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

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 the criteria for FRET induction, and the fluorescence lifetime of the donor is shortened during the process. When the endocytic vesicles release the drug intracellularly, the distance between the cell membrane and nano drug is altered, and the FRET effect diminishes or disappears.In this study, we used a two-photon excitation fluorescence lifetime microscopic imaging system(TP-FLIM) to monitor the FRET effect during this process, to distinguish between the diffusion movement of nanoparticles being endocytosed into cells and smallmolecule drugs and to monitor the endocytosis process of cells in real time. We used this method to verify the upregulation of the endocytosis movement of cells under starvation conditions.Results and Discussions In this study, BSA was used to wrap DOX into nanoparticles that could be endocytosed into cells,resulting in the formation of BSA-DOX nanoparticles with a particle size below 100 nm. The process of cellular uptake of nanoparticles by endocytosis is long, which enables a more in-depth study of microscopic physiological processes. In addition, the endocytosis pathway of the nanocarriers was evaluated using four endocytosis pathway inhibitors. BSA-DOX nanoparticles entered the cells via clathrin-mediated endocytosis. The associated dynamic process was elucidated. Our study shows that the FLIM-FRET technique combined with quantitative analysis methods can be used to study the similarities and differences between small-molecule drugs and nanoparticle-cell interactions.Conclusions In this study, we present a new method for the qualitative and quantitative analysis of endocytosis of nanomedicine in OVCAR-3 cells. We synthesized BSA-DOX nanoparticles by desolvation using a material that allows us to use its own fluorescence to form FRET pairs with sfGFP proteins transfected onto the cell membrane. We used the TP-FLIM system for qualitative analysis of cellular endocytosis by two-photon fluorescence and interference-free monitoring of donor lifetime during FRET. Quantitative analysis was performed by FLIM. The experimental results show that the distance between the cell membrane and nano molecule can be accurately reflected by detecting FRET efficiency as the nanomedicine is endocytosed by the cells and released within the cells. We also used this method to verify that starvation-treated cells upregulated endocytosis motility.