A drug delivery system based on nucleic acid chemical modification and its preparation method and application

Abstract

The present application relates to a kind of drug delivery system based on nucleic acid chemical modification and its preparation method and application.The drug delivery system includes nucleic acid aptamer coupling drug and antisense nucleic acid coupling drug, and the nucleic acid aptamer coupling drug and antisense nucleic acid coupling drug are co-assembled to form nanoparticle;The nucleic acid aptamer coupling drug includes nucleic acid aptamer and the chemotherapeutic drug coupled therewith;The antisense nucleic acid coupling drug includes antisense nucleic acid and the photosensitizer coupled therewith.The present application can reduce the up-regulation of the expression level of anti-apoptotic gene caused by chemotherapy and photodynamic therapy in tumor site by assembling tumor-targeting nucleic acid aptamer, chemotherapeutic drug, photosensitizer and antisense nucleic acid into drug delivery system, realizes the combination of chemotherapy, photodynamic therapy and gene therapy, efficiently inhibits tumor growth, and preparation method is simple and easy to operate, with universality, large drug loading, high production efficiency, it is expected to be applied in the treatment of various diseases, and promote the popularization and use of related products.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to a drug delivery system based on nucleic acid chemical modification, its preparation method and application. Background Technology

[0002] Prostate cancer is one of the leading causes of cancer death in men. Androgen deprivation therapy is considered the standard treatment for metastatic prostate cancer, but patients may develop castration resistance, leading to treatment failure. Furthermore, the overall efficacy of chemotherapy and hormone therapy is not ideal. Therefore, comprehensively managing castration resistance and drug resistance in prostate cancer is crucial for its treatment.

[0003] HSP27, a member of the heat shock protein (HSP) family, is highly expressed in various types of tumors and widely participates in tumorigenesis and development, becoming a major molecular marker for assessing treatment efficacy and prognosis. In prostate cancer patients, HSP27 is a key protein in tumor progression, promoting the folding and transport of androgen receptors. Increased HSP27 expression exacerbates castration resistance in prostate cancer, enhances tumor anti-apoptosis and drug resistance, and reduces the effectiveness of tumor treatment. Therefore, targeting HSP27 in prostate cancer is a very promising tumor treatment strategy. Gene therapy, which treats or prevents diseases by supplementing or correcting related genes, is receiving increasing attention. Utilizing antisense nucleic acids to reduce HSP27 expression is of great significance for addressing castration resistance and drug resistance in prostate cancer.

[0004] Nucleic acid nanostructures possess precise structural controllability and good biocompatibility, showing great potential in molecular diagnostics and drug delivery. However, nucleic acid nanostructures internalized into cells often require an endosome escape process to ultimately exert their efficacy. Endosomes contain a large number of nucleolytic enzymes that degrade the nucleic acid structure and nucleic acid drugs, thereby reducing their therapeutic effect.

[0005] In conclusion, developing a nucleic acid nano-drug delivery system capable of endosome escape is of great significance in the field of drug development. Summary of the Invention

[0006] To address the shortcomings of existing technologies and practical needs, this invention provides a drug delivery system based on nucleic acid chemical modification, its preparation method, and its applications. The drug delivery system is co-assembled from nucleic acid aptamer-conjugated drugs and antisense nucleic acid-conjugated drugs, enabling efficient delivery of chemotherapeutic drugs, photosensitizers, and antisense nucleic acids. It exhibits good biosafety, promotes endosome escape under laser irradiation, and improves the combined therapeutic effect of chemotherapy, photodynamic therapy, and gene therapy.

[0007] To achieve this objective, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides a drug delivery system based on nucleic acid chemical modification, the drug delivery system comprising a nucleic acid aptamer-conjugated drug and an antisense nucleic acid-conjugated drug, wherein the nucleic acid aptamer-conjugated drug and the antisense nucleic acid-conjugated drug are co-assembled to form nanoparticles; the nucleic acid aptamer-conjugated drug comprises a nucleic acid aptamer and a chemotherapeutic drug conjugated thereto; the antisense nucleic acid-conjugated drug comprises an antisense nucleic acid and a photosensitizer conjugated thereto.

[0009] In this invention, through rational sequence and structural design, the drug delivery system is mainly formed by the co-assembly of two nucleic acid-conjugated drugs. The nucleic acid aptamer-conjugated drug uses a branched organic molecule as its core, covalently integrating multiple chemotherapeutic drug molecules and nucleic acid aptamers into a single structure to form a nucleic acid aptamer-conjugated drug. This significantly enhances the hydrophobicity of the chemotherapeutic drug end and promotes the formation of nucleic acid nanomedicines. The antisense nucleic acid-conjugated drug is formed by antisense nucleic acid conjugated with a photosensitizer. Under laser irradiation, the drug delivery system can generate a large amount of reactive oxygen species (ROS), disrupting the endosome membrane, promoting the endosome escape of nucleic acid drugs, and enhancing the gene therapy effect.

[0010] In this invention, the target of the nucleic acid aptamer can be a variety of disease-related biomarkers. It is understood that nucleic acid aptamers commonly used in the field are applicable to this invention.

[0011] Preferably, the receptor for the nucleic acid aptamer may include at least one of nucleolin, angiopoietin, vascular endothelial growth factor, mucin 1, or integrin α6β4.

[0012] Preferably, the chemotherapy drug may include at least one of doxorubicin, paclitaxel, docetaxel, oxaliplatin, cabazitaxel, and hydroxycamptothecin (CPT).

[0013] Preferably, the molar ratio of the nucleic acid aptamer to the chemotherapeutic drug is 1:(1-5), which can be 1:1, 1:2, 1:3, 1:4 or 1:5, and more preferably 1:5.

[0014] In this invention, the targets of the antisense nucleic acid include tumor cell apoptosis-related targets. It is understood that antisense nucleic acids commonly used in the art are applicable to this invention.

[0015] Preferably, the tumor cell apoptosis-related target includes the cellular heat shock protein HSP27.

[0016] Preferably, the photosensitizer includes at least one of dihydroporphyrin, pyrophyll-a, and chlorophyll-a.

[0017] Preferably, the molar ratio of the antisense nucleic acid to the photosensitizer is 1:1.

[0018] Preferably, the receptor for the nucleic acid aptamer is integrin α6β4, the chemotherapeutic drug is hydroxycamptothecin, the target of the antisense nucleic acid is tumor cell apoptosis-associated heat shock protein HSP27, and the photosensitizer is clopidogrel.

[0019] Preferably, the nucleic acid sequence of the nucleic acid aptamer includes the sequence shown in SEQ ID NO:1.

[0020] Preferably, the nucleic acid sequence of the antisense nucleic acid includes the sequence shown in SEQ ID NO:2.

[0021] SEQ ID NO:1 (NIA-B, aptamer chain targeting integrin α6β4):

[0022] CGTGCGTATTCGTACTGGAACTGATATCGATGTCCCTTTTTTTTTTTTTTTTTT.

[0023] SEQ ID NO:2 (Antisense nucleic acid strand targeting HSP27):

[0024] TTGGGACGCGGCGCTCGGTCATTT.

[0025] Preferably, the molar ratio of the nucleic acid aptamer conjugate to the antisense nucleic acid conjugate is 1:(1-10), more preferably 1:(2-9), further preferably 1:(3-8), and even more preferably 1:(4-7), such as 1:4, 1:5, 1:6, 1:7, or 1:8.

[0026] In a second aspect, the present invention provides a method for preparing the drug delivery system based on nucleic acid chemical modification as described in the first aspect, the method comprising:

[0027] Preparation of nucleic acid aptamer-conjugated drugs; preparation of antisense nucleic acid-conjugated drugs;

[0028] A drug delivery system is obtained by co-assembling nucleic acid aptamer-conjugated drugs and antisense nucleic acid-conjugated drugs.

[0029] The preparation method provided by this invention is simple, the materials have good repeatability, and it can be mass-produced.

[0030] Preferably, the preparation of the nucleic acid aptamer-conjugated drug specifically includes:

[0031] Synthetic hydrophobic core DBCO-PEG4-(SS-CPT)5;

[0032] The nucleic acid aptamer with an azide-terminated group at the end was subjected to a copper-free click reaction with the hydrophobic core DBCO-PEG4-(SS-CPT)5 to obtain the nucleic acid aptamer-conjugated drug.

[0033] In this invention, a branched hydrophobic drug, DBCO-PEG4-(SS-CPT)5 (Formula I), containing five disulfide-linked camptothecin (CPT) molecules modified with DBCO at the ends, is synthesized. This is then reacted with an azide-modified nucleic acid aptamer (Apt-N3) in a click chemistry reaction to obtain the nucleic acid aptamer-conjugated drug Apt-DBCO-PEG4-(SS-CPT)5. Chemotherapy drugs such as CPT molecules, with their hydroxyl groups coupled to disulfide-linked functional groups, exhibit reduction responsiveness. Under the influence of high concentrations of glutathione (GSH) within tumor cells, they release the parent drug, thus exerting an anti-tumor effect.

[0034]

[0035] The nucleic acid sequence of the aptamer (Apt-N3) with an azide group modified at the 3' end is shown below:

[0036] CGTGCGTATTCGTACTGGAACTGATATCGATGTCCCTTTTTTTTTTTTTTTTTT-N3.

[0037] Preferably, the molar ratio of the nucleic acid aptamer with the terminal modified azide group to the hydrophobic core DBCO-PEG4-(SS-CPT)5 is 1:(1 to 20), for example, it can be 1:1, 1:2, 1:5, 1:10, 1:15 or 1:20, etc., preferably 1:15.

[0038] Preferably, the reaction temperature of the copper-free click reaction is 40-60°C, for example, 40°C, 45°C, 50°C, 55°C or 60°C, preferably 50°C.

[0039] Specifically, the reaction conditions for click chemistry are as follows: Apt-N3 (1.0 nmol, 50 μM) dissolved in 20 μL ddH2O is mixed with DBCO-PEG4-(SS-CPT)5 (15 nmol, 1 mM) dissolved in 15 μL DMSO, and the mixture is stirred and shaken for 24 h. DMSO in the reaction system is removed through a dialysis bag, and then excess DBCO-PEG4-(SS-CPT)5 is removed by high-speed centrifugation (14000 rpm, 10 min) using a separation column. The coupling product is then purified and recovered by polyacrylamide gel electrophoresis.

[0040] Preferably, the preparation of the antisense nucleic acid conjugate drug includes:

[0041] The antisense nucleic acid with terminal modifications of disulfide bonds and amino groups is subjected to a dehydration condensation reaction with a photoclox molecule (Formula II) activated by N-hydroxysuccinimide to obtain the antisense nucleic acid conjugate drug.

[0042] Preferably, the 3' end of the antisense nucleic acid is modified with -SS- and NH2-C6, and its nucleic acid sequence is as follows: TTGGGACGCGGCGCTCGGTCATTT-SS-C6-NH2.

[0043] Preferably, the molar ratio of the antisense nucleic acid with terminal disulfide bonds and amino groups to the photoclone molecule activated by N-hydroxysuccinimide is 1:(1-200), for example, it can be 1:1, 1:10, 1:50, 1:100 or 1:200, preferably 1:100.

[0044] Specifically, the antisense nucleic acid of heat shock protein modified with terminal disulfide bonds and amino groups (AS-SS-NH2) (10 nmol, 0.2 mM) can be dissolved in 50 μL ddH2O, and the activated N-hydroxysuccinimide-modified photoclox HPPH (1.0 μmol, 10 mM) can be dissolved in 100 μL DMSO. The two can then be mixed evenly and 1.0 μL triethylamine can be added. The reaction can be maintained at room temperature for 12 h. Then, the excess activated HPPH can be removed by ethanol precipitation, and AS-SS-HPPH can be purified and recovered by gel electrophoresis.

[0045]

[0046] Preferably, after co-assembly, the drug delivery system is harvested using dialysis.

[0047] Preferably, the molar ratio of the nucleic acid aptamer conjugate to the antisense nucleic acid conjugate is 1:(1 to 10), for example, it can be 1:1, 1:2, 1:5 or 1:10, etc., preferably 1:5.

[0048] Specifically, the co-assembly conditions can be as follows: dissolve two nucleic acid-conjugated drugs in DMSO, remove the DMSO by dialysis, and simultaneously obtain the nucleic acid nanomedicine. The morphology of the co-assembled product is characterized using atomic force microscopy (AFM). The AFM sample scanning mode can be solid-phase or liquid-phase mode, preferably solid-phase scanning mode.

[0049] Thirdly, the present invention provides the application of the nucleic acid chemical modification-based drug delivery system described in the first aspect in the preparation of drugs for treating tumors.

[0050] Preferably, the tumor includes any one of prostate cancer, breast cancer, liver cancer, ovarian cancer, non-small cell carcinoma, or cervical cancer.

[0051] Compared with the prior art, the present invention has at least the following beneficial effects:

[0052] (1) The drug delivery system of the present invention integrates a nucleic acid aptamer, a chemotherapeutic drug, a photosensitizer, and an antisense nucleic acid onto a single nucleic acid nanostructure, achieving precise loading of the chemotherapeutic drug, photosensitizer, and antisense nucleic acid drug. Utilizing the nucleic acid aptamer and EPR effect, the nucleic acid nanomedicine can target tumors. Under laser irradiation, the ROS generated by the photosensitizer can disrupt the endosome membrane, promoting the endosome escape of the antisense nucleic acid drug. The nucleic acid nanomedicine enhances the targeting and delivery efficiency of the drug.

[0053] (2) The target gene in the drug delivery system of the present invention is arbitrary and highly versatile. It does not involve complex organic synthesis or other chemical reaction processes. It can be obtained by simple nucleic acid chemical modification and co-assembly. The preparation method is simple and can achieve large-scale production.

[0054] (3) From the perspective of biosafety, the nucleic acid nanostructure of the present invention has good biocompatibility and low toxicity compared with carriers such as polymers, liposomes and inorganic nanoparticles. It has great potential in drug delivery and can be applied to a wider range of biological disease models. Attached Figure Description

[0055] Figure 1 This is a schematic diagram illustrating the construction method and working principle of a drug delivery system based on nucleic acid chemical modification.

[0056] Figure 2 Flowcharts for the preparation of nucleic acid aptamer-conjugated drugs and antisense nucleic acid-conjugated drugs;

[0057] Figure 3 The image shows the gel electrophoresis results of the nucleic acid conjugate drug prepared in Example 1. In the image, lane 1 is a double-stranded DNA marker of known length, lane 2 is an antisense nucleic acid, lane 3 is an antisense nucleic acid conjugate drug, lane 4 is a nucleic acid aptamer, and lane 5 is a nucleic acid aptamer conjugate drug.

[0058] Figure 4 The image shows an atomic force microscopy characterization of the drug delivery system prepared in Example 1 (scale bar = 50 nm).

[0059] Figure 5 This is a chemotherapy drug release curve of the nucleic acid nanomedicine prepared in Example 1;

[0060] Figure 6 This image shows the internalization effect of nucleic acid nanomedicines on tumor cells.

[0061] Figure 7 A statistical graph showing the internalization results of nucleic acid nanomedicines by tumor cells;

[0062] Figure 8 This image shows the escape effect of nucleic acid nanomedicines in tumor cell lysosomes.

[0063] Figure 9 A statistical graph showing the escape efficiency of nucleic acid nanomedicines in tumor cell lysosomes;

[0064] Figure 10 This is a graph showing the results of the inhibition of tumor cell activity by nucleic acid nanomedicines;

[0065] Figure 11 This is a graph showing the results of apoptosis induction in tumor cells by nucleic acid nanomedicines.

[0066] Figure 12 This image shows the silencing effect of nucleic acid nanomedicine on HSP27 mRNA in tumor cells. Detailed Implementation

[0067] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments. However, the following examples are merely simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention is determined by the claims.

[0068] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased from legitimate channels.

[0069] The instruments and materials used in specific embodiments of the present invention are as follows:

[0070] Equipment: Microcentrifuge (ThermoFisher, USA), UV-Vis spectrophotometer (Shimadzu, Japan), multimode scanning probe microscope (Bruker, Germany), confocal microscope (Zeiss 710, Germany), flow cytometer (Agilent, Germany), full-wavelength microplate reader (TECAN, Switzerland), 671nm laser (Changchun New Industries, China), real-time PCR instrument (Real-4, Germany).

[0071] Raw materials: Terminally chemically modified deoxyribonucleotide sequences were purchased from Shanghai Sangon Biotech Co., Ltd.

[0072] Hydroxycamptothecin (CPT) was purchased from Shanghai Bid Pharmaceutical Technology Co., Ltd.

[0073] The HPPH light was purchased from Beijing Innocare Technology Co., Ltd.

[0074] DBCO-PEG4-(SS-CPT)5 was synthesized according to the reference (Angewandte Chemie International Edition, 2015, 54, 1946-1949).

[0075] Reagents: Cell viability assay kit was purchased from Dojin Chemicals, Japan; apoptosis assay kit was purchased from Beijing Dongge Boye Biotechnology Co., Ltd.; qPCR assay kit was purchased from Beijing Yuanchang Zhihe Biotechnology Co., Ltd.; Hoechst33342 and LysoTracker Green were purchased from Shanghai Beyotime Biotechnology Co., Ltd.

[0076] Cells: The human prostate cancer cell line PC-3 was purchased from the Cell Center of the Institute of Basic Medical Sciences, Peking Union Medical College.

[0077] Culture media: 1640 medium, fetal bovine serum and double antibodies were purchased from ThermoFisher Scientific.

[0078] Example 1

[0079] Figure 1 This is a schematic diagram illustrating the preparation method and working principle of a drug delivery system based on nucleic acid chemical modification. Figure 2 This is a flowchart illustrating the preparation of nucleic acid aptamer-conjugated drugs and antisense nucleic acid-conjugated drugs. Based on the above diagram, the specific preparation steps of the nucleic acid chemically modified drug delivery system are as follows:

[0080] (1) Preparation of nucleic acid aptamer-conjugated drugs

[0081] The receptor for the nucleic acid aptamer (IDA-B) is integrin α6β4, which is highly expressed in the human prostate cancer cell line PC-3. Azide-modified nucleic acid aptamer Apt-N3 (SEQ ID NO:1, 1.0 nmol, 50 μM) dissolved in 20 μL ddH2O was mixed with DBCO-PEG4-(SS-CPT)5 (15 nmol, 1 mM) dissolved in 15 μL DMSO (molar ratio 1:15), and the mixture was stirred and reacted at 50 °C for 24 h. DMSO was removed from the reaction system using a dialysis bag, followed by centrifugation at high speed (14000 rpm, 10 min) using a separation column to remove excess DBCO-PEG4-(SS-CPT)5. Finally, the nucleic acid aptamer-conjugated drug Apt-PEG4-(SS-CPT)5 was purified and recovered by polyacrylamide gel electrophoresis.

[0082] (2) Preparation of antisense nucleic acid conjugate drugs

[0083] Antisense nucleic acid AS-SS-NH2 (SEQ ID NO:2, 10 nmol, 200 μM) with terminal disulfide and amino groups was dissolved in 50 μL ddH2O, and HPPH (1.0 μmol, 10 mM) activated by N-hydroxysuccinimide was dissolved in 100 μL DMSO. The two solutions (molar ratio 1:100) were then uniformly mixed and 1.0 μL triethylamine was added. The reaction was carried out at room temperature for 12 h. Excess HPPH activated by N-hydroxysuccinimide was then removed by ethanol precipitation, and AS-SS-HPPH was subsequently purified and recovered by gel electrophoresis.

[0084] The above-mentioned nucleic acids and nucleic acid-conjugated drugs were detected by electrophoresis in 8% polyacrylamide, and the results are as follows: Figure 3 As shown, lane 1 represents a double-stranded DNA marker of known length, with electrophoretic speeds from fastest to slowest: 20, 40, 60, 80, 100, 120, 140, 160, 180, and 200 base pairs. Lane 2 contains antisense nucleic acid, lane 3 contains antisense nucleic acid-conjugated drugs, lane 4 contains nucleic acid aptamers, and lane 5 contains nucleic acid aptamer-conjugated drugs. Compared to the nucleic acid precursor bands, the electrophoretic speed of the nucleic acid-conjugated drugs is slower, indicating successful synthesis of the nucleic acid-conjugated drugs.

[0085] (3) Co-assembly of aptamer-conjugated drugs and antisense nucleic acid-conjugated drugs

[0086] The molar ratio of nucleic acid aptamer-conjugated drug and antisense nucleic acid conjugated drug is 1:5. The two nucleic acid conjugated drugs are dissolved in DMSO and the DMSO is removed by dialysis to obtain a co-assembled drug delivery system.

[0087] The morphology of nucleic acid nanomedicines was characterized using atomic force microscopy (AFM) in ScanAsyst Air scanning mode. The results are as follows: Figure 4 As shown, spherical nucleic acid nanomedicines were successfully assembled, with regular morphology and good dispersibility.

[0088] Example 2

[0089] Compared to Example 1, this example synthesizes a nucleic acid conjugate drug without targeting nucleic acid aptamer modification during the synthesis of the nucleic acid aptamer drug. The nucleic acid sequence of the nucleic acid aptamer is SEQ ID NO:3. It is then co-assembled with the same antisense nucleic acid conjugate drug to form a nucleic acid nanomedicine without targeting modification, under the same conditions as in Example 1.

[0090] SEQ ID NO:3 (Untargeted group): TTTTTTTTTTTTTTTTTTT-N3.

[0091] Example 3

[0092] Compared with Example 1, in step (1), the molar ratio of the nucleic acid aptamer with terminal azide group to DBCO-PEG4-(SS-CPT)5 was 1:1, and the reaction temperature was 40℃. In step (2), the molar ratio of the antisense nucleic acid with terminal disulfide bond and amino group to HPPH molecule was 1:1. In step (3), the molar ratio of nucleic acid aptamer conjugate drug to antisense nucleic acid conjugate drug was 1:1. Other conditions were the same as in Example 1.

[0093] This embodiment simply increases the molar ratio of reactants in each step and lowers the reaction temperature, yet it can still efficiently obtain nucleic acid-conjugated drugs for use in assembling drug delivery systems.

[0094] Example 4

[0095] Compared with Example 1, in step (1), the molar ratio of the nucleic acid aptamer with terminal azide group to DBCO-PEG4-(SS-CPT)5 was 1:20, and the reaction temperature was 60℃. In step (2), the molar ratio of the antisense nucleic acid with terminal disulfide bond and amino group to HPPH molecule was 1:200. In step (3), the molar ratio of nucleic acid aptamer conjugate drug to antisense nucleic acid conjugate drug was 1:10. Other conditions were the same as in Example 1.

[0096] This embodiment simply reduces the molar ratio of reactants in each step and increases the reaction temperature, yet it can still efficiently obtain nucleic acid-conjugated drugs for use in assembling drug delivery systems.

[0097] Example 5

[0098] This embodiment investigates the ability of a drug delivery system to release chemotherapy drugs, and the steps are as follows:

[0099] 500 μL of nucleic acid nanomedicine (prepared in Example 1) was placed in a dialysis bag (3000 Da). The dialysis bag containing the nucleic acid nanomedicine was then placed in a centrifuge tube containing 10 mL of glutathione GSH (10 mM) solution. The mixture was shaken at 120 rpm and 37°C. 100 μL of buffer solution was collected at 1, 2, 4, 8, and 12 h for analysis. The samples taken at each time point were analyzed for fluorescence using an ELISA reader to quantify the efficiency of chemotherapeutic drug release.

[0100] The results are as follows Figure 5 As shown, nucleic acid nanomedicines can rapidly release chemotherapy drugs under GSH reduction conditions, releasing more than 60% of the chemotherapy drugs within 12 hours, which is beneficial to enhancing the effect of chemotherapy.

[0101] Example 6

[0102] This embodiment investigates the targeted cell internalization efficiency of a drug delivery system, using the drug delivery system as a nucleic acid nanomedicine. The steps are as follows:

[0103] Count 5×10 4 PC-3 cells were seeded in confocal imaging dishes and incubated overnight at 37°C. The culture medium was discarded, and unmodified nucleic acid nanomedicine (prepared in Example 2) and targeted nucleic acid nanomedicine (prepared in Example 1) were added, with a final HPPH concentration of 1 μM in both experiments. After incubation for 4 h, the drug-containing culture medium was removed, and the cells were washed three times with PBS and stained with Hoechst 33342 for 5 min. Then, the cells were washed three times with PBS, placed in PBS solution, and the uptake of the nucleic acid nanomedicine by PC-3 cells was observed using a laser confocal microscope.

[0104] The results are as follows Figure 6 As shown, targeted modification significantly enhanced the uptake of nucleic acid nanomedicines by PC-3 cells. The intracellular fluorescence intensity of CPT and HPPH in the targeted modified nucleic acid nanomedicines was significantly enhanced compared with the untargeted modification group.

[0105] PC-3 cells were loaded at 5 × 10 4 Cells were seeded at a density of / well in 24-well plates and incubated overnight in a cell culture incubator. After culture, the culture medium was discarded, and untargeted nucleic acid nanomedicine (prepared in Example 2) and targeted nucleic acid nanomedicine (prepared in Example 1) were added, respectively. The final concentration of HPPH in both experiments was 1 μM. After incubation for 4 h, the drug-containing culture medium was removed, the cells were washed three times with PBS, digested with trypsin, and cell uptake was detected by flow cytometry (HPPH fluorescence).

[0106] The results are as follows Figure 7 As shown, targeted modification significantly enhanced the uptake of nucleic acid nanomedicines by PC-3 cells, with the uptake of targeted modified nucleic acid nanomedicines by cells being 1.5 times that of the untargeted modification group.

[0107] Example 7

[0108] This embodiment investigates the lysosomal escape effect of nucleic acid nanomedicines, and the steps are as follows:

[0109] PC-3 cells were loaded at 5 × 10 4 Cells were seeded at a density of / wells in confocal imaging dishes and cultured overnight in a cell culture incubator. Targeted nucleic acid nanomedicine (prepared in Example 1, HPPH 1 μM) was added to the confocal imaging dishes and co-incubated with the cells for 4 h. The drug-containing medium was then removed, and the cells were washed three times with PBS. For the targeted nucleic acid nanomedicine + light irradiation group, laser irradiation was applied for 1 min (671 nm, 200 mW / cm²). 2Subsequently, Lyso tracker Green (1000×) was added and incubated for 45 min, followed by Hoechst 33342 (1000×) staining for 5 min. Then, the mixture was washed three times with PBS, placed in PBS solution, and the lysosomal escape of the nucleic acid nanomedicine was observed using a laser confocal microscope.

[0110] The results are as follows Figure 8 As shown, after nucleic acid nanomedicines are taken up by cells, fluorescence is mainly located in lysosomes. However, after laser irradiation, a large amount of fluorescence appears outside the lysosomes, indicating that ROS generated by laser irradiation of photosensitizers can promote the lysosomal escape of nucleic acid nanomedicines.

[0111] The results were analyzed using ImageJ software. The results are as follows: Figure 9 As shown, after laser irradiation, the Mandel colocalization coefficient of lysosomes and nucleic acid nanomedicines decreased from 0.89 to 0.35, indicating that the ROS generated by the photosensitizer by laser irradiation can promote the lysosomal escape of nucleic acid nanomedicines.

[0112] Example 8

[0113] This embodiment investigates the inhibitory effect of nucleic acid nanomedicines on the activity of human prostate cancer cell line PC-3. The steps are as follows:

[0114] PC-3 cells were seeded at a density of 3500 cells / well in 96-well plates and cultured overnight. Drugs were added to the 96-well plates according to the following groups: nucleic acid aptamer-conjugated drug group, antisense nucleic acid-conjugated drug group, nucleic acid nanomedicine group, and nucleic acid nanomedicine + light irradiation group (prepared in Example 1). All four groups had the same drug concentration, with the nucleic acid aptamer-conjugated drug concentration gradients of 150 nM, 300 nM, and 600 nM. For the nucleic acid nanomedicine + light irradiation group, after 12 hours of incubation, the culture medium was replaced, and the cells were irradiated using a 671 nm laser at 200 mW / cm². 2 Cells were irradiated with high intensity for 1 min. After laser irradiation, the medium was replaced with drug-containing medium, and incubation continued for 48 h. 100 μL of 1640 medium containing cell viability assay reagent was added to each well, and incubation continued for 1 h. The OD values ​​of each well at 450 nm and 620 nm were measured using a microplate reader. The survival rate of tumor cells was calculated based on the OD values ​​using the formula: Survival rate % = Experimental group (OD value of experimental group) / ... 450 -OD 620 ) / Control group (OD 450 -OD 620 )×100%.

[0115] The results are as follows Figure 10As shown, the nucleic acid nanomedicine + light irradiation group has a significant killing effect on tumor cells, resulting in a decrease in tumor cell viability of more than 80%, indicating that the co-assembled nucleic acid nanomedicine significantly enhances the combined therapeutic effect of chemotherapy, photodynamic therapy and gene therapy.

[0116] Example 9

[0117] This embodiment investigates the apoptosis-inducing effect of nucleic acid nanomedicines on the human prostate cancer cell line PC-3. The steps are as follows:

[0118] PC-3 cells were seeded at a density of 3500 cells / well in 96-well plates and cultured overnight. Drugs were added to the 96-well plates according to the following groups: nucleic acid aptamer-conjugated drug group, antisense nucleic acid-conjugated drug group, nucleic acid nanomedicine group, and nucleic acid nanomedicine + light irradiation group (prepared in Example 1), with the same drug concentration for all four groups. The concentration of the nucleic acid aptamer-conjugated drug was 600 nM. For the nucleic acid nanomedicine + light irradiation group, after incubation for 12 hours, the culture medium was replaced, and a 671 nm laser was used at 200 mW / cm². 2 Cells were irradiated with laser light for 1 minute, then the medium was replaced with drug-containing medium, and incubation continued for 48 hours. Cells were stained using an apoptosis detection kit, and the fluorescence intensity of each group of cells was detected using flow cytometry.

[0119] The results are as follows Figure 11 As shown, nucleic acid aptamer-conjugated drugs, antisense nucleic acid conjugated drugs, and nucleic acid nanomedicines induced apoptosis in 60%, 11%, and 76% of tumor cells, respectively, while the nucleic acid nanomedicine + light irradiation group induced apoptosis in 85% of tumor cells. This demonstrates the application potential of the co-assembled nucleic acid nanomedicines described in this invention in tumor targeted therapy, and is expected to be used for targeted therapy of other related diseases.

[0120] Example 10

[0121] This embodiment investigates the effect of nucleic acid nanomedicines on the expression of HSP27 mRNA in the human prostate cancer cell line PC-3. The steps are as follows:

[0122] PC-3 cells were seeded at a density of 3500 cells / well in 96-well plates and cultured overnight. Drugs were added to the 96-well plates according to the following groups: nucleic acid aptamer-conjugated drug group, antisense nucleic acid-conjugated drug group, nucleic acid nanomedicine group, and nucleic acid nanomedicine + light irradiation group (prepared in Example 1), with the same drug concentration for all four groups. The concentration of the nucleic acid aptamer-conjugated drug was 600 nM. For the nucleic acid nanomedicine + light irradiation group, after incubation for 12 hours, the culture medium was replaced, and a 671 nm laser was used at 200 mW / cm². 2Cells were irradiated with laser light for 1 min, then the medium was replaced with drug-containing medium, and incubation continued for 48 h. The effect of globular nucleic acid on HSP27 mRNA expression in the human prostate cancer cell line PC-3 was detected using qPCR.

[0123] The results are as follows Figure 12 As shown, the nucleic acid nanomedicine + light irradiation group significantly downregulated the expression of HSP27 mRNA in tumor cells, decreasing it by more than 70%, indicating that nucleic acid nanomedicine significantly reduced the expression of HSP27 mRNA under laser irradiation, which helps to enhance the therapeutic effect of tumors.

[0124] In summary, this invention integrates tumor-targeting nucleic acid aptamers, chemotherapeutic drugs, photosensitizers, and antisense nucleic acids to form a drug delivery system that can serve as a nucleic acid nanomedicine. Under laser irradiation, the ROS generated by the photosensitizer disrupts the endosomal membrane, promotes the release of antisense nucleic acids, and reduces the upregulation of antiapoptotic gene expression levels in tumor sites caused by chemotherapy and photodynamic therapy. This achieves targeted delivery and controlled release of drugs, realizing the combination of chemotherapy, photodynamic therapy, and gene therapy. It exhibits good biosafety and broad application prospects.

[0125] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. A drug delivery system based on chemical modification of nucleic acids, characterized in that, The drug delivery system includes a nucleic acid aptamer-conjugated drug and an antisense nucleic acid-conjugated drug, which are co-assembled to form nanoparticles. The nucleic acid aptamer-conjugated drug includes a nucleic acid aptamer and a chemotherapy drug conjugated thereto; The antisense nucleic acid conjugate includes an antisense nucleic acid and a photosensitizer conjugated thereto; The receptor for the nucleic acid aptamer is integrin α6β4, the chemotherapeutic drug is hydroxycamptothecin, the target of the antisense nucleic acid is tumor cell apoptosis-associated heat shock protein HSP27, and the photosensitizer is chloroquine. The nucleic acid sequence of the nucleic acid aptamer is the sequence shown in SEQ ID NO: 1; The nucleic acid sequence of the antisense nucleic acid is the sequence shown in SEQ ID NO:

2.

2. The drug delivery system based on nucleic acid chemical modification according to claim 1, characterized in that, The molar ratio of the nucleic acid aptamer to the chemotherapy drug is 1:(1~5).

3. The drug delivery system based on nucleic acid chemical modification according to claim 1, characterized in that, The molar ratio of the antisense nucleic acid to the photosensitizer is 1:

1.

4. The drug delivery system based on nucleic acid chemical modification according to claim 1, characterized in that, The molar ratio of the nucleic acid aptamer conjugate to the antisense nucleic acid conjugate is 1:(1~10).

5. A method for preparing a drug delivery system based on nucleic acid chemical modification as described in any one of claims 1-4, characterized in that, The preparation method includes: Preparation of nucleic acid aptamer-conjugated drugs; preparation of antisense nucleic acid-conjugated drugs; A drug delivery system is obtained by co-assembling nucleic acid aptamer-conjugated drugs and antisense nucleic acid-conjugated drugs.

6. The method for preparing a drug delivery system based on nucleic acid chemical modification according to claim 5, characterized in that, The preparation of the nucleic acid aptamer-conjugated drug specifically includes: Synthetic hydrophobic core DBCO-PEG4-(SS-CPT)5; The nucleic acid aptamer with an azide-terminated group at the end was subjected to a copper-free click reaction with the hydrophobic core DBCO-PEG4-(SS-CPT)5 to obtain the nucleic acid aptamer-conjugated drug.

7. The method for preparing a drug delivery system based on nucleic acid chemical modification according to claim 6, characterized in that, The molar ratio of the nucleic acid aptamer with the terminal modified azide group to the hydrophobic core DBCO-PEG4-(SS-CPT)5 is 1:(1~20).

8. The method for preparing a drug delivery system based on nucleic acid chemical modification according to claim 6, characterized in that, The reaction temperature for the copper-free click reaction is 40~60℃.

9. The method for preparing a drug delivery system based on nucleic acid chemical modification according to claim 5, characterized in that, The preparation of the antisense nucleic acid conjugate includes: The antisense nucleic acid with terminal modifications of disulfide bonds and amino groups was subjected to a dehydration condensation reaction with a photoclox molecule activated by N-hydroxysuccinimide to obtain the antisense nucleic acid conjugate drug.

10. The method for preparing a drug delivery system based on nucleic acid chemical modification according to claim 9, characterized in that, The molar ratio of the antisense nucleic acid with terminal modifications of disulfide bonds and amino groups to the light clox molecule activated by N-hydroxysuccinimide is 1:(1~200).

11. The method for preparing a drug delivery system based on nucleic acid chemical modification according to claim 5, characterized in that, The molar ratio of the nucleic acid aptamer conjugate to the antisense nucleic acid conjugate is 1:(1~10).

12. The use of the nucleic acid chemically modified drug delivery system as described in any one of claims 1-4 in the preparation of a drug for treating prostate cancer.

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