A Biomimetic Nanodecoy Traps Zika Virus To Prevent Viral Infection

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A Biomimetic Nanodecoy Traps Zika Virus to Prevent Viral Infection and Fetal Microcephaly Development Lang Rao, Wenbiao Wang, Qian-Fang Meng, Mingfu Tian, Bo Cai, Yingchong Wang, Aixin Li, Minghui Zan, Feng Xiao, Lin-Lin Bu, Geng Li, Andrew Li, Yingle Liu, Shi-Shang Guo, Xingzhong Zhao, Tza-Huei Wang, Wei Liu, and Jianguo Wu Nano Lett., Just Accepted Manuscript • DOI: 10.1021/acs.nanolett.8b03913 • Publication Date (Web): 13 Dec 2018 Downloaded from http://pubs.acs.org on December 13, 2018

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A Biomimetic Nanodecoy Traps Zika Virus to Prevent Viral Infection and Fetal Microcephaly Development Lang Rao†,‡,□,#, Wenbiao Wang†,┴,#, Qian-Fang Meng‡, Mingfu Tian†, Bo Cai‡, Yingchong Wang†, Aixin Li†, Minghui Zan‡, Feng Xiao†, Lin-Lin Bu‡, Geng Li§, Andrew Li∆, Yingle Liu†, Shi-Shang Guo‡, Xing-Zhong Zhao‡, Tza-Huei Wang∆, Wei Liu‡,*, and Jianguo Wu†,┴,* †

State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan,

Hubei 430072, China. ‡ Key

Laboratory of Artificial Micro- and Nano-Structures of Ministry of Education, School

of Physics and Technology, Wuhan University, Wuhan, Hubei 430072, China. ┴ Institute

of Medical Microbiology, Jinan University, Guangzhou, Guangdong 510632,

China. §

School of Chinese Pharmaceutical Science, Guangzhou University of Chinese Medicine,

Guangzhou, Guangdong 510006, China. ∆

Department of Biomedical Engineering, Johns Hopkins University, Baltimore, Maryland

21205, USA. # These

authors contributed equally to this work.

* Corresponding e-mail: [email protected] (W.L.); [email protected] (J.W.).

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ABSTRACT Zika virus (ZIKV) has emerged as a global health threat due to its unexpected causal link to devastating neurological disorders such as fetal microcephaly. However, to date, no approved vaccine or specific treatment is available for ZIKV infection. Here, we develop a biomimetic nanodecoy (ND) that can trap ZIKV, divert ZIKV away from its intended targets, and inhibit ZIKV infection. The ND, which is composed of a gelatin nanoparticle core camouflaged by mosquito medium host cell membranes, effectively adsorbs ZIKV and inhibits ZIKV replication in ZIKV-susceptible cells. Using a mouse model, we demonstrate that NDs significantly attenuate the ZIKV-induced inflammatory responses and degenerative changes and thus improve the survival rate of ZIKV-challenged mice. Moreover, by trapping ZIKV, NDs successfully prevent ZIKV from passing through physiologic barriers into fetal brain and thereby mitigate ZIKV-induced fetal microcephaly in pregnant mice. We anticipate that this study will provide new insights on the development of safe and effective protection against ZIKV and various other viruses that threaten public health. KEYWORDS: cell membrane-coated nanomaterials; biomimetic nanoparticles; Zika virus; fetal microcephaly; host-cell-mimicking nanodecoys


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Zika virus (ZIKV) is a mosquito-borne flavivirus that was originally discovered in 1947 in the serum of a rhesus macaque in Uganda.1 In humans, ZIKV infection generally manifests in the form of mild symptoms such as rash, fever, arthralgia, and headache.2 However, the severity of the situation has intensified due to evolutionary increases of the infectivity of ZIKV to its mosquito medium hosts (e.g., Aedes albopictus and Aedes aegypti).3, 4 Since 2015, the outbreak of ZIKV has spread to more than 20 American countries, rapidly becoming a global public health issue.5,

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ZIKV epidemic is linked to devastating neurological

complications, including microcephaly in human fetuses,7 Guillain-Barre syndrome and meningoencephalitis in human adults,8, 9 and testis damage and male infertility in mice.10, 11 Rapid and promising progress has been made towards effective control of ZIKV infection,1216

but so far, no licensed vaccine or specific treatment is available.17 Safe and effective

protection against ZIKV infection remains to be challenging and of paramount importance. During the replication process, ZIKV first bind to the host cell (HC) membrane receptors and trigger endocytosis.18 Meanwhile, utilization of biomimetic cell membranes for surface disguise of nanomaterials presents a novel strategy that endow nanomaterials with various unique properties from the source cells,19, 20 such as long systematic circulation from erythrocytes,21 tumor targeting ability from cancer cells,22, 23 and pathogen binding capability form platelets.21 In this work, an anti-ZIKV host-mimicking nanodecoy (ND) was constructed by wrapping a polymeric core with HC membranes (Figure 1a). The HC membrane shell acts as a substrate mimic for ZIKV adsorption,24 while the polymeric nanoparticle core stabilizes the HC membrane shell improving survival in circulation essential for sequestration of ZIKV in the bloodstream.21 Such structural characteristic of NDs can effectively divert ZIKV away from its intended targets. Using ZIKV-infectious cells, we demonstrated that NDs can prevent ZIKV replication in vitro. In a mouse model, we observed that NDs effectively negated ZIKV-induced inflammatory and degenerative 3 ACS Paragon Plus Environment

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changes, and thereby improved the survival rate of mice. Moreover, ZIKV can pass through physiologic barriers (i.e., the placental blood barrier (PBB) of pregnant mice and the brainblood barrier (BBB) of fetal mice) and cause fetal microcephaly,25, 26 while it is difficult for nanoparticles to enter into immune privileged sites.27 Thus, by trapping ZIKV, NDs effectively diverted ZIKV away from the fetal brain and suppressed the ZIKV-induced fetal microcephaly in pregnant mice. In addition, our biomimetic NDs exhibited superior biocompatibility, effective immune evasion and long circulation time, ensuring the safety and effectiveness of NDs in complex in vivo environments. This biologically inspired ND presents an antiviral application that have the potential to treat a variety of diseases induced by ZIKV and other viruses. The NDs were fabricated by fusing mosquito medium host Aedes albopictus (C6/36) cell membrane-derived vesicles (HC-vesicles) onto Food and Drug Administration (FDA)approved gelatin nanoparticles (GNPs) via a cell membrane cloaking technique (Figure S1, Supporting Information).21,

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Specifically, C6/36 cells were subjected to lysis, sonication,

and extrusion, yielding HC-vesicles with a mean diameter of ~200 nm and zeta potential of ~ -12 mV (Figure S2). The NDs were then obtained by repeated extrusion of HC-vesicles and GNPs through 200-nm pores. Transmission electron microscopy (TEM) clearly showed a gelatin nanoparticle core of ~80 nm in diameter and a lipid bilayer shell of ~9 nm in thickness (Figure 1b). Dynamic light scattering (DLS) characterizations indicated that the resulting NDs were ~18 nm larger than bare GNPs and exhibited an isovalent surface potential when compared to that of HCvesicles (Figure 1c). Furthermore, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) suggested that the membrane proteins of HCs were successfully translocated onto the NDs (Figure 1d). Notably, enhanced colloidal stability was observed with the NDs when compared to uncoated GNPs (Figure S3a) and could be ascribed to the stabilizing effect


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of the hydrophilic glycans of the cell membranes.29,

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The membrane coating was further

optimized and the lowest shell-to-core ratio at which the NDs retained a stable size was determined to be approximately 1 mg membrane protein per 1 mg GNPs (Figure S3b). It is also important to note that the HC membrane shell provides cloaking ability for the GNPs allowing them to reduce non-specific clearance (Figure S4) while the inner GNP core stabilizes the HC membrane shell enhancing survival in the circulatory system (Figure S5). After confirming the structure of NDs, we investigated the adsorption of ZIKV onto the NDs. First, ZIKV was assembled in C6/36 cells and then purified. TEM confirmed the presence of spherical ZIKV with the diameter of ~36 nm (Figure S6). Then, 108 plaqueforming units (PFU) of ZIKV were incubated with 20 μg of NDs for 4 h and excess ZIKV was removed from the NDs. TEM image demonstrated that ZIKV was closely attached to the core-shell ND (Figure 1e) and DLS results reflected that the size of NDs increased ~72 nm after the adsorption of ZIKV (Figure S7). For further validation of the adsorption, the envelope (E) protein, a major protein involved in ZIKV receptor binding and fusion,31 was analyzed and observed by western blot analysis to be associated with NDs after incubation with ZIKV (Figure 1f). Finally, confocal laser scanning microscopy (CLSM) imaging confirmed co-localization and tight interactions between NDs and ZIKV even after they were internalized by human embryonic kidney (HEK 293T) cells (Figure 1g). It is noteworthy that, compared to other HC membrane-coated NDs, C6/36 cell membrane-coated NDs exhibited the highest adsorption of ZIKV (Figure S8), aiding in the selection of membranes for ND construction. We also quantitatively analyzed the ability of C6/36 cell membrane-coated NDs to adsorb ZIKV and found that 1 ug NDs can adsorb 5 × 105 PFU of ZIKV at most (Figure S9). Like other flavivirus, ZIKV can induce cytopathic effects (CPE).32 To examine if the NDs can adsorb ZIKV and protect cellular targets, African green monkey kidney epithelial


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(Vero) cells were incubated with 106 PFU of ZIKV or with ZIKV and quantities of NDs. CPE became apparent at 48 h and NDs markedly alleviated the CPE caused by ZIKV (Figure 2a). The culture media from the transfected cells were also collected and viral titers were determined by plaque assays. The group containing ZIKV and NDs displayed significantly less plaques than the group containing just ZIKV (Figure 2b). In the latter group containing just ZIKV, the viral titer of ZIKV was about 2.3 × 105 mL-1, which was approximately 10fold higher than that of the group containing ZIKV and 20 ug of NDs (Figure 2c). Tissue culture infective dose (TCID50) assay also reflected a similar trend (Figure 2d), suggesting that the NDs successfully diverted ZIKV away from its targets and inhibited ZIKV infection. We then quantified the ZIKV RNA levels in Vero and human uterine cancer (HeLa) cells by real-time polymerase chain reaction (RT-PCR). The viral RNA level in the group containing ZIKV and NDs was about half of that of the ZIKV group for both cell types (Figure 2e,f). CLSM imaging showed less viral particles in the ZIKV + NDs group as well (Figure 2g). It is worth mentioning that, though NDs can inhibit ZIKV infection in a certain degree, NDsadsorbed ZIKV can infect cells still (Figure S10). Also, we found the side effects of NDs on various cells were negligible (Figure S11), ensuring the reliability of cellular studies. The replication and infection of ZIKV is a complete and complex process, including interaction between viral particles and host receptors, fusion of the virion envelope with cell membranes, uncoating of the nucleocapsid, releasing RNA genome into the cytoplasm.6 After the adsorption by NDs, which were two-fold larger than ZIKV, the ability of ZIKV to replicate and infect in vitro was reduced. Recent advances in microbiology have demonstrated that type I interferon (IFN-α/β) receptor deficient (A129) mice challenged with ZIKV exhibit several serious symptoms including widespread viral RNA presence and distinct histological changes.33 In this work, A129 mice were used as a model to systemically assess the anti-ZIKV efficacy of NDs. The


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mice received an intravenous (i.v.) injection of phosphate buffer solution (PBS) at day 0 or PBS containing 200 µg of NDs at day -2, 0, 2, and 4. All PBS or NDs treated mice received an intraperitoneal (i.p.) injection of 106 PFU of ZIKV at day 0. Mock-infected mice were used as control. Survival analysis demonstrated that all animals in the ZIKV-infected group and ZIKV + NDs (-2 day) group met humane clinical endpoints at day 10 and 11 after the injection of ZIKV (Figure 3a). Remarkably, all animals in the ZIKV + NDs (0 day) and ZIKV + NDs (2 day) groups survived by day 13, demonstrating that i.v. injection with NDs at day 0 and 2 after the ZIKV infection resulted in a significant reduction in disease mortality. Fluctuation in body weight is a direct indicator of ZIKV infection.26 It was observed that the ZIKV-challenged mice started to lose weight rapidly after day 4, whereas the mice in the ZIKV + NDs (0 day) and ZIKV + NDs (2 day) groups exhibited stabilization from weight loss after 8 days post challenge (Figure 3b), indicating that the mice gradually maintained their health. At endpoints, we collected the mice brains for viral RNA quantification and other organs for histological analysis and immunofluorescence assay. Compared to the control, significantly higher viral RNA levels were detected in the brain of ZIKV-challenged mice (Figure 3c). The levels of viral RNA in the ZIKV + NDs (-2 day) and ZIKV + NDs (4 day) groups were not different from that of the ZIKV group. However, remarkably, the levels of viral RNA in the ZIKV + NDs (0 day) and ZIKV + NDs (2 day) groups were comparable to that of the control group, demonstrating that injection of NDs at day 0 and 2 could effectively inhibit ZIKV infection in vivo. From histopathology, we observed that the ZIKV + NDs (-2 day) and ZIKV + NDs (4 day) groups exhibited ZIKV-induced inflammatory and degenerative changes, including polymorphonuclear cells (PMNs) near blood vessels in the brain (Figure 3d), large and poorly defined corpuscle structures in the spleen, and enhanced vascular permeability in the liver and kidney. In contrast, for the mice in the ZIKV + NDs (0


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day) and ZIKV + NDs (2 day) groups, lesions associated with ZIKV infection were overall not observed in the brain, spleen, liver and kidney (Figure 3d), suggesting that the timely injection of NDs successfully diverted ZIKV away from its targets and effectively suppressed ZIKV infectivity. Immunofluorescence results further supported the notion that ZIKV can infect A129 mice (Figure 3e) but NDs can efficiently divert ZIKV away from its hosts and prevent ZIKV infection as seen in the ZIKV + NDs (0 day) and ZIKV + NDs (2 day) groups. Furthermore, we found that compared to single injection, repeated injection of NDs can further protect adult A129 mice from viral infection and inhibit ZIKV replication in the blood and brain (Figure S12). After confirming the anti-ZIKV effects of NDs in vitro and in vivo, we further investigated the ability of NDs prevent ZIKV to pass through the BBB in A129 mice. Mice received an i.p. injection of 106 PFU of ZIKV which was immediately followed by an i.v. injection of PBS or PBS containing 200 µg of NDs. Mock-infected mice were used as control. At day 2, 4, 6, and 8 after the injection, we measured the viral RNA levels in the blood and brain. Compared to the ZIKV group, higher accumulation of ZIKV in the blood was detected in the ZIKV + NDs group at day 6 (Figure S13a), which may be attributed to the effective adsorption of ZIKV and the long blood circulation time of NDs. Remarkably, we found that the NDs efficiently reduced the ZIKV accumulation in the brain at all time points (Figure S13b). Lastly, considering the ability of ZIKV to pass through the BBB and into the brain26 and the difficulty for 100-nm nanoparticles to do the same,27 we attribute the reduction in brain accumulation to the sequestration of ZIKV by the NDs. ZIKV infection of pregnant women is associated with severe fetal neurological damage and fetal microcephaly.7 Considering that NDs can effectively prevent ZIKV from passing through the BBB, we speculate that NDs may also prevent ZIKV from passing through the PBB and into the mice fetuses. To test this, pregnant A129 mice received an i.p.


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injection of 106 PFU of ZIKV which was immediately followed by an i.v. injection of PBS or PBS containing 200 µg of NDs at embryonic day 13.5 (E13.5). Mock-infected mice were used as control. The embryos of each pregnant mouse were collected for analysis at E18. Fetuses from ZIKV-infected pregnant mice showed obvious whole-body growth delay (Figure 4a and Figure S14). The fetal body length (Figure 4b) and weight (Figure 4c) were significantly reduced in the the ZIKV group while the NDs treatment effectively protected the fetus from ZIKV-induced defects (Figure 4a-c). We also found that the fetal brains from the ZIKV-infected pregnant mice were significantly smaller than these of the control group (Figure 4d-f); this deficiency was also inhibited by treatment with NDs. In terms of ZIKV RNA levels, the ZIKV + NDs group exhibited lower viral RNA levels in the fetal brain than the ZIKV-infected group (Figure 4g). Immunoﬂuorescence assay for viral E protein further demonstrated that NDs successfully mitigated ZIKV infection in the fetal brain (Figure 4h). In all, the results support the notion that NDs can suppress ZIKV-induced fetal microcephaly in pregnant A129 mice. Systematic toxicity is a major consideration for nanomaterials used for biomedical applications,34 particularly considering that the employment of C6/36 cells for cell membrane derivation and nanoparticle coating may induce potential safety issues (e.g., toxicity and immunogenicity) due to the hurdles across different species. In this work, in order to determine the systematic toxicity, Institute for Cancer Research (ICR) mice received i.v. injection of PBS or PBS containing 500 μg of NDs every three day. Neither death nor obvious weight difference was monitored between control and the NDs group after 30 days (Figure S15a), suggesting that no significant side effects were caused by the NDs. At 30 days post injection, all mice were sacrificed and organs including blood were harvested for blood and histology analysis. No significant differences were observed in tissue slices and blood parameters (Figure S15b,c and Table S1), implying in vivo compatibility. Considering that


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C6/36 cells are exogenous cells, acute host response from mice upon the injection of NDs was also investigated. ICR mice received an i.v. injection of PBS or PBS containing NDs. At day 1, 2, 7, and 15 after the injection, we detected the levels of pro-inflammatory cytokines in the blood. No differences were observed in tumor necrosis factor-α (TNF-α) and interleukin6 (IL-6) levels (Figure S16). We further tested the antibody reaction by measuring the IgM and IgG levels in the blood samples after administration of a first and second dose of NDs. The antibody levels of the mice that received C6/36 cells were ~3 times higher than the control (Figure S17). In contrast, the administration of NDs resulted in no noticeable increase in IgM and IgG, further demonstrating good in vivo compatibility of NDs. Although systematical research is needed to further investigate the short- and long-term in vivo toxicity, our small-scale pilot safety study could reassure further exploration of these bio-inspired nanoparticles. In addition, the removal of nuclear components from the final formation can help to mitigate the potential genetic risk of the cell membrane-coated nanoparticles in a certain degree. It should also be noted that, due to the cell membrane coating, NDs acquire immune evasion capability and long blood circulation time (Figure S18) comparable to that of gold standard red blood cell (RBC) membrane-coated nanoparticles.21 In short, the combination of superior biocompatibility, immune evasion capability, and long blood circulation time of NDs significantly enhances their effectiveness in vivo. In summary, based on a bio-inspired design strategy, a safe and effective anti-ZIKV ND was developed. The NDs, composed of GNP-stabilized HC membranes, can adsorb ZIKV and divert ZIKV away from its targets. Furthermore, we demonstrated that NDs could significantly inhibit ZIKV infection of cells, improve the mice survival rate and negate the ZIKV-induced inflammatory and degenerative changes in adult mice, and prevent ZIKV from passing through the physiologic barriers by sequestration of ZIKV and suppress the ZIKVinduced fetal microcephaly in pregnant mice. Our biomimetic NDs exhibited superior


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biocompatibility, effective immune evasion and long circulation time, ensuring the safety and effectiveness of NDs in complex in vivo environments. The use of HC membranes for coating GNPs as a strategy for camouflaging nanoparticles in vivo represents a novel and promising nanotechnology that has great clinical translation potential. To realize this, further developments and detailed studies need to be conducted. Scalability is always a key issues that need to be considered to translate any nanomaterial-based platform into clinical use.19,

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Previous works on the biomimetic cell

membrane coating technique have guided optimization and led to efficient coating and robust storage strategies.36-38 By adopting large-scale purification and dispersion techniques widely used in biologics development, reliable and efficient production of NDs can be potentially realized. Looking to the future, the employment of cell membrane-camouflaged nanoparticles may open up an exciting field in the personalized diagnosis and therapy. Anthropogenic HCs can be separated from a patient and the derived HC membranes can be wrapped onto multifunctional theranostic nanoparticles before injecting them back into the source patient. Autologous administration of cell membranes and cell membrane-coated nanoparticles would maximize immune tolerance to the delivered agent.39 Moving forward, the host-virus affinity is also a crucial factor that needs to be accounted for. Although recent reports have demonstrated that the E protein is a major protein in ZIKV involved in receptor binding and fusion, the identity of the receptor for the E protein on HCs remains to be a mystery. Once the receptor is characterized, approaches including genetic engineering and bioconjugate chemistry can be used to improve the receptor expression which may further enhance the platform’s efficacy.40 Another promising feature of this kind of cell membrane-wrapped nanomaterials is that it offers a high freedom in customizability due to myriad choices in core and shell


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materials. While the current design employs a polymer as the core material, we can easily envision this technique to be generalized to many other types of nanostructures, such as magnetic nanoparticles (MNs).

Under an external magnetic field, HC membrane-

camouflaged MNs can be magnetically localized away from tissues that are highly susceptible to ZIKV infection to further reduce viral burden.39 Although traditional vaccine have been widely used for antiviral application, there are still some inherent flaws, such as instability, leading to potential virus atavistic, and causing potential infection or spread. Compared to the traditional vaccination, our cell membrane-coated nanoparticle strategy possess unique advantages including robust production and storage and no cross infection risk. The endless selection of cell types for shell construction can also be exploited to target the vast viral repertoire. By customizing this novel nanoplatform, it may one day be possible to develop a wide array of nanomedicine for safe and effective protection against ZIKV and various other viruses.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: Experimental details; preparation and optimization of nanoparticles; in vitro and in vivo toxicity evaluation; in vitro immune evasion evaluation; pharmacokinetic studies and in vivo biodistribution; TEM image and size analysis of ZIKV; western blot protein analysis (PDF).

AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected] *E-mail: [email protected] 12 ACS Paragon Plus Environment

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Present Address □ Simmons

Comprehensive Cancer Center, University of Texas Southwestern Medical Center,

Dallas, Texas 75390, USA. Author Contribution # L.R.

and W.W. contributed equally to this work.

ORCID Lang Rao: 0000-0001-5010-0729 Wei Liu: 0000-0003-4789-362X Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was supported by National Mega Project on Major Infectious Disease Prevention (No. 2017ZX10103005), National Key R&D Program (No. 2016YFC1000700), National Key R&D Program for Major Research Instruments (No. 81527801), National Natural Science Foundation for Outstanding Youth Foundation (No. 61722405), and National Natural Science Foundation of China (No. 31200134, 31230005, 31270206, 61474084, 81471942, and 81730061).

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(22) Rao, L.; Bu, L.-L.; Cai, B.; Xu, J.-H.; Li, A.; Zhang, W.-F.; Sun, Z.-J.; Guo, S.-S.; Liu, W.; Wang, T.-H.; Zhao, X.-Z. Adv. Mater. 2016, 28, 3460-3466. (23) Yu, G.-T.; Rao, L.; Wu, H.; Yang, L.-L.; Bu, L.-L.; Deng, W.-W.; Wu, L.; Nan, X.; Zhang, W.-F.; Zhao, X.-Z.; Liu, W.; Sun, Z.-J. Adv. Funct. Mater. 2018, 28, 1801389. (24) Hu, C.-M. J.; Fang, R. H.; Copp, J.; Luk, B. T.; Zhang, L. Nat. Nanotechnol. 2013, 8, 336-340. (25) Platt, D. J.; Smith, A. M.; Arora, N.; Diamond, M. S.; Coyne, C. B.; Miner, J. J. Sci. Transl. Med. 2018, 10, eaao7090. (26) Wang, W.; Li, G.; De, W.; Luo, Z.; Pan, P.; Tian, M.; Wang, Y.; Xiao, F.; Li, A.; Wu, K.; Liu, X.; Rao, L.; Liu, F.; Liu, Y.; Wu, J. Nat. Commun. 2018, 9, 106. (27) Kreuter, J. Adv. Drug Delivery Rev. 2001, 47, 65-81. (28) Li, J.; Zhen, X.; Lyu, Y.; Jiang, Y.; Huang, J.; Pu, K. ACS Nano 2018, 12, 8520-8530. (29) Rao, L.; Bu, L.-L.; Xu, J.-H.; Cai, B.; Yu, G.-T.; Yu, X.; He, Z.; Huang, Q.; Li, A.; Guo, S.-S.; Zhang, W.-F.; Liu, W.; Sun, Z.-J.; Wang, H.; Wang, T.-H.; Zhao, X.-Z. Small 2015, 11, 6225-6236. (30) Hu, C.-M. J.; Fang, R. H.; Wang, K.-C.; Luk, B. T.; Thamphiwatana, S.; Dehaini, D.; Nguyen, P.; Angsantikul, P.; Wen, C. H.; Kroll, A. V.; Carpenter, C.; Ramesh, M.; Qu, V.; Patel, S. H.; Zhu, J.; Shi, W.; Hofman, F. M.; Chen, T. C.; Gao, W.; Zhang, K.; Chien, S.; Zhang, L. Nature 2015, 526, 118-121. (31) Dai, L.; Song, J.; Lu, X.; Deng, Y.-Q.; Musyoki, Abednego M.; Cheng, H.; Zhang, Y.; Yuan, Y.; Song, H.; Haywood, J.; Xiao, H.; Yan, J.; Shi, Y.; Qin, C.-F.; Qi, J.; Gao, George F. Cell Host Microbe 2016, 19, 696-704. (32) Savidis, G.; Perreira, J. M.; Portmann, J. M.; Meraner, P.; Guo, Z. R.; Green, S.; Brass, A. L. Cell Rep. 2016, 15, 2323-2330.


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Figure 1. Preparation of NDs for ZIKV adsorption. (a) Schematic of a ND that adsorbs ZIKV. (b) TEM images of single GNP (i), HC-vesicle (ii), single ND (iii), and multiple NDs (iv). Scale bars, 50 nm in i-iii; 100 nm in iv. (c) Mean diameter and zeta potential of GNPs, HC-vesicles and NDs. Data points represent as mean ± s.d. (n = 3). (d) SDS-PAGE protein analysis of GNPs, HC-vesicles, and NDs. (e) TEM image of single ND that adsorbs ZIKV. Scale bar, 50 nm. Red arrows indicate ZIKV. (f) Western blot analysis of the E protein in the samples of NDs before and after incubation with ZIKV. (g) CLSM image of HEK 293T cell after incubation with NDs and ZIKV. Scale bar, 10 µm. Cell nuclei, NDs, and the E protein of ZIKV were labeled with DAPI (blue), FITC (green), and Dylight 649 (red), respectively.


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Figure 2. NDs prevent ZIKV infection in cells. (a) Bright-field images of Vero cells after 48-h incubation with ZIKV alone or with ZIKV and various amounts of NDs (i.e., 5, 10, or 20 μg). The cells without any treatments were used as control. Scale bar, 100 µm. (b) Plaque morphology of ZIKV on Vero cells without or with the addition of various amounts of NDs. (c) PFU and (d) TCID50 assay analysis of Vero extracellular ZIKV titer without or with the addition of various amounts of NDs. Data points represent as mean ± s.e.m. (n = 3). As compared with the ZIKV group, ns, *, **, and *** indicates no statistical difference, P < 0.05, P < 0.01, and P < 0.001, respectively. RT-PCR detection of ZIKV RNA levels in (e) Vero and (f) HeLa cells without or with the addition of NDs. (g) CLSM images of Vero cells after incubation with ZIKV alone or with ZIKV and NDs. Scale bar, 50 µm. Cell nuclei and the E protein of ZIKV were labeled with DAPI (blue) and Dylight 649 (red), respectively.


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Figure 3. NDs prevent ZIKV infection in adult mice. (a) Survival rates of A129 mice. (b) Mice body weight change curves. (c) RT-PCR detection of ZIKV RNA levels in the brain. Data points represent as mean ± s.e.m. (n = 4).

ns

and *** indicates no statistical difference

and P < 0.001, respectively. (d) H&E-stained slices of the brain, spleen, liver, and kidney. Scale bars, 500 µm for brain and spleen and 200 µm for liver and kidney. (e) Immunoﬂuorescence images of the sections of brain, spleen, liver, and kidney. Scale bars, 200 µm. Cell nuclei and the E protein of ZIKV were labeled with DAPI (blue) and Dylight 649 (red), respectively. The mice received an i.v. injection of PBS at day 0 or PBS containing 200 µg of NDs at day -2, 0, 2, and 4. All the PBS or NDs treated mice received an i.p. injection of 106 PFU of ZIKV at day 0. Mock-infected mice were used as control.


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Figure 4. NDs suppress ZIKV-induced fetal microcephaly in pregnant mice. (a) Representative images of the fetuses. (b) The length of fetuses. (c) The weight of fetuses. (d) The length of fetal heads. (e) Representative images of the fetal brains. (f) The weight of fetal brains. (g) RT-PCR detection of ZIKV RNA levels in the fetal brains. Data points represent as mean ± s.e.m. (n = 8 for the control group; n = 5 for the ZIKV and ZIKV + NDs groups). *, **, and *** indicates P < 0.05, P < 0.01, and P < 0.001, respectively. (h) Fluorescence images of the fetal brain sections. Scale bar, 200 µm. Cell nuclei and the E protein of ZIKV were labeled with DAPI (blue) and Dylight 649 (red), respectively. The pregnant mice received an i.p. injection of 106 PFU of ZIKV which was immediately followed by an i.v. injection of PBS or PBS containing 200 µg of NDs at E13.5. Mock-infected pregnant mice were used as control. The embryos of each pregnant mouse were collected for analysis at E18.


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A Biomimetic Nanodecoy Traps Zika Virus To Prevent Viral Infection

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