Armin W. Opitz, University of Delaware, Newark, DE, Kirk J. Czymmek, University of Delaware, Newark, DE, Eric Wickstrom, Thomas Jefferson University, Philadelphia, PA, and Norman J. Wagner, University of Delaware, Newark, DE.
A method to study the kinetics of internalization and release of fluorescently labeled nanoparticles into and out of mammalian cells using confocal microscopy is introduced. The technique is established using a third generation poly(amido amine) (PAMAM) dendrimer labeled with an Alexa Fluor 555 dye and Capan-1 pancreatic cancer cells labeled with green cell tracker dye. Using fluorescence confocal microscopy, the uptake of the labeled dendrimer into a cluster of cells is recorded as a function of time. The cell tracker dye allows for a clear boundary of the cell membrane in the image as well as quantification of the intensity values in the green and red channel recorded by the microscope. A dilution curve of the fluorescently labeled PAMAM dendrimer allows for conversion of the recorded intensity values inside the cell to actual concentration values. A simple mass transfer model is proposed to describe the uptake and release behavior of the PAMAM dendrimer. Using the model a mass transfer coefficient as well as rate constant of the PAMAM dendrimer into the Capan-1 cells is obtained. The release kinetics of the PAMAM dendrimer from the cell is complicated by a bound fraction.