Fluorescence in situ hybridization to monitor the intracellular location and accessibility of plasmid DNA delivered by cationic polymer-based gene carriers.

Information about the intracellular trafficking of exogenous DNA delivered by nonviral gene delivery systems is of major importance for optimization of such gene carriers. We used fluorescence in situ hybridization (FISH) as a tool to visualize polyplex-delivered pDNA inside cells. This avoids the need to directly label DNA inside the polyplexes, which may influence their cellular behavior and fate. Using FISH the introduced plasmid DNA could be detected in the cytosol and nucleus of different cell lines. The FISH probe itself did not interact with cells nor different polymers used for condensing the DNA. We further demonstrate differences in accessibility of polyplex-delivered DNA when different polymers were used for DNA complexation. Therefore, FISH is a valuable tool to detect location and accessibility of exogenous plasmid DNA delivered in the cell by cationic polymers.

Cellular uptake of cationic polymer-DNA complexes via caveolae plays a pivotal role in gene transfection in COS-7 cells.

PURPOSE: Knowledge about the uptake mechanism and subsequent intracellular routing of non-viral gene delivery systems is important for the development of more efficient carriers. In this study we compared two established cationic polymers pDMAEMA and PEI with regard to their transfection efficiency and mechanism of cellular uptake. MATERIALS AND METHODS: The effects of several inhibitors of particular cellular uptake routes on the uptake of polyplexes and subsequent gene expression in COS-7 cells were investigated using FACS and transfection. Moreover, cellular localization of fluorescently labeled polyplexes was assessed by spectral fluorescence microscopy. RESULTS: Both pDMAEMA- and PEI-complexed DNA showed colocalization with fluorescently-labeled transferrin and cholera toxin after internalization by COS-7 cells, which indicates uptake via the clathrin- and caveolae-dependent pathways. Blocking either routes of uptake with specific inhibitors only resulted in a marginal decrease in polyplex uptake, which may suggest that uptake routes of polyplexes are interchangeable. Despite the marginal effect of inhibitors on polyplex internalization, blocking the caveolae-mediated uptake route resulted in an almost complete loss of polyplex-mediated gene expression, whereas gene expression was not negatively affected by blocking the clathrin-dependent route of uptake. CONCLUSIONS: These results show the importance of caveolae-mediated uptake for successful gene expression and have implications for the rational design of non-viral gene delivery systems.

An NLS peptide covalently linked to linear DNA does not enhance transfection efficiency of cationic polymer based gene delivery systems.

BACKGROUND: Transfection with non-viral gene delivery vectors, such as cationic polymers, generally results in low transgene expression in vivo. This is likely due to poor cytoplasmic transport and intra-nuclear DNA delivery. METHODS: In this study two strategies to improve nuclear import were investigated. Linear DNA constructs with or without an NLS peptide were prepared by PCR. Alternatively, linear DNA obtained by enzymatic cleavage followed by capping of both ends with DNA-hairpins was used. An NLS peptide was attached to one of the capped ends of the linear DNA. Both biodegradable (pDMAEAppz) and non-degradable polymers (PEI or pDMAEMA) were used to complex the DNA. Several cell types, dividing and non-dividing, were transfected with the linear DNA constructs containing a SV40-derived NLS peptide. Nuclear import of the DNA constructs was studied using digitonin-permeabilized cells. RESULTS: Linear DNA prepared by PCR proved not useful as it was degraded from the 3'end. Linear DNA capped with hairpins was more successful with regard to stability. However, Cells transfected with linear DNA constructs by electroporation or by using cationic polymers with linear DNA containing a NLS peptide, failed to show significantly higher luciferase expression levels when compared to cells transfected with plasmid DNA or linear DNA without an NLS peptide attached. No nuclear localization was observed in digitonin-permeabilized cells. CONCLUSION: Taken together, these data demonstrate that this nuclear localisation signal when attached to DNA is neither able to improve transfection efficiency of cationic polymers nor the nuclear import of the DNA constructs.