CN103656757A - Double-layer intravascular stent carrying heparin and preparation method thereof - Google Patents
Double-layer intravascular stent carrying heparin and preparation method thereof Download PDFInfo
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Abstract
The invention provides a double-layer intravascular stent carrying heparin and a preparation method thereof. The stent is divided into an internal structure and an external structure which are independent. The external structure is in a porous polymer nanofiber structure. The internal structure is a compact stuffed polymer layer. The internal structure is prepared from a lactic acid-caprolactone copolymer. The external structure of the stent is prepared from a lactic acid-glycolic acid copolymer. The stent can bear large expansionary force and can recover to the original form after being bended and squeezed. The stent is divided into the two independent structures, wherein the external structure is in the porous polymer nanofiber structure and the internal structure is the compact stuffed polymer layer. Smooth muscle cell can adhere to the surface of the external structure, and the cell relative growth rate (RGR) is at 1 degrade. Platelet adhesion experiments show that the stent is free of obvious platelet adhesion.
Description
Technical field
The present invention relates to a kind of Biodegradable high-molecular intravascular stent with double-deck load heparin and preparation method thereof, belong to intravascular tissue engineering field.
Background technology
The peripheral blood vessels such as coronary atherosclerosis are one of important diseases of harm humans.Effective therapy of a lot of these class severe diseases is to use Replacement pathological tissues, but the clinical manifestation of the transplant operation of traditional autologous quiet, tremulous pulse is always undesirable.Along with the development of macromolecular material, artificial blood vessel's exploitation has obtained very much progress.Use has the material of biocompatibility and makes vessel-like support, more than wall is implanted Smooth Muscle Cell surrounding within it, make it produce substrate to replace the support of degraded or to fill up the space between support, shaping is endotheliocyte on vascular inner surface is planted afterwards, has so just formed the double-layer structure artificial blood vessel of similar natural blood vessel.
Because artificial blood vessel lacks the active substance that natural blood vessel surface exists, often cause a large amount of hemocytees to show enrichment at it, finally form thrombosis.For addressing the above problem, the surface-functionalized artificial blood vessel bracket of a large class occurs.Large quantity research finds, the mixture of the polysaccharide chain that the repetition disaccharide unit that heparin couples together with 1-4 key as a class alduronic acid and glucamine forms, and the activation that can lower complement coacervated system and hemocyte, finally prevents hemocyte and platelet adhesion reaction.
Therefore, many degradation materials are for the preparation of the intravascular stent that becomes tubulose, and by its surface carry out heparin physical modification.As polylactic-co-glycolic acid mix with heparin after the multilamellar of preparation mix blood vessel (quiet etc., 2009); After the heparinization of polycaprolactone and polylactic-co-glycolic acid for myocardial blood transport reconstruction (Zhao Jian etc., 2008).Also occur simultaneously some by heparin altogether block or conjugation at degradable macromolecule surface or inner report (CN200910228706.2; 201110453941.7 etc.).But the degradable macromolecule intravascular stent of current heparinization adopts electrostatic spinning technique preparation more, bring under the prerequisite of many electrostatic spinning advantages and also have the defects such as mechanical strength deficiency, the thrombosed problem (stone Yun etc., 2005) of can not avoiding subsiding.Therefore need a kind of preparation method that can greatly promote heparinization intravascular stent mechanical strength, retain the feature of electrostatic spinning simultaneously.
Summary of the invention
The object of the invention is to provide a kind of Biodegradable high-molecular intravascular stent with double-deck load heparin, applies to intravascular tissue engineering research.
A double-deck intravascular stent for load heparin, is characterized in that, support is divided into inside and outside two structures independently, and its external structure has the high polymer nanometer fiber structure of porous, and inner structure is fine and close solid macromolecule layer; Wherein, inner structure is prepared by lactic acid-caprolactone copolymer, and the external structure of support is prepared by poly lactic coglycolic acid.
Described lactic acid-caprolactone copolymer weight average molecular weight is 40 ~ 60kDa, and the mol ratio of lactic acid and caprolactone is 8:2; Poly lactic coglycolic acid weight average molecular weight is 50 ~ 100kDa, and the mol ratio of lactic acid and hydroxyacetic acid is 5:5.
Described nanofiber average diameter is 0.8～1.2 micron.
A preparation method for the double-deck intravascular stent of load heparin, is characterized in that, comprises the steps:
(1) lactic acid-caprolactone copolymer is dissolved in dichloromethane, and heating condensing reflux, to dissolving, insert in above-mentioned lactic acid-caprolactone copolymer organic solution with bar-shaped mould, after appropriateness stirs, vertically takes out at once, at room temperature, volatilizees;
(2) poly lactic coglycolic acid is dissolved in dichloromethane and N-dimethylformamide mixed solvent, heating condensing reflux, to dissolving completely, pack in glass syringe;
(3) build electrostatic spinning apparatus, glass syringe is fixed on to jet deck, bar-shaped mould is vertically fixed on metal rotation device, under the state of average rate rotation, receive Electrospun;
(4) by the support of preparation after lyophilization is processed, be immersed in heparin solution, after taking out, continue lyophilization and process 6 hours;
(5) support is separated with bar-shaped mould, can obtain the Biodegradable high-molecular intravascular stent of double-deck load heparin.
The concentration of described lactic acid-caprolactone copolymer organic solution is 10～20g/100mL; The concentration of poly lactic coglycolic acid organic solution is 2～4g/100mL; Described dichloromethane and N-dimethylformamide mixed solvent, its volume ratio is 8:2～7:3.
Described bar-shaped mould is smooth stainless steel bar, and length is 10～20 centimetres, and diameter is 2～4 millimeters.
Described syringe needle is 5-6 syringe needle.
In described electrostatic spinning and receiving system, injection rate is 0.4～0.7mL/h, injection volume 0.5～~ 1 milliliter, and the distance of bar-shaped mould and syringe needle is 5～20 centimetres, its rotary speed is 50～200rpm.
Described heparin solution concentration is 2～6g/100mL.
Before support soaks in heparin solution, need lyophilization to process 6 hours; Support soaks in heparin solution, and its time is 1～2 hour; After immersion, take out and continue lyophilization processing 6 hours.
The prepared intravascular stent of the present invention has completely by biodegradable Polymer materialspreparation, has good biocompatibility and absorbability; Intravascular stent has independently inside and outside double-layer structure, and external structure has the 3 D stereo supporting structure of porous, can be for cell attachment and growth; Endothecium structure is solid membrane structure, for whole support provides necessary mechanical property; By solution soaking, process, heparin compositions in support load, stops the adhesion of hemocyte.Intravascular stent preparation technology provided by the invention is simple, and equipment requirements is low, preparation with low cost, and preparation time is short.
Intravascular stent can bear powerful expansionary force, after bending and extruding, can recover original form, smooth muscle cell can be attached on external structure surface, the relative rate of increase of its cell (RGR) is classified as 1 grade, platelet adhesion experiment shows that support does not have obvious platelet adhesion reaction, the hot strength of intravascular stent is 7～12MPa, and stretch-proof shape becomes 5～8%.
accompanying drawing explanation:
Fig. 1 is the form schematic diagram of the double-deck intravascular stent of the load heparin prepared of the present invention.1 is the inner structure of intravascular stent, 2 external structures that are intravascular stent.
Fig. 2 is the structural representation of the double-deck intravascular stent of the load heparin prepared of the present invention.1 is the inner structure of intravascular stent, 2 external structures that are intravascular stent, and 3 is heparin.
Fig. 3 is the scanning electron microscope diagram of nanofiber of external structure of the double-deck intravascular stent of the load heparin prepared of the present invention.
The specific embodiment
Embodiment 1:
The preparation process of the double-deck intravascular stent of load heparin is as follows:
(1) take in the dichloromethane that 2g poly-dl-lactide is dissolved in 10mL, heating condensing reflux to material dissolve completely, be cooled to room temperature, with diameter, be 3mm, long be immersed in completely in above-mentioned lactic acid-caprolactone copolymer organic solution for the bar-shaped stainless steel mould of 15cm, immersion length is 8cm, vertically takes out, room temperature volatilization 3 hours after appropriateness stirs at once;
(2) take 0.3g poly lactic coglycolic acid solution in the dichloromethane and N-dimethylformamide (volume ratio is 7:3) mixed solvent of 10mL, heating condensing reflux dissolve 1 hour, and the poly lactic coglycolic acid solution obtaining is packed in the body of glass syringe;
(3) build electrostatic spinning apparatus, glass syringe is fixed on to jet deck, with No. 6 syringe needles, injection rate is 0.7mL/h, injection volume 1mL; Bar-shaped mould is vertically fixed on metal rotation device, and the distance of bar-shaped mould and syringe needle is 10cm, under the state rotating, receives Electrospun with the average rate of 200rpm.
(4) support of preparation is processed after 6 hours through lyophilization, be immersed in the heparin solution of 6g/100mL and soak 2 hours, after taking out, continue lyophilization and process 6 hours.
(5) support is separated with bar-shaped mould, can obtain the Biodegradable high-molecular intravascular stent of double-deck load heparin.
The hollow form that the double-deck intravascular stent of the load heparin of gained is tubulose, has inside and outside two structures.Stent length is 80mm, can bear powerful expansionary force, after bending and extruding, can recover original form, and its hot strength is 10MPa; Stretch-proof shape becomes 7%.Through sem observation, the average fibre diameter of external structure is 1.01 μ m.
Embodiment 2:
The preparation process of the double-deck intravascular stent of load heparin is as follows:
(1) take in the dichloromethane that 2g lactic acid-caprolactone copolymer is dissolved in 10mL, heating condensing reflux to material dissolve completely, be cooled to room temperature, with diameter, be 3mm, long be immersed in completely in above-mentioned lactic acid-caprolactone copolymer organic solution for the bar-shaped stainless steel mould of 15cm, immersion length is 3cm, vertically takes out, room temperature volatilization 3 hours after appropriateness stirs at once;
(2) take 0.2g poly lactic coglycolic acid solution in the dichloromethane and N-dimethylformamide (volume ratio is 8:2) mixed solvent of 10mL, heating condensing reflux dissolve 1 hour, and the poly lactic coglycolic acid solution obtaining is packed in the body of glass syringe;
(3) build electrostatic spinning apparatus, glass syringe is fixed on to jet deck, adopt No. 6 syringe needle, injection rate is 0.4mL/h, injection volume 0.8mL; Bar-shaped mould is vertically fixed on metal rotation device, and the distance of bar-shaped mould and syringe needle is 5cm, under the state rotating, receives Electrospun with the average rate of 50rpm.
(4) support of preparation is processed after 6 hours through lyophilization, be immersed in the heparin solution of 5g/100mL and soak 1 hour, after taking out, continue lyophilization and process 6 hours.
(5) support is separated with bar-shaped mould, can obtain the Biodegradable high-molecular intravascular stent of double-deck load heparin.
The hollow form that the double-deck intravascular stent of the load heparin of gained is tubulose, has inside and outside two structures.Stent length is 30mm, can bear powerful expansionary force, after bending and extruding, can recover original form, and its hot strength is 12MPa; Stretch-proof shape becomes 5%.Through sem observation, the average fibre diameter of external structure is 0.83 μ m.
Embodiment 3:
The preparation process of the double-deck intravascular stent of load heparin is as follows:
(1) take in the dichloromethane that 1g lactic acid-caprolactone copolymer is dissolved in 10mL, heating condensing reflux to material dissolve completely, be cooled to room temperature, with diameter, be 3mm, long be immersed in completely in above-mentioned lactic acid-caprolactone copolymer organic solution for the bar-shaped stainless steel mould of 15cm, immersion length is 10cm, vertically takes out, room temperature volatilization 3 hours after appropriateness stirs at once;
(2) take 0.2g poly lactic coglycolic acid solution in the dichloromethane and N-dimethylformamide (volume ratio is 8:2) mixed solvent of 10mL, heating condensing reflux dissolve 1 hour, and the poly lactic coglycolic acid solution obtaining is packed in the body of glass syringe;
(3) build electrostatic spinning apparatus, glass syringe is fixed on to jet deck, adopt No. 5 syringe needle, injection rate is 0.7mL/h, injection volume 0.5mL; Bar-shaped mould is vertically fixed on metal rotation device, and the distance of bar-shaped mould and syringe needle is 20cm, under the state rotating, receives Electrospun with the average rate of 200rpm.
(4) support of preparation is processed after 6 hours through lyophilization, be immersed in the heparin solution of 6g/100mL and soak 2 hours, after taking out, continue lyophilization and process 6 hours.
(5) support is separated with bar-shaped mould, can obtain the Biodegradable high-molecular intravascular stent of double-deck load heparin.
The hollow form that the double-deck intravascular stent of the load heparin of gained is tubulose, has inside and outside two structures.Stent length is 30mm, can bear powerful expansionary force, after bending and extruding, can recover original form, and its hot strength is 7MPa; Stretch-proof shape becomes 8%.Through sem observation, the average fibre diameter of external structure is 0.99 μ m.
Embodiment 4:
Double-deck intravascular stent to load heparin prepared in embodiment 1 ~ 3 carries out Study on biocompatibility.
The double-deck intravascular stent of load heparin prepared in embodiment 1-3 is cut into the segment that length is 0.5cm left and right with scalpel.Ultraviolet sterilization was processed after 2 hours, was placed on respectively in 48 orifice plates.Every hole inoculates respectively 10
5individual vascular smooth muscle cell, cultivates in cell culture incubator 24 hours, with CCK-8(days roots of cytoactive test kit, China) carry out cytoactive test, the OD value under the optical filter of 450nm wavelength has been reacted cell gross activity.10
5individual vascular smooth muscle cell is seeded in behind a hole, do not have the cell of support and equal number in incubator, cultivate 24 hours be matched group.Calculate the relative rate of increase of cell (RGR) and use following formula:
RGR (%)=(sample OD/ contrasts OD) * 100%
The relative rate of increase of cell and toxic reaction classification are in Table 1.By table 1, can find, the double-deck intravascular stent biocompatibility of prepared load heparin is good.
The relative rate of increase of cell and the toxic reaction classification of the double-deck intravascular stent of table 1 load heparin
| ? | The relative rate of increase RGR(% of cell) | Toxic |
| Embodiment | ||
| 1 | 91.09 | 1 |
| Embodiment | ||
| 2 | 87.26 | 1 |
| Embodiment | ||
| 3 | 89.11 | 1 grade |
Note: toxic reaction classification: the toxic reaction classification of material is as follows according to RGR: 0 grade is RGR >=100%; 1 grade is 80%-99%; 2 grades is 50%-79%; 3 grades is 30%-49%; 4 grades is 0%-29%
Embodiment 5:
Double-deck intravascular stent to load heparin prepared in embodiment 1 ~ 3 carries out Study on biocompatibility.
Bull rat is got blood 20mL from aorta, adds after anticoagulin, and centrifugal 10min under 1500rpm, draws the supernatant, obtains platelet rich plasma.
By the double-deck intravascular stent of load heparin prepared in embodiment 1 ~ 3, be cut into the segment of 0.8cm left and right, therefrom cut open with the body of glass syringe and pressing and putting into 24 orifice plates, every hole adds 1mL platelet blood plasma, and in cell culture incubator, constant temperature is placed 1 hour.Take out orifice plate, pour out residue PRP, use PBS washing by soaking 3 times, each 2mL.The double-deck intravascular stent that take does not have heparin to process is contrast.
Antiplatelet ability with the double-deck intravascular stent of electron microscope observation load heparin, refers to table 2.By table 2, can find, the double-deck platelet adhesion reaction of prepared load heparin is not obvious.
The platelet adhesion experiment of the double-deck intravascular stent of table 2 load heparin
| ? | Platelet |
| Embodiment | |
| 1 | Platelet adhesion reaction is not |
| Embodiment | |
| 2 | Platelet adhesion reaction is not |
| Embodiment | |
| 3 | Platelet adhesion reaction is not obvious |
| Matched group | Platelet adhesion reaction is obvious |
Claims (10)
1. a double-deck intravascular stent for load heparin, is characterized in that, support is divided into inside and outside two structures independently, and its external structure has the high polymer nanometer fiber structure of porous, and inner structure is fine and close solid macromolecule layer; Wherein, inner structure is prepared by lactic acid-caprolactone copolymer, and the external structure of support is prepared by poly lactic coglycolic acid.
2. the double-deck intravascular stent of load heparin according to claim 1, is characterized in that, described lactic acid-caprolactone copolymer weight average molecular weight is 40 ~ 60kDa, and the mol ratio of lactic acid and caprolactone is 8:2; Poly lactic coglycolic acid weight average molecular weight is 50 ~ 100kDa, and the mol ratio of lactic acid and hydroxyacetic acid is 5:5.
3. the double-deck intravascular stent of load heparin according to claim 1, is characterized in that, described nanofiber average diameter is 0.8～1.2 micron.
4. according to the preparation method of the double-deck intravascular stent of load heparin described in claim 1-3 any one, it is characterized in that, comprise the steps:
(1) lactic acid-caprolactone copolymer is dissolved in dichloromethane, and heating condensing reflux, to dissolving, insert in above-mentioned lactic acid-caprolactone copolymer organic solution with bar-shaped mould, after appropriateness stirs, vertically takes out at once, at room temperature, volatilizees;
(2) poly lactic coglycolic acid is dissolved in dichloromethane and N-dimethylformamide mixed solvent, heating condensing reflux, to dissolving completely, pack in glass syringe;
(3) build electrostatic spinning apparatus, glass syringe is fixed on to jet deck, bar-shaped mould is vertically fixed on metal rotation device, under the state of average rate rotation, receive Electrospun;
(4) by the support of preparation after lyophilization is processed, be immersed in heparin solution, after taking out, continue lyophilization and process 6 hours;
(5) support is separated with bar-shaped mould, can obtain the Biodegradable high-molecular intravascular stent of double-deck load heparin.
5. the preparation method of the double-deck intravascular stent of load heparin according to claim 4, is characterized in that, the concentration of described lactic acid-caprolactone copolymer organic solution is 10～20g/100mL; The concentration of poly lactic coglycolic acid organic solution is 2～4g/100mL; Described dichloromethane and N-dimethylformamide mixed solvent, its volume ratio is 8:2～7:3.
6. the preparation method of the double-deck intravascular stent of load heparin according to claim 4, is characterized in that, described bar-shaped mould is smooth stainless steel bar, and length is 10～20 centimetres, and diameter is 2～4 millimeters.
7. the preparation method of the double-deck intravascular stent of load heparin according to claim 4, is characterized in that, described syringe needle is 5-6 syringe needle.
8. the preparation method of the double-deck intravascular stent of load heparin according to claim 4, it is characterized in that, in described electrostatic spinning and receiving system, injection rate is 0.4～0.7mL/h, injection volume 0.5～~ 1 milliliter, the distance of bar-shaped mould and syringe needle is 5～20 centimetres, and its rotary speed is 50～200rpm.
9. the preparation method of the double-deck intravascular stent of load heparin according to claim 4, is characterized in that, described heparin solution concentration is 2～6g/100mL.
10. the preparation method of the double-deck intravascular stent of load heparin according to claim 4, is characterized in that, before support soaks in heparin solution, needs lyophilization to process 6 hours; Support soaks in heparin solution, and its time is 1～2 hour; After immersion, take out and continue lyophilization processing 6 hours.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104888287A (en) * | 2015-05-13 | 2015-09-09 | 东华大学 | Method for preparing heparinized lipidosome loaded double-layered intravascular stent |
| CN106421921A (en) * | 2015-11-10 | 2017-02-22 | 江苏工程职业技术学院 | Preparation method of double-layer intravascular stent |
| CN107789666A (en) * | 2016-08-30 | 2018-03-13 | 北京航空航天大学 | A kind of inwall micro-patterning small-caliber artificial blood vessel |
| CN108888384A (en) * | 2018-07-18 | 2018-11-27 | 广州迈普再生医学科技股份有限公司 | A kind of tubular bracket and preparation method thereof with double-layer structure |
| CN109663148A (en) * | 2018-12-17 | 2019-04-23 | 太阳雨林（厦门）生物医药有限公司 | A kind of extracellular matrix high molecular material biology composite vascular |
| CN109966548A (en) * | 2019-03-05 | 2019-07-05 | 太阳雨林（厦门）生物医药有限公司 | A kind of bilayer polymer Material cladding blood vessel |
| CN111135346A (en) * | 2019-12-20 | 2020-05-12 | 厦门大学附属中山医院 | Human-derived cell biological composite blood vessel |
| CN111467582A (en) * | 2020-03-03 | 2020-07-31 | 昆明医科大学 | Intravascular stent and preparation method thereof |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104888287A (en) * | 2015-05-13 | 2015-09-09 | 东华大学 | Method for preparing heparinized lipidosome loaded double-layered intravascular stent |
| CN106421921A (en) * | 2015-11-10 | 2017-02-22 | 江苏工程职业技术学院 | Preparation method of double-layer intravascular stent |
| CN107789666A (en) * | 2016-08-30 | 2018-03-13 | 北京航空航天大学 | A kind of inwall micro-patterning small-caliber artificial blood vessel |
| CN108888384A (en) * | 2018-07-18 | 2018-11-27 | 广州迈普再生医学科技股份有限公司 | A kind of tubular bracket and preparation method thereof with double-layer structure |
| CN109663148A (en) * | 2018-12-17 | 2019-04-23 | 太阳雨林（厦门）生物医药有限公司 | A kind of extracellular matrix high molecular material biology composite vascular |
| CN109966548A (en) * | 2019-03-05 | 2019-07-05 | 太阳雨林（厦门）生物医药有限公司 | A kind of bilayer polymer Material cladding blood vessel |
| CN111135346A (en) * | 2019-12-20 | 2020-05-12 | 厦门大学附属中山医院 | Human-derived cell biological composite blood vessel |
| CN111467582A (en) * | 2020-03-03 | 2020-07-31 | 昆明医科大学 | Intravascular stent and preparation method thereof |
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Application publication date: 20140326 |