Toward a Radically Simple Multi‐Modal Nasal Spray for Preventing Respiratory Infections

RESEARCH ARTICLE

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Toward a Radically Simple Multi-Modal Nasal Spray for Preventing Respiratory Infections

John Joseph, Helna Mary Baby, Joselyn Rojas Quintero, Devin Kenney,

Yohannes A Mebratu, Eshant Bhatia, Purna Shah, Kabir Swain, Dongtak Lee,

Shahdeep Kaur, Xiang-Ling Li, John Mwangi, Olivia Snapper, Remya Nair, Eli Agus, Sruthi Ranganathan, Julian Kage, Jingjing Gao, James N Luo, Anthony Yu,

Dongsung Park, Florian Douam, Yohannes Tesfaigzi,* Je rey M Karp,* and Nitin Joshi*

Introduction

Nasal sprays for pre-exposure prophylaxis against respiratory infections show

limited protection ( — %), largely due to their single mechanism of Respiratory infections result in signi cant action either neutralizing pathogens or blocking their entry at the morbidity and mortality worldwide.[ ] The

past few decades have witnessed numer- nasal lining, and a failure to maximize the capture of respiratory

ousoutbreaks,oftenleadingtoepidemicsor droplets, allowing them to potentially rebound and reach deeper airways. unanticipated pandemics such as COVID-

This report introduces the Pathogen Capture and Neutralizing Spray (PCANS), . Although vaccines are available against which utilizes a multi-modal approach to enhance e cacy. PCANS coats In uenza A virus (IAV), severe acute respi- the nasal cavity, capturing large respiratory droplets from the air, and serving ratory syndrome coronavirus (SARS-CoV- as a physical barrier against a broad spectrum of viruses and bacteria, while ), respiratory syncytial virus (RSV) and

Streptococcus pneumoniae, the emergence rapidly neutralizing them with over . % e ectiveness. The formulation of mutants often reduces the e cacy of

consists of excipients identi ed from the FDA s Inactive Ingredient Database vaccines.[ ] Additionally, there are several and Generally Recognized as Safe list to maximize e cacy for each step in the pathogens, including adenovirus, Klebsiella multi-modal approach. PCANS demonstrates nasal retention for up to hours pneumoniae, Staphylococcus aureus, and Es-

in mice. In a severe In uenza A mouse model, a single pre-exposure dose cherichia coli, which can cause severe res-

piratory diseases, but do not have clinically of PCANS leads to a> . % reduction in lung viral titer and ensures %

available vaccines, as of now. In the face of survival,comparedto %inthecontrolgroup.PCANSsuppressespathological an unforeseen pandemic, the timeline for

manifestations and o ers protection for at least hours. This data suggest developing vaccines targeting a pathogen PCANS as a promising daily-use prophylactic against respiratory infections. can range from to years, contingent upon the specic nature of the pathogen.[ , ]

J. Joseph, H. M. Baby, P. Shah, K. Swain, D. Lee, S. Kaur, X.-L. Li,

J. Mwangi, O. Snapper, E. Agus, S. Ranganathan, J. Kage, J. Gao, A. Yu,

J. M Karp, N. Joshi

Center for Accelerated Medical Innovation

Department of Anesthesiology

Perioperative and Pain Medicine

Brigham and Women s Hospital

Boston, MA , USA E-mail:jmkarp@bwh.harvard.edu;njoshi@bwh.harvard.edu

J.Joseph,H.M.Baby,P.Shah,D.Lee,S.Kaur,X.-L.Li,J.Mwangi, O.Snapper,E.Agus,S.Ranganathan,J.Kage,J.Gao,A.Yu,J.MKarp, N.Joshi

CenterforNanomedicine

DepartmentofAnesthesiology

PerioperativeandPainMedicine

BrighamandWomen sHospital

Boston,MA ,USA

The ORCID identi cation number(s) for the author(s) of this article can be found under

DOI: . /adma.

J. Joseph, J. R. Quintero, Y. A Mebratu, D. Lee, S. Kaur, R. Nair, J. Gao,

J. N Luo, A. Yu, Y. Tesfaigzi, J. M Karp, N. Joshi Harvard Medical School

Boston, MA , USA

E-mail:ytesfaigzi@bwh.harvard.edu

J.R.Quintero,Y.AMebratu,Y.Tesfaigzi DivisionofPulmonology BrighamandWomen sHospital Boston,MA ,USA

D.Kenney,F.Douam NationalEmergingInfectiousDiseasesLaboratories DepartmentofMicrobiology

BostonUniversity Chobanian&AvedisianSchoolofMedicine Boston,MA ,USA

E.Bhatia IndianInstituteofTechnology Mumbai ,India

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The rapid creation of e cacious COVID- vaccines stands as

an unparalleled scienti c achievement. However, it took several months for the vaccine to become available, during which nu- merous hospitalizations and deaths were reported.[ ] Addition- ally, multiple obstacles, such as production complexities, vac- cine nationalism, and the emergence of novel variants, collec- tively posed major challenges around the world. A large percent- age of the population did not consent to vaccination for vari- ous reasons, which posed a signi cant hurdle in minimizing the transmission of pathogens. Another concern pertaining to vaccines is their partial mitigation of the pathogen burden,[ , ] which implies that vaccinated people can still contract and dis- seminate the infection, albeit at a reduced rate compared to those who are unvaccinated. Thus, there is a critical need to develop a pre-exposure prophylactic approach that can be eas- ily and rapidly deployed either independently or in tandem with vaccines, serving as the primary safeguard against cur- rent and emerging respiratory pathogens. Such an approach should e ciently reduce pathogen load, and be radically sim- ple to scale up and manufacture to ensure widespread global adoption.

Transmission of most respiratory pathogens predominantly occurs through inhalation of contaminated respiratory droplets and their subsequent deposition in the nasal cavity, which has an entry checkpoint.[ ] For instance, SARS-CoV- virus binds to the angiotensin-convertingenzyme (ACE )locatedinnasalepithe- lial cells via its receptor-binding domain (RBD). The nasal cavity is a primary target for SARS-CoV- infection due to high expres- sion of ACE ,[ — ] which decreases towards the lower respiratory tract.[ ] The infection spreads to the deeper airways via virus- laden extracellular vesicles secreted by infected cells in the nasal cavity.[ ] Similarly, bacteria, including

S. pneumoniae

and

S. au- reus

adhere to nasal mucin via a speci c adhesin receptor.[ , ] Considering the vulnerability of nasal cavity and its critical role inthetransmissionofrespiratorypathogens,chemoprophylactic nasal sprays have been developed to o er pre-exposure prophy- laxis against respiratory infections. This approach utilizes chem- ical agents, including small molecule drugs, antiseptics, or nitric oxide, to deactivate the pathogen in the nasal cavity or a polymer that acts as a physical barrier to prevent pathogen entry through

J. N Luo

Department of Surgery Brigham and Women s Hospital Boston, MA , USA

D. Park

Center for Functional Nanomaterials Brookhaven National Laboratory Upton, NY , USA

J. M Karp

Harvard—Massachusetts Institute of Technology Division of Health Sciences and Technology

Massachusetts Institute of Technology

Cambridge, MA , USA J. M Karp

Broad Institute Cambridge, MA , USA

1. M Karp

Harvard Stem Cell Institute Cambridge, MA , USA

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thenasallining.[ , ]Althoughmultiplepre-exposurechemopro- phylactic approaches have been previously developed,[ — ] they have resulted in sub-optimal e cacy with only — % protec- tion achieved in pre-clinical and clinical studies.[ — ] We con- tend that the sub-optimal clinical e cacy of previous chemo- prophylactic nasal sprays can be attributed, at least in part, to their dependence on a single mode of action, typically centered aroundeitherpathogenneutralizationorhinderingpathogenen- try through the nasal lining. Additionally, as pathogens are de- posited in the nasal cavity through the impaction of large respi- ratory droplets,[ ] the potential for these droplets to bounce o the cavity wall and reach the deeper airways has been overlooked in previous approaches. A comprehensive prophylactic strategy shouldprioritizethee ectivecaptureofpathogen-ladendroplets, preventing them from bouncing o a critical consideration ne- glected in prior e orts. Previous chemoprophylactic approaches also target a limited type/class of pathogen,[ , — ] which could potentially compromise their e ectiveness against newly emerg- ing pathogens. Finally, many chemoprophylactic nasal sprays face limitations for repeated/daily application due to toxicity concerns.[ , ]

Herein, we report a Pathogen Capture and Neutralizing Spray (PCANS), which, unlike previously developed chemoprophylac- tic approaches, acts via a multi-modal approach that involves three key steps (Figure ). First, PCANS enhances the capture of pathogen-laden respiratory droplets from inspired air by pre- ventingthemfrombouncingo thenasallining.Second,PCANS provides a physical barrier over nasal mucosa to intercept inva- sion/colonization of di erent pathogens. Last, PCANS rapidly neutralizes a wide range of pathogens. To ensure safety during daily or repeated use and achieve design simplicity, PCANS was meticulouslydesignedasa drug-free formulation,utilizingma- terialsfrominactiveingredientdatabase(IID)orgenerallyrecog- nized as safe (GRAS) list of the Food and Drug Administration (FDA) that are present as excipients in commercially available nasal/topicalformulationsandareavailablecommerciallyintons of quantities. Using a highly iterative approach, we performed rigorous screening of these excipients, their di erent concentra- tions and combinations to identify optimal agents and their con- centrations that maximize the e cacy of each step of the multi- modalapproach,whilemaximizingtheresidencetimeofPCANS inthenasalcavity.Consequently,the nalformulationofPCANS comprises a blend of multiple active agents, each contributing to the overall e ectiveness through a synergistic multi-modal ap- proach. Given the diverse active agents in PCANS, we also con- ducted comprehensive studies to ascertain the optimal concen- tration for each agent, ensuring that their individual activities re- mained unaltered. This meticulous investigation was imperative to guarantee the e cacy at every stage of the multi-modal ap- proach.

In vitro, PCANS demonstrated excellent physical barrier prop- erty against multiple viruses and bacteria preventing their trans- port by > . %. The unique composition of PCANS enabled broad-spectrum neutralization activity against a range of viruses (both enveloped and non-enveloped) and bacteria, resulting in

• . % reduction in the pathogen load. On the other hand, iota/kappa carrageenan, the key component of a commercially available chemoprophylactic nasal spray- AGOVIRAX, resulted in only % reduction in the viral loads of pathogens includ-

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Figure . Pathogen Capture and Neutralizing Spray (PCANS) acts via a multi-modal approach against respiratory pathogens. An aqueous, drug-free solution of PCANS, comprising mucoadhesive biopolymers, surfactants, and alcohol, is administered using a pocket-sized nasal spray device and undergoesaphasetransitiontoformahydrogellayerovernasalmucosa.SurfactantsinPCANSreduceinterfacialtensionofthenasalliningandincrease wettabilitytoenhancethecaptureorreducethebounce-o ofpathogen-ladenrespiratorydropletsfromtheinhaledair.PCANSlayersasaphysicalbarrier preventing the transport of pathogens through the nasal lining. Finally, pathogens are neutralized by biopolymers and surfactants present in PCANS. PCANS is cleared via the native mucosal clearance mechanism and is eliminated through the digestive route.

ing IAV and SARS-CoV- viruses. Coating a D-model of hu- man nasal cavity with PCANS signi cantly increased the cap- ture of large respiratory droplets, compared to an only mucus- coated nasal cavity. Intranasal administration of PCANS-loaded with a uorescent dye resulted in at least h of residence time in the mouse nasal cavity, measured as the retention of uo- rescence signal over time. No discernible tissue in ammation was observed in mouse nasal turbinate after repeated dosing of PCANS, establishing its safety in mice. In a proof-of-concept in vivo study performed in a mouse model of severe In uenza A infection induced by a supra-lethal dose of PR virus (a mouse- adapted H N In uenza virus), PCANS showed superior pro- phylactic e ect compared to the e cacy reported for previous approaches.[ ]Pre-exposureprophylacticadministrationofasin- gle dose of PCANS was e ective within min, resulting in

• . % reduction in lung viral titer, and % survival by day

ascomparedto %observedinthePBS-treatedgroup.PCANS also suppressed pathological manifestations, and o ered protec- tion for at least h. Overall, PCANS holds promise as a pre- exposureprophylacticapproachtopreventcurrentandemerging respiratory infections. The straightforward and readily scalable manufacturingprocessofPCANS,combinedwithits drug-free compositionandrobuststabilitydemonstratedinthisstudy,posi- tionsitfavorablyforwidespreadadoptionandglobaldistribution. Toourknowledge,thisisthe rststudytodescribeamulti-modal chemoprophylactic nasal spray, as well as the rst to demon- stratebroad-spectrumactivityofachemoprophylacticnasalspray against both bacteria and viruses. Additionally, this nasal spray is the rst to achieve an -hour residence time in mouse nasal cav- ity, and the rst chemoprophylactic strategy to show % pro- tectioninanimalswithinapre-clinicalmodelofrespiratoryvirus infection.

• Results
• . Leveraging Biopolymers to Restrict Pathogen Entry via

Formation of a Physical Barrier

We selected mucoadhesive biopolymers that are listed in the IID orGRASlistoftheFDAandarepresentasexcipientsincommer- cially available nasal/topical formulations. Speci cally, gellan, pectin, hydroxypropyl methylcellulose (HPMC), carboxymethyl cellulose sodium salt (CMC), carbopol, and xanthan gum were selected. The biopolymers were screened for their ability to im- part physical barrier property to PCANS. Since a metered spray device would be used to administer PCANS, we rst identi ed sprayable concentration of each biopolymer by performing rhe- ological measurements (Figure a—f). Dynamic viscosity curves were generated using a rotational rheometer by varying shear rates up to s- , which is within the lower limits of shear ratesencounteredwhiledispensingformulationsthroughanasal spray device. Concentrations that exhibited a viscosity of less than . Pa.s were considered sprayable .[ , ] Next, we deter- mined the mechanical strength of each biopolymer at the high- est sprayable concentration before and after the addition of sim- ulated nasal uid (SNF). SNF was added to mimic the physio- logical environment in the nasal cavity. Mechanical strength was measured using a rotational rheometer and quanti ed as storage modulus (G ), which represents the amount of structure present

in a material.[ ] In the presence of SNF, gellan showed the high- est G as compared to other biopolymers (Figure g), indicating its superior mechanical strength. Gellan showed a -fold in- crease in its G in the presence of SNF (Figure g), which is con- sistent with its ability to undergo in situ gelation under phys- iological conditions. Mono and divalent cations present in the

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Figure . Biopolymers were screened for physical barrier property against pathogen entry. Viscosity as a function of shear rate up to s− at ¡C for di erentconcentrationsofa)gellan,b)pectin,c)hydroxypropylmethylcellulose(HPMC),d)carboxymethylcellulose(CMC),e)Carbopolandf)xanthan gum in water. The sprayable viscosity window is shown below the dashed line. g) Storage modulus (G ) of . % (w/v) gellan, % (w/v) pectin, . % (w/v) HPMC, . % (w/v) CMC, . % (w/v) Carbopol, and . % (w/v) xanthan gum, without and with simulated nasal uid (SNF). Amplitude sweep

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SNFcomplexwithglucuronicmonomericunitsofgellantoform

a crosslinked hydrogel.[ ] Compared to gellan, other biopoly- mers showed minimal or no increase in their storage modulus, suggesting poor in situ gelation. To investigate physical barrier property of biopolymers, a trans-membrane assay was devised (Figure S , Supporting Information), which involved evaluating the transport of IAV through an SNF-coated cell strainer (pore size m)oracellstrainercoatedwithsimulatedmucus/SNF mixture or a biopolymer/SNF mixture. After h, the viral titer in the chamber below the strainer was quanti ed by performing a plaque assay in Madin-Darby canine kidney (MDCK) host cells. Consistent with its excellent mechanical strength, Gellan/SNF reduced the transport of IAV particles by > -log fold ( . %) as compared to only SNF-coated or mucus/SNF-coated strain- ers (Figure h). Xanthan/SNF, CMC/SNF and HPMC/SNF also signi cantly reduced the IAV transport, but not as e ciently as gellan/SNF. Interestingly, despite signi cantly lower mechani- cal strength of pectin/SNF as compared to gellan/SNF, it inter- cepted the IAV transport with similar e ciency as gellan/SNF. Carrageenan,abiopolymerusedinpreviouslyreportedandcom- mercially available chemoprophylactic nasal sprays,[ ] was used as a control and did not reduce IAV transport in the presence of SNF.

Reduction in the transport of IAV particles by anionic biopoly- mers could be a result of their physical barrier property and/or electrostatic interactions between their negatively charged poly- meric chains and the positively charged capsid of IAV. To decou- ple the e ects of physical barrier property and electrostatic inter- actions, we studied the transport of a low molecular weight dye, rhodamine B isothiocyanate, which does not exhibit electrostatic interactions with any of the biopolymers evaluated, as con rmed usinganinsilicostudy(Figure S ,SupportingInformation).Gel- lan/SNF resulted in % reduction in the transport of the dye, con rmingexcellentphysicalbarrierproperty(Figure i).Incon- trast, other biopolymers did not reduce the transport of the dye, indicatingtheirpoorphysicalbarrierproperty.Thiscon rmsthat the reduction in IAV transport by pectin was primarily mediated via electrostatic interaction of pectin s chains with the virus cap- sid.

Next, we aimed to investigate the underlying mechanism re- sponsible for the observed reduction in the transport of rho- damine B isothiocyanate by the gellan/SNF system. Data from the in silico study exploring the intermolecular interactions of rhodamine B isothiocyanate with di erent biopolymers (Figure

S and Table S , Supporting Information) indicated that the dye interacts with gellan primarily through hydrogen bonds, similar

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to its interactions with other biopolymers. Notably, rhodamine

B isothiocyanate does not display hydrophobic interactions with gellanbutdoeswithcertainotherbiopolymers.Therefore,thein- teractions between the dye and gellan do not appear to be unique and are unlikely to be the primary factor contributing to the ob- served inhibition of the dye transport with gellan/SNF. Interest- ingly, gellan/SNF reduced the transport of rhodamine B isothio- cyanate in a concentration-dependent manner (Figure j), with a minimum of . % w/v gellan required. Higher polymer con- centration in a gel can increase tortuosity.[ ] As the concentra- tion of the polymer increases, the network of cross-linked poly- mer chains becomes denser. This increased tortuosity creates more complex and convoluted di usion pathways, thereby re- ducing the e ective di usion coe cient. Previous studies have shown that in systems with large pores, where the pore diameter

is approximately — times the size of the di using molecule, geometric tortuosity is the primary factor in uencing di usive transport.[ ]Incontrast,forporeswithdiametersbarely times

the molecular size, intermolecular interactions are more pro- nounced. Given the small size of rhodamine B isothiocyanate

( nm) relative to the micron-sized pores in the gellan gel,[ ] the di usion of the dye through gellan gel aligns with the for- mer scenario, suggesting that the observed transport reduction could be attributed to the increased tortuosity caused by the gel s cross-linking.Anotherphysicalfactorresponsibleforthereduced transport of the dye could be the mechanical strength of the gel, measured as the storage modulus (G ), which increased with the concentration of gellan (Figure S , Supporting Information). An increase in the mechanical strength of gels results in re- ducedswellingandhencereducesthedi usivityofmolecules.[ ] Therefore, taken together, the primary drivers for the reduction

in dye transport by the gellan/SNF can be attributed to both in- creased tortuosity and gel strength at speci c gellan concentra- tions.

Gellan/SNF also showed excellent physical barrier property againstbacteria.A . %w/vconcentrationofgellanalsoreduced the transport of E.colibacteria by > -log fold ( %) (Figure S , Supporting Information), suggesting it s broad spectrum physi- cal barrier property to limit the transport of both viruses and bac- teria. Mucus, on the other hand, only showed a -log fold ( %) reduction.Toconclude,gellanataconcentrationof . %w/vand aboveimpededthetransportofrhodamineBdye, E.coli,andIAV by %.

To ensure maximum coverage of the nasal cavity, we evalu- ated the spray characteristics of gellan at a concentration of . % w/v or higher when sprayed using a nasal spray pump (Aptar).

measurements were performed at ¡C by varying oscillatory strain between . % to % at Hz frequency. ****P < . , *P < . . n.s., not signi cant.h)AmountofIn uenzaAvirus(IAV)thatpermeatedwithin hthroughasimulatednasal uid(SNF)-coatedcellstrainer(poresize m) or a cell strainer coated with simulated mucus/SNF mixture or a biopolymer/SNF mixture. Permeation of viral particles was quanti ed by evaluating the viral titer in the chamber below the strainer using plaque assay performed in MDCK host cells. Results are expressed in plaque-forming units (PFU/mL). **P< . , *P < . compared to mucus/SNF, n.s, not signi cant. Percentage permeation of a uorescent dye, rhodamine B isothiocyanate through i) an SNF-coated cell strainer or a cell strainer coated with simulated mucus/SNF mixture or a biopolymer/SNF mixture. ****P< . compared to mucus/SNF and j) an SNF-coated strainer or strainer coated with gellan/SNF at di erent concentrations of gellan. ****P< . compared to . % w/v gellan/SNF. k) Percentage drip length of free brilliant green dye or mucoadhesive polymers mixed with brilliant green dye on sheep mucosal tissue. Driplengthfromthesprayareawasmeasuredasthedistancetraversedin hbythebiopolymerorfreedyefromthepointofdeposition.Thepercentage drip length of each biopolymer was calculated with respect to the drip length of the free dye. ****P< . compared to free dye. For g and h, P-values were determined using two-way ANOVA with Tukey s multiple comparisons tests. For i-k, P-values were determined using one-way ANOVA with Tukey s post hoc analysis. Data in a-f are from a single experiment (experiment repeated three times). Data in g—k are means – SD of technical repeats (n = , each experiment performed at least twice).

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Plume geometry and spray coverage were measured with a high- speed image acquisition system. Increasing gellan concentration resulted in a signi cant reduction in the angle of emitted plume ofthespray(de nedas plumeangle )andcoveragearea(Figure

S a—d, Supporting Information). Henceforth, we used . % w/v gellan due to its superior physical barrier property, plume angle, and coverage area, as compared to other concentrations.

Next, we evaluated the retention ability of gellan and other biopolymers at the mucosal tissue upon spraying. Mucosal re- tention was measured as the drip length, de ned as the distance traversedin hbythebiopolymerfromthepointofdepositionon sheep sintestinalmucosaplacedvertically.Tovisualizedripping, biopolymers were mixed with a brilliant green dye. The percent- age driplength ofeach biopolymer was calculated with respect to the drip length of the free dye. Gellan ( . % w/v) demonstrated excellent mucosal retention with zero drip length (Figure k; Figure S , Supporting Information). Other biopolymers, includ- ing carrageenan, which was used as a control showed > % drip length, indicating poor mucosal retention. Gellan s superior mucosal retention is attributed to its ability to strongly entangle with mucin glycoprotein in the mucosal tissue during the sol-gel transition.[ ]

• . Identifying Agents for Neutralizing a Broad-Spectrum of

Respiratory Pathogens

To impart PCANS a broad-spectrum pathogen neutralization ability, we screened agents from three di erent classes of com- pounds,includingbiopolymers,surfactants,andalcohols.Wede- ne neutralization as a process that impedes pathogen entry into host cells by either destabilizing the pathogen cell mem- brane or blocking the receptor-mediated binding/fusion of the pathogenthroughchemicalinteractions.Thesecompoundswere selected based on their previously reported ability to neutralize di erent types of pathogens.[ — ] To maximize safety and trans- latability of PCANS, we only selected agents that are listed in the IIDorGRASlistoftheFDAandarepresentasexcipientsincom- mercially available nasal/topical formulations (Figure a). We rst evaluated the neutralization ability of these agents against viruses (Figure b—k). Neutralization was studied in vitro by in- cubating each agent individually with either IAV or SARS-CoV- for or min, followed by -min centrifugation and subse- quent infection of target cells with the supernatant evaluated us- ing plaque forming or focus-forming assay. We chose IAV and SARS-CoV- due to their high prevalence worldwide as respi- ratory viruses and also due to a di erence in their capsid pro- teins and charge.[ , ] Biopolymers were evaluated at their high- est sprayable concentration, except for gellan and carrageenan. Gellan was evaluated at . % w/v due to its superior physical barrier property compared to . % w/v concentration and su- perior spray pattern compared to . % w/v concentration. Car- rageenan, used as a control, was evaluated at . % w/v, as this concentration is present in a commercially available chemopro- phylactic nasal spray.[ , ] Surfactants and alcohols were evalu- ated at the highest concentration previously used in humans via nasal route.[ , ] Compared to carrageenan, pectin exhibited su- perior neutralization of IAV, regardless of the incubation time, and demonstrated a -log fold ( . %) reduction in viral titer in

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the host cells in comparison to PBS (Figure b). Ten min of in- cubation with carbopol did not reduce the IAV titer, but a -log fold ( . %) reduction was observed with min of incubation. GellanexhibitedsimilarneutralizationofIAVascarrageenan,re- sultinginonlya -logfold( %)reductioninviralloadinthehost cells. For SARS-CoV- , both pectin and carrageenan showed less thana -logfolddecreaseinviralloadinthehostcells(Figure g). Gellan showed a -log fold ( . %) reduction in the viral titer,

but only with h incubation time. Among surfactants, tween and benzalkonium chloride (BKC) showed a -log log fold reduc- tion in IAV titer in the host cells, regardless of the incubation time (Figure c). Rapid neutralization of SARS-CoV- was ob- served with BKC, resulting in a -log fold (> . %) reduction

in viral load in the host (Figure h). Alcohols did not neutralize SARS-CoV- ,andminimumneutralizationwasobservedforIAV, resulting in less than -log fold ( %) reduction in viral load for chlorobutanolandphenethylalcohol(PEA)(Figure d,i).Overall, this extensive screening identi ed pectin and BKC as the most

e ective agents for rapid neutralization of IAV and SARS-CoV- , respectively. Neutralization ability of pectin and BKC was found to be dose-dependent (Figure e,j). Minimum concentrations of

• % w/v and . % w/v were required for pectin and BKC, re-

spectively, to achieve > -log fold (> . %) reduction in the viral load with min of incubation time.

To elucidate the viral neutralization mechanism of pectin and BKC, we performed in silico modeling to determine their bind- ing a nity with the receptor binding domains (RBD) of IAV and SARS-CoV- , respectively. For IAV, anionic pectin targets RBD at the distal part of hemagglutinin, which is positively charged,thusavertingthevirusentryintothehostcell(Figure l). Compared to the host ligand sialic acid present in mucosal ep- ithelia, pectin showed stronger binding to RBD through dis- tant hydrogen bonding with Se , Ser , and Thr and hy- drophobic linkage with Ser and Glu (Figure S , Support- ing Information). BKC was found to exhibit hydrophobic inter- actions with the ACE binding motif of spike protein of SARS- CoV- (Figure m). BKC also showed hydrophobic interactions with Phe and Phe in membrane helices via pi-pi stacking (Figure m), which can distort the helical conformation of adja- cent helices, as aromatic stacking of Phe and Phe is a pre- requisite to stabilizing helix-helix interface of the envelope trans- membrane protein. BKC ts into the pentameric ion channels

at the N terminus of the transmembrane domain through in- teraction with Thr and potentially blocks the in ux/e ux of ions(Figure n).Todeterminetheroleofelectrostaticinteraction in pectin- and BKC-mediated neutralization of IAV and SARS- CoV- , respectively, we performed a neutralization assay by pre- treatingpectinandBKCwithcounterionstoo setthecharge.As anticipated, anionic pectin in the presence of positively charged polyethyleneimine lost its neutralization activity and failed to show a signi cant reduction in the viral load compared to PBS (Figure f). Likewise, the pretreatment of BKC with negatively charged bovine serum albumin diminished the ability of BKC to reduce the SARS-CoV- titer in the host cells (Figure k).

Next, we investigated whether ionic interactions between an- ionic gellan or pectin with cationic BKC, when present to- gether in a formulation, would impact the neutralization abil- ity of pectin or BKC. Notably, the neutralization e ciency of pectin ( . % w/v) against IAV remained conserved even with a

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dose-dependent increase in BKC up to a concentration of . % w/v (Figure o). Neutralization e ciency of BKC ( . % w/v) againstSARS-CoV- wasnotimpactedbygellanorpectinat . % w/v or . % w/v concentrations, respectively, but reduced at higherconcentrations(Figure p,q).Theseresultsfurtherunder- score that the concentration of each agent is critical for e cient neutralization.

Finally, we also screened surfactants and alcohols to assess their neutralization ability against bacteria, including E. coliand

1. pneumoniae. Neutralization was determined by measuring the bactericidal activity. Each agent was individually incubated with either E. colior K. pneumoniae for or min, followed by - min centrifugation, and then evaluating the bacterial load in the supernatant using a colony-forming assay. BKC was more e ec- tive than non-ionic surfactants, resulting in a -log fold ( . %)

and -log fold ( . %) reduction in colony-forming units (CFU)

of E. coli and K. pneumoniae, respectively, with an incubation time of min (Figure r,t). Previously reported molecular mod- eling studies have shown that BKC incorporates into the bacte- rial membrane, resulting in its destabilization.[ ] This involves BKC s attachment to the bacterial cell surface followed by its quick integration into the lipid bilayer, where it stays for at least several nanoseconds, likely causing membrane destabilization. Alcohols had a negligible bactericidal e ect over the exposure periods of or min (Figure s,u). Altogether, our data on physical barrier property, spray pattern, mucosal retention, and neutralization indicate gellan, pectin, and BKC as the three crit- ical components to formulate PCANS. However, we also incor- porated phenethyl alcohol (PEA), as it is commonly added as a stabilizer to nasal formulations to prevent the growth of gram- negative bacteria,[ ] thereby ensuring a long shelf-life.

• . Utilizing Surfactants to Promote the Capture of Respiratory

Droplets

Pulmonary surfactant layers the alveolar epithelium to en- hance wettability and trap airborne particles.[ ] We adopted this biomimetic approach to capture pathogen-laden respiratory droplets in the nasal cavity. Speci cally, we identi ed surfactants

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to reduce interfacial tension of PCANS and reduce the bounce o /escapeofrespiratorydroplets.Weevaluatedsurfactantslisted in the IID list, including Tween- , Tween- , and BKC. Screen- ing was performed using a twin impinger, which is a glass appa- ratus that can be used to assess the deposition of aerosolized par- ticles in di erent regions of the respiratory tract (Figure a).[ , ] Simulated mucus or a biopolymer mixture of gellan ( . % w/v) and pectin ( . % w/v) without or with di erent concentrations ofsurfactantswassprayedintotheSNF-coatedoropharyngealre- gionoftheimpinger(Figure a).Dropletswithmassmedialaero- dynamic diameter > m and laden with rhodamine B-loaded liposomes (size nm) were generated using a jet nebulizer to mimic pathogen-laden large respiratory droplets. Droplet cap- ture was determined by quantifying the uorescence intensity of rhodamineBinthebiopolymer/surfactantmixtureorthemucus layer. Biopolymer mixture without any surfactant showed simi- lar uorescence intensity as mucus (Figure b). Combining the biopolymer mixture with Tween- or Tween- at a concentra- tion higher than . % w/v or with BKC at a concentration higher than . % w/v resulted in a signi cant increase in the

uorescence intensity as compared to mucus or only biopoly- mer mixture, suggesting increased capture of droplets due to surfactants. Compared to Tween- , BKC and Tween- resulted

in a signi cantly higher fold increase in the uorescence inten- sity when added to the biopolymer mixture at a concentration of . % w/v or higher (Figure b). At . % w/v concentration, bothBKCandTween- containingbiopolymermixturesshowed similar uorescence intensity, which was -fold higher than the

uorescenceintensityofmucusorbiopolymermixturewithouta surfactant. Since . % w/v is the most commonly used concen- trationofBKCincommerciallyavailablenasalformulations,[ , ] and also showed excellent neutralization activity against SARS- CoV- , we decided to use this concentration in PCANS, even though BKC didn t increase the capture of respiratory droplets at this concentration.

To impart respiratory droplet-capturing ability, we decided to proceed with Tween- and determined its safe concentration that would not compromise the permeability or metabolic activ- ity of nasal epithelium. To that end, we performed an in vitro

Figure . Biopolymers, surfactants and alcohols were screened for neutralization of di erent respiratory pathogens. a) Table summarizes di erent components and their concentrations to determine the neutralization ability against respiratory pathogens. Each component was individually evaluated

for its pathogen neutralization potential. IAV and SARS-CoV- viral loads in the host cells after or min incubation of the virus with b, g) di erent biopolymers, c, h) di erent surfactants, and d, i) di erent alcohols. Viable viral titer was quanti ed using plaque assay in MDCK host cells for IAV and focus-forming assay in Vero E cells for SARS-CoV- virus. Results are expressed in plaque-forming units (PFU/mL) or focus-forming units (FFU/mL). ****P < . , ***P < . , **P < . , *P < . compared to minutes of incubation with PBS. n.s, not signi cant. Viral loads in the host cells

after min incubation of e) IAV and j) SARS-CoV- with di erent concentrations of pectin and BKC, respectively. **P < . , *P < . compared to PBS.Viralloadsinthehostcellsafter minincubationoff)IAVandk)SARS-CoV- withpectin( . %w/v) + polyethylenimineandBKC( . %w/v) + bovine serum albumin, respectively. **P< . compared to PBS. n.s, not signi cant. l) Pectin (yellow) binds to the receptor binding site of IAV (purple)

at the distal part of hemagglutinin monomer (colored in purple) through hydrophobic interactions with Ser and Glu , and hydrogen bonding with Ser , Ser , and Thr . Blue and red dots in hydrogen bonding maps represent carbon and oxygen atoms, respectively. m) Chemical interaction of BKC (green) with ACE binding motif (red) in the spike protein of SARS-CoV- . Interaction map reveals the hydrogen bonding of BKC with Tyr and Gly . n) Aromatic pi-pi interaction of BKC (green) with Phe (purple) in the transmembrane domain and with Thr (brown) membrane helices. Interaction analysis shows hydrophobic bonds with Phe and hydrophobic bonds with Phe . o) Viral load in host cells after min incubation of IAV with pectin ( . % w/v) in the presence of di erent concentrations of BKC. ****P < . compared to PBS. Viral load in the host cells after

min incubationof SARS-CoV- with BKC ( . % w/v) in thepresence of di erent concentrations of p) gellanand q) pectin. ****P < . compared

to PBS. E ect of surfactants (r, t) and alcohols (s, u) against gram-negative bacteria E. coliand K. pneumoniae using colony-forming unit (CFU) plate count method after and minutes of exposure. Viable bacterial colonies are expressed in CFU/mL. *P< . compared to PBS. For b—d, P values were determined using two-way ANOVA with Tukey s post hoc analysis. For e, f, j, k, o—q, P values were determined using one-way ANOVA. Data in b-k and o-u are presented as Means – S.D of technical repeats (n = , each experiment performed at least twice). Ser, serine; Thr, threonine; Glu, glutamic acid; Phe, phenylalanine; Tyr, tyrosine; Gly, glycine.

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Figure . PCANS enhances the capture of respiratory droplet-mimicking aerosol and exhibits prolonged nasal residence time in mice. a) Experimental design for measuring the capture of respiratory droplet-mimicking aerosol. A twin impinger was used to simulate the aerodynamics of the human respiratory tract. Mucus or gellan ( . % w/v) and pectin ( . % w/v) solution (G+P), without or with di erent concentrations of Tween- , Tween- orBKCwascoatedontheinnersurfaceofthethroatregionoftheimpingerusinganasalspraydevice.Dropletswithmassmedialaerodynamicdiameter

• m and laden with rhodamine B-loaded liposomes (size nm) were generated using a jet nebulizer and administered into the impinger under vacuum ( L min− ). Droplet capture was determined by quantifying the uorescence intensity of rhodamine B in the biopolymer/surfactant mixture or

mucuslayer.b)Foldincreasein uorescenceintensitywithrespecttomucus.****P< . ,*P< . comparedtomucus.c)Transepithelialelectrical resistance (TEER) across the human nasal epithelial cell (RPMI- )-based monolayer at di erent time points after treatment with only medium or medium containing Triton-X ( . % w/v) or di erent concentrations of Tween- . Surfactant-containing medium was replaced at h with fresh medium toexamineimpedancerecovery.****P< . ,***P< . comparedtountreatedcontrol.n.s,notsigni cant.d)Experimentaldesignformeasuring the capture of respiratory droplet-mimicking aerosol using a D human nasal cavity model (Koken cast). The inner surface of the nasal cavity was coated with mucus, G+P solution or PCANS (the nal formulation) using a nasal spray device. The throat part of the model was coupled to a vacuum pump for simulating the respiratory air ow ( L min− ). Nostrils were then exposed to nebulized rhodamine B-loaded liposomes for minute. Droplet capture was determined by quantifying the uorescence intensity of rhodamine B in the nasal cavity. e) Fold increase in uorescence intensity with respect to mucus. **P < . compared to mucus. n.s, not signi cant. f) Experimental outline for the evaluation of the nasal residence time of PCANS in mice. C BL/ mice were intranasally administered with L of free DiR or DiR-loaded PCANS (PCANS/DiR) into each nostril. Mice were euthanized at di erent time points over h, and nasal cavity was harvested and imaged using an in vivo imaging system (IVIS). g) Representative images of the

nasal cavity excised at di erent time points. h) Quanti cation of uorescence intensity in the nasal cavity at di erent time points. i) Fold change in total ux at h in the nasal cavity relative to G+P. *P< . , compared to G+P. n.s, not signi cant. j) Experimental design to assess the biocompatibility of PCANS in mouse nasal cavity. L PCANS or PBS was administered into each nostril of C BL/ mice once daily for consecutive days. Animals were euthanized on day and nasal cavity was analyzed histologically. k) Representative images of H&E-stained sections of nasal turbinate from mice captured using a X objective. Insets represent healthy olfactory epithelium (i) and (iii), and lamina propria (ii) and (iv) captured at X objective. For

b, e and i, P values were determined by one-way ANOVA using Tukey s post hoc analysis. For b, concentrations for each surfactant were compared individually. For c,Pvalues were determined by two-way ANOVA with Tukey s multiple comparison test. Data in b,c, and e are presented as Means – S.D of biological repeats (n = , each experiment performed at least twice). Data in h and i are presented as Means – SEM (n = mice/group).

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assay evaluating the transepithelial electrical resistance (TEER) across the human nasal epithelial cell (RPMI- )-based mono- layer upon treatment with di erent concentrations of tween- .

A transient dip of less than % in TEER was observed in the monolayer immediately after the addition of Tween- , irrespec- tive of the concentrations evaluated in this study. However, the TEER reversed rapidly to the original value in less than h af- ter replacing tween- -containing medium with fresh medium (Figure c). The drop in the TEER for Tween- was signi - cantly less compared to Triton-X (negative control), which re- sulted in a permanent change in the TEER. Second, we evalu- ated the e ect of di erent concentrations of Tween- on the metabolic activity of RPMI- cells upon or h of incuba-

tion. Cells incubated with . % or . % w/v tween- showed similar metabolic activity as cells incubated in medium. How- ever, Tween- ( . % w/v) resulted in a signi cant reduction in

the metabolic activity of RPMI cells (Figure S , Supporting In- formation). Thus, we decided to use . % w/v as the nal con- centration of Tween- in PCANS. Overall, based on our data for physical barrier property, spray pattern, mucosal retention, neutralization, droplet capture, and nasal epithelial cell toxicity, we decided on gellan, pectin, BKC, PEA, and Tween- as the - nalcomponentsforPCANS,andvalidatedtherespiratorydroplet capturing ability of the nal formulation using a D- model of human nasal cavity (Koken cast) with the anatomical intricacies (Figure d).[ ] Consistent with the twin impinger results, there was no signi cant di erence in the uorescence intensity be- tweengellanandpectinmixture,andmucus(Figure e).PCANS, on the other hand, showed a -fold higher uorescence com- pared to mucus, suggesting the potential of PCANS to increase the capture of pathogen-laden respiratory droplets from inhaled air.

• . Prolonged Nasal Retention of PCANS and Safety Upon

Repeated Administration

Next, we evaluated the retention of PCANS in the nasal cavity of mice (Figure f). PCANS ( L) mixed with a uorescent dye — (DiIC ( ) ( , -Dioctadecyl- , , , - Tetramethylindotricarbocyanine Iodide) (DiR) was administered into both nostrils of C /BL mice. Free DiR was used as a control. Mice were euthanized at di erent time points over h,

and nasal cavity was harvested and imaged using an in vivo imaging system (IVIS) to quantify the uorescence signal from DiR. Free DiR resulted in negligible uorescence signal, even

at min after administration, suggesting its rapid clearance (Figure g,h). Interestingly, mice administered with DiR-loaded PCANS showed signi cant uorescence for up to h, sug- gesting prolonged nasal retention of PCANS (Figure g,h). We hypothesized that prolonged retention of PCANS is attributed to the presence of surfactants, including Tween- and BKC, which have previously been shown to reduce cilia beat frequency in the nasal cavity.[ ] To test our hypothesis, we compared nasal reten- tion of DiR-loaded mixture of gellan and pectin without or with tween- or BKC. The addition of both BKC or tween- signi - cantly enhanced the nasal retention of gellan and pectin mixture

at h post-nasal administration, as evident from the uorescent signal of DiR in the nasal cavity (Figure i). However, tween-

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resulted in signi cantly higher nasal retention than BKC. The ability of tween- to enhance the nasal retention of gellan and pectin mixture was found to be concentration-dependent (Figure S , Supporting Information). However, considering irreversible nasal epithelial permeabilization and cytotoxicity at . % w/v or higher concentration of tween- , we maintained . % w/v in PCANS for further experiments. Notably, nasal administration of DiR-loaded PCANS only showed uorescence signals in the nasal cavity and stomach, suggesting no systemic absorption. PCANS was fully cleared at h (Figure g,h;Figure S , Supporting Information), resulting in negligible uorescence signal in both the nasal cavity and the stomach. To con rm safety of PCANS, we performed a repeat-dose toxicity study in healthy mice intranasally administered with PCANS or PBS once daily for consecutive days (Figure j). Hematoxylin and eosin (H&E) stained sections of nasal cavity from both PBS or PCANS-administered mice did not show any in ammation or other gross evidence of toxicity, as evident by a de ned lamina propria (Figure k). This connotes the safety of PCANS for daily administration.

• . Broad-Spectrum Activity, Spray Characteristics, and Shelf

Stability of PCANS

Having identi ed the nal components of PCANS, along with their optimal concentrations, we sought to demonstrate the physical barrier property and neutralization ability of PCANS against a broad spectrum of respiratory pathogens, including en- veloped viruses (IAV, SARS-CoV- , RSV), a non-enveloped virus (adenovirus), and bacteria (E. coli and K. pneumoniae). Physi- cal barrier property was evaluated by assessing the transport of pathogens through an SNF-coated cell strainer or a cell strainer coated with simulated mucus/SNF mixture or PCANS/SNF mix- ture. PCANS/SNF prevented the transport of all the pathogens

by > -log fold (> . %) (Figure a-f), suggesting its broad- spectrum physical barrier property. For all pathogens, except RSV, mucus/SNF mixture showed signi cantly less prevention ofpathogentransportcomparedtoPCANS/SNF.PCANSalso ef-

ciently neutralized all the tested pathogens within or min

of incubation time, resulting in > -log fold (> . %) reduction

in pathogen load (Figure g—l). Excitingly, PCNAS demonstrated remarkable e cacy in co-neutralizing both bacterial and viral pathogens (Figure S , Supporting Information), achieving > - fold (> . %) reduction in the load of E. coliand SARS-CoV- , used as proof-of-concept pathogens in this experiment. This

nding is clinically signi cant, as respiratory droplets can carry both bacterial and viral pathogens, highlighting PCNAS s poten- tialinprovidingcomprehensiveprotectionagainstrespiratoryin- fections. We also evaluated the spray characteristics of PCANS sprayed through a standard and commercially used VP multi- dose nasal spray pump (Aptar, USA). The droplet distribution data showed that % of PCANS droplets had size > m, and

• had size < m (Figure m), which is desirable to maxi-

mize the deposition in nasal cavity, while minimizing deposition into deep lungs. PCANS resulted in a wide plume angle within the ideal range of — ¡, an ovality close to , covering a circu- lar area of up to %, which is in line with the commercial nasal sprays (Figure m).[ , ]

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Figure . PCANS exhibits broad-spectrum physical barrier property with pathogen neutralization, and is sprayable and shelf-stable. a—d) Amount of di erentvirusesthatpermeatedwithin hthroughasimulatednasal uid(SNF)-coatedcellstraineroracellstrainercoatedwithsimulatedmucus/SNF mixtureorPCANS/SNFmixture.Viruspermeationwasquanti edbyplaqueassayinMDCKcells(IAV),VeroE cells(SARS-CoV- ),andHep- cells(RSV and adenovirus). ****P < . , ***P < . , **P < . , *P < . . n.s, not signi cant. Amount of e) E. coliand f) K. pneumoniaethat permeated within hthroughanSNF-coatedcellstraineroracellstrainercoatedwithsimulatedmucus/SNFmixtureorPCANS/SNFmixture.Bacterialpermeation was quanti ed using a CFU plate count method. ****P < . , ***P < . , **P < . . n.s, not signi cant. g—j) Viral titer for IAV, SARS-CoV- , adenovirus, and RSV after treatment with PBS or PCANS. IAV and SARS-CoV- were incubated with PCANS for min, while adenovirus and RSV were treated for min. Anti-bacterial activity of PCANS against k) E. coliand l) K. pneumoniaeusing CFU plate count method after and min incubation, respectively. ***P < . , **P< . , *P < . . m) Spray characteristics of PCANS. The droplet size distribution of PCANS was analyzed using a laser di raction system. Representative images of single time delay plume angle and ovality ratio, captured using a high-speed digital camera and laser light sheet. n) Experimental design to assess the stability of PCANS in accelerated temperature conditions ( ¡C). PCANS was stored in glass amber bottles. Aliquots were taken at di erent time points to investigate spray characteristics and pathogen neutralization e cacy. o) Plume angle, p) ovality, q) mean droplet diameter and r) spray deposition area over a period of days. ***P < . , **P< . compared to day , n.s, not signi cant. s) Percent reduction in the viral load of IAV and SARS-CoV- in their respective host cells after min incubation with PCANS aliquoted at di erent time points in the stability study. *P < . compared to day . n.s, not signi cant. For a—f and o—s, P values were determined using one-way ANOVA with Tukey s post-hoc analysis. For g—l,P values were determined using a two-tailed t-test. Data are presented as Means – SD of biological repeats (n = , each experiment performed at least twice).

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Shelf-stability is a key attribute governing the translational po- tential of formulations. We tested shelf-stability of PCANS over days at ¡C temperature, as per the International Confer- ence on Harmonization (ICH) guidelines for stability testing un- deracceleratedstorageconditions(Figure n).Overaperiodof days,weobservednosubstantialvariationsinthespraycharacter- istics, including plume angle, ovality, coverage area, and droplet size distribution (Figure o—r; Figure S , Supporting Informa- tion). PCANS also displayed no changes in its neuralization ac- tivity over days, resulting in > . % reduction in In uenza

A and SARS-CoV- viral loads in the host cells upon min of incubation (Figure s). Interestingly, in a separate stability study, we found that PEA s presence in PCANS is critical for minimiz- ing bacterial growth after the formulation was challenged with E.coli(Figure S , Supporting Information). This is consistent with previous reports,[ ] which have shown that PEA prevents bacterial growth in nasal sprays. Collectively, these data con rm the shelf-stability of PCANS.

• . PCANS Exhibits Prophylactic Activity In Vivo

Next, in a proof-of-concept study, we investigated the prophylac- tic e cacy of PCANS against respiratory infection in vivo. PR ,

a mouse-adapted strain of H N In uenza virus, was used to in- duce infection. PR is a highly virulent strain that induces severe respiratory infection in mice,[ ] and can be lethal at a dose of

PFU.[ ] In vitro assay revealed excellent potency of PCANS to neutralize PFUofPR within minofincubation,resulting

in > -log fold (> . %) reduction of the viral load in host cells (Figure S ,SupportingInformation).Todemonstratee cacyin vivo, PCANS or PBS ( L) was administered prophylactically to both the nostrils of healthy mice on day (Figure a). Fifteen minutes later, animals were challenged intranasally with a free-

owingPR solution( L/nostril),exposingmicetoatotaldose of PFU,whichhasbeenpreviouslyusedbyothergroups.[ , ] Intranasalinstillationof Lofa uorescentdye(DiR)solution into each nostril of mice showed a stronger uorescent signal in

the lungs compared to the nasal cavity (Figure S , Supporting Information) at h post-administration. This con rms that the liquid reached the lungs and was not primarily retained in the nasal cavity, suggesting that the viral suspension administered

as a liquid bolus also reached the deep lungs.

Remarkably, all mice in the PCANS-treated group survived for atleast daysaftertheinfection,whereasthePBS-treatedgroup showed % lethality by day (Figure b). Over days, no dis- cernible change was observed in the body weight of the PCANS- treated animals, while signi cant weight loss was observed for PBS-treated ones after days post-infection (Figure c). PCANS also curtailed the lung viral titer to undetectable levels on days

and post-infection, resulting in > -log fold (> . %) re- duction compared to PBS-treated mice (Figure d, e). Com- pared to healthy mice, mice infected with PR and treated with PBS showed signi cant di erences in the levels of in amma-

tory cells, including leukocytes, neutrophils, lymphocytes, and macrophagesinbronchoalveolarlavage(BAL) uid(Figure f—i). ProphylactictreatmentofmicewithPCANSrestoredthelevelsof in ammatory cells in BAL uid to normal. Additionally, cytokine pro le from lung homogenate showed a signi cant reduction of

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IL- and TNF-a levels in PCANS-treated mice, as compared to the PBS-treated group (Figure j—l). No reduction was, however, observed in the levels of IL- . Histological examination of lung sections revealed a substantial reduction in leukocyte in ltrates

in PCANS-treated mice, as compared to the PBS-treated group, which showed an abundant presence of bronchial and alveolar in ltrates (Figure m). Overall, compared to PBS-treated mice, we observed a signi cant reduction in pulmonary in ammation score for PCANS-treated group (Figure S , Supporting Infor- mation). An escalated dose challenge was performed to deter- mine the potency of PCANS to neutralize a higher viral load of PR ( PFU). Compared to the PBS-treated group, prophylac-

tic treatment with PCANS signi cantly improved survival and body weight and reduced lung viral titer on days and post- infection(Figure S ,SupportingInformation).PCANSalsopro- tected mice against the PR challenge performed after and h

of prophylactic treatment, as evident from signi cant reduction observed in lung viral titer on day , as compared to the PBS- treated group (Figure n—p). Speci cally, average reductions of

• and % in lung viral titer were observed for and h

challengegroups,respectively;however,therewasnostatistically signi cant di erence between PCANS-mediated reductions ob- servedinanimalschallengedafter versus h.Thesedataclearly indicate the potential of nasally administered PCANS to protect against respiratory infection in mice for at least h.

• Discussion

We report PCANS — a radically simple and multi-modal pre- exposure prophylactic nasal spray to o er protection against res- piratorypathogens.Unlikevaccines,whicharepathogen-speci c and exhibit reduced e cacy as the pathogen mutates,[ ] PCANS has the potential to o er broad spectrum protection against a wide range of pathogens. In a proof-of-concept study performed in mice, a single intranasal dose of PCANS was e ective against supra-lethal dosages of a highly virulent mouse-adapted strain of H N In uenza virus (PR ), and e ciently reduced the lung

viral titer providing protection for at least hours. This under- scores the potential utility of PCANS as an additional layer of protection in conjunction with vaccines to minimize pathogen load,consideringthatvaccinesaloneoftenachieveonlypartialre- duction. For example, in a clinical study, participants vaccinated with BNT b and mRNA- had only percent less de- tectable virus compared to those who were unvaccinated when infected.[ ]

To our knowledge, PCANS is the rst multi-modal chemo- prophylactic approach, equipped with three critical attributes that involve ) capturing pathogen-laden respiratory droplets in thenasalcavity, )preventingpathogentransportvianasallining and ) neutralizing a broad spectrum of pathogens. This is the

rst report of a chemoprophylactic nasal spray that exhibits neu- tralizationactivityagainstabroadspectrumofpathogens,includ- ing both bacteria and viruses (enveloped and non-enveloped), and the rst that has shown > log-fold reduction in pathogen load across multiple bacteria and viruses. Previously devel- oped nasal sprays have demonstrated limited e ectiveness with only a — . -log fold reduction in pathogen load. For instance, compared to xylitol-based formulations pHOXWELL and Xlear, which reduced viral titer of SARS-CoV- by and . log fold,

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Figure . Pre-exposure prophylactic treatment with intranasal PCANS reduces respiratory infection in mice. a) Experimental outline for the prophylac-

tic e cacy study. C B/ mice received a single dose ( L) of PCANS or PBS before minutes of intranasal inoculation with PFU In uenza A/PR/ / . One cohort of animals was followed for body weight changes and survival for a period of days. Animals from a second cohort were eutha- nized on day or after infection to enumerate lung viral titer, in ammatory cell count in bronchoalveolar lavage (BAL) uid, and in ammatory cytokine

levels in lung homogenate. Hematoxylin and eosin (H&E) stained lung tissue sections from animals euthanized were assessed for in ammation. b) Survival and c) body weight change of mice over a period of days post-infection. P = . compared to the PBS-treated group for Kaplan-Meier survival curve. *P< . compared to PBS-treated group for body weight change curves. d) Viral titer from lung homogenate of mice and e) percentage reduction in viral load in the lungs on day and post-infection, as quanti ed by plaque assay performed in MDCK cells. **P= . . f—i) In ammatory

cell count in BAL on day and after infection. ****P < . , **P < . . Levels of j) IL- , k) TNF- and l) IL- in lung tissues. ****P < . , **P = . . n.s, non-signi cant, n.d, not detected. m) Representative images of H&E-stained lung tissue sections of virus-challenged mice that were prophylactically treated with PBS or PCANS. Histology images were captured using X and X objectives. Scale bar: m. High-magni ed insets depict the di erence in the extent of in ammatory in ltrates. Scale bar: m. n) Experimental outline to evaluate time-dependent nasal protection by PCANS. Mice received a single dose of PCANS at or h prior to intranasal inoculation with PFU in uenza A/PR/ / . Animals were euthanized

on day post-infection to enumerate lung viral titer o) Viral titer quanti ed from lung homogenate and p) percentage reduction in viral load in the lungs

on day post-infection for animals challenged after or h after prophylactic treatment. **P < . , *P < . , n.s., not signi cant. For b, P values

were determined using the Gehan-Breslow-Wilcoxon test. For c, P values were determined using one-way ANOVA with Brown-Forsythe. For d and f—l, P values were determined using two-way ANOVA with Tukey s post hoc analysis. For o, P values were determined using one-way ANOVA with Tukey s post hoc analysis. n = mice/group for b. Data in c are presented as Means – SEM (n = mice/group). Data in d—l are presented as Means – SEM (n

• mice/group). Data in o, p are presented as Means – SEM (n = mice/group).

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respectively,[ , ] PCANS demonstrated a remarkable log fold reduction. In our study, iota + kappa carrageenan, the key com- ponent of a commercially available nasal spray- AGOVIRAX¤, resulted in only -log fold reduction in the viral loads of both IAV and SARS-CoV- within min, while PCANS showed > log fold reduction for both IAV and SARS-CoV- within min. The superior prophylactic e ect of PCANS was also demon- strated in vivo. Compared to a previous study,[ ] where multi- ple dosages of iota + kappa carrageenan showed % survival of mice in a PR model of infection, a single dose of our for- mulation showed % survival of mice. To our knowledge, this

is the rst chemoprophylactic strategy to show % protection within a pre-clinical model of respiratory virus infections. Addi- tionally, we also demonstrated > . % reduction in lung viral titer. On the other hand, iota + kappa carrageenan only showed

• reduction.PCANS also exhibits an unprecedented resi-

dence time of h in the mouse nasal cavity and provides protec- tionforatleast h.Toourknowledge,thisisthelongestnasalres- idence time that has been reported for nasal sprays in mice. The interaction of PCANS with mucosal tissue and its prolonged retention time involve three key mechanisms: ( ) mucoadhe- sive forces between the biopolymers (gellan and pectin) and the mucin proteins in the mucosal layer,[ , ] ( ) gelation of PCANS, facilitated by cross-linking of gellan in the presence of physio- logical electrolytes in nasal uid, which intercalates with mu- copolysaccharides to form a hydrogel mesh, preventing nasal dripping,and( )thepresenceoftween- ,whichhasbeenprevi- ously shown to slow mucociliary clearance by reducing cilia beat frequency,[ ] thereby enhancing the nasal retention of PCANS. Such a long nasal residence time would potentially minimize dosage frequency in future clinical studies, o ering an advan- tage over previously developed chemoprophylactic approaches, including SaNOtize, which require — doses per day.[ , ] Fi- nally,toourknowledge,thisisthe rstnasalformulationthatcan

e ectively trap large respiratory droplets from the air we breathe in, which would be essential for maximizing its preventive e ec- tiveness in future clinical studies.

The drug-free nature of PCANS is favorable for the regula- tory process, which could be tedious for chemoprophylactic ap- proaches based on investigational new drugs such as IgM- .[ ] Also, since all the components used in PCANS are commercially available o -the-shelf and require simple mixing without chem- ical modi cations, our approach is amenable to scale-up and large-scale manufacturing. PCANS is also safe for daily admin- istration, as demonstrated in mice, which is a signi cant advan- tage over previously developed povidone iodine-based anti-viral nasal sprays,[ , ] which are associated with iodine burns, thy- roid toxicity, and disruption of the mucosal barrier, constrain- ing repeated administration. Similarly, frequent use of a nitric oxide (NO)-inducing nasal spray (SaNOtize), which has shown potential in post-exposure prophylaxis of SARS-CoV- infection, canresultinelevatedTh cytokines,whichmediateautoimmune disorders.[ ] In addition, excessive NO can cause tissue dam- age and cell death.[ ] The incidence of such adverse e ects with PCANS is likely to be low, as the formulation is devoid of im- munomodulatory molecules such as NO and steroids.

Our study has several strengths. First, the components consti- tuting PCANS were identi ed via rigorous in vitro and in vivo screenings of excipients from the IID and GRAS list of the FDA,

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and their di erent concentrations and combinations. These ex- tensive screening experiments were aimed to optimize the key parameters, including sprayability, mucoadhesiveness, capture of respiratory droplets, physical barrier property, broad spectrum pathogen neutralization activity, and nasal residence time. Our comprehensive investigations allowed us to construct an un- precedented, multi-modal chemoprophylactic strategy. Second, to evaluate the respiratory droplet capturing ability of PCANS, we used a D- model of human nasal cavity (Koken cast), which has been previously used for in vitro evaluation of nasal drug delivery, as it replicates all the anatomical intricacies of human nasal cavity.[ ] We measured capture e ciency by comparing the fold increase in rhodamine B uorescence intensity in the PCANS-coated nasal cavity compared to the one coated with mu- cus. This provided a robust and reproducible measurement. We also attempted to measure droplet capture e ciency by counting the droplets that passed through the model. To achieve this, we connected the nasal cavity cast to an aerosol monitoring system. However, the readings were inconsistent due to technical issues likeambientdustin owthroughgapsinthenebulizer-nasalcav- ity connector and non-speci c droplet deposition in the connec- tor, leading to high variability. Third, physical barrier property of di erent biopolymers was evaluated by two complementary techniques — quanti cation of viral and bacterial transport using plaque-forming and colony forming assays as well as quanti ca- tion of the transport of small molecule dye using uorescence spectroscopy. Fourth, we demonstrated broad spectrum physical barrier property and neutralization ability of PCANS in ve dif- ferent pathogens — three enveloped viruses (IAV, SARS-CoV- , RSV),onenon-envelopedvirus(adenovirus),andtwobacteria(E. coliand K. pneumoniae). Lastly, to demonstrate prophylactic e - cacy of PCANS in vivo, we used a highly virulent mouse-adapted strain of H N In uenza virus (PR ) that induces severe respi- ratory infections in mice.[ ] Prophylactic e cacy of PCANS was demonstrated against three di erent dosages of the virus, which were — times higher than the previously established lethal dose for PR in mice.[ ] Due to its prolonged nasal residence time, PCANS was e ective for at least h after nasal administra- tion.

Ourstudyalsohascertainlimitations,andthereareadditional questions that need to be answered. First, for the in vivo e cacy study,weinstilledtheviralsuspensionintranasallyasaliquidbo- lus. Although aerosol administration of virus more closely reca- pitulatesreal-lifeexposureinhumans,intranasalinoculationasa liquid bolus is a widely accepted standard in mouse models.[ , ] Studies have shown that both methods result in comparable lev- els of morbidity, mortality, and viral titers in lung and nasal tis- sues for di erent in uenza viruses as well as SARS-CoV- .[ , ] Thus, our choice of intranasal inoculation is scienti cally justi-

ed. Excitingly, with intranasal inoculation where the virus s contact time with PCANS is limited to just a few seconds we observed a greater than . % reduction in lung viral titers and improved animal survival for PFU dosage of PR virus. This underscores the signi cance of our ndings. However, the

PFU group showed higher viral titer on day and an early mortality compared to PFU. We believe that reduced e - cacy at PFU might be partly, if not entirely, attributed to the limited contact time of virus particles with PCANS. Inhala- tion administration of the virus would deposit pathogen-laden

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respiratory droplets on the PCANS layer, providing signi cant contact time between the pathogen and PCANS, potentially en- hancing its prophylactic e cacy. Similar to the virus inoculation, we also instilled PCANS as a liquid bolus intranasally, while in humans PCANS would be sprayed using a metered dose spray pump,whichistechnically di culttorecapitulateinmicedueto signi cant di erence in their respiratory tract anatomy as com- pared to humans. To overcome these limitations, future studies should evaluate the e cacy of PCANS in large animal models, for example non-human primates (NHPs) that allow the admin- istrationofPCANSasanasalsprayandcanbesubsequentlychal- lenged with virus-laden respiratory droplets as an aerosol. Sec- ond, compared to > . % reduction in lung viral titer achieved by PCANS for PR challenge after min, a % reduction was observed when the challenge was performed after h. This can be largely explained by clearance of PCANS in the nasal cav- ity, resulting in signi cantly less formulation available to cap- ture/neutralize the virus at h as compared to min. The res- idence time study with DiR-loaded PCANS showed % clear- ance of PCANS in the rst h. This was also the reason for not evaluating the prophylactic e cacy of PCANS beyond h in this study. Since mucociliary clearance in NHPs and humans is slower than mice,[ ] future e cacy studies in large animal mod-

els are warranted to accurately determine the local pharmacoki- netics of PCANS and duration of protection following a single administration. Notably, the % reduction in lung viral titer, as observed in our study after h of prophylactic treatment is still an improvement over % reduction observed with an inhalable formulation — spherical hydrogel inhalation for enhanced lung defense(SHIELD),developedrecentlyasaprophylacticapproach againstSARS-CoV- .[ ]Finally,althoughwedemonstratedbroad spectrum activity of PCANS against multiple pathogens in vitro, our proof-of-concept in vivo e cacy study only focused on PR . Future studies should also evaluate the prophylactic e cacy of PCANS against other pathogens to con rm the broad-spectrum activity in vivo, and this should be ideally performed in NHPs to mimic the virus challenge and PCANS administration in humans.

• Conclusion

PCANS presents a promising chemoprophylactic approach against respiratory infections. Besides its potential to act as a rst line of defense against respiratory pathogens and emerging variants for which there are no vaccines available, our approach couldalsobepotentiallyusedasanaddedlayerofprotectionwith existing vaccines. Given its broad-spectrum prophylactic activity and shelf stability, we anticipate PCANS holds the potential for global distribution, especially in countries with low vaccination rates against respiratory pathogens. Alongside, the bene ts of PCANS can also be extended to immunocompromised patients, high-risk individuals with co-morbidities, and vaccine-hesitant populations. Its pocket-sized spray format allows for easy porta- bility, making it convenient to carry during social gatherings and travel. With these signi cant bene ts, we believe PCANS will ex- perience rapid widespread adoption, enhancing the accessibility ofrespiratoryinfectionprevention.Byenablingpeopletobreathe cleanandminimizingthetransmissionofrespiratoryinfections,

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PCANS could potentially play a pivotal role in safeguarding pub- lic health worldwide.

• Experimental Section

PreparationofBiopolymerSolutionsandPCANS: Biopolymersolutions were prepared by the addition of the biopolymer ( . to % w/v) to ul- trapure deionized sterile water (Invitrogen). The solution was then mixed

to attain a homogenous mixture with slight heating at ¡C. Biopoly- mersincludinggellan(Gelzan),pectin,carboxymethylcellulose(CMC),hy- droxypropylmethylcellulose(HPMC),carrageenan,xanthangum,andCar- bopol were purchased from Sigma Aldrich. To prepare PCANS, . % w/v gellanand . % w/v pectin solutionswere mixed in aratioof : , followed

by the addition of tween- (Sigma Aldrich) to obtain a nal concentra-

tion of . % w/v. The solution was then supplemented with benzalko- nium chloride (BKC) (Sigma Aldrich) and subjected to immediate mixing bypipettingseveraltimestoyield . %w/vinthesolution.Finally, . %

w/v phenethyl alcohol (Sigma Aldrich) was added, and the pH of the so- lution was adjusted to . . For cell culture experiments and in vivo e - cacystudy,theindividualcomponentsofPCANSweresterile lteredusing

• m PVDF syringe lters (EMD Millipore) and combined as described

above.

PreparationofSimulatedNasalFluid(SNF)andSimulatedMucus: SNF was prepared by dissolving . g sodium chloride ( mM), mg potassiumchloride( . mM),and . mgcalciumchloride( . mM)in mLultrapuredeionizedsterilewaterand lteredusing . m lter.[ ] The healthy simulated mucus was formulated by dissolving . mg mucin fromporcinestomachTypeII(SigmaAldrich), . mgmucinfromporcine stomach Type III (Sigma Aldrich), . mg bovine serum albumin (Sigma Aldrich) in mL ultrapure deionized water containing mM HEPES

bu er and mM sodium chloride solution.[ ] The mixture was stirred vigorously under slight heating to attain a homogenous solution.

Rheological Measurements: Dynamic viscosity behavior of biopolymer solutions was evaluated using a rotational rheometer (Discovery HR- ,

TA Instruments) using a mm diameter cone with a geometry angle of

• Samples were subjected to a linear shear rate ramp up to s− at

¡ C to mimic the strain encountered by the formulation when actuated through the nozzle of the spray device. The viscosity of the biopolymer solution was measured during the upward ramp in triplicates. The sol-gel transition of biopolymer solutions with and without the presence of SNF was evaluated by rotational rheology. The mechanical strength in terms of storagemoduluswasassessedbyapplyingamplitudesweepwithavarying oscillatory strain at Hz at ¡ C.

ExVivoMucosalRetentionStudy: Tissueharvestedfromsheepwascut open to expose the mucosal surface and trimmed down to × mm. Mucosal tissue was then mounted on a glass slide facing upwards and positioned at to align it with the spray actuation angle. The tissue was initially moistened with SNF using a generic nasal spray device, and ex- cessive uid was removed with sterile wipes. Brilliant green dye (Sigma Aldrich) loaded polymeric solution was sprayed, keeping the spray nozzle tip at a distance of cm from the slide surface. The slides were examined

for runo /drip after h of spraying. The distance traveled by the poly- mer solution down the glass slide from the bottom end of formulation deposited on mucosal tissue was measured as drip length. Drip length of

free dye was considered %.

CellCulture: Madin-Darbycaninekidneycells(ATCC¤)werecultured in T- asks (CELLTREAT) at ¡C and % CO in DMEM (Gibco) sup- plemented with % fetal bovine serum (FBS) (Gibco) and % penicillin- (streptomycin (Invitrogen). Hep cells and Vero E cells (ATCC¤) were cultured in T- asks at ¡C and % CO in EMEM supplemented with

• FBS and % penicillin-streptomycin. Human nasal epithelial cells

(ATCC¤) were cultured in T- asks at ¡C and % CO in EMEM supplemented with % FBS and % penicillin-streptomycin.

Production of NanoLuc Luciferase Expressing Recombinant SARS-CoV- : All replication-competent SARS-CoV- experiments were performed in a BSL- facility at the Boston University National Emerging Infectious Dis- eases Laboratories. A recombinant SARS-CoV- virus expressing a Neon-

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Green uorescent protein (rSARS-CoV- mNG) was generously provided bytheLaboratoryofPei-YongShei.[ ]Topropagatethevirus, × Vero

E cellswereseededinaT- askonedaypriortopropagation.Thenext

day, L of rSARS-CoV- mNG virus stock was diluted in mL of Opti- MEM,addedtocells,andthenincubatedfor hat ¡C.Afterincubation, mLofDMEMcontaining %FBSand %penicillin/streptomycinwas added to cells. The next morning, media was removed, cells were washed with X PBS and mL of fresh DMEM containing % FBS was added. Virus was incubated for an additional h. The supernatant was collected

at h, ltered through a . m lter, and stored at − ¡C. The vi- ral stock was thawed and concentrated by ultracentrifugation (Beckman CoulterOptimaL- k;SW Ti rotor)ona %sucrosecushion(Sigma- Aldrich) at x g for h at ¡C. Media and sucrose were then dis- carded, pellets were dried for min at room temperature, and viral pellets wereresuspendedin Lofcold XPBSat ¡Covernight.Thenextday, concentrated virus was combined, aliquoted and stored at − ¡C.

In Vitro Physical Barrier Assay: A m pore size mesh cell strainer was coated with L of mucus, or a biopolymer solution, or PCANS. The formulationwasspreadevenlyusingasterilestainless-steelspatulawitha taperedend.Tofacilitateinsitugelation, LofSNFwasadded,covering the entire surface of the strainer. The strainer was placed in a -well plate containing . mL of serum-free DMEM (for virus/bacteria penetration) orultrapuredeionizedwater(forrhodamineBisothiocyanatepenetration)

in each well, and . mL of diluted virus ( × PFU mL− )/bacteria

( × CFU mL− ) stock or rhodamine B isothiocyanate ( mg mL− ) was added to the upper compartment of the strainer. After h of incu- bation at ¡C, medium or deionized water from the bottom reservoir was retrieved, andthe viral titer permeated through thehydrogel layer was quanti ed using plaque assay for IAV performed in MDCK cells, crystal violet staining for RSV performed in Hep- cells, immunostaining for ade- novirusperformedinVeroE cells,focusformingassayforSARS-CoV- in VeroE cells,andcolonyformingunit(CFU)platecountmethodforbacte- ria, as described in the following sections. The permeation of dye through biopolymersolution/mucuswasquanti edbymeasuringthe uorescence intensity using a microplate reader.

In Vitro Neutralization Assay with In uenza A: Neutralization activity of di erent excipients and PCANS was evaluated by plaque assay. MDCK cells were seeded at a density of — million cells per well in a -well plate and then incubated at ¡C to achieve — % con uency one day before infection. On the day of infection, L of HKx In uenza A virus (H N , × — × PFU mL− ) (BEI Resources) in infection media (serum-free DMEM containing mg mL− TPCK-trypsin) was pre- treated with L of PCANS, biopolymer solution, surfactant solution, alcohol solution or PBS. Samples were vortexed for seconds and in- cubated at ¡C for or min. After incubation, samples were cen- trifuged for min at RPM, and the supernatant was subjected to a

• foldserialdilutionuntileighthdilutionusinginfectionmedium.MDCK cells were then exposed to pre-treated virus dilutions for h. After infec- tion, an overlay growth medium containing X DMEM with % agarose

( : ) was poured onto the top of the cell monolayer and incubated for

h. The overlay was removed, and cells were then xed using mL

of % formalin and left for h at room temperature, followed by the addition of % crystal violet for — min. Wells were washed with wa- ter and left to dry out and PFUs were counted to determine the viral titer.

In Vitro Neutralization Assay with SARS-CoV- : The day prior to infec- tion experiment, × Vero E cells/well were plated in a -well plate. To perform neutralization assay, L of PCANS, biopolymer solution, surfactant solution, alcohol solution or PBS was mixed with × PFU of SARS-CoV- mNG in L of infection media (OptiMEM (Gibco) con- taining mg mL− TPCK-trypsin), vortexed and centrifuged brie y prior to incubation at ¡C for or min. After incubation, samples were centrifuged for min at RPM and serially diluted -fold until eighth dilution with infection medium. Of each dilution, L was then plated into a -well plate and incubated for h at ¡C prior addition of L of . % Avicel (Dupont). Following a h incubation period at ¡C, Avi- cel was removed, cells were washed with X PBS and xed for h with

%neutral bu ered formalin.Focalforming units(FFU) per mL werede-

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termined by counting NeonGreen expressing foci using an Evos M

uorescent microscope (Thermo Scienti c).

In Vitro Neutralization Assay with Adenovirus and Respiratory Syncytial Virus: The broad-spectrum neutralization potency of PCANS was evalu- ated against adenovirus type (ADV- , ATCC, VR- ) and respiratory syncytial virus strain A (RSV-A , ATCC, VR- ) using plaque assay. Brie y,thedaypriortotheinfection, × VeroE cells/wellor . × Hep- cells/ well were plated in a -well plate for ADV- and RSV-A , re- spectively. On the day of infection, L of PCANS was mixed with L

of virus ( × PFU/mL of ADV- and × PFU/mL of RSV-A ) in the infection media and incubated at ¡C for min. The pre-treated mix- ture was -fold serially diluted in infection media after the incubation. Cells were washed with serum-free media before infection and L of each dilution was transferred to the cells for a h incubation prior to the addition of a mL overlay medium containing methylcellulose. Following

a h incubation, the overlay layer was removed, and cells were xed us-

ing % formalin with subsequent immunostaining for Vero E cells and crystalvioletstainingforHep- cells.Plaqueswerecountedusingaplaque reader (Bioreader- -Va).

In Vitro Neutralization Assay with Bacteria: The neutralization potency

of components and PCANS was studied against gram-negative bacteria including E. coliand K. pneumoniae. An overnight culture of bacteria was preparedin mLtrypticsoybroth(TSB,SigmaAldrich)media.Ontheday oftheexperiment,bacteriasuspensionwasadjustedtoobtainanOD nm

• . , which corresponds to CFU/mL. A L of bacterial suspension

in TSB media was incubated with L of PCANS, biopolymer solution, surfactantsolutionoralcoholsolutionat ¡Cfor or min.Afterincu- bation, the sample/bacteria mixture was -fold serially diluted in X PBS,

and L of each dilution was plated onto pre-poured LB (Luria Broth, HiMedia Laboratories Pvt Ltd) agar plates followed by an incubation of

— h at ¡C, % CO . The plates were then counted for CFUs.

In Vitro Assay for Co-Neutralization of Bacteria and Virus: An overnight culture of E. coliwas prepared in tryptic soy broth (TSB, Sigma Aldrich) media, and the bacterial suspension was adjusted to obtain a titer cor- responding to CFU/mL. A L bacterial suspension was added to

mL of PCANS at ¡C for mins. Following the incubation, a L PCANS/bacteriamixtureunderwenta -foldserialdilutionin xPBS,and

a CFU assay was performed. The remaining PCNAS/bacteria mixture was syringe ltered using a . m lter to remove the E. coli, and neutraliza- tion of SARS-CoV- was performed as described above.

Molecular Docking Simulation for Rhodamine B Isothiocyanate: Molec- ular docking simulations were conducted using Biovia Discovery Stu- dio Client and PyRx (version . ) software to investigate the bind-

ing a nities and interaction types between rhodamine B isothiocyanate

and various biopolymers. Initially, the D structures of rhodamine B isothiocyanate (CID: ), gellan gum (SID: ), pectin (CID: ), HPMC (CID: ), CMC (CID: ), xanthan gum (CID: ), Carbopol (CID: ), and -carrageenan (SID:

) was obtained in.sdf format from the PubChem database. These structures were imported into PyRx, wherethe OpenBabel toolkit was employed for energy minimization and geometric con rmation. The molecules were then converted to.pdbqt format and categorized either

as macromolecules (rhodamine B isothiocyanate) or as ligands (biopoly- mers). Docking analyses were carried out using AutoDock Vina within PyRx, employing a grid box method with dimensions of × × . Af-

ter the docking process, the binding free energies (expressed in kcal/mol)

of the rhodamine B isothiocyanate-biopolymercomplexes were evaluated. These docked complex les were subsequently imported into Biovia Dis- covery Studio to analyze various interaction types, including hydrogen bonds, carbon-hydrogen bonds, and hydrophobic interactions between

the dye molecule and biopolymers.

In Silico Modeling of Viral Protein Interactions Neutralizing Agents: In- silico binding analysis was conducted using AutoDock Vina (https:// vina.scripps.edu/). The receptor binding domains of in uenza hemag- glutinin (PDBID: WE ), SARS-CoV- spike receptor-binding domain (PDBID: M J) and Envelope Protein Transmembrane Domain (PDBID:

K G) were sourced from the Protein Data Bank (PDB) available at RCSB (https://www.rcsb.org/). To prepare for docking simulations, molecular

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les of the ligands were rst generated using Marvin Sketch and then con- verted into PDB format using PyMOL for D structural visualization. The protein and ligand les were converted into AutoDock-readable formats (.pdbqt)tofacilitatedockingsimulations.Thesimulationswereperformed with the receptor binding domains of the proteins to predict the most fa- vorable binding conformations. AutoDock Vina and PyMOL were used to visualize and analyze these conformations. The binding energies associ- atedwiththepredictedconformationswerereported.Furthermore,togain insightsintothepotentialinteractionsbetweenproteinandligand,LigPlot (https://www.ebi.ac.uk/thornton-srv/software/LigPlus/)softwarewasuti- lized. This tool provided a detailed visual representation of the molecular interactions, highlighting the key residues involved in binding.

TEER Assay and In Vitro Cytotoxicity of tween- : RPMI cells were seeded on the apical part of Transwell inserts ( . mm polyester mem- brane . m pore size, Corning) at a density of . × cells/cm in . mL EMEM. The basolateral compartment of the insert was lled with . mL EMEM media supplemented with % FBS followed by in- cubation at ¡C. On day , the medium was removed from the top of the inserts, and media volume in the bottom well was reduced to

1. Every days the medium was changed, and TEER was measured.

An epithelial volt ohmmeter (World Precision Instrument) was used to measure the impedance. Until the monolayer formed with a constant impedance around , cells were grown with an air-liquid interface. On day , TEER was measured prior to the treatment of cells with surfac- tants. L of medium containing Triton X- ( . % w/v) or tween-

at di erent concentrations was added to the insert. Plate was incu- bated at ¡C for h. After incubation, wells were replenished with fresh medium, and TEER was measured after , , , and h. The cytotoxic

e ect of tween- at di erent concentrations was also studied on RPMI

cells. Brie y, cells/well were seeded in a -well plate and in- cubatedat ¡Covernighttoachieve — %con uency.Tween- ( . ,

• , and . % w/v) solution in . mL EMEM media was added to the

wells, followed by an incubation for and h. The metabolic activity of RPMI cellswasmeasuredusinganXTT( , -bis( -methoxy- -nitro- - sulfophenyl)− H-tetrazolium- -carboxanilide) assay kit (ATCC¤) accord- ing to the manufacturer s protocol.

Capture of Respiratory Droplets: The inner surface of a glass twin im- pinger s (Copley Scienti c) oropharyngeal region (denoted by red arrows in Figure a) was coated with SNF followed by spraying the gellan ( . % w/v) and pectin ( . % w/v) mixture without or with di erent concentra- tionsoftween- ,tween- orBKCusingaVP nasalspraypump(Aptar). Droplets with mass median aerodynamic diameter > m and laden with rhodamine B-loaded liposomes (size nm) were generated using a

jet nebulizer. Nebulized droplets were administered into the impinger un- der vacuum at a ow rate of L min− for min. The gel was retrieved,

and uorescence intensity was quanti ed at an excitation and emission wavelength of and nm. Rhodamine B-loaded liposomes were syn- thesized using the thin- lm hydration method.[ , ] Brie y, the lipids, DSPE-PEG ( ) amine (Avanti Polar lipids), cholesterol (Sigma) and L- -phosphatidylcholine, hydrogenated (Soy) (HPC, Avanti Polar lipids) were dissolved in chloroform to prepare a mg mL− lipid stock solu- tion in : : molar ratio. A mL of lipid stock solution was added to a round-bottom askcontaining . mLofrhodamineBisothiocyanatefrom

a mg mL− stock. The organic solvent was then evaporated using a ro- tary evaporator for min to form a thin lipid layer. The lipid lm was then hydrated using mL ultrapure water (Invitrogen) and silica glass beads were added to the ask to suspend the lipid in the solution with vigorous shaking using the rotary evaporator at ¡C for min. The hydrated lipid suspension was sonicated (Probe sonicator) at % amplitude for min with a sec pulse on and o condition. The size of liposomes was then analyzed using a Zeta Analyzer (Malvern).

To emulate the capture of pathogen-laden droplets in the human nasal cavity, a D transparent, silicone human nose model (Koken Co, Ltd) was used. The anterior region of the Koken model was deposited with SNF followed by the gellan ( . % w/v) and pectin ( . % w/v) mixture or PCANS with a single actuation using a nasal spray pump (Aptar). Koken model was connected to a vacuum pump at an air ow rate of L min−

and rhodamine B-loaded liposomes were then nebulized for min. The

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model was disassembled to retrieve the formulation and captured dye- loaded droplets after nebulization. The capture of droplets was measured by quantifying the uorescence intensity at an excitation and emission wavelength of and nm.

Spray Characterization: Multi-dose nasal spray vials were lled with water or gellan solution or PCANS. The pump ( L) with an insertion depth of . cm (Aptar) was used to study the spray characteristics in- cludingplumegeometry,sprayplume,anddropletsizedistribution.Three replicate measurements were performed for each sample. Plume geome- try and spray pattern were measured using a Spray-View measurement system (Proveris Scienti c, Hudson, MA) at a distance of mm from the nozzle ori ce of the actuator. This acquisition system employs a high- speed digital camera and laser light sheet to capture images. Data were analyzedusinganimageprocessingsoftware,Viota.Actuationparameters includingvelocity,accelerationandholdtime,andsettingsforcameraand laserwerekeptidenticalacrossallthesamples.Plumegeometrymeasures the angle of plume ejected from the nozzle ori ce. Ovality and plume area were evaluated to quantify the spray pattern of the samples. Ovality was de ned as the ratio of maximum to minimum cross-sectional diameter

of the spray plume. A uniform circular plume with an ovality close to can be considered an optimal condition for nasal sprays.[ ] Droplet size

analysis of samples was inspected using a Malvern Spraytec laser di rac- tion system. The FDA recommends reporting the measurements of size distributiondataatD(v, . ),D(v, . ),andD(v, . )thresholdswhichcorre- spond to the size of %, %, and % droplets by volume distribution, respectively.[ ] It was suggested to have droplet population with D(v, . )

• m, D(v, . ) between — m and D(v, . ) < m. Droplet pop- ulations smaller than m have a propensity to induce a non-targeted deposition at the lungs, and droplets greater than m tend to drip/ run o the nasal cavity.[ ]

Shelf-Stability Study: PCANS ( mL) was lled in a sterile multi-dose nasal sprays (Aptar) capped with the actuator. The nasal spray vials were stored at an accelerated temperature condition ( ¡ C). Aliquots were re- trieved at di erent time points and evaluated for neutralization activity against IAV and SARS-CoV- using plaque forming and focus forming as- says, respectively, as described above. Aliquots were collected from three di erent vials. Similarly, mL aliquots were used to evaluate the spray features, including spray pattern, plume geometry, and droplet size distri- bution.

Mice: Animalexperimentswereconductedaccordingtoethicalguide- lines approved by the Institutional Animal Care and Use Committee (IACUC)ofBrighamandWomen sHospital.Experimentswereconducted in — weeks-old C BL/ mice (Jackson Laboratories, USA). Mice were maintainedunderpathogen-freeconditionsandrandomlyassignedtovar- ious experiment groups, irrespective of gender. The group size of animals in experiments was decided based on the minimum number of animals required to attain a statistical signi cance of P< . among di erent test groups. For mouse model of in uenza infection, experiments were con- ducted in Biosafety Level according to ethical guidelines approved by the Institutional Animal Care and Use Committee (IACUC-A - ) of Brigham and Women s Hospital.

In Vivo Biodistribution and Nasal Retention: Nasal retention of the for- mulationwasperformedinmice.Brie y,C BL/ micewereadministered with L per nostril of free DiR (Thermo sher) or PCANS mixed with DiR at a nal concentration of g mL− ). Mice were euthanized at dif- ferent time points and nasal cavity was harvested and imaged using IVIS (Bruker s In-Vivo Xtreme optical and x-ray in vivo imaging system) at an excitation and emission wavelength of / nm. Vital organs such as lung, liver, spleen, kidney, and heart, were also imaged at and h time points.Todeterminethemechanismoflongresidencetime,animalswere intranasallyinstilledwithDiR-mixedgellan( . %w/v)andpectin( . % w/v) mixture without or with BKC and tween- . After h, animals were euthanized to harvest and image the nasal cavity using Perkin Elmer IVIS Lumina II and the total ux was expressed in (p/sec/m /sr).

In Vivo Prophylactic Activity of PCANS: Mice were intranasally instilled with L PCANS or PBS into each nostril under brief anesthesia using iso urane. After min, animals were challenged with or PFU

of PR intranasally. One cohort of animals was followed for body weight

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changes and survival for a period of days. Animals were euthanized when the body weight was reduced to %. Animals from a second cohort were euthanized either on day or after infection to enumerate lung viral titer, in ammatory cell count in bronchoalveolar lavage (BAL) uid, and in ammatory cytokine levels in lung homogenate. BAL uid was iso- lated by gently instilling saline solution into bronchioles with a catheter inserted through the trachea. The total cells and immune cell types from the collected BAL uid were quanti ed using Di -quik kit as per man- ufacturer s protocol. For lung viral titer and cytokine pro ling, left lung was homogenized and centrifuged at g for min at ¡C to collect the supernatant. The obtained supernatant was further used for down- stream assays. Viral titer was enumerated using plaque assay with MDCK cells,asdetailedabove.Cytokinepro lingwasperformedusingrespective ELISA kits of IL- , TNF-a, and IL- b (BioLegend) according to the manu- facturer sprotocol.Histopathologyoftherightlungwasdeterminedusing hematoxylinand eosin staining,andin ammationscoring was performed

as reported previously.[ ] To evaluate the time-dependent protection of PCANS,animalswerechallengedwith PFUofPR- viaintranasalroute after or h of PCANS or PBS treatment and euthanized on day post- infection to quantify lung viral titer using plaque assay.

Statistics: Statistical analysis and graphing were conducted using Graphpad Prism. A one-way ANOVA with Tukey s post hoc analysis was used to compare multiple groups. Two-way ANOVA with Tukey s multi- ple comparison tests was used to analyze the data with two variables. To evaluate the e ciency of PCANS, survival plots were generated using the Kaplan-Meier survival curve, and the statistical signi cance of the re- sults was analyzed using the Gehan-Breslow-Wilcoxon test. P values for the body weight changes were determined using one-way ANOVA with Brown-Forsythe post hoc analysis. A P-value of less than . was con- sidered statistically signi cant.

Supporting Information

Supporting Information is available from the Wiley Online Library or from the author.

Acknowledgements

The authors acknowledge the use of BioRender for creating schematic illustrations - Fig. , Figs. a, f, j, Fig. n, and Figs. a, n. in the manuscript. The authors acknowledge funding support from Gillian Reny Stepping Strong Center for Trauma Innovation at the Brigham and Women s Hospital (to NJ and JMK), Department of Anesthesiology, Peri- operative, and Pain Medicine at the Brigham and Women s Hospital (to NJ), Fulbright-Nehru Postdoctoral Fellowship (to JJ), and Boston Univer- sity (to FD) and the Peter Paul Career Development Award (to FD). The metered dose spray pumps were generously gifted by Aptar Inc. The au- thors acknowledge Integrated BioTherapeutics (IBT) Bioservices for eval- uating the neutralization activity of PCANS against RSV and adenovirus.

Con ict of Interest

J.J., H.M.B, Y.T., and J.M.K have one pending patent based on the PCANS formulation described in this manuscript. N.J. and J.M.K are paid con- sultants, scienti c advisory board members, and hold equity in Akita Bio- sciences, a company that has licensed IP generated by N.J. and J.MK. that may bene t nancially if the IP was further validated. The interests of N.J. and J.MK. were reviewed and overseen by their institution in accordance with its con ict of interest policies.

Author Contributions

J.J., H.M.B., and J.R.Q contributed equally to this work. J.J., H.M.B., J.R.Q., Y.T., J.M.K., N.J. performed conceptualization. J.J, H.M.B., J.R.Q., D.K.,

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E.B., D.L., D.P. was performed data curation. J.J., H.M.B., J.R.Q., D.K., E.B., D.L., D.P. performed data analysis. J.M.K., N.J. performed funding acquisition. J.J., H.M.B., J.R.Q., Y.M., E.B., P.S., K.S., O.S., R.N., E.A., S.R., J.K. performed investigation. J.J., H.M.B., J.R.Q., D.K., S.K., X.L.L., J.M., J.G., J.N.L, A.Y., F.D. performed methodology. Y.T., J.M.K., N.J. performed project administration. F.D., Y.T., J.M.K., N.J. performed supervision. J.J., H.M.B., J.R.Q., J.M.K., N.J. performed validation. J.J., H.M.B., S.R., N.J., wrote manuscript — original draft: J.J., H.M.B., D.K., F.D., Y.T., J.M.K, N.J. edited the manuscript.

Data Availability Statement

The data that support that ndings of this study are available from the corresponding author upon reasonable request.

Keywords

antibacterial, antiviral, broad spectrum protection, nasal prophylaxis, nasal spray, pathogen capture, respiratory infections, virus neutralization

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