Characterization and enhancement of interstitial gene transport during electric field-mediated gene delivery to solid tumor

In this dissertation, electric field-induced interstitial transport was characterized using a single molecule detection method in vitro, and quantified in vivo using an intravital confocal microscopy technique. In addition, novel combination therapies were investigated to further enhance interstitial gene transport during electric field-mediated gene delivery.;The electromobility behavior of single DNA molecules was investigated in the presence of pulsed, high field strength electric fields, typical of those used during electric field-mediated gene delivery. The observation of a single molecule provided a qualitative understanding of the electromobility behavior and provided insight into the mechanisms behind the electromobility dependence on several of the applied pulsing parameters that has been seen elsewhere. The electromobility dependence on pulse strength, pulse duration, and interstitial matrix pore size were investigated. In addition, results of this study revealed a strong electromobility dependence on pulse interval, and the existence of a minimum pulse amplitude required to initiate electrophoretic transport. These findings provide important insight into the mechanisms of DNA electromobility in tissues, which are useful for improving electric field-mediated gene delivery in tumors.;To understand how electrophoresis influences pDNA delivery in vivo, the magnitude of electric field-induced interstitial transport of pDNA was quantified in 4T1 and B16.F10 tumors implanted in mouse dorsal skin-fold chamber (DSC) models. Four different electric pulse sequences were used in this study, each consisted of 10 identical pulses that were 100 or 400 V/cm in strength and 20 or 50 ms in duration. The interval between consecutive pulses was 1 s. The largest distance of transport was obtained with the 400 V/cm and 50 ms pulse, and was 0.23 and 0.22 μm/pulse in 4T1 and B16.F10 tumors, respectively. There were no significant differences in transport distances between 4T1 and B16.F10 tumors. Histological examination and a hydroxyproline assay correlated interstitial transport with tissue collagen content. Results from in vivo mapping and numerical simulations revealed an approximately uniform intratumoral electric field that was predominantly in the direction of the applied field. The data in the study suggested that interstitial transport of pDNA induced by a sequence of 10 electric pulses was ineffective for macroscopic delivery of genes in tumors, and that additional treatment must be given to the tissue if such transport is to be achieved. However, it is important to note that the induced transport was more efficient than passive diffusion, and may still play a critical role in electric field-mediated gene delivery.;Since electric field treatment with a 10-pulse sequence alone was deemed insufficient to elicit in vivo electrophoresis beyond 2.5 μm, two combination treatments, to be used with pulsed electric field, were investigated for their ability to further enhance interstitial gene transport. The first combination treatment investigated attempted to increase the magnitude of in vivo electrophoresis by treating tumor bearing mice with recombinant human relaxin (rh-Rlx), a peptide hormone known to induce interstitial collagen remodeling in solid tumor. In addition, this study suggested that pDNA-collagen binding may contribute to the limited interstitial mobility of pDNA, and the correlation of mobility with interstitial collagen content.;The second combination treatment investigated to improve the electric field-induced interstitial pDNA transport targeted cells of the tumor using the hyperosmotic agent, mannitol. In vitro and ex vivo kinetic studies showed 4T1 and B16.F10 tumor cells, suspended in a 1% agarose gel matrix, were reduced to 50 and 54 %, respectively, of their original volume 20 mintues following exposure to 1 M mannitol. In vivo gene expression studies indicated electric field treatment significantly increased gene expression in 4T1 and B16.F10 hind leg tumor. Furthermore, pretreatment of tumor with 1M manntiol solution resulted in a further enhancement of gene expression in both 4T1 and B16.F10 tumor, beyond that obtained with electric field treatment alone. In vitro expression studies, which did not show mannitol to effect gene expresson with electric field treatment of cells in suspension, indicated that the improved expression observed was due to an extracellular event, most likely enhanced intersitial transport. However, in vivo electromobility studies using the DSC tumor model were unable to directly correlate improved gene expression with enhanced interstitial transport.