CN118576787A - A macroscopic cell membrane coating with high interface stability and preparation method and application thereof - Google Patents

Abstract

The present invention discloses a macro-scale cell membrane coating with high interface stability, a preparation method and an application, and belongs to the technical field of biomedical materials. The present invention first prepares a cell membrane vesicle solution; then mixes the cell membrane vesicle solution with a cross-linking agent solution, and finally coats it on the surface of a hydrophilic substrate, and after drying treatment, obtains a macro-scale cell membrane coating with high interface stability. The cell membrane coating prepared by the present invention can form a complete coating on the surface of a variety of substrate materials by methods such as drip coating, perfusion, spin coating, spraying or dipping, thereby improving the anti-coagulation and anti-inflammatory properties. In addition, the cell membrane coating prepared by the present invention also has the function of promoting endothelial repair, and can be applied to the surface modification of cardiovascular implantable interventional devices such as vascular stents, heart occluders, artificial heart valves and artificial blood vessels, as well as cardiopulmonary circulation pipelines and intravenous catheters for dialysis, which significantly reduces the risk of thrombosis and the degree of inflammatory response after device implantation.

Description

A macroscopic cell membrane coating with high interface stability and preparation method and application thereof

Technical Field

The present invention relates to the technical field of biomedical materials, and in particular to a macroscopic cell membrane coating with high interface stability, a preparation method and an application thereof.

Background Art

At present, cardiovascular implants such as vascular stents, occluders, artificial heart valves and artificial blood vessels, as well as cardiopulmonary circulation circuits and intravenous catheters for dialysis are prone to problems such as poor blood compatibility and strong immune rejection reactions due to the lack of appropriate natural biological surface functions, which leads to the occurrence of various clinical complications. Improving the biocompatibility of blood-contact materials through surface modification methods is an important research direction. Although a large number of studies have been conducted to achieve biological functions such as anticoagulation and anti-inflammatory by physically and chemically modifying biomaterials, they still face problems such as poor biocompatibility, severe foreign body reactions and poor biological activity.

As a natural component in the body, the cell membrane has both good blood compatibility and diverse biological functions. The diverse membrane surface proteins give it a variety of biological functions such as immune escape and cell adhesion. Therefore, bionic cell membrane is one of the research hotspots of biomaterial surface modification. However, due to the tiny size of cell membrane vesicles, few studies have successfully achieved the assembly of cell membrane coatings at a macroscopic scale. This process depends on the fusion between cell membrane vesicles and with the material substrate. Affected by surface properties such as charge, hydrophilicity and micromorphology, it is difficult to form a complete and stable macroscopic cell membrane coating. Although the prior art has successfully achieved the formation of a complete cell membrane coating on the surface of large-scale medical devices, it still faces the problems of cumbersome pre-coating operation, weak cohesion of the cell membrane coating, and unstable connection between the cell membrane and the substrate. Under the scouring of the fluid, the integrity of the cell membrane coating is easily damaged, which limits the wide application of the cell membrane coating in blood contact materials and other medical devices.

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 macro-scale cell membrane coating with high interface stability and a preparation method and application thereof, so as to solve the problems of weak cohesion of the existing cell membrane coating and unstable connection between the cell membrane and the substrate.

The technical solution of the present invention to solve the above technical problems is as follows: a method for preparing a macroscopic cell membrane coating with high interface stability is provided, comprising the following steps:

(1) Extracting and separating cells, washing, resuspending and sonicating to obtain a cell membrane vesicle suspension;

(2) adding a water-soluble polymer to the cell membrane vesicle suspension obtained in step (1) to obtain a cell membrane vesicle solution;

(3) The cell membrane vesicle solution obtained in step (2) is mixed with a cross-linking agent solution, coated on the surface of a hydrophilic base material, and dried to obtain a cross-linking agent.

Furthermore, the cells in step (1) are at least one of red blood cells, platelets, macrophages, neutrophils and endothelial cells.

Furthermore, the water-soluble polymer is polyvinyl alcohol, sodium hyaluronate or polyacrylamide; and the concentration of the water-soluble polymer is 0.2-4 mg/mL.

Furthermore, in step (3), the hydrophilic substrate material is a metal-based biomaterial, a polymer-based biomaterial or a decellularized biological tissue; and the water contact angle of the hydrophilic substrate surface is less than 60°.

Furthermore, the cross-linking agent is at least one of polyphenol compounds, carbodiimide compounds, glutaraldehyde and genipin.

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

Furthermore, the coating method in step (3) is drip coating, pouring, spin coating, spray coating or dipping.

The invention provides a macroscopic-scale cell membrane coating with high interface stability, which is prepared by adopting the above-mentioned preparation method.

The present invention also provides an application of a macroscopic-scale cell membrane coating with high interface stability in the preparation of blood contact materials/devices.

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

The present invention has the following beneficial effects

(1) The present invention mixes a crosslinking agent solution and a cell membrane vesicle solution on the surface of a hydrophilic base material and then applies the mixture. The hydrophilic surface ensures that the mixed solution is fully infiltrated. The interaction between the crosslinking agents and between the crosslinking agents and various biological macromolecules is utilized to effectively drive the combination of cell membranes and cell membranes and cell membranes and matrix materials, thereby forming a cell membrane coating with complete coverage and good stability on the surface of large-scale devices.

(2) In the present invention, cell membrane vesicles and water-soluble polymers are mixed to obtain a cell membrane vesicle solution. The water-soluble polymer prevents spontaneous polymerization between cell membrane vesicles and increases the uniformity of cell membrane vesicle dispersion, which is beneficial to the subsequent generation of a smooth and uniform coating.

(3) The preparation method of the macro-scale cell membrane coating with high interface stability of the present invention is simple and requires mild conditions. It can inherit the natural cell membrane function and retain the functional proteins on the cell membrane surface. It can be widely used in a variety of medical device materials, greatly enhancing the anti-coagulant and anti-inflammatory properties of the material, and has good cell compatibility, supporting the growth of endothelial cells on the surface of the material.

(4) When the cell membrane vesicle solution and the cross-linking agent solution are mixed and incubated together, the quinone group formed by the oxidation of the polyphenolic compounds can undergo a covalent reaction with the free thiol group of the cell membrane surface protein, and then undergo oxidative cross-linking between the polyphenolic compounds; the carbodiimide compounds can directly couple the free carboxylic acids on the surfaces of different cell vesicles with the primary amines; glutaraldehyde and genipin can undergo a Schiff base reaction with the free amino groups on the cell membrane surface, connecting the cell membranes in the form of a five-carbon bridge, thereby forming a firm macroscopic cell membrane coating, enhancing the stability of the coating, and promoting its long-term function after implantation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a schematic diagram of the preparation process of the cell membrane coating of Examples 1-4;

FIG2 is a scanning electron microscope image of the cell membrane coating prepared in Example 1 and the comparative example; wherein FIGA is a scanning electron microscope image of the cell membrane coating prepared in the comparative example; and FIGB is a scanning electron microscope image of the cell membrane coating prepared in Example 1;

FIG3 is a scanning electron microscope image of the cell membrane coating retention after the fluid flushing test for different days in Example 1;

FIG4 is a platelet fluorescence staining image of the cell membrane coating and the naked polylactic acid material prepared in Example 2; wherein FIGA is a platelet fluorescence staining image of the naked polylactic acid material, and FIGB is a platelet fluorescence staining image of the cell membrane coating prepared in Example 2;

FIG5 is a fluorescence staining image of endothelial cells of the cell membrane coating and the naked polylactic acid material prepared in Example 2; wherein FIGA is a fluorescence staining image of endothelial cells of the naked polylactic acid material, and FIGB is a fluorescence staining image of endothelial cells of the cell membrane coating prepared in Example 2;

Figure 6 is a macrophage fluorescence staining image of the cell membrane coating and naked polylactic acid material prepared in Example 2; wherein, Figure A is a macrophage fluorescence staining image of the naked polylactic acid material, and Figure B is a macrophage fluorescence staining image of the cell membrane coating prepared in Example 2.

DETAILED DESCRIPTION

The following examples 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 conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

The preparation process of the macro-scale cell membrane coating with high interface stability of Examples 1-4 is shown in FIG1 , and the specific implementation process is as follows:

Example 1

A method for preparing a macroscopic cell membrane coating with high interface stability 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 inhibitor cocktail (EDTA-Free, 100X in DMSO) was added at a volume ratio of 1:100 and the platelets were repeatedly frozen and thawed 5 times. The platelets were washed 3 times with PBS solution, then resuspended and sonicated to obtain a platelet membrane vesicle suspension.

(2) adding polyvinyl alcohol to the platelet membrane vesicle suspension obtained in step (1) to obtain a platelet membrane vesicle solution; wherein the molecular weight of the polyvinyl alcohol is 20,000 and the final concentration is 0.5 mg/mL;

(3) The platelet membrane vesicle solution prepared in step (2) was mixed with an equal volume of a Tris-HCl buffer solution (50 mM, pH=8.5) containing 2 mg/mL epigallocatechin gallate, and the mixture was drop-coated on the cleaned titanium foil surface with a drop volume of 0.1 mL/cm ² , followed by incubation at 40°C for 2 h, and then washed with deionized water for 3 times to obtain a film, the scanning electron micrograph of which is shown in Figure 2B. As can be seen from Figure 2B, the surface of the substrate material is completely covered with a relatively flat and uniform cell membrane coating, indicating that Example 1 successfully prepared a macroscopic cell membrane coating with high interface stability.

Example 2

A method for preparing a macroscopic cell membrane coating with high interface stability 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 inhibitor cocktail (EDTA-Free, 100X in DMSO) was added at a volume ratio of 1:100 and the platelets were repeatedly frozen and thawed 5 times. The platelets were washed 3 times with PBS solution, then resuspended and sonicated to prepare a platelet membrane vesicle suspension.

(2) adding sodium hyaluronate to the platelet membrane vesicle suspension obtained in step (1) to obtain a platelet membrane vesicle solution; wherein the molecular weight of sodium hyaluronate is 10,000 and its final concentration is 0.5 mg/mL;

(3) The cleaned polylactic acid material was placed in a Tris-HCl buffer solution (50 mM, pH = 8.5) containing 2 mg/mL dopamine hydrochloride, reacted at 30 °C for 2 h, and then washed with deionized water three times;

(4) The cell membrane vesicle solution obtained in step (2) was mixed with an equal volume of a Tris-HCl buffer solution (50 mM, pH = 8.5) containing 1 mg/mL epigallocatechin gallate, and the mixture was dropwise coated on the surface of the polylactic acid material in step (3) at a drop volume of 0.1 mL/cm ² . The mixture was incubated at 40° C. for 2 h, and washed with deionized water three times to obtain a product.

Example 3

A method for preparing a macroscopic cell membrane coating with high interface stability comprises the following steps:

(1) The collected endothelial cells were dispersed in TM buffer solution (pH 7.4; 10 mM Tris + 1 mMMgCl ₂ ) at 4°C, transferred to a glass homogenizer and homogenized 30 times. Subcellular organelles were removed by multiple gradient centrifugation, washed 3 times with PBS solution, then resuspended and sonicated to obtain endothelial cell membrane vesicle suspension;

(2) adding polyacrylamide to the endothelial cell membrane vesicle suspension obtained in step (1) to obtain an endothelial cell membrane vesicle solution; wherein the molecular weight of the polyacrylamide is 10,000 and the final concentration is 0.5 mg/mL;

(3) The endothelial cell membrane vesicle solution prepared in step (2) was mixed with equal volumes of Tris-HCl buffer solution (50 mM, pH = 8.5) containing 1 mg/mL tannic acid, and the decellularized porcine pericardium material was immersed in the mixed solution at a speed of 10 mm/s, left for 30 s, then taken out at a speed of 10 mm/s, dried at 37 °C, and the above operation was repeated 5 times, and then washed with deionized water 3 times to obtain.

Example 4

A method for preparing a macroscopic cell membrane coating with high interface stability 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 inhibitor cocktail (EDTA-Free, 100X in DMSO) was added at a volume ratio of 1:100 and the platelets were repeatedly frozen and thawed 5 times. The platelets were washed 3 times with PBS solution, then resuspended and sonicated to obtain a platelet membrane vesicle suspension.

(2) adding polyvinyl alcohol to the platelet membrane vesicle suspension obtained in step (1) to obtain a platelet membrane vesicle solution; wherein the molecular weight of the polyvinyl alcohol is 20,000 and the final concentration is 0.5 mg/mL;

(3) The cleaned polylactic acid vascular stent was placed in a Tris-HCl buffer solution (50 mM, pH = 8.5) containing 2 mg/mL dopamine hydrochloride, reacted at 30 °C for 2 h, and washed with deionized water three times;

(4) The cell membrane vesicle solution obtained in step (2) was mixed with equal volumes of a Tris-HCl buffer solution (50 mM, pH = 8.5) containing 1 mg/mL epigallocatechin gallate, and the surface of the polylactic acid scaffold in step (3) was immersed in the mixed solution at a speed of 10 mm/s, left for 30 s, and taken out at a speed of 10 mm/s, dried at 37 °C, and the above operation was repeated 5 times, and then washed with deionized water 3 times to obtain a polylactic acid scaffold.

Comparative Example

A cell membrane coating without adding a water-soluble high molecular polymer 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 inhibitor cocktail (EDTA-Free, 100X in DMSO) was added at a volume ratio of 1:100 and the platelets were repeatedly frozen and thawed 5 times. The platelets were washed 3 times with PBS solution, then resuspended and sonicated to obtain a platelet membrane vesicle suspension.

(2) The platelet membrane vesicle suspension prepared in step (1) was mixed with an equal volume of a Tris-HCl buffer solution (50 mM, pH = 8.5) containing 2 mg/mL epigallocatechin gallate, and the mixture was drop-coated on the cleaned titanium foil surface with a drop volume of 0.1 mL/cm ² . The mixture was then incubated at 40°C for 2 h and then washed with deionized water for 3 times to obtain a titanium foil, the scanning electron micrograph of which is shown in Figure 2A. As can be seen from Figure 2A, the platelet vesicles aggregate together, resulting in uneven coverage of the cell membrane coating on the surface of the substrate material.

As can be seen from Figure 2, the cell membrane coating with the addition of water-soluble polymer (Example 1) covers the material surface more evenly than the cell membrane coating without the addition of water-soluble polymer, indicating that the added water-soluble polymer prevents the spontaneous polymerization between cell membrane vesicles and increases the uniformity of the dispersion of cell membrane vesicles, making the generated cell membrane coating smoother and more uniform.

Test Example 1 Simulated blood fluid flushing test

The cell membrane coating prepared in Example 1 was rolled up and placed in a 16# PVC pipe, connected to a silicone tube, 1× PBS solution was added, and the peristaltic pump rate was adjusted to a PBS solution flow rate of 100 cm/s in the PVC pipe. A fluid flushing experiment was performed, and the titanium foil was taken out at 8, 16, and 32 days, respectively. The retention of the cell membrane coating on the titanium foil surface was directly observed by scanning electron microscopy. The results are shown in Figure 3.

As shown in FIG3 , after being flushed with simulated fluid for different days, a large amount of cell membrane coating on the titanium foil surface still remains after 32 days. Therefore, the macroscopic cell membrane coating with high interface stability prepared in Example 1 has excellent stability.

Test Example 2 Platelet Adhesion Test

The cell membrane coating and naked polylactic acid material prepared in Example 2 were mixed with platelet-rich plasma, respectively, and incubated at 37° C. for 1 h. The adhesion of platelets on the cell membrane coating and naked polylactic acid material prepared in Example 2 was observed under a fluorescence microscope by fluorescence staining. The results are shown in FIG4 .

As can be seen from Figure 4, compared with the naked polylactic acid material, the platelets adhered to the surface of the high interface stability macro-scale cell membrane coating prepared in Example 2 are significantly reduced. Therefore, the high interface stability macro-scale cell membrane coating prepared by the preparation method of the present invention has better anti-coagulation performance.

Experimental Example 3 Endothelial cell adhesion assay

The cell membrane coating and naked polylactic acid material prepared in Example 2 were co-cultured with endothelial cells for 3 days respectively, and the growth of endothelial cells on the surface of the macroscopic cell membrane coating with high interface stability and naked polylactic acid material prepared in Example 2 was observed. The results are shown in FIG5 .

As can be seen from Figure 5, compared with the naked polylactic acid material, the number of endothelial cells growing on the surface of the high interface stability macro-scale cell membrane coating prepared in Example 2 is increased. Therefore, the high interface stability macro-scale cell membrane coating prepared by the preparation method of the present invention has better cell compatibility and endothelial cell growth promoting function.

Experimental Example 4 Macrophage Adhesion Assay

The cell membrane coating and naked polylactic acid material prepared in Example 2 were co-cultured with endothelial cells for 1 day, respectively, and the activation of macrophages on the surface of the macroscopic cell membrane coating with high interface stability and naked polylactic acid material prepared in Example 2 was observed. The results are shown in FIG6 .

As can be seen from Figure 6, compared with the naked polylactic acid material, the macrophages grown on the surface of the high interface stability macro-scale cell membrane coating prepared in Example 2 are not in a polarized state. Therefore, the high interface stability macro-scale cell membrane coating prepared by the preparation method of the present invention has better anti-inflammatory properties.

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 macroscopic cell membrane coating with high interface stability, characterized in that it comprises the following steps: (1) Extracting and separating cells, washing, resuspending and sonicating to obtain a cell membrane vesicle suspension; (2) adding a water-soluble polymer to the cell membrane vesicle suspension obtained in step (1) to obtain a cell membrane vesicle solution; (3) The cell membrane vesicle solution obtained in step (2) is mixed with a cross-linking agent solution, coated on the surface of a hydrophilic base material, and dried to obtain a cross-linking agent. 2. The method for preparing a macro-scale cell membrane coating with high interface stability according to claim 1, characterized in that the cells in step (1) are at least one of red blood cells, platelets, macrophages, neutrophils and endothelial cells. 3. The method for preparing a macro-scale cell membrane coating with high interface stability according to claim 1, characterized in that the water-soluble polymer is polyvinyl alcohol, sodium hyaluronate or polyacrylamide; and the concentration of the water-soluble polymer is 0.2-4 mg/mL. 4. The method for preparing a macro-scale cell membrane coating with high interface stability according to claim 1 is characterized in that the hydrophilic substrate material in step (3) is a metal-based biomaterial, a polymer-based biomaterial or a decellularized biological tissue; and the water contact angle of the hydrophilic substrate surface is less than 60°. 5. The method for preparing a macro-scale cell membrane coating with high interface stability according to claim 1, wherein the cross-linking agent is at least one of a polyphenol compound, a carbodiimide compound, glutaraldehyde and genipin. 6. The method for preparing a macro-scale cell membrane coating with high interface stability according to claim 5, characterized in that the polyphenol compound is pyrogallol, tannic acid or epigallocatechin gallate; and the carbodiimide compound is carbodiimide hydrochloride or N-cyclohexyl-N′-(2-morpholinoethyl)carbodiimide methyl p-toluenesulfonate. 7. The method for preparing a macro-scale cell membrane coating with high interface stability according to claim 1, characterized in that the coating method in step (3) is drip coating, perfusion, spin coating, spray coating or dipping. 8. A macroscopic cell membrane coating with high interface stability, characterized in that it is prepared by the preparation method described in any one of claims 1 to 7. 9. Use of the macroscopic cell membrane coating with high interface stability as claimed in claim 8 in the preparation of blood contact materials/devices. 10. Use of the macro-scale cell membrane coating with high interfacial stability according to claim 9 in the preparation of blood contact materials/devices, characterized in that the blood contact materials/devices are vascular stents, heart occluders, artificial heart valves, artificial blood vessels, cardiopulmonary circulation circuits or intravenous catheters for dialysis.