Cellular uptake study of non-viral vectors for gene therapy.

Abstract

Gene therapy is fast gaining prominence as a highly promising treatment option for genetic disorders or diseases in humans. Gene therapy seeks to deliver nucleic acids into the body to modulate gene expression and achieve therapeutic outcomes. For gene delivery into cells, viral vectors have been widely utilized for its high transfection efficiency but doubts on its immunogenicity paved the way for the use of non-viral vectors that can circumvent the risks involving the use of viral vectors. In order to fulfill the full potential of gene therapy, the complex first barrier of successful gene delivery into the cells must be overcome, which is why the aim of this work is to study the underlying mechanism of cellular uptake of novel non-viral LbL-HA nanoparticles. Functionality studies of the LbL-HA nanoparticles using confocal microscopy confirmed successful cellular uptake and transfection in HEK 293T cells. Further transfection study confirmed the postulation of layer depth- and time-dependent intracellular defoliation of the multilayer nanoparticles. Inhibitor studies using chemical inhibitors suggest preliminary findings that uptake of LbL-HA nanoparticles occurred predominantly through clathrin-mediated endocytosis. Uptake and transfection at 4˚C presents possible non-endocytic uptake that may be a result of CPP-resembling properties of the LbL-HA nanoparticles conferred by the pARG layers. Results show that LbL-HA nanoparticles are effective gene delivery vectors and the present study gives a valuable basic understanding of the complex cellular uptake process. Future studies can build on these findings to achieve a more thorough mechanistic understanding of the uptake pathways.