The
Lung cancer is among the most frequently occurring and deadly cancers affecting both sexes [
Lung cancers are divided into two major types of small-cell lung carcinoma (SCLC) and non-small-cell lung carcinoma (NSCLC). NSCLC accounts for 80% to 85%, and SCLC is responsible for 15% to 20% of lung cancers [
Membrane proteins are the characteristic feature of a cancer cell. The overexpressed membrane receptors have critical importance in cancer diagnosis and therapy. It is possible to select and design new molecules that can be attached specifically to these cell-surface proteins and detect/destroy cancer cells [
Molecular imaging targets used in the clinic must have high affinity for the overexpressed receptors and great tumor-to-organ ratio to deliver imaging probes to the tumor sites. Biological tumor-targeting agents include antibodies, proteins, peptides, and aptamers [
Among the aforementioned tumor-targeting ligands, peptides benefit from high selectivity, high potency, fast blood clearance, and low toxicity, and nonimmunogenicity (excellent tolerability by patients), broad range of targets, better intratumoral diffusion owning to their lower molecular weight, and peptides are easy and quite inexpensive to synthesize and allow numerous conjugation possibilities for targeted delivery [
Radiolabeled peptides have been extensively studied, and various peptide receptor imaging and targeted therapies have been performed. Peptides as radiotracer show appropriate characteristic such as metabolic stability and tolerance to changes during radiolabeling process [
The integrin family regulates critical cellular functions in solid tumor progression and consequently made them an attractive target for cancer therapy/diagnosis. They are transmembrane
Two of the major integrins involved in NSCLC proliferation and metastasis are
In the last decade, various radiolabeled RGD peptides targeting integrin
From early studies, various diagnostic and therapeutic radiopharmaceuticals have been developed. Radiometals are an important component of most radiopharmaceuticals because of their nuclear properties. Their nuclear emissions, energies, half-lives’ importance, availability, and cost are determining factors in their application for regular medical use [
Here, we assessed the radiosynthesis, quality control, and
DOTA-E(cRGDfK)2 was purchased from FutureChem (Seol, Korea) (Figure
All chemicals were obtained from Aldrich (Germany) and used without further purification. Normal saline, sodium acetate, methanol, ammonium acetate, trifluoroacetic acid (TFA), acetonitrile, acetone, hydrochloric acid, and sodium acetate used for radiolabeling were of high purity. Milli-Q water (ultrapure water (Type 1), resistivity 18.2 MΩ·cm at 25°C) was obtained from a Direct Q system (Millipore) and used for the preparation of all aqueous solutions and buffers.
Cartridges, sterile collection vials, and all cold standards were purchased from ABX (Advanced Biochemical Compounds, Germany).
Radioactivity was determined by an ionization chamber (PTW CURIEMENTOR 4). Radio-TLC was performed using chromatography paper impregnated with silica-gel (ITLC-SG, Agilent Technologies, Santa Clara, California). Analysis was carried out with a TLC scanner (miniGita; Raytest, Straubenhardt, Germany). Radio-HPLC was performed using an Agilent 1260 reverse-phase HPLC system equipped with a NaI (Tl) radiodetector (Gabi, Raytest, Germany) and a PC interface running service pack 2 software (Raytest, Straubenhardt, Germany). pH was measured using a pH meter (Knick, 765 Laboratory pH Meter, Germany). NaI (Tl) gamma detector (Delshid, Tehran, Iran) was applied for radioactivity measurements during animal biodistribution studies.
Non-small-cell lung carcinoma (NSCLC) cell line (A549) and Swiss mouse embryo fibroblast (NIH-3T3) cells were obtained from Pasture Institute of Iran.
Animals’ studies were approved by the Research committee of Tehran University of Medical Sciences. PET images were obtained using the Siemens Biograph6 True-Point (trueV) PET/CT scanner (Siemens AG, Erlangen, Germany).
Sequences of various RGD-containing peptides were retrieved from the protein databank (
To simulate the interaction of integrin-binding motifs with integrin
The MD simulations were carried out for 20 ns using GROMACS 5.0.7 software package with CHARM36 force field in a cubic 15
After energy minimization, the system was equilibrated, and 20 ns MD simulations were carried out at the NPT. In all simulations, to keep all hydrogen bonds rigid, the SHAKE algorithm was used.
Analyses of RMSD, radius of gyration, temperature, density, and pressure have been carried out to confirm the stability of each MD simulation.
After ensuring the stability of MD simulations, MM-PBSA analysis for each complex was carried out to calculate the total energy and estimate the affinity of binding.
68Ga was obtained from a 68Ge/68Ga-sterile generator. Labeling was performed using a modular synthesizer (GRP 4V) (Scintomics, Fürstenfeldbruck, Germany) with single-use cassettes. For radiolabelling, 68Ga[Ga3+] eluate was added to a mixture of 1.5 ml HEPES buffer (1.5 M in H2O, pH 4.5–5) and 70
The radiochemical purity was determined via reverse-phase HPLC and ITLC. HPLC was performed on an Agilent 1260 system using a C18 column 150 × 3 mm, 3
ITLC was performed by chromatography paper impregnated with silica-gel (ITLC-SG, Agilent Technologies) using ammonium acetate (1 M) and methanol (1 : 1) as the mobile phase. The strips were analyzed using the thin-layer chromatography scanner (miniGita, Raytest, Germany).
In a 5 ml Eppendorf tube, 100
The stability of [68Ga]Ga-DOTA-E(cRGDfK)2 peptides was determined by incubating the compounds in 0.01 M phosphate buffered saline (PBS), pH 7.4, %5 human serum albumin, and 0.1 M sodium acetate, pH 5.5. All the test tubes were incubated at 37°C with mild shaking in Thermomixer and analyzed by ITLC at different time intervals. Results were expressed as percent radiochemical purity (%RCP) yield.
To determine the peptide stability at 4°C, the compounds were mixed with human serum albumin and incubated for different time points followed by ITLC analysis. Results were expressed as percent radiochemical purity (%RCP) yield.
The protein binding properties of [68Ga]Ga-DOTA-E(cRGDfK)2 in blood were investigated by protein precipitation. 1ml of the labeled complex and 3ml of human plasma were mixed and incubated for 1 hour at 37°C. Then, an equal volume of 10% trichloroacetic acid (TCA) was added, and samples were centrifuged at 3,000 rpm for 10 min to separate serum from cells. Precipitate was resuspended in 5% TCA and centrifuged at 3,000 rpm for 10 min. Precipitate and the supernatant fractions were analyzed for radioactivity in a gamma counter. Protein binding of [68Ga]Ga-DOTA-E(cRGDfK)2 was expressed as the fraction of radioactivity bound to the protein, in percentage of the total radioactivity.
1
2 × 106 human lung adenocarcinoma A549 cells were seeded in a 24-well plate (105 per well). After 24 hours of incubation at 37°C in a cell culture incubator, cells were treated by 1
Cells were incubated with 0.5 ml/well of acid wash buffer (50 mM glycine buffer, 100 mM NaCl, pH = 2.8) at room temperature to remove surface-bound radioactivity. This step determines the membrane-bound radioligand and internalized radioligand. Then, the cells were lysed using 1N NaOH at 37°C for 10 min and harvested. The two fractions were measured in a gamma counter. Each experiment was done in triplicate.
For the determination of [68Ga]Ga-DOTA-E(cRGDfK)2 specific binding, A549 cells were pretreated with 500-fold of unlabeled DOTA-E(cRGDfK)2 30 min before the addition of labeled peptides.
The binding affinity of [68Ga]Ga-DOTA-E(cRGDfK)2 was determined by the saturation radioligand binding assay. [68Ga]Ga-DOTA-E(cRGDfK)2 with high radiochemical purity was prepared. Human lung adenocarcinoma A549 was seeded in cell culture plates (1
100
Human lung adenocarcinoma A549 (NCBI No: C137) and Swiss mouse embryo fibroblast NIH-3T3 (NCBI No: C156) cells were acquired from Pasteur Institute of Iran. Cells were grown to confluence at 37°C with %5 CO2 and %85 humidity in DMEM/F12 and RMPI 1640 supplemented with %10 fetal bovine serum (Gibco) and 1% penicillin/streptomycin, respectively.
100
6–8 weeks female BALB/c mice (with body weights of 20–25 g) were purchased from Royan Insitute (Amol, Iran). Animals were housed in wire cages under controlled conditions of temperature at 25°C, relative humidity around 50%, and 12/12 h light/dark cycles with food, and water was given ad libitum. 2 × 106 cell suspensions of human lung adenocarcinoma A549 in 100
All values were expressed as mean ± standard deviation (SD) with statistical significance analyzed using one-way analysis of variance or
Various selected sequences for docking are summarized in Table
Root-mean-square deviation of the C
Radius of gyration (Rg) correlates to the compactness of the structures, and if a protein is stably folded, it will likely maintain a relatively steady value. Radius of gyration in each MD simulation alters over time which is due to conformational changes to create a more compact/tense structure. The radius of gyration becomes relatively fixed and stable in the last 2 nanoseconds (Figure
The temperatures and densities ultimately became fixed and stable, representing further proof of simulation stability (Figures
MM-PBSA analysis after MD simulations in GROMACS was performed to estimate the affinity of binding. The results are summarized in Table
As the E(cRGDfK)2 sequence in the complex with
Nonradioactive DOTA-E(cRGDfK)2 (Figure
[68Ga]Ga-DOTA-E(cRGDfK)2 was prepared in a high yield (%>98). The radiochemical purity determined by radio-TLC was >99.5% (Figure
ITLC analysis of [68Ga]Ga-DOTA-E(cRGDfK)2. The radiochemical purity determined by radio-TLC was 99.7%.
HPLC profile of [68Ga]Ga-DOTA-E(cRGDfK)2. The peaks at 2.54 and 10.59 min are related to free 68Ga and [68Ga]Ga-DOTA-E(cRGDfK)2, respectively.
The log
The stability of [68Ga]Ga-DOTA-E(cRGDfK)2 was determined at different time intervals by ITLC, as described above. The radiolabeled complex remained stable at 37°C, for up to 120 min for [68Ga]Ga-DOTA-E(cRGDfK)2 (Figure
The
The ITLC radiochromatograms of the [68Ga]Ga-DOTA-E(cRGDfK)2 peptide compared to appropriate urine samples showed good
Metabolic stability of 68Ga-DOTA-E(cRGDfK)2.
For cell-binding assay, A549 cells (human adenocarcinoma cell line) were used. The human lung A549 cell that overexpressed
The total binding, surface-bound, and internalized radioactivity of [68Ga]Ga-DOTA-E(cRGDfK)2 at different time points.
Compared to the A549 cell line, the binding of [68Ga]Ga-DOTA-E(cRGDfK)2 to NIH-3T3 (normal cell line) was low. Data from the competitive assay using 500-fold excess of the unlabeled peptide showed a very significant reduction in binding of [68Ga]Ga-DOTA-E(cRGDfK)2 to the A549 cell line which confirms the specific binding through the peptide (Figure
Cell uptake studies using human lung cancer A449 (
Dissociation constant (
[68Ga]Ga-DOTA-E(cRGDfK)2 saturation binding assay curve.
Over time after injection, the radioactivity in tissues and organs decreased gradually (Figure
The
Biodistribution of [68Ga]Ga-DOTA-E(cRGDfK)2 in percentage of ID/g of organs at 30, 60, and 90 min postinjection in
All acquisitions using [68Ga]Ga-DOTA-E(cRGDfK)2 were carried out in A549 tumor-bearing BALB/c mice. Fused PET/CT scans are shown in Figure
PET imaging of [68Ga]Ga-DOTA-E(cRGDfK)2 in normal mice at (a) 1 hour and (b) 2 hour after IV injection, 1 h (c) and 2 h (d) blocking using DOTA-E(cRGDfK)2, and PET images of tumor-bearing BALB/c mice at (e) 1 h and (f) 2 h after IV injection of [68Ga]Ga-DOTA-E(cRGDfK)2. White arrows are the indication of the tumor position.
Radiolabeled peptides have shown great promise for cancer therapy [
In the last decade, various radiolabeled RGD peptides targeting integrin
Therefore, in recent years, many studies have focused on describing novel radiolabeled RGD peptides for molecular-based imaging [
In this work, the best RGD-containing peptide was selected. Sequences of various RGD-containing peptides were retrieved from the protein databank (
In the present study, we selected 68Ga for DOTA-E(cRGDfK)2 labeling due to its favorable physicochemical characteristics for imaging. 68Ga, a positron emitter radionuclide, has a suitable half-life of 68 min and is a generator-produced radionuclide. 68Ga-labeled peptides can be used by diagnostic imaging using PET. The 68Ga decay mode results in superior imaging in positron emission tomography (PET).
We synthesized [68Ga]Ga-DOTA-E(cRGDfK)2 in high radiochemical and radionuclide purity. The log
[68Ga]Ga-DOTA-E(cRGDfK)2 showed very good
68Ga-DOTA-E(cRGDfK)2 recognizes specifically
Dissociation constant of [68Ga]Ga-DOTA-E(cRGDfK)2 was 15.38 ± 3.42 which demonstrates that only a low concentration of the ligand is required to occupy the receptors, indicative of high binding affinity. Based on previous studies, multimeric cyclic RGD has higher (20–100 more)
PET/CT imaging of [68Ga]Ga-DOTA-E(cRGDfK)2 showed very high tumor uptake.
In conclusion, this study provides the first evaluation of the potential of [68Ga]Ga-DOTA-E(cRGDfK)2 for non-small-cell lung cancer imaging. Single dose of [68Ga]Ga-DOTA-E(cRGDfK)2 would be a promising diagnostic biological-based drug for cancer imaging, tumor treatment response monitoring, and follow-up imaging. Therefore, [68Ga]Ga-DOTA-E(cRGDfK)2 can serve as a great radiotracer for accurate and early detection of lung lesions.
The data used to support the findings of this study are available from the corresponding author upon request.
The authors declare no conflicts of interest.
The authors thank Dr. Samira Soltani Ghooshkhane and Razavi Hospital, Mashhad, for technical assistance. This study was part of a PhD thesis supported by Tehran University of Medical Sciences (grant no. 98-3-104-45895).
Figure S1. Schematic representation for the extracellular part of integrin