CN120053759A

CN120053759A - Polypeptide grafted cell membrane and preparation method thereof

Info

Publication number: CN120053759A
Application number: CN202311627272.XA
Authority: CN
Inventors: 袁宁; 张堃; 李建霖
Original assignee: Imeik Technology Development Co ltd
Current assignee: Imeik Technology Development Co ltd
Priority date: 2023-11-30
Filing date: 2023-11-30
Publication date: 2025-05-30

Abstract

The present invention provides a polypeptide grafted decellularized membrane and a preparation method thereof, which specifically comprises (1) preparing an amino-blocked decellularized membrane; (2) activating the carboxyl groups in the decellularized membrane; (3) modifying the decellularized membrane obtained in step (2) with thiol groups; (4) grafting the polypeptide onto the decellularized membrane: reacting the thiol-modified decellularized membrane obtained in step (3) with a maleimide-modified polypeptide to obtain a polypeptide grafted decellularized membrane. The decellularized membrane prepared by the present invention has no cytotoxicity and can effectively promote the adhesion of cells on the surface of the decellularized membrane.

Description

Polypeptide grafted cell membrane and preparation method thereof

Technical Field

The invention relates to the field of biological medicine, in particular to a polypeptide grafted cell membrane and a preparation method thereof.

Background

In tissue engineering, promoting cell adhesion to the surface of biological materials is an effective strategy to increase the material's cellular compatibility, improve tissue integration and host response. Cell adhesion can significantly affect cell fate, such as cell signaling, migration, proliferation, and differentiation. In order to impart good biocompatibility to biological materials, the most common approach is to modify extracellular matrix (ECM) proteins or polypeptides on the surface of the materials, which can promote cell adhesion. Some proteins from ECM, such as fibronectin, have been used in biological materials to promote cell adhesion, however, these naturally derived proteins have problems of immunogenicity, high preparation costs, and limited applications. Thus, some extracellular matrix protein-derived peptides, such as RGD, REDV, IKVAV, and the like, are widely used as substitutes for ECM proteins to promote cell adhesion. Some biological materials used in tissue engineering have relatively poor cell affinity, can not provide proper support for cell adhesion, have relatively weak ability to promote tissue regeneration, after the surface of the materials is modified by adopting the cell adhesion polypeptide, the biological activity molecular change of the surface can improve the interaction of specific integrin receptors on cells, so that the cells generate different behaviors and influence the activity of the cells. More importantly, cell adhesion is the starting point for various subsequent cell behaviors, and good adhesion of cells to biological materials can further promote proliferation and differentiation of cells, such as osteogenic differentiation and angiogenic differentiation, and the like, depending on cell adhesion.

The cell membrane removal is a material prepared by removing cells and antigen components from tissues and organs of animals or humans through physical, chemical or biological methods, and the like, and is widely applied to clinical repair of tissues such as esophagus, urethra, bladder, abdomen, dura mater and the like. The risk of adverse reactions such as inflammation and immune rejection at the transplanted site can be reduced after removing cells and antigen components. However, after repeated washing and complicated physical and chemical decellularization treatments, bioactive molecules such as growth factors which are originally present in tissues and promote adhesion and proliferation of endothelial cells and epithelial cells are hardly retained, so that biocompatibility of the decellularized membrane material and the capability of promoting tissue regeneration are reduced.

Patent document CN114917413B discloses a method for preparing amniotic membrane loaded with recombinant polypeptide, which comprises constructing recombinant polypeptide by fusing polypeptide RRTTTKKRRT or RRTTTKKRRTKL with specific collagen binding ability and CAG, REDV or YIGSR for promoting cell adhesion, and binding polypeptide to amniotic membrane by physical adsorption after treating amniotic membrane with aqueous solution containing recombinant polypeptide. In the invention, the epidermal cells can adhere and proliferate on the modified amniotic membrane, so that the multifaceted performance of the human amniotic membrane serving as a biomedical material is improved, including the function of promoting epithelialization. Similarly, patent document CN113490517a discloses a chimeric peptide modified decellularized porcine small intestine mucosa (SIS) material by constructing chimeric peptides using type I collagen binding peptide TKKTLRT and type III collagen binding peptide KELNLVY in combination with Hst1 peptide having a certain degree of healing promoting activity and JH8194 peptide having a certain degree of osteogenic activity and antibacterial activity, respectively, through flexible linkers. By immersing the SIS membrane in the chimeric solution, the polypeptide can physically adsorb to the membrane. The chimeric peptide modified cell membrane can effectively promote proliferation and osteogenic differentiation of BMSC cells and inhibit bacteria from growing on the surface of SIS membrane. Although physical adsorption is a feasible polypeptide grafting cell membrane removing preparation method, on one hand, the sequence length of collagen binding peptide is more than 5 amino acids, which can be even longer than that of some cell adhesion promoting peptide sequences, and the preparation cost of chimeric peptide is obviously improved, on the other hand, when the cell is combined with biological materials, the cell is firstly contacted with the surface of the materials, and most of polypeptide of the cell membrane removing prepared by the physical adsorption method is adsorbed in the materials, and the modified polypeptide presented on the surface of the cell membrane removing is limited, so that the physical adsorption method is not the optimal modification method.

Chemical modification is a method that allows more specific, more durable modification of active polypeptides to the surface of the cell membrane. Patent document CN101385870B discloses a preparation method of a polypeptide grafted decellularized tissue engineering valve/vascular stent, which uses epoxy groups of epichlorohydrin to crosslink and fix RGD (YGRDSP) polypeptide on the decellularized membrane engineering valve/vascular stent, and simultaneously, the epichlorohydrin can also crosslink with collagen, so that the finally improved decellularized tissue engineering valve/vascular stent has better biological and mechanical properties, and the adhesion and growth of seed cells are obviously improved. However, in this method, the RGD polypeptide reacts with an epoxy group mainly through an amino group at its N-terminal, and if lysine is contained in the active polypeptide, this method may also react with an amino group on lysine, so that this method lacks selectivity. And for polypeptide sequences in which some lysines have an important role in polypeptide activity, this modification approach is not applicable and would significantly reduce polypeptide activity.

In addition, regarding the loading of polypeptides in the prior art, the polypeptides can be loaded on the base materials such as collagen, hyaluronic acid and the like, but compared with other soluble polymer base materials with specific structures such as collagen, hyaluronic acid and the like, the cell-removing membrane material is composed of a plurality of components such as collagen, polysaccharide, glycoprotein, fibronectin and the like, has fine assembly and arrangement, forms a complex three-dimensional structure, is obviously different from other soluble polymer base materials such as collagen, hyaluronic acid and the like, has high difficulty in modifying functional groups on the surface of the cell-removing membrane by adopting heterogeneous reaction due to the complex structure and non-water solubility of the cell-removing membrane, has low modification efficiency, and is mainly directly combined with the cell-removing material by adopting physical adsorption or using epichlorohydrin, but the physical adsorption loading is generally only capable of retaining the polypeptides in a compact layer inside the cell-removing membrane by virtue of charge interaction, size effect and the like, is unfavorable for the exposure of active polypeptides, and has poor cell adhesion effect, and the modified polypeptides are grafted on the cell-removing membrane by using epichlorohydrin, so that the graft rate is low on the one hand, has more limitation of polypeptide types, and is difficult to be suitable for various types of polypeptides.

Disclosure of Invention

In order to overcome the defects in the prior art, the application provides a polypeptide grafted cell membrane removing active polypeptide modification method based on sulfhydryl modified cell membrane removing, which has good selectivity and compatibility with different polypeptide sequences, can efficiently graft active polypeptide onto the cell membrane removing surface, has high activity, can effectively promote the adhesion of cells on the cell membrane removing surface, and can regulate and endow different new functions of the cell membrane removing material by changing the category of the active polypeptide.

In a first aspect of the invention, there is provided a polypeptide grafted cell membrane having the structure:

wherein P is a polypeptide residue, C is an amino-blocked cell membrane,

L ₁ is absent or is

L ₂ is- (PEG) _m -or

R is selected from H, COOH, COOCH ₃ or COOCH ₂CH₃,

Y is selected from integers from 1 to 6, for example 1,2,3,4, 5 or 6;

r ₁、R₂ is independently selected from H or C ₁-C₄ alkyl (e.g., methyl, ethyl);

x is an integer from 1 to 6 (e.g., 1, 2, 3, 4, 5, 6),

N is an integer from 1 to 10 (e.g., 1,2, 3, 4, 5, 6, 7, 8, 9, 10).

Further, the polypeptide grafted cell membrane has the following structure:

In a second aspect of the present invention, there is provided a method for preparing a polypeptide graft cell membrane, comprising the steps of:

(1) Preparing amino-closed cell membrane;

(2) Activating carboxyl groups in the cell membrane;

(3) Sulfhydryl modification of the cell membrane obtained in step (2);

(4) And (3) polypeptide grafting cell membrane removal, namely reacting the sulfhydryl modified cell membrane obtained in the step (3) with maleimide modified polypeptide to obtain the polypeptide grafting cell membrane.

Further, the cell membrane is one of decellularized bovine pericardium, decellularized porcine dermal matrix or decellularized porcine small intestine mucosa.

Further, in the step (1), the amino group of the cell membrane is blocked by using an epoxy compound.

Further, the epoxy compound is selected from ethylene oxide, propylene oxide, epichlorohydrin, 1, 2-butylene oxide, 1, 4-butylene oxide, propylene oxide, and may be a diepoxide or a polyepoxide.

In one embodiment, in step (1), the deprotected cell membrane is amino-blocked with a sodium carbonate solution of glycidol.

Further, the mass ratio of the epoxy compound to the cell membrane is 1:5-15, preferably 1:10.

Further, the reaction temperature in the step (1) is 20-40 ℃ and the reaction time is 12-36h.

Further, the amino group blocking reaction is followed by a neutralization and/or washing step.

In one specific embodiment, the step (1) comprises placing the cell membrane in a 0.1M sodium carbonate solution containing 4% glycidol, stirring and reacting for 24 hours at 30 ℃ at 50rad/min, taking out the cell membrane after the reaction is completed, cleaning 3 times by using ultrapure water for 5-10 minutes each time, placing the cleaned cell membrane in a 0.1M phosphate buffer solution with pH of 5.8 for neutralization, stirring for 80-90 minutes at 25 ℃ at 50r/min +/-10 r/min, taking out the cell membrane after the completion, cleaning 3 times by using ultrapure water each time for 5-10 minutes, and freeze-drying the cell membrane for later use after the cleaning is completed.

The invention uses the amino group of the cell-free membrane to carry out covalent reaction, so as to avoid the competitive participation of the amino group of the cell-free membrane in modification reaction, facilitate the accurate modification of sulfhydryl and improve the subsequent polypeptide grafting efficiency, and simultaneously can effectively reduce the immunogenicity of the cell-free membrane and improve the safety, the effectiveness and the clinical medical value of the cell-free membrane product.

Further, the activating reagent used for activating the carboxyl groups in the cell membrane in the step (2) is 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS), the ratio of the amount of the substances of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide to the amount of the substances of N-hydroxysuccinimide is 1:0.5-2, preferably, the ratio of the amount of the substances of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide to the amount of the substances of N-hydroxysuccinimide is 1:1.

Further, the mass ratio of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide to cell membrane removal is (0.5-2.0): 1, preferably (0.75-1.6): 1.

Further, the pH of the system at the time of the activation reaction is 4.0 to 7.0 (e.g., 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0), preferably 5.5.

Further, the activation reaction time is 6 to 24 (e.g., 6, 7, 8, 10, 12, 18, 20, 24) hours, preferably 8 to 12 hours.

In one embodiment, step (2) comprises taking the amino-blocked cell membrane prepared in step (1), immersing in 10mM 2-morpholinoethanesulfonic acid (MES) buffer for 30 minutes, adding EDC and NHS, and reacting at room temperature. Then, the cell membrane was removed and placed in ultrapure water, and washed three times, with the ultrapure water being replaced every 1 hour.

The step (2) is used for activating the carboxyl of the cell removal membrane, and aims to prepare N-hydroxysuccinimide activated ester in advance, and then clean and remove redundant activating reagent, so that the reaction of sulfhydryl and unsaturated C=N double bond in carbodiimide is avoided, side reaction is reduced, and the reaction efficiency of the cell removal membrane and amino in the sulfhydryl-containing compound is improved.

Further, in the step (3), a sulfhydryl-containing compound or a salt thereof is adopted for sulfhydryl modification, wherein the sulfhydryl-containing compound is a compound containing amino groups and sulfhydryl groups at the same time, and specifically comprises sulfhydryl-containing amino acid, aliphatic amine or polyethylene glycol.

Further, the fatty amine refers to a compound containing amino groups and mercapto groups and connected by saturated fatty chains, wherein the carbon number of the fatty chains is 2-6 (such as 2, 3, 4, 5 and 6), preferably, the fatty amine containing mercapto groups comprises cysteamine or hydrochloride thereof, 3-mercapto-1-propylamine or hydrochloride thereof and 6-amino-1-mercaptohexane hydrochloride thereof;

The polyethylene glycol refers to linear or multi-branched polyethylene glycol with terminal groups comprising sulfhydryl groups and amino groups. Preferably, the thiol-group-containing polyethylene glycol structure is HS- (PEG) _m-NH₂, m=an integer from 5 to 20 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20).

The amino acid comprises cysteine or hydrochloride thereof, cysteine ethyl ester or hydrochloride thereof, homocysteine or hydrochloride thereof, penicillamine or hydrochloride thereof;

Still more preferably, the thiol-group-containing compound is preferably a thiol-group-containing amino acid or a salt thereof, and still more preferably cysteine ethyl ester hydrochloride.

Further, the mass ratio of the mercapto group-containing compound to the cell membrane removal is (0.1-2.0): 1, preferably (0.5-1.6): 1;

Further, the pH of the thiol modification reaction is 5.0 to 7.0 (e.g., 5.0, 5.5, 6.0, 6.5, 7.0), preferably 6.5, and the reaction time is 24 to 72 (e.g., 24, 48, 72) hours, preferably 24 hours.

In one embodiment, the preparation of the thiol-modified cell membrane in step (3) comprises the steps of:

Under the protection of inert gas, dissolving cysteine ethyl ester hydrochloride containing sulfhydryl compound into 10mM MES buffer solution, regulating the pH value of the solution to be between 5.0 and 7.0 by using 5N sodium hydroxide, adding the carboxyl prepared in the step (2) to activate and de-cell membrane, and stirring for reaction for 24 to 72 hours. After completion of the reaction, the reaction mixture was repeatedly washed with a solution of 12M hydrochloric acid in ultrapure water containing 0.01% v/v, and the washing liquid was replaced every 1 hour.

In particular, the inventor finds that cysteine ethyl ester hydrochloride has the advantages of proper steric hindrance, difficult oxidation, good affinity, high final sulfhydryl modification rate and the like in the reaction process of sulfhydryl modification and cell membrane removal.

Further, in the step (4), the maleimide modified polypeptide is a maleimide-containing molecule directly linked to the N-terminus of the polypeptide, or may be linked to the N-terminus of the polypeptide via an H ₂N-(PEG)_n -COOH linker, where n=1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10), preferably n=1 to 3.

Further, the maleimide modified polypeptide has the following structure:

wherein P is a polypeptide residue,

L ₁ is absent or is

X is an integer from 1 to 6 (e.g., 1, 2, 3, 4, 5, 6),

N is an integer from 1 to 10 (e.g., 1,2, 3, 4, 5, 6, 7, 8, 9, 10).

Further, the maleimide-containing molecules include 2-maleimidoacetic acid, 3-maleimidopropionic acid, 4-maleimidobutyric acid, 5-maleimidopentanoic acid or 6-maleimidocanoic acid, preferably 3-maleimidopropionic acid.

Further, the mass ratio of maleimide modified polypeptide to cell membrane removal is 5-20:1 (mg/g), preferably 10:1 (mg/g).

Further, the pH of the reaction is 4.5-7.0 (e.g., 4.5, 5.0, 5.5, 6.0, 6.5, 7.0), preferably 6.5.

Preferably, the concentration of the reactive polypeptide is 0.2-1.0mg/ml.

In one specific embodiment, the step (4) of preparing polypeptide grafted cell membrane removing step comprises the steps of dissolving the polypeptide of N-terminal modified maleimide molecule in phosphate buffer, adjusting the pH of the solution by using sodium hydroxide solution, soaking the sulfhydryl modified cell membrane obtained in the step (3) in the prepared polypeptide solution, and carrying out shaking reaction for 12-24 hours in a constant temperature shaking table. After the reaction is completed, repeatedly cleaning and eluting the cell membrane by using ultrapure water for 5-15 times, and freeze-drying to obtain the polypeptide grafted cell membrane.

Further, polypeptide grafted cell membrane is obtained by the preparation method.

Furthermore, polypeptide grafted cell membrane removal can improve cell adhesion.

Further, the length of the polypeptide is 3 amino acids or more.

Further, the polypeptide sequence comprises one of RGD (SEQ ID NO: 1), REDV (SEQ ID NO: 2) or IKVAV (SEQ ID NO: 3).

In a third aspect, the present invention provides an application of the polypeptide graft decellularized membrane or the polypeptide graft decellularized membrane prepared by the preparation method, where the application includes:

1) Preparing artificial biological tissue, artificial organ, wound dressing, tissue engineering scaffold or human body graft;

2) For tissue or organ repair.

The terms "comprising" or "includes" are used in this specification to be open-ended, having the specified components or steps described, and other specified components or steps not materially affected.

The invention has the beneficial effects that:

(1) Compared with the method, the invention develops a set of brand-new polypeptide grafting cell membrane removing process aiming at the specificity of the cell membrane removing material structure, and the characteristic that heterogeneous covalent modification reaction preferentially occurs on the cell membrane removing material surface is utilized, after the thiol active site is modified on the cell membrane removing surface, the active polypeptide is specifically modified on the cell membrane removing surface through thiol-Michael addition covalent reaction, thereby being more beneficial to the interaction of cells and the active polypeptide and being capable of effectively promoting the adhesion of cells on the cell membrane removing surface.

(2) In the prior art, covalent reaction is usually carried out by utilizing amino groups of a cell removal membrane, on one hand, the amino groups are easy to be completely reacted, on the other hand, carboxyl groups in the cell removal membrane can also competitively participate in the reaction, and the residual amino groups can always cause immunogenicity.

(3) By carrying out the stepwise reaction of carboxyl activation and mercapto compound substitution on the cell membrane with blocked amino, the reaction of mercapto with unsaturated C=N double bond in carbodiimide during one-pot condensation can be avoided, the reaction efficiency of the cell membrane with amino in mercapto compound can be improved, the mercapto modification efficiency can be improved, and the byproducts can be reduced.

(4) The sulfhydryl modified cell membrane prepared by the method adopts sulfhydryl compounds such as cysteine, cysteine ethyl ester, cysteamine and the like containing amino and sulfhydryl, has good biological compatibility, and the modified cell membrane has no cytotoxicity.

(5) The preparation method of the polypeptide grafted cell membrane has universality, high selectivity and high polypeptide activity, and can regulate and endow the cell membrane removal material with different new functions by changing the type of the active polypeptide.

Drawings

FIG. 1 is a schematic diagram of the reaction principle of the invention for preparing polypeptide graft cell membrane.

FIG. 2 is a view of FITC-labeled polypeptide graft decoon membrane, FITC-labeled polypeptide direct load at blank decoon membrane under UV (FIG. 2 a-FITC-labeled polypeptide graft decoon membrane, FIG. 2 b-blank decoon membrane directly loaded with FITC-labeled polypeptide).

Fig. 3 is a linear regression graph of test example 2.

FIG. 4 is a graph of the post-reaction solutions of examples 1 and 5 (FIG. 4 a-post-reaction solution of example 1, FIG. 4 b-post-reaction solution of example 5).

FIG. 5 is a Scanning Electron Microscope (SEM) image of the surface cell adhesion of the blank cell membrane, the polypeptide graft cell membrane of examples 8, 17-19 and the loaded polypeptide cell membrane prepared in comparative examples 1-2 (FIG. 5 a-blank cell membrane, FIG. 5 b-example 8 polypeptide graft cell membrane, FIG. 5 c-example 17 polypeptide graft cell membrane, FIG. 5 d-example 18 polypeptide graft cell membrane, FIG. 5 e-example 19 polypeptide graft cell membrane, FIG. 5 f-comparative example 1 loaded polypeptide cell membrane, FIG. 5 g-comparative example 2 loaded polypeptide cell membrane).

Detailed Description

In order to better understand the technical solutions of the present invention, the following description will clearly and completely describe the technical solutions of the embodiments of the present invention, and it is obvious that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the present invention without making any inventive effort, shall fall within the scope of the present invention.

Materials, reagents, instruments and the like used in the examples described below are commercially available unless otherwise specified.

In the embodiment of the invention, EDC is 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrogen chloride (EDC. HCl), NHS is N-hydroxysuccinimide, and MES is 2- (N-morpholinyl) ethanesulfonic acid.

Maleimide modified RGD polypeptides (MAL-GGRGDS (SEQ ID NO: 1) -NH ₂), REDV polypeptides (MAL-GGREDV (SEQ ID NO: 2) -NH ₂) and IKVAV polypeptides (MAL-GGSIKVAVSR (SEQ ID NO: 3) -NH ₂) and FITC fluorescent molecular labeled REDV peptides (GGRGDS (SEQ ID NO: 1) -FITC) were purchased from Nanjing peptide industry Biotechnology Co., ltd, using conventional solid-phase polypeptide synthesis methods. Wherein the structural formula of the REDV peptide marked by FITC fluorescent molecules is as follows:

The principle of the polypeptide grafting cell membrane removal prepared by the invention is shown in figure 1.

Preparation of thiol-modified cell membrane

Example 1:

(1) Preparing amino-closed cell membrane-removing material by taking bovine pericardium, and making it undergo the processes of sheet skin, degreasing and alkali treatment. 1 piece of 3.3g (about 3 cm. Times.3 cm, wet weight) of the cell membrane was taken and placed in a 0.1M sodium carbonate solution containing 4% glycidol, wherein the mass ratio of epichlorohydrin to cell membrane was 1:10, and the reaction was stirred at 30℃for 24 hours at 50 rad/min. After the reaction is completed, the cell membrane is removed, and the cell membrane is washed with ultrapure water for 3 times, each time for 5-10 minutes. Neutralizing the cleaned cell membrane in 0.1M phosphate buffer solution with pH of 5.8, stirring for 80-90 min at 25 ℃ at 50 r/min+/-10 r/min, taking out the cell membrane after the completion, and cleaning with ultrapure water for 3 times, each time for 5-10 min. After the washing is finished, the cell membrane is removed and freeze-dried for later use.

(2) Activation of the carboxyl groups in the cell membranes obtained in step (1) were immersed in 50ml of 10mM MES buffer pH5.5 for 30 minutes, 2.5g of EDC (50 mg/ml) was added to the solution, and 1.5g of NHS (30 mg/ml) was reacted at room temperature for 12 hours. Then, the substrate was taken out and placed in ultrapure water, and washed three times, with the ultrapure water being replaced every 1 hour, to obtain an activated cell membrane.

(3) Thiol-modification of the cell membrane obtained in step (2), taking the cell membrane obtained in step (2), placing the cell membrane in 50ml of 10mM MES buffer solution containing 100mg/ml L-cysteine ethyl ester hydrochloride and having pH of 6.5, reacting for 24 hours, taking out, and washing with ultrapure water (12M hydrochloric acid for adjusting pH to about 3.0) for 8 times, each for 1 hour. And (3) performing vacuum freeze drying to obtain sulfhydryl modified cell membrane, which is marked as A1.

Example 2

The procedure was the same as in example 1 except that "2.5 g of EDC (50 mg/ml) and 1.5g of NHS (30 mg/ml)" in step (2) were replaced with "5 g of EDC (50 mg/ml) and 3g of NHS (30 mg/ml)", and the resulting mercapto-modified cell membrane was designated as A2.

Example 3

The procedure was the same as in example 1 except that "pH6.5" in step (3) was replaced with "pH5.5", and the resulting thiol-modified cell membrane was designated A3.

Example 4

The procedure was the same as in example 1 except that "100mg/ml L-cysteine ethyl ester hydrochloride" in step (3) was replaced with "30mg/ml L-cysteine ethyl ester hydrochloride", and the resulting thiol-modified cell membrane was designated A4.

Example 5

The procedure was the same as in example 1 except that "L-cysteine ethyl ester hydrochloride" in step (3) was replaced with "cysteine hydrochloride", and the resulting thiol-modified cell membrane was designated A5.

Example 6

The procedure was the same as in example 1 except that "L-cysteine ethyl ester hydrochloride" in step (3) was replaced with "cysteamine hydrochloride", and the resulting thiol-modified cell membrane was designated A6.

Example 7

The procedure was the same as in example 1 except that "L-cysteine ethyl ester hydrochloride" in step (3) was replaced with "HS-PEG ₅-NH₂ (MW=200)", and the resulting thiol-modified cell membrane was designated A7.

(II) preparation of polypeptide graft cell membrane

Example 8

Polypeptide grafting cell membrane removal prepared by preparing 66ml of 10mM MES buffer solution containing REDV polypeptide (MAL-GGREDV (SEQ ID NO: 2) -NH ₂, modified by N-terminal 3-maleimidopropionic acid) at 0.5mg/ml, and adjusting pH to 5.5 with 1N NaOH. To the polypeptide solution was added 3.3g (about 3 cm. Times.3 cm, wet weight) of the thiol-modified cell membrane A1 prepared in example 1, and the reaction was stirred at 150rad/min for 16 hours, followed by removal of the cell membrane. Placing the cell membrane in a 1L Schottky bottle, adding 400ml of ultrapure water, putting into a 25 ℃ constant temperature shaking table 150rad/min, repeatedly cleaning for 10 times, cleaning for 1 hour each time, taking out the cell membrane, and performing vacuum freeze drying to obtain the final polypeptide grafted cell membrane.

Example 9

The procedure was the same as in example 8, except that "thiol-modified cell membrane A1 prepared in example 1 was added" was replaced with "thiol-modified cell membrane A2 prepared in example 2" to obtain the final thiol-modified cell membrane.

Example 10

The procedure was the same as in example 8, except that "thiol-modified cell membrane A1 prepared in example 1 was added" was replaced with "thiol-modified cell membrane A3 prepared in example 3 was added", to obtain the final thiol-modified cell membrane.

Example 11

The procedure was the same as in example 8, except that "thiol-modified cell membrane A1 prepared in example 1 was added" was replaced with "thiol-modified cell membrane A4 prepared in example 4", to obtain the final thiol-modified cell membrane.

Example 12

The procedure was the same as in example 8, except that "thiol-modified cell membrane A1 prepared in example 1 was added" was replaced with "thiol-modified cell membrane A5 prepared in example 5", to obtain the final thiol-modified cell membrane.

Example 13

The procedure was the same as in example 8, except that "thiol-modified cell membrane A1 prepared in example 1 was added" was replaced with "thiol-modified cell membrane A6 prepared in example 6", to obtain the final thiol-modified cell membrane.

Example 14

The procedure was the same as in example 8, except that "thiol-modified cell membrane A1 prepared in example 1 was added" was replaced with "thiol-modified cell membrane A7 prepared in example 7", to obtain the final thiol-modified cell membrane.

Example 15

The procedure was the same as in example 8 except that 0.5mg/ml of "REDV polypeptide (MAL-GGREDV (SEQ ID NO: 2) -NH ₂, N-terminal 3-maleimidopropionic acid modification) was replaced with 66ml of" REDV polypeptide (MAL-GGREDV (SEQ ID NO: 2) -NH ₂, N-terminal 3-maleimidopropionic acid modification) 1.0mg/ml, 33ml in total ", to obtain the final thiol-modified cell membrane.

Example 16

The procedure was as in example 8, except that "pH was adjusted to 5.5" was replaced with "pH was adjusted to 7", to obtain the final thiol-modified cell membrane.

Example 17

The procedure was the same as in example 8 except that the "REDV polypeptide (MAL-GGREDV (SEQ ID NO: 2) -NH ₂, modified with 3-maleimidopropionic acid at the N-terminus)" was replaced by "3-maleimidopropionic acid molecule at the N-terminus of REDV polypeptide was linked to the N-terminus of polypeptide via H ₂N-(PEG)_n -COOH linker, where n=3", to give the final thiol-modified cell membrane.

Example 18

The procedure was as in example 8, except that the "REDV polypeptide (MAL-GGREDV (SEQ ID NO: 2) -NH ₂, modified at the N-terminus with 3-maleimidopropionic acid)" was replaced with the "RGD polypeptide (MAL-GGRGDS (SEQ ID NO: 1) -NH ₂, 3-maleimidopropionic acid molecule at the N-terminus of RGD polypeptide was linked to the N-terminus of polypeptide via H ₂N-(PEG)_n -COOH linker, where n=3", to obtain the final thiol-modified cell membrane.

Example 19

The procedure was the same as in example 8, except that the "REDV polypeptide (MAL-GGREDV (SEQ ID NO: 2) -NH ₂, modified at the N-terminus by 3-maleimidopropionic acid)" was replaced with the "IKVAV polypeptide (MAL-GGSIKVAVSR (SEQ ID NO: 3) -NH ₂, modified at the N-terminus by 4-maleimidobutyric acid", to give the final thiol-modified cell membrane.

Comparative example 1

(1) Activating carboxyl in cell membrane, taking bovine pericardium, and making routine procedures of sheet skin, degreasing, alkali treatment, etc. to obtain the cell-free matrix material. 1 piece of 3.3g (about 3 cm. Times.3 cm, wet weight) of the cell membrane was immersed in 50ml of 10mM MES buffer for 30 minutes, 2.5g of EDC (50 mg/ml), 1.5g of NHS (30 mg/ml) and 5g of L-cysteine ethyl ester hydrochloride were added, the pH of the solution was adjusted to 5.5, and the reaction was carried out at room temperature for 24 hours. The matrix was then removed and placed in ultrapure water, and washed 8 times with ultrapure water (12M hydrochloric acid to adjust the pH to about 3.0) for 1 hour each. Vacuum freeze drying to obtain sulfhydryl modified cell membrane, designated as a1.

(2) Polypeptide grafting cell membrane removal 50ml of 10mM MES buffer solution containing REDV polypeptide (MAL-GGREDV (SEQ ID NO: 2) -NH ₂, modified by N-terminal 3-maleimidopropionic acid) 0.5mg/ml was prepared, and pH was adjusted to 5.5 using 1N NaOH. The thiol-modified cell membrane a1 prepared in comparative example 1 was added to the polypeptide solution, and the reaction was stirred for 16 hours at 150rad/min, followed by removal of the cell membrane. Placing the cell membrane in a 1L Schottky bottle, adding 400ml of ultrapure water, putting into a 25 ℃ constant temperature shaking table 150rad/min, repeatedly cleaning for 10 times, cleaning for 1 hour each time, taking out the cell membrane, and performing vacuum freeze drying to obtain the final polypeptide grafted cell membrane.

Comparative example 2

10MM MES buffer 50ml containing REDV polypeptide (MAL-GGREDV (SEQ ID NO: 2) -NH ₂, N-terminal 3-maleimidopropionic acid modification) 0.5mg/ml was prepared and pH was adjusted to 5.5 using 1N NaOH. Adding unmodified blank cell membrane to the polypeptide solution, stirring for reaction at 150rad/min for 16h, and taking out the cell membrane. Placing the cell membrane in a 1L Schottky bottle, adding 400ml of ultrapure water, putting into a 25 ℃ constant temperature shaking table 150rad/min, repeatedly cleaning for 10 times, cleaning for 1 hour each time, taking out the cell membrane, and performing vacuum freeze drying to obtain the final polypeptide grafted cell membrane.

(III) Performance detection

Test example 1 detection of thiol-modified cell membrane thiol-modified Rate

As a working solution, a 0.1M phosphate buffer solution having a pH of 8.0 and containing 1mM ethylenediamine tetraacetic acid (EDTA) was prepared. 185.7mg of L-cysteine ethyl ester hydrochloride is weighed and dissolved in 10ml of working solution to prepare standard solution with the thiol content of 100mM, and standard solutions with the thiol contents of 0.2, 0.5, 0.8, 1.0, 1.2 and 1.5mM are obtained through dilution and used for measuring a calibration curve. Then weighing 60.6mg of 5,5' -dithiobis (2-nitrobenzoic acid (DTNB) to be dissolved in 15ml of working solution to be used as reaction solution, in a 15ml centrifuge tube, weighing 2.5ml of working solution, 250 mu l of thiol-containing standard solution and 50 mu l of DTNB reaction solution, fully mixing uniformly by vortex, reacting for 15min, measuring absorbance at 412nm by an enzyme-labeling instrument, recording absorbance value, taking the thiol concentration in a final detection system as an abscissa, taking the absorbance value as an ordinate, carrying out linear regression analysis, calculating correlation coefficient, and obtaining a linear regression curve, wherein the regression coefficient R ² of a linear equation is 0.998, the calculated formula of curve fitting is y=A+Bx, the estimated value of parameter A is 0, the estimated value of parameter B is 13.691, R ² =0.998.

The thiol-modified cell membrane samples prepared in examples 1 to 7 and comparative example 1 were taken for thiol content detection, respectively. A predetermined amount of buffer (0.1M phosphate buffer containing 1mM EDTA, pH=8.0) was measured, and 2% v/v of Ellman reagent reaction solution (4 mg/ml) was added and mixed uniformly to prepare a detection solution. 5-10mg of thiol-modified cell membranes of examples 1-7 and comparative example 1 were weighed and placed in a 50ml centrifuge tube, the centrifuge tube was placed in a 37℃shaker (180 rad/min) to react for 24 hours after 5ml of detection solution was added, absorbance of the reaction solution at 412nm was detected, and thiol-modified rates were calculated, and the detection results are shown in Table 1.

TABLE 1 thiol content and thiol modification ratio of thiol-modified cell membranes

Test sample	Mercapto content (mmol/g)	Sulfhydryl modification rate (%)
			Blank film	0.003	/
Example 1	0.150	71%
			Example 2	0.137	65%
Example 3	0.123	58%
			Example 4	0.116	55%
Example 5	0.112	53%
			Example 6	0.099	47%
Example 7	0.108	51%
			Comparative example 1	0.015	7%

The main carboxyl reaction sites in the cell membrane are aspartic acid and glutamic acid, and the total content of the two amino acids in the dry cell membrane is about 0.35mmol/g. Since the modification of the cell membrane into a heterogeneous reaction, the reaction efficiency is low and usually occurs on the surface of the cell membrane, and usually about 60% of carboxyl sites (0.21 mmol/g) are distributed on the surface of the membrane, the thiol modification ratio is calculated according to the data.

As can be seen from Table 1, the thiol-modified cell membranes prepared in examples 1 to 7 have high thiol content, the thiol modification rate is up to 45% or more, wherein the thiol modification rate in example 1 is up to 71%, and the thiol modification rate in comparative example 1 without amino blocking is only 7%, which is far lower than that in example 1, showing that the thiol-modified cell membrane is treated with amino blocking and then reacted with thiol-containing compound, so that the participation of the amino group of the cell membrane in the modification reaction can be avoided, the accurate modification of the thiol is facilitated, the thiol modification rate is improved, and the subsequent polypeptide grafting reaction is facilitated.

It can be seen from table 1 that the thiol-group-containing compound was changed to cysteine hydrochloride (example 1) and then the amount of thiol groups in the cell membrane removed was somewhat decreased, and that after the completion of the reaction, as shown in fig. 4, a more remarkable precipitate was observed in example 5, indicating that cysteine hydrochloride was oxidized to form insoluble cystine, whereas cysteine ethyl ester hydrochloride was more resistant to steric hindrance than cysteine hydrochloride, and was less oxidized, and had the advantages of good affinity, and a high final thiol modification rate, and therefore the reaction solution was always a transparent liquid.

The cysteine ethyl ester hydrochloride (example 1) is replaced by the fatty chain sulfhydryl compound cysteamine hydrochloride (example 6), the polyethylene glycol sulfhydryl compound HS-PEG ₅-NH₂ (example 7) has lower sulfhydryl modification effect than the amino acid sulfhydryl compound (example 1, example 5), and the sulfhydryl modification effect is reduced along with the increase of the molecular weight mainly because the introduction of fatty chains in the fatty chain sulfhydryl compound affects the molecular solubility to a certain extent, so that the sulfhydryl modification amount is reduced, and the polyethylene glycol sulfhydryl compound has good solubility in water.

Overall, the above results indicate that the oxidizing property of the thiol group in the thiol-containing compound and its steric hindrance are the main factors affecting the modification effect, and that the compound is oxidized to form byproducts during the thiol oxidizing process too strongly, while the molecular steric hindrance is too large to be favorable for reacting with NHS active ester on the cell membrane surface through heterogeneous reaction. The thiol modification rate can be improved by screening specific thiol-containing compounds, such as cysteine ethyl ester hydrochloride, so that the subsequent grafting modification of the polypeptide is facilitated.

Test example 2 detection of polypeptide grafting

The maleimide amount in the polypeptide solutions prepared in examples 8 to 19 and the maleimide amount in the reaction solution from which the cell membrane was removed after the polypeptide graft cell membrane removal reaction were detected, respectively, using Amplite maleimide quantitative kit, and the polypeptide graft ratio was calculated, and the detection results are shown in Table 2.

TABLE 2 grafting yield of polypeptide graft cell membrane removal

As can be seen from Table 2, the grafting rates of the polypeptides in examples 8 to 19 are all higher than 50%, wherein the grafting rate of example 1 is highest and reaches 86%, and the grafting rate of comparative example 1 with low thiol modification rate is only 10%, which is far lower than that of example 1, which indicates that the modification rate of thiol can be improved after cell membrane removal by amino blocking treatment, and the grafting rate of the polypeptide can be improved in favor of the subsequent polypeptide grafting reaction. The amino is adopted to seal the cell membrane, so that the immunogenicity of the cell membrane can be effectively reduced, the safety and the effectiveness of the cell membrane product can be improved, and the clinical medical value of the cell membrane product can be facilitated.

In addition, as can be seen from table 2, 3-maleimidopropionic acid molecules at the N-terminus of REDV polypeptides are linked to the N-terminus of the polypeptides using H ₂N-(PEG)₃ -COOH linker (example 17), which on the one hand can increase polypeptide solubility and to some extent increase the grafting rate of the polypeptides, but on the other hand is less likely to react with NHS active ester sites in the pores of the partially decellularized membrane due to the easy entanglement of the chain length of the molecules, resulting in a lower grafting rate for example 17 than for example 8 without the use of PEG linker.

In addition, examples 18 and 19, which used RGD polypeptide and IKVAV polypeptide, respectively, showed a somewhat lower grafting rate than example 17, which was probably due to the difficulty in charge interactions between RGD polypeptide, IKVAV polypeptide and cell membrane removal, which was detrimental to the grafting reaction.

In general, the results show that the grafting efficiency can be further regulated and controlled by screening the connecting molecules at the N end of the polypeptide and the specific types of the polypeptide, and the polypeptide with the N end modified 3-maleimide propionic acid is directly used for cell membrane removal grafting, so that the grafting efficiency is higher.

Test example 3 visualization of polypeptide graft cell Membrane removal

Under ultrasonic conditions, 10mg of 3-maleimidopropionic acid modified REDV polypeptide (GGRGDS (SEQ ID NO: 1) -FITC) with FITC label was dissolved in 20ml of PBS (pH=6.9) to prepare a reaction solution for use. The thiol-modified cell membrane prepared in example 1 and the unmodified blank cell membrane were placed in 10ml of the above-described reaction solution, and stirred at room temperature overnight. After the completion of the reaction, the reaction mixture was washed with ultrapure water 5 times for 30 minutes each. The cell membrane was then placed in N, N-Dimethylformamide (DMF) and shaken on a 180rad/min shaker at 37℃overnight. Then washed with ultrapure water 2 times for 30min each. After washing, the fluorescence intensity is observed under the irradiation condition of an ultraviolet lamp, and the observation result is shown in fig. 2, and it can be seen from fig. 2 that compared with blank cell membrane without modification, the thiol-modified cell membrane is more tightly combined with REDV polypeptide, and fluorescence is more obvious, mainly because the thiol-modified cell membrane is loaded with REDV polypeptide through covalent coupling, and the blank cell membrane without modification is only loaded with REDV polypeptide through physical adsorption, so that fluorescence response is weak.

Test example 4 cytotoxicity test

Blank cell membrane removal, polypeptide grafting cell membrane removal prepared in examples 8-19 and polypeptide cell membrane removal of comparative examples 1-2 are sheared, then added into a culture medium according to a surface area ratio of 6cm ²/ml for leaching, L929 cells are cultured by taking a leaching liquid culture medium after 72 hours, cell morphology is observed under a microscope after 72 hours, and then MTT colorimetric method is used for detecting cell survival. Comparing the relative absorbance values of the cells cultured by the leaching solution and the blank culture medium, wherein the relative absorbance values are higher than 70%, so that no cytotoxicity is proved. The test results are shown in Table 3.

TABLE 3 cytotoxicity assay results

As can be seen from Table 4, the relative absorbance values of the polypeptide graft decellularized membranes of examples 8-19 were above 70% and were close to those of the blank cell membranes, indicating that the chemical reaction of polypeptide graft after amino blocking, activation and thiolation did not result in cytotoxicity of the final decellularized membrane. In addition, the relative absorbance value of the decellularized membrane in comparative examples 1-2 was also above 70%, indicating that the physically adsorbed decellularized membrane was also not cytotoxic.

Test example 5 cell proliferation assay

Blank cell membrane removal, polypeptide graft cell membrane removal prepared in examples 8-19, and polypeptide cell membrane removal of comparative examples 1-2 were cut into 12 well plate sizes and placed at the bottom of the well plate. Approximately 3 ten thousand epidermal cells were inoculated on the surface of each sample, the medium was removed after incubation in a cell culture incubator for 2 days, and the samples were rinsed 3 times with PBS for 5 minutes each. 1ml of complete medium containing 10% CCK-8 reagent was then added and incubated in the cell incubator for 1 hour. The absorbance of the medium at 450nm was then measured. High absorbance indicates a high number of surviving cells.

The results of the measurements are shown in Table 4

TABLE 4 proliferation rate of epidermal cells

As can be seen from Table 4, the polypeptides of examples 8 to 19 were grafted to remove cell membranes, and all had good epidermal cell proliferation rate, and the higher the grafting rate was, the higher the cell proliferation rate was. In addition, the RGD polypeptide grafts (example 18) and the IKVAV polypeptide (example 19) showed a decrease in the epidermal cell proliferation rate compared to the REDV polypeptide (example 17), because both of them had a lower grafting rate than example 17, but the RGD polypeptide was similar to the REDV polypeptide, and the effect of the IKVAV polypeptide grafts on promoting cell proliferation was not obvious, and therefore, the epidermal cell proliferation rate was lower than that of the RGD polypeptide. Whereas the rate of proliferation of the epidermal cells in comparative examples 1-2 was low, mainly due to poor loading effect of the polypeptide, thus resulting in low rate of proliferation of the final epidermal cells.

Test example 6 cell adhesion assay

Based on the experimental results of test example 5, we selected blank cell membrane removal, polypeptide graft cell membrane removal prepared in example 8 and examples 17 to 19, and polypeptide cell membrane removal of comparative examples 1 to 2, and carried out cell adhesion detection. The cell membrane was cut to a size of 12 well plate and placed at the bottom of the well plate. After 2 days of surface culture of L929 cells, the 12-well plate was removed, the blotted medium was discarded, gently rinsed 3 times with PBS buffer, slowly added with 2ml of 4% paraformaldehyde solution, and after 2 hours fixation at room temperature, dehydrated with 30%, 50%, 70%, 80%, 90%, 95% and 100% ethanol gradient for 60min each time, then freeze-dried, and then observed for cell adhesion and growth on the sample surface using a scanning electron microscope, and the experimental results are shown in fig. 5 (area 100 x 100 μm ² in red frame). The number of cells adhered to a surface area of about 100 x 100 μm ² in each group of cell membrane removal statistics was averaged, and the results are shown in table 5.

TABLE 5 statistics of adherent cell count per unit area (100 x 100 μm ²) in SEM pictures of cell adhesion

	Number of adherent cells
		Blank film	5
Example 8	14
		Example 17	12
Example 18	9
		Example 19	7
Comparative example 1	5
		Comparative example 2	2

As can be seen from FIGS. 5 and Table 5, the number of cells that could be adhered per unit area on the cell membrane surface of comparative examples 1-2 was 5 and 2, respectively, which were lower than the number of adhered cells of the samples of examples, mainly because the amount of polypeptide loading was low, and therefore the number of adhered cells was small. In addition, the use of REDV polypeptide grafted cell membrane (example 8, example 17) showed that more cells can adhere per unit area, indicating that the surface has better adhesion effect, and the adhesion effect increases with the increase of REDV polypeptide grafting rate. The number of cell adhesion of RGD polypeptide grafts (example 18) and IKVAV polypeptides (example 19) was lower than that of REDV polypeptides (example 17), and the number of cell adhesion was lower because the grafting rate of RGD polypeptide and IKVAV polypeptides was lower than that of RGD polypeptide grafts.

Although the present invention has been described in detail by way of preferred embodiments, the present invention is not limited thereto. Various equivalent modifications and substitutions may be made in the embodiments of the present invention by those skilled in the art without departing from the spirit and scope of the present invention, and it is intended that all such modifications and substitutions be within the scope of the present invention/be within the scope of the present invention as defined by the appended claims.

Claims

1. A polypeptide grafted cell membrane, characterized by the following structure:

wherein P is a polypeptide residue, C is an amino-blocked cell membrane,

L ₁ is absent or is

L ₂ is- (PEG) _m -or

R is selected from H, COOH, COOCH ₃ or COOCH ₂CH₃,

Y is selected from integers from 1 to 6;

R ₁、R₂ is independently selected from H or C ₁-C₄ alkyl;

x is an integer of 1 to 6,

N is an integer of 1 to 10,

M is an integer of 5 to 20.

2. The polypeptide graft decellularized membrane of claim 1, having the structure:

3. a method for producing a polypeptide graft-decellularized membrane according to any one of claims 1-2, comprising the steps of:

(1) Preparing amino-closed cell membrane;

(2) Activating carboxyl groups in the cell membrane;

(3) Sulfhydryl modification of the cell membrane obtained in step (2);

4. The method according to claim 3, wherein the step (1) comprises amino-blocking the cell membrane with an epoxy compound, preferably, the mass ratio of the epoxy compound to the cell membrane is 1:5-15, preferably, the reaction temperature in the step (1) is 20-40 ℃, the reaction time is 12-36h, and preferably, the amino-blocking reaction is followed by a neutralization and/or washing step.

5. The method according to claim 3, wherein the reagent used for activating the carboxyl group in the cell membrane in the step (2) is 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide and N-hydroxysuccinimide.

6. The process according to claim 5, wherein the mass ratio of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide to cell membrane-removed in the step (2) is (0.5-2.0): 1, preferably the pH of the system at the time of the activation reaction in the step (2) is 4.0-7.0, and preferably the activation reaction time in the step (2) is 6-24 hours.

7. The method according to claim 3, wherein the thiol-group-containing compound or a salt thereof is used for thiol-modification in the step (3), the thiol-group-containing compound is a compound containing both an amino group and a thiol group, preferably the thiol-group-containing compound is a thiol-group-containing amino acid, a fatty amine or a polyethylene glycol, preferably the thiol-group-containing compound is selected from the group consisting of cysteamine or a hydrochloride thereof, 3-mercapto-1-propylamine or a hydrochloride thereof, 6-amino-1-mercaptohexane hydrochloride, cysteine or a hydrochloride thereof, cysteine ethyl ester or a hydrochloride thereof, homocysteine or a hydrochloride thereof, penicillamine or a hydrochloride thereof, and HS- (PEG) _m-NH₂, wherein m is an integer of 5 to 20.

8. The method according to claim 7, wherein the mass ratio of the thiol-group-containing compound to the cell membrane-removed compound in the step (3) is (0.1-2.0) 1, preferably the pH of the thiol-modification reaction in the step (2) is 5.0-7.0, and preferably the reaction time in the step (2) is 24-72 hours.

9. The method of any one of claims 3-8, wherein the maleimide modified polypeptide of step (4) has the structure:

wherein P is a polypeptide residue,

L is absent or is

X is an integer of 1 to 6,

N is an integer of 1 to 10.

10. The method according to claim 9, wherein the mass ratio of maleimide modified polypeptide to cell membrane removal in step (4) is in the range of 5 to 20:1, preferably the reaction pH in step (4) is in the range of 4.5 to 7.0, and preferably the concentration of the reaction polypeptide is in the range of 0.2 to 1.0mg/ml.

11. The method according to claim 9, wherein the decellularized membrane is one of decellularized bovine pericardium, decellularized porcine dermal matrix or decellularized porcine intestinal mucosa, and preferably the polypeptide sequence comprises one of SEQ ID NO. 1, SEQ ID NO.2 or SEQ ID NO. 3.

12. Use of a polypeptide graft de-cellular membrane according to any one of claims 1-2 or a polypeptide graft de-cellular membrane according to any one of claims 3-11, wherein said use comprises:

2) For tissue or organ repair.