Novel protein micro-nano particle system based on supramolecular self-assembly as well as preparation method and application of novel protein micro-nano particle system

Abstract

The invention belongs to the technical field of nano medicine, and particularly relates to a novel protein micro-nano particle system based on supramolecular self-assembly as well as a preparation method and application of the novel protein micro-nano particle system. According to the protein micro-nano particle system, protein or polypeptide is driven to be self-assembled through non-covalent and hydrophobic interaction among fatty acid compounds, long-chain alkyl mercaptan compounds and protein ligands so as to form super-stable protein nano particles. According to the method provided by the invention, the protein does not need to be subjected to chemical modification or genetic engineering modification, self-assembly of various proteins or polypeptides can be promoted, and the self-assembly method has good universality and simplicity; the prepared protein micro-nano particle system has high protein encapsulation efficiency, and when the system is used for delivering antigens, the cross presentation efficiency of protein antigens can be remarkably improved. The protein micro-nano particle system provided by the invention is simple in preparation process, is suitable for the fields of tumor vaccines, antiviral vaccines and the like, and has a wide clinical application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of nanomedicine, and specifically relates to a novel protein micro-nano particle system based on supramolecular self-assembly, and a preparation method and application thereof. Background Art

[0002] Proteins, as therapeutic drugs, have high biological activity and specificity and are widely used in various therapeutic methods such as antibodies, hormones, and enzyme replacement. However, the application of protein drugs is largely limited by the delivery system. For example, poor stability in the body, short half-life, low bioavailability, and immunogenicity all limit their efficiency in clinical use. To overcome these obstacles, current solutions usually include chemical modification, genetic engineering modification, and loading protein drugs on nanocarriers. However, chemical modification and genetic modification will change the structure of the protein, affecting its activity and function, and the production process is relatively complicated. Traditional protein delivery carriers face problems such as in vivo degradation, low biocompatibility, and low delivery efficiency.

[0003] In addition, vaccine adjuvants are key components for enhancing the immunogenicity of vaccines. Although traditional aluminum adjuvants (such as aluminum hydroxide) are safe and effective, they mainly induce Th2 humoral immunity and are difficult to activate Th1 cellular immunity, which limits their application in tumor therapeutic vaccines. In recent years, although new adjuvants (such as MF59, AS01, and CpG) can stimulate cellular immunity, they face problems such as complex preparation processes, toxicity risks, and insufficient stability. Nanovaccines deliver antigens through carriers (such as liposomes and polymers), but their antigen encapsulation efficiency is low (usually <10%), the preparation process is complex, and there is potential toxicity. On the other hand, existing protein carriers rely on covalent modification or genetic engineering, which may destroy protein activity and have low lysosomal escape efficiency.

[0004] Nanovaccines can achieve efficient delivery of antigens, prolong the in vivo retention time, and improve the uptake and processing efficiency of antigen presenting cells (APCs) by combining tumor-specific antigens (such as tumor-associated antigens, new antigens) with nanocarriers (such as liposomes, polymer nanoparticles, metal organic frameworks, etc.). For example, liposome-based nanovaccines can directly target lymph nodes and activate dendritic cells (DCs) by mimicking the size and surface properties of pathogens, thereby inducing strong CD8 +T cell response. However, the clinical transformation of nanovaccines still faces multiple challenges: low protein antigen loading efficiency (usually less than 10%), complex preparation process and difficulty in standardization, and potential toxicity of nanomaterials (such as inflammatory response, organ damage, etc.). To address these problems, researchers are exploring solutions through strategies such as developing biomimetic nanocarriers, optimizing microfluidics technology and surface functionalization modification. Despite this, how to improve antigen delivery efficiency and simplify the production process while ensuring safety remains one of the key challenges for tumor nanovaccines to move towards clinical practice.

[0005] The existing technology lacks a universal protein self-assembly nanoplatform with high stability, high encapsulation efficiency, targeted delivery and lysosomal escape capabilities. Summary of the Invention

[0006] In order to overcome the above-mentioned deficiencies in the prior art, the present invention proposes a novel protein micro-nanoparticle system based on supramolecular self-assembly, and a preparation method and application thereof.

[0007] According to the first aspect of the present invention, a novel protein micro-nanoparticle system based on supramolecular self-assembly is provided; the components of the protein micro-nanoparticle system include: a fatty acid compound 1, a long-chain alkyl thiol compound 2 and a protein ligand 3; the molar ratio of the protein ligand 3 to the fatty acid compound 1 is 1:20-600; the molar ratio of the protein ligand 3 to the long-chain alkyl thiol compound 2 is 1:3-400; the protein ligand 3 is at least one of a protein, a polypeptide, a protein derivative or a polypeptide derivative.

[0008] Specifically, the novel protein micro-nanoparticle system based on supramolecular self-assembly provided by the present invention is a protein nanoparticle (stable colloidal particle) formed by the hydrophobic interaction of the non-covalent coassembly of a fatty acid compound 1, a long-chain alkylthiol compound 2, and a protein ligand 3. This novel protein micro-nanoparticle system can be used for the efficient delivery of protein drugs or antigens and stimulate specific cellular immune responses, making it particularly suitable for tumor immunotherapy and the development of anti-infective vaccines.

[0009] In some embodiments, the molar ratio of the protein ligand 3 to the fatty acid compound 1 is 1:400; the molar ratio of the protein ligand 3 to the long-chain alkyl thiol compound 2 is 1:200.

[0010] In some embodiments, the fatty acid compound 1 is a fatty acid or a corresponding fatty acid salt compound; the chemical formula of the fatty acid is CH3(CH2) n COOH, n is a natural number ranging from 6 to 21; the long-chain alkylthiol compound 2 is a long-chain alkylthiol or a corresponding long-chain alkylthiolate compound; the chemical formula of the long-chain alkylthiol is CH3(CH2) mSH, m is a natural number, ranging from 5 to 20.

[0011] In some embodiments, the value range of n is 10-16; the value range of m is 9-16.

[0012] In some embodiments, the fatty acid compound 1 is sodium myristate; and the long-chain alkyl thiol compound 2 is sodium dodecyl mercaptan.

[0013] In some embodiments, the protein ligand 3 is at least one of albumin, a tumor antigen, a viral antigen, a bacterial antigen, or a mycoplasma antigen.

[0014] In some embodiments, the albumin is at least one of chicken egg albumin and serum albumin.

[0015] In some embodiments, the viral antigen is at least one of envelope glycoprotein (HIV gp120 / gp41, influenza virus HA hemagglutinin), capsid protein (p24, L1), new coronavirus surface antigen, human papillomavirus HPV protein antigen and EBV antigen.

[0016] In some embodiments, the bacterial antigen is at least one of a tuberculosis antigen, α-hemolysin, Staphylococcal enterotoxin B, and streptolysin O.

[0017] According to a second aspect of the present invention, a method for preparing a novel protein micro-nanoparticle system based on supramolecular self-assembly is provided, comprising the following steps: preparing a fatty acid compound 1, a protein ligand 3, and a long-chain alkyl thiol compound 2 into solutions using a solvent; then mixing the fatty acid compound 1 solution and the protein ligand 3 solution to obtain a primary complex (protein / polypeptide-fatty acid complex); mixing the primary complex with the long-chain alkyl thiol compound 2 solution to obtain a complex; and dialyzing the complex with a buffer solution having a pH of 3.0-6.5 (through hydrophobic interaction and disulfide bond cross-linking to form nanoparticles with a particle size of 50-300 nm) to obtain a novel protein micro-nanoparticle system based on supramolecular self-assembly.

[0018] In some embodiments, the solvent is one of water, anhydrous ethanol, dimethyl sulfoxide, phosphate buffered saline (PBS buffer) or physiological saline.

[0019] In some embodiments, the method of mixing the fatty acid compound 1 solution and the protein ligand 3 solution comprises the following steps: adding the fatty acid compound 1 solution to the protein ligand 3 solution, and stirring for 10-30 minutes at a stirring rate of 30-100 rpm.

[0020] In some embodiments, the method of mixing the primary complex with the long-chain alkyl thiol compound 2 solution comprises the following steps: adding the long-chain alkyl thiol compound 2 solution to the primary complex, and stirring at a stirring rate of 100-800 rpm for 30-60 minutes.

[0021] In some embodiments, the dialysis treatment comprises the following steps: placing the complex in a dialysis bag, dialyzing in a buffer solution with a pH of 3.0-6.5 for 4-12 hours, and collecting the retained solution in the dialysis bag; the molecular weight cut-off of the dialysis bag is 50-100 kDa.

[0022] In some embodiments, after the complex is dialyzed, the following steps are further included: centrifugation and freeze-drying to obtain a novel protein micro-nanoparticle system based on supramolecular self-assembly (protein nanoparticles with an encapsulation rate ≥ 80%); the centrifugation speed is 12000-15000 rpm, and the centrifugation time is 20-30 minutes; the freeze-drying temperature is -20°C to -80°C, and the freeze-drying time is 36-72 hours.

[0023] In some embodiments, the freeze-drying temperature is -60°C, and the freeze-drying time is 42 hours.

[0024] In some embodiments, the centrifugal rotation speed is 15000 rpm, and the centrifugal treatment time is 20 minutes.

[0025] In some embodiments, the centrifugal rotation speed is 12000 rpm, and the centrifugal treatment time is 20 minutes.

[0026] During the formation of the novel protein micro-nanoparticle system based on supramolecular self-assembly provided by the present invention, an amphiphilic fatty acid compound 1 and a long-chain alkyl thiol compound 2 are first adsorbed on the surface of a protein ligand 3 respectively through hydrophobic interactions; subsequently, the long-chain alkyl thiol compound 2 molecules on the surface of the protein ligand 3 promote the formation of disulfide bonds (-SS-) between thiol groups (-SH) through an oxidation reaction, so that the long-chain alkyl thiol compound 2 can serve as a molecular bridge to connect the complex (primary complex) formed by the protein ligand 3 and the fatty acid compound 1, and at the same time, the amphiphilic long-chain alkyl thiol compound 2 is converted into a hydrophobic molecule; finally, under weakly acidic conditions, the protonation of the amphiphilic fatty acid compound 1 converts it from a soluble substance into a hydrophobic molecule, further promoting the hydrophobic interaction between the protein-molecular bridge, thereby triggering their phase separation and promoting the self-assembly of the protein-fatty acid compound-long-chain alkyl thiol compound intermediate into an ultra-stable protein nanoaggregate.

[0027] The present invention can well control the size, morphology and stability of the protein micro-nanoparticle system by precisely adjusting the hydrophobic interaction mediated by fatty acid compounds (by adjusting the acidic pH value of the buffer) and the formation of disulfide bonds.

[0028] According to a third aspect of the present invention, there is provided the use of a novel protein micro-nanoparticle system based on supramolecular self-assembly in the preparation of vaccines.

[0029] In some embodiments, the vaccine is a tumor vaccine or an anti-infective vaccine.

[0030] The novel protein micro-nanoparticle system based on supramolecular self-assembly provided by the present invention is a universal protein delivery platform and vaccine antigen delivery system that does not require chemical modification or genetic modification, and is based on amphiphilic molecules that induce self-assembly by regulating the hydrophobic interactions between proteins. It has higher delivery efficiency and good protein activity retention. The novel protein micro-nanoparticle system based on supramolecular self-assembly provided by the present invention is a protein nanoparticle platform based on a non-covalent co-assembly strategy, which can effectively solve the problems of low antigen encapsulation efficiency, poor stability, and inability to effectively activate CD8 in existing adjuvants. + T cell-mediated cellular immune response and other issues, and to achieve efficient delivery and cross-presentation of tumor or other protein (epitope) antigens, and to efficiently induce antigen-specific CD8 + T cell-mediated cellular immune response.

[0031] The novel protein micro-nanoparticle system based on supramolecular self-assembly provided by the present invention has the following technical effects:

[0032] (a) When protein ligand 3 was ovalbumin, the novel protein micro-nanoparticle system based on supramolecular self-assembly exhibited significant antitumor effects: in the B16-OVA melanoma model, the tumor inhibition rate reached 88%, significantly superior to the control group (normal saline and free antigen);

[0033] (b) Simple preparation process: no genetic engineering or covalent chemical modification is required, making it suitable for industrial production;

[0034] (c) Through modular design, it has good universality: it can adapt to the self-assembly and self-delivery of a variety of antigens (such as new antigens, viral epitopes, and bacterial antigens), and can be expanded to the field of infectious disease vaccines;

[0035] (d) High encapsulation efficiency: The protein encapsulation efficiency reached over 80% (determined by the Bradford method), which is significantly better than that of traditional nanocarriers (<10%).

[0036] (e) Good lysosomal escape performance: cytoplasmic delivery of antigens is achieved through lysosomal membrane disruption, promoting cross-presentation of antigens to the MHC-I pathway;

[0037] (f) Effective activation and induction of antigen-specific CD8+ T cells and humoral immune responses: It can induce the production of high levels of IgG antibodies and CD8 + T cell-mediated cellular immune response.

[0038] Compared with the prior art, the present invention has the following beneficial effects and advantages:

[0039] (1) The present invention realizes efficient and controllable self-assembly of various polypeptides or proteins through non-covalent and hydrophobic interactions between straight-chain fatty acid compounds 1 (such as sodium myristate), long-chain alkyl thiol compounds 2 (such as sodium dodecyl mercaptan) and protein ligands 3. Traditional protein self-assembly methods rely on covalent chemical modification or genetic engineering, which not only has cumbersome steps but may also destroy the natural conformation of the protein. The method provided by the present invention does not require chemical modification or genetic engineering modification of the protein itself, and can promote the self-assembly of various proteins or polypeptides. The self-assembly method has good universality and simplicity, and ensures its biological activity.

[0040] (2) The preparation method provided by the present invention is that proteins, polypeptides or their corresponding derivatives with different functions form nano-scale aggregates of controllable size through self-assembly, and the particle size is in the range of 50-300nm. By regulating conditions such as pH value, the ratio of fatty acid compounds and long-chain alkyl thiol compounds, the size of protein particles can be controlled to meet different application requirements.

[0041] (3) The preparation method provided by the present invention only requires steps such as mixing and dialysis, does not require complex equipment or high-purity raw materials, has controllable costs, and is suitable for large-scale production. Therefore, it has good prospects for large-scale production.

[0042] (4) The traditional antigen adjuvant aluminum mainly induces Th2 type humoral immunity, and the Th1 type cellular immune response, which is crucial for tumor treatment, is weak. However, the novel protein micro-nanoparticle system based on supramolecular self-assembly provided by the present invention can achieve humoral and cellular immunity (antigen-specific CD8 + Dual activation of T cell responses).

[0043] (5) The novel protein micro-nanoparticle system based on supramolecular self-assembly provided by the present invention can be used to deliver antigens. The fatty acid compound 1 can act as a surfactant to destroy the cell membrane of the lysosome, thereby promoting the lysosomal escape of the antigen and promoting the cross-presentation of the antigen. Therefore, it can efficiently induce antigen-specific CD8 + T cell response.

[0044] (6) The preparation method provided by the present invention constructs a novel protein micro-nanoparticle system based on supramolecular self-assembly through precise regulation of non-covalent hydrophobic interactions. Compared with traditional technologies, it has significantly improved antigen delivery efficiency and immune activation ability, simple process, and industrial feasibility, providing a new solution for tumor immunotherapy and infectious disease vaccine development. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 The particle size distribution diagram of the protein nanoaggregates (HRP PCD) prepared when the self-assembly ratio is 1:400:50 in Example 1;

[0046] Figure 2 This is the particle size distribution diagram of the protein nanoaggregates (HRP PCD) prepared when the self-assembly ratio is 1:400:100 in Example 1;

[0047] Figure 3 This is the particle size distribution diagram of the protein nanoaggregates (HRP PCD) prepared when the self-assembly ratio is 1:400:200 in Example 1;

[0048] Figure 4 The particle size distribution diagram of the protein nanoaggregates (HRP PCD) prepared when the self-assembly ratio is 1:400:300 in Example 1;

[0049] Figure 5 This is an electron microscope image of the HRP PCD prepared when the self-assembly ratio is 1:400:200 in Example 1;

[0050] Figure 6 is the particle size distribution diagram of OVA PCD synthesized in Example 2;

[0051] Figure 7 is an electron micrograph of free OVA (OVA solution) in Example 2;

[0052] Figure 8 is an electron micrograph of the OVA PCD synthesized in Example 2;

[0053] Figure 9 Figure 2 is the particle size stability result of OVA PCD synthesized in Example 2 within 12 days;

[0054] Figure 10 This is a graph showing the PDI coefficient of the OVA PCD synthesized in Example 2 within 12 days;

[0055] Figure 11 This is the result of detecting EE% in OVA PCD by Bradford method in Example 2;

[0056] Figure 12 This is a graph showing the cumulative release of OVA from OVA PCD within 144 hours as determined by the Bradford assay in Example 2;

[0057] Figure 13 Representative fluorescence microscopy images of OVA PCD achieving cytoplasmic delivery via lysosomal escape in Example 3;

[0058] Figure 14 This is a graph showing the results of the in vitro cytotoxicity evaluation of OVA PCD in Example 4;

[0059] Figure 15 The graphs show the results of in vivo fluorescence signal acquisition of draining inguinal lymph nodes (LNs) using a small animal in vivo fluorescence imaging system (FX Pro) at 12 hours and 24 hours after injection in Example 5;

[0060] Figure 16 This is a graph showing the results of in vitro fluorescence signal collection of lymph nodes removed at corresponding time points in Example 5;

[0061] Figure 17 The relative quantitative analysis results of the in vitro fluorescence signals of lymph nodes removed at the corresponding time points in Example 5 are shown (**** indicates p≤0.0001);

[0062] Figure 18 This is a graph showing the results of detecting total IgG antibody titer in mouse serum using the ELISA method in Example 6 (**** indicates p≤0.0001, *** indicates p≤0.001);

[0063] Figure 19 This is the tumor growth curve within 18 days after mice were inoculated with B16-OVA tumor cells in Example 7;

[0064] Figure 20 The weight change curves of each group of mice after tumor inoculation in Example 7;

[0065] Figure 21 These are pictures of in vitro tumors on the 18th day after tumor inoculation in each group of mice in Example 7. × indicates death before the end of the experiment;

[0066] Figure 22 This is a graph showing the statistical analysis results of the in vitro tumor weights of each group of tumors grown to day 18 in Example 7;

[0067] Figure 23 This is the growth curve of a single tumor in each group after subcutaneous inoculation of B16-OVA tumor cells in Example 7;

[0068] Figure 24This is a diagram of the flow cytometry results in Example 8; Figure 24 A is the result of flow cytometry analysis of DC maturation rate in the spleen of mice receiving different treatments; Figure 24 B is IFNγ + CD8 + T cell percentage; Figure 24 C is the OVA antigen peptide-specific CD8 after SIINFEKL polypeptide (8ug / ml) restimulation in vitro + relative quantitative analysis of T cells by flow cytometry; Figure 24 D is the OVA antigen peptide-specific CD8 after SIINFEKL polypeptide (8ug / ml) restimulation in vitro + Representative results of T cell flow cytometry analysis;

[0069] Figure 25 This is a graph showing the results of statistical analysis of the number of IFNγ spot-forming cells in splenocytes after in vitro restimulation in Example 9;

[0070] Figure 26 This is a representative ELISPOT image of IFNγ spot-forming cells in splenocytes after in vitro restimulation in Example 9;

[0071] Figure 27 This is a graph showing the results of the toxic effect of effector T cells targeting and killing tumor cells in vitro in Example 10. DETAILED DESCRIPTION

[0072] The present invention will be further described in detail below with reference to the examples, but the embodiments of the present invention are not limited thereto. It should be noted that if there are any processes or parameters not specifically described below, they can be understood or implemented by those skilled in the art with reference to the prior art.

[0073] The mice used in the following examples were female C57BL / 6 mice (6-8 weeks old) purchased from the Guangdong Provincial Laboratory Animal Center. Mice were housed in a room maintained at 22°C-25°C with artificial light on a 12-hour light / 12-hour dark cycle. Feed and water were available ad libitum. This study complied with relevant ethical regulations for animal testing and research.

[0074] The following examples utilize the following cell culture methods: RAW264.7 and 293T cells were cultured in DMEM (Gibco) supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin at 37°C and 5% (v / v) CO2. B16-OVA cells were cultured in RPMI-1640 (Gibco) supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin under the same culture conditions. RAW 264.7 and 293T cells were obtained from ATCC, catalog numbers TIB-71 and CRL-3216, respectively. B16-OVA cells are a stably expressing cell line constructed by lentiviral transduction of the exogenous gene OVA into B16 cells (purchased from VigenCell, catalog number VGC-0106-007P).

[0075] The in vitro release experiments conducted in the following examples specifically included the following steps: To determine the release characteristics of OVA PCD in PBS buffer (pH 7.4), 1 ml of purified OVA PCD solution (OVA concentration of 1.33 mg / mL) was sealed in a dialysis bag (MW cutoff of 50 kDa) and placed in 10 ml of PBS buffer (pH 7.4). The solution containing the dialysis bag was shaken at 70 rpm at 4°C. At 3, 12, 24, 48, 72, 96, 120, and 144 hours, 100 μL of PBS outside the dialysis bag was removed and fresh PBS buffer was added to replenish the removed dialysate. The OVA content in the PBS solution at each time point was determined using the Bradford method.

[0076] Example 1 Self-assembly ratio optimization

[0077] In the self-assembly process of a novel protein micro-nanoparticle system (protein nano-aggregates) based on supramolecular self-assembly, the content of amphiphilic molecules will affect the morphology and size of the final nano-aggregates. Example 1 of the present invention first uses horseradish peroxidase (HRP) as a model protein, sets different self-assembly ratios (molar ratios), namely protein: sodium myristate (SAM): sodium dodecyl mercaptan (SDT) = 1:400:50, 1:400:100, 1:400:200, 1:400:300, and prepares HRP protein nano-aggregates using the same preparation process. And measure its particle size distribution and morphology. The experimental steps are as follows:

[0078] (1) Dissolve 0.88 mg of HRP in deionized water to obtain a stock solution with a concentration of 0.88 mg / mL for later use;

[0079] (2) Weigh 2 mg of sodium myristate and dissolve it in 1 mL of deionized water to prepare a 2 mg / mL stock solution for later use.

[0080] (3) Dissolve 0.1012 g of dodecyl mercaptan and 0.02 g of NaOH in 5 mL of anhydrous ethanol to prepare a 0.1 mol / L sodium dodecyl mercaptan solution for later use;

[0081] (4) 500 μl of each of the HRP solution and sodium myristate solution prepared in step (1) and step (2) were taken and mixed. The molar ratio of HRP to sodium myristate was 1:400. The mixture was stirred for 20 minutes at a stirring speed of 100 rpm to obtain a mixed solution A. Four portions of mixed solution A were prepared at the same time.

[0082] (5) adding the sodium dodecyl mercaptan solution prepared in step (3) to the mixed solution A of step (4) at a protein: sodium dodecyl mercaptan molar ratio of 1:50, 1:100, 1:200, and 1:300, respectively, with the corresponding volumes of the sodium dodecyl mercaptan solution being 5 μL, 10 μL, 20 μL, and 30 μL, respectively, and stirring for 30 minutes at a stirring speed of 200 rpm to obtain 4 portions of mixed solution B;

[0083] (6) transferring the four portions of mixed solution B obtained in step (5) into four dialysis bags with a molecular cutoff of 50 kDa for dialysis treatment to obtain four portions of protein nanoaggregates (i.e., a novel protein micro-nanoparticle system based on supramolecular self-assembly, labeled as HRP PCD);

[0084] (7) In step (6), the dialysis conditions are: the dialysis bag is placed in a PBS solution with a pH of 5.0, and the dialysis time is 12 h;

[0085] (8) Evaluation of particle size and morphology: The average particle size and physical morphology of the obtained nanoaggregates were measured using a nanometer size potentiometer (Malvern Nano-ZS) and a transmission electron microscope.

[0086] (9) Figure 1 、 Figure 2 、 Figure 3 and Figure 4 As shown in Example 1, by adjusting the ratio of sodium dodecyl mercaptan, HRP nanoaggregates of different particle sizes can be obtained. However, when the ratio is 1:400:200 (protein: sodium myristate: sodium dodecyl mercaptan), stable, monodisperse nanoparticles can be formed. TEM imaging further shows that at this ratio, uniform, spherical nanoparticles are formed, with a size of approximately 100 nm ( Figure 5 No obvious nanoparticle formation was detected in the soluble HRP sample. Therefore, 1:400:200 (protein: sodium myristate: sodium dodecyl mercaptan) was determined to be the optimal self-assembly ratio for the preparation of PCD in the present invention.

[0087] Example 2 Preparation and Preliminary Characterization of OVA Nanoaggregates (OVA PCD)

[0088] In this example, ovalbumin (OVA) was used as the raw material to construct nanoscale protein aggregates (OVA PCD, a novel protein micro-nanoparticle system based on supramolecular self-assembly) through non-covalent self-assembly of the amphiphilic molecules sodium myristate (SAM) and sodium dodecylthiolate (SDT).

[0089] (1) First, according to the self-assembly ratio and preparation steps optimized in Example 1, a concentration of 1×10 -5 A 500 μL mol / L OVA solution was mixed with 500 μL of sodium myristate (2 mg / mL) at a 1:400 molar ratio (protein:sodium myristate molar ratio was 1:400) on a magnetic stirrer at room temperature. Stirring was continued for 20 minutes at 50 rpm to form a SMA-OVA hybrid system. This step utilizes the interaction between the long hydrophobic alkyl chains in the SAM molecule and the hydrophobic regions on the surface of the OVA molecule to form a primary SMA-OVA complex.

[0090] (2) Subsequently, the pre-prepared SDT solution (preparation process refers to Example 1) was added to the SMA-OVA mixed system in step (1) at a ratio of 1:200 (molar ratio of protein: sodium dodecyl mercaptan is 1:200), and stirring was continued for 30 minutes at a stirring speed of 200 rpm so that the SDT molecules were further embedded in the complex through their dodecyl chains, thereby strengthening the construction of the hydrophobic core and forming an OVA PCD hybrid.

[0091] (3) The OVA PCD mixture from step (2) was transferred to a dialysis bag with a molecular weight cutoff of 50 kDa and dialyzed against a PBS solution (pH = 5.0) for 12 hours to remove free components, thereby obtaining OVA nanoaggregates (OVA PCD). Dialysis induces the protonation of SMA molecules, converting them to hydrophobic properties, leading to sulfhydryl oxidative coupling between SDT molecules to form stable disulfide bonds, inducing solid-liquid phase separation in the system, and ultimately forming ultrastable OVA PCD of a certain nanometer size.

[0092] The obtained OVA PCD was centrifuged at a rotation speed of 12000 rpm for 20 minutes and freeze-dried to obtain a purified novel protein micro-nanoparticle system based on supramolecular self-assembly, which was stored at -20°C.

[0093] (4) The prepared OVA PCD samples were observed for morphology and size using transmission electron microscopy (TEM). They were negatively stained with a 2% phosphotungstic acid solution before imaging. The OVA PCD solution was then placed at 4°C and its particle size was monitored using dynamic light scattering (DLS) for 12 days.

[0094] Dynamic light scattering (DLS) measured the hydrated particle size to be 133.5±21.8nm (PDI<0.2) (e.g. Figure 6 shown).

[0095] (5) Figure 7 、 Figure 8 As shown in Figure 2, TEM images show that the OVA PCD particles are about 50-100 nm in size, spherical, and monodispersed. In contrast, there is no obvious nanoparticle formation in the free OVA. Further particle size stability evaluation results (DLS analysis) show that (e.g. Figure 9 and Figure 10 Over 12 days, OVAPCD maintained a relatively stable particle size, with a PDI of 0.174 ± 0.007. This result demonstrates the high stability of nanoaggregates formed in the 1:400:200 self-assembly system. This result further confirms that the optimized ratio (1:400:200) in Example 1 is the optimal self-assembly ratio.

[0096] In vitro release experiment: In a simulated physiological environment (PBS buffer, pH 7.4), the OVA released by OVAPCD within 144 hours was detected by Bradford method. The protein encapsulation efficiency was also measured by Bradford method. The protein encapsulation efficiency (EE%) was calculated by measuring the OVA content in the resuspended OVA nanoaggregates (OVAPCD) particles after centrifugation. The formula for calculating protein encapsulation efficiency is EE% = (actual encapsulated protein amount / total amount of protein initially added) × 100%. The results are shown in Figure 2. Figure 11 As shown in Figure 2, the protein encapsulation efficiency can reach about 80%. In vitro release experiments showed that the cumulative release rate of OVA-PCD in a simulated physiological environment (PBS, pH 7.4) within 144 hours was only 8.88±0.41% (as shown in Figure 2). Figure 12 ), indicating that it has good structural stability and sustained-release properties, which may effectively protect the antigen from degradation and prolong its action time in the body. Figure 10 It can be seen that OVA PCD forms stable, monodisperse nanoparticles.

[0097] Example 3 In vitro cellular uptake and lysosomal escape experiments

[0098] 1×10 5RAW cells (RAW 264.7, from ATCC) were cultured in a confocal culture dish at a density of 10 cells, and then OVA PCD (2.5 μg / mL, prepared in Example 2 with a molar ratio of OVA, SMA, and SDT of 1:400:200) labeled with Cy3-NHS (Cyanine 3-NHS) was added and incubated for 12 hours. The cells were then stained with LysoTrackerGreen (50 nM) (from Thermo Fisher Scientific, product number L7526-100) and Hoechst 33342 (5 μg / mL) (from Beyotime, product number C1028). The green fluorescent probe Lysotracker Green was used to label lysosomes, and Hoechst33342 was used to stain the cell nucleus. After each treatment, the cells were rinsed three times with PBS buffer and then observed using a laser confocal microscope (LSM 880). The results are shown in Figure 2. Figure 13 As shown, the red fluorescence (Cy3-NHS-labeled OVA PCD) was almost completely separated from the green fluorescence (Lysotracker Green-labeled lysosomes), indicating that OVA PCD was successfully taken up into cells and was able to escape from lysosomes into the cytoplasm, thereby achieving cytoplasmic delivery.

[0099] Example 4 OVAPCD in vitro cytotoxicity assessment

[0100] Example 4 The OVA PCD prepared in Example 2 with a molar ratio of OVA, SMA, and SDT of 1:400:200 was used for the experiment.

[0101] In Example 4 of the present invention, a CCK-8 detection kit (purchased from APExBIO Technology, product number K1018) was used to evaluate the in vitro cytotoxicity of OVAPCD. The specific steps are as follows:

[0102] (1) 293T cells (from ATCC cell bank) were cultured at 5×10 3 Cells were seeded into 96-well plates at a density of 100 cells / well and cultured for 24 hours.

[0103] (2) Different concentrations of OVA PCD (10 μg / mL, 30 μg / mL, 50 μg / mL, 80 μg / mL, 100 μg / mL) were added to step (1) and incubated with 293T cells for 24 hours.

[0104] (3) Six replicate wells were set for each concentration in step (2).

[0105] (4) After removing the culture medium containing OVAPCD in the wells prepared in step (2), 100 μL of complete culture medium containing 10 μL of CCK-8 was added to each well and incubated at 37°C for 30 minutes.

[0106] (5) After the incubation in step (4) is completed, the absorbance is measured at a wavelength of 450 nm.

[0107] (6) The relative cell viability was calculated according to the following formula:

[0108] Cell viability (%) = [(At-Ab) / (Ac-Ab)] × 100%.

[0109] (7) In step (6), At, Ab, and Ac represent the absorbance of the tested cells, no cells, and untreated cells, respectively.

[0110] (8) Figure 14 As shown, OVAPCD did not show significant cytotoxicity within different concentration ranges, indicating that the present invention meets the safety requirements of biomedical materials and exhibits good clinical translation potential.

[0111] Example 5 OVAPCD lymphatic targeting delivery characteristics

[0112] Example 5 The OVA PCD prepared in Example 2 with a molar ratio of OVA, SMA, and SDT of 1:400:200 was used for the experiment.

[0113] For lymph node drainage studies, OVA PCD and Free OVA were pre-labeled with the near-infrared fluorescent dye Cy5.5 to obtain Cy5.5-OVA PCD and Cy5.5-OVA. C57BL / 6 mice were treated with equal doses of Cy5.5-OVA PCD or Cy5.5-OVA (injection dose was 25 μg OVA / mouse) by subcutaneous injection through the tail. Under isoflurane anesthesia, in vivo near-infrared fluorescence imaging was performed using a multimodal small animal in vivo imaging system (FX Pro) 12h and 24h after injection. At the corresponding time points, the mice were euthanized and the inguinal lymph nodes were removed for ex vivo fluorescence signal acquisition. Figure 15 、 Figure 16 、 Figure 17As shown in the figure, at the 12-hour time point, both Cy5.5-OVA PCD and Cy5.5-OVA exhibited significant fluorescence signals in peripheral lymph nodes (LNs), but the fluorescence signal intensity of the Cy5.5-OVA PCD group in the inguinal lymph nodes was significantly higher than that of the Free OVA group. Quantitative analysis of relative fluorescence in lymph nodes further confirmed that OVA PCD had approximately 3.8-fold higher lymph node targeting efficiency than Free OVA, and maintained significant signal intensity even after 24 hours, while the fluorescence signal in the Free OVA group had essentially disappeared, demonstrating that OVA PCD possesses excellent specific lymphatic delivery capabilities.

[0114] Example 6 OVA PCD Enhancement of Humoral Immunity Experiment

[0115] Example 6 The OVA PCD prepared in Example 2 with a molar ratio of OVA, SMA, and SDT of 1:400:200 was used for the experiment.

[0116] Healthy C57BL / 6 mice (6-8 weeks old, purchased from Guangdong Experimental Animal Center) were selected and randomly divided into three groups (n=6): experimental group: OVA PCD was used as the immunogen; control group: free OVA was used as the immunogen; blank group: the antigen was replaced by an equal volume of normal saline.

[0117] Mice were immunized twice by subcutaneous injection (sc), 7 days apart. The primary immunization dose was 50 μg (calculated as OVA antigen content), and the secondary immunization dose was adjusted to 25 μg.

[0118] On the 14th day after the last immunization, whole blood samples were collected through the orbital venous plexus, allowed to stand at room temperature for 30 min, and then centrifuged at 3000 rpm for 10 min at 4°C to separate the serum.

[0119] The titer of total anti-OVA IgG antibodies in serum was determined using an enzyme-linked immunosorbent assay (ELISA) (ELISA kit manufacturer: Crystal Chem; Cat. No. 80644). The experiment was repeated three times, and the results are presented as the mean ± SD.

[0120] like Figure 18 As shown, the total IgG antibody titer in the OVA PCD group reached approximately 1×10 4 , which was significantly higher than that of the free OVA group (antibody titer was approximately 8×10 2 )(P<0.001), the antibody titer increased by 12.5 times.

[0121] The above results indicate that the OVA PCD prepared in Example 2 of the present invention can not only achieve efficient delivery of antigens, but also effectively activate B cells through antigen multimerization, significantly improving the immunogenicity of the antigen.

[0122] Example 7 OVA PCD in vivo anti-tumor immunotherapy and effect evaluation

[0123] Example 7 The OVA PCD prepared in Example 2 with a molar ratio of OVA, SMA, and SDT of 1:400:200 was used for the experiment.

[0124] This example systematically evaluates the effectiveness of OVA PCD in anti-tumor immunotherapy by establishing a B16-OVA melanoma mouse model. The specific implementation plan is as follows:

[0125] First, several healthy C57BL / 6 mice (6-8 weeks old, purchased from Guangdong Experimental Animal Center) were selected and randomly divided into three groups with equal numbers (n=5) in each group, namely, normal saline control group, free OVA (free antigen group) and OVA PCD (complex group).

[0126] A three-injection immunization schedule was used, with subcutaneous injections at the tail on days 0, 7, and 21. The initial immunization dose was 50 μg OVA equivalent, and the second and third immunization doses were gradually reduced to 25 μg. Seven days after the last immunization, 5×10 5 B16-OVA melanoma cells.

[0127] Tumor growth was monitored continuously for 18 days after inoculation. The long diameter (L) and short diameter (W) of the tumor were measured every 2 days using a vernier caliper. The formula V = 0.5 × L × W was used. 2 Tumor volume was calculated. The experiment was terminated on day 18. Mice were euthanized, and subcutaneous tumors were removed for weight, recording, and photographing. The spleens were sterilely removed and ground, and splenocytes were collected for subsequent flow cytometry analysis, IFN-γ ELISPOT assay, and CCK-8 assay to assess the functional activity of effector T cells.

[0128] The experimental results are as follows Figure 19 、 Figure 20 、 Figure 21 、 Figure 22 and Figure 23 shown.

[0129] Figure 19 The tumor growth curve within 18 days after mice were inoculated with B16-OVA tumor cells in Example 7 is shown in Figure 1. Figure 19 It can be seen that the tumor volumes of the saline group and the free OVA group showed an exponential growth trend, and the average volumes reached (1.04±0.52)×10 3 mm 3 and (1.09±0.51)×10 3 mm 3There was no statistical significance between the two groups. However, the tumor growth in the OVA PCD group was significantly inhibited, with the final volume being only (0.12±0.08)×10 3 mm 3 This result confirms that the OVA PCD constructed in Example 2 of the present invention can effectively activate anti-tumor immune response and significantly delay the progression of solid tumors, and has potential clinical application value.

[0130] Figure 20 The weight change curves of mice in each group after tumor inoculation are shown in Figure 2. Figure 20 As shown in the figure, there was no significant change in body weight in the OVA PCD group, indicating that it had no obvious systemic toxicity, while the body weights of the saline group and the free OVA group showed an upward trend due to the growth of their tumors.

[0131] Figure 21 These are pictures of in vitro tumors on the 18th day after tumor inoculation in each group of mice (5 mice in each group). × indicates death before the end of the experiment; Figure 22 This is the statistical analysis result of the in vitro tumor weight of each group at day 18 of tumor growth; Figure 23 is the growth curve of a single tumor in each group after subcutaneous injection of B16-OVA cells; Figure 21 、 Figure 22 and Figure 23 It can be seen that OVA PCD can effectively delay the progression of solid tumors.

[0132] Example 8 Study on the anti-tumor immune mechanism of OVA PCD in vivo

[0133] In order to further explore the anti-tumor immune mechanism of OVA PCD, based on Example 7, spleen cells of each group of mice were collected at the end of the experiment, and the DC maturation ratio, CD8 + The ratio of specific OVA tetramer-positive cells and IFN-γ-positive cells in T cells.

[0134] (1) Detection of CD8 + The specific experimental steps for the proportion of specific OVA tetramer-positive cells in T cells are as follows:

[0135] At the end of the experiment, mice were sacrificed and their spleens were aseptically removed and ground. Splenocytes were collected and filtered through a 70 μm cell sieve. Red blood cells were removed using an erythrocyte lysis buffer (model R1010, provided by Beijing Solebeau Technology Co., Ltd.) to obtain splenic lymphocytes. Isolated splenic lymphocytes were cultured in RPMI-1640 medium (Gibco) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin at 37°C and 5% (v / v) CO2.

[0136] 1×10 6 Splenocytes were seeded in 6-well plates and 8 μg / mL SIINFEKL (OVA 257-264 ) polypeptide, SIINFEKL(OVA 257-264 ) Peptides were provided by Shanghai Bioengineering Co., Ltd. and stimulated in a 37°C, 5% CO2 incubator for 72 hours.

[0137] The cells were collected into a 1.5 mL centrifuge tube, washed with 200 μL PBS, and centrifuged at 500 × g for 5 min;

[0138] Add 50 μL FACS buffer (PBS containing 2% FBS) and resuspend;

[0139] Mouse CD16 / 32 antibody (1:100 dilution, FcR blocking reagent, from Biolegend, cat. no. 101319) was added to block Fc receptors at room temperature for 10 min;

[0140] Add 5 μL of PE-labeled OVA tetramer (H-2Kb / SIINFEKL, derived from MBL, Catalog No. TS-5001-1C) and incubate at 4°C in the dark for 30 min;

[0141] After washing with PBS, 1 μL Pacific Blue-CD3 antibody (clone number: 17A2, purchased from Biolegend, cat. no. 100213) and 0.5 μL APC-CD8 antibody (clone number: 53-6.7, purchased from Biolegend, cat. no. 100712) were added respectively;

[0142] Incubate at 4°C in the dark for 20 min, wash with PBS buffer and filter through a 200-mesh nylon mesh

[0143] Flow cytometric analysis: BD FACSCanto II flow cytometer was used for detection and FlowJo V10 software was used for analysis.

[0144] (2) Methods for detecting the proportion of DC maturation in the spleen and CD8 + The method for determining the proportion of specific OVA tetramer-positive cells in T cells is basically the same and can be performed according to the above method. The difference lies in the different detection antibody combinations. The detection antibody combination for DC maturation ratio is as follows:

[0145] APC-labeled anti-CD11c monoclonal antibody (clone number: N418, product number 117310, Biolegend);

[0146] PE-labeled anti-CD86 monoclonal antibody (clone number: GL-1, product number 105007, Biolegend);

[0147] FITC-labeled anti-CD80 monoclonal antibody (clone number: 16-10A1, product number 104705, Biolegend).

[0148] (3) Methods for detecting the proportion of IFN-γ positive cells in the spleen and CD8 + The method for determining the proportion of specific OVA tetramer-positive cells in T cells is basically the same and can be performed according to the above method. The difference lies in the different detection antibody combinations. The detection antibody combination for detecting IFN-γ-positive cells is as follows:

[0149] APC-labeled anti-CD3 monoclonal antibody (clone number: 17A2, product number 100235, Biolegend);

[0150] FITC-labeled anti-CD8 monoclonal antibody (clone number: 53-6.7, product number 100705, Biolegend);

[0151] PE-labeled anti-IFN-γ monoclonal antibody (clone number: XMG1.2, product number 505807, Biolegend).

[0152] Flow cytometry showed that CD11c + CD80 + CD86 + The proportion of mature dendritic cells reached (13.4±2.0)%, which was significantly higher than that of the free OVA group (3.81±1.39)% and the normal saline group (3.62±0.72)%. Figure 24 This indicates that OVA PCD promotes DC cell maturation and the expression of co-stimulatory molecules by enhancing the ability of antigen cross-presentation. + CD8 + The proportion of T cells in the total CD8 positive T cells was (25.7±2.0)% and OVA tetramer + CD8 + The proportion of T cells in the total CD8 positive T cells was (19.7±2.7)%, which was significantly higher than that in the normal saline group ((4.7±1.2)%, (0.73±0.05)%) and the free OVA group ((5.1±1.3)%, (0.80±0.23)%) (such as Figure 24 B, C, and D of the Figure 3), indicating that it can induce potent antigen-specific cytotoxic T lymphocyte (CTL) responses, thereby exerting anti-tumor effects.

[0153] Example 9 IFN-γ ELISPOT assay analysis

[0154] This example further validates the T cell function induced by OVA PCD by detecting cytokine secretion capacity. The specific implementation is as follows (refer to the kit instructions, the ELISPOT kit used is purchased from Diaclone, catalog number 862.031.001S):

[0155] Plate pretreatment: ELISPOT plates were treated with 35% ethanol for 5 min and washed five times with sterile water;

[0156] Antibody coating: Add anti-mouse IFN-γ capture antibody (5 μg / mL) and coat at 4°C overnight (12 hours). Wash with sterile water to remove unbound antibody.

[0157] Cell stimulation: The spleen cells prepared in Example 8 (2.5×10 5 Cells (cells / well) were seeded onto pre-coated plates, and 8 μg / mL SIINFEKL peptide (provided by Shanghai Bioengineering Co., Ltd.) was added. The cells were incubated at 37°C, 5% CO2 for 20-24 hours to serve as the experimental group. A non-stimulated control group and a PMA / ConA positive control group (PMA at a concentration of 1 ng / mL and ConA at a concentration of 10 μg / mL) were also established.

[0158] Spot detection: After removing the cells, add biotinylated detection antibody (1 μg / mL, room temperature for 2 hours) and streptavidin-alkaline phosphatase (1:1000 dilution, room temperature for 1 hour) in sequence.

[0159] Color development: Add BCIP / NBT substrate for 10-15 minutes, and terminate the reaction with deionized water;

[0160] Plate reading: spot-forming cells (SFC) were counted using an IRIS ELISpot / Fluorospot plate reader (Mabtech);

[0161] like Figure 25 、 Figure 26 As shown, the OVA PCD group had an IFN-γ SFC of (107 ± 11) per 250,000 splenocytes, which was significantly higher than the free OVA group (6 ± 1) and the saline group (11 ± 4) (P < 0.0001). This indicates that the OVA PCD of the present invention can not only amplify the number of antigen-specific CTLs, but also significantly enhance their functional IFN-γ secretion capacity.

[0162] Example 10 Evaluation of the functional activity of effector T cells after immunization

[0163] To evaluate the functional activity of effector T cells after immunization, the splenocytes (E) of each group of mice obtained in Example 8 were co-cultured with B16-OVA-GFP tumor cells (T) in vitro at a gradient E / T ratio (6.25:1 to 100:1) for 24 hours, and then their effector T cell cytotoxicity was evaluated.

[0164] Specific operations include:

[0165] Take 50 μl containing 1×10 4 The target B16-OVA-GFP cells (T) of the cell were added to the wells of a 96-well plate, and then different numbers of effector cells (E) were added to the wells at 50 μl / well (E / T = 100:1, 50:1, 25:1, 12.5:1, 6.25:1). The cells were gently pipetted to mix, avoiding bubbles, so that the target cells and effector cells were fully in contact. At the same time, only B16-OVA-GFP or effector spleen cells were set as controls, and 4 replicates were set for each ratio. The 96-well plate was then placed in an incubator (37°C, 5% CO2) and incubated for 24 hours. The results of the cytotoxicity test are shown in Figure 2. Figure 27 As shown in the figure, at the highest E / T ratio (100:1), the tumor cell killing efficiency of effector T cells in the OVAPCD group reached (66.8±3.3)%, which was significantly higher than that of the free OVA group (23.6±2.1)% and the normal saline group (37.4±3.5)%, and at the lowest ratio (6.25:1), it could still achieve 30% cytotoxic killing.

[0166] In summary, through the combined detection of ELISPOT experiment and flow cytometry, the present invention systematically reveals the significant advantages of the OVAPCD platform in activating immune cells: it can not only significantly expand the antigen-specific IFN-γ secreting T cell clones, but also enhance the specific CD8 + These results provide molecular and cellular evidence for the mechanism of OVAPCD in anti-tumor immunotherapy and lay the foundation for subsequent clinical translational research.

[0167] The above embodiments are only preferred implementation modes of the present invention and are only used to explain the present invention rather than to limit the present invention. Any changes, substitutions, modifications, etc. made by those skilled in the art without departing from the spirit of the present invention should fall within the scope of protection of the present invention.

Claims

1. A novel protein micro-nanoparticle system based on supramolecular self-assembly, characterized by: The invention comprises the following components: a fatty acid compound 1, a long-chain alkyl thiol compound 2 and a protein ligand 3; the molar ratio of the protein ligand 3 to the fatty acid compound 1 is 1:20-600; the molar ratio of the protein ligand 3 to the long-chain alkyl thiol compound 2 is 1:3-400; the protein ligand 3 is at least one of a protein, a polypeptide, a protein derivative or a polypeptide derivative.

2. The novel protein micro-nanoparticle system based on supramolecular self-assembly according to claim 1, characterized in that: The fatty acid compound 1 is a fatty acid or a corresponding fatty acid salt compound; the chemical formula of the fatty acid is CH3(CH2) n COOH, n is a natural number ranging from 6 to 21; the long-chain alkyl mercaptan compound 2 is a long-chain alkyl mercaptan or a corresponding long-chain alkyl mercaptan salt compound; the chemical formula of the long-chain alkyl mercaptan is CH3(CH2) m SH, m is a natural number, ranging from 5 to 20.

3. The novel protein micro-nanoparticle system based on supramolecular self-assembly according to claim 2, characterized in that: The value range of n is 10-16; the value range of m is 9-16.

4. The novel protein micro-nanoparticle system based on supramolecular self-assembly according to claim 3, characterized in that: The fatty acid compound 1 is sodium myristate; the long-chain alkyl mercaptan compound 2 is sodium dodecyl mercaptan.

5. The novel protein micro-nanoparticle system based on supramolecular self-assembly according to any one of claims 1 to 4, characterized in that: The protein ligand 3 is at least one of albumin, tumor antigen, viral antigen, bacterial antigen or mycoplasma antigen.

6. The novel protein micro-nanoparticle system based on supramolecular self-assembly according to claim 5, characterized in that: The protein ligand 3 is at least one of chicken ovalbumin, serum albumin, HIV gp120 / gp41, Mycobacterium tuberculosis antigen, Staphylococcal enterotoxin B, streptolysin O, influenza virus HA hemagglutinin, new coronavirus surface antigen, human papillomavirus HPV antigen and EBV antigen.

7. The method for preparing a novel protein micro-nanoparticle system based on supramolecular self-assembly according to any one of claims 1 to 6, characterized in that: The method comprises the following steps: preparing a fatty acid compound 1, a protein ligand 3, and a long-chain alkyl thiol compound 2 into solutions respectively using solvents; then mixing the fatty acid compound 1 solution and the protein ligand 3 solution to obtain a primary complex; mixing the primary complex with the long-chain alkyl thiol compound 2 solution to obtain a complex; and dialyzing the complex using a buffer solution with a pH of 3.0-6.5 to obtain the novel protein micro-nanoparticle system based on supramolecular self-assembly.

8. The preparation method according to claim 7, characterized in that The solvent is one of water, anhydrous ethanol, dimethyl sulfoxide, phosphate buffer or normal saline; The method for mixing the fatty acid compound 1 solution and the protein ligand 3 solution comprises the following steps: adding the fatty acid compound 1 solution to the protein ligand 3 solution, and stirring for 10-30 minutes at a stirring rate of 30-100 rpm; The method of mixing the primary complex with the long-chain alkyl thiol compound 2 solution comprises the following steps: adding the long-chain alkyl thiol compound 2 solution to the primary complex, and stirring for 30-60 minutes at a stirring rate of 100-800 rpm; The dialysis treatment comprises the following steps: placing the complex in a dialysis bag, dialysing in a buffer solution with a pH of 3.0-6.5 for 4-12 hours, and taking the retained liquid in the dialysis bag; the molecular weight cut-off of the dialysis bag is 30-400 kDa.

9. The preparation method according to any one of claims 7-8, characterized in that After the complex is dialyzed, the following steps are further included: centrifugal treatment and freeze drying to obtain the novel protein micro-nanoparticle system based on supramolecular self-assembly; the centrifugal treatment speed is 12000-15000 rpm, and the centrifugal treatment time is 20-30 minutes; the freeze drying temperature is -20°C to -80°C, and the freeze drying time is 36-72 hours.

10. Use of the novel protein micro-nanoparticle system based on supramolecular self-assembly according to any one of claims 1 to 6 in the preparation of vaccines, characterized in that: The vaccine is a tumor vaccine or an anti-infection vaccine.

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