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. 2021 Jan 8;6(3):1846–1856. doi: 10.1021/acsomega.0c03991

Synthesis of Radiolabeled Technetium- and Rhenium-Luteinizing Hormone-Releasing Hormone (99mTc/Re-Acdien-LHRH) Conjugates for Targeted Detection of Breast Cancer Cells Overexpressing the LHRH Receptor

Lindsay E Calderon †,*, Carrie A Black ‡, Joseph D Rollins †, Brittany Overbay ‡, Semekidus Shiferawe †, Andrew Elliott ‡, Sara Reitz ‡, Shu Liu §, Junling Li ∥, Chin K Ng ∥, Margaret W Ndinguri ‡,*
PMCID: PMC7841779  PMID: 33521425

Abstract

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Currently, 186/188Re and 99mTc are widely used radionuclides for cancer detection and diagnosis. New advancements in modalities and targeting strategies of radiopharmaceuticals will provide an opportunity to enhance imagery and detection of smaller colonies of cancer cells while lowering false-positive diagnoses. To understand the chemistry of agents derived from fac-[99mTc(CO)3(H2O)3]+ species, the nonradioactive [Re(CO)3(H2O)3]+ analogue was used. We have designed and synthesized Re-Acdien-LHRH, Re-Acdien-peg-LHRH, and a radiolabeled 99mTc-Acdien-LHRH (rhenium- and technetium-luteinizing hormone-releasing hormone) conjugates using a tridentate linker to detect cancers overexpressing the LHRH receptor. Re-Acdien-LHRH and Re-Acdien-peg-LHRH were synthesized from non-PEGylated and PEGylated LHRH-Acdien, respectively. Cellular uptake of the compounds 99mTc-Acdien-LHRH, Re-Acdien-LHRH, and Re-Acdien-peg-LHRH was found to be significantly enhanced compared to that of untargeted 99mTc alone and unlabeled [Re(CO)3(H2O)3]+. In addition, the conjugate compounds showed no difference in cellular toxicity compared to untargeted 99mTc alone or unlabeled [Re(CO)3(H2O)3]+. Further, a competition assay using LHRH indicated selective targeting of Re-Acdien-peg-LHRH toward the LHRH receptor (p < 0.05) compared to that of [Re(CO)3(H2O)3]+ alone. Together, our data show the design paradigm and synthesis of targeting radionuclides using the LHRH peptide. Our data suggests that utilizing the LHRH peptide can lead to selective targeting and diagnosis of breast cancers expressing the LHRH receptor.

Introduction

Continual advancements in early detection and imaging modalities have contributed to improved breast cancer survival outcomes and overall quality of life in patients over the past few decades. However, despite recent advances in therapeutic and diagnostic medicine toward breast cancer, cancer imaging development remains minimal. Triple-negative breast cancer (TNBC) is of utmost concern, as it is noted to be the most lethal and aggressive form of breast cancer, leading to high rates of metastasis beyond the breast and the highest rate of recurrence within the first 5 years after diagnosis than any other form.1 About 10–15% of breast cancer cases are diagnosed as TNBC, which lack the estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 (HER2/neu) expression rendering it unresponsive to hormone therapy and HER2/neu-targeted medications.2 Subsequently, women with TNBC have higher rates of death within the first 5 years of diagnosis than any other form of breast cancer with only a 77% survival rate for TNBC compared to 93% for other types.3−5

Currently used diagnostic techniques are not selective for TNBC detection or imaging, and improvements on these technologies are still needed. Mammogram screening has been critical to lowering mortality rates; however, this testing modality is limited in efficacy by high false-positive rates, 121.2 per 1000, as well as high false-negative rates at 1.0–1.5 per 1000. This is especially prevalent in women with dense fibrous breast tissue.6 Positron emission tomography (PET) imaging provides a combination of sensitivity and quantitative accuracy through the injection of radioactive compounds, which accumulate at the tumor sites due to their high vascularization and cellular accumulation.7,8 Though PET scans provide a more precise image of tumor locations than X-rays or MRIs, their accuracy can be limited by a few critical factors. The tumor’s position and its characteristics can impede PET imaging as can limitations associated with the radiopharmaceuticals used and their required quantification protocols. Recent innovations have focused on the use of targeted radiopharmaceuticals for more precise detection of specific cancers and accurate stage determination.9,10

Two metals of interest utilized in radiopharmaceuticals are radiolabeled technetium (99mTc) and rhenium (186/188Re), both are transition metals of the same group on the periodic table (group 7) and therefore share similar chemical and structural properties.11,12 Their properties make them excellent analogues for each other and their decay characteristics make them suitable for both imaging and therapy.13 A nonradioactive Re analogue is often used as a model for [99mTc] because the radioactive properties of [99mTc] are often difficult to work with in certain lab situations. [99mTc] is the most widely used radionuclide in radiopharmaceutical imaging due to its ideal physical properties.14 Nuclear imaging using 99mTc has been used in bone scans, vascular perfusions, brain imaging, and cardiac perfusion. The increased use of 99mTc in nuclear medicine is due to its ideal physical properties including emitting detectable gamma rays (γ ray = 142 keV) and a half-life of 6.02 h with almost full decay at 24 h.9,12,15 The relatively short half-life of 99mTc allows for the preparation of the radiopharmaceutical for injection and subsequent imaging with minimal radiation exposure to the patient.12,16

Due to its small size and stability, fac-[99mTc(CO)3]+ is regarded as an excellent core to introduce [99mTc] into biomolecules.17 These characteristics have also made [99mTc] an excellent candidate for use in conjunction with receptor-specific peptide moieties. Significant progress with promising results has been made over the past several years in this area of research using [99mTc] complexes with several different peptides, including bombesin (BBS) analogues,18 the folate receptor,19 ανβ3-integrin receptor, CCK2 receptor, GLP-1 receptor, and the GRP receptor.14

Luteinizing hormone-releasing hormone (LHRH) receptor, also referred to as gonadotropin-releasing hormone (GnRH) receptor, has been found to be overexpressed in breast, prostate, endometrial, and ovarian cancers in comparison to normal cells making it a good candidate for drug targeting.20−23 An examination of the literature to date yields studies exploring the synthesis of rhenium and its analogue technetium with the LHRH peptide using different combinatory methods.24,25 For instance, several groups have investigated the use of dithia-bisphosphine chelation agents and noted the high affinity of alkyl phosphines toward oxidation as the limitation in these kinds of syntheses.25−29 Other studies have used a hydrazino nicotinamide (HYNIC) chelation system to radiolabel DLys6 LHRH analogues; however, HYNIC can only coordinate as a monodentate or bidentate ligand, thereby requiring a coligand to complete the coordination sphere of technetium.30−34 In another study, Guo et al. extensively looked at conjugating the LHRH peptide analogue with a DOTA chelator labeled with 111In and researched the cellular and biological effects of these radiopeptides.24,35,36 Some studies have shown that while DOTA chelates are stable, the labeling kinetics can be slow and dependent on the radiolabeling conditions.30,34

An increase in both sensitivity and specificity of radiotracers would advance the nuclear medicine field and patient care allowing for earlier diagnosis (subclinical), small metastatic colony imaging, and specific cancer cell targeting. Peptide motifs have beneficial properties for use as radiolabeled tracers, mainly due to rapid plasma clearance and high receptor affinity for targeting.9,15 Different cancers are characterized by overexpression of specific receptors. For example, in the area reproductive cancers, there are numerous cancer types including breast, genital, uterine, ovary, uvula, prostate, testicular, endometrial, etc., that express the LHRH receptor.9,37 TNBC, which is highly aggressive and metastatic, has also been shown to overexpress the LHRH receptor.37 Hence, the targeting of overexpressed receptors in tumors using small peptides is of considerable interest, especially in nuclear medicine.9 Furthermore, there is a need for high uptake and selective diagnostic tracers in tumors that cannot be surgically treated to provide earlier and more precise cancer detection, especially for small colony formation. Accurate and precise cancer regression tracking and tumor positioning imagery will aid in patient treatment paradigms.

Subsequently, this paper focuses on the design and synthesis of novel targeting radiopharmaceutical conjugates for cancers that overexpress LHRH receptors. We demonstrate the chemical synthesis of Re-Acdien-LHRH, Re-Acdien-peg-LHRH, and 99mTc-Acdien-LHRH (Figure 1) by attaching LHRH (targeting moiety) with Bocdienac, a tridentate linker, and report data on cellular uptake and selectivity toward TNBC. This project focuses on the development of radiolabeled 99mTc-Acdien-LHRH and Re-Acdien-LHRH conjugates and the ability of these conjugates to detect breast cancers overexpressing the LHRH receptor. By linking LHRH to the radiotracers using a simple tridentate linker, we alleviate the need of other coligands for the technetium core, thereby enhancing cellular uptake and targeting of cells that overexpress the LHRH receptor.

Figure 1.

Figure 1

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Chemical structure of 99mTc/Re-Acdien-LHRH with or without pegylation. The diagram represents the final general structure where X can be 99mTc/Re-Acdien-LHRH in a Peg or non-PEGylated form.

Methods

Reagents

All starting reagents listed below were obtained from commercial sources and used without further purification: trifluoroacetic acid (TFA), N-methylmorpholine (NMM), triisopropylsilane (TIPS), acetic acid, dichloromethane, dry solvents, AgNO3, and Re2(CO)10 were obtained from Fisher Scientific or Sigma-Aldrich; Fmoc-11-amino-3,6,9-trioxaundecanoic acid (miniPEG, mPEG3) was obtained from Peptides International, and all amino acids and activators were from Novabiochem or AGTC Bioproducts. N,N″-Bis(tert-butyloxycarbonyl)diethylenetriaminyl-N′-glycine (Bocdienac) and 2-(bis(2-aminoethyl)amino)acetic acid (Acdien) were prepared by a known method.38

High-Performance Liquid Chromatography (HPLC)

HPLC was performed using an Agilent 1260 Infinity with a quaternary solvent delivery system and a 1260 Infinity Diode Array Detector HS controlled by Agilent OpenLab CDS ChemStation Edition software with detection at 220 nm. Different columns were used for analysis and purification of the peptides. Analytical and semipreparative chromatography was performed on an Agilent C18 (2.7 μm; 120 Å) reverse-phase column (3.0 mm × 150 mm) at 1 mL/min (column 1). Preparative HPLC was performed on an Agilent PLRP-S (8 μm; 100 Å) reverse-phase column cross-linked polymer column (250 mm × 4.6 mm) at 10 mL/min (column 2). HPLC for radiolabeled 99mTc-Acdien-LHRH was on a Varian prostar 210 with a UV–visible detector and a NaI (Tl) scintillation flow-through radiation detector controlled by and processed by Galaxie software. Chromatography was performed on a Kinetex column (5 μm; 100 Å) reverse-phase C18 column (250 mm × 4.6 mm) at 1 mL/min (column 3).

NMR Spectroscopy

1H NMR was used to analyze and confirm the different steps of linker formation. All 1H NMR spectra were recorded on a 400 MHz JEOL Eclipse+ NMR Spectrometer and processed with Delta NMR software.

Mass Spectrometry

Analysis was done on a Thermo Scientific LTQ XL Mass Spectrometer using a direct analysis in real time (DART) or electrospray ionization (ESI) on an Agilent Technologies instrument processed with Analyst QS1.1 (Applied Biosystems) or Mass Hunter (Agilent).

Synthesis of Bocdienac Tridentate Linker

The N,N″-bis(tert-butyloxycarbonyl)diethylenetriaminyl-N′-glycine (Bocdienac) tridentate linker was synthesized as previously described by Ndinguri et al.38 All analyses matched the reported NMR and mass spectral values.

Synthesis of Acdien-LHRH

Using the standard Fmoc chemistry, the peptide Acdien-LHRH was synthesized as described by Calderon et al.20 Briefly, 10 amino acids were assembled on an Fmoc-Rink amide-AM resin using a PS3 peptide synthesizer (Scheme 1.). The Alloc protecting group on the d-lysine group was selectively removed using tetrakis(triphenylphosphine)palladium(0) along with a 37:2:1 mixture of methylene chloride, acetic acid, and NMM for 2 h, followed by washing and double coupling of the Bocdienac linker. The fully assembled peptide was cleaved from the solid support using a TFA/water/TIPS cleavage cocktail to give the target compound Acdien-LHRH. Acdien-LHRH was purified by HPLC or gel filtration using Sephadex G-10. The purified samples were lyophilized before use in the next step. The crude Acdien-LHRH (see the Supporting Information) was purified by HPLC on a reverse-phase C18 column 1 with a linear gradient from 10 to 90% B eluent in 10 min; tR = 5.5 min. The purity of Acdien-LHRH was also confirmed using mass spectrometry. Yield, 30%. ESI-MS (M + H)+, calcd for C65H99N21O15 1414.61; found 1414.7664 (see the Supporting Information).

Scheme 1. Synthesis of 99mTc/Re-Acdien-LHRH.

Scheme 1

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Synthesis of Acdien-LHRH using solid-phase chemistry and conjugation with Re and Tc analogues.

Synthesis of Re-Acdien-LHRH Conjugate

Acdien-LHRH was dissolved in water, and the pH of the solution was adjusted to 7, by titration with 1 M NaOH. The [Re(CO)3(H2O)3]+ core was prepared as reported in the literature.39 Equimolar amount of [Re(CO)3(H2O)3]+ was reacted with Acdien-LHRH (30 mg, 0.02 mmol) to give the crude Re-Acdien-LHRH conjugate. Re-Acdien-LHRH was purified by HPLC or gel filtration using Sephadex G-10. The purified samples were lyophilized before use in the next step. The crude Re-Acdien-LHRH conjugate was purified by HPLC on a reverse-phase C18 column with a linear gradient from 10 to 90% B eluent in 10 min; tR = 7.3 min (see the Supporting Information). The purity of Re-Acdien-LHRH was confirmed using mass spectrometry. Yield, 20%. ESI-MS (TOF) (M + H)+ calcd for C69H102N21O17Re 1684.73; found 1684.7023 (see the Supporting Information).

Synthesis of Acdien-peg-LHRH

Complete synthesis of Acdien-peg-LHRH complex was achieved using the same procedure described for Acdien-LHRH with slight variation. The LHRH peptide was assembled as described by Calderon et al.20 The Alloc protecting group was selectively removed using palladium as described above, and Fmoc-11-amino-3,6,9-trioxaundecanoic acid (mPEG3) was conjugated to the free amine end. The Fmoc group on mPEG3 was deprotected with 20% piperidine in N,N-dimethylformamide (DMF) for 3 min followed by coupling of the peptide to Bocdienac to give the target peptide. The fully assembled Acdien-peg-LHRH peptide was cleaved from the solid support using a TFA/water/TIPS cleavage cocktail to give the crude target compound Acdien-peg-LHRH. Intermediate products were washed between reactions with DMF. Acdien-peg-LHRH was purified by HPLC or gel filtration using Sephadex G-10. The purified samples were lyophilized before use in the next step. The crude Acdien-peg-LHRH (see the Supporting Information) peptide was purified by HPLC on a reverse-phase C18 column with a linear gradient from 10 to 90% B eluent in 10 min; tR = 6.5 min. The purity of Acdien-peg-LHRH was also confirmed using mass spectrometry. Yield, 31%. ESI-MS (M + H)+, calcd for C73H114N22O19 1602.86; found 1602.87 (see the Supporting Information).

Synthesis of Re-Acdien-peg-LHRH Conjugate

Complete synthesis of Re-Acdien-peg-LHRH conjugate was achieved using the same procedure described for Re-Acdien-LHRH above. Re-Acdien-peg-LHRH was purified by HPLC or gel filtration using Sephadex G-10. The purified samples were lyophilized before use in the next step. The crude Re-Acdien-peg-LHRH (see the Supporting Information) was purified by HPLC on a reverse-phase C18 column with a linear gradient from 10 to 90% B eluent in 10 min; tR = 8.3 min. The purity of Re-Acdien-peg-LHRH was confirmed using mass spectrometry. Yield, 25%. ESI-MS (TOF) (M + H)+, calcd for C76H115N22O22Re 1874.81; found 1874.8023. The most intense peaks (base peak) at 625.2813 and 937.4119 are the triply charged and doubly charged peaks, respectively (see the Supporting Information).

Radiolabeling of 99mTc-Acdien-LHRH

LHRH was labeled with 99mTc based on a previously reported method.40 [99mTcO4]− solution was first prepared with the Isolink kit (Paul Scherrer Institute, Switzerland) according to the manufacturer’s instructions to give [99mTc(CO)3]+ and then further reacted with Acdien-LHRH. Typically, 2 mCi 99mTcO4– was added into one vial of [99mTcO4]− solution and heated at 100 °C for 20 min using the Isolink kit protocol and reagents. pH was adjusted to about 7–7.4 with HCl. Next, 100 μL of Acdien-LHRH from a stock solution of 1 mg/mL was then added and incubated at 75 °C for 60 min. Afterward, the labeled peptide was purified by HPLC (C18 column, 10% MeCN + 0.1% HOAc 0–10 min, 90% MeCN + 0.1% HOAc 10–20 min. 200 nm, 1 mL/min). The retention time was ∼16 min (Figure 2). Nondecay corrected yield was calculated based on (100 × collected radioactivity at 15–17 min)/(total injected radioactivity).

Figure 2.

Figure 2

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Representative HPLC chromatographs for purified 99mTc-Acdien-LHRH (top: radioactive peak, bottom: UV). 2 mCi 99mTcO4– was added into one vial of the Isolink kit and heated at 100 °C for 20 min. pH was adjusted to about 7–7.4 with HCl. Chelator conjugated LHRH (1 mg in 10 μL water) was then added and incubated at 75 °C for 60 min. The labeled peptide was purified with HPLC (C18 column, 10% MeCN + 0.1% HOAc 0–10 min, 90% MeCN + 0.1% HOAc 10–20 min. 200 nm, 1 mL/min). The retention time was ∼16 min. 99mTcO4– and 99mTc(CO)3(H2O)3+ retention times were 3–5 min. 51% nondecay corrected yield was obtained based on (100 × collected radioactivity at 15–17 min)/(total injected radioactivity).

Cell Culture

4T1 mouse mammary tumor and MDA-MB-231 human mammary tumor cell lines were purchased from ATCC. Both cell lines were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin, incubated at 37 °C in a humidified atmosphere of 95% air and 5% CO2.

Re-Acdien-LHRH and Re-Acdien-peg-LHRH Cell Viability Assay

To assess if Re-Acdien-LHRH and Re-Acdien-peg-LHRH affected cell viability, an MTT assay was utilized. 4T1 or MDA-MB-231 cells were seeded at 2000 cells/100 μL in a 96-well plate and treated with either [Re(CO)3(H2O)3]+, Re-Acdien-LHRH, or Re-Acdien-peg-LHRH from a range of 0.1–100 μM for 24 h. Cells were washed 3 times with phosphate-buffered saline (PBS) and incubated in DMEM supplemented with 10% fetal bovine serum (FBS) for 48 h. Afterward, the cells were incubated for 4 h in a 10 μL MTT solution obtained from the Vybrant MTT Cell Proliferation Assay Kit (Life Technologies). Finally, cells were solubilized and mixed with sodium dodecyl sulfate (SDS), and absorbance was read at 595 nm on a Phenix Genios Tecon 96-well plate reader.

Re-Acdien-LHRH and Re-Acdien-peg-LHRH Drug Uptake Assay

To determine the cellular uptake of Re-Acdien-LHRH and Re-Acdien-peg-LHRH, 4T1 or MDA-MB-231 cells were seeded 1 × 106 in 6-well plates and treated with either unbound [Re(CO)3(H2O)3]+ (100 μM), Re-Acdien-LHRH (100 μM), or Re-Acdien-peg-LHRH (100 μM) for 24 h. Cells were washed 3 times with PBS, harvested, and metal (rhenium) concentration (mg/L) was measured by ICP-mass spectrometry at Louisiana State University as previously described.41

Re-Acdien-peg-LHRH Receptor Binding Assay

To determine the binding of Re-Acdien-peg-LHRH to the LHRH receptor for entry into the cell, a receptor binding assay was conducted. 4T1 cells were seeded at 1 × 106 per well in 6-well plates and treated with the free LHRH peptide (100 μM) for 30 min and then concurrently with Re-Acdien-peg-LHRH (100 μM) and incubated for 24 h at 37 °C. Cells were washed 3 times with PBS, harvested, and the metal (rhenium) concentration (mg/L) was quantified by ICP-mass spectrometry as described above.

Cell Uptake and Competition Assay of 99mTc-Acdien-LHRH at Various Incubation Times

MDA-MB-231 cells were cultured in Dulbecco’s minimum essential medium supplemented with 5 mM l-glutamine, 1% penicillin/streptomycin, and 10% fetal bovine serum (Invitrogen) at 37 °C in 5% CO2 conditions. The cells were seeded at 2 × 105 cells per well in a 12-well plate with 2.0 mL of culture medium and incubated for 48 h at 37 °C; 1 μCi (0.037 MBq) of conjugated 99mTc-Acdien-LHRH or unbound [99mTc(CO)3(H2O)3]+ was then added to corresponding wells and incubated at 37 °C for 15, 30, 60, and 120 min (n = 3). For the LHRH receptor competition, there was a cotreatment of 100 μg of LHRH with 99mTc-Acdien-LHRH at 37 °C. At the end of each designated time point, the medium was discarded and the cells were washed 3 times with a 2 mL PBS buffer. Trypsin (0.5 mL) was added to each well to detach the cells for counting. An automated γ counter was used to quantify radioactivity levels in the cells (Perkin Elmer, MA). Cell uptake was calculated by counts in cells divided by the total counts and reported as percent.

Statistics

Data was illustrated as mean ± standard error of the mean (SEM), and statistical analyses were carried out using GraphPad, Prism 6 (San Diego, CA). Unpaired t-tests and two-way analysis of variances (ANOVAs) were used where appropriate.

Results

Synthesis of Re-Acdien-LHRH and Re-Acdien-peg-LHRH Conjugates

For complete synthesis of Re-Acdien-LHRH, a bifunctional chelator, Bocdienac, containing a functional unit for conjugation to the targeting vector LHRH and a coordination moiety for the metal or radionuclide to securely bind, was first synthesized.38 Use of the Bocdienac linker for chelation of the radionuclide is advantageous as studies on [99mTc(CO)3(H2O)3]+ have demonstrated that tridentate ligands are optimal in substituting the aqua ligands to provide robust chemical complexes.42 Starting from an amide-based resin and using Fmoc chemistry, the peptide was assembled, the Alloc protecting group was removed followed by conjugation of the Bocdienac. The fully assembled peptide was cleaved from the solid support using a cleavage cocktail to give the target compound, crude Acdien-LHRH. The crude Acdien-LHRH was purified by either gel filtration or HPLC, and the fractions collected were identified by ESI-MS and lyophilized to give the pure Acdien-LHRH peptide. Chelation of Acdien-LHRH peptide was achieved using an equal molar ratio with activated fac-[Re(CO)3(H2O)3]+ using a standard procedure to give Re-Acdien-LHRH peptide as shown in Scheme 1. The rhenium conjugate was also purified by gel filtration or HPLC, and the pure fraction was lyophilized and identified by ESI-MS to give the target compound at an 18–20% yield.

In a similar manner, Re-Acdien-peg-LHRH was synthesized using the methodology described for the synthesis of Re-Acdien-LHRH, modified with the addition of mPEG3 on the peptide motif. The mPEG3 on the peptide motif was added to increase solubility, additional spacing, and stability. Many studies have shown that pegylation has several positive effects, such as reduced immunogenicity43,44 and increased stability that reduces drug efflux.45 Using the standard Fmoc chemistry, 10 amino acids were assembled on a Rink amide resin. The last amino acid on the sequence (glutamic acid) utilized a Boc protecting group on the side chain to allow further modification of the peptide (Scheme 1.). The Alloc protecting group on the d-lysine side chain was removed and its N terminus was coupled with Fmoc-11-amino-3,6,9-trioxaundecanoic acid. The mPEG3’s Fmoc group was removed using 20% piperidine. The presence of the Boc group on the glutamic acid side chain allowed for selective deprotection of the Fmoc group on the mPEG3 availing the amine end, which was coupled with Bocdienac. Similarly, Acdien-peg-LHRH peptide was purified using analytical HPLC and the identity was confirmed by mass spectroscopy. Chelation of Acdien-peg-LHRH was achieved using the same protocol described above to give the Re-peg-LHRH peptide as shown in Figure 1 and Supporting Information. The rhenium conjugate was purified by gel filtration or by HPLC, and the identity was confirmed by ESI-MS to give the desired compound.

Radiochemistry of 99mTc-Acdien-LHRH

Nondecay corrected yield was 51% for 99mTc-Acdien-LHRH. All injected radioactivity was completely eluted off the HPLC column during a separation period of 20 min, which allowed the use of the HPLC method for radiochemical and chemical purity analyses. Both radiochemical and chemical purities were greater than 99% after HPLC purification (Figure 2).

No Alteration in Cell Viability Was Found with Re-Acdien-LHRH or Re-Acdien-peg-LHRH Treatment

Previous experimental and clinical data indicate the overexpression of the LHRH receptor on various human breast carcinomas. To ensure that Re-Acdien-LHRH or Re-Acdien-peg-LHRH did not have cytotoxicity or safety concerns upon administration, cell viability was measured by MTT assay. Cell viability was assessed with MDA-MB-231 breast cancer cells treated with [Re(CO)3(H2O)3]+ or Re-Acdien-LHRH from 0.1 to 100 μM (Figure 3A). Re-Acdien-LHRH was not found to significantly inhibit MDA-MB-231 viability compared to the non-LHRH peptide-bound Re. Similar results were found with Re-Acdien-peg-LHRH treatment of MDA-MB-231 and 4T1 cells, in which no significant attenuation in cell viability was found compared to [Re(CO)3(H2O)3]+ treatment (Figure 4A,B).

Figure 3.

Figure 3

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Re-Acdien-LHRH shows increased breast cancer cellular uptake compared to Re with no alteration in cell viability. (A) MDA-MB-231 cells were treated with [Re(CO)3(H2O)3]+ or Re-Acdien-LHRH from 0.1 to 100 μM for 24 h. Viability rates were analyzed by an MTT assay after 48 h incubation. (B) MDA-MB-231 cells were treated with 100 μM of [Re(CO)3(H2O)3]+ or Re-Acdien-LHRH for 24 h. Cells were collected and Re concentration mg/L was measured using ICP-MS. n = 3, two-way ANOVA and unpaired t-test; ****p < 0.0001.

Figure 4.

Figure 4

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Re-Acdien-peg-LHRH shows increased cellular uptake with no difference in cell viability compared to Re. (A) MDA-MB-231 and (B) 4T1 cells were treated with [Re(CO)3(H2O)3]+ or Re-Acdien-peg-LHRH from 0.1 to 100 μM for 24 h. Viability rates were analyzed by an MTT assay after 48 h incubation. (C) MDA-MB-231 and (D) 4T1 cells were treated with 100 μM of [Re(CO)3(H2O)3]+ or Re-Acdien-peg-LHRH for 24 h. Cells were collected, and Re concentration mg/L was measured using ICP-MS. n = 3, two-way ANOVA and unpaired t-test; ****p < 0.0001.

Cellular Uptake of Re-Acdien-LHRH and Re-Acdien-peg-LHRH Was Significantly Increased

To examine if bound Re-Acdien-LHRH and Re-Acdien-peg-LHRH had increased cellular entry, an uptake assay was performed. A significant increase in Re was found in cells treated with Re-Acdien-LHRH compared to that of unbound [Re(CO)3(H2O)3]+ (Figure 3), indicating that Re-Acdien-LHRH is a targeting conjugate. Subsequently, a significant increase in the cellular uptake of Re-Acdien-peg-LHRH was found in both MDA-MB-231 and 4T1 cell lines compared to that of unconjugated [Re(CO)3(H2O)3]+ (Figure 4C,D). The 4T1 cell line was used to verify the LHRH targeting results found in the MDA-MB-231 cell line, indicating that the uptake is not a cell-line-specific phenomenon but the result of the receptor presence and expression.

LHRH Peptide Mediates Conjugate Targeting to the LHRH Receptor

A receptor binding assay was used to confirm that the targeting ability of the conjugate, Re-Acdien-peg-LHRH, is mediated via binding of the LHRH-bound peptide to LHRH receptor expressed on breast cancer cells (Figure 5). 4TI cells were pretreated with the LHRH peptide for 30 min prior to addition of the Re-Acdien-peg-LHRH compound. Cellular uptake of Re-Acdien-Peg-LHRH was significantly attenuated with LHRH peptide pretreatment, indicating that the conjugated LHRH peptide is biologically active.

Figure 5.

Figure 5

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Re-Acdien-peg-LHRH targets the LHRH receptor. 4T1 cells (1 × 106) were treated with or without LHRH (100 μM) for 30 min prior to concurrent Re-Acdien-peg-LHRH (100 μM) treatment for 24 h. Cells were collected, and Re concentration mg/L was measured using ICP-MS. n = 3, unpaired t-test; *p < 0.05.

Cell Uptake of 99mTc-Acdien-LHRH

Cell uptake of 99mTc-Acdien-LHRH increased steadily from 15 to 120 min (Figure 6). This data indicates that 99mTc-Adien-LHRH gains entry into the cells and the uptake is dependent on the incubation time. Further, the application of LHRH used to compete for the LHRH receptor showed a decrease in the 99mTc-Acdien-LHRH cellular uptake. This data indicates that 99mTc-Acdien-LHRH utilizes the LHRH receptor for cellular uptake. In addition, there is a significant increase in the cellular uptake of 99mTc-Acdien-LHRH compared to that of [99mTc(CO)3(H2O)3]+ alone, indicating bioactivity of LHRH and significant cellular targeting.

Figure 6.

Figure 6

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Cell uptake at different incubation times. A total of 2 × 105 MDA-MB-231 cells were seeded in a 12-well plate for 48 h at 37 °C. 1 μCi of 99mTc-Acdien-LHRH or [99mTc(CO)3(H2O)3]+ was then added to the corresponding wells and incubated at 37 °C for 15, 30, 60, and 120 min. Further, LHRH (100 μg) was cotreated with 99mTc-Acdien-LHRH in the competition wells. After cells were washed 3 times with PBS buffer, cells were isolated. Radioactivity in the cells was counted in an automated γ counter (Perkin Elmer, MA). Cell uptake was calculated by % (counts in cells)/total counts. n = 3, two-way ANOVA; **p < 0.005 and ****p < 0.0001.

Discussion

The development of technetium-based imaging agents requires a design of suitable ligands that are robust and effective molecular imaging probes. In this study, we have designed new LHRH peptide conjugates that allow chelation of Re and Tc metals. Re-Acdien-LHRH and Re-Acdien-peg-LHRH were synthesized to facilitate the development of the 99mTc-Acdien-LHRH complex as a potential radiopharmaceutical. We synthesized targeting radioimaging agents to detect cancer cells that overexpress the LHRH receptor relative to normal cells. Studies have shown that several cancers overexpress LHRH receptors such as breast, prostate, endometrial, and ovarian cancers among others. To form 99mTc-Acdien-LHRH, we have combined two moieties (LHRH and 99mTc(CO)3+) using a tridentate linker making the new conjugate target-specific. The tridentate chelator combines with the LHRH peptide on position 6 to ensure that the intrinsic properties of the LHRH peptide are maintained, which allows the delivery of the radiometal to the tumor cells. Symmetrical tridentate ligands such as those utilized for conjugation of these complexes have been shown to be suitable linkers because they are less likely to form racemic or diastereoisomeric mixtures, thereby increasing stability.46,47

Literary analysis demonstrates the use of LHRH for targeted delivery in the fields of therapeutic and diagnostic medicines. The predominant use of LHRH is in the development of chemotherapeutic compounds including curcumin-LHRH, lytic peptide-LHRH, paclitaxel-LHRH, and platinum-LHRH conjugates.48−50 The few diagnostic studies that utilize the LHRH receptors are unique in their architecture and differ significantly from our compound. For instance, LHRH conjugates with indium-111 have been used to target and image prostate cancer.35 Even though the results were promising, the shorter half-life, high specific activity, excellent imaging characteristics, widespread availability, and low cost of technetium make our conjugate an excellent option.15 In another study, LHRH was successfully conjugated with the radiometal chelator indium-111DOTA; however, the DOTA conjugate radiolabeling process is dependent on several conditions such as buffering agents and the presence of metal ions, which can make the labeling kinetic process slow.30,34,36 Another promising approach has been the use of HYNIC ligands for conjugation of Tc and LHRH; however, the ligand can only coordinate as a monodentate or bidentate ligand, thereby requiring a coligand to complete the coordination sphere of technetium.30,51,52 Published studies utilizing 99mTcO4– in combination with LHRH and other various combinatory methods for tumor imaging differ significantly in the architecture and mode of delivery from the 99mTc-Acdien-LHRH complex reported in this research.53−55 To produce a conjugated product, a specialized linker is designed to allow the targeting peptide to retain bioactivity and bind receptors at a high affinity. To date, there are no approved technetium LHRH-conjugated radiometal diagnostic agents, indicating a dire clinical need for optimal targeting diagnostic radiotracers.

The data contained in this manuscript demonstrate the design paradigm and synthesis of both radiolabeled 99mTc/Re-Acdien-LHRH to be used as effective diagnostic agents for cancers overexpressing the LHRH receptor. Scientific focus is needed in the realm of diagnostic compounds, specifically the field of radionuclide medicine to be used in combination with chemotherapeutic LHRH compounds. Further, a conjugation approach to target imaging agents that can detect cancer earlier could lead to a better prognosis for cancer patients and tracking of therapeutic effectiveness.

Numerous cancers have been shown to overexpress the LHRH receptor, making it a good candidate for diagnostic targeting. Both reproductive cancers including breast, prostate, and ovarian have been shown to overexpress the receptor,21,22,56,57 along with nonreproductive cancers including lung, bladder, and pancreatic.58−60 Specifically, about 60% of all human breast cancers and 74% of triple-negative cancers have been shown to express the LHRH receptor, including the 4T1 and MDA-MB-231 cell lines.61 The expression of LHRH in the 4TI and MDA-MB-231 cell lines has been abundantly identified and confirmed in numerous research studies using various techniques including but not limited to FITC staining, reverse transcriptase quantitative polymerase chain reaction (RT-qPCR), protein quantification, etc.62−65 This makes utilizing the LHRH peptide for diagnosis of triple-negative breast cancer a logical design method.

Further, the triple-negative cell lines MDA-MB-231 (human mammary adenocarcinoma) and 4T1 (mouse mammary carcinoma) were experimentally used to determine compound uptake by the LHRH receptor. To indicate that LHRH can be used to deliver radiolabeled metals to cancer cells overexpressing the LHRH receptor, 99mTc-Acdien-LHRH was synthesized. For ease of use through experimental procedures, Re-Acdien-LHRH was not radio-tagged. Both 99mTc-Acdien-LHRH and Re-Acdien-LHRH showed cellular uptake by the MDA-MB-231 cell line (Figures 6 and 4B). Furthermore, neither 99mTc-Acdien-LHRH nor Re-Acdien-LHRH requires mPEG3 to be soluble or effective; however, PEGylation is a common technique used to enhance compound solubility and bioavailability upon in vivo administration.66,67 To study the synthesis and formulation of Peg incorporation, Re-Acdien-peg-LHRH was synthesized.

Both Re-Acdien-LHRH and Re-Acdien-peg-LHRH showed enhanced cellular uptake compared to [Re(CO)3(H2O)3]+ treatment alone (Figures 3B and 4C,D). Subsequently, to show that the LHRH-conjugated peptide binds and uses the LHRH receptor for cellular entry, a competition assay was employed. Figure 5 shows that pretreatment of the LHRH peptide competed with Re-Peg-LHRH for binding to the LHRH receptor, as there was a significant decrease in the cellular uptake of Re-Peg-LHRH compared to that of the unconjugated Re alone. This demonstrates that the uptake of our conjugated compounds is mediated by binding to the LHRH receptor. In addition, our compound toxicity was investigated. Both Re-Acdien-LHRH and Re-Acdien-peg-LHRH did not induce cytotoxicity even at the highest dose (100 μM), compared to unconjugated [Re(CO)3(H2O)3]+ (Figures 3A and 4A,B). This provides preliminary data that Re-Acdien-LHRH and Re-Acdien-peg-LHRH can safely be administered in vivo. Further, data from our investigation suggest that LHRH and LHRH analogues can act as clinically relevant and safe antiproliferative treatments in androgen-dependent carcinomas.68,69 Hence, both Re/Tc and LHRH alone are already used in patients and the safety profile is thoroughly investigated providing support for combinational application.

Conclusions

Our results can lead the way and provide a foundation for enhanced in vivo imaging by targeting the LHRH receptors on tumor cells. Through the use of targeting radionuclides, (1) we can target radiolabeled tracers to deposits of cancer cells or any tumor microenvironment that overexpress these receptors and (2) the targeted conjugate will help monitor or image the tumor microenvironment, an important condition for subsequent radionuclide therapy. Taken together, we have demonstrated the synthesis of a new diagnostic agent 99mTc/Re-Acdien-LHRH and its subsequent PEGylated form, designed to selectively target cancer cells overexpressing the LHRH receptor. Our preliminary in vitro results demonstrate enhanced cellular uptake of the diagnostic agents by use of the conjugated LHRH peptide while showing no indication of toxicity. Hence, our results indicated that these compounds have the potential to be clinically used as diagnostic agents.

Acknowledgments

We would like to thank Dr. Thomas Blanchard for his help with the ICP analysis and Connie David for her help with mass spectroscopy, both from Louisiana State University.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.0c03991.

  • Synthetic scheme, HPLC analysis spectra (Acdien-LHRH, Re-Acdien-LHRH, Acdien-peg-LHRH, and Re-Acdien-peg-LHRH), and mass spectra analysis (Acdien-LHRH, Re-Acdien-LHRH, Acdien-peg-LHRH, and Re-Acdien-peg-LHRH) (PDF)

Kentucky Biomedical Research Infrastructure Network (KBRIN) grant (P20GM103436) and EKU University Research Grant.

The authors declare no competing financial interest.

Supplementary Material

ao0c03991_si_001.pdf (328.4KB, pdf)

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