CN116515164B - 一种贻贝蛋白抗菌水凝胶及其制备方法与应用 - Google Patents
一种贻贝蛋白抗菌水凝胶及其制备方法与应用 Download PDFInfo
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- mussel protein
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Abstract
本发明公开了一种贻贝蛋白抗菌水凝胶及其制备方法与应用，将聚乙烯醇水溶液和海藻酸钠水溶液按照不同比例混合均匀，经脱气处理，冷冻‑解冻两次转变为复合水凝胶，随后浸泡在贻贝蛋白乙酸溶液中，最后取出放在室温里吹干得到均匀的抗菌水凝胶。本发明制备工艺简单，易操作，制得的贻贝蛋白‑聚乙烯醇‑海藻酸钠抗菌水凝胶不仅能提高水凝胶的细胞黏附和增殖性能，还能增加一定的抗菌性能和抗氧化性能。该水凝胶呈现多孔结构，能够模拟人体皮肤细胞外基质(ECM)的环境，具有优异的生物相容性，对细胞黏附和生长的有促进作用，对人体无害，有作为理想的敷料的潜能。
Description
技术领域
本发明属于医用材料技术领域，涉及一种贻贝蛋白抗菌水凝胶及其制备方法与应用。
背景技术
在日常生活中，人们会不可避免的受到一些皮肤上的创伤，创伤修复成为了皮肤临床医学上难以解决的问题，特别是大面积不规则的伤口，往往愈合速度较慢，机体不能自主愈合，需要借助外部辅助达到修复的目的。伤口敷料是一种可以起到暂时保护伤口、促进愈合的医用材料。水凝胶具有疏松多孔的结构，具备一定抗压强度，能为伤口提供湿润的环境，具有优异的生物相容性，能够促进伤口修复，可以作为伤口敷料，有着极好的应用前景。
聚乙烯醇(PVA)作为一种人工合成高分子材料，合成方便、价格便宜、对人体安全，具有良好的生物相容性。海藻酸钠(SA)是一种从褐海藻中分离得到的天然多糖，可被广泛应用于食品、制药、组织工程等领域，具有极高的安全性。通过反复冻融制得的聚乙烯醇-海藻酸钠水凝胶，不用添加有一定毒害的交联剂，生物相容性较好，但是聚乙烯醇-海藻酸钠水凝胶有较强的亲水性，不利于细胞粘附，使用的广泛性受到限制。
发明内容
发明目的：本发明所要解决的技术问题是针对现有技术的不足，提供一种强粘附性、抗氧化、抗菌的贻贝蛋白-聚乙烯醇-海藻酸钠水凝胶，以改善传统聚乙烯醇水凝胶的组织粘附性和抗菌性。
为了实现上述目的，本发明采取的技术方案如下：
一种贻贝蛋白抗菌水凝胶的制备方法，包括如下步骤：
(1)将聚乙烯醇溶解于去离子水中，获得聚乙烯醇水溶液；将海藻酸钠溶解于去离子水中，获得海藻酸钠水溶液；
(2)将步骤(1)中聚乙烯醇水溶液和海藻酸钠水溶液混合均匀，得到复合水溶液；
(3)将步骤(2)得到的复合水溶液进行脱气处理，冷冻-解冻两次转变为复合水凝胶；
(4)将贻贝蛋白溶解于乙酸溶液中，获得贻贝蛋白乙酸溶液；
(5)将步骤(3)得到的复合水凝胶充分浸泡在步骤(4)贻贝蛋白乙酸溶液中，取出后在室温下吹干即得。
优选地，步骤(1)中，所述的聚乙烯醇为高黏度的可溶性聚乙烯醇，醇解度为99.0％～99.4％，数均分子量为1～2万；在80～100℃下，将聚乙烯醇溶解于去离子水中，得到质量浓度为5～20％的聚乙烯醇水溶液。
优选地，步骤(1)中，所述的海藻酸钠为可溶性海藻酸钠，聚合度80～750；在45～60℃下，将海藻酸钠溶解于去离子水中，获得质量浓度为0.1～2％的海藻酸钠水溶液。
优选地，步骤(2)中，所述聚乙烯醇水溶液和海藻酸钠水溶液混合体积比1～5:1。
优选地，步骤(3)中，所述的脱气处理的方式为采用超声处理30～60min，静置15～30min；所述的冷冻温度为-80～-20℃，所述的解冻温度为20～25℃。
优选地，步骤(4)中，所述的贻贝蛋白为贻贝足丝蛋白，贻贝足丝蛋白为基因工程菌发酵获得的人工合成贻贝粘附蛋白；所述乙酸溶液的体积浓度为2～5％，2～4℃下将贻贝蛋白溶解于乙酸溶液中，得到浓度为2～10mg/mL的贻贝蛋白乙酸溶液，即贻贝蛋白乙酸溶液中贻贝蛋白的浓度为2～10mg/mL。
本发明中，贻贝蛋白(MFP)是一种生物相容性好，粘附性特别强大的原料。贻贝蛋白带正电荷，能与带负电荷的多糖海藻酸钠静电组装，且贻贝蛋白本身也具备良好的抗菌和抗氧化性能，可以广泛应用于组织粘合剂等粘附性材料的研发中。
优选地，步骤(5)中，所述的复合水凝胶在贻贝蛋白乙酸溶液中的浸泡时间至少为6h，取出后在室温下至少吹干24h。
进一步地，上述制备方法制备得到的贻贝蛋白抗菌水凝胶也在本发明的保护范围之中。
更进一步地，本发明还要求保护上述贻贝蛋白抗菌水凝胶在用于制备伤口敷料中的应用。
更进一步地，本发明还要求保护上述贻贝蛋白抗菌水凝胶在用于制备伤口敷料防止大肠杆菌和金黄色葡萄球菌感染的应用。
有益效果：
本发明以微生物发酵制得的贻贝蛋白、聚乙烯醇海藻酸钠为原料，均为生物相容性好的材料，本发明制备过程无需添加毒副作用的试剂，绿色环保，成本低廉，工艺简单，易于操作，在组织工程、医学等领域具有广阔的应用前景。本发明的方法制得的水凝胶含水量大，机械性能好，生物相容性优异，本发明制得的抗菌水凝胶敷料对大肠杆菌和金黄色葡萄球菌均具有优良的抑制作用，能有效防止伤口感染，扩大了其使用范围，能够作为理想的伤口敷料。
附图说明
下面结合附图和具体实施方式对本发明做更进一步的具体说明，本发明的上述和/或其他方面的优点将会变得更加清楚。
图1本发明实施实例1制备的贻贝蛋白-聚乙烯醇-海藻酸钠水凝胶的扫描电镜图片。
图2本发明实施实例1制备的贻贝蛋白-聚乙烯醇-海藻酸钠水凝胶的抗氧化性能。
图3本发明实施实例1制备的贻贝蛋白-聚乙烯醇-海藻酸钠水凝胶的对大肠杆菌(E.coli)和金黄色葡萄球菌(S.aureus)的抗菌效果。
图4本发明实施实例1制备的贻贝蛋白-聚乙烯醇-海藻酸钠水凝胶的对大肠杆菌(E.coli)和金黄色葡萄球菌(S.aureus)的抗菌率。
图5本发明实施实例1制备的贻贝蛋白-聚乙烯醇-海藻酸钠水凝胶的细胞生长情况。
图6本发明实施实例1制备的贻贝蛋白-聚乙烯醇-海藻酸钠水凝胶的细胞存活率。
具体实施方式
根据下述实施例，可以更好地理解本发明。
实施例1贻贝蛋白-聚乙烯醇-海藻酸钠水凝胶的制备。
100℃下，将100g聚乙烯醇溶解于1L去离子水中，获得10％的聚乙烯醇水溶液；60℃下，将15g海藻酸钠溶解于1L去离子水中，获得1.5％的海藻酸钠水溶液；将聚乙烯醇水溶液：海藻酸钠水溶液＝5:1、3:1和1:1的比例混合均匀，获得复合水溶液；将复合水溶液进行超声脱气处理，-80℃冷冻1h后25℃解冻1h，两次循环后转变为聚乙烯醇-海藻酸钠(PVA-SA)水凝胶；4℃下，将40mg贻贝蛋白溶解于10ml的5％乙酸溶液中，获得4mg/mL的贻贝蛋白乙酸溶液；将PVA-SA水凝胶浸泡在贻贝蛋白乙酸溶液12h，随后取出放在室温里吹干36h得到贻贝蛋白-聚乙烯醇-海藻酸钠(PVA-SA-MFP)抗菌水凝胶。图1是实施例1制备的贻贝蛋白-聚乙烯醇-海藻酸钠水凝胶的扫描电镜图片，可以看出：贻贝蛋白-聚乙烯醇-海藻酸钠水凝胶的内部结构是紧密多孔的，可储存营养物与水分，以及为细胞提供了生长和繁殖的空间，是一个良好的皮肤组织工程领域的生物支架。
实施例2贻贝蛋白-聚乙烯醇-海藻酸钠水凝胶的制备。
85℃下，将75g聚乙烯醇溶解于1L去离子水中，获得7.5％的聚乙烯醇水溶液；45℃下，将10g海藻酸钠溶解于1L去离子水中，获得1％的海藻酸钠水溶液；将聚乙烯醇水溶液：海藻酸钠水溶液＝5:1、3:1和1:1的比例混合均匀，获得复合水溶液；将复合水溶液进行超声脱气处理，-40℃冷冻2h后20℃解冻1.5h，两次循环后转变为聚乙烯醇-海藻酸钠水凝胶；4℃下，将20mg贻贝蛋白溶解于10ml的2％乙酸溶液中，获得2mg/mL的贻贝蛋白乙酸溶液；将PVA-SA水凝胶浸泡在贻贝蛋白乙酸溶液10h，随后取出放在室温里吹干40h得到贻贝蛋白-聚乙烯醇-海藻酸钠抗菌水凝胶敷料。
实施例3贻贝蛋白-聚乙烯醇-海藻酸钠(PVA-SA-MFP)水凝胶的抗氧化性能测试。
称取0.40g DPPH溶解于100ml去离子水中，将实施例1制备的不同比例的PVA-SA-MFP水凝胶用均质器打碎，过滤得到澄清液体。将样品溶液(2mL)放入试管中，加入2mL DPPH(0.1mmol/L)无水乙醇溶液。摇匀后，25℃避光反应30分钟，517nm紫外波长扫描。本实施例对制备的水凝胶的抗氧化性能进行考察，其结果如图2所示。可以观察到经过贻贝蛋白溶液浸泡过的水凝胶的抗氧化性能更好，且在聚乙烯醇水溶液：海藻酸钠水溶液＝1:1的比例时，贻贝蛋白溶液浸泡过的水凝胶抗氧化率最高达到79.6％，这些结果充分说明，本发明的水凝胶在抗氧化上表现优异。
实施例4贻贝蛋白-聚乙烯醇-海藻酸钠水凝胶的抗菌性能测试。
称取15克琼脂粉溶解于100毫升LB中，随后放在立式高压蒸汽灭菌锅，参数设置为：121℃灭菌30分钟。随即在超净台中将其趁热倒入细胞培养皿，风干备用。将活化后的大肠杆菌(ATCC 25933)和金黄色葡萄球菌(ATCC 6538)培养液用PBS缓冲液稀释至106CFU/mL。将实施例3中最佳抗氧化性能比例的的贻贝蛋白-聚乙烯醇-海藻酸钠(PVA-SA-MFP)水凝胶与菌液共同培养，放置于37℃恒温培养箱中孵育12小时，吸取100微升稀释的菌液均匀滴在LB培养板中，用涂布棒均匀涂布至菌液完全吸收。观察菌落生长情况，并用相机拍照记录。本实施例对制备的水凝胶的抗菌性能进行考察，其结果如图3所示。可以观察到，经过贻贝蛋白溶液浸泡过的水凝胶，接触部位更加透明，细菌生长较少，抗菌效果更好。结合图4中水凝胶对大肠杆菌和金黄葡萄球菌的抗菌率更充分说明了本发明的水凝胶在抗菌上表现优异。
实施例5贻贝蛋白-聚乙烯醇-海藻酸钠水凝胶的生物相容性测试。
利用噻唑蓝(MTT)法检测实施例3中最佳抗氧化性能比例的的贻贝蛋白-聚乙烯醇-海藻酸钠(PVA-SA-MFP)水凝胶对L929小鼠成纤维细胞的细胞毒性。将水凝胶浸泡在基础细胞培养基中，在4℃冰箱中保存1、2和3天。在96孔板中每孔接种细胞，并置于37℃恒温培养箱中孵育36小时，吸出孔中的培养基，将其替换水凝胶预处理的培养基，以未经任何处理的DMEM培养基作为对照组。在37℃培养箱中分别孵育24和48小时后，吸出培养基，用PBS缓冲液轻轻冲洗每个孔，该步骤重复三次。向孔中加入0.2毫升MTT的DMEM溶液(0.5毫克/毫升)，37℃恒温培养箱中孵育4小时。轻轻吸出培养基，向孔中加入0.2毫升二甲基亚砜以溶解蓝紫色结晶甲臜。15分钟后用荧光倒置显微镜拍照记录，其结果如图5所示。该结果表明了复合水凝胶处理下的小鼠细胞72h的生长状况，复合水凝胶对小鼠成纤维细胞几乎没有任何毒性作用，并且结合图6的水凝胶的细胞存活率进一步说明对细胞生长有一定促进作用。
本发明提供了一种贻贝蛋白抗菌水凝胶及其制备方法与应用的思路及方法，具体实现该技术方案的方法和途径很多，以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。本实施例中未明确的各组成部分均可用现有技术加以实现。
Claims (7)
1.一种贻贝蛋白抗菌水凝胶的制备方法，其特征在于，包括如下步骤：
（1）将聚乙烯醇溶解于去离子水中，获得聚乙烯醇水溶液；将海藻酸钠溶解于去离子水中，获得海藻酸钠水溶液；
（2）将步骤（1）中聚乙烯醇水溶液和海藻酸钠水溶液混合均匀，得到复合水溶液；
（3）将步骤（2）得到的复合水溶液进行脱气处理，冷冻-解冻两次转变为复合水凝胶；
（4）将贻贝蛋白溶解于乙酸溶液中，获得贻贝蛋白乙酸溶液；
（5）将步骤（3）得到的复合水凝胶充分浸泡在步骤（4）贻贝蛋白乙酸溶液中，取出后在室温下吹干即得；
所述的聚乙烯醇为高黏度的可溶性聚乙烯醇，醇解度为99.0%～99.4 %，数均分子量为1~2万；在80~100℃下，将聚乙烯醇溶解于去离子水中，得到质量浓度为5~20%的聚乙烯醇水溶液；
所述的海藻酸钠为可溶性海藻酸钠，聚合度80~750；在45~60℃下，将海藻酸钠溶解于去离子水中，获得质量浓度为0.1~2%的海藻酸钠水溶液；
所述的贻贝蛋白为贻贝足丝蛋白，贻贝足丝蛋白为基因工程菌发酵获得的人工合成贻贝粘附蛋白；所述乙酸溶液的体积浓度为2~5%，2~4℃下将贻贝蛋白溶解于乙酸溶液中，得到浓度为2~10mg/mL的贻贝蛋白乙酸溶液。
2.根据权利要求1所述的贻贝蛋白抗菌水凝胶的制备方法，其特征在于，步骤（2）中，所述聚乙烯醇水溶液和海藻酸钠水溶液混合体积比1~5:1。
3.根据权利要求1所述的贻贝蛋白抗菌水凝胶的制备方法，其特征在于，步骤（3）中，所述的脱气处理的方式为采用超声处理30~60min，静置15~30min；所述的冷冻温度为-80~-20℃，所述的解冻温度为20~25℃。
4.根据权利要求1所述的贻贝蛋白抗菌水凝胶的制备方法，其特征在于，步骤（5）中，所述的复合水凝胶在贻贝蛋白乙酸溶液中的浸泡时间至少为6h，取出后在室温下至少吹干24h。
5.权利要求1~4中任意一项所述制备方法制备得到的贻贝蛋白抗菌水凝胶。
6.权利要求5所述的贻贝蛋白抗菌水凝胶在用于制备伤口敷料中的应用。
7.根据权利要求5所述的贻贝蛋白抗菌水凝胶在用于制备伤口敷料防止大肠杆菌和金黄色葡萄球菌感染的应用。
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| CN109354886A (zh) * | 2018-11-07 | 2019-02-19 | 中国科学院烟台海岸带研究所 | 复合水凝胶及其制备方法 |
| CN109942905A (zh) * | 2019-03-01 | 2019-06-28 | 昆明理工大学 | 一种复合水凝胶材料及其制备方法 |
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| KR20100118860A (ko) * | 2009-04-29 | 2010-11-08 | 한남대학교 산학협력단 | 생체적합성 폴리비닐알콜계 하이드로겔의 제조방법 |
| CN105477674A (zh) * | 2014-10-10 | 2016-04-13 | 北京铂铱电气科技有限公司 | 一种基于壳聚糖与海洋贻贝粘蛋白的止血复合材料及其制备方法 |
| WO2018093161A1 (ko) * | 2016-11-16 | 2018-05-24 | 포항공과대학교 산학협력단 | 홍합 접착 단백질을 포함하는 신규한 지혈제 및 이의 제조 방법 |
| CN109354886A (zh) * | 2018-11-07 | 2019-02-19 | 中国科学院烟台海岸带研究所 | 复合水凝胶及其制备方法 |
| CN109942905A (zh) * | 2019-03-01 | 2019-06-28 | 昆明理工大学 | 一种复合水凝胶材料及其制备方法 |
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