(Download) "Drug Transport in Cell Preparations with Diffusional Dosing and Temporal Ratiometry" by Prasad Oruganti # Book PDF Kindle ePub Free
eBook details
- Title: Drug Transport in Cell Preparations with Diffusional Dosing and Temporal Ratiometry
- Author : Prasad Oruganti
- Release Date : January 19, 2013
- Genre: Medical,Books,Professional & Technical,
- Pages : * pages
- Size : 19883 KB
Description
The introduction of specific molecules into single cells is a widely used approach to probe cellular mechanisms. For example, to better understand the complex phenomenon of cancer drug resistance due to sequestration, binding and efflux, drug molecules are introduced into cancer cells. To probe cell-cell communication tracer molecules are delivered into connected cells and their junctional permeabilities are estimated.Existing methods for delivering molecules into cells include the injection of discrete boluses of material using mechanical or electro-mechanical principles, and continuous perfusion from a patch pipette in the whole cell configuration. However with these methods delivery cannot be quantified, or can introduce stress to the cells due to volume addition, or can cause excessive washout of cytosolic content.In this work we report on the use of a diffusional microburet that is a micropipette with a diffusion port at its tip to deliver molecules into an impaled target cell, quantifiably and with reduced washout of intracellular constituents. A new ratiometry technique called temporal ratiometry has also been developed in this work to provide dynamic time resolved concentration maps of the delivered molecules within the cell. These methods have been used to determine junctional permeabilities between connected A7r5 cells over a 30 min time frame. The DMB has also been used to dose the anticancer drug doxorubicin into the human breast cancer cell line MCF-7 and the rat epithelial cell line NRKE to titrate intracellular drug binding sites. Doxorubicin was found to accumulate in the nucleus and slow propagation at binding sites at the rate of ~2.0 ± 0.8 micrometers/min (n=4 cells) were observed for the first time. The methods presented in this work can make new types of experiment, such as continuous and quantitative titrations of binding sites, sequestration processes, and efflux pathways on single cells feasible.As an offshoot of diffusive dosing, we also report on the development of a delivery device that uses hydrodynamic resistance in the path of flow to effect very slow deliveries in the order of nL/s to make ultra low concentration solutions in a single step.