Cellular Nanosponges Inhibit SARS-CoV‑2 Infectivity

The research reported cellular nanosponges as an effectivemedical countermeasure to the SARS-CoV-2 virus. Two types ofcellular nanosponges are made of the plasma membranes derivedfrom human lung epithelial type II cells or human macrophages.These nanosponges display the same protein receptors, bothidentified and unidentified, required by SARS-CoV-2 for cellularentry. It is shown that, following incubation with the nanosponges,SARS-CoV-2 is neutralized and unable to infect cells. Crucially, thenanosponge platform is agnostic to viral mutations and potentiallyviral species, as well. As long as the target of the virus remains theidentified host cell, the nanosponges will be able to neutralize thevirus.

Figure 1.Fabrication and characterization of cellular nanosponges. (A) Schematic mechanism of cellular nanosponges inhibiting SARS-CoV-2infectivity. The nanosponges were constructed by wrapping polymeric nanoparticle (NP) cores with natural cell membranes from target cells suchas lung epithelial cells and macrophages (MΦs). The resulting nanosponges (denoted“Epithelial-NS”and“MΦ-NS”, respectively) inherit thesurface antigen profiles of the source cells and serve as decoys to bind with SARS-CoV-2. Such binding interaction blocks viral entry and inhibitsviral infectivity. (B) Dynamic light scattering measurements of hydrodynamic size (diameter, nm) and surface zeta-potential (ζ, mV) of polymericNP cores before and after coating with cell membranes (n= 3; mean + standard deviation). (C) Selective protein bands of cell lysate, cellmembrane vesicles, and cellular nanosponges resolved with Western blotting analysis. (D) Comparison of thefluorescence intensity measured fromcellular nanosponges (100μL, 0.5 mg/mL membrane protein concentration) or source cells (100μL, approximately 2.5×106cells) containingequal amounts of membrane content and stained withfluorescently labeled antibodies (n= 3; mean + standard deviation; n.s.: not significant;statistical analysis was performed with paired two-tailedt-test). (E) Stability of cellular nanosponges in 1×phosphate-buffered saline determined bymonitoring particle size (diameter, nm) over a span of 7 days (n= 3; mean±standard deviation).

The emergence of severe acute respiratory syndromecoronavirus 2 (SARS-CoV-2) has caused an outbreak ofcoronavirus disease (COVID-19), and the pandemic hasunfolded into a severe global public health crisis.1,2Remdesiviris currently the most advanced antiviral drug for COVID-19treatment, which received an emergency-use authorization inthe United States for patients with severe disease, but themortality benefit is unproven.3The search for new drugsrequires a clear understanding of the underlying molecularmechanisms of viral infection, which is a particular challengewith emerging viruses such as SARS-CoV-2.4,5Moreover,antiviral medicine often targets a specific viral species thatcannot be deployed across different species or families ofviruses and may be rendered ineffective as the virusaccumulates mutations and escapes treatments.6Therefore,an effective therapeutic agent to inhibit SARS-CoV-2infectivity, as well as its potential mutated species, would bea significant game changer in the battle against this publichealth crisis.Early understanding of the clinical manifestation of COVID-19 is severe viral pneumonia. Emerging data are clear thatSARS-CoV-2 elicits significant damage on other organ systemseither directly or indirectly through downstream immuno-logical effects.7Up to 75% of COVID-19 patients present withsome renal involvement, with a significant portion of patientsdeveloping acute kidney injury.8Acute respiratory distresssyndrome (ARDS) is a common and deadly manifestation ofCOVID-19 and is associated with prolonged intubation andhigh mortality.9Typically, COVID-19 patients initially presentmild symptoms, yet a subset of patients rapidly developcomplications such as ARDS and multiorgan failure andultimately death. The rapid clinical deterioration is thought tobe closely related to the cytokine storm.10Recently,coagulopathy has been described as a critical morbidity inCOVID-19 patients and is associated with worse outcomes.11All of these clinical complications speak to the complexity ofthis disease and that the consequence of immune response tothe viral infection may be the main driver of morbidity andmortality of COVID-19.A novel approach to drug development is to place the focuson the affected host cells instead of targeting the causativeagent. Inspired by the fact that the infectivity of SARS-CoV-2relies on its binding with the protein receptors, either knownor unknown, on the target cells, we create cellular nanospongesas a medical countermeasure to the coronavirus. Thesenanosponges are made of human-cell-derived membranes,which are sourced from cells that are naturally targeted bySARS-CoV-2 (Figure 1A). The nanosponges display the samereceptors that the viruses depend on for cellular entry. Wehypothesize that, upon binding with nanosponges, thecoronaviruses are unable to infect their usual cellular targets.SARS-CoV-2 uses angiotensin-converting enzyme 2 (ACE2)and CD147 expressed on the host cells, such as human alveolarepithelial type II cells, as receptors for cellular entry.12Human macrophages both express CD147 and have been reported toplay a significant role in the infection by frequent interactionswith virus-targeted cells through chemokines and phagocytosissignaling pathways.13Based upon the current knowledge of SARS-CoV-2, wefabricated two types of cellular nanosponges, human lungepithelial type II cell nanosponge (denoted“Epithelial-NS”)and human macrophage nanosponge (denoted“MΦ-NS”).The resulting cellular nanosponges were thoroughly charac-terized for their physicochemical and biological properties,followed byin vivoevaluation of their safety in the lungs. Then,these samples were independently tested in a biosafety level 4(BSL-4) laboratory for inhibitory effects on human SARS-CoV-2 virus and demonstrated clear antiviral efficacyin vitro.To prepare cellular nanosponges, cell membranes of humanlung epithelial cells and macrophages were derived with adifferential centrifugation method and verified for purity. Themembranes were then coated onto polymeric nanoparticlecores made from poly(lactic-co-glycolic acid) (PLGA) with asonication method to form Epithelial-NS and MΦ-NS,respectively. When examined with dynamic light scattering,both Epithelial-NS and MΦ-NS showed hydrodynamicdiameters larger than that of the uncoated PLGA cores(Figure 1B). The surface zeta-potential of the nanospongeswas less negative than that of the PLGA cores but comparableto that of the source cells (Table S1). These changes areconsistent with the addition of a bilayer cell membrane. Cellmembrane coating allows nanosponges to inherit the viralreceptors related to coronavirus entry into the host cells. Forverification, Western blot analysis showed the presence of viralreceptors such as ACE2, transmembrane serine protease 2(TMPRSS2), and dipeptidyl peptidase IV (DPP4) on theEpithelial-NS, and ACE2, C-type lectin domain family 10(CLEC10), and CD147 on the MΦ-NS (Figure 1C).12,13Theresults also showed that the nanosponge preparation facilitatedmembrane protein retention and enrichment on the nano-sponges, without contamination from intracellular proteins(Figure S1). For viral neutralization, right-side-out membraneorientation, driven by the asymmetric repulsion between thecores and the extracellular membrane versus the intracellularmembrane, is essential.14To examine the membrane sidedness,we stained cellular nanosponges and their source cellscontaining equal amounts of membrane content usingfluorescently labeled antibodies against select membraneantigens. After the removal of free antibodies, cellularnanosponge samples showedfluorescence intensities compa-rable with those of the cell samples (Figure 1D). This indicatesthat the nanosponges adopted a right-side-out membraneorientation because inside-out membrane coating wouldreduce antibody staining.15The membrane coating alsoprovided cellular nanosponges with extended colloidal stabilityin 1×phosphate-buffered saline (Figure 1E).After confirming the successful fabrication of Epithelial-NSand MΦ-NS, we sought to evaluate their acute toxicity afterinvivoadministration in mice. Given that our intended use is thedeployment of cellular nanosponges for the treatment ofcoronavirus infections that predominantly affect the respiratorytract,9we elected to study the intratracheal route ofadministration using the highest feasible dose of Epithelial-NS or MΦ-NS (300μg, based on membrane protein, in asuspension of 20μL). Histopathological analysis of lung tissue3 days after nanosponge administration revealed that immuneinfiltration was similar to baseline levels, and there was noevidence of lesion formation or tissue damage (Figure 2A).Furthermore, we examined multiple blood parameters,including a comprehensive serum chemistry panel and bloodcell counts, 3 days after nanosponge administration (Figure2B,C). All of the blood markers that were studied, in additionto red blood cells, platelets, and white blood cell counts, wereconsistent with baseline levels, confirming the short-term safetyof the cellular nanosponges.We next evaluated the neutralization of infectivity byauthentic SARS-CoV-2 with a plaque reduction neutralizationtest. In the study, a low passage sample of SARS-CoV-2 (USA-WA1/2020, World Reference Center for Emerging Viruses andArboviruses)16was amplified in Vero E6 cells to make aworking stock of the virus. Vero E6 cells were seeded at 8×105cells per well in 6-well plates the day prior to theexperiment. Serial quarter-log dilutions of the nanospongeswere mixed with 200 plaque-forming units (PFU) of SARS-CoV-2. The mixture was incubated at 37°C for 1 h and thenadded to the cell monolayers followed by an additional 1 h ofincubation. Mock-infected and diluent-only infected wellsserved as negative and positive controls, respectively.Monolayers were overlaid and incubated for 2 days followedby viral plaque enumeration. Following the incubation, cultureswithout adding Epithelial-NS showed a viral count comparableto that in the negative control, confirming viral entry andinfection of the host cells. Inhibition of the infectivity increasedas the concentration of Epithelial-NS increased, suggesting adose-dependent neutralization effect (Figure 3A). Based on theresults, a half-maximal inhibitory concentration (IC50) value of 827.1μg/mL for Epithelial-NS was obtained. In parallel, asimilar dose-dependent inhibition of the viral infectivity wasobserved with MΦ-NS (Figure 3B). In this case, an IC50valueof 882.7μg/mL was obtained. These results indicate that theEpithelial-NS and MΦ-NS have comparable ability to inhibitviral infectivity of SARS-CoV-2. To further verify that theinhibition was indeed due to epithelial cell or macrophagemembrane coating, control nanosponges made from mem-branes of red blood cells (denoted“RBC-NS”) were also testedin parallel for viral inhibition but were not effective inn

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