Peptide-Directed Synthesis of Aggregation-Induced Emission Enhancement-Active Gold Nanoclusters for Single- and Two-Photon Imaging of Lysosome and Expressed αvß3 Integrin Receptors.

This study explores the synthesis and characterization of aggregation-induced emission enhancement (AIEE)-active gold nanoclusters (AuNCs), focusing on their near-infrared luminescence properties and potential applications in biological imaging. These AIEE-active AuNCs were synthesized via the NaBH4-mediated reduction of HAuCl4 in the presence of peptides. We systematically investigated the influence of the peptide sequence on the optical features of the AuNCs, highlighting the role of glutamic acid in enhancing their quantum yield (QY). Among the synthesized peptide-stabilized AuNCs, EECEE-stabilized AuNCs exhibited the maximum QY and a pronounced AIEE effect at pH 5.0, making them suitable for the luminescence imaging of intracellular lysosomes. The AIEE characteristic of the EECEE-stabilized AuNCs was demonstrated through examinations using transmission electron microscopy, dynamic light scattering, zeta potential analysis, and single-particle imaging. The formation of the EECEE-stabilized AuNCs was confirmed by size-exclusion chromatography and mass spectrometry. Spectroscopic and electrochemical examinations uncover the formation process of EECEE-stabilized AuNCs, comprising EECEE-mediated reduction, NaBH4-induced nucleation, complex aggregation, and subsequent cluster growth. Furthermore, we demonstrated the utility of these AuNCs as luminescent probes for intracellular lysosomal imaging, leveraging their pH-responsive AIEE behavior. Additionally, cyclic arginylglycylaspartic acid (RGD)-modified AIEE dots, derived from cyclic RGD-linked peptide-induced aggregation of EECEE-stabilized AuNCs, were developed for single- and two-photon luminescence imaging of αvß3 integrin receptor-positive cancer cells.

In Situ Self-Assembly of Bispecific Peptide for Cancer Immunotherapy.

Precise and effective manipulation of protein functions still faces tremendous challenges. Herein we report a programmable peptide molecule, consisted of targeting and self-assembly modules, that enables specific and highly efficient assembly governed by targeting receptor proteins. Upon binding to the cell membrane receptor, peptide conformation is somewhat stabilized along with decreased self-assembly activation energy, promoting peptide-protein complex oligomerization. We first design a GNNQQNY-RGD peptide (G7-RGD) to recognize integrin αV ß3 receptor for proof-of-concept study. In the presence of αV ß3 protein, the critical assembly concentration of free G7-RGD decreases from 525 to 33 µM and the resultant G7-RGD cluster drives integrin receptor oligomerization. Finally, a bispecific assembling peptide antiCD3-G7-RGD is rationally designed for cancer immunotherapy, which validates CD3 oligomerization and concomitant T cell activation, leading to T cell-mediated cancer cell cytolysis.

MR/NIRF Dual-Mode Imaging of αvß3 Integrin-Overexpressing Tumors Using a Lipopeptide-Based Contrast Agent.

Early diagnosis and noninvasive detection of hepatocellular carcinoma have profound clinical implications for treatment quality and improved prognosis. To obtain high-resolution macroscopic anatomical information and high-sensitivity microscopic optical signals to detect tumors, it is highly desirable to develop dual-mode magnetic resonance imaging (MRI) and near-infrared fluorescent (NIRF) probes. An MR/NIRF dual-mode targeted contrast agent was created by encapsulating cyclic arginine-glycine-aspartate (cRGD) and Cy5.5 in liposomes and characterized by the particle size distribution, cytotoxicity, targeting, and MRI relaxivity. The MR T2 intensity and fluorescence intensity were evaluated in the tumors, livers, and muscles after the injection of cRGD-Liposome-Cy5.5 and Liposome-Cy5.5 at different time points. The average size of cRGD-Liposome-Cy5.5 was 62.33 ± 4.648 nm. The transverse relaxivity (R2) values had a negative correlation with the concentration of molecular probes. The MR signal intensity was enhanced in tumors after the cRGD-Liposome-Cy5.5 injection and not enhanced in liver parenchyma and muscles at the same time. The fluorescence intensity was enhanced in tumors after cRGD-Liposome-Cy5.5 injection in the targeted group. cRGD -Liposome-Cy5.5 as an entirely organic T2-positive dual-mode MR/NIRF targeted contrast agent is therefore able to detect early-stage hepatocellular carcinoma by targeting integrin αvß3, providing advantages for potential clinical utility and ease of clinical transformation.

Super-resolution Surface-Enhanced Raman Scattering Imaging of Single Particles in Cells.

The ability to locate and identify molecular interactions in cells has significant importance for understanding protein function and molecular biology. Functionalized metallic nanoparticles have been used as probes for protein tracking and drug delivery because of their ability to carry therapeutic agents and readily functionalized surfaces. In this work, we present a super-resolution surface-enhanced Raman scattering (SERS) approach for imaging and tracking membrane receptors interacting with peptide-functionalized gold nanostars (AuNS). The αvß3 integrin receptors in colon cancer cells are successfully targeted and imaged using AuNS with the high-affinity amino acid sequence arginine-glycine-aspartic acid-phenylalanine-cysteine (RGDFC) attached. The RGDFC peptide interaction with the integrin receptor provides a bright and fluctuating SERS signal that can be analyzed with localization microscopy algorithms. Additionally, the observed SERS spectrum is used to confirm protein-peptide interaction. Experiments with functionalized and bare AuNS illustrate specific and nonspecific binding events. Specific binding is monitored with a localization precision of ∼6 nm. The observed spatial resolution is associated with tight binding, which was confirmed by the slower diffusion coefficient measured from 4.4 × 10-11 cm2/s for the AuNS-RGDFC compared to 7.8 × 10-10 cm2/s for the bare AuNS. Super-resolution SERS images at different focal planes show evidence of internalized particles and suggest insights into protein orientation on the surface of cells. Our work demonstrates super-resolution SERS imaging to probe membrane receptor interactions in cells, providing chemical information and spatial resolution with potential for diverse applications in life science and biomedicine.

Visualisation of interstitial lung disease by molecular imaging of integrin αvß3 and somatostatin receptor 2.

OBJECTIVE: To evaluate integrin αvß3 (alpha-v-beta-3)-targeted and somatostatin receptor 2 (SSTR2)-targeted nuclear imaging for the visualisation of interstitial lung disease (ILD). METHODS: The pulmonary expression of integrin αvß3 and SSTR2 was analysed in patients with different forms of ILD as well as in bleomycin (BLM)-treated mice and respective controls using immunohistochemistry. Single photon emission CT/CT (SPECT/CT) was performed on days 3, 7 and 14 after BLM instillation using the integrin αvß3-targeting 177Lu-DOTA-RGD and the SSTR2-targeting 177Lu-DOTA-NOC radiotracer. The specific pulmonary accumulation of the radiotracers over time was assessed by in vivo and ex vivo SPECT/CT scans and by biodistribution studies. RESULTS: Expression of integrin αvß3 and SSTR2 was substantially increased in human ILD regardless of the subtype. Similarly, in lungs of BLM-challenged mice, but not of controls, both imaging targets were stage-specifically overexpressed. While integrin αvß3 was most abundantly upregulated on day 7, the inflammatory stage of BLM-induced lung fibrosis, SSTR2 expression peaked on day 14, the established fibrotic stage. In agreement with the findings on tissue level, targeted nuclear imaging using SPECT/CT specifically detected both imaging targets ex vivo and in vivo, and thus visualised different stages of experimental ILD. CONCLUSION: Our preclinical proof-of-concept study suggests that specific visualisation of molecular processes in ILD by targeted nuclear imaging is feasible. If transferred into clinics, where imaging is considered an integral part of patients' management, the additional information derived from specific imaging tools could represent a first step towards precision medicine in ILD.

Clinicopathological and prognostic values of fibronectin and integrin αvß3 expression in primary osteosarcoma.

BACKGROUND: Osteosarcoma is a malignant bone tumor with a high potential for lung metastasis, and the prognosis for patients with metastatic disease is very poor. The interaction between fibronectin (FN) and integrin αvß3 in soft-tissue sarcoma promotes cell migration, invasion, and lung metastasis. This study aimed to investigate the prognostic significance of FN and αvß3 in osteosarcoma. METHODS: Immunohistochemistry and western blotting were used to detect the expression of FN and αvß3 in 60 osteosarcoma specimens and in 30 osteochondroma specimens. Furthermore, correlations of FN and αvß3 with the clinicopathological features of osteosarcoma patients were analyzed using the χ2 test and Fisher's exact test. Disease-free survival and overall survival of osteosarcoma patients were assessed using the Kaplan-Meier method and Cox proportional hazards model. The predictive accuracy of the model was determined by the Harrell concordance index. RESULTS: FN (P < 0.05) and αvß3 (P < 0.05) were overexpressed in osteosarcoma specimens compared with osteochondroma specimens. High FN expression was associated with a poor response to chemotherapy (P = 0.001) and poor disease-free (P < 0.001) and overall (P < 0.001) survival. High expression of αvß3 was linked to an advanced surgical stage (P = 0.028), a poor response to chemotherapy (P = 0.002), and both poor disease-free survival (P < 0.001) and overall survival (P < 0.001). FN and αvß3 co-expression were associated with sex (P = 0.011), an advanced surgical stage (P = 0.013), and a poor response to chemotherapy (P = 0.002). Moreover, high expression of both proteins can serve as an independent prognostic value for reduced survival time in osteosarcoma patients. CONCLUSIONS: The results of this study suggest that FN and αvß3 expression is associated with an unfavorable clinical outcome of osteosarcoma, and these molecules may constitute attractive therapeutic targets for osteosarcoma treatment. To improve the survival of osteosarcoma patients, further investigations are required to clarify their prognostic values in a larger population.

Novel 64Cu Labeled RGD2-BBN Heterotrimers for PET Imaging of Prostate Cancer.

Bombesin receptor 2 (BB2) and integrin αvß3 receptor are privileged targets for molecular imaging of cancer because of their overexpression in a number of tumor tissues. The most recent developments in heterodimer-based radiopharmaceuticals concern BB2- and integrin αvß3-targeting compounds, consisting of bombesin (BBN) and cyclic arginine-glycine-aspartic acid peptides (RGD), connected through short length linkers. Molecular imaging probes based on RGD-BBN heterodimer design exhibit improved tumor targeting efficacy compared to the single-receptor targeting peptide monomers. However, their application in clinical study is restricted because of inefficient synthesis or unfavorable in vivo properties, which could depend on the short linker nature. Thus, the aim of the present study was to develop a RGD2-BBN heterotrimer, composed of (7-14)BBN-NH2 peptide (BBN) linked to the E[ c(RGDyK)]2 dimer peptide (RGD2), bearing the new linker type [Pro-Gly]12. The heterodimer E[c(RGDyK)]2-PEG3-Glu-(Pro-Gly)12-BBN(7-14)-NH2 (RGD2-PG12-BBN) was prepared through conventional solid phase synthesis, then conjugated with 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) or 1,4,7-triazacyclononane-1-glutaric acid-4,7-diacetic acid (NODA-GA). In 64Cu labeling, the NODA-GA chelator showed superior radiochemical characteristics compared to DOTA (70% vs 40% yield, respectively). Both conjugates displayed dual targeting ability, showing good αvß3 affinities and high BB2 receptor affinities which, in the case of the NODA-GA conjugate, were in the same range as the best RGD-BBN heterodimer ligands reported to date ( Ki = 24 nM). 64Cu-DOTA and 64Cu-NODA-GA probes were also found to be stable after 1 h incubation in mouse serum (>90%). In a microPET study in prostate cancer PC-3 xenograft mice, both probes showed low tumor uptake, probably due to poor pharmacokinetic properties in vivo. Overall, our study demonstrates that novel RGD-BBN heterodimer with long linker can be prepared and they preserve high binding affinities to BB2 and integrin αvß3 receptor binding ability. The present study represents a step forward in the design of effective heterodimer or heterotrimer probes for dual targeting.

Click Conjugation of Cloaked Peptide Ligands to Microbubbles.

Interest in the use of targeted microbubbles for ultrasound molecular imaging (USMI) has been growing in recent years as a safe and efficacious means of diagnosing tumor angiogenesis and assessing response to therapy. Of particular interest are cloaked microbubbles, which improve specificity by concealing the ligand from blood components until they reach the target vasculature, where the ligand can be transiently revealed for firm receptor-binding by ultrasound acoustic radiation force pulses. Herein, a bio-orthogonal "click" conjugation chemistry is introduced to decorate the surface of cloaked 4-5-µm-diameter microbubbles as part of a sterile and reproducible production process. Azido-functionalized antagonists for the angiogenic biomarkers αVß3 integrin (cRGD) and VEGFR2 (A7R) proteins were conjugated to bimodal-brush microbubbles via strain-promoted [3 + 2] azide-alkyne cycloaddition (SPAAC) click chemistry. Ligand conjugation was validated by epifluorescent microscopy, flow cytometry, and Fourier-transform infrared spectroscopy. Sterility was validated by bacterial culture and endotoxin analysis. Additionally, clinically normal dogs receiving escalating microbubble doses were shown to experience no pathologic changes in physical examination, complete blood count, serum biochemistry profile, or coagulation panel. This bio-orthogonal microbubble conjugation process for cloaked peptide ligands may be leveraged for future USMI studies of tumor angiogenesis for translation to preclinical and clinical applications.

Coordination-Mediated Synthesis of Purification-Free Bivalent 99mTc-Labeled Probes for in Vivo Imaging of Saturable System.

In the synthesis of technetium-99m (99mTc) labeled target-specific ligands, the presence of a large excess of unlabeled ligands over 99mTc in the injectate hinders target accumulation of 99mTc-labeled ligands by competing for target molecules. To circumvent the problem, we recently developed a concept of the metal coordination-mediated multivalency, and proved the concept with a 99mTc-labeled trivalent compound [99mTc(CO)3(CN-RGD)3]+. In this study, D-penicillamine (Pen) was selected as a chelating molecule and a cyclic RGDfK peptide was conjugated to Pen via a hexanoic linkage (Pen-Ahx-c(RGDfK)). 99mTc complexation reaction, and the stability, integrin αvß3 binding affinity, and biodistribution of the 99mTc-labeled probe were investigated to evaluate the applicability of the concept to bivalent probes. 99mTc-[Pen-Ahx-c(RGDfK)]2 was obtained over 95% radiochemical yields under low Pen-Ahx-c(RGDfK) concentration (50 µM). 99mTc-[Pen-Ahx-c(RGDfK)]2 showed approximately 10-times higher integrin αvß3 binding affinity than the monovalent compounds, Pen-Ahx-c(RGDfK) and c(RGDyV). In biodistribution studies, the tumor accumulation of 99mTc-[Pen-Ahx-c(RGDfK)]2 was decreased to 77% and 43% of HPLC-purified (Pen-Ahx-c(RGDfK)-free) 99mTc-[Pen-Ahx-c(RGDfK)]2 by the presence of 5 nmol of unlabeled Pen-Ahx-c(RGDfK) and Re-[Pen-Ahx-c(RGDfK)]2, respectively. 99mTc-[Pen-Ahx-c(RGDfK)]2 provided tumor image without removing unlabeled ligand, while a 99mTc-labeled monovalent probe prepared from a monovalent ligand could not. These findings indicate the availability of the design concept to prepare 99mTc-labeled bivalent probes with a variety of 99mTc core and other metallic radionuclides of clinical relevance.

"Polymultivalent" Polymer-Peptide Cluster Conjugates for an Enhanced Targeting of Cells Expressing αvß3 Integrins.

A new class of "polymultivalent" ligands combining several ligand clusters and a water-soluble biocompatible polymer is introduced. These original conjugates bear two levels of multivalency. They are prepared by covalent coupling of a controlled number of tetrameric cRGD peptide clusters along a well-defined copolymer synthesized by RAFT polymerization. The presence of multiple copies of peptide clusters on the same polymer backbone resulted in a much-higher relative potency than the free cluster reference. Thanks to the "polymultivalency", up to ∼2 orders of magnitude potency enhancement was reached in a competitive cell adhesion assay (nanomolar-range IC50 values). In addition, confocal microscopy and flow cytometry demonstrated that fluorescent "polymultivalent" conjugates (emitting in the far-red/near-infrared region) were able to specifically and selectively label cells expressing αvß3-integrin, the natural receptor of cRGD.

Enhancing PET Signal at Target Tissue in Vivo: Dendritic and Multimeric Tris(hydroxypyridinone) Conjugates for Molecular Imaging of αvß3 Integrin Expression with Gallium-68.

Tris(hydroxypyridinone) chelators conjugated to peptides can rapidly complex the positron-emitting isotope gallium-68 (68Ga) under mild conditions, and the resulting radiotracers can delineate peptide receptor expression at sites of diseased tissue in vivo. We have synthesized a dendritic bifunctional chelator containing nine 1,6-dimethyl-3-hydroxypyridin-4-one groups (SCN-HP9) that can coordinate up to three Ga3+ ions. This derivative has been conjugated to a trimeric peptide (RGD3) containing three peptide groups that target the αvß3 integrin receptor. The resulting dendritic compound, HP9-RGD3, can be radiolabeled in 97% radiochemical yield at a 3-fold higher specific activity than its homologues HP3-RGD and HP3-RGD3 that contain only a single metal binding site. PET scanning and biodistribution studies show that [68Ga(HP9-RGD3)] demonstrates higher receptor-mediated tumor uptake in animals bearing U87MG tumors that overexpress αvß3 integrin than [68Ga(HP3-RGD)] and [68Ga(HP3-RGD3)]. However, concomitant nontarget organ retention of [68Ga(HP9-RGD3)] results in low tumor to nontarget organ contrast in PET images. On the other hand, the trimeric peptide homologue containing a single tris(hydroxypyridinone) chelator, [68Ga(HP3-RGD3)], clears nontarget organs and exhibits receptor-mediated uptake in mice bearing tumors and in mice with induced rheumatoid arthritis. PET imaging with [68Ga(HP3-RGD3)] enables clear delineation of αvß3 integrin receptor expression in vivo.

Selective Detection of RGD-Integrin Binding in Cancer Cells Using Tip Enhanced Raman Scattering Microscopy.

Ligand-receptor interactions play important roles in many biological processes. Cyclic arginine-glycine-aspartic acid (RGD) containing peptides are known to mimic the binding domain of extracellular matrix protein fibronectin and selectively bind to a subset of integrin receptors. Here we report the tip enhanced Raman scattering (TERS) detection of RGD-functionalized nanoparticles bound to integrins produces a Raman scattering signal specific to the bound protein. These results demonstrate that this method can detect and differentiate between two different integrins (α5ß1 and αvß3) bound to RGD-conjugated gold nanoparticles both on surfaces and in a cancer cell membrane. In situ measurements of RGD nanoparticles bound to purified α5ß1 and αvß3 receptors attached to a glass surface provide reference spectra for a multivariate regression model. The TERS spectra observed from nanoparticles bound to cell membranes are analyzed using this regression model and the identity of the receptor can be determined. The ability to distinguish between receptors in the cell membrane provides a new tool to chemically characterize ligand-receptor recognition at molecular level and provide chemical perspective on the molecular recognition of membrane receptors.

Integrin αvß3-Targeted C-Dot Nanocomposites as Multifunctional Agents for Cell Targeting and Photoacoustic Imaging of Superficial Malignant Tumors.

With a cocktail formulation of soybean milk as a green carbon source and TTDDA as a capping agent, integrin αvß3-targeted C-dot nanocomposites (MB-CDs@NH-RGD) have been successfully fabricated via a facile microwaving protocol. Modification of the surface coating and RGD-conjugation endow their superior biocompatibility as well as highly specific targeting profile to αvß3-overexpressed cell lines of MDA-MB231 and B16 as representative superficial malignant tumors. Meanwhile, the significant photothermal effect has been generated on irradiation of these targeted C-dot nanocomposites by a pulsed laser, which proved their eligibility for potential thermal ablation therapy. In vivo photoacoustic imaging using these C-dot nanocomposites as novel imaging probes verified their excellent targeting sensitivity and contrast enhancement. This exciting evidence implies a promising strategy to utilize them for multifunctional nanotheranostic purposes in combination with precision diagnosis and photothermal treatment against superficial malignant tumors.

Rapid synthesis of cyclic RGD conjugated gold nanoclusters for targeting and fluorescence imaging of melanoma A375 cells.

A simple, rapid, and inexpensive method for the synthesis of cyclic arginine-glycine-aspartic acid (RGD) peptide conjugated gold nanoclusters (RGD-GNCs) was reported. The nanoclusters were synthesized with chloraurate as precursor and cyclic RGD peptides as both reducing and protecting agent directly under alkali condition, and the whole synthetic process only took 15 min at room temperature. The properties of the nanoclusters were characterized by means of ultraviolet-visible spectra, Fourier transform infrared spectroscopy (FTIR), fluorescence, transmission electron microscopy (TEM), and X-ray photoelectron spectroscopy (XPS). The prepared gold nanoclusters were successfully used as a contrast agent in fluorescence imaging of the melanoma A375 cells, which overexpress the integrin αvß3. The results demonstrated that our nanoclusters possess good biocompatibility, stability, and low toxicity. Moreover, the method is expected to be applicable to the synthesis of nanoclusters conjugated with other biomolecules.

Molecular imaging of tumor-associated angiogenesis using a novel magnetic resonance imaging contrast agent targeting αvß 3 integrin.

BACKGROUND: The recent introduction of biological anticancer therapy has renewed the interest in functional imaging of tumor-associated angiogenesis (TAA) as a tool to monitor early therapy response. The present study evaluated imaging of TAA using P1227, a novel, small molecular magnetic resonance imaging (MRI) probe targeting αvß3 integrin. METHODS: HT29 human colorectal cancers were grown in athymic mice. Dynamic MRI was performed using a three-dimensional VIBE sequence up to 110 min after injection of P1227 or gadolinium-tetraazacyclododecane tetraacetic acid (Gd-DOTA). Specificity was assessed by using P1227 1 h after intravenous administration of the αvß3 inhibitor cilengitide. Regions of interest were drawn encompassing the tumor rim and normal muscle. Imaging data were compared with microvessel density and αvß3 expression. RESULTS: Using P1227, specific enhancement of the angiogenic tumor rim, but not of normal muscle, was observed, whereas Gd-DOTA enhanced tumor and normal muscle. After administering cilengitide, enhancement with P1227, but not with DOTA, was significantly suppressed during the first 20 min. When using P1227, a significant correlation was observed between normalized enhancement of the tumor rim and immunohistochemical αvß3 integrin expression. CONCLUSIONS: Molecular MRI using a small monogadolinated tracer targeting αvß3 integrin and moderate magnetic field strength holds promise in assessing colorectal TAA.

TERS detection of αVß3 integrins in intact cell membranes.

Integrins are important membrane receptors that form focal adhesions with the extracellular matrix and are transmembrane signaling proteins. We demonstrate that nanoparticles functionalized with c-RGDfC ligands bind to intact cell membranes and selectively enhance the amino acid signals of the integrin receptor when coupled with tip-enhanced Raman scattering (TERS) detection. Controlling the plasmonic interaction between the functionalized nanoparticle and the TERS tip provides a clear Raman signal from αVß3 integrins in the cell membrane that matches the signal of the purified integrin receptor. Random aggregation of nanoparticles on the cell does not provide the same spectral information. Chemical characterization of membrane receptors in intact cellular membranes is important for understanding membrane signaling and drug targeting. These results provide a new method to investigate the chemical interactions associated with ligand binding to membrane receptors in cells.

[18F-FPRGD2 PET/CT imaging of integrin αVß3]. / Imagerie de l'intégrine αVß3 par PET/CT au 18F-FPRGD2.

PET/CT imaging of 15F-FPRGD2 allows the visualization and quantification of integrin αVß3 in tissues. This imaging technique was developed with the purpose of quantifying tumor angiogenesis and of assessing the efficacy of antiangiogenic treatments. However, the PET signal of 18F-FPRGD2 appears more complex as various tumor cell types, inflammatory cells and osteoclasts express the integrin αVß3 regulating cell interactions with the extracellular matrix. This article provides data of clinical studies evaluating 18F-FPRGD2 PET/CT imaging in patients with a renal mass or a locally advanced rectal carcinoma and finally reports on the incidental discovery of 18F-FPRGD2 uptake in osteoarticular processes such as osteoarthritis.

Micro-positron emission tomography/contrast-enhanced computed tomography imaging of orthotopic pancreatic tumor-bearing mice using the αvß3 integrin tracer 64Cu-labeled cyclam-RAFT-c(-RGDfK-)4.

The purpose of this study was to develop a clinically relevant orthotopic xenotransplantation model of pancreatic cancer and to perform a preclinical evaluation of a new positron emission tomography (PET) imaging probe, 64Cu-labeled cyclam-RAFT-c(-RGDfK-)4 peptide (64Cu-RAFT-RGD), using this model. Varying degrees of αvß3 integrin expression in several human pancreatic cancer cell lines were examined by flow cytometry and Western blotting. The cell line BxPC-3, which is stably transfected with a red fluorescence protein (RFP), was used for surgical orthotopic implantation. Orthotopic xenograft was established in the pancreas of recipient nude mice. An in vivo probe biodistribution and receptor blocking study, preclinical PET imaging coregistered with contrast-enhanced computed tomography (CECT) comparing 64Cu-RAFT-RGD and ¹8F-fluoro-2-deoxy-d-glucose (¹8F-FDG) accumulation in tumor, postimaging autoradiography, and histologic and immunohistochemical examinations were done. Biodistribution evaluation with a blocking study confirmed that efficient binding of probe to tumor is highly αvß3 integrin specific. 64Cu-RAFT-RGD PET combined with CECT provided for precise and easy detection of cancer lesions. Autoradiography, histologic, and immunohistochemical examinations confirmed the accumulation of 64Cu-RAFT-RGD in tumor versus nontumor tissues. In comparative PET studies, 64Cu-RAFT-RGD accumulation provided better tumor contrast to background than ¹8F-FDG. Our results suggest that 64Cu-RAFT-RGD PET imaging is potentially applicable for the diagnosis of αvß3 integrin-expressing pancreatic tumors.

Quantitative positron emission tomography imaging of angiogenesis in rats with forelimb ischemia using (68)Ga-NOTA-c(RGDyK).

Gallium-68-labeled 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA)-cyclic Arg-Gly-Asp-D-Tyr-Lys (c(RGDyK)) was developed for αvß3 targeting, and is a promising agent for imaging of cancer and disorders related to angiogenesis. In this study, we performed kinetic analysis of (68)Ga-NOTA-c(RGDyK) in rats with surgically induced forelimb ischemia, and immunohistochemical analysis was also performed to assess αvß3 immuno-staining level. Animal models were created by excision of the left brachial vessels, and a sham operation was performed on the right brachial region under 2 % isoflurane anesthesia. Using an animal positron emission tomography/computed tomography (PET/CT) scanner, a list mode PET scan (120 min) was started with the injection of (68)Ga-NOTA-c(RGDyK) via the tail vein at 3, 5 and 7 days after ischemic surgery. Volumes of interest were drawn on the left ventricle, sham operation, control, and ischemic regions. Compartmental and two graphical analyses (Logan and RE plots) were performed for kinetic parameter estimation. The immunohistochemical analysis was also performed after the last PET scan, and cell components were scored on a six point scale for quantification of immuno-staining level (0-negative to 5-very high). A 3-compartment model with reversible binding best described the tissue time-activity curves. The distribution volume of the ischemic region was significantly higher than that of the sham operation (P < 10(-6)) and control region (P < 10(-9)). Both the Logan and RE plots showed high correlation with compartmental analysis (R(2) = 0.96 and 0.95 for Logan and RE, respectively). The temporal changes in distribution volume and binding potential were not significant. The immuno-staining level of the ischemic region was significantly higher than that of sham operation (P < 10(-4)) and control region (P < 10(-8)). Kinetic modeling studies with dynamic (68)Ga-NOTA-c(RGDyK) PET scan are feasible based on an image-derived input function in a rat ischemia model. The kinetic modeling analysis performed in this study will be useful for the quantitative evaluation of (68)Ga-NOTA-c(RGDyK) binding to αvß3 in angiogenic tissues.

Real-time near infrared fluorescence (NIRF) intra-operative imaging in ovarian cancer using an α(v)ß(3-)integrin targeted agent.

BACKGROUND: In ovarian cancer, optimal cytoreductive surgery is of the utmost importance for long-term survival. The ability to visualize minuscule tumor deposits is important to ensure complete resection of the tumor. The purpose of our study was to estimate the in vivo sensitivity, specificity and diagnostic accuracy of an intra-operative fluorescence imaging system combined with an α(v)ß(3)-integrin targeted near-infrared fluorescent probe. METHOD: Tumor bearing mice were injected intravenously with a fluorescent probe targeting α(v)ß(3) integrins. Fluorescent spots and non-fluorescent tissue were identified and resected. Standard histopathology and fluorescence microscopy were used as gold-standard for tumor detection. RESULTS: Fifty-eight samples excised with support of intra-operative image-guided surgery were analyzed. The mean target to background ratio was 2.2 (SD 0.5). The calculated sensitivity of the imaging system was 95%, and the specificity was 88% with a diagnostic accuracy of 96.5%. CONCLUSION: Near-infrared image-guided surgery in this model has a high diagnostic accuracy and a fair target to background ratio that supports the development towards clinical translation of α(v)ß(3)-integrin targeted imaging.