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).

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, &#946;+ = 89%, and EC = 11%) was obtained from the 68Ga/68Ge generator.
<ol>
	<li>Elute the 68GaCl3 from the generator with 4 mL of 0.05 M HCl.</li>
	<li>Purge with nitrogen gas at 80 &#176;C for 30 min to dry 68GaCl3 (333 kBq, 1 mL) in a 5 mL reaction vial.</li>
	<li>Add a solution of RGD-peptide (100 &#181;g)in 1 M sodium acetate (100 &#181;L, pH 5 - 6) to the reaction vial containing 68GaCl3 from step 1.2.</li>
	<li>Heat the reaction mixture at 80 &#176;C for 5 min. Then, cool it down to room temperature.</li>
	<li>Purify the crude product with high-performance liquid chromatography (HPLC). Use the following system: a C-18 column, a flow rate of 0.5 mL/min, a gradient slope of acetonitrile of 1.17%/min (5% - 40% in 30 min), and elution components: A = 0.1% trifluoroacetic acid (TFA) in acetonitrile, B = 0.1% TFA in water. 
	NOTE: The HPLC is equipped with a photodiode array detector and a radioactivity detector. The 68Ga-RGD-peptide was collected at a retention time of 12.5 min (Figure 2).</li>
	<li>Purify the resulting 68Ga-RGD-peptide using a solid phase extraction system.
	<ol>
		<li>Pass the solution through a C18 reverse-phase cartridge and wash with 2 mL of saline.</li>
		<li>Elute 68Ga-RGD-peptide with 0.7 mL of 95% ethanol. Remove the solvent at 80 &#176;C under nitrogen gas for 20 min and reconstitute with phosphate-buffered saline (PBS) before use.</li>
		<li>Filter the radiolabeled product through a 0.22 &#181;m sterile filter and formulate in 1 mL of sterile saline solution.</li>
	</ol>
	</li>
	<li>Check the radiochemical yield by radio-thin-layer chromatography (TLC).
	<ol>
		<li>Spot 1 &#181;L on an instant thin layer chromatography plate (ITLC, 10 cm in length). Develop the plate in a chamber containing the eluent (aqueous 0.1 M citric acid, pH 5.0) until 9 cm away from the spot. 
		NOTE: The retention factor for 68Ga-RGD-peptide is 0 and the retention factor for unreacted 68Ga3+ is 1.</li>
	</ol>
	</li>
	<li>Calculate the final specific activity from the ratio of radioactivity corresponding to the non-radioactivity as MBq/nmol. 
	NOTE: After the injection of 100 &#181;L of the formulated 68Ga-RGD-peptide to HPLC, the amount of non-radioactive component was calculated from the standard calibration curve using nonradioactive Ga-RGD-peptide.</li>
</ol>
2. In Vitro Cellular Uptake
Note: Uppsala 87 Malignant Glioma (U87MG) human glioblastoma cells were grown in Dulbecco&#39;s modified Eagle&#39;s media (DMEM), supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. Cells were grown in 150 mm dishes at 37 &#176;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 &#176;C for 3 - 5 min.
<ol>
	<li>Seed U87MG cells into 6-well plates at a density of 1 x 106 cells/well.</li>
	<li>Incubate the cells with 68Ga-RGD-peptide (111 kBq) at 37 &#176;C for 30, 60, 90, and 120 min. Prepare samples in triplicate.</li>
	<li>Wash the cells 2x with 2 mL of PBS and harvest by trypsinization. Use 0.25% (w/v) trypsin and 0.02% (w/v) ethylenediaminetetraacetic acid (EDTA) in PBS at 37 &#176;C for 3 - 5 min.</li>
	<li>Collect the cell suspension (500 &#181;L) and measure in a &#947;-counter.</li>
	<li>Calculate the percent uptake of the compound by the cells by % (counts in cells/total counts).</li>
</ol>
3. In Vitro Serum Stability
<ol>
	<li>Add 500 &#181;L of freshly prepared mouse serum, 500 &#181;L of human serum, and 500 &#181;L of PBS. Incubate the mixture at 37 &#176;C for 2 h.</li>
	<li>Evaluate by ITLC at the specified time intervals (30, 60, 90, and 120 min). Spot 1 - 2 &#181;L aliquot of the mixture to the ITLC plate (mobile phase: 0.1 M citric acid). Develop the plate as in step 1.7. 
	NOTE: 68Ga3+ is expected to move with the solvent front, whereas the labeled compound will remain at the origin.</li>
</ol>
4. Determination of Lipophilicity
<ol>
	<li>Add 68Ga-RGD-peptide (3.7 MBq, 3.7 &#181;L) to the octanol-PBS system (1:1, v/v, total 1 mL).</li>
	<li>Mix the vials vigorously for 5 min at room temperature and centrifuge at 10,000 x g for 5 min at room temperature.</li>
	<li>Take 100 &#181;L samples from each layer and measure the radioactivity with a &#947;-counter. The reported log P value is based on the average of three samples.</li>
</ol>
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).
<ol>
	<li>Load tumor cells into 28 G, 1/2 inch insulin syringes.</li>
	<li>Inject U87MG cells (5 x 106) in 100 &#181;L of PBS into the left arm region.</li>
	<li>Anesthetize the mouse with 2% isoflurane in oxygen gas during cell injection.
	<ol>
		<li>Ensure that the mouse has been anesthetized by the loss of the pedal withdrawal reflex following pinching with forceps between the toes of the right hind foot. Do not leave an animal unattended until it has regained sufficient consciousness to maintain sternal recumbency.</li>
	</ol>
	</li>
</ol>
6. In Vivo Quantification of &#945;v&#946;3 Integrin Using PET
<ol>
	<li>Anesthetize the mice with 2% isoflurane in oxygen.
	<ol>
		<li>Ensure that the mouse has been anesthetized by the loss of the pedal withdrawal reflex following pinching with forceps between the toes of the right hind foot. Do not leave an animal unattended until it has regained sufficient consciousness to maintain sternal recumbency.</li>
	</ol>
	</li>
	<li>Place the head in the center of the PET gantry.</li>
	<li>Intravenously administer the 68Ga-RGD-peptide solution (7.4 MBq, 200 &#181;L) to the xenograft mouse model via the tail vein for 1 min.</li>
	<li>At the same time, perform a PET scan in list mode (dynamic scan) for 150 min. 
	NOTE: The raw PET data were reconstructed by a user-defined time frame (i.e., every 30 min). After the PET scan, a micro-computed tomography (CT) scan (50 kVp of X-ray, 0.16 mA) was conducted for attenuation correction.</li>
</ol>
7. Ex Vivo Biodistribution
<ol>
	<li>Inject 68Ga-RGD-peptide (0.37 MBq, 200 &#181;L) into the tail vein of the xenograft mouse model. Anesthetize the mouse with 2% isoflurane in oxygen gas during the injections. 
	Note: BALB/c nude mice, as described in section 5, were divided into four groups and sacrificed at different time points (n = 5 per group).</li>
	<li>Wake the mice immediately after the administration of 68Ga-RGD-peptide and sacrifice them at 30, 60, 90, and 120 min postinjection with carbon dioxide euthanasia. 
	NOTE: The tissues of interest were extracted. Selected targets were the blood, muscle, heart, lung, liver, spleen, stomach, intestine, kidneys, bone, and tumor.</li>
	<li>Weigh the tissue and measure the radioactivity with a &#947;-counter. 
	NOTE: Results were expressed as the percentage injected dose per gram of tissue (% ID/g).</li>
</ol>

<ol><li>Friedlander, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Definition+of+Two+Angiogenic+Pathways+by+Distinct+alpha+v+integrins%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Definition of Two Angiogenic Pathways by Distinct alpha v integrins.</a> Science. 270 (5241), 1500-1502 (1995).</li>
<li>Janssen, M. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tumor+Targeting+with+Radiolabeled+alpha+v+beta+3+Integrin+Binding+Peptides+in+a+Nude+Mouse+Model%5BTitle%5D)+AND+%22Cancer+Research%22%5BJournal%5D)">Tumor Targeting with Radiolabeled alpha v beta 3 Integrin Binding Peptides in a Nude Mouse Model.</a> Cancer Research. 62, 6146-6151 (2002).</li>
<li>Kok, R. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Preparation+and+functional+evaluation+of+RGD-modified+proteins+as+%CE%B1v%CE%B23+integrin+directed+therapeutics%5BTitle%5D)+AND+%22Bioconjugate+Chemistry%22%5BJournal%5D)">Preparation and functional evaluation of RGD-modified proteins as αvβ3 integrin directed therapeutics.</a> Bioconjugate Chemistry. 13 (1), 128-135 (2002).</li>
<li>Garanger, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((New+multifunctional+molecular+conjugate+vector+for+targeting%2C+imaging%2C+and+therapy+of+tumors%5BTitle%5D)+AND+%22Molecular+Therapy%22%5BJournal%5D)">New multifunctional molecular conjugate vector for targeting, imaging, and therapy of tumors.</a> Molecular Therapy. 12 (6), 1168-1175 (2005).</li>
<li>Dijkgraaf, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((PET+imaging+of+%CE%B1v%CE%B23+integrin+expression+in+tumours+with+68Ga-labelled+mono-%2C+di-+and+tetrameric+RGD+peptides%5BTitle%5D)+AND+%22European+Journal+of+Nuclear+Medicine+and+Molecular+Imaging%22%5BJournal%5D)">PET imaging of αvβ3 integrin expression in tumours with 68Ga-labelled mono-, di- and tetrameric RGD peptides.</a> European Journal of Nuclear Medicine and Molecular Imaging. 38 (1), 128-137 (2011).</li>
<li>Liu, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((68Ga-labeled+cyclic+RGD+dimers+with+Gly3and+PEG4linkers%3A+Promising+agents+for+tumor+integrin+%CE%B1v%CE%B23+PET+imaging%5BTitle%5D)+AND+%22European+Journal+of+Nuclear+Medicine+and+Molecular+Imaging%22%5BJournal%5D)">68Ga-labeled cyclic RGD dimers with Gly3and PEG4linkers: Promising agents for tumor integrin αvβ3 PET imaging.</a> European Journal of Nuclear Medicine and Molecular Imaging. 36 (6), 947-957 (2009).</li>
<li>Li, Z. B., Chen, K., Chen, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((68Ga-labeled+multimeric+RGD+peptides+for+microPET+imaging+of+integrin+%CE%B1v%CE%B23expression%5BTitle%5D)+AND+%22European+Journal+of+Nuclear+Medicine+and+Molecular+Imaging%22%5BJournal%5D)">68Ga-labeled multimeric RGD peptides for microPET imaging of integrin αvβ3expression.</a> European Journal of Nuclear Medicine and Molecular Imaging. 35 (6), 1100-1108 (2008).</li>
<li>Liu, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isomerism+and+solution+dynamics+of+90Y-labeled+DTPA-biomolecule+conjugates%5BTitle%5D)+AND+%22Bioconjugate+Chemistry%22%5BJournal%5D)">Isomerism and solution dynamics of 90Y-labeled DTPA-biomolecule conjugates.</a> Bioconjugate Chemistry. 12 (1), 84-91 (2001).</li>
<li>Haubner, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Glycosylated+RGD-containing+peptides%3A+tracer+for+tumor+targeting+and+angiogenesis+imaging+with+improved+biokinetics%5BTitle%5D)+AND+%22Journal+of+Nuclear+Medicine%22%5BJournal%5D)">Glycosylated RGD-containing peptides: tracer for tumor targeting and angiogenesis imaging with improved biokinetics.</a> Journal of Nuclear Medicine. 42 (2), 326-336 (2001).</li>
<li>Sivolapenko, G. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Imaging+of+metastatic+melanoma+utilising+a+technetium-99m+labelled+RGD-containing+synthetic+peptide%5BTitle%5D)+AND+%22Euroean+Journal+of+Nuclear+Medicine%22%5BJournal%5D)">Imaging of metastatic melanoma utilising a technetium-99m labelled RGD-containing synthetic peptide.</a> Euroean Journal of Nuclear Medicine. 25 (10), 1383-1389 (1998).</li>
<li>Haubner, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Noninvasive+Imaging+of+%CE%B1v%CE%B23+Integrin+Expression+Using+18+F-labeled+RGD-containing+Glycopeptide+and+Positron+Emission+Tomography%5BTitle%5D)+AND+%22Cancer+Research%22%5BJournal%5D)">Noninvasive Imaging of αvβ3 Integrin Expression Using 18 F-labeled RGD-containing Glycopeptide and Positron Emission Tomography.</a> Cancer Research. 61, 1781-1785 (2001).</li>
<li>Clarke, E. T., Martell, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stabilities+of+trivalent+metal+ion+complexes+of+the+tetraacetate+derivatives+of+12-%2C+13-+and+14-membered+tetraazamacrocycles%5BTitle%5D)+AND+%22Inorganica+Chimica+Acta%22%5BJournal%5D)">Stabilities of trivalent metal ion complexes of the tetraacetate derivatives of 12-, 13- and 14-membered tetraazamacrocycles.</a> Inorganica Chimica Acta. 190 (1), 37-46 (1991).</li>
<li>Clarke, E. T., Martell, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stabilities+of+the+Fe%28III%29%2C+Ga%28III%29+and+In%28III%29+chelates+of+N%2CN%E2%80%B2%2CN%E2%80%B3-triazacyclononanetriacetic+acid%5BTitle%5D)+AND+%22Inorganica+Chimica+Acta%22%5BJournal%5D)">Stabilities of the Fe(III), Ga(III) and In(III) chelates of N,N′,N″-triazacyclononanetriacetic acid.</a> Inorganica Chimica Acta. 181 (2), 273-280 (1991).</li>
<li>Shetty, D., Lee, Y. S., Jeong, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((68Ga-labeled+radiopharmaceuticals+for+positron+emission+tomography%5BTitle%5D)+AND+%22Nuclear+Medicine+Molecular+Imaging%22%5BJournal%5D)">68Ga-labeled radiopharmaceuticals for positron emission tomography.</a> Nuclear Medicine Molecular Imaging. 44 (4), 233-240 (2010).</li>
<li>Shin, U. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Synthesis+and+Preliminary+Evaluation+of+68Ga-NOTA-Biphenyl-c%28RGDyK%29+for+the+Quantification+of+Integrin+%CE%B1v%CE%B23%5BTitle%5D)+AND+%22Bulletin+of+the+Korean+Chemical+Society%22%5BJournal%5D)">Synthesis and Preliminary Evaluation of 68Ga-NOTA-Biphenyl-c(RGDyK) for the Quantification of Integrin αvβ3.</a> Bulletin of the Korean Chemical Society. 38 (12), 1415-1418 (2017).</li>
<li>Cai, W., Chen, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multimodality+Molecular+Imaging+of+Tumor+Angiogenesis%5BTitle%5D)+AND+%22Journal+of+Nuclear+Medicine%22%5BJournal%5D)">Multimodality Molecular Imaging of Tumor Angiogenesis.</a> Journal of Nuclear Medicine. 49, suppl2 113-128 (2008).</li>
</ol>

The authors have nothing to disclose.

In the present study, we introduced a protocol to prepare a radiopeptide targeting &#945;v&#946;3 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 (&#62; nM) and selectivity (&#62; 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).

Angiogenesis is a biological process that is characterized by the development of new blood vessels. Among many angiogenetic factors, &#945;v&#946;3 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 &#945;v&#946;3 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&#39;-di(2-hydroxybenzyl)ethylenediamine-N,N&#39;-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.

<table><tbody><tr><td>&lt;sup&gt;68&lt;/sup&gt;Ga/&lt;sup&gt;68&lt;/sup&gt;Ge generator</td><td>ITG Company</td><td>-</td><td>10 mCi&amp;nbsp;</td></tr><tr><td>Hydrogen chloride solution</td><td>Sigma-aldrich</td><td>84429</td><td></td></tr><tr><td>Sodium acetate</td><td>Sigma-aldrich</td><td>S2889</td><td></td></tr><tr><td>C18 reverse-phase cartridge</td><td>Waters</td><td>WAT020515</td><td></td></tr><tr><td>0.22-&amp;mu;m sterile filter</td><td>Milllipore</td><td>SLGV033RS</td><td></td></tr><tr><td>Radio-TLC scanner</td><td>Bioscan</td><td>AR2000</td><td></td></tr><tr><td>ITLC paper</td><td>Agilent</td><td>SGI001</td><td></td></tr><tr><td>Citric acid</td><td>Sigma-aldrich</td><td>251275</td><td></td></tr><tr><td>HPLC</td><td>Waters</td><td>-</td><td>Waters 1525 system containing binary pump, photo diode array (Waters 2998), radioactivity detector (Raytest, Gabi)</td></tr><tr><td>Acetonitrile</td><td>J.T. Baker</td><td>14-650-359</td><td></td></tr><tr><td>Trifluoroacetic acid</td><td>Sigma-aldrich</td><td>302031</td><td></td></tr><tr><td>Dulbecco&#039;s modified Eagle media&amp;nbsp;</td><td>Thermo fisher scientific</td><td>11965092</td><td></td></tr><tr><td>fetal bovine serum</td><td>Thermo fisher scientific</td><td>16000044</td><td></td></tr><tr><td>T175 flasks&amp;nbsp;</td><td>Corning</td><td>CLS431080</td><td></td></tr><tr><td>Trypsin-EDTA (0.25%)</td><td>Thermo fisher scientific</td><td>25200072</td><td></td></tr><tr><td>penicillin-streptomycin</td><td>Thermo fisher scientific</td><td>15240112</td><td></td></tr><tr><td>&amp;gamma;-counter</td><td>Perkin Elmer</td><td>-</td><td>1480 Wizard 3</td></tr><tr><td>Insunlin syringe</td><td>Becton Dickinson</td><td>326105</td><td></td></tr><tr><td>Synringe pump</td><td>Harvard Apparatus</td><td>70-4500</td><td></td></tr><tr><td>micro-PET/CT</td><td>Siemens Inveon</td><td>-</td><td></td></tr></tbody></table>

preparing a 68ga-labeled arginine glycine aspartate (rgd)-peptide for angiogenesis

The &#945;v&#946;3 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 &#945;v&#946;3 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 &#945;v&#946;3 integrin. Here, we introduce a protocol to prepare a 68Ga-RGD-peptide and the evaluation of its biological efficacy.

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 (&#62; 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).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58218/58218fig1v2.jpg" /> 
Figure 1: Flow diagram of the experimental procedures. This figure shows a schematic overview of the development of radiopharmaceutical. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/58218/58218fig1v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58218/58218fig2.jpg" /> 
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. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/58218/58218fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58218/58218fig3.jpg" /> 
Figure 3: Structure of 68Ga-RGD-peptide and its radiochemical purity. The ITLC of 68Ga-RGD-peptide showed high radiochemical purity. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/58218/58218fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58218/58218fig4.jpg" /> 
Figure 4:&#160;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 &#177; the standard deviation from five mice at each time point. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/58218/58218fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Preparing a 68Ga-labeled Arginine Glycine Aspartate RGD-peptide for Angiogenesis at JoVE.com

Preparing a 68Ga-labeled Arginine Glycine Aspartate RGD-peptide for Angiogenesis

The &#945;v&#946;3 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.

Preparing a 68Ga-labeled Arginine Glycine Aspartate (RGD)-peptide for Angiogenesis

The &#945;v&#946;3 integrin is a heterodimeric adhesion molecule involved in tumor cell migration and angiogenesis. The integrin is overexpressed in ...

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6.9K Views.  Korea Institute of Radiological and Medical Sciences. 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.

Video: Preparing a 68Ga-labeled Arginine Glycine Aspartate RGD-peptide for Angiogenesis

Investigations on the Ga(III) Complex of EOB-DTPA and Its 68Ga Radiolabeled Analogue

We demonstrate a method for the isolation of EOB-DTPA (3,6,9-triaza-3,6,9-tris(carboxymethyl)-4-(ethoxybenzyl)-undecanedioic acid) from its Gd(III) complex and protocols for the preparation of its novel non-radioactive, i.e., natural Ga(III) as well as radioactive 68Ga complex. The ligand as well as the Ga(III) complex were characterized by nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry and elemental analysis. 68Ga was obtained by a standard elution method from a 68Ge/68Ga generator. Experiments to evaluate the 68Ga-labeling efficiency of EOB-DTPA at pH 3.8&#8211;4.0 were performed. Established analysis techniques radio TLC (thin layer chromatography) and radio HPLC (high performance liquid chromatography) were used to determine the radiochemical purity of the tracer. As a first investigation of the 68Ga tracers&#39; lipophilicity the n-octanol/water distribution coefficient of 68Ga species present in a pH 7.4 solution was determined by an extraction method. In vitro stability measurements of the tracer in various media at physiological pH were performed, revealing different rates of decomposition.

Investigations on the GaIII Complex of EOB-DTPA and Its 68Ga Radiolabeled Analogue

A procedure for the isolation of EOB-DTPA and subsequent complexation with natural Ga(III) and 68Ga is presented herein, as well as a thorough analysis of all compounds and investigations on labeling efficiency, in vitro stability and the n-octanol/water distribution coefficient of the radiolabeled complex.

We demonstrate a method for the isolation of EOB-DTPA (3,6,9-triaza-3,6,9-tris(carboxymethyl)-4-(ethoxybenzyl)-undecanedioic acid) from its Gd(III) ...

Chemistry

MR Molecular Imaging of Prostate Cancer with a Small Molecular CLT1 Peptide Targeted Contrast Agent

Tumor extracellular matrix has abundance of cancer related proteins that can be used as biomarkers for cancer molecular imaging. In this work, we demonstrated effective MR cancer molecular imaging with a small molecular peptide targeted Gd-DOTA monoamide complex as a targeted MRI contrast agent specific to clotted plasma proteins in tumor stroma. We performed the experiment of evaluating the effectiveness of the agent for non-invasive detection of prostate tumor with MRI in a mouse orthotopic PC-3 prostate cancer model. The targeted contrast agent was effective to produce significant tumor contrast enhancement at a low dose of 0.03 mmol Gd/kg. The peptide targeted MRI contrast agent is promising for MR molecular imaging of prostate tumor.

To demonstrate MR cancer molecular imaging with a small peptide targeted MRI contrast agent specific to clotted plasma proteins in tumor stroma in a mouse prostate cancer model.

Tumor extracellular  matrix has abundance of cancer related proteins that can be used as biomarkers  for cancer molecular imaging. In this work,  we ...

Development of a 68Gallium-Labeled D-Peptide PET Tracer for Imaging Programmed Death-Ligand 1 Expression

The development of immune checkpoint blockade therapy based on programmed cell death-protein 1 (PD-1)/programmed death-ligand 1 (PD-L1) has revolutionized cancer therapies in recent years. However, only a fraction of patients responds to PD-1/PD-L1 inhibitors, owing to the heterogeneous expression of PD-L1 in tumor cells. This heterogeneity presents a challenge in the precise detection of tumor cells by the commonly used immunohistochemistry (IHC) approach. This situation calls for better methods to stratify patients who will benefit from immune checkpoint blockade therapy, to improve treatment efficacy. Positron emission tomography (PET) enables real-time visualization of the whole-body PD-L1 expression in a noninvasive way. Therefore, there is a need for the development of radiolabeled tracers to detect PD-L1 distribution in tumors through PET imaging.
Compared to their L-counterparts, dextrorotary (D)-peptides have properties such as proteolytic resistance and remarkably prolonged metabolic half-lives. This study designed a new method to detect PD-L1 expression based on PET imaging of 68Ga-labeled PD-L1-targeted D-peptide, a D-dodecapeptide antagonist (DPA), in tumor-bearing mice. The results showed that the [68Ga]DPA can specifically bind to PD-L1-overexpressing tumors in vivo, and showed favorable stability as well as excellent imaging ability, suggesting that [68Ga]DPA-PET is a promising approach for the assessment of PD-L1 status in tumors.

Development of a 68Gallium-Labeled D-Peptide PET Tracer for Imaging Programmed Death-Ligand 1 Expression

This study developed a noninvasive and real-time method to evaluate the distribution of programmed death-ligand 1 in the whole body, based on positron emission tomographic imaging of [68Ga] D-dodecapeptide antagonist. This technique has advantages over conventional immunohistochemistry and improves the efficiency of identifying appropriate patients who will benefit from immune checkpoint blockade therapy.

The development of immune checkpoint blockade therapy based on programmed cell death-protein 1 (PD-1)/programmed death-ligand 1 (PD-L1) has revolutionized ...

Cancer Research

The Synthesis of RGD-functionalized Hydrogels as a Tool for Therapeutic Applications

The use of polymers as biomaterials has provided significant advantages in therapeutic applications. In particular, the possibility to modify and functionalize polymer chains with compounds that are able to improve biocompatibility, mechanical properties, or cell viability allows the design of novel materials to meet new challenges in the biomedical field. With the polymer functionalization strategies, click chemistry is a powerful tool to improve cell-compatibility and drug delivery properties of polymeric devices. Similarly, the fundamental need of biomedicine to use sterile tools to avoid potential adverse-side effects, such as toxicity or contamination of the biological environment, gives rise to increasing interest in the microwave-assisted strategy.
The combination of click chemistry and the microwave-assisted method is suitable to produce biocompatible hydrogels with desired functionalities and improved performances in biomedical applications. This work aims to synthesize RGD-functionalized hydrogels. RGD (arginylglycylaspartic acid) is a tripeptide that can mimic cell adhesion proteins and bind to cell-surface receptors, creating a hospitable microenvironment for cells within the 3D polymeric network of the hydrogels. RGD functionalization occurs through Huisgen 1,3-dipolar cycloaddition. Some PAA carboxyl groups are modified with an alkyne moiety, whereas RGD is functionalized with azido acid as the terminal residue of the peptide sequence. Finally, both products are used in a copper catalyzed click reaction to permanently link the peptide to PAA. This modified polymer is used with carbomer, agarose and polyethylene glycol (PEG) to synthesize a hydrogel matrix. The 3D structure is formed due to an esterification reaction involving carboxyl groups from PAA and carbomer and hydroxyl groups from agarose and PEG through microwave-assisted polycondensation. The efficiency of the gelation mechanism ensures a high degree of RGD functionalization. In addition, the procedure to load therapeutic compounds or biological tools within this functionalized network is very simple and reproducible.

We present a protocol for the synthesis of RGD-functionalized hydrogels as devices for cell and drug delivery. The procedure involves copper catalyzed alkyne-azide cycloaddition (CuAAC) between alkyne-modified polyacrylic acid (PAA) and a RGD-azide derivative. The hydrogels are formed using microwave-assisted polycondensation and their physicochemical properties are investigated.

The use of polymers as biomaterials has provided significant advantages in therapeutic applications. In particular, the possibility to modify and ...

Bioengineering

Radiosynthesis and PET/CT Imaging of 68Ga-Labelled Nanobody in Xenograft Models

Radiosynthesis, Quality Control, and Small Animal Positron Emission Tomography Imaging of 68Ga-Labelled Nano Molecules

Small animal Positron Emission Tomography (PET) and Single Photon Emission Computed Tomography (SPECT) imaging techniques are crucial in preclinical cancer research, necessitating meticulous attention to radiotracer synthesis, quality assurance, and in vivo injection protocols. This study presents a comprehensive workflow tailored to enhance the robustness and reproducibility of small animal PET experiments. The synthesis process in the radiochemistry laboratory using 68Ga is detailed, highlighting stringent quality control and assurance protocols for each radiotracer production. Parameters such as concentration, molar activity, pH, and purity are rigorously monitored, aligning with standards applicable to human studies. This methodology introduces streamlined syringe preparation and a custom-designed 30G cannula for precise intravenous injections into mice. Monitoring of animal health during scanning, including temperature and heart rate, ensures their well-being throughout the procedure. Dosages for PET and SPECT scans are predetermined to balance data acquisition with minimizing radiation exposure to animals and researchers. Similarly, CT scans employ pre-programmed settings to limit radiation exposure, especially pertinent in long-term studies assessing treatment effects. By optimizing these steps, the workflow aims to standardize procedures, reduce variability, and enhance the quality of small animal PET/SPECT/CT imaging. This resource provides valuable insights for researchers seeking to improve the accuracy and reliability of preclinical investigations in molecular imaging, ultimately advancing the field.

This paper describes the radiosynthesis, formulation, quality control of a new radiolabeled probe (i.e., 68Ga-labeled nanobody NM-02), and its use for small animal PET/CT imaging in a xenograft model.

Small animal Positron Emission Tomography (PET) and Single Photon Emission Computed Tomography (SPECT) imaging techniques are crucial in preclinical ...

Automated Preparation of [68Ga]Ga-3BP-3940 on a Synthesis Module for PET Imaging of the Tumor Microenvironment

A fast, efficient method has been developed on the GAIA synthesis module for automated gallium-68 radiolabeling of 3BP-3940, a molecular imaging probe targeting the fibroblast activation protein for positron emission tomography imaging of the tumor microenvironment. The reaction conditions involved acetate buffer (final concentration: 0.1 M), methionine as an anti-radiolysis agent (final concentration: 5.4 mg/mL), and 30 &#181;g of 3BP-3940, with heating for 8 min at 98 &#176;C. A final purification step on a C18 cartridge was necessary to obtain a radiolabeled product of high purity. In contrast, the generator-produced 68Ga was used directly without a concentration step on a cation exchange cartridge. The production of three validation batches confirmed the method&#39;s reliability, allowing the synthesis of [68Ga]Ga-3BP-3940 in 22.3 &#177; 0.6 min with high radiochemical purity (RCP), as determined by both radio-HPLC (99.1% &#177; 0.1%) and radio-TLC (99.2% &#177; 0.1%). The average radiochemical yield, based on RCP values measured by radio-HPLC, was 74.4% &#177; 3.3%. The stability of the radiolabeled product was demonstrated for up to 4 h after preparation. This protocol provides a reliable, rapid, and efficient methodology for the preparation of [68Ga]Ga-3BP-3940, which can easily be transposed to a clinical setting.

This research describes the automated process for [68Ga]Ga-3BP-3940 production with the GAIA V2 synthesizer, for PET imaging of fibroblast activation protein. The results of quality control tests performed on three test batches are also presented.

A fast, efficient method has been developed on the GAIA synthesis module for automated gallium-68 radiolabeling of 3BP-3940, a molecular imaging probe ...

Rat Mesentery Angiogenesis Assay

The adult rat mesentery window angiogenesis assay is biologically appropriate and is exceptionally well suited to the 
study of sprouting angiogenesis in vivo [see review papers], which is the dominating form of angiogenesis in human tumors and non-tumor 
tissues, as discussed in invited review papers1,2. Angiogenesis induced in the membranous mesenteric parts by intraperitoneal (i.p.) injection of a pro-angiogenic factor can be modulated
by subcutaneous (s.c.), intravenous (i.v.) or oral (p.o.) treatment with modifying agents of choice. Each membranous part of the mesentery is 
translucent and framed by fatty tissue, giving it a window-like appearance.
The assay has the following advantageous features: (i) the test tissue is natively vascularized, albeit 
sparsely, and since it is extremely thin, the microvessel network is virtually two-dimensional, which allows the entire network 
to be assessed microscopically in situ; (ii) in adult rats the test tissue lacks significant physiologic angiogenesis, which characterizes
 most normal adult mammalian tissues; the degree of native vascularization is, however, correlated with age, as discussed in1; 
(iii) the negligible level of trauma-induced angiogenesis ensures high sensitivity; (iv) the 
assay replicates the clinical situation, as the angiogenesis-modulating test drugs are administered systemically and the responses 
observed reflect the net effect of all the metabolic, cellular, and molecular alterations induced by the treatment; (v) the assay 
allows assessments of objective, quantitative, unbiased variables of microvascular spatial extension, density, and network pattern 
formation, as well as of capillary sprouting, thereby enabling robust statistical analyses of the dose-effect and molecular 
structure-activity relationships; and (vi) the assay reveals with high sensitivity the toxic or harmful effects of treatments in 
terms of decreased rate of physiologic body-weight gain, as adult rats grow robustly.
Mast-cell-mediated angiogenesis was first demonstrated using this assay3,4. The model demonstrates a high level of 
discrimination regarding dosage-effect relationships and the measured effects of systemically administered chemically or functionally closely related drugs and proteins, 
including: (i) low-dosage, metronomically administered standard chemotherapeutics that yield diverse, drug-specific effects (i.e., 
angiogenesis-suppressive, neutral or angiogenesis-stimulating activities5); (ii) natural iron-unsaturated human lactoferrin, which stimulates 
VEGF-A-mediated angiogenesis6, and natural iron-unsaturated bovine lactoferrin, which inhibits VEGF-A-mediated angiogenesis7; and (iii) 
low-molecular-weight heparin fractions produced by various means8,9. Moreover, the assay is highly suited to studies of the combined 
effects on angiogenesis of agents that are administered systemically in a concurrent or sequential fashion.
The idea of making this video originated from the late Dr. Judah Folkman when he visited our laboratory and witnessed the methodology being demonstrated.
Review papers (invited) discussing and appraising the assay
Norrby, K. In vivo models of angiogenesis. J. Cell. Mol. Med. 10, 588-612 (2006).
Norrby, K. Drug testing with angiogenesis models. Expert Opin. Drug. Discov. 3, 533-549 (2008).

Normal adult vascularized mammalian tissue that lacks physiologic angiogenesis and that has not been exposed to surgical intervention is used to study: (i) the initiation and development of angiogenesis following intraperitoneal administration of test agents; and (ii) modification of angiogenesis following systemic administration of selected test agents.

The adult rat mesentery window angiogenesis assay is biologically appropriate and is exceptionally well suited to the 
study of sprouting angiogenesis in ...

Biology

Preparation and Characterization of Targeted Microbubbles

Targeting of microbubbles (ultrasound contrast agents for molecular imaging) has been researched for more than two decades. However, methods of microbubble preparation and targeting ligand attachment are cumbersome, complicated, and lengthy. Therefore, there is a need to simplify the targeted microbubble preparation procedure to bring it closer to clinical translation. The purpose of this publication is to provide a detailed description and explanation of the steps necessary for targeted microbubble preparation, functional characterization and testing. A sequence of the optimized and simplified procedures is presented for two systems: a biotin-streptavidin targeting pair model, and a cyclic RGD peptide targeting the recombinant &#945;v&#946;3 protein, which is overexpressed on the endothelial lining of the tumor neovasculature.
Here, we show the following: covalent coupling of the targeting ligand to a lipid anchor, assessment of the reagent quality, and tests that confirm the successful completion of the reaction; preparation of the aqueous precursor medium containing microbubble shell components, followed by microbubble preparation via amalgamation; assessment of the efficacy of lipid transfer onto the microbubble stabilizer shell; adjustment of microbubble size distribution by flotation at normal gravity to remove larger microbubbles that might be detrimental for in vivo use; assessment of microbubble size distribution by electrozone sensing; evaluation of targeted binding of the microbubbles to receptor-coated surface in a static binding assay test (in an inverted dish); and evaluation of targeted binding of the microbubbles to receptor-coated surface in a parallel plate flow chamber test.

The goal of this protocol is to prepare, purify and characterize gas-filled microbubbles (targeted contrast agents for ultrasound molecular imaging). Two targeting systems are described: biotinylated bubbles adherent to streptavidin, and cyclic RGD peptide microbubbles that bind to &#945;v&#946;3, a known tumor neovasculature biomarker.

Targeting of microbubbles (ultrasound contrast agents for molecular imaging) has been researched for more than two decades. However, methods of ...

Screening Peptides that Activate MRGPRX2 using Engineered HEK Cells

Identifying ligands specific to therapeutically significant cell receptors is crucial for many applications, including the design and development of new therapeutics. Mas related G-protein receptor-X2 (MRGPRX2) is an important receptor that regulates mast cell activation and, thus, directs the general immune response. Numerous ligands for MRGPRX2 have been identified and include endogenous peptides like PAMPs, defensins, LL-37 and other protein fragments (i.e., degraded albumin). Further identification of MRGPRX2 specific ligands requires the screening of a large number of peptides (i.e., peptide library); however, mast cells are difficult and expensive to maintain in vitro and, therefore, not economical to use for screening large numbers of molecules. The present paper demonstrates a method to design, develop, and screen a library of small peptide molecules using MRGPRX2 expressing HEK cells. This cell line is relatively easy and inexpensive to maintain and can be used for in vitro high-throughput analysis. A calcium sensitive Fura-2 fluorescent dye to mark intracellular calcium flux upon activation was used to monitor the activation. The ratio of fluorescence intensity of Fura-2 at 510 nm against excitation wavelengths of 340 and 380 nm was used to calculate calcium concentration. The peptide library used to verify this system was based on the endogenous proadrenomedullin N-terminal 12 (PAMP-12) secretagogue, which is known to bind MRGPRX2 with high specificity and affinity. Subsequent peptides were generated through amino acid truncation and alanine scanning techniques applied to PAMP-12. The method described here is simple and inexpensive yet robust for screening a large library of compounds to identify binding domains and other important parameters that play an important role in receptor activation.

Techniques for generating a library of short peptides that can activate mast cells via the MRGPRX2 receptor are described. Associated techniques are easy, inexpensive, and can be extended to other cell receptors.

Identifying ligands specific to therapeutically significant cell receptors is crucial for many applications, including the design and development of new ...

Immunology and Infection

Synthesis of 68Ga Core-doped Iron Oxide Nanoparticles for Dual Positron Emission Tomography /(T1)Magnetic Resonance Imaging

Here, we describe a microwave synthesis to obtain iron oxide nanoparticles core-doped with 68Ga. Microwave technology enables fast and reproducible synthetic procedures. In this case, starting from FeCl3 and citrate trisodium salt, iron oxide nanoparticles coated with citric acid are obtained in 10 min in the microwave. These nanoparticles present a small core size of 4.2 &#177; 1.1 nm and a hydrodynamic size of 7.5 &#177; 2.1 nm. Moreover, they have a high longitudinal relaxivity (r1) value of 11.9 mM-1&#183;s-1 and a modest transversal relaxivity value (r2) of 22.9 mM-1&#183;s-1, which results in a low r2/r1 ratio of 1.9. These values enable positive contrast generation in magnetic resonance imaging (MRI) instead of negative contrast, commonly used with iron oxide nanoparticles. In addition, if a 68GaCl3 elution from a 68Ge/68Ga generator is added to the starting materials, a nano-radiotracer doped with 68Ga is obtained. The product is obtained with a high radiolabeling yield (&#62; 90%), regardless of the initial activity used. Furthermore, a single purification step renders the nano-radiomaterial ready to be used in vivo.

Synthesis of 68Ga Core-doped Iron Oxide Nanoparticles for Dual Positron Emission Tomography /T1Magnetic Resonance Imaging

Here, we present a protocol to obtain 68Ga core-doped iron oxide nanoparticles via fast microwave-driven synthesis. The methodology renders PET/(T1)MRI nanoparticles with radiolabeling efficiencies higher than 90% and radiochemical purity of 99% in a 20-min synthesis.

Here, we describe a microwave synthesis to obtain iron oxide nanoparticles core-doped with 68Ga. Microwave technology enables fast and reproducible ...

Preparing a ⁶⁸Ga-labeled Arginine Glycine Aspartate (RGD)-peptide for Angiogenesis

Summary

Explore More Videos

Investigations on the Ga(III) Complex of EOB-DTPA and Its ⁶⁸Ga Radiolabeled Analogue

MR Molecular Imaging of Prostate Cancer with a Small Molecular CLT1 Peptide Targeted Contrast Agent

Development of a ⁶⁸Gallium-Labeled D-Peptide PET Tracer for Imaging Programmed Death-Ligand 1 Expression

The Synthesis of RGD-functionalized Hydrogels as a Tool for Therapeutic Applications

Radiosynthesis, Quality Control, and Small Animal Positron Emission Tomography Imaging of ⁶⁸Ga-Labelled Nano Molecules

Automated Preparation of [⁶⁸Ga]Ga-3BP-3940 on a Synthesis Module for PET Imaging of the Tumor Microenvironment

Rat Mesentery Angiogenesis Assay

Preparation and Characterization of Targeted Microbubbles

Screening Peptides that Activate MRGPRX2 using Engineered HEK Cells

Synthesis of ⁶⁸Ga Core-doped Iron Oxide Nanoparticles for Dual Positron Emission Tomography /(T₁)Magnetic Resonance Imaging