Encapsulation of siRNA-FAM and PTX into NPsA, NPsB and NPsAB
The drug-loaded nanoparticles NPsA/siRNA/PTX, NPsB/siRNA/PTX and
NPsAB/siRNA/PTX NPsAB/siRNA/PTX were prepared based on the previous
report with minor modification (Perez et al., 2001a).
Briefly, to synthesis of NPsA/siRNA/PTX, 0.5 ml water solution
containing siRNA-FAM (250μg) was added dropwise into 4 ml of aceton/DCM
solution containing NPsA (30 mg) and PTX (5 mg)The reaction mixture was
sonicated on ice for 30 seconds (Sonicator® XL, Misonix, NY), then
slowly added to 6 mL of PVA solution (1% w/v) and sonicated again for
two minutesNext, the reaction mixture was poured dropwise into a stirred
PVA solution (30ml, 0.3% w/v).
After 5 min, the solvent (DCM and acetone) from the nanoparticles
fraction was evaporated using a rotary vacuum evaporator. Then, the
NPsA/siRNA/PTX were collected were collected from the solution by
centrifugation at 15000 rpm (16602 ×g) for 30 min (Sigma 1-14K), and
after washed several times with deionized water to remove the unloaded
drug, the the resultant were filtered using a 0.1 and 0.45 micrometer
membrane filter to remove large scattering particles and free NPsA, and
finally, the product was lyophilized. The NPsB/siRNA/PTX and
NPsAB/siRNA/PTX were also prepared as described above.
The encapsulation efficiency of siRNA-FAM and PTX in nanoparticles was
determined by measuring the amount of siRNA-FAM and PTX that was not
encapsulated in nanoparticles. Therefore, the amount of siRNA-FAM and
PTX in the supernatant of the nanoparticle were measured using
spectrophotometer at 490 (siRNA-FAM) and 230 (PTX) nm respectively.
Then, the amount of siRNA-FAM and PTX in the supernatant was compared
with the total amount of siRNA-FAM and PTX used in the encapsulation
process (Son & Kim, 2010). encapsulation efficiency (EE) of siRNA-FAM
and PTX was determined as follow Eq. (1):
(%EE)
=\(\ \frac{\ Amount\ of\ used\ drug-Amount\ of\ drug\ in\ supernatant\ }{\text{Amount\ of\ used\ drug}}\)×100
(1)
The PTX and siRNA release kinetics from the nanoparticles was assessed
in PBS buffer (pH 7.4). The nanoparticles were treated separately with 5
ml of PBS buffer (1 mg/ml) then, collected via centrifugation (16602×g
for 30 minutes) at predetermined time intervals (i.e. 30 minutes - 30
days after inoculation). After each collection, the supernatants were
employed to determine the amount of siRNA and PTX released from the
nanoparticles. Afterwards, nanoparticles were re-suspended in 5 ml of
fresh PBS buffer (pH 7.4), and incubated at 37 °C until the next
scheduled time interval. The total DNA release ratio for each sample was
calculated using the following Eq. (2):
Cumulative Release (%) =\(\frac{\mathrm{\text{Total}}\mathrm{\text{\ drug}}\mathrm{\text{\ content\ in}}\mathrm{\ }\mathrm{\text{supernatant}}}{\mathrm{\text{The\ amount\ of\ encapsulated\ drug}}}\mathrm{\times 100}\)(2)