Biomedical Engineering Reference
In-Depth Information
(HLB value in the range of 3-6). The resulting w/o droplets are
dispersed in an external aqueous phase containing PVA, allowing
the formation of secondary emulsion (w/o/w). Singh et al.
reported the preparation of Flt23k PLGA nanoparticles and their
functionalization with RGD peptide and transferrin to treat laser-
induced CNV by intravenous delivery [
7
]. Luo et al. [
19
] and Cho
et al. [
20
] also described the use of PLGA nanoparticles entrapping
Flt23k plasmid, for controlling angiogenesis and fibrosis associated
with CNV. The step-by-step procedure for preparing nanoparticles
entrapping hydrophilic drugs like plasmids is described below:
1. 100 mg polymer (PLGA-Resomer RG 503H) is dissolved in
2 ml DCM in a 5 ml glass vial (
see Note 27
).
2. Drug (e.g., Flt23k plasmid 4 mg in buffer) is dissolved in about
500
l distilled, de-ionized water.
3. The drug solution is added to the polymer solution in a glass vial
under probe sonication (Misonix Sonicator
®
3000, Farming-
dale, NY) for 1 min at a power of 6-10 W (
see Notes 11
-
15
).
4. The primary w/o emulsion obtained in step 3 is added under
probe sonication to 10 ml of prechilled 2 % w/v PVA solution
taken in a 50ml glass beaker and sonicated for 3min at 24-30W
power to obtain the (w/o/w) emulsion (
see Note 28
).
5. The secondary emulsion thus obtained is kept under stirring
at room temperature for 3 h to evaporate the organic solvent.
Residual solvent, if any, is further evaporated by using a rotary
evaporator (Buchi Rotavapor R-200, Buchi Corporation, New
Castle, DE, USA) for 2 h at 40
C(
see Notes 17
-
20
).
6. The nanoparticles thus formed are centrifuged using a Sorvall
RC 6 plus centrifuge (Thermo Scientific, Asheville, NC, USA)
at ~25,000-30,000
μ
g
for 15-20 min to obtain a pellet of the
nanoparticles (
see Notes 21
and
22
).
7. The supernatant is removed and the pellet is redispersed in
25 ml distilled, de-ionized water and centrifuged again. This
washing step ensures further removal of the free drug and
additives like emulsifiers (
see Note 23
).
8. Another roundof washing followed by centrifugation is repeated.
9. The pellet is redispersed in 10 ml distilled, de-ionized water and
frozen by storing at
80
C for 30 min.
10. After the primary freezing step, the nanoparticles are lyophi-
lized in a Labconco freeze dryer (Labconco Corporation,
Kansas city, MO) at
80
C and at a pressure of 0.1 mBar for
24 h (
see Notes 24
-
26
).
