CN107998453A - The acellular matrix and its method of modifying that a kind of surface is modified - Google Patents
The acellular matrix and its method of modifying that a kind of surface is modified Download PDFInfo
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- CN107998453A CN107998453A CN201711321409.3A CN201711321409A CN107998453A CN 107998453 A CN107998453 A CN 107998453A CN 201711321409 A CN201711321409 A CN 201711321409A CN 107998453 A CN107998453 A CN 107998453A
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3683—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
- A61L27/3687—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment characterised by the use of chemical agents in the treatment, e.g. specific enzymes, detergents, capping agents, crosslinkers, anticalcification agents
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
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Abstract
The invention belongs to field of tissue engineering technology, and in particular to a kind of surface modifying method of acellular matrix.The method of modifying includes procedure below：(1) activated carboxylic is carried out to acellular matrix；(2) carboxy protective is carried out to phenylalanine or phenylalanine derivative, the phenylalanine of carboxy protective or the derivative of phenylalanine will be have passed through graft modification is carried out by the carboxyl of its amino and acellular matrix；(3) acellular matrix after graft modification is subjected to post-processing, that is, obtains the acellular matrix of surface modification.The acellular matrix of the modification has the function of that antitumor cell and inflammatory cell infiltration, anti-infective, anti-matrix metalloproteinase and lactic acid degrade and improve inflammatory environment, can be used for the postoperative tissue repair of oncotherapy.
Description
Technical field
The present invention relates to field of tissue engineering technology, and in particular to the acellular matrix and its method of modifying that a kind of surface is modified.
Background technology
Acellular matrix material has abundant bioactive ingredients, day as a kind of natural biodegradable material
Right three-dimensional net structure, relatively low immunogenicity, have been widely used in field of tissue engineering technology.In terms of oncotherapy,
Acellular matrix material has been used for solving the problems, such as that the postoperative viscous mucosa injury of throat tumor resection is repaired, (Zhang Qingquan, Sun Yan, king
By force, xenogenesis (ox) acellular dermal matrix is waited to repair recruitment evaluation [J] the China group of the postoperative Mucosa Defect of throat tumor resection
Knit engineering research, 2008,12 (06):1081-1084.), there is an urgent need for for solving the problems, such as the de- thin of the postoperative reparation of malignant tumour at present
Cytoplasmic matrix material preparation technology.
The postoperative special physiological environment of malignant tumour has following requirement to the performance for being implanted into acellular matrix：
1. tumour cell has metastatic and latency, Partial tumors cell is transferred in blood vessel and perienchyma, and with
The form of normal cell is hidden.Can be according to regulation expanded scope during oncotherapy, the tumour finally killed in therapeutic domain is thin
Born of the same parents, but tumour cell latent outside therapeutic domain still remains；Some tumour growths are in important organ or big blood vessel, nerve week
Enclose, it is impossible to cut off in the lump.These factors cause the postoperative possibility there are neoplasm in situ recurrence.When tumour recurs at other positions,
Huge pain can be brought to patient, but can still take Retreatment；But if tumour is answered in the original position do not repaired completely
Hair, not only increases the difficulty of operation, but also add the difficulty of recovery from illness, in some instances it may even be possible to direct threat to life.Therefore.If implantation
Acellular matrix material have the function of to prevent tumor recurrence, it will expand its application range, improve clinical therapeutic efficacy.
2. the high expression matrix metalloproteinase of tumour cell (a kind of zinc dependence neutral endopeptidase, participation inflammatory reaction,
Substrate degradation, metastases), extracellular matrix can be degraded and reconstruct, this is also that tumour cell has metastatic basis, and
Acellular matrix material main component is extracellular matrix.Preferable acellular matrix implantation material should have antitumor cell
Infiltration and the function of reconstruct extracellular matrix.
3. tumour cell mainly obtains energy by glycolysis mode, metabolism can produce a large amount of lactic acid, make the environment on periphery
In faintly acid, therefore, the degraded of acellular matrix can be accelerated.Once tumour recurs in the original location, the acellular matrix material of implantation
If having the function of anti-lactic acid degraded, it can not only extend the degradation time of itself, tumour cell can also be reduced to normal
The infiltration of tissue.
4. high inflammatory conditions are presented after tumor locus treatment, a large amount of inflammatory cells in periphery can move into damaged part.If plant
The acellular matrix entered has the ability of anti-inflammatory cellular infiltration, can slow down the degraded of itself, while improve damaged part
Inflammatory environment.
Therefore, compared to other wound repairs, the postoperative reparation of malignant tumour faces more acute rush degraded environment, and same
When the anti-degradation capability of acellular matrix material that is implanted into tissue reparation and reduce the infiltration of tumour cell normal tissue
Aspect has prior meaning.The acellular matrix material for the postoperative tissue repair of malignant tumour is obtained, it is necessary to solve to take off
Cell matrix materials degradation-resistant problem under tumour cell, inflammatory cell, lactic acid effect.If modified acellular matrix
Material has the function of to prevent tumor recurrence, it will has bigger application value.
At present, for the anti-degradation problem of acellular matrix material, existing method of modifying include chemical method, Physical,
Bioanalysis.(1) chemical method is to carry out internal crosslinking to acellular matrix using chemical cross-linking agent.Use chemical cross-linking agent bag earliest
Include glutaraldehyde, formaldehyde, 1- (3- dimethylamino-propyls) -3- ethyl carbodiimides (EDC), n-hydroxysuccinimide (NHS), ring
Oxide, two acid diamides, diisocyanate, polyethylene glycol etc..But modified acellular matrix material can be discharged in degradation process
Go out these crosslinking agents, influence normal physiological function.Natural biological crosslinking agent is gradually available for the cross-linking modified of acellular matrix.Specially
Sharp (201110165428.8 and 201610284706.4) are then using natural biologicals such as anthocyanidin, riboflavin, ribose and Geniposides
Crosslinking agent is crosslinked acellular matrix；Patent (201410459064.8) devises the special crosslinking agent (10~20% of cornea
(w/v) dextran solution, 0.1~0.2% (w/v) riboflavin) solve the problems, such as the crosslinking of cell-eliminating coanea matrix.(2) physics
Method is using the physical factor such as irradiation, heat, solution environmental, activates the group in acellular matrix, realizes acellular matrix oneself
Crosslinking.Such as：Patent (201611209243.1) proposes that improving acellular matrix is freezed and be heat-treated, and realizes de- cell base
The heat cross-linking of matter.Patent (201110199312.6) changes the dynamic electricity of collagen by sodium polyaspartate and water soluble chitosan
Current potential, realizes and adjusts collagen binding structure and pattern, repairs the natural structure of collagen.(3) bioanalysis refers to use transglutamin-ase 9
Self-crosslinking occurs for the catalysis acellular matrix such as enzyme, horseradish peroxidase, tyrosinase, lysyloxidase.Above-mentioned these change
Property method improve the connection of collagen inside acellular matrix, extend the degradation time of acellular matrix material, but can not
Avoid the immersional wetting of the matrix metalloproteinase of tumour cell, lactic acid and inflammatory cell.
In addition, inflammation and easy infection feature for damage location, researcher is by loading inflammation or antibacterials
Mode is modified acellular matrix material.Patent (200810017603.7) coats load mould on acellular matrix surface
The gel of element, for inflammation, ulcer, burns the treatment of defect of skin caused by reason such as wound, iatrogenic.Patent
(200910200933.4) in the nanoparticle of acellular matrix surface crosslinked antimicrobial agent, its anti-infectious ability is assigned.Patent
(201610395479.2) it is broken in the fiber surfaces such as fibroin albumen, poly (glycolide-co-lactide) or polycaprolactone coating acellular matrix
Piece and aspirin, Ketoprofen, Shu Tian lead to or the gel of the preparation such as Finasteride, realizes the sustained release of anti-inflammatory drugs.Use
Similar method of modifying, even if addition tumour medicine, can only reduce the direct effect of tumour cell and inflammatory cell, it is impossible to shield
The degradation of matrix metalloproteinase and lactic acid to material.And the method for these carrying medicaments, tumour medicine can not be avoided
Damage of the thing to normal cell.
For amino acid as the elementary cell for forming protein, itself has carboxyl and amino, can be used as crosslinking agent to de-
Cellular matrix is modified.Patent (200910048157.0) adds free amino acid into acellular dermal matrix and promotes to take off
The vascularization of cellular matrix, amino acid used include institute's tryptophan, methionine, threonine, valine, lysine, histidine,
Leucine, isoleucine, alanine, phenylalanine, cystine, cysteine, arginine, glycine, serine, tyrosine,
Glutamine, proline, aspartic acid and arginine etc..Lai is reported respectively using glycine (neutrality), lysine (alkali
Property), the modified de- cell amnion of glutamic acid (acidity), have it is good cells infiltrating, for corneal transplantation (Lai
JY.Carbodiimide cross-linking of amniotic membranes in the presence of amino
acid bridges.Mat SciEng C-Mater.2015；51:28-36.).Patent (201610040990.0) uses acid
Amino acid (aspartic acid, glutamic acid, histidine) and basic amino acid (lysine, arginine, proline) are to taking off cell cornea
It is modified, the water lock ability of amino acid used improves the moisture content of de- cell cornea, is conducive to growing into for cell, Ke Yijia
The reparation of fast cornea function.However, the purpose of above-mentioned amino acid crosslinks method is for promoting cell to grow into de- cell base
Matter, and in order to more preferably promote cell to grow into, amino acid used in patent (200910048157.0) requirement is free ammonia
Base acid.It is on the contrary tumour if the acellular matrix implantation tumour that above-mentioned amino acid crosslinks method of modifying is obtained cuts off position
Cell provides more preferable nutrition and living environment.
In short, existing acellular matrix material modification method solves the problems, such as many clinical practices, but face pernicious
The physiological environment of tumor post-operation is, it is necessary to solve the anti-degraded under tumour cell, inflammatory cell, lactic acid effect of acellular matrix material
The problem of, it is therefore desirable to design new acellular matrix method of modifying.
The content of the invention
Based on this, it is an object of the present invention to providing a kind of surface modifying method of acellular matrix, realize de-
Cellular matrix it is surface-functionalized, can be used for preparing and meet the acellular matrix material of the postoperative reparation demand of malignant tumour.
Realize that the concrete technical scheme of above-mentioned purpose is as follows.
A kind of surface modifying method of acellular matrix, comprises the following steps：
(1) activated carboxylic is carried out to acellular matrix；
(2) carboxy protective is carried out to phenylalanine or phenylalanine derivative, the phenylalanine of carboxy protective will be have passed through
Or the derivative of phenylalanine carries out graft modification by the carboxyl of its amino and acellular matrix；
(3) the acellular matrix decarboxylate after graft modification is protected；
(4) carboxy protective group is eluted；
(5) it is carboxylic acid halides by the converting carboxylate groups of the acellular matrix after elution carboxy protective group, that is, obtains surface modification
Acellular matrix.
The surface modifying method of the acellular matrix of the present invention, by the inventor research to acellular matrix and reality for a long time
Test, repair demand for satisfaction property tumor post-operation, dexterously used phenylalanine or derivatives thereof to acellular matrix carry out table
Face is modified.The carboxyl of acellular matrix is activated first, then the carboxyl of phenylalanine and its derivative is protected,
The amino and acellular matrix activated carboxyl for reusing the derivative after carboxy protective are grafted, and afterwards, remove carboxy protective,
It is acyl chlorides by the converting carboxylate groups of acellular matrix, from last grafting antitumor drug, antibacterials, anti-inflammatory medicaments, so that
Acellular matrix material is modified to clinical surface is really suitable for.The modified acellular matrix in obtained surface, which can be realized, to be borrowed
The carboxyl of phenylalanine, load antitumor drug, antibacterials, anti-inflammatory medicaments are helped, and realize medicament slow release, are extended to tumour
The inhibitory action of cell and inflammatory cell；By tumour cell to phenylalanine psychological need, the targeting of medicine is improved；By
The hydrophobic effect of phenylalanine phenyl ring, suppresses cellular infiltration, anti-matrix metalloproteinase and lactic acid degraded, improves medicament slow release effect
Fruit.
Step (1) is the purpose of acellular matrix activated carboxylic, improves its amino with the derivative of phenylalanine
The yield of reaction.
Acellular matrix described in step (1) include the matrix after de- cell processing or it is cross-linking modified after de- cell base
Matter.
In one of the embodiments, the activating reagent used in the activated carboxylic described in step (1) is 1- (3- dimethylaminos
Propyl group) -3- ethyl carbodiimides and/or n-hydroxysuccinimide.Cell-eliminating coanea matrix is carried out to pH during activated carboxylic
For 4-8.
Step (2) is the purpose of phenylalanine carboxy protective, avoids the carboxyl and acellular matrix of phenylalanine
Amino reacts, and retains reaction site for final grafting medicine；Make the amino and acellular matrix of the derivative of phenylalanine
Carboxyl is grafted.
In one of the embodiments, in step (2), the carboxy protective of the phenylalanine described in step (2) uses ester
Change method, acid amides or hydrazides method, or using the amino acid derivativges with carboxy protective group, obtain the derivative of phenylalanine.
The purpose of the decarboxylate protection of the step (3) is the carboxyl of phenylalanine being exposed, for follow-up
Medicine is grafted.
Carboxyl deprotection processing method in the step (3) includes ester, acid amides, hydrazides and is hydrolyzed under the catalysis of acid or alkali
Or acidolysis under pyrolysis, temperate condition, and the hydrogenolysis of benzyl ester.
In wherein one is implemented, the reagent used of the hydrogenolysis of the benzyl ester includes H2/ Pd-C/ ethanol, H2/Pd
(OH)2- C/ ethanol, beta -mercaptoethanol/BF3/ ether, HBr/ glacial acetic acid/dichloroacetic acid, the one of Iodotrimethylsilane/carbon tetrachloride
Group or more than one group.
The purpose of step (4) the elution carboxy protective group is to wash away carboxy protective group.
The method of elution carboxy protective group in the step (4) includes extraction, dialysis.The step (5) will be grafted
The purpose that-the OH of the carboxyl of modified acellular matrix is converted into halogen is, is acyl chlorides by converting carboxylate groups, raising and medicine
The speed and yield of grafting.
In wherein one is implemented, the carboxyl by the acellular matrix after graft modification described in the step (5)-
OH is converted into halogen, preferably chlorine element, and preparation method is acellular matrix and phosphorus trichloride, phosphorus pentachloride, thionyl chloride, sulfurous
Acyl chlorides or oxalyl chloride reaction.
Another object of the present invention is to provide acellular matrix material and its application that a kind of surface is modified.
The acellular matrix material that a kind of surface is modified, is prepared by the above method.
The acellular matrix material that above-mentioned surface is modified, can be used for preparing load antitumor drug, antibacterials and/
Or the preparation of anti-inflammatory medicaments.
Acellular matrix material load of the present invention has tumour medicine to be simultaneously sustained for a long time, is expected to reduce tumour
Recurrence rate；Material surface has the ability of antitumor cell and inflammatory cell infiltration, and can resist the long-term erosion of lactic acid etc.,
And there is anti-infective, improvement inflammatory environment.
The technical principle of the present invention is as follows：
1. growth of the phenylalanine to tumour cell has a very important role, phenylalanine load tumour medicine, can
To improve the probability that tumour medicine enters tumour cell, and reduce the influence to normal cell.
2. the phenyl ring that phenylalanine carries has very strong hydrophobic forces, the phenylalanine contained in protein is to maintaining
Protein tridimensional and quaternary structure have great importance.Hydrophobic bond active force scope can reach more than 100nm, and hydrogen bond,
Within 5nm, the phenylalanine that modified acellular matrix surface is contained has the scope of Van der Waals force, ionic bond and covalent bond etc.
There is larger sphere of action.
3. pair phenylalanine carries out carboxy protective using esterification process, acid amides or hydrazides method, phenylpropyl alcohol ammonia in modifying process is avoided
Amino reaction in the carboxyl and acellular matrix of acid, reduces final load drug effect fruit.After modification, by phenylalanine
Ester, acid amides, the hydrazides of formation are hydrolyzed or are pyrolyzed under the catalysis of acid or alkali, acidolysis under temperate condition, and the hydrogenolysis of benzyl ester, extensive
Multiple carboxyl.
4. often with tumour medicine (such as cis-platinum, adriamycin), anti-inflammatory drug (such as glucocorticoid), antibacterial medicines (ten thousand
Ancient mycin etc.) be respectively provided with amino or hydroxyl, the carboxyl on modified acellular matrix surface, acyl chlorides by ionic adsorption, altogether
The effect absorption said medicine that valence link closes and (forms amido link or ester bond).
5. the reactivity of acyl chlorides and amino and carboxyl is higher than carboxyl ,-the OH of carboxyl is substituted using halogen, can be improved
The speed and quantity of medicine grafting, facilitate Clinical practice.
Compared with prior art, the present invention having the following advantages that and beneficial effect：
1. it is swollen can make it that tumour medicine efficiently enters to the different demands of phenylalanine for tumour cell and normal cell
Oncocyte reduces the injury to normal cell at the same time.Due to phenylalanine and essential amino acid, modified de- cell base
Have no adverse effects after matter degraded to perienchyma.
2. modified acellular matrix surface hydrophobic of the present invention can reduce periphery tumour cell and inflammatory
The infiltration of cell.
3. modified acellular matrix surface hydrophobicity active force of the present invention avoids matrix metalloproteinase, lactic acid
Enter Deng material inside acellular matrix, while also avoid the outflow of acellular matrix internal water-soluble biomolecule, slow down de-
The degraded of cellular matrix.
4. conventional tumour medicine, anti-inflammatory drug, antibacterials are mostly liposoluble substance, the hydrophobicity of phenylalanine is favourable
In combining and load these medicines；The ionic bond or covalent bond of phenylalanine can and medicine form stable combination, realize medicine
Thing is sustained, and is avoided violent release and is extended drug effect.
5. surrounding aqueous environment interacts with modified acellular matrix surface hydrophobicity key, formed " clathrate hydrate ",
Tumour medicine is pinned, strengthens the slow release effect of medicine.
After 6. the converting carboxylate groups on modified acellular matrix surface are acyl chlorides, the efficiency with medicine grafting is improved, can be with
Half an hour contains medicine before use, facilitates Clinical practice.
7. modified acellular matrix surface can load different medicines, for suppressing tumour cell, preventing bacterium
Infection, improve inflammatory environment.
Brief description of the drawings
Fig. 1 is the Fourier transformation decay Total Reflection Infrared spectrogram before and after the accellular pericardial membrane modifying in embodiment 1；
Fig. 2 is the release profiles of the modified cis-platinum of accellular pericardial membrane matrix in embodiment 1；
Fig. 3 is the modified collagen content change of accellular pericardial membrane matrix in embodiment 1.
Embodiment
In order to be more clearly understood that the technology contents of the present invention, it is described with reference to the accompanying drawings especially exemplified by following embodiments.
It is to be understood that these embodiments are only illustrative of the invention and is not intended to limit the scope of the invention.It is not specified in the following example
The experimental method of actual conditions, usually according to normal condition, such as Sambrook et al., molecular cloning：Laboratory manual (New
York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or built according to manufacturer
The condition of view.Used various common chemical reagent, are commercial product in embodiment.Following reagent used in the present invention can
It is commercially available by conventional route.
Unless otherwise defined, all of technologies and scientific terms used here by the article is with belonging to technical field of the invention
The normally understood implication of technical staff is identical.Term used in the description of the invention herein is intended merely to description tool
The purpose of the embodiment of body, it is not intended that in the limitation present invention.Term as used herein "and/or" includes one or more phases
The arbitrary and all combination of the Listed Items of pass.
Each technical characteristic of embodiment described above can be combined arbitrarily, to make description succinct, not to above-mentioned reality
Apply all possible combination of each technical characteristic in example to be all described, as long as however, the combination of these technical characteristics is not deposited
In contradiction, the scope that this specification is recorded all is considered to be.
In method of modifying of the present invention, to improve the efficiency of the reaction, phase in version catalyst is included the use of, light is urged
The one or more of change, microwave, ultrasonic wave, irradiation etc..Adjust power, the frequency of ultrasound；The power of microwave；Irradiate agent
The parameters such as amount, time, improve modified effect.
The phase transfer catalyst includes polyethers (such as chain polyethylene glycol, chain dialkylethers), ring-type is preced with
Ethers (such as cyclodextrin), quaternary ammonium salt (such as tetrabutylammonium bromide, dodecyl trimethyl ammonium chloride), tertiary amine (such as tri-n-butylamine), season
Ammonium alkali, quaternary alkylphosphonium salt.
The power of the ultrasonic wave is preferably 100~600W, and frequency is preferably 30-80kHz.
The power of the microwave is preferably 80-120W.
The irradiation dose be preferably 10-30KyG, time be preferably 2-10min.
Embodiment 1
The modification accellular pericardial film of the present embodiment is prepared by following methods：
(1) activated carboxylic of accellular pericardial film
10cm × 5cm accellular pericardials film (DPP) is taken, immerses 8mL2- (N- morpholines) second sulphur (MES) solution (pH=
6.5) in, 0.85g EDC, 0.51g NHS are sequentially added, 12h is stirred, the carboxyl of accellular pericardial film surface is activated.
Dialyse (bag filter molecular cut off 500Da) 24h in ultra-pure water, removes activator, spare.
(2) surface of accellular pericardial film is modified
Contain the L-phenylalanine benzyl ester hydrochloric acid of carboxy protective group to 10mL MES solutions (pH=6.5) addition 0.3g
Salt, 0.57g tetrabutylammonium bromide, adds the accellular pericardial film after activated carboxylic, stirs 48h, make phenylpropyl alcohol ammonia after being completely dissolved
The amino of acid and the amino graft reaction on acellular matrix surface.Dialysed 3 days using bag filter (molecular cut off 500Da).Obtain
Obtain modified accellular pericardial film (M-DPP).
(3) the decarboxylate protection of modified accellular pericardial film
M-DPP is taken out, is immersed in 5mL di-chloroacetic acid solutions, ice bath, the 33% HBr/ glacial acetic acid for adding 5 times of volumes is molten
Liquid, lucifuge reaction 3h, protects phenylalanine decarboxylate, exposes carboxyl.
(4) elution of carboxy protective group
Acellular matrix lucifuge dialysis (bag filter molecular cut off 500Da) 3 days in ultra-pure water are taken out, elution carboxyl is protected
Group is protected, is freezed.
(5)-the OH of the carboxyl of modified accellular pericardial film is converted into chlorine element
Lyophilized M-DPP is taken, is added in 2mL dichloromethane (being steamed using front overhang, remove moisture removal), 0.5mL grass is slowly added dropwise
Acyl chlorides, is stirred at room temperature 1h, and-the OH of M-DPP surface carboxyl groups is converted into chlorine element.Freeze-drying removes oxalyl chloride and dichloromethane
Alkane.
(6) load of antitumor drug
Immersed after obtained Weighed product in 5mL 3mg/mL cisplatin solutions, stir 15min, obtain load antitumor drug
M-DPP.
The modification accellular pericardial film that the present embodiment is prepared carries out following performance test：
1. chemical structure analysis
Use Fourier transformation infrared spectrometer (EQUINOX-70, Bruker, Germany) detection accellular pericardial membrane modifying
Front and rear chemical constitution, as a result as shown in Figure 1.In figure as it can be seen that compared to DPP, the 1631cm of M-DPP-1The amido link C=at place
O stretching vibration peaks strengthen, 3284cm-1Locate the increase of secondary amide NH stretching vibrations peak intensity, show that phenylalanine has accessed the de- cell heart
Envelope membrane surface.
2. surface hydrophilic and hydrophobic detects
Accellular pericardial membrane material after taking before modified respectively, is placed on the glass slide of cleaning.Use contact angle tester
(Kruss DSA100) detects material surface static contact angle.28 DEG C of test temperature, humidity 80%, every group randomly selects 10 points
Test.The result shows that DPP contact angles are 93 ± 3.21 °, M-DPP contact angles are 126.1 ± 1.37 °, are shown modified de- thin
Born of the same parents' pericardium has stronger hydrophobicity.
3. it is modified the detection of accellular pericardial film drugloading rate
Cis-platin concentrations gradient solution is set, and collects the cisplatin solution after immersing modified accellular pericardial film, ultraviolet spectrometry
Photometer detects 301nm absorbing wavelengths, calculates drugloading rate.The result shows that the drugloading rate of M-DPP is 74.9 ± 11 μ g/mg.
4. it is modified the detection of accellular pericardial film insoluble drug release
The M-DPP of 5mg load cis-platinums is taken, is moved into bag filter (molecular cut off 500Da), is added in 40mLPBS solution.
Insoluble drug release is carried out in 37 DEG C, the air blast shaking table of 100rad/min, takes out 1mL release liquids within 1 week, 2 weeks, 3 weeks, 4 weeks, at the same time plus
Enter isometric PBS solution.Concentration detection method is the same, and the results are shown in Figure 2.In Fig. 2 as it can be seen that in 1 week M-DPP load it is suitable
Platinum has faster rate of release, and plateau is entered after 2 weeks, and 4 weeks total release percentages are 71 ± 1.19%.
5. it is modified the anti-lactic acid degraded detection of acellular matrix
Using ultra-pure water dissolving MRS culture mediums (the rich biology in Qingdao sea), fully after dissolving, pH=6.2 is adjusted, uses 120
Autoclaving 20min at DEG C.Culture medium is wrapped up using unmodified accellular pericardial film and modified accellular pericardial film respectively,
Surgical thread sutures.Lactic acid bacteria culturers are internally injected, are laid in culture dish (diameter 10cm), periphery adds 3mL culture mediums, 37 DEG C
Constant temperature plate shaker culture.Observe pericardium periphery one week, bacterium colony formational situation after two weeks, the quality for detecting pericardium becomes
Change.The result shows that there is bacterium colony to be formed on unmodified accellular pericardial film periphery, and it is existing without this on modified accellular pericardial film periphery
As there was no significant difference for the two quality, these results suggest that lactic acid can ooze out from unmodified pericardium and change cell
Compact texture inside film, and the modified infiltration that can reduce lactic acid of phenylalanine, and reduce the destruction of lactic acid.
6. it is modified the anti-matrix metalloproteinase degraded detection of acellular matrix
(pH=7.4, contains matrix metalloproteinase (MMP-1, Sigma, article No. SRP3117) solution of configuration 500mg/L
The CaCl of 10mmol/L2).The unmodified accellular pericardial films of 3mg and modified accellular pericardial film are taken, adds 5mL matrix gold
In Proteases solution, it is incubated at 37 DEG C.Collagen content change (kit in 100 μ L solution detection solution is taken out per 30min：
Sirius Red Total Collagen Detection Kit, article No.：9062).The results are shown in Figure 3, modified de- cell
The collagen content that matrix discharges under the action of metalloproteinases is far below unmodified acellular matrix.
Embodiment 2
The modification of the present embodiment takes off cell amnion and is prepared by following methods：
(1) activated carboxylic of cell amnion is taken off
Take 5cm × 5cm to take off cell amnion, immerse in 10mL 2- (N- morpholines) second sulphur (MES) solution (pH=6.5), add
Enter 0.57g EDC, stir 12h, activating surface carboxyl is spare.
(2) surface for taking off cell amnion is modified
The carboxyl of phenylalanine is protected using esterification process, preparation method bibliography (Zeng Guangxiang, Lu Huibang, grandson
Win the tinkling of pieces of jade, wait synthesis and bacteriostatic activity [J] the food industry science and technology of sorbic acid phenylalanine ethyl esters, 2013,34 (24):321-
325.) L-phenylalanine ethyl ester, is obtained.
0.5g L-phenylalanine ethyl esters are added into the solution 10mL of step 1, ice bath stirs 12h, makes the ammonia of phenylalanine
Base and the carboxyl on de- cell amnion surface are reacted.Dialysis (molecular cut off 3000Da) 3 days in PBS.
(3) the decarboxylate protection of modified de- cell amnion
Modified de- cell amnion is taken out, is added in 5mL PBS, addition 30mg NaOH, ultrasonic 4h (37Hz, 580W),
Realize the carboxyl deprotection of phenylalanine.
(4) elution of carboxy protective group
Modified de- cell amnion is taken out, after PBS is rinsed, dialyse (bag filter molecular cut off 500Da) 3 in ultra-pure water
My god, the carboxy protective group of lower phenylalanine is eluted, is freeze-dried.
(5)-the OH of modified de- cell amnion carboxyl is converted into chlorine element
De- cell amnion is added in 3mL dichloromethane, 0.8mL thionyl chlorides are added dropwise, 200 μ L dimethylformamides are added dropwise
(DMF), ice bath stirring 48h, chlorine element is converted into by-the OH of the carboxyl on modified de- cell amnion surface.It is outstanding to boil off except two
Chloromethanes, thionyl chloride, dimethylformamide.
(6) load of tumour medicine
Obtain product to immerse in 5mL 1mg/mL Doxorubicin solutions, stir 5min, obtain the modification of load antitumor drug
De- cell amnion.
The modification that the present embodiment is prepared takes off cell amnion and carries out following performance test：
1. surface hydrophilic and hydrophobic detects
Test method is the same, the results showed that de- cell amnion contact angle is 73 ± 1.01 °, and modified de- cell amnion connects
Feeler is 116.3 ± 4.87 °, shows the hydrophobicity enhancing of modified de- cell amnion.
2. it is modified de- cell amnion drugloading rate detection
Test method is the same, Detection wavelength 253nm.The result shows that the drugloading rate of de- cell amnion is 100.6 ± 4.2 μ
g/mg。
3. phenylalanine loads tumour medicine targeting effect assessment
The 3mg phenylalanines into 10mL 1mg/mL Doxorubicin solutions, after reacting at room temperature 24h, are using molecular cut off
600Da bag filters remove phenylalanine and adriamycin small molecule, the copolymer of adriamycin and phenylalanine are obtained, after freeze-drying
Weigh 8.3mg.Take out 4mg copolymers to add in MES solution (pH=6.8), add 1.31mg EDC.Fully after dissolving, lucifuge
Add the fluorescein isothiocynate ester (CAS of 0.7mg：3326-32-7) labeled copolymer, same mode mark adriamycin, keep away
Light freezes, and weighs.Take tumor of prostate DU145 cell seedings (every group of 3 holes), culture dish 80% to be covered with 6 orifice plates
Afterwards, the adriamycin and the copolymer of adriamycin and phenylalanine of fluorescent marker, 37 DEG C of 5%CO are separately added into2Lucifuge in incubator
6h (apoptosis does not activate completely) is cultivated, cell is cleaned repeatedly 3 times using PBS under the conditions of lucifuge, uses flow cytomery
The quantity of fluorescent marker.The result shows that adriamycin is Ah mould with cell quantity of the phenylalanine polymer group with fluorescent marker
3.1 ± 0.02 times of element group.
Embodiment 3
The modification of the present embodiment takes off cell peritonaeum and is prepared by following methods：
(1) cell peritonaeum activated carboxylic is taken off
Take 10cm × 10cm to take off cell peritonaeum, immerse in 8mL 2- (N- morpholines) second sulphur (MES) solution (pH=5.8),
1.51g NHS are added, stir 12h, realize the activated carboxylic of de- cell peritonaeum.Dialysis (bag filter molecular cut off in ultra-pure water
It is spare 500Da) after 6h.
(2) surface for taking off cell peritonaeum is modified
MES solution (pH=6.5) addition 0.8g to 70% dichloromethane of 20mL contains the L- phenylpropyl alcohols of carboxy protective group
Propylhomoserin benzyl ester hydrochloride, 1g tetrabutylammonium bromide, adds the de- cell peritonaeum after activated carboxylic, ultrasonic 2h after being completely dissolved
(37Hz, 580W), makes the amino of phenylalanine and the carboxyl reaction of de- cell peritoneal surface.Use bag filter (molecular cut off
500Da) dialyse 3 days.
(3) modified de- cell peritonaeum decarboxylate protection
To L-phenylalanine benzyl ester hydrogenolysis：Modified de- cell peritonaeum is taken out, is immersed in 8mL ethanol, adds 2g Pd-
C, is passed through hydrogen (pressure, 1MPa) using hydrogen pillow, reacts 3h, realizes the decarboxylate protection of phenylalanine.
(4) elution of carboxy protective group
Modified de- cell peritonaeum is taken out, after PBS is rinsed, toluene is extracted using chloroform, removes carboxy protective group, then
Rinsed, freezed repeatedly using PBS.
(5)-the OH of modified de- cell peritonaeum carboxyl is converted into bromo element
Lyophilized modified de- cell peritonaeum is added in 2.5mL dichloromethane, and 1mL POBr are added dropwise3, it is stirred at room temperature
48h, bromo element is converted into by-the OH of modified de- cell peritonaeum carboxyl.It is outstanding to boil off except dichloromethane and POBr3。
(6) antibacterial and antitumor drug are loaded
Immerse in 10mL 5mg/mL vancomycin solution, stir 30min, the cry of certain animals of 5mL 3mg/mL first ammonia butterfly is immersed after taking-up
In solution, 20min is stirred, the modification for obtaining load antibacterial/antitumor drug takes off cell peritonaeum.
The modification that the present embodiment is prepared takes off cell peritonaeum and carries out following performance test：
1. surface hydrophilic and hydrophobic detects
Test method is the same, the results showed that de- cell peritonaeum contact angle is 87 ± 1.29 °, and modified de- cell peritonaeum connects
Feeler is 119.3 ± 2.47 °, shows the hydrophobicity enhancing of modified de- cell peritonaeum.
2. it is modified de- cell peritonaeum drugloading rate detection
Test method is the same, and vancomycin Detection wavelength is 236nm, and the cry of certain animals of first ammonia butterfly is 302nm.The result shows that de- cell abdomen
Film vancomycin and first ammonia butterfly cry of certain animals drugloading rate are respectively 57 ± 2.8 μ g/mg, 31 ± 0.7 μ g/mg.
Embodiment 4
The modification of the present embodiment takes off cell cornea and is prepared by following methods：
(1) cell cornea activated carboxylic is taken off
Take 1cm × 1cm to take off cell cornea (method of modifying is shown in patent 201610040990.0) after being crosslinked, immerse 3mL 2-
In (N- morpholines) second sulphur (MES) solution (pH=6.8), 0.47g EDC are sequentially added, stir 2h, realize de- cell cornea
Activated carboxylic.
(2) surface for taking off cell cornea is modified
Carboxy protective group is carried out to phenylalanine, synthetic method is shown in that (Zhang Wenjun, Liang Li, Li Hui .L- phenylalanines are chiral
Preparation [J] fine chemistry industries of stationary phase, 2012,29 (3):69-72), phenyalanine methyl ester is obtained.
100mg L-phenylalanine methyl esters is added into above-mentioned solution, (100W, 60 DEG C) reacts 4h under microwave condition, makes
The amino of phenylalanine and the carboxyl reaction of accellular pericardial film.Dialysed 3 days using bag filter (molecular cut off 8000Da).
(3) modified de- cell cornea decarboxylate protection
Take out modified de- cell cornea, add 5mL MES solutions (pH=5.6), be added dropwise 200 μ L concentrated hydrochloric acids, 40 DEG C
3h is reacted, makes phenyalanine methyl ester acidolysis, realizes the decarboxylate protection of phenylalanine.
(4) elution of carboxy protective group
Modified de- cell cornea is taken out, after PBS is rinsed, (bag filter molecular cut off of dialysing in ultra-pure water
500Da) 3 days, carboxy protective group is eluted, is freezed.
(5)-the OH of modified de- cell cornea carboxyl is converted into chlorine element
Modified de- cell cornea is added in 2.5mL dichloromethane, and 1mL phosphorus trichlorides are added dropwise, 48h is stirred at room temperature, will
- the OH of modified de- cell cornea carboxyl is converted into chlorine element.It is outstanding to boil off except dichloromethane and phosphorus trichloride.
(6) load of anti-inflammatory drug
Immerse in 3mL 10mg/mL dexamethasone solutions, stir 2h, obtain the de- cell cornea base of load anti-inflammatory drug
Matter.
The modification that the present embodiment is prepared takes off cell cornea and carries out following performance test：
1. surface hydrophilic and hydrophobic detects
Test method is the same, the results showed that de- cell Corneal Contact angle is 67 ± 1.29 °, and modified de- cell peritonaeum connects
Feeler is 99.3 ± 2.47 °, shows the hydrophobicity enhancing of modified de- cell cornea.
2. it is modified de- cell cornea drugloading rate detection
Test method is the same, Detection wavelength 240nm.The result shows that the drugloading rate of de- cell cornea is 118 ± 4.8 μ g/
mg。
Each technical characteristic of embodiment described above can be combined arbitrarily, to make description succinct, not to above-mentioned reality
Apply all possible combination of each technical characteristic in example to be all described, as long as however, the combination of these technical characteristics is not deposited
In contradiction, the scope that this specification is recorded all is considered to be.
Embodiment described above only expresses the several embodiments of the present invention, its description is more specific and detailed, but simultaneously
Cannot therefore it be construed as limiting the scope of the patent.It should be pointed out that come for those of ordinary skill in the art
Say, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to the protection of the present invention
Scope.Therefore, the protection domain of patent of the present invention should be determined by the appended claims.
Claims (10)
1. a kind of surface modifying method of acellular matrix, it is characterised in that comprise the following steps：
(1) activated carboxylic is carried out to acellular matrix；
(2) carboxy protective is carried out to phenylalanine or phenylalanine derivative, the phenylalanine or benzene of carboxy protective will be have passed through
The derivative of alanine carries out graft modification by the carboxyl of its amino and acellular matrix；
(3) the acellular matrix decarboxylate after graft modification is protected；
(4) carboxy protective group is eluted；
(5) it is carboxylic acid halides by the converting carboxylate groups of the acellular matrix after elution carboxy protective group, that is, obtains the de- thin of surface modification
Cytoplasmic matrix.
2. the surface modifying method of acellular matrix according to claim 1, it is characterised in that the acellular matrix bag
Containing de- cell processing after matrix or it is cross-linking modified after acellular matrix；The activation used in activated carboxylic described in step (1)
Reagent is 1- (3- dimethylamino-propyls) -3- ethyl carbodiimides and/or n-hydroxysuccinimide.
3. the surface modifying method of acellular matrix according to claim 1, it is characterised in that described in step (2)
The carboxy protective of phenylalanine uses esterification process, acid amides or hydrazides method, or using amino acid derived with carboxy protective group
Thing, obtains the derivative of phenylalanine.
4. the surface modifying method of acellular matrix according to claim 1, it is characterised in that described to have passed through carboxyl guarantor
The phenylalanine of shield or the derivative of phenylalanine are L-phenylalanine benzyl ester hydrochloride or L-phenylalanine ethyl ester or phenylpropyl alcohol ammonia
Sour methyl esters.
5. the surface modifying method of acellular matrix according to claim 1, it is characterised in that
The de-protected processing method of carboxyl in the step (3) include ester, acid amides, hydrazides under the catalysis of acid or alkali hydrolysis or
Acidolysis under pyrolysis, temperate condition, and at least one of hydrogenolysis of benzyl ester.
6. the surface modifying method of acellular matrix according to claim 1, it is characterised in that the hydrogenolysis of the benzyl ester
The reagent used include H2/ Pd-C/ ethanol, H2/Pd(OH)2- C/ ethanol, beta -mercaptoethanol/BF3/ ether, HBr/ glacial acetic acid/
Dichloroacetic acid, Iodotrimethylsilane/carbon tetrachloride it is a group or more of.
7. the surface modifying method of acellular matrix according to claim 1, it is characterised in that in the step (4)
Eluting the method for carboxy protective group includes extraction, dialysis.
8. according to the surface modifying method of any acellular matrixes of claim 1-6, it is characterised in that the step
(5) carboxyl, which turns carboxylic acid halides, in includes：By the acellular matrix and phosphorus trichloride, phosphorus pentachloride, thionyl chloride, thionyl chloride or
Oxalyl chloride is reacted.
9. the acellular matrix that the surface being prepared according to any one of the claim 1-8 surface modifying methods is modified.
10. the acellular matrix that surface described in claim 9 is modified prepare load antitumor drug, antibacterials, and/or
Application in anti-inflammatory medicaments preparation.
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