90
1. Create a standard curve at pH 7.4 and 4.6 based on the
concentration- dependent UV-vis absorption peak of the
employed model drug (e.g., doxorubicin hydrochloride is at
480 nm).
2. Redisperse × mg of loaded NCs in water.
3. Introduce the dispersion into the dialysis bag and seal it.
4. Place the dialysis bag in a vessel containing the desired buffer
solution (pH 7.4 and/or 4.6) at different temperatures.
5. Remove aliquots of 0.5 mL at different time points and add
0.5 mL to maintain the total volume.
6. Measure characteristic absorption wavelength of released drug
at each aliquot removed.
7. Quantify the release percentage by the standard curve equation created in step 1.
8. Investigate the mechanism of release according to Korsmeyer–
Peppas equation:
R t
kt
n
%
R%(t) is the percent of drug released at time t, while k and n are
the kinetics constant and the release exponent, respectively.
In order to assess the optimum frequency related to the appropriate concentration for in vitro and in vivo experiments, first the
mNCs are tested in aqueous solutions ex vitro.
1. Create 1 mL dispersions of consecutive concentrations of
mNCs in ddH 2 O in glass vials of the same dimensions.
2. Insert glass vials in the coil of the magnetic hyperthermia
apparatus (see Note 3).
3. Apply alternating magnetic field of various frequencies for
30 min.
4. Record temperature fluctuations.
The optimum sample and frequency to be used for further biological evaluation should have a thermal response plateau between
40 and 45 °C at the shortest time interval.
Comparative studies should be performed where the tested sample
should include NCs and/or mNCs, drug-loaded NCs, and/or
drug-loaded mNCs and pure drug. The concentrations at which all
the samples will be evaluated are tested in a range around the IC50
of the pure drug used for drug loading in order to assess the novelty of the DDS compared to the free drug.
In order to perform an in vitro biological evaluation proper human
cell lines should be cultured according to American Tissue Type
3.2 Hyperthermia
Measurements
3.3 Cytotoxicity
and Biocompatibility
Evaluation
3.3.1 Cell Culture
Maria Theodosiou et al.
1. Create a standard curve at pH 7.4 and 4.6 based on the
concentration- dependent UV-vis absorption peak of the
employed model drug (e.g., doxorubicin hydrochloride is at
480 nm).
2. Redisperse × mg of loaded NCs in water.
3. Introduce the dispersion into the dialysis bag and seal it.
4. Place the dialysis bag in a vessel containing the desired buffer
solution (pH 7.4 and/or 4.6) at different temperatures.
5. Remove aliquots of 0.5 mL at different time points and add
0.5 mL to maintain the total volume.
6. Measure characteristic absorption wavelength of released drug
at each aliquot removed.
7. Quantify the release percentage by the standard curve equation created in step 1.
8. Investigate the mechanism of release according to Korsmeyer–
Peppas equation:
R t
kt
n
%
R%(t) is the percent of drug released at time t, while k and n are
the kinetics constant and the release exponent, respectively.
In order to assess the optimum frequency related to the appropriate concentration for in vitro and in vivo experiments, first the
mNCs are tested in aqueous solutions ex vitro.
1. Create 1 mL dispersions of consecutive concentrations of
mNCs in ddH 2 O in glass vials of the same dimensions.
2. Insert glass vials in the coil of the magnetic hyperthermia
apparatus (see Note 3).
3. Apply alternating magnetic field of various frequencies for
30 min.
4. Record temperature fluctuations.
The optimum sample and frequency to be used for further biological evaluation should have a thermal response plateau between
40 and 45 °C at the shortest time interval.
Comparative studies should be performed where the tested sample
should include NCs and/or mNCs, drug-loaded NCs, and/or
drug-loaded mNCs and pure drug. The concentrations at which all
the samples will be evaluated are tested in a range around the IC50
of the pure drug used for drug loading in order to assess the novelty of the DDS compared to the free drug.
In order to perform an in vitro biological evaluation proper human
cell lines should be cultured according to American Tissue Type
3.2 Hyperthermia
Measurements
3.3 Cytotoxicity
and Biocompatibility
Evaluation
3.3.1 Cell Culture
Maria Theodosiou et al.