CN112694618B - 一种线性多聚抗菌肽及其制备方法与应用 - Google Patents

一种线性多聚抗菌肽及其制备方法与应用 Download PDF

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CN112694618B
CN112694618B CN202011417894.6A CN202011417894A CN112694618B CN 112694618 B CN112694618 B CN 112694618B CN 202011417894 A CN202011417894 A CN 202011417894A CN 112694618 B CN112694618 B CN 112694618B
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王淼
陈旭
潘国庆
王云龙
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Abstract

本发明属于生物医学高分子材料领域，涉及一种线性多聚抗菌肽及其制备方法与应用。本发明将含炔基的线性聚合物对游离肽（RW）3‑N3进行修饰，通过可逆断裂链转移聚合得到聚合物，将抗菌肽修饰到聚合物上形成多聚肽，制备出以多价效应为基础的线性多聚抗菌肽聚合物。其在保持良好的生物相容性的基础上大大提高了抗菌性能。本发明通过引入纳米片和多聚抗菌肽在水中强静电作用交联形成线性多聚肽纳米水凝胶，不仅提高传统水凝胶的力学性能，而且具备优异的自愈合、可注射性能。因此，在伤口敷料、种植体涂层、抗生物膜和组织工程等生物医学领域具有广阔的应用前景。

Description

一种线性多聚抗菌肽及其制备方法与应用
技术领域
本发明属于生物医学高分子材料领域，涉及一种线性多聚抗菌肽及其制备方法与应用。
背景技术
细菌是化脓性感染、菌血症、脓毒症的等疾病的罪魁祸首，长久以来，细菌感染一直是一个困扰生物医学领域的难题，危害着人类生命健康。目前，清创引流是治疗临床化脓性感染的常用方法，但由此引起组织的继发性损伤给患者带来了巨大的痛苦。抗生素作为有效的抗菌药物也一直扮演着重要的角色，但随着抗生素的滥用导致了超级耐药菌的出现，而新型抗生素的研发又存在周期长、成本高的缺点。因此，寻求一种新型有效的抗菌药物成为了生物医学领域的焦点课题。
近年来，具有优异抗菌性能的抗菌肽引起了人们的广泛关注。抗菌肽多为具有两亲性的阳离子多肽，它能够通过与菌膜磷脂层静电作用结合从而破坏菌膜已达到杀菌的效果。不同于小分子药物的单一靶向作用，抗菌肽独特的抗菌机制，使得细菌难以产生耐药性。抗菌肽水凝胶能够应用在伤口敷料、种植体涂层、抗生物膜和组织工程等生物医学领域。良好的抗菌水凝胶材料应该具备不易膨胀、力学性能优异、生物相容性好、可自愈合、可注射的优点。但大多数新型的抗菌水凝胶在生理条件下易膨胀，从而使力学性能急剧下降。此外，大部分水凝胶合成的方法都使用了与生物不相容的化学实验条件，如自由基引发剂和紫外线辐射。因此，目前新型优异抗菌水凝胶的开发仍然存在着许多挑战。
发明内容
本发明旨在至少解决现有技术中存在的技术问题之一。
本发明提供了一种线性多聚抗菌肽及其制备方法与应用。所述的线性多聚抗菌肽采用含炔基的线性聚合物PRMA-HEMA对游离肽（RW）3-N3进行修饰，具有更高的抗菌性能。基于线性多聚抗菌肽制备的线性多聚肽纳米水凝胶在提高传统水凝胶力学性能的同时具备优异的自愈合、可注射性，在伤口敷料、种植体涂层、抗生物膜和组织工程等生物医学领域具有广阔的应用前景。
为实现上述发明目的，本发明采用以下技术方案：
一种线性多聚抗菌肽的制备方法，具体包括步骤如下：
（1）将炔丙基丙烯酰胺和羟乙基丙烯酰胺溶于二甲基甲酰胺中，加入引发剂和链转移剂；冰水浴中通入惰性气体除氧密封，振荡反应后通氧停止反应，加入去离子水溶解产物后透析，冻干得到线性聚合物聚炔丙基丙烯酰胺-羟乙基丙烯酰胺。
（2）将步骤（1）中得到的聚炔丙基丙烯酰胺-羟乙基丙烯酰胺与游离肽（RW）3-N3共同溶解于二甲亚砜水溶液中，加入五水硫酸铜作为催化剂，通入惰性气体除氧，加入抗坏血酸钠，密封，室温下振荡反应后离心，取上清液过滤，滤液于弱酸性去离子水中透析，冻干得到线性多聚抗菌肽。
本发明，步骤（1）中炔丙基丙烯酰胺、羟乙基丙烯酰胺和二甲基甲酰胺的用量关系为260~280mg：1000~1200m：6~9ml。
步骤（1）中所述的引发剂为偶氮二异丁腈，所述的链转移剂为4-氰基-4（硫代苯甲酸）戊酸。
步骤（1）中所述的炔丙基丙烯酰胺与引发剂和链转移剂的用量比例关系为260~280mg：10~12mg：32~35mg。
步骤（1）中所述的振荡反应的温度为55~60℃，振荡反应的时间为48~60小时。
步骤（2）中所述聚炔丙基丙烯酰胺-羟乙基丙烯酰胺、游离肽（RW）3-N3与二甲亚砜水溶液的用量关系为50~60mg：100~120mg：6~9ml；所述二甲亚砜水溶液中二甲亚砜与水的体积比为1:2。
步骤（2）中所述聚炔丙基丙烯酰胺-羟乙基丙烯酰胺与五水硫酸铜、抗坏血酸钠的用量比为50~60mg：1.0~1.5mg：18~22mg。
步骤（2）中所述弱酸性为pH值在5.5~6.5之间。
本发明还提供了一种基于所述线性多聚抗菌肽制备的线性多聚肽纳米水凝胶，所述线性多聚肽纳米水凝胶的制备方法为：
（1）将硅酸镁锂盐无机纳米片溶于水中磁子搅拌，加入聚丙烯酸钠继续搅拌形成CNS溶液；
（2）将线性多聚抗菌肽溶于水中后缓慢地加入步骤（1）得到的CNS溶液中，停止搅拌溶液数分钟后形成自立的线性多聚肽纳米水凝胶。
进一步地，步骤（1）中硅酸镁锂盐无机纳米片、水、聚丙烯酸钠的用量关系为55~60mg ：2.0~2.5ml ：1.2~1.8mg 。
进一步地，步骤（2）中线性多聚抗菌肽、水与CNS溶液的用量关系为30~45mg：0.5ml：2.0~2.5ml 。
本发明还提供了所制备的线性多聚肽纳米水凝胶在伤口敷料、种植体涂层和/或抗生物膜领域中的应用。
与现有技术相比，本发明的有益效果：
本发明将含炔基的线性聚合物PRMA-HEMA对游离肽（RW）3-N3进行修饰，以炔丙基丙烯酰胺为单体通过可逆断裂链转移聚合得到聚合物，通过点击反应将抗菌肽修饰到聚合物上形成多聚肽，制备出以多价效应为基础的线性多聚抗菌肽聚合物是一种温敏性肿瘤细胞特异性分子印迹聚合物。相比于游离肽（RW）3-N3，在保持良好的生物相容性的基础上大大提高了抗菌性能。本发明通过引入纳米片和多聚抗菌肽在水中强静电作用交联形成线性多聚肽纳米水凝胶，不仅提高传统水凝胶的力学性能，而且具备优异的自愈合、可注射性能。制备过程中不需要自由基引发剂和紫外线辐射，具有良好的安全性，在伤口敷料、种植体涂层、抗生物膜和组织工程等生物医学领域具有广阔的应用前景。
附图说明
图1是制备的线性多聚肽纳米水凝胶实物图及SEM图像；其中，图a、b为实物图，图c为SEM图；
图2为多聚肽和游离肽的琼脂板培养实物图；
图3为多聚肽和游离肽培养下的细菌存活率图；
图4是制备的线性多聚抗菌肽加入大肠杆菌前后的SEM图，图a为完好的大肠杆菌SEM图，图b为加入线性多聚抗菌肽之后的大肠杆菌SEM图；
图5是线性多聚肽纳米水凝胶的自愈合性能及可注射性能图；其中，a是自愈合性能图，b是可注射性能图。
具体实施方式
通过下面的实施例可以对本发明进行进一步的描述，然而，本发明的范围并不限于下述实施例。本发明对试验中所使用到的材料以及试验方法进行一般性和/或具体的描述。本领域技术人员清楚，在下文中，如果未特别说明，本发明所用材料和操作方法是本领域公知的，所使用的实验方法均为常规方法，所用材料、试剂等均可从化学试剂公司购买。本发明中游离肽（RW）3-N3购自上海强耀生物科技有限公司。
实施例1
（1）聚炔丙基丙烯酰胺-羟乙基丙烯酰胺（PRMA-HEMA）的制备
将280mg炔丙基丙烯酰胺（PRMA）和1200mg羟乙基丙烯酰胺（HEMA）溶于6ml二甲基甲酰胺（DMF），加入11mg引发剂偶氮二异丁腈（AIBN）和35mg链转移剂4-氰基-4（硫代苯甲酸）戊酸（CTPA）；冰水浴中通入氮气除氧30分钟，通气结束后密封，在60℃下振荡反应48小时；反应结束后通氧停止反应，加入15ml去离子水溶解，随后透析10天，冻干得到线性聚合物PRMA-HEMA。
（2）线性多聚抗菌肽的合成
取52mg步骤（1）中得到的PRMA-HEMA，与115mg游离肽（RW）3-N3溶解于6ml水和二甲亚砜（DMSO）的混合溶液（体积比为1:2）中，加入1.25mg五水硫酸铜（CuSO4·5H2O）作为催化剂，随后通氮气除氧30分钟，中途加入20mg抗坏血酸钠，通气结束后密封，在室温下振荡反应24小时；反应结束后离心，取上清液过滤，滤液于PH 为5.5的弱酸性去离子水中透析5天，冻干得到线性多聚抗菌肽。
实施例2
将60mg硅酸镁锂盐无机纳米片（CNS）溶于2.5ml水中，用磁子搅拌5分钟，再加入1.8mg聚丙烯酸钠（ASAP）使原本缠结的纳米片分散开来，继续搅拌20分钟形成CNS溶液；取45mg多聚抗菌肽溶于0.5ml的水中，随后缓慢均匀地加入到CNS溶液中，停止搅拌溶液数分钟后形成自立的线性多聚肽纳米水凝胶。
图1是制备的线性多聚肽纳米水凝胶实物图及SEM图像；其中，图a、b为实物图，图c为SEM图；由图1、b可以看出制备的抗菌水凝胶失去了流动性、为可以自立的水凝胶形态，由图c可以看到制备的抗菌水凝胶具有多孔的三维网状的微观结构。
实施例3
本实施例以大肠杆菌（E.coli）为测试菌种，采用琼脂涂布法测定实施例1所制备的线性多聚抗菌肽（多聚肽）的最低抑菌浓度（MIC），探究线性多聚抗菌肽的抗菌性能。
配置浓度为134μM的多聚抗菌肽的PBS溶液，其中多聚肽(RW)3-N3浓度为800μM。取0.4mL多聚抗菌肽稀释到1.6mL的PBS溶液中，然后依次进行倍比稀释为抗菌液，稀释后各个抗菌液所含多聚肽(RW)3-N3浓度依次为160μM、80μM、40μM、20μM、10μM、5μM。取1mL稀释后的抗菌液与1mL的1×106 CFU/ml的E.coli菌液混合，最终保证所含多聚肽（(RW)3-N3浓度依次为80μM、40μM、20μM、10μM、5μM、2.5μM，并以PBS溶液作为空白对照组。将混合溶液放置在摇床中培养12h，温度为37℃，转速为160r/min。取0.1mL培养后的混合菌液稀释到5mLPBS溶液中，混合充分后取0.1mL的混合液均匀涂布到琼脂板上，每个浓度涂布三个琼脂板。最后将涂布好的琼脂板放在37℃的生物培养箱中培养24h，随后进行计数以确定最低抑菌浓度（MIC）。
作为对照，在保证游离肽(RW)3-N3与多聚肽浓度相同的情况下，采用同样的方法测定游离肽(RW)3-N3的最低抑菌浓度（MIC）。
图2为多聚肽和游离肽的琼脂板培养实物图，图3为多聚肽和游离肽培养下的细菌存活率图。综合图2、3分析，多聚肽与游离肽相比具有更为明显优异的抗菌性能，由图3可见，多聚肽 (RW)3-N3 MIC为5μM，而游离肽(RW)3-N3 MIC为40μM，多聚肽浓度为2.5~80μM时均具有明显的抑菌性能，而游离肽的浓度达到20μM时才具有较为明显的抑菌能力，可见多聚肽的抗菌性能相比于普通的游离肽提升了8倍。
图4是制备的线性多聚抗菌肽加入大肠杆菌前后的SEM图，图a为完好的大肠杆菌SEM图，图b为加入线性多聚抗菌肽之后的大肠杆菌SEM图。可以清晰地看出加入线性多聚抗菌肽之后大肠杆菌变得干瘪，周围有内容物流出，证明菌膜已经被破坏达到了杀菌的效果。
实施例4
本实施例探究制备的线性多聚肽纳米水凝胶的自愈合性能及可注射性能。
将制备好的线性多聚肽纳米水凝胶用手术刀一分为二，一半用甲基橙溶液浸泡染色，一半用甲基蓝溶液浸泡染色，分别浸染12h后，将两块经过不同染色的水凝胶沿接口处并拢，静置观察1 h后水凝胶的愈合情况。
另将制备好的线性多聚肽纳米水凝胶放入注射针筒内，针头型号为23G，随后在培养皿中注射，观察水凝胶的可注射性能。是否能连续不断。
图5是线性多聚肽纳米水凝胶的自愈合性能及可注射性能图；其中，a是自愈合性能图，b是可注射性能图；如图5所示，两块经过不同染色的水凝胶沿接口处并拢,1h后断开的水凝胶愈合成一块水凝胶，且无明显的断口，施加一定的外力后，水凝胶也依然保持完整。由此证明，该线性多聚肽纳米水凝胶具备良好的自愈合性能。注射出来的水凝胶能够保持连续不断，且能够随意的注射成型。可见，制备的线性多聚肽纳米水凝胶具备良好的拉伸性能，可自愈合，并具有良好的注射性。
实施例5
（1）聚炔丙基丙烯酰胺-羟乙基丙烯酰胺（PRMA-HEMA）的制备
将280mg炔丙基丙烯酰胺（PRMA）和1200mg羟乙基丙烯酰胺（HEMA）溶于6ml二甲基甲酰胺（DMF），加入12mg引发剂偶氮二异丁腈（AIBN）和35mg链转移剂4-氰基-4（硫代苯甲酸）戊酸（CTPA）；冰水浴中通入氩气除氧30分钟，通气结束后密封，在60℃下振荡反应48小时；反应结束后通氧停止反应，加入15ml去离子水溶解，随后透析10天，冻干得到线性聚合物PRMA-HEMA。
（2）线性多聚肽的合成
取60mg步骤（1）中得到的PRMA-HEMA，与120mg游离肽（RW）3-N3溶解于6ml水和二甲亚砜（DMSO）的混合溶液（体积比为1:2）中，加入1. 5mg五水硫酸铜（CuSO4·5H2O）作为催化剂，随后通氮气除氧30分钟，中途加入22mg抗坏血酸钠，通气结束后密封，在室温下振荡反应24小时；反应结束后离心，取上清液过滤，滤液于PH 为6.5的弱酸性去离子水中透析5天，冻干得到线性多聚肽。
（3）线性多聚肽纳米水凝胶的制备
将55mg硅酸镁锂盐无机纳米片（CNS）溶于2.0ml水中，用磁子搅拌5分钟，再加入1.2mg聚丙烯酸钠（ASAP）使原本缠结的纳米片分散开来，继续搅拌20分钟形成CNS溶液；取40mg步骤（2）得到的线性多聚肽溶于0.5ml的水中，随后缓慢均匀地加入到先前搅拌的CNS溶液中，停止搅拌溶液数分钟后形成自立的线性多聚肽纳米水凝胶。
实施例6
（1）PRMA-HEMA的制备
将260mg PRMA和1000mg HEMA溶于4ml DMF，加入10mg AIBN和32mg链转移剂CTPA；冰水浴中通入氩气除氧20分钟，通气结束后密封，在55℃下振荡反应60小时；反应结束后通氧停止反应，加入15ml去离子水溶解，随后透析10天，冻干得到线性聚合物PRMA-HEMA；
（2）线性多聚肽的合成
取50mg步骤（1）中得到的PRMA-HEMA，与100mg游离肽（RW）3-N3溶解于9ml水和DMSO的混合溶液（体积比为1:2）中，加入1mg CuSO4·5H2O作为催化剂，随后通氮气除氧20分钟，中途加入18mg抗坏血酸钠，通气结束后密封，在室温下振荡反应20小时；反应结束后离心，取上清液过滤，滤液于PH 为6.5的弱酸性去离子水中透析7天，冻干得到线性多聚肽。
（3）线性多聚肽纳米水凝胶的制备
将58mg CNS溶于2.5ml水中，用磁子搅拌8分钟，再加入1.6mg ASAP使原本缠结的纳米片分散开来，继续搅拌16分钟形成CNS溶液；取30mg步骤（2）得到的线性多聚肽溶于0.5ml的水中，随后缓慢均匀地加入到CNS溶液中，停止搅拌溶液数分钟后形成自立的线性多聚肽纳米水凝胶。
以上显示和描述了本发明的基本原理、主要特征以及本发明的优点。本行业的技术人员应该了解，本发明不受上述实施例的限制，上述实施例和说明书中描述的只是说明本发明的原理，在不脱离本发明精神和范围的前提下，本发明还会有各种变化和改进，这些变化和改进都落入要求保护的本发明范围内。本发明要求保护范围由所附的权利要求书及其等效物界定。

Claims (10)

1.一种线性多聚抗菌肽的制备方法，其特征在于，具体包括步骤如下：
（1）将炔丙基丙烯酰胺和羟乙基丙烯酰胺溶于二甲基甲酰胺中，加入引发剂和链转移剂；冰水浴中通入惰性气体除氧密封，振荡反应后通氧停止反应，加入去离子水溶解产物后透析，冻干得到线性聚合物聚炔丙基丙烯酰胺-羟乙基丙烯酰胺；
（2）将步骤（1）中得到的聚炔丙基丙烯酰胺-羟乙基丙烯酰胺与游离肽（RW）3-N3共同溶解于二甲亚砜水溶液中，加入五水硫酸铜作为催化剂，通入惰性气体除氧，加入抗坏血酸钠，密封，室温下振荡反应后离心，取上清液过滤，滤液于弱酸性去离子水中透析，冻干得到线性多聚抗菌肽。
2.根据权利要求1所述的制备方法，其特征在于，步骤（1）中炔丙基丙烯酰胺、羟乙基丙烯酰胺和二甲基甲酰胺的用量关系为260~280mg：1000~1200m：6~9ml。
3.根据权利要求1所述的制备方法，其特征在于，步骤（1）中所述的引发剂为偶氮二异丁腈，所述的链转移剂为4-氰基-4（硫代苯甲酸）戊酸；所述的炔丙基丙烯酰胺与引发剂和链转移剂的用量比例关系为260~280mg：10~12mg：32~35mg。
4.根据权利要求1所述的制备方法，其特征在于，步骤（1）中所述的振荡反应的温度为55~60℃，振荡反应的时间为48~60小时。
5.根据权利要求1所述的制备方法，其特征在于，步骤（2）中所述聚炔丙基丙烯酰胺-羟乙基丙烯酰胺、游离肽（RW）3-N3与二甲亚砜水溶液的用量关系为50~60mg：100~120mg：6~9ml；所述二甲亚砜水溶液中二甲亚砜与水的体积比为1:2。
6.根据权利要求1所述的制备方法，其特征在于，步骤（2）中所述聚炔丙基丙烯酰胺-羟乙基丙烯酰胺与五水硫酸铜、抗坏血酸钠的用量比为50~60mg：1.0~1.5mg：18~22mg。
7.根据权利要求1所述的制备方法，其特征在于，步骤（2）中所述弱酸性为pH值在5.5~6.5之间。
8.一种线性多聚肽纳米水凝胶的制备方法，其特征在于，包含以下步骤：
（1）将硅酸镁锂盐无机纳米片溶于水中磁子搅拌，加入聚丙烯酸钠继续搅拌形成CNS溶液；
（2）将根据权利要求1~7任一项所述的制备方法制备的线性多聚抗菌肽溶于水中后缓慢地加入步骤（1）得到的CNS溶液中，停止搅拌溶液数分钟后形成自立的线性多聚肽纳米水凝胶。
9.根据权利要求8所述的制备方法，其特征在于，进一步地，步骤（1）中硅酸镁锂盐无机纳米片、水、聚丙烯酸钠的用量关系为55~60mg ：2.0~2.5ml ：1.2~1.8mg 。
10.根据权利要求8所述的制备方法，其特征在于，进一步地，步骤（2）中线性多聚抗菌肽、水与CNS溶液的用量关系为30~45mg：0.5ml ：2.0~2.5ml。
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