key: cord-0948216-ih25u0tt
authors: Chen, Min; Rosenberg, Jillian; Cai, Xiaolei; Lee, Andy Chao Hsuan; Shi, Jiuyun; Nguyen, Mindy; Wignakumar, Thirushan; Mirle, Vikranth; Edobor, Arianna Joy; Fung, John; Donington, Jessica Scott; Shanmugarajah, Kumaran; Chang, Eugene; Randall, Glenn; Penaloza-MacMaster, Pablo; Tian, Bozhi; Madariaga, Maria Lucia; Huang, Jun
title: Nanotraps for the containment and clearance of SARS-CoV-2
date: 2021-02-01
journal: bioRxiv
DOI: 10.1101/2021.02.01.428871
sha: 48c453fc2581e23ee665932c4af4bd6dd0aedf4e
doc_id: 948216
cord_uid: ih25u0tt

SARS-CoV-2 enters host cells through its viral spike protein binding to angiotensin-converting enzyme 2 (ACE2) receptors on the host cells. Here we show functionalized nanoparticles, termed “Nanotraps”, completely inhibited SARS-CoV-2 infection by blocking the interaction between the spike protein of SARS-CoV-2 and the ACE2 of host cells. The liposomal-based Nanotrap surfaces were functionalized with either recombinant ACE2 proteins or anti-SARS-CoV-2 neutralizing antibodies and phagocytosis-specific phosphatidylserines. The Nanotraps effectively captured SARS-CoV-2 and completely blocked SARS-CoV-2 infection to ACE2-expressing human cell lines and primary lung cells; the phosphatidylserine triggered subsequent phagocytosis of the virus-bound, biodegradable Nanotraps by macrophages, leading to the clearance of pseudotyped and authentic virus in vitro. Furthermore, the Nanotraps demonstrated excellent biosafety profile in vitro and in vivo. Finally, the Nanotraps inhibited pseudotyped SARS-CoV-2 infection in live human lungs in an ex vivo lung perfusion system. In summary, Nanotraps represent a new nanomedicine for the inhibition of SARS-CoV-2 infection. Highlights Nanotraps block interaction between SARS-CoV-2 spike protein and host ACE2 receptors Nanotraps trigger macrophages to engulf and clear virus without becoming infected Nanotraps showed excellent biosafety profiles in vitro and in vivo Nanotraps blocked infection to living human lungs in ex vivo lung perfusion system Progress and Potential To address the global challenge of creating treatments for SARS-CoV-2 infection, we devised a nanomedicine termed “Nanotraps” that can completely capture and eliminate the SARS-CoV-2 virus. The Nanotraps integrate protein engineering, immunology, and nanotechnology and are effective, biocompatible, safe, stable, feasible for mass production. The Nanotraps have the potential to be formulated into a nasal spray or inhaler for easy administration and direct delivery to the respiratory system, or as an oral or ocular liquid, or subcutaneous, intramuscular or intravenous injection to target different sites of SARS-CoV-2 exposure, thus offering flexibility in administration and treatment. More broadly, the highly versatile Nanotrap platform could be further developed into new vaccines and therapeutics against a broad range of diseases in infection, autoimmunity and cancer, by incorporating with different small molecule drugs, RNA, DNA, peptides, recombinant proteins, and antibodies.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused the global pandemic of coronavirus disease 2019 (COVID- 19) . As of January 8, 2021, SARS-CoV-2 has spread to over 180 countries and has resulted in more than 88.2 million infections and over 1.9million deaths globally (Dong et al., 2020) . Despite tremendous efforts devoted to drug development, safe and effective medicines to treat SARS-CoV-2 infection are largely lacking. Given that the virus is within nanoscale, nanomaterial based delivery systems are expected to play a paramount role in the success of prophylactic or therapeutic approaches (Florindo et al., 2020; Shin et al., 2020) . To combat this highly contagious virus, here we set out to devise a nanomedicine termed "Nanotraps" to inhibit SARS-CoV-2 infection.

To gain entry to host cells for infection, SARS-CoV-2 surface spike protein binds to its receptor human angiotensin-converting enzyme 2 (ACE2) with high affinity (Shang et neutralizing antibodies with high surface density. This design endowed the Nanotraps with high-avidity to outperform its soluble ACE2 or antibody counterparts to capture and contain SARS-CoV-2. Thus, the high avidity, small size, and high diffusivity of our newly engineered Nanotraps efficiently blocked the binding of SARS-CoV-2 to ACE2expressing host cells including epithelial cells in the respiratory system, resulting in abrogation of SARS-CoV-2 entry.

Furthermore, we aimed to clear the virus after containment by the Nanotrapmediated macrophage phagocytosis. The role of macrophages in the control of infections has long been documented (Gordon, 2016) , and recent single-cell RNA set out to engineer a family of nano-enabled virus-trapping particles, termed "Nanotraps", to contain and clear SARS-CoV-2 ( Figure 1A ). We used an FDAapproved, biodegradable PLA polymeric core and liposome shell materials to synthesize the Nanotraps. Nanotraps with different diameters (200, 500, and 1200 nm) were synthesized by varying polymer concentrations (see Methods for details) ( Figure   S1A ). The solid PLA core acts as a 'cytoskeleton' to provide mechanical stability, controlled morphology, biodegradability, and large surface area for nanoscale membrane coating and surface modification. The lipid shell enveloping the PLA core exhibits behavior similar to that of cell membranes. The lipid shell provides a nanoscopic platform and can interact with a wide variety of molecules(Allen and Cullis, 2013; Riley et al., 2019; Torchilin, 2005 ) either within the membrane or on the surface (Suk et al., 2016; Torchilin, 2014) . Thus, we aimed to functionalize the Nanotrap surface with a molecular bait (a recombinant ACE2 protein or an anti-SARS-CoV-2 neutralizing antibody) and a phagocytosis-inducing ligand (phosphatidylserine). We hypothesized that (1) the high-density ACE2 or neutralizing antibodies on the Nanotraps can outcompete low-expression ACE2 on host cells in capturing SARS-CoV-2, thus enabling selective virus containment by the Nanotraps, and that (2) surface phosphatidylserine ligands on suitably sized Nanotraps can trigger subsequent phosphatidylserine-mediated phagocytosis by professional phagocytes, such as macrophages, thus enabling viral clearance ( Figure 1A ). The resultant structures were monodispersed and significantly smaller than mammalian cells, yet still large enough to bind several SARS-CoV-2 virions ( Figure 1B-F) .

To characterize the Nanotraps, we first used dynamic light scattering to measure the size dispersity of the constituent nanoparticles. Controlling particle size is important for tuning the phagocytosis efficacy, reproducible mechanical characteristics, and material biocompatibility (Champion et al., 2008; He et al., 2010) . The hydrolyzed diameter of the Nanotraps was measured by dynamic light scattering, which increased with the addition of each molecule ( Figure 1B ). The zeta potential, which reflects the surface charges of the Nanotraps (Doane et al., 2012) , was found to change slightly with the addition of each molecule to the Nanotrap surface ( Figure 1C ). We next used fluorescent labeling and total internal reflection fluorescence microscopy (TIRFM) to confirm the presence of the ACE2 moiety on the Nanotraps. The PLA polymeric core of each Nanotrap was labelled with a lipophilic carbocyanine dye: 1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate salt (DiD, red).

The Nanotrap-ACE2 were further stained with an anti-ACE2 antibody labelled with Alexa fluor-488 dye (AF488, green). The TIRFM images clearly showed excellent colocalization between the Nanotrap core and surface ACE2 at the single particle level, confirmed by line scans of the fluorescent channels corresponding to each component ( Figure 1D ). These results demonstrated that we have successfully functionalized the Nanotraps with recombinant ACE2 protein. Finally, we employed scanning electron microscopy (SEM) to image the Nanotraps at the sub-nanometer level. SEM images showed that the Nanotraps were spherical and well-dispersed ( Figure S1B ). The Nanotraps appeared slightly crenellated in the SEM images, as the lipid layer may have shrunk due to the drying sample preparation procedure before imaging ( Figure 1E ). As expected, after incubation with the pseudotyped SARS-CoV-2 for 1 hour at 37 ℃, the Nanotraps effectively captured the virus as evidenced by single virions clearly visualized on the surface of a Nanotrap; no freestanding virions were observed outside of the Nanotrap ( Figure 1F ).

Macrophages are a class of phagocytes that engulf and clear cell debris, pathogens, microbes, cancer cells and other foreign intruders (Gordon, 2016) Thus, we utilize the 500-nm core size and 15% surface phosphatidylserine Nanotraps for the following experiments to maximize viral capture and macrophage phagocytosis.

We next generated multiple types of Nanotraps to test their efficacy. First, avi-tagged biotinylated ACE2 was conjugated to the Nanotrap surface via biotin-streptavidin interactions to make Nanotrap-ACE2. In addition, we synthesized Nanotrap-Antibody by conjugating a SARS-CoV-2 neutralizing antibody to the Nanotrap surface via Nhydroxysuccinimide (NHS) esters (see details in Experimental procedures). Finally, in order to test the specificity of the Nanotraps, we made Nanotrap-Blank without a virusbinding epitope.

We next examined whether the Nantraps could effectively capture and contain SARS-CoV-2 in vitro. All three Nanotraps were incubated with SARS-CoV-2 spike pseudotyped lentivirus or vesicular stomatitis virus (VSV) for 1 hour before adding to HEK293T-ACE2 cells for 24 hours and 72 hours, respectively. Both Nanotrap-ACE2

and Nanotrap-Antibody completely blocked SARS-CoV-2 pseudovirus infection, while the Nanotrap-Blank did not, indicating both the specificity and functionality of our Nanotraps ( Figure 3A -B and Figure S3A macrophages. These data suggest that macrophages significantly reduced the viral infection but could not completely eradicate it ( Figure 3E -F, comparing "E+V" with "E+ MФ+V"). Finally, we determined whether our engineered Nanotraps triggered phosphatidylserine-mediated phagocytosis by dTHP-1 macrophages for the clearance of virus. After adding Nanotrap-ACE2 into the co-culture of epithelial cells, macrophages, and SARS-CoV-2 spike pseudotyped VSV, the viral infection was completely inhibited ( Figure 3E -F, comparing "E+V+NT" and "E+MФ +V+NT" with "E+MФ+V"). We further observed incorporation of Nanotrap-ACE2 into the macrophage cell body, indicating successful phagocytosis ( Figure 2E , Figure 3E "E+MФ+V+NT" inset, Supplemental Video 1).

Nanotraps not only served as a sponge to capture and contain SARS-CoV-2, but also utilized the phagocytosis and sterilization machinery of macrophages to defend the host cells from infection, as we depicted in our original experimental design ( Figure 1A ).

In order to assess the safety of Nanotrap treatment, we first examined in vitro cytotoxicity on human cell lines. Neither A549 nor HEK293T-ACE2 cells displayed significant cytotoxicity with the addition of Nanotrap-Blank, Nanotrap-ACE2, or Nanotrap-Antibody, as evaluated by a CCK8 cytotoxicity assay ( Figure S4 ). We next examined the delivery of Nanotraps to mouse lungs and evaluated the biosafety of Nanotraps in vivo. We intratracheally injected immunocompetent mice with Nanotrap-ACE2 (labelled with DiD) at a dose of 10 mg/kg. Mice were sacrificed 3 days post-injection. Delivery of Nanotraps to mouse lungs was confirmed with cryosectioned mouse lung tissues: significant Nanotrap accumulation and distribution were found in the lung tissues, particularly in regions around bronchioles in the respiratory tracts. As expected, no Nanotraps were found in the lungs of PBS-treated mice ( Figure 4A -B).

In vivo safety was next analyzed. Hematoxylin and eosin (H&E) staining of major organs including lung, heart, liver, spleen, and kidney showed no histological differences in the Nanotrap-treated mice when compared to the PBS-treated control group ( Figure 4C ). Furthermore, complete blood counts were performed to evaluate white blood cells (WBCs), red blood cells (RBCs) and platelets (PLTs). The cell counts were similar between Nanotrap-and PBS-treated groups ( Figure 4D ). Next, comprehensive metabolic panels of mouse blood sera were examined to provide an overall picture of the chemical balance and metabolism. No statistical differences were found between Nanotrap-and PBS-treated mice for glucose levels, electrolyte and fluid balance, kidney function, or liver function ( Figure 4E ). These results demonstrated the safety of Nanotraps when delivered in vivo.

We next examined their therapeutic efficacy in inhibiting pseudotyped SARS-CoV-2 infection in healthy, non-transplantable human donor lungs using an ex vivo lung perfusion (EVLP) system ( Figure 5A and Supplemental Video 2). EVLP allows a lung to be perfused and ventilated ex vivo after organ retrieval by maintaining lungs at normothermic physiologic conditions and is thus an excellent platform to model lung diseases (Doane et al., 2012) .

The infection potential of the SARS-CoV-2 spike pseudotyped lentivirus at different doses over time in primary human lung cells was first tested in vitro, and infection was observed within 8 hours ( Figure S5A -B). After confirming infection potential, we tested our Nanotraps on an EVLP system with a pair of healthy lungs.

Static lung compliance and oxygenation capacity was measured over time ( Figure S5C ). SARS-CoV-2 pseudovirus carrying a luciferase reporter gene was injected into the lingula of left upper lung lobe, and pseudovirus plus Nanotrap-Antibody was injected into the right middle lobe; the right upper lung lobe was used as an untreated control ( Figure 5C , arrows). Human lung tissue samples were collected after perfusing for 8

hours. Single-cell suspensions were generated, and luciferase expression was determined ( Figure 5B ). The results showed that (1) the pseudovirus infected the lung tissues and (2) the Nanotraps completely inhibited the viral infection. Furthermore, H&E staining showed significant RBC infiltration in the virus-treated sample, which was not present in the virus plus Nanotrap-treated region ( Figure 5C ). As our EVLP system maintains lung viability for less than 12 hours, we treated single-cell suspensions of healthy, untreated lung from the right upper lobe in vitro for 48 hours to confirm the Nanotraps can function for longer term incubations in human tissue. Again, Nanotrap-Antibody was able to fully inhibit the virus ( Figure 5D ). Finally, since the EVLP could not be conducted under BSL-3 conditions in order to use authentic SARS-CoV-2, we tested the ability of the Nanotraps to prevent authentic SARS-CoV-2 from infecting Vero E6 cells, which are highly susceptible to SARS-CoV-2 infection (Matsuyama et al., 2020) . Indeed, Nanotrap-Antibody was able to completely inhibit infection of authentic SARS-CoV-2, as expected ( Figure 5E ).

Taken together, our EVLP experiments demonstrated that (1) SARS-CoV-2 pseudovirus can infect human lung, and (2) our newly engineered Nanotraps can completely block the viral infection, thus paving the way for future clinical trials using Nanotraps for the inhibition of SARS-CoV-2 infection.

The highly contagious SARS-CoV-2 has caused the global COVID-19 pandemic, so effective and safe treatments are urgently needed. Remdesivir has been approved by the To block the interaction between the SARS-CoV-2 spike protein and the host ACE2 receptors, we coated the Nanotrap surfaces with a high molecular density of either recombinant ACE2 proteins or anti-SARS-CoV-2 neutralizing antibodies ( Figure   1A ). In principle, the high binding avidity, high diffusivity, and small size of Nanotraps Furthermore, our Nanotraps harness the immune system to clear the SARS-CoV-2 ( Figure 2 and Figure 3 ). By incorporating the phagocyte-specific phosphatidylserine ligands onto the Nanotrap surfaces, macrophages readily engulfed the virus-bound Nanotraps without becoming infected themselves ( Figure 3E-F) . While macrophages were used as a proof-of-principle in this study, other professional phagocytes such as neutrophils, monocytes, and dendritic cells should be able to similarly clear the virus-bound Nanotraps. In particular, macrophages and dendritic cells are professional antigen presenting cells, which present engulfed antigens to the adaptive immune system (Janeway, 2008 ). Since the Nanotraps are able to engage antigen presenting cells, it is possible that they may also elicit virus-specific adaptive immune responses. Future studies will evaluate whether Nanotraps can prime adaptive immune responses, thereby promoting vaccine-like protection (Klichinsky et al., 2020) .

In addition, we purposely designed Nanotraps to be biocompatible, As current biosafety regulations preclude the testing of authentic SARS-CoV-2 in the EVLP, we further confirmed our Nanotraps can inhibit authentic virus in vitro ( Figure   5E ). These experiments together suggest our Nanotraps could potentially be used to treat SARS-CoV-2 infection in the clinic.

In summary, we developed a new type of potent, effective nanomedicine "Nanotraps" to contain and clear SARS-CoV-2 by harnessing and integrating the power of nanotechnology and immunology. The Nanotraps completely inhibited the SARS-CoV-2 infection to human cells and lung organs. The Nanotraps are effective, biocompatible, safe, stable, feasible for mass production. It is reasonable to hypothesize that the Nanotraps could be easily formulated into a nasal spray or inhaler for easy administration and direct delivery to the respiratory system, or as an oral or ocular liquid, or subcutaneous, intramuscular or intravenous injection to target different sites of SARS-CoV-2 exposure, thus offering flexibility in administration. Furthermore, the design of our Nanotrap is highly versatile: they can be modified to incorporate small molecule drugs or protein/mRNA vaccines to their core, and different human ACE2 

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Jun Huang (huangjun@uchicago.edu).

The materials generated in this study are available from the corresponding author upon request.

The data used to support the findings of this study are available from the corresponding author upon request.

To synthesize the Nanotraps, we employed a two-step method developed for polymer- Table S1 . 

The sizes of Nanotraps were measured by a dynamic light scattering particle size analyzer (Malvern Zetasizer). Briefly, 1 µL of Nanotraps were dispersed in 0.1× PBS and further dispersed in ultrasonic water bath for 10 minutes before testing. The size measurement was carried at 25 ℃ with count rates within 300-500 kcps and measured 3 times. The zeta potentials of Nanotraps were performed by a Möbiuζ system (Wyatt Technology). The data were presented as mean ± SD.

AF488-labeled anti-ACE2 antibody (Santa Cruz Biotechnology) was added to the Nanotrap-ACE2 for 30 minutes on ice and centrifuged at 5,000×g for 10 min, the pellet was washed with PBS for 3 times. The resulting Nanotraps were resuspended in 50% glycol and imaged by total internal reflection fluorescence microscopy (Nikon) with 488 nm and 647 nm excitation lasers and 200 milliseconds exposure. Line scans were performed in Fiji.

The sizes and morphologies of the Nanotraps were studied by scanning electron microscopy. Briefly, 10 μL of Nanotrap-ACE2 were diluted in MiliQ water and further dispersed on ultrasonic water bath for 10 minutes before adding onto a silicon chip. 40

μL of SARS-CoV-2 spike pseudotyped lentivirus was fixed by 4% PFA at 37 ℃ for 30 minutes, the virus was then washed by PBS 3 times using an Amicon Ultra-15 Centrifugal Filter (pore size 100 KDa) at 3,000×g, 10 minutes. The resulting fixed virus was incubated with 10 μL Nanotraps-ACE2 at 37 ℃ for 1 hour and added onto the 1 cm 2 silicon chip followed by airdrying overnight. After dehydration, the samples were coated with 8 nm platinum/palladium by sputter coater (Cressington 208HR). The scanning electronic microscope (Carl Zeiss Merlin) was used to image the morphology of the Nanotraps with an accelerating voltage of 2.0 kV. For each sample, more than 10 measurements with different magnification were performed to ensure the repeatability of the results.

Macrophage differentiation was conducted as follows (Hsu et al., 1996) For SARS-CoV-2 spike pseudotyped lentivirus neutralizing assay, 1×10 4 HEK293T-ACE2 cells were seeded onto a 384-well plate overnight. Nanotraps or ACE2 was added to SARS-CoV-2 pseudovirus (4 µL per well) and incubated for 1 hour at 37 C. The virus-Nanotrap solution was added to each well (n = 3 for each group). 72 hours later, the plate was centrifuged for 5 minutes at 500×g to prevent cell loss. Supernatant was aspirated and 35 µL of PBS was added. PBS was carefully aspirated, leaving ~15 µL of liquid behind. Renilla-Glo assay substrate was added to the Assay Buffer at a 1:100 dilution, then 15 µL of the substrate:buffer was added to each well of a 384-well plate.

The bioluminescence was recorded by a microplate reader (Fisher Scientific BioTek Cytation 5) with an exposure of 200 milliseconds. Wells infected with pseudovirus only were normalized as 100%.

THP-1 cells were differentiated into macrophages as described above. Coculture was carried out in a macrophage to A549 cell ratio of 1:5. 4×10 4 A549 cells were seeded in an 18-well microslide (Vivid) overnight and 8×10 3 dTHP-1 macrophages were added onto the A549 cells for another 6 hours. 500 FFU SARS-CoV-2 spike pseudotyped VSV was incubated with Nanotraps or PBS in 37 °C for 1 hour before adding to the coculture cells. 24 hours later, the cells were fixed with 4% PFA and stained with CF532 Wheat Germ Agglutinin (WGA) Conjugates (Biotium) and DAPI and imaged under a confocal microscope (Leica SP8). Percent infectivity was quantified in FIJI by dividing GFP + cells by total cell number (DAPI-stained nuclei). Each channel was processed as follows: Image>Threshold ("Huang" preset(Huang and Wang)); Image>Binary>Fill Holes; Image>Binary>Watershed; Analyze>Count Particles.

A549 or HEK293T-ACE2 cells (both maintained in DMEM supplemented with 10% FBS and 1% Penicillin-Streptomycin) were seed in a 96-well plate at a density of 1×10 4 cells/well in 100 μL of culture medium overnight. Nanotraps (3.8×10 7 particles/mL) were added into cells and the cells were cultured in a CO2 incubator at 37 °C for 72 hours.10 μL of CCK-8 (MedChem Express) solution was added to each well of the plate.

The plate was incubated for 2 hours in the incubator. Then it was put into a microplate reader (Fisher Scientific BioTek Cytation 5) and the plate was gently shaken for 1 minute before measuring the absorbance at 450 nm. The cytotoxicity was calculated by cell viability, that the relative absorbance from the control wells without Nanotraps were normalized as 100%. 

Non-transplantable human lungs were obtained from deceased individuals provided by the organ procurement organization Gift of Hope. All specimens and data were deidentified prior to receipt. This study was deemed exempt by the University of Chicago Institutional Review Board (IRB19-1942).

Lung Harvest. Living lungs unsuitable for transplantation were harvested in standard clinical fashion (Hsu et al., 1996) from deceased patients. Figure 5A shows the lung of a 56-year-old male patient (87.2 kg, cause of death: brain death). Lungs were transported to the laboratory at 4 C. Sample Processing. After 8 hours perfusion, tissues were harvested as described above. 

All SARS-CoV-2 infections were performed in biosafety level 3 conditions at the University of Chicago Howard T. Ricketts Regional Biocontainment Laboratory.

African green monkey kidney (Vero E6) cells were maintained in DMEM supplemented with 10% FBS and 1% Penicillin-Streptomycin. Nanotraps or neutralizing antibodies were serially diluted 2-fold and mixed with 400 PFU of SARS-CoV-2 (nCoV/Washington/1/2020, kindly provided by the National Biocontainment Laboratory, Galveston, TX) for one hour at 37 ºC, then used to infect Vero E6 cells for three days. Cells were fixed with 3.7% formalin and stained with 0.25% crystal violet.

Crystal violet stained cells were then quantified by absorbance at (595 nm) with a Tecan m200 microplate reader. Cell survival was calculated by normalizing untreated cells to 100%.

Supplemental information can be found online at https://doi.org/xxxxxxxxxx 

The University of Chicago is in the process of filing a patent based on some of the findings described in this manuscript.

showing the process of the Nanotraps with polymeric core coated with lipid-bilayer functionalized with ACE2 protein/neutralizing antibody. Following intratracheal administration, Nanotraps efficiently accumulated and trapped SARS-CoV-2 virionsin the lung tissue forming virus-Nanotrap complexes, which can be cleared by macrophages via phagocytosis, thereby blocking viral cellentry. (B-C) Dynamic light scattering (B) and Zeta-potential measurements (C) during different stages of Nanotrap preparation. (D) Fluorescent images of the prepared Nanotraps with PLA polymeric core (DiD, red) and ACE2 (anti-ACE2-AF488, green). Scale bar represents 5 µm. Dotted lines represent displayed plot profile below. (E-F) Pseudocolored SEM images of Nanotraps alone (E, orange) or with SARS-CoV-2 pseudovirus (F, cyan). To better visualize the selectivity for viral binding, larger Nanotraps were imaged. Scale bar represents 300 nm. and Nanotrap-ACE2, Nanotrap-Antibody, or Nanotrap-Blank for 72 and 24 hours, respectively. Data are presented as mean ± SD and fitted with a two-phase decay model. (C-D) HEK293T-ACE2 cells were treated with SARS-CoV-2 spike pseudotyped lentivirus (C) or VSV (D) and Nanotrap-ACE2 or soluble ACE2 for 72 and 24 hours, respectively. Data are presented as mean ± SD and fitted with a trend curve. For both SARS-CoV-2 spike pseudotyped lentivirus (C) and VSV (D), the Nanotrap-ACE2 and soluble ACE2 curves differ with p<0.0001, as tested by sum-of-squares F tests. (E) Confocal microscopy of pseudotyped VSV infection (GFP, green) in dTHP1 macrophages (WGA, red) and A549 epithelial cells (DAPI, blue); Nanotrap-ACE2 displayed in yellow. Scale bars represent 100 µm, with inset scale bars representing 40 µm. MФ: macrophages; V: virus; E: epithelial cells; NT: Nanotraps. (F) Quantification of (E). Data are shown as mean ± SD; unpaired t tests were conducted from three independent experiments. 

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