Amino acid zwitterionic nanogel and application thereof

Abstract

The invention discloses amino acid zwitterionic nanogel and application thereof. The nanogel is prepared by using methylacryloyl lysine as a monomer and disulfide bond-containing N, N '-bis (acryloyl) cystamine as a cross-linking agent through a dispersion polymerization method. The surface of the nanogel is rich in amino groups and carboxyl groups, and the nanogel has typical zwitter-ion characteristics and is easy to modify targeting groups. The nanogel has excellent biocompatibility, colloidal stability and remarkable protein adsorption resistance, and meanwhile, due to an internal disulfide bond structure, the nanogel can be subjected to reduction responsive degradation in a tumor microenvironment of high-concentration glutathione, so that intelligent controlled release of drugs is realized. Experiments show that the doxorubicin-loaded nanogel has an efficient killing effect on tumor cells, is very low in toxicity to normal cells, and shows huge application potential in a drug delivery system, especially in the aspects of targeted delivery and controlled release of anti-tumor drugs.

Description

Technical Field

[0001] This invention belongs to the field of biomedical polymer materials technology, specifically relating to an amino acid-based zwitterionic nanogel. Furthermore, this invention relates to a method for preparing this nanogel, and its application as a drug carrier in the biomedical field, particularly in tumor treatment. Background Technology

[0002] Nanoparticle drug delivery systems can precisely deliver drugs to diseased tissues or targets, thereby improving efficacy, reducing side effects, and optimizing drug distribution and pharmacokinetics. However, proteins in the physiological environment often adsorb onto the surface of nanocarriers, forming a "protein crown," which affects the stability, drug release characteristics, and targeting selectivity of nanoparticle drug delivery systems. Forming an antifouling surface to prevent non-specific protein adsorption is key to solving these problems. Strong hydration is considered an important factor determining protein antifouling properties. Polyethylene glycol (PEG) and its derivatives are the most widely used antifouling materials, but their long-term in vivo application is greatly limited due to structural instability and immunogenicity.

[0003] Zwitterionic polymers carry oppositely charged groups and exhibit overall charge neutrality. Through ionic solvation, they generate strong hydration, forming an antifouling surface with properties such as anti-protein adsorption, interfacial lubrication, and protein stabilization. Compared to PEG, zwitterionic polymers have unique advantages in immunogenicity, targeting, responsive release, and biodegradability.

[0004] Amino acids are considered zwitterionic compounds across a wide pH range because they have a carboxyl group and a primary amino group directly attached to their central α-carbon. Their derived polymers exhibit pH responsiveness, displaying cationic or anionic properties at low or high pH values, while maintaining their zwitterionic state at physiological pH. To retain the zwitterionic moiety, amino acid monomers with zwitterionic structures can be prepared by esterification or amidation of hydroxyl or amino groups with methacrylate. Polymerization of these monomers forms polyamino acids, such as ornithine, lysine, serine, histidine, aspartic acid, glutamic acid, and cysteine. Compared to other zwitterionic polymers, these polymers retain the corresponding amino acid activity while exhibiting good anti-protein adsorption properties. Furthermore, the amino and carboxyl terminal groups in the molecules readily couple with other biomolecules to achieve specific functions, such as active targeting and fluorescent labeling.

[0005] Amino acid-based zwitterionic polymer nanogels offer advantages such as high drug loading, intelligent release, and low toxicity as drug delivery carriers, showing broad application prospects in the treatment of diseases such as tumors. Common methods for preparing amino acid zwitterionic polymer nanogels include physical crosslinking and reverse emulsion polymerization. The Mandal team functionalized poly(acryloyl-L-serine) with L-cysteine ​​to form zwitterionic peptides with an isoelectric point of 2.7. Near the isoelectric point, the aqueous solution of the zwitterionic peptides exhibits pH responsiveness (pH 2.2–4.1), transforming from a transparent solution into an insoluble, turbid suspension of hierarchical nanoaggregates with various dendritic morphologies through electrostatic interactions. The Liu Lingyun team prepared ultra-low-pollution polyamino acid zwitterionic nanogels with fluorescent properties via water-hexane reverse emulsion polymerization using the amino acid zwitterionic monomer methacryloylornithine and a fluorescent carbon dot crosslinking agent. However, gels formed by physical crosslinking methods exhibit poor stability, and their performance is highly dependent on the crosslinking agent, pH, temperature, and other variables. Reverse emulsion polymerization is mainly suitable for monomers with strong hydrophilicity. It has high polymerization efficiency and is easy to control and adjust, but it also has problems such as complex post-processing, surfactant residue, and insufficient stability of the polymerization system.

[0006] Dispersion polymerization refers to a method of preparing a polymer dispersion by dissolving monomers, initiators, and stabilizers in a specific solvent or mixed solvent to form a homogeneous mixture. When the polymerization reaches a critical chain length, the initially unstable polymer chains begin to aggregate until a stable polymer dispersion is formed. Dispersion polymerization is applicable to a wider range of monomers, and by optimizing the mixing solvent ratio and controlling the composition of the reaction mixture, nanogels with different particle sizes and functions can be obtained. Therefore, amino acid monomers with zwitterionic structures can be prepared by esterification or amidation of hydroxyl or amino groups with methacrylate. After adding functional crosslinking agents and initiators, polyamino acid zwitterionic nanogels with good stability, excellent biocompatibility, and drug controlled release capabilities can be formed through free radical polymerization. At the same time, the abundant amino and carboxyl groups on the surface can couple with other biomolecules, endowing the nanogels with functions such as active targeting and fluorescent labeling, making them widely applicable in the treatment of diseases such as tumors.

[0007] Therefore, developing a nanogel based on a specific amino acid monomer, using a dispersion polymerization process, and possessing the aforementioned excellent properties is an urgent problem to be solved in this field. Summary of the Invention

[0008] Lysine is one of the essential amino acids that make up human proteins, and it mainly participates in physiological processes such as protein synthesis, immune regulation, and calcium absorption. It cannot be synthesized by the body itself and must be obtained through diet or supplements, commonly found in meat, legumes, dairy products, and other foods. Its core functions include promoting growth and development, enhancing immunity, and maintaining bone health. Acrylonitrifying one amino group of lysine yields the easily polymerizable zwitterionic monomer LysAA. Polymer nanogels based on acryloyllysine zwitterionic monomers possess advantages such as long circulation, enhanced mucosal penetration and cellular uptake, excellent biocompatibility, stimulus-responsive drug release, multifunctional synergistic therapy, and high drug loading capacity, showing broad application prospects in the field of drug delivery.

[0009] This invention involves acylation of one amino group of lysine to obtain methacryloyllysine (LysAA), which is then used as a monomer. N,N'-bis(acryloyl)cysteine ​​(BAC), containing disulfide bonds, is used as a crosslinking agent to prepare a reductively degradable amino acid zwitterionic nanogel drug delivery carrier resistant to protein adsorption via dispersion polymerization. Because the surface of the amino acid nanogel is rich in carboxyl and amino groups, it readily binds to drugs or biomolecules, achieving high drug loading capacity and active targeting, fluorescent labeling, and other capabilities. When used as a nanomedicine carrier, the nanogel exhibits excellent stability, long cycling time, and anti-protein adsorption capacity. Through targeted delivery to tumor cells, the disulfide bonds within the crosslinking agent are reduced and broken into thiol groups, causing the nanogel to degrade into low-molecular-weight linear polymers, releasing the loaded drug. Therefore, PlysAA nanogels possess controlled drug release properties and good biodegradability.

[0010] The structure, morphology, degradability, drug loading, and drug release behavior of the products were characterized using various instruments such as transmission electron microscopy (TEM), dynamic light scattering (DLS), Fourier transform infrared spectroscopy (FTIR), Raman spectroscopy (RS), nuclear magnetic resonance (NMR), gel permeation chromatography (GPC), and ultraviolet spectroscopy (UV) to improve experimental conditions and strive to prepare a drug delivery system with optimal performance. Cytotoxicity was determined using CCK-8 assays to characterize the biocompatibility and tumor cell killing effect of the materials.

[0011] To achieve the above objectives, the present invention includes the following technical solutions: An amino acid zwitterionic nanogel, wherein the nanogel is formed by free radical polymerization crosslinking of methacryloyl lysine monomer and a disulfide-containing crosslinking agent; the surface of the nanogel is rich in amino and carboxyl groups.

[0012] Furthermore, in the above-mentioned amino acid zwitterionic nanogel, the crosslinking agent containing disulfide bonds is N,N'-bis(acryloyl)cystamine.

[0013] Furthermore, in the above-mentioned amino acid zwitterionic nanogel, the mass ratio of the methacryloyl lysine monomer to the crosslinking agent is (60~80): (20~40).

[0014] The preparation method of the above-mentioned amino acid zwitterionic nanogel includes the following steps: (1) Dissolve the methacryloyl lysine monomer and the stabilizer in water to form an aqueous phase; (2) Dissolve the crosslinking agent and initiator containing disulfide bonds in polar solvents such as methanol, ethanol, tetrahydrofuran, and 2-methoxyethanol to form an organic phase; (3) The organic phase is added dropwise to the aqueous phase, mixed evenly, and then heated to 75~85℃ under an inert atmosphere to carry out the polymerization reaction; (4) After the reaction is complete, the product is centrifuged, washed and freeze-dried to obtain the amino acid zwitterionic nanogel.

[0015] Furthermore, in the above preparation method, the stabilizer is polyvinylpyrrolidone K30 or K90, and its amount is 20% to 40% of the monomer mass; the initiator is azobisisobutyronitrile, and its amount is 2% to 4% of the monomer mass.

[0016] Furthermore, in the above preparation method, in step (3), the volume ratio of water to 2-methoxyethanol is 3.1:16.9 ~ 3.8:16.2.

[0017] This invention also discloses the application of the above-mentioned amino acid zwitterionic nanogels in the preparation of drug delivery carriers.

[0018] Furthermore, in the above applications, the drug delivery carrier is used to deliver antitumor drugs.

[0019] Furthermore, in the above application, the antitumor drug is doxorubicin.

[0020] The present invention also discloses a pharmaceutical composition comprising the amino acid zwitterionic nanogel described in any one of the above claims and a pharmaceutically active ingredient loaded thereon.

[0021] Compared with the prior art, the present invention has the following outstanding advantages: 1. Excellent biocompatibility: The nanogel uses lysine as its basic building block, exhibiting high biocompatibility. Cell experiments show that after co-incubation with normal human liver cells, even at high concentrations, the cell survival rate of the nanogel remains above 90%, demonstrating low intrinsic cytotoxicity.

[0022] 2. Stability and Anti-fouling Ability: The nanogel structure formed through covalent cross-linking is stable, and the particle size did not change significantly within one week in a physiological environment. Its zwitterionic properties enable it to effectively resist the non-specific adsorption of bovine serum albumin, which helps maintain the stability of the nanocarrier in blood circulation.

[0023] 3. Possesses reduction-responsive degradation capability: Due to the introduction of disulfide bonds in the cross-linking network, this nanogel can degrade in a high-concentration reducing environment (such as GSH) that simulates tumor cells. The gel structure disintegrates, and the degraded polymer has a low molecular weight, which is easily metabolized by the human body, thus providing a basis for the controlled release of drugs.

[0024] 4. Drug loading capacity and functionalization potential: This nanogel exhibits good loading capacity for the model drug doxorubicin, with a high encapsulation efficiency. Furthermore, its abundant amino and carboxyl groups on the surface provide the possibility for subsequent coupling with other functional molecules (such as targeting ligands), facilitating the realization of more complex functions. Attached Figure Description

[0025] Figure 1 NMR spectrum of methacryloyl lysine (LysAA); Figure 2 Infrared spectrum of methacryloyl lysine (LysAA); Figure 3 Infrared spectrum of PLysAA nanogel; Figure 4 Raman spectrum of PLysAA nanogel; Figure 5 Transmission electron microscopy (TEM) image of PLysAA(P6); Figure 6 Transmission electron microscopy (TEM) image of PLysAA(P11); Figure 7 Transmission electron microscope (TEM) image of PLysAA(P15); Figure 8 Potential and particle size of PlysAA nanogels under different pH conditions; Figure 9 PlysAA nanogel (P15) particle size changes over one week in PBS pH 7.4 buffer and BSA solution; Figure 10 The reductive degradation properties of PLysAA nanogel (a) and the molecular weight after reductive degradation (b); Figure 11 PLysAA nanogel exhibits 24-hour cytotoxicity in LO2 cells; Figure 12PLysAA nanogel showed cytotoxicity of human breast cancer cells (MCF-7) at 24 h (a) and 48 h (b). Detailed Implementation

[0026] (1) Synthesis and structural characterization of methacryloyl lysine Dissolve 7.30–8.20 g of L-lysine acid salt in 70–100 mL of water at 85–95 °C. Add 4.8–5.7 g of basic copper carbonate to the solution and stir for 5–15 min. After filtering out the insoluble residue, add 30–45 mL of acetone, followed by 17–26 mL of 2 M KOH aqueous solution. Cool to 0–4 °C and simultaneously add 4–6 mL of methacryloyl chloride and 18–28 mL of 2 M KOH aqueous solution dropwise over 15–30 min in an ice bath. After the addition is complete, warm to room temperature and stir overnight at room temperature. Filter to obtain a filter cake, which is a blue precipitate of methacryloyl lysine copper complex, and wash successively with water, methanol, and diethyl ether. 4.5–6.0 g of the obtained intermediate product, methacryloyl lysine copper complex powder, was added to 60–70 mL of chloroform solution containing 1.6–2.2 g of 8-hydroxyquinoline, followed by the addition of 60–70 mL of water. The mixture was shaken overnight. The green precipitate of 8-hydroxyquinoline copper complex in the chloroform layer was removed by filtration, and the aqueous phase was obtained. The product in the aqueous phase was added to THF for precipitation and recrystallization. The product was then centrifuged to obtain LysAA crystals, which were lyophilized into a white powder.

[0027] (2) Synthesis of PlysAA nanogel Weigh 60–80 mg of methacryloyllysine (LysAA) and 20–40 mg of polyvinylpyrrolidone (PVP) K30 or K90 and dissolve them in 3.1–3.8 mL of pure water; separately weigh 20–40 mg of N,N'-bis(acryloyl)cysteine ​​(BAC) and 2–4 mg of azobisisobutyronitrile (AIBN) and dissolve them in 16.2–16.9 mL of 2-methoxyethanol. Then, slowly add the 2-methoxyethanol solution containing BAC and AIBN dropwise to the aqueous solution containing LysAA and K30 or K90, and after ultrasonic dispersion, transfer the solution to a 100 mL three-necked flask. While continuously stirring at 150–200 rpm, introduce N2 for 20–30 min, and then begin heating to 75–85 °C. The reaction was stopped after 30-60 min. The resulting emulsion was centrifuged at 8000-12000 rpm for 10-20 min and washed with water 2-3 times under the same centrifugation conditions. The solid obtained by centrifugation was dispersed with a small amount of pure water and then freeze-dried to obtain a white lyophilized powder.

[0028] (3) Analysis of the structure, morphology, particle size and potential of PlysAA nanogel The chemical structure of the synthesized PlysAA nanogel was analyzed by infrared spectroscopy and Raman spectroscopy, its morphology was observed by transmission electron microscopy (TEM), and its potential, particle size, and monodispersity were determined by dynamic light scattering (DLS). To investigate the effect of pH changes on the properties of the nanogel, it was dispersed in a series of buffer solutions with pH values ​​ranging from 0.5 to 1.0 mg / mL, and the changes in potential and particle size under each pH condition were measured.

[0029] (4) Stability test of PlysAA nanogel To evaluate the long-term stability of PlysAA nanogels in a physiologically simulated environment, they were dispersed at concentrations of 0.1–1.0 mg / mL in 0.01–0.02 M PBS 7.4 buffer or in 0.01–0.02 M PBS 7.4 buffer containing 1–3 mg / mL bovine serum albumin (BSA). Subsequently, dynamic light scattering technology was used to monitor the particle size changes at different time points within one week.

[0030] (5) Reductive degradation experiment of PlysAA nanogel PlysAA nanogels were dispersed in 0.01–0.02 M PBS pH 7.4 buffer solutions with GSH concentrations ranging from 0 to 20 mmol / mL, and degradation experiments were conducted at 30–40 °C. The molecular weight of the degraded nanogels was determined by gel permeation chromatography (GPC). Changes in scattered light intensity were tracked using dynamic light scattering (DLS) to investigate its redox degradation performance. The test temperature was set at 30–40 °C, and the scattered light intensity of the samples was recorded at set time points. 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; changes in the relative turbidity reflected the degree of degradation.

[0031] (6) Drug loading experiment of PlysAA nanogel Doxorubicin (DOX), a first-line clinical oncology drug, was selected as a model drug. DOX and nanogel were thoroughly dispersed in 2–5 mL of pH 7.4 PBS buffer at a mass ratio of 1:10–2:5. After stirring at room temperature for 12–24 h, the mixture was centrifuged to collect the DOX-loaded nanogel, which was then washed twice with pure water to remove surface-adsorbed DOX. The supernatant was collected, and the drug content in the supernatant was analyzed by UV absorption spectroscopy to calculate the drug loading and encapsulation efficiency of the nanogel.

[0032] (7) Cytotoxicity test Cytotoxicity was determined using the CCK-8 assay. Human normal hepatocytes (LO2 cells) were used to evaluate the biocompatibility of the nanogel, and human breast cancer cells (MCF-7 tumor cells) were used to evaluate the ability of free DOX and DOX-loaded nanogels to kill tumor cells. The cell culture methods and specific procedures are as follows: cells were digested, counted, and prepared to a concentration of 1×10⁻⁶. 4 ~1×10 5 Add 50-200 μL of cell suspension (5 × 10⁶ cells / mL) to each well of a 96-well cell culture plate. 3 ~1×10 4 Cells were cultured in a 37°C, 5% CO2 incubator for 24 h. A working solution containing 0–1000 μg / mL nanogel was prepared using complete culture medium (80–90% RPMI 1640 medium + 10–20% fetal bovine serum) for LO2 cells. A working solution containing 0–20 μg / mL free DOX and an equal amount of drug-loaded nanogel was prepared using complete culture medium (80–90% DMEM medium + 10–20% fetal bovine serum) for MCF-7 tumor cells. 50–200 μL of the corresponding working solution was added to each well, with three replicates for each concentration. Cells were cultured at 37°C and 5% CO2 incubators for 24 h and 48 h, respectively, and the supernatant was discarded. The 96-well plate was stained with CCK-8, and the OD value was measured at λ=450 nm.

[0033] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0034] Example 1 (1) Synthesis and structural characterization of methacryloyl lysine (LysAA) L-lysine acid salt (8.00 g, 43.80 mmol) was dissolved in 80 mL of water at 90 °C. Basic copper carbonate (5.344 g, 24.3 mmol) was added to the solution and stirred for 10 min. After filtering out the insoluble residue, 38.4 mL of acetone was added, followed by 21.9 mL of 2 M KOH aqueous solution. The mixture was cooled to 0 °C, and methacryloyl chloride (5.25 mL, 54.78 mmol) and 24.32 mL of 2 M KOH aqueous solution were added dropwise over 20 min in an ice bath. After the addition was complete, the mixture was warmed to room temperature and stirred overnight at room temperature. The mixture was filtered to obtain a filter cake, which was a blue precipitate of methacryloyl lysine copper complex. The cake was washed successively with water, methanol, and diethyl ether. The obtained intermediate product, methacryloyl lysine copper complex powder (5.2 g, 10.29 mmol), was added to 64 mL of chloroform solution of 8-hydroxyquinoline (1.87 g, 12.89 mmol), followed by the addition of 64 mL of water. The mixture was shaken overnight. The green precipitate of 8-hydroxyquinoline copper complex in the chloroform layer was removed by filtration, and the aqueous phase was obtained. The product in the aqueous phase was added to THF for precipitation, recrystallized, and centrifuged to obtain LysAA crystals, which were then lyophilized into a white powder. The structure of LysAA was characterized by NMR and IR spectroscopy.

[0035] (2) Synthesis of PlysAA nanogel 70.0 mg of methacryloyllysine (LysAA) and 30 mg of polyvinylpyrrolidone (PVP) K90 were weighed and dissolved in 3.6 mL of pure water; 30.0 mg of N,N'-bis(acryloyl)cysteine ​​(BAC) and 2.0 mg of azobisisobutyronitrile (AIBN) were separately weighed and dissolved in 16.4 mL of 2-methoxyethanol. The 2-methoxyethanol solution containing BAC and AIBN was then slowly added dropwise to the aqueous solution containing LysAA and K90. After ultrasonic dispersion, the solution was transferred to a 100 mL three-necked flask. Under continuous stirring at 160 rpm, N2 was introduced for 30 min, and the temperature was initially raised to 78 °C. After 30 min of reaction, the reaction was stopped. The resulting emulsion was centrifuged at 12000 rpm for 15 min and washed twice with water under the same centrifugation conditions. The solid obtained from centrifugation was dispersed in a small amount of pure water and then freeze-dried to obtain a white lyophilized powder.

[0036] (3) Analysis of the structure, morphology, particle size and potential of PlysAA nanogel The structure of the synthesized PlysAA nanogel was characterized by infrared and Raman spectroscopy, the morphology of the nanogel was characterized by transmission electron microscopy (TEM), and the particle size and monodispersity of the nanogel were characterized by dynamic light scattering (DLS). Buffer solutions with pH values ​​ranging from 3.0 to 9.2 were prepared, and the nanogel was dispersed at a concentration of 0.5 mg / mL in these buffer solutions. The particle size and potential values ​​at different pH values ​​were analyzed.

[0037] (4) Stability test of PlysAA nanogel PlysAA nanogels were dispersed at a concentration of 0.1 mg / mL in 0.01 M PBS 7.4 buffer or in 0.01 M PBS 7.4 buffer with a bovine serum albumin (BSA) concentration of 2 mg / mL. The particle size changes of the nanogels at different time intervals over one week were monitored by DLS.

[0038] (5) Reductive degradation experiment of PlysAA nanogel The nanogels were placed in 0.01M pH 7.4 buffer solutions of reduced glutathione at concentrations of 0 and 20 mM, respectively. The molecular weight of the degraded nanogels was determined by gel permeation chromatography (GPC). Changes in scattered light intensity were tracked using dynamic light scattering (DLS) to investigate their redox degradation performance. The test temperature was set at 37℃, and the scattered light intensity of the samples was recorded at set times. The ratio of the scattered light intensity of the sample at different time points to that of the initial sample was defined as the relative turbidity of the sample; changes in the relative turbidity reflected the degree of degradation.

[0039] (6) Drug loading Doxorubicin (DOX), a first-line clinical oncology drug, was selected as a model drug. DOX and PlysAA nanogel were thoroughly dispersed in 2 mL of pH 7.4 PBS buffer at a mass ratio of 1:5. After stirring at room temperature for 24 h, the mixture was centrifuged to collect the DOX-loaded nanogel, which was then washed three times with pure water to remove surface-adsorbed DOX. The supernatant was collected, and the drug loading of the nanogel was determined by UV absorption spectroscopy to be 15.59%, with an encapsulation efficiency of 92.38%.

[0040] (7) Cytotoxicity test Cytotoxicity was determined using the CCK-8 assay. Human normal hepatocytes (LO2 cells) were used to evaluate the biocompatibility of the nanogel, and human breast cancer cells (MCF-7 tumor cells) were used to evaluate the ability of free DOX and DOX-loaded nanogels to kill tumor cells. The cell culture methods and specific procedures are as follows: cells were digested, counted, and prepared to a concentration of 1×10⁻⁶. 5Add 100 μL of cell suspension (1 × 10⁶ cells / mL) to each well of a 96-well cell culture plate. 4 Cells were cultured in a 37°C, 5% CO2 incubator for 24 h. Working solutions containing 0–1000 μg / mL nanogels were prepared using complete culture medium (90% RPMI 1640 medium + 10% fetal bovine serum). Working solutions containing 0–20 μg / mL free DOX and equal DOX content were prepared using complete culture medium (90% DMEM medium + 10% fetal bovine serum). 100 μL of the corresponding working solution was added to each well, with three replicates for each concentration. Cells were cultured at 37°C and 5% CO2 incubators for 24 h and 48 h, respectively, and the supernatant was discarded. The 96-well plates were stained with CCK-8, and the OD value was measured at λ=450 nm.

[0041] Example 2 Test and verification.

[0042] (1) The NMR results of methacryloyl lysine (LysAA) are as follows: Figure 1 As shown (using deuterium water as solvent): 1 H NMR(LysAA): d=5.68 (s, 1H, C=C H 2 ), 5.45 (s, 1H, C=C H 2 ), 3.74 (t, 1H, C H ), 3.29(t, 2H, C H 2 ), 1.93 (s, 3H, C H 3 ), 1.88 (m, 2H, C H 2 ), 1.60 (m, 2H, C H 2 ), 1.36–1.49 (m, 2H, C) H 2 ).

[0043] (2) The structure of LysAA was analyzed using Fourier transform infrared spectroscopy (FTIR). Figure 2 3320 cm -1 The peak at 3077 cm⁻¹ is attributed to the NH stretching vibration in the -NH₂ group of lysine. -1 The peak at 2950–2864 cm⁻¹ is attributed to the OH stretching vibration from the carboxyl group (-COOH) of lysine. -1The peak at 1655 cm⁻¹ is attributed to the stretching vibration of the saturated alkyl group (C−H). -1 The peak at 1618 cm⁻¹ is mainly attributed to the C=O stretching vibration of the amide bond in the amide I band, and may also include contributions from the C=O vibration of incompletely deprotonated carboxylic acid groups. -1 The peak at 1525 cm⁻¹ is attributed to the C=O stretching vibration of the amide bond in amide I. -1 The peak at 1415 cm⁻¹ is attributed to the NH bending vibration of amide II with amide bonds. -1 This is attributed to the symmetric stretching vibration of the deprotonated carboxylic acid group in LysAA.

[0044] (3) The infrared spectrum of PLYsAA nanogel is as follows: Figure 3 As shown, 3382 cm -1 The peak at 1633 cm⁻¹ is attributed to the stretching vibration of the NH group in the -NH₂ group of lysine. -1 The peak at 1536 cm⁻¹ is attributed to the C=O stretching vibration of the amide bond in amide I and the asymmetric stretching vibration of the deprotonated carboxylic acid group in LysAA; -1 The peak at 1407 cm⁻¹ is attributed to the NH bending vibration of amide II with amide bonds. -1 This is attributed to the symmetric stretching vibration of the deprotonated carboxylic acid group in LysAA.

[0045] (4) The Raman spectrum of PLYsAA nanogel is shown below. Figure 4 As shown in the figure, at approximately 506 cm -1 An absorption peak corresponding to the SS bond appeared at approximately 637 cm⁻¹. -1 An absorption peak corresponding to the CS bond appeared at the location, indicating that the disulfide bond was successfully introduced into the polymer network.

[0046] (5) Under the condition of keeping the total volume of the mixed solvent constant (20 mL), PlysAA nanogels with different particle sizes and dispersibility were prepared by changing the volume ratio of water to 2-methoxyethanol while keeping other conditions constant. As shown in Table 1, when the volume of water is 3.1~3.3 mL, precipitation occurs in the reaction system, and stable microspheres cannot be obtained. With the increase of water volume, the particle size of the microspheres first decreases and then increases, the particle size dispersibility improves, and a uniform emulsion can be formed. When the water volume increases to 3.6 mL, the particle size is smaller and the dispersibility is the best (P6, Figure 5 As the volume of water continues to increase, the dispersibility of the microspheres deteriorates (PDI increases).

[0047] (6) Under the condition that the total monomer concentration is 5 mg / mL, the total volume of the mixed solvent is 20 mL, and the amount of water is 3.6 mL, the effects of reaction conditions on the particle size and dispersibility of PlysAA nanogels were analyzed by changing the amount of AIBN, the type and amount of stabilizer. As shown in Table 2, when the amount of AIBN changes from 4% to 2%, the particle size of PlysAA nanogels decreases (from 743.8 nm to 600.4 nm) and has good dispersibility; when the stabilizer K30 is changed to K90, the PlysAA nanogels further decrease in size (from 600.4 nm to 561.3 nm) and maintain good dispersibility and uniformity. When the amount of K90 increases from 20% to 30%, the particle size changes from 561.3 nm to 339.4 nm and has excellent dispersibility and uniformity (PDI=0.034). Figure 6 Further increasing the amount of K90 did not significantly change the particle size and dispersibility of PlysAA nanogel.

[0048] (7) Under the conditions of keeping the total monomer concentration at 5 mg / mL, the total volume of the mixed solvent at 20 mL, the amount of water at 3.6 mL, the amount of initiator at 2%, and the amount of K90 at 30%, the reaction temperature was changed, and the effect of the reaction temperature on the particle size and dispersibility of PlysAA nanogel was analyzed. As can be seen from Table 3, when the temperature was reduced from 85 ℃ to 78 ℃, the particle size decreased from 339.4 nm to 283.2 nm, while maintaining good dispersibility and stability, indicating that reducing the temperature helps to reduce the particle size. Figure 7 The electron microscopy image shows the PlysAA nanogel synthesized at a reaction temperature of 78 °C. It exhibits a uniform spherical morphology and good dispersibility. Considering that excessively low temperatures would affect the initiation efficiency and polymerization rate of the polymerization reaction, 78 °C was chosen as the optimal reaction temperature.

[0049] (8) Using sample P15 as the research object, the potential values ​​under pH conditions of 3.0–9.2 were measured. The results are shown in Table 4 and Figure 8Experimental results show that as the pH increases from 3.0 to 9.2, the potential value changes from 3.44 mV to -14.41 mV, indicating that the potential decreases with increasing pH. This is because the increase in pH increases the negative charge on the surface of the PlysAA nanogel, thus lowering the potential. Regarding particle size changes, as the pH increases from 3.0 to 9.2, the particle size shows a trend of first decreasing and then increasing. At pH 3.0 and 4.0, the nanogel particles are larger and less stable, because at pH 3.0 they carry a small amount of positive charge, and at pH 4.0 they carry a small amount of negative charge, approaching the isoelectric point. When the pH increases to 6.0, the particle size is smallest, at which point the nanogel carries a certain amount of negative charge (potential -5.78 mV) and exhibits better stability. When the pH changes from 6.0 to 9.2, the particle size gradually increases and tends to level off. This is because with increasing pH, the negative charge on the surface of the nanogel increases, and the electrostatic repulsion between the negative charges causes the nanogel to expand, thus increasing the particle size.

[0050] (9) Good colloidal stability is crucial for the application of polymer nanogels as drug delivery carriers. Therefore, we tracked the particle size change of PlysAA nanogel (P15) for one week, and the results showed that almost no particle size change was observed. Figure 9 This indicates that PLYsAA nanogel possesses excellent stability and anti-protein adsorption capacity. The anti-protein adsorption property of nanocarriers plays an important role in blood circulation, prolonging blood circulation time. Utilizing this property, nanocarriers can evade the surveillance of the immune system and the clearance by the reticuloendothelial system.

[0051] (10) Due to the introduction of the crosslinking agent BAC containing disulfide bonds in the polymerization reaction, PlysAA nanogels exhibit reductive degradation properties. They can be reduced to thiol groups by the reducing agent (GSH) in tumor cells, causing the crosslinking points to break and the nanogels to degrade. Dynamic light scattering (DLS) was used to track the changes in the relative turbidity of PlysAA nanogels in GSH-free and 20 mM GSH-reducing media to study their reductive degradation behavior. Figure 10 (a) shows the change in relative turbidity of the PlysAA nanogel dispersion system over time under 0 mM and 20 mM GSH conditions. It can be seen that the scattered light intensity hardly changes when GSH is absent; however, in the presence of GSH, the relative turbidity of the nanogel gradually decreases over time, indicating that GSH can reduce the disulfide bonds inside the nanogel to thiol groups, thus promoting the degradation of the PlysAA nanogel.

[0052] GPC results indicate that ( Figure 10(b) The polymer nanogels after GSH reduction and degradation have a low average molecular weight (M). w 3679) indicates 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 narrower distribution.

[0053] (11) From Figure 11 It can be seen that after PLYsAA nanogel was co-incubated with normal human hepatocytes LO2 cells for 24 h, the cell survival rate was still over 90% even at a concentration of 1000 μg / mL, indicating that the nanogel has good biocompatibility.

[0054] (12) When human breast cancer cells (MCF-7) were co-incubated with pure DOX and PlysAA-DOX-loaded nanogels for 24 h and 48 h respectively, the drug-loaded nanogels and free DOX showed similar cytotoxicity. Figure 12 When MCF-7 cells were co-incubated with 20 μg / mL pure DOX and an equal amount of DOX-loaded nanogels for 24 h, the cell viability was 32.08% and 40.52%, respectively; when co-incubated with MCF-7 cells for 48 h, the cell viability was 19.29% and 23.69%, respectively. These results indicate that the DOX-loaded PlysAA nanogel has significant cytotoxicity against MCF-7 tumor cells, thus possessing a strong ability to kill tumor cells.

[0055] It is worth noting that the above description of the embodiments focuses on illustrating the technical solution of the present invention, rather than precisely defining its scope of protection. Those skilled in the art should understand that appropriate adjustments and optimizations can be made based on the technical details disclosed in the embodiments of the present invention, or equivalent substitutions can be implemented for individual or even all technical elements. Such adjustments and substitutions will not deviate from the core essence of the technical solution of the present invention and should be included within the technical protection scope of the embodiments of the present invention. In short, the protection of the present invention should not be limited to the concrete presentation of the above embodiments, but broadly covers all equivalent changes and improvements that do not depart from its basic concept. In summary, the protection definition of the present invention should be based on the statement of the claims, and the above embodiments are only used as a reference guide for understanding the present invention.

Claims

1. An amino acid zwitterionic nanogel, characterized in that, The nanogel is formed by radical polymerization cross-linking of amino acid monomers and disulfide bond-containing cross-linking agents; the surface of the nanogel is rich in amino groups and carboxyl groups.

2. The amino acid zwitterionic nanogel of claim 1, wherein, The amino acid monomer is a lysine amino acid monomer; the lysine amino acid monomer is methacryloyl lysine; and the disulfide bond-containing cross-linking agent is N,N'-bis(acryloyl) cystamine.

3. The amino acid zwitterionic nanogel of claim 1 or 2, wherein, The mass ratio of the amino acid monomer to the cross-linking agent is (60-80):(20-40).

4. The method of preparing an amino acid zwitterionic nanogel according to any one of claims 1 to 3, wherein, The method comprises the following steps: (1) dissolving the amino acid monomer and the stabilizer in water to form an aqueous phase; (2) dissolving the disulfide bond-containing cross-linking agent and the initiator in a polar solvent such as methanol, ethanol, tetrahydrofuran, 2-methoxyethanol, etc. to form an organic phase; (3) adding the organic phase to the aqueous phase, mixing uniformly, and then heating to 75-85°C under an inert atmosphere to perform a polymerization reaction; (4) after the reaction is completed, centrifuging, washing, and freeze-drying the product to obtain the amino acid zwitterionic nanogel.

5. The preparation method according to claim 4, characterized in that, The solvent is a polar solvent such as methanol, ethanol, tetrahydrofuran, 2-methoxyethanol, etc., preferably 2-methoxyethanol; the stabilizer is polyvinylpyrrolidone K90 or K30, and the amount used is 20%-40% of the mass of the monomer; and the initiator is azobisisobutyronitrile, and the amount used is 2%-4% of the mass of the monomer.

6. The production method according to claim 4 or 5, characterized by, In step (3), the volume ratio of water to solvent is 3.1:16.9-3.8:16.

2.

7. Use of the amino acid zwitterionic nanogel according to any one of claims 1-3 in the preparation of a drug delivery carrier.

8. Use according to claim 7, characterized in that, The drug delivery carrier is used for delivering an antitumor drug.

9. Use according to claim 8, characterized in that, The antitumor drug is doxorubicin.

10. A pharmaceutical composition, characterized by, The amino acid zwitterionic nanogel according to any one of claims 1-3 and a pharmaceutically active ingredient loaded thereon.

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