Image Cytometry: Versatile Tool for Probing Cellular Uptake Kinetics of Biodegradable Nanoparticles

Biodegradable nanoparticles have been extensively investigated to facilitate intracellular delivery of therapeutics. Uptake kinetic studies and subsequent quantitative analysis are important in order to develop efficient drug delivery vehicle. In the present work, image cytometry is used for studying intra cellular uptake kinetics of biodegradable nanoparticles with two model systems- HeLa and Caco-2 cells. Nanoparticles (NPs) were formulated using biodegradable Poly D, L-lactic- co -Glycolic Acid (PLGA) polymer containing 6-coumarin as a fluorescent marker and characterized for size, surface morphology, surface charge, loading efficiency and encapsulation efficiency. Cells were incubated with coumarin loaded NPs for defined time intervals (0 to 2 hours) and at different temperature conditions (4°C, 25°C and 37°C). Intracellular amount of nanoparticles (in terms of its fluorescence intensity) was quantified by analyzing the captured fluorescent images of cells in MATLAB (using self-written algorithms). Both types of cells took up PLGA NPs quite readily with similar uptake profile and saturation was observed after one hour incubation period. The internalization of NPs was found to be more in Caco-2 cells when compared with HeLa, but the initial rate of uptake was found to be higher in HeLa cells. Also, no statistical difference in uptake kinetics was observed between synchronized and non-synchronized cells. The internalization of NPs was confirmed by confocal laser scanning microscopy. We report for the first time rigorous image analysis using MATLAB for studying uptake kinetics of NPs thus providing a new quantitative tool in studying drug delivery. Also, the results described herein will enhance our basic understanding of the cellular uptake of biodegradable NPs with cells in vitro which in turn could help in developing efficient nanocarrier systems for intracellular delivery of with Cellular nanoparticle uptake studies: Both HeLa and Caco-2 cells (in separate sets of experiments) were seeded in 35 mm diameter tissue culture petridishes and were allowed to grow till 80% confluency. A suspension of nanoparticles (2 mg/ml) in DMEM was prepared as stock The medium in the petri dishes was replaced with the suspension of the nanoparticles (100µg/ml) and incubated for different time intervals 0 min, 15 min, 30 min, 60 min, 90 min and 120 min. After specific time durations the cells were washed three times with DPBS (with Ca/Mg) to remove any non- cell associated nanoparticles [10,19]. Then the cells were fixed with 4% formaldehyde [22] and were observed using an inverted fluorescence microscope IX51 Olympus Inc., Japan). Green channel data for Coumarin (Co) visualization was acquired by using the mirror unit U-MWB2 (Olympus Inc., with excitation filter of 460-490 nm, emission filter of 520 nm and a dichromatic mirror at 500 nm. The images were acquired using a cooled CCD camera (DP70, Olympus Inc., Tokyo, Japan). The uptake studies were performed at temperatures 4°C, 25°C and 37°C independently for both cell lines. For each case the exposure time for image acquisition (using DP70) was kept same (fixed manually) to allow signal comparisons. drug delivery. We present comparison of cellular uptake kinetics in two model systems of fluorescent marker loaded biodegradable NPs using image cytometry. Data collected in our work demonstrates that PLGA nanoparticles were internalized in two different types of epithelial cell lines with similar uptake profile however initial uptake rate was observed to be high in HeLa cells than Caco-2 cells. An observed plateau effect of the cellular uptake efficiency against cell incubation time suggested that the cellular uptake of PLGA NPs is saturable suggesting receptor mediated endocytosis to be the pathway mitigating intracellular localization of PLGA NPs. No previous studies have shown such rigorous quantitative image analysis (in terms of its population and single cell assays) using MATLAB for studying intracellular uptake of PLGA NPs. Also proper controls like synchronization studies, comparison between live and fixed cells image data are not yet reported. The findings of our image analysis studies provides further insights in proving that PLGA nanoparticles have great potential to be an efficient drug delivery vehicle for different types of cells thus making image cytometry a versatile tool for performing quantitative analysis in drug delivery research.