Polyelement polyelectrolyte nanogel as well as preparation method and application thereof

Abstract

The invention belongs to the field of biotechnology and material synthesis, and discloses polyelectrolyte nanogel as well as a preparation method and application thereof. The multi-element polyelectrolyte nanogel is formed by copolymerizing a first comonomer and a second comonomer, the first comonomer is a first cationic monomer, and the second comonomer is selected from one or more of a second cationic monomer, an anionic monomer, a zwitterionic monomer and a neutral monomer. By adding different types of monomers, the structure of the nanogel can be regulated and controlled, and a neutral or amphoteric shell layer structure or a core-free shell structure is formed. The multi-element polyelectrolyte nanogel is uniform in particle size distribution, low in toxicity and excellent in biocompatibility, and the transfection rate of the multi-element polyelectrolyte nanogel to various nucleic acids is higher than that of commercial reagents.

Description

Technical Field

[0001] The invention belongs to the fields of biotechnology and material synthesis, and relates to a multi-component polyelectrolyte nanogel and a preparation method and application thereof. Background Art

[0002] In recent years, gene therapy based on nucleic acid drugs has been widely used to intervene in and treat a variety of diseases. However, due to their large molecular weight, susceptibility to degradation in the body, and poor autonomous transmembrane transport, nucleic acids typically require safe and efficient delivery systems to improve their bioavailability and therapeutic efficacy. Cationic polymers and their derivatives are an important class of nucleic acid drug delivery vehicles. Existing cationic polymer carriers mostly use a single monomer, which has structural and functional limitations.

[0003] Based on this, the present invention integrates the advantages of multiple monomers through a multi-monomer copolymerization strategy to synergistically optimize the application performance of nucleic acid delivery vectors. Summary of the Invention

[0004] The present invention addresses the problems of the prior art and proposes a synthesis process and application of a multi-electrolyte nanogel. Based on an electrostatic template polymerization method, the present invention copolymerizes a cationic monomer with one or more cationic, anionic, zwitterionic, or neutral monomers to obtain a multi-electrolyte nanogel that can selectively have a shell structure. The multi-electrolyte nanogel of the present invention is designed to combine the functional advantages of different monomers to synergistically enhance the nucleic acid delivery performance of the vector and mediate the efficient transfection of different nucleic acid molecules.

[0005] One aspect of the present invention provides a multi-electrolyte nanogel, which includes a copolymerization product of a first comonomer and a second comonomer, wherein the first comonomer is a first cationic monomer, and the second comonomer is selected from one or more of a second cationic monomer, an anionic monomer, a zwitterionic monomer and a neutral monomer.

[0006] In one or more embodiments, the multi-electrolyte nanogel has a core-shell structure or does not have a core-shell structure.

[0007] In one or more embodiments, the shell layer of the multi-electrolyte nanogel having a core-shell structure comprises a neutral polymer or an amphoteric polymer.

[0008] In one or more embodiments, the surface of the multi-electrolyte nanogel is grafted with targeting molecules or is not grafted with targeting molecules.

[0009] In one or more embodiments, the first cationic monomer and the second cationic monomer are different and are each independently a compound of Formula I:

[0010]

[0011] in,

[0012] R1 is -H or -CH3;

[0013] R2 is -C(=O)-NH-, -C(=O)-O- or -C6H4-;

[0014] R3 is -N-R4R5, -P-R6R7 or -NH-C(=NH)-NH2, wherein R4, R5, R6, and R7 are each independently H or C1-C5 alkyl;

[0015] l is 0, 1, 2, 3, 4 or 5.

[0016] In one or more embodiments, the anionic monomer is a compound of formula II:

[0017]

[0018] in,

[0019] R8 is -H or -CH3;

[0020] R9 is -C(=O)-NH-, -C(=O)-O-, -C6H4- or a covalent bond;

[0021] R 10 It is -COOH, -SO3H, -B(OH)2 or -PO3H2.

[0022] In one or more embodiments, the zwitterionic monomer is a compound of formula III:

[0023]

[0024] in,

[0025] R 11 is -H or -CH3;

[0026] R 12 is -C(=O)-NH-, -C(=O)-O- or -C6H4-;

[0027] R 13 -N + (CH3)2-or-OP(=O)(-OH)-O-;

[0028] R 14 -COOH-, -N + (CH3)3 or -SO3H;

[0029] m is 0, 1, 2, 3, 4 or 5, and n is 0, 1, 2, 3, 4 or 5.

[0030] In one or more embodiments, the neutral monomer is selected from one or more of acrylamide and fluorine-containing monomers.

[0031] In one or more embodiments, the fluorine-containing monomer is a compound of formula IV:

[0032]

[0033] in,

[0034] R 15 is -H or -CH3;

[0035] R 16 is -C(=O)-NH-, -C(=O)-O-, -C6H4- or a covalent bond;

[0036] R 17 C n F 2n+1 , n is 1, 2, 3, 4, 5, 6, 7, 8 or 9.

[0037] In one or more embodiments, the molar ratio of the first comonomer to the second comonomer is 1:(0.01-100).

[0038] In one or more embodiments, the neutral polymer in the shell layer is selected from one or more of polyethylene glycol, polyethylene, and polytetrafluoroethylene.

[0039] In one or more embodiments, the amphoteric polymer in the shell layer is selected from one or both of poly (2-methacryloyloxyethyl) phosphorylcholine and poly (methacryloyloxyethyl) sulfobetaine.

[0040] In one or more embodiments, the targeting molecule is selected from one or more of folic acid, alendronate sodium, transferrin, hyaluronic acid, polypeptide, mannose and biotin.

[0041] In one or more embodiments, the multi-electrolyte nanogel has a particle size of 40-400 nm.

[0042] Another aspect of the present invention provides a method for preparing multi-electrolyte nanogel.

[0043] In one or more embodiments, when the multi-electrolyte nanogel has a shell layer, the method includes the following steps:

[0044] (1) mixing a first comonomer, a second comonomer, an anionic homopolymer template, a crosslinking agent, a composite chain transfer agent, and an initiator, and adjusting the pH to electroneutrality, wherein the composite chain transfer agent comprises (a) a neutral polymer or an amphoteric polymer and (b) a chain transfer agent;

[0045] (2) obtaining nanogel composites by photo- or thermal-induced polymerization;

[0046] (3) removing the anionic homopolymer template in the nanogel complex to obtain the multi-electrolyte nanogel.

[0047] In one or more embodiments, when the multi-electrolyte nanogel does not have a shell layer, the method includes the following steps:

[0048] (1') mixing a first comonomer, a second comonomer, a block polymer template, a crosslinker, and an initiator, and adjusting the pH to electroneutrality, wherein the block polymer template comprises (a') a neutral polymer or an amphoteric polymer and (b') a polyanion connected thereto;

[0049] (2') obtaining a nanogel complex by photo- or thermal-initiated polymerization;

[0050] (3') removing the block polymer template in the nanogel complex to obtain the multi-electrolyte nanogel.

[0051] In one or more embodiments, in step (1), the anionic homopolymer template is selected from one or more of polyacrylic acid, sodium poly (2-acrylamido-2-methylpropanesulfonate), sodium poly (p-styrenesulfonate), poly (dimethylaminoethyl methacrylate), poly (methacryloyloxyethyl trimethylammonium chloride), poly (2-aminoethyl methacrylate), polyanion-polyethylene glycol block copolymer and polyanionic copolymer.

[0052] In one or more embodiments, in step (1), the neutral polymer of the composite chain transfer agent is selected from one or more of polyethylene glycol, polyethylene and polytetrafluoroethylene, and the amphoteric polymer in the composite chain transfer agent is selected from one or more of poly-2-methacryloyloxyethyl phosphorylcholine, poly-methacryloylethyl sulfobetaine, poly-3-[[2-(methacryloyloxy)ethyl]dimethylammonium]propionate and poly-3-[(3-acrylamidopropyl)dimethylammonium]propionate.

[0053] In one or more embodiments, in step (1), the chain transfer agent in the composite chain transfer agent is selected from one or more of methyl (phenyl) aminodithiocarboxylic acid cyanomethyl ester, S-cyanomethyl-S-dodecyl trithiocarbonate, dithiobenzoic acid cyanoisopropyl ester, 4-cyano-4-(thiobenzoyl) pentanoic acid, S-(2-cyano-2-propyl)-S-dodecyl trithiocarbonyl ester, 4-cyano-4-[[(dodecylthio)thioketomethyl]thio] pentanoic acid and 2-(dodecyl trithiocarbonate)-2-methylpropionic acid.

[0054] In one or more embodiments, in step (1), the cross-linking agent is selected from one or more of diallyl disulfide, N,N'-bis(acryloyl)cystamine, 2,2-dithiodiethanol diacrylic acid, and N,N-methylenebisacrylamide.

[0055] In one or more embodiments, in step (1), the initiator is selected from one or more of a photoinitiator and a thermal initiator.

[0056] In one or more embodiments, in step (1), the initiator is selected from one or more of 2-hydroxy-2-methyl-1-phenyl-1-propanone, 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone, lithium phenyl-2,4,6-trimethylbenzoylphosphinate, persulfate and water-soluble azo compounds.

[0057] In one or more embodiments, in step (1'), the neutral polymer in the block polymer template is selected from one or more of polyethylene glycol, polyethylene, and polytetrafluoroethylene.

[0058] In one or more embodiments, in step (1'), the amphoteric polymer in the block polymer template is selected from one or both of poly (2-methacryloyloxyethyl) phosphorylcholine and poly (methacryloyloxyethyl) sulfobetaine.

[0059] In one or more embodiments, in step (1'), the polyanion in the block polymer template is selected from one or more of polyacrylic acid, sodium poly (2-acrylamido-2-methylpropanesulfonate), sodium poly (p-styrenesulfonate), poly (dimethylaminoethyl methacrylate), poly (methacryloyloxyethyl trimethylammonium chloride), poly (2-aminoethyl methacrylate), and polyanion-polyethylene glycol block copolymer.

[0060] In one or more embodiments, in step (1'), the cross-linking agent is selected from one or more of diallyl disulfide, N,N'-bis(acryloyl)cystamine, 2,2-dithiodiethanol diacrylic acid, and N,N-methylenebisacrylamide.

[0061] In one or more embodiments, in step (1'), the initiator is selected from one or more of a photoinitiator and a thermal initiator.

[0062] In one or more embodiments, in step (1'), the initiator is selected from one or more of 2-hydroxy-2-methyl-1-phenyl-1-propanone, 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone, lithium phenyl-2,4,6-trimethylbenzoylphosphinate, persulfate and water-soluble azo compounds.

[0063] In one or more embodiments, in step (1) and step (1'), the charge concentration of the monomer in the mixture system is 5-500 mmol / L.

[0064] In one or more embodiments, in step (1), the charge concentration of the anionic homopolymer template in the mixture system is 5-500 mmol / L.

[0065] In one or more embodiments, in step (1'), the charge concentration of the block polymer template in the mixture system is 5-500 mmol / L.

[0066] In one or more embodiments, in step (1) and step (1'), the amount of the cross-linking agent is 1% to 50% of the amount of the ionic monomer.

[0067] In one or more embodiments, in step (1), the composite chain transfer agent is grafted with or not grafted with a targeting molecule.

[0068] In one or more embodiments, in step (2) and step (2'), the photoinitiation is to initiate polymerization of a mixture system containing a photoinitiator under irradiation with an ultraviolet lamp at 200-400 nm for 1-12 hours.

[0069] In one or more embodiments, in step (2) and step (2'), the thermal initiation is to initiate polymerization of a mixture system containing a thermal initiator at 60°C-80°C for 1-12 hours.

[0070] In one or more embodiments, in step (3) and step (3'), the anionic homopolymer template or block polymer template in the nanogel complex is removed by ultrafiltration, centrifugation or dialysis using an inorganic salt solution.

[0071] In one or more embodiments, the inorganic salt in the inorganic salt solution is selected from one or more of sodium chloride, potassium chloride, sodium nitrate, potassium nitrate, sodium sulfate and potassium sulfate.

[0072] In one or more embodiments, the concentration of the inorganic salt in the inorganic salt solution is 0.1-5 mol / L.

[0073] The present invention provides use of the multi-electrolyte nanogel according to any embodiment of the present invention in nucleic acid transfection.

[0074] The present invention provides a nanogel-nucleic acid complex, which comprises nucleic acid and a multi-electrolyte nanogel prepared by the method described in any embodiment of the present invention.

[0075] In one or more embodiments, the nanogel-nucleic acid complex has a nitrogen to phosphorus ratio of 0.1-1000.

[0076] In one or more embodiments, the method for preparing the nanogel-nucleic acid complex comprises mixing the multi-electrolyte nanogel with nucleic acid in a culture medium to obtain the nanogel-nucleic acid complex.

[0077] In one or more embodiments, the nucleic acid is selected from one or more of small interfering RNA (siRNA), messenger RNA (mRNA), and plasmid (pDNA).

[0078] In one or more embodiments, the culture medium is Dulbecco's Modified Eagle Medium (DMEM), Minimum Essential Medium (MEM), or RPMI-1640 medium.

[0079] Another aspect of the present invention provides use of the nanogel-nucleic acid complex in nucleic acid transfection. BRIEF DESCRIPTION OF THE DRAWINGS

[0080] Figure 1 This is the particle size distribution curve of nanogel NG1 with different particle sizes in Preparation Example 2.

[0081] Figure 2 The particle size and polymer dispersibility index (PDI) of the nanogel NG1 with different crosslinking degrees in Preparation Example 4 are shown.

[0082] Figure 3 This is the particle size distribution curve of nanogels NG3-NG10 with different components in Preparation Example 5.

[0083] Figure 4 This is a curve showing the change in light intensity over time for the nanogel NG1 in Preparation Example 2 in a 10 mmol / L GSH solution.

[0084] Figure 5 The cytotoxicity comparison results of the nanogel NG1 prepared in Preparation Example 2 and the commercial transfection reagent Lipo 3000 are shown.

[0085] Figure 6These are the fluorescence images of siRNA delivered by NG1 and NG10 prepared in Preparation Examples 2 and 5, and the nucleic acid transfection rates of NG1, NG10 and Lipo 3000 in Hela cells.

[0086] Figure 7 These are the fluorescence images of mRNA delivery by NG1 and NG10 prepared in Preparation Examples 2 and 5, and the nucleic acid transfection rates of NG1, NG10 and Lipo 3000 in Hela cells.

[0087] Figure 8 These are the fluorescence images of pDNA delivered by NG1 and NG10 prepared in Preparation Examples 2 and 5, and the nucleic acid transfection rates of NG1, NG10 and Lipo 3000 in Hela cells. DETAILED DESCRIPTION

[0088] To facilitate understanding of the features and effects of the present invention by those skilled in the art, the following provides a general description and definition of the terms and expressions used herein. Unless otherwise indicated, all technical and scientific terms used herein have the ordinary meanings as understood by those skilled in the art regarding the present invention. In the event of conflict, the definitions in this specification shall prevail.

[0089] The theories or mechanisms described and disclosed herein, whether correct or incorrect, should not limit the scope of the present invention in any way, that is, the present invention can be implemented without being limited by any specific theory or mechanism.

[0090] Herein, “comprising,” “including,” “containing” and similar terms encompass the meanings of “consisting essentially of” and “consisting of,” for example, when “A comprises B and C” is disclosed herein, “A consists essentially of B and C” and “A consists of B and C” should be deemed to have been disclosed herein.

[0091] Herein, all features such as values, amounts, amounts, and concentrations defined in numerical ranges or percentage ranges are for brevity and convenience only. Accordingly, the description of numerical ranges or percentage ranges should be considered to include and specifically disclose all possible subranges and individual values ​​within the range (including integers and fractions).

[0092] In this document, unless otherwise specified, percentage refers to mass percentage and ratio refers to mass ratio.

[0093] Herein, the sum of the percentages of the various components of the composition is 100%.

[0094] Herein, when describing embodiments or examples, it should be understood that they are not intended to limit the present invention to these embodiments or examples. On the contrary, all substitutes, modifications and equivalents of the methods and materials described herein are encompassed within the scope of the present invention.

[0095] In this document, for the sake of brevity, not all possible combinations of the various technical features in each embodiment or example are described. Therefore, as long as there are no contradictions in the combination of these technical features, the various technical features in each embodiment or example can be combined in any way, and all possible combinations should be considered to be within the scope of this specification.

[0096] The present invention found that adding a second cationic monomer to the nanogel can improve the efficiency of drug entry into cells, induce a "proton sponge" effect, promote endosome escape, and thus improve the transfection efficiency of the multi-electrolyte nanogel.

[0097] The present invention also found that adding anionic monomers, zwitterionic monomers and neutral monomers to the nanogel can reduce the charge density, reduce the damage to the cell membrane and organelles, and reduce cytotoxicity.

[0098] The present invention provides a multi-electrolyte nanogel, a preparation method, and applications thereof. The multi-electrolyte nanogel comprises a copolymerization product of a first comonomer and a second comonomer, wherein the first comonomer is a first cationic monomer and the second comonomer is selected from one or more of a second cationic monomer, an anionic monomer, a zwitterionic monomer, and a neutral monomer.

[0099] In some embodiments, the multi-electrolyte nanogel has a core-shell structure or does not have a core-shell structure.

[0100] In some embodiments, the shell layer of the multi-electrolyte nanogel having a core-shell structure comprises a neutral polymer or an amphoteric polymer.

[0101] In some embodiments, the surface of the multi-electrolyte nanogel is grafted with targeting molecules or is not grafted with targeting molecules.

[0102] In some embodiments, the first cationic monomer and the second cationic monomer are different and are each independently a compound of Formula I:

[0103]

[0104] in,

[0105] R1 is -H or -CH3;

[0106] R2 is -C(=O)-NH-, -C(=O)-O- or -C6H4-;

[0107] R3 is -N-R4R5, -P-R6R7 or -NH-C(=NH)-NH2, wherein R4, R5, R6, and R7 are each independently H or C1-C5 alkyl;

[0108] l is 0, 1, 2, 3, 4 or 5.

[0109] In some embodiments, the anionic monomer is a compound of formula II:

[0110]

[0111] in,

[0112] R8 is -H or -CH3;

[0113] R9 is -C(=O)-NH-, -C(=O)-O-, -C6H4- or a covalent bond;

[0114] R 10 It is -COOH, -SO3H, -B(OH)2 or -PO3H2.

[0115] In some embodiments, the zwitterionic monomer is a compound of Formula III:

[0116]

[0117] in,

[0118] R 11 is -H or -CH3;

[0119] R 12 is -C(=O)-NH-, -C(=O)-O- or -C6H4-;

[0120] R 13 -N + (CH3)2-or-OP(=O)(-OH)-O-;

[0121] R 14 -COOH-, -N + (CH3)3 or -SO3H;

[0122] m is 0, 1, 2, 3, 4 or 5, and n is 0, 1, 2, 3, 4 or 5.

[0123] In some embodiments, the neutral monomer is selected from one or more of acrylamide and a fluorine-containing monomer.

[0124] In some embodiments, the fluorine-containing monomer is a compound of formula IV:

[0125]

[0126] in,

[0127] R 15 is -H or -CH3;

[0128] R 16 is -C(=O)-NH-, -C(=O)-O-, -C6H4- or a covalent bond;

[0129] R 17 C n F 2n+1 , n is 1, 2, 3, 4, 5, 6, 7, 8 or 9.

[0130] In some embodiments, the amount of the first comonomer to the second comonomer is in a ratio of 1:(0.01-100), such as 1:0.01, 1:0.05, 1:0.1, 1:0.2, 1:0.5, 1:1, 1:1.5, 1:2, 1:5, 1:10, 1:15, 1:20, 1:25, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, 1:100.

[0131] In some embodiments, the neutral polymer in the shell layer is selected from one or more of polyethylene glycol, polyethylene, and polytetrafluoroethylene.

[0132] In some embodiments, the amphiphilic polymer in the shell layer is selected from one or both of poly-2-methacryloyloxyethyl phosphorylcholine and poly-methacryloyloxyethyl sulfobetaine.

[0133] In some embodiments, the targeting molecule is selected from one or more of folic acid, alendronate, transferrin, hyaluronic acid, a polypeptide, mannose, and biotin.

[0134] In some embodiments, the particle size of the multi-electrolyte nanogel is 40-400 nm, such as 40 nm, 40-100 nm, 50 nm, 50-100 nm, 60 nm, 50-100 nm, 70 nm, 70-100 nm, 70-110 nm, 70-120 nm, 70-150 nm, 80 nm, 80-150 nm, 80-200 nm, 80-300 nm, 90 nm, 90-150 nm, 90-180 nm, 90-200 nm, 90-300 nm, 90-400 nm, 100 nm, 110 nm, 120 nm, 120-250 nm, 120-200 nm, 120-250 nm, 120-250 nm, 120-250 nm, 120-250 nm, 120-250 nm, 120-250 nm, 120-250 nm, 120-250 nm, 0nm, 120-300nm, 120-400nm, 130nm, 140nm, 150nm, 150-200nm, 150-30 0nm, 160nm, 170nm, 180nm, 190nm, 200nm, 200-300nm, 210nm, 220nm, 23 0nm, 240nm, 250nm, 260nm, 270nm, 280nm, 290nm, 300nm, 300-400nm, 31 0nm, 320nm, 330nm, 340nm, 350nm, 360nm, 370nm, 380nm, 390nm, 400nm,.

[0135] In some embodiments, when the multi-electrolyte nanogel has a shell layer, the method for preparing the multi-electrolyte nanogel comprises the following steps:

[0136] (1) mixing a first comonomer, a second comonomer, an anionic homopolymer template, a crosslinking agent, a composite chain transfer agent, and an initiator, and adjusting the pH to electroneutrality, wherein the composite chain transfer agent comprises (a) a neutral polymer or an amphoteric polymer and (b) a chain transfer agent;

[0137] (2) obtaining nanogel composites by photo- or thermal-induced polymerization;

[0138] (3) The anionic homopolymer template in the nanogel complex is removed to obtain a multi-electrolyte nanogel.

[0139] In some embodiments, when the multi-electrolyte nanogel does not have a shell layer, the method for preparing the multi-electrolyte nanogel comprises the following steps:

[0140] (1') mixing a first comonomer, a second comonomer, a block polymer template, a crosslinker, and an initiator, and adjusting the pH to electroneutrality, wherein the block polymer template comprises (a') a neutral polymer or an amphoteric polymer and (b') a polyanion connected thereto;

[0141] (2') obtaining a nanogel complex by photo- or thermal-initiated polymerization;

[0142] (3') Removing the block polymer template from the nanogel complex yields a multi-electrolyte nanogel.

[0143] In some embodiments, in step (1), the anionic homopolymer template is selected from one or more of polyacrylic acid, sodium poly (2-acrylamido-2-methylpropanesulfonate), sodium poly (p-styrenesulfonate), poly (dimethylaminoethyl methacrylate), poly (methacryloyloxyethyl trimethylammonium chloride), poly (2-aminoethyl methacrylate), polyanion-polyethylene glycol block copolymer and polyanionic copolymer.

[0144] In some embodiments, in step (1), the neutral polymer of the composite chain transfer agent is selected from one or more of polyethylene glycol, polyethylene, and polytetrafluoroethylene.

[0145] In some embodiments, in step (1), the amphoteric polymer in the composite chain transfer agent is selected from one or more of poly 2-methacryloyloxyethyl phosphorylcholine, poly methacryloylethyl sulfobetaine, poly 3-[[2-(methacryloyloxy)ethyl]dimethylammonium]propionate, and poly 3-[(3-acrylamidopropyl)dimethylammonium]propionate.

[0146] In some embodiments, in step (1), the chain transfer agent in the composite chain transfer agent is selected from one or more of methyl(phenyl)aminodithiocarboxylic acid cyanomethyl ester, S-cyanomethyl-S-dodecyl trithiocarbonate, dithiobenzoic acid cyanoisopropyl ester, 4-cyano-4-(thiobenzoyl) pentanoic acid, S-(2-cyano-2-propyl)-S-dodecyl trithiocarbonyl ester, 4-cyano-4-[[(dodecylthio)thioketomethyl]thio] pentanoic acid and 2-(dodecyltrithiocarbonate)-2-methylpropionic acid.

[0147] In some embodiments, in step (1), the cross-linking agent is selected from one or more of diallyl disulfide, N,N'-bis(acryloyl)cystamine, 2,2-dithiodiethanol diacrylic acid, and N,N-methylenebisacrylamide.

[0148] In some embodiments, in step (1), the initiator is selected from one or more of a photoinitiator and a thermal initiator.

[0149] In some embodiments, in step (1), the initiator is selected from one or more of 2-hydroxy-2-methyl-1-phenyl-1-propanone, 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone, lithium phenyl-2,4,6-trimethylbenzoylphosphinate, persulfate and water-soluble azo compounds.

[0150] In some embodiments, in step (1'), the neutral polymer in the block polymer template is selected from one or more of polyethylene glycol, polyethylene, and polytetrafluoroethylene.

[0151] In some embodiments, the amphiphilic polymer in the block polymer template is selected from one or both of poly (2-methacryloyloxyethyl) phosphorylcholine and poly (methacryloyloxyethyl) sulfobetaine.

[0152] In some embodiments, in step (1'), the polyanion in the block polymer template is selected from one or more of polyacrylic acid, sodium poly (2-acrylamido-2-methylpropanesulfonate), sodium poly (p-styrenesulfonate), poly (dimethylaminoethyl methacrylate), poly (methacryloyloxyethyl trimethylammonium chloride), poly (2-aminoethyl methacrylate), and polyanion-polyethylene glycol block copolymer.

[0153] In some embodiments, in step (1'), the cross-linking agent is selected from one or more of diallyl disulfide, N,N'-bis(acryloyl)cystamine, 2,2-dithiodiethanol diacrylic acid, and N,N-methylenebisacrylamide.

[0154] In some embodiments, in step (1'), the initiator is selected from one or more of a photoinitiator and a thermal initiator.

[0155] In some embodiments, in step (1'), the initiator is selected from one or more of 2-hydroxy-2-methyl-1-phenyl-1-propanone, 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone, lithium phenyl-2,4,6-trimethylbenzoylphosphinate, persulfate and water-soluble azo compounds.

[0156] In some embodiments, in step (1) and step (1'), the charge concentration of the monomer in the mixture system is 5-500mmol / L, such as 5mmol / L, 10mmol / L, 15mmol / L, 20mmol / L, 25mmol / L, 30mmol / L, 40mmol / L, 50mmol / L, 60mmol / L, 70mmol / L, 80mmol / L, 90mmol / L, 100mmol / L, 120mmol / L, 150mmol / L, 200mmol / L, 250mmol / L, 300mmol / L, 350mmol / L, 400mmol / L, 450mmol / L, 500mmol / L.

[0157] In some embodiments, in step (1), the charge concentration of the anionic homopolymer template in the mixture system is 5-500mmol / L, such as 5mmol / L, 10mmol / L, 15mmol / L, 20mmol / L, 25mmol / L, 30mmol / L, 40mmol / L, 50mmol / L, 60mmol / L, 70mmol / L, 80mmol / L, 90mmol / L, 100mmol / L, 120mmol / L, 150mmol / L, 200mmol / L, 250mmol / L, 300mmol / L, 350mmol / L, 400mmol / L, 450mmol / L, 500mmol / L.

[0158] In some embodiments, in step (1'), the charge concentration of the block polymer template in the mixture system is 5-500mmol / L, such as 5mmol / L, 10mmol / L, 15mmol / L, 20mmol / L, 25mmol / L, 30mmol / L, 40mmol / L, 50mmol / L, 60mmol / L, 70mmol / L, 80mmol / L, 90mmol / L, 100mmol / L, 120mmol / L, 150mmol / L, 200mmol / L, 250mmol / L, 300mmol / L, 350mmol / L, 400mmol / L, 450mmol / L, 500mmol / L.

[0159] In some embodiments, in step (1) and step (1'), the amount of the cross-linking agent is 1%-50% of the amount of the ionic monomer, such as 1%, 2%, 5%, 10%, 12%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%.

[0160] In some embodiments, in step (1), the composite chain transfer agent is grafted with or not grafted with a targeting molecule.

[0161] In some embodiments, in step (2) and step (2'), photoinitiation is the polymerization of a mixture system containing a photoinitiator under 200-400 nm ultraviolet light for 1-12 hours, such as 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 8 hours, 10 hours, 11 hours, or 12 hours.

[0162] In some embodiments, in step (2) and step (2'), thermal initiation is to initiate polymerization of a mixture system containing a thermal initiator at 60°C-80°C for 1-12 hours, for example, the temperature is 60°C, 65°C, 70°C, 75°C, 80°C, and the polymerization time is 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 8 hours, 10 hours, 11 hours, 12 hours.

[0163] In some embodiments, in step (3) and step (3'), the anionic homopolymer template or block polymer template in the nanogel complex is removed by ultrafiltration, centrifugation or dialysis using an inorganic salt solution.

[0164] In some embodiments, the inorganic salt in the inorganic salt solution is selected from one or more of sodium chloride, potassium chloride, sodium nitrate, potassium nitrate, sodium sulfate, and potassium sulfate.

[0165] In some embodiments, the concentration of the inorganic salt in the inorganic salt solution is 0.1-5 mol / L, such as 0.1mmol / L, 0.2mmol / L, 0.25mmol / L, 0.5mmol / L, 0.6mmol / L, 0.7mmol / L, 0.8mmol / L, 0.9mmol / L, 1mmol / L, 1.2mmol / L, 1.5mmol / L, 1.8mmol / L, 2mmol / L, 2.5mmol / L, 3mmol / L, 3.5mmol / L, 4mmol / L, 4.5mmol / L, 5mmol / L.

[0166] The multi-electrolyte nanogel of the present invention can be used to prepare nanogel-nucleic acid complexes.

[0167] In some embodiments, the nanogel-nucleic acid complex comprises a nucleic acid and a multi-polymer polyelectrolyte nanogel.

[0168] In some embodiments, the nitrogen to phosphorus ratio of the nanogel-nucleic acid complex is 0.1-1000, for example, the nitrogen to phosphorus ratio can be 1, 2, 5, 10, 20, 50, 100, 200, 500.

[0169] In some embodiments, the method for preparing the nanogel-nucleic acid complex comprises mixing a multi-component polyelectrolyte nanogel with nucleic acid in a culture medium to obtain the nanogel-nucleic acid complex.

[0170] In some embodiments, the nucleic acid is selected from one or more of small interfering RNA (siRNA), messenger RNA (mRNA), and plasmid (pDNA).

[0171] In some embodiments, the culture medium is Dulbecco's Modified Eagle Medium (DMEM), Minimum Essential Medium (MEM), or RPMI-1640 medium.

[0172] The multi-electrolyte nanogel and nanogel-nucleic acid complex of the present invention can be used for nucleic acid transfection.

[0173] The present invention will be described below using specific examples. It should be understood that these examples are merely illustrative and are not intended to limit the scope of the present invention. The methods, reagents, and materials used in the examples and comparative examples are conventional methods, reagents, and materials in the art, unless otherwise stated. The starting compounds in the examples and comparative examples can all be purchased from commercial sources.

[0174] Preparation Example 1

[0175] Preparation of composite chain transfer agent

[0176] The composite chain transfer agent is folic acid-polyethylene glycol-4-cyano-4-(thiobenzoyl)valeric acid. 500.0 mg of NH2-terminated polyethylene glycol and 21.8 mg of di-tert-butyl dicarbonate were weighed and reacted in a tert-butyl alcohol solution containing 10 wt% sodium hydroxide at room temperature for 8 hours. The resulting reactant was mixed with 44.1 mg of folic acid, 25.8 mg of dicyclohexylcarbodiimide, and 1.8 mg of 4-dimethylaminopyridine. After reacting at room temperature for 8 hours, the mixture was washed with 0.1 mol / L hydrochloric acid to obtain the folic acid-modified polyethylene glycol. Then, 279.4 mg of 4-cyano-4-(thiobenzoyl)valeric acid and 2.4 mg of 4-dimethylaminopyridine were weighed and mixed with 500.0 mg of the folic acid-modified polyethylene glycol, deoxygenated by nitrogen evacuation, and stirred for 20 minutes. Add 206.3 mg of dicyclohexylcarbodiimide dropwise through a constant pressure funnel at a rate of 3 seconds per drop. The mixed solution was allowed to react at 25°C for 48 hours. Insoluble matter was removed by filtration, and the solid was precipitated with ether. After vacuum drying, the folic acid- and chain transfer agent-modified polyethylene glycol was obtained. The weight-average molecular weight of the polyacrylic acid was 6984, while the weight-average molecular weights of the polyethylene glycol and polyacrylic acid in the polyethylene glycol-polyacrylic acid were 3784 and 10768, respectively.

[0177] Preparation Example 2

[0178] Preparation of core-shell NG1 nanogels

[0179] Weigh 44.7 mg of folic acid-polyethylene glycol-4-cyano-4-(thiobenzoyl)valeric acid, 29.0 mg of polyacrylic acid, 64.5 mg of N-4-vinylphenyl-N,N-dimethylamine (monomer concentration is 20 mmol / L), 84.7 mg of benzylethyltrimethylammonium chloride (monomer concentration is 20 mmol / L), 41.7 mg of N,N'-bis(acryloyl)cystamine (20 mol% of ionic monomer concentration), and 3.0 mg of 2-hydroxy-2-methyl-1-phenyl-1-propanone (2 wt% of monomer mass), and dissolve them in 20 mL of deionized water. Adjust the pH of the solution to electroneutrality, pump nitrogen to remove nitrogen, and react under 300 nm ultraviolet light for 10 hours with continuous stirring. After the reaction, a multi-electrolyte nanogel complex can be obtained. Add 2 mol / L sodium chloride solution to the above solution, use this concentration of sodium chloride as eluent to wash 8 times with an ultrafiltration centrifuge to remove the template and recycle it, then dialyze with deionized water to remove sodium chloride, and finally obtain multi-electrolyte nanogel NG1 with different particle sizes. Figure 1 shown.

[0180] Preparation Example 3

[0181] Preparation of shell-free nanogel NG2

[0182] 21.8 mg of polyethylene glycol-polyacrylic acid, 64.5 mg of N-4-vinylphenyl-N,N-dimethylamine (monomer concentration 20 mmol / L), 84.7 mg of benzylethyltrimethylammonium chloride (monomer concentration 20 mmol / L), 41.7 mg of N,N'-bis(acryloyl)cystamine (20 mol% of the ionic monomer concentration), and 3.0 mg of 2-hydroxy-2-methyl-1-phenyl-1-propanone (2 wt% of the monomer mass) were weighed and dissolved in 20 mL of deionized water. The solution pH was adjusted to electroneutrality, and nitrogen was removed by purging. The reaction was carried out under 300 nm ultraviolet light for 10 hours with continuous stirring. A multi-electrolyte nanogel complex was obtained. A 2 mol / L sodium chloride solution was added to the solution, and the solution was washed eight times with this concentration of sodium chloride as the eluent using an ultrafiltration centrifuge to remove the template and recycle it. The sodium chloride was then dialyzed against deionized water to remove the sodium chloride, resulting in a purified multi-electrolyte nanogel.

[0183] Preparation Example 4

[0184] Preparation of core-shell nanogels NG1 with different cross-linking degrees

[0185] Weigh 44.7 mg of folic acid-polyethylene glycol-4-cyano-4-(thiobenzoyl)valeric acid, 29.0 mg of polyacrylic acid, 64.5 mg of N-4-vinylphenyl-N,N-dimethylamine (monomer concentration is 20 mmol / L), 84.7 mg of benzylethyltrimethylammonium chloride (monomer concentration is 20 mmol / L), 20.8-104.2 mg of N,N'-bis(acryloyl)cystamine (10 mol%-50 mol% of ionic monomer concentration), and 3.0 mg of 2-hydroxy-2-methyl-1-phenyl-1-propanone (2 wt% of monomer mass), dissolve them in 20 mL of deionized water, adjust the pH of the solution to electroneutrality, pump nitrogen to remove nitrogen, and react under 300 nm ultraviolet light for 10 hours with continuous stirring. After the reaction, a multi-electrolyte nanogel complex can be obtained. Add 2 mol / L sodium chloride solution to the above solution, use this concentration of sodium chloride as eluent to wash 6-8 times with an ultrafiltration centrifuge to remove the template and recycle it, then dialyze with deionized water to remove sodium chloride, and finally obtain purified multi-electrolyte nanogel. The particle size and PDI of nanogel NG1 with different cross-linking degrees are as follows: Figure 2 shown.

[0186] Preparation Example 5

[0187] Preparation of nanogels NG3-NG10 with different components

[0188] Nanogels NG3-NG10 were prepared using folic acid-polyethylene glycol-4-cyano-4-(thiobenzoyl)valeric acid as a composite chain transfer agent, polyacrylic acid as an anionic homopolymer template, N-4-vinylphenyl-N,N-dimethylamine as the first comonomer (monomer concentration was 20 mmol / L), N,N'-bis(acryloyl)cystamine as a crosslinker (crosslinker concentration was 20 mol% of the ionic monomer concentration), and 2-hydroxy-2-methyl-1-phenyl-1-propanone as an initiator (2 wt% of the monomer mass). Different second comonomers were added (monomer concentration was 20 mmol / L). The masses of each substance and the second comonomer are shown in Table 1.

[0189] Dissolve the substances in each preparation group in Table 1 in 20 mL of deionized water, adjust the solution pH to electroneutrality, pump nitrogen gas to remove nitrogen, and react under 300 nm ultraviolet light for 10 hours with continuous stirring. After completion, a multi-electrolyte nanogel complex is obtained. Add 2 mol / L sodium chloride solution to the above solution and wash 6-8 times using an ultrafiltration centrifuge with this concentration of sodium chloride as the eluent to remove the template and recycle it. Then, dialyze against deionized water to remove the sodium chloride, ultimately obtaining purified multi-electrolyte nanogels NG3-NG10. Figure 3 is the particle size distribution of the prepared nanogel NG3-NG10.

[0190] Table 1: Mass of various substances used to prepare nanogels NG3-NG10

[0191]

[0192] Test Example 1

[0193] Evaluation of the degradability of nanogel NG1

[0194] The nanogel NG1 prepared in Example 2 was diluted with a 20 mmol / L PB solution at a pH of 7.4. GSH was then added to the solution to make the GSH concentration in the solution 10 mmol / L. Dynamic light scattering (DLS) was used to monitor the changes in the light intensity and particle size of the NG1-GSH complex over time. Figure 4 shown.

[0195] Depend on Figure 4 It can be seen that after the addition of GSH, the scattered light intensity of NG1 decreased rapidly within 10 minutes, confirming that the nanogel NG1 can be degraded in response to the GSH concentration, indicating that NG1 can be rapidly degraded in a reducing environment (such as inside the cell) and has low cytotoxicity.

[0196] Test Example 2

[0197] Cytotoxicity evaluation of nanogel NG1

[0198] HeLa cells were seeded into 96-well plates in advance and incubated overnight at 37°C and 5% CO2. The culture medium was removed, and 100 μL of complete culture medium solution containing different concentrations of NG1 or 100 μL of complete culture medium solution containing the commercial reagent Lipofectamine 3000 (referred to as Lipo 3000) was added to each well, and the cells were incubated for 24 hours. After incubation, the 96-well plate was taken out, the culture medium was removed, and 10 μL of CCK-8 reagent was added to each well. The cell viability was detected according to the standard steps of the CCK-8 method. Six samples were tested in each group of experiments, and the concentration of the commercial reagent Lipo 3000 was the concentration used for transfection in the product manual. The nanogel NG1 prepared in Preparation Example 2 was subjected to a cytotoxicity test with the commercial transfection reagent Lipo 3000, and the results are as follows. Figure 5 shown.

[0199] Depend on Figure 5 As can be seen, when the concentration of NG1 reaches 100 μg / mL, the survival rate of HeLa cells treated with NG1 exceeds 90%, significantly higher than the survival rate of HeLa cells treated with commercial reagents. This shows that the multi-electrolyte nanogel prepared by the present invention has low cytotoxicity, is superior to common commercial reagents, and has good biocompatibility.

[0200] Application Example 1

[0201] Multi-component polyelectrolyte nanogels NG1 and NG10 for delivery and transfection of siRNA

[0202] The method for delivering siRNA is as follows: HeLa cells are seeded into a 24-well plate and placed in an incubator at 37°C and 5% CO2 overnight. The cells are observed under a microscope. When the confluence of the HeLa cells reaches more than 80%, the nucleic acid delivery experiment is started.

[0203] NG1 prepared in Preparation Example 2 or NG10 prepared in Preparation Example 5 was added to 500 μL of complete culture medium and mixed evenly by pipetting. The concentration of NG1 (or NG10) was 20 μg / mL. 0.5 μg of siRNA-FAM was then added to the complete culture medium containing NG1 (or NG10) and vortexed for 10 seconds. The old cell culture medium was removed, and after washing twice with phosphate buffered saline (PBS), a culture medium solution containing the siRNA complex was added and incubated in an incubator for 24 hours. Afterwards, the nucleic acid delivery was qualitatively observed using a fluorescence microscope, or the mean fluorescence intensity of HeLa cells was quantitatively analyzed using a flow cytometer. The specific method is as follows: after the cells are treated with the culture medium solution containing the siRNA complex, the culture medium is removed, the cells are washed twice with PBS, and the cells are placed under a fluorescence microscope to observe the fine green fluorescence intensity; or after removing the culture medium and washing twice with PBS, the cells are collected by trypsin digestion, centrifuged and resuspended with PBS, and the green fluorescence intensity in the cells is detected by flow cytometry. Figure 6 Fluorescence images of NG1 and NG10 transfected with siRNA, and the nucleic acid transfection rates of NG1, NG10, and Lipo 3000 in Hela cells.

[0204] The specific procedure for siRNA transfection is as follows: HeLa cells are pre-seeded into a 96-well plate and placed in an incubator at 37°C and 5% CO2 overnight. NG1 prepared in Preparation Example 2 or NG10 prepared in Preparation Example 5 is added to complete culture medium and mixed thoroughly using a pipette. The concentration of NG1 (or NG10) is 20 μg / mL. A negative control small interfering RNA (siNC) or siVEGF is then added to the complete culture medium containing NG1 (or NG10), and the cells are vortexed for 10 seconds. The concentration of siNC or siVEGF is 100 nmol / L. The culture medium is removed, and 100 μL of complete culture medium containing the NG1 (or NG10)-siRNA complex is added. The cells are incubated for 24 hours. After the incubation period, the cell plate is removed, the culture medium is removed, and 10 μL of CCK-8 reagent is added to each well. Cell viability is determined using the standard CCK-8 assay. The difference between the cell viability in the siNC experimental group and the cell viability in the siVEGF experimental group is the apoptosis rate. Each experiment was repeated for 6 samples. The commercial reagent Lipo3000 was used as a control and transfection was performed according to the product manual.

[0205] Application Example 2

[0206] Multi-electrolyte nanogels NG1 and NG10 were used for transfection of green fluorescent protein mRNA (GFP mRNA) and plasmid (GFP pDNA)

[0207] HeLa cells were seeded into 24-well plates and placed in an incubator at 37°C and 5% CO2 overnight. The cells were observed under a microscope. When the confluence of HeLa cells reached more than 80%, the nucleic acid delivery experiment was started.

[0208] Add NG1 prepared in Preparation Example 2 or NG10 prepared in Preparation Example 5 to 500 μL of complete culture medium and mix them evenly by pipetting. The concentration of NG1 (or NG10) is 20 μg / mL. Then add 2.5 μg of GFP mRNA or GFP pDNA to the complete culture medium containing NG1 (or NG10) and vortex for 10 seconds. Remove the old cell culture medium in the well plate, wash it twice with PBS, add the culture medium solution containing the nucleic acid complex, and incubate it in an incubator for 24 hours. Then use a fluorescence microscope to qualitatively observe the nucleic acid transfection situation, or use a flow cytometer to quantitatively analyze the average fluorescence intensity of HeLa cells. The specific method is: after the cells are treated with the culture medium solution containing the nucleic acid complex, remove the culture medium, wash the cells twice with PBS, and observe the green fluorescence intensity in the cells with a fluorescence microscope; or after removing the culture medium and washing the cells twice with PBS, use trypsin to digest and collect the cells, resuspend them with PBS after centrifugation, and use a flow cytometer to detect the green fluorescence intensity in the cells. Figure 7Fluorescence images of NG1 and NG10 when transfected with mRNA, and the nucleic acid transfection rates of NG1, NG10 and Lipo 3000 in Hela cells. Figure 8 Fluorescence images of NG1 and NG10 transfected with pDNA, and the nucleic acid transfection rates of NG1, NG10, and Lipo 3000 in Hela cells.

[0209] Figure 1 The particle size distribution curve of the multi-component copolymer nanogel NG1 with different particle sizes in Preparation Example 2 is shown. The particle size distribution of the six particle sizes of NG1 nanogels R1-R6 is between 40-400 nm.

[0210] Figure 2 The particle size and PDI of the nanogel NG1 with different crosslinking degrees in Preparation Example 4. As can be seen from the figure, the particle size R of the nanogel NG1 with a crosslinking degree of 10-50 is h The particle size distribution is between 80-120 nm, and the polydispersity index PDI is mainly between 0.1-0.2, indicating that the nanogel NG1 with different cross-linking degrees has a stable particle size structure and uniform particle size.

[0211] Figure 3 This is the particle size distribution curve of the nanogels NG3-NG10 with different components in Preparation Example 5. The particle size distribution of the 8 nanogels with different components is between 40-400 nm.

[0212] Figure 4 The light intensity of the nanogel NG1 in a 10 mmol / L antioxidant GSH solution changes over time. The curve shows that the light intensity of the NG1-GSH complex decreases rapidly within 10 minutes, indicating that the nanogel NG1 of the present invention can be rapidly degraded in a reducing environment (such as within a cell) and has low cytotoxicity.

[0213] Figure 5 The cytotoxicity comparison results of the nanogel NG1 prepared in Preparation Example 2 and the commercial transfection reagent Lipo 3000 are shown. When NG1 was added at a concentration of 10-100 μg / mL, the survival rate of HeLa cells treated with NG1 consistently exceeded 90%, while the survival rate of HeLa cells treated with the commercial reagent Lipo 3000 was only 80%. This demonstrates that the multi-electrolyte nanogel prepared in this invention has low cytotoxicity, surpasses common commercial reagents, and has good biocompatibility.

[0214] from Figure 6-Figure 8 From the fluorescence images of NG1 and NG10 delivering siRNA, mRNA and pDNA, it can be seen that NG1 and NG10 showed good delivery effects in different nucleic acid delivery, indicating that the multi-electrolyte nanogel of the present invention has certain versatility in nucleic acid drug delivery.

[0215] from Figure 6-Figure 8 As shown in the bar graph of the transfection rates of NG1, NG10 and Lipo 3000 for three kinds of nucleic acids, the nucleic acid transfection rates of NG1 and NG10 of the present invention for siRNA, mRNA and pDNA are higher than the transfection rate of the commercial reagent Lipo 3000, indicating that the multi-electrolyte nanogel of the present invention has excellent transfection performance.

Claims

1. A multi-electrolyte nanogel, comprising a copolymerization product of a first comonomer and a second comonomer, wherein the first comonomer is a first cationic monomer, and the second comonomer is selected from one or more of a second cationic monomer, an anionic monomer, a zwitterionic monomer, and a neutral monomer; The multi-electrolyte nanogel has a core-shell structure or does not have a core-shell structure, and the shell layer of the multi-electrolyte nanogel with a core-shell structure comprises a neutral polymer or an amphoteric polymer; The surface of the multi-electrolyte nanogel is grafted with targeting molecules or is not grafted with targeting molecules; The first cationic monomer and the second cationic monomer are different and are each independently a compound of formula I: in, R1 is -H or -CH3; R2 is -C(=O)-NH-, -C(=O)-O- or -C6H4-; R3 is -N-R4R5, -P-R6R7 or -NH-C(=NH)-NH2, wherein R4, R5, R6, and R7 are each independently H or C1-C5 alkyl; l is 0, 1, 2, 3, 4 or 5; The anionic monomer is a compound of formula II: in, R8 is -H or -CH3; R9 is -C(=O)-NH-, -C(=O)-O-, -C6H4- or a covalent bond; R 10 is -COOH, -SO3H, -B(OH)2 or -PO3H2; The zwitterionic monomer is a compound of formula III: in, R 11 is -H or -CH3; R 12 is -C(=O)-NH-, -C(=O)-O- or -C6H4-; R 13 -N + (CH3)2-or-OP(=O)(-OH)-O-; R 14 -COOH-, -N + (CH3)3 or -SO3H; m is 0, 1, 2, 3, 4 or 5, n is 0, 1, 2, 3, 4 or 5; The neutral monomer is selected from one or more of acrylamide and a fluorine-containing monomer, and the fluorine-containing monomer is a compound of formula IV: in, R 15 is -H or -CH3; R 16 is -C(=O)-NH-, -C(=O)-O-, -C6H4- or a covalent bond; R 17 C n F 2n+1 , n is 1, 2, 3, 4, 5, 6, 7, 8 or 9.

2. The multi-electrolyte nanogel according to claim 1, wherein The multi-electrolyte nanogel has one or more of the following characteristics: The molar ratio of the first comonomer to the second comonomer is 1:(0.01-100); The neutral polymer in the shell layer is selected from one or more of polyethylene glycol, polyethylene and polytetrafluoroethylene, The amphoteric polymer in the shell layer is selected from one or both of poly-2-methacryloyloxyethyl phosphorylcholine and poly-methacryloyloxyethyl sulfobetaine; The targeting molecule is selected from one or more of folic acid, alendronate sodium, transferrin, hyaluronic acid, polypeptide, mannose and biotin.

3. The multi-electrolyte nanogel according to claim 1, wherein The particle size of the multi-electrolyte nanogel is 40-400nm.

4. A method for preparing the multi-electrolyte nanogel according to any one of claims 1 to 3, characterized in that: When the multi-electrolyte nanogel has a shell layer, the method comprises the following steps: (1) mixing a first comonomer, a second comonomer, an anionic homopolymer template, a crosslinking agent, a composite chain transfer agent, and an initiator, and adjusting the pH to electroneutrality, wherein the composite chain transfer agent comprises (a) a neutral polymer or an amphoteric polymer and (b) a chain transfer agent; (2) obtaining nanogel composites by photo- or thermal-induced polymerization; (3) removing the anionic homopolymer template in the nanogel complex to obtain the multi-electrolyte nanogel; When the multi-electrolyte nanogel does not have a shell layer, the method comprises the following steps: (1') mixing a first comonomer, a second comonomer, a block polymer template, a crosslinker, and an initiator, and adjusting the pH to electroneutrality, wherein the block polymer template comprises (a') a neutral polymer or an amphoteric polymer and (b') a polyanion connected thereto; (2') obtaining a nanogel complex by photo- or thermal-initiated polymerization; (3') removing the block polymer template in the nanogel complex to obtain the multi-electrolyte nanogel.

5. The method according to claim 4, wherein The method has one or more of the following characteristics: In step (1), the anionic homopolymer template is selected from one or more of polyacrylic acid, sodium poly (2-acrylamido-2-methylpropanesulfonate), sodium poly (p-styrenesulfonate), poly (dimethylaminoethyl methacrylate), poly (methacryloyloxyethyl trimethylammonium chloride), poly (2-aminoethyl methacrylate), polyanion-polyethylene glycol block copolymer and polyanionic copolymer; In step (1), the neutral polymer of the composite chain transfer agent is selected from one or more of polyethylene glycol, polyethylene and polytetrafluoroethylene, and the amphoteric polymer in the composite chain transfer agent is selected from one or more of poly-2-methacryloyloxyethyl phosphorylcholine, poly-methacryloylethyl sulfobetaine, poly-3-[[2-(methacryloyloxy)ethyl]dimethylammonium]propionate and poly-3-[(3-acrylamidopropyl)dimethylammonium]propionate; In step (1), the chain transfer agent in the composite chain transfer agent is selected from one or more of methyl (phenyl) aminodithiocarbamate, S-cyanomethyl-S-dodecyl trithiocarbonate, dithiobenzoic acid cyanoisopropyl ester, 4-cyano-4-(thiobenzoyl) valeric acid, S-(2-cyano-2-propyl)-S-dodecyl trithiocarbonyl ester, 4-cyano-4-[[(dodecylthio)thioketonemethyl]thio] valeric acid and 2-(dodecyl trithiocarbonate)-2-methylpropionic acid; In step (1), the cross-linking agent is selected from one or more of diallyl disulfide, N,N'-bis(acryloyl)cystamine, 2,2-dithiodiethanol diacrylate and N,N-methylenebisacrylamide; In step (1), the initiator is selected from one or more of a photoinitiator and a thermal initiator; preferably, in step (1), the initiator is selected from one or more of 2-hydroxy-2-methyl-1-phenyl-1-propanone, 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone, lithium phenyl-2,4,6-trimethylbenzoylphosphinate, persulfate and water-soluble azo compounds.

6. The method according to claim 4, wherein: The method has one or more of the following characteristics: In step (1'), the neutral polymer in the block polymer template is selected from one or more of polyethylene glycol, polyethylene and polytetrafluoroethylene, and the amphoteric polymer in the block polymer template is selected from one or both of poly (2-methacryloyloxyethyl) phosphorylcholine and poly (methacryloyloxyethyl) sulfobetaine; In step (1'), the polyanion in the block polymer template is selected from one or more of polyacrylic acid, sodium poly (2-acrylamido-2-methylpropanesulfonate), sodium poly (p-styrenesulfonate), poly (dimethylaminoethyl methacrylate), poly (methacryloyloxyethyl trimethylammonium chloride), poly (2-aminoethyl methacrylate), and polyanion-polyethylene glycol block copolymer; In step (1'), the cross-linking agent is selected from one or more of diallyl disulfide, N,N'-bis(acryloyl)cystamine, 2,2-dithiodiethanol diacrylate and N,N-methylenebisacrylamide; In step (1'), the initiator is selected from one or more of a photoinitiator and a thermal initiator; preferably, in step (1'), the initiator is selected from one or more of 2-hydroxy-2-methyl-1-phenyl-1-propanone, 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone, lithium phenyl-2,4,6-trimethylbenzoylphosphinate, persulfate and water-soluble azo compounds.

7. The method according to claim 4, wherein The method has one or more of the following characteristics: In step (1) and step (1'), the charge concentration of the monomer in the mixture system is 5-500 mmol / L; In step (1), the charge concentration of the anionic homopolymer template in the mixture system is 5-500 mmol / L; In step (1'), the charge concentration of the block polymer template in the mixture system is 5-500 mmol / L; In step (1) and step (1'), the amount of the cross-linking agent is 1% to 50% of the amount of the ionic monomer; In step (1), the composite chain transfer agent is grafted with or not grafted with a targeting molecule; In step (2) and step (2'), the photoinitiation is to initiate polymerization of a mixture system containing a photoinitiator under irradiation of an ultraviolet lamp at 200-400nm for 1-12 hours; In step (2) and step (2'), the thermal initiation is to initiate polymerization of a mixture system containing a thermal initiator at 60°C-80°C for 1-12 hours; In step (3) and step (3'), an anionic homopolymer template or block polymer template in the nanogel complex is removed by ultrafiltration, centrifugation or dialysis using an inorganic salt solution; preferably, the inorganic salt in the inorganic salt solution 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.

8. Use of the multi-electrolyte nanogel according to any one of claims 1 to 3 or the multi-electrolyte nanogel prepared by the method according to any one of claims 4 to 7 in nucleic acid transfection.

9. A nanogel-nucleic acid complex, characterized in that: The nanogel-nucleic acid complex comprises nucleic acid and the multi-electrolyte nanogel according to any one of claims 1 to 3 or the multi-electrolyte nanogel prepared by the method according to any one of claims 4 to 7; The nitrogen to phosphorus ratio of the nanogel-nucleic acid complex is 0.1-1000; Preferably, the method for preparing the nanogel-nucleic acid complex comprises: mixing a multi-electrolyte nanogel with nucleic acid in a culture medium to obtain the nanogel-nucleic acid complex; Preferably, the nucleic acid is selected from one or more of small interfering RNA (siRNA), messenger RNA (mRNA) and plasmid (pDNA); Preferably, the culture medium is Dulbecco's Modified Eagle Medium (DMEM), Minimum Essential Medium (MEM) or RPMI-1640 medium.

10. Use of the nanogel-nucleic acid complex according to claim 9 in nucleic acid transfection.

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