Technical Briefs

Freezing-Assisted Intracellular Drug Delivery to Multidrug Resistant Cancer Cells

[+] Author and Article Information
Ka Yaw Teo

Department of Mechanical and Aerospace Engineering, University of Texas at Arlington, Arlington, TX 76019

Bumsoo Han1

Department of Mechanical and Aerospace Engineering, University of Texas at Arlington, Arlington, TX 76019bhan@uta.edu


Corresponding author.

J Biomech Eng 131(7), 074513 (Jun 29, 2009) (4 pages) doi:10.1115/1.3153325 History: Received September 01, 2008; Revised April 27, 2009; Published June 29, 2009

The efficacy of chemotherapy is significantly impaired by the multidrug resistance (MDR) of cancer cells. The mechanism of MDR is associated with the overexpression of certain adenosine triphosphate-binding cassette protein transporters in plasma membranes, which actively pump out cytotoxic drugs from the intracellular space. In this study, we tested a hypothesis that freezing and thawing (F/T) may enhance intracellular drug delivery to MDR cancer cells via F/T-induced denaturation of MDR-associated proteins and/or membrane permeabilization. After a human MDR cancer cell line (NCI/ADR-RES) was exposed to several F/T conditions, its cellular drug uptake was quantified by a fluorescent calcein assay using calcein as a model drug. After F/T to 20°C, the intracellular uptake of calcein increased by 70.1% (n=5, P=0.0004). It further increased to 118% as NCI/ADR-RES cells were frozen/thawed to 40°C (n=3, P=0.009). These results support the hypothesis, and possible mechanisms of F/T-enhanced intracellular drug delivery were proposed and discussed.

Copyright © 2009 by American Society of Mechanical Engineers
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Figure 1

Sequence of quantitative fluorescence microscopy: (a) The contrast of bright field image was enhanced; (b) cell borders were highlighted; (c) the image was thresholded to distinguish cells from the background; (d) cell areas were chosen as regions of interest, and intensity values were extracted from the regions of interest in the corresponding fluorescent image

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Figure 2

Micrographs of cellular calcein accumulation and propidium iodide uptake after F/T. Intracellular calcein fluoresces green under FITC filter, and necrotic cells stained with propidium iodide appear red under TRITC filter. In drug sensitive cancer cells, calcein AM easily diffuses into the cells and is then converted into impermeable fluorescent calcein (FITC row-DS column). In MDR cancer cells, Pgp extrudes calcein AM, and only a small amount of fluorescent calcein accumulates in the cells (FITC row-MDR (unfrozen) column). After F/T, more fluorescent calcein accumulates in the MDR cancer cells, as well as cryoinjury is augmented (FITC and TRITC rows-MDR (−20°C and −40°C) columns).

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Figure 3

Quantitative intracellular calcein fluorescence intensity and cell viability after F/T. The average calcein fluorescence intensity per DS cancer cell is higher than that of MDR cancer cell (P∗∗∗<10−6). MDR cancer cells retain significantly more calcein after F/T to −20°C(P∗∗=0.0004). F/T to −40°C induces a significant increase in calcein accumulation in MDR cancer cells (P∗=0.009).

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Figure 4

Schematic of drug transport around a multidrug resistant cancer cell (modified from Ref. 2). Freezing and thawing are thought to facilitate intracellular drug delivery by targeting both drug influx and efflux mechanisms simultaneously. Increased drug influx may be associated with the permeabilization of lipid bilayer, and decreased drug efflux may be caused by the denaturation of MDR-associated proteins (Pgp).



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