Polymer-metal ion compound nanoparticle with endoplasmic reticulum targeting and immune activation functions

Abstract

The invention discloses a polymer-metal ion compound nanoparticle with endoplasmic reticulum targeting and immune activation functions, the structural formula of a zwitterionic polymer containing oxidized tertiary amine is # imgabs0 # as shown in I, X is a polymerization unit, including but not limited to (methyl) acrylate (or) amide, amino acid and the like; y is an N-oxidized tertiary amine linking group, R1 and R2 are selected from methyl, ethyl, propyl, butyl, amyl and hexyl, and n is 3-300; the metal ions with the immune agonist function are selected from one of manganese ions, calcium ions, zinc ions, iron ions and platinum ions. The nanoparticles are effectively delivered to the endoplasmic reticulum and activate the cGAS-STING pathway, so that the anti-tumor immune response is enhanced, and the tumor growth is inhibited.

Description

Technical Field

[0001] The present invention relates to the technical field of drug preparation, and particularly relates to a polymer-metal ion complex nanoparticle with endoplasmic reticulum targeting and immune activation functions. Background Art

[0002] In recent years, the immunomodulatory function of metal ions has received increasing attention. It has been found that divalent manganese ions Mn 2+ can significantly activate the cGAS-STING signaling pathway. The high concentration of Mn in the cytoplasm 2+ makes the cGAS-STING pathway of cells in a hyperactivated state. Therefore, manganese ions have a certain immunomodulatory function and are excellent tumor immune agonists. Platinum-containing drugs can release Pt ions in cancer cells, induce DNA damage, activate the STING signaling pathway, stimulate innate and adaptive immune responses, and improve the efficacy of chemoimmunotherapy. However, high doses of metal ions can cause certain toxicity in the body. At the same time, free metal ions have poor targeting in the body and are difficult to accumulate and penetrate at the tumor site, resulting in unsatisfactory treatment effects. There is an urgent need to develop a safe and highly immunologically active metal ion delivery system.

[0003] The cGAS-STING pathway is involved in regulating the innate immune response against tumors. The activation of the STING protein promotes the release of type I interferons and triggers a series of immune responses. Among them, the STING protein is located on the endoplasmic reticulum membrane, and the accumulation of metal ions in the endoplasmic reticulum can effectively activate the cGAS-STING pathway and generate an immune response. Summary of the Invention

[0004] The purpose of the present invention is to provide a polymer-metal ion complex nanoparticle with endoplasmic reticulum targeting and immune activation functions, which can effectively deliver metal ions to the endoplasmic reticulum, activate the cGAS-STING pathway in vivo, enhance the anti-tumor immune response, and inhibit tumor growth.

[0005] The technical solution adopted by the present invention to solve its technical problems is: A polymer-metal ion complex nanoparticle with endoplasmic reticulum targeting and immune activation functions, wherein the polymer-metal ion complex nanoparticle is formed by complexing an immune agonist using an amphoteric ion polymer containing an oxidized tertiary amine as a carrier; the structural formula of the amphoteric ion polymer containing an oxidized tertiary amine is shown as I, wherein, X is a polymerization unit, including but not limited to acrylate, methacrylate, acrylamide, methacrylamide, vinylimine, vinylpyridine, vinylimidazole or amino acid; Y is an alkyl group with 1-5 carbon atoms, R 1 and R2 Each is independently selected from methyl, ethyl, propyl, butyl, pentyl, hexyl, and n = 3 - 300; The immune agonist is a metal ion selected from one of manganese ions, calcium ions, zinc ions, iron ions, and platinum ions.

[0006] Amino acids include glycine, alanine, valine, leucine, isoleucine, methionine, proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, histidine, N,N-dimethyl-lysine.

[0007] The present invention utilizes the characteristic that the zwitterionic polymer containing an oxidized tertiary amine can complex with metal ions having an immune activation function, and utilizes the ability of this polymer to adhere to tumor capillaries and rapidly extravasate into tumor tissues from them (Enhanced tumour penetration and prolonged circulation in blood of polyzwitterion–drug conjugates with cell-membrane affinity, Nature Biomedical Engineering 5 (9), 1019 - 1037), as well as the ability to be targeted to the endoplasmic reticulum after endocytosis by cells, to prepare polymer-metal ion complex nanoparticles having endoplasmic reticulum targeting and immune activation functions.

[0008] The present invention discovers that after the zwitterionic polymer containing an oxidized tertiary amine complexes with metal ions, nanoparticles are formed, and the characteristics of zwitterions are utilized, including prolonging blood circulation, enhancing tumor accumulation and penetration. Most importantly, the rapid cell internalization and endoplasmic reticulum targeting ability can reduce the in vivo toxicity of metal ions, and at the same time utilize the endoplasmic reticulum targeting function to efficiently deliver metal ions, significantly improving the effect of metal ions activating the anti-tumor immune response.

[0009] The zwitterionic polymer containing an oxidized tertiary amine is preferably OPDMA (R is methyl) or OPDEA (R is ethyl); OPDMA: poly(2-(N-oxide-N,N-dimethylamino)ethyl methacrylate), OPDEA: poly(2-(N-oxide-N,N-diethylamino)ethyl methacrylate).

[0010] The molar ratio of the zwitterionic polymer containing an oxidized tertiary amine to the immune agonist is 10:1 - 1:1.

[0011] The source of manganese ions is selected from MnSO 4 , MnCl 2 , Mn(NO 3) 2 , Mn(ClO 4 ) 2 or a combination of one or more of them.

[0012] The source of platinum ions is selected from one or a combination of several of cisplatin, carboplatin, cycloplatin, nedaplatin, oxaliplatin, and lobaplatin.

[0013] The preparation method is as follows: The zwitterionic polymer containing tertiary amine oxide and the immune agonist are respectively dissolved in a solvent and dispersed and mixed evenly. Under stirring conditions, the immune agonist solution is dropped into the zwitterionic polymer solution. After the dropping is completed, stir at room temperature for 2 - 4 h to obtain nanoparticles, and ultrafilter to remove free metal ions.

[0014] The solvent is selected from one or a combination of several of deionized water, 0.9 wt% sodium chloride solution, and 5 wt% glucose solution.

[0015] In the zwitterionic polymer solution, the amount of solvent used is 2 - 3 mL per 10 mg of zwitterionic polymer.

[0016] In the immune agonist solution, the amount of solvent used is 2 - 5 mL per 10 mg of immune agonist.

[0017] The cut-off molecular weight of ultrafiltration is 1 KD - 1000 KD.

[0018] The beneficial effects of the present invention are as follows: Compared with free metal ions, the novel polymer-metal ion complex nanoparticles of the present invention can reduce in vivo toxicity, and at the same time have the functions of tumor enrichment of metal ions and endoplasmic reticulum targeting, effectively improving the curative effect of anti-tumor immunity. Tumors include liver cancer, melanoma, colorectal cancer, lung cancer, breast cancer and other tumor types responsive to STING treatment, and prolong the survival time of advanced cancer patients. Description of the Drawings

[0019] Figure 1 It is the particle size and PDI diagram of the manganese-based nanoparticles in Example 3 of the present invention; Figure 2 It is the particle size and PDI diagram of the manganese-based nanoparticles in Example 4 of the present invention; Figure 3 It is the particle size and PDI diagram of the manganese-based nanoparticles in Example 5 of the present invention; Figure 4 It is the scanning electron microscope diagram of the nanoparticles; Figure 5 It is the stability particle size diagram of the nanoparticles; Figure 6 It is the complexation constant of isothermal titration; Figure 7are the results of cytotoxicity experiments on different cell lines; in each figure, each concentration consists of a group of three bar graphs, and in each group, the three bar graphs represent MnCl 2 , Mn / OPDMA, OPDMA in sequence from left to right; Figure 8 is the observation of the uptake of the material by tumor cells under a laser confocal microscope; Figure 9 is the observation of the subcellular distribution of manganese-based nanoparticles in cells under a biological electron microscope; Figure 10 is the enrichment of manganese-based nanoparticles in mouse tumors at different time points; Figure 11 is the graph of the change in the volume of mouse tumors after administration; Figure 12 is the graph of the size of mouse tumors at the end of the experiment; Figure 13 is the graph of the change in the body weight of mice after administration; Figure 14 is the graph of the change in immune cells detected by flow cytometry for manganese-based nanoparticles; Figure 15 is the graph of the particle size and PDI of the platinum-based nanoparticles in Example 11 of the present invention. Detailed implementation manners

[0020] The technical solutions of the present invention will be further specifically described below through specific examples.

[0021] In the present invention, unless otherwise specified, the raw materials and equipment used can be purchased from the market or are commonly used in the art. The methods in the following examples are all conventional methods in the art unless otherwise specified.

[0022] Example 1: Preparation of zwitterionic polymer OPDMA containing tertiary amine oxide (1) Synthesis of polymer PDMA RAFT polymerization was selected for polymer synthesis. 72.8 mg of RAFT chain transfer agent trithiocarbonate (TMA) (0.2 mmol), 3.3 mg of azobisisobutyronitrile (AIBN, 0.02 mmol), and 1.67 g of monomer N,N-dimethylaminoethyl methacrylate (DMAM, 10 mmol) were weighed in sequence and placed in a 50 mL round-bottom flask, and 15 mL of anhydrous tetrahydrofuran solution was added. The mixture was purged with nitrogen for 30 minutes by the bubbling method to remove the oxygen in the bottle and the oxygen in the solvent. The flask was placed in an oil bath at 65 °C and reacted for eight hours. After the reaction, the reaction flask was quickly placed in liquid nitrogen to terminate the polymerization. The reaction solution was concentrated by rotary evaporation, precipitated with frozen n-hexane, and then dried in vacuo to obtain the product PDMA.

[0023] (2) Synthesis of Polymer OPDMA Weigh 100 mg of the polymer PDMA obtained by the above method and place it in a round-bottom flask. Add 5 mL of 30% hydrogen peroxide aqueous solution and stir at room temperature for 2 hours. During the reaction, the solid gradually dissolves to obtain a clear aqueous solution. Transfer this solution to a dialysis bag with a molecular weight of 3500 KDa and dialyze it in ultrapure water for 24 hours, changing the water every 4 hours. After dialysis is completed, lyophilize the obtained aqueous solution to obtain the final product OPDMA.

[0024] Example 2: Preparation of Zwitterionic Polymer OPDEA Containing Oxidized Tertiary Amine The difference between this example and Example 1 is that 10 mmol of ethyl N,N-diethylaminomethyl acrylate monomer is used instead of 10 mmol of N,N-dimethylaminomethyl acrylate monomer (DMAM) to first synthesize PDEA and then synthesize OPDEA, and the others are the same as in Example 1.

[0025] Example 3: Preparation of Manganese-Based Nanoparticles (1) Dissolve 10 mg of OPDMA and 10 mg of manganese chloride tetrahydrate in 3 mL of deionized water respectively, and stir to obtain a clear and transparent solution; (2) Under stirring conditions, add the manganese ion aqueous solution dropwise into the OPDMA solution according to the molar ratio of manganese ion:OPDMA monomer = 1:1. After the addition is completed, stir at room temperature for 2 h to obtain nanoparticles. Ultrafiltration (cut-off molecular weight is 1 KD) is used to concentrate and remove free metal ions to obtain nanoparticles with a manganese ion concentration of 1 mg / mL ( Figure 1 ).

[0026] Example 4: Preparation of Manganese-Based Nanoparticles The difference between this example and Example 3 is that the molar ratio of manganese ion:OPDMA monomer = 1:2, and the others are the same as in Example 3, to obtain nanoparticles with a manganese ion concentration of 1 mg / mL ( Figure 2 ), and the transmission electron microscope image of the nanoparticles is shown in Figure 4 .

[0027] Example 5: Preparation of Manganese-Based Nanoparticles The difference between this example and Example 3 is that the molar ratio of manganese ion:OPDMA monomer = 1:5, and the others are the same as in Example 3, to obtain nanoparticles with a manganese ion concentration of 1 mg / mL ( Figure 3 ).

[0028] Example 6: Preparation of Manganese-Based Nanoparticles (1) Dissolve 10 mg of OPDEA and 10 mg of manganese chloride tetrahydrate in 3 mL of deionized water respectively, and stir to obtain a clear and transparent solution; (2) Under stirring conditions, an aqueous solution of manganese ions was added dropwise to the polymer solution according to a molar ratio of manganese ions: OPDEA monomer = 1:1. After the addition was completed, the mixture was stirred at room temperature for 2 h to obtain nanoparticles. Ultrafiltration (with a molecular weight cut-off of 1 KD) was used to concentrate and remove free metal ions, and nanoparticles with a concentration of 1 mg / mL were obtained.

[0029] Example 7: Preparation of Manganese-based Nanoparticles The difference between this example and Example 6 is that the molar ratio of manganese ions: OPDEA monomer = 1:2, and the others are the same as in Example 6, and nanoparticles with a manganese ion concentration of 1 mg / mL were obtained.

[0030] Example 8: Preparation of Manganese-based Nanoparticles The difference between this example and Example 6 is that the molar ratio of manganese ions: OPDEA monomer = 1:5, and the others are the same as in Example 6, and nanoparticles with a manganese ion concentration of 1 mg / mL were obtained.

[0031] Example 9: Preparation of Manganese-based Nanoparticles (1) Dissolve 10 mg of OPDEA and 10 mg of MnSO 4 10 in 3 mL of deionized water respectively, and stir to obtain a transparent and clear solution; (2) Under stirring conditions, an aqueous solution of manganese ions was added dropwise to the polymer solution according to a molar ratio of manganese ions: OPDEA monomer = 1:6. After the addition was completed, the mixture was stirred at room temperature for 3 h to obtain nanoparticles. Ultrafiltration was used to concentrate and remove free metal ions, and nanoparticles with a concentration of 1 mg / mL were obtained.

[0032] Example 10: Preparation of Manganese-based Nanoparticles (1) Dissolve 10 mg of OPDEA and 10 mg of Mn(NO 3 ) 2 10 in 3 mL and 5 mL of deionized water respectively, and stir to obtain a transparent and clear solution; (2) Under stirring conditions, an aqueous solution of manganese ions was added dropwise to the polymer solution according to a molar ratio of manganese ions: OPDEA monomer = 1:10. After the addition was completed, the mixture was stirred at room temperature for 4 h to obtain nanoparticles. Ultrafiltration was used to concentrate and remove free metal ions, and nanoparticles with a concentration of 1 mg / mL were obtained.

[0033] Example 11: Preparation of Platinum-based Nanoparticles (1) Dissolve 10 mg of OPDMA and 10 mg of oxaliplatin in 2 mL of deionized water and 5 mL of glucose solution respectively, and stir to obtain a transparent and clear solution; (2)Under stirring conditions, an oxaliplatin solution was added dropwise to the polymer solution according to the molar ratio of platinum: OPDMA monomer = 1:5. After the addition was completed, the mixture was stirred at room temperature for 4 h to obtain nanoparticles. Ultrafiltration and concentration were used to remove free metal ions, and nanoparticles with a concentration of 1 mg / mL were obtained ( Figure 15 ).

[0034] Experiment 1. Nanoparticle stability experiment The stabilities of Mn / OPDMA (Examples 3-5) and Mn / OPDEA (Examples 6-8) nanoparticles were detected in different solvents respectively. Samples obtained by diluting with dd water or high-glucose medium DMEM were placed at room temperature for 24 h, and then the change in particle size was detected using a dynamic light scattering nanoparticle size analyzer ( Figure 5 ).

[0035] Experiment 2. Isothermal titration experiment ITC is a calorimeter with continuously changing heat. By balancing the temperature change caused by the energy release or absorption during the formation of the complex, the signal is recorded, where the energy required for the temperature shift is represented in the form of a peak. OPDMA (10 mM) and MnCl 2 (10 mM) were dissolved in deionized water respectively to obtain clear aqueous solutions. The OPDMA solution was added dropwise into the MnCl 2 solution at a rate of 5 μL per drop for 10 s, and the time interval between each drop injection was 120 s. The solution was stirred at a rate of 1000 rpm to ensure complete mixing. After the titration was completed, the original data was collected and analyzed using Origin 7.0 software. A suitable model was determined by non-linear regression to obtain thermodynamic parameters (n, K, ΔH, ΔS).

[0036] We obtained the complexation constant of the zwitterionic polymer with manganese using isothermal titration calorimetry ( Figure 6 ). Further explanation shows that complexation occurred between manganese ions and the zwitterionic polymer, rather than simple mixing. The estimated binding site N of the obtained fitting curve was 0.10, and the equilibrium dissociation constant Kd was 1.45×10 -4 M.

[0037] Experiment 3. In vitro cytotoxicity experiment (using the manganese-based nanoparticles in Example 4) 1000 - 10000 cells were seeded in each well of a 96-well plate (mouse liver cancer cell lines including Hepa1-6, H22, etc., mouse colon cancer cell lines including MC38, CT26, etc., normal mouse hepatocytes AML12), and incubated overnight until the cells adhered. The prepared manganese chloride aqueous solution (MnCl 2at 1 mg / mL), the OPDMA aqueous solution (17.3 mg / mL), and the solution containing Mn / OPDMA nanoparticles (Example 4, MnCl 2 at 1 mg / mL) were serially diluted with media (RPMI-1640, DMEM-high glucose, DMEM-low glucose, DMEM / F12, MEM, etc.) to corresponding concentrations. After the cells adhered, the medium was discarded, and the above-mentioned drug-containing media with different concentration gradients were added. After continued incubation for 24 h, CCK8 was added, and the absorbance was measured with an enzyme-linked immunosorbent assay (ELISA) reader. The IC50 value was calculated based on the absorbance.

[0038] It can be seen from the in vitro cytotoxicity experiment ( Figure 7 ), Mn / OPDMA can significantly reduce the cytotoxicity of divalent manganese ions at the cellular level. In the Hepa1-6 cell line, the IC 50 of free manganese and nanoparticles were 0.075 mM and 0.89 mM, respectively. In the AML12 cell line, the IC 50 of free manganese and nanoparticles were 0.084 mM and 0.24 mM, respectively. In the MC 38 cell line, the IC 50 of free manganese and nanoparticles were 0.072 mM and 0.34 mM, respectively. At the same time, OPDMA itself had basically no cytotoxicity. When the drug administration concentration reached 1 mM in the three cell lines, good cell viability could still be maintained.

[0039] Experiment 4. Laser confocal microscopy was used to observe the uptake of the material by tumor cells (using the manganese-based nanoparticles in Example 4) Hepatocarcinoma cells with a density of 2×10 5 were added to a confocal imaging dish, and 2 mL of DMEM medium was added to each dish. The cells were cultured in a constant temperature incubator for 24 h. The medium was replaced with a new medium containing 1 mg / mL of Mn / Cy5 OPDMA solution labeled with cy5, and incubation was continued for 1 h and 4 h. Before using laser confocal microscopy for observation, the nuclear dye Hoechst33342 was added dropwise and incubated for 15 min. After incubation, the medium was discarded, and the cells were washed 3 times with PBS. The excitation wavelength of Hoechst 33342 in the cells was 405 nm, and the emission wavelength was 425 - 475 nm. The excitation wavelength of Cy5 labeling was 649 nm, and the emission wavelength was 662 - 737 nm.

[0040] Cell uptake experiment Figure 8 It can be seen that the Mn / OPDMA nanoparticles can quickly enter the cells and accumulate. This indicates that the nanoparticles retain the function of zwitterions.

[0041] Experiment 5. Subcellular distribution of manganese-based nanoparticles in cells observed by biological electron microscopy (using the manganese-based nanoparticles in Example 4) Add THP-1 cells at a density of 5×10 5 to each well of a six-well plate, and add 2 mL of 1640 medium (containing 10 ng / mL PMA) to each dish. Incubate in a constant temperature incubator for 24 h. Replace with fresh medium containing 1 mg / mL Mn / OPDMA solution and continue to incubate for 4 h. Collect the cells, fix them with 2.5% glutaraldehyde in the dark at room temperature for 30 minutes, and place them at 4°C for embedding and staining.

[0042] Subcellular distribution experiment of cells Figure 9 It can be seen that the Mn / OPDMA nanoparticles co-localize with the endoplasmic reticulum in the cells, showing the characteristics of endoplasmic reticulum targeting.

[0043] Experiment 6. In vivo and organ distribution in mice (using the manganese-based nanoparticles in Example 4) Select 5 C57BL / 6J black mice aged 6-8 weeks, subcutaneously inoculate Hepa1-6 tumor cells, and wait until a tumor of a certain volume grows (200-300 mm 3 ). Then, inject 200 μL of 1 mg / mL Mn / Cy5 OPDMA solution via the tail vein. Anesthetize the mice with an isoflurane gas anesthesia system at 1 h, 2 h, 6 h, 12 h, and 24 h respectively, place them in a small animal in vivo imaging system for imaging, and sacrifice the mice at 6 h, 12 h, and 24 h respectively. Take the main organs, heart, liver, spleen, lung, kidney and tumor for imaging.

[0044] In vivo experiment Figure 10 It can be seen that the Mn / OPDMA nanoparticles can be effectively enriched at the tumor site in mice, indicating that the nanoparticles can improve the bioavailability of free metal ions.

[0045] Experiment 7. In vivo anti-tumor experiment in animals (using the manganese-based nanoparticles in Example 4) Establish a subcutaneous tumor model in mice by subcutaneously inoculating tumor cells. Select 12 C57BL / 6J black mice aged 6-8 weeks, subcutaneously inoculate MC38 tumor cells, and wait until a tumor of a certain volume grows. Then randomly divide them into 4 groups, with 3 mice in each group. The three groups are: ① PBS; ② MnCl 2 ; ③ OPDMA; ④ Mn / OPDMA. Administer the drugs via the tail vein, once every two days for a total of four times. Record the body weight of the mice and the size of the tumors. After the drug administration is completed, sacrifice the mice according to ethical requirements, and dissect each main organ and the tumor to detect various indicators.

[0046] Figures 11 - 14The results showed that during the whole treatment process, there was no significant change in the body weight of the mice. In the groups administered with free manganese ions and nanoparticles, the tumor growth of the mice was inhibited. At the end of the experiment, the tumor inhibition rate of the nanoparticle administration group was 71.8%. Meanwhile, compared with free manganese ions, the nanoparticles could well activate DC and CTL. The positive proportion of DC (CD11c + MHCII + ) was significantly up-regulated, rising from 24.3% (Mn group) to 35.5% (Mn / OPDMA NP group). The proportion of cytotoxic CD8 + T (IFN-γ + ) rose from 2.63% (PBS group) to 8.46% (Mn / OPDMA NP group), achieving efficient anti-tumor immunity.

[0047] The above-described embodiments are only a preferred solution of the present invention and do not impose any form of limitation on the present invention. There are other variations and modifications without exceeding the technical solutions recorded in the claims.

Claims

1. A polymer-metal ion complex nanoparticle with endoplasmic reticulum targeting and immune activation function, characterized in that: The polymer-metal ion complex nanoparticles are formed by using zwitterionic polymers containing tertiary amine oxides as carriers to complex immune agonists; the structural formula of the zwitterionic polymers containing tertiary amine oxides is shown in I, Wherein, X is a polymerized unit, including but not limited to acrylate, methacrylate, acrylamide, methacrylamide, ethyleneimine, vinylpyridine, vinylimidazole or amino acid; Y is a C1-C5 alkyl group, R1 and R2 are both selected from methyl, ethyl, propyl, butyl, pentyl, hexyl, and n=3-300; The immunostimulant is a metal ion, and the metal ion is selected from one of manganese ion, calcium ion, zinc ion, iron ion and platinum ion.

2. The metal nanoparticles according to claim 1, characterized in that The molar ratio of the zwitterionic polymer containing oxidized tertiary amine to the immune agonist is 10:1-1:

1.

3. The metal nanoparticles according to claim 1, characterized in that The source of manganese ions is selected from one or a combination of MnSO4, MnCl2, Mn(NO3)2, and Mn(ClO4)2.

4. The metal nanoparticles according to claim 1, characterized in that The platinum ion supply source is selected from one or a combination of cisplatin, carboplatin, cyclothioplatin, nedaplatin, oxaliplatin, and lobaplatin.

5. The metal nanoparticles according to claim 1, 2, 3 or 4, characterized in that: The preparation method is as follows: a zwitterionic polymer containing tertiary amine oxide and an immunostimulant are respectively dissolved in a solvent and dispersed and mixed, the immunostimulant solution is dropped into the zwitterionic polymer solution under stirring, and after the dropwise addition is completed, the solution is stirred at room temperature for 2-4 hours to obtain nanoparticles, and free metal ions are removed by ultrafiltration.

6. The metal nanoparticles according to claim 5, characterized in that The solvent is selected from deionized water, 0.9wt% sodium chloride solution, 5wt% glucose solution, or a combination of the two.

7. The metal nanoparticles according to claim 5, characterized in that In the zwitterionic polymer solution, the amount of solvent used is 2-3 mL per 10 mg of zwitterionic polymer.

8. The metal nanoparticles according to claim 5, characterized in that In the immunostimulant solution, the amount of solvent used is 2-5 mL per 10 mg of immunostimulant.

9. The metal nanoparticles according to claim 5, characterized in that The molecular weight cut-off of ultrafiltration is 1KD-1000KD.

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