pH 8.0), then against 3 L of buffer G
(0.01 M Tris-HCl, 0.15 M NaCl, pH 8.0) for 24 hours each.
The dialyzed protein was concentrated on the cell membrane
Amicon PM-10 ("Millipore", USA) to 400 mL. The concentrate
was fractionated on a column Toyopearl TM DEAE ("Tosoh
Bioscience LLC") by elution with a gradient of 0-0.5 M NaCl
with a flow rate of 1 mL/min and detection at 280 nm. The
target fracti on ( monomer) was concen trated on the cell Amico n
(PM-10) to 1.5 mL and stored at -70°C. Protein concentration
was determined by the BCA assay (Bicinchoninic Acid Kit,
Sigma).
2.3. Investigation of AFP 3D Binding and Endocytosis
by Flow Cytofluorometry
1) Synthesis of fluorescein isothiocyanate (FITC)-labeled
AFP3D: 6.5 µL (4-fold molar excess) of FITC solution in
DMSO (dimethyl sulfoxide) (10 mg/mL) was added to AFP3D
solution (1 mg in 0.1 M bicarbonate buffer, pH 8.5) and the
mixture was stirred in the dark for 1 hour at room temperature,
then for 18 hours at +4°C. FITC-labeled protein was applied to
a column with Sephadex G-25 equilibrated with phosphate
buffered saline (PBS), pH 7.4. Protein fraction was eluted with
the same buf fer at a flow rate o f 1 mL/min and detection at 280
nm in the void volume and concentrated on an Amicon cell
with PM-10 membrane to 300 µL. The protein concentration
was measured using the BCA kit. Concentration of FITC was
determined by spectrophotometry assuming the molar extinc-
tion coefficient of FITC epsilon 498 = 68,000 M-1•cm-1. The
protein/FITC ratio was 1: 1.4.
2) Cell cultivation: Breast adenocarcinoma cells MCF-7
were cultured in plastic bottles ("Corning-Costar", USA) in
DMEM medium containing 10% FBS (fetal bovine serum) and
gentamicin (50 µg/ml) in incubator at 37 °C and humidified
atmosphere containing 5% CO2.
3) Flow cytometry: One day before the experiment, cells
were transferred to 6-well plates ("Corning-Costar", USA).
Before starting the experiment, cells were incubated for 2 hours
in DMEM medium without FBS. To analyze the binding,
AFP3D -F ITC was added to a suspension of cells in the concen-
tratio n range 30-4000 nM and incubated at 4 °C for 1 hour. To
analyze the endocytosis, incubation was performed at 37 °C,
and t hen cells were w ashed three times with cold PBS and fixed
with 2% paraformaldehyde (PF) in PBS. F luorescence in tensi ty
was measured using an EPICS-C flow cytofluorometer ("Coul-
ter Electronics", USA). Fluorescence was excited by an argon
laser (wavelength 488 nm, emission bandwidth 520 nm). For
every sample (10^5 cells) the median fluorescence intensity
(MFI) and percentage o f cells that exceeded the flu orescen ce of
autofluorescence were determined. The specificity of AFP3D-
FITC binding to AFP receptor was determined by incubating
the cells with AFP3D-FITC i n the presence of a 30-fold excess
of AFP.
2.4. Conjugation of AFP3D with PAMAM Dendrimer
and Doxor ubicin
1) Conjugation in the aqueous medium: 172 mg of a con-
densing agent, EDC (1-Ethyl-3 -[3-dimethylaminopropyl] car-
bodiimide hydrochloride) was dissolved in 3 ml of PBS to ob-
tain a co ncent ration of 0.3 M (buffer A). To this buffer 3 mg of
dendrimer PAMAM-COOH 3.5 G (37 ml of 10% methanol
solution) was added and solution was stirred for 30 min at room
temperature. Sulfo-NHS (Sulfo-N-hydroxysuccinimide) was
added in catalytic amounts. Protein solution, concentrated to 4
mg in 500 ml, was then added dropwise, with constant stirring.
After 10 min of incubation at room temperature, 2 mL of dox-
orubicin (Dox) with a concentration of 1mg/ml and buffer A up
to concentration of 0.2 M EDC were added. The mixture was
incubated for 1 hour at room temperature with stirring.
2) Conjugation in non-aqueous medium: To PAMAM den-
drimer (1.8 mg), dissolved in 500 mL of DMFA (dimethylfor-
mamide) with 10% of D-MAP (dimethylaminopyridine), a
14-fold excess of EDC (1 mg) was added. Mixture was incu-
bated for 30 min at room temperature. Then catalytic amounts
of the Sulfo-NHS and 2 mg of AFP3D concentrated in 500 mL
were added. Then 4 mL of 0.1% doxorubicin solution was
added dropwise. The mixture was incubated for 2 hours with
stirring. The solution was clarified by centrifugation. Unbound
doxorubicin and dendrimer were removed by dialysis against 3
L of buffer 0.05 M NaHCO3, pH 8.7, for 2 days. Resulting
solution was centrifuged at 14,000 rev/min for 30 min at 15°C.
Supernatant was collected, filtered through a sterilizing filter
Chromafil-Pet-20/15 MS with a pore diameter of 0.20 µm and
frozen at -70°C.
2.5. Determination of Antiproliferative Activity of
AFP3D-PAMAM-Dox Using the MTT-test
Breast carcin oma cells MC F-7 were cultured in plastic flasks in
RPMI medium containing 10% of FBS, 100 u/mL penicillin
and 100 µg/mL streptomycin in a humidified atmosphere of 5%
CO2 at 37°C. Cells were transferred into 96-well plates by 10
000 cell s per well. C onjugates b eing in vestigated were add ed at
various concentrations and cells were incubated for 72 h. Four
hours before the incubation finish, 50 µL of MTT solution at
concentration of 1 mg/mL in the culture medium was added to
each well. After color d evelopment , the medium was re moved,
the sedimented formazan crystals were dissolved in 150 µL of
DMSO, and the color intensity was measured by absorption at
540 nm on a plate spectrophotometer (Labsystem). The cell
survivab ility was est imated as percentage of untr eated control.
YU. F. MIGAL ET AL.
Copyright © 2012 SciRes. ENG
82
3. Results
3.1. Expression of AFP3D in E. Coli
Polyacrylamide gel electrophoresis (PAGE) confirmed that the
target protein is synthesized in high yield and accumulated in
inclus ion bodi e s (Figure 1).
3.2. Biological Activity of Recombinant AFP3D
To determine the biological activity of obtained recombinant
AFP3D, we examined the binding at 4°C and endocytosis at
37°C of FITC-labeled AFP3D by MCF-7 human breast cancer
cells using flow cytofluometry. To study the binding, the cells
were incubated with AFP3D-FITC at +4 °C for one hour,
washed, fixed, and then the fluo rescence of the cells was meas-
ured. It was shown that AFP3D-FITC binds to MCF-7 cells. To
investigate the specificity of binding, MCF-7 cells were incu-
bated with AFP3D-FITC in the presence of a 30-fold excess of
human AFP for one hour at +4 °C. Incubation of MCF-7 under
these conditions led to a significant reduction of AFP3D-FITC
binding to tumor cells (Figure 2). To study the endocytosis,
MCF-7 cells were in cubated with AFP3D -FITC for one hour at
+37 °C. AFP3D binding to tumor cells correlates with endocy-
tosis (Figure 3), which means that these processes are interre-
lated.
3.3. The Co mponent Ratio in the Resulting
Conjugates
The protein concentration was measured using the BCA kit.
Concentration of doxorubicin was determined by spectropho-
tometric method. In the conjugate synthesized in aqueous me-
dia, the protein: cytostatic ratio was 1 : 3.5, i.e. one molecul e of
the recombinant protein was associated with dendrimer carry-
ing 3.5 molecules of doxorubicin. In the conjugate synthesized
in nonaqueous media, the protein : cytostatic ratio was 1 : 7.9.
3.4. Cytotoxic Activity of AFP3D-PAMAM-Dox
Conjugates
To determinate the cytotoxic activity of conjugates, we ex-
amined their ability to influence survival of human breast car-
cinoma cell li ne MCF-7 . Tumor cells were in cubated with con-
jugates for 72 hours. The data shown in Figure 4 demonstrates
M 1 2
kDa
66
45
36
24
29
Figure 1. AFP3D gene expression. The results of electrophore si s i n
13% polyacrylamide gel in the presence of SDS. Total cell proteins
of E.coli, transformed with plasmid pAFP28D3: before the induc-
tion (1), after the induction with IPTG (2), inclusion bodies (3),
M-molecular weigh t markers.
0,0 0,1 0,2 0,3 0,4 0,5
0,00
0,05
0,10
0,15
0,20
0,25
0,30
0,35
Сблок
AFP3D concentration (mkM)
without AFP blocking
with AFP blocki ng
Fig ure 2. 30-fold excess of AFP inhibits binding of AFP3D-FITC
to MCF-7 cells .
AFP3D concentration (mkM)
Fluorescence intensity
00,03 0,52
1,0
1,5
2,0
2,5
3,0
3,5
Эндоц
binding
endocytosis
Fig ure 3. Correlation between binding and endocytosis of AFP3D-
FITC by MCF -7 cells.
that both of the conjugates decreased survival of tumor cells,
and the inhibitory effect was dose-dependent. The inhibitory
effect of the conjugate, obtained in non-aqueous conditions,
was higher than inhibitory effect of the aqueous-synthesized
compound, but lower than that of pure doxorubicin.
4. Discussion
In this study, we have synthesized an AFP3D conjugate with
doxorubicin-loaded PAMAM dendrimer nanoparticles and
investigated its cytotoxic activity.
To produce the recombinant AFP3D, E. coli strain, containing
plasmid pAFP28D3, was used. The final yield of highly puri-
fied AFP3D, obtained by this method, is 23 mg per liter of bac-
terial culture, which is a sufficient quantity for preparative use.
The excess of native AFP suppressed AFP3D binding to tu-
mor cells, which shows that recombinant AFP3D competes
with native AFP for binding to the specific AFP recep tor. Cor-
relation between AFP3D binding and endocytosis shows that
endocytosis is receptor-mediated. Thus, AFP3D obtained by
this method could be used as vector molecule.
YU. F. MIGAL ET AL.
Copyright © 2012 SciRes. E NG
83
IC
50
, µg/mL
AFP3D-PAMAM-3.5 Dox (aqueous conjugation) 1.270
AFP3D-PAMAM-7.9 Dox (non-aqueous conjugation) 0.291
Doxorubicin 0.080
Dox orubicin co ncentr ation (mkg/mL)
MCF-7 cells sur vival (%)
Figure 4. The cytotoxic activity of conjugates AFP3D-PAMAM-
Dox agains t brea s t cancer cell lin e M C F-7.
To carry out conjugation reactions the water-soluble carbo-
diimide was used. EDC was chosen as cross-linking agent be-
cause of its ability to form a direct covalent amide bond be-
tween two molecules without additional atoms. That prevents
the possibility of immune response and formation of antibodies
specific to th e linker. No n-aqu eous co nju gatio n was carried in a
polar aprotonic solvent DMFA with the addition of DMAP as a
nucl eophilic catalyst.
Our experiments have shown that the conjugation carried in
non-aqueous mediu m is more than t wice efficient th en the con-
jugation carried in water. In the conjugate synthesized in
non-aqueous reacti on, the AFP3D : doxorubicin ratio was 1:7.9,
while the ratio obtained in aqueo us r eaction was 1:3.5.
Both drugs showed dose-related cytotoxic activity (CTA)
against tumor cells. CTA level of the conjugate, obtained in
non-aqueous medium, was higher than that of the conjugate,
obtained in aqueous medium, but lower than CTA of pure dox-
orubicin. These results propose that higher load of conjugate
with doxorubicin increases it's CTA. Lower CTA of conjugates
than pure doxorubicin's one is due to difficult dissociation of
doxorubicin from the dendrimer within the cell.
It should be noted that in vivo anti-tumor drug compounds
(including doxorubicin) easily penetrate not only the tumor, but
also all types o f nor mal cells, cau sin g d ifferen t sid e effects. The
presented results indicate that the AFP3D-PAMAM-Dox con-
jugate has directed action on the cells of malignant tumors.
Therefore, despite the lower values of conjugates’ CTA com-
pared with the CTA of free doxorubicin in vitro, the therapeutic
use of the conjugates in vivo can possibly minimize the nega-
tive side effects of antitumor drugs alone. This approach allows
increasing the dosage of the drug, as well as improves the safe-
ty of anti cancer th er apy.
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