Insights into the mechanisms of plasmidic DNA nuclear delivery by confocal microscopy in living cells

Transfection allows the introduction of foreign genetic material into cells to study gene function and regulation, investigate protein function and, potentially, cure diseases by delivering the gene of interest into the target cells of patients. Although virus-mediated gene delivery methods show high transfection efficiency, they are mostly limited by their hazardous immunogenicity. That is the reason why, lately, chemical and physical approaches have been developed to overcome this huge disadvantage, though displaying a low efficiency. The use of cationic lipids as a chemical method is very common today because of their ease to use: such a system just exploits the capability of liposomes to trap hydrophilic molecules, like DNA, and facilitate their delivery into the cells. More recently, complexes made of lipids and polycations (e.g. protamine) have been deeply investigated because of a promising improvement in transfection efficiency as compared to cationic lipids alone. A general feeling coming from the recent literature is that the mechanisms involved in the cellular delivery of DNA with non-viral gene delivery methods are not clear, especially those concerning the intracellular trafficking and transport into the nucleus. In this Master’s thesis project two transfection nanoformulations are compared to give a deeper insight into one of the most important cellular barrier that plasmidic DNA encounters at the end of its intracellular trafficking: the entry into the nucleus. The first nanoformulation chosen is Lipofectamine, a gold standard for transfection; the second one is the complex between cationic lipids and the polycationic agent protamine, which is used to condense DNA before formulation with lipids. Observations coming from laser scanning confocal microscopy and flow cytometry allowed studying the different mechanisms of DNA nuclear delivery among these two nanoformulations. The most important finding is the understanding of the crucial role played by the cell cycle during transfection: data show that all the cells go through mitosis before being transfected. This prompts us to speculate that the breakdown of the nuclear envelope during the mitotic phase may facilitate trapping of plasmidic DNA within the nucleus. Another important observation is the establishment of a different transfection phenotype after mitosis, between the two transfection formulations: cells transfected with Lipofectamine show high transfection efficiency and symmetry of the fluorescent signal between the two daughter cells after cell division; by contrast, cells transfected with the lipids/protamine complexes typically show a lower characteristic transfection efficiency, and marked asymmetry of the fluorescent signal between the two daughter cells (along with the existence of big clusters of plasmidic DNA colocalizing inside the nucleus). From the literature, it is known that condensation of DNA with protamine before mixing with cationic liposomes increases transfection efficiency (as measured by the luciferase assay) in comparison with the use of liposomes only. The observations coming from my experiments may help elucidating the differences between the two formulations: transfection with lipids/protamine could yield lower transfection efficiency, compared to Lipofectamine, because of a lower bioavailability of plasmidic DNA, which is clustered in a few “big” aggregates. These clusters may explain the asymmetry described before: in other words, a limited number of plasmidic DNA molecules can be segregated into the two daughter nuclei after the nuclear envelope breakdown, giving rise to a pronounced asymmetry of the fluorescence signal between daughter cells. These insights provided by confocal microscopy and flow cytometry on the relationship between transfection and cell division may help guiding the development of a new class of non-viral gene delivery systems with higher performances