In vitro skin permeation studies

The mice were sacrificed by cervical dislocation, and abdominal hairs were removed carefully using an electric shaver. The skin from the abdominal surface was excised, and the adherent fat and subcutaneous tissue were removed carefully. Next, the isolated skin was washed with phosphate-buffered saline (PBS, pH 7.40), and its integrity was assured during the experiment.

The percutaneous permeability of LN-NLCs and LN-NLCs loaded with various amounts of cell penetrating peptide R11 (0.01%, 0.02% and 0.04% respectively) was investigated in in vitro skin permeation studies using the Franz diffusion method.

Briefly, 1 mL (0.75 mg of LN) of LN-NLC or R11-coated LN-NLC formulations was applied onto the stratum corneum (SC), which faced the donor compartment of Franz diffusion cells. The receptor chamber was filled with 16 mL of PBS (pH 7.40) under the condition of 400 r/min and 37±0.5 °C to maintain the physiological activity of the skin. The occlusive condition was sustained to prevent water from evaporating throughout the experiment. To draw the percutaneous permeability curve, all receiver fluid was collected and the fresh PBS (pH 7.40) with an equal volume and temperature was added directly into the receptor compartment at predetermined time intervals. The content of LN in receiver fluid was measured using HPLC (Agilent 1260) and the cumulative amount of permeated LN was calculated as follows.

Here, Q (μg/cm²) is the cumulative penetration amount per cm², C_n (μg/mL) is the LN concentration of the nth sample, V (mL) is the volume of the receiver fluid, and A (cm²) is the infiltrating area.

After 24 h of skin permeation, the fluid in the receptor compartment was collected, and the surface of the mouse skin was washed carefully with PBS (pH 7.4) to remove excess formulations and sucked dry. Thereafter, the mouse skin dosing area was cut off and then cut into pieces to a mixture with 0.4 mL of PBS (pH 7.4). After homogenization in the vortex, 0.6 mL of methanol was added to the mixture. The mixture was then disposed using an ultrasonic cell disruptor (Scientz-II D; Ningbo Scientz Biotechnology Co., Ltd., China) for 10 min to destroy skin tissue maximally. Subsequently, the mixture was centrifuged at 13,500 rpm for 10 min to collect the supernatant, and then the residues were mixed again with 0.5 mL of methanol, homogenized and subjected to centrifugation as described above. After 3 rounds of homogenization and centrifugation, the total supernatant was analyzed using HPLC to determine the drug content, and the skin deposition of LN per unit area Q_s (μg/cm²) was calculated