CN116196472A - 一种双导电载药复合神经导管及其制备方法 - Google Patents
一种双导电载药复合神经导管及其制备方法 Download PDFInfo
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Abstract
本发明公开了一种双导电载药复合神经导管，该导管为中空管状结构，导管内层为含有导电物质二维碳化钛的聚乳酸静电纺丝纤维；导管外层为导电物质聚吡咯涂层，导管中间层为含齐墩果的聚乳酸静电纺丝纤维，本发明实现了导电的持续性，通过齐墩果酸的缓慢释放进一步促进受损神经元的修复及其轴突的再生，在神经修复治疗中具有很好的应用前景。
Description
技术领域
本发明涉及一种周围神经修复生物材料，具体涉及一种双导电载药复合神经导管及其制备方法。
背景技术
周围神经损伤是由各种原因引起的受该神经支配的区域无法接收近端神经的调控，从而造成相应的功能障碍。目前，自体神经移植是治疗的金标准，但是存在神经供体的来源有限，供体不足和手术时间过长等问题。导电神经导管是人工制成的用以桥接神经断端的组织工程管状物，研究人员发现，电刺激可促进神经突生长、神经元迁移、神经再生和部分早期功能恢复，此外，电刺激还可增强神经再生过程中轴突再生的速度和方向。
二维过渡金属碳氮化物(MXene)是一种二维层状纳米材料,一般是通过化学刻蚀的方法将三元层状化合物MAX相中的A层选择性刻蚀掉作为一种新型二维材料，二维碳化钛作为其中一种，拥有接近金属的电导性。二维碳化钛除了具备石墨烯等传统二维材料的性能外，还同时具有良好的亲水性以及表面官能团(-F、-OH、-О等)可调控性。
聚吡咯由单体吡咯通过电化学法或化学氧化聚合法聚合得到合成。原始状态的聚吡咯是不导电的，但是经过掺杂后，聚吡咯的电导率可急剧上升。聚吡咯在具有良好的导电性的同时，还具有极佳的化学稳定性。体外和体内实验表明，聚吡咯同细胞和组织都具有相当出色的生物相容性,并且在体内长时间植入时不会对机体产生明显的损伤，是一种十分有潜力的组织工程支架材料。
齐墩果酸是一种普遍存在的天然五环三萜化合物，广泛存在于食用水果、蔬菜和药用植物中。近年来，齐墩果酸在治疗治疗支气管炎、肺炎、急性扁桃体炎、牙周炎、菌痢、急性肠胃炎、泌尿系统感染等抗击炎症方面取得一定进展，但并未发现其应用于周围神经炎症的报导。
静电纺丝是一种适用于多种聚合物的通用技术，静电纺丝法制备的静电纺丝纤维支架可以从成分和结构上实现对天然细胞外基质的仿生；支架具有高孔隙率和高比表面积的特点,利于细胞的黏附、迁移、生长及分化。
发明内容
本发明的目的是提供一种双导电载药复合神经导管及其制备方法，旨在提高周围神经损伤的修复效果。
上述目的是通过以下技术方案实现的：
本发明的第一个目的是提供一种双导电载药复合神经导管，该导管为中空管状结构，导管内层为含有导电物质二维碳化钛的聚乳酸静电纺丝纤维；导管外层为导电物质聚吡咯涂层，导管中间层为含齐墩果的聚乳酸静电纺丝纤维。
其中，所述导管内层的厚度为0.1-0.3mm，导管外层的厚度为0.08-0.15mm，导管中间层的厚度为0.5-2mm。
本发明的第二个目的是提供一种双导电载药复合神经导管的制备方法，该方法包括以下步骤：
1)用有机溶剂溶解聚乳酸和平均粒径100-1000nm的二维碳化钛，制成聚乳酸浓度5-10％、二维碳化钛浓度0.1-1％的纺丝溶液，进行静电纺丝，得到导电定向静电纺丝纤维；
2)用有机溶剂溶解聚乳酸和齐墩果酸，制成聚乳酸浓度5-10％、齐墩果酸浓度0.05-0.2％的纺丝溶液，将步骤1)制备的导电定向静电纺丝纤维裁剪成小块并包覆在接收器上，用含有齐墩果的纺丝溶液进行静电纺丝，形成载药神经导管；
3)用有机溶剂溶解聚吡咯，制成聚吡咯浓度1-5％的喷涂溶液，均匀喷涂到步骤2)制备的载药神经导管表面，室温干燥后脱模，得双导电载药复合神经导管。
优选地，所述静电纺丝的工艺参数为：接收器转速1000-1500r/min，推注速度为0.1-1mm/min，电压10-15KV，接收距离20-30cm，温度20-30℃，湿度30-50％。
优选地，所述二维碳化钛的平均粒径为300-500nm，在使用前通过研磨使其达到所需的粒径。
优选地，所述纺丝溶液中，聚乳酸的浓度为6-8％。
优选地，所述纺丝溶液中，二维碳化钛的浓度为0.5-0.8％；齐墩果酸的浓度为0.05-0.1％。
根据本发明的一个实施例，其中一种较佳的制备方法如下：
1)导电定向静电纺丝纤维的制备
用二氯甲烷溶解聚乳酸和平均粒径500nm的二维碳化钛，制成聚乳酸浓度8％、二维碳化钛浓度0.8％的纺丝溶液，进行静电纺丝，得到导电定向静电纺丝纤维；
2)载药神经导管的制备
用二氯甲烷溶解聚乳酸和齐墩果酸，制成聚乳酸浓度8％、齐墩果酸浓度0.08％的纺丝溶液，将步骤1)制备的导电定向静电纺丝纤维裁剪成小块并包覆在接收器上，用含有齐墩果的纺丝溶液进行静电纺丝，形成载药神经导管；
3)双导电载药复合神经导管的制备
用乙醇溶解聚吡咯，制成聚吡咯浓度3％的喷涂溶液，均匀喷涂到步骤2)制备的载药神经导管表面，室温干燥后脱模，得双导电载药复合神经导管，
步骤1)、2)中，所述静电纺丝的工艺参数为：接收器转速为1400r/min，推注速度为0.5mm/min，电压12KV，接收距离25cm，温度25℃，湿度40％。
本发明使用聚乳酸为纺丝材料，通过负载二维碳化钛并进行定向纺丝来制备导电定向静电纺丝纤维膜并以此为内层，通过负载齐墩果酸纺丝纤维形成中间层导管，最后采用静电喷涂在神经导管的表面涂覆聚吡咯导电层，最终制备成双导电载药复合神经导管。两种导电物质可以保证导管导电的持续性，纤维的定向排列和药物的释放也可以减轻周围神经损伤导致的炎症，进一步促进受损神经的修复。
神经缺损中巨噬细胞的特定表型对周围神经的再生发挥重要作用。这些活化的巨噬细胞主要分为促炎(M1)和促愈合(M2)两种表型。其中M1型巨噬细胞通过产生高水平的促炎细胞因子来支持炎症，而M2型巨噬细胞通过分泌抗炎细胞因子和生长因子来促进组织修复，这些因子介导细胞增殖、血管生成和基质重塑。研究结果表明，本发明制备的神经导管可显著诱导巨噬细胞(RAW264.7细胞)极化，促进其向M2表型分化，且极化的巨噬细胞在体外具有更好的促进雪旺细胞增殖和迁移的能力，进而促进受损神经元的存活及其轴突的再生。
本发明的有益效果是：
1)本发明使用了两种导电材料，以掺入纺丝和静电喷涂两种方式负载在神经导管上，内外两层都有导电率，极大增加了导管的机械强度和细胞的黏附性，同时也满足了电刺激的需要及导电的连续性，延长了神经再生的进程。
2)本发明使用了齐墩果酸和定向排列的纺丝纤维，已有研究表明，定向排列的纺丝纤维具有促进神经细胞再生的作用，而齐墩果酸则可以通过促巨噬细胞向M2极化来减轻周围神经损伤后导致的炎症反应，发挥与定向排列纺丝纤维的协同作用，进一步促进受损神经元的修复及其轴突的再生。
3)本发明所使用的材料包括聚乳酸、聚吡咯、二维碳化钛等均具有良好的生物相容度及可降解性，且廉价易得，适用于大规模生产。
附图说明
图1为导电定向静电纺丝纤维膜的扫描电镜图。
图2为双导电载药神经导管的外观示意图。
具体实施方式
为了更好的理解本发明，下面结合实施例进一步阐述本发明的内容，但本发明的内容不仅仅局限于下面的实施例。实施例中所使用的单层碳化钛和聚吡咯均为市售产品，分别从中科雷鸣(北京)科技有限公司和湖北诺纳科技有限公司购买获得，也能使用以下方法自制：
导电物质二维碳化钛合成：取0.5g钛碳化铝，加入到1mol/L的氟化氢铵水溶液中，刻蚀铝层，反应5天后，离心洗涤干燥得到多层碳化钛粉末。取0.1g多层碳化钛粉末加入20ml N,N-二甲基甲酰胺中，快速搅拌，降低材料层层间的相互作用力，超声离心洗涤干燥得到单层碳化钛粉末。
导电物质聚吡咯合成：取3.5g十二烷基硫酸钠均匀分散在70ml去离子水中，向其中缓慢滴入1ml吡咯，搅拌均匀后先其中25ml六水合氯化铁水溶液转移到低温反应釜中反应，在-5℃下反应12h后离心洗涤干燥得聚吡咯。
实施例1
双导电载药复合神经导管的制备方法如下：
1)导电定向静电纺丝纤维膜的制备
用7.5g二氯甲烷溶解0.6g聚乳酸，将0.06g二维碳化钛研磨至平均粒径500nm，然后均匀分散于聚乳酸溶液中，制成聚乳酸浓度8％、二维碳化钛浓度0.8％的纺丝溶液，进行静电纺丝，得到导电定向静电纺丝纤维，置于烘箱中50℃干燥后备用。
2)载药神经导管的制备
用12.5g二氯甲烷溶解1g聚乳酸和0.01g齐墩果酸，制成聚乳酸浓度8％、齐墩果酸浓度0.08％的纺丝溶液，将步骤1)制备的导电定向静电纺丝纤维裁剪为5cm×5cm的正方形，缠绕包覆在接收器上，用含有齐墩果的纺丝溶液进行静电纺丝，形成载药神经导管。
3)双导电载药神经导管的制备
将0.3g聚吡咯均匀分散于10ml无水乙醇中，制成聚吡咯浓度3％的喷涂溶液，均匀喷涂到载药神经导管表面，室温干燥后从接收器上脱模，得双导电载药神经导管。
步骤1)、2)中，所述静电纺丝的工艺参数为：接收器转速为1400r/min，推注速度为0.5mm/min，电压12KV，接收距离25cm，温度25℃，湿度40％。
图1为导电定向静电纺丝纤维膜的扫描电镜图，导电纺丝纤维定向排列，直径1-10μm。
图2为双导电载药神经导管的外观结构示意图，所制备的产品为中空管状结构，表面为黑色。内层为含有导电物质二维碳化钛的聚乳酸静电纺丝纤维，厚度约0.2mm；外层为导电物质聚吡咯涂层，厚度约0.1mm；中间层为含齐墩果的聚乳酸静电纺丝纤维，厚度约1mm。
将制备的双导电载药神经导管用四探针电阻率仪测导电率，该导管在生理盐水中放置7周后的导电率仍可达到1.9×10-3S/cm，依然满足最低电刺激10-4S/cm的要求。本发明使用了二维碳化钛和聚吡咯两种导电材料，并通过适当的工艺将其复合，所制备的神经导管导电持续更久，更有利于神经细胞的恢复与再生。
实施例2
双导电载药复合神经导管的制备方法如下：
1)导电定向静电纺丝纤维膜的制备
用二氯甲烷溶解聚乳酸，将二维碳化钛研磨至平均粒径200nm，然后均匀分散于聚乳酸溶液中，制成聚乳酸浓度6％、二维碳化钛浓度1％的纺丝溶液，进行静电纺丝，得到导电定向静电纺丝纤维，置于烘箱中50℃干燥后备用。
2)载药神经导管的制备
用二氯甲烷溶解聚乳酸和齐墩果酸，制成聚乳酸浓度6％、齐墩果酸浓度0.05％的纺丝溶液，将步骤1)制备的导电定向静电纺丝纤维裁剪为5cm×5cm的正方形，缠绕包覆在接收器上，用含有齐墩果的纺丝溶液进行静电纺丝，形成载药神经导管。
3)双导电载药神经导管的制备
将聚吡咯均匀分散于无水乙醇中，制成聚吡咯浓度5％的喷涂溶液，均匀喷涂到载药神经导管表面，室温干燥后从接收器上脱模，得双导电载药神经导管。
步骤1)、2)中，所述静电纺丝的工艺参数为：接收器转速为1200r/min，推注速度为1mm/min，电压10KV，接收距离25cm，温度25℃，湿度40％。
实施例3
双导电载药复合神经导管的制备方法如下：
1)导电定向静电纺丝纤维膜的制备
用二氯甲烷溶解聚乳酸，将二维碳化钛研磨至平均粒径800nm，然后均匀分散于聚乳酸溶液中，制成聚乳酸浓度10％、二维碳化钛浓度0.5％的纺丝溶液，进行静电纺丝，得到导电定向静电纺丝纤维，置于烘箱中50℃干燥后备用。
2)载药神经导管的制备
用二氯甲烷溶解聚乳酸和齐墩果酸，制成聚乳酸浓度10％、齐墩果酸浓度0.12％的纺丝溶液，将步骤1)制备的导电定向静电纺丝纤维裁剪为5cm×5cm的正方形，缠绕包覆在接收器上，用含有齐墩果的纺丝溶液进行静电纺丝，形成载药神经导管。
3)双导电载药神经导管的制备
将聚吡咯均匀分散于无水乙醇中，制成聚吡咯浓度1％的喷涂溶液，均匀喷涂到载药神经导管表面，室温干燥后从接收器上脱模，得双导电载药神经导管。
步骤1)、2)中，所述静电纺丝的工艺参数为：接收器转速为1500r/min，推注速度为0.3mm/min，电压15KV，接收距离25cm，温度25℃，湿度40％。
试验例
试验例1、神经导管对PC12细胞生长的影响
将本发明制备的双导电载药神经导管与PC12细胞共培养并施加电刺激，培养后的第5天进行CCK-8细胞增殖活力检测，具体方法如下：
为了研究电刺激(ES)与神经支架对神经轴突生长的影响，制备双导电神经导管放入孔板中，支架两端穿透铂丝用来连接电刺激装置电极。以1×106个的细胞密度在孔板中接种PC12细胞，每孔填充3mL新鲜培养基，并在37℃下培养24小时以使细胞贴壁，之后每天施加10μA的电流ES处理1h，连续培养5天，隔天换一次新鲜培养液。于第5天进行检测，先在倒置荧光显微镜下观察细胞生长状态，然后向孔板中加入10μLCCK-8，轻轻晃动使其混合均匀，在细胞培养箱培养4小时，取出培养板，弃去上清液，酶标仪上检测其OD值，通过观察各组的OD值可以初步判断神经导管对细胞的作用，OD值越高，说明细胞增殖活力越强。重复试验5次，同时设置空白组和以下对照组：
对照组1：制备静电纺丝纤维膜时未向纺丝溶液中加入二维碳化钛，得到仅有一种导电材料即聚吡咯的单导电载药神经导管。
对照组2：制备载药神经导管后未使用聚吡咯喷涂，室温干燥后直接脱模，得到仅有一种导电材料即二维碳化钛的单导电载药神经导管。
对照组3：步骤2)中未向聚乳酸纺丝液中加入齐墩果酸，得到不载药的双导电神经导管。
对照组1-3中，未予说明的工艺步骤均与实施例1相同。
表1双导电神经导管对PC12细胞的影响(n＝5)
| 分组 | OD(450nm) |
| 空白组 | 1.33±0.34 |
| 实施例1 | 1.92±0.62** |
| 实施例2 | 1.71±0.57* |
| 实施例3 | 1.84±0.61** |
| 对照组1 | 1.48±0.45 |
| 对照组2 | 1.59±0.41* |
| 对照组3 | 1.65±0.50* |
与空白组相比，*P<0.05；**P<0.01
从试验结果可以得出，本发明制备的双导电载药复合神经导管促进了PC12细胞的增殖，与单导电载药神经导管或不载药的双导电神经导管相比，本发明对pc12细胞的增殖效果更好。
试验例2、神经导管对药物释放的影响
将实施例制备的双导电载药神经导管放入pH值为7.4的磷酸盐缓冲盐水(PBS)中，放在摇床中(37℃，70rpm)，并在特定时间收集释放介质。以齐墩果酸标准品制备对照品溶液，在210nm波长下，用HPLC方法测定齐墩果酸的药物释放量。
本发明制备的神经导管，齐墩果酸10天的释放量在25％左右，20天的累积释放量约55％，30天左右接近峰值，累积释放量约80％。本发明将齐墩果酸负载于聚乳酸纺丝纤维中，且位于复合神经导管的中间层，实现了较好的缓释效果。研究表明，齐墩果酸通过促巨噬细胞向M2极化来减轻周围神经损伤后导致的炎症反应，能为神经细胞的再生创造更加有利的外部环境，而缓慢释放的齐墩果酸有利于延长作用时间。
Claims (7)
1.一种双导电载药复合神经导管，其特征在于：为中空管状结构，导管内层为含有导电物质二维碳化钛的聚乳酸静电纺丝纤维；导管外层为导电物质聚吡咯涂层，导管中间层为含齐墩果的聚乳酸静电纺丝纤维。
2.如权利要求1所述的双导电载药复合神经导管，其特征在于：所述导管内层的厚度为0.1-0.3mm，导管外层的厚度为0.08-0.15mm，导管中间层的厚度为0.5-2mm。
3.一种制备权利要求1或2所述双导电载药复合神经导管的方法，其特征在于包括以下步骤：
1)用有机溶剂溶解聚乳酸和平均粒径100-1000nm的二维碳化钛，制成聚乳酸浓度5-10％、二维碳化钛浓度0.1-1％的纺丝溶液，进行静电纺丝，得到导电定向静电纺丝纤维；
2)用有机溶剂溶解聚乳酸和齐墩果酸，制成聚乳酸浓度5-10％、齐墩果酸浓度0.05-0.2％的纺丝溶液，将步骤1)制备的导电定向静电纺丝纤维裁剪成小块并包覆在接收器上，用含有齐墩果的纺丝溶液进行静电纺丝，形成载药神经导管；
3)用有机溶剂溶解聚吡咯，制成聚吡咯浓度1-5％的喷涂溶液，均匀喷涂到步骤2)制备的载药神经导管表面，室温干燥后脱模，得双导电载药复合神经导管。
4.如权利要求3所述的制备方法，其特征在于，所述静电纺丝的工艺参数为：接收器转速1000-1500r/min，推注速度为0.1-1mm/min，电压10-15KV，接收距离20-30cm，温度20-30℃，湿度30-50％。
5.如权利要求3所述的制备方法，其特征在于，所述二维碳化钛的平均粒径为300-500nm，在使用前通过研磨使其达到所需的粒径。
6.如权利要求3所述的制备方法，其特征在于：所述纺丝溶液中，聚乳酸的浓度为6-8％。
7.如权利要求3所述的制备方法，其特征在于：所述纺丝溶液中，二维碳化钛的浓度为0.5-0.8％；齐墩果酸的浓度为0.05-0.1％。
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