Intracellular Trafficking of DNA/Polymer Polyplexes

In this thesis, we designed and developed a simple method to prepare longer structure-defined linear polyethylenimine (PEI) chains with degradable disulfide linkages (lPEIs-s). We avoided the use of branched PEI chains because the inter-chain coupling normally leads to ill-defined structures and purposely chose our initial linear chain (lPEIi) with a length of ~15 nm to test whether the extension of the chain length with degradable disulfide linkage can indeed promote the gene transfection. Our results indicated that the extension of the chain length makes longer lPEIs-s chains slightly more cytotoxic but has nearly no effect on the gene transfection, very different from our previous studies in which the gene transfection efficiency increased ~5-7 orders when three-to-four small branch PEI chains (Mw ~ 2 kg/mol) were coupled together to form a larger one (> 15 nm). Such a drastic difference supported our recent hypothesis that the enhancement of the gene transfection was mainly due to the length of those cationic PEI chains free in the solution mixture of PEI and DNA.;Further, we designed and synthesized a rhodamine B labeled linear PEI as a probe to investigate the intracellular trafficking of the PEI/DNA polyplexes and when and where the disulfide bonds are broken in living cells. In this approach, a self-made linear PEI (Mn ≈ 3000 g/mol) was used as a starting material. One end of each PEI chain was modified with a rhodamine B molecule with a mercapto-group so that they can be coupled together with a disulfide bond in the middle and one rhodamine B molecule on each side of the disulfide bond, followed by one linear PEI chain on each side, where fluorescence is self-quenched because two rhodamine B molecules are too closely linked by a short disulfide bond. As expected, the cleavage of the disulfide bond will lead to the separation of two rhodamine B molecules and significantly enhance the fluorescent intensity. Further, plasmid DNA was modified with bodipy, a FRET pair of rhodamine B. FRET happens once DNA is condensed by the rhodamine B-modified PEI. Armed with these specially prepared PEI chains, we have investigated the self-quenching and FRET property of the PEI/DNA polyplexes during the intracellular trafficking by using the flow cytometry and confocal laser scanning microscopy. We found that the fluorescent intensity of rhodamine B is greatly enhanced in the living cells, especially in lysosome due to the cleavage of the disulfide bonds. At the same time, the fluorescent intensity of bodipy gradually increases, indicating DNA is released from the PEI/DNA polyplexes.;Our results indicate that 1) once the polyplexes enter the cell, DNA is continuously dissociated and released prior to the cleavage of the disulfide bonds; 2) most of the polyplexes can escape from the endolysosomes; and 3) the disulfide bonds on those un-escaped polyplexes are cleaved inside the lysosomes. Our results further confirm that the disulfide bonds do not play a critical role in promoting the gene transfection efficiency. It is those PEI chains free in the solution mixture of DNA and PEI that interact with those anionic signal proteins on the cell and endosome membranes, blocking the inter-vesicular fusion and inducing the escape from endosomes so that the ingested PEI/DNA polyplexes have a less chance to be degraded inside the lysosome. (Abstract shortened by UMI.).