CN114133420B - 兼具黏附细菌和光动力杀伤双功能aie光敏剂及用途 - Google Patents
兼具黏附细菌和光动力杀伤双功能aie光敏剂及用途 Download PDFInfo
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Abstract
本发明公开了一种兼具黏附细菌和光动力杀伤双功能AIE光敏剂及用途。在AIE光敏剂基团引入糖基，在水溶液中基于自组装形成表面含多糖基的AIE光敏剂聚集体，兼具黏附细菌和光动力杀伤双功能。所发明的多糖基AIE聚集体能通过其表面糖基与细菌表面凝集素结合实现对细菌的黏附并促进其聚集。在光照下，所发明AIE光敏剂能高效产生活性氧。基于黏附作用和光动力的协同，实现对病原菌高效地杀伤。
Description
技术领域
本发明属于生物应用材料领域，具体涉及一种兼具黏附细菌和光动力杀伤双功能AIE光敏剂及用途。
背景技术
致病菌无处不在，严重威胁人类生命安全和健康。尤其近年来，细菌耐药性的加剧，甚至出现多重耐药的“超级细菌”，进一步加大了解决医疗卫生难题的挑战。因此，迫切需要开发一种高效且不易产生细菌耐药性的新型抗菌策略。
光动力疗法(PDT)因其具有高时空分辨性、低耐药性等特点受到广泛关注。光动力疗法是基于在光照下光敏剂产生活性氧，实现对病原菌的杀伤。传统光敏剂如卟啉、酞菁等在生物水环境中存在聚集诱导荧光猝灭和活性氧产生性能降低等问题，从而降低光动力治疗效果。相比之下，近年来新兴的AIE材料在单分子状态下通常不发光或弱发光，但在生物水环境中组装形成聚集体后能发出强的荧光便于可视化与病原菌作用，同时产生活性氧的性能也明显提高。因此，AIE分子聚集体作为光动力治疗的光敏剂具有大的应用前景。但活性氧的寿命短和有效作用距离短，将大大降低光动力治疗效果。因此，从长远角度，将光动力治疗与其他抗菌策略协同作用将具有更大的应用前景和临床价值。
发明内容
本发明所要解决的技术问题为：如何提供一种兼具黏附细菌和光动力杀伤双功能AIE光敏剂。
本发明的技术方案为：一种化合物，具有式1所示的结构：
式1
其中，糖基G选自能与病原菌表面凝集素特异作用的糖基基团；抗衡阴离子X-选自具有一个电荷的阴离子；其中，每个R独立选自氢、羟基、氨基、烷基的一种或几种。
进一步地，所述化合物具有式2所示的结构：
式2
本发明的新化合物在AIE光敏剂基团引入糖基，在水溶液中基于自组装形成表面含多糖基的AIE光敏剂聚集体，兼具黏附细菌和光动力杀伤双功能。所发明的多糖基AIE聚集体能通过其表面糖基与细菌表面凝集素结合实现对细菌的黏附并促进其聚集。在光照下，所发明AIE光敏剂能高效产生活性氧。基于黏附作用和光动力的协同，实现对病原菌高效地杀伤。
研究表明，多糖基体系可以通过与细菌表面凝集素结合实现对细菌的黏附。更重要的是，通过这种糖基体系与细菌的结合可以阻断细菌与细胞糖配体间的特异性作用，也能对治疗细菌感染提供很好的辅助作用。由此可见，开发的含糖基的AIE光敏剂体系，既能够满足黏附细菌需求又可以有效实现黏附和光动力协同杀菌目的。
作为本发明的另一目的，本发明的化合物作为病原菌杀菌剂的用途。
进一步地，所述病原菌为与AIE光敏剂所带糖基基团特异性作用的菌种。
与现有技术相比，本发明具有以下有益效果：
在AIE光敏剂基团引入糖基，在水溶液中基于自组装形成表面含多糖基的AIE光敏剂聚集体，兼具黏附细菌和光动力杀伤双功能。所发明的多糖基AIE聚集体能通过其表面糖基与细菌表面凝集素结合实现对细菌的黏附并促进其聚集。在光照下，所发明AIE光敏剂能高效产生活性氧。基于黏附作用和光动力的协同，实现对病原菌高效地杀伤。
附图说明
图1示出了糖基AIE分子TPEPy-Gal的合成路线。
图2示出了(a)浓度为50μM的TPEPy-Gal在DMSO溶液中的紫外可见吸收光谱；(b)浓度为50μM的TPEPy-Gal在PBS溶液中的荧光光谱，激发波长420nm。
图3示出了(a)不同浓度TPEPy-Gal在PBS溶液中的荧光光谱，激发波长420nm；(b)在PBS溶液中，TPEPy-Gal在576nm处的荧光发射强度随浓度变化关系图。
图4示出了浓度为20μM的TPEPy-Gal在PBS溶液中形成聚集体(a)粒径分布；(b)透射电镜图；(c)电位结果。
图5示出了以9,10-蒽二基-双(亚甲基)二丙二酸(ABDA)为探针检测在白光照射下(20mW/cm2)TPEPy-Gal的活性氧产生性能。(a)50μM的ABDA在水溶液中紫外可见吸收光谱随光照时间变化；(b)加入TPEPy-Gal后，ABDA紫外可见吸收光谱随光照时间变化；(c)有无TPEPy-Gal存在，ABDA在380nm处的吸光度随白光照射时间的变化而变化，其中A0和A分别是ABDA在照射前后380nm处的吸光度。TPEPy-Gal的浓度：20μM，ABDA的浓度：50μM。
图6示出了(a)和(b)分别为铜绿假单胞菌与TPEPy-Gal作用15min后的荧光成像和明场图，激发波长：460-550nm。
图7示出了(a)不同浓度TPEPy-Gal在暗处或光照下对铜绿假单胞菌的抗菌性能；(b)在暗处或光照下，与不同浓度TPEPy-Gal作用后的铜绿假单胞菌在琼脂板生长菌落照片。
图8示出了(a)50μM的TPEPy-Et在DMSO溶液中的紫外可见吸收光谱；(b)50μM的TPEPy-Et在PBS溶液中的荧光光谱，激发波长420nm；(c)不同浓度TPEPy-Et在PBS溶液中的荧光光谱，激发波长420nm；(d)在PBS溶液中，TPEPy-Et在584nm处的荧光发射强度随浓度变化关系图；(e)在白光照射下TPEPy-Et加入后，ABDA紫外可见吸收光谱随光照时间变化；(f)有无TPEPy-Et存在，ABDA在380nm处的吸光度随白光照射时间的变化而变化，其中A0和A分别是ABDA在照射前后378nm处的吸光度。TPEPy-Et：20μM，ABDA的浓度：50μM。
图9示出了(a)不同浓度TPEPy-Et在暗处和光照下对铜绿假单胞菌的抗菌性能；(b)在暗处或光照下，与不同浓度TPEPy-Et作用后的铜绿假单胞菌在琼脂板生长菌落照片。
图10示出了不同浓度TPEPy-Gal在暗处或光照下对人正常肝细胞Lo2的毒性。
图11示出了TPEPy-Gal的1H NMR谱(400MHz,DMSO-d6)。
图12示出了TPEPy-Gal的MALDI-TOF高分辨质谱图。
具体实施方式
下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的试验材料，如无特殊说明，均为从商业渠道购买得到的。
实施例1
TPETy-Gal通过简便的合成路线被制备(图1)
(1)将1,2,3,4,6-五-O-乙酰基-β-D-吡喃半乳糖(2.1g，5.37mmol)和2-溴乙醇(0.6mL，8.46mmol)溶于超干二氯甲烷(10mL)中，在冰水浴及N2保护下，滴加三氟化硼乙醚(3.3mL，26.0mmol)反应2h后，将溶液温度升至室温反应20h。反应结束后，将溶液加入冰水中，用二氯甲烷萃取三次。合并萃取物，依次用水，饱和碳酸氢钠溶液，水进行洗涤，用硫酸钠干燥过滤后将所得滤液旋蒸。残余物通过色谱纯化(乙酸乙酯∶正己烷＝1∶3)，收集产物进行旋蒸，真空干燥后得白色粉末IX。
(2)以化合物IX(500mg,1.09mmol)和TPEPy(300mg,0.605mmol)为原料，用甲苯作溶剂，在氮气保护下将温度升至120℃反应20h。反应结束后通过色谱纯化(CH2Cl2∶CH3OH＝15∶1),收集产物进行旋蒸，真空干燥后得橙红色粉末X。
(3)将化合物X(27.2mg,0.0286mmol)溶于甲醇中，加入K2CO3(15.81mg，0.1143mmol),室温反应1h。反应结束后加入三氯甲烷，饱和NaCl溶液，进行萃取，溶液分三层，取下层溶液用无水Na2SO4干燥后，离心进行旋蒸，从含有石油醚，二氯甲烷的溶剂中重结晶，真空干燥后得到最终产物TPEPy-Gal。
TPEPy-Gal结构的确认
结合TPEPy-Gal的氢谱(图11)以及高分辨质谱图(图12)，可以确认该分子的结构如下图所示：
TPEPy-Gal的光物理性质表征
如图2中a的紫外-可见吸收光谱所示，TPEPy-Gal的最大吸收在417nm处，最大发射波长在580nm左右(图2)。TPEPy-Gal呈现典型的AIE效应性质，如图3中a所示，随着TPEPy-Gal溶液浓度的增大，其荧光强度逐渐增大。基于该分子的AIE性质，可以确定其临界聚集浓度，如图3中b所示，在浓度大于8.4μM时，TPEPy-Gal在580nm的荧光强度明显增强，表明聚集体的形成，因而，确定其临界聚集浓度为8.4μM。
TPEPy-Gal的聚集形貌表征
动态光散射(DLS)测量TPEPy-Gal的粒径和电位，使用透射电镜观察样品的形貌。如图4所示，20μM的TPEPy-Gal溶液电位值为10mV，粒径分布在100nm左右，在透射电镜下观察到粒径大约为100nm的球形聚集体。
TPEPy-Gal产生单线态氧能力评估
利用商业探针9,10-蒽二基-双(亚甲基)二丙二酸(ABDA)对TPEPy-Gal产生单线态氧能力进行检测。ABDA被单线态氧氧化后特征吸收峰下降，因而基于ABDA特征吸收峰降低程度来评估TPEPy-Gal产生单线态氧的能力。取10μL探针ABDA(10mM)至2mLTPEPy-Gal(20μM)的PBS溶液中，混匀后，用20mW/cm2的白光照射，每隔30s测定一次吸光值。如图5所示，光照下，在无TPEPy-Gal的对照组，ABDA的特征吸收峰强度基本没有变化，而加入TPEPy-Gal分子后，ABDA的吸光度随着光照时间延长不断下降，证实该TPEPy-Gal可以有效地产生单线态氧。
TPEPy-Gal与细菌作用，促进其聚集
将TPEPy-Gal溶液与菌液浓度为OD600＝1.0的铜绿假单胞菌暗处培养15min后，离心取走上清液，加10μL PBS将沉淀混匀，取2μL至干净的载玻片上，盖上盖玻片进行观察。
利用荧光显微镜观察TPEPy-Gal与铜绿假单胞菌的相互作用。如图6所示，TPEPy-Gal聚集体发射橙色荧光，观察到细菌团聚在聚集体的周围，而没有聚集体的地方，菌落分布均匀且数量较少。表明该TPEPy-Gal形成的聚集体对铜绿假单胞菌有粘附作用，促进其聚集。
TPEPy-Gal抗菌活性评估
本实验以铜绿假单胞菌为例，用传统铺平板法评估了TPEPy-Gal在暗处和光照下的抗菌效果，并以未进行糖基修饰的TPEPy-Et作为对照。把单克隆菌落放在10mL的LB液体培养基中，在37℃，180rpm下摇床振荡培养6-8h。取一定体积菌液，离心除去培养基(7100rpm,2min)，用PBS洗涤2次，最后悬于PBS中并调菌液浓度为OD600＝1.0。暗室培养，将菌液分别与不同浓度TPEPy-Gal在37℃下作用40min；光照组则是将菌液分别与不同浓度TPEPy-Gal在37℃下作用10min，采用90mW/cm2的白光照射30min；然后分别用PBS稀释104倍,取100μL稀释菌液均匀涂于LB固体培养基上(固体培养基平板规格为90mm)，每个浓度均进行三次平行实验，在37℃下培养14-16h，统计菌斑个数。根据以下公式计算TPEPy-Gal的杀菌活性(IR)： 其中A为对照组菌落形成单位数(cfu)，B为实验组的菌落形成单位数(cfu)。
如图7所示，在暗处孵育40min，浓度为10μM的TPEPy-Gal对铜绿假单胞菌的抗菌活性为15.9％，当用90mW/cm2的白光照射30min后，其抗菌活性由暗处15.9％提高到73.9％；当TPEPy-Gal浓度增加到20μM时，暗处活性提高到32.6％，结合光照后，抗菌活性高达99.7％。表明在光照下单线态氧的产生结合糖基对细菌的粘附作用，TPEPy-Gal聚集体对细菌呈现高效地杀伤活性。
为了进一步证实TPEPy-Gal糖基存在对细菌黏附对其抗菌活性的作用，选用未进行糖基修饰的TPEPy-Et作为对照。如图8所示，对照分子TPEPy-Et最大吸收在416nm处(图8中a)，最大发射波长在585nm左右(图8中b)。TPEPy-Et也具有典型的AIE性质，随着分子浓度增大，在585nm处的荧光逐渐增强(图8中c)，其临界聚集浓度大约在6.4μM(图8中d)。图8中e和f所示，以ABDA为单线态氧探针证实，加入TPEPy-Et，光照1min后，ABDA完全降解，其速度要大于TPEPy-Gal，表明对照分子TPEPy-Et具有优异的单线态氧产生性能。
TPEPy-Gal与对照分子TPEPy-Et具有相似的光物理性质和聚集能力，但抗菌结果表明(图9)，在暗处和光照下，相同浓度的TPEPy-Et对铜绿假单胞菌无明显的的抗菌活性。这证实引入糖基对细菌的粘附作用在光敏剂TPEPy-Gal展现抗菌活性中发挥重要作用。
TPEPy-Gal生物相容性
如图10所示，细胞存活率实验是通过CCK8法来测定的。TPEPy-Gal具有好的生物相容性。在TPEPy-Gal浓度高达128μM时，在暗处和光照下，人正常肝细胞细胞存活率都保持在接近100％，证明该分子具有低的细胞毒性。
Claims (3)
1.一种化合物，其特征在于，所述化合物具有式2所示的结构：
式2。
2.权利要求1所述的化合物制备病原菌杀菌剂的用途。
3.根据权利要求2所述的用途，其特征在于，所述病原菌为与AIE光敏剂所带糖基基团特异性作用的菌种。
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| CN110950779A (zh) * | 2018-09-25 | 2020-04-03 | 天津理工大学 | 集细菌荧光成像与光动力杀菌于一体的光敏剂及其制备方法和应用 |
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| CN110950779A (zh) * | 2018-09-25 | 2020-04-03 | 天津理工大学 | 集细菌荧光成像与光动力杀菌于一体的光敏剂及其制备方法和应用 |
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