Anti-inflammatory and anti-cancer activity of citral: Optimization of citral-loaded solid lipid nanoparticles (SLN) using experimental factorial design and LUMiSizer®

Essential oils containing monoterpenes are widely used in pharmaceuticals and cosmetic products on account of their wide range of bioactive properties (including anti-cancer activity). Two monoterpenes (citral and geraniol) were firstly tested for their anti-inflammatory activity in a RAW 264.7 cell line, demonstrating citral to have enhanced capacity to inhibit NO production (ca. 84% for citral and 52% for geraniol at the lowest tested concentration of 5 mu g/ml). As citral showed higher NO inhibitory activity than geraniol, to measure the level of cytotoxicity of citral, AlamarBlue reduction assay was run in two cell models (non-tumoral HaCaT and tumoral A431). Citral exhibited a strong cytotoxic effect in both cell lines, i.e. cell viability lower that 10% after 24 h exposure at 100 mu g/ml of monoterpene. An optimized solid lipid nanoparticles (SLNs) formulation for citral was further developed by design of experiments (2(2) factorial design), followed by accelerated stability testing (LUMiSizer (R)). An optimal SLN composed of 1 wt% of citral, 4 wt% of lipid and 2.5 wt% surfactant were successfully produced by hot high pressure homogenization (hot HPH) showing a mean particle size (Z-Ave) of 97.7 nm and polydispersity index of 0.249. The produced formulations were analyzed in a high-end dispersion analyzer LUMiSizer (R) to characterize any demixing phenomena, demonstrating to be long-term stable at room temperature (25 degrees C), exhibiting very low instability indices (0.032 after production and 0.042 after one month of storage).