CN116036387A - 一种外包型载药可视纳米纤维人工血管支架的制备方法 - Google Patents
一种外包型载药可视纳米纤维人工血管支架的制备方法 Download PDFInfo
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Abstract
本发明涉及一种外包型载药可视纳米纤维人工血管支架的制备方法,包括:1)制备含碳点内层纺丝溶液:将葡萄皮粉末与尿素混合加热,冷却后超声溶解在去离子水,透析膜,真空冷冻干燥;将PEO、丝素蛋白、壳聚糖中的一种以上溶解在水、乙酸或乙醚中,再加入葡萄皮基碳点;2)制备载药的外层纺丝溶液;将PI、PCL、PLGA、PLA、PU中的一种溶解在含黄芩苷的DMF、DCM或HFIP中的一种以上;3)以1)制备的溶液为芯,以2)制备的溶液为壳制备纳米纤维膜;用模具将得到的纳米纤维膜卷成纳米纤维管,纳米纤维管包裹于患处血管,有效的防止支架对血管内膜的损伤,增强支架与血管的物理相容性和生物相容性。
Description
技术领域
本发明属于血管支架领域,尤其涉及一种外包型载药可视纳米纤维人工血管支架的制备方法。
背景技术
心脑血管疾病(CVD)是全球主要的死亡原因之一。本项目拟研究纳米纤维的外包血管支架,与传统的内植入血管支架大不相同。目前大多数支架均为内植入支架,其结构基础是金属裸支架。但是金属裸支架本身的组织不相容性导致了对患处的长期刺激,特别是表面处理不够等问题破坏血管内壁平滑结构。同时炎症反应刺激内皮细胞,导致血管再狭窄和支架内血栓,相关术后恢复问题依旧突出。为解决这一问题,药物洗脱支架应运而生。药物洗脱支架以金属裸支架作为结构基础,在其上附着有生物相性较好的涂层和缓释药物,减少对血管壁刺激的同时抑制内膜纤维细胞增生。但是,现有的内植入支架在植入体在身体里容易被免疫系统识别为异物,产生强烈的免疫反应(如免疫球蛋臼的吸附等),致使支架类免疫反应和血栓的形成。
为此,采取外包血管支架的形式,这样可以有效的防止支架对血管内膜的损伤,增强支架与血管的物理相容性和生物相容性;进而通过植入药物纳米纤维以及药物的持续原位释放,可以实现抑制支架植入部位免疫反应;另外,纳米纤维管由弹性优异的纳米纤维毯层层包裹组成,有着一定的自支撑强度,可以完全方便配合支架植入装置的植入过程。但外包型支架对于小血管病变以外的大血管原发病变、开口病变、分叉病变、慢性闭塞病变、严重钙化病变和急性心肌梗死病变等需要机械强制扩张血管的病症起着较为有限的效果,更适用于配合手术后的给药治疗以及预防开放血管手术后的再狭窄,内膜增生(IH),或透析用动脉血管移植。只有选择专业的介入团队通过专业的判断和操作,才能使其发挥到最佳疗效。
在外包型血管支架中,包裹在血管周的支架材料进行药物释放,缓释药物主要针对于包裹位点进行治疗,但无法避免其流失到周围组织,且针对体内的药物缓释速率往往受多因素影响,与体外测试理论值往往不大相同。因此需要进行荧光可视监测碳点监测,及时进行医疗方案的调整。生物成像技术可以通过探针和检测器以实时和非侵入性的方式直接可视化生物方式。荧光成像作为一种成像方式,由于其方便,低成本,高灵敏度,无创性和长期观察等优点,已成为一种强有力的临床诊断方法。然而,常规的荧光团如传统量子点(QDs)和有机染料受毒性问题或荧光性能差的困扰,激起了碳点CDs(Carbon dots,CDs)的快速发展。碳点的光稳定性能优越,生物相容性良好,合成简便快捷,灵活的可设计性,多色发射,深红色/NIR发射和两光/多光子光致发光(PL)使CDs成为用于体外和体内生物成像的下一代荧光探针。除了体外成像外,利用组织的最小自发荧光和光散射,提供了优秀的成像对照度和空间辨识度,具有红色/NIR发射或两光子/多光子光致发光(PL)的CDs是体内荧光跟踪剂的极佳候选者,这为通过实时跟踪预防和治疗脑疾病提供了一种新的方法。
发明内容
本发明的目的是提供一种外包型载药可视纳米纤维人工血管支架的制备方法,利用同轴静电纺技术制备包裹碳点的载药纳米纤维,通过层层自支撑方式构造,可灵活调控直径、管壁厚、载药量、释放速率以及可视化药物追踪,可以应用于临床人工血管支架中,
为实现上述目的,本发明通过以下技术方案实现:
一种外包型载药可视纳米纤维人工血管支架的制备方法,包括以下步骤:
1)制备葡萄皮基碳点
将葡萄皮粉末与尿素混合,在190~220℃下加热6~10h冷却至室温后,将黑色产物超声溶解在去离子水中,离心处理,用透析对收集的上清液进行透析膜48~72h,真空冷冻干燥;
2)制备含碳点内层纺丝溶液
将PEO、丝素蛋白、壳聚糖中的一种或几种溶解在水、乙酸或乙醚中,得到高聚物溶液,再加入葡萄皮基碳点;
3)制备载药的外层纺丝溶液
将PI、PCL、PLGA、PLA、PU中的一种溶解在含黄芩苷的DMF、DCM或HFIP中的一种或几种中;
4)以步骤2)制备的含碳点内层纺丝溶液为芯,步骤2)制备的载药的外层纺丝溶液为壳采用同轴共轭静电纺丝方法制备纳米纤维膜;外层流速为内层流速的10~15倍;
5)利用直径1~6mm的棒状模具,将步骤4)得到的纳米纤维膜卷成所需壁厚的纳米纤维管,室温干燥;
6)将步骤5)制备的纳米纤维管包裹于患处血管,通过连接位点固定,并通过纳米纤维管的层层自支撑塑形血管释放药物。
步骤1)中所述的葡萄皮粉末的制备方法,葡萄皮用去离子水和酒精超声清洗两次以上,干燥并研磨成粉。
步骤1)中葡萄皮粉末与尿素按照质量比1︰(2~3)。
步骤2)中所述的PEO溶液中,PEO质量浓度为10%~18%。
步骤2)中所述的葡萄皮基碳点占PEO溶液的质量浓度为0.1%~0.5%。
步骤3)中所述的载药的外层纺丝溶液中黄芩苷的质量浓度为1%~5%。
步骤3)中所述的将PI、PCL、PLGA、PLA、PU中的一种溶解在DMF、DCM或HFIP中的一种或几种中,配制浓度为15%~20%的PI或PCL、或PLGA、或PLA、或PU溶液。
步骤1)中真空冷冻干燥为-20~-40℃温度下干燥。
与现有技术相比,本发明的有益效果是:
本发明通过同轴静电纺丝方式将零维结构光致发光碳点包裹在轴壳介孔纳米纤维的中空芯结构内,最终形成形态良好的纳米纤维膜,以用于制备外包型血管支架。具体优点是:
1、本发明制备的包裹碳点的载药血管支架,可以有效的防止支架对血管内膜的损伤,增强支架与血管的物理相容性和生物相容性;
2、通过植入药物纳米纤维以及药物的持续原位释放,可以实现药物直接作用于患处,解决黄芩苷存在的稳定性不佳,脂溶性和水溶性都较差,口服生物利用度低,不易透过血脑屏障等问题。
3、位于介孔纳米纤维中空内芯的碳点,在“可视化”检测药物释放速率,并将药物的副作用降至最小化起着至关重要的作用。
4、碳点CDs通过其荧光特性在可视化病理部位的药物蓄积和活性方面具有优势,这在评估药物的治疗效果方面起着不可估量的作用。
5、制备方法简单可靠,且制备材料成本较低,可应用于工业制备的工艺和临床医疗。
附图说明
图1为静电纺同轴纳米纤维设备的示意图;
图2为实施例1中制备的含碳点载药PI-PEO同轴纳米纤维膜的扫描电镜图;
图3为实施例1中制备的含碳点载药PI-PEO同轴纳米纤维膜的荧光显微镜图。
图1中,1-注射器一2-注射器二3-同轴喷丝头4-接收器。
具体实施方式
下面结合说明书附图对本发明进行详细地描述,但是应该指出本发明的实施不限于以下的实施方式。
应用以黄芩苷为模板药物,拟制备生物相容性好的中空包裹碳点的轴壳结构纳米多孔纤维,将其作载药纳米纤维管进行血管外包支架植入,黄芩苷分子离开载体,接触附着于介孔纳米纤维中空内芯的碳点,通过碳点荧光成像“可视化”实时反映药物释放速率,开发一类黄芩苷荧光可视药物递送系统。通过碳点荧光检测药物控释放位点、释放量,达到实时监测从而及时调整医疗方案的临床监测目的。
具体的,外包型载药可视纳米纤维人工血管支架的制备方法,包括以下步骤:
1)制备葡萄皮基碳点
将葡萄皮粉末与尿素按照质量比1︰(2~3)混合,在190~220℃下加热6~10h冷却至室温后,将黑色产物超声溶解在去离子水中,离心处理,用透析对收集的上清液进行透析膜48~72h,真空冷冻干燥;
葡萄皮粉末的制备方法,葡萄皮用去离子水和酒精超声清洗两次以上,干燥并研磨成粉,得到葡萄皮基碳点。
2)制备含碳点内层纺丝溶液
将PEO、丝素蛋白、壳聚糖中的一种或几种溶解在水、乙酸或乙醚中,得到PEO溶液,PEO质量浓度为10%~18%,再加入葡萄皮基碳点,葡萄皮基碳点占PEO溶液的质量浓度为0.1%~0.5%。
3)制备载药的外层纺丝溶液
将可溶性PI(聚酰亚胺)、PCL(聚己内酯)、PLGA(聚乳酸-羟基乙酸共聚物))、PLA(聚乳酸)、PU(聚氨酯)中的一种溶解在DMF、DCM或HFIP中的一种或几种中,配制质量浓度为15%~20%的PI或PCL、或PLGA、或PLA、或PU溶液,再向其中加入黄芩苷,得到载药的外层纺丝溶液。载药的外层纺丝溶液中黄芩苷的质量浓度为1%~5%。
含碳点内层纺丝使用不溶于载药的外层纺丝的溶液,这样才可以制备出同轴纳米纤维,否则内层结构的溶液和外层结构的溶液易相溶的话,高压形成泰勒锥的时候无法分层,纳米纤维不成同轴共轭结构。芯结构的PEO、丝素蛋白等因为有着较好的生物相容性和可降解性,常作为药物缓释的载体使用。同时壳结构溶液所使用的高聚物均有着较好的生物相容性,但不容易降解,可以长期存在于体内,不至于长时间使用之后机械性能下降。
4)以步骤2)制备的含碳点内层纺丝溶液为芯,步骤2)制备的载药的外层纺丝溶液为壳采用同轴共轭静电纺丝方法制备纳米纤维膜;外层流速为内层流速的10~15倍。
见图1,静电纺同轴纳米纤维设备包括注射器一(带有内芯溶液导管)、注射器二(带有外壳溶液导管)、同轴喷丝头、滚筒接收器,同轴喷丝头为内外同心圆结构的空心双管,注射器一的注射口与同轴喷丝头的内管连接,注射器二的注射口与同轴喷丝头的外管连接,接收器用于收集同轴纳米纤维。内管内径0.2~0.3mm,外管内壁直径为0.8~1.2mm,电压为7kV~25kV伏,针头与接受滚轮的距离为10~30厘米,滚轮转速为100~150rpm,在滚筒接收器上形成纳米纤维膜,见图2、图3,内层纺丝溶液形成芯结构,外层纺丝溶液形成壳结构。
制备纳米纤维膜时,将载药内层纺丝溶液含银离子外层纺丝溶液充入注射器二,将载药内层纺丝溶液含银离子外层纺丝溶液注入同轴喷丝头的外管,形成的纳米纤维膜由接收器收集。
5)将纳米纤维膜取下,裁剪至所需要的长度、宽度。利用直径1~6mm的棒状模具,棒状模具为外套PDMS(聚二甲基硅氧烷)层的不锈钢棒的,将纳米纤维膜卷成所需壁厚的纳米纤维管,并不封口,即该纳米纤维管横截面为圆弧形,室温干燥24h以上。
6)将步骤5)制备的纳米纤维管以绷带状包裹于患处血管,通过连接位点(可生物降解的微针(MN))固定,并通过纳米纤维管的层层自支撑塑形血管,起到释放药物。药物可视化追踪,防止血管萎缩的三层作用。
【实施例1】
外包型载药可视纳米纤维人工血管支架的制备方法,包括以下步骤:
1)制备葡萄皮基碳点,葡萄皮用去离子水和酒精超声清洗两次,然后干燥并研磨成粉。将2.0g葡萄皮粉末和4.0g尿素混合并转移到50mL坩埚中,然后在200℃下加热8小时冷却至室温后,将黑色产物超声溶解在20mL去离子水中,并离心以10000rpm离心20分钟以除去不溶性和较大的颗粒。用透析对收集的上清液进行透析膜(MWCO 500)48小时以去除未反应的原料以及其他杂质。最后,在50℃真空冷冻干燥48小时后,获得葡萄皮基碳点。
2)制备含碳点内层纺丝溶液,将PEO溶解在水或乙醚中,质量浓度为18%,加入葡萄皮基碳点检测药物的荧光传感器,用量占溶液质量的0.5%,得到含碳点内层纺丝溶液。
3)制备载药的外层纺丝溶液,将可溶性PI粉末溶解在DMF溶液中,使用黄芩苷为药物源,调节黄芩苷占溶液质量百分比1%~5%;
4)用如图1所示的静电纺同轴纳米纤维设备制造纳米纤维膜,并根据需要进行长度、宽度的裁剪;利用直径1~6mm的棒状模具,棒状模具为外套PDMS(聚二甲基硅氧烷)层的不锈钢棒的,纳米纤维膜卷成所需壁厚的纳米纤维管成型备用;该纳米纤维管并不封口,即该纳米纤维管横截面为圆弧形,室温干燥24h以上。
5)根据临床实际需求,将纳米纤维管以绷带状包裹于患处,通过可生物降解的微针(MN)作为连接位点固定,并通过纳米纤维膜的层层自支撑塑形血管,起到释放药物、药物可视化追踪,防止血管萎缩的三重作用。
【实施例2】
外包型载药可视纳米纤维人工血管支架的制备方法,包括以下步骤:
1)采用与实施例1相同的工艺方法制备葡萄皮基碳点。
2)将PEO溶解在水或乙醚中得到PEO溶液,PEO质量浓度为10%,向PEO溶液加入葡萄皮基碳点得到含碳点内层纺丝溶液,其中,葡萄皮基碳点用量占溶液质量的0.5%。
3)将PCL溶解在DCM与DMF的混合溶液中得到质量浓度为10%的PCL溶液,混合溶液中DCM与DMF的质量比为4:6,再加入黄芩苷为药物源,黄芩苷占PCL溶液质量百分比为1%~5%。
4)用如图1所示的静电纺同轴纳米纤维设备制造纳米纤维膜,并根据需要进行长度、宽度的裁剪;利用直径1~6mm的棒状模具,棒状模具为外套PDMS(聚二甲基硅氧烷)层的不锈钢棒的,纳米纤维膜卷成所需壁厚的纳米纤维管状成型备用;该纳米纤维管并不封口,即该纳米纤维管横截面为圆弧形,室温干燥24h以上。
5)根据临床实际需求,将纳米纤维管以绷带状包裹于患处,通过通过可生物降解的微针(MN)为连接位点固定,并通过纳米纤维膜的层层自支撑塑形血管,起到释放药物、药物可视化追踪,防止血管萎缩的三重作用。
【实施例3】
外包型载药可视纳米纤维人工血管支架的制备方法,包括以下步骤:
1)采用与实施例1相同的工艺方法制备葡萄皮基碳点。
2)制备含碳点内层纺丝溶液,将PEO溶解在水或乙醚中,质量浓度为15%,加入葡萄皮基碳点检测药物的荧光传感器,用量占溶液质量的0.3%,得到含碳点内层纺丝溶液。
3)将PU溶解在HFIP中得到质量浓度为20%的PU溶液,再加入黄芩苷为药物源,黄芩苷占PU溶液质量百分比为1%~5%。
4)用如图1所示的静电纺同轴纳米纤维设备制造纳米纤维膜,并根据需要进行长度、宽度的裁剪;利用直径1~6mm的棒状模具,棒状模具为外套PDMS(聚二甲基硅氧烷)层的不锈钢棒的,纳米纤维膜卷成所需壁厚的纳米纤维管状成型备用;该纳米纤维管并不封口,即该纳米纤维管横截面为圆弧形,室温干燥24h以上。
5)根据临床实际需求,将纳米纤维管以绷带状包裹于患处,通过可生物降解的微针(MN)为连接位点固定,并通过纳米纤维膜的层层自支撑塑形血管,起到释放药物、药物可视化追踪,防止血管萎缩的三重作用。
【实施例4】
外包型载药可视纳米纤维人工血管支架的制备方法,包括以下步骤:
1)采用与实施例1相同的工艺方法制备葡萄皮基碳点。
2)制备含碳点内层纺丝溶液,将丝素蛋白溶解在乙酸中,质量浓度为18%,加入葡萄皮基碳点检测药物的荧光传感器,用量占溶液质量的0.3~0.5%,得到含碳点内层纺丝溶液。
3)将PCL溶解在DCM与DMF的混合溶液中得到质量浓度为10%的PCL溶液,混合溶液中DCM与DMF的质量比为4:6,再加入黄芩苷为药物源,黄芩苷占PCL溶液质量百分比为1%~5%。
4)用如图1所示的静电纺同轴纳米纤维设备制造纳米纤维膜,并根据需要进行长度、宽度的裁剪;利用直径1~6mm的棒状模具,棒状模具为外套PDMS(聚二甲基硅氧烷)层的不锈钢棒的,纳米纤维膜卷成所需壁厚的纳米纤维管状成型备用;该纳米纤维管并不封口,即该纳米纤维管横截面为圆弧形,室温干燥24h以上。
5)根据临床实际需求,将纳米纤维管以绷带状包裹于患处,通过可生物降解的微针(MN)作为连接位点固定,并通过纳米纤维膜的层层自支撑塑形血管,起到释放药物、药物可视化追踪,防止血管萎缩的三重作用。
【实施例5】
与实施例4的区别在于制备含碳点内层纺丝溶液不同,具体为:
制备含碳点内层纺丝溶液,将壳聚糖溶解在乙酸中,质量浓度为20%,加入葡萄皮基碳点检测药物的荧光传感器,用量占溶液质量的0.3~0.5%,得到含碳点内层纺丝溶液。
Claims (8)
1.一种外包型载药可视纳米纤维人工血管支架的制备方法,其特征在于,包括以下步骤:
1)制备葡萄皮基碳点
将葡萄皮粉末与尿素混合,在190~220℃下加热6~10h冷却至室温后,将黑色产物超声溶解在去离子水中,离心处理,用透析对收集的上清液进行透析膜48~72h,真空冷冻干燥;
2)制备含碳点内层纺丝溶液
将PEO、丝素蛋白、壳聚糖中的一种或几种溶解在水、乙酸或乙醚中,得到高聚物溶液,再加入葡萄皮基碳点;
3)制备载药的外层纺丝溶液
将PI、PCL、PLGA、PLA、PU中的一种溶解在含黄芩苷的DMF、DCM或HFIP中的一种或几种中;
4)以步骤2)制备的含碳点内层纺丝溶液为芯,步骤2)制备的载药的外层纺丝溶液为壳采用同轴共轭静电纺丝方法制备纳米纤维膜;外层流速为内层流速的10~15倍;
5)利用直径1~6mm的棒状模具,将步骤4)得到的纳米纤维膜卷成所需壁厚的纳米纤维管,室温干燥;
6)将步骤5)制备的纳米纤维管包裹于患处血管,通过连接位点固定,并通过纳米纤维管的层层自支撑塑形血管释放药物。
2.根据权利要求1所述的一种外包型载药可视纳米纤维人工血管支架的制备方法,其特征在于,步骤1)中所述的葡萄皮粉末的制备方法,葡萄皮用去离子水和酒精超声清洗两次以上,干燥并研磨成粉。
3.根据权利要求1所述的一种外包型载药可视纳米纤维人工血管支架的制备方法,其特征在于,步骤1)中葡萄皮粉末与尿素按照质量比1︰(2~3)。
4.根据权利要求1所述的一种外包型载药可视纳米纤维人工血管支架的制备方法,其特征在于,步骤2)中所述的PEO溶液中,PEO质量浓度为10%~18%。
5.根据权利要求1所述的一种外包型载药可视纳米纤维人工血管支架的制备方法,其特征在于,步骤2)中所述的葡萄皮基碳点占PEO溶液的质量浓度为0.1%~0.5%。
6.根据权利要求1所述的一种外包型载药可视纳米纤维人工血管支架的制备方法,其特征在于,步骤3)中所述的载药的外层纺丝溶液中黄芩苷的质量浓度为1%~5%。
7.根据权利要求1所述的一种外包型载药可视纳米纤维人工血管支架的制备方法,其特征在于,步骤3)中所述的将PI、PCL、PLGA、PLA、PU中的一种溶解在DMF、DCM或HFIP中的一种或几种中,配制浓度为15%~20%的PI或PCL、或PLGA、或PLA、或PU溶液。
8.根据权利要求1所述的一种外包型载药可视纳米纤维人工血管支架的制备方法,其特征在于,步骤1)中真空冷冻干燥为-20~-40℃温度下干燥。
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