A nanogel, a nanogel-nucleic acid complex, a preparation method and application thereof

Abstract

The present application belongs to the field of biotechnology, and particularly relates to a nanogel, a nanogel-nucleic acid complex, a preparation method and application thereof. The nanogel has a controllable core-shell structure or no core-shell structure, and the material forming the core structure includes a compound of formula I, wherein the definitions of each group are as described herein. The material forming the shell structure of the nanogel includes a neutral polymer and an amphoteric polymer. The nanogel has a uniform particle size, and compared with bPEI 25k and mainstream commercial reagents which are widely used as performance benchmarks in the field of gene transfection, the nanogel can significantly reduce cytotoxicity while improving the transfection efficiency of various nucleic acids.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a nanogel, a nanogel-nucleic acid complex, its preparation method, and its application. Background Technology

[0002] Gene therapy refers to the process of introducing exogenous nucleic acids into target cells to replace or correct the expression of defective genes, or to induce cells to express specific functional proteins, thereby regulating their biological behavior and ultimately achieving the goal of treating diseases. This technology has shown great potential in the treatment of various diseases such as hereditary diseases, malignant tumors, diabetes, and Alzheimer's disease, as well as in vaccine development. However, nucleic acid drugs typically have characteristics such as large molecular weight, negative charge, and strong hydrophilicity, making it difficult to effectively cross the cell membrane barrier. Therefore, developing safe and efficient delivery vectors is crucial for their clinical application.

[0003] Currently, commonly used nucleic acid delivery systems mainly fall into two categories: viral vectors and non-viral vectors. While viral vectors offer high transfection efficiency, their high production costs and potential immunogenicity and carcinogenic risks limit their widespread application. Non-viral vectors, such as cationic liposomes and cationic polymers, have become the main direction for the development of commercial transfection reagents due to their advantages such as good biocompatibility, ease of large-scale production, and functional modification. However, existing non-viral vectors generally face problems such as limited delivery efficiency, high cytotoxicity, and complex operating procedures, and their delivery effects vary significantly for different types of nucleic acid drugs. To reduce the cytotoxicity of non-viral vectors, common strategies include using biodegradable materials, introducing hydrophilic modifications (such as polyethylene glycolation), optimizing the vector structure and composition to improve biocompatibility, and developing stimulus-responsive release systems. However, these methods generally struggle to balance toxicity and nucleic acid delivery efficiency. There is an urgent need in the field to develop novel nucleic acid delivery systems that combine high delivery efficiency, low toxicity, ease of operation, and versatility. Summary of the Invention

[0004] In response to the aforementioned problems in existing technologies, this invention proposes a non-viral vector that can significantly reduce cytotoxicity and enhance gene silencing efficiency, thereby addressing the common challenges faced by existing delivery systems in terms of safety and effectiveness.

[0005] Specifically, one aspect of the present invention provides a nanogel comprising a phosphonic cationic polymer and an optional second polymer; wherein the monomer of the phosphonic cationic polymer comprises one or more compounds of formula I; , In Formula I, R1 is selected from hydrogen atoms or methyl groups; R2 is selected from -C(=O)-NH-, -C(=O)-O-, or -C6H4-; R3 is selected from -P+ -(CH3)3、-P + -(CH2CH3)3、-P + -(CH2CH2CH3)3 or -P + -(CH2CH2CH2CH3)3; n is 1, 2 or 3.

[0006] In one or more embodiments, the nanogel includes a nanogel core and a shell coating the outer surface of the nanogel core, wherein the nanogel core is a phosphonic cationic polymer and the shell is a second polymer; or, the nanogel does not have a shell.

[0007] In one or more embodiments, the monomer of the phosphonic cationic polymer further includes a crosslinking agent.

[0008] In one or more embodiments, the crosslinking agent is one or more selected from diallyl disulfide, N,N'-bis(acryloyl)cysteine, N,N'-(1,2-dihydroxyethylene)bisacrylamide glycol dimethacrylate and 2,2-dithiodiethanol diacrylate.

[0009] In one or more embodiments, the molar ratio of the crosslinking agent to the compound of formula I is (0.01:1) to (0.5:1).

[0010] In one or more embodiments, the molar ratio of the crosslinking agent to the compound of formula I is (0.1:1) to (0.5:1).

[0011] In one or more embodiments, the second polymer is a neutral polymer or an amphoteric polymer.

[0012] In one or more embodiments, the second polymer is one or more selected from polyethylene glycol, polyethylene, polytetrafluoroethylene, poly-2-methacryloyloxyethyl phosphocholine, polymethacryloylethyl sulfobetaine, poly-3-[[2-(methacryloyloxy)ethyl]dimethylammonium]propionate and poly-3-[(3-acrylamidopropyl)dimethylammonium]propionate.

[0013] In one or more embodiments, the second polymer is modified with a chain transfer agent.

[0014] In one or more embodiments, the chain transfer agent is selected from one or more of methyl (phenyl)aminodithiocarbamate, S-cyanomethyl-S-dodecyl trithiocarbonate, cyanoisopropyl dithiobenzoate, 4-cyano-4-(thiobenzoyl)valerate, S-(2-cyano-2-propyl)-S-dodecyl trithiocarbonyl ester, 4-cyano-4-[[(dodecylthio)thionylmethyl]thio]valerate, and 2-(dodecyl trithiocarbonate)-2-methylpropionic acid.

[0015] In one or more embodiments, the molar ratio of the chain transfer agent to the second polymer is (1:1) to (2:1).

[0016] In one or more embodiments, the second polymer is modified with a targeting molecule, said targeting molecule being folic acid, galactose, sodium alendronate, transferrin, hyaluronic acid, peptide, mannose, or biotin.

[0017] In one or more embodiments, the molar ratio of the target molecule to the second polymer is (0.02:1) to (1:1).

[0018] In one or more embodiments, the hydrodynamic diameter of the nanogel in water, as measured by dynamic light scattering, is 40-500 nm.

[0019] In one or more embodiments, the PDI of the nanogel is 0.02-0.3.

[0020] In one or more embodiments, the mass ratio of the phosphonic cationic polymer to the second polymer is (2:1) to (20:1).

[0021] In one or more embodiments, in Formula I, R1 is selected from hydrogen atoms, R2 is selected from -C6H4-, and R3 is selected from -P + -(CH3)3、-P + -(CH2CH3)3、-P + -(CH2CH2CH3)3 or -P + -(CH2CH2CH2CH3)3; n is 1, 2 or 3.

[0022] In one or more embodiments, the compound of Formula I is selected from one or more of trimethyl-(4-vinylbenzyl)phosphonium chloride, triethyl-(4-vinylbenzyl)phosphonium chloride, tri-n-propyl-(4-vinylbenzyl)phosphonium chloride, and tri-n-butyl-(4-vinylbenzyl)phosphonium chloride.

[0023] Another aspect of the present invention provides a method for preparing the nanogel described in any embodiment of the present invention, the method comprising: using an anionic polymer as a template and reacting it with one or more compounds of formula I ( The crosslinking agent, initiator, and optional second polymer are mixed, and the pH of the solution is adjusted to electroneutrality; polymerization is initiated to obtain a nanogel precursor; the anionic polymer in the nanogel precursor is removed to obtain the nanogel; in Formula I, R1, R2, R3, and n are defined as defined in any embodiment of the present invention.

[0024] In one or more embodiments, the method includes: (1) using an anionic polymer as a template and reacting it with one or more compounds of formula I ( (1) Mix the crosslinking agent, the second polymer and the initiator, and adjust the pH of the mixed solution to be electrically neutral; (2) Initiate polymerization to obtain a nanogel precursor; (3) Remove the anionic polymer in the nanogel precursor to obtain the nanogel; In Formula I, R1, R2, R3 and n are defined as defined in any embodiment of the present invention.

[0025] In one or more embodiments, the method includes: (1) using an anionic polymer as a template and reacting it with one or more compounds of formula I ( (1) Mix the crosslinking agent and the initiator, and adjust the pH of the mixed solution to be electrically neutral; (2) Initiate polymerization to obtain a nanogel precursor; (3) Remove the anionic polymer in the nanogel precursor to obtain the nanogel; In Formula I, R1, R2, R3 and n are defined as defined in any embodiment of the present invention.

[0026] In one or more embodiments, the anionic polymer is selected from one or more of polyacrylic acid, sodium poly(2-acrylamido-2-methylpropanesulfonate), sodium poly(p-styrene sulfonate), poly(dimethylaminoethyl methacrylate), poly(methacryloyloxyethyltrimethylammonium chloride), and poly(2-aminoethyl methacrylate).

[0027] In one or more embodiments, the initiator includes a photoinitiator and / or a thermal initiator.

[0028] In one or more embodiments, the photoinitiator is selected from one or more of 2-hydroxy-2-methyl-1-phenyl-1-propanone, 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylphenylpropanone, and phenyl-2,4,6-trimethylbenzoylphosphinic acid lithium, and the thermal initiator is selected from persulfate and / or water-soluble azo compounds.

[0029] In one or more embodiments, in step (1), the charge concentration of the Formula I compound is 5-500 mM, and the charge concentration of the Formula I compound and the anionic polymer are the same.

[0030] In one or more embodiments, in step (1), the amount of the crosslinking agent is 1-50% of the charge concentration of the compound of formula I.

[0031] In one or more embodiments, in step (1), the amount of the second polymer used is 0.1-5% of the charge concentration of the compound of formula I.

[0032] In one or more embodiments, in step (1), the amount of the initiator is 1-5% of the charge concentration of the compound of formula I.

[0033] In one or more embodiments, in step (2), the conditions for initiating polymerization are: the photoinitiator initiates polymerization under ultraviolet light for 1-12 hours; or the thermal initiator initiates polymerization at 60-80°C for 1-12 hours.

[0034] In one or more embodiments, in step (3), the polyanionic template in the nanogel precursor is removed by ultrafiltration, centrifugation or dialysis using an inorganic salt solution to obtain the nanogel.

[0035] In one or more embodiments, the inorganic salt is selected from one or more of sodium chloride, potassium chloride, sodium nitrate, potassium nitrate, sodium sulfate, and potassium sulfate.

[0036] In one or more embodiments, the concentration of the inorganic salt in the inorganic salt solution is 0.1-5 mol / L.

[0037] Another aspect of the present invention provides a nanogel-nucleic acid complex, the nanogel-nucleic acid complex comprising the nanogel described in any embodiment of the present invention or the nanogel and nucleic acid prepared by the method described in any embodiment of the present invention.

[0038] In one or more embodiments, the nucleic acid is selected from one or more of siRNA, mRNA, and pDNA.

[0039] Another aspect of the present invention provides a method for preparing the nanogel-nucleic acid complex according to any embodiment of the present invention, the method comprising: mixing the nanogel according to any embodiment of the present invention or the nanogel prepared by the method described in any embodiment of the present invention with nucleic acid in a culture medium to obtain the nanogel-nucleic acid complex.

[0040] Another aspect of the present invention provides the application of the nanogels described in any embodiment of the present invention, the nanogels prepared by the methods described in any embodiment of the present invention, or the nanogel-nucleic acid complexes described in any embodiment of the present invention in nucleic acid transfection.

[0041] The present invention has the following beneficial technical effects: 1. The nanogel transfection reagent provided by the present invention has high delivery efficiency, low cytotoxicity, convenient operation, and is applicable to a variety of nucleic acid drugs and cell lines, and its performance is significantly better than that of commercially available transfection reagents.

[0042] 2. The nanogel proposed in this invention exhibits high delivery efficiency for siRNA, mRNA, and pDNA, making it a highly efficient and versatile nucleic acid delivery platform. Furthermore, the toxicity of the nanogel is significantly lower than that of commonly used amine cationic transfection reagents (bPEI). 25k The cell survival rate of the nucleic acid transfection is higher than 90% under the experimental conditions of Lipofectamine 3000, which has high safety and biocompatibility. At the same time, the transfection operation is simple and avoids the cumbersome operation of conventional cationic polymer nucleic acid delivery, which requires transfection in serum-free medium first and then changing to complete medium several hours later.

[0043] 3. The nucleic acid transfection vector proposed in this invention is a low-toxicity, high-efficiency, easy-to-operate, and universal transfection platform, which has potential value in in vitro transfection and clinical application in non-viral gene vectors. Attached Figure Description

[0044] Figure 1 Particle size distribution of NG1 nanogels of different sizes.

[0045] Figure 2 The polydispersity index represents the hydrodynamic radius and particle size distribution of NG1 nanogels with different degrees of crosslinking.

[0046] Figure 3 Particle size distribution diagrams of NG1-NG5 nanogels with different chemical compositions.

[0047] Figure 4 The light intensity of NG1 nanogel in 10 mM GSH solution changes over time.

[0048] Figure 5 For NG1 and bPEI 25k Comparison of cytotoxicity results with the commercially available transfection reagent Lipo3000.

[0049] Figure 6 Fluorescence images, flow cytometry results, and results of mediated apoptosis for siRNA transfected into nanogels NG1 and NG5.

[0050] Figure 7 Fluorescence images and flow cytometry results of GFP mRNA transfected with NG1 and NG5 nanogels.

[0051] Figure 8Fluorescence images and flow cytometry results of GFP pDNA transfection with nanogels NG1 and NG5.

[0052] Figure 9 The results show the siRNA delivery of the nanogel NG1 in various cell lines. Detailed Implementation

[0053] To enable those skilled in the art to understand the features and effects of the present invention, the terms and expressions used herein are explained and defined in general terms below. Unless otherwise specified, all technical and scientific terms used herein have the common meaning understood by those skilled in the art regarding the present invention, and in case of conflict, the definitions in this specification shall prevail.

[0054] The theories or mechanisms described and disclosed herein, whether right or wrong, should not in any way limit the scope of the invention, that is, the contents of the invention can be implemented without being limited by any particular theory or mechanism.

[0055] In this document, the terms “contains,” “includes,” “containing,” and similar terms encompass the meanings of “basically composed of” and “composed of.” For example, when this document discloses “A contains B and C,” “A is basically composed of B and C” and “A is composed of B and C” should be considered as having been disclosed in this document.

[0056] In this document, all features defined by numerical ranges or percentage ranges, such as numerical values, quantities, contents, and concentrations, are for the sake of brevity and convenience only. Accordingly, descriptions of numerical ranges or percentage ranges should be considered as covering and specifically disclosing all possible sub-ranges and individual numerical values ​​(including integers and fractions) within those ranges.

[0057] Unless otherwise specified, percentages refer to mass percentages and proportions refer to mass ratios in this article.

[0058] In this article, the sum of the percentages of all components in the composition is 100%.

[0059] In this document, when describing embodiments or examples, it should be understood that it is not intended to limit the invention to those embodiments or examples. Rather, all alternatives, modifications, and equivalents of the methods and materials described herein are covered within the scope of this invention.

[0060] For the sake of brevity, not all possible combinations of the technical features in each implementation scheme or embodiment are described herein. Therefore, as long as there is no contradiction in the combination of these technical features, the technical features in each implementation scheme or embodiment can be combined arbitrarily, and all possible combinations should be considered within the scope of this specification.

[0061] In this invention, the relationship between charge concentration and molecular concentration (molar concentration of compound I) should be: charge concentration = molecular concentration In this invention, the compound of formula I carries only one charge, so the charge concentration of the compound of formula I is its molecular concentration.

[0062] This invention provides a highly efficient, safe, convenient, and versatile nucleic acid drug delivery carrier. The nanogel of this invention contains a large number of positively charged molecules, enabling efficient loading of various nucleic acid drug molecules; simultaneously, the nanogel core possesses a good biocompatible environment, effectively maintaining the structure and bioactivity of nucleic acids; compared to amine-based cationic polymers bPEI... 25k Compared to commonly used commercial transfection reagents, its cytotoxicity is significantly reduced; its cross-linking agent can respond to the intracellular environment, mediating the escape and controlled release of nucleic acid drugs. For nanogels with a core-shell structure, the shell is a neutral polymer, which can further weaken the positive charge on the gel surface and reduce cytotoxicity; at the same time, targeting molecules can be selectively modified on the neutral polymer shell to achieve precise delivery of nucleic acid drugs.

[0063] Specifically, the present invention provides a nanogel comprising a phosphonic cationic polymer and a second polymer; wherein the monomer of the phosphonic cationic polymer comprises one or more compounds of formula I; , In Formula I, R1 is selected from hydrogen atoms or methyl groups; R2 is selected from -C(=O)-NH-, -C(=O)-O-, or -C6H4-; R3 is selected from -P + -(CH3)3、-P + -(CH2CH3)3、-P + -(CH2CH2CH3)3 or -P + -(CH2CH2CH2CH3)3; n is 1, 2 or 3.

[0064] It should be understood that in this article, the carbonyl moiety (-C(=O)-NH-) in -C(=O)-O- is different from that in the compound of formula I. Partially connected, among which, This represents the site that is attached to the carbonyl group.

[0065] In some embodiments, the nanogel has a core-shell structure, comprising a nanogel core and a shell covering the outer surface of the nanogel core.

[0066] In some implementations, the nanogel does not have a core-shell structure and does not have a shell.

[0067] In some embodiments, the monomers of the phosphonic cationic polymer further include a crosslinking agent. Crosslinking agents suitable for use in this invention include, but are not limited to, one or more of diallyl disulfide, N,N'-bis(acryloyl)cysteine, N,N'-(1,2-dihydroxyethylene)bisacrylamide glycol dimethacrylate, and 2,2-dithiodiethanol diacrylate. In this invention, the molar ratio of the crosslinking agent to the compound of formula I is (0.01:1)-(0.5:1), for example 0.01:1, 0.02:1, 0.03:1, 0.04:1, 0.05:1, 0.06:1, 0.07:1, 0.08:1, 0.09:1, 0.1:1, 0.15:1, 0.2:1, 0.25:1, 0.3:1, 0.35:1, 0.4:1, 0.45:1, 0.5:1, or any range between two values, preferably (0.1:1)-(0.5:1).

[0068] In this invention, the second polymer is a neutral polymer or an amphoteric polymer; neutral polymers suitable for this invention include, but are not limited to, polyethylene glycol, polyethylene, polytetrafluoroethylene, etc.; amphoteric polymers suitable for this invention include, but are not limited to, poly(2-methacryloyloxyethyl)phosphocholine, poly(methacryloylethyl)sulfobetaine, poly(3-[[2-(methacryloyloxy)ethyl]dimethylammonium]propionate and poly(3-acrylamidopropyl)dimethylammonium]propionate, etc.

[0069] The second polymer of the present invention may be modified with a chain transfer agent, wherein suitable chain transfer agents include, but are not limited to, one or more of the following: methyl cyanomethyl dithiocarbamate, S-cyanomethyl-S-dodecyl trithiocarbonate, cyanoisopropyl dithiobenzoate, 4-cyano-4-(thiobenzoyl)valerate, S-(2-cyano-2-propyl)-S-dodecyl trithiocarbonyl ester, 4-cyano-4-[[(dodecylthio)thionylmethyl]thio]valerate, and 2-(dodecyl trithiocarbonate)-2-methylpropionic acid. In the present invention, the molar ratio of the chain transfer agent to the second polymer is (1:1)-(2:1), for example, 1:1, 1.2:1, 1.4:1, 1.6:1, 1.8:1, 2:1, or any range between two values. It should be understood that the molar ratio of the second polymer can be calculated based on the mass of the second polymer / the number-average molecular weight of the polymer.

[0070] Depending on the application, the second polymer of this invention can be grafted with targeting molecules, such as folic acid, galactose, sodium alendronate, transferrin, hyaluronic acid, polypeptides, mannose, biotin, etc. In this invention, the molar ratio of the targeting molecule to the second polymer is (0.02:1)-(1:1), for example 0.02:1, 0.04:1, 0.06:1, 0.08:1, 0.1:1, 0.12:1, 0.14:1, 0.16:1, 0.18:1, 0.2:1, 0.22:1, 0.24:1, 0.26:1, 0.28:1, 0.3:1, 0.32:1, 0.34:1, 0.36:1, 0.38:1, 0.4:1, 0.42:1, 0.44:1, 0.46:1, 0 The molar ratios are 0.48:1, 0.5:1, 0.52:1, 0.54:1, 0.56:1, 0.58:1, 0.6:1, 0.62:1, 0.64:1, 0.66:1, 0.68:1, 0.7:1, 0.72:1, 0.74:1, 0.76:1, 0.78:1, 0.8:1, 0.82:1, 0.84:1, 0.86:1, 0.88:1, 0.9:1, 0.92:1, 0.94:1, 0.96:1, 0.98:1, 1:1, or any range between two values. It should be understood that the molar ratio of the second polymer can be calculated based on the mass of the second polymer divided by the number-average molecular weight of the polymer.

[0071] In some embodiments, for target molecules containing -COOH, the grafting method is as follows: a neutral segment (neutral polymer) with -NH2 at both ends is selected. First, the neutral segment is protected with BOC, then the target molecule is added and linked by an amidation reaction; subsequently, BOC removal is performed, a chain transfer agent is added, and after dehydration condensation, the neutral segment grafted with the target molecule and chain transfer agent is obtained.

[0072] In some embodiments, for the target molecule containing -NH2, the grafting method is as follows: a neutral segment (neutral polymer) with -OH and -COOH ends respectively is selected. First, the neutral segment is activated with NHS, then the target molecule is added and linked via an NHS / NH2 reaction; subsequently, a chain transfer agent is added, and after esterification, the neutral segment grafted with the target molecule and chain transfer agent is obtained.

[0073] In some embodiments, the diameter of the nanogel of the present invention is 40-500 nm, such as 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 150 nm, 200 nm, 250 nm, 300 nm, 350 nm, 400 nm, 450 nm or any range between two values, such as 180-300 nm.

[0074] In some embodiments, the PDI of the nanogel of the present invention is 0.02-0.3.

[0075] In some embodiments, the mass ratio of the phosphonic cationic polymer and the second polymer in the nanogel of the present invention is (2:1)-(20:1), for example 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, 5:1, 5.5:1, 6:1, 6.5:1, 7:1, 7.5:1, 8:1, 8.5:1, 9:1, 9.5:1, 10:1, 10.5:1, 11:1, 11.5:1, 12:1, 12.5:1, 13:1, 13.5:1, 14:1, 14.5:1, 15:1, 15.5:1, 16:1, 16.5:1, 17:1, 17.5:1, 18:1, 18.5:1, 19:1, 19.5:1, 20:1, or any range between two values.

[0076] In some embodiments, in Formula I, R1 is selected from hydrogen atoms, R2 is selected from -C6H4-, and R3 is selected from -P. + -(CH3)3、-P + -(CH2CH3)3、-P + -(CH2CH2CH3)3 or -P + -(CH2CH2CH2CH3)3; n is 1, 2 or 3.

[0077] In some embodiments, in Formula I, R1 is selected from hydrogen atoms, R2 is selected from -C6H4-, and R3 is selected from -P. + -(CH3)3、-P + -(CH2CH3)3、-P + -(CH2CH2CH3)3 or -P + -(CH2CH2CH2CH3)3; n is 1.

[0078] In some embodiments, the compound of formula I is selected from one or more of trimethyl-(4-vinylbenzyl)phosphonium chloride, triethyl-(4-vinylbenzyl)phosphonium chloride, tri-n-propyl-(4-vinylbenzyl)phosphonium chloride, and tri-n-butyl-(4-vinylbenzyl)phosphonium chloride.

[0079] This invention also provides a method for preparing nanogels via electrostatic template method, specifically comprising: using an anionic polymer as a template and reacting it with one or more compounds of formula I ( The crosslinking agent, initiator, and optional second polymer are mixed, and the pH of the solution is adjusted to electroneutrality; polymerization is initiated to obtain a nanogel precursor; the anionic polymer in the nanogel precursor is removed to obtain the nanogel; in Formula I, R1, R2, R3, and n are defined as defined in any embodiment of the present invention.

[0080] In some embodiments, the method for preparing nanogels according to the present invention specifically includes: (1) Using an anionic polymer as a template, and combining it with one or more compounds of formula I ( The crosslinking agent, the second polymer, and the initiator are mixed, and the pH of the mixed solution is adjusted to electroneutrality. (2) Initiate polymerization to obtain nanogel precursors; (3) Remove the anionic polymer from the nanogel precursor to obtain the nanogel; In Formula I, R1, R2, R3 and n are defined as in any embodiment of the present invention.

[0081] In some embodiments, the method for preparing nanogels according to the present invention specifically includes: (1) Using an anionic polymer as a template, and combining it with one or more compounds of formula I ( The crosslinking agent and initiator are mixed, and the pH of the mixed solution is adjusted to electroneutrality; (2) Initiate polymerization to obtain nanogel precursors; (3) Remove the anionic polymer from the nanogel precursor to obtain the nanogel; In Formula I, R1, R2, R3 and n are defined as in any embodiment of the present invention.

[0082] The anionic polymers suitable for use in this invention include, but are not limited to, one or more of polyacrylic acid, sodium polyacrylamido-2-methylpropanesulfonate, sodium poly(p-styrene sulfonate), poly(dimethylaminoethyl methacrylate), poly(methacryloyloxyethyltrimethylammonium chloride), and poly(2-aminoethyl methacrylate).

[0083] Initiators suitable for use in this invention include photoinitiators and / or thermal initiators. Photoinitiators suitable for use in this invention include, but are not limited to, one or more of 2-hydroxy-2-methyl-1-phenyl-1-propanone, 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylphenylpropanone, and phenyl-2,4,6-trimethylbenzoyl lithium phosphinate; thermal initiators suitable for use in this invention include, but are not limited to, persulfates and water-soluble azo compounds.

[0084] In the method of the present invention, in step (1), the charge concentration of the compound of formula I is 5-500mM, for example 10-100mM, 15-400mM, 30-200mM, 25-300mM, 50-350mM, etc.; and the charge concentration of the compound of formula I and the anionic polymer are the same.

[0085] In the method of the present invention, in step (1), the amount of crosslinking agent is 1-50% of the molar concentration (charge concentration) of compound I, for example 5-40%, 15-4%, 25-35%, etc., preferably 3-30%.

[0086] In the method of the present invention, in step (1), the amount of the second polymer is 0.1-5% of the molar concentration (charge concentration) of the compound of formula I, for example, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, or any range between two values.

[0087] In the method of the present invention, in step (1), the amount of initiator is 1-5% of the molar concentration of the compound of formula I, for example 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, or any range between two values.

[0088] In the method of the present invention, in step (2), the conditions for initiating polymerization are: the photoinitiator initiates polymerization under ultraviolet light for 1-12 hours, for example 2-9 hours or 5-7 hours; or the thermal initiator initiates polymerization at 60-80°C (for example 65-75°C, 70-80°C, etc.) for 1-12 hours, for example 2-9 hours or 5-7 hours.

[0089] In the method of the present invention, in step (3), the polyanionic template in the nanogel precursor is removed by ultrafiltration, centrifugation or dialysis using an inorganic salt solution to obtain the nanogel.

[0090] In some embodiments, the inorganic salt is selected from one or more of sodium chloride, potassium chloride, sodium nitrate, potassium nitrate, sodium sulfate, and potassium sulfate.

[0091] In some implementations, the concentration of the inorganic salt is 0.1-5 mol / L, such as 0.5-3 mol / L, 1-4 mol / L, 2-3.5 mol / L, etc.

[0092] In some preferred embodiments, the nanogel comprises a nanogel core and a shell coating the outer surface of the nanogel core, wherein the nanogel core is a phosphonic cationic polymer and the shell is a second polymer; the synthesis method includes the following steps: using an anionic polymer as a template, adding a compound of formula I, a crosslinking agent, a neutral polymer-chain transfer agent, and an initiator; adjusting the pH of the solution to electrically neutral; then removing oxygen by purging with nitrogen to initiate polymerization and obtain a nanogel precursor. The polyanionic template in the nanogel precursor is removed by ultrafiltration, centrifugation, or dialysis with an inorganic salt solution to obtain the nanogel of the present invention.

[0093] In some preferred embodiments, the nanogel has a core-shell-free structure, comprising a phosphonic cationic polymer without a shell. The synthesis method in this case includes the following steps: using an anionic polymer as a template, adding a compound of formula I, a crosslinking agent, and an initiator; adjusting the pH of the solution to electrically neutral; then removing oxygen by purging with nitrogen to initiate polymerization and obtain a nanogel precursor; removing the polyanionic template from the nanogel precursor by ultrafiltration, centrifugation, or dialysis with an inorganic salt solution to obtain a core-shell-free nanogel.

[0094] The present invention also provides a nanogel-nucleic acid complex, which includes the nanogel described in any embodiment of the present invention or the nanogel and nucleic acid prepared in any embodiment of the present invention.

[0095] Nucleic acids applicable to this invention include, but are not limited to, small interfering RNA (siRNA), messenger RNA (mRNA), plasmids (pDNA), etc.

[0096] The present invention also provides a method for preparing the nanogel-nucleic acid complex according to any embodiment of the present invention, specifically comprising: mixing the nanogel according to any embodiment of the present invention or the nanogel prepared by the method described in any embodiment of the present invention with nucleic acid in a culture medium to obtain the nanogel-nucleic acid complex.

[0097] In some embodiments, the method for preparing the nanogel-nucleic acid complex includes: adding the nanogel and nucleic acid to a buffer solution and vortexing for 5-30 seconds; then adding complete culture medium and mixing by pipetting to obtain the nanogel-nucleic acid complex.

[0098] In the buffer solution, the concentration of nucleic acid is 0.1-20 μg / mL, and the concentration of nanogel is 5-100 μg / mL. Preferably, the concentration of nucleic acid is 1-10 μg / mL, and the concentration of phosphonic cationic nanogel is 10-50 μg / mL.

[0099] The present invention also provides the application of the nanogel described in any embodiment of the present invention, the nanogel prepared by the method described in any embodiment of the present invention, or the nanogel-nucleic acid complex described in any embodiment of the present invention in nucleic acid transfection; wherein, nucleic acid transfection is the delivery of nucleic acid from outside the cell to inside the cell and the successful expression of nucleic acid.

[0100] The present invention will be described below by way of specific embodiments. It should be understood that these embodiments are merely illustrative and are not intended to limit the scope of the invention. The methods, reagents, and materials used in the embodiments and comparative examples are conventional methods, reagents, and materials in the art, unless otherwise stated. The starting material compounds in the embodiments and comparative examples are all commercially available.

[0101] The polyethylene glycol modified with folic acid and chain transfer agent in the following preparation examples was obtained by the following method: Weigh 500.0 mg of NH2-terminated polyethylene glycol (number average molecular weight 5000) and 21.8 mg of di-tert-butyl dicarbonate, and react at room temperature for 8 h in a 10% wt sodium hydroxide solution in tert-butanol. Mix the reactants with 44.1 mg of folic acid, 25.8 mg of dicyclohexylcarbodiimide, and 1.8 mg of 4-dimethylaminopyridine, and react at room temperature for 8 h. Wash with 0.1 M hydrochloric acid to obtain folic acid-modified polyethylene glycol. Then weigh 279.4 mg of 4-cyano-4-(thiobenzoyl)valerate, 2.4 mg of 4-dimethylaminopyridine, and mix with 500.0 mg of folic acid-modified polyethylene glycol. After purging with nitrogen to remove oxygen, stir for 20 min. Add 206.3 mg of dicyclohexylcarbodiimide dropwise through a constant pressure funnel at a rate of 3 drops per second. After reacting the mixed solution at a constant temperature of 25°C for 48 hours, the insoluble matter was removed by filtration, and the solid was precipitated with diethyl ether. After vacuum drying, polyethylene glycol modified with folic acid and a chain transfer agent was obtained. The molar ratio of 4-cyano-4-(thiobenzoyl)valerate (chain transfer agent) to polyethylene glycol (second polymer) was 1:1; the molar ratio of folic acid (targeting molecule) to polyethylene glycol (second polymer) was also 1:1.

[0102] Preparation Example 1

[0103] In this preparation example, nanogels of different sizes, NG1, were prepared using the following method.

[0104] Weigh out 28.8 mg of polyacrylic acid (charge concentration of the anionic polymer template is 20 mM), 91.5 mg of trimethyl-(4-vinylbenzyl)phosphonium chloride (monomer molar concentration (for this monomer, the monomer molar concentration equals the charge concentration) is 20 mM), 10.4 mg of N,N'-bis(acryloyl)cysteine ​​(10% of the monomer molar concentration), 1.3 mg of 2-hydroxy-2-methyl-1-phenyl-1-propanone (2% of the monomer molar concentration), and folic acid- 21.0 mg of polyethylene glycol-4-cyano-4-(thiobenzoyl)valerate (1% of monomer molar concentration) was dissolved in an appropriate amount of deionized water. Different amounts of NaCl were added to achieve final concentrations of 0, 5 mM, 10 mM, 15 mM, 20 mM, and 30 mM in the reaction solution. Deionized water was then added to a total volume of 20 mL, and the pH of the solution was adjusted to electroneutrality. The reaction system was purged with nitrogen to remove oxygen, and the reaction was carried out under ultraviolet light for 8 hours to obtain the nanogel precursor. 1.753 g of sodium chloride was added to the obtained nanogel precursor to adjust the salt concentration to 1.5 M. Using 1.5 M sodium chloride as the eluent, the nanogel precursor was washed six times by ultrafiltration centrifugation to remove the anionic polymer template. The nanogel was then dialyzed with deionized water to remove sodium chloride, yielding the nanogel. The particle size distribution of the obtained nanogels of different sizes is shown in the figure. Figure 1 As shown, their different dimensions are represented as NG1-R1, NG1-R2, NG1-R3, NG1-R4, NG1-R5 and NG1-R6 respectively.

[0105] Preparation Example 2

[0106] In this preparation example, nanogels NG1 with different degrees of crosslinking were prepared by the following method.

[0107] Weigh out 29.0 mg of polyacrylic acid (charge concentration of anionic polymer template is 20 mM), 74.2 mg of trimethyl-(4-vinylbenzyl)phosphonium chloride (monomer molar concentration is 20 mM), 10.4 mg, 20.8 mg, 31.2 mg, 41.7 mg, and 52.1 mg of N,N'-bis(acryloyl)cysteine ​​(10%, 20%, 30%, 40%, and 50% of monomer molar concentration, respectively), 1.1 mg of 2-hydroxy-2-methyl-1-phenyl-1-propanone (2% of monomer molar concentration), and 21.0 mg of folic acid-polyethylene glycol-4-cyano-4-(thiobenzoyl)valerate. Dissolve in 20 ml of deionized water, adjust the pH of the solution to electrically neutral, purge the reaction system with nitrogen to remove oxygen, and react under ultraviolet light for 8 h to obtain the nanogel precursor. Add 1.753 g of sodium chloride to the obtained nanogel precursor to adjust the salt concentration of the solution to 1.5 M. Use 1.5 M sodium chloride as the eluent and wash the nanogel precursor 6 times by ultrafiltration centrifugation to remove the anionic polymer template. Then, dialyze with deionized water to remove sodium chloride to obtain nanogels with different degrees of crosslinking. The corresponding hydrodynamic radii R are shown below. h And the polydispersity index (PDI) of particle size distribution, such as Figure 2 As shown.

[0108] Preparation Example 3

[0109] This preparation example demonstrates the preparation of nanogels NG1-5 with different chemical compositions using the following method.

[0110] NG1: Weigh 29.0 mg of polyacrylic acid (charge concentration of anionic polymer template is 20 mM), 74.2 mg of trimethyl-(4-vinylbenzyl)phosphonium chloride (monomer molar concentration is 20 mM), 20.8 mg of N,N'-bis(acryloyl)cystamine (monomer molar concentration is 20%), 1.1 mg of 2-hydroxy-2-methyl-1-phenyl-1-propanone (monomer molar concentration is 2%), and 21.0 mg of folic acid-polyethylene glycol-4-cyano-4-(thiobenzoyl)valerate, dissolve them in 20 ml of deionized water, adjust the pH of the solution to electrical neutrality, purge the reaction system with nitrogen to remove oxygen, and react under ultraviolet light for 8 h to obtain the nanogel precursor. Add 1.753 g of sodium chloride to the nanogel precursor obtained above, adjust the salt concentration of the solution to 1.5 M, and use 1.5 M sodium chloride as the eluent. Wash the nanogel precursor 6 times by ultrafiltration centrifugation to remove the anionic polymer template, and then dialyze with deionized water to remove sodium chloride to obtain the nanogel, named NG1.

[0111] NG2 was prepared using a similar method to that used to prepare NG1, but the difference was that the monomer trimethyl-(4-vinylbenzyl)phosphonium chloride was replaced with 87.9 mg of triethyl-(4-vinylbenzyl)phosphonium chloride (monomer concentration of 20 mM).

[0112] NG3 was prepared using a similar method to that used to prepare NG1, but the difference was that the monomer trimethyl-(4-vinylbenzyl)phosphonium chloride was replaced with 101.5 mg of tri-n-propyl-(4-vinylbenzyl)phosphonium chloride (monomer concentration of 20 mM).

[0113] NG4 was prepared using a similar method to that used to prepare NG1, but the difference was that the monomer trimethyl-(4-vinylbenzyl)phosphonium chloride was replaced with 115.2 mg of tri-n-butyl-(4-vinylbenzyl)phosphonium chloride (monomer concentration of 20 mM).

[0114] NG5 was prepared using a similar method to that used to prepare NG1, but with the difference that folic acid-polyethylene glycol-4-cyano-4-(thiobenzoyl)valerate was replaced with an equal mass of unmodified folic acid-polyethylene glycol-4-cyano-4-(thiobenzoyl)valerate.

[0115] The particle size distributions of the nanogels with different chemical compositions prepared in Example 3 are as follows: Figure 3 As shown.

[0116] like Figure 1-3 As shown, according to the synthesis method of the present invention, phosphonic cationic nanogels with independently adjustable particle size and crosslinking degree can be prepared. The resulting nanogels have a narrow particle size distribution, and the method is applicable to at least four phosphonic cationic monomers. Experimental results demonstrate that the synthesis method of the present invention is a versatile and controllable polymerization method, and the resulting phosphonic cationic nanogels possess adjustable structural parameters such as size and stiffness.

[0117] Test Example 1

[0118] Evaluation of the degradability of NG1 nanogels

[0119] The NG1 prepared in Example 1 of this invention was diluted in a 20 mM HEPES solution at pH 7.4, and then glutathione (GSH) was added to the solution to bring the GSH concentration to 10 mM. The changes in light intensity and particle size of NG1 over time were monitored using dynamic light scattering (DLS), and the results are as follows: Figure 4 As shown.

[0120] Figure 4 The results show that after the addition of GSH, the light intensity of NG1 decreased rapidly within 1 hour, and the particle size was greatly reduced, confirming that NG1 can respond to GSH and degrade as the GSH concentration increases.

[0121] Test Example 2

[0122] Cytotoxicity evaluation of NG1 nanogels

[0123] HeLa cells were pre-seeded into 96-well plates and incubated overnight at 37°C with 5% CO2. The culture medium was then removed, and 100 μL of DMEM complete medium solution containing different concentrations of NG1 (10 μg / mL, 20 μg / mL, 50 μg / mL, 80 μg / mL, 120 μg / mL) or 100 μL of bPEI solution containing different concentrations were added to each well. 25k Cells were incubated for 24 hours with complete culture medium solutions of 10 μg / mL, 20 μg / mL, 50 μg / mL, 80 μg / mL, or 120 μg / mL, or commercially available Lipofectamine 3000 (Lipo3000). Afterward, the culture medium was removed, and 10 μL of CCK-8 was added to each well. Cell viability was assessed according to the standard CCK-8 assay procedure. Six replicates were performed for each experiment. The concentration of commercially available Lipo3000 used was the concentration specified in the product manual for transfection. Test results are as follows: Figure 5 As shown.

[0124] Figure 5 The results showed that at concentrations up to 120 μg / mL, the survival rate of HeLa cells treated with NG1 was higher than 90%, significantly higher than that treated with bPEI. 25k The nanogel prepared in this invention exhibits low cytotoxicity compared to commonly used transfection reagents and good biocompatibility. The results show that the nanogel prepared in this invention has better biocompatibility than commonly used transfection reagents.

[0125] Application Example 1

[0126] siRNA transfected with nanogels NG1 and NG5

[0127] Using small interfering RNA (siRNA-FAM) labeled with the green fluorescent molecule 5-carboxyfluorescein (FAM) as a model nucleic acid, the efficiency of nanogel delivery was evaluated on HeLa cells by detecting intracellular fluorescence, and the successful transfection of vascular endothelial growth factor small interfering RNA (siVEGF) was mediated.

[0128] The specific method for siRNA delivery is as follows: HeLa cells are seeded into 24-well plates and incubated overnight at 37°C with 5% CO2. When the HeLa cell confluence reaches 80% or more, the nucleic acid delivery experiment begins. NG1 (or NG5) prepared in Example 3 of this invention is added to 50 μL of 20 mM HEPES solution at pH 7.4 and mixed thoroughly by pipetting. Then, 0.5 μg of siRNA-FAM is added, vortexed for 10 seconds, incubated for 10 minutes, and then 450 μL of complete culture medium is added and mixed thoroughly by pipetting. The mass ratio of NG1 (or NG5) to siRNA is W... NG / W siRNA =20. Remove the old cell culture medium from the well plate, wash twice with PBS, add culture medium solution containing siRNA complex, and incubate in an incubator for 24 hours. Afterwards, qualitatively observe nucleic acid delivery using fluorescence microscopy, or quantitatively analyze the average fluorescence intensity of HeLa cells using flow cytometry. Specifically: after treating cells with culture medium solution containing siRNA complex, remove the culture medium, wash cells twice with PBS, and observe the green fluorescence intensity within the cells using fluorescence microscopy; or after removing the culture medium and washing cells twice with PBS, collect cells by trypsin digestion, centrifuge, resuspend in PBS, and detect the green fluorescence intensity within the cells using flow cytometry.

[0129] The specific procedure for siRNA transfection is as follows: HeLa cells are pre-seeded into 96-well plates and incubated overnight at 37°C and 5% CO2. NG1 (or NG5) prepared in Example 3 of this invention is added to the complete culture medium and mixed thoroughly by pipetting. The mass ratio of NG1 (or NG5) to siRNA is W... NG / W siRNA =20. Then, add negative control small interfering RNA (siNC) or siVEGF to the complete medium containing NG1 (or NG5), vortex for 10 s, where the concentration of siNC or siVEGF is 100 nM. Remove the medium, add 100 μL of complete medium solution containing NG1 (or NG15)-siRNA complex, and incubate the cells for 24 hours. Afterward, remove the medium and add 10 μL of CCK-8 to each well. Detect cell viability according to the standard CCK-8 assay procedure. The difference between the cell viability of the siNC experimental group and the siVEGF experimental group is the apoptosis rate. Six replicates were tested for each experiment. bPEI 25k The commercially available reagent Lipo3000 was used as a control, with transfection performed according to the product manual.

[0130] Figure 6This describes the results of delivering siRNA-FAM and transfecting siVEGF with materials NG1 and NG5 obtained in the preparation examples of this invention on HeLa cells. The mass ratio of NG1 (or NG5) to siRNA is W. NG / W siRNA =20. The results showed that NG1 and NG5 could efficiently deliver nucleic acids to 100% of cells and efficiently transfect siVEGF, inducing apoptosis. The intracellular delivery of siRNA and the level of apoptosis mediated by NG1 and NG5 were both higher than those mediated by bPEI. 25k Compared with the commercial reagent Lipo3000, NG1-mediated delivery and transfection efficiency was slightly higher than that of NG5, indicating that folic acid molecules further promoted receptor-mediated endocytosis.

[0131] Application Example 2

[0132] Nanogels NG1 and NG5 were used to transfect green fluorescent protein mRNA (GFP mRNA) and plasmid (GFP pDNA).

[0133] HeLa cells were seeded into 24-well plates and incubated overnight at 37°C with 5% CO2. Once cell confluence reached 80% or higher, nucleic acid transfection experiments were initiated. NG1 (or NG5) prepared in Example 3 of this invention was added to 50 μL of 20 mM HEPES solution at pH 7.4 and mixed thoroughly by pipetting. Then, 2.5 μg of GFP mRNA or GFP pDNA was added, vortexed for 10 s, incubated for 10 minutes, and then 450 μL of complete culture medium was added and mixed thoroughly by pipetting. The mass ratio of NG1 (or NG5) to 2.5 μg of GFP mRNA or GFP pDNA was W. NG / W mRNA =5 and W NG / W mRNA =5, vortex for 10s. Remove the old cell culture medium from the well plate, wash twice with PBS, add culture medium solution containing nucleic acid complex, and incubate in an incubator for 24 hours. Afterwards, qualitatively observe nucleic acid transfection using fluorescence microscopy, or quantitatively analyze the average fluorescence intensity of HeLa cells using flow cytometry. Specifically: after treating cells with culture medium solution containing nucleic acid complex, remove the culture medium, wash cells twice with PBS, and observe the green fluorescence intensity within the cells using fluorescence microscopy; or after removing the culture medium and washing cells twice with PBS, collect cells by trypsin digestion, centrifuge, resuspend in PBS, and detect the green fluorescence intensity within the cells using flow cytometry.

[0134] Figure 7 , 8The results show the effects of NG1 and NG5 transfecting GFP mRNA or GFP pDNA in HeLa cells. The results indicate that NG1 and NG5 are significantly more efficient than bPEI in transfecting mRNA and pDNA in HeLa cells. 25k Using the commercially available reagent Lipo3000, efficient expression of green fluorescent protein was achieved. The transfection efficiency of NG1 was significantly higher than that of NG5, demonstrating that folic acid molecules further promoted receptor-mediated endocytosis. The nanogel of this invention serves as a universal nucleic acid transfection platform.

[0135] Application Example 3

[0136] NG1 nanogel transfects siRNA in multiple cell lines

[0137] Human alveolar adenocarcinoma basal epithelial cells (A549), human umbilical vein endothelial cells (HUVEC), and human osteosarcoma cells (143B) were seeded into 24-well plates and incubated overnight at 37°C and 5% CO2. When the cell confluence reached over 80%, nucleic acid delivery experiments were initiated. NG1 prepared in Example 3 of this invention was added to 50 μL of 20 mM HEPES solution at pH 7.4 and mixed thoroughly by pipetting. Then, 0.5 μg of siRNA-FAM was added, vortexed for 10 s, incubated for 10 minutes, and then 450 μL of complete culture medium was added and mixed thoroughly by pipetting. The mass ratio of NG1 to siRNA was W / W. NG / W siRNA =20. Remove the old cell culture medium from the well plate, wash twice with PBS, add culture medium solution containing siRNA complex, and incubate in an incubator for 24 hours. Afterwards, qualitatively observe nucleic acid delivery using fluorescence microscopy, or quantitatively analyze the average fluorescence intensity of the cells using flow cytometry. Specifically: after treating cells with culture medium solution containing siRNA complex, remove the culture medium, wash the cells twice with PBS, and observe the green fluorescence intensity within the cells using fluorescence microscopy; or after removing the culture medium and washing the cells twice with PBS, collect the cells by trypsin digestion, centrifuge, resuspend in PBS, and detect the green fluorescence intensity within the cells using flow cytometry.

[0138] Figure 9 The results show the delivery of siRNA by NG1, the material obtained in Example 3 of this invention, in A549, HUVEC, and 143B cells. The results indicate that NG1 can efficiently deliver siRNA in a variety of cell lines.

Claims

1. A nanogel, characterized in that, The nanogel comprises a phosphonic cationic polymer and an optional second polymer; the monomers of the phosphonic cationic polymer comprise one or more compounds of formula I; ， In Formula I, R1 is selected from hydrogen atoms or methyl groups; R2 is selected from -C(=O)-NH-, -C(=O)-O-, or -C6H4-; R3 is selected from -P + -(CH3)3、-P + -(CH2CH3)3、-P + -(CH2CH2CH3)3 or -P + -(CH2CH2CH2CH3)3; n is 1, 2 or 3.

2. The nanogel as described in claim 1, characterized in that, The nanogel comprises a nanogel core and a shell coating the outer surface of the nanogel core, wherein the nanogel core is a phosphonic cationic polymer and the shell is a second polymer; or, the nanogel does not have a shell.

3. The nanogel as described in claim 1, characterized in that, The nanogel has one or more of the following characteristics: The monomer of the phosphonic cationic polymer further includes a crosslinking agent. Preferably, the crosslinking agent is one or more selected from diallyl disulfide, N,N'-bis(acryloyl)cysteine, N,N'-(1,2-dihydroxyethylene)bisacrylamide glycol dimethacrylate, and 2,2-dithiodiethanol diacrylate. Preferably, the molar ratio of the crosslinking agent to the compound of formula I is (0.01:1)-(0.5:1), and more preferably (0.1:1)-(0.5:1). The second polymer is a neutral polymer or an amphoteric polymer. Preferably, the second polymer is one or more selected from polyethylene glycol, polyethylene, polytetrafluoroethylene, poly-2-methacryloyloxyethyl phosphocholine, polymethacryloylethyl sulfobetaine, poly-3-[[2-(methacryloyloxy)ethyl]dimethylammonium]propionate and poly-3-[(3-acrylamidopropyl)dimethylammonium]propionate. The second polymer is modified with a chain transfer agent. Preferably, the chain transfer agent is selected from one or more of the following: methyl cyano(phenyl)aminodithiocarbamate, S-cyanomethyl-S-dodecyl trithiocarbonate, cyanoisopropyl dithiobenzoate, 4-cyano-4-(thiobenzoyl)valerate, S-(2-cyano-2-propyl)-S-dodecyl trithiocarbonyl ester, 4-cyano-4-[[(dodecylthio)thionylmethyl]thio]valerate, and 2-(dodecyl trithiocarbonate)-2-methylpropionic acid. Preferably, the molar ratio of the chain transfer agent to the second polymer is (1:1) to (2:1). The second polymer is modified with a targeting molecule, which is folic acid, galactose, sodium alendronate, transferrin, hyaluronic acid, polypeptide, mannose, or biotin; preferably, the molar ratio of the targeting molecule to the second polymer is (0.02:1) to (1:1).

4. The nanogel as described in claim 1, characterized in that, The hydrodynamic diameter of the nanogel, as measured by dynamic light scattering in water, is 40-500 nm; and / or, the PDI of the nanogel is 0.02-0.

3.

5. The nanogel as described in claim 1, characterized in that, The mass ratio of the phosphonic cationic polymer to the second polymer is (2:1)-(20:1).

6. The nanogel according to claim 1, characterized in that, In Formula I, R1 is selected from hydrogen atoms, R2 is selected from -C6H4-, and R3 is selected from -P. + -(CH3)3、-P + -(CH2CH3)3、-P + -(CH2CH2CH3)3 or -P + -(CH2CH2CH2CH3)3; n is 1, 2, or 3; Preferably, the compound of Formula I is selected from one or more of trimethyl-(4-vinylbenzyl)phosphonium chloride, triethyl-(4-vinylbenzyl)phosphonium chloride, tri-n-propyl-(4-vinylbenzyl)phosphonium chloride, and tri-n-butyl-(4-vinylbenzyl)phosphonium chloride.

7. A method for preparing the nanogel according to any one of claims 1-6, characterized in that, The method includes: using an anionic polymer as a template, and reacting it with one or more compounds of formula I (… The crosslinking agent, initiator, and optional second polymer are mixed, and the pH of the solution is adjusted to electroneutrality; polymerization is initiated to obtain a nanogel precursor; the anionic polymer in the nanogel precursor is removed to obtain the nanogel; in Formula I, R1, R2, R3, and n are defined as defined in claim 1 or 4.

8. The method as described in claim 7, characterized in that, The method includes: (1) Using an anionic polymer as a template, and combining it with one or more compounds of formula I ( The crosslinking agent, the second polymer, and the initiator are mixed, and the pH of the mixed solution is adjusted to electroneutrality. (2) Initiate polymerization to obtain nanogel precursors; (3) Remove the anionic polymer from the nanogel precursor to obtain the nanogel; In Formula I, R1, R2, R3 and n are defined as described in claim 1 or 4.

9. The method as described in claim 7, characterized in that, The method includes: (1) Using an anionic polymer as a template, and combining it with one or more compounds of formula I ( The crosslinking agent and initiator are mixed, and the pH of the mixed solution is adjusted to electroneutrality; (2) Initiate polymerization to obtain nanogel precursors; (3) Remove the anionic polymer from the nanogel precursor to obtain the nanogel; In Formula I, R1, R2, R3 and n are defined as described in claim 1 or 4.

10. The method according to any one of claims 7-9, characterized in that, The method has one or more of the following characteristics: The anionic polymer is selected from one or more of polyacrylic acid, sodium poly(2-acrylamido-2-methylpropanesulfonate), sodium poly(p-styrene sulfonate), poly(dimethylaminoethyl methacrylate), poly(methacryloyloxyethyltrimethylammonium chloride), and poly(2-aminoethyl methacrylate). The initiator includes a photoinitiator and / or a thermal initiator. Preferably, the photoinitiator is selected from one or more of 2-hydroxy-2-methyl-1-phenyl-1-propanone, 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylphenylpropanone, and phenyl-2,4,6-trimethylbenzoyl lithium phosphinate, and the thermal initiator is selected from persulfates and / or water-soluble azo compounds. In step (1), the charge concentration of the compound of formula I is 5-500 mM, and the charge concentration of the compound of formula I and the anionic polymer are the same; In step (1), the amount of the crosslinking agent used is 1-50% of the charge concentration of the compound of formula I; In step (1), the amount of the second polymer used is 0.1-5% of the charge concentration of the compound of formula I; In step (1), the amount of the initiator is 1-5% of the charge concentration of the compound of formula I; In step (2), the conditions for initiating polymerization are: photoinitiator initiating polymerization under ultraviolet light for 1-12 hours; or, thermal initiator initiating polymerization at 60-80°C for 1-12 hours. In step (3), the polyanionic template in the nanogel precursor is removed by ultrafiltration, centrifugation or dialysis using an inorganic salt solution to obtain the nanogel; preferably, the inorganic salt is selected from one or more of sodium chloride, potassium chloride, sodium nitrate, potassium nitrate, sodium sulfate and potassium sulfate; preferably, the concentration of the inorganic salt in the inorganic salt solution is 0.1-5 mol / L.

11. A nanogel-nucleic acid complex, characterized in that, The nanogel-nucleic acid complex comprises the nanogel according to any one of claims 1-6 or the nanogel prepared by the method according to any one of claims 7-10 and nucleic acid; preferably, the nucleic acid is selected from one or more of siRNA, mRNA and pDNA.

12. A method for preparing the nanogel-nucleic acid complex of claim 11, characterized in that, The method includes: mixing the nanogel according to any one of claims 1-6 or the nanogel prepared by the method according to any one of claims 7-10 with nucleic acid in a culture medium to obtain a nanogel-nucleic acid complex.

13. The application of the nanogel according to any one of claims 1-6, the nanogel prepared by the method according to any one of claims 7-10, or the nanogel-nucleic acid complex according to claim 11 in nucleic acid transfection.

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