The αvβ3 integrin is a type of adhesion protein that is highly expressed on activated endothelial cells undergoing angiogenesis. Thus, evaluating the integrity of the integrin is of great interest in oncology. Here, we introduce a method to prepare 68Ga-labeled radiopeptides and a method to assess its biological effectiveness.
The αvβ3 integrin is a heterodimeric adhesion molecule involved in tumor cell migration and angiogenesis. The integrin is overexpressed in angiogenic tumor endothelial cells, where it typically has a low concentration. This specific expression of αvβ3 makes it a valid biomarker for antiangiogenic and imaging drugs. As a functional imaging modality, positron emission tomography (PET) provides information about biochemical and physiological changes in vivo, due to its unique high sensitivity at the nanomolar scale. Hence, radiometal-based PET radiopharmaceuticals have received great attention for the non-invasive quantification of tumor angiogenesis. This paper provides a systemic protocol to prepare a new radiometal-labeled peptide for the evaluation of angiogenesis. This protocol contains information about radiochemical reliability, lipophilicity, cell uptake, serum stability, and pharmacokinetic properties. The 68Ga-RGD-peptide is one of the representative PET ligands toward αvβ3 integrin. Here, we introduce a protocol to prepare a 68Ga-RGD-peptide and the evaluation of its biological efficacy.
Angiogenesis is a biological process that is characterized by the development of new blood vessels. Among many angiogenetic factors, αvβ3 integrin is associated with invasiveness, because the integrin is highly expressed in angiogenic tumor vessels but is absent in normal tissue1.
Radiolabeled receptor-binding peptides with the arginine glycine aspartate (RGD) domain, which has a high affinity toward αvβ3 integrin receptors, are considered promising angiogenesis imaging agents2,3,4,5,6,7. Several radiopharmaceuticals have been created for PET and its biological properties have been validated in various animal models8,9,10,11. In terms of a radionuclide, 68Ga has several advantages over other radioisotopes. Firstly, it has a high accessibility for users and is economically advantageous because a cyclotron is not required. Secondly, 68Ga-based radiopharmaceuticals produce high spatial resolution compared with single-photon emission computed tomography (SPECT), allowing more accurate quantification. Lastly, the 67.71 minutes half-life of 68Ga may be sufficient for the preparation of small peptides or proteins.
To produce a stable complex with 68Ga, many chelators have been developed. Representative chelators are 1,4,8,11-tetraazacyclotetradecanetetraacetic acid (TETA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), diethylenetriaminepentaacetic acid (DTPA), and N,N'-di(2-hydroxybenzyl)ethylenediamine-N,N'-diacetic acid (HBED). NOTA has been reported to form a highly stable complex with 68Ga (log stability constant 30.98)12,13,14.
The purpose of the present study is to provide a concise protocol for the development of a new radiopeptide (Figure 1). As an example, we prepare 68Ga-labeled RGD-peptides and present methods for the biological evaluation of these analogues in a xenograft model.
All animal experiments were conducted in compliance with the Guidelines for the Care and Use of Research Animals under protocols approved by the Korea Institute of Radiological and Medical Sciences Animal Studies Committee. All reagents and solvents were purchased and used without further purification. NOTA-RGD-peptides were prepared according to literature methods15.
CAUTION: 68Ga emits both positron and gamma rays. All experiments, including direct or indirect contact with radioactive substances, must be undertaken by trained and permitted personnel only. When handling radioactive materials, proper protective equipment, shielding, radiation dosimeter badge and rings, and a survey meter should be used.
1. Radiolabeling RGD-peptides with 68GaCl3
Note: 68Ga (t1/2 = 68 min, β+ = 89%, and EC = 11%) was obtained from the 68Ga/68Ge generator.
2. In Vitro Cellular Uptake
Note: Uppsala 87 Malignant Glioma (U87MG) human glioblastoma cells were grown in Dulbecco's modified Eagle's media (DMEM), supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. Cells were grown in 150 mm dishes at 37 °C in a humidified atmosphere of 5% CO2. Cells were harvested or split by trypsinization: 0.25% (w/v) trypsin and 0.02% (w/v) ethylenediaminetetraacetic acid (EDTA) in PBS at 37 °C for 3 – 5 min.
3. In Vitro Serum Stability
4. Determination of Lipophilicity
5. Tumor Model
Note: BALB/c nude mice (6 – 8 weeks old, female, n = 23) were used for this study. The mice were subsequently used for PET studies (n = 3) and biodistribution (n = 20) when the tumor volumes reached 200 – 300 mm3 (1 – 2 weeks after implantation).
6. In Vivo Quantification of αvβ3 Integrin Using PET
7. Ex Vivo Biodistribution
The chelation of 68GaCl3 with the NOTA-RGD-peptide was straightforward, and the radiolabeling yield was 99%. Reaction impurities were successfully removed as shown in Figure 2. The radiochemical purity of 68Ga-RGD-peptide was greater than 99%, and specific activity at the end of the synthesis was 90 – 130 MBq/nmol (Figure 3).
The cell uptake values for 68Ga-RGD-peptide were 1.49%, 0.85%, 0.36%, and 0.39% at 30, 60, 90, and 120 min, respectively. Serum stability showed that 68Ga-RGD-peptide remained almost intact after 2 h of incubation with human or mouse serum as well as PBS (> 92% stability at 2 h). The partition coefficient (log P) was 2.96, indicating high lipophilicity. PET showed an initial high uptake in the major organs, including the liver, kidney, heart, muscle, and tumor. However, in the late period (90 – 150 min), the tumor region was clearly visualized. The tumor-to-muscle ratio at 90 min was 17.57 and remained unchanged, indicating kinetic stability. The ex vivo biodistribution showed that the accumulated radioactivity in the tumor was 6.19, 4.96, 4.44, and 4.39 (% ID/g) at 30, 60, 90, and 120 min, respectively. The results of the ex vivo experiment were in accordance with the in vivo PET findings (Figure 4).
Figure 1: Flow diagram of the experimental procedures. This figure shows a schematic overview of the development of radiopharmaceutical. Please click here to view a larger version of this figure.
Figure 2: Purification of 68Ga-RGD-peptide by HPLC. Blue is radioactivity signal and black is ultraviolet (UV) signal. The UV wavelength is 314 nm. The X-axis is time and the Y-axis is absorbance unit (AU). The 68Ga-RGD-peptide has 12.4 min of retention time. Please click here to view a larger version of this figure.
Figure 3: Structure of 68Ga-RGD-peptide and its radiochemical purity. The ITLC of 68Ga-RGD-peptide showed high radiochemical purity. Please click here to view a larger version of this figure.
Figure 4: PET imaging (upper) and ex vivo biodistribution data for 68Ga-RGD-peptide (lower). PET data were expressed on the SUV scale from 0 to 5. Biodistribution data shown are the mean ± the standard deviation from five mice at each time point. Please click here to view a larger version of this figure.
In the present study, we introduced a protocol to prepare a radiopeptide targeting αvβ3 integrin and its biological evaluation. Traditional drug development involves a complicated procedure. It requires a large quantity of reference material and a relatively long evaluation time. Although the suggested methodology cannot replace the delicate evaluation process, this system can be used for screening purposes. This proposed system would considerably reduce the time and cost.
Over the past decade, many radiolabeled RGD-peptides have been extensively studied as radiotracers for imaging tumors16. To obtain promising radiopharmaceuticals for clinical trials, systemic approaches for drug development should be provided. Radiochemical feasibility, high selectivity-affinity to the target, metabolic stability, and proper pharmacokinetics are four major concerns. For a routine PET study, a reasonable radiochemical yield ensures the reliability of the radiopharmaceuticals. The issues of high affinity (> nM) and selectivity (> 100x) to the target protein are also satisfied. In terms of pharmacokinetics, the candidate PET tracer is rapidly excreted from the non-target tissue and has a long retention time in the tumor, allowing a high target-to-reference ratio. Candidate radiopharmaceuticals should not have troublesome metabolites in vivo that could increase non-specific binding and provide low contrast imaging. It is important to assess the comprehensive characteristics because each term influences the other properties, which are not independent.
The radiopeptide introduced in this research has suitable drug-like properties. The 68Ga-RGD-peptide has a high radiochemical yield of 99%, metabolic stability, and proper lipophilicity. In the in vivo experiment, the radiopeptide exhibited high selectivity (tumor-to-reference ratio = 17.57), and the ex vivo biodistribution data also showed significant tumor uptake (up to 6.19% ID/g).
The authors have nothing to disclose.
This work was supported by a Nuclear Research and Development Program of the National Research Foundation of Korea (NRF) grant funded by the Korean government (No. 2017M2A2A6A02019904).
68Ga/68Ge generator | ITG Company | – | 10 mCi |
Hydrogen chloride solution | Sigma-aldrich | 84429 | |
Sodium acetate | Sigma-aldrich | S2889 | |
C18 reverse-phase cartridge | Waters | WAT020515 | |
0.22-μm sterile filter | Milllipore | SLGV033RS | |
Radio-TLC scanner | Bioscan | AR2000 | |
ITLC paper | Agilent | SGI001 | |
Citric acid | Sigma-aldrich | 251275 | |
HPLC | Waters | – | Waters 1525 system containing binary pump, photo diode array (Waters 2998), radioactivity detector (Raytest, Gabi) |
C-18 colunm | Develosil | HG31546150W | ODS-HG-5, 150 ⅹ 4.6 mm, 5μm |
Acetonitrile | J.T. Baker | 14-650-359 | |
Trifluoroacetic acid | Sigma-aldrich | 302031 | |
Dulbecco's modified Eagle media | Thermo fisher scientific | 11965092 | |
fetal bovine serum | Thermo fisher scientific | 16000044 | |
T175 flasks | Corning | CLS431080 | |
Trypsin-EDTA (0.25%) | Thermo fisher scientific | 25200072 | |
penicillin-streptomycin | Thermo fisher scientific | 15240112 | |
γ-counter | Perkin Elmer | – | 1480 Wizard 3 |
Insunlin syringe | Becton Dickinson | 326105 | |
Synringe pump | Harvard Apparatus | 70-4500 | |
micro-PET/CT | Siemens Inveon | – |