RGD Probe Research Articles

Improved synthesis of DA364, an NIR fluorescence RGD probe targeting αvβ3 integrin.

Optical imaging (OI) is gaining increasing attention in medicine as a non-invasive diagnostic imaging technology and as a useful tool for image-guided surgery. OI exploits the light emitted in the near-infrared region by fluorescent molecules able to penetrate living tissues. Cyanines are an important class of fluorescent molecules and by their conjugation to peptides it is possible to achieve optical imaging of tumours by selective targeting. We report here the improvements obtained in the synthesis of DA364, a small fluorescent probe (1.5 kDa) prepared by conjugation of pentamethine cyanine Cy5.5 to an RGD peptidomimetic, which can target tumour cells overexpressing integrin αvβ3 receptors.

Aryl isocyanide derivative for one-pot synthesis of purification-free 99mTc-labeled hexavalent targeting probe

Introduction99mTc-labeled hexavalent probes can be readily synthesized by the coordination of six equivalent isocyanide ligands towards TcI, and alkyl isocyanide ligands have been extensively used for preparing such probes. However, high ligand concentration (>1 mM) is generally required due to their insufficient coordination ability to TcI. Methods and resultsIn this study, we revealed that aryl isocyanide ligands, which have greater π-accepting ability compared with alkyl ones, provided 99mTc-labeled hexavalent probes in high radiochemical yields (>95%) even at low ligand concentration (50 μM). We applied this finding to the synthesis of a 99mTc-labeled hexavalent RGD probe, targeting integrin αvβ3. This 99mTc-labeled probe was prepared in a 5 min reaction at ligand concentration of 50 μM, and exhibited high tumor localization in vivo without post-labeling purification. ConclusionThe present findings indicate that aryl isocyanide ligands would be a useful precursor to a variety of 99mTc-labeled hexavalent targeting probes for molecular imaging of saturable systems. Advances in knowledgeAryl isocyanide is a better precursor than alkyl isocyanide for preparing 99mTc-labeled hexavalent targeting probe. Implication for patient careThis work provides a straightforward method to prepare molecular imaging agents of high target uptake, which would facilitate nuclear medicine imaging in clinical settings.

The synthesis of a 99mTc-labeled tetravalent targeting probe upon isonitrile coordination to 99mTcI for enhanced target uptake in saturable systems.

The presence of excess unlabeled ligands in the injectate hinders the target uptake of 99mTc-labeled targeting vectors. To address the issue, we previously developed a chemical design which provides a 99mTc-labeled trivalent RGD probe upon CN-βAla-Gly-Gly-c(RGDfK) (Lβ) coordination to [99mTc][Tc(CO)3]+ core at pH 6.0. In this study, we extended our coordination mediated synthesis of the trivalent RGD probe to that of a tetravalent one. Our initial attempts reacting Lβ with [99mTc][Tc(CO)3]+ core at pH 8.0 failed to provide [99mTc][Tc(CO)2(Lβ)4]+ due to the formation of multiple side products. A γ-aminobutylic acid (GABA) based isonitrile ligand CN-GABA-Gly-Gly-c(RGDfK) (LG), on the other hand, avoided the side reaction and selectively provided [99mTc][Tc(CO)2(LG)4]+ (99mTc-[LG]4) at pH 8.0. 99mTc-[LG]4 exhibited higher binding affinity to integrin αvβ3 than its unlabeled ligand, and visualized U87MG tumor without tedious post-labeling purification. These results indicate that the metal coordination-mediated syntheses of 99mTc-labeled multivalent probes have been successfully applied to a tetravalent one, which would allow a wider range of choices for designing novel 99mTc-labeled multivalent probes of high in vivo target uptake.

The diagnosis of hepatic fibrosis by magnetic resonance and near-infrared imaging using dual-modality nanoparticles.

Hepatic fibrosis (HF), as the only reversible process of chronic liver disease, remains a big diagnostic challenge. Development of noninvasive and effective methods to assess quantitatively early-stage HF is of great clinical importance. Compared with conventional diagnostic methods, near-infrared fluorescence imaging (NIR) and magnetic resonance imaging (MRI) could offer highly sensitive and spatial resolution signals for HF detection. However, precise detection using contrast agents is not possible. Superparamagnetic iron oxide (SPIO) nanoparticles have low toxicity, high sensitivity and excellent biocompatibility. Integration of Fe3O4 nanoparticles and indocyanine green (ICG), coupled with targeting ligand of integrin αvβ3, arginine–glycine–aspartic acid (RGD) expressed on hepatic stellate cells (HSCs), were used to detect HF. Both in vivo and in vitro results showed that the SPIO@SiO2–ICG–RGD had high stability and low cytotoxicity. The biodistribution of SPIO@SiO2–ICG–RGD was significantly different between mice with HF and healthy controls. SPIO@SiO2–ICG–RGD was characterized and the results of imaging in vitro and in vivo demonstrated the expression of integrin αvβ3 on activated HSCs. These data suggest that our SPIO@SiO2–ICG–RGD probe could be used for the diagnosis of early-stage HF. This new nanoprobe with a dual-modality imaging approach holds great potential for the diagnosis and classification of HF.

F-18 Labeled RGD Probes Based on Bioorthogonal Strain-Promoted Click Reaction for PET Imaging.

A series of fluorine-substituted monomeric and dimeric cRGD peptide derivatives, such as cRGD-ADIBOT-F (ADIBOT = azadibenzocyclooctatriazole), di-cRGD-ADIBOT-F, cRGD-PEG5-ADIBOT-F, and di-cRGD-PEG5-ADIBOT-F, were prepared by strain-promoted alkyne azide cycloaddition (SPAAC) reaction of the corresponding aza-dibenzocyclooctyne (ADIBO) substituted peptides with a fluorinated azide 3. Among these cRGD derivatives, di-cRGD-PEG5-ADIBOT-F had the highest binding affinity in a competitive binding assay compared to other derivatives and even the original cRGDyk. On the basis of the in vitro study results, di-cRGD-PEG5-ADIBOT-(18)F was prepared from a SPAAC reaction with (18)F-labeled azide and subsequent chemo-orthogonal scavenger-assisted separation without high performance liquid chromatography (HPLC) purification in 92% decay-corrected radiochemical yield (dcRCY) with high specific activity for further in vivo positron emission tomography (PET) imaging study. In vivo PET imaging study and biodistribution data showed that this radiotracer allowed successful visualization of tumors with good tumor-to-background contrast and significantly higher tumor uptake compared to other major organs.

Effects of integrin-targeted photodynamic therapy on pancreatic carcinoma cell

Objective To investigate the anti carcinoma role of integrin targeted photodynamic therapy (PDT) on pancreatic carcinoma cells in vitro.Methods Pancreatic carcinoma cells SW1990 were divided into four groups:cells without quantum dots (QDs) and light-treated as blank control group,pure light-treated group,photosensitizer group and PDT group.The targeting of QDs-arginine,glycine,aspartic acid (RGD) and integrin probe was confirmed by laser confocal microscopy.And as a photosensitizer for photodynamic therapy,after treated for 48 hours the morphology changes of pancreatic carcinoma cells of each group were observed.After 48 hours,the cell proliferation,apoptosis and cell cycle changes were detected by methyl thiazolyl tetrazolium (MTT) assay and flow cytometry (FCM).The expressions of myeloid cell leukemia-1 (Mcl-1),protein kinase B(Akt) and tumor necrosis factor-related apoptosis inducing ligand (TRAIL) were detected by reverse transcriptase polymerase chain reaction(RT-PCR).The amount of reactive oxygen species (ROS) of each group were evaluated by fluorescence probe.One-way ANOVA was performed for comparison between groups to analyze the treatment effects of PDT group.Results The QDs RGD probe could effectively targeting pancreatic carcinoma cells.The MTT results indicated that the relative inhibition rate of pancreatic carcinoma cells proliferation of PDT group was statistically higher than that of the other groups at 24,48,72 h (F=73.00,85.10,126.58; all P＜0.01).The FCM results revealed that the cell apoptosis rate of PDT group (17.860％ ±1.230％) was higher than that of the other groups (F=130.617,P＜0.01) and cell cycle G0/G1 phase (69.14％±2.63％) and S phase (24.41％ ± 2.67 ％) retardance was also significant (all P＜0.05).The expression of proliferation and apoptosis related gene Mcl-1 and Akt at mRNA level was lower than that of the other groups however the expression of apoptosis-inducing ligand TRAIL at mRNA level was higher than that of the other groups (F=567.456,446.817,145.238; all P＜0.05).The ROS level of PDT group was higher than that of the other groups (F=3262.559,P＜0.01).Conclusion PDT with a QDs-RGD probe could significantly inhibit pancreatic carcinoma cell proliferation and promote cell apoptosis. Key words: Pancreatic neoplasms; Integrins; Molecular probes; Photochemotherapy; Apoptosis

High Efficiency Diffusion Molecular Retention Tumor Targeting

Here we introduce diffusion molecular retention (DMR) tumor targeting, a technique that employs PEG-fluorochrome shielded probes that, after a peritumoral (PT) injection, undergo slow vascular uptake and extensive interstitial diffusion, with tumor retention only through integrin molecular recognition. To demonstrate DMR, RGD (integrin binding) and RAD (control) probes were synthesized bearing DOTA (for 111 In3+), a NIR fluorochrome, and 5 kDa PEG that endows probes with a protein-like volume of 25 kDa and decreases non-specific interactions. With a GFP-BT-20 breast carcinoma model, tumor targeting by the DMR or IV methods was assessed by surface fluorescence, biodistribution of [111In] RGD and [111In] RAD probes, and whole animal SPECT. After a PT injection, both probes rapidly diffused through the normal and tumor interstitium, with retention of the RGD probe due to integrin interactions. With PT injection and the [111In] RGD probe, SPECT indicated a highly tumor specific uptake at 24 h post injection, with 352%ID/g tumor obtained by DMR (vs 4.14%ID/g by IV). The high efficiency molecular targeting of DMR employed low probe doses (e.g. 25 ng as RGD peptide), which minimizes toxicity risks and facilitates clinical translation. DMR applications include the delivery of fluorochromes for intraoperative tumor margin delineation, the delivery of radioisotopes (e.g. toxic, short range alpha emitters) for radiotherapy, or the delivery of photosensitizers to tumors accessible to light.

The PEG-Fluorochrome Shielding Approach for Targeted Probe Design

We provide a new approach for fluorescent probe design termed "PEG-fluorochrome shielding", where PEGylation enhances quantum yields while blocking troublesome interactions between fluorochromes and biomolecules. To demonstrate PEG-fluorochrome shielding, fluorochrome-bearing peptide probes were synthesized, three without PEG and three with a 5 kDa PEG functional group. In vitro, PEG blocked the interactions of fluorochrome-labeled peptide probes with each other (absorption spectra, self-quenching) and reduced nonspecific interactions with cells (by FACS). In vivo, PEG blocked interactions with biomolecules that lead to probe retention (by surface fluorescence). Integrin targeting in vivo was obtained as the differential uptake of an (111)In-labeled, fluorochrome-shielded, integrin-binding RGD probe and a control RAD. Using PEG to block fluorochrome-mediated interactions, rather than synthesizing de novo fluorochromes, can yield new approaches for the design of actively or passively targeted near-infrared fluorescent probes.

Serial non-invasive monitoring of renal disease following immune-mediated injury using near-infrared optical imaging.

BackgroundNon-invasive monitoring of disease progression in kidney disease is still a major challenge in clinical practice. In vivo near-infrared (NIR) imaging provides a new tool for studying disease mechanisms and non-invasive monitoring of disease development, even in deep organs. The LI-COR IRDye® 800CW RGD optical probe (RGD probe) is a NIR fluorophore, that can target integrin alpha v beta 3 (αvβ3) in tissues.ObjectiveThis study aims to monitor renal disease progression in an anti-glomerular basement membrane (GBM) nephritis mouse model.MethodsAnti-GBM nephritis was induced in 129x1/svJ mice by anti-GBM serum challenge. The expression of integrin αvβ3 in the diseased kidney was examined by immunohistochemistry and quantitative polymerase chain reaction. The RGD probe and control fluorophores, the 800CW dye, and the BSA-conjugated 800CW dye, were administered into anti-GBM nephritic mice. LI-COR Pearl® Impulse imaging system was used for in vivo imaging; while ex vivo organ imaging was acquired using the MaestroTM imaging system.ResultsKidney tissue from anti-GBM nephritic mice showed higher levels of integrin αvβ3 expression at both the protein and the mRNA level compared to normal mice. The RGD probe allowed in vivo renal imaging and the fluorescent signal could be specifically captured in the diseased kidneys up to 14 days, reflecting longitudinal changes in renal function.ConclusionThe infrared RGD molecular probe that tracks integrin expression can be successfully used to monitor renal disease progression following immune-mediated nephritis.

Strain-Promoted Catalyst-Free Click Chemistry for Rapid Construction of (64)Cu-Labeled PET Imaging Probes.

A rapid, efficient, and catalyst-free click chemistry method for the construction of (64)Cu-labeled PET imaging probes was reported based on the strain-promoted aza-dibenzocyclooctyne ligation. This new method was exemplified in the synthesis of (64)Cu-labeled RGD peptide for PET imaging of tumor integrin αvβ3 expression in vivo. The catalyst-free click chemistry reaction proceeded with a fast rate and eliminated the contamination problem of the catalyst Cu(I) ions interfering with the (64)Cu radiolabeling procedure under the conventional Cu-catalyzed 1,3-dipolar cycloaddition condition. The new strategy is simple and robust, and the resultant (64)Cu-labeled RGD probe was obtained in an excellent yield and high specific activity. PET imaging and biodistribution studies revealed significant, specific uptake of the "click" (64)Cu-labeled RGD probe in integrin αvβ3-positive U87MG xenografts with little uptake in nontarget tissues. This new approach is versatile, which warrants a wide range of applications for highly diverse radiometalated bioconjugates for radioimaging and radiotherapy.

Fast clearing RGD‐based near‐infrared fluorescent probes for in vivo tumor diagnosis

A fast clearing hydrophilic near-infrared (NIR) dye ICG-Der-02 was used to constitute tumor targeting contrast agents. Cell adhesion molecule integrin α(v)β(3) served as the target receptor because of its unique expression on almost all sprouting tumor vasculatures. The purpose of this study was to synthesize and compare the properties of integrin α(v)β(3)-targeted, fast clearing NIR probes both in vitro and in vivo for tumor diagnosis. ICG-Der-02 was covalently conjugated to three kinds of RGD peptide including linear, monoeric cyclic and dimeric RGD to form three RGD-based NIR probes. The integrin receptor specificities of these probes were evaluated in vitro by confocal microscopy. The dynamic bio-distribution and elimination ratse were in vivo real-time monitored by a near-infrared imaging system in normal mice. Further, the in vivo tumor targeting abilities of the RGD-based NIR probes were compared in α(v)β(3) -positive MDA-MB-231, U87MG and α(v)β(3)-negtive MCF-7 xenograft mice models. Three RGD-based NIR probes were successfully synthesized with good optical properties. In vitro cellular experiments indicated that the probes have a clear binding affinity to α(υ)β(3) -positive tumor cells, with a cyclic dimeric RGD probe owing the highest integrin affinity. Dynamic bio-distributions of these probes showed a rapid clearing rate through the renal pathway. In vivo tumor targeting ability of the RGD-based porbes was demonstrated on MDA-MB-231 and U87MG tumor models. As expected, the c(RGDyK)(2)-ICG-Der-02 probe displayed the highest tumor-to-normal tissue contrast. The in vitro and in vivo block experiments confirmed the receptor binding specificity of the probes. The hydrophilic dye-labeled NIR probes exhibited a fast clearing rate and deep tissue penetration capability. Further, the α(υ)β(3) receptor affinity of the three RGD-based NIR probes followed the order of dimer cyclic > monomer cyclic > linear. The results demonstrate potent fast clearing probes for in vivo early tumor diagnosis.

Abstract 5288A: Multivalent fluorescent probes for in vivo tumor targeting

Abstract Conventional fluorescence probes are monovalent molecules that interact with cellular or extra-cellular target molecules through affinity binding. To improve the binding affinity and sensitivity of detection of the fluorescence probe, we describe a novel strategy for the synthesis designed to contain multiple targeting moieties and a near infrared fluorescent dye Iflour750 for optimal in vivo detection. In this study, we applied several multivalent probes to in vivo tumor targeting and monitored the targeting process using fluorescence imaging. Deoxyglucose based fluorescence probes have been applied to tumor labeling as the high metabolic rate of the tumor cells causes preferential labeling of the tumor mass. With a multivalent 2-deoxy-D- glucose Iflour750 (2-DG-IF750) probe, we demonstrated sensitive in vivo targeting as compared to a control mono-valent 2DG probe in several tumor models, including PC3M-luc2 prostate tumor cells, MDA-MB-231-luc2 mammary fat tumor cells as well as LL/2-luc and H460-luc2 lung cancer cells. Distinct tumor signal was visualized within a few hours after intravenous delivery and reached the peak of signal/background ratio between 6-24 hours. With a multivalent cyclooxygenase (COX) binding fluorescence probe Indomethacin-IF750, we observed an interesting targeting of COX2 negative colon tumor cells HCT116-luc2, but not the COX2 positive HT29-luc2, hypothetically due to selective binding to COX1. We also tested a multivalent RGD probe for targeting integrin avb3, the expression of which is associated with tumor angiogenesis and metastasis. With our iFlur750-(RGD)4 probe, we demonstrated in vivo targeting of U87-MG-luc2, HCT116-luc2 and HT29-luc2 tumor cells. Finally, we demonstrated that optical imaging was conveniently applied to evaluating the PK/PD profile of the probes. Our multivalent probes showed a fast clearance with a half life of less than 1 hour, which is a desirable feature for achieving maximal tumor/background contrast. In summary, we have developed a new class of multi-valent fluorescence probes with improved specificity and sensitivity of tumor targeting as compared to mono-valent analogs. Utility of this class of multivalent probes is likely to pass beyond pre-clinical models, as imaging guided tumor surgery is on the verge of clinical applications. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5288A. doi:10.1158/1538-7445.AM2011-5288A

Tumor Uptake of the RGD Dimeric Probe 99mTc-G3-2P4-RGD2 is Correlated with Integrin αvβ3 Expressed on both Tumor Cells and Neovasculature

Integrin αvβ3 has been well-documented as one of the key players in the process of tumor angiogenesis. Radiolabeled RGD (Arg-Gly-Asp) peptides that specifically target integrin αvβ3 have great potential for tumor early detection and noninvasively monitoring the status of tumor angiogenesis. We developed a cyclic RGD dimeric probe (99m)Tc-HYNIC-Gly3-E[PEG4-c(RGDfK)]2 ((99m)Tc-G3-2P4-RGD2) (using tricine and TPPTS as the coligands, TPPTS = trisodium triphenylphosphine-3,3',3''-trisulfonate), and investigated whether it could be used to noninvasively visualize and quantify integrin αvβ3 expression in vivo. HYNIC-Gly3-E[PEG4-c(RGDfK)]2 was synthesized and labeled with (99m)Tc. The biodistribution and planar γ-imaging studies of (99m)Tc-G3-2P4-RGD2 were performed in both U87MG (human integrin αvβ3 positive/murine integrin αvβ3 positive) and HT-29 (human integrin αvβ3 negligible /murine integrin αvβ3 positive) tumor-bearing nude mouse models. The correlation of (99m)Tc-G3-2P4-RGD2 tumor uptake values (measured by ex vivo biodistribution) with expression levels of human integrin αvβ3 or murine integrin αvβ3 (measured by Western blot) were determined in U87MG and HT-29 tumor models, respectively. (99m)Tc-G3-2P4-RGD2 exhibited increased receptor binding affinity and in vivo tumor uptake as compared with previously reported RGD dimeric tracer (99m)Tc-RGD2 (without Gly3 and PEG4 spacers). The tumor uptake of (99m)Tc-G3-2P4-RGD2 was related to the expression levels of both human integrin αvβ3 (expressed on tumor cells) and murine integrin αvβ3 (expressed on newborn tumor vasculature). Our results demonstrate that (99m)Tc-G3-2P4-RGD2 is a useful agent for integrin αvβ3 imaging. The relationship between (99m)Tc-G3-2P4-RGD2 uptake and integrin αvβ3 expression level as determined by this study would provide useful information for clinical translation of RGD probes.