CN118557814B - A cell membrane coating with a pit-shaped topological structure and its preparation method and application - Google Patents

Abstract

The invention discloses a cell membrane coating with a pit-shaped topological structure, a preparation method and application thereof, and belongs to the technical field of biomedical materials. The method comprises the steps of preparing cell membrane vesicle suspension, and then spraying the surface of a substrate material by using ultrasonic atomization spraying equipment to prepare the cell membrane coating with a pit-shaped topological structure. The preparation method of the cell membrane coating is simple and has strong universality, not only keeps the anticoagulation performance and good biocompatibility of the cell membrane coating, but also further endows the cell behavior guided by the topological structure of the coating, such as cell adhesion, proliferation, migration and differentiation, and can be used for preparing blood contact materials/devices, such as vascular stents, heart occluders, artificial heart valves, artificial blood vessels and other cardiovascular implantation interventional devices, cardiopulmonary circulation pipelines and the serving effect of venous catheters for dialysis.

Description

A cell membrane coating with a pit-shaped topological structure and its preparation method and application

Technical Field

The invention relates to the technical field of biomedical materials, and in particular to a cell membrane coating with a pit-shaped topological structure and a preparation method and application thereof.

Background Art

The cell membrane-derived top-down modification strategy makes full use of the functional properties of the natural membrane interface, giving the material surface good anti-fouling properties, immunomodulatory functions and good biocompatibility. At present, cell membrane-derived bionic coatings have been widely used for surface coating and targeted delivery of drug-loaded nanoparticles, and have great potential in the fields of drug delivery, bioimaging, cancer immunotherapy and detoxification. In addition, studies have explored the use of cell membrane-derived bionic coatings for surface modification of macroscopic implant devices, such as vascular stents, dental implants, and decellularized extracellular matrix scaffolds. However, the current coating assembly process relying on drip coating and dip coating consumes a large amount of cell membrane materials and has poor coating stability. At the same time, the cell membrane coatings prepared on the macroscopic surface are generally two-dimensional planar structures, and it is difficult to achieve the topological structure functionalization of the coating surface by simple methods to enhance the adhesion, proliferation and differentiation of cells on the surface.

Summary of the invention

In order to solve the above-mentioned deficiencies in the prior art, the purpose of the present invention is to provide a cell membrane coating with a pit-like topological structure and a preparation method and application thereof, so as to solve the problems of large consumption of cell membrane materials and poor coating stability in the existing coating assembly process.

The technical solution of the present invention to solve the above technical problem is as follows: a method for preparing a cell membrane coating having a pit-shaped topological structure is provided, comprising the following steps:

(1) extracting and separating cells, resuspending them, and then breaking the cells, collecting cell membrane fragments after washing, resuspending the cell membrane fragments and performing ultrasonication to obtain a cell membrane vesicle suspension;

(2) mixing the cell membrane vesicle suspension with a cross-linking agent to obtain a cell membrane vesicle mixed solution;

(3) adding the cell membrane vesicle mixed solution into the syringe of the ultrasonic atomization spraying equipment, performing a first ultrasonic spraying on the surface of the substrate material, and then cleaning the surface of the substrate material after the spraying;

(4) performing a second ultrasonic spraying of the surface of the substrate material obtained in step (3) with an impact solution;

(5) Drying and solidifying the cell membrane coating obtained in step (4) to obtain.

Furthermore, in step (1), the cells are at least one of red blood cells, platelets, macrophages, neutrophils and T lymphocytes; and the protein concentration of the cell membrane vesicle suspension is 0.5-8 mg/mL.

Furthermore, in step (2), the cross-linking agent is at least one of a polyphenol compound, a carbodiimide compound, glutaraldehyde and genipin; and the mass ratio of the cell membrane vesicle suspension to the cross-linking agent is 1-20:1.

Furthermore, the polyphenol compound is pyrogallol, tannic acid or epigallocatechin gallate.

Furthermore, the carbodiimide compound is carbodiimide hydrochloride or N-cyclohexyl-N'-(2-morpholinoethyl)carbodiimide methyl p-toluenesulfonate.

Furthermore, the ultrasonic spraying in step (3) includes the following steps: controlling the distance between the nozzle and the surface of the substrate material to be 2-10 cm, and then spraying 20-100 layers on the surface of the substrate material at an ultrasonic frequency of 25-250 kHz, an ultrasonic power of 0.5-10 W, a solution propulsion rate of 0.01-0.1 mL/min and a gas pressure of 0.5-1 psi.

Furthermore, the shock solution in step (4) is deionized water, phosphate buffer or physiological saline.

Furthermore, the ultrasonic spraying in step (4) includes the following steps: controlling the distance between the nozzle and the surface of the substrate material to be 2-10 cm, and then spraying 1-100 layers on the surface of the substrate material at an ultrasonic frequency of 25-250 kHz, an ultrasonic power of 0.5-10 W, a solution propulsion rate of 0.01-0.1 mL/min and a gas pressure of 1-3 psi.

Furthermore, in step (3) and step (4), the substrate materials are metal-based biomaterials, polymer-based biomaterials or decellularized biological tissues.

Furthermore, in step (5), the drying and curing temperature is 30-60°C and the time is 10-60 min.

The present invention provides a cell membrane coating with a pit-shaped topological structure, which is prepared by adopting the above-mentioned preparation method.

The present invention also provides an application of a cell membrane coating having a pit-shaped topological structure in the preparation of cardiovascular materials/devices.

Furthermore, the blood contact material/device is a cardiopulmonary circulation circuit, a dialysis intravenous catheter, a vascular stent, a heart occluder, an artificial heart valve or an artificial blood vessel.

The present invention has the following beneficial effects:

(1) The present invention uses ultrasonic spraying to construct a cell membrane coating with a pit-shaped topological structure, which has the advantages of simple method, strong versatility and low cost. There is no need to etch the surface of the substrate in advance, avoiding harsh and expensive photoetching, plasma etching and other technologies, reducing costs and increasing the selectivity of the substrate.

(2) The present invention utilizes the difference in rigidity before and after cross-linking of the cell membrane coating, and uses ultrafine droplets generated by ultrasonic spraying to impact the coating surface to obtain a cell membrane coating with a pit-shaped topological structure. This method completely retains the biological function of the cell membrane coating and enhances the anti-coagulation performance and biocompatibility of the material.

(3) By utilizing the impact of ultrafine droplets generated by ultrasonic spraying, a pit-like structure of about 2-20 μm can be generated on the surface of the cell membrane coating, and as the number of impacts increases, the height of the pit structure changes from nanoscale (80 nm) to submicron (600 nm). The cell membrane coating with a pit-like topological structure expands the interface area and can effectively enhance the adhesion and growth of cells on the surface. At the same time, the adhered cells are stimulated by the enhanced mechanical signals on the surface of the pit-like topological structure coating, and the cytoskeleton transmits the force from the membrane to the cell nucleus, directly affecting the expression of genes and proteins through complex biological pathways, thereby affecting cell morphology, differentiation and proliferation behavior.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a schematic diagram of the preparation process of a cell membrane coating having a pit-shaped topological structure according to the present invention;

FIG2 is a scanning electron microscope image of a naked polylactic acid substrate, a comparative example, and a cell membrane coating prepared in Example 1-2; wherein FIGA is a naked polylactic acid substrate; FIGB is a cell membrane coating prepared in the comparative example; FIGC is a cell membrane coating prepared in Example 1; and FIGD is a cell membrane coating prepared in Example 2;

Figure 3 is a fluorescence staining diagram of platelet adhesion of a naked polylactic acid substrate, a cell membrane coating-modified polylactic acid substrate obtained in a comparative example and Examples 1-2; wherein Figure A is a naked polylactic acid substrate; Figure B is a cell membrane coating-modified polylactic acid substrate obtained in a comparative example; Figure C is a cell membrane coating-modified polylactic acid substrate obtained in Example 1; and Figure D is a cell membrane coating-modified polylactic acid substrate obtained in Example 2;

Figure 4 is a fluorescence staining image of endothelial cells of a naked polylactic acid substrate, a polylactic acid substrate modified with a cell membrane coating prepared in the comparative example and Examples 1-2; wherein, Figure A is a naked polylactic acid substrate; Figure B is a polylactic acid substrate modified with a cell membrane coating prepared in the comparative example; Figure C is a polylactic acid substrate modified with a cell membrane coating prepared in Example 1; and Figure D is a polylactic acid substrate modified with a cell membrane coating prepared in Example 2.

DETAILED DESCRIPTION

The principles and features of the present invention are described below in conjunction with the examples, which are only used to explain the present invention and are not intended to limit the scope of the present invention. If no specific conditions are specified in the examples, the conditions are carried out according to conventional conditions or the conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased commercially.

The preparation method of the cell membrane coating with topological structure of Examples 1-4 is shown in FIG1 .

Example 1

A method for preparing a cell membrane coating having a pit-shaped topological structure comprises the following steps:

(1) Whole blood was centrifuged to obtain red blood cells, which were washed three times with PBS solution and resuspended in pre-cooled 0.25× PBS solution (containing 0.5 mM EDTA, 0.1 mM PMSF) and placed in a 4°C refrigerator for 2 h. The solution was centrifuged at 10,000 g for 20 min, the supernatant was discarded and washed three times with PBS solution, and finally resuspended and sonicated to obtain a milky white red blood cell membrane vesicle suspension, where the protein concentration of the cell membrane vesicle suspension was 4 mg/mL;

(2) mixing the cell membrane vesicle suspension obtained in step (1) with an aqueous solution of 2 mg/mL EGCG in equal volumes to prepare a cell membrane vesicle mixed solution;

(3) Add the cell membrane vesicle mixed solution obtained in step (2) into a gas-tight syringe, adjust the nozzle to 5 cm away from the surface of the polylactic acid substrate, and spray it layer by layer on the surface of the cleaned substrate material at an ultrasonic frequency of 120 kHz, an ultrasonic power of 1 W, and a gas pressure of 1 psi, wherein the propulsion rate of the cell membrane vesicle mixed solution is 0.03 mL/min, spray 40 layers, and rinse with deionized water 3 times;

(4) Replace the cell membrane vesicle mixed solution with deionized water and add it to the airtight syringe of the ultrasonic atomization spraying equipment. Adjust the nozzle to 5 cm away from the material surface and spray it layer by layer on the material surface at an ultrasonic frequency of 120 kHz, an ultrasonic power of 1 W, and a gas pressure of 3 psi. The propulsion rate of the deionized water solution is 0.01 mL/min, and 3 layers are sprayed.

(5) The cell membrane coating obtained in step (4) was dried and cured at 40°C for 30 min to obtain a cell membrane coating having a pit-like topological structure, and its electron microscope scanning image is shown in Figure 2 C. As shown in Figure 2 C, a randomly distributed pit-like structure is formed on the surface of the cell membrane coating obtained in this embodiment, with a diameter of 2-20 μm and a pit depth of 80 nm.

Example 2

A method for preparing a cell membrane coating having a pit-shaped topological structure comprises the following steps:

(1) Whole blood was centrifuged to obtain red blood cells, which were washed three times with PBS solution and resuspended in pre-cooled 0.25× PBS solution (containing 0.5 mM EDTA and 0.1 mM PMSF protease inhibitor), placed in a 4°C refrigerator for 2 hours, and then centrifuged at 10,000 g for 20 min. The supernatant was discarded and washed three times with PBS solution. Finally, a milky white red blood cell membrane vesicle suspension was obtained after resuspending and sonication. The protein concentration of the red blood cell membrane vesicle suspension was 4 mg/mL.

(2) mixing equal volumes of the cell membrane vesicle suspension obtained in step (1) and a 2 mg/mL EGCG-containing Tris-HCl buffer solution (50 mM, pH = 8.5) to prepare a cell membrane vesicle mixed solution;

(3) Add the cell membrane vesicle mixed solution obtained in step (2) into a gas-tight syringe, adjust the nozzle to 5 cm away from the surface of the polylactic acid substrate, and spray it layer by layer on the surface of the cleaned substrate material at an ultrasonic frequency of 120 kHz, an ultrasonic power of 1 W, and a gas pressure of 1 psi, wherein the propulsion rate of the cell membrane vesicle mixed solution is 0.03 mL/min, spray 100 layers, and rinse with deionized water 3 times;

(4) Replace the cell membrane vesicle mixed solution with deionized water and add it to the gas-tight syringe. Adjust the nozzle to 5 cm away from the material surface and spray it layer by layer on the material surface at an ultrasonic frequency of 120 kHz, an ultrasonic power of 1 W, and a gas pressure of 3 psi. The propulsion rate of the deionized water solution is 0.01 mL/min, and 50 layers are sprayed.

(5) The cell membrane coating obtained in step (4) was dried and cured at 40°C for 30 min to obtain a cell membrane coating having a pit-like topological structure, the electron microscope scanning image of which is shown in Figure 2D. As can be seen from Figure 2D, the depth of the pit-like structure is further increased to the submicron level (600 nm), and the diameter of the pit-like structure is 5-10 μm.

Example 3

A method for preparing a cell membrane coating having a pit-shaped topological structure comprises the following steps:

(1) Whole blood was centrifuged to obtain platelets, which were then resuspended in a PBS solution containing 1 mM EDTA. Protease inhibitors were then added and the platelets were repeatedly frozen and thawed three times. The platelet membrane vesicle suspension was obtained by washing, resuspending and sonicating three times in sequence. The protein concentration of the platelet membrane vesicle suspension was 2 mg/ml.

(2) mixing the cell membrane vesicle suspension obtained in step (1) with a 0.5 mg/ml EGCG-containing Tris-HCl buffer solution (50 mM, pH = 8.5) in equal volumes to prepare a cell membrane vesicle mixed solution;

(3) Add the cell membrane vesicle mixed solution obtained in step (2) into a gas-tight syringe, adjust the nozzle to 5 cm away from the surface of the titanium foil material, and spray it layer by layer on the surface of the cleaned substrate material at an ultrasonic frequency of 120 kHz, an ultrasonic power of 1.5 W, and a gas pressure of 1 psi. The propulsion rate of the cell membrane vesicle mixed solution is 0.02 mL/min. Spray 50 layers and rinse with deionized water 3 times.

(4) Replace the cell membrane vesicle mixed solution with deionized water and add it to the gas-tight syringe. Adjust the nozzle to 5 cm away from the surface of the titanium foil material, and spray it layer by layer on the surface of the material at an ultrasonic frequency of 120 kHz, an ultrasonic power of 1 W, and a gas pressure of 3 psi. The propulsion rate of the deionized water solution is 0.01 mL/min, and 3 layers are sprayed.

(5) The cell membrane coating obtained in step (4) is dried and cured at 40°C for 30 min to obtain a cell membrane coating with a pit-like topological structure. Randomly distributed pit-like structures are formed on the surface of the coating, with a diameter of 2-20 μm and a pit depth of 80 nm.

Example 4

A method for preparing a cell membrane coating having a pit-shaped topological structure comprises the following steps:

(1) The collected neutrophils were resuspended in 0.25× PBS buffer solution (containing 0.5 mM EDTA and 0.1 mM PMSF) and homogenized 30 times using a glass homogenizer. The subcellular organelles were removed by multiple gradient centrifugation. The precipitate was resuspended in PBS solution and then ultrasonicated to obtain a neutrophil vesicle suspension, wherein the protein concentration of the neutrophil membrane vesicle suspension was 1.5 mg/mL;

(2) mixing the cell membrane vesicle suspension obtained in step (1) with an aqueous solution of 1 mg/mL EGCG in equal volumes to prepare a cell membrane vesicle mixed solution;

(3) Add the cell membrane vesicle mixed solution obtained in step (2) into a gas-tight syringe, adjust the nozzle to 3.5 cm away from the surface of the decellularized bovine pericardium material, and spray it layer by layer on the surface of the cleaned substrate material at an ultrasonic frequency of 120 kHz, an ultrasonic power of 1.4 W, and a gas pressure of 1 psi. The propulsion rate of the cell membrane vesicle mixed solution is 0.02 mL/min, and 100 layers are sprayed. Rinse with deionized water three times;

(4) Replace the cell membrane vesicle mixed solution with deionized water and add it to the airtight syringe of the ultrasonic atomization spraying equipment. Adjust the nozzle to 5 cm away from the surface of the bovine pericardium material. Spray it layer by layer on the surface of the material at an ultrasonic frequency of 120 kHz, an ultrasonic power of 1.4 W, and a gas pressure of 3 psi. The propulsion rate of the deionized water solution is 0.01 mL/min, and 50 layers are sprayed.

(5) The cell membrane coating obtained in step (4) is dried and cured at 40°C for 30 min to obtain a cell membrane coating having a pit-like topological structure. The depth of the pit-like structure is further increased to the submicron level (600 nm), and the size of the pit-like structure is 5-10 μm.

Comparative Example

A method for preparing a polyphenol cross-linked cell membrane coating comprises the following steps:

(1) Whole blood was centrifuged to obtain red blood cells, which were washed three times with PBS solution and resuspended in pre-cooled 0.25× PBS solution (containing 0.5 mM EDTA and 0.1 mM PMSF) and placed in a 4°C refrigerator for 2 h. The solution was centrifuged at 10,000 g for 20 min, the supernatant was discarded and washed three times with PBS solution, and finally resuspended and sonicated to obtain a milky white red blood cell membrane vesicle suspension, where the protein concentration of the red blood cell membrane vesicle suspension was 4 mg/mL;

(2) mixing the cell membrane vesicle suspension obtained in step (1) with an aqueous solution of 2 mg/mL EGCG in equal volumes to prepare a cell membrane vesicle mixed solution;

(3) The cell membrane vesicle mixed solution obtained in step (2) was added to a gas-tight syringe, and the nozzle was adjusted to 5 cm away from the surface of the polylactic acid substrate. The mixed solution was sprayed layer by layer on the surface of the cleaned substrate material at an ultrasonic frequency of 120 kHz, an ultrasonic power of 1 W, and a gas pressure of 1 psi. The propulsion rate of the mixed solution of cell membrane vesicles was 0.03 mL/min. 40 layers were sprayed and washed with deionized water for 3 times to obtain a polyphenol-crosslinked red blood cell membrane coating. The electron microscope scanning image is shown in Figure 2 B. The coating surface is flat and no excess cell membrane vesicles are adsorbed on the surface.

Test Example 1 Platelet Adhesion Test

The bare polylactic acid substrate, the polylactic acid substrate modified with the erythrocyte membrane coating having a pit-like topological structure obtained in Example 1-2, and the polylactic acid substrate modified with the polyphenol cross-linked erythrocyte membrane coating obtained in the comparative example were mixed with platelet-rich plasma, respectively, and incubated at 37°C for 1 h. After the incubation, the incubated polylactic acid substrate was washed 3 times with PBS solution, and the adhesion of platelets was observed under a fluorescence microscope by fluorescence staining. The results are shown in Figure 3.

As shown in Figure 3, compared with the naked polylactic acid substrate and the polylactic acid substrate modified with the polyphenol cross-linked erythrocyte membrane coating obtained in the comparative example (see Figure A and Figure B in Figure 3), the platelets adhering to the surface of the polylactic acid substrate modified with the erythrocyte membrane coating with the pit-shaped topological structure obtained in Example 1-2 are significantly reduced (see Figure C and Figure D in Figure 3). The erythrocyte membrane coating with the pit-shaped topological structure obtained in Example 1-2 inherits the natural anticoagulant properties of the polyphenol cross-linked erythrocyte membrane coating in the comparative example, indicating that the introduction of the pit-shaped topological structure does not change the good biocompatibility of the coating.

Experimental Example 2 Endothelial cell adhesion assay

The bare polylactic acid substrate, the polylactic acid substrate modified with the erythrocyte membrane coating having the pit-like topology structure obtained in Example 1-2, and the polylactic acid substrate modified with the polyphenol cross-linked erythrocyte membrane coating obtained in the comparative example were co-cultured with endothelial cells in an incubator at 37°C and 5% _CO2 for 1 day, and the adhesion of the endothelial cells and the differences in cell morphology were observed and compared by fluorescence staining. The results are shown in Figure 4.

As shown in Figure 4, compared with the naked polylactic acid substrate and the polylactic acid substrate modified with the polyphenol cross-linked erythrocyte membrane coating obtained in the comparative example (see Figure A and Figure B in Figure 4), the surface of the polylactic acid substrate modified with the erythrocyte membrane coating with the pit-shaped topology structure obtained in Example 1-2 has good biological activity, and promotes the adhesion and growth of more endothelial cells on the surface (see Figure C and Figure D in Figure 4). The number of endothelial cells adhering to the erythrocyte membrane coating with the pit-shaped topology structure obtained in Example 1-2 is significantly increased, and the cells on the coating surface have a significantly expanded morphology, indicating that the pit-shaped topology structure affects the adhesion state of the cells on the surface, which may promote the diffusion and migration of cells on the surface of the cell membrane coating with the pit-shaped topology structure.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (10)

1. A method for preparing a cell membrane coating having a pit-like topological structure, characterized in that it comprises the following steps: (1) extracting and separating cells, resuspending them, and then breaking the cells, collecting cell membrane fragments after washing, resuspending the cell membrane fragments and performing ultrasonication to obtain a cell membrane vesicle suspension; (2) mixing the cell membrane vesicle suspension with a cross-linking agent to obtain a cell membrane vesicle mixed solution; (3) adding the cell membrane vesicle mixed solution into the syringe of the ultrasonic atomization spraying equipment, performing a first ultrasonic spraying on the surface of the substrate material, and then cleaning the surface of the substrate material after the spraying; (4) performing a second ultrasonic spraying of the surface of the substrate material obtained in step (3) with an impact solution; (5) Drying and solidifying the cell membrane coating obtained in step (4) to obtain. 2. The method for preparing a cell membrane coating with a pit-shaped topological structure according to claim 1, characterized in that the cells in step (1) are at least one of red blood cells, platelets, macrophages, neutrophils and T lymphocytes; and the protein concentration of the cell membrane vesicle suspension is 0.5-8 mg/mL. 3. The method for preparing a cell membrane coating with a pit-shaped topological structure according to claim 1, characterized in that the cross-linking agent in step (2) is at least one of a polyphenol compound, a carbodiimide compound, glutaraldehyde and genipin; and the mass ratio of the cell membrane vesicle suspension to the cross-linking agent is 1-20:1. 4. The method for preparing a cell membrane coating with a pit-shaped topological structure according to claim 1 is characterized in that the ultrasonic spraying in step (3) comprises the following steps: controlling the distance between the nozzle and the surface of the substrate material to be 2-10 cm, and then spraying 20-100 layers on the surface of the substrate material at an ultrasonic frequency of 25-250 kHz, an ultrasonic power of 0.5-10 W, a solution propulsion rate of 0.01-0.1 mL/min and a gas pressure of 0.5-1 psi. 5. The method for preparing a cell membrane coating having a pit-shaped topological structure according to claim 1, characterized in that the shock solution in step (4) is deionized water, phosphate buffer or physiological saline. 6. The method for preparing a cell membrane coating with a pit-shaped topological structure according to claim 1 is characterized in that the ultrasonic spraying in step (4) comprises the following steps: controlling the distance between the nozzle and the surface of the substrate material to be 2-10 cm, and then spraying 1-100 layers on the surface of the substrate material at an ultrasonic frequency of 25-250 kHz, an ultrasonic power of 0.5-10 W, a solution propulsion rate of 0.01-0.1 mL/min and a gas pressure of 1-3 psi. 7. The method for preparing a cell membrane coating having a pit-shaped topological structure according to claim 1 is characterized in that the substrate materials in step (3) and step (4) are metal-based biomaterials, polymer-based biomaterials or decellularized biological tissues. 8. The method for preparing a cell membrane coating having a pit-shaped topological structure according to claim 1, characterized in that the drying and curing in step (5) is carried out at a temperature of 30-60°C and for a time of 10-60 min. 9. A cell membrane coating having a pit-shaped topological structure, characterized in that it is prepared by the preparation method described in any one of claims 1 to 8. 10. Use of the cell membrane coating with a pit-like topological structure as claimed in claim 9 in the preparation of cardiovascular materials/devices.