No abstract

The impact of a peptide that contains a nuclear localisation sequence (NLS) on intracellular DNA trafficking was studied. We used the adenoviral core peptide mu and an SV40 NLS peptide to condense plasmid DNA (pDNA) prior to formulation with 3beta-[N-(N', N'-dimethylaminoethane)carbamoyl]cholesterol/dioleoyl-L-alpha-phosphatidyl ethanolamine (DC-Chol/DOPE) liposomes to give LMD and LND vectors, respectively. Fluorescent-labelled lipid and peptides plus dye-labelled pDNA components were used to investigate gene delivery in dividing and S-phase growth-arrested cells. Confocal microscopic analyses reveal little difference in intracellular trafficking events. Strikingly, mu peptide associates with nuclei and nucleoli of cells within less than 15 mins incubation of LMD with cells, which suggests that mu peptide has an NLS function. These NLS properties were confirmed by cloning of a mu-beta-galactosidase fusion protein that localises in the nuclei of cells after cytosolic translation. In dividing cells both LMD and LND deliver pDNA(Cy3) to nuclei within 30-45 min incubation with cells. By contrast, pDNA is detected only in the cytoplasm in growth-arrested cells over the period of time investigated, and not in the nuclei. LD systems prepared from DC-Chol/DOPE cationic liposomes and pDNA(Cy3) behave similarly to LMD systems, which suggests that mu peptide is unable to influence trafficking events in this current LMD formulation, in spite of its strong NLS capacity. We further describe the effect of polyethyleneglycol (PEG) on cellular uptake. "Stealth" systems obtained by post-coating LMD particles with fluorescent-labelled PEG molecules (0.5, 5 and 10 mol % fluorescein-PEG(5000)-N-hydroxysuccinimide) were prepared and shown to be internalised rapidly (mins) by cells, without detectable transgene expression. This result indicates that PEG blocks intracellular trafficking of pDNA.

In this work, we demonstrated that the hydrogel obtained from a very simple and single synthetic molecule, N-heptyl-galactonamide was a suitable scaffold for the growth of neuronal cells in 3D. We evidenced by confocal microscopy the presence of the cells into the gel up to a depth of around 200 μm, demonstrating that the latter was permissive to cell growth and enabled a true 3D colonization and organization. It also supported successfully the differentiation of adult human neuronal stem cells (hNSCs) into both glial and neuronal cells and the development of a really dense neurofilament network. So the gel appears to be a good candidate for neural tissue regeneration. In contrast with other molecular gels described for cell culture, the molecule can be obtained at the gram scale by a one-step reaction. The resulting gel is very soft, a quality in accordance with the aim of growing neuronal cells, that requires low modulus substrates similar to the brain. But because of its fragility, specific procedures had to be implemented for its preparation and for cell labeling and confocal microscopy observations. Notably, the implementation of a controlled slow cooling of the gel solution was needed to get a very soft but nevertheless cohesive gel. In these conditions, very wide straight and long micrometric fibers were formed, held together by a second network of flexible narrower nanometric fibers. The two kinds of fibers guided the neurite and glial cell growth in a different way. We also underlined the importance of a tiny difference in the molecular structure on the gel performances: parent molecules, differing by a one-carbon increment in the alkyl chain length, N-hexyl-galactonamide and N-octyl-galactonamide, were not as good as N-heptyl-galactonamide. Their differences were analyzed in terms of gel fibers morphology, mechanical properties, solubility, chain parity, and cell growth.