CN110917410B - Cardiovascular stent coating based on double-layer heterogeneous structure and preparation method thereof - Google Patents
Cardiovascular stent coating based on double-layer heterogeneous structure and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a preparation method of a cardiovascular stent coating based on a double-layer heterogeneous structure, which comprises the following steps: 1) constructing a drug coating containing an antiproliferative drug on the cardiovascular stent; 2) mixing a double-bond end-capped polymer and a water-soluble polymer, dissolving the mixture in a solvent, spraying the mixture on a drug coating, and soaking the drug coating in water to obtain a porous coating; 3) and covering the surface of the porous coating with sulfhydrylation biomacromolecule solution containing a photoinitiator, irradiating by ultraviolet light to obtain a heparinized modified porous coating, and finally loading biological factors to obtain the cardiovascular stent coating. The invention also discloses a cardiovascular stent coating prepared by the method. The method is simple and efficient, the preparation steps are greatly simplified, and the prepared coating can enable the biological factors to be loaded with high activity, so that the compounding of the growth factors and the medicines is realized, the intimal hyperplasia can be inhibited, the rapid repair of the endothelial layer is promoted, and the better reconstruction performance of the vascular tissue is achieved.
Description
Technical Field
The invention relates to the field of biomedical materials, in particular to a cardiovascular stent coating based on a double-layer heterogeneous structure and a preparation method thereof.
Background
Cardiovascular disease (CVD) is currently the first killer to threaten human life. According to the investigation and research of the national cardiovascular disease center, the number of CVD patients in China currently reaches 2.9 hundred million, and the number of patients with coronary atherosclerotic heart disease (coronary heart disease) with high mortality rate is about 1100 million, which becomes a great public health problem.
With the development of modern medical technology, Drug-eluting stent (DES) implantation having a function of inhibiting neointimal hyperplasia has become the gold standard for treating cardiovascular diseases. However, with the widespread use of DES, adverse events such as Late Stent Thrombosis (LST) are continuously reported, which raises concerns of researchers and patients about the risk of Late DES. Clinical studies have shown that in more than 50% of patients with advanced thrombosis, stenting dislocation (mallaposis) and incomplete coverage (unover) are present, and delayed healing of post-vascular injury caused by DES is directly related to LST. Researchers speculate that the antiproliferative drugs used at present not only inhibit the growth of smooth muscle cells, but also inhibit the growth of endothelial cells, thereby delaying the remodeling of the endothelial layer and further influencing the function of the endothelial layer including anticoagulation. Therefore, the design of the existing stent drug coating does not affect or even promote the repair and functional reconstruction of the endothelial layer while satisfying the requirement of inhibiting the intimal hyperplasia, and has very important significance for reconstructing normal vascular tissues.
In recent years, in the research field, researchers adsorb or fix macromolecules with biological activity on the surface of a coating from the bionic aspect, and further realize the function of promoting the rapid reconstruction of an endothelial layer. By covalently immobilizing an active polypeptide comprising the specific affinity sequence REDV on an inert coating, the adhesion proliferation capacity of vascular endothelial cells on its surface is much greater than that of smooth muscle cells, thereby achieving rapid remodeling of the endothelial layer. The antibody anti-34, the vascular endothelial cell growth factor VEGF and the like are fixed on the surface of the material in a layer-by-layer self-assembly mode, so that the affinity of the material and endothelial cells can be obviously improved, the growth of the endothelial cells is regulated, and the aims of rapidly reconstructing an endothelial layer on the surface of an implant and reducing the probability of adverse events of restenosis and thrombus are also achieved.
Although bioactive molecules are widely developed and utilized in research fields, exhibiting remarkable prospects, designs of bioactive functional coatings that are actually applied to clinical treatment are rarely reported. Among them, antibody-modified coating represented by the Genous scaffold developed by orbus neich corporation is the only bioactive coating scaffold at present. However, in actual clinical comparative studies, the antibody anti-CD34 modified Genous stent did not exhibit the expected superior effect compared to the conventional drug eluting stent. There are many possible reasons, on one hand, the irreversible damage to the bioactive molecule, which is caused by the sterilization and storage process indispensable to the preparation process of the scaffold, makes the bioactive molecule partially or totally ineffective, thereby greatly weakening the action of the bioactive molecule; on the other hand, the vascular loss of the stent at the initial stage of implantation initiates the self-repair mechanism of the human body, and therefore, the use of anti-proliferative drugs at the early stage is indispensable.
In conclusion, the traditional drug eluting stent coating delays or even hinders the reconstruction of the endothelial layer of the blood vessel, thereby increasing the risk of late thrombosis; bioactive molecules with endothelial cell specificity can achieve the goal of accelerating endothelial layer repair, however, how to deal with the destruction of bioactive molecules by sterilization and storage during stent preparation remains challenging. More importantly, the use of antiproliferative drugs in stent coatings is inevitable because the stent implantation process inevitably causes the loss of blood vessels, thereby activating the self-repair mechanism of blood vessels.
Therefore, the stent coating is designed to realize the combination of the antiproliferative drugs and the bioactive functional molecules, on one hand, the slow release of the antiproliferative drugs can be met, on the other hand, the bioactivity of the bioactive molecules can be maintained, and the stent coating has great significance for further improving the stent curative effect, reducing complications and improving the life quality of patients.
Disclosure of Invention
The invention aims to provide a cardiovascular stent coating for promoting endothelial layer repair based on a double-layer heterogeneous structure and a preparation method thereof, which realize high activity load of biological factors, further utilize the synergistic effect of the biological factors and medicaments, realize inhibition of intimal hyperplasia and simultaneously quickly promote vascular endothelial layer repair.
In order to achieve the above object, the present invention provides a method for preparing a cardiovascular stent coating based on a double-layer heterogeneous structure, comprising the following steps:
(1) constructing a drug coating containing an antiproliferative drug on the cardiovascular stent;
(2) mixing a double-bond end-capped polymer and a water-soluble polymer, dissolving the mixture in a solvent, spraying the mixture on a drug coating, and immersing the drug coating in water for corrosion to obtain a porous coating;
(3) and covering the surface of the porous coating with sulfhydrylation biomacromolecule solution containing a photoinitiator, irradiating by ultraviolet light to obtain a biomacromolecule modified porous coating, and finally loading biological factors to obtain the cardiovascular stent coating.
The cardiovascular stent coating provided by the invention has a double-layer heterogeneous structure, the bottom layer of the coating is a degradable compact coating containing an antiproliferative drug, the top layer is a degradable porous coating modified by biomacromolecules, and the biological factor is completed before a stent implantation operation by utilizing the porous coating, so that the problem that the activity of the biological factor in the existing coating is difficult to store is solved, the effective combination of the biological factor and the antiproliferative drug is realized, and the prepared stent not only can inhibit intimal hyperproliferation, but also can rapidly promote the repair of the vascular endothelial layer.
In addition, because growth factors have strong specific interaction with some biological macromolecules (such as heparin, hyaluronic acid and the like), how to realize biological macromolecule modification of the porous coating is very important. In the prior literature, a self-assembly method is adopted to fix biological macromolecules on the surface of a substrate, but the method has complicated steps and is difficult to efficiently and stably prepare in industrial preparation. The invention firstly discovers that: the method is characterized in that the biomacromolecule is subjected to sulfydryl modification to prepare sulfydryl biomacromolecule solution, and then the light click reaction of sulfydryl and double bonds is utilized to covalently modify the biomacromolecule into the porous coating layer in one step, so that the preparation steps are greatly simplified, and the industrial efficient and stable preparation can be realized.
The preparation method of the cardiovascular stent coating based on the double-layer heterogeneous structure comprises the following three steps: constructing a drug coating, constructing a porous coating and modifying a porous coating biological macromolecule.
In the step (1), the construction of the drug coating layer:
the medicine coating is obtained by dissolving the antiproliferative medicine and the degradable polymer in a solvent and then spraying the solution on the cardiovascular stent by ultrasonic atomization.
The degradable polymer is one of poly-L-lactic acid (PLLA), poly-racemic-lactic acid (PDLLA), poly-lactic-co-glycolic acid (PLGA) or Polycaprolactone (PCL); the number average molecular weight is 1 to 30 ten thousand, and the number average molecular weight is preferably 5 to 15 ten thousand in consideration of the degradation time of the coating layer.
Preferably, the degradable polymer is PLGA with a Lactic Acid (LA) content of 50-75% in the copolymer, because the degradation rate of PLGA is related to the proportion of the copolymer inside thereof, and the degradation effect is better in the proportion range.
The antiproliferative drug comprises paclitaxel or derivatives thereof, and rapamycin or derivatives thereof. The usage amount of the antiproliferative drug is 1-15 mug/mm bracket, preferably 3-10 mug/mm bracket.
The solvent comprises one or more of acetone, ethyl acetate, dichloromethane, chloroform and tetrahydrofuran, and preferably one or more of acetone, ethyl acetate and chloroform.
Preferably, the medicine coating is obtained by spraying the medicine coating on the cardiovascular stent by an ultrasonic atomization spraying mode. The thickness of the drug coating is 5-20 μm, preferably 5-10 μm.
In the second step (2), the porous coating is constructed:
the double-bond-terminated polymer is methyl acrylate-terminated poly-L-lactic acid (PLLA), poly-racemic-lactic acid (PDLLA), poly-lactic-co-glycolic acid (PLGA) or Polycaprolactone (PCL), the number average molecular weight is 1-30 ten thousand, and considering the degradation time of the coating, the number average molecular weight is preferably 5-15 ten thousand.
Since the degradation rate of PLGA is related to its internal copolymer ratio, the double bond terminated polymer is preferably PLGA with a methacrylate-based terminated Lactic Acid (LA) content of 50% to 75%.
The construction method of the porous coating is a water etching method, wherein the water-soluble polymer comprises polyethylene glycol or polyvinylpyrrolidone, and the number average molecular weight is 1-20 ten thousand. In view of the fact that the water solubility of the water-soluble polymer is closely related to the molecular weight, the number average molecular weight is preferably 1 to 8 ten thousand.
The solvent selected comprises one or more of acetone, ethyl acetate, dichloromethane, chloroform, tetrahydrofuran, and preferably dichloromethane or chloroform in view of co-solubility of the two polymers.
The water-soluble polymer accounts for 20-80% of the total polymer mass fraction. However, when the proportion of the erosion phase is too low, the uniformity of the porous structure obtained by phase separation is poor; when the proportion of the erosion phase is too high, the stability of the porous coating layer is affected; therefore, in consideration of uniformity of the porous structure and stability of the coating layer, it is preferable that the water-soluble polymer accounts for 40 to 60 mass% of the total polymer.
Preferably, the coating is sprayed on the drug coating by ultrasonic atomization spraying. The corrosion time of the water is 4-6 minutes per time, and the corrosion times are 2-3 times. The thickness of the porous coating is 2-20 μm, preferably 5-10 μm.
In the third step (3), the porous coating biomacromolecule modification:
the biological macromolecule is heparin or hyaluronic acid, and the molecular weight is 0.2-10 ten thousand.
The photoinitiator is photoinitiator I2959, the concentration of the photoinitiator is 250-350ppm, and the concentration of the sulfhydrylation biomacromolecule solution is 4-6 mg/ml.
The conditions of the ultraviolet irradiation are as follows: irradiating in 365nm LED ultraviolet curing instrument for 1-10 min with ultraviolet intensity of 50-200mW/cm2。
The growth factor biological factor is quickly adsorbed on the porous coating through capillary action to prepare the cardiovascular stent coating. The total concentration of the biological factors is 20-200 mug/ml, and the biological factors comprise human Vascular Endothelial Growth Factor (VEGF) or/and human Hepatocyte Growth Factor (HGF). The biological factor loading amount on the cardiovascular stent coating is 10-1000ng/cm2。
Because the growth factors VEGF and HGF have the specific growth promotion capability of endothelial cells, the growth of smooth muscle cells on the composite coating is obviously inhibited by utilizing the active loading mode of the invention, and the growth of the endothelial cells can be well maintained. And further, in practical application, the reconstruction of the vascular endothelial layer is promoted while the intimal hyperplasia is prevented, so that the risk of late thrombosis is reduced.
The invention also provides a cardiovascular stent coating based on the double-layer heterogeneous structure prepared by the preparation method.
Compared with the prior art, the invention has the following beneficial effects:
(1) the invention solves the problems of poor vascular healing risk and serious influence of the activity of biological factors caused by a plurality of processes of coating preparation, sterilization, storage and the like of a single-layer drug-coated stent. The basal layer of the coating is a drug-loaded coating, so that the slow release of the antiproliferative drugs is realized, and then the porous coating with the affinity for the growth factors is further constructed on the drug-loaded coating, so that the biological factors can be highly loaded with activity only by adsorbing the biological factors of the growth factors in real time before surgical implantation, thereby achieving the compounding of the growth factors and the drugs, not only inhibiting the intimal hyperplasia, but also promoting the rapid repair of the endothelial layer and achieving better reconstruction performance of the vascular tissue.
(2) According to the method, the biomacromolecule is subjected to sulfydryl modification to prepare sulfydryl biomacromolecule solution, and then the light click reaction of sulfydryl and double bonds is utilized, so that the biomacromolecule can be subjected to covalent modification into the porous coating layer in one step, the preparation steps are greatly simplified, and the industrial efficient and stable preparation of the cardiovascular stent coating layer can be realized.
Drawings
FIG. 1 is an appearance (a) and micrograph (b) of an uncoated growth factor-loaded coated stent according to the present invention;
FIG. 2 is a fluorescence micrograph and a three-dimensional reconstruction of the coating after loading with a fluorescently labeled growth factor of the present invention;
FIG. 3 is a graph comparing the proliferation behavior of endothelial cells in coculture with smooth muscle cells before (a) and after (b) loading VEGF in example 1;
FIG. 4 is a graph comparing the proliferation density of endothelial cells before loading VEGF (-VEGF) and after loading VEGF (+ VEGF) in example 1 with that of smooth muscle cells.
Detailed Description
It should be noted that the coating of the present invention can be used for stents of various materials, such as metal stents and degradable stents, for convenience of description, the present invention in the following specific examples uses poly-L-lactic acid (PLLA) as a model substrate to construct the coating, but the actual stent material includes, but is not limited to, the materials in the examples.
The preparation of thiolated biomacromolecules described in the following examples is based on literature (references: Wang, L.M.; Chang, H.; Zhang, H.; Ren, K.F.; Li, H.; Hu, M.; Li, B.C.; Martins, M.C.L.; Barbosa, M.A.; Ji, J., Dynamic stability of polyalkylene glycol multi layer based on dispersed fibers for in situ control of cell adhesive. JMater Chem B2015, 3(38),7546-7553.), and thiolated heparin is exemplified as an example, and detailed preparation methods are as follows: 0.2g of heparin sodium (average molecular weight 12000) was dissolved in an acetic acid buffer solution having a pH of 5.5, 0.42g of 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC) was added, and after stirring at room temperature for 1 hour, 0.25g of N-hydroxysuccinimide (NHS) and 0.49g of cystamine dihydrochloride were added, and the reaction was stirred at room temperature for 24 hours, followed by removing unreacted cystamine dihydrochloride and the catalyst EDC/NHS by dialysis. Adding 0.34g of Dithiothreitol (DTT) into the obtained solution, stirring at room temperature for reacting for 6 hours, then removing residual DTT and small molecular cysteine obtained by the reaction through dialysis, and preparing the residual solution into sulfhydryl modified heparin through freeze drying.
Example 1: VEGF/rapamycin co-loaded stent coating
(1) rapamycin/PDLLA drug layer preparation: PDLLA with the number average molecular weight of 10 ten thousand is adopted as a degradable coating material, rapamycin is adopted as an antiproliferative drug, chloroform is adopted as a solvent, a drug-carrying substrate layer is prepared by an ultrasonic atomization spraying mode, the thickness of the drug coating layer is 10 mu m, and when the substrate is a stent, the rapamycin content is 6 mu g/mm.
(2) Preparing a heparinized PDLLA porous coating: number average with double bond cappingPDLLA with the molecular weight of 10 ten thousand is used as a coating material, polyvinylpyrrolidone (PVP) with the weight-average molecular weight of 4 ten thousand is used as an erosion phase, the mass fraction of the erosion phase is 55%, chloroform is used as a solvent, the blend is coated on a drug-carrying layer of a substrate by adopting an ultrasonic spraying method, and a porous coating with the thickness of 5 mu m is obtained by erosion of sterile ultrapure water. Subsequently, the surface of the coating was covered with a thiolated heparin aqueous solution (concentration 5mg/ml) containing 300ppm of photoinitiator I2959 using an LED 365nm light source ultraviolet lamp with a light intensity of 100mW/cm2And irradiating for 5 minutes, cleaning with ultrapure water, and drying in vacuum to obtain the heparin-modified double-layer heterogeneous structure coating.
(3) VEGF loading: immersing the coating in a VEGF sterile aqueous solution with the concentration of 50 mu g/ml, taking out after fully immersing for 2 minutes, and cleaning by using sterile water to obtain the VEGF/rapamycin co-loaded coating, wherein the loading amount of VEGF is 560 +/-40 ng/cm2。
FIG. 1(a) is an appearance image of the growth factor-unloaded coated stent obtained in step (2), and FIG. 1(b) is a micrograph of the growth factor-unloaded coated stent obtained in step (3).
Fig. 2 is a fluorescence micrograph and a three-dimensional reconstruction of the coating after loading the growth factor with the fluorescence label, and it can be seen from fig. 2 that the adsorbed biological factor is uniformly distributed in the coating.
Fig. 3 and 4 are graphs comparing the proliferation behaviors of endothelial cells and smooth muscle cells before and after loading VEGF, and it can be seen that the growth of smooth muscle cells on the composite coating is significantly inhibited and endothelial cells can better maintain the growth after the active loading of growth factors according to the present invention.
Example 2: VEGF/paclitaxel co-loaded stent coating
(1) Preparing a paclitaxel/PLGA medicine layer: PLGA (LA: GA 75:25) with the number average molecular weight of 10 ten thousand is used as a degradable coating material, paclitaxel is used as an antiproliferative drug, chloroform is used as a solvent, and a drug-carrying basal layer is prepared by ultrasonic spraying, wherein the thickness of the drug-carrying basal layer is 10 mu m, and the drug-carrying capacity is 5 mu g/mm.
(2) Preparing a hyaluronic acid modified PLGA porous coating: PLGA with the number average molecular weight of 10 ten thousand and end-capped double bonds is adopted as a coating material, and the coating material is heavyPolyvinylpyrrolidone (PVP) with the average molecular weight of 4 ten thousand is used as an erosion phase, the mass fraction of the erosion phase is 60 percent, chloroform is used as a solvent, the mixture is coated on the drug-carrying layer of the substrate by adopting an ultrasonic spraying method, and a porous coating with the thickness of 5 mu m is obtained by erosion of sterile ultrapure water. Then, the surface of the coating is covered with a thiolated hyaluronic acid aqueous solution (molecular weight 5 ten thousand, concentration 5mg/ml) containing 300ppm of photoinitiator I2959, an LED 365nm light source ultraviolet lamp is adopted, and the light intensity is 80mW/cm2And irradiating for 5 minutes, cleaning with ultrapure water, and drying in vacuum to obtain the heparin-modified double-layer heterogeneous structure coating.
(3) VEGF loading: immersing the coating in a VEGF sterile water solution with the concentration of 100 mu g/ml, taking out after fully immersing for 2 minutes, and cleaning by using sterile water to obtain a VEGF/paclitaxel co-loaded coating, wherein the loading amount of VEGF is 860 +/-65 ng/cm2。
Through the combined action of VEGF and paclitaxel, the coating obviously inhibits the proliferation of smooth muscle cells, and specifically reduces the influence of paclitaxel on endothelial cells, wherein the density of the endothelial cells is 15 times that of the smooth muscle cells.
Example 3: VEGF/zotarolimus co-loaded stent coating
(1) Preparation of zotarolimus/PDLLA drug layer: PDLLA with the number average molecular weight of 15 ten thousand is adopted as a degradable coating material, paclitaxel is adopted as an antiproliferative drug, chloroform is adopted as a solvent, and a drug-carrying basal layer is prepared by ultrasonic spraying, wherein the thickness of the drug-carrying coating is 5 mu m, and the drug-carrying capacity is 5 mu g/mm.
(2) Preparing a heparinized PLGA porous coating: PDLLA with the number average molecular weight of 10 ten thousand and end-capped double bonds is used as a coating material, polyethylene glycol (PEG) with the weight average molecular weight of 1 ten thousand is used as an erosion phase, the mass fraction of the erosion phase is 50%, chloroform is used as a solvent, the blend is coated on a drug-loaded layer of a substrate by adopting an ultrasonic spraying method, and a porous coating with the thickness of 5 mu m is obtained by erosion of sterile ultrapure water. Subsequently, the surface of the coating was covered with a thiolated heparin solution (concentration 5mg/ml) containing 300ppm of photoinitiator I2959 using an LED 365nm light source UV lamp at a light intensity of 100mW/cm2And irradiating for 5 minutes, cleaning with ultrapure water, and drying in vacuum to obtain the heparin-modified double-layer heterogeneous structure coating.
(3) VEGF loading: immersing the coating in a VEGF sterile water solution with the concentration of 50 mu g/ml, taking out after fully immersing for 2 minutes, and cleaning by using sterile water to obtain a VEGF/paclitaxel co-loaded coating, wherein the loading amount of VEGF is 520 +/-45 ng/cm2。
Through the combined action of VEGF and zotarolimus, the coating obviously inhibits the proliferation of smooth muscle cells, and specifically reduces the influence of paclitaxel on endothelial cells, wherein the density of the endothelial cells is 20 times that of the smooth muscle cells.
Example 4: HGF/rapamycin co-loaded stent coating
(1) rapamycin/PDLLA drug layer preparation: PDLLA with the number average molecular weight of 15 ten thousand is adopted as a degradable coating material, rapamycin is adopted as an antiproliferative drug, chloroform is adopted as a solvent, and a drug-carrying basal layer is prepared by ultrasonic spraying, wherein the thickness of the drug-carrying coating is 5 mu m, and the drug-carrying capacity is 4 mu g/mm.
(2) Preparing a hyaluronic acid modified PDLLA porous coating: PDLLA with the number average molecular weight of 15 ten thousand and terminated by double bonds is used as a coating material, polyvinylpyrrolidone (PVP) with the weight average molecular weight of 4 ten thousand is used as an erosion phase, the mass fraction of the erosion phase is 55%, chloroform is used as a solvent, the blend is coated on a drug-carrying layer of a substrate by adopting an ultrasonic spraying method, and a porous coating with the thickness of 5 mu m is obtained by erosion of sterile ultrapure water. Then, the surface of the coating is covered with a thiolated hyaluronic acid solution (molecular weight 10 ten thousand, concentration 5mg/ml) containing 300ppm of photoinitiator I2959, an LED 365nm light source ultraviolet lamp is adopted, and the light intensity is 100mW/cm2And irradiating for 5 minutes, cleaning with ultrapure water, and drying in vacuum to obtain the heparin-modified double-layer heterogeneous structure coating.
(3) HGF loading: immersing the coating in 100 mug/ml HGF sterile water solution, taking out after fully immersing for 1 minute, and cleaning with sterile water to obtain the VEGF/rapamycin co-loaded coating, wherein the loading amount of HGF is 760 +/-55 ng/cm2。
Through the combined action of HGF and rapamycin, the coating obviously inhibits the proliferation of smooth muscle cells, and specifically reduces the influence of paclitaxel on endothelial cells, wherein the density of the endothelial cells is 12 times that of the smooth muscle cells.
Example 5: HGF/zotarolimus co-loaded stent coating
(1) Preparation of zotarolimus/PLLA drug layer: the preparation method comprises the steps of adopting PLLA with the number average molecular weight of 8 ten thousand as a degradable coating material, zotarolimus as an antiproliferative drug, chloroform as a solvent, and preparing a drug-carrying substrate layer by ultrasonic spraying, wherein the thickness of the drug coating is 5 mu m, and the drug carrying capacity is 4 mu g/mm.
(2) Preparing a heparinized PDLLA porous coating: PDLLA with the number average molecular weight of 10 ten thousand and terminated by double bonds is used as a coating material, polyvinylpyrrolidone (PVP) with the weight average molecular weight of 4 ten thousand is used as an erosion phase, the mass fraction of the erosion phase is 55%, chloroform is used as a solvent, the blend is coated on a drug-carrying layer of a substrate by adopting an ultrasonic spraying method, and a porous coating with the thickness of 5 mu m is obtained by erosion of sterile ultrapure water. Subsequently, the surface of the coating was covered with a thiolated heparin aqueous solution (concentration 5mg/ml) containing 300ppm of photoinitiator I2959 using an LED 365nm light source ultraviolet lamp with a light intensity of 100mW/cm2And irradiating for 5 minutes, cleaning with ultrapure water, and drying in vacuum to obtain the heparin-modified double-layer heterogeneous structure coating.
(3) HGF loading: immersing the coating in an HGF sterile aqueous solution with the concentration of 50 mu g/ml, taking out after fully immersing for 1 minute, and cleaning by using sterile water to obtain the VEGF/rapamycin co-loaded coating, wherein the loading amount of VEGF is 580 +/-60 ng/cm2。
Through the combined action of HGF and zotarolimus, the coating obviously inhibits the proliferation of smooth muscle cells, and specifically reduces the influence of paclitaxel on endothelial cells, wherein the density of the endothelial cells is 14 times that of the smooth muscle cells.
Claims (9)
1. A preparation method of a cardiovascular stent coating based on a double-layer heterogeneous structure comprises the following steps:
(1) constructing a drug coating containing an antiproliferative drug on the cardiovascular stent;
(2) mixing a double-bond end-capped polymer and a water-soluble polymer, dissolving the mixture in a solvent, spraying the mixture on a drug coating, and immersing the drug coating in water for corrosion to obtain a porous coating; the double-bond end capping is methacrylate group end capping;
(3) and (2) immersing the sulfhydrylation biomacromolecule solution containing the photoinitiator on the surface of the porous coating, irradiating by ultraviolet light to obtain the biomacromolecule modified porous coating, and finally loading biological factors to obtain the cardiovascular stent coating.
2. The method for preparing the cardiovascular stent coating based on the two-layer heterogeneous structure according to claim 1, wherein in the step (1), the drug coating is prepared by dissolving the antiproliferative drug and the polymer in a solvent and then performing ultrasonic atomization spraying on the cardiovascular stent.
3. The method for preparing the cardiovascular stent coating based on the two-layer heterogeneous structure according to claim 1 or 2, wherein the antiproliferative drug comprises paclitaxel or its derivatives, rapamycin or its derivatives.
4. The method for preparing cardiovascular stent coating based on two-layer heterogeneous structure according to claim 1, wherein in step (2), the water-soluble polymer is polyethylene glycol or polyvinylpyrrolidone, and the number average molecular weight is 1-20 ten thousand.
5. The method for preparing cardiovascular stent coating based on two-layer heterogeneous structure according to claim 1, wherein in step (2), the water-soluble polymer accounts for 20% -80% of the total polymer mass fraction.
6. The method for preparing a cardiovascular stent coating based on a two-layer heterogeneous structure according to claim 1, wherein in the step (3), the photoinitiator is photoinitiator I2959, and the concentration of the photoinitiator is 250-350 ppm.
7. The method for preparing cardiovascular stent coating based on two-layer heterogeneous structure according to claim 1, wherein in step (3), the biomacromolecule is heparin or hyaluronic acid, the molecular weight is 0.2-10 ten thousand, and the concentration of the thiolated biomacromolecule solution is 4-6 mg/ml.
8. The method for preparing a cardiovascular stent coating layer based on a double-layer heterogeneous structure according to claim 1, wherein in the step (3), the biological factor comprises human vascular endothelial growth factor or/and human hepatocyte growth factor.
9. A cardiovascular stent coating based on a double-layer heterogeneous structure, which is prepared by the preparation method of any one of claims 1 to 8.
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