Radiolabeling and in vitro evaluation of a new 5-fluorouracil derivative with cell culture studies

Article abstract of DOI:10.1002/jlcr.3804

The clinical impact and accessibility of ^99mTc tracers for cancer diagnosis would be greatly enhanced by the availability of a new, simple, and easy labeling process and radiopharmaceuticals. 5-Fluorouracil is an antitumor drug, which has played an important role for the treatment of breast carcinoma. In the present study, a new derivative of 5-Fluorouracil was synthesized as (1-[{1′-(1′′-deoxy-2′′,3′′:4′′,5′′-di-O-isopropylidene-β-D-fructopyranose-1′′-yl)-1′H-1′,2′, 3′-triazol-4′-yl}methyl]-5-fluorouracil) (E) and radiolabeled with ^99mTc. It was analyzed by radio thin layer chromatography for quality control and stability. The radiolabeled complex was subjected to in vitro cell-binding studies to determine healthy and cancer cell affinity using HaCaT and MCF-7 cells, respectively. In addition, in vitro cytotoxicity studies of compound E were performed with HaCaT and MCF-5 cells. The radiochemical purity of the [^99mTc]TcE was found to be higher than 90% at room temperature up to 6 hours. The radiolabeled complex showed higher specific binding to MCF-7 cells than HaCaT cells. IC₅₀ values of E were found 31.5 ± 3.4 μM and 20.7 ± 2.77 μM for MCF-7 and HaCaT cells, respectively. The results demonstrated the potential of a new radiolabeled E with ^99mTc has selective for breast cancer cells.

Full text of DOI:10.1002/jlcr.3804

Atlihan-Gundogdu Evren (Orcid ID: 0000-0002-4095-2714)
Radiolabeling and in vitro evaluation of a new 5-Fluorouracil derivative with cell
culture studies
a
a
a
b
c
Derya Ilem-Ozdemir, Evren Atlihan-Gundogdu, Meliha Ekinci, Erkan Halay, Kadir
d
a
Ay, Tamer Karayildirim, Makbule Asikoglu
a. Department of Radiopharmacy, Faculty of Pharmacy, Ege University, Bornova, Izmir,
Turkey.
b. Scientific Analysis and Technological Application and Research Center, Usak University,
Usak, Turkey.
c. Department of Chemistry, Faculty of Art and Sciences, Manisa Celal Bayar University,
Yunusemre, Manisa, Turkey.
d. Department of Chemistry, Faculty of Sciences, Ege University, Bornova, Izmir, Turkey.
This article has been accepted for publication and undergone full peer review but has not been
through the copyediting, typesetting, pagination and proofreading process which may lead to
differences between this version and the Version of Record. Please cite this article as doi:
10.1002/jlcr.3804
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ABSTRACT
The clinical impact and accessibility of 99mTc tracers for cancer diagnosis would be greatly
enhanced by the availability of a new, simple and easy labeling process and
radiopharmaceuticals.
5
-Fluorouracil (5-FU) is an anti-tumor drug which has played an important role for the
treatment of breast carcinoma. In the present study, a new derivative of 5-FU was synthesized
as (1-[{1′-(1′′-deoxy-2′′,3′′:4′′,5′′-di-O-isopropylidene-β-D-fructopyranose-1′′-yl)-1′H-1′,2′,3′-
triazol-4′-yl}methyl]-5-fluorouracil) (E), and radiolabeled with 99mTc. It was analyzed by radio
thin layer chromatography (RTLC) for quality control and stability. The radiolabeled complex
was subjected to in vitro cell binding studies to determine healthy and cancer cell affinity using
HaCaT and MCF-7 cells, respectively. In addition, in vitro cytotoxicity studies of compound
E were performed with HaCaT and MCF-5 cells.
The radiochemical purity of the [99mTc]TcE was found to be higher than 90 % at room
temperature up to 6 h. The radiolabeled complex showed higher specific binding to MCF-7
cells than HaCaT cells. IC50 values of E were found 31.5±3.4 and 20.7±2.77 µM for MCF-7
and HaCaT cells, respectively.
The results demonstrated the potential of a new radiolabeled E with 99mTc has selective for
breast cancer cells.
Key words: 5-fluorouracil derivative, Radiolabeled compound, Technetium-99m, Breast
cancer, Cell binding.
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INTRODUCTION
Breast cancer is the most common cancer among women worldwide [1]. Prognosis is
very good if the disease is detected in early stages, however, in the presence of metastatic
disease, 5-year survival drops dramatically. Since early diagnosis is critical for patient survival,
there is an acute need for developing novel sensitive breast cancer imaging agents and
techniques [2, 3]. Nuclear medicine imaging provides noninvasive functional information at
molecular and cellular level that contributes to the determination of health status by measuring
the uptake and turnover of target-specific radiotracers in the tissue. By using nuclear imaging
procedures, abnormalities are often identified in very early stages. The early detection allows
more successful prognosis possibility for the treatment of disease [1].
Cancer imaging studies by using radiopharmaceuticals targeted to specific receptors
have been employed successfully up to now [4]. Radiopharmaceuticals consist of two
components, a biomolecule and a radionuclide with a specific radiation [4, 5]. For diagnostic
purposes, gamma emitter radionuclides are preferred because of their low linear energy transfer
which results in low tissue damage in the targeted organ [5]. The ideal nuclear properties of
99mTc likewise; 140 keV pure gamma photon emission, 6 h physical half-life, low cost, readily
availability are optimum for preparing radiopharmaceuticals for diagnostic purposes [6-8]. The
biomolecules of the radiopharmaceuticals are responsible for the accumulation of the
radionuclide in the targeted tissue. High accumulation in the target and low accumulation in
non-target tissue is preferred to increase the resolution and sensitivity of the images and to
reduce radiation damage in the rest of the body. The better target/non target ratios are achieved
with radiolabeled receptor-specific molecules for cancer imaging and therapy.
5
-Fluorouracil (5-FU) is an effective anti-tumor drug, which has been used
either as a single agent or in combination with other chemotherapeutic agents for the treatment
of tumors such as breast and colorectal carcinoma [9-12]. 5-FU is an antimetabolite drug and
works by inhibiting essential biosynthetic processes, or by being incorporated into
macromolecules, such as DNA and RNA. Also, 5-FU is an analogue of uracil with a fluorine
atom at the C-5 position in place of hydrogen. It rapidly enters the cell using the same facilitated
transport mechanism as uracil and is converted intracellularly to several active metabolites:
Fluorodeoxyuridine monophosphate (FdUMP), fluorodeoxyuridine triphosphate (FdUTP) and
fluorouridine triphosphate (FUTP)— which disrupt RNA synthesis. There are some studies
showing a higher selective potential for cancer cells than normal cells. The mechanism of
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cytotoxicity of 5-FU on cancer cells has been ascribed to the misincorporation of
fluoronucleotides into RNA and DNA and to the inhibition of the nucleotide synthetic enzyme
thymidylate synthase [13, 14]. Since 5-FU is poorly tumor selective, high incidences of bone
marrow, gastrointestinal tract and central nervous system toxicity are common in its therapeutic
use. To tackle these problems, numerous structural modifications of the 5-FU have been
performed. Thus, a series of 5-FU prodrugs in which 5-FU is attached to amino acids, peptides,
phospholipids and polymers have been reported [15-23]. These N-1 and/or N-3 substituted
derivatives have exhibited improved pharmacological and pharmacokinetic properties,
including increased bioactivity, selectivity, metabolic stability, absorption and lower toxicity
[
20]. According to this strategy, in this study a new derivative of 5-FU that contains nucleobase
and sugar molecules, 1-[{1′-(1′′-deoxy-2′′,3′′:4′′,5′′-di-O-isopropylidene-β-D-fructopyranose-
′′-yl)-1′H-1′,2′,3′-triazol-4′-yl}methyl]-5-florouracil (Compound E), was reported [20] and
1
synthesized for higher specificity to breast cancer.
The synthesis of new derivatives of 5-FU could be beneficial for receptor mediated
uptake of 5-FU by utilizing these elevated receptors on cancer cells. In this study, we propose
that radiolabeled compound of 5-FU derivative bearing pyranose sugar and nucleobase groups
could be beneficial for increasing ‘MFC-7/HaCaT cellular uptake ratios’ and aim to radiolabel
compound E with 99mTc and show cellular binding capacity for different types of cells by using
in vitro cell culture studies. For this purpose, compound E was radiolabeled with 99mTc under
varying conditions. Radiochemical purity was determined with radioactive thin layer
chromatography (RTLC). After determining the optimum labeling conditions for maximum
labeling efficiency and stability, the in vitro cell incorporation affinity of developed
formulation to breast carcinoma (MCF-7) and the human keratinocyte (HaCaT) cell lines was
investigated.
MATERIALS AND METHODS
Materials
Stannous chloride dehydrate was purchased from Sigma-Aldrich (USA). [99mTc]TcO -
4
was eluted from the Molybdenum-99 ( Mo)/99mTc generator. All solvents were obtained from
Merck (Germany). Cell culture reagents and supplies were obtained from American Type
Culture Collection (ATCC). Radioactive samples counted in a counting unit (Atomlab 100
Dose Calibrator Biodex Medical Systems). All other materials were of analytical grade.
99
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Experimental
Synthesis of (1-[{1′-(1′′-deoxy-2′′,3′′:4′′,5′′-di-O-isopropylidene-β-D-fructopyranose-1′′-
yl)-1′H-1′,2′,3′-triazol-4′-yl}methyl]-5-fluorouracil) (E)
The azido-fructopyranose (C) and alkynated-5-fluorouracil (D) derivatives obtained in
the first two steps and were reacted in THF:t-BuOH:H O solvent system in the presence of
2
suitable catalysts to give the desired target compound E in high yield via copper-catalyzed
azide-alkyne cycloadditon (CuAAC), namely click reaction as previously reported (Figure 1)
[
24].
Radiolabeling Studies
Radiolabeling was obtained with [99mTc]TcO -
compound E (1 mg) was dissolved in 1 mL saline. Stannous chloride was dissolved in 1 mL of
.02 N HCl and kept under an atmosphere of nitrogen. Reduction of [99mTc]TcO -
was
performed by adding different amount of 1 mL stannous chloride solution (10, 50, 250 and
4
(37 MBq) in saline (0.1 mL). The
0
4
5
1
00 µg/mL) to 1 mg/mL E solution. and the solution was left to stand at room temperature for
5 min prior to radiochemical analyses. The final volume of reaction medium was 2.1 mL.
To determine and optimize the required concentration of reducing agent and pH value
for efficient labeling, radiolabeling process was performed with different concentrations of
stannous chloride as reducing agent at different pH values (pH 5 and 7.4). In each optimization,
radiochemical purity was assessed by RTLC up to 6 h.
Effect of Reducing Agent on Radiolabeling
Reduction of [99mTc]TcO
-
was performed with different amount of stannous chloride
4
-1
in 0.02 N HCl (10, 50, 250 and 500 µg.mL ). Compound E was dissolved in 1 mL saline.
Radiolabeling was performed with [99mTc]TcO -
(37 MBq) in saline (0.1 mL) and solution was
4
allowed to stand at room temperature for 15 min prior to radiochemical analyses.
Effect of pH on Radiolabeling
The radiochemical purity of 99mTc radiopharmaceutical is highly dependent on the pH
[
6]. The effect of pH on the radiochemical purity of [99mTc]TcE was examined for pH 5.0 and
7
.4. The potassium phosphate monobasic dibasic solution was used to adjust the pH 7.4. The
sodium acetate buffer was used to adjust the pH 5.
Stability of [99mTc]TcE in Different Media
The saline, human serum and cell medium were used to evaluate the stability of
radiolabeled complex at different media. [99mTc]TcE reaction medium (37 MBq-100 µL) was
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added to 900 µL of each media. The mixtures were incubated at 37 °C at different time intervals
up for 6 h and 24 h. Aliquots were taken out and spotted on chromatographic strips and
analyzed by RTLC [25].
For cell medium stability assay, 37 MBq of radiolabeled compound was added into cell
medium. The mixture was incubated at 37°C and samples were taken and spotted on
chromatographic papers at 15, 60 and 120 min after incubation. The radiochemical purity of
samples was analyzed with RTLC.
RTLC Procedure
RTLC was performed using Whatman No:3 paper as stationary phase. Free 99mTc was
determined by using acetone as the mobile phase. Reduced/Hydrolyzed (R/H) 99mTc was
determined by Whatman No:3 paper which developed in saline. The radioactivity on
chromatographic papers was measured using a TLC scanner (Bioscan AR 2000), and %
radiochemical purity (RP) of [99mTc]TcE was calculated from the following equation
(Equation 1) by subtracting from 100 the sum of measured impurities percentages.
RP (%) = 100 – [Free 99mTc (%) + R/H 99mTc (%)]
Equation 1
Cell Culture Studies
MCF-7 (breast carcinoma) and HaCaT (human keratinocyte) cells were in McCoy’s 5A
supplemented with 10% fetal bovine serum. Cell culture was maintained at 37°C under 90%
humidity and 5% CO . Subculturing was performed employing a 0.25% Trypsin–0.1% EDTA
2
solution. Cell monolayers were prepared by seeding cells on six wells.
After 24 h seeding, the integrity of each cell monolayer was checked by measuring its
transepithelial electrical resistance (TEER) with an epithelial voltammeter (EVOM, World
Precision Instrument, Sarasota, FL, USA). The TEER value was calculated from the following
equation (Equation 2):
TEER

RmonolayerRblank

 A
Equation
2
R
monolayer is the resistance of the cell monolayer along with the filter membrane, Rblank is the
2
resistance of the filter membrane and A is the surface area of the membrane (4.7 cm in six
well plates) [25, 26].
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Cell Binding Studies
In vitro cell binding experiments were performed in triplicate. The cells (5 × 105
cells/well) were distributed in 6-well plates and incubated at 37 °C in cell culture medium. The
medium was removed, and cells were washed with phosphate-buffered saline (PBS; 1 mL) and
incubated at 37 °C in 0.5 mL of McCoy’s 5A supplemented with 10% fetal bovine serum. After
2
h, 0.5 mL of 37 MBq [99mTc]TcE was added and incubation was continued. The incubation
medium was removed and cells were washed and harvested at 30, 60, 90 and 120 min. After
removal of the radiolabel-containing medium, the isolated cells were consecutively washed
with 1 mL of cell medium and 1 mL of PBS to remove free radiolabeled complex. The
activities, which in the tubes containing sediment cells and in the tubes containing culture
medium were both counted by a gamma counter (Sesa Uniscaller). The cellular 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 equation by
dividing the radioactivity of cells to the total radioactivity (radioactivity of cells plus
radioactivity of cell medium) (Equation 3).
Radioactivity of Cells = (Radioactivity of Cells / Total radioactivity) x 100
Equation 3
In vitro cytotoxicity studies
The cytotoxicity of E was determined by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-
5
-
tetrazolium bromide (MTT) assay. MCF-7 and HaCaT cells (1 × 10 /well) were plated in
1
mL of medium/well in 24-well plates (Costar Corning, Rochester,NY). After the cell reaches
the confluence, they were incubated in the presence of various concentrations (10, 50, 100,
00, 1000 and 2000 µg/mL) of E in 0.1% DMSO for 48 h at 37 ° C. The sample solutions
5
were removed and 1 mg/mL MTT in phosphate- buffered saline solution was added. Viable
cells were determined by the absorbance at 570 nm. Measurements were performed and the
concentration required for a 50 % inhibition of viability (IC50) was determined with GraphPad
Prism and fitted using a ‘one-site total binding’ algorithm program. In this program, the
concentration of E was varied and IC50 was calculated. In this program, XY data table was
created. The E concentration is entered as X values, and cellular uptake was entered as Y
values. Nonlinear regression was chosen and IC50 values were calculated and results viewed
in the scene.
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Statistical Analysis
The calculation of means and standard deviations were made on Microsoft Excel.
Oneway Anova was used to determine statistical significance. Differences at the 95%
confidence level (p<0.05) were considered significant. Experiments were performed in
triplicate unless stated otherwise. Results are reported as mean ± standard error.
RESULTS
Synthesis of (1-[{1′-(1′′-deoxy-2′′,3′′:4′′,5′′-di-O-isopropylidene-β-D-fructopyranose-1′′-
yl)-1′H-1′,2′,3′-triazol-4′-yl}methyl]-5-fluorouracil) (E)
The structural elucidation of compound E was performed by spectroscopic methods and
all obtained data are compatible with our previous study [24].
Radiolabeling Studies
A novel, simple, rapid and efficient direct method for labeling of compound E with
99mTc was developed and conducted at room temperature. Labeling efficiency of the
[99m
Tc]TcE was assessed by RTLC studies. Two solvents were used to distinguish and quantify
the amounts of radioactive impurities.
According to RTLC results, The R
f
values of [99mTc]TcE mobile phases were presented
in Table 1. The RTLC chromatogram of [99mTc]TcE was presented in Figure 2. In RTLC, using
acetone as the solvent, free 99mTc migrated with the solvent front, while [99mTc]TcE and R/H
99mTc remained at the spotting point. R/H 99m
Tc was determined by using saline as the mobile
phase where the R/H 99mTc remained at the point of spotting while free 99mTc and [99mTc]TcE
moved with the solvent front. Under optimized conditions, [99mTc]TcE was prepared and at a
radioactivity around 37 MBq and radiochemical purity over 90%.
Effect of Reducing Agent on Radiolabeling
Radiolabeling studies were performed with different concentrations of reducing agent
to determine the optimum conditions. The effect of reducing agent concentration on the
radiochemical purity was evaluated and optimum reducing agent amount was found to be 50
µg. Under these conditions labeling efficiency was around 90 % and did not change
significantly during 6 h at room temperature (p˃0.05) (Table 2).
Effect of pH on Radiolabeling
The effect of pH on radiochemical purity was examined at two different pH values (pH
5
.0 and 7.4). The radiochemical purity of 99mTc radiolabeled complex was found to be greater
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than 96 % and 95 % at pH 5 and 7.4, respectively. Our results demonstrate that while keeping
other reaction conditions constant and varying the pH of the reaction from 5.0 and 7.4,
radiochemical purity was not changed significantly (p>0.05) (Figure 3) and the radiolabeled
complex was not affected by pH alteration.
Stability of [99mTc]TcE in Different Media
The stability of [99mTc]TcE was evaluated in different media (saline, human serum and
cell medium). The radiochemical purity of the [99mTc]TcE was found to be higher than 95 %,
9
3
0 % and 80 % for 6 h, 24 h and 2 h in saline, human serum and cell medium, respectively at
7 ° C (p>0.05) (Figure 4).
Cell Binding Studies
Radiolabeled samples were tested for their cell binding capacity. Due to the available
half-life of 99mTc, radiolabeled complexes were assessed at 2 h. The cell binding results of
[99m
Tc]TcE for MCF-7 and HaCaT cell lines were illustrated in Fig. 5. Apparently, high cell
binding capacity of % [99mTc]TcE was obtained on MCF-7 cell line. While the cell binding %
[99mTc]TcE ranged from 0.18±0.01 to 0.96±0.06 for MCF-7, the cell binding % [99mTc]TcE
ranged from 0.052±0.03 to 0.35±0.22 for HaCaT cells (Table 3). The radioactivity levels of
[99m
Tc]TcE in MCF-7 was approximately four times higher than HaCaT cell lines and
statistically significant differences were observed between the two cell lines at 30 and 120 min.
Transepithelial electrical resistance measurements (TEER)
The TEER values of cells were found to be between 1028±168.65 and 1302±49.52
-1
ohms.cm at the beginning of seeding period (Figure 6). After 24 h, the TEER values were
-1
measured and found to be between 1112±14.75 and 1537±43.72 ohms.cm (Figure 7).
In vitro cytotoxicity studies
Six different concentrations were used for cytotoxicity studies. While 2000 µg/mL of
E produces 75.5 % cell viability, 1000 µg/mL produces 89.9 % cell viability, 500 µg/mL
produces 93.5 % cell viability, 100 µg/mL produces 96.3 % cell viability, 50 µg/mL produces
9
7.8 % cell viability, 10 µg/mL produces 99 % cell viability for MCF-7 cells, 2000 µg/mL of
E produces 73.5 % cell viability, 1000 µg/mL produces 89 % cell viability, 500 µg/mL
produces 94.5 % cell viability, 100 µg/mL produces 97.5 % cell viability, 50 µg/mL produces
9
8 % cell viability and 10 µg/mL produces 99.5 % cell viability for HaCaT cells. The IC50
values of E for MCF-7 and HaCaT cells were found to be 31.5±3.4 and 20.7±2.77 µM,
respectively.
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DISCUSSION
Selection of radionuclide and drug/biomolecule is important in the development of new
radiopharmaceuticals. While selecting radionuclide as well as biomolecule for the development
of a new radiopharmaceutical, compatibility, half-life of the radioisotope, energy emission,
stability, stoichiometry and molecular size should always be kept in mind. In this study, 5-FU
derivate- compound E was used as a pharmaceutical part and 99m Tc was used as radionuclide
part of developed radiolabeled compound. Compound E contains pyranose sugar and
nucleobase groups (Figure 1). These two groups are important role in terms of higher affinity
to cancer cells [24]. Because of this, we thought that modification of 5-FU with these groups
can affect the cell binding capacity of new radiolabeled compound.
The reducing agent is very crucial for 99mTc radiopharmaceuticals. The colloid structure
occurs in the radiolabeled compound and radiochemical purity ratio begins to decrease by
adding reducing agent in high concentrations. With lower concentrations of reducing agent,
free technetium ratio decreases. In both cases, radiochemical purity of radiolabeled compound
is adversely affected. Mostly stannous salts are preferred to use as reducing agents in 99mTc
radiopharmaceuticals. Technetium normally exists as stable pertechnatate in the +7 oxidative
state. While pertechnetate is the most stable state in aqueous solution, technetium compounds
have been prepared with oxidation states from 1- to 7+. To promote binding of 99mTc with the
synthesized molecule, reducing agents were used to reduce 99mTc from the +7 oxidation state
to more reactive +5 oxidation state [27,28]. In this study, [99mTc]TcE was prepared by using
stannous chloride. The effect of reducing agent concentration on the labeling efficiency was
evaluated, and optimum reducing agent amount was found to be 50 μg (Table 2).
pH can influence the radiolabeling efficiency [29-31]. Generally, ideal pH of a
radiopharmaceutical is 7.4 or can vary between 2 and 9. According to the Young et al. study,
the pH of the reaction medium was found to play unimportant role in the labeling process of 5-
FU [9]. While reaction conditions are keeping constant and the reaction pH is varied from 5 to
7
.4, no significant differences were observed on labeling efficiency (Figure 3). According to
result, the pH of the reaction medium was found to play insignificant role in this study too.
Jung et al. was radiolabeled 5-FU by using the stannous chloride reduction method with
5
55 MBq of 99mTc. Radiochemical purity of the complex was determined by chromatographic
techniques. The overall labeling yield of the99mTc-5-FU complex was calculated to be more
than 98.1±1.2% [10]. Furthermore, Young et al. used simple basic radiochemical reactions for
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the radiolabeling of the 5-FU. The authors have observed >95% radiochemical purity of this
radiolabeled complex [9]. Herein, although the chemical modifications on the main molecule
(
5-FU), the radiochemical purity of [99mTc]TcE was found to be greater than 90%. The various
complexes of 99mTc may be formed by interactions between electron donor atoms and reduced
technetium. In order to form bonds with technetium, the structure must contain electron donors
such as oxygen, nitrogen and sulfur. Although the exact complex structure is not known, results
showed that molecule E is coordinated with 99mTc because of its electron donor atoms such as
oxygen in its structure [32].
The stability of developed radiopharmaceutical is also a very significant factor as the
instability will result in an in vivo dispersion and poor target/non target ratio. In this study,
[99m
Tc]TcE was found to be stable in saline, human serum and cell culture media (Figure 4).
The retention of radioactivity in tumor cells is following binding of a radiolabeled
molecule with factors including the nature of the pharmaceutical part, the chemical structure
of radiolabeled group and the properties of the radionuclide [33]. Dar et al. [17] evaluated the
potential of 5-FU as a diagnostic radiolabeled compound in advanced breast cancer. Their study
showed that [99mTc]Tc-5-FU was a promising agent for diagnosing advanced breast cancer with
optimum visualization at 1h. In this study, we used cell culture models to quantitatively
evaluate the binding capacity of [99mTc]TcE at the breast cancer and healthy cells and we found
that [99mTc]TcE is more bound to MCF-7 cell than HaCaT cell (Figure 5) which offer
advantages. While optimum visualization of breast cancer with 99mTc-5-FU was obtained at 1h
in Dar et. al. studies, high binding capacity with [99mTc]TcE was achieved for breast cancer
cells in the first 30 minutes. The obtained TEER data supports this binding without damaging
healthy cells in this study.
Johnston et al. [34] investigated the cellular interaction of 5-fluorouracil and cisplatin
in a human colon carcinoma cell line and found that exposure to 5-FU is resulted in a IC50 value
of 24.2 ±4.5 µM. Another study, Ando et al. [35] examined 5-fluorouracil sensitivity in
esophageal carcinoma cell lines and found that IC50 value for 5-FU was 39.81 µM. In this
study, IC50 values of E were found to be 31.5±3.4 and 20.7±2.77 µM for MCF-7 and HaCaT
cells, respectively. There is a small change for IC50 values. This finding suggests that the
biological inhibition mechanism of 5-FU and molecule E can be similar and molecule E may
act via thymidylate synthase (TS) inhibition through its active anabolites such as
fluorodeoxyuridine monophosphate (FdUMP) [36].
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A high target to non-target ratio is always preferred as it shall result in superior quality
imaging of the target organ because there would be little interference from the non-target
organs. So, the radiopharmaceutical should have high specificity for the target organ, only then
it will result in high target to non-target ratio. If this ratio is not high enough, drug uptake in
non-target areas can damage the healthy tissues [4, 12, 32, 33, 37, 38]. Although the exact
reason is not explained, the changes have been observed in obtained cell culture results at some
time intervals. These changes may be due to the effect of molecule E on the cell. Therefore,
the evaluation of cellular uptake ratio will be more accurate for cell culture studies. According
to cell culture study results, the highest MCF-7/HaCaT cellular uptake ratio was observed with
[99m
Tc]TcE at 30 min (15.123±9.151%) and gradually decreased at other times (Table 3).
The use of tracer compounds (drug molecules, radiopharmaceuticals) can interfere with
the binding/transport process under study and can also affect the cell integrity. Also, the use of
chemical dyes renders the tested cells unusable for further experiments. Therefore, non-
invasive techniques are best suited to continuously monitor the cell integrity. TEER is the
measurement of electrical resistance across a cellular monolayer and is a very sensitive and
reliable method to confirm the integrity and permeability of the monolayer. In this study, the
resistance of the cells at the beginning of seeding and after 24 h was measured by TEER. This
method addresses different aspects of cell functionally and is widely used as a general criterion
of cell viability and integrity [39]. No significant decrease in the TEER values was observed
to start binding studies (Figure 6 and 7). The TEER variations were not higher than 40%. If the
TEER variation was never higher than 40% the cells were not damaged and the cells were
viable and ready for experiments [40].
CONCLUSION
The presented study aimed to develop a new radiolabeled compound which is able to
detect the breast cancer. To improve the accumulation percentage of the pharmacocophore in
the desired tissue, a new derivative of 5-FU, that contains nucleobase and sugar molecule, was
synthesized as (1-[{1′-(1′′-deoxy-2′′,3′′:4′′,5′′-di-O-isopropylidene-β-D-fructopyranose-1′′-yl)-
1
′H-1′,2′,3′-triazol-4′-yl}methyl]-5-fluorouracil) and optimized.
An easy method of radiolabeling method for novel 5-FU derivative was obtained with
high labeling yields at pH 5 and 7.4. To estimate the cancer cell imaging capability, in vitro
cell binding studies were performed and the incorporation ratios of the labeled compound to
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normal and cancer cells were evaluated. 30 min after incubation the affinity of labeled
compound to cancer cell lines was found higher than normal cells.
In conclusion, radiolabeled compound has promising properties for detection of breast
cancer. Further, investigations are needed for clinical validation via animal experiments as well
as clinical studies.
Acknowledgements
This study was supported by The Scientific and Technological Research Council of
Turkey (TUBITAK-115/S/935). The authors also would like to thank to the T.R. Prime
Ministry State Planning Organization Foundation (Project Number: 09/DPT/001). The authors
would like to acknowledge the support of Ege University Nuclear Medicine Department to
obtain the 99mTc radionuclide.
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Table 1. Rf values of 99mTc-E in mobile phases
Whatman 3MM
Acetone
0.8-1.0
0.0
Saline
0.8-1.0
0.0
Free 99mTc
R/H 99mTc
99mTc–E
0.0
0.8-1.0
Table 2. Radiochemical purity of 99mTc-E which prepared with different amounts of stannous
chloride.
99mTc-E (%)
Stannous Chloride (µg)
Time (Hour)
10
50
250
500
0
1
2
3
4
5
6
93.47 ± 1.10
92.94 ± 1.26
90.51 ± 1.75
90.59 ± 1.46
87.46 ± 5.60
82.77 ± 11.49
82.47 ± 7.01
97.23 ± 0.16
96.43 ± 0.65
96.50 ± 0.54
96.48 ± 0.30
95.87 ± 0.68
96.17 ± 0.67
96.28 ± 1.08
94.11 ± 1.33
93.78 ± 0.72
93.97 ± 1.84
93.75 ± 1.26
92.85 ± 0.51
94.25 ± 0.73
94.89 ± 0.91
95.91 ± 0.47
96.12 ± 0.74
94.64 ± 2.08
95.53 ± 1.64
95.69 ± 0.91
94.75 ± 1.63
95.29 ± 0.63
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Table 3. Cell binding ratios of 99mTc-E to MCF-7 and HaCaT cell lines
Time (Min)
MCF-7/HaCaT ratio
15.123 ± 9.151
1.638 ± 1.907
3
6
9
0
0
0
1.521 ± 0.386
1
20
1.479 ± 0.709
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