CN107998453B

CN107998453B - Surface-modified acellular matrix and modification method thereof

Info

Publication number: CN107998453B
Application number: CN201711321409.3A
Authority: CN
Inventors: 沈靖南; 尹军强; 武征; 林熙
Original assignee: Jinan University; First Affiliated Hospital of Sun Yat Sen University
Current assignee: Jinan University; First Affiliated Hospital of Sun Yat Sen University
Priority date: 2017-12-12
Filing date: 2017-12-12
Publication date: 2020-09-25
Anticipated expiration: 2037-12-12
Also published as: CN107998453A

Abstract

The invention belongs to the field of tissue engineering, and particularly relates to a surface modification method of an acellular matrix. The modification method comprises the following steps: (1) performing carboxyl activation on the acellular matrix; (2) carrying out carboxyl protection on phenylalanine or phenylalanine derivatives, and carrying out graft modification on the phenylalanine or phenylalanine derivatives subjected to carboxyl protection and carboxyl of acellular matrixes through amino groups of the phenylalanine or phenylalanine derivatives; (3) and performing post-treatment on the grafted and modified acellular matrix to obtain the surface-modified acellular matrix. The modified acellular matrix has the functions of resisting infiltration of tumor cells and inflammatory cells, resisting infection, resisting degradation of matrix metalloproteinase and lactic acid and improving inflammatory environment, and can be used for postoperative tissue repair of tumor treatment.

Description

Surface-modified acellular matrix and modification method thereof

Technical Field

The invention relates to the field of tissue engineering, in particular to a surface modified acellular matrix and a modification method thereof.

Background

The acellular matrix material is used as a natural biodegradable material, has rich bioactive components, a natural three-dimensional network structure and lower immunogenicity, and has wide application in the field of tissue engineering. In the aspect of tumor treatment, acellular matrix materials have been used for solving the problem of repairing mucous membrane injury after throat tumor resection, (Zhang Qingquan, Sun rock, Wangqiang, etc.. evaluation on the effect of xenogenic (bovine) acellular dermal matrix in repairing mucous membrane defect after throat tumor resection [ J ] Chinese tissue engineering research, 2008, 12(06):1081 and 1084.), and a preparation technology of acellular matrix materials for solving the problem of repairing malignant tumor after surgery is urgently needed at present.

The special physiological environment after malignant tumor operation has the following requirements on the performance of implanting the acellular matrix:

1. tumor cells are metastatic and latent, and some of the tumor cells are metastasized to blood vessels and peripheral tissues and latent in the form of normal cells. The range can be expanded according to the specification during the tumor treatment, the tumor cells in the treatment range are killed finally, but the latent tumor cells outside the treatment range still exist; some tumors grow around important organs, large vessels and nerves, and cannot be excised together. These factors lead to the possibility of in situ recurrence of the tumor after surgery. When the tumor recurs at other parts, the patient can be suffered from huge pain, but secondary treatment can still be adopted; however, if the tumor is recurrent in situ without complete repair, not only the difficulty of the operation is increased, but also the difficulty of recovery is increased, and even the life can be directly endangered. Thus. If the implanted acellular matrix material has the function of preventing tumor recurrence, the application range of the acellular matrix material can be expanded, and the clinical treatment effect can be improved.

2. Tumor cells highly express matrix metalloproteinase (a zinc-dependent neutral endopeptidase which participates in inflammatory reaction, matrix degradation and tumor metastasis), can degrade and reconstruct extracellular matrix, which is also the basis of metastatic tumor cells, and the main component of the acellular matrix material is the extracellular matrix. The ideal acellular matrix implant material should have the functions of resisting tumor cell infiltration and reconstructing extracellular matrix.

3. Tumor cells obtain energy mainly through glycolysis, and metabolism can generate a large amount of lactic acid, so that the surrounding environment is weakly acidic, and degradation of acellular matrixes can be accelerated. Once the tumor recurs in situ, if the implanted acellular matrix material has the function of resisting lactic acid degradation, the degradation time of the implanted acellular matrix material can be prolonged, and the infiltration of tumor cells to normal tissues can be reduced.

4. After treatment of the tumor site, a high inflammatory state is present, and a large number of inflammatory cells in the periphery will migrate into the damaged site. If the implanted acellular matrix has the capacity to resist infiltration of inflammatory cells, the degradation of the matrix itself can be slowed down, and the inflammatory environment of the damaged part can be improved.

Therefore, compared with other wound repairs, the postoperative repair of malignant tumor is faced with a more severe degradation-promoting environment, and the degradation-resistant capability of the implanted acellular matrix material has more important significance in repairing tissues and reducing infiltration of tumor cells to normal tissues. The acellular matrix material for repairing the postoperative tissue of the malignant tumor needs to solve the problem that the acellular matrix material is resistant to degradation under the action of tumor cells, inflammatory cells and lactic acid. If the modified acellular matrix material has the function of preventing tumor recurrence, the modified acellular matrix material has greater application value.

At present, aiming at the problem of degradation resistance of acellular matrix materials, the existing modification methods comprise a chemical method, a physical method and a biological method. (1) The chemical method is to internally crosslink the acellular matrix using a chemical crosslinking agent. The earliest used chemical crosslinkers include glutaraldehyde, formaldehyde, 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC), N-hydroxysuccinimide (NHS), epoxides, diamines, diisocyanates, polyethylene glycols and the like. However, these cross-linking agents are released from the modified acellular matrix material during the degradation process, which affects the normal physiological functions. Natural biological cross-linking agents are increasingly used for cross-linking modification of acellular matrices. Patents (201110165428.8 and 201610284706.4) use natural biological crosslinkers such as anthocyanins, riboflavin, ribose, and genipin to crosslink acellular matrices; the patent (201410459064.8) designs a special corneal cross-linking agent (10-20% (w/v) dextran solution, 0.1-0.2% (w/v) riboflavin) to solve the cross-linking problem of acellular corneal stroma. (2) The physical method is to activate the groups in the acellular matrix by using physical factors such as radiation, heat, solution environment and the like to realize the self-crosslinking of the acellular matrix. For example: the patent (201611209243.1) proposes enhancing the lyophilization and heat treatment of acellular matrices to achieve thermal crosslinking of the acellular matrices. The patent (201110199312.6) changes the electrokinetic potential of collagen by means of poly-sodium aspartate and water-soluble chitosan, realizes the regulation of collagen bundle structure and appearance, and repairs the natural structure of collagen. (3) The biological method is to use transglutaminase, horseradish peroxidase, tyrosinase, lysyl oxidase and other catalytic acellular matrixes to perform self-crosslinking. The modification methods improve the connection of collagen in the acellular matrix and prolong the degradation time of the acellular matrix material, but cannot avoid the infiltration of matrix metalloproteinase, lactic acid and inflammatory cells of tumor cells.

In addition, researchers modify acellular matrix materials by loading inflammatory or antibacterial drugs against the inflammatory and infectious characteristics of the injury site. The patent (200810017603.7) coats penicillin-loaded gel on the surface of acellular matrix, and is used for treating skin defects caused by inflammation, ulcer, burn wound, iatrogenic sources and the like. The patent (200910200933.4) crosslinks nanoparticles of the antimicrobial agent at the surface of the acellular matrix, imparting resistance to infection. The patent (201610395479.2) coats acellular matrix fragments and gel prepared from aspirin, ketoprofen, sulbactam or finasteride on the surfaces of fibers such as silk fibroin, polyglycolide lactide or polycaprolactone, and realizes the slow release of inflammatory drugs. Using a similar modification method, even if tumor drugs are added, only the direct action of tumor cells and inflammatory cells can be reduced, and the degradation effect of matrix metalloproteinase and lactic acid on materials cannot be shielded. Moreover, these methods of loading drugs cannot avoid the damage of tumor drugs to normal cells.

Amino acids, which are basic units constituting proteins, have a carboxyl group and an amino group, and can be used as a crosslinking agent to modify an acellular matrix. The addition of free amino acids to the acellular dermal matrix of patent (200910048157.0) promotes vascularization of the acellular matrix, the amino acids used including tryptophan, methionine, threonine, valine, lysine, histidine, leucine, isoleucine, alanine, phenylalanine, cystine, cysteine, arginine, glycine, serine, tyrosine, glutamine, proline, aspartic acid, arginine, and the like. Lai reported that the use of glycine (neutral), lysine (alkaline), glutamic acid (acidic) modified acellular amniotic membrane, respectively, had good cellular infiltration for corneal transplantation (Lai JY. carbohydrate cross-linking of ammoniatic membranes in the presence of aminoacid bridges. Mat Scieng C-mater. 2015;51: 28-36.). The patent (201610040990.0) modifies the acellular cornea with acidic amino acids (aspartic acid, glutamic acid and histidine) and basic amino acids (lysine, arginine and proline), and the water-locking capacity of the amino acids improves the water content of the acellular cornea, is beneficial to the growth of cells and can accelerate the restoration of the corneal function. However, the above-mentioned amino acid crosslinking methods are all intended to promote the growth of cells into acellular matrix, and in order to promote the growth of cells more effectively, patent (200910048157.0) requires that the amino acid used is a free amino acid. If the acellular matrix obtained by the amino acid crosslinking modification method is implanted into a tumor resection part, better nutrition and living environment are provided for tumor cells.

In summary, the existing acellular matrix material modification method solves many problems in clinical application, but in the physiological environment after malignant tumor operation, the problem of degradation resistance of the acellular matrix material under the action of tumor cells, inflammatory cells and lactic acid needs to be solved, so a new acellular matrix modification method needs to be designed.

Disclosure of Invention

Based on the above, one of the objectives of the present invention is to provide a surface modification method of an acellular matrix, which realizes surface functionalization of the acellular matrix and can be used for preparing an acellular matrix material meeting the postoperative repair requirement of malignant tumors.

The specific technical scheme for achieving the purpose is as follows.

A method of surface modification of an acellular matrix comprising the steps of:

(1) performing carboxyl activation on the acellular matrix;

(2) carrying out carboxyl protection on phenylalanine or phenylalanine derivatives, and carrying out graft modification on the phenylalanine or phenylalanine derivatives subjected to carboxyl protection and carboxyl of acellular matrixes through amino groups of the phenylalanine or phenylalanine derivatives;

(3) removing carboxyl protection from the grafted and modified acellular matrix;

(4) eluting the carboxyl protecting group;

(5) and (3) converting the carboxyl of the acellular matrix after the carboxyl protecting group is eluted into acyl halide to obtain the surface modified acellular matrix.

According to the surface modification method of the acellular matrix, through long-term research and experiments on the acellular matrix by the inventor, in order to meet the postoperative repair requirement of the sexual tumor, phenylalanine or derivatives thereof are skillfully used for carrying out surface modification on the acellular matrix. Activating carboxyl of acellular matrix, protecting carboxyl of phenylalanine and derivatives thereof, grafting amino of the derivatives after carboxyl protection with the activated carboxyl of the acellular matrix, removing carboxyl protection, converting the carboxyl of the acellular matrix into acyl chloride, and finally grafting an anti-tumor drug, an antibacterial drug and an anti-inflammatory drug to obtain the surface modified acellular matrix material which is really suitable for clinical use. The obtained acellular matrix after surface modification can realize the loading of antitumor drugs, antibacterial drugs and anti-inflammatory drugs by virtue of the carboxyl of phenylalanine, realize the slow release of the drugs and prolong the inhibition effect on tumor cells and inflammatory cells; the targeting property of the medicament is improved by means of the physiological requirement of tumor cells on phenylalanine; by means of the hydrophobic effect of phenylalanine benzene ring, cell infiltration is inhibited, matrix metalloproteinase and lactic acid degradation are resisted, and the drug slow release effect is improved.

The purpose of activating the carboxyl group of the acellular matrix in the step (1) is to improve the yield of the reaction between the carboxyl group and the amino group of the phenylalanine derivative.

The acellular matrix in the step (1) comprises an acellular treated matrix or a crosslinking modified acellular matrix.

In one embodiment, the activating reagent used for activating the carboxyl group in the step (1) is 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide and/or N-hydroxysuccinimide. The pH of the acellular corneal stroma is 4-8 when subjected to carboxyl activation.

The aim of protecting phenylalanine carboxyl in the step (2) is to avoid the reaction between the carboxyl of phenylalanine and amino of acellular matrix and reserve a reaction site for the final grafted drug; the amino group of the phenylalanine derivative is grafted with the carboxyl group of the acellular matrix.

In one embodiment, in step (2), the carboxyl group protection of phenylalanine described in step (2) is performed by using an esterification method, an amide or hydrazide method, or by using an amino acid derivative having a carboxyl group protecting group, to obtain a derivative of phenylalanine.

The decarboxylation protection in the step (3) aims to expose the carboxyl of phenylalanine for subsequent drug grafting.

The carboxyl deprotection treatment method in the step (3) comprises hydrolysis or pyrolysis of ester, amide and hydrazide under catalysis of acid or alkali, acidolysis under mild conditions and hydrogenolysis of benzyl ester.

In one embodiment, the reagent used for the hydrogenolysis of benzyl ester comprises H₂Pd-C/ethanol, H₂/Pd(OH)₂-C/ethanol, β -mercaptoethanol/BF₃One or more groups of ethyl ether, HBr/glacial acetic acid/dichloroacetic acid, and trimethyl iodosilane/carbon tetrachloride.

The purpose of the elution of the carboxyl protecting group in the step (4) is to wash away the carboxyl protecting group.

The method for eluting the carboxyl protecting group in the step (4) comprises extraction and dialysis. The step (5) of converting-OH of the carboxyl of the acellular matrix after grafting modification into halogen aims to convert the carboxyl into acyl chloride and improve the grafting speed and yield with the drug.

In one embodiment, in the step (5), the-OH of the carboxyl group of the graft-modified acellular matrix is converted into a halogen, preferably chlorine, by reacting the acellular matrix with phosphorus trichloride, phosphorus pentachloride, thionyl chloride, or oxalyl chloride.

Another object of the present invention is to provide a surface-modified acellular matrix material and applications thereof.

A surface modified acellular matrix material is prepared by the method.

The surface-modified acellular matrix material can be used for preparing preparations loaded with antitumor drugs, antibacterial drugs and/or anti-inflammatory drugs.

The acellular matrix material is loaded with tumor drugs and can be slowly released for a long time, so that the recurrence rate of tumors is expected to be reduced; the surface of the material has the capacity of resisting infiltration of tumor cells and inflammatory cells, can resist long-term erosion of lactic acid and the like, and has the functions of resisting infection and improving inflammatory environment.

The technical principle of the invention is as follows:

1. phenylalanine has a very important effect on the growth of tumor cells, and the phenylalanine loads tumor drugs, so that the probability of the tumor drugs entering the tumor cells can be improved, and the influence on normal cells is reduced.

2. The benzene ring carried by phenylalanine has strong hydrophobic acting force, and phenylalanine contained in the protein has important significance for maintaining the three-dimensional and four-dimensional structures of the protein. The range of the acting force of the hydrophobic bond can reach more than 100 nm, the ranges of hydrogen bond, van der waals force, ionic bond, covalent bond and the like are within 5 nm, and the modified acellular matrix has a large action range of phenylalanine contained on the surface.

3. The phenylalanine is protected by carboxyl by using an esterification method, an amide method or a hydrazide method, the carboxyl of the phenylalanine is prevented from reacting with amino in the acellular matrix in the modification process, and the final drug loading effect is reduced. After the modification is finished, the carboxyl is recovered through hydrolysis or pyrolysis of ester, amide and hydrazide formed by the phenylalanine under the catalysis of acid or alkali, acidolysis under mild conditions and hydrogenolysis of benzyl ester.

4. Common tumor drugs (such as cisplatin, adriamycin and the like), anti-inflammatory drugs (such as glucocorticoid and the like) and antibacterial drugs (such as vancomycin and the like) all have amino or hydroxyl, and carboxyl and acyl chloride on the surface of the modified acellular matrix adsorb the drugs through the actions of ion adsorption and covalent bonding (forming amido bond or ester bond).

5. The reactivity of acyl chloride and amino is higher than that of carboxyl, and-OH of the carboxyl is replaced by halogen, so that the grafting speed and quantity of the medicine can be increased, and the clinical use is facilitated.

Compared with the prior art, the invention has the following advantages and beneficial effects:

1. the different requirements of the tumor cells and the normal cells for phenylalanine can ensure that the tumor medicine can efficiently enter the tumor cells and simultaneously reduce the damage to the normal cells. As phenylalanine is also an essential amino acid for human bodies, the modified acellular matrix has no adverse effect on peripheral tissues after being degraded.

2. The modified acellular matrix surface hydrophobicity can reduce infiltration of peripheral tumor cells and inflammatory cells.

3. The hydrophobic acting force on the surface of the modified acellular matrix prevents substances such as matrix metalloproteinase, lactic acid and the like from entering the acellular matrix, and simultaneously prevents water-soluble biomolecules in the acellular matrix from flowing out, so that the degradation of the acellular matrix is slowed down.

4. Most of conventional tumor drugs, anti-inflammatory drugs and antibacterial drugs are fat-soluble substances, and the hydrophobicity of phenylalanine is favorable for combining and loading the drugs; the ionic bond or covalent bond of phenylalanine can form stable combination with the drug, thereby realizing the slow release of the drug, avoiding burst release and prolonging the drug effect.

5. The surrounding water environment interacts with the hydrophobic bond on the surface of the modified acellular matrix to form a cage-shaped hydrate, so that the tumor drug is locked, and the slow release effect of the drug is enhanced.

6. After the carboxyl on the surface of the modified acellular matrix is converted into acyl chloride, the grafting efficiency with the medicine is improved, the medicine can be loaded within half an hour before use, and the clinical use is convenient.

7. The surface of the modified acellular matrix can be loaded with different medicines, and is used for inhibiting tumor cells, preventing bacterial infection and improving inflammatory environment.

Drawings

FIG. 1 is a Fourier transform attenuated total reflection infrared spectrum before and after modification of the decellularized pericardium in example 1;

FIG. 2 is a graph showing the release of cisplatin following modification of the acellular pericardial matrix of example 1;

FIG. 3 is the change in collagen content after modification of the acellular pericardial matrix of example 1.

Detailed Description

In order to clearly understand the technical contents of the present invention, the following embodiments are described in detail with reference to the accompanying drawings. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's recommendations. The various chemicals used in the examples are commercially available. The following reagents used in the present invention can be purchased from conventional sources.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

In the modification method, in order to improve the efficiency of the reaction, one or more of phase transition catalyst, photocatalysis, microwave, ultrasonic wave, irradiation and the like are used. Adjusting the power and frequency of the ultrasound; the power of the microwaves; the parameters such as irradiation dose, time and the like improve the modification effect.

The phase transfer catalyst comprises polyether (such as chain polyethylene glycol and chain polyethylene glycol dialkyl ether), cyclic crown ether (such as cyclodextrin), quaternary ammonium salt (such as tetrabutylammonium bromide and dodecyl trimethyl ammonium chloride), tertiary amine (such as tributylamine), quaternary ammonium base and quaternary phosphonium salt.

The power of the ultrasonic wave is preferably 100-600W, and the frequency is preferably 30-80 kHz.

The power of the microwave is preferably 80-120W.

The irradiation dose is preferably 10-30 KyG, and the time is preferably 2-10 min.

Example 1

The modified acellular pericardium of this example was prepared by the following method:

(1) carboxyl activation of decellularized pericardium

10 cm × 5 cm of decellularized pericardium (DPP) is immersed in 8 mL of 2- (N-Morpholine) Ethanesulfonic (MES) solution (pH = 6.5), 0.85 g of EDC and 0.51 g of NHS are sequentially added, and stirring is performed for 12 h, so that carboxyl groups on the surface of the decellularized pericardium are activated. Dialyzing (dialysis bag molecular weight cut-off 500 Da) in ultrapure water for 24 h, and removing the activating agent for later use.

(2) Surface modification of acellular pericardium

To 10mL of MES solution (pH = 6.5), 0.3 g of benzyl L-phenylalanine hydrochloride containing a carboxyl-protecting group and 0.57 g of tetrabutylammonium bromide were added, and after complete dissolution, the acellular pericardium after carboxyl activation was added, and stirred for 48 hours to graft-react the amino group of phenylalanine with the carboxyl group on the surface of the acellular matrix. Dialyzing with dialysis bag (molecular weight cut-off 500 Da) for 3 days. Obtaining the modified acellular pericardium (M-DPP).

(3) Decarboxylation protection of modified decellularized pericardium

And taking out the M-DPP, immersing the M-DPP into 5 mL of dichloroacetic acid solution, carrying out ice bath, adding 33% HBr/glacial acetic acid solution with the volume 5 times that of the solution, carrying out a light-shielding reaction for 3 hours, carrying out carboxyl protection on phenylalanine, and exposing carboxyl.

(4) Elution of the carboxyl protecting group

Taking out the acellular matrix, dialyzing in ultrapure water in the dark (the molecular weight cut-off of a dialysis bag is 500 Da) for 3 days, eluting the carboxyl protecting group, and freeze-drying.

(5) Conversion of-OH of carboxyl group of modified acellular pericardium into chlorine element

The freeze-dried M-DPP is taken and added into 2 mL of dichloromethane (pre-suspension evaporation is used for removing water), 0.5 mL of oxalyl chloride is slowly and dropwise added, and the mixture is stirred for 1 h at room temperature, so that-OH of carboxyl on the surface of the M-DPP is converted into chlorine. Freeze drying removed oxalyl chloride and dichloromethane.

(6) Loading of antitumor drugs

The obtained product is weighed and then immersed in 5 mL of 3 mg/mL cisplatin solution, and stirred for 15 min, so as to obtain M-DPP loaded with the antitumor drug.

The modified acellular pericardium prepared in this example was subjected to the following performance tests:

1. chemical structure analysis

The chemical structures before and after modification of the decellularized pericardium were examined using a Fourier transform infrared spectrometer (EQUINOX-70, Bruker, Germany), and the results are shown in FIG. 1. As can be seen, 1631 cm of M-DPP compared to DPP^-1Increase of C ═ O stretching vibration peak of amido bondStrong, 3284 cm^-1The intensity of the secondary amide NH stretching vibration peak is increased, which indicates that phenylalanine is grafted to the surface of acellular pericardium.

2. Surface hydrophilicity and hydrophobicity detection

The acellular pericardium materials before and after modification are respectively taken and placed on a clean glass slide. The material surface static contact angle was measured using a contact angle tester (Kruss DSA 100). The test temperature is 28 ℃, the humidity is 80%, and 10 points are randomly selected for each group to test. The result shows that the DPP contact angle is 93 +/-3.21 degrees, the M-DPP contact angle is 126.1 +/-1.37 degrees, and the modified acellular pericardium has stronger hydrophobicity.

3. Modified acellular pericardium drug loading detection method

And (3) setting a cisplatin concentration gradient solution, collecting the cisplatin solution immersed in the modified acellular pericardium, detecting the absorption wavelength of 301 nm by using an ultraviolet spectrophotometer, and calculating the drug loading rate. The results show that the drug loading of M-DPP is 74.9 +/-11 mu g/mg.

4. Modified acellular pericardium drug release detection

5 mg of cisplatin-loaded M-DPP was transferred into a dialysis bag (molecular weight cut-off 500 Da) and added to 40 ml PBS solution. Drug release was performed in a100 rad/min air-blown shaker at 37 deg.C, and 1mL of the release solution was removed at 1 week, 2 weeks, 3 weeks, and 4 weeks while adding an equal volume of PBS solution. The results of the concentration detection method are shown in FIG. 2. As can be seen in FIG. 2, M-DPP-loaded cisplatin released more rapidly within 1 week, entering a plateau after 2 weeks, with a cumulative percentage release at 71. + -. 1.19% at 4 weeks.

5. Modified acellular matrix lactic acid degradation resistance detection method

The MRS medium (Islands Mariots) was dissolved in ultrapure water, and after sufficient dissolution, pH =6.2 was adjusted, and autoclaving was performed at 120 ℃ for 20 min. Respectively using unmodified acellular pericardium and modified acellular pericardium to wrap the culture medium, and suturing with surgical thread. Injecting lactobacillus strain into the inner part, spreading on a culture dish (diameter 10 cm), adding 3 mL culture medium at the periphery, and culturing at 37 deg.C with a constant temperature flat shaking table. The colony formation condition after one week or two weeks around the pericardium is observed, and the change of the quality of the pericardium is detected. The results show that the bacterial colony is formed around the unmodified acellular pericardium, but not formed around the modified acellular pericardium, the quality of the bacterial colony and the modified acellular pericardium is not obviously different, and the results show that the lactic acid can seep out from the unmodified pericardium and change the compact structure inside the cell membrane, and the phenylalanine can reduce the penetration of the lactic acid and reduce the destructive effect of the lactic acid after modification.

6. Detection of modified acellular matrix for resisting matrix metalloproteinase degradation

500 mg/L matrix metalloproteinase (MMP-1, Sigma, cat # SRP 3117) solution (pH =7.4, containing 10 mmol/L CaCl) was prepared₂). 3mg of unmodified acellular pericardium and modified acellular pericardium are added into 5 mL of matrix metalloproteinase solution and incubated at 37 ℃. Each 30 min 100. mu.L of the solution was removed to detect changes in the Collagen content of the solution (Kit: Sirius Red Total Collagen Detection Kit, cat # 9062). The results are shown in FIG. 3, where the modified acellular matrix released much less collagen than the unmodified acellular matrix under the action of metalloproteases.

Example 2

The modified acellular amniotic membrane of this example was prepared by the following method:

(1) carboxyl activation of decellularized amniotic membrane

5 cm × 5 cm of decellularized amniotic membrane was immersed in 10mL of 2- (N-Morpholino) Ethanesulfonic (MES) solution (pH = 6.5), 0.57 g of EDC was added thereto, and the mixture was stirred for 12 hours to activate surface carboxyl groups for use.

(2) Surface modification of acellular amniotic membrane

Protecting carboxyl of phenylalanine by using an esterification method, and obtaining L-phenylalanine ethyl ester by using a preparation method reference (Zengguang, Luhuibang, Sunshunling, and the like, synthesis and antibacterial activity of sorbic acid phenylalanine ethyl ester [ J ] food industry science and technology, 2013, 34(24): 321-.

And (3) adding 0.5 g L-phenylalanine ethyl ester into 10mL of the solution in the step (1), and stirring in ice bath for 12 h to enable the amino group of phenylalanine to react with the carboxyl group on the surface of the acellular amniotic membrane. Dialyzed against PBS (molecular weight cut-off 3000 Da) for 3 days.

(3) Decarboxylation protection of modified decellularized amniotic membrane

And taking out the modified acellular amniotic membrane, adding the modified acellular amniotic membrane into 5 mL of PBS, adding 30 mg of NaOH, and performing ultrasonic treatment for 4h (37 Hz, 580W) to realize the carboxyl deprotection of phenylalanine.

(4) Elution of the carboxyl protecting group

Taking out the modified acellular amniotic membrane, washing with PBS, dialyzing in ultrapure water (with a dialysis bag molecular weight cutoff of 500 Da) for 3 days, eluting the carboxyl protecting group of phenylalanine, and freeze-drying.

(5) the-OH of the modified acellular amniotic membrane carboxyl is converted into chlorine element

Adding the acellular amniotic membrane into 3 mL of dichloromethane, dropwise adding 0.8 mL of thionyl chloride, dropwise adding 200 mu L of dimethyl formamide (DMF), stirring in ice bath for 48 hours, and converting carboxyl-OH on the surface of the modified acellular amniotic membrane into chlorine. And (3) performing suspension evaporation to remove dichloromethane, thionyl chloride and dimethylformamide.

(6) Loading of tumor drugs

And immersing the obtained product into 5 mL of 1 mg/mL adriamycin solution, and stirring for 5 min to obtain the modified acellular amnion loaded with the anti-tumor drug.

The modified acellular amniotic membrane prepared in this example was subjected to the following performance tests:

1. surface hydrophilicity and hydrophobicity detection

The test method is the same as the previous test method, and the result shows that the contact angle of the acellular amniotic membrane is 73 +/-1.01 degrees, the contact angle of the modified acellular amniotic membrane is 116.3 +/-4.87 degrees, and the hydrophobicity of the modified acellular amniotic membrane is enhanced.

2. Modified acellular amniotic membrane drug loading detection method

The test method is the same as that of the previous method, and the detection wavelength is 253 nm. The result shows that the drug loading rate of the acellular amniotic membrane is 100.6 +/-4.2 mu g/mg.

3. Evaluation of target effect of phenylalanine loaded tumor drug

Adding 3mg phenylalanine into 10mL of 1 mg/mL adriamycin solution, reacting at room temperature for 24 h, and removing small molecules of phenylalanine and adriamycin by using a dialysis bag with the molecular weight cutoff of 600 Da to obtain the copolymer of adriamycin and phenylalanineAfter freeze-drying, 8.3 mg was weighed. 4 mg of copolymer were taken out and added to the MES solution (pH = 6.8) and 1.31 mg of EDC were added. After sufficient dissolution, 0.7 mg of fluorescein isothiocyanate (CAS: 3326-32-7) labeled copolymer was added in the dark, doxorubicin was labeled in the same manner, lyophilized in the dark, and weighed. Planting the prostate tumor DU145 cells on a 6-well plate (3 wells in each group), adding fluorescent labeled adriamycin and copolymer of adriamycin and phenylalanine after the culture dish is filled with 80 percent of the cells, and adding 5 percent of CO at 37 DEG C₂Culturing in an incubator for 6 h in the dark (apoptosis is not completely activated), repeatedly washing the cells for 3 times by using PBS in the dark condition, and detecting the number of the fluorescent markers by using a flow cytometer. The result shows that the number of cells with fluorescent labels in the adriamycin and phenylalanine polymer group is 3.1 +/-0.02 times of that in the adriamycin group.

Example 3

The modified decellularized peritoneum of this example was prepared by the following method:

(1) acellular peritoneal carboxyl activation

10 cm × 10 cm of decellularized peritoneum was immersed in 8 mL of 2- (N-Morpholino) Ethanesulfonic (MES) solution (pH = 5.8), and 1.51 g of NHS was added thereto and stirred for 12 hours to activate the carboxyl group in the decellularized peritoneum. Dialyzing (molecular weight cut-off of 500Da in dialysis bag) in ultrapure water for 6 h, and reserving.

(2) Surface modification of acellular peritoneum

To 20 mL of 70% methylene chloride MES solution (pH = 6.5) was added 0.8 g benzyl L-phenylalanine hydrochloride containing a carboxyl protecting group, 1 g tetrabutylammonium bromide, and after complete solubilization, the carboxyl-activated decellularized peritoneum was added and sonicated for 2 h (37 Hz, 580W) to react the amino groups of phenylalanine with the carboxyl groups on the surface of the decellularized peritoneum. Dialyzing with dialysis bag (molecular weight cut-off 500 Da) for 3 days.

(3) Modified decellularized peritoneal decarboxylation protection

P-benzyl L-phenylalanine hydrogenolysis: taking out the modified acellular peritoneum, immersing the acellular peritoneum into 8 mL of ethanol, adding 2 g of Pd-C, introducing hydrogen (pressure, 1 MPa) by using a hydrogen pillow, and reacting for 3 h to realize decarboxylation protection of phenylalanine.

(4) Elution of the carboxyl protecting group

And taking out the modified acellular peritoneum, washing with PBS, extracting toluene with chloroform, removing carboxyl protecting groups, repeatedly washing with PBS, and freeze-drying.

(5) Conversion of-OH of modified decellularized peritoneal carboxyl groups to elemental bromine

The lyophilized modified acellular peritoneum was added to 2.5 mL of dichloromethane, and 1mL of POBr was added dropwise₃Stirring at room temperature for 48h to convert the-OH of the modified acellular peritoneal carboxyl into bromine. Suspension evaporation for removing dichloromethane and POBr₃。

(6) Loaded with antibacterial and antitumor drugs

Immersing into 10mL of 5 mg/mL vancomycin solution, stirring for 30 min, taking out, immersing into 5 mL of 3 mg/mL methotrexate solution, and stirring for 20 min to obtain the modified acellular peritoneum loaded with the antibacterial/antitumor drugs.

The modified acellular peritoneum prepared in this example was subjected to the following performance tests:

1. surface hydrophilicity and hydrophobicity detection

The test method is the same as the previous test method, and the result shows that the contact angle of the acellular peritoneum is 87 +/-1.29 degrees, the contact angle of the modified acellular peritoneum is 119.3 +/-2.47 degrees, and the hydrophobicity of the modified acellular peritoneum is enhanced.

2. Modified acellular peritoneal drug loading detection

The test method is the same as that of the previous method, the detection wavelength of vancomycin is 236 nm, and the detection wavelength of methotrexate is 302 nm. The results show that the drug-loading rates of the decellularized peritoneum vancomycin and the methotrexate are 57 +/-2.8 mu g/mg and 31 +/-0.7 mu g/mg respectively.

Example 4

The modified acellular cornea of this example was prepared by the following method:

(1) acellular corneal carboxyl activation

The acellular cornea after cross-linking is taken to be 1 cm multiplied by 1 cm (the modification method is shown in patent 201610040990.0), the acellular cornea is immersed into 3 mL of 2- (N-Morpholine) Ethanesulfonic (MES) solution (pH = 6.8), 0.47 g of EDC is added in sequence, and the mixture is stirred for 2 h, so that the carboxyl activation of the acellular cornea is realized.

(2) Surface modification of acellular cornea

The synthesis method of the carboxyl protecting group of phenylalanine is shown in (preparation of chiral stationary phase of L-phenylalanine [ J ]. fine chemical industry, 2012, 29(3): 69-72) by virtue of the army, Liangli and Li Hui), and the phenylalanine methyl ester is obtained.

100 mg of L-phenylalanine methyl ester is added into the solution and reacts for 4 hours under the microwave condition (100W, 60 ℃) to ensure that the amino of phenylalanine reacts with the carboxyl of the acellular pericardium. Dialyzing with dialysis bag (molecular weight cut-off 8000 Da) for 3 days.

(3) Modified decellularized corneal decarboxylation protection

And taking out the modified acellular cornea, adding 5 mL of MES solution (pH = 5.6), dropwise adding 200 mu L of concentrated hydrochloric acid, reacting at 40 ℃ for 3 h to acidolyze methyl phenylalanine, and realizing decarboxylation protection of phenylalanine.

(4) Elution of the carboxyl protecting group

Taking out the modified acellular cornea, washing with PBS, dialyzing in ultrapure water (molecular weight cut-off of dialysis bag is 500 Da) for 3 days, eluting carboxyl protecting group, and freeze-drying.

(5) Modified acellular corneal carboxyl-OH converted into chlorine element

And adding the modified acellular cornea into 2.5 mL of dichloromethane, dropwise adding 1mL of phosphorus trichloride, stirring at room temperature for 48 hours, and converting the-OH of the carboxyl group of the modified acellular cornea into chlorine. Methylene chloride and phosphorus trichloride were removed by suspension evaporation.

(6) Loading of anti-inflammatory drugs

Immersing into 3 mL of 10 mg/mL dexamethasone solution, and stirring for 2 h to obtain the acellular corneal stroma loaded with the anti-inflammatory drug.

The modified acellular cornea prepared in this example was subjected to the following performance tests:

1. surface hydrophilicity and hydrophobicity detection

The test method is the same as the previous test method, and the result shows that the contact angle of the acellular cornea is 67 +/-1.29 degrees, the contact angle of the modified acellular peritoneum is 99.3 +/-2.47 degrees, and the hydrophobicity of the modified acellular cornea is enhanced.

2. Modified acellular cornea drug loading detection

The test method is the same as that of the previous method, and the detection wavelength is 240 nm. The results show that the drug loading of the acellular cornea is 118 +/-4.8 mu g/mg.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims

1. A surface modification method of an acellular matrix is characterized by comprising the following steps:

(1) performing carboxyl activation on the acellular matrix;

(4) eluting the carboxyl protecting group;

2. The method of surface modification of an acellular matrix according to claim 1, wherein the acellular matrix comprises an acellular treated matrix or a cross-linked modified acellular matrix; the activating reagent used for activating the carboxyl in the step (1) is 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide and/or N-hydroxysuccinimide.

3. The method for surface modification of acellular matrix according to claim 1, wherein the carboxyl protection of phenylalanine in step (2) is performed by esterification, amide or hydrazide method, or by using amino acid derivatives with carboxyl protecting group, to obtain phenylalanine derivatives.

4. The method of surface modification of an acellular matrix according to claim 1, wherein the method comprises

The derivative of phenylalanine or phenylalanine protected by carboxyl is L-phenylalanine benzyl ester hydrochloride or L-phenylalanine ethyl ester or phenylalanine methyl ester.

5. The method of surface modification of an acellular matrix according to claim 1,

the treatment method for carboxyl deprotection in the step (3) comprises at least one of hydrolysis or pyrolysis of ester, amide and hydrazide under catalysis of acid or base, acidolysis under mild conditions and hydrogenolysis of benzyl ester.

6. The method of claim 5, wherein the reagent used for the hydrogenolysis of benzyl ester comprises H₂Pd-C/ethanol, H₂/Pd(OH)₂-C/ethanol, β -mercaptoethanol/BF₃More than one group of ethyl ether, HBr/glacial acetic acid/dichloroacetic acid, and trimethyl iodosilane/carbon tetrachloride.

7. The method for modifying the surface of an acellular matrix according to claim 1, wherein the method for eluting carboxyl protecting groups in step (4) comprises extraction and dialysis.

8. The method for surface modification of an acellular matrix according to any one of claims 1 to 6, wherein the carboxyl acyl halide in the step (5) comprises: reacting the acellular matrix with phosphorus trichloride, phosphorus pentachloride, thionyl chloride or oxalyl chloride.

9. A surface-modified acellular matrix prepared according to the surface modification method of any one of claims 1 to 8.

10. Use of the surface-modified acellular matrix of claim 9 in the preparation of a loaded anti-tumor, anti-bacterial, or anti-inflammatory pharmaceutical formulation.