Biomedical Engineering Reference
In-Depth Information
3. The organic solvent is evaporated under nitrogen and the drug
in residue is reconstituted using a suitable solvent. For instance,
for the budesonide loaded PLA nanoparticles, 1ml acetonitrile--
water (70:30) mixture was used for extraction. For Flt23k plas-
mid loaded, RGD and transferrin-functionalized PLGA
nanoparticles, TE buffer was employed for plasmid extraction
from the residue [
7
] and for Flt23k loaded RGD-functionalized
PLGA nanoparticles, 1 ml water was used [
20
](
see Note 40
).
4. The dispersion in step 3 is vortexed for about 1-2 h (
see
Note 41
).
5. The extract is used for drug quantification. Quantification can
be done by a suitable method like absorbance [
7
,
20
] or fluo-
rescence spectroscopy, HPLC [
16
] or LC-MS (
see Note 42
).
These studies are performed in a buffer, which acts as the release
medium and dissolves the drug. In vitro release studies are typically
carried out at 37
C and employ a dialysis membrane for small
molecule drugs, peptides, and small nucleic acids. For large mole-
cules such as proteins and plasmids, a membrane free method such
as ultracentrifugation for collection of full amount of dispersion
medium at various time points is more appropriate. For the small
molecule drugs, the drug-loaded nanoparticles are added to a
dialysis bag, sealed, placed in a release medium. The drug released
from the particles penetrates across the dialysis membrane into the
release medium. Aliquots are withdrawn from the bulk medium at
specific pre-determined time points. The drug released into the
dissolution medium is then quantified using a suitable technique
like absorbance, fluorescence, or mass spectroscopy. PBS at pH 7.4
is generally chosen as the buffer because it simulates physiological
conditions.
Detailed procedure to be followed while performing release
study by the dialysis method for small molecule drugs is as follows:
3.2.5 In Vitro Drug
Release Studies
1. 1-2 mg of drug-loaded PLA/PLGA particles are weighed in
1.5 ml eppendorf tubes (
see Note 43
).
2. 1 ml water is added to the particles in the eppendorf tubes.
3. PBS (pH 7.4) containing 0.05 % w/v sodium azide as preser-
vative is prepared (
see Note 44
).
4. 50 ml buffer from step 3 is added to centrifuge tubes
(
see Note 45
).
5. Dialysis membrane with a suitable molecular weight cut off in
the range of 6,000-50,000 Da is chosen based on the difference
between the molecular weights of the drug and the polymer
used to prepare nanoparticles (
see Note 46
). The membrane is
sealed at one end to form a bag.
