Zwitterionic nanogel and application thereof

Abstract

The invention belongs to the field of biological medicine, and discloses zwitterionic nanogel and application thereof.According to the method, N-vinyl caprolactam and methacryloyl ornithine are copolymerized, a cross-linking agent containing disulfide bonds is introduced, and the zwitterionic polymer nanogel with reductive degradability is prepared. The nanogel is good in stability, high in protein adsorption resistance and excellent in biocompatibility, can be kept stable in a physiological environment, and is degraded in tumor cells under the action of reducing substances, so that controlled release of drugs is realized; meanwhile, amino and carboxyl are formed on the surface, so that targeting or fluorescent molecules can be easily connected. According to the invention, the structure, morphology, temperature responsiveness, stability, reductive degradation performance and drug loading and releasing capability of the nanogel are systematically characterized; cytotoxicity experiments prove the biocompatibility and the capability of killing tumor cells; the nanogel disclosed by the invention is used as a drug carrier, is suitable for loading and delivering various drugs, and particularly shows a wide application prospect in tumor treatment.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine and relates to a zwitterionic nanogel and application thereof. Background Art

[0002] With the development of nanotechnology, polymer nanogels, as a new type of biomedical material, have shown great application potential in drug delivery systems due to their unique properties such as good stability and easy functionalization. Polymer nanogels are usually formed by hydrophilic polymers through physical or chemical crosslinking, and have the characteristics of nano-sized dispersed particles. They can be endowed with surface functional groups, biodegradability and active targeting by introducing functional monomers and crosslinkers, modifying targeting ligands, and achieving controlled release of drugs.

[0003] Poly(N-vinyl caprolactam) (PVCL) is an ideal choice for drug delivery due to its good solubility, biocompatibility, high absorption capacity and thermal responsiveness. However, despite its outstanding performance in the field of drug delivery, PVCL nanogels still face challenges such as poor resistance to protein adsorption and poor long circulation. The ability to resist protein adsorption is crucial for the circulation stability and bioavailability of nanocarriers in the body, and existing PVCL nanogels are insufficient in this regard.

[0004] At present, one of the main strategies to improve the ability of nanomaterials to resist protein adsorption is to introduce zwitterionic polymers. Zwitterionic polymers carry a pair of oppositely charged groups in their repeating units, exhibit overall charge neutrality when evenly distributed at the molecular level, and produce strong hydration through ion solvation, thereby effectively preventing nonspecific protein adsorption. Although zwitterionic polymers are widely used in the biomedical field, research on combining them with PVCL to prepare nanogels with multiple advantages is still insufficient.

[0005] The defect of the prior art is that the traditional PVCL nanogel is difficult to meet the application requirements in complex biological environments, especially in terms of anti-protein adsorption and long circulation. The zwitterionic polymer obtained by polymerization of amino acids after acrylation has a pair of oppositely charged groups, amino and carboxyl groups, which not only have good biocompatibility, but also have anti-protein adsorption properties. Therefore, the development of a new type of degradable amino acid zwitterionic nanogel, combining the advantages of PVCL and zwitterionic polymers, has become a hot topic in current research and a problem to be solved urgently. Summary of the invention

[0006] To solve the above problems, we disclose a zwitterionic nanogel and its application.

[0007] The present invention includes the following specific technical solutions:

[0008] A method for preparing zwitterionic nanogel comprises the following steps:

[0009] (1) Synthesis of methacryloyl ornithine: react L-ornithine hydrochloride with basic copper carbonate in water to obtain a methacryloyl ornithine copper complex; react the methacryloyl ornithine copper complex with 8-hydroxyquinoline in chloroform-water to obtain methacryloyl ornithine OrnAA after recrystallization;

[0010] (2) Synthesis of P(VCL-ss-OrnAA) nanogel: N-vinyl caprolactam, initiator, crosslinker and surfactant were mixed and dissolved in water, nitrogen was introduced and the temperature was raised to react, and methacryloyl ornithine OrnAA was added for copolymerization to obtain degradable amino acid zwitterionic nanogel P(VCL-ss-OrnAA).

[0011] In the above scheme, ornithine is a naturally occurring amino acid that plays a wide range of important roles in the human body. It can promote the release of growth hormone, improve the body's immunity, protect the liver, and repair damaged tissues. After one amino group in ornithine is acrylated, an easily polymerizable amino acid zwitterionic monomer (OrnAA) can be obtained. N-vinyl caprolactam (VCL) is a thermosensitive monomer with excellent biocompatibility. By copolymerizing the above two monomers, the advantages of both can be combined to prepare a stimuli-responsive nanogel drug delivery carrier that resists protein adsorption. Since the concentration of the reducing substance glutathione in tumor cells is about 10 times that of normal cells, in the precipitation polymerization process of OrnAA and VCL, N,N'-bis(acryloyl)cystamine (BAC) containing disulfide bonds can be used as a crosslinker to prepare a disulfide-crosslinked reduction-degradable nanogel (P(VCL-ss-OrnAA)). When used as a nanocarrier, the nanogel has excellent stability and anti-protein adsorption ability. When it reaches the tumor cells, the disulfide bonds are reduced and broken into thiol groups, and the nanogel is degraded into low-molecular linear polymers, so that the drugs loaded therein are released. Therefore, P(VCL-ss-OrnAA) nanogel has drug controlled release properties and good biocompatibility.

[0012] Furthermore, in the method for preparing the zwitterionic nanogel, in step (1), the reaction temperature of L-ornithine hydrochloride and basic copper carbonate is 80-95° C., and the reaction time is 10-15 min; the molar ratio of L-ornithine hydrochloride to basic copper carbonate is 1:1 to 3:1.

[0013] Furthermore, in the method for preparing the zwitterionic nanogel, in step (2), optionally: the initiator is azobisisobutyramidine hydrochloride, potassium persulfate and ammonium persulfate, the crosslinker is N,N'-bis(acryloyl)cystamine containing a disulfide bond, and the surfactant is sodium dodecyl sulfate.

[0014] Furthermore, in the method for preparing the zwitterionic nanogel, in step (2), the reaction temperature in the copolymerization step is 60-80° C., and the reaction time is 2-6 h.

[0015] Furthermore, the preparation method of the zwitterionic nanogel comprises the following steps:

[0016] (1) Synthesis and structural characterization of methacryloylornithine:

[0017] (1.1) Dissolve 4.22-5.06 g of L-ornithine hydrochloride in 40-60 mL of water at 80-95°C, then add 3.05-3.66 g of basic copper carbonate to the solution and stir for 10-15 min;

[0018] (1.2) After filtering the insoluble residue, add 20-30 mL of acetone, and then add 12.5-15 mL of 2M KOH aqueous solution;

[0019] (1.3) Cool to 0°C, add 3.00-3.59 mL of methacryloyl chloride and 13.8-16.6 mL of 2M KOH aqueous solution dropwise over 10-30 min in an ice bath, and after the addition is complete, warm to room temperature and stir at room temperature overnight;

[0020] (1.4) Filter to obtain a filter cake, i.e., a blue precipitate of methacryloylornithine copper complex, which is washed with water, methanol and ether in sequence;

[0021] (1.5) Add 6.0-8.0 g of the intermediate product methacryloyl ornithine copper complex powder to 80-120 mL of chloroform solution containing 2.5-3.4 g of 8-hydroxyquinoline, add an equal volume of water, and shake overnight;

[0022] (1.6) filtering and removing the green precipitate 8-hydroxyquinoline copper complex in the chloroform layer, separating the aqueous phase; adding the product in the aqueous phase to THF at a volume ratio of 1:5 to 1:15 for recrystallization to obtain OrnAA crystals, centrifuging and discarding the supernatant, and lyophilizing the precipitate to obtain white powder methacryloyl ornithine, OrnAA; characterizing the structure of OrnAA by nuclear magnetic resonance;

[0023] (2) Synthesis of P(VCL-ss-OrnAA) Nanogel

[0024] (2.1) In a 100 mL three-necked flask equipped with a spherical condenser and mechanical stirring, weigh 160-200 mg of N-vinyl caprolactam (VCL), 2-10 mg of NaHCO3, 2-10 mg of sodium dodecyl sulfate (SDS) and 10-30 mg of N,N'-bis(acryloyl)cystamine (BAC), add 10-30 mL of ultrapure water, and dissolve by ultrasonication until there are no particles;

[0025] (2.2) After N2 is introduced for 20 to 40 minutes, an aqueous solution of initiators such as azobisisobutylamidine hydrochloride (AIBA) and potassium persulfate (KPS) is injected into the three-necked flask. The aqueous solution is prepared by dissolving 5 to 10 mg of the initiator in 1 mL of ultrapure water. The temperature is initially raised to 60 to 80°C. After reacting for 20 to 40 minutes, 0 to 40 mg of methacryloylornithine OrnAA is added. After 2 to 6 hours, a white emulsion is obtained.

[0026] (23) The obtained emulsion was dialyzed in pure water for 3–5 days, and the dialyzed emulsion was freeze-dried to obtain a white freeze-dried powder, namely, P(VCL-ss-OrnAA) nanogel.

[0027] The invention also discloses a zwitterionic nanogel, which is prepared by the above-mentioned preparation method.

[0028] The invention also discloses the use of the nanogel in preparing a drug delivery carrier.

[0029] Furthermore, in the above use, the drug is a broad-spectrum drug for treating tumors, and preferably, the drug is doxorubicin.

[0030] The present invention also discloses a drug preparation for treating tumors, comprising a drug delivery carrier and a drug, wherein the drug carrier is the nanogel as claimed in claim 6; and the drug is a drug for treating tumors, preferably doxorubicin.

[0031] The present invention also discloses a method for preparing the above-mentioned drug preparation for treating tumors, comprising the following steps: fully dispersing DOX and nanogel in 2-5 mL of pH 7.4 PBS buffer solution at a mass ratio of 1:10-3:10; after stirring at room temperature for 12-24 hours, the above-mentioned mixture is centrifuged to collect the nanogel loaded with DOX, and the mixture is centrifuged and washed twice with pure water to remove the DOX adsorbed on the surface, so as to obtain the drug preparation.

[0032] Compared with the prior art, the present invention has the following outstanding beneficial effects:

[0033] The invention discloses the preparation and drug-carrying application of a zwitterionic nanogel. After initiating the reaction for 20 to 40 minutes, an amino acid zwitterionic monomer (such as methacryloylornithine) is introduced to combine with poly (N-vinyl caprolactam) (PVCL), so that the surface of the formed nanogel is rich in amino acid zwitterionic polymer chains, which significantly improves the anti-protein adsorption ability and enhances the circulation stability and bioavailability of the nanocarrier in the body; at the same time, the cross-linking agent containing disulfide bonds introduced into the nanogel enables it to respond to reducing substances (such as glutathione) in tumor cells and degrade, thereby realizing the controlled release of drugs, improving the targeting and efficacy of drugs, and reducing side effects; in addition, the nanogel is prepared from materials with good biocompatibility, ensuring its safety in biomedical applications; the preparation method is simple and flexible, the performance of the nanogel can be regulated by adjusting parameters, and it is easy to functionalize to meet different needs; finally, the nanogel of the invention shows broad application prospects in multiple fields such as drug delivery, tissue engineering, and biosensing, especially in complex biological environments such as tumor treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 NMR spectrum of methacryloylornithine;

[0035] Figure 2 Schematic diagram of the synthesis of P(VCL-ss-OrnAA) nanogels;

[0036] Figure 3 IR spectra of PVCL and P(VCL-ss-OrnAA) nanogels;

[0037] Figure 4 Raman spectra of cross-linker BAC and P(VCL-ss-OrnAA) nanogel;

[0038] Figure 5 Particle size of P(VCL-ss-OrnAA) nanogels with different OrnAA ratios;

[0039] Figure 6 Transmission electron microscopy (TEM) image of P(VCL-ss-OrnAA) nanogel with 10 mg initiator dosage and 10% OrnAA ratio;

[0040] Figure 7 The particle size of P(VCL-ss-OrnAA) nanogel with initiator dosage of 10 mg and OrnAA ratio of 10% changes with temperature;

[0041] Figure 8 The particle size of P(VCL-ss-OrnAA) nanogel with 10 mg initiator dosage and 10% OrnAA ratio changes with time;

[0042] Fig. 9 Particle size distribution of P(VCL-ss-OrnAA) nanogels with different initiator concentrations;

[0043] Fig.10 Particle size changes of P(VCL-ss-OrnAA) nanogels in PBS pH 7.4 buffer solution and PBS pH 7.4 buffer solution containing BSA for 7 days;

[0044] Fig.11 Reductive degradability of P(VCL-ss-OrnAA) nanogels;

[0045] Fig.12 The molecular weight of P(VCL-ss-OrnAA) nanogel after reduction and degradation;

[0046] Fig.13 Drug release curves of P(VCL-ss-OrnAA) nanogels under different media conditions;

[0047] Fig.14 Cytotoxicity of P(VCL-ss-OrnAA) nanogel to normal human umbilical vein endothelial cells (HUVEC) at 24h (a) and 48h (b);

[0048] Fig.15 Cytotoxicity of P(VCL-ss-OrnAA) nanogels to human colon cancer cells (HCT116) at 24h (a) and 48h (b). DETAILED DESCRIPTION

[0049] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0050] The main experimental schemes include:

[0051] (1) Synthesis and structural characterization of methacryloylornithine

[0052] Dissolve 4.22-5.06g of L-ornithine hydrochloride in 40-60mL of water at 80-95°C, then add 3.05-3.66g of basic copper carbonate to the above solution and stir for 10-15min. After filtering the insoluble residue, add 20-30mL of acetone, and then add 12.5-15mL of 2M KOH aqueous solution. Cool to 0°C, add 3.00-3.59mL of methacryloyl chloride and 13.8-16.6mL of 2M KOH aqueous solution simultaneously in an ice bath for 10-30min, and after the addition is complete, warm to room temperature and stir overnight at room temperature. Filter to obtain a filter cake, i.e., a blue precipitate of methacryloyl ornithine copper complex, and wash with water, methanol and ether in turn. Add 6.0-8.0 g of the intermediate product methacryloyl ornithine copper complex powder to 80-120 mL of chloroform solution containing 2.5-3.4 g of 8-hydroxyquinoline, add an equal volume of water, and shake overnight. Filter and remove the green precipitate 8-hydroxyquinoline copper complex in the chloroform layer, and separate the aqueous phase. Add the product in the aqueous phase to THF at a volume ratio of 1:5-1:15 for recrystallization to obtain OrnAA crystals, centrifuge and discard the supernatant, and freeze-dry the precipitate to obtain a white powder (methacryloyl ornithine, OrnAA). The structure of OrnAA was characterized by nuclear magnetic resonance.

[0053] (2) Synthesis of P(VCL-ss-OrnAA) Nanogel

[0054] In a 100mL three-necked flask equipped with a spherical condenser and mechanical stirring, weigh 160-200mg of N-vinyl caprolactam (VCL), 2-10mg of NaHCO3, 2-10mg of sodium dodecyl sulfate (SDS) and 10-30mg of N,N'-bis(acryloyl)cystamine (BAC), add 10-30mL of ultrapure water, and dissolve by ultrasound until there are no particles. After passing N2 for 20-40min, inject azobisisobutyramidine hydrochloride (AIBA) aqueous solution (5-10mg AIBA dissolved in 1mL ultrapure water) into the three-necked flask, start heating to 60-80℃, react for 20-40min, add 0-40mg of methacryloyl ornithine (OrnAA), and obtain a white emulsion after 2-6h. The obtained emulsion is dialyzed in pure water for 3-5 days, and the dialyzed emulsion is freeze-dried to obtain a white freeze-dried powder.

[0055] (3) Analysis of the structure, morphology, particle size and potential of P(VCL-ss-OrnAA) nanogel

[0056] The structure of the synthesized P(VCL-ss-OrnAA) nanogel was characterized by infrared and Raman spectroscopy, the morphology of the nanogel was characterized by transmission electron microscopy (TEM), and the particle size, potential and monodispersity of the nanogel were characterized by dynamic light scattering (DLS).

[0057] (4) Temperature responsiveness experiment of P(VCL-ss-OrnAA) nanogel

[0058] The concentration of the nanogel dispersed in pH 7.4 buffer solution was 0.5-1.5 mg / mL, the temperature was set from 20°C to 65°C, the temperature increase interval was 2.5°C, and the particle size was measured after 3-8 minutes of constant temperature. The dynamic light scattering light source was He-Ne laser, the scattering angle was 90°, and the wavelength was 632.8nm.

[0059] (5) Stability test of P(VCL-ss-OrnAA) nanogel

[0060] The nanogel was added at a concentration of 0.5-1.0 mg mL -1 Dispersed in 0.01M PBS 7.4 buffer solution or human serum albumin (HSA) content of 1-3 mg mL -1 The particle size changes of the nanogels were monitored by DLS at different time intervals within 1 month in 0.01 M PBS 7.4 buffer solution.

[0061] (6) Reduction and degradation experiment of P(VCL-ss-OrnAA) nanogel

[0062] The nanogels were placed in reduced glutathione (GSH) solutions with a concentration of 0 to 20 mmol. The molecular weight of the degraded nanogels was determined by gel permeation chromatography (GPC). The changes in scattered light intensity and particle size were tracked by dynamic light scattering (DLS) to study its redox degradation performance. The test temperature was set at 20 to 30 ° C, and the scattered light intensity and particle size of the sample were recorded every 5 minutes. The ratio of the scattered light intensity of the sample at different time points to the initial sample was defined as the relative turbidity of the sample, and the degree of degradation was reflected by the change in the relative turbidity of the sample.

[0063] (7) Drug loading and release experiments

[0064] Adriamycin (DOX), a first-line clinical tumor drug, was selected as a model drug. DOX and nanogel were fully dispersed in 2-5 mL of pH 7.4 PBS buffer solution at a mass ratio of 1:10-3:10. After stirring at room temperature for 12-24 hours, the mixture was centrifuged to collect the DOX-loaded nanogel, and washed twice with pure water to remove the DOX adsorbed on the surface. The supernatant was collected and the drug loading and encapsulation efficiency of the nanogel were quantitatively analyzed by ultraviolet absorption spectroscopy.

[0065] In order to simulate the normal physiological environment and tumor reduction environment, drug release experiments were carried out in the following five different buffer solutions: phosphate buffer solutions of pH 7.4, 6.5 and 5.5 (37°C) and phosphate buffer solutions of pH 6.5 containing 10mM GSH (37°C and 42°C). The release experiment operation steps are as follows: 1-2 mg of DOX-loaded nanogels were weighed and dispersed in 1-2 mL of the corresponding buffer solution, and then transferred to a dialysis bag with a molecular weight cutoff of 14000Da, and then the dialysis bag was quickly immersed in the above simulated release environment (volume 50-200mL) and slightly magnetically stirred. According to the set time (0-24h), 1-2 mL of solution was taken out from the release medium, and 1-2 mL of the corresponding fresh buffer solution was added to the release medium to keep the volume of the release medium unchanged. The concentration of released DOX was determined by UV spectroscopy. The release experiments in all different release media were repeated 3 times and the average value was taken.

[0066] (8) Cytotoxicity assay

[0067] The cytotoxicity was determined using the CCK-8 method. Normal human umbilical vein endothelial cells (HUVEC cells) were used to evaluate the biocompatibility of the nanogels, and human colon cancer cells (HCT-116 cells) were used to evaluate the ability of free DOX and DOX-loaded nanogels to kill tumor cells. The cell culture method and specific operation steps are as follows: the cells were digested, counted, and prepared to a concentration of 5×10 4 ~1×10 5 50-200 μL of cell suspension (5×10 cells / mL) was added to each well of a 96-well cell culture plate. 3 ~1×10 4cells); the cell culture plate was placed in a 37°C, 5% CO2 incubator for 24 hours; a complete culture medium (80% to 90% RPMI1640 culture medium + 10% to 20% fetal bovine serum + 1% to 5% double antibody) was used to prepare a nanogel working solution containing a concentration of 0 to 1000 μg / mL (HUVEC cells), and a complete culture medium (80% to 90% DMEM culture medium + 10% to 20% fetal bovine serum) was used to prepare a free DOX of 0 to 20 μg / mL and a drug-loaded nanogel working solution with an equal DOX content (HCT-116 cells); 50 to 200 μL of the corresponding working solution was added to each well, and 3 replicates were set for each concentration; the cells were cultured in a 37°C, 5% CO2 incubator for 24 and 48 hours, respectively, and the supernatant was discarded; the 96-well plate was stained with CCK-8, and the OD value was measured at λ=450nm.

[0068] Example 1

[0069] (1) Synthesis and structural characterization of methacryloylornithine

[0070] 4.62g L-ornithine hydrochloride was dissolved in 50mL water at 90°C, and then 3.34g basic copper carbonate was added to the above solution and stirred for 10min. After filtering the insoluble residue, 24mL acetone was added, and then 13.7mL 2M KOH aqueous solution was added. The temperature was lowered to 0°C, and 3.28mL methacryloyl chloride and 15.2mL 2M KOH aqueous solution were added dropwise at the same time under an ice bath for 20min. After the addition was completed, the temperature was raised to room temperature and stirred at room temperature overnight. Filter to obtain a filter cake, i.e., a blue precipitate of methacryloyl ornithine copper complex, which was washed with water, methanol and ether in turn. 6.0g of the intermediate product methacryloyl ornithine copper complex powder was added to 88mL chloroform solution of 2.55g 8-hydroxyquinoline, and then an equal volume of water was added and shaken overnight. The green precipitate 8-hydroxyquinoline copper complex in the chloroform layer was filtered to separate the aqueous phase. The product in the aqueous phase was added to THF at a volume ratio of 1:10 for recrystallization to obtain OrnAA crystals, the supernatant was discarded by centrifugation, and the precipitate was freeze-dried to obtain a white powder (OrnAA). The structure of OrnAA was characterized by nuclear magnetic resonance.

[0071] The results of NMR are as follows Figure 1 As shown (deuterated water as solvent): 1 HNMR(OrnAA): d=5.60(s,1H,C=CH2),5.34(s,1H,C=CH2),3.65(t,1H,CH),3.20 (t,2H,CH2),1.83(s,3H,CH3),1.70–1.80(m,2H,CH2),1.45–1.60(m,2H,CH2).

[0072] Example 2

[0073] (2) Synthesis of P(VCL-ss-OrnAA) Nanogel

[0074] Schematic diagram of the synthesis of P(VCL-ss-OrnAA) nanogels Figure 2 As shown. The specific experimental steps are as follows; in a 100mL three-necked flask equipped with a spherical condenser and mechanical stirring, weigh 180mg of N-vinyl caprolactam (VCL), 5mg NaHCO3, 5mg sodium dodecyl sulfate (SDS) and 20mg N,N'-bis(acryloyl)cystamine (BAC), add 20mL ultrapure water, and ultrasonically dissolve until there are no particles. After N2 is introduced for 30 minutes, azobisisobutyramidine hydrochloride (AIBA) aqueous solution (5.5mg AIBA dissolved in 1mL ultrapure water) is injected into the three-necked flask, and the temperature is raised to 70°C. After reacting for 30 minutes, 1 ml of aqueous solution containing 20mg methacryloyl ornithine (OrnAA) is injected, and a white emulsion is obtained after reacting for 2 hours. The obtained emulsion is dialyzed in pure water for 3 days, and the dialyzed emulsion is freeze-dried to obtain a white freeze-dried powder.

[0075] Example 3

[0076] (3) Characterization of the structure, particle size, potential, and morphology of P(VCL-ss-OrnAA) nanogels

[0077] The structure of the synthesized P(VCL-ss-OrnAA) nanogel was characterized by infrared and Raman spectroscopy, the morphology of the nanogel was characterized by transmission electron microscopy (TEM), and the particle size, potential and monodispersity of the nanogel were characterized by dynamic light scattering (DLS).

[0078] Fourier transform infrared spectroscopy (FTIR) was used to analyze the structures of PVCL nanogel and P(VCL-ss-OrnAA) nanogel. Figure 3 As shown, for P(VCL-ss-OrnAA) nanogel, 3500~3300cm -1 The typical double peak at 3069 cm-1 corresponds to the NH stretching vibration of NH2. -1 The peaks at 2928 and 2854 cm are attributed to the OH stretching vibration of (-COOH) in OrnAA. -1 The stretching and deformation vibration peaks of -CH2 appeared at 1724cm -1 The carbonyl stretching vibration peak of the carboxyl group in OrnAA appeared at 1626 cm -1The stretching vibration peak of amide I band appeared at 1541 cm -1 There is an obvious absorption peak at , which is attributed to the coupling of the NH in-plane bending vibration of the amide bond (-CONH-) of OrnAA and the partial CN stretching vibration in P(VCL-ss-OrnAA) nanogel. The above results show that POrnAA was successfully introduced into the polymer network by copolymerization of OrnAA.

[0079] Figure 4 This is the Raman spectrum of the crosslinker BAC and P(VCL-ss-OrnAA) nanogel. It can be seen from the figure that at 510 cm -1 Absorption peaks corresponding to SS bonds appeared, indicating that disulfide bonds were successfully introduced into the polymer network.

[0080] By changing the amount of OrnAA, while keeping other conditions unchanged, P(VCL-ss-OrnAA) nanogels with different OrnAA contents were prepared. Figure 5 It can be seen that with the increase of the OrnAA ratio, the gel particle size increases. This is because OrnAA is a hydrophilic zwitterionic monomer with strong hydration ability. With the increase of the mass fraction of OrnAA, the number of hydrophilic POrnAA chains on the surface of the polymer nanogel increases, resulting in the enhancement of the hydrophilicity of the polymer chains in the gel particles, and the particles swell more in the aqueous solution, so the particle size increases.

[0081] Table 1 Particle size of P(VCL-ss-OrnAA) nanogels with different amounts of acryloyl-ornithine

[0082]

[0083] a. OrnAA content in monomer %

[0084] b. Polydispersity index, used to characterize the distribution of particle size

[0085] according to Figure 6 It can be seen that the P(VCL-ss-OrnAA) nanogel with an initiator dosage of 10 mg and an OrnAA proportion of 10% presents a regular spherical shape, forming a core-shell structure. This is because VCL first reacts with the cross-linker to form an initial core and then reacts with the monomer OrnAA, thus forming a structure rich in POrnAA on the surface.

[0086] Depend on Figure 7It can be seen that in pH 7.4 buffer solution, for P(VCL-ss-OrnAA) nanogels with an initiator dosage of 10 mg and an OrnAA ratio of 10%, the particle size decreases with increasing temperature, changing from 20°C (about 258nm) to 60°C (about 207nm), indicating that P(VCL-ss-OrnAA) nanogels have significant temperature responsiveness. After fitting calculation, the volume phase transition temperature (VPTT) is 39.3°C, slightly higher than the human body temperature and close to the tumor tissue temperature. When entering the tumor tissue, the volume of the nanogel shrinks, which is conducive to drug release, so the OrnAA ratio is selected to be 10%.

[0087] Figure 8 The particle size of P(VCL-ss-OrnAA) nanogel with 10mg initiator and 10% OrnAA as a function of reaction time. In the initial polymerization hour, the particle size increases rapidly with the growth of time. This may be because in the initial polymerization reaction, the concentration of free radicals and monomers is large, the frequency of collision between them is high, and the reaction rate is fast, so the particle size grows very fast in the initial reaction; after 1 to 2 hours, the particle size increases slowly, because the concentration of free radicals and monomers decreases and the reaction rate decreases; after 2 hours, the particle size hardly changes, because the monomers are almost consumed and cannot provide raw materials for the growth of particle size, so the particle size hardly increases. In summary, the best reaction time is 2h.

[0088] The effect of different amounts of initiator on the particle size of P(VCL-ss-OrnAA) nanogel was investigated. Fig. 9 It can be seen that as the amount of initiator increases, the particle size becomes larger and larger. The more initiators there are, the faster the reaction rate becomes, the more monomer free radicals are generated, the longer the length of the polymer chain becomes during the polymerization process, the larger the particles formed, and the larger the particle size. Considering the size and monodispersity of the particle size, the initiator amount is selected to be 5.5 mg.

[0089] Table 2 Particle size of P(VCL-ss-OrnAA) nanogels with different initiator dosages

[0090]

[0091] a. Polydispersity index, used to characterize the distribution of particle size

[0092] Example 4

[0093] (4) Stability test of P(VCL-ss-OrnAA) nanogel

[0094] The nanogel with an initiator dosage of 5.5 mg and an OrnAA monomer ratio of 10% was prepared at a concentration of 1.0 mg mL-1 Dispersed in 0.01M PBS 7.4 buffer solution or bovine serum albumin (BSA) content of 2 mg mL -1 The particle size changes of the nanogels were monitored by DLS at different time intervals within 1 month in 0.01 M PBS 7.4 buffer solution.

[0095] Good colloidal stability is crucial for the application of polymer nanogels as drug delivery carriers. Therefore, we tracked the particle size changes of P(VCL-ss-OrnAA) nanogels with 10% OrnAA content and 5.5 mg initiator for one week. The results showed that almost no particle size changes were observed ( Fig.10 ), indicating that the nanogel has excellent stability. Zwitterionic polymers can resist nonspecific protein adsorption for a long time due to their neutral surface charge. The anti-protein adsorption of nanocarriers plays an important role in blood circulation and can prolong blood circulation time. By using this property, nanocarriers can evade the monitoring of the immune system and the clearance of the reticuloendothelial system.

[0096] Example 5

[0097] (5) Reduction and degradation experiment of P(VCL-ss-OrnAA) nanogel

[0098] The nanogels were placed in reduced glutathione (GSH) pH 7.4 buffer solutions with concentrations of 0, 10 and 20 mmol, respectively. The molecular weight of the nanogels after degradation was determined by gel permeation chromatography (GPC). The changes in scattered light intensity and particle size were tracked by dynamic light scattering (DLS) to study its redox degradation performance. The test temperature was set at 25°C, and the scattered light intensity and particle size of the sample were recorded every 5 minutes. The ratio of the scattered light intensity of the sample at different time points to the initial sample was defined as the relative turbidity of the sample, and the degree of degradation was reflected by the change in the relative turbidity of the sample.

[0099] Dynamic light scattering (DLS) was used to track the relative turbidity changes of P(VCL-ss-OrnAA) nanogels in different concentrations of GSH reducing media to study their reduction-responsive degradation behavior ( Fig.11 Here, three different concentrations (0, 10, 20 mM) of pH 7.4 phosphate buffer solutions were prepared. Fig.11 The figure shows the change of relative turbidity of nanogel dispersion system with time under different GSH concentrations. It can be seen that when there is no GSH, the scattered light intensity hardly changes; in the presence of GSH, the relative turbidity of nanogel gradually decreases with time, and the higher the GSH concentration, the faster the relative turbidity decreases, indicating that high concentration of GSH promotes the degradation of nanogel.

[0100] The GPC results show that ( Fig.12 ), the polymer nanogel after GSH reduction degradation has a lower average molecular weight (Mw, 1530), indicating that the disulfide bonds inside the gel are converted into thiol groups through reduction cleavage, forming a linear polymer with a smaller molecular weight and a narrower distribution.

[0101] Example 6

[0102] (6) Drug loading and release experiments

[0103] Adriamycin (DOX), a first-line clinical tumor drug, was selected as a model drug. DOX and P(VCL-ss-OrnAA) nanogels were fully dispersed in 2mL pH 7.4 PBS buffer solution at a mass ratio of 2:10. After stirring at room temperature for 24 hours, the mixture was centrifuged to collect the DOX-loaded nanogels, and washed twice with pure water to remove the DOX adsorbed on the surface. The supernatant was collected, and the drug loading of the nanogel was determined to be 14.37% by ultraviolet absorption spectroscopy, and the encapsulation efficiency was 85.6%.

[0104] In order to simulate the normal physiological environment and tumor reduction environment, drug release experiments were carried out in the following five different buffer solutions: pH 7.4, 6.5 and 5.5 phosphate buffer solutions (37°C) and pH 6.5 phosphate buffer solutions containing 10mM GSH (37°C and 42°C). The release experiment operation steps are as follows: 2mg of DOX-loaded nanogels were weighed and dispersed in 1mL of the corresponding buffer solution, and then transferred to a dialysis bag with a molecular weight cutoff of 14000Da, followed by rapid immersion of the dialysis bag in the above simulated release environment (volume 100mL) with slight magnetic stirring. According to the set time, 1mL of solution was taken out from the release medium, and then an equal volume of the corresponding fresh buffer solution was added to the release medium to keep the volume of the release medium unchanged. The concentration of released DOX was measured by UV spectroscopy. The release experiments in all different release media were repeated 3 times and the average value was taken.

[0105] Since the cross-linking agent BAC containing disulfide bonds was introduced into the polymerization reaction, the P(VCL-ss-OrnAA) nanogel has reduction degradation and can be reduced to sulfhydryl groups by reducing substances (GSH) in tumor cells, causing the cross-linking points to break and the nanogel to degrade. The drug release results showed that ( Fig.13), under the physiological condition of pH 7.4, the drug release rate within 24 hours was 18.36%, indicating that the drug-loaded gel can remain stable in the blood circulation; in the tumor cytoplasm environment of pH 6.5, the drug release rate within 24 hours was 40.73%; in the tumor cell lysosome environment of pH 5.5, the drug release rate within 24 hours was 67.81%; the above results indicate that the release of the anti-tumor drug DOX is pH responsive. In a simulated tumor cell reducing environment (pH 6.5, 10 mM GSH, 37°C), 83.22% of DOX was released from the drug delivery system after 24 hours, indicating that in the cell reducing environment, the nanogel degraded due to the reduction and cleavage of the internal disulfide bonds, thereby promoting the rapid and full release of the drug; under the same conditions, when the temperature was raised to 42°C, 90.36% of DOX was released from the drug delivery system after 24 hours, indicating that when the temperature was greater than the volume phase transition temperature of the nanogel (VPTT, 39.3°C), the volume of the nanogel shrank, promoting the release of the drug.

[0106] Example 7

[0107] (7) Cytotoxicity assay

[0108] The cytotoxicity was determined using the CCK-8 method. Normal human umbilical vein endothelial cells (HUVEC cells) were used to evaluate the biocompatibility of the nanogels, and human colon cancer cells (HCT-116 cells) were used to evaluate the ability of free DOX and DOX-loaded nanogels to kill tumor cells. The cell culture method and specific operation steps are as follows: the cells were digested, counted, and prepared to a concentration of 8×10 4 100 μL of cell suspension (8×10 cells / mL) was added to each well of a 96-well cell culture plate. 3 cells); the cell culture plate was placed in a 37°C, 5% CO2 incubator for 24 hours; a complete culture medium (89% RPMI1640 + 10% FBS + 1% double antibody) was used to prepare a nanogel working solution containing a concentration of 0-1000 μg / mL (HUVEC cells), and a complete culture medium (90% DMEM culture medium + 10% fetal bovine serum) was used to prepare a free DOX of 0-20 μg / mL and a drug-loaded nanogel working solution with an equal DOX content (HCT-116 cells); 100 μL of the corresponding working solution was added to each well, and 3 replicates were set for each concentration; the cells were cultured in a 37°C, 5% CO2 incubator for 24 hours and 48 hours, respectively, and the supernatant was discarded; the 96-well plate was stained with CCK-8, and the OD value was measured at λ = 450nm.

[0109] from Fig.14It can be seen that after P(VCL-ss-OrnAA) nanogels were co-incubated with normal HUVEC cells for 24 h and 48 h, respectively, the cell survival rate was still over 90% even at a concentration of 1000 μg / mL, indicating that the nanogels have good biocompatibility.

[0110] like Fig.15 As shown, when pure drug DOX and DOX@P(VCL-ss-OrnAA) loaded nanogels were co-incubated with human colon cancer cells (HCT116) for 24h and 48h, the loaded nanogels and free DOX showed similar cytotoxicity. When 20μg / mL pure DOX and the loaded nanogels with the same amount of DOX were co-incubated with HCT116 cells for 24h, the cell survival rates were 27.59% and 32.76%, respectively; when they were co-incubated with HCT116 cells for 48h, the cell survival rates were 12.93% and 16.30%, respectively. The above results show that P(VCL-ss-OrnAA) nanogels loaded with DOX have obvious cytotoxicity to tumor cells HCT116 cells and have a strong ability to kill tumor cells.

[0111] It can be seen from the above embodiments that the present invention has the following outstanding substantiality and remarkable progress:

[0112] 1) Successfully synthesized novel nanogels: The present invention successfully synthesized the zwitterionic P (VCL-ss-OrnAA) nanogels of copolymerized methacryloyl-ornithine (OrnAA) and N-vinyl caprolactam (VCL), and achieved the reduction degradation of the nanogels by introducing a disulfide bond crosslinker.

[0113] 2) Nanogels have significant temperature responsiveness: the particle size decreases with increasing temperature, and the volume phase transition temperature (VPTT) is 39.3°C (pH 7.4). After entering the tumor tissue, the nanogels shrink in volume, which is beneficial to drug release.

[0114] 3) Nanogel has excellent stability: P(VCL-ss-OrnAA) nanogel showed excellent stability in both PBS buffer solution and PBS buffer solution containing bovine serum albumin (BSA), and the particle size remained almost unchanged over a long period of time, which is conducive to its application in drug delivery systems.

[0115] 4) Reduction-responsive degradation performance: Nanogels can be degraded in the presence of reduced glutathione (GSH), and the higher the GSH concentration, the faster the degradation rate. This property enables nanogels to achieve rapid degradation in the high GSH environment in tumor cells, thereby releasing drugs.

[0116] 5) Efficient drug loading and release capacity: P(VCL-ss-OrnAA) nanogel has a high drug loading and encapsulation rate for doxorubicin (DOX). In a simulated tumor cell reduction environment, the nanogel can release the drug quickly and fully, while remaining stable under physiological conditions, reducing the premature release of the drug.

[0117] 6) Good biocompatibility and cytotoxicity: Cytotoxicity experiments showed that P(VCL-ss-OrnAA) nanogels had good biocompatibility to normal cells (HUVEC cells), but showed obvious cytotoxicity to tumor cells (HCT-116 cells), and had a strong ability to kill tumor cells.

[0118] In summary, the surface of the P(VCL-ss-OrnAA) nanogel synthesized in the present invention contains equal amounts of amino and carboxyl groups, has excellent stability, temperature responsiveness, resistance to protein adsorption, reduction-responsive degradation performance, efficient drug loading and release capabilities, good biocompatibility and cytotoxicity, and is a potential drug delivery carrier, particularly suitable for targeted delivery and controlled release of tumor drugs.

[0119] The above are only a few preferred embodiments of the present invention, which are described in a more specific and detailed manner, but they cannot be understood as limiting the scope of the present invention. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. A method for preparing zwitterionic nanogel, characterized in that: The following steps are involved: (1) Synthesis of methacryloyl ornithine: react L-ornithine hydrochloride with basic copper carbonate in water to obtain methacryloyl ornithine copper complex; react the methacryloyl ornithine copper complex with 8-hydroxyquinoline in chloroform-water to obtain methacryloyl ornithine OrnAA after recrystallization; (2) P(VCL- ss Synthesis of the degradable amino acid zwitterionic nanogel P(VCL-OrnAA): N-vinyl caprolactam, initiator, crosslinker and surfactant were mixed and dissolved in water, nitrogen was introduced and the temperature was raised to react, and methacryloyl ornithine OrnAA was added for copolymerization to obtain the degradable amino acid zwitterionic nanogel P(VCL- ss -OrnAA).

2. The preparation method according to claim 1, characterized in that: In the step (1), the reaction temperature of L-ornithine hydrochloride and basic copper carbonate is 80-95° C., and the reaction time is 10-15 min. The molar ratio of L-ornithine hydrochloride to basic copper carbonate is 1:1-3:

1.

3. The preparation method according to claim 1, characterized in that: In the step (2), the initiator may be azobisisobutylamidine hydrochloride, potassium persulfate, ammonium persulfate, etc., the crosslinking agent may be N, N'-bis(acryloyl)cystamine containing a disulfide bond, and the surfactant may be sodium dodecyl sulfate.

4. The preparation method according to claim 1, characterized in that: In the step (2), the reaction temperature in the copolymerization step is 60-80°C and the reaction time is 2-6 h.

5. The preparation method according to claim 1, characterized in that: The following steps are involved: (1) Synthesis and structural characterization of methacryloylornithine: (1.1) Dissolve 4.22-5.06 g of L-ornithine hydrochloride in 40-60 mL of water at 80-95 °C. Add 3.05-3.66 g of basic copper carbonate to the solution and stir for 10-15 min. (1.2) After filtering the insoluble residue, add 20-30 mL of acetone, and then add 12.5-15 mL of 2 M KOH aqueous solution; (1.3) Cool to 0 °C, and add 3.00-3.59 mL of methacryloyl chloride and 13.8-16.6 mL of 2 M KOH aqueous solution dropwise over 10-30 min in an ice bath. After the addition is complete, warm to room temperature and stir at room temperature overnight. (1.4) Filter to obtain a filter cake, i.e., a blue precipitate of methacryloylornithine copper complex, which is washed with water, methanol and ether in sequence; (1.5) Add 6.0-8.0 g of the intermediate product methacryloylornithine copper complex powder to 80-120 mL of chloroform solution containing 2.5-3.4 g of 8-hydroxyquinoline, add an equal volume of water, and shake overnight; (1.6) Filter and remove the green precipitate 8-hydroxyquinoline copper complex in the chloroform layer, and separate the aqueous phase; add the product in the aqueous phase to THF at a volume ratio of 1:5 to 1:15 for recrystallization to obtain OrnAA crystals, centrifuge and discard the supernatant, and freeze-dry the precipitate to obtain white powder methacryloyl ornithine (OrnAA); The structure of OrnAA was characterized by NMR; (2) P(VCL- ss Synthesis of -OrnAA) nanogels (2.1) In a 100 mL three-necked flask equipped with a spherical condenser and mechanical stirring, weigh 160-200 mg of N-vinyl caprolactam (VCL), 2-10 mg of NaHCO3, 2-10 mg of sodium dodecyl sulfate (SDS) and 10-30 mg of N,N'-bis(acryloyl)cystamine (BAC), add 10-30 mL of ultrapure water, and sonicate to dissolve until there are no particles; (2.2) After N2 is introduced for 20-40 min, an aqueous solution of initiators such as azobisisobutylamidine hydrochloride AIBA and potassium persulfate (KPS) is injected into the three-necked flask. The aqueous solution is prepared by dissolving 5-10 mg of initiator in 1 mL of ultrapure water. The temperature is raised to 60-80 °C. After reacting for 20-40 min, 0-40 mg of methacryloylornithine OrnAA is added. After 2-6 h, a white emulsion is obtained. (2.3) The obtained emulsion was dialyzed in pure water for 3 to 5 days, and the dialyzed emulsion was freeze-dried to obtain a white freeze-dried powder, namely P(VCL- ss -OrnAA) nanogel.

6. A zwitterionic nanogel, characterized in that: The compound is prepared by the preparation method described in any one of items 1-5.

7. Use of the nanogel as claimed in claim 6 in preparing a drug delivery carrier.

8. The use according to claim 7, characterized in that The drug is a broad-spectrum drug for treating tumors, and preferably, the drug is doxorubicin.

9. A pharmaceutical preparation for treating tumors, characterized in that: It comprises a drug delivery carrier and a drug, wherein the drug carrier is the nanogel as claimed in claim 6; and the drug is a drug for treating tumors, preferably doxorubicin.

10. The method for preparing the pharmaceutical preparation according to claim 9, characterized in that: The following steps are involved: DOX and nanogel are fully dispersed in 2-5 mL pH 7.4 PBS buffer solution at a mass ratio of 1:10-3:10; after stirring at room temperature for 12-24 hours, the mixture is centrifuged to collect the DOX-loaded nanogel, and the mixture is centrifuged and washed twice with pure water to remove the DOX adsorbed on the surface, thereby obtaining the pharmaceutical preparation.

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