CN116808284A - 一种用于伤口愈合光催化二重纳米酶凝胶敷料 - Google Patents
一种用于伤口愈合光催化二重纳米酶凝胶敷料 Download PDFInfo
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- CN116808284A CN116808284A CN202310838291.0A CN202310838291A CN116808284A CN 116808284 A CN116808284 A CN 116808284A CN 202310838291 A CN202310838291 A CN 202310838291A CN 116808284 A CN116808284 A CN 116808284A
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Abstract
本发明公开了一种用于伤口愈合光催化二重纳米酶凝胶敷料，本发明用水热法制备了铁掺杂硒化钼纳米材料，利用液体部分具有还原性及光催化活性、固体部分具有纳米酶模拟过氧化酶活性特点，将液体部分作为还原剂，结合具有收敛止血特性白及提取液，具有抗菌壳聚糖及聚乙二醇为凝胶分子骨架，固体部分纳米酶催化氧化白及提取液酚类物质，制备壳聚糖银纳米凝胶敷料。制备的铁掺杂硒化钼具有的光催化缺陷纳米酶的强模拟过氧化酶活性，产生有毒的羟基自由基，及银纳米及凝胶基底抗菌作用，达到高效抗菌效果；同时铁掺杂硒化钼具有模拟超氧歧化酶活性，结合白及提取液具有的收敛止血作用，凝胶敷料能够对细菌感染的伤口起到有效的愈合作用。
Description
技术领域
本发明涉及纳米材料抗菌技术领域，具体为一种用于伤口愈合光催化二重纳米酶凝胶敷料。
背景技术
皮肤作为人体最大的器官，是人体自然防御系统的第一道防线，可以防止外界微生物的入侵、保护内部 组织等。然而，皮肤容易受到各种类型的创伤，如烧伤、烫伤、撕裂等，虽然能自我修复，但是有一定的局限性，因而伤口敷料成为促进皮肤组织修复或愈合的有效辅助手段。水凝胶因其能提供湿润的环境、吸收伤口渗出物、抗细菌等特性，一直被认为是很有前途的伤口敷料。由于细菌和活性氧自由基会引发严重的炎症反应，进而延缓伤口愈合，因此抗菌和抗氧化是伤口敷料的重要指标。白及，为兰科植物白及[Bletill astriata(Thunb.) Reichb. f.]的干燥块茎，其味甘、苦、 涩，性偏寒，归胃经、肺经、肝经，具有敛疮、补肺、消肿、止血及生肌等功效。壳聚糖具有良好的生物相容性、生物降解性、低毒性、无抗原性、抗微生物活性、凝血和止血作用的优点成为伤口敷料重要的基材。纳米酶因具有模拟过氧化物酶（POD）活性而被广泛探索以达到更好的抗菌效果。它们可以催化过氧化氢（H2O2）生成羟基自由基（·OH），具有更高的抗菌活性，可以最大限度地降低高浓度H2O2的毒性。特别地，·OH可以对细胞壁和细胞膜造成初始的氧化损伤，当与光热治疗（PTT），即近红外光（NIR）的转换来诱导热疗相结合时，克服了每个单独的抗菌模型的缺陷，从而表现出增强的抗菌活性。碳点作为一种具有还原性的纳米材料，同时可以在光照射下活化产生光致电荷进而得到活性中间体，增加其光催化效应。硒化钼（MoSe2）作为一种二维纳米材料，具有比表面积大、表面易修饰、生物相容性好等优点。MoSe2在NIR范围内的优异吸收和类POD纳米酶特性使其可以应用于非侵入性抗菌治疗方法。然而，将天然产物提取物与无机纳米材料结合，充分发挥其优异抗菌、抗氧化性能、光催化性能用于制备伤口愈合凝胶敷料仍然是十分必要的。
发明内容
本发明提供了一种伤口愈合光催化二重纳米酶凝胶敷料，该方法利用了纳米酶产生的双重纳米酶活性，一是在弱酸性条件下Fe-MoSe2/AgNPs催化H2O2产生有毒羟基自由基（·OH），起到杀菌活性，二是Fe-MoSe2/AgNPs在中性条件下具有模拟超氧歧化酶（SOD）活性，结合白及提取液具有的收敛止血作用，在近红外光（NIR）辐射下，抗菌、抗氧化、消炎作用得到进一步加强。体外抗菌及动物试验得到充分证明。
本发明公开了一种用于伤口愈合光催化二重纳米酶凝胶敷料，本发明用水热法制备了铁掺杂硒化钼纳米材料（Fe-MoSe2），利用液体部分（碳点）具有还原性及光催化活性、固体部分具有纳米酶模拟过氧化酶（POD）活性特点，将液体部分与具有抗氧化及抗炎特性石榴皮水提液作为还原剂、具有抗菌壳聚糖（CS）及聚乙二醇（PEG）为凝胶分子骨架，固体部分Fe-MoSe2（纳米酶）催化氧化白及提取液酚类物质，制备银纳米（AgNPs）凝胶敷料（Fe-MoSe2/ AgNPs）。利用Fe-MoSe2具有的光催化缺陷纳米酶的强模拟过氧化酶（POD）活性，结合低浓度的过硫酸纳有效地转化为有毒的羟基自由基（·OH），碳点的引入加强了光电子的转移，提高了光催化作用，同时利用AgNPs及凝胶基底抗菌作用，达到高效抗菌效果；同时Fe-MoS2具有模拟超氧歧化酶（SOD）活性，结合白及提取液具有的收敛止血作用，Fe-MoSe2/AgNPs凝胶敷料能够对细菌感染的伤口起到有效的愈合作用。
本发明制备用于伤口愈合光催化二重纳米酶凝胶敷料，按以下步骤进行：
（1）铁掺杂硒化钼纳米材料（Fe-MoSe2）制备：0.3 - 0.5 g硒酸钠、1.5 - 2.0 gL-半胱氨酸、0.1 - 0.2g FeSO4·7H2O溶于50 - 80 mL去离子水中，超声处理10 - 15min后，转移至聚四氟乙烯罐中，马弗炉200 - 220℃烧18-24 h，离心，经0.22μm滤膜过滤，得到上清液，即液体部分（Fe,Mo,Se-CDs），固体部分依次用0.1mol/L稀硫酸、无水乙醇、去离子水洗涤，真空干燥，即得Fe-MoSe2；
（2）Fe-MoSe2/AgNPs凝胶敷料制备：在聚乙二醇400水溶液中，加入AgNO3溶液及过硫酸纳溶液，搅拌40 min，冷却至室温，作为A液；将Fe,Mo,Se-CDs与白及提取液按体积比1:10混合搅拌，同时将 Fe-MoSe2搅拌分散到其中，作为B液；将5重量份A液和1重量份B液混合，常温避光搅拌30 min，得C液；用1%的醋酸配制壳聚糖CS溶液，取CS溶液和C液按4:1的体积比混合搅拌10 min，即得Fe-MoSe2/AgNPs凝胶敷料。条件完全相同制备Fe,Mo,Se-CDs、Fe-MoSe2凝胶敷料。其中，白及提取液浓度为2-3mg/mL，聚乙二醇400浓度为10-12%（w/v），AgNO3溶液浓度为2-3mg/mL，过硫酸纳溶液浓度为0.001-0.005mg/mL，CS浓度为2-3%（w/v），Fe,Mo,Se-CDs浓度为0.1-0.3mg/mL，Fe-MoSe2浓度为0.1-0.5mg/mL。
所述的白及提取液按以下方法制备：将干燥白及粉碎，过40-60目筛，称取白及粉末0.1-0.2g，加入800-100mL 40%-50%乙醇，在50℃条件下超声25-30min，离心，取上清液，烘箱60-70℃烘1-2h后，得白及提取液，根据需求用纯水配制为所需浓度。
所述的离心是在4000-6000r/min处理10-15min。
所制备的光催化二重纳米酶凝胶敷料，将其在抗细菌感染伤口产品中应用。
本发明的优点在于：
1、本发明制备的铁掺杂硒化钼纳米材料（Fe-MoSe2），既利用具有碳点特性液体部分的还原性及光催化活性，又利用固体部分具有模拟过氧化酶（POD）及超氧歧化酶（SOD）特性，具有抗氧化的碳点作为还原剂，结合具有收敛止血特性白及提取液，抗菌壳聚糖（CS）及聚乙二醇（PEG）为凝胶分子骨架，固体部分Fe-MoSe2（纳米酶）催化氧化白及提取液酚类物质，制备银纳米（AgNPs）凝胶敷料（Fe-MoSe2/ AgNPs）。具有过氧化物酶活性的Fe-MoSe2将低浓度的过硫酸纳有效地转化为有毒的羟基自由基（·OH），起到杀菌作用，在近红外光（NIR）作用下，Fe-MoSe2/AgNPs的POD及SOD活性得到进一步加强，对于细菌和活性氧自由基会引发严重的炎症反应伤口，其愈合效果非常明显，因为抗菌和抗氧化是伤口敷料的重要指标；
2、本发明将天然产物活性物质与纳米材料有效结合，进行细菌感染伤口凝胶敷料制备，充分利用抗菌、抗氧化、收敛止血及光催化特性，同时利用凝胶无刺激、安全、无毒副作用特点，能够促进伤口愈合，防止伤口感染；
3、MoSe2/AgNPs材料具有低毒性、良好生物相容性、稳定性。
附图说明
图1为实施例1中Fe,Mo,Se-CDs的TEM图；
图2为实施例1中Fe-MoSe2的TEM图；
图3为实施例1中Fe-MoSe2/AgNPs凝胶敷料的TEM图；
图4为实施例1中Fe,Mo,Se-CDs、Fe-MoSe2及Fe-MoSe2/AgNPs凝胶敷料的Zeta电位图；
图5为实施例1 Fe-MoSe2/AgNPs凝胶敷料氧化TMB（拟POD）有NIR或没有NIR的紫外可见吸收光谱图，图中（1）为单独TMB，（2）TMB和Fe-MoSe2/AgNPs凝胶敷料有NIR辐射，（3）TMB和Fe-MoSe2/AgNPs凝胶敷料没有NIR辐射；
图6为实施例1 Fe-MoSe2/AgNPs凝胶敷料光还原硝基蓝四氮唑（NBT）的抑制率，清除能力O2 ·-的能力（拟SOD）有NIR或没有NIR的紫外可见吸收光谱图，图中（1）为单独NBT，（2）NBT和Fe-MoSe2/AgNPs凝胶敷料有NIR辐射，（3）NBT和Fe-MoSe2/AgNPs凝胶敷料没有NIR辐射；
图7为EDTA对Fe-MoSe2/AgNPs凝胶敷料氧空位捕获效果图，图中Blank是不添加Fe-MoSe2/AgNPs凝胶敷料的EDTA，+Fe-MoSe2/AgNPs凝胶敷料为不含EDTA；
图8为实施例1 纳米酶凝胶敷料细胞毒性实验结果；
图9为实施例1 纳米酶凝胶敷料抗菌实验结果，图中空白为不加凝胶敷料；
图10 为实施例1 纳米酶凝胶敷料处理细菌感染小鼠伤口愈合照片图；
图11 为实施例1 纳米酶凝胶敷料处理细菌感染小鼠伤口愈合率。
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1：二重纳米酶凝胶敷料制备及性能
（1）铁掺杂硒化钼纳米材料（Fe-MoSe2）制备：0.4 g硒酸钠、1.7 gL-半胱氨酸、0.2g FeSO4·7H2O溶于60 mL去离子水中，超声处理15min后，转移至聚四氟乙烯罐中，马弗炉200℃烧20 h，6000r/min离心处理15min，经0.22μm滤膜过滤，得到上清液，即液体部分（Fe,Mo,Se-CDs），固体部分依次用0.1mol/L稀硫酸、无水乙醇、去离子水洗涤，真空干燥，即得Fe-MoSe2；
（2）白及提取液制备：将干燥白及粉碎，过40-60目筛，称取白及粉末0.1g，加入50mL 50%乙醇，在50℃条件下超声25 min，4000 r/min处理15min，取上清液，烘箱70℃烘2h后，得白及提取物，取100mg白及提取液溶于10mL去离子水中，制备10mg/mL白及提取液；
（3）Fe-MoSe2/AgNPs凝胶敷料制备：用纯净水配制12%的PEG400溶液，加入浓度为2.3 mg/mL的AgNO3溶液及0.005mg/mL过硫酸纳溶液，搅拌40 min，冷却至室温，作为A液；0.2 mg/mL的 Fe,Mo,Se-CDs与1 mg/mL白及提取液按体积比1:10混合搅拌，0.2 mg/mL Fe-MoSe2搅拌分散到其中，作为B液；将5重量份A液和1重量份B液混合，常温避光搅拌30 min，得C液；用1%的醋酸配制3%的壳聚糖（CS）溶液，取3%CS溶液和C液按4:1的体积比混合搅拌10min，即得Fe-MoSe2/AgNPs凝胶敷料。条件完全相同制备Fe,Mo,Se-CDs、Fe-MoSe2凝胶敷料；
（4）纳米材料的TEM及Zeta电位测试：将合成的Fe,Mo,Se-CDs、Fe-MoSe2及Fe-MoSe2/AgNPs凝胶敷料，冷冻干燥后进行透射电镜（TEM）及Zeta电位测试（图1-4），从TEM图（图1）看出，Fe,Mo,Se-CDs呈现良好的分散性带平均尺寸为2.7±0.7 nm的准球形形貌，晶格空间为0.21 nm，对应于石墨的（100）晶面；Fe-MoSe2为纳米片状结构，大小均匀，平均尺寸为50 - 200 nm（图2）；Fe-MoSe2/AgNPs凝胶敷料呈现高度多孔结构（图3）；Fe,Mo,Se-CDs及Fe-MoSe2的Zeta电位为负，而Fe-MoSe2/AgNPs凝胶敷料为正（图4），因为CS的存在使得材料表面带正电荷，从而有利于与带负电荷的细胞膜结合，改变细胞膜通透性，有利于抗菌；
（5）采用TMB显色反应测定纳米酶的拟过氧化酶（POD）活性
将100µg/mL Fe-MoSe2/AgNPs凝胶敷料100μL、100mmol/L的TMB 50µL，40mmol/LH2O2 50µL，加入到pH5.0醋酸盐缓冲溶液2mL中，经1.0 W/cm2 808nm红外光照射15min后，4000r/min离心处理10min，取上层清液用紫外-可见分光光度计在654 nm处测量吸光度，每个样品测量3次，取平均值，且设置没有经过1.0 W/cm2 808nm红外光照射的作为空白对照，吸收光谱图见图5，由图可知红外光作用下，Fe-MoSe2/AgNPs凝胶敷料的过氧化物酶活性得到进一步提高；
（6）采用纳米酶对还原硝基蓝四氮唑（NBT）抑制反应测定拟超氧歧化酶（SOD）活性
将100µg/mL Fe-MoSe2/AgNPs凝胶敷料100μL与5 mg/mL NBT 100 μL 、100μL 5mg/mL核黄素和pH = 3磷酸盐缓冲液2mL中，经1.0 W/cm2 808nm红外光照射15min后，4000r/min离心处理10min，取上层清液用紫外-可见分光光度计在580 nm处测定吸光度，每个样品测量3次，取平均值，且设置没有经过808nm红外光照射的作为空白对照，结果如图6；从图6中可以看出，由图可知红外光作用下，Fe-MoSe2/AgNPs凝胶敷料表现出相当高的拟SOD活性；
（7）EDTA为氧空位捕获剂，在0.5、1.0、5.0 μg/mL的EDTA加入100 µg/mL Fe-MoSe2/AgNPs凝胶敷料100μL、10 mmol/L TMB 50µL、40mmol/L H2O2 50µL，4000 r/min离心处理15 min，取上层清液用紫外-可见分光光度计在654 nm处测量吸光度，结果见图7，随着EDTA浓度的增加，吸光度随之下降，表明MoSe2/AgNPs凝胶敷料存在氧空位，氧空位的存在可提高表面氧气的吸附与活化，进而对底物的氧化有促进作用；
（8）细胞毒性测试：采用CCK-8细胞活力试剂盒检测纳米酶的细胞毒性，具体实验，将人脐静脉内皮细胞（HUVECs, 北纳创联生物科技有限公司）接种于96孔板中培养24h后，分别用不同浓度的Fe-MoSe2/AgNPs凝胶敷料（0、5、10、20、40、80、160 μg/mL，以Ag+含量计算）分别孵育12、24、36h，分次用PBS漂洗细胞，每孔加入CCK-8溶液至10%浓度，37℃孵育，在450nm处测定吸光度；CCK-8分析（图8）显示MoSe2/AgNPs凝胶敷料对细胞均无明显毒性；
（9）MoSe2/AgNPs凝胶敷料抗菌试验
以下菌种从北纳创联生物科技有限公司获得；
实验方法：以耐甲氧西林金黄色葡萄球菌（MRSA, ATCC 43300）和大肠杆菌（E. coli，ATCC 25922）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MoSe2/AgNPs凝胶敷料的抗菌性能。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 h，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12h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，Fe,Mo,Se-CDs、Fe-MoSe2及Fe-MoSe2/AgNPs凝胶敷料组。将培养的细菌加入磷酸盐缓冲液中作为空白对照组，其他组与不同凝胶敷料混合，凝胶敷料浓度为50μg/mL，分别经1.0 W/cm2 808nm红外光照射10min后和不经红外光照射处理，然后在37℃下将孵育60min，菌悬液经过稀释后（100 μL）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24 h，计算菌落数，判断抗菌性能；
结果如图9所示，空白对照组几乎没有抗菌性能，没有经红外光照射Fe,Mo,Se-CDs、Fe-MoSe2及Fe-MoSe2/AgNPs凝胶敷料组下，MRSA和E. coli的抑菌率分别约为26.7%、38.1%及 68.4%，24.8%、34.2%及 60.0%，经红外光照射Fe,Mo,Se-CDs、Fe-MoSe2及Fe-MoSe2/AgNPs凝胶敷料组对MRSA和E. coli的抑菌率分别约为52.0%、70.5%及 99.9%，46.1%、64.7%及 99.0%，，Fe-MoSe2/AgNPs凝胶敷料经红外光照射对MRSA和E. coli有近100%的杀菌率；
（10）小鼠伤口愈合试验
小鼠背部创面模型：所有动物实验均符合《动物护理指导原则》。选取6~8周龄、体重18~20g的雄性ICR小鼠作为实验动物。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 MRSA或E. coli菌悬液（1×108 CFU/mL）均匀涂于创面，用纱布和医用胶带包扎。小鼠感染菌24h后，随机分为4组（每组 5只小鼠）：对照组、Fe,Mo,Se-CDs凝胶敷料、Fe-MoSe2凝胶敷料及Fe-MoSe2/AgNPs凝胶敷料，对照涂抹无菌PBS，其余凝胶浓度为50μg/mL，将上述凝胶（各300μL）注入小鼠伤口，经1.0 W/cm2 808nm红外光照射15min；每24h更换小鼠创面水凝胶中，在第0、2、4、6、8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1天出现脓液，持续至第8天，提示伤口感染。第8天，Fe-MoSe2/AgNPs凝胶敷料组创面面积明显减少，为8.31%（愈合率为91.69%），在其他治疗组创面面积中最低，创面基本愈合，而对照组创面未愈合，创面面积90.15%（愈合率为9.85%）（图10、图11）。
以上结果表明，本发明制备的纳米酶Fe-MoSe2/AgNPs凝胶敷料有二重酶活性，通过纳米酶模拟POD活性起到抗菌、石榴皮水提物抗氧化、收敛作用及纳米酶模拟SOD活性清除自由起到的消炎作用，使细菌感染小鼠伤口具有好的愈合效果。
Claims (4)
1.一种用于伤口愈合光催化二重纳米酶凝胶敷料，其特征在于，制备步骤如下：
（1）铁掺杂硒化钼纳米材料（Fe-MoSe2）制备：0.3 - 0.5 g硒酸钠、1.5 - 2.0 gL-半胱氨酸、0.1 - 0.2g FeSO4·7H2O溶于50 - 80 mL去离子水中，超声处理10 - 15min后，转移至聚四氟乙烯罐中，马弗炉200 - 220℃烧18-24 h，离心，经0.22μm滤膜过滤，得到上清液，得铁掺杂硒化钼纳米材料液体部分即Fe,Mo,Se-CDs，固体部分依次用0.1mol/L稀硫酸、无水乙醇、去离子水洗涤，真空干燥，得铁掺杂硒化钼纳米材料，即Fe-MoSe2；
（2）Fe-MoSe2/AgNPs凝胶敷料制备：在聚乙二醇400水溶液中，加入AgNO3溶液及过硫酸纳溶液，搅拌40 min，冷却至室温，作为A液；将Fe,Mo,Se-CDs与白及提取液按体积比1:10混合搅拌，同时将 Fe-MoSe2搅拌分散到其中，作为B液；将5重量份A液和1重量份B液混合，常温避光搅拌30 min，得C液；用1%的醋酸配制壳聚糖CS溶液，取CS溶液和C液按4:1的体积比混合搅拌10 min，即得Fe-MoSe2/AgNPs凝胶敷料。条件完全相同制备Fe,Mo,Se-CDs、Fe-MoSe2凝胶敷料。其中，白及提取液浓度为2-3mg/mL，聚乙二醇400浓度为10-12%（w/v），AgNO3溶液浓度为2-3mg/mL，过硫酸纳溶液浓度为0.001-0.005mg/mL，CS浓度为2-3%（w/v），Fe,Mo,Se-CDs浓度为0.1-0.3mg/mL，Fe-MoSe2浓度为0.1-0.5mg/mL。
2.根据权利要求1所述的一种用于伤口愈合光催化二重纳米酶凝胶敷料，其特征在于：所述的白及提取液按以下方法制备：将干燥白及粉碎，过40-60目筛，称取白及粉末0.1-0.2g，加入800-100mL 40%-50%乙醇，在50℃条件下超声25-30min，离心，取上清液，烘箱60-70℃烘1-2h后，得白及提取液，根据需求用纯水配制为所需浓度。
3.根据权利要求1所述的一种用于伤口愈合光催化二重纳米酶凝胶敷料，其特征在于：离心是在4000- 6000r/min处理10-15min。
4.根据权利要求1制备的光催化二重纳米酶凝胶敷料，将其在抗细菌感染伤口产品中应用。
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