CN105536057A - Preparation method and application of polylactic acid-glycolic acid grafted RGD peptide - Google Patents
Preparation method and application of polylactic acid-glycolic acid grafted RGD peptide Download PDFInfo
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- CN105536057A CN105536057A CN201610016991.1A CN201610016991A CN105536057A CN 105536057 A CN105536057 A CN 105536057A CN 201610016991 A CN201610016991 A CN 201610016991A CN 105536057 A CN105536057 A CN 105536057A
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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
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/14—Macromolecular materials
- A61L27/18—Macromolecular materials obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
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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
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
- C08G63/00—Macromolecular compounds obtained by reactions forming a carboxylic ester link in the main chain of the macromolecule
- C08G63/91—Polymers modified by chemical after-treatment
- C08G63/912—Polymers modified by chemical after-treatment derived from hydroxycarboxylic acids
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Abstract
The invention discloses a preparation method and application of polylactic acid-glycolic acid grafted RGD peptide. The preparation method mainly includes the following steps that firstly, -COOH on PLGA molecules is activated, and hexamethylenediamine is grafted to the surface of PLGA to obtain aminated PLGA; secondly, aminated PLGA is treated with glutaraldehyde for aldehyde treatment; thirdly, aldehyde-treated PLGA is reacted with -NH2 on RGD peptide molecules, and RGD is grafted to the surface of PLGA. Conditions in the preparation process are mild, the preparation process is simple and controllable, the bioactivity and osteoinductivity of PLGA can be promoted, and the preparation method has broad application prospects.
Description
Technical field
The present invention relates to a kind of preparation method and application of polylactic-co-glycolic acid grafting RGD peptide, belong to biological medical polymer material technical field.
Background technology
In recent years, biological medical polymer material is with its good mechanical performance, degradation cycle can control, and the small-molecule substance that in degradation process, hydrolysis or enzymolysis produce can be absorbed by the body or excrete, and can not damage the extensive concern receiving research worker to human body.Polylactic-co-glycolic acid (PLGA) material is a kind of synthesising biological medical macromolecular materials that present range of application is wider, its advantage is to have good biocompatibility, degradability and more moderate mechanical performance, is widely used in organizational project.But because the chemical constitution of polylactic-co-glycolic acid itself is simple, the hydrophobicity on surface is strong, but shortage cell recognition signal, cellular affinity are poor, and degraded is in acid, easily causes and organizes bland necrosis.Thus it can only be applied in every field as a kind of inert material of universality.But, in many field of tissue engineering technology, wish that timbering material can identify different cells, adsorb some seed cell selectively and can promote that the growth of cell, propagation and the direction to hope break up.For overcoming the above-mentioned shortcoming of polylactic acid, then need to carry out modification to polylactic-co-glycolic acid.
In order to improve the biological activity of PLGA, just needing to change its surface characteristic, introducing bioactive molecule, thus reaching promotion cell adhesion, breed and increase the object of its anti-nonspecific proteins absorbability.RGD is as a kind of important cell recognition site and signal enabling molecule, important regulatory function is played in many vital movements, apply comparatively extensive in the treatment of tumor, there is the effect of direct killing tumor cell and cell death inducing, and be the minimum identification short peptide sequence of many extracellular matrix proteins, be also attachment proteins and the cell surface specially interactional recognition site of receptor protein.Therefore surface RGD being grafted to polylactic-co-glycolic acid is that one comparatively simply improves polylactic-co-glycolic acid surface bioactive and hydrophilic method, is also simultaneously in polylactic-co-glycolic acid class material will be for one of human tissue engineering direction having development prospect.But existing method preparation technology is comparatively complicated, need to carry out under high temperature or catalytic condition, thus need to improve existing preparation method, to improve the range of application of polylactic-co-glycolic acid grafting RGD peptide.
Summary of the invention
The object of the present invention is to provide a kind of preparation method of polylactic-co-glycolic acid grafting RGD peptide.The polylactic-co-glycolic acid grafting RGD peptide that this preparation method obtains solves the poor problem of polylactic-co-glycolic acid biological activity, improve the problem that polylactic-co-glycolic acid hydrophilic is poor simultaneously, prepared material shows good biological activity, is specially adapted to the application in organizational project.
For achieving the above object, the present invention is by the following technical solutions:
A preparation method for polylactic-co-glycolic acid grafting RGD peptide, step is as follows:
(1) polylactic-co-glycolic acid containing end carboxyl is dissolved in anhydrous methylene chloride obtains PLA solution, then by N, N'-carbonyl dimidazoles (CDI) joins in PLA solution, after activation 0.5-1h is carried out to carboxyl, hexamethylene diamine is joined in PLA solution, reaction 8-12h, by reacted product alcohol settling out, washing, dry, obtain the polylactic-co-glycolic acid (PLGA-NH2) of surface amination;
(2) compound concentration is the glutaraldehyde water solution of 5mol/L, then joins in glutaraldehyde water solution by PLGA-NH2, reaction 6-8h, is carried out by reacted product washing, dry, obtains the polylactic-co-glycolic acid (PLGA-CHO) of surface aldehydes;
(3) compound concentration is the RGD peptide aqueous solution of 5mol/L, is joined by PLGA-CHO in RGD peptide aqueous solution, and reaction 4-6h, washs reacted product, lyophilization, namely obtain the PLGA(PLGA-RGD of surface grafting RGD peptide).
For the N of activated carboxyl in described step (1), N'-carbonyl dimidazoles is 2-3:1. with the mol ratio of the polylactic-co-glycolic acid containing end carboxyl
The moisture content <20ppm of anhydrous methylene chloride in described step (1).
Be 1:1-1.5 containing the polylactic-co-glycolic acid of end carboxyl and the mol ratio of hexamethylene diamine in described step (1).
In described step (2), the mol ratio of PLGA-NH2 and glutaraldehyde is 1:1-2.
The mol ratio of PLGA-CHO and the RGD peptide in described step (3) is 1:1-1.5.
In described step (3), lyophilization is dry 24 ~ 48h under the condition of-50 ~-40 DEG C.
The application of polylactic-co-glycolic acid in organizational project of the surface grafting RGD peptide that the preparation method of described polylactic-co-glycolic acid grafting RGD peptide prepares.
Compared with prior art, the advantage of novel polylactic-co-glycolic acid grafting RGD peptide that prepared by the present invention is: 1. the polylactic-co-glycolic acid grafting RGD peptide that prepared by the present invention improves biological activity and the hydrophilic of polylactic-co-glycolic acid; 2. the material aftertreatment technology prepared of the present invention is simple, and can not residual organic solvent, and the biocompatibility of material is good; 3. the present invention compared with the prior art: institute responds and all can carry out at normal temperatures, and mild condition is simple to operate, substantially reduces the response time, improves preparation efficiency, be conducive to industrialized production; 4. present invention improves biological activity and the hydrophilic of polylactic-co-glycolic acid, add the osteoinductive of polylactic-co-glycolic acid simultaneously, make the application of PLGA in organizational project more extensive.
Detailed description of the invention
Embodiment 1
The preparation method of the polylactic-co-glycolic acid grafting RGD peptide of the present embodiment, step is as follows:
(1) the polylactic-co-glycolic acid 0.5mol containing end carboxyl is dissolved in anhydrous methylene chloride (moisture content < 20ppm) and obtains PLA solution, then by N, N'-carbonyl dimidazoles 1mol joins in PLA solution, after activation 0.5h is carried out to carboxyl, then hexamethylene diamine 0.5mol is joined in PLA solution, reaction 8h, by product with alcohol settling out, washing, dry, obtain the polylactic-co-glycolic acid (PLGA-NH2) of surface amination;
(2) by PLGA-NH
20.5mol joins in 100mL glutaraldehyde water solution (5mol/L), reaction 6h, is carried out by reacted product washing, dry, namely obtains the polylactic-co-glycolic acid (PLGA-CHO) of surface aldehydes;
(3) joined by PLGA-CHO0.5mol in the aqueous solution (5mol/L) of 100mLRGD peptide, reaction 4h, washs reacted product, lyophilization 24h under the condition of-50 DEG C, namely obtains the PLGA(PLGA-RGD of surface grafting RGD peptide).
Embodiment 2
The preparation method of the polylactic-co-glycolic acid grafting RGD peptide of the present embodiment, step is as follows:
(1) the polylactic-co-glycolic acid 0.5mol containing end carboxyl is dissolved in anhydrous methylene chloride (moisture content < 20ppm) and obtains PLA solution, then by N, N'-carbonyl dimidazoles 1.5mol joins in PLA solution, after activation 1h is carried out to carboxyl, then hexamethylene diamine 0.5mol is joined in PLA solution, reaction 12h, by product with alcohol settling out, washing, dry, obtain the polylactic-co-glycolic acid (PLGA-NH2) of surface amination;
(2) by PLGA-NH
20.5mol joins in 125mL glutaraldehyde water solution (5mol/L), reaction 6h, is carried out by reacted product washing, dry, namely obtains the polylactic-co-glycolic acid (PLGA-CHO) of surface aldehydes;
(3) joined by PLGA-CHO0.5mol in the aqueous solution (5mol/L) of 150mLRGD peptide, reaction 4h, washs reacted product, lyophilization 36h under the condition of-45 DEG C, namely obtains the PLGA(PLGA-RGD of surface grafting RGD peptide).
Embodiment 3
The preparation method of the polylactic-co-glycolic acid grafting RGD peptide of the present embodiment, step is as follows:
(1) the polylactic-co-glycolic acid 0.5mol containing end carboxyl is dissolved in anhydrous methylene chloride (moisture content < 20ppm) and obtains PLA solution, then by N, N'-carbonyl dimidazoles 1.25mol joins in PLA solution, after activation 1h is carried out to carboxyl, then hexamethylene diamine 0.6mol is joined in PLA solution, reaction 9h, by product with alcohol settling out, washing, dry, obtain the polylactic-co-glycolic acid (PLGA-NH2) of surface amination;
(2) by PLGA-NH
20.5mol joins (5mol/L) in 250mL glutaraldehyde water solution, reaction 7h, is carried out by reacted product washing, dry, namely obtains the polylactic-co-glycolic acid (PLGA-CHO) of surface aldehydes;
(3) joined by PLGA-CHO0.5mol in the aqueous solution (5mol/L) of 150mLRGD peptide, reaction 5h, washs reacted product, lyophilization 24h under the condition of-50 DEG C, namely obtains the PLGA(PLGA-RGD of surface grafting RGD peptide).
Embodiment 4
The preparation method of the polylactic-co-glycolic acid grafting RGD peptide of the present embodiment, step is as follows:
(1) the polylactic-co-glycolic acid 0.5mol containing end carboxyl is dissolved in anhydrous methylene chloride (moisture content < 20ppm) and obtains PLA solution, then by N, N'-carbonyl dimidazoles 1.5mol joins in PLA solution, after activation 0.5h is carried out to carboxyl, then hexamethylene diamine 0.75mol is joined in PLA solution, reaction 10h, by product with alcohol settling out, washing, dry, obtain the polylactic-co-glycolic acid (PLGA-NH2) of surface amination;
(2) by PLGA-NH
20.5mol joins containing in 100mL glutaraldehyde water solution (5mol/L), reaction 8h, is carried out by reacted product washing, dry, namely obtains the polylactic-co-glycolic acid (PLGA-CHO) of surface aldehydes;
(3) joined by PLGA-CHO0.5mol in the aqueous solution (5mol/L) of 150mLRGD peptide, reaction 6h, washs reacted product, lyophilization 48h under the condition of-40 DEG C, namely obtains the PLGA(PLGA-RGD of surface grafting RGD peptide).
More than show and describe ultimate principle of the present invention and principal character and advantage of the present invention.The technical staff of the industry should understand; the present invention is not restricted to the described embodiments; what describe in above-described embodiment and description just illustrates principle of the present invention; without departing from the spirit and scope of the present invention; the present invention also has various changes and modifications, and these changes and improvements all fall in the claimed scope of the invention.Application claims protection domain is defined by appending claims and equivalent thereof.
Claims (8)
1. a preparation method for polylactic-co-glycolic acid grafting RGD peptide, is characterized in that step is as follows:
(1) polylactic-co-glycolic acid containing end carboxyl is dissolved in anhydrous methylene chloride obtains PLA solution, then by N, N'-carbonyl dimidazoles joins in PLA solution, after activation 0.5-1h is carried out to carboxyl, hexamethylene diamine is joined in PLA solution, reaction 8-12h, by reacted product alcohol settling out, washing, dry, obtain the polylactic-co-glycolic acid of surface amination;
(2) compound concentration is the glutaraldehyde water solution of 5mol/L, the polylactic-co-glycolic acid of the surface amination then step (1) obtained joins in glutaraldehyde water solution, reaction 6-8h, is undertaken reacted product washing, dry, obtains the polylactic-co-glycolic acid of surface aldehydes;
(3) compound concentration is the RGD peptide aqueous solution of 5mol/L, the polylactic-co-glycolic acid of surface aldehydes step (2) obtained joins in RGD peptide aqueous solution, and reaction 4-6h, washs reacted product, lyophilization, namely obtains the polylactic-co-glycolic acid of surface grafting RGD peptide.
2. the preparation method of polylactic-co-glycolic acid grafting RGD peptide according to claim 1, is characterized in that: for the N of activated carboxyl in described step (1), and N'-carbonyl dimidazoles is 2-3:1 with the mol ratio of the polylactic-co-glycolic acid containing end carboxyl.
3. the preparation method of polylactic-co-glycolic acid grafting RGD peptide according to claim 1, is characterized in that: the moisture content <20ppm of anhydrous methylene chloride in described step (1).
4. the preparation method of polylactic-co-glycolic acid grafting RGD peptide according to claim 1, is characterized in that: be 1:1-1.5 containing the polylactic-co-glycolic acid of end carboxyl and the mol ratio of hexamethylene diamine in described step (1).
5. the preparation method of polylactic-co-glycolic acid grafting RGD peptide according to claim 1, is characterized in that: in described step (2), the polylactic-co-glycolic acid of surface amination and the mol ratio of glutaraldehyde are 1:1-2.
6. the preparation method of polylactic-co-glycolic acid grafting RGD peptide according to claim 1, is characterized in that: the polylactic-co-glycolic acid of the surface aldehydes in described step (3) and the mol ratio of RGD peptide are 1:1-1.5.
7. the preparation method of polylactic-co-glycolic acid grafting RGD peptide according to claim 1, is characterized in that: in described step (3), lyophilization is dry 24 ~ 48h under the condition of-50 ~-40 DEG C.
8. the application of polylactic-co-glycolic acid in organizational project of surface grafting RGD peptide that prepare of the preparation method of the arbitrary described novel polylactic-co-glycolic acid grafting RGD peptide of claim 1 ~ 7.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106432623A (en) * | 2016-10-09 | 2017-02-22 | 湖南科技大学 | Method for preparing ethidene diamine and RGD polypeptide modified high molecular weight polylactide-glycolide |
| CN106480537A (en) * | 2016-12-21 | 2017-03-08 | 哈尔滨理工大学 | A kind of method that gelatin is grafted PLGA electrospinning fibre |
| CN106913904A (en) * | 2017-03-06 | 2017-07-04 | 苏州大学附属第医院 | A kind of micrometer-nanometer tissue engineering support with immunization therapy function and preparation method thereof |
| CN106963984A (en) * | 2017-03-02 | 2017-07-21 | 复旦大学 | A kind of preparation method of gelatin/carboxy apatite composite coating |
| CN114634634A (en) * | 2022-03-22 | 2022-06-17 | 陈凌卉 | Biological function composite porous polyester microsphere and preparation method thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1919360A (en) * | 2006-09-14 | 2007-02-28 | 同济大学 | Polylactic acid base/nano hydroxyapatite biological material and preparation method thereof |
| CN104927319A (en) * | 2015-06-29 | 2015-09-23 | 河南工程学院 | Preparation method of novel hydroxyapatite graft polylactic acid |
-
2016
- 2016-01-12 CN CN201610016991.1A patent/CN105536057A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1919360A (en) * | 2006-09-14 | 2007-02-28 | 同济大学 | Polylactic acid base/nano hydroxyapatite biological material and preparation method thereof |
| CN104927319A (en) * | 2015-06-29 | 2015-09-23 | 河南工程学院 | Preparation method of novel hydroxyapatite graft polylactic acid |
Non-Patent Citations (2)
| Title |
|---|
| GU SHUYING等: "Synthesis and evaluation of a biodegradable material with cell recognition motives", 《CARBOHYDRATE POLYMERS》 * |
| 陶春等: "ＲＧＤ修饰的聚乳酸－羟基乙酸骨组织工程材料的研究进展", 《第二军医大学学报》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106432623A (en) * | 2016-10-09 | 2017-02-22 | 湖南科技大学 | Method for preparing ethidene diamine and RGD polypeptide modified high molecular weight polylactide-glycolide |
| CN106480537A (en) * | 2016-12-21 | 2017-03-08 | 哈尔滨理工大学 | A kind of method that gelatin is grafted PLGA electrospinning fibre |
| CN106963984A (en) * | 2017-03-02 | 2017-07-21 | 复旦大学 | A kind of preparation method of gelatin/carboxy apatite composite coating |
| CN106913904A (en) * | 2017-03-06 | 2017-07-04 | 苏州大学附属第医院 | A kind of micrometer-nanometer tissue engineering support with immunization therapy function and preparation method thereof |
| CN114634634A (en) * | 2022-03-22 | 2022-06-17 | 陈凌卉 | Biological function composite porous polyester microsphere and preparation method thereof |
| CN114634634B (en) * | 2022-03-22 | 2024-08-09 | 陈凌卉 | Biological function composite porous polyester microsphere and preparation method thereof |
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Application publication date: 20160504 |