CN114053473A - 一种四氧化三铁复合纳米酶抗菌剂的制备方法及应用 - Google Patents
一种四氧化三铁复合纳米酶抗菌剂的制备方法及应用 Download PDFInfo
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- CN114053473A CN114053473A CN202111324140.0A CN202111324140A CN114053473A CN 114053473 A CN114053473 A CN 114053473A CN 202111324140 A CN202111324140 A CN 202111324140A CN 114053473 A CN114053473 A CN 114053473A
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- ferroferric oxide
- nanoenzyme
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- antibacterial agent
- oxide composite
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Abstract
本发明公开了一种四氧化三铁复合纳米酶抗菌剂的制备方法及其应用，该方法是采用碳纳米管修饰四氧化三铁与血红素、三磷酸腺苷形成复合纳米酶，该酶具有拟过氧化酶活性，利用三磷酸腺苷及血红素对Fe3O4‑CNT过氧化酶活性的调节作用，使Fe3O4‑CNT‑Hemin/ATP纳米酶活性增强，同时三磷酸腺苷能在中性条件下调节催化活性，产生更多的活性氧（ROS）；在过硫酸钾存在下，Fe3O4‑CNT‑Hemin/ATP对致病菌有强的抑菌杀菌效果；该纳米酶具有良好生物相容性，将其作为创可贴的用药，不仅快速促进伤口愈合，防止伤口感染，对伤口无刺激、安全、无毒副作用；与阳性对照药相比，对耐药致病菌的抗菌效果更加优异。
Description
技术领域
本发明涉及纳米材料抗菌技术领域，具体为一种四氧化三铁复合纳米酶抗菌剂的制备方法及应用。
背景技术
微生物污染和耐药细菌的流行被认为是一个世界性的公共卫生问题，由细菌引发的相关疾病和环境污染等问题引起了人们的高度重视，同时随着抗生素的使用，细菌的耐药性逐渐增强，人们急需开发新型抗菌剂。纳米酶是新一代人工模拟酶，兼具纳米材料独特的理化性质和类酶催化活性，因其结构稳定、生产成本低等优点受到广泛关注。纳米酶通过自身具有的类天然酶活性，分别以催化产生强氧化性活性氧自由基破坏细菌生物膜成分、产生次卤酸干扰细菌生存重要的群体感应***和降解细菌生物膜中外源性DNA 的途径，而达到抗菌的目的。相比于天然酶和传统抗菌剂，纳米酶具有稳定性好、成本低、易于功能化等优点。纳米酶作为新型抗菌剂研究已有相关报道，但也存在抗菌性能有待加强，尤其是对耐药菌的抗菌性能、生物安全性能提高方面的问题。目前报道的纳米酶产生的抗菌酶活大都集中在拟过氧化酶活性方面，也就是说，需要H2O2的参与，尽管H2O2的浓度很低，但没有H2O2的存在，抗菌效果大大降低，但由于H2O2为液体，这给后续在固体载体上的应用带来不便。
发明内容
针对现有技术的不足，本发明提供了一种四氧化三铁复合纳米酶抗菌剂的制备方法，该方法是将碳纳米管修饰四氧化三铁与血红素、三磷酸腺苷形成复合纳米酶（Fe3O4-CNT-Hemin/ATP），该纳米酶具有的拟过氧化酶特性，在过硫酸钾存在下，产生更多的活性氧（ROS），达到好的抗菌效果。
本发明复合纳米酶（Fe3O4-CNT-Hemin/ATP），具有拟过氧化酶活性，由于一般过氧化酶的pH依赖活性仅限于酸性环境，在近中性pH条件下抗菌活性急剧下降；本发明利用三磷酸腺苷能在中性pH (7.4)下调节催化活性，同时三磷酸腺苷及血红素对Fe3O4-CNT过氧化酶活性的调节作用，使Fe3O4-CNT-Hemin/ATP纳米酶活性增强，用过硫酸钾替代H2O2，发挥拟过氧化酶活性。Fe3O4-CNT-Hemin/ATP对致病菌，包括金黄色葡萄球菌、耐药金黄色葡萄球菌、大肠杆菌、耐药大肠杆菌、绿脓假单胞菌等有强的抑菌杀菌效果。该纳米酶具有良好生物相容性，将其用于创可贴的用药，不仅快速促进伤口愈合，防止伤口感染，对伤口无刺激、安全、无毒副作用。与阳性对照药相比，对耐药致病菌的抗菌效果更加优异。
本发明四氧化三铁复合纳米酶抗菌剂的制备方法如下：
（1）碳纳米管-四氧化三铁-血红素纳米酶的制备
取80-100重量份碳纳米管、30重量份乙二醇、10重量份聚乙二醇混合后，加入1.5-2.0重量FeCl3∙6H2O、3.5-4.0重量份NaAc及0.02-0.05重量份氯化血红素，超声处理1h，转入马弗炉200℃烧制10-12h，冷却至室温后，用水和无水乙醇交替洗三次后，真空干燥得碳纳米管-四氧化三铁-血红素纳米酶Fe3O4-CNT-Hemin；
（2）碳纳米管-四氧化三铁-血红素-三磷酸腺苷纳米酶的制备
将步骤（1）纳米酶Fe3O4-CNT-Hemin的水溶液与三磷酸腺苷溶液等体积混合，搅拌5-6h后，外加磁铁分离，固体用乙醇洗2-4次，真空干燥得四氧化三铁复合纳米酶抗菌剂；其中纳米酶Fe3O4-CNT-Hemin水溶液的浓度为20-50µg/mL，三磷酸腺苷溶液的浓度为2-4mmol/L。
所述真空干燥条件为40-60℃干燥24-48h。
所述碳纳米管包括单壁碳纳米管、多壁碳纳米管。
所述聚乙二醇包括聚乙二醇400、聚乙二醇600。
本发明另一目的是将上述方法制得的四氧化三铁复合纳米酶抗菌剂应用在制备创可贴中，具体是将四氧化三铁复合纳米酶抗菌剂与过硫酸钾加入到创可贴中药层中，其中四氧化三铁复合纳米酶抗菌剂与过硫酸钾重量比为1:0.1-0.5。
本发明的优点在于：
1、本发明采用碳纳米管修饰四氧化三铁与血红素、三磷酸腺苷形成复合纳米酶，该酶具有拟过氧化酶活性，利用三磷酸腺苷及血红素对Fe3O4-CNT过氧化酶活性的调节作用，使Fe3O4-CNT-Hemin/ATP纳米酶活性增强，同时三磷酸腺苷能在中性pH (7.4)下调节催化活性，产生更多的活性氧（ROS），用过硫酸钾替代H2O2，产更多的活性氧（ROS），使四氧化三铁复合纳米酶抗菌剂能达到好的抗菌效果；
2、该纳米酶抗菌剂具有对耐药性金黄色葡萄球菌、耐药大肠杆菌等较好抗菌效果，与阳性对照药相比，杀菌率提高了60-70%；
3、将该纳米酶抗菌剂应用到创可贴用药，不仅能快速促进伤口愈合，防止伤口感染，且对伤口无刺激、安全、无毒副作用；
本发明产品适于工业化生产和市场推广应用。
附图说明
图1为纳米酶氧化底物TMB的光谱图；
图2为活性氧（ROS）产生监控结果示意图；
图3为纳米酶及抗生素对金黄色葡萄球菌耐药菌抗菌效果图；
图4为纳米酶及抗生素对铜绿假单胞菌耐药菌抗菌效果图。
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：Fe3O4-CNT-Hemin/ATP+K2S2O8抗菌剂的制备及抗菌作用
1、多壁碳纳米管-四氧化三铁-血红素纳米酶（Fe3O4-MWCNT-Hemin）的制备
取10g多壁碳纳米管、3g乙二醇、1g聚乙二醇400混合，然后加入0.15g FeCl3∙6H2O、0.35g NaAc及0.002g氯化血红素，超声处理1h，转入马弗炉200℃烧制10h，冷却至室温后，用水和无水乙醇交替洗三次后，40℃真空干燥48h，得Fe3O4-MWCNT-Hemin纳米酶；
2、多壁碳纳米管-四氧化三铁-血红素-三磷酸腺苷纳米酶（Fe3O4-MWCNT-Hemin/ATP）的制备
将浓度50µg/mL纳米酶Fe3O4-CNT-Hemin的水溶液5mL添加到4mmol/L的三磷酸腺苷（ATP）水溶液5mL中，搅拌5h后，外加磁铁分离，用乙醇洗3次，移出多余的ATP，40℃真空干燥得Fe3O4-MWCNT-Hemin/ATP纳米酶；
3、采用TMB显色反应测定Fe3O4-MWCNT-Hemin/ATP纳米酶的过氧化酶活性
将100µg/mLFe3O4-MWCNT-Hemin/ATP纳米酶100μL、100mmol/L的TMB50µL、12.5µmol/L K2S2O850µL，加入到pH7.4磷酸盐缓冲溶液2mL中，充分混匀，室温孵育10min后，外加磁铁分离，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2~3次，取平均值，结果如图1；从图1中可以看出，在中性条件下，纳米酶Fe3O4-MWCNT-Hemin/ATP表现出相当高的过氧化酶活性。
4、活性氧（ROS）产生量的监控：以抗坏血酸（AA）为探针进行监测，AA在266nm有吸收，但被ROS氧化生成脱氢抗坏血酸后，吸收峰消失。在磷酸缓冲盐溶液( PBS )中共孵育1h后，纳米酶Fe3O4-MWCNT-Hemin/ATP+K2S2O8对抗坏血酸在266nm处的吸光度有很大的降低，且降低程度结果见图2，Fe3O4-MWCNT-Hemin/ATP+K2S2O8＞Fe3O4-MWCNT-Hemin/ATP＞K2S2O8。
5、纳米酶对细菌平板的杀灭实验
（1）实验方法：以金黄色葡萄球菌(S. aureus，ATCC 25923)和枯草杆菌(B. subtilis，ATCC 6051)为代表革兰氏阳性菌株，大肠杆菌(E. coli，ATCC 25922)和铜绿假单胞菌(P.aeruginosa，ATCC 27853)为代表革兰氏阴性菌株。此外，耐甲氧西林金黄色葡萄球菌(MRSA) (ATCC BAA-1720)被用作抗生素耐药菌株的代表。对于每个菌株，将3-5个单菌落接种到新鲜的胰蛋白胨大豆肉汤(TSB)中，37℃培养16-18h至稳定期。取40μL菌液用新鲜TSB稀释100倍，37℃培养至对数中期(OD600=0.5-0.7)。细菌细胞收获后，用无菌PBS离心洗涤1次，用无菌PBS调至1.5×106CFU/mL；用无菌PBS缓冲液对纳米酶分散液进行2倍系列稀释，每个纳米酶稀释液(100μL)被加入到96孔微孔板的每个零稀释孔中，将50μL调整好的菌悬液接种到预置的微孔板的每个零稀释孔中，使每孔达到5×105 CFU/mL (150μL)；然后将微孔板置于37℃孵育3h。随后用无菌PBS缓冲液进行10倍系列稀释，然后将稀释后的菌液(20μL)涂布于TSB琼脂平板上，37℃培养过夜，形成肉眼可见的菌落。接种物大小由含同样处理但不含纳米酶的细菌的对照样品指示。每项试验一式3次，试验的结果是两个独立试验的平均值。所述的最小杀菌浓度(MBC)值定义为抗菌药物或纳米酶抑制99.9 %细菌生长的最小浓度；最低抑菌浓度（MIC）值定义为抗生素或纳米酶抑制90 %细菌生长的最低浓度。
耐药菌培养：通过循环抑制实验研究药物耐药性，以铜绿假单胞菌作为革兰氏阴性菌的代表菌株，金黄色葡萄球菌作为革兰氏阳性菌的代表菌株，分别用头孢羟氨苄、庆大霉素、纳米酶处理，观察耐药性；具体方法：使用前面抑制试验（300μL，每孔100μL）的½MIC处理的细菌培养物，稀释至OD600=0.001，作为下一种生长抑制试验的最终细菌接种物，进行循环抑制试验，每项试验一式3次，试验的结果是两个独立试验的平均值。
（2）抗菌结果：纳米酶的最小杀菌浓度(MBC)值见表1；
表1纳米酶杀菌效果
药物耐药性试验结果表明，8次循环后，头孢羟氨苄对金黄色葡萄球菌的抑制作用显著降低，细菌存活率由初始的5.9%提高到78.6%，而纳米酶对细菌存活率仅由3.6%提高到58.5%；同样，庆大霉素对铜绿假单胞菌的抑制作用也在循环后明显下降，细菌存活率从开始的9.1%上升到74.4%，而纳米酶对细菌存活率仅从7.9%上升到47.1%，见图3、4。
结果表明，纳米酶不仅在抑制耐药菌方面表现出优异的性能，而且在减缓细菌耐药性的发展方面也表现出优异的性能。产生ROS的纳米酶可以规避细菌的耐药机制，因为ROS同时破坏了对细胞功能至关重要的多种小球物质(如核酸、蛋白质、脂类等)，而不是像抗生素那样靶向特定的细胞内代谢途径。
实施例2：Fe3O4-SWCNT-Hemin/ATP+K2S2O8抗菌剂的制备和用于创可贴
1、单壁碳纳米管-四氧化三铁-血红素纳米酶（Fe3O4-SWCNT-Hemin）的制备
取8g单壁碳纳米管、3g乙二醇、1g聚乙二醇400混合后，加入0.2g FeCl3∙6H2O、0.4gNaAc及0.005g氯化血红素，超声处理1h，转入马弗炉200℃烧制12h，冷却至室温后，用水和无水乙醇交替洗三次后，60℃真空干燥24h，得Fe3O4-SWCNT-Hemin纳米酶；
2、单壁碳纳米管-四氧化三铁-血红素-三磷酸腺苷纳米酶（Fe3O4-SWCNT-Hemin/ATP）的制备
将步骤（1）20µg/mL纳米酶Fe3O4-SWCNT-Hemin/ATP水溶液5mL添加到2mmol/L的三磷酸腺苷（ATP）水溶液5mL中，搅拌6h后，外加磁铁分离，用乙醇洗3次，移出多余的ATP，50℃真空干燥得Fe3O4-SWCNT-Hemin/ATP纳米酶；
3、纳米酶抗菌创可贴的制备：将步骤2制备的Fe3O4-SWCNT-Hemin/ATP纳米酶与K2S2O8按重量比1:0.1的比例混合，组成抗菌剂用于创可贴用药，按与创可贴重量比制成0.05mg/g的纳米酶抗菌创可贴，按常规创可贴制备方法制备；
4、纳米酶抗菌创可贴伤口愈合试验
伤口愈合试验采用6-8周健康ICR雌性小鼠，在小鼠背部制作直径1cm的圆形伤口，感染铜绿假单胞菌和金黄色葡萄球菌24h后，分别设置纳米酶抗菌创可贴组、白药创可贴组及未作处理组，每24h更换一次创可贴并观察伤口愈合情况；结果显示在24h后，纳米酶抗菌创可贴及白药创可贴处理的伤口开始愈合，而未作处理的小鼠在72h后，才有伤口愈合迹象，在第7天，使用纳米酶抗菌创可贴小鼠伤口基本愈合，在第9天，使用白药创可贴小鼠伤口基本愈合，未而未作处理的小鼠在第16天鼠伤口才基本愈合。
Claims (5)
1.一种四氧化三铁复合纳米酶抗菌剂的制备方法，其特征在于，包括以下步骤：
（1）碳纳米管-四氧化三铁-血红素纳米酶的制备
取80-100重量份碳纳米管、30重量份乙二醇、10重量份聚乙二醇混合后，加入1.5-2.0重量FeCl3∙6H2O、3.5-4.0重量份NaAc及0.02-0.05重量份氯化血红素，超声处理1h，转入马弗炉200℃烧制10-12h，冷却至室温后，用水和无水乙醇交替洗三次后，真空干燥得碳纳米管-四氧化三铁-血红素纳米酶Fe3O4-CNT-Hemin；
（2）碳纳米管-四氧化三铁-血红素-三磷酸腺苷纳米酶的制备
将步骤（1）纳米酶Fe3O4-CNT-Hemin的水溶液与三磷酸腺苷溶液等体积混合，搅拌5-6h后，外加磁铁分离，固体用乙醇洗2-4次，真空干燥得四氧化三铁复合纳米酶抗菌剂；其中纳米酶Fe3O4-CNT-Hemin水溶液的浓度为20-50µg/mL，三磷酸腺苷溶液的浓度为2-4mmol/L。
2.根据权利要求1所述的四氧化三铁复合纳米酶抗菌剂的制备方法，其特征在于：碳纳米管为单壁碳纳米管、多壁碳纳米管中的一种。
3.根据权利要求1所述的四氧化三铁复合纳米酶抗菌剂的制备方法，其特征在于：聚乙二醇为聚乙二醇400、聚乙二醇600中的一种。
4.根据权利要求1所述的四氧化三铁复合纳米酶抗菌剂的制备方法，其特征在于：真空干燥条件为40-60℃干燥24-48h。
5.权利要求1-4中任一项所述的四氧化三铁复合纳米酶抗菌剂的制备方法制得的四氧化三铁复合纳米酶抗菌剂在制备创可贴中的应用，其特征在于：将四氧化三铁复合纳米酶抗菌剂与过硫酸钾混合后加入到创可贴中药层中，其中四氧化三铁复合纳米酶抗菌剂与过硫酸钾的重量比为1:0.1-0.5。
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