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Sep 13, 2016 - In Vivo Targeting and Imaging of Atherosclerosis Using. Multifunctional Virus-Like Particles of Simian Virus 40. Xianxun Sun,. †,§,â...
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In Vivo Targeting and Imaging of Atherosclerosis Using Multifunctional Virus-Like Particles of Simian Virus 40 Xianxun Sun,†,§,∥ Wei Li,† Xiaowei Zhang,† Mi Qi,†,§ Zhiping Zhang,† Xian-En Zhang,*,‡ and Zongqiang Cui*,† †

State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China § Graduate University of Chinese Academy of Sciences, Beijing 100049, China ∥ College of Life Science, Jiang Han University, Wuhan 430056, China ‡

S Supporting Information *

ABSTRACT: Atherosclerosis is a leading cause of death globally. Targeted imaging and therapeutics are desirable for the detection and treatment of the disease. In this study, we developed trifunctional Simian virus 40 (SV40)-based nanoparticles for in vivo targeting and imaging of atherosclerotic plaques. These novel trifunctional SV40-based nanoparticles encapsulate near-infrared quantum dots and bear a targeting element and a drug component. Using trifunctional SV40based nanoparticles, we were able to noninvasively fluorescently image atherosclerotic plaques in live intact ApoE(−/ −) mice. Near-infrared quantum dots encapsulated in the SV40 virus-like particles showed prominent optical properties for in vivo imaging. When different targeting peptides for vascular cell adhesion molecule-1, macrophages, and fibrin were used, early, developmental, and late stages of atherosclerosis could be targeted and imaged in live intact ApoE(−/−) mice, respectively. Targeted SV40 virus-like particles also delivered an increased concentration of the anticoagulant drug Hirulog to atherosclerosis plaques. Our study provides novel SV40-based nanoparticles with multivalency and multifunctionality suitable for in vivo imaging, molecular targeting, and drug delivery in atherosclerosis. KEYWORDS: SV40 multifunctional nanoparticle, in vivo targeted imaging, quantum dots, Hirulog, atherosclerosis

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One of the main obstacles to molecular imaging in vivo is the opacity of tissues. To reduce tissue absorbance and scattering, excitation and emission light should match the tissue optical window of between 700 and 1200 nm. Near-infrared quantum dots (NIR QDs) provide a good candidate to construct fluorescent probes for in vivo imaging because of the brightness, good photostability, and high detection sensitivity in deep tissues.14 Another concern for atherosclerosis imaging is in vivo targeting and molecular epitope recognition. Atherosclerosis can begin in the first decade of life, when arterial endothelial cells subject to irritating stimuli and express vascular cell adhesion molecule-1 (VCAM-1), which capture leukocytes on their surfaces and enter the inner layers of arteries.15−17 In the tunica intima, monocytes in plaques differentiate into tissue macrophages. Macrophages are key components of vascular inflammation and contribute to the development and complications of atherosclerosis.3,18−21 When plaques develop,

ardiovascular disease results in nearly a third of all deaths worldwide each year.1−3 Atherosclerosis, a systemic disease affecting arteries, is the leading cause of cardiovascular disease. Atherosclerosis results in the formation of plaques in arterial walls and narrows the lumen of arteries, and can cause myocardial infarction and stroke when atherosclerotic plaques rupture. Atherosclerosis development often remains asymptomatic until a clinical event, so atherosclerosis detection is important.4−6 Traditionally, diagnosis of atherosclerosis was possible only at advanced stages of the disease by invasive detection of luminal narrowing and intima-media thickening. Currently, noninvasive molecular imaging methods such as computed tomography, single-photon emission computed tomography, positron emission tomography, magnetic resonance imaging, and optical imaging are in development or clinical testing for accurate diagnosis of the composition of atherosclerotic plaques.7−10 Of these modalities, optical molecular imaging is meaningful and promising because of its high sensitivity, excellent spatial resolution, and fast acquisition and image processing, which may allow real-time diagnosis.8,11−13 However, an appropriate method for optical imaging of atherosclerosis in vivo remains to be developed. © XXXX American Chemical Society

Received: June 10, 2016 Revised: September 12, 2016

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Figure 1. Schematic of study design. Enlarged view of the structure of an engineered VP1 unit (left) shows a blue section that represents the inserted targeting peptide, and a green section that represents the Hirulog peptide at the free N-terminal. Twelve VP1 pentamers with targeting peptides and Hirulog peptides package one QD800 to form trifunctional virus-like particles (VLPs). Trifunctional VLPs were injected into ApoE(−/−) mice via the tail vein for in vivo imaging.

Figure 2. Characterization of trifunctional virus-like particles (VLPs). (a) Purified Hir-M-VP1 examined by SDS-PAGE. (b) Image of a sucrose density gradient centrifugation tube after separation of trifunctional VLPs. Note that F6 was a clear band. (c) Fluorescence spectra of trifunctional MH-QDs compared with quantum dots (QDs). (d) Dynamic light scattering volume distribution vs hydrodynamic diameter for VLP-QDs and MHQDs. (e, f, g, h, i, j) Transmission electron microscopy images of negatively stained MH-QDs, M-QDs, FH-QDs, V-QDs, VLP-QDs, and H-QDs. Scale bars: 50 nm.

fibrin-containing blood clots are deposited on the surface of plaques.21,22 Therefore, VCAM-1, macrophages, and fibrin can be used as targets for atherosclerosis origination, development, and the late stage, respectively.16,18,22 Our own work and the previous work of others has shown that some protein nanoparticles, such as heat shock protein,18 virus capsids,16,23 and ferritins,24,25 can be developed as platforms for molecular targeting and imaging. For example,

we have generated Simian virus 40 (SV40)-based nanoparticles containing quantum dots (QDs) and used the hybrid particles to visualize viral behavior and organelle targeting in live cells.26 Because of their controllable self-assembly, nanoparticle load capacity, and ease of modification, SV40 virus-like particles (VLPs) show great potential as platforms for complex multivalent structures and biomedical applications. B

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Figure 3. Fluorescence imaging of atherosclerosis in live mice. (a) Fluorescence in ApoE(−/−) mice injected with MH-QDs, VLP-QDs, or QDs, respectively, or in wild-type mice injected with MH-QDs (left to right). (b) Fluorescence in ApoE(−/−) mice was quantified by measuring fluorescence intensity (n = 3 per group). (c) ApoE(−/−) mice injected with V-QDs or FH-QDs.

nm, and MH-QDs had the same emission wavelength as that of QDs alone (Figure 2c). Dynamic light scattering analysis demonstrated that MH-QDs were highly monodispersed in aqueous solution and were homogeneous in size, with a diameter of 26 ± 1.2 nm (Figure 2d). In the negatively stained sample (Figure 2e), MH-QDs appeared as protein shells, with almost each having one electron dense core. MH-QDs are morphologically indistinguishable from M-QDs, which have no Hirulog peptides (Figure 2f), or VLP-QDs (Figure 2i) assembled from wild-type VP1 protein, or H-QDs (Figure 2j), which have Hirulog without the targeting peptides. Together, these results showed that the multifunctional SV40 VNPs were successfully developed, and CGNKRTRGC substitution for the HI loop of the VP1 unit and incorporation of the Hirulog peptide into the N-terminus of the VP1 subunit did not interfere with self-assembly of the SV40 VLPs architecture. Further, when replacing the DE loop of VP1 protein with CREKA peptide, we also obtained another trifunctional SV40 VNP, FH-QDs, which should target fibrin at the late stage of atherosclerosis (Figure 2g). A type of SV40 VNPs encapsulating QD800 and presenting a VHSPNKK peptide, V-QDs, was also constructed for targeting VCAM-1, the marker for the original stage of atherosclerosis (Figure 2h). Because the anticoagulant Hirulog is mainly used to treat thrombin at the developmental and late stages of atherosclerosis, we made VQDs without Hirulog. We found no obvious morphological difference between SV40-based VNPs with or without targeting peptides and the anticoagulant Hirulog. Multifunctional MH-QDs were used for atherosclerosis targeting and imaging in vivo. We used ApoE(−/−) mice as

In this study, we developed multifunctional SV40 VLPs encapsulating NIR QDs (QD800) and bearing atherosclerotic targeting elements and the thrombin inhibitor Hirulog. With these multifunctional SV40 virus-based nanoparticles (VNPs), we were able to noninvasively target and fluorescently image atherosclerotic plaques in live intact ApoE(−/−) mice. When different molecular recognition elements were used, atherosclerosis markers at different stages, such as VCAM-1, macrophages, and fibrin, could be targeted in arteries. Targeted SV40 VNPs also delivered a greater concentration of the anticoagulant drug Hirulog to atherosclerotic plaques. A schematic of SV40 VNPs construction is shown in Figure 1. The SV40 major capsid protein VP1 was genetically incorporated with the atherosclerosis-targeting peptide by substitution of its HI loop and a fused anticoagulant peptide into its N-terminus. The engineered VP1 fusion proteins then undergo self-assembly with QD800 to form trifunctional SV40 VLPs encapsulating NIR QDs, and carry the atherosclerosistargeting element and the thrombin inhibitor Hirulog. We first inserted the CGNKRTRGC peptide, which targets macrophage cells in the vessel wall,18−20 into the HI loop region of VP1 protein. Hirulog peptide was fused to the Nterminal of VP1 protein. The fusion protein Hir-M-VP1 was expressed, purified (Figure 2a), and then assembled with QD800. The assembled VNPs, called MH-QDs, were purified through sucrose density gradient centrifugation. In the sucrose density gradient centrifugation tube, F6 was the major fluorescence band observed that contained the most uniform MH-QDs, as examined by transmission electron microscopy (Figure 2b). Fluorescence measurement showed that fluorescence emission of purified MH-QDs in F6 peaked at ∼780 C

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Figure 4. Ex vivo imaging of the aortic tree of atherosclerotic mice. (a) In situ fluorescence images of aortas taken from mice injected with MH-QDs, VLP-QDs, and QDs. (b) Ex vivo fluorescence of aortas from ApoE(−/−) mice injected with MH-QDs, VLP-QDs, and QDs. (c) Quantitative image analysis showing average fluorescence intensity per sample of virus-like particle (n = 3 per group). (d) In vitro fluorescence images of various tissues and organs taken from mice injected with MH-QDs.

in atherosclerotic mice (Figure 3c). Quantitative image analysis indicated a 3.3-fold increase in the accumulation of V-QDs vs nontargeted VLP-QDs and a 6.5-fold increase vs QDs (Figure S1a). For FH-QDs, a 3.8-fold increase in accumulation vs nontargeted VLP-QDs and a 7.2-fold increase vs QDs were observed (Figure S1b). Interestingly, these two SV40-based VNPs showed different fluorescence localization patterns in mice. FH-QDs showed concentrated patches, which might target fibrin-containing blood clots, whereas V-QDs showed a fluorescence signal along the back of the spine, similar to that for MH-QDs. In our experiments, we also detected the fluorescence signals of the targeted VNP-QDs in live mice at different postinjection time periods. As shown in Figure S2, the fluorescence signals of V-QDs at 12 or 24 h postinjection were lower compared with those at 3 h postinjection, but could still be detected at 72 h postinjection. The fluorescence signal from the targeting SV40 VNPs was imaged and evaluated with in situ and ex vivo imaging. As shown in Figure 4a, a greater signal for targeted MH-QDs compared with nontargeted VLP-QDs was observed in regions where the brachiocephalic artery and lower aortic arch are located. The brachiocephalic artery and lower aortic arch contained most of the atherosclerotic lesions. The aortic tree was excised after perfusion and imaged ex vivo (Figure 4b). Quantitative fluorescence comparison of the isolated aortic tree indicated a 3.4-fold increase in accumulation of MH-QDs vs nontargeted VLP-QDs and an 8.8-fold increase in accumulation of MH-QDs vs QDs (Figure 4c). These results indicate that MH-QDs are able to specifically target the diseased vasculature in atherosclerotic mice. Similar targeting results were also acquired for the other two types of multifunctional SV40 VNPs, V-QDs (Figure S3a) and FH-QDs (Figure S3c), which target VCAM-1 and fibrin, respectively. Targeting SV40 VLP-QDs did not bind substantially to other tissues, including the lung,

the animal model. The developmental stages of atherosclerotic plaques were induced in ApoE(−/−) mice by keeping them on a high-fat diet for 8−16 weeks as described previously.27 Mice were intravenously injected with 150 μL MH-QDs (2 mg/mL) in phosphate-buffered saline. Fluorescence signals of NIR QDs were detected at 3 h postinjection. Fluorescence images are shown in Figure 3a. Results showed that mice intravenously injected with MH-QDs had a strong and obvious fluorescence signal along the back of the spine (Figure 3a). The aortic tree spans the arch, thoracic, and abdominal regions of the spine. It is well known that the aortic arch, thoracic aorta, and celiac artery easily develop atherosclerotic plaques. As a control, mice injected with nontargeted VLP-QDs at the same concentration only showed a very weak fluorescence signal (Figure 3a). Mice injected with QDs also showed no obvious fluorescence signal along the spine. Additionally, healthy mice injected with MHQDs showed no obvious increase in fluorescence signal along the spine. Quantitative comparison of in vivo fluorescence signals revealed a large difference among mice injected with target MH-QDs (fluorescence intensity in photons/s/cm2: 2659 ± 120), those injected with nontargeted VLP-QDs (photons/s/cm2: 742 ± 80), and those injected with naked QDs (photons/s/cm2: 389 ± 50) at equal concentrations. The difference was statistically significant (P ≤ 0.05). Quantitative image analysis indicated a 3.6-fold increase in the accumulation of MH-QDs vs nontargeted VLP-QDs and a 6.8-fold increase vs QDs (Figure 3b). These results indicate that MH-QDs are able to specifically target and concentrate in areas that are prone to atherosclerotic plaque formation in atherosclerotic mice. Multifunctional V-QDs and FH-QDs, which target VCAM-1 and fibrin, respectively, were also examined for atherosclerosis targeting and imaging in the early and late stages of atherosclerotic ApoE(−/−) mice.27 The results showed that both V-QDs and FH-QDs could specifically target and concentrate in areas prone to atherosclerotic plaque formation D

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Figure 5. Representative confocal images of cryosectioned aortas from ApoE(−/−) mice. (a) Histology analyses of VNPs homed to different part of aortas. Fluorescence colocalization (yellow) showed that V-QDs targeted to the endothelial surface of the aortas, MH-QDs accumulated inside the plaque tissue, and FH-QDs homed to the surface of the plaques Images were taken at a 10× magnification. (b) Serial cross sections (5 μm thick) were stained with antibodies for CD68 to show macrophages (green). Quantum dots (QDs) are shown in red and cell nuclei (stained with DAPI) are shown in blue. Images were taken at a 60× magnification.

kidney, and pancreas, but small quantities were found in the liver, spleen, and heart (Figure 4d). Localization of targeted SV40 VNPs in atherosclerotic plaques was further confirmed by immunofluorescenece imaging of cross-sectioned aortas. As shown in Figure 5a, histology analyses of aortas demonstrated that V-QDs targeted to the endothelial surface of the aortas. MH-QDs accumulated inside the plaque tissue, and FH-QDs homed to the surface of the plaques (Figure 5a). Representative images of sectioned and stained aortas from mice injected with MH-QDs are shown in Figure 5b. Aorta sections were stained for macrophages (with a CD68 antibody) to confirm the presence of plaques.

Fluorescence from MH-QDs was seen to colocalize with macrophages in plaques. As the control, sectioned aortas from ApoE(−/−) mice injected with VLP-QDs had little QDs signal in the plaques. Co-staining of the V-QDs or FH-QDs with the macrophage marker for the early and late stages of atherosclerotic plaques also showed that there was no obvious colocalization for the V-QDs and FH-QDs nanoparticles with macrophages. These results demonstrate the selective accumulation of MH-QDs in macrophage-rich vascular lesions. MHQDs specifically targeted atherosclerotic plaques. Similarly, VQDs and FH-QDs were found to target atherosclerotic plaques using immunohistochemistry (Figure S4). E

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Figure 6. Targeting of Hirulog to atherosclerotic plaques. (a) MH-QDs were tested for antithrombin activity by comparison with equal molar concentrations of Hirulog peptide MH-QDs. Hirulog peptide showed similar activity in a chromogenic assay. (b) MH-QDs and H-QDs were injected intravenously into ApoE(−/−) mice or wild-type mice and allowed to circulate for 3 h. The aortic tree was excised and analyzed for bound Hirulog (n = 3 per group). (c) FH-QDs and H-QDs were injected intravenously into ApoE(−/−) mice or wild-type mice and allowed to circulate for 3 h. The aortic tree was excised and analyzed for bound Hirulog (n = 3 per group).

assembly of VLPs was not affected. Additionally, two types of targeting SV40 VNPs bearing the anticoagulant drug were developed, which might be used to treat thrombin at the developmental and late stages of atherosclerosis. Our results demonstrate that SV40 VLPs are a good platform for the development of in vivo targeting and imaging agents. Because one SV40 VNP presents 60 copies of the targeting peptide on its exterior surface and bears 60 copies of Hirulog peptide in the cavity, these multivalent particles would beneficially enhance the modest affinity of the targeting peptide and increase the drug concentration at the targeted region. Multifunctional SV40 VNPs were used for targeting and imaging of atherosclerotic plaques in ApoE(−/−) mice. QD800 encapsulated in VNPs produced a sufficient signal to visualize plaques in mice. Compared with traditional fluorescence dyes, CdTe QD800 can emit in the near-infrared range, which penetrates deeper into tissues with very little absorbance by the tissues and very little signal interference from autofluorescence, making it a powerful tool for fluorescence imaging of atherosclerotic plaques in mice. To our knowledge, this is the first time that fluorescence imaging of atherosclerosis in live atherosclerotic mice has been realized. Considering the fact that one SV40 VNP presents 60 copies of the targeting peptide on its exterior, the significant increase in sensitivity can be explained by multivalent display molecular targeting in combination with near-infrared fluorescence. More importantly, exploitation of SV40 VLPs as platforms for imaging probes is not limited to QDs fluorescence imaging. SV40 VLPs could be ideal containers to load agents for a variety of imaging techniques such as magnetic resonance imaging,31,32 computed tomography,33,34 positron emission tomography,35,36 and single-photon emission computed tomography,34,37 and combinations of these techniques are promising for multimodal detection of atherosclerosis.17,38 The specificity of VNPs targeting diseased vasculature was evident from a number of observations. First, in vivo imaging showed obvious and extensive fluorescence signals along the aorta line in the backs of atherosclerotic mice injected with targeted VNPs. There was weak or no fluorescence signals in atherosclerotic mice injected with nontargeted VNPs or in wildtype mice. Second, fluorescence signals from targeting VLPQDs in the aortic tree of atherosclerotic mice were found to be localized to known areas of plaque formation, and no

Multifunctional SV40 VNPs also contained an anticoagulant peptide drug, Hirulog, in addition to NIR QDs and the targeting element. Hirulog is a 20-amino-acid peptide comprising an anion-binding exosite of thrombin recognition sequence, a polymeric linker, and a thrombin-active-site specificity sequence.28 Hirulog can inhibit thrombin catalytic activity and prevent thrombus formation and is therefore used for patients with angina.22 Here, the Hirulog was fused to the VP1 unit N terminal and retained antithrombin activity in a chromogenic assay (Figure 6a). When multifunctional SV40 VNPs was injected into atherosclerotic mice, Hirulog was delivered to the atherosclerotic plaques accompanying the fluorescence accumulation in atherosclerotic aortas. As shown in Figure 6b, the antithrombin activity of Hirulog in the excised aortic tree was significantly greater in aortas of mice injected with MH-QDs compared with mice injected with nontargeted VHLP-QDs (3.3 and 1.2 μg Hirulog per mg of tissue; Figure 6b). Targeted MH-QDs also caused significantly greater antithrombin activity in aortas of atherosclerotic mice compared with wild-type mice (0.9 μg/mg of tissue; Figure 6b). As with MH-QDs, FH-QDs also caused significantly greater antithrombin activity in aortas of atherosclerotic mice (Figure 6c). Targeted multifunctional SV40 VNPs are able to selectively deliver Hirulog to atherosclerotic plaques. It was reported that high antithrombin activity in atherosclerotic aortas (1.8 μg/mg of tissue) had been obtained by using the targeting micelles.22 Here, we acquired a higher effective concentration at atherosclerotic plaques than previous reports.29 The aim of this study was to use SV40 VLPs as a platform to encapsulate NIR QDs for use as a targeting element and therapeutic compound for in vivo targeting and imaging of atherosclerosis. On the basis of the crystal structure of SV40 VLP, the HI and DE loops of VP1 are exposed on the exterior surface and can be substituted by foreign sequences.30 We selected three typical peptides targeting different atherosclerosis markers, VCAM-1, macrophages, and fibrin, and inserted them into VP1. Through the assembly of VP1 proteins and NIR QD800, we successfully obtained three SV40 VNPs targeting atherosclerotic markers at the early, developmental, and late stages of atherosclerosis. When the peptide Hirulog, which can inhibit thrombin activity and prevent thrombus formation, was further fused to the free N-terminal of the VP1 protein, the selfF

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fluorescence was observed in corresponding control groups. Third, immunohistochemistry confirmed that targeted VLPQDs colocalized with their targeting atherosclerosis markers. SV40 VNPs bearing different molecular recognition elements specifically targeted different atherosclerosis markers in the artery: V-QDs targeted VCAM-1, MH-QDs targeted macrophages, and FH-QDs targeted fibrin. These three targeted particles showed different fluorescence localization patterns in atherosclerotic mice, and provide targeting imaging agents for the origin, developmental, and late stages of atherosclerosis, which is promising for clinical diagnosis and treatment. We were also able to use targeting VLPs to deliver an anticoagulant. When targeting SV40 VNPs bearing the anticoagulant drug Hirulog were injected into mice, an increased concentration of Hirulog at atherosclerotic plaques was observed. Antithrombin activity was significantly greater in aortas of mice injected with targeting VLPs compared with nontargeting VLPs. In our study, SV40 VNPs targeting macrophages and fibrin were fused with Hirulog, which could be used to treat thrombin at the developmental and late stages of atherosclerosis. These multifunctional VNPs could potentially also reduce the risk of thrombus formation on plaque rupture. Therapeutic drugs such as statins for atherosclerosis could be incorporated into the cage constructs or via loading into the cavity of VPLs for targeted delivery.39 Therefore, SV40 VLPs can be good “theranostic” platforms that could be conveniently incorporated with both imaging probes and therapeutic drugs. More detailed biocompatibility assessment of SV40 VLPs, such as blood half-life and immunogenicity, and comparisons with other protein cages, such as human ferritin or viral capsids, are required. In summary, we successfully constructed multifunctional SV40 VLPs encapsulating NIR QDs and bearing atherosclerotic targeting peptides and the anticoagulant drug Hirulog. These multifunctional SV40 VNPs can be used to specifically target the atherosclerosis markers VACM-1, macrophages, and fibrin for imaging atherosclerosis at different stages in live ApoE(−/ −) mice. The trifunctional SV40 VNPs bearing Hirulog can increase the anticoagulant concentration in plaques. Our work suggests that SV40-VLP is a good “theranostic” platform.

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ACKNOWLEDGMENTS

Z.Q.C. is supported by the National Nano Project (no. 2011CB933600), the National Natural Science Foundation of China (NSFC) (no. 31470269) and the Youth Innovation Promotion Association CAS. X.E.Z. is grateful for support from the Chinese Academy of Sciences (KJZD-EW-TZ-L04). We thank the Core Facility and Technical Support, Wuhan Institute of Virology for excellent technical support.

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ABBREVIATIONS SV40, Simian virus 40; VCAM-1, vascular cell adhesion molecule-1; VNPs, virus-based nanoparticles; QDs, quantum dots; VLPs, SV40 virus-like particles; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis

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REFERENCES

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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.nanolett.6b02386.

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*E-mail: [email protected]. Telephone: +86 27 8719 9115. *E-mail: [email protected]. Telephone: +86 10 6488 8148. Author Contributions

All authors contributed to the experimental data and writing of the manuscript. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest. G

DOI: 10.1021/acs.nanolett.6b02386 Nano Lett. XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.nanolett.6b02386 Nano Lett. XXXX, XXX, XXX−XXX