Nanoparticles have been assessed in preclinical models of atherosclerosis for detection of plaque complexity and treatment. However, their successful clinical translation has been hampered by less than satisfactory plaque detection and lack of a general strategy for assessing the translational potential of nanoparticles. Herein, nanoparticles based on comb-co-polymer assemblies were synthesized through a modular construction approach with precise control over the conjugation of multiple functional building blocks for in vivo evaluation. This high level of design control also allows physicochemical properties to be varied in a controllable fashion. Through conjugation of c-atrial natriuretic factor (CANF) peptide and radiolabeling with 64Cu, the 64Cu-CANF-comb nanoparticle was assessed for plaque imaging by targeting natriuretic peptide clearance receptor (NPRC) in a double-injury atherosclerosis model in rabbits. The prolonged blood circulation and enhanced binding capacity of 64Cu-CANF-comb nanoparticles provided sensitive and specific imaging of NPRC overexpressed in atherosclerotic lesions by positron emission tomography at intervals during the progression of the disease. Ex vivo tissue validation using autoradiography and immunostaining on human carotid endarterectomy specimens demonstrated specific binding of 64Cu-CANF-comb to human NPRC receptors. Taken together, this study not only shows the potential of NPRC-targeted 64Cu-CANF-comb nanoparticles for increased sensitivity to an epitope that increases during atherosclerosis plaque development but also provides a useful strategy for the general design and assessment of the translational potential of nanoparticles in cardiovascular imaging.

Enzyme active sites afford an intricate interplay of functional groups to mediate complex organic and inorganic reactions. Many hydrolytic enzymes use a catalytic triad comprising three different functional residues—(Ser(-OH), Hist(-imidazole), Asp(-CO2H))—that catalyze the hydrolysis of numerous unique substrates. Inspired by this design, we have developed a simple one-step synthesis for preparing a new supported catalytic system in which the three reactive groups of the catalytic triad (alcohol, imidazole, and carboxylate) are incorporated into a single functional unit. These artificial active sites can be coupled to a solid-phase support (Merrifield resin) by copper(I)-catalyzed azide-alkyne cycloaddition “click chemistry,” and their effectiveness as esterolysis catalysts was demonstrated. Furthermore, tuning the local hydrophobicity of the resin particles with an approach analogous to the native enzyme hydrophobic pocket increased the catalytic efficiency. Quantum mechanics and molecular dynamics computational modeling were used to probe the catalytic effect and suggested a concerted two-step mechanism and hydrophobic nanoenvironment similar to that of hydrolytic enzymes.

UNLABELLED:Atherosclerosis is inherently an inflammatory process that is strongly affected by the chemokine-chemokine receptor axes regulating the trafficking of inflammatory cells at all stages of the disease. Of the chemokine receptor family, some specifically upregulated on macrophages play a critical role in plaque development and may have the potential to track plaque progression. However, the diagnostic potential of these chemokine receptors has not been fully realized. On the basis of our previous work using a broad-spectrum peptide antagonist imaging 8 chemokine receptors together, the purpose of this study was to develop a targeted nanoparticle for sensitive and specific detection of these chemokine receptors in both a mouse vascular injury model and a spontaneously developed mouse atherosclerosis model. METHODS:The viral macrophage inflammatory protein-II (vMIP-II) was conjugated to a biocompatible poly(methyl methacrylate)-core/polyethylene glycol-shell amphiphilic comblike nanoparticle through controlled conjugation and polymerization before radiolabeling with (64)Cu for PET imaging in an apolipoprotein E-deficient (ApoE(-/-)) mouse vascular injury model and a spontaneous ApoE(-/-) mouse atherosclerosis model. Histology, immunohistochemistry, and real-time reverse transcription polymerase chain reaction were performed to assess the plaque progression and upregulation of chemokine receptors. RESULTS:The chemokine receptor-targeted (64)Cu-vMIP-II-comb showed extended blood retention and improved biodistribution. PET imaging showed specific tracer accumulation at plaques in ApoE(-/-) mice, confirmed by competitive receptor blocking studies and assessment in wild-type mice. Histopathologic characterization showed the progression of plaque including size and macrophage population, corresponding to the elevated concentration of chemokine receptors and more importantly increased PET signals. CONCLUSION:This work provides a useful nanoplatform for sensitive and specific detection of chemokine receptors to assess plaque progression in mouse atherosclerosis models.

To assess the physicochemical properties, pharmacokinetic profiles, and in vivo positron emission tomography (PET) imaging of natriuretic peptide clearance receptors (NPRC) expressed on atherosclerotic plaque of a series of targeted, polymeric nanoparticles.

353 Objectives There is intense interest to develop a molecular imaging probe to detect stages of atherosclerotic plaque stability. Natriuretic peptide receptor C (NPRC) is an attractive target because it is up-regulated in various plaque components. We have developed a nanoparticle-based radiopharmaceutical, 64Cu-25%-CANF-Comb, that targets this receptor and permits noninvasive plaque detection in pre-clinical models of atherosclerosis. We have now begun safety, biodistribution and dosimetry testing in human subjects. Methods After obtaining eIND approval from the FDA for 64Cu-25%-CANF-Comb, we intravenously injected ~5.6 mCi of the radiopharmaceutical into the first 2 of 8 normal volunteers (1M, 1F; average 35 yrs). Subjects underwent a whole body PET-CT at 1-4, 5-10 and 24 hours p.i., for evaluation of biodistribution and radiation dosimetry, with vital signs (heart rate, respiratory rate, blood pressure), serum laboratory assessment, urinalysis and EKG at baseline, and p.i at imaging time points. Results There were no significant differences in vital signs, blood or urine assays, or EKG results between baseline and post-injection values. Activity was observed to predominantly accumulate in the liver and spleen with a long blood retention time, 15% excreted through urine. Dosimetry showed the critical organ was liver at ~1 rad/mCi with an Effective Dose of 0.14 rem/mCi. Pre-clinical studies showed critical organs to be bone surface, 0.307 rad/mCi and heart, 0.245 rad/mCi. Conclusions 64Cu-25%-CANF-Comb, a nanoparticle PET radiopharmaceutical for atherosclerosis imaging has been translated into humans. Although requiring further study, initial toxicity, biodistribution and dosimetry data suggest that this radiopharmaceutical will be safe for human use in setting the stage for more extensive studies to evaluate its diagnostic performance. Research Support This work is supported by the National Institutes of Health as a Program of Excellence in Nanotechnology (HHSN268201000046C).

1234 Objectives The goal of this study was to determine the in vivo stability, distribution, and metabolic pathway of C-atrial natriuretic factor (CANF) conjugated to comb-like nanoparticles. The 64Cu-CANF-Comb has been shown to bind to the natriuretic peptide receptor-C (NPR-C) upregulated in atherosclerosis. Methods The radiolabeling stability of 64Cu-CANF-Comb nanoparticles was determined by incubating the particles with mouse serum and analyzing with fast protein liquid chromatography (FPLC). After intravenous injection of 64Cu-CANF-Comb in C57 mice, blood samples were collected and separated into red blood cells, platelet-rich plasma, platelet-poor plasma, and the platelet pellet via centrifugation and counted with a gamma counter. The platelet-poor plasma was analyzed with FPLC to assess the stability of 64Cu-CANF-Comb. The longitudinal distribution of CANF-Comb in vivo was performed via 67Cu radiolabeling up to 2 weeks. The excretion of 67Cu-CANF-Comb was collected to evaluate its metabolic pathway. Results FPLC analysis showed 71.4±0.8% (n=3) intact 64Cu-CANF-Comb after 48h incubation with mouse serum. The longitudinal in vivo evaluation of 67Cu-CANF-Comb showed Conclusions The in vivo evaluation of radiolabeled CANF-Comb showed radiolabeling stability for PET imaging with minimal non-specific binding to blood cells. The longitudinal stability and significant clearance of CANF-Comb suggests its potential for translational research. Research Support This work is supported by the National Institutes of Health as a Program of Excellence in Nanotechnology (HHSN268201000046C).

1236 Objectives We have demonstrated the specific PET imaging of CCR5 important to atherosclerosis with 64Cu-DAPTA peptide tracer in a vascular injury model. Herein, we aim to demonstrate improved CCR5 detection specificity and sensitivity with DAPTA peptides conjugated nanoparticles in a clinically relevant model of atherosclerosis. Methods The DAPTA-Comb nanoparticles were prepared with controlled conjugation of DAPTA peptides and DOTA. Biodistribution of 64Cu-DAPTA-Comb was performed in C57 mice. Aortic artery (AA) CCR5 detection efficiency and specificity was assessed in ApoE-/- mice on western diet and age-matched C57 mice on normal chow with PET/CT, respectively. Histopathological characterization of CCR5 was performed on the AA after scan. PET blocking studies were performed to evaluate CCR5 targeting specificity. Results Biodistribution showed extended blood circulation and reduced hepatic and splenic accumulation of 64Cu-DAPTA-Comb relative to 64Cu-Comb and 64Cu-DAPTA peptide. 64Cu-DAPTA-Comb showed significantly higher AA uptake (7.5 ± 1.0 %ID/g, p Conclusions The 64Cu-DAPTA-Comb affords sensitive and specific imaging of CCR5 that is superior to 64Cu-DAPTA peptide in atherosclerotic lesion. Its superior pharmacokinetics and targeting efficiency make it a promising candidate tracer for further evaluation. Research Support This work is supported by the NHLBI Program of Excellence in Nanotechnology (HHSN268201000046C).

461 Objectives The goal of this study was to assess the imaging capability of C-type atrial natriuretic factor (CANF) conjugated nanoparticles to target the natriuretic peptide clearance receptor (NPR-C) up-regulated in a clinically relevant model of atherosclerosis. Methods CANF-Comb nanoparticles were prepared through modular construction with controlled functionalities for PET imaging of NPR-C receptor via radiolabeling with 64Cu. The efficiency of the construct was demonstrated in ApoE-/- mice fed a high cholesterol diet to form spontaneous atherosclerotic lesions in the aorta. Age-matched C57BL6 mice fed a normal diet were the control animals. PET/CT imaging was performed at multiple time points to follow the progression of disease. The aortic arteries were collected post scan for histopathological examination and RT-PCR analysis. Blocking studies were also performed to confirm targeting specificity. Results PET studies show greater uptake of 64Cu-CANF-Comb in the ApoE-/- mice compared to the C57BL6 mice on 18 weeks of diet with standard uptake values (SUV) of 5.35 (SD = 1.01) and 3.30 (SD = 1.13), respectively (P Conclusions PET/CT imaging showed the capability of the 64Cu-CANF-Comb to image plaque in a pre-clinical model of spontaneous atherosclerosis development. Its in vivo pharmacokinetics and plaque uptake indicate translational potential as a PET imaging agent for early-stage atherosclerosis in humans. Research Support This work is supported by the National Institutes of Health as a Program of Excellence in Nanotechnology (HHSN268201000046C).

Inflammation plays important roles at all stages of atherosclerosis. Chemokine systems have major effects on the initiation and progression of atherosclerosis by controlling the trafficking of inflammatory cells in vivo through interaction with their receptors. Chemokine receptor 5 (CCR5) has been reported to be an active participant in the late stage of atherosclerosis and has the potential as a prognostic biomarker for plaque stability. However, its diagnostic potential has not yet been explored. The purpose of this study was to develop a targeted nanoparticle for sensitive and specific PET/CT imaging of the CCR5 receptor in an apolipoprotein E knock-out (ApoE(-/-)) mouse vascular injury model. Methods: The D-Ala1-peptide T-amide (DAPTA) peptide was selected as a targeting ligand for the CCR5 receptor. Through controlled conjugation and polymerization, a biocompatible poly(methyl methacrylate)-core/polyethylene glycol-shell amphiphiliccomblike nanoparticle was prepared and labeled with Cu-64 for CCR5 imaging in the ApoE(-/-) wire-injury model. Immunohistochemistry, histology, and real-time reverse transcription polymerase chain reaction (RT-PCR) were performed to assess the disease progression and upregulation of CCR5 receptor. Results: The Cu-64-DOTA-DAPTA tracer showed specific PET imaging of CCR5 in the ApoE-/- mice. The targeted Cu-64-DOTA-DAPTA-comb nanoparticles showed extended blood signal and optimized biodistribution. The tracer uptake analysis showed significantly higher accumulations at the injury lesions than those acquired from the sham-operated sites. The competitive PET receptor blocking studies confirmed the CCR5 receptor-specific uptake. The assessment of Cu-64-DOTA-DAPTA-comb in C57BL/6 mice and Cu-64-DOTA-comb in ApoE(-/-) mice verified low nonspecific nanoparticle uptake. Histology, immunohistochemistry, and RT-PCR analyses verified the upregulation of CCR5 in the progressive atherosclerosis model. Conclusion: This work provides a nanoplatform for sensitive and specific detection of CCR5's physiologic functions in an animal atherosclerosis model.

Atrial natriuretic peptide has been recently discovered to have anticancer effects via interaction with cell surface natriuretic peptide receptor A (NPRA) and natriuretic peptide clearance receptor (NPRC). In a preclinical model, NPRA expression has been identified during tumor angiogenesis and may serve as a potential prognostic marker and target for prostate cancer (PCa) therapy. However, the presence of NPRC receptor in the PCa model has not yet been assessed. Furthermore, there is still no report using nanoparticle for PCa positron emission tomography (PET) imaging. Herein, an amphiphilic comb-like nanoparticle was synthesized with controlled properties through modular construction containing C-atrial natriuretic factor (CANF) for NPRC receptor targeting and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) chelator for high specific activity Cu-64 radiolabeling. The pharmacokinetics of 64Cu-CANF-Comb exhibited tuned biodistribution and optimized in vivo profile in contrast to the nontargeted 64Cu-Comb nanoparticle. PET imaging with 64Cu-CANF-Comb in CWR22 PCa tumor model showed high blood pool retention, low renal clearance, enhanced tumor uptake, and decreased hepatic burden relative to the nontargeted 64Cu-Comb. Immunohistochemistry staining confirmed the presence of NPRC receptor in tumor tissue. Competitive PET receptor blocking study demonstrated the targeting specificity of 64Cu-CANF-Comb to NPRC receptor in vivo. These results establish a new nanoagent for prostate cancer PET imaging.