Drug transport in cell preparations with diffusional dosing and temporal ratiometry

Delivery of molecules into cells is of importance in many areas of biological research to probe cellular function and to evaluate their pharmacological effectiveness at target sites. One example of this is in the development of new drugs for cancer treatment. At the single cell level cancers have been found to exhibit resistance to anticancer drugs. Intense research into the causes of drug resistance has been carried out. Traditionally the efficacy of drug molecules have been determined by incubating cells in medium containing the drug which provides valuable information about cytotoxicity, membrane permeability etc. However these experiments do not provide information about localization as a function of the amount of drug that enters each cell. For information regarding localization of drug molecules within different parts of the cell, biochemical fractionation of intracellular organelles and analysis of drug content is carried out. Such biochemical fractionation and analysis have problems with time taken for the various steps to be carried out and possibly any fundamental changes to the properties of the cells due to the fractionation processes.;Here, we show the use of a microscopic diffusion port mounted at the tip of a micropipette, called the diffusional microburet (DMB) to effect continuous delivery of molecules into single live cells. Molecules and drugs are introduced into cells using many methods, e.g., incubation and microinjection, but common drawbacks of these methods are the inability to assess actual amounts delivered into cells, determining concentrations everywhere, perturbation to the cell due to added volume or washout of intracellular contents. The DMB delivers molecules purely by diffusion, without any addition of volume with reduced washout of cytosolic components and whose delivery is quantifiable.;Chapters 1 to 3 of this thesis cover the development and characterization of this delivery methodology together with the development of a novel method to determine dynamics of concentrations of the delivered molecule within the cell. In Chapter 1 tunable nature of the delivery which allows different rates of deliveries to be achieved is presented. Further, evidence to show that washout of intracellular constituents is reduced when using the DMB method to deliver molecules into cells over long time periods and validation of DMB delivery by comparing to incubation based methods of drug loading are presented. In Chapter 2 electrical measurements are added to the DMB delivery methodology to allow quantitative estimation of the actual mass of molecules delivered into a cell. The total mass balance of the delivered molecules is reconstructed at every point in time. In Chapter 3 a new analytical method, called temporal ratiometry, to determine concentrations of delivered fluorescent molecules as they are dosed into cells is presented. This method allows concentration maps within the cell to be created at each point in time. The methods presented in Chapters 2 and 3 allow analysis of amounts of the molecule of interest to be quantified at every location within the cell in real time, without involving fractionation or lysing of the cell.;The developed DMB approach and concentration measurement scheme is applied to two specific areas to demonstrate its use. The first application described in Chapter 4 is titration of intracellular binding sites of the anticancer drug doxorubicin (DOX) using delivery with the DMB. Temporal ratiometry is utilized to analyze concentrations of DOX and its dynamics in single cells during the loading process. In addition an analytical method to determine absolute DOX concentrations is detailed. For the first time dynamics of DOX concentrations during uptake and its slow diffusion are detailed. The second application pertains to the use of the DMB methodology to study cell-cell communication over sustained time periods to elucidate cell junctional permeabilities as described in Chapter 5.;As an offshoot of diffusive delivery, an idea to deliver substances using convective delivery was also developed. This is described in Chapter 6. With this technology flow rates down to nL/s can be achieved, which is very useful in preparation of solutions at very low concentrations and also to microinject solutions into biological samples such as cells.