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VCAM-Targeted MS2 Viral Capsids for the Detection of Early-Stage Atherosclerotic Plaques Ioana Laura Aanei, Tony Huynh, Youngho Seo, and Matthew B Francis Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00453 • Publication Date (Web): 30 Jul 2018 Downloaded from http://pubs.acs.org on July 31, 2018
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VCAM-Targeted MS2 Viral Capsids for the Detection of Early-Stage Atherosclerotic Plaques Ioana L. Aanei,†,‡ Tony Huynh,§ Youngho Seo,§ and Matthew B. Francis †,‡,* Department of Chemistry, University of California, Berkeley, California 94720, USA. ‡Materials Sciences Division, Lawrence Berkeley National Laboratories, Berkeley, California 94720, USA. §Department of Radiology and Biomedical Imaging, University of California, San Francisco, California 94143, USA. †
*Corresponding author. Email:
[email protected] ABSTRACT
descending aorta
Atherosclerosis is a cardiovascular disease characterized by the foraortic arch mation of lipid-rich plaques within the walls of large arteries. Over time, a portion of these lesions can detach and lead to serious complications, such as strokes or heart attacks. Currently, there is no clinically effective way to plaques detect the presence of atherosclerosis in patients until it has reached a relatively advanced stage. Furthermore, increasing evidence suggests that the pathobiological behavior of plaques is determined mainly by their composi27 nm aortic arch tion, and not their size, which is the parameter usually monitored with current Early detection of atherosclerotic imaging techniques. In this work, we report protein-based agents that target plaques with VCAM-targeted protein nanoparticles the vascular cell adhesion molecule (VCAM1), a protein that plays a crucial brightfield fluorescence role in atherosclerosis progression. In vivo experiments with murine atherosclerosis models indicated that the targeted protein nanoparticles were successful in detecting plaques of various sizes in the descending aorta and the aortic arch. This finding encourages the further development of these nanoscale agents for applications in the imaging, diagnosis and treatment of cardiovascular diseases. descending aorta
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Despite several decades of considerable therapeutic advances for cardiovascular diseases, these conditions remain the leading causes of death worldwide.1,2 The aging population and changes in dietary habits have expanded the number of individuals with risk factors for atherosclerosis, a disease characterized by the narrowing of the arterial lumen3 and the development of fibrous caps.4 These caps can rupture and prevent blood flow to critical organs, such as the heart (myocardial infarction) or the brain (stroke)2 and therefore can be fatal. It is now appreciated that atherosclerosis involves an inflammation of the blood vessels that occurs in several distinct stages.5 Traditionally, diagnosis of atherosclerosis was only possible at advanced stages of the disease by detecting morphological changes in the arteries through the use of contrast-enhanced X-ray imaging,6,7 magnetic resonance imaging (MRI),8,9 or ultrasound.10 These approaches yield little information about the molecular composition of plaques, the stages of disease progression, or the risk for fibrous cap rupture.4 More recent work in the field has focused on developing methods to detect specific components or events taking place in arterial plaques, such as macrophage11 and lipid accumulation12 or calcification.3
sels, as well as on macrophages present in atherosclerotic lesions.13,14 Its role is to regulate the cell adhesion through interaction with the integrin very late antigen-4 (VLA-4).14,15 It is increasingly recognized that the imaging efficiency of VCAM1-binding constructs could be augmented by the use of nanoscale carriers that can present multiple copies of both the targeting groups and the reporters.16 In addition to increasing the binding affinity through multivalency, such platforms could produce improved levels of signal and allow multimodal detection as tracers are combined in various ways. Successful examples of this strategy include work done by the Steinmetz lab17 to develop a peptide-targeted tobacco mosaic virus nanoparticle. The agents were modified with near-infrared dyes and chelated Gd ions to allow for dual-modal fluorescence and magnetic resonance imaging. In addition, previous work by Yang and coworkers18 studied the uptake of targeted iron oxide nanoparticles in activated human umbilical vein endothelial cells (HUVECs), and work by the Weissleder lab validated the ability of VCAM1-targeted CLIO nanoparticles to accumulate in an ear inflammation model.19-21 These studies and others22-27 serve to validate the ability of nanoparticles bearing VCAM1-binding groups to engage and identify areas of inflammation in vivo.
Our lab has focused on the development of genome-free Targeting moieties such as peptides and antibodies can MS2 viral capsids as versatile nanocarrier platforms for imdetect the presence of markers for the different stages of aging and drug delivery applications. The viral capsid of the atherosclerosis and thus provide a wealth of information. As MS2 is a 27 nm icosahedron comprising 180 sequence-idenan early marker of inflammation, several previous studies tical coat proteins.28 The advantages of this platform for dehave focused on targeting the vascular cell adhesion mole13,14 livery applications include monodispersity, ease of produccule 1 (VCAM1 or CD106). The VCAM1 receptor is extion, the ability to tailor the interior and exterior surfaces pressed on the activated endothelial cells that line blood vesACS Paragon Plus Environment
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Figure 1. Synthesis of VCAM-targeted MS2 agents. (a) Cysteine residues engineered on the interior surface (red) can be modified with AlexaFluor680 (AF680) fluorescent dye with a maleimide functionality (orange spheres). (b) A reaction scheme is shown for the external oxidative coupling strategy. (c) The lysine residues on the αVCAM antibody were coupled to nitrophenol groups using NHS-ester chemistry. The antibody was then attached to the MS2 VLP using an oxidative coupling. NP = nitrophenol, NHS = N-hydroxysuccinimidyl, AP = aminophenol. differentially (Figure 1a,b), and the ability to protect cargo including familial hypercholesterolemia and type III hyperlipfrom degradation. In previous work, we have shown that the idemia, and patients with these conditions have increased capsid can be modified with up to 180 copies of a desired susceptibility to atherosclerosis.15 ApoE-deficient mice show reporter group inside the protein shell with minimal-to-no ef- elevated plasma cholesterol levels, and develop atherofect on biodistribution.29 We have also shown that the in vivo sclerotic plaques either spontaneously (after 3-8 months) or stability of the capsids is excellent, and that a large amount more predictably when fed a high fat diet (plaques form after remains in the blood at 24 h and even 48 h post-injection.30 14 weeks).15 The resistance of MS2 capsids to clearance from the blood To verify that the experimental conditions used led to stream is in sharp contrast to other viral capsids that have the formation of atherosclerotic plaques, descending aortas been evaluated, which are typically cleared completely in from ApoE KO mice and control mice were harvested. The minutes to a few hours.31-34 presence of numerous plaques (opaque white spots) was
To convert these agents into early-stage delivery vehicles for atherosclerotic lesions, we have attached antibodies specific to VCAM to the capsid surfaces (Figure 1c) using protocols reported previously.35 The vast array of antibodies that is already available, combined with their unrivaled affinity and specificity, makes them desirable targeting moieties. We then evaluated the ability of these agents to accumulate in plaques in mouse models, with the finding that high levels of specific binding were observed.
We used optical imaging to determine if the VCAM-targeted nanoparticles could reach the atherosclerotic plaque targets. A fluorescent dye with near-IR properties (AlexaFluor680, AF680) and excellent photostability was chosen to limit the tissue autofluorescence issues commonly encountered at shorter wavelengths. The free antibody labeled with the fluorescent dye served as a positive control. The characterization of the agents is described in Supplementary Figure 1.
observed in the case of ApoE mice (Supplementary Figure S2a). The blood vessels were cryopreserved and sectioned. Hematoxylin/eosin (H&E) staining was performed to investigate the cellular composition of the blood vessel walls. Oil Red O was used to detect the plaques. In Supplementary Figure S2b, it can be observed that the blood vessels harvested from ApoE KO mice have a thicker layer of cells, and Oil Red O staining reveals the presence of a lesion. Therefore, the designed aging process was determined to be successful in developing plaques in ApoE KO mice.
ApoE KO and control mice were injected intravenously (IV) with fluorescently-labeled MS2 agents, PBS or an antibody control. At 24 h post-injection, the mice were sacrificed and perfused with phosphate buffered saline (PBS) and neutral buffered 10% formalin. The main internal organs (liver, pancreas, spleen, kidneys, heart and fat) were harvested and imaged using an IVIS Xenogen 50 system. Figure 2 and Supplementary Figure S3 shows the photographs and The most commonly used animal model for atheroscle- fluorescent channel readings. The count range was adjusted rosis consists of mice with a knockout of the gene encod- to limit the autofluorescence signal, based on the PBS-ining for apolipoprotein E (ApoE), a protein important for the jected control mice. The agents mainly accumulated in the clearance of cholesterol and triglyceride-rich lipoprotein par- liver, kidneys and spleen. Fat tissue was included as a refticles from the blood.15 In humans, mutations in ApoE are erence, given that accumulation in the fat tissue is unlikely. associated with several hereditary hyperlipidemic disorders, The antibody-dye was dosed at the same number of moles ACS Paragon Plus Environment
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Figure 2. Biodistribution of agents. An IVIS imaging system was used to determine the biodistribution of the fluorescently-labeled agents at the 24 h time point. The fluorescent signal was quantified using regions of interest. The average and standard deviation are shown. The main organs where the signal accumulated were the liver (L), kidneys (K) and spleen (S). The pancreas (P), heart (H) and fat (F) had very small amounts of agents still present at 24 h.
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Figure 3e shows the average fluorescence intensities for regions including the blood vessels for n=3 animals (see Supplementary Figure S5 for the specific regions of interest used). These data further confirm the significant accumulation differences between the targeted and untargeted agents. This result is very encouraging for the potential of MS2-based agents to target markers in the cardiovascular system for use in non-invasive imaging, diagnosis, and drug delivery applications.
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After excising the descending aortas and removing the surrounding connective tissue, the blood vessels were imaged using an IVIS Spectrum system. A bin size of 1 was selected for image analysis in order to achieve maximum resolution. The photographs and fluorescent channel are shown in Figure 3a-d for representative samples from each group. In the case of VCAM-targeted agents, the fluorescent signal clearly colocalizes with the opaque spots representing the atherosclerotic plaques, including the small plaques that formed at the branching of arteries from the main aorta. A small amount of signal was colocalized with the plaques in the case of MS2-treated mice. This observation could be explained by the fact that macrophages present in the plaques could interact with the MS2 capsids. In in vitro experiments (data not shown), we observed that activated macrophages can phagocytose the capsid after prolonged incubation. Supplementary Figure S4 shows all the replicates for the MS2- and MS2-aVCAM-treated mice.
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as displayed on the MS2 capsids but did not have the same number of dyes attached, and thus the signal was much weaker.
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Figure 3. Colocalization of agents with atherosclerotic plaques. (a-d) IVIS imaging of representative descending aortas from ApoE KO mice. The photographs are presented juxtaposed to the fluorescent signal represented in the reverse rainbow color scheme. The untargeted MS2 capsids show limited accumulation within the lesions. This accumulation could be explained by interaction with macrophages. For the VCAM-targeted agent, the localization within the lesions is much more pronounced and even In this work, we have synthesized and characterized small lesions on the branching arteries can be observed. The aortic VCAM-targeted MS2 agents that can be used for the detecarch, which is the most likely location for plaque formation, is tion of plaques in the early stages of atherosclerosis developclearly containing the fluorescently-labeled MS2 agents. (e) ment. The MS2 viral capsid has several characteristics that Quantification of average fluorescent signal intensity in the blood are advantageous: the ease of synthesis and modification, vessels harvested (n=3). ACS Paragon Plus Environment
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control over the sites of attachment for the targeting group, solubility, stability and long circulation time in vivo. In addition, this platform is a versatile scaffold that allows numerous combinations of targeting groups and cargo for imaging and drug delivery applications. Future work will focus on developing agents targeting other atherosclerotic markers using the MS2 platform. Imaging agents that can detect markers for the different stages of atherosclerosis could be used for detection of the plaques, as well as for monitoring the effects of treatments on disease progression. Further applications could include the delivery of anti-inflammatory drugs, such as statins or methotrexate, to these lesions using the same carrier platform.36 ASSOCIATED CONTENT The Supporting Information is available free of charge on the ACS Publications website at DOI: . Synthesis and characterization of agents used; Pathology of control and ApoE knockout mice; Biodistribution of agents; Colocalization of agents with atherosclerotic plaques. ACKNOWLEDGEMENTS This work was supported by the NIH grant R21 EB018055. The authors would like to acknowledge Dr. Tovo David for providing invaluable insight regarding the experimental details. We are grateful to Dr. Adel ElSohly for helpful discussions. REFERENCES 1. Mozaffarian, D., Benjamin, E.J., Go, A.S., Arnett, D.K., Blaha, M.J., Cushman, M., Das, S.R., de Ferranti, S., Després, J.P., Fullerton, H.J., et al. (2016) Heart Disease and Stroke Statistics-2016 Update: A Report From the American Heart Association. Circulation 133, e38-360. 2. Libby, P. (2002) Inflammation in Atherosclerosis. Nature 420, 868-874. 3. Otsuka, F., Sakakura, K., Yahaigi, K., Joner, M. and Virmani, R. (2014) Has Our Understanding of Calcification in Human Coronary Atherosclerosis Progressed?, Atherioscler Thromb Vasc Biol. 34, 724-736. 4. Yamashita, A. and Asada, Y. (2011) A Rabbit Model of Thrombosis on Atherosclerotic Lesions. J Biomed Biotechnol. 2011, 424929. 5. Libby, P., Ridker, P.M. and Hansson, G.K. (2011) Progress and Challenges in Translating the Biology of Atherosclerosis. Nature 473, 317-325. 6. Choudhury, R.P., Fuster, V. and Fayad, Z.A. (2004) Molecular, Cellular and Functional Imaging of Atherothrombosis. Nat Rev Drug Discov. 3, 913-925.
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