CN111286810B - 积雪草苷生物相容可降解电纺丝及其制备方法和应用 - Google Patents
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Abstract
一种纺丝,由PLGA、PEO和积雪草苷经稳定射流静电纺丝技术混纺制得,具有相同的取向性。其中,PLGA与PEO的重量比为4∶1,积雪草苷用量为PLGA的10wt%。本发明的纺丝,其亲水性显著提高,具有调控体内巨噬细胞表型转化和抗炎的作用,可用于制备调控体内巨噬细胞表型转化和抗炎的药物或医疗器械(如:支架)。
Description
技术领域
本发明涉及一种生物材料,尤其涉及一种含有积雪苷的生物材料,具有生物可降解和生物相容性,以及其制备方法和应用。
背景技术
积雪草苷又称积雪草甙,淡黄色至淡棕黄色的粉末;无臭,味苦,稍具有引湿性,有促进创伤愈合作用。其主要用于治疗外伤,手术创伤,烧伤,疤痕疙瘩及硬皮病等症。
PLGA(即聚乳酸羟基乙酸酯)具有优异的生物相容性,良好的机械性能和可调节的生物降解时间,作为组织工程合成材料被广泛应用。PLGA电纺丝和支架已经广泛应用于组织工程的组织构建,例如:骨、软骨、皮肤和神经再生,以及用于药物递送系统。然而,PLGA本身及其植入体内后引发的降解反应、降解产物都能导致体内严重的炎症反应,导致炎症细胞例如树突细胞,肥大细胞,粒细胞和巨噬细胞等的高度浸润,致使PLGA降解加速,病理细胞过度增生,从而影响PLGA支架介导的再生能力。
发明内容
本发明的一个目的在于提供一种纺丝,其采用稳定射流静电纺丝技术,经混纺制得。
本发明的另一个目的在于提供一种纺丝,其以PLGA/PEO为材料,加入积雪草苷,经稳定射流静电纺丝混纺制得,具有生物相容和生物可降解特性。
本发明的再一个目的在于提供一种纺丝,作为组织工程材料构件用于制备调控体内巨噬细胞表型转化和抗炎的作用的药物或医疗器械。
本发明的又一个目的在于提供一种纺丝,作为支架材料用于制备抗炎和诱导组织再生的医疗器械。
一种纺丝,由PLGA、PEO和积雪草苷经稳定射流静电纺丝技术混纺制得,其中PLGA与PEO的重量比为4∶1,积雪草苷用量为PLGA的10wt%。
另一种纺丝的具体实施方式,其直径为1269±188.2纳米。
另一种纺丝的具体实施方式,其亲水角为17.47°±0.5360°。
另一种纺丝的具体实施方式,其在傅立叶变换的红外光谱下,于1756厘米-1和2886厘米-1具有特征峰。
一种纺丝,由如下方法制备:
分子量100,000的PLGA和分子量500万以上的聚环氧乙烷(PEO)(重量比为4∶1)溶解在2,2,2-三氟乙醇(TFE)中,质量浓度为5%,然后将积雪草苷DMSO溶液和PLGA混合(积雪草苷用量为PLGA的10wt%),得纺丝原液。采用稳定射流静电纺丝技术,将纺丝原液以1.0毫升/小时的速率进行混纺,电压为5千伏~6千伏,喷丝头尖端和收集鼓旋转之间的距离为20厘米,收集鼓的旋转速度为1000转/分钟,在静电纺丝过程中,环境温度为20℃,相对湿度为50%。
本发明的纺丝,其亲水性显著提高,具有调控体内巨噬细胞表型转化和抗炎的作用,可用于制备调控体内巨噬细胞表型转化和抗炎的药物或医疗器械(如:支架)。
本发明的纺丝,其亲水性显著提高,可用作为体内植入后抗炎和诱导组织再生诱导的一种支架材料。
附图说明
图1为本发明的方法制得纺丝在扫描电镜下形态示意图;
图2为本发明制得纺丝直径分布示意图;
图3为本发明制得纺丝所具有的傅立叶变换的红外光谱图;
图4为本发明制得纺丝亲水角统计图;
图5A为本发明制得纺丝埋植入受试动物14天后,经CD86免疫组化染色观察M1巨噬细胞的变化情况;
图5B为本发明制得纺丝埋植入受试动物28天后,经CD86免疫组化染色观察M1巨噬细胞的变化情况;
图6A为本发明制得纺丝埋植入受试动物14天后,经CD163免疫组化染色观察M2巨噬细胞的变化情况;
图6B为本发明制得纺丝埋植入受试动物28天后,经CD163免疫组化染色观察M2巨噬细胞的变化情况;
图7为本发明制得纺丝基质对成纤维细胞、M1和M2巨噬细胞共培养48小时后,对三种细胞基因表达的影响,其中“(a)”图为纺丝对成纤维细胞基因表达的影响,“(b)”图纺丝对M1巨噬细胞基因表达的影响,“(c)”图纺丝对M2巨噬细胞基因表达的影响。
具体实施方式
以下结合附图详细描述本发明的技术方案。本发明实施例仅用以说明本发明的技术方案而非限制,尽管参照较佳实施例对本发明进行了详细说明,本领域的普通技术人员应当理解,可以对发明的技术方案进行修改或者等同替换,而不脱离本发明技术方案的精神和范围,其均应涵盖在本发明的权利要求范围中。
本实施例的纺丝,采用如下方法制得:
PLGA和聚环氧乙烷(PEO)(重量比为4∶1)溶解在2,2,2-三氟乙醇(TFE)中,质量浓度为5%,然后积雪草苷DMSO溶液和PLGA混合(积雪草苷与PLGA的重量比为10wt%),得纺丝原液。采用稳定射流静电纺丝技术,将纺丝原液以1.0毫升/小时的速率进行混纺,电压为5千伏~6千伏,喷丝头尖端和收集鼓旋转之间的距离为20厘米,收集鼓的旋转速度为1000转/分钟,在静电纺丝过程中,环境温度为20℃,相对湿度为50%。
本实施例制取的纺丝,具有高度取向结构(参见图1),能很好地仿生一些具有各向异性结构的天然组织,如:肌腱,韧带和血管等,对于细胞的功能表达以及组织的修复效果具有重要的作用。因此,通过稳定射流技术制备的取向纤维负载积雪草苷对于再生具有各向异性结构的组织过程中的缓解炎症反应有着更好的优势。
实施例1纺丝特性考察
制得的纺丝喷金后,使用扫描电子显微镜观察。如图1所示,未载药的PLGA电纺丝(PLGA)和载药PLGA电纺丝(AS-PLGA)的形态。如图2所示,发现未载药的PLGA电纺丝直径为1265±170.5纳米,载药PLGA的平均直径为1269±188.2纳米,两者相似。该直径能显著地促进成纤维细胞地增殖、铺展,以及胶原蛋白的表达,更利于创伤的修复。
机械分析两者最大负载,杨氏模量、机械张力以及应力-应变曲线,发现载药后的PLGA机械强度和性能稍稍减弱。
应用傅立叶变换的红外光谱(FTIR)来鉴定未载药和载药电纺丝的性质。分析显示载药PLGA的特征峰在1756厘米-1和2886厘米-1,相当于羧基键(-COO-)和碳氢键(CH),这些数据表明积雪苷成功载入到PLGA纳米纤维中。
通过滴水至两种PLGA表面,观察其接触角以计算两者的亲水性,并拍下液滴在垂直于纤维轴线的照片。如图4所示,观察发现载药PLGA的亲水角(17.47°±0.5360°)远高于未载药PLGA亲水角(40.56°±2.208°),表面积雪苷增加PLGA电纺丝亲水性。材料经功能化后((即对材料实施改性或者引入其他材料和药物)),亲水性得到增强,其表面形貌,化学结构的变化,能很好地改善材料与细胞之间的相互作用,比如:黏附,铺展等,从而提高材料的生物相容性,对于创伤修复具有积极作用。
实施例2纺丝功能验证
将SD大鼠随机分为两组:分别植入未载药的PLGA电纺丝和载药PLGA电纺丝。SD大鼠用10%水合氯醛(4毫升/公斤)麻醉。两种PLGA电纺丝被压缩成圆筒状(直径9毫米,厚度1毫米)。在大鼠脊柱的两侧制作两个长度为1cm的切口,分离皮下组织后将PLGA和载药PLGA电纺丝分别埋入,然后用缝线进行伤口闭合。在手术后2周和4周收集材料通过HE染色和免疫组化染色观察。结果发现,载药PLGA组宿主产生的炎症反应明显减弱,材料降解也稍有减轻。通过对巨噬细胞表型蛋白的染色发现,如图5A和图5B所示,载药PLGA电纺丝周围组织中浸润的促炎型M1型巨噬细胞明显减少在14天和28天结果都是如此。如图6A和图6B所示,抗炎型M2型巨噬细胞明显增多,在14天和28天结果都是如此。
在电纺丝上培养成纤维细胞(人皮肤成纤维细胞取自包皮环切手术患者,已得到患者同意和书面告知)和或巨噬细胞。将PLGA电纺丝负载在圆形载玻片(直径35mm,厚度为0.45mm)上,。将原代巨噬细胞系(RAW 264.7)以2×106细胞/孔的密度接种到6孔板上,在含有10%胎牛血清的RPMI1640培养液中培养24小时。然后用脂多糖(LPS,100纳克/毫升)和重组IL-4(20纳克/毫升)分别刺激RAW 264.7细胞12小时,使其分别分化为M1和M2巨噬细胞,收集极化的M1和M2巨噬细胞用于随后的实验。将1×106的成纤维细胞,M1/M2巨噬细胞分别接种到普通6孔培养板,铺好PLGA电纺丝或载药PLGA电纺丝的6孔培养板上,用2.5毫升含有10%胎牛血清的相应培养基培养48小时后进行PCR检测基因表达,包括白细胞介素类生长因子(IL-1β、IL-4、IL-6、IL-8、IL-10、IL-12、IL-17、IL-18)、血小板衍生生长因子(PDGF)、血管内皮生长因子(VEGF)、诱导型一氧化氮合成酶(INOS)、肿瘤坏死因子α(TNF-α)、Kruppel样因子4(KLF4)、细胞标记物CD206和转化生长因子-β1(TGF-β1)。
如图7所示,48小时后,培养在未载药PLGA电纺丝上的成纤维细胞较培养在普通培养皿上的细胞炎症因子表达明显升高,但培养在载药PLGA电纺丝的细胞这种情况可明显改善。
培养在未载药PLGA电纺丝上的M1型巨噬细胞较培养在普通培养皿上的细胞炎症因子表达明显升高,但载药PLGA电纺丝可明显改善这种促炎情况。
培养在未载药的PLGA电纺丝上的M2型巨噬细胞和培养在普通培养皿上的细胞表达的抗炎基因基本无差异,但载药PLGA电纺丝可明显提高M2型巨噬细胞和抗炎基因表达。
Claims (5)
1.一种纺丝在制备调控体内巨噬细胞表型转化和抗炎的药物或医疗器械中的应用,其特征在于所述的纺丝由PLGA、PEO和积雪草苷经稳定射流静电纺丝技术混纺制得,具有相同的取向性;
所述的PLGA分子量为10万;
所述的PEO分子量大于500万;
所述的PLGA与所述的PEO的重量比为4∶1;
所述的积雪草苷用量为所述PLGA的10wt%。
2.根据权利要求1所述的纺丝在制备调控体内巨噬细胞表型转化和抗炎的药物或医疗器械中的应用,其特征在于所述的纺丝直径为1269±188.2纳米。
3. 根据权利要求1所述的纺丝在制备调控体内巨噬细胞表型转化和抗炎的药物或医疗器械中的应用,其特征在于所述的纺丝亲水角为17.47 ±0.5360。
4.根据权利要求1所述的纺丝在制备调控体内巨噬细胞表型转化和抗炎的药物或医疗器械中的应用,所述的纺丝用于制成支架材料。
5.根据权利要求1所述的纺丝在制备调控体内巨噬细胞表型转化和抗炎的药物或医疗器械中的应用,制取所述纺丝的方法包括如下步骤:
PLGA和PEO按重量比为4∶1溶解在2,2,2-三氟乙醇中,质量浓度为5%,然后将积雪草苷DMSO溶液和PLGA混合,得纺丝原液;
采用稳定射流静电纺丝技术,将纺丝原液以1.0毫升/小时的速率进行混纺,电压为5千伏~6千伏,喷丝头尖端和收集鼓旋转之间的距离为20厘米,收集鼓的旋转速度为1,000转/分钟,在静电纺丝过程中,环境温度为20℃,相对湿度为50%。
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