ANALYSIS OF DOXORUBICIN ENCAPSULATION IN APOFERRITIN CAGE
BY CAPILLARY ELECTROPHORESIS
WITH LASER-INDUCED FLUORESCENCE DETECTION
Maja Stanisavljevića, Marketa Ryvolovaa,b, Pavel Kopela,b, Vojtech Adama,b, Tomas Eckschlagerc, Rene Kizeka,b
aDepartment
of Chemistry and Biochemistry, Faculty of Agronomy, Mendel University in Brno, Zemedelska 1, CZ-613 00 Brno, Czech Republic
bCentral European Institute of Technology, Brno University of Technology, Technicka 3058/10, CZ-616 00 Brno, Czech Republic
cDepartment of Paediatric Haematology and Oncology, 2nd Faculty of Medicine Charles University in Prague and University hospital Motol, Prague,Czech Republic
RESULTS AND DISCUSSION
INTRODUCTION
Doxorubicin belongs to anthracycline antibiotics group and is an effective
cytostatic drug used for cancer treatments. It is known for his cardiotoxicity
which limit his application.
The ferritins/apoferritin are a family of proteins which in biological systems
are used to store iron and prevent its toxic effect to the cells.[1]
Ferritin/apoferritin cages are stable in physiological conditions and
applicable as a drug delivery system.[2] Using apoferritin cages as carrier
of doxorubicin there is possibility to reduce its toxicity and improve
efficiency. Doxorubicin was loaded into the apoferritin cages using
characteristic of apoferritin to dissociate at low pH (pH 2) into 24 subunits,
allowing loading of the drug and reassemble by changing pH to basic (pH
8.5).[3]
pH =2
Fig.2: Emission spectra of APODOX and its dependence on the
concentration of encapsulated doxorubicin (0, 6.25, 12.5, 25, 100, 200
µg/ml). The emission maximum of APODOX is the same as the maximum
of non-encapsulated doxorubicin - 600 nm and the dependence of the
fluorescence on the doxorubicin concentration is linear.
pH=7
Apoferritin
1
APODOX
Apoferritin subunit
Fig.1: Loading doxorubicin into apoferritin, by decreasing the pH to 2 causing
disassemble of the apoferritin and increasing the pH to 7 to captive doxorubicin in
the apoferritin cavity
6.25µg/ml
12.5µg/ml
0.8
Fluorescence (a.u.)
Doxorubicin
Peak y
Apoferritin
0 µg/ml
25 µg/ml
100 µg/ml
0.6
Height of peak (a.u.)
1.2
0.8
0.6
0.4
0.2
0
6.25 12.5
25
100
200
Concentration of doxorubicin (mg/ml)
200 µg/ml
0.4
Peak x
Peak y
1
Peak x
0.2
0
0
ACKNOWLEDGEMENTS
The financial support by CYTORES GA ČR P301/10/0356 and CEITEC
CZ.1.05/1.1.00/02.0068 is highly acknowledged.
4
6
8
Migration time (min)
10
12
1
1
Height of peak
pH
6.2
Apodox-2,
pH 6,2
pH 4.4
Apodox-2,
pH 4,4
0.8
pH 3.8
Apodox-2,
pH 3,8
Peak y
0.6
Peak x
Peak y
0.8
0.6
0.4
0.2
0
6.2
4.4
pH
3.8
Peak x
0.4
0.2
0
0
-0.2
REFERENCES
[1] Zhao, Z., Malik, A., Lee, M. L., Watt, G. D., Anal. Biochem. 1994, 218, 47-54.
[2] Dospivova, D., Hynek, D., Kopel, P., Bezdekova, A., et al., Int. J. Electrochem.
Sci. 2012, 7, 6378-6395.
[3] Ma-Ham, A. H., Wu, H., Wang, J., Kang, X. H., et al., J. Mater. Chem. 2011, 21,
8700-8708.
2
Fig.3: CE-LIF of APODOX solutions with increasing concentraation of
doxorubicin encapsulated in apoferritin, inset: dependence of the peak height
on the concentration of doxorubicin in APODOX. Peak x present
doxorubicin itself, and peak y present APODOX, which grows with
concentration of doxorubicin. Concentration over 100µg/ml change the
shape of peak.
Fluorescence (a.u.)
EXPERIMENTAL
Fluorimetric analysis
- measurements preformed by multifunctional microplate reader
Tecan Infinite 200PRO (Tecan, Switzerland)
-sample were placed in transparent 96 well microplate with flat
bottom by Nunc (ThermoScientific, USA)
-fluorescene was measured with λex=480nm and λem was in the
range of 510nm to 850nm per 5nm steps. Each value is avarage
of 5 measurements.
-sample volume of APODOX (6.25, 12.5, 25, 100, 200µg/ml)
and apoferritin(1mg/ml) as a blank probe was 50µl
Capillary electrophoresis
- measurement was done by using capillary electrophoresis
system (Beckman P/ACE 5500) with laser-induced
fluorescence detection (λex=488 nm, λem= 600 nm)
- uncoated fused silica capillary was used - ltot = 47 cm,
leff = 40 cm and ID = 75 µm
-injection conditions: hydrodynamic injection 3.4kPa for 20 sec
-separation conditions: 20kV, background electrolyte 50mM
Tris-HCl (pH 8.2)
- samples: APODOX (0, 6.25, 12.5, 25, 100, 200 µg/ml) and
apoferritin (1mg/ml)
2
4
6
8
10
12
Migration time (min)
Fig.4: CE-LIF of APODOX sample in different pH, inset: increase of the
peak x height depending on the pH decrease.Decreasing pH causes growing
of peak x. Low pH causes dissociation of apoferritin and releases
doxorubicin into solution, which is seen like growth of peak x.
CONCLUSION
It was found that apoferritin encapsulated doxorubicin is providing intensive
fluorescence and its behavior can be investigated by CE-LIF. The structural
changes of this nanocarrier under different conditions can be monitored by the
peak shape changes. Based on the results can be concluded that complex
processes are taking place during the structure opening and closing. This needs to
be investigated in more details in the future.