The MTT assay for cellular metabolic activity is almost ubiquitous to studies of cell toxicity; however, it is commonly applied and interpreted erroneously. We investigated the applicability and limitations of the MTT assay in representing treatment toxicity, cell viability, and metabolic activity. We evaluated the effect of potential confounding variables on the MTT assay measurements on a prostate cancer cell line (PC-3) including cell seeding number, MTT concentration, MTT incubation time, serum starvation, cell culture media composition, released intracellular contents (cell lysate and secretome), and extrusion of formazan to the extracellular space. We also assessed the confounding effect of polyethylene glycol (PEG)-coated gold nanoparticles (Au-NPs) as a tested treatment in PC-3 cells on the assay measurements. We additionally evaluated the applicability of microscopic image cytometry as a tool for measuring intracellular MTT reduction at the single-cell level. Our findings show that the assay measurements are a result of a complicated process dependant on many of the above-mentioned factors, and therefore, optimization of the assay and rational interpretation of the data is necessary to prevent misleading conclusions on variables such as cell viability, treatment toxicity, and/or cell metabolism. We conclude, with recommendations on how to apply the assay and a perspective on where the utility of the assay is a powerful tool, but likewise where it has limitations.

Nanoparticle radiosensitization has been well demonstrated to enhance effects of radiotherapy, motivated to improve therapeutic ratios and decrease morbidity in cancer treatment. A significant challenge exists in optimizing formulations and translation due to insufficient knowledge of the associated mechanisms which have historically been limited to physical concepts. Here we investigated a concept for the role of biological mechanisms. The mere presence of gold nanoparticles led to a down regulation of thymidylate synthase, important for DNA damage repair in the radioresistant S phase cells. By developing a cross-correlative methodology to reveal probabilistic gold nanoparticle uptake by cell sub-populations and the associated sensitization as a function of the uptake, a number of revealing observations have been achieved. Surprisingly, for low numbers of nanoparticles a desensitization action was observed. Sensitization was discovered to preferentially impact S phase cells where impairment of the DNA damage response by the homologous recombination pathway dominates. This small but radioresistant cell population correlates with much greater proliferative ability. Thus a paradigm is presented whereby enhanced DNA damage is not necessarily due to an increase in the number of DNA Double Strand Breaks (DSBs) created, but can be from a nanoparticle-induced impairment of the damage response by down regulating repair proteins such as thymidylate synthase.

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Zinc oxide nanoparticles (ZnO NPs) are a key constituent of many commercial broad-spectrum sunscreens. Studies have shown that these NPs are retained on the superficial layers of the skins' barrier layer, the stratum corneum, and solubilized zinc species from the ZnO NPs have been shown qualitatively to penetrate intact human skin. The cytotoxicity of zinc is concentrationand species-dependent; however, to date, the amount of zinc permeating the skin strata is yet to be determined. Here, we applied commercial ZnO NPs to intact and impaired ex vivo barrier human skin. Artificial human sweat (to provide an electrolyte solution) and caprylic capric triglyceride (CCT; a common sunscreen formulation base) suspensions were applied to encompass potential "inuse" scenarios. A state-of-the-art multimodal approach analyzed zinc permeation. Our data show that elevated zinc concentrations within the skin are dependent on a number of variables, with barrier impairment and time being the most important factors followed by the vehicle, where sweat was more impactful than CCT. When ZnO NPs were applied to impaired barrier skin for 24 h, there was a 60−65-fold increase in zinc in the viable epidermis for both CCT and sweat compared to the control, increasing >100-fold after 48 h. Importantly, we identify that the localized cutaneous zinc concentration increase is not present as the nano ZnO that is used in sunscreens but only after dissolution and permeation as a different solubilized zinc species.

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