Polymer Technology for Improving Gene Delivery

Gene therapy has great potential as a treatment for many human diseases by the transfer of therapeutic genes to cells. However, the lack of safe and efficient gene carrier systems is one of the primary limiting obstacles for the introduction of gene therapy into practical medicine. Current research focuses on two possible gene carrier systems: virus-based ( viral ) and non-viral vectors. While viral vectors have extremely high gene delivery performance, they carry a risk of causing adverse reactions. Non-viral vectors, in particular those formulated using synthetic polymers, have attracted the interest of an increasing number of researchers as safer alternatives that carry a greatly reduced risk of the adverse reactions that have plagued the viral approach. Despite its great potential, the polymer-based approach has yet been limited in its application in large part due to their poor biological activities. Here, barriers exist in various aspects: insufficient protection against agglomeration, enzymatic degradation, and non-specific adhesion to cells, tissues and proteins, during the extracellular delivery stage; relatively low efficiency of gene transfer and expression during the intracellular delivery stage. In this work, the knowledge of polymer technology is applied to improve our understanding of the extracellular and intracellular barriers associated with the polymer-based gene delivery systems and therefore provide a set of essential criteria for future design and development of better polymer-based gene carriers. (1) The role of proton-absorbing properties of positively-charged polymers ( polycations ) in the endosomal release process was investigated by potentiometric titration experiments and ab initio density functional theory (DFT) simulations. The results demonstrate that the proton-absorbing capacity of polycations for the pH change relevant to the endocytosis is not correlated with the endosomal escape potential of the corresponding polycation/DNA complexes. These results make an interesting contrast to the so-called proton sponge hypothesis that the higher the proton-absorbing capacity, the greater the endosomal escape potential. These findings led us to develop a novel hypothesis that the endosomal release process requires, in addition to the proton-sponge effect, the hydrophobic and electrostatically-driven adsorption of polycations to the endosomal membrane. (2) The role of DNA unpackaging in the transgene expression and nuclear entry was investigated using a photolytic gene carrier that is degradable upon exposure to mild UV irradiation. The results showed that the photo-degradation of the carrier, though it does not influence the nuclear localization, improves the transgene expression especially when the polymer/DNA complexes are located near (or inside) the cell nuclei. The improved transgene expression is possibly mediated by facilitated association of transcriptional machinery with DNA due to hydrophobic and anionic byproducts of the photo-degradation. (3) The role of poly(ethylene glycol) brush coating ( PEGylation ) in preventing aggregation of the coated particles was investigated by x-ray and neutron reflectivity measurements, self-consistent field theory calculations. The results showed that PEG chains are not hydrophilic when they exist as polymer brush chains. Even though the grafted PEG chains experience a poor solvent environment, the PEG brush layer exhibits positive surface pressures because the hydrophobicity of the PEG brush chains is insufficient to overcome the opposing effect of the chain conformational entropy. This finding gives a valuable insight into why the PEG brush chains in water are in general so effective in preventing aggregation of the coated particles