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Synthesis, Radiolabeling and Bioevaluation of 99mTc (CO)3-Folate Azide Complex for Tumor Detection.

Byline: Irfan Ullah Khan, Abubaker Shahid, Fayyaz Ahmad, Ume Kalsoom Dar, Ijaz Ullah Khan, Muhammad Javed and Mahera Roohi

Summary: Folic acid is a common dietary vitamin. Various cancer lines exhibit folate receptors on their surface. Many studies have been carried out to radiolabel the folic acid for diagnostic and therapeutic study of tumor. The aim of the present work is to develop 99mTc(CO)3-folate azide complex for tumor scintigraphy. 1,2,3-Triazole-containing folate azide complex was synthesized by using click chemistry strategy.

The product was characterized by matrix-assisted LASER desorption ionization (MALDI). The radiolabeling was carried out by using isolink kit for stable complexation of organometallic core with folate azide complex. The radiochemical purity of the product was observed by using paper chromatography (PC). The protein binding assay in human blood was also studied. Normal distribution and tumoral uptake study of the drug was carried out in Swiss Webster mice.

MALDI data showed molecule ion peak of the folate azide complex at m/z 636.279, which confirmed the successful synthesis of the ligand. Radiochemical analysis showed 98.2 +- 1.6 % binding of radiometal with the ligand, which remained (greater than)96% up to 5h. Drug showed 70.1 +- 2.8 % binding with blood plasma proteins. Biodistribution results exhibited high uptake in targeted tumor and significantly low accumulation in non targeted organs. These data indicate that 99mTc(CO)3- folate azide complex might prove a successful tumor scintigraphic agent in future clinical applications.

Key words: Tumor, folate azide, click chemistry, scintigraphy, biodistribution, protein binding.


The radiolabeling of biologically active molecules with 99mTc is now of great interest for scientists [1-4]. Folic acid (vitamin B12) is successfully used as a "Trojan horse" for the targeted delivery of bioactive drugs to those tumors which show high-affinity folate receptors (FR-(Alpha)) on the cell surface [5]. Various type of cancer cell lines show folate receptors (FR) on their cell surface.

The expression of receptors is usually restricted to normal tissues. In previous studies, folic acid conjugates have been successfully used to deliver diagnostic and therapeutic agents to folate receptor-positive tumors [6, 7]. It was obtained through attachment of the radiometal chelate to folate derivative for diagnostic and therapeutic applications [8-14].

Click chemistry has great significance for biological conjugation [15, 16], ultimately, leading to target-oriented synthesis [17, 18] and building molecular library [19, 20]. 1, 2, 3-Triazoles (e.g., formed during click chemistry reaction) shows high biological activity because of its ability to well mimic natural peptides and heterocycles in geometrical shape and interaction functions [17, 21, 22].

The technetium-99m (99mTc) is a widely used radioisotope for diagnostic study in nuclear medicine. The new precursor, e.g., organometallic complex [99mTc(H2O)3(CO)3]+ has recently drawn much attention because of its high affinity for the large variety of donor atoms and convenient synthesis procedure [23]. Click chemistry has significantly been applied for the efficient and rapid functionalization of tumor targeting biomolecules by developing the labeling strategy with 99mTc, particularly, with [99mTc(H2O)3(CO)3]+ [17].

In the present work, we have used click chemistry strategy for the synthesis of folate azide complex. The folate azide complex was labeled with 99mTc through isolink kit. Various parameters like in vitro stability of drug, protein binding and bioevaluation were studied to evaluate the feasibility of drug as a potential future tumor diagnostic agent.

Results and Discussion

Folic acid is another name of vitamin B12. It is an essential dietary supplement, which is required for the synthesis of nucleotide bases [24]. In the present study, radiolabeling of folate azide complex was performed to develop it as a feasible candidate for targeted tumor scintigraphy. Folate azide complex helps us to deliver high dose of radiation to selected malignant sites in targeted tumor or tissue, while minimizing the radiation dose to surrounding healthy cells.

The synthesis of folic acid derivative by using click chemistry is summarized in Scheme-1. The reaction proceeds specifically by combining the azide and alkyne in copper(1) catalyzed azide alkyne cycloaddition. It is one-pot conversion of the reactants to the required 1, 4 disubstituted 1,2,3- triazoles.

Synthesis of Folate Azide Complex by using Click


As stated earlier, click chemistry is an ideal approach to achieve an ideal and innovative functionalization for biomolecules due to its selectivity and efficiency. Folate receptors are usually expressed by most of the cancer cells; its expression to normal cells is restricted, which makes folate receptor an ideal target for receptor based expression. The Cu(1)-catalyzed 1,3-dipolar cycloaddition of folate azide and alkyne is shown in Scheme-1. The final product was characterized by elemental analysis and MALDI.

Elemental analysis data predicted that the molecular formula of ligand should be C27H32N12O7 (C, 50.93 %; H, 5.034 %; N, 26.42%), which is consistent with the theoretically calculated formula i.e., C27H32N12O7: (C, 50.94 %; H, 5.03 %; N, 26.41%). The MALDI of the product gave peak at m/z 636.279, consistent with the molecular formula C27H32N12O7 (M + H+). In this reaction, aqueous solution of folate azide and alkyne (propargyl glycine) in the presence of copper (1) catalyst was heated in boiling water bath for 60 min. The elemental analysis data are summarized in Table-1.

Table-1 Elemental Analysis Data of Folate Azide complex

###C (%)###H (%)###N (%)



Radiolabeling and Quality Control

The radiolabeling was carried out by using isolink kit. The radiochemical purity of the [99mTc(CO)3(H2O)3] was determined by PC. In the next step, folate azide complex was labeled with [99mTc(CO)3(H2O)3]. The radiochemical purity of the 99mTc(CO)3-folate azide complex was also determined by PC.

During the radiolabeling of the drug, two species were formed, the bound 99mTc complex, and free pertechnetate (99mTcO4-), which were separated by PC. In PC, 99mTcO had an Rf of 0.8 - 0.9, while the 99mTc-ligand complex appeared at Rf = 0.00 - 0.01. The overall labeling yield of 99mTc(CO)3-ligand complex, as calculated by this method, was more than 98.2 +- 1.6%. These data are summarized in Table-2.

Table-2: Radiochemical Purity Analysis of Folate Azide complex

Species###Rf###% Binding

99m Tc-folate complex###0.00 0.1###98.2 +- 1.6

99m TcO4-###0.8 0.9###1.8 +- 1.4

In Vitro Stability and Protein Binding

In vitro stability of the 99mTc-labeled drug at room temperature was evaluated as a function of time by calculating the percentage of free pertechnetate with constant intervals of time up to 5h. Fig. 1 shows the graphical presentation of labeling efficacy of the radiolabeled drug up to 5h. The labeling efficacy remained 96.1 +- 1 % after 5h.

Fig. 1: In vitro stability of 99mTc(CO)3-labeled folate azide complex at room temperature at various time intervals.

The protein binding of the radiopharmaceutical gives us information about the efficacy of the drug inside the body, thus making it necessary to evaluate the blood protein binding in vitro before applying the drug in vivo. Protein binding is usually evaluated by measuring degree of transchelation of radiopharmaceutical to plasma proteins. The interaction of drug with blood proteins affects the pharmacokinetic characteristics such as volume of distribution, excretion of drug, metabolism and dosage [25].

The radiopharmaceuticals with high blood protein binding will stay in the blood stream for long time and the free constituents will be extracted, called active part of the radiopharmaceutical which causes pharmacological changes. In our study, protein binding of 99mTc(CO)3- folate azide complex in plasma proteins was 70.1 +- 2.843 %. This feature of the drug might be helpful to retain the drug in blood, thus decreasing chance of any pharmacological change [26].

Partition Coefficient

Partition coefficient is an essential parameter to be studied for evaluating pharmacokinetics of the drug. The data for partition coefficient of the radiolabeled drug are shown in the Table-3.

Table-3: Partition coefficient data of 99mTc(CO)3- click folate azide complex

Hydrophilic media###Lipophilic###99m

###Tc(CO)3-click folate azide

###(H)###media (L)###Activity in (H) Activity in (L)

PO4-3 buffer pH = 6.6###98.2 +- 1.5###1.8 +- 0.04


PO4 buffer pH = 7.0###98.0 +- 0.9###2.0 +- 0.09


PO4-3 buffer pH = 7.6###98.1 +- 0.7###1.9 +- 0.08

###Saline###98.8 +- 1.1###1.2 +- 0.09

Histopathology of Tumor

After staining the tumor tissues with hematoxylin-eosin (HE) (Fig. 2), histopathological data revealed that neuroendocrine tumor of intestine of diverse histopathology type might be present.

Fig. 2: Immunohistochemistry data of tumor tissue samples obtained after dissection of mice.

Bioevaluation in Normal and Tumor Bearing Mice

For studying the in vivo behavior of 99mTc(CO)3-folate azide complex, time dependent biodistribution studies were performed with normal and tumor bearing Swiss webster mice (Fig. 3).

In vivo stability of drug was confirmed by absence of activity in stomach and thyroid. Our radiopharm- aceutical revealed a very similar biodistribution pattern to the previous work reported by Thomas et al. [24]. The data indicate that drug showed rapid clearance from the blood and low retention in nontargeted organs, which was generally below 1% ID/g. The high accumulation of the radiolabeled complex in liver is also concordant with findings already reported by Thomas et al [24].

The %ID/g of tumor uptake of 99mTc(CO)3-folate azide complex at 15 min of post injection was 5.01 +- 0.09% and 6.94 +- 0.08% at 1h of post injection, respectively. The probable route of excretion was through kidney and bladder. Data are shown in Tables 4 and 5.

Table-4: ID/g (%) of 99mTc(CO)3-labeled folate azide complex in various organs of normal mice

###% ID/g at 15 min of###% ID/g at 60 min of

Organs###post injection###post injection

Brain###0.01 +- 0.001###0.01 +- 0.001

Spleen###0.3 +- 0.004###0.6 0+- 0.061

Liver###3.06 +- 0.009###1.05 +- 0.307

lungs###0.24 +- 0.211###0.40 +- 0.710

Heart###0.29 +- 0.211###0.12 +- 0.144

Muscles###0.29 +- 0.021###0.32 +- 0.154

Blood###1.05 +- 0.041###0.21 +- 0.094

Kidneys###8.5 +- 0.911###17.36 +- 1.347

Bladder###2.95 +- 0.077###8.78 +- 1.969

Tail###41.04 +- 3.889###20.69 +- 2.787

(injection site)

Table-5: %ID/g of 99mTc(CO)3-labeled folate azide complex in various organs of tumor bearing mice

Organs###%ID/g at 15 min of###%ID/g at 60 min of

###post injection###post injection

Tumor###5.01 +- 0.09###6.94 +- 0.08

Liver###2.08 +- 0.11###3.18 +- 0.15

Heart###0.22 +- 0.05###0.76 +- 0.06

Spleen###0.15 +- 0.03###0.35 +- 0.02

Lungs###0.22 +- 0.01###0.072 +- 0.01

Muscles###0.29 +- 0.021###0.32 +- 0.154

Blood###1.55 +- 0.041###0.12 +- 0.094

Kidneys###6.75 +- 0.15###36.75 +- 1.95

Bladder###1.2 +- 0.06###13.2 +- 1.25

Tail###42.54 +- 2.09###27.54 +- 3.05

Experimental Materials and Methods Synthesis and Radiolabeling

Before starting the radiolabeling procedure, we synthesized the folate azide complex by using click chemistry. As an experimental procedure, 500(Mu)l propagyl glycine (0.01M in H2O), 75 (Mu)l copper acetate (0.01M in H2O), 150(Mu)l sodium ascorbate (0.01M in H2O) and 650 (Mu)l folate azide (0.01M in H2O) were taken in a sterilized serum vial. The vial was sealed and placed in boiling water bath for 1h. After 1h, the vial was allowed to cool at room temperature. The characterization data include: MALDI, calculated for C27H32N12O7 (M + H+):

636.619; found: 636.279. Scheme-1 shows the formation of folate azide complex.


Scheme-1: Complex formation of folate azide by using click chemistry We used carbonyl method for radiolabeling of folate azide complex. It involves two steps reaction. In the first step, we radiolabeled the carbonyl kit (isolink kit) with 99mTc and in the second step, we radiolabeled folate azide complex through isolink kit.

Step 1: 10mCi/ml of Na99mTcO4 was added to the serum vial containing isolink kit. The isolink kit contains the following lyophilized formulation:

4.5 mg sodium boranocarbonate, 2.85 mg sodium tetraborate.10H2O, 8.5 mg sodium tartrate.2H2O and 7.15 mg sodium carbonate. The sealed vial was placed in a boiling water bath for 20 min, allowed to cool to room temperature and neutralization of the radiolabeled isolink kit was performed by adding 170 (Mu)l 1N HCl. The radiochemical purity (RCP) of the 99mTc-labeled isolink kit [99mTc(CO)3(H2O)3]+ was tested by paper chromatography (PC).

Step 2: 140 (Mu)l folate azide (0.01M) was taken in a sterilized serum vial and 0.5 ml from [99mTc(CO)3(H2O)3]+ kit was added in it. The reaction mixture was placed in a boiling water bath for 1h. The radiochemical purity (RCP) of the labeled products was again tested by paper chromatography (PC). Scheme-2 shows the radiolabeling of folate azide complex with [99mTc(CO)3(H2O)3]+.

Quality Control

The radiochemical purity of the labeled isolink kit and folate azide complex was observed by using paper chromatography (PC), which was used to evaluate the free pertechnetate by using acetone as a mobile phase. Small aliquots from the kit were spotted on 3MM Whatman paper (20 x 1 cm). After elution, the strip was cut in fractions of 1 cm and counted for radioactivity.

In vitro Stability

In vitro stability of the radiocomplex was studied at room temperature for time intervals varying from 0, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4 and 5h. The purpose to determine the in vitro stability was to detect any dissociation of the complex at room temperature. For this purpose, paper chromatography (PC) was again used as a standard technique. The percentage dissociation of the complex at a particular time interval was detected by the percentage of free pertechnetate (%) at that time. In case of significant loss of metal-complex stability, it is not advisable to use the radiopharmaceutical for clinical applications.

Protein Binding

Fresh and healthy human blood was taken in a test tube. A volume of 500 (Mu)l (3 mCi) 5 ml from the kit was added in the blood and incubated at room temperature till 1h. The reaction was brought to physiological conditions by incubating the tube in water bath for 10 min, pre-set at 37oC. The tubes were centrifuged for 10 min at 3000 rpm, followed by separation of serum and blood cells in two separate layers. Equal volume of 10% trichloroacetic acid (TCA) was added into separated serum.

After 10 min shaking, the mixture was centrifuged at 3000 rpm for another 10 min. Residues were separated from supernatant and counted for radioactivity.


Scheme-2: Radiolabeling of folate complex by using technitium-99m labeled isolink kit.

Partition Coefficient

200 (Mu)l Phosphate buffer of pH 6.6, 7.0 and 7.6 was taken in separate vials and same quantity of octanol was added in each vial. Radiolabeled kit (200 (Mu)l)was added in each vial. These vials were sealed and shaked for 10 min. After vortex mixing, two separate layers were obtained. They were separated and counted for radioactivity. Same procedure was repeated by using saline instead of phosphate buffer.

Safety Measurements

Before determining bioevaluation of the radiopharmaceutical in animals, few safety measures were made. The whole apparatus for kit formulation was sterilized in oven for 2h at 200oC. The laminar flow hood was sterilized with spirit and UV light exposure for 24h. The finally prepared kit was passed through 0.22 (Mu)m membrane filter.


For evaluating the biological uptake of drug, firstly we studied normal distribution of 99mTc(CO)3- labeled folate azide complex in healthy Swiss Webster mice. As an experimental procedure, 1 mCi/ 200 (Mu)l 99mTc(CO)3-labeled folate azide complex was injected intravenously in the tail of mice. Mice were dissected at 15min and 60min of post injection.

Organs were weighed and %ID/g of the drug was measured in various body organs by counting each organ in scintillation gamma counter.

For studying the potential of drug as a tumor diagnostic agent, we injected the drug in tumor bearing mice. For this purpose, 1 mCi/ 200 (Mu)l drug was injected intravenously in the tail of mice. Mice were dissected at 15min and 60min of post injection. Organs and tumor were weighed, %ID/g of the drug was measured in various body organs including tumor by counting in a scintillation gamma counter.

Fig. 3: (a) Disected tumor-bearing Swiss webster mouse showing tumor in thorex region (b) Disected tumor-bearing Swiss webster mouse having tumor in abdomen region. Histopathological Studies of Tumor.

Specimen (2.1 x 2.1 cm diameter) having smooth, reddish appearance was dissected and entirely submitted on tissue cassette. All samples were processed in the processor for 14 - 15h. Specimens were embedded, blocks prepared with wax and frozen. After cutting slides on microtome (in size of 3 micron), samples were prepared for staining and reporting.

The blocks were cut; slides dewaxed on hot plate, and dipped in xylene. The process was repeated three times, and placed the slides in 95% ethanol for 10 - 20 min followed by 75% ethanol for 5 - 7 min. The process was again repeated three times, placed slides in haematoxlyne for 15 min followed by placement in running tab water for 10 min. Decolorization was developed by placing the slides for 1 - 2 second in standard acid decolorizer.

The rinsing was carried out in standard ammonia solution containing 2 ml NH3 in 8 ml water, followed by rinsing in water and placing in Eosin for 5 min. The slides were further rinsed through ethanol by using increasing concentrations of 75%, 80% and 95%. Slides were finally placed in xylene container, cleaned, put in mounting media and covered with slips.


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1Radiopharmacy and PET Radiochemistry Division, Institute of Nuclear Medicine and Oncology (INMOL), New Campus Road, P. O. Box 10068, Lahore- 54600, Pakistan. 2Institute of Chemistry, University of the Punjab, Quaid-e-Azam Campus, Lahore 54600, Pakistan. 3Gujranwala Institute of Nuclear Medicine (GINUM), Gujranwala, Pakistan.
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Publication:Journal of the Chemical Society of Pakistan
Date:Dec 31, 2013
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