Nanomedicine strategies to target coronavirus

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ContentslistsavailableatScienceDirect

Nano

Today

j o ur na l ho me p ag e :w w w . e l s e v i e r . c o m / l o c a t e / n a n o t o d a y

Nanomedicine

strategies

to

target

coronavirus

Marcel

Alexander

Heinrich

a

_,

_Byron

_Martina

b

_,

_Jai

_Prakash

a,∗

a_Department_of_Biomaterials_Science_and_Technology,_Section_Targeted_{Therapeutics,}_Technical_Medical_Centre,_University_of_Twente,_7500AE,_Enschede,

theNetherlands

b_Artemis_One_Health_Research_Institute,_2629JD,_Delft,_the_Netherlands

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received14June2020

Receivedinrevisedform7August2020

Accepted26August2020 Keywords: Coronavirus SARS-CoV-2 Nanomedicine Nanoparticle-basedvaccine

Nasaldrugdelivery

Virus-mimickingnanoparticles

a

b

s

t

r

a

c

t

Withthesevereacuterespiratorysyndromecoronavirus(SARS-CoV)in2002,themiddleeastrespiratory syndromeCoV(MERS-CoV)in2012andtherecentlydiscoveredSARS-CoV-2inDecember2019,the21st firstcenturyhassofarfacedtheoutbreakofthreemajorcoronaviruses(CoVs).Inparticular, SARS-CoV-2spreadrapidlyovertheglobeaffectingnearly25.000.000peopleuptodate.Recentevidences pointingtowardsmutationswithintheviralspikeproteinsofSARS-CoV-2thatareconsideredthecause forthisrapidspreadandcurrentlyaround300clinicaltrialsarerunningtofindatreatmentfor SARS-CoV-2infections.Nanomedicine,theapplicationofnanocarrierstodeliverdrugsspecificallytoatarget sites,hasbeenappliedfordifferentdiseases,suchascancerbutalsoinviralinfections.Nanocarriers canbedesignedtoencapsulatevaccinesanddeliverthemtowardsantigenpresentingcellsorfunction asantigen-presentingcarriersthemselves.Furthermore,drugscanbeencapsulatedintosuchcarriers todirectlytargetthemtoinfectedcells.Inparticular,virus-mimickingnanoparticles(NPs)suchas self-assembledviralproteins,virus-likeparticlesorliposomes,areabletoreplicatetheinfectionmechanism andcannotonlybeusedasdeliverysystembutalsotostudyviralinfectionsandrelatedmechanisms. ThisreviewwillprovideadetaileddescriptionofthecompositionandreplicationstrategyofCoVs,an overviewofthetherapeuticscurrentlyevaluatedinclinicaltrialsagainstSARS-CoV-2andwilldiscuss thepotentialofNP-basedvaccines,targeteddeliveryoftherapeuticsusingnanocarriersaswellasusing NPstofurtherinvestigateunderlyingbiologicalprocessesingreaterdetail.

Contents

Introduction...2

Viralbiologyandclinicalfeatures...2

Replicationstrategy...2

Clinicalfeaturesofcoronavirusinfections...3

Therapeuticinterventionsandnanomedicinestrategies ... 4

VaccinesagainstCoVsandtheroleofnanomedicine...6

NanomedicinestodelivervaccinesagainstCoVs...7

TargetingCoV’slife-cycleandnanomedicinestrategies...11

Blockingreceptor-mediatedcellentry(extracellular)...11

Blockingcellentryandfusionofthevirusenvelopwiththecellmembrane(intracellular)...11

Blockingviralproteases...12

InhibitingtheRNA-dependentRNApolymerase...12

Nanomedicinestrategytotargetpathologicalcells...12

Targetingtheimmunesystemandnanomedicinestrategies...14

Supportingtheimmunesystem...14

∗ Correspondingauthor.

E-mailaddress:j.prakash@utwente.nl(J.Prakash).

https://doi.org/10.1016/j.nantod.2020.100961

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2 M.A.Heinrich,B.MartinaandJ.Prakash/NanoToday35(2020)100961

Inhibitingtheinﬂammatoryresponse...14

Nanomedicinestrategiestotargetimmunesystem...15

NanomedicinestounderstandCoV’smechanisms...15

Conclusionsandfutureoutlook...16

CRediTauthorshipcontributionstatement...18

References...18

Introduction

The21stcenturyhassofarexperienced theemergence and epidemicofthreemajorcoronaviruses(CoVs):i)thesevereacute respiratorysyndromecoronavirus(SARS-CoV)in2002,ii)the mid-dleeastrespiratory syndromecoronavirus(MERS-CoV)in2012, andiii)therecentlydiscovered SARS-CoV-2in December2019, whichwasfirst reportedinthecityof Wuhan,HubeiProvince, China[1–4].Aftertheirdiscoveryin1960upuntil2002,human CoVs(hCoVs)weremostlytreatedasaminorratherthana seri-ousthreatduetothefactthatthesevirusesonlycausedminor respiratorysymptoms.However,startingwithSARS-CoVin2002, differentCoVsemergedthatwereassociatedwithserious respi-ratorydiseasesincludingMERS-CoVandtherecentlydiscovered SARS-CoV-2.WhereastheincidenceofSARS-CoVandMERS-CoV werecomparablylowwith8.096and2.494reportedcases, respec-tively,theincidenceof SARS-CoV-2is significantlyhigher [5,6]. UptoSeptember1st,2020,around25.251.334 caseshavebeen reportedworldwidewithatotalof846.841relateddeaths[7].In particular,therapidincreaseincasesoverthelastmonthscauses immensechallengestohealthcaresystemsandsocieties, result-inginmorethan50countriesworldwideannouncinga‘lockdown’ periodtolimitsocialcontactanddeceleratethespreadingofthe virus.Thehighincidenceandthedrasticmeasurestakenbythe gov-ernmentsunderlinetheneedfortherapeuticinterventionsagainst SARS-CoV-2.Differenttherapeuticsandvaccinesarebeing devel-opedandtestedatrapidspeedtotreatorcontainedSARS-CoV-2, ofwhichsomearealreadyreachingclinicaltrials,stokingthehope forafastsolution[8].Thisincludesrepurposingtheuseof exist-ingtherapeutics developed for the treatmentof other diseases suchasRemdesivir,Ivermectin,andothers[8,9].However, differ-entpromisingtherapeutics,suchashydroxychloroquinefailedas potentialtreatmentforSARS-CoV-2intheclinicduetosevereside effectsorlackofefficacy[10],whereasothertherapeuticssuchas Remdesivirstillneedmoreevaluationbeforebeingconsideredas successfultreatment[11].

Intherecentyears,nanomedicineofferedpromisingstrategies toovercomelimitationsofcurrenttherapeuticsbyprovidinga plat-formthatincreasestreatmentefficacy,reduced sideeffects and enablingspecifictargetingtoachievethedesiredresponseson cel-lularlevel[12–18].Ontheonehand,nanocarrierscanherebybe usedtofunctionaseitherdeliveryvehiclesofaspecificvaccine toachieveimmunizationofthehost[12–14],orasnanocarriersto deliverdifferenttherapeuticstothetargetsiteprolongingtheir cir-culationtimewhileprotectingthetherapeuticsfromdegradation orbyreducingsideeffectsofnon-encapsulateddrugs[15,13–18]. Inparticulartheuseofliposomesasnanocarriersformsa promis-ingapproach that mimictheinfection profileofvirusesdue to theirhighresemblanceinstructure,presentingalipidbilayer sur-faceandinhighsimilaritiesin cellularuptake [19–21].Another strategy canbe theuseof empty virusparticles themselvesto functionasnanocarriersforadifferentloadingsuchastheuseof genetherapy[22,23].Thecombinationofaforementioneddrugs ornewlydevelopedtherapeuticswithnanocarriersystemscould formapromisingstrategytodelivertherapeuticsorvaccine

anti-genstotargetcellsandtherebystopthelife-cycleofCoVsorprevent disease.

Inthisreview,wewillcriticallydiscusshownanomedicinecan playacrucialroletodevelopeffectivetreatmentsforCoVs.First, wewillprovideadescriptionandclassificationofCoVsandtheir specificreplication strategy within thehost. We then focus on therapeuticsthatarecurrentlytestedtotreatCoVsandtheir spe-cificmechanismofactionwithinthecellaswellasdescribingthe potentialcombination ofthesetherapeuticswithnanomedicine toincreasetheirefficacy,reducesideeffectsandenabledirected treatmentofSARS-CoV-2targetsitesandcells(Fig.1).

Viralbiologyandclinicalfeatures

CoVs wereﬁrst discoveredin the1960s and were classiﬁed underthesubfamilyof Orthocoronavirinae,withinthefamily of Coronaviridae,whichthemselvesformthelargestfamilywithinthe orderNidovirales[4,24,25].SARS-CoV,MERS-CoVandSARS-CoV-2 andthelesserknownHCoV-HKU1andHCoV−OC43,allbelongto theso-called␤-CoVs,havingahighpotentialtoinfecthumans.CoVs areenvelopedvirusescontainingasingle-strandedpositive-sense ribonucleicacid((+)ssRNA)of27–

32kb[24–26].Thegenomeisprotectedwithinthenucleocapsid andencodesforfourtoﬁvestructuralproteins,dependingonthe typeofCoV:spike(S),membrane(M),envelope(E),hemagglutinin esterase(HE)andnucleocapsid(N)proteins(Fig.2).Whereonly the␤-CoVsHCoV-HKU1andHCoV−OC43encodefortheadditional HEprotein.ItisspeculatedthatdifferencesintheSproteinandin particulardifferencesinthecleavagesites,includinganadditional furincleavagesite,causestherapidspreadofthenovelSARS-CoV-2 whencomparedtoothertypesofCoVs[25,27–30].

Replicationstrategy

The replicationprocess of CoVs withintarget cells is a cru-cialstepintheinfectionanddiseaseprogressionandoneofthe majortargetsforpossibletherapeuticintervention.Astheexact mechanismofCoVinfectionandreplicationisalreadydiscussed elsewhere,thissectionwillfocusonkeyaspectsandtargetsinthis processthatformthebasisfortherapeuticinterventionsthatwill bedescribedinthenextsectionofthisreview[1,25,28,31–33].

ThelifecycleofCoVsinhostcellsissimilar(Fig.3),although certainaspectsbetweenSARS-CoV,MERS-CoVandSARS-CoV-2are differentresultinginslightlydifferentuptakemechanismsand pro-cessingwithinthehostcells.Theinitialattachmentofthevirion withthehostcellisdrivenbyinteractionbetweentheSproteinand thespeciﬁcreceptor[28].SARS-CoVandSARS-CoV-2bindtothe cellularreceptorangiotensin-convertingenzyme2(ACE2)present primarilyinthelung,butalsoinendothelialcellsofarteriesand veins,theintestine mucosalcells, thetubularepithelialcells of thekidneyandtherenaltubulesaswellascerebralneuronsand immunecells[28,33–40].MERS-CoVbindstothecellularreceptor dipeptidylpeptidase4(DPP4),alsoknownasclusterof differenti-ation26(CD26),presentonepithelialcellsinthekidney,alveoli, smallintestine,liver,prostateandonactivatedleukocytesaswell ashumandendriticcells, T-cellsandmacrophages,

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demonstrat-Fig.1.Schematicoverviewofthedifferenttopicscoveredinthisreview.

Fig.2.Structureofcoronaviruses.Schematicrepresentationofacoronavirus

describingstructuralproteinsandtheviralgenomeandrepresentativeelectron

microscopyimageofSARS-CoVdisplayingthecharacteristiccrown-likesurface

(arrowindicatingsinglevirion).PhotocredittoDr.FredMurphy,reproducedunder

CreativeCommonAttributionLicenseCC-BY4.0fromD.N.Valencia30_.

inghowthisvirusmightbeabletoaffecttheimmunesystemand facilitateimmuneevasion[28,41–49].

Aftersuccessfulfusionoftheviralenvelopewiththehostcell membrane,theviralgenomeisreleasedintothecytoplasmofthe host,wheretheviralRNAistranslatedresultinginthe produc-tionoftheRNAreplicase-transcriptasecomplexalsoreferredtoas RNA-dependentRNApolymerase(RdRP)[28].Thiscomplex gener-atesintermediatefull-lengthnegative-sense(-)RNAcopies,which arelaterusedastemplatesforfull-lengthpositivesense(+)RNA genomes.Furthertranslationleadstotheproductionofviral pro-teins.Next,viralnucleocapsidsareformedfromviralgenomicRNA andN-proteinsinthecytoplasmfollowedbybuddingofthe nucle-ocapsidandstructuralproteinsinthelumenoftheendoplasmatic reticulum–golgiintermediatecompartment(ERGIC)toformnovel virions,whicharethenreleased(egressed)byexocytosis.This pro-cessisreportedtobesimilarforalltypesofCoVs,includingthe novelSARS-CoV-2.

Clinicalfeaturesofcoronavirusinfections

GeneralsymptomsofaninfectionwithCoVs,and in particu-larwiththenovelSARS-CoV-2(relateddiseasenames‘coronavirus disease’or COVID-19)includefever, cough,shortnessof breath and fatigue,which aresimilar toSARSand MERS,but can also include headache, haemoptysis or diarrhea [50–52]. So far an exceedingly highnumberof patientsexperience(severe) pneu-monia uptoacute respiratory distress(ARDS), severesepsis or multipleorgandysfunction[11,50,52].Moreovernewevidences arefoundthatCOVID-19isalsorelatedtogastrointestinal symp-toms,which might directlyberelated toviralinfections of the intestineduetothehighexpressionofACE2receptorwithin gas-trointestinalepithelialcellsformingadirecttargetforthevirus [53,54].RecentstudiesfurthermoredisplayedthepotentialofCoVs toinfectthecentralnervoussystem,explainingsymptomssuchas headache,nauseaandvomitinginsomepatients[55,56].

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Remark-4 M.A.Heinrich,B.MartinaandJ.Prakash/NanoToday35(2020)100961

Fig.3.ReplicationstrategyofSARS-CoV,MERS-CoVandSARS-CoV-2.Schematicrepresentationof(i)attachmentofthevirustothespeciﬁcreceptor(ACE2forSARS-CoV

andSARS-CoV-2andDPP4forMERS-CoV),(ii)viruscellentryviaendocytosis,(iii)fusionoftheviralenvelopwiththeendosomalmembraneandreleaseofviralRNA,(iv)

translationofnon-structuralproteinsincludingtheRdRPandRNAreplication,(v)translationofstructuralviralproteinsattheendoplasmicreticulumandtheassemblyofa

newvirionintheERGIC,(vi)packagingofreplicatedRNAintothenovelvirionsandﬁnally(vii)viralegress.

ableforCOVID-19patientsisalsothehighamountofinﬂammatory cytokinesandchemokines,whicheventuallyleadstothecytokine releasesyndrome(CRS)[57–59].SevereformsofCRS,inparticular incombinationwithARDS,canleadtoseveremulti-organfailure andeventuallythepatient’sdeath.AlthoughCRSisalife-threating andseverediagnosisforthepatient,theunderlyingmechanisms andcytokinesandchemokinesinvolved,suchasinterleukins1or 6(IL-1,IL6)ortumornecrosisfactor␣(TNF␣)arewellunderstood, facilitatingtheuseofalreadyknowndrugsinthetreatmentofthis syndrome.

Therapeuticinterventionsandnanomedicinestrategies

Todate,therearenoexistingantiviraldrugsthatareknown toefﬁcientlytreatCOVID-19progression,although,thereare sev-eraltherapeuticsinclinicaltrials thatshowthepotentialtobe effectiveagainstthevirusprogression.Mostofthesedrugswere originally designed for the treatment of other infections and are now evaluatedfor theirpotential tobe usedfor COVID-19 (re-purposing).Currently,around300clinicaltrialsfallingunder the EuropeanUnion Drug Regulating Authorities Clinical Trials

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Fig.4. SchematicrepresentationofpotentialtargetsitesforSARS-CoV-2therapeuticscurrentlyevaluatedinongoingclinicaltrials.Startingwith(i)prophylactic

treatments,(ii)vaccines,(iii)therapiesthattargetthelifecycleofSARS-CoV-2beforeandafterenteringthehostcells,(iv)treatmenttosupporttheimmunesystemand(v)

therapeuticstoreduceCOVID-19symptomsincludinganti-inﬂammatorytherapeuticstoinhibitcytokinereleasesyndromeaswellastherapeuticstoavertlong-termtissue

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Database (EudraCT)are listed in theEU Clinical Trials Register focusingonthetreatmentorpreventionofCOVID-19[8].Ingeneral, thesetreatmentscanbedividedintofourcategories:(i)vaccines againstSARS-CoV-2,(ii)therapies targetingtheCoV’s life-cycle, eitherblockingcellentryorinhibitingtheviralcyclewithinthe hostcell,(iii) therapiestotargettheimmuneresponse and(iv) othertreatments,includingprophylactictreatmentsorpreventing long-termlungdamage(Fig.4).

There-purposingofsuchdrugsdoesnotonlydisplayamajor reductionin thenecessarytime todevelopa treatmentagainst COVID-19asthesedrugshaveoftenbeentestedextensivelybefore, butscientistscanalsobeneﬁtfromthealreadyexistingknowledge ofhowthesedrugsfunctioninpatientsandtheriskofpotential side-effects.Forinstance,hydroxychloroquine,whichwas consid-eredoneofthemostpromisingtherapeuticsagainstSARS-CoV-2 infections,failedinrecentclinicaltrialsduetosevereside-effects, whichhavebeenknownandreportedforyears[60–62]. Nonethe-less, the mechanism of function of this drug makes it highly interestingfortheuseinCoVinfections.Whereas,thedrugalone didnotsucceed,thecombinationofthisdrugwithnanomedicine strategiesmightbeabletoreduceknownside-effectsand over-comelimitationsinefﬁcacytomakethisdruginterestingagainfor thetreatmentofSARS-CoV-2[63].

Overthelastdecade,nanomedicine,describingtheuseof col-loidalcarriers,alsocallednanocarriersystems, hasfoundbroad applicationinpharmacologyandaimedtoimprovethetreatment ofseveraldiseases[64].Inparticularcancerresearchhasrapidly facilitatedthe useofsuchnanocarriers torenderdrugdelivery moreefficientandspecific[15].Thereareseveraladvantagesinthe useofnanocarriersforthedeliverydrugs,including(i)enhancing thesolubilityofcertaindrugs,(ii)thecontrolledsustained-release ofdrugsprovidingalong-termtreatmentorhighdrugexposure, (iii)thesuitabilitytodelivermacromolecules,whichareprotected fromdegradation within thebody or protected from clearance by theimmune system,(iv) a general decrease in side-effects, (v) the potential increase drug internalization by cells as well as (vi)the capability to target specific cells [65]. Furthermore, nanocarrierscan be altered in theirsize, shape, charge or sur-facechemistryfacilitatingthefabricationoftailorablebiological properties.Moreover,nanocarriersystemscanbeadministeredvia differentroutes,suchassub-cutaneousormuscularinjections,via oralorintranasaladministrationandarecapableofpenetrating capillariesandmucosalsurfaces[66].Thesecharacteristicsmake nanocarriers also highly interesting for the treatmentof CoVs, eitherbydeliveringvaccinesandstimulatetheimmuneresponse, orbydeliveringdrugstoinfectedcellstoimprovetheirefficiency andspecificity.

Overthelastyearsseveraldifferentmaterialshavebeen exten-sivelyinvestigatedfortheuseasdrugdeliverysystemsincluding polymericnanoparticles(NPs),facilitatingtheuseofpolymerssuch aspoly(lactic-co-glycolic acid(PLGA), poly (␥-glutamic acid)( ␥-PGA),polystyreneorpoly-alkylacrylate;inorganicNPs,basedon gold,silicaorcarbon;liposomesandlipid-basedNPsorvirus-like particles(VLPs), whichis based ontheimplementationofviral proteinstoformcarriers(Fig.5).Theadvantagesand disadvan-tagesofthedifferentsystemsareextensivelydiscussedelsewhere [13,22,66].Thisreviewwillmainlyfocusonthepotentialof lipid-basedNPsaswellasvirus-likeNPsfortheuseinCoV-targeted NP-basedvaccine(NPb-V)duetothehighsimilarityofsuch sys-temswithnaturalviruses.Suchvirus-mimickingNPsareableto displaysimilarcharacteristicsasvirusesintermofsizeand per-formancewithinthebody,promotingtheircapabilitytofollowthe samemechanismofenteringandspreadingthroughoutthebody asviruses.Inthissection,wewillsummarizedifferentdrugsand therapeuticsthatarecurrentlyevaluatedforthetreatmentof SARS-CoV-2infectionsandtakeadetailedlookondifferentnanocarrier

Fig.5. Differentnanoparticlesforvaccine/drugdelivery.Schematic

represen-tationofdifferentnanoparticlesabletodelivervaccinesortherapeuticstowards

targetcells,highlightingnanoparticlesthatdisplayvirus-mimickingproperties.

systemswiththepotentialtorenderthesetherapeuticsmore efﬁ-cientandsafer.

VaccinesagainstCoVsandtheroleofnanomedicine

Arguablytheearliestpoint oftherapeuticinterventionisthe clearance of the virusbefore it can even infect target cells or spread throughout thebody. Vaccines allowfor sucha type of intervention withthe potentialto provide a long-lastingeffect againstCoVs,however,thedevelopmentofvaccinescantake sev-eralmonthsuptoyearstoreachthemarket,leavingvaccinesas astrategytopreventoutbreakofSARS,MERSorCOVID-19inthe future.Nonetheless,duetotherapidsequencingoftheSARS-CoV-2 genome,differentvaccinecandidateshavealreadybeendeveloped thatareevaluatedinclinicaltrials.

Vaccinesbasicallyintroducespeciﬁcviralantigenstothebody, whicharealsoproducedinpatientsundergoingthedisease, how-ever, in a safe fashion for the patient [67]. Such antigens are oftenpresentedonthecellsurfaceofsocalledantigen present-ingcells(APCs),inparticulardendriticcells,embodiedinthemajor histocompatibilitycomplex(MHC)IandII[68].Thesepresented antigensarecrucialfortheadaptiveimmunesystem,which

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rec-ognizedsuchantigensas hostileinvadersand furtherproduces antibodiesortriggerTcells tokilltheinvader. MemoryBcells furthermoredevelopvirusspeciﬁcantibodiesonitscellsurface, whichuponrecognitionofthevirus,triggerafastimmuneresponse tocleartheviralinfection.Severaldifferenttypesofvaccinesare currentlyinusedependingonthetypeofinfection,suchaslive attenuatedvaccines,inactivatedvaccines,conjugatedvaccines,or morerecentlyDNAorRNAvaccines[26].Despiteitspresencefor nearly20years,sofarnoeffectivevaccineagainsthumanCoVs couldbedevelopedduetoinefﬁciencyofthevaccineorinduced side-effectsupontreatment, demonstrating theneed ofa novel strategytotargetCoVs[69].

In particulartheimportance oftheSprotein in CoVs,made it a promisingtarget for vaccines[70]. Currently different clin-icaltrialsarerunningtoinvestigatetheefficacyofmRNA, DNA andnon-replicatingadenovirusvector-basedvaccines.Moderna’s mRNA-1273,BioNTech’sBNT162a1,b1,b2andc2,Arcturus Ther-apeutics’LUNAR−COV19and anunnamedvaccinecandidateby CureVacaremRNAvaccinesthattargettheSproteinofCoVsor spe-cificregionswithin.Similarly,InovioPharmaceuticals’INO-4800, Genexine’sGX-19andZydusCadila’sZyCoV-DareDNAvaccines targetingtheSproteinofCoVs[8].Both,RNAandDNAvaccines have severaladvantages compared toconventional vaccines, in particularlowerproductionscostsandsimplepurification, how-ever,thedeliveryofsuchvaccinetothetargetcellsisachallenge due tostability and specificity,which makes them suitable for nanomedicine-baseddeliverysystemswhichwillbediscussedat alaterstageinthis review.TheUniversityofOxfordin collabo-rationwithAstraZenecarecentlypresentedadifferentvaccineto tackleCoVscomposedonanon-replicatingadenovirusvectorable toreplicatetheSproteinofSARS−CoV-2calledAZD1222(formerly ChAdOX1)demonstratingadifferentwayforvaccinedevelopment. SimilarlyAd5-nCoVfromCanSinoBiologicsandGam−COVID-Vac fromtheGamaleyaResearchInsititutefacilitatethesamestrategy tofindavaccinecandidateagainstSARS_−CoV-2.Recently,theuse ofinactivatedCoVshasalsoshownefficacytoinduceimmunity andarecurrentlyinphaseIIIclinicaltrialsincludingCoronaVac fromSinovacortwovaccinecandidatesdevelopedbySinopahrm incollaborationwiththeWuhanInstituteofBiologicalProductsor theBeijingInstituteofBiologicalProducts.Furthermore, protein-basedvaccinessuchasNVX-CoV2373fromNovavaxorCOVAX-19 fromVaxinePTYLtd.arecurrentlyinevaluation.Inaddition,several othercandidatesarecurrentlydevelopedthathavenotyetreach clinicalphases[71].

NanomedicinestodelivervaccinesagainstCoVs

WiththeincreasinginterestinRNAandDNA-basedvaccines, thecombinationofsuchvaccineswithnanocarriershasbecomean interestingstrategytoovercomelimitationsofthecurrent deliv-eryofthesevaccines.ThecombinationofRNAwithnanocarriers hasbeenaneffectiveapproachtodeliversmallinterferingRNA (siRNA)forthetreatmentofseveraldiseasessuchasmalignancies, infections,autoimmunediseaseandneurologicaldiseases[72].For instance,theaforementionedRNA-basedvaccinemRNA-1273from ModernaisbasedonthecombinationoftheRNAdrugsubstance witha nanocarriersystem,which willbediscussedlater inthis sectioningreaterdetail. Similarly,nanocarrierscanbeusedfor thedeliveryofantigensavoidingprematuredegradationofsuch moleculesinthebody,aswellasassistinthetranslationofthese moleculesinto functional immunogens avoiding potential side-effectscausedbythetreatment[12].

Ingeneral,thedeliveryofvaccinesusingNPssystems(NPb-Vs) canbedividedintotwomainstrategies:(i)NPb-Vswherethe anti-genorRNA/DNAisencapsulatedwithinthenanocarrierand (ii) attachingantigensonthenanocarriersurfaceexposingittothe sur-rounding(Fig.6)[13,66].TheencapsulationofantigensorRNA/DNA

vaccinewithinnanocarriersmainlyaimsonprotectingthe anti-gensfromproteolyticdegradationandtoallowdirectedtargeting ofthevaccinetowardsAPCs[13].TheseAPCstakeuptheNPb-V andeitherprocesstheinducedantigenstowardsthecellsurface, ortranslatetheinfusedRNAorDNAtotherespectiveantigenbefore implementingitintothesurface[73].Hereby,RNAmightbe prefer-ablewhencomparingtoDNAasRNAcanbedirectlytranslatedin thecellcytoplasmwhileDNAmustreachthenucleusofthe tar-getcellfirst[73].EncapsulatingRNAorDNAintonanocarrierscan beapromisingapproachtocreateNPb-VagainstCoVs.As afore-mentioned,differentRNAorDNAvaccinesarecurrentlyevaluated inclinicaltrials,whichmakesthecombinationofthese ribonucle-aseswithnanocarriersahighlypromisingstrategy.Furthermore, NPb-Vbasedonantigenencapsulationalsoshowthepotentialto exertalocaldepoteffect,whichprolongstheexposureofantigens towardstheimmunecells[74].Thesecondstrategytocreate NPb-Vsistodirectlyattachorconjugateantigensontothenanocarrier surface[66].InsuchwaytheNPb-Visnotdirectlyaimedtobring a cargotoAPCsbuttomimicthevirusitself.Forinstance, pre-sentingSproteinspecificantigensontopofananocarriercould triggeraspecificimmuneresponsetowardstheseantigens, form-ingapromisingstrategyagainstCoVs.Inparticular,thepurification ofimmunoglobulinsfrompatientplasma,asaforementioned,in combinationwithananocarriercouldcreateapromisingNPb-V againstCoVs.

DesignconsiderationsofNP-basedvaccines. CoVsarethoughttobe spreadfromhumanhosttohostbyrespiratorydroplets,mainly produced whenaninfected personsneezes or coughs.Recently ithasbeenshownthatthemajorrouteforSARS-CoV-2toenter thebodyisthroughthenasalcavity,inparticulartroughmucosal epithelialcellsincludingmucus-producinggobletcellsandciliated cells,formingthemaintargetsiteofcoronainfections[75,76].The presentlymphoidtissue,callednasal-associatedlymphoidtissue (NALT),comprisesoflymphoidfollicles(B-cellareas), interfollic-ularareas(T-cellareas),macrophagesanddendriticcells,which when activatedortriggeredsupporttheclearance ofinfectious agenteitherbyactivatedkillercellsorbytheproductionof antigen-speciﬁcantibodiespresentedinthemucuslayer,whichformsa liquidlayerontopoftheepithelialcellsofthemucosa[66,77]. Asaresultofsuchresponse,theNALTis apromisingtargetfor vaccinesagainstrespiratoryvirusandmoreinterestinglyforCoVs. Ideally,NPb-VsagainstCoVsshouldfollowthesamepathasCoVsto reachtheNALTandtriggeraspeciﬁcmucosalimmuneresponse.To achievethis,NPb-Vshavetobedesignedpresentingsimilar kinet-icsasviruseswithinthehost,whichcanbedependentonthesize, shape,chargeandgeneralsurfaceproperties.ThesizeofCoVshas beenmeasuredtobebetween50–150nmusingelectron cryomi-croscopy,withtheaveragediametersizeof82–94nm(excluding thespikes)[78].Mostnanocarriersystemsareaimedtoachievea sizebetween20–200nm,makingthemsuitabletomimicthesize ofCoVsaswelltofollowthesamemechanismofinfectiontothe NALT[14,15].

AnothercrucialaspectwhentargetingtheNALT,besidesthe size,istherouteofNPb-Vadministration.Ithasbeenshownthat intravenousorintramuscularinjectionsofvaccinesinducea sys-temicimmunity,however,onlyaweakmucosalresponse[66].The applicationof vaccinesviathenasalroute,similartothe infec-tionmechanismofCoVs,isthereforemorepromisingtotriggera systemicimmunityontheonehand,whilealsoinducing immu-nityonthemucosalsurfaces.It hasbeen shownthat thenasal routeofvaccineadministrationresultedinincreasedproliferation ofantigen-speciﬁclymphocytes,increasedcytokineproductionas wellasinductionofantigen-speciﬁcantibodieswhencomparedto subcutaneouslyorsystematicadministration[79–81].Apromising strategytoadministerNPb-Vsistheuseofanasalspraythat

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deliv-8 M.A.Heinrich,B.MartinaandJ.Prakash/NanoToday35(2020)100961

Fig.6.StrategiestodelivervaccinesagainstCoVsusingnanoparticles.Schematicrepresentationof(i)nanoparticle-basedvaccinesencapsulatingageneorantigenfor

thedeliverytowardsantigen-presentingcellswhichcausesanimmunereactionor(ii)nanoparticle-basedvaccineswithanantigenconjugatedtothenanocarriersurfaceto

functionasantigen-presentingcarriersthemselves.

ersthevaccinedirectlytothetargetsite,however,suchsystems currentlydisplayseverallimitationsincludinginsufﬁcientantigen uptakeinthemucosa,rapidmucociliaryclearanceortoxicityof theadministeredtherapeuticsaswellaschallengesinstable fab-ricationofNPb-Vsfornasaldelivery[82].Nonetheless,different strategieshaveemergedoverthelastdecadesfacilitatingtheuseof anasalsprayforvaccinedelivery.Oneoftheﬁrstnasalvaccinesthat reachedclinicaltrialswasdevelopedin2010,describingtheuseof driedliveattenuatedmeaslesvaccines[83,84].Althoughthis vac-cinedidnotinvolvenanocarrierssystem,thisstudydemonstrated thepotentialusenasalsprayforapplication.Besidessprays,nasal applicationoftherapeuticscanalsoinvolvedroppersorneedleless syringes,whicharelesscomplicatedinthefabricationprocess[85]. RemarkablefortheclinicalsymptomsoftherecentSARS-CoV-2 outbreakisalsothehighinfectionpotentialoftheintestine,central nervoussystemaswellasindicationsforinfectionsofendothelial cellsinbloodvesselsandcapillariesasaforementioned.Although thenasalrouteofaccessisconsideredtheprimaryinfectionsite ofSARS-CoV-2andpotentiallythemostpromisingroutetodesign

avaccine,sofaritisunknownifotherpotentialinfectionroutes are possible. In suchway, besides targeting the NATL, NPb-Vs mightalsobedesignedtotargetlymphoidsystemsinthe intes-tineortoreachimmunecellsinthebraintoblockviralinfections ofthecentralnervoussystem,ofwhichthenasaladministration couldbeapromisingstrategyasdifferentdrugdeliveryapproaches usenasaladministrationtoreachthebrainenvironment[86].To potentiallytreatalltargetsites,acombinationofintranasallyand subcutaneously or intravenously injected NPb-Vs could form a promisingstrategy forSARS-CoV-2.For instance,arecentstudy demonstrated thatintranasal combinedwithintravenous injec-tionoftheantigenkeyholelimpethemocyanininmiceresulted intheincreasedproductionantibodiesagainstthisantigenwhen comparedtoeitherinjectionalone[87].However,subcutaneous injectiondidnotdemonstratesimilarbeneficialeffects.Asimilar studytoinvestigatedthecombinedinjectionofintranasaland sub-cutaneousvaccineinjectionagainstinfluenzaalsodidnotshowa significantadditionalvalueofsubcutaneousinjectioncomparedto thenasaladministrationalone[88],demonstratinginparticularthe

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combinationwithanintravenousinjectionformsapromising strat-egy.Suchcombinationalstrategiesshouldbetakenintoaccountin SARS-CoV-2duetotherapidspreadofthevirusawayfromthe maintargetsitetowardsothersecondarysites.

Promisingnanocarriersforefficientvaccinedelivery. Sincethe out-breakofSARS-CoVin2002,severaldifferentvaccinecandidatesin combinationofnanocarriershavebeendeveloped(Table1), how-ever,onlywiththerecentoutbreakofSARS-CoV-2,suchvaccines arenowreachingclinicalphasesduetohighurgencytodevelop anovelvaccine.Arguably,themoststraight-forwardstrategyto fabricateaNPb-Vtomimicanactualvirus’mechanism ofbody entry,istheuseofviralcomponentsasbasisforthenanocarrier. ANPb-Vapproachthathasrecentlybeenusedtotarget respira-toryviruses,inparticularhumanrespiratorysyncytialvirus(RSV), istheuseofself-assemblingproteinNPs(SAPNs)whichare20–100 nmsized nanocarriersthat arebasedontheoligomerizationof monomericproteins[89].Forinstance,theNproteinofRSVhas beenusedtocreateself-assembliedsub-nucleocapsidring struc-turesthatprovoked anantigen-specificT-cellresponsetowards theprotein.Invivo examinationin a RSVBALB/cmouse model displayedenhancedimmunitytowardsRSVaftertreatment[90]. Inalaterstage,theseNproteinstructureswerecombinedwith palivizumab,aFsII-proteinantagonist,tofurtherenhance immu-nityagainstRSV [91].Ina similarfashion,theNproteinofRSV wascombinedwiththeectodomainoftheinfluenzavirusAmatrix protein2(M2e)toinduceimmunitytowardstheinfluenza(H1N1) virus[92].Furthermore,thisstudydemonstratedtheadvantageof nasaladministrationwhencompared tosubcutaneousinjection. The useof SAPNs hasalsoshown application in CoVs, demon-stratingthepotentialofsuchstrategiestobeusedinthenovel SARS-CoV-2aswell.Forexample,thepurifiedSproteinofCoVs self-assemblingintomicellularNPsincombinationwithaMatrix M1adjuvantwasusedtoinduceimmunitytowardsCoVsinBALB/c mice.Itwasfoundthatthemicedisplayedenhancedpresenceof neutralizingantibodiesaftervaccination[93].Inarecentapproach, a similarstrategy ofusingpurified Sprotein-based NPs formu-latedwithanadjuvant(Aluminum)displayedasimilarresponse inMERS-CoV[94].CurrentlytheusemicellullarNPsbasedonthe SproteinincombinationwithaMatrixM1adjuvants,inastudy ledbyNovavax(NVX-CoV2373),isevaluatedaspotentialvaccine forSARS-CoV-2demonstratingthepotentialuseofSprotein-based vaccinesforCoVs[8,95].

SimilartoSAPNs,VLPsarebasedonviralproteins,inparticular viralcapsidproteins,thatself-assembleintospherical nanocarri-ersof20–200nm.SuchVLPshavesuccessfullybeentestedfortheir vaccinationcapabilityininfluenzaaswellasRSV.Forinstance,VLPs comprised ofA/PR8/34 (H1N1) hemagglutininand matrix (M1) wereinvestigated for theirimmunogenic potentialin influenza afternasaladministrationinmice[96].Similarly,VPLscomprising ofmultipleectodomainsofmatrixprotein2(M2e5xVLPs)have beenevaluatedforinductionofinfluenzaimmunityusingnasal administration[97].Bothstudiesdisplayed enhancedimmunity towardsinfluenzaaftertreatment,whichshowedthepotentialof VLPsinvaccineapplications.ChimericVLPs,comprisingofproteins fromdifferenttypesofviruses,havealsoshownthepotentialto induceimmunity.AVLPcomprisedoftheFproteinorGproteinof RSVandM1proteinofinfluenzadisplayedenhancedimmunogenic potentialagainst RSV in mice[98]. Althoughnot demonstrated inSARS-CoV,VLPsbasedonMERS-CoVviralproteinshavebeen demonstratedtoinduceimmunityinmice.Inthisstudy,MERS-CoV VLPsweregeneratedbyusingachimericapproachofcombining canineparvovirusVP2structuralgenewiththereceptorbinding domain(RBD)ofMERS-CoVtomimictheSproteinbindingsiteof thisvirus[99].IntramuscularinjectionofthesechimericVLPs dis-playedincreasedimmunityagainstMERS-CoV,demonstratingthe

potentialofthisVLPagainstMERS-CoVinfections.Inamorerecent study,MERS-CoVVLPswerepreparedincludingthefullSprotein ofMERS-CoVtoinduceimmunitybygeneratingVLPsusinginsect cells[100].Althoughthisapproachlackstheevaluationinvivoso far,VLPsexpressingthefullSproteinofCoVs,mightdemonstrate increasedimmunitycomparedtospeciﬁcsequencessuchasthe RBD.CurrentlyMedicago,acompanyfromQuebecCity,Canada, isevaluatingaVLP-basedvaccinecandidateagainstSARS-CoV-2 aimingtostartaphaseIclinicaltrialincluding180patientssoon [101].

Lipid-basedNPs,suchasliposomesorsolid-lipidNPs(SLNs), dis-playhighsimilaritiestovirusparticlesresemblingasimilarsurface structureasviruses,whichcanadditionallybechemically modi-fied.AlthoughtheuseofliposomesinNPb-Vagainstrespiratory virusesislimited,theuseofthesesystemsishighlyfavorablein vaccinedevelopmentbecausethecarrierscanbealteredtoachieve similarcharacteristicsasviruseswithouttheuseofvirus-specific peptides.1,2-dilauroyl-sn-glycero-3-phosphocholine(DLPC) lipo-somescontainingeithertheadjuvantsmonophosphoryllipidaor trehalose6,6’dimycolateorshortsyntheticpeptideswhichhave beenderivedfromconservedregionsofpathogen-derivedproteins havebeenshowntoinduceimmunityagainstinfluenzainmice whennasallyadministered,demonstratingtheuseofliposomes asNPb-Vfor nasaladministration[102]. Thesamestrategy has beenappliedtoinduceimmunityagainstSARS-CoVbycoupling syntheticpeptidesmimickingtheNproteinofSARS-CoVthatwere basedonHLA-A*0201transgenicmiceandrecombinantadenovirus forexpressingthesepeptidesontothesurfaceofliposomes[103]. TheyobservedinducedCoV-specificTlymphocyteactivityaswell asenhancedviralclearanceinHLA-A*0201transgenicmiceafter treatment, demonstrating a potentialstrategy touseliposomes combinedwithvirus-specificantigensasNPb-V.Inparticular anti-genspurifiedfrompatientplasmacombinedwithlipid-basedNPs formsapromisingstrategy tocreateimmunitytowardsCoVsin thenearfuture.AdifferentadvantageofusingtheseNPsforthe developmentofNPb-Vs,istheirunmetcapabilitytoencapsulate RNAandDNA,oneofthemajorinvestigatedtypeofvaccinesat themoment.Theuseofliposomeshasbeenwidelyappliedfor thedeliveryofdifferentRNAcargos,forexampleofsiRNAaswell asmessengerRNA(mRNA),demonstratinghowthesesystemare abletoprojectthecargoencapsulatedwhilebeingrapidlytaken upbytargetcells[72,73].IthasbeenproventhatmRNAliposome complexeswereabletoinfectthelung tissue andtranslatethe inducedmRNAintofunctionalproteins,demonstratingtheuseof liposomesforefficientgenedelivery[104]. Currentlyoneofthe mostpromisingstrategiestodevelopavaccineagainst SARS-CoV-2isbasedonnanocarriersystemsencapsulatingRNAtoenhance theirstability andperformance suchas Moderna’smRNA-1273. ThistherapeuticisbasedontheencapsulationoftheRNAdrug substancewithinaSLN.Althoughtheirtherapeuticisadministered intramuscularlyandnotvianasaladministration,itdemonstrates thepotentialofcombiningRNA-basedvaccineswithnanocarrier systemstoenhancetheirperformance.Furthermore,acurrent clin-icaltrialledbyArcturusTherapeuticsevaluatesthecombinationof self-replicatingRNAwithlipidNPsaspotentialvaccinecandidate forSARS-CoV-2 followinga similarstrategyasModernafurther demonstratingthehighpotentialoflipid-basedsystemsfor vac-cinedelivery[105].TwoadditionalstudiesledbytheUniversity ofWashington(HDT-301)andCanSinoBiologicals(NP-based Ad5-nCoV),respectively,facilitatethesamestrategytocombineRNA vaccinewithlipid-basednanocarrierstoeffectivelydeliverthe vac-cinecandidatestowardsthetargetsite[106].

Adifferentnanocarrier,whichhasrecentlybeeninvestigated fortheuseasNPb-V,areextracellularvesiclesorexosomes[107]. Exosomesarevesiclesthatarereleasedfromcellsuponfusionof multivesicularbodieswiththeplasmamembrane,liberating

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intra-10 M.A. Heinrich, B. Martina and J. Prakash / Nano Today 35 (2020) 100961 Table1

Overviewofdifferentnanoparticle-basedvaccinesforcoronavirusesandtheadvantagesanddisadvantagesoftherespectivenanoparticleplatform.

Nanocarrier Characteristics&Pros/Cons Target Formulation Vaccine Stage Ref

Self-assembling proteinNPs (SAPNs)

(Puriﬁed)proteinsthatself-assembleintoNPs

(e.g.micelles). SARS-CoV&MERS-CoV

Protein-proteinmicellular

nanoparticlesbasedonCoVSprotein, administeredwithMatrixM1adjuvant

Sprotein EarlyStage [93]

Pro:Simplepreparation,surface chargeandsizepreventclearance,

kineticallystable. MERS-CoV

Protein-proteinmicellular

nanoparticlesbasedonCoVSprotein, formulatedwithaluminumadjuvant

Con:Highcostsprevent industrialupscaling, short-termstabilityinvivo.

SARS-CoV-2 Protein-proteinmicellularnanoparticles basedonCoVSprotein

Sprotein PhaseIC.T. Novavax,[95]

Virus-likeparticles (VLPs)

Basedonstructuralviralproteins(e.g. capsidproteins)thatformspherical NPs.Oftenobtainedfromplants, bacteriaorinactivehumanviruses.

MERS-CoV VP2structuralproteinofcanineparvovirus withreceptorbindingdomain(RBD)of MERS-CoV

RBD(Sprotein) EarlyStage [99]

MERS-CoV

NanovesiclesbasedonstructuralE,M andSproteinsobtainedfrom expressingBm5cells.

Pro:Highbiocompatibilityand

biodegradability,mimicsvirus’sdistribution invivoduetosimilarcharacteristics.

Con:Difﬁculttoscaleupindustrially, dependingonsourcecancause side-effectsorunwantedimmune reactions.

SARS-CoV-2 Plant-based Notclariﬁed PhaseIC.T. Medicago,[101]

Lipid-basedNPs

NPsbasedonalipidbilayerwithahydrophilic

compartmentinsideorassolid-lipidNPs. SARS-CoV

Liposomescomposedofdioleoyl phosphatidylcholine,dioleoyl phosphatidylethanolamine,dioleoyl phosphatidylglycerolacidand cholesterolwithsyntheticpeptide conjugatedtosurface

Nprotein EarlyStage [103]

Pro:Simplepreparation,longphysical stability,sustainedrelease,high controlonsurfacepropertiesandsize, highfeasibilityforindustrialupscaling,

highlysuitableforRNA/DNAdelivery. SARS-CoV, MERS-CoV& SARS-CoV-2

Solid-lipidNPscomposedofionizable lipid,SM-102,withcholesterol, 1,2-distearoyl-sn-glycero-3-phosphocholine(DSPC)and

1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000(DMG-PEG2000)

RNA(encodesforS protein)

PhaseIIIC.T.(for

SARS-CoV-2) Moderna

Con:Limitedloadingcapacity,burst releaseofload.

SARS-CoV-2 Solid-lipidNPscomposedofionizablelipid, ATX,cholesterol,DSPC,DMG-PEG2000

RNA(notclariﬁed) PhaseI/IIC.T. ArcturusTherap. SARS-CoV-2 Lipid-inorganicNPscomposedof

superparamagneticironoxideNPswithina hydrophobicsqualenecore

RNA(encodesS protein)

Pre-clinical Uni.OfWash., [106] SARS-CoV-2 UnspeciﬁedlipidNP Non-replicating

adenovirustype5 (Sprotein)

EarlyStage CanSino

Exosomes

Vesiclesreleasedfromcellsuponfusionof multivesicularbodieswiththeplasma membrane.

SARS-CoV

Exosomesobtainedfrom293Tcells transfectedwithCoVS

protein-expressingplasmid.

Pro:Highbiologicalrelevanceand compatibility,canbemodiﬁedtoexpress speciﬁcproteininmembrane,highlysuitable forRNA/DNAdelivery.

Con:Complexfabricationprocess,notsuitable (yet)forindustrialupscaling,highcostsand laborintensive.

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luminal vesiclesinto theextracellular milieuwhich are further calledexosomes[108].Asthecompositionsofexosomesaresimilar to the body’s own cells, they are widely considered non-immunogenic,whichmakesthethemhighlypromisingforgene ordrugdelivery[109].Furthermore,exosomeshaverecentlybeen demonstrating theirpotentialforthedeliveryof RNAand DNA forvaccinationpurposes[107].Furthermore,similartoliposomes, exosomescanbemodiﬁedtoexpressviralantigens.In 2007,it wasdemonstrated thattransfecting293Tcells withS protein-expressing plasmids resulted in exosomes that included the S proteinintotheircompositions[110].Animalexperimentsusing BL57/6miceshowedinducedneutralizing antibodylevelsupon treatment,clearlyshowinghowexosomescanbeusedasNPb-V. However,asexosomesareproducedbycells,theamountsthatcan befabricatedarecurrentlylimited.

TargetingCoV’slife-cycleandnanomedicinestrategies

TherapiesthatdirectlytargettheCoVslife-cyclecanhave dif-ferenttargetsorsitesofaction,includingblockingtheCoV-specific receptortopreventCoVbinding,thefusionoftheviralenvelopwith thecellmembrane,theviralproteaseswhichamongothers facili-tatethegenerationoftheRdRP,ordirectlyinhibittheRdRP.Inthis subsection,wewilldescribethedifferenttherapeuticscategorized basedontheirspecificsiteofaction,beforediscussingthepotential ofnanocarriersystemstomakethedeliveryofthesetherapeutics morespecificandefficient.

Blockingreceptor-mediatedcellentry(extracellular)

Arguablytheearlieststrategyofinterventionbesides govern-mental lockdownor avaccine,is avoiding thecellentryofthe virusbyblockingtheCoV-specificreceptors.Twodrugcandidates arecurrentlyevaluatedthatcanblocktheentryofCoVsintothe hostcells–Umifenovir(brandname:Arbidol)andCamostat mesy-late.Umifenovir,whichsofarisonlylicensedasabroad-spectrum antiviraldruginRussiaand China,hasshowntoblockdifferent life-cyclestepsoftheinfluenzavirusincludingblockingthevirion attachmenttothecellbyinteractingwiththeviralSprotein.This makes Umifenovir particularly interesting for blocking the cell entryforSARS-CoV,MERS-CoVandSARS-CoV-2[111].Camostat mesylateisanothertherapeuticthatshowspromisingapplication forthetreatmentforSARS-CoV,MERS-CoVandSARS-CoV-2by inhibitingTMPRSS2,eventuallyblockingtheSproteincleavageand thereforethefusionwiththecellmembrane[112].Furthermore, Camostatmesylatehasalsoshownsuitabilitytobecombinedwith nanocarriersfordrugdelivery,makingitapromisingcandidatefor futuretargeteddeliveryapplications[113].Similarlytheinhibition offurincouldalsoblockthecleavageoftheSproteinandthe medi-atedcellentry,howeversuchapproachesaresofarnotinvestigated inclinicaltrials.Nonetheless,inparticularduetotheadditional furin-dependentcleavagesiteinthenovelSARS-CoV-2,the inhibi-tionoffurincouldformapromisingapproachtoblockSARS-CoV-2 cell entry.Starting in 1994,different studies have investigated thepotentialoffurininhibitioninviralinfections,however,such approacheshavenotreachedclinicaltrials[114].Alsoastudywhich successfullyblockedfurininEbolainfections,didnotshowa reduc-tionintheviralreplicationinvitro,demonstratingthatEbloamight havedifferentmechanismsofcellentrybesidesfurin-mediatedS proteincleavage[115].However,asdifferencesbetweenEbolaand SARS-CoV-2mightaltertheefficiencyoftherapeutics,the inhi-bitionoffurinmightformafunctionalapproachinCoVs,which needsfurtherinvestigation.Forinstance,Remdesivir, oneofthe mostpromisingtherapeuticsagainstSARS-CoV-2,which willbe discussedinmoredetailinalatersectionofthisreview,didnot showefficacyinEbolabutpromisingresultsinSARS-CoV-2clinical trials.

Besides drugcandidates,neutralizing monoclonal antibodies (mAbs)form anotherpromising therapeutic to preventcellular entryofthevirus.SincetheoutbreakofSARS-CoVandMERS-CoV severaldifferentantibodycandidateshavebeen developedthat showthecapabilitytoblockcellentrybybindingtodifferentsites oftheSproteinandattenuatebindingoftheviriontothespecific ACE2orDPP4receptorforSARS-CoV/SARS-CoV-2orMERS-CoV respectively.SuchcandidatesforSARS-CoVand MERS-CoVhave beendiscussedindetailelsewhere[24].Duetothehighsimilarities incellentryandbindingsite,mAbsthatshowpromising applica-tionforSARS-CoVmightalsoformapromisingstrategytotarget SARS-CoV-2[116].Inparticular,themAbs80R,CR3014,201and68 haveshowneffectiveblockingofSARS-CoVtotheACE2receptorin smallanimalmodels[117–123].PromisingmAbsforMERS-CoV, whichhaveproventheirperformanceinvivo,include4C2,m336, MERS-GD27,MCA1,CDC2-C2and7D10[124–134].Recently,the humanmonoclonal47D11antibodyhasbeenidentifiedtobindto theSARS2-S-S1BdomainoftheSproteininSARS-CoVand SARS-CoV-2,inhibitingthebindingtothecellreceptorACE2[135].Invitro studieshaveshownthatthismonoclonalantibodycaninhibitviral uptake in Verocells transfected withSARS-CoV and SARS-CoV-2,however,didnotaffecttheuptakeofMERS-CoV.Clinicaltrials involvingsuchneutralizingantibodiesarecurrentlyperformedto ensurepatient’ssafetyandefficacy.Inparticularthecombination ofantibodieswithnanocarriersmightformapromisingstrategy toenhancethestabilityoftheseantibodiesinvivoaswellasallow foratargetingapproachtothesiteofinterest.Suchcombinations havealreadyshownbeneficialcharacteristicsforthetreatmentof differentdiseasescomparedtoantibodiesalone[136].

Anotherapproach,thatiscurrentlyevaluatedinclinicaltrials, is theuseof solublehumanACE2calledAPN01.Recent studies haveshownthathumanrecombinantACE2canblocktheuptakeof SARS-CoV-2inhumanbloodvesselorganoidsandhumankidney organoidsaswellasreducetherecoveryofSARS-CoV-2fromVero cellsbyafactorof1.000–5.000displayingpromisingstrategiesto blockearlystagesofSARS-CoV-2infections[137].Furthermore,as SARS-CoVisemployingtheACE2receptor,theoverallACE/ACE2 balanceisdisruptedleadingtohigherlevelsofangiotensinII(Ang II)inCOVID-19patients,whichgoesalongwithseverelunginjury andARDS[138].APN01hasshownthepotentialtoreduceAngII levelsinthebloodbycoveringthevirusandthereby reconstitut-ingtheACE/ACE2balance[139].Duetothecombinedworkingof APN01itmightformapromisingtherapeuticforuseinSARS-CoV-2 applications.

Blockingcellentryandfusionofthevirusenvelopwiththecell membrane(intracellular)

Chloroquine(CQ)anditsmoresolubleandlesstoxic metabo-lite hydroxychloroquine (HCQ) were ﬁrst used as prophylactic treatmentformalariaandwerelongconsideredapromising treat-ment for SARS-CoV-2 due to its broad spectrum of functions [30,140–146].HoweverduetoseveresideeffectsofCQ/HCQ sev-eralclinicaltrialsfailedandeventuallystudieswerediscontinued bytheWHO[147–149].Despitethisfact,thecombinationofHCQ andnanocarriers,mightreducethefoundsideeffectsandfacilitate theuseofHCQinthetreatmentofSARS-CoV-2.Thecombination ofCQwithSLNsforexamplehasshownpromisingperformancein thetreatmentofCQ-resistantmalariainthepast[150].Meﬂoquine, adrugwhichisconsideredtobesimilartoCQiscurrently evalu-atedasprophylactictreatmentforSARS-CoV-2infections,however, theexactmechanismofactionisnotcompletelyunderstoodwhich limitsitsdirectuse[140].

Otherpotentialdrugsthatareabletoinhibitvirusendocytosis areImatinibandBaricitinib.Imatinibontheonehandis protein-kinaseinhibitorthatinhibitsthebcr-abltyrosinekinase,whichhas beenshowntobeinvolvedinthecellentryandfusionoftheviral

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envelopeandthecellmembraneinhepatitisCandebola,andmight besimilarly involvedin thelife-cycleofCoVs [151–153]. Baric-itinib,ontheotherhand,isa Januskinaseinhibitor,whichwas previouslyusedinthetreatmentofrheumatoidarthritis.Itmight alsobeusedforblockingCoVcytosisduetoitsafﬁliationwiththe AP2-associatedproteinAAK1[154,155].

Blockingviralproteases

Viralproteasesarecrucialforthegenerationofnon-structural viralproteins,suchastheRdRPaswellastransportofviralproteins tothenucleus[9].Ivermectinisananti-parasiticagent,whichhas previouslybeenusedforhumanimmunodeﬁciencyvirus(HIV)and denguevirus[156].ItdissociatestheIMP␣/␤1homodimerrelated withthenucleartransportofviralproteins.Ivermectinadditionally showedtoreduceviralRNAinVerocellsupto5000fold demon-stratingitspotentialfortheuseinCoVs[157].Anothertherapeutic thatwasmentionedinrelationwiththetreatmentofSARS-CoV-2 istheHIVtherapeuticDarunavir,which,however,isnotconﬁrmed aspotentialtreatmentforCoVsandisstillinprematuretrialsto showitspotentialasstatedbythemanufacturerJanssen[158]. InhibitingtheRNA-dependentRNApolymerase

TheRdRPisacrucialproteininthelife-cycleofCoVsandthereby blockingtheRdRPisapromisingstrategytostoptheviral spread-ingwithinthebodyand treatCoVs.Oneofthemostpromising treatmentsagainstSARS-CoV-2andotherCoVs isRemdesivir, a therapeuticdevelopedbyGileadSciences(USA)underthebrand nameGS-5734[159].Althoughitdidnotshowefficacyagainstits originaltargetEbola,itwasproventobesafeforpatientuse,which facilitatedrapid clinicaltestingagainstCoV[160].Furthermore, studieshaveprovenitsefficacytoinhibitthereplicationSARS-CoV andMERS-CoVinprimaryhumanairwayepithelialcellculturesas wellasbeingeffectiveagainstbatCoVsandcirculating contempo-raryhumanCoVs.Itsefficacyhasalsobeendemonstratedinvivo inaSARS-CoVmousemodel[161].Remdesivirhasbeenshownto beeffectiveinvitrousingSARS-CoV-2infectedVerocells.Arecent studyperformedintheUSwasabletoshowarecoverytimeof patientsfrom15daysto11daysinthetreatmentgroupsaswell asadropinpatientmortalityfrom11.6%intheplaceboto8%in thetreatmentgroup,demonstratingitspotentialtoalsotreatthe novelSARS-CoV-2[162].

Favipiravir,atherapeuticdesignedfortreatinginﬂuenza, dis-playsasimilarmechanismofactionasRemdesivirbyinhibiting theRdRP,however,itislessexperimentallysupportedcompared toRemdesivir[163]. Nonetheless, Favipiravirhasbeenrecently approvedbytheNationalMedicalProductsAdministrationofChina forthetreatmentofCOVID-19patientsafteraclinicaltrialwhere Favipiravirwascombinedwithinterferon-␣showedclinical ben-eﬁtsofthedrugcombination[164,165].Furthermore,Tenofovir,a differentRNAtranscriptaseinhibitor,iscurrentlyinevaluationfor theuseaprophylactictreatment[166].

Nanomedicinestrategytotargetpathologicalcells

Asaforementioned,nanocarriersarepromisingforthestable deliveryof drugcandidatestowards target cells avoiding rapid clearance by theimmune system while preventingside-effects [18,64].Duetovarietyofdifferentnanocarriersavailableandthe possibilitytotunethesetoﬁtthedrugloadaswellasthe tar-get,mostoftheaforementioneddrugcandidatesagainstCoVcan beincluded into nanocarriers totarget the lung, preventearly degradationorclearanceaswellasavertsideeffects[167].Infact, differentstudiesofdrug-loadednanocarriershavebeenreportedin thepast,involvingCQ-loadedSLNstotreatCQ-resistancemalaria [150], Tenofovir-loaded PLGA NPs to induce long- acting pre-ventionofHIV vaginal transmission[168] orIvermectin-loaded SLNstofacilitatetransdermaldeliveryforthetreatmentof

sca-bies[169]. Similarlydifferentnanocarriers havebeenevaluated forthedeliveryofantibodies[170,171].Thesestudiesdemonstrate thatthecombinationofcurrentlyevaluateddrugcandidateswith nanocarriersystemsisfeasibleandcapabletoimprovethedrug characteristicsandproperties.Duetothebigvarietyofpossible candidatesanddrug-nanocarriercombinations,thisreviewwill focusonpossibledeliverystrategiesthatarecapabletodelivery drugstowardsCoVinfectedcellsaswellascellsinvolvedinthe COVID-19diseaseprogression.

Targetingthe nasalmucosa. Asaforementioned,themain target cellsofCoVsaremucus-producinggobletcellsandciliatedcells locatedinthenasalmucosa.Targetingthesecellswithnanocarriers isapromisingstrategytodeliverdrugcandidatestotheinfected cells(Fig.7).Aparticularbarrierfoundinthenasal administra-tionoftherapeuticsisthemucus.Themucusforms anaqueous layerontopthemucosalendothelial cells,which based onthe secretionofmucin,ciliaactionandcoughiskeptinconstantflow facilitating rapidclearance ofagents present in themucusfilm [172,173].Additionallythemucuscontainsseveralsecretory anti-bodiesforminga firstlineofdefenseagainstpathogens, which, asaforementioned,makesthenasalmucosa apromising target forvaccinesaimedagainstrespiratoryviruses[174].Althoughthe rapidclearancefromthemucosaisfavorableagainstpathogens,it createsahardtopenetratebarrierfornanocarrierstoreach under-lyingepithelialcells.Inthenose,themucusisreportedtohave athicknessofaround15␮m,includingarapidlyflowingloosely adherentmucuslayerontopofafirmlyadherentslowlyflowing layercoveringtheepithelialcells[175].Clearanceofagentsform themucuscanoccurwithin30min,givingappliednanocarriersa limitedtimetoovercomethebarrierandbeingtakenupbyorpass theepitheliallining[175].Nonethelessseveralmucus-penetrating NPs(MPPs)havebeendevelopedoverthelastyears,asextensively discussedelsewhere[173,175,176].Ageneralinspiration forthe design ofsuchMPPsareviruses,suchasCoVs, thatareableto penetratethemucuswithsimilardiffusionratescompared diffu-sioninwater.Differentstudiessuggestedthatthesurfacecharge andrelatedhydrophilicityaswellasthesizeplayacrucialrolein thepenetrationofvirusesthroughthemucus[175].Furthermore, glycolyzedvirusesorparticlesdisplayenhancedpenetrating prop-erties,indicatingthePEGylation(theconjugationofpolyethylene glycolontoananocarriersurface)canincreasetheefficacyofMPPs andexplainshowCoVs,withtheirheavilyglycolyzedSproteins canpenetratethemucusaswell[177].Generatingnanocarriersfor thedeliveryoftherapeuticsshouldthereforebegreatlyinspired bysuchvirus-likeproperties.Asdescribedaforementioned,viral particlesorliposomescanbetunedintermsofcharge,sizeand surfacechemistry (conjugationofPEGorotherpeptides to cre-ateamuco-inertsurface)toreachthesecharacteristicsfacilitating deliveryoftherapeuticstowardsgobletcellsorciliatedcellsinthe mucosa.Forinstance,nanocarriersmodifiedwiththegoblet cell-targetedpeptide(CSKSSDYQC(CSK)),havedemonstratedenhanced mucus penetrating characteristics, reduced mucus clearance as wellasspecificuptakeintogobletcells.Studiesdemonstratethe CSKpeptideenhancednanocarrieruptakeintheoralmucosafor thedeliveryofinsulinorexenatideforthetreatmentofdiabetes [178–181],aswellasenhanceduptakeofgemcitabineinintestinal gobletcellsforthetreatmentofbreastcancerafteroral adminis-tration[182].Thecombinationofnanocarriersloadedwithdrug candidatesfocusingonthetreatmentofCoVscombinedwithsuch surfacepeptidescouldformapromisingstrategyforthenasal deliv-erytowardsCoV-infectedepithelialcells.

Anadditionalstrategytorapidlyallowtargetingofthemucosal epithelialcellsisdisruptingthemucustoenhancedrugdelivery towardsunderlyingcells.Suchadjuvanttherapiescanbe particu-larlyadvantageousindiseasesincludinga changeinthemucus,

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Fig.7. Strategiestodelivertherapeuticstotargetcellsusingnanoparticles.Schematicrepresentationofdifferentnanocarriersdeliveringtherapeuticstowardsnasal

mucosa(primarytargetsite)aswellassecondarytargetsitesincludingthecentralnervoussystem,intestineorbloodvesselendothelium.Nanocarriersincludemucus

penetratingparticlesandtheircharacteristics,speciﬁcgobletcelltargetednanoparticles,DNAsestrategiestoincreasemucuspenetrationofnanoparticlesanddifferent

strategiestodirectlytargettheACE2/DPP4receptorincludingvirus-likeparticlesbasedoncoronavirusesortargetingligandsinvolvingsyntheticSproteins,targeting

peptidesandantibodies.

rendering it highly viscous, such as cystic ﬁbrosis or chronic obstructivepulmonary disease(COPD). Given thesymptoms of coughandsometimesbloodymucusinCOVID-19patients[183], it is likely that the mucus of these patients present abnormal mechanicalproperties whichcandirectlyaffectthepenetration ofnanocarrierstowardsmucosalepithelialcells.Dornasealfa,a recombinanthumanDNAse(rhDNAse),hasshowntohydrolyze DNAinvolvedinthedensecrosslinksofglycoproteinswithinthe mucus[184].ItisalsocurrentlyevaluatedinCOVID-19patients relatedtoreductionofARDS [185].Dornasealfaisanadjuvant, whichcanbeadministeredinformanaerosol,enablingittobe com-binedwithanti-CoVtherapeuticsandbeingadministeredusingthe nasaldeliveryrouteinformofasprayorsyringe[186].

NanocarriersystemsspecificallytargetingtheACE2/DPP4receptor. AlthoughthenasalmucosaistheprimarytargetsiteofCoVs, sev-eralothersitesandtissuesaffectedbyCoVshavebeenannounced recentlywiththepotentialthatothertissuescanalsobeaffected, whichareyettobediscovered.Targetingthevirus-specific recep-tors,ACE2andDPP4isapromisingstrategytodelivertherapeutics toallCoV-infectedcellsthroughoutthehost’sbody.Efficient target-ingofthesereceptorscanbeachievedbydifferentmeansincluding theconjugationofa targetingagent(peptide orantibody) onto ananocarriersurfaceorbytheuseofaVLPscomprisedof CoV-basedproteins.Usingthevirus’owncharacteristicsandbehavior totargetACE2expressinghasbeendemonstratedrecently,using structuralproteins(M,E,S)fromHCoV-NL63togenerateVLPsthat couldeffectivelytransfectciliatedcellsofthenasalmucosa[187]. Furthermore,theseVLPscouldbeloadedwithafluorescentcargo

demonstrating thecapability ofthese VLPsfor efﬁcientprotein delivery.AlthoughthisissofartheonlyattempttouseaCoV-based VLPforthedeliveryoftherapeuticstowardsinfectedcells,theuse ofVLPmightformoneofthemostpromisingstrategiesforefﬁcient drugdelivery,astheseVLPs,similartothevirus,areabletoescape immuneclearance,penetratethemucusandmightdisplaysimilar characteristicsininfectingnotonlythemaintargetsiteofthelung butalsosecondarytargetsites,thatarealsoaffectedby SARS-CoV-2.

AdifferentstrategytotargettheACE2orDPP4receptorisusing atargetingligand,suchaspeptidesormAbs,andconjugatethese ontothesurfaceofnanocarriers,enablingtheactivetargetingof thesereceptorsasfrequentlyappliedinrecentnanomedicine appli-cations[188,189].Thesepeptidesarespeciﬁcallydesignedtobind toCoV-speciﬁcreceptorssimilartotheactualSARS-CoV, MERS-CoVorSARS-CoV-2.Inrecentstudies,suchpeptidesormAbswere mostly applied toworkas ACE2 or DPP4antagonist and block thefunctionofthereceptortopreventcellentryofCoVsduring theirlife-cycle[190,191].However,theseinhibitorsalsoshowthe potentialtobeusedastargetingligandcombinedwith nanocar-riersto directtherapeuticstowardsinfected cells. In suchway, thesenanocarriermightexertadouble-sidedeffectbydelivering therapeuticstowardsinfectcells,whileblockingthevirusentry onnon-infectedcells. Nonetheless,astheseandpotentialnovel inhibitorsarenotdirectlydesignedastargetingligandsandhave been extensively described elsewhere for ACE2 [190–193] and DPP4[194–197],respectively,thisreviewwillnotfocusonsuch inhibitorsingreaterdetail.Sofar,nostudiesdescribetheuseACE2 orDPP4antagonisticpeptidesormAbsinthecontextoftargeted

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deliveryof drugstowardsSARS-CoV,MERS-CoVorSARS-CoV-2. However,arecentstudydescribesthetargetingoftheangiotensin IIreceptortype1tospecificallydeliverPLA/PLGA-PEGNPstowards mesangialcellsforthepotentialtreatmentordetectionofdiabetic nephropathy.Hereby,angiotensin-Iwascovalentlyboundontothe surfaceoftheNPstoallowbindingtoACEreceptorsofthefirst tar-getcell.ThebindingtoACEallowedforenzymaticprocessingof angiotensin-Itowardsangiotensin-II,whichfurtheralloweduptake oftheseNPsthroughAT1Rmediatedendocytosisbyasecond tar-getcell.Thisstrategywasmainlyinspiredbythecellularuptake ofinfluenzaAviruses,whichfirstundergoectoenzymatic activa-tionofhemagglutininbyonecellwhichfacilitatestheuptakeofthe virusbyasecondarytargetcell.Althoughtheirstudydidnotaimfor ACE-mediatedcellularuptake,thestrategyofusing angiotensin-coatednanocarriersisapromisingstrategytospecificallytarget ACE2expressingcells.Adifferentstrategyisbasedontherecent sequencingoftheSprotein,enablingdeeperunderstandingofthe proteinstructureoftheSprotein,whichfurtherallowstogenerate asyntheticpeptidemimickingthisstructure.ThesyntheticS pro-teincanpotentiallybeconjugatedontothesurfaceofnanocarriers functioningastargetingligand.Studieshavereportedthepotential ofsuchsyntheticSproteinsfortheirantiviralcapabilityin SARS-CoV-2byblockingtheACE2receptorsimilartotheactualvirus [198].Suchsyntheticproteinswouldallowforthetargetingofthe ACE2receptorwithouttheneedforpurificationoftheSprotein fromactualCoVs.

Thesurfacepropertiesofnanocarriershavealsoshownto facili-tatebindingtoCoV-relatedreceptors.Ithasbeendescribedrecently that cationic NPs, in particular polyamidoamines (PAMAMs), bind to ACE2 receptor, subsequently blocking the cleavage of angiotensin,which cancauseARDS [199]. Interestingly,anionic nanocarriersdidnotdisplaysuchbindingproperties, demonstrat-ing how the surface charge of nanocarriers can alsofacilitates speciﬁcreceptorbinding.However,itisunclearwhethersuch char-acteristicswouldallowforthetargetingofACE2-expressingciliated orgobletcellsinthenasalmucosa,ascationicnanocarriersmight notbeabletosuccessfullypenetratemucuslayer.

Thisstudyalsodemonstratesaparticularchallengewhen tar-getingtheACE2receptor.Theblockingofthisreceptorhasshown toreducetheenzymaticcleavageofangiotensin-II,disruptingthe renin-angiotensinsystem(RAS)andeventuallyincreasingitsblood levels,whichsubsequentlycanpromoteARDS[38,200,201]. Addi-tionaltargetingofthisreceptormightfurtherblockACE2receptor levelswhichcanenhancethiscascade.Nonethelessbyapplyinga multilayeredtherapy,suchasforinstanceatreatmentusing ACE2-targeteddeliveryoftherapeuticsfollowedbytheadministrationof thepreviouslydescribedAPN01,asolubleformofACE2,couldavert suchsideeffects,however,thesehypotheticalstrategiesremainto beproven inexperimentalsettings.Anotherpotentialchallenge inthetargetingofACE2orDPP4receptorsforthedirected deliv-eryoftherapeuticstoinfectedcells,isthereducedpresenceand expressionofthesereceptorafterinfectionswithSARS-CoV, MERS-CoVandSARS-CoV-2,whicheventuallymightreducetheoverall amountoftherapeuticsthatcanbedeliveredtowardstargetcells [38,200].Similarly,despitethatDPP4targetingmightallowforthe targeteddeliveryofMERStreatmenttowardsinfectedcells,DPP4 blockingor inhibitionhasshown profound inﬂuenceon T cell-speciﬁcimmunity,downregulatingTcellactivityandmaturation. AlthoughtheexactconsequencesofDPP4blockingontheimmune systemaresofarnotwellunderstood,theprobableside-effects ofDPP4targetedpeptidesormAbsmustbetakeninto considera-tionwhendesigningtargetedsystemsfordeliverytowardsACE2 orDPP4expressingcells.

Despitethesedifferentchallenges,directtargetingofACE2or DPP4mightformapromisingstrategytonotonlydeliver thera-peuticstothemajortargetsitebutalsospeciﬁcallyreachsecondary

sitesofinfections.Theproperinvivoevaluationofsuchstrategiesis crucialtoavertpotentialsideeffectsandensurethetargeted deliv-eryoftreatments.Adifferentstrategytoavertsideeffectsmight bethesearchfordifferentsharedsurfacereceptorsexceptACE2 orDPP4,however,duetothevarietyofaffectedcells,acommon receptorthatdoesnotexertsideeffectsupontargetingmightbe challengingtoﬁnd.

Targetingtheimmunesystemandnanomedicinestrategies

Asdescribed before, SARS-CoV-2 ischaracterized bya rapid progression of thedisease where an exceedingly high number of patientsexperiences severe pneumonia orARDS. In particu-larpatientswithaweakimmunesystembasedonageorother pre-existingconditionsaremorepronetoalethaloutcomeofthe diseasecomparedtoyoungerhealthierpatients.Currently differ-entstrategiesaredevelopedtoinhibittheprogressionofCOVID-19 andreducepatientmortality.Suchstrategiesmainlyfocuson sup-portingtheimmunesystemtoenhancetheclearanceofthevirus fromthebodyortoinhibittheinﬂammatoryresponsecausedby theviralinfectionandrelatedtoARDSandseverelungdamage. Supportingtheimmunesystem

A strategythat is currentlyevaluated for theusein COVID-19patientsistheadministrationof interferonbeta1a(IFN␤),a body-owncytokinewithextensiveantiviralfunctions[202,203]. The administrationof interferon hasbeen investigatedto treat SARS-CoVandMERS-CoVdemonstratingpromisingresultsinvitro andinvivowhich,however,couldnotbetranslatedtoclinical set-tingsinthepast[203–205].Alsotheexactroleofinterferonsin viralinfectionscanbehighlydependentontheinfectiontypeand progressasinterferons arealsoreportedtopromote inﬂamma-toryresponsesandincreasepatientmortality[202].Nonetheless, administrationofIFN␤mightformapromisingstrategyforcertain COIVD-19patients.

Anotherstrategy tofacilitateantibodytherapy inthe target-ingof CoVs, is theuse ofpurified plasma for patientsthat are alreadyrecoveredfromCoVs[206].Theplasmaofsuchpatients, whenprovensafetohavenotracesofthespecificpathogenleft, mightincludesufficientantibodies/immunoglobulinstosupport thebody’sownimmunesystemagainsttheCoVs.Astheplasma fromSARS,MERSofCOVID-19patientswouldincludespecificAbs againsttheseviruses,itmightformapromisingstrategytosupport viralclearancebeforeenteringthehostcellsandwhengiven intra-venously(referredtoasintravenousimmunoglobulins(IVIGs))in combinationwithantiviraldrugscouldformapromisingtreatment againstCoVs.

Inhibitingtheinﬂammatoryresponse

As mentioned before, COVID-19 patients often experience increasedlevelsofinflammatorycytokinesandchemokines(CRS) related toARDS, which forms one of themain reasonsfor the highmortalityofpatients[57,58].Inparticular,ARDSmightalso causeseverelong-lastingdamagetothelungsinvolvingscarring ofthelungtissueandsubstantialreductionofthepatient’squality oflife,whichiswhyatreatmenttoattenuatetheinflammatory response leading to ARDS is a promising strategy to treat the consequencesof aCoVinfection[207].Such treatmentsinvolve cell-basedtreatments,treatmentswithintravenous immunoglob-ulins,generalimmunomodulatorydrugsordrugstargetingspecific pathwaysinvolvedintheCRS.

Besides the use of antibodies or immunoglobulins to tar-get and neutralize CoVs, IVIGs, isolated from healthy donors, have also shown the potential to exert anti-inﬂammatory properties in patients with severe pneumonia by suppressing

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inﬂammatory cells, inhibit phagocytosis and interfering with antibody-dependentcytotoxicity[206,208,209].

A commonstrategy toattenuate theinflammatory response is the use of already known therapeutics that function as immunomodulators.Hereby,itcanbedifferentiatedbetweendrugs thatingeneralshowimmunomodulatorypropertiesordrugsthat targetaspecificcytokinethatisrelatedtoCRS.Therapeuticsthat showimmunomodulatorypropertiescurrentlyevaluatedcomprise Thalidomide[210],Methylprednisolone[209],Fingolimod[211], Ruxolitinib [212], Eculizumab [213], Leukine [214], Colchicine [215],Vafidemstat[216],Melatonin[217],Nivolumab[218], Dex-amethasone[219]orCyclosporineA[220,221].

Targeting specific pathways and cytokines forms another promising, more focused, strategy to treat the inflammatory responsesinpatients.Inparticular,IL-6,IL-1andTNF␣havebeen identified as the key players in CRS and form the most pro-inflammatorycytokinesinthehumanbody [57–59].Recently it hasbeen alsoshown that IL-6 is directly involved in theneed formechanicalventilationofpatientsandpoorsurvival progno-sis[222].SarilumabisamAbfunctiongasaIL-6antagonistthatis currentlyevaluatedinclinicaltrials,whichbindstosolubleaswell asmembraneboundIL-6receptors(sIL-6RandmIL-6R),blocking therelatedreceptormediatedIL-6signaling[223,224].Originally applied in rheumatoid arthritis, Sarilumab is now a promising agentfor thetreatmentof COVID-19patients. Similarly,the IL-6antagonistsTocilizumab,atherapeuticalsousedinrheumatoid arthritis,andSiltuximab,originallyappliedinmulticentric Castle-man’sdisease,arecurrentlyevaluatedfortheirefficacyinCOVID-19 [225].BesidesIL-6,IL-1isalsodirectlyrelatedtoCRSandshown tobehighlyexpressedinCOVID-19patients[226].Anakinraand CanakinumabarebothIL-1antagonists,whicharecurrently inves-tigatedfortheirefficacyandtheirpotentialuseforthetreatmentof COVID-19[227,228].Lastly,targetingTNF␣,acytokinewell-known foritspro-inflammatorypotential,isalsocurrentlyevaluatedin clinicaltrialsforthetreatmentofCOVID-19usingAdalimumab,a TNF␣antagonist,whichhasbeenusedtotreatrheumatoidarthritis orMorbusCrohninthepast[229].

Nanomedicinestrategiestotargetimmunesystem

Besides facilitating targeting of the infected cells in CoVto preventthevirusfromspreadingthroughoutthebody, nanocarri-ersalsoshowpromisingcapabilitiestodeliveranti-inflammatory drugs towards immune cells to avert CRS. Different from the nanocarriers discussed before that aimed to reach the NALT and provoke an immune response towards presented antigens causing immunity,suchnanocarriersare aimedtodeliver anti-inflammatory agentstowardsinflammatory macrophages andT cells,whicharemainlyinvolvedinCRSandblocktheproduction ofIL-6,IL-1,TNF␣and othercytokines.Inparticularthe target-ingofmacrophageshasbeenrecentlyinfocusofcountlessstudies, duetotheinvolvementofmacrophagesininflammatorydiseases suchasrheumatoidarthritis,inflammatoryboweldisease,systemic lupuserythematosus,multiplesclerosisordiabetes,cardiovascular diseases,cardiacdiseasesaswellascancerassummarized else-where[230].Similartotherapeuticsaimedforthetreatmentof CoV- infected cells, several studieshave been described in the pastthatcombinedcurrentlyevaluatedanti-inflammatory thera-peuticswithnanocarrierstoenhancetheirstabilityandresistance todegradation,prolongdrugexposureorallowtargeteddelivery ofthesetherapeutics. Forinstance,Tocilizumab,anIL-6 antago-nist,wascombinedwithhyaluronate-goldNPsforthetreatment ofrheumatoidarthritis[231],dexamethasoneacetatewasloaded intoSLNsforthedeliverytothelung[232],orcolchicine-loaded lipidbilayer-coatedmesoporousNPsforthetreatmentofcancer [233].

Although different strategies to target macrophages have been emerged over the last years, directed targeting of pro-inflammatorymacrophagesremainschallengingandstudiesthat target macrophages with context of the CRS are rare [230]. Nonetheless, a few approaches have demonstrated the use of nanocarriersdesignedtotargetpro-inflammatoryimmunecells. Apromisingtargetforspecificdeliverytowardsmacrophagesis the presence of mannose receptor (also known as CD206) on thesurfaceofsuchmacrophages.Forinstance,NPswere gener-atedcomprisedofmannosylatedbioreduciblecationicpolymers facilitatingthepresenceofmannosereceptorforcellularuptake [234].ThesenanocarriersweredesignedtodeliversiRNAagainst TNF␣expressioninmacrophages,whicheventuallyreducedthe pro-inflammatoryresponseininflammatoryboweldisease. How-ever,mannosereceptorisnotonlyexpressedonpro-inflammatory macrophages,andinfact,displaysoverexpressioninanti- inflam-matorymacrophages aswell.Thishighlightsa challengein the specific targeteddelivery oftherapeutics towardsmacrophages – macrophage specific markers might be sharedby both, pro-and anti-inflammatory markers, making the design of specifi-callytargetednanocarriersparticularlychallenging[230].Different potential surface markers for the targeting of inflammatory macrophages and averting CRS are CD80 or CD86, also being reportedassurfacemarkersforinflammatorymacrophages[235]. Ingeneral,nanocarriersthatareaimedforthedeliveryof anti-inflammatorytherapeuticsforpreventingCRSfacesimilarbarriers asNPb-Vsandotherlung-aimednanocarriersdescribedindetail beforeincludingthemucuslayeroftopofthepulmonarylining aswellastheneedtocrossthepulmonarybarriertoreachthe capillariesandeventuallyinhibitthesecretionofpro-inflammatory cytokinesfrommacrophagesandTcells.

NanomedicinestounderstandCoV’smechanisms

Remarkableforalltheeffortsoffindinganefficienttreatment or vaccineagainstthe novelSARS-CoV-2or therelated disease COVID-19isthefactthatmostdrugscurrentlyinclinicaltrialsare repurposedfromotherdiseasetargetsincludingotherviral infec-tions,suchEbolaorHIV,butalsocancerorrheumatoidarthritis. Withtheincreasingnumberofcasesandrelateddeaths,afast treat-mentwascrucialandtherapeuticsalreadyeffectiveagainstknown pathwaysoralreadyevaluatedforsafetyinclinicaltrialsarethe mostrapidstrategytofindatreatmentagainstSARS-CoV-2 infec-tions.Nonetheless,SARS-CoVandMERS-CoVhavebeenknownfor nearly20years,andsofar,noefficienttreatmentagainstthese dis-easesisavailableandremarkableforthecurrentoutbreakandthe relatedclinicaltrialsisthatnodrugiscurrentlyevaluatedthatis directlyaimedtotreatSARSorMERS.AsSARS-CoVandMERS-CoV onlyhadlimitedcasenumbers,whichledtotheoutbreaksbeing controlledcomparablyfast,fundingforfindingavaccineor treat-mentagainsttheseviruseswaslimitedandeffortstofindavaccine wereshelvedaftergovernmentalfundingwasstopped[236,237]. Nonetheless,thefactthatafternearly2decadesnoefficient treat-mentisavailable,alsodemonstratesthatitisalsocrucialtofurther investigateand understandthevirus, and therelatedprocesses involvingcellular uptake and replication,to design an efficient treatmentdirectlytargetedtoSARS-CoV-2,andpotentiallyother CoVs.Recently,awiderangestudyanalyzingproteininteractions involvedinhostcell-virusresponseshasidentified332possible protein-proteininteractionsofwhich66demonstratedadrugable profile[238].Theseinteractionscanbetargetedby69knowndrugs whichareeitheralreadyFDA-approvedfordifferentdiseasesorin clinicaltrials.Inparticular,therapeuticstargetingmRNA transla-tionandpredictedregulatorsoftheSigma1andSigma2receptors demonstratedpromisingprofilesforthetreatmentofSARS-CoV-2