SINGLE CELL MANIPULATION BY FEMTOSECOND LASERS AND VOLUMETRIC ANALYSIS OF CONVECTION ENHANCED DRUG DELIVERY

Abstract

Optoporation allows the minimal invasive transfer of extracellular molecules into cells. The use of a low repetition rate laser system (100 fs at 790 nm and 1 kHz repetition rate) can enable the future transfer of this technique to in vivo applications. However, it is essential to understand the biophysical parameter space of this technique beforehand. We examined the optoporation dependence on the extracellular calcium concentration in HeLa cells. We observed a low cell recovery rate in calcium free medium and varying cell viabilities with different calcium concentrations, possibly related to calcium dependent repair processes. Also we followed the calcium rise in the cell by a fluorescent calcium indicator. We extended our previous model of pore formation model and applied different sized dextrans to verify our assumptions on pore radius, and resealing time. Therefore, this study provides a better understanding of the biophysical processes accompanying single cell laser transfection. Drug delivery into the brain is inhibited by the blood brain barrier and limited by the structure of the brain interstitium. Convection-enhanced delivery (CED) is a technique that delivers therapeutics by infusing directly into brain parenchyma through a needle inserted into the brain. We developed a fast scanning, real time volumetric imaging platform to image transport of nanoparticles in the rat cortex in vivo with high temporal and spatial resolution and to study transport mechanisms for different sized nanoparticles in the brain interstitium and the perivascular space (PVS). Fluorescent polystyrene beads of sizes ranging from 20nm to 200nm were infused directly into a rat cortex. Particles sized >100nm had distribution routes along the perivascular spaces and were relatively hindered in the extracellular spaces in comparison to particles in the 20nm – 40nm size range that prefered transport in extracellular space. Thus, we can study the alterations in the shape, size and movement of the infusion in the brain, allowing for better control for potential clinical design and optimization