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- W2039857555 abstract "Purpose We have previously shown that a novel non-viral gene delivery system (VIPER) composed of FDA-approved agents (2% lipiodol, 5.5% iopamidol, and 50:1 protamine:plasmid DNA) can selectively transfect hepatocellular carcinoma (HCC). In this study we test the hypothesis that VIPER utilizes receptor-mediated endocytosis (RME) and subsequent lysosomal trafficking to gain access to intracellular components, in particular the nuclear transcription machinery. Materials and Methods Intracellular VIPER localization experiments were conducted in rat hepatoma cells (McARH7777) utilizing multiphoton confocal microscopy in conjunction with three cellular stains and an exogenous DNA stain: Hoechst 33342 nuclear stain (Abs: 350, Em: 461nm), WGA Alexa Fluor 350 plasma membrane stain (Abs: 346, Em: 442nm), LysoTracker DND-26 lysosomal stain (Abs: 504, Em: 511nm), and Label-It Cy5 DNA stain (Abs: 649, Em: 670nm). Intracellular localization of Cy5-labeled firefly luciferase plasmid DNA (Cy5-FLuc) combined with VIPER was evaluated as a function of VIPER incubation time (0.5, 1, 1.5, 2, 2.5, 3, 6, 12, and 24h). Effects of RME inhibition on internalization were assessed by pretreating with 100μM chlorpromazine, an RME inhibitor. RME-specific inhibition was verified with a transferrin Alexa Fluor 647 conjugated control. Results Intracellular co-localization of Cy5-FLuc and LysoTracker fluorescence spectra was identifiable at all time points. Cy5-FLuc fluorescence was greatest at 30min, and decreased with prolonged VIPER incubation. Co-localization of Cy5-FLuc and Hoechst nuclear stain was observable as early as 6h. RME inhibition led to significantly decreased intracellular Cy5-FLuc fluorescence at 30min post-VIPER treatment as compared to the uninhibited controls. Conclusion Using co-localization techniques, we determined that VIPER, a non-viral gene delivery system, mediates uptake of exogenous DNA into HCC cells through RME, as RME inhibition significantly decreases intracellular Cy5-FLuc fluorescence. Once internalized, our co-localization results suggest that VIPER-associated plasmid DNA gains access to lysosomal compartments and eventually the nucleus." @default.
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- W2039857555 date "2013-04-01" @default.
- W2039857555 modified "2023-09-26" @default.
- W2039857555 title "Cellular uptake and trafficking of a non-viral gene delivery system: analysis using multiphoton confocal microscopy" @default.
- W2039857555 doi "https://doi.org/10.1016/j.jvir.2013.01.230" @default.
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