CN113304333B - 一种溶栓涂层的制备方法及溶栓涂层 - Google Patents
一种溶栓涂层的制备方法及溶栓涂层 Download PDFInfo
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Abstract
本申请提供了一种溶栓涂层的制备方法,包括以下步骤:提供一种基材;将所述基材浸入抗凝溶液中,干燥后得到第一预制品;将紫外光引发剂加入到ε‑赖氨酸‑聚乙二醇‑丙烯酸酯溶液中,得到混合溶液,将所述第一预制品浸入所述混合溶液中,干燥后得到第二预制品;将所述第二预制品重复浸入所述抗凝溶液和所述混合溶液中,得到第三预制品;对所述第三预制品进行紫外灯照射,从而在所述基材表面制得所述溶栓涂层。本申请提供了一种操作简单、条件温和的溶栓涂层的制备方法。本申请还提供了一种溶栓涂层。
Description
技术领域
本申请涉及生物医药的技术领域,尤其涉及一种溶栓涂层的制备方法及溶栓涂层。
背景技术
心血管植介入治疗器械主要包括支架、球囊扩张导管以及配套的导丝、导管、栓塞等部件,这些器械与血液接触后主要通过凝血因子激活、血小板粘附与聚集、红细胞粘附以及补体系统激活等四个相互关联而又相互影响的途径导致凝血过程的发生。而材料表面血浆蛋白吸附层存在导致血小板粘附而出现的凝血行为是导致血栓形成最核心的途径。目前构建抗凝血材料表面主要包括抗表面修饰生物惰性聚合物(如聚乙二醇(PEG)类聚合物和两性离子类聚合物)、表面修饰抗血栓活性分子(如抗凝剂和抗血小板分子)以及构建表面多孔结构促进微量血栓形成进而促进血管伪内皮的重建等。其中抗凝血活性表面的设计主要是一些具有抗凝血活性或纤溶活性的生物分子修饰到心血管材料的表面,当其与血液中发生接触时,材料表面的活性分子能够启动抗凝或栓溶系统而实现材料表面抗凝血的目的。然而大量实践证明,异体材料在接触血液时,凝血反应是不可避免的。因此,这些策略应用于临床的成功性仍十分有限。
“纤溶表面”概念的提出和实践突破了传统抗血栓思路,从“溶解纤维蛋白”的角度设计材料表面,使其在接触血液环境时,充分调动人体纤溶系统,从而迅速溶解初步形成但尚未对人体造成危害的血栓。然而,由于表面修饰ε-赖氨酸的过程十分复杂且反应条件苛刻,导致重复性和可控性差,从而限制了纤溶概念在商品化材料表面的应用。
发明内容
有鉴于此,本申请提供一种操作简单、条件温和、可控性好的溶栓涂层的制备方法。
另外,还有必要提供一种由所述的溶栓涂层制备方法制备的溶栓涂层。
为实现上述目的,本申请提供了一种溶栓涂层的制备方法,包括以下步骤:提供一种基材;将所述基材浸入抗凝溶液中,干燥后得到第一预制品;将紫外光引发剂加入到ε-赖氨酸-聚乙二醇-丙烯酸酯溶液中,得到混合溶液,将所述第一预制品浸入所述混合溶液中,干燥后得到第二预制品;将所述第二预制品重复浸入所述抗凝溶液和所述混合溶液中,得到第三预制品;对所述第三预制品进行紫外灯照射,从而在所述基材表面制得所述溶栓涂层。
在一些可能的实现方式中,所述抗凝溶液为肝素钠、水蛭素、比伐卢定、阿加曲班、3-三氟甲基-苯磺酰-精氨酸中的一种。
在一些可能的实现方式中,所述紫外光引发剂的浓度为0.01-5%(w/v)。
在一些可能的实现方式中,所述抗凝溶液的浓度为0.1-100mg/mL。
在一些可能的实现方式中,所述ε-赖氨酸-聚乙二醇-丙烯酸酯溶液的浓度为1-1000mg/L。
在一些可能的实现方式中,所述基材在浸入所述抗凝溶液之前还经过预处理,所述预处理包括:将所述基材浸入聚乙烯亚胺溶液中,然后用pH=7.4的磷酸盐缓冲溶液进行清洗,氮气吹干。
在一些可能的实现方式中,所述基材浸入抗凝溶液的时间为5-360min;第一预制品浸入混合溶液的时间为5-360min。
在一些可能的实现方式中,所述ε-赖氨酸-聚乙二醇-丙烯酸酯的制备包括以下步骤:
将活性酯-聚乙二醇-羟基溶解于二甲基亚砜中,加入N(e)-叔丁氧羰基-L-赖氨酸和三乙胺进行反应,将反应液倒入冰乙醚中沉淀,过滤收集产物,真空干燥,得到N(e)-叔丁氧羰基-L-赖氨酸-聚乙二醇-羟基;
将所述N(e)-叔丁氧羰基-L-赖氨酸-聚乙二醇-羟基溶于氯仿中,加入丙烯酰氯和三乙胺进行反应,减压浓缩后倒入冰乙醚中沉淀,过滤收集产物,真空干燥,得到N(e)-叔丁氧羰基-L-赖氨酸-聚乙二醇-丙烯酸酯;
将所述N(e)-叔丁氧羰基-L-赖氨酸-聚乙二醇-丙烯酸酯溶于二氯甲烷中,加入三氟乙酸进行反应,倒入冰乙醚中沉淀,过滤收集产物,真空干燥得到ε-赖氨酸-聚乙二醇-丙烯酸酯。
在一些可能的实现方式中,所述ε-赖氨酸-聚乙二醇-丙烯酸酯中所述聚乙二醇的分子量为600、800、1000或2000。
本申请还提供一种溶栓涂层,包括所述的溶栓涂层的制备方法制备的溶栓涂层。
本申请中提供的溶栓涂层的制备方法,通过将溶栓涂层复合于基材上,基材表面上得到具有溶解初期血栓性能的涂层,再经过紫外灯照射,增强涂层与基材表面的结合力,在涂层中的抗凝溶液还对基材表面有抗凝血作用。本申请的制备方法简单,条件温和,可控性强,适合规模工业生产。
附图说明
图1是实施例2和对比例1-2的凝血酶原时间测试图。
图2是实施例2和对比例1的血小板粘附率的示意图。
图3中,A图是对比例1中基材粘附血小板的扫描电子显微镜图,B图是实施例2中基材粘附血小板的扫描电子显微镜图。
具体实施方式
下面详细描述本发明的实施例。下面通过参考附图描述的实施例是示例性的,仅用于解释本发明,而不能理解为对本发明的限制。
在本说明书的描述中,参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中,对上述术语的示意性表述不一定指的是相同的实施例或示例。而且,描述的具体特征、结构、材料或者特点可以在任何的一个或多个实施例或示例中以合适的方式结合。
本申请提供一种溶栓涂层的制备方法,包括以下步骤:
步骤一:提供一种基材;
步骤二:将所述基材浸入抗凝溶液中,浸入时间为5-360min,然后用醋酸缓冲溶液冲洗所述基材,氮气吹干,得到第一预制品S-H;
步骤三:将紫外光引发剂加入到ε-赖氨酸-聚乙二醇-丙烯酸酯溶液(ε-Lys-PEG-DA)中,得到混合溶液,将所述第一预制品S-H浸入所述混合溶液中,浸入时间为5-360min,然后用醋酸缓冲溶液清洗,氮气吹干,得到第二预制品S-H-ε-Lys;
重复多次步骤二和步骤三,得到第三预制品S-(H-ε-Lys)n,所述n为重复次数。
步骤四:紫外灯照射所述第三预制品S-(H-ε-Lys)n,时间为3-60min,紫外灯的功率为200-2000W,从而在所述基材表面制得所述溶栓涂层。
在本申请中,将基材浸入抗凝溶液中,使得抗凝物质通过化学键结合方式粘附于所述基材上,使得基材表面具有抗凝血作用;另外通过将具有抗凝血作用的基材浸入所述ε-赖氨酸-聚乙二醇-丙烯酸酯溶液中并进行紫外光照射,使得紫外光引发剂产生的自由基能够促进ε-赖氨酸-聚乙二醇-丙烯酸酯溶液、抗凝溶液与基材表面发生交联聚合反应,从而在基材表面制得溶栓涂层。其中,ε-赖氨酸可以与血液中的纤溶酶原及组织型纤溶酶原激活剂(t-PA)来产生纤溶酶,从而实现纤维蛋白的溶解,溶解血栓,使得基材表面具有溶解血栓的作用。另外,由于ε-赖氨酸-聚乙二醇-丙烯酸酯溶液、抗凝溶液与基材表面发生交联聚合反应,从而能够提高涂层与基材表面的结合力。
在一些实施例中,在步骤二中,所述抗凝溶液中加入有pH为4的醋酸缓冲溶液。在一些实施例中,所述醋酸缓冲溶液中含有0.018mol/L的醋酸钠、0.082mol/L的醋酸和0.14mol/L的氯化钠溶液。
紫外光引发剂采用紫外光引发剂2959(化工型号)。
在一些实施例中,所述抗凝溶液为肝素钠、水蛭素、比伐卢定、阿加曲班、3-三氟甲基-苯磺酰-精氨酸中的一种。
在一些实施例中,所述紫外光引发剂的浓度为0.01-5%(w/v)。
当紫外光引发剂的浓度小于0.01%(w/v),使得ε-赖氨酸-聚乙二醇-丙烯酸酯溶液、抗凝溶液与基材表面交联不充分,降低涂层与基材的结合力;当紫外光引发剂的浓度大于5%(w/v),会使得ε-赖氨酸-聚乙二醇-丙烯酸酯溶液、抗凝溶液与基材表面交联过度,得到的涂层发黄发脆,另外紫外光引发剂具有一定毒性,加入过多也会产生危害。
在一些实施例中,所述抗凝溶液的浓度为0.1-100mg/mL。
在一些实施例中,所述ε-赖氨酸-聚乙二醇-丙烯酸酯溶液的浓度为1-1000mg/L。
在一些实施例中,在所述步骤一中,所述基材在浸入所述抗凝溶液之前还经过预处理,所述预处理包括:将所述基材浸入聚乙烯亚胺溶液中,然后用pH=7.4的磷酸盐缓冲溶液进行清洗,氮气吹干。
将所述基材表面进行预处理,可以使得基材表面带有正电,再与所述抗凝溶液作用时,可以提高基材与抗凝溶液的作用结合力。在一些实施例中,聚乙烯亚胺的分子量为600-70000M.W.,浓度为0.5-100mg/mL。
在一些实施例中,所述基材的材质为聚对苯二甲酸乙二醇酯(PET)、聚四氟乙烯(PTFE)、聚氨酯(PU)、聚醚醚酮(PEEK)、聚乳酸(PLLA)、聚己内酯(PCL)、不锈钢、钴镍合金、钴铬合金以及硅胶中的至少一种。
在一些实施例中,所述ε-赖氨酸-聚乙二醇-丙烯酸酯的制备包括以下步骤:
(1)将活性酯-聚乙二醇-羟基溶解于二甲基亚砜中,加入N(e)-叔丁氧羰基-L-赖氨酸和三乙胺完全溶解,40℃下反应2h,随后将反应液倒入冰乙醚中沉淀,过滤收集产物,真空干燥,得到N(e)-叔丁氧羰基-L-赖氨酸-聚乙二醇-羟基;
(2)将所述N(e)-叔丁氧羰基-L-赖氨酸-聚乙二醇-羟基溶于氯仿中,加入丙烯酰氯和三乙胺完全溶解,40℃下反应4h,减压浓缩后倒入冰乙醚中沉淀,过滤收集产物,真空干燥,得到N(e)-叔丁氧羰基-L-赖氨酸-聚乙二醇-丙烯酸酯;
(3)将所述N(e)-叔丁氧羰基-L-赖氨酸-聚乙二醇-丙烯酸酯溶于二氯甲烷中,加入三氟乙酸,常温下反应30min,水洗3次,无水硫酸钠干燥,倒入冰乙醚中沉淀,过滤收集产物,真空干燥得到ε-赖氨酸-聚乙二醇-丙烯酸酯。
下面化学式为ε-赖氨酸-聚乙二醇-丙烯酸酯聚合物合成线路图。
在一些实施例中,所述ε-赖氨酸-聚乙二醇-丙烯酸酯中所述聚乙二醇的分子量为600、800、1000或2000。
本申请还提供一种溶栓涂层,包括所述的溶栓涂层的制备方法制备的溶栓涂层。
下面将结合实施例对本发明的方案进行解释。本领域技术人员将会理解,下面示例仅用于解释本发明,而不能理解为对本发明的限制。除另有交待,以下实施例中涉及的未特别交待的试剂、软件及仪器,都是常规市售产品或者开源的。
实施例1
(1)基材表面正电处理
将表面羟基化的316L不锈钢浸入PEI溶液中1h,PEI的浓度为3mg/mL。PEI的分子量为25000,然后用PBS溶液进行清洗,每次3min,清洗3次,氮气吹干,处理后的表面记为S。
(2)S表面接枝抗凝物质
用醋酸钠缓冲溶液配置1mg/mL肝素钠溶液A,将S浸入A溶液15min,然后用醋酸缓冲溶液进行清洗,每次3min,清洗3次,氮气吹干,处理后的表面记为S-H。
(3)S-H表面接枝ε-赖氨酸-PEG1000-丙烯酸酯(ε-Lys-PEG1000-DA)
用超纯水配置50mg/mL的ε-赖氨酸-PEG-丙烯酸酯溶液,并且加入紫外光引发剂2959,浓度为0.5%(w/v),得到混合溶液B。将S-H浸入B混合溶液中15min,然后用醋酸缓冲溶液进行清洗,氮气吹干,记为S-H-ε-Lys。然后循环重复(2)和(3),次数为5次,记为S-(H-ε-Lys)5。
(4)紫外光固化S-(H-ε-Lys)5
用紫外灯照射S-(H-ε-Lys)5表面5min,得到溶栓抗凝涂层。
实施例2
(1)基材表面正电处理
将PET材质的基材浸入PEI溶液,其他条件如实施例1的步骤(1)。
(2)S表面接枝抗凝物质
用醋酸钠缓冲溶液配置1mg/mL水蛭素溶液A,将S浸入A溶液15min,然后用醋酸缓冲溶液进行清洗,每次3min,清洗3次,氮气吹干,处理后的表面记为S-H。
步骤(3)和步骤(4)同实施例1中的步骤(3)和步骤(4)。
实施例3
(1)基材表面正电处理
将PLLA无纺布浸入浓度为10mg/mL的PEI溶液中3h,PEI的分子量为600。然后用PBS溶液进行清洗,每次3min,一共清洗3次,氮气吹干,处理后的表面记为S。
(2)S表面接枝抗凝药物
用醋酸钠缓冲溶液配置1mg/mL肝素钠溶液A,将S浸入A溶液30min,然后用醋酸缓冲溶液进行清洗,每次3min,一共清洗3次,氮气吹干,处理后的表面记为S-H。
(3)S-H表面接枝ε-赖氨酸-PEG2000-丙烯酸酯(ε-Lys-PEG2000-DA)
用超纯水配置100mg/mL的ε-赖氨酸-PEG-丙烯酸酯溶液,并且加入紫外光引发剂2959,浓度为0.5%(w/v),得到混合溶液B。将S-H浸入B混合溶液中30min,然后用醋酸缓冲溶液进行清洗,氮气吹干,记为S-H-ε-Lys。然后循环重复(2)和(3),次数为8次,记为S-(H-ε-Lys)8。
步骤(4)同实施例1步骤(4)。
对比例1
对比例1与实施例2的不同之处在于:提供一种PET材质的基材,未进行其他处理。
对比例2
对比例2与实施例3的不同之处在于:提供一种PLLA无纺布,未进行其他处理。
凝血酶原时间测试:将缺乏血小板的血浆和活化部分凝血活酶时间测定试剂滴加于基材表面,在37℃下孵育一段时间后,取一定量的上述样品于检测管中并加入氯化钙,通过血凝仪检测血浆凝血时间。
血小板粘附测试:
取白兔耳缘静脉血配置新鲜抗凝血[兔血与3.8%枸橼酸钠以9:1(v:v)配制]在4℃,100g条件下离心10min,取上清液,得到富血小板血浆(PRP)。
将0.5cm×0.5cm大小的待测样品用去离子水洗净、干燥、灭菌后,平铺于96孔板底部,每孔加入100μL PRP,于37℃孵育2h后,移除PRP,用PBS反复清洗10次,加入4%多聚甲醛,于4℃固定12h。随后移除4%多聚甲醛,用PBS反复清洗3次,利用梯度酒精(乙醇溶液)进行脱水,每次10min。脱水后将样品干燥,喷金(真空度控制在6mmHg,电流控制在6mA,喷金时间约为30s)后利用扫描电子显微镜(SEM,荷兰皇家飞利浦电子公司)进行表面观察并拍照。利用血小板计数的方法评价血小板粘附率(plts/mm3)。
对测试结果进行统计计算,采用平均数±标准差表示。统计分析采用Turkey多重比较方法进行单因素方差分析,其中*p<0.05和**p<0.01代表具有统计学差异。
参阅图1,本申请还对上述实施例2对比例1-2的凝血酶原时间进行测试。相对于对比例1,实施例2中具有溶栓涂层的PET基材的凝血时间延长了3倍以上,由于溶栓涂层中具有ε-赖氨酸和抗凝物质,增强基材表面的溶栓涂层的抗凝和溶解血栓的效果。
参阅图2,实施例2中血小板粘附率为81.48±11.31plts/mm3,对比例1中PET基材的血小板粘附率为368.72±11.99plts/mm3,实施例2中血小板粘附率显著低于对比例1中血小板粘附率。这说明含有实施例2中含溶栓涂层的PET表面能够降低血小板的粘附率,能够更好的抗凝血性能,具有抑制血栓的能力。为了进一步验证血小板粘附情况,还对实施例2和对比例1进行了扫描电镜测试。请参阅图3,在A图中,对比例1中未进行处理的PET基材表面粘附有大量的血小板,在B图中,实施例2中具有溶栓涂层的PET表面粘附有少量的血小板。
以上实施方式仅用以说明本发明的技术方案而非限制,尽管参照以上实施方式对本发明进行了详细说明,本领域的普通技术人员应当理解,可以对本发明的技术方案进行修改或等同替换都不应脱离本发明技术方案的精神和范围。
Claims (9)
1.一种溶栓涂层的制备方法,其特征在于,包括以下步骤:
提供一种基材;
将所述基材浸入抗凝溶液中,干燥后得到第一预制品;
将紫外光引发剂加入到ε-赖氨酸-聚乙二醇-丙烯酸酯溶液中,得到混合溶液,将所述第一预制品浸入所述混合溶液中,干燥后得到第二预制品,所述ε-赖氨酸-聚乙二醇-丙烯酸酯的制备包括以下步骤:将活性酯-聚乙二醇-羟基溶解于二甲基亚砜中,加入N(e)-叔丁氧羰基-L-赖氨酸和三乙胺进行反应,将反应液倒入冰乙醚中沉淀,过滤收集产物,真空干燥,得到N(e)-叔丁氧羰基-L-赖氨酸-聚乙二醇-羟基;将所述N(e)-叔丁氧羰基-L-赖氨酸-聚乙二醇-羟基溶于氯仿中,加入丙烯酰氯和三乙胺进行反应,减压浓缩后倒入冰乙醚中沉淀,过滤收集产物,真空干燥,得到N(e)-叔丁氧羰基-L-赖氨酸-聚乙二醇-丙烯酸酯;将所述N(e)-叔丁氧羰基-L-赖氨酸-聚乙二醇-丙烯酸酯溶于二氯甲烷中,加入三氟乙酸进行反应,倒入冰乙醚中沉淀,过滤收集产物,真空干燥得到ε-赖氨酸-聚乙二醇-丙烯酸酯;
将所述第二预制品重复浸入所述抗凝溶液和所述混合溶液中,得到第三预制品;
对所述第三预制品进行紫外灯照射,从而在所述基材表面制得所述溶栓涂层。
2.如权利要求1所述的溶栓涂层的制备方法,其特征在于,所述抗凝溶液为肝素钠、水蛭素、比伐卢定、阿加曲班、3-三氟甲基-苯磺酰-精氨酸中的一种。
3.如权利要求1所述的溶栓涂层的制备方法,其特征在于,所述紫外光引发剂的浓度为0.01-5%(w/v)。
4.如权利要求1所述的溶栓涂层的制备方法,其特征在于,所述抗凝溶液的浓度为0.1-100mg/mL。
5.如权利要求1所述的溶栓涂层的制备方法,其特征在于,所述ε-赖氨酸-聚乙二醇-丙烯酸酯溶液的浓度为1-1000mg/L。
6.如权利要求1所述的溶栓涂层的制备方法,其特征在于,所述基材在浸入所述抗凝溶液之前还经过预处理,所述预处理包括:
将所述基材浸入聚乙烯亚胺溶液中,然后用pH=7.4的磷酸盐缓冲溶液进行清洗,氮气吹干。
7.如权利要求1所述的溶栓涂层的制备方法,其特征在于,所述基材浸入抗凝溶液的时间为5-360min;第一预制品浸入混合溶液的时间为5-360min。
8.如权利要求1所述的溶栓涂层的制备方法,其特征在于,所述ε-赖氨酸-聚乙二醇-丙烯酸酯中的聚乙二醇的分子量为600、800、1000或2000。
9.一种溶栓涂层,其特征在于,包括如权利要求1-8任一项所述的溶栓涂层的制备方法制备的溶栓涂层。
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