Cell-penetrating peptides enhance gene delivery by lipid-DNA complexes

Objective: (i) To test the hypothesis that cell-penetrating peptides (HIV-Tat-peptide) and microtubule disrupting agents (vinblastine) can facilitate cytoplasmic delivery and nuclear entry of DNA, and (ii) to observe the intracellular localization of cationic liposome-DNA complexes (lipoplexes) in HSC-3 and H413 human oral squamous cell carcinoma (OSCC) cells. Methods: HSC-3 and H413 cells were maintained in DME and DME/F12 media with 10% FBS. Cells were seeded in 48-well plates the day before transfection and used at approx. 70% confluency. The plasmid pCMV.luc expressing luciferase under the control of the cytomegalovirus promoter was complexed with optimal volumes Metafectene, with or without the HIV-Tat-peptide, and incubated with the cells for 4 h. Transgene expression was assayed 48 h after transfection, using the Promega Luciferase Assay System. For fluorescence microscopy, cells were seeded in fibronectin-coated Lab Tek II chambered cover glasses. Cells were transfected with rhodamine-labeled Fluo-Metafectene-DNA plasmid complexes. Lysosensor and Hoechst dye were used to stain the lysosomes and the nucleus, respectively. Fluorescence was observed 2, 4 and 48 h after transfection using a Zeiss LSM-510 Confocal Fluorescence Microscope. Results: HIV-Tat-peptide, at 1 and 5 µg, mixed with 1 µg DNA, and then complexed with Metafectene, enhanced gene expression in HSC-3 cells by 4- and 5-fold, respectively. Treatment of HSC-3 cells with 2-4 µg/ml vinblastine also enhanced gene expression 2-fold. HSC-3 cells displayed a higher level of cell-associated rhodamine fluorescence compared to H413 cells. Metafectene-DNA complexes co-localized with lysosomes in the perinuclear region. Vinblastine treatment enhanced perinuclear localization of lipoplexes. Conclusion: HSC-3 cells internalize Fluo-Metafectene more efficiently than H413 cells, that are resistant to transfection. Cell penetrating peptides and microtubule-disrupting agents may be useful for gene delivery to OSCC cells. Funded by an AADR Student Research Fellowship, and a Research Pilot Project Award 03 Activity 072 from the Dugoni School of Dentistry (J.F).