A nucleic acid-encapsulating and delivering material having enzyme and pH responsiveness and a method for preparing the same

Abstract

This invention discloses an enzyme- and pH-responsive nucleic acid loading and delivery material and its preparation method. The material can be used for efficient loading and delivery of nucleic acid molecules. The preparation method uses amphiphilic zwitterionic monomers to improve the reverse emulsion polymerization system, enhancing polymerization at the two-phase interface and avoiding the extrusion effect of polymerization in the aqueous core on nucleic acid molecules, thereby improving the loading efficiency of nucleic acid molecules. Further preparation of cross-linking agents MP-CL and CB-CL, which can cleave in response to matrix metalloproteinase II or cathepsin B, endows the nanogel with the ability to respond to charge reversal in the tumor matrix and to release nucleic acid molecules from tumor cells. The acid-sensitive blocks on the amphiphilic monomers give the nanogel lysosomal escape capability. The delivery material can efficiently deliver nucleic acid molecules into cells and exhibits excellent stability and biosafety.

Description

Technical Field

[0001] This invention belongs to the field of polymer chemistry and biomedical engineering, and more specifically, relates to a nucleic acid loading and delivery material with enzyme and pH responsiveness and its preparation method. Background Technology

[0002] There are two main methods for delivering biological macromolecules: viral vectors and non-viral vectors. Viral vectors have limitations in clinical application due to issues such as potential immunogenicity, random gene integration, limited loading capacity, high preparation costs, and inability to deliver mRNA and protein drugs. Non-viral delivery vectors are of great significance for the delivery of biological macromolecule drugs, but they also face some challenges:

[0003] 1. Cationic toxicity and potential inflammatory responses

[0004] Most nucleic acid delivery materials are cationic materials, which combine nucleic acid molecules through positive and negative electrostatic interactions to form nanoparticles. To maintain the stability of the complex in blood circulation, nucleic acid nanoparticles need to be prepared at a high N / P ratio. This not only results in a large amount of residual positive charge on the nanoparticles but also in a large amount of free cationic material in the complex system, which is the main source of cationic toxicity of nucleic acid delivery materials. In addition, during the storage of nucleic acid / cationic complexes, cationic materials may dissolve due to kinetic instability, which also increases cationic toxicity. To address this, some strategies have been developed to try to reduce the risks of cationic toxicity. For example, we have protonated the acid-sensitive groups under acidic conditions and deprotonated them under neutral physiological conditions to load nucleic acids under acidic conditions, and then used polymer chain crosslinking or hydrophobic aggregation to stabilize the nanocomplex, so that the nucleic acid-loaded nanoparticles are electrically neutral or weakly negatively charged under neutral conditions, thereby reducing the toxic side effects of positive charge; or we have linked the cationic groups to the main chain through acid-sensitive bonds, and after combining the nucleic acid molecules, we have used disulfide bonds to crosslink and stabilize the nanoparticles, allowing the positively charged groups to be dialyzed out under acidic conditions. While these strategies can reduce cationic toxicity to some extent, they still cannot completely eliminate it. Furthermore, cationic materials (including ionizable lipid particles) also present potential inflammatory challenges. Therefore, there is a need to develop novel nucleic acid loading strategies and materials that are independent of cationic components and electrostatic interactions.

[0005] 2. In vivo stability issues of biomacromolecule / carrier complexes

[0006] Cationic polymers / lipids load nucleic acid molecules through positive and negative electrostatic interactions. The entire loading process is a complex kinetic and thermodynamic process: a competition between short-range electrostatic attraction (between nucleic acid and cationic materials) and long-range electrostatic repulsion (between nucleic acid molecules). Because the primary complex formed instantaneously after mixing nucleic acids and cationic materials is not in a state of minimum free energy, many unneutralized ring-shaped loop structures are generated, which adjust over time. These complex processes lead to secondary aggregation and phase separation precipitation of the complex, resulting in unstable particle size, uneven distribution, and dissolution of free cationic polymer / lipid molecules. For example, the size of the DNA / cationic material complex gradually increases over time. Due to these complex processes, the complexation of nucleic acid molecules with cationic materials is affected by multiple factors. The complexation is related not only to the N / P ratio and the polymer's inherent properties (such as chemical composition, molecular weight, charge density, topology, and hydrophobicity), but also to the order, method, and rate of mixing. Furthermore, the ionic strength and pH of the solvent also affect the formation of the complex. These factors make the complexation process variable and difficult to precisely replicate.

[0007] 3. The problem of difficulty in particle size control

[0008] The particle size of drug-loaded complexes is crucial for the blood circulation time, in vivo distribution, and deep tissue penetration of biomolecules. The breadth of the particle size distribution also plays a key role in improving drug accumulation in target tissues and reducing side effects on normal tissues. Therefore, controlling the particle size and narrowing its distribution is essential for improving the delivery capability of biomolecules. As mentioned earlier, nucleic acid-loaded nanoparticles prepared based on cationic materials exhibit instability, leading to difficulty in controlling particle size and uneven distribution. For microcapsules loaded with biomolecules prepared by the dual emulsion method, spontaneous assembly into primary liposome microparticles during aqueous hydration results in unsatisfactory size and distribution. Post-processing methods such as ultrasonication, homogenization, and membrane extrusion are required to obtain particles with uniform size. These processing steps are cumbersome and have limited ability to control the particle size distribution.

[0009] Current research utilizes reverse emulsion polymerization to prepare nanogels for nucleic acid delivery. There are two common methods for preparing nanogels: direct polymer crosslinking and heterogeneous polymerization of monomers and crosslinking agents. Polymer crosslinking is divided into physical crosslinking and chemical crosslinking. Physical crosslinking generally involves polymers self-assembling in water through non-covalent bonds such as hydrophilic-hydrophobic interactions to form crosslinked structures. Chemical crosslinking involves polymers forming a crosslinked network through polymerization or other chemical reactions to prepare nanogels. Monomer polymerization involves dispersing monomers and crosslinking agents in emulsions or reverse emulsions for polymerization to obtain nanogels. These nanogels exhibit high stability and loading capacity, capable of loading not only hydrophobic drugs but also hydrophilic bioactive reagents, and are widely used by researchers.

[0010] Inverse emulsion polymerization (IEP) is a heterogeneous polymerization method in which an aqueous phase containing monomers and crosslinking agents is stably dispersed in an organic continuous phase under the action of surfactants and mechanical shear forces to form a water-in-oil (W / O) dispersion. After the addition of an initiator, a polymerization reaction occurs in the aqueous phase to form a nanogel. Hydrophilic drugs can achieve higher loading capacity due to hydrophilic-hydrophobic interactions. The design of the chemical structure of the crosslinking agent can also enable the gel to achieve different stimulus-response functions.

[0011] As mentioned earlier, assembling nucleic acid nanoparticles through positive and negative electrostatic interactions presents several insurmountable drawbacks. Therefore, new strategies for loading nucleic acid molecules without relying on electrostatic interactions are needed. Reverse fine emulsions can encapsulate nucleic acid molecules within aqueous droplets, offering the possibility of developing new materials and methods for loading nucleic acid molecules without electrostatic interactions. While free radical polymerization of reverse fine emulsions can encapsulate large water-soluble nucleic acid molecules and small chemical drugs within nanogels, the encapsulation efficiency for large molecular weight nucleic acids is low, failing to meet the requirements of practical applications. This is because polymerization reactions begin at multiple centers within the aqueous phase of the emulsion droplet, and polymerization at the interface offers no advantage. Consequently, the multiple cross-linked gel networks formed within the aqueous phase easily expel nucleic acids from the nanogel, resulting in low nucleic acid encapsulation efficiency. Currently, there are no literature reports on materials and methods that overcome these drawbacks. Therefore, it is urgent to improve the material composition and preparation methods for preparing nanogels with nucleic acid molecules using reverse fine emulsion polymerization to enhance their nucleic acid encapsulation efficiency. Summary of the Invention

[0012] The purpose of this invention is to overcome the above-mentioned defects and deficiencies in the prior art and to provide a method for preparing nanogels for loading and delivering nucleic acid molecules.

[0013] A second objective of this invention is to provide a nanogel material for loading and delivering nucleic acid molecules.

[0014] The above-mentioned objective of this invention is achieved through the following technical solution:

[0015] This invention first provides a method for preparing nanogel materials for loading and delivering nucleic acid molecules, comprising the following steps:

[0016] S1. Obtain an organic dispersion by dissolving the surfactant in an organic solvent;

[0017] S2. An aqueous mixture is obtained by dissolving a water-soluble monomer, an amphiphilic zwitterionic monomer, and an enzyme-responsive crosslinking agent in water;

[0018] S3. Add the aqueous mixture to the organic dispersion to form a water-in-oil system, add an initiator to cause emulsion polymerization of the water-in-oil system to form a nanogel.

[0019] This invention introduces amphiphilic zwitterionic monomers into a reverse microemulsion polymerization system, thereby improving the monomer type. The introduced amphiphilic zwitterionic monomers allow nucleic acid molecules to be more readily located at the interface, thus leveraging the polymerization advantages of the interface to rapidly and preferentially form a cross-linked network, confining the nucleic acid molecules within the aqueous core of the nanogel. This improves the nanogel's encapsulation efficiency for nucleic acid molecules (up to 90.24%), achieving efficient and safe encapsulation and delivery of nucleic acid molecules. Furthermore, environmentally sensitive components can be introduced to endow the nanogel with tumor matrix-responsive charge reversal and tumor cell disintegration and release of nucleic acid molecules, thereby enhancing the nanogel's nucleic acid loading and delivery capabilities.

[0020] Furthermore, the nucleic acid molecules include, but are not limited to, siRNA.

[0021] Further, the surfactant mentioned in step S1 includes, but is not limited to, one or more of sodium bis(2-ethylhexyl)sulfosuccinate (AOT), Span-80, polyoxyethylene lauryl ether (Brij 30), and potassium monododecyl phosphate (MAEPK).

[0022] Preferably, the surfactant is sodium bis(2-ethylhexyl)sulfosuccinate (AOT) and Span-80.

[0023] Furthermore, the organic solvent in step S1 includes, but is not limited to, n-hexane, cyclohexane, isopentane, and dichloromethane.

[0024] Preferably, the organic solvent is n-hexane.

[0025] Further, the water-soluble monomers in step S2 include, but are not limited to, any one or more of 3-[(3-acrylamidopropyl)dimethylammonium]acetate (CBAA), 3-[N,N-dimethyl-[2-(2-methylprop-2-enoyloxy)ethyl]ammonium]propane-1-sulfonic acid inner salt (SBMA), and [2-(methacryloxy)ethyl]dimethyl-(3-sulfonic acid propyl)ammonium hydroxide (DMAPS).

[0026] Preferably, the water-soluble monomer is 3-[(3-acrylamidopropyl)dimethylammonium]acetate (CBAA).

[0027] Further, the amphiphilic zwitterionic monomers in step S2 include, but are not limited to, any one or more of the following: poly(diisopropylaminoethyl methacrylate)-poly(3-[(3-acrylamidopropyl)dimethylammonium]acetate-dimethylacetyl-methylpropenylamine (PPAA), poly(diisopropylaminoethyl methacrylate)-polyethylene glycol-dimethylacetyl-methylpropenylamine (PPGAA), and poly(diisopropylaminoethyl methacrylate)-poly(3-[N,N-dimethyl-[2-(2-methylprop-2-enoyloxy)ethyl]ammonium]propane-1-sulfonic acid inner salt]-dimethylacetyl-methylpropenylamine (PPSBAA).

[0028] Preferably, the amphiphilic zwitterionic monomer is poly(diisopropylaminoethyl methacrylate)-poly(3-[(3-acrylamidopropyl)dimethylammonium]acetate-dimethylacetyl-methacrylamine (PPAA).

[0029] Preferably, the preparation method of PPAA is as follows: Raft reagent, 2-dodecyl trithiocarbonate-2-methylpropionic acid (DDAT), diisopropylaminoethyl methacrylate (DPA), and azobisisobutyronitrile (AIBN) initiator are dissolved in a mixed solvent of DMF and water. Under nitrogen protection at room temperature, the reaction system is transferred to an oil bath for reaction, followed by the addition of 3-[(3-acrylamidopropyl)dimethylammonium]acetate (CBAA), and the reaction continues. After cooling to room temperature, AIBN is added, and the mixture is bubbled with nitrogen for 45 min, then reacted at 70°C. This process is repeated twice to remove the thiocarbonate groups from the polymer. The reaction system is then precipitated twice in anhydrous diethyl ether, filtered, washed, and vacuum dried to obtain a white solid. The solid from the previous step is taken, and 3-methylpropenamine, N-hydroxysuccinimide (NHS), and N,N-dicyclohexylcarbodiimide (DCC) are added, followed by DMSO and reaction at room temperature. The white precipitate 1,3-dicyclohexylurea (DCU) in the reaction system was removed by filtration using an oily filter membrane. The resulting filtrate was precipitated in anhydrous diethyl ether, centrifuged, and the precipitate was washed twice with anhydrous diethyl ether. The precipitate was then dried under vacuum for 12 hours to obtain a pale yellow solid (PPAA).

[0030] Preferably, the structural formula of the PPAA is shown in formula (Ⅰ):

[0031]

[0032] Preferably, the PPGAA and PPSBAA can be synthesized by Raft polymerization, referring to the PPAA synthesis steps.

[0033] Furthermore, the enzyme-responsive crosslinking agent in step S2 is a crosslinking agent that can be cleaved by an enzyme.

[0034] Furthermore, the enzyme-responsive crosslinking agent is one or two of the following: crosslinking agents that can be cleaved by matrix metalloproteinases (MMP2) and crosslinking agents that can be cleaved by cathepsin B (CTSB).

[0035] Preferably, the enzyme-responsive crosslinking agent is a crosslinking agent that can be cleaved by matrix metalloproteinases (MMP2) and cathepsin B (CTSB).

[0036] Preferably, in step S2, the mass ratio of the water-soluble monomer, the amphiphilic zwitterionic monomer, the crosslinking agent that can be cleaved by matrix metalloproteinases, and the crosslinking agent that can be cleaved by cathepsin B is 30:1:(0.1-0.5):2. More preferably, it is 30:1:0.25:2.

[0037] Preferably, the cross-linking agent (MP-CL) cleavable by matrix metalloproteinases comprises the peptide segment: Ac-PLGLAGGKG-GKGGALGLP-Ac, wherein the amino acid sequence "PLGLA" can be cleaved and hydrolyzed by MMP2 enzyme, and a carbon-carbon double bond is introduced at the lysine "K" position; the cross-linking agent (CB-CL) cleavable by cathepsin B enzyme comprises the peptide segment: Ac-GLKGFLG KLG-NH2, wherein the amino acid sequence "GFLG" can be cleaved and hydrolyzed by cathepsin B, and a carbon-carbon double bond is introduced at the lysine "K" position.

[0038] Preferably, the cross-linking agent that can be cleaved by matrix metalloproteinases has a peptide segment that is an inverted repeat sequence of a matrix metalloproteinase substrate.

[0039] Preferably, the crosslinking agent (MP-CL) that can be cleaved by matrix metalloproteinases is synthesized by the following method: utilizing the amidation reaction between the primary amine and acyl chloride in the polypeptide, the MMP2 responsive polypeptide (sequence Ac-PLGLAGGKG-GKGGALGLP-Ac) is mixed with methacryloyl chloride, dissolved in DMSO, stirred and reacted overnight at room temperature, the reaction solution is dialyzed sequentially with methanol and ultrapure water, the dialysate is lyophilized, and finally a white powder MP-CL is obtained.

[0040] Preferably, the structure of the MP-CL is as shown in formula (II):

[0041]

[0042] Preferably, the cross-linking agent (CB-CL) that can be cleaved by cathepsin B enzyme is synthesized by the following method: CTSB enzyme-responsive polypeptide (sequence Ac-GLKGFLGKLG-NH2) is mixed with methacryloyl chloride, dissolved in DMSO, stirred and reacted overnight at room temperature, the reaction solution is dialyzed sequentially with methanol and ultrapure water, the dialysate is lyophilized, and finally a white powder CB-CL is obtained.

[0043] Preferably, the structural formula of the CB-CL is shown in formula (Ⅲ):

[0044]

[0045] Further, the initiator mentioned in step S3 is any one or any combination of ammonium persulfate and tetramethylethylenediamine (APS and TEMED), ammonium persulfate and sodium sulfite, azobisisobutyronitrile (AIBN), and azobisisobutyramidoline hydrochloride (AIBI).

[0046] Preferably, the initiator is ammonium persulfate and tetramethylethylenediamine (APS and TEMED).

[0047] The present invention also provides nanogel materials for loading and delivering nucleic acid molecules prepared by the above-described preparation method.

[0048] This invention also provides a nanogel loaded with nucleic acid molecules and exhibiting triple responsiveness to MMP2 enzyme, CTSB enzyme, and pH. It is based on the above-mentioned preparation method for nanogel materials used to load and deliver nucleic acid molecules, wherein step S2 involves dissolving a water-soluble monomer, an amphiphilic zwitterionic monomer, an enzyme-responsive crosslinking agent, and nucleic acid molecules in water to obtain an aqueous mixture; the enzyme-responsive crosslinking agent is a crosslinking agent that can be cleaved by matrix metalloproteinase MMP2 and a crosslinking agent that can be cleaved by cathepsin B. The specific steps are as follows:

[0049] S1. Obtain an organic dispersion by dissolving the surfactant in an organic solvent;

[0050] S2. An aqueous mixture is obtained by dissolving a water-soluble monomer, an amphiphilic zwitterionic monomer, an enzyme-responsive crosslinking agent, and a nucleic acid molecule in water; wherein the enzyme-responsive crosslinking agent is a crosslinking agent that can be cleaved by matrix metalloproteinase MMP2 and a crosslinking agent that can be cleaved by cathepsin B;

[0051] S3. Add the aqueous mixture to the organic dispersion to form a water-in-oil system, add an initiator, and cause the water-in-oil system to undergo emulsion polymerization to form a nanogel loaded with nucleic acid molecules.

[0052] Furthermore, the enzyme-responsive cross-linking agent is MP-CL and CB-CL.

[0053] Furthermore, the average particle size of the nanogel material is 60–70 nm.

[0054] Preferably, the average particle size of the nanogel material is 65 nm.

[0055] Furthermore, the nucleic acid molecule may be siRNA.

[0056] The aforementioned nanogel with triple responsiveness to MMP2, CTSB, and pH was prepared by modifying the reverse emulsion polymerization system with amphiphilic zwitterionic monomers and simultaneously introducing two cross-linking agents (MP-CL and CB-CL) responsive to both MMP2 and CTSB enzymes. Nucleic acid molecules were dissolved in the aqueous phase, and finally, an initiator was added to induce emulsion polymerization in a water-in-oil system to form the nanogel. The resulting nanogel's interfacial cross-linking layer is a cross-linked structure of amphiphilic PPAA and enzyme-sensitive cross-linking agents (MP-CL and CB-CL), with internal voids that can be used to load siRNA. The amphiphilic zwitterionic monomers, MP-CL, and CB-CL synergistically enhance the cellular uptake capacity, while the simultaneous presence of low pH, MMP2, and CTSB synergistically promotes the release of nucleic acid molecules from the nanogel.

[0057] Furthermore, the siRNA includes, but is not limited to, siRNAs that can effectively inhibit the expression of the PLK1 gene. Taking siRNA targeting PLK1 as an example, the present invention provides a nanogel delivery material loaded with siRNA that can efficiently deliver siRNA to the cell wall, effectively inhibit the expression of the PLK1 gene, and exhibits excellent stability and biosafety.

[0058] This invention provides the application of any of the above-described nanogel materials for loading and delivering nucleic acid molecules in the preparation of tumor therapeutic drugs.

[0059] The present invention also provides the application of the above-described nanogel loaded with nucleic acid molecules and possessing triple responsiveness to MMP2 enzyme, CTSB enzyme and pH in the preparation of tumor therapeutic drugs.

[0060] Compared with the prior art, the present invention has the following beneficial effects:

[0061] This invention provides a nanogel with high loading and delivery efficiency. The material can be used for the efficient loading and delivery of nucleic acid molecules. Its preparation method improves the reverse emulsion polymerization system with amphiphilic zwitterionic monomers, enhancing polymerization at the two-phase interface and avoiding the extrusion effect of polymerization on nucleic acid molecules in the aqueous core, thereby improving the loading efficiency of the nanogel for nucleic acid molecules. Its encapsulation efficiency for siRNA reaches 90.24%, and the drug loading rate is approximately 1.60%. Furthermore, the introduction of cross-linking agents MP-CL and CB-CL, which can cleave in response to matrix metalloproteinase II (MMP2) or cathepsin B (CTSB), endows the nanogel with the ability to respond to charge reversal in the tumor matrix and release nucleic acid molecules from tumor cells. Simultaneously, the introduction of the amphiphilic zwitterionic monomers, MP-CL, and CB-CL synergistically enhances the ability of the nanogel to be taken up by cells; the acid-sensitive blocks on the amphiphilic monomers give the nanogel lysosomal escape capability. The delivery material can efficiently deliver siRNA to the cellular matrix, effectively inhibiting the expression of the target gene, and exhibits excellent stability and biosafety. Attached Figure Description

[0062] Figure 1 The synthesis flowcharts are shown for MP-CL(a), CB-CL(b), and PPAA(c).

[0063] Figure 2 The NMR spectrum of MP-CL, an enzyme-responsive cross-linking agent for MMP2.

[0064] Figure 3 The NMR spectrum of CB-CL, a CTSB enzyme-responsive crosslinking agent.

[0065] Figure 4 This is the NMR spectrum of the amphiphilic molecule PPAA.

[0066] Figure 5 This is a transmission electron microscope image of PMC-NG / siRNA.

[0067] Figure 6 This is the standard curve for siRNA.

[0068] Figure 7 The in vitro simulated release curves of siRNA in PMC-NG / siRNA under different pH, MMP2 and CTSB conditions are shown.

[0069] Figure 8 The particle size potential results of nanogels with different MP-CL input amounts under different conditions (without MMP2 enzyme and with 10nM MMP2 enzyme).

[0070] Figure 9 This figure shows the stability results of the nanogel in in vitro simulated serum.

[0071] Figure 10 Confocal microscopy and flow cytometry images of polymer nanogels incubated with cells for 4 hours.

[0072] Figure 11 This is a schematic diagram illustrating the preparation and operation of siRNA-loaded nanogels.

[0073] Figure 12 The results of cytotoxicity of different concentrations of empty nanogels against hepatocellular carcinoma cells SMMC7721 and normal human hepatocytes LO2 are shown in (a) and (b) respectively.

[0074] Figure 13 Tumor volume curves for different groups of mice.

[0075] Figure 14 HE-stained images of tumor (a) and major organ tissue (b) sections from different groups of mice. Detailed Implementation

[0076] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0077] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0078] Example 1: Synthesis of related compounds

[0079] (1) Synthesis of MMP2 enzyme-responsive cross-linking agent (MP-CL): 0.1 g of MMP2-sensitive peptide (sequence Ac-PLGLAGGKG-GKGGALGLP-Ac) and 0.11 g of methacryloyl chloride were added to a 25 mL round-bottom flask, dissolved in 10 mL of DMSO, and stirred overnight at room temperature. The reaction solution was then transferred to a dialysis bag (MWCO: 0.5 kDa), dialyzed with methanol for 24 h, and then dialyzed with ultrapure water for 24 h. The dialysate was lyophilized to obtain a white powder (MP-CL). The synthesis process is as follows: Figure 1 As shown in (a).

[0080] (2) Synthesis of CTSB enzyme-responsive cross-linking agent (CB-CL): Specifically, 0.10 g of peptide (containing the GFLG sequence Ac-GLKGFLGKLG-NH2) and 0.101 g of methacryloyl chloride were added to a 25 mL round-bottom flask, dissolved in 10 mL of DMSO, and stirred at room temperature for 12 h. The reaction solution was then transferred to a dialysis bag with a molecular weight cutoff (MWCO) of 0.5 kDa, dialyzed with methanol for 1 day, followed by dialyzed with ultrapure water for 1 day, and the dialysate was lyophilized to obtain a white powder (CB-CL). The synthesis process is as follows: Figure 1 As shown in (b).

[0081] (3) Synthesis of the amphiphilic molecule (PPAA): Specifically, 0.5 g of Raft reagent 2-dodecyltrithiocarbonate-2-methylpropionic acid (DDAT) was weighed, 1.74 g of diisopropylaminoethyl methacrylate (DPA) was added, and 22.49 mg of initiator azobisisobutyronitrile (AIBN) was added to a 100 mL reaction flask and dissolved in a 50 mL mixture of reaction-grade DMF and water (95:5, v / v). After purging with nitrogen at room temperature for 45 min, the reaction system was transferred to a 70 °C oil bath and reacted for 24 h. Then, 0.43 g of 3-[(3-acrylamidopropyl)dimethylammonium]acetate (CBAA) was added, and the reaction was continued for another 24 h. Then, the mixture was rapidly cooled to room temperature, 4.51 g of AIBN was added, and the mixture was bubbled with nitrogen for 45 min before being placed at 70 °C for another 24 h. This process was repeated twice to remove the thiocarbonate groups from the polymer. The reaction system was then precipitated twice in anhydrous diethyl ether, filtered, and the solid was washed with acetone and dried under vacuum to obtain a white solid. Then, 1 g of the solid obtained in the previous step was taken, and 0.071 g of 3-methylpropenylamine, 0.144 g of N-hydroxysuccinimide (NHS), and 0.258 g of N,N-dicyclohexylcarbodiimide (DCC) were added, along with 25 mL of DMSO. The reaction was carried out at room temperature for 24 h. Subsequently, the white precipitate 1,3-dicyclohexylurea (DCU) in the reaction system was removed by filtration through an oily filter membrane. The resulting filtrate was precipitated in anhydrous diethyl ether, centrifuged, and the precipitate was washed twice with anhydrous diethyl ether. The precipitate was dried under vacuum for 12 h to finally obtain a pale yellow solid (denoted as PPAA). The synthesis process is as follows: Figure 1 As shown in (c).

[0082] The obtained products were subjected to NMR analysis. The NMR spectra of the MMP2 enzyme-responsive cross-linking agent MP-CL, the CTSB enzyme-responsive cross-linking agent CB-CL, and the amphiphilic molecule PPAA are shown below. Figure 2As shown in Figures 3 and 4, the main NMR peaks have good assignments. The number of repeating units in PCBAA can be calculated to be approximately 2 and the number of repeating units in PDPA can be calculated to be 3, based on the ratio of the NMR integral area of ​​the carbon-carbon double bond hydrogen at the end of the PPAA main chain to the integral area of ​​the -CH3 hydrogen connected to the quaternary amine in the repeating unit of PCBAA and the integral area of ​​the -CH(CH3)2 hydrogen connected to the tertiary amine in PDPA.

[0083] Example 2: Preparation of siRNA-loaded nanogels PMC-NG / siRNA

[0084] Weigh out sodium bis(2-ethylhexyl)sulfosuccinate (AOT, 160 mg) and Span-80 (640 mg); add n-hexane (20–30 mL), stir vigorously until the surfactant is completely dissolved, and purge with nitrogen for 10 min. Dissolve the monomers CBAA (30 mg, dissolved in 150 μL water), PPAA (1 mg, dissolved in 500 μL water), CB-CL (2 mg, dissolved in 20 μL DMSO), MP-CL (0.25 mg, dissolved in 5 μL DMSO), and siRNA (33 μg, dissolved in 33 μL water) in water, respectively. Purge with nitrogen into the aqueous phase for 5–10 min, then slowly add the aqueous mixture dropwise to the organic dispersion system. After stirring for 5–10 min, add 15 μL of 20% ammonium persulfate (APS) solution to the aqueous phase. After stirring for another 5 minutes, 8 μL of tetramethylethylenediamine (TEMED) was added to initiate the polymerization reaction. The reaction was then rapidly magnetically stirred in an ice-water bath for 2 hours. Three excess volumes of pre-cooled acetone were added to precipitate the product, which was then placed at -20°C for 30 minutes. The product was then washed three times with acetone and vacuum-dried for approximately 24 hours to obtain a lyophilized product for storage or use. The nanogel was reconstituted in PBS buffer at pH 7.4, centrifuged to obtain a clear liquid, and then purified using a 100 kDa ultrafiltration tube to remove free CBAA, siRNA, cross-linking agents, and other molecular substances, yielding the siRNA-loaded nanogel PMC-NG / siRNA.

[0085] The nanogel PMC-NG / siRNA was characterized by TEM, and the results are as follows: Figure 5 As shown, the nanogel PMC-NG / siRNA has a spherical structure with a particle size of approximately 65 nm.

[0086] Example 3: Determination of encapsulation efficiency and drug loading rate of siRNA on nanogel PMC-NG / siRNA

[0087] Determination of siRNA content in the nanogel prepared in Example 2: The following absorbance values ​​were obtained by detecting the UV absorption of RNA at 260 nm using a Unico UV-2000 UV-Vis spectrophotometer. First, aqueous solutions of siRNA with known concentration gradients (10, 15, 20, 25, and 30 μg / mL) were prepared. The absorbance values ​​were measured for each concentration. A standard curve for siRNA was then established using Origin simulation. The standard curve for siRNA is shown below. Figure 6 As shown. A certain amount of siRNA-loaded nanogel was then lyophilized, its mass was measured using an electronic balance, and then reconstituted with 1 mL of water. The absorbance of the resulting solution at 260 nm was measured using a UV-Vis spectrophotometer. The mass of the siRNA was calculated using the previously simulated standard curve. PMC-NG / siRNA samples were lyophilized, and the sample mass was measured after lyophilization. Simultaneously, siRNA-loaded nanogels without introduced PPAA were prepared and measured. The encapsulation efficiency and drug loading rate of the sample for siRNA can then be calculated using the following formulas:

[0088] The encapsulation efficiency (LE) and drug loading rate (LC) of siRNA were calculated as follows: Encapsulation efficiency (LE) = (mass of siRNA loaded on the nanogel) ÷ (mass of added siRNA) × 100%; Drug loading rate (LC) = (mass of siRNA loaded on the nanogel) ÷ (mass of lyophilized nanogel sample) × 100%. The nanogels measured using the above methods achieved an encapsulation efficiency of 90.24% for siRNA and a drug loading rate of approximately 1.60%. In contrast, the nanogels without PPAA showed an encapsulation efficiency of only 61.25% and a drug loading rate of 1.06% for siRNA.

[0089] Example 4: PMC-NG / siRNA nanogel siRNA in vitro simulated release curve

[0090] First, the siRNA release assay was divided into 10 groups (Group 1: pH 7.4 PBS; Group 2: pH 6.5 PBS; Group 3: pH 5.0 PBS; Group 4: pH 7.4, 10 nM MMP2; Group 5: pH 6.5, 10 nM MMP2; Group 6: pH 7.4, 1 mM papain; Group 7: pH 5.0, 1 mM papain; Group 8: pH 7.4, 10 nM MMP2 and 1 mM papain; Group 9: pH 6.5, 10 nM MMP2 and 1 mM papain; Group 10: pH 5.0, 10 nM MMP2 and 1 mM papain). All solutions were prepared using 50 mM PBS. First, the nanogels were assembled according to the optimized conditions described in the previous section. Then, the samples were divided into 10 groups, with each group having 6 time points (0, 2, 4, 8, 12, and 24 hours). Each of the 10 solutions was further divided into 6 equal aliquots, and the solutions were added at the designated time points. Finally, the samples were placed in a 37°C constant-temperature shaker to simulate the in vivo environment for drug release testing. At each designated time point, 2 mL of buffer medium was removed, and an equal volume of fresh PBS was added. All samples from each time point, after 24 hours of release, were simultaneously subjected to agarose gel electrophoresis. Gray-scale analysis was used to calculate the siRNA release amount at different time points for each group, and the release was recorded.

[0091] like Figure 7 As shown, the release rate of siRNA molecules from the nanogel was lowest at pH 7.4 and 6.5, with a cumulative release rate not exceeding 8% at both pH levels after 24 hours of incubation. However, at pH 5.0, the release of siRNA increased to approximately 15%, attributed to the PDPA block in PPAA changing from hydrophobic to hydrophilic due to protonation of the tertiary amine group. At pH 6.5, the addition of MMP2 further increased the release rate of siRNA to approximately 20%, because MP-CL in the gel was cleaved by the MMP2 enzyme, and the breakage of the cross-linking agent weakened the binding effect on siRNA to some extent. However, due to the low proportion of MP-CL in the nanogel, the release of siRNA from the nanogel was limited at this point. Conversely, at pH 7.4 and 6.5, the addition of CTSB enzyme increased the siRNA release rate to approximately 42% and 50%, respectively. In the presence of both MMP2 and CTSB, the cumulative 24-hour release rate of siRNA at pH 5.0 exceeded 80%, indicating that low pH, MMP2, and CTSB synergistically promoted the release of siRNA from the nanogel.

[0092] Example 5: Particle size potential test of nanogels with different MP-CL dosages under different conditions (without MMP2 enzyme and with 10 nM MMP2 enzyme).

[0093] Following the method in Example 2, the amount of MP-CL added was varied (0.1 mg, 0.25 mg, 0.5 mg) to prepare siRNA-loaded nanogels PMC-NG / siRNA (denoted as NG 0.1, NG 0.25, and NG 0.5, respectively). Three nanogel samples were taken, each divided into two equal portions, and 10 nM MMP2 enzyme solution and pH 7.4 PBS were added to each portion. The hydrated particle size and zeta surface potential were measured using a 90 Plus / BI-MAS zeta potential and particle size analyzer. Each sample was measured in triplicate.

[0094] like Figure 8 As shown, the particle sizes of NG 0.1 and NG 0.25 are approximately 68–69 nm, with good uniformity in distribution and a Zeta potential of approximately -1 mV. In contrast, the particle size and potential of NG 0.5 are 473.43 nm and -0.36 mV, respectively, with poor particle size uniformity and unstable test results. This may be due to excessive MP-CL usage, leading to a higher degree of cross-linking of the polymer chains in the NG 0.5 structure, resulting in larger and less uniform particles. Furthermore, the effect of MMP2 enzyme on the surface potential of the nanogels was investigated. Compared to samples without MMP2 enzyme (-0.95 mV, -1.19 mV, and -0.36 mV), the surface potentials of NG0.1, NG 0.25, and NG 0.5 hydrogels changed to +6.52 mV, +15.13 mV, and +22.40 mV, respectively, after the addition of 10 nM MMP2 enzyme. This is attributed to the cleavage of MP-CL by the MMP2 enzyme. When the MMP2 enzyme cleaves the reverse-linked MMP2 substrate peptide, the primary amine group remains in the nanogel, while the end linked to the carboxyl group detaches, making the nanogel positively charged. This also verifies that the prepared nanogel possesses good MMP2 enzyme responsiveness, and can achieve charge reversal in response to the high expression of MMP2 enzyme in the tumor matrix microenvironment, thereby theoretically promoting the uptake of siRNA by tumor cells.

[0095] Example 6: Stability study of nanogels in in vitro simulated serum

[0096] Following the method described in Example 2, empty nanogels PMC-NG (without loading), PMC-NG-SCR (loaded with common primer SCR), and PMC-NG-siPLK1 (loaded with siRNA) were prepared. The changes in hydrated particle size of the drug-loaded nanogels in a simulated physiological environment (PBS + 10% FBS) were measured using a 90Plus / BI-MAS Zeta potential and particle size analyzer (Brookhaven, USA). 1 mg / mL of each group of nanogels was added to 10% FBS in PBS, and the hydrated particle size of each group was measured at different incubation times (4, 8, 12, 24, and 48 h).

[0097] like Figure 9 As shown, under DMEM + 10% FBS conditions, the particle size of different groups of nanogels fluctuated between 55 and 74 nm within 50 h, with essentially no change in particle size. This is due to the good water solubility and biocompatibility of the nanogels, providing a basis for subsequent cell and animal experiments.

[0098] Example 7: Study on the uptake and intracellular distribution of polymer nanogels

[0099] Referring to the method in Example 2, the loaded siRNA was replaced with primers labeled with Cy3 to prepare four experimental groups: CB-NG-Cy3 nanogels (containing no PPAA or MP-CL), PC-NG-Cy3 nanogels (containing no MP-CL), MC-NG-Cy3 nanogels (containing no PPAA), and PMC-NG-Cy3 nanogels (containing PPAA, MP-CL, and CB-CL). PBS was used as the control group. Tumor cell uptake of the nanogels was observed using CLSM confocal electron microscopy. The specific steps are as follows: Prepare approximately 1×10⁻⁶ ppm of siRNA. 5 SMMC7721 cells at a concentration of / mL were seeded in each confocal culture dish with DMEM complete medium at pH 7.4 at / mL, and then cultured overnight in a humidified incubator (37℃ and 5% CO2). The next day, the cells were co-incubated with the same concentration of nanogel (50 ng / mL) for 4 h, washed twice with PBS, and then co-incubated with Hoechst 33342 (5 μg / mL) to stain the cell nuclei for 15 min. The original medium was then removed, and the cells were washed three times with PBS. The incubation of the drug with cells was observed using CLSM, fluorescence was captured in each group, and the efficiency of cell uptake of the nanogel was detected by flow cytometry.

[0100] like Figure 10 As shown, to detect the uptake of siPLK1 loaded on a nanogel carrier by cells, a primer-loaded nanogel PMC-NG-Cy3 was first designed to simulate PMC-NG-siRNA for experimental research. Firstly, this invention uses confocal microscopy to detect the results as shown below. Figure 10 Compared to the control group, all cells in the experimental groups exhibited red fluorescence, and the red fluorescence intensity of the groups containing PPAA, MMP2, and CTSB enzyme-sensitive peptides was significantly higher than that of the other experimental groups. Flow cytometry was used to detect the uptake effect of Cy3 primer-loaded nanogels in different groups after 4 hours, further confirming the uptake of the nanogels. As... Figure 10As shown, when the control group and five groups of nanogel materials (CB-NG-Cy3, PC-NG-Cy3, MC-NG-Cy3, and PMC-NG-Cy3) were co-incubated with cells for 4 hours, their uptake efficiencies and fluorescence intensities were 0.27%, 23.1%, 55.84%, 73.4%, 94.3% and 1453±128, 5020±114, 7550±290, 12299±1592, 20949±1366, respectively. Although varying degrees of red fluorescence were detected in the cells of all nanogel-loaded primer Cy3 experimental groups, the PMC-NG-Cy3 nanogel showed the highest uptake by cells. This indicates that, under the same concentration of nanogel incubation, the introduction of PPAA, MMP2, and CTSB enzyme-sensitive peptides into the nanogel can synergistically enhance the effects of cellular uptake and drug release. On the one hand, the introduction of PPAA, due to its amphiphilic nature, causes it to tend to distribute at the oil-water interface during the polymerization of nanogels, which to some extent increases the void space in the gel core and improves the loading efficiency of siRNA. On the other hand, the amphiphilic PPAA can enhance the interaction between the nanogel and the cell membrane, thus facilitating uptake by tumor cells. Furthermore, the introduction of two cross-linking agents (MP-CL and CB-CL) responsive to MMP2 and CTSB enzymes allows the nanogel to respond hierarchically to the highly expressed MMP2 enzyme in tumor tissue and the intracellular CTSB enzyme, enhancing drug uptake and release. In tumor tissues with MMP2 enzyme overexpression, the peptide sequence of MP-CL breaks down, exposing a large amount of positive charge on the surface of the nanoparticles, converting the surface potential of the nanoparticles to positive, and promoting cellular uptake. After entering the cell, the CTSB enzyme-specific peptides constituting the cross-linking layer respond to the highly expressed CTSB enzyme in the lysosomes of tumor cells, breaking down the siRNA and releasing it from the nanogel. The tertiary amine of the PDPA block in the PPAA of the nanogel can respond to the acidic environment within the lysosome, promoting lysosomal rupture through the proton sponge effect, thereby releasing the siRNA. The preparation and working schematic diagram of siRNA-loaded nanogels are shown below. Figure 11 As shown.

[0101] Example 8: Biosafety evaluation of nanogels and cytotoxicity detection of siRNA-loaded nanogels

[0102] Hepatocellular carcinoma cells (SMMC7721) and normal human hepatocytes (LO2) were cultured. On the second day, the cells were washed twice with PBS and digested with 0.25% EDTA trypsin to form a cell suspension, with a cell count concentration of approximately 3 × 10⁻⁶. 4Cells were cultured at a density of approximately 3000 cells / well using 100 μL of the pre-prepared empty nanogel (added according to a specific polymer concentration gradient) under the same conditions overnight. The original culture medium was then removed, and pre-prepared empty nanogel was added to each well (with three replicates per group). After co-culturing the samples and cells for 48 hours, the culture medium was discarded, and 100 μL of medium containing 5 mg / mL MTT was added to each well. The cells were cultured under the same conditions for another 4 hours. The culture medium was then discarded, and 150 μL of dimethyl sulfoxide (DMSO) was added to each well with shaking to dissolve the cells. The absorbance at 570 nm was measured using a microplate reader. Cell viability was calculated as follows: Cell viability = (Experimental group - Blank background group) ÷ (Control group - Blank background group) × 100%. PMC-NG / siRNA and PMC-NG / SCR were evaluated for their cytotoxicity using the same method.

[0103] like Figure 12 As shown in (a), the cell viability decreased slightly with increasing concentration of empty nanogels. At a polymer concentration of 50 μg / mL, the viability of LO2 and SMMC7721 cells approached 100% and exceeded 90%, respectively; even at a concentration of 200 μg / mL, the viability of LO2 and SMMC7721 cells remained as high as 90% and 80%, respectively. These results indicate that the carrier itself has almost no toxicity to cells.

[0104] As Figure 12 As shown in (b), for PMC-NG / siRNA, the inhibitory effect of PLK1siRNA on tumor cells is significant with increasing siRNA concentration. However, since PLK1 is also expressed in non-tumor cells, excessive siRNA can also have some impact on normal cells. To reduce side effects while still achieving therapeutic effects against tumors, this study explored a reasonable siRNA concentration range of approximately 4.5 μg / mL. This concentration can effectively kill tumor cells while minimizing damage to other normal cells.

[0105] Example 9: Treatment of tumor-bearing mice with siRNA-loaded nanogels.

[0106] Tumor-bearing nude mice were randomly divided into three groups (PBS control group, PMC-NG / SCR group, and PMC-NG / siPLK1 group), with 12 mice in each group. The mice were administered the drug every two days. After each administration, the long and short diameters of the tumor (tumor long diameter a, tumor short diameter b) were measured using digital calipers, and the tumor volume was calculated using the formula: V = ab 2 / 2. When the tumor volume exceeds 2000 mm 3 Those who survive to the 39th day will all be euthanized.

[0107] Tumor tissue and major organs were removed and fixed with 4% paraformaldehyde. Major organs (heart, liver, spleen, kidney, and lung) and tumors were collected, cut into paraffin sections, and stained with hematoxylin-eosin (HE). After staining, the sections were washed with running water, then separated with 1% dilute acid for 30 seconds, washed with running water, stained with eosin for 2 minutes, and washed with running water. The samples were then sequentially immersed in 100%, 90%, 80%, and 70% ethanol for 5 minutes each time for hydration. Finally, the sections were dried, sealed with neutral adhesive, and photographed and recorded under a microscope.

[0108] like Figure 13 The tumor growth curves in mice showed that both the PBS control group and the PMC-NG-SCR control group experienced significant increases in tumor volume, with the average tumor volume reaching 1800 mm² after treatment on day 18. 3 With 1600mm 3 The mean volume of the PMC-NG-siPLK1 treatment group was only 600 mm. 3 The difference between the control group and the control group was statistically significant (p<0.05), indicating that the PMC-NG / siPLK1 treatment group had a certain inhibitory effect on tumor growth in mice.

[0109] In addition, from Figure 14 HE staining images of tumor tissues showed that the PBS control group and the PMC-NG-SCR group exhibited high-density cell nuclei, clearly defined cytoplasm, and tight junctions, indicating high tumor cell proliferative activity. In the PMC-NG / siPLK1 group, the cell morphology showed, to some extent, lightly stained nuclei, rounded or even shrunken nuclei, intercellular fat filling, and widened intercellular spaces, indicating apoptosis. This suggests that PMC-NG / siPLK1 has an inhibitory effect on tumor growth in mice. Furthermore, no significant pathological changes were observed in the major organs (heart, liver, spleen, lung, and kidney) of the three groups of mice, indicating that the nanogel possesses good biocompatibility.

Claims

1. A method for preparing nanogels for loading and delivering nucleic acid molecules, characterized in that, Includes the following steps: S1. Obtain an organic dispersion by dissolving the surfactant in an organic solvent; S2. An aqueous mixture is obtained by dissolving a water-soluble monomer, an amphiphilic zwitterionic monomer, and an enzyme-responsive crosslinking agent in water; S3. Add the aqueous mixture to the organic dispersion to form a water-in-oil system, add an initiator to cause emulsion polymerization of the water-in-oil system to form a nanogel; The amphiphilic zwitterionic monomer in step S2 is poly(diisopropylaminoethyl methacrylate)-poly(3-[(3-acrylamidopropyl)dimethylammonium]acetate-dimethylacetyl-methylpropenamine; The enzyme-responsive cross-linking agent in step S2 is MP-CL and CB-CL; the structural formula of MP-CL is shown in formula (II): ； The structural formula of the CB-CL is shown in formula (Ⅲ): 。 2. The preparation method according to claim 1, characterized in that, The surfactant in step S1 is any one or more of sodium bis(2-ethylhexyl)sulfosuccinate, Span 80, polyoxyethylene lauryl ether, or potassium monododecyl phosphate.

3. The preparation method according to claim 1, characterized in that, The organic solvent in step S1 is any one or more of n-hexane, cyclohexane, isopentane, or dichloromethane.

4. The preparation method according to claim 1, characterized in that, The water-soluble monomer in step S2 is any one or more of 3-[(3-acrylamidopropyl)dimethylammonium]acetate, 3-[N,N-dimethyl-[2-(2-methylprop-2-enoyloxy)ethyl]ammonium]propane-1-sulfonic acid inner salt, and [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfonic acid propyl)ammonium hydroxide.

5. Nanogel materials for loading and delivering nucleic acid molecules prepared by any of the preparation methods described in claims 1 to 4.

6. A nanogel loaded with nucleic acid molecules and exhibiting triple responsiveness to MMP2 enzyme, CTSB enzyme, and pH, characterized in that, The nanogel is prepared by the preparation method of claim 1, wherein step S2 is to dissolve the water-soluble monomer, the amphiphilic zwitterionic monomer, the enzyme-responsive crosslinking agent, and the nucleic acid molecule in water to obtain an aqueous mixture; the enzyme-responsive crosslinking agent is a crosslinking agent that can be cleaved by matrix metalloproteinase MMP2 and a crosslinking agent that can be cleaved by cathepsin B.

7. The nanogel according to claim 6, characterized in that, The nucleic acid molecule in question is siRNA.

8. The use of the nanogel according to claim 6 or 7 in the preparation of tumor therapeutic drugs.

Images