CN114225122A - 一种含有抗菌肽的壳芯结构纳米纤维膜的制备方法 - Google Patents
一种含有抗菌肽的壳芯结构纳米纤维膜的制备方法 Download PDFInfo
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Abstract
本发明公开了一种含有抗菌肽的壳芯结构纳米纤维膜的制备方法，包括以下步骤：取PLGA粉末溶于DMF溶液搅拌溶解，得到浓度为10wt％～18wt％的壳层PLGA/DMF溶液备用；取KSL‑W粉末与4mL～5mL的蒸馏水混合得到芯层KSL‑W水溶液备用；静电纺丝机分别吸取壳层PLGA/DMF溶液和芯层KSL‑W水溶液形成纳米纤维膜；收集厚度为0.1mm～0.3mm的纳米纤维膜，而后将纳米纤维膜折叠并放置入温度为40℃～70℃的烘箱中烘干，得到含有抗菌肽的壳芯结构纳米纤维膜。利用同轴静电纺丝技术，保证了纤维膜的屏障性能，又提高了力学性能，抗菌肽使纳米纤维膜具有抗菌性，解决了现有膜性材料易与外界交通发生细菌感染而影响预后的缺点；在病损区有效地释放抗菌药物，提高复层纤维膜组织亲和性，同时降低药物的初期突释现象。
Description
技术领域
本发明属于根尖周炎修复材料技术领域，尤其是涉及一种含有抗菌肽的壳芯结构纳米纤维膜的制备方法。
背景技术
根尖周炎是一种由牙髓内微生物感染引起的根尖周组织炎症和骨破坏的口腔常见疾病，临床上常用根管治疗去除牙髓内的感染，促进病损区愈合。现代根管治疗技术已愈发成熟，但仍有一部分患牙经过治疗后牙槽骨的破坏未停止。
随着牙髓病治疗技术的不断发展，再生性技术(如引导再生骨/组织再生等)越来越多的应用于较大范围的根尖周骨缺损的治疗中。引导骨/组织再生技术是指将生物膜材料作为屏障，排除上皮细胞和结缔组织的干扰，使受损的组织得以修复和替代，恢复组织原有的结构和功能。但是，使用不可吸收的生物膜需二次手术取出；使用可吸收的生物膜(例如Geistlich
膜)，该生物膜的质地软，易塌陷移位，代谢较慢等，导致现有的生物膜在临床应用中存在缺陷。另外，对于伴有牙周病的手术适应症患者，术后生物膜易与外界交通而发生细菌感染，影响手术预后。
抗菌肽具有广谱抗菌活性，通过在细菌的细胞膜积累并发生构象的变化进而破坏膜结构的完整性，导致细胞内容物外流，细菌细胞膜破裂，也可能同时存在一些胞内抗菌机制，如抑制核酸合成，干扰蛋白质和抑制细胞膜合成等。抗菌肽的抗菌过程迅速，在接触微生物数秒里就能发生，这是传统抗生物无法相比拟的。此外，抗菌肽多数都是带有正电荷的，而细菌的细胞膜富含磷脂酰甘油或丝氨酸磷脂之类的磷脂而使其带有负电荷，两者之间极易发生电荷作用，使抗菌肽产生杀灭细菌的生物学效应。但是天然抗菌肽的抗菌活性相对较低，且容易水解，用于体内时稳定性差。其中抗菌肽KSL-W是根据天然抗菌肽的特性合成的具有较强抑菌性能的新型抗菌肽，其既具备天然抗菌肽广谱性和高效性的特点，还具有分子量小、成本低、组织毒性小、不易导致口腔菌群失调和不易产生耐药性的优点。
因此，为了解决上述技术问题，需要研制制备出具有良好生物相容性、优良的力学性能、适当降解性、具有抗菌性能的生物膜材料。
发明内容
本发明的目的是提供一种结构简单、保持抗菌肽KSL-W活性、具有良好的抗菌性能、屏障作用和成骨活性的一种含有抗菌肽的壳芯结构纳米纤维膜的制备方法。
本发明的技术方案如下：
一种含有抗菌肽的壳芯结构纳米纤维膜的制备方法，包括以下步骤：
(1)从-20℃的冰箱内取1.0g～1.8g的PLGA粉末放置至室温，将PLGA粉末溶于8.2g～9.0g的DMF溶液中，并用磁力搅拌器搅拌溶解10s～30s，磁力搅拌器的转速为1000rpm/s，而后静置24h脱泡，得到浓度为10wt％～18wt％的壳层PLGA/DMF溶液，将壳层PLGA/DMF溶液置于4℃的环境下储存备用；
(2)从-20℃的冰箱内取出10mg～15mg的KSL-W粉末并放置至室温，将KSL-W粉末与4mL～5mL的蒸馏水混合，得到浓度为3.75mg/mL～4.75mg/mL的芯层KSL-W水溶液，并置于4℃的环境下储存备用；
(3)同轴静电纺丝机的注射器分别吸取壳层PLGA/DMF溶液和芯层KSL-W水溶液，并将注射器固定在静电纺丝机上，装有壳层PLGA/DMF溶液的注射器及装有芯层KSL-W水溶液的注射器分别通过输液软管与同轴纺丝头的壳芯连接孔连接，将金属板固定在接收装置上，同轴纺丝头与高压电连接，排空输液软管中的空气使输液软管内充满溶液，同轴静电纺丝机运行开始纺丝，壳层PLGA/DMF溶液的推进速率为1.0mL/h～2.0mL/h，芯层KSL-W水溶液的推进速率为0.5mL/h～1.0mL/h，电压为20V～26kV，同轴纺丝头与金属板的距离为35cm～40cm，纺丝在金属板上接收并形成纳米纤维膜；
(4)当收集厚度为0.1mm～0.3mm的纳米纤维膜后，将纳米纤维膜折叠并放置入温度为40℃～70℃的烘箱中，烘干0.5min～2min，最终得到含有抗菌肽的壳芯结构纳米纤维膜。
在上述技术方案中，步骤(1)中的PLGA粉末的分子量为5.0×104～50×104。
在上述技术方案中，步骤(4)得到的所述壳芯结构纳米纤维膜中，抗菌肽占壳芯结构纳米纤维膜总重量百分比为0.025％～0.25％，PLGA占壳芯结构纳米纤维膜总重量百分比为99.975％～97.5％。
本发明具有的优点和积极效果是：
1.本发明的制备方法，利用同轴静电纺丝技术，在保证了纳米纤维膜的屏障性能的同时提高了其力学性能，并且抗菌肽使纳米纤维膜具有抗菌性，解决了现有膜性材料易与外界交通发生细菌感染而影响预后的缺点；同时壳芯结构使纳米纤维膜可以在病损区有效地释放抗菌药物，在提高复层纤维膜组织亲和性的同时降低药物的初期突释现象，使药物更加平缓、稳定的持久释放。
附图说明
图1是本实施例1制备的壳芯结构纳米纤维膜的电镜照片，其中，图1A是10.0k倍下的电镜照片，图1B是5.0k倍下的电镜照片；
图2是本实施例1制备的壳芯结构纳米纤维膜的横断面电镜照片；
图3是本实施例1制备的壳芯结构纳米纤维膜的透射电镜照片(显示出壳芯结构)；
图4是本实施例1制备的壳芯结构纳米纤维膜对粪肠球菌的杀菌效果图，其中，图4A是空白组，图4B是Bio-Gide对照组、图4C是实施例1制备的壳芯结构纳米纤维膜缓释液组；
图5是本实施例1制备的壳芯结构纳米纤维膜在修复兔模拟根尖周炎骨缺损模型的Micro-CT图像，其中，图5A是4周时空白组Micro-CT图像，图5B是4周时Bio-Gide对照组Micro-CT图像，图5C是4周时壳芯结构纳米纤维膜Micro-CT图像，图5D是8周时空白组Micro-CT图像，图5E是8周时Bio-Gide对照组Micro-CT图像，图5F是8周时壳芯结构纳米纤维膜Micro-CT图像。
具体实施方式
以下结合具体实施例对本发明作进一步详细说明。应当理解，此处所描述的具体实施例仅仅用于解释本发明，并不用于限定本发明，决不限制本发明的保护范围。
下述实施例中，PLGA粉末购买于济南岱罡生物有限公司、KSL-W粉末购买于上海吉尔生化、同轴静电纺丝机购买于聚纳达科技有限公司。
实施例1
本发明的一种含有抗菌肽的壳芯结构纳米纤维膜的制备方法，包括以下步骤：
(1)从-20℃的冰箱内取出1.5g的PLGA粉末并放置至室温，将PLGA粉末溶于8.5g的DMF溶液，并用磁力搅拌器搅拌溶解20s，磁力搅拌器转速为1000rpm/s，静置24h脱泡，得到浓度为15wt％的壳层PLGA/DMF溶液，将壳层PLGA/DMF溶液置于4℃的环境下储存备用；
(2)从-20℃的冰箱内取出15mg的KSL-W粉末并放置至室温，将4mL的KSL-W粉末与蒸馏水混合，得到浓度为3.75mg/mL的芯层KSL-W水溶液，并置于4℃的环境下储存备用；
(3)同轴静电纺丝机的注射器各吸取5mL的壳层PLGA/DMF溶液和5mL的芯层KSL-W水溶液，并将注射器固定在静电纺丝机上，装有壳层PLGA/DMF溶液的注射器及装有芯层KSL-W水溶液的注射器分别通过输液软管与同轴纺丝头的壳芯连接孔连接，将金属板固定在接收装置上，同轴纺丝头与高压电连接，排空输液软管中的空气使输液软管内充满溶液，同轴静电纺丝机运行开始纺丝，壳层PLGA/DMF溶液的推进速率为1.5mL/h，芯层KSL-W水溶液的推进速率为0.6mL/h，电压为20kV，同轴纺丝头与金属板的距离为38cm，纺丝在金属板上接收并形成纳米纤维膜；
(4)当收集厚度为0.1mm的纳米纤维膜后，将纳米纤维膜折叠并放置入温度为60℃的烘箱中，烘干2min，最终得到含有抗菌肽的壳芯结构纳米纤维膜。
芯层溶液与壳层溶液分别加入同轴静电纺丝机的两个注射器中，在高压电场的作用下形成“泰勒锥”，并在同轴静电纺丝机的接收装置上得到具有壳芯结构的纳米纤维。使用同轴静电纺丝技术能将两种或多种类型的聚合物溶液合成形态和用途多样化的纳米纤维。芯层为KSL-W、壳层PLGA/DMF制成的壳芯结构纳米纤维膜，使制得的纳米材料最大程度地保持原材料的生物活性，保证了其基础屏障作用，使其在病损区可以排除上皮细胞和结缔组织干扰，为成骨细胞提供空间，以恢复组织原有结构和功能；同时抗菌肽的加入赋予纳米纤维膜抗菌性能，解决了传统膜性材料易与外界交通引起细菌干扰而影响预后的问题。
以实施例1制备出的所述壳芯结构的纳米纤维膜作为实验组、膜性材料“Geistlich
膜”为对照组、不做任何处理作为空白对照组进行粪肠球菌实验兔体内骨缺损修复实验。
实验结果如下：
如图4所示，PLGA/KSL-W纳米纤维膜具有明显的粪肠球菌杀菌效果，而对照组的Bio-Gide膜不具有杀菌效果；
如图5所示，在4周、8周时分别拍摄CBCT，由图可以看出，PLGA/KSL-W纳米纤维膜对骨修复能力可与现有的Bio-Gide膜达到相似的效果。
综上所述，本实施例制备的纳米纤维膜既具有良好的抗菌性，又可以提高复层纤维膜组织亲和性，达到与现有的Bio-Gide膜相似的缺损修复效果。
实施例2
本发明的一种含有抗菌肽的壳芯结构纳米纤维膜的制备方法，包括以下步骤：
(1)从-20℃的冰箱内取出1.2g的PLGA粉末并放置至室温，将PLGA粉末溶于8.8g的DMF溶液，并用磁力搅拌器搅拌溶解20s，磁力搅拌器的转速为1000rpm/s，并静置24h脱泡，得到浓度为14wt％的壳层PLGA/DMF溶液，将壳层PLGA/DMF溶液置于4℃的环境下储存备用；
(2)从-20℃的冰箱内取出15mg的KSL-W粉末并放置至室温，将所述KSL-W粉末与4mL的蒸馏水混合，得到浓度为3.75mg/mL的芯层KSL-W水溶液，并置于4℃的环境下储存备用；
(3)同轴静电纺丝机的注射器各吸取5mL的壳层PLGA/DMF溶液和5mL的芯层KSL-W水溶液，并将注射器固定在静电纺丝机上，装有壳层PLGA/DMF溶液的注射器及装有芯层KSL-W水溶液的注射器分别通过输液软管与同轴纺丝头的壳芯连接孔连接，将金属板固定在接收装置上，同轴纺丝头与高压电连接，排空输液软管中的空气使输液软管内充满溶液，同轴静电纺丝机运行开始纺丝，壳层PLGA/DMF溶液的推进速率为1.5mL/h，芯层KSL-W水溶液的推进速率为0.6mL/h，电压为20kV，同轴纺丝头与金属板的距离为38cm，纺丝在金属板上接收并形成纳米纤维膜；
(4)当收集厚度为0.1mm的纳米纤维膜后，将纳米纤维膜折叠并放置入温度为60℃的烘箱中，烘干1min，最终得到含有抗菌肽的壳芯结构纳米纤维膜。
实施例3
本发明的一种含有抗菌肽的壳芯结构纳米纤维膜的制备方法，包括以下步骤：
(1)从-20℃的冰箱内取出1.0g的PLGA粉末(分子量为40w)并放置至室温，将PLGA粉末溶于9.0g的DMF溶液，并用磁力搅拌器搅拌溶解20s，磁力搅拌器的转速为1000rpm/s，而后静置24h脱泡，得到浓度为10wt％的壳层PLGA/DMF溶液，将壳层PLGA/DMF溶液置于4℃的环境下储存备用；
(2)从-20℃的冰箱内取出15mg的KSL-W粉末并放置至室温，所述KSL-W粉末与4mL的蒸馏水混合，得到浓度为3.75mg/mL的芯层KSL-W水溶液，并置于4℃的环境下储存备用；
(3)同轴静电纺丝机的注射器各吸取5mL的壳层PLGA/DMF溶液和5mL的芯层KSL-W水溶液，并将注射器固定在静电纺丝机上，装有壳层PLGA/DMF溶液的注射器及装有芯层KSL-W水溶液的注射器分别通过输液软管与同轴纺丝头的壳芯连接孔连接，将金属板固定在接收装置上，同轴纺丝头与高压电连接，排空输液软管中的空气使输液软管内充满溶液，同轴静电纺丝机运行开始纺丝，壳层PLGA/DMF溶液的推进速率为1.0mL/h，芯层KSL-W水溶液的推进速率为0.5mL/h，电压为20kV，同轴纺丝头与金属板的距离为35cm，纺丝在金属板上接收并形成纳米纤维膜；
(4)当收集厚度为0.1mm的纳米纤维膜后，将纳米纤维膜折叠并放置入温度为60℃的烘箱中，烘干1min，最终得到含有抗菌肽的壳芯结构纳米纤维膜。
芯层溶液与壳层溶液分别加入同轴静电纺丝机的两个注射器中，在高压电场的作用下形成“泰勒锥”，并在同轴静电纺丝机的接收装置上得到具有壳芯结构的纳米纤维。使用同轴静电纺丝技术能将两种或多种类型的聚合物溶液合成形态和用途多样化的纳米纤维。芯层为KSL-W、壳层PLGA/DMF制成的壳芯结构纳米纤维膜，使制得的纳米材料最大程度地保持原材料的生物活性，保证了其基础屏障作用，使其在病损区可以排除上皮细胞和结缔组织干扰，为成骨细胞提供空间，以恢复组织原有结构和功能；同时抗菌肽的加入赋予纳米纤维膜抗菌性能，解决了传统膜性材料易与外界交通引起细菌干扰而影响预后的问题。
以上对本发明做了示例性的描述，应该说明的是，在不脱离本发明的核心的情况下，任何简单的变形、修改或者其他本领域技术人员能够不花费创造性劳动的等同替换均落入本发明的保护范围。
Claims (3)
1.一种含有抗菌肽的壳芯结构纳米纤维膜的制备方法，包括以下步骤：
(1)从-20℃的冰箱内取1.0g～1.8g的PLGA粉末放置至室温，将PLGA粉末溶于8.2g～9.0g的DMF溶液中，并用磁力搅拌器搅拌溶解10～30s，磁力搅拌器的转速为1000rpm/s，而后静置24h脱泡，得到浓度为10wt％～18wt％的壳层PLGA/DMF溶液，将壳层PLGA/DMF溶液置于4℃的环境下储存备用；
(2)从-20℃的冰箱内取出15mg～19mg的KSL-W粉末并放置至室温，将KSL-W粉末与4mL蒸馏水混合，得到浓度为3.75mg/mL～4.75mg/mL的芯层KSL-W水溶液，并置于4℃的环境下储存备用；
(3)用同轴静电纺丝机的注射器分别吸取壳层PLGA/DMF溶液和芯层KSL-W水溶液，并将注射器固定在静电纺丝机上，装有壳层PLGA/DMF溶液的注射器及装有芯层KSL-W水溶液的注射器分别通过输液软管与同轴纺丝头的壳芯连接孔连接，将金属板固定在接收装置上，同轴纺丝头与高压电连接，排空输液软管中的空气使输液软管内充满溶液，同轴静电纺丝机运行开始纺丝，壳层PLGA/DMF溶液的推进速率为1.0～2.0mL/h，芯层KSL-W水溶液的推进速率为0.5～1.0mL/h，电压为20～26kV，同轴纺丝头与金属板的距离为35～40cm，纺丝在金属板上接收并形成纳米纤维膜；
(4)当收集厚度为0.1mm～0.3mm的纳米纤维膜后，将纳米纤维膜折叠并放置入温度为40℃～70℃的烘箱中，烘干0.5min～2min，最终得到含有抗菌肽的壳芯结构纳米纤维膜。
2.根据权利要求1所述的制备方法，其特征在于：步骤(1)中的PLGA粉末的分子量为5.0×104～50×104。
3.根据权利要求1所述的制备方法，其特征在于：步骤(4)得到的所述壳芯结构纳米纤维膜中，抗菌肽占壳芯结构纳米纤维膜总重量百分比为0.025％～2.5％，PLGA占壳芯结构纳米纤维膜总重量百分比为99.975％～97.5％。
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| 徐小茵等: "PLGA/KSL-W纳米纤维膜用于根尖周炎骨缺损修复的研究", 《2020年中华口腔医学会老年口腔医学专业委员会第十五次全国老年口腔医学学术年会论文集》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115887762A (zh) * | 2022-10-11 | 2023-04-04 | 苏州大学 | 一种微流控静电纺纤维膜微球及其制备方法与在血管修复方面的应用 |
| CN115887762B (zh) * | 2022-10-11 | 2024-03-15 | 苏州大学 | 一种微流控静电纺纤维膜微球及其制备方法与在血管修复方面的应用 |
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