| In this work, the transport of synthetic gene vectors is investigated quantitatively in live cells for the first time with high-resolution real-time particle tracking. Using this novel technology, we show nonviral polyethylenimine-based gene vectors are actively transported to the perinuclear region of cells, disproving the common belief that the intracellular transport of drug and gene vectors may be critically hindered due to difficult transport through the crowded cytoplasm.; Next, we developed live-cell confocal particle tracking (CPT) to directly correlate the biophysical transport of therapeutic colloids with intracellular biological location, providing new insights into the mechanism of particle trafficking in live cells. Multicolor CPT is four-dimensional particle tracking with the dimensions of x, y, time, and color, with color indicating the biological location of gene vectors. The real-time simultaneous imaging of gene vectors and cellular organelles in mesenchymal stem cells revealed the synthetic particles are actively transported through the endo-lysosomal pathway of cells, leading to the accumulation of vectors in perinuclear vesicles.; If gene vectors successfully escape perinuclear vesicles, results suggest they will face undesirable adhesion to microtubules that are in high concentration around the cell nucleus. This may prevent the efficient transport of gene vectors from the perinuclear region to the nuclear pore complexes---the entryway into the nucleus. As a method of overcoming this potential barrier, nanoparticles are modified with the hydrophilic polymer, polyethyleneglycol (PEG), which has been shown to improve particle transport through other complex biological environments. Results indicate that PEGylation of gene vectors improves intracellular particle transport rates. Lastly, the intracellular transport and trafficking of nonviral gene vectors is investigated in human blood-derived dendritic cells. These studies have highlighted ways to improve gene delivery into dendritic cells, with the ultimate goal of designing high efficiency nonviral vectors for immunotherapy against HIV/AIDS. (Abstract shortened by UMI.)... |
