubicin hydrochloride (0.79 mg, 1.354 μmoL) and dissolve it in DEPC water (1.0 mL) and PBS buffer (1.25 mL). Add 4% paraformaldehyde aqueous solution (0.25 mL) and mix well under ice-water bath cooling. Mix the mixture with EGFR-4 Bio-DNA targeting vector (1 mg) and react at 20°C for 48 hours under dark conditions. Control the reaction pH value at 7.

[0569] (2) The reaction solution was washed with chloroform (10 mL × 3), and 25 mL of anhydrous ethanol was added to the aqueous phase. After mixing, the mixture was placed in the dark at 4°C to allow the product to precipitate completely (4 hours). Centrifugation was performed (4000 rpm), and the supernatant was transferred. The solid product was washed again with ethanol (50 mL), and the supernatant was removed. The residue was evaporated to dryness under reduced pressure at low temperature to obtain a dark red solid product, which is the epirubicin targeted drug EGFR-4Bio-DNA-epirubicin.

[0570] (4) Calculation of mount rate:

[0571] 1. Prepare 100 μL of epirubicin-PBS standard solutions of known concentrations: 2 μM, 4 μM, 6 μM, 8 μM, and 10 μM;

[0572] 2. Dissolve Apt EGFR-4*Bio-DNA-epirubicin in 100ul PBS;

[0573] 3. Place the standard solution and Apt EGFR-4*Bio-DNA-epirubicin in a PCR plate, heat at 85°C for 5 minutes, and then cool to room temperature;

[0574] 4. Use a microplate reader to measure the absorbance of epirubicin at 492 nm, draw a standard curve, and calculate the molar concentration of epirubicin in the mounted product;

[0575] 5. Use a spectrophotometer to measure the absorbance of DNA at 260 nm to obtain the mass concentration of DNA vector in each sample;

[0576] 6. Calculate the loading efficiency based on the measured epirubicin molar concentration and the mass concentration of the DNA vector. The theoretical maximum loading efficiency of the epirubicin-targeted drug Apt EGFR-4*Bio-DNA-epirubicin is approximately 23%. Testing has shown that the actual average loading efficiency is 20%.

[0577] Example 3

[0578] In this example, the epirubicin targeted drug Apt AS1411-4*Bio-DNA-epirubicin was synthesized.

[0579] Step 1: Synthesize the Apt AS1411-4* Bio-DNA targeting vector, the single-stranded sequence of which is shown in Table 3 below:

[0580] Table 3

[0581] 1. Experimental materials and reagents:

[0582] (1)A sequence;

[0583] (2) B sequence;

[0584] (3) C sequence;

[0585] (4) magnesium chloride aqueous solution;

[0586] (5) TBM (Tris-borate magnesium) buffer

[0587] 2. Experimental methods:

[0588] Sequence A, sequence B, and sequence C were dissolved in a magnesium chloride aqueous solution at a molar ratio of 1.05:1.05:1; after mixing, the pH of the reaction solution was controlled to 8. The mixture was heated to 90°C, maintained for 5 minutes, then cooled to 65°C at a rate of 2°C / min and maintained for 5 minutes, and then cooled to 5°C at a rate of 2°C / min to obtain a crude product.

[0589] The product was loaded onto an 8% (m / v) non-denaturing PAGE gel and the vector was purified by electrophoresis at 100 V in TBM buffer at 4°C. The target band was excised and eluted in DNA elution buffer at 37°C, followed by ethanol precipitation overnight and evaporation at low temperature under reduced pressure to obtain the self-assembled product - Apt AS1411-4*Bio-DNA targeting vector.

[0590] Step 2: Mount Epirubicin

[0591] 1. Experimental materials and reagents:

[0592] (1) Apt AS1411-4*Bio-DNA targeting vector prepared in step 1;

[0593] (2)DEPC water.

[0594] (3) PBS buffer (6.5).

[0595] (4) 4% paraformaldehyde

[0596] (5) The appearance is softer than the star.

[0597] (6) Toluene.

[0598] (7) Anhydrous ethanol.

[0599] 2. Experimental methods:

[0600] (1) Accurately weigh epirubicin hydrochloride (0.79 mg, 1.354 μmoL) and dissolve it in DEPC water (1.0 mL) and PBS buffer (1.25 mL). Add 4% paraformaldehyde aqueous solution (0.25 mL) and mix well under ice-water bath cooling. Mix the mixture with AS1411-4Bio-DNA targeting vector (1 mg) and react at 15°C for 72 hours under dark conditions. Control the reaction pH to 8.

[0601] (2) The reaction solution was washed with toluene (10 mL × 3), and 25 mL of anhydrous ethanol was added to the aqueous phase. After mixing, the mixture was placed in the dark at 4°C to allow the product to precipitate completely (4 hours). Centrifugation was performed (4000 rpm), and the supernatant was transferred. The solid product was washed again with ethanol (50 mL). The supernatant was removed, and the residue was evaporated to dryness under reduced pressure at low temperature to obtain a dark red solid product, which is the epirubicin targeted drug AS1411-4Bio-DNA-epirubicin.

[0602] (4) Calculation of mount rate:

[0603] 1. Prepare 100 μL of epirubicin-PBS standard solutions of known concentrations: 2 μM, 4 μM, 6 μM, 8 μM, and 10 μM;

[0604] 2. Dissolve Apt AS1411-4*Bio-DNA-epirubicin in 100 μl PBS;

[0605] 3. Place the standard solution and Apt AS1411-4*Bio-DNA-epirubicin in a PCR plate, heat at 85°C for 5 minutes, and then cool to room temperature;

[0606] 4. Use a microplate reader to measure the absorbance of epirubicin at 492 nm, draw a standard curve, and calculate the molar concentration of epirubicin in the mounted product;

[0607] 5. Use a spectrophotometer to measure the absorbance of DNA at 260 nm to obtain the mass concentration of DNA vector in each sample;

[0608] 6. Calculate the loading efficiency based on the measured epirubicin molar concentration and the mass concentration of the DNA vector. The maximum theoretical loading efficiency of the epirubicin-targeted drug Apt AS1411-4*Bio-DNA-epirubicin is approximately 23. Testing has shown that the average loading efficiency is 20.

[0609] Example 4

[0610] In this example, the epirubicin targeted drug Apt A1-Apt A15-DNA-epirubicin was synthesized.

[0611] Step 1: Synthesize the Apt A1-Apt A15-DNA targeting vector, the single-stranded sequence of which is shown in Table 4 below:

[0612] Table 4

[0613] 1. Experimental materials and reagents:

[0614] (1)A sequence;

[0615] (2) B sequence;

[0616] (3) C sequence;

[0617] (4)DEPC water;

[0618] (5) TBM (Tris-borate magnesium) buffer

[0619] 2. Experimental methods:

[0620] Sequences A, B, and C were dissolved in DEPC water at a molar ratio of 1.1:1:1. After mixing, the pH of the reaction solution was controlled to 5.4. The mixture was heated to 95°C for 5 minutes, then cooled to 55°C at a rate of 2°C / min and incubated for 10 minutes. The temperature was then slowly cooled to 8°C at a rate of 2°C / min.

[0621] The product was loaded onto an 8% (m / v) non-denaturing PAGE gel and purified by electrophoresis at 100 V in TBM buffer at 4°C. The target band was excised and eluted in DNA elution buffer at 37°C, followed by ethanol precipitation overnight and evaporation at low temperature under reduced pressure to obtain the self-assembled product—Apt A1-Apt A15-DNA targeting vector.

[0622] Step 2: Mount Epirubicin

[0623] 1. Experimental materials and reagents:

[0624] (1) the Apt A1-Apt A15-DNA targeting vector prepared in step 1;

[0625] (2)DEPC water.

[0626] (3) PBS buffer (6.6).

[0627] (4) 4% paraformaldehyde

[0628] (5) The appearance is softer than the star.

[0629] (6) Ether.

[0630] (7) Anhydrous ethanol.

[0631] 2. Experimental methods:

[0632] (1) Accurately weigh epirubicin hydrochloride (0.79 mg, 1.354 μmoL) and dissolve it in DEPC water (1.0 mL) and PBS buffer (1.25 mL). Add 4% paraformaldehyde aqueous solution (0.25 mL) and mix well under ice-water bath cooling. Mix the mixture with A1-A15-DNA targeting vector (1 mg) and react at 5°C for 96 hours under dark conditions. Control the reaction pH value between 6 and 8.

[0633] (2) The reaction solution was washed with ether (10 mL x 3), and 25 mL of anhydrous ethanol was added to the aqueous phase. After mixing, the mixture was placed in the dark at 4°C to allow the product to precipitate completely (4 hours). The mixture was centrifuged (4000 rpm), and the supernatant was transferred. The solid product was washed again with ethanol (50 mL). The supernatant was removed, and the residue was evaporated to dryness under reduced pressure at low temperature to obtain a dark red solid product, which is the epirubicin targeted drug Apt A1-Apt A15-DNA-epirubicin.

[0634] (4) Calculation of mount rate:

[0635] 1. Prepare 100 μL of epirubicin-PBS standard solutions of known concentrations: 2 μM, 4 μM, 6 μM, 8 μM, and 10 μM;

[0636] 2. Dissolve Apt A1-Apt A15-DNA-epirubicin in 100 μl PBS;

[0637] 3. Place the standard solution and Apt A1-Apt A15-DNA-epirubicin in a PCR plate, heat at 85°C for 5 minutes, and then cool to room temperature;

[0638] 4. Use a microplate reader to measure the absorbance of epirubicin at 492 nm, draw a standard curve, and calculate the molar concentration of epirubicin in the mounted product;

[0639] 5. Use a spectrophotometer to measure the absorbance of DNA at 260 nm to obtain the mass concentration of DNA vector in each sample;

[0640] 6. Calculate the loading efficiency based on the measured epirubicin molar concentration and the mass concentration of the DNA vector. The average theoretical maximum loading efficiency of epirubicin in the epirubicin-targeted drug Apt A1-Apt A15-DNA-epirubicin is approximately 20. Testing revealed an average loading efficiency of 18.

[0641] Example 5

[0642] In this embodiment, the epirubicin targeted drug Apt AS1411-Apt EGFR-Apt A15-2*Bio-DNA-epirubicin was synthesized.

[0643] Step 1: Synthesize the Apt AS1411-Apt EGFR-Apt A15-2*Bio-DNA targeting vector, the single-stranded sequence of which is shown in Table 5 below:

[0644] Table 5

[0645] 1. Experimental materials and reagents:

[0646] (1)A sequence;

[0647] (2) B sequence;

[0648] (3) C sequence;

[0649] (4) TMS aqueous solution;

[0650] (5) TBM (Tris-borate magnesium) buffer

[0651] 2. Experimental methods:

[0652] Sequence A, sequence B, and sequence C were dissolved in a TMS aqueous solution at a molar ratio of 1:1:1. After mixing, the pH of the reaction solution was controlled to 8.8. The mixture was heated to 99°C, maintained for 5 minutes, then cooled to 60°C at a rate of 2°C / min and maintained for 5 minutes. The temperature was then cooled to 2°C at a rate of 2°C / min to obtain a crude product.

[0653] The product was loaded onto a SEC column (in series), the main fraction was intercepted, the column was desalted, and the product was evaporated to dryness under reduced pressure at low temperature to obtain a self-assembled product - Apt AS1411-Apt EGFR-Apt A15-2*Bio-DNA targeting vector.

[0654] Step 2: Mount Epirubicin

[0655] 1. Experimental materials and reagents:

[0656] (1) Apt AS1411-Apt EGFR-Apt A15-2*Bio-DNA targeting vector prepared in step 1;

[0657] (2)DEPC water.

[0658] (3) PBS buffer (6.8).

[0659] (4) 4% paraformaldehyde

[0660] (5) Epirubicin hydrochloride.

[0661] (6) Dichloromethane.

[0662] (7) Anhydrous ethanol.

[0663] 2. Experimental methods:

[0664] (1) Accurately weigh epirubicin hydrochloride (0.79 mg, 1.354 μmoL) and dissolve it in DEPC water (1.0 mL) and PBS buffer (1.25 mL). Add 4% paraformaldehyde aqueous solution (0.25 mL) and mix well under ice-water bath cooling. Mix the mixture with Apt AS1411-Apt EGFR-Apt A15-2*Bio-DNA targeting vector (1 mg) and react at 4°C for 72 hours in the dark. Control the reaction pH to 7.

[0665] (2) The reaction solution was washed with dichloromethane (10 mL × 3), and 25 mL of anhydrous ethanol was added to the aqueous phase. After mixing, the mixture was placed in the dark at 4°C to allow the product to precipitate completely (4 hours). Centrifugation was performed (4000 rpm), and the supernatant was transferred. The solid product was washed again with ethanol (50 mL). The supernatant was removed, and the residue was evaporated to dryness under reduced pressure at low temperature to obtain a dark red solid product, which was the epirubicin targeted drug Apt AS1411-Apt EGFR-Apt A15-2*Bio-DNA-epirubicin.

[0666] (4) Calculation of mount rate:

[0667] 1. Prepare 100 μL of epirubicin-PBS standard solutions of known concentrations: 2 μM, 4 μM, 6 μM, 8 μM, and 10 μM;

[0668] 2. Dissolve Apt AS1411-Apt EGFR-Apt A15-2*Bio-DNA-epirubicin in 100 μl PBS;

[0669] 3. Place the standard solution and Apt AS1411-Apt EGFR-Apt A15-2*Bio-DNA-epirubicin in a PCR plate, heat at 85°C for 5 minutes, and then cool to room temperature;

[0670] 4. Use a microplate reader to measure the absorbance of epirubicin at 492 nm, draw a standard curve, and calculate the molar concentration of epirubicin in the mounted product;

[0671] 5. Use a spectrophotometer to measure the absorbance of DNA at 260 nm to obtain the mass concentration of DNA vector in each sample;

[0672] 6. Calculate the loading efficiency based on the measured epirubicin molar concentration and the mass concentration of the DNA vector. The maximum theoretical loading efficiency of the epirubicin-targeted drug Apt AS1411-Apt EGFR-Apt A15-2*Bio-DNA-epirubicin is approximately 30%. Testing has shown an average loading efficiency of 25%.

[0673] Example 6

[0674] In this embodiment, the epirubicin targeted drug Apt AS1411-Apt EGFR-AptA15-DNA-epirubicin was synthesized.

[0675] Step 1: Synthesize the Apt AS1411-Apt EGFR-Apt A15-DNA targeting vector, the single-stranded sequence of which is shown in Table 6 below:

[0676] Table 6

[0677] 1. Experimental materials and reagents:

[0678] (1)A sequence;

[0679] (2) B sequence;

[0680] (3) C sequence;

[0681] (4) TMS aqueous solution;

[0682] (5) TBM (Tris-borate magnesium) buffer

[0683] 2. Experimental methods:

[0684] Sequences A, B, and C were dissolved in a TMS aqueous solution at a molar ratio of 1:1:1. After mixing, the reaction mixture was controlled to a pH of 8.8. The mixture was heated to 99°C for 5 minutes, then cooled to 65°C at a rate of 5°C / min for 3 minutes, and then cooled to 2°C at a rate of 5°C / min to obtain a crude product. Mass spectrometry analysis of the crude targeted nucleic acid vector after sequence assembly is shown in Figure 1 (analysis conditions are as follows: chromatographic column: Biosep SEC-S2000, 5 μm, 7.8 × 300 mm, two columns connected in series; mobile phase: water (50 mM Tris-HCl, 100 mM NaCl, pH 8); wavelength: 260 nm; flow rate: 0.6 ml / min; column temperature: 35°C; sample preparation: dissolved in 2 mg / ml mobile phase, incubated at 37°C with gentle shaking for 30 minutes, and then filtered through a 0.22 μm nylon filter; injection volume: 100 μL).

[0685] The product was loaded onto a SEC column (in series), the main fraction was cut, ultrafiltered to remove salt, and evaporated to dryness under reduced pressure at low temperature. The HPLC chromatography (SEC) detection results of the targeted nucleic acid vector after HPLC purification are shown in Figure 2.

[0686] Step 2: Mount Epirubicin

[0687] 1. Experimental materials and reagents:

[0688] (1) Apt AS1411-Apt EGFR-AptA15-DNA targeting vector prepared in step 1;

[0689] (2)DEPC water.

[0690] (3) PBS buffer (7.0).

[0691] (4) 4% paraformaldehyde

[0692] (5) Epirubicin hydrochloride.

[0693] (6)Chloroform.

[0694] (7) Anhydrous ethanol.

[0695] 2. Experimental methods:

[0696] (1) Accurately weigh epirubicin hydrochloride (0.79 mg, 1.354 μmoL) and dissolve it in DEPC water (1.0 mL) and PBS buffer (1.25 mL). Add 4% paraformaldehyde aqueous solution (0.25 mL) and mix well under ice-water bath cooling. Mix the mixture with Apt AS1411-Apt EGFR-AptA15-DNA targeting vector (1 mg) and react at 4°C for 72 hours in the dark. Control the reaction pH to 7.

[0697] (2) The reaction solution was washed with chloroform (10 mL x 3), 25 mL of anhydrous ethanol was added to the aqueous phase, mixed and placed in the dark at 4 ° C to allow the product to be fully precipitated (4 hours). Centrifuge (4000 rpm), transfer the supernatant, and wash the solid product with ethanol (50 mL) again. The supernatant was removed and the residue was evaporated to dryness under reduced pressure at low temperature to obtain a dark red solid product, which is the epirubicin targeted drug Apt AS1411-Apt EGFR-AptA15-DNA-epirubicin. The HPLC chromatography (reverse phase) detection results of the crude targeted chemical drug after epirubicin was mounted are shown in Figure 3, and the HPLC chromatography (reverse phase) detection results of the crude targeted chemical drug after purification by HPLC (reverse phase) chromatography column are shown in Figure 4 (test conditions are as follows: chromatographic column: Aeris widepore XB-C18, 3.6 μm, 4.6×250mm; Mobile phase: A: Water (1% HFIP, TEA adjusted to pH 8) B: Acetonitrile; Flow rate: 0.8mL / min; Wavelength: 260nm; Column temperature: 40°C)

[0698] (4) Calculation of mount rate:

[0699] 1. Prepare 100 μL of epirubicin-PBS standard solutions of known concentrations: 2 μM, 4 μM, 6 μM, 8 μM, and 10 μM;

[0700] 2. Dissolve Apt AS1411-Apt EGFR-AptA15-DNA-epirubicin in 100 μl PBS;

[0701] 3. Place the standard solution and Apt AS1411-Apt EGFR-AptA15-DNA-epirubicin in a PCR plate, heat at 85°C for 5 minutes, and then cool to room temperature;

[0702] 4. Use a microplate reader to measure the absorbance of epirubicin at 492 nm, draw a standard curve, and calculate the molar concentration of epirubicin in the mounted product;

[0703] 5. Use a spectrophotometer to measure the absorbance of DNA at 260 nm to obtain the mass concentration of DNA vector in each sample;

[0704] 6. Calculate the loading efficiency based on the measured epirubicin molar concentration and the mass concentration of the DNA vector. The maximum theoretical loading efficiency of epirubicin in the aforementioned epirubicin-targeted drug, Apt AS1411-Apt EGFR-AptA15-DNA-epirubicin, is approximately 30%. Testing revealed an average loading efficiency of 25%.

[0705] This example also verifies the trend of the final Apt AS1411-Apt EGFR-AptA15-DNA-epirubicin drug loading amount as the degree of excess of the epirubicin small molecule varies, as shown in Figures 5 to 7. Figure 5 shows an HPLC reverse-phase chromatography detection graph of the drug obtained at a molar ratio of epirubicin to carrier of 10:1; Figure 6 shows an HPLC reverse-phase chromatography detection graph of the drug obtained at a molar ratio of epirubicin to carrier of 20:1; and Figure 7 shows an HPLC reverse-phase chromatography detection graph of the drug obtained at a molar ratio of epirubicin to carrier of 30:1. As can be seen from the figures, 1) the distribution of epirubicin loading efficiency (DAR value) is a range; 2) as the amount of epirubicin added increases, the characteristic peak area in the HPLC reverse-phase chromatogram increases significantly, indicating that the loading efficiency DAR value of epirubicin is approaching the theoretical loading efficiency.

[0706] Example 7

[0707] In this embodiment, the epirubicin targeted drug Apt AS1411-Apt EGFR-Apt A15-2*Bio-AmiR-21-DNA-epirubicin was synthesized.

[0708] Step 1: Synthesize the Apt AS1411-Apt EGFR-Apt A15-2*Bio-AmiR-21-DNA targeting vector, the single-stranded sequence of which is shown in Table 7 below:

[0709] Table 7

[0710] 1. Experimental materials and reagents:

[0711] (1)A sequence;

[0712] (2) B sequence;

[0713] (3) C sequence;

[0714] (4)DEPC water;

[0715] (5) TBM (Tris-borate magnesium) buffer

[0716] 2. Experimental methods:

[0717] Sequences A, B, and C were dissolved in DEPC water at a molar ratio of 1:1:1. After mixing, the pH of the reaction solution was controlled to 7. The mixture was heated to 85°C, maintained for 5 minutes, then cooled to 60°C at a rate of 2°C / min and maintained for 5 minutes. The temperature was then cooled to 4°C at a rate of 2°C / min.

[0718] The product was loaded onto an 8% (m / v) non-denaturing PAGE gel and the vector was purified by electrophoresis at 100 V in TBM buffer at 4°C. The target band was excised and eluted in DNA elution buffer at 37°C, followed by ethanol precipitation overnight and evaporation under reduced pressure at low temperature to obtain the self-assembled product - Apt AS1411-Apt EGFR-Apt A15-2*Bio-AmiR-21-DNA targeting vector.

[0719] Step 2: Mount Epirubicin

[0720] 1. Experimental materials and reagents:

[0721] (1) Apt AS1411-Apt EGFR-Apt A15-2*Bio-AmiR-21-DNA targeting vector prepared in step 1;

[0722] (2)DEPC water.

[0723] (3) PBS buffer (6.8).

[0724] (4) 4% paraformaldehyde

[0725] (5) Epirubicin hydrochloride.

[0726] (6)Chloroform.

[0727] (7) Methanol.

[0728] 2. Experimental methods:

[0729] (1) Accurately weigh epirubicin hydrochloride (0.79 mg, 1.354 μmoL) and dissolve it in DEPC water (1.0 mL) and PBS buffer (1.25 mL). Add 4% paraformaldehyde aqueous solution (0.25 mL) and mix well under ice-water bath cooling. Mix the mixture with Apt AS1411-Apt EGFR-Apt A15-2*Bio-AmiR-21-DNA targeting vector (1 mg) and react at 4°C for 72 hours in the dark. Control the reaction pH at 6.

[0730] (2) The reaction solution was washed with chloroform (10 mL × 3), and 25 mL of methanol was added to the aqueous phase. After mixing, the mixture was placed in the dark at 4°C to allow the product to precipitate completely (4 hours). Centrifugation was performed (4000 rpm), and the supernatant was transferred. The solid product was washed again with methanol (50 mL). The supernatant was removed, and the residue was evaporated to dryness under reduced pressure at low temperature to obtain a dark red solid product, which is the epirubicin targeted drug Apt AS1411-Apt EGFR-Apt A15-2*Bio-AmiR-21-DNA-epirubicin.

[0731] (4) Calculation of mount rate:

[0732] 1. Prepare 100 μL of epirubicin-PBS standard solutions of known concentrations: 2 μM, 4 μM, 6 μM, 8 μM, and 10 μM;

[0733] 2. Dissolve Apt AS1411-Apt EGFR-Apt A15-2*Bio-AmiR-21-DNA-epirubicin in 100 μl PBS;

[0734] 3. Place the standard solution and Apt AS1411-Apt EGFR-Apt A15-2*Bio-AmiR-21-DNA-epirubicin in a PCR plate, heat at 85°C for 5 minutes, and then cool to room temperature;

[0735] 4. Use a microplate reader to measure the absorbance of epirubicin at 492 nm, draw a standard curve, and calculate the molar concentration of epirubicin in the mounted product;

[0736] 5. Use a spectrophotometer to measure the absorbance of DNA at 260 nm to obtain the mass concentration of DNA vector in each sample;

[0737] 6. Calculate the loading efficiency based on the measured epirubicin molar concentration and the mass concentration of the DNA vector. The average maximum theoretical loading efficiency of the epirubicin-targeted drug Apt AS1411-Apt EGFR-Apt A15-2*Bio-AmiR-21-DNA-epirubicin is approximately 27. Testing revealed an average loading efficiency of 23.

[0738] Example 8

[0739] In this example, the epirubicin-targeted drug Apt TfRA4-AptAS1411-Apt A15-DNA-epirubicin (1) was synthesized.

[0740] Step 1: Synthesize the Apt TfRA4-AptAS1411-Apt A15-DNA targeting vector, the single-stranded sequence of which is shown in Table 8 below:

[0741] Table 8

[0742] 1. Experimental materials and reagents:

[0743] (1)A sequence;

[0744] (2) B sequence;

[0745] (3) C sequence;

[0746] (4)DEPC water;

[0747] (5) TBM (Tris-borate magnesium) buffer

[0748] 2. Experimental methods:

[0749] Sequence A, sequence B, and sequence C were dissolved in DEPC water at a molar ratio of 1:1:1. After mixing, the pH of the reaction solution was controlled to 7. The mixture was heated to 99°C, maintained for 5 minutes, then cooled to 65°C at a rate of 2°C / min and maintained for 15 minutes. The temperature was then cooled to 4°C at a rate of 2°C / min.

[0750] The product was loaded onto an 8% (m / v) non-denaturing PAGE gel and the vector was purified by electrophoresis at 100V in TBM buffer at 4°C. The target band was cut out and eluted in DNA elution buffer at 37°C, followed by ethanol precipitation overnight and evaporation at low temperature under reduced pressure to obtain the self-assembled product - Apt TfRA4-AptAS1411-Apt A15-DNA targeting vector (1).

[0751] Step 2: Mount Epirubicin

[0752] 1. Experimental materials and reagents:

[0753] (1) Apt TfRA4-AptAS1411-Apt A15-DNA targeting vector (1) prepared in step 1;

[0754] (2)DEPC water.

[0755] (3) PBS buffer (6.6).

[0756] (4) 4% paraformaldehyde

[0757] (5) Epirubicin hydrochloride.

[0758] (6)Chloroform.

[0759] (7) Isopropyl alcohol.

[0760] 2. Experimental methods:

[0761] (1) Accurately weigh epirubicin hydrochloride (0.79 mg, 1.354 μmoL) and dissolve it in DEPC water (1.0 mL) and PBS buffer (1.25 mL). Add 4% paraformaldehyde aqueous solution (0.25 mL) and mix well under ice-water bath cooling. Mix the mixture with AS1411-TfRA4-A15-DNA targeting vector (1 mg) and react at 4°C for 72 hours in the dark. Control the reaction pH to 7.

[0762] (2) The reaction solution was washed with chloroform (10 mL × 3), and 25 mL of isopropanol was added to the aqueous phase. After mixing, the mixture was placed in the dark at 4°C to allow the product to precipitate completely (4 hours). Centrifugation was performed (4000 rpm), and the supernatant was transferred. The solid product was washed again with isopropanol (50 mL), and the supernatant was removed. The residue was evaporated to dryness under reduced pressure at low temperature to obtain a dark red solid product, which was the epirubicin targeted drug AS1411-TfRA4-A15-DNA-epirubicin (1).

[0763] (4) Calculation of mount rate:

[0764] 1. Prepare 100 μL of epirubicin-PBS standard solutions of known concentrations: 2 μM, 4 μM, 6 μM, 8 μM, and 10 μM;

[0765] 2. Dissolve Apt TfRA4-AptAS1411-Apt A15-DNA-epirubicin in 100 μl PBS;

[0766] 3. Place the standard solution and Apt TfRA4-AptAS1411-Apt A15-DNA epirubicin in a PCR plate, heat at 85°C for 5 minutes, and then cool to room temperature;

[0767] 4. Use a microplate reader to measure the absorbance of epirubicin at 492 nm, draw a standard curve, and calculate the molar concentration of epirubicin in the mounted product;

[0768] 5. Use a spectrophotometer to measure the absorbance of DNA at 260 nm to obtain the mass concentration of DNA vector in each sample;

[0769] 6. Calculate the loading efficiency based on the measured epirubicin molar concentration and the mass concentration of the DNA vector. The maximum theoretical loading efficiency of epirubicin of the epirubicin targeted drug Apt TfRA4-AptAS1411-Apt A15-DNA-epirubicin (1) is about 36. After testing, the average loading efficiency is 30.

[0770] Example 9

[0771] In this example, the epirubicin targeted drug Apt TfRA3-Apt AS1411-Apt A15-DNA-epirubicin was synthesized.

[0772] Step 1: Synthesize Apt TfRA3-Apt AS1411-Apt A15-DNA-targeting vector, the single-stranded sequence of which is shown in Table 9 below:

[0773] Table 9

[0774] 1. Experimental materials and reagents:

[0775] (1)A sequence;

[0776] (2) B sequence;

[0777] (3) C sequence;

[0778] (4)DEPC water;

[0779] (5) TBM (Tris-borate magnesium) buffer

[0780] 2. Experimental methods:

[0781] Sequence A, sequence B, and sequence C were dissolved in DEPC water at a molar ratio of 1:1:1. After mixing, the pH of the reaction solution was controlled to 7. The mixture was heated to 99°C, maintained for 5 minutes, then cooled to 65°C at a rate of 2°C / min and maintained for 15 minutes. The temperature was then cooled to 4°C at a rate of 2°C / min.

[0782] The product was loaded onto an 8% (m / v) non-denaturing PAGE gel and the vector was purified by electrophoresis at 100 V in TBM buffer at 4°C. The target band was excised and eluted in DNA elution buffer at 37°C, followed by ethanol precipitation overnight and evaporation under reduced pressure at low temperature to obtain the self-assembled product - Apt TfRA3-Apt AS1411-Apt A15-DNA-targeting vector.

[0783] Step 2: Mount Epirubicin

[0784] 1. Experimental materials and reagents:

[0785] (1) Apt TfRA3-Apt AS1411-Apt A15-DNA-targeting vector prepared in step 1;

[0786] (2)DEPC water.

[0787] (3) PBS buffer (6.6).

[0788] (4) 4% paraformaldehyde

[0789] (5) Epirubicin hydrochloride.

[0790] (6)Chloroform.

[0791] (7) Isopropyl alcohol.

[0792] 2. Experimental methods:

[0793] (1) Accurately weigh epirubicin hydrochloride (0.79 mg, 1.354 μmoL) and dissolve it in DEPC water (1.0 mL) and PBS buffer (1.25 mL). Add 4% paraformaldehyde aqueous solution (0.25 mL) and mix well under ice-water bath cooling. Mix the mixture with Apt AS1411-Apt TfRA3-Apt A15-DNA-targeting vector (1 mg) and react at 4°C for 72 h in the dark. Control the reaction pH to 7.

[0794] (2) The reaction solution was washed with chloroform (10 mL × 3), and 25 mL of isopropanol was added to the aqueous phase. After mixing, the mixture was incubated at 4°C in the dark to allow the product to precipitate completely (4 hours). The mixture was centrifuged (4000 rpm), and the supernatant was removed. The solid product was washed again with isopropanol (50 mL). The supernatant was removed, and the residue was evaporated to dryness under reduced pressure at low temperature to obtain a dark red solid product, which was the epirubicin targeted drug Apt AS1411-Apt TfRA3-Apt A15-DNA--epirubicin.

[0795] (4) Calculation of mount rate:

[0796] 1. Prepare 100 μL of epirubicin-PBS standard solutions of known concentrations: 2 μM, 4 μM, 6 μM, 8 μM, and 10 μM;

[0797] 2. Dissolve Apt TfRA3-Apt AS1411-Apt A15-DNA-epirubicin in 100 μl PBS;

[0798] 3. Place the standard solution and Apt TfRA3-Apt AS1411-Apt A15-DNA-epirubicin in a PCR plate, heat at 85°C for 5 minutes, and then cool to room temperature;

[0799] 4. Use a microplate reader to measure the absorbance of epirubicin at 492 nm, draw a standard curve, and calculate the molar concentration of epirubicin in the mounted product;

[0800] 5. Use a spectrophotometer to measure the absorbance of DNA at 260 nm to obtain the mass concentration of DNA vector in each sample;

[0801] 6. Calculate the loading efficiency based on the measured epirubicin molar concentration and the mass concentration of the DNA vector. The maximum theoretical loading efficiency of epirubicin for the aforementioned epirubicin-targeted drug, Apt TfRA3-Apt AS1411-Apt A15-DNA-epirubicin, is approximately 27%. Testing has shown an average loading efficiency of 25%.

[0802] Example 10

[0803] In this example, the epirubicin targeted drug CPG2006 (DNA)-Apt CD40-Apt PD-L1-DNA-epirubicin was synthesized.

[0804] Step 1: Synthesize the CPG2006 (DNA)-Apt CD40-Apt PD-L1-DNA targeting vector, the single-stranded sequence of which is shown in Table 10 below:

[0805] Table 10

[0806] Note: The adjacent bases at positions 1-24 at the 5' end of sequence A are all thiolated, and the adjacent bases at positions 1-3 at the 3' end are all thiolated; the adjacent bases at positions 1-3 at the 5' end and 3' end of sequences B and C are all thiolated.

[0807] 1. Experimental materials and reagents:

[0808] (1)A sequence;

[0809] (2) B sequence;

[0810] (3) C sequence;

[0811] (4)DEPC water;

[0812] (5) TBM (Tris-borate magnesium) buffer

[0813] 2. Experimental methods:

[0814] Sequence A, sequence B, and sequence C were dissolved in DEPC water at a molar ratio of 1:1:1. After mixing, the pH of the reaction solution was controlled to 7. The mixture was heated to 99°C, maintained for 5 minutes, then cooled to 65°C at a rate of 2°C / min and maintained for 15 minutes. The temperature was then cooled to 4°C at a rate of 2°C / min.

[0815] The product was loaded onto an 8% (m / v) non-denaturing PAGE gel and the vector was purified by electrophoresis at 100 V at 4°C in TBM buffer. The target band was excised and eluted in DNA elution buffer at 37°C, followed by ethanol precipitation overnight and evaporation at low temperature under reduced pressure to obtain the self-assembled product - CPG2006 (DNA) - Apt CD40 - Apt PD-L1 - DNA targeting vector.

[0816] Step 2: Mount Epirubicin

[0817] 1. Experimental materials and reagents:

[0818] (1) CPG2006(DNA)-Apt CD40-Apt PD-L1-DNA targeting vector prepared in step 1;

[0819] (2)DEPC water.

[0820] (3) PBS buffer (6.6).

[0821] (4) 4% paraformaldehyde

[0822] (5) Epirubicin hydrochloride.

[0823] (6)Chloroform.

[0824] (7) Isopropyl alcohol.

[0825] 2. Experimental methods:

[0826] (1) Accurately weigh epirubicin hydrochloride (0.79 mg, 1.354 μmoL) and dissolve it in DEPC water (1.0 mL) and PBS buffer (1.25 mL). Add 4% paraformaldehyde aqueous solution (0.25 mL) and mix well under ice-water bath cooling. Mix the mixture with CPG2006 (DNA)-Apt CD40-Apt PD-L1-DNA targeting vector (1 mg) and react at 4°C for 72 hours in the dark. Control the reaction pH at 7.

[0827] (2) The reaction solution was washed with chloroform (10 mL x 3), and 25 mL of isopropanol was added to the aqueous phase. After mixing, the mixture was placed in the dark at 4°C to allow the product to precipitate completely (4 hours). Centrifugation was performed (4000 rpm), and the supernatant was transferred. The solid product was washed again with isopropanol (50 mL). The supernatant was removed, and the residue was evaporated to dryness under reduced pressure at low temperature to obtain a dark red solid product, which was the epirubicin targeted drug CPG2006(DNA)-Apt CD40-Apt PD-L1-DNA-epirubicin.

[0828] (4) Calculation of mount rate:

[0829] 1. Prepare 100 μL of epirubicin-PBS standard solutions of known concentrations: 2 μM, 4 μM, 6 μM, 8 μM, and 10 μM;

[0830] 2. Dissolve CPG2006(DNA)-Apt CD40-Apt PD-L1-DNA-epirubicin in 100ul PBS;

[0831] 3. Place the standard solution and CPG2006(DNA)-Apt CD40-Apt PD-L1-DNA-epirubicin in a PCR plate, heat at 85°C for 5 minutes, and then cool to room temperature;

[0832] 4. Use a microplate reader to measure the absorbance of epirubicin at 492 nm, draw a standard curve, and calculate the molar concentration of epirubicin in the mounted product;

[0833] 5. Use a spectrophotometer to measure the absorbance of DNA at 260 nm to obtain the mass concentration of DNA vector in each sample;

[0834] 6. Calculate the loading efficiency based on the measured epirubicin molar concentration and the mass concentration of the DNA vector. The theoretical maximum loading efficiency of the epirubicin-targeted drug CPG2006(DNA)-Apt CD40-Apt PD-L1-DNA-epirubicin is approximately 25%. Testing revealed an average loading efficiency of 21.

[0835] Example 11

[0836] In this example, the epirubicin targeted drug 2*CPG2006(DNA)-Apt CD40-Apt PD-L1-Apt C12-DNA-epirubicin was synthesized.

[0837] Step 1: Synthesize 2*CPG2006(DNA)-Apt CD40-Apt PD-L1-Apt C12-DNA targeting vector, the single-stranded sequence of which is shown in Table 11 below:

[0838] Table 11

[0839] Note: The adjacent bases at positions 1-24 on the 5' end of sequences A and C are all thiolated; the adjacent bases at positions 1-3 on the 5' end of sequences B, D, and E are all thiolated.

[0840] 1. Experimental materials and reagents:

[0841] (1)A sequence;

[0842] (2) B sequence;

[0843] (3) C sequence;

[0844] (4) D sequence;

[0845] (5)E sequence;

[0846] (6)DEPC water;

[0847] (7) TBM (Tris-borate magnesium) buffer

[0848] 2. Experimental methods:

[0849] Sequences A, B, C, D, and E were dissolved in DEPC water at a molar ratio of 1:1:1:1:1; after mixing, the pH of the reaction solution was controlled to 7. The mixture was heated to 99°C, maintained for 5 minutes, then cooled to 65°C at a rate of 2°C / min and maintained for 15 minutes, and then cooled to 4°C at a rate of 2°C / min.

[0850] The product was loaded onto an 8% (m / v) non-denaturing PAGE gel and the vector was purified by electrophoresis at 100 V in TBM buffer at 4°C. The target band was excised and eluted in DNA elution buffer at 37°C, followed by ethanol precipitation overnight and evaporation at low temperature under reduced pressure to obtain the self-assembled product - 2*CPG2006(DNA)-Apt CD40L-Apt PD-L1-Apt C12-DNA targeting vector.

[0851] Step 2: Mount Epirubicin

[0852] 1. Experimental materials and reagents:

[0853] (1) 2*CPG2006(DNA)-Apt CD40-Apt PD-L1-Apt C12-DNA targeting vector prepared in step 1;

[0854] (2)DEPC water.

[0855] (3) PBS buffer (6.6).

[0856] (4) 4% paraformaldehyde

[0857] (5) Epirubicin hydrochloride.

[0858] (6)Chloroform.

[0859] (7) Isopropyl alcohol.

[0860] 2. Experimental methods:

[0861] (1) Accurately weigh epirubicin hydrochloride (0.79 mg, 1.354 μmoL) and dissolve it in DEPC water (1.0 mL) and PBS buffer (1.25 mL). Add 4% paraformaldehyde aqueous solution (0.25 mL) and mix well under ice-water bath cooling. Mix the mixture with 2*CPG2006(DNA)-Apt CD40-Apt PD-L1-Apt C12-DNA targeting vector (1 mg) and react at 4°C for 72 hours in the dark. Control the reaction pH at 7.

[0862] (2) The reaction solution was washed with chloroform (10 mL x 3), and 25 mL of isopropanol was added to the aqueous phase. After mixing, the mixture was placed in the dark at 4°C to allow the product to precipitate completely (4 hours). Centrifugation was performed (4000 rpm), and the supernatant was transferred. The solid product was washed again with isopropanol (50 mL), and the supernatant was removed. The residue was evaporated to dryness under reduced pressure at low temperature to obtain a dark red solid product, which was the epirubicin targeted drug 2*CPG2006(DNA)-Apt CD40-Apt PD-L1-Apt C12-DNA-epirubicin.

[0863] (4) Calculation of mount rate:

[0864] 1. Prepare 100 μL of epirubicin-PBS standard solutions of known concentrations: 2 μM, 4 μM, 6 μM, 8 μM, and 10 μM;

[0865] 2. Dissolve 2*CPG2006(DNA)-Apt CD40-Apt PD-L1-Apt C12-DNA-epirubicin in 100ul PBS;

[0866] 3. Place the standard solution and 2*CPG2006(DNA)-Apt CD40-Apt PD-L1-Apt C12-DNA-epirubicin in a PCR plate, heat at 85°C for 5 minutes, and then cool to room temperature;

[0867] 4. Use a microplate reader to measure the absorbance of epirubicin at 492 nm, draw a standard curve, and calculate the molar concentration of epirubicin in the mounted product;

[0868] 5. Use a spectrophotometer to measure the absorbance of DNA at 260 nm to obtain the mass concentration of DNA vector in each sample;

[0869] 6. Calculate the loading efficiency based on the measured epirubicin molar concentration and the mass concentration of the DNA vector. The theoretical maximum loading efficiency of the epirubicin-targeted drug 2*CPG2006(DNA)-Apt CD40-Apt PD-L1-Apt C12-DNA-epirubicin is approximately 36. Testing revealed an average loading efficiency of 30.

[0870] Example 12

[0871] In this example, the epirubicin targeted drug CPG2006 (DNA)-Apt C12-CD47 siRNA-Apt PD-L1-PD-L1 siRNA-DNA-epirubicin was synthesized.

[0872] Step 1: Synthesize the CPG2006 (DNA)-Apt C12-CD47 siRNA-Apt PD-L1-PD-L1 siRNA-DNA targeting vector, the single-stranded sequence of which is shown in Table 12 below:

[0873] Table 12

[0874] Note: The adjacent bases at positions 1-24 of the 5' end of sequence A are all thiolated, and the adjacent bases at positions 1-3 of the 3' end are all thiolated; the adjacent bases at positions 1-3 of the 5' end and 3' end of sequence B are all thiolated, and the adjacent bases at positions 19-21 of the 5' end of sequence B are all thiolated; the adjacent bases at positions 1-3 of the 5' end and 3' end of sequence C are all thiolated, and the adjacent bases at positions 19-21 of the 5' end of sequence C are all thiolated. The 5'-end of sequence D contains thiosulfate modifications between adjacent bases 19-21; the 5'-end and 3'-end of sequence D contain thiosulfate modifications between adjacent bases 19-21; the 5'-end and 3'-end of sequence E contain thiosulfate modifications between adjacent bases 1-3, and the 3'-end of sequence E contain thiosulfate modifications between adjacent bases 19-21. The C / U2'F modifications are underlined in the RNA sequence.

[0875] 1. Experimental materials and reagents:

[0876] (1)A sequence;

[0877] (2) B sequence;

[0878] (3) C sequence;

[0879] (4) D sequence;

[0880] (5)E sequence;

[0881] (6)DEPC water;

[0882] (7) TBM (Tris-borate magnesium) buffer

[0883] 2. Experimental methods:

[0884] Sequences A, B, C, D, and E were dissolved in DEPC water at a molar ratio of 1:1:1:1:1; after mixing, the pH of the reaction solution was controlled to 7. The mixture was heated to 99°C, maintained for 5 minutes, then cooled to 65°C at a rate of 2°C / min and maintained for 15 minutes, and then cooled to 4°C at a rate of 2°C / min.

[0885] The product was loaded onto an 8% (m / v) non-denaturing PAGE gel, and the vector was purified by electrophoresis at 100 V at 4°C in TBM buffer. The target band was excised and eluted in DNA elution buffer at 37°C, followed by ethanol precipitation overnight and evaporation at low temperature under reduced pressure to obtain the self-assembled product - CPG2006 (DNA)-Apt C12-CD47 siRNA-Apt PD-L1-PD-L1 siRNA-DNA targeting vector.

[0886] Step 2: Mount Epirubicin

[0887] 1. Experimental materials and reagents:

[0888] (1) CPG2006 (DNA)-Apt C12-CD47 siRNA-Apt PD-L1-PD-L1 siRNA-DNA targeting vector prepared in step 1;

[0889] (2)DEPC water.

[0890] (3) PBS buffer (6.6).

[0891] (4) 4% paraformaldehyde

[0892] (5) Epirubicin hydrochloride.

[0893] (6)Chloroform.

[0894] (7) Isopropyl alcohol.

[0895] 2. Experimental methods:

[0896] (1) Accurately weigh epirubicin hydrochloride (0.79 mg, 1.354 μmoL) and dissolve it in DEPC water (1.0 mL) and PBS buffer (1.25 mL). Add 4% paraformaldehyde aqueous solution (0.25 mL) and mix well under ice-water bath cooling. Mix the mixture with CPG2006 (DNA)-Apt C12-CD47 siRNA-Apt PD-L1-PD-L1 siRNA-DNA targeting vector (1 mg) and react at 4°C for 72 hours in the dark. Control the reaction pH at 7.

[0897] (2) The reaction solution was washed with chloroform (10 mL x 3), and 25 mL of isopropanol was added to the aqueous phase. After mixing, the mixture was placed in the dark at 4°C to allow the product to precipitate completely (4 hours). Centrifugation (4000 rpm) was performed, and the supernatant was transferred. The solid product was washed again with isopropanol (50 mL). The supernatant was removed, and the residue was evaporated to dryness under reduced pressure at low temperature to obtain a dark red solid product, which was the epirubicin targeted drug CPG2006 (DNA)-Apt C12-CD47 siRNA-Apt PD-L1-PD-L1 siRNA-DNA-epirubicin.

[0898] (4) Calculation of mount rate:

[0899] 1. Prepare 100 μL of epirubicin-PBS standard solutions of known concentrations: 2 μM, 4 μM, 6 μM, 8 μM, and 10 μM;

[0900] 2. Dissolve CPG2006 (DNA)-Apt C12-CD47 siRNA-Apt PD-L1-PD-L1 siRNA-DNA-epirubicin in 100 μl PBS;

[0901] 3. Place the standard solution and CPG2006 (DNA)-Apt C12-CD47 siRNA-Apt PD-L1-PD-L1 siRNA-DNA-epirubicin in a PCR plate, heat at 85°C for 5 minutes, and then cool to room temperature;

[0902] 4. Use a microplate reader to measure the absorbance of epirubicin at 492 nm, draw a standard curve, and calculate the molar concentration of epirubicin in the mounted product;

[0903] 5. Use a spectrophotometer to measure the absorbance of DNA at 260 nm to obtain the mass concentration of DNA vector in each sample;

[0904] 6. Calculate the loading efficiency based on the measured epirubicin molar concentration and the mass concentration of the DNA vector. The theoretical maximum loading efficiency of epirubicin for the epirubicin-targeted drug CPG2006(DNA)-Apt C12-CD47 siRNA-Apt PD-L1-PD-L1 siRNA-DNA-epirubicin is approximately 30%. Testing revealed an average loading efficiency of 26.

[0905] Example 13

[0906] In this example, the epirubicin targeted drug A-miR-21-TGF-β1siRNA-Apt TfRA4-Apt PD-L1-DNA-epirubicin was synthesized.

[0907] Step 1: Synthesize the A-miR-21-TGF-β1siRNA-Apt TfRA4-Apt PD-L1-DNA targeting vector, the single-stranded sequence of which is shown in Table 13 below:

[0908] Table 13

[0909] Note: The 5' end of sequence B contains thiolation between bases 1-3; the 5' end of sequence C contains thiolation between bases 29-31 and between bases 1-3; the 3' end of sequence D contains thiolation between bases 1-3 and between bases 19-21. C / U2'F modifications are underlined in the RNA sequence.

[0910] 1. Experimental materials and reagents:

[0911] (1)A sequence;

[0912] (2) B sequence;

[0913] (3) C sequence;

[0914] (4) D sequence;

[0915] (5)DEPC water;

[0916] (6) TBM (Tris-borate magnesium) buffer

[0917] 2. Experimental methods:

[0918] Sequences A, B, C, and D were dissolved in DEPC water at a molar ratio of 1:1:1:1. After mixing, the pH of the reaction solution was controlled to 7. The mixture was heated to 99°C for 5 minutes, then cooled to 65°C at a rate of 2°C / min and incubated for 15 minutes. The temperature was then cooled to 4°C at a rate of 2°C / min.

[0919] The product was loaded onto an 8% (m / v) non-denaturing PAGE gel and the vector was purified by electrophoresis at 100 V at 4°C in TBM buffer; the target band was excised and eluted in DNA elution buffer at 37°C, followed by ethanol precipitation overnight and evaporation at low temperature under reduced pressure to obtain the self-assembled product - A-miR-21-TGF-β1siRNA-Apt TfRA4-Apt PD-L1-DNA targeting vector.

[0920] Step 2: Mount Epirubicin

[0921] 1. Experimental materials and reagents:

[0922] (1) A-miR-21-TGF-β1siRNA-Apt TfRA4-Apt PD-L1-DNA prepared in step 1;

[0923] (2)DEPC water.

[0924] (3) PBS buffer (6.6).

[0925] (4) 4% paraformaldehyde

[0926] (5) Epirubicin hydrochloride.

[0927] (6)Chloroform.

[0928] (7) Isopropyl alcohol.

[0929] 2. Experimental methods:

[0930] (1) Accurately weigh epirubicin hydrochloride (0.79 mg, 1.354 μmoL) and dissolve it in DEPC water (1.0 mL) and PBS buffer (1.25 mL). Add 4% paraformaldehyde aqueous solution (0.25 mL) and mix well under ice-water bath cooling. Mix the mixture with A-miR-21-TGF-β1siRNA-Apt TfRA4-Apt PD-L1-DNA targeting vector (1 mg) and react at 4°C for 72 hours in the dark. Control the reaction pH at 7.

[0931] (2) The reaction solution was washed with chloroform (10 mL x 3), and 25 mL of isopropanol was added to the aqueous phase. After mixing, the mixture was placed in the dark at 4°C to allow the product to precipitate completely (4 hours). Centrifugation was performed (4000 rpm), and the supernatant was transferred. The solid product was washed again with isopropanol (50 mL). The supernatant was removed, and the residue was evaporated to dryness under reduced pressure at low temperature to obtain a dark red solid product, which was the epirubicin targeted drug A-miR-21-TGF-β1siRNA-Apt TfRA4-Apt PD-L1-DNA-epirubicin.

[0932] (4) Calculation of mount rate:

[0933] 1. Prepare 100 μL of epirubicin-PBS standard solutions of known concentrations: 2 μM, 4 μM, 6 μM, 8 μM, and 10 μM;

[0934] 2. Dissolve A-miR-21-TGF-β1siRNA-Apt TfRA4-Apt PD-L1-DNA-epirubicin in 100ul PBS;

[0935] 3. Place the standard solution and A-miR-21-TGF-β1siRNA-Apt TfRA4-Apt PD-L1-DNA-epirubicin in a PCR plate, heat at 85°C for 5 minutes, and then cool to room temperature;

[0936] 4. Use a microplate reader to measure the absorbance of epirubicin at 492 nm, draw a standard curve, and calculate the molar concentration of epirubicin in the mounted product;

[0937] 5. Use a spectrophotometer to measure the absorbance of DNA at 260 nm to obtain the mass concentration of DNA vector in each sample;

[0938] 6. Calculate the loading efficiency based on the measured epirubicin molar concentration and the mass concentration of the DNA vector. The theoretical maximum loading efficiency of the epirubicin-targeted drug A-miR-21-TGF-β siRNA-Apt TfRA4-Apt PD-L1-DNA-epirubicin is approximately 33%. Testing revealed an average loading efficiency of 28.

[0939] Example 14

[0940] In this example, the epirubicin targeting drug Apt IL-4Ra-CD47 siRNA-Apt TfRA4-PD-L1 siRNA-Apt GPC-1-DNA-epirubicin was synthesized.

[0941] Step 1: Synthesize the Apt IL-4Ra-CD47 siRNA-Apt TfRA4-PD-L1 siRNA-Apt GPC-1-DNA targeting vector, the single-stranded sequence of which is shown in Table 14 below:

[0942] Table 14

[0943] Note: The adjacent bases at positions 1-3 of the 3' end of sequence A are all thiolated, and the adjacent bases at positions 19-21 of the 3' end of sequence A are all thiolated; the adjacent bases at positions 1-3 of the 5' end of sequence B are all thiolated, and the adjacent bases at positions 19-21 of the 5' end of sequence B are all thiolated; the adjacent bases at positions 1-3 of the 3' end of sequence C are all thiolated, and the adjacent bases at positions 19-21 are all thiolated; the adjacent bases at positions 1-4 of the 5' end of sequence D are all thiolated, and the adjacent bases at positions 1-3 of the 3' end of sequence D are all thiolated, and the adjacent bases at positions 19-21 of the 3' end of sequence D are all thiolated. In sequences A, B, and C, the underlined RNA sequences are C / U2'F modified; in sequence D, the first 44 bases are C / U2'F and A / G2'-OMe modified, and bases 45-64 are C / U2'F modified.

[0944] 1. Experimental materials and reagents:

[0945] (1)A sequence;

[0946] (2) B sequence;

[0947] (3) C sequence;

[0948] (4) D sequence;

[0949] (5)DEPC water;

[0950] (6) TBM (Tris-borate magnesium) buffer

[0951] 2. Experimental methods:

[0952] Sequences A, B, C, and D were dissolved in DEPC water at a molar ratio of 1:1:1:1. After mixing, the pH of the reaction solution was controlled to 7. The mixture was heated to 99°C for 5 minutes, then cooled to 65°C at a rate of 2°C / min and incubated for 15 minutes. The temperature was then cooled to 4°C at a rate of 2°C / min.

[0953] The product was loaded onto an 8% (m / v) non-denaturing PAGE gel, and the vector was purified by electrophoresis at 100 V at 4°C in TBM buffer; the target band was excised and eluted in DNA elution buffer at 37°C, followed by ethanol precipitation overnight and evaporation at low temperature under reduced pressure to obtain the self-assembled product - Apt IL-4Ra-CD47 siRNA-Apt TfRA4-PD-L1 siRNA-Apt GPC-1-DNA targeting vector.

[0954] Step 2: Mount Epirubicin

[0955] 1. Experimental materials and reagents:

[0956] (1) Apt IL-4Ra-CD47 siRNA-Apt TfRA4-PD-L1 siRNA-Apt GPC-1-DNA targeting vector prepared in step 1;

[0957] (2)DEPC water.

[0958] (3) PBS buffer (6.6).

[0959] (4) 4% paraformaldehyde

[0960] (5) Epirubicin hydrochloride.

[0961] (6)Chloroform.

[0962] (7) Isopropyl alcohol.

[0963] 2. Experimental methods:

[0964] (1) Accurately weigh epirubicin hydrochloride (0.79 mg, 1.354 μmoL) and dissolve it in DEPC water (1.0 mL) and PBS buffer (1.25 mL). Add 4% paraformaldehyde aqueous solution (0.25 mL) and mix well under ice-water bath cooling. Mix the mixture with Apt IL-4Ra-CD47 siRNA-Apt TfRA4-PD-L1 siRNA-Apt GPC-1-DNA targeting vector (1 mg) and react at 4°C for 72 hours in the dark. Control the reaction pH at 7.

[0965] (2) The reaction solution was washed with chloroform (10 mL x 3), 25 mL of isopropanol was added to the aqueous phase, mixed, and placed in the dark at 4°C to allow the product to precipitate completely (4 hours). Centrifuged (4000 rpm), the supernatant was transferred, and the solid product was washed again with isopropanol (50 mL). The supernatant was removed, and the residue was evaporated to dryness under reduced pressure at low temperature to obtain a dark red solid product, which is the epirubicin targeted drug Apt IL-4Ra-CD47 siRNA-Apt TfRA4-PD-L1 siRNA-Apt GPC-1-DNA-epirubicin.

[0966] (4) Calculation of mount rate:

[0967] 1. Prepare 100 μL of epirubicin-PBS standard solutions of known concentrations: 2 μM, 4 μM, 6 μM, 8 μM, and 10 μM;

[0968] 2. Dissolve Apt IL-4Ra-CD47 siRNA-Apt TfRA4-PD-L1 siRNA-Apt GPC-1-DNA-epirubicin in 100 μl PBS;

[0969] 3. Place the standard solution and Apt IL-4Ra-CD47 siRNA-Apt TfRA4-PD-L1 siRNA-Apt GPC-1-DNA-epirubicin in a PCR plate, heat at 85°C for 5 minutes, and then cool to room temperature;

[0970] 4. Use a microplate reader to measure the absorbance of epirubicin at 492 nm, draw a standard curve, and calculate the molar concentration of epirubicin in the mounted product;

[0971] 5. Use a spectrophotometer to measure the absorbance of DNA at 260 nm to obtain the mass concentration of DNA vector in each sample;

[0972] 6. Calculate the loading efficiency based on the measured epirubicin molar concentration and the mass concentration of the DNA vector. The theoretical maximum loading efficiency of the epirubicin-targeted drug (Apt IL-4Ra-CD47 siRNA-Apt TfRA4-PD-L1 siRNA-Apt GPC-1-DNA-epirubicin) is approximately 32. Testing revealed an average loading efficiency of 27.

[0973] Example 15

[0974] In this example, the gemcitabine targeted drug Apt AS1411-Apt EGFR-Apt A15-DNA-gemcitabine was synthesized.

[0975] Apt AS1411-Apt EGFR-Apt A15-DNA-gemcitabine targeted drug was synthesized, and the single-stranded sequence is shown in Table 15 below:

[0976] Table 15

[0977] Note: The J above represents gemcitabine, which is directly synthesized in the sequence in the form of base substitution and base extension during sequence synthesis, which is equivalent to 36 gemcitabine molecules connected to a single DNA targeting vector.

[0978] 1. Experimental materials and reagents:

[0979] (1)A sequence;

[0980] (2) B sequence;

[0981] (3) C sequence;

[0982] (4)DEPC water;

[0983] (5) TBM (Tris-borate magnesium) buffer

[0984] 2. Experimental methods:

[0985] Sequence A, sequence B, and sequence C were dissolved in DEPC water at a molar ratio of 1:1:1. After mixing, the pH of the reaction solution was controlled to 7. The mixture was heated to 99°C, maintained for 5 minutes, then cooled to 65°C at a rate of 2°C / min and maintained for 15 minutes. The temperature was then cooled to 4°C at a rate of 2°C / min.

[0986] The product was loaded onto an 8% (m / v) non-denaturing PAGE gel and the vector was purified by electrophoresis at 100V in TBM buffer at 4°C. The target band was excised and eluted in DNA elution buffer at 37°C, followed by ethanol precipitation overnight and evaporation at low temperature under reduced pressure to obtain the self-assembled product - Apt AS1411-Apt EGFR-Apt A15-DNA-gemcitabine targeted drug.

[0987] Example 16

[0988] In this example, epirubicin and gemcitabine targeted drugs Apt MUC1-Apt AS1411-Apt GPC1-gemcitabine-DNA epirubicin were synthesized.

[0989] Step 1: Synthesize Apt MUC1-Apt AS1411-Apt GPC1-gemcitabine-DNA targeting vector, the single-stranded sequence of which is shown in Table 16 below:

[0990] Table 16

[0991] Note: The J above represents gemcitabine, which was directly synthesized into the sequence during sequence synthesis through base substitution and base extension. Each adjacent base of gemcitabine is thiolated, equivalent to 36 gemcitabine residues linked to a single DNA targeting vector. Sequences A, B, and C above are all thiolated between bases 1 and 2 at the 5' end.

[0992] 1. Experimental materials and reagents:

[0993] (1)A sequence;

[0994] (2) B sequence;

[0995] (3) C sequence;

[0996] (4)DEPC water;

[0997] (5) TBM (Tris-borate magnesium) buffer

[0998] 2. Experimental methods:

[0999] Sequence A, sequence B, and sequence C were dissolved in DEPC water at a molar ratio of 1:1:1. After mixing, the pH of the reaction solution was controlled to 7. The mixture was heated to 99°C, maintained for 5 minutes, then cooled to 65°C at a rate of 2°C / min and maintained for 15 minutes. The temperature was then cooled to 4°C at a rate of 2°C / min.

[1000] The product was loaded onto an 8% (m / v) non-denaturing PAGE gel and the vector was purified by electrophoresis at 100 V at 4°C in TBM buffer. The target band was excised and eluted in DNA elution buffer at 37°C, followed by ethanol precipitation overnight and evaporation at low temperature under reduced pressure to obtain the self-assembled product - Apt MUC1-Apt AS1411-Apt GPC1-gemcitabine - DNA targeting vector.

[1001] Step 2: Mount Epirubicin

[1002] 1. Experimental materials and reagents:

[1003] (1) Apt MUC1-Apt AS1411-Apt GPC1-gemcitabine-DNA targeting vector prepared in step 1;

[1004] (2)DEPC water.

[1005] (3) PBS buffer (6.6).

[1006] (4) 4% paraformaldehyde

[1007] (5) Epirubicin hydrochloride.

[1008] (6)Chloroform.

[1009] (7) Isopropyl alcohol.

[1010] 2. Experimental methods:

[1011] (1) Accurately weigh epirubicin hydrochloride (0.79 mg, 1.354 μmoL) and dissolve it in DEPC water (1.0 mL) and PBS buffer (1.25 mL). Add 4% paraformaldehyde aqueous solution (0.25 mL) and mix well under ice-water bath cooling. Mix the mixture with Apt MUC1-Apt AS1411-Apt GPC1-gemcitabine-DNA targeting vector (1 mg) and react at 4°C for 72 hours in the dark. Control the reaction pH at 7.

[1012] (2) The reaction solution was washed with chloroform (10 mL x 3), and 25 mL of isopropanol was added to the aqueous phase. After mixing, the mixture was placed in the dark at 4°C to allow the product to precipitate completely (4 hours). Centrifugation was performed (4000 rpm), and the supernatant was transferred. The solid product was washed again with isopropanol (50 mL). The supernatant was removed, and the residue was evaporated to dryness under reduced pressure at low temperature to obtain a dark red solid product, which was the Apt MUC1-Apt AS1411-Apt GPC1-gemcitabine-DNA-epirubicin targeted drug.

[1013] (4) Calculation of mount rate:

[1014] 1. Prepare 100 μL of epirubicin-PBS standard solutions of known concentrations: 2 μM, 4 μM, 6 μM, 8 μM, and 10 μM;

[1015] 2. Dissolve Apt MUC1-Apt AS1411-Apt GPC1-gemcitabine-DNA-epirubicin in 100 μl PBS;

[1016] 3. Place the standard solution and Apt MUC1-Apt AS1411-Apt GPC1-gemcitabine-DNA-epirubicin in a PCR plate, heat at 85°C for 5 minutes, and then cool to room temperature;

[1017] 4. Use a microplate reader to measure the absorbance of epirubicin at 492 nm, draw a standard curve, and calculate the molar concentration of epirubicin in the mounted product;

[1018] 5. Use a spectrophotometer to measure the absorbance of DNA at 260 nm to obtain the mass concentration of DNA vector in each sample;

[1019] 6. Calculate the loading efficiency based on the measured epirubicin molar concentration and the mass concentration of the DNA vector. The maximum theoretical loading efficiency of epirubicin in the Apt MUC1-Apt AS1411-Apt GPC1-gemcitabine-DNA-epirubicin targeted drug is approximately 36, and the maximum theoretical loading efficiency of gemcitabine is 28. Testing showed that the average loading efficiency of epirubicin was 30, and the average loading efficiency of gemcitabine was 28.

[1020] Example 17

[1021] In this example, a 5-fluorouracil targeted drug Apt AS1411-Apt EGFR-Apt A15-RNA-5'-fluorouracil was synthesized.

[1022] Apt AS1411-Apt EGFR-Apt A15-RNA-5' fluorouracil targeted drug was synthesized, and the sequence of its single chain is shown in Table 17 below:

[1023] Table 17

[1024] Note: The N above represents 5'-fluorouracil, which is directly synthesized into the sequence during sequence synthesis via base substitution and base extension. Each base between two adjacent 5'-fluorouracil residues is thiolated, equivalent to 36 5-fluorouracil residues attached to a single DNA targeting vector. Sequence A is thiolated between the first two bases at the 5' end; sequence B is thiolated between the 5' and 3' ends of sequence B; and sequence C is thiolated between the 5' end and 3' ends of sequence C. RNA C / U2F and A / G2'_OMe are thiolated between the 5' and 3' ends of sequence C. The C / U2'F and A / G2'_OMe modifications are underlined in the RNA sequence.

[1025] 1. Experimental materials and reagents:

[1026] (1)A sequence;

[1027] (2) B sequence;

[1028] (3) C sequence;

[1029] (4)DEPC water;

[1030] (5) TBM (Tris-borate magnesium) buffer

[1031] 2. Experimental methods:

[1032] Sequence A, sequence B, and sequence C were dissolved in DEPC water at a molar ratio of 1:1:1. After mixing, the pH of the reaction solution was controlled to 7. The mixture was heated to 99°C, maintained for 5 minutes, then cooled to 65°C at a rate of 2°C / min and maintained for 15 minutes. The temperature was then cooled to 4°C at a rate of 2°C / min.

[1033] The product was loaded onto an 8% (m / v) non-denaturing PAGE gel and the vector was purified by electrophoresis at 100V at 4°C in TBM buffer. The target band was excised and eluted in DNA elution buffer at 37°C, followed by ethanol precipitation overnight and evaporation at low temperature under reduced pressure to obtain the self-assembled product - Apt AS1411-Apt EGFR-Apt A15-RNA-5' fluorouracil targeted drug.

[1034] Example 18

[1035] In this embodiment, the paclitaxel targeted drug Apt AS1411-Apt EGFR-Apt A15-DNA-paclitaxel was synthesized.

[1036] Step 1: Synthesize the Apt AS1411-Apt EGFR-Apt A15-DNA targeting vector, the single-stranded sequence of which is shown in Table 18 below:

[1037] Table 18

[1038] Note: During the synthesis of each sequence, the Oligo synthesizer was programmed to add a long-chain primary amine linker containing a phosphoramidite monomer (N4-benzoyl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxycytidine-3'-(6-aminohexyl-2-hydroxymethyl)-N,N'-diisopropylphosphoramidite) at the designated site (between the underlined base pairs in the table, with a theoretical number of 12 sites) for solid-phase synthesis. Following completion of the reaction, subsequent procedures were performed to complete the synthesis and modification of the entire single strand. Sequences A, B, and C were all thiolated between bases 1-2 at the 5' and 3' ends. The 35th, 39th, 54th and 59th bases from the 5' end of the above-mentioned A sequence are amino-modified (-NH2), the 50th, 55th, 66th and 70th bases from the 5' end of the above-mentioned B sequence are amino-modified (-NH2), and the 24th and 28th bases from the 5' end of the above-mentioned C sequence are amino-modified (-NH2).

[1039] 1. Experimental materials and reagents:

[1040] (1)A sequence;

[1041] (2) B sequence;

[1042] (3) C sequence;

[1043] (4)DEPC water;

[1044] (5) TBM (Tris-borate magnesium) buffer

[1045] 2. Experimental methods:

[1046] Sequence A, sequence B, and sequence C were dissolved in DEPC water at a molar ratio of 1:1:1. After mixing, the pH of the reaction solution was controlled to 7. The mixture was heated to 99°C, maintained for 5 minutes, then cooled to 65°C at a rate of 2°C / min and maintained for 15 minutes. The temperature was then cooled to 4°C at a rate of 2°C / min.

[1047] The product was loaded onto an 8% (m / v) non-denaturing PAGE gel and the vector was purified by electrophoresis at 100 V in TBM buffer at 4°C. The target band was excised and eluted in DNA elution buffer at 37°C, followed by ethanol precipitation overnight and evaporation at low temperature under reduced pressure to obtain the self-assembled product - Apt AS1411-Apt EGFR-Apt A15-DNA targeting vector.

[1048] Step 2: Paclitaxel modification

[1049] Dissolve 1.2 g (0.004 mol) of triphosgene in 25 mL of dichloromethane. Add 8.5 g (0.01 mol) of paclitaxel and 2.45 g (0.02 mol) of 4-dimethylaminopyridine in 100 mL of dichloromethane slowly dropwise over approximately 30 minutes with stirring at room temperature. Continue the reaction for 10 minutes. Then, add 1.53 g (0.01 mol) of bis(2-hydroxyethyl) disulfide in 10 mL of dichloromethane. Continue stirring at room temperature for 30 minutes. Evaporate to dryness, add 40 mL of ethyl acetate, and wash sequentially with 10% KHSO₄, 5% NaHCO₃, and a saturated sodium chloride solution. Dry the organic phase over anhydrous magnesium sulfate and evaporate to dryness to yield 9.1 g of paclitaxel-hydroxyethyl disulfide (88% yield). Recrystallize from methanol to yield 8.2 g of a white crystalline solid.

[1050] Dissolve 8.2 g (0.008 mol) of paclitaxel-hydroxyethyl disulfide in 50 mL of dichloroethane. Add a solution of 1.5 g (0.012 mol) of N,N-diisopropylethylamine in 10 mL of dichloroethane dropwise with stirring at room temperature. Reflux the mixture for 3 hours. Cool to room temperature, add 40 mL of water, and stir for 5 minutes. Transfer the mixture to a separatory funnel and allow the layers to separate. Separate the lower organic phase and evaporate to dryness to yield 7.7 g of paclitaxel disulfide succinimidyl ester (81% yield).

[1051] Step 3: Loading Paclitaxel

[1052] 1. Experimental materials and reagents:

[1053] (1) Apt AS1411-Apt EGFR-Apt A15-DNA targeting vector prepared in step 1;

[1054] (2)DEPC water.

[1055] (3) PBS buffer (6.6).

[1056] (4) 4% paraformaldehyde

[1057] (5) Paclitaxel disulfide succinimidyl ester prepared in step 2.

[1058] (6)Chloroform.

[1059] (7) Isopropyl alcohol.

[1060] 2. Experimental methods:

[1061] The Apt AS1411-Apt EGFR-Apt A15-DNA targeting vector and paclitaxel disulfide succinimidyl ester were added to a 30-fold volume of water at a molar ratio of 1:12 and allowed to react at room temperature for 48 hours. The mixture was cooled and distilled to near dryness under reduced pressure. Ethanol was added with stirring, dispersed, filtered, and washed with ethanol. The filter cake was dried under reduced pressure to obtain Apt AS1411-Apt EGFR-Apt A15-DNA-paclitaxel.

[1062] Example 19

[1063] In this embodiment, the maytansine targeted drug Apt AS1411-Apt EGFR-Apt A15-DNA-maytansine was synthesized.

[1064] Step 1: Synthesize the Apt AS1411-Apt EGFR-Apt A15-DNA targeting vector, the single-stranded sequence of which is shown in Table 19 below:

[1065] Table 19

[1066] Note: During the synthesis of each sequence, the Oligo synthesizer was programmed to add a long-chain primary amine linker containing a phosphoramidite monomer (N4-benzoyl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxycytidine-3'-(6-aminohexyl-2-hydroxymethyl)-N,N'-diisopropylphosphoramidite) at the designated site (underlined base pairs in the table, with a theoretical maximum of 6 sites) for solid-phase synthesis. After the reaction, subsequent procedures were performed to complete the synthesis and modification of the entire single strand. Sequences A, B, and C were all thiolated between bases 1-2 at the 5' and 3' ends. The 43rd and 61st bases from the 5' end of the above-mentioned A sequence are amino-modified (-NH2), the 48th and 63rd bases from the 5' end of the above-mentioned B sequence are amino-modified (-NH2), and the 24th and 43rd bases from the 5' end of the above-mentioned C sequence are amino-modified (-NH2).

[1067] 1. Experimental materials and reagents:

[1068] (1)A sequence;

[1069] (2) B sequence;

[1070] (3) C sequence;

[1071] (4)DEPC water;

[1072] (5) TBM (Tris-borate magnesium) buffer

[1073] 2. Experimental methods:

[1074] Sequence A, sequence B, and sequence C were dissolved in DEPC water at a molar ratio of 1:1:1. After mixing, the pH of the reaction solution was controlled to 7. The mixture was heated to 99°C, maintained for 5 minutes, then cooled to 65°C at a rate of 2°C / min and maintained for 15 minutes. The temperature was then cooled to 4°C at a rate of 2°C / min.

[1075] The product was loaded onto an 8% (m / v) non-denaturing PAGE gel and the vector was purified by electrophoresis at 100 V in TBM buffer at 4°C. The target band was excised and eluted in DNA elution buffer at 37°C, followed by ethanol precipitation overnight and evaporation at low temperature under reduced pressure to obtain the self-assembled product - Apt AS1411-Apt EGFR-Apt A15-DNA targeting vector.

[1076] Step 2: Mounting Maytansine

[1077] 1. Experimental materials and reagents:

[1078] (1) Apt AS1411-Apt EGFR-Apt A15-DNA targeting vector prepared in step 1;

[1079] (2)DEPC water.

[1080] (3) PBS buffer (6.6).

[1081] (4) 4% paraformaldehyde.

[1082] (5) Maytansine SMCC-DM1:C 51 H 66 ClN5O 16 S, molecular weight 1072.61, CAS No.: 1228105-51-8

[1083] (6)Chloroform.

[1084] (7) Isopropyl alcohol.

[1085] 2. Experimental methods:

[1086] The Apt AS1411-Apt EGFR-Apt A15-DNA targeting vector and maytansine SMCC-DM1 were added to a 30-fold volume of water relative to maytansine SMCC-DM1 at a molar ratio of 1:6 and allowed to react at room temperature for 48 hours. The mixture was cooled and distilled to near dryness under reduced pressure. Ethanol was added and dispersed with stirring, filtered, and washed with ethanol. The filter cake was dried under reduced pressure to obtain Apt AS1411-Apt EGFR-Apt A15-DNA-maytansine.

[1087] Example 20

[1088] In this embodiment, the epirubicin targeted drug Apt AS1411-Apt EGFR-Apt A15-AmiR-21-DNA-epirubicin was synthesized.

[1089] Step 1: Synthesize the Apt AS1411-Apt EGFR-Apt A15-AmiR-21-DNA targeting vector, the single-stranded sequence of which is shown in Table 20 below:

[1090] Table 20

[1091] 1. Experimental materials and reagents:

[1092] (1)A sequence;

[1093] (2) B sequence;

[1094] (3) C sequence;

[1095] (4)DEPC water;

[1096] (5) TBM (Tris-borate magnesium) buffer

[1097] 2. Experimental methods:

[1098] Sequences A, B, and C were dissolved in DEPC water at a molar ratio of 1:1:1. After mixing, the pH of the reaction solution was controlled to 7. The mixture was heated to 85°C, maintained for 5 minutes, then cooled to 60°C at a rate of 2°C / min and maintained for 5 minutes. The temperature was then cooled to 4°C at a rate of 2°C / min.

[1099] The product was loaded onto an 8% (m / v) non-denaturing PAGE gel and the vector was purified by electrophoresis at 100 V in TBM buffer at 4°C. The target band was excised and eluted in DNA elution buffer at 37°C, followed by ethanol precipitation overnight and evaporation under reduced pressure at low temperature to obtain the self-assembled product - Apt AS1411-Apt EGFR-Apt A15-2*Bio-AmiR-21-DNA targeting vector.

[1100] Step 2: Mount Epirubicin

[1101] 1. Experimental materials and reagents:

[1102] (1) Apt AS1411-Apt EGFR-Apt A15-AmiR-21-DNA targeting vector prepared in step 1;

[1103] (2)DEPC water.

[1104] (3) PBS buffer (6.8).

[1105] (4) 4% paraformaldehyde

[1106] (5) Epirubicin hydrochloride.

[1107] (6)Chloroform.

[1108] (7) Methanol.

[1109] 2. Experimental methods:

[1110] (1) Accurately weigh epirubicin hydrochloride (0.79 mg, 1.354 μmoL) and dissolve it in DEPC water (1.0 mL) and PBS buffer (1.25 mL). Add 4% paraformaldehyde aqueous solution (0.25 mL) and mix well under ice-water bath cooling. Mix the mixture with Apt AS1411-Apt EGFR-Apt A15-AmiR-21-DNA targeting vector (1 mg) and react at 4°C for 72 hours in the dark. Control the reaction pH at 6.

[1111] (2) The reaction solution was washed with chloroform (10 mL x 3), and 25 mL of methanol was added to the aqueous phase. After mixing, the mixture was placed in the dark at 4°C to allow the product to precipitate completely (4 hours). Centrifugation was performed (4000 rpm), and the supernatant was transferred. The solid product was washed again with methanol (50 mL). The supernatant was removed, and the residue was evaporated to dryness under reduced pressure at low temperature to obtain a dark red solid product, which is the epirubicin targeted drug Apt AS1411-Apt EGFR-Apt A15-AmiR-21-DNA-epirubicin.

[1112] (4) Calculation of mount rate:

[1113] 1. Prepare 100 μL of epirubicin-PBS standard solutions of known concentrations: 2 μM, 4 μM, 6 μM, 8 μM, and 10 μM;

[1114] 2. Dissolve Apt AS1411-Apt EGFR-Apt A15-AmiR-21-DNA-epirubicin in 100 μl PBS;

[1115] 3. Place the standard solution and Apt AS1411-Apt EGFR-Apt A15-AmiR-21-DNA-epirubicin in a PCR plate, heat at 85°C for 5 minutes, and then cool to room temperature;

[1116] 4. Use a microplate reader to measure the absorbance of epirubicin at 492 nm, draw a standard curve, and calculate the molar concentration of epirubicin in the mounted product;

[1117] 5. Use a spectrophotometer to measure the absorbance of DNA at 260 nm to obtain the mass concentration of DNA vector in each sample;

[1118] 6. Calculate the loading efficiency based on the measured epirubicin molar concentration and the mass concentration of the DNA vector. The average maximum theoretical loading efficiency of epirubicin for the epirubicin-targeted drug Apt AS1411-Apt EGFR-Apt A15-AmiR-21-DNA-epirubicin is approximately 27. Testing revealed an average loading efficiency of 23.

[1119] Example 21

[1120] In this example, the epirubicin targeting drug Apt TfRA4-AptAS1411-Apt A15-DNA-epirubicin (2) was synthesized.

[1121] Step 1: Synthesize the Apt TfRA4-AptAS1411-Apt A15-DNA targeting vector (2), the single-stranded sequence of which is shown in Table 21 below:

[1122] Table 21

[1123] 1. Experimental materials and reagents:

[1124] (1)A sequence;

[1125] (2) B sequence;

[1126] (3) C sequence;

[1127] (4)DEPC water;

[1128] (5) TBM (Tris-borate magnesium) buffer

[1129] 2. Experimental methods:

[1130] Sequence A, sequence B, and sequence C were dissolved in DEPC water at a molar ratio of 1:1:1. After mixing, the pH of the reaction solution was controlled to 7. The mixture was heated to 99°C, maintained for 5 minutes, then cooled to 65°C at a rate of 2°C / min and maintained for 15 minutes. The temperature was then cooled to 4°C at a rate of 2°C / min.

[1131] The product was loaded onto an 8% (m / v) non-denaturing PAGE gel and the vector was purified by electrophoresis at 100V in TBM buffer at 4°C. The target band was cut out and eluted in DNA elution buffer at 37°C, followed by ethanol precipitation overnight and evaporation at low temperature under reduced pressure to obtain the self-assembled product - Apt TfRA4-AptAS1411-Apt A15-DNA targeting vector (2).

[1132] Step 2: Mount Epirubicin

[1133] 1. Experimental materials and reagents:

[1134] (1) Apt TfRA4-AptAS1411-Apt A15-DNA targeting vector (2) prepared in step 1;

[1135] (2)DEPC water.

[1136] (3) PBS buffer (6.6).

[1137] (4) 4% paraformaldehyde

[1138] (5) Epirubicin hydrochloride.

[1139] (6)Chloroform.

[1140] (7) Isopropyl alcohol.

[1141] 2. Experimental methods:

[1142] (1) Accurately weigh epirubicin hydrochloride (0.79 mg, 1.354 μmoL) and dissolve it in DEPC water (1.0 mL) and PBS buffer (1.25 mL). Add 4% paraformaldehyde aqueous solution (0.25 mL) and mix well under ice-water bath cooling. Mix the mixture with AS1411-TfRA4-A15-DNA targeting vector (1 mg) and react at 4°C for 72 hours in the dark. Control the reaction pH to 7.

[1143] (2) The reaction solution was washed with chloroform (10 mL x 3), and 25 mL of isopropanol was added to the aqueous phase. After mixing, the mixture was placed in the dark at 4°C to allow the product to precipitate completely (4 hours). Centrifugation was performed (4000 rpm), and the supernatant was transferred. The solid product was washed again with isopropanol (50 mL), and the supernatant was removed. The residue was evaporated to dryness under reduced pressure at low temperature to obtain a dark red solid product, which was the epirubicin targeted drug Apt TfRA4-AptAS1411-Apt A15-DNA-epirubicin (2).

[1144] (4) Calculation of mount rate:

[1145] 1. Prepare 100 μL of epirubicin-PBS standard solutions of known concentrations: 2 μM, 4 μM, 6 μM, 8 μM, and 10 μM;

[1146] 2. Dissolve Apt TfRA4-AptAS1411-Apt A15-DNA-epirubicin in 100 μl PBS;

[1147] 3. Place the standard solution and Apt TfRA4-AptAS1411-Apt A15-DNA epirubicin in a PCR plate, heat at 85°C for 5 minutes, and then cool to room temperature;

[1148] 4. Use a microplate reader to measure the absorbance of epirubicin at 492 nm, draw a standard curve, and calculate the molar concentration of epirubicin in the mounted product;

[1149] 5. Use a spectrophotometer to measure the absorbance of DNA at 260 nm to obtain the mass concentration of DNA vector in each sample;

[1150] 6. Calculate the loading efficiency based on the measured epirubicin molar concentration and the mass concentration of the DNA vector. The maximum theoretical loading efficiency of epirubicin for the epirubicin-targeted drug Apt TfRA4-AptAS1411-Apt A15-DNA-epirubicin is approximately 36. Testing revealed an average loading efficiency of 31.

[1151] Performance characterization:

[1152] 1. In vivo efficacy test of the epirubicin targeted drug Apt EGFR-DNA-epirubicin prepared in Example 1 on the N87 mouse gastric cancer model:

[1153] experimental animals

[1154] Species and strain: Balb / c nude

[1155] Age: 6 to 8 weeks

[1156] Gender: male or female

[1157] Weight: 18~22g

[1158] Experimental methods

[1159] 1. Cell Culture: Tumor cells were cultured in RPMI-1640 medium containing 10% inactivated fetal bovine serum, 100 U / ml penicillin, 100 μg / ml streptomycin, and 2 mM glutamine at 37°C in a 5% CO2 incubator. Cells were passaged every 3 to 4 days after confluence. Tumor cells in the logarithmic growth phase were used for inoculation of tumors in vivo.

[1160] 2. Tumor cell inoculation and grouping:

[1161] PBS: 1x10 tumor cells resuspended in Matrigel 7 After inoculation into the right flank of the experimental animal, the tumor grew to 80-100 mm. 3 The drugs were divided into groups and administered at 14:00 and 18:00. The specific administration schedule is shown in the table below (PBS and original epirubicin were used for comparison).

[1162] 3. Dosage regimen:

[1163] Table 22

[1164] Note: If the animal loses 20% of its body weight during the experiment, stop dosing. Continue dosing after the animal regains its weight.

[1165] The dosage volume is 200ul / 20g mouse, and the specific dosage is calculated based on the weight of the mouse; the observation period is 60 days (depending on the mouse and tumor condition)

[1166] Animal husbandry and management

[1167] Feeding and management: The experimental animals were housed in an SPF-grade laminar flow clean room with constant temperature and humidity, using independently ventilated cages (IVC), with 3 mice per cage.

[1168] Temperature / degrees: controlled within the range of (23±3)℃ / 40-70%.

[1169] Cages: Made of polycarbonate. Dimensions: 370 mm x 155 mm x 135 mm. Bedding: autoclaved soft corncob, changed twice weekly. Each cage has a cage label indicating the number of animals, sex, strain, date of receipt, group, and start date of the experiment.

[1170] Feed and drinking water: SPF grade mouse feed, Co 60 Irradiation sterilization. Drinking water was ultrafiltration purified water and autoclaved. Animals had free access to autoclaved food and water.

[1171] Animal ID: Ear piercing.

[1172] Detection indicators

[1173] 1. Detection indicators

[1174] a. Tumor volume: Tumor volume was measured twice weekly using a vernier caliper. The long and short diameters of the tumor were measured. The volume was calculated using the formula: Volume = 0.5 × Long Diameter × Short Diameter 2 .

[1175] b. Animal response after drug administration: While measuring tumor volume, weigh the mice. Record changes in weight relative to drug administration time. Observe the survival and health of the mice, including general activity and feeding status, during drug administration.

[1176] c. Tumor weight and photography: After administration, the mice were euthanized, photographed, and fixed or frozen for later use.

[1177] a) Each animal was sacrificed and the tumor was photographed;

[1178] b) The subcutaneous tumor of each animal was removed and weighed;

[1179] c) All tumors were placed on a whiteboard according to the grouping table and photographed together. All tumors were sectioned longitudinally and placed on a whiteboard according to the grouping table and photographed together.

[1180] d) Cryopreservation or formalin fixation of the tumor as required;

[1181] d. Dissect animals, take organs, photograph, weigh and image them:

[1182] a) Simultaneously remove organs (heart, liver, spleen, lungs, and kidneys) from each animal and weigh and record them. Calculate the organ / body weight ratio. Mark and photograph any macroscopic metastases. Fix or cryopreserve the organs, and record their condition.

[1183] b) Organs (heart, liver, spleen, lung, kidney) and tumors of one mouse in each experimental group were imaged using Epirubicin wavelengths to detect drug residues.

[1184] 2. Drug evaluation indicators:

[1185] a. Tumor growth inhibition rate (%)

[1186] Tumor growth inhibition rate (%) = (1-T / C) × 100%

[1187] Relative tumor growth rate T / C = RTV of treatment group / RTV of control group, RTC: relative tumor volume

[1188] Tumor growth inhibition rate ≥ 60% and p < 0.05 after statistical analysis was considered effective.

[1189] b. Tumor volume ratio T / C (%) of treatment group / control group

[1190] Tumor volume ratio of treatment group / control group T / C (%) = RTV of treatment group / RTV of control group × 100%.

[1191] Therapeutic efficacy evaluation criteria: T / C (%)>40% was considered ineffective, and T / C (%)≤40% and p<0.05 was considered effective.

[1192] c. Tumor weight inhibition rate IR TW

[1193] The calculation formula is as follows: Tumor weight inhibition rate IR TW (%)=(W 溶剂对照组 -W 治疗组 ) / W 溶剂对照组 ×100, W refers to tumor weight; IR TW %>60% is effective.

[1194] The test results are shown in the table below:

[1195] Table 23

[1196] II. In vivo efficacy test of the epirubicin targeted drug Apt AS1411-Apt EGFR-Apt A15-2*Bio-DNA-epirubicin prepared in Example 5 on the N87 mouse gastric cancer model:

[1197] experimental animals

[1198] Species and strain: Balb / c nude

[1199] Age: 5 weeks

[1200] Gender: Male

[1201] Weight: ~16g

[1202] Experimental methods

[1203] 1. Cell culture and preparation: Cells were revived and inoculated into corresponding culture medium (1640 basal medium + 10% FBS + 1% P / S).

[1204] Passaging method:

[1205] a) Discard the culture medium and gently rinse the cells twice with PBS.

[1206] b) Add appropriate amount of 0.25% trypsin and digest in a 37 degree incubator for about 1-2 minutes. After the cells shrink significantly, add complete culture medium (1640 basal medium + 10% FBS + 1% P / S) to neutralize the trypsin and gently pipette the cells to make them

[1207] The cells are passaged at a certain ratio.

[1208] c) Passaging ratio: 1:2-1:4. The ratio in our laboratory is 1:3.

[1209] d) Passaging frequency: every 2-3 days, depending on the cell seeding density and growth conditions.

[1210] collect:

[1211] a) Take cells in the logarithmic growth phase (preferably at a cell density of about 80-90%);

[1212] b) After trypsinization of the cells, complete medium was added to stop the digestion, and the cells were collected into a centrifuge tube and centrifuged at 1000 rpm for 5 minutes;

[1213] c) Wash twice with pre-chilled PBS and centrifuge;

[1214] d) Pipette the cell pellet with PBS or serum-free medium to an appropriate concentration (2×10^7 cells / ml).

[1215] 2. Vaccination:

[1216] Injection site: Subcutaneous in the right axillary dorsum

[1217] Inoculation volume: 3×10^6 cells / mouse, inoculate 30 mice

[1218] Inoculation volume: 0.1 ml

[1219] Inoculation method: cell inoculation

[1220] Resuscitate N87 cells to an appropriate cell count and adjust the cell concentration to 3 × 107 cells / ml with sterile PBS. Place on ice until ready to use. Use a sterile, disposable 1ml syringe to draw up 0.1ml of the cell suspension and inoculate subcutaneously under the right axilla of the mouse. The day of inoculation is designated as D0.

[1221] Vaccination process:

[1222] a) Before inoculation, use a gun to fully disperse the tumor suspension to prevent cells from clumping and reducing cell survival rate;

[1223] b) Pinch the skin on the back of the mouse's neck with your left thumb and index finger, and pinch the mouse's tail with your pinky finger;

[1224] c) Use a disposable 1 ml syringe to inject an appropriate amount of tumor suspension into the subcutaneous tissue of the mouse. During inoculation, insert the needle slightly deeper, approximately 1 cm, to minimize spillage of the cell suspension from the needle eye.

[1225] 3. Dosage regimen:

[1226] Table 24

[1227] Detection indicators

[1228] 1. General clinical observations:

[1229] The clinical status of the mice was observed every day during the experiment, including the rupture of tumor nodules, the animal's mental state, hair, behavior, diet and excretion.

[1230] 2. Weight:

[1231] The mice were weighed three times a week, and the data were recorded and analyzed. The relative growth curves of body weight of each group are shown in Figure 8.

[1232] 3. Tumor volume (V):

[1233] Tumor volume was measured three times a week using a vernier caliper, and the long and short diameters of the tumor were recorded. The volume was calculated using the formula: volume (V) = 0.5 × long diameter (a) × short diameter (b) 2 .

[1234] 4.Relative tumor volume (RTV),

[1235] The calculation formula is: RTV = V t / V0

[1236] V0 is the tumor volume measured at the time of group administration (ie, D0), and Vt is the tumor volume at each measurement.

[1237] 5.Tumor growth inhibition rate:

[1238] The calculation formula is: Tumor growth inhibition rate (%) = (1-T / C) × 100%

[1239] T / C = RTV of treatment group / RTV of control group

[1240] If the tumor growth inhibition rate is ≥60%, and the tumor volume of the treatment group is significantly lower than that of the solvent control group (P<0.05) after statistical processing, it is considered effective, that is, it has a significant inhibitory effect on tumor growth.

[1241] 6. Tumor weight inhibition rate IR TW

[1242] The calculation formula is as follows: Tumor weight inhibition rate IR TW (%)=(W 溶剂对照组 -W 治疗组 ) / W 溶剂对照组 ×100, W refers to tumor weight; IR TW %>60% is effective.

[1243] The test results are shown in Table 25 below, and the tumor growth curves of each group are shown in Figure 9:

[1244] Table 25

[1245] Tumor weight results and tumor weight inhibition rate IR TW The results are shown in Table 26 below, and the changes in tumor volume in each group are shown in Figure 10:

[1246] Table 26

[1247] 3. Comparison of the in vivo efficacy of the epirubicin targeted drug Apt AS1411-Apt EGFR-Apt A15-2*Bio-DNA-epirubicin prepared in Example 5 and the epirubicin targeted drug Apt AS1411-Apt EGFR-Apt A15-2*Bio-AmiR-21-DNA-epirubicin prepared in Example 7 on the N87 mouse gastric cancer model:

[1248] experimental animals

[1249] Species and strain: Balb / c nude

[1250] Age: 6 to 8 weeks

[1251] Gender: Male

[1252] Weight: 17~19g

[1253] Animals were housed in an SPF-grade barrier environment at the Institute of Materia Medica, Chinese Academy of Medical Sciences, in isolators according to the facility's standards. Six animals were kept per cage in an animal room with ample lighting, good ventilation and air conditioning, a room temperature of 18-25°C, and a relative humidity of 50-70%.

[1254] Experimental methods

[1255] 1. Inoculation and grouping: The mouse gastric cancer N87 model was established using male BALB / c-nu mice weighing 17.0-19.0 g. N87 tumor tissue was removed under sterile conditions and cut into 2.0 mm × 2.0 mm × 2.0 mm tumor masses. The tumor masses were evenly inoculated subcutaneously on the left axillary dorsum of the nude mice. The day of inoculation was designated as D0. When the tumor volume reached 250.0 mm 3 Around day 6, animals with poor tumor growth were eliminated and randomly divided into groups according to tumor volume. Drug administration began on the day of grouping.

[1256] 2. Dosage regimen:

[1257] Table 27

[1258] Detection indicators

[1259] 1. General clinical observations:

[1260] The clinical status of the mice was observed every day during the experiment, including the rupture of tumor nodules, the animal's mental state, hair, behavior, diet and excretion.

[1261] 2. Weight:

[1262] The mice were weighed three times a week, and the data were recorded and analyzed. The weight change curve of the mice is shown in Figure 11.

[1263] 3. Tumor volume (V):

[1264] Tumor volume was measured three times a week using a vernier caliper, and the long and short diameters of the tumor were recorded.

[1265] The formula is: Volume (V) = 0.5 × Long diameter (a) × Short diameter (b) 2 .

[1266] 4.Relative tumor volume (RTV),

[1267] The calculation formula is: RTV = V t / V0

[1268] V0 is the tumor volume measured at the time of group administration (ie, D0), and Vt is the tumor volume at each measurement.

[1269] 5.Tumor growth inhibition rate:

[1270] The calculation formula is: Tumor growth inhibition rate (%) = (1-T / C) × 100%

[1271] T / C = RTV of treatment group / RTV of control group

[1272] If the tumor growth inhibition rate is ≥60%, and the tumor volume of the treatment group is significantly lower than that of the solvent control group (P<0.05) after statistical processing, it is considered effective, that is, it has a significant inhibitory effect on tumor growth.

[1273] 6. Tumor weight inhibition rate IR TW

[1274] The calculation formula is as follows: Tumor weight inhibition rate IR TW (%)=(W 溶剂对照组 -W 治疗组 ) / W 溶剂对照组 ×100, W refers to tumor weight; IR TW %>60% is effective.

[1275] All animals were closely monitored for tumor growth during the experiment. Tumors were measured twice weekly, and the results were recorded and used to calculate the tumor growth inhibition rate. The results of the tumor growth inhibition rate test are shown in Table 28 below, the tumor volume change curve is shown in Figure 12, and the relative tumor volume change curve is shown in Figure 13.

[1276] Table 28

[1277] The results of the T / C test for the tumor volume ratio of the treatment group and the control group are shown in the table below:

[1278] Table 29

[1279] 41 days after N87 inoculation, all animals were killed by cervical dislocation, and the tumors were removed and weighed. The tumor weight inhibition rate (IR) TW％ The data is shown in the table below:

[1280] Table 30

[1281] From the above data, it can be seen that when epirubicin raw drug was injected through the tail vein, the tumor growth inhibition rate (calculated based on tumor weight and tumor volume) was less than 60% and the T / C ratio was greater than 40% at the end of the experiment (D41). When the targeted drugs Apt AS1411-Apt EGFR-Apt A15-2*Bio-DNA-epirubicin and Apt AS1411-Apt EGFR-Apt A15-2*Bio-AmiR-21-DNA-epirubicin were injected through the tail vein, the tumor growth inhibition rates (calculated based on tumor weight and tumor volume) were both greater than 60% and the T / C ratio was less than 40% at the end of the experiment (D41). These results suggest that tail vein injection of Apt AS1411-Apt EGFR-Apt A15-2*Bio-DNA-epirubicin and Apt AS1411-Apt EGFR-Apt A15-2*Bio-AmiR-21-DNA-epirubicin targeted drugs have a significant growth inhibitory effect on N87.

[1282] IV. In vivo efficacy test of the epirubicin targeted drug Apt AS1411-4*Bio-DNA-epirubicin prepared in Example 3 on A549 mouse lung cancer model:

[1283] experimental animals

[1284] Species and strain: Balb / c nude

[1285] Age: 6-7 weeks

[1286] Gender: Male

[1287] Weight: 16~18g

[1288] Experimental methods

[1289] 1. Inoculation and grouping: Mouse lung cancer A549 model was established. The animals used were male Balb / c-nu mice weighing 16.0-18.0 g. A549 tumor cells were collected under sterile conditions and the cell concentration was adjusted to 1×10 7 Take 0.2 ml and inoculate it subcutaneously in the axillary dorsum of nude mice. Wait until the tumor grows to a diameter of 1000-1500 mm. 3 The tumor was removed under sterile conditions and cut into 2.0 mm × 2.0 mm × 2.0 mm tumor pieces, which were evenly inoculated subcutaneously in the axillary dorsum of nude mice. The day of inoculation was recorded as D0. When the tumor volume reached 100.0 mm 3 Around day 5, animals with poor tumor growth were eliminated and randomly divided into groups according to tumor volume. Drug administration began on the day of grouping.

[1290] 2. Dosage regimen:

[1291] Table 31

[1292] Detection indicators

[1293] 1. General clinical observations:

[1294] The clinical status of the mice was observed every day during the experiment, including the rupture of tumor nodules, the animal's mental state, hair, behavior, diet and excretion.

[1295] 2. Weight:

[1296] The mice were weighed twice a week, and the data were recorded and analyzed.

[1297] 3. Tumor volume (V):

[1298] The tumor volume was measured twice a week using a vernier caliper, and the long and short diameters of the tumor were recorded. The volume was calculated using the formula: volume (V) = 0.5 × long diameter (a) × short diameter (b) 2 .

[1299] 4.Relative tumor volume (RTV),

[1300] The calculation formula is: RTV = V t / V0

[1301] V0 is the tumor volume measured at the time of group administration (ie, D0), and Vt is the tumor volume at each measurement.

[1302] 5.Tumor growth inhibition rate:

[1303] The calculation formula is: Tumor growth inhibition rate (%) = (1-T / C) × 100%

[1304] T / C = RTV of treatment group / RTV of control group

[1305] If the tumor growth inhibition rate is ≥60%, and the tumor volume of the treatment group is significantly lower than that of the solvent control group (P<0.05) after statistical processing, it is considered effective, that is, it has a significant inhibitory effect on tumor growth.

[1306] All animals were closely monitored for tumor growth during the experiment. Tumors were measured twice weekly, and the results were recorded and used to calculate the tumor growth inhibition rate. The results of the tumor growth inhibition rate test are shown in Table 32 below, the tumor volume change curve is shown in Figure 14, and the relative tumor volume change curve is shown in Figure 15:

[1307] Table 32

[1308] The results of the T / C test for the tumor volume ratio of the treatment group and the control group are shown in the table below:

[1309] Table 33

[1310] The above data show that, with tail vein injection of the original epirubicin drug, the tumor growth inhibition rate (calculated by tumor weight and volume) was less than 60% and the T / C ratio was greater than 40% at the end of the experiment (D32). With tail vein injection of the high- and medium-dose targeted drug Apt AS1411-4*Bio-DNA-epirubicin, the tumor growth inhibition rate (calculated by tumor weight and volume) exceeded 60% and the T / C ratio was less than 40% at the end of the experiment (D32 or D25). With tail vein injection of the low-dose targeted drug AS1411-4Bio-DNA-epirubicin, the tumor growth inhibition rate was also significantly higher than that of the original epirubicin drug at the same dose at the end of the experiment (D32). This suggests that tail vein injection of the targeted drug Apt AS1411-4*Bio-DNA-epirubicin has a significant growth inhibitory effect on lung cancer A549 cells.

[1311] V. In vivo efficacy test of the epirubicin targeted drug Apt AS1411-Apt EGFR-Apt A15-DNA-epirubicin prepared in Example 6 and the epirubicin targeted drug Apt AS1411-Apt EGFR-Apt A15-AmiR-21-DNA-epirubicin prepared in Example 20 on the A549 mouse lung cancer model:

[1312] experimental animals

[1313] Species and strain: BALB / cAnSlacNifdc-nu

[1314] Gender: Male

[1315] Weight: 17-21g

[1316] Experimental methods

[1317] 1. Inoculation and grouping: Mouse lung cancer A549 model was established. Male BALB / cAnSlacNifdc-nu mice weighing 17.0-21.0 g were used. A549 tumor tissue was removed under sterile conditions and cut into 2.0 mm × 2.0 mm × 2.0 mm tumor masses. The tumor masses were evenly inoculated subcutaneously on the left axillary dorsum of nude mice. The day of inoculation was designated as D0. When the tumor volume reached 150.0 mm 3 Around day 7, animals were randomly divided into four groups based on tumor volume: A (solvent control), B, C, and D, with eight animals in each group. On the same day of grouping, all groups began receiving medication via tail vein injection. A, B, C, and D1 were administered once weekly for a total of three doses; D2 was administered twice weekly for a total of six doses. Details of animal vaccination and grouping are shown in the table below.

[1318] 2. Dosage regimen:

[1319] Table 34

[1320] Detection indicators

[1321] 1. General clinical observations:

[1322] The clinical status of the mice was observed every day during the experiment, including the rupture of tumor nodules, the animal's mental state, hair, behavior, diet and excretion.

[1323] 2. Weight:

[1324] The mice were weighed twice a week, and the data were recorded and analyzed.

[1325] 3. Tumor volume (V):

[1326] The tumor volume was measured twice a week using a vernier caliper, and the long and short diameters of the tumor were recorded. The volume was calculated using the formula: volume (V) = 0.5 × long diameter (a) × short diameter (b) 2 .

[1327] 4.Relative tumor volume (RTV),

[1328] The calculation formula is: RTV = V t / V0

[1329] V0 is the tumor volume measured at the time of group administration (ie, D0), and Vt is the tumor volume at each measurement.

[1330] 5.Tumor growth inhibition rate:

[1331] The calculation formula is: Tumor growth inhibition rate (%) = (1-T / C) × 100%

[1332] T / C = RTV of treatment group / RTV of control group

[1333] If the tumor growth inhibition rate is ≥60%, and the tumor volume of the treatment group is significantly lower than that of the solvent control group (P<0.05) after statistical processing, it is considered effective, that is, it has a significant inhibitory effect on tumor growth.

[1334] All animals were closely monitored for tumor growth during the experiment. Tumors were measured twice weekly, and the results were recorded and used to calculate the tumor growth inhibition rate. The results of the tumor growth inhibition rate test are shown in Table 35 below, the tumor volume change curve is shown in Figure 16, and the relative tumor volume change curve is shown in Figure 17:

[1335] Table 35

[1336] The results of the T / C test for the tumor volume ratio of the treatment group and the control group are shown in the table below:

[1337] Table 36

[1338] From the above data, it can be seen that when epirubicin raw drug was injected into the tail vein, the tumor growth inhibition rate (calculated by tumor volume) was less than 60% and the T / C ratio was greater than 40% at the end of the experiment (D29). When the targeted drug Apt AS1411-Apt EGFR-Apt A15-DNA-epirubicin was injected intravenously, the tumor growth inhibition rate (calculated by tumor volume) exceeded 60% and the T / C ratio was less than 40% at the end of the experiment (D29), indicating that the targeted drug Apt AS1411-Apt EGFR-Apt A15-DNA-epirubicin injected into the tail vein has a significant growth inhibitory effect on lung cancer A549.

[1339] VI. In vivo efficacy test of the epirubicin targeted drug Apt AS1411-Apt EGFR-AptA15-DNA-epirubicin prepared in Example 6 on the HepG2 mouse liver cancer model:

[1340] experimental animals

[1341] Species and strain: BALB / cAnSlacNifdc-nu

[1342] Gender: Male

[1343] Weight: 16~18g

[1344] Experimental methods

[1345] 1. Inoculation and grouping: The mouse liver cancer HepG2 model was established using male BALB / cAnSlacNifdc-nu nude mice weighing 16.0-18.0 g. HepG2 tumor tissue was removed under sterile conditions and cut into 2.0 mm × 2.0 mm × 2.0 mm tumor masses. The tumors were evenly inoculated subcutaneously on the left axillary dorsum of the nude mice. The day of inoculation was designated as D0. When the tumor volume reached 250.0 mm 3 Around day 7, the animals were randomly divided into 3 groups according to tumor volume: A (solvent control), B, and C, with 8 animals in each group. On the day of grouping, all groups of animals began to receive the drug via tail vein injection, once a week, for a total of 3 times.

[1346] 2. Dosage regimen:

[1347] Table 37

[1348] Detection indicators

[1349] 1. General clinical observations:

[1350] The clinical status of the mice was observed every day during the experiment, including the rupture of tumor nodules, the animal's mental state, hair, behavior, diet and excretion.

[1351] 2. Weight:

[1352] The mice were weighed twice a week, and the data were recorded and analyzed.

[1353] 3. Tumor volume (V):

[1354] The tumor volume was measured twice a week using a vernier caliper, and the long and short diameters of the tumor were recorded. The volume was calculated using the formula: volume (V) = 0.5 × long diameter (a) × short diameter (b) 2 .

[1355] 4.Relative tumor volume (RTV),

[1356] The calculation formula is: RTV = V t / V0

[1357] V0 is the tumor volume measured at the time of group administration (ie, D0), and Vt is the tumor volume at each measurement.

[1358] 5.Tumor growth inhibition rate:

[1359] The calculation formula is: Tumor growth inhibition rate (%) = (1-T / C) × 100%

[1360] T / C = RTV of treatment group / RTV of control group

[1361] If the tumor growth inhibition rate is ≥60%, and the tumor volume of the treatment group is significantly lower than that of the solvent control group (P<0.05) after statistical processing, it is considered effective, that is, it has a significant inhibitory effect on tumor growth.

[1362] 6. Tumor Weight: At the end of the experiment (D23), the remaining animals were sacrificed by cervical dislocation. Tumors were removed, weighed, and photographed. The tumors were then calculated. An IRTW (%) > 60% was considered an efficacy indicator using the following formula: IRTW (%) = (W vehicle control group - W treatment group) / W vehicle control group × 100, where W represents tumor weight.

[1363] All animals were closely monitored for tumor growth during the experiment. Tumors were measured twice weekly, and the results were recorded and used to calculate the tumor growth inhibition rate. The results of the tumor growth inhibition rate test are shown in Table 38 below, the tumor volume change curve is shown in Figure 18, and the relative tumor volume change curve is shown in Figure 19:

[1364] Table 38

[1365] The results of the T / C test for the tumor volume ratio of the treatment group and the control group are shown in the table below:

[1366] Table 39

[1367] 23 days after HepG2 inoculation, all animals were killed by cervical dislocation, and the tumors were removed and weighed. The tumor weight inhibition rate (IR) TW％ The data is shown in the table below:

[1368] Table 40

[1369] The above data show that, with tail vein injection of the original epirubicin drug, the tumor growth inhibition rate (calculated by tumor weight and tumor volume) was less than 60%, and the T / C ratio was greater than 40% at the end of the experiment (D23). When the targeted drug Apt AS1411-Apt EGFR-Apt A15-DNA-epirubicin was injected intravenously, the tumor growth inhibition rate (calculated by tumor volume) exceeded 60%, and the T / C ratio was less than 40% at the end of the experiment (D23), indicating that tail vein injection of the targeted drug Apt AS1411-Apt EGFR-Apt A15-DNA-epirubicin has a significant growth inhibitory effect on HepG2 liver cancer cells.

[1370] VII. In vivo efficacy test of the epirubicin targeted drug Apt TfRA3-Apt AS1411-Apt A15-DNA-epirubicin prepared in Example 9 on a U251 glioma mouse model:

[1371] Cell Information

[1372] U251-RFP, a human glioma cell line expressing red fluorescent protein, source: Antaikang Biotechnology (Beijing) Co., Ltd.

[1373] experimental animals

[1374] Species and strain: Balb / c nude (immunodeficient mice)

[1375] Age: 4 weeks

[1376] Gender: Female

[1377] Weight: 15~18g

[1378] Animals were housed in an SPF-grade barrier environment at Antaikang Biotechnology (Beijing) Co., Ltd. using laminar flow cabinets and maintained according to the animal facility's standards. Three animals were kept per cage in an animal room with ample lighting, good ventilation and air conditioning, a room temperature of 18-25°C, and a relative humidity of 50-70%.

[1379] Experimental methods

[1380] 1. Inoculation and grouping: 32 4-week-old female BALB / c-nu mice were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd. (Laboratory Animal Quality Certificate No. SCXK Jing 2016-0006). U251-RFP tumor tissue was obtained from the subcutaneous tissue of the same strain of mice using the surgical orthotopic transplantation method. Necrotic tissue was removed under a dissecting microscope and the tumor tissue was cut into 1 mm 3 Tissue blocks of uniform size. After isoflurane anesthesia, under aseptic surgical conditions, surgical orthotopic implantation technology was used to implant the excised tumor tissue blocks into the skull of experimental mice. The skull was repositioned using the mosaic method, and the mouse scalp was sutured interruptedly with 6-0 surgical sutures (ETHICON.INC) to establish an orthotopic mouse model of human glioma (U251-RFP) expressing red fluorescent protein. The day of inoculation was recorded as D0. On D3, non-invasive real-time imaging of brain tumors in all mice was performed using a FluorVivo Model-100 (INDEC BioSystems, CA, USA) fluorescence imager. The animals were randomly divided into groups, and all groups began to receive medication on the day of grouping.

[1381] 2. Dosage regimen:

[1382] Table 41

[1383] The dosage of all test drugs is 200ul / 20g mouse, and the actual dosage needs to be calculated based on the weight of the mouse at the time of administration.

[1384] Detection indicators

[1385] a) Tumor area: The fluorescence area was measured by fluorescence imaging twice a week.

[1386] The inhibition rate was calculated using the formula: tumor growth inhibition rate (%) = (1-T / C) x 100%, T / C = RTV of treatment group / RTV of control group, wherein the volume data used the fluorescence area.

[1387] b) Animal Responses After Dosing: Weigh the mice twice weekly while measuring tumor volume. Record changes in weight and the relationship between dosing time and the mice's survival and health, including general activity and feeding, during dosing.

[1388] c) Tumor and Organ Imaging: After dosing, mice were euthanized, and the brain, heart, liver, spleen, lung, and kidney organs were harvested and arranged alongside the corresponding orthotopic tumors. Images were taken and then imaged using both tumor fluorescence and epirubicin wavelengths.

[1389] d) Taking photos of the tumor:

[1390] The in situ tumors were isolated, weighed, and photographed (camera, tumor autoluminescence wavelength and epirubicin wavelength imaging).

[1391] Tumor and Organ Imaging: One mouse from each group was selected for imaging of the tumor (already removed), heart, liver, spleen, lung, kidney, and large intestine. Images were taken using both the tumor's autoluminescence wavelength and the Epirubicin wavelength. After the experiment, the mouse carcasses and organs were placed in a -20°C freezer and discarded.

[1392] Calculate tumor weight inhibition rate IR TW %, with IRTW%>60% as the reference index of effectiveness, and the calculation formula is as follows: tumor weight inhibition rate IR TW (%)=(W solvent control group-W treatment group) / W solvent control group×100, where W refers to tumor weight.

[1393] result

[1394] The experimental animals were generally in good condition, including activity and feeding, during the dosing period. Around D10 after dosing, the weight of mice in Group 3 began to decrease, and their spontaneous activity decreased, which was analyzed to be possibly due to drug toxicity.

[1395] All animals were closely monitored for tumor growth during the experiment. Tumors were measured twice a week, and the results were recorded and the tumor growth inhibition rate was calculated. The results of the tumor growth inhibition rate test are shown in the table below:

[1396] Table 42

[1397] Tumor weight results and tumor weight inhibition rate IR TW The results are shown in Table 43 below, and the tumor photos of Groups 1, 2, and 4 are shown in Figure 20:

[1398] Table 43

[1399] As shown in the table above, the epirubicin-targeted drug Apt TfRA3-Apt AS1411-Apt A15-DNA-epirubicin provided by the present invention exhibits a more pronounced inhibitory effect on U251-RFP tumor growth than the original epirubicin drug. Furthermore, compared to the positive control group, temozolomide, the present invention achieves a higher tumor growth inhibition rate at a lower dose.

[1400] 8. In vivo efficacy test of the epirubicin targeted drug Apt TfRA4-Apt AS1411-Apt A15-DNA-epirubicin (1) prepared in Example 8 on the GL261-Luc brain glioma mouse model:

[1401] Cell culture

[1402] GL261-Luc cells were purchased from Ningbo Mingzhou Biotechnology Co., Ltd. and were identified as a mouse cell line, free of rat or human cell contamination. Cells were cultured in DMEM supplemented with 10% fetal bovine serum, 1% glutamine, and 1% HEPES at 37°C in a 5% CO2 incubator. Cells were passaged every 3 to 4 days after confluence. Tumor cells in the logarithmic growth phase were used for inoculation of in vivo tumors.

[1403] experimental animals

[1404] Species and strain: C57BL / 6

[1405] Grade: SPF

[1406] Age: 6-8 weeks

[1407] Gender: Female

[1408] Weight: 18~22g

[1409] Animal Housing: Experimental animals were housed at Youji (Tianjin) Pharmaceutical Technology Co., Ltd. All animals were housed in an IVC system within a barrier enclosure. Upon receipt, animals were quarantined for at least three days, and only after all animals passed quarantine could they enter the formal experiment. Throughout the experiment, the animals were housed at a temperature of 20.5-24.5°C, a humidity of 40-70%, and a photoperiod of 12 hours light and 12 hours dark. Three to five animals were housed in each cage, and adequate water and feed were provided throughout the experiment.

[1410] Experimental methods

[1411] 1. Inoculation and grouping: Resuspend GL261-Luc cells in PBS at a concentration of 1×10 9 +M / ml, injected orally into the brain, 5μL / mouse. Seven days after tumor cell inoculation, the mice were divided into two groups, each with 6 mice. The specific dosing schedule is as follows:

[1412] 2. Dosage regimen:

[1413] Table 44

[1414] Detection indicators

[1415] (1) After grouping, the mice in each group were imaged and the BLI values ​​were detected using a small animal in vivo imaging device. The mice were weighed twice a week, and the relationship between the changes in the weight of the mice and the time of administration was recorded. At the same time, the survival and health status of the mice, such as the general status of animal activity and eating during the administration period, were observed.

[1416] (2) The survival status of the tumor-bearing mice in each group was checked and recorded daily. When the mice became moribund, they were euthanized and their survival was recorded. The longest observation period was 138 days after tumor cell inoculation. At the end of the experiment, the median survival time (MST) of the tumor-bearing mice in each group was calculated. The survival extension rate (ILS%) of the tumor-bearing mice in the treatment group was calculated according to the following formula:

[1417] The extension rate of survival of tumor-bearing mice in the treatment group (ILS%) = (median survival days of the treatment group / median survival days of the solvent control group-1) × 100%.

[1418] (3) The median survival time and survival extension rate of each group of mice were calculated and statistically analyzed. P < 0.05 was considered to be a significant difference. BLI value curves of each group of mice were drawn and statistically analyzed. P < 0.05 was considered to be a significant difference.

[1419] (4) When the mice died, the brain tissue was fixed with paraformaldehyde, embedded in paraffin, and pathological sections were prepared and stained with HE and observed and photographed.

[1420] (5) At the end of the observation, the surviving mice were anesthetized, their eyeballs were removed, and blood was collected. The mice were euthanized, and their brain tissue was removed. The left and right leg bones were removed, and the bone marrow cells in the bone marrow cavity were flushed with PBS. The brain tissue was fixed with paraformaldehyde and embedded in paraffin. After preparing pathological sections, they were stained with HE and observed and photographed. The proportions of CD4+ T cells, CD8+ T cells, and Treg cells in peripheral blood and bone marrow cells were detected by flow cytometry.

[1421] Data processing and statistical analysis

[1422] IBM SPSS Statistics 21.0 statistical software was used to perform inter-group statistical analysis of BLI values ​​using One-Way ANOVA or Kruskal-Wallis test. The Kaplan-Meier test was used to perform inter-group statistical analysis of the survival time of tumor-bearing mice. P < 0.05 was considered to indicate a significant difference.

[1423] result

[1424] (1) Reactions and weight changes of experimental animals after administration

[1425] After the start of drug administration, the body weight change curves of mice in each group are shown in FIG21 , and the body weight change percentages are shown in FIG22 .

[1426] As can be seen from the figure, the body weight of the control group dropped to less than 15% after 39 days, while the mice in the Apt TfRA4-Apt AS1411-Apt A15-DNA-epirubicin group were generally in good condition, with no significant weight loss, no drug withdrawal, and no other abnormal manifestations or abnormal deaths.

[1427] (2) In vivo imaging results of mice

[1428] On day 5 after tumor inoculation, the animals were imaged using a small animal in vivo imaging instrument and dosed in groups. Thereafter, imaging was performed twice a week, and BLI curves were plotted and statistically analyzed. Bioluminescence imaging images of each group of mice after tumor inoculation are shown in Figures 23 to 26 (Figure 23 is a dorsal in vivo imaging image of the mouse, Figure 24 is a left in vivo imaging image of the mouse, Figure 25 is a right in vivo imaging image of the mouse, and Figure 26 is a ventral in vivo imaging image of the mouse). The BLI value change curves of each group of mice are shown in Figure 27 (...

Claims

1. A targeted chemical drug, characterized in that: The targeted chemical drug comprises a targeted nucleic acid vector and a chemical small molecule drug mounted on the targeted nucleic acid vector, and the targeted nucleic acid vector comprises a nucleic acid vector and a targeting molecule connected to the nucleic acid vector; wherein the nucleic acid vector is a DNA vector or an RNA vector, and the nucleic acid vector comprises a sequence, b sequence and c sequence that form a self-assembly structure, The a sequence is SEQ ID NO: 1: 5'-ACGAGCGTTCCG-3'; The b sequence is SEQ ID NO:2: 5'-CGGTTCGCCG-3'; The c sequence is SEQ ID NO:3: 5'-CGGCCATAGCCGT-3'; or, The a sequence is SEQ ID NO:4: 5'-ACGAGCGUUCCG-3'; The b sequence is SEQ ID NO:5:5'-CGGUUCGCCG-3'; The c sequence is SEQ ID NO:6: 5'-CGGCCAUAGCCGU-3'; Optionally, at least one base is substituted, inserted or deleted in the a sequence, the b sequence and the c sequence.

2. The targeted chemical drug according to claim 1, characterized in that: The 5' end and 3' end of the a sequence, the b sequence and the c sequence are each independently connected to an extended base segment, and the extended base segment includes 0 to 14 bases; preferably, the nucleic acid vector comprises the following sequence that forms a self-assembly structure: The first set of sequences: a Sequence: SEQ ID NO:7: 5'-GCGGCGCCCACGAGCGTTCCGGGAGC-3'; b sequence: SEQ ID NO:8: 5'-GCTCCCGGTTCGCCGCCAGCCGCC-3'; c sequence: SEQ ID NO:9: 5'-GGCGGCAGGCGGCCATAGCCGTGGGCGCCGC-3'; or The second set of sequences: a Sequence: SEQ ID NO: 10: 5'-GCGGCGCCCACGAGCGTTCCGGGAGAGGAGC-3'; b sequence: SEQ ID NO: 11: 5'-GCTCCTCTCCCGGTTCGCCGCGAGCCGCG-3'; c sequence: SEQ ID NO: 12: 5'-CGCGGCTCGCGGCCATAGCCGTGGGCGCCGC-3'; or The third sequence: a Sequence: SEQ ID NO: 10: 5'-GCGGCGCCCACGAGCGTTCCGGGAGAGGAGC-3'; b Sequence: SEQ ID NO: 13: 5'-GCTCCTCTCCCGGTTCGCCAGCCAGCCGCGG-3'; c sequence: SEQ ID NO: 14: 5'-GGCGGCTGGCGGCCATAGCCGTGGGCGCCGC-3'; or The fourth sequence: a Sequence: SEQ ID NO: 15: 5'-GCGGCGCCCACGAGCGTTCCGGGAGAGGCC-3'; b sequence: SEQ ID NO: 16: 5'-GGCCTCTCCCGGTTCGCCGCCAGCCGCC-3'; c sequence: SEQ ID NO: 14: 5'-GGCGGCTGGCGGCCATAGCCGTGGGCGCCGC-3'; or The fifth sequence: a Sequence: SEQ ID NO: 17: 5'-GCGGCGCCCACGAGCGUUCCGGGAGAGGCC-3'; b sequence: SEQ ID NO: 18: 5'-GGCCUCUCCCGGUUCGCCGCCAGCCGCC-3'; c sequence: SEQ ID NO: 19: 5'-GGCGGCUGGCGGCCAUAGCCGUGGGCGCCGC-3'; or The sixth sequence: a Sequence: SEQ ID NO: 20: 5'-GCGGCGCCCACGAGCGTTCCGGGAGAGGAGGCC-3'; b sequence: SEQ ID NO: 21: 5'-GGCCTCCTCTCCCGGTTCGCCGCCAGCCGCC-3'; c sequence: SEQ ID NO: 14: 5'-GGCGGCTGGCGGCCATAGCCGTGGGCGCCGC-3'; or The seventh sequence: a Sequence: SEQ ID NO: 10: 5'-GCGGCGCCCACGAGCGTTCCGGGAGAGGAGC-3'; b sequence: SEQ ID NO: 22: 5'-GCTCCTCTCCCGGTTCGCCGCCAGCCGCC-3'; c sequence: SEQ ID NO: 14: 5'-GGCGGCTGGCGGCCATAGCCGTGGGCGCCGC-3'; or The eighth sequence: a Sequence: SEQ ID NO: 23: 5'-GCGACGCCCACGAGCGTTCCGGGAGAGGAG-3'; b sequence: SEQ ID NO: 24: 5'-CTCCTCTCCCGGTTCGCCGCGAGCCGCG-3'; c sequence: SEQ ID NO: 25: 5'-CGCGGCACGCGGCCATAGCCGTGGGCGTCGC-3'; or The ninth sequence: a Sequence: SEQ ID NO: 26: 5'-GCGACGCCCACGAGCGTTCCGGGAGAGGAGC-3'; b sequence: SEQ ID NO: 11: 5'-GCTCCTCTCCCGGTTCGCCGCGAGCCGCG-3'; c sequence: SEQ ID NO: 27: 5'-CGCGGCTCGCGGCCATAGCCGTGGGCGTCGC-3'; or The tenth sequence: a Sequence: SEQ ID NO: 28: 5'-GACGCCCACGAGCGTTCCGGGAGAGG-3'; b sequence: SEQ ID NO: 29: 5′-CCTCTCCCGGTTCGCCGCGAGCCT-3′; c sequence: SEQ ID NO: 30: 5'-GGCTCGCGGCCATAGCCGTGGGCGTCTGCTGCTGCTGCTG-3'; or Eleventh sequence: a Sequence: SEQ ID NO: 31: 5'-GCCCACGAGCGTTCCGGGAGA-3'; b sequence: SEQ ID NO: 32: 5'-TCTCCCGGTTCGCCGCCAGCCGCC-3'; c sequence: SEQ ID NO: 33: 5'-GGCGGCTGGCGGCCATAGCCGTGGGC-3'.

3. The targeted chemical drug according to claim 2, characterized in that: The targeted chemical drug also includes an oligonucleotide effector molecule and / or an immunostimulant mounted on the targeted nucleic acid carrier; Preferably, the oligonucleotide effector molecule comprises one or more of ASO, siRNA, miRNA, and nucleic acid aptamer; More preferably, the siRNA includes one or more of ASAP1, ATAD2, CD24, CD47, EGFR, HBV, HSP, HS70, PD-L1, PAPP-1, Survivin, TAP, TIM-3, TGF-β1, and VEGF-C; further preferably, the siRNA includes one or more of ASAP1, CD47, PD-L1, and TGF-β1; More preferably, the miRNA includes one or more of A-miR21, A-miR-10a, A-miR-30c, miR-34, miR-542, miR-126-3p, and miR-122; further preferably, the miRNA is A-miR21; More preferably, the nucleic acid aptamer comprises a nucleic acid aptamer in the form of DNA and / or a nucleic acid aptamer in the form of RNA. The aptamer is preferably selected from A1, A15, AS1411, AFP, ATP, Act-12c, A18, BAF7-1, C-Met-SL1, CH6, CA2, C12, CRAC Orail, CEA, CEA-18, CEA-T84, CSC1, CSC13, CD40, CD16a, CD19, CD3-4, CD44, CD12 (HDLBP), CD20, CD24, CD33, CD38, CD105, CD117, CD63, CD123, EGFR, EpCAM, EcR, FAP, GPC-1, GSK836, GPC3 (APS63-1), Her 2. Her3, HMGA2, H2, HbsAg, IFN-y(B4), IL-4Ra, IL-17, LZH8, MUC1, M5, M7, M1, N5, NG-Dua, NKG2D(20- N-15), NSE, Np-A15, Np-A48, Np-A58, Np-A61, OX40, PSMA, PDGFRβ, PDGF, PD-L1, PD-1, PTK-7, ProGRP- 48. SF, TBA15, TBA29, TfRA4, TfRA3, TTA1, TLS9a, TGF-βII(S58), TNF-a, TNF, T1, VEGF, VCAM-1, VCAM -12d, CH6, PL-45, EP66, AGC, Karpas299, SW620, MDA-MB-231, MCF-7, PC-3, BCMA, CTLA-4, CCL1, CD4-3 , CD28, FGF2 (F2), FGF2, FGF5, LAG-3, MRP1, TIM3, TIMC-11, VEGF165, 4-1BB or more; Further preferably, the nucleic acid aptamer includes: A1, A15, AS1411, C12, CD40L, EGFR, GPC-1, IL-4Ra, MUC1, OX40, PD-L1, TTA1, TfRA3, TfRA4 or more; Preferably, the immunostimulant comprises one or more of CPG2006, CPG1826, CPG2216, CPG2395, CPG-ODNT7, and CPG-ODN-PCIF1; more preferably, the immunostimulant is CPG2006.

4. The targeted chemical drug according to claim 3, characterized in that: The sequence structure of the sense strand and antisense strand of each siRNA from the 5' end to the 3' end is as follows: ASAP1: the sense strand thereof is SEQ ID NO:34: 5'-UGAUAUUAUGGAAGCAAAUUU-3', and the antisense strand thereof is SEQ ID NO:35: 5'-AUUUGCUUCCAUAAUAUCAUU-3'; or the sense strand thereof is SEQ ID NO:36: 5'-UUAGGUUUGGGGUUGGAUCUU-3', and the antisense strand thereof is SEQ ID NO:37: 5'-GAUCCAACCCCAAACCUAAUU-3'; ATAD2: its sense strand is SEQ ID NO:38: 5'-AAUCCUACAACUUCGACGCUU-3', and its antisense strand is SEQ ID NO:39: 5'-GCGUCGAAGUUGUAGGAUUUU-3'; CD24: its sense strand is SEQ ID NO:40: 5'-UGUUUACAUUGUUGAGGUAUU-3', and its antisense strand is SEQ ID NO:41: 5'-UACCUCAACAAUGUAAACUUU-3'; CD47: Its sense strand is SEQ ID NO:42: 5'-GGUGAUUACCCAGAGAUAUTT-3', and its antisense strand is SEQ ID NO:43: 5'-AUAUCUCUGGGUAAUCACCTT-3'; or, its sense strand is SEQ ID NO:44: 5'-UGGUGAAAGAGGUCAUUCCUU-3', and its antisense strand is SEQ ID NO:45: 5'-GGAAUGACCUCUUUCACCAUU-3'; or, its sense strand is SEQ ID NO:46: 5'-GGAAUGACCUCUUUCACCATT-3', and its antisense strand is SEQ ID NO:47: 5'-UGGUGAAAGAGGUCAUUCCTT-3'; or, its sense strand is SEQ ID NO:48: 5'-GGUGAUUACCCAGAGAUAUUU-3', and its antisense strand is SEQ ID NO:49:5'-AUAUCUCUGGGUAAUCACCU-3'; Preferably, the base modifications in the CD47 are as follows: a thiolation modification is made between two adjacent bases in the 2nd to 4th bases from the 5' end of the sense strand, a thiolation modification is made between two adjacent bases in the 1st to 3rd bases from the 3' end of the sense strand, a thiolation modification is made between two adjacent bases in the 1st to 2nd bases from the 5' end of the antisense strand, and a thiolation modification is made between two adjacent bases in the 1st to 3rd bases from the 3' end of the antisense strand; EGFR: Its sense strand is SEQ ID NO:50: 5'-GGCUGGUUAUGUCCUCAUUUU-3', and its antisense strand is SEQ ID NO:51: 5'-AAUGAGGACAUAACCAGCCUU-3'; or, its sense strand is SEQ ID NO:52: 5'-UUAGAUAAGACUGCUAAGGUU-3', and its antisense strand is SEQ ID NO:53: 5'-UUAGAUAAGACUGCUAAGGUU-3'; or, its sense strand is SEQ ID NO:54: 5'-UGCCUUAGCAGUCUUAUCUAAUU-3', and its antisense strand is SEQ ID NO:55: 5'-UUAGAUAAGACUGCUAAGGCAUU-3'; or, its sense strand is SEQ ID NO:78: 5'-UGCCUUAGCAGUCUUAUCUAAUUUU-3', and its antisense strand is SEQ ID NO:79:5'-AAUUAGAUAAAGACUGCUAAGGCAUU-3'; HBV: its sense strand is: SEQ ID NO:56: 5'-GGACUUCUCUCAAUUUUCUUU-3', and its antisense strand is: SEQ ID NO:57: 5'-AGAAAAUUGAGAGAAGUCCUU-3'; preferably, C and U in the HBV are modified to 2'-F; HSP: its sense strand is SEQ ID NO:58: 5'-CGCAGAACACCGUGUUCGAUU-3', and its antisense strand is SEQ ID NO:59: 5'-UCGAACACGGUGUUCUGCGUU-3'; preferably, in the HSP, the thiocyanate modification is between two adjacent bases in the first to third bases from the 5' end of the sense strand, and the thiocyanate modification is between two adjacent bases in the first to third bases from the 3' end of the antisense strand; HS70: Its sense strand is SEQ ID NO:60: 5'-GGCCAACAAGAUCACCAUCUU-3', and its antisense strand is SEQ ID NO:61: 5'-GAUGGUGAUCUUGUUGGCCUU-3'; preferably, in the HS70, the antisense strand has a thiolation modification between two adjacent bases in the first to third bases from the 5' end, and has a thiolation modification between two adjacent bases in the first to third bases from the 3' end; PD-L1: its sense strand is SEQ ID NO:62: 5'-CCAGCACACUGAGAAUCAAUU-3', and its antisense strand is SEQ ID NO:63: 5'-UUGAUUCUCAGUGUGCUGGUU-3'; or, its sense strand is SEQ ID NO:64: 5'-AGACGUAAGCAGUGUUGAATT-3', and its antisense strand is SEQ ID NO:65: 5'-UUCAACACUGCUUACGUCUTT-3'; PAPP-1: its sense strand is SEQ ID NO:66: 5'-GAGGAAGGUAUCAACAAAUTT-3', and its antisense strand is SEQ ID NO:67: 5'-AUUUGUUGAUACCUUCCUCTT-3'; Survivin: The sense strand is SEQ ID NO:70: 5'-GCAGGUUCCUUAUCUGUCACAUU-3', and the antisense strand is SEQ ID NO:71: 5'-UGUGACAGAUAAGGAACCUGCAGUU-3'; or, The sense strand is SEQ ID NO:72: 5'-GGAAUUGGAAGGCUGGGAACCUU-3', and the antisense strand is SEQ ID NO:73: 5'-GGUUCCCAGCCUUCCAAUUCCUU-3'; preferably, the sense strand 5' The thiolation between two adjacent bases from the 1st to 3rd bases from the end is modified, and the thiolation between two adjacent bases from the 1st to 3rd bases from the 5' end of the antisense strand is modified, and the thiolation between the 1st to 2nd bases from the 3' end is modified; or, The sense strand is: SEQ ID NO:74: 5'-UGCAGGUUCCUUAUCUGUCATT-3', and the antisense strand is: SEQ ID NO:75: 5'-UGACAGAUAAGGAACCUGCTT-3'; or, The sense strand is: SEQ ID NO:76: 5'-CUGCAGGUUCCUUAUCUGUCACAUU-3', and the antisense strand is: SEQ ID NO:77: 5'-UGUGACAGAUAAGGAACCUGCAGUU-3'; preferably, the sense strand has a thiolation modification between two adjacent bases from the 1st to 3rd bases from the 5' end, and a thiolation modification between two adjacent bases from the 1st to 3rd bases from the 3' end, and the antisense strand has a thiolation modification between two adjacent bases from the 1st to 3rd bases from the 5' end, and a thiolation modification between the 1st to 3rd bases from the 3' end; TAP: The sense strand is SEQ ID NO:80: 5'-GCUGCACACGGUUCAGAAUUU-3', and the antisense strand is SEQ ID NO:81: 5'-AUUCUGAACCGUGUGCAGCUU-3'; preferably, the sense strand is thiolated between two adjacent bases in the first to third bases from the 5' end, and the antisense strand is thiolated between two adjacent bases in the first to third bases from the 5' end, and is thiolated between two adjacent bases in the first to third bases from the 3' end; or, The sense strand thereof is SEQ ID NO:82: 5'-CAGGAUGAGUUACUUGAAAUU-3', and the antisense strand is SEQ ID NO:83: 5'-UUUCAAGUAACUCAUCCUGUU-3'; TIM-3: its sense strand is SEQ ID NO:86: 5'-GUGCUCAGGACUGAUGAAATT-3', and its antisense strand is SEQ ID NO:87: 5'-UUUCAUCAGUCCUGAGCACTT-3'; TGF-β1: the sense strand thereof is SEQ ID NO:88: 5'-GUCAACUGUGGAGCAACACUU-3', and the antisense strand thereof is SEQ ID NO:89: 5'-GUGUUGCUCCACAGUUGACUU-3'; or the sense strand thereof is SEQ ID NO:90: ​​5'-GCAACAACGCCAUCUAUGATT-3', and the antisense strand thereof is SEQ ID NO:91: 5'-UCAUAGAUGGCGUUGUUGCTT-3'; VEGF-C: The sense strand is SEQ ID NO:92: 5'-GCAAGACGUUGUUUGAAAUUAUU-3', and the antisense strand is SEQ ID NO:93: 5'-UAAUUUCAAACAACGUCUUGCUU-3'; preferably, the sense strand is thiolated between two adjacent bases from the 1st to 3rd bases at the 5' end, and the antisense strand is thiolated between two adjacent bases from the 1st to 3rd bases at the 5' end, and is thiolated between two adjacent bases from the 1st to 3rd bases at the 3' end; or, The sense strand thereof is SEQ ID NO:84: 5'-CAGCAAGACGUUGUUUGAAAUUAUU-3', and the antisense strand is SEQ ID NO:85: 5'-UAAUUUCAAACAACGUCUUGCUGUU-3'; or, The sense strand is SEQ ID NO:94: 5'-CAGGAUGGUAAAGACUACAUU-3', and the antisense strand is SEQ ID NO:95: 5'-UGUAGUCUUUACCAUCCUGUU-3'; preferably, the 5' end of the sense strand is The thiolation between two adjacent bases in the first to third bases is modified, and the thiolation between two adjacent bases in the first to third bases from the 5' end of the antisense strand is modified, and the thiolation between two adjacent bases in the first to third bases from the 3' end is modified; Preferably, the miRNA includes one or more of miR-34, miR-542, miR-126-3p and miR-122; Preferably, the ASO includes one or more of A-miR21, A-miR-10a, A-miR-30c and A-miR-1306; The sequence structure of each miRNA and ASO from 5' to 3' end is as follows: A-miR21: 5'-GATAAGCT-3'; preferably, each base thereof is modified with a locked nucleic acid; or, 5'-GAUAAGCU-3'; preferably, each base thereof is modified with a locked nucleic acid; or, SEQ ID NO:96: 5'-GTCAACATCAGTCTGATAAGCTA-3'; or, SEQ ID NO:97: 5'-GUCAACAUCAGUCUGAUAAGCUA-3'; or, SEQ ID NO:98: 5'-TCAACATCAGTCTGATAAGCTA; or, 5'-GATAAGCT-3'; preferably, thiolation modification is performed between two adjacent bases; A-miR-10a: 5'-ACAGGGTA-3'; preferably, each base thereof is modified with a locked nucleic acid; A-miR-30c: SEQ ID NO:99: 5'-GCTGAGAGTGTAGGATGTTTACA-3'; miR-34: 5′-TGTGACAG-3′; miR-542: 5′-TGGCAGTGT-3′; miR-126-3p: 5'-UCGUACC-3'; preferably, the adjacent bases are thiolated; miR-122: 5′-GGAAGTGT-3; AmiR-1306: SEQ ID NO: 100: 5'-CATCACCACCAGAGCCAACGTC-3'; preferably, the 5' end of the 5'-base sequence has a thiolation modification between two adjacent bases; The sequence structure of each of the nucleic acid aptamers from the 5' end to the 3' end is as follows: A1: SEQ ID NO: 101: 5'-GGTTGCATGCCGTGGGGAGGGGGGTGGGTTTTATAGCGTACTCAG-3'; A15: SEQ ID NO: 102: 5'-CCCTCCTACATAGGG-3'; or, SEQ ID NO: 227: 5'-CCCUCCUACAUAGGG-3'; AS1411: SEQ ID NO:103: 5'-GGTGGTGGTGGTTGTGGTGGTGGTGG-3'; or, SEQ ID NO:228: 5'-GGUGGUGGUGGUUGUGGUGGUGGUGG-3'; or, SEQ ID NO:229: AUUCUGAACCGUGUGCAGCACCACGCUGCACACGGUUCAGAAUACACACGAGGCTATCTAGAATGTAC-3'; AFP: SEQ ID NO: 104: 5'-GGCAGGAAGACAAACAAGCTTGGCGGCGGGAAGGTGTTTAAATTCCCGGGTCTGCGTGGTCTGTGGTGCTGT-3'; or, SEQ ID NO: 105: 5'-ACCTGGGGAGTATTGGGGAGGAAGG-3'; ATP: SEQ ID NO: 106: 5'-ACCTGGGGAGTATTGGGGAGGAAGG-3'; or, SEQ ID NO: 107: 5'-GGGAGGACGATGCGGAGGAAGGGTAGG-3', preferably, the 5' end of the nucleotide sequence is thiolated between two adjacent bases in the first to fourth bases; Act-12c: SEQ ID NO: 108: 5'-CGGGGAAAGTCACGGGGGGTTTCAGATGTTCTGATCGGTGTGGAG-3'; A18: SEQ ID NO:109: 5'-CCAGATAGTCCCTGG-3'; BAF7-1: SEQ ID NO: 110: 5'-GATAACGGGCACGAATTCGGAGTG-3'; C-Met-SL1: SEQ ID NO: 111: 5'-ATCAGGCTGGATGGTAGCTCGGTCGGGGTGGGTGGGTTGGCAAGTCTGAT-3'; CH6: SEQ ID NO: 112: 5'-AGTCTGTTGGACCGAATCCCGTGGACGCACCCTTTGGACG-3'; CA2: SEQ ID NO: 113: 5'-CCCACCGTCTGCGCTTAGCTCCTGGGCCTGGATGGGC-3'; C12: SEQ ID NO: 114: 5'-GTGGATTGTTGTGTTCTGTTGGTTTTTGTGTTGTC-3'; preferably, the 5' end of the 1st to 3rd bases are thiolated between two adjacent bases; CRAC Orail: SEQ ID NO:115: 5’-CCAGTAGCCATACCGGTTTGTGGATGGGGTGTATGCGAGT-3’; CEA: SEQ ID NO:116: 5’-CTAGGATCCCCACTCACCATCTCTCAGCTTGCTTCCTAGC-3’; or, SEQ ID NO:234: 5’-CUAGGAUCCCCACUCACCAUCUCUCAGCUUGCUUCCUAGC-3’; CEA-18: SEQ ID NO:117: 5’-TTAACTTATTCGACCATA-3’; CEA-T84: SEQ ID NO:118: 5’-TCGCGCGAGTCGTCTGGGGAACCATCGAGTTACACCGACCTTCTATGTGCGGCCC CCCGCATCGTCCTCCC-3’; CSC1: SEQ ID NO:119: 5’-ACCTTGGCTGTCGTGTTGTAGGTGGTTTGCTGCGGTGGGCTCAAGAAGAAAGCGCAAAGAGGTCAGTGGTCAGAGCGT-3’; CSC13: SEQ ID NO:120: 5’-ACCTTGGCTGTCGTGTTGTGGGGTGTCGTATCTTTCGTGTCTTATTATTTTCTAGGT GGAGGTCAGTGGTCAGAGCGT-3’; CD40: SEQ ID NO:121: 5’-CCAACGAGTAGGCGATAGCGCGTGG-3’; or, SEQ ID NO:122: 5’-AGAGACGATGCGGCCAACGAGTAGGCGATAGCGCGTGGCAGAGCGTCGCT-3’; CD16a: SEQ ID NO:123: 5’-CCACTGCGGGGGTCTATACGTGAGGAAGAAGTGG-3’; CD19: SEQ ID NO:124: 5’-TGCGTGTGTAGTGTGTGTCGTTTCTCCTTTTTTTGGTTGCTGCTCTTAGGGATTTGGGCGG-3’; or, SEQ ID NO:125: 5’-TGCGTGTGTAGTGTGTCTGTTCTCCTTTTTTTGGTTGCTGCTCTTAGGGATTTGGGCGG-3’; CD3-4: SEQ ID NO: 126: 5'-TCTCGGACGCGTGTGGTCGGCCGAGTGGCCCACGGTAGAAGGGTTAGAACTGCTGGTTGGTGAATCTCGCTGCCTGGCCCTAGAGTG-3'; CD44: SEQ ID NO: 127: 5'-CCAAGGCCTGCAAGGGAACCAAGGACACAG-3'; or, SEQ ID NO:128: 5'-CCAAGGCCTGCAAGGGAACCAAGGACACAG-3'; preferably, the 5' end is thiolated between two adjacent bases among the 3rd to 5th bases, the 5' end is thiolated between two adjacent bases among the 12th to 14th bases, the 3' end is thiolated between the 1st to 2nd bases, the 3' end is thiolated between the 3rd to 4th bases, the 3' end is thiolated between the 5th to 6th bases, and the 3' end is thiolated between two adjacent bases among the 12th to 14th bases; or, SEQ ID NO: 237: 5'-GGGAUGGAUCCAAGCUUACUGGCAUCUGGAUUUGCGGCGUGCCAGAAUAAAGAGUAUAACGUGUGAAUGGGAAGCUUCGAUAGGAAUUCGG-3'; CD12 (HDLBP): SEQ ID NO: 129: 5'-GTGGATTGTTGTGTTCTGTTGGTTTTTGTGTTGTC-3'; CD20: SEQ ID NO: 130: 5'-TGCGTGTGTAGTGTGTCTGTTTTTTATCTTCTTTTATCTACTCTTAGGGATTTGGGCGG-3'; CD24: SEQ ID NO: 131: 5'-TATGTGGGTGGGTGGGCGGTTATGCTGAGTCAGCCTTGCT-3'; or, SEQ ID NO: 132: 5'-ATCCAGAGTGACGCAGCATATGTGGGTGGGTGGGCGGTTATGCTGAGTCAGCCTTGCTTGGACACGGTGGCTTAGT-3'; CD33: SEQ ID NO: 133: 5'-TACCAGTGCGATGCTCAGCACGCTTATAGGGGCTGGACAAAATTCTACCCAGCCTTT-3'; CD38: SEQ ID NO: 134: 5'-TACGTGAATCTCGTACGATACTCTGTAAGCGT-3'; CD105: SEQ ID NO: 135: 5'-GATCAGTTTTCCATGCCAGTTGGTATTCCGCGACAGTTTGATCTC-3'; CD117: SEQ ID NO: 136: 5'-GGGGCCGGGGCAAGGGGGGGGTACCGTGGTAGGAC-3'; CD63: SEQ ID NO: 137: 5'-CACCCCACCTCGCTCCCGTGACACTAATGCTA-3'; CD123: SEQ ID NO: 138: 5'-TGCGTGTGTAGTGTGTCTGGGCTACATCGATGAGCTGCCTAGGGTCCCTCTTAGGGATTTGGGCGG-3'; EGFR: SEQ ID NO: 139: 5'-GCCTTAGTAACGTGCTTTGATGTCGATTCGACAGGAGGC-3'; or, SEQ ID NO: 239: 5'-GCCUUAGUAACGUGCUUUGAUGUCGAUUCGACAGGAGGC-3'; or, SEQ ID NO:240:5'-UGCCGCUAUAAUGCACGGAUUUAAUCGCCGUAGAAAAGCAUGUCAAAGCCGUU-3'; EpCAM: SEQ ID NO: 140: 5'-CACTACAGAGGTTGCGTCTGTCCCACGTTGTCATGGGGGGTTGGCCTG-3'; or, SEQ ID NO: 141: 5'-GACAAACGGGGGAAGATTTGACGTCGACGAC-3'; or, SEQ ID NO: 241: 5'-GCGACUGGUUACCCGGUCG-3'; EcR: SEQ ID NO: 142: 5'-GCAGGTCCACTGCGGGGGTCTATACGTGAGGAAGAAGTGGGCAGGTC-3'; FAP: SEQ ID NO: 143: 5'-TGGGGGTTGAGGCTAAGCCGA-3'; Anti-FAP: SEQ ID NO: 144: 5'-CCGCTCGAGCTAGTCTGACAAAGAGAAACAC-3'; GPC-1: SEQ ID NO: 145: 5'-AACGGAGTGTGGCTAACTCGA-3'; GSK836: SEQ ID NO: 147: 5'-GCAGAGGTGAAGCGAAGTCG-3'; preferably, the 5' end of the nucleotide sequence of bases 1 to 6 is thiolated between two adjacent bases; GPC3(APS63-1):SEQ ID NO:148: 5'-TAACGCTGACCTTAGCTGCATGGCTTTACATGTTCCA-3'; preferably, the 5' end and the 3' end are thiolated between two adjacent bases in the 1st to 4th bases; Her2: SEQ ID NO: 149: 5'-AGCCGCGAGGGGAGGGATAGGGTAGGGCGCGGCT-3'; or, SEQ ID NO: 150: 5'-GGGAGCTCAGAATAAACGCTCAAAGGGTCAAGCTGATTACACTTTGTCCACTATTGGGTCCTTCGACATGAGGCCCGGATC-3'; or, SEQ ID NO: 246: 5'-AGCCGCGAGGGGAGGGAUAGGGUAGGGCGCGGCU-3'; Her3: SEQ ID NO: 151: 5'-GGGAGCTCAGAATAAACGCTCAAGGCTAACAGCACGCAACGGGGGGGAGTAATCGTGTCTGTTCGACATGAGGCCCGGATC-3'; or, SEQ ID NO: 247: 5'-CAGCGAAAGUUGCGUAUGGGUCACAUCGCAGGCACAUGUCAUCUGGGCG-3'; or, SEQ ID NO: 248: 5'-GAAUUCCGCGUGUGCCAGCGAAAGUUGCGUAUGGGCCACAUCGCAGGCACAUGUCAUCUGGGCGGUCCGUUCGGGAUCC-3'; HMGA2: SEQ ID NO: 152: 5'-GGAAAAAATTTTTTAAAAAACCC-3'; preferably, the bases between two adjacent bases are thiolated; H2: SEQ ID NO: 146: 5'-GGGCCGTCGAACACGAGCATGGTGCGTGGACCTAGGATGACCTGAGTACTGTCC-3'; HBsAg: SEQ ID NO: 153: 5'-CACAGCGAACAGCGGCGGACATAATAGTGCTTACTACGAC-3'; preferably, the 5' end of the 1st to 4th bases are thiolated between two adjacent bases; IFN-y(B4): SEQ ID NO:154: 5'-CCGCCCAAATCCCTAAGAGAAGACTGTAATGACATCAAACCAGACACACACACTACACACGCA-3'; IL-4Ra: SEQ ID NO:155: 5’-GGAGGACGAUGCGGAAAAAGCAACAGGGUGCUCCAUGCGCAUGGAACCUGCGCGCAGACGACUCGCUGAGGAUCCGAGA-3’; or, SEQ ID NO:156: 5’-AAAAAGCAACAGGGUGCUCCAUGCGCAUGGAACCUGCGCG-3’; IL-17: SEQ ID NO:157: 5’-CTTGGATCACCATAGTCGCTAGTCGAGGCT-3’; or, SEQ ID NO:158: 5’-GCGGCATCCTATCACGCATTGACC-3’; LZH8: SEQ ID NO:159: 5’-ATCCAGAGTGACGCAGCATATTAGTACGGCTTAACCCCATGGTGGACACGGTGGCTTAGT-3’; MUC1: SEQ ID NO:160: 5’-GCAGTTGATCCTTTGGATACCCTGG-3’; or, SEQ ID NO:161: 5’-GAAGTGAAAATGACAGAACACAACA-3’; or, SEQ ID NO:162: 5’-AACCGCCCAAATCTCTAAGAGTCGGACTGCAACCTATGCTATCGTTGATGTCTGTCCAAGCAACACAGACACACTACACACACGCACA-3’; or, SEQ ID NO:163: 5’-AATGACAGAACACAACATT-3’; or, SEQ ID NO:250: 5’-GCAGUUGAUCCUUUGGAUACCCUGG-3’; M5: SEQ ID NO:164: 5’-AGCAGCACAGAGGTCAGATGCTTGGTTCCACCGTACTGACTGTAGTAAAATCTGATCACTCCTATGCGTGCTACCGTGAA-3’; M7: SEQ ID NO:165: 5’-AGCAGCACAGAGGTCAGATGTAGTCGGTCTTCTTGTTTGAAACTGCTAATTTTGAAAAAACCTATGCGTGCTACCGTGAA-3’; M1: SEQ ID NO:166: 5’-AGCAGCACAGAGGTCAGATGATATAACCTTAATAAATAAAATATAAATTATTTAATCTTACCTATGCGTGCTACCGTGAA-3’; N5:SEQ ID NO:167:5’-GATTGAGTAGATAGTGGTTCTGTACGTAGTGAAAGAGTGG-3’; N-G-Dua:SEQ ID NO:168:5’-CAAGTTGCTCGTCGCGATACGTTTGGTTGGTGTGGTTGGCAGTATCGCAGGTCCAAGTTGCTCGTCGCGATACAACGGAGTGTGGCTAACTCGA-3’; NKG2D(20-N-15):SEQ ID NO:169:5’-CAAGTTGCTCGTCGCGATACGTTTGGTTGGTGTGGTTGGCAGTATC-3’; NSE:SEQ ID NO:170:5’-TCACACGGACCTCTCTCTACATTAATTGCGCATTTCGTT-3’; Np-A15:SEQ ID NO:171:5’-GCTGGATGTTCATGCTGGCAAAATTCCTTAGGGGCACCGTTACTTTGACACATCCAGC-3’; Np-A48:SEQ ID NO:172:5’-GCTGGATGTCGCTTACGACAATATTCCTTAGGGGCACCGCTACATTGACACATCCAGC-3’; Np-A58:SEQ ID NO:173:5’-GCTGGATGTCACCGGATTGTCGGACATCGGATTGTCTGAGTCATATGACACATCCAGC-3’; Np-A61:SEQ ID NO:174: 5’-GCTGGATGTTGACCTTTACAGATCGGATTCTGTGGGGCGTTAAACTGACACATCCAGC-3’; OX40: SEQ ID NO:175: 5’-GGGAGGACGATGCGGCAGTCTGCATCGTAGGAATCGCCACCGTATACTTTCCCACCAGACGACTCGCTGAGGATCCGAGA-3’; or, SEQ ID NO:176: 5’-CAGTCTGCATCGTAGGATTAGCCACCGUATCTTTCCCAC-3’; or, SEQ ID NO:177: 5’-CCAACGAGTAGGCGATAGCGCGTGG-3’; or, SEQ ID NO:252: 5’-GGGAGGACGAUGCGGCAGUCUGCAUCGUAGGAAUCGCCACCGUAUACUUUCCCACCAGACGACUCGCUGAGGAUCCGAGA-3’; or, SEQ ID NO:253: 5’-GGGAGGACGAUGCGGCAGUCUGCAUCGUAGGAAUCGCCACCGUAUACUUUCCCACCAGACGACUCGCUG-3’; or, SEQ ID NO:254: 5’-CAGUCUGCAUCGUAGGAAUCGCCACCGUAUACUUUCCCAC-3’; or, SEQ ID NO:255: 5’-GGGAUGCGGAAAAAAGAACACUUCCGAUUAGGGCCCACCCUAACGGCCGCAGAC-3’; PSMA: SEQ ID NO:179: 5’-GGGAGGACGATGCGGATCAGCCATGTTTACGTCACTCCT-3’; or, SEQ ID NO:180: 5’-GCGTTTTCGCTTTTGCGTTTTGGGTCATCTGCTTACGATAGCAATGCT-3’; or, SEQ ID NO:256: 5’-GGGACCGAAAAAGACCUGACUUCUAUACUAAGUCUACGUUCCC-3’; or, SEQ ID NO:257: 5’-GGGAGGACGAUGCGGAUCAGCCAUGUUUACGUCACUCCU-3’; PDGFRβ: SEQ ID NO:181: 5’-TGTCGTGGGGCATCGAGTAAATGCAATTCGACA-3’; or, SEQ ID NO:258: 5’-UGUCGUGGGGCAUCGAGUAAAUGCAAUUCGACA-3’; PDGF: SEQ ID NO:182: 5’-CAGGCTACGGCACGTAGAGCATCACCATGATCCTG-3’; PD-L1: SEQ ID NO:183: 5’-AACAACGTATAACAATGCCCACGTCACCAGAGTACTATGG-3’; or, SEQ ID NO:184: 5’-GCCCCAGTTATGCTTTCCCCCTCTGTCTCTTTG-3’; or, SEQ ID NO:185: 5’-ATCGCCCGCAGCACCCATTTGTTTTTTTTTG-3’; or, SEQ ID NO:69: ACGGGCCACATCAACTCATTGATAGACAATGCGTCCACTGCCCGT; or, SEQ ID NO:186: 5’-TGCCCGCACATCAACTCATTGATAGACAATGCGTCCACTCGGGCA-3’; or, SEQ ID NO:187: 5’-TGCCCGCACATCAACTCATTGATAGACAATGCGTCCACTACGGGC-3’; or, SEQ ID NO:188: 5’-CGGGCACACATCAACTCATTGATAGACAATGCGTCCACTGCCCGT-3’; or, SEQ ID NO:189: 5’-GTTGGTCACATCAACTCATTGATAGACAATGCGTCCACTACCAAC-3’; or, SEQ ID NO:190: 5’-GGGCCACATCAACTCATTGATAGACAATGCGTCCACTGCCC-3’; or, SEQ ID NO:191: 5’-TGGTTGCACATCAACTCATTGATAGACAATGCGTCCACTCAACCA-3’; or, SEQ ID NO:200: 5’-TACAGGTTCTGGGGGGTGGGTGGGGAACCTGTT-3’; or, SEQ ID NO:238: 5’-CTACGAGACGAACTTATGCGTAATAATGACTGTCGTAG-3’; PD-1: SEQ ID NO:192: 5’-GACGATAGCGGTGACGGCACAGACGGTACAGTTCCCGTCCCTGCACTACACGTATGCCGCTTCCGTCCGTCGCTC-3’; or, SEQ ID NO:193: 5’-GAGCGACGGACGGAAGCGGCATACGTGTAGTGCAGGGACGGGAACTGTACCGTCTGTGCCGTCACCGCTATCGTC-3’; or, SEQ ID NO:194: 5’-GGATCCTAGACGCATTGACCCGCTGCCTCTACTGAGGCTGTGTCAGTGTGCGGCTCGGACTGTTGAATTC-3’; or, SEQ ID NO:195: 5’-AGCGGTGACGGCACAGACGGTACAGTTCCCGTCCCTGCACTACACGTATGCCGCTGAGAGAGAGGGAGGC-3’; or, SEQ ID NO:196: 5’-ACCGACAGTGAAGGACTCAGCGAACTCTCAGACTCGGTTC-3’; PTK-7: SEQ ID NO:197: 5’-ATCTAACTGCTGCGCCGCCGGGAAAATACTGTACGGTTAGA-3’; ProGRP-48: SEQ ID NO:198: 5’-CATGCGGAGTAGAGCGAGCCCAGATAGTCCCTGGTTATTTCCTTAGG-3’; SF: SEQ ID NO:199: 5’-GATCTCTCTCTGCCCTAAGTCCGCACCCGTGCTTCCCTGT-3’; TBA15: SEQ ID NO:201: 5’-GGTTGGTGTGGTTGG-3’; TBA29: SEQ ID NO:202: 5’-AGTCCGTGGTAGGGCAGGTTGGGGTGACT-3’; TfRA4: SEQ ID NO:203: 5’-GCGTGGTACCACGC-3’; or, SEQ ID NO:259: 5’-GCGUGGUACCACGC-3’; TfRA3:SEQ ID NO:204:5'-GCGTGGTCACACGC-3':seq ID NO:205:5'-GCGGCGCCCACGAGCGTTCGCGTGGTCACACGCGTTCCGCCCTCCTACATAGGGCGCATAGCCGTGGGCGCGC-3',SEQ ID NO:260:5'-GCGUGGUCACACGC-3' TTA1:SEQ ID NO:206:5′-CCTGCACTTGGCTTGGATTTCAGAAGGGAGACCC-3′,split SEQ ID NO:286,5′-CTGCACTTGGCTTGGATTTCAGAAGGGAGACCC-3′,spring SEQ ID NO:261: 5'-CCUGCACUUGGCTTGGAUUUCAAGGGAGACCC-3' TLS9a:SEQ ID NO:207:5'-AGTCCATTTTATTCCTGAATATTTGTTAACCTCATGGAC-3'; TGF-βII(S58):SEQ ID NO:208:5'-ACATTGCTGCGTGATCGCTCACATGGGTTTGTCTGGTCGATTTGGAGGTGGTGGGTGGC-3'; TNF-a:SEQ ID NO:209:5'-GCGGCCGATAAGGTCTTTCCAAGCGAACGAAAA-3' TNF:SEQ ID NO:210:5′-GCGCCACTACAGGGGAGCTGCCATTCGAATAGGTGGGCCGC-3′; T1:SEQ ID NO:211:5'-CGCTCGATAGATCGAGCTTCGCTCGATGTGGTGTTGTGGGGGCTTGTATTGGTCGATCACGCTCTAGAGCACTG-3' VEGF:SEQ ID NO:212:5′-TGTGGGGGTGGACTGGGTGGGGTACC-3′,spring,SEQ ID NO:213:5′-TGTGGGGGTGGACGGGCCGGGTAGA-3′,SEQ ID NO:214:5′-GGTGGGGGTGGACGGGCCGGGTAGA-3′,FRENCH,SEQ ID NO:266:5'-AUGCAGUUUGAGAAGUCGCGCAU-3' and a 5' fragment of 6 / 7 fragment. VCAM-1: SEQ ID NO:215: 5’-ATACCAGCTTATTCAATTGGACACGGCAAAGGGGTATAGCCTACCGGACCGTGAACATGGAATGGTGTGCTGCGTGGAGATAGTAAGTGCAATCT-3’; or, SEQ ID NO:216: 5’-GGACACGGCAAAGGGGTATAGCCTACCGGACCGTGAACATGGAATGGTGTGCTGCGTGG-3’; VCAM-1 2d: SEQ ID NO:217: 5’-AGGGAATCTTGCCTAGGGAGGGAGTAGCGAAAGGGCTCA-3’; CH6: SEQ ID NO:218: 5’-AGTCTGTTGGACCGAATCCCGTGGACGCACCCTTTGGACG-3’; PL-45: SEQ ID NO:219: 5’-ACTCATAGGGTTAGGGGCTGCTGGCCAGATACTCAGATGGTAGGGTTACTATGAGC-3’; EP66: SEQ ID NO:220: 5’-AACAGAGGGACAAACGGGGGAAGATTTGACGTCGACGACA-3’; AGC: SEQ ID NO:221: 5’-CGACCCGGCACAAACCCAGAACCATATACACGATCATTCGTCTCCTGGGCCG-3’; Karpas299: SEQ ID NO:222: 5’-ATCCAGATGACGCAGCACCACCACCGTACAATTTTTTCATTACCTACTCGGC-3’; SW620: SEQ ID NO:223: 5’-CCCATCAATGTTACGACCCGCTAGGGCTGCTGTGCCATCGGGTAA-3’; MDA-MB-231: SEQ ID NO:224: 5’-AGAATTCAGTCGGACAGCGAAGTAGTTTTCCTTCTAACCTAAGAACCCGCGGCAGTTTAATGTAGA-3’; MCF-7: SEQ ID NO:225: 5’-GCATGGGGTTTCGGCGTTTCGTCTATCTTGTTTCTGTTAGCGTCT-3’; PC-3: SEQ ID NO:226: 5’-TGCCACTACAGCTGGTTCGGTTTGGTGACTTCGTTCTTCGTTGTGGTGCTTAGTGGC-3’; BCMA: SEQ ID NO:230: 5’-AGUGCAAGACGUUCGCAGAUUAGCGAAAAGAGGGUCUCAUUGACUAGUAC-3’; CTLA-4: SEQ ID NO:231: 5’-GGUGGAAGAGGGAUGGGCCGACGUGCCGCAU-3’; or, SEQ ID NO:232: 5’-TCCCTACGGCGCTAACGATGGTGAAAATGGGCCTAGGGTGGACGGTGCCACCGT GCTACAAC-3’; CCL1: SEQ ID NO:233: 5’-UGACUCCUCUGACAGCCUAAUUUCUCCCGAUUACCCUG-3’; CD4-3: SEQ ID NO:235: 5’-GGGAGGACGAUGCGGUUUGGGGUUUUCCCGUGCCCCAGACGACUCGCCCGA-3’; CD28: SEQ ID NO:236: 5’-GGGAGAGAGGAAGAGGGAUGGGGAUUAGACCAUAGGCUCCCAACCCCCCCCGGGAGAGAGGAAGAGGGAUGGGGAUUAGACCAUAGGCUCCCAACCCCCGGG-3’; FGF2(F2): SEQ ID NO:242: 5’-GGGAUACUAGGGCAUUAAUGUUACCAGUGUAGUCCC-3’; FGF2: SEQ ID NO:243: 5’-GGGAAACUAGGGCGUUAACGUGACCAGUGUUUCUCGA-3’; or, SEQ ID NO:244: 5’-GGGAAACUAGGGCGUUAACGUGACCAGUGUUUCCC-3’; FGF5: SEQ ID NO:245: 5’-GGGCGACCUCUCCGUACUGACCUACAGAGCGACAUACUAGUGUAUCCAGAUC GCCC-3’; LAG-3: SEQ ID NO:249: 5’-GGGAGAGAGAUAUAAGGGCCUCCUGAUACCCGCUGCUAUCUGGACCGAUCCCAUUACCAAAUUCUCUCCC-3’; MRP1: SEQ ID NO:251: 5’-GGGAGAAUAGUCAACAAAUCGUUUGGGGCGACUUCUCCUUCCUUUCUCCC-3’; TIM3: SEQ ID NO:262: 5'-GGGAGAGGACCAGUA-GCCACUAUGGUGUUGGAGCUAGCGG-CAGAGCGUCGCGGUCCUCCC-3'; or, SEQ ID NO:263: 5'-GGGAGAGGACCAGUA-CUGGUAGUUCUCUGUGCGGACUCCUA-CAGAGCGUCGCGGUCCUCCC-3'; TIMC-11: SEQ ID NO: 264: 5'-AAGCAACACUUAGUCGCGAUUGAUACGUGCGCAGUCAU-3'; or, SEQ ID NO: 265: 5'-GGAGGACGAUGCGGGGAAGCAACACUUAGUCGCGAUUGAUACGUGCGCAGUCAUCAGACGACUCGCUGAGGAUCCGAGA-3'; VEGF165: SEQ ID NO:267: 5'-CGGAAUCAGUGAAUGCUUAUACAUCCG-3'; 4-1BB: SEQ ID NO:268: 5'-GGGAGAGAGGAAGAGGGAUGGGCGACCGAACGUGCCCUUCAAAGCCGUUCACUAACCAGUGGCAUAACCCAGAGGUCGAUAGUACUGGAUCCCCCC-3'; The sequence structure of each of the immunostimulants from the 5' end to the 3' end is as follows: CPG2006: SEQ ID NO:269: 5'-TCGTCGTTTTGTCGTTTTGTCGTT-3'; preferably, a thiolation modification between two adjacent bases; or, SEQ ID NO:270: 5'-UCGUCGUUUUGUCGUUUUGUCGUU-3'; CPG1826: SEQ ID NO: 271: TCCATGACGTTCCTGACG-3'; preferably, the two adjacent bases are thiolated; CPG2216: SEQ ID NO:272: 5'-GGGGGACGATCGTCGGGGGG-3'; CPG2395: SEQ ID NO: 273: TCGTCGTTTTCGGCGCGCGCCG-3'; CPG-ODNT7: SEQ ID NO:274: 5'-TCGTCGTCGTCGTCGTCGTCG-3'; or, SEQ ID NO:275: 5'-TCGTCGTCGTCGTCGTCGTCGTCG-3'; CPG-ODN-PCIF1:5'-AGCGAA-3'.

5. The targeted chemical drug according to claim 3 or 4, characterized in that: When the targeted chemical drug includes the nucleic acid aptamer, the targeting molecule includes the nucleic acid aptamer and / or a small molecule target head; when the targeted chemical drug does not include the nucleic acid aptamer, the targeting molecule is a small molecule target head, wherein the small molecule target head is selected from one or more of folic acid, biotin, vitamin B12 and mannose.

6. The targeted chemical drug according to claim 5, characterized in that: The chemical small molecule drug is selected from anthracycline drugs, pyrimidine drugs, platinum drugs, glutamic acid derivative drugs, flavonoid drugs, botanical drugs and their derivatives, folic acid drugs, salicylic acid drugs or acridine drugs; Preferably, the anthracycline drug is selected from doxorubicin, epirubicin, pirarubicin, daunorubicin, idarubicin, mitoxantrone, valrubicin, or the free base or hydrochloride of the above drugs; Preferably, the pyrimidine drug is selected from gemcitabine, 5-fluorouracil, cytarabine or capecitabine; Preferably, the platinum chemical is selected from cisplatin, oxaliplatin, carboplatin, nedaplatin or lobaplatin; Preferably, the glutamic acid derivative drug is selected from lenalidomide, thalidomide or pomalidomide; Preferably, the flavonoid chemical is selected from flavones, flavonols, dihydroflavones, isoflavones or chalcones; Preferably, the botanical drug and its derivatives are selected from vincristine, dihydroartemisinin, paclitaxel, maytansine, docetaxel or 10-hydroxycamptothecin; Preferably, the folic acid chemical is selected from methotrexate; Preferably, the salicylic acid chemical is selected from aspirin or sodium salicylate; Preferably, the acridine chemical drug is selected from tacrine or amsacrine; More preferably, the chemical small molecule drug is selected from one or more of epirubicin or its free base or its hydrochloride, gemcitabine, 5-fluorouracil, paclitaxel, and maytansine.

7. The targeted chemical drug according to claim 6, characterized in that: When the small molecule target head, the oligonucleotide effector molecule and the immunostimulant are connected to the nucleic acid vector, they are directly connected to the terminal bases of each sequence of the nucleic acid vector, or connected to the terminal bases of each sequence of the nucleic acid vector through a base bridge containing 1 to 10 bases; preferably, When the chemical small molecule drug is the anthracycline drug or the acridine drug, the chemical small molecule drug is inserted between adjacent GC base pairs of the targeted nucleic acid vector in a specific intercalation form; When the chemical small molecule drug is the pyrimidine drug, the small molecule target head and the oligonucleotide effector The molecule and the immunostimulant are connected to the terminal bases of each sequence of the nucleic acid vector via a base connection bridge comprising 1 to 10 bases, and the chemical small molecule drug is connected to the nucleic acid vector in the form of at least partially replacing the bases in the base connection bridge and / or as an extended base and / or a carrier backbone base replacement; When the chemical small molecule drug is the platinum-based drug, the glutamic acid derivative-based drug, the flavonoid-based drug, the botanical drug and its derivatives, the folic acid-based drug or the salicylic acid-based drug, the chemical small molecule drug is covalently linked to the nucleic acid vector via a linker; preferably, the linker is selected from a long-chain primary amine compound, more preferably a long-chain primary amine compound containing C2 to C14, and further preferably 5-bromomethyl-6-bromo-1-hexylamine.

8. The targeted chemical drug according to claim 7, characterized in that: The oligonucleotide effector molecule, the immunostimulant and the small molecule target head are connected to one or more sequences in the nucleic acid vector by chain extension or chain complementary pairing; preferably, the targeted chemical drug is any one of the following: A) The targeted chemical drug comprises the DNA vector, the chemical small molecule drug, and the nucleic acid aptamer EGFR and optional biotin connected to the DNA vector, preferably the chemical small molecule drug is epirubicin or its free base or its hydrochloride; preferably, the DNA vector comprises the first group of sequences, the nucleic acid aptamer EGFR is connected to the 5' end of the b sequence, and the molar ratio of the nucleic acid aptamer EGFR to the DNA vector is 1:1; preferably, the nucleic acid aptamer EGFR is connected to the 5' end of the b sequence through a base bridge TTTTT; when the targeted chemical drug comprises the DNA vector, the chemical small molecule drug, the nucleic acid aptamer EGFR and the biotin connected to the DNA vector, 2 to 4 biotins are connected to each of the DNA vectors, and each biotin is respectively connected to the 5' end or 3' end of the a sequence or the c sequence; or, B) The targeted chemical drug comprises the DNA vector, the chemical small molecule drug, and nucleic acid aptamer AS1411 in DNA form, nucleic acid aptamer EGFR, nucleic acid aptamer A15 in DNA form and optional biotin connected to the DNA vector, preferably the chemical small molecule drug is epirubicin or its free base or its hydrochloride; preferably, the DNA vector comprises the first group of sequences, the molar ratio of the nucleic acid aptamer AS1411 in DNA form to the DNA vector is 1:1, and the nucleic acid aptamer AS1411 is connected to the 5' end of the a sequence; preferably, the nucleic acid aptamer AS1411 is connected to the 5' end of the a sequence via a base bridge TTTTT; the molar ratio of the nucleic acid aptamer EGFR to the DNA vector is 1:1, and the nucleic acid aptamer EGFR is connected to the 5' end of the b sequence; Preferably, the nucleic acid aptamer EGFR is connected to the 5' end of the b sequence through a base bridge TTTTT; the molar ratio of the nucleic acid aptamer A15 in DNA form to the DNA vector is 1:1, and is connected to the 5' end of the c sequence; preferably, the nucleic acid aptamer A15 is connected to the 5' end of the c sequence through a base bridge TTTTT; when the targeted chemical drug includes the DNA vector, the chemical small molecule drug, and the nucleic acid aptamer AS1411 in DNA form, the nucleic acid aptamer EGFR, the nucleic acid aptamer A15 in DNA form and the biotin connected to the DNA vector, each of the DNA vectors is connected to 2 biotins, and each of the biotins is respectively connected to the 3' end of the a sequence and the c sequence; or, C) The targeted chemical drug comprises the DNA vector, the chemical small molecule drug, and the nucleic acid aptamer AS1411 and biotin in the form of DNA connected to the DNA vector, preferably the chemical small molecule drug is epirubicin or its free base or its hydrochloride; preferably, the DNA vector comprises the first group of sequences, the DNA form The nucleic acid aptamer AS1411 of the formula is connected to the 5' end of the b sequence, and the molar ratio of the nucleic acid aptamer AS1411 in the form of DNA to the DNA vector is 1:1; preferably, the nucleic acid aptamer AS1411 is connected to the 5' end of the b sequence through a base bridge TTTTT; 2 to 4 biotins are connected to each of the DNA vectors, and each biotin is respectively connected to the 5' end or 3' end of the a sequence or the c sequence; or, D) The targeted chemical drug includes the DNA vector, the chemical small molecule drug, and the nucleic acid aptamer A1 and the nucleic acid aptamer A15 in DNA form connected to the DNA vector, preferably the chemical small molecule drug is epirubicin or its free base or its hydrochloride; preferably, the DNA vector contains the first group of sequences, the molar ratio of the nucleic acid aptamer A1 to the DNA vector is 1:1, and the aptamer A1 is connected to the 5' end of the b sequence; preferably, the nucleic acid aptamer A1 is connected to the 5' end of the b sequence through a base bridge TTTTT; the molar ratio of the nucleic acid aptamer A15 in DNA form to the DNA vector is 1:1, and the aptamer A15 is connected to the 5' end of the c sequence; preferably, the nucleic acid aptamer A15 is connected to the 5' end of the b sequence through a base bridge TTTTT; or, E) The targeted chemical drug includes the DNA vector, the chemical small molecule drug, and the nucleic acid aptamer AS1411 in DNA form, the nucleic acid aptamer EGFR, the nucleic acid aptamer A15 in DNA form, optional biotin and oligonucleotide A-miR-21 connected to the DNA vector, preferably the chemical small molecule drug is epirubicin or its free base or its hydrochloride; preferably, the DNA vector contains the first group of sequences, the molar ratio of the nucleic acid aptamer AS1411 in DNA form to the DNA vector is 1:1, and is connected to the 5' end of the a sequence; preferably, the nucleic acid aptamer AS1411 is connected to the 5' end of the a sequence via a base bridge TTTTT; the molar ratio of the nucleic acid aptamer EGFR to the DNA vector is 1:

1. The molar ratio of the nucleic acid aptamer A15 to the DNA vector is 1:1, and is connected to the 5' end of the b sequence; preferably, the nucleic acid aptamer EGFR is connected to the 5' end of the b sequence through a base bridge TTTTT; the molar ratio of the nucleic acid aptamer A15 in DNA form to the DNA vector is 1:1, and is connected to the 5' end of the c sequence; preferably, the nucleic acid aptamer A15 is connected to the 5' end of the c sequence through a base bridge TTTTT; the molar ratio of the oligonucleotide A-miR-21 to the DNA vector is 1:1, and is connected to the 3' end of the c sequence; optionally, each of the DNA vectors is connected to 2 biotins, and each of the biotins is respectively connected to the 3' end of the a sequence and the end of the oligonucleotide A-miR-21 away from the c sequence; or, F) The targeted chemical drug comprises the DNA vector, the chemical small molecule drug, and the DNA-based nucleic acid aptamer TfRA4, the DNA-based nucleic acid aptamer AS1411, and the DNA-based nucleic acid aptamer A15 connected to the DNA vector, preferably the chemical small molecule drug is epirubicin or its free base or its hydrochloride; preferably, the DNA vector comprises the first group of sequences, the molar ratio of the DNA-based nucleic acid aptamer TfRA4 to the DNA vector is 1:1, and the nucleic acid aptamer TfRA4 is connected to the 5' end of the a sequence; preferably, the nucleic acid aptamer TfRA4 The nucleic acid aptamer AS1411 is connected to the 5' end of the a sequence via a base bridge TTTTT; the molar ratio of the nucleic acid aptamer AS1411 in DNA form to the DNA vector is 1:1, and is connected to the 5' end of the b sequence; preferably, the nucleic acid aptamer AS1411 is connected to the 5' end of the b sequence via a base bridge TTTTT; the nucleic acid aptamer A15 in DNA form is connected to the 5' end of the c sequence via a base bridge TTTTT; preferably, the nucleic acid aptamer A15 is connected to the 5' end of the c sequence via a base bridge TTTTT; or, G) The targeted chemical drug includes the DNA vector, the chemical small molecule drug, and the DNA-form nucleic acid aptamer TfRA4, the DNA-form nucleic acid aptamer AS1411 and the DNA-form nucleic acid aptamer AS1411 connected to the DNA vector. The nucleic acid aptamer A15, the chemical small molecule drug is epirubicin or its free base or its hydrochloride; the DNA vector comprises the eleventh group of sequences, the molar ratio of the nucleic acid aptamer TfRA4 in DNA form to the DNA vector is 1:1, and is connected to the 5' end of the a sequence; preferably, the nucleic acid aptamer TfRA4 is connected to the 5' end of the a sequence through a transition base segment (transition base segment) SEQ ID NO:276: 5'-TTGCGGCGAGCGGCGA-3', and the targeted chemical drug also includes a complementary sequence (complementary sequence) SEQ ID NO:277: 5'-TCGCCGCTCGCCGCTT-3', which is connected to the transition base segment SEQ ID NO:276: 5'-TTGCGGCGAGCGGCGA-3' is complementary and connected, and its 3' end is connected to the 5' segment of the nucleic acid aptamer TfRA4; the molar ratio of the nucleic acid aptamer AS1411 in DNA form to the DNA vector is 1:1, and it is connected to the 5' end of the b sequence; preferably, the nucleic acid aptamer AS1411 is connected to the 5' end of the b sequence through the base bridge TTTTT; the molar ratio of the nucleic acid aptamer A15 in DNA form to the DNA vector is 1:1, and it is connected to the 5' end of the c sequence; preferably, the nucleic acid aptamer A15 is connected to the 5' end of the c sequence through the base bridge TTTTT; or, H) The targeted chemical drug comprises the DNA vector, the chemical small molecule drug, and the nucleic acid aptamer AS1411 in DNA form, the nucleic acid aptamer TfRA3 in DNA form, and the nucleic acid aptamer A15 in DNA form connected to the DNA vector, preferably the chemical small molecule drug is epirubicin or its free base or its hydrochloride; preferably, the DNA vector comprises the first group of sequences, the nucleic acid aptamer AS1411 in DNA form and the DNA vector have a molar ratio of 1:1, and are connected to the 5' end of the a sequence; preferably, the nucleic acid aptamer AS1411 in DNA form and the nucleic acid aptamer TfRA3 in DNA form and the nucleic acid aptamer A15 in DNA form ... 11 is connected to the 5' end of the a sequence through a base bridge TTTTT; the molar ratio of the DNA-based nucleic acid aptamer TfRA3 to the DNA vector is 1:1, and is connected to the 5' end of the b sequence; preferably, the nucleic acid aptamer TfRA3 is connected to the 5' end of the b sequence through a base bridge TTTTT; the molar ratio of the DNA-based nucleic acid aptamer A15 to the DNA vector is 1:1, and is connected to the 5' end of the c sequence; preferably, the nucleic acid aptamer A15 is connected to the 5' end of the b sequence through a base bridge TTTTT; or, I) The targeted chemical drug includes the DNA vector, the chemical small molecule drug, and the immunostimulant CPG2006, the nucleic acid aptamer CD40 and the nucleic acid aptamer PD-L1 in DNA form connected to the DNA vector, preferably the chemical small molecule drug is epirubicin or its free base or its hydrochloride; preferably, the DNA vector comprises the ninth group of sequences, the molar ratio of the immunostimulant CPG2006 to the DNA vector is 1:1, and they are connected to the 5' end of the a sequence; preferably, the immunostimulant CPG2006 is connected to the 5' end of the a sequence through a base The connecting bridge TTTTT is connected to the 5' end of the a sequence; the molar ratio of the nucleic acid aptamer CD40 to the DNA vector is 1:1, and the nucleic acid aptamer CD40 is connected to the 5' end of the b sequence; preferably, the nucleic acid aptamer CD40 is connected to the 5' end of the b sequence through the base connecting bridge TTTTT; the DNA form of the nucleic acid aptamer PD-L1 and the DNA vector have a molar ratio of 1:1, and are connected to the 5' end of the c sequence; preferably, the nucleic acid aptamer PD-L1 is connected to the 5' end of the c sequence through the base connecting bridge TTTTTT; or, J) The targeted chemical drug includes the DNA vector, the chemical small molecule drug, and the immunostimulant CPG2006, nucleic acid aptamer CD40, nucleic acid aptamer PD-L1 in DNA form, and nucleic acid aptamer C12 connected to the DNA vector; wherein the DNA vector also includes a d sequence (d sequence) and an e sequence (e sequence) as complementary sequences, the d sequence is SEQ ID NO: 278: 5'-GCCACCGTGCTACA-3', the e sequence is SEQ ID NO: 279: 5'-CAGCAGCAGCAGCA-3'; the 3' of the b sequence in the DNA vector The end of the DNA vector is connected with a transition base segment SEQ ID NO:280: 5'-GTAGCACGGTGGC-3', the d sequence is complementary to the transition base segment SEQ ID NO:280: 5'-GTAGCACGGTGGC-3' by strand complementation, and the e sequence is complementary to the c sequence of the DNA vector by strand complementation; preferably, the chemical small molecule drug is epirubicin or its free base or its hydrochloride; preferably, the DNA vector comprises the tenth group of sequences, the molar ratio of the immunostimulant CPG2006 to the DNA vector is 2:1, and they are connected to the 5' end of the a sequence and the 5' end of the c sequence respectively; preferably, the immunostimulant CPG2006 is connected to the 5' end of the a sequence by a base bridge TTTTTT, and is connected to the 5' end of the c sequence by a base bridge TTTTT; the nucleic acid aptamer C The molar ratio of D40 to the DNA vector is 1:1, and D40 is connected to the 5' end of the b sequence; preferably, the nucleic acid aptamer CD40 is connected to the 5' end of the b sequence through a base bridge TTTTT; the molar ratio of the nucleic acid aptamer PD-L1 to the DNA vector is 1:1, and the aptamer PD-L1 is connected to the 5' end of the d sequence; preferably, the nucleic acid aptamer PD-L1 is connected to the 5' end of the d sequence through a base bridge TTTTTT; the molar ratio of the nucleic acid aptamer C12 to the DNA vector is 1:1, and the aptamer C12 is connected to the 5' end of the e sequence; preferably, the nucleic acid aptamer C12 is connected to the 5' end of the e sequence through a base bridge TTTTT; or, K) The targeted chemical drug includes the DNA vector, the chemical small molecule drug, and the immunostimulant CPG2006, nucleic acid aptamer C12, siRNA oligonucleotide CD47, nucleic acid aptamer PD-L1 in DNA form and siRNA oligonucleotide PD-L1 connected to the DNA vector; preferably, the chemical small molecule drug is epirubicin or its free base or its hydrochloride; preferably, the DNA vector contains the ninth group of sequences, the molar ratio of the siRNA oligonucleotide PD-L1 to the DNA vector is 1:1, and its sense chain is connected to the 5' end of the c sequence in the DNA vector as a transition base segment, and the antisense chain is complementary to the sense chain as a complementary sequence, and the nucleic acid aptamer PD-L1 in DNA form is connected to the 5' end of the antisense chain of the siRNA oligonucleotide PD-L1; preferably, the nucleic acid aptamer PD-L1 is linked to the 5' end of the antisense chain of the siRNA oligonucleotide PD-L1 through a base linker. The bridge TTTTT is connected to the 5' end of the antisense strand of the siRNA oligonucleotide PD-L1; the molar ratio of the immunostimulant CPG2006 to the DNA vector is 1:1, and it is connected to the 5' end of the a sequence; preferably, the immunostimulant CPG2006 is connected to the 5' end of the a sequence through the base bridge TTTTT; the molar ratio of the siRNA oligonucleotide CD47 to the DNA vector is 1:1, and its sense strand is connected to the 5' end of the b sequence as a transition base segment, and the antisense strand is complementary to the sense strand as a complementary sequence; the molar ratio of the nucleic acid aptamer C12 to the DNA vector is 1:1, and it is connected to the 5' end of the antisense strand of the siRNA oligonucleotide CD47; preferably, the nucleic acid aptamer C12 is connected to the 5' end of the antisense strand of the siRNA oligonucleotide CD47 through the base bridge TTTTT; or, L) The targeted chemical drug includes the DNA vector, the chemical small molecule drug, and the miRNA oligonucleotide A-miR-21, siRNA oligonucleotide TGF-β1, nucleic acid aptamer TfRA4 and nucleic acid aptamer PD-L1 connected to the DNA vector; preferably, the chemical small molecule drug is epirubicin or its free base or its hydrochloride; preferably, the DNA vector contains the second group of sequences, the molar ratio of the siRNA oligonucleotide TGF-β1 to the DNA vector is 1:1, and its antisense strand is used as a transition base The base segment is connected to the 3' end of the c sequence, and the sense strand is complementary to the antisense strand as a complementary sequence; the molar ratio of the nucleic acid aptamer TfRA4 to the DNA vector is 1:1, and it is connected to the 5' end of the a sequence; preferably, the nucleic acid aptamer TfRA4 is connected to the 5' end of the a sequence through a base bridge TTTTT; the molar ratio of the nucleic acid aptamer PD-L1 to the DNA vector is 1:1, and it is connected to the 5' end of the b sequence; preferably, the nucleic acid aptamer PD-L1 is connected to the 5' end of the b sequence through a base bridge TTTTT At the 5' end of the b sequence; the molar ratio of the miRNA oligonucleotide A-miR-21 to the DNA vector is 2:1, and is sequentially connected to the 5' end of the sense strand of the siRNA oligonucleotide TGF-β1; or, M) The targeted chemical drug includes the DNA vector, the chemical small molecule drug, and the nucleic acid aptamer IL-4Ra, siRNA oligonucleotide CD47, DNA-formed nucleic acid aptamer TfRA4, siRNA oligonucleotide PD-L1 and nucleic acid aptamer GPC-1 connected to the DNA vector; preferably, the chemical small molecule drug is epirubicin or its free base or its hydrochloride; the DNA vector contains the third group of sequences, the nucleic acid aptamer TfRA4 and the DNA vector have a molar ratio of 1:1, and are connected to the 5' end of the a sequence; preferably, the nucleic acid aptamer TfRA4 is connected to the 5' end of the a sequence through a base bridge TTTTT; the nucleic acid aptamer GPC-1 and the DNA vector have a molar ratio of 1:1, and are connected to the 5' end of the c sequence. end; the molar ratio of the siRNA oligonucleotide PD-L1 to the DNA vector is 1:1, and its sense chain is connected to the 5' end of the b sequence as a transition base segment, and the antisense chain is complementary to the sense chain as a complementary sequence and connected to the 3' end of the a sequence; the molar ratio of the siRNA oligonucleotide CD47 to the DNA vector is 1:1, and its sense chain is connected to the 3' end of the c sequence as a transition base segment, and the antisense chain is complementary to the sense chain as a complementary sequence; the molar ratio of the nucleic acid aptamer IL-4Ra to the DNA vector is 1:1, and is connected to the 5' end of the antisense chain of the siRNA oligonucleotide CD47; preferably, the nucleic acid aptamer IL-4Ra is connected to the 5' end of the antisense chain of the siRNA oligonucleotide CD47 through a base bridge AAAA; or, N) The targeted chemical drug includes the DNA vector, the chemical small molecule drug, and the DNA-formed nucleic acid aptamer AS1411, the DNA-formed nucleic acid aptamer EGFR, and the DNA-formed nucleic acid aptamer A15 connected to the DNA vector, preferably the chemical small molecule drug is gemcitabine; preferably, the DNA vector contains the fourth group of sequences, the molar ratio of the DNA-formed nucleic acid aptamer AS1411 to the DNA vector is 1:1, and the aptamer AS1411 is connected to the 5' end of the a sequence; the molar ratio of the DNA-formed nucleic acid aptamer EGFR to the DNA vector is 1:1 , and connected to the 5' end of the b sequence; the molar ratio of the DNA-based nucleic acid aptamer A15 to the DNA vector is 1:1, and connected to the 5' end of the c sequence; preferably, the gemcitabine is connected in the form of at least one base insertion at one or more of the following positions: between the DNA-based nucleic acid aptamer AS1411 and the a sequence, at the 3' end of the a sequence, between the DNA-based nucleic acid aptamer EGFR and the b sequence, at the 3' end of the b sequence, between the DNA-based nucleic acid aptamer A15 and the c sequence, at the 3' end of the c sequence; or, O) The targeted chemical drug includes the RNA vector, the chemical small molecule drug, and the nucleic acid aptamer AS1411 in DNA form, the nucleic acid aptamer EGFR in DNA form, and the nucleic acid aptamer A15 in DNA form connected to the RNA vector, preferably the chemical small molecule drug is 5-fluorouracil; preferably, the RNA vector contains the fifth group of sequences, the nucleic acid aptamer AS1411 in DNA form and the RNA vector have a molar ratio of 1:1, and are connected to the 5' end of the a sequence; the nucleic acid aptamer EGFR in DNA form and the RNA vector have a molar ratio of 1:1, and are connected to the 5' end of the a sequence; The molar ratio of the DNA aptamer AS1411 to the RNA vector is 1:1, and the aptamer is connected to the 5' end of the b sequence; the molar ratio of the DNA aptamer A15 to the RNA vector is 1:1, and the aptamer is connected to the 5' end of the c sequence; preferably, the 5-fluorouracil is connected to one or more of the following positions in the form of at least one base insertion: between the DNA aptamer AS1411 and the a sequence, at the 3' end of the a sequence, between the DNA aptamer EGFR and the b sequence, at the 3' end of the b sequence, between the DNA aptamer A15 and the c sequence between the columns, the 3' end of the c sequence; or, P) The targeted chemical drug includes the DNA vector, the chemical small molecule drug, and the DNA-formed nucleic acid aptamer AS1411, the DNA-formed nucleic acid aptamer EGFR, and the DNA-formed nucleic acid aptamer A15 connected to the DNA vector, preferably the chemical small molecule drug is paclitaxel; preferably, the DNA vector contains the sixth group of sequences, the molar ratio of the DNA-formed nucleic acid aptamer AS1411 to the DNA vector is 1:1, and the aptamer AS1411 is connected to the 5' end of the a sequence; preferably, the nucleic acid aptamer AS1411 is connected to the 5' end of the a sequence by alkali The base bridge TTTTT is connected to the 5' end of the a sequence; the molar ratio of the DNA form of the nucleic acid aptamer EGFR to the DNA vector is 1:1, and it is connected to the 5' end of the b sequence; preferably, the nucleic acid aptamer EGFR is connected to the 5' end of the b sequence through the base bridge TTTTT; the molar ratio of the DNA form of the nucleic acid aptamer A15 to the DNA vector is 1:1, and it is connected to the 5' end of the c sequence; preferably, the nucleic acid aptamer A15 is connected to the 5' end of the c sequence through the base bridge TTTTT; or, Q) The targeted chemical drug includes the DNA vector, the chemical small molecule drug, and the DNA-formed nucleic acid aptamer AS1411, the DNA-formed nucleic acid aptamer EGFR, and the DNA-formed nucleic acid aptamer A15 connected to the DNA vector, preferably the chemical small molecule drug is maytansine; preferably, the DNA vector comprises the seventh group of sequences, the DNA-formed nucleic acid aptamer AS1411 and the DNA vector have a molar ratio of 1:1, and are connected to the 5' end of the a sequence; preferably, the nucleic acid aptamer AS1411 is connected to the 5' end of the a sequence by alkali The base bridge TTTTT is connected to the 5' end of the a sequence; the molar ratio of the DNA form of the nucleic acid aptamer EGFR to the DNA vector is 1:1, and it is connected to the 5' end of the b sequence; preferably, the nucleic acid aptamer EGFR is connected to the 5' end of the b sequence through the base bridge TTTTT; the molar ratio of the DNA form of the nucleic acid aptamer A15 to the DNA vector is 1:1, and it is connected to the 5' end of the c sequence; preferably, the nucleic acid aptamer A15 is connected to the 5' end of the c sequence through the base bridge TTTTT; or, R) The targeted chemical drug includes the DNA vector, the chemical small molecule drug, and the nucleic acid aptamer MUC1, nucleic acid aptamer AS1411 and nucleic acid aptamer GPC-1 connected to the DNA vector, preferably the chemical small molecule drug is gemcitabine; preferably, the DNA vector contains the ninth group of sequences, the molar ratio of the nucleic acid aptamer MUC1 to the DNA vector is 1:1, and it is connected to the 5' end of the a sequence; the molar ratio of the nucleic acid aptamer AS1411 to the DNA vector is 1:1, and it is connected to the 5' end of the b sequence; the molar ratio of the nucleic acid aptamer GPC-1 to the DNA vector is 1:1, and it is connected to the 5' end of the c sequence.

9. A pharmaceutical composition, characterized in that The invention comprises the targeted chemical drug according to any one of claims 1 to 8 and a pharmaceutically acceptable carrier and / or excipient.

10. Use of the targeted chemical drug according to any one of claims 1 to 8 in the preparation of a drug for treating tumors.

11. The use according to claim 10, characterized in that: The tumor is a male tumor, a gynecological tumor, a respiratory system tumor, a digestive system tumor, a hematological tumor, a urinary system tumor, a bone marrow tumor, a neurological tumor, a dermatological tumor, a general surgical tumor or an ENT tumor; preferably, the male tumor is prostate cancer, penile cancer, testicular tumor or male urethral cancer, the gynecological tumor is ovarian cancer, cervical cancer, mesothelioma, uterine fibroids, vulvar cancer or malignant hydatidiform mole, the respiratory system tumor is lung cancer, non-small cell lung cancer, small cell lung cancer, nasopharyngeal carcinoma, tracheal tumor, Lung cancer metastasis, inflammatory pseudotumor or radiation lung cancer, the digestive system tumor is liver cancer, gastric cancer, colorectal cancer, gallbladder cancer, esophageal cancer, rectal cancer, pancreatic cancer or colon cancer, the hematological tumor is leukemia, lymphoma, lymphosarcoma or multiple myeloma, the kidney cancer, bladder cancer or urinary cancer, the bone marrow tumor is giant cell tumor of bone, osteochondroma or osteosarcoma, the neurological tumor is brain tumor, meningioma, brain tuberculoma, pituitary tumor, neuroblastoma, glioblastoma or brain glioma, the dermatological tumor is skin cancer or melanoma, the general surgical tumor is breast cancer, lipoma, thyroid cancer or thyroid tumor, the ENT tumor is oral cancer, tongue cancer, laryngeal cancer, middle ear cancer, gingival cancer or orbital tumor; Preferably, the drug is administered by tumor administration, intravenous administration or intraperitoneal administration during the application process; Preferably, the daily dosage of the drug during the application process is 0.1 μg / Kg to 100 mg / Kg.

12. A method for preparing the targeted chemical drug according to any one of claims 1 to 8, characterized in that: The following steps are involved: A targeted nucleic acid vector with an optional oligonucleotide effector molecule and an optional immunostimulant is formed by sequence self-assembly; and a small chemical molecule drug is connected to the targeted nucleic acid vector to obtain the targeted chemical drug.

13. The preparation method according to claim 12, characterized in that: The steps of forming the targeted nucleic acid vector with optional oligonucleotide effector molecules and optional immunostimulants by sequence self-assembly include: Dissolving at least three sequences with optional oligonucleotide effector molecules, optional immunostimulants and targeting molecules in an assembly solution for denaturation reaction to form a self-assembly crude product; purifying, eluting and evaporating the self-assembly crude product in sequence to obtain the targeting nucleic acid vector with optional oligonucleotide effector molecules and optional immunostimulants; Preferably, the at least three sequences include at least the following three sequences: Sequence A: The end is directly connected or connected through a base bridge to the optional oligonucleotide effector molecule, the optional immunostimulatory agent and the optional targeting molecule a sequence; Sequence B: The end is directly connected or connected through a base bridge to the optional oligonucleotide effector molecule, the optional immunostimulatory agent and the optional targeting molecule b sequence; Sequence C: The end is directly connected or connected through a base bridge to the c sequence of an optional oligonucleotide effector molecule, an optional immunostimulatory agent and an optional targeting molecule; The molar ratio of the A sequence, the B sequence and the C sequence is 0.90-1.10: 0.90-1.10: 0.90-1.10, more preferably 1:1:1; when the at least three sequences include sequences other than the a sequence, the b sequence and the c sequence, the molar ratio of each sequence to the A sequence is 0.90-1.10: 0.90-1.10, more preferably 1:1; preferably, when the at least three sequences include sequences other than the A sequence, the B sequence and the C sequence, they are recorded as complementary sequences, at which time any 5' segment or 3' segment of the a sequence, the b sequence and the c sequence is independently connected with a transition base segment, and the complementary sequence and the transition base segment present a base complementary structure, so that the complementary sequence and the transition base segment are complementarily connected during the self-assembly process; more preferably, the complementary sequence itself is a siRNA oligonucleotide effector The complementary sequence is used to provide a mounting position for a small molecule target head, a targeting aptamer or an immunostimulant; Further preferably, when the at least three sequences include the complementary sequence, the complementary sequence carrying an optional small molecule target head, an optional targeting aptamer, and an optional immunostimulant is added to the assembly solution at the same time to perform the denaturation reaction; Preferably, the assembly liquid is TMS aqueous solution, sodium chloride aqueous solution, magnesium chloride aqueous solution or purified water; Preferably, the temperature of the denaturation reaction is 80 to 99°C, more preferably 85 to 99°C, and further preselected to be 90 to 99°C; Preferably, after the denaturation reaction is completed, the reaction system is cooled to a holding temperature for holding reaction, and finally cooled to obtain the group of products; wherein the holding temperature is 70 to 50° C., more preferably 65 to 55° C., and further preferably 63 to 57° C.; the holding reaction time is 3 to 15 min, more preferably 3 to 10 min, and further preferably 3 to 5 min; Preferably, in the process of cooling the reaction system to the insulation temperature, the cooling rate is 2 to 10°C / min, more preferably 2 to 6°C / min, and further preferably 2 to 3°C / min; Preferably, the cooling end point temperature is 0 to 25°C, more preferably 0 to 15°C, and further preferably 0 to 4°C; Preferably, the pH value of the denaturation reaction is 5.4-8.

8.

14. The preparation method according to claim 13, characterized in that: When the chemical small molecule drug is an anthracycline drug or an acridine drug, the step of mounting the chemical small molecule drug on the targeted nucleic acid carrier comprises: dissolving the chemical small molecule, a coupling agent, the targeted nucleic acid carrier with an optional oligonucleotide effector molecule and an optional immunostimulant in a first solvent and reacting under light-proof conditions, so that the chemical small molecule is specifically inserted into the GC adjacent base pairs of the targeted nucleic acid carrier to obtain a crude reaction product; and sequentially crystallizing, washing and evaporating the crude reaction product to obtain the targeted chemical drug; Preferably, the coupling agent is formaldehyde and / or paraformaldehyde; Preferably, the reaction temperature is 0 to 37°C, more preferably 4 to 20°C, and further preferably 4 to 8°C; Preferably, the pH value of the reaction is 6 to 8; Preferably, the reaction time is 24 to 96 hours, more preferably 48 to 72 hours, and further preferably 72 hours; Preferably, the first solvent is purified water and / or PBS buffer; Preferably, the washing agent used in the washing process is a halogenated alkane solvent, an ether solvent or a benzene solvent, more preferably chloroform, dichloromethane, ether, toluene or xylene; Preferably, the solvent used in the crystallization process is a lower alcohol solvent, more preferably methanol, ethanol or isopropanol; Preferably, each of the targeting nucleoside-containing oligonucleotide effector molecules, optional immunostimulatory agents, The average number of small molecule drugs mounted on the acid carrier is 1 to 50, more preferably 10 to 40, and further preferably 20 to 35; When the chemical small molecule drug is a pyrimidine drug, the small molecule target head, the oligonucleotide effector molecule and the immunostimulant are connected to the terminal bases of each sequence of the nucleic acid vector through a base connection bridge containing 1 to 10 bases, and the step of connecting the chemical small molecule drug to the targeted nucleic acid vector with optional oligonucleotide effector molecules and optional immunostimulants includes: in the step of forming the targeted nucleic acid vector with optional oligonucleotide effector molecules and optional immunostimulants by sequence self-assembly, the chemical small molecule drug is connected to at least one sequence of the nucleic acid vector in the form of at least partially replacing the base in the base connection bridge and / or as an extended base and / or a carrier backbone base replacement, and then the connection of the chemical small molecule drug is completed by sequence self-assembly; preferably, the average number of chemical small molecule drugs mounted on each targeted nucleic acid vector molecule is 1 to 30, more preferably 10 to 20, and further preferably 12 to 18; When the chemical small molecule drug is a platinum drug, a glutamic acid derivative drug, a flavonoid drug, a botanical drug and its derivatives, a folic acid drug or a salicylic acid drug, the step of connecting the chemical small molecule drug to the targeted nucleic acid vector with an optional oligonucleotide effector molecule and an optional immunostimulant comprises: in the process of synthesizing each sequence of the nucleic acid vector connected with the targeting molecule, a linker is bound to at least one sequence of the nucleic acid vector with the optional oligonucleotide effector molecule, the optional immunostimulant and the targeting molecule to obtain a targeted nucleic acid vector connected with a linker; the targeted nucleic acid vector connected with the linker and the chemical small molecule drug are dissolved in a second solvent for reaction, and then cooled, purified, washed and evaporated to obtain the targeted chemical drug; Preferably, when the chemical small molecule drug itself carries a succinimide lipid group, the targeted nucleic acid vector connected with a linker and the chemical small molecule drug are dissolved in the second solvent for reaction; when the chemical small molecule drug itself does not carry a succinimide lipid group, before dissolving the chemical small molecule drug in the second solvent for reaction, the method further includes: reacting the chemical small molecule drug with bis(2-hydroxyethyl) disulfide and N,N-diisopropylethylamine in sequence to modify the succinimide lipid group on the chemical small molecule drug.

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