J Radioanal Nucl Chem
DOI 10.1007/s10967-015-4210-6
Radiolabeling and in vitro evaluation of
on breast cancer cell line
Emre Ozgenc1 Meliha Ekinci1 Derya Ilem-Ozdemir1 Evren Gundogdu1
Makbule Asikoglu1
Received: 11 March 2015
Akademiai Kiado, Budapest, Hungary 2015
Abstract In the present study 99mTc-MTX was prepared
with high labeling yield by a new simple and easy formu-
lation method. According to cell culture studies, 99mTc-
MTX incorporated with both MCF-7 and CRL8798 cells,
with significant differences in the uptake percentages. Since
99m
Tc-MTX highly uptake in cancer cell line, the results
demonstrated that radiolabeled MTX may be promising for
breast cancer diagnosis of oncological patients.
Keywords Methotrexate  99mTc-Methotrexate  Breast
cancer diagnosis  Radiolabeling  Radiopharmaceuticals
Introduction
Cancer has been one of the leading causes of death in the
world [1]. According to the World Health Organization
(WHO), the number of deaths from cancer worldwide is
expected to keep increasing and is estimated to be 12
million in 2030. Early diagnosis and effective treatment of
cancer significantly reduced risk of the mortality and
morbidity [2]. Diagnose the cancer at an early stage is an
important health problem in the world. Many researchers
study on developing new radiopharmaceuticals which are
specific to cancer cells. Recent studies showed that folate
receptors in tumor tissue compared with healthy tissue
indicates that they have been high amount in tumor tissue.
For this reason, folate receptor conjugates can be used as a
potential tumor targeting agent for tumor tissue [37].
& Derya Ilem-Ozdemir
deryailem@gmail.com
1
Department of Radiopharmacy, Faculty of Pharmacy,
University of Ege, Bornova, 35100 Izmir, Turkey
99m
Tc-methotrexate
The folate receptors are overexpressed on a variety of
human tumors, such as ovarian, breast, colorectal, cervical
and renal tumors but occur in a limited number of normal
tissues, thus making folate receptors a potential molecular
target for tumor imaging. Methotrexate (MTX) is one of
the most widely and effectively use anticancer drug in
human malignancies such as acute lymphoblastic leukemia,
malignant lymphoma, osteosarcoma, breast cancer and
neck cancer. Also, MTX acts as an antagonist of folic acid,
which is necessary for DNA synthesis, and has a
therapeutic effect on many types of cancer cells that
overexpress folate receptors on their surfaces [813].
Radiopharmaceuticals are drugs which contain ra-
dionuclide for diagnosis or treatment of a disease. In ad-
dition, radiopharmaceuticals are important for early
diagnosis of cancer and examination of cancer treatment.
The evaluation of diagnostic radiopharmaceuticals can be
attributed to the existence and chemical versatility of
Technetium-99m (99mTc) as ideal radiotracer. 99mTc is the
radionuclide of choice in the development of diagnostic
imaging agents by virtue of convenient half life, ideal en-
ergy for imaging, short enough to minimize absorbed ra-
diation dose [1416].
The aim of this study was to radiolabeled MTX with
99m
Tc in a simple radiochemical method and investigated
incorporation of 99mTc-MTXMCF-7 and CRL8798 cell
lines.
Materials and methods
MTX was obtained from Kocak Pharma (Turkey). Stannous
tartrate was purchased from Sigma-Aldrich (USA) and
ascorbic acid was purchased from Sigma-Aldrich (United
Kingdom). 99mTc was eluted from the Molybdenum-99
123

(99Mo)/99mTc generator (Nuclear Medicine Department of
Ege University). The syringe filter with 0.22 lm pore size
GH Polypro membrane was purchased from Pall Life Sci-
ence. All solvents were obtained from Merck (Germany).
Radioactive samples counted in a counting unit (Atomlab
100 Dose Calibrator Biodex Medical Systems). Experiments
were performed in triplicate unless stated otherwise. Results
are reported as mean standard error.
Radiolabeling studies
The radiolabeling of 99mTc-MTX was tested with different
concentrations of reducing and antioxidant agent. Radio-
chemical purity (RP) was determined with radio thin layer
chromatography (RTLC) analysis.
Effect of reducing agent amount on labeling
99m
Tc pertechnetate was eluted from 99Mo/99mTc generator
in99m
Tc(VII) oxidation state which does not able to label
with any compound on direct addition. So prior to labeling
procedure, reduction of 99mTc is required for converting
99m
Tc from the 99mTc(VII) state to a desired lower oxida-
tion state, which can make complexes with the ligand to
form the radiopharmaceutical. Different types of reduction
agents are used for this reason [16]. In this study, stannous
tartrate was used as reducing agent. To examine the effect
of reducing agent concentration on labeling; MTX (1 mg)
was dissolved in 0.9 % sodium chloride solution (0.5 mL).
To this stock solution, stannous tartrate was added under an
atmosphere of bubbling nitrogen. Reduction of 99mTc was
performed at acid pH (1 mg reducing agent dissolved in
1 mL 0.1 N HCl) with different amount of stannous tartrate
(500, 750, 1000, 1250 and 1500 lg). Although 99mTc-
MTX was needed to adjust the body pH, preparations in
small volume were adjusted to pH stable. Furthermore the
pH of the solution was adjusted at pH 9 before labeling.
Radiolabeling was performed with 99mTc (37 MBq) in
0.9 % sodium chloride solution (0.1 mL) and the solution
was allowed to stand at room temperature for 5 min prior
to radiochemical analysis.
Effect of antioxidant agent on labeling
Radiopharmaceuticals which contain 99mTc may undergo
auto radiolysis during preparation, release and storage. De-
composition of radiopharmaceutical will decrease the target-
ing capability before or after administration. Therefore, it is
very important to use a stabilizer to minimize the auto radi-
olysis. Radiolytic stabilizers are often antioxidants, such as
ascorbic acid, gentisic acid and p-aminobenzoic acid [17]. To
examine the effect of antioxidant agent on labeling efficiency
and stability of the complex, labeling studies were performed
123
J Radioanal Nucl Chem
in the absence and presence of an antioxidant agent. In this
study, ascorbic acid was used as an antioxidant agent. MTX
(1 mg) was dissolved in 0.9 % sodium chloride solution
(0.5 mL) in five groups of vials. 500, 750, 1000, 1250 and
1500 lg stannous tartrate was added to each individual group.
Each group has three vials and labeling was performed in the
absence and presence (0.05 and 0.1 mg) of ascorbic acid. pH
of the solution was maintained at pH 9. Freshly eluted
37 mBq 99mTc was added to each vial. The vials were shaken
for 1 min, filtered through a 0.22 lm pore size membrane
filter and incubated for 5 min at room temperature. After that,
the labeling efficiency was analyzed by RTLC.
99m
Stability of Tc-MTX in saline
100 lL of 99mTc-MTX reaction medium was added to
1 mL of saline. The mixture was incubated at 37 C for
24 h. One drop of sample was spotted on chromatographic
strips at different time intervals up to 24 h and RTLC
studies were performed to determine the stability of 99mTc-
MTX in saline.
99m
Stability of Tc-MTX in human serum
100 lL of 99mTc-MTX reaction medium was added to
1 mL of freshly prepared human serum. The mixture was
incubated at 37 C for 24 h. One drop of sample was
spotted on chromatographic strips at different time inter-
vals up to 24 h and RTLC studies were performed to de-
termine the stability of 99mTc-MTX in human serum.
Radio thin layer chromatography (RTLC) analysis
For RTLC studies, Whatman 3MM papers and instant thin
layer chromatography-silica gel coated plates (ITLC-SG) were
used as stationary phases. Free 99mTc was determined by using
Whatman 3MM papers as stationary phase and acetone as the
mobile phase. Reduced/Hydrolyzed (R/H) 99mTc was deter-
mined by ITLC-SG plates which developed in Acetonitrile/
Water/Trifluoroacetic acid (ACN/W/TFA; 50/25/1.5) solvent
system. The radioactivity on plates was measured using a TLC
scanner (Bioscan AR 2000), and RP (%) of 99mTc was cal-
culated from the following Eq. (1) by subtracting from 100 the
sum of measured impurities percentages.
 
RP % 100  Free99m Tc % R=H99m Tc %
1
Effect of radioactivity doses on labeling
In vitro radiolabeling studies were performed with 37 mBq
99m
Tc for the regard to radiation safety of personnel and the
J Radioanal Nucl Chem
environment. Since, human studies with radiopharmaceu-
ticals are performed with higher radiation doses; the RP of
MTX which prepared 37 and 370 MBq 99mTc was
examined.
Cell culture studies
The MCF-7 and CRL8798 cells were cultured in Dulbec-
cos modified essential medium (DMEM) supplemented
with 10 % fetal bovine serum. Cell monolayers were pre-
pared by seeding 2 9 105 cells on six wells with transwell
insert filter. Cell culture was maintained at 37 C under
90 % humidity and 5 % CO2. 24 h after seeding, the in-
tegrity of each cell monolayer was checked by measuring
its TEER with an epithelial voltameter (EVOM, World
Precision Instrument, Sarasota, FL, USA) during test pe-
riod. The TEER value was calculated from the following
Eq. (2):

statistical significance. Differences at the 95 % confidence
TEER Rmonolayer  Rblank  A 2
Where Rmonolayer is the resistance of the cell monolayer
along with the filter membrane, Rblank is the resistance of
the filter membrane and A is the surface area of the
membrane (4.7 cm2 in six well plates) [18].
In vitro incorporation studies
Incorporation of 99mTc-MTX to human breast (CRL8798)
and human breast carcinoma (MCF-7) cell was investi-
gated. Before the experiments, cell cultures were tryp-
sinzed and to remove trypsin, washed once in the
phosphate buffered saline (PBS). MTX-cell culture medi-
um solution was labeled with 1 mCi 99mTc and then in-
cubated for 30, 60, 90 and 120 min at 37 C. The activities,
which in the apical side containing sediment cells and in
the basolateral side containing culture medium were both
counted by a gamma counter (Sesa Uniscaller). The cel-
lular uptake was calculated as the percentage of the activity
counted in the cells relative to the total activity counted.
The percentage radioactivity of cells was calculated from
the following Eq. 3 by dividing the radioactivity of cells to
the total radioactivity. The total radioactivity is radioac-
tivity of cells plus radioactivity of cell culture medium.
Radioactivity of Cells 100  Radioactivity of Cells
= Total radioactivity 3 To investigate the effect of reducing agent amount on la-
Sterility test
The sterility of the MTX solution was tested by direct
inoculation method according to the European Pharma-
copeia 6.0 recommendations. The MTX solution was
aseptically inoculated to the sterilized terrific broth medi-
um (TB medium) and triptic soy broth (TSB medium)
vials, and incubated at 35 2 C during 7 days. At the
end of the incubation time, the vials were evaluated ac-
cording to growth of microorganisms.
Isotonicitiy test
The isotonicity test of the MTX solution was tested by
using osmometer. The samples within eppendorf tube were
analyzed in the device which was calibrated with calibra-
tion solutions.
Statistical analysis
The calculation of means and standard deviations were
made on Microsoft Excel. t test was used to determine
level (p [ 0.05) were considered significant.
Results and discussion
Radiolabeling studies
A new, simple, rapid and efficient direct method for la-
beling of MTX with 99mTc was developed. Labeling effi-
ciency of the 99mTc-MTX was assessed by RTLC studies.
Two solvent systems were used to distinguish and quantify
the amounts of radioactive contaminants (Free 99mTc, R/H
99m
Tc). RP and stability of 99mTc-MTX were assessed by
RTLC studies. In RTLC using acetone as the solvent, free
99m
Tc moved with the solvent front, while 99mTc-MTX and
R/H 99mTc remained at the spotting point. R/H 99mTc was
determined by using (ACN/W/TFA; 50/25/1.5) as the
mobile phase where the R/H 99mTc remained at the point of
spotting while free 99mTc and 99mTc-MTX moved with the
solvent front. The RTLC chromatogram of 99mTc-MTX
was presented in Figs. 1 and 2. The RP of 99mTc-MTX was
95 %, acquired via RTLC.
Effect of reducing agent amount on labeling
beling yield, labeling experiments were performed. By
increasing the amount of stannous tartrate, the labeling
efficiency was decreased while the amounts of colloid in-
creased. By increasing the reducing agent concentration,
the labeling yield was slightly decreased (Fig. 3). Ac-
cording to radiolabeling studies, no significant differences
between groups were assessed (p \ 0.05).
123
 J Radioanal Nucl Chem

Fig. 1 RTLC chromatogram of

99m
Tc-MTX in acetonitrile/
water/trifloroacetic acid (50/25/
1.5)

Table 1 500 lg stannous tartrate including formulations radio-

chemical purity in the absence and presence of ascorbic acid
Time (h) Radiochemical purity
Ascorbic acid (mg)
0 0.05 0.1

0 89.55 8.72 95.27 2.32 95.44 2.90

1 81.82 10.34 89.38 5.05 89.86 7.29
2 73.29 8.19 86.96 8.54 76.56 19.76
3 66.11 13.87 75.52 25.57 56.79 33.17
4 60.96 20.64 62.34 25.07 41.67 25.98
5 54.94 24.01 60.80 33.56 41.36 23.85
6 55.25 25.29 49.76 42.03 40.54 31.83
99m
Fig. 2 RTLC chromatogram of Tc-MTX in acetone

99m
Fig. 3 Variation of Tc-MTX as a function of stannaous tartrate

123
J Radioanal Nucl Chem

Effect of antioxidant agent amount on labeling Table 4 1250 lg stannous tartrate including formulations radio-
chemical purity in the absence and presence of ascorbic acid
In the presence of ascorbic acid, stability of the complex Time (h) Radiochemical purity
was increase slightly while labeling efficiency for early
Ascorbic acid (mg)
hours was not affected significantly. Increasing of the la-
beling efficiency takes place due to a decreasing of the R/H 0 0.05 0.1
99m
Tc percentage. The results were shown in Tables 15 0 91.27 4.47 96.27 0.42 87.67 8.37
and revealed that, maximum RP was obtained with 1 87.30 3.16 91.83 4.84 91.28 5.23
1000 lg stannous tartrate and 0.1 mg ascorbic acid in- 2 80.49 9.84 90.44 3.94 81.98 4.63
cluding formulations. Effect of incubation time, pH and
3 74.59 16.57 88.77 3.99 83.83 9.16
stability of these formulations were investigated to evaluate
4 66.79 24.87 86.46 5.63 79.13 7.38
labeling conditions for MTX.
5 62.17 38.77 84.36 7.75 71.81 17.99
6 56.05 40.38 79.76 10.72 60.68 18.94
Effect of radioactivity doses on labeling

MTX solutions were labeled with 37 and 370 mBq 99mTc.

Table 5 1500 lg stannous tartrate including formulations radio-
Slightly decrease in RP of 99mTc- MTX complex was ob- chemical purity in the absence and presence of ascorbic acid
served with increasing of radioactivity (Table 6).
Time (h) Radiochemical purity
Ascorbic acid (mg)
0 0.05 0.1
Table 2 750 lg stannous tartrate including formulations radio-
chemical purity in the absence and presence of ascorbic acid 0 86.67 6.08 94.51 0.79 94.65 2.06
Time (h) Radiochemical purity 1 76.65 6.76 87.55 4.89 93.30 3.81
2 79.83 2.65 86.65 4.29 90.17 6.05
Ascorbic acid (mg)
3 69.24 6.14 86.16 5.50 83.07 9.24
0 0.05 0.1
4 50.94 18.35 81.95 8.84 84.94 6.09
0 92.2 4.07 93.77 4.16 96.20 1.76 5 61.31 21.84 80.71 5.41 76.12 15.43
1 89.09 0.52 89.39 4.85 89.78 6.96 6 57.71 31.43 76.17 9.00 83.32 17.63
2 77.38 3.71 90.07 4.75 85.99 6.04
3 64.64 14.60 85.52 7.89 79.19 14.95 99m
Table 6 The effect of radioactivity doses (37 and 370 mBq Tc)
4 65.79 15.85 53.99 20.24 68.86 13.64 on radiochemical purity of 99mTc-MTX complex
5 60.42 15.92 48.69 36.46 47.73 37.60
Time (h) Radiochemical purity
6 42.39 27.30 20.29 4.74 42.80 35.04
Radioactivity doses (MBq)
37 370

0 97.37 1.01 96.43 1.46

Table 3 1000 lg stannous tartrate including formulations radio- 1 94.62 2.76 96.54 0.47
chemical purity in the absence and presence of ascorbic acid 2 93.99 3.87 92.60 0.99
Time (h) Radiochemical purity 3 94.27 3.41 91.94 2.69
Ascorbic acid (mg) 4 90.56 4.16 85.07 1.29
5 87.86 3.38 76.88 2.48
0 0.05 0.1
6 92.43 3.72 17.43 2.66
0 91.20 6.21 95.84 2.03 97.37 1.01
1 89.86 1.96 93.95 2.23 94.62 2.76
99m
2 84.62 5.66 93.65 1.04 93.99 3.87 Stability of Tc-MTX in saline
3 66.64 29.25 91.93 0.35 94.27 3.41
4 67.33 31.37 89.79 0.97 90.56 4.16 The 99mTc-MTX complex stability in saline was controlled
5 59.97 31.79 82.68 10.07 87.86 3.38 to 24 h at room temperature. During the incubation period
6 53.64 29.43 70.37 29.38 92.43 3.72 the compound were found stable as determined by RTLC
(Table 7).

123
 J Radioanal Nucl Chem

99m
Table 7 Radiochemical purity of Tc-MTX complex in saline
Time (h) Radiochemical purity (%)

0 95.30 1.10
1 94.00 1.05
2 94.56 0.33
3 93.54 1.47
4 92.69 0.66
5 92.83 0.30
6 92.68 0.45
24 59.61 3.86

99mTc
Stability of -MTX in human serum
Fig. 4 Incorporation percentage of 99mTc-MTX to the MCF-7 and
The 99mTc-MTX complex stability in human serum was CRL8798 cell lines for 30, 60, 90 and 120 min (p \ 0.05)
controlled to 24 h at room temperature. During the incu-
bation period the compound were found stable as deter-
Cell binding studies
mined by RTLC (Table 8).
Okarvi et al. labeled MTX and folic acid molecule
In this study, the incorporation capacity of 99mTc-MTX on
(15 mg) with 200 lL 99mTc containing 370555 MBq of
MCF-7 and CRL8798 cells has been observed. Incorpora-
radioactivity and applied heating on labeling complex at
tion percentage of 99mTc-MTX to MCF-7 and CRL8798
908 C for 1015 min. They used stannous chloride as re-
cell lines for 30, 60, 90 and 120 min incubation periods are
ducing agent. According to their results, the optimal ra-
shown in Fig. 4. The cell incorporation percentage after the
diolabeling conditions were achieved at pH of 10 and
99m application of 99mTc-MTX were found to be between
Tc-MTX conjugates remained stable (up to 60 %) at
38.2 4.49 % and 30.6 5.67 % for CRL8798 cell
room temperature for up to 18 h after labeling [15]. In this
during experiment time. On the other hand, after exposure
study, MTX was labeled 99mTc with simple method and
99m to 99mTc-MTX for MCF-7, the cell incorporation percent-
Tc-MTX complex was quite stable and radiolabeling
age were found to be between 56.7 0.64 % and
yield [95 % was maintained for up to 6 h. In addition,
50.57 1.26 % during 30, 60, 90 and 120 min (Fig. 4).
stability of 99mTc-MTX in human serum and saline was
Evaluation of the results revealed that 99mTc-MTX has a
obtained during 24 h.
greater incorporation activity on MCF-7 than CRL8798.
Dar et al. were labeled MTX with 99mTc containing
555 MBq, using stannous tartrate as a reducing agent. They
Transepithelial electrical resistance measurements
found 99mTc-MTX complex was stable up to 5 h at room
temperature (RP = 98.2 0.5 %) [13]. In our study, we
The resistance of the cells before and after the experiments
studied with 37 and 370 MBq 99mTc doses and found
99m was measured by TEER. This method addresses different
Tc-MTX was more stable in lower radioactivity dose.
aspects of cell functionally and is widely used as a general
criterion of cytotoxicity [19]. In this study, MCF-7 and
Table 8 Radiochemical purity of 99mTc-MTX complex in human
CRL8798 were used for examining the binding affinity of
serum 99m
Tc-MTX. The TEER values of all experiments were
Time (h) Radiochemical purity (%) found to be between 1207 51.76 and 1278 70.95
0 75.78 3.39 ohms/cm at the beginning of experiments for all cell lines
1 75.93 5.31 (Table 9). After each test period, the TEER values were
2 75.10 5.83 found to be between 1206 39.16 and 1218 65.84
3 71.81 7.49
ohms/cm. No significant decrease in the TEER values was
4 62.34 5.99
observed during the experiments. The TEER variations
were not higher than 40 %. If the TEER variation was
5 61.4 9.66
never higher than 40 % the cells were not damaged [20].
6 61.87 5.67
This demonstrated that the MCF-7 and CRL8798 were still
24 59.3 1.43
viable after the completion of all experiments.

123
J Radioanal Nucl Chem
Table 9 The TEER values of MCF-7 and CRL8798 cell lines during
the test period (p \ 0.05)
TEER values
Time (min) MCF-7 CRL8798
0 1207 51.76 1278 70.95
30 1206 39.16 1218 65.84
60 1198 22.45 1203 45.60
90 1201 12.56 1178 33.44
120 1179 41.78 1194 50.71
Sterility test
The sterility of the MTX solution was tested. At the end of
the sterility test, absence of clearly visible growth of mi-
croorganisms in the vials exhibited the sterility.
Isotonicity test
The isotonizing of the MTX solution was found
300 mOsm/mL. This value was available for injectable
preparation according to European Pharmacopeia 6.0
recommendation.
Conclusion
MTX is a well-known adjuvant for the treatment of various
cancers including leukemia, esophageal, gastric, pancreat-
ic, ovarian, bladder, cervical, colorectal, penile carcinomas,
lymphoma, head and neck cancer, and breast tumors. MTX
was labeled with 99mTc. 99mTc-MTX was developed and
standardized under varying conditions of reducing and
antioxidant agent concentration. Labeling studies were
performed by changing the selected parameters one by one
and optimum labeling conditions were determined.
Simple method for radiolabeling of MTX with 99mTc
has been developed and standardized. Labeling efficiency
of 99mTc-MTX was assessed by both RTLC and RHPLC.
The resulting complex was quite stable and labeling yield
[90 % was maintained for up to 6 h. According to cell
culture results, radiolabeled MTX proved a useful tool for
assessing the in vitro affinity of the normal and cancer
breast cell binding. 99mTc-MTX is incorporated into the
MCF-7 and CRL8798 cells, especially the MCF-7 which is
a cancer cell line. Consequently 99mTc-MTX can be can-
didate for breast cancer diagnosis, offering potential for
developing new selective therapies for this resilient cancer.
Acknowledgments This study was supported by 14-ECZ-036. The
authors would like to acknowledge the support of T.R. Prime Ministry
State Planning Organization Foundation Grant Project Number:
09DPT001 for TLC Scanner. Also the authors thank to Ege Univer-
sity Nuclear Medicine Department technicians for obtaining 99mTc.
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