Permeability Study

This section aimed to investigate the ability of both carrier systems to penetrate the cell membrane of the oral cell tissue. Human oral epithelial cells (OEC), Applied Biological Materials Inc. (Richmond, BC, Canada) were cultured and seeded (1×10⁶ viable cells) on T25 flasks. The culture medium was replaced daily. The cells suitability for the experiment was tested by examining their viability and confluence using the inverted light microscope (Carl Zeiss AG, Oberkochen, Germany).

The OEC was divided into three groups. The first group was exposed to 0.1 mg/mL SMV in the form of inclusion complex dispersion in dimethyl sulfoxide (DMSO) of 0.1%. The second group was treated with the same drug concentration in the form of MM nanoparticles. The third group was subjected to the same concentration of pure SMV in DMSO. Blank OEC containing only the culture medium without drug was used as a reference. The experiment was conducted in triplicate. The OEC was incubated under standard conditions at 37°C in a humidified atmosphere containing 5% CO₂. The studied cells were collected at predetermined specified times and washed twice with ice-cold phosphate buffer saline. The collected cell pellets were suspended in 1 mL hypotonic saline solution (0.3%) to allow cell swelling. Cell pellets suspensions were subjected to three repeated cycles of freezing in −80 °C freezer for about 15 minutes and thawing at room temperature for 15 minutes. The cells were subsequently exposed to ultrasonic homogenization for 10 minutes, using Sonics Vibra cell, VCX 750 Ultrasonic probe sonicator (Sonics and Materials Inc., Newton, CT, USA) at an output of 250 W and a frequency of 40 kHz, to ensure complete rupture of the cells. Finally, cell lysates were subjected to centrifugation at 15,000× g for 60 minutes at 4°C using 3K30 sigma laboratory centrifuge (Osterode am Harz, Germany). The supernatant was collected, filtered and the concentration of SMV was calculated using high-performance liquid chromatography (HPLC) method.7,33 Agilent 1200 HPLC system of Agilent Technologies, Palo Alto (CA, USA) equipped with a UV diode array detector was used. The chromatographic analysis was performed using methanol- 0.05 M potassium dihydrogen orthophosphate (pH 5) (80/20 v/v) as a mobile phase. The flow rate was adjusted at 1.2 mL/min and the absorbance was detected at 239 nm. SMV retention time was detected at 9.7 min. Drug standards containing known weight of SMV in the OEC were prepared, treated as mentioned above and assayed for drug content before determination of the unknown SMV concentrations in the tested samples.