Correction for photobleaching in dynamic fluorescence microscopy: application in the assessment of pharmacokinetic parameters in ultrasound-mediated drug delivery

M Derieppe,M De Greef,C Moonen,C Bos,B Denis De Senneville

doi:10.1088/0031-9155/61/2/588

Abstract

We have previously demonstrated the feasibility of monitoring ultrasound-mediated uptake of a hydrophilic model drug in real time with dynamic confocal fluorescence microscopy. In this study, we evaluate and correct the impact of photobleaching to improve the accuracy of pharmacokinetic parameter estimates.To model photobleaching of the fluorescent model drug SYTOX Green, a photobleaching process was added to the current two-compartment model describing cell uptake. After collection of the uptake profile, a second acquisition was performed when SYTOX Green was equilibrated, to evaluate the photobleaching rate experimentally.Photobleaching rates up to 5.0 10−3 s−1 were measured when applying power densities up to 0.2 W.cm−2. By applying the three-compartment model, the model drug uptake rate of 6.0 10−3 s−1 was measured independent of the applied laser power.The impact of photobleaching on uptake rate estimates measured by dynamic fluorescence microscopy was evaluated. Subsequent compensation improved the accuracy of pharmacokinetic parameter estimates in the cell population subjected to sonopermeabilization.

Highlights

Local drug delivery in oncology aims at increasing the dose of an anticancer agent at the tumour site while limiting its concentration in the general circulation [1,2]
We demonstrated in vitro the feasibility to monitor in real-time the USand microbubble-mediated permeabilization of a SYTOX Green model drug using a fibered confocal fluorescence microscopy (FCFM) system that avoids the geometrical constraints of standard microscope setups
In the range of uptake rates and PB rates obtained experimentally using the 3CMref (Eq 3), the sensitivity of the three-compartment model was evaluated for 3 time windows

Summary

Introduction

Local drug delivery in oncology aims at increasing the dose of an anticancer agent at the tumour site while limiting its concentration in the general circulation [1,2]. To increase the permeability of these biological barriers towards compounds exhibiting a low lipophilicity, strategies exploiting acoustic cavitation generated by ultrasound waves (US) have been devised in a localized and non-ionizing fashion [3,4,5,6,7] Injected intravenously, these gas-filled micrometric particles remain located in the vasculature and induce permeability of the endothelial cells, facilitating drug extravasation of therapeutic agents that exhibit poor transendothelial transport. These gas-filled micrometric particles remain located in the vasculature and induce permeability of the endothelial cells, facilitating drug extravasation of therapeutic agents that exhibit poor transendothelial transport Monitoring of these processes in vitro at the cellular level and in vivo at the endothelialbarrier level is of special interest to optimize US-mediated delivery protocols and gain insight in US bioeffects. With a molecular weight close to that of anticancer agents, these fluorophores, e.g. SYTOX Green, 600 Da or TO-PRO-3, 671 Da, can be considered models for low molecular weight anticancer drugs

Methods

Results

Conclusion