CN111150876A - 耐药性可视化的创可贴及其制备方法 - Google Patents
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Abstract
本发明提供一种耐药性可视化的创可贴及其制备方法,属于创可贴制备技术领域。该方法先合成了一个基于卟啉的金属有机框架材料的PCN‑224,然后合成壳聚糖包裹的PCN‑224纳米粒子,最后制备耐药性可视化的创可贴。本发明还提供上述制备方法得到的耐药性可视化的创可贴。本发明的耐药性可视化的创可贴不仅能够快速检测细菌的感染并实时监测耐药性的产生,而且能有效地杀伤大肠杆菌的药敏型(DS)和耐药型(DR)菌株,有效地防止伤口感染,加快伤口愈合,还能防止水果的染菌、保证新鲜储存。
Description
技术领域
本发明属于创可贴制备技术领域,具体涉及一种耐药性可视化的创可贴及其制备方法。
背景技术
目前,每年超过七万次的死亡是与抗菌剂耐性有关的。耐性菌种的加速出现和广泛发展已经构成对全世界人类健康的最严重威胁之一。然而,商品化的抗生素,因其具有明确的抗菌机制,仍旧是最为人们接受的治疗模式。由于抗菌剂的“自动淘汰”性质,如何合理利用现有抗生素,克服耐药性是当前抗菌领域的一个重要课题。
及早感知细菌感染并追踪耐药性的出现是选择抗菌方案的重要前提。一方面,为了检测到细菌感染,细菌独特的微环境(pH、毒力因子、酶等)给人们带来启示。其中,大多数致病菌通过糖代谢产酸的性质常被用于细菌感染的检测。另一方面,为了追踪耐药性,耐药细菌产生的酶,尤其是β-内酰胺酶,通常用作标志物。由于β-内酰胺抗生素的广泛使用,对它们的耐药性被认为是巨大的威胁。因此,β-内酰胺酶作为识别耐药细菌的标志物具有普遍性和实用性。
为了削弱耐药性,基于活性氧簇(ROS)的策略,包括光动力学疗法和化学动力学疗法,是很好的候选方案。它们主要的优势在于多靶标效应,即与细菌耐药性相关的因素如细菌细胞壁、核酸和蛋白质等,都可以被破坏。值得注意的是,ROS没有特定的靶向性,其滥用可能会对健康组织产生不良影响。因此,合理使用ROS策略来抵抗耐药性是有效的措施。
受到世界卫生组织在全球行动计划中对抵抗抗菌素耐药性的便携、低廉设备的需求的启发,试纸型设备(PBD)是很有前景的抗菌平台。由于环保、生物安全、廉价和易于修饰等优势,PBD广泛应用于便携式生物传感器和灭菌纸。然而,PBD还没有同时实现“检测和治疗”的功能。因此,我们旨在构建基于纸的便携式创可贴(PBA)来检测和对抗细菌的耐药性。
发明内容
本发明的目的是提供一种耐药性可视化的创可贴及其制备方法,该耐药性可视化的创可贴不仅能快速地检测并有效地杀伤大肠杆菌(E.coli)的药敏型(DS)和耐药型(DR)菌株,而且有效地防止伤口感染,加快伤口愈合,还能防止水果的染菌、保证新鲜储存。
本发明提供一种耐药性可视化的创可贴的制备方法,该方法包括:
步骤一:PCN-224纳米粒子合成
将ZrOCl2·8H2O溶液、四(4-羧基苯基)卟啉溶液和苯甲酸溶液在90℃下搅拌5h或120℃下反应24h,得到PCN-224纳米粒子;
步骤二:壳聚糖包裹的PCN-224纳米粒子合成
将步骤一得到的PCN-224纳米粒子溶解于MES缓冲液中,然后加入1-[3-(二甲胺基)丙基]-3-乙基碳二亚胺盐酸盐和N-羟基硫代琥珀酰亚胺钠盐进行活化,再加入壳聚糖搅拌,得到壳聚糖包裹的PCN-224纳米粒子;
步骤三:耐药性可视化的创可贴的制备
将滤纸样品浸入氧化物溶液中,并将溶液温度在60℃维持1-2h,经过水洗干燥消毒后切成小块,将步骤二中壳聚糖包裹的PCN-224纳米粒子浸泡在含有β-内酰胺类抗生素药物和头孢硝噻的缓冲液中,得到载药PCN-224,然后将载药PCN-224和NaCNBH3的HEPES缓冲溶液滴到小块试纸上,将试纸进行培养,再将含有PEG的BTB溶液覆盖在试纸上,得到耐药性可视化的创可贴。
优选的是,所述步骤一的ZrOCl2·8H2O溶液、四(4-羧基苯基)卟啉溶液和苯甲酸溶液的体积比为1:2:2。
优选的是,所述的ZrOCl2·8H2O溶液的浓度为15mg/mL,四(4-羧基苯基)卟啉溶液的浓度为1-3mg/mL,苯甲酸溶液的浓度为59.3-88.9mg/mL。
优选的是,所述的步骤二中PCN-224纳米粒子、1-[3-(二甲胺基)丙基]-3-乙基碳二亚胺盐酸盐N-羟基硫代琥珀酰亚胺钠盐和壳聚糖(mg)的质量比为10:1:1:1。
优选的是,所述的步骤二的活化时间为2-4h,搅拌时间为12-48h。
优选的是,所述的步骤三中氧化物溶液为将氯化锂和高碘酸钠溶解于水中后得到的。
优选的是,所述的步骤三中含有β-内酰胺类抗生素药物为氨苄西林钠。
优选的是,所述的步骤三中载药PCN-224的质量mg:氰基硼氢化钠(NaCNBH3)μmol为1:10;
优选的是,所述的步骤三的培养温度为25-37℃,培养时间为1-3h。
本发明还提供上述制备方法得到的耐药性可视化的创可贴。
本发明的有益效果
本发明提供一种耐药性可视化的创可贴及其制备方法,该方法先合成了一个基于卟啉的金属有机框架材料的PCN-224,它比游离的卟啉分子具有更好的光动力能力,伴随着光的照射,PCN-224产生的ROS很大程度上破坏了细菌且减弱了其耐药性,本发明将上述材料经过壳聚糖包裹后整合到纤维素纸上并制备了耐药性可视化的创可贴,我们通过颜色的变化即可判断是否有耐药型(DR)细菌,对于药敏型(DS)细菌,本发明利用细菌微环境的酸性:一方面,溴百里香酚蓝(BTB)响应感染部位的酸性环境,颜色由绿色(中性)变化为黄色(酸性);另一方面,将负载氨苄西林的纳米材料用壳聚糖包裹,以引诱带负电的细菌并实现酸响应性药物释放,从而杀死DS细菌。对于耐药型(DR)细菌,在耐药细菌分泌的β-内酰胺酶的作用下,头孢硝噻的颜色从黄色变为红色。通过颜色的变化区分不同的治疗方案,实验结果表明:本发明的耐药性可视化的创可贴不仅能够快速检测(4h)细菌的感染并实时监测耐药性的产生,而且能有效地杀伤大肠杆菌(E.coli)的DS和DR菌株,有效地防止伤口感染,加快伤口愈合,还能防止水果的染菌、保证新鲜储存。
附图说明
图1是本发明耐药性可视化创可贴的电镜照片。
图2是本发明耐药性可视化创可贴通过显色法感知细菌感染和细菌的耐药性的照片。
图3为对于DS E.coli和DR E.coli不同治疗方式后对比柱形图;
图4是耐药性可视化创可贴低温保存一段时间后的性能测试图。
图5分别为感染DS E.coli和DR E.coli的小鼠在经过不同处理方式后0-3天的伤口愈合照片;
图6是耐药性可视化创可贴治疗人工伤口后的组织学分析图。
图7为小番茄在感染DS E.coli、DR E.coli和DS E.coli与DR E.coli混合(99:1)后经过不同处理方式在0-3天的照片;
图8为用涂布平板法定量测量小番茄的细菌存活图。
具体实施方式
本发明提供一种耐药性可视化的创可贴的制备方法,该方法包括:
步骤一:PCN-224纳米粒子合成
将ZrOCl2·8H2O溶液、四(4-羧基苯基)卟啉溶液和苯甲酸溶液在90℃下搅拌5h或120℃下反应24h,得到PCN-224纳米粒子;所述的ZrOCl2·8H2O溶液、四(4-羧基苯基)卟啉溶液和苯甲酸溶液的体积比优选为1:2:2,所述的ZrOCl2·8H2O溶液的浓度优选为15mg/mL,四(4-羧基苯基)卟啉溶液的浓度优选为1-3mg/mL,更优选为2.5mg/mL,苯甲酸溶液的浓度优选为59.3-88.9mg/mL,更优选为70mg/mL;
步骤二:壳聚糖包裹的PCN-224纳米粒子合成
将步骤一得到的PCN-224纳米粒子溶解于MES缓冲液中,然后加入1-[3-(二甲胺基)丙基]-3-乙基碳二亚胺盐酸盐和N-羟基硫代琥珀酰亚胺钠盐进行活化,所述的活化温度优选为室温,时间优选为2-4h,再加入壳聚糖搅拌,所述的搅拌时间优选为12-48h,得到壳聚糖包裹的PCN-224纳米粒子;所述的PCN-224纳米粒子、1-[3-(二甲胺基)丙基]-3-乙基碳二亚胺盐酸盐N-羟基硫代琥珀酰亚胺钠盐和壳聚糖的质量比优选为10:1:1:1;
步骤三:耐药性可视化的创可贴的制备
将滤纸样品浸入氧化物溶液中,所述的氧化物溶液将氯化锂和高碘酸钠溶解于水中后得到的,所述的氯化锂和高碘酸钠的质量比优选为1:2.8;
将上述溶液温度在60℃下维持1-2h,优选为1h,经过水洗干燥消毒后切成小块,所述的小块尺寸优选为5mm×5mm,将步骤二中壳聚糖包裹的PCN-224纳米粒子浸泡在含有β-内酰胺类抗生素药物和头孢硝噻的缓冲液中,得到载药PCN-224,所述的缓冲液中,头孢硝噻的浓度优选为0.5-2mmol/L,含有β-内酰胺类抗生素药物的浓度优选为10-25mg/mL,所述的含有β-内酰胺类抗生素药物优选为氨苄西林钠;
然后将载药PCN-224和NaCNBH3的HEPES缓冲溶液滴到小块试纸上,将试纸进行培养,所述的培养温度优选为25-37℃,培养时间优选为1-3h,所述的载药PCN-224的质量mg:氰基硼氢化钠(NaCNBH3)μmol为1:10;再将含有5wt%PEG的BTB溶液(0.5mg/mL)覆盖在试纸上,得到耐药性可视化的创可贴。
下面结合具体实施例对本发明做进一步详细的描述,实施例中涉及到的原料均为商购获得。
实施例1
步骤一:PCN-224纳米粒子合成。
将ZrOCl2·8H2O溶液(10mL,15mg/mL的N,N-二甲基甲酰胺溶液)、四(4-羧基苯基)卟啉溶液(20mL,2.5mg/mL的N,N-二甲基甲酰胺溶液)和苯甲酸溶液(20mL,70mg/mL的N,N-二甲基甲酰胺溶液)加入圆底烧瓶中,在90℃下搅拌5h,离心(13000rpm)后用N,N-二甲基甲酰胺溶液洗涤三次得到PCN-224纳米粒子;
步骤二:壳聚糖包裹的PCN-224纳米粒子合成
将上述100mgPCN-224纳米粒子溶解于5mL的MES缓冲液(25mM,pH 6.0)中,经10mg1-[3-(二甲胺基)丙基]-3-乙基碳二亚胺盐酸盐和10mg N-羟基硫代琥珀酰亚胺钠盐活化2h,然后室温下加入10mg壳聚糖,并连续搅拌20h,离心后水洗三次获得壳聚糖修饰的PCN-224纳米粒子;
步骤三:可视化创可贴的制备
将纤维素纸样品浸入氧化物溶液中(0.03g氯化锂和0.084g高碘酸钠溶解于15mL水),并将溶液温度在60℃下维持60min。随后用去离子水冲洗两次,在37℃的烘箱中干燥,然后对处理过的试纸进行消毒,并将纸切成5mm×5mm的小块,将修饰了壳聚糖的PCN-224浸泡在含有1mmol/L的头孢硝噻和20mg/mL的氨苄西林钠的HEPES缓冲溶液(pH 6.0)中,搅拌24h后,离心并用HEPES缓冲液(pH 7.4)洗涤以除去过量的药物。用移液管将含有5mg载药PCN-224和50μmol氰基硼氢化钠的1mL的HEPES缓冲溶液(50mmol/L,pH=8.0)滴到小块试纸上,在37℃下反应1h,随后进行清洗,最后用含有5wt%PEG的BTB溶液(0.5mg/mL)覆盖在试纸上,即制备得到耐药性可视化的创可贴。
实施例1制备得到的耐药性可视化的创可贴的扫描电镜照片如图1所示,其中A为纤维素纸的照片,B为耐药性可视化的创可贴的照片,从图中可以看出,PCN-224均匀地修饰到纤维素上;图c为耐药性可视化的创可贴用pH 7.4的缓冲液浸润后的照片,从图c可以看出,颜色为绿色,表明无细菌感染。
将实施例1制备得到的耐药性可视化的创可贴进行性能检测,如下:
1、耐药性可视化创可贴检测细菌及耐药性
将琼脂培养基上的药敏型大肠杆菌(DS E.coli,ATCC5922)和耐药型大肠杆菌(DRE.coli,ATCCBAA-2193)转移到Luria–Bertani液体培养基内,并放置在细菌摇床内以转速180rpm在37℃条件下培养12h。将可视化创可贴放置在培养孔的底部,取不同浓度(0~106CFU/mL)的50μL DS E.coli或DR E.coli菌液滴至该可视化创可贴上,反应4h来观察试纸的颜色变化。为了证明该可视化创可贴可以指示酸性环境,我们用缓冲液(pH 8.0~6.0)与创可贴反应。为了证明大肠杆菌内膜分泌的β-内酰胺酶导致红色的显色反应,我们将DR细菌分泌物、DR细菌提取物和β-内酰胺酶与该可视化创可贴反应,如图2所示,绿色表示无明显细菌感染,黄色表示细菌代谢产酸,红色表示存在对β-内酰胺类抗生素的耐药性。表明本发明制备的创可贴可以检测细菌及耐药性。
2、抗菌性能测定
将实施例1制备的创可贴放置在培养孔的底部,用培养液稀释细菌至106CFU/mL。反应4h后,若试纸呈黄色,对于DS E.coli,则继续培养12h后,用平板计数法计算细菌的存活率;若试纸呈红色,对于DR E.coli,则进行光动力学的策略,用638nm激光照射10min(0.2W/cm2),暗处共培养12h后,用平板计数法计算细菌的存活率。
图3为对于DS E.coli和DR E.coli不同治疗方式后对比柱形图,图3说明,对于DSE.coli,仅用本发明的创可贴单独处理,即可实现较高的杀伤率。对于DR E.coli,仅用本发明的创可贴处理,细菌的存活率并没有显著下降,然而加入光照射后,其存活率变得极低。结果表明,仅基于抗生素的化学治疗几乎能根除DS E.coli,然而只有抗生素治疗和光动力治疗的结合可以有效地杀死DR E.coli。此外,与对照组相比,光动力治疗(45.3%)和抗生素(72.4%)对杀伤DR E.coli有显著的协同效应。
4、耐药性可视化创可贴低温保存后性能测定
将实施例1制备的可视化创可贴在4℃下分别密封保存一周、一个月后,取出用于细菌和耐药性的显色反应(如图4A),以及根据颜色,分别进行抗生素治疗(图4B)和光动力治疗(图4C)。图4说明,本发明的可视化创可贴保存一个月后,仍维持有较好地显色反应和杀菌能力。
5、耐药性可视化创可贴对人工伤口的检测和治疗效果
利用脱毛膏对实验小鼠的背部进行脱毛处理。接着,用灭菌、消毒后的手术剪在小鼠背部剪出伤口。将背部有伤口的小鼠分为两大组,分别感染DS E.coli和DR E.coli。每一大组再分为三组,分别给与可视化创可贴(无抗生素)、可视化创可贴、可视化创可贴+光照的处理。将可视化创可贴贴在小鼠的伤口表面,并用纱布包裹,防止掉落,反应4h后,观察试纸颜色,若为黄色,则不进行其他处理;若为红色,则用638nm激光照射10min。最后,在伤口上缠上白色医用胶带以避免脱落。每隔24h,对小鼠的伤口进行观察、拍照,并更换创可贴。
如图5所示,图5分别为感染DS E.coli和DR E.coli的小鼠在经过不同处理方式后0-3天的伤口愈合照片,图5说明,仅用本发明制备的可视化创可贴治疗的伤口在第三天几乎完全愈合,表明抗生素对于治愈DS E.coli引起的伤口感染是有效的。当引入光照时,伤口出现长时间肿胀,表明基于单线态氧的治疗引起了副作用。对于DR E.Coli引发的感染,创可贴变红表明其作为耐药性传感器的可行性。只有在光照和可视化创可贴联合治疗时,伤口才能愈合得较好,表明光动力治疗和化疗结合对伤口愈合更有效。因此,可视化创可贴有望作为创可贴用于检测耐药性并治疗伤口感染。
6、耐药性可视化创可贴治疗伤口的组织学分析
在用可视化创可贴治疗伤口3天后,提取小鼠伤口的皮肤组织。并固定在10%多聚甲醛中,然后将其包埋在石蜡中,切片厚度约4μm,最后用苏木精-伊红(H&E)染色。观察皮肤组织的表皮再生情况。如图6所示,对于DS E.Coli,仅使用本发明制备的创可贴治疗后的小鼠伤口的皮肤组织显示出了完整的表皮。对于DR E.Coli引发的感染,只有在光照和本发明制备的可视化创可贴联合治疗时,伤口处的表皮较完整。
7、耐药性可视化创可贴对染菌水果的保鲜效果
分别固定DS E.coli、DR E.coli和DS E.coli与DR E.coli混合(99:1)菌液浓度为OD600nm=0.5,然后取无菌棉签蘸取菌液,分别涂抹到小番茄的外表皮上,感染面积约为0.25cm2,并保持感染处湿润。将本发明的可视化创可贴和对照组(可视化创可贴中无抗生素)贴于感染处,4h后观察试纸颜色。试纸显红色时,对感染处辅以光照治疗。每24h对感染处进行观察、拍照,并更换创可贴。三天处理后,用生理盐水冲洗感染处表皮,收集洗液,用涂布平板法观察细菌存活情况。
如图7所示,图7为小番茄在感染DS E.coli、DR E.coli和DS E.coli与DR E.coli混合(99:1)后经过不同处理方式在0-3天的照片,其中A组为感染DS E.Coli后经过本发明的耐药性可视化创可贴变化照片,B组为感染DR E.coli后经过本发明的耐药性可视化创可贴+光照治疗的变化照片,C组为感染DS E.coli与DR E.coli混合(99:1)后经过本发明的耐药性可视化创可贴+光照治疗的变化照片,D组为感染DS E.coli与DR E.coli混合(99:1)后经过本发明的耐药性可视化创可贴的变化照片,E组为感染DS E.Coli后经过无抗生素的创可贴变化照片,图8为用涂布平板法定量测量小番茄的细菌存活图,其中图A-E是和图7中的A-E对应,从图7和8可以看出,小番茄分别被DS E.coli、DR E.coli及其混合细菌(99:1)感染,创可贴在感染的第一天分别显示黄色、红色和黄色,进一步证明该创可贴可用于检测细菌的感染。针对创可贴的颜色,分别对小番茄进行化学治疗和光动力学治疗。而对于混合菌种的感染,因未进行光照处理,在第二天创可贴显示了红色,证明该创可贴可用于实时监测耐药性的产生。如果继续不进行光照的处理,小番茄的感染部位会变软和内陷(D组),由涂布平板法定量,仍有大量细菌存活,证明光动力治疗对于耐药菌的杀伤是有必要的。
Claims (10)
1.一种耐药性可视化的创可贴的制备方法,其特征在于,该方法包括:
步骤一:PCN-224纳米粒子合成
将ZrOCl2·8H2O溶液、四(4-羧基苯基)卟啉溶液和苯甲酸溶液在90℃下搅拌5h或120℃下反应24h,得到PCN-224纳米粒子;
步骤二:壳聚糖包裹的PCN-224纳米粒子合成
将步骤一得到的PCN-224纳米粒子溶解于MES缓冲液中,然后加入1-[3-(二甲胺基)丙基]-3-乙基碳二亚胺盐酸盐和N-羟基硫代琥珀酰亚胺钠盐进行活化,再加入壳聚糖搅拌,得到壳聚糖包裹的PCN-224纳米粒子;
步骤三:耐药性可视化的创可贴的制备
将滤纸样品浸入氧化物溶液中,并将溶液温度在60℃维持1-2h,经过水洗干燥消毒后切成小块,将步骤二中壳聚糖包裹的PCN-224纳米粒子浸泡在含有β-内酰胺类抗生素药物和头孢硝噻的缓冲液中,得到载药PCN-224,然后将载药PCN-224和NaCNBH3的HEPES缓冲溶液滴到小块试纸上,将试纸进行培养,再将含有PEG的BTB溶液覆盖在试纸上,得到耐药性可视化的创可贴。
2.根据权利要求1所述的一种耐药性可视化的创可贴的制备方法,其特征在于,所述步骤一的ZrOCl2·8H2O溶液、四(4-羧基苯基)卟啉溶液和苯甲酸溶液的体积比为1:2:2。
3.根据权利要求1所述的一种耐药性可视化的创可贴的制备方法,其特征在于,所述的ZrOCl2·8H2O溶液的浓度为15mg/mL,四(4-羧基苯基)卟啉溶液的浓度为1-3mg/mL,苯甲酸溶液的浓度为59.3-88.9mg/mL。
4.根据权利要求1所述的一种耐药性可视化的创可贴的制备方法,其特征在于,所述的步骤二中PCN-224纳米粒子、1-[3-(二甲胺基)丙基]-3-乙基碳二亚胺盐酸盐N-羟基硫代琥珀酰亚胺钠盐和壳聚糖(mg)的质量比为10:1:1:1。
5.根据权利要求1所述的一种耐药性可视化的创可贴的制备方法,其特征在于,所述的步骤二的活化时间为2-4h,搅拌时间为12-48h。
6.根据权利要求1所述的一种耐药性可视化的创可贴的制备方法,其特征在于,所述的步骤三中氧化物溶液为将氯化锂和高碘酸钠溶解于水中后得到的。
7.根据权利要求1所述的一种耐药性可视化的创可贴的制备方法,其特征在于,所述的步骤三中含有β-内酰胺类抗生素药物为氨苄西林钠。
8.根据权利要求1所述的一种耐药性可视化的创可贴的制备方法,其特征在于,所述的步骤三中载药PCN-224的质量mg:NaCNBH3的μmol为1:10。
9.根据权利要求1所述的一种耐药性可视化的创可贴的制备方法,其特征在于,所述的步骤三的培养温度为25-37℃,培养时间为1-3h。
10.权利要求1所述的制备方法得到的耐药性可视化的创可贴。
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