Preparation method and application of a tumor microenvironment-responsive dual-targeted gene delivery system

Abstract

The present invention relates to a preparation method and application of a tumor microenvironment-responsive dual-targeted gene delivery system, which can effectively solve the problem of in situ reprogramming of tumor cells and T cells, improve the anti-tumor immunotherapy effect, and ensure the cancer treatment effect. The technical solution is to react polyethyleneimine and 1H-pyrazole-1-formamidine hydrochloride at room temperature, then modify a tumor cell-targeting polypeptide and a T cell-targeting polypeptide through an amide reaction, and then condense them with an sgLSD1 plasmid respectively, and then physically mix them, add glutaraldehyde for cross-linking, and finally wrap poly(2-ethyl-2-oxazoline) in an outer layer to form a stable core-shell structure to obtain a tumor microenvironment-responsive dual-targeted gene delivery system. The dual-targeted gene delivery system prepared by the present invention has good biocompatibility and a long retention time, can effectively improve the efficiency of in situ programming of tumor cells and T cells, and efficiently activate anti-tumor immune responses, and is an innovation in tumor treatment drugs.

Description

1. Technical Field

[0001] The present invention relates to the field of medicine, and in particular to a preparation method and application of a tumor microenvironment-responsive dual-targeting gene delivery system. 2. Background Technology

[0002] CD8 + T cells are key effector cells for killing tumors. + Insufficient T cell infiltration and CD8 + T cell dysfunction will reduce its tumor killing ability. + T cell infiltration, reversal of CD8 + The exhaustion of T cells has become the key to improving anti-tumor immunotherapy. The latest research shows that knocking out gastric cancer cells or CD8 + LSD1 in T cells can promote CD8 + T cells infiltrate the tumor site and downregulate the expression of PD-L1 and PD-1, reversing the CD8 + Compared with knocking out LSD1 in one cell, it also affects gastric cancer cells and CD8 + LSD1 in T cells better restores CD8 + Therefore, it is necessary to design a dual-targeted gene delivery system to deliver the LSD1 knockout plasmid (sgLSD1) into the two cell types, thereby producing a synergistic anti-tumor immunotherapy effect.

[0003] Polyethyleneimine (PEI) is the most widely studied cationic polymer gene carrier. However, it suffers from low transfection efficiency, necessitating modification to improve it. Studies have shown that guanidinium modification is an effective method for increasing transfection efficiency. Furthermore, to target both T cells and tumor cells, separate peptides targeting both cells can be modified on the PEI surface. However, achieving co-delivery of two plasmids in a single system remains a challenge. Aldehyde groups and primary amino groups readily form imine bonds, acid-sensitive bonds that remain stable under physiological conditions (pH = 7.4) but undergo hydrolytic cleavage in the tumor microenvironment (pH = 6.5). Therefore, two nanoparticles with different targeting properties can be cross-linked using imine bonds formed by the aldehyde groups at the ends of glutaraldehyde and the amino groups on the polyethyleneimine nanoparticles. However, even after cross-linking, the nanoparticles still retain a strong positive charge, susceptible to nonspecific binding to serum proteins, and are rapidly cleared from the blood, severely hindering their further application in vivo. Studies have shown that poly (2-ethyl-2-oxazoline) carries a negative charge under physiological conditions, but undergoes protonation at pH 6.5 to convert it to a positive charge. Using PEOz as the shell of the nanosystem is expected to solve the above problems. In summary, targeted knockout of LSD1 in tumor cells and T cells, respectively, can better restore CD8 + The ability of T cells to kill tumors and achieve excellent anti-tumor immunotherapy effects is a technical problem that urgently needs to be solved. 3. Summary of the Invention

[0004] In view of the above situation and to address the deficiencies of the prior art, the purpose of the present invention is to provide a preparation method and application of a tumor microenvironment-responsive dual-targeted gene delivery system, which can effectively solve the problem of in situ reprogramming of tumor cells and T cells, improve the anti-tumor immunotherapy effect, and ensure the cancer treatment effect.

[0005] The technical solution provided by the present invention is to react polyethyleneimine and 1H-pyrazole-1-carboxamidine hydrochloride at room temperature, then modify the tumor cell-targeting polypeptide and the T cell-targeting polypeptide respectively through an amide reaction, and then condense them with the sgLSD1 plasmid respectively, and then physically mix them, add glutaraldehyde for cross-linking, and finally wrap poly(2-ethyl-2-oxazoline) in the outer layer to form a stable core-shell structure, thereby obtaining a tumor microenvironment-responsive dual-targeting gene delivery system, which specifically includes the following steps:

[0006] 1) Preparation of guanidinized polyethyleneimine (GPEI)

[0007] 1-6 g of polyethyleneimine was dissolved in 3-8 mL of solvent and added to a three-necked flask under nitrogen protection. 1-6 g of 1H-pyrazole-1-carboxamidine hydrochloride was dissolved in 5 mL of solvent and added to the polyethyleneimine solution. The mixture was stirred at room temperature for 10-35 min. 1-6 g of triethylamine was added to the mixed solution and stirred at room temperature for 24 h. The reaction solution was placed in a 500 Da dialysis bag and dialyzed for 48 h. The product was freeze-dried to obtain guanidinated polyethyleneimine (GPEI).

[0008] 2) Preparation of T cell targeting vector (T-GPEI)

[0009] 10-60 mg of T cell targeting peptide was dissolved in 1-6 mL of solvent and placed in a three-necked flask, which was filled with nitrogen for protection. 3-8 mg of N-hydroxysuccinimide and 6-11 mg of (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) were dissolved in 1 mL of solvent and added to the above three-necked flask and stirred at room temperature for 12 hours. 20-70 mg of guanidinated polyethyleneimine was added to 2-7 mL of solvent and fully dissolved. The mixture was added to the three-necked flask and stirred at room temperature for 24 hours. After that, the mixture was placed in a 1500 Da dialysis bag and dialyzed for 48 hours to fully remove unreacted substances. The dialyzed product was freeze-dried for 48 hours to obtain T cell targeting peptide-modified guanidinated polyethyleneimine (T-GPEI).

[0010] The T cell targeting peptide is a polypeptide chain containing the sequence AKMGEGGWGANDY;

[0011] 3) Preparation of tumor cell targeting vector (M-GPEI)

[0012] Take 20-70 mg of tumor cell targeting peptide and dissolve it in 3 mL of solvent, then add it to a three-necked flask and fill it with nitrogen for protection. Then add a solvent solution containing 3-8 mg of N-hydroxysuccinimide and 8-13 mg of (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) to the three-necked flask and activate the carboxyl group at room temperature for 12 hours. Finally, add a solvent solution containing 30-80 mg of guanidinated polyethyleneimine to the above reaction system and react at room temperature for 24 hours. The reacted solution is placed in a 2500 Da dialysis bag and dialyzed for 48 hours. The product is freeze-dried for 48 hours to obtain tumor cell targeting peptide-modified guanidinated polyethyleneimine (M-GPEI);

[0013] The tumor cell targeting peptide is a polypeptide chain containing the sequence SGQYASYHCWCWRDPGRSGGSK;

[0014] 4) Prepare T cell targeting vector solution and tumor cell targeting vector solution (TG, MG)

[0015] Dissolve 30-80 mg of the T cell-targeting vector prepared in step 2) and the tumor cell-targeting vector prepared in step 3) in 40 mL of solvent, adjust the pH of the two solutions to 7.4 with 1 mol / L NaOH solution, transfer to a 50 mL volumetric flask, dilute to 50 mL with solvent, and shake well to obtain a T cell-targeting vector solution and a tumor cell-targeting vector solution with a concentration of 1 mg / mL. Filter the prepared solutions through a 0.22 μm microporous filter membrane;

[0016] 5) Synthesis of T cell targeting vector / plasmid complexes and tumor cell targeting vector / plasmid complexes (TG / sg, MG / sg)

[0017] Take two aliquots of plasmid, 4-9 mL and 7-12 mg, respectively, and dilute them to 0.01 mg / mL with solvent. Take 50 mL of the T cell-targeting vector solution and tumor cell-targeting vector solution prepared in step 4) and dilute them to 0.1 mg / mL with solvent. Add them dropwise to the diluted plasmid solutions, shake continuously to mix, and let stand at room temperature for 10 minutes to obtain T cell-targeting vector / plasmid complexes and tumor cell-targeting vector / plasmid complexes.

[0018] 6) Tumor microenvironment-responsive dual-targeted gene delivery system (P@cPG / sg)

[0019] Take the T cell targeting vector / plasmid complex and the tumor cell targeting vector / plasmid complex in step 5), mix the two solutions in equal amounts, add 170-220 mL of 25% glutaraldehyde solution, mix well, and let stand at room temperature for 5 minutes. Take 30-80 mg of poly (2-ethyl-2-oxazoline) and prepare it with a solvent to a concentration of 1 mg / mL solution. 300-800 mL is added dropwise to the above solution and allowed to stand at room temperature for 10 minutes to obtain a tumor microenvironment-responsive dual-targeting gene delivery system.

[0020] The solvent in steps 1) to 6) is one of ultrapure water, PBS buffer with pH=7.4, and formamide.

[0021] Application of the tumor microenvironment-responsive dual-targeted gene delivery system of the present invention in the preparation of anti-tumor drugs.

[0022] Application of the tumor microenvironment-responsive dual-targeted gene delivery system of the present invention in the preparation of highly efficient gene delivery.

[0023] The invention is easy to operate, the method is stable and reliable, the prepared dual-target gene delivery system has good biocompatibility and long retention time, and can be used in situ to reprogram tumor cells and T cells, increase CD8 +It has multiple functions such as T cell infiltration and reversal of T cell exhaustion, which can effectively improve the efficiency of in situ programming of tumor cells and T cells and efficiently activate anti-tumor immune responses. It is an innovation in tumor treatment drugs with huge economic and social benefits. IV. Description of the Figures

[0024] Figure 1 TEM images of P@cPG / sg prepared in the present invention at pH = 7.4 and pH = 6.5.

[0025] Figure 2 AB Western Blot detection of MFC cells (1, 2) and CD8 + LSD1 expression before and after transfection of T cells (1, 3).

[0026] Figure 3 CD8 + Expression of LSD1 in T cells.

[0027] Figure 4 These are the live cell imaging images of the untreated group and the P@cPG / sg group of the present invention.

[0028] Figure 5 These are in vivo fluorescence images of mice taken at different drug administration time points using the in vivo imaging method of the present invention.

[0029] Figure 6 The flow cytometry analysis of CD8 + Uptake of the formulation by T cells (A) and MFC cells (B).

[0030] Figure 7 The flow cytometry method of the present invention is used to detect tumor cells (A) and CD8 + LSD1 expression in T cells (B).

[0031] Figure 8 Flow cytometry was used to detect CD8 + T cell infiltration (A) and its function-related indicators (BD). V. Specific Implementation Methods

[0032] The specific embodiments of the present invention are further described in detail below with reference to the examples.

[0033] Example 1

[0034] The present invention, when implemented, specifically comprises the following steps:

[0035] 1) Preparation of guanylated polyethyleneimine

[0036] Dissolve 1g of polyethyleneimine in 3mL of ultrapure water, add the mixture to a three-necked flask and fill it with nitrogen. Dissolve 1g of 1H-pyrazole-1-carboxamidine hydrochloride in 5mL of ultrapure water, add the mixture to the polyethyleneimine solution, and stir at room temperature for 10 minutes. Add 1g of triethylamine to the mixed solution and stir at room temperature for 24 hours. The resulting solution is placed in a 500Da dialysis bag and dialyzed for 48 hours. The product is then freeze-dried to obtain guanylated polyethyleneimine.

[0037] 2) Preparation of T cell targeting vector

[0038] Dissolve 10 mg of T cell targeting peptide in 1 mL of formamide and place in a three-necked flask, then fill with nitrogen for protection. Dissolve 3 mg of N-hydroxysuccinimide and 6 mg of (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) in 1 mL of formamide and add to the above three-necked flask. Stir at room temperature for 12 hours. Dissolve 20 mg of the guanidinated polyethyleneimine prepared in step 1) in 2 mL of formamide and add to the three-necked flask. Stir and react at room temperature for 24 hours, then place in a 1500 Da dialysis bag and dialyze for 48 hours to fully remove unreacted substances. The dialyzed product is freeze-dried for 48 hours to obtain a T cell targeting vector.

[0039] 3) Preparation of tumor cell targeting vector

[0040] 20 mg of tumor cell targeting peptide was dissolved in 3 mL of formamide, then added to a three-necked flask and filled with nitrogen for protection. A formamide solution containing 3 mg of N-hydroxysuccinimide and 8 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride was then added to the three-necked flask and the carboxyl groups were activated at room temperature for 12 hours. Finally, a formamide solution containing 30 mg of guanidinated polyethyleneimine was added to the above reaction system and reacted at room temperature for 24 hours. The reaction solution was placed in a 2500 Da dialysis bag and dialyzed for 48 hours. The product was freeze-dried for 48 hours to obtain a tumor cell targeting vector.

[0041] 4) Prepare T cell targeting vector solution and tumor cell targeting vector solution

[0042] Dissolve 30 mg of each of the T cell-targeting vector prepared in step 2) and the tumor cell-targeting vector prepared in step 3) in 40 mL of ultrapure water. Adjust the pH of the two solutions to 7.4 with 1 mol / L NaOH solution. Transfer the solutions to a 50 mL volumetric flask, dilute to 50 mL with ultrapure water, and shake well to obtain two vector solutions with a concentration of 1 mg / mL. Filter the prepared solutions through a 0.22 μm microporous filter membrane.

[0043] 5) Synthesis of T cell targeting vector / plasmid complexes and tumor cell targeting vector / plasmid complexes

[0044] Take two aliquots of 4 mL and 7 mg of plasmid, respectively, and dilute them to 0.01 mg / mL with ultrapure water. Take 50 mL of the T cell-targeting vector solution and tumor cell-targeting vector solution prepared in step 4) (corresponding to 4 mL and 7 mg of plasmid, respectively), dilute them to 0.1 mg / mL with ultrapure water, and add them dropwise to the diluted plasmid solutions. Mix by continuous shaking and let stand at room temperature for 10 minutes to obtain T cell-targeting vector / plasmid complexes and tumor cell-targeting vector / plasmid complexes.

[0045] 6) Tumor microenvironment-responsive dual-targeted gene delivery system

[0046] Take the T cell targeting vector / plasmid complex and the tumor cell targeting vector / plasmid complex prepared in step 5), mix the two solutions in equal amounts, add 170 mL of 25% glutaraldehyde solution, mix well and let stand at room temperature for 5 minutes, take 30 mg of poly (2-ethyl-2-oxazoline) and prepare it with ultrapure water to a concentration of 1 mg / mL solution, measure 500 mL and add it dropwise to the above solution, and let stand at room temperature for 10 minutes to obtain a tumor microenvironment-responsive dual-targeting gene delivery system.

[0047] Example 2

[0048] The present invention may further comprise the following steps when it is specifically implemented:

[0049] 1) Preparation of guanylated polyethyleneimine

[0050] 2 g of polyethyleneimine was dissolved in 4 mL of ultrapure water and added to a three-necked flask under nitrogen protection. 2 g of 1H-pyrazole-1-carboxamidine hydrochloride was dissolved in 5 mL of ultrapure water and added to the polyethyleneimine solution. The mixture was stirred at room temperature for 15 minutes. 2 g of triethylamine was added to the mixed solution and stirred at room temperature for 24 hours. The resulting solution was placed in a 500 Da dialysis bag and dialyzed for 48 hours. The product was then freeze-dried to obtain guanylated polyethyleneimine.

[0051] 2) Preparation of T cell targeting vector

[0052] 20 mg of T cell targeting peptide was dissolved in 2 mL of formamide and placed in a three-necked flask, which was filled with nitrogen for protection. 4 mg of N-hydroxysuccinimide and 7 mg of (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) were dissolved in 2 mL of formamide and added to the above three-necked flask and stirred at room temperature for 12 h. 30 mg of the guanidinated polyethyleneimine prepared in step 1) was added to 3 mL of formamide and fully dissolved. The mixture was added to the three-necked flask and stirred at room temperature for 24 h. After that, the mixture was placed in a 1500 Da dialysis bag and dialyzed for 48 h to fully remove unreacted substances. The dialyzed product was freeze-dried for 48 h to obtain a T cell targeting vector.

[0053] 3) Preparation of tumor cell targeting vector

[0054] 30 mg of tumor cell targeting peptide was dissolved in 3 mL of formamide, then added to a three-necked flask and filled with nitrogen for protection. A formamide solution containing 5 mg of N-hydroxysuccinimide and 9 mg of (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) was then added to the three-necked flask and the carboxyl groups were activated at room temperature for 12 hours. Finally, a formamide solution containing 40 mg of guanidinated polyethyleneimine was added to the above reaction system and reacted at room temperature for 24 hours. The reaction solution was placed in a 2500 Da dialysis bag and dialyzed for 48 hours. The product was freeze-dried for 48 hours to obtain a tumor cell targeting vector.

[0055] 4) Prepare T cell targeting vector solution and tumor cell targeting vector solution

[0056] Dissolve 40 mg of the T cell-targeting vector prepared in step 2) and the tumor cell-targeting vector prepared in step 3) in 50 mL of ultrapure water, adjust the pH of the two solutions to 7.4 with 1 mol / L NaOH solution, transfer the solutions to a 50 mL volumetric flask, dilute to 50 mL with ultrapure water, and shake well to obtain 1 mg / mL T cell-targeting vector solutions and tumor cell-targeting vector solutions. Filter the prepared solutions through a 0.22 μm microporous filter membrane.

[0057] 5) Synthesis of T cell targeting vector / plasmid complexes and tumor cell targeting vector / plasmid complexes

[0058] Take two aliquots of 5 mL and 8 mg of plasmid, respectively, and dilute them to 0.01 mg / mL with ultrapure water. Take 50 mL of the T cell-targeting vector solution and tumor cell-targeting vector solution prepared in step 4), dilute them to 0.1 mg / mL with ultrapure water, and add them dropwise to the diluted plasmid solution, respectively. Mix by continuous shaking, and let stand at room temperature for 10 minutes to obtain T cell-targeting vector / plasmid complexes and tumor cell-targeting vector / plasmid complexes;

[0059] 6) Tumor microenvironment-responsive dual-targeted gene delivery system

[0060] Take the T cell targeting vector / plasmid complex and the tumor cell targeting vector / plasmid complex prepared in step 5), mix the two solutions in equal amounts, add 180 mL of 25% glutaraldehyde solution, mix well and let stand at room temperature for 5 minutes, take 40 mg of poly (2-ethyl-2-oxazoline) and prepare it with ultrapure water to a concentration of 1 mg / mL solution, measure 600 mL and add it dropwise to the above solution, and let stand at room temperature for 10 minutes to obtain a tumor microenvironment-responsive dual-targeting gene delivery system.

[0061] Example 3

[0062] The present invention may further comprise the following steps when it is specifically implemented:

[0063] 1) Preparation of guanylated polyethyleneimine

[0064] Dissolve 3g of polyethyleneimine in 5mL of ultrapure water and add it to a three-necked flask under nitrogen. Dissolve 3g of 1H-pyrazole-1-carboxamidine hydrochloride in 5mL of ultrapure water and add it to the polyethyleneimine solution. Stir at room temperature for 20 minutes. Add 3g of triethylamine to the mixture and stir at room temperature for 24 hours. Place the resulting solution in a 500Da dialysis bag and dialyze for 48 hours. Then, lyophilize the product to obtain guanylated polyethyleneimine.

[0065] 2) Preparation of T cell targeting vector

[0066] Dissolve 30 mg of T cell targeting peptide in 3 mL of formamide and place in a three-necked flask, then fill with nitrogen for protection. Dissolve 5 mg of N-hydroxysuccinimide and 10 mg of (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) in 3 mL of formamide and add to the above three-necked flask, stir at room temperature for 12 hours, take 40 mg of the guanidinated polyethyleneimine prepared in step 1) and add 4 mL of formamide to fully dissolve it, add it to the three-necked flask, stir at room temperature for 24 hours, then place it in a 1500 Da dialysis bag and dialyze for 48 hours to fully remove unreacted substances. The dialyzed product is freeze-dried for 48 hours to obtain a T cell targeting vector.

[0067] 3) Preparation of tumor cell targeting vector

[0068] 40 mg of tumor cell targeting peptide was dissolved in 3 mL of formamide, and then the solution was added to a three-necked flask and filled with nitrogen for protection. Then, a formamide solution containing 7 mg of N-hydroxysuccinimide and 10 mg of (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) was added to the three-necked flask, and the carboxyl groups were activated at room temperature for 12 hours. Finally, a formamide solution containing 50 mg of guanidinated polyethyleneimine was added to the above reaction system and reacted at room temperature for 24 hours. The reaction solution was placed in a 2500 Da dialysis bag and dialyzed for 48 hours. The product was freeze-dried for 48 hours to obtain a tumor cell targeting vector.

[0069] 4) Prepare T cell targeting vector solution and tumor cell targeting vector solution

[0070] Dissolve 50 mg of the T cell-targeting vector prepared in step 2) and the tumor cell-targeting vector prepared in step 3) in 50 mL of ultrapure water, adjust the pH of the two solutions to 7.4 with 1 mol / L NaOH solution, transfer the solutions to a 50 mL volumetric flask, dilute to 50 mL with ultrapure water, and shake well to obtain the T cell-targeting vector solution and the tumor cell-targeting vector solution. Filter the prepared solutions through a 0.22 μm microporous filter membrane.

[0071] 5) Synthesis of T cell targeting vector / plasmid complexes and tumor cell targeting vector / plasmid complexes

[0072] Take two aliquots of 6 mL and 9 mg of plasmid, respectively, and dilute them to 0.01 mg / mL with ultrapure water. Take 50 mL of the T cell-targeting vector solution and tumor cell-targeting vector solution prepared in step 4), dilute them to 0.1 mg / mL with ultrapure water, and add them dropwise to the diluted plasmid solutions, respectively. Mix by continuous shaking, and let stand at room temperature for 10 minutes to obtain T cell-targeting vector / plasmid complexes and tumor cell-targeting vector / plasmid complexes;

[0073] 6) Tumor microenvironment-responsive dual-targeted gene delivery system

[0074] Take the T cell targeting vector / plasmid complex and the tumor cell targeting vector / plasmid complex in step 5), mix the two solutions in equal amounts, add 190 mL of 25% glutaraldehyde solution, mix well, and let stand at room temperature for 5 minutes. Take 50 mg of poly (2-ethyl-2-oxazoline) and prepare it with ultrapure water to a concentration of 1 mg / mL solution. Measure 700 mL and add it dropwise to the above solution. Let it stand at room temperature for 10 minutes to obtain a tumor microenvironment-responsive dual-targeting gene delivery system.

[0075] Example 4

[0076] The present invention may further comprise the following steps when it is specifically implemented:

[0077] 1) Preparation of guanylated polyethyleneimine: 4 g of polyethyleneimine was dissolved in 6 mL of ultrapure water, added to a three-necked flask and filled with nitrogen. 4 g of 1H-pyrazole-1-carboxamidine hydrochloride was dissolved in 5 mL of ultrapure water and added to the polyethyleneimine solution. The mixture was stirred at room temperature for 25 minutes. 4 g of triethylamine was added to the mixed solution and stirred at room temperature for 24 hours. The reaction solution was placed in a 500 Da dialysis bag, dialyzed for 48 hours, and then freeze-dried to obtain guanylated polyethyleneimine.

[0078] 2) Preparation of T cell targeting vector

[0079] 40 mg of T cell targeting peptide was dissolved in 4 mL of formamide and placed in a three-necked flask, which was filled with nitrogen for protection. 6 mg of N-hydroxysuccinimide and 9 mg of (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) were dissolved in 3 mL of formamide and added to the above three-necked flask and stirred at room temperature for 12 h. 50 mg of the guanidinated polyethyleneimine prepared in step 1) was added to 5 mL of formamide and fully dissolved. The mixture was added to the three-necked flask and stirred at room temperature for 24 h. After that, the mixture was placed in a 1500 Da dialysis bag and dialyzed for 48 h to fully remove unreacted substances. The dialyzed product was freeze-dried for 48 h to obtain a T cell targeting vector.

[0080] 3) Preparation of tumor cell targeting vector

[0081] 50 mg of tumor cell targeting peptide was dissolved in 4 mL of formamide, and then the solution was added to a three-necked flask and filled with nitrogen for protection. Then, a formamide solution containing 7 mg of N-hydroxysuccinimide and 11 mg of (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) was added to the three-necked flask, and the carboxyl groups were activated at room temperature for 12 hours. Finally, a formamide solution containing 60 mg of guanidinated polyethyleneimine was added to the above reaction system and reacted at room temperature for 24 hours. The reaction solution was placed in a 2500 Da dialysis bag and dialyzed for 48 hours. The product was freeze-dried for 48 hours to obtain a tumor cell targeting vector.

[0082] 4) Prepare T cell targeting vector solution and tumor cell targeting vector solution

[0083] Dissolve 60 mg of the T cell-targeting vector prepared in step 2) and the tumor cell-targeting vector prepared in step 3) in 50 mL of ultrapure water, adjust the pH of the two solutions to 7.4 with 1 mol / L NaOH solution, transfer the solutions to a 50 mL volumetric flask, dilute to 50 mL with ultrapure water, and shake well to obtain 1 mg / mL T cell-targeting vector solutions and tumor cell-targeting vector solutions. Filter the prepared solutions through a 0.22 μm microporous filter membrane.

[0084] 5) Synthesis of T cell targeting vector / plasmid complexes and tumor cell targeting vector / plasmid complexes

[0085] Take two aliquots of 7 mL and 10 mg of plasmid, respectively, and dilute them to 0.01 mg / mL with ultrapure water. Take 50 mL of the T cell-targeting vector solution and tumor cell-targeting vector solution prepared in step 4), dilute them to 0.1 mg / mL with ultrapure water, and add them dropwise to the diluted plasmid solution, respectively. Mix by continuous shaking, and let stand at room temperature for 10 minutes to obtain T cell-targeting vector / plasmid complexes and tumor cell-targeting vector / plasmid complexes;

[0086] 6) Tumor microenvironment-responsive dual-targeted gene delivery system

[0087] Take the T cell targeting vector / plasmid complex and the tumor cell targeting vector / plasmid complex prepared in step 5), mix the two solutions in equal amounts, add 200 mL of 25% glutaraldehyde solution, mix well and let stand at room temperature for 5 minutes, take 60 mg of poly (2-ethyl-2-oxazoline) and prepare it with ultrapure water to a concentration of 1 mg / mL solution, measure 700 mL and add it dropwise to the above solution, and let stand at room temperature for 10 minutes to obtain a tumor microenvironment-responsive dual-targeting gene delivery system.

[0088] Example 5

[0089] The present invention may further comprise the following steps when it is specifically implemented:

[0090] 1) Preparation of guanylated polyethyleneimine

[0091] 5 g of polyethyleneimine was dissolved in 7 mL of ultrapure water and added to a three-necked flask under nitrogen protection. 5 g of 1H-pyrazole-1-carboxamidine hydrochloride was dissolved in 5 mL of ultrapure water and added to the polyethyleneimine solution. The mixture was stirred at room temperature for 30 min. 5 g of triethylamine was added to the mixed solution and stirred at room temperature for 24 h. The resulting solution was placed in a 500 Da dialysis bag and dialyzed for 48 h. The product was then freeze-dried to obtain guanylated polyethyleneimine.

[0092] 2) Preparation of T cell targeting vector

[0093] 50 mg of T cell targeting peptide was dissolved in 5 mL of formamide and placed in a three-necked flask, which was protected by nitrogen. 7 mg of N-hydroxysuccinimide and 10 mg of (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) were dissolved in 3 mL of formamide and added to the above three-necked flask and stirred at room temperature for 12 h. 60 mg of the guanidinated polyethyleneimine prepared in step 1) was added to 6 mL of formamide and fully dissolved. The mixture was added to the three-necked flask and stirred at room temperature for 24 h. After that, the mixture was placed in a 1500 Da dialysis bag and dialyzed for 48 h to fully remove unreacted substances. The dialyzed product was freeze-dried for 48 h to obtain a T cell targeting vector.

[0094] 3) Preparation of tumor cell targeting vector

[0095] 60 mg of tumor cell targeting peptide was dissolved in 4 mL of formamide, and then the solution was added to a three-necked flask and filled with nitrogen for protection. Then, a formamide solution containing 7 mg of N-hydroxysuccinimide and 12 mg of (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) was added to the three-necked flask, and the carboxyl groups were activated at room temperature for 12 hours. Finally, a formamide solution containing 70 mg of guanidinated polyethyleneimine was added to the above reaction system and reacted at room temperature for 24 hours. The reaction solution was placed in a 2500 Da dialysis bag and dialyzed for 48 hours. The product was freeze-dried for 48 hours to obtain a tumor cell targeting vector.

[0096] 4) Prepare T cell targeting vector solution and tumor cell targeting vector solution

[0097] 70 mg each of the T cell-targeting vector prepared in step 2) and the tumor cell-targeting vector prepared in step 3) were dissolved in 50 mL of ultrapure water. The pH of the two solutions was adjusted to 7.4 with 1 mol / L NaOH solution. The solutions were then transferred to a 50 mL volumetric flask, made up to 50 mL with ultrapure water, and shaken to obtain 1 mg / mL T cell-targeting vector solutions and tumor cell-targeting vector solutions. The prepared solutions were filtered through a 0.22 μm microporous membrane.

[0098] 5) Synthesis of T cell targeting vector / plasmid complexes and tumor cell targeting vector / plasmid complexes

[0099] Take two aliquots of 8 mL and 11 mg of plasmid, respectively, and dilute them to 0.01 mg / mL with ultrapure water. Take 50 mL of the T cell-targeting vector solution and tumor cell-targeting vector solution prepared in step 4), dilute them to 0.1 mg / mL with ultrapure water, and add them dropwise to the diluted plasmid solutions, respectively. Mix by continuous shaking, and let stand at room temperature for 10 minutes to obtain T cell-targeting vector / plasmid complexes and tumor cell-targeting vector / plasmid complexes;

[0100] 6) Tumor microenvironment-responsive dual-targeted gene delivery system

[0101] Take the T cell targeting vector / plasmid complex and the tumor cell targeting vector / plasmid complex prepared in step 5), mix the two solutions in equal amounts, add 210 mL of 25% glutaraldehyde solution, mix well and let stand at room temperature for 5 minutes, take 70 mg of poly (2-ethyl-2-oxazoline) and prepare it with ultrapure water to a concentration of 1 mg / mL solution, measure 700 mL and add it dropwise to the above solution, and let stand at room temperature for 10 minutes to obtain a tumor microenvironment-responsive dual-targeting gene delivery system.

[0102] Example 6

[0103] The present invention may further comprise the following steps when it is specifically implemented:

[0104] 1) Preparation of guanylated polyethyleneimine

[0105] 6 g of polyethyleneimine was dissolved in 8 mL of ultrapure water and added to a three-necked flask under nitrogen protection. 6 g of 1H-pyrazole-1-carboxamidine hydrochloride was dissolved in 5 mL of ultrapure water and added to the polyethyleneimine solution. The mixture was stirred at room temperature for 35 minutes. 6 g of triethylamine was added to the mixed solution and stirred at room temperature for 24 hours. The resulting solution was placed in a 500 Da dialysis bag and dialyzed for 48 hours. The product was then freeze-dried to obtain guanylated polyethyleneimine.

[0106] 2) Preparation of T cell targeting vector

[0107] 60 mg of T cell targeting peptide was dissolved in 6 mL of formamide and placed in a three-necked flask, which was filled with nitrogen for protection. 8 mg of N-hydroxysuccinimide and 11 mg of (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) were dissolved in 3 mL of formamide and added to the above three-necked flask and stirred at room temperature for 12 h. 70 mg of the guanidinated polyethyleneimine prepared in step 1) was added to 7 mL of formamide and fully dissolved. The mixture was added to the three-necked flask and stirred at room temperature for 24 h. After that, the mixture was placed in a 1500 Da dialysis bag and dialyzed for 48 h to fully remove unreacted substances. The dialyzed product was freeze-dried for 48 h to obtain a T cell targeting vector.

[0108] 3) Preparation of tumor cell targeting vector

[0109] 70 mg of tumor cell targeting peptide was dissolved in 4 mL of formamide, and then the solution was added to a three-necked flask and filled with nitrogen for protection. Then, a formamide solution containing 8 mg of N-hydroxysuccinimide and 13 mg of (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) was added to the three-necked flask, and the carboxyl groups were activated at room temperature for 12 hours. Finally, a formamide solution containing 80 mg of guanidinated polyethyleneimine was added to the above reaction system and reacted at room temperature for 24 hours. The reaction solution was placed in a 2500 Da dialysis bag and dialyzed for 48 hours. The product was freeze-dried for 48 hours to obtain a tumor cell targeting vector.

[0110] 4) Prepare T cell targeting vector solution and tumor cell targeting vector solution

[0111] 80 mg of each of the T cell-targeting vector prepared in step 2) and the tumor cell-targeting vector prepared in step 3) were dissolved in 50 mL of ultrapure water. The pH of the two solutions was adjusted to 7.4 with 1 mol / L NaOH solution. The solutions were then transferred to a 50 mL volumetric flask, made up to 50 mL with ultrapure water, and shaken to obtain 1 mg / mL T cell-targeting vector solutions and tumor cell-targeting vector solutions. The prepared solutions were filtered through a 0.22 μm microporous membrane.

[0112] 5) Synthesis of T cell targeting vector / plasmid complexes and tumor cell targeting vector / plasmid complexes

[0113] Take two aliquots of 9 mL and 12 mg of plasmid, respectively, and dilute them to 0.01 mg / mL with ultrapure water. Take 50 mL of the T cell-targeting vector solution and tumor cell-targeting vector solution prepared in step 4), dilute them to 0.1 mg / mL with ultrapure water, and add them dropwise to the diluted plasmid solution, respectively. Mix by continuous shaking, and let stand at room temperature for 10 minutes to obtain T cell-targeting vector / plasmid complexes and tumor cell-targeting vector / plasmid complexes;

[0114] 6) Tumor microenvironment-responsive dual-targeted gene delivery system

[0115] Take the T cell targeting vector / plasmid complex and the tumor cell targeting vector / plasmid complex prepared in step 5), mix the two solutions in equal amounts, add 220 mL of 25% glutaraldehyde solution, mix well and let stand at room temperature for 5 minutes, take 80 mg of poly (2-ethyl-2-oxazoline) and prepare it with ultrapure water to a concentration of 1 mg / mL solution, measure 700 mL and add it dropwise to the above solution, and let stand at room temperature for 10 minutes to obtain a tumor microenvironment-responsive dual-targeting gene delivery system.

[0116] The tumor microenvironment-responsive dual-targeted gene delivery system prepared in the above-mentioned Examples 1-6 and other Examples of the present invention can play multiple roles in tumor treatment, such as targeting tumors, improving the effect of anti-tumor immunotherapy, and reversing T cell exhaustion. It is effectively used in the preparation of anti-tumor drugs, and can be used in the preparation of anti-tumor drugs (injections), in the preparation of drugs that reduce the PD-1 / PD-L1 axis, and in the preparation of immunotherapy drugs based on in situ reprogramming of tumor cells and T cells.

[0117] The present invention uses 1800Da polyethyleneimine, a less toxic material, to condense a plasmid that knocks out LSD1. However, it suffers from a low transfection efficiency and, therefore, requires modification and transformation to improve its transfection efficiency. To address this, the 1800Da polyethyleneimine is guanidated, which improves the transfection efficiency of polyethyleneimine in multiple pathways, including plasmid condensation, cellular uptake, lysosomal escape, and nuclear entry. To achieve cellular selectivity, two polypeptides targeting tumor cells (SGQYASYHCWCWRDPGRSGGSK) and T cells (AKMGEGGWGANDY) are modified, respectively, and two small-size nanoparticles with different targeting properties are obtained after plasmid condensation. To deliver them simultaneously to the tumor site without affecting the subsequent separate targeting of the two cells, glutaraldehyde is proposed as a linker. The aldehyde group and the primary amino group easily form an imine bond, an acid-sensitive bond that remains structurally stable under physiological conditions (pH = 7.4) but undergoes hydrolysis and cleavage in the tumor microenvironment (pH = 6.5). Therefore, the aldehyde groups at both ends of the imine bond and the amino groups on the polyethyleneimine in the nanoparticle form an imine bond to crosslink the two nanoparticles with different targeting properties. After crosslinking, the nanoparticles still have a strong positive charge, easily nonspecifically binding to serum proteins and being rapidly cleared from the blood, seriously hindering their further application in vivo. To shield the positive charge, the present invention chooses to use poly(2-ethyl-2-oxazoline) as the outer shell. It has a negative charge under physiological conditions, but can be protonated to a positive charge at pH = 6.5, thereby generating charge repulsion with the core and achieving the effect of shedding from the nanoparticle surface. When the dual-targeted gene delivery system reaches the tumor microenvironment, the outer layer (2-ethyl-2-oxazoline) is reversed from negative charge to positive charge, and the inner layer with positive charge is repelled and detached. At the same time, the carbon-nitrogen double bond in the cross-linked core is broken in response to acid, and the two nanoparticles with different targeting properties are separated. Under the modification of the target head and guanidine group, the LSD1 knockout plasmid can be efficiently and accurately delivered to the tumor cells and T cell nuclei, knocking out LSD1 in both cells, thereby increasing CD8 + T cell infiltration, restoration of CD8 + T cell proliferation and differentiation ability, reduce the expression of PD-L1 and PD-1, activate CD8 + T cell killing response stimulates a powerful anti-tumor immune response.

[0118] The gene delivery vector prepared by the present invention has a simple synthesis method. The dual-targeted nanogene delivery system can respond to the acidic environment of the tumor microenvironment. The poly (2-ethyl-2-oxazoline) shell is reversed from a negative charge to a positive charge, generating charge repulsion with the positively charged core and falling off from the surface of the nanoparticle. The cross-linked core also responds to the acidic environment at the same time, the nitrogen-carbon double bond breaks, and disintegrates into nanoparticles with different targeting properties, respectively accurately targeting tumor cells and T cells and knocking out LSD1 in both cells, thereby increasing CD8+ T cell infiltration and activation of its killing response, exerting a powerful anti-tumor immunotherapy, and repeated multiple tests have achieved consistent or similar results. Taking Example 2 as an example, the relevant experimental data are as follows:

[0119] 1. Characterization of a Tumor Microenvironment-Responsive Dual-Targeting Gene Delivery System

[0120] 1. Determination of the guanidine grafting rate in guanylated polyethyleneimine;

[0121] Appropriate amounts of polyethyleneimine before and after guanidine modification were weighed, and the C, N, H, and S content of the two analytes was determined using an organic element analyzer using a high-temperature combustion method. The grafting rate was calculated to be approximately 28.9%.

[0122] 2. Determination of particle size and potential of tumor microenvironment-responsive dual-targeted gene delivery system;

[0123] Appropriate amounts of dual-targeted gene delivery system (pH = 7.4) and dual-targeted gene delivery system (pH = 6.5) were taken, and their hydrated particle sizes and potentials were measured using a laser nanoparticle size analyzer to be 200 nm and 60 nm, and the potentials were -5 mV and 35 mV, respectively.

[0124] 3. Transmission electron microscopy characterization of the tumor microenvironment-responsive dual-targeted gene delivery system:

[0125] Take the complex at pH = 7.4 and pH = 6.5, add one drop on a common carbon support film, wait for the liquid to evaporate, and take a picture with a transmission electron microscope, as shown in the figure. Figure 1 As shown in AB, it can be seen that the dual-targeted gene delivery system successfully disintegrated into small-sized nanoparticles before cross-linking under the acidic conditions of the tumor microenvironment.

[0126] 2. In vitro targeting of the tumor microenvironment-responsive dual-targeting gene delivery system

[0127] 1. Detection of LSD1 knockout in tumor cells by western blot technology:

[0128] MFC gastric cancer cells were cultured, and tumor cell protein samples were extracted 48 hours after administration. LSD1 protein expression was detected by western blot. Figure 2 AB results showed that the dual-targeted gene delivery system effectively knocked out LSD1 in tumor cells.

[0129] 2. Detection of LSD1 knockout in T cells by flow cytometry:

[0130] T cells were extracted from 615 mice, plated and collected 48 hours after administration, incubated with antibody working solution on ice, washed, filtered and loaded into flow tubes for detection by flow cytometry. The results showed that the dual-targeted gene delivery system effectively reduced the expression of LSD1-positive T cells ( Figure 3 ).

[0131] 3. Detect T cell function recovery in the co-culture system using live cell imaging:

[0132] The extracted T cells were co-incubated with MFC gastric cancer cells, and the viability of tumor cells in the co-incubation system was detected by live cell imaging 48 hours after administration. Figure 4 The results showed that after treatment with the dual-targeted gene delivery system, tumor cells were significantly killed, indicating that the T cell function of this group was best restored.

[0133] 3. In vivo targeting of the tumor microenvironment-responsive dual-targeting gene delivery system

[0134] 1. In vivo imaging to investigate tumor targeting:

[0135] When the tumor volume of mice reached 200 mm 3 The preparation containing Cy5.5 labeled plasmid was injected into the tail vein. After 1, 4, 8, 12, 24, and 36 hours of administration, the mice were anesthetized and placed in the in vivo imaging instrument to collect images. The results showed that the tumor microenvironment responsive dual-targeted gene delivery system can effectively accumulate in the tumor site ( Figure 5 ).

[0136] 2. Flow cytometry to investigate the uptake of the preparation by target cells:

[0137] When the tumor volume of mice reached 200 mm 3 At 14:00, a preparation containing a DiYO-1 labeled plasmid was injected into the tail vein. Twelve hours later, the tumor tissue was dissected out and lysed to obtain a single cell suspension, which was then stained, washed, filtered, and transferred to a flow cytometer for detection. Figure 6 The results showed that the preparation was mainly taken up by two target cells: T cells and tumor cells.

[0138] 4. Flow cytometry to investigate the effect of LSD1 knockout in tumor cells and T cells

[0139] MFC gastric cancer cells were cultured and inoculated subcutaneously into 615 mice. When the tumor volume reached 100 mm3, the drug was administered once every three days for a total of five times. Tumor tissues of tumor-bearing mice were extracted and single cell suspensions were obtained. After staining, washing, filtering, and transfer to flow tubes, flow cytometry was used for detection. Figure 7 AB found that LSD1 was effectively knocked out in both tumor cells and T cells.

[0140] 5. Flow cytometry to investigate the infiltration effect of CD8+ T cells and the expression of their exhaustion markers in tumor tissues

[0141] After administration, the tumor tissue of the tumor-bearing mice was extracted and a single cell suspension was obtained. After staining, washing, filtering, the suspension was transferred to a flow tube and detected by flow cytometry. Figure 8 AD results showed that after treatment with the dual-targeted gene delivery system, CD8 + The proportion of T infiltration was significantly increased, and exhaustion markers were significantly reduced.

[0142] The present invention conducted the same experiments on other embodiments according to the above experimental method, and obtained the same or similar results, which will not be listed here one by one.

[0143] It can be seen from the above experiments that compared with the prior art, the present invention has the following outstanding beneficial technical effects:

[0144] 1) The tumor microenvironment-responsive dual-targeted gene delivery system provided by the present invention has excellent biocompatibility and stability, and can program tumor cells and T cells in situ to exert immunotherapy;

[0145] 2) The tumor microenvironment-responsive dual-targeted gene delivery system provided by the present invention has sensitive tumor microenvironment acid responsiveness and can effectively knock out LSD1 in tumor cells and T cells in vitro and in vivo, thereby promoting CD8 + T cell infiltration, reducing the intensity of the PD-1 / PD-L1 axis, and reversing the CD8 + T cell depletion.

[0146] The present invention is easy to operate, the method is stable and reliable, and the prepared tumor microenvironment-responsive dual-targeted gene delivery system has good biocompatibility. It can realize the particle size reduction from large to small under the action of the acidic pH in the tumor microenvironment, separate targeting, and enhance the delivery of LSD1 gene by nanoparticles to tumor cells and T cells, effectively restore the function of effector T cells, and enhance the anti-tumor immunotherapy effect. It is an innovation in in situ reprogramming cells, has practical clinical significance and good application and promotion value, and has huge economic and social benefits.

[0147] It should be pointed out that the above is only a preferred embodiment of the present invention and does not impose any form of limitation on the present invention. Any technician familiar with this profession can make changes or modify the technical content disclosed above into equivalent embodiments without departing from the scope of the technical solution of the present invention, and all of them fall within the scope of protection of the present invention.

Claims

1. A method for preparing a tumor microenvironment-responsive dual-targeting gene delivery system, characterized in that: Polyethyleneimine and 1H-pyrazole-1-carboxamidine hydrochloride are reacted at room temperature, and then modified with a tumor cell-targeting peptide and a T cell-targeting peptide through an amide reaction, respectively. Subsequently, they are condensed with an sgLSD1 plasmid, physically mixed, and cross-linked with glutaraldehyde. Finally, poly(2-ethyl-2-oxazoline) is wrapped in the outer layer to form a stable core-shell structure, thereby obtaining a tumor microenvironment-responsive dual-targeting gene delivery system. The sgLSD1 plasmid is a plasmid for knocking out LSD1; the T cell-targeting peptide is a polypeptide chain with the sequence AKMGEGGWGANDY; and the tumor cell-targeting peptide is a polypeptide chain with the sequence SGQYASYHCWCWRDPGRSGGSK.

2. The method for preparing the tumor microenvironment-responsive dual-targeting gene delivery system according to claim 1, wherein: The specific steps include: 1) Preparation of guanylated polyethyleneimine Dissolve 1-6 g of polyethyleneimine in 3-8 mL of solvent, add the mixture to a three-necked flask and protect with nitrogen. Dissolve 1-6 g of 1H-pyrazole-1-carboxamidine hydrochloride in 5 mL of solvent, add the mixture to the polyethyleneimine solution, and stir at room temperature for 10-35 minutes. Add 1-6 g of triethylamine to the mixed solution and stir at room temperature for 24 hours. The resulting solution is placed in a 500 Da dialysis bag and dialyzed for 48 hours. The product is then freeze-dried to obtain guanylated polyethyleneimine. 2) Preparation of T cell targeting vector 10-60 mg of T cell targeting peptide was dissolved in 1-6 mL of solvent and placed in a three-necked flask, which was filled with nitrogen for protection. 3-8 mg of N-hydroxysuccinimide and 6-11 mg of (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) were dissolved in 1 mL of solvent and added to the above three-necked flask and stirred at room temperature for 12 hours. 20-70 mg of guanidinated polyethyleneimine was added to 2-7 mL of solvent and fully dissolved. The mixture was added to the three-necked flask and stirred at room temperature for 24 hours. After that, the mixture was placed in a 1500 Da dialysis bag and dialyzed for 48 hours to fully remove unreacted substances. The dialyzed product was freeze-dried for 48 hours to obtain a T cell targeting vector. 3) Preparation of tumor cell targeting vectors 20-70 mg of tumor cell targeting peptide was dissolved in 3 mL of solvent and then added to a three-necked flask and filled with nitrogen for protection. A solvent solution containing 3-8 mg of N-hydroxysuccinimide and 8-13 mg of (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) was then added to the three-necked flask and the carboxyl groups were activated at room temperature for 12 hours. Finally, a solvent solution containing 30-80 mg of guanidinated polyethyleneimine was added to the above reaction system and reacted at room temperature for 24 hours. The reaction solution was placed in a 2500 Da dialysis bag and dialyzed for 48 hours. The product was freeze-dried for 48 hours to obtain a tumor cell targeting vector. 4) Prepare T cell targeting vector solution and tumor cell targeting vector solution Dissolve 30-80 mg of each of the T cell-targeting vector prepared in step 2) and the tumor cell-targeting vector prepared in step 3) in 40 mL of solvent, adjust the pH of both solutions to 7.4 with 1 mol / L NaOH solution, transfer the solutions to a 50 mL volumetric flask, dilute to 50 mL with solvent, and shake well to obtain 1 mg / mL T cell-targeting vector and tumor cell-targeting vector solutions. Filter the prepared solutions through a 0.22 μm microporous filter membrane. 5) Synthesis of T cell targeting vector / plasmid complexes and tumor cell targeting vector / plasmid complexes Take two aliquots of plasmid, 4-9 mL and 7-12 mg respectively, and dilute them to 0.01 mg / mL with solvent. Take 50 mL of the T cell-targeting vector solution and tumor cell-targeting vector solution prepared in step 4) and dilute them to 0.1 mg / mL with solvent. Add them dropwise to the diluted plasmid solution, shake continuously to mix, and let stand at room temperature for 10 minutes to obtain T cell-targeting vector / plasmid complexes and tumor cell-targeting vector / plasmid complexes. 6) Tumor microenvironment-responsive dual-targeted gene delivery system Take the T cell targeting vector / plasmid complex and the tumor cell targeting vector / plasmid complex from step 5), mix the two solutions in equal amounts, add 170-220 mL of 25% glutaraldehyde solution, mix well, and let stand at room temperature for 5 minutes. Take 30-80 mg of poly(2-ethyl-2-oxazoline) and prepare it with a solvent to a solution with a concentration of 1 mg / mL. 300-800 mL is added dropwise to the above solution and let stand at room temperature for 10 minutes to obtain a tumor microenvironment-responsive dual-targeting gene delivery system.

3. The method for preparing the tumor microenvironment-responsive dual-targeting gene delivery system according to claim 2, characterized in that: The specific steps include: The solvent in steps 1) to 6) is one of ultrapure water, PBS buffer with pH=7.4, and formamide.

4. Use of the tumor microenvironment-responsive dual-targeting gene delivery system prepared by the preparation method according to any one of claims 1 to 3 in the preparation of anti-gastric cancer drugs.

5. Use of the tumor microenvironment-responsive dual-targeting gene delivery system prepared by the preparation method according to any one of claims 1 to 3 in the preparation of gene delivery drugs.

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