Evaluation of the ROS Inhibiting Activity and Mitochondrial Targeting of Phenolic Compounds in Fibroblast Cells Model System and Enhancement of Efficiency by Natural Deep Eutectic Solvent (NADES) Formulation.

PURPOSE: Many phenolics have already been tested for their antioxidant activities using in vitro methods. However, such assays do not consider the complexity of real cellular systems, and most of the phenolics characterized with such assays shows disappointing results when evaluated in cells. Accordingly, there is a need to develop effective screening methods. METHODS: Antioxidants were first evaluated by CAT assay and then, evaluated for their ability (i) to reduce the level of ROS using fluorescent probe, (ii) to cross fibroblast cell membranes using confocal microscopy, and (iii) to target mitochondria. Antioxidants were also formulated in NADES. RESULTS: Correlation was obtained when comparing CAT results with short term inhibition (2 h) in the fibroblast cells. On the contrary, it was difficult to anticipate ROS inhibiting efficiency at long term (24 h) from both the CAT assay and the short term inhibition measurements. Indeed, some molecules displayed activity rapidly but lost it over time. In contrast, other molecules were better for long term. The comparable efficiency at long term of Bis-Ethylhexyl Hydroxydimethoxy Benzylmalonate (Bis-EHBm) and decyl rosmarinate, prompted us to further investigate the potential mitochondrial targeting of the former. Using mitochondrial probes, our results confirmed its mitochondrial location. Finally, the formulation of antioxidants in NADES could greatly improve their activity. CONCLUSIONS: Combinations of fast acting and slow acting molecules could be promising strategies to identify a performant antioxidant system. Bis-EHBm behaves as decyl rosmarinate with a confirmed mitochondrial location. Finally, the formulation of antioxidants in NADES could greatly improve their activity for ROS inhibition.

Factorial design approach to evaluate interactions between electrically assisted enhancement and skin stripping for delivery of tacrine.

The objective of this work was to study the mechanisms of action of iontophoresis and electroporation and their interaction effects on delivery of tacrine hydrochloride in vitro across intact and stripped rat skin. Experiments were done according to a full factorial design, to quantify the effects of iontophoresis (X1), electroporation (X2) and stripped skin (X3) alone and in combination on cumulative drug delivery at 6 h. Mathematical model eliciting the main effects of the factors and their interaction on cumulative tacrine delivery in 6 h shows that all three techniques examined alone have a positive impact on the permeation of tacrine, the greatest enhancement in delivery achieved by iontophoresis. However, iontophoresis in combination with electroporation or skin stripping yielded no improvement in delivery compared to iontophoresis alone. The most significant enhancement is seen when all three methods of assisted delivery are done in combination. Iontophoresis appears to control drug delivery to the exclusion of other enhancement methods. Electroporation appears to cause formation of a large depot of tacrine in the skin.

Response surface methodology to investigate the iontophoretic delivery of tacrine hydrochloride.

PURPOSE: The objective of this work was to apply response surface approach to investigate the main and interaction effects of delivery parameters for iontophoretic delivery of tacrine HCl in vitro. METHODS: Iontophoresis was used to deliver tacrine HCl across rat skin. Experiments were performed according to Box-Behnken design to evaluate effects of drug concentration (X1), current density (X2), and donor buffer molarity (X3) on cumulative drug delivered in 24 h (Y1), 6 h (Y2), iontophoretic flux (Y3), and post-iontophoretic flux (Y4). RESULTS: Mathematical model for Y1 was Y1 = 0.653 + 0.163 * X1 + 0.456 * X2 - 0.156 * X3 + 0.190 * X1X2 + 0.139* X3X3. Response surface plot indicated that at low level of X2 (0.1mA/cm2), X1 had little effect on Y1. However, at high level of X2 (0.5 mA/cm2), Y1 significantly increased from 0.75 mg/cm2 to 1.46 mg/cm2 when X1 increased from 1% to 9%. Regression equations predicted responses for Y1 to Y4, for optimal formulation, which were in reasonably good agreement with experimental values. CONCLUSIONS: Experimental design methodology revealed an interaction between drug concentration and current density, which would have been difficult to predict from one factor at a time classic experimental approach.