ABSTRACT

One of the challenging requirements in cancer disease treatment today is maintaining high enough drug concentrations in order to obtain effective killing of cancer cells. Many attempts have been made to increase the intracellular concentrations of drugs that are based on "push" mechanism. In our novel approach we use a "pull" mechanism, meaning that we express a molecule inside the cell that has been selected specifically to bind with the drug molecule. The molecule expressed inside the cell is an aptamer. Aptamers are short, single stranded nucleic acid molecules ranging from about 15-100 nucleotides long. These small aptamer molecules are mobile inside the cells. Mathematical modeling of the effect of aptamer expression for increasing intra cellular drug concentration showed that this was possible. The initial model was developed for mammalian cells and the initial experimental testing of the hypothesis was done with Escherichia coli bacterial cells. Therefore now a new mathematical model for prokaryotic cells has been developed in parallel with the work described here. To test the hypothesis, E. coli BL21 strain cells were transformed with plasmids from which aptamers were expressed. The effect of expressing several aminoglycoside aptamers on the toxicity of a range of aminoglycosides was observed over a 12 h period. Then neomycin-B aptamer and the neomycin-B were selected for future studies. The half maximal inhibitory concentration (IC50), which is the concentration of the drug required for 50% growth inhibition, were calculated to be decreased by 2 fold with aptamer expression. The Minimum Inhibitory Concentration (MIC) was also ~2 fold lower for cells with aptamer expression as compared to cells with control RNA expression. To determine the effect of aptamer expression on the intracellular drug concentration we used Cy3-paromomycin. Cells with aptamer expression and cells with control RNA expression were incubated with Cy3-paromomycin and imaged directly or cell lysates were isolated and analyzed for fluorescence content by fluorimetry. Both the images and fluorimetry showed that cells with aptamer expression concentrate more Cy3- paromomycin compared to cells with control RNA expression. In summary, this study gives insight towards a novel approach to increase the intracellular drug concentration by the use of small, mobile RNA aptamers.