Scheme 1. Probe structure and schematic illustration of proposed strategy of Gram-positive bacteria diagnosis and therapy by in situself-assembly of AIEgen-peptide conjugate.抗生素滥用以及由此产生的细菌抗生素耐药是人类面临的重大公共卫生问题。对抗抗生素耐药,合理使用抗生素是关键。传统的细菌诊断方法操作复杂、耗时长、特异性差,新型诊断试剂研发是对抗抗生素耐药重要手段和措施。自组装肽近年来在生物工程、药物输送、疾病治疗等研究领域发挥着越来越重要的作用。体外预组装的纳米材料由于其本身的纳米尺度,体内应用时无法避免纳米材料易于被巨噬系统吞噬而造成的生物利用度降低、肝脾毒副作用等问题。最近几年,研究者开始构筑在活细胞内以及动物体内特殊刺激下响应的原位自组装(In Situ Self-Assembly)短肽材料,通过精细调控短肽结构和组装策略实现生物功能,为肿瘤等多种重大疾病的诊疗提供了新的研究思路。与传统荧光分子相比,聚集诱导发光分子(AIEgens)可以在聚集时开启荧光,基于AIEgens的探针和纳米Dots展示了实时、长效监测的特点和兼顾成像和治疗的多重功能,被广泛应用于生物医学领域。受AIEgens独特的荧光开启特性和基于多肽的体内自组装所提供的高效组织选择性启发,课题组设计合成了一组AIEgen-肽探针,通过探针在革兰氏阳性细菌表面原位自组装实现细菌感染的敏感检测,并通过光动力杀伤治疗细菌感染(Scheme 1)。以N3-DFDFDYDEnGDK作为自组装因子,将万古霉素共价修饰到K侧链上,再通过click反应将AIEgen(TPE-Py)连接到自组装肽上,合成了AIEgen-peptide-Van,该分子具有良好的组装能力、自组装诱导发光性能和优越的ROS产生能力(Figure 1)。在体外研究中,AIEgen-peptide-Van实现了革兰氏阳性细菌的高特异性和高灵敏性靶向诊断(Figure 2)。在体内研究中,该探针可以快速、精准地检测金黄色葡萄球菌引起的小鼠肌炎(Figure 3)。经两次光动力治疗后,第四天探针治疗组的小鼠炎症抑制率为98.2%(Figure 4)。其优异的诊疗功能和良好的体内外生物相容性表明,此类探针分子在细菌感染的可视化治疗和诊疗一体化中具有潜在的应用价值。
Figure 1. Characterization of E-probe. (a) UV–vis absorption spectra of AIEgen and E-probe in water. (b) Critical assembly concentration of E-probe. (c) PL spectra of E-probe (2 μM) at different ratios of self-assembling peptide (Nap-GDFDFG) in PBS buffer (λex = 405 nm). (d)1O2 detection of E-probe (10 μM) under light irradiation (100 mW cm-2) by absorption spectra of 9,10-Anthracenediyl-bis(methylene)dimalonic acid (ABDA). (e) Superoxide detection of E-probe (10 μM) under irradiation (100 mW cm-2) by fluorescence spectra of dihydroethidium (λex = 518 nm). (f) Relative 1O2 generation of E-probe at 5 μM (percentage of decrease of abs of ABDA at 378 nm) with different ratios of self-assembling peptide (Nap-GDFDFG) in PBS buffer.
Figure 2. In vitro bacteria imaging of E-probe. (a) PL spectra of E-probe, and E. coli, MRSA, S. aureus Xen 36 stained with E-probe at the concentration of 2 mM for 0.5 h in PBS buffer. Excitation wavelength: 405 nm; Concentration of bacteria: 107 CFU mL-1. (b) Detection sensitivity of E-probe to S. aureus Xen 36. (c) Confocal images of E. coli, MRSA and S. aureus Xen 36 (2×108 CFU mL-1 incubated with E-probe at the concentration of 2 μM for 0.5 h. Insertions: 10×enlarged AIE fluorescence of E-probe treated bacteria, Red: E-Probe, Blue: DAPI. (d) TEM images of nanoaggregates on the bacterial surface after 0.5 h of incubation with solutions of E-probe in PBS buffer at the concentration of 2 μM.
Figure 3. In vivo diagnosis by fluorescence imaging. (a) Time-dependent in vivo fluorescence images of bacteria-bearing mice pretreated with the AIEgen and E-probe at the concentration of 20μM by i.v. injection. (b) The average fluorescence intensity of infected skin in (a) at differents time points. (c) Ex vivo fluorescence images of main organs and infected skin of AIEgen and E-probe treated S. aureus Xen 36-infected mice at 2 h post injection (1: heart; 2: liver; 3: spleen; 4: lung; 5: kidney; 6: infected skin).
Figure 4. In vivo bacterial infection inhibition by i.v. injection of PBS, vancomycin, AIEgen and E-probe at day 1 and day 3 for two injections. (a) Time-dependent in vivo luminescence images of S. aureus Xen 36-bearing mice which indicated the inhibition of S. aureus Xen 36 by E-probe treatment. (b) The relative S. aureus Xen 36inhibition by different treatments at the end of experiment (at day 4, *P<0.05, **P<0.01). (c) The average bioluminescence intensity of infected region with different treatments before and after treatment.论文共同第一作者为中国医学科学院放射医学研究所杨翠红副研究员和新加坡国立大学胡方博士,新加坡国立大学刘斌教授和中国医学科学院放射医学研究所刘鉴峰研究员为该文章的共同通讯作者
Paper information
Yang C#, Hu F#, Zhang X, Ren C, Huang F, Liu J, Zhang Y, Yang L, Gao Y, Liu B*, Liu J*. Combating Bacterial Infection by In Situ Self-Assembly of AIEgen-Peptide Conjugate. Biomaterials 2020, 119972.
来源:Biomaterials