Page 10 - PROPOSAL LATIP Zanamivir_Neat 2

The free drug and nanoparticles will be separated through centrifugal

ultrafiltration using. Briefly, 15 mL of fresh nanoparticles will be loaded
into the filter device and centrifuged at 4000Xg for 20 min. The filtrate

containing solely free drug will collected for HPLC assay, while the
concentrate was freeze-dried. The freeze-dried powders will be weighed

and dissolve in an organic solvent for HPLC assay of the drug content in

the nanoparticle. The drug loading (DL) and encapsulation efficiency (EE)
will be calculated using the following equations:

EE (%) = total amount of drug - amount of free drug X 100%
total amount of drug

DL (%) = total amount of drug in nanoparticles X 100%

total amount of nanoparticles

3.6 Mechanism of Zanamivir transport via transepithelial electrical resistance (TEER)
analysis.

The electrodes will be equilibrated and sterilized before the analysis
according to the manufacturer’s recommendations. Two hundred microliters of

culture medium will be added in the upper compartment of the cell culture

system. The ohmic resistance of a blank (culture insert without cells) will be
measured in parallel. To obtain the sample resistance, the blank value will be

subtracted from the total resistance of the sample. The final unit area resistance
(Ω*cm2) will be calculated by multiplying the sample resistance by the effective

area of the membrane (0.33 cm2 for 24-well Millicell inserts)

3.7 In vitro diffusion chamber method.

The transport of Zanamivir across the rat intestinal membrane will
be conducted using in vitro diffusion chamber method (K.A. Hamid et al., 2017).

This study will be performed in accordance with the guidelines and approval from

the animal ethics committee of Universiti Teknologi MARA (UiTM) (UiTM
Care:53/2014). Male Sprague Dawley rats, weighing 230–250 g will be

anesthesize with a mixture of ketamin and xylazil (3:2), intraperitoneally (i.p.)
after being fasted overnight. By doing a midline abdominal incision, the intestine

will be exposed and removed and then wash with phosphate buffered saline