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
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