CN115025044B - 一种抗菌聚鞣酸纳米粒PTA NPs及其制备方法 - Google Patents
一种抗菌聚鞣酸纳米粒PTA NPs及其制备方法 Download PDFInfo
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Abstract
本发明属于纳米材料技术领域,具体涉及一种抗菌聚鞣酸纳米粒(PTA NPs)及其制备方法。在碱性条件下,鞣酸(TA)通过一锅法合成聚鞣酸纳米粒(PTA NPs)。其具有良好的抗菌活性,能够与细菌细胞膜相互作用,导致微生物细胞膜损伤,从而能够有效杀灭革兰氏阳性(金黄色葡萄球菌)和阴性(大肠杆菌)菌株,且对两种菌株均具有良好的生物膜抑制和破坏能力。本发明制备的抗菌聚鞣酸纳米粒表现出较高的水稳定性和优异的生物相容性,在抗菌性能上优于游离鞣酸,是杀灭细菌和促进伤口愈合的一种安全高效的工具。
Description
技术领域
本发明属于纳米材料技术领域,具体涉及一种抗菌聚鞣酸纳米粒PTA NPs及其制备方法。
背景技术
微生物作为人类生存的重要组成部分,其中作为古老微生物的细菌,对人类的日常生活有着极大的影响。大多数细菌引起的疾病可以直接或间接(通过媒介)从一个人传播给另一个人成为传染病,对人类健康构成威胁。抗生素的发现是现代医学最伟大的突破之一。但是,抗生素的广泛应用导致动物微生物群中抗菌素耐药性的增加,后来还产生了耐药基因从动物菌株转移到人类菌株,抗生素处方错误和过度处方一起导致了多种耐药致病菌的发展。
纳米医学是发展最迅速的前沿研究之一,不同于抗生素,不同类型和功能的纳米药物可通过多种机制显著抑制细菌感染。鞣酸(TA)是一种广泛分布于自然界的多酚类分子。据报道,TA对许多致病菌具有活性,包括金黄色葡萄球菌(革兰氏阳性菌株)和大肠杆菌(革兰氏阴性菌株)。因此,利用TA作为治疗细菌感染的活性成分已被许多纳米医学研究提出。但是,游离TA抗菌性能较差,稳定性也较差。
发明内容
为了制备稳定的聚鞣酸纳米粒,本发明采用一锅法合成了PTA NPs,本发明制备的抗菌PTA NPs具有良好的生物安全性,在抑菌、杀菌领域均有广泛的应用前景。
抗菌PTA NPs的制备方法,步骤如下:
(1)将100mg鞣酸溶于1mL超纯水中,待其完全溶解后加入5mL无水乙醇,搅拌10-15min后,逐滴注入200μL NaOH(2.5M),滴加过程可观察到溶液由黄色变为灰白色,反应20-30min;
聚鞣酸纳米粒前体水合粒径1618±42nm,多分散系数0.562。
(2)将不同体积(400-1200μL)H2O2(30%)逐滴注入步骤(1)搅拌的溶液中,反应30min,得到多种不同粒径的聚鞣酸纳米粒。
(3)收集反应液离心(10000-13000rpm,5-10min),沉淀物使用无水乙醇反复洗涤3次除去杂质。最后沉淀重悬于水中,再次离心(5000rpm,5min)去除大颗粒沉淀,获得澄清的黄色溶液即PTA NPs水溶液。
合成的聚鞣酸纳米粒子水合粒径101±5nm,多分散系数0.221。
上述方法制备的抗菌PTA NPs具有良好的抗菌活性,能够与细菌细胞膜相互作用,导致微生物细胞膜损伤。PTA NPs可用于金黄色葡萄球菌体内体外杀菌,抑制和破坏生物膜,以及对皮肤伤口愈合具有积极影响。
本发明具有以下有益效果:
本发明先制备聚鞣酸纳米粒前体,水合粒径为1618±42nm,以此粒径的PTA NPs前体为模板,使用一定体积的H2O2(30%)进行还原,然后离心洗涤形成水合粒径为101±5nm近似球形结构的PTA NPs。该纳米粒不需要严苛的反应条件,能与细菌细胞膜相互作用,破坏细菌细胞膜,从而达到抗菌作用。体内实验证明,PTA NPs对皮肤细菌感染有一定的抑制作用,并且能够促进皮肤伤口愈合。
此外,本发明仅仅采用了100mg鞣酸,在制备过程中几乎全部反应,后续处理简单便捷,制得的PTA NPs稳定性好,生物相容性高。本发明公开的制备工艺简单,原料来源广泛,反应条件温和,易于合成,适于推广使用。
附图说明
图1为PTA NPs粒径分布图;
图2为游离TA与PTA NPs zeta电位变化图;
图3为PTA NPs 7天内粒径变化图;
图4为PTA NPs透射电镜图;
图5为游离TA与PTA NPs的紫外吸收光谱图;
图6为不同浓度PTA NPs的紫外吸收光谱图;
图7为不同浓度PTA NPs的体外细胞毒性结果图;
图8为溶血实验结果图;
图9为PTA NPs对S.aureus存活率的影响图(琼脂板计数法);
图10为PTA NPs对S.aureus存活率的影响图(柱状图);
图11为PTA NPs对S.aureus存活率的影响图(浊度法);
图12为PTA NPs对E.coli存活率的影响图(琼脂板计数法);
图13为PTA NPs对E.coli存活率的影响图(柱状图);
图14为PTA NPs对E.coli存活率的影响图(浊度法);
图15为游离TA与PTA NPs对S.aureus存活率的影响图(浊度法);
图16为游离TA与PTA NPs对E.coli存活率的影响图(浊度法);
图17为S.aureus的live/dead染色图;
图18为E.coli的live/dead染色图;
图19为PTA NPs对S.aureus扫描电镜图;
图20为PTA NPs对E.coli扫描电镜图;
图21为PTA NPs抑制S.aureus生物膜的形成图;
图22为PTA NPs抑制E.coli生物膜的形成图;
图23为PTA NPs破坏已形成的S.aureus生物膜图;
图24为PTA NPs破坏已形成的E.coli生物膜图;
图25为S.aureus感染小鼠的伤口图;
图26为小鼠的伤口动态变化图;
图27为小鼠伤口面积变化百分比柱状图;
图28为治疗期间小鼠体重变化图;
图29为S.aureus感染小鼠伤口组织涂板结果图;
图30为细胞迁移图;
图31为三组小鼠的皮肤切片的H&E染色图;
图32为三组小鼠的皮肤切片的Masson染色图;
图33为三组小鼠的皮肤切片的免疫荧光染色图;
图34为三组小鼠的主要内脏器官切片的H&E染色图。
具体实施方式
以下结合实施例对本发明进行详细阐述,但本发明不局限于这些实施例。
实施例1
1、PTA NPs的合成及纯化
将100mg鞣酸溶于1mL超纯水中,待其完全溶解后加入5mL无水乙醇,搅拌15min后,逐滴注入200μL NaOH(2.5M),滴加过程可观察到溶液由黄色变为灰白色,室温反应20min。此时形成粒径较大的PTA NPs前体。
为了找出制备PTA NPs的最佳配方,分别量取400,600,800,1000,1200μL过氧化氢(30%),快速注入剧烈搅拌的溶液中,室温反应30min后收集反应液,得到聚鞣酸纳米粒。
2.对照实验
未添加无水乙醇,以水(6mL)为溶剂,后续滴加过氧化氢反应获得粒径数据如表2所示。
3、PTA NPs的表征
1)PTA NPs的水合粒径
取聚鞣酸纳米粒1mL用于测水合粒径。样品平行测定三次,同时监测整个过程的变化。测量结果见表1所示。
表1为不同用量H2O2制备的PTA NPs的水合粒径和多分散系数变化情况表。
表1
根据测得的水合粒径结果显示,制备的PTA NPs分散性差,平均水合粒径为1618±42nm,多分散性指数为0.562。随着H2O2(30%)剂量的不断增加,能够获得多种尺寸的PTANPs。前期加入量增加的同时,粒径减小,在600μL处出现转折。当H2O2(30%)的剂量为600μL时,水合粒径稳定在了300nm左右。因此,筛选出H2O2(30%)最佳剂量为600μL,水合粒径大小均匀,除非另有说明,否则将用于以下研究。
表2
以这3种过氧化氢添加量为例,从所得数据上可看出添加过氧化氢并未出现明显的粒径变化,3例数值接近且波动较大极为不稳定。
因所得样品稳定性较差,改进实验过程,在加入H2O2(30%)反应结束后,追加离心洗涤步骤。将反应液离心(13000rpm,5min),沉淀物使用无水乙醇反复洗涤3次除去杂质。最后沉淀重悬于水中,再次离心(5000rpm,5min)去除大颗粒沉淀,获得澄清的黄色溶液即PTANPs水溶液。
2)PTA NPs水溶液水合粒径和Zeta电位的测定实验
最终使用600μL H2O2(30%)合成PTA NPs的水合粒径和Zeta电位图如附图1和2所示。
3)PTA NPs的粒径稳定性
使用PTA NPs水溶液进行为期7天的粒径稳定性测定,每次测定均使用1mL纳米粒水溶液,结果如附图3所示,PTA NPs的水动力粒径基本一致,具有较高的水稳定性。
4)PTA NPs的透射电镜实验
分别取30μL PTA NPs于4mL离心管中,加入1970μL去离子水稀释,水浴超声15min,吸取10μL超声后的样品滴于铜网上,铜网置于室温干燥过夜,制备透射电子显微镜(TEM)样品。
由TEM观察到的结果如附图4所示,PTA NPs为近球形纳米粒子,真实直径约为90nm。
5)PTA NPs的紫外吸收光谱
为了进一步证实PTA NPs的成功制备,测定了游离TA溶液和PTA NPs水溶液的紫外-可见吸收光谱。在比色皿中依次分别加入200μL游离TA溶液,PTA NPs水溶液,用紫外分光光度计在200-800nm处进行扫描,得到游离TA溶液和PTA NPs的紫外吸收光谱,如附图5所示。在相同的浓度下,游离TA的吸收峰位于315nm,而PTA NPs的吸收峰红移至370nm左右。如附图6所示,不同浓度PTA NPs的紫外光谱分析也显示,增加的PTA NPs可以使吸收峰发生红移。结果表明,随着纳米颗粒浓度的增加,其紫外吸光度增大,表明PTA NPs在水溶液中具有良好的分散性能,也证实了PTA NPs的成功制备。
3、PTA NPs的生物相容性实验
1)细胞毒性(MTT)
细胞毒性是决定生物功能材料生物安全性的重要因素之一。选用小鼠成纤维细胞(L929)以及人脐静脉内皮细胞(HUVEC)进行评估。实验需要铺设细胞,在96孔板中以8000cell/孔进行铺设,放置于培养箱中(37℃,CO2)培养过夜,然后使用不同浓度的PTANPs(最终浓度:0、0.05、0.1、0.2、0.4、0.8、1.6mg/mL)处理,每组重复3孔。放置于培养箱中再次孵育24h后,加入MTT试剂(20μL/孔),避光静置4h,使用适量DMSO溶解,最后测定溶液在590nm处吸光度。附图7显示,随着PTA NPs浓度的增加,细胞活性并未降低,在多数浓度下呈现增殖。即使当浓度高达1.6mg/mL时,细胞活性仍旧与对照组相似,说明PTA NPs具有较低的细胞毒性。
2)溶血实验
为进一步检测PTA NPs的生物相容性,进行了溶血实验。需从健康小鼠的眼眶静脉丛采集新鲜血液,获得红细胞。使用预冷PBS清洗沉淀2次,随后将红细胞沉淀稀释至20%。然后取用20μL细胞悬液与1mL样品,以1%曲拉通为阳性对照,PBS为阴性对照以及不同浓度的PTA NPs(最终浓度:0、0.05、0.1、0.2、0.4、0.8、1.6mg/mL)为实验组。静置于37℃培养箱孵育2h,最后离心(2000rpm,10min),测定溶液在590nm处的吸光度。附图8显示,实验组均未表现出较高溶血,即便使用浓度高达1.6mg/mL的PTA NPs与红细胞进行孵育时,其结果依旧低于药典规定5%的溶血率。这些结果无疑进一步证明PTA NPs具有良好的生物相容性。
4、PTA NPs的IC50及IC90值测定
1)PTA NPs对金黄色葡萄球菌(S.aureus)的IC50及IC90值测定
用超纯水配制0,15,30,45,60,75μg/mL的PTA NPs溶液。取培养6h的S.aureus菌液,用PBS稀释80倍备用。接下来取200μL稀释后的菌液加入2mL离心管中,再加入200μL浓度分别是0,15,30,45,60,75μg/mL的PTA NPs溶液,放置于摇床(250rpm,37℃)共孵育30min。培养完成后,将细菌悬液稀释2×104倍,吸取100μL混合溶液于固体琼脂板上孵化18h。最后对固体琼脂上的菌落计数,并重复3次。如附图9所示,PTA NPs对S.aureus具有剂量依赖性的抗菌作用,PTA NPs浓度越高抑制效果越好。与对照相比,实际抗菌浓度为15μg/mL时,S.aureus的存活率仅为50%,即IC50为15μg/mL,实际抗菌浓度为30μg/mL时,仅10%S.aureus存活,即IC90为30μg/mL(附图10)。使用IC50及IC90的PTA NPs溶液进行生长曲线测定。取培养过夜的S.aureus菌液,用PBS稀释100倍备用。接下来取100μL稀释后的菌液加入96孔板中,再加入100μL浓度分别是0,30,60μg/mL的PTA NPs溶液,每组重复加三个孔。共孵育后,每间隔1h用酶标仪检测OD600nm,持续测定12h,通过培养基的浊度判断细菌存活率,吸光度值越大,说明细菌存活率越高。如附图11所示,PTA NPs对S.aureus的存活率有明显的抑制作用,与固体琼脂板菌落计数法测定结果一致。
2)PTA NPs对大肠杆菌(E.coli)的IC50及IC90值测定
用超纯水配制0,150,300,450,600,750μg/mL的PTA NPs溶液。取培养6h的E.coli菌液,用PBS稀释30倍备用。接下来取200μL稀释后的菌液加入2mL离心管中,再加入200μL浓度分别是0,150,300,450,600,750μg/mL的PTA NPs溶液,之后放置于摇床(250rpm,37℃)共孵育30min。培养完成后,将细菌悬液稀释2×104倍,吸取100μL混合溶液于固体琼脂板孵化18h。最后对固体琼脂上的菌落计数,并重复3次。如附图12所示,PTA NPs对E.coli具有剂量依赖性的抗菌作用,PTA NPs浓度越高抑制效果越好。与对照相比,实际抗菌浓度为150μg/mL时,E.coli的存活率仅为50%,即IC50为150μg/mL,实际抗菌浓度为375μg/mL时,仅10%E.coli存活,即IC90为375μg/mL(附图13)。使用IC50及IC90的PTA NPs溶液进行生长曲线测定。取培养过夜的E.coli菌液,用PBS稀释50倍备用。接下来取100μL稀释后的菌液加入96孔板中,再加入100μL浓度分别是0,300,750μg/mL的PTA NPs溶液,每组重复加三个孔。共孵育后,每间隔1h用酶标仪检测OD600nm,持续测定12h,通过培养基的浊度判断细菌存活率,吸光度值越大,说明细菌存活率越高。如附图14所示,PTA NPs对E.coli的存活率有明显的抑制作用,与固体琼脂板菌落计数法测定结果一致。
5、游离TA与PTA NPs抗菌对比实验
使用不同浓度的PTA NPs溶液进行生长曲线测定。取培养6h的S.aureus/E.coli菌液,用PBS稀释80/50倍备用。接下来取100μL稀释后的菌液加入96孔板中,对于S.aureus再加入100μL浓度分别是0,30μg/mL的PTA NPs溶液,对于E.coli则使用浓度为0,150μg/mL的PTA NPs溶液,每组重复加三个孔。共孵育后,每间隔1h用酶标仪检测OD600nm,持续测定12h。对于S.aureus(附图15)和E.coli(附图16),PTA NPs对于细菌的杀伤抑制效果明显优于游离TA,这可能是由于PTA NPs局部浓度增加增强了抗菌效果。
6、PTA NPs处理后细菌的live/dead染色实验
使用live/dead染色试剂盒探究PTA NPs作用于S.aureus/E.coli细菌前后的存活情况。将过夜培养的S.aureus/E.coli细菌稀释8倍,取1mL稀释后菌液于EP管中,5000rpm,4℃,冷冻离心5min,弃上清,加入200μL无菌PBS溶液重悬备用。加入200μL PTA NPs(对于S.aureus终浓度为15,30μg/mL;对于E.coli终浓度为150,375μg/mL),其中对照组加入无菌PBS溶液。共孵育30min后,5000rpm,4℃,冷冻离心5min,弃上清。于沉淀中加入40μL live/dead试剂进行染色,将其涡旋混匀,避光静置20min。吸取10μL样品滴于载玻片上,加盖盖玻片,荧光显微镜观察拍照。
为了探索PTA NPs的抗菌机理,使用双重荧光染料SYTO9/碘化丙啶(PI)对S.aureus和E.coli进行了染色。碘化丙啶(PI)是一种具有低渗透性的荧光染料,因此只有当细胞膜被破坏,完整性受损的情况下,它才能与DNA结合呈现红色荧光。与之不同,SYTO9是一种可渗透的荧光染料进入所有细胞并表达为绿色荧光。当两种染料同时出现时,SYTO9的荧光强度会变弱,因此具有完整膜结构的细菌呈现绿色荧光,膜结构受损的细菌呈现红色荧光。如图显示了用PTA NPs处理后的S.aureus(附图17)和E.coli(附图18)的荧光图像。以PBS组为对照,其中PBS组中无论是S.aureus还是E.coli细菌均呈现出明亮的绿色,仅少量细菌被PI染成红色,说明细菌细胞膜结构完整,保持着较好的细菌活力,但是加入PTANPs后细菌呈现明显的浓度依赖性,随着PTA NPs浓度增加红色荧光点也逐步增加,即细菌活力减弱。在IC90组中,仅可检测到微弱的绿色荧光,表明此时具有完整膜结构的细菌数量减少。推测是PTA NPs可与细胞膜相互作用,破坏细菌细胞膜的完整性,从而杀死细菌。
7、PTA NPs处理后细菌的SEM成像
为探究PTA NPs作用于S.aureus/E.coli细菌前后的形态变化,进行细菌SEM成像实验。取过夜培养的S.aureus/E.coli细菌400μL于EP管中,5000rpm,4℃,冷冻离心5min,弃上清,加入200μL PTA NPs(对于S.aureus终浓度为30μg/mL;对于E.coli终浓度为375μg/mL),其中对照组加入无菌PBS溶液。置于恒温摇床(37℃,250rpm)孵育30min,5000rpm,4℃,冷冻离心5min,弃上清,使用无菌PBS清洗2次。加入200μL 4%多聚甲醛固定细菌,避光静置2h,5000rpm,4℃,冷冻离心5min,使用1mL乙醇溶液(50%,70%,90%,100%)进行梯度脱水。脱水完成后使用无菌PBS离心清洗2次,随后使用无菌PBS稀释,取10μL样品滴加于硅片上,硅片室温干燥后喷金,利用扫描电子显微镜SEM成像。如图显示了用PTA NPs处理后的S.aureus(附图19)和E.coli(附图20)的SEM图像。由对照可看出正常细菌表面光滑,但经过PTA NPs处理后的S.aureus和E.coli菌体出现皱缩变形,细菌细胞壁和细胞膜受到破坏,无疑表明PTA NPs对细菌造成了不可逆的伤害。
8、PTA NPs对生物膜的抑制和破坏实验
1)PTA NPs抑制生物膜的形成实验
在96孔板中加入过夜培养的S.aureus或者E.coli细菌菌液10μL,再加入20μL PTANPs,用TSB或者LB培养基稀释PTA NPs,对S.aureus终浓度约为15,30μg/mL,对E.coli终浓度约为150,375μg/mL,以PBS组作为对照,每组重复3个孔,置于培养箱中孵育48h。孵育完成后缓缓取出孔板,使用注射器贴壁去除上层培养基,随后使用PBS清洗3次,操作过程尽量轻柔。静置风干10min后,加入100μL 1%的结晶紫染色20min,然后将上层结晶紫去除,再次用PBS清洗3遍,静置风干后加入200μL 80%的乙醇溶液,置于摇床振荡2h使得结晶紫完全溶解,用酶标仪测定溶解后溶液在590nm处的吸光度。为评估PTA NPs对生物膜的抑制能力,不同浓度的PTA NPs与生物膜共孵育48h,然后评价生物膜的抑制情况。如附图21所示,在IC90浓度下,PTA NPs对S.aureus生物膜的抑制率高达68%,其对E.coli生物膜的抑制率达到59%(如附图22),这些结果表明PTA NPs在抑制生物膜形成方面极具潜能。
2)PTA NPs消除已生成的生物膜实验
取过夜培养的S.aureus或者E.coli细菌菌液10μL于96孔板中,再加入200μL TSB或者LB培养基,于培养箱中孵育48h,待其形成完整的生物膜,孵育完成后取出,加入20μLPTA NPs溶液,对S.aureus终浓度约为15,30μg/mL,对E.coli终浓度约为150,375μg/mL,以PBS组作为对照,每组平行3个孔,置于摇床孵育1h。孵育结束后去除上层培养基,并用PBS清洗3次,操作过程尽量轻柔。静置风干后加入100μL 1%的结晶紫染色20min,随后将上层结晶紫吸走,再次使用PBS清洗3遍,静置风干后加入200μL 80%的乙醇溶液,置于摇床振荡2h使得结晶紫完全溶解,用酶标仪测OD590nm处的吸光度。为评估PTA NPs对已经形成的生物膜的破坏能力,先培养生成生物膜,随后加入不同浓度的PTA NPs与生物膜共孵育1h,然后评价生物膜的破坏情况。共孵育1h后,PTA NPs对S.aureus(如附图23)和E.coli(如附图24)生成的生物膜均具有破坏作用,在IC90浓度下对S.aureus以及E.coli的破坏率分别达到63%和73%。
9、PTA NPs体内抗菌实验
1)建立小鼠S.aureus细菌伤口感染模型
为探究PTA NPs对小鼠S.aureus细菌感染的皮肤伤口处的抗菌效果,首先建立小鼠S.aureus细菌感染模型。提前一天下午剃除小鼠背毛留用。次日上午实验时,将小鼠背部向上,使用打孔器在背部打出直径约为10mm的圆形全厚伤口。吸取50μL S.aureus菌液(107CFU/mL)滴在小鼠背部伤口位置进行感染,每只滴加两次,待伤口上菌液风干即可,10h后观察伤口感染情况,若未成功感染上S.aureus,则再次复染2次。
2)PTA NPs对S.aureus感染小鼠伤口的治疗
将感染成功的小鼠随机分为3组(PBS组,IC50组,IC90组),每组4只,所有小鼠均分笼饲养,防止互相舔舐伤口。于PBS组感染后的伤口上滴加50μLPBS;IC50组滴加50μL PTANPs(15μg/mL);IC90组组滴加50μL PTA NPs(30μg/mL)。第一天被认为是为感染后的第一天,持续治疗四天,四天后将不再进行治疗。每天于相同时间观察伤口愈合情况,并拍摄背部伤口照片,使用Photoshop绘制不同时间伤口动态变化图,最后用ImageJ定量伤口面积,用以计算每天伤口面积与初始伤口面积的百分比,并记录小鼠体重。
实验过程中伤口面积变化如附图25,26所示,所有组分S.aureus感染的伤口均随着时间的增长逐步愈合伤口逐渐缩小。如附图27所示,第4天IC90组面积已经缩小50%然而PBS组仅仅减小10%,相较于缓慢愈合的对照组,实验组可谓是高效快速的进行着伤口愈合。并且在治疗期间,小鼠的体重无明显波动基本平稳(附图28),无疑表明PTA NPs具有良好的安全性。在第8天处死小鼠,取背部伤口进行涂板,伤口浸泡于2mL的无菌PBS中,并将组织研磨一下,置于摇床6h后,取组织液涂布于固体培养基上。结果如附图29,在PBS组仍旧存在大量S.aureus,而IC90组中无S.aureus,PTA NPs可通过杀灭细菌促进伤口愈合。
3)细胞迁移实验
采用L929进行划痕实验以模拟体外伤口。在6孔板中铺设细胞(5×106cell/孔)置于培养箱(37℃,CO2)中培养过夜,使用划痕枪头进行划痕,然后用无菌PBS清洗3次以去除细胞碎片,操作过程需轻柔,最后加入2mL的PTA NPs(浓度:0,15,30μg/mL)处理。细胞迁移状态在0h,24h,48h时使用共聚焦显微镜拍照记录。每组L929最初(0h)以相同宽度切开,经过培养所有划痕间隙均减少(附图30),表明细胞迁移。与对照相比,PTA NPs存在的情况下细胞表现出更强的迁移能力,可观察到更窄的划痕间隙。无疑这些图像表明,PTA NPs能促进细胞迁移和生长。
4)创面皮肤组织染色
伤口愈合是一个极其复杂的过程,存在多种生物因素影响伤口愈合,例如炎症、细菌感染、胶原蛋白沉积、血管生成等。因此,在第8天处死小鼠并将小鼠背部皮肤组织进行H&E染色、Masson染色和免疫组织荧光染色,以研究PTA NPs促进伤口愈合过程背后的潜在机制。
经S.aureus感染后,伤口在第2天出现炎症,过度炎症是延迟伤口愈合另一重要因素。H&E染色结果如附图31所示,PBS组伤口未愈合完全且可以观察到炎症细胞的浸润,而PTA NPs处理后可明显改善这种情况。其中IC90组伤口已恢复平整,极少观察到炎症细胞浸润,这些结果表明皮肤伤口完全恢复,且伤口愈合较好。
胶原蛋白对于伤口愈合过程举足轻重。附图32显示的是Masson染色结果,胶原纤维被染成蓝绿色。从图中可以看出,对照组完全愈合伤口处胶原纤维偏少,伤口仍在愈合,未完全形成真皮层。PTA NPs治疗过的组分可观察到更多的胶原纤维(蓝绿色染色),结构紧密互相交织,说明伤口愈合良好,IC90组已经形成完整的真皮层。
据报道,细胞因子可间接反应伤口愈合的质量。因此,通过免疫荧光染色分析伤口中典型促炎因子(TNF-α)的分泌来评估纳米粒预防感染的效果。如附图33所示,在PBS组观察到高TNF-α表达,其含量约为IC50组的3倍,IC90组的7倍,表明出严重的炎症反应。除此以外,以血小板内皮细胞粘附因子(CD31)作为评估再生期间血管重建的指标。据结果显示,所有组分均有血管生成,而PTA NPs IC90组显著优于其他组分。总结而言,免疫荧光分析表明纳米粒IC90组能够下调TNF-α的表达和上调CD31的表达,以达到促进伤口愈合的效果。
10、PTA NPs体内安全性评价
1)内脏安全性评价
PTA NPs对小鼠背部感染伤口治疗8天后,处死小鼠,取所有组分小鼠主要内脏(心,肝,脾,肺,肾)进行H&E染色,通过病理分析检验PTA NPs是否对小鼠内脏器官具有毒性。结果如附图34所示,将实验组与对照组主要器官的H&E染色进行比较,所有组分主要器官之间并未观察到明显的组织形态学差异,如上所述,在减少炎症、抑制细菌、促进胶原蛋白沉积、增加血管生成等方面,PTA NPs组比PBS组表现出更好的伤口愈合性能且不会对生物体产生伤害。PTA NPs可以通过协同杀菌有效防止伤口感染,显著促进伤口愈合。所有结果表明,PTA NPs具有良好的生物相容性,强大的细菌抑制作用,有效促进伤口愈合,并可与其他活性成分联合,具有可观的发展前景。
Claims (2)
1.一种抗菌聚鞣酸纳米粒PTA NPs水溶液,其特征在于,所述
抗菌聚鞣酸纳米粒PTA NPs水溶液的制备方法步骤如下:
(1)将鞣酸溶于超纯水中,待其完全溶解后加入无水乙醇,搅拌10-15 min后,逐滴注入2.5 M NaOH,滴加过程可观察到溶液由黄色变为灰白色,反应20 -30 min,得到聚鞣酸纳米粒前体;
制备的聚鞣酸纳米粒前体水合粒径1618±42 nm,多分散系数0.562;
(2)将600 μL 30% H2O2逐滴注入步骤(1)搅拌的溶液中,反应30 min后收集反应液,得到聚鞣酸纳米粒;
聚鞣酸纳米粒子水合粒径294±22 nm,多分散系数0.642;
(3)将反应液离心,沉淀物使用无水乙醇反复洗涤3次除去杂质,最后沉淀重悬于水中,再次离心去除大颗粒沉淀,获得澄清的黄色溶液即PTA NPs水溶液;
将合成的聚鞣酸纳米粒子离心后,得到的聚鞣酸纳米粒水合粒径101±5 nm,多分散系数0.221;
抗菌聚鞣酸纳米粒PTA NPs用于制备金黄色葡萄球菌体内外杀菌,抑制和破坏生物膜的药剂。
2.根据权利要求1所述的抗菌聚鞣酸纳米粒PTA NPs水溶液,其特征在于,步骤(1)所述鞣酸和2.5 M NaOH 的质量体积比为1:2 mg/μL。
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