89
of enhanced therapeutic efficacy due to their stimulus-sensitive
nature. Above their volume-phase transition temperatures they
shrink; in acidic pH they are protonated and at high intracellular
glutathione (GSH) levels they suffer from disulfide bond collapse.
Finally, multisensitive polymeric nanocontainers modified with iron
oxide nanoparticles (mNCs) possess an extra sensitivity related to
heat in the concept of inducing cancer cell apoptosis and enhancement of drug release under the application of magnetic hyperthermia. Combination of the aforementioned materials promotes drug
release and in general targeted intracellular drug delivery.
1. Create a standard curve at pH 7.4 based on the concentrationdependent UV-vis absorption peak of the employed model
drug (e.g., doxorubicin hydrochloride is at 480 nm) (see
Note 1).
2. In a container of known mass, disperse 5 mg of hollow NCs or
mNCs in 5 mL of phosphate-buffered solution at pH 7.4
(PBS) (see Note 2).
3. Add 5 mg of drug and sonicate for 5 min at 25 °C.
4. Stir for 24–72 h under gentle agitation at 25 °C.
5. Centrifuge the mixture in order to remove the unloaded drug.
Resuspend the material by vortexing and centrifuge at
6080 × g until supernatant is clear or measured absorption is
close to zero.
6. Measure the absorption of centrifugations supernatants containing the unloaded drug.
7. Freeze-dry, weight, and store the precipitate at 25 °C for a few
days until the release study.
8. Determine the amount of loaded drug via standard curve
methodology.
9. Calculate the loading content using the equations below:
Loading capacity
Weight of the drug in NCs
Total Weight o
%
f f the NCs
u100
Encapsulation efficiency
Weight of the drug in NCs
Weight
%
of the feeding drug
u100
Generally in vitro release studies are performed at 37 °C (physiological temperature), though in some cases testing is performed at
elevated temperatures for exploring and characterizing drug release
using a variety of dosage forms. The most renowned and versatile
method of assessing drug release from nano-sized dosage forms is
the dialysis method. Using a dialysis membrane, which is permeable by the desired drug, physical separation will take place through
diffusion. The protocol for this is as follows:
3.1.1 Drug Loading
in Nanocontainers
3.1.2 Drug Release
from Nanocontainers
Multisensitive Polymeric Nanocontainers as Drug Delivery Systems: Biological Evaluation
of enhanced therapeutic efficacy due to their stimulus-sensitive
nature. Above their volume-phase transition temperatures they
shrink; in acidic pH they are protonated and at high intracellular
glutathione (GSH) levels they suffer from disulfide bond collapse.
Finally, multisensitive polymeric nanocontainers modified with iron
oxide nanoparticles (mNCs) possess an extra sensitivity related to
heat in the concept of inducing cancer cell apoptosis and enhancement of drug release under the application of magnetic hyperthermia. Combination of the aforementioned materials promotes drug
release and in general targeted intracellular drug delivery.
1. Create a standard curve at pH 7.4 based on the concentrationdependent UV-vis absorption peak of the employed model
drug (e.g., doxorubicin hydrochloride is at 480 nm) (see
Note 1).
2. In a container of known mass, disperse 5 mg of hollow NCs or
mNCs in 5 mL of phosphate-buffered solution at pH 7.4
(PBS) (see Note 2).
3. Add 5 mg of drug and sonicate for 5 min at 25 °C.
4. Stir for 24–72 h under gentle agitation at 25 °C.
5. Centrifuge the mixture in order to remove the unloaded drug.
Resuspend the material by vortexing and centrifuge at
6080 × g until supernatant is clear or measured absorption is
close to zero.
6. Measure the absorption of centrifugations supernatants containing the unloaded drug.
7. Freeze-dry, weight, and store the precipitate at 25 °C for a few
days until the release study.
8. Determine the amount of loaded drug via standard curve
methodology.
9. Calculate the loading content using the equations below:
Loading capacity
Weight of the drug in NCs
Total Weight o
%
f f the NCs
u100
Encapsulation efficiency
Weight of the drug in NCs
Weight
%
of the feeding drug
u100
Generally in vitro release studies are performed at 37 °C (physiological temperature), though in some cases testing is performed at
elevated temperatures for exploring and characterizing drug release
using a variety of dosage forms. The most renowned and versatile
method of assessing drug release from nano-sized dosage forms is
the dialysis method. Using a dialysis membrane, which is permeable by the desired drug, physical separation will take place through
diffusion. The protocol for this is as follows:
3.1.1 Drug Loading
in Nanocontainers
3.1.2 Drug Release
from Nanocontainers
Multisensitive Polymeric Nanocontainers as Drug Delivery Systems: Biological Evaluation