阅读说明：本技术 一种聚合物泡沫涂层支架及其制备方法 (Polymer foam coating support and preparation method thereof ) 是由 郑兆柱 刘静宜 李刚 吕强 王晓沁 于 2021-09-13 设计创作，主要内容包括：本发明公开了一种聚合物泡沫涂层支架,包括支架本体和形成于所述支架本体表面的聚合物多孔泡沫涂层,所述聚合物多孔泡沫涂层中负载有药物和/或活性物质；所述聚合物多孔泡沫涂层不溶于水性液体或部分溶于水性液体,其原料为天然聚合物材料；所述药物和/或活性物质是将聚合物泡沫涂层支架灭菌后,通过后载药的方式负载于所述聚合物多孔泡沫涂层中的。本发明还公开了所述聚合物泡沫涂层支架的制备方法。本发明的聚合物泡沫涂层支架,采用后载药的方式将药物、活性物质负载于聚合物多孔泡沫涂层中,从而有效地解决了现有涂层技术中灭菌过程药物和活性物质活力损失的问题。(The invention discloses a polymer foam coating stent, which comprises a stent body and a polymer porous foam coating formed on the surface of the stent body, wherein a medicament and/or an active substance are loaded in the polymer porous foam coating; the polymer porous foam coating is insoluble or partially soluble in aqueous liquid, and the raw material of the polymer porous foam coating is a natural polymer material; the drug and/or active substance is loaded in the polymer porous foam coating in a post-drug loading mode after the polymer foam coating stent is sterilized. The invention also discloses a preparation method of the polymer foam coating bracket. According to the polymer foam coating stent, a drug and an active substance are loaded in the polymer porous foam coating in a post-drug loading manner, so that the problem of activity loss of the drug and the active substance in a sterilization process in the prior coating technology is effectively solved.)

1. A polymer foam coating stent is characterized by comprising a stent body and a polymer porous foam coating formed on the surface of the stent body, wherein the polymer porous foam coating is loaded with a medicament and/or an active substance; the polymer porous foam coating is insoluble or partially soluble in aqueous liquid, and the raw material of the polymer porous foam coating is a natural polymer material; the drug and/or active substance is loaded in the polymer porous foam coating in a post-drug loading mode after the polymer foam coating stent is sterilized.

2. The polymer foam coated stent of claim 1 wherein the porous polymer foam coating has an elastic recovery capability that is pre-compressed prior to placement in the body and then springs back after placement in the body so that the porous polymer foam coating does not detach from the stent body.

3. The polymer foam coated stent of claim 1 or 2, wherein the drug loading in the polymer porous foam coating is 1-5000 ng/mm, and the active substance loading is 1-5000 ng/mm.

4. The polymer foam coated stent of claim 1 or 2, wherein the polymer porous foam coating has a thickness of 1nm to 1 mm.

5. A preparation method of a polymer foam coating bracket is characterized by comprising the following steps:

s1, providing a support body, and forming a polymer porous foam coating on the support body, wherein the raw material of the polymer porous foam coating is a natural polymer material;

s2, sterilizing the stent body with the polymer porous foam coating, and then loading medicines and/or active substances in the polymer porous foam coating in a medicine loading mode to obtain the polymer foam coating stent.

6. The method of claim 5, wherein the stent body is a metal stent or a polymer stent; the material of the metal bracket comprises stainless steel, nickel-titanium alloy and cobalt-chromium alloy, and the material of the polymer bracket comprises nylon, polymethyl methacrylate, polyethylene, polytetrafluoroethylene, orlon, Everron and terylene; the stent body is a non-absorbable stent or an absorbable stent.

7. The method of claim 5, wherein the step of forming the porous polymer foam coating comprises the steps of:

(i) preparing a coating mother solution from polymer raw materials, wherein the polymer raw materials comprise one or more of hyaluronic acid, collagen, alginate, chitosan, silk fibroin, gelatin, albumin and derivatives thereof;

(ii) loading the coating mother solution on the surface of the stent body by means of dipping or coating;

(iii) and forming water-insoluble porous foam by using the coating mother liquor on the surface of the stent body to obtain the polymer porous foam coating.

8. The method of claim 7, wherein the method of forming the water-insoluble cellular foam from the coating dope comprises one or more of the following methods: (1) forming a water-insoluble gel, and then forming a water-insoluble porous foam by a freeze-drying process; (2) directly forming a water insoluble porous foam; (3) directly freeze-drying to form water-soluble or partially water-soluble porous foam, and performing post-treatment to form water-insoluble porous foam;

wherein, the method (1) comprises a physical crosslinking method and a chemical crosslinking method;

the method (2) is that under the condition that the coating mother liquor is frozen, the polymer in the coating mother liquor is chemically crosslinked to form frozen gel;

the method (3) comprises one or more of steam fumigation, solvent soaking and chemical crosslinking.

9. The method of claim 8, wherein the polymer material further comprises a chemical cross-linking agent and/or a plasticizer, wherein the chemical cross-linking agent comprises formaldehyde, glutaraldehyde, EDC-NHS, horseradish peroxidase/H₂O₂、BDDE、Ca²⁺And the photosensitizer comprises one or more of glycerol and linear polymer molecular materials.

10. The method for preparing a polymer foam coated stent according to claim 5, wherein the method for loading the drug and/or the active substance in the polymer porous foam coating comprises the following steps:

dipping the stent body with the polymer porous foam coating into a solution containing the drug and/or the active substance, so that the drug and/or the active substance are loaded in the polymer porous foam coating;

or immersing the stent body with the polymer porous foam coating into a gel-forming precursor containing the medicine and/or the active substance, so that the gel precursor containing the medicine and/or the active substance is filled in the pores of the polymer porous foam coating and forms a gel; the gel precursor material comprises one or more of hyaluronic acid, collagen, alginate, chitosan, silk fibroin, gelatin, albumin, and derivatives thereof.

Technical Field

The invention relates to the technical field of tissue engineering materials, in particular to a polymer foam coating support and a preparation method thereof.

Background

The bracket is a medical appliance commonly used in the operation of lumen tissues and organs and has the function of dredging body fluid. At present, stents are mainly classified into metal stents made of stainless steel, nitinol, or cobalt-chromium alloy, etc., and polymer stents made of polymer materials.

Metal stents mainly made of stainless steel, cobalt-chromium alloy, nickel-titanium alloy and other materials are widely applied to the treatment of cardiovascular diseases, and achieve better treatment effect. However, these cardiovascular stents are not degradable in vivo during the treatment process, are easy to cause cardiovascular restenosis, embolism-causing reaction, chronic inflammation reaction and release toxic metal ions in vivo. To address this series of problems, researchers have coated stent surfaces with drugs, growth factors, or other active substances. After the coating stent is implanted into a body, active substances such as medicines and/or growth factors and the like can be slowly released, and the regeneration of tissues can be promoted while the occurrence of side effects is inhibited.

However, the existing active substances such as drugs and/or growth factors are generally unstable in properties, and the release rate in vivo is difficult to control. More importantly, terminal sterilization process is commonly used in the stent preparation process, which easily causes the inactivation of active substances such as drugs and/or growth factors, and the like, and the inactivation is in a very high proportion, and the coating properties of the active substances such as the drugs and/or the growth factors are obviously different from those before sterilization. The Chinese invention patent CN 106913915B discloses a self-healing stent composite coating, which is prepared by synthesizing degradable double-bond end-capping block polymers, coating the polymers on the surface of a stent, crosslinking and freezing to obtain a self-healing porous coating, wherein the self-healing porous coating can realize the healing of a porous structure by changing the temperature, so that the self-healing stent composite coating loaded with drugs and/or active molecules is prepared. This patent uses synthetic polymers and achieves drug loading by inducing the healing of the porous structure through a change in temperature. The Chinese invention patent CN 108485512B discloses a patterned porous polymer coating, which is obtained by introducing photo-crosslinking groups into a coating material, combining the dynamic characteristics of the coating, realizing the regulation and control of local microphase separation, and realizing the selective pore-forming of areas with different shapes and sizes. However, the coating materials in these patents use synthetic polymers, on one hand, the synthetic polymer uses organic solvents in the synthesis process, which not only causes environmental pollution, but also causes unpredictable biological safety risks due to residual reactants or intermediate products; on the other hand, the degradation of synthetic polymers in vivo is complicated, and both the polymer itself and the degradation products pose unpredictable biosafety risks. In addition, the use of self-healing polymers and photocrosslinking polymers limits the materials from which their coatings can be formed and their range of applications. Chinese patent CN00137207.6 discloses a vascular stent with a restenosis preventing function, which is characterized in that a polymer carrier is prepared into dispersion liquid to be coated on the surface of the stent, a polymer coating with a porous structure is formed after vacuum drying, and then a medicament is adsorbed in the polymer coating in a dipping mode. Although gelatin is disclosed as the coating material, the gelatin coatings prepared according to the method of the examples (drying at 60 ℃ C. for 1 hour) are all solid coatings and no porous foam coating is obtained, and therefore no protection of the active substance is achieved. Therefore, how to use natural polymers with high biocompatibility to prepare porous stent coatings to protect active substances is a problem to be solved urgently in the field of stent research and application at present.

Disclosure of Invention

The invention aims to solve the technical problem of providing a polymer foam coating stent, which effectively loads medicines and/or active substances in a polymer porous foam coating in a post-loading mode and has a slow release effect, so that the problems of medicine slow release and activity loss of the medicines and the active substances in the sterilization process in the prior coating technology are effectively solved.

In order to solve the technical problems, the invention provides the following technical scheme:

the invention provides a polymer foam coating stent, which comprises a stent body and a polymer porous foam coating formed on the surface of the stent body, wherein a medicament and/or an active substance are loaded in the polymer porous foam coating; the polymer porous foam coating is insoluble or partially soluble in aqueous liquid, and the raw material of the polymer porous foam coating is a natural polymer material; the drug and/or active substance is loaded in the polymer porous foam coating in a post-drug loading mode after the polymer foam coating stent is sterilized. The polymeric porous foam has a sustained release effect on the drug and/or active substance carried.

In the present invention, the natural polymer material includes, but is not limited to, one or more of hyaluronic acid, collagen, alginate, chitosan, silk fibroin, gelatin, albumin, and derivatives thereof.

Preferably, the polymeric porous foam coating has an elastic recovery capability that is pre-compressed prior to placement in the body; after being placed in the body, the polymer porous foam coating rebounds, so that the polymer porous foam coating can not be separated from the stent body.

In the present invention, the polymeric porous foam coating has elastic recovery capability, i.e., the polymeric porous foam coating is deformable while maintaining its physical integrity. The polymeric cellular foam coating may be pre-compressed prior to and/or during placement in the tissue to be repaired or expanded. And then placed in the tissue, where the compressed polymer foam coating can expand and recover its original shape over a period of time.

In the present invention, the expansion of the polymer foam coating within the tissue to reach a plateau volume can be spontaneous (e.g., within 5 seconds or less), or can occur over a period of time (e.g., seconds, minutes, and hours). While the polymer foam (before compression) may be adapted to be any size, depending on the size of the stent pore size and/or the nature of the polymer foam. The rate of expansion of the polymer foam within the tissue may depend on several factors including, but not limited to, hydration state, pressure, volume of void space, and foam structural properties (including porosity), and interactions between fluids and structures.

In the present invention, the loading of the drug and active substance on the polymer porous foam coating is not limited. Preferably, in the polymer porous foam coating, the loading capacity of the medicine is 1-5000 ng/mm, and the loading capacity of the bioactive molecules is 1-5000 ng/mm.

The thickness of the polymer foam coating can affect the release properties of the loaded drug. In the present invention, the thickness of the polymer porous foam coating layer is not limited, and preferably, the thickness of the polymer porous foam coating layer is 1nm to 1 mm.

The invention also provides a preparation method of the polymer foam coating bracket, which comprises the following steps:

s1, providing a support body, and forming a polymer porous foam coating on the support body, wherein the raw material of the polymer porous foam coating is a natural polymer material;

s2, sterilizing the stent body with the polymer porous foam coating, and then loading medicines and/or active substances in the polymer porous foam coating in a medicine loading mode to obtain the polymer foam coating stent.

In the invention, the stent body is distinguished from materials and can be divided into a metal stent or a polymer stent. As the metal stent, materials thereof include, but are not limited to, stainless steel, nickel titanium alloy, and cobalt chromium alloy. As the polymer scaffold, a degradable scaffold or a non-degradable scaffold may be used. The material of the polymer scaffold includes, but is not limited to, nylon, polymethylmethacrylate, polyethylene, polytetrafluoroethylene, orlon, efloren, and dacron. In addition, the stent body may comprise a stent which has been coated by a reported chemical crosslinking or layer-by-layer self-assembly method, and may also comprise a stent which has been coated with a drug and/or active substance which is susceptible to inactivation by a non-sterilization process by a reported chemical crosslinking or layer-by-layer self-assembly method.

In the present invention, the drug may be a hydrophobic drug including, but not limited to, rapamycin and its derivatives, paclitaxel and its derivatives, everolimus, novel sirolimus or a hydrophilic drug including, but not limited to, a nucleophilic NO donor. Coating drugs can be divided into five major classes according to their main pharmacological actions: (1) antithrombotic drugs such as heparin, hirudin, prostacyclin, abciximab, and the like. (2) Anti-inflammatory drugs, such as Dexamethasone (DXM), methylprednisolone, diphosphate liposome, etc. (3) anti-VSMC proliferation drugs such as Rapamycin (RAPM), Paclitaxel (PTX), angiopeptin, Mycophenoic Acid, Tracolimus, Everolimus, Cyclosporin A, methyl-RAPM, and the like. (4) anti-VSMC migratory drugs such as batimastat, etc. (5) The medicine for promoting endothelial healing, such as 17 beta estradiol, vascular endothelial growth factor, etc.

In the present invention, the active substance includes, but is not limited to, vascular endothelial cell growth factor, vascular endothelial cell specific antibody anti-CD34, etc., vascular endothelial cell growth factor gene, etc.

In the present invention, the method of forming the polymeric porous foam coating comprises the steps of:

(i) preparing a coating mother solution from a polymer raw material;

(ii) loading the coating mother solution on the surface of the stent body by means of dipping or coating;

(iii) and forming the coating mother liquor on the surface of the stent body into a porous foam shape, thus obtaining the polymer porous foam coating.

In the present invention, the polymer material comprises a natural polymer material, the natural polymer including, but not limited to, hyaluronic acid, collagen, alginate, chitosan, silk fibroin, gelatin, albumin, and one or more of their derivatives. Such natural polymers not only have good biocompatibility, but also can gradually degrade in an in vivo environment.

Preferably, the natural polymer material is silk fibroin. The silk fibroin is protein extracted from silk, accounts for 75-80% of the mass of the silk, and has low immunogenicity, no immunogenicity, excellent and adjustable mechanical properties. The ratio of hydrophobic amino acid to hydrophilic amino acid in silk fibroin 6: 1, and may form a gel or scaffold material through a variety of crosslinking mechanisms. Various acting forces (hydrophilic and hydrophobic effects, hydrogen bonds, van der waals forces and the like) exist between the material interface and the medicine, the bioactive factors and even metal ions after medicine loading, the slow release effect on the loaded preparation can be effectively realized, and the acting force on the loaded preparation can be regulated and controlled in a water-insoluble treatment mode, so that the effective controlled release of the loaded preparation is realized.

Further, the method for forming the water-insoluble porous foam-like coating mother liquor comprises one or more of the following methods: (1) forming water insoluble gel, and freeze drying to form water insoluble porous foam. (2) Forming insoluble porous foam directly. (3) Directly freeze-drying to form water-soluble or partially water-soluble porous foam, and performing post-treatment to form water-insoluble porous foam.

Among them, the method of forming the water-insoluble gel includes a physical crosslinking method and a chemical crosslinking method.

Direct formation of an insoluble porous foam refers to chemically cross-linking the polymer in the coating mother liquor to form a cryogel under low temperature conditions, i.e., temperatures below the ice crystal formation temperature of the coating mother liquor.

The water-insoluble porous foam is formed by post-treating a water-soluble or partially water-soluble porous foam by one or more of steam fumigation, solvent soaking and chemical cross-linking.

Furthermore, the polymer raw material also comprises a chemical cross-linking agent, and the existence of the chemical cross-linking agent enables the high molecular material in the polymer raw material to be chemically cross-linked, so that the water solubility of the coating is reduced, the stability of the coating is improved, and the release period of the active substance is prolonged. Wherein the chemical cross-linking agent includes but is not limited to formaldehyde, glutaraldehyde, EDC-NHS, horseradish peroxidase (HRP)/H₂O₂、BDDE、Ca²⁺And a photosensitizer.

Further, the polymer raw material also comprises a plasticizer, and the plasticity of the polymer material is improved by adding the plasticizer, so that the toughness and the elasticity of the coating are improved. The plasticizer comprises one or more of glycerin and linear polymer molecular materials, and the linear polymer molecular materials comprise but are not limited to polyether.

In the present invention, the sterilization method may employ sterilization methods commonly used in the art, including, but not limited to, autoclaving, ethylene oxide sterilization, and radiation sterilization.

In the invention, the method for loading the medicine and/or the active substance in the polymer porous foam coating comprises the following steps: the stent body with the polymer porous foam coating is immersed in a solution containing a drug and/or an active substance so that the drug and/or the active substance is supported in the polymer porous foam coating.

In the present invention, the thickness of the polymer porous foam coating layer is preferably 1nm to 1 mm. The rate of release of the drug and/or active agent from the polymer foam coating is directly related to the volume and thickness of the coating, i.e., the thicker the polymer foam coating, the faster the drug release rate and vice versa. The surface physical and chemical properties of the polymer species and polymer derivatives, and the forces of drugs and active substances are diversified. Therefore, the invention can control the slow release effect of the loaded drug and the active substance by controlling the thickness and the volume of the porous foam coating and the selection of the polymer type and the polymer derivative.

In addition, the volume of the polymeric foam coating can be controlled in part by the degradation and/or dissolution characteristics of the polymeric porous foam, thereby further controlling the release of the drug, active agent. The polymer foam coating may be adapted to degrade at a predetermined rate such that the polymer foam coating gradually decreases in volume while still providing sufficient support as the polymer-coated stent is applied in a human body.

In a preferred embodiment, to further control the drug release profile, allowing for sustained release of the drug (release of the pharmaceutically active drug over a period of about 6 months or more), the stent body with the polymeric porous foam coating can be dipped into a drug/active containing gel precursor with or without a viscosity inducing component, surfactant and/or lubricant containing fluid (e.g., saline) such that the drug/active containing gel precursor fills the pores of the polymeric porous foam coating and forms a gel, thereby extending the release cycle of certain growth factors or cytokines and stabilizing their functionality. Wherein the gel precursor material includes, but is not limited to, one or more of hyaluronic acid, collagen, alginate, chitosan, silk fibroin, gelatin, albumin, and derivatives thereof.

Compared with the prior art, the invention has the beneficial effects that:

1. the invention adopts a mode of post-loading drugs to load active substances in the polymer porous foam coating, thereby effectively solving the problem of activity loss of drugs and active substances in the sterilization process in the prior coating technology.

2. In the polymer foam coating bracket, the coating is prepared from natural polymers, and compared with synthetic polymers, the natural polymers do not need artificial synthesis, so that the harm of reactants and intermediate products to the environment in the synthesis process is avoided; meanwhile, the natural polymer has good biocompatibility, no/low immunogenicity and controllable degradation property, does not cause potential biosafety risk, and is beneficial to promoting cell proliferation and tissue healing.

3. In the polymer foam coating stent, acting forces such as physical confinement effect, electrostatic effect, hydrophilic and hydrophobic effect, ionic effect and the like exist between the coating on the stent and the drug or the active substance, and the polymer foam coating stent can play a role in slowly releasing the loaded drug or the active substance.

4. In the polymer foam coating stent, the coating on the stent and the medicament or active substance are protected by the polymer, so that the influence of temperature, pH value, osmotic pressure and the like on the activity of the carried coating and the medicament or active substance is avoided, and the action of the medicament or active substance can be better played.

Drawings

FIG. 1 is a graph showing activity changes in a drug sterilization process when four modes, i.e., whole-process sterilization, post-drug loading, and gel-like post-drug loading, are respectively adopted;

FIG. 2 is a graph showing activity changes during the sterilization process of growth factors when four modes of whole-process sterilization, post-drug loading and gel-like post-drug loading are respectively adopted;

fig. 3 is a drug release profile of the lyophilized silk fibroin scaffolds, fully sterilized scaffolds, and post-drug loaded sterilized scaffolds prepared in example 1;

fig. 4 is a drug release profile of the silk fibroin lyophilized scaffold prepared in example 2;

fig. 5 is a drug release profile of the silk fibroin lyophilized scaffold prepared in example 3;

fig. 6 is a drug release profile of the silk fibroin lyophilized scaffold prepared in example 4;

fig. 7 is a drug release profile of the silk fibroin scaffolds prepared in examples 1-2 and 5;

FIG. 8 is a drug release profile of the hyaluronic acid stent prepared in example 6;

fig. 9 is a drug release profile of the silk fibroin/hyaluronic acid lyophilized scaffold prepared in example 7.

Detailed Description

The present invention is further described below in conjunction with specific examples to enable those skilled in the art to better understand the present invention and to practice it, but the examples are not intended to limit the present invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

The experimental methods used in the following examples are conventional methods unless otherwise specified, and materials, reagents and the like used therein are commercially available without otherwise specified.

Example 1: silk fibroin freeze-dried stent coating

1. Preparation of Silk fibroin solution

Placing appropriate amount of silk or silkworm cocoon (pupa removed) in 0.02M Na₂CO₃Boiling the solution for 1.5 hours to degum, then repeatedly washing the degummed silk by distilled water, and air-drying the degummed silk by a fume hood to obtain the degummed silk. And (3) degumming the degummed silk according to the mass volume ratio of 1: 10 into the mixed solution (CaCl)₂/H₂O/C₂H₅OH 1/8/2, molar ratio), stirring continuously at 75-80 deg.C water bath temperature until the fibroin fiber is completely dissolved (about 2 hr), dialyzing the solution with distilled water for 72 hr, and centrifuging to remove insoluble particles to obtain fibroin solution with concentration of about 50-60 mg/ml.

2. Impregnation

The stent body (nickel-titanium alloy) is soaked in silk fibroin solution (0.1-30% wt/v) to obtain the water-soluble primary silk fibroin coating.

3. Freeze-drying

The stent body coated with the silk fibroin coating is directly pre-frozen for 2 to 48 hours at the temperature of about-5, 6, 7, 9, 10, 20, 40, 80 and 196 ℃ or pre-frozen and quenched for 2 to 72 hours at various temperature combinations. And after maintaining for 3 days, freeze-drying the water-soluble silk fibroin coating stent body for 48-96 hours. The surface freeze-dried silk fibroin coating becomes a foam-like material with a very uniform, interconnected fine pore structure. And (3) further crosslinking the silk fibroin foam coating: steam fumigation of water, formaldehyde, ethanol, diphenylethanol, etc. or direct soaking in alcohol solvent is adopted to induce beta-sheet formation. Table 1 shows the relationship between the concentration of silk fibroin solution and the thickness of the coating layer.

TABLE 1

Serial number	Concentration of silk fibroin solution	Single coat thickness	Number of coats up to 1mm in thickness
				1	0.1％w/v	1nm	100
2	1％w/v	75nm	60
				3	3％w/v	150nm	55
4	6％w/v	500nm	45
				5	10％w/v	750nm	30
6	15％w/v	0.001mm	10
				7	20％w/v	0.05mm	4
8	25％w/v	0.1mm	2
				9	30％w/v	1mm	1

4. Sterilization

And (3) carrying out ethylene oxide fumigation, high-temperature high-pressure sterilization and gamma-ray sterilization on the prepared cardiovascular stent with the silk fibroin dipping coating. Table 2 shows the variation of rigidity and toughness after sterilization of coatings coated with different concentrations of silk fibroin solutions.

TABLE 2

As can be seen from table 2, the rigidity and toughness of the coatings coated with the silk fibroin solutions of different concentrations after the sterilization treatment varied by less than 15%.

5. After-loading medicine

And (3) dissolving glutamine in triple distilled water to obtain a glutamine solution, wherein the concentration of the glutamine solution is 0.2 mol/mL. Subsequently, the above scaffold coated with the silk fibroin coating was immersed in a glutamine solution at room temperature for 30 minutes. Then the mixture is directly encapsulated under aseptic conditions, and can be directly used clinically after being encapsulated. The glutamine loading on the final scaffold was 1. mu. mol/mL. The loading capacity of the medicine can be adjusted to be 1-5000 ng/mm by adjusting the concentration of the medicine mother liquor.

Vascular Endothelial Growth Factor (VEGF) was dissolved in water to give an aqueous VEGF solution at a concentration of 2000 pg/ml. Subsequently, the above scaffold coated with the silk fibroin coating was immersed in the mixed solution at room temperature for 30 minutes. Then the mixture is directly encapsulated under aseptic conditions, and can be directly used clinically after being encapsulated. The loading of endothelial growth factor on the final scaffold was 200 ng/mm. The loading capacity of the medicine can be adjusted to be 1-5000 ng/mm by adjusting the concentration of the medicine mother liquor.

Fig. 1-2 are activity change diagrams of the sterilization process of drugs and growth factors when the mode of carrying drugs by layer-by-layer self-assembly method (whole sterilization), layer-by-layer self-assembly method (sterilization after drug loading) and polymer stent coating (post drug loading) under aseptic conditions is adopted, respectively.

As can be seen from the figure, the sterilization process has a very large effect on the activity of the drug as well as the growth factors, which will also affect the clinical efficacy. In addition, the total drug activity of the drug-loaded coating layer by layer self-assembly method under aseptic condition is lower than that of the polymer stent coating layer in a drug-loaded mode, which shows that the polymer stent coating layer has protective effect on the loaded drug and does not influence the complete release of the drug.

Drug release test:

(1) drug release amount measurement operation and calculation steps:

preparing a leaching medium: 100g SDS (sodium dodecyl sulfate), 120g ethanol and 780g acetic acid buffer pH6.0 were weighed into a suitable closed container with a magnetic stir bar and stirred until all solids were completely dissolved, taking care to minimize foaming.

Preparing a test solution: 8mL of the extraction medium was pipetted into a brown vial, the holder was transferred to the extraction medium (the holder had to be completely immersed in the extraction medium), and the vial was capped with a Teflon-lined screw cap. The sample bottle was placed in a shaker at a shaker temperature of (37. + -. 0.5). degree.C.and 100 r/mim. The scaffolds were removed at the indicated time points (1 day, 7 days, 15 days, 30 days, 45 days, 60 days, 90 days and 180 days), the extract was filtered through a 0.45 μm filter membrane and the filtrate was directly analyzed by injection.

And (3) measurement operation: samples are taken, and the solution to be detected is prepared according to the preparation method of the test sample solution. The holder was removed from the vial by a hook (or equivalent) made of 0.4064mm stainless steel wire. When the rack is positioned on the hook, in order to prevent cross contamination at each time point, a conical lint-free tip part is used to only touch the bottom of the tail end of the rack, the residual liquid in the rack is wiped off, then the rack is moved to the next glass bottle containing eluent, the glass bottle taking out the rack is covered, the mixture is uniformly mixed, and the elution test is continued to the next time point. Sampling 80 mu L of sample, measuring peak area, calculating accumulated content, and recording chromatogram. And calculating by peak area according to an external standard method to obtain a measurement result of the sustained release amount of the sample.

(2) Measuring and calculating the release amount of the growth factor:

the scaffolds were placed in phosphate buffered saline (PBS, pH7.4) and then placed on a shaker at 80rpm at room temperature (20 ℃ to 25 ℃). After a certain time of slow release (1 day, 7 days, 15 days, 30 days, 45 days, 60 days, 90 days and 180 days), 10000rcf centrifugates for 5 minutes, takes the supernatant for storage, and adds an equivalent amount of fresh PBS buffer (pH is 7.4) to continue slow release; multiple supernatant samples were taken sequentially in this manner during the sustained release period. The concentration of growth factor in the supernatant samples was determined using an ELISA kit and the cumulative amount released of growth factor was calculated. Each group tested 3 samples and the results averaged.

Fig. 3 is a drug release curve of the mode of layer-by-layer self-assembly drug-loaded coating (whole sterilization), layer-by-layer self-assembly coating post-drug-loaded sterilization (post-drug-loaded sterilization) and polymer stent coating post-drug-loaded under aseptic conditions. As can be seen from the figure, the drug carried by the silk fibroin foam coating of example 1 has the slowest release speed, the best slow release effect and the more effective slow release effect.

Similar results and conclusions as in fig. 1-3 can be obtained when the stent body is a metal stent or a polymer stent.

Similar results and conclusions as shown in fig. 1-3 can be obtained when the polymer material is one or more of hyaluronic acid, collagen, alginate, chitosan, silk fibroin, gelatin, albumin, and derivatives thereof.

Example 2: silk fibroin freeze-dried stent coating

1. Preparation of Silk fibroin solution

A silk fibroin solution was prepared according to the method of example 1.

2. Impregnation

Soaking the stent body in a mixed solution of silk fibroin solution (0.1-30% wt/v) and PEG400 with the concentration of 5-20% wt/wt to obtain the water-soluble primary silk fibroin coating.

3. Gel-forming

And (3) placing the stent coated with the silk fibroin mixed solution in a container with the temperature of 37 ℃ and the humidity of more than or equal to 60% until the silk fibroin becomes gel.

4. Washing with water

The PEG was removed by washing with water for 72 hours.

5. Freeze-drying

And (3) directly pre-freezing the stent body coated with the silk fibroin coating for 48 hours at the temperature of about-20 ℃, and freeze-drying for 48-96 hours. The surface freeze-dried silk fibroin coating becomes a foam-like material with a very uniform, interconnected fine pore structure.

6. Sterilization

And (3) carrying out ethylene oxide fumigation, high-temperature high-pressure sterilization and gamma-ray sterilization on the prepared cardiovascular stent with the silk fibroin dipping coating.

7. After-loading medicine

Curcumin was dissolved in PEG-400 and 0.1% propyl gallate was added as a stabilizer, the curcumin solution concentration was 37.5 mg/ml. Subsequently, the above scaffold coated with the silk fibroin coating was immersed in a curcumin solution at room temperature for 30 minutes. Then the mixture is directly encapsulated under aseptic conditions, and can be directly used clinically after being encapsulated. The drug loading of curcumin on the final scaffold was 1 μmol/mL.

Dissolving a proper amount of goat anti-pig CD34 antibody in 0.01mo 1/L PBS (pH7.4), adding 20 mmo/L SPDP solution in a ratio of 1:15, reacting in a refrigerator at 4 ℃ for 60min, adding the reaction mixture into a microcon YM 230 ultrafiltration tube, centrifuging and washing 3 times (7000 rpm, 10 min/time) to remove the residual SPDP, adding an excessive dithiothreitol acetic acid solution (DTT) (pH 4.5) into the obtained goat anti-pig CD34 antibody-PDP solution, reacting in a refrigerator at 4℃ for 30min, centrifuging and washing 3 times (the same method is adopted) by the micon YM ultrafiltration tube again to remove the residual dithiothreitol, and finally obtaining the goat anti-pig CD34 antibody-PDP-SH solution. Chemically coupling 125I-labeled anti-porcine CD34 antibody IgG to the scaffold coated with the silk fibroin coating by using an SPDP coupling agent, adding 20mmol/L SPDP solution into the polymer, reacting in a refrigerator at 4 ℃ for 60min, washing the reaction solution by 0.01mmol/LPBS (pH7.4) solution by adopting a floating method to obtain a polymer-PDP scaffold, immersing the polymer-PDP scaffold into goat anti-porcine CD34 antibody-PDP-SH solution, reacting in a refrigerator at 4 ℃ for 10h, and washing by using the floating method again to finally obtain the antibody-coated scaffold. Then the mixture is directly encapsulated under aseptic conditions, and can be directly used clinically after being encapsulated. The loading of endothelial growth factor on the final stent was (0.64325. + -. 0.1325) μ g.

The prepared stent was subjected to a drug release test in the same manner as in example 1 to obtain a drug release profile as shown in fig. 4. As can be seen from the figure, the silk fibroin lyophilized scaffold of example 2 has a good sustained release effect on the drug.

Example 3: silk fibroin freeze-dried stent coating

1. Preparation of Silk fibroin solution

A silk fibroin solution was prepared according to the method of example 1.

2. Impregnation

Adding cross-linking agents such as EDC-NHS, glutaraldehyde, HRP and H into about 1-30% of silk fibroin solution₂O₂One or more of genipin and the like are used as chemical cross-linking agents, mixed to obtain mixed solution, and the cardiovascular stent is soaked in the mixed solution to obtain the primary water-soluble silk fibroin cross-linked coating.

3. Gel-forming

And standing the scaffold body coated with the silk fibroin and the cross-linking agent until the scaffold body becomes gel.

4. Washing with water

Washing with water to remove unreacted chemical crosslinking agent.

5. Freeze-drying

Pre-freezing the fibroin protein chemical cross-linked cardiovascular scaffold at about-20 deg.C overnight, and freeze-drying for 48-72 hr. After removal from the lyophilizer, the freeze-dried silk fibroin chemically cross-linked coating becomes a foam-like material with a very consistent, interconnected fine pore structure.

6. Sterilization

And (3) carrying out ethylene oxide fumigation, high-temperature high-pressure sterilization and gamma-ray sterilization on the prepared cardiovascular stent with the silk fibroin dipping coating.

7. After-loading medicine

And (3) dissolving glutamine in triple distilled water to obtain a glutamine solution, wherein the concentration of the glutamine solution is 0.2 mol/mL. Subsequently, the above scaffold coated with the silk fibroin coating was immersed in a glutamine solution at room temperature for 30 minutes. Then the mixture is directly encapsulated under aseptic conditions, and can be directly used clinically after being encapsulated. The glutamine loading on the final scaffold was 1 umol/mL.

Dissolving a proper amount of goat anti-pig CD34 antibody in 0.01mo 1/L PBS (pH7.4), adding 20 mmo/L SPDP solution in a ratio of 1:15, reacting in a refrigerator at 4 ℃ for 60min, adding the reaction mixture into a microcon YM 230 ultrafiltration tube, centrifuging and washing 3 times (7000 rpm, 10 min/time) to remove the residual SPDP, adding an excessive dithiothreitol acetic acid solution (DTT) (pH 4.5) into the obtained goat anti-pig CD34 antibody-PDP solution, reacting in a refrigerator at 4℃ for 30min, centrifuging and washing 3 times (the same method is adopted) by the micon YM ultrafiltration tube again to remove the residual dithiothreitol, and finally obtaining the goat anti-pig CD34 antibody-PDP-SH solution. Chemically coupling 125I-labeled anti-porcine CD34 antibody IgG to the scaffold coated with the silk fibroin coating by using an SPDP coupling agent, adding 20mmol/L SPDP solution into the polymer, reacting in a refrigerator at 4 ℃ for 60min, washing the reaction solution by 0.01mmol/LPBS (pH7.4) solution by adopting a floating method to obtain a polymer-PDP scaffold, immersing the polymer-PDP scaffold into goat anti-porcine CD34 antibody-PDP-SH solution, reacting in a refrigerator at 4 ℃ for 10h, and washing by using the floating method again to finally obtain the antibody-coated scaffold. Then the mixture is directly encapsulated under aseptic conditions, and can be directly used clinically after being encapsulated. The loading of endothelial growth factor on the final stent was (0.64325. + -. 0.1325) μ g.

The prepared stent was subjected to a drug release test in the same manner as in example 1 to obtain a drug release profile as shown in fig. 5. As can be seen from the figure, the silk fibroin lyophilized scaffold of example 3 has good controlled-release effect and sustained-release effect on the drug.

Example 4: silk fibroin freeze-dried stent coating

1. Preparation of Silk fibroin solution

Degummed silk was prepared according to the method of example 1.

2. Impregnation

Dissolving degummed silk in 9.3M lithium bromide to obtain 1-30% of silk fibroin aqueous solution, and mixing the silk fibroin aqueous solution with diglycidyl ether (BDDE) 5: 1, adding a chemical cross-linking agent BDDE, and then soaking the cardiovascular stent in the mixed solution to obtain the primary water-soluble silk fibroin cross-linked coating. Wherein, the chemical crosslinking agent can also be one or more than two of diglycidyl ether BDDE, divinyl sulfone, 1,2,7, 8-diepoxyoctane, 1, 3-diepoxybutane and sodium trimetaphosphate.

3. Gel-forming

And standing the scaffold body coated with the silk fibroin and the cross-linking agent until the scaffold body becomes gel.

4. Washing with water

And washing with water to remove unreacted chemical crosslinking agent and lithium bromide.

5. Freeze-drying

Pre-freezing the fibroin protein chemical cross-linked cardiovascular scaffold at about-20 deg.C overnight, and freeze-drying for 48-72 hr. After removal from the lyophilizer, the freeze-dried silk fibroin chemically cross-linked coating becomes a foam-like material with a very consistent, interconnected fine pore structure.

6. Sterilization

And (3) carrying out ethylene oxide fumigation, high-temperature high-pressure sterilization and gamma-ray sterilization on the prepared cardiovascular stent with the silk fibroin dipping coating.

7. After-loading medicine

And (3) dissolving glutamine in triple distilled water to obtain a glutamine solution, wherein the concentration of the glutamine solution is 0.2 mol/mL. Subsequently, the above scaffold coated with the silk fibroin coating was immersed in a glutamine solution at room temperature for 30 minutes. Then the mixture is directly encapsulated under aseptic conditions, and can be directly used clinically after being encapsulated. The glutamine loading on the final scaffold was 1 umol/mL.

Vascular Endothelial Growth Factor (VEGF) was dissolved in water to give an aqueous VEGF solution at a concentration of 2000 pg/ml. Subsequently, the above scaffold coated with the silk fibroin coating was immersed in the mixed solution at room temperature for 30 minutes. Then the mixture is directly encapsulated under aseptic conditions, and can be directly used clinically after being encapsulated. The loading of endothelial growth factor on the final scaffold was 200 ng/mm.

The prepared stent was subjected to a drug release test in the same manner as in example 1 to obtain a drug release profile as shown in fig. 6. As can be seen from the figure, the silk fibroin lyophilized scaffold of example 4 has good controlled-release effect and sustained-release effect on the drug.

Example 5: silk fibroin freeze-dried stent coating

1. A silk fibroin solution was prepared, impregnated, lyophilized, and sterilized according to the method of example 1 or example 2.

2. After-loading medicine

Dissolving curcumin in PEG-400, adding 0.1% propyl gallate as a stabilizer to obtain a drug mother liquor, adding the drug mother liquor into the concentration of 1-10% silk fibroin, and adding 80% PEG400 with the same volume to the mixed liquor to obtain a coating mother liquor. The stent body is dipped in the coating mother liquor, taken out and stood at 37 ℃ until the gel is formed.

Dissolving a growth factor in a PBS (phosphate buffer solution) solution to obtain an active substance mother solution, adding the active substance mother solution into a 1-10% silk fibroin concentration solution, and adding 80% PEG400 with the same volume to the mixed solution to obtain a coating mother solution. The stent body is dipped in the coating mother liquor, taken out and stood at 37 ℃ until the gel is formed.

The stents prepared in examples 1-2, 5 were subjected to a drug release test in the same manner as in example 1 to obtain a drug release profile as shown in FIG. 7. As can be seen from the figure, compared with examples 1-2, the gel drug loading is adopted in the present example, and the release control effect and the release effect of the drug are better.

Example 6: hyaluronic acid stent coating

1. Impregnation

Dissolving hyaluronic acid in 9.3M lithium bromide to obtain 1-30% hyaluronic acid aqueous solution, wherein the weight ratio of the silk fibroin to the BDDE is 5: 1, and soaking the cardiovascular stent in the mixed solution to obtain the primary water-soluble hyaluronic acid cross-linked coating.

2. Gel-forming

And standing the stent body coated with the hyaluronic acid and the cross-linking agent until the stent body becomes gel.

3. Washing with water

And washing with water to remove unreacted chemical crosslinking agent and lithium bromide.

4. Freeze-drying

Pre-freezing hyaluronic acid chemically cross-linked cardiovascular stent at-20 deg.C overnight, and freeze-drying for 48-72 hr. After removal from the lyophilizer, the freeze-dried silk fibroin chemically cross-linked coating becomes a foam-like material with a very consistent, interconnected fine pore structure.

5. Sterilization

The prepared cardiovascular stent with the hyaluronic acid dipping coating is subjected to ethylene oxide fumigation sterilization, high-temperature high-pressure sterilization and gamma-ray sterilization.

6. After-loading medicine

Curcumin was dissolved in PEG-400 and 0.1% propyl gallate was added as a stabilizer, the curcumin solution concentration was 37.5 mg/ml. Subsequently, the above scaffold coated with the silk fibroin coating was immersed in a curcumin solution at room temperature for 30 minutes. Then the mixture is directly encapsulated under aseptic conditions, and can be directly used clinically after being encapsulated. The drug loading of curcumin on the final scaffold was 1 umol/mL.

Dissolving a proper amount of goat anti-pig CD34 antibody in 0.01mo 1/L PBS (pH7.4), adding 20 mmo/L SPDP solution in a ratio of 1:15, reacting in a refrigerator at 4 ℃ for 60min, adding the reaction mixture into a microcon YM 230 ultrafiltration tube, centrifuging and washing 3 times (7000 rpm, 10 min/time) to remove the residual SPDP, adding an excessive dithiothreitol acetic acid solution (DTT) (pH 4.5) into the obtained goat anti-pig CD34 antibody-PDP solution, reacting in a refrigerator at 4℃ for 30min, centrifuging and washing 3 times (the same method is adopted) by the micon YM ultrafiltration tube again to remove the residual dithiothreitol, and finally obtaining the goat anti-pig CD34 antibody-PDP-SH solution. Chemically coupling 125I-labeled anti-porcine CD34 antibody IgG to the scaffold coated with the silk fibroin coating by using an SPDP coupling agent, adding 20mmol/L SPDP solution into the polymer, reacting in a refrigerator at 4 ℃ for 60min, washing the reaction solution by 0.01mmol/LPBS (pH7.4) solution by adopting a floating method to obtain a polymer-PDP scaffold, immersing the polymer-PDP scaffold into goat anti-porcine CD34 antibody-PDP-SH solution, reacting in a refrigerator at 4 ℃ for 10h, and washing by using the floating method again to finally obtain the antibody-coated scaffold. Then the mixture is directly encapsulated under aseptic conditions, and can be directly used clinically after being encapsulated. The loading of endothelial growth factor on the final stent was (0.64325. + -. 0.1325) μ g.

The stent prepared in example 6 was subjected to a drug release test in the same manner as in example 1, to obtain a drug release profile as shown in fig. 8. As can be seen from the figure, the hyaluronic acid stent of the embodiment has good release control effect and release effect on the drug.

Similar results and conclusions can be obtained when one or more of collagen, alginate, chitosan, gelatin, albumin, and their derivatives are used as coating materials.

Example 7: silk fibroin/hyaluronic acid freeze-dried stent coating

1. Preparation of Silk fibroin solution

Degummed silk was prepared according to the method of example 1.

2. Impregnation

Dissolving degummed silk and hyaluronic acid in 9.3M lithium bromide to obtain 1% -30% of silk fibroin and hyaluronic acid aqueous solution, wherein the ratio of silk fibroin, hyaluronic acid and BDDE is 5: 1, and soaking the cardiovascular stent in the mixed solution to obtain the primary water-soluble silk fibroin and hyaluronic acid cross-linked coating.

3. Gel-forming

And standing the scaffold body coated with the silk fibroin, the hyaluronic acid and the cross-linking agent until the scaffold body becomes gel.

4. Washing with water

And washing with water to remove unreacted chemical crosslinking agent and lithium bromide.

5. Freeze-drying

Pre-freezing the fibroin protein chemical cross-linked cardiovascular scaffold at about-20 deg.C overnight, and freeze-drying for 48-72 hr. After removal from the lyophilizer, the freeze-dried silk fibroin and hyaluronic acid chemically cross-linked coating becomes a foam-like material with a very consistent, interconnected fine pore structure.

6. Sterilization

The prepared cardiovascular stent with the silk fibroin and hyaluronic acid dipping coating is subjected to ethylene oxide fumigation sterilization, high-temperature high-pressure sterilization and gamma-ray sterilization.

7. After-loading medicine

And (3) dissolving glutamine in triple distilled water to obtain a glutamine solution, wherein the concentration of the glutamine solution is 0.2 mol/mL. Subsequently, the above scaffold coated with the silk fibroin coating was immersed in a glutamine solution at room temperature for 30 minutes. Then the mixture is directly encapsulated under aseptic conditions, and can be directly used clinically after being encapsulated. The glutamine loading on the final scaffold was 1 umol/mL.

Vascular Endothelial Growth Factor (VEGF) was dissolved in water to give an aqueous VEGF solution at a concentration of 2000 pg/ml. Subsequently, the above scaffold coated with the silk fibroin coating was immersed in the mixed solution at room temperature for 30 minutes. Then the mixture is directly encapsulated under aseptic conditions, and can be directly used clinically after being encapsulated. The loading of endothelial growth factor on the final scaffold was 200 ng/mm.

The stent prepared in example 7 was subjected to a drug release test in the same manner as in example 1, to obtain a drug release profile as shown in fig. 9. As can be seen from the figure, the silk fibroin/hyaluronic acid lyophilized scaffold of this example has good release control effect and sustained release effect on the drug.

Similar results and conclusions can be obtained when various combinations of one or more of hyaluronic acid, collagen, alginate, chitosan, silk fibroin, gelatin, albumin, and derivatives thereof are used as coating materials.

Animal experiments

Experiments are carried out according to relevant regulations, and 12 healthy hybrid dogs with the weight of 15-20 Kg are selected. A blood vessel stent coating of silk fibroin coating. About 8cm long (according to the test standard of the national food and drug administration YY0500-2004, IS 07198: the length of the implanted blood vessel IS equal to the diameter of the blood vessel multiplied by 10), and the blood vessel IS directly implanted into the abdominal aorta of the dog without autologous blood pre-coagulation operation.

The labels of the samples are shown in table 3, wherein label 1 is the silk fibroin lyophilized scaffold prepared in example 1, the concentration of the silk fibroin solution used is 1% w/v, label 2 is the silk fibroin lyophilized scaffold prepared in example 2, the concentration of the silk fibroin solution used is 1% w/v, and label 16 is an uncoated control group, and autologous blood is used for pre-coagulation. The meaning of the other reference numerals is analogized.

TABLE 3

Reference numerals	1	2	3	4	5	6	7
								1％w/v	Example 1	Example 2	Example 3	Example 4	Example 5	Example 6	Example 7
Reference numerals	8	9	10	11	12	13	14
								3％w/v	Example 1	Example 2	Example 3	Example 4	Example 5	Example 6	Example 7
Reference numerals	15
								6％w/v	Example 5
Reference numerals	16
								Uncoated	Control group

The sustained release effect of the drug was tested for each experimental group on days 1, 7, 15 and 30 of implantation, respectively, and the results are shown in table 4.

TABLE 4

The results show that the artificial blood vessel operation of the silk fibroin chemical crosslinking coating has no obvious bleeding and only a small amount of inflammatory reaction, and the effective release time of the drug loaded on the coating and the Vascular Endothelial Growth Factor (VEGF) is measured to be more than 7 days.

The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitution or change made by the technical personnel in the technical field on the basis of the invention is all within the protection scope of the invention. The protection scope of the invention is subject to the claims.