Abstract
Triggered release and targeted drug delivery of potent anti-cancer agents using hyperthermia-mediated focused-ultrasound (FUS) is gaining momentum in the clinical setting. In early phase studies, tissue biopsy samples may be harvested to assess drug delivery efficacy and demonstrate lack of instantaneous cell death due to FUS exposure. We present an optimised tissue cell recovery method and a cell viability assay, compatible with intra-cellular doxorubicin. Flow cytometry was used to determine levels of cell death with suspensions comprised of: (i) HT29 cell line exposed to hyperthermia (30 min at 47 °C) and/or doxorubicin, or ex-vivo bovine liver tissue exposed to (ii) hyperthermia (up to 2 h at 45 °C), or (iii) ablative high intensity FUS (HIFU). Flow cytometric analysis revealed maximal cell death in HT29 receiving both heat and doxorubicin insults and increases in both cell granularity (p < 0.01) and cell death (p < 0.01) in cells recovered from ex-vivo liver tissue exposed to hyperthermia and high pressures of HIFU (8.2 MPa peak-to-peak free-field at 1 MHz) relative to controls. Ex-vivo results were validated with microscopy using pan-cytokeratin stain. This rapid, sensitive and highly quantitative cell-viability method is applicable to the small masses of liver tissue typically recovered from a standard core biopsy (5–20 mg) and may be applied to tissues of other histological origins including immunostaining.
Highlights
Triggered release and targeted drug delivery of potent anti-cancer agents using hyperthermiamediated focused-ultrasound (FUS) is gaining momentum in the clinical setting
Flow cytometric cell viability analysis of HT29 exposed to doxorubicin and/or hyperthermia
Preliminary work demonstrated that doxorubicin did not interfere with the L/D stain emission spectra, which could clearly be distinguished by the far-red detector (660 ± 16 nm) of the flow cytometer and demonstrated absence of significant cell death at two hours with 97.5% of cells being viable, as demonstrated by only weak L/D staining (Fig. 3)
Summary
Triggered release and targeted drug delivery of potent anti-cancer agents using hyperthermiamediated focused-ultrasound (FUS) is gaining momentum in the clinical setting. The lyso-thermosensitive liposomal doxorubicin (LTLD) formulation (ThermoDox), a thermally activated liposomal drug delivery system developed under Celsion’s investigational drug program, has demonstrated relative stability at body temperature (37 °C) allied to the capacity to release free drug at temperatures greater than 39.5 °C5,6 Such formulations may be ideal oncological drug-delivery systems when used in combination with FUS-mediated mild tumour hyperthermia (increases of around 3–5 °C), thereby achieving targeted intratumoral drug release. This approach relies on sub-ablative levels of hyperthermia and methods of validating the mechanism of subsequent cell death are great importance in early studies; significant over-heating will cause direct tissue ablation, as with conventional HIFU, rather than desired chemo-ablation due to localized release from LTLD. Early ablation may even damage tumour vasculature to the extent that it can no longer support supply therapeutic levels of LTLD to the tumour
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