Universal cell membrane nanoparticle coating method based on solvent balance and thiol anchoring and application thereof

Abstract

The application belongs to the technical field of nanobiomaterials, and particularly relates to a preparation method of cell membrane-coated nanoparticles based on solvent balance and thiol anchoring. The method is characterized in that thiol-containing lipids are added to the membrane system to form covalent anchoring bonds with the substrate surface to enhance the membrane-substrate binding force, and a small amount of organic solvent is added to improve the lipid fluidity, promote the spontaneous fusion and uniform spreading of the membrane layer on the particle surface. After centrifugation and washing, the cell membrane nanoparticles with complete surface coating can be obtained. The method does not require high-energy ultrasonic or extrusion treatment, can realize stable coating while keeping the integrity of the membrane protein and lipid composition, and significantly improves the adhesion stability of the membrane layer in a high-salt and shearing environment. The method is suitable for various nano substrates and different morphologies, and the obtained particles show higher antibody titer and excellent safety in an anti-alpha-hemolysin vaccine model. The application provides a mild, efficient and widely applicable strategy for constructing biomimetic nanomaterials.

Description

Technical Field

[0001] This invention relates to the field of nanobiomaterials technology, and in particular to a method for preparing biofunctionalized nanomaterials by utilizing solvent incubation equilibrium and thiol-anchored cell membranes and nanoparticles to achieve stable coating, belonging to the field of biofunctionalized nanomaterials technology. Background Technology

[0002] Cell membrane coating technology is an emerging and versatile strategy for functionalizing the surface of nanoparticles. This technology utilizes the self-assembly properties of the cell membrane lipid bilayer to transfer natural functional components (including proteins, lipids, and carbohydrates) from the source cell membrane to the surface of inorganic or organic nanoparticles, thereby endowing the particles with physiological functions such as biological recognition, immune escape, or antitoxins. Existing membrane coating methods typically involve using ultrasound or extrusion to induce the rupture of cell membrane vesicles and their fusion with nanoparticles. Similar principles are also widely used in the preparation of supported lipid bilayers (SLBs). However, unlike artificially synthesized lipid systems with simple composition and controllable properties, natural cell membranes are complex systems composed of various lipids and membrane proteins, exhibiting significant heterogeneity and phase separation characteristics. These characteristics present unique challenges in forming stable, uniform membrane coatings.

[0003] In addition, membrane proteins (such as CD9, Caveolin-1, etc.) Due to their different curvature stability properties, membranes may introduce additional instabilities when they need to adapt to the surfaces of nanoparticles with high positive curvature. Desorption of the membrane has two consequences: first, the loss of functional molecules from the membrane will lead to the loss of nanoparticle function; second, the steric hindrance provided by the cell membrane coating itself is crucial for maintaining the colloidal stability of nanoparticles under high ionic strength conditions. Once the membrane is desorbed, the particles are prone to aggregation, exceeding the physiologically usable size range, thus losing their expected biological function.

[0004] Summary of the Invention

[0005] To overcome the problems of poor stability, easy membrane detachment, and limited substrate adaptability of existing cell membrane-coated nanoparticles in physiological environments, this invention provides a stable cell membrane coating method based on solvent equilibrium and thiol anchoring. This method achieves stable coating of nanomaterials with different morphologies and material types by establishing a covalent anchoring structure at the membrane-substrate interface and introducing solvent-assisted membrane component rearrangement.

[0006] To address the technical problem of this invention, the proposed technical solution is as follows: a method for preparing cell membrane-coated nanoparticles based on solvent equilibrium and thiol anchoring, comprising the following steps:

[0007] Preparation of cell membrane vesicles: Red blood cell membranes are isolated from red blood cells by hypotonic lysis and formed into membrane vesicles;

[0008] Solvent equilibrium-induced membrane fusion: Gold or silica nanoparticles were dispersed in an organic solvent with a volume ratio of 2%–50%, and erythrocyte membrane vesicles were added to the gold or silica nanoparticle solution;

[0009] Thiolized lipid incorporation: Thiolized lipid molecules 1,2-dipalmitoyl-sn-glycerol-3-phosphate thioethanol, which can form covalent bonds with the nanoparticle substrate, are added to the above solution of gold or silica nanoparticles containing erythrocyte membrane vesicles, so that the thiolated lipid molecules are incorporated into the membrane lipid bilayer; under mild conditions, the erythrocyte membrane vesicles spontaneously fuse on the surface of the nanoparticles to form a continuous membrane coating.

[0010] Washing and purification: Unbound membrane vesicles were removed by centrifugation and washing to obtain nanoparticles with a uniform surface coating of cell membrane.

[0011] The thiolized lipids form gold-sulfur covalent anchoring bonds with the surface of gold nanoparticles; the disulfur covalent bonds formed between the thiolized modified silica surface and the thiol ligands enhance the membrane-substrate interaction; and the organic solvent is used to improve the fluidity of the membrane lipids and promote the uniform fusion of the membrane layers.

[0012] Preferably, it includes the following steps:

[0013] Preparation of cell membrane vesicles: Red blood cell membranes are isolated from red blood cells by hypotonic lysis and formed into membrane vesicles;

[0014] Solvent equilibrium-induced membrane fusion: Gold nanoparticles were dispersed in 16% acetonitrile by volume, and erythrocyte membrane vesicles were added to the gold nanoparticle solution;

[0015] Thiolized lipid incorporation: 1,2-dipalmitoyl-sn-glycerol-3-phosphate thioethanol, which can form covalent bonds with the nanoparticle substrate, was added to the above solution of gold nanoparticles containing erythrocyte membrane vesicles to incorporate thiolated lipid molecules into the membrane lipid bilayer; the solution was then incubated at 37°C for 2 h to allow the erythrocyte membrane vesicles to spontaneously fuse on the surface of the nanoparticles to form a continuous membrane coating.

[0016] Washing and purification: Unbound membrane vesicles were removed by centrifugation and washing to obtain nanoparticles with a uniform surface coating of cell membrane.

[0017] Preferably, the organic solvent is selected from acetonitrile (ACN), ethanol (EtOH), dimethyl sulfoxide (DMSO), or a mixture thereof in any proportion.

[0018] Preferably, the substrate material for the nanoparticles is gold or silicon oxide nanoparticles of different sizes.

[0019] Preferably, the morphology of the nanoparticles is spherical, triangular nanosheet, cubic or other polyhedral structures with positive curvature, as well as other complex morphological nanoparticles with both positive and negative curvature on their surfaces.

[0020] Preferably, the incubation temperature is 0-100°C and the incubation time is 5 minutes to 24 hours.

[0021] Preferably, the proportion of thiolated lipids incorporated is 0.1%–10% of the total mass of the erythrocyte membrane.

[0022] The cell membrane-coated nanoparticles prepared by the method retain the protein, lipid, and carbohydrate components of the original cell membrane, and exhibit enzyme activity comparable to that of the erythrocyte-derived membrane in the acetylcholinesterase (AChE) activity test.

[0023] The cell membrane-coated nanoparticles described herein are used to construct vaccines, drug delivery systems, or biomimetic protective materials. The surface of the nanoparticles is covered with a continuous membrane layer formed by the cell membrane, and the membrane layer is anchored to the surface of the nanoparticles through thiol-metal covalent bonds. The membrane layer maintains the protein and lipid composition of the source cell membrane and remains structurally stable under physiological salt concentration and fluid shear conditions, without aggregation or membrane detachment.

[0024] The cell membrane-coated nanoparticles are used in the preparation of anti-α-hemolysin immune vaccines, targeted drug delivery systems, or biotoxin neutralization nanoformulations.

[0025] Beneficial effects

[0026] The core technical points of the method of this invention include the following two aspects:

[0027] 1. Thioylated lipid incorporation and covalent anchoring to the substrate

[0028] Incorporating thiol-containing (-SH) lipid molecules into the cell membrane system enables them to form stable gold-sulfur covalent bonds with metal surfaces (such as gold) at the membrane-nanoparticle interface; and to form stable disulfur covalent bonds with thiol-modified silica surfaces. This anchoring effect significantly enhances the interaction force between the membrane layer and the substrate, and improves the adhesion stability of the membrane layer under external disturbance conditions.

[0029] 2. Solvent balance promotes membrane fusion and uniform coating.

[0030] Introducing a small amount of organic solvent (such as acetonitrile, ethanol, or dimethylformamide) into the membrane-particle composite system increases the fluidity of membrane lipids, promotes the redistribution of lipid molecules and their fusion with the membrane layer, thereby forming a complete, continuous, and uniform cell membrane coating layer on the surface of nanoparticles. This process does not require drastic physical disturbances (such as ultrasound or extrusion), thus avoiding the inactivation of membrane proteins or the destruction of membrane components.

[0031] This invention uses gold nanoparticles (AuNPs) and erythrocyte membranes (RBCMs) as model systems for validation. Acetylcholinesterase (AChE) activity assays, dynamic light scattering (DLS), and lipidomics analysis demonstrate that this method can achieve complete membrane reconstruction and coating while maintaining the chemical composition and biological function of the erythrocyte membrane. Transmission electron microscopy (TEM) results further confirm that this method has good adaptability to gold nanoparticles of different morphologies (including spherical, triangular nanosheets, and cubic shapes) and thiol-modified silica particles, exhibiting excellent substrate versatility.

[0032] Theoretical modeling and energy stability analysis show that covalent anchoring significantly increases the energy barrier required for membrane desorption, thereby enhancing overall coating stability. This conclusion is verified by comparing results from centrifugation shear perturbation experiments and α-hemolysin (αHL) insertion experiments: AuNP@RBCM prepared using the conventional ultrasonic method exhibits severe aggregation under physiological saline conditions, while samples prepared using the method of this invention maintain good dispersibility under the same conditions.

[0033] At the application level, when this stable coating system is used in an anti-α-hemolysin vaccine model, AuNP@RBCM can more efficiently deliver α-HL antigens to antigen-presenting cells such as macrophages, thereby inducing a stronger specific antibody response. Mouse experiments showed that the vaccine group prepared by this method had a higher anti-α-HL antibody titer than the conventionally prepared group and exhibited stronger protective effects in a subcutaneous toxin challenge model. Meanwhile, safety evaluations showed that particles prepared by the conventional ultrasonic method experienced antigen shedding during the immunization phase due to membrane instability, leading to local skin damage, while the particles prepared by the method of this invention do not pose such safety risks.

[0034] This invention relates to a method for preparing cell membrane-coated nanoparticles based on solvent equilibrium and thiol-anchored structure. The method includes the following steps: first, cell membrane vesicles are obtained through hypotonic lysis; second, thiol-containing lipid molecules are incorporated into the membrane system to form covalent anchoring bonds with the metal surface at the membrane-nanoparticle interface, thereby enhancing the adhesion between the membrane layer and the substrate; subsequently, a small amount of organic solvent is added to the mixture of membrane vesicles and nanoparticles to improve lipid fluidity, promoting spontaneous fusion and uniform spreading of the membrane layer on the particle surface; finally, nanoparticles with intact cell membranes are obtained by centrifugation and washing. This method eliminates the need for high-energy sonication or extrusion treatment, achieving stable coating while maintaining the integrity of membrane proteins and lipids, and significantly improving the adhesion stability of the membrane layer in high ionic strength and fluid shear environments. Experimental results show that the method of this invention is applicable to metal or oxide nanosubstrates and different morphologies (spherical, triangular, cubic, etc.), and the resulting particles exhibit higher antibody titers and better safety in an anti-α-hemolysin vaccine model. This invention provides a universal, mild, and efficient cell membrane coating strategy, offering a new approach for the construction of biomimetic nanomedicines and vaccine carriers.

[0035] In summary, this invention provides a cell membrane coating method that combines universality and high stability. This method achieves stable coating of various nanoparticle materials by regulating the fluidity of membrane components through solvent balance and enhancing membrane-substrate binding through thiol anchoring. It provides a universal and scalable preparation route for next-generation biomimetic nanotherapy systems, possessing significant scientific and application value. Attached Figure Description

[0036] The present invention will be further described below with reference to the accompanying drawings.

[0037] Figure 1 (A) AuNP@RBCM prepared by sonication stabilizes the cell membrane on gold nanoparticles through weak non-covalent substrate interactions. In the equilibrium incubation method, PSH doping in the erythrocyte membrane enables it to form covalent Au-S bonds with the gold nanoparticle substrate, resulting in strong substrate interactions. (B) The addition of organic solvents promotes the fusion of cell membranes onto the gold nanoparticle substrate, forming a uniform and complete invisible layer.

[0038] Figure 2 Quantitative analysis of acetylcholinesterase (AChE) activity on erythrocyte membranes after treatment with different volume fractions of dimethyl sulfoxide (DMSO), ethanol (EtOH), or acetonitrile (ACN).

[0039] Figure 3 (A) UV-Vis absorption spectra of AuNP@RBCM solutions prepared using different organic solvents via a balanced incubation method. Before absorption measurement, the nanoparticles were transferred to 1×PBS.

[0040] (B) UV-Vis absorption spectra of AuNP@RBCM solutions prepared using PSH-doped or undoped erythrocyte membranes. The nanoparticles were transferred to 1×PBS before absorption measurement.

[0041] Figure 4 The effect of different amounts of thiolipic acid incorporated on coating stability.

[0042] Figure 5 (A) Dynamic light scattering (DLS) measurement of the hydrodynamic diameter and surface zeta potential of gold nanoparticles before and after erythrocyte membrane coating. (B) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of AuNP@RBCM or erythrocyte vesicle membrane proteins.

[0043] Figure 6 (A) Distribution of major lipid classes in erythrocyte vesicles and AuNP@RBCM. (B) Relative abundance of specific phospholipid classes in erythrocyte vesicles and AuNP@RBCM.

[0044] Figure 7 Volcano plot of differential lipid types between erythrocyte vesicles and AuNP@RBCM.

[0045] Figure 8 Representative transmission electron microscopy (TEM) images of spherical gold nanoparticles with diameters of 15, 40, and 75 nm in uncoated (A), sonicated (B), and balanced-coated (C) states. Samples in B and C were negatively stained with 1% ammonium molybdate to enhance film contrast. Scale bar: 100 nm. Scale bar in image insets: 5 nm.

[0046] Figure 9 Transmission electron microscopy (TEM) images of anisotropic gold nanoparticles (including nanorods, nanotriangles, and nanocubes) with different geometries in uncoated (A), ultrasonically coated (B), and balanced coated (C) states. Samples in B and C were negatively stained with 1% ammonium molybdate to enhance film contrast. Scale bar: 100 nm. Scale bar in image insets: 5 nm.

[0047] Figure 10 A lipid membrane (B) was coated onto the surface of thiol-modified 80 nm silica particles (A). The samples were negatively stained with 1% ammonium molybdate to enhance the contrast of the membrane. Scale bar: 100 nm.

[0048] Figure 11(A) UV-Vis spectra of AuNP@RBCM prepared by solvent equilibration after centrifugation at 0, 1.1, and 2.2 Pa, respectively. (B) Transmission electron microscopy images show that the solvent-equilibrated AuNP@RBCM remains monodisperse and the membrane shell is visible (scale bar 200 nm, inset showing a single particle, scale bar 25 nm). (C) UV-Vis spectra of AuNP@RBCM prepared by solvent equilibration in phosphate-buffered saline (PBS) after three centrifugation shearings. (D) Dynamic light scattering (DLS) analysis shows that the solvent-equilibrated AuNP@RBCM has a stable hydrodynamic diameter (average 55 nm) and polydispersity index (PDI, average 0.2) in phosphate-buffered saline (PBS) for 15 days.

[0049] Figure 12 (A) UV-Vis spectra of AuNP@RBCM prepared by sonication in PBS after centrifugation at 0, 1.1, and 2.2 Pa shear forces. (B) Transmission electron microscopy images show extensive aggregation of the sonicated particles and a lack of discernible membrane coverage (scale bar 200 nm, inset showing a single particle, scale bar 25 nm). (C) UV-Vis spectra of AuNP@RBCM prepared by solvent equilibrium (red line) and sonication (black line) in PBS after incubation with α-HL at progressively increasing concentrations show that the equilibrium-prepared AuNP@RBCM maintained dispersion and had stable plasmon peaks; while the sonicated control group showed a weakened plasmon peak at approximately 530 nm and broad absorption at 600–700 nm, indicating aggregation. (DF) Transmission electron microscopy images of AuNP@RBCM prepared by solvent equilibrium (D) and sonication (F) after α-HL treatment. The solvent-equilibrium-prepared AuNP@RBCM shows an intact and continuous erythrocyte membrane shell. The ultrasound control group showed significant membrane shedding (scale bar 50 nm).

[0050] Figure 13 Schematic diagram of α-HL-AuNP@RBCM. α-HL is a protein toxin secreted by Staphylococcus aureus, which can assemble into transmembrane channels on the erythrocyte membrane layer of AuNP@RBCM.

[0051] Figure 14 Confocal fluorescence images of RAW 264.7 macrophages incubated with α-HL-AuNPs@RBCM. α-HL was labeled with Alexa Fluor 488. Intracellular fluorescence signals were evaluated to compare the uptake efficiency of nanoparticles prepared by two methods (solvent equilibration incubation and sonication), scale bar 10 μm.

[0052] Figure 15 Quantitative analysis of the average Alexa Fluor 488 intensity per cell. Statistical significance was determined by t-test (0.0069, p<0.01).

[0053] Figure 16 (A) Mice received the initial dose on day 0, followed by two booster doses on days 7 and 14. 100 μL of peripheral blood was collected before each booster dose. On day 21, 5 μg of α-HL was injected subcutaneously. Lesion size was monitored for 4 days. (B, C) Quantitative analysis of anti-α-HL antibody titers in peripheral blood. Blood was collected from mice treated with PBS or immunized with α-HL-AuNP@RBCM at specified time points (n=6). Data from day 21 are presented in the bar chart in Figure C.

[0054] Figure 17 Representative images and quantitative results of skin lesions in mice 2 days after subcutaneous injection of α-HL. The size of the lesions was quantified using images (scale bar: 1 cm).

[0055] Figure 18 H&E staining and TUNEL staining of mouse skin in each group. Scale bar: 1.25 μm.

[0056] Figure 19 (A) shows a representative image of the skin of healthy mice on day 13 after subcutaneous injection of PBS, ultrasound-prepared α-HL-AuNP@RBCM, and solvent-equilibrated α-HL-AuNP@RBCM. The quantitative results of lesion size are plotted in Figure B. (C) shows the body weight of the three groups of immunized mice on day 21.

[0057] Figure 20 : Schematic diagram of the present invention Specific implementation methods

[0058] The technical solution of the present invention will be further described below with reference to embodiments, but the present invention is not limited to the following embodiments. Any equivalent substitutions or modifications made based on the concept of the present invention should be considered to fall within the protection scope of the present invention.

[0059] Example 1

[0060] 1. Required materials and reagents

[0061] Nanoparticle substrates include gold nanospheres (40 nm in diameter), gold triangular nanosheets (approximately 150 nm in side length), and gold cubes (approximately 80 nm in side length). Other metal or oxide nanoparticles (such as silver, iron oxide, and silicon dioxide) can also be used as substrates.

[0062] Cell membrane source: Freshly isolated red blood cells (RBCs) or other cell sources (such as white blood cells, platelets, tumor cells, stem cells, etc.). Cell membrane vesicles are obtained after hypotonic lysis and cryopreserved for later use.

[0063] Thiolized lipids: Phospholipid molecules containing thiol groups, such as 1,2-dipalmitoyl-sn-glycerol-3-phosphothioethanol (PSH), are used to form gold-sulfur covalent bonds between membranes and metal surfaces.

[0064] Organic solvents: acetonitrile (ACN), ethanol (EtOH), dimethylformamide (DMF) or mixtures thereof, used to promote the fluidity and uniform fusion of membrane lipids.

[0065] Buffer solution: Phosphate buffer (PBS, 1×, pH 7.4) for rapid assessment of coating stability.

[0066] 2. Implementation Steps

[0067] 2.1. Preparation of cell membrane vesicles

[0068] Whole blood was collected from 5-8 week old female ICR mice. After centrifugation at 800g for 5 min at 4°C, the erythrocyte sedimentation rate (ESR) layer containing leukocytes and platelets was removed and discarded, and the lower layer of red blood cells was collected. The red blood cells were washed three times with 1×PBS. To remove intracellular substances, a hypotonic treatment was performed: the red blood cells were immersed in 0.25×PBS and placed in an ice bath for 20 min, followed by high-speed centrifugation (30,000×g, 5 min) to collect red blood cell membrane vesicles. The red blood cell membrane vesicles were washed twice with 1×PBS and stored at -80°C for later use.

[0069] 2.2. Preparation of 40 nm sodium citrate-stabilized gold nanoparticles

[0070] 100 mL of a 0.1 mg / mL tetrachloroauric acid (HAuCl4) aqueous solution was placed in a three-necked flask and heated to boiling. Then, 3 mL of a 10 mg / mL sodium citrate aqueous solution was quickly added. The mixture was kept boiling and stirred continuously for 1 hour to reduce gold ions into gold nanospheres of approximately 15 nm. After cooling to room temperature, the resulting solution was the 15 nm sodium citrate-stabilized gold nanoparticle seed crystal solution. 10 mL of the above 15 nm gold nanoparticle seed crystal solution was diluted to 50 mL of deionized water and placed in a 250 mL flask. The solution was heated under reflux in an oil bath and stirred vigorously for 30 minutes to stabilize the system. Subsequently, 0.25 mL of a 10 mg / mL sodium citrate solution and 0.3 mL of a 10 mg / mL tetrachloroauric acid aqueous solution were quickly added to the boiling solution. The mixture was kept boiling for 30 minutes to allow gold to continue to be reduced and deposited on the seed crystal surface, promoting particle growth. After completion, repeat the above addition and heating steps twice to obtain sodium citrate stabilized gold nanoparticles (AuNPs) with an average particle size of approximately 40 nm, which can be stored at room temperature for later use.

[0071] 2.3. Nanoparticle Pretreatment

[0072] 1 mL of 40 nm gold nanoparticles were separated from the original stored excess sodium citrate ligand aqueous solution by centrifugation at 6000 rpm for 10 min. After removing the supernatant, the gold nanoparticle precipitate was redispersed in a mixed solution of water and acetonitrile (water / ACN: 5 / 1) with a total volume of 1.12 mL and placed in a clean reaction flask for later use.

[0073] 2.4. Addition of membrane source and anchoring molecules

[0074] Under stirring conditions, 2 μL of erythrocyte membrane solution (concentration 2 mg / mL) and 0.5 μL of thiophospholipid (PSH) solution (concentration 2 mg / mL) were added sequentially to the above solution. After PSH is incorporated into the membrane system, its thiol groups can form stable Au-S covalent anchoring bonds with the metal surface in subsequent steps.

[0075] 2.5 Solvent-induced coating equilibrium reaction

[0076] The above mixture was incubated at 37°C for 2 hours to allow the cell membrane vesicles to fully fuse and rearrange on the particle surface in 16% acetonitrile solvent, thereby forming a continuous and uniform cell membrane coating layer on the surface of gold nanoparticles.

[0077] 2.6 Purification and Collection

[0078] After incubation, the solution was allowed to cool to room temperature, then centrifuged at 6000 rpm for 10 min to remove excess cell membrane components and acetonitrile solvent. The supernatant was discarded, and the product was repeatedly dispersed with 200 μL PBS to obtain the final AuNP@RBCM. The obtained sample was stored at 4°C for subsequent characterization and applications.

[0079] Example 2

[0080] Screening and determination of the effects of organic solvents on the activity of erythrocyte membrane proteins:

[0081] Considering that the introduction of organic solvents may disrupt the hydrophobic interactions that maintain the structure and function of membrane proteins, thereby affecting the stability and biological function of the cell membrane coating system, this embodiment first screened the biocompatibility of different organic solvents. The enzyme activity of acetylcholinesterase (AChE) in erythrocyte membranes was used as the evaluation index. AChE is a typical membrane-bound enzyme that catalyzes the hydrolysis of the neurotransmitter acetylcholine, and its activity changes can reflect the conformational retention of membrane proteins under solvent conditions. In the experiment, purified erythrocyte membrane samples were mixed and incubated with three organic solvents—dimethyl sulfoxide (DMSO), ethanol (EtOH), and acetonitrile (ACN)—at different volume fractions, and their AChE activity was measured. The results showed that the AChE activity of all three groups of samples decreased with increasing organic solvent volume fraction. The ethanol system showed the best biocompatibility, with AChE activity remaining relatively stable at a volume fraction not exceeding 32%. However, the AChE activity decreased more significantly in the DMSO and acetonitrile system, with enzyme activity decreasing by approximately 20% at a solvent volume fraction of 16%. Figure 2 Based on these results, subsequent erythrocyte membrane coating experiments used three organic solvents with a volume fraction of 16% to balance membrane lipid fluidity and membrane protein structural stability, thereby achieving a better coating effect.

[0082] Verification of the integrity and stability of gold nanoparticles coated with erythrocyte membrane solvent equilibrium method:

[0083] This embodiment aims to verify whether the solvent equilibrium method can form a complete and stable erythrocyte membrane coating layer on the surface of gold nanoparticles. Gold nanospheres with a diameter of approximately 40 nm were used as a model substrate, exhibiting a typical surface plasmon resonance absorption peak at 530 nm, indicating extreme sensitivity to particle aggregation. In the experiment, acetonitrile (ACN), dimethyl sulfoxide (DMSO), and ethanol (EtOH) were used as solvents to prepare AuNP@RBCM samples via the solvent equilibrium method. After coating, the purified samples were transferred to 1×PBS solution to evaluate their stability under physiological saline conditions. The results showed that the AuNP@RBCM sample prepared using the acetonitrile system maintained a single and strong absorption peak at 530 nm in PBS, indicating good particle dispersion and a complete membrane layer; while the samples prepared using the DMSO and ethanol systems showed a significant decrease in absorption at 530 nm and a new absorption peak at approximately 630 nm, indicating particle aggregation due to incomplete membrane coating. Figure 3 A). This result indicates that the fluidity of membrane lipids in the solvent system is crucial for the formation of complete membrane coating. In parallel experiments, to further investigate the role of thiolated phospholipids in membrane-substrate binding, erythrocyte membranes with and without 1,2-dipalmitoyl-sn-glycerol-3-phosphate thioethanol (PSH) were used to coat 40 nm gold nanospheres in an acetonitrile system. The purified samples were also transferred to 1×PBS to test their stability. The results showed that the 530 nm characteristic peak of the undoped sample completely disappeared, indicating severe particle aggregation; while the absorption peak of the PSH-doped sample remained sharp and unchanged in intensity, indicating that the system was still monodisperse. Figure 3 B).

[0084] Besides PSH, we used other thiolipic acids (e.g., 11-mercaptoundecanoic acid, MUA; 16-mercaptohexadecanoic acid, MHA) and dinaphthalenethiol (2-NT) molecules for anchoring, but the results showed ( Figure 4 ), 3.66 mM PSH is most effective in anchoring cell membranes and granules.

[0085] The results in summary indicate that the covalent anchoring synergy between PSH and acetonitrile in 16% acetonitrile solvent is a key condition for obtaining intact and stable cell membrane-coated gold nanoparticles.

[0086] Example 3

[0087] Verification of the structure and composition of gold nanoparticles coated on erythrocyte membranes:

[0088] The particle size of gold nanoparticles coated with erythrocyte membranes (AuNP@RBCM) was determined using dynamic light scattering (DLS). The results showed that the average particle size of AuNP@RBCM increased by approximately 10 nm compared to uncoated gold nanoparticles. This increase is comparable to the thickness of the lipid bilayer, indicating that a complete cell membrane coating layer was formed on the nanoparticle surface. Further analysis of the particle surface electrical properties revealed a zeta potential of approximately −30 mV, reflecting the successful transfer of the negative charge characteristic of the erythrocyte membrane to the particle surface. Figure 5 A). To confirm the retention of membrane protein components, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed on AuNP@RBCM samples and primitive erythrocyte membranes. The results showed that the protein band distributions were almost identical in both samples, indicating that the types and relative proportions of membrane proteins were effectively preserved. Figure 5 B). The combined results of DLS and SDS-PAGE confirm that the erythrocyte membrane was successfully transferred to the surface of gold nanoparticles during the coating process, forming a biomimetic membrane coating system with complete structure and faithful composition.

[0089] Example 4

[0090] Lipomics analysis of AuNP@RBCM prepared by solvent equilibration method

[0091] The lipid composition of AuNP@RBCM prepared by solvent equilibrium method was analyzed using liquid chromatography-tandem mass spectrometry (LC-MS / MS) lipidomics and compared with that of primitive erythrocyte membrane. The results showed that the lipid profile of AuNP@RBCM was highly consistent with that of RBCM, indicating that membrane lipids were effectively transferred and retained during the coating process. The lipids in both samples were mainly composed of glycerophospholipids (approximately 75%–80% of total lipids), followed by fatty acyls (approximately 15%–20%), with sphingolipids accounting for approximately 5%–10%, while glycerols were almost undetectable. Compared to the primitive erythrocyte membrane, the sphingolipid content in AuNP@RBCM was slightly increased, while the relative ratio of glycerophospholipids to fatty acyls remained basically unchanged. Figure 6 A). Further analysis of specific phospholipid subclasses revealed that phosphatidylcholine (PC) was the most abundant component in both sample groups, accounting for over 80%, while phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS), and lysophosphatidylcholine (LPC) had relatively lower proportions; phosphatidic acid (PA) and ether-bonded PE were almost undetectable. Figure 6 B). The above results indicate that the erythrocyte membrane not only maintains the stability of the overall lipid composition during the coating process but also preserves the proportional distribution of various phospholipids, thus proving that this method can achieve complete transfer of membrane lipids and preserve structural fidelity. However, a small amount of lipids with poor mobility is still lost. Figure 7 )

[0092] Example 5

[0093] Preparation of gold nanoparticles coated with erythrocyte membrane solvent equilibrium method and comparative sample ( Figure 1 ):

[0094] Washed and purified gold nanoparticles (which can be spherical, triangular, or cubic structures, etc.) were redispersed in a mixture of 1.12 mL of water and acetonitrile (1:0.2, v / v). Then, 2 μL of erythrocyte membrane solution (2 mg / mL) and 0.5 μL of a thioglycolic acid (PSH) solution (2 mg / mL) were added sequentially under stirring. This thioglycolic lipid can form stable Au–S covalent anchoring bonds with the gold surface in subsequent steps, thereby enhancing the binding strength between the membrane and the substrate. The mixture was gently stirred and incubated at 37 °C for 2 hours to promote the spontaneous fusion of membrane vesicles on the particle surface and achieve uniform spreading under solvent equilibrium. After the reaction, excess membrane components and acetonitrile were removed by centrifugation at 6000 rpm for 10 minutes, and the mixture was washed twice with deionized water. The resulting precipitate was redispersed in 200 μL of deionized water to obtain a fully coated AuNP@RBCM, which was stored at 4 °C for later use. Figure 8 C, 9 C)

[0095] As a comparative example, a erythrocyte membrane-coated sample was prepared using a conventional ultrasonic method: the same number of gold nanoparticles were mixed with 2 μL of erythrocyte membrane solution (2 mg / mL), and then ultrasonicated at 108 W power for 5 minutes to induce the rupture of membrane vesicles and their attachment to the particle surface. Figure 8 B, 9 B).

[0096] Example 6

[0097] Thiol-modified silica nanoparticles coated with erythrocyte membrane lipid solvent equilibrium method

[0098] Thiol-modified silica nanoparticles were dispersed in a 1.12 mL mixture of water and acetonitrile (volume ratio 1:0.2). Then, under stirring, 2 μL of erythrocyte membrane solution (2 mg / mL) and 0.5 μL of a thiophospholipid (PSH) solution (2 mg / mL) were added sequentially. This thiolated lipid can form stable S–S covalent anchoring bonds with the surface of the thiolated silica nanoparticles in subsequent steps, thereby enhancing the adhesion between the membrane and the substrate.

[0099] The mixed solution was gently stirred and incubated at a constant temperature of 37 °C for 2 hours. After the reaction, excess membrane components and acetonitrile were removed by centrifugation at 6000 rpm for 2 minutes, and the solution was washed twice with deionized water. The resulting precipitate was redispersed in 200 μL of deionized water to obtain fully coated SiO2@RBCM, which was then stored at 4 °C for later use. Figure 10 )

[0100] Example 7

[0101] Stability of AuNP@RBCM under environmental disturbances:

[0102] PBS was added to the system to induce aggregation of particles exposed on the gold surface after the film layer was damaged, thereby assessing the integrity of the coating through changes in light absorption. The results showed that both groups of untreated samples exhibited typical single-peak absorption at 530 nm, indicating that the particles maintained good dispersion in PBS. When shear forces (1.1 Pa and 2.2 Pa) were applied, the absorption peak positions and intensities of the AuNP@RBCM samples prepared by the solvent equilibrium method remained essentially unchanged, indicating that their structure was stable and no significant aggregation occurred. Figure 11 A). Transmission electron microscopy (TEM) further confirmed that the sample surface still retained an intact erythrocyte membrane coating. Figure 11 B), this stability was maintained after three cycles of 1.7 Pa shear. Figure 11 C), and no signs of aggregation were observed during the 15-day observation period (C). Figure 11 D). Conversely, the absorption peak of the AuNP@RBCM sample prepared by ultrasonication was significantly weakened under a shear condition of 1.1 Pa and completely disappeared at 2.2 Pa, indicating loss of particle dispersibility and severe aggregation. Figure 12 A). TEM results showed that a large number of aggregates formed in the sample, and under high magnification, film peeling was visible, exposing the metal surface. Figure 12 B). The above results demonstrate that the Au–S covalent anchoring achieved through PSH incorporation via the solvent equilibrium method significantly enhances the membrane-substrate bonding, resulting in excellent structural stability of the coating layer under fluid shear conditions. Notably, the tested shear stresses (1.1–2.2 Pa) fall within the physiological range for humans (approximately 0.01–0.5 Pa in venous circulation and approximately 1–7 Pa in arterial circulation). Therefore, the AuNP@RBCM obtained via the solvent equilibrium method can maintain structural integrity and functional stability under physiological blood flow conditions, exhibiting promising application potential.

[0103] When co-incubated with α-HL toxin, the stability of AuNP@RBCM prepared by the solvent equilibrium method did not change significantly with increasing toxin concentration, but particles prepared by the ultrasonic method showed obvious particle instability and aggregation. Figure 12 C). Differences in the cell membrane layers on the surfaces of the two types of particles can be observed in the transmission electron microscopy images. Figure 12 (D, 12E). This further illustrates the application potential of AuNP@RBCM toxin neutralization prepared by solvent equilibrium method and related vaccines.

[0104] Application Example 1

[0105] Application of AuNP@RBCM obtained by solvent equilibrium method in toxin neutralization and immune enhancement

[0106] Alpha-hemolysin (α-HL) is a key virulence factor secreted by Staphylococcus aureus. It can disrupt host cell membranes through perforation and promote bacterial immune escape, thus it is widely considered an important target for bacterial vaccine development. Red blood cell membrane-coated nanoparticles, due to their excellent biocompatibility and biomimetic properties, provide an ideal platform for α-HL neutralization and immune response enhancement. On the one hand, after α-HL is embedded in the surface of α-HL-AuNP@RBCM, its toxicity is directly neutralized, thereby achieving a safe vaccine configuration. Figure 13 On the other hand, this coating structure can maintain the native conformation of α-HL protein, which helps to induce a stronger and more specific immune response. Furthermore, the nanoscale particle size facilitates uptake by antigen-presenting cells and retention in lymph nodes, thereby promoting antigen presentation and the formation of adaptive immune responses. To verify the in vivo immunomodulatory effects of α-HL-AuNP@RBCM prepared by the solvent equilibrium method, its uptake behavior in macrophages was first investigated. Macrophages, as typical antigen-presenting cells (APCs), can engulf exogenous antigens and process them into short peptides for presentation to T cells, thereby triggering subsequent immune responses. In the experiment, α-HL-AuNP@RBCM was prepared using both the solvent equilibrium method and the traditional sonication method, where α-HL was labeled with green fluorescence to track intracellular distribution (…). Figure 14 Confocal fluorescence microscopy results showed that macrophages prepared by the solvent equilibration method exhibited significantly higher intracellular fluorescence signals, while the intracellular fluorescence intensity of samples prepared by the sonication method was only about 25% of the former. Figure 15 This result indicates that the solvent equilibrium method effectively maintains the structure and binding state of α-HL through stable membrane coating, thereby significantly improving the delivery efficiency of antigens in macrophages and laying the foundation for subsequent induction of stronger anti-α-HL specific immune responses.

[0107] Application Example 2

[0108] In vivo immunization and protection with α-HL-AuNP@RBCM vaccine:

[0109] To evaluate the immunogenicity and protective effect of α-hemolysin-coated nanovaccines prepared using different methods, α-HL-AuNP@RBCM were prepared using both solvent equilibration and conventional sonication methods, and immunization experiments were conducted in a mouse model. Mice received the first immunization on day 1, and two booster immunizations on days 7 and 14, respectively. Figure 16 A). Blood samples were collected from the mandibular vein before the first injection and before each booster immunization to monitor changes in serum anti-α-HL antibody titers. Results showed that, compared to the ultrasonic method preparation group, the solvent equilibrium method preparation group exhibited higher antibody levels throughout the immunization process, and the antibody titer increased with the number of immunizations. Figure 16 B). After the second booster immunization, the anti-α-HL antibody titer in the solvent equilibrium group was approximately 1.65 times higher than that in the ultrasound group ( Figure 16 (C) indicates that this method can significantly enhance antigen-specific humoral immune responses.

[0110] Subsequently, a challenge experiment was conducted by subcutaneous injection of 5 μg α-HL to evaluate the protective effect of the vaccine. Mice in the unimmunized control group showed significant skin necrosis and ulceration after injection (average lesion area approximately 1.1 cm²), consistent with the cytolytic characteristics of α-HL toxin; while mice immunized with α-HL-AuNP@RBCM showed significantly reduced skin damage. Figure 17 A). Although the difference in lesion area between the two preparation methods did not reach statistical significance, the average lesion area of ​​the solvent equilibrium method group was smaller (approximately 0.03 cm²), which was significantly better than that of the ultrasound method group (approximately 0.39 cm²). Figure 17 B). Histopathological results (H&E and TUNEL staining) further confirmed that the degree of apoptosis and necrosis in the skin tissue of the solvent balance group was significantly reduced, showing stronger toxin neutralization and tissue protection capabilities. Figure 18 ).

[0111] It is worth noting that the vaccine preparation method also has a significant impact on safety. Mice immunized with α-HL-AuNP@RBCM prepared by ultrasound still developed local skin lesions even without exposure to exogenous α-HL, while the solvent balance group did not show similar adverse reactions. Figure 19 A, B). This phenomenon is presumably related to the instability of the sample membrane prepared by ultrasonication and the shedding of α-HL in vivo, which then restores its hemolytic activity. Apart from local skin damage, no significant abnormalities were observed in the body weight and hematological parameters of the mice in each group, indicating that the vaccine system has good systemic safety. Figure 19 C). In summary, the α-HL-AuNP@RBCM prepared by the solvent equilibrium method significantly improved immunogenicity and protective effect while maintaining antigen conformation and stable binding, and effectively avoided the risk of toxin release caused by membrane instability, demonstrating excellent immunoprotection and biosafety performance.

[0112] The present invention is not limited to the specific technical solutions described in the above embodiments. All technical solutions formed by equivalent substitutions are within the scope of protection claimed by the present invention.

Claims

1. A method for preparing cell membrane-coated nanoparticles based on solvent equilibration and thiol anchoring, characterized by: Includes the following steps: Preparation of cell membrane vesicles: Red blood cell membranes are isolated from red blood cells by hypotonic lysis and formed into membrane vesicles; Solvent equilibrium-induced membrane fusion: Gold nanoparticles were dispersed in acetonitrile with a volume fraction of 16%, and erythrocyte membrane vesicles were added to the gold nanoparticle solution; Thiol-based lipid incorporation: 3.66 mM 1,2-dipalmitoyl-sn-glycerol-3-phosphate thioethanol, which can form covalent bonds with the nanoparticle substrate, was added to a solution of gold nanoparticles containing erythrocyte membrane vesicles, thereby incorporating thiolated lipid molecules into the membrane lipid bilayer; after static incubation at 37 °C for 2 h, the erythrocyte membrane vesicles spontaneously fused on the nanoparticle surface to form a continuous membrane coating; Washing and purification: Unbound membrane vesicles were removed by centrifugation and washing to obtain nanoparticles with a uniform surface coating of cell membranes; The thiolated lipids form gold-sulfur covalent anchoring bonds with the surface of the gold nanoparticles; the acetonitrile is used to improve the fluidity of the membrane lipids and promote the uniform fusion of the membrane layers.

2. The method of claim 1, wherein: The substrate material for the nanoparticles is gold nanoparticles of different sizes.

3. The method according to claim 1, characterized in that: The morphology of the nanoparticles includes spherical, triangular nanosheets, cubic or other polyhedral structures with positive curvature, as well as other complex morphological nanoparticles with both positive and negative curvature on their surfaces.

4. The cell membrane-coated nanoparticles prepared according to any one of claims 1-3, characterized in that: The resulting nanoparticles with cell membrane coatings retain the protein, lipid, and carbohydrate components of the original cell membrane and exhibit enzyme activity comparable to that of the erythrocyte-derived membrane in acetylcholinesterase (AChE) activity assays.

5. The application of the cell membrane-coated nanoparticles according to claim 4 in the preparation of an anti-α-hemolysin immunization vaccine.

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