CN113384737A - 一种吸渗控释抗菌肽水凝胶双层敷料及其制备方法和应用 - Google Patents

一种吸渗控释抗菌肽水凝胶双层敷料及其制备方法和应用 Download PDF

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CN113384737A
CN113384737A CN202110691435.5A CN202110691435A CN113384737A CN 113384737 A CN113384737 A CN 113384737A CN 202110691435 A CN202110691435 A CN 202110691435A CN 113384737 A CN113384737 A CN 113384737A
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王万能
曹武龙
成福
唐一山
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Suzhou 30 Billion Technology Co ltd
Yantai Baofuling Biotechnology Co ltd
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Abstract

本发明涉及一种吸渗控释抗菌肽水凝胶双层敷料及其制备方法和应用，属于生物医用材料领域。所述水凝胶双层敷料包括抗菌肽控释水凝胶层和吸渗凝胶层，本发明首次将壳聚糖凝胶膜作为吸渗材料用于药物敷料中吸收伤口感染渗液，实现渗液自调节的作用，上层抗菌肽水凝胶释放层控制抗菌肽proSP‑B的持续释放，从而长效抑制伤口处的MRSA等多重耐药菌，加快伤口感染愈合。

Description

一种吸渗控释抗菌肽水凝胶双层敷料及其制备方法和应用
技术领域
本发明属于生物医用材料领域，涉及一种吸渗控释抗菌肽水凝胶双层敷料及其制备方法和应用。
背景技术
抗菌肽(AMPs)是无数动植物自然免疫系统的重要组成部分，因为它们不易产生耐药性，因此被认为有潜力成为新型治疗药物。肺表面活性蛋白是哺乳动物肺的呼吸上皮分泌的脂蛋白复合物。肺表面活性蛋白B(SP-B)是该复合物的一部分，其可以降低在肺中表面张力，增加肺顺应性，参与稳定肺环境。缺乏SP-B可引起肺表面活动功能障碍，呼吸功能异常和呼吸失常。此外，它还具有抗菌、调节机体免疫反应等防御作用。SP-B是由381-氨基酸(42-kDa)的ProSP-B前体蛋白经过至少三个蛋白水解步骤成熟的。研究发现SP-B的前体ProSP-B也具有潜在的抗菌活性。还指出SP-B蛋白属于SAPLIPs家族具有“saponsin”结构，而且SAPLIP家族具有抗菌活性。研究发现，proSP-B对革兰氏阳性菌和革兰氏阴性菌以及红色毛廯菌、白色念珠菌都具有良好的抑菌效果，且不具备细胞毒性。
水凝胶代表一类材料，广泛用于皮肤，血管，肌肉和脂肪的软组织工程。水凝胶是由亲水性聚合物的物理或化学交联键组成的三维(3D)网络。不溶性亲水结构显示出吸收伤口渗出液的显著潜力，并允许氧扩散以促进愈合。重要的是，水凝胶具有高度水合的3D聚合物网络，与它们的干重相比，可以结合多倍的水，从而可以保持伤口处的高水分含量。由于这些独特的物理特性，水凝胶网络可以铸成各种尺寸和形状加以应用。因此，基于水凝胶的材料是最适合覆盖皮肤伤口的敷料。
在灾难，事故和战争等紧急情况下，严重的出血是导致高死亡率的主要原因。产生的伤口可能给患者带来严重的生理和心理创伤，并带来巨大的社会经济负担。因此，快速止血和伤口愈合技术已成为挽救生命的治疗的关键举措。尽管依靠绷带和纱布的传统方法在控制出血方面是有效的，但它们也存在一些局限性：不可生物降解性，易感染，不适合不规则伤口，继发性组织损伤以及对伤口愈合几乎无效。随着时间的推移，聚合物等敷料材料或将成为修复皮肤组织和愈合伤口的必然趋势。
良好的敷料应具有以下特征：(1)易于适应复杂的伤口轮廓和体积；(2)具有机械保护；(3)保持潮湿的环境；(4)具有理想的气体渗透性；(5)能够吸收渗出液；(6)保护伤口免受细菌感染；(7)易于无创伤地进行更换和去除；(8)能够在极端环境中长时间保存；(9)昂贵但商业上可接受，(10)质量较轻，(11)无毒、无过敏、生物相容性好、可生物降解和具有一定弹性。
发明内容
有鉴于此，本发明的目的之一在于提供一种吸渗控释抗菌肽水凝胶双层敷料，本发明的目的之二在于提供所述抗菌肽双层敷料在治疗创伤后多重耐药菌引起的伤口感染方面的应用。
为达到上述目的，本发明提供如下技术方案：
1、一种吸渗控释抗菌肽水凝胶双层敷料，所述敷料包括双层结构，上层为抗菌肽控释水凝胶层，下层为吸渗凝胶层，所述抗菌肽控释水凝胶层与所述吸渗凝胶层通过物理或化学的方法贴合。
作为优选的技术特征之一，所述吸渗凝胶层的厚度为1-2mm，抗菌肽控释水凝胶层的厚度为3-5mm。
作为优选的技术特征之一，所述抗菌肽包括但不限于pro-SP-B、K11、LL-37、DPK-060、RP557、DRGN-1或天蚕素A中的一种或几种。
作为优选的技术特征之一，所述抗菌肽控释水凝胶层的制备方法为，将聚乙烯醇和聚乙二醇混合，加入抗菌肽，混合均匀后，-80℃冷冻10-14h，取出后冻融一次，得到抗菌肽控释水凝胶层。
作为优选的技术特征之一，所述壳聚糖、聚乙二醇和甘油的质量分数分别为1-3％、0.6-1％和1-3％。
作为优选的技术特征之一，所述抗菌肽的终浓度为10-100μM。
作为优选的技术特征之一，所述吸渗凝胶层的制备方法为，将壳聚糖溶于冰醋酸中，200-500r/min搅拌10-14h，过滤后加入聚乙二醇和甘油，室温下搅拌1-4h，脱气处理后再倒入培养皿中，﹣80℃冷冻10-14h，真空冷冻干燥，即得吸渗凝胶层。
作为优选的技术特征之一，所述聚乙烯醇和聚乙二醇的质量分数分别为6-8％和0.6-2％。
2、所述水凝胶双层敷料在治疗多重耐药菌导致的伤口感染中的应用。
本发明的有益效果在于：
1、本发明制备出吸渗控释抗菌肽水凝胶双层敷料综合了抗菌肽、壳聚糖、聚乙烯醇、聚乙二醇等的优点。其中，抗菌肽是天然的蛋白，对MRSA等多重耐药菌有良好的抑制效果，除此之外抗菌肽可以通过促进再上皮形成和肉芽组织形成来加速体内伤口愈合。抗菌肽还可以通过诱导内皮细胞管形成，上调血管生成蛋白，增加血管内皮生长因子的产生来支持血管生成。复合聚乙烯醇、聚乙二醇通过物理交联制备的水凝胶，实现了抗菌肽proSP-B的缓释可控释释放；再通过复合壳聚糖、聚乙二醇、甘油制备的吸渗膜，完善了整体敷料的功能，使该双层敷料不仅具有调节渗液，还可以起到持续释放抗菌肽，作用于伤口感染部位的目的。
2、本发明以抗菌肽、聚乙烯醇、壳聚糖、聚乙二醇等为原料，通过复合和贴合制备得到吸渗控释抗菌肽水凝胶，在复合过程中，吸渗层与抗菌肽释放层的材料组分之间存在着氢键的结合。该双层敷料在满足良好生物相容性的基础上，具有促进MRSA感染伤口快速愈合的作用。本发明的吸渗控释抗菌肽水凝胶双层敷料是一种理想的新型治疗多重耐药菌引起伤口感染难愈合的皮肤修复材料，具有良好的应用前景。
3、本发明制备的吸渗控释抗菌肽水凝胶，具备良好的生物相容性和与满足伤口敷料匹配的力学性能以及凝胶的回弹性。相比于其他同类水凝胶敷料，采用抗菌肽作为治疗药物，制备工艺简单，易于操作和控制，具有广阔的应用前景。
附图说明
为了使本发明的目的、技术方案和优点更加清楚，下面将结合附图对本发明作优选的详细描述，其中：
图1为吸渗控释抗菌肽水凝胶双层敷料外观图；
图2为制备的吸渗膜的红外光谱图；
图3为制备抗菌肽释放水凝胶的红外光谱图；
图4为吸渗控释proSP-B抗菌肽凝胶对MRSA的抑菌圈，1为抗菌肽proSP-B控释水凝胶；2为吸渗控释水凝胶双层敷料；3为吸渗控释抗菌肽水凝胶双层敷料；
图5吸渗控释抗菌肽水凝胶与兔红细胞接触；
图6为吸渗控释抗菌肽水凝胶的血液相容性；
图7为SD大鼠皮肤刺激性实验，1为阳性对照10％的甲醛溶液；2为生理盐水；3为实验组，双层敷料的浸提液；
图8为第一组不同时间点创面形态学照片；Control(左)/吸渗控释抗菌肽水凝胶双层敷料(右)；
图9为第二组不同时间点创面形态学照片；纳米银凝胶组(左)/吸渗控释抗菌肽水凝胶敷料(右)；
图10为SD大鼠不同时间的伤口愈合程度；与空白对照组比：*P<0.05，**P<0.01；与市售敷料组比：#P<0.05，##P<0.01；
图11为H&E染色结果，Control：纱布；阳性对照：纳米银离子凝胶；实验组:吸渗控释抗菌肽水凝胶。
具体实施方式
以下实施例以proSP-B抗菌肽为例，具体说明本发明的实施方式，本领域技术人员可由本说明书所揭露的内容轻易地了解本发明的其他优点与功效。本发明还可以通过另外不同的具体实施方式加以实施或应用，本说明书中的各项细节也可以基于不同观点与应用，在没有背离本发明的精神下进行各种修饰或改变。
实施例1
吸渗控释抗菌肽水凝胶双层敷料的制备与表征
(1)吸渗控释抗菌肽水凝胶双层敷料的制备方法
A、将CS(壳聚糖)溶解在冰醋酸中，300r/min过夜搅拌，四层无菌纱布过滤出未溶解CS，向壳聚糖溶液中加入聚乙二醇和甘油，室温下搅拌2h，1500r/min离心10min，再将混合液倒入培养皿中，﹣80℃冷冻过夜，真空冷冻干燥，即得下层吸渗凝胶；CS、PEG4000和甘油的质量分数分别为3％、0.6％和1％；
B、将聚乙烯醇和聚乙二醇混合，加入proSP-B抗菌肽粉末，使proSP-B抗菌肽的终浓度为1000μg/mL，混合均匀后，-80℃冷冻过夜，冻融一次，得到上层proSP-B抗菌肽控释水凝胶；聚乙烯醇和聚乙二醇的质量分数分别为6％和1％；
C、将下层吸渗凝胶与上层proSP-B抗菌肽控释水凝胶剪成相应大小、物理贴合，得到吸渗控释抗菌肽水凝胶双层敷料。
对所制备的吸渗控释抗菌肽水凝胶双层敷料采用微距拍摄，结果如图1所示，吸渗凝胶膜层约1mm，抗菌肽控释水凝胶层约3mm。
(2)吸渗控释抗菌肽水凝胶双层敷料的表征
采用傅里叶变换衰减全反射红外光谱法(ATR–FTIR)表征吸渗膜在制备过程中(CS-PEG)，抗菌肽控释水凝胶(不含抗菌肽proSP-B)在制备过程中(PVA，PVA-PEG)官能团的改变。
吸渗凝胶层红外表征结果如图2所示，CS/PEG表面在3350cm-1的-OH吸收峰，相比于PEG有明显的增强，说明壳聚糖的加入使吸渗膜表面羟基含量明显增多，从而提高了吸渗膜的亲水能力。CS和PEG在2883-2886cm-1具有吸收峰，为C-H强振动吸收峰，C-H强振动吸收峰的峰值减小。以上表明各组分之间存在一定的氢键作用力，使各个材料成分结合成膜。
抗菌肽控释水凝胶红外表征结果如图3所示，PVA表面在3277cm-1的-OH吸收峰，PVA与PEG共混之后在3304cm-1出现了-OH的伸缩振动峰，表明PVA的加入使得整个PVA/PEG材料的亲水性能得到了增强。此外，在1100-842cm-1出现的峰，均为C-H强振动吸收峰。PVA与PEG结合之后C-H强振动吸收峰同样在减小。
实施例2
吸渗控释抗菌肽水凝胶双层敷料对MRSA的抑菌活性
按照实施例1中方法制备吸渗凝胶层和proSP-B抗菌肽控释水凝胶，经过高温灭菌后置于烘箱中干燥，紫外杀菌2h以上。将两层凝胶物理贴合制备得到吸渗控释抗菌肽水凝胶双层敷料。用pH为7.2的无菌PBS溶液将吸渗凝胶层浸润，模仿伤口感染处pH值，然后将吸渗控释抗菌肽水凝胶双层敷料覆盖在涂满MRSA的平板上，37℃正置培养18h，用直尺测量抑菌圈大小，统计分析。
结果如图4所示，双层敷料具有良好的抑制MRSA效果，单一的控释抗菌肽proSP-B水凝胶的抗菌效果(抑菌圈13±2.3mm)比吸渗控释抗菌肽水凝胶双层敷料(抑菌圈0.8±0.67mm)要强p<0.05。
实施例3
吸渗控释抗菌肽水凝胶双层敷料溶血性评价
1、样品准备：
实验组：0.05g冻干敷料/10mL生理盐水
阳性对照组：10mL蒸馏水
阴性对照组：10mL生理盐水
2、新鲜抗凝血：草酸钾抗凝兔血
3、稀释抗凝血的制备：4mL抗凝血+0.2mL草酸钾溶液(20g/L)+5mL生理盐水，将三者充分摇匀，即得到稀释抗凝血。
4、实验步骤：取6支干净的离心管(15mL规格)，每组设3个平行组，分别编号为阳性对照、阴性对照、吸渗液层、释放层、双层(不含proSP-B)、双层(含1000μg/mL proSP-B)。实验组的每个离心管中分别加入不同编号的敷料0.05g，再加10mL生理盐水；阴性对照组直接加入10mL生理盐水；阳性对照组加入10ml去离子水。将各管在37℃恒温条件下孵育0.5h，再加入250μL稀释抗凝血，混合均匀，37℃静置1h。将各组溶液转移到新的已编号的离心管中，1000r/min，离心5min，取上清液200μL于96孔板中，测其在540nm下的OD值，并计算每组的平均值。由下式计算溶血程度：
Figure BDA0003126917510000051
其中，A1为实验组OD值，A2为阳性对照组OD值，A3为阴性对照OD值(按照国标要求，溶血度不得大于5％)
阳性对照组充分破坏了红细胞(图5)，导致离心之后毫无红细胞沉淀产生，而反观阴性对照组和实验组，离心之后，上清液的颜色几乎没有红色，表明红细胞并没有被破坏。且实验组的溶血度均未超过1％(图6，表1)，符合国标要求。然后评价了双层敷料的溶血性，跟阳性和阴性相比，双层敷料不具备溶血性，可以跟血液直接接触。
表1吸渗控释水凝胶的血液相容性
Figure BDA0003126917510000061
实施例4
吸渗控释抗菌肽水凝胶双层敷料皮肤刺激性测试
1、材料浸提液的制备:在37℃水浴条件下，按0.2g冻干敷料/mL生理盐水的比例，浸提24h，浸提完成后，在超净工作台里用0.22μm的滤膜对浸提液进行充分的过滤除菌，除菌之后转移至灭菌后的EP管中，备用。
2、取三只健康的SD大鼠，采用同体自身对比法，每只体重200±10g，正常饮食，使动物适应环境3天。
3、实验前24h，对大鼠背部皮肤进行检查，并确保完好无损，用理发器对大鼠背部剃毛，剃毛范围脊柱两侧各6cm×5cm，检查皮肤无因剃毛而受损，背部皮肤划分为4个区域。将纱布剪成2cm×2cm的方块，高温灭菌后备用。吸渗控释抗菌肽水凝胶(实验组)，10％的甲醛溶液(阳性对照组)，生理盐水(阴性对照组)，按照1mL液体/块纱布，分别将无纺布贴敷于新西兰兔背部的1、2、3、4区，涂药面接触皮肤，其中1、3区为实验组；2、4区为阳性对照和阴性对照。负覆盖后用压敏胶固定纱布。并在8h、16h、24h，三个不同时间观察皮肤反应，根据表2对皮肤红斑和水肿情况记分。
表2皮肤反应积分系统
Figure BDA0003126917510000071
SD大鼠皮肤刺激性结果如图7和表3所示，吸渗控释抗菌肽水凝胶对大鼠皮肤无刺激作用，红斑、水肿均未发生。而阳性对照的红斑水肿等情况较为明显，双层敷料对皮肤的刺激性记为0分，实验结果理想。
表3皮肤刺激反应记分
Figure BDA0003126917510000072
实施案例5
吸渗控释抗菌肽水凝胶双层敷料对MRSA感染伤口的愈合作用
1、SPF级SD雄性大鼠20只，体重约200g，适应性喂养一周，实验当天禁食，制备大鼠皮肤创伤模型，随机将其分为2组，纱布(阴性对照组)和实验组(含1000μg/mL抗菌肽proSP-B的双层敷料组)；阳性对照组(纳米银抗菌凝胶)和实验组(含1000μg/mL抗菌肽proSP-B双层敷料组)。
2、菌悬液的制备：向已培养好的MRSA斜面中加入4mL无菌生理盐水，用灭菌后的接种环刮取MRSA，然后将菌液转入无菌的10mL离心管中，在涡旋振荡器上混匀1min后静置，无菌生理盐水调整菌悬液的浓度为1×108CFU/mL。
3、大鼠预处理：将SD大鼠用水合氯醛(每公斤体重30mg的剂量)麻醉大鼠进行麻醉后，用理发器在其背部剃出4cm×4cm的面积，尽量把面积内的毛发剃干净。
4、然后，在每只大鼠的主干的左右两侧各创建一个局部厚度的伤口(直径＝7毫米)。将50μL MRSA 1×108CFU/mL细菌悬浮液直接涂在每只动物的伤口上。经过一天的全层皮肤损伤细菌攻击后，使用无菌医用胶带将两组伤口用相应的敷料紧密覆盖。第一组左侧为纱布敷料，右侧为proSP-B抗菌肽双层敷料；第二组左侧为纳米银离子凝胶，右侧为proSP-B抗菌肽双层敷料组。每隔一天更换一次敷料。在不同的时间点(1、3、5、7、9、11天)对不同组的伤口拍照。对于每只大鼠，追踪伤口边缘并使用图像分析软件(Image J，NIH，Bethesda，MD，USA)量化伤口面积。以第0天初始伤口面积的百分比计算伤口面积。在第4、8和11天处死大鼠后，移除伤口部位和周围健康皮肤进行HE染色。
5、大鼠创伤模型建立后，在第1、3、5、7、9d给大鼠伤口进行拍照，并采用Image J图像分析软件计算大鼠伤口面积。大鼠伤口愈合率计算公式如下：
Figure BDA0003126917510000081
各组在伤后创面形态结果如图8、9、10所示，1天后，各组创面具有明显的创伤特点，伤口周围皱缩；5天后伤口愈合各组达50％以上，到第七天的时候伤口愈合速率减慢，到11天之后伤口愈合度都在85％以上。H&E染色结果如图11所示，在第4天，所有组均形成上皮层，但对照纳米银离子凝胶和纱布组在伤口周围表现出更多的炎性细胞。与对照组纳米银离子凝胶和纱布组相比，用双层抗菌肽凝胶覆盖的伤口中的炎症细胞较少，这表明双层抗菌肽凝胶可以减轻伤口周围的炎症。到第8天时，与双层抗菌肽凝胶接触的组织中出现了一些毛囊和血管，而纱布处理组的炎症细胞仍比其余两组多。尽管在用纳米银凝胶处理的伤口中肉芽组织得以再生，但未观察到毛囊和上皮。在第11天，所有组仍显示出轻度的急性炎症反应，而双层抗菌肽凝胶组中出现了大量的毛囊和血管，并出现了完整的表皮，表明双层抗菌肽凝胶具有优异的伤口愈合能力。
最后说明的是，以上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，可以对本发明的技术方案进行修改或者等同替换，而不脱离本技术方案的宗旨和范围，其均应涵盖在本发明的权利要求范围当中。

Claims (9)

1.一种吸渗控释抗菌肽水凝胶双层敷料，其特征在于，所述敷料包括双层结构，上层为抗菌肽控释水凝胶层，下层为吸渗凝胶层，所述抗菌肽控释水凝胶层与所述吸渗凝胶层通过物理或化学的方法贴合。
2.如权利要求1所述的水凝胶双层敷料，其特征在于，所述吸渗凝胶层的厚度为1-2mm，抗菌肽控释水凝胶层的厚度为3-5mm。
3.如权利要求1所述的水凝胶双层敷料，其特征在于，所述抗菌肽包括但不限于pro-SP-B、K11、LL-37、DPK-060、RP557、DRGN-1或天蚕素A中的一种或几种。
4.如权利要求1～3任一项所述的水凝胶双层敷料，其特征在于，所述抗菌肽控释水凝胶层的制备方法为，将聚乙烯醇和聚乙二醇混合，加入抗菌肽，混合均匀后，-80℃冷冻10-14h，取出后冻融一次，得到抗菌肽控释水凝胶层。
5.如权利要求4所述的水凝胶双层敷料，其特征在于，所述壳聚糖、聚乙二醇和甘油的质量分数分别为1-3％、0.6-1％和1-3％。
6.如权利要求4所述的水凝胶双层敷料，其特征在于，所述抗菌肽的终浓度为10-100μM。
7.如权利要求1～3任一项所述的水凝胶双层敷料，其特征在于，所述吸渗凝胶层的制备方法为，将壳聚糖溶于冰醋酸中，200-500r/min搅拌10-14h，过滤后加入聚乙二醇和甘油，室温下搅拌1-4h，脱气处理后再倒入培养皿中，﹣80℃冷冻10-14h，真空冷冻干燥，即得吸渗凝胶层。
8.如权利要求7所述的水凝胶双层敷料，其特征在于，所述聚乙烯醇和聚乙二醇的质量分数分别为6-8％和0.6-2％。
9.权利要求1～8任一项所述的水凝胶双层敷料在治疗多重耐药菌导致的伤口感染中的应用。
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