Optimization of resveratrol nanoformulation and evaluation of its cytotoxicity on cochlear derived cells

Purpose: The aim of the work is to develop resveratrol (RES)-loaded nanocarriers (NCs) intended for cochlear drug delivery. Furthermore, in vitro toxicity of the synthesized NCs is evaluated in cochlear cell lines. Methods: RES-NCs are prepared by solvent-diffusion technique without surfactant. RES, PLGA and PCL-PEG di-block are mixed in different ratios, dissolved in acetone and added dropwise to aqueous phase under constant stirring (acetone/water ratio 1/20). RES-NCs are washed twice and freeze-dried. Box-Behnken design (BBD) is used to study the influence of formulation variables on Z-size, Polydispersity index (PDI), Zeta-potential, drug encapsulation efficiency (EE%) and ratio between Z-size before and after freeze-drying (S f/Si). To increase RES-NC stability during freeze-drying, lactose, mannitol, sucrose and trehalose are tested at different concentrations (1%, 5%, 10%, 15%, 20%w/v). Finally, MTS and LDH assays are carried out to check RES and Blank NCs in vitro toxicity after 24h incubation in an organ of Corti (HEI-OC1) and a stria vascularis (SVK-1) cell lines. Results: BBD model is validated since all experimental responses fit with predicted values. Checkpoint analyses (bias < 10%) and Monte Carlo simulation (response defect values < 10%) show good robustness of the model to predict RES-NCs properties. The optimal formulation (desirability: 0.86) corresponds to Z-size of 135.5±37.3nm, PDI of 0.126±0.080, Z- potential of -26.84±3.31mV, EE% of 99.83±17.59% and Sf/Si of 3.30±0.92. RES-NCs stability is increase when the selected cryoprotectants are used during the freeze-drying. However, only trehalose (up to 15%w/v) can maintain the Z-size and PDI within the model space. In vitro toxicity studies show that high concentrations of RES (>500μM) and blank NCs (>800μg/mL) affect the cell viability. Conclusion: RES-NCs are successfully synthesized and optimized by BBD. In addition, in vitro studies show that RES (<500μM) and blank NCs (< 800μg/mL) are non-toxic for HEI-OC1 and SVK-1 cells.