In-situ cross-linked high-molecular polymer nucleic acid delivery carrier as well as preparation method and application thereof

Abstract

The invention relates to the field of biomedical materials, in particular to an in-situ cross-linked high-molecular polymer nucleic acid delivery carrier as well as a preparation method and application thereof. The preparation method comprises the following steps: partially deacetylating poly (2-ethyl-2-oxazoline) to obtain a poly (2-ethyl-2-oxazoline)-polyethyleneimine random copolymer; 4-carboxybenzaldehyde and dihydroxy polyethylene glycol are subjected to a reaction to prepare dialdehyde polyethylene glycol; dissolving the two in water for in-situ cross-linking to form an in-situ cross-linked high-molecular product; the pH-sensitive polymer nucleic acid carrier can be used as a pH-sensitive polymer nucleic acid carrier, and has excellent transfection efficiency and good biocompatibility in different cells.

Description

Technical Field

[0001] The present invention relates to the field of biomedical materials, and in particular to an in-situ cross-linked high molecular polymer nucleic acid delivery carrier and a preparation method and application thereof. Background Art

[0002] As an emerging treatment method, gene therapy is used in clinical research on tumor treatment, accounting for about 65% of the total number of tumor clinical trials, and is expected to completely cure major diseases such as cancer. However, simple genes cannot enter cells on their own to play a role, so the participation of gene carriers is needed.

[0003] The current vectors are mainly divided into viral vectors and non-viral vectors. The use of viral vectors to deliver genes has disadvantages such as high safety risks and limited nucleic acid carrying capacity. Among non-viral vectors, polymer vectors have attracted extensive attention from scientific researchers due to their advantages of good biosafety, large nucleic acid carrying capacity, and easy functionalization.

[0004] Among the many polymer carriers, polyethyleneimine is a widely studied non-viral gene delivery vector and one of the most effective gene carriers to date. However, although low molecular weight polyethyleneimine has low toxicity, its transfection effect is poor. Although high molecular weight polyethyleneimine has a high transfection effect, its high cytotoxicity also limits its application. Among them, polyethyleneimine with a molecular weight of 25k (PEI25k) is known as the "gold standard" for polymer carrier transfection efficiency. It shows stable and efficient gene transfection effects under different cells and transfection conditions, but its high cytotoxicity also limits the application of LPEI. It can be seen that it is difficult to have both high transfection efficiency and low cytotoxicity. How to balance the transfection effect and cytotoxicity of the material and develop safe and efficient polymer gene delivery vectors is urgent. Researchers have developed a series of polymer carriers around polyethyleneimine, but its gene delivery efficiency and biosafety still need to be further improved to meet clinical applications. Summary of the invention

[0005] In view of this, the present invention provides an in situ cross-linked polymer nucleic acid delivery vector and a preparation method and application thereof. The in situ cross-linked polymer nucleic acid delivery vector prepared by the present invention has both excellent transfection efficiency and good biocompatibility.

[0006] The present invention provides a method for preparing an in situ cross-linked polymer nucleic acid delivery vector, comprising the following steps:

[0007] A) deacetylation of poly(2-ethyl-2-oxazoline) to obtain a poly(2-ethyl-2-oxazoline)-polyethyleneimine random copolymer represented by formula (1);

[0008] B) reacting 4-carboxybenzaldehyde with bishydroxy polyethylene glycol in the presence of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 4-dimethylaminopyridine to obtain a bisaldehyde polyethylene glycol represented by formula (2);

[0009] C) dissolving the poly(2-ethyl-2-oxazoline)-polyethyleneimine random copolymer obtained in step A) and the dialdehyde polyethylene glycol obtained in step B) in water to obtain an in situ cross-linked polymer nucleic acid delivery vector;

[0010] There is no order restriction for step A) and step B);

[0011]

[0012] Preferably, in step A), the molecular weight of the poly(2-ethyl-2-oxazoline) is 300 to 500,000 Dalton;

[0013] The reaction temperature of the deacetylation is 130°C.

[0014] Preferably, step A) specifically comprises:

[0015] A1) mixing poly(2-ethyl-2-oxazoline) with an acid solution, heating to perform deacetylation, and obtaining a reaction solution;

[0016] A2) adjusting the pH value of the reaction solution obtained in step A1) to a weak alkaline state;

[0017] A3) dialyzing and freeze-drying the solution obtained in step A2) to obtain a poly(2-ethyl-2-oxazoline)-polyethyleneimine random copolymer represented by formula (1).

[0018] Preferably, in step A1), the acid solution is at least one of a hydrochloric acid aqueous solution, a sulfuric acid aqueous solution and a trifluoroacetic acid aqueous solution;

[0019] The concentration of the acid solution is 2-6 mol / L.

[0020] Preferably, in step A2), the pH value is adjusted to 7.4-8.0;

[0021] In step A3), the molecular weight of the dialysis is 500 to 7000 Dalton, and the dialysis time is 48 to 96 hours.

[0022] Preferably, in step B), the reaction temperature is 20-40°C.

[0023] Preferably, step B) specifically comprises:

[0024] B1) mixing 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 4-dimethylaminopyridine, 4-carboxybenzaldehyde, bishydroxy polyethylene glycol and a solvent to obtain a reaction solution;

[0025] B2) removing the solvent from the reaction solution obtained in step B1), adding water to dissolve, dialyzing and freeze-drying to obtain the dialdehyde polyethylene glycol represented by formula (2).

[0026] Preferably, in step B2), the dialysis molecular weight is 500 to 7000 Dalton and the dialysis time is 48 to 96 hours.

[0027] The present invention also provides an in-situ cross-linked high molecular polymer nucleic acid delivery vector prepared by the preparation method described in the above technical solution.

[0028] The present invention also provides an application of the in-situ cross-linked high molecular polymer nucleic acid delivery vector described in the above technical solution in the preparation of a nucleic acid vector.

[0029] The preparation method of the present invention uses poly(2-ethyl-2-oxazoline) to partially deacetylate to obtain poly(2-ethyl-2-oxazoline)-polyethyleneimine random copolymers with different degrees of deacetylation; 4-carboxybenzaldehyde is reacted with dihydroxy polyethylene glycol to prepare dialdehyde polyethylene glycol; the two are then dissolved in water and cross-linked in situ to form an in situ cross-linked polymer product; the polyethylene glycol is used to improve the long-circulation ability of the carrier / gene complex in vivo, and the pH sensitivity of the Schiff base bond formed by the aldehyde group and the amino group is used to achieve the controlled release of the gene in vivo, so as to improve the comprehensive performance of the polymer carrier. The in situ cross-linked nucleic acid delivery carrier OHC-PEG prepared by the present invention m -CHO / PEtOx n(1-x) -r-PEI nx It has excellent transfection efficiency and good biocompatibility in different cells. Its preparation steps are simple and reproducible, providing new ideas for the development of efficient and safe polymer nucleic acid vectors. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0031] Figure 1 A schematic diagram of one of the crosslinking conditions in the in-situ crosslinked product of the present invention;

[0032] Figure 2Schematic diagram of the evolution of pH sensitivity of the cross-linked product obtained in the present invention. DETAILED DESCRIPTION

[0033] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application belongs. The terms used herein in the specification of this application are only for the purpose of describing specific embodiments and are not intended to limit this application.

[0034] In this article, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0035] As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0036] Herein, when it comes to numerical ranges, unless otherwise specified, the numerical ranges are deemed to be continuous and include the minimum and maximum values ​​of the range, as well as every value between such minimum and maximum values. Further, when a range refers to an integer, every integer between the minimum and maximum values ​​of the range is included. In addition, when multiple ranges are provided to describe features or characteristics, the ranges can be merged. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges included therein.

[0037] In this article, when referring to the unit of a data range, if there is a unit only after the right endpoint, it means that the units of the left and right endpoints are the same. For example, 2~6mol / L means that the units of the left endpoint "2" and the right endpoint "6" are both mol / L.

[0038] The present invention provides a method for preparing an in situ cross-linked polymer nucleic acid delivery vector, comprising the following steps:

[0039] A) deacetylation of poly(2-ethyl-2-oxazoline) to obtain a poly(2-ethyl-2-oxazoline)-polyethyleneimine random copolymer represented by formula (1);

[0040] B) reacting 4-carboxybenzaldehyde with bishydroxy polyethylene glycol in the presence of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 4-dimethylaminopyridine to obtain a bisaldehyde polyethylene glycol represented by formula (2);

[0041] C) dissolving the poly(2-ethyl-2-oxazoline)-polyethyleneimine random copolymer obtained in step A) and the dialdehyde polyethylene glycol obtained in step B) in water to obtain an in situ cross-linked polymer nucleic acid delivery vector;

[0042] There is no order restriction for step A) and step B);

[0043]

[0044]

[0045] [About Step A]:

[0046] A) Poly(2-ethyl-2-oxazoline) is deacetylated to obtain a poly(2-ethyl-2-oxazoline)-polyethyleneimine random copolymer represented by formula (1).

[0047] The reaction scheme of this step is as follows:

[0048]

[0049] In the present invention, step A) preferably specifically comprises:

[0050] A1) mixing poly(2-ethyl-2-oxazoline) with an acid solution, heating to perform deacetylation, and obtaining a reaction solution;

[0051] A2) adjusting the pH value of the reaction solution obtained in step A1) to a weak alkaline state;

[0052] A3) dialyzing and freeze-drying the solution obtained in step A2) to obtain a poly(2-ethyl-2-oxazoline)-polyethyleneimine random copolymer represented by formula (1).

[0053] Regarding step A1):

[0054] In the present invention, the poly (2-ethyl-2-oxazoline) (abbreviated as PEtOx n ) has a molecular weight of preferably 300 to 500,000 Dalton, specifically 300, 500, 1000, 1500, 3000, 5000, 10000, 20000, 50000, 100000, 200000, 300000, 400000, 500000 Dalton.

[0055] In the present invention, the acid solution is preferably at least one of hydrochloric acid aqueous solution, sulfuric acid aqueous solution and trifluoroacetic acid aqueous solution, more preferably hydrochloric acid aqueous solution. In the present invention, the concentration of the acid solution is preferably 2 to 6 mol / L, specifically 2 mol / L, 3 mol / L, 4 mol / L, 5 mol / L, 6 mol / L. In the present invention, when the poly (2-ethyl-2-oxazoline) is mixed with the acid solution, the acid solution can be excessive; more preferably, taking the acid solution with a concentration of 6 mol / L as an example, the amount ratio of poly (2-ethyl-2-oxazoline) to the acid solution is preferably 1 g: 5 mL, and the acid solutions of other concentrations can be converted according to this amount relationship.

[0056] In the present invention, the deacetylation by heating is specifically deacetylation by heating under reflux. The temperature of the heating under reflux is preferably 130° C. The time of the heating under reflux is preferably 2 to 24 hours, specifically 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours.

[0057] After the above deacetylation reaction, a poly(2-ethyl-2-oxazoline)-polyethyleneimine random copolymer represented by formula (1) is generated in the system, and a reaction solution containing the compound represented by formula (1) is obtained. The reaction solution is preferably cooled, specifically to room temperature, before proceeding to the next step.

[0058] Regarding step A2):

[0059] In the present invention, the regulator used to adjust the pH value is preferably an alkaline regulator, more preferably a NaOH aqueous solution. In the present invention, the concentration of the NaOH aqueous solution is preferably 2-6 mol / L, specifically 2 mol / L, 3 mol / L, 4 mol / L, 5 mol / L, 6 mol / L.

[0060] In the present invention, the weak alkalinity is preferably such that the pH value reaches 7.4 to 8.0, specifically 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0.

[0061] Regarding step A3):

[0062] In the present invention, the molecular weight of the dialysis is preferably 500 to 7000 Dalton. The dialysis time is preferably 48 to 96 hours. After the product of the above molecular weight is intercepted by dialysis, it is freeze-dried to obtain the poly (2-ethyl-2-oxazoline) - polyethyleneimine random copolymer (abbreviated as PEtOx nx -r-PEI n(1-x) , or [PEtOx x -r-PEI (1-x) ] n ).

[0063] In formula (1), 0<x<1, specifically 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, more preferably 0.2. 1-x corresponding to the polyethyleneimine unit represents the degree of deacetylation. For example, the degree of deacetylation is 80%, that is, 1-x=0.8, and the product is PEtOx n0.2 -r-PEI n0.8 or [PEtOx 0.2 -r-PEI 0.8 ] n .

[0064] [About step B]:

[0065] B) In the presence of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 4-dimethylaminopyridine, 4-carboxybenzaldehyde reacts with bishydroxy polyethylene glycol to obtain bisaldehyde polyethylene glycol represented by formula (2).

[0066] The reaction scheme of this step is as follows:

[0067]

[0068] In the present invention, step B) preferably specifically comprises:

[0069] B1) mixing 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 4-dimethylaminopyridine, 4-carboxybenzaldehyde, bishydroxy polyethylene glycol and a solvent to obtain a reaction solution;

[0070] B2) removing the solvent from the reaction solution obtained in step B1), adding water to dissolve, dialyzing and freeze-drying to obtain the dialdehyde polyethylene glycol represented by formula (2).

[0071] Regarding step B1):

[0072] In the present invention, the 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (C8H 17 N3·HCl) and 4-dimethylaminopyridine (DMAP) act as catalysts in the reaction and also react with carboxylic acid to generate active intermediates. These two substances are generated again after the main reaction is completed.

[0073] In the present invention, the molar ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride to 4-carboxybenzaldehyde is preferably (5-50):(5-50), i.e. (0.1-10):1, and specifically can be 0.1:1, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1. The above ratio range refers to the ratio of the lowest value in the left value range / the highest value in the right value range to the ratio of the highest value in the left value range / the lowest value in the right value range, i.e. (5:50) to (50:5) (i.e. (0.1-10):1). In this article, the same applies to other places.

[0074] In the present invention, the molar ratio of 4-dimethylaminopyridine to 4-carboxybenzaldehyde is preferably (1-5):(5-50), that is, (0.02-1):1, specifically 0.02:1, 0.05:1, 0.1:1, 0.5:1, 1:1.

[0075] In the present invention, the molar ratio of the 4-carboxybenzaldehyde to -OH in the dihydroxy polyethylene glycol is preferably (5-50):2, specifically 5:2, 10:2, 15:2, 20:2, 25:2, 30:2, 35:2, 40:2, 45:2, 50:2.

[0076] In the present invention, the solvent is preferably an aprotic solvent, more preferably at least one of dichloromethane, N,N-dimethylformamide and acetonitrile. The amount of the solvent is not particularly limited, as long as it can fully dissolve and mix the above materials.

[0077] In the present invention, the reaction temperature is preferably 20-40°C, specifically 20°C, 25°C, 30°C, 35°C, 40°C. The reaction time is preferably 48-96h, specifically 48h, 72h, 96h. After the reaction, a reaction solution containing the dialdehyde polyethylene glycol represented by formula (2) is obtained.

[0078] Regarding step B2):

[0079] In the present invention, the method of removing the solvent is preferably rotary evaporation. After the solvent is dried, water is added to dissolve it, preferably deionized water. There is no special restriction on the amount of water used, as long as it can fully dissolve the material. After dissolution, dialysis and lyophilization are performed. Among them, the dialysis molecular weight is preferably 500 to 7000 Dalton. The dialysis time is preferably 48 to 96 hours. After the dialysis intercepts the product of the above molecular weight, lyophilization is performed to obtain the dialdehyde polyethylene glycol (abbreviated as OHC-PEG) shown in formula (2) m -CHO).

[0080] [About Step C]:

[0081] C) dissolving the poly(2-ethyl-2-oxazoline)-polyethyleneimine random copolymer obtained in step A) and the dialdehyde polyethylene glycol obtained in step B) in water to obtain an in situ cross-linked high molecular weight nucleic acid delivery vector.

[0082] In the present invention, the water is preferably deionized water.

[0083] In the present invention, the concentration of the poly(2-ethyl-2-oxazoline)-polyethyleneimine random copolymer obtained in step A) in water is preferably 0.25 to 2 mg / mL, specifically 0.25 mg / mL, 0.30 mg / mL, 0.35 mg / mL, 0.40 mg / mL, 0.45 mg / mL, 0.50 mg / mL, 0.55 mg / mL, 0.60 mg / mL, 0.65 mg / mL, 0.70 mg / mL, 0.75 mg / mL, 0.80 mg / mL, 0.85 mg / mL, 0.90 mg / mL, 0.95 mg / mL, 1.00mg / mL, 1.05mg / mL, 1.10mg / mL, 1.15mg / mL, 1.20mg / mL, 1.25mg / mL, 1.30mg / mL, 1.35mg / mL, 1.40mg / mL, 1.45mg / mL, 1.50m g / mL, 1.55mg / mL, 1.60mg / mL, 1.65mg / mL, 1.70mg / mL, 1.75mg / mL, 1.80mg / mL, 1.85mg / mL, 1.90mg / mL, 1.95mg / mL, 2.00mg / mL.

[0084] In the present invention, the concentration of the bisaldehyde polyethylene glycol obtained in step B) in water is preferably 0.125-1 mg / mL, specifically 0.125 mg / mL, 0.15 mg / mL, 0.20 mg / mL, 0.25 mg / mL, 0.30 mg / mL, 0.35 mg / mL, 0.40 mg / mL, 0.45 mg / mL, 0.50 mg / mL, 0.55 mg / mL, 0.60 mg / mL, 0.65 mg / mL, 0.70 mg / mL, 0.75 mg / mL, 0.80 mg / mL, 0.85 mg / mL, 0.90 mg / mL, 0.95 mg / mL, 1.00 mg / mL.

[0085] In the present invention, the poly(2-ethyl-2-oxazoline)-polyethyleneimine random copolymer obtained in step A) and the dialdehyde polyethylene glycol obtained in step B) are dissolved in water. The temperature condition is not particularly limited and can be at room temperature. The two naturally undergo a cross-linking reaction in the aqueous solution to form an in situ cross-linked polymer nucleic acid delivery vector.

[0086] The cross-linking process is relatively complicated, and many dialdehyde polyethylene glycols and PEtOx may appear. n(1-x) -r-PEI nx The amino groups on the main chain undergo cross-linking reactions. Take one of the cases as an example, Figure 1 As shown, the red structure in the middle is dialdehyde polyethylene glycol, and the two blue structures on the left and right are random copolymers PEtOx n(1-x) -r-PEI nx The amino groups on the main chain of the random copolymer react with the aldehyde groups on the dialdehyde polyethylene glycol to form a structure as shown in the figure. After cross-linking, the N atom is positively charged. The above is just an example. In the actual cross-linking process, the two aldehyde groups at both ends of the dialdehyde polyethylene glycol may cross-link with the amino groups on the same main chain, and one aldehyde group of the dialdehyde polyethylene glycol may cross-link with the amino group, resulting in a cross-linked product with a complex structure.

[0087] The present invention also provides an in-situ cross-linked polymer nucleic acid delivery vector prepared by the preparation method described in the above technical solution.

[0088] The present invention also provides an application of the in situ cross-linked polymer nucleic acid delivery vector described in the above technical solution in the preparation of nucleic acid delivery vectors. The nucleic acid type that can be carried by the in situ cross-linked polymer nucleic acid delivery vector prepared by the present invention is DNA, mRNA or siRNA.

[0089] The preparation method of the present invention comprises the following steps: partially deacetylating poly(2-ethyl-2-oxazoline) to obtain poly(2-ethyl-2-oxazoline)-polyethyleneimine random copolymers with different deacetylation degrees; further utilizing 4-carboxybenzaldehyde to react with dihydroxy polyethylene glycol to prepare dialdehyde polyethylene glycol; then dissolving the two in water and in situ cross-linking them to form an in situ cross-linked polymer product; improving the in vivo long circulation ability of the carrier / gene complex by polyethylene glycol, and realizing the in vivo controlled release of the gene (such as lipase) by the pH sensitivity of the Schiff base bond formed by the aldehyde group and the amino group. Figure 2 As shown, as the pH changes, the Schiff base of the carrier breaks to release the nucleic acid it carries), thereby improving the comprehensive performance of the polymer carrier. The in situ cross-linked nucleic acid delivery carrier OHC-PEG prepared by the present invention m -CHO / PEtOx n(1-x) -r-PEI nx It has excellent transfection efficiency and good biocompatibility in different cells. Its preparation steps are simple and reproducible, providing new ideas for the development of efficient and safe polymer nucleic acid vectors.

[0090] OHC-PEG with a molecular weight of 2000 Dalton 2000-CHO), PEtOx with a molecular weight of 50,000 Dalton and a deacetylation degree of 80% to form a random copolymer PEtOx n(1-x) -r-PEI nx Polymer carrier (PEtOx 50,0.2 -r-PEI 50,0.8 ) as an example, the preparation process is as follows:

[0091] A1) dissolving 50000 Dalton poly(2-ethyl-2-oxazoline) in a 6 mol / L aqueous solution of HCl, heating under reflux at 130° C. for deacetylation, to obtain a reaction solution;

[0092] A2) adjusting the pH value of the reaction solution obtained in step A1) to 7.5;

[0093] A3) dialyzing and freeze-drying the solution obtained in step A2) to obtain a poly(2-ethyl-2-oxazoline)-polyethyleneimine random copolymer PEtOx having a deacetylation degree of 80% 50,0.2 -r-PEI 50,0.8 ,

[0094] B1) 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 4-dimethylaminopyridine, 4-carboxybenzaldehyde, dihydroxy polyethylene glycol (OH-PEG 2000 -OH) is added into dichloromethane to react to obtain a reaction solution;

[0095] B2) The reaction solution obtained in step B1) is subjected to rotary evaporation to dry the solvent, dissolved in deionized water, dialyzed and freeze-dried to obtain the dialdehyde polyethylene glycol OHC-PEG represented by formula (2) 2000 -CHO;

[0096] C) Poly(2-ethyl-2-oxazoline)-polyethyleneimine random copolymer PEtOx obtained in step A) 50,0.2 -r-PEI 50,0.8 and the dialdehyde polyethylene glycol OHC-PEG obtained in step B) 2000 -CHO was dissolved in deionized water to obtain the in situ cross-linked polymer nucleic acid delivery carrier OHC-PEG 2000 -CHO / PEtOx 50,0.2 -r-PEI 50，0.8 .

[0097] 1. OHC-PEG m -CHO / PEtOx n(1-x) -r-PEI nxAs a polymer vector of pDNA, it is used in a variety of cell lines (such as B16F10, HeLa, etc.); as a polymer vector of pLUC (luciferase plasmid), the steps and conditions are as follows:

[0098] (1) Cell culture

[0099] Taking B16F10 cells as an example, the cells were cultured with a culture medium containing 10% fetal bovine serum by volume, and cultured in a constant temperature incubator at a culture temperature of 37° C. and a CO 2 volume fraction of 5%.

[0100] (2) Cell transfection experiment

[0101] B16F10 cells in the logarithmic phase were selected and digested with trypsin, neutralized with cell culture medium, centrifuged, washed twice with PBS, and suspended in fresh cell culture medium at a rate of 1×10 cells per well. 4 The cells were plated at a density of 100 μg / well in a 96-well plate and cultured in a constant temperature incubator at 37°C and 5% CO2 volume fraction until the cell confluence reached 80-90%. During cell transfection, the vector / pDNA was incubated for 20 minutes according to the set mass ratio, and then 0.2 μg pDNA was added to each well of the 96-well plate and incubated in a constant temperature incubator for 48 hours.

[0102] (3) Determination of transfection efficiency

[0103] Luciferase (LUC) expression detection

[0104] Take out the cells after transfection for 48 hours from the incubator, remove the cell culture medium, wash twice with PBS, add 25 μL of cell lysis solution to each well, place in a -80°C refrigerator for freezer lysis for 20 minutes, add 25 μL of luciferase substrate to each well, and use an enzyme marker to detect the cell transfection efficiency of the nucleic acid vector.

[0105] (4) Detection of cytotoxicity (CCK8)

[0106] The cells in logarithmic growth phase were obtained and digested with trypsin. Then, 10% fetal bovine serum culture medium was added to neutralize the cells. The cells were centrifuged, washed twice with PBS, and suspended in fresh cell culture medium. The cells were then incubated at 1×10 4 The density of cells / well was plated in a 96-well plate and cultured in a constant temperature incubator at 37°C and 5% CO2 until the cell confluence reached 80-90%. Different concentrations of polymer carriers were added to the cell plate and cultured for 24 hours, the cell culture medium was removed, and then CCK8 / 1640 solution was added at a volume ratio of 1:9 of CCK8:1640 culture medium, and cultured at 37°C for 30-45 minutes. The absorption of the cell plate was detected by an ELISA instrument with a detection wavelength of 450nm. The cell survival rate was calculated according to the following formula:

[0107] Cell survival rate = [(As-Ab) / (Ac-Ab)] × 100%

[0108] Among them, As is the absorbance value of the test group, Ab is the absorbance value of the blank group, Ac is the absorbance value of the control group, and each group of materials contains 3 replicate wells.

[0109] 2. OHC-PEG m -CHO / PEtOx n(1-x) -r-PEI nx As a polymer carrier of mRNA, it is used in various cell lines (such as B16F10, HeLa, etc.); as a carrier of mLUC (mRNA encoding luciferase), the steps and conditions are as follows:

[0110] (1) Cell culture

[0111] The cells were placed in a culture medium containing 10% by volume of fetal bovine serum and cultured in a constant temperature incubator at 37° C. and 5% by volume of CO 2 .

[0112] (2) Cell transfection

[0113] The cells in the logarithmic phase were obtained, digested with trypsin, neutralized with 10% fetal bovine serum culture medium, centrifuged, washed twice with PBS, and suspended in fresh cell culture medium at 1×10 4 Cells / well were plated in 96-well plates and cultured in a constant temperature incubator at 37°C and 5% CO2 until the cell confluence reached 80-90%. During transfection, the vector / mRNA complex was added for 20 minutes, 0.2 μg mRNA / well was added to the 96-well cell plate, and incubated for 24 hours.

[0114] (3) Determination of cell transfection efficiency

[0115] Luciferase expression detection

[0116] Take out the cells after transfection for 24 hours from the incubator, remove the cell culture medium, wash twice with PBS, add 25 μL of cell lysis solution to each well, place in a -80℃ refrigerator for freezer lysis for 20 minutes, add 25 μL of luciferase substrate to each well, and quantitatively determine the cell transfection efficiency by microplate reader.

[0117] 3. OHC-PEG m -CHO / PEtOx n(1-x) -r-PEI nxAs a polymer carrier of siRNA, it is used in various cell lines (such as HeLa LUC, B16F10 LUC, etc.); as a LUC siRNA (siRNA encoding silencing luciferase) carrier, the steps and conditions are as follows:

[0118] (1) The sequence of siRNA is 5'-CUUACGCU GAGUACUUCGAdTdT-3', which is LUC siRNA that can silence luciferase.

[0119] (2) Cell culture

[0120] The cells were cultured in a culture medium containing 10% by volume fetal bovine serum in a constant temperature incubator at 37° C. and 5% by volume CO 2 .

[0121] (3) Cell transfection

[0122] Logarithmic phase cells were obtained, digested with trypsin, neutralized with culture medium containing 10% fetal bovine serum, centrifuged, washed twice with PBS, suspended in fresh cell culture medium, and concentrated to 1×10 4 The cells were plated at a density of 100 cells / well in a 96-well cell plate and cultured in a constant temperature incubator at 37°C and 5% CO2 until the cell confluence reached 80-90%. During transfection, the vector / siRNA complex was added to the 96-well cell plate at 0.2 μg siRNA / well after 20 minutes of incubation, and the cells were incubated for 48 hours.

[0123] (4) Determination of cell transfection efficiency

[0124] After incubation for 48 h, the cells were removed from the cell culture incubator, the cell culture medium was removed, the cells were washed twice with PBS, 25 μL of cell lysis solution was added, and the cells were placed in a -80°C refrigerator for lysis for 20 min. Then, 25 μL of luciferase substrate was added to each well, and the gene silencing efficiency of the vector material was determined by an enzyme reader.

[0125] 4. The present invention provides an in situ cross-linked polymer nucleic acid carrier OHC-PEG m -CHO / PEtOx n(1-x) -r-PEI nx In vivo application is achieved through the following scheme:

[0126] (1) Culture of B16F10 cells

[0127] The B16F10 cells were placed in a culture medium containing 10% by volume of fetal bovine serum and cultured in a constant temperature incubator at 37° C. and 5% by volume of CO 2 .

[0128] (2) Tumor inoculation

[0129] C57BL / 6J mice aged 6 to 8 weeks and weighing about 20 g were purchased. B16F10 cells in the logarithmic phase were obtained before tumor inoculation. After trypsin digestion, trypsin was neutralized with cell culture medium and the cells were heated to 1×10 3 The cells were centrifuged at rpm for 3 min, washed twice with PBS, and finally suspended in PBS. 6 The cells were inoculated in the armpits of mice. After 8 days, when the tumor diameter reached 8 mm, pDNA was transfected in vivo.

[0130] (3) In vivo transfection

[0131] When the tumor diameter reached 8mm, pDNA was transfected in vivo. pRFP expressing red fluorescent protein was selected as the reporter gene, and the vector / pRFP complex was injected into the tumor-bearing mice through the tail vein at a volume of 200μL with a pRFP dosage of 20μg per mouse. After 48h of in vivo transfection, the mice were killed, and the tumors, heart, liver, spleen, lungs, and kidneys of each group were removed and imaged using small animal in vivo imaging technology.

[0132] (4) Distribution in the body

[0133] When the tumor diameter reached 8 mm, the in vivo distribution of pDNA was performed. Cy5 DNA was selected as the reporter gene, and the vector / Cy5 DNA complex was injected into the tumor-bearing mice through the tail vein at a volume of 200 μL with a Cy5 DNA dosage of 20 μg per mouse. After 24 hours, the mice were killed, and the tumors, heart, liver, spleen, lungs, and kidneys of each group were removed and imaged using small animal in vivo imaging technology.

[0134] (5) In vivo treatment

[0135] The therapeutic genes used were plasmid pSpam1, which can express hyaluronidase, and promote T cells to penetrate into tumor tissues by destroying the extracellular matrix of tumor cells, thereby achieving T cell-mediated tumor killing; and pshVEGF, which can express shRNA that silences vascular endothelial growth factor VEGF. The vector / pDNA complex (volume 100μL) was injected into the tail vein of tumor-bearing mice with 20ug pSpam1 or 20ug pshVEGF per mouse, and the drug was given once every 2 days for a total of 4 times.

[0136] (6) Determination of tumor size

[0137] Starting from the first administration of the drug to the mice, the changes in tumor size and body weight of the tumor-bearing mice were measured every day.

[0138] OHC-PEG provided by the present invention 2000 -CHO / PEtOx 50,0.2 -r-PEI 50,0.8It is an efficient, safe and multifunctional polymer carrier. The optimal transfection efficiency of pLUC carried by it in B16F10 cells is comparable to that of PEI22k. 2000 -CHO / PEtOx 50,0.2 -r-PEI 50,0.8 At the optimal transfection ratio, the cell survival rate is higher than that of PEI22k; at the same time, the prepared polymer carrier can achieve a step-by-step increase in transfection efficiency and cell uptake efficiency under the conditions of pH 7.4 (simulating physiological environment), pH 6.8 (simulating tumor acidic microenvironment), and pH 5.4 (simulating lysosome). The polymer carrier has better cell uptake efficiency and less cytotoxicity. In addition, the carrier can not only achieve good in vitro DNA transfection, but also has excellent transfection effect in in vitro mRNA transfection. And using OHC-PEG 2000 -CHO / PEtOx 50,0.2 -r-PEI 50,0.8 Tail vein administration of therapeutic genes pshVEGF and pSpam1 can significantly inhibit tumor growth.

[0139] In order to further understand the present invention, preferred embodiments of the present invention are described below in conjunction with examples. However, it should be understood that these descriptions are only for further illustrating the features and advantages of the present invention, rather than limiting the claims of the present invention.

[0140] Examples 1 to 16

[0141] 1. Preparation of polymers of formula (1) with different molecular weights and different degrees of deacetylation

[0142] A1) dissolving poly(2-ethyl-2-oxazoline) of different molecular weights (10000, 20000, 50000, 100000 Dalton) in a 6 mol / L aqueous solution of HCl, heating under reflux at 130° C. for deacetylation to obtain a reaction solution;

[0143] A2) adjusting the pH value of the reaction solution obtained in step A1) to 7.5;

[0144] A3) dialyzing and freeze-drying the solution obtained in step A2) to obtain a series of partially deacetylated poly(2-ethyl-2-oxazoline)-polyethyleneimine random copolymers PEtOx represented by formula (1) nx -r-PEI n(1-x) , see Table 1 (16 products in total).

[0145] Table 1: Types of polymers represented by formula (1)

[0146] The polymer of formula (1) is referred to as Deacetylation degree / % <![CDATA[PEtOx 10,0.5 -r-PEI 10,0.5 ]]> 50 <![CDATA[PEtOx 10,0.3 -r-PEI 10,0.7 ]]> 70 <![CDATA[PEtOx 10,0.2 -r-PEI 10,0.8 ]]> 80 <![CDATA[PEtOx 10,0 -r-PEI 10,1 ]]> 100 <![CDATA[PEtOx 20,0.5 -r-PEI 20,0.5 ]]> 50 <![CDATA[PEtOx 20,0.3 -r-PEI 50,0.7 ]]> 70 <![CDATA[PEtOx 20,0.2 -r-PEI 20,0.8 ]]> 80 <![CDATA[PEtOx 20,0 -r-PEI 20,1 ]]> 100 <![CDATA[PEtOx 50,0.5 -r-PEI 50,0.5 ]]> 50 <![CDATA[PEtOx 50,0.3 -r-PEI 50,0.7 ]]> 70 <![CDATA[PEtOx 50,0.2 -r-PEI 50,0.8 ]]> 80 <![CDATA[PEtOx 50,0 -r-PEI 50,1 ]]> 100 <![CDATA[PEtOx 100,0.5 -r-PEI 100,0.5 ]]> 50 <![CDATA[PEtOx 100,0.3 -r-PEI 100,0.7 ]]> 70 <![CDATA[PEtOx 100,0.2 -r-PEI 100,0.8 ]]> 80 <![CDATA[PEtOx 100,0 -r-PEI 100,1 ]]> 100

[0147] 2. In vitro DNA transfection of polyoxazolines with different molecular weights and different degrees of deacetylation

[0148] B16F10 cells were added to a culture medium containing 10% fetal bovine serum and cultured in a constant temperature incubator set at 37°C and 5% CO2. Before transfection, logarithmic phase cells were obtained, digested with trypsin, and diluted with a culture medium containing 10% fetal bovine serum to a concentration of 1×10 4 The cells / well were plated in a 96-well plate and cultured in a constant temperature incubator at 37°C and 5% CO2 until the cell confluence reached 80-90%. During transfection, polymers with different molecular weights and different degrees of deacetylation were loaded with pLUC. After the carrier / LUC complex was complexed for 20 minutes, 0.2 μg pDNA / well was added to the 96-well plate and cultured for 48 hours.

[0149] The cell transfection efficiency was determined as follows:

[0150] The cells were removed from the cell culture incubator, the cell culture medium was removed, and the cells were washed twice with PBS. 25 μL of cell lysis solution was added, and the cells were placed in a -80°C refrigerator for lysis for 20 min. Then 25 μL of luciferase substrate was added to each well, and the transfection efficiency of the vector / pLUC complex in B16F10 cells was detected by an ELISA instrument. Table 2 shows the transfection efficiency of the vector / pLUC complex.

[0151] Table 2: In vitro transfection efficiency of luciferase plasmid mediated by polymers of different molecular weights and different deacetylation degrees

[0152]

[0153] It can be seen from the test results in Table 2 that the use of polyoxazoline carriers with different molecular weights and different deacetylation degrees to load DNA all achieved a certain transfection efficiency, among which the polymer PEtOx 50,0.2 -r-PEI 50,0.8 Achieved the highest DNA transfection efficiency.

[0154] 3. In vitro mRNA transfection of polyoxazolines with different molecular weights and different degrees of deacetylation

[0155] B16F10 cells were added to a culture medium containing 10% fetal bovine serum and cultured in a constant temperature incubator at 37°C and 5% CO2. Logarithmic phase cells were obtained, digested with trypsin, and diluted with a culture medium containing 10% fetal bovine serum to a concentration of 1×10 4Cells / well were plated in a 96-well plate and cultured in a constant temperature incubator at 37°C and 5% CO2 until the cell confluence reached 80-90%. Polymers with different molecular weights and different degrees of deacetylation were used to load mLUC (mRNA encoding luciferase). During transfection, the vector / mRNA complex was compounded for 20 minutes, and then added to the 96-well plate at a dosage of 0.2 μg mRNA / well, and cultured for 24 hours.

[0156] Luciferase activity assay:

[0157] The cells were removed from the cell culture incubator, the cell culture medium was removed, and the cells were washed twice with PBS, 25 μL of cell lysis solution was added, and the cells were placed in a -80°C refrigerator for lysis for 20 min. Then, 25 μL of luciferase substrate was added to each well, and the transfection efficiency of the complex in B16F10 cells was measured by an ELISA reader. Table 3 shows the transfection efficiency of the vector / mL UC complex.

[0158] Table 3: In vitro transfection efficiency of luciferase mRNA mediated by polymers of different molecular weights and different deacetylation degrees

[0159]

[0160] It can be seen from the test results in Table 3 that the use of polyoxazoline carriers with different molecular weights and different deacetylation degrees to carry mRNA all achieved a certain transfection efficiency, among which the polymer PEtOx 50,0.2 -r-PEI 50,0.8 Achieved the highest mRNA transfection efficiency.

[0161] 4. In vitro cytotoxicity of polyoxazolines with different molecular weights and different degrees of deacetylation

[0162] B16F10 cells were added to a 96-well plate containing 10% fetal bovine serum culture medium and cultured in a CO2 constant temperature incubator set at 37°C and 5% volume fraction for 24 hours. After 24 hours, the culture medium was aspirated, different concentrations of polyoxazoline were added and the culture medium was used to fill up to 200 μL of liquid per well until the final material concentration was 0, 25, 50, 75, 100, 125, 150, 175 μg / μL, respectively, and cultured in a CO2 constant temperature incubator set at 37°C and 5% volume fraction for 4 hours. After the culture was completed, all the liquid in the well plate was aspirated, and a mixture of CCK8 and culture medium was added in a volume ratio of CCK8:1640 culture medium of 1:9 in the dark, and placed in a CO2 constant temperature incubator set at 37°C and 5% volume fraction for 45 minutes, and the absorbance at 450nm was measured by an ELISA reader. Table 4 shows the cell viability of B16F10 under different materials.

[0163] Table 4: Cell viability of B16F10 under different materials (%)

[0164]

[0165] It can be seen from the test results in Table 4 that polyoxazolines with different molecular weights and different degrees of deacetylation all have certain cytotoxicity, but the polymer PEtOx 50,0.2 -r-PEI 50,0.8 Minimal cytotoxicity.

[0166] Example 2

[0167] 1. Preparation of polymer of formula (2)

[0168] B1) 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 4-dimethylaminopyridine, 4-carboxybenzaldehyde, dihydroxy polyethylene glycol (OH-PEG 2000 -OH) is added into dichloromethane to react to obtain a reaction solution;

[0169] B2) The reaction solution obtained in step B1) is subjected to rotary evaporation to dry the solvent, dissolved in deionized water, dialyzed and freeze-dried to obtain the dialdehyde polyethylene glycol OHC-PEG represented by formula (2) 2000 -CHO.

[0170] 2. Preparation of in situ cross-linked polymer nucleic acid delivery carrier OHC-PEG m -CHO / PEtOx n(1-x) -r-PEI nx C) OHC-PEG 2000 -CHO / PEtOx 50,0.2 -r-PEI 50,0.8 For example:

[0171] The poly(2-ethyl-2-oxazoline)-polyethyleneimine random copolymer PEtOx obtained in step A) of Example 1 50,0.2 -r-PEI 50,0.8 and the dialdehyde polyethylene glycol OHC-PEG obtained in step B) of Example 2 2000 -CHO was dissolved in deionized water to obtain the in situ cross-linked polymer nucleic acid delivery carrier OHC-PEG 2000 -CHO / PEtOx 50,0.2 -r-PEI 50,0.8 .

[0172] 3. OHC-PEG m -CHO / PEtOx n(1-x) -r-PEI nx In vitro DNA transfection

[0173] OHC-PEG 2000 -CHO / PEtOx 50,0.2 -r-PEI 50,0.8 For example, B16F10 cells were added to a culture medium containing 10% fetal bovine serum and cultured in a constant temperature incubator set at 37°C and 5% CO2. Before transfection, logarithmic phase cells were obtained, digested with trypsin, and diluted with a culture medium containing 10% fetal bovine serum to a concentration of 1×10 4 Cells / well were plated in a 96-well plate and cultured in a constant temperature incubator at 37°C and 5% CO2 until the cell confluence reached 80-90%. The culture medium in the 96-well plate was then replaced with fresh culture medium with pH values ​​of 7.4, 6.8, and 5.4, respectively. After 2 hours of treatment, the medium was replaced with normal culture medium for subsequent transfection experiments. 50,0.2 -r-PEI 50,0.8 pLUC was loaded, and OHC-PEG was added after 20 min of carrier / pLUC complexation. 2000 -CHO was cross-linked and added into a 96-well plate at a dosage of 0.2 μg pDNA / well and cultured for 48 h.

[0174] The cell transfection efficiency was determined as follows:

[0175] OHC-PEG 2000 -CHO / PEtOx 50,0.2 -r-PEI 50,0.8 For example, cells were taken out from the cell culture incubator, the cell culture medium was removed, 25 μL of cell lysis solution was added, and the cells were placed in a -80°C refrigerator for lysis for 20 min. Then, 25 μL of luciferase substrate was added to each well, and the transfection efficiency of the vector / pLUC complex in B16F10 cells was detected by an ELISA instrument. Table 5 shows the transfection efficiency of the vector / pLUC complex.

[0176] Table 5: OHC-PEG m -CHO / PEtOx n(1-x) -r-PEI nx Mediate in vitro transfection efficiency of luciferase plasmid

[0177]

[0178] As can be seen from the test results in Table 5, the use of OHC-PEG m -CHO / PEtOx n(1-x) -r-PEI nx As a carrier to carry DNA, it can achieve a higher transfection efficiency; among them, OHC-PEG 2000 -CHO / PEtOx 50,0.2-r-PEI 50,0.8 The mRNA transfection efficiency was optimal when the PEG / PEI / DNA mass ratio was 2.5:5:1, and the pH change also brought about the greatest improvement in mRNA transfection efficiency.

[0179] 4. OHC-PEG m -CHO / PEtOx n(1-x) -r-PEI nx In vitro mRNA transfection

[0180] B16F10 cells were added to a culture medium containing 10% fetal bovine serum and cultured in a constant temperature incubator set at 37°C and 5% CO2. Before transfection, logarithmic phase cells were obtained, digested with trypsin, and diluted with a culture medium containing 10% fetal bovine serum to a concentration of 1×10 4 Cells / well were plated in a 96-well plate and cultured in a constant temperature incubator at 37°C and 5% CO2 until the cell confluence reached 80% to 90%. The culture medium in the 96-well plate was then replaced with fresh culture medium with pH values ​​of 7.4, 6.8, and 5.4, respectively. After 2 hours of treatment, the medium was replaced with normal culture medium for subsequent transfection experiments. 50,0.2 -r-PEI 50,0.8 mLUC was loaded, and OHC-PEG was added after 20 min of carrier / mLUC complexation. 2000 -CHO were cross-linked and added into 96-well plates at a dosage of 0.2 μg mRNA / well, and cultured for 48 h.

[0181] The cell transfection efficiency was determined as follows:

[0182] The cells were taken out from the cell culture incubator, the cell culture medium was removed, 25 μL of cell lysis solution was added, and the cells were placed in a -80°C refrigerator for lysis for 20 min. Then 25 μL of luciferase substrate was added to each well, and the transfection efficiency of the vector / mLUC complex in B16F10 cells was detected by an ELISA instrument. Table 6 shows the transfection efficiency of the vector / mLUC complex.

[0183] Table 6: OHC-PEG m -CHO / PEtOx n(1-x) -r-PEI nx In vitro transfection efficiency of mLUC

[0184]

[0185] As can be seen from the test results in Table 6, the use of OHC-PEG m -CHO / PEtOx n(1-x) -r-PEI nxAs a carrier to carry mRNA, it can achieve a higher transfection efficiency; among them, OHC-PEG 2000 -CHO / PEtOx 50,0.2 -r-PEI 50,0.8 The mRNA transfection efficiency was optimal when the PEG / PEI / DNA mass ratio was 2.5:5:1, and the pH change also brought about the greatest improvement in mRNA transfection efficiency.

[0186] 5. OHC-PEG m -CHO / PEtOx n(1-x) -r-PEI nx In vitro siRNA transfection

[0187] Using OHC-PEG 2000 -CHO / PEtOx 50,0.2 -r-PEI 50,0.8 Transfection of Luc siRNA carrying silencing luciferase in B16F10 LUC cell line. B16F10 LUC cells were placed in a culture medium containing 10% fetal bovine serum and placed in a constant temperature incubator at 37°C and 5% CO2. Logarithmic phase cells were obtained, digested with trypsin, neutralized with 10% fetal bovine serum culture medium, and then incubated with 1×10 4 Cells / well were plated in a 96-well plate and cultured in a 37°C, 5% CO2 incubator until the cell confluence reached 80-90%. During transfection, the vector / siRNA complex was added to the 96-well cell plate at 0.2 μg siRNA / well after 20 minutes of incubation and continued to incubate for 48 hours.

[0188] Cell transfection efficiency determination:

[0189] The cells were taken out from the constant temperature incubator, the cell culture medium was removed, washed twice with PBS, 25 μL of cell lysis solution was added, and placed in a -80°C refrigerator for lysis for 20 min, and then 25 μL of luciferase substrate was added to each well, and the cell transfection efficiency was measured by an ELISA instrument. Table 7 lists the silencing efficiency of luciferase.

[0190] Table 7: OHC-PEG m -CHO / PEtOx n(1-x) -r-PEI nx In vitro transfection efficiency of Luc siRNA mediating luciferase silencing

[0191]

[0192] As can be seen from the test results in Table 7, the use of OHC-PEG m -CHO / PEtOx n(1-x)-r-PEI nx As a carrier to carry siRNA, it can achieve a higher silencing ability; among them, OHC-PEG 2000 -CHO / PEtOx 50,0.2 -r-PEI 50,0.8 The siRNA silencing was highest when the PEG / PEI / DNA mass ratio was 2.5:5:1, and the pH change also brought about the greatest improvement in the siRNA silencing ability.

[0193] 6. OHC-PEG m -CHO / PEtOx n(1-x) -r-PEI nx Application in in vivo pDNA transfection

[0194] (1) B16F10 cell culture

[0195] The B16F10 cells were placed in a culture medium containing 10% by volume of fetal bovine serum and cultured in a constant temperature incubator at 37° C. and 5% by volume of CO 2 .

[0196] (2) Tumor inoculation

[0197] C57BL / 6J mice aged 6 to 8 weeks and weighing about 20 g were ordered. Before tumor inoculation, B16F10 cells in the logarithmic phase were obtained and digested with trypsin and then added into cell culture medium to neutralize trypsin. 1×10 3 Centrifuge at rpm for 5 min, wash 3 times with PBS, and suspend the cells with PBS. 6 Six days after the cells were inoculated in the mouse armpits, in vivo gene administration was performed when the tumor diameter reached an average of 6 mm.

[0198] (3) In vivo treatment

[0199] The therapeutic genes used were plasmid pSpam1, which can express hyaluronidase, and promote T cells to penetrate into tumor tissues by destroying the extracellular matrix of tumor cells, thereby achieving T cell-mediated tumor killing; and pshVEGF, which can express shRNA that silences vascular endothelial growth factor VEGF. The vector / pDNA complex (volume 100μL) was injected into the tail vein of tumor-bearing mice with 20ug pSpam1 or 20ug pshVEGF per mouse, and the drug was given once every 2 days for a total of 4 times.

[0200] (4) Determination of tumor growth in mice

[0201] From the first administration of the mice, the changes in tumor size and body weight were measured every day. Table 8 lists the volume of tumor-bearing mice after 14 days of administration.

[0202] Table 8: OHC-PEG2000 -CHO / PEtOx 50,0.2 -r-PEI 50,0.8 Mediating tumor size after pSpam1 and pshVEGF treatment in vivo

[0203] Group Name <![CDATA[Tumor diameter size, mm 3 > PBS 1442.5 pshVEGF+pSpam1 860.7 PEI22k / pshVEGF+pSpam1 662.5 <![CDATA[PEtOx 50,0.2 -r-PEI 50,0.8 / pshVEG]]> 512.2 <![CDATA[PEtOx 50,0.2 -r-PEI 50,0.8 / pSpam1]]> 371.6 <![CDATA[PEtOx 50,0.2 -r-PEI 50,0.8 / pshVEGF+pSpam1]]> 272.9 <![CDATA[OHC-PEG 2000 -CHO / PEtOx 50,0.2 -r-PEI 50,0.8 / pshVEGF+pSpam1]]> 66.5

[0204] As can be seen from the test results in Table 8, the use of OHC-PEG 2000 -CHO / PEtOx 50,0.2 -r-PEI 50,0.8 As a carrier to mediate therapeutic genes, it can effectively deliver therapeutic genes, thereby effectively inhibiting tumor growth and achieving better therapeutic effects.

[0205] Specific examples are used herein to illustrate the principles and implementation methods of the present invention. The description of the above embodiments is only used to help understand the method of the present invention and its core ideas, including the best mode, and also enable any technician in the field to practice the present invention, including making and using any device or system, and implementing any combined method. It should be pointed out that for ordinary technicians in this technical field, without departing from the principle of the present invention, the present invention can also be improved and modified, and these improvements and modifications also fall within the scope of protection of the claims of the present invention. The scope of patent protection of the present invention is defined by the claims and may include other embodiments that can be thought of by those skilled in the art. If these other embodiments have structural elements similar to the literal expression of the claims, or if they include equivalent structural elements that are not substantially different from the literal expression of the claims, then these other embodiments should also be included in the scope of the claims.

Claims

1. A method for preparing an in situ cross-linked polymer nucleic acid delivery vector, characterized in that: The following steps are involved: A) deacetylation of poly(2-ethyl-2-oxazoline) to obtain a poly(2-ethyl-2-oxazoline)-polyethyleneimine random copolymer represented by formula (1); B) reacting 4-carboxybenzaldehyde with bishydroxy polyethylene glycol in the presence of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 4-dimethylaminopyridine to obtain a bisaldehyde polyethylene glycol represented by formula (2); C) dissolving the poly(2-ethyl-2-oxazoline)-polyethyleneimine random copolymer obtained in step A) and the dialdehyde polyethylene glycol obtained in step B) in water to obtain an in situ cross-linked polymer nucleic acid delivery vector; There is no order restriction for step A) and step B); 2. The preparation method according to claim 1, characterized in that: In step A), the molecular weight of the poly(2-ethyl-2-oxazoline) is 300 to 500,000 Dalton; The reaction temperature of the deacetylation is 130°C.

3. The preparation method according to claim 1, characterized in that: Step A) specifically includes: A1) mixing poly(2-ethyl-2-oxazoline) with an acid solution, heating to perform deacetylation, and obtaining a reaction solution; A2) adjusting the pH value of the reaction solution obtained in step A1) to a weak alkaline state; A3) dialyzing and freeze-drying the solution obtained in step A2) to obtain a poly(2-ethyl-2-oxazoline)-polyethyleneimine random copolymer represented by formula (1).

4. The preparation method according to claim 3, characterized in that: In step A1), the acid solution is at least one of a hydrochloric acid aqueous solution, a sulfuric acid aqueous solution and a trifluoroacetic acid aqueous solution; The concentration of the acid solution is 2-6 mol / L.

5. The preparation method according to claim 3, characterized in that: In step A2), the pH value is adjusted to 7.4-8.0; In step A3), the molecular weight of the dialysis is 500 to 7000 Dalton and the dialysis time is 48 to 96 hours.

6. The preparation method according to claim 1, characterized in that: In step B), the reaction temperature is 20-40°C.

7. The preparation method according to claim 1, characterized in that: Step B) specifically includes: B1) mixing 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 4-dimethylaminopyridine, 4-carboxybenzaldehyde, bishydroxy polyethylene glycol and a solvent to obtain a reaction solution; B2) removing the solvent from the reaction solution obtained in step B1), adding water to dissolve, dialyzing and freeze-drying to obtain the dialdehyde polyethylene glycol represented by formula (2).

8. The preparation method according to claim 7, characterized in that: In step B2), the dialysis molecular weight is 500 to 7000 Dalton and the dialysis time is 48 to 96 hours.

9. An in situ cross-linked high molecular polymer nucleic acid delivery vector prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the in situ cross-linked high molecular polymer nucleic acid delivery vector according to claim 9 in the preparation of a nucleic acid vector.

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