Cationic nanogel and its preparation method and use

Abstract

The present invention discloses a cationic nanogel, a preparation method, and uses thereof. The cationic nanogel has a core-shell structure, wherein the core structure comprises one or more cationic monomers having a structure as shown in Formula I, and the shell structure comprises a neutral polymer.

Description

Technical Field

[0001] The present invention relates to the field of polymer chemistry and biotechnology, and in particular to a method for preparing a core-shell cationic nanogel and an application thereof as a nucleic acid transfection carrier. Background Art

[0002] Gene therapy involves the introduction of exogenous nucleic acids into cells to replace or modify the expression of defective genes or to express specific functional proteins, thereby regulating cellular biological activities and treating diseases. Gene therapy holds great promise for the treatment of a variety of diseases, including genetic disorders, cancer, type 2 diabetes, and Alzheimer's disease, as well as for vaccine development. However, due to their large molecular weight, negative charge, and high hydrophilicity, nucleic acids are difficult to directly uptake by cells. Therefore, the design and development of safe and efficient delivery vectors is crucial for the effective use of nucleic acid drugs.

[0003] Currently, commonly used nucleic acid delivery vectors can be divided into viral vectors and non-viral vectors. Viral vectors have high delivery efficiency, but high production costs and potential carcinogenicity. Non-viral vectors mainly include cationic liposomes and cationic polymers. These vectors have good biosafety, wide applicability, and are easy to prepare on a large scale and functionally modify, making them the mainstream direction of commercial transfection reagent development. However, existing non-viral vectors generally have common problems such as low delivery efficiency, high cytotoxicity, and cumbersome operation methods, and the delivery effects of various nucleic acid drugs are uneven.

[0004] Therefore, there is an urgent need in this field to design efficient, safe, easy-to-operate and universal nucleic acid drug delivery vectors. Summary of the Invention

[0005] The present invention aims to provide a low-toxic, high-efficiency, easy-to-operate, and universal cationic nanogel nucleic acid transfection vector. Another object of the present invention is to provide a preparation method of the anionic nanogel and its application in nucleic acid transfection.

[0006] In a first aspect of the present invention, a cationic nanogel is provided, wherein the cationic nanogel has a core-shell structure, wherein the substance forming the core structure comprises one or more cationic monomers having a structure as shown in Formula I, and the substance forming the shell structure comprises a neutral polymer;

[0007]

[0008] in,

[0009] R1 represents -H or -CH3;

[0010] R2 represents -C(=O)-NH-, -C(=O)-O- or -C6H4-;

[0011] R3 represents -NH2, -NH-CH3, -N-(CH3)2, -N + -(CH3)3 or -NH-C(=NH)-NH2;

[0012] n is an integer between 1 and 3.

[0013] In another embodiment, the neutral polymer is selected from one or more of the following: polyethylene glycol, polyethylene, polytetrafluoroethylene, poly-2-methacryloyloxyethyl phosphorylcholine, poly-methacryloylethyl sulfobetaine, poly-3-[[2-(methacryloyloxy)ethyl]dimethylammonium]propionate, and poly-3-[(3-acrylamidopropyl)dimethylammonium]propionate.

[0014] In another embodiment, the cationic nanogel has a diameter of 40-500 nm.

[0015] In a second aspect of the present invention, a method for preparing the cationic nanogel provided by the present invention as described above is provided, the method comprising the steps of:

[0016] (1) Using an anionic polymer as a template, one or more cationic monomers having a structure as shown in Formula I, a crosslinking agent, a neutral polymer-chain transfer agent, and an initiator are added, mixed, and the pH of the solution is adjusted to neutrality;

[0017] (2) initiating polymerization to obtain a nanogel complex;

[0018] (3) removing the polyanion template in the nanogel complex to obtain the cationic nanogel provided by the present invention as described above;

[0019]

[0020] in,

[0021] R1 represents -H or -CH3;

[0022] R2 represents -C(=O)-NH-, -C(=O)-O- or -C6H4-;

[0023] R3 represents -NH2, -NH-CH3, -N-(CH3)2, -N + -(CH3)3 or -NH-C(=NH)-NH2;

[0024] n is an integer between 1 and 3.

[0025] In another embodiment, the anionic polymer template is selected from one or more of the following: polyacrylic acid, sodium poly (2-acrylamido-2-methylpropanesulfonate), sodium poly (p-styrenesulfonate), poly (dimethylaminoethyl methacrylate), poly (methacryloyloxyethyl trimethylammonium chloride), and poly (2-aminoethyl methacrylate).

[0026] In another embodiment, the charge concentrations of the cationic monomer and the anionic polymer template are 5-500 mM, respectively.

[0027] In another embodiment, the cross-linking agent is selected from one or more of the following: diallyl disulfide, N,N'-bis(acryloyl)cystamine, and 2,2-dithiodiethanol diacrylic acid.

[0028] In another embodiment, the cross-linking agent is used in an amount of 1-50 mol% of the cationic monomer concentration.

[0029] In another embodiment, the neutral polymer is selected from one or more of the following: polyethylene glycol, polyethylene, polytetrafluoroethylene, poly-2-methacryloyloxyethyl phosphorylcholine, poly-methacryloylethyl sulfobetaine, poly-3-[[2-(methacryloyloxy)ethyl]dimethylammonium]propionate, and poly-3-[(3-acrylamidopropyl)dimethylammonium]propionate.

[0030] In another embodiment, the neutral polymer is grafted with or not grafted with a targeting molecule, and the grafted targeting molecule is selected from folic acid, sodium alendronate, transferrin, hyaluronic acid, polypeptide, mannose, or biotin.

[0031] In another embodiment, the chain transfer agent is selected from one or more of the following: 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.

[0032] In another embodiment, the initiator includes a photoinitiator and a thermal initiator.

[0033] In another embodiment, the initiator is selected from one or more of the following: 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.

[0034] In another embodiment, step (2) is to initiate polymerization with a photoinitiator under ultraviolet light for 1-12 hours; or to initiate polymerization with a thermal initiator at 60-80° C. for 1-12 hours.

[0035] In another embodiment, the step (3) uses ultrafiltration, centrifugation or dialysis with an inorganic salt solution to remove the polyanion template in the nanogel complex.

[0036] In another embodiment, the inorganic salt is selected from one or more of the following: sodium chloride, potassium chloride, sodium nitrate, potassium nitrate, sodium sulfate, and potassium sulfate.

[0037] In another embodiment, the concentration of the inorganic salt is 0.1-5 mol / L.

[0038] In a third aspect of the present invention, a use of the cationic nanogel provided by the present invention as described above in nucleic acid transfection is provided.

[0039] In a fourth aspect of the present invention, a nanogel-nucleic acid complex is provided, which is formed by the cationic nanogel provided by the present invention as described above and nucleic acid.

[0040] In another embodiment, the nucleic acid is selected from one or more of the following: small interfering RNA (siRNA), messenger RNA (mRNA), and plasmid (pDNA).

[0041] In a fifth aspect of the present invention, a method for preparing the nanogel-nucleic acid complex provided by the present invention as described above is provided, the method comprising the steps of: mixing the cationic nanogel provided by the present invention as described above with nucleic acid in a culture medium to obtain the nanogel-nucleic acid complex provided by the present invention as described above.

[0042] In a sixth aspect of the present invention, a use of the nanogel-nucleic acid complex provided by the present invention as described above in nucleic acid transfection is provided.

[0043] Based on this, the present invention provides a highly efficient, safe, easy-to-operate, and universal nucleic acid drug delivery vector. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 is the particle size of the core-shell cationic nanogel in the preparation example; wherein,

[0045] (a) Particle size distribution of NG1 nanogels with different particle sizes.

[0046] (b) Particle size and PDI of the prepared NG1 nanogels with different cross-linking degrees;

[0047] (c) Particle size distribution of nanogels NG1-NG15 prepared from different monomers.

[0048] Figure 2 This is the time-dependent change of light intensity of NG1 prepared in the preparation example in 10 mM GSH solution.

[0049] Figure 3 The results are a comparison of the cytotoxicity of NG1 prepared in the preparation example and commercial transfection reagents Lipo3000 and Lipo8000.

[0050] Figure 4 The results of nucleic acid transfection in Hela cells using NG1 and NG15 prepared in the preparation examples and commercial transfection reagents Lipo3000 and Lipo8000 are shown; wherein,

[0051] (a) Fluorescence images, flow cytometry results, and mediated cell apoptosis of siRNA delivery;

[0052] (b) Fluorescence images and flow cytometry results of mRNA transfection;

[0053] (c) Fluorescence images and flow cytometry results of pDNA transfection.

[0054] Figure 5 The figures show the siRNA delivery results of NG1 and NG15 prepared in the examples in various cell lines. DETAILED DESCRIPTION

[0055] After extensive and in-depth research, the inventors developed a core-shell cationic nanogel carrier. The cationic nanogel contains a large amount of positive charge, enabling efficient loading of a variety of nucleic acid drug molecules. Furthermore, the cationic nanogel core possesses a biocompatible environment, effectively preserving the structure and bioactivity of nucleic acids. Its crosslinker can respond to the intracellular environment, mediating the escape and controlled release of nucleic acid drugs. The gel shell is a neutral polymer, significantly weakening the positive charge on the gel surface and reducing cytotoxicity. Furthermore, it can selectively modify targeting molecules, enabling precise delivery of nucleic acid drugs. This foundation led to the present invention.

[0056] Specifically, the present invention provides a core-shell cationic nanogel, the shell of which is a neutral polymer and the core of which is a polycationic monomer and a cross-linker; the nanogel is then directly mixed with nucleic acid in a complete culture medium to prepare a nucleic acid delivery complex, which is then transferred into a well plate pre-seeded with cells. After 24 hours, the transfection effect is qualitatively observed using a fluorescence microscope, or the transfection results are quantitatively detected using a flow cytometer.

[0057] The present invention also provides a method for synthesizing cationic nanogels, comprising the following steps:

[0058] Using an anionic polymer as a template, a cationic monomer, a crosslinking agent, a neutral polymer-chain transfer agent, and an initiator are added. The solution pH is adjusted to electroneutrality, and then nitrogen is pumped in and out to deoxygenate and initiate polymerization to produce a nanogel composite. The polyanionic template in the nanogel composite is removed by ultrafiltration, centrifugation, or dialysis using an inorganic salt solution to obtain the cationic nanogel.

[0059] The general structural formula of the cationic monomer is shown in Formula I, and is a combination of one or more monomers described in Formula I:

[0060]

[0061] In Formula I,

[0062] R1 is a side chain functional group, including but not limited to -H, -CH3;

[0063] R2 is a connecting bond, including but not limited to -C(=O)-NH-, -C(=O)-O-, and -C6H4-;

[0064] R3 is a chemical functional group, including but not limited to -NH2, -NH-CH3, -N-(CH3)2, -N + -(CH3)3, -NH-C(=NH)-NH2;

[0065] n is an integer between 1 and 3;

[0066] Furthermore, the charge concentrations of the cationic monomer and the anionic polymer template are both 5-500 mM; for example, but not limited to, the charge concentrations of the cationic monomer and the anionic polymer template are both 10-100 mM, 15-400 mM, 30-200 mM, 25-300 mM, 50-350 mM, etc.

[0067] The cross-linking agent includes, but is not limited to, diallyl disulfide, N,N'-bis(acryloyl)cystamine, and 2,2-dithiodiethanol diacrylic acid.

[0068] The anionic polymer template includes, but is not limited to, polyacrylic acid, sodium poly (2-acrylamido-2-methylpropanesulfonate), sodium poly (p-styrenesulfonate), poly (dimethylaminoethyl methacrylate), poly (methacryloyloxyethyl trimethylammonium chloride) or poly (2-aminoethyl methacrylate).

[0069] The neutral polymer includes, but is not limited to, polyethylene glycol, polyethylene, polytetrafluoroethylene, poly-2-methacryloyloxyethyl phosphorylcholine, poly-methacryloylethyl sulfobetaine, poly-3-[[2-(methacryloyloxy)ethyl]dimethylammonium]propionate, and poly-3-[(3-acrylamidopropyl)dimethylammonium]propionate.

[0070] Wherein, the chain transfer agent includes but is not limited to methyl (phenyl) amino dithiocarboxylic acid cyanomethyl ester, 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.

[0071] Wherein, the neutral polymer can be grafted with a targeting molecule to form a targeting molecule-neutral polymer-chain transfer agent; the targeting molecule includes but is not limited to folic acid, sodium alendronate, transferrin, hyaluronic acid, polypeptide, mannose, and biotin.

[0072] Wherein, the grafting method of the targeting molecule is:

[0073] For targeting molecules containing -COOH, a neutral chain segment (neutral polymer) with -NH2 at both ends is selected. This neutral chain segment is first BOC-protected, and then the targeting molecule is added and linked via amidation. Subsequently, the BOC is removed, and a chain transfer agent is added. After dehydration condensation, the neutral chain segment grafted with the targeting molecule and chain transfer agent is obtained.

[0074] For targeting molecules containing -NH2, a neutral chain segment (neutral polymer) with -OH and -COOH ends is selected. This neutral chain segment is first activated with NHS, and then the targeting molecule is added and linked via an NHS / NH2 reaction. Subsequently, a chain transfer agent is added, and an esterification reaction is performed to produce the neutral chain segment grafted with the targeting molecule and chain transfer agent.

[0075] Wherein, the initiator is a photoinitiator or a thermal initiator, including but not limited to 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;

[0076] Furthermore, the reaction conditions are: a photoinitiator initiates polymerization under ultraviolet light for 1-12 hours; or a thermal initiator initiates polymerization at 60-80° C. for 1-12 hours;

[0077] The polymerization time is, for example but not limited to, 2-9 hours, 5-7 hours, etc.;

[0078] When a thermal initiator is used, the temperature is, for example but not limited to, 65-75° C., 70-80° C., etc.

[0079] Furthermore, the content of the initiator is 0.1-10 wt% of the cationic monomer, for example but not limited to, 0.5-4 wt%, 1-3 wt%, 0.7-7 wt%, 5-8 wt%, etc.

[0080] Wherein, the inorganic salt is one or more of sodium chloride, potassium chloride, sodium nitrate, potassium nitrate, sodium sulfate or potassium sulfate;

[0081] Furthermore, the concentration of the inorganic salt is 0.1-5 mol / L, for example but not limited to, 0.5-3 mol / L, 1-4 mol / L, 2-3.5 mol / L, etc.

[0082] Wherein, the particle size of the cationic nanogel is 40-500 nm; preferably, 180-300 nm.

[0083] The crosslinking agent of the cationic nanogel is 1-50 mol% of the cationic monomer concentration, for example but not limited to, 5-40 mol%, 15-45 mol%, 25-35 mol%, etc.; preferably, 3-30 mol%.

[0084] The present invention also provides application of the cationic nanogel in nucleic acid transfection.

[0085] The nucleic acid transfection is to deliver the nucleic acid from outside the cell into the cell and successfully express the nucleic acid.

[0086] The nucleic acid includes but is not limited to small interfering RNA (siRNA), messenger RNA (mRNA), plasmid (pDNA), etc.

[0087] The present invention also provides a complex comprising the cationic nanogel and nucleic acid.

[0088] The present invention also provides a method for preparing the complex, comprising the steps of: adding the cationic nanogel to a serum-containing DMEM complete culture medium and beating the culture medium evenly; then adding nucleic acid and vortexing the culture medium for 5-30 seconds to obtain the complex.

[0089] The concentration of the nucleic acid is 0.1-20 μg / mL, and the concentration of the cationic nanogel is 5-100 μg / mL. Preferably, the concentration of the nucleic acid is 1-10 μg / mL, and the concentration of the cationic nanogel is 10-50 μg / mL.

[0090] The present invention also provides use of the complex in nucleic acid transfection.

[0091] 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 in the specification and claims. 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.

[0092] 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.

[0093] Throughout this document, all features, such as values, amounts, amounts, and concentrations, defined in numerical ranges or percentage ranges are provided for simplicity and convenience only. Accordingly, the description of numerical ranges or percentage ranges should be considered to encompass and specifically disclose all possible subranges and individual values ​​within those ranges (including integers and fractions).

[0094] The above-mentioned features mentioned in the present invention or the features mentioned in the embodiments can be combined in any combination. All features disclosed in this specification can be used in any combination form. As long as there is no contradiction in the combination of these features, all possible combinations should be considered to be within the scope of this specification. Each feature disclosed in the specification can be replaced by any alternative feature that can provide the same, equal or similar purpose. Therefore, unless otherwise specified, the disclosed features are only general examples of equal or similar features.

[0095] The main advantages of the present invention are:

[0096] 1. The cationic nanogel transfection reagent provided by the present invention has high delivery efficiency, low cytotoxicity, convenient operation, and is universally applicable to a variety of nucleic acid drugs and cell lines. Its performance is significantly better than commercially available transfection reagents.

[0097] 2. The cationic nanogel proposed in the present invention has a high delivery efficiency for siRNA, mRNA, and pDNA, and is an efficient and universal nucleic acid delivery platform; the toxicity of the cationic nanogel is much lower than that of commonly used commercial reagents such as Lipofectamine 3000 (Lipo3000) and Lipofectamine 8000 (Lipo8000), and the cell survival rate under the experimental conditions of nucleic acid transfection is higher than 90%, with high safety and biocompatibility; at the same time, the transfection operation is extremely simple, avoiding the cumbersome operations commonly used with commercial reagents, such as pre-mixing of nucleic acid and transfection reagent in serum-free culture medium or buffer and the need for incubation time.

[0098] 3. The nucleic acid transfection vector proposed in the present invention is a low-toxic, high-efficiency, easy-to-operate, and universal transfection platform, which has potential in vitro transfection and clinical application value in terms of non-viral gene vectors.

[0099] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods for which specific conditions are not specified in the following examples are generally based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, all percentages, ratios, proportions, or parts are by weight. The units in the weight-volume percentages in the present invention are well known to those skilled in the art, for example, refer to the weight (grams) of the solute in 100 milliliters of solution. Unless otherwise defined, all professional and scientific terms used herein have the same meaning as those familiar to those skilled in the art. In addition, any method and material similar or equivalent to the described content can be applied to the inventive method. The preferred embodiments and materials described herein are for demonstration purposes only.

[0100] Preparation Example

[0101] Preparation of core-shell cationic nanogels

[0102] The polyethylene glycol modified with folic acid and chain transfer agent in the following examples was obtained by the following method:

[0103] 500.0 mg of NH2-terminated polyethylene glycol and 21.8 mg of di-tert-butyl dicarbonate were weighed and reacted in a 10% wt solution of sodium hydroxide in tert-butanol at room temperature for 8 h. The reactants were 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 h, the mixture was washed with 0.1 M hydrochloric acid to obtain the folic acid-modified polyethylene glycol. Next, 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. The mixture was deoxygenated by nitrogen evacuation and stirred for 20 min. 206.3 mg of dicyclohexylcarbodiimide was added dropwise using a constant pressure funnel at a rate of 3 seconds per drop. The mixed solution was reacted at a constant temperature of 25° C. for 48 hours, and then the insoluble matter was removed by filtration. The solid was precipitated with ether and vacuum-dried to obtain polyethylene glycol modified with folic acid and a chain transfer agent.

[0104] Example 1

[0105] Preparation of NG1 nanogels

[0106] Weigh 29.0 mg of polyacrylic acid (the charge concentration of the anionic polymer template is 20 mM), 53.3 mg of 4-vinylbenzylamine (monomer concentration (for monomers, monomer concentration is equal to charge concentration) is 20 mM), 10.4 mg of N,N'-bis(acryloyl)cystamine (10 mol% of monomer concentration), 1.1 mg of 2-hydroxy-2-methyl-1-phenyl-1-propanone (2 wt% of monomer concentration), and 21.0 mg of folic acid-polyethylene glycol-4-cyano-4-(thiobenzoyl)valeric acid and dissolve them in 20 ml of deionized water. Adjust the pH of the solution to electroneutrality, pump nitrogen into the reaction system to deoxygenate, and react under ultraviolet light for 8 hours to obtain a nanogel composite. 0.1753 g of sodium chloride was added to the nanogel complex obtained above to adjust the salt concentration of the solution to 1.5 M. 1.5 M sodium chloride was used as the eluent. The nanogel complex was centrifuged and washed six times in an ultrafiltration centrifuge tube to remove the anionic polymer template. The sodium chloride was then dialyzed with deionized water to remove the sodium chloride. A nanogel was obtained, named NG1. The particle size distribution of the prepared nanogels of different sizes is shown in FIG. Figure 1 As shown in (a).

[0107] Example 2

[0108] Preparation of NG1 nanogels with different cross-linking degrees

[0109] Weigh 29.0 mg of polyacrylic acid (the charge concentration of the anionic polymer template is 20 mM), 53.3 mg of 4-vinylbenzylamine (monomer concentration is 20 mM), 10.4-52.0 mg of N,N'-bis(acryloyl)cystamine (10-50 mol% of the monomer concentration), 1.1 mg of 2-hydroxy-2-methyl-1-phenyl-1-propanone (2 wt% of the monomer concentration), and 21.0 mg of folic acid-polyethylene glycol-4-cyano-4-(thiobenzoyl)valeric acid and dissolve them in 20 ml of deionized water. Adjust the pH of the solution to electroneutrality, pump nitrogen into the reaction system to deoxygenate, and react under ultraviolet light for 8 hours to obtain a nanogel composite. 0.1753 g of sodium chloride was added to the nanogel complex obtained above to adjust the salt concentration of the solution to 1.5 M, and 1.5 M sodium chloride was used as the eluent. The nanogel complex was centrifuged and washed 6 times with an ultrafiltration centrifuge tube to remove the anionic polymer template, and then dialyzed with deionized water to remove sodium chloride. Nanogels with different crosslinking degrees were obtained. The particle size and PDI were as follows: Figure 1 (b) shown.

[0110] Example 3

[0111] Preparation of NG1-15 nanogels

[0112] Weigh 29.0 mg of polyacrylic acid (the charge concentration of the anionic polymer template is 20 mM), 53.3 mg of 4-vinylbenzylamine (or 84.7 mg of benzylethyltrimethylammonium chloride; 64.5 mg of N-4-vinylbenzyl-N,N-dimethylamine; 58.9 mg of 4-vinyl-N-methylbenzylamine; 83.1 mg of methacryloyloxyethyltrimethylammonium chloride; 77.5 mg of acryloyloxyethyltrimethylammonium chloride; N,N,N-trimethyl- 3-(2-methylallylamino)-1-propylammonium chloride 88.3 mg; (3-acrylamidopropyl)trimethylammonium chloride 82.7 mg; 2-(dimethylamino)ethyl methacrylate 62.9 mg; 2-(dimethylamino)ethyl acrylate 57.3 mg; N-[(3-(dimethylamino)propyl]methacrylamide 68.1 mg; N-[(3-dimethylamino)propyl]acrylamide 62.5 mg; 2-aminoethyl methacrylate 66.2 mg; 2-guanidinoethyl methacrylate 68.9 mg), monomer concentrations of both were 20 mM, N,N'-bis(acryloyl)cystamine 20.8 mg (20 mol% of the monomer concentration), 2-hydroxy-2-methyl-1-phenyl-1-propanone 1.1 mg (2 wt% of the monomer concentration), and folic acid-polyethylene glycol-4-cyano-4-(thiobenzoyl)valeric acid 21.0 mg were dissolved in 20 ml of deionized water, the pH of the solution was adjusted to electroneutrality, the reaction system was evacuated with nitrogen to deoxygenate, and the reaction was carried out under ultraviolet light for 8 hours to obtain a nanogel composite. 0.1753 g of sodium chloride was added to the nanogel composite obtained above to adjust the salt concentration of the solution to 1.5 M, and 1.5 M sodium chloride was used as the eluent. The nanogel composite was centrifuged and washed six times in an ultrafiltration centrifuge tube to remove the anionic polymer template, and then dialyzed with deionized water to remove sodium chloride to obtain nanogels, designated NG1-NG14.

[0113] Specifically, NG15 was prepared according to the above-mentioned operation process using 4-vinylbenzylamine as a monomer and polyethylene glycol-4-cyano-4-(thiobenzoyl)valeric acid without folic acid modification as a chain transfer agent.

[0114] The particle size distribution of the prepared nanogels with different chemical compositions is as follows Figure 1 (c) shown.

[0115] like Figure 1 The results show that the synthesis method of the present invention can produce cationic nanogels with independently adjustable particle size and cross-linking degree. The resulting nanogels have a narrow particle size dispersion and are applicable to at least 14 cationic monomers. The experimental results demonstrate that the method is a highly versatile, controllable polymerization method, and the resulting nanogels possess adjustable structural parameters such as size and stiffness.

[0116] Test Examples

[0117] Example 1

[0118] Evaluation of the biodegradability of core-shell cationic nanogel NG1

[0119] NG1 prepared in Example 1 of the present invention was diluted in a 20 mM PB solution (pH 7.4). Dynamic light scattering (DLS) was used to characterize the light intensity and size of the complex. GSH was then added to the solution to a GSH concentration of 10 mM. DLS was used to monitor the changes in light intensity and particle size of the complex over time.

[0120] Figure 2 The results shown indicate that after the addition of GSH, the light intensity of NG1 decreased rapidly within 2 h and the particle size was greatly reduced, confirming that NG1 can be degraded in response to the GSH concentration.

[0121] Example 2

[0122] Cytotoxicity evaluation of core-shell cationic nanogel NG1

[0123] HeLa cells were pre-seeded into 96-well plates and incubated overnight at 37°C in a 5% CO2 environment. The culture medium was removed, and 100 μL of complete culture medium containing NG1 at different concentrations, or 100 μL of complete culture medium containing commercial reagents Lipo3000 and Lipo8000, was added to each well and the cells were incubated for 24 hours. The culture medium was then removed, and 10 μL of CCK-8 was added to each well. Cell viability was determined using the standard CCK-8 assay. Six replicates were tested for each experiment, and the concentrations of the commercial reagents were those specified in the product manual for transfection.

[0124] Figure 3 The results show that at concentrations up to 100 μg / mL, the viability of HeLa cells treated with NG1 exceeded 90%, significantly higher than that of commercial reagents. These results demonstrate that the cationic nanogels prepared in this invention exhibit low cytotoxicity, superior to common commercial reagents, and possess good biocompatibility.

[0125] Application Examples

[0126] Example 1

[0127] siRNA transfection with core-shell cationic nanogels NG1 and NG15

[0128] Small interfering RNA (siRNA-FAM) labeled with the green fluorescent molecule FAM was used as a model nucleic acid. The efficiency of cationic nanogel delivery was evaluated on Hela cells by detecting intracellular fluorescence, and the delivery of vascular endothelial growth factor small interfering RNA (siVEGF) was successfully mediated.

[0129] The specific method of siRNA delivery is as follows: Hela cells are seeded into a 24-well plate and placed in an environment of 37°C and 5% CO2 overnight. When the confluence of the Hela cells reaches more than 80%, the nucleic acid delivery experiment is started. NG1 prepared in Example 1 of the present invention (or NG15 prepared in Example 3 of the invention) is added to 500 μL of complete culture medium and pipetted to mix. The concentration of NG1 (or NG15) is 20 μg / mL. Then 0.5 μg siRNA-FAM is added to the complete culture medium containing NG1 (or NG15) and vortexed for 10 seconds. The old cell culture medium in the well plate is removed, washed twice with PBS, and then a culture medium solution containing the siRNA complex is added and incubated in an incubator for 24 hours. Afterwards, a fluorescence microscope is used to qualitatively observe the nucleic acid delivery, or a flow cytometer is used to quantitatively analyze the mean fluorescence intensity of the Hela cells. 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 green fluorescence intensity in the cells is observed using a fluorescence microscope; or after removing the culture medium and washing the cells 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 using a flow cytometer.

[0130] The specific operation method of siRNA transfection is as follows: Hela cells are pre-seeded into a 96-well plate and incubated overnight at 37°C and 5% CO2. NG1 (or NG15) prepared in Example 1 of the present invention is added to the complete culture medium and pipetted to mix. The concentration of NG1 (or NG15) is 20 μg / mL. Then, negative control small interfering RNA (siNC) or siVEGF is added to the complete culture medium containing NG1 (or NG15) and vortexed for 10 seconds, wherein the concentration of siNC or siVEGF is 100 nM. The culture medium is removed, 100 μL of complete culture medium solution containing NG1 (or NG15)-siRNA complex is added, and the cells are incubated for 24 hours. Thereafter, the culture medium is removed, and 10 μL of CCK-8 is added to each well. The cell viability is detected according to the standard steps of the CCK-8 method. The difference between the cell viability of the siNC experimental group and the cell viability of the siVEGF experimental group is the apoptosis rate. Six replicate samples were tested in each group of experiments. Commercial reagents Lipo3000 and Lipo8000 were used as controls, and transfection was performed according to the product manual.

[0131] Figure 4(a) siRNA-FAM delivery and siVEGF transfection results using NG1 and NG15, materials obtained in Examples 1 and 3 of the present invention, at varying concentrations in HeLa cells. The results demonstrate that NG1 and NG15 efficiently delivered nucleic acids to 100% of cells, efficiently transfected siVEGF, and induced apoptosis. The levels of siRNA intracellular delivery and apoptosis mediated by NG1 and NG15 were higher than those achieved by the commercial reagents Lipo3000 and Lipo8000. NG1 also exhibited slightly higher delivery and transfection efficiencies than NG15, indicating that folic acid further promotes receptor-mediated endocytosis.

[0132] Example 2

[0133] Core-shell cationic nanogels NG1 and NG15 were transfected with green fluorescent protein mRNA (GFP mRNA) and plasmid (GFP pDNA)

[0134] Hela cells were seeded into a 24-well plate and incubated overnight at 37°C and 5% CO2. When the cell confluence reached more than 80%, the nucleic acid transfection experiment was started. NG1 prepared in Example 1 of the present invention (or NG15 prepared in Example 3 of the present invention) was added to 500 μL of complete culture medium and pipetted to mix. The concentration of NG1 (or NG15) was 20 μg / mL. 2.5 μg of GFP mRNA or GFP pDNA was then added to the complete culture medium containing NG1 (or NG15) and vortexed for 10 seconds. The old cell culture medium in the well plate was removed, washed twice with PBS, and then a culture medium solution containing the nucleic acid complex was added and incubated in an incubator for 24 hours. Afterwards, the nucleic acid transfection was qualitatively observed using a fluorescence microscope, or the mean fluorescence intensity of the 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 nucleic acid complex, the culture medium is removed, the cells are washed twice with PBS, and the green fluorescence intensity in the cells is observed using a fluorescence microscope; or after removing the culture medium and washing the cells 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 using a flow cytometer.

[0135] Figure 4 (b) and (c) show the results of NG1 and NG15 transfection of GFP mRNA or GFP pDNA in Hela cells. The results demonstrate that NG1 and NG15 were significantly more efficient in transfecting mRNA and pDNA in Hela cells than commercial reagents Lipo3000 and Lipo8000, achieving efficient expression of green fluorescent protein. The significantly higher transfection efficiency of NG1 than that of NG15 demonstrates that folic acid further promotes receptor-mediated endocytosis. This cationic nanogel is a versatile platform for nucleic acid transfection.

[0136] Example 3

[0137] Core-shell cationic nanogels NG1 and NG15 for transfection of siRNA in various cell lines

[0138] Human alveolar adenocarcinoma basal epithelial cells (A549), human umbilical vein endothelial cells (HUVEC), and mouse embryonic fibroblasts (NIH-3T3) were respectively inoculated into 24-well plates and kept overnight at 37°C and 5% CO2. When the cell confluence reached more than 80%, the nucleic acid delivery experiment was started. NG1 prepared in Example 1 of the present invention (or NG15 prepared in Example 3 of the present invention) was added to 500 μL of complete culture medium and pipetted to mix. The concentration of NG1 (or NG15) was 20 μg / mL. Then, 0.5 μg siRNA-FAM was added to the complete culture medium containing NG1 (or NG15) and vortexed for 10 seconds. The old cell culture medium in the well plate was removed, washed twice with PBS, and then 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 the 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 green fluorescence intensity in the cells is observed using a fluorescence microscope; or after removing the culture medium and washing the cells 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 using a flow cytometer.

[0139] Figure 5 The results of siRNA delivery of materials NG1 and NG15 obtained in the preparation examples of the present invention on A549, HUVEC, and NIH-3T3 cells are shown. The results show that NG1 and NG15 can efficiently deliver siRNA in a variety of cell lines.

[0140] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of the substantive technical content of the present invention. The substantive technical content of the present invention is broadly defined in the scope of the claims of the application. Any technical entity or method completed by others, if it is exactly the same as that defined in the scope of the claims of the application or is an equivalent variation, will be deemed to be included in the scope of the claims.

Claims

1. A cationic nanogel, characterized in that: The cationic nanogel has a core-shell structure, the substance forming the core structure includes one or more cationic monomers having a structure as shown in Formula I, and the substance forming the shell structure includes a neutral polymer; in, R1 represents -H or -CH3; R2 represents -C(=O)-NH-, -C(=O)-O- or -C6H4-; R3 represents -NH2, -NH-CH3, -N-(CH3)2, -N + -(CH3)3 or -NH-C(=NH)-NH2; n is an integer between 1 and 3; The preparation method of the cationic nanogel comprises the steps of: (1) Using an anionic polymer as a template, one or more cationic monomers having a structure as shown in Formula I, a crosslinking agent, a neutral polymer grafted with a chain transfer agent, and an initiator are added, mixed, and the pH of the solution is adjusted to neutrality; (2) initiating polymerization to obtain a nanogel complex; (3) removing the polyanion template in the nanogel complex to obtain the cationic nanogel; The neutral polymer is selected from one or more of the following: polyethylene glycol, polyethylene, polytetrafluoroethylene, poly-2-methacryloyloxyethyl phosphorylcholine, poly-methacryloylethyl sulfobetaine, poly-3-[[2-(methacryloyloxy)ethyl]dimethylammonium]propionate, and poly-3-[(3-acrylamidopropyl)dimethylammonium]propionate.

2. The cationic nanogel according to claim 1, wherein The diameter of the cationic nanogel is 40-500 nm.

3. A method for preparing the cationic nanogel according to claim 1 or 2, characterized in that: The method comprises the steps of: (1) Using an anionic polymer as a template, one or more cationic monomers having a structure as shown in Formula I, a crosslinking agent, a neutral polymer grafted with a chain transfer agent, and an initiator are added, mixed, and the pH of the solution is adjusted to neutrality; (2) initiating polymerization to obtain a nanogel complex; (3) removing the polyanion template in the nanogel complex to obtain the cationic nanogel as claimed in claim 1 or 2; in, R1 represents -H or -CH3; R2 represents -C(=O)-NH-, -C(=O)-O- or -C6H4-; R3 represents -NH2, -NH-CH3, -N-(CH3)2, -N + -(CH3)3 or -NH-C(=NH)-NH2; n is an integer between 1 and 3; The neutral polymer is selected from one or more of the following: polyethylene glycol, polyethylene, polytetrafluoroethylene, poly-2-methacryloyloxyethyl phosphorylcholine, poly-methacryloylethyl sulfobetaine, poly-3-[[2-(methacryloyloxy)ethyl]dimethylammonium]propionate, and poly-3-[(3-acrylamidopropyl)dimethylammonium]propionate.

4. The preparation method according to claim 3, wherein The anionic polymer template is selected from one or more of the following: polyacrylic acid, sodium poly (2-acrylamido-2-methylpropanesulfonate), sodium poly (p-styrenesulfonate), poly (dimethylaminoethyl methacrylate), poly (methacryloyloxyethyl trimethylammonium chloride), and poly (2-aminoethyl methacrylate).

5. The preparation method according to claim 3, wherein The charge concentrations of the cationic monomer and the anionic polymer template are 5-500 mM, respectively.

6. The preparation method according to claim 3, wherein The cross-linking agent is selected from one or more of the following: diallyl disulfide, N,N'-bis(acryloyl)cystamine, and 2,2-dithiodiethanol diacrylate.

7. The preparation method according to claim 3, wherein The amount of the cross-linking agent used is 1-50 mol% of the cationic monomer concentration.

8. The preparation method according to claim 3, wherein The neutral polymer is grafted with or not grafted with a targeting molecule, and the grafted targeting molecule is selected from folic acid, sodium alendronate, transferrin, hyaluronic acid, polypeptide, mannose, or biotin.

9. The preparation method according to claim 3, wherein The chain transfer agent is selected from one or more of the following: 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.

10. The preparation method according to claim 3, wherein The initiator includes a photoinitiator and a thermal initiator.

11. The preparation method according to claim 3, wherein The initiator is selected from one or more of the following: 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.

12. The preparation method according to claim 3 or 10, characterized in that: Step (2) is to initiate polymerization with a photoinitiator under ultraviolet light for 1-12 hours; or to initiate polymerization with a thermal initiator at 60-80° C. for 1-12 hours.

13. The preparation method according to claim 3, wherein The step (3) uses ultrafiltration, centrifugation or dialysis with an inorganic salt solution to remove the polyanion template in the nanogel complex.

14. The preparation method according to claim 13, wherein The inorganic salt is selected from one or more of the following: sodium chloride, potassium chloride, sodium nitrate, potassium nitrate, sodium sulfate, and potassium sulfate.

15. The preparation method according to claim 13, wherein The concentration of the inorganic salt is 0.1-5 mol / L.

16. Use of the cationic nanogel according to claim 1 or 2 in nucleic acid transfection.

17. A nanogel-nucleic acid complex, characterized in that: The cationic nanogel according to claim 1 or 2 is formed with nucleic acid.

18. The composite according to claim 17, wherein The nucleic acid is selected from one or more of the following: small interfering RNA (siRNA), messenger RNA (mRNA), and plasmid (pDNA).

19. A method for preparing the nanogel-nucleic acid complex according to claim 17 or 18, characterized in that: The method comprises the steps of: mixing the cationic nanogel according to claim 1 or 2 with nucleic acid in a culture medium to obtain the nanogel-nucleic acid complex according to claim 17 or 18.

20. Use of the nanogel-nucleic acid complex according to claim 17 or 18 in nucleic acid transfection.

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