A polyamino acid carrier-based nucleic acid drug delivery system, and a preparation method and application thereof

Abstract

The application provides a nucleic acid drug delivery system based on a polyamino acid carrier and a preparation method and application thereof, wherein the polyamino acid carrier has a structural formula as shown in the specification formula (I), the polyamino acid carrier is modified by using a small molecule anhydride on the basis of arginine grafting to form a polyamino acid carrier with a specific R1 group, and the transfection efficiency is greatly improved; moreover, the polyamino acid carrier is self-assembled with a nucleic acid drug to form a nanoparticle, the targeting property of the nucleic acid drug is significantly improved, and the curative effect is improved; in addition, the polyamino acid drug carrier has no cytotoxicity and has good biocompatibility.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceuticals, specifically relating to a nucleic acid drug delivery system based on a polyamino acid carrier, its preparation method, and its application. Background Technology

[0002] With economic development and improved living standards, people are paying increasing attention to their health. Technological advancements have also enabled scientists to delve deeper into the pathogenesis of various diseases, moving from the individual and organ levels to the genetic level. Researchers have discovered that many diseases are caused by alterations in genetic material, such as the addition, deletion, or mutation of a gene, ultimately leading to abnormal expression of proteins that perform related functions, thus causing pathological changes. Currently common intractable diseases such as cancer and cardiovascular diseases involve changes in nucleic acids at the gene level. Therefore, current clinical treatments such as surgery and chemotherapy only address the surface level and cannot resolve the root cause, thus only treating the symptoms and not the underlying problem, leaving a high risk of disease recurrence.

[0003] In the 1960s, scientists first proposed the concept of gene therapy for diseases. This novel conceptual strategy aims to achieve long-term therapeutic effects, and even cure diseases, by introducing exogenous genetic material into the patient's body. Gene therapy is effective not only for hereditary diseases such as thalassemia, but also for some acquired diseases, such as cancer. In recent years, gene therapy has received increasing attention, but it also faces many bottlenecks in its application. For example, nucleic acid drugs are easily degraded in vitro and in vivo, have low targeting specificity after entering the body, and have low transfection efficiency, all of which hinder the further development of gene therapy and its widespread clinical application. Therefore, developing a good gene delivery vector is an urgent problem to be solved. Viral vectors were among the earliest gene vectors used, but their strong immunogenicity and the side effects they cause in vivo have driven researchers to develop non-viral gene vectors.

[0004] Due to the negatively charged nature of nucleic acids, positively charged materials are excellent nucleic acid delivery systems, with cationic liposomes and polyethyleneimine being typical examples. Cationic liposomes consist mainly of three parts: a cationic head group, a middle linker group, and a hydrophobic tail group. This allows them to bind to DNA or RNA through electrostatic interactions to form complexes, and their good lipid solubility enables them to better enter cells, facilitating gene delivery and therapy. Polyethyleneimine is a cationic polymer, primarily with branched and linear structures, and possesses a high positive charge density. It can tightly bind to nucleic acids, preventing their degradation in the environment and transporting them into cells.

[0005] While non-viral vectors are playing an increasingly important role in gene therapy, and research on them is booming, they also have their own unavoidable drawbacks. Although cationic liposomes can efficiently deliver genes in various cell types, their gene therapy efficacy in the in vivo environment is less than satisfactory. Regarding targeted delivery, apart from high targeting efficiency in the liver, cationic liposomes show poor targeting effects in other tissues and organs. The biggest drawback of polyethyleneimine is its high toxicity, primarily due to its high density of positive charges, which can lead to gangrene and cell apoptosis. Furthermore, polyethyleneimine's targeting ability is relatively weak.

[0006] How to design and construct a novel nucleic acid drug delivery system based on polyamino acid carriers with low biotoxicity, high transfection efficiency, and high targeting, so as to treat or even cure diseases from the root cause, is the technical problem to be solved and the goal to be achieved by this invention. Summary of the Invention

[0007] To overcome the technical problems of poor therapeutic effects and high toxicity of existing drugs, this invention proposes a nucleic acid drug delivery system based on polyamino acid carriers, its preparation method and application. This delivery system can encapsulate therapeutic nucleic acid drugs, which can enter cells with high targeting, escape from lysosomes and release nucleic acid drugs to achieve gene therapy. It has the advantages of high transfection efficiency and low biotoxicity.

[0008] Therefore, the present invention provides a polyamino acid carrier having the structural formula shown in formula (I):

[0009] (I); Where m and n are the number-average aggregation degree, and y is an integer selected from 2 to 16;

[0010] R1 is selected from, and R2 and R3 are independently selected from H or C1-C6 alkyl groups; for or .

[0011] Furthermore, R1 is selected from

[0012] ,or.

[0013] Furthermore, n is 10-500, m is 10-200, and n≥m.

[0014] The present invention also provides a method for preparing any of the above-described polyamino acid carriers, comprising the following steps: (1) Lysine with an amino acid protecting group reacts with triphosgene or phosgene in the presence of a solvent to prepare L-lysine-N-carboxylated intracyclic anhydride with an amino acid protecting group; (2) The L-lysine-N-carboxy ring anhydride with amino acid protecting group is ring-opened polymerized with the monomer shown in formula (II) in the presence of solvent, and then the amino acid protecting group is removed to obtain polylysine with alkyl chain; (3) Arginine with an amino acid protecting group is used as a graft monomer and reacted with an amidation catalyst in the presence of a solvent. The reaction product is then grafted with polylysine with an alkyl chain in the presence of an acid-binding agent, and then the amino acid protecting group is removed. (4) Modify the polymer product obtained in step (3) by removing the amino acid protecting group with the small molecule acid anhydride shown in formula (III) to obtain a polyamino acid carrier.

[0015] (II) (III) Wherein, y is selected from an integer of 2-16, and R2 and R3 are independently selected from H or C1-C6 alkyl groups;

[0016] for or Preferably, the small molecule anhydride is selected from one or more of maleic anhydride (MA), succinic anhydride (SSA), or dimethylmaleic anhydride (DMA).

[0017] Further, the small molecule anhydride is dimethylmaleic anhydride (DMA); wherein, n is 60-120, the grafting rate of arginine is greater than 0 and less than or equal to 100%; preferably 25-100%, and the modification ratio of DMA is greater than 0 and less than or equal to 100%; preferably 10-100%; more preferably, n is 90, the grafting rate of arginine is 50%, and the modification ratio of DMA is 100%.

[0018] The grafting rate of the arginine is greater than 0 and less than or equal to 100%, including but not limited to 10%, 20%, 30%, 50%, 80%, and 100%. The modification ratio of the dimethylmaleic anhydride is greater than 0 and less than or equal to 100%, including but not limited to 10%, 20%, 30%, 50%, 80%, and 100%.

[0019] Further, in step (1), the reaction temperature is 50-60℃; and / or, the solvent is tetrahydrofuran; and / or, the lysine with the amino acid protecting group is selected from one or both of Nε-Boc-L-lysine and Nε-benzyloxycarbonyl-L-lysine. In step (2), the reaction time is 30-40℃; the reaction time is 48-96 h; and / or the solvent is DMF; and / or the amino acid protecting group is removed by hydrolysis in an ice-water bath using trifluoroacetic acid and acetic acid solution containing hydrogen bromide. In step (3), the amidation catalyst includes EDCI and HOBT; and / or, the solvent is DMF; and / or, the arginine with the amino acid protecting group is selected from Nα-Boc-Nω-(2,2,4,6,7-pentamethyl-dihydrobenzofuran-5-sulfonyl)-L-arginine; and / or, the acid binding agent is selected from N,N-diisopropylethylamine; and / or, the graft polymerization reaction is carried out at room temperature for a reaction time of 24-96 h; and / or, the amino acid protecting group is removed by hydrolysis with trifluoroacetic acid at room temperature. In step (4), a solution of small molecule acid anhydride as shown in formula (III) is added dropwise to an alkaline solution containing the polymer product from which the amino acid protecting group has been removed in step (3), and the reaction is carried out for 4-6 hours.

[0020] The present invention also provides a nucleic acid drug delivery system based on a polyamino acid carrier, comprising any of the polyamino acid carriers described above or a polyamino acid carrier prepared by any of the methods described above, and further comprising a nucleic acid drug; Preferably, the nucleic acid drug is siRNA; more preferably, the nucleic acid drug is selected from one or more of siCamk2g, siCCR2, siEpsin1 / 2, siScr, and siGFP. Preferably, the molar ratio between nitrogen in the polyamino acid carrier and phosphorus in the nucleic acid drug is 0.25-16:1; more preferably, it is 4:1. Preferably, the average particle size of the nucleic acid drug delivery system based on polyamino acid carriers is 40-80 nm; more preferably, it is 50-60 nm.

[0021] This invention also tested a nucleic acid drug delivery system using the polyamino acid carrier of this invention to encapsulate the nucleic acid drugs siGFP and siScr (a control siRNA). The results showed an average particle size of 50-60 nm. This system is applicable to various nucleic acid drugs.

[0022] The present invention also provides a method for preparing a nucleic acid drug delivery system based on a polyamino acid carrier, comprising mixing a polyamino acid carrier and a nucleic acid drug in the presence of an aqueous solution; preferably, the molar ratio of the polyamino acid carrier to the nucleic acid drug is 0.25 µg-0.03 mg: 80-500 pmol; preferably, the mixing time is 30 s-5 min.

[0023] The aqueous solution is a conventional aqueous solution such as water, PBS solution, glucose solution, or culture medium.

[0024] The present invention also provides the application of the above-described polyamino acid carrier-based nucleic acid drug delivery system or the preparation method thereof in the preparation of drugs for the prevention or treatment of tumors, cardiovascular diseases or genetic diseases.

[0025] The genetic diseases mentioned above are non-tumor and non-cardiovascular genetic diseases.

[0026] Furthermore, the tumor is selected from one or more of the following: glioma, pancreatic cancer, liver cancer, lung cancer, bladder cancer, bone cancer, breast cancer, colorectal cancer, endometrial cancer, esophageal cancer, kidney cancer, melanoma, ovarian cancer, prostate cancer, and stomach cancer; the cardiovascular disease is selected from one or more of the following: hyperlipidemia, diabetes, atherosclerosis, hypertension, arteritis, coronary heart disease, and heart disease; the genetic disease is selected from one or more of the following: thalassemia, albinism, color blindness, intellectual disability, mental developmental disorder, flat feet, and deformities.

[0027] Furthermore, the drug further includes other pharmaceutically acceptable carriers; preferably, the pharmaceutically acceptable excipients are selected from at least one of pharmaceutically acceptable solvents, solubilizers, co-solvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, binding agents, penetration enhancers, pH adjusters, buffers, plasticizers, surfactants, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants, anti-flocculation agents, filter aids, release inhibitors, polymeric framework materials, and film-forming materials.

[0028] Furthermore, the dosage form of the drug is selected from injections, oral preparations, or topical preparations; preferably, the injection is selected from injection solutions or powder injections; the oral preparation is selected from tablets, solutions, capsules, powders, pills, granules, syrups, suspensions, or oral sustained-release preparations; and the topical preparation is selected from ointments, sprays, or patches.

[0029] Furthermore, the molecular weight of the polyamino acid carrier is 1500-8000 g / mol, for example, the molecular weight of the polyamino acid carrier prepared in Example 1 below is 32397 g / mol.

[0030] Compared with the prior art, the above-mentioned technical solution of the present invention has the following advantages: 1. Polylysine (PLL) has been used for gene transfection, but it suffers from low transfection efficiency and high cytotoxicity. The polyamino acid carrier provided by this invention has the structural formula shown in formula (I). This polyamino acid carrier is modified with small molecule anhydrides after arginine grafting to form a polyamino acid carrier with a specific R1 group, greatly improving transfection efficiency. Furthermore, the polyamino acid carrier of this invention can efficiently load nucleic acid drugs and self-assemble to form a nucleic acid drug delivery system, significantly improving the targeting of nucleic acid drugs and enhancing efficacy. It also possesses strong lysosomal escape capability, allowing the nucleic acid drug delivery system to enter cells with high targeting, escape from the lysosomes, and release nucleic acids, achieving highly efficient gene therapy. In addition, the polyamino acid drug carrier of this invention has high safety in vivo, is biodegradable, and does not produce cytotoxicity.

[0031] The specific R1 group formed after modification with small molecule anhydrides can shield or partially shield the positive charge of the polyamino acid carrier provided by this invention under normal physiological conditions, thereby reducing cytotoxicity and improving bioavailability; it can break in the acidic environment of cell lysosomes, thereby exposing the positive charge of the polyamino acid carrier, amplifying the protonated sponge effect to achieve efficient lysosomal escape of nucleic acid drugs, thereby improving the transfection efficiency of polylysine gene carriers.

[0032] 2. The polyamino acid carrier provided by this invention, compared with other options of this invention, shows a significant improvement in transfection efficiency when R1 is selected from [specific ingredient] or when the small molecule anhydride is DMA; the reason is... Succinic anhydride lacks acid responsiveness, maleic anhydride has a weak acid responsiveness, dimethylmaleic anhydride has the strongest acid responsiveness, and finally DMA-modified PLL-Arg has the strongest lysosomal escape ability and the best transfection performance.

[0033] 3. The preparation method of the polyamino acid carrier provided by this invention is simple, convenient, and low in cost, and can be industrialized. Furthermore, by studying the effects of the number-average degree of polymerization of polylysine, the arginine grafting rate, and the DMA modification ratio on the transfection efficiency of the polylysine gene carrier, it was found that the optimal transfection efficiency of the polylysine gene carrier was achieved under the following conditions: L-lysine degree of polymerization of 90%, arginine grafting rate of 50%, and DMA modification ratio of 100%.

[0034] 4. The method for preparing the polyamino acid carrier provided by the present invention, wherein the lysine with the amino acid protecting group is selected from one or both of Nε-Boc-L-lysine and Nε-benzyloxycarbonyl-L-lysine, but compared with Nε-Boc-L-lysine, Nε-benzyloxycarbonyl-L-lysine has a more stable chemical structure, is less prone to racemization during the reaction, and reduces by-products.

[0035] 5. The nucleic acid drug delivery system based on polyamino acid carriers provided by this invention differs significantly from ordinary chemical drugs in that nucleic acids have a large number of phosphate groups, resulting in a negative charge. Using the cationic polymer of this invention, nucleic acid drugs can be encapsulated through electrostatic interactions, resulting in high drug loading capacity.

[0036] The average particle size of the nucleic acid drug delivery system based on polyamino acid carrier of the present invention is 40-80 nm (preferably 50-60 nm), which is significantly smaller than the particle size (>100 nm) reported in the literature.

[0037] 6. The nucleic acid drug delivery system based on a polyamino acid carrier provided by this invention can efficiently deliver different nucleic acid drugs (e.g., but not limited to siCamk2g, siGFP) into different cells. After being taken up by the cells and entering lysosomes, the polyamino acid carrier can generate a protonated sponge effect, promoting the lysosomal escape of genes. It is expected to have stronger penetration capabilities in certain lesion sites with many biological barriers and difficult penetration, such as atherosclerotic plaques, solid tumors, and the blood-brain barrier, and may have better therapeutic effects in diseases including, but not limited to, glioma, pancreatic cancer, and lung cancer. Attached Figure Description

[0038] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein: Figure 1 NMR spectra of polyamino acid carriers LAD with different DMA modification ratios prepared in Experimental Example 1; Figure 2 The particle size characterization results for different nucleic acid drug delivery systems based on polyamino acid carriers are shown. The vertical axis, Diameter, represents the particle size. Figure 3 Gel electrophoresis images of nucleic acid drug delivery systems based on polyamino acid carriers with different N / P ratios loaded onto polyamino acid carriers LAD with different DMA modification ratios. Figure 4 The results of the cytotoxicity experiment for the polyamino acid carrier are shown, with Cell viability on the ordinate. Figure 5The results of cytotoxicity experiments for different types of nucleic acid drug delivery systems based on polyamino acid carriers are shown. The vertical axis represents cell viability. Figure 6 The images show the lysosomal escape ability detection results for the siRNA group and the LAD@siRNA group. Original is the original image; Pseudo colour is the pseudo-color image. Figure 7 The results show the test results of the gene silencing efficiency of macrophages reprogrammed by the polyamino acid vector prepared in Example 1; Figure 8 The results show the HepG 2-GFP cell gene transfection capability of the nucleic acid drug delivery system prepared using the polyamino acid carrier of Example 1. Figure 9 This is a confocal image of nitric oxide production in macrophages by the nucleic acid drug delivery system based on polyamino acid carriers in Experiment Example 3. Figure 10 The results of Western blot analysis were used to detect the gene silencing efficiency of a nucleic acid drug delivery system based on a polyamino acid carrier. Figure 11 The results show the effectiveness of a nucleic acid drug delivery system based on polyamino acid carriers in promoting endothelial cell migration. Figure 12 The results show the in vivo targeting effect of a nucleic acid drug delivery system based on polyamino acid carriers on atherosclerotic mice. Figure 13 The results show the in vivo therapeutic effect of a nucleic acid drug delivery system based on polyamino acid carriers on atherosclerotic mice. Figure 14 The results show the detection of in situ editing of macrophages in atherosclerotic mice using a nucleic acid drug delivery system based on polyamino acid carriers. Figure 15 The results show the effect of a nucleic acid drug delivery system based on polyamino acid carriers on the repair of the endothelial barrier in atherosclerotic mice; in Figure B, the vertical axis represents the intensity of EB in the aorta, which is the intensity of EB in the aorta. This indicates that p < 0.05. Detailed Implementation

[0039] The technical solution of the present invention will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0040] The method for preparing the polyamino acid carrier provided in this invention includes the following steps: (1) Take 0.5-20 g of Nε–benzyloxycarbonyl-L-lysine, preferably 8 g; dissolve it in 4-100 mL of anhydrous tetrahydrofuran, preferably 40 mL; then take 0.4-15 g of triphosgene, preferably 4 g; dissolve it in 4-100 mL of anhydrous tetrahydrofuran, preferably 20 mL; add it dropwise at a constant pressure dropping funnel and reflux it at 50-60℃, preferably 50℃; when the reaction solution becomes clear and transparent, precipitate it with n-hexane, filter the solid obtained, recrystallize it in THF / n-hexane = 1:1-4, preferably 1:2; dry the recrystallized solid under reduced pressure at room temperature to obtain Nε-benzyl–benzyloxycarbonyl-L-lysine-(N-carboxyl ring anhydride) (i.e. ZLL(NCA)).

[0041] (2) Take 1-100 mg of hexadecylamine solid into a round-bottom flask, preferably 7 mg; then add 0.3-30 g of ZLL (NCA) solid, preferably 1 g; and 1-50 mL of anhydrous DMF, preferably 5 mL; react at 30-40℃ for 48-96 h, preferably 37℃ for 72 h; then precipitate the reaction mixture twice with ice-cold diethyl ether to obtain a solid, which is then dried under vacuum overnight to obtain hexadecylamine-PZLL. 90 Subsequently, hexadecylamine-PZLL was obtained. 90 Add 1-50 mL of trifluoroacetic acid to dissolve the solution completely, preferably 5 mL; then add 2-20 mL of 33% HBr / acetic acid solution, preferably 3 mL; hydrolyze in an ice-water bath for 3-6 h, preferably 4 h; precipitate the resulting solution with ice-cold ether and centrifuge to obtain a white solid hexadecylamine-PLL. 90 Vacuum dry overnight.

[0042] (3) PLL 90 -Arg synthesis Take 0.2-40 g of Nα-Boc-Nω-(2,2,4,6,7-pentamethyl-dihydrobenzofuran-5-sulfonyl)-L-arginine, preferably 1 g; 0.3-30 g of EDCI, preferably 0.75 g; and 0.2-20 g of HOBT, preferably 0.5 g; and dissolve them in 5-800 mL of DMF, preferably 20 mL. Then take 0.1-20 g of hexadecylamine-PLL90, preferably 0.5 g; and 0.2-20 g of N,N-diisopropylethylamine, preferably 0.5 g; dissolve them in 4-400 mL of deionized water, preferably 10 mL, and then add and maintain the reaction at room temperature for 12-96 h, preferably 72 h; permeate the resulting solution with deionized water (dialysis bag molecular weight cutoff 3500 Da) for 24-96 h, preferably 72 h; freeze-dry to obtain a white solid. The white solid was then passed through trifluoroacetic acid to obtain PLL. 90 -Arg.

[0043] (4) PLL 90 Synthesis of Arg-DMA (abbreviated as LAD) 0.1-10 g of PLL 90 -Arg, preferably 0.5 g; dissolved in 2-200 mL of NaOH solution with pH=8.0, preferably 10 mL; then take out DMA greater than 0 and less than or equal to 30 g, preferably 1.5 g; dissolved in 1-20 mL of 2,4-dioxane, preferably 5 mL, and react for 4-6 h, preferably 5 h. Dialyze (dialysis bag molecular weight cutoff 3500 Da) for 24-96 h, preferably 72 h; lyophilize to obtain PLL. 90 -Arg-DMA white solid (polyamino acid carrier).

[0044] Using a similar method, maleic anhydride (MA) or succinic anhydride (SSA) can be used to replace DMA to prepare maleic anhydride or succinic anhydride-modified polymers (PLLs). 90 -Arg-MA (abbreviated as LAM) and PLL 90 -Arg-SSA (abbreviation: LAS).

[0045] The siCamk2g in this invention comes from Guangzhou Ruibo Biotechnology Co., Ltd.; the sequence (5'-3') is as follows: 5'-AAC GUG GUA CAU AAU GCU ACA-3'.

[0046] In the following examples and comparative examples, the grafting rate or modification rate of DMA was tested and calculated according to item (1) of Experimental Example 1; the grafting rate of arginine was measured by nuclear magnetic resonance hydrogen spectroscopy, and the number-average degree of polymerization was measured by nuclear magnetic resonance hydrogen spectroscopy.

[0047] Example 1: Preparation of polyamino acid carrier This embodiment provides a method for preparing a polyamino acid carrier, comprising the following steps: (1) Synthesis of ZLL(NCA) 8 g of Nε–benzyloxycarbonyl-L-lysine was dissolved in 40 mL of anhydrous tetrahydrofuran to obtain the reaction base solution. Then, 4 g of triphosgene was dissolved in 20 mL of anhydrous tetrahydrofuran and added dropwise at a constant pressure dropping funnel to the reaction base solution, which was then refluxed at 50 °C. Once the reaction solution became clear and transparent, it was precipitated with n-hexane, filtered, and recrystallized at a THF / n-hexane ratio of 1:2 (v / v). The recrystallized solid was dried under reduced pressure at room temperature to obtain ZLL (NCA), full Chinese name: Nε-benzyl–benzyloxycarbonyl-L-lysine-N-carboxyl ring anhydride.

[0048] (2) Synthesis of hexadecylamine-PLL 7 mg of hexadecylamine solid was placed in a round-bottom flask; then 1 g of ZLL(NCA) solid and 5 mL of anhydrous DMF were added, and the mixture was reacted at 37 °C for 72 h; the reaction product was then precipitated twice with ice-cold diethyl ether to obtain a solid, which was dried under vacuum overnight to obtain hexadecylamine-PLL. 90 Subsequently, hexadecylamine-PLL was obtained. 90 Add 5 mL of trifluoroacetic acid to dissolve it completely. Then add 3 mL of 33% HBr / acetic acid solution and hydrolyze in an ice-water bath for 4 h. The resulting solution is then precipitated with ice-cold diethyl ether and centrifuged to obtain a white solid, hexadecylamine-PLL. 90 Vacuum dry overnight.

[0049] (3) PLL 90 -Arg synthesis 1 g of Nα-Boc-Nω-(2,2,4,6,7-pentamethyl-dihydrobenzofuran-5-sulfonyl)-L-arginine, 0.75 g of EDCI (1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride), and 0.5 g of HOBT (1-hydroxybenzotriazole) were dissolved in 20 mL of DMF and reacted at room temperature for 30 min to obtain the reaction solution. Subsequently, 0.5 g of hexadecylamine-PLL was... 900.5 g of N,N-diisopropylethylamine was dissolved in 10 mL of deionized water, and then added to the above reaction solution, and the reaction was maintained at room temperature for 72 h. The resulting solution was dialyzed against deionized water (molecular weight cutoff 3500 Da) for 72 h; lyophilized to obtain a white solid. The white solid was then added to 6 mL of trifluoroacetic acid; stirred at room temperature for 4 h. The mixture was then centrifuged by sedimentation with anhydrous diethyl ether, and the resulting solid was reconstituted with DMSO, dialyzed (molecular weight cutoff 3500 Da) for 72 h; then 2 mL of mercaptoethanol was added; the mixture was stirred overnight at room temperature, dialyzed (molecular weight cutoff 3500 Da) for 72 h; and then lyophilized to obtain PLL. 90 -Arg.

[0050] (4) PLL 90 Synthesis of Arg-DMA (abbreviated as LAD) 0.5 g of PLL 90 Arg was dissolved in 10 mL of NaOH solution at pH 8.0 to obtain the reaction solution. Then, 1.5 g of dimethylmaleic anhydride (DMA) was dissolved in 5 mL of 2,4-dioxane and added to the above reaction solution. The reaction was allowed to proceed for 5 h. The solution was then dialyzed (dialysis bag molecular weight cutoff 3500 Da) for 72 h; lyophilized to obtain PLL. 90 Arg-DMA is a white solid (polyamino acid carrier, abbreviated as "LAD carrier"). In this example, the polyamino acid carrier prepared has a number-average degree of polymerization of 90 for L-lysine, a DMA grafting rate (or modification ratio) of 100%, and an arginine grafting rate of 50%. This LAD carrier is denoted as: LAD. 100 .

[0051] Example 2 Preparation of polyamino acid carrier This embodiment provides a method for preparing a polyamino acid carrier, which is basically the same as the method in Example 1, except that in step (4), 1.5 g of maleic anhydride (MA) is used instead of dimethylmaleic anhydride (DMA) to finally obtain PLL. 90 -Arg-MA white solid (abbreviated as LAM carrier).

[0052] Example 3 Preparation of polyamino acid carrier This embodiment provides a method for preparing a polyamino acid carrier, which is basically the same as the method in Example 1, except that in step (4), 1.5 g of succinic anhydride (SSA) is used instead of dimethylmaleic anhydride (DMA) to finally obtain PLL. 90 -Arg-SSA white solid (abbreviated as: LAS carrier).

[0053] Example 4 Preparation of polyamino acid carrier This embodiment provides a method for preparing a polyamino acid carrier, which is basically the same as the method in Example 1, except that the amount of dimethylmaleic anhydride (DMA) in step (4) is adjusted from 1.5 g to 0.1 g. The polyamino acid carrier prepared in this embodiment has a number-average degree of polymerization of 90 for L-lysine, a DMA grafting rate of 10%, and an arginine grafting rate of 50%. This LAD carrier is denoted as: LAD 10 .

[0054] Example 5 Preparation of polyamino acid carrier This embodiment provides a method for preparing a polyamino acid carrier, which is basically the same as the method in Example 1, except that the amount of dimethylmaleic anhydride (DMA) in step (4) is adjusted from 1.5 g to 0.2 g. The polyamino acid carrier prepared in this embodiment has a number-average degree of polymerization of 90 for L-lysine, a DMA grafting rate of 20%, and an arginine grafting rate of 50%. This LAD carrier is denoted as: LAD 20 .

[0055] Example 6 Preparation of polyamino acid carrier This embodiment provides a method for preparing a polyamino acid carrier, which is basically the same as the method in Example 1, except that the amount of dimethylmaleic anhydride (DMA) in step (4) is adjusted from 1.5 g to 0.5 g. The polyamino acid carrier prepared in this embodiment has a number-average degree of polymerization of 90 for L-lysine, a DMA grafting rate of 50%, and an arginine grafting rate of 50%. This LAD carrier is denoted as: LAD 50 .

[0056] Example 7: Preparation of a nucleic acid drug delivery system based on a polyamino acid carrier This embodiment provides a method for preparing a nucleic acid drug delivery system based on a polyamino acid carrier, comprising the following steps: The LAD carrier prepared in Example 1 was dissolved in water to prepare a polyamino acid carrier concentration of 1 mg / mL. 20 μL of the LAD carrier solution was added to 460 pmol of the nucleic acid drug siCamk2g and mixed for 1 min to obtain a nucleic acid drug delivery system based on the polyamino acid carrier. This system was then diluted to 1 mL of water to obtain a suspension containing the nucleic acid drug delivery system based on the polyamino acid carrier (hereinafter referred to as "LAD@siCamk2g", "LAD..."). 100 @siCamk2g or "LAD" 100 @siRNA”).

[0057] Example 8: Preparation of a nucleic acid drug delivery system based on a polyamino acid carrier This embodiment provides a method for preparing a nucleic acid drug delivery system based on a polyamino acid carrier, which is basically the same as that in Example 7, except that the LAM carrier of Example 2 is used instead of the polyamino acid carrier prepared in Example 1 to obtain a nucleic acid drug delivery system based on a polyamino acid carrier and a suspension containing the nucleic acid drug delivery system based on a polyamino acid carrier (hereinafter referred to as "LAM@siCamk2g").

[0058] Example 9: Preparation of a nucleic acid drug delivery system based on a polyamino acid carrier This embodiment provides a method for preparing a nucleic acid drug delivery system based on a polyamino acid carrier, which is basically the same as that in Example 7, except that the LAS carrier of Example 3 is used instead of the polyamino acid carrier prepared in Example 1 to obtain a nucleic acid drug delivery system based on a polyamino acid carrier and a suspension containing the nucleic acid drug delivery system based on a polyamino acid carrier (hereinafter referred to as "LAS@siCamk2g").

[0059] Example 10: Preparation of a nucleic acid drug delivery system based on a polyamino acid carrier This embodiment provides a method for preparing a nucleic acid drug delivery system based on a polyamino acid carrier, which is basically the same as that in Example 7, except that it uses the LAD from Example 4. 10 Instead of the polyamino acid carrier prepared in Example 1, a nucleic acid drug delivery system based on a polyamino acid carrier and a suspension containing the nucleic acid drug delivery system based on a polyamino acid carrier (hereinafter referred to as "LAD") were prepared. 10 @siCamk2g or "LAD" 10 @siRNA”).

[0060] Example 11 Preparation of a nucleic acid drug delivery system based on a polyamino acid carrier This embodiment provides a method for preparing a nucleic acid drug delivery system based on a polyamino acid carrier, which is basically the same as that in Example 7, except that it uses the LAD from Example 5. 20 Instead of the polyamino acid carrier prepared in Example 1, a nucleic acid drug delivery system based on a polyamino acid carrier and a suspension containing the nucleic acid drug delivery system based on a polyamino acid carrier (hereinafter referred to as "LAD") were prepared. 20 @siCamk2g or "LAD" 20 @siRNA”).

[0061] Example 12 Preparation of a nucleic acid drug delivery system based on a polyamino acid carrier This embodiment provides a method for preparing a nucleic acid drug delivery system based on a polyamino acid carrier, which is basically the same as that in Example 7, except that it uses the LAD from Example 6. 50Instead of the polyamino acid carrier prepared in Example 1, a nucleic acid drug delivery system based on a polyamino acid carrier and a suspension containing the nucleic acid drug delivery system based on a polyamino acid carrier (hereinafter referred to as "LAD") were prepared. 50 @siCamk2g or "LAD" 50 @siRNA”).

[0062] Comparative Example 1 siCamk2g was encapsulated using a commercial liposome carrier (specifically, Lipofectamine 2000, purchased from Thermo Fisher Scientific, catalog number 11668019). The specific method was as follows: 8 μL of liposomes were mixed with 250 μL of serum-free culture medium for 5 min to prepare a dispersion of the liposome carrier. 460 pmol of the nucleic acid drug siCamk2g was mixed with 250 μL of serum-free culture medium for 5 min to prepare a dispersion of the nucleic acid drug siCamk2g. The above dispersion of the liposome carrier and the dispersion of the nucleic acid drug siCamk2g were mixed for 10 min, and then diluted in 1 mL of water to obtain a suspension containing drug-loaded liposome particles (hereinafter referred to as "Lipo@siCamk2g").

[0063] The particle size distribution of Lipo@siCamk2g was measured by a particle size analyzer. The results showed that the average particle size of Lipo@siCamk2g was 110 nm and the PDI was 0.24.

[0064] Comparative Example 2 This comparative example provides a method for preparing a polyamino acid carrier, which is basically the same as the method in Example 1, except that step (4) is omitted, and in step (3), the amount of Nα-Boc-Nω-(2,2,4,6,7-pentamethyl-dihydrobenzofuran-5-sulfonyl)-L-arginine is adjusted from 1 g to 0.5 g. The polyamino acid carrier prepared in this comparative example has a number-average degree of polymerization of L-lysine of 90, a DMA grafting rate of 0%, and an arginine grafting rate of 25%.

[0065] This comparative example also provides a method for preparing a nucleic acid drug delivery system based on a polyamino acid carrier, which is basically the same as that in Example 7, except that the carrier of this comparative example is used instead of the polyamino acid carrier prepared in Example 1 to obtain a nucleic acid drug delivery system based on a polyamino acid carrier and a suspension containing the nucleic acid drug delivery system based on a polyamino acid carrier.

[0066] Comparative Example 3 This comparative example provides a method for preparing a polyamino acid carrier, which is basically the same as the method in Example 1, except that step (4) is not performed. The polyamino acid carrier prepared in this example has a number-average degree of polymerization of 90 for L-lysine, a DMA grafting rate of 0%, and a arginine grafting rate of 50%. This polyamino acid carrier is not grafted with DMA and is denoted as LAD0.

[0067] This comparative example also provides a method for preparing a nucleic acid drug delivery system based on a polyamino acid carrier, which is basically the same as that in Example 7, except that the carrier of this comparative example is used instead of the polyamino acid carrier prepared in Example 1 to obtain a nucleic acid drug delivery system based on a polyamino acid carrier and a suspension containing the nucleic acid drug delivery system based on a polyamino acid carrier (hereinafter referred to as "LAD0@siCamk2g" or "LAD0@siRNA").

[0068] Comparative Example 4 This comparative example provides a method for preparing a polyamino acid carrier, which is basically the same as the method in Example 1, except that step (4) is omitted. In step (3), the amount of Nα-Boc-Nω-(2,2,4,6,7-pentamethyl-dihydrobenzofuran-5-sulfonyl)-L-arginine is adjusted from 1 g to 1.4 g. In the polyamino acid carrier prepared in this example, the number-average degree of polymerization of L-lysine is 90, the grafting rate of DMA is 0%, and the grafting rate of arginine is 70%.

[0069] This comparative example also provides a method for preparing a nucleic acid drug delivery system based on a polyamino acid carrier, which is basically the same as that in Example 7, except that the carrier of this comparative example is used instead of the polyamino acid carrier prepared in Example 1 to obtain a nucleic acid drug delivery system based on a polyamino acid carrier and a suspension containing the nucleic acid drug delivery system based on a polyamino acid carrier.

[0070] Comparative Example 5 This comparative example provides a method for preparing a polyamino acid carrier, which is basically the same as the method in Example 1, except that step (4) is omitted. In step (3), the amount of Nα-Boc-Nω-(2,2,4,6,7-pentamethyl-dihydrobenzofuran-5-sulfonyl)-L-arginine is adjusted from 1 g to 2 g. The polyamino acid carrier prepared in this example has a number-average degree of polymerization of 90 for L-lysine, a DMA grafting rate of 0%, and a arginine grafting rate of 100%.

[0071] This comparative example also provides a method for preparing a nucleic acid drug delivery system based on a polyamino acid carrier, which is basically the same as that in Example 7, except that the carrier of this comparative example is used instead of the polyamino acid carrier prepared in Example 1 to obtain a nucleic acid drug delivery system based on a polyamino acid carrier and a suspension containing the nucleic acid drug delivery system based on a polyamino acid carrier.

[0072] Comparative Example 6 This comparative example provides a method for preparing a polyamino acid carrier, which is basically the same as the method in Example 1, except that steps (3) and (4) are not performed, and the amount of ZLL (NCA) solid in step (2) is adjusted from 1 g to 0.67 g. The polyamino acid carrier prepared in this example has a number-average degree of polymerization of L-lysine of 60, a DMA grafting rate of 0%, and an arginine grafting rate of 0%.

[0073] This comparative example also provides a method for preparing a nucleic acid drug delivery system based on a polyamino acid carrier, which is basically the same as that in Example 7, except that the carrier of this comparative example is used instead of the polyamino acid carrier prepared in Example 1 to obtain a nucleic acid drug delivery system based on a polyamino acid carrier and a suspension containing the nucleic acid drug delivery system based on a polyamino acid carrier.

[0074] Comparative Example 7 This comparative example provides a method for preparing a polyamino acid carrier, which is basically the same as the method in Example 1, except that steps (3) and (4) are not performed. The polyamino acid carrier prepared in this example has a number-average degree of polymerization of 90 for L-lysine, a grafting rate of 0% for DMA, and a grafting rate of 0% for arginine.

[0075] This comparative example also provides a method for preparing a nucleic acid drug delivery system based on a polyamino acid carrier, which is basically the same as that in Example 7, except that the carrier of this comparative example is used instead of the polyamino acid carrier prepared in Example 1 to obtain a nucleic acid drug delivery system based on a polyamino acid carrier and a suspension containing the nucleic acid drug delivery system based on a polyamino acid carrier.

[0076] Comparative Example 8 This comparative example provides a method for preparing a polyamino acid carrier, which is basically the same as the method in Example 1, except that steps (3) and (4) are not performed. In step (2), the amount of ZLL (NCA) solid is adjusted from 1 g to 1.34 g. The polyamino acid carrier prepared in this example has a number-average degree of polymerization of L-lysine of 120, a DMA grafting rate of 0%, and an arginine grafting rate of 0%.

[0077] This comparative example also provides a method for preparing a nucleic acid drug delivery system based on a polyamino acid carrier, which is basically the same as that in Example 7, except that the carrier of this comparative example is used instead of the polyamino acid carrier prepared in Example 1 to obtain a nucleic acid drug delivery system based on a polyamino acid carrier and a suspension containing the nucleic acid drug delivery system based on a polyamino acid carrier.

[0078] Example 1 Characterization (1) DMA modification ratio in polyamino acid carrier The polyamino acid carriers LAD0 and LAD prepared in Comparative Examples 3, Examples 4-6, and Example 1 were analyzed using nuclear magnetic resonance imaging. 10 LAD 20 LAD 50 LAD 100 The test was conducted, and the results are shown below. Figure 1 As shown.

[0079] Depend on Figure 1 It can be seen that, compared to the polyamino acid carrier LAD0, LAD... 10 LAD 20 LAD 50 LAD 100 The peak area gradually increases in the chemical shift range of 1.6–2.0 ppm. The characteristic peak in the chemical shift range of 1.6–2.0 ppm is the characteristic peak of the dimethylmaleic acid group in the polyamino acid carrier, denoted as peak a.

[0080] The DMA modification ratio in each group of polyamino acid carriers was calculated using the following formula: Modification ratio = 1 / 6 (Integrated area of ​​peak a - area of ​​peak a0) / integrated area of ​​peak at chemical shift of 4.2 ppm × 100%, where: the area of ​​peak a0 is the integrated area of ​​LAD0 at peak a. The polyamino acid carriers LAD0 and LAD... 10 LAD 20 LAD 50 LAD 100 The modification ratios were 0%, 10%, 20%, 50%, and 100%, respectively. The successful synthesis of polyamino acid carriers with different modification ratios was confirmed.

[0081] (2) Particle size of nucleic acid drug delivery system based on polyamino acid carrier The particle size distribution of suspensions containing polyamino acid-based nucleic acid drug delivery systems prepared in Examples 7, 10-12, and Comparative Example 3 was measured using a particle size analyzer. The results are shown in [Figure number missing]. Figure 2 As shown.

[0082] from Figure 2 It can be seen that the average particle size of each drug delivery system is 40-80 nm.

[0083] Experimental Example 2: Nucleic acid loading capacity of polyamino acid carriers with different DMA modification ratios (1) Preparation of nucleic acid drug delivery system based on polyamino acid carrier The polyamino acid carrier and nucleic acid drug were mixed for 1 min at the following ratios: nitrogen in the polyamino acid carrier and phosphorus in the nucleic acid drug (nitrogen-to-phosphorus ratio, N / P): 0:1, 0.5:1, 1:1, 2:1, 4:1, 8:1, and 16:1. (Specifically, the polyamino acid carriers LAD0 and LAD0 prepared in Comparative Examples 3, Examples 4-6, and Example 1 were mixed respectively.) 10 LAD 20 LAD 50 LAD 100 Using LAD vectors, 0g, 0.5μg, 0.9μg, 1.9μg, 3.7μg, 7.4μg, and 14.8μg of LAD vectors were mixed with 92 pmol of the nucleic acid drug siCamk 2g for 1 min to obtain nucleic acid drug delivery systems based on polyamino acid vectors with different N / P ratios.

[0084] (2) Preparation of agarose gel Dissolve 2 g of agarose in 100 mL of 1×TAE solution; then heat in a microwave oven on medium-high for 1 min to dissolve completely. After it cools slightly, add 1 / 10000 of the total solution volume of nucleic acid dye (Yisheng Biotechnology (Shanghai) Co., Ltd., 10204ES76), then pour it into a mold and let it cool completely to set.

[0085] (3) Electrophoresis Add 5 μL loading buffer to the above-prepared nucleic acid drug delivery systems based on polyamino acid carriers with different N / P ratios, mix for 1 min, and obtain the loading system.

[0086] Add 2 L of 1×TAE solution to the electrophoresis tank, immerse the gel in the TAE solution, add the prepared sample loading system to the gel wells, and start electrophoresis. Select a voltage of 140 V and an electrophoresis time of 30 min. After electrophoresis, place the gel in an imaging system for imaging. The results are shown in [Figure 1]. Figure 3 As shown.

[0087] from Figure 3 It can be seen that when the N / P ratio is 4:1, the nucleic acid drug becomes less negatively charged due to complete binding with the polyamino acid carrier, and thus cannot migrate downwards; this indicates that the polyamino acid carrier can efficiently load nucleic acid drugs, achieving 100% full loading of the nucleic acid drug.

[0088] Experiment Example 3: Cytotoxicity Test Test 1: (1) Test method ① Preparation of test sample solutions: Before testing, the polyamino acid carrier prepared in Example 1 was dissolved in water to prepare a polyamino acid carrier solution with a concentration of 1 mg / mL. 0, 5 μL, 10 μL, 15 μL, and 20 μL of the above polyamino acid carrier solution were added to 460 pmol of nucleic acid drug (i.e., siCamk2g), mixed for 1 min, and a nucleic acid drug delivery system based on the polyamino acid carrier was obtained. This system was then diluted to 1 mL of culture medium to obtain test samples with carrier concentrations of 0, 5, 10, 15, and 20 μg / mL, respectively.

[0089] ② Cell treatment and testing: according to 3×10 4 Raw 264.7 cells in logarithmic growth phase were seeded in 96-well plates and allowed to adhere. The cells were then resuspended in the culture medium of the test samples described above, with five replicates per sample and 200 μL of test sample added to each well. A negative control group was prepared by replacing the culture medium in the cells with 200 μL of fresh culture medium. A blank control group was prepared by using 200 μL of culture medium without cells or test substances. All groups were incubated for 48 h, and the absorbance at 450 nm was measured using a CCK8 assay reader. The results were analyzed.

[0090] (2) Test results The results are as follows Figure 4 As shown, the toxicity of the drug delivery system with different concentrations to cells was similar to that without the drug, with no significant difference in cell viability (p>0.05), indicating that the polyamino acid carrier of the present invention has very low toxicity.

[0091] Test 2: (1) Test method ① Preparation of test samples: Before testing, the polyamino acid carriers prepared in Comparative Examples 3, Examples 4-6, and Example 1 were dissolved in water to prepare a polyamino acid carrier solution with a concentration of 1 mg / mL. 20 μL of the above polyamino acid carrier solution was added to 460 pmol of the nucleic acid drug siRNA (i.e., siCamk2g), and mixed for 1 min to obtain a nucleic acid drug delivery system based on the polyamino acid carrier. This system was then diluted to 1 mL of culture medium to obtain test samples with a carrier concentration of 20 μg / mL, denoted as LAD0@siRNA, LAD0@siRNA, and LAD0@siRNA, respectively. 10 @siRNA、LAD 20 @siRNA、LAD 50 @siRNA、LAD 100 @siRNA.

[0092] ② Cell treatment and testing: according to 3×10 4 HUVEC cells in logarithmic growth phase were seeded in 96-well plates and allowed to adhere. The cells were then resuspended in the culture medium of the test samples described above, with five replicates per sample. 200 μL of the test sample was added to each well. 200 μL of fresh culture medium was used as a negative control group. 200 μL of culture medium without cells and test substances was used as a blank control group. All groups were incubated for 48 h, and the absorbance at 450 nm was measured using a CCK8 assay kit. The results were analyzed.

[0093] (2) Test results The results are as follows Figure 5 As shown, compared to Comparative Example 3, the polyamino acid nanoparticles loaded with siRNA formed by the carriers of Examples 1 and 4-6 showed reduced toxicity to HUVEC cells, with no significant difference (p>0.05), indicating that the polyamino acid carrier of the present invention has very low toxicity.

[0094] Experiment Example 4: Detection of Lysosomal Escape Ability (1) Test method ① Preparation of test samples: Dissolve the polyamino acid carrier obtained in Example 1 in PBS to prepare a PBS solution containing 1 mg / mL of polyamino acid. Take 10 μL of the above PBS solution and add 230 pmol of siRNA (i.e., siCamk2g) and mix for 1 min to form a nucleic acid drug delivery system based on polyamino acid carrier, which is the test sample.

[0095] ② Cell treatment and testing: according to 1×10 5 293T cells (purchased from Heyuan Biotechnology (Shanghai) Co., Ltd.) genetically modified to express Gal8 protein in logarithmic growth phase were seeded in eight-well chambers. After cell attachment, the complete culture medium was replaced with serum-free medium to resuspend the cells. The cell suspension was randomly divided into two groups: siRNA group and LAD@siRNA group, with three replicates per group. 200 μL of cell suspension was added to each well. 4 μL of the sample to be tested was added to the LAD@siRNA group cells; 2 μL of DEPC (diethyl pyrocarbonate) aqueous solution containing 230 pmol of siRNA was added to the siRNA group cells. Both groups were incubated for 8 h. After removing the culture medium, the cells were washed three times with PBS solution. Finally, spot fluorescence within the cells was captured using a laser confocal microscope.

[0096] See results Figure 6As shown, the average fluorescence intensity of the punctate fluorescence was 0.06±0.02, while that of the LAD@siRNA group was 15.75±3.39, with a p<0.001 between the two groups. Compared to the siRNA group, the LAD@siRNA group provided by this invention has good lysosomal escape ability, which is beneficial for the subsequent functioning of the target gene.

[0097] Experiment Example 5: Test of gene silencing efficiency in reprogrammed macrophages (1) Test method The silencing efficiency of the polyamino acid carriers prepared in Examples 1-6 and Comparative Examples 2-8 was tested.

[0098] ① Preparation of test samples: Dissolve each group of polyamino acid carriers in PBS solution to prepare a 1 mg / mL polyamino acid carrier PBS solution. Add 460 pmol of nucleic acid drug siRNA (i.e., siCamk2g) to 20 μL of the 1 mg / mL polyamino acid carrier PBS solution and mix for 1 min to obtain the nucleic acid drug delivery system based on the polyamino acid carrier.

[0099] ② Cell treatment: according to 7×10 5 Raw 264.7 cells in logarithmic growth phase were seeded in 6-well plates and allowed to adhere. The cells were resuspended in serum-free medium, and the cell suspension was randomly divided into 14 groups: experimental groups 1-13 and a PBS group, with three replicates per group. 2 mL of cell suspension was added to each well. For experimental groups 1-13, 40 μL of the sample to be tested was added to the cell suspension, while the PBS group received the same volume of PBS solution. The cells were incubated for 8 h. After incubation, the supernatant was removed and replaced with complete medium, and the cells were cultured for another 16 h. The cells were then collected.

[0100] ③ Test: Before collecting the cultured adherent cell samples, the culture medium needs to be removed. Add 1 mL of TRIzol to lyse the cells, and repeatedly pipette until there is no obvious precipitate in the lysate. Place on ice for 5 min. Add 200 μL of chloroform to 1 mL of sample; vortex for 15 s, and place on ice for 3 min. Centrifuge at 12000 g for 15 min at 4 ℃, and carefully remove the sample and place it on a test tube rack. Take 500 μL of the supernatant into a new centrifuge tube, and mark the tube cap. Add 500 μL of isopropanol; mix gently, and place on ice for 10 min; centrifuge at 12000 g for 10 min at 4 ℃; slowly discard the supernatant, add 1 mL of 75% ethanol; gently tap the precipitate, centrifuge at 7500 g for 5 min at 4 ℃, and slowly discard the supernatant; then briefly centrifuge at 7500 g for 30 s; try to remove as much residual liquid as possible; invert the centrifuge tube on a clean PE glove and let it cool until the edge of the precipitate becomes transparent. Add 40 μL of RNase-Free H2O; incubate at room temperature for 10 min; dissolve the RNA precipitate, and finally mix thoroughly by repeatedly pipetting several times; assess the quality of the extracted RNA by agarose gel electrophoresis, using three ribosomal RNA bands (28 S, 18 S, 5 S) to evaluate RNA quality. Take 1.5 μL of total RNA and determine the RNA concentration of the sample using a micro-spectrophotometer. Then label the tube wall and cap, and place... Store at 80℃ for later use. Perform reverse transcription according to the RT-qPCR kit instructions to synthesize cDNA. Finally, perform qPCR experiments according to the qPCR kit instructions to detect the intracellular target mRNA content. The CT method is used to calculate the siRNA silencing efficiency, and the calculation formula is as follows: CT 样本 =CT 靶基因 -CT 内参基因 CT 对照 =CT 靶基因 -CT 内参基因 CT= CT 样本 - CT 对照 Target gene expression = 2- CT (2) Test results The results are shown in Table 1 below: Table 1. Effects of different lysine polymerization degrees, arginine grafting rates, and DMA modification ratios on siRNA silencing efficiency.

[0101] As can be seen from the table above, compared with Comparative Examples 2-8, the silencing efficiency of the polyamino acid carriers prepared in Examples 1 and 4-6 of the present invention is significantly improved, especially Example 7.

[0102] The results are as follows Figure 7 As shown, the polyamino acid nucleic acid delivery vector of Example 1 of the present invention has a high gene silencing efficiency and can achieve protein expression silencing in macrophages.

[0103] Table 2. Effects of different small molecule anhydrides on siRNA silencing efficiency

[0104] As can be seen from the table above, compared with Examples 2 and 3, the silencing efficiency of the polyamino acid carrier prepared in Example 1 of the present invention is significantly improved.

[0105] Experiment Example 6: Inhibition of GFP protein expression in liver cancer cells (1) Test method ① Preparation of test samples: Dissolve each group of polyamino acid carriers in PBS solution to prepare a 1 mg / mL polyamino acid carrier PBS solution. Take 20 μL of the 1 mg / mL polyamino acid carrier PBS solution and add 460 pmol of nucleic acid drug siRNA (i.e., siGFP, manufacturer: Guangzhou Ruibo Biotechnology Co., Ltd.; sequence (5'-3') as follows: 5'-GCACCAUCUUCUUCAAGGA dTdT-3'). Mix for 1 min to obtain a nucleic acid drug delivery system based on polyamino acid carriers.

[0106] ② Cell treatment and testing: according to 1.2×10 5 HepG 2-GFP cells (human liver cancer cells highly expressing green fluorescent protein, purchased from Heyuan Biotechnology (Shanghai) Co., Ltd.) were seeded in 12-well plates and allowed to adhere to the wells. The cells were resuspended in serum-free medium after replacing the complete culture medium. The cell suspension was randomly divided into two groups: the siGFP group and the LAD@siGFP group, with three replicates per group. 1 mL of cell suspension was added to each well. The LAD@siGFP group had 20 μL of the sample to be tested added to the cell suspension, while the siGFP group had an equal volume of PBS containing 2g of siCamk (460 pmol). Cells were incubated for 8 h. After incubation, the supernatant was removed and replaced with complete culture medium, and the cells were cultured for another 16 h. The cells were then collected. The fluorescence intensity of GFP in HepG 2-GFP cells was captured using a fluorescence microscope.

[0107] (2) Test results The results are as follows Figure 8 As shown, the average fluorescence intensity of the punctate fluorescence was calculated, and the fluorescence intensity of the siRNA group was (1.6±0.43)×10⁻⁶. 8 The fluorescence intensity of the LAD@siRNA group was (4.3±0.29)×10⁻⁶. 8 The p-value between the two groups was <0.0001. Compared to the siRNA group, the polyamino acid nucleic acid nanoparticles of this invention can effectively silence the expression of GFP protein in cells, and may have the potential to prevent or treat liver cancer.

[0108] Experimental Example 7: Evaluation of the Nitric Oxide Generation Efficiency of a Polyamino Acid Carrier-Based Nucleic Acid Drug Delivery System in Macrophages 1. Preparation of the test drug The LAD vector prepared in Example 1, the LAM vector prepared in Example 2, and the LAS vector prepared in Example 3 were assembled with control siRNA (hereinafter referred to as "scr", purchased from Guangzhou Ruibo Biotechnology Co., Ltd., sequence (5'-3') as follows: 5'-UUGGGAAAAAGUUGAGUGGUU-3') to form nucleic acid drug delivery systems based on polyamino acid carriers: LAD@scr, LAM@scr, and LAS@scr. Specifically, the polyamino acid carrier was dissolved in water to prepare an aqueous solution with a concentration of 1 mg / mL. 20 μL of the polyamino acid carrier aqueous solution was added to 460 pmol of scr, and mixed for 1 min to obtain the nucleic acid drug delivery systems LAD@scr, LAM@scr, and LAS@scr, respectively.

[0109] 2. Cell Culture and Drug Administration According to 5×10 4 Raw 264.7 cells were seeded in 8-well cell culture chambers at a density of cells / well. After cell attachment, the cells were resuspended in cell culture medium and the cell suspension was randomly divided into 6 groups: PBS group, LPS group, LPS+Arg group, LPS+LAS@scr group, LPS+LAM@scr group and LPS+LAD@scr group, with 3 parallel wells for each group and 200 μL per well.

[0110] PBS group: Add 2 μL of PBS buffer to the cells; LPS group: 0.2 μL of 1 mg / mL LPS (lipopolysaccharide) was added to the cells; LPS+Arg group: 2 μL of arginine solution (solvent: PBS, concentration 0.7 mg / mL) was added to the cells, followed by 0.2 μL of 1 mg / mL LPS; LPS+LAS@scr group: 4 μL of LAS@scr, a nucleic acid drug delivery system based on polyamino acid carriers, was added to the cells, followed by 0.2 μL of 1 mg / mL LPS.

[0111] LPS+LAM@scr group: 4 μL of LAM@scr, a nucleic acid drug delivery system based on polyamino acid carriers, was added to the cells, followed by 0.2 μL of 1 mg / mL LPS.

[0112] LPS+LAD@scr group: 4 μL of LAD@scr, a nucleic acid drug delivery system based on polyamino acid carriers, was added to the cells, followed by 0.2 μL of 1 mg / mL LPS.

[0113] 3. Detect the efficiency of nitric oxide production in macrophages by the polyamino acid carrier-based nucleic acid drug delivery system. All groups were incubated at 37℃ for 24 h. 2 μL of LDAF-FM DA (green fluorescence, nitric oxide indicator, Beyotime, S0019S) was added to each well and incubated at 37℃ for 0.5 h. After washing with PBS, the samples were stained with 1 µg of DAPI (nuclear dye) for 10 min, washed with PBS, and then observed using a confocal microscope.

[0114] 4. Test Results The results are as follows Figure 9 As shown, the fluorescence intensity of the PBS group was (1.52 ± 0.15) × 10⁻⁶. 3 The fluorescence intensity of the LPS group was (1.47±0.14)×10⁻⁶. 3 The fluorescence intensity of the LPS+Arg group was (6.01 ± 0.54) × 10⁻⁶. 3 The fluorescence intensity of the LPS+LAS@scr group was (1.53 ± 0.01) × 10⁻⁶. 3 The fluorescence intensity of the LPS+LAM@scr group was (2.5 ± 0.58) × 10⁻⁶. 3 The fluorescence intensity of the LPS+LAD@scr group was (6.16 ± 1.35) × 10⁻⁶. 3Compared to the PBS and LPS groups, the fluorescence intensity of the LPS+LAD@scr group was significantly increased (p<0.0001); while compared to the LPS group, the fluorescence intensity of the LPS+LAM@scr and LPS+LAS@scr groups was slightly increased, but the difference was not statistically significant. The results indicate that the LAD@scr treatment group can more effectively react with inducible nitric oxide synthase (iNOS) in macrophages to produce nitric oxide.

[0115] Experimental Example 8: Gene Silencing Efficiency 1. Cell Culture and Drug Delivery According to 7×10 5 Raw 264.7 cells were seeded in 6-well plates at a density of cells / well. After cell attachment, the complete culture medium was replaced with serum-free medium to resuspend the cells. The cell suspension was randomly divided into two groups, with 3 parallel wells per group and 2 mL per well. 40 μL of LAD@siCamk2g and Lipo@siCamk2g prepared in Example 4 and Comparative Example 1, respectively, were added to each group. The cells were incubated at 37°C for 8 h. After incubation, the supernatant was removed and replaced with 2 mL of complete culture medium per well. The cells were cultured at 37°C for another 40 h. The culture medium was then discarded, and the cells were collected.

[0116] 2. Western blot test Add an appropriate amount of lysis buffer to the wells and pipette several times to remove the cells. Centrifuge at 12000 rpm at 4℃ for 10 min. After centrifugation, aspirate the protein and place it in an EP tube. Quantify the protein using a BCA kit (30 μg protein loading amount). Add loading buffer and heat in a metal bath at 100℃ for 10 min to denature the protein and further separate and degrade the DNA bound to the protein. The tube should no longer be viscous after heating.

[0117] Prepare an 8% polyacrylamide gel and place it in the assembled electrophoresis tank. Add the sample sequentially to the gel wells, pour in the electrophoresis buffer, and begin electrophoresis. Electrophoresis is performed at a constant voltage of 80 V for 30 min, followed by a constant voltage of 120 V for 1.5 h. After electrophoresis, remove the gel and place it in the assembled transfer system for transfer. Transfer is performed at a constant current of 200 mA for 40 min. After transfer, block with 5% skim milk for 2 h. After blocking, wash with TBST, then add primary antibodies for GAPDH and CaMKIIγ protein and incubate overnight at 4°C. After primary antibody incubation, wash with TBST, then add secondary antibody and incubate at room temperature for 2 h. After secondary antibody incubation, develop with a developing agent.

[0118] 3. Test Results The results are as follows Figure 10As shown in the results, both Lipo@siCamk2g and LAD@siCamk2g drug delivery systems can efficiently silence the expression of CaMKIIγ protein in macrophages.

[0119] Experimental Example 9: Cell Migration 1. Testing Method Use a marker to draw lines on the back of the 6-well plate for positioning, and then seed 6×10 wells in the plate. 5 HUVEC cells per well; then seed 1×102 cells. 5 264.7 cells / well Raw; after the cells adhered, a 200 μL pipette tip was used to make a mark in the well plate, and then the cells that had fallen off were washed off with PBS. The cells were then resuspended in fresh complete culture medium. The cell suspensions were randomly divided into PBS group, LPS group, LPS+Arg group and LPS+LAD@siCamk2g group, with 3 parallel wells for each group and 2 mL per well.

[0120] PBS group: Add 20 μL of PBS buffer to the cells; LPS group: 2 μL of 1 mg / mL LPS was added to the cells; LPS+Arg group: 20 μL of arginine solution (solvent: PBS, concentration 0.7 mg / mL) was added to the cells, followed by 2 μL of 1 mg / mL LPS; LPS+LAD@siCamk2g group: 40 μL of LAD@siCamk2g prepared in Example 4 was added to the cells, followed by 2 μL of LPS at a concentration of 1 mg / mL.

[0121] All groups were incubated at 37℃ for 24 hours, and scratches were photographed at the same location at 0 h, 8 h, and 24 h.

[0122] 2. Test Results The results are as follows Figure 11 As shown, the cell migration area in the PBS group was (6.7±0.39)×10⁻⁶. 4 The cell migration area in the LPS group was (8.42±0.33)×10 pixels. 4 The cell migration area in the LPS+Arg group was (11.45±1.35)×10 pixels. 4 The cell migration area in the LPS+LAD@siCamk2g group was (12.36±1.85)×10 pixels. 4pixels. The p-value between the PBS group and the LPS+LAD@siCamk2g group was <0.0001. The results show that compared to the PBS group, the migration area of ​​cells in the LPS+Arg group and the LPS+LAD@siCamk2g group was significantly increased (p<0.0001), indicating that significant cell migration occurred in both groups. This demonstrates that the nucleic acid drug delivery system based on polyamino acid carriers can efficiently generate nitric oxide gas intracellularly to promote cell migration.

[0123] Example 10: Investigation of the targeted accumulation ability of a nucleic acid drug delivery system based on a polyamino acid carrier in mice. 1. Preparation of the sample to be tested ① The polyamino acid carrier prepared in Example 1 was dissolved in water to prepare a polyamino acid carrier aqueous solution with a concentration of 1 mg / mL. 62.5 μL of the above polyamino acid carrier aqueous solution was mixed with 62.5 μL of Cyanine 5-labeled siCamk2g (purchased from Guangzhou Ruibo Biotechnology Co., Ltd., solvent: DEPC water, molar concentration of 230 pmol / μL) for 30s-2min (1min in this experimental example) to obtain Cyanine 5-labeled LAD@siCamk2g.

[0124] ② Take 31.25 μL of liposomes (specific name: Lipofectamine2000, purchased from Thermo Fisher Scientific, model Lipo2000) and 31.25 μL of PBS solution and incubate at room temperature for 5 minutes. Then mix it with 62.5 μL of Cyanine 5-labeled siCamk2g (purchased from Guangzhou Ruibo Biotechnology Co., Ltd., solvent: DEPC water, molar concentration: 230 pmol / μL) for 10 minutes to obtain Cyanine 5-labeled Lipo@siCamk2g.

[0125] 2. Animal grouping, drug administration and testing Apoe at 12 weeks old - / - Six mice (model group) were used. Three 9-week-old wild-type C57BL / 6J mice served as the control group (WT). The model group was randomly divided into two subgroups of three mice each. The model groups received 125 μL of Cyanine 5-labeled LAD@siCamk2g and Cyanine 5-labeled Lipo@siCamk2g via tail vein injection, respectively. The control group received 125 μL of Cyanine 5-labeled LAD@siCamk2g via tail vein injection. All three groups were fed for another 24 h. The aorta of the mice was removed and imaged using a small animal in vivo imaging system.

[0126] 3. Test Results The results are as follows Figure 12 As shown, the fluorescence intensity of the WT group was (1.21±0.86)×10⁻⁶. 6 The fluorescence intensity of the Lipo@siCamk2g group was (7.63±3.21)×10⁻⁶. 6 The fluorescence intensity of the LAD@siCamk2g group was (18.07±4.88)×10⁻⁶. 6 The p-value between the Lipo@siCamk2g group and the LAD@siCamk2g group was <0.01. Compared with the WT group and the model treatment group injected with Lipo@siCamk2g, the fluorescence intensity of the mouse aorta was significantly increased after the LAD@siCamk2g provided by this invention was injected into the model group (p<0.01), indicating that the nucleic acid drug delivery system based on polyamino acid carrier provided by this invention can accumulate more precisely at the lesion plaque without affecting normal tissue cells, and shows excellent targeting ability for atherosclerotic plaques.

[0127] Experiment Example 11: Investigation of the therapeutic effect of a nucleic acid drug delivery system based on polyamino acid carriers on atherosclerotic mice. 1. Animal grouping and administration Apoe at 12 weeks old - / - Nine mice (model group) were randomly divided into three groups of three mice each: PBS group, LAD@siCamk2g treatment group, and Lipo@siCamk2g treatment group.

[0128] LAD@siCamk2g treatment group: 125 μL of LAD@siCamk2g prepared in Example 4 was injected into mice via tail vein injection. Lipo@siCamk2g treatment group: 125 μL of Lipo@siCamk2g prepared in Comparative Example 1 was injected into mice via tail vein injection. Injections were given twice weekly for a total of 4 weeks. The PBS group received parallel injections of an equal volume of PBS solution.

[0129] 2. Testing Methods After treatment, the aortas of mice in each group were removed, washed twice with PBS, and the vessels were carefully longitudinally dissected along the vessel wall using dissecting scissors. The dissected vessels were then immersed in Oil Red O staining solution at 37°C for 60 min; after removal, they were differentiated with 75% ethanol until the fatty plaques inside the lumen turned orange-red or bright red, while other areas remained nearly colorless. The vessels were then washed twice with distilled water. The staining results were photographed.

[0130] 3. Test Results The results are as follows Figure 13As shown, the percentage of Oil Red O area in the PBS group was 61.79±18.73% of the total area, the percentage in the Lipo@siCamk2g group was 27.73±11.46%, and the percentage in the LAD@siCamk2g group was 8.47±3.17%. The p-value between the PBS group and the LAD@siCamk2g group was <0.05. Compared to the PBS group and the Lipo@siCamk2g treatment group, the LAD@siCamk2g treatment group showed a significantly smaller intraluminal fatty plaque area (red area) after LAD@siCamk2g injection (p<0.05), indicating a significantly higher therapeutic effect than the other two groups. This demonstrates that in situ gene-edited macrophages combined with nitric oxide gas therapy can achieve good therapeutic effects in treating atherosclerosis.

[0131] Example 12: Investigation into the in situ editing effect of a nucleic acid drug delivery system based on polyamino acid carriers on macrophages in vivo. 1. Animal grouping and administration Apoe at 12 weeks old - / - Nine mice (model group) were randomly divided into three groups of three mice each: PBS group, LAD@siCamk2g treatment group, and Lipo@siCamk2g treatment group.

[0132] LAD@siCamk2g treatment group: 125 μL of LAD@siCamk2g prepared in Example 4 was injected into mice via tail vein injection. Lipo@siCamk2g treatment group: 125 μL of Lipo@siCamk2g prepared in Comparative Example 1 was injected into mice via tail vein injection. Injections were given twice weekly for a total of 4 weeks. The PBS group received the same volume of PBS solution in parallel injections.

[0133] 2. Testing Methods After treatment, the aortas of mice in each group were removed, embedded in OTC immunofluorescence, and then frozen sections were prepared. The frozen sections were stained with TUNEL (red) and F8 / 40 (green) immunofluorescence. The staining results were observed using a slide scanner. The fluorescence staining imaging results are shown below. Figure 14 As shown. The white arrows indicate the co-localization of macrophages (green) and apoptotic cells (red).

[0134] 3. Test Results The results are as follows Figure 14 As shown, the colocalization fluorescence intensity of the PBS group was (3.95±0.68)×10⁻⁶. 5 The colocalization fluorescence intensity of the Lipo@siCamk2g group was (6.44±0.7)×10⁻⁶. 5The colocalization fluorescence intensity of the LAD@siCamk2g group was (14.65±2.85)×10⁻⁶. 5 The p-value between the Lipo@siCamk2g group and the LAD@siCamk2g group was <0.001. Compared with the PBS group and the Lipo@siCamk2g treatment group, the LAD@siCamk2g treatment group showed a significantly higher degree of co-localization (yellow) of macrophages (green) and apoptotic cells (red) after injection of LAD@siCamk2g (p<0.001). This indicates that it can significantly genetically modify macrophages, restore their phagocytic capacity, clear apoptotic cells, and its therapeutic effect is significantly higher than the other two groups.

[0135] Example 13: Investigation into the effect of a nucleic acid drug delivery system based on polyamino acid carriers on the repair of the endothelial barrier in vivo. Evans blue (EB) dye is commonly used to test vascular endothelial cell barrier permeability. Under physiological conditions, albumin cannot permeate the endothelium; however, under pathological conditions, dysfunction of intercellular junction proteins leads to increased barrier permeability, in which case Evans blue-albumin leakage can be detected in a monolayer endothelial cell model.

[0136] 1. Animal grouping and administration Apoe at 12 weeks old - / - Nine mice (model group) were randomly divided into three groups of three mice each: PBS group, LAD@siCamk2g treatment group, and Lipo@siCamk2g treatment group.

[0137] LAD@siCamk2g treatment group: 125 μL of LAD@siCamk2g prepared in Example 4 was injected into mice via tail vein injection. Lipo@siCamk2g treatment group: 125 μL of Lipo@siCamk2g prepared in Comparative Example 1 was injected into mice via tail vein injection. Injections were given twice weekly for a total of 4 weeks. The PBS group received the same volume of PBS solution in parallel injections.

[0138] 2. Testing Methods After treatment, 2% Evans blue dye was injected into mice in each group via the tail vein. Thirty minutes after injection, the mice were perfused with 4% paraformaldehyde solution. The aorta was then harvested and longitudinally opened for photographing and staining intensity analysis. The heart was then embedded in OTC (over-the-counter) material, and frozen sectioning was used to extract sections from the aortic root for staining observation.

[0139] 3. Test Results The results are as follows Figure 15As shown, the blood vessels of atherosclerotic model mice treated with PBS were significantly stained by Evans blue. The staining was lighter in the Lipo@siCamk2g treatment group, and the staining was the least in the LAD@siCamk2g treatment group. Therefore, compared with the Lipo@siCamk2g group (i.e., without nitric oxide gas treatment), the LAD@siCamk2g group, through gene editing combined with nitric oxide gas treatment, can significantly promote the proliferation and migration of vascular endothelial cells (p<0.05), ultimately achieving the repair of the vascular endothelial barrier.

[0140] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A polyamino acid carrier, characterized in that, The polyamino acid carrier has the structural formula shown in formula (I): （I）； Where m and n are the number-average aggregation degree, y is selected from integers 15-16; n is 10-500, m is 10-200, and n≥m; R1 is selected from .

2. A method for preparing the polyamino acid carrier according to claim 1, characterized in that, Includes the following steps: (1) Lysine with an amino acid protecting group reacts with triphosgene or phosgene in the presence of a solvent to prepare L-lysine-N-carboxylated intracyclic anhydride with an amino acid protecting group; (2) The L-lysine-N-carboxy ring anhydride with amino acid protecting group is ring-opened polymerized with the monomer shown in formula (II) in the presence of solvent, and then the amino acid protecting group is removed to obtain polylysine with alkyl chain. (3) Arginine with an amino acid protecting group is used as a graft monomer and reacted with an amidation catalyst in the presence of a solvent. The reaction product is then grafted with polylysine with an alkyl chain in the presence of an acid-binding agent, and then the amino acid protecting group is removed. (4) Modify the polymer product obtained in step (3) by removing the amino acid protecting group with a small molecule acid anhydride to obtain a polyamino acid carrier; (II) Wherein, y is selected from an integer between 15 and 16; the small molecule anhydride is selected from dimethylmaleic anhydride (DMA).

3. The method for preparing the polyamino acid carrier according to claim 2, characterized in that, n is 60-120, the grafting rate of arginine is greater than 0 and less than or equal to 100%, and the modification ratio of DMA is 100%.

4. The method for preparing the polyamino acid carrier according to claim 3, characterized in that, The grafting rate of arginine is 25-100%, and the modification ratio of DMA is 100%.

5. The method for preparing the polyamino acid carrier according to claim 4, characterized in that, n is 90, the grafting rate of arginine is 50%, and the modification ratio of DMA is 100%.

6. The method for preparing the polyamino acid carrier according to claim 2, characterized in that, In step (1), the reaction temperature is 50-60℃; and / or the solvent is tetrahydrofuran; and / or the lysine with the amino acid protecting group is selected from one or both of Nε-Boc-L-lysine and Nε-benzyloxycarbonyl-L-lysine. In step (2), the reaction temperature is 30-40℃; the time is 48-96 h; and / or the solvent is DMF; and / or the amino acid protecting group is removed by hydrolysis in an ice-water bath using a solution of trifluoroacetic acid and acetic acid containing hydrogen bromide. In step (3), the amidation catalyst includes EDCI and HOBT; and / or, the solvent is DMF; and / or, the arginine with the amino acid protecting group is selected from Nα-Boc-Nω-(2,2,4,6,7-pentamethyl-dihydrobenzofuran-5-sulfonyl)-L-arginine; and / or, the acid binding agent is selected from N,N-diisopropylethylamine; and / or, the graft polymerization reaction is carried out at room temperature for a reaction time of 24-96 h; and / or, the amino acid protecting group is removed by hydrolysis with trifluoroacetic acid at room temperature. In step (4), a solution containing small molecule acid anhydride is added dropwise to an alkaline solution containing the polymer product from which the amino acid protecting groups have been removed in step (3), and the reaction is carried out for 4-6 hours.

7. A nucleic acid drug delivery system based on a polyamino acid carrier, characterized in that, The polyamino acid carrier includes the polyamino acid carrier as described in claim 1 or the polyamino acid carrier prepared by any of the methods described in claims 2-6, and also includes nucleic acid drugs.

8. The nucleic acid drug delivery system based on a polyamino acid carrier according to claim 7, characterized in that, The nucleic acid drug is siRNA.

9. The nucleic acid drug delivery system based on a polyamino acid carrier according to claim 7, characterized in that, The nucleic acid drug is selected from one or more of siCamk2g, siCCR2, siEpsin1 / 2, siScr, and siGFP.

10. The nucleic acid drug delivery system based on a polyamino acid carrier according to claim 7, characterized in that, The molar ratio between nitrogen in the polyamino acid carrier and phosphorus in the nucleic acid drug is 0.25-16:

1.

11. The nucleic acid drug delivery system based on a polyamino acid carrier according to claim 10, characterized in that, The molar ratio between nitrogen in the polyamino acid carrier and phosphorus in the nucleic acid drug is 4:

1.

12. The nucleic acid drug delivery system based on a polyamino acid carrier according to claim 7, characterized in that, The average particle size of the nucleic acid drug delivery system based on polyamino acid carriers is 40-80 nm.

13. The nucleic acid drug delivery system based on a polyamino acid carrier according to claim 12, characterized in that, The average particle size of the nucleic acid drug delivery system based on polyamino acid carriers is 50-60 nm.

14. A method for preparing a nucleic acid drug delivery system based on a polyamino acid carrier as described in any one of claims 7-13, characterized in that, include: It is prepared by mixing polyamino acid carriers and nucleic acid drugs in the presence of aqueous solution.

15. The method for preparing a nucleic acid drug delivery system based on a polyamino acid carrier according to claim 14, characterized in that, The ratio of the mass of the polyamino acid carrier to the molar amount of the nucleic acid drug is 0.25 µg-0.03 mg: 80-500 pmol.

16. The method for preparing a nucleic acid drug delivery system based on a polyamino acid carrier according to claim 14, characterized in that, The mixing time is 30 s to 5 min.

17. The use of the nucleic acid drug delivery system based on polyamino acid carriers as described in any one of claims 7-13 or the nucleic acid drug delivery system based on polyamino acid carriers prepared by any one of the preparation methods described in claims 14-16 in the preparation of drugs for the prevention or treatment of liver cancer and atherosclerosis.

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