A drug carrier based on nucleic acid aptamer modification

Abstract

This invention discloses a drug carrier based on nucleic acid aptamer modification, belonging to the field of biomedical materials technology. The carrier uses L-cysteine-modified chitosan as a substrate, and introduces the nucleic acid aptamer L-AS1411, combined with functional agents and single-stranded DNA. The functional agents include tetrahydrofurfuryl methacrylate and sec-butyl methacrylate, which are then photopolymerized to form a hydrogel. Compared to natural D-AS1411, L-AS1411 is resistant to degradation by nucleases and proteases, improving the stability and targeting duration of the carrier in serum and in vivo circulation. The resulting carrier has a dense three-dimensional network structure, high drug loading capacity, low in vitro degradation rate, low cytotoxicity, and excellent biocompatibility, enabling efficient targeting of tumor cells and providing a novel and efficient drug delivery system for targeted tumor therapy.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, specifically relating to a drug carrier based on nucleic acid aptamer modification. Background Technology

[0002] The targeting and stability of drug carriers are core factors determining drug delivery efficiency and therapeutic efficacy. In the construction of targeted drug carriers, cell-targeting recognition molecules must possess high specificity and strong affinity. Nucleic acid aptamers, with their precise recognition capabilities of target molecules, have demonstrated unique advantages in targeted therapy for diseases such as tumors. Among them, the nucleic acid aptamer AS1411, due to its specific binding to nucleolin highly expressed on the surface of tumor cells, has become a hot molecule in tumor-targeting carrier design.

[0003] However, the natural D-type aptamer, D-AS1411, faces severe challenges in the physiological environment: degradation by nucleases in vitro, destruction by proteases in serum, and rapid clearance in vivo circulation significantly limit its targeting efficiency and duration of action, greatly restricting its application as a drug carrier. In contrast, the L-type aptamer, L-AS1411, through conformational inversion, forms a spatial structure complementary to D-AS1411, effectively resisting degradation by nucleases and biological enzymes, and significantly improving its stability in serum and in vivo environments. This provides a new approach for extending the targeting time of the carrier and improving therapeutic efficacy.

[0004] Hydrogels, as a class of biomaterials with a three-dimensional network structure, have attracted much attention in the field of drug delivery due to their excellent biocompatibility, tunable swelling properties, and sustained drug release capabilities. Combining nucleic acid aptamers with hydrogels can endow carriers with targeted recognition functions, further expanding their applications in precision medicine. However, existing nucleic acid aptamer-modified hydrogel carriers still suffer from insufficient stability and limited targeting efficiency, especially the easily degradable nature of D-AS1411, which makes it difficult for the carrier to maintain a long-term targeting effect in vivo. Therefore, developing hydrogel drug carriers with excellent stability (L-AS1411) as the targeting unit, possessing both high biocompatibility and structural stability, is of great significance for promoting the development of targeted cancer therapy. Summary of the Invention

[0005] The purpose of this invention is to provide a drug carrier based on nucleic acid aptamer modification with excellent stability and good biocompatibility, which solves the problems of natural D-AS1411 being easily degraded by nucleases and proteases in physiological environment, having low targeting efficiency and short duration of action. The invention utilizes the dense three-dimensional network structure of hydrogel to improve drug loading rate and reduce in vitro degradation rate, while ensuring that the carrier has low cytotoxicity and excellent biocompatibility, thus providing a novel and efficient drug delivery system for tumor targeted therapy.

[0006] The technical solution adopted by the present invention to achieve the above objectives is as follows:

[0007] A drug carrier, wherein the drug carrier is a hydrogel comprising L-cysteine-modified chitosan and a nucleic acid aptamer, wherein the nucleic acid aptamer comprises L-AS1411, and the mass ratio of the nucleic acid aptamer to L-cysteine-modified chitosan is 250-2500 μg: 5-50 mg.

[0008] Preferably, the hydrogel includes a functional agent.

[0009] Preferably, the functional agents include tetrahydrofurfuryl methacrylate and sec-butyl methacrylate.

[0010] Preferably, the mass ratio of tetrahydrofurfuryl methacrylate to modified chitosan is 2-20:5-50.

[0011] Preferably, the mass ratio of sec-butyl butyl ester to modified chitosan is 2-20:5-50.

[0012] Tetrahydrofurfuryl methacrylate and sec-butyl methacrylate form covalent crosslinks through photopolymerization with modified chitosan, jointly regulating the physicochemical properties of the three-dimensional network structure of the hydrogel. Under their synergistic effect, they can form a dense and stable network skeleton, reducing the swelling and degradation of the hydrogel in the physiological environment, and retain drugs through the pore structure and intermolecular forces, thereby improving the structural stability, anti-degradation ability and drug loading efficiency of the carrier.

[0013] Preferably, the reactive monomers for L-cysteine-modified chitosan include chitosan and L-cysteine.

[0014] Preferably, the mass ratio of chitosan to L-cysteine ​​is 5-50:3.5-35.

[0015] Preferably, the catalyst for preparing L-cysteine-modified chitosan includes 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide.

[0016] Preferably, the hydrogel comprises single-stranded DNA.

[0017] Preferably, the single-stranded DNA includes DNA-A and DNA-B, the nucleotide sequence of DNA-A is shown in SEQ ID No. 3, and the nucleotide sequence of DNA-B is shown in SEQ ID No. 4.

[0018] Preferably, the mass ratio of single-stranded DNA to modified chitosan is 500-5000 μg: 5-50 mg.

[0019] A method for preparing a drug carrier includes: reacting an L-cysteine-modified chitosan solution with a functional agent at room temperature; adding a DNA solution and a photoinitiator and then reacting under ultraviolet light in a deoxygenated, sealed environment; adding a nucleic acid aptamer solution and then reacting under vacuum to obtain the drug carrier.

[0020] Preferably, the DNA solution comprises single-stranded DNA and Tris-HCl buffer solution.

[0021] Preferably, the nucleic acid aptamer solution comprises a nucleic acid aptamer and a Tris-HCl buffer solution.

[0022] Preferably, the drug carrier targets and binds to nucleolin on the surface of tumor cells.

[0023] More preferably, the functional agent includes ethyl 4-aminocrotonic acid (-2-methoxy) ester, and the mass ratio of ethyl 4-aminocrotonic acid (-2-methoxy) ester to modified chitosan is 2-20:5-50. After ethyl 4-aminocrotonic acid (-2-methoxy) ester participates in the construction of the hydrogel network as a crosslinking monomer, the hydrophilic groups in its molecular structure enhance the adsorption and retention capacity of drugs through hydrogen bonds; it increases the crosslinking sites, making the three-dimensional network structure of the hydrogel more compact, further reducing drug leakage; the presence of hydrophilic groups improves the stability of the hydrogel in the buffer solution, reduces structural damage caused by swelling, thereby synergistically improving the drug loading rate and anti-degradation performance of the carrier.

[0024] The present invention also provides a method for preparing modified chitosan, comprising: dissolving L-cysteine ​​in deionized water, adding 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide, activating for 1-3 h to obtain an activated L-cysteine ​​solution; dissolving chitosan in 0.5-1.5 wt% glacial acetic acid solution, stirring evenly, adding the activated L-cysteine ​​solution, stirring evenly, reacting at room temperature for 20-25 h, transferring the reacted solution to a dialysis bag, dialyzing for 70-80 h, and freeze-drying the dialyzed solution to obtain modified chitosan.

[0025] Preferably, the mass-to-volume ratio of L-cysteine ​​to deionized water is 3.5-35g:150-1500mL.

[0026] Preferably, the mass ratio of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride to L-cysteine ​​is 5.75-57.5:3.5-35.

[0027] Preferably, the mass ratio of N-hydroxysuccinimide to L-cysteine ​​is 3.5-35:3.5-35.

[0028] Preferably, the mass-to-volume ratio of chitosan to glacial acetic acid solution is 5-50g:350-3500mL.

[0029] Preferably, the mass of the activated L-cysteine ​​solution is measured by the mass of L-cysteine ​​therein, and the mass ratio of chitosan to L-cysteine ​​is 5-50:3.5-35.

[0030] Preferably, the molecular weight cutoff of the dialysis bag is 3400-3600 Da, and the deionized water is changed every 4-8 hours.

[0031] The present invention also provides a method for preparing a nucleic acid aptamer solution, comprising: dissolving the nucleic acid aptamer in a Tris-HCl buffer solution with a pH of 7.3-7.5 to obtain a nucleic acid aptamer solution.

[0032] Preferably, the nucleic acid aptamer is purchased from Sangon Biotech and includes AS1411 and a connecting arm.

[0033] Preferably, the nucleotide sequence of AS1411 is as shown in SEQ ID No.1, which is 5'-GGTGGTGGTGGTTGTGGTGGTGGTGG-3'.

[0034] Preferably, the configuration of AS1411 is L-AS1411.

[0035] Preferably, the nucleotide sequence of the nucleic acid aptamer is as shown in SEQ ID No. 2, which is 5'-ACTCATCTGTGAATCTCGGTGGTGGTGGTTGTGGTGGTGGTGG-3'.

[0036] Preferably, the mass-to-volume ratio of nucleic acid aptamer to Tris-HCl buffer solution is 250-2500 μg: 125-1250 μL.

[0037] This invention also provides a method for preparing a DNA solution, comprising:

[0038] Single-stranded DNA was dissolved in Tris-HCl buffer solution at pH 7.3-7.5 to obtain DNA solution.

[0039] Preferably, the single-stranded DNA is purchased from Sangon Biotech and includes DNA-A and DNA-B.

[0040] Preferably, the nucleotide sequence of DNA-A is as shown in SEQ ID No. 3, which is 5'-acrydite-AAAACACCACCGAGAT-3'.

[0041] Preferably, the nucleotide sequence of DNA-B is as shown in SEQ ID No. 4, which is 5'-acrydite-AAAATCACAGATGAGT-3'.

[0042] Preferably, the mass-to-volume ratio of single-stranded DNA to Tris-HCl buffer solution is 500-5000 μg: 125-1250 μL.

[0043] Preferably, the mass ratio of DNA-A to DNA-B is 250-2500:250-2500.

[0044] The present invention also provides a method for preparing a drug carrier, comprising: reacting an L-cysteine-modified chitosan solution with a functional agent at room temperature, adding a DNA solution and a photoinitiator and then reacting under ultraviolet light in an oxygen-free, sealed environment, and then reacting under vacuum after adding a nucleic acid aptamer solution to obtain a drug carrier.

[0045] Preferably, the preparation method of the drug carrier specifically includes: dissolving modified chitosan in PBS solution at pH 7.3-7.5, degassing by sonication for 5-15 min, adding functional agent, stirring evenly at room temperature, adding DNA solution, mixing, adding 0.5-0.7 w / v% of photoinitiator 1173, bubbling with nitrogen to remove oxygen for 3-10 min, sealing, irradiating with ultraviolet light at 364-366 nm for 20-40 min, rinsing with PBS 2-4 times, adding nucleic acid aptamer solution, and reacting under vacuum conditions at 36-38℃ for 20-40 min to obtain the drug carrier.

[0046] Preferably, the mass-to-volume ratio of modified chitosan to PBS solution is 5-50 mg: 100-1000 mL.

[0047] Preferably, the functional agents include tetrahydrofurfuryl methacrylate and sec-butyl methacrylate.

[0048] Preferably, the mass ratio of tetrahydrofurfuryl methacrylate to modified chitosan is 2-20:5-50.

[0049] Preferably, the mass ratio of sec-butyl butyl ester to modified chitosan is 2-20:5-50.

[0050] Preferably, the mass of the DNA solution is measured by the mass of the single-stranded DNA therein, and the mass ratio of single-stranded DNA to modified chitosan is 500-5000 μg: 5-50 mg;

[0051] Preferably, the mass of the nucleic acid aptamer solution is measured by the mass of the nucleic acid aptamer therein, and the mass ratio of nucleic acid aptamer to modified chitosan is 250-2500 μg: 5-50 mg.

[0052] Preferably, the mass ratio of photoinitiator 1173 to modified chitosan is 0.5-5:5-50.

[0053] More preferably, the functional agent includes ethyl 4-aminocrotonic acid (-2-methoxy) ester.

[0054] More preferably, the mass ratio of 4-aminocrotonic acid (-2-methoxy) ethyl ester to modified chitosan is 2-20:5-50.

[0055] This invention utilizes L-AS1411 as the targeting unit and modified chitosan as the substrate, crosslinking it with tetrahydrofurfuryl methacrylate, sec-butyl methacrylate, etc., to form a hydrogel with a dense three-dimensional network structure, serving as a drug carrier. Therefore, it has the following beneficial effects: the prepared drug carrier enhances its anti-swelling and anti-degradation capabilities, and also improves the drug loading rate through pore retention and intermolecular forces; moreover, the material itself exhibits low cytotoxicity to Caco-2 cells and excellent biocompatibility. Therefore, this invention is a stable and biocompatible drug carrier based on nucleic acid aptamer modification. Attached Figure Description

[0056] Figure 1 This is a schematic diagram showing the stability test results of L-AS1411 in simulated serum.

[0057] Figure 2 This is a schematic diagram showing the stability test results of D-AS1411 in simulated serum.

[0058] Figure 3 This is a schematic diagram showing the stability test results of nucleic acid aptamers in a living environment.

[0059] Figure 4 This is a schematic diagram of a scanning electron microscope image of a drug carrier.

[0060] Figure 5 This is a schematic diagram of the in vitro degradation test results of the drug carrier.

[0061] Figure 6 This is a schematic diagram showing the drug loading test results for the drug carrier.

[0062] Figure 7 This is a schematic diagram of the biosafety test results for drug carriers. Detailed Implementation

[0063] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0064] The concepts involved in this application will first be described with reference to the accompanying drawings. It should be noted that the following descriptions of various concepts are only for the purpose of making the content of this application easier to understand and do not constitute a limitation on the scope of protection of this application; furthermore, the embodiments and features in the embodiments of this application can be combined with each other unless otherwise specified. This application will now be described in detail with reference to the accompanying drawings and embodiments.

[0065] Example 1: A drug carrier based on nucleic acid aptamer modification is provided, specifically including the following preparation steps.

[0066] Preparation of modified chitosan: L-cysteine ​​was dissolved in deionized water, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide were added. The mixture was activated for 2 hours to obtain an activated L-cysteine ​​solution. Chitosan was dissolved in 1 wt% glacial acetic acid solution and stirred until homogeneous. The activated L-cysteine ​​solution was added and stirred until homogeneous. The mixture was reacted at room temperature for 24 hours. The reacted solution was transferred to a dialysis bag and dialyzed for 72 hours. The dialyzed solution was freeze-dried to obtain modified chitosan. The mass-to-volume ratio of L-cysteine ​​to deionized water was 7 g:300 mL; the mass ratio of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride to L-cysteine ​​was 11.5:7; the mass ratio of N-hydroxysuccinimide to L-cysteine ​​was 7:7; and the mass-to-volume ratio of chitosan to glacial acetic acid solution was 10 g:700 mL. The mass of the activated L-cysteine ​​solution was measured by the mass of L-cysteine ​​contained therein, and the mass ratio of chitosan to L-cysteine ​​was 10:7. The molecular weight cutoff of the dialysis bag was 3500 Da, and the deionized water was changed every 6 hours.

[0067] Preparation of nucleic acid aptamer solution: The nucleic acid aptamer was dissolved in Tris-HCl buffer solution at pH 7.4 to obtain the nucleic acid aptamer solution. The nucleic acid aptamer was purchased from Sangon Biotech and included AS1411 and a linker arm; the nucleotide sequence of AS1411 is shown in SEQ ID No. 1, which is 5'-GGTGGTGGTGGTTGTGGTGGTGGTGG-3', and the conformation of AS1411 is L-AS1411; the nucleotide sequence of the nucleic acid aptamer is shown in SEQ ID No. 2, which is 5'-ACTCATCTGTGAATCTCGGTGGTGGTGGTTGTGGTGGTGGTGG-3'; the mass-to-volume ratio of nucleic acid aptamer to Tris-HCl buffer solution was 500 μg: 250 μL.

[0068] Preparation of DNA solution: Single-stranded DNA was dissolved in Tris-HCl buffer solution at pH 7.4 to obtain DNA solution. The single-stranded DNA was purchased from Sangon Biotech and included DNA-A and DNA-B; the nucleotide sequence of DNA-A is shown in SEQ ID No. 3, which is 5'-acrydite-AAAACACCACCGAGAT-3'; the nucleotide sequence of DNA-B is shown in SEQ ID No. 4, which is 5'-acrydite-AAAATCACAGATGAGT-3'; the mass-to-volume ratio of single-stranded DNA to Tris-HCl buffer solution was 1000 μg:250 μL; the mass ratio of DNA-A to DNA-B was 500:500.

[0069] Preparation of drug carrier: Modified chitosan was dissolved in PBS solution at pH 7.4 and degassed by sonication for 10 min. Tetrahydrofurfuryl methacrylate and sec-butyl methacrylate were added and stirred evenly at room temperature. DNA solution was added and mixed. 0.6 w / v% of photoinitiator 1173 was added and the mixture was bubbled with nitrogen for 5 min to remove oxygen. After sealing, it was irradiated with 365 nm ultraviolet light for 30 min. The mixture was washed 3 times with PBS. Nucleic acid aptamer solution was added and reacted under vacuum at 37 °C for 30 min to obtain hydrogel, which is the drug carrier. The mass-to-volume ratio of modified chitosan to PBS solution was 10 mg:200 mL; the mass ratio of tetrahydrofurfuryl methacrylate to modified chitosan was 4:10; the mass ratio of sec-butyl methacrylate to modified chitosan was 4:10; the mass of DNA solution was measured by the mass of single-stranded DNA, with a mass ratio of single-stranded DNA to modified chitosan of 1000 μg:10 mg; the mass of nucleic acid aptamer solution was measured by the mass of nucleic acid aptamers, with a mass ratio of nucleic acid aptamers to modified chitosan of 500 μg:10 mg; and the mass ratio of 0.6 w / v% photoinitiator 1173 to modified chitosan was 1:10.

[0070] Example 2: The only difference between this example and Example 1 is the preparation of the drug carrier.

[0071] Preparation of drug carrier: Modified chitosan was dissolved in PBS solution at pH 7.4 and degassed by sonication for 10 min. Tetrahydrofurfuryl methacrylate and sec-butyl methacrylate were added and stirred evenly at room temperature. DNA solution was added and mixed. 0.6 w / v% of photoinitiator 1173 was added and the mixture was bubbled with nitrogen for 5 min to remove oxygen. After sealing, it was irradiated with 365 nm ultraviolet light for 30 min. The mixture was washed 3 times with PBS. Nucleic acid aptamer solution was added and reacted under vacuum at 37 °C for 30 min to obtain hydrogel, which is the drug carrier. The mass-to-volume ratio of modified chitosan to PBS solution was 10 mg:200 mL; the mass ratio of tetrahydrofurfuryl methacrylate to modified chitosan was 6:10; and the mass ratio of sec-butyl methacrylate to modified chitosan was 4:10. The mass of the DNA solution was measured by the mass of single-stranded DNA, with a mass ratio of single-stranded DNA to modified chitosan of 1000 μg:10 mg. The mass of the nucleic acid aptamer solution was measured by the mass of nucleic acid aptamers, with a mass ratio of nucleic acid aptamers to modified chitosan of 500 μg:10 mg. The mass ratio of 0.6 w / v% photoinitiator 1173 to modified chitosan was 1:10.

[0072] Example 3: The only difference between this example and Example 1 is the preparation of the drug carrier.

[0073] Preparation of drug carrier: Modified chitosan was dissolved in PBS solution at pH 7.4 and degassed by sonication for 10 min. Tetrahydrofurfuryl methacrylate, sec-butyl methacrylate, and ethyl 4-aminocrotonate (-2-methoxy) ester were added and stirred evenly at room temperature. DNA solution was added and mixed. 0.6 w / v% of photoinitiator 1173 was added and the mixture was bubbled with nitrogen to remove oxygen for 5 min. After sealing, it was irradiated with 365 nm ultraviolet light for 30 min. The mixture was washed three times with PBS. Nucleic acid aptamer solution was added and reacted under vacuum at 37 °C for 30 min to obtain hydrogel, which is the drug carrier. The mass-to-volume ratio of modified chitosan to PBS solution was 10 mg:200 mL; the mass ratio of tetrahydrofurfuryl methacrylate to modified chitosan was 4:10; the mass ratio of sec-butyl butyl methacrylate to modified chitosan was 4:10; the mass ratio of 4-aminocrotonic acid (-2-methoxy)ethyl ester to modified chitosan was 4:10; the mass of the DNA solution was based on the mass of single-stranded DNA, with a mass ratio of single-stranded DNA to modified chitosan of 1000 μg:10 mg; the mass of the nucleic acid aptamer solution was based on the mass of nucleic acid aptamers, with a mass ratio of nucleic acid aptamers to modified chitosan of 500 μg:10 mg; and the mass ratio of 0.6 w / v% photoinitiator 1173 to modified chitosan was 1:10.

[0074] Example 4: The only difference between this example and Example 1 is the preparation of the drug carrier.

[0075] Preparation of drug carrier: Modified chitosan was dissolved in PBS solution at pH 7.4 and degassed by sonication for 10 min. Tetrahydrofurfuryl methacrylate, sec-butyl methacrylate, and ethyl 4-aminocrotonate (-2-methoxy) ester were added and stirred evenly at room temperature. DNA solution was added and mixed. 0.6 w / v% of photoinitiator 1173 was added and the mixture was bubbled with nitrogen to remove oxygen for 5 min. After sealing, it was irradiated with 365 nm ultraviolet light for 30 min. The mixture was washed three times with PBS. Nucleic acid aptamer solution was added and reacted under vacuum at 37 °C for 30 min to obtain hydrogel, which is the drug carrier. The mass-to-volume ratio of modified chitosan to PBS solution was 10 mg:200 mL; the mass ratio of tetrahydrofurfuryl methacrylate to modified chitosan was 4:10; the mass ratio of sec-butyl butyl methacrylate to modified chitosan was 4:10; and the mass ratio of 4-aminocrotonic acid (-2-methoxy)ethyl ester to modified chitosan was 6:10. The mass of the DNA solution was based on the mass of single-stranded DNA, with a mass ratio of single-stranded DNA to modified chitosan of 1000 μg:10 mg. The mass of the nucleic acid aptamer solution was based on the mass of nucleic acid aptamers, with a mass ratio of nucleic acid aptamers to modified chitosan of 500 μg:10 mg. The mass ratio of 0.6 w / v% photoinitiator 1173 to modified chitosan was 1:10.

[0076] Comparative Example 1: The only difference between this comparative example and Example 1 is that the configuration of AS1411 in the preparation of the nucleic acid aptamer solution is D-AS1411.

[0077] Comparative Example 2: The only difference between this comparative example and Example 1 is that tetrahydrofurfuryl methacrylate was not used in the preparation of the drug carrier.

[0078] Comparative Example 3: The only difference between this comparative example and Example 1 is that sec-butyl butyl ester was not used in the preparation of the drug carrier.

[0079] Comparative Example 4: The only difference between this comparative example and Example 1 is that tetrahydrofurfuryl methacrylate and sec-butyl methacrylate were not used in the preparation of the drug carrier.

[0080] Experimental Example 1: Stability test of nucleic acid aptamers.

[0081] Test samples: nucleic acid aptamer solutions of Example 1 and Comparative Example 1.

[0082] Test method: Nucleic acid aptamer solution was added to a culture medium containing 10% fetal bovine serum and cultured at 37℃. Samples were collected at 0h, 2h, 4h, 8h, 12h, 24h, and 48h. The degradation of nucleic acid aptamers in simulated serum was obtained by Western blotting. The nucleic acid aptamer solution was fluorescently labeled, and a random chain was set up as a control. The labeled nucleic acid aptamer solution was injected into mice of the corresponding groups. The mice were imaged using a small animal in vivo imaging system at 0min, 5min, 30min, 1h, 2h, 4h, and 5h to detect the distribution of fluorescence signals in vivo. The changes in fluorescence intensity at the tumor site were observed to obtain the degradation of nucleic acid aptamers in the in vivo environment.

[0083] The stability test results of L-AS1411 prepared in this invention in simulated serum are as follows: Figure 1 As shown, the stability test results of D-AS1411 prepared in this invention in simulated serum are as follows. Figure 2 As shown, with the time extended from 0h to 48h, the band intensity of the protein immunoblot in Comparative Example 1 gradually decreased, indicating that D-AS1411 was degraded in simulated serum, and the stability of Example 1 was better than that of Comparative Example 1; the stability test results of the nucleic acid aptamers prepared in this invention in the in vivo environment are as follows. Figure 3 As shown, in the control group, the fluorescence intensity at the tumor site remained weak and showed little change at different time points, indicating that the random chain did not have a significant targeting and enrichment effect on tumor tissue in mice. Example 1 and Comparative Example 1 demonstrated the ability to target tumor tissue. Compared to Comparative Example 1, Example 1 showed a significant increase in fluorescence intensity at the tumor site after injection, indicating better stability in the in vivo environment. These test results demonstrate that L-AS1411, through its complementary spatial structure to D-AS1411, can effectively resist degradation by nucleases and biological enzymes, significantly improving its stability in serum and in vivo environments, prolonging the carrier's targeting time, and thus enhancing therapeutic efficacy.

[0084] Experimental Example 2: Microstructural characterization of drug carriers.

[0085] Test sample: Drug carrier prepared in Example 1.

[0086] Test method: The surface of the drug carrier was sputtered with gold and observed using a scanning electron microscope at 25°C with an accelerating voltage of 20 kV.

[0087] Scanning electron microscope image of the drug carrier prepared in this invention is shown below. Figure 4 As shown, the surface structure and regular morphology are dense, indicating that the drug carrier shown in the figure has been successfully obtained.

[0088] Experimental Example 3: In vitro degradation test of drug carrier.

[0089] Test samples: Drug carriers prepared in each example and comparative example.

[0090] Test method: Dissolve lysozyme in PBS buffer at pH 6.8 to obtain a degradation solution; cut the drug carrier into 2cm×2cm pieces, weigh them to obtain mass m0, immerse them in the degradation solution, remove them after 3 hours, blot the surface moisture with filter paper, dry them and weigh them to obtain m1. Calculate the in vitro degradation rate (%) according to (m0-m1) / m0×100%.

[0091] The in vitro degradation test results of the drug carrier prepared in this invention are as follows: Figure 5 As shown, in Example 1, tetrahydrofurfuryl methacrylate and sec-butyl methacrylate were crosslinked with modified chitosan in equal proportions, forming a three-dimensional network structure with good density, effectively blocking the penetration of nucleases and serum components, and reducing hydrogel swelling and degradation. In Example 2, the amount of tetrahydrofurfuryl methacrylate was increased, which enhanced the rigidity of the crosslinked network, reduced network porosity, and further reduced the in vitro degradation rate of the drug carrier. In Example 3, 4-aminocrotonic acid (-2-methoxy)ethyl ester was introduced as a crosslinking monomer, which increased the hydrophilicity of the network, making the hydrogel more stable in the buffer solution, and further reducing the in vitro degradation rate compared with Example 2. In Example 4, 4-aminocrotonic acid (-2-methoxy)ethyl ester was added. The amount of crotonic acid (-2-methoxy)ethyl ester increased the number of cross-linking sites, resulting in a denser network structure and the lowest in vitro degradation rate. Comparative Example 1 used D-AS1411, which had poor stability, and the in vitro degradation rate of the drug carrier formed was higher than that of Example 1. Comparative Example 2 lacked tetrahydrofurfuryl methacrylate, resulting in a network structure that was too flexible and easily swelled and deformed in the buffer solution, and the in vitro degradation rate was higher than that of Example 1. Comparative Example 3 lacked sec-butyl butyl methacrylate, resulting in a network that was too rigid but lacked toughness, and the in vitro degradation rate was higher than that of Example 1. Comparative Example 4 completely lacked both cross-linking monomers, and the modified chitosan and nucleic acid aptamers dissolved rapidly in solution, resulting in the highest in vitro degradation rate.

[0092] Experimental Example 4: Drug Loading Test of Drug Carrier.

[0093] Test samples: Drug carriers prepared in each example and comparative example.

[0094] Test method: Doxorubicin was dispersed in PBS buffer at pH 7.4 and mixed evenly to obtain a doxorubicin solution with a concentration of C0. Each drug carrier was cut into small pieces of 2cm × 2cm, weighed and recorded as m0, and V mL of doxorubicin solution was added. After sealing, the mixture was incubated in a constant temperature shaker at 37℃ and 100 rpm for 24 h. The supernatant was taken, and the absorbance was measured at a wavelength of 480 nm using a UV-Vis spectrophotometer to obtain the concentration of remaining doxorubicin in the supernatant, C1. The drug loading rate was calculated as follows: Drug loading rate (%) = (C0 - C1) × V / m0 × 100%, where C0 is the concentration of doxorubicin in the initial doxorubicin solution, C1 is the concentration of doxorubicin in the supernatant, V is the volume of doxorubicin solution added, and m0 is the mass of the drug carrier.

[0095] The drug loading test results of the drug carrier prepared in this invention are as follows: Figure 6 As shown, in Example 1, the equal crosslinking of tetrahydrofurfuryl methacrylate and sec-butyl methacrylate resulted in a three-dimensional network structure with moderate density, which could retain a certain amount of doxorubicin through the pores. In Example 2, increasing the amount of tetrahydrofurfuryl methacrylate enhanced the network rigidity, reduced the pores, and improved the doxorubicin loading capacity, resulting in a higher drug loading rate than in Example 1. In Example 3, the introduction of ethyl 4-aminocrotonic acid (-2-methoxy) ester, whose hydrophilic groups could bind to doxorubicin through hydrogen bonds, further improved the loading rate. In Example 4, increasing the amount of ethyl 4-aminocrotonic acid (-2-methoxy) ester increased the number of crosslinking sites, resulting in a denser network structure, and the hydrophilic groups interacted with each other. The stronger D-AS1411 resulted in the highest loading rate. Comparative Example 1, using D-AS1411, showed weakened binding force with the hydrogel network during incubation, and some doxorubicin leaked due to pores caused by nucleic acid aptamer degradation, resulting in a lower loading rate than Example 1. Comparative Examples 2 and 3 lacked a cross-linking monomer, leading to insufficient network structure integrity, larger pores, and easy leakage of doxorubicin from the network, thus reducing the loading rate. Comparative Example 4 completely lacked a cross-linking monomer, resulting in a loose and easily swollen hydrogel network, which could not effectively retain doxorubicin, thus resulting in the lowest loading rate. This demonstrates that the high stability and dense cross-linked network structure of L-AS1411 are key to improving the drug loading capacity of hydrogels as drug carriers.

[0096] Experimental Example 5: Biosafety test of drug carrier.

[0097] Test samples: Drug carriers prepared in each example and comparative example.

[0098] Test method: Caco-2 cells in logarithmic growth phase were washed twice with PBS, digested with 0.25 w / v% trypsin to prepare a cell suspension, centrifuged, and then resuspended in IMDM medium containing 10% fetal bovine serum. Cell counts were precisely adjusted to obtain a cell density of 102. 5Cell suspension was prepared at 100 μL / mL. The cell suspension was seeded into 96-well plates at 100 μL per well, and 100 μL of IMDM medium containing 10% fetal bovine serum was added. The plates were pre-cultured at 37°C with 5% CO2 for 24 h. 10 mg of the drug carrier was added to each well as the experimental group, and a blank control group was also set up. After 12 h of culture, 10 μL of 5 mg / mL MTT solution was added to each well, the plate was gently shaken to mix, and the plate was incubated for another 5 h. The supernatant was discarded, and 110 μL of LDMSO was added to each well to dissolve Formazan crystals. The plates were stored at room temperature in the dark, and the absorbance of each well was measured at 490 nm using a microplate reader. Cell viability was calculated as: Cell viability (%) = (A0 - A1) / A0 × 100%, where A0 is the absorbance of the blank control group and A1 is the absorbance of the experimental group.

[0099] The biosafety test results of the drug carrier prepared in this invention are as follows: Figure 7 As shown, the cell survival rates of each embodiment and comparative example are high, indicating that the drug carrier prepared by the present invention has good biocompatibility and low toxicity to Caco-2 cells.

[0100] The embodiments and / or implementation methods described above are merely preferred embodiments and / or implementation methods for implementing the technology of the present invention, and are not intended to limit the implementation methods of the technology of the present invention in any way. Any person skilled in the art can make some modifications or alterations to other equivalent embodiments without departing from the scope of the technical means disclosed in the content of the present invention, but they should still be regarded as the technology or embodiments that are substantially the same as the present invention.

[0101] This document uses specific examples to illustrate the principles and implementation methods of this application. The descriptions of the above embodiments are only for the purpose of helping to understand the methods and core ideas of this application. The above descriptions are only preferred embodiments of this application. It should be noted that due to the limitations of written expression, while there are objectively infinite specific structures, those skilled in the art can make several improvements, modifications, or changes without departing from the principles of this application, and can also combine the above technical features in an appropriate manner. These improvements, modifications, changes, or combinations, or the direct application of the inventive concept and technical solution to other situations without modification, should all be considered within the scope of protection of this application.

Claims

1. A drug carrier, wherein the drug carrier is a hydrogel, characterized in that: The hydrogel comprises L-cysteine-modified chitosan, nucleic acid aptamers, and single-stranded DNA. The nucleic acid aptamers include L-AS1411, and the mass ratio of the nucleic acid aptamers to L-cysteine-modified chitosan is 250-2500 μg: 5-50 mg. The hydrogel includes a functional agent, which is tetrahydrofurfuryl methacrylate and sec-butyl methacrylate or tetrahydrofurfuryl methacrylate, sec-butyl methacrylate and 4-aminocrotonic acid (-2-methoxy) ethyl ester. The mass ratio of tetrahydrofurfuryl methacrylate to L-cysteine-modified chitosan is 2-20:5-50. The mass ratio of sec-butyl butyrate to L-cysteine-modified chitosan is 2-20:5-50; The single-stranded DNA includes DNA-A and DNA-B, the nucleotide sequence of DNA-A is shown in SEQ ID No. 3, and the nucleotide sequence of DNA-B is shown in SEQ ID No. 4; The mass ratio of the single-stranded DNA to L-cysteine-modified chitosan is 500-5000 μg: 5-50 mg.

2. The drug carrier according to claim 1, characterized in that: The reactive monomers of the L-cysteine-modified chitosan include chitosan and L-cysteine, and the mass ratio of chitosan to L-cysteine ​​is 5-50:3.5-35.

3. A drug carrier according to claim 1, characterized in that: The catalyst in the L-cysteine-modified chitosan includes 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide.

4. The method for preparing the drug carrier according to any one of claims 1-3, characterized in that... include: L-cysteine-modified chitosan solution was reacted with functional agents at room temperature, followed by the addition of DNA solution and photoinitiator, and then reacted under ultraviolet light in a deoxygenated and sealed environment. After adding nucleic acid aptamer solution, the reaction was carried out under vacuum to obtain a drug carrier. The DNA solution included single-stranded DNA and Tris-HCl buffer solution.

5. The method according to claim 4, characterized in that: The nucleic acid aptamer solution comprises a nucleic acid aptamer and a Tris-HCl buffer solution.

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