Polypeptide grafted decellularized membrane and preparation method thereof

Abstract

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

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and particularly to a polypeptide-grafted acellular membrane and a preparation method thereof. Background Art

[0002] In tissue engineering, promoting cell adhesion on the surface of biomaterials is an effective strategy to improve the cell compatibility of materials, and to enhance tissue integration and host response. Cell adhesion significantly affects cell fate, such as cell signal transduction, migration, proliferation and differentiation. In order to endow biomaterials with good biocompatibility, the most common method is to modify the surface of the materials with extracellular matrix (ECM) proteins or polypeptides that can promote cell adhesion. Some proteins from ECM, such as fibronectin, have been used in biomaterials to promote cell adhesion. However, these proteins of natural origin have problems such as immunogenicity and high preparation costs, and their applications are limited. Therefore, some extracellular matrix protein-derived peptides, such as RGD, REDV, and IKVAV, etc., are widely used as substitutes for ECM proteins to promote cell adhesion. Although some biomaterials applied in tissue engineering have good mechanical properties, their cell affinity is poor, and they cannot provide appropriate support for cell adhesion, and their ability to promote tissue regeneration is relatively weak. After modifying the surface of these materials with cell adhesion polypeptides, the change of bioactive molecules on this surface can improve the interaction with specific integrin receptors on cells, enabling cells to exhibit different behaviors and affecting cell activity. More importantly, cell adhesion is the starting point of various subsequent cell behaviors. Good attachment of cells on biomaterials can further promote cell proliferation and differentiation. For example, osteogenic differentiation and angiogenic differentiation both depend on cell adhesion.

[0003] An acellular membrane is a material prepared by removing cells and antigen components from animal or human tissues and organs through physical, chemical or biological methods, etc., and has currently been widely used in clinical repairs of tissues such as esophagus, urethra, bladder, abdomen, dura mater, etc. After removing cells and antigen components, the risk of adverse reactions such as inflammation and immune rejection at the transplantation site can be reduced. However, after repeated washing and complex physical and chemical decellularization treatments, almost no bioactive molecules such as growth factors that originally existed in the tissue and promoted the adhesion and proliferation of endothelial cells and epithelial cells can be retained, resulting in a decline in the biocompatibility and tissue regeneration-promoting ability of acellular membrane materials.

[0004] Patent document CN114917413B discloses a method for preparing amniotic membrane loaded with recombinant polypeptides. The method is to construct recombinant polypeptides by fusing the polypeptide RRTTTKKRRT or RRTTTKKRRTKL with specific collagen-binding ability with CAG, REDV or YIGSR that promotes cell adhesion. After treating the amniotic membrane with an aqueous solution containing the recombinant polypeptide, the polypeptide binds to the amniotic membrane by physical adsorption. In this invention, epidermal cells can adhere and proliferate on the modified amniotic membrane, improving various properties of human amniotic membrane as a biomedical material, including promoting the epithelialization function. Similarly, patent document CN113490517A discloses a decellularized porcine small intestinal submucosa (SIS) material modified with chimeric peptides. The method is to construct chimeric peptides by binding the type I collagen-binding peptide TKKTLRT and the type III collagen-binding peptide KELNLVY with the Hst1 peptide with a certain degree of healing-promoting activity and the JH8194 peptide with a certain degree of osteogenic activity and antibacterial activity through a flexible linker respectively. Immersing the SIS membrane in a solution containing the chimera, the polypeptide can be physically adsorbed into the membrane. The decellularized membrane modified with chimeric peptides in this invention can effectively promote the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs), and can also inhibit the growth of bacteria on the surface of the SIS membrane. Although physical adsorption is a feasible method for preparing polypeptide-grafted decellularized membranes, on the one hand, the sequence length of the collagen-binding peptide is more than 5 amino acids, and it may even be longer than some cell adhesion-promoting peptide sequences, significantly increasing the preparation cost of the chimeric peptide; on the other hand, when cells bind to biomaterials, they first come into contact with the material surface, and most of the polypeptides in the decellularized membrane prepared by the physical adsorption method are adsorbed inside the material, and the modified polypeptides presented on the surface of the decellularized membrane are limited. Therefore, the physical adsorption method is not the best modification method.

[0005] The chemical modification method is a method that can modify active polypeptides onto the surface of decellularized membranes more specifically and durably. Patent document CN101385870B discloses a method for preparing a polypeptide-grafted decellularized tissue engineering valve / vascular scaffold. This method uses the epoxy group of epichlorohydrin to crosslink and fix the RGD (YGRGDSP) polypeptide on the decellularized membrane engineering valve / vascular scaffold. At the same time, epichlorohydrin can also crosslink with collagen, making the finally modified decellularized tissue engineering valve / vascular scaffold have better biological and mechanical properties, significantly improving the adhesion and growth of seeded cells. However, in this method, the RGD polypeptide mainly reacts with the epoxy group through the amino group at its N-terminus. If the active polypeptide contains lysine, this method may also react with the amino group on lysine. Therefore, this method lacks selectivity. And for some polypeptide sequences where lysine plays an important role in polypeptide activity, this modification method is not applicable and will significantly reduce polypeptide activity.

[0006] In addition, in the prior art regarding the loading of polypeptides, it can be loaded onto substrates such as collagen and hyaluronic acid. However, compared with other soluble polymer substrates with specific structures such as collagen and hyaluronic acid, the decellularized membrane material is composed of multiple components such as collagen, polysaccharides, glycoproteins, and fibronectin, with fine assembly and arrangement, forming a complex three-dimensional structure, which is significantly different from other soluble polymer substrates such as collagen and hyaluronic acid. Due to the complex structure and non-water solubility of the decellularized membrane, it is difficult to modify functional groups on the surface of the decellularized membrane by heterogeneous reaction, and the modification efficiency is low. Therefore, most of them directly use physical adsorption or use epichlorohydrin to bind polypeptides to the decellularized material. However, for physical adsorption loading, polypeptides can usually only be retained in the dense layer inside the decellularized membrane through charge interaction, size effect, etc., which is not conducive to the exposure of active polypeptides and the cell adhesion effect is poor; when using epichlorohydrin to modify polypeptides and then graft them onto the decellularized membrane, on the one hand, the grafting rate is low, and on the other hand, there are many restrictions on the types of polypeptides, making it difficult to apply to various types of polypeptides. Summary of the Invention

[0007] To overcome the deficiencies in the prior art, the present application provides a polypeptide-grafted decellularized membrane and a method for modifying active polypeptides based on thiol-modified decellularized membranes. This method has good selectivity, is compatible with different polypeptide sequences, can efficiently graft active polypeptides onto the surface of the decellularized membrane. In the obtained polypeptide-grafted decellularized membrane, the polypeptide has high activity, can effectively promote cell adhesion on the surface of the decellularized membrane, and can regulate and endow the decellularized membrane material with different new functions by changing the types of active polypeptides.

[0008] In the first aspect of the present invention, there is provided a polypeptide-grafted decellularized membrane having the following structure:

[0009]

[0010] Wherein, P is a polypeptide residue, and C is an amino-blocked decellularized membrane.

[0011] L 1 Does not exist or is

[0012] L 2 -(PEG) m - or

[0013] R is selected from H, COOH, COOCH 3 or COOCH 2 CH 3 ,

[0014] y is an integer from 1 to 6, for example, 1, 2, 3, 4, 5, or 6;

[0015] R1 , R 2 is independently selected from H or C 1 -C 4 alkyl (such as methyl, ethyl);

[0016] x is an integer from 1 to 6 (such as 1, 2, 3, 4, 5, 6),

[0017] n is an integer from 1 to 10 (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10).

[0018] Furthermore, the polypeptide-grafted decellularized membrane has the following structure:

[0019]

[0020] In a second aspect of the present invention, a method for preparing a polypeptide-grafted decellularized membrane is provided, comprising the following steps:

[0021] (1) Prepare an amino-blocked decellularized membrane;

[0022] (2) Activate the carboxyl groups in the decellularized membrane;

[0023] (3) Modify the decellularized membrane obtained in step (2) with a thiol group;

[0024] (4) Polypeptide grafting on the decellularized membrane: React the thiol-modified decellularized membrane obtained in step (3) with a maleimide-modified polypeptide to obtain a polypeptide-grafted decellularized membrane.

[0025] Furthermore, the decellularized membrane is one of a decellularized bovine pericardium, a decellularized porcine dermal matrix, or a decellularized porcine small intestinal submucosa.

[0026] Furthermore, in step (1), an epoxide is used to block the amino groups of the decellularized membrane.

[0027] Furthermore, the epoxide is selected from ethylene oxide, propylene oxide, epichlorohydrin, 1,2-epoxybutane, 1,4-epoxybutane, epipropanol, and can also be a bicyclic epoxide or a polycyclic epoxide.

[0028] In one embodiment, in step (1), a sodium carbonate solution of epipropanol is used to block the amino groups of the decellularized membrane.

[0029] Furthermore, the mass ratio of the epoxide to the decellularized membrane is 1:5 - 15; preferably 1:10.

[0030] Furthermore, in step (1), the reaction temperature is 20 - 40 °C and the reaction time is 12 - 36 h.

[0031] Furthermore, after the amino group blocking reaction, a neutralization and / or washing step is also included.

[0032] In a specific embodiment, step (1) includes placing the decellularized membrane in a 0.1 M sodium carbonate solution containing 4% glycidol, stirring and reacting at 50 rad / min at 30 °C for 24 hours. After the reaction is completed, the decellularized membrane is taken out and washed 3 times with ultrapure water for 5 - 10 minutes each time. The washed decellularized membrane is placed in a 0.1 M phosphate buffer solution with a pH of 5.8 for neutralization, and stirred at 50 r / min ± 10 r / min at 25 °C for 80 - 90 min. After completion, the decellularized membrane is taken out and washed 3 times with ultrapure water for 5 - 10 minutes each time. After the washing is completed, the decellularized membrane is freeze-dried and reserved for use.

[0033] The prior art usually uses the amino groups of the decellularized membrane for covalent reactions. On the one hand, it is easy for the amino groups not to be completely reacted. On the other hand, the carboxyl groups in the decellularized membrane will also competitively participate in the reaction, and the remaining amino groups often cause immunogenicity. In the present invention, by first blocking the amino groups of the decellularized membrane and using the carboxyl groups of the decellularized membrane for covalent reactions, the competitive participation of the amino groups of the decellularized membrane itself in the modification reaction is avoided, which is beneficial to the precise modification of thiol groups and improves the subsequent polypeptide grafting efficiency; at the same time, it can also effectively reduce the immunogenicity of the decellularized membrane and improve the safety, effectiveness and clinical medical value of the decellularized membrane product.

[0034] Furthermore, the activating reagent used to activate the carboxyl groups in the decellularized membrane in step (2) is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS). The molar ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide to N-hydroxysuccinimide is 1:0.5 - 2. Preferably, the molar ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide to N-hydroxysuccinimide is 1:1.

[0035] Furthermore, the mass ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide to the decellularized membrane is (0.5 - 2.0):1, preferably (0.75 - 1.6):1.

[0036] Furthermore, the pH of the system during the activation reaction is 4.0 - 7.0 (such as 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0), preferably 5.5.

[0037] Furthermore, the activation reaction time is 6 - 24 (such as 6, 7, 8, 10, 12, 18, 20, 24) hours, preferably 8 - 12 hours.

[0038] In a specific embodiment, step (2) includes taking the amino-group-blocked de-cellularized membrane prepared in step (1), soaking it in a 10 mM 2-morpholinoethanesulfonic acid (MES) buffer for 30 minutes, adding EDC and NHS, and reacting at room temperature. Then, take out the de-cellularized membrane and place it in ultrapure water, wash it three times, and change the ultrapure water every 1 hour.

[0039] The purpose of carboxyl activation of the de-cellularized membrane in step (2) is to pre-prepare N-hydroxysuccinimide-activated ester, and then remove the excess activation reagent through washing, to avoid the reaction of the thiol group with the unsaturated C=N double bond in carbodiimide, reduce side reactions and improve the reaction efficiency between the de-cellularized membrane and the amino group in the thiol-containing compound.

[0040] Furthermore, in step (3), a thiol-containing compound or its salt is used for thiol modification. The thiol-containing compound is a compound containing both an amino group and a thiol group, specifically including thiol-containing amino acids, fatty amines or polyethylene glycols.

[0041] Furthermore, the fatty amine refers to a compound in which the amino group and the thiol group are connected by a saturated fatty chain, and the number of carbon atoms in the fatty chain is 2-6 (such as 2, 3, 4, 5, 6); preferably, the thiol-containing fatty amine includes cysteamine or its hydrochloride, 3-mercapto-1-propylamine or its hydrochloride, 6-amino-1-mercaptohexane hydrochloride;

[0042] The polyethylene glycol refers to a linear or multi-branched polyethylene glycol with thiol and amino groups at the end groups. Preferably, the structure of the thiol-containing polyethylene glycol is HS-(PEG) m -NH 2 , where m is an integer from 5 to 20 (such as 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20).

[0043] The amino acids include cysteine or its hydrochloride, cysteine ethyl ester or its hydrochloride, homocysteine or its hydrochloride, penicillamine or its hydrochloride;

[0044] Even more preferably, the thiol-containing compound is preferably a thiol-containing amino acid or its salt, and further preferably cysteine ethyl ester hydrochloride.

[0045] Furthermore, the mass ratio of the thiol-containing compound to the de-cellularized membrane is (0.1-2.0):1, preferably (0.5-1.6):1;

[0046] Furthermore, the pH of the thiol modification reaction is 5.0-7.0 (such as 5.0, 5.5, 6.0, 6.5, 7.0), preferably 6.5, and the reaction time is 24-72 (such as 24, 48, 72) hours, preferably 24 hours.

[0047] In a specific embodiment, the preparation of the thiol-modified acellular membrane in step (3) includes the following steps:

[0048] Under the protection of an inert gas, dissolve the thiol-containing compound cysteine ethyl ester hydrochloride in 10 mM MES buffer, adjust the pH value of the solution to between 5.0 and 7.0 using 5N sodium hydroxide, add the carboxyl-activated acellular membrane prepared in step (2), and stir and react for 24-72 hours. After the reaction is completed, repeatedly wash with an ultrapure aqueous solution of 12M hydrochloric acid containing 0.01% v / v, and change the washing solution every 1 hour.

[0049] In particular, the inventors found that cysteine ethyl ester hydrochloride has advantages such as appropriate steric hindrance, being not easily oxidized, good affinity, and a high final thiol modification rate during the reaction of thiol-modifying the acellular membrane.

[0050] Further, in step (4), the maleimide-modified polypeptide is such that the maleimide-containing molecule is directly connected to the N-terminus of the polypeptide, or can be connected to the N-terminus of the polypeptide through an H 2 N-(PEG) n -COOH linker, where n = 1-10 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10), preferably n = 1-3.

[0051] Further, the maleimide-modified polypeptide has the following structure:

[0052]

[0053] Among them, P is a polypeptide residue,

[0054] L 1 is absent or is

[0055] x is an integer from 1 to 6 (for example, 1, 2, 3, 4, 5, 6),

[0056] n is an integer from 1 to 10 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10).

[0057] Further, the maleimide-containing molecule includes 2-maleimidoglycolic acid, 3-maleimidopropionic acid, 4-maleimidobutyric acid, 5-maleimidovaleric acid, or 6-maleimidohexanoic acid, preferably 3-maleimidopropionic acid.

[0058] Further, the mass ratio of the maleimide-modified polypeptide to the acellular membrane is 5-20:1 (mg / g), preferably 10:1 (mg / g).

[0059] Further, the pH of the reaction is 4.5 - 7.0 (such as 4.5, 5.0, 5.5, 6.0, 6.5, 7.0), preferably 6.5.

[0060] Preferably, the concentration of the reaction polypeptide is 0.2 - 1.0 mg / ml.

[0061] In a specific embodiment, the steps for preparing the polypeptide-grafted acellular membrane in step (4) are as follows: Dissolve the polypeptide with a maleimide molecule modified at the N-terminus in phosphate buffer. After adjusting the pH of the solution with sodium hydroxide solution, immerse the thiol-modified acellular membrane prepared in step (3) in the prepared polypeptide solution, and place it in a constant temperature shaker for oscillating reaction for 12 - 24 hours. After the reaction is completed, wash the acellular membrane repeatedly with ultrapure water for 5 - 15 times, and prepare the polypeptide-grafted acellular membrane after freeze-drying.

[0062] Further, the polypeptide-grafted acellular membrane is prepared by the above preparation method.

[0063] Further, the acellular membrane grafted with polypeptide can improve cell adhesion performance.

[0064] Further, the polypeptide has a length of 3 amino acids or more.

[0065] Further, the polypeptide sequence includes one of RGD (SEQ ID NO:1), REDV (SEQ ID NO:2), or IKVAV (SEQ ID NO:3).

[0066] In the third aspect of the present invention, there is provided the application of the above polypeptide-grafted acellular membrane or the polypeptide-grafted acellular membrane prepared by the above preparation method, and the application includes:

[0067] 1) Preparing artificial biological tissues, artificial organs, wound dressings, tissue engineering scaffolds, or human grafts;

[0068] 2) For tissue or organ repair.

[0069] The term "comprising" or "including" in the present invention is an open-ended description, including the specified components or steps described, as well as other specified components or steps that will not be substantially affected.

[0070] The beneficial effects of the present invention:

[0071] (1) For the physical adsorption method in the prior art to load polypeptides, usually only through charge interaction, size effect, etc., the polypeptides can only be retained in the dense layer inside the decellularized cell membrane, which is not conducive to the exposure of active polypeptides and results in poor cell adhesion effect. Compared with this method, in view of the particularity of the structure of the decellularized cell membrane material, the present invention has developed a brand-new polypeptide grafting decellularized cell membrane process. By utilizing the characteristic that the heterogeneous covalent modification reaction preferentially occurs on the surface of the decellularized cell membrane material, after modifying the thiol active sites on the surface of the decellularized cell membrane, the active polypeptides are specifically modified on the surface of the decellularized cell membrane through the thiol-Michael addition covalent reaction, which is more conducive to the interaction between cells and active polypeptides and can effectively promote cell adhesion on the surface of the decellularized cell membrane.

[0072] (2) In the prior art, the amino groups of the decellularized cell membrane are usually used for covalent reactions. On the one hand, it is easy for the amino groups not to be completely reacted. On the other hand, the carboxyl groups in the decellularized cell membrane will also competitively participate in the reaction, and the residual amino groups often cause immunogenicity. Compared with this method, the present invention first adopts amino group blocking and uses the carboxyl groups of the decellularized cell membrane for covalent reactions, thereby avoiding the competitive participation of the amino groups of the decellularized cell membrane itself in the modification reaction, facilitating the precise modification of thiols, improving the subsequent polypeptide grafting efficiency; at the same time, it can also effectively reduce the immunogenicity of the decellularized cell membrane and improve the safety, effectiveness and clinical medical value of the decellularized cell membrane products.

[0073] (3) By carrying out stepwise reactions of carboxyl activation and thiol compound substitution on the decellularized cell membrane after amino group blocking, it is possible to avoid the reaction of thiols with the unsaturated C=N double bond in carbodiimide during the one-pot condensation method, improve the reaction efficiency between the decellularized cell membrane and the amino groups in the thiol-containing compound, improve the thiol modification efficiency and reduce by-products.

[0074] (4) For the prepared thiol-modified decellularized cell membrane, thiol compounds such as cysteine, cysteine ethyl ester and cysteamine that contain both amino groups and thiols have good biocompatibility, and the modified decellularized cell membrane has no cytotoxicity.

[0075] (5) The preparation method of the polypeptide grafting decellularized cell membrane described in the present invention has universality and high selectivity, and the polypeptides have high activity. By changing the types of active polypeptides, different new functions can also be regulated and imparted to the decellularized cell membrane material. Brief Description of the Drawings

[0076] Figure 1 It is a schematic diagram of the reaction principle for preparing the polypeptide grafting decellularized cell membrane of the present invention.

[0077] Figure 2 It is the observation diagram under ultraviolet light of the FITC-labeled polypeptide grafting decellularized cell membrane and the FITC-labeled polypeptide directly loaded on the blank decellularized cell membrane ( Figure 2 a - FITC-labeled polypeptide grafting decellularized cell membrane, Figure 2b - Blank cell membrane - removed carriers directly loaded with FITC - labeled polypeptides

[0078] Figure 3 It is the linear regression curve graph of Test Example 2.

[0079] Figure 4 It is the solution graph after the reactions of Example 1 and 5 ( Figure 4 a - The solution after the reaction of Example 1, Figure 4 b - The solution after the reaction of Example 5).

[0080] Figure 5 It is the scanning electron microscope (SEM) graph of the surface cell adhesion of blank cell membrane - removed carriers, polypeptide - grafted cell membrane - removed carriers prepared in Example 8, Examples 17 - 19, and loaded - polypeptide cell membrane - removed carriers prepared in Comparative Examples 1 - 2 ( Figure 5 a - Blank cell membrane - removed carriers, Figure 5 b - Polypeptide - grafted cell membrane - removed carriers of Example 8, Figure 5 c - Polypeptide - grafted cell membrane - removed carriers of Example 17, Figure 5 d - Polypeptide - grafted cell membrane - removed carriers of Example 18, Figure 5 e - Polypeptide - grafted cell membrane - removed carriers of Example 19, Figure 5 f - Loaded - polypeptide cell membrane - removed carriers of Comparative Example 1, Figure 5 g - Loaded - polypeptide cell membrane - removed carriers of Comparative Example 2). Detailed implementation manners

[0081] In order to enable those skilled in the art to better understand the technical solutions in the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts shall fall within the protection scope of the present invention.

[0082] The materials, reagents, instruments, etc. used in the following embodiments can be obtained from commercial channels without special instructions.

[0083] In the embodiments of the present invention, EDC is 1 - (3 - dimethylaminopropyl) - 3 - ethylcarbodiimide hydrochloride (EDC·HCl); NHS is N - hydroxysuccinimide; MES is 2 - (N - morpholino)ethanesulfonic acid.

[0084] Maleimide - modified RGD polypeptide (MAL - GGRGDS(SEQ ID NO:1) - NH 2 ) and REDV polypeptide (MAL - GGREDV(SEQ ID NO:2) - NH 2) and the IKVAV polypeptide (MAL-GGSIKVAVSR (SEQ ID NO:3)-NH 2 ) and the REDV peptide labeled with FITC fluorescent molecule (GGRGDS (SEQ ID NO:1)-FITC) were purchased from Nanjing Peptide Biotechnology Co., Ltd. and prepared by the conventional solid-phase polypeptide synthesis method. The structural formula of the REDV peptide labeled with FITC fluorescent molecule is:

[0085] The principle of preparing the polypeptide grafted acellular membrane of the present invention is as Figure 1 shown.

[0086] (I) Preparation of thiol-modified acellular membrane

[0087] Example 1:

[0088] (1) Preparation of amino-blocked acellular membrane: Take bovine pericardium, and prepare the acellular membrane material after slicing, degreasing, and alkali treatment. Take 1 piece of acellular membrane (about 3 cm×3 cm, wet weight 3.3 g) and place it in a 0.1 M sodium carbonate solution containing 4% epichlorohydrin, where the mass ratio of epichlorohydrin to acellular membrane is 1:10. Stir and react at 30°C at 50 rad / min for 24 hours. After the reaction is completed, take out the acellular membrane and wash it 3 times with ultrapure water, 5 - 10 minutes each time. Place the washed acellular membrane in a 0.1 M phosphate buffer solution with pH 5.8 for neutralization, stir at 50 r / min ± 10 r / min at 25°C for 80 - 90 min, and after completion, take out the acellular membrane and wash it 3 times with ultrapure water, 5 - 10 minutes each time. After washing, freeze-dry the acellular membrane for later use.

[0089] (2) Activate the carboxyl groups in the acellular membrane: Take the acellular membrane obtained in step (1) and soak it in 50 ml of 10 mM MES buffer solution with pH 5.5 for 30 minutes, add 2.5 g of EDC (50 mg / ml) and 1.5 g of NHS (30 mg / ml), and react at room temperature for 12 hours. Then take out the matrix and place it in ultrapure water, wash it three times, and change the ultrapure water every 1 hour to obtain the activated acellular membrane.

[0090] (3) Thiol-modify the acellular membrane obtained in step (2): Take the acellular membrane obtained in step (2) and place it in 50 ml of 10 mM MES buffer solution with pH 6.5 containing 100 mg / ml L-cysteine ethyl ester hydrochloride, react for 24 hours, then take it out and wash it 8 times with ultrapure water (adjust the pH to about 3.0 with 12 M hydrochloric acid), 1 hour each time. Vacuum freeze-dry to obtain the thiol-modified acellular membrane, denoted as A1.

[0091] Example 2

[0092] The process steps are the same as those in Example 1, except that in step (2), "add 2.5 g of EDC (50 mg / ml) and 1.5 g of NHS (30 mg / ml)" is replaced with "add 5 g of EDC (50 mg / ml) and 3 g of NHS (30 mg / ml)", and the obtained thiol-modified decellularized membrane is denoted as A2.

[0093] Example 3

[0094] The process steps are the same as those in Example 1, except that in step (3), "pH 6.5" is replaced with "pH 5.5", and the obtained thiol-modified decellularized membrane is denoted as A3.

[0095] Example 4

[0096] The process steps are the same as those in Example 1, except that in step (3), "100 mg / ml L-cysteine ethyl ester hydrochloride" is replaced with "30 mg / ml L-cysteine ethyl ester hydrochloride", and the obtained thiol-modified decellularized membrane is denoted as A4.

[0097] Example 5

[0098] The process steps are the same as those in Example 1, except that in step (3), "L-cysteine ethyl ester hydrochloride" is replaced with "cysteine hydrochloride", and the obtained thiol-modified decellularized membrane is denoted as A5.

[0099] Example 6

[0100] The process steps are the same as those in Example 1, except that in step (3), "L-cysteine ethyl ester hydrochloride" is replaced with "cysteamine hydrochloride", and the obtained thiol-modified decellularized membrane is denoted as A6.

[0101] Example 7

[0102] The process steps are the same as those in Example 1, except that in step (3), "L-cysteine ethyl ester hydrochloride" is replaced with "HS-PEG 5 -NH 2 (MW = 200)", and the obtained thiol-modified decellularized membrane is denoted as A7.

[0103] (II) Preparation of polypeptide-grafted decellularized membrane

[0104] Example 8

[0105] Polypeptide-grafted decellularized membrane: Prepare a solution containing REDV polypeptide (MAL-GGREDV (SEQ ID NO:2)-NH 2, 10 mM MES buffer (N-terminal 3-maleimidopropionic acid modified) 66 ml at 0.5 mg / ml, adjust the pH to 5.5 using 1 N NaOH. Add 3.3 g (about 3 cm × 3 cm, wet weight) of the thiol-modified cell membrane-depleted A1 prepared in Example 1 to the polypeptide solution, stir and react at 150 rad / min for 16 h, and then take out the cell membrane-depleted material. Place the cell membrane-depleted material in a 1 L Schott bottle, add 400 ml of ultrapure water, and put it in a constant temperature shaker at 25°C and shake at 150 rad / min for 10 times, 1 hour each time. Then take out the cell membrane-depleted material and vacuum freeze-dry to obtain the final polypeptide-grafted cell membrane-depleted material.

[0106] Example 9

[0107] The process steps are the same as those in Example 8, except that "add the thiol-modified cell membrane-depleted A1 prepared in Example 1" is replaced with "add the thiol-modified cell membrane-depleted A2 prepared in Example 2" to obtain the final thiol-modified cell membrane-depleted material.

[0108] Example 10

[0109] The process steps are the same as those in Example 8, except that "add the thiol-modified cell membrane-depleted A1 prepared in Example 1" is replaced with "add the thiol-modified cell membrane-depleted A3 prepared in Example 3" to obtain the final thiol-modified cell membrane-depleted material.

[0110] Example 11

[0111] The process steps are the same as those in Example 8, except that "add the thiol-modified cell membrane-depleted A1 prepared in Example 1" is replaced with "add the thiol-modified cell membrane-depleted A4 prepared in Example 4" to obtain the final thiol-modified cell membrane-depleted material.

[0112] Example 12

[0113] The process steps are the same as those in Example 8, except that "add the thiol-modified cell membrane-depleted A1 prepared in Example 1" is replaced with "add the thiol-modified cell membrane-depleted A5 prepared in Example 5" to obtain the final thiol-modified cell membrane-depleted material.

[0114] Example 13

[0115] The process steps are the same as those in Example 8, except that "add the thiol-modified cell membrane-depleted A1 prepared in Example 1" is replaced with "add the thiol-modified cell membrane-depleted A6 prepared in Example 6" to obtain the final thiol-modified cell membrane-depleted material.

[0116] Example 14

[0117] The process steps are the same as those in Example 8, except that "add the thiol-modified cell membrane-depleted A1 prepared in Example 1" is replaced with "add the thiol-modified cell membrane-depleted A7 prepared in Example 7" to obtain the final thiol-modified cell membrane-depleted material.

[0118] Example 15

[0119] The process steps are the same as those in Example 8, except that “REDV polypeptide (MAL-GGREDV (SEQ ID NO: 2)-NH 2 , N-terminally modified with 3-maleimidopropionic acid) 0.5 mg / ml, a total of 66 ml” is replaced with “REDV polypeptide (MAL-GGREDV (SEQ ID NO: 2)-NH 2 , N-terminally modified with 3-maleimidopropionic acid) 1.0 mg / ml, a total of 33 ml” to obtain the final thiol-modified decellularized membrane.

[0120] Example 16

[0121] The process steps are the same as those in Example 8, except that “Adjust the pH to 5.5” is replaced with “Adjust the pH to 7” to obtain the final thiol-modified decellularized membrane.

[0122] Example 17

[0123] The process steps are the same as those in Example 8, except that “REDV polypeptide (MAL-GGREDV (SEQ ID NO: 2)-NH 2 , N-terminally modified with 3-maleimidopropionic acid)” is replaced with “The 3-maleimidopropionic acid molecule at the N-terminus of the REDV polypeptide is linked to the N-terminus of the polypeptide through an H 2 N-(PEG) n -COOH linker, where n = 3” to obtain the final thiol-modified decellularized membrane.

[0124] Example 18

[0125] The process steps are the same as those in Example 8, except that “REDV polypeptide (MAL-GGREDV (SEQ ID NO: 2)-NH 2 , N-terminally modified with 3-maleimidopropionic acid)” is replaced with “RGD polypeptide (MAL-GGRGDS (SEQ ID NO: 1)-NH 2 , and the 3-maleimidopropionic acid molecule at the N-terminus of the RGD polypeptide is linked to the N-terminus of the polypeptide through an H 2 N-(PEG) n -COOH linker, where n = 3” to obtain the final thiol-modified decellularized membrane.

[0126] Example 19

[0127] The process steps are the same as those in Example 8, except that “REDV polypeptide (MAL-GGREDV (SEQ ID NO: 2)-NH 2" replaced with "IKVAV polypeptide (MAL-GGSIKVAVSR (SEQ ID NO: 3)-NH 2 " and the N-terminus was modified with 4-maleimidobutyric acid" to obtain the final thiol-modified acellular membrane.

[0128] Comparative Example 1

[0129] (1) Activation of carboxyl groups in the acellular membrane: Bovine pericardium was taken and processed through conventional processes such as slicing, degreasing, and alkali treatment to obtain an acellular matrix material. One piece of acellular membrane weighing 3.3 g (about 3 cm × 3 cm, wet weight) was placed in 50 ml of 10 mM MES buffer and soaked for 30 minutes. Then, 2.5 g of EDC (50 mg / ml), 1.5 g of NHS (30 mg / ml), and 5 g of L-cysteine ethyl ester hydrochloride were added, and the pH of the solution was adjusted to 5.5. The reaction was carried out at room temperature for 24 hours. Then, the matrix was taken out and placed in ultrapure water, and washed 8 times with ultrapure water (adjusted to about pH 3.0 with 12 M hydrochloric acid), 1 hour each time. Vacuum freeze-drying was performed to obtain the thiol-modified acellular membrane, denoted as a1.

[0130] (2) Polypeptide grafting onto the acellular membrane: Prepare 50 ml of 10 mM MES buffer containing REDV polypeptide (MAL-GGREDV (SEQ ID NO: 2)-NH 2 " and the N-terminus was modified with 3-maleimidopropionic acid) at a concentration of 0.5 mg / ml, and adjust the pH to 5.5 using 1N NaOH. Add the thiol-modified acellular membrane a1 prepared in Comparative Example 1 to the polypeptide solution, and stir the reaction at 150 rad / min for 16 h, then take out the acellular membrane. Place the acellular membrane in a 1 L Schott bottle, add 400 ml of ultrapure water, and place it in a constant temperature shaker at 25 °C and stir at 150 rad / min for 10 times, 1 hour each time. Then, take out the acellular membrane and perform vacuum freeze-drying to obtain the final polypeptide-grafted acellular membrane.

[0131] Comparative Example 2

[0132] Prepare 50 ml of 10 mM MES buffer containing REDV polypeptide (MAL-GGREDV (SEQ ID NO: 2)-NH 2 " and the N-terminus was modified with 3-maleimidopropionic acid) at a concentration of 0.5 mg / ml, and adjust the pH to 5.5 using 1N NaOH. Add the unmodified blank acellular membrane to the polypeptide solution, and stir the reaction at 150 rad / min for 16 h, then take out the acellular membrane. Place the acellular membrane in a 1 L Schott bottle, add 400 ml of ultrapure water, and place it in a constant temperature shaker at 25 °C and stir at 150 rad / min for 10 times, 1 hour each time. Then, take out the acellular membrane and perform vacuum freeze-drying to obtain the final polypeptide-grafted acellular membrane.

[0133] (3) Performance Detection

[0134] Test Example 1: Detection of the Thiol Modification Rate of Thiol-Modified Decellularized Membranes

[0135] Prepare a 0.1M phosphate buffer solution with pH 8.0 containing 1 mM ethylenediaminetetraacetic acid (EDTA) as the working solution. Weigh 185.7 mg of L-cysteine ethyl ester hydrochloride and dissolve it in 10 ml of the working solution to prepare a standard solution with a thiol content of 100 mM, and obtain standard solutions with thiol contents of 0.2, 0.5, 0.8, 1.0, 1.2, and 1.5 mM by dilution for determining the calibration curve. Then weigh 60.6 mg of 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) and dissolve it in 15 ml of the working solution as the reaction solution. In a 15 ml centrifuge tube, measure 2.5 ml of the working solution, 250 μl of the thiol-containing standard solution, and 50 μl of the DTNB reaction solution. After thoroughly mixing with a vortex for 15 min, measure the absorbance at 412 nm using an enzyme-linked immunosorbent assay (ELISA) reader and record the absorbance value. Using the thiol concentration in the final detection system as the abscissa and the absorbance value as the ordinate, perform linear regression analysis, calculate the correlation coefficient, and refer to the linear regression curve as shown in Figure 3 shown. Among them, the regression coefficient R of the linear equation 2 is 0.998, the calculation formula for curve fitting is y = A + Bx, the estimated value of parameter A is 0, the estimated value of parameter B is 13.691, and R 2 = 0.998. It can be seen from the figure that the detection result of the absorbance value is proportional to the thiol concentration, and the linear relationship is good.

[0136] Respectively take the blank decellularized membranes, the thiol-modified decellularized membrane samples prepared in Examples 1-7, and Comparative Example 1 for thiol content detection. Measure a certain amount of buffer solution (0.1M phosphate buffer solution containing 1 mM EDTA, pH = 8.0), add 2% v / v of the Ellman reagent reaction solution (4 mg / ml), and mix evenly to prepare the detection solution. Weigh 5-10 mg of the thiol-modified decellularized membranes in Examples 1-7 and Comparative Example 1 respectively and place them in 50 ml centrifuge tubes. After adding 5 ml of the detection solution, place the centrifuge tubes in a shaker at 37°C (180 rad / min) for 24 hours, measure the absorbance of the reaction solution at 412 nm, and calculate the thiol modification rate. The detection results are shown in Table 1.

[0137] Table 1 Thiol Content and Thiol Modification Rate of Thiol-Modified Decellularized Membranes

[0138] Test sample Thiol content (mmol / g) Thiol modification rate (%) Blank membrane 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％

[0139] The main carboxyl reaction sites in the decellularized membrane are aspartic acid and glutamic acid, and the total content of the two amino acids in the dried decellularized membrane is about 0.35 mmol / g. Since the modification of the decellularized membrane is a heterogeneous reaction, the reaction efficiency is low and usually occurs on the surface of the decellularized membrane. Generally, about 60% of the carboxyl sites (0.21 mmol / g) are distributed on the membrane surface. The present invention calculates the thiol modification rate based on this data.

[0140] As can be seen from Table 1, the thiol content in the thiol-modified decellularized membranes prepared in Examples 1-7 is high, and the thiol modification rates all reach more than 45%. Among them, the thiol modification rate of Example 1 is the highest, reaching 71%. For Comparative Example 1 without amino blocking, its thiol modification rate is only 7%, far lower than that of Example 1. This shows that after treating the decellularized membrane by amino blocking and then reacting with the thiol-containing compound, it is possible to avoid the participation of the amino group of the decellularized membrane itself in the modification reaction, which is beneficial to the precise modification of thiol, improves the thiol modification rate, and thus is beneficial to the subsequent polypeptide grafting reaction.

[0141] In addition, as can also be seen from Table 1, compared with ethyl cysteine hydrochloride (Example 1), after replacing the thiol-containing compound with cysteine hydrochloride (Example 5), the thiol amount of the decellularized membrane decreases to a certain extent, and after the reaction is completed, as Figure 4 shown, it can be observed that there is a relatively obvious precipitate in Example 5, indicating that cysteine hydrochloride is oxidized to form insoluble cystine. Compared with cysteine hydrochloride, ethyl cysteine hydrochloride has advantages such as appropriate steric hindrance, not easily oxidized, good affinity, and high final thiol modification rate. Therefore, the reaction solution is always a transparent liquid.

[0142] When ethyl cysteine hydrochloride (Example 1) is respectively replaced with the fatty chain thiol-containing compound cysteamine hydrochloride (Example 6) and the polyethylene glycol thiol-containing compound HS-PEG 5 -NH 2 (Example 7), the thiol modification effects of the two are lower than those of the amino acid thiol-containing compounds (Example 1, Example 5). This is mainly because the introduction of the fatty chain in the fatty chain thiol-containing compound affects the molecular solubility to a certain extent, resulting in a decrease in the thiol modification amount; and although the polyethylene glycol thiol-containing compound has good solubility in water, with the increase of the molecular weight, the thiol modification effect will also decrease.

[0143] Generally speaking, the above results indicate that the oxidizing property and steric hindrance of the mercapto group in the mercapto-containing compound are the main factors affecting the modification effect. If the oxidizing property of the mercapto group is too strong, the compound will be oxidized to form by-products during the reaction process, while too large molecular steric hindrance is not conducive to the reaction with the NHS active ester on the surface of the decellularized membrane through heterogeneous reaction. By screening specific mercapto-containing compounds, such as cysteine ethyl ester hydrochloride, the mercapto modification rate can be increased, which is more conducive to the subsequent graft modification of polypeptides.

[0144] Test Example 2 Detection of Polypeptide Grafting Rate

[0145] Use the Amplite maleimide quantification kit to detect the amount of maleimide in the polypeptide solutions prepared in Examples 8-19 and the amount of maleimide in the reaction solution of the decellularized membrane taken out after the polypeptide grafting decellularization reaction respectively, and calculate the polypeptide grafting rate. The detection results are shown in Table 2.

[0146] Table 2 Grafting Rate of Polypeptide Grafting Decellularized Membrane

[0147]

[0148]

[0149] It can be seen from Table 2 that the grafting rates of the polypeptides in Examples 8-19 are relatively high, all above 50%. Among them, the grafting rate of Example 1 is the highest, reaching 86%. While for Comparative Example 1 with a low mercapto modification rate, its grafting rate is only 10%, far lower than that of Example 1, indicating that after decellularizing the membrane through amino group blocking treatment, the mercapto modification rate can be increased, which is conducive to the subsequent polypeptide grafting reaction and improves the polypeptide grafting rate. Using amino group blocking for decellularization can also effectively reduce the immunogenicity of the decellularized membrane and improve the safety and effectiveness of the decellularized membrane product, which is beneficial to the clinical medical value of the decellularized membrane product.

[0150] In addition, it can also be seen from Table 2 that the 3-maleimide propionic acid molecule at the N-terminus of the REDV polypeptide is connected to the N-terminus of the polypeptide using an H 2 N-(PEG) 3 -COOH linker (Example 17). On the one hand, it can improve the solubility of the polypeptide and to a certain extent improve the polypeptide grafting rate. However, on the other hand, due to the easy entanglement after the molecular chain becomes longer, it is not conducive to the reaction with the NHS active ester sites in some pores of the decellularized membrane. Finally, the grafting rate of Example 17 is lower than that of Example 8 without using the PEG linker.

[0151] In addition, in Examples 18 and 19 using RGD polypeptide and IKVAV polypeptide respectively, compared with Example 17, their grafting rates also decreased to a certain extent. This may be because it is difficult for RGD polypeptide and IKVAV polypeptide to have charge interactions with the acellular membrane, which is not conducive to the grafting reaction.

[0152] Generally speaking, the above results indicate that by screening the linking molecules at the N-terminus of the polypeptide and the specific types of polypeptides, the grafting efficiency can be further regulated. Directly using the polypeptide with 3-maleimidopropionic acid modified at the N-terminus for acellular membrane grafting has a relatively high grafting efficiency.

[0153] Test Example 3 Visualization effect of polypeptide grafted acellular membrane

[0154] Under ultrasonic conditions, 10 mg of 3-maleimidopropionic acid modified REDV polypeptide (GGRGDS(SEQ ID NO:1)-FITC) with FITC label was dissolved in 20 ml of PBS (pH = 6.9) to prepare a reaction solution for standby. The thiol-modified acellular membrane prepared in Example 1 and the unmodified blank acellular membrane were respectively placed in 10 ml of the above-prepared reaction solution, and stirred overnight at room temperature. After the reaction was completed, it was washed 5 times with ultrapure water, 30 minutes each time. Then the acellular membrane was placed in N,N-dimethylformamide (DMF) and shaken on a shaker at 180 rad / min at 37 °C overnight. Then it was washed 2 times with ultrapure water, 30 minutes each time. After washing, under the condition of ultraviolet lamp irradiation, the fluorescence intensity was observed respectively, and the observation results are as Figure 2 shown. It can be seen from Figure 2 that compared with the unmodified blank acellular membrane, the binding between the thiol-modified acellular membrane and REDV polypeptide is tighter and the fluorescence performance is more obvious. This is mainly because after the acellular membrane is modified with thiol, it is loaded with REDV polypeptide by covalent coupling, while for the unmodified blank acellular membrane, it is only physically adsorbed with REDV polypeptide for loading. Therefore, the fluorescence response is weak.

[0155] Test Example 4: Cytotoxicity detection

[0156] The blank acellular membrane, the polypeptide grafted acellular membranes prepared in Examples 8 - 19, and the polypeptide acellular membranes of Comparative Examples 1 - 2 were cut into pieces and added to the culture medium for extraction according to the surface area ratio of 6 cm 2 / ml. After 72 hours, the extraction medium was taken to culture L929 cells. After 72 hours, the cell morphology was observed under a microscope, and then the MTT colorimetric method was used to detect the cell survival situation. The relative absorbance values of the cells cultured in the extraction medium and the blank culture medium were compared. If the relative absorbance value was greater than 70%, it proved that there was no cytotoxicity. The detection results are shown in Table 3.

[0157] Table 3 Results of cytotoxicity detection

[0158]

[0159]

[0160] As can be seen from Table 4, for the polypeptides grafted onto the acellular membranes in Examples 8 - 19, their relative absorbance values are all above 70%, and are close to the relative absorbance value of the blank cell membrane, indicating that the chemical reaction of polypeptide grafting after amino group blocking, activation and mercapto group treatment will not cause the final acellular membrane to have cytotoxicity. In addition, the relative absorbance values of the acellular membranes in Comparative Examples 1 - 2 are also above 70%, indicating that the physically adsorbed acellular membranes also have no cytotoxicity.

[0161] Test Example 5: Cell proliferation detection

[0162] The blank acellular membranes, the polypeptide-grafted acellular membranes prepared in Examples 8 - 19, and the polypeptide acellular membranes in Comparative Examples 1 - 2 were cut into the size of a 12-well plate and placed at the bottom of the well plate. Approximately 30,000 epidermal cells were inoculated on the surface of each sample. After incubation in a cell culture incubator for 2 days, the culture medium was removed, and the samples were rinsed 3 times with PBS for 5 minutes each time. Subsequently, 1 ml of complete medium containing 10% CCK-8 reagent was added, and the samples were incubated in a cell culture incubator for 1 hour. Subsequently, the absorbance of the culture medium at 450 nm was measured. A high absorbance indicates a large number of surviving cells.

[0163] The detection results are shown in Table 4

[0164] Table 4 Epidermal cell proliferation rate

[0165]

[0166]

[0167] As can be seen from Table 4, the polypeptide-grafted acellular membranes in Examples 8 - 19 all have good epidermal cell proliferation rates, and as the grafting rate increases, the cell proliferation rate is higher. In addition, compared with the REDV polypeptide (Example 17), the RGD polypeptide grafting (Example 18) and the IKVAV polypeptide (Example 19) have lower epidermal cell proliferation rates. This is because the grafting rates of both are lower than that of Example 17. However, the RGD polypeptide is similar to the REDV polypeptide and can promote cell proliferation to a certain extent, while the effect of IKVAV polypeptide grafting on promoting cell proliferation is not obvious. Therefore, its epidermal cell proliferation rate is lower than that of the RGD polypeptide. The epidermal cell proliferation rates in Comparative Examples 1 - 2 are low, mainly because the loading effect of the polypeptide is poor, resulting in a low final epidermal cell proliferation rate.

[0168] Test Example 6: Cell adhesion detection

[0169] Based on the experimental results of Test Example 5, we selected blank decellularized membranes, the polypeptide-grafted decellularized membranes prepared in Example 8 and Examples 17-19, and the polypeptide decellularized membranes of Comparative Examples 1-2 for cell adhesion detection. The decellularized membranes were cut into the size of a 12-well plate and placed at the bottom of the well plate. After culturing L929 cells on the surface for 2 days, the 12-well plate was taken out, the culture medium was sucked out and discarded, gently rinsed 3 times with PBS buffer, slowly added 2 ml of 4% paraformaldehyde solution, fixed at room temperature for 2 h, dehydrated with gradients of 30%, 50%, 70%, 80%, 90%, 95% and 100% ethanol for 60 min each time, then freeze-dried, and then the cell adhesion and growth on the sample surface were observed using a scanning electron microscope. The experimental results are as Figure 5 shown (the area in the red frame is 100*100μm 2 ). The number of cells adhered to the surface area of about 100*100μm 2 in not less than 3 SEM pictures of each group of decellularized membranes was counted and averaged. The results are shown in Table 5.

[0170] Table 5 Statistics of the number of adhered cells per unit area (100*100μm 2 ) in SEM pictures of cell adhesion

[0171] Number of adherent cells (pcs) Blank membrane 5 Example 8 14 Example 17 12 Example 18 9 Example 19 7 Comparative example 1 5 Comparative example 2 2

[0172] Combined Figure 5 with Table 5, it can be seen that the number of cells that can be adhered per unit area on the surface of the decellularized membranes of Comparative Examples 1-2 are 5 and 2 respectively, both lower than the number of adhered cells in the samples of the examples. This is mainly due to the low loading amount of the polypeptide, so the number of adhered cells is small. In addition, the surface of the decellularized membrane grafted with REDV polypeptide (Example 8, Example 17) can adhere more cells per unit area, indicating that it has a better adhesion effect on the surface, and the adhesion effect increases with the increase of the REDV polypeptide grafting rate. The number of cell adhesions of RGD polypeptide grafting (Example 18) and IKVAV polypeptide (Example 19) are both lower than that of REDV polypeptide (Example 17), also because the grafting rates of RGD polypeptide and IKVAV polypeptide are lower than that of REDV polypeptide grafting rate, so the number of cells adhered to their surfaces is lower.

[0173] Although the present invention has been described in detail by way of preferred embodiments, the present invention is not limited thereto. Without departing from the spirit and essence of the present invention, those of ordinary skill in the art can make various equivalent modifications or substitutions to the embodiments of the present invention, and these modifications or substitutions should all be within the scope of the present invention / Any person skilled in the art within the technical scope disclosed by the present invention can easily think of changes or substitutions, which should all be covered by the protection scope of the present invention.

Claims

1. A polypeptide-grafted decellularized cell membrane, characterized in that, it has the following structure: wherein, P is a polypeptide residue, C is an amino-blocked decellularized cell membrane, L 1 Absent or L 2 is -(PEG) m - or R is selected from H, COOH, COOCH 3 or COOCH 2 CH 3 , y is an integer selected from 1 to 6; R 1 、R 2 is independently selected from H or C 1 -C 4 alkyl; x is an integer from 1 to 6, n is an integer from 1 to 10, m is an integer from 5 to 20.

2. The polypeptide-grafted decellularized cell membrane according to claim 1, characterized in that, it has the following structure:

3. A method for preparing the polypeptide-grafted decellularized cell membrane according to any one of claims 1-2, characterized in that, the preparation method includes the following steps: (1) Prepare amino-blocked decellularized cell membrane; (2) Activate the carboxyl groups in the decellularized cell membrane; (3) Modify the decellularized cell membrane obtained in step (2) with a thiol group; (4) Polypeptide grafting to the decellularized cell membrane: React the thiol-modified decellularized cell membrane obtained in step (3) with a maleimide-modified polypeptide to obtain a polypeptide-grafted decellularized cell membrane.

4. The preparation method according to claim 3, characterized in that, in step (1), an epoxide is used to block the amino groups of the decellularized cell membrane; preferably, the mass ratio of the epoxide to the decellularized cell membrane is 1:5-15; preferably, the reaction temperature in step (1) is 20-40°C and the reaction time is 12-36h; preferably, the amino-blocking reaction is followed by a neutralization and / or washing step.

5. The preparation method according to claim 3, characterized in that, the reagent used to activate the carboxyl groups in the decellularized cell membrane in step (2) is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide.

6. The preparation method according to claim 5, characterized in that, the mass ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide to the decellularized cell membrane in step (2) is (0.5-2.0):1; preferably, the pH of the activation reaction system in step (2) is 4.0-7.0; preferably, the activation reaction time in step (2) is 6-24 hours.

7. The preparation method according to claim 3, characterized in that, In step (3), thiol modification is carried out using a thiol-containing compound or its salt, and the thiol-containing compound is a compound containing both an amino group and a thiol group; preferably, the thiol-containing compound is a thiol-containing amino acid, fatty amine or polyethylene glycol; preferably, the thiol-containing compound is selected from cysteamine or its hydrochloride, 3-mercapto-1-propylamine or its hydrochloride, 6-amino-1-mercaptohexane hydrochloride, cysteine or its hydrochloride, cysteine ethyl ester or its hydrochloride, homocysteine or its hydrochloride, penicillamine or its hydrochloride, and HS-(PEG). m -NH 2 , where m is an integer from 5 to 20.

8. The preparation method according to claim 7, characterized in that, the mass ratio of the thiol-containing compound to the decellularized cell membrane in step (3) is (0.1-2.0):1; preferably, the pH of the thiol modification reaction in step (2) is 5.0-7.0; preferably, the reaction time in step (2) is 24-72 hours.

9. The preparation method according to any one of claims 3-8, characterized in that, the maleimide-modified polypeptide in step (4) has the following structure: wherein, P is a polypeptide residue, L does not exist or is x is an integer from 1 to 6, n is an integer from 1 to 10.

10. The preparation method according to claim 9, characterized in that, the mass ratio of the maleimide-modified polypeptide to the decellularized cell membrane in step (4) is 5-20:1 mg / g; preferably, the reaction pH in step (4) is 4.5-7.0; preferably, the concentration of the reaction polypeptide is 0.2-1.0 mg / ml.

11. The preparation method according to claim 9, characterized in that, The decellularized membrane is one of decellularized bovine pericardium, decellularized porcine dermal matrix or decellularized porcine small intestinal submucosa; preferably, the sequence of the polypeptide includes one of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO:

3.

12. Use of the polypeptide grafted decellularized membrane according to any one of claims 1-2 or the polypeptide grafted decellularized membrane according to any one of claims 3-11, characterized in that, the use includes: 1) Preparation of artificial biological tissues, artificial organs, wound dressings, tissue engineering scaffolds or human grafts; 2) For tissue or organ repair.

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