CN112778399A - 一类源自毒性淀粉样纤维纳米抗菌肽的制备及性质表征方法 - Google Patents
一类源自毒性淀粉样纤维纳米抗菌肽的制备及性质表征方法 Download PDFInfo
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- CN112778399A CN112778399A CN202110079562.XA CN202110079562A CN112778399A CN 112778399 A CN112778399 A CN 112778399A CN 202110079562 A CN202110079562 A CN 202110079562A CN 112778399 A CN112778399 A CN 112778399A
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Abstract
一类源自毒性淀粉样纤维纳米抗菌肽的制备及性质表征方法,共包含三个多肽序列,是基于金黄色葡萄球菌分泌强毒性PSMα3的序列通过理性分子设计得到的,分别是直接从PSMα3截短得到的十一肽、将序列中第九、十二位的赖氨酸残基用精氨酸替换和将第九位、十一、十二位的赖氨酸和苯丙氨酸分别用精氨酸、色氨酸替换。形貌表征表明截短肽以及突变体分别形成纳米管、扭曲的纳米带结构。细菌实验表明这两种突变体对细菌具有显著抗菌活性,组装多肽可导致细菌膜的破坏。本发明的优点是:三种抗菌肽具有很低的临界胶束浓度,能够在较低的浓度下组装成纳米结构并作用于细菌的细胞膜造成细菌的死亡,同时三种多肽在较高浓度下仍保持较低的细胞毒性。
Description
技术领域
本发明涉及超分子纳米药物技术领域,具体涉及三个具有抗菌效果的多肽纳米材料的制备方法。
技术背景
由于多重耐药菌对人类健康的严重威胁,开发新型抗菌药物势在必行。在新开发的传统抗生素的抗菌替代品中,源于宿主防御肽的抗菌肽是通过调节先天免疫系统来防御病原体的天然生物活性肽,已被证明在开发新一代抗生素方面具有巨大的潜力。不同于常规抗生素的蛋白靶向机制,抗菌肽原则上是通过膜扰动机制杀灭细菌,这导致其具有广谱抗菌活性且不易产生耐药性。迄今为止,许多来自天然蛋白或人工设计的肽主要集中于强化抗菌活性、减弱细胞毒性以及优化抗菌肽的药理作用。一方面,位点突变、多肽库筛选或计算机模拟等方法已被用于发现具有良好生物相容性的抗菌肽。另一方面,一些化学方法包括引入非天然氨基酸,多肽的脂化或环化,以及使用递送载体被应用于解决药物动力学问题。尽管在过去的20年里取得了进展,开发既具有抗菌活性又能延长半衰期的天然肽仍然具有挑战性。
将多肽自组装成明确的纳米结构已被证明是一种简便的生物材料开发策略,在组织工程和疾病诊断和治疗方面具有巨大的潜力。组装的肽对蛋白酶降解具有抗性,延长了肽治疗的半衰期,这意味着组装的抗菌肽可以优化其药物动力学。事实上,许多抗菌肽是由天然产生的肽(包括毒素肽和宿主防御肽)发展而来的,其中大多数抗菌肽的组装性能尚不明确或研究较少。然而,强毒性淀粉样蛋白表现出严重的细胞毒性和很强的组装倾向,这表明来自强毒性淀粉样蛋白的抗菌肽的开发有可能同时解决抗菌活性和药物动力学问题。淀粉样蛋白是由蛋白质聚集形成的纤维状纳米结构,有助于许多神经退行性疾病的发病机制,包括阿尔茨海默病和帕金森病。与致病性淀粉样蛋白破坏基本蛋白功能不同,在金黄色葡萄球菌中发现的毒性淀粉样蛋白,即酚可溶性调控蛋白α3(PSMα3),是造成侵袭性细菌感染的原因。在PSM蛋白质家族,二十二肽PSMα3具有较强的细胞毒性和裂解细胞膜的能力,形式具有α-螺旋构象的细长纤维。
发明内容
本发明目的是克服现有抗菌药物存在的上述缺陷,提供三个具有抗菌效果的多肽纳米材料的制备方法,在具有良好生物相容性的前提下,能够对革兰氏阳性和革兰氏阴性细菌具有显著的抗菌活性,它们以高于其临界聚集浓度的浓度破坏细菌膜,从而形成两种组装的纳米抗菌药物。该制备方法简单,反应条件温和,操作简便。
为实现上述目的,本发明提供的技术方案为:
一类源自毒性淀粉样纤维纳米抗菌肽的制备方法:
抗菌肽UP、UP-RR、UP-RWR的制备
通过标准Fmoc固相多肽合成(SPPS)方法,采用2-氯三酰氯树脂合成了三个不同序列的十一肽,分别为UP(EFVAKLFKFFK)、UP-RR(EFVAKLFRFFR)和UP-RWR(EFVAKLFRWFR),并通过高效液相色谱法纯化。纯化后的多肽溶液通过冷冻干燥机冻干成为多肽粉末,并使用质谱和超高效液相色谱对UP、UP-RR和UP-RWR进行了纯度表征。将冻干十一肽粉末使用超纯水溶解后,用氨水调节其pH值为7.4,最终十一肽溶液浓度为2mM。将配置好的多肽溶液进行退火处理,即加热到80℃并保持30分钟,然后自然冷却到室温。退火后的溶液用于后续实验中的构象和形貌表征以及生物实验。在研究多肽自组装之前,通过记录尼罗红的最大发射波长来估算UP、UP-RR、UP-RWR的临界聚集浓度(CAC)。研究表明,多肽UP、UP-RR、UP-RWR聚集浓度分别为6.89、4.89和7.32μM。抗菌肽UP、UP-RR、UP-RWR的二级结构通过圆二色(CD)光谱进行了表征。抗菌肽UP的CD光谱分别在190和203nm处显示最小或最大强度。抗菌肽UP-RR和UP-RWR的CD光谱均在192和207nm处显示最小或最大强度。这些信号表明抗菌肽UP、UP-RR、UP-RWR形成了β-折叠构象。此外,通过傅里叶变换红外光谱进一步研究十一肽的构象。抗菌肽UP的FTIR光谱分别在1624和1692cm-1处显示高强度信号和低强度信号。同时,肽UP-RR和UP-RWR均在1626和1693cm-1处显示两个信号,这些结果证实了三个抗菌肽均形成的反平行β-折叠二级结构。进一步进行了硫黄素T(Th-T)分析,实验结果显示将肽UP,UP-RR和UP-RWR添加到Th-T溶液中导致染料Th-T的荧光强度增加,分别是单独的Th-T的2.01、1.23和1.72倍,因此证实了肽形成β-折叠。随后,进行了广角X射线散射研究,以深入了解由三种十一肽形成的聚集体的内部结构特征。这三种肽均显示出强的WAXS信号,表明它们组织成有序的纳米结构。所有三种肽的WAXS谱图均显示在约处有很强的布拉格反射,这与传统β-折叠的沿生长轴方向单体单元的周期间隔一致。这直接证实了三个抗菌肽形成β-折叠。对于UP,UP-RR和UP-RWR三个肽,在7.6、8.2和处检测到另一个布拉格反射带。这潜在地对应于垂直于β-折叠的生长轴的肽单体之间的距离。利用原子力显微镜(AFM)和透射电子显微镜(TEM)进一步表征了三种抗菌肽形成组装体的形貌。结合AFM和TEM图像可知,抗菌肽UP形成外径为10nm,壁厚为3.8nm左右的纳米管结构。UP-RR和UP-RWR均形成左旋扭曲的纳米带形貌,螺距分别为70和100nm。对于UP-RR形成的扭曲纳米带的最小和最大高度估计分别为4.9和8.8nm,对于肽UP-RWR形成的扭曲纳米带的最小和最大高度分别为9.3和13.6nm。由肽UP-RR和UP-RWR形成的扭曲纳米带的最小高度大致接近单分子肽伸直的长度或两倍长度,表明UP-RR和UP-RWR分别形成的单体和双层组装结构。
本发明的优点和有益效果:
(1)本发明所形成的多肽组装体具有较好的生物相容性、较低的临界聚集浓度、易于组装等优点。(2)本发明所有的反应条件非常温和,制备方法简单,操作简便。(3)本发明得到的抗菌肽UP能够对解淀粉芽孢杆菌和变形链球菌有抑菌效果,抗菌肽UP-RR和UP-RWR能够对大肠杆菌、金黄色葡萄球菌、解淀粉芽孢杆菌和变形链球菌有抑菌效果。
附图说明
图1.抗菌肽UP、UP-RR、UP-RWR的化学结构式。
图2.抗菌肽UP、UP-RR、UP-RWR的结构模型,以及它们在伸直状态下的长度。
图3.抗菌肽UP(A)、UP-RR(B)、UP-RWR(C)中的尼罗红的最大荧光发射波长(λmax)与多肽浓度之间的函数曲线图(0.1至100μM)。
图4.抗菌肽UP、UP-RR、UP-RWR在pH 7.4下的CD光谱(A),傅里叶变换红外(FTIR)光谱(B),抗菌肽/ThT溶液的荧光光谱图(C),抗菌肽UP(D)、UP-RR(E)和UP-RWR(F)的广角x射线散射图。
图5.抗菌肽UP(A)、UP-RR(B)、UP-RWR(C)在pH 7.4下的形成的一维纳米结构的CLSM图像。
图6.抗菌肽UP(A)、UP-RR(B)、UP-RWR(C)的AFM图像,抗菌肽UP(E)、UP-RR(F)、UP-RWR(G)的TEM图像,抗菌肽UP(H)、UP-RR(I)、UP-RWR(G)通过反平行β-折叠组装示意图。
图7.与抗菌肽UP、UP-RR、UP-RWR共孵育后的293T细胞(A)和3T3细胞(B)的细胞活力,与抗菌肽共孵育后的大肠杆菌(C)、金黄色葡萄球菌(D)、解淀粉芽孢杆菌(E)和变形链球菌(F)的生长抑制曲线,与PBS、UP-RR、UP-RWR共孵育后大肠杆菌(G)、金黄色葡萄球菌(H)的AO/EB染色CLSM图像
图8.经过PBS(A)、UP-RR(B)、UP-RWR(C)处理后的大肠杆菌SEM图像以及经过PBS(D)、UP-RR(E)、UP-RWR(F)处理后的金黄色葡萄球菌SEM图像
具体实施方式
下面将通过实例描述,阐述本发明的优点和效果。
抗菌肽UP、UP-RR、UP-RWR的制备及表征:
通过标准Fmoc固相多肽合成(SPPS)方法,采用2-氯三酰氯树脂合成了三个不同序列的十一肽,分别为UP(EFVAKLFKFFK)、UP-RR(EFVAKLFRFFR)和UP-RWR(EFVAKLFRWFR),并通过高效液相色谱法纯化。所得抗菌肽UP、UP-RR、UP-RWR的化学结构式见附图1;图2示出了抗菌肽UP、UP-RR、UP-RWR的结构模型,以及它们在伸直状态下的长度,其中UP长度为4.5nm、UP-RR和UP-RWR长度为4.66nm。纯化后的多肽溶液通过冷冻干燥机冻干成为多肽粉末,并使用质谱和超高效液相色谱对UP、UP-RR和UP-RWR进行了纯度表征。将冻干十一肽粉末使用超纯水溶解后,用氨水调节其pH值为7.4,最终十一肽溶液浓度为2mM。将配置好的多肽溶液进行退火处理,即加热到80℃并保持30分钟,然后自然冷却到室温。退火后的溶液用于后续实验中的构象和形态表征以及生物实验。在研究多肽自组装之前,通过记录尼罗红的最大发射波长来估算UP、UP-RR、UP-RWR的临界聚集浓度(CAC)。根据尼罗红发射波长因微环境疏水性的变化而改变的原理,通过记录尼罗红的最大发射波长来估算十一肽的临界聚集浓度。将退火后的2mM十一肽溶液(UP、UP-RR、UP-RWR)用磷酸缓冲溶液稀释(PBS,10mM,pH=7.4)得到一系列浓度范围从0.1μM到100μM的稀溶液。随后,将尼罗红乙醇溶液(2μL,100μM)加入的多肽稀溶液中,避光静置12小时。然后,在使用荧光分光光度计(Agilent Cary Eclipse)测量所有含有多肽和尼罗红的样品的荧光光谱。荧光光谱记录的激发波长为550nm,范围为600-700nm。将尼罗红的最大荧光发射波长绘制为多肽浓度的函数,以估算CAC值。研究表明,多肽UP、UP-RR、UP-RWR聚集浓度分别为6.89、4.89和7.32μM(图3)。抗菌肽UP、UP-RR、UP-RWR的二级结构通过圆二色(CD)光谱进行了表征。十一肽溶液(UP、UP-RR、UP-RWR)的CD光谱通过分光光度计记录(JASCO-715)。将浓度为2mM的多肽溶液用超纯水稀释至浓度为1mM后,用移液枪吸取40μL溶液并转移到两片0.1mm的石英载玻夹片中间进行扫描。波长扫描范围为190nm至250nm,间隔为1.0nm,狭缝宽度为2nm。测量前样品前,去除超纯水溶液介质的背景信号。抗菌肽UP的CD光谱分别在190和203nm处显示最小或最大强度。抗菌肽UP-RR和UP-RWR的CD光谱均在192和207nm处显示最小或最大强度(图4A)。这些信号表明抗菌肽UP、UP-RR、UP-RWR形成了β-折叠构象。此外,通过傅里叶变换红外光谱进一步研究十一肽的构象。在室温下,将浓度为2mM的多肽溶液滴加在石英检测器上,在4000和400cm-1的波数范围内采集红外光谱信号。抗菌肽UP的FTIR光谱分别在1624和1692cm-1处显示高强度信号和低强度信号。同时,肽UP-RR和UP-RWR均在1626和1693cm-1处显示两个信号,这些结果证实了三个抗菌肽均形成的反平行β-折叠二级结构(图4B)。进一步进行了硫黄素T(Th-T)分析,将ThT(20μM)加入到十一肽溶液(UP、UP-RR、UP-RWR)中放置12h,然后用于测量。在测量期间,使用光程为1cm的石英池,设置激发波长为421nm,激发和发射光源的狭缝宽度为20nm,记录以450至600nm范围内的荧光光谱。实验结果显示将肽UP,UP-RR和UP-RWR添加到Th-T溶液中导致染料Th-T的荧光强度增加,分别是单独的Th-T的2.01、1.23和1.72倍,因此证实了肽形成β-折叠(图4C)。通过荧光共聚焦显微镜观察与Th-T共孵育24h后的抗菌肽UP、UP-RR、UP-RWR,记录十一肽(UP、UP-RR、UP-RWR)的CLSM图像。将ThT(20μM)加入到十一肽溶液中并避光放置24h,然后用移液枪吸取40μL溶液滴加在载玻片上,用载玻片覆盖后,用于测量。可知三个抗菌肽都形成细长的纳米结构(图5)。随后,进行了广角X射线散射研究,以深入了解由三种十一肽形成的聚集体的内部结构特征。使用广角X射线散射结构分析仪(Xenocs,France)进行十一肽(UP、UP-RR、UP-RWR)的广角X射线散射测试。将退火后的溶液十一肽溶液进行冷冻干燥处理,将得到的十一肽样品的粉末用于测试。这三种肽均显示出强的WAXS信号,表明它们组织成有序的纳米结构。所有三种肽的WAXS谱图均显示在约处有很强的布拉格反射,这与传统β-折叠的沿生长轴方向单体单元的周期间隔一致,这直接证实了三个抗菌肽形成β-折叠(图4D-F)。对于UP,UP-RR和UP-RWR三个肽,在7.6、8.2和处检测到另一个布拉格反射带。这潜在地对应于垂直于β-折叠的生长轴的肽单体之间的距离。利用原子力显微镜(AFM)和透射电子显微镜(TEM)进一步表征了三种抗菌肽形成组装体的形貌(图6A-F)。在敲击模式下,使用Bruker ICON仪器记录UP、UP-RR、UP-RWR的AFM图像。首先,将浓度为2mM的多肽溶液用超纯水稀释至浓度为100μM后,用移液枪吸取10μL溶液滴加在云母片表面,静置5分钟。残留的液体用滤纸吸干,在自然晾干后用于测试。十一肽UP、UP-RR、UP-RWR的TEM图像是从Philips Tecnai G220 S-TWIN显微镜测试得到的。首先,将浓度为2mM的多肽溶液用超纯水稀释至浓度为100μM后,用移液枪吸取10μL溶液滴加在到碳涂层铜网格的表面,静置5分钟。残留的液体用滤纸吸干,随后将10μL的2wt%乙酸双氧铀滴加在铜网表面,静置3分钟后用滤纸将其除去。结合AFM和TEM图像可知,抗菌肽UP形外径为10nm,壁厚为3.8nm左右的纳米管结构。UP-RR和UP-RWR均形成左旋扭曲的纳米带形貌,螺距分别为70和100nm。对于UP-RR形成的扭曲纳米带的最小和最大高度估计分别为4.9和8.8nm,对于肽UP-RWR形成的扭曲纳米带的最小和最大高度分别为9.3和13.6nm。由肽UP-RR和UP-RWR形成的扭曲纳米带的最小高度大致接近单分子肽伸直的长度或两倍长度,表明UP-RR和UP-RWR分别形成的单体和双层组装结构(图6G-I)。
体外细胞毒性分析
通过使用MTT比色法评估了十一肽(UP、UP-RR、UP-RWR)的细胞毒性。选择了正常细胞人肾上皮293T细胞和小鼠成纤维细胞3T3细胞两种类型的细胞,以考察三个十一肽对不同正常细胞的毒性。首先使用DMEM培养基(10%胎牛血清(FBS),1%青霉素-链霉素(PS))稀释复苏后的细胞,然后加入细胞溶液至96孔细胞培养板中(每孔100μL,每孔6000个细胞),置于细胞培养箱(37℃,5%CO2)中培养24小时。用DMEM培养基稀释三种多肽溶液至浓度为128、64、32、16、8、4、2、1μM。用移液枪移除96孔板中原始培养基溶液,然后将一系列不同浓度多肽溶液(每孔100μL)添加到96孔细胞培养板中,并在培养箱中培养24小时。随后,向细胞中加入MTT溶液(每孔10μL,5mg/mL),孵育4小时后除去培养基并添加DMSO(每孔100μL)显色。最后通过酶标仪(Thermo scientific,USA)测量492nm波长处的吸光度来评估细胞的活力。每组实验至少平行重复3次,以保证实验结果的准确性。实验结果表明UP、UP-RR和UP-RWR在1到128μM浓度范围内作用于正常细胞只有少量的细胞生存能力下降,表明三个抗菌肽对正常细胞具有良好的生物相容性(图7A,B)。
S3:
细菌生长抑制试验
在96孔板中采用梯度稀释法测定三种十一肽抗菌材料的细菌生长抑制曲线。一种革兰氏阴性菌(大肠杆菌(ATCC 8739))和三种革兰氏阳性菌(金黄色葡萄球菌(ATCC6538)、解淀粉芽孢杆菌(ATCC 23842)、变形链球菌(CGMCC1.2499))作为测试菌种。将培养到对数生长期的菌液用LB培养基进行稀释,通过紫外分光光度计测试得到OD600值为1.0的细菌悬浮液,然后继续用LB培养基稀释1000倍。取50μL配好的菌液加入96孔板的各个孔中,然后用LB培养基稀释三种多肽溶液至浓度为512、256、128、64、32、16、8、4、2μM。将50μL配好的稀溶液依次加入96孔板中与菌液混合,放置在37℃恒温细菌培养箱中过夜培养。通过酶标仪测试得到96孔板每个孔在OD600的吸光度,以不加多肽溶液的菌液为对照组,每组实验至少平行重复3次,以保证实验结果的准确性。
通过将不同浓度的肽分别与大肠杆菌、金黄色葡萄球菌、解淀粉芽孢杆菌和变形链球菌共孵育,评估了这3个肽的抗菌活性(图7C-F和表1)。UP对解淀粉芽孢杆菌和变形链球菌的最小抑菌浓度分别为为128μM,64μΜ。UP-RR对大肠杆菌、金黄色葡萄球菌、解淀粉芽孢杆菌和变形链球菌的最小抑菌浓度分别为为32,32,16和32μΜ。UP-RWR对四种菌的最小抑菌浓度分别为为64,64,32,32μΜ。
表1
S4:
死/活细菌荧光染色
将处于快速增殖期的大肠杆菌(ATCC 8739)、金黄色葡萄球菌(ATCC 6538)悬浮液离心(5000rpm,5min),用无菌PBS(10mM,pH=7.4)重新分散,并将细菌浓度调至OD600值为1.0。取1mL上述细菌液悬浮液离心(5000rpm,5min)后与浓度为128μM的UP-RR、UP-RWR多肽溶液共孵育,空白组与灭菌PBS共孵育,放置在37℃恒温细菌培养箱中培养4h。用AO和EB染料(10μL,1.0mg/mL)避光染色15min,用无菌PBS洗涤三次,染色后用激光共聚焦显微镜观察细菌形态(图7G,H)。吖啶橙(AO)是一种渗透细菌膜的染料,能够与细菌中的dsDNA结合,从而产生绿色荧光信号,而溴化乙锭(EB)只能插入受损细胞或死亡细菌的核酸,并显示红色荧光信号。因此,在此实验中使用这两种染料来揭示细菌的存活状态。与PBS共孵育的空白组显示出的较强绿色荧光信号,与UP-RR、UP-RWR共孵育的实验组具有显著的红色荧光信号。这些结果表明,UP-RR和UPRWR可诱导大肠杆菌和金黄色葡萄球菌死亡,从而进一步说明抗菌肽的抗菌活性是通过使细菌死亡,而不是通过抑制其生长。
S5:
细菌扫描电子显微镜(SEM)实验
取1.0mL大肠杆菌、金黄色葡萄球菌悬浮液(OD600=1.0)用无菌PBS缓冲液(10mM,pH=7.4)洗涤两遍,并用128μM的UP-RR、UP-RWR多肽溶液重新分散细菌,放置在37℃恒温细菌培养箱中培养4h。将PBS作为阴性对照组。离心(5000rpm,5min)并用无菌PBS洗涤三遍,以除去多余的材料。将处理后的细菌重新分散在1mL无菌PBS中,取适量均匀涂布于硅片上,用戊二醛溶液(2.5%,v/v)固定4h。去除多余的戊二醛溶液,用无菌PBS洗涤三遍。将细菌样品用一系列的乙醇水溶液(30%、50%、70%、90%、95%和100%,v/v)依次脱水10min,随后在室温下干燥过夜。将样品固定并喷金后通过扫描电子显微镜(SEM,FEI Quanta 250 FEG)观察细菌形态。
通过对与抗菌肽UP-RR和UP-RWR共孵育后的大肠杆菌和金黄色葡萄球菌进行扫描电子显微镜(SEM)实验来研究抗菌肽的抗菌机制(图8)。通过PBS缓冲液处理的大肠杆菌和金黄色葡萄球菌分别表现出具有光滑的表面的棒状和球形。相比之下,用多肽UP-RR和UP-RWR处理的细菌SEM图像显示出明显的塌陷和融合的形态,这表明细菌膜被破坏,从而证实了细菌死亡的膜靶向机制。多肽UP-RR和UP-RWR抗菌活性的增强表明,用精氨酸取代赖氨酸会增强肽和细菌膜之间的结合,这是由于精氨酸的胍基部分和膜磷脂上的磷酸基团之间可能存在相互作用导致的。
Claims (6)
1.一类源自毒性淀粉样纤维纳米抗菌肽的制备方法,其特征是包括如下步骤:
通过标准Fmoc固相多肽合成(SPPS)方法,采用2-氯三酰氯树脂合成了三个不同序列的十一肽,分别为UP(EFVAKLFKFFK)、UP-RR(EFVAKLFRFFR)和UP-RWR(EFVAKLFRWFR),并通过高效液相色谱法纯化,纯化后的多肽溶液通过冷冻干燥机处理成为多肽粉末,并使用质谱和超高效液相色谱对UP、UP-RR和UP-RWR进行了纯度表征;将十一肽粉末使用超纯水溶解后,用氨水调节其pH值为7.4,最终十一肽溶液浓度为2mM;将配置好的多肽溶液进行退火处理,即加热到80℃并保持30分钟,然后自然冷却到室温。
2.一种权利要求1所述的源自毒性淀粉样纤维纳米抗菌肽的性质表征方法,其特征是包括:
临界聚集浓度(CAC)
多肽UP、UP-RR、UP-RWR聚集浓度分别为6.89、4.89和7.32μM;
圆二色性(CD)光谱
抗菌肽UP的CD光谱分别在190和203nm处显示最小或最大强度,抗菌肽UP-RR和UP-RWR的CD光谱均在192和207nm处显示最小或最大强度;
傅里叶变换红外(FTIR)光谱
抗菌肽UP、UP-RR、UP-RWR形成了β-折叠构象;
硫黄素T(Th-T)结合测定
将肽UP,UP-RR和UP-RWR添加到Th-T溶液中导致染料Th-T的荧光强度增加,分别是单独的Th-T的2.01、1.23和1.72倍,证实了肽形成β-折叠;
激光共聚焦扫描显微镜(CLSM)和广角X射线散射实验(WAXS)
抗菌肽UP、UP-RR、UP-RWR都形成细长的纳米结构,组织成有序的纳米结构;
原子力显微镜(AFM)和透射电子显微镜(TEM)
抗菌肽UP形外径为10nm,壁厚为3.8nm左右的纳米管结构;UP-RR和UP-RWR均形成左旋扭曲的纳米带形貌,螺距分别为70和100nm;其中UP-RR形成的扭曲纳米带的最小和最大高度估计分别为4.9和8.8nm,肽UP-RWR形成的扭曲纳米带的最小和最大高度分别为9.3和13.6nm;UP-RR和UP-RWR分别形成的单体和双层组装结构。
3.一种权利要求1所述的源自毒性淀粉样纤维纳米抗菌肽的性质表征方法,其特征是:
体外细胞毒性分析
通过使用MTT比色法评估了十一肽UP、UP-RR、UP-RWR的细胞毒性,选择了正常细胞人肾上皮293T细胞和小鼠成纤维细胞3T3细胞两种类型的细胞,首先使用10%胎牛血清(FBS)和1%青霉素-链霉素(PS)的DMEM培养基稀释复苏后的细胞,然后加入细胞溶液至每孔100μL、每孔6000个细胞的96孔细胞培养板中,置于37℃、5%CO2的细胞培养箱中培养24小时;用DMEM培养基稀释三种多肽溶液至浓度为128、64、32、16、8、4、2、1μM;用移液枪移除96孔板中原始培养基溶液,然后将一系列不同浓度多肽溶液每孔100μL添加到96孔细胞培养板中,并在培养箱中培养24小时;随后,向细胞中加入每孔10μL,5mg/mL的MTT溶液,孵育4小时后除去培养基并添加每孔100μL的DMSO显色;最后通过酶标仪测量492nm波长处的吸光度来评估细胞的活力,每组实验至少平行重复3次。
4.一种权利要求1所述的源自毒性淀粉样纤维纳米抗菌肽的性质表征方法,其特征是:
细菌生长抑制试验
在96孔板中采用梯度稀释法测定三种十一肽抗菌材料的细菌生长抑制曲线,一种革兰氏阴性菌:大肠杆菌(ATCC8739)和三种革兰氏阳性菌:金黄色葡萄球菌(ATCC6538)、解淀粉芽孢杆菌(ATCC23842)、变形链球菌(CGMCC1.2499)作为测试菌种,将培养到对数生长期的菌液用LB培养基进行稀释,通过紫外分光光度计测试得到OD600值为1.0的细菌悬浮液,然后继续用LB培养基稀释1000倍;取50μL配好的菌液加入96孔板的各个孔中,然后用LB培养基稀释三种多肽溶液至浓度为512、256、128、64、32、16、8、4、2μM;将50μL配好的稀溶液依次加入96孔板中与菌液混合,放置在37℃恒温细菌培养箱中过夜培养;通过酶标仪测试得到96孔板每个孔在OD600的吸光度,以不加多肽溶液的菌液为对照组,每组实验至少平行重复3次。
5.一种权利要求1所述的源自毒性淀粉样纤维纳米抗菌肽的性质表征方法,其特征是:
死/活细菌荧光染色
将处于快速增殖期的大肠杆菌(ATCC8739)、金黄色葡萄球菌(ATCC6538)5000rpm悬浮液离心5min,用10mM,pH=7.4的无菌PBS重新分散,并将细菌浓度调至OD600值为1.0;取1mL上述细菌液5000rpm,悬浮液离心5min后用浓度为128μM的UP-RR、UP-RWR多肽溶液重新分散细菌,放置在37℃恒温细菌培养箱中培养4h;用AO和10μL,1.0mg/mL的EB染料避光染色15min,用无菌PBS洗涤三次,最后用CLSM观察细菌形态;PBS处理的细菌作为对照组。
6.一种权利要求1所述的源自毒性淀粉样纤维纳米抗菌肽的性质表征方法,其特征是:
细菌扫描电子显微镜(SEM)实验
取1.0mL大肠杆菌、OD600=1.0金黄色葡萄球菌悬浮液用10mM、pH=7.4的无菌PBS缓冲液洗涤两遍,并用128μM的UP-RR、UP-RWR多肽溶液重新分散细菌,放置在37℃恒温细菌培养箱中培养4h;将PBS作为阴性对照组;5000rpm离心5min并用无菌PBS洗涤三遍,以除去多余的材料;将处理后的细菌重新分散在1mL无菌PBS中,取适量均匀涂布于硅片上,用体积比2.5%的戊二醛溶液固定4h;去除多余的戊二醛溶液,用无菌PBS洗涤三遍;将细菌样品用一系列的乙醇水溶液,体积比30%、50%、70%、90%、95%和100%,依次脱水10min,随后在室温下干燥过夜;将样品固定并喷金后通过扫描电子显微镜观察细菌形态。
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