CN111643724B - 一种载有噬菌体和酸性成纤维细胞生长因子的水凝胶伤口敷料的制备方法及其应用 - Google Patents
一种载有噬菌体和酸性成纤维细胞生长因子的水凝胶伤口敷料的制备方法及其应用 Download PDFInfo
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Abstract
本发明公开了一种载有噬菌体和酸性成纤维细胞生长因子(aFGF)的水凝胶伤口敷料的制备方法及其应用,该伤口敷料由水凝胶包裹噬菌体而形成。该伤口敷料具有以下特征:本发明中应用的噬菌体来源广泛,扩增简单,制备成本低;特异性强,在杀死致病菌的同时,不会影响有益菌群;安全性好,目前没有对动物细胞产生影响的报道;本发明中的水凝胶,以天然高分子为原料,安全无毒,有良好的吸水性和机械性能。通过一系列实验评价,此种伤口敷料可以有效杀死伤口组织中的细菌,并明显促进伤口愈合。这为对抗伤口耐药菌感染提供了一种新的思路。
Description
技术领域
本发明涉及生物医用材料领域,具体地说是一种载有噬菌体和酸性成纤维细胞生长因子(aFGF)的水凝胶伤口敷料的制备方法及其应用。
背景技术
由于抗生素的滥用,耐药菌感染已成为人类健康的主要威胁之一。由耐药菌造成的伤口感染,不仅给患者带来了无限的痛苦,也带来了巨大的经济损失。目前对抗耐药菌的主要策略是研究开发新型的抗生素药物,而目前的新型药物多是对已有药物的再设计,缺乏新型的抗菌机理,从而导致成果转化效率低,难以满足不断增长的临床需求。
噬菌体是细菌的天然杀手,并且在自然界中拥有庞大的数量。噬菌体曾是人类对抗细菌感染的重要武器之一,并成功应用于多种疾病的防治。青霉素的发现和临床应用,使人类抗细菌感染步入了抗生素时代。抗生素治疗的有效性和广谱性使得噬菌体疗法逐渐淡出人们的视野。近年来愈演愈烈的耐药菌问题,使得人们开始重新审视噬菌体疗法的价值,许多研究都证实了噬菌体疗法的有效性和安全性。
酸性成纤维细胞生长因子(aFGF)是一种具有多种作用的生长因子,会在伤口愈合的前期大量合成。它与相应的酪氨酸激酶型受体结合后,会引起下游的酪氨酸磷酸化级联反应,进而促进细胞迁移和增殖,因此,aFGF是一种促伤口愈合因子。
以天然高分子材料为原料的水凝胶,由于其良好的生物相容性、较高的安全性等,已广泛应用于药物递送、组织工程、整形美容等领域。水凝胶作为伤口敷料,也从传统的物理保护,向着功能化发展。
现有的功能性伤口敷料大多只有单一的功能,加入噬菌体和促伤口修复因子的复合水凝胶,不仅可以抑制耐药菌增长,还可以促进伤口愈合,是一种多功能的伤口敷料,具有生物安全性好,抗菌性能高,以及促伤口愈合能力强等特点。
发明内容
本发明的目的在于提供一种载有噬菌体和酸性成纤维细胞生长因子(aFGF)的水凝胶伤口敷料,所述伤口敷料由水凝胶包裹噬菌体及aFGF构成。
所述的水凝胶以明胶、氧化海藻酸钠、透明质酸为原料制备。
所述的噬菌体包括但不限于T4噬菌体。
本发明还提供一种载有噬菌体和酸性成纤维细胞生长因子(aFGF)的水凝胶伤口敷料的制备方法,该方法包括如下步骤:
用双蒸水分别将透明质酸和氧化海藻酸钠配置成溶液。按照一定浓度配置明胶溶液,将其放入60度的水浴中保温,直至明胶溶解。将三种溶液加入圆形模具中,并迅速搅拌均匀。加入一定量的噬菌体和aFGF溶液,搅拌后室温敞口放置直至其形成固态凝胶。
所述明胶、氧化海藻酸钠、透明质酸的最终质量比为1∶1-4∶1。
本发明还提供了一种载有噬菌体和酸性成纤维细胞生长因子(aFGF)的水凝胶伤口敷料的应用,实验结果表明,该载有噬菌体的水凝胶伤口敷料可以抑制耐药菌生长并大幅减少伤口中的细菌数量,并且有效促进伤口的愈合。
本发明的水凝胶伤口敷料具有以下有益效果:
本发明中应用的噬菌体来源广泛,扩增简单,制备成本低;特异性强,在杀死致病菌的同时,不会影响有益菌群;安全性好,目前没有对动物细胞产生影响的报道,因而具有广阔的临床应用前景。本发明中的水凝胶,以天然高分子为原料,安全无毒,有良好的吸水性和机械性能。在动物模型评价中,可以有效抑菌和存进伤口愈合;制备方式简单,成本低,可批量生产。
附图说明
图1为本发明水凝胶的SEM表征图;
图2为本发明水凝胶对MEF细胞的细胞毒性实验图;
图3为本发明水凝胶的溶胀率实验图;
图4为本发明水凝胶的药物释放实验图;
图5为本发明水凝胶的体外抑菌实验图;
图6为本发明水凝胶的糖尿病小鼠伤口愈合实验图;
图7为本发明糖尿病小鼠伤口愈合率及组织菌落数图;
图8为本发明糖尿病小鼠伤口组织切片图
具体实施方式
通过下述实施例将有助于进一步理解本发明,并非因此限制本发明的专利范围。
实施例1载有噬菌体的水凝胶的制备及表征
用双蒸水分别将透明质酸和氧化海藻酸钠配置成溶液。按照一定浓度配置明胶溶液,将其放入60度的水浴中保温,直至明胶溶解。将三种溶液加入圆形模具中,并迅速搅拌均匀。加入一定量的噬菌体溶液,搅拌后室温敞口放置直至其形成固态凝胶。所述明胶和氧化海藻酸钠的质量比为1∶1-4∶1。将所得水凝胶冷冻干燥后,用SEM观察其形貌。SEM形貌如图1所示。
小鼠胚胎成纤维细胞(MEF)接种在96孔细胞培养板中,加入DMEM完全培养基培养24小时。用含有水凝胶浸提液的培养基替换原培养基并继续培养24小时。而后加入MTT溶液并孵育4小时,用DMSO溶解结晶并测其在570nm处的吸收。MTT实验结果如图2所示,各浓度的水凝胶浸提液无细胞毒性。
制备好的水凝胶称重后放入离心管中,管中加入PBS没过水凝胶。在特定时间点取出凝胶,尽量擦干表面的水,然后称重。计算水凝胶的吸水溶胀率。水凝胶的溶胀率如图3所示,该种水凝胶有较好的吸水性,溶胀率可以达到600%以上。
用罗丹明B标记噬菌体,然后将载有标记噬菌体的水凝胶浸入10mL的PBS中。在特定的时间点,用酶标仪测定上清液在625nm处的吸光度,然后计算噬菌体的释放率。实验结果如图4所示,噬菌体可以持续从水凝胶中释出,促进伤口愈合。
实施例2载有噬菌体的水凝胶的体外抑菌评价
水凝胶的体外抑菌评价采用抑菌圈法。细菌培养过夜后稀释一定倍数,然后取50-200μL滴加于固体琼脂糖培养基的表面。用涂布棒涂布均匀。将水凝胶放在固体培养基上,37℃孵育4-12h,观察抑菌圈的情况。实验结果如图5所示。载有噬菌体的水凝胶周围有明显的抑菌圈出现,说明此水凝胶可以抑制细菌生长。
实施例3载有噬菌体的水凝胶促进伤口的评价
小鼠放入气体麻醉机中用异氟烷进行麻醉,然后用脱毛膏涂抹背部并用剃刀退毛。用酒精消毒后,用手术剪在背部造成直径约1cm的全皮层切除伤口。对伤口拍照以供后续处理。将菌液滴加在伤口处,并随机分组:1.只用医用透明固定胶带覆盖伤口;2.注射氨苄青霉素,并用医用透明胶带包裹伤口;3.将水凝胶覆盖在伤口上,并用医用胶带固定;4.将添加噬菌体的水凝胶覆盖在伤口上,并固定。
在1,3,7,14天用异氟烷将小鼠麻醉,对伤口拍照用以计算面积。而后取伤口处组织,取一部分进行菌落计数,其他进行组织学检测。伤口愈合率结果如图6、7所示。载有噬菌体的水凝胶处理的实验组伤口愈合率明显高与对照组,说明此水凝胶可以促进伤口愈合。
取伤口处组织称重,按一定的比例加入PBS。用匀浆机打成匀浆。各组分别进行梯度稀释,然后取50-200μL滴加在含有琼脂的固体培养基上,用涂布棒涂匀,倒置放入37℃恒温培养箱中培养24h。选取菌落数量合适的浓度进行菌落计数。每个浓度做3组平行试验,并计算菌落数。计数结果如图7所示,实验组的组织菌落数明显低于对照组,说明水凝胶中有噬菌体释出并杀死了细菌。
取下的组织用4%的多聚甲醛固定,用石蜡包埋,做成4μm左右的切片,然后进行H&E染色。样本用光学显微镜观察。组织切片结果如图8所示。根据对组织切片的分析可以看出,此水凝胶可以促进伤口愈合。
Claims (3)
1.一种载有噬菌体和酸性成纤维细胞生长因子(aFGF)的水凝胶伤口敷料,其特征在于,所述伤口敷料由水凝胶包裹噬菌体构成,所述的水凝胶以明胶、氧化海藻酸钠、透明质酸为原料制备。
2.如权利要求1所述的载有噬菌体和酸性成纤维细胞生长因子(aFGF)的水凝胶伤口敷料,其特征在于,所述的噬菌体包括但不限于T4噬菌体。
3.权利要求1-2任一项所述的载有噬菌体和酸性成纤维细胞生长因子(aFGF)的水凝胶伤口敷料的制备方法,其特征在于,该方法包括如下步骤:
用双蒸水分别将透明质酸和氧化海藻酸钠配置成溶液,按照一定浓度配置明胶溶液,将其放入60度的水浴中保温,直至明胶溶解,将三种溶液加入圆形模具中,并迅速搅拌均匀,加入一定量的噬菌体和酸性成纤维细胞生长因子(aFGF)溶液,搅拌后室温敞口放置直至其形成固态凝胶,
所述明胶和氧化海藻酸钠最终质量比为1∶1-4∶1。
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