Continuous and quantitative delivery of molecules into individual cells with a diffusional microburet.

Abstract

Direct delivery of molecules into the cytosol of live cells is required in many areas of biology and clinical research. Molecules of interest include indicator dyes, biomolecules, and pharmacological agents. In this work we describe continuous delivery of molecules into single cells using a diffusional microburet, DMB. The DMB is a pulled glass micropipette with a fine tip that contains a microscopic plug made of a hydrogel such as agar or polyacrylamide. This plug prevents flow but allows diffusive delivery of the molecule of interest from the DMB body into the cytosol, driven by its concentration gradient. This leads to a scheme of sustained intracellular dosing that is highly reproducible and quantifiable yet does not require the addition of solution volume to the cell. Potential loss of biomolecules from the cytosol through the plug of the DMB can be greatly reduced by proper choice of the pore size and tortuosity of the hydrogel in the DMB tip. The intracellular concentration of fluorescent molecules during delivery can be obtained calibration free. In this work we demonstrate dosing of Lucifer Yellow CH, LY, a charged fluorescent dye, into individual a7r5 vascular smooth muscle cells with a DMB. New types of quantitative analytical experiments on single live cells that the DMB technology enables are titration of intracellular ions and ligands, binding sites, and efflux pathways such as those that are involved in drug resistance.