Elucidating the cellular uptake mechanisms of heptamethine cyanine dye analogues for their use as an anticancer drug-carrier molecule for the treatment of glioblastoma.

The development of chemotherapies for glioblastoma is hindered by their limited bioavailability and toxicity on normal brain function. To overcome these limitations, we investigated the structure-dependent activity of heptamethine cyanine dyes (HMCD), a group of tumour-specific and BBB permeable near-infrared fluorescent dyes, in both commercial (U87MG) and patient-derived GBM cell lines. HMCD analogues with strongly ionisable sulphonic acid groups were not taken up by patient-derived GBM cells, but were taken up by the U87MG cell line. HMCD uptake relies on a combination of transporter uptake through organic anion-transporting polypeptides (OATPs) and endocytosis into GBM cells. The uptake of HMCDs was not affected by p-glycoprotein efflux in GBM cells. Finally, we demonstrate structure-dependent cytotoxic activity at high concentrations (EC50 : 1-100 µM), likely due to mitochondrial damage-induced apoptosis. An in vivo orthotopic glioblastoma model highlights tumour-specific accumulation of our lead HMCD, MHI-148, for up to 7 days following a single intraperitoneal injection. These studies suggest that strongly ionisable groups like sulphonic acids hamper the cellular uptake of HMCDs in patient-derived GBM cell lines, highlighting cell line-specific differences in HMCD uptake. We envisage these findings will help in the design and structural modifications of HMCDs for drug-delivery applications for glioblastoma.

ß-Galactosidase-Catalyzed Fluorescent Reporter Labeling of Living Cells for Sensitive Detection of Cell Surface Antigens.

The ability to detect cell surface proteins using fluorescent-dye-labeled antibodies is crucial for the reliable identification of many cell types. However, the different types of cell surface proteins used to identify cells are currently limited in number because they need to be expressed at high levels to exceed background cellular autofluorescence, especially in the shorter-wavelength region. Herein we report on a new method, quinone methide-based catalyzed labeling for signal amplification (CLAMP), in which the fluorescence signal is amplified by an enzymatic reaction that strongly facilitates the detection of cell surface proteins on living cells. We used ß-galactosidase as an amplification enzyme and designed a substrate for it, called MUGF, that contains a fluoromethyl group. Upon removal of the galactosyl group in MUGF by ß-galactosidase labeling of the target cell surface proteins, the resulting product containing the quinone methide group was found to be both cell-membrane-permeable and reactive with intracellular nucleophiles, thereby providing fluorescent adducts. Using this method, we successfully detected several cell surface proteins, including programmed death ligand 1 protein, which is difficult to detect using conventional fluorescent-dye-labeled antibodies.