key: cord-0734281-52lj9gw7 authors: Cai, Xiaolei; Prominski, Aleksander; Lin, Yiliang; Ankenbruck, Nicholas; Rosenberg, Jillian; Chen, Min; Shi, Jiuyun; Chang, Eugene B.; Penaloza-MacMaster, Pablo; Tian, Bozhi; Huang, Jun title: A Neutralizing Antibody-Conjugated Photothermal Nanoparticle Captures and Inactivates SARS-CoV-2 date: 2020-11-30 journal: bioRxiv DOI: 10.1101/2020.11.30.404624 sha: 23699ad14f0f37a33729b801e655515f33c90f61 doc_id: 734281 cord_uid: 52lj9gw7 The outbreak of 2019 coronavirus disease (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has resulted in a global pandemic. Despite intensive research including several clinical trials, currently there are no completely safe or effective therapeutics to cure the disease. Here we report a strategy incorporating neutralizing antibodies conjugated on the surface of a photothermal nanoparticle to actively capture and inactivate SARS-CoV-2. The photothermal nanoparticle is comprised of a semiconducting polymer core and a biocompatible polyethylene glycol surface decorated with neutralizing antibodies. Such nanoparticles displayed efficient capture of SARS-CoV-2 pseudoviruses, excellent photothermal effect, and complete inhibition of viral entry into ACE2-expressing host cells via simultaneous blocking and inactivating of the virus. This photothermal nanoparticle is a flexible platform that can be readily adapted to other SARS-CoV-2 antibodies and extended to novel therapeutic proteins, thus providing a broad range of protection against multiple strains of SARS-CoV-2. Several nanomaterial-based approaches have been investigated for virus detection, [36] [37] [38] [39] vaccine delivery, 40, 41 and viral capture, [42] [43] [44] however, the lack of a method to effectively capture and inactivate the virus after binding-which may lead to ADE-remains to be addressed. Herein we report the development of a strategy utilizing photothermal nanoparticles decorated with highaffinity neutralizing antibodies in order to effectively capture and inactivate SARS-CoV-2 ( Figure 1a ). Each photothermal nanoparticle contains a semiconducting polymer core, poly[2,6-(4,4-bis- as the matrix to encapsulate PCPDTBT through self-assembly. 47 Subsequently, an anti-SARS-CoV-2 neutralizing antibody was crosslinked to the nanoparticles via reaction of NHS esters with amines on the antibody surface. Using dynamic light scattering (DLS), we determined the hydrodynamic diameters of the photothermal nanoparticles to be ~ 90 nm, which was validated by transmission electron microscopy (TEM) (Figure 1b ). In addition, the photothermal nanoparticles exhibited good stability without forming any aggregation or precipitation after being stored in aqueous dispersions at 4 °C for several weeks ( Figure S1 ) Since the photothermal nanoparticles exhibited excellent absorption in the near-infrared region, we hypothesized that they could effectively generate local heating after light excitation with a suitable wavelength. To test this hypothesis, we applied a 650-nm LED with a power density of 250 mW/cm 2 to excite the photothermal nanoparticles, because it would fit the absorption The measurement of UV-vis absorption spectra was carried out using a UV-vis spectrophotometer (Thermo Scientific NanoDrop™ 2000). The sizes of the nanoparticles were measured by dynamic light scattering (DLS) particle size analyzer (Malvern Zetasizer). The sizes and morphologies of the nanoparticles were studied by transmission electron microscopy (TEM, JEM-2010F, JEOL, Japan). The photothermal nanoparticles (100 µg/mL, 10 µL) was incubated with the secondary Alexa Fluor 488-labeled anti-IgG2b antibody (100 µg/mL, 10 µL) for 1 h. Single particle fluorescent images were captured by a fluorescence microscopy with a 100×/1.49 numerical aperture (NA) objective using a Nikon Ti-E inverted microscope. A 7-color solid state LED light source was connected with a liquid light guide to the microscope; the light then passes through a quad bandpass filter (ZET405-488-532-647m). The LED excitation wavelengths used were 470 ± 25 nm for green channel and 740 ± 20 nm for red channel (Spectra X, Lumencor). The emissions from the nanoparticles and Alexa Fluor-488 were captured by an Andor iXon Ultra 888 back-illuminated electron multiplying CCD (EMCCD) camera (Oxford Instruments). Photothermal nanoparticles (100 µg/mL in 1 × PBS) were added into a well in a 96-well plate (flat bottom, GenClone®). A 650-nm LED (Spectra X, Lumencor) was applied to excite the nanoparticle solution for 10 min with a power density of 250 mW/cm 2 . The temperature changes from 0 to 10 min were recorded by a probe thermometer. Each measurement was repeated three times. Temperature changes of pure 1 × PBS were also measured using the same method. Photothermal response measurements were performed on an upright microscope (Olympus, BX61WI) with a 20×/0.5 NA water-immersion objective. An LED light source (Lumencore Spectra X) with a 650 nm filter was used for excitation and was electronically controlled using transistor-transistor logic signals delivered from a digitizer (Molecular Devices, Digidata 1550). Temperature was inferred using a temperature-resistance calibration curve measured for the pipette. The authors declare no competing interests. Critical supply shortages-the need for ventilators and personal protective equipment during the Covid-19 pandemic COVID-19 pandemic: perspectives on an unfolding crisis Real-time tracking of self-reported symptoms to predict potential COVID-19 COVID-19: consider cytokine storm syndromes and immunosuppression COVID-19 and the cardiovascular system Structural basis for the recognition of SARS-CoV-2 by full-length human ACE2 Angiotensin-converting enzyme 2 (ACE2) as a SARS-CoV-2 receptor: molecular mechanisms and potential therapeutic target An mRNA Vaccine against SARS-CoV-2 -preliminary report Safety and immunogenicity of the ChAdOx1 nCoV-19 vaccine against SARS-CoV-2: a preliminary report of a phase 1/2, single-blind, randomised controlled trial The COVID-19 vaccine development landscape Phase I/II study of COVID-19 RNA vaccine BNT162b1 in adults COVID-19 vaccine development pipeline gears up Developing Covid-19 Vaccines at Pandemic Speed Repurposing anticancer drugs for COVID-19-induced inflammation, immune dysfunction, and coagulopathy Hydroxychloroquine for the prevention of Covid-19 -searching for evidence Remdesivir in adults with severe COVID-19: a randomised, double-blind, placebo-controlled, multicentre trial Treatment of 5 critically Ill patients with COVID-19 with convalescent plasma Serosurveys and convalescent plasma in COVID-19 A perspective on potential antibody-dependent enhancement of SARS-CoV-2 The potential danger of suboptimal antibody responses in COVID-19 Implications of antibody-dependent enhancement of infection for SARS Molecular mechanism for antibody-dependent enhancement of coronavirus entry Potent neutralizing antibodies from COVID-19 patients define multiple targets of vulnerability Potent Neutralizing antibodies against SARS-CoV-2 identified by highthroughput single-cell sequencing of convalescent patients' B cells Isolation of potent SARS-CoV-2 neutralizing antibodies and protection from disease in a small animal model Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor Structural basis of receptor recognition by SARS-CoV-2 Structural and functional basis of SARS-CoV-2 entry by using human ACE2 Human monoclonal antibodies block the binding of SARS-CoV-2 spike protein to angiotensin converting enzyme 2 receptor A noncompeting pair of human neutralizing antibodies block COVID-19 virus binding to its receptor ACE2 A neutralizing human antibody binds to the N-terminal domain of the Spike protein of SARS-CoV-2 Potent neutralizing antibodies against multiple epitopes on SARS-CoV-2 spike A human neutralizing antibody targets the receptor-binding site of SARS-CoV-2 Dual-Functional Plasmonic Photothermal Biosensors for Highly Accurate Severe Acute Respiratory Syndrome Coronavirus 2 Detection Multiplexed Nanomaterial-Based Sensor Array for Detection of COVID-19 in Exhaled Breath Selective Naked-Eye Detection of SARS-CoV-2 Mediated by N Gene Targeted Antisense Oligonucleotide Capped Plasmonic Nanoparticles Rapid Detection of COVID-19 Causative Virus (SARS-CoV-2) in Human Nasopharyngeal Swab Specimens Using Field-Effect Transistor-Based Biosensor Self-amplifying RNA SARS-CoV-2 lipid nanoparticle vaccine candidate induces high neutralizing antibody titers in mice Leveraging mRNA Sequences and Nanoparticles to Deliver SARS-CoV-2 Antigens In Vivo Nanoparticle-based strategies to combat COVID-19 Cellular nanosponges inhibit SARS-CoV-2 infectivity Toward Nanotechnology-Enabled Approaches against the COVID-19 Plasmonic photothermal therapy (PPTT) using gold nanoparticles Noble metals on the nanoscale: optical and photothermal properties and some applications in imaging, sensing, biology, and medicine Identifying glioblastoma margins using dual-targeted organic nanoparticles for efficient in vivo fluorescence image-guided photothermal therapy Lowlevel laser (light) therapy (LLLT) in skin: stimulating, healing, restoring Photodynamic and photobiological effects of lightemitting diode (LED) therapy in dermatological disease: an update Nongenetic optical neuromodulation with silicon-based materials Rational design of silicon structures for optically controlled multiscale biointerfaces Heterogeneous silicon mesostructures for lipid-supported bioelectric interfaces Establishment and validation of a pseudovirus neutralization assay for SARS-CoV-2 Development and optimization of a sensitive pseudovirus-based assay for HIV-1 neutralizing antibodies detection using A3R5 cells Neutralizing antibody response during acute and chronic hepatitis C virus infection Teicoplanin inhibits Ebola pseudovirus infection in cell culture Nanomaterials for the photothermal killing of bacteria Nanomaterial applications in photothermal therapy for cancer