Characterizing the dynamics of fluorescent molecule delivery using reversible membrane permeabilization with streptolyosin-O

This study quantifies the delivery of single molecule-sensitive probes with the use of Steptolysin-O (SLO) reversible membrane permeabilization in the cytosol of A549 lung carcinoma cells. RSVcy3b MTRIPs (multiply labeled tetravalent RNA imaging probes) were delivered into noninfected A549 cells in increasing concentrations using a fixed concentration of SLO. Increase in MTRIP delivery concentrations positively correlated to high MTRIP concentrations in the cytoplasm of these cells. Conversely, the use of varying SLO concentrations with a fixed MTRIP concentration exhibited no distinct change in intracellular MTRIP concentrations, except for high SLO concentrations specifically at 50µ mL-1. The effects of MTRIP delivery with SLO were also observed in MDCK, Vero, U2OS, and HEp2 cells. For any noticeable differences in untargeted MTRIP delivery, a higher concentration of SLO was used than in the A549 delivery cases. Significant differences in intracellular MTRIP concentrations were observed in MDCK, U2OS, and HEp2 cells. However, even at high SLO concentrations, the untargeted, intracellular MTRIP concentration was not significant in Vero cells. This understanding of the dynamics of SLO delivery for fluorescent probes is crucial to targeting endogenous RNA and proteins to improve in vitro imaging techniques.Faculty Mentor: Santangelo, Phili