- Open Access
99mTc-HYNIC-(tricine/EDDA)-FROP peptide for MCF-7 breast tumor targeting and imaging
© The Author(s). 2018
- Received: 18 October 2017
- Accepted: 8 February 2018
- Published: 19 February 2018
Breast cancer is the most common malignancy among women in the world. Development of novel tumor-specific radiopharmaceuticals for early breast tumor diagnosis is highly desirable. In this study we developed 99mTc-HYNIC-(tricine/EDDA)-Lys-FROP peptide with the ability of specific binding to MCF-7 breast tumor.
The FROP-1 peptide was conjugated with the bifunctional chelator hydrazinonicotinamide (HYNIC) and labeled with 99mTc using tricine/EDDA co-ligand. The cellular specific binding of 99mTc-HYNIC-FROP was evaluated on different cell lines as well as with blocking experiment on MCF-7 (human breast adenocarcinoma). The tumor targeting and imaging of this labeled peptide were performed on MCF-7 tumor bearing mice.
Radiochemical purity for 99mTc-HYNIC-(tricine/EDDA)-FROP was 99% which was determined with ITLC method. This radiolabeled peptide showed high stability in normal saline and serum about 98% which was monitored with HPLC method. In saturation binding experiments, the binding constant (Kd) to MCF-7 cells was determined to be 158 nM. Biodistribution results revealed that the 99mTc-HYNIC-FROP was mainly exerted from urinary route. The maximum tumor uptake was found after 30 min post injection (p.i.); however maximum tumor/muscle ratio was seen at 15 min p.i. The tumor uptake of this labeled peptide was specific and blocked by co-injection of excess FROP. According to the planar gamma imaging result, tumor was clearly visible due to the tumor uptake of 99mTc-HYNIC-(tricine/EDDA)-FROP in mouse after 15 min p.i.
The 99mTc-HYNIC-(tricine/EDDA)-FROP is considered a promising probe with high specific binding to MCF-7 breast cancer cells.
- Breast cancer
- Tumor targeting
Breast cancer is the second most common malignancy with high risk of metastasis and death among women in the world . The 5-year survival rate for women diagnosed with metastatic breast cancer is only about 27.4% . In order to overcome mortality and morbidity in breast cancer, diagnosis of this cancer in the early stage is necessary. The early detection can dramatically improve the survival rate of breast cancer patients . So far traditional imaging methods, such as x-ray computed tomography (CT), ultrasound and magnetic resonance imaging (MRI), have been used for breast cancer diagnosis . Mammography is a commonly used screening method for breast cancer. This method can be problematic due to low sensitivity especially in the case of dense breasts [5–7]. Therefore, high sensitive methods for detecting of breast cancer at its earliest presymptomatic stages are necessary. Single-photon emission computed tomography (SPECT) or positron emission tomography (PET) imaging techniques represent an appropriate tool with high sensitivity for breast cancer imaging. In recent years developing a peptide-based radiopharmaceuticals due to certain favorable properties for the detection of breast cancer are increased. Previously technetium-99m labeled alpha-M2 , H6F , TP1623 , PI  and EC peptide  are used as a molecular imaging agent for breast cancer.
A 12-amino-acid FROP-1 peptide as tumor cell binding peptide was identified through phage display system by Sabine Zitzmann research group . FROP-1 peptide consists of Glu1-As2-Tyr3-Glu4-Leu5-Met6-Asp7-Leu8-Leu9-Ala10-Tyr11-Leu12 amino acids that significantly has ability binding to the various type of the cancer cell lines, especially thyroid cancer cells FRO82–2 and MCF-7 breast cancer cells. Previously this research group labeled FROP-1 peptide with iodide radioisotopes (131I and 125I)  and also 111In radionuclide  for in vitro and in vivo evaluation of FROP-1 binding to MCF-7 cell line.
99mTc is one of the most commonly used radionuclides in the clinic. The popularity of this radionuclide is due to its appropriate half-life and excellent radiochemical properties that make it suitable for peptide-based nuclear imaging. The use of a HYNIC as a bifunctional chelating ligand for the labeling of a various biomolecules, including antibodies and peptides has received considerable interest [15, 16].
In radiolabeling method, the nature and location of a chelating moiety within a peptide molecule can influence on its receptor-binding. To avoid possible interference of the radionuclide with the binding region of peptides, the spacer is incorporated between the binding site and the chelating moiety. For completing the coordination sphere of the technetium core in 99mTc-HYNIC the presence of the co-ligand is required. The stability, lipophilicity, clearance property, and protein binding potency of the radiolabeled compounds are affected by the type of a co-ligand . In the labeling process, co-ligands tricine and EDDA could be used separately or together via the exchange labeling method.
In Zitzmann and Mire research, since the labeling method and pharmacokinetics characterization of FROP-1 radiopeptide derivatives have problems of slow binding capacity and insufficient in vivo accumulation, and furthermore with respect to the effect of the type of chelator and co-ligand on radiopharmaceutical’s pharmacokinetic [18, 19] and tumor targeting in animal model , we decided to modify FROP-1 peptide through practicing HYNIC chelator. This modification is leading to some advantages including a well-defined chemistry, ability for post conjugation labeling, the ability of storing for later radiolabeling process and production of a stable complex with 99mTc radionuclide  which has high popularity in nuclear medicine due to the suitable photonic characterization. Therefore in this study, we radiolabeled FROP-1 peptide through practicing HYNIC as a chelating moiety (at the C-terminus) and Lys residue as a spacer in order to develop a 99mTc-HYNIC-(tricine-EDDA)-Lys-FROP for the targeting of the MCF-7 breast cancer cells.
The 12-amino acid FROP peptide with HYNIC conjugation at C-terminus (EDYELMDLLAYLK-(HYNIC)) was purchased from Prote-Genix (France). Tricine (N-[tris-(hydroxymethyl) methyl] glycine), ethylene diamino diacetic acid (EDDA), trifluoroacetic acid (TFA) and tin (II)-chloride dihydrate, were obtained from Sigma (St. Louis, MO, USA). Acetonitrile (HPLC grade), sodium succinate dihydrate and methyl ethyl ketone (MEK) were purchased from Merck (Darmstadt, Germany). Solutions were prepared by standard procedures and using high quality water. Sodium pertechnetate (99mTcO4−Na) was obtained from commercial 99Mo/99mTc generator (Pars Isotope, Tehran, Iran). The radiochemical purity was performed by instant thin layer chromatography (ITLC) with different mobile phases using a Lablogic mini scan TLC scanner (Sheffield, UK) and Laura image analysis software. A NaI (Tl) gamma detector (Delshid, Tehran, Iran) was used for measurement of radioactivity. Reversed phase-HPLC was performed on a Knauer HPLC systems (Knauer HPLC, Berlin, Germany) using a Lablogic radioactivity gamma detector with a Eurospher 100–5 C-18, 4.6 × 250 mm (Knauer, Berlin, Germany) column with a pre-column (Knauer HPLC, Berlin, Germany). MCF-7 (breast cancer), SKOV-3 (ovarian cancer), PC-3 (prostate cancer), A-549 (non-small cell lung cancer), T47D (breast cancer) and HFFF2 (normal human fibroblast) cell lines were obtained from the Iranian Pasteur Institute and the National Center of Genetic and Biological Reserves of Iran. Dulbecco’s DMEM high glucose culture, fetal bovine serum (FBS), penicillin–streptomycin and trypsin solution were purchased from Gibco (UK) and Biosera (UK). All cell lines were grown in Dulbecco’s DMEM high glucose culture media with 10% fetal bovine serum (FBS) in tissue culture flasks in humidified air containing 5% CO2 at 37 °C until a sufficient number of cells were available. Confluent cultures were harvested by employing a trypsin solution (Biosera, UK).
For radiolabeling, HYNIC-FROP peptide (10 μg) was incubated with 50 μL tricine solution (10 mg tricine in 50 μL succinate buffer with pH 6.5) and 100 μL succinate buffer. This mixture reaction was shaken and then 25 μL of tin (II) solution (2 mg/1 mL in nitrogen-purged 0.1 M HCL) was added. After adding of the 5 mCi (185 MBq) fresh 99mTcO4−Na and then 150 μL EDDA solution (5 mg EDDA) the mixture reaction heated for 20 min at 95 °C.
The radiochemical purity was determined by instant thin layer chromatography (ITLC) in different mobile phases such as methyl ethyl ketone (MEK), acetonitrile/water (50/50) and citrate solvent (0.1 M, pH 5.2) using a Lablogic mini scan TLC scanner (Sheffield, UK) and Laura image analysis software. In MEK, free 99mTcO4− migrated with the solvent front, while radiolabeled peptide remained at the application point. In the acetonitrile/water system, the colloids remained at the application point, while radiolabeled peptide migrated in front of solvent. The amount of labeled co-ligand was determined by citrate solvent, that free 99mTcO4 Na and labeled co-ligand migrate in front of solvent while both RHT and labeled peptide (99mTc-FROP) remind at the origin. A NaI (Tl) gamma detector (Delshid, Tehran, Iran) was used to for measurement of radioactivity.
Reversed phase-HPLC was performed on a Knauer HPLC systems (Knauer HPLC, Berlin, Germany) using a Lablogic radioactivity gamma detector with a Eurospher 100–5 C-18, 4.6 × 250 mm (Knauer, Berlin, Germany) column with a pre-column. (Knauer HPLC, Berlin, Germany) HPLC was run using a gradient of 0.1% TFA in H2O (solvent A) and 0.1% TFA in CH3CN (solvent B) at a flow rate of 1 mL/min. A gradient with solvents A and B was run as follows: 0 min, 10% B; 0–10 min, 10–30% B; 10–20 min, 30–80% B; and 20–25 min, 80–100% B, for a total time of 30 min.
Stability in normal saline and serum
The stability of 99mTc-HYNIC-FROP in normal saline was evaluated using ITLC at various times (2, 4, 6 and 24 h) at room temperature. For serum stability, 99mTc-HYNIC-FROP (30 μL) was incubated with freshly human plasma serum (90 μL) in triplicate at 37 °C. At 1 h of incubation, the plasma proteins were precipitated with a mixture of acetonitrile/ethanol (1:1 v/v) and centrifugation. The supernatant layer was filtered through a 0.22 μm filter and then the fraction was analyzed by RP-HPLC. Partition coefficient of 99mTc-HYNIC-FROP was determined via method that previously was described .
In vitro binding experiments
The cellular uptake studies of 99mTc-HYNIC-(tricine/EDDA)-FROP were performed on MCF-7, A-549 SKOV-3, PC-3, T47D and HFFF2 cell lines. The cells (5 × 105) were seeded in 12-well plates overnight and incubated with 99mTc-HYNIC-FROP (40 nM) at 37 °C for 1 h. Incubation of the samples was terminated by removing medium and washing with cold serum-free medium. Thereafter, the cells were detached by trypsin and the radioactivity in the cell suspension was quantified.
For specific binding experiment, the MCF-7 cells were saturated with 500-fold excess of unlabeled peptide prior adding of the 99mTc-HYNIC-FROP to cells for 30 min.
For internalization experiment, MCF-7 cells (1 × 106 cells) were seeded on the single plates and incubated with 99mTc-HYNIC-FROP (40 nM) at 4 °C. After 1 h incubation, medium was removed and cells were washed with ice-cold serum free medium. Then 1 mL complete medium was added to each dish and cells were further incubated for 0.5, 1, 2, 6 and 24 h at 37 °C. After incubation, medium was removed from cellular plate and then was washed once with cold serum free medium. Then supernatant was collected, and for removing radioactivity bound to the surface of the cells, the acid wash with urea buffer (pH 2.5) was used. The internalized radiolabeled peptide was recovered by solubilizing the cells with 1 M NaOH at 37 °C for 5 min, and the surface-bound (acid-wash) and internalized (acid-resistant) radioactivity were measured with the gamma counter.
The cell-binding affinity of 99mTc-HYNIC-FROP was assessed with saturation method. For determining of dissociation rate constant (Kd) and total number of binding sites (Bmax), 99mTc-HYNIC-FROP was prepared in high radiochemical purity. MCF-7 cells were seeded into 24-well plates and incubated overnight. Then cells were incubated with various concentrations of radiolabeled peptide (30–250 nM) for 30 min. One dish was added blocking solution containing of 500-fold excess of highest concentration of 99mTc-HYNIC-FROP. The cells were then washed with cold serum free medium to remove free radioactivity. The cells with bound radioactivity detached with trypsin and collected and radioactivity was measured. The (Kd) and (Bmax) value were calculated using Graphpad prism software based on specific binding of 99mTc-HYNIC-FROP on MCF-7 cells.
Biodistribution in MCF-7 tumor-bearing nude mice
The all animal studies were approved by Research and Ethical Committees of Mazandaran University of Medical Sciences. For xenografting, female C57 nu/nu mice were injected subcutaneously on the right upper back with a suspension of 1 × 107 MCF-7 cells and Matrigel® (BD, USA) and the tumors were allowed to grow for about 7–8 weeks. Estradiol valerate was injected subcutaneously into nude mice for 1 week. After tumor size reached in 1 cm3, this tumor was removed from mouse in aseptic condition and was mechanically squelched with a mortar and with adding complete medium. Squelch of tumor tissue was centrifuged at 1500 rpm for 7 min at 4 °C. The upper medium was removed from tube and then complete medium and estradiol were added to cells pellet. Cell suspension (200 μL) was injected to nude mouse implantation site and it was observed at regular intervals for tumor formation and progression. Xenografts were allowed to develop during 2 months and the average tumor size was 1.01 ± 0.24 g. The in vivo biodistribution of 99mTc-HYNIC-(tricine/EDDA)-Lys-FROP was performed on tumor bearing mice at 15 and 30 min post-injection. The mice (four mice in each time group) were injected with 100 μL of HYNIC-(tricine/EDDA)-Lys-FROP (1 μg) via a lateral tail vein. Mice were sacrificed with injection of a lethal dose of ketamine/xylazine after 15 and 30 min. The blood was collected from the heart puncture and the mice were dissected. Major organs (tumor, blood, heart, lung, liver, kidney, spleen, salivary gland, stomach, intestine, muscle and bone) were placed into pre weighed gamma counter tubes and radioactivity in each organ and tissue was measured using a gamma counter and expressed as a percent injected dose (% ID/g).
To confirm that the radiolabeled peptide was taken up specifically by MCF-7 tumor in vivo, the biodistribution of 99mTc-HYNIC-FROP was performed with co-injection of a 500-fold (0.5 mg) excess non-radiolabelled FROP-1 and radiolabelled peptide in mice (N = 3 mice in this group). The unlabeled peptide was injected at 30 min before the injection of radiolabeled peptide.
For gamma camera imaging, MCF-7 tumor bearing mouse was intravenously injected through the tail with 99mTc-HYNIC-FROP. After 15 min p.i. the mouse immediately was anesthetized using ketamine/xylazine and whole-body dynamic imaging was conducted. The imaging was performed at the department of radiology at Mazandaran Hospital by a dual-head e.cam gamma camera (Siemens Medical Systems, Inc.), equipped with a low-energy, high-resolution collimator. The evaluation of the image was performed using an e-soft (Siemens Medical Systems).
All statistical analyses were performed using Excel software (2011, Microsoft Office). The in vitro experiments were performed three-times and the final values were presented as mean ± standard deviation (SD). The t-test was used for comparison of groups and p < 0.05 was considered as statistical significance level between groups. The binding data were analyzed with nonlinear regression using Prism 5 software (Version 5.04, 2010, USA).
In Vitro Stability of 99mTc-HYNIC-(tricine/EDDA)-FROP in succinate buffer and normal saline at room temperature (n = 3)
%RCP in succinate buffer (peptide reaction mixture)
%RCP in normal saline
−2.03 ± 0.03
99.6 ± 0.2
99.5 ± 0.4
99.1 ± 0.9
96.8 ± 2.4
99.5 ± 0.4
99.1 ± 0.9
99 ± 0.8
94.6 ± 2.7
In vivo animal biodistribution
Biodistribution and tumor-to-tissue ratios of 99mTc-HYNIC-(tricine/EDDA)-FROP in MCF-7 female tumor-bearing nude at 15 and 30 min p.i. Data expressed as % ID/g organ
0.48 ± 0.09
0.35 ± 0.10
0.69 ± 0.16
0.49 ± 0.08
0.26 ± 0.11
0.45 ± 0.03
0.45 ± 0.08
0.41 ± 0.26
0.60 ± 0.04
0.25 ± 0.03
0.32 ± 0.22
0.25 ± 0.01
0.20 ± 0.02
0.14 ± 0.00
0.26 ± 0.04
0.15 ± 0.01
0.28 ± 0.11
0.23 ± 0.07
2.22 ± 0.16
1.73 ± 0.15
2.97 ± 0.23
0.21 ± 0.01
0.33 ± 0.12
0.26 ± 0.01
0.08 ± 0.01
0.21 ± 0.16
0.15 ± 0.01
0.18 ± 0.02
0.24 ± 0.12
0.22 ± 00
0.34 ± 0.09
0.79 ± 0.25
0.64 ± 0.09
0.25 ± 0.02
0.38 ± 0.21
0.09 ± 0.02
3.03 ± 0.12
1.94 ± 0.46
0.60 ± 0.12
0.53 ± 0.15
1.09 ± 0.47
0.14 ± 0.06
1.61 ± 0.22
1.56 ± 1.04
0.42 ± 0.14
1.36 ± 0.28
1.57 ± 0.48
0.40 ± 0.09
1.24 ± 0.28
2.62 ± 1.38
0.36 ± 0.13
Breast cancer is one of the most common causes of cancer with high mortality among women in the world . It is necessary to mention that diagnosis of breast cancer at early stage leads to significantly improve the efficacy of therapy and an overall reduction of breast cancer mortality. Nuclear imaging techniques, namely, PET and SPECT enable to detect tumors at early stage or metastasis . However various radiopharmaceuticals with high selectivity have been used for molecular imaging in breast cancer [24, 25], but radiolabeled peptide has advantages such as small size, rapid distribution, clearance and high priority target for cancer. Radiolabeled peptide which was used for the targeting of MCF-7 cells is highly interesting. MCF-7 is a commonly used breast cancer cell line that isolated from the pleural effusion of a woman with metastatic disease and is used ubiquitously in research for breast cancer cell experiments . MCF-7 is estrogen receptor (ER) positive cell line with moderate EGFR-expressing and HER-2 negative . MCF-7 parental cells also maintain the expression of epithelial markers, such as E-cadherin, β-catenin and cytokeratin 18 (CK18) and other specific molecular markers of natural epithelial layers, such as claudins and zona occuldens protein 1 (ZO-1), among other proteins that constitute the intercellular junctions. Furthermore, MCF-7 are mesenchymal markers negative, these markers are including vimentin and smooth muscle actin (SMA) [28, 29]. FROP-1 peptide was identified by phage display technology by Sabine Zitzmann research group in 2007 . This research group demonstrated the specific binding of iodinated derivative (131I and 125I radionuclide) of the FROP-1 peptide for MCF-7 breast tumor cells both in vitro and in vivo models. FROP-1 peptide is not a derivative of cholecystokinin-B or its family which was confirmed by the Basic Linear Alignment and Search Tool against protein databases. Though so far no attempt was made to identify a receptor for the FROP-1 peptide, further more study seems to be needed to find out the ability of targeting characteristics of this molecule specially targeting of the epithelial markers . Although this agent exhibited a specific binding in cellular experiment, its in vivo tumor uptake was limited due to poor stability and pharmacokinetics. This limitation was due to the fact that the iodination of tyrosine occurred in radiolabeling reaction. The mixture of different by-products not only was difficult to purification as a single product but also the existence of this impurity affected on binding of the peptide to the receptor. However, several methods were conducted for improvement the stability and affinity of the FROP-1 molecule such as coupling of FROP-1 peptide to chelator  and its conjugating with ethylene glycol (PEG) oligomer . In comparison to the FROP-1 peptide without chelator, coupling of a FROP-1 peptide to the chelator 1, 4, 7, 10-tetraazacyclododecane-1, 4, 7, 10-tetraacetic acid (DOTA) led to enhancing cellular uptake kinetics and increasing in vivo stability. High stability of FROP-DOTA is attributed to the different secondary structure between FROP-DOTA and free FROP-1 peptide in solution which was conformed by circular dichroism (CD) spectra . However the binding kinetic was found to be too slow and its fast excretion by urinary system forestalled on tumor accumulation. To solve this problem, PEGylation of 111In-DOTA-FROP was proposed by Walter Mire. The conjugation of FROP-DOTA with PEG oligomer increased a time dependent tumor uptake, but unfortunately the binding kinetics of the PEGylated 111In-DOTA-FROP was even slower than that of 111In-DOTA-FROP . With respect to the effect of changing chelator on pharmacokinetic of radiopharmaceuticals [18, 19, 31], in this research study we developed a new radiolabeled FROP peptide derivative using the HYNIC as a chelating moiety at C-terminus and EDDA-tricine as co-ligand to form a stable complex with 99mTc by ligand exchange method. Mainspring choosing of C-terminus for labeling was that the attachment of the chelator to the C-terminal end stabilize the peptide against enzymatic degradation and induce structural constrains that resemble those in the phage-bound structure . FROP-1 peptide was labeled with 99mTc at high radiochemical purity (≥99.5%). It is necessary to mention that the type of buffer which was used for labeling reaction affected the radiochemical purity. The maximum radiochemical purity was found for succinate buffer (pH 6.5) without any further purification. In comparison to the other buffer it is most likely that succinate buffer assemble good situation for facilitating the exchange radiolabeling reaction. Also 99mTc-HYNIC-FROP showed a good in vitro stability in normal saline and human serum and after 1 h incubation with freshly human serum that the major radioactivity was still associated with 99mTc-FROP. This in vitro and in vivo stability for FROP peptide attributed to the attachment of HYNIC chelator like that was seen for DOTA-FROP. To validate the ability of 99mTc-HYNIC-Lys-FROP as a ligand to detect MCF-7 breast cancer cells, we evaluated the cellular uptake of the peptide in various types of a cancer cell lines and normal human fibroblast cell line. Data analysis has indicated that 99mTc-HYNIC-FROP has higher uptake in MCF-7 cells in comparison especially to the normal cells and other cancerous cell lines. Similar to 111In-DOTA-FROP the cell binding characteristics of 99mTc-HYNIC-FROP is not affected by the addition of a chelating moiety and chelation with 99mTc. However in comparison to the 131I–FROP-1 and DOTA-FROP, the modification of our proposed molecule is leading to not only the improvement of binding capacity and binding kinetics but also the in vivo tumor accumulation. This binding was specific and significantly blocked in the presence of cold peptide as a competitor. The internalization experiment showed that the fast internalization was occurred after 30 min the exposure of MCF-7 cell with radiolabeled peptide. However, the amount of internalization for 99mTc-HYNIC-FROP was greater than both 125I–FROP-1and 111In-DOTA-FROP at 30 min by a factor of approximately 2. 99mTc-HYNIC-FROP revealed a high rate of internalization between 4 and 6 h after incubation perhaps due to the both slow internalization kinetics and the attachment of the HYNIC chelator leading to high stability of the FROP-1 peptide against the peptidases of the MCF-7 cell surface . This delayed internalization was previously seen for 111In-DOTA-FROP in Mire research but not for 131I–FROP-1 .99mTc-HYNIC-FROP exhibited a higher affinity in the nanomolar range (158 nM) for MCF-7 cell than 125I–FROP-1 that was in micromolar range (8 μM). The dissociation constant in the nanomolar range for 99mTc-HYNIC-FROP demonstrates the high specific affinity to the MCF-7 cells. Therefore, conjugation of the HYNIC moiety to FROP-1 peptide increases the affinity by a factor of approximately 78. However the binding affinity of 111In-DOTA-FROP to FRO82–2 cells was reported 494 nM . The in vivo results showed the radiolabeled peptide exhibited a rapid clearance from the blood. The excretion pathway was mainly through renal system which possibly was attributed to the nature of peptide sequence and the amphiphilic character of the FROP-1 peptide with a log p = − 2.03. The same excretion pathway was seen for 131I–FROP-1 . In comparison with 131I–FROP-1, both DOTA-FROP and HYNIC-FROP showed a faster blood clearance. This rapid blood clearance is caused by the hydrophilic DOTA and HYNIC conjugates that have high polarity. A low uptake of radioactivity was seen in the stomach and thyroid at all times, indicating a high in vivo stability for 99mTc core in 99mTc-HYNIC-FROP peptide. The tumor uptake of 99mTc-HYNIC-FROP peptide was found to be 0.25 ± 0.02% ID/g after 15 min that this tumor uptake relatively good (in comparison the kidneys uptake). However, this tumor uptake was specific and significantly blocked in the presence of the cold peptide (FROP-1) as a competitor in block mouse group. In most organs (except the excretion organs) the excess of unlabeled peptide did not lead to a change in radiolabeled peptide accumulation. Both radiolabeled HYNIC-FROP and DOTA-FROP (For MCF-7 cell line) peptides showed similar kinetics for tumor accumulation, as the steady decrease of radioactivity in tumor was detected after 15 min p.i. . Unfortunately, the fast wash out of 99mTc-HYNIC-FROP peptide from tumor caused low tumor uptake compared to lung and heart which is a limiting factor for this radiolabeled peptide. The maximum tumor/muscle ratio was obtained after 15 min p.i. of 99mTc-HYNICFROP the same time achieved by DOTA-FROP; while this ratio was found for 131I–FROP-1 after 135 min. Although previously reported PEGylation of DOTA-FROP increased the tumor/muscle ratio to 4.1 in female Balb/c nu/nu mice bearing FRO82–2 tumor after 24 h p.i. This time is not suitable for imaging goal especially regarding to the short half-life radionuclide. Despite fast washing out from tumor, 99mTc-HYNIC-Lys-FROP has a good ability for tumor visualization after 15 min p.i. This ability can be attributed to the both high Bmax value and high affinity for MCF-7 cells. Also the fast internalization of 99mTc-HYNICFROP in tumor organ immediately after the injection can help to this aim. Though attachment of the HYNIC chelator to FROP-1 peptide improved the both binding capacity and binding kinetics, the further studies are recommended for improvement the in vivo differentiation of tumor and other normal organs. The other co-ligands, spacers and chelators can be practiced for labeling of FROP peptide with 99mTc to achieve better in vivo tumor targeting and imaging.
In this study, FROP-1 peptide was radiolabeled with 99mTc via HYNIC chelator and formed a stable complex. 99mTc-labeled peptide exhibited highly specific binding on MCF-7 cells that were confirmed in vitro and in vivo. The high affinity of FROP-1 peptide on MCF-7 cells is retained due to attachment of HYNIC chelator and labeling with 99mTc. Biodistribution in mice exhibited a rapid clearance from the blood and excretion pathway was mainly through renal system. Moreover 99mTc-HYNIC-(tricine/EDDA)-FROP accumulated in MCF-7 tumor rapidly and it was observed a high tumor-non-targets ratios. The result of this study is promising for imaging and diagnosis of MCF-7 breast cancer with 99mTc-HYNIC-(tricine/EDDA)-FROP peptide, however future experiments are needed for improvement of in vivo tumor targeting.
The authors thank M Asori and R Behzadi, from the North Branch of Institute of Pasture, Amol, Iran, for the Nude mice bearing tumor assistance.
This work was supported financially by Mazandaran University of Medical Sciences, Sari, Iran. Seyed Jalal Hosseinimehr has received this research grant (Grant number: 1926).
Availability of data and materials
All data and materials are available.
SA and SJH designed the study concept and experiments. SA, ZN, SA and SJH performed the experiments. SA and SJH wrote the manuscript. SA and SJH read and finalized the manuscript. All authors read and approved the final manuscript.
Ethics approval and consent to participate
Animal studies were approved by ethical and research committee of Mazandaran University of Medical Sciences. This article does not describe any studies with human participants performed by any of the authors.
Consent for publication
All the authors have consented for publication.
The authors declare that they have no competing interests.
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