Titanium dioxide nanoparticles (TiO₂NPs) suspensions (concentration 1.0 g/L) in synthetic sweat solution were applied on Franz cells for 24 h using intact and needle-abraded human skin. Titanium content into skin and receiving phases was determined. Cytotoxicity (MTT, AlamarBlue^® and propidium iodide, PI, uptake assays) was evaluated on HaCat keratinocytes after 24 h, 48 h, and seven days of exposure. After 24 h of exposure, no titanium was detectable in receiving solutions for both intact and damaged skin. Titanium was found in the epidermal layer after 24 h of exposure (0.47 ± 0.33 μg/cm²) while in the dermal layer, the concentration was below the limit of detection. Damaged skin, in its whole, has shown a similar concentration (0.53 ± 0.26 μg/cm²). Cytotoxicity studies on HaCaT cells demonstrated that TiO₂NPs induced cytotoxic effects only at very high concentrations, reducing cell viability after seven days of exposure with EC₅₀s of 8.8 × 10⁻⁴ M (MTT assay), 3.8 × 10⁻⁵ M (AlamarBlue^® assay), and 7.6 × 10⁻⁴ M (PI uptake, index of a necrotic cell death). Our study demonstrated that TiO₂NPs cannot permeate intact and damaged skin and can be found only in the stratum corneum and epidermis. Moreover, the low cytotoxic effect observed on human HaCaT keratinocytes suggests that these nano-compounds have a potential toxic effect at the skin level only after long-term exposure.

Skin absorption and toxicity on keratinocytes of cobalt oxide nanoparticles (Co₃O₄NPs) have been investigated. Co₃O₄NPs are commonly used in industrial products and biomedicine. There is evidence that these nanoparticles can cause membrane damage and genotoxicity in vitro, but no data are available on their skin absorption and cytotoxicity on keratinocytes. Two independent 24 h in vitro experiments were performed using Franz diffusion cells, using intact (experiment 1) and needle-abraded human skin (experiment 2). Co₃O₄NPs at a concentration of 1000 mg/L in physiological solution were used as donor phase. Cobalt content was evaluated by Inductively Coupled–Mass Spectroscopy. Co permeation through the skin was demonstrated after 24 h only when damaged skin protocol was used (57 ± 38 ng·cm⁻²), while no significant differences were shown between blank cells (0.92 ± 0.03 ng cm⁻²) and those with intact skin (1.08 ± 0.20 ng·cm⁻²). To further investigate Co₃O₄NPs toxicity, human-derived HaCaT keratinocytes were exposed to Co₃O₄NPs and cytotoxicity evaluated by MTT, Alamarblue^® and propidium iodide (PI) uptake assays. The results indicate that a long exposure time (i.e., seven days) was necessary to induce a concentration-dependent cell viability reduction (EC₅₀ values: 1.3 × 10⁻⁴ M, 95% CL = 0.8–1.9 × 10⁻⁴ M, MTT essay; 3.7 × 10⁻⁵ M, 95% CI = 2.2–6.1 × 10⁻⁵ M, AlamarBlue^® assay) that seems to be associated to necrotic events (EC₅₀ value: 1.3 × 10⁻⁴ M, 95% CL = 0.9–1.9 × 10⁻⁴ M, PI assay). This study demonstrated that Co₃O₄NPs can penetrate only damaged skin and is cytotoxic for HaCat cells after long term exposure.