Unveiling role of serum albumin in disaggregation and cellular delivery of a near-infrared chlorophyll-based photosensitizer in breast cancer cells.

The efficacy of photosensitizer (PS) for photodynamic therapy of malignant tumors depends significantly on its interaction with serum proteins. Albumin binds non-covalently with hydrophilic and amphiphilic PSs. However, a clear understanding on role of albumin in delivery of PS in cancer cells is still lacking. We explored the role of albumin in disaggregation and cellular uptake of Cycloimide Purpurin-18 (CIPp-18), an amphiphilic near-infrared PS, in human breast carcinoma (MCF-7) cells. Results show that CIPp-18 added to fetal bovine serum (FBS) associates mainly with albumin and its aggregation in neat buffer is completely reversed by addition of bovine serum albumin (BSA). Under serum-enriched condition, CIPp-18 accumulated efficiently in cells and localized mainly in cell membrane and ER but not in lysosomes. Accumulation of CIPp-18 in cells was not affected by inhibitors of metabolic energy but partially inhibited at cold temperature. Confocal microscopy studies on uptake of CIPp-18 complexed to FITC-labeled BSA revealed significant colocalization of CIPp-18 and FITC-BSA in the distinct regions of cell membrane at 15 min after incubation, whereas at 90 min, FITC albumin localized independently in endocytic vesicles. Studies on binding of CIPp-18 with albumin using intrinsic tryptophan fluorescence of BSA and in silico docking reveal that CIPp-18 binds near Sudlow site I but away from tryptophan residue. Significant quenching of fluorescence of BSA-bound CIPp-18 by iodide ions further confirms that CIPp-18 binds at the surface of albumin. These results together demonstrate that the cellular uptake of albumin-bound CIPp-18 is mediated via facilitated diffusion involving dissociation of CIPp-18 from albumin prior to endocytosis. Further, the binding of CIPp-18 on the surface of albumin appears to play a crucial role in disaggregation and cellular uptake of CIPp-18.