基于FLIM-FRET技术揭示OVCAR-3细胞膜和BSA-DOX纳米颗粒的相互作用  

Elucidation of Interaction Between OVCAR-3 Cell Membranes and BSADOX Nanoparticles Based on FLIM-FRET

作　　者：李慧娴 林方睿 徐云剑 李艳萍 王柯欣 王诗琪 邹炎华 胡睿 屈军乐[1] 刘丽炜 Li Huixian;Lin Fangrui;Xu Yunjian;Li Yanping;Wang Kexin;Wang Shiqi;Zou Yanhua;Hu Rui;Qu Junle;Liu Liwei(Key Laboratory of Optoelectronic Devices and Systems of Guangdong Province and Ministry of Education,College of Physics and Optoelectronic Engineering,Shenzhen University,Shenzhen 518060,Guangdong,China)

机构地区：[1]深圳大学物理与光电工程学院教育部/广东省光电子器件与系统重点实验室,广东深圳518060

出　　处：《中国激光》2023年第3期130-140,共11页Chinese Journal of Lasers

基　　金：国家自然科学基金(62175163,62225505,61935012);深圳市杰青项目(RCJC20210706091949022);深圳市重点项目(JCYJ20200109105404067)。

摘　　要：荧光寿命显微成像(FLIM)常用来检测活细胞内荧光基团的寿命信息,以实现微观定量分析。荧光共振能量转移(FRET)可用来表征能量从供体荧光分子到受体荧光分子的传递过程。将FLIM技术与FRET结合(FLIMFRET),可以监测活细胞中蛋白质的相互作用、亚细胞器的动态过程等。构建了以细胞膜上转染的绿色荧光蛋白(sfGFP)为供体、以阿霉素(DOX)为受体的FRET纳米体系,利用双光子激发荧光寿命显微成像(TPFLIM)系统,通过监测FRET纳米体系中供体荧光寿命的变化,研究了药物DOX在细胞中的递送机制和运输效率。此外,进一步采用四种内吞途径抑制剂,对纳米药物的内吞途径进行了评估。结果证明,牛血清白蛋白(BSA)包裹的DOX(BSADOX)纳米颗粒通过网格蛋白介导的内吞作用进入细胞。揭示了BSADOX纳米颗粒通过网格蛋白介导的内吞作用进入细胞的动态过程。研究表明,FLIMFRET技术结合定量分析方法可用于区分小分子药物和纳米颗粒与细胞作用的异同。Objective In typical tumor therapy, the drug must reach the tumor site via blood vessels and access the cell membrane of the tumor cells to act on a certain target. The drug recognizes the target molecules and then enters the tumor cells in a specific manner that facilitates the release of the drug without toxic side effects on normal cells. Numerous membrane proteins and receptors on the cell membrane can be considered targets during drug carrier design. Corresponding biochemical studies, such as immunoblotting and flow cytometry, are often conducted, supplemented by fluorescence co-staining imaging. However, intensity-based fluorescence imaging has considerable limitations, both in terms of its inability to distinguish small-molecule drugs from nanomedicine drugs and to monitor endocytosis in living cells in real time. Fluorescence lifetime imaging microscopy(FLIM) is commonly used to evaluate the lifetime of fluorescent moieties in living cells for quantitative microscopic analysis. F?rster resonance energy transfer(FRET) can be used to characterize the transfer of energy from a donor fluorescent molecule to an acceptor fluorescent molecule. FLIM technology with FRET(FLIM-FRET) can monitor protein interactions and the dynamic processes of subcellular organelles in living cells.Methods In this study, doxorubicin(DOX) nanoparticles encapsulated in bovine serum albumin(BSA) were synthesized. Albumin nanoparticles demonstrate good biocompatibility and inherent passive targeting in living organisms and can be effective drug carriers for slow release and reduced toxic side effects. DOX is an amphiphilic molecule that is not completely encapsulated in the nanoparticles and is attached to the nanoparticle surface. Superfolder GFP(sfGFP) was transfected into the cell membrane as a donor,and the BSA-DOX nanoparticles were used as acceptor molecules. Together, both molecules constituted the FRET nanosystem.During the uptake of nano drugs by cells via endocytosis, the distance between the cell membrane and nano drug meets

关 键 词：生物光学 荧光共振能量转移 荧光寿命 细胞膜 

分 类 号：Q334[生物学—遗传学]