CN111939307B - 一种医用复合纳米纤维敷料及其制备方法与应用 - Google Patents
一种医用复合纳米纤维敷料及其制备方法与应用 Download PDFInfo
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Abstract
本发明涉及一种医用复合纳米纤维敷料及其制备方法。该敷料分为内外两层，其中内层含有聚乙烯醇、胶原和季铵化壳聚糖，外层含有聚己内酯和十二烷基取代的季铵化硅酮，将聚乙烯醇、胶原和季铵化壳聚糖混合后，通过静电纺丝法制备内层PCQC5；以聚己内酯和十二烷基取代的季铵化硅酮QP12为原料，在内层PCQC5的基础上，通过静电纺丝法制备外层。该敷料可用于组织修复或瘢痕修复中，在抗菌材料或医疗器械领域中具有广泛的应用。
Description
技术领域
本发明属于医疗器械领域，具体涉及一种具有止血、抗菌抗炎、创伤修复功能、瘢痕修复功能的医用复合纳米纤维敷料及其制备方法与应用。
背景技术
创伤和术后止血、预防感染和瘢痕治疗一直是临床亟需解决的重点和难点问题。由于创伤和术后伤口修复时间长、病情复杂所致的愈合不良，甚至发生重症感染、脓毒血症以及溃疡癌变等并发症，可能会危及患者生命。
创面愈合是机体对皮肤组织缺陷的修复反应。正常的皮肤创伤愈合是四个不同但重叠的阶段，分别为止血期、炎症期、增殖期和重塑期。创面愈合的四个阶段中断时，将产生慢性难愈创面。医用材料对于慢性难愈创面可发挥修复作用，目前国内外市场上各类创面敷料已达2400种，但都在功能单一、易感染等问题。据世界卫生组织(WHO)颁布的《院内感染防治实用手册》数据显示，全球60％的临床感染与医疗器械使用有关，为了应对医疗器械及植入物造成的感染，目前临床上多采用抗生素系统治疗，但存在明显的不良反应、过敏反应和耐药性。临床上，具有抗菌性能的功能材料成为最佳选择，然而临床现用的伤口敷料等医疗器械要么抗菌活性不够，要么功能单一，导致创伤感染频发、创伤和瘢痕修复效果差等一系列问题。
发明内容
针对以上问题，本发明以季铵化壳聚糖与季铵化硅酮这两种生物安全性高的材料为切入点，基于静电纺丝纤维基医用复合材料制备技术，赋予材料“止血修复-抗菌抗炎-瘢痕修复”等多重功能。
本发明提供一种利用静电纺丝方法制备具有止血、抗菌抗炎、创伤修复和瘢痕修复功能的含季铵化壳聚糖/季铵化硅酮的医用复合纳米纤维敷料。该医用复合纳米纤维敷料具备良好的力学性能，且内外层兼具多种不同功能，内层能够快速止血、促进伤口修复、加速皮肤创伤愈合；外层阻止细菌侵袭、保湿、透气、抑制瘢痕增生。
本发明的医用复合纳米纤维敷料是通过以下技术方案实现的：
本发明的医用复合纳米纤维敷料，即MPC12，该敷料通过静电纺丝法制得，含有内外双层结构，所述内层含有聚乙烯醇、胶原和季铵化壳聚糖，所述外层含有聚己内酯和十二烷基取代的季铵化硅酮；其中，辅料聚己内酯(PCL)的相对分子质量为8万；辅料聚乙烯醇(PVA)为PVA-1788型；辅料胶原(COL)来源于海洋鱼皮Ⅰ型胶原；其中十二烷基取代的季铵化硅酮(QP12)和季铵化壳聚糖(QACS)的结构式如下：
本发明的医用复合纳米纤维敷料的制备路线：
将聚乙烯醇、胶原和季铵化壳聚糖混合后，通过静电纺丝法制备内层PCQC5；以聚己内酯和十二烷基取代的季铵化硅酮QP12为原料，在内层PCQC5的基础上，通过静电纺丝法制备外层。
本发明的医用复合纳米纤维敷料的具体制备方法如下：
(1)内层纳米纤维的制备：称取将聚乙烯醇溶于溶剂中，待完全溶解后，称取胶原、季铵化壳聚糖加入溶液中共混，使充分溶胀，后继续磁力搅拌均匀，离心脱泡得到纺丝液；采用静电纺丝得到复合膜内层PCQC5纳米纤维；其中纺丝液聚乙烯醇和胶原的质量比60-80：40-20，聚乙烯醇和胶原的总质量浓度为5-10％，季铵壳聚糖的质量浓度为0.25-0.5％；所述溶剂是指乙酸或六氟异丙醇；静电纺丝中纺丝参数为：电压15KV，流速0.1mL/h，接收距离17cm，环境参数温度28±3℃，湿度40±10％；
(2)外层纳米纤维的制备：称取一定量的聚己内酯粉末和十二烷基取代的季铵化硅酮，室温条件下磁力搅拌溶解于六氟异丙醇中，离心脱泡得到均相纺丝液；采用静电纺丝得到外层纳米纤维；纺丝液中聚己内酯/十二烷基取代的季铵化硅酮质量比为80-40：20-60，聚己内酯和十二烷基取代的季铵化硅酮的总质量浓度为5-10％；纺丝参数为：电压15KV,流速1mL/h,接收距离15cm，环境参数温度28±2℃，湿度40±2％。
十二烷基取代的季铵化硅酮的制备方法包括以下步骤：
(1)惰性气体(氮气或氩气)保护下，在干燥的实验装置中，取聚甲基氢硅氧烷、适量6-溴-1-己烯溶解于无水甲苯中形成混合溶液，常温下向上述混合溶液中滴加适量的催化剂，在40～80℃下反应24～48小时，所得溶液与冷无水甲醇混合，置于-50℃冰箱下静置过夜，倒出上层清液，残余物用0.20～0.60μm的滤膜进行超滤，滤液减压蒸除溶剂得到中间体；
(2)将上述得到的中间体和十二烷基二乙醇胺溶于体积比1:1的三氯甲烷/N,N-二甲基甲酰胺混合溶液中，60-80℃搅拌反应12～24小时，反应完成后，冷却至室温，混合物倾倒入无水乙醚中沉淀，沉淀抽滤，用冷的无水乙醚洗涤三次，真空干燥得到目标产物十二烷基取代的季铵化硅酮；
其中，6-溴-1-己烯加入量为聚甲基氢硅氧烷中硅氢键摩尔数的1.0～2.0倍；
所述催化剂为1,3-二乙烯基-1,1,3,3-四甲基二硅氧烷铂的二甲苯溶液，其中铂元素含量为2％。
季铵化壳聚糖(QACS)来源于ZL2015100806169的十二烷基取代的季铵化壳聚糖。
本发明的医用复合纳米纤维敷料在抗菌材料或医疗器械领域中的应用。
本发明的医用复合纳米纤维敷料在组织修复或瘢痕修复中的应用。
本发明的医用复合纳米纤维敷料用作伤口敷料、组织工程敷料。
本发明具有以下的创新性：
(1)多种功能性医用敷料有效复合，得到多功能医用复合纳米纤维敷料。该纳米纤维敷料含有季铵化修饰硅酮、季铵化壳聚糖、深海鱼皮胶原与无毒、无刺激高分子聚合物PCL、PVA，既能弥补硅酮凝胶抗菌抗炎效果弱、刺痛感强的缺陷，又能达到创伤止血-创伤愈合-瘢痕修复等功效。多种组分协调作用，既能保证膜的亲水性、通透性、止血性能和抗菌性能，又能体现硅酮凝胶的疏水性、瘢痕修复和抗菌抗炎性能，隔绝外界的感染，减少瘢痕的增生，达到瘢痕修复的作用。
(2)采用静电纺丝法制备的医用复合纳米纤维敷料具有双层仿生结构，物理化学性能和生物学性能优良，总体兼备止血修复-抗菌抗炎-瘢痕修复等多重功能。一方面，该纳米纤维敷料，纤维直径在纳米级，与天然细胞外基质形态相似，有利于瘢痕组织和各人体器官的加速修复，加之仿生医用复合纳米纤维敷料比其他纤维类材料比表面积大、孔隙率高等优良的特性，大大地提升了瘢痕组织修复的效果、生物化学相容性及生物可降解性。另一方面，该纳米纤维敷料具有双层结构，内层为PVA、胶原、季铵化壳聚糖制备的创伤修复膜(PCQC5)，能够快速止血、促进伤口修复、加速皮肤创伤愈合，同时具有抗菌抗炎作用；外层为PCL、季铵化硅酮制备的瘢痕修复膜(MQP12)，能够外层阻止细菌侵袭、保湿、透气、抑制瘢痕增生。总体兼备止血修复-抗菌抗炎-瘢痕修复等多重功能。
本发明技术水平国内领先、国际先进，有望打通新型抗菌创伤修复产品成果转化和产业化技术通道，将为提升我国高端医用材料自主创新水平，促进我国高端医疗器械产业结构调整、提升高端医疗器械产业整体竞争力和水平起到积极的带动作用。
附图说明
图1：纳米纤维敷料的纤维形貌；
图2：纳米纤维敷料的体外增殖活性考察；
图3：PCL纳米纤维敷料抗微生物侵袭作用(A，金葡菌B，大肠杆菌C白色念珠菌)；
图4：MQP12纳米纤维敷料抗微生物侵袭作用(A，金葡菌B，大肠杆菌C白色念珠菌)；
图5：MPC12瘢痕修复性能考察。
具体实施方式
以下给出本发明的具体实施方式，用来对本发明的构成作进一步的说明，但并不认为本发明仅局限于下述的实施方式。
实施例1.本发明含季铵化壳聚糖/季铵化硅酮(QP12)的多组分纳米复合纤维敷料的制备方法如下：
(1)称取一定量的PVA-1788型粉末，溶于1M的稀醋酸中，室温下磁力搅拌均匀溶解成PVA溶液，待完全溶解后，称取胶原、季铵化壳聚糖加入以一定质量比共混，使充分溶胀，后继续磁力搅拌均匀，离心脱泡。PVA和胶原的总质量分数为8％，季铵化壳聚糖质量分数为0.4％，PVA/胶原质量比为80:20；静电纺丝中纺丝参数为：电压15KV，流速0.1mL/h，接收距离17cm，环境参数温度28±3℃，湿度40±10％；所得电纺纤维敷料的纤维形态良好，直径分布均一。
(2)按质量比为70:30称取聚己内酯粉末和十二烷基取代的季铵化硅酮，室温条件下磁力搅拌溶解于六氟异丙醇中，获得均相溶液；该均相溶液中聚己内酯和十二烷基取代的季铵化硅酮的总质量分数为7％；纺丝参数为：电压15KV,流速1mL/h,接收距离15cm，环境参数温度28±2℃，湿度40±2％。经力学性能测试结果显示，制得的MPC12复合膜的断裂伸张率为47.5±1.4％，与人体的断裂伸张率(54±17％)相近。MPC12的扫面电镜可以观察到内外层的纤维形貌良好，直径分布均一(如图1所示)，该纳米复合膜具有成为敷贴材料的潜力。
本发明方法制备的复合纤维敷料，具有良好的生物医学性能：
(1)本发明的复合纤维敷料体外细胞毒性合格
MPC12纳米纤维复合膜通过Hacat细胞增殖率间接反映其毒性。MQP12、PCQC5分别为复合膜的内外层纳米膜，其中PCL与QP12制备成MQP12纳米膜后，相对增值率从87.05±5.36降低至83.42±5.43，根据《GB/T16886.5》中细胞毒性评价等级表1中的毒性等级划分，MQP12为1级细胞毒性，属于合格；MQP12与PCQC5相结合成的MPC12纳米纤维复合膜细胞增值率为100.5±4.39，属于0级细胞毒性，合格(表2)。以上结果显示出MPC12及其组分纳米膜的毒性均属于合格，表明了良好的细胞相容性。
表1《GB/T16886.5》中细胞毒性评价等级表
表2纳米膜的Hacat细胞毒性研究
(2)本伤口敷料具有较好的体外细胞增殖活力
本实验考察Hacat细胞在不同纳米纤维敷料浸提液中的增殖情况，MTT法测试培养24h、36h、72h后细胞数，衡量不同纳米纤维敷料促进Hacat细胞增殖能力，实验结果如图2所示。阳性对照组为0.6％-苯酚的培养液溶液，与其他组别相同操作后培养至第24h，活细胞数较其他组别明显减少，测量的细胞OD值也无明显变化，增殖测试结果显示该组细胞与0.6％-苯酚接触培养后失去细胞的增殖能力。阴性对照组是以不含其他药液的空白培养基与细胞相互接触，表现出随时间延长而由MTT法检测出的活细胞数OD值逐渐上升的趋势，主要是由于细胞的繁殖能力增加所致。实验组OD值也呈现随时间增大而增大。排除阳性对照组，Hacat细胞与浸提液相互接触24h后，其他组别之间统计学分析无明显差异(P>0.05)，该现象出现的原因为Hacat细胞与培养液接触后首先以附着为主，故增殖较少，OD值相近；阳性药组别在于Hacat细胞接触后24h后，检测得出较低的OD值，同其他组别统计学分析具有显著性差异(P<0.05)，表明了实验组中各纳米膜具有较低的细胞毒性，该结果同细胞毒性测试结果一致。
实验MQP12组培养至72h的OD值稍有增加，但复合后的MPC12组培养至72h的OD值明显增加，其良好的促细胞增殖能力可能来源于复合膜中PCQC5纳米膜。
(3)本复合纤维敷料溶血性合格
根据《GB/T 16886.4-2003/ISO 10993-4：2002，医疗器械生物学评价第4部分：与血液相互作用试验选择》中的要求，测试了4种静电纺丝膜的溶血性(表3)，实验结果显示均无溶血作用。溶血率＝(试验组OD-阴性组OD)/(阳性组OD-阴性组OD)×100％。结果评价：阴性组吸光度OD值，阳性组吸光度OD值为，实验组溶血率<5％，则材料符合要求。
表3静电纺丝膜的溶血率
(4)本复合纤维敷料皮内刺激性
根据《GB/T 16886.10—2005/ISO 10993-10:2002，医疗器械生物学评价第10部分：刺激与迟发型超敏反应试验》要求，测试了MPC12纤维敷料的皮内刺激性，分别在三个时间点24h、48h、72h观察记录各激发部位的反应情况，按记分系统(表4)对各激发部位的皮内刺激反应进行评分。评分结束计算刺激指数，分别将试验组和对照组出现的红斑和水肿的记分相加，再除以12[2(动物数)×3(观察期)×2(记分类型)]，得试验样品和对照品的综合平均计分。两者之差不大于1，则符合实验要求。膜材料符合皮内刺激毒性要求。
表4复合纤维敷料MPC12皮内刺激性
(5)本复合纤维敷料具备微生物抵御能力
本发明利用扫描电镜观察了细菌在其外表面培养8h、12h、24h后，纳米纤维敷料MQP12内外两侧微生物的生长情况，设PCL纳米纤维敷料为对照组，直观的考察了MQP12膜的微生物抵御能力(图3、图4)。一方面，在相同实验时间内，对照组内侧的微生物数量明显高于实验组，说明了MQP12优良的微生物抵御能力来源于纤维中的季铵化硅酮；另一方面，实验时间24h，MQP12内侧也出现微生物增殖迹象，结果表明MQP12可维持24h内部无菌或少菌的状态，另与内膜PCQC5相结合能保持伤口长时间的无菌环境，降低感染率。
(6)MPC12促伤口愈合性能
促伤口愈合实验以市售硅酮类产品芭克、舒疤灵为阳性对照组，设置空白对照组，在手术创伤后的1d、3d、7d、10d、14d分别以数码相机纪录伤口图片，并通过Image J图形处理软件对所得图片进行处理，计算并统计伤口的愈合率(表5)。MPC12在实验10天愈合率已达51.56％，其愈合率是其他同时间组别的近两倍，实验14天愈合率高达90.05％，创口已修复完毕，表现出优良的促伤口愈合能力。与MPC12纳米纤维敷料相比，芭克为阳性药的组别其愈合率实验14天愈合率仅为41.24％且低于空白对照组，说明芭克难以促进伤口愈合，甚至减缓伤口愈合。而舒疤灵组愈合率为73.58％，高于空白组愈合率，原因可能为舒疤灵中含有部分促伤口愈合的组分。
表5 MPC12对于伤口的愈合率(n＝6,x±s,％)
(7)MPC12瘢痕修复性能
实验测试了纳米纤维复合膜与市售相关产品(芭克、舒疤灵)的瘢痕修复性能，记录了各组在不同时间点的创口的愈合情况及瘢痕的生长情况(图5)。实验1天，随机分组的家兔予以7mm直径大小的创口，并定时更换创口处待测样品。
图5中所示，空白对照组从实验1天至50天，创口经历愈合、瘢痕增生，表明了该创伤与瘢痕模型建立成功。PCQC5组功能主要为促伤口愈合，图5中显示实验7天该组的创口的面积明显小于空白组及阳性药组，该组实验50天瘢痕组织凸出皮肤表面，表明了该纳米复合膜无瘢痕修复功能；MQP12主要功能为瘢痕修复功能，创口实验14天修复后的组织未凸出皮肤表面，并且随时间的延长，组织表面变平且与周围组织相似，表明了MQP12纳米纤维敷料良好的瘢痕修复能力；MPC12是结合具有创伤修复功能的PCQC5和瘢痕修复功能的MQP12的纳米纤维复合膜，实验7天显示MPC12组与PCQC5组的创口面积大小相近，表明了两组具有相近的创口的修复效果。实验38天MPC12组创口未形成瘢痕组织，修复后的组织与周围组织呈现相似的红润，该种现象与MQP12修复后效果相近，表明了两组具有相似的瘢痕修复功能。阳性药组别为相关的市售产品，包括两种芭克、舒疤灵含PDMS的瘢痕治疗药物，与MPC12纳米纤维敷料相比，两种市售产品实验50天未能有效地消除瘢痕，并且舒疤灵使得创口形成萎缩性瘢痕。以上结果显示了MPC12纳米纤维敷料具有优于市售产品芭克、舒疤灵的瘢痕修复性能。
Claims (8)
1.一种医用复合纳米纤维敷料，其特征在于：该敷料含有内外双层纳米纤维结构，所述内层纳米纤维含有聚乙烯醇、胶原和季铵化壳聚糖，所述外层纳米纤维含有聚己内酯和十二烷基取代的季铵化硅酮，其中十二烷基取代的季铵化硅酮和季铵化壳聚糖的结构式如下：
该敷料通过静电纺丝法制得：内层纳米纤维的纺丝液为：聚乙烯醇和胶原的质量比为60-80:40-20，聚乙烯醇和胶原的总质量浓度为5-10%，季铵化壳聚糖的质量浓度为0.25-0.5%；外层纳米纤维的纺丝液为：聚己内酯与十二烷基取代的季铵化硅酮质量比为80-40:20-60，聚己内酯和十二烷基取代的季铵化硅酮的总质量浓度为5-10%；
十二烷基取代的季铵化硅酮的制备方法包括以下步骤：
(1)惰性气体氮气或氩气的保护下，在干燥的实验装置中，取聚甲基氢硅氧烷、6-溴-1-己烯溶解于无水甲苯中形成混合溶液，常温下向混合溶液中滴加催化剂，在40~80℃下反应24~48小时，所得溶液与冷无水甲醇混合，置于-50℃冰箱静置过夜，倒出上层清液，残余物用0.20~0.60μm的滤膜进行超滤，滤液减压蒸除溶剂得到中间体；6-溴-1-己烯加入量为聚甲基氢硅氧烷中硅氢键摩尔数的1.0~2.0倍；催化剂为1,3-二乙烯基-1,1,3,3-四甲基二硅氧烷铂的二甲苯溶液，其中铂元素含量为2%；
(2)将得到的中间体和十二烷基二乙醇胺溶于体积比1:1的三氯甲烷与N,N-二甲基甲酰胺混合溶液中，60-80℃搅拌反应12~24小时，反应完成后，冷却至室温，混合物倾倒入无水乙醚中沉淀，沉淀抽滤，用冷的无水乙醚洗涤三次，真空干燥得到目标产物十二烷基取代的季铵化硅酮。
2.根据权利要求1所述的医用复合纳米纤维敷料，其特征在于：聚己内酯的相对分子质量为8万；聚乙烯醇为PVA-1788型；胶原来源于海洋鱼皮Ⅰ型胶原。
3.一种如权利要求1所述的医用复合纳米纤维敷料的制备方法，其特征在于：将聚乙烯醇、胶原和季铵化壳聚糖混合后，通过静电纺丝法制备内层纳米纤维；以聚己内酯和十二烷基取代的季铵化硅酮为原料，通过静电纺丝法在内层纳米纤维的基础上制备外层纳米纤维。
4.根据权利要求3所述的医用复合纳米纤维敷料的制备方法，其特征在于：包括以下步骤：
(1) 内层纳米纤维的制备：称取将聚乙烯醇溶于溶剂中，待完全溶解后，称取胶原、季铵化壳聚糖加入溶液中共混，使充分溶胀，后继续磁力搅拌均匀，离心脱泡得到纺丝液；采用静电纺丝得到复合膜内层纳米纤维；其中纺丝液中聚乙烯醇和胶原的质量比为60-80:40-20，聚乙烯醇和胶原的总质量浓度为5-10%，季铵化壳聚糖的质量浓度为0.25-0.5%；所述溶剂是指乙酸或六氟异丙醇；静电纺丝中纺丝参数为：电压15 KV，流速0.1 mL/h，接收距离17 cm，环境参数温度28±3℃，湿度40±10%；
(2) 外层纳米纤维的制备：称取聚己内酯粉末和十二烷基取代的季铵化硅酮，室温条件下磁力搅拌溶解于六氟异丙醇中，离心脱泡得到均相纺丝液；采用静电纺丝得到外层纳米纤维；纺丝液中聚己内酯与十二烷基取代的季铵化硅酮质量比为80-40:20-60，聚己内酯和十二烷基取代的季铵化硅酮的总质量浓度为5-10%；纺丝参数为：电压15 KV, 流速1 mL/h, 接收距离15 cm，环境参数温度28±2℃，湿度40±2%。
5.根据权利要求4所述的医用复合纳米纤维敷料的制备方法，其特征在于，步骤（2）中，所述十二烷基取代的季铵化硅酮的制备方法包括以下步骤：
(1)惰性气体氮气或氩气的保护下，在干燥的实验装置中，取聚甲基氢硅氧烷、6-溴-1-己烯溶解于无水甲苯中形成混合溶液，常温下向混合溶液中滴加催化剂，在40~80℃下反应24~48小时，所得溶液与冷无水甲醇混合，置于-50℃冰箱静置过夜，倒出上层清液，残余物用0.20~0.60μm的滤膜进行超滤，滤液减压蒸除溶剂得到中间体；
(2)将得到的中间体和十二烷基二乙醇胺溶于体积比1:1的三氯甲烷与N,N-二甲基甲酰胺混合溶液中，60-80℃搅拌反应12~24小时，反应完成后，冷却至室温，混合物倾倒入无水乙醚中沉淀，沉淀抽滤，用冷的无水乙醚洗涤三次，真空干燥得到目标产物十二烷基取代的季铵化硅酮；
其中，6-溴-1-己烯加入量为聚甲基氢硅氧烷中硅氢键摩尔数的1.0~2.0倍；
所述催化剂为1,3-二乙烯基-1,1,3,3-四甲基二硅氧烷铂的二甲苯溶液，其中铂元素含量为2%。
6.如权利要求1所述的医用复合纳米纤维敷料在抗菌材料或医疗器械领域中的应用。
7.如权利要求1所述的医用复合纳米纤维敷料在制备组织修复或瘢痕修复材料中的应用。
8.如权利要求1所述的医用复合纳米纤维敷料在制备伤口敷料、组织工程敷料材料中的应用。
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