Drug targeting delivery system and application thereof in preparation of antitumor drugs

Abstract

The invention relates to the technical field of medicines, in particular to a drug targeting delivery system and application thereof in preparation of antitumor drugs. Compared with the prior art, the drug targeting delivery system provided by the invention realizes fixed-point quantitative integration of chemotherapeutic drugs through a nucleic acid solid-phase synthesis technology, and the aptamer drug can target overexpression receptor protein on the tumor surface, so that effective enrichment of the drug at the tumor part is realized. The system can synergistically induce death of immunogenic cells, and by enhancing antigen presentation and promoting maturation of dendritic cells and infiltration of effector T lymphocytes, tumor proliferation is remarkably inhibited, and an immunosuppressive tumor microenvironment is effectively relieved. The problems that existing chemotherapy-immune combined therapy is poor in targeting property, uncontrollable in medicine proportion and the like are solved, and the application prospect in antitumor medicine development is achieved.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to a drug targeted delivery system and its application in the preparation of antitumor drugs. Background Technology

[0002] Currently, malignant tumors have surpassed cardiovascular diseases to become one of the leading causes of death worldwide, seriously threatening human health and life. In recent years, groundbreaking advancements in tumor immunotherapy have brought about a revolutionary change in cancer treatment. Unlike traditional therapies that directly kill tumor cells, tumor immunotherapy, as a novel treatment approach, activates the body's own immune system, relying on the host's immune function to specifically kill tumor cells. It possesses core advantages such as high specificity, significant curative potential, and long-lasting efficacy. Based on clinical cancer immunotherapy strategies, it can be divided into five categories: immune checkpoint inhibitors, tumor vaccines, cytokine therapy, adoptive cell transplantation, and oncolytic virus therapy.

[0003] Despite its promising prospects, tumor immunotherapy still faces numerous serious challenges. Currently, some tumor types still exhibit extremely low response rates or even no response to immunotherapy. Insufficient tumor immunogenicity and weak anti-tumor immune responses are the core reasons for the limited efficacy of many immunotherapies. Therefore, developing effective strategies to enhance tumor immunogenicity, thereby improving the efficacy of immunotherapy and overcoming tumor immune tolerance and immune escape, has become a critical issue that urgently needs to be addressed in the field of cancer treatment.

[0004] To overcome the inherent limitations of single immunotherapy, combination therapy strategies have gradually become a research hotspot in the field, with chemotherapy-immunotherapy synergistic anti-tumor combination therapy being particularly prominent. The core logic of this strategy lies in achieving the dual effects of directly killing tumors and activating the body's anti-tumor immunity through the synergistic action of chemotherapy and immune responses, significantly improving treatment efficacy and reducing the risk of drug resistance. However, traditional chemotherapy-immunotherapy combination regimens still suffer from problems such as insufficient targeting, low bioavailability of immunomodulators, and difficulty in precisely matching the timing windows and dosage ratios of chemotherapy and immune responses. This not only leads to insufficient release of the synergistic anti-tumor effect but also easily induces systemic immune-related adverse reactions. Therefore, developing a drug delivery system capable of precisely co-delivering chemotherapeutic drugs and immune-active components, achieving targeted enrichment and synergistic release of both at the tumor site, has become a key technological direction for overcoming the aforementioned technical bottlenecks and efficiently improving the efficacy of synergistic anti-tumor immunotherapy. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides a targeted drug delivery system and its application in the preparation of antitumor drugs. This invention overcomes the inherent limitations of single immunotherapy and single chemotherapy, achieving the dual effects of directly killing tumor cells and activating the body's antitumor immunity, significantly improving the treatment efficacy of malignant tumors and reducing the risk of drug resistance.

[0006] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a drug-targeted delivery system comprising a nucleic acid aptamer embedded with a chemotherapeutic drug and a CpG oligonucleotide; wherein the nucleic acid aptamer embedded with the chemotherapeutic drug is a phosphoramidic active monomer of fluorouracil in which the thymine deoxyribonucleotide is replaced; the nucleic acid aptamer includes EpCAM nucleic acid aptamer, AS1411 nucleic acid aptamer, Sgc8c nucleic acid aptamer, VEGF nucleic acid aptamer or HER2 nucleic acid aptamer; the nucleic acid aptamer embedded with the chemotherapeutic drug is covalently linked to the CpG oligonucleotide via a 3'-5' phosphodiester bond.

[0007] Preferably, the nucleotide sequence of the EpCAM aptamer is shown in SEQ ID NO.2; the nucleotide sequence of the AS1411 aptamer is shown in SEQ ID NO.10; the nucleotide sequence of the Sgc8c aptamer is shown in SEQ ID NO.11; the nucleotide sequence of the VEGF aptamer is shown in SEQ ID NO.12; and the nucleotide sequence of the HER2 aptamer is shown in SEQ ID NO.13.

[0008] Preferably, the nucleotide sequence of the EpCAM aptamer containing the chemotherapeutic drug is shown in SEQ ID NO.6.

[0009] Preferably, the nucleotide sequence of the CpG oligonucleotide is shown in SEQ ID NO.1.

[0010] Preferably, when the nucleic acid aptamer is the EpCAM nucleic acid aptamer, the nucleotide sequence of the drug targeted delivery system is shown in SEQ ID NO.8.

[0011] This invention provides a method for preparing the drug targeted delivery system described above, comprising the following steps: Using nucleic acid solid-phase synthesis technology, all thymine deoxyribonucleotides in the nucleic acid aptamer were replaced with phosphoramidite active monomers of fluorouracil and linked with oligonucleotide CpG to obtain a primary drug targeted delivery system. The primary drug targeted delivery system was annealed at 90°C for 5 min and then cooled to 25-30°C to obtain the drug targeted delivery system.

[0012] This invention provides the application of the drug targeted delivery system described above in the preparation of antitumor drugs.

[0013] Preferably, the tumor includes malignant tumors that overexpress EpCAM receptor, nucleolin receptor, protein tyrosine kinase 7 receptor, VEGF receptor, or HER2 receptor on their surface.

[0014] Preferably, the malignant tumors that overexpress the EpCAM receptor on their surface include one or more of triple-negative breast cancer, colorectal cancer, and gastric cancer.

[0015] This invention provides an anti-tumor drug, the active ingredient of which includes the drug targeted delivery system described in the above technical solution.

[0016] Beneficial effects: This invention provides a targeted drug delivery system comprising a nucleic acid aptamer embedded with a chemotherapeutic drug and a CpG oligonucleotide; wherein the nucleic acid aptamer embedded with the chemotherapeutic drug is a phosphoramidic active monomer of fluorouracil in which the thymine deoxyribonucleotide of the nucleic acid aptamer is replaced; the nucleic acid aptamer includes EpCAM nucleic acid aptamer, AS1411 nucleic acid aptamer, Sgc8c nucleic acid aptamer, VEGF nucleic acid aptamer, or HER2 nucleic acid aptamer; the nucleic acid aptamer embedded with the chemotherapeutic drug and the CpG oligonucleotide are covalently linked via a 3'-5' phosphodiester bond. This invention utilizes the ability of nucleic acid aptamers to accurately identify specific markers on the surface of tumor cells and the structural characteristics of nucleoside analogue chemotherapeutic drugs (fluorouracil) to couple chemotherapeutic drugs and immunomodulators together, achieving targeted and quantitative release of chemotherapeutic drugs and immunomodulators, enhancing tumor immunogenicity, reducing the toxic side effects of drug molecules, improving drug bioavailability, and compensating for the shortcomings of monotherapy. The drug-targeted delivery system provided by this invention retains the structure and function of its natural nucleic acid aptamer. Therefore, it eliminates the need for introducing additional carriers to achieve targeted tumor therapy, avoiding the problems of toxic side effects and immunogenicity associated with carrier introduction. It overcomes the inherent limitations of single immunotherapy and single chemotherapy, achieving the dual effects of directly killing tumors and activating the body's anti-tumor immunity, significantly improving the treatment efficacy of malignant tumors and reducing the risk of drug resistance.

[0017] Furthermore, the drug-targeted delivery system provided by this invention is prepared using a solid-phase synthesis method. The synthesis method is simple, and nucleic acid aptamers, fluorouracil molecules, and oligonucleotide CpG can be precisely prepared in proportion. This solves the problems in the prior art, such as the difficulty in precisely co-delivering chemotherapy drugs and immune-active components, the low bioavailability of immunomodulators, and the difficulty in precisely matching the timing window and dosage ratio of chemotherapy and immune effects. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0019] Figure 1 A schematic diagram of the F-EpCAM-CpG targeted drug delivery system prepared by solid-phase synthesis technology and its synergistic anti-tumor immunotherapy effect; Figure 2 Polyacrylamide gel images of oligonucleotide sequences including scramble, EpCAM, F-scramble, F-EpCAM, F-scramble-CPG, and F-EpCAM-CpG; where 1 represents the DNA marker; 2 represents the scramble; 3 represents the EpCAM aptamer; 4 represents the F-scramble with fluorouracil; 5 represents the F-EpCAM aptamer with fluorouracil; 6 represents the F-scramble-CpG with fluorouracil and CpG conjugation; and 7 represents the F-EpCAM-CpG aptamer with fluorouracil and CpG conjugation. Figure 3 Mass spectra of scramble and EpCAM oligonucleotide sequences; Figure 4 Mass spectra of F-EpCAM and F-EpCAM-CpG oligonucleotide sequences; Figure 5 Mass spectra of F-scramble and F-scramble-CPG oligonucleotide sequences; Figure 6 This is an in vitro stability diagram of EpCAM, F-EpCAM, and F-EpCAM-CpG analyzed by polyacrylamide gel electrophoresis under simulated physiological conditions; lane 1 is the DNA marker, and lanes 2-8 are the in vitro stability diagrams at 0 h, 1 h, 2 h, 4 h, 6 h, 8 h, and 24 h, respectively. Figure 7 The binding affinity maps of F-EpCAM nucleic acid drug aptamer and EpCAM nucleic acid aptamer to 4T1 cells, as determined by flow cytometry, are shown. Figure 8 The endocytic uptake map (A) of scramble, EpCAM, F-EpCAM, scramble-CpG, EpCAM-CpG and F-EpCAM-CpG after incubation in 4T1 cells for 2 h and the corresponding mean fluorescence intensity (MFI) change analysis diagram (B). Figure 9Confocal microscopy images of F-scramble, EpCAM, F-EpCAM, and F-EpCAM-CpG oligonucleotide sequences after incubation in 4T1 cells for 2 hours; Figure 10 The internalization flow cytometry uptake maps of scramble, EpCAM, F-EpCAM, scramble-CpG, EpCAM-CpG and F-EpCAM-CpG after incubation for 2 h in MDA-MB-231 cells, HT-29 cells and HepG2 cells and the corresponding changes in mean fluorescence intensity (MFI) are shown. Figure 11 MTT assay diagram showing the cytotoxicity of free fluorouracil and different drug-containing oligonucleotide sequences against 4T1 tumor cells; Figure 12 Figure 1 shows the apoptosis experiment of 4T1 tumor cells induced by the free drug fluorouracil and different drug-containing oligonucleotide sequences. Figure 13 MTT assay results showing the cytotoxicity of free fluorouracil and different drug-containing oligonucleotide sequences to MDA-MB-231, HT-29, and HepG2 cells, respectively. Figure 14 The diagram shows the in vivo biodistribution of different drug-treated groups in the 4T1 tumor mouse model. A represents in vivo targeted fluorescence imaging of mice in different groups at different time points after injection of different drugs (tumor locations are marked with red circles); B represents in vitro fluorescence imaging of mouse tumors and major organs 3 h and 24 h after injection of different drugs; C represents quantitative analysis of F-EpCAM-CpG distribution in tumors and organs 3 h and 24 h after injection of different drugs. Figure 15 The graph shows the correlation between the in vivo anticancer efficacy of different drug administration groups in the 4T1 tumor mouse model; where A is a line graph of tumor growth dynamics in each group of mice; B is a photograph of the tumor tissue separated from the mice after drug administration; C is a graph of the weight of the tumor tissue separated from the mice in each group; and D is a graph of the dynamic changes in the body weight of each group of mice during the entire experiment. Figure 16 Representative immunofluorescence images of CRT and HMGB1 expression in tumor tissues of mice in each treatment group; the scale bar is 50 μm. Figure 17 Figure A shows the correlation between different drugs inducing anti-tumor immune responses in mice; A represents the infiltrating CD8+ cells in mouse tumor tissue after different drug treatments. +B is a flow cytometry analysis of T cells; C is a flow cytometry analysis of the maturity of dendritic cells (DCs) in mouse lymph nodes after different drug treatments; D is a graph showing the concentrations of IL-6, TNF-α, IL-10 and IL-12 inflammatory cytokines in mouse serum after different drug treatments. Figure 18 The graph shows the results of blood biochemical indicators related to liver and kidney function in mice after different drug treatments. Among them, ALT (alanine aminotransferase), AST (aspartate aminotransferase), and ALP (alkaline phosphatase) are the core indicators for detecting liver function; BUN (blood urea nitrogen), CREA (creatinine), and UA (uric acid) are the core indicators for detecting kidney function. Figure 19 Chemical synthesis route diagram for phosphoramide active monomers of fluorourea; in, express P <0.05, express P <0.01, express P <0.001, ns represents P >0.05. Detailed Implementation

[0020] This invention provides a drug-targeted delivery system comprising a nucleic acid aptamer embedded with a chemotherapeutic drug and a CpG oligonucleotide; wherein the nucleic acid aptamer embedded with the chemotherapeutic drug is a phosphoramidic active monomer of fluorouracil in which the thymine deoxyribonucleotide is replaced; the nucleic acid aptamer includes EpCAM nucleic acid aptamer, AS1411 nucleic acid aptamer, Sgc8c nucleic acid aptamer, VEGF nucleic acid aptamer, or HER2 nucleic acid aptamer; the nucleic acid aptamer embedded with the chemotherapeutic drug and the CpG oligonucleotide are covalently linked via a 3'-5' phosphodiester bond. As one embodiment, the 5' end of the nucleic acid aptamer embedded with the chemotherapeutic drug is coupled to the 3' end of the CpG oligonucleotide chain.

[0021] In one embodiment, the nucleotide sequence of the EpCAM aptamer is shown in SEQ ID NO.2; the nucleotide sequence of the AS1411 aptamer is shown in SEQ ID NO.10; the nucleotide sequence of the Sgc8c aptamer is shown in SEQ ID NO.11; the nucleotide sequence of the VEGF aptamer is shown in SEQ ID NO.12; and the nucleotide sequence of the HER2 aptamer is shown in SEQ ID NO.13, as detailed below: SEQ ID NO.10: 5'-GGTGGTGGTGGTTGTGGTGGTGGTGG-3'; SEQ ID NO.11: 5'-ATCTAACTGCTGCGCCGCCGGGAAAATACTGTACGGTTAGA-3'; SEQ ID NO.12: 5'-TGTGGGGGTGGACGGGCCGGGTAGA-3'; SEQ ID NO.13: 5'-CGTTACGCCCTGCCACTTGAAGATTGACCATTAAGAGCTA-3'.

[0022] In one embodiment, the nucleotide sequence of the EpCAM aptamer in which the chemotherapeutic drug is embedded is shown in SEQ ID NO. 6. In another embodiment, the nucleotide sequence of the CpG oligonucleotide is shown in SEQ ID NO. 1. In yet another embodiment, when the aptamer is an EpCAM aptamer, the nucleotide sequence of the drug-targeted delivery system is shown in SEQ ID NO. 8.

[0023] The drug-targeted delivery system provided by this invention selects the phosphoramidic active monomer of fluorouracil, a nucleoside analog drug molecule with a structure similar to that of natural thymine deoxyribonucleotide (T), to replace the thymine deoxyribonucleotide in the nucleic acid aptamer. This achieves targeted and quantitative integration of chemotherapy drugs. The nucleic acid aptamer embedded with the chemotherapy drug retains the targeting function of the original nucleic acid aptamer, enabling specific targeting of tumor cells. It can accurately deliver chemotherapy drugs and immune activators to the tumor site, and achieve programmed drug release at the tumor site with targeted and quantitative release while reducing toxic side effects. Among them, fluorouracil directly kills tumor cells by interfering with DNA / RNA synthesis and also promotes the release of tumor-associated antigens (TAAs) as an immunogenic cell death (ICD) inducer. CpG oligonucleotides reverse the tumor immunosuppressive microenvironment by binding to Toll-like receptor 9 (TLR9) on the surface of immune cells, and produce a synergistic anti-tumor effect with fluorouracil. Figure 1 The nucleic acid aptamer sequence in the targeted drug delivery system provided by this invention can be replaced with a nucleic acid aptamer sequence that can target other tumor cells, depending on the needs of treating different cancers, making it suitable for targeted therapy of various malignant tumors. This targeted drug delivery system can also achieve co-delivery of different chemotherapeutic drugs and immunomodulators by changing the type of nucleic acid aptamer or natural nucleoside analog chemotherapeutic drug molecule, making it suitable for combined anti-tumor immunotherapy of different types of tumors.

[0024] The drug-targeted delivery system provided by this invention can synergistically induce immunogenic cell death by enhancing antigen presentation, promoting dendritic cell maturation, and infiltrating effector T lymphocytes (CD8+). + This invention significantly increases T-cell infiltration density, inhibits tumor proliferation, and effectively alleviates the immunosuppressive tumor microenvironment. It addresses the problems of poor targeting and uncontrollable drug ratios in existing chemotherapy-immunotherapy combinations, and shows promise for application in anti-tumor drug development.

[0025] This invention provides a method for preparing the drug targeted delivery system described above, comprising the following steps: Using nucleic acid solid-phase synthesis technology, all thymine deoxyribonucleotides in the nucleic acid aptamer were replaced with phosphoramidite active monomers of fluorouracil and linked with oligonucleotide CpG to obtain a primary drug targeted delivery system. The primary drug targeted delivery system was annealed at 90°C for 5 min and then cooled to 25-30°C to obtain the drug targeted delivery system.

[0026] As one implementation method, the preparation method of the phosphorus amide active monomer of fluorouracil refers to the synthetic route of the phosphorus amide active monomer of fluorouracil in the existing literature [Mou, Q.; Ma, Y.; Pan, G.; Xue, B.; Yan, D.; Zhang, C.; Zhu, X. DNA Trojan Horses: Self‐Assembled Floxuridine‐Containing DNA Polyhedra for Cancer Therapy. Angew. Chem. Int. Ed., 2017, 129, 12702-12706.]. Figure 19 The drug-targeted delivery system provided by this invention is prepared using a solid-phase synthesis method, which is simple. The nucleic acid aptamer, which is prepared using solid-phase synthesis technology, retains the targeting function of the original nucleic acid aptamer and can specifically bind to tumor cells overexpressing the corresponding receptor on their surface, thereby achieving targeted drug delivery.

[0027] Based on the above advantages, the present invention provides the application of the drug targeted delivery system described in the above technical solution in the preparation of anti-tumor drugs.

[0028] In one embodiment, the tumor includes malignant tumors that overexpress EpCAM receptor, nucleolin receptor, protein tyrosine kinase 7 receptor, VEGF receptor, or HER2 receptor on their surface.

[0029] As one implementation, the malignant tumors that overexpress the EpCAM receptor on their surface include one or more of triple-negative breast cancer, colorectal cancer, and gastric cancer.

[0030] Based on the above advantages, the present invention provides an anti-tumor drug, the active ingredient of which includes the drug targeted delivery system described in the above technical solution.

[0031] To further illustrate the present invention, the following detailed description, in conjunction with embodiments and accompanying drawings, describes a drug targeted delivery system provided by the present invention and its application in the preparation of antitumor drugs, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0032] Example 1 Fluorouracil, a nucleoside analog drug molecule with a structure similar to that of natural thymine deoxyribonucleotide (T), was selected. The phosphorus amide active monomer of fluorouracil was prepared by referring to the existing literature [Mou, Q.; Ma, Y.; Pan, G.; Xue, B.; Yan, D.; Zhang, C.; Zhu, X. DNA Trojan Horses: Self‐Assembled Floxuridine‐Containing DNA Polyhedra for Cancer Therapy. Angew. Chem. Int. Ed., 2017, 129, 12702-12706.] for the synthetic route of the phosphorus amide active monomer of fluorouracil. Figure 19 The phosphoramid activated monomer was prepared with aminobenzyl protection, 3' OH protected by DMT, and 2' OH protected by TBDMS.

[0033] In this embodiment, EpCAM nucleic acid aptamers were selected as the parent compound. Solid-phase synthesis was used to construct EpCAM nucleic acid aptamers (EpCAM), EpCAM nucleic acid drug aptamers intercalated with fluorouracil (F-EpCAM), EpCAM nucleic acid drug aptamers intercalated with fluorouracil and conjugated with CpG (F-EpCAM-CpG), scrambled strands, scrambled strands intercalated with fluorouracil (F-scramble), and scrambled strands intercalated with fluorouracil and conjugated with CpG (F-scramble-CpG). The nucleotide sequences are shown in Table 1. Taking F-EpCAM-CpG as an example, the preparation method of the aptamer intercalated with fluorouracil is as follows: The phosphoramidite active monomer of fluorouracil was dissolved in anhydrous acetonitrile. Using solid-phase nucleic acid synthesis, all natural thymine deoxyribonucleotides (T) in the EpCAM nucleic acid aptamer sequence were replaced with the phosphoramidite active monomer of fluorouracil, resulting in a targeted drug delivery system (F-EpCAM-CpG) with a specific sequence intercalated into the fluorouracil molecule and linked to the oligonucleotide CpG. The F-EpCAM-CpG nucleic acid drug aptamer was dissolved in 1×PBS solution, annealed at 90°C for 5 min, and then cooled to room temperature to form a secondary structure similar to the parent EpCAM nucleic acid aptamer, thus obtaining a targeted drug delivery system capable of specifically targeting the overexpression of the EpCAM receptor on the cell surface.

[0034] Table 1. List of different oligonucleotide sequences

[0035] Note: N represents the phosphoramidite monomer of fluorouracil (5'-FdUR), EpCAM represents the EpCAM aptamer strand, and scramble represents the disordered DNA strand.

[0036] Through polyacrylamide gel ( Figure 2 ) and mass spectrometry characterization ( Figures 3-5 The structures and molecular weights of the synthesized EpCAM, F-EpCAM, F-EpCAM-CpG, scramble, F-scramble, and F-scramble-cPG oligonucleotide sequences were characterized. The results showed that both the oligonucleotides intercalated with fluorouracil and their unmodified original sequences exhibited single, clear bands. Due to the increased molecular weight, the electrophoretic migration rate of the nucleic acid sequences intercalated with fluorouracil was slower than that of the unmodified sequences. Furthermore, the measured molecular weights of all constructs were in good agreement with the theoretical values, indicating the successful synthesis of the nucleic acid drug aptamers.

[0037] Example 2 1. In vitro stability of F-EpCAM-CpG conjugate The in vivo efficacy of nucleic acid drugs is closely related to their stability. Therefore, this embodiment uses polyacrylamide gel electrophoresis (PAGE) to evaluate the serum stability of F-EpCAM and F-EpCAM-CpG under physiological conditions, including: using DMEM medium containing 10% fetal bovine serum (FBS) to simulate in vitro physiological conditions, and analyzing by 20% denaturing polyacrylamide gel electrophoresis. 5 μL each of EpCAM, F-EpCAM, and F-EpCAM-CpG solutions (containing 1 μg of nucleic acid drug) were mixed with 5 μL of DMEM medium containing 10% FBS and incubated at 37°C. Samples were collected at 0 h, 1 h, 2 h, 4 h, 6 h, 8 h, and 24 h for 20% denaturing polyacrylamide gel electrophoresis analysis. Gel electrophoresis was performed in 1×TBE buffer at a constant voltage of 300 V. After 1-2 h of electrophoresis, the gel was stained with GelRed for 5 min and analyzed using a Bio-Rad gel imaging system. The results are shown in the figure. Figure 6 .

[0038] Electrophoresis results showed that new bands appeared in the sample after 4 hours of incubation, indicating that all oligonucleotides underwent partial degradation. However, even with an extended incubation time of 24 hours, more than 50% of the original oligonucleotides retained their structural integrity, demonstrating the system's significant serum stability.

[0039] 2. In vitro affinity of F-EpCAM nucleic acid drug aptamer for triple-negative breast cancer 4T1 cells To evaluate the binding affinity of the F-EpCAM nucleic acid drug aptamer to its target, this embodiment used flow cytometry to determine the dissociation constant (Kd) value, with the EpCAM nucleic acid aptamer used as a control. Figure 7 As shown, a single-point binding model (Y=BmaxX / (Kd+X)) was used for nonlinear regression fitting. The results showed that the affinity of F-EpCAM for binding to target proteins on the surface of 4T1 cells was comparable to that of the EpCAM aptamer, confirming that the fluorouracil integration technology did not weaken its binding ability to the target.

[0040] 3. In vitro targeting of F-EpCAM-CpG conjugate Epithelial cell adhesion molecule (EpCAM) is a transmembrane glycoprotein widely expressed on the surface of epithelial cells. Due to its high specific expression in tumor cells and key biological functions, it has become one of the core targets for tumor targeted therapy, diagnosis, and drug delivery systems. After binding to its aptamer and ligand, EpCAM mediates receptor-mediated endocytosis, enabling targeted delivery systems to efficiently enter tumor cells and achieve intracellular release of chemotherapeutic drugs, immunomodulators, and other payloads. In normal tissues, EpCAM is mainly expressed at low levels on the basolateral membrane of epithelial cells; however, in various epithelial tumors such as colorectal cancer, breast cancer, pancreatic cancer, lung cancer, and gastric cancer, its expression level is significantly upregulated (usually 10-100 times that of normal tissues), and the expression level is closely related to tumor stage, differentiation degree, and prognosis.

[0041] This example evaluates the targeting ability of F-EpCAM-CpG on 4T1 cells highly expressing EpCAM receptor protein. 200 μL of Cy5-labeled sample (1 μM Cy5 concentration) was incubated with 4T1 cells (80,000 cells / well) at 37°C for 2 h, followed by flow cytometry analysis. Figure 8 ) and laser confocal ( Figure 9 The cellular uptake of Cy5-labeled F-EpCAM-CpG was analyzed. Figure 8 As shown, the results indicated that F-EpCAM and the EpCAM aptamer exhibited similar uptake levels, both significantly higher than the out-of-order control group, confirming that the insertion of fluorouracil into the EpCAM aptamer sequence did not affect its targeting and endocytosis efficiency on 4T1 cells. Furthermore, although the uptake of EpCAM-CpG and F-EpCAM-CpG conjugates was slightly lower than their parent structures (EpCAM and F-EpCAM drug aptamers), no significant difference was observed between the two conjugates. This suggests that conjugation of negatively charged oligonucleotides (such as CpG) may slightly hinder the internalization process. In addition, the experimental results also showed that the uptake in all targeted groups was still significantly higher than the out-of-order control group, confirming the targeting ability of F-EpCAM-CpG. Results of laser confocal microscopy (…) Figure 9 The results are consistent with those obtained by flow cytometry.

[0042] In addition, this embodiment also used flow cytometry to analyze the uptake of Cy5-labeled F-EpCAM-CpG by human breast cancer cells MDA-MB-231, colorectal cancer cells HT-29, and gastric cancer cells HepG2. The results are as follows: Figure 10 As shown, the results indicate that the F-EpCAM-CpG conjugate in this invention has good targeting ability against breast cancer, colorectal cancer, and gastric cancer cells that overexpress EpCAM receptors on their cell surface, and can be used for targeted therapy of these tumors.

[0043] 4. In vitro cytotoxicity of F-EpCAM-CpG conjugate First, in this embodiment, the in vitro cytotoxicity of F-EpCAM-CpG on 4T1 cells was evaluated using the MTT assay, with free fluorouracil (5'-FdUR) as the control group. The results are as follows... Figure 11 As shown in Table 2, Table 2. In vitro cytotoxicity of different drug-containing oligonucleotide sequences on 4T1 cells.

[0044] F-EpCAM-CpG exhibits toxicity to 4T1 tumor cells comparable to that of free fluorouracil, with a half-maximal inhibitory concentration (IC50) of [value missing]. 50 The effective concentrations were 1.19 ± 0.24 μM and 1.01 ± 0.18 μM, respectively. The slightly lower cytotoxicity of F-EpCAM-CpG may be due to differences in cellular uptake mechanisms: free fluorouracil enters cells via passive diffusion, while F-EpCAM-CpG requires endocytosis and depends on intracellular degradation for drug release, resulting in a delayed effect.

[0045] This embodiment also used Annexin V-FITC / PI double staining to detect the apoptosis level of tumor cells after co-incubation of F-EpCAM-CpG with 4T1 cells for 72 h. The results are as follows: Figure 12 As shown, the results indicated that the F-EpCAM-CpG treatment group exhibited a significant apoptosis-inducing effect on 4T1 cells, with an apoptosis rate comparable to that of the free fluorouracil group and higher than that of the F-EpCAM treatment group and the disordered chain group. These results confirm that F-EpCAM-CpG can achieve targeted delivery and effective intracellular release of chemotherapeutic drugs, providing direct experimental evidence for its anti-tumor therapeutic efficacy by efficiently inducing tumor cell apoptosis.

[0046] Based on the above in vitro cytotoxicity results, this embodiment also used the MTT assay to evaluate the in vitro cytotoxicity of F-EpCAM-CpG against human breast cancer cells MDA-MB-231, colorectal cancer cells HT-29, and gastric cancer cells HepG2. Figure 13 As shown in Table 3.

[0047] Table 3. In vitro cytotoxicity of different drug-containing oligonucleotide sequences on different cell types.

[0048] Compared with the free drug fluorouracil, the F-EpCAM-CpG conjugate also showed significant cytotoxicity against human breast cancer cells MDA-MB-231, colorectal cancer cells HT-29, and gastric cancer cells HepG2.

[0049] 5. In vivo targeting of F-EpCAM-CpG conjugate Based on in vitro results, this study further investigated the in vivo targeting ability of F-EpCAM-CpG. In BALB / c mice with 4T1 tumor transplantation, fluorescence imaging was performed after intravenous injection of 200 μL of free Cy5 (0.01 μg / μL), Cy5-labeled F-scramble, Cy5-labeled F-EpCAM, and Cy5-labeled F-EpCAM-CpG, respectively. Figure 14 (A) Compared to free Cy5 and Cy5-labeled F-scramble, Cy5-labeled F-EpCAM and F-EpCAM-CpG showed significantly higher accumulation at tumor sites, confirming the in vivo targeting properties of the F-EpCAM drug modifier. Both reached peak fluorescence intensity at the tumor site 3 hours after injection, followed by gradual decay. 24 hours after injection, the fluorescence intensity at the tumor site in all groups decreased to its lowest level, indicating that F-EpCAM-CpG was essentially cleared from the body within 24 hours.

[0050] To assess in vivo biodistribution, tumor-bearing mice (tumor volume approximately 500 mm²) were used. 3 The patients were randomly divided into four groups and received different drug formulations: free Cy5, Cy5-labeled F-scramble, Cy5-labeled F-EpCAM, and Cy5-labeled F-EpCAM-CpG (n=3 per group). Tumor and organ imaging was performed 3 h and 24 h after injection (Cy5 dosage was 100 μg / kg). Figure 14 (B) The tumor accumulation of F-EpCAM-CpG was comparable to that of F-EpCAM, confirming its strong tumor targeting ability. Quantitative analysis results ( Figure 14 (C) The results show that F-EpCAM-CpG is mainly metabolized in the liver and kidneys, while maintaining continuous tumor accumulation. In summary, these results validate the potent in vivo tumor-targeting efficacy of F-EpCAM-CpG.

[0051] 6. In vivo antitumor effects of F-EpCAM-CpG conjugate To evaluate the in vivo antitumor efficacy of F-EpCAM-CpG, a 4T1 tumor transplantation BALB / c mouse model was established. When the tumor volume in the mice was approximately 150 mm... 3At that time, participants were randomly divided into seven groups (n=4 per group). Different drugs (fluorouracil at a dose of 5 mg / kg) were injected via the tail vein every three days, and tumor volume and body weight changes were recorded daily. The experimental groups were: saline group (G1), fluorouracil group (G2), CpG group (G3), F-scramble group (G4), F-scramble-CpG group (G5), F-EpCAM (G6), and F-EpCAM-CpG (G7). Figure 15 In Figure A, tumor growth kinetics are represented in each treatment group. The results showed that the F-EpCAM-CPG group (G7) exhibited the strongest inhibitory effect, with a mean tumor volume of 189.6 mm on day 18. 3 This demonstrates the synergistic effect of chemotherapy and immunotherapy. After treatment, all mice were euthanized and dissected, and the tumors were weighed and photographed. Figure 15 (Middle B and 15 Middle C). The results showed that the F-EpCAM-CpG treatment group had the smallest tumor volume and weight compared with other groups, confirming its superior efficacy. In addition, no significant weight fluctuations were observed in any of the experimental and control groups, indicating that this targeted drug delivery system has extremely low toxicity. Figure 15 (D).

[0052] 7. In vivo antitumor immunomodulatory effects of F-EpCAM-CpG conjugate To evaluate the in vivo antitumor immune response induced by the F-EpCAM-CpG targeted drug delivery system, 4T1 tumor mice (tumor volume: approximately 200 mm) were used in this example. 3 The mice were randomly divided into seven groups (n=3 per group). Each group received an intravenous injection of the following formulations every other day (5 mg / kg of fluorouracil): saline, free fluorouracil (5'-FdUR), CpG, F-scramble, F-scramble-CpG, F-EpCAM, and F-EpCAM-CpG. The dose of fluorouracil was standardized to 5 mg / kg in all groups. Tumor and inguinal lymph node samples were collected on day 18 to assess immunogenic cell death (ICD) biomarkers and CD4 counts. + / CD8 + T lymphocyte infiltration and dendritic cell (DC) maturation.

[0053] Since the chemotherapeutic drug fluorouracil is an immunogenic inducer, it can induce immunogenic cell death (ICD) and activate T cell responses by releasing damage-associated molecular patterns (DAMPs). In this embodiment, immunofluorescence was used to detect the expression of classic DAMP markers calreticulin (CRT) and high-mobility family box 1 (HMGB1) to assess the ICD-inducing effect. Figure 16As shown, both the F-EpCAM and F-EpCAM-CpG groups significantly upregulated CRT surface exposure levels (facilitating dendritic cell phagocytosis of tumor debris and providing antigens to promote DC maturation), and increased exogenous HMGB1 levels (red fluorescence), confirming that targeting EpCAM can enhance the release of cell death-related antigens. The F-EpCAM-CpG group had the highest CRT and HMGB1 levels, suggesting a synergistic effect between EpCAM targeted delivery and CpG immune stimulation, which can significantly activate downstream immune pathways. In conclusion, F-EpCAM-CpG can effectively enhance tumor immunogenicity and amplify the systemic immune response, highlighting its potential for anti-tumor immunotherapy.

[0054] To assess the tumor immune microenvironment after treatment, this embodiment used flow cytometry to quantitatively analyze the proportion of dendritic cells (DCs) and tumor-infiltrating immune cells in the inguinal lymph nodes. Figure 17 As shown in Figure A, the F-EpCAM-CpG group exhibits the highest maturity DC (CD11c). + CD80 + CD86 + The proportion of F-EpCAM-CpG was 56.6%, which was 2.9, 2.0, 2.6, 1.7, and 2.0 times higher than that of the free 5'-FdUR, CpG, F-scramble, F-scramble-CpG, and F-EpCAM groups, respectively. This result highlights the superior immunostimulatory capacity of F-EpCAM-CpG. Subsequently, mature dendritic cells presented tumor-associated antigens to T cells, thereby generating cytotoxic T lymphocytes (CTLs), which infiltrated tumor tissue and exerted anti-tumor effects. To investigate CTL infiltration, tumor tissue was collected and analyzed by flow cytometry. Figure 17 As shown in Figure B, after F-EpCAM-CpG processing, CTL(CD3) + CD8 + The proportion of F-EpCAM-CpG was significantly increased, increasing 5.9 times compared to the saline group. This indicates that F-EpCAM-CpG can enhance T lymphocyte-dependent antitumor immunity.

[0055] In addition, the levels of inflammatory cytokines in serum after treatment were detected using an ELISA kit in the examples. Figure 17 (C). Pro-inflammatory cytokines interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and interleukin-12 (IL-12) were significantly elevated after F-EpCAM-CpG administration, while the anti-inflammatory cytokine IL-10 was significantly inhibited. These results also confirm that F-EpCAM-CpG can induce the release of cell death-related antigens and promote the formation of a potent anti-tumor immune microenvironment.

[0056] 8. In vivo biocompatibility of F-EpCAM-CpG conjugates To assess the in vivo biosafety of F-EpCAM-CpG, this embodiment systematically evaluated liver and kidney function in mice using serum biochemical markers. Figure 18 As shown, the levels of serum biochemical markers related to liver and kidney function in mice treated with F-EpCAM-CpG, including the core liver function indicators alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP), and the key kidney function indicators blood urea nitrogen (BUN), creatinine (CREA), and uric acid (UA), were not statistically significantly different from those in the saline control group. These results indicate that F-EpCAM-CpG has no significant toxic effects on the liver and kidney tissues of mice and possesses excellent in vivo biocompatibility.

[0057] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A targeted drug delivery system, characterized in that, The invention comprises a nucleic acid aptamer embedded with a chemotherapeutic drug and a CpG oligonucleotide; wherein the nucleic acid aptamer embedded with the chemotherapeutic drug is a phosphoramidic active monomer in which the thymine deoxyribonucleotide in the nucleic acid aptamer is replaced with fluorouracil; the nucleic acid aptamer includes EpCAM nucleic acid aptamer, AS1411 nucleic acid aptamer, Sgc8c nucleic acid aptamer, VEGF nucleic acid aptamer or HER2 nucleic acid aptamer; the nucleic acid aptamer embedded with the chemotherapeutic drug is covalently linked to the CpG oligonucleotide via a 3'-5' phosphodiester bond.

2. The drug targeted delivery system according to claim 1, characterized in that, The nucleotide sequence of the EpCAM aptamer is shown in SEQ ID NO.2; the nucleotide sequence of the AS1411 aptamer is shown in SEQ ID NO.10; the nucleotide sequence of the Sgc8c aptamer is shown in SEQ ID NO.11; the nucleotide sequence of the VEGF aptamer is shown in SEQ ID NO.12; and the nucleotide sequence of the HER2 aptamer is shown in SEQ ID NO.

13.

3. The drug targeted delivery system according to claim 1 or 2, characterized in that, The nucleotide sequence of the EpCAM aptamer embedded with a chemotherapy drug is shown in SEQ ID NO.

6.

4. The drug targeted delivery system according to claim 1, characterized in that, The nucleotide sequence of the CpG oligonucleotide is shown in SEQ ID NO.

1.

5. The drug targeted delivery system according to claim 1, characterized in that, When the nucleic acid aptamer is the EpCAM nucleic acid aptamer, the nucleotide sequence of the drug targeted delivery system is shown in SEQ ID NO.

8.

6. A method for preparing the drug targeted delivery system according to any one of claims 1 to 5, characterized in that, Includes the following steps: Using nucleic acid solid-phase synthesis technology, all thymine deoxyribonucleotides in the nucleic acid aptamer were replaced with phosphoramidite active monomers of fluorouracil and linked with oligonucleotide CpG to obtain a primary drug targeted delivery system. The primary drug targeted delivery system was annealed at 90°C for 5 min and then cooled to 25-30°C to obtain the drug targeted delivery system.

7. The application of the drug-targeted delivery system according to any one of claims 1 to 5 or the drug-targeted delivery system prepared by the preparation method of claim 6 in the preparation of antitumor drugs.

8. The application according to claim 7, characterized in that, The tumors include malignant tumors that overexpress EpCAM receptors, nucleolin receptors, protein tyrosine kinase 7 receptors, VEGF receptors, or HER2 receptors on their surface.

9. The application according to claim 8, characterized in that, The malignant tumors that overexpress the EpCAM receptor on their surface include one or more of triple-negative breast cancer, colorectal cancer, and gastric cancer.

10. An antitumor drug, characterized in that, The active ingredient includes the drug targeted delivery system according to any one of claims 1 to 5 or the drug targeted delivery system prepared by the preparation method according to claim 6.

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