Phenazine Cations as Anticancer Theranostics†.

The biological properties of two water-soluble organic cations based on polypyridyl structures commonly used as ligands for photoactive transition metal complexes designed to interact with biomolecules are investigated. A cytotoxicity screen employing a small panel of cell lines reveals that both cations show cytotoxicity toward cancer cells but show reduced cytotoxicity to noncancerous HEK293 cells with the more extended system being notably more active. Although it is not a singlet oxygen sensitizer, the more active cation also displayed enhanced potency on irradiation with visible light, making it active at nanomolar concentrations. Using the intrinsic luminescence of the cations, their cellular uptake was investigated in more detail, revealing that the active compound is more readily internalized than its less lipophilic analogue. Colocalization studies with established cell probes reveal that the active cation predominantly localizes within lysosomes and that irradiation leads to the disruption of mitochondrial structure and function. Stimulated emission depletion (STED) nanoscopy and transmission electron microscopy (TEM) imaging reveal that treatment results in distinct lysosomal swelling and extensive cellular vacuolization. Further imaging-based studies confirm that treatment with the active cation induces lysosomal membrane permeabilization, which triggers lysosome-dependent cell-death due to both necrosis and caspase-dependent apoptosis. A preliminary toxicity screen in the Galleria melonella animal model was carried out on both cations and revealed no detectable toxicity up to concentrations of 80 mg/kg. Taken together, these studies indicate that this class of synthetically easy-to-access photoactive compounds offers potential as novel therapeutic leads.

Development and validation of ELISA for herceptin detection in human serum.

Herceptin, a humanized anti-human epidermal growth factor receptor-2 (HER2) monoclonal antibody, is used for treatment of metastatic breast cancer patients overexpressing HER2 on tumor cells, and is being studied in clinical trials for therapy of other types of cancer. In the present work, we developed a Herceptin enzyme-linked immunosorbent assay (ELISA) from commercially available reagents to meet the growing needs of clinical studies. In this immunoassay, a mixture of monoclonal antibodies specific for the cytoplasmic domain of human HER2 (676-1255 amino acids) is adsorbed onto a microtiter plate, followed by addition of full-length HER2 protein, which is captured by the antibodies. Herceptin in the serum that is added to the microwells binds to the extracellular domain (ECD) of the captured HER2. The Herceptin bound to HER2 is detected by an antihuman IgG-horse radish peroxidase (HRP) conjugate and a (3, 5, 3', 5')-tetramethylenbenzidine (TMB) substrate. The calibration range of the assay is 5-100 ng/mL after 1:2000 sample dilution corresponding to 10-200 microg/mL Herceptin in undiluted serum. The intra- and interassay CVs ranged from 4.56% to 13.3% and from 9.9% to 18.9%, respectively. The assay shows dilutional linearity and specificity. Soluble p105 HER2, which can be shed into serum does not interfere with the assay. The analytical performance of the Herceptin ELISA indicates that this assay can be used for monitoring concentration levels of Herceptin in human serum.