Universal cell membrane nanoparticle coating method based on solvent equilibrium and sulfydryl anchoring and application of universal cell membrane nanoparticle coating method

Abstract

The invention belongs to the technical field of nano biological materials, and particularly relates to a preparation method of cell membrane coated nanoparticles based on solvent equilibrium and sulfydryl anchoring. According to the method, sulfydryl-containing lipid is doped into a membrane system, so that the sulfydryl-containing lipid and the surface of a substrate form a covalent anchor bond to enhance the membrane-substrate binding force, and a small amount of organic solvent is added to improve the fluidity of the lipid and promote spontaneous fusion and uniform spreading of a membrane layer on the surfaces of particles. And centrifuging and washing to obtain the cell membrane nanoparticles with completely coated surfaces. According to the method, high-energy ultrasonic or extrusion treatment is not needed, stable coating can be realized on the premise of maintaining complete composition of membrane protein and lipid, and the adhesion stability of a membrane layer in a high-salt and shearing environment is remarkably improved. The method is suitable for various nano substrates and different morphological structures, and the obtained particles show higher antibody titer and excellent safety in an anti-alpha-hemolysin vaccine model. The invention provides a mild and efficient bionic nano material construction strategy with wide applicability.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of nanobiomaterials, and particularly to a method for preparing biofunctionalized nanomaterials by using solvent incubation balance and thiol anchoring cell membranes and nanoparticles to achieve stable coating, and belongs to the technical field of biofunctionalized nanomaterials. BACKGROUND

[0002] Cell membrane coating technology is a new and general nanoparticle surface functionalization strategy. This technology takes advantage of the self-assembly characteristics of cell membrane lipid bilayers to transfer natural functional components (including proteins, lipids and sugars) in 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 antitoxin. Existing membrane coating methods usually use ultrasonic or extrusion means to cause cell membrane vesicles to rupture and fuse with nanoparticles. Similar principles are also widely used in the preparation of supported lipid bilayers (SLB). However, unlike the single-component and controllable-property artificial synthetic lipid system, natural cell membranes are complex systems composed of multiple lipids and membrane proteins, and have significant heterogeneity and phase separation characteristics. These characteristics pose unique challenges in forming stable and uniform membrane coatings.

[0003] In addition, membrane proteins (such as CD9, Caveolin-1, etc.) Due to their different curvature stability characteristics, when the membrane layer needs to adapt to the surface of nanoparticles with high positive curvature, additional instability factors may be triggered. The detachment of the membrane layer will have two consequences: first, the loss of functional molecules on the membrane will lead to the loss of function of the nanoparticles; second, the steric hindrance effect provided by the cell membrane coating layer is crucial for maintaining the colloidal stability of the nanoparticles under high ionic strength conditions. Once the membrane layer is detached, the particles are prone to aggregation, the size exceeds the physiological range, and thus the expected biological function is lost.

[0004] SUMMARY

[0005] To overcome the problems of poor stability in physiological environment, easy detachment of the membrane layer, and limited substrate adaptability of existing cell membrane coated nanoparticles, the present application provides a cell membrane stable coating method based on solvent balance and thiol anchoring. This method establishes a covalent anchoring structure at the membrane-substrate interface and introduces solvent-assisted membrane component rearrangement, achieving stable coating of nanoparticles with different morphologies and material types.

[0006] To solve the technical problems of the present application, the technical solution is as follows: A preparation method of cell membrane coated nanoparticles based on solvent balance and thiol anchoring, comprising the following steps:

[0007] Cell membrane vesicle preparation: red blood cell membranes are separated from red blood cells by hypotonic lysis, and membrane vesicles are formed;

[0008] Solvent balance induced membrane fusion: gold or silicon oxide nanoparticles are dispersed in an organic solvent with a volume ratio of 2%-50%, and red blood cell membrane vesicles are added to the gold or silicon oxide nanoparticle solution;

[0009] Thiolated lipid incorporation: 1,2-dipalmitoyl-sn-glycero-3-phosphothioethanol, which can form a covalent bond with the nanoparticle substrate, is added to the above gold or silicon oxide nanoparticle solution containing red blood cell membrane vesicles, and the thiolated lipid molecules are incorporated into the membrane lipid bilayer; incubation under mild conditions allows the red blood cell membrane vesicles to spontaneously fuse on the surface of the nanoparticles to form a continuous membrane coating;

[0010] Washing and purification: unbound membrane vesicles are removed by centrifugation and washing to obtain uniformly surface-coated nanoparticles with cell membranes.

[0011] Wherein, the thiolated lipid and the gold nanoparticle surface form a gold-sulfur covalent anchoring bond; the double sulfur covalent bond formed between the thiol-modified silicon oxide surface and the thiol ligand enhances the membrane-substrate interaction; the organic solvent is used to improve the membrane lipid fluidity and promote uniform fusion of the membrane layer.

[0012] Preferably, the following steps are included:

[0013] Cell membrane vesicle preparation: red blood cell membranes are separated from red blood cells by hypotonic lysis, and membrane vesicles are formed;

[0014] Solvent balance induced membrane fusion: gold nanoparticles are dispersed in acetonitrile with a volume of 16%, and red blood cell membrane vesicles are added to the gold nanoparticle solution;

[0015] Thiolated lipid incorporation: 1,2-dipalmitoyl-sn-glycero-3-phosphothioethanol, which can form a covalent bond with the nanoparticle substrate, is added to the above gold nanoparticle solution containing red blood cell membrane vesicles, and the thiolated lipid molecules are incorporated into the membrane lipid bilayer; incubation at 37°C for 2h allows the red blood cell membrane vesicles to spontaneously fuse on the surface of the nanoparticles to form a continuous membrane coating;

[0016] Washing and purification: unbound membrane vesicles are removed by centrifugation and washing to obtain uniformly surface-coated nanoparticles with cell membranes.

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

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

[0019] Preferably, the morphology of the nanoparticles is spherical, triangular nanoplate, cubic or other polyhedral structures with positive curvature, and other complex morphology nanoparticles with both positive and negative curvatures.

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

[0021] Preferably, the incorporation ratio of the thiolated lipid is 0.1%-10% of the total mass of the red blood cell membrane.

[0022] The cell membrane-coated nanoparticles prepared by the method maintain the protein, lipid and carbohydrate components of the source cell membrane, and exhibit enzyme activity comparable to that of red blood cell-derived membranes in acetylcholinesterase (AchE) activity tests.

[0023] The cell membrane-coated nanoparticles 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 cell membranes, which is anchored to the surface of the nanoparticles by 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 concentrations and fluid shear conditions without aggregation or membrane shedding.

[0024] The cell membrane-coated nanoparticles are used to prepare anti-alpha-hemolysin immune vaccines, targeted drug delivery systems or biological toxin neutralization nanofactories.

[0025] Advantages

[0026] The core technical points of the method of the present application include the following two aspects:

[0027] 1. Thiolated lipid incorporation and covalent anchoring

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

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

[0030] A small amount of organic solvent (such as acetonitrile, ethanol or dimethylformamide, etc.) is introduced into the membrane-particle composite system to increase the fluidity of the membrane lipid, promote the redistribution of lipid molecules and the fusion of the membrane layer, and thus form a complete, continuous and uniform cell membrane coating layer on the surface of the nanoparticles. This process does not require severe physical disturbance (such as ultrasonic or extrusion), avoiding the inactivation of membrane proteins or the destruction of membrane components.

[0031] The present application is verified by using gold nanoparticles (AuNPs) and red blood cell membranes (RBCM) as a model system. The acetylcholinesterase (AchE) activity detection, dynamic light scattering (DLS) and lipidomics analysis show that this method can realize the reconstruction and coating of the complete membrane layer while maintaining the chemical composition and biological function of the red blood cell membrane. Transmission electron microscopy (TEM) results further prove that this method has good adaptability to gold nanoparticles of different morphologies (including spherical, triangular nanosheets and cubes) and thiol-modified silicon oxide particles, and exhibits excellent substrate versatility.

[0032] Theoretical modeling and energy stability analysis show that covalent anchoring significantly increases the energy barrier required for membrane layer detachment, thereby enhancing the overall coating stability. The comparison results of centrifugal shear disturbance experiment and alpha-hemolysin (alpha-HL) insertion experiment verify this conclusion: AuNP@RBCM prepared by traditional ultrasonic method aggregates seriously under physiological salt conditions, while the sample prepared by the method of the present application still maintains good dispersibility under the same conditions.

[0033] In the application layer, when the stable coating system is used for anti-alpha-hemolysin vaccine model, AuNP@RBCM can more efficiently deliver alpha-HL antigen to antigen-presenting cells such as macrophages, thereby inducing a stronger specific antibody response. Mouse experiment results show that the vaccine group prepared by the method has higher anti-alpha-HL antibody titer than the traditional preparation group, and shows stronger protective effect in the subcutaneous toxin challenge model. At the same time, safety evaluation shows that particles prepared by traditional ultrasonic method cause antigen shedding due to unstable membrane layer during the immune stage, resulting in local skin damage, while particles prepared by the method of the present application do not have such safety hazards.

[0034] The application is based on a preparation method of cell membrane-coated nanoparticles by solvent equilibration and thiol anchoring. The method comprises the following steps: firstly, obtaining cell membrane vesicles by low-osmotic lysis; secondly, incorporating thiol-containing lipid molecules into the membrane system, so that the thiol-containing lipid molecules form covalent anchoring bonds with the metal surface on the membrane-nanoparticle interface, thereby enhancing the binding force between the membrane layer and the substrate; subsequently, adding a small amount of organic solvent into the mixed system of the membrane vesicles and the nanoparticles to improve the lipid fluidity, promote the spontaneous fusion and uniform spreading of the membrane layer on the surface of the nanoparticles; finally, obtaining the nanoparticles coated with the complete cell membrane on the surface through centrifugation and washing. The method does not require high-energy ultrasonic or extrusion treatment, can realize stable coating on the premise of keeping the integrity of the membrane protein and lipid composition, and significantly improves the attachment stability of the membrane layer in a high ionic strength and fluid shear environment. Experimental results show that the method is suitable for metal or oxide nanoparticle substrates and different morphological structures (spherical, triangular flake, cubic, etc.), and the obtained particles exhibit higher antibody titer and better safety in an anti-alpha-hemolysin vaccine model. The application provides a universal, mild and efficient cell membrane coating method, and provides a new way for the construction of a biomimetic nanodrug and vaccine carrier.

[0035] In summary, the application provides a cell membrane coating method with universality and high stability. The method realizes stable coating of various nanoparticle materials by regulating the fluidity of membrane components through solvent equilibration and enhancing the membrane-substrate combination through thiol anchoring, provides a universal and scalable preparation approach for a new generation of biomimetic nanotherapeutic systems, and has significant scientific and application value. BRIEF DESCRIPTION OF DRAWINGS

[0036] The application will be further described below in conjunction with the drawings.

[0037] Figure 1 : (A) AuNP@RBCM prepared by the ultrasonic method relies on weak non-covalent substrate interaction to stabilize the cell membrane on the gold nanoparticles. In the equilibrium incubation method, the PSH doping in the red blood cell membrane enables it to form a covalent Au-S bond with the gold nanoparticle substrate, thereby producing strong substrate interaction. (B) The addition of organic solvent promotes the fusion of the cell membrane to the gold nanoparticle substrate, forming a uniform and complete stealth layer.

[0038] Figure 2 : Quantitative analysis of acetylcholinesterase (AchE) activity on the red blood cell membrane after treatment of the red blood cell membrane with different volume fractions of dimethyl sulfoxide (DMSO), ethanol (EtOH) or acetonitrile (ACN).

[0039] Figure 3 : (A) UV-visible absorption spectra of AuNP@RBCM solutions prepared using different organic solvents by the equilibrium incubation method. Before measurement, the nanoparticles were transferred to 1xPBS.

[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 solvent equilibrated AuNP@RBCM after centrifugal shear at 0, 1.1 and 2.2 Pa, respectively. (B) TEM images show that solvent equilibrated AuNP@RBCM remain monodispersed and the presence of the membrane shell (scale bar, 200 nm, inset shows a single particle, scale bar, 25 nm). (C) UV-Vis spectra of solvent equilibrated AuNP@RBCM in phosphate buffered saline (PBS) after three centrifugal shear treatments. (D) Dynamic light scattering (DLS) analysis shows that solvent equilibrated AuNP@RBCM have stable hydrodynamic diameter (average 55 nm) and polydispersity index (PDI, average 0.2) in PBS for 15 days.

[0049] Figure 12 (A) UV-Vis spectra of ultrasonically prepared AuNP@RBCM in PBS after centrifugal shear treatment at 0, 1.1 and 2.2 Pa. (B) TEM images show that ultrasonically prepared particles have undergone extensive aggregation and lack discernible membrane coverage (scale bar, 200 nm, inset shows a single particle, scale bar, 25 nm). (C) UV-Vis spectra of AuNP@RBCM prepared by solvent equilibration (red line) and ultrasonication (black line) incubated with increasing concentrations of a-HL in PBS show that equilibrated AuNP@RBCM remain dispersed with stable plasmonic peaks, while the ultrasonic control group has a diminished plasmonic peak at -530 nm and a broad absorption between 600-700 nm, indicating aggregation. (D-F) TEM images of solvent equilibrated (D) and ultrasonically prepared (F) AuNP@RBCM after a-HL treatment. Solvent equilibrated AuNP@RBCM show intact and continuous red blood cell membrane shells. The ultrasonic control group shows extensive membrane shedding (scale bar, 50 nm).

[0050] Figure 13 Schematic of a-HL-AuNP@RBCM. a-HL is a protein toxin secreted by Staphylococcus aureus that assembles into a transmembrane pore on the red blood cell membrane layer of AuNP@RBCM.

[0051] Figure 14 Confocal fluorescence images of RAW 264.7 macrophages incubated with a-HL-AuNPs@RBCM. a-HL was labeled with Alexa Fluor 488. Intracellular fluorescence signal was evaluated to compare the uptake efficiency of nanoparticles prepared by two methods (solvent equilibration incubation and ultrasonication), 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 primary dose injection at day 0, and two booster injections at day 7 and day 14, respectively. 100 microliters of peripheral blood was collected before the primary injection and each booster injection. At day 21, 5 pg of a-HL was injected subcutaneously. The lesion size was monitored for 4 days. (B, C) Quantitative analysis of anti-a-HL antibody titers in peripheral blood. Blood was collected from PBS-treated or a-HL-AuNP@RBCM-immunized mice at the indicated time points (n=6). Data at day 21 were presented as a histogram in panel C.

[0054] Figure 17 Representative images and quantification of skin lesions in mice 2 days after subcutaneous injection of a-HL. Lesion size was quantified by images (scale bar, 1 cm).

[0055] Figure 18 H&E staining and TUNEL staining of skin in mice of each group. Scale bar, 1.25 pm.

[0056] Figure 19 Representative images of skin at day 13 (scale bar, 1 cm) in healthy mice after subcutaneous injection of PBS, ultrasonically prepared a-HL-AuNP@RBCM, and solvent-equilibrated a-HL-AuNP@RBCM. Quantification of lesion size is plotted in panel B. (C) Body weight of the three groups of immunized mice at day 21.

[0057] Figure 20 Schematic diagram of the present application Detailed implementation method

[0058] The technical solutions of the present application are further described below in conjunction with examples, but the present application is not limited to the following embodiments. Any equivalent replacement or modification based on the idea of the present application shall be considered to fall within the scope of protection of the present application.

[0059] Example 1

[0060] 1. Required materials and reagents

[0061] Nanoparticle substrate: gold nanospheres (diameter 40 nm), gold triangular nanoplatelets (edge length about 150 nm), gold cubes (edge length about 80 nm), etc. Other metal or oxide nanoparticles (such as silver, iron oxide, silicon dioxide, etc.) can also be used as substrates.

[0062] Cell membrane source: Freshly isolated red blood cells (RBC) or other source cells (such as white blood cells, platelets, tumor cells, stem cells, etc.). Cell membrane vesicles were obtained after hypotonic lysis and stored at -80°C for later use.

[0063] Thiolated lipids: Phospholipid molecules containing thiol groups, 1,2-dipalmitoyl-sn-glycero-3-phosphoric acid thioethanol (PSH), etc., used to form gold-sulfur covalent bonds between the membrane and the metal surface.

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

[0065] Buffer solution: Phosphate buffer (PBS, 1x, pH 7.4), used to quickly assess the stability of the coating.

[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 4°C at 800g for 5 min, the buffy coat containing white blood cells and platelets was extracted and discarded, and the lower layer of red blood cells was collected. The red blood cells were washed three times with 1x PBS. To remove intracellular material, hypotonic treatment was used: the red blood cells were immersed in 0.25x PBS and placed in an ice bath for 20 minutes, followed by high-speed centrifugation (30,000xg for 5 minutes) to collect the red blood cell membrane vesicles. The red blood cell membrane vesicles were washed twice with 1x PBS and stored at -80°C for later use.

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

[0070] Take 100 mL of 0.1 mg / mL aqueous solution of tetrachloroauric acid (HAuCl4) in a three-necked flask, heat to boiling, then quickly add 3 mL of 10 mg / mL aqueous solution of sodium citrate. Keep boiling and continue stirring the reaction for 1 hour to reduce gold ions to form about 15 nm gold nanospheres. After cooling to room temperature, the resulting solution is a 15 nm sodium citrate stabilized gold nanoparticle seed solution. Take 10 mL of the above 15 nm gold nanoparticle seed solution, dilute to 50 mL of deionized water, and place it in a 250 mL flask. Heat the solution to reflux in an oil bath and stir vigorously for 30 minutes to stabilize the system. Then, quickly add 0.25 mL of 10 mg / mL sodium citrate solution and 0.3 mL of 10 mg / mL aqueous solution of tetrachloroauric acid to the boiling solution. Keep boiling for 30 minutes to continue reducing and depositing gold on the surface of the seeds to promote particle growth. After the end, repeat the above addition and heating steps twice to obtain sodium citrate stabilized gold nanoparticles (AuNPs) with an average particle size of about 40 nm, which are stored at room temperature for future use.

[0071] 2.3. Nanoparticle pretreatment

[0072] Centrifuge 1 mL of 40 nm gold particles from the original stored excess sodium citrate ligand aqueous solution at 6000 rpm for 10 min. After removing the supernatant, the gold nanoparticle precipitate is redispersed in a mixed solution of water and acetonitrile (water / ACN: 5 / 1) with a total volume of 1.12 mL in a clean reaction bottle for future use.

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

[0074] Under stirring conditions, 2 μL of red blood cell membrane solution (concentration 2 mg / mL) and 0.5 μL of thiol phospholipid (PSH) solution (concentration 2 mg / mL) are sequentially added to the above solution. After the PSH is incorporated into the membrane system, its thiol group can form a stable Au-S covalent anchoring bond with the metal surface in the subsequent step.

[0075] 2.5 Solvent-induced coating equilibrium reaction

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

[0077] 2.6 Purification and collection

[0078] After the incubation, the solution was allowed to cool down to room temperature, then centrifuged at 6000 rpm for 10 min to remove the excess cell membrane components and acetonitrile solvent. After discarding the supernatant, the product was resuspended in 200 μΐ^of PBS to obtain the final AuNP@RBCM. The obtained sample was stored at 4 °C for subsequent characterization and application.

[0079] Example 2

[0080] Screening and determination of the effect of organic solvents on the activity of red blood cell membrane proteins:

[0081] Considering that the introduction of organic solvents can destroy 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 example first screened the biocompatibility of different organic solvents. The enzyme activity of acetylcholinesterase (AChE) in red blood cell membranes was used as an evaluation index. AChE is a typical membrane-bound enzyme that can catalyze the hydrolysis of the neurotransmitter acetylcholine, and changes in its activity can reflect the conformational maintenance of membrane proteins under the action of solvents. In the experiment, the purified red blood cell membrane samples were mixed with three organic solvents, dimethyl sulfoxide (DMSO), ethanol (EtOH), and acetonitrile (ACN), at different volume fractions and incubated, and then the AChE activity was measured. The results showed that with the increase of the volume fraction of organic solvents, the AChE activity of the three groups of samples showed a downward trend. Among them, the biocompatibility of the ethanol system was the best, and when the volume fraction was not more than 32%, the AChE activity remained basically stable; while the AChE activity in the DMSO and acetonitrile systems decreased more significantly, and when the volume fraction of the solvent was 16%, the enzyme activity had decreased by about 20% ( Figure 2 ). Based on this result, the volume fraction of the three organic solvents was selected as 16% in the subsequent red blood cell membrane coating experiment to balance the membrane lipid fluidity and membrane protein structure stability, so as to obtain a better coating effect.

[0082] Verification of the integrity and stability of gold nanoparticles coated by red blood cell membrane solvent equilibration method:

[0083] This example aims to verify whether the solvent equilibration method can form a complete and stable red blood cell membrane coating on the surface of gold nanoparticles. Gold nanospheres with a particle size of about 40 nm were used as a model substrate, which has a typical surface plasmon resonance absorption peak at 530 nm and is extremely sensitive to particle aggregation. In the experiment, acetonitrile (ACN), dimethyl sulfoxide (DMSO) and ethanol (EtOH) were used as solvents, respectively, to prepare AuNP@RBCM samples by solvent equilibration method. After coating, the purified samples were transferred to 1xPBS solution to evaluate their stability in a physiological salt environment. The results show that the AuNP@RBCM sample prepared by acetonitrile system still maintains a single and strong 530 nm absorption peak in PBS, indicating that the particles have good dispersion and the membrane layer is complete; while the samples prepared by DMSO and ethanol system have significantly weakened absorption at 530 nm, and a new absorption peak appears at about 630 nm, indicating that the particles have aggregated due to incomplete membrane coating Figure 3 A). This result shows that the fluidity of membrane lipids in the solvent system is crucial for forming a complete membrane coating. In parallel experiments, to further explore the role of thiolated phospholipids in membrane-substrate binding, red blood cell membranes with or without 1,2-dipalmitoyl-sn-glycero-3-phosphothioethanol (PSH) were used to coat 40 nm gold nanospheres in the acetonitrile system. The purified samples were also transferred to 1xPBS to detect their stability. The results show that the characteristic peak at 530 nm of the sample without PSH completely disappeared, indicating that the particles were severely aggregated; while the sample with PSH maintained sharp and unchanged absorption peak, showing that the system was still in a single dispersed state Figure 3 B).

[0084] In addition to PSH, we selected other thiolated lipid acids (such as 11-mercaptoundecanoic acid, MUA; 16-Mercaptohexadecanoic acid, MHA), and 2-naphthalene thiol (2-NT) molecules for anchoring, but the results show that Figure 4 ), 3.66 mM of PSH works best in anchoring the cell membrane and particles.

[0085] The above results show that the covalent anchoring of PSH in 16% acetonitrile solvent is a key condition for obtaining a complete and stable cell membrane-coated gold nanoparticle.

[0086] Example 3

[0087] Structure and composition verification of red blood cell membrane-coated gold nanoparticles:

[0088] The particle size of AuNP@RBCM was determined by dynamic light scattering (DLS). The results showed that the average particle size of AuNP@RBCM increased by about 10 nm compared with the uncoated gold nanoparticles, which was equivalent to the thickness of the double lipid membrane, indicating that an intact cell membrane coating layer was formed on the surface of the nanoparticles. Further detection of the electrical properties of the particle surface showed that the zeta potential was about -30 mV, reflecting that the negative charge characteristics of the red blood cell membrane had been successfully transferred to the surface of the particles Figure 5 A). To confirm the preservation of membrane protein components, SDS-PAGE analysis was performed on AuNP@RBCM samples and original red blood cell membranes. The results showed that the protein band distribution of the two was almost completely consistent, indicating that the types and relative proportions of membrane proteins were effectively preserved Figure 5 B). Based on the DLS and SDS-PAGE results, it can be confirmed that the red blood cell membrane was successfully transferred to the surface of the gold nanoparticles during the coating process, forming a complete and component-faithful biomimetic membrane-coated system.

[0089] Example 4

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

[0091] The lipid composition of AuNP@RBCM prepared by the solvent equilibration method was analyzed by LC-MS / MS lipidomics technology, and compared with the original red blood cell membrane. The results showed that the lipid spectrum of AuNP@RBCM was highly consistent with RBCM, indicating that the membrane lipids were effectively transferred and maintained during the coating process. The lipids of the two groups of samples were mainly composed of glycerophospholipids (about 75%-80% of the total lipids), followed by fatty acyls (about 15%-20%), and about 5%-10% of sphingolipids, while glycerol lipids were almost not detected Figure 6 A). In further analysis of specific phospholipid subclasses, it was found that phosphatidylcholine (PC) was the main component in both groups of samples, accounting for more than 80%, and the proportions of phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS) and lysophosphatidylcholine (LPC) were relatively low; phosphatidic acid (PA) and ether PE were almost not detected Figure 6 B). The above results showed that the red blood cell membrane not only maintained the stability of the overall lipid composition during the coating process, but also preserved the proportion distribution of various phospholipids, thereby proving that this method could achieve complete transfer and structure fidelity of membrane lipids. However, there was still a small amount of loss of lipids with poor fluidity Figure 7 ​

[0092] Example 5

[0093] Preparation of gold nanoparticle coated by red blood cell membrane solvent equilibrium method and comparative example samples Figure 1

[0094] The washed and purified gold nanoparticles (which can be spherical, triangular or cubic structure, etc.) were re-dispersed in 1.12 mL of a mixture of water and acetonitrile with a volume ratio of 1:0.2, and 2 μL of red blood cell membrane solution (2 mg / mL) and 0.5 μL of thiol phospholipid (PSH) solution (2 mg / mL) were added in turn under stirring. The thiolated lipid can form a stable Au-S covalent anchor bond with the gold surface in the subsequent step, thereby enhancing the binding strength of the membrane layer and the substrate. The mixed solution was incubated at 37 °C under gentle stirring 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 was completed, the excess membrane components and acetonitrile were removed by centrifugation at 6000 rpm for 10 minutes, and washed twice with deionized water. The obtained precipitate was re-dispersed in 200 μL of deionized water to obtain AuNP@RBCM coated with intact membranes, and stored at 4 °C for standby use. Figure 8 C, 9 C)

[0095] As a comparative example, a red blood cell membrane coated sample was prepared by a conventional ultrasonic method: the same amount of gold nanoparticles was mixed with 2 μL of red blood cell membrane solution (2 mg / mL), and ultrasonicated at a power of 108 W for 5 minutes to promote the rupture of membrane vesicles and adhere to the particle surface Figure 8 B, 9 B).

[0096] Example 6

[0097] Preparation of thiol-modified silica nanoparticles coated by red blood cell membrane lipid solvent equilibrium method

[0098] The thiol-modified silica nanoparticles were dispersed in 1.12 mL of a mixture of water and acetonitrile with a volume ratio of 1:0.2, and 2 μL of red blood cell membrane solution (2 mg / mL) and 0.5 μL of thiol phospholipid (PSH) solution (2 mg / mL) were added in turn under stirring. The thiolated lipid can form a stable S-S covalent anchor bond with the surface of the thiol-modified silica nanoparticles in the subsequent step, thereby enhancing the binding strength of the membrane layer and the substrate.

[0099] ​The mixed solution was incubated at 37 °C for 2 hours under constant temperature and gentle stirring. After the reaction was completed, the excess membrane components and acetonitrile were removed by centrifugation at 6000 rpm for 2 minutes, and then washed twice with deionized water. The obtained precipitate was redispersed in 200 μL deionized water to obtain the intact SiO2@RBCM, and stored at 4 °C for standby use. Figure 10 )

[0100] Example 7

[0101] Stability of AuNP@RBCM in environmental disturbance:

[0102] PBS was added to the system to induce aggregation of the particles with exposed gold surfaces after the membrane layer was damaged, so as to evaluate the coating integrity by the change of light absorption. The results show that the two groups of samples without shear treatment all exhibit typical single-peak absorption at 530 nm, indicating that the particles remain good dispersion state in PBS. When shear force (1.1 Pa and 2.2 Pa) is applied, the absorption peak position and intensity of the AuNP@RBCM sample prepared by the solvent equilibration method are basically unchanged, indicating that the structure is stable and no obvious aggregation occurs ( Figure 11 A). Transmission electron microscopy (TEM) observation further confirms that the sample still retains the intact red blood cell membrane coating layer on the surface ( Figure 11 B), which remains stable after three cycles of 1.7 Pa shear cycles ( Figure 11 C), and no aggregation is observed within a 15-day observation period ( Figure 11 D). In contrast, the absorption peak of the AuNP@RBCM sample prepared by the ultrasonic method is significantly weakened at 1.1 Pa shear, and completely disappears at 2.2 Pa, indicating that the particle dispersion is lost and serious aggregation occurs ( Figure 12 A). TEM results show that a large number of aggregates are formed in the sample, and the membrane layer is peeled off under high magnification observation, and the metal surface is exposed ( Figure 12 B). The above results show that the Au–S covalent anchoring realized by PSH incorporation by the solvent equilibration method significantly enhances the membrane–substrate binding force, so that the coating layer has excellent structural stability in a fluid shear environment. It is worth noting that the shear stress tested (1.1–2.2 Pa) is within the physiological range of the human body (about 0.01–0.5 Pa for venous circulation and about 1–7 Pa for arterial circulation), therefore, the AuNP@RBCM obtained by the solvent equilibration method can maintain structural integrity and functional stability under physiological blood flow conditions, showing good application potential.

[0103] When co-incubated with α-HL toxin, the stability of the AuNP@RBCM prepared by the solvent equilibration method does not change significantly with the increase of the toxin concentration, but the particles prepared by the ultrasonic method show obvious aggregation due to particle instability.Figure 12 C). The difference of cell membrane layer on the surface of two kinds of particles can be seen in the transmission electron microscopy image Figure 12 D, 12E). Further illustrate the application potential of AuNP@RBCM toxin neutralization and related vaccine prepared by solvent equilibration method.

[0104] Application Example 1

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

[0106] Alpha-hemolysin (a-HL) is one of the key virulence factors secreted by Staphylococcus aureus, which can destroy the host cell membrane through pore-forming effect and promote the immune escape of bacteria, and is therefore widely regarded as an important target for bacterial vaccine development. Red blood cell membrane-coated nanoparticles provide an ideal platform for a-HL neutralization and immune response enhancement due to their excellent biocompatibility and biomimetic properties. On the one hand, the toxicity of a-HL is directly neutralized after a-HL is embedded on the surface of a-HL-AuNP@RBCM, thereby realizing a safe vaccine configuration Figure 13 ); on the other hand, the coating structure can maintain the natural conformation of a-HL protein, which helps to induce a stronger and more specific immune response. In addition, the nanoscale particle size is conducive to being taken up by antigen-presenting cells and retained in lymph nodes, thereby promoting antigen presentation and the formation of adaptive immune response. To verify the immune effect of a-HL-AuNP@RBCM prepared by solvent equilibration method in vivo, the uptake behavior of macrophages was first studied. As a typical antigen-presenting cell (APC), macrophages can phagocytose exogenous antigens and process them into short peptides to present to T cells, thereby triggering subsequent immune response. In the experiment, a-HL-AuNP@RBCM was prepared by solvent equilibration method and traditional ultrasonic method, respectively, and a-HL was labeled with green fluorescence for tracking intracellular distribution Figure 14 ). Confocal fluorescence microscopic imaging results show that macrophages treated with samples prepared by solvent equilibration method exhibit significantly higher intracellular fluorescence signals, while the intracellular fluorescence intensity of samples prepared by ultrasonic method is only about 25% of that of the former Figure 15 ). This result shows that the solvent equilibration method effectively maintains the structure and binding state of a-HL through stable membrane coating, thereby significantly improving the delivery efficiency of antigens in macrophages, laying a foundation for subsequent induction of stronger anti-a-HL specific immune response.

[0107] Application Example 2

[0108] In vivo immunity and protection of a-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 application is not limited to the specific technical solutions described in the above embodiments, and any technical solution formed by equivalent replacement is within the scope of protection of the present application.

Claims

1. A method for preparing cell membrane-coated nanoparticles based on solvent equilibrium and thiol anchoring, characterized in that, 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 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; 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. Washing and purification: Unbound membrane vesicles were removed by centrifugation and washing to obtain nanoparticles with a uniform surface coating of cell membrane. The thiolized lipids form gold-sulfur covalent anchoring bonds with the surface of the gold nanoparticles. The disulfide covalent bonds formed between the thiol-modified silica surface and the thiol ligand enhance the membrane-substrate interaction; the organic solvent is used to improve the fluidity of the membrane lipids and promote the uniform fusion of the membrane layers.

2. The method according to claim 1, characterized in that: 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; 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. Washing and purification: Unbound membrane vesicles were removed by centrifugation and washing to obtain nanoparticles with a uniform surface coating of cell membrane.

3. The method according to claim 1, characterized in that: The organic solvent is selected from acetonitrile (ACN), ethanol (EtOH), dimethyl sulfoxide (DMSO), or mixtures thereof in any proportion.

4. The method according to claim 1, characterized in that: The substrate material for the nanoparticles is gold or silicon oxide nanoparticles of different sizes.

5. 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.

6. The method according to claim 1, characterized in that: The incubation temperature is 0-100°C, and the incubation time is from 5 minutes to 24 hours.

7. The method according to claim 1, characterized in that: The incorporation ratio of thiolized lipids is 0.1%–10% of the total mass of the erythrocyte membrane.

8. The cell membrane-coated nanoparticles prepared by the method according to any one of claims 1-7, 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.

9. The application of cell membrane-coated nanoparticles according to claim 8, characterized in that: The resulting cell membrane-coated nanoparticles 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.

10. The use of the cell membrane-coated nanoparticles according to claim 8 in the preparation of anti-α-hemolysin immune vaccines, targeted drug delivery systems, or biotoxin neutralization nanoformulations.

Images