WO2019042261A1 - 复合肝素抗凝涂层液、涂层用微球及制备方法与应用 - Google Patents

复合肝素抗凝涂层液、涂层用微球及制备方法与应用 Download PDF

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WO2019042261A1
WO2019042261A1 PCT/CN2018/102568 CN2018102568W WO2019042261A1 WO 2019042261 A1 WO2019042261 A1 WO 2019042261A1 CN 2018102568 W CN2018102568 W CN 2018102568W WO 2019042261 A1 WO2019042261 A1 WO 2019042261A1
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Prior art keywords
heparin
curcumin
composite
coating liquid
coating
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PCT/CN2018/102568
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English (en)
French (fr)
Inventor
严拓
欧阳晨曦
刘雅文
刘思诗
刘园
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武汉杨森生物技术有限公司
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Priority claimed from CN201710748805.8A external-priority patent/CN107519544A/zh
Priority claimed from CN201710748820.2A external-priority patent/CN107376037A/zh
Application filed by 武汉杨森生物技术有限公司 filed Critical 武汉杨森生物技术有限公司
Publication of WO2019042261A1 publication Critical patent/WO2019042261A1/zh
Priority to US16/691,688 priority Critical patent/US11083824B2/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L33/00—Antithrombogenic treatment of surgical articles, e.g. sutures, catheters, prostheses, or of articles for the manipulation or conditioning of blood; Materials for such treatment
    • A61L33/0005—Use of materials characterised by their function or physical properties
    • A61L33/0011—Anticoagulant, e.g. heparin, platelet aggregation inhibitor, fibrinolytic agent, other than enzymes, attached to the substrate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L33/00—Antithrombogenic treatment of surgical articles, e.g. sutures, catheters, prostheses, or of articles for the manipulation or conditioning of blood; Materials for such treatment
    • A61L33/04—Use of organic materials, e.g. acetylsalicylic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L33/00—Antithrombogenic treatment of surgical articles, e.g. sutures, catheters, prostheses, or of articles for the manipulation or conditioning of blood; Materials for such treatment
    • A61L33/06—Use of macromolecular materials
    • A61L33/068—Use of macromolecular materials obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L33/00—Antithrombogenic treatment of surgical articles, e.g. sutures, catheters, prostheses, or of articles for the manipulation or conditioning of blood; Materials for such treatment
    • A61L33/06—Use of macromolecular materials
    • A61L33/12—Polypeptides, proteins or derivatives thereof, e.g. degradation products thereof
    • A61L33/128—Other specific proteins or polypeptides not covered by A61L33/122 - A61L33/126
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
    • A61L2300/42—Anti-thrombotic agents, anticoagulants, anti-platelet agents

Definitions

  • the invention relates to a composite heparin anti-coagulation coating liquid, a microsphere for coating, a preparation method and application thereof, and belongs to the technical field of biomedical materials.
  • cardiovascular diseases such as atherosclerosis, vascular embolism and aneurysms pose great threats to human health.
  • blood vessels of the human body cannot supply blood normally due to the above diseases and the like, artificial blood vessels are required for surgical replacement surgery.
  • the diameter of the blood vessels in the human body ranges from 2mm to 30mm.
  • small caliber diameter ⁇ 6mm
  • intimal hyperplasia and thrombosis which leads to low patency rate of small-caliber vessels.
  • most of the artificial blood vessel materials used in hospital operations are ePTFE, PU and other materials, but these materials do not have good anticoagulant properties themselves, and anticoagulant coatings are required on the surface of the materials.
  • the commonly used anticoagulant coatings have heparin coatings, and the coating methods vary, and the properties of the heparin coating materials prepared by different methods are also different.
  • the object of the present invention is to provide a composite heparin anticoagulant coating liquid, a microsphere for coating, a preparation method and application thereof.
  • the purpose comprises two parts, the first part of the composite heparin anticoagulant coating liquid and the preparation method and application thereof, the second part of the composite heparin anti-coagulation coating microsphere, preparation method and application thereof
  • the composite heparin anticoagulant coating liquid provided by the present invention contains a bi-amino curcumin-heparin in a crosslinked state.
  • the active part of the composite heparin anticoagulant coating liquid of the present invention is a crosslinked state of bi-aminocurcumin-heparin.
  • the diamino curcumin is crosslinked with heparin via an amino group thereon.
  • the invention also provides a preparation method of the above composite heparin anticoagulant coating liquid, the steps are as follows:
  • the weight ratio of the combined amino curcumin and heparin is from 1 to 10:40.
  • the ratio is a preferred reaction ratio of the combination of curcumin and heparin.
  • the active ingredient in the present invention is a bi-amino curcumin-heparin in a crosslinked state, so that any ratio of the addition of the bi-aminocurcumin and the heparin can be produced. It is all usable.
  • the above coating liquid is not effective when used alone as a coating liquid, and it needs to function in combination with a biologically active protein.
  • a biologically active protein such as collagen, silk fibroin, and fibronectin.
  • the inventors found that in a certain ratio, the combination of diamino curcumin-heparin and silk fibroin has a good effect.
  • the present invention also provides a bio-anticoagulating coating liquid of diamino curcumin-heparin and silk fibroin, wherein the bio-anticoagulating coating liquid contains a bi-amino curcumin-heparin in a crosslinked state, Included in the biotin anticoagulant coating liquid, wherein the bi-aminocurcumin-heparin is at a concentration of 0.5 to 5% by weight, the silk fibroin concentration is 5 to 15% by weight, and the bi-amino turmeric The concentration ratio of the hormone-heparin to the silk fibroin is 1:1 to 10.
  • the microspheres for the composite heparin anticoagulant coating provided by the present invention are microspheres in which the linked amino curcumin-heparin is a content of PLA-PEG-PLA as a carrier.
  • the diamino curcumin is crosslinked with heparin via an amino group thereon.
  • the invention also provides a preparation method of the above composite heparin anticoagulant coating microsphere, characterized in that the steps are as follows:
  • the composite heparin anticoagulant coating liquid is prepared into a lyophilized powder and then emulsified together with PLA-PEG-PLA to form microspheres.
  • the weight ratio of the diamino curcumin and the heparin is from 1 to 10:40.
  • the ratio is a preferred reaction ratio of the combination of curcumin and heparin.
  • the active ingredient in the present invention is a bi-amino curcumin-heparin in a crosslinked state, so that any ratio of the addition of the bi-aminocurcumin and the heparin can be produced. It is all usable.
  • the above-mentioned composite heparin anticoagulant coating microspheres are not effective when used alone as a coating liquid, and it is required to function in combination with biologically active proteins.
  • biologically active proteins such as collagen, silk fibroin, and fibronectin.
  • the inventors found that in a certain ratio, the combination of diamino curcumin-heparin and silk fibroin has a good effect.
  • the present invention also provides a silk fibroin composite heparin microsphere anticoagulant microsphere coating liquid, the coating liquid comprising the above composite heparin anticoagulant coating microsphere, and a silk wrapped around the microsphere
  • concentration of the silk fibroin is 5 to 15% by weight, and the ratio of the concentration of the microspheres to the silk fibroin of the composite heparin anticoagulant coating is 1:1 to 10.
  • the invention utilizes the anti-tissue proliferative and anti-inflammatory reaction properties of curcumin, and combines it with heparin (using the PLA-PEG-PLA drug-loaded sustained-release microsphere property to further improve the biological anticoagulant stability),
  • implanted medical devices such as artificial blood vessels
  • tissue hyperplasia and inflammatory reactions can also cause rejection
  • the blood vessels are narrowed, and eventually the biocompatibility of the implanted device such as the artificial blood vessel is deteriorated and the implantation fails.
  • the beneficial effects of the invention modern pharmacological studies show that curcumin components have various pharmacological effects such as anticoagulation, anti-tissue proliferation, anti-inflammatory reaction, etc.
  • the invention combines curcumin and heparin to enhance heparin coating.
  • Anticoagulant function using PLA-PEG-PLA drug-loaded sustained-release microspheres to further improve stability, and play a role in anti-tissue proliferation and anti-inflammatory reaction that can not be achieved by coatings such as heparin or protein.
  • Implanted devices such as artificial blood vessels, vascular stents, and blood vessel patches reduce thrombosis in the human body, reduce postoperative complications, and improve product lifespan.
  • the artificial blood vessel (ePTFE) used in the following examples is pre-treated before coating to improve the adhesion to the coating, so that the bio-coating can stably and effectively exert anticoagulant functions. .
  • IPA isopropyl alcohol
  • PEI polyethyleneimine
  • the treated artificial blood vessel is immersed in a 0.05% glutaraldehyde solution for crosslinking, and after the reaction is completed, the artificial blood vessel is again immersed in a 0.5% PEI solution, and the constructed artificial blood vessel is soaked and rinsed with deionized water. . After the rinsing is completed, the artificial blood vessel is immersed in a sodium cyanoborohydride solution, and the reaction is completed, and then rinsed with deionized water.
  • the third layer of the treated artificial blood vessel was immersed in a 0.3% PEI solution for 0.5 to 1 hour, and the artificial blood vessel was soaked and washed with 50 g/L of sodium chloride solution, and then rinsed with deionized water to obtain an artificial blood vessel (ePTFE) base. material.
  • ePTFE artificial blood vessel
  • a composite heparin anticoagulant coating liquid comprising a crosslinked state of bi-aminocurcumin-heparin.
  • the preparation method of the composite heparin anticoagulant coating liquid has the following steps:
  • a method for preparing a bio-anticoagulation coated artificial blood vessel mixed with diamino curcumin-heparin and silk fibroin is as follows:
  • Example 1 1) The lyophilized powder prepared in Example 1 is dissolved in a silk fibroin macromolecular solution, and the obtained mixed coating solution has a diamino curcumin-heparin concentration of 1% by weight and a silk fibroin concentration of 10% by weight.
  • step 2) Immerse the artificial blood vessel (ePTFE) substrate in the bio-an anti-coagulant coating liquid of step 1) for 10 to 30 minutes, then take it out in a ventilated place, and after immersing it completely, soak it in 30-75% methanol solution 2 ⁇ 4h, after soaking, place it in a ventilated place to dry.
  • ePTFE artificial blood vessel
  • step 2) twice to apply a second and third coating to the artificial blood vessel (ePTFE).
  • the silk fibroin preparation method of the step 1) 20 to 30 g of raw silk is placed in 10 to 12 L of a solution containing Na 2 CO 3 (2.12 g / L) in boiling water for 20 to 30 mins, and then repeatedly rinsed with deionized water. The resulting degummed silk is used to remove residual sericin.
  • the washed silk fibroin fibers are spread in a ventilated place to be dried. Weigh 15 ⁇ 25g of dried silk fiber dissolved in LiBr solution (9.3M) at 100°C to 60°C. After the dissolution is completed, the silk protein solution is sneaked for 36-72h with dialysis bag. After the dialysis is completed, the solution is centrifuged. Impurity, a silk fibroin macromolecular solution is obtained.
  • This example is basically the same as Example 2 except that the concentration of the bi-aminocurcumin-heparin in the bio-anticoagulant coating liquid of the bi-amino curcumin-heparin and silk fibroin is 0.7% by weight, and the silk fibroin concentration is 5.6%. Wt.
  • This example is basically the same as Example 2 except that the concentration of the bi-aminocurcumin-heparin in the bio-an anticoagulant coating liquid of the bi-amino curcumin-heparin and silk fibroin is 5% wt, and the silk fibroin concentration is 15%. Wt.
  • Heparin/poly-L-lysine coated artificial blood vessel the preparation steps of the method are as follows:
  • the heparin/poly-L-lysine lyophilized powder 0.9g was dissolved in 200mL of deionized water to prepare a heparin macromolecular solution, and the artificial blood vessel treated by the three treatments was soaked in the heparin macromolecular solution prepared above. After reacting at °C for 10-20 min, 572 uL of 2.5% sodium cyanoborohydride was added to continue the reaction for 2 to 3 h. After all the reactions are completed, the artificial blood vessels are rinsed off with deionized water and borate buffer, and lyophilized.
  • Silk fibroin heparin coated artificial blood vessel the preparation steps are as follows:
  • the silk fibroin macromolecule solution was blended with heparin/poly-L-lysine to obtain a silk fibroin concentration of 10% solution and a heparin/poly-L-lysine concentration of 4%.
  • the first layer the artificial blood vessel (ePTFE) substrate is immersed in the above mixture for 10 to 30 mins, and then taken out and placed in a ventilated place. After being completely dried, immersed in 30 to 75% methanol solution for 2 to 4 hours. After the immersion is completed. Place in a ventilated place to dry.
  • ePTFE artificial blood vessel
  • Second/third layer Repeat the first layer operation.
  • the method for preparing a fibronectin heparin coated artificial blood vessel is as follows:
  • Adhesin is blended with heparin/poly-L-lysine solution, the concentration of laminin in the mixture is 10-30%wt, and the concentration of heparin/poly-L-lysine is 0.5-5%wt, 37 °C reaction 1 ⁇ 2h, then an EDC/NHS/MES crosslinker with a molar ratio of 1M:2M:2M was added, and the volume ratio of heparin/PLL to crosslinker was 1:2-15.
  • the artificial blood vessel (ePTFE) substrate was immersed in the mixed solution and reacted at 37 ° C for 1 to 5 hours.
  • the method for preparing a collagen coated artificial blood vessel is as follows:
  • the collagen is placed in a concentration of 10 to 30% by weight with glacial acetic acid, and the heparin lyophilized powder is added to 200 mL of the collagen mixture to make the concentration of heparin 0.5 to 5% by weight.
  • the artificial blood vessel ePTFE
  • the substrate was infiltrated in the mixed solution, fully wetted, and lyophilized for 24 to 48 hours. After the freeze-drying is completed, the collagen is cross-linked by ultraviolet irradiation.
  • the surface collagen coating polymer material can be obtained by repeatedly infiltrating and lyophilizing three times.
  • the preparation method of polydopamine coated artificial blood vessel is as follows:
  • the artificial blood vessel (ePTFE) substrate was immersed in a concentration of 8 mg/mL dopamine solution for 12 h at room temperature, then ultrasonically washed in deionized water, and then immersed in the dopamine solution again. After 4 times of reaction, the artificial blood vessel was placed at 100. Heat treatment at °C for 1 h. After the treatment is completed, the artificial blood vessel is washed with deionized water and dried at normal temperature.
  • the cytotoxicity of the coated artificial blood vessel was detected by the extraction method.
  • the specific experimental steps are as follows:
  • the coated artificial blood vessel or coating material prepared in the comparative example and the examples 2 to 9 was extracted with a high glucose cell culture solution containing fetal bovine serum at 0.2 g/mL for 24 to 72 hours.
  • MTT reaction 10 uL of 5 mg/mL MTT solution was added to each well, and incubation was continued for 4 hours in a culture tank.
  • the 96-well plate was placed on a microplate reader to measure the absorbance at 570 nm.
  • Partial thromboplastin time (APTT) experiment The coated artificial blood vessels prepared in Examples 2 to 9 were taken, and 100 to 200 ⁇ L of platelet-poor plasma was added thereto at 37 ° C for 0.5 h in a water bath. After the heating is completed, 100 ⁇ L of a 100- ⁇ L partial thromboplastin solution and a calcium chloride solution are added, and after mixing uniformly, the APTT is detected by an automatic coagulation apparatus.
  • APTT Partial thromboplastin time
  • Prothrombin time (PT) experiment 100-200 ⁇ L of platelet-poor plasma was added to the artificial blood vessel sample and a water bath at 37 ° C for 0.5 h. After the heating is completed, the PT reagent is added thereto, and the PT is detected by an automatic coagulation instrument.
  • TT Thrombin time experiment: 100-200 ⁇ L of platelet-poor plasma was added to the artificial blood vessel sample and a water bath at 37 ° C for 0.5 h. After the completion of the heating, the TT reagent was added thereto, and the TT was detected by an automatic coagulation apparatus.
  • the uncoated artificial blood vessel clotting time is similar to the normal plasma clotting time, and the artificial blood vessel clotting time coated by the curcumin-heparin silk fibroin blending solution (Example 2) ⁇ 4)
  • the APTT time of the substrate artificial blood vessels is extended by about 3 to 16 s
  • the PT time is extended by about 4 to 7 s
  • the TT time is prolonged.
  • the above test shows that the curcumin-heparin-coated artificial blood vessel coating has more obvious anticoagulant properties than other uncoated artificial blood vessels, and anticoagulation for other artificial blood vessels using heparin and macromolecular protein coating.
  • the performance has also improved, indicating that curcumin and heparin have been used to enhance anticoagulant effect, and the coating effect is better.
  • the invention relates to a microsphere for compound heparin anticoagulant coating, which is a PLA-PEG-PLA heparin microsphere, which is a microsphere of a content of a cross-linked diamino curcumin-heparin which is a carrier of PLA-PEG-PLA.
  • the composite heparin anticoagulant coating lyophilized powder was prepared by the method of Example 1 using the method for preparing the composite heparin anticoagulant coating microspheres.
  • Example 11 Silk fibroin composite heparin microsphere anticoagulant coating
  • the method for preparing the silk fibroin compound heparin microsphere anticoagulant coating artificial blood vessel is as follows:
  • Example 2 The compound heparin anticoagulating coating prepared in Example 10 is dissolved in the silk fibroin macromolecular solution, and the mixed heparin anticoagulating coating has a microsphere concentration of 1% by weight.
  • the protein concentration was 10%wt, which was a silk fibroin complex heparin microsphere anticoagulant coating solution.
  • the coating solution was ultrasonically shaken for 30-60 s to change the silk fibroin structure.
  • step 3) Repeat step 3) twice to apply a second and third coating to the artificial blood vessel (ePTFE).
  • ePTFE artificial blood vessel
  • Example 12 Silk fibroin composite heparin microsphere anticoagulant coating
  • This embodiment is basically the same as the embodiment 11, except that the concentration of the microspheres of the composite heparin anticoagulant coating in the silk fibroin composite heparin microsphere anticoagulant coating liquid is 0.7% by weight, and the silk fibroin concentration is 5.6% by weight.
  • Example 13 Silk fibroin composite heparin microsphere anticoagulant coating
  • This embodiment is basically the same as the embodiment 11, except that the concentration of the composite heparin anticoagulant coating in the silk fibroin composite heparin microsphere anticoagulant coating liquid is 5% wt, and the silk fibroin concentration is 15% wt.
  • the cytotoxicity of the coated artificial blood vessel was detected by the extracting liquid method, and the experimental subjects were the comparative examples and the examples 11 to 13, 5 to 9.
  • the specific experimental procedure was the same as that of the experimental example 1.
  • the clotting time test subjects were the control examples and Examples 11 to 13, 5 to 9, and the procedure was the same as Experimental Example 2.
  • the clotting time of the uncoated artificial blood vessel was similar to that of the normal plasma, and the artificial blood vessel clotting time coated with the curcumin-heparin silk fibroin blending solution (Example 11) ⁇ 13)
  • the APTT time of the substrate artificial blood vessels is extended by about 11-18s compared with other different coatings
  • the PT time is extended by about 5-9s
  • the TT time is prolonged.
  • the above test shows that the curcumin-heparin-coated artificial blood vessel coating has more obvious anticoagulant properties than other uncoated artificial blood vessels, and anticoagulation for other artificial blood vessels using heparin and macromolecular protein coating.
  • the performance has also improved, indicating that curcumin and heparin have been used to enhance anticoagulant effect, and the coating effect is better.
  • the ePTFE artificial blood vessel is used as the substrate, and in fact, other materials such as PU, PE, polyester, polylactic acid, polysilicone, polyglycolic acid, silicone rubber, etc., may also be used in the coating liquid of the present invention.
  • other materials such as PU, PE, polyester, polylactic acid, polysilicone, polyglycolic acid, silicone rubber, etc.
  • PU polyethylene glycol
  • PE polyethylene glycolity
  • polyester polylactic acid
  • polysilicone polyglycolic acid
  • silicone rubber etc.

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Abstract

本发明公开了一种复合肝素抗凝涂层液、涂层用微球及制备方法与应用。本发明将姜黄素与肝素两者连用不仅可以增强肝素涂层的抗凝血功能，利用PLA-PEG-PLA载药缓释微球特性，进一步提升稳定性，起到了单纯用肝素或蛋白等涂层所无法达到的抗组织增生、抗炎性反应等功能，对于人造血管、血管支架、血管补片等植入器械在人体内减少血栓形成，降低术后并发症，提高产品的使用寿命至关重要。

Description

复合肝素抗凝涂层液、涂层用微球及制备方法与应用 技术领域
本发明涉及一种复合肝素抗凝涂层液、涂层用微球及制备方法与应用，属于生物医用材料技术领域。
背景技术
随着人们生活水平的提高，动脉粥样硬化，血管栓塞以及动脉瘤等心血管疾病对人类的健康造成了极大的威胁。因上述疾病等原因导致人体自身血管无法正常供血时，则需采用人工血管进行外科更换手术。人体内血管口径由2mm至30mm不等，在这其中，小口径(直径<6mm)极易发生内膜增生以及血栓形成，进而导致小口径血管远期通畅率低。目前，医院手术中使用的人造血管材料多为ePTFE，PU等材料，但这些材料本身不具备良好的抗凝血性能，需在材料表面进行抗凝血涂层。
现有常用的抗凝血涂层有肝素涂层，其涂层方法各异，不同的方法所制备的肝素涂层材料性能也有所差异。
发明内容
本发明的目的在于提供一种复合肝素抗凝涂层液、涂层用微球及制备方法与应用。该目的包括两部分，第一部分复合肝素抗凝涂层液及制备方法与应用，第二部分复合肝素抗凝涂层用微球及制备方法与应用
为实现上述第一部分，本发明所提供的复合肝素抗凝涂层液中包含有交联状态的联氨基姜黄素-肝素。
本发明的复合肝素抗凝涂层液中起效的部分为交联状态的联氨基姜黄素-肝素。所述联氨基姜黄素通过其上的氨基与肝素交联。
本发明还提供了一种上述复合肝素抗凝涂层液的制备方法，步骤如下：
将联氨基姜黄素溶解于乙醇溶液中，配置成姜黄素醇溶液，向 姜黄素醇溶液中加入MES缓冲液，再加入肝素，然后再加入EDC和NHS交联剂，搅拌1～2h，使联氨基姜黄素中的氨基与肝素充分反应，将反应完成的溶液放入透析袋透析24～72h，透析完成后，得到复合肝素抗凝涂层液。
上述方案中，所述联氨基姜黄素和肝素的添加重量比为1～10:40。该比例是联安姜黄素和肝素的较佳反应比值，本发明中的有效成分为交联状态的联氨基姜黄素-肝素，所以只要能够生成它的联氨基姜黄素和肝素任意配比添加量都是可以使用的。
需要说明的是，上述涂层液在单独作为涂层液使用时效果并不理想，它需要和生物活性蛋白联用发挥功效。如胶原蛋白、丝素蛋白、粘连蛋白。在研究过程中，发明人发现在一定的配比下，联氨基姜黄素-肝素和丝素蛋白联用有较好的效果。
因此，本发明还提供了一种联氨基姜黄素-肝素及丝素蛋白的生物抗凝涂层液，所述生物抗凝涂层液中包含有交联状态的联氨基姜黄素-肝素，还包括丝素蛋白；所述生物抗凝涂层液中所述联氨基姜黄素-肝素在的浓度为0.5～5％wt，所述丝素蛋白的浓度为5～15％wt，且联氨基姜黄素-肝素与丝素蛋白的浓度比值为1:1～10。
为实现上述第二部分，本发明所提供的复合肝素抗凝涂层用微球，是以PLA-PEG-PLA为载体，交联状态的联氨基姜黄素-肝素为内容物的微球。
所述联氨基姜黄素通过其上的氨基与肝素交联。
本发明还提供了一种上述复合肝素抗凝涂层用微球的制备方法，其特征在于，步骤如下：
1)将联氨基姜黄素溶解于乙醇溶液中，配置成姜黄素醇溶液，向姜黄素醇溶液中加入MES缓冲液，再加入肝素，然后再加入EDC和NHS交联剂，搅拌1～2h，使联氨基姜黄素中的氨基与肝素充分反应，将反应完成的溶液放入透析袋透析24～72h，透析完成后，得到复合肝素抗凝涂层液；
2)以PLA-PEG-PLA为材料，将复合肝素抗凝涂层液制备成冻干粉之后与PLA-PEG-PLA一并乳化制成的微球。
上述制备方法中，所述联氨基姜黄素和肝素的添加重量比为1～10:40。该比例是联安姜黄素和肝素的较佳反应比值，本发明中的有效成分为交联状态的联氨基姜黄素-肝素，所以只要能够生成它的联氨基姜黄素和肝素任意配比添加量都是可以使用的。
需要说明的是，上述复合肝素抗凝涂层用微球在单独作为涂层液使用时效果并不理想，它需要和生物活性蛋白联用发挥功效。如胶原蛋白、丝素蛋白、粘连蛋白。在研究过程中，发明人发现在一定的配比下，联氨基姜黄素-肝素和丝素蛋白联用有较好的效果。
因此，本发明还提供了一种丝素蛋白复合肝素微球抗凝微球涂层液，所述涂层液中包含有上述复合肝素抗凝涂层用微球，以及包裹住微球的丝素蛋白；所述丝素蛋白的浓度为5～15％wt，所述复合肝素抗凝涂层用微球与丝素蛋白的浓度比值为1:1～10。
本发明利用姜黄素具有的抗组织增生、抗炎症反应等特性，将它和肝素结合在一起(利用PLA-PEG-PLA载药缓释微球特性，可以进一步提高其生物抗凝稳定性)，以克服植入性医疗器械(如人造血管)在植入人体之后易发生的问题(如凝血作用会导致血小板聚集引起血栓导致血管再次狭窄，而组织过度增生和炎性反应也会引起排异反应而导致血管狭窄，最终使人造血管等植入器械的生物相容性变差而植入失败)。
本发明的有益效果：现代药理研究表明姜黄素类成分具有抗凝血、抗组织增生、抗炎性反应等多种药理作用，本发明将姜黄素与肝素两者连用不仅可以增强肝素涂层的抗凝血功能，利用PLA-PEG-PLA载药缓释微球特性，进一步提升稳定性，起到了单纯用肝素或蛋白等涂层所无法达到的抗组织增生、抗炎性反应等功能，对于人造血管、血管支架、血管补片等植入器械在人体内减少血栓形成，降低术后并发症，提高产品的使用寿命至关重要。
具体实施方式
以下结合具体实施例对本发明作进一步的详细描述。
需要说明的是，下述例子中使用的人造血管(ePTFE)在涂层之前均经过前处理，以提高其对涂层的结合力，使生物涂层能够稳定且有效的发挥抗凝血等功能。
人造血管(ePTFE)基材，前处理过程如下：
第一层
将ePTFE人造血管浸没于异丙醇(IPA)5～10mins，然后用镊子取出再浸泡于4％聚乙烯亚胺(PEI)和IPA混合液(体积比PEI:IPA＝1:1)15～30mins后，取出浸泡的人造血管，用去离子水浸泡冲洗人造血管。待冲洗完后，将人造血管放置于0.05％戊二醛溶液中15～20mins进行交联，待反应完成后将人造血管再次浸泡在0.5％PEI溶液中15～30mins，用去离子水对构造后的人造血管进行浸泡冲洗。冲洗完成后，将人造血管浸泡在氰基硼氢化钠溶液中反应15～30mins，反应完成后用去离子水浸泡冲洗。
第二层
将上述处理后的人造血管浸泡于0.05％戊二醛溶液中进行交联，待反应完成后将人造血管再次浸泡在0.5％PEI溶液中反应，用去离子水对构造后的人造血管进行浸泡冲洗。冲洗完成后，将人造血管浸泡在氰基硼氢化钠溶液中反应反应完成后用去离子水浸泡冲洗。
第三层
将0.15g硫酸葡聚糖和100gNaCl溶解于1L去离子水中，将上述第二层处理后的人造血管浸泡于混合液中，在60℃反应1～2h，待反应完成后，用去离子水对人造血管反复冲洗。制备中间电荷层。
第四层
将第三层处理后的人造血管浸泡于0.3％PEI溶液中0.5～1h，用50g/L的氯化钠溶液对人造血管浸泡冲洗，再用去离子水浸泡冲 洗，得到人造血管(ePTFE)基材。
第一部分复合肝素抗凝涂层液的制备及实验
实施例1联氨基姜黄素-肝素
一种复合肝素抗凝涂层液，涂层液中包含有交联状态的联氨基姜黄素-肝素。
该复合肝素抗凝涂层液的制备方法，步骤如下：
将联氨基姜黄素溶解于乙醇溶液中，配置成浓度为5mg/mL的姜黄素醇溶液，向姜黄素醇溶液中加入MES缓冲液使姜黄素浓度降低为2.5mg/mL，向300mL姜黄素溶液中加入10g肝素后，再加入4g EDC和4g NHS，搅拌1～2h，使联氨基姜黄素中的氨基与肝素充分反应，将反应完成的溶液放入透析袋透析24～72h，透析完成后，得到复合肝素抗凝涂层液。可以对该涂层液进行冷冻干燥制得复合肝素抗凝涂层冻干粉，长期保存。
实施例2联氨基姜黄素-肝素与丝素蛋白涂层
联氨基姜黄素-肝素与丝素蛋白混合的生物抗凝涂层人造血管制备方法，步骤如下：
1)将实施例1制得的冻干粉溶于丝素蛋白大分子溶液，得到的混合涂层溶液中联氨基姜黄素-肝素浓度为1％wt，丝素蛋白浓度为10％wt，为联氨基姜黄素-肝素及丝素蛋白的生物抗凝涂层液。
2)将人造血管(ePTFE)基材浸没在步骤1)的生物抗凝涂层液中10～30mins，随后取出置于通风处，待干燥完全后，浸泡在30～75％甲醇溶液中2～4h，浸泡完成后放置于通风处干燥。
3)重复步骤2)两次，给人造血管(ePTFE)涂第二、三层涂层。
其中，步骤1)的丝素蛋白制备方法：将20～30g生丝放入10～12L含有Na 2CO 3(2.12g/L)溶液中沸水煮20～30mins，随后用去离子 水反复冲洗煮好后的脱胶丝以去除残余丝胶。将洗净后的蚕丝蛋白纤维平铺于通风处干燥。称取15～25g干燥蚕丝纤维溶于60℃100～150mL的LiBr溶液中(9.3M)，待溶解完成后，用透析袋对蚕丝蛋白溶液进行偷袭36～72h，透析完成后对溶液进行离心去除杂质，得到丝素蛋白大分子溶液。
实施例3联氨基姜黄素-肝素与丝素蛋白涂层
本实施例和实施例2基本相同，区别在于，联氨基姜黄素-肝素及丝素蛋白的生物抗凝涂层液中联氨基姜黄素-肝素浓度为0.7％wt，丝素蛋白浓度为5.6％wt。
实施例4联氨基姜黄素-肝素与丝素蛋白涂层
本实施例和实施例2基本相同，区别在于，联氨基姜黄素-肝素及丝素蛋白的生物抗凝涂层液中联氨基姜黄素-肝素浓度为5％wt，丝素蛋白浓度为15％wt。
实施例5肝素/聚-L-赖氨酸涂层
肝素/聚-L-赖氨酸涂层人造血管，其制备方法步骤如下：
1)肝素大分子制备
将0.176g聚-L-赖氨酸溶解于300mL MES缓冲液中，再向其中加入4g磺酸-NHS和4g盐酸EDC，待上述混合液在室温中反应1h后，加入10g肝素钠粉末反应4h，反应完成后，用透析袋透析混合液24～36h。透析完成后，向透析液中加入10mg亚硝酸钠和2mL乙酸，在0℃反应2h，反应完成后，继续对溶液进行透析24h，透析完成后，对溶液进行冷冻、干燥得到肝素/聚-L-赖氨酸冻干粉。
2)涂层制备
取肝素/聚-L-赖氨酸冻干粉0.9g溶解于200mL去离子水，制得肝素大分子溶液，将三中处理后的人造血管浸泡于前述制得的肝素 大分子溶液中于60℃反应10～20min后，再加入572uL 2.5％氰基硼氢化钠继续反应2～3h。所有反应完成后，用去离子水和硼酸盐缓冲液将人造血管冲洗干净，冷冻干燥处理即可。
实施例6肝素与丝素蛋白涂层
丝素蛋白肝素涂层人造血管，制备方法步骤如下：
1)肝素大分子制备(同实施例5)，丝素蛋白大分子溶液制备(同实施例2)
2)丝素蛋白与肝素涂层
丝素蛋白大分子溶液与肝素/聚-L-赖氨酸共混，得到的混合液中的丝素蛋白浓度为10％溶液，肝素/聚-L-赖氨酸浓度为4％。
3)涂层制备
第一层：取人造血管(ePTFE)基材浸没于上述混合液中10～30mins，随后取出置于通风处，待干燥完全后，浸泡在30～75％甲醇溶液中2～4h，浸泡完成后放置于通风处干燥。
第二/三层：重复第一层操作。
实施例7粘连蛋白与肝素涂层
粘连蛋白肝素涂层人造血管制备方法，步骤如下：
将粘连蛋白与肝素/聚-L-赖氨酸溶液共混，混合液中粘连蛋白浓度为10～30％wt，肝素/聚-L-赖氨酸浓度为0.5～5％wt，37℃反应1～2h，随后加入摩尔比为1M:2M:2M的EDC/NHS/MES交联剂，肝素/PLL与交联剂体积比为1:2～15。将人造血管(ePTFE)基材浸没在混合液中于37℃反应1～5h。
待上述反应完成后，取100～1000uL浓度为200ng/mL的SDF-1α浸润基底材料，该反应在4℃中反应12～24h，待反应完成后，用磷酸盐缓冲液浸泡冲洗15～30mins，再用去离子水浸泡冲洗30mins，干燥即可。
实施例8胶原蛋白涂层
胶原蛋白涂层人造血管制备方法，步骤如下：
用冰醋酸将胶原蛋白配置成浓度为10～30％wt，向200mL胶原蛋白混合液中加入肝素冻干粉，使肝素浓度为0.5～5％wt，待充分溶解后，将人造血管(ePTFE)基材浸润在混合液中，充分浸润后，并对其进行冷冻干燥24～48h。待冷冻干燥完成后，采用紫外线照射对胶原蛋白进行交联。如此反复浸润、冻干三次即可得表面胶原蛋白涂层聚合物材料。
实施例9聚多巴胺涂层
聚多巴胺涂层人造血管制备方法，步骤如下：
将人造血管(ePTFE)基材浸入浓度为8mg/mL多巴胺溶液中室温反应12h后，至于去离子水中超声清洗，然后后再次浸入多巴胺溶液中反应，如此反应4次后，将人造血管置于100℃中热处理1h。处理完成后用去离子水清洗人造血管，常温下干燥即可。
实验例1细胞毒性检测
以浸提液法检测涂层人造血管细胞毒性，具体实验步骤如下：
1)取对照例及实施例2～9中制备的涂层人造血管或涂层材料按照0.2g/mL用含胎牛血清的高糖细胞培养液浸提24～72h。
2)取正常生长对数期细胞(NIH/3T3)，消化吹打，用培养液配置成1×10 5个/mL浓度的细胞悬液，接种于96孔板，每孔100μL。放置于二氧化碳恒温培养箱24h后，弃去原培养液。
3)每组分别加入DMEM完全培养液(空白组)，聚乙烯浸提液(阴性对照)，含5％DMSO培养液(阳性对照)，人造血管样品浸提液(样品组)。继续培养24h后弃去孔内液体。
4)MTT反应，每孔中加入10uL 5mg/mL的MTT溶液，在培 养箱内继续培养孵育4h。
5)每孔加入100μL的Formanzan溶解液，在细胞培养箱内继续孵育，直至普通光学显微镜下Formanzan全部溶解。
6)将96孔板放置于酶标仪上测量570nm处的吸光度值。
依据各组细胞浓度计算细胞相对增殖度(RGD)：
Figure PCTCN2018102568-appb-000001
表1细胞相对增殖度分级表
分级 相对增殖度(％)
0 ≥100
1 75～99
2 50～74
3 25～49
4 1～24
5 0
实验结果：
表2各组细胞毒性检测结果
Figure PCTCN2018102568-appb-000002
Figure PCTCN2018102568-appb-000003
实验例2：凝血时间实验
1)部分凝血活酶时间(APTT)实验：取实施例2～9中制备的涂层人造血管，向其中加入100～200μL贫血小板血浆在37℃水浴0.5h。待加热完成后，加入100～200μL部分凝血活酶试剂液100μL和氯化钙溶液，混合均匀后，利用自动凝血仪检测APTT。
2)凝血酶原时间(PT)实验：向人造血管样品中加入100～200μL贫血小板血浆并在37℃水浴0.5h。待加热完成后，向其中加入PT试剂，用自动凝血仪检测PT。
3)凝血酶时间(TT)实验：向人造血管样品中加入100～200μL贫血小板血浆并在37℃水浴0.5h。待加热完成后，向其中加入TT试剂，用自动凝血仪检测TT。
表3人造血管或材料在涂层前后凝血实验结果
Figure PCTCN2018102568-appb-000004
Figure PCTCN2018102568-appb-000005
如表3所示，如表3所示，未涂层的人造血管凝血时间与正常血浆凝血时间相近，经姜黄素-肝素丝素蛋白共混溶液涂层过的人造血管凝血时间(实施例2～4)相比未涂层人造血管有显著提高，且相比于其他不同涂层的基材人造血管的APTT时间延长了约3～16s，PT时间延长了约4～7s，TT时间延长了约5～8s。以上试验表明经姜黄素-肝素涂层的人造血管涂层比其他未涂层的人造血管具有更明显的抗凝血性能，同时对于其他采用肝素及大分子蛋白涂层的人造血管在抗凝血性能上也有提升，表明姜黄素与肝素连用起到了增强抗凝血的作用，涂层效果更好。
第二部分复合肝素抗凝涂层用微球的制备及实验
实施例10复合肝素抗凝涂层用微球
一种复合肝素抗凝涂层用微球，它是PLA-PEG-PLA肝素微球，是以PLA-PEG-PLA为载体，交联状态的联氨基姜黄素-肝素为内容物的微球。
该复合肝素抗凝涂层用微球的制备方法，采用实施例1的方法制备复合肝素抗凝涂层冻干粉。
2)按照重量比1:1的比例称取复合肝素抗凝涂层冻干粉和PLA-PEG-PLA溶于二氯甲烷烧杯中，搅拌10～30min使混合物全部溶解，然后在溶液中加入乳化剂PVA水溶液，继续搅拌30～60min，随后吸取带有微球的液相滴入500～1000mL超纯水中继续搅拌洗涤干燥。洗涤3次后，高速离心收集溶液底部微球沉淀，冷冻干燥收集微球，得到复合肝素抗凝涂层用微球。
实施例11丝素蛋白复合肝素微球抗凝涂层
丝素蛋白复合肝素微球抗凝涂层人造血管制备方法，步骤如下：
1)丝素蛋白制备方法：同实施例2。
2)将实施例10制得的复合肝素抗凝涂层用微球溶于丝素蛋白大分子溶液，得到的混合涂层溶液中复合肝素抗凝涂层用微球浓度为1％wt，丝素蛋白浓度为10％wt，为丝素蛋白复合肝素微球抗凝涂层液，对涂层液进行超声振荡30～60s以改变丝素蛋白结构。
3)将人造血管(ePTFE)基材浸没在丝素蛋白复合肝素微球抗凝涂层液中10～30mins，随后取出置于通风处，待干燥完全后，浸泡在30～75％甲醇溶液中2～4h，浸泡完成后放置于通风处干燥。
4)重复步骤3)两次，给人造血管(ePTFE)涂第二、三层涂层。
实施例12丝素蛋白复合肝素微球抗凝涂层
本实施例和实施例11基本相同，区别在于，丝素蛋白复合肝素微球抗凝涂层液中复合肝素抗凝涂层用微球浓度为0.7％wt，丝素蛋白浓度为5.6％wt。
实施例13丝素蛋白复合肝素微球抗凝涂层
本实施例和实施例11基本相同，区别在于，丝素蛋白复合肝素微球抗凝涂层液中复合肝素抗凝涂层用微球浓度为5％wt，丝素蛋白浓度为15％wt。
实验例3细胞毒性检测
以浸提液法检测涂层人造血管细胞毒性，实验对象为对照例和实施例11～13、5～9，具体实验步骤同实验例1。
表4细胞相对增殖度分级表
分级 相对增殖度(％)
0 ≥100
1 75～99
2 50～74
3 25～49
4 1～24
5 0
实验结果：
表5各组细胞毒性检测结果
Figure PCTCN2018102568-appb-000006
实验例4：凝血时间实验
凝血时间实验对象为对照例和实施例11～13、5～9，步骤同实验例2。
表6人造血管或材料在涂层前后凝血实验结果
Figure PCTCN2018102568-appb-000007
如表3所示，如表6所示，未涂层的人造血管凝血时间与正常血浆凝血时间相近，经姜黄素-肝素丝素蛋白共混溶液涂层过的人造血管凝血时间(实施例11～13)相比未涂层人造血管有显著提高，且相比于其他不同涂层的基材人造血管的APTT时间延长了约11～18s，PT时间延长了约5～9s，TT时间延长了约7～11s。以上试验表明经姜黄素-肝素涂层的人造血管涂层比其他未涂层的人造血管具有更明显的抗凝血性能，同时对于其他采用肝素及大分子蛋白涂层的人造血管在抗凝血性能上也有提升，表明姜黄素与肝素连用起到了增强抗凝血的作用，涂层效果更好。
本发明实施例中均以ePTFE人造血管为基材，实际上其他材料 如PU、PE、涤纶、聚乳酸、聚硅酮、聚羟基乙酸、硅橡胶等，也可以采用本发明中的涂层液进行涂覆，只需要处理好基材，使生物涂层能够稳定结合，同样能获得较好的凝血效果。

Claims (11)

  1. 一种复合肝素抗凝涂层液，其特征在于：所述涂层液中包含有交联状态的联氨基姜黄素-肝素。
  2. 根据权利要求1所述的复合肝素抗凝涂层液，其特征在于：所述联氨基姜黄素通过其上的氨基与肝素交联。
  3. 一种权利要求1所述复合肝素抗凝涂层液的制备方法，其特征在于，步骤如下：
    将联氨基姜黄素溶解于乙醇溶液中，配置成姜黄素醇溶液，向姜黄素醇溶液中加入MES缓冲液，再加入肝素，然后再加入EDC和NHS交联剂，搅拌1～2h，使联氨基姜黄素中的氨基与肝素充分反应，将反应完成的溶液放入透析袋透析24～72h，透析完成后，得到复合肝素抗凝涂层液。
  4. 一种权利要求3所述复合肝素抗凝涂层液的制备方法，其特征在于：所述联氨基姜黄素和肝素的添加重量比为1～10:40。
  5. 一种联氨基姜黄素-肝素及丝素蛋白的生物抗凝涂层液，其特征在于：所述生物抗凝涂层液中包含有交联状态的联氨基姜黄素-肝素，还包括丝素蛋白；所述生物抗凝涂层液中所述联氨基姜黄素-肝素在的浓度为0.5～5％wt，所述丝素蛋白的浓度为5～15％wt，且联氨基姜黄素-肝素与丝素蛋白的浓度比值为1:1～10。
  6. 根据权利要求5所述联氨基姜黄素-肝素及丝素蛋白的生物抗凝涂层液，其特征在于：所述联氨基姜黄素通过其上的氨基与肝素交联。
  7. 一种复合肝素抗凝涂层用微球，其特征在于：它是以PLA-PEG-PLA为载体，交联状态的联氨基姜黄素-肝素为内容物的微球。
  8. 根据权利要求7所述的复合肝素抗凝涂层用微球，其特征在于：所述联氨基姜黄素通过其上的氨基与肝素交联。
  9. 一种权利要求7所述复合肝素抗凝涂层用微球的制备方法， 其特征在于，步骤如下：
    1)将联氨基姜黄素溶解于乙醇溶液中，配置成姜黄素醇溶液，向姜黄素醇溶液中加入MES缓冲液，再加入肝素，然后再加入EDC和NHS交联剂，搅拌1～2h，使联氨基姜黄素中的氨基与肝素充分反应，将反应完成的溶液放入透析袋透析24～72h，透析完成后，得到复合肝素抗凝涂层液；
    2)以PLA-PEG-PLA为材料，将复合肝素抗凝涂层液制备成冻干粉之后与PLA-PEG-PLA一并乳化制成的微球。
  10. 根据权利要求9所述复合肝素抗凝涂层用微球的制备方法，其特征在于：所述联氨基姜黄素和肝素的添加重量比为1～10:40。
  11. 一种丝素蛋白复合肝素微球抗凝微球涂层液，其特征在于：所述涂层液中包含有权利要求7所述复合肝素抗凝涂层用微球，以及包裹住微球的丝素蛋白；所述丝素蛋白的浓度为5～15％wt，所述复合肝素抗凝涂层用微球与丝素蛋白的浓度比值为1:1～10。
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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101940803A (zh) * 2010-09-26 2011-01-12 复旦大学附属中山医院 肝素壳聚糖层层自组装小口径涤纶人工血管及其生产方法
CN103087218A (zh) * 2011-12-30 2013-05-08 北京大学 直链形肝素修饰的生物型人造血管
WO2013095270A1 (en) * 2011-12-22 2013-06-27 Corline Systems Ab An aqueous solution comprising a macromolecular conjugate of heparin for the treatment of blood vessels
EP3068454B1 (en) * 2013-11-14 2017-04-19 pfm medical titanium gmbh Polyurethane having an antithrombogenic coating
CN107376037A (zh) * 2017-08-28 2017-11-24 武汉杨森生物技术有限公司 复合肝素生物抗凝涂层液及制备方法与应用
CN107519544A (zh) * 2017-08-28 2017-12-29 武汉杨森生物技术有限公司 复合肝素抗凝涂层用微球及制备方法与应用

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2508212A1 (en) * 2011-04-05 2012-10-10 Universitätsklinikum Freiburg Biocompatible and biodegradable gradient layer system for regenerative medicine and for tissue support
CN102988999B (zh) 2012-05-09 2015-04-08 中国药科大学 姜黄素-多糖类偶联物及其制备方法与应用
CN103566414B (zh) 2013-08-13 2016-01-13 重庆大学 一种核壳结构纳微粒涂层血管支架及其制备方法

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101940803A (zh) * 2010-09-26 2011-01-12 复旦大学附属中山医院 肝素壳聚糖层层自组装小口径涤纶人工血管及其生产方法
WO2013095270A1 (en) * 2011-12-22 2013-06-27 Corline Systems Ab An aqueous solution comprising a macromolecular conjugate of heparin for the treatment of blood vessels
CN103087218A (zh) * 2011-12-30 2013-05-08 北京大学 直链形肝素修饰的生物型人造血管
EP3068454B1 (en) * 2013-11-14 2017-04-19 pfm medical titanium gmbh Polyurethane having an antithrombogenic coating
CN107376037A (zh) * 2017-08-28 2017-11-24 武汉杨森生物技术有限公司 复合肝素生物抗凝涂层液及制备方法与应用
CN107519544A (zh) * 2017-08-28 2017-12-29 武汉杨森生物技术有限公司 复合肝素抗凝涂层用微球及制备方法与应用

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