Limited utility of acetoxymethyl (AM)-based intracellular delivery systems, in vivo: interference by extracellular...

The use of acetoxymethyl (AM) groups to deliver and trap exogenous optical probes inside cells is an established tool in cell biology/physiology, however, these probes have not been used extensively in vivo. In this study, the use of the acetoxymethyl delivery system for optical probes was evaluated, in vivo. Initial studies revealed very little trapped probe in intact tissues even when near saturating levels of probe were injected in living animals. We tested the hypothesis that extracellular esterases rapidly cleave the acetoxymethyl groups preventing the probes from entering cells, in vivo. The rates of hydrolysis of 11 acetoxymethyl probes in diluted porcine plasma revealed an essentially first order high rate dye cleavage with half times on the order of minutes or less. Studies on mice and rabbits revealed rates 10- to 2-fold higher, respectively. These plasma studies suggested that the acetoxymethyl probes were being cleaved before having a chance to enter cells in tissues in vivo. This was confirmed using intravital 2-photon excitation microscopy in muscle tissue where several acetoxymethyl probes were found to rapidly cleave in the vascular space during infusion and not be trapped in the muscle cells. Studies with succinimidyl esters that should quickly bind to proteins on cleavage also failed to enter cells, in vivo, consistent with the notion that the cleavage was occurring in the extracellular space. These data suggest that the high level of plasma and extracellular esterase activity render the classical acetoxymethyl probes ineffective for monitoring intracellular events, in vivo. Different approaches to trapping exogenous probes will need to be explored for physiological studies using intravital microscopy.