Topical delivery of hydrophilic macromolecules from various formulations.

Abstract

The advantages of topical delivery of gene vectors to cells within the skin include the possibility of direct <italic>in vivo</italic> transfer of the gene to epidermal and dermal cells. However, plasmid DNA delivery into skin across intact stratum corneum is highly unlikely due to the large size of the molecules, hydrophilicity and the presence of numerous negatively charged groups. In addition, the degradative effects of bacterial exonuleases in and on the skin on plasmid integrity introduces a more serious hurdle to successful transfection following topical application. Previous studies with nonionic lipid-based liposomal formulations had indicated their ability to facilitate transport of a variety of compounds into skin <italic>via</italic> a transfollicular pathway. Their failure to improve transfection characteristics following topical application of nonionic-based liposomal plasmid formulations containing cationic lipids was found to be related to: (a) the inability of these liposomal formulations to prevent plasmid degradation by skin and (b) severe interparticle aggregation of the liposome-plasmid complexes upon partial dehydration of the liposomes. The latter prevented efficient delivery of the plasmid into hair follicles. It therefore became necessary to develop and design non-liposomal formulations that would address plasmid integrity characteristics as well as facilitate transport of the plasmid into the sebum-filled hair follicle environment. The results of studies presented in this dissertation indicate that water-in-oil microemulsions possess attributes that not only allow for efficient plasmid incorporation and for stability upon storage for extended periods of time but also provide substantial protection of the plasmid from nuclease degradation upon contact with the skin. The results of disposition and preliminary microautoradiographic studies following topical application of these plasmid formulations to hairless mouse and rhino mouse skin suggest that transport of the plasmid from water-in-oil plasmid microemulsions occurs <italic>via</italic> a transfollicular pathway. These findings are also supported by <italic>in situ</italic> PCR studies that indicate the presence of plasmid in perifollicular cells and/or distributed around hair follicles of hairy mice following topical <italic>in vivo</italic> application of water-in-oil plasmid microemulsions. Finally, positive results of studies of transfection following topical <italic> in vivo</italic> application of a water-in-oil pCF1CAT microemulsion to hairless mouse skin demonstrated the utility of water-in-oil plasmid microemulsions to induce elevated and reproducible levels of expression of an exogenous gene marker in skin tissues. Neither simple plasmid aqueous solutions nor cationic liposomal plasmid formulations or water-in-oil microemulsions containing an irrelevant plasmid were able to elicit CAT expression beyond background levels following their topical application to hairless mouse skin.