Pressure-mediated oligonucleotide transfection of rat and human cardiovascular tissues

The application of gene therapy to human disease is currently restricted by the relatively low efficiency and potential hazards of methods of oligonucleotide or gene delivery. Antisense or transcription factor decoy oligonucleotides have been shown to be effective at altering gene expression in cell culture expreriments, but their in vivo application is limited by the efficiency of cellular delivery, the intracellular stability of the compounds, and their duration of activity. We report herein the development of a highly efficient method for naked oligodeoxynucleotide (ODN) transfection into cardiovascular tissues by using controlled, nondistending pressure without the use of viral vectors, lipid formulations, or exposure to other adjunctive, potentially hazardous substances. In this study, we have documented the ability of ex vivo, pressure-mediated transfection to achieve nuclear localization of fluorescent (FITC)-labeled ODN in approximately 90% and 50% of cells in intact human saphenous vein and rat myocardium, respectively. We have further documented that pressure-mediated delivery of antisense ODN can functionally inhibit target gene expression in both of these tissues in a sequence-specific manner at the mRNA and protein levels. This oligonucleotide transfection system may represent a safe means of achieving the intraoperative genetic engineering of failure-resistant human bypass grafts and may provide an avenue for the genetic manipultation of cardiac allograft rejection, allograft vasculopathy, or other transplant diseases.

Drying Increases Intracellular Partitioning of Amphiphilic Substances into the Lipid Phase1 : Impact on Membrane Permeability and Significance for Desiccation Tolerance

Previously we proposed that endogenous amphiphilic substances may partition from the aqueous cytoplasm into the lipid phase during dehydration of desiccation-tolerant organ(ism)s and vice versa during rehydration. Their perturbing presence in membranes could thus explain the transient leakage from imbibing organisms. To study the mechanism of this phenomenon, amphiphilic nitroxide spin probes were introduced into the pollen of a model organism, Typha latifolia, and their partitioning behavior during dehydration and rehydration was analyzed by electron paramagnetic resonance spectroscopy. In hydrated pollen the spin probes mainly occurred in the aqueous phase; during dehydration, however, the amphiphilic spin probes partitioned into the lipid phase and had disappeared from the aqueous phase below 0.4 g water g−1 dry weight. During rehydration the probes reappeared in the aqueous phase above 0.4 g water g−1 dry weight. The partitioning back into the cytoplasm coincided with the decrease of the initially high plasma membrane permeability. A charged polar spin probe was trapped in the cytoplasm during drying. Liposome experiments showed that partitioning of an amphiphilic spin probe into the bilayer during dehydration caused transient leakage during rehydration. This was also observed with endogenous amphipaths that were extracted from pollen, implying similar partitioning behavior. In view of the fluidizing effect on membranes and the antioxidant properties of many endogenous amphipaths, we suggest that partitioning with drying may be pivotal to desiccation tolerance, despite the risk of imbibitional leakage.