A polypolypeptide gene vector and its preparation method and application

Abstract

The present invention provides a polypolypeptide gene vector and a preparation method and application thereof, wherein the polypolypeptide gene vector comprises nanospheres formed by self-assembly of triblock polypolypeptides, wherein the triblock polypolypeptides comprise polyethylene glycol, polylysine and polyleucine connected in sequence; one end of the polylysine is connected to the polyethylene glycol via an amide bond, and the other end of the polylysine is connected to the polyleucine via a peptide bond. The polypolypeptide gene vector of the present invention can efficiently load the target gene in the polypolypeptide gene vector, and can form stable gene particles with high encapsulation rate and high load; the obtained polypolypeptide gene vector can produce a protonated sponge effect inside the lysosome to promote the lysosomal escape of the target gene and prevent the target gene from being degraded in the lysosome. It has important application prospects in the fields of gene editing, gene diagnosis, gene therapy and gene vaccines.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical materials, and specifically relates to a polypolypeptide gene carrier and a preparation method and application thereof. Background Art

[0002] Gene therapy is the process of intervening in the expression of specific genes in target cells to treat pathological diseases by introducing exogenous RNA or DNA. Gene therapy can be used to treat a variety of diseases, including cancer, cystic fibrosis, heart disease, diabetes, and hemophilia. Unlike traditional small molecule drugs and antibody drugs, the design of gene drugs based on nucleotide sequences is simpler and more convenient, and is less restricted by drug targets. It has the advantages of high efficiency, practicality, simplicity, and low cost. However, the clinical application of gene drugs still needs to overcome many obstacles. Naked nucleic acids are easily degraded by nucleases, and foreign RNA and DNA are easily recognized and cleared by the immune system, resulting in a very short half-life of nucleic acid drugs. For nucleic acid drugs to take effect, they must not only overcome the extracellular barriers (macrophages and serum endonucleases, etc.) of the systemic delivery process, but also overcome the intracellular barriers (cellular endocytosis and endosomal escape, etc.) to reach the cytoplasm (siRNA and mRNA) or nucleus (ASOs, DNA, CRISPR) of the target cell. Therefore, developing a safe and efficient nucleic acid vector system and improving the targeted delivery efficiency and transfection efficiency of gene drugs are the cornerstones and key technical challenges for the success of gene therapy.

[0003] The rapid development of nanotechnology has provided new ideas for solving the problems faced in gene therapy. Nano drug delivery systems can further improve the transfection efficiency and targeting of genes. In particular, the effective delivery of therapeutic genes to extrahepatic host cells will have a huge impact on future gene-based therapies.

[0004] Gene therapy has shown excellent results in the treatment of rare diseases. At present, two major types of gene vector systems have been developed: viral vectors and non-viral vectors. However, the expensive production costs of transgenic cells or viral vectors and potential biosafety risks have greatly reduced their clinical applicability. Physical gene delivery methods include direct DNA injection, gene guns, electric pulse-mediated methods, etc., which are prone to tissue damage, limiting their clinical application. Non-viral gene vectors include lipid nanoparticles (LNPs), cationic polymers, etc., which have unique advantages such as simple preparation, low immunogenicity, difficulty in integrating with host genes, and unrestricted cloning ability, making them important candidates for nucleic acid drug delivery. However, the LNP delivery system still has defects such as toxicity, complement activation and poor biodistribution. LNPs delivery is mainly limited to the liver and reticuloendothelial system, and its low targeting ability to organs other than the liver has always been a problem that the industry has not been able to overcome.

[0005] Therefore, providing a transfection reagent with the advantages of low toxicity, high transfection ability, high targeting, etc. has important application value in the fields of gene editing, gene diagnosis, gene therapy and gene vaccines. Summary of the invention

[0006] In view of the deficiencies in the prior art, the object of the present invention is to provide a polypolypeptide gene carrier and a preparation method and application thereof. The polypolypeptide gene carrier of the present invention can efficiently load the target gene in the polypolypeptide gene carrier to form stable gene particles with high encapsulation rate and high load; the obtained polypolypeptide gene carrier can produce a protonated sponge effect inside the lysosome to promote the lysosomal escape of the target gene and prevent the target gene from being degraded in the lysosome. The present invention also provides a polypolypeptide gene transfection system with simple preparation process, stable properties, high transfection efficiency and good biocompatibility. The polypolypeptide gene transfection reagent provided by the present invention has the advantages of low toxicity, high transfection and high targeting, which can effectively overcome the problems existing in the current gene delivery system and has important application prospects in the fields of gene editing, gene diagnosis, gene therapy and gene vaccines.

[0007] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides a polypolypeptide gene vector, which comprises nanospheres formed by self-assembly of triblock polypolypeptides, wherein the triblock polypolypeptides comprise polyethylene glycol, polylysine and polyleucine connected in sequence; one end of the polylysine is connected to the polyethylene glycol via an amide bond, and the other end of the polylysine is connected to the polyleucine via a peptide bond.

[0009] The polypolypeptide gene carrier of the present invention is a complex with a three-layer structure, wherein the polyleucine constitutes the inner layer of the complex, the polyethylene glycol constitutes the outer layer of the complex, and the middle layer of the complex includes the polylysine.

[0010] Preferably, the number average molecular weight of the polyethylene glycol is 1000-5000, for example, 1000, 2000, 3000, 4000 or 5000.

[0011] Preferably, the number average molecular weight of the polylysine is 4500-17000, for example, 4500, 5000, 8000, 10000, 12000, 14000, 15000 or 17000, etc., preferably 13000.

[0012] Preferably, the number average molecular weight of the polyleucine is 1500-5000, for example, 1500, 2000, 2500, 3000, 3500, 4000, 4500 or 5000, etc., preferably 2000.

[0013] Preferably, the particle size of the nanospheres is 50-200 nm, for example, 50 nm, 80 nm, 100 nm, 120 nm, 150 nm, 180 nm or 200 nm.

[0014] In a second aspect, the present invention provides a method for preparing the polypolypeptide gene vector described in the first aspect, wherein a triblock polypolypeptide of polyethylene glycol-polylysine-polyleucine is synthesized by monomer modification, ring-opening polymerization and hydrolysis, and the obtained triblock polypolypeptide is dissolved in a solvent to allow it to self-assemble into nanospheres, thereby obtaining the polypolypeptide gene vector.

[0015] Preferably, the solvent comprises water and / or PBS.

[0016] The polypolypeptide gene carrier obtained by the preparation method of the present invention can efficiently load the target gene in the polypolypeptide gene carrier to form stable gene particles with high encapsulation rate and high loading amount; the obtained polypolypeptide gene carrier can produce a protonated sponge effect inside the lysosome to promote the lysosomal escape of the target gene and prevent the target gene from being degraded in the lysosome.

[0017] Preferably, the polyethylene glycol-polylysine-polyleucine triblock polypeptide is prepared by a method comprising the following steps:

[0018] (1) respectively modifying Nε-benzyloxycarbonyl-L-lysine and leucine to obtain modified Nε-benzyloxycarbonyl-L-lysine and modified L-leucine;

[0019] (2) mixing amino polyethylene glycol monomethyl ether and modified L-lysine to carry out a ring-opening polymerization reaction to obtain polyethylene glycol-poly Nε-benzyloxycarbonyl-L-lysine;

[0020] (3) mixing the obtained polyethylene glycol-poly Nε-benzyloxycarbonyl-L-lysine and modified L-leucine to carry out a ring-opening polymerization reaction to obtain polyethylene glycol-poly Nε-benzyloxycarbonyl-L-lysine-polyleucine;

[0021] (4) The obtained polyethylene glycol-poly Nε-benzyloxycarbonyl-L-lysine-polyleucine is sequentially mixed with trifluoroacetic acid and aqueous solution of hydrobromic acid to carry out hydrolysis reaction, thereby obtaining a triblock polypeptide of polyethylene glycol-polylysine-polyleucine.

[0022] In the present invention, in step (1), the step of modifying Nε-benzyloxycarbonyl-L-lysine comprises: subjecting Nε-benzyloxycarbonyl-L-lysine, anhydrous tetrahydrofuran and triphosgene to condensation reflux reaction, obtaining a yellow solid by n-hexane precipitation, then recrystallizing using n-hexane and tetrahydrofuran, and vacuum drying to obtain modified Nε-benzyloxycarbonyl-L-lysine (NCA).

[0023] The mass volume ratio of the Nε-benzyloxycarbonyl-L-lysine, anhydrous tetrahydrofuran and triphosgene is (7-9 g):(28-32 mL):(3.6-4 g), wherein "7-9 g" can be, for example, 7 g, 8 g or 9 g, and "28-32 mL" can be, for example, 28 mL, 29 mL, 30 mL, 31 mL or 32 mL.

[0024] Triphosgene is added to the Nε-benzyloxycarbonyl-L-lysine and anhydrous tetrahydrofuran at 50-52° C. (eg, 50° C., 51° C., or 52° C.).

[0025] The condensation reflux reaction time is 4-5 hours, for example, 4 hours, 4.5 hours or 5 hours.

[0026] In the recrystallization step, the volume ratio of n-hexane to tetrahydrofuran is (1.8-2.2):1, for example, it can be 1.8:1, 2:1 or 2.2:1.

[0027] In the present invention, in step (1), the step of modifying leucine comprises: subjecting leucine, anhydrous tetrahydrofuran and triphosgene to condensation reflux reaction, obtaining a yellow solid by n-hexane precipitation, and then recrystallizing using n-hexane and tetrahydrofuran to obtain modified L-leucine (NCA).

[0028] The mass volume ratio of the leucine, anhydrous tetrahydrofuran and triphosgene is (0.8-1.2g):(28-32mL):(2.8-3.2g), wherein "0.8-1.2g" can be, for example, 0.8g, 1g or 1.2g, etc., "28-32mL" can be, for example, 28mL, 30mL or 32mL, etc., and "2.8-3.2g" can be, for example, 2.8g, 3.0g or 3.2g, etc.

[0029] The leucine and anhydrous tetrahydrofuran are added with triphosgene at 50-52° C. (eg, 50° C., 51° C., or 52° C.).

[0030] The condensation reflux reaction time is 4-5 hours, for example, 4 hours, 4.5 hours or 5 hours.

[0031] In the recrystallization step, the volume ratio of n-hexane to tetrahydrofuran is (4.8-5.2):1, for example, it can be 4.8:1, 5:1 or 5.2:1.

[0032] Preferably, in step (2), the temperature of the ring-opening polymerization reaction is 28-32°C, for example, 28°C, 30°C or 32°C, etc., the time of the ring-opening polymerization reaction is 70-74h, for example, 70h, 72h or 74h, etc., and the rotation speed of the ring-opening polymerization reaction is 250-750rpm, for example, 250rpm, 300rpm, 500rpm, 600rpm or 750rpm, etc.

[0033] Preferably, in step (3), the temperature of the ring-opening polymerization reaction is 28-32°C, for example, 28°C, 30°C or 32°C, etc., the time of the ring-opening polymerization reaction is 70-74h, for example, 70h, 72h or 74h, etc., and the rotation speed of the ring-opening polymerization reaction is 250-750rpm, for example, 250rpm, 300rpm, 500rpm, 600rpm or 750rpm, etc.

[0034] Preferably, in step (4), the mass volume ratio of the polyethylene glycol-poly Nε-benzyloxycarbonyl-L-lysine-polyleucine, trifluoroacetic acid and hydrobromide acetic acid aqueous solution is (0.8-1.2 g):(5-7 mL):(3-5 mL), wherein "0.8-1.2 g" can be, for example, 0.8 g, 1 g or 1.2 g, etc., "5-7 mL" can be, for example, 5 mL, 6 mL or 7 mL, etc., and "3-5 mL" can be, for example, 3 mL, 4 mL or 5 mL, etc.

[0035] Preferably, in step (4), the volume concentration of the aqueous solution of hydrobromide acetic acid is 30-36%, for example, 30%, 32%, 33% or 36%.

[0036] Preferably, in step (4), the hydrolysis reaction is carried out in an ice-water bath, the hydrolysis reaction is carried out for 4-6 hours, for example, 4 hours, 5 hours or 6 hours, etc., and the hydrolysis reaction is carried out at a speed of 250-750 rpm, for example, 250 rpm, 300 rpm, 500 rpm, 600 rpm or 750 rpm, etc.

[0037] Preferably, in steps (2), (3) and (4), the ring-opening polymerization reaction or hydrolysis reaction further comprises the steps of precipitation with glacial ether and vacuum drying.

[0038] Preferably, the vacuum drying conditions are: vacuum degree -0.1Mpa, constant temperature of 30°C.

[0039] As a preferred technical solution of the present invention, the method for preparing the polypolypeptide gene vector comprises the following steps:

[0040] (1) Nε-benzyloxycarbonyl-L-lysine and leucine are modified to obtain modified Nε-benzyloxycarbonyl-L-lysine and modified L-leucine respectively.

[0041] Nε-benzyloxycarbonyl-L-lysine and anhydrous tetrahydrofuran are mixed, triphosgene is added dropwise at 50-52° C. using a constant pressure dropping funnel, and the mixture is condensed and refluxed for 4-5 hours; the mass volume ratio of the Nε-benzyloxycarbonyl-L-lysine, anhydrous tetrahydrofuran and triphosgene is (7-9 g):(28-32 mL):(3.6-4 g); a yellow solid is obtained by precipitation with n-hexane, and then recrystallization is performed using n-hexane and tetrahydrofuran in a volume ratio of (1.8-2.2):1, and vacuum drying is performed to obtain modified Nε-benzyloxycarbonyl-L-lysine (NCA);

[0042] Leucine and anhydrous tetrahydrofuran are mixed, and a triphosgene solution is added dropwise at 50-52° C. using a constant pressure dropping funnel, and the mixture is condensed and refluxed for 4-5 hours; the mass volume ratio of leucine, anhydrous tetrahydrofuran and triphosgene is (0.8-1.2 g):(28-32 mL):(2.8-3.2 g); a yellow solid is obtained by precipitation with n-hexane, and then recrystallized using n-hexane and tetrahydrofuran in a volume ratio of (4.8-5.2):1 to obtain modified L-leucine (NCA).

[0043] (2) mixing amino polyethylene glycol monomethyl ether and modified L-lysine for ring-opening polymerization to obtain polyethylene glycol-poly Nε-benzyloxycarbonyl-L-lysine; the temperature of the ring-opening polymerization reaction is 28-32° C., the time is 70-74 h, and the rotation speed is 250-750 rpm; after the reaction is completed, precipitating with glacial ether and vacuum drying to obtain polyethylene glycol-poly Nε-benzyloxycarbonyl-L-lysine.

[0044] (3) mixing the obtained polyethylene glycol-poly Nε-benzyloxycarbonyl-L-lysine and modified L-leucine for ring-opening polymerization to obtain polyethylene glycol-poly Nε-benzyloxycarbonyl-L-lysine-polyleucine; the temperature of the ring-opening polymerization reaction is 28-32° C., the time is 70-74 h, and the speed is 250-750 rpm; after the reaction is completed, precipitating with ice ether and vacuum drying to obtain polyethylene glycol-poly Nε-benzyloxycarbonyl-L-lysine-polyleucine.

[0045] (4) The obtained polyethylene glycol-poly Nε-benzyloxycarbonyl-L-lysine-polyleucine is subjected to a hydrolysis reaction with trifluoroacetic acid and an aqueous solution of hydrogen bromide acetic acid in a mass volume ratio of (0.8-1.2 g):(5-7 mL):(3-5 mL), wherein the volume concentration of the aqueous solution of hydrogen bromide acetic acid is 30-36%; the hydrolysis reaction is carried out in an ice water bath, the hydrolysis reaction time is 4-6 h, and the hydrolysis reaction speed is 250-750 rpm; the mixture is precipitated with ice ether and vacuum dried to obtain a triblock polypeptide of polyethylene glycol-polylysine-polyleucine.

[0046] In a third aspect, the present invention provides a gene delivery system, comprising the polypolypeptide gene vector described in the first aspect, a target gene loaded on the vector, and sodium hyaluronate coated on the vector.

[0047] Preferably, the target gene comprises any one of plasmid, DNA, mRNA or siRNA, or a combination of at least two of them.

[0048] Preferably, the number average molecular weight of the sodium hyaluronate is 3000-100000, for example, it can be 3000, 10000, 20000, 35000, 50000, 70000, 90000 or 100000, etc., preferably 35000.

[0049] The present invention provides a method for preparing a polypolypeptide gene transfection reagent with high transfection efficiency and its application in plasmid and RNA transfection, aiming to provide a polypolypeptide gene transfection system with simple preparation process, stable properties, high transfection efficiency and good biocompatibility. The specific preparation method is: first, a triblock polypolypeptide of polyethylene glycol-polylysine-polyleucine is synthesized by monomer modification, ring-opening polymerization and hydrolysis. Then the obtained triblock polypolypeptide is dissolved in a PBS solution, and the gene (plasmid or RNA) to be transfected is added thereto to obtain a polypolypeptide gene complex; at the same time, sodium hyaluronate is wrapped in the outer layer of the polypolypeptide gene complex by layer-by-layer self-assembly, and finally a polypolypeptide gene vector is obtained. Subsequently, after the gene-loaded polypolypeptide gene vector and cells are co-incubated, the gene-loaded polypolypeptide gene vector successfully enters the target cell, and the target gene successfully intervenes in the expression of specific genes in the target cell.

[0050] The present invention has found that polylysine is an excellent gene transfection substrate, which can be further modified and modified to become an efficient gene transfection reagent. The polypolypeptide gene carrier described in the present invention has the following advantages: ① The target gene can be efficiently loaded in the polypolypeptide gene carrier to form stable, high encapsulation rate, and high load gene particles; ② Polylysine and sodium hyaluronate have good biocompatibility, biodegradability, and non-toxicity; ③ The polypolypeptide gene carrier can produce a protonated sponge effect inside the lysosome to promote the lysosomal escape of the target gene and prevent the target gene from being degraded in the lysosome; ④ The transfection efficiency of the target gene is greatly increased by introducing leucine and sodium hyaluronate on the polylysine substrate. ⑤ Through the coating of sodium hyaluronate, the surface positive charge of the polypolypeptide gene carrier can be greatly reduced, increasing the long circulation time in the body or the ability to target tumor tissue.

[0051] In a fourth aspect, the present invention provides a method for preparing the gene delivery system described in the third aspect, the preparation method comprising the following steps: loading the target gene on the polypolypeptide gene carrier, coating it with sodium hyaluronate, and obtaining the polypolypeptide gene carrier loaded with the target gene.

[0052] The polylysine and sodium hyaluronate used in the present invention have good biocompatibility, biodegradability, and are non-toxic; during the preparation process, the transfection efficiency of the target gene is greatly increased by introducing leucine and sodium hyaluronate on the polylysine substrate; the surface positive charge of the polypolypeptide gene carrier can be greatly reduced by coating with sodium hyaluronate, and the long circulation time in the organism is increased. The preparation process of the present invention is simple, the cost is low, and large-scale preparation is easy to achieve.

[0053] Preferably, loading the target gene and coating sodium hyaluronate are carried out by a method comprising the following steps: preparing a solution of a triblock polypeptide of polyethylene glycol-polylysine-polyleucine, adding the target gene to mix, letting it stand, and then adding sodium hyaluronate to mix to obtain the gene delivery system.

[0054] Preferably, the mass ratio of the triblock polypeptide, the target gene and sodium hyaluronate is (0.8-1.2 mg):(100-300 μg):(100-1000 μg), wherein "0.8-1.2 mg" can be, for example, 0.8 mg, 1 mg or 1.2 mg, etc., "100-300 μg" can be, for example, 100 μg, 200 μg or 300 μg, etc., and "100-1000 μg" can be, for example, 100 μg, 200 μg, 500 μg, 800 μg or 1000 μg, etc.

[0055] Preferably, the time for adding the target gene and mixing is 10-30s, for example, 10s, 15s, 20s, 25s or 30s, etc., and mechanical pipetting and mixing are performed 3-10 times, for example, 3, 5, 8 or 10, etc., and the mixing temperature is 20-25°C, for example, 20°C, 22°C or 25°C, etc.

[0056] Preferably, the standing time is 4-6 min, for example, 4 min, 5 min or 6 min, etc.; the standing temperature is 20-25°C, for example, 20°C, 22°C or 25°C, etc.

[0057] Preferably, the mixing time of adding sodium hyaluronate is 10-30s, for example, 10s, 15s, 20s, 25s or 30s, etc., and mechanical blowing and mixing are performed 3-10 times, for example, 3, 5, 8 or 10, etc., and the mixing temperature is 20-25°C, for example, 20°C, 22°C or 25°C, etc.

[0058] As a preferred technical solution of the present invention, the preparation method of the gene delivery system comprises the following steps:

[0059] A PBS solution of a triblock polypeptide of polyethylene glycol-polylysine-polyleucine is prepared, and the target gene is added and mixed. The mixing time is 10-30 seconds, and the mixture is mechanically blown and mixed for 3-10 times at a temperature of 20-25°C; the mixture is allowed to stand at 20-25°C for 4-6 minutes; sodium hyaluronate is then added and mixed to obtain the gene delivery system; the sodium hyaluronate is added and mixed for 10-30 seconds, and the mixture is mechanically blown and mixed for 3-10 times at a temperature of 20-25°C; the mass ratio of the triblock polypeptide, the target gene and the sodium hyaluronate is (0.8-1.2 mg):(100-300 μg):(100-1000 μg).

[0060] In a fifth aspect, the present invention provides the use of any one or a combination of at least two of the polypolypeptide gene vector described in the first aspect, the method for preparing the polypolypeptide gene vector described in the second aspect, the gene delivery system described in the third aspect, or the method for preparing the gene delivery system described in the fourth aspect in the preparation of a nucleic acid transfection reagent.

[0061] The numerical range described in the present invention not only includes the point values ​​listed above, but also includes any point values ​​between the above numerical ranges that are not listed. Due to space limitations and for the sake of simplicity, the present invention no longer exhaustively lists the specific point values ​​included in the range.

[0062] Compared with the prior art, the present invention has the following beneficial effects:

[0063] (1) Advantages of the preparation method of the polypolypeptide gene vector described in the present invention: ① The target gene can be efficiently loaded into the polypolypeptide gene vector to form stable gene particles with high encapsulation rate and high loading capacity; ② Polylysine and sodium hyaluronate have good biocompatibility, biodegradability and non-toxicity; ③ The polypolypeptide gene vector can produce a protonated sponge effect inside the lysosome to promote the lysosomal escape of the target gene and prevent the target gene from being degraded in the lysosome; ④ The transfection efficiency of the target gene is greatly increased by introducing leucine and sodium hyaluronate on the polylysine substrate; ⑤ The surface positive charge of the polypolypeptide gene vector can be greatly reduced by coating with sodium hyaluronate, thereby increasing the long circulation time in the body; The preparation process of the polypolypeptide gene vector is simple, the cost is low, and large-scale preparation is easy to achieve.

[0064] (2) Advantages of the polypolypeptide gene vector system prepared by the present invention: ① The polypolypeptide gene vector has stable properties, good biocompatibility, and low toxicity, which can overcome the shortcomings of the current mainstream polycationic transfection reagent (PEI); ② The high transfection ability of the polypolypeptide gene vector can effectively overcome the disadvantage of low transfection efficiency of the current gene delivery system, and the high transfection efficiency also has greater application prospects; ③ The polypolypeptide gene vector has good targeting. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] Figure 1 It is the H NMR spectrum of polyethylene glycol-polylysine-polyleucine (PEG-PLL-PLLeu) triblock polypeptide;

[0066] Figure 2 This is the gel electrophoresis diagram of the polypolypeptide gene vector;

[0067] Figure 3 is the particle size distribution diagram of the polypolypeptide gene carrier;

[0068] Figure 4 It is a transmission electron micrograph of a polypolypeptide gene vector;

[0069] Figure 5 This is a fluorescence microscope image of a polypolypeptide gene vector;

[0070] Figure 6 This is the flow cytometry fluorescence statistics of the polypolypeptide gene vector. DETAILED DESCRIPTION

[0071] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0072] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0073] The sources of materials used in the following specific embodiments are as follows:

[0074] Aminopolyethylene glycol monomethyl ether: source: MacLean Company, product number M813480-5g;

[0075] Nε-benzyloxycarbonyl-L-lysine: from MacLean, catalog number: N812371-25g;

[0076] Leucine: source: MacLean, product number L812333-25g;

[0077] Sodium hyaluronate: Source: Shanghai Yuanye Company, item number S22166.

[0078] Example 1 Preparation of Polypeptide Gene Vector

[0079] (1) 8.0 g of Nε-benzyloxycarbonyl-L-lysine and 30 mL of anhydrous tetrahydrofuran were added to a round-bottom flask, and 20 mL of triphosgene (3.8 g) was gradually dissolved therein at 50° C. The mixture was condensed and refluxed for 4-5 h until the solution became clear. A yellow solid was obtained by precipitation with n-hexane, and then recrystallization was performed in a 2:1 ratio of n-hexane to tetrahydrofuran to obtain a white solid, which was then dried in vacuum to obtain the modified Nε-benzyloxycarbonyl-L-lysine (NCA).

[0080] 1.0 g of leucine and 30 mL of anhydrous tetrahydrofuran were added to a round-bottom flask, and 20 mL of triphosgene (3.0 g) was gradually dissolved therein at 50°C, and the mixture was condensed and refluxed for 4-5 hours until the solution became clear. A yellow solid was obtained by precipitation with n-hexane, and then a white solid was obtained by recrystallization in 5:1 n-hexane:tetrahydrofuran, and vacuum drying was performed to obtain the modified L-leucine (NCA).

[0081] (2) Add 100 mg of amino polyethylene glycol monomethyl ether (PEG-NH2, molecular weight: 2000) and 0.7 g of L-lysine (NCA) to a round-bottom flask, then add 10 mL of anhydrous N,N-dimethylformamide (DMF), and then stir at 30°C and 500 rpm for 72 h. After the reaction is completed, precipitate with glacial ether and vacuum dry to obtain polyethylene glycol-poly Nε-benzyloxycarbonyl-L-lysine.

[0082] (3) Add 1.2 g of polyethylene glycol-poly (Nε-benzyloxycarbonyl)-L-lysine and 100 mg of L-leucine (NCA) to a round-bottom flask, and then add 10 mL of anhydrous N,N-dimethylformamide (DMF), followed by stirring at 30°C and 500 rpm for 72 h. After the reaction is completed, precipitate with glacial ether and vacuum dry to obtain polyethylene glycol-poly (Nε-benzyloxycarbonyl)-L-lysine-polyleucine.

[0083] (4) Add 1.0 g of polyethylene glycol-poly Nε-benzyloxycarbonyl-L-lysine-polyleucine to a round-bottom flask and add 6 mL of trifluoroacetic acid to completely dissolve it, then add 4 mL of 33% aqueous solution of hydrobromide acetic acid to react in an ice-water bath for 5 hours at a speed of 500 rpm; precipitate with ice ether, and vacuum dry to obtain a triblock polypeptide of polyethylene glycol-polylysine-polyleucine (PEG-PLL-PLLeu). The number average molecular weight of polylysine in the triblock polypeptide is 10,000, and the number average molecular weight of polyleucine is 2,000.

[0084] The obtained triblock polypeptide of polyethylene glycol-polylysine-polyleucine (PEG-PLL-PLLeu) was characterized by hydrogen nuclear magnetic resonance spectroscopy. The results of hydrogen nuclear magnetic resonance spectroscopy are as follows: Figure 1 As shown, the results of H-NMR spectrum analysis show that this example successfully synthesized polyethylene glycol-polylysine-polyleucine with uniform and controllable degree of polymerization, and the degree of polymerization calculated according to the H-NMR area is PEG44-PLL45-PLLeu15.

[0085] Example 2 Preparation of Polypeptide Gene Vectors

[0086] (1) 7.0 g of Nε-benzyloxycarbonyl-L-lysine and 28 mL of anhydrous tetrahydrofuran were added to a round-bottom flask, and 20 mL of triphosgene (3.6 g) was gradually dissolved therein at 50° C. The mixture was condensed and refluxed for 4 h until the solution became clear. A yellow solid was obtained by precipitation with n-hexane, and then recrystallization was performed in a 1.8:1 ratio of n-hexane to tetrahydrofuran to obtain a white solid, which was then dried in vacuum to obtain the modified Nε-benzyloxycarbonyl-L-lysine (NCA).

[0087] 0.8 g of leucine and 28 mL of anhydrous tetrahydrofuran were added to a round-bottom flask, and 20 mL of triphosgene (2.8 g) was gradually dissolved therein at 50°C, and the mixture was condensed and refluxed for 4 h until the solution became clear. A yellow solid was obtained by precipitation with n-hexane, and then a white solid was obtained by recrystallization in 4.8:1 n-hexane:tetrahydrofuran, and vacuum drying was performed to obtain the modified L-leucine (NCA).

[0088] (2) Add 80 mg of amino polyethylene glycol monomethyl ether (PEG-NH2, molecular weight: 1000) and 0.5 g of L-lysine (NCA) to a round-bottom flask, and then add 8 mL of anhydrous N,N-dimethylformamide (DMF), followed by stirring at 28°C and 250 rpm for 74 h. After the reaction is completed, precipitate with glacial ether and vacuum dry to obtain polyethylene glycol-poly Nε-benzyloxycarbonyl-L-lysine.

[0089] (3) Add 0.5 g of polyethylene glycol-poly (Nε-benzyloxycarbonyl)-L-lysine and 120 mg of L-leucine (NCA) to a round-bottom flask, and then add 8 mL of anhydrous N,N-dimethylformamide (DMF), and then stir at 28°C and 250 rpm for 74 h. After the reaction is completed, precipitate with ice ether and vacuum dry to obtain polyethylene glycol-poly (Nε-benzyloxycarbonyl)-L-lysine-polyleucine.

[0090] (4) Add 0.8 g of polyethylene glycol-poly Nε-benzyloxycarbonyl-L-lysine-polyleucine to a round-bottom flask and add 5 mL of trifluoroacetic acid to completely dissolve it, then add 3 mL of 36% aqueous hydrobromide acetic acid solution to react in an ice-water bath for 5 hours at a speed of 250 rpm; precipitate with ice ether, and vacuum dry to obtain a triblock polypeptide of polyethylene glycol-polylysine-polyleucine (PEG-PLL-PLLeu). The number average molecular weight of polylysine in the triblock polypeptide is 4500, and the number average molecular weight of polyleucine is 1500.

[0091] Example 3 Preparation of Polypeptide Gene Vectors

[0092] (1) 9.0 g of Nε-benzyloxycarbonyl-L-lysine and 32 mL of anhydrous tetrahydrofuran were added to a round-bottom flask, and 20 mL of triphosgene (4 g) was gradually dissolved therein at 50° C. The mixture was condensed and refluxed for 5 h until the solution became clear. A yellow solid was obtained by precipitation with n-hexane, and then recrystallization was performed in a 2.2:1 ratio of n-hexane to tetrahydrofuran to obtain a white solid, which was then dried in vacuum to obtain the modified Nε-benzyloxycarbonyl-L-lysine (NCA).

[0093] 1.2 g of leucine and 32 mL of anhydrous tetrahydrofuran were added to a round-bottom flask, and 20 mL of triphosgene (3.2 g) was gradually dissolved therein at 50°C, and the mixture was condensed and refluxed for 5 h until the solution became clear. A yellow solid was obtained by precipitation with n-hexane, and then recrystallization was performed in 5.2:1 n-hexane:tetrahydrofuran to obtain a white solid, which was then dried in vacuum to obtain the modified L-leucine (NCA).

[0094] (2) Add 120 mg of amino polyethylene glycol monomethyl ether (PEG-NH2, molecular weight: 5000) and 0.8 g of L-lysine (NCA) to a round-bottom flask, and then add 12 mL of anhydrous N,N-dimethylformamide (DMF), followed by stirring at 32°C and 750 rpm for 70 h. After the reaction is completed, precipitate with glacial ether and vacuum dry to obtain polyethylene glycol-poly Nε-benzyloxycarbonyl-L-lysine.

[0095] (3) Add 0.6 g of polyethylene glycol-poly (Nε-benzyloxycarbonyl)-L-lysine and 120 mg of L-leucine (NCA) to a round-bottom flask, and then add 12 mL of anhydrous N,N-dimethylformamide (DMF), and then stir at 32°C and 750 rpm for 70 h. After the reaction is completed, precipitate with ice ether and vacuum dry to obtain polyethylene glycol-poly (Nε-benzyloxycarbonyl)-L-lysine-polyleucine.

[0096] (4) Add 1.2 g of polyethylene glycol-poly Nε-benzyloxycarbonyl-L-lysine-polyleucine to a round-bottom flask and add 7 mL of trifluoroacetic acid to completely dissolve it, then add 5 mL of 30% aqueous hydrobromide acetic acid solution to react in an ice-water bath for 4 hours at a speed of 750 rpm; precipitate with ice ether, and vacuum dry to obtain a triblock polypeptide of polyethylene glycol-polylysine-polyleucine (PEG-PLL-PLLeu). The number average molecular weight of polylysine in the triblock polypeptide is 15,000, and the number average molecular weight of polyleucine is 5,000.

[0097] Example 4

[0098] This embodiment provides a method for preparing a polypolypeptide gene vector, which is different from that of Example 1 only in that the number average molecular weight of polyethylene glycol in the prepared triblock polypolypeptide is 10,000, the number average molecular weight of polylysine is 10,000, and the number average molecular weight of polyleucine is 2,000. The remaining steps refer to Example 1.

[0099] Example 5

[0100] This example provides a method for preparing a polypolypeptide gene vector, which differs from Example 1 only in that the number average molecular weight of polyethylene glycol in the prepared triblock polypolypeptide is 500, the number average molecular weight of polylysine is 10000, and the number average molecular weight of polyleucine is 2000. The remaining steps refer to Example 1.

[0101] Example 6

[0102] This example provides a method for preparing a polypolypeptide gene vector, which differs from Example 1 only in that the number average molecular weight of polyethylene glycol in the prepared triblock polypolypeptide is 2000, the number average molecular weight of polylysine is 1500, and the number average molecular weight of polyleucine is 2000. The remaining steps refer to Example 1.

[0103] Example 7

[0104] This example provides a method for preparing a polypolypeptide gene vector, which differs from Example 1 only in that the number average molecular weight of polyethylene glycol in the prepared triblock polypolypeptide is 2000, the number average molecular weight of polylysine is 25000, and the number average molecular weight of polyleucine is 2000. The remaining steps refer to Example 1.

[0105] Example 8

[0106] This embodiment provides a method for preparing a polypolypeptide gene vector, which is different from that of Example 1 only in that the number average molecular weight of polyethylene glycol in the prepared triblock polypolypeptide is 2000, the number average molecular weight of polylysine is 10000, and the number average molecular weight of polyleucine is 10000. The remaining steps refer to Example 1.

[0107] Example 9

[0108] This example provides a method for preparing a polypolypeptide gene vector, which differs from Example 1 only in that the number average molecular weight of polyethylene glycol in the prepared triblock polypolypeptide is 2000, the number average molecular weight of polylysine is 10000, and the number average molecular weight of polyleucine is 0. The remaining steps refer to Example 1.

[0109] The polypolypeptide gene vector in Example 1-9 was mixed with the EGFP plasmid, and the polypolypeptide gene vector loaded with the EGFP plasmid was used to perform a transfection experiment. The transfection efficiency is shown in Table 1.

[0110] Table 1

[0111] Carrier Transfection efficiency Example 1 80-95％ Example 2 70-80％ Example 3 70-85％ Example 4 50-60％ Example 5 60-75％ Example 6 20-40％ Example 7 50-75％ Example 8 5-10％ Example 9 0-5％

[0112] It can be seen from the results of Example 1 and Examples 4-5 that changes in the length of the PEG segment will affect the transfection efficiency of the gene vector.

[0113] The results of Example 1 and Examples 6-7 show that if the functional segment polylysine is too short, the transfection efficiency will be significantly reduced, and if it is too long, the cell toxicity will be increased and the transfection efficiency will be slightly affected.

[0114] It can be seen from the results of Example 1 and Examples 8-9 that an overly long polyleucine segment will greatly affect the transfection efficiency of the gene vector. Similarly, the transfection efficiency without a leucine segment is also very low.

[0115] Example 10 Preparation method of gene delivery system

[0116] (1) Prepare a PBS solution (1 mg / mL) of a triblock polypeptide of polyethylene glycol-polylysine-polyleucine (PEG-PLL-PLLeu).

[0117] (2) Take 10 μL of the above solution, add 0 μg, 1.2 μg, 3 μg, 6 μg, 12 μg, and 30 μg of EGFP plasmid thereto, mix evenly, mix for 20 s, mix by mechanical blowing for 5 times, and the temperature is 22°C; let stand at 20°C for 5 min; then add 50 μg of sodium hyaluronate (Mw=35000) thereto, mix evenly to obtain the gene delivery system, mix for 20 s, mix by mechanical blowing for 5 times, and the temperature is 22°C.

[0118] (3) The nucleic acid loading capacity of the polypolypeptide gene vector was then characterized by gel electrophoresis. The characterization results are as follows: Figure 2 As shown, from Figure 2 It can be seen that according to the results of gel electrophoresis, the polypolypeptide gene vector can completely load the EGFP plasmid when the nitrogen-phosphorus ratio is 0.2, and has excellent nucleic acid loading capacity.

[0119] Example 11 Preparation method of gene delivery system

[0120] (1) Prepare a PBS solution (1 mg / mL) of a triblock polypeptide of polyethylene glycol-polylysine-polyleucine (PEG-PLL-PLLeu).

[0121] (2) Take 10 μL of the above solution, add EGFP plasmid thereto, mix evenly, mix for 30 seconds, mix by mechanical blowing 10 times, and the temperature is 25°C; let stand at 20°C for 5 minutes; then add 50 μg of sodium hyaluronate (Mw=35000) thereto, mix evenly, the mass ratio of the triblock polypeptide, the target gene and sodium hyaluronate in the mixed solution is 1.2 mg:300 μg:1000 μg, mix for 30 seconds, mix by mechanical blowing 10 times, and the temperature is 25°C.

[0122] Example 12 Preparation method of gene delivery system

[0123] (1) Prepare a PBS solution (1 mg / mL) of a triblock polypeptide of polyethylene glycol-polylysine-polyleucine (PEG-PLL-PLLeu).

[0124] (2) Take 10 μL of the above solution, add EGFP plasmid thereto, mix evenly, mix for 10 s, mix by mechanical blowing for 3 times, and the temperature is 20°C; let stand at 20°C for 5 min; then add sodium hyaluronate (Mw=10000) thereto, mix evenly, the mass ratio of the triblock polypeptide, the target gene and sodium hyaluronate in the mixed solution is 0.8 mg:100 μg:100 μg, mix for 10 s, mix by mechanical blowing for 3 times, and the temperature is 20°C.

[0125] Example 13 Preparation method of gene delivery system

[0126] This example provides a method for preparing a gene delivery system, which differs from Example 8 only in that the mass ratio of the triblock polypeptide, target gene and sodium hyaluronate (Mw=35000) in the mixed solution is 0.5 mg:300 μg:1000 μg. The remaining steps refer to Example 11.

[0127] Example 14 Preparation method of gene delivery system

[0128] This example provides a method for preparing a gene delivery system, which differs from Example 8 only in that the mass ratio of the triblock polypeptide, the target gene and sodium hyaluronate (Mw=35000) in the mixed solution is 2 mg:300 μg:1000 μg. The remaining steps refer to Example 11.

[0129] Example 15 Preparation method of gene delivery system

[0130] This example provides a method for preparing a gene delivery system, which differs from Example 8 only in that the number average molecular weight Mw of the sodium hyaluronate in the mixed solution is 3000. The remaining steps refer to Example 11.

[0131] Example 16 Preparation method of gene delivery system

[0132] This example provides a method for preparing a gene delivery system, which differs from Example 8 only in that the number average molecular weight Mw of the sodium hyaluronate in the mixed solution is 100000. The remaining steps refer to Example 11.

[0133] Example 17 Preparation method of gene delivery system

[0134] This example provides a method for preparing a gene delivery system, which differs from Example 8 only in that the number average molecular weight Mw of the sodium hyaluronate in the mixed solution is 2000. The remaining steps refer to Example 11.

[0135] Example 18 Preparation method of gene delivery system

[0136] This example provides a method for preparing a gene delivery system, which differs from Example 8 only in that the number average molecular weight Mw of the sodium hyaluronate in the mixed solution is 150000. The remaining steps refer to Example 11.

[0137] Example 19 Preparation method of gene delivery system

[0138] This embodiment provides a method for preparing a gene delivery system. The method is different from that of Embodiment 8 only in that sodium hyaluronate is replaced with glutamic acid to prepare the gene delivery system. The remaining steps refer to Embodiment 11.

[0139] Example 20 Preparation method of gene delivery system

[0140] This embodiment provides a method for preparing a gene delivery system. The method is different from that of Embodiment 8 only in that sodium hyaluronate is replaced by cell membrane to prepare the gene delivery system. The remaining steps refer to Embodiment 11.

[0141] Example 21 Preparation method of gene delivery system

[0142] This embodiment provides a method for preparing a gene delivery system. The method is different from that of Example 8 only in that dopc (dioleoylphosphatidylcholine) is used to replace sodium hyaluronate to prepare the gene delivery system. The remaining steps refer to Example 11.

[0143] Example 22 Preparation method of gene delivery system

[0144] This embodiment provides a method for preparing a gene delivery system. The method is different from that of Embodiment 8 only in that the EGFP plasmid is replaced by linear DNA to prepare the gene delivery system. The remaining steps refer to Embodiment 11.

[0145] Example 23 Preparation method of gene delivery system

[0146] This example provides a method for preparing a gene delivery system, which differs from Example 8 only in that mRNA is used to replace the EGFP plasmid to prepare the gene delivery system. The remaining steps refer to Example 11.

[0147] Effect detection of the gene delivery system in Examples 11-23:

[0148] Table 2

[0149]

[0150]

[0151] By comparing Example 11 with Examples 13-14, it can be seen that the ratio of the triblock polypolypeptide affects the transfection efficiency. If the amount of the triblock polypolypeptide is too much, the cytotoxicity is greater and the transfection efficiency decreases. If the amount of the triblock polypolypeptide is too little, the gene loading efficiency is low and the transfection efficiency decreases.

[0152] By comparing Example 11 with Examples 15-18, it can be seen that the number average molecular weight of sodium hyaluronate also affects the transfection efficiency. When the molecular weight of sodium hyaluronate is too low, it is difficult to form an effective coating; when the molecular weight of sodium hyaluronate is too high, the particle size of the particles is too large; both will affect the transfection efficiency.

[0153] By comparing Example 11 with Examples 19-21, it can be seen that the introduction of sodium hyaluronate is a key factor in improving the uptake efficiency of gene vectors, thereby further improving the transfection efficiency.

[0154] By comparing Example 10 with Examples 21-23, it can be seen that the gene delivery system of the present invention can achieve efficient delivery of genes including EGFP plasmid, linear DNA, mRNA, etc.

[0155] Example 24 Particle size distribution and zeta potential detection of polypolypeptide gene vector loaded with EGFP plasmid

[0156] The particle size distribution of the polypolypeptide gene vector carrying the EGFP plasmid was measured by Malvern particle size analyzer, and the Zeta potential of the polypolypeptide gene vector carrying the EGFP plasmid was detected. Figure 3 As shown in FIG. 1 , the particle size of the polypolypeptide gene carrier is about 120 nm. The Zeta potential of the polypolypeptide gene carrier is about -10 mV. The polypolypeptide gene carrier was then characterized using a transmission electron microscope. The results are shown in FIG. Figure 4 As shown, from Figure 4 It can be seen that according to the transmission electron microscope, the dry particle size of the obtained polypolypeptide gene carrier is about 100 nm.

[0157] Example 25 Gene transfection efficiency test of polypolypeptide gene vector loaded with EGFP plasmid

[0158] Human embryonic kidney cells (293T cells) were seeded in 12-well plates (10 5 30 μL of EGFP plasmid-loaded polypeptide gene vector (Nano-PLL), EGFP plasmid-loaded linear polyethyleneimine (LPEI25k), EGFP plasmid-loaded liposome and EGFP plasmid-loaded polylysine (PLL) were co-incubated with U87 cells for 24 h, and then the cell fluorescence intensity was detected by fluorescence microscopy and flow cytometry. The results are shown in Figure 2. Figure 5-6 As shown, from Figure 5and Figure 6 It can be seen that according to Figure 5-6 The results show that the transfection ability of the polypolypeptide gene vector of this example for pEGFP at the cellular level is basically equivalent to that of Lipsome8000.

[0159] In summary, the present invention provides a method for preparing a polypolypeptide gene transfection reagent with high transfection efficiency and its application in plasmid and RNA transfection, aiming to provide a polypolypeptide gene transfection system with simple preparation process, stable properties, high transfection efficiency and good biocompatibility.

[0160] The applicant declares that the above is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention are within the protection scope and disclosure scope of the present invention.

Claims

1. A gene delivery system, characterized in that The gene delivery system comprises a polypolypeptide gene carrier, a target gene loaded on the carrier, and sodium hyaluronate coated on the carrier; The polypolypeptide gene carrier comprises a nanosphere formed by self-assembly of a triblock polypolypeptide, wherein the triblock polypolypeptide comprises polyethylene glycol, polylysine and polyleucine connected in sequence; one end of the polylysine is connected to the polyethylene glycol via an amide bond, and the other end of the polylysine is connected to the polyleucine via a peptide bond; The number average molecular weight of the sodium hyaluronate is 3000-35000; The mass ratio of the triblock polypeptide, the target gene and sodium hyaluronate is (0.8-1.2 mg):(100-300 μg):(100-1000 μg); The number average molecular weight of the polyethylene glycol is 1000-5000; the number average molecular weight of the polylysine is 2200-7200; and the number average molecular weight of the polyleucine is 1500-5000.

2. The gene delivery system according to claim 1, characterized in that The target gene includes any one of plasmid, DNA, mRNA or siRNA, or a combination of at least two of them.

3. The gene delivery system according to claim 1, characterized in that The particle size of the polypolypeptide gene carrier is 50-200nm.

4. A method for preparing a gene delivery system according to any one of claims 1 to 3, characterized in that: The preparation method comprises the following steps: loading the target gene on the polypolypeptide gene carrier and coating it with sodium hyaluronate to obtain the gene delivery system.

5. The method for preparing the gene delivery system according to claim 4, characterized in that: The triblock polypeptide of polyethylene glycol-polylysine-polyleucine is synthesized by monomer modification, ring-opening polymerization and hydrolysis, and the obtained triblock polypeptide is dissolved in a solvent to self-assemble into nanospheres to obtain the polypeptide gene carrier.

6. The method for preparing the gene delivery system according to claim 5, characterized in that: The solvent includes water and / or PBS.

7. The method for preparing the gene delivery system according to claim 5, characterized in that: The polyethylene glycol-polylysine-polyleucine triblock polypeptide is prepared by a method comprising the following steps: (1) respectively modifying Nε-benzyloxycarbonyl-L-lysine and leucine to obtain modified Nε-benzyloxycarbonyl-L-lysine and modified L-leucine; (2) mixing amino polyethylene glycol monomethyl ether and modified L-lysine to carry out a ring-opening polymerization reaction to obtain polyethylene glycol-poly Nε-benzyloxycarbonyl-L-lysine; (3) mixing the obtained polyethylene glycol-poly Nε-benzyloxycarbonyl-L-lysine and modified L-leucine to carry out a ring-opening polymerization reaction to obtain polyethylene glycol-poly Nε-benzyloxycarbonyl-L-lysine-polyleucine; (4) The obtained polyethylene glycol-poly Nε-benzyloxycarbonyl-L-lysine-polyleucine is sequentially mixed with trifluoroacetic acid and aqueous solution of hydrobromic acid to carry out hydrolysis reaction, thereby obtaining a triblock polypeptide of polyethylene glycol-polylysine-polyleucine.

8. The method for preparing the gene delivery system according to claim 7, characterized in that: In step (2), the temperature of the ring-opening polymerization reaction is 28-32° C., the time of the ring-opening polymerization reaction is 70-74 h, and the rotation speed of the ring-opening polymerization reaction is 250-750 rpm.

9. The method for preparing the gene delivery system according to claim 7, characterized in that: In step (3), the temperature of the ring-opening polymerization reaction is 28-32° C., the time of the ring-opening polymerization reaction is 70-74 h, and the rotation speed of the ring-opening polymerization reaction is 250-750 rpm.

10. The method for preparing the gene delivery system according to claim 7, characterized in that: In step (4), the mass volume ratio of the polyethylene glycol-poly Nε-benzyloxycarbonyl-L-lysine-polyleucine, trifluoroacetic acid and hydrobromide acetic acid aqueous solution is (0.8-1.2 g):(5-7 mL):(3-5 mL).

11. The method for preparing a gene delivery system according to claim 7, characterized in that: In step (4), the volume concentration of the aqueous solution of hydrogen bromide and acetic acid is 30-36%.

12. The method for preparing the gene delivery system according to claim 7, characterized in that: In step (4), the hydrolysis reaction is carried out in an ice-water bath, the hydrolysis reaction time is 4-6 hours, and the hydrolysis reaction speed is 250-750 rpm.

13. The method for preparing the gene delivery system according to claim 7, characterized in that: In steps (2), (3) and (4), the ring-opening polymerization reaction or the hydrolysis reaction further comprises the steps of precipitation with glacial ether and vacuum drying.

14. The method for preparing the gene delivery system according to claim 4, characterized in that: The target gene is loaded and the sodium hyaluronate is coated by a method comprising the following steps: preparing a solution of a triblock polypeptide of polyethylene glycol-polylysine-polyleucine, adding the target gene to mix, letting it stand, and then adding sodium hyaluronate to mix to obtain the gene delivery system.

15. The method for preparing the gene delivery system according to claim 14, characterized in that: The target gene is added and mixed for 10-30 seconds, and mechanical mixing is performed 3-10 times, and the mixing temperature is 20-25°C.

16. The method for preparing the gene delivery system according to claim 14, characterized in that: The standing time is 4-6 minutes; the standing temperature is 20-25°C.

17. The method for preparing the gene delivery system according to claim 14, characterized in that: The sodium hyaluronate is added and mixed for 10-30 seconds, and mechanical blowing and mixing are performed for 3-10 times. The mixing temperature is 20-25°C.

18. Use of any one of the gene delivery systems according to any one of claims 1 to 3 or the methods for preparing the gene delivery systems according to any one of claims 4 to 17, or a combination of at least two of them, in preparing a nucleic acid transfection reagent.

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