Before TEWL measurements, rats were anesthetized by intraperitoneal injection of ketamine (30 mg/kg). After recording pre-treatment TEWL values, 0.5 g of formulation (Na-DOC Gel, nanoparticle colloidal suspension incorporated in chitosan Gel or commercial cream) was spread uniformly on 2 × 2 cm area of shaven rat abdomen (4 cm2) and left for 1 h. Transepidermal water loss (TEWL) measurements were obtained a Tewameter TM 300 (Courage + Khazaka electronic GmbH, Köln, Germany) applied on the skin for 1 min. TEWL values were recorded 5 min, 2 and 4 h after the removal of the formulation applied on the skin by means of a dry cotton pad. The laboratory temperature and relative humidity were kept constant in the range of 24–25 °C and 45%–50% RH, respectively.