CN115227864A - 一种多功能纳米酶原位水凝胶的制备方法及应用 - Google Patents
一种多功能纳米酶原位水凝胶的制备方法及应用 Download PDFInfo
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Abstract
本发明公开了一种多功能纳米酶原位水凝胶的制备方法及应用，本发明制备了锌掺杂碳点与抗坏血酸作为还原剂及稳定剂的具有拟氧化物酶活性的银纳米（AgNPs），通过化学硼酯键和氢键交联制备了AgNPs与微晶纤维素/单宁酸/聚乙烯醇（CNCs/TA/PVA）水凝胶，本发明制得的纳米酶抗菌水凝胶具有好的机械性能、粘附性能，好的生物相容性、抗菌性能及伤口愈合性能，这主要取决于水凝胶中化学硼酯键和氢键交联网络结构，同时保留了丰富的酚羟基，维持了酚醌的氧化还原动态平衡，为水凝胶提供了长期性和可重复性的黏附性，同时纳米酶的抗菌性能及清除自由基的性能，为伤口愈合提供了保障，该纳米酶抗菌水凝胶用于伤口愈合是安全、有效的。
Description
技术领域
本发明涉及纳米材料抗菌技术领域，具体为一种多功能纳米酶原位水凝胶的制备方法及应用。
背景技术
由细菌引起的传染病一直是日益严重的健康问题之一，每年导致数以百万计的疾病和死亡。细菌感染导致的慢性皮肤缺损严重影响生活质量，甚至威胁人们的生活，常规抗生素治疗由于抗生素误用而出现抗菌耐药性，疗效越来越差。伤口愈合是一个复杂的过程，需要具有多功能特性的伤口敷料，包括组织粘附以保持伤口湿润，快速自愈以保持原形，超塑性以快速适应多种伤口形状。具有网络结构的水凝胶可以保留水分，保持伤口湿润，阻止细菌入侵。特别是形状适应型水凝胶可以填充整个不规则创面的空洞，保证其以最大的效率实现其功能。为此，具有抗菌和抗氧化性能的水凝胶伤口敷料是非常理想的。
然而，目前的伤口敷料材料在研究和临床应用过程中已经显现出其缺点，如抗菌能力差、缺乏抗炎性能、机械强度不理想、对体液染色表面粘附能力弱，同时，生物利用度较差限制了其实际应用。近年来，活性氧（reactive oxygen species，ROS）是包括H2O2、超氧自由基（O2 •-）和羟基自由基（·OH）在内的小分子反应的统称，被证明是抗耐药菌的有效替代品。通常，具有拟过氧化物酶（POD）或氧化酶（OXD）活性的纳米酶能特异性地催化H2O2或O2转化为高毒性的ROS。
聚乙烯醇（PVA）水凝胶由于其优良的组织粘弹性和生物相容性，被广泛应用于组织工程等生物医学领域。然而，它们较差的力学性能极大地限制了它们的应用。微晶纤维素（CNCs）来源于天然材料，由于其较高的纤维素含量和较高的结晶度，已被报道作为交联剂来改善水凝胶的力学性能，但对提高水凝胶强度贡献不大，可能归因于其物理相互作用较弱，如CNCs与聚合物之间的氢键作用。为了建立多羟基聚合物之间的强氢键作用，利用天然多酚类化合物单宁酸（TA）与邻苯二酚、邻苯三酚等多官能团形成强氢键，增强PVA水凝胶的力学性能。
发明内容
本发明提供了一种多功能纳米酶原位水凝胶的制备方法及应用，该方法利用了纳米酶的催化性、抗菌性，通过化学硼酯键和氢键交联制备了多功能纳米酶原位水凝胶，用于伤口创面愈合。
本发明制备了锌掺杂碳点与抗坏血酸作为还原剂及稳定剂的具有拟氧化物酶（OXD）活性的银纳米（AgNPs），通过化学硼酯键和氢键交联制备了AgNPs与微晶纤维素/单宁酸/聚乙烯醇（CNCs/TA/PVA）水凝胶，该水凝胶通过CNCs、TA和PVA分子骨架中的羟基(-OH)与硼砂水解的B (OH)4 -反应，形成硼酸酯键和氢键，能够发生动态的缔合和解缔，从而赋予水凝胶优异的自修复能力。同时利用AgNPs具有的内在OXD纳米酶活性，维持TA酚醌的动态氧化还原平衡，为水凝胶提供了长期性和可重复性的黏附性，类似于贻贝的黏附性。酚羟基还提供了纳米酶在水凝胶网络中均匀分布，从而提高了其力学性能，此外，纳米酶通过OXD类催化反应产生的活性氧与AgNPs的固有杀菌活性协同作用赋予水凝胶抗菌活性。该多功能水凝胶形成网络结构具有黏附性、自修复性和形状适应性，作为伤口敷料，在防止感染的同时加速组织再生，可填充不规则创面的空洞，促进纳米酶发挥最大效能。
本发明多功能纳米酶原位水凝胶的制备如下：
（1）锌掺杂碳点（Zn,CDs）的合成：称取0.1-0.3g ZnCl2、0.5-1.0g多巴胺、1.0-2.0g柠檬酸及0.5-1.0g乙二胺溶于30-40mL超纯水中，超声混合均匀，将溶液转移至聚四氟乙烯内衬反应器中，置于微波消解炉中，设置温度1分钟升至150℃，在150℃、850W下水解40-60分钟，反应完成后自然冷却至室温，得棕色溶液；将棕色溶液用0.22μm滤膜除去大颗粒杂质，再经高速离心，上清液真空干燥，得到锌掺杂碳点；
所述高速离心是在10000r/min下处理15-20分钟；
（2）银纳米（AgNPs）合成：10-15mg的Zn,CDs与20-25mg抗坏血酸溶于30mL超纯水中，油浴加热至90-100℃，加入AgNO3，AgNO3在混合液中的浓度为20-25mg/mL，避光搅拌60分钟，冷却至室温，高速离心，上清液冷冻干燥，得到银纳米；
所述高速离心是在10000r/min下处理15-20分钟；
（3）水凝胶的合成：将0.4-0.6 g微晶纤维素（CNCs）分散于40mL pH=8.5的磷酸盐缓冲溶液中，搅拌均匀后，加入0.4-0.5g单宁酸（TA），搅拌均匀后，按0.02-0.04mg/mL的添加比例加入银纳米，搅拌30-40分钟后，离心分离，用纯水洗涤固体后2-3次，重新分散于纯水中，配制成5-7%的Zn-Ag/TA@CNCs分散液；将10-11g聚乙烯醇（PVA）、15-18mL Zn-Ag/TA@CNCs分散液加入62-65mL纯水中，于90-95℃加热搅拌溶解后加入质量浓度5-10%的硼砂溶液5-10mL，剧烈搅拌混匀后，将其倒入模具，冷却后即得纳米酶原位水凝胶；
所述离心分离是在4000-6000r/min下处理10-15分钟。
本发明另一目的是将上述方法制得的纳米酶原位水凝胶应用在制备用于伤口愈合的试剂中。
本发明的优点在于：
1、本发明利用碳点银纳米具有拟氧化物酶（OXD）特性，与含多酚单宁酸形成酚醌的动态氧化还原平衡，为制备的水凝胶提供了长期性和可重复性的黏附性，类似于贻贝的黏附性；
2、通过化学硼酯键和氢键交联制备了AgNPs与微晶纤维素/单宁酸/聚乙烯醇（CNCs/TA/PVA）水凝胶，该水凝胶能够发生动态的缔合和解缔，从而赋予水凝胶优异的自修复能力，酚羟基还提供了纳米酶在水凝胶网络中均匀分布，从而提高了其力学性能；
3、纳米酶水凝胶通过OXD类催化反应产生的活性氧与AgNPs的固有杀菌活性协同作用赋予水凝胶抗菌活性；
4、该水凝胶用于伤口创面愈合，具有防止感染的同时加速组织再生，可填充不规则创面的空洞。
附图说明
图1为不同水凝胶氧化TMB的紫外可见吸收光谱图，图中（1）为单独TMB，（2）TMB和凝胶基底，（3）为Zn,CDs水凝胶，（4）为实施例1制得的水凝胶；
图2为不同水凝胶过氧化氢酶活结果示意图；
图3为不同水凝胶的黏附强度结果示意图；
图4为不同水凝胶的含水量结果；
图5为不同水凝胶的溶胀率结果；
图6为本发明水凝胶细胞毒性实验结果；
图7为不同水凝胶抗菌实验结果；
图8为不同纳米酶凝胶片对小鼠伤口愈合照片；
图9为不同纳米酶凝胶片对小鼠伤口愈合率统计结果。
具体实施方式
下面将结合具体的实施例对本发明的技术方案作进一步详细地描述说明，但本发明的保护范围并不仅限于此。
实施例1：多功能纳米酶原位水凝胶的制备及性能
（1）锌掺杂碳点（Zn,CDs）的合成：称取0.2g ZnCl2、0.5g多巴胺、1.0g柠檬酸及0.5g乙二胺溶于30mL超纯水中，超声混合均匀，将溶液转移至聚四氟乙烯内衬反应器中，置于微波消解炉中，设置温度1分钟升至150℃，在150℃、850W下水解40分钟，反应完成后自然冷却至室温，得棕色溶液；将棕色溶液用0.22μm滤膜除去大颗粒杂质，再10000 r/min下处理15分钟，上清液真空干燥，得到锌掺杂碳点；
（2）银纳米（AgNPs）合成：10mg的Zn,CDs与20mg抗坏血酸溶于30mL超纯水中，油浴加热至100℃，加入AgNO3，AgNO3在混合液中的浓度为20mg/mL，避光搅拌60分钟，冷却至室温，10000 r/min下处理15分钟，上清液冷冻干燥，得到银纳米；
（3）水凝胶（Zn-Ag/TA@CNCs/PVA）的合成：将0.4g微晶纤维素（CNCs）分散于40mLpH=8.5的磷酸盐缓冲溶液中，搅拌均匀后，加入0.4-g单宁酸（TA），搅拌均匀后，按0.02mg/mL的添加比例加入银纳米，搅拌30分钟后，4000r/min下处理15分钟，用纯水洗涤固体后3次，重新分散于纯水中，配制成质量浓度6%的Zn-Ag/TA@CNCs分散液；将10g聚乙烯醇（PVA）和15mL Zn-Ag/TA@CNCs分散液加入62mL纯水中，于90℃加热搅拌溶解后加入5%的硼砂溶液6mL，剧烈搅拌混匀后，将其倒入模具，冷却后即得纳米酶原位水凝胶；
用Zn,CDs替代银纳米在完全相同条件下制备Zn,CDs水凝胶，以及不添加银纳米制得的凝胶基底，作为对照；
（4）采用TMB显色反应测定水凝胶的拟氧化酶（OXD）活性
将100µg/mL 水凝胶100μL、100mmol/L的TMB 50µL，加入到pH7.4磷酸盐缓冲溶液2mL中，充分混匀，室温孵育10分钟后，离心分离，取上层清液用紫外-可见分光光度计在655nm处测量吸光度，每个样品测量3次，取平均值，结果如图1；从图1中可以看出，在中性条件下，本实施例制得的水凝胶表现出相当高的拟氧化酶活性；
（5）采用 TiCl4显色反应测定水凝胶的拟过氧化氢酶（CAT）活性
将100µg/mL水凝胶100μL与1mL、50mM的 H2O2反应10分钟，加入200μL10% TiCl4，充分混匀，室温孵育10分钟后，离心分离，取上层清液用紫外-可见分光光度计在415nm处测量吸光度，每个样品测量3次，取平均值；用Zn,CDs替代银纳米在完全相同条件下制备Zn,CDs水凝胶，以及不添加银纳米制得的凝胶基底，作为对照；
结果如图2；从图2中可以看出，本实施例制得的水凝胶表现出好的拟过氧化氢酶活性。
（6）水凝胶的性能测试
参照文献（Polym. Chem., 2015, 6, 2204–2213方法，崔春燕等“一种高强度速黏纳米杂化水凝胶“创可贴”，高分子学报，2019，50（6）：613-622）测试纳米酶凝胶片对玻璃的粘附性能，图3显示了Zn,CDs水凝胶与Zn-Ag/TA@CNCs水凝胶的粘附强度，Zn,CDs水凝胶的粘附强度仅为0.75kPa，说明凝胶与载玻片之间的相互作用较弱，对于与Zn-Ag/TA@CNCs/PVA水凝胶，其粘附强度为3.9kPa。结果表明，Zn-Ag/TA@CNCs/PVA水凝胶中的酚基，显著提高了水凝胶的粘附强度5倍，其中的酚醌动态平衡改善黏附性能。
参照文献（Shengbo Li等，Calcium ion cross-linked sodium alginatehydrogels containingdeferoxamine and copper nanoparticles for diabetic woundhealing.International Journal of Biological Macromolecules 202 (2022) 657–670）进行含水量及溶胀率测定，水凝胶的湿重（W湿）和在真空环境中冻干12小时后的干重（W干），根据以下公式计算水凝胶的含水量：含水量（%）= （W湿− W干）/ W湿× 100%；
将冻干水凝胶称重后浸入pH7.4、0.01M、37℃磷酸盐缓冲液中，浸泡后的水凝胶定期称重，直至重量保持恒定（3次），记为W溶胀，根据以下公式计算水凝胶的溶胀率：
溶胀率（%）= （W溶胀− W干）/ W溶胀× 100%；
结果如图4和5所示，水凝胶之间的含水量和溶胀比没有显著差异，所有水凝胶均含有90~95 %的水，溶胀率约为3100 %。
以下菌种分别从北纳创联生物科技有限公司、云南大学微生物研究所及昆明理工大学生命科学与技术学院获得；
实验方法：以金黄色葡萄球菌(S.aureus，ATCC25923)和和铜绿假单胞菌(P.aeruginosa，ATCC27853)为代表革兰氏阴性菌株。采用平板计数法，通过计数CFU数来判定水凝胶的抗菌性能。首先，将上述菌种在固体Luria-Bertani（LB）培养基和固体营养肉汤培养基孵育24小时，用接种环挑取少量形成的菌落，接种到对应液体培养基（5mL）中，然后，在37℃、180rpm恒温摇床下震荡孵育12小时后即可获得细菌悬浮液（1×108 CFU/mL），用无菌磷酸盐缓冲液（PBS）稀释到1×105 CFU/mL。材料分为四组：空白对照组，水凝胶基质空白组、Zn,CDs水凝胶组和Zn-Ag/TA@CNCs/PVA水凝胶组。将培养的细菌加入磷酸盐缓冲液中作为空白对照组，其他组与不同水凝胶混合，水凝胶浓度为100μg/mL，然后在37℃下将孵育60min，菌悬液经过稀释后（100μL）均匀涂布在LB固体培养基和营养肉汤固体培养基上，在37℃下培养24h，计算菌落数，判断抗菌性能。
结果如图7所示，空白对照组几乎没有抗菌性能，凝胶基底对铜绿假单胞菌和金黄色葡萄球菌的捕获效率分别约为27.3%和37.1%，这一结果证明水凝胶能有效捕获细菌，有利于抗菌，而Zn,CDs水凝胶对铜绿假单胞菌和金黄色葡萄球菌有57.4%和60.1 %的抗菌率，Zn-Ag/TA@CNCs/PVA水凝胶对铜绿假单胞菌和金黄色葡萄球菌有近100%的杀菌率。
（7）小鼠伤口愈合试验
小鼠背部全厚度切除创面模型：选取6~8周龄、体重18~20g的雄性ICR小鼠作为实验动物。首先，用医用剪刀在乙醚麻醉的小鼠背部切开直径1cm的圆形手术伤口。然后，将100μL金黄色葡萄球菌或铜绿假单胞菌悬液（1×108CFU/mL）均匀涂于创面，用纱布和医用胶带包扎。小鼠感染菌24小时后，随机分为4组（每组 5只小鼠）：对照组、水凝胶基底组、Zn,CDs水凝胶组和Zn-Ag/TA@CNCs/PVA水凝胶组，对照涂抹无菌PBS，其余凝胶浓度为100μg/mL，将上述凝胶（各300μL）注入小鼠伤口，每24小时更换小鼠创面水凝胶中，在第0、2、5、8、10天测量小鼠创面情况，通过愈合率(%) = (A0 - At) / (A0×100)计算创面愈合率，其中A0为初始创面面积，At为各时间点的残余创面面积。
试验结果表明，PBS处理组于第2天出现脓液，持续至第8天，提示伤口感染。第8天，Zn-Ag/TA@CNCs/PVA水凝胶组创面面积明显减少，为26.12%，在其他治疗组创面面积中最低，第10天Zn-Ag/TA@CNCs/PVA水凝胶组创面基本愈合，创面面积减少至4.09%，而对照组创面未愈合，创面面积40.15%（图8、图9）。
以上结果表明，本发明制备的纳米酶抗菌水凝胶具有好的机械性能、粘附性能，好的生物相容性、抗菌性能及伤口愈合性能，这主要取决于水凝胶中化学硼酯键和氢键交联网络结构，同时保留了丰富的酚羟基，维持了酚醌的氧化还原动态平衡，为水凝胶提供了长期性和可重复性的黏附性，同时纳米酶的抗菌性能及清除自由基的性能，为伤口愈合提供了保障，该纳米酶抗菌水凝胶用于伤口愈合是安全、有效的。
Claims (4)
1.一种多功能纳米酶原位水凝胶的制备方法，其特征在于，制备步骤如下：
（1）锌掺杂碳点Zn,CDs的合成：称取0.1-0.3g ZnCl2、0.5-1.0g多巴胺、1.0-2.0g柠檬酸及0.5-1.0g乙二胺溶于30-40mL超纯水中，超声混合均匀，将溶液转移至聚四氟乙烯内衬反应器中，置于微波消解炉中，1分钟升至150℃，在150℃、850W下水解40-60分钟，反应完成后自然冷却至室温，得棕色溶液；将棕色溶液用0.22μm滤膜除去大颗粒杂质，再经高速离心，上清液真空干燥，得到锌掺杂碳点Zn,CDs；
（2）银纳米AgNPs合成：将10-15mg的Zn,CDs与20-25mg抗坏血酸溶于30mL超纯水中，油浴加热至90-100℃，加入AgNO3，AgNO3在混合液中的浓度为20-25mg/mL，避光搅拌60分钟，冷却至室温，高速离心，上清液冷冻干燥，得到银纳米；
（3）水凝胶Zn-Ag/TA@CNCs/PVA的合成：将0.4-0.6g微晶纤维素CNCs分散于40mL pH=8.5的磷酸盐缓冲溶液中，搅拌均匀后，加入0.4-0.5g单宁酸TA，搅拌均匀后，按0.02-0.04mg/mL的添加比例加入银纳米，搅拌30-40分钟后，离心分离，用纯水洗涤固体2-3次后，重新分散于纯水中，配制成5-7%的Zn-Ag/TA@CNCs分散液；将10-11g聚乙烯醇、15-18mL Zn-Ag/TA@CNCs分散液、62-65mL纯水混合，于90-95℃加热搅拌溶解后加入质量浓度5-10%的硼砂溶液5-10mL，剧烈搅拌混匀后，将其倒入模具，冷却后即得纳米酶原位水凝胶。
2.根据权利要求1所述的多功能纳米酶原位水凝胶的制备方法，其特征在于：步骤（1）、步骤（2）中高速离心是在10000r/min下处理15-20分钟。
3.根据权利要求1所述的多功能纳米酶原位水凝胶的制备方法，其特征在于：步骤（3）中离心分离是在4000-6000r/min下处理10-15分钟。
4.权利要求1-3中任一项所述的多功能纳米酶原位水凝胶的制备方法制得的纳米酶原位水凝胶在制备用于伤口愈合的试剂中的应用。
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| CN116370406B (zh) * | 2023-06-02 | 2023-08-15 | 云南伦扬科技有限公司 | 一种用于治疗痔疮纳米酶可注射水凝胶 |
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