| Synthetic gene therapy vectors must be designed to safely and efficiently escort DNA from outside the cell to the nucleus, overcoming a number of physical barriers. Researchers have relied almost exclusively on quantitation of gene expression as a measure of a polymer's efficacy; hence the synthetic vector development has been largely trial-and-error. A quantitative understanding of gene delivery mechanisms may allow more efficient design of new vectors.; First, a flow cytometry-based assay for investigating endocytic trafficking was developed and applied to polyethylenimine (PEI). PEI is “proton-sponge” polymer; conventional wisdom says that PEI buffers endosomal acidification and hastens the release of the carrier into the cytoplasm.{09}However, I found that, PEI does not buffer endocytic compartments well. In fact, endosomal acidification appears to be necessary for PEI to efficiently deliver genetic material to the nucleus.; Since acidification is necessary for PEI's efficiency, I modified the buffering capacity of PEI to see what effect this had. Primary and secondary amines were acetylated, reducing the buffering capacity in the endosomal range (pH 6–7). The transfection efficiency was increased by up to 30 fold in some cell 1ines and polymer cytotoxicity was reduced.; PEI toxicity increases with molecular weight; however, low molecular weight PEI, essentially non-toxic, induces little gene expression. No intracellular degradation or secretion pathways exist for high molecular weight (HMW) PEI; therefore, a biodegradable crosslinked polymer was developed using ester crosslinkers. The resulting polymers were 14–30 kDa and biodegradable; up to a 10 fold improvement in transfection efficiency over HMW PEI was achieved in some cell lines with no measurable cytotoxicity.; Finally, for polymeric devices to be made safe and efficient for in vivo usage there must be some means to target them to specific cells or tissues. Various cyclodextrins were conjugated to PEI using an amide linkage. The hydrophobic interior of the cyclodextrin allows the facile addition of small targeting and lysomotropic agents to the polyplex without chemical conjugation. By applying a hydrophobic analog of human insulin to the cyclodextrin polyplexes, I was able demonstrate a 2–3 fold enhancement in transfection efficiency in insulin receptor-rich cell lines. |
