Page 9 - PROPOSAL LATIP Zanamivir_Neat 2

and fetal bovine serum at 37 °C under a 5 % CO2 atmosphere. The cells will be seeded

on permeable polycarbonate Transwell membranes at a density of 1.0×105 cells/cm2 and
will be cultured for 21–28 days. Before and after the experiments, the integrity of the

Caco-2 cell monolayers will be evaluated by measuring the transepithelial electrical
resistance (TEER) using a Voltohmmeter and only Caco-2 cell monolayers with a TEER

value of>200 Ω·cm2 at pre-and post-incubations will be use for the experiments. The

apparent experimental permeability coefficients (Papp, nm/s) will be calculated for time-
dependent absorption in vitro from the apical side of the Caco-2 monolayer in HBSS with

10mM MES (pH 6.0) to the basal side of the Caco-2 monolayer in HBSS with 10mM
HEPES (pH 7.4). 1–100 μM (dependent on the solubility of each substrate) of test

substance in a final concentration of<0.1 % dimethyl sulfoxide (originally dissolved in

dimethyl sulfoxide and diluted with Hank’s balanced salt solution) will be applied to the
apical side of Caco-2 cells cultured on Transwell plates. Caffeine and lucifer yellow were

used as positive and negative permeability controls, respectively. The amounts of the test
substances in permeation samples from the basal sides were measured by high

performance liquid chromatography.

3.5 In vitro drug release study and encapsulation efficiency study (EE)

3.5.1 In vitro drug release study
The release studies will be using nanoparticles that will be conducted in

triplicates using dialysis membrane (DM). Samples of 2.5 or 5 mL of
nanoparticles with 1.25 mg drug will be loaded into Spectra/Por® 3

standard regenerated cellulose dialysis tubing with an MWCO of 3.5 kDa

and clipped by standard closures. The dialysis tube was immersed into 450
mL release medium at 37ºC with a magnetic stirrer stirring at 75 rpm. At

5, 15, 30, 45, 60, 75, 90, 120, 180, and 360 min, 3 mL of the dissolution
medium will be withdrawn and replaced with an equal volume of fresh

medium. The sample solution was treated with EtOH to free the entrapped

drug and assayed by HPLC for drug content. Then, the nanoparticles will
be centrifugally ultrafiltrate to obtain filtrate for HPLC assay of free drug

content.

3.5.2 Encapsulation Efficiency Study (EE)