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Radiolabeled cyclic RGD peptides as integrin alpha(v)beta(3)-targeted radiotracers: maximizing binding affinity via bivalency.

ABSTRACT: Integrin alpha(v)beta(3) plays a significant role in tumor angiogenesis and is a receptor for the extracellular matrix proteins with the exposed arginine-glycine-aspartic (RGD) tripeptide sequence. These include vitronectin, fibronectin, fibrinogen, lamin, collagen, Von Willibrand's factor, osteoponin, and adenovirus particles. Integrin alpha(v)beta(3) is expressed at low levels on epithelial cells and mature endothelial cells, but it is overexpressed on the activated endothelial cells of tumor neovasculature and some tumor cells. The restricted expression of integrin alpha(v)beta(3) during tumor growth, invasion, and metastasis presents an interesting molecular target for both early detection and treatment of rapidly growing solid tumors. Over the past decade, many radiolabeled linear and cyclic RGD peptide antagonists have been evaluated as integrin alpha(v)beta(3)-targeted radiotracers. Significant progress has been made on their use for imaging integrin alpha(v)beta(3)-positive tumors by SPECT or PET. Among the radiotracers evaluated in preclinical tumor-bearing models, [18F]Galacto-RGD (2-[18F]fluoropropanamide c(RGDfK(SAA); SAA = 7-amino-L-glyero-L-galacto-2,6-anhydro-7-deoxyheptanamide) and [18F]-AH111585 are currently under clinical investigation for visualization of integrin alpha(v)beta(3) expression in cancer patients. However, their low tumor uptake, high cost, and lack of preparative modules for routine radiosynthesis will limit their continued clinical application. Thus, there is a continuing need for more efficient integrin alpha(v)beta(3)-targeted radiotracers that are readily prepared from a kit formulation without further postlabeling purification. This article will focus on different approaches to maximize the targeting capability of cyclic RGD peptides and to improve the radiotracer excretion kinetics from noncancerous organs. Improvement of tumor uptake and tumor-to-background ratios is important for early detection of integrin alpha(v)beta(3)-positive tumors and/or noninvasive monitoring of therapeutic efficacy of antiangiogenic therapy.

SUBMITTER: Liu S

PROVIDER: S-EPMC2795072 | biostudies-literature | 2009 Dec

REPOSITORIES: biostudies-literature

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Radiolabeled cyclic RGD peptides as integrin alpha(v)beta(3)-targeted radiotracers: maximizing binding affinity via bivalency.

Liu Shuang S

Bioconjugate chemistry 20091201 12

Integrin alpha(v)beta(3) plays a significant role in tumor angiogenesis and is a receptor for the extracellular matrix proteins with the exposed arginine-glycine-aspartic (RGD) tripeptide sequence. These include vitronectin, fibronectin, fibrinogen, lamin, collagen, Von Willibrand's factor, osteoponin, and adenovirus particles. Integrin alpha(v)beta(3) is expressed at low levels on epithelial cells and mature endothelial cells, but it is overexpressed on the activated endothelial cells of tumor ...[more]

PMID: 19719118

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Project description:This study sought to evaluate FITC-conjugated cyclic RGD peptides (FITC-RGD2, FITC-3P-RGD2, and FITC-Galacto-RGD2) as fluorescent probes for in vitro assays of integrin αvβ3/αvβ5 expression in tumor tissues. FITC-RGD2, FITC-3P-RGD2, and FITC-Galacto-RGD2 were prepared, and their integrin αvβ3/αvβ5 binding affinity was determined using the displacement assay against (125)I-echistatin bound to U87MG glioma cells. IC50 values of FITC-Galacto-RGD2, FITC-3P-RGD2, and FITC-RGD2 were calculated to be 28 ± 8, 32 ± 7, and 89 ± 17 nM, respectively. The integrin αvβ3/αvβ5 binding affinity followed a general trend: FITC-Galacto-RGD2 ∼ FITC-3P-RGD2 > FITC-RGD2. The xenografted tumor-bearing models were established by subcutaneous injection of 5 × 10(6) tumor cells into shoulder flank (U87MG, A549, HT29, and PC-3) or mammary fat pad (MDA-MB-435) of each athymic nude mouse. Three to six weeks after inoculation, the tumor size was 0.1-0.3 g. Tumors were harvested for integrin αvβ3/αvβ5 staining, as well as hematoxylin and eosin (H&E) staining. Six human carcinoma tissues (colon cancer, pancreatic cancer, lung adenocarcinoma, squamous cell lung cancer, gastric cancer, and esophageal cancer) were obtained from recently diagnosed cancer patients. Human carcinoma slides were deparaffinized in xylene, rehydrated with ethanol, and then used for integrin αvβ3/αvβ5 staining, as well as H&E staining. It was found that the tumor staining procedures with FITC-conjugated cyclic RGD peptides were much simpler than those with the fluorescence-labeled integrin αvβ3 antibodies. Since FITC-RGD2, FITC-3P-RGD2, and FITC-Galacto-RGD2 were able to co-localize with the fluorescence-labeled integrin β3 antibody, their tumor localization and tumor cell binding are integrin αvβ3-specific. Quantification of the fluorescent intensity in five xenografted tumors (U87MG, MDA-MB-435, A549, HT29, and PC-3) and six human carcinoma tissues revealed an excellent linear relationship between the relative integrin αvβ3/αvβ5 expression levels determined with FITC-Galacto-RGD2 and those obtained with the fluorescence-labeled anti-human integrin β3 antibody. There was also an excellent linear relationship between the tumor uptake (%ID/g) of (99m)Tc-3P-RGD2 (an integrin αvβ3/αvβ5-targeted radiotracer) and the relative integrin αvβ3/αvβ5 expression levels from the quantification of fluorescent intensity in the tumor tissues stained with FITC-Galacto-RGD2. These results suggest that FITC-conjugated cyclic RGD peptides might be useful to correlate the in vitro findings with the in vivo imaging data from an integrin αvβ3/αvβ5-targeted radiotracer. The results from this study clearly showed that the FITC-conjugated cyclic RGD peptides (particularly FITC-3P-RGD2 and FITC-Galacto-RGD2) are useful fluorescent probes for assaying relative integrin αvβ3/αvβ5 expression levels in tumor tissues.

Project description:Molecular probes targeting bacteria provide opportunities to target bacterial infections in vivo for both imaging and therapy. In the current study, we report the development of positron emission tomography (PET) probes for imaging of live bacterial infection based on the small molecules HLys-DOTA, a polycationic peptide synthesized as the D-isomer (RYWVAWRNRG) conjugated to 1, 4, 7, 10-tetraazacyclododecane-N',N″,N‴,N-tetraacetic acid (DOTA) and AB1-HLys-DOTA, which includes an unnatural amino acid AB1 that preferentially binds to bacteria membrane lipids with amine groups via formation of iminoboronates. HLys-DOTA and AB1-HLys-DOTA peptides were radiolabeled with 64Cu and investigated as PET imaging agents to track bacterial infection in vitro and in intramuscularly infected (IM) mice models. Cell uptake studies at 37°C in Staphylococcus aureus (SA) show higher uptake of 64Cu-AB1-HLys-DOTA; 98.47 ± 3.54% vs 64Cu-HLys-DOTA; 39.12 ± 3.27% at 24 h. Standard uptake values (SUV) analysis of the PET images resulted in mean SUV of 0.70 ± 0.08, 0.49 ± 0.04, and 0.31 ± 0.01 for 64Cu-AB1-HLys-DOTA and 0.17 ± 0.06, 0.16 ± 0.02, and 0.13 ± 0.01 for 64Cu-HLys-DOTA at 1, 4, and 24 h post injection, respectively, in the infected muscles. Similarly, in the biodistribution studies, dose uptake in the infected muscles was 4 times higher in the targeted 64Cu-AB1-HLys-DOTA group than in the 64Cu-HLys-DOTA group and 2-3 times higher than in the PBS control group at 1, 4, and 24 h post injection. 64Cu-AB1-HLys-DOTA was able to distinguish between SA-infected muscle and Pseudomonas aeruginosa (PA) infected muscle with lower mean SUV of 0.28 ± 0.10 at 1 h post injection. This illustrates the utility of the AB1 covalently targeting group in synergy with the HLys peptide, which noncovalently binds to bacterial membranes. These results suggest that 64Cu-labeled AB1-HLys-DOTA peptide could be used as an imaging probe for detection of bacterial infection in vivo with specificity for Gram-positive bacteria.

Project description:PurposeWe previously showed that intravitreal injection of the sFLT morpholino-oligomer (FLT-MO) suppresses laser-induced choroidal neovascularization (CNV) in mice by decreasing the membrane bound form of Flt-1 while increasing the soluble form of Flt-1 via alternative splicing shift. In this study, we examined whether cyclic RGD peptide (cRGD) can promote morpholino-oligomer accumulation in CNV following tail vein injection, and whether systemic cRGD conjugated FLT-MO (cRGD-FLT-MO) suppresses CNV growth.MethodscRGD conjugated fluorescent morpholino-oligomer (cRGD-F-MO) was injected via tail vein into mice with previous retinal laser photocoagulation and examined for cRGD-F-MO accumulation in CNV. To examine whether cRGD-FLT-MO suppresses CNV growth, mice were tail-vein injected with cRGD-FLT-MO, cRGD conjugated standard morpholino-oligomer (cRGD-STD-MO), or Dulbecco's Phosphate-Buffered Saline (DPBS) 1 and 4 days postlaser photocoagulation. Seven days postlaser photocoagulation, eyes were harvested and laser CNV was stained with isolectin GS-IB4, allowing quantification of CNV size by confocal microscopy.ResultscRGD-F-MO accumulation in CNV commenced immediately after tail vein injection and could be observed even 1 day after injection. cRGD-FLT-MO tail vein injection significantly suppressed CNV size (2.7 × 105 ± 0.3 × 105 ?m3, P < 0.05 by Student's t-test) compared with controls (DPBS: 5.1 × 105 ± 0.6 × 105 ?m3 and cRGD-STD-MO: 5.5 × 105 ± 0.8 × 105 ?m3).ConclusionscRGD peptide facilitates morpholino-oligomer accumulation in CNV following systemic delivery. cRGD-FLT-MO suppressed CNV growth after tail-vein injection, demonstrating the potential utility of cRGD peptide for morpholino-oligomer delivery to CNV.Translational relevanceCurrent therapy for neovascular age-related macular degeneration involves intravitreal injection of anti-vascular endothelial growth factor drugs. Our results indicate that CNV can be treated systemically, thus eliminating risks and hazards associated with intravitreal injection.

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Radiolabeled cyclic RGD peptides as integrin alpha(v)beta(3)-targeted radiotracers: maximizing binding affinity via bivalency.

Publications

Radiolabeled cyclic RGD peptides as integrin alpha(v)beta(3)-targeted radiotracers: maximizing binding affinity via bivalency.

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