CN106049026A - 一种负载姜黄素的胶原‑pcl‑pvp复合微纳纤维膜及其制备方法 - Google Patents

一种负载姜黄素的胶原‑pcl‑pvp复合微纳纤维膜及其制备方法 Download PDF

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CN106049026A
CN106049026A CN201610552326.4A CN201610552326A CN106049026A CN 106049026 A CN106049026 A CN 106049026A CN 201610552326 A CN201610552326 A CN 201610552326A CN 106049026 A CN106049026 A CN 106049026A
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pcl
curcumin
pvp
collagen
composite micro
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但卫华
刘新华
但年华
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Sichuan University
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Abstract

本发明公开了一种负载姜黄素的胶原‑PCL‑PVP复合微纳纤维膜及其制备方法，其特点是将一定体积比的二氯甲烷和乙醇混合，制成静电纺丝溶剂，接着将一定质量比例的姜黄素、PCL和PVP混合加入到静电纺丝溶剂中，室温下搅拌至透明，配制成浓度为6%～12%的纺丝母液，将纺丝母液注入静电纺丝装备中进行纺丝，得到负载姜黄素的PCL‑PVP复合微纳纤维膜，再将复合膜放置于超声波发生器中实施间歇性超声处理，最后将复合膜浸渍在I型胶原溶液中，经自然晾干后，制得负载姜黄素的胶原‑PCL‑PVP复合微纳纤维膜。该材料具有良好的生物相容性和可生物降解性，能促进细胞生长、增殖、粘附，又具备抗炎、抗氧化、促进伤口愈合等功效，可用作组织工程支架、高级敷料、止血材料等。

Description

一种负载姜黄素的胶原-PCL-PVP复合微纳纤维膜及其制备 方法
技术领域
本发明涉及了一种负载姜黄素的胶原-PCL-PVP复合微纳纤维膜及其制备方法，属于生物医用材料制备领域。
背景技术
以静电纺丝技术制备的微纳纤维膜材料因其比表面积大、孔隙率高以及良好的物理机械性能等优点，已广泛应用于药物缓/控释放、滤纸、半透膜、生物传感器、能源储备、细胞载体、环境工程、组织工程支架等多种领域。聚己内酯（PCL）是一种半晶型高分子聚合物，具有优异的生物可降解性，目前美国FDA批准PCL用作手术缝线、药物控释、骨折固定和组织工程支架材料等的基材。然而，研究表明，PCL存在降解率慢、亲水性差等缺点，限制了其生物医学领域应用。聚乙烯吡咯烷酮（PVP）是一种无定形物，具有良好的亲水性、溶解性、成膜性、粘合性和配合性等，在生物医学领域常常用作润滑涂层、药物成分、外伤包扎带等。研究者常将PVP和PCL有机复合，取长补短。二者的复合静电纺丝也有研究，刘青青等人以二氯甲烷为溶剂，通过静电纺丝获得了PCL-PVP微纳纤维，亲水性得到了很大的提高（刘青青，贾永堂，朱湘英，等. PCL/PVP电纺复合纤维膜的制备及其亲疏水性能研究[J]. 产业用纺织品，2012（3）：11-15）；Kim G M等以氯仿和甲醇为溶剂，制备了PCL-PVP纤维，通过降解性实验和细胞实验发现，PVP的加入不仅加速了PCL纤维的降解还增强了它的细胞黏附性（Kim G M,Le K H T, Giannitelli S M, et al. Electrospinning of PCL/PVP blends fortissue engineering scaffolds[J]. Journal of Materials Science: Materials inMedicine, 2013, 24(6): 1425-1442）。然而，他们所获得的PCL-PVP复合纤维直径在1.5μm左右，纤维表面凹凸不平，呈“树皮”状，不利于细胞的增殖和迁移，再者，PVP与PCL本身的生物惰性也在一定程度上影响了其在生物材料领域中的应用。
型胶原具有低免疫性、促进细胞增殖、促进血小板凝聚、良好的生物相容性、生物可降解性等诸多生物特性，已被广泛应用于各型生物材料的制备。近年来，静电纺丝胶原制备组织工程支架材料日益受到关注，然而已发现，胶原在目前报道的常用静电纺丝溶剂中其三股螺旋结构基本不能完整保留，极大地降低了其优越的生物活性，限制了其医学应用，在静电纺丝后将胶原直接引入到微纳纤维支架中可能是保持其三股螺旋结构的最为简单的途径。
综上所述，本发明为了解决目前PVP-PCL静电纺丝微纳纤维膜平均直径大、纤维表面凹凸不平、生物惰性及静电纺丝胶原其三股螺旋结构不能保持等的问题，以二氯甲烷和乙醇为混合溶剂，通过静电纺丝制备得到纤维表面光滑的PVP-PCL复合微纳纤维膜，添加一定量的姜黄素使复合膜具有抗炎、抗氧化、抗菌等生物学功能，再将I型胶原通过浸渍法直接引入到复合膜中，使完整地保持了胶原本身的三股螺旋结构，该膜材料具有良好的生物相容性、亲水性、可生物降解性等优良性能，又兼具一定的抗炎、抗氧化、抗菌等功效，生物活性好，可用作组织工程支架、高级敷料、止血材料等。
发明内容
本发明的目的是针对现有技术的不足而提供的一种负载姜黄素的胶原-PCL-PVP复合微纳纤维膜。该膜材料具有良好的生物相容性、亲水性、可生物降解性等优良性能，又兼具一定的抗炎、抗氧化、抗菌等功效，生物活性好，能促进创面愈合和修复，可用作组织工程支架、高级敷料、止血材料等。
为实现上述目的，本发明采用如下技术方案：
（1）静电纺丝制备负载姜黄素的PCL-PVP复合微纳纤维膜：将以不同体积比例的二氯甲烷和乙醇混合，制备得到静电纺丝母液，其中二氯甲烷含量体积百分比为 50%～70%；接着将按质量比为8～2∶2～8将PCL与PVP共混，室温下搅拌至透明，配制成质量浓度为 6%～12%的混合液，再向上述PCL-PVP混合液中添加浓度为5～20mg/ml的姜黄素，室温下快速搅拌至完全溶解，得到静电纺丝母液，将静电纺丝母液注入静电纺丝机中进行静电纺丝，得到负载姜黄素的PCL-PVP复合微纳纤维膜；
（2）超声处理负载姜黄素的PCL-PVP复合微纳纤维膜：将步骤（1）制备得到的负载姜黄素的PCL-PVP复合微纳纤维膜浸泡在装有超纯水的超声波发生器中，在超声波频率为20～40 kHz、功率为50～100w下，对微纳膜实施间歇性超声波处理，每超声作用5～10min，停止作用5～10min，超声处理总时间为30～60min；
（3）负载姜黄素的胶原-PCL-PVP复合微纳纤维膜的制备：在4-10℃恒温条件下，将I型胶原溶解在pH为7.2～7.4的PBS缓冲液中，配制成5mg/ml～10mg/ml的I型胶原溶液，再将超声处理后的负载姜黄素的PCL-PVP复合微纳纤维膜浸渍在I型胶原溶液中2～10h，最后在无菌洁净工作台上晾干，再经剂量为 6～30KGy/h60Co所产生的γ射线消毒灭菌，成型包装，得到负载姜黄素的胶原-PCL-PVP复合微纳纤维膜。
在上述的制备方法中，步骤（1）中的PCL与PVP均为市售；步骤（3）中姜黄素为市售；步骤（1）中的负载姜黄素的PCL-PVP复合微纳纤维膜的纤维直径可以通过调节静电纺丝过程中的纺丝参数（静电场电压、喷丝孔直径、滚筒的转速、纺丝距离等）来有效控制。
负载姜黄素的胶原-PCL-PVP复合微纳纤维膜的关键性能应符合以下要求：
外观：微黄色膜状，无肉眼可见之杂质；
水分含量：≤20%（wt）；
重金属含量：≤10μg/g；
纤维直径:0.1nm～1μm;
孔隙率：≥90%；
拉伸强度:≥2.5MPa;
断裂伸长率:100~170%;
细胞毒性：细胞毒性反应不大于1级；
无菌试验：无菌。
本技术与现有技术相比，具有如下优点：
（1）与已有报道的PCL-PVP复合微纳纤维膜不同，本发明以二氯甲烷和乙醇为混合溶剂，通过静电纺丝制备得到的PVP-PCL复合微纳纤维膜，纤维表面光滑、直径均一，有利于细胞的增殖与迁移；
（2）本发明使用浸渍法将I型胶原直接引入到复合膜中，胶原完整地保持了其本身的三股螺旋结构；再者，将I型胶原引入到PCL-PVP复合膜，通过协同作用使复合膜具有较高的生物活性；
（3）作为支架材料，其孔尺寸、孔隙率和孔形态对细胞的生长行为有很大的影响，一定程度上决定了细胞长入支架的程度，也能对细胞的生长、增殖及细胞外基质的形成、营养的传输等方面起着决定作用。本发明使用超声法是利用超声波的振动使支架中的纤维分离，产生较大的孔隙，能有效增加纤维膜孔径的尺寸同时增大纤维膜的孔隙，有利于细胞长入、生长和增殖，且超声法易于实现，适合工业化应用；
（4）本发明为首次制备得到了负载姜黄素的胶原-PCL-PVP复合微纳纤维膜，姜黄素具有良好的抗氧化作用、抗菌活性、抗癌/抗肿瘤作用、抗病毒作用等性能，根据协同理论，复合膜同时具备了PCL-PVP复合膜良好的亲水性、可生物降解性等性能，又具有了胶原优异的生物活性，更兼具姜黄素一定的抗炎、抗氧化、抗菌等功效，综合性能优良，功能多样，易于形成规模化产业链，取得规模化效益的良好前景。
附图说明
图1为PCL-PVP复合微纳纤维膜的扫描电镜（SEM）图。
具体实施方式
下面通过实施对本发明进行具体的描述，有必要在此指出的是本实施例只用于对本发明进行进一步说明，而不能理解为对本发明保护范围的限制，该领域的技术熟练人员可以根据上述发明的内容作出非本质的改进和调整。
实施例1
（1）静电纺丝制备负载姜黄素的PCL-PVP复合微纳纤维膜：将以一定体积比例的二氯甲烷和乙醇混合，制备得到静电纺丝母液，其中二氯甲烷含量体积百分比为 50%；接着将按质量比为4∶1将PCL与PVP共混，室温下搅拌至透明，配制成质量浓度为6%的混合液，再向上述PCL-PVP混合液中添加浓度为5mg/ml的姜黄素，室温下快速搅拌至完全溶解，得到静电纺丝母液，将静电纺丝母液注入静电纺丝机中，按电压10kv、溶液流量为0.04mm/min、接收距离为10cm、转速为15r/min的纺丝条件进行静电纺丝，得到负载姜黄素的PCL-PVP复合微纳纤维膜；
（2）超声处理负载姜黄素的PCL-PVP复合微纳纤维膜：将步骤（1）制备得到的负载姜黄素的PCL-PVP复合微纳纤维膜浸泡在装有超纯水的超声波发生器中，在超声波频率为20kHz、功率为50w下，对微纳膜实施间歇性超声波处理，每超声作用10min，停止作用10min，超声处理总时间为60min；
（3）负载姜黄素的胶原-PCL-PVP复合微纳纤维膜的制备：在4℃恒温条件下，将I型胶原溶解在pH为7.2的PBS缓冲液中，配制成5mg/ml的I型胶原溶液，再将超声处理后的负载姜黄素的PCL-PVP复合微纳纤维膜浸渍在I型胶原溶液中2h，最后在无菌洁净工作台上晾干，再经剂量为 6KGy/h60Co所产生的γ射线消毒灭菌，成型包装，得到负载姜黄素的胶原-PCL-PVP复合微纳纤维膜。
实施例2
（1）静电纺丝制备负载姜黄素的PCL-PVP复合微纳纤维膜：将以一定体积比例的二氯甲烷和乙醇混合，制备得到静电纺丝母液，其中二氯甲烷含量体积百分比为60%；接着将按质量比为3∶1将PCL与PVP共混，室温下搅拌至透明，配制成质量浓度为8%的混合液，再向上述PCL-PVP混合液中添加浓度为12mg/ml的姜黄素，室温下快速搅拌至完全溶解，得到静电纺丝母液，将静电纺丝母液注入静电纺丝机中，按电压15kv、溶液流量为0.1mm/min、接收距离为15cm、转速为20r/min的纺丝参数进行静电纺丝，得到负载姜黄素的PCL-PVP复合微纳纤维膜；
（2）超声处理负载姜黄素的PCL-PVP复合微纳纤维膜：将步骤（1）制备得到的负载姜黄素的PCL-PVP复合微纳纤维膜浸泡在装有超纯水的超声波发生器中，在超声波频率为30kHz、功率为60w下，对微纳膜实施间歇性超声波处理，每超声作用8min，停止作用8min，超声处理总时间为45min；
（3）负载姜黄素的胶原-PCL-PVP复合微纳纤维膜的制备：在8℃恒温条件下，将I型胶原溶解在pH为7.3的PBS缓冲液中，配制成8mg/ml的I型胶原溶液，再将超声处理后的负载姜黄素的PCL-PVP复合微纳纤维膜浸渍在I型胶原溶液中5h，最后在无菌洁净工作台上晾干，再经剂量为 15KGy/h60Co所产生的γ射线消毒灭菌，成型包装，得到负载姜黄素的胶原-PCL-PVP复合微纳纤维膜。
实施例3
（1）静电纺丝制备负载姜黄素的PCL-PVP复合微纳纤维膜：将以不同体积比例的二氯甲烷和乙醇混合，制备得到静电纺丝母液，其中二氯甲烷含量体积百分比为70%；接着将按质量比为1∶1将PCL与PVP共混，室温下搅拌至透明，配制成质量浓度为12%的混合液，再向上述PCL-PVP混合液中添加浓度为20mg/ml的姜黄素，室温下快速搅拌至完全溶解，得到静电纺丝母液，将静电纺丝母液注入静电纺丝机中，按电压20kv、溶液流量为0.2mm/min、接收距离为20cm、转速为30r/min的纺丝参数进行静电纺丝，得到负载姜黄素的PCL-PVP复合微纳纤维膜；
（2）超声处理负载姜黄素的PCL-PVP复合微纳纤维膜：将步骤（1）制备得到的负载姜黄素的PCL-PVP复合微纳纤维膜浸泡在装有超纯水的超声波发生器中，在超声波频率为40kHz、功率为100w下，对微纳膜实施间歇性超声波处理，每超声作用5min，停止作用5min，超声处理总时间为30min；
（3）负载姜黄素的胶原-PCL-PVP复合微纳纤维膜的制备：在10℃恒温条件下，将I型胶原溶解在pH为7.4的PBS缓冲液中，配制成10mg/ml的I型胶原溶液，再将超声处理后的负载姜黄素的PCL-PVP复合微纳纤维膜浸渍在I型胶原溶液中10h，最后在无菌洁净工作台上晾干，再经剂量为 30KGy/h60Co所产生的γ射线消毒灭菌，成型包装，得到负载姜黄素的胶原-PCL-PVP复合微纳纤维膜。

Claims (3)

1.一种负载姜黄素的胶原-PCL-PVP复合微纳纤维膜的制备方法，其特征在于该方法包括以下步骤：
（1）静电纺丝制备负载姜黄素的PCL-PVP复合微纳纤维膜：将以不同体积比例的二氯甲烷和乙醇混合，制备得到静电纺丝母液，其中二氯甲烷含量体积百分比为 50%～70%；接着将按质量比为8～2∶2～8将PCL与PVP共混，室温下搅拌至透明，配制成质量分数为6%～12%的混合液，再向上述PCL-PVP混合液中添加浓度为5～20mg/ml的姜黄素，室温下快速搅拌至完全溶解，得到静电纺丝母液，将静电纺丝母液注入静电纺丝机中进行静电纺丝，得到负载姜黄素的PCL-PVP复合微纳纤维膜；
（2）超声处理负载姜黄素的PCL-PVP复合微纳纤维膜：将步骤（1）制备得到的负载姜黄素的PCL-PVP复合微纳纤维膜浸泡在装有超纯水的超声波发生器中，在超声波频率为20～40 kHz、功率为50～100w下，对微纳膜实施间歇性超声波处理，每超声作用5～10min，停止作用5～10min，超声处理总时间为30～60min；
（3）负载姜黄素的胶原-PCL-PVP复合微纳纤维膜的制备：在4-10℃恒温条件下，将I型胶原溶解在pH为7.2～7.4的PBS缓冲液中，配制成5mg/ml～10mg/ml的I型胶原溶液，再将超声处理后的负载姜黄素的PCL-PVP复合微纳纤维膜浸渍在I型胶原溶液中2～10h，最后在无菌洁净工作台上晾干，再经剂量为 6～30KGy/h60Co所产生的γ射线消毒灭菌，成型包装，得到负载姜黄素的胶原-PCL-PVP复合微纳纤维膜；所制得的负载姜黄素的胶原-PCL-PVP复合微纳纤维膜，其关键性能指标如下：
外观：微黄色膜状，无肉眼可见之杂质；
水分含量：≤20%（wt）；
重金属含量：≤10μg/g；
纤维直径:0.1nm～1μm;
孔隙率：≥90%；
拉伸强度:≥2.5MPa;
断裂伸长率:100~170%;
细胞毒性：细胞毒性反应不大于1级；
无菌试验：无菌。
2.如权利要求1所述的一种负载姜黄素的胶原-PCL-PVP复合微纳纤维膜的制备方法，其特征在于所述的I型胶原是从猪皮、猪腱、牛皮、牛腱、驴皮、驴腱、马皮、马腱、鼠皮或鼠尾腱中的任一种制备获得的。
3.如权利要求1所述的一种负载姜黄素的胶原-PCL-PVP复合微纳纤维膜的制备方法，其特征在于所述的静电纺丝条件为：电压10～20kv、溶液流量为0.04～0.2mm/min、接收距离为10～20cm、转速为15-30r/min。
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