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764 G. Mishra et al.
(D2O): 1.35 (t, 3H); 1.87 (mixed d and t, 2H); 2.41 (t, 2H);
2.51–3.29 (m, 22H), 4.07 (q, 2H). 13C NMR (D2O): 23.12
(C-18); 48.01 (C-17); 50.10 (C9, C-11); 52.4 to 52.7 (C-2 to
C-6); 56.62 (C-8, C-12); 57.21 (C-13); 173.59 (C-14).
fresh human serum at physiological conditions, i.e. at
30°C at a concentration of 100 nM/ml and then analyzed
by instant thin layer chromatography (ITLC-SG) at differ-
ent time intervals to detect any dissociation of complex.
Percentage of free pertchnetate at a particular time point
that was estimated using saline and acetone as mobile
phase, represented percentage dissociation of the com-
plex at that particular time point in serum.
Conjugation of DO3A-EA to folate
A solution of 5 (0.38 g, 0.88 mmol), folic acid (0.2 g, 0.88
mmol), NMM (0.18 ml, 1.6 mmol) and HOBt (0.13 g, 0.97
mmol) in DMF (5 ml) was stirred at 0–5°C for 15min and
then EDC (0.19 g, 0.97 mmol) was added. e reaction
mixture was stirred overnight at room temperature. e
progress of reaction was monitored by thin layer chro-
matography. After completion, the reaction mixture
was poured in water, extracted with DCM (3× 100 ml),
organic layer was dried over Na2SO4, filtered, filtrate
was evaporated under reduced pressure and purified
by column chromatography (silica gel, 10% MeOH in
dichloromethane) to give 0.32 g (55%) of 6 as dark yel-
low viscous oil. ESI-HRMS (+): calcd C29H51N7O8S: m/z
658.35926 (M+H)+; found 658.35913 (M+H)+.
Receptor binding studies
Exponentially growing cells (KB, OAW and U-87MG)
0.1 × 106 cells/PD were plated at a uniform cell density
and incubated overnight. Monolayer culture of the cell
lines were washed twice for 2 min with ice cold binding
buffer (25 mM HEPES, 10 mM MgCl2 and 1% BSA). e
cell line culture were then incubated for 40 min with
labeled DO3A-EA-Folate (varying concentration) in
the absence and presence of the 100-folds excess unla-
beled DO3A-EA-Folate for estimation of total binding
and nonspecific binding, respectively. Specific binding
was obtained by subtracting nonspecific binding from
total binding. At the end of each experiment, the cells
were washed with ice cold binding buffer three times for
3 min. e cells were lysed with 200 μl of Lysis buffer. e
cell-associated radioactivity was determined by gamma
scintillation counting. Scatchard plot analysis was done
using Equilibrate software from graph pad.
Radiolabeling and radiochemical purity
Radiolabeling was carried out as per the reference 24 and
25. 2mg solution of the compounds dissolved in water was
taken in a shielded vial and 100 µl of 1×10−2 M SnCl2.2H2O
(dissolved in N2 purged 1ml (10% acetic acid) was added
followed by addition of (<1h) freshly eluted saline solu-
−
tion of sodium pertchnetate (NaTcO4 ) (82 MBq, 200ml).
e pH of the reaction mixture was adjusted to 6.5 with
0.1M NaHCO3 solution and shook to mix the contents. e
vial was allowed to stand for 15min at room temperature.
Labeling of the compound, radiochemical purity as well
as Rf of the 99mTc complexes was determined by ITLC-SG
strips using 0.9% NaCl aqueous solution (saline) as devel-
oping solvent and simultaneously in acetone and PAW
(Pyridine, acetic acid and water in 3:5:1.5 ratio) as men-
tioned in previous literature. Each TLC was cut in 0.1cm
segments and counts of each segment were taken. By
Cytotoxicity of folate and DO3A-EA-Folate
Cytotoxicity was determined using the MTT [3-(4,
5-dimethylthiazole-2-yl)-2, 5-diphenyl-2H-tetrazolium
bromide] assay. Exponentially growing cells were
plated in a 96-well microtitre plate at a uniform cell
density of 10,000 cells/well 24 h before treatment. Cells
were treated with varying concentrations of conjugate
(mM–μM range) for various time intervals viz., 24h, 48 h,
72 h and MTT assays were performed. At the end of treat-
ment, negative control and treated cells were incubated
with MTT at a final concentration of 0.05mg/ml for 2 h at
37°C and the medium was removed. e cells were lysed
and the formazan crystals were dissolved using 150 μl of
DMSO. Optical density is measured on 150 µl of extracts
at 570 nm (reference filter: 630 nm). Mitochondrial activ-
ity was expressed as percentage of viability compared
to negative control (mean SD of triplicate cultures). %
of viability = [OD (570–630 nm) test product/OD (570–
630 nm) negative control] × 100. Percentage viability at 1
μM and 10 μM concentrations was plotted against time
for folic acid and DO3A-EA-Folate.
using this method percentage of free Na99mTcO4 , reduced
−
99mTc and the complex formed between 99mTc and folate
conjugate could be calculated. 99mTc-DO3A-EA-Folate
conjugate remained at the origin and free technetium
migrated with the solvent front in acetone.
e radiolabeled drug was injected on HPLC using
solvent gradient (eluant A, 10 mM ammonium acetate
buffer at pH 8; eluant B, acetonitrile gradient, 0 min at
4% B, 10 min at 12% B and 15 min 15% B at a flow rate of
0.5 ml/min) and fractions were collected and radioactiv-
ity was counted on automated gamma counter.
In vitro serum stability assay
Macrocolony assay
e fresh human serum was prepared by allowing blood
collected from healthy volunteers to clot for 1 h at 37°C
in a humidified incubator maintained at 5% carbon diox-
ide, 95% air. en the sample was centrifuged at 400 rpm
and the serum was filtered through 0.22 micron syringe
filter into sterile plastic culture tubes. e above freshly
prepared technetium radio complex were incubated in
Monolayer cultures of OAW and U-87MG cell lines were
trypsinized and 100–1000 cells were plated depending
upon the concentrations of the drug in 60-mm petridishes
and incubated at 37°C in 5% CO2 humidified atmosphere
for 8 days. Colonies were fixed in methanol and stained
with 1% crystal violet. Colonies containing more than 50
cells were counted.
Journal of Drug Targeting