Extracellular vesicles as a next-generation drug delivery platform.

1 Re iew Manusc ip 1 2 Ex acellula esicles as a nex -gene a ion d ug deli e y pla o m 3 4 Inge Ka in He mann1,2*, Ma hew John And ew Wood3 and G ego Fuh mann4,5,6* 5 1Nanopa icle Sys ems Enginee ing Labo a o y, Ins i u e o Ene gy and P ocess Enginee ing, 6 Depa men o Mechanical and P ocess Enginee ing, ETH Zu ich, Sonneggs asse 3, CH-8092 Zu ich, 7 Swi ze land. 8 2Pa icles Biology In e ac ions, Depa men Ma e ials Mee Li e, Swiss Fede al Labo a o ies o 9 Ma e ials Science and Technology (Empa), Le chen elds asse 5, CH-9014 S . Gallen, Swi ze land. 10 3Depa men o Paedia ics and Ox o d Ha ing on Ra e Disease Cen e, Uni e si y o Ox o d, OX1 11 3QX, Uni ed Kingdom. 12 4Helmhol z Cen e o In ec ion Resea ch (HZI), Biogenic Nano he apeu ics G oup (BION), 13 Helmhol z Ins i u e o Pha maceu ical Resea ch Saa land (HIPS), Campus E8.1, Saa b ücken 66123, 14 Ge many 15 5Depa men o Pha macy, Saa land Uni e si y, Campus E8.1, Saa b ücken 66123, Ge many 16 6P esen add ess: Chai o Pha maceu ical Biology, Depa men o Biology, F ied ich-Alexande -17 Uni e si y E langen Nu embe g, 91058 E langen, Ge many 18 19 *Co esponding au ho s: 20 IKH: phone: +41 58 765 7153, email: [email p o ec ed] 21 GF: phone: +49 68198806 1500, email: [email p o ec ed] 22 23 24 ORCID iDs: Inge K. He mann: 0000-0002-3018-6796; Ma hew Wood: 0000-0002-5436-6011; 25 G ego Fuh mann: 0000-0002-6688-5126 26 27 28 29 2 Abs ac 30 Ex acellula esicle (EV)-based cell- o-cell communica ion is conse ed ac oss all kingdoms o li e. 31 The e is compelling e idence ha EVs a e in ol ed in majo (pa ho)physiological p ocesses, 32 including cellula homeos asis, in ec ion p opaga ion, cance de elopmen , and ca dio ascula 33 diseases. Va ious s udies sugges ha EVs ha e se e al ad an ages o e con en ional syn he ic 34 ca ie s, opening new on ie s o mode n d ug deli e y. Despi e ex ensi e esea ch, clinical 35 ansla ion o EV-based he apies emains challenging. He e, we discuss he uniqueness o EVs along 36 wi h c i ical design and de elopmen s eps equi ed o exploi hei ull po en ial as d ug ca ie s, 37 including loading me hods, in-dep h cha ac e isa ion, and la ge-scale manu ac u ing. We compa e he 38 p ospec s o EVs o hose o well-es ablished liposomes and p o ide guidelines o di ec he p ocess o 39 de eloping esicle-based d ug deli e y sys ems. 40 41 3 As he ield o a ge ed d ug deli e y has expanded, nano echnology has con ibu ed subs an ially o 42 he de elopmen o sma ca ie s in ecen decades.1 In pa icula , lipid-based nanoca ie s o e a 43 e sa ile pla o m o d ug encapsula ion, which has led o clinical ansla ion o se e al o mula ions. 44 In addi ion o syn he ic nanoca ie s, cell-de i ed ex acellula esicle (EV)-based ca ie sys ems 45 ha e a ac ed conside able in e es .2 46 EVs a e a he e ogeneous g oup o small, lipid-bound nanopa icles ac ing as key media o s o many 47 (pa ho)physiological p ocesses.3 They a e also being explo ed o he deli e y o he apeu ic payloads 48 o speci ic cells o issues, ha nessing hei in insic issue homing capabili ies.4 F om a d ug deli e y 49 pe spec i e, EVs a e compa able o liposomes, gi en ha bo h a e phospholipid-based. Howe e , EVs 50 a e assembled om a complex mix u e o a ious lipids and su ace and memb ane p o eins; some o 51 hese componen s aid issue a ge ing, while o he s ensu e minimal non-speci ic in e ac ions.5,6 These 52 unique p o ein-deco a ed phospholipid esicles ha e been pos ula ed o con ain he speci ic ba codes 53 needed o ind hei a ge bo h locally and a dis an si es. Despi e ex ensi e esea ch, he supe io i y 54 o EV-based d ug deli e y o e deli e y ia enginee ed nanoca ie s, such as liposomes, and he 55 associa ed isk-bene i a io emain ma e o deba e.7 56 He e, we c i ically discuss he p ospec s o EVs as d ug deli e y ehicles and as nex -gene a ion 57 he apeu ics. We ou line he ad an ages o EVs o e s anda d deli e y me hods, discuss cu en 58 obs acles ela ed o hei clinical and indus ial ansla ion and highligh syne gies wi h o he eme ging 59 ields, such as cell he apeu ics (EVs a e some imes conside ed ‘cell- ee cell he apeu ics’). We also 60 p opose a colou code guideline ega ding expe imen al equi emen s and scien i ic needs o acili a e 61 he de elopmen o EVs as d ug ca ie s o e alua e hei deli e y e icacy and allow benchma king 62 agains al e na i es. 63 64 Uniqueness o ex acellula esicles biology and unc ion 65 Composi ion o EVs. EV sec e ion appea s o be an e olu iona ily conse ed p ocess p esen 66 h oughou all kingdoms o li e.8 Rega ding undamen al biology, EV esea ch ocuses on 67 unde s anding he biogenesis and elease o hese na u al ca ie s and hei a e upon in e ac ion wi h 68 4 a ge cells. This also comp ise he geno ypic and pheno ypic esponses ha EVs induce and he 69 mechanisms by which EVs media e cell- o-cell communica ion.5,9 70 Se e al sub ypes o EVs, including exosomes, ec osomes, mic o esicles, memb ane esicles, and 71 apop o ic bodies, ha e been iden i ied.10 These EVs ha e been isola ed om a ious sou ces, 72 including mammalian and p oka yo ic cell cul u es, blood plasma, bo ine milk, and plan s.8 Each EV 73 subpopula ion may be de i ed ia dis inc biogenesis pa hways, and because hei p ecise biogenic 74 o igin is impossible o asce ain in mos cases, a comp ehensi e cha ac e isa ion o he esicles is 75 c ucial. In addi ion, di e en EV o mula ions may ha e subs an ially di e en size dis ibu ions; hus, 76 s anda dised cha ac e isa ion is challenging.11 The gene al ecommenda ion in he ield is o use ‘EVs’ 77 as a gene al e m. Impo an ly, he gene al concep o ‘ he EV’ does no exis —cu en ly, he e m 78 ‘EV’ comp ises a he e ogeneous popula ion, as indica ed in he Minimal In o ma ion o S udies o 79 Ex acellula Vesicles (MISEV) guidelines.12 P o eomic e idence sugges s ha an EV co e p o ein 80 signa u e (e.g., CD63, CD9, o CD81) o highly exp essed esicula p o eins is commonly sha ed 81 be ween EVs o di e se pa en cell o igins.13 Va ious e aspanins a e commonly used as molecula 82 ma ke s o EVs. In con as o he p e ious MISEV guidelines, he e a e no ypical EV ma ke s ha 83 need o be iden i ied on EVs, bu ca e ul disc imina ion o EVs om con aminan s, such as p o ein 84 agg ega es and i uses, is impo an . To add an addi ional laye o complexi y, esicles s ill ca y 85 pa en cell-speci ic signa u es, which a e c ucial componen s pe mi ing a ge cell in e ac ions in 86 dis inc ly di e en manne s.14 In addi ion o he co e signa u e o highly exp essed and highly en iched 87 esicula p o eins, o he ypically low-abundance and less-en iched p o ein componen s a e p esen ; 88 hese p o eins e lec he speci ic pa en cell o igin o he EVs and may also a y depending on he 89 na u e and biogenesis o di e en EV subpopula ions.15 F om a d ug deli e y pe spec i e, his 90 complexi y needs o be unde s ood ia comp ehensi e (mul i)omics s udies16 and add essed in all 91 cha ac e isa ion and p oduc ion p ocesses (Fig. 1). He e, we elucida e he assembly o EVs on he 92 cellula /molecula le el and hei media ion o selec i e in e cellula signalling ac i i ies o ex end 93 biomedical esea ch.17 94 Up ake and biological ole o EVs Unde physiological condi ions, EVs a e signal ca ie s in ol ed in 95 he homeos asis o se e al p ocesses and o e en s du ing cell de elopmen , e.g., cell di e en ia ion.18 96 5 EV-media ed c oss- alk may occu unidi ec ionally o ecip ocally, i.e., one cell sends in o ma ion o 97 ano he wi h o wi hou ecip ocal signal ansmission om he ecipien cell, espec i ely, o e en ia 98 sys emic communica ion, du ing which EVs a ic o a ious issues and o gans. This in e ac ion may 99 in ol e no only he elease and deli e y o EV ca go bu also cell su ace in e ac ions and a ge cell 100 modula ion, such as immune cell ac i a ion by majo his ocompa ibili y complex-pep ide in e ac ions. 101 The mechanisms by which EVs a e aken up by hei a ge cells a e s ill poo ly unde s ood, and 102 examples om he li e a u e a e o en speci ic o a ce ain ype o esicle.5 Cu en ly known cellula 103 en y ou es o EVs ange om ecep o -media ed endocy osis, lipid a in e ac ions, cla h in 104 in e ac ions, phagocy osis, mac opinocy osis, and possibly di ec usion.9 Like many o he 105 nanoca ie s, EVs a e aken up in o endosomes need o escape he endosome o elease hei ca go in o 106 he cy osol. Endosomal escape is associa ed wi h deg ada ion in acidic compa men s o he lysosomal 107 pa hway, which could impai he in eg i y o EV ca goes.19 Al hough EVs we e ini ially pos ula ed o 108 be an unp eceden ed ou e o di ec cell memb ane usion and cy osolic deli e y,20 esicle up ake has 109 been con i med o be a e y complex mechanism, which equi es mo e in-dep h e alua ion explo ing 110 subcellula analyses based on high- esolu ion mic oscopy o no el li e-cell epo e s.21 On he o he 111 hand, he biological e ec s induced by EVs a e cu en ly well known. Du ing oncogenesis, umou 112 cells inc ease hei yield o EVs, allowing no only he modula ion o su ounding heal hy cells, 113 immune cell dys egula ion, and umou p oli e a ion bu also communica ion wi h dis an issues, e.g., 114 du ing angiogenesis.22 Glioblas oma cells we e shown o sec e e EVs capable o immunosupp ession 115 by blocking T cell ac i a ion and ecep o s imula ion.23 Mo eo e , widely used cy o oxic d ugs, such 116 as axanes, may also induce shedding o EVs wi h p o-me as a ic p ope ies.24 Al hough he ole o 117 EVs in umou biology has been in es iga ed ex ensi ely, he de elopmen o new ools o ea men 118 and diagnos ics is s ill hampe ed by he absence o umou -speci ic EV ma ke s. 119 A compa able modula o y ole o EVs has been obse ed in he p og ession o esis ance o in ec ions. 120 In he con ex o i al in ec ions, some EVs may ca y i al p o eins om in ec ed cells and ollow 121 compa able biogenesis pa hways.25 Fu he mo e, bac e ia u ilise EVs o he ansmission o esis ance 122 genes and i ulence ac o s,26 which has spa ked in e es in he de elopmen o bac e ial esicles o 123 accina ion applica ions.27 Bac e ial EVs om non-pa hogenic o p obio ic bac e ial sou ces may also 124 6 be ha nessed as po en ial EV-based deli e y ca ie s, and hei p oduc ion may be eadily scalable by 125 cul i a ion o EV-p oducing bac e ia in small e men e s.28,29 This is a p omising a enue o he 126 manu ac u ing o EVs wi h no el unc ionali ies and in conjunc ion wi h bioma e ials.30,31 Howe e , 127 immunogenici y equi es mo e de ailed e alua ion o bac e ial esicles han o mammalian EVs 128 owing o he po en ial p esence o lipopolysaccha ides, as ecen ly discussed in de ail.32 129 130 Ex acellula esicle-based d ug ca ie s 131 De elopmen . Wi h he de elopmen o new analy ical ool, i has been ound ha many p e iously 132 applied isola ion echniques a e no speci ic o EVs and lead o he inclusion o con aminan s. 133 Me hods a e cons an ly e ined, bu hey o en expose he limi a ions in he ield, making i di icul 134 o new esea che s o ollow p og ess in he s a e-o - he-a me hods. Fo e e y d ug nanoca ie , a 135 comp ehensi e physico-chemical cha ac e isa ion and i s in e ac ions in biological en i onmen s mus 136 be in es iga ed o he apeu ic de elopmen . While liposomes ha e been ex ensi ely e alua ed o 137 e icacy and biocompa ibili y bo h in i o and in i o, me hodologies well adap ed o he conside ably 138 mo e complex EVs a e lacking. These na u al esicles a e assembled and packaged in a cell-speci ic 139 manne , e.g., cance -de i ed EVs, ca y molecula in o ma ion dis inc om ha ca ied by s em cell- 140 o blood cell-de i ed EVs. While challenging om he pe spec i e o d ug ca ie de elopmen , hese 141 p ope ies make EVs a p omising bioma ke o liquid biopsies in se e al applica ions.33 142 In egene a i e medicine, EVs de i ed om mesenchymal s em cells (MSCs) a e al eady unde 143 clinical assessmen 34 o u u e use in nanodeli e y (Table 1). S em cell-de i ed EVs can induce 144 immune cells o unde go modula ion om an ac i a ed in lamma o y s a e o a ole an egula o y 145 s a e. Some o he s a egies used o s imula e EV shedding and enhance yield can also be used o 146 MSCs. N-me hyldopamine and no epineph ine induced an inc ease in MSC-de i ed EV p oduc ion 147 wi hou al e ing hei modula o y capaci y.35 O he app oaches apply physical s imuli such as pH 148 a ia ions o low oxygen condi ions, bu hei long- e m e ec on he physiological p ope ies o EVs 149 needs o be e alua ed. In a mu ine wound healing model, MSC-EVs we e associa ed wi h sec e ion o 150 an in e leukin-1 ecep o an agonis and induced apid gingi al healing.36 Compa able e ec s ha e 151 been shown o sys emic applica ion o MSC-EVs in pa ien ials, which has un o una ely led o he 152 7 use o ‘exosome’ p oduc s in unapp o ed applica ions. The Food and D ug Adminis a ion ecen ly 153 s a ed ha se ious ad e se e ec s we e expe ienced by pa ien s in Neb aska ea ed wi h unapp o ed 154 p oduc s ma ke ed as con aining exosomes37. The agency emphasised ha he e a e cu en ly no 155 egula o y app o ed EV p oduc s and ha some clinics “decei e pa ien s wi h unsubs an ia ed claims 156 abou he po en ial o hese p oduc s o p e en , ea o cu e a ious diseases o condi ions”. 157 Impo an ly, any he apeu ic applica ion o EVs equi es anspa en epo ing o da a on esicle 158 manu ac u ing and cha ac e isa ion, sui able quali y con ol p o isions, p eclinical sa e y, and 159 e icacy.38 Mo eo e , a a ional clinical ial design and egula o y moni o ing a e impo an o ensu e 160 pa ien sa e y, as ecen ly indica ed by he in e na ional socie ies on s em cells and EVs.39 To suppo 161 he use o MSC-EVs, unc ional assays ha allow in i o-in i o co ela ion o he he apeu ic 162 po ency o di e en s em cell p epa a ions mus be de eloped.40 Despi e hese ca ea s, ongoing e o s 163 o p oduce EVs om MSCs unde good manu ac u ing p ocess-like condi ions41,42 and o design 164 upscaling app oaches43 will be ins umen al o hei de elopmen as d ug ca ie s. 165 8 Table 1. Ongoing clinical ials wi h ex acellula esicles 166 # Name* S a us Condi ion Type o ex acellula esicles§ Loca ion NCT numbe S em cell-de i ed ex acellula esicles 1 A Clinical S udy o Mesenchymal P ogeni o Cell Exosomes Nebulize o he T ea men o Pulmona y In ec ions Rec ui ing; Phase 1/2 D ug- esis an in ec ions Mesenchymal s em/p ogeni o cell-de i ed exosomes Shanghai, China NCT04544215 2 E ec o Mic o esicles and Exosomes The apy on β-Cell Mass in Type I Diabe es Melli us (T1DM) Unknown s a us Diabe es melli us ype 1 Mesenchymal s em cell- de i ed exosomes Sahel, Egyp NCT04213248 3 E alua ion o Sa e y and E iciency o Exosome Inhala ion in SARS- CoV-2 Associa ed Pneumonia. En olling by in i a ion; Phase 1/2 SARS-CoV-2 pneumonia Mesenchymal s em cell- de i ed exosomes Sama a, Russia NCT04213248 4 A Pilo Clinical S udy on Inhala ion o Mesenchymal S em Cells Exosomes o he T ea men o Se e e No el Co ona i us Pneumonia Comple ed; Phase 1 SARS-CoV-2 pneumonia Mesenchymal s em cell- de i ed exosomes Shanghai, China NCT04213248 5 Sa e y and E iciency o Me hod o Exosome Inhala ion in COVID-19 Associa ed Pneumonia En olling by in i a ion; Phase 2 SARS-CoV-2 pneumonia Mesenchymal s em cell- de i ed exosomes Sama a, Russia NCT04602442 6 E ec o UMSCs De i ed Exosomes on D y Eye in Pa ien s Wi h cGVHD Rec ui ing; Phase 1/2 D y eye Umbilical mesenchymal s em cell- de i ed exosomes Guangzhou, China NCT04213248 7 MSC-Exos P omo e Healing o MHs Rec ui ing; ea ly Phase 1 Macula holes Mesenchymal s em cell- de i ed exosomes Tianjin, China NCT04213248 8 A Tole ance Clinical S udy on Ae osol Inhala ion o Mesenchymal S em Cells Exosomes in Heal hy Volun ee s Rec ui ing; Phase 1 Sa e y and ole ance s udies Mesenchymal s em cell- de i ed exosomes Shanghai, China NCT04213248 9 Allogenic Mesenchymal S em Cell De i ed Exosome in Pa ien s wi h Acu e Ischemic S oke Comple ed; Phase 1/2 Ce eb o ascul a diso de s Mesenchymal s omal cell- de i ed exosomes Teh an, I an NCT03384433 10 E alua ion o Adipose De i ed S em Cells Exosomes in T ea men o Pe iodon i is Rec ui ing; ea ly Phase 1 Pe iodon i is Adipose-de i ed s em cell- de i ed exosomes Cai o, Egyp NCT04213248 Allogenic and au ologous ex acellula esicles 9 Table 1. Con inued 167 # Name* S a us Condi ion Type o ex acellula esicles§ Loca ion NCT numbe 11 Sa e y and E icacy E alua ion o Allogenic Adipose MSC-Exosomes in Pa ien s wi h Alzheime 's Disease Rec ui ing; Phase 1/2 Alzheime ’s disease Allogenic adipose mesenchymal s em cell- de i ed exosomes Shanghai, China NCT04213248 12 A Clinical S udy o Mesenchymal S em Cell Exosomes Nebulize o he T ea men o ARDS No ye ec ui ing; Phase 1/2 Acu e espi a o y dis ess synd ome Allogeneic human mesenchymal s em cell- de i ed exosomes Ruijin, China NCT04602104 13 MSC ex acellula esicles in Dys ophic Epide molysis Bullosa No ye ec ui ing; Phase 1 Dys ophic epide molysis bullosa Allogeneic mesenchymal s em cell- de i ed ex acellula esicles Aegle The apeu ics NCT04173650 14 E ec o Plasma De i ed Exosomes on Cu aneous Wound Healing En olling by in i a ion; ea ly Phase 1 Ulce Au ologous exosome- ich plasma Kumamo o, Japan NCT04213248 O he cells o ex acellula esicle sou ces 15 COVID-19 Speci ic T Cell De i ed Exosomes Ac i e; Phase 1 SARS-CoV-2 pneumonia T cell-de i ed exosomes Kayse i, Tu key NCT04213248 16 Ex acellula Vesicle In usion The apy o Se e e COVID-19 No ye ec ui ing; Phase 2 SARS-CoV-2 pneumonia, acu e espi a o y dis ess synd ome Bone ma ow de i ed ex acellula esicles Di ec Biologics NCT04493242 17 Edible Plan Exosome Abili y o P e en O al Mucosi is Associa ed wi h Chemo adia ion T ea men o Head and Neck Cance Ac i e; Phase 1 Head and neck cance , o al mucosi is G ape exosomes and en anyl pa ch Louis ille, USA NCT04213248 D ug loaded ex acellula esicles 18 iExosomes in T ea ing Pa icipan s wi h Me as a ic Panc eas Cance wi h K asG12D Mu a ion No ye ec ui ing; Phase 1 Me as a ic panc ea ic adenoca cino ma, panc ea ic duc al adenoca cino ma Mesenchymal s omal cell- de i ed exosomes loaded wi h siRNA agains K asG12D Hous on, USA NCT04213248 19 S udy In es iga ing he Abili y o Plan Exosomes o Deli e Cu cumin o No mal and Colon Cance Tissue Ac i e; Phase 1 Colon cance Plan exosomes loaded wi h cu cumin Louis ille, USA NCT04213248 20 T ial o a Vaccina ion wi h Tumo An igen-loaded Dend i ic Cell- de i ed Exosomes Comple ed; Phase 2 Non-small cell lung cance Dend i ic cell- de i ed exosomes loaded wi h an igen Villejui , F ance NCT04213248 *T ials cu en ly lis ed on clinical ials.go ; o consis ency, ial names a e indica ed as lis ed on clinical ials.go . 168 §The ype and/o sou ce o ex acellula esicles a e indica ed as gi en by he s udy sponso and we e no e i ied ega ding 169 sui able cha ac e isa ion (e.g., exosomes and mic o esicles). 170 16 homing p ope ies o EVs and on po en ial immunogenic and oncogenic conside a ions. Addi ionally, 344 gene ic s abili y, hos cell impu i ies (such as pa hogens, and especially i uses) and EV yields play a 345 majo ole in he pa en cell selec ion p ocess.81 Once selec ed, hese hos cells a e cul u ed o 346 e icien ly yield EVs wi h he app op ia e pheno ype. Sugges ed me hods include mul i-laye ed cul u e 347 lasks, bio eac o s, and hollow ib e ca idges. Fo small-scale manu ac u ing, cells can be expanded 348 in shake lasks, spinne s, olle bo les, wa e bags, o bio eac o s. Fo la ge-scale cell cul u e, cells can 349 be g own in s ainless s eel bio eac o s (up o 20,000 L scale), pla o m- ocke wa e bags (up o 500 L 350 scale), o e en in disposable bio eac o s (up o 2,000 L scale).81 F om a p ocess sa e y pe spec i e, 351 closed sys ems a e p e e ed; howe e , hese sys ems a e mo e di icul o moni o han open 352 sys ems.42 Gene ic d i and con amina ion need o be moni o ed closely, ollowing p o ocols used in 353 he plan s p oducing biologicals and cell he apies. Accumula ing e idence indica es ha bo ine milk-354 de i ed EVs may be a alid al e na i e sou ce o ob aining la ge amoun s o biocompa ible esicles. 355 Al hough easibili y s udies using milk EVs as d ug ca ie s a e unde way,82 la ge-scale isola ion o 356 pu e esicles om complex milk s ills needs op imisa ion.62 357 Op ional endogenous d ug loading. Pa en cells may be enginee ed o boos EV p oduc ion and/o 358 yield EVs wi h enhanced p ope ies. Se e al me hods o load EVs wi h di e en molecules ha e been 359 expe imen ally e alua ed.83 These me hods include (i) endogenous echniques in which he EV-360 p oducing cells also equip esicles wi h d ug ca go o modi ied s uc u al p o ein/RNA componen s o 361 (ii) exogenous app oaches in which d ugs a e loaded in o EVs pos isola ion. The endogenous loading 362 echnique has a lowe deg ee o complexi y i he p oduce cells di ec ly shed EVs con aining he 363 desi ed molecule.84 Endogenous loading app oaches ha e also been used o he encapsula ion o 364 nanopa icle-based d ugs in o EVs.85 Because he loading e iciency is ypically limi ed, cells may be 365 gene ically enginee ed o p oduce EVs con aining he desi ed ac i e componen s. Howe e , his 366 app oach is limi ed o biologically accessible d ugs, such as nucleic acid and p o ein d ugs. Recen ly, 367 an op ogene ically enginee ed exosome sys em ha in eg a es a blue-ligh esponsi e memb ane 368 module o con ollable p o ein–p o ein in e ac ions o encapsula e la ge quan i ies o an i-369 in lamma o y p o eins in o EVs was p esen ed.86 While highly in e es ing, gene ic enginee ing o EV-370 p oducing cells esul s in mo e complex p oduc ion upscaling han isola ion o esicles om naï e 371 cells and is less elabo a e han pos -isola ion enginee ing. 372 17 373 EV Ha es ing and Enginee ing 374 P oduc sepa a ion and cha ac e isa ion o p epa ed EVs. Fo EV isola ion, he p ima y sepa a ion o 375 p oduc s om cells can be accomplished by well-es ablished p ocedu es o he isola ion o biologics, 376 including cen i uga ion, dep h il a ion (mechanical sie ing and adso p ion) o angen ial low (c oss 377 low) il a ion . While se e al s a egies o EV isola ion and pu i ica ion, such as di e en ial 378 ul acen i uga ion (dUC), p ecipi a ion, size exclusion ch oma og aphy , a ini y ch oma og aphy, and 379 angen ial low il a ion, ha e been e alua ed, he e is no consensus on an app op ia e EV isola ion 380 echnique o la ge-scale manu ac u ing.87 This lack o consensus is mos ly because some p ocedu es 381 may nega i ely a ec he in eg i y and quali y o EVs. Addi ionally, yield and p oduc pu i y a y 382 among me hods and epo s, and he o e all EV pu i y gene ally appea s o be low.88 Mo eo e , o 383 he e ogeneous EV popula ions, he isola ion me hod may lead o selec i e isola ion o one speci ic 384 subpopula ion wi h highe o lowe biological ac i i y han he o al popula ion. Densi y g adien 385 cen i uga ion and dUC o e a decen comp omise in e ms o yield and pu i y. dUC is b oadly 386 accep ed as a common me hod o ob ain ‘pu e’ EVs, especially when using chemically de ined media 387 wi hou oe al bo ine se um. Howe e , ca e ul e alua ion o a e ac s in oduced by he cul u e 388 medium is pi o al, as i was ecen ly shown ha co-p ecipi a ion o nucleic acid impu i ies is possible 389 e en unde hese condi ions.89 In ac , ecen s udies sugges ha ul a il a ion and size exclusion 390 ch oma og aphy (alone o in combina ion) ou pe o m dUC in e ms o bo h yield and pu i y.90,91 391 Howe e , depending on he equi ed EV pu i y, o hogonal me hods ha a ge di e en EV 392 cha ac e is ics may need o be in eg a ed a he expense o yield.89 Ano he in insic p oblem a ises 393 because mos o he cu en ly used pu i ica ion echniques we e o iginally de eloped o he 394 pu i ica ion o i uses,42,92 which a e amongs he mos c i ical po en ial con aminan s o EVs. 395 Recen ly, Ba one e al. comp ehensi ely analysed he isks, cos s, and implica ions o i al 396 con amina ion in biological manu ac u ing.93 Vi us isk mi iga ion s a egies o gene al 397 biopha maceu ical manu ac u ing a e based on h ee ene s: (i) p e en ion o i al en y by selec ing 398 low- isk s a ing and aw ma e ials and using manu ac u ing con ols; (ii) es ing o in-p ocess 399 ma e ials o ensu e hey a e ee o i us and enable lo ejec ion; and (iii) clea ance o i al 400 con aminan s ( ia inac i a ion and/o emo al) om he p oduc . While bo h ca e ul selec ion o cells 401 18 and aw ma e ials and es ing (based on polyme ase chain eac ion o in i o i us assays) can educe 402 isk, he unique size o EVs makes he inac i a ion and/o emo al o i al con aminan s e en mo e 403 challenging o EVs han o o he biopha maceu ical p oduc s. Cu en s a egies a e based on a ini y 404 a he han size exclusion. Ex ensi e mul i-s ep downs eam p ocessing may, howe e , c i ically o ce 405 manu ac u ing cos s o inc ease. In addi ion o he challenges associa ed wi h he comple e 406 cha ac e isa ion o con en s (including con aminan s), he spa ial and con o ma ional o ganisa ion o 407 he cons i uen s is ex emely challenging o assess wi h exis ing analy ical echniques. E en small 408 changes in composi ion o a chi ec u e can signi ican ly a ec e icacy and sa e y. The use o 409 unc ional ac i i y assays is o u mos impo ance because isola ion me hods and he p esence o o he 410 EV popula ions o con aminan s may decisi ely in luence p oduc ac i i y and o - a ge e ec s. 411 Exogenous d ug loading. Fo clinical EV ansla ion, ep oducible and echnologically accessible 412 me hods a e needed o load hem wi h he desi ed d ugs.94 While loading me hods o liposomes ha e 413 been op imised and applied in indus ial p oduc ion, such se ings o EVs a e s ill lacking. Exogenous 414 loading me hods wo k passi ely by he associa ion o d ugs wi h he lipid bilaye memb ane a e 415 incuba ion,83 by a aching he apeu ics o he EV su ace,95 and by mechanical o chemical echniques 416 o ansien ly open he EV memb ane o allow di usion o compounds in o he esicle. The mos 417 common app oaches o empo a ily pe meabilize he memb ane include sonica ion, elec opo a ion, 418 saponin ea men , and passi e incuba ion.96 The ad an ages and disad an ages o each app oach 419 depend on he expe imen al se ings, he ypes o d ugs and sou ce o EVs, and hey can be scaled 420 (Fig. 4c). Passi e incuba ion is a e y simple me hod in which pu i ied EVs a e incuba ed wi h d ugs 421 o allow inco po a ion in o he esicle memb ane. Many ea ly loading p o ocols ollowed his me hod 422 because i exhibi s excellen pe o mance o inco po a ion o hyd ophobic compounds such as 423 cu cumin.97 Howe e , he s abili y o d ugs loaded by passi e inco po a ion ac oss he EV memb ane 424 is s ill unclea . Fo hyd ophilic compounds, loading may be enhanced by he addi ion o saponin, 425 which has been shown o be e ec i e o la ge p o eins.98 Saponins a e mild su ac an s ha induce 426 ansien memb ane des abilisa ion and may also a ec biomolecules; hus, ca e ul pu i ica ion is 427 needed when saponins a e used on a la ge scale. Mechanical me hods o pe meabilising he EV 428 memb ane, such as elec opo a ion o sonica ion, ha e been shown o be success ul o bo h small 429 molecules and mac omolecules.50 E en hough hese me hods can be scaled up, hei po en ial 430 19 in luence on p o ein and nucleic acid d ugs equi es ca e ul conside a ion.99 In addi ion o conce ns 431 wi h main aining he s abili y o biomac omolecules, he size o he d ugs poses ano he challenge 432 du ing EV-loading p ocedu es. La ge enzymes >200 kDa ha e been success ully loaded in o EVs 433 using saponin p e ea men .98,100 As he size o nucleic acids ha may be encapsula ed exogenously 434 in o EVs is also limi ed, a cell nanopo a ion me hod o la ge-scale p oduc ion o unc ional EVs has 435 been de eloped.101 EV yield and mRNA loading we e enhanced by his me hod; howe e , he equi ed 436 addi ional s eps o ans ec ion and elec ical s imula ion ende i s indus ial adop ion ela i ely 437 di icul . 438 Recen ly, an al e na i e me hod based on liposome usion has been p oposed.102 Liposomes con aining 439 usogenic lipids we e incuba ed wi h EVs, and he ca go o he syn he ic liposomes was me ged wi h 440 ha om he EVs. Such an app oach may pa e he way o e icien loading o la ge molecules 441 wi hou comp omising he EV memb ane.95 442 443 Downs eam 444 Pu i ica ion. In addi ion o he EV isola ion and pu i ica ion s eps p io o d ug loading, addi ional 445 pu i ica ion s eps may be necessa y o emo e he ee d ug and exclude he po en ial con aminan s 446 in oduced du ing pos -p ocessing (Fig. 4b and d). Magne ic immunoa ini y pu i ica ion has gained 447 inc easing a en ion owing o he high pu i y o he ob ained p oduc s. Howe e , low yields a e 448 gene ally ob ained, al hough heo e ical yields may be unde es ima ed due o con aminan s. 449 Quali y con ol—(minimal) cha ac e isa ion o enginee ed EVs. Fo ou ine manu ac u ing and 450 p oduc moni o ing, c i ical quali y a ibu es need o be de ined. Me hods o assess hese a ibu es 451 may include e alua ions o pa en cell p ope ies (e.g., e alua ion o iabili y and su ace ma ke 452 exp ession o assess he pheno ype), EV cha ac e is ics (e.g., e alua ion o quan i y, size, and su ace 453 ma ke exp ession), assessmen o mic obial con amina ion (e.g., de ec ion o endo oxin and 454 mycoplasma) and (applica ion-speci ic) unc ional ac i i y.42 Fo assessmen o ba ch- o-ba ch 455 a ia ions, we sugges he use o a concep simila o ha applicable o biosimila s— he analy ical 456 cha ac e is ics o he p oduc s should be highly simila o hose o he e e ence p oduc . While non-457 cell cul u e me hods (such as isola ion om plasma o milk) o e s in e es ing al e na i es o cell 458 cul u e and access o po en ially la ge amoun s o EVs, hese sou ces con ain EVs o igina ing om 459 20 many cell ypes103 ha canno be sepa a ed easily. The e o e, cha ac e isa ion o he unc ional ac i i y 460 o EVs is e en mo e c ucial in quan i ying on- a ge and o - a ge e ec s. 461 462 Fo mula ion and shel li e. Recen s udies ha e ocused on he o mula ion and s o age condi ions 463 (e.g., –80 °C s. 4 °C) o EVs by assessing he size, cha ge, and numbe o EVs, bu s ong 464 co ela ions ha e no been ound.95,104 S o age a 4 °C has been shown o cause agg ega ion and 465 damage o he EV s uc u e.105 Mo eo e , e en hough he size and numbe o EVs emained 466 unchanged a −80 °C, al e a ions in biological ac i i y we e de ec ed.106 Lyophilisa ion has been 467 in es iga ed as an al e na i e o long- e m s o age; howe e , i s impac on esicle in eg i y du ing 468 econs i u ion depends on he use o c yop o ec an s.105 Al hough s o age a −80 °C is ecommended, i 469 is logis ically mos challenging and cos ly me hod (Fig. 4e). 470 Sa e y by design and p ocess de- isking. While a ew yea s ago, he mammalian cell o igin o EVs 471 was a majo hu dle o hei clinical ansla ion, conside able ad ances ha e been made in cell-based 472 he apeu ics. Rega ding sa e y, EVs de i ed om au ologous cells a e associa ed wi h lowe isks han 473 EVs de i ed om he e ologous cells (including cell lines). Howe e , he ime needed o p oduce 474 au ologous EVs is o en incompa ible wi h he ime a ailable o ini ia ing ea men . Du ing he ime 475 equi ed o he manu ac u ing and quali y con ol o pa ien -speci ic EVs, he clinical condi ion o he 476 pa ien may wo sen, making i impossible o adminis e he pe sonalised p oduc . Despi e he se e al 477 examples o au ologous p oduc s de eloped and comme cialised by pha maceu ical companies, he 478 cu en amewo ks seem o be p edominan ly sui ed o small-scale academic p oduc ion a he han 479 o la ge-scale pha maceu ical p oduc ion, and p oduc ion cos s may be p ohibi i e. While he use o 480 allogenic EVs appea s gene ally easible, he selec ion o pa en cells, assessmen o immunologic and 481 oncogenic e ec s, and isk o i al con amina ion need o be minimised by con inuous moni o ing. 482 Selec ion o assays o moni o ing, pa icula ly hei sensi i i y, is a key challenge in de e mining he 483 ime equi ed o clinical ansla ion o EV-based d ug ca ie s. Regula o s ha e ye o elease 484 guidance on how he sa e y and po ency o hese EVs should be es ed. Cu en ly, EVs a e es ed ba ch 485 by ba ch, wi h each labo a o y and company using di e en assays.107 486 487 21 Pe spec i es 488 EVs may be used as ca ie sys ems o a ious d ug deli e y applica ions. Compa ed o s anda d 489 deli e y me hods, EVs ha e been shown o deli e unc ional ca go wi h dec eased immune clea ance 490 when adminis e ed sys emically o oden s. Howe e , mo e e alua ion in clinically ele an sys ems 491 and di ec , quan i a i e compa ison wi h liposome-based al e na i es a e equi ed o comp ehensi ely 492 assess he isk/bene i a io.59 Success ul ansla ion o EVs depends on he a ailabili y o cos -493 e ec i e la ge-scale p oduc ion, isola ion, and cha ac e isa ion me hods wi h high sensi i i y o assess 494 ba ch- o-ba ch a ia ions (and hei biological consequences), and he a ailabili y o widely applicable 495 me hods o loading d ugs (Fig. 4 ). The inc easing a ailabili y o new analy ical echniques is 496 expec ed o p o ide new insigh s in o he uniqueness o EVs and may inspi e he enginee ing o nex -497 gene a ion syn he ic sys ems. The p oduc ion o a i icial EVs o EV mimics can o e come challenges 498 ela ed o s e ili y, mass p oduc ion and egula ion. Exci ing new a enues, including he usion o 499 d ug-loaded liposomes wi h EVs o imp o e d ug loading capabili ies, a e al eady being explo ed.102 500 No ably, he p oduc ion o designe EVs by implan ed cells has ecen ly been epo ed. This echnique 501 o e s a new ou e o in i o p oduc ion o enginee ed exosomes inside he body.108 Despi e hese 502 p omising esul s, mo e insigh s in o he mechanisms ha make EVs so e ec i e a in il a ing cells 503 and e ading immune de ec ion a e needed o unlock hei ull po en ial. 504 505 506 22 Re e ences 507 1 an de Meel, R. e al. Sma cance nanomedicine. Na Nano echnol 14, 1007-1017, 508 doi:10.1038/s41565-019-0567-y (2019). 509 2 Elsha kasy, O. M. e al. Ex acellula esicles as d ug deli e y sys ems: Why and how? Ad 510 D ug Deli Re , doi:10.1016/j.add .2020.04.004 (2020). 511 3 Mölle , A. & Lobb, R. J. The e ol ing ansla ional po en ial o small ex acellula esicles in 512 cance . Na Re Cance , doi:10.1038/s41568-020-00299-w (2020). 513 4 El Andaloussi, S., Mage , I., B eake ield, X. O. & Wood, M. J. A. Ex acellula esicles: 514 biology and eme ging he apeu ic oppo uni ies. Na Re D ug Disco 12, 347-357, 515 doi:10.1038/n d3978 (2013). 516 5 Ma hieu, M., Ma in-Jaula , L., La ieu, G. & Thé y, C. Speci ici ies o sec e ion and up ake o 517 exosomes and o he ex acellula esicles o cell- o-cell communica ion. Na Cell Biol 21, 9-518 17, doi:10.1038/s41556-018-0250-9 (2019). 519 6 Hopps äd e , J. e al. Toll-like ecep o 2 elease by mac ophages: an an i-in lamma o y 520 p og am induced by glucoco icoids and lipopolysaccha ide. F on Immunol, 521 doi:doi.o g/10.3389/ immu.2019.01634 (2019). 522 7 Kooijmans, S. A. A. e al. PEGyla ed and a ge ed ex acellula esicles display enhanced cell 523 speci ici y and ci cula ion ime. J Con ol Release 224, 77-85, 524 doi:10.1016/j.jcon el.2016.01.009 (2016). 525 8 Woi h, E., Fuh mann, G. & Melzig, M. F. Ex acellula Vesicles—Connec ing Kingdoms. In 526 J Mol Sci 20, 5695 (2019). 527 9 Kallu i, R. & LeBleu, V. S. The biology, unc ion, and biomedical applica ions o exosomes. 528 Science 367, eaau6977, doi:10.1126/science.aau6977 (2020). 529 10 Wi we , K. W. & Thé y, C. Ex acellula esicles o exosomes? On p imacy, p ecision, and 530 popula i y in luencing a choice o nomencla u e. J Ex acell Vesicles 8, 1648167-1648167, 531 doi:10.1080/20013078.2019.1648167 (2019). 532 11 Cabeza, L. e al. Cance he apy based on ex acellula esicles as d ug deli e y ehicles. J 533 Con ol Release 327, 296-315, doi:doi.o g/10.1016/j.jcon el.2020.08.018 (2020). 534 12 Thé y, C. e al. Minimal in o ma ion o s udies o ex acellula esicles 2018 (MISEV2018): 535 a posi ion s a emen o he In e na ional Socie y o Ex acellula Vesicles and upda e o he 536 MISEV2014 guidelines. J Ex acell Vesicles 7, 1535750, 537 doi:10.1080/20013078.2018.1535750 (2018). 538 13 O’B ien, K. e al. RNA deli e y by ex acellula esicles in mammalian cells and i s 539 applica ions. Na Re Mol Cell Biol, doi:10.1038/s41580-020-0251-y (2020). 540 14 Tkach, M., Kowal, J. & Thé y, C. Why he need and how o app oach he unc ional di e si y 541 o ex acellula esicles. Philos T ans R Soc Lond, Se B: Biol Sci 373, 542 doi:10.1098/ s b.2016.0479 (2018). 543 15 Hu wi z, S. N. e al. P o eomic p o iling o NCI-60 ex acellula esicles unco e s common 544 p o ein ca go and cance ype-speci ic bioma ke s. Onco a ge 7, 86999-87015, 545 doi:10.18632/onco a ge .13569 (2016). 546 16 Rocha, S. e al. 3D Cellula A chi ec u e A ec s Mic oRNA and P o ein Ca go o 547 Ex acellula Vesicles. Ad Sci 6, 1800948, doi:doi.o g/10.1002/ad s.201800948 (2019). 548 17 Fuh mann, G., He mann, I. & S e ens, M. M. Cell-de i ed esicles o d ug he apy and 549 diagnos ics: oppo uni ies and challenges. Nano Today 10, 397-409, 550 doi:doi:10.1016/j.nan od.2015.04.004 (2015). 551 18 G oss, J. C., Chaudha y, V., Ba sche e , K. & Bou os, M. Ac i e Wn p o eins a e sec e ed 552 on exosomes. Na Cell Biol 14, 1036-1045, doi:10.1038/ncb2574 (2012). 553 19 Smi h, S. A., Selby, L. I., Johns on, A. P. R. & Such, G. K. The Endosomal Escape o 554 Nanopa icles: Towa d Mo e E icien Cellula Deli e y. Bioconj Chem 30, 263-272, 555 doi:10.1021/acs.bioconjchem.8b00732 (2019). 556 20 an den Boo n, J. G., Schlee, M., Coch, C. & Ha mann, G. SiRNA deli e y wi h exosome 557 nanopa icles. Na Bio echnol 29, 325-326 (2011). 558 21 Sung, B. H. e al. A li e cell epo e o exosome sec e ion and up ake e eals pa h inding 559 beha io o mig a ing cells. Na Comm 11, 2092, doi:10.1038/s41467-020-15747-2 (2020). 560 22 Xu, R. e al. Ex acellula esicles in cance — implica ions o u u e imp o emen s in 561 cance ca e. Na Re Clin Oncol 15, 617-638, doi:10.1038/s41571-018-0036-9 (2018). 562 23 23 Rickle s, F. L. e al. Immune e asion media ed by PD-L1 on glioblas oma-de i ed 563 ex acellula esicles. Sci Ad 4, eaa 2766, doi:10.1126/sciad .aa 2766 (2018). 564 24 Keklikoglou, I. e al. Chemo he apy elici s p o-me as a ic ex acellula esicles in b eas 565 cance models. Na Cell Biol 21, 190-202, doi:10.1038/s41556-018-0256-3 (2019). 566 25 Nol e-‘ Hoen, E., C eme , T., Gallo, R. C. & Ma golis, L. B. Ex acellula esicles and 567 i uses: A e hey close ela i es? P oc Na l Acad Sci USA 113, 9155-9161, 568 doi:10.1073/pnas.1605146113 (2016). 569 26 Toyo uku, M., Nomu a, N. & Ebe l, L. Types and o igins o bac e ial memb ane esicles. Na 570 Re Mic obiol 17, 13-24, doi:10.1038/s41579-018-0112-2 (2019). 571 27 Mehanny, M., Koch, M., Leh , C.-M. & Fuh mann, G. S ep ococcal Ex acellula Memb ane 572 Vesicles A e Rapidly In e nalized by Immune Cells and Al e Thei Cy okine Release. F on 573 Immunol 11, 10.3389/ immu.2020.00080, doi:10.3389/ immu.2020.00080 (2020). 574 28 Goes, A. e al. Myxobac e ia-De i ed Ou e Memb ane Vesicles: Po en ial Applicabili y 575 Agains In acellula In ec ions. Cells 9, 194 (2020). 576 29 Guj a i, V. e al. Bioenginee ed Bac e ial Ou e Memb ane Vesicles as Cell-Speci ic D ug-577 Deli e y Vehicles o Cance The apy. ACS Nano 8, 1525-1537, doi:10.1021/nn405724x 578 (2014). 579 30 Kuhn, T., Koch, M. & Fuh mann, G. P obiomime ics—No el Lac obacillus-Mimicking 580 Mic opa icles Show An i-In lamma o y and Ba ie -P o ec ing E ec s in Gas oin es inal 581 Models. Small 16, 2003158, doi:doi.o g/10.1002/smll.202003158 (2020). 582 31 Mu ali, V. P. & Holmes, C. A. Bioma e ial-based ex acellula esicle deli e y o he apeu ic 583 applica ions. Ac a Bioma e , doi:doi.o g/10.1016/j.ac bio.2021.01.010 (2021). 584 32 Pou alebi Jah omi, L. & Fuh mann, G. Bac e ial Ex acellula Vesicles: Unde s anding 585 Biology P omo es Applica ions as Nanopha maceu icals. Ad D ug Deli Re , 586 doi:doi.o g/10.1016/j.add .2021.03.012 (2021). 587 33 Aye s, L., Pink, R., Ca e , D. R. F. & Nieuwland, R. Clinical equi emen s o ex acellula 588 esicle assays. J Ex acell Vesicles 8, doi:10.1080/20013078.2019.1593755 (2019). 589 34 Nassa , W. e al. Umbilical co d mesenchymal s em cells de i ed ex acellula esicles can 590 sa ely amelio a e he p og ession o ch onic kidney diseases. Bioma e Res 20, 21-21, 591 doi:10.1186/s40824-016-0068-0 (2016). 592 35 Wang, J., Bonacquis i, E. E., B own, A. D. & Nguyen, J. Boos ing he Biogenesis and 593 Sec e ion o Mesenchymal S em Cell-De i ed Exosomes. Cells 9, 660 (2020). 594 36 Kou, X. e al. The Fas/Fap-1/Ca -1 complex egula es IL-1RA sec e ion in mesenchymal 595 s em cells o accele a e wound healing. Sci T ansl Med 10, eaai8524, 596 doi:10.1126/sci anslmed.aai8524 (2018). 597 37 FDA. Public Sa e y No i ica ion on Exosome P oduc s, <www. da.go / accines-blood- 598 biologics/sa e y-a ailabili y-biologics/public-sa e y-no i ica ion-exosome-p oduc s> (2019). 599 38 Lene , T. e al. Applying ex acellula esicles based he apeu ics in clinical ials - an ISEV 600 posi ion pape . J Ex acell Vesicles (2015). 601 39 Bö ge , V. e al. In e na ional Socie y o Ex acellula Vesicles and In e na ional Socie y o 602 Cell and Gene The apy s a emen on ex acellula esicles om mesenchymal s omal cells 603 and o he cells: conside a ions o po en ial he apeu ic agen s o supp ess co ona i us 604 disease-19. Cy o he apy 22, 482-485, doi:doi.o g/10.1016/j.jcy .2020.05.002 (2020). 605 40 Galipeau, J. The mesenchymal s omal cells dilemma - does a nega i e phase III ial o 606 andom dono mesenchymal s omal cells in s e oid- esis an g a - e sus-hos disease 607 ep esen a dea h knell o a bump in he oad? Cy o he apy 15, 2-8, 608 doi:10.1016/j.jcy .2012.10.002 (2013). 609 41 Wi we , K. W. e al. De ining mesenchymal s omal cell (MSC)-de i ed small ex acellula 610 esicles o he apeu ic applica ions. J Ex acell Vesicles 8, 611 doi:10.1080/20013078.2019.1609206 (2019). 612 42 Rohde, E., Pachle , K. & Gimona, M. Manu ac u ing and cha ac e iza ion o ex acellula 613 esicles om umbilical co d-de i ed mesenchymal s omal cells o clinical es ing. 614 Cy o he apy 21, 581-592, doi:10.1016/j.jcy .2018.12.006 (2019). 615 43 Zipkin, M. Exosome edux. Na Bio echnol 37, 1395-1400, doi:10.1038/s41587-019-0326-5 616 (2019). 617 44 Chuo, S. T.-Y., Chien, J. C.-Y. & Lai, C. P.-K. Imaging ex acellula esicles: cu en and 618 eme ging me hods. J Biomed Sci 25, 91, doi:10.1186/s12929-018-0494-5 (2018). 619 24 45 Zee -Ben-Mo dehai, T. e al. Ex acellula esicles: a pla o m o he s uc u e de e mina ion 620 o memb ane p o eins by C yo-EM. S uc u e 22, 1687-1692, doi:10.1016/j.s .2014.09.005 621 (2014). 622 46 K eime , S. e al. Mass-Spec ome y-Based Molecula Cha ac e iza ion o Ex acellula 623 Vesicles: Lipidomics and P o eomics. J P o eome Res 14, 2367-2384, doi:10.1021/p 501279 624 (2015). 625 47 Van Deun, J. e al. EV-TRACK: anspa en epo ing and cen alizing knowledge in 626 ex acellula esicle esea ch. Na Me h 14, 228-232, doi:10.1038/nme h.4185 (2017). 627 48 Welsh, J. A. e al. Towa ds de ining e e ence ma e ials o measu ing ex acellula esicle 628 e ac i e index, epi ope abundance, size and concen a ion. J Ex acell Vesicles 9, 1816641, 629 doi:10.1080/20013078.2020.1816641 (2020). 630 49 Valadi, H. e al. Exosome-media ed ans e o mRNAs and mic oRNAs is a no el mechanism 631 o gene ic exchange be ween cells. Na Cell Biol 9, 654-659, doi:doi.o g/10.1038/ncb1596 632 (2007). 633 50 Al a ez-E i i, L. e al. Deli e y o siRNA o he mouse b ain by sys emic injec ion o 634 a ge ed exosomes. Na Bio ech 29, 341-345, doi:10.1038/nb .1807 (2011). 635 51 Mi elb unn, M. e al. Unidi ec ional ans e o mic o RNA-loaded exosomes om T cells o 636 an igen-p esen ing cells. Na Commun 2, 282, doi:doi.o g/10.1038/ncomms1285 (2011). 637 52 Mu phy, D. E. e al. Na u al o Syn he ic RNA Deli e y: A S oichiome ic Compa ison o 638 Ex acellula Vesicles and Syn he ic Nanopa icles. Nano Le 21, 1888-1895, 639 doi:10.1021/acs.nanole .1c00094 (2021). 640 53 Hoshino, A. e al. Tumou exosome in eg ins de e mine o gano opic me as asis. Na u e 527, 641 329-335, doi:10.1038/na u e15756 (2015). 642 54 Qiao, L. e al. Tumo cell-de i ed exosomes home o hei cells o o igin and can be used as 643 T ojan ho ses o deli e cance d ugs. The anos ics 10, 3474-3487, doi:10.7150/ hno.39434 644 (2020). 645 55 Dai, J. e al. Exosomes: key playe s in cance and po en ial he apeu ic s a egy. Signal 646 T ansduc Ta ge The 5, 145, doi:10.1038/s41392-020-00261-0 (2020). 647 56 Wiklande , O. P. B. e al. Ex acellula esicle in i o biodis ibu ion is de e mined by cell 648 sou ce, ou e o adminis a ion and a ge ing. J Ex acell Vesicles 4, 26316 (2015). 649 57 Lai, C. P. e al. Dynamic Biodis ibu ion o Ex acellula Vesicles in Vi o Using a 650 Mul imodal Imaging Repo e . ACS Nano 8, 483-494, doi:10.1021/nn404945 (2014). 651 58 Hen iksen, J. R. e al. Remo e Loading o 64Cu2+ in o Liposomes wi hou he Use o Ion 652 T anspo Enhance s. ACS Appl Ma e In e aces 7, 22796-22806, 653 doi:10.1021/acsami.5b04612 (2015). 654 59 Johnsen, K. B. e al. On he use o liposome con ols in s udies in es iga ing he clinical 655 po en ial o ex acellula esicle-based d ug deli e y sys ems – A commen a y. J Con ol 656 Release 269, 10-14, doi:10.1016/j.jcon el.2017.11.002 (2018). 657 60 Lenzini, S., Ba gi, R., Chung, G. & Shin, J.-W. Ma ix mechanics and wa e pe mea ion 658 egula e ex acellula esicle anspo . Na Nano echnol 15, 217-223, doi:10.1038/s41565-659 020-0636-2 (2020). 660 61 Geige , J. in The Challenge o CMC Regula o y Compliance o Biopha maceu icals and 661 O he Biologics (ed John Geige ) 221-237 (Sp inge New Yo k, 2013). 662 62 Somiya, M., Yoshioka, Y. & Ochiya, T. Biocompa ibili y o highly pu i ied bo ine milk-663 de i ed ex acellula esicles. J Ex acell Vesicles 7, 1440132, 664 doi:10.1080/20013078.2018.1440132 (2018). 665 63 McVey, M. J. e al. Pla ele ex acellula esicles media e ans usion- ela ed acu e lung inju y 666 by imbalancing he sphingolipid heos a . Blood 137, 690-701, doi:10.1182/blood.2020005985 667 (2021). 668 64 Balachand an, B. & Yuana, Y. Ex acellula esicles-based d ug deli e y sys em o cance 669 ea men . Cogen Medicine 6, 1635806, doi:10.1080/2331205X.2019.1635806 (2019). 670 65 Robbins, P. D. & Mo elli, A. E. Regula ion o immune esponses by ex acellula esicles. 671 Na Re Immunol 14, 195-208, doi:10.1038/n i3622 (2014). 672 66 Mehanny, M., Leh , C.-M. & Fuh mann, G. Ex acellula esicles as an igen ca ie s o no el 673 accina ion a enues. Ad D ug Deli Re , doi:doi.o g/10.1016/j.add .2021.03.016 (2021). 674 25 67 Zhu, X. e al. Comp ehensi e oxici y and immunogenici y s udies e eal minimal e ec s in 675 mice ollowing sus ained dosing o ex acellula esicles de i ed om HEK293T cells. J 676 Ex acell Vesicles 6, 1324730, doi:10.1080/20013078.2017.1324730 (2017). 677 68 Thippabho la, S., Zhong, C. & He, M. 3D cell cul u e s imula es he sec e ion o in i o like 678 ex acellula esicles. Sci Rep 9, 13012, doi:10.1038/s41598-019-49671-3 (2019). 679 69 Li, Y.-J. e al. Eme ging s a egies o labeling and acking o ex acellula esicles. J 680 Con ol Release 328, 141-159, doi:doi.o g/10.1016/j.jcon el.2020.08.056 (2020). 681 70 de Ab eu, R. C. e al. Na i e and bioenginee ed ex acellula esicles o ca dio ascula 682 he apeu ics. Na Re Ca diol, doi:10.1038/s41569-020-0389-5 (2020). 683 71 Ikeda, G. e al. Mi ochond ia-Rich Ex acellula Vesicles F om Au ologous S em Cell–684 De i ed Ca diomyocy es Res o e Ene ge ics o Ischemic Myoca dium. J Am Coll Ca diol 77, 685 1073-1088, doi:doi.o g/10.1016/j.jacc.2020.12.060 (2021). 686 72 Villa, A. e al. T ansplan a ion o au ologous ex acellula esicles o cance -speci ic 687 a ge ing. The anos ics 11, 2034-2047, doi:10.7150/ hno.51344 (2021). 688 73 Escudie , B. e al. Vaccina ion o me as a ic melanoma pa ien s wi h au ologous dend i ic cell 689 (DC) de i ed-exosomes: esul s o he i s phase I clinical ial. J T ansl Med 3, 10, 690 doi:10.1186/1479-5876-3-10 (2005). 691 74 Clemmens, H. & Lambe , D. W. Ex acellula esicles: ansla ional challenges and 692 oppo uni ies. Biochem Soc T ans 46, 1073-1082, doi:10.1042/BST20180112 (2018). 693 75 Vul o, A. G. & Jaquez, O. A. The p ocess de ines he p oduc : wha eally ma e s in 694 biosimila design and p oduc ion? Rheuma ology (Ox o d) 56, i 14-i 29, 695 doi:10.1093/ heuma ology/kex278 (2017). 696 76 Pa el, D. B. e al. Impac o cell cul u e pa ame e s on p oduc ion and ascula iza ion 697 bioac i i y o mesenchymal s em cell-de i ed ex acellula esicles. Bioeng T ansl Med 2, 698 170-179, doi:10.1002/b m2.10065 (2017). 699 77 Sil e man, L. I. e al. Iden i ying and Managing Sou ces o Va iabili y in Cell The apy 700 Manu ac u ing and Clinical T ials. Regen Eng T ansl Med 5, 354-361, doi:10.1007/s40883-701 019-00129-y (2019). 702 78 Iancu, E. M. & Kandala , L. E. Challenges and ad an ages o cell he apy manu ac u ing 703 unde Good Manu ac u ing P ac ices wi hin he hospi al se ing. Cu Opin Bio echnol 65, 704 233-241, doi:doi.o g/10.1016/j.copbio.2020.05.005 (2020). 705 79 Wilhelm, S. e al. Analysis o nanopa icle deli e y o umou s. Na Re Ma e 1, 16014, 706 doi:10.1038/na e ma s.2016.14 (2016). 707 80 Geige , J. in The Challenge o CMC Regula o y Compliance o Biopha maceu icals and 708 O he Biologics (ed John Geige ) 105-137 (Sp inge New Yo k, 2013). 709 81 Geige , J. in The Challenge o CMC Regula o y Compliance o Biopha maceu icals and 710 O he Biologics (ed John Geige ) 139-178 (Sp inge New Yo k, 2013). 711 82 Munagala, R., Aqil, F., Jeyabalan, J. & Gup a, R. C. Bo ine milk-de i ed exosomes o d ug 712 deli e y. Cance Le 371, 48-61, doi:doi.o g/10.1016/j.canle .2015.10.020 (2016). 713 83 Fuh mann, G. e al. Ac i e loading in o ex acellula esicles signi ican ly imp o es he 714 cellula up ake and pho odynamic e ec o po phy ins. J Con ol Release 205, 35-44, 715 doi:doi:10.1016/j.jcon el.2014.11.029 (2015). 716 84 Schulz, E. e al. Biocompa ible bac e ia-de i ed esicles show inhe en an imic obial ac i i y. 717 J Con ol Release 290, 46-55 (2018). 718 85 Pi oux, M. e al. Ex acellula Vesicle P oduc ion Loaded wi h Nanopa icles and D ugs in a 719 T ade-o be ween Loading, Yield and Pu i y: Towa ds a Pe sonalized D ug Deli e y Sys em. 720 Ad Biosys 1, 1700044, doi:10.1002/adbi.201700044 (2017). 721 86 Choi, H. e al. Exosome-based deli e y o supe - ep esso IκBα elie es sepsis-associa ed 722 o gan damage and mo ali y. Sci Ad 6, eaaz6980, doi:10.1126/sciad .aaz6980 (2020). 723 87 Meng, W. e al. P ospec s and challenges o ex acellula esicle-based d ug deli e y sys em: 724 conside ing cell sou ce. D ug Deli 27, 585-598, doi:10.1080/10717544.2020.1748758 725 (2020). 726 88 Lee, Y. X. F., Johansson, H., Wood, M. J. A. & El Andaloussi, S. Conside a ions and 727 Implica ions in he Pu i ica ion o Ex acellula Vesicles – A Cau iona y Tale. F on Neu osci 728 13, 1067 (2019). 729