| The delivery of plasmid DNA into cells using the cationic polymers poly- L-lysine (PLL), polyethyleneimine (PEI), PLL and PEI conjugated with either or both poly(ethylene)glycol (PEG) and the nuclear localization sequence (NLS) adopted from the SV40 virus was investigated. In addition, fluorescent markers, fluorescein and indocarocyanine, were covalently bound to the gene delivery carriers and plasmid DNA to study cellular uptake and trafficking.; Conjugation of PEG and NLS sequence to PLL and PEI was accomplished using conjugation chemistry such as isothiocyanates, N-hydroxysuccinimide esters, and vinyl sulfones. The order of the conjugation chemistry was important so as to create graft co-polymers of PEG, PEG-fluorescein, PEG-NLS, and PEG-NLS-fluorescein off of the primary amines of PLL or PEI. Size exclusion chromatography using an FPLC system was used for separation and 1H-NMR was used for characterization; Epi-fluorescent, laser confocal microscopy was used to visualize the trafficking of the gene delivery complex in HT1080, human fibrosarcoma, cells via labeling of a different fluorescent dye to both the polymeric carrier and to the plasmid DNA. Fluorescence was observed in the nuclei of cells under those conditions where the complex was also toxic to the cells. Otherwise, fluoresence was not seen in the nuclei of cells under nontoxic, transfecting conditions. Based on microscopy data, all complexes seemed to be trapped inside the endosomes/lysosomes near the nuclei of cells.; Conjugating PEG to both PLL and PEI decreased the toxicity of the overall carrier. However, no transfection conjugates were better than the control: unmodified PEI (25 kDa). Even pegylation of PEI and PLL did not improve transfection. This is likely due to the PEG masking the positive surface charge of the complexes; preventing the non-specific interaction of the complex with the cell surface. However, PLL-PEG-NLS showed higher transfection in the 293T, human kidney, cell line to that of PLL. And PLL-NLS showed equivalent transfection to that of PLL. |
