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Biological and Medical Applications of Materials and Interfaces
Assessment of Targeted Nanoparticle Assemblies for Atherosclerosis Imaging with Positron Emission Tomography and Potential for Clinical Translation Yongjian Liu, Hannah Luehmann, Lisa Detering, Eric D Pressly, Alaina J McGrath, Deborah Sultan, Annie Nguyen, Susannah Grathwohl, Monica Shokeen, Mohamed Zayed, Robert J Gropler, Dana Abendschein, Craig J. Hawker, and Pamela K Woodard ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b02750 • Publication Date (Web): 10 Apr 2019 Downloaded from http://pubs.acs.org on April 11, 2019
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ACS Applied Materials & Interfaces
Assessment of Targeted Nanoparticle Assemblies for Atherosclerosis Imaging with Positron Emission Tomography and Potential for Clinical Translation
Yongjian Liu,*† Hannah P. Luehmann,† Lisa Detering,† Eric D. Pressly,‡ Alaina J. McGrath, ‡ Deborah H. Sultan,† Annie Nguyen,§ Susannah Grathwohl, § Monica Shokeen,† Mohamed Zayed,, Robert J. Gropler,† Dana Abendschein,§ Craig J. Hawker,‡ Pamela K. Woodard*†
† Mallinckrodt Institute of Radiology, Washington University, St. Louis, MO 63110, United States ‡ Materials Department, University of California, Santa Barbara, CA 93106, United States § Department of Medicine, Washington University, St. Louis, MO 63110, United States
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ABSTRACT: 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-copolymer 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 (PET) at intervals during the progression of 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 translational potential of nanoparticles in cardiovascular imaging.
KEYWORDS: Nanoparticle, NPRC, atherosclerosis, positron emission tomography, translation
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ACS Applied Materials & Interfaces
Introduction Cardiovascular disease (CVDs) is the leading killer worldwide with atherosclerosis being the primary cause. It is a chronic inflammatory disease characterized by the accumulation of lipids, immune cells and cell debris in the vessel wall. It is the main underlying cause of CVDs including myocardial infarction, stroke, and peripheral arterial disease.1-4 Due to the chronic nature and long asymptomatic phase, the clinical challenge of atherosclerosis is predicting when a plaque may cause vessel occlusion and associated management of treatment to avoid fatal consequences.5 Although significant progresses have been achieved in risk factor modification, anatomic imaging techniques, new therapeutics, and revascularization procedures, the prevalence of CVDs and its complications are not only projected to increase by 12% by 2030,6 but also result in a significant financial burden.6 Complex plaque pathophysiology results from the dynamic expression and often low abundance of disease-related targets on advanced plaques, a scenario that limits clinically available anatomic imaging modalities from reliably visualizing specific cellular features or biological processes that identify highrisk individuals for targeted treatment. Thus, nanoparticles have demonstrated the advantages for early and sensitive detection of plaque and played more roles in drug discovery for cardiovascular diseases.5, 7-9 Of various imaging modalities, radionuclide-based molecular imaging, especially positron emission tomography (PET), has been extensively used for CVDs research due to its superior sensitivity, quantification, functional detection, non-invasive nature, and well-established pathways for human translation.10-12 Compared to small molecules or peptide-based imaging platforms with rapid in vivo pharmacokinetics, nanoparticles provide unique advantages for atherosclerosis imaging and therapy including extended blood circulation, improved sensitivity and specificity, and elevated drug loading capacity for theranostics.13-19 Previously, we reported a polymeric nanoparticle (64Cu-CANF-Comb) for the detection of natriuretic peptide clearance receptor (NPRC) that is overexpressed on plaques in a mouse apoE knock-out (apoE-/-) model using PET.20 This polymeric nanoparticle can be prepared using modular strategy that enables the
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synthesis in large quantify necessary for translational studies, we wanted to further assess this nanoprobe for plaque imaging in rabbits with advanced atherosclerosis.21 Moreover, based on our previous report showing up-regulation of NPRC receptor in the intima of advanced atherosclerotic lesions in the carotid arteries of patients who underwent carotid endarterectomy (CEA),22 we characterized the binding profile of
64Cu-CANF-Comb
to human NPRC receptors expressed on CEA specimens using ex vivo
autoradiography. We hypothesized that the combination of an advanced preclinical model and human CEA specimens would be a useful strategy to determine the potential of
64Cu-CANF-Comb
and other
agents for future assessment of atherosclerosis severity in patients using PET imaging. RESULTS AND DISCUSSION Synthesis of CANF-Comb nanoparticle The CANF-Comb nanoparticle was synthesized following a previous report to afford control over the conjugation of CANF on the nanostructure.20 The methacrylate-based functional monomers including PEG-methacrylate (PEGMA), CANF-PEG-methacrylate (CANF-PEGMA), and DOTA-methacrylate (DOTA-MA) were copolymerized with methyl methacrylate to prepare the CAN-Comb copolymer as outlined in Figure 1a. The controlled radical polymerization strategy enables the precise control of functional components incorporated in CANF-Comb by varying the feed ratios of the functional monomers, affording desirable physicochemical properties and targeting capability of the final CANFComb nanoparticles after assembly. As shown in Table 1, the targeted CANF-Comb nanoparticle has ~35 copies of CANF peptide on the surface for NPRC receptor targeting and ~105 copies of DOTA chelator buried in the core for 64Cu radiolabeling. PET imaging in rabbit atherosclerosis model At 4 weeks after air desiccation-induced injury of the femoral arteries in hypercholesterolemic rabbits, 64Cu-CANF-Comb
PET showed significant uptake in the injured artery with minimal signal observed in
the sham-operated artery (Figure 2) at 24 h post injection. Quantification showed that the SUV of 64Cu-
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ACS Applied Materials & Interfaces
CANF-Comb at the injury site was 4.93±0.98 (n=12), which was 2.5 times as much as that determined from the sham site (1.96 ± 0.66, n=12, p< 0.001). A competitive receptor blocking study, using an excess amount of non-radiolabeled CANF-Comb, resulted in more than 50% decrease of
64Cu-CANF-Comb
uptake at the injured site (2.36 ± 0.62, n=4) to a level not significantly different from the sham artery (1.53± 0.21, n=4) (Figure 2E, G), demonstrating the specificity of
64Cu-CANF-Comb
targeting NPRC
receptor expressed on plaque. Compared to NPRC-targeted
64Cu-CANF-Comb,
without the conjugation of CANF peptide, the non-
targeted 64Cu-Comb showed significantly lower tracer accumulation at the injury site at TP 1 (1.99 ± 0.88, p
