CN105713152A - Polyethylene glycol bipolymer with endothelial cell selectivity and preparation method and application method of composite coating - Google Patents
Polyethylene glycol bipolymer with endothelial cell selectivity and preparation method and application method of composite coating Download PDFInfo
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- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F283/00—Macromolecular compounds obtained by polymerising monomers on to polymers provided for in subclass C08G
- C08F283/06—Macromolecular compounds obtained by polymerising monomers on to polymers provided for in subclass C08G on to polyethers, polyoxymethylenes or polyacetals
- C08F283/065—Macromolecular compounds obtained by polymerising monomers on to polymers provided for in subclass C08G on to polyethers, polyoxymethylenes or polyacetals on to unsaturated polyethers, polyoxymethylenes or polyacetals
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- A—HUMAN NECESSITIES
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- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
- A61L31/08—Materials for coatings
- A61L31/10—Macromolecular materials
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
- A61L31/14—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
- A61L31/14—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L31/16—Biologically active materials, e.g. therapeutic substances
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F220/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride ester, amide, imide or nitrile thereof
- C08F220/02—Monocarboxylic acids having less than ten carbon atoms; Derivatives thereof
- C08F220/10—Esters
- C08F220/26—Esters containing oxygen in addition to the carboxy oxygen
- C08F220/28—Esters containing oxygen in addition to the carboxy oxygen containing no aromatic rings in the alcohol moiety
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
- A61L2300/42—Anti-thrombotic agents, anticoagulants, anti-platelet agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2420/00—Materials or methods for coatings medical devices
- A61L2420/02—Methods for coating medical devices
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F220/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride ester, amide, imide or nitrile thereof
- C08F220/02—Monocarboxylic acids having less than ten carbon atoms; Derivatives thereof
- C08F220/10—Esters
- C08F220/26—Esters containing oxygen in addition to the carboxy oxygen
- C08F220/28—Esters containing oxygen in addition to the carboxy oxygen containing no aromatic rings in the alcohol moiety
- C08F220/285—Esters containing oxygen in addition to the carboxy oxygen containing no aromatic rings in the alcohol moiety and containing a polyether chain in the alcohol moiety
- C08F220/286—Esters containing oxygen in addition to the carboxy oxygen containing no aromatic rings in the alcohol moiety and containing a polyether chain in the alcohol moiety and containing polyethylene oxide in the alcohol moiety, e.g. methoxy polyethylene glycol (meth)acrylate
Abstract
The invention relates to a polyethylene glycol bipolymer with endothelial cell selectivity and a preparation method and an application method of a composite coating. The polyethylene glycol bipolymer is synthesized by a cellular carbohydrate coat-bionic biocompatible monomer and a polymerizable monomer containing reactive active functional groups through a free radical polymerization method, and bioactive molecules for promoting endothelial cell adhesion is introduced through a surface fixing method to enable the coating to have endothelial cell selectivity. The polyethylene glycol composite coating with excellent reactivity is capable of keeping non-specificity impedance performance of stent surfaces and achieving in-vivo in-situ endothelial cell capture capability, is stable in structure and can be applicable to an internal environment of a human body, thereby having a promising prospect on cardiovascular restenosis, cancers and the like.
Description
Technical field
The technical field of the invention is the interdisciplinary field of the subjects such as material, biology, physics, chemistry, the preparation method relating to a kind of endothelial cell-selective polyethylene glycols bipolymer and this copolymer, moreover, it relates to the application in preparing composite coating of this bipolymer and application process.
Background technology
Widely using of bracket for eluting medicament, provides good means for efficiently, minimally invasively treating cardiovascular disease.But, the self-contained medicine of bracket for eluting medicament is while the restenosis that treatment is caused by smooth muscle cell hyperplasia, and they also can reduce migration and the multiplication capacity of endotheliocyte, makes Ink vessel transfusing cortex be destroyed, adds the risk of advanced thrombus.The endothelialization caused by the non-selective depression effect of medicine postpones and advanced thrombus is still that the key issue restricting its application.In order to solve this key issue, people have carried out a series of trial.
After vessel endothelialisation degree and stenter to implant, vascular restenosis has close contacting, and therefore has become as the New Policy of cardiovascular embedded material prevention of restenosis based on the research of endothelialization.The fixing of bioactive molecule is realized by function interface material, the selectivity of In-sltu reinforcement endotheliocyte sticks and competitive growth, realize the blood vessel endothelium original position quickly-healing at implant site, be one of important channel solving cardiovascular embedded material biocompatibility.Not only can be effectively improved the blood compatibility of material based on the monomer material that cell saccharic clothing is bionical, and can effectively realize the non-specific impedance including protein, platelet and cell, it has also become one of effective means of cardiovascular embedded material modifying interface.Polymerisable monomer containing reacdon-activity group can more specific binding biomolecule, there is good reactivity.
Summary of the invention
The application is to be the further research on 2010102490045 patent of invention bases in the patent No., the coating material of this patent of invention requires over three kinds of monomer polymerizations and obtains, and the application only requires over two kinds of monomers, it is about to that there is bioactive endothelium selectivity peptide molecule combine with biocompatibility monomer, a kind of novel complex function coating material can be built, and then obtain the Novel cardiovascular bracket coating with endothelial cell-selective.This coating with new function can effectively facilitate the original position endothelialization effect of support, and compared with traditional bracket for eluting medicament, this method rapid original position can catch the endotheliocyte in blood circulation in vivo, forms the process of quick endothelialization;Suppress the growth of smooth muscle cell, it is to avoid the generation of the untoward reaction such as thrombosis, restenosis simultaneously.This polyethylene glycols complex function coating material can keep the non-specific impedance behavior of rack surface, realize internal in-situ endotheliocyte capture ability simultaneously, and the coating structure obtained is stably, adapt to the environment of human body, have a good application prospect in cardiovascular restenosis, cancer etc..
First purpose of the present invention is to provide a kind of endothelial cell-selective polyethylene glycols bipolymer, this bipolymer, the biocompatibility monomer bionical by cell saccharic clothing and containing the polymerisable monomer of reacdon-activity group being synthesized by the method for radical polymerization;
The biocompatibility monomer that described cell saccharic clothing is bionical is selected from monomer:
Wherein R=H or CH3, n=1-50;
Described containing can the polymerisable monomer of reacdon-activity group as follows:
Preferably, the biocompatibility monomer that described cell saccharic clothing is bionical, R=H or CH3, n=5-30.
Second purpose of the present invention is the preparation method that above-mentioned endothelial cell-selective polyethylene glycols bipolymer is claimed, and comprises the following steps:
(1) by biocompatibility monomer bionical for cell saccharic clothing with containing can the polymerisable monomer of reacdon-activity group dissolve in a solvent according to the ratio of weight ratio 10:1~1:10, solute and solvent quality ratio is for 1:5~1:50, it is thus achieved that solution A;
(2) weigh radical initiator azodiisobutyronitrile according in step (1) the 0.2%~2% of two kinds of monomer gross masses, join in solution A, obtain solution B;
(3) with nitrogen or argon to solution B deoxygenation 5~30min;
(4) solution after deoxygenation is heated to 50~90 DEG C, reacts 6~48h;
(5) removal of solvent under reduced pressure, by ice ether precipitating 2~3 times, obtains having endothelial cell-selective polyethylene glycols bipolymer.
Further, step (1) described solvent is the one in oxolane, chloroform, dichloromethane, isopropanol, ethanol or methanol.
The 3rd purpose of the present invention is the composite coating being claimed and having polyethylene glycols bipolymer of the present invention.This composite coating comprises endothelial cell-selective polyethylene glycols bipolymer and is fixed on the bioactive molecule of substrate surface.
The expansion of bioactive molecule kind makes can there be wider range of degree of freedom when selecting, and carries out selecting most suitable biomolecule according to practical situation, such as factors such as the compatibility, substrate surface, internal milieu or costs.
Preferably, bioactive molecule of the present invention is the rgd peptide containing arginine-glycine-aspartic acid sequence, REDV polypeptide containing arginine-glutamic acid-aspartic acid-valine sequence, cRGD cyclic peptide containing ring arginine-glycine-aspartic acid sequence, containing Tyrosine-Isoleucine-glycine-serine-arginic YIGSR polypeptide, CD34 antibody or the one in CD133 antibody.
Preferably, base material is the one in PET substrate's film, substrate of glass film, stainless steel-based counterdie or rustless steel angiocarpy bracket.
The 4th purpose of the present invention is the application above-mentioned endothelial cell-selective polyethylene glycols composite coating being claimed on angiocarpy bracket.
The method of this application comprises the steps:
A () will have endothelial cell-selective polyethylene glycols bipolymer and be dissolved in tetrahydrofuran solvent, be configured to the solution that mass concentration is 0.01~5%, and by supersound process, obtain the solution C being uniformly dissolved;
(b) by base material successively with acetone, methanol, the ultrasonic pretreatment of tri-distilled water 10~30 minutes;
C () takes 1,6-hexamethylene diamine solid and is dissolved in dehydrated alcohol, be configured to the solution of 10wt%, is immersed in 1,6-hexamethylene diamine solution by the base material anticipated, and reacts 24h at normal temperatures, then rinses 3 times with tri-distilled water, N2Dry up, save backup;
D () adopts dip coating that solution C is coated in the substrate surface that step (c) processed;
E the substrate surface nitrogen having been coated with material is dried up by () after, immerse the phosphate buffer that mass concentration is 0.01~0.5% bioactive molecule, 24~72h is reacted at 4~25 DEG C, terminate by the PBS solution of lysine, vacuum drying, obtains endothelial cell-selective polyethylene glycols composite coating.
Step (c) is key one step formed a connecting link.By this step, making covalent modification amino group on pretreated basement membrane, then the epoxide group in coating material (bipolymer) with the amino reaction modified from the teeth outwards, can make coating material be grafted on basement membrane surface.
Preferably, the processing method of step (b) is: with the concentrated sulphuric acid that volume ratio is 7:3 and hydrogen peroxide Grape berry 30~60 minutes, then rinses with tri-distilled water.
The present invention compared with prior art has the beneficial effect that
1) synthetic method of polymers is simple, it is only necessary to two kinds of monomer polymerizations form;
2) polymer synthesized has good reactivity, is conducive to the fixing of biomolecule, it is possible to keep the non-specific impedance behavior of rack surface, realizes internal in-situ endotheliocyte capture ability simultaneously, and the coating structure obtained is stable;
3) institute's fixing biological molecules is in extensive range, including polypeptide and antibody, range of choice width;
4) in composite coating, the synergism of the bionical monomer of cell saccharic clothing and biomolecule can effectively prevent restenosis and thrombosis from simultaneously facilitating the formation of surface endothelialization, and the endothelialization deficiency of existing angiocarpy bracket is well improved.
Accompanying drawing explanation
Fig. 1 is the nuclear-magnetism structure spectrogram of PEGMA-GMA;Wherein (a) n=19, (b) n=6;
Fig. 2 is the composite coating structure schematic diagram of PEGMA (n=19)-GMA-REDV;
Fig. 3 is the composite coating structure schematic diagram of PEGMA (n=6)-GMA-RGD;
Fig. 4 is endotheliocyte and the smooth muscle cell proliferation comparison diagram of PEGMA (n=19)-GMA-REDV composite coating;
Fig. 5 is endotheliocyte and the smooth muscle cell proliferation comparison diagram of PEGMA (n=6)-GMA-RGD composite coating.
Detailed description of the invention
The present invention is described in detail by the examples below, and if no special instructions, the chemical reagent related in following embodiment is all commercially.
Embodiment 1
(1) endothelial cell-selective polyethylene glycols bipolymer is prepared
(1.1) by biocompatibility monomer PEGMA (n=19) bionical for 0.5g cell saccharic clothing, 1.5g containing can reacdon-activity group polymerisable monomer GMA 30ml ethanol dissolve, obtain solution A;
(1.2) 0.04g initiator A IBN is joined in solution A, obtain solution B;
(1.3) with argon to solution B deoxygenation 30 minutes, it is heated to 60 DEG C, reacts 24 hours;
(1.4) decompression is except solvent, ice ether precipitating 2 times, obtains having endothelial cell-selective polyethylene glycols bipolymer PEGMA-GMA.Nuclear-magnetism result confirms that the product obtained has intended structure.As shown in Fig. 1 (a).
(2) endothelial cell-selective polyethylene glycols composite coating is prepared
(2.1) will have endothelial cell-selective polyethylene glycols bipolymer PEGMA-GMA and be dissolved in tetrahydrofuran solvent, and be configured to the solution that mass concentration is 0.05%, and by supersound process, obtain the solution C being uniformly dissolved;
(2.2) by PET base film successively with acetone, methanol, the ultrasonic pretreatment of tri-distilled water 30 minutes, tri-distilled water rinses;
(2.3) take appropriate 1,6-hexamethylene diamine solid and be dissolved in dehydrated alcohol, be configured to the solution of 10%, the PET base film anticipated be immersed in 1, the 6-hexamethylene diamine solution of 10%, and react 24h at normal temperatures, then rinse 3 times with tri-distilled water, N2Dry up, save backup.
(2.4) adopt dip coating that solution C is coated in the PET base film surface that step (2.3) processed;
(2.5) after the PET base film surface nitrogen having been coated with material being dried up, immersing mass concentration is the phosphate buffer of 0.02%REDV, 24h is reacted at 4 DEG C, terminate by the PBS solution of lysine, vacuum drying, obtain the endothelial cell-selective composite coating for angiocarpy bracket, as shown in Figure 2.
(3) cell selective evaluation
Polymer membrane circular stamping knife is prepared into the circular membrane (suitable in 96 porocyte culture plates) of 6mm, sample is moved into super-clean bench, ultra-vioket radiation sterilizing 2h, is then dipped in sterilizing 2h in 75% alcoholic solution by sample, finally move into the PBS solution of sterilizing is soaked standby.
The endotheliocyte digested and smooth muscle cell are planted on 96 orifice plate samples according to the density in 2000, every hole respectively, after cultivating 24h, is respectively adopted fluorescent staining method (FDA) the test cell rate of increase.
Endotheliocyte and smooth muscle cell test result indicate that, prepared polymer coating can promote the growth of endotheliocyte and suppress the growth of smooth muscle cell, and as shown in Figure 4, after 24h propagation, endothelial cell density is 140/mm to experimental result2, and the density of smooth muscle cell is 40/mm2。
Embodiment 2
(1) endothelial cell-selective polyethylene glycols bipolymer is prepared
(1.1) by biocompatibility monomer PEGMA (n=6) bionical for 0.8g cell saccharic clothing, 1.6g containing can reacdon-activity group polymerisable monomer GMA 50ml ethanol dissolve, obtain solution A;
(1.2) 0.048g initiator A IBN is joined in solution A, obtain solution B;
(1.3) with argon to solution B deoxygenation 20 minutes, it is heated to 70 DEG C, reacts 36 hours;
(1.4) decompression is except solvent, ice ether precipitating 2 times, obtains having endothelial cell-selective polyethylene glycols bipolymer PEGMA-GMA.Nuclear-magnetism result confirms that the product obtained has intended structure.As shown in Fig. 1 (b).
(2) endothelial cell-selective polyethylene glycols composite coating is prepared
(2.1) will have endothelial cell-selective polyethylene glycols bipolymer PEGMA-GMA and be dissolved in tetrahydrofuran solvent, and be configured to the solution that mass concentration is 0.1%, and by supersound process, obtain the solution C being uniformly dissolved;
(2.2) by the circular thin glass sheet H of diameter 8mm size2SO4/H2O2=7:3 (V/V) soaks, at 80 DEG C, and heat treated 30min;Fully rinse with tri-distilled water, dry, save backup;
(2.3) take appropriate 1,6-hexamethylene diamine solid and be dissolved in dehydrated alcohol, be configured to the solution of 10%, the glass substrate surface anticipated be immersed in 1, the 6-hexamethylene diamine solution of 10%, and react 24h at normal temperatures, then rinse 3 times with tri-distilled water, N2Dry up, save backup.
(2.4) adopt dip coating that solution C is coated in the glass substrate surface that step (2.3) processed;
(2.5) after the glass substrate surface nitrogen having been coated with material being dried up, immerse in the RGD-phosphate buffer that mass concentration is 0.05%, 24h is reacted at 25 DEG C, terminate by the PBS solution of lysine, vacuum drying, obtain the endothelial cell-selective composite coating for angiocarpy bracket, as shown in Figure 3.
(3) cell selective evaluation
Circular glass sheet is put in 48 orifice plates, ultra-vioket radiation sterilizing 2h, then sample is dipped in sterilizing 2h in 75% alcoholic solution, finally move into the PBS solution of sterilizing is soaked standby.
The endotheliocyte digested and smooth muscle cell are planted on 48 orifice plate samples according to the density in 8000, every hole respectively, after cultivating 24h, is respectively adopted fluorescent staining method (FDA) the test cell rate of increase.
Endotheliocyte and smooth muscle cell test result indicate that, prepared polymer coating can promote the growth of endotheliocyte and suppress the growth of smooth muscle cell, experimental result as it is shown in figure 5,24h propagation after endothelial cell density be 240/mm2, and the density of smooth muscle cell is 125/mm2。
Embodiment 3
(1) endothelial cell-selective polyethylene glycols bipolymer is prepared
(1.1) by biocompatibility monomer PEGMA (n=30) bionical for 2g cell saccharic clothing, 1g containing can reacdon-activity group polymerisable monomer GMA 70ml ethanol dissolve, obtain solution A;
(1.2) 0.01g initiator A IBN is joined in solution A, obtain solution B;
(1.3) with argon to solution B deoxygenation 20 minutes, it is heated to 80 DEG C, reacts 48 hours;
(1.4) decompression is except solvent, ice ether precipitating 2 times, obtains having endothelial cell-selective polyethylene glycols bipolymer PEGMA-GMA.Nuclear-magnetism result confirms that the product obtained has intended structure.
(2) endothelial cell-selective polyethylene glycols composite coating is prepared
(2.1) will have endothelial cell-selective polyethylene glycols bipolymer PEGMA-GMA and be dissolved in tetrahydrofuran solvent, and be configured to the solution that mass concentration is 2%, and by supersound process, obtain the solution C being uniformly dissolved;
(2.2) by the circular stainless steel substrates H of diameter 14mm size2SO4/H2O2=7:3 (V/V) soaks, at 80 DEG C, and heat treated 60min;Fully rinse with tri-distilled water, dry, save backup.
(2.3) take appropriate 1,6-hexamethylene diamine solid and be dissolved in dehydrated alcohol, be configured to the solution of 10%, the stainless steel-based counterdie anticipated be immersed in 1, the 6-hexamethylene diamine solution of 10%, and react 24h at normal temperatures, then rinse 3 times with tri-distilled water, N2Dry up, save backup.
(2.4) adopt dip coating that solution C is coated in the rustless steel basement membrane surface that step (2.3) processed;
(2.5) after the stainless steel substrate surface nitrogen having been coated with material being dried up, immerse in the YIGSR-phosphate buffer that mass concentration is 0.05%, 24h is reacted at 25 DEG C, terminate by the PBS solution of lysine, vacuum drying, obtains the endothelial cell-selective composite coating for angiocarpy bracket.
(3) cell selective evaluation
Stainless steel substrates is put in 24 orifice plates, ultra-vioket radiation sterilizing 2h, then sample is dipped in sterilizing 2h in 75% alcoholic solution, finally move into the PBS solution of sterilizing is soaked standby.
The endotheliocyte digested and smooth muscle cell are planted on 24 orifice plate samples according to the density in 15000, every hole respectively, after cultivating 24h, is respectively adopted fluorescent staining method (FDA) the test cell rate of increase.
Endotheliocyte and smooth muscle cell test result indicate that, prepared polymer coating can promote the growth of endotheliocyte and suppress the growth of smooth muscle cell, and after 24h propagation, endothelial cell density is 135/mm2, and the density of smooth muscle cell is 55/mm2。
Embodiment 4
(1) endothelial cell-selective polyethylene glycols bipolymer is prepared
(1.1) by biocompatibility monomer PEGMA (n=15) bionical for 2.5g cell saccharic clothing, 1g containing can reacdon-activity group polymerisable monomer GMA 100ml ethanol dissolve, obtain solution A;
(1.2) 0.07g initiator A IBN is joined in solution A, obtain solution B;
(1.3) with argon to solution B deoxygenation 30 minutes, it is heated to 70 DEG C, reacts 24 hours;
(1.4) decompression is except solvent, ice ether precipitating 3 times, obtains having endothelial cell-selective polyethylene glycols bipolymer PEGMA-GMA.Nuclear-magnetism result confirms that the product obtained has intended structure.
(2) endothelial cell-selective polyethylene glycols composite coating is prepared
(2.1) will have endothelial cell-selective polyethylene glycols bipolymer PEGMA-GMA and be dissolved in tetrahydrofuran solvent, and be configured to the solution that mass concentration is 1%, and by supersound process, obtain the solution C being uniformly dissolved;
(2.2) by stainless steel stent H2SO4/H2O2=7:3 (V/V) soaks, at 80 DEG C, and heat treated 40min;Fully rinse with tri-distilled water, dry, save backup;
(2.3) take appropriate 1,6-hexamethylene diamine solid and be dissolved in dehydrated alcohol, be configured to the solution of 10%, the stainless steel stent anticipated be immersed in 1, the 6-hexamethylene diamine solution of 10%, and react 24h at normal temperatures, then rinse 3 times with tri-distilled water, N2Dry up, save backup.
(2.4) adopt dip coating that solution C is coated in the stainless steel stent surface that step (2.3) processed;
(2.5) after the stainless steel stent surface nitrogen having been coated with material being dried up, immerse in the CD34-phosphate buffer that mass concentration is 0.05%, 24h is reacted at 25 DEG C, terminate by the PBS solution of lysine, vacuum drying, obtains the endothelial cell-selective composite coating for angiocarpy bracket.
(3) cell selective evaluation
Stainless steel stent is put in 24 orifice plates, ultra-vioket radiation sterilizing 2h, then sample is dipped in sterilizing 2h in 75% alcoholic solution, finally move into the PBS solution of sterilizing is soaked standby.
The endotheliocyte digested and smooth muscle cell are planted on 24 orifice plate samples according to the density in 15000, every hole respectively, after cultivating 24h, is respectively adopted fluorescent staining method (FDA) the test cell rate of increase.
Endotheliocyte and smooth muscle cell test result indicate that, prepared polymer coating can promote the growth of endotheliocyte and suppress the growth of smooth muscle cell, and after 24h propagation, endothelial cell density is 120/mm2, and the density of smooth muscle cell is 50/mm2。
Embodiment 5
(1) endothelial cell-selective polyethylene glycols bipolymer is prepared
(1.1) by biocompatibility monomer PEGMA (n=15) bionical for 1.8g cell saccharic clothing, 1.8g containing can reacdon-activity group polymerisable monomer GMA 80ml ethanol dissolve, obtain solution A;
(1.2) 0.02g initiator A IBN is joined in solution A, obtain solution B;
(1.3) with argon to solution B deoxygenation 30 minutes, it is heated to 65 DEG C, reacts 24 hours;
(1.4) decompression is except solvent, ice ether precipitating 2 times, obtains having endothelial cell-selective polyethylene glycols bipolymer PEGMA-GMA.Nuclear-magnetism result confirms that the product obtained has intended structure.
(2) endothelial cell-selective polyethylene glycols composite coating is prepared
(2.1) will have endothelial cell-selective polyethylene glycols bipolymer PEGMA-GMA and be dissolved in tetrahydrofuran solvent, and be configured to the solution that mass concentration is 0.4%, and by supersound process, obtain the solution C being uniformly dissolved;
(2.2) by PET base film successively with acetone, methanol, the ultrasonic pretreatment of tri-distilled water 30 minutes, tri-distilled water rinses;
(2.3) take appropriate 1,6-hexamethylene diamine solid and be dissolved in dehydrated alcohol, be configured to the solution of 10%, the PET base film anticipated be immersed in 1, the 6-hexamethylene diamine solution of 10%, and react 24h at normal temperatures, then rinse 3 times with tri-distilled water, N2Dry up, save backup.
(2.4) adopt dip coating that solution C is coated in the PET base film surface that step (2.3) processed;
(2.5) after the PET base film surface nitrogen having been coated with material being dried up, immerse in the cRGD-phosphate buffer that mass concentration is 0.05%, 48h is reacted at 4 DEG C, terminate by the PBS solution of lysine, vacuum drying, obtains the endothelial cell-selective composite coating for angiocarpy bracket.
(3) cell selective evaluation
Polymer membrane circular stamping knife is prepared into the circular membrane (suitable in 96 porocyte culture plates) of 6mm, sample is moved into super-clean bench, ultra-vioket radiation sterilizing 2h, is then dipped in sterilizing 2h in 75% alcoholic solution by sample, finally move into the PBS solution of sterilizing is soaked standby.
The endotheliocyte digested and smooth muscle cell are planted on 96 orifice plate samples according to the density in 2000, every hole respectively, after cultivating 24h, is respectively adopted fluorescent staining method (FDA) the test cell rate of increase.
Endotheliocyte and smooth muscle cell test result indicate that, prepared polymer coating can promote the growth of endotheliocyte and suppress the growth of smooth muscle cell, and after 24h propagation, endothelial cell density is 220/mm2, and the density of smooth muscle cell is 95/mm2。
Embodiment 6
(1) endothelial cell-selective polyethylene glycols bipolymer is prepared
(1.1) by biocompatibility monomer PEGMA (n=10) bionical for 0.8g cell saccharic clothing, 2.0g containing can reacdon-activity group polymerisable monomer GMA 60ml ethanol dissolve, obtain solution A;
(1.2) 0.015g initiator A IBN is joined in solution A, obtain solution B;
(1.3) with argon to solution B deoxygenation 30 minutes, it is heated to 80 DEG C, reacts 18 hours;
(1.4) decompression is except solvent, ice ether precipitating 2 times, obtains having endothelial cell-selective polyethylene glycols bipolymer PEGMA-GMA.Nuclear-magnetism result confirms that the product obtained has intended structure.
(2) endothelial cell-selective polyethylene glycols composite coating is prepared
(2.1) will have endothelial cell-selective polyethylene glycols bipolymer PEGMA-GMA and be dissolved in tetrahydrofuran solvent, and be configured to the solution that mass concentration is 1.5%, and by supersound process, obtain the solution C being uniformly dissolved;
(2.2) by the circular thin glass sheet H of diameter 8mm size2SO4/H2O2=7:3 (V/V) soaks, at 80 DEG C, and heat treated 30min;Fully rinse with tri-distilled water, dry, save backup;
(2.3) take appropriate 1,6-hexamethylene diamine solid and be dissolved in dehydrated alcohol, be configured to the solution of 10%, the glass baseplate anticipated be immersed in 1, the 6-hexamethylene diamine solution of 10%, and react 24h at normal temperatures, then rinse 3 times with tri-distilled water, N2Dry up, save backup.
(2.4) adopt dip coating that solution C is coated in the glass substrate surface that step (2.3) processed;
(2.5) after the glass substrate surface nitrogen having been coated with material being dried up, immerse in the CD133-phosphate buffer that mass concentration is 0.05%, 24h is reacted at 25 DEG C, terminate by the PBS solution of lysine, vacuum drying, obtains the endothelial cell-selective composite coating for angiocarpy bracket.
(3) cell selective evaluation
Circular glass sheet is put in 48 orifice plates, ultra-vioket radiation sterilizing 2h, then sample is dipped in sterilizing 2h in 75% alcoholic solution, finally move into the PBS solution of sterilizing is soaked standby.
The endotheliocyte digested and smooth muscle cell are planted on 48 orifice plate samples according to the density in 8000, every hole respectively, after cultivating 24h, is respectively adopted fluorescent staining method (FDA) the test cell rate of increase.
Endotheliocyte and smooth muscle cell test result indicate that, prepared polymer coating can promote the growth of endotheliocyte and suppress the growth of smooth muscle cell, and after 24h propagation, endothelial cell density is 215/mm2, and the density of smooth muscle cell is 105/mm2。
Claims (10)
1. an endothelial cell-selective polyethylene glycols bipolymer, it is characterised in that the biocompatibility monomer bionical by cell saccharic clothing and containing the polymerisable monomer of reacdon-activity group being synthesized by the method for radical polymerization;
The biocompatibility monomer that described cell saccharic clothing is bionical is selected from monomer:
Wherein R=H or CH3, n=1-50;
Described containing can the polymerisable monomer of reacdon-activity group as follows:
2. a kind of endothelial cell-selective polyethylene glycols bipolymer according to claim 1, it is characterised in that the biocompatibility monomer that described cell saccharic clothing is bionical, R=H or CH3, n=5-30.
3. the preparation method of an endothelial cell-selective polyethylene glycols bipolymer as claimed in claim 1, it is characterised in that comprise the following steps:
(1) by biocompatibility monomer bionical for cell saccharic clothing with containing can the polymerisable monomer of reacdon-activity group dissolve in a solvent according to the ratio of weight ratio 10:1~1:10, solute and solvent quality ratio is for 1:5~1:50, it is thus achieved that solution A;
(2) weigh radical initiator azodiisobutyronitrile according in step (1) the 0.2%~2% of two kinds of monomer gross masses, join in solution A, obtain solution B;
(3) with nitrogen or argon to solution B deoxygenation 5~30min;
(4) solution after deoxygenation is heated to 50~90 DEG C, reacts 6~48h;
(5) removal of solvent under reduced pressure, by ice ether precipitating 2~3 times, obtains having endothelial cell-selective polyethylene glycols bipolymer.
4. the preparation method of endothelial cell-selective polyethylene glycols bipolymer according to claim 3, it is characterised in that step (1) described solvent is the one in oxolane, chloroform, dichloromethane, isopropanol, ethanol or methanol.
5. an endothelial cell-selective polyethylene glycols composite coating, it is characterised in that there is endothelial cell-selective polyethylene glycols bipolymer as claimed in claim 1 and be fixed on the bioactive molecule of substrate surface.
6. endothelial cell-selective polyethylene glycols composite coating according to claim 5, it is characterised in that bioactive molecule is rgd peptide, REDV polypeptide, cRGD cyclic peptide, YIGSR polypeptide, CD34 antibody or the one in CD133 antibody.
7. endothelial cell-selective polyethylene glycols composite coating according to claim 5, it is characterised in that base material is PET substrate's film, substrate of glass film, stainless steel-based counterdie or the one in rustless steel angiocarpy bracket.
8. the endothelial cell-selective polyethylene glycols composite coating application on angiocarpy bracket.
9. the application process of an endothelial cell-selective polyethylene glycols composite coating as claimed in claim 8, it is characterised in that comprise the steps:
A () will have endothelial cell-selective polyethylene glycols bipolymer and be dissolved in tetrahydrofuran solvent, be configured to the solution that mass concentration is 0.01~5%, and by supersound process, obtain the solution C being uniformly dissolved;
B base material is used acetone, methanol, the ultrasonic pretreatment 10~30min of tri-distilled water by () successively;
C () takes 1,6-hexamethylene diamine solid and is dissolved in dehydrated alcohol, be configured to the solution of 10wt%, is immersed in 1,6-hexamethylene diamine solution by the base material anticipated, and reacts 24h at normal temperatures, then rinses 3 times with tri-distilled water, N2Dry up, save backup;
D () adopts dip coating that solution C is coated in the substrate surface that step (c) processed;
E the substrate surface nitrogen having been coated with material is dried up by () after, immerse the phosphate buffer that mass concentration is 0.01~0.5% bioactive molecule, 24~72h is reacted at 4~25 DEG C, terminate by the PBS solution of lysine, vacuum drying, obtains endothelial cell-selective polyethylene glycols composite coating.
10. application process according to claim 9, it is characterised in that the processing method of step (b) is: with the concentrated sulphuric acid that volume ratio is 7:3 and hydrogen peroxide Grape berry 30~60min, then rinse with tri-distilled water.
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