New Approaches in Nanomedicine for Ischemic Stroke

pha maceu ics
Re iew
New App oaches in Nanomedicine o Ischemic S oke
Cla a Co ea-Paz 1,†, And és da Sil a-Candal 2,†, Es e Polo 3,4, Jé ôme Pa cq 5, Denis Vi ien 6,7,
Dusica Maysinge 8, Bea iz Pelaz 3,9 and F ancisco Campos 1,*


Ci a ion: Co ea-Paz, C.; da
Sil a-Candal, A.; Polo, E.; Pa cq, J.;
Vi ien, D.; Maysinge , D.; Pelaz, B.;
Campos, F. New App oaches in
Nanomedicine o Ischemic S oke.
Pha maceu ics 2021,13, 757. h ps://
doi.o g/10.3390/pha maceu ics
13050757
Academic Edi o : Leena Pel onen
Recei ed: 31 Ma ch 2021
Accep ed: 11 May 2021
Published: 20 May 2021
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1Clinical Neu osciences Resea ch Labo a o y (LINC), Heal h Resea ch Ins i u e o San iago de
Compos ela (IDIS), 15706 San iago de Compos ela, Spain; [email p o ec ed]
2Neu ology Se ice, Uni e si y Hospi al Complex o A Co uña, A Co uña Biomedical Resea ch Ins i u e,
15006 A Co uña, Spain; and [email p o ec ed]
3Cen o Singula de In es igación en Química Biolóxica e Ma e iais Molecula es (CiQUS), Uni e sidade de
San iago de Compos ela, 15782 San iago, Spain; es e [email p o ec ed] (E.P.); [email p o ec ed] (B.P.)
4
G upo de Física de Coloides y Políme os, Depa amen o de Física de Pa ículas, Uni e sidade de San iago de
Compos ela, 15782 San iago, Spain
5Op2Lysis SAS, 14000 Caen, F ance; je ome.pa [email p o ec ed]
6
UNICAEN, INSERM U1237, E ablissemen F ançais du Sang, Physiopa hology and Imaging o Neu ological
Diso de s (PhIND), Cyce on, Ins i u Blood and B ain @ Caen-No mandie (BB@C), No mandie Uni e si y,
14000 Caen, F ance; [email p o ec ed]
7Depa men o clinical esea ch, Caen-No mandie Uni e si y Hospi al, CHU, A enue de la cô e de Nac e,
14000 Caen, F ance
8Depa men o Pha macology & The apeu ics, McGill Uni e si y, Mon éal, QC H3G 1Y6, Canada;
[email p o ec ed]
9
G upo de Física de Coloides y Políme os, Depa amen o de Química Ino gánica, Uni e sidade de San iago de
Compos ela, 15782 San iago, Spain
*Co espondence: ancisco.campos.pe ez@se gas.es; Tel.: +34-981951097; Fax: +34-981951086
† These au ho s con ibu ed equally o his wo k.
Abs ac :
Ischemic s oke, caused by he in e up ion o blood low o he b ain and subsequen
neu onal dea h, ep esen s one o he main causes o disabili y in de eloped coun ies. The apeu ic
me hods such as ecanaliza ion app oaches, neu op o ec i e d ugs, o eco e y s a egies ha e been
widely de eloped o imp o e he pa ien ’s ou come; howe e , impo an limi a ions such as a na ow
he apeu ic window, he abili y o each b ain a ge s, o d ug side e ec s cons i u e some o he main
aspec s ha limi he clinical applicabili y o he cu en ea men s. Nano echnology has eme ged as
a p omising ool o o e come many o hese d ug limi a ions and imp o e he e icacy o ea men s
o neu ological diseases such as s oke. The use o nanopa icles as a con as agen o as d ug
ca ie s o a speci ic a ge a e some o he mos common app oaches de eloped in nanomedicine o
s oke. Th oughou his e iew, we ha e summa ized ou expe ience o using nano echnology ools
o he s udy o s oke and he sea ch o no el he apies.
Keywo ds:
cell acking; diagnosis; d ug ca ie s; d ug con ol elease; nanomedicine; neu op o ec-
ion; eco e y; s oke; issue plasminogen ac i a o
1. In oduc ion
1.1. S oke
S oke is a ce eb o ascula disease esul ing om dis u bance in no mal ce eb al blood
low (CBF), which causes a ansien o pe manen de ici in he unc ion o one o mo e
pa s o he b ain, and is becoming one o he leading causes o disabili y in de eloped
coun ies. The dis u bance o no mal CBF induces me abolic and cellula changes ha
can lead o cell dea h and dis up ion o he ne ous sys em [
1
]. S oke can be classi ied
in o wo ypes: hemo hagic and ischemic. A hemo hagic s oke is due o a blood essel
up u e, ep esen s up o 15% o all s oke cases, and is associa ed wi h high mo ali y. On
he con a y, an ischemic s oke is caused by an obs uc ion in he essel, o en due o he
p esence o a clo , and cons i u es app oxima ely 85% o s oke e en s [2] (Figu e 1).
Pha maceu ics 2021,13, 757. h ps://doi.o g/10.3390/pha maceu ics13050757 h ps://www.mdpi.com/jou nal/pha maceu ics
Pha maceu ics 2021,13, 757 2 o 26
Pha maceu ics 2021, 13, x FOR PEER REVIEW 2 o 27
due o he p esence o a clo , and cons i u es app oxima ely 85% o s oke e en s [2] (Fig-
u e 1)
Figu e 1. Rep esen a ion o ischemic s oke and hemo hagic s oke. In an ischemic s oke, he clo
blocks blood low o an a ea o he b ain; meanwhile, hemo hagic s oke happens when a blood
essel b eaks and bleeds in o he b ain. Figu e c ea ed wi h BioRende .com.
The ischemic in a c is a dynamic egion in which mul iple molecula and cellula
p ocesses a e in ol ed (de ined as an ischemic cascade), which s a s immedia ely a e
he ischemic insul and changes e en weeks and mon hs la e . Based on he blood pe u-
sion le el and me abolic ac i i y in he in a c b ain, wo egions ha e been ex ensi ely
desc ibed: he ischemic co e and he penumb a [3] (Figu e 2). The co e is he egion mos
a ec ed by he lack o blood low and is i e e sibly inju ed in a ew minu es. The penum-
b a is a ulne able, hypo-pe used, and me abolically comp omised egion ha can be
eco e ed i he blood low supply is e-es ablished in he i s ew hou s a e a s oke.
The penumb a is also he a ge o mul iple p o ec i e d ugs aimed a educing o block-
ing he p og ession o cell dea h [4,5].
Figu e 2. Illus a ion o he penumb a concep . Ischemic co e ep esen s he in a c ed issue and
he penumb a egion he sal ageable issue a isk o in a c ion in case o pe sis ence essel occlu-
sion. While neu ons in he ischemic co e a e conside ed beyond escue, neu ons in he penumb a
a e po en ial a ge s o he apeu ic in e en ion. Figu e c ea ed wi h BioRende .com.
The apeu ic app oaches a e s oke onse may be classi ied in o h ee main g oups:
epe usion, p o ec i e, and eco e y s a egies (Figu e 3).
Figu e 1.
Rep esen a ion o ischemic s oke and hemo hagic s oke. In an ischemic s oke, he clo
blocks blood low o an a ea o he b ain; meanwhile, hemo hagic s oke happens when a blood
essel b eaks and bleeds in o he b ain. Figu e c ea ed wi h BioRende .com.
The ischemic in a c is a dynamic egion in which mul iple molecula and cellula
p ocesses a e in ol ed (de ined as an ischemic cascade), which s a s immedia ely a e
he ischemic insul and changes e en weeks and mon hs la e . Based on he blood pe u-
sion le el and me abolic ac i i y in he in a c b ain, wo egions ha e been ex ensi ely
desc ibed: he ischemic co e and he penumb a [
3
] (Figu e 2). The co e is he egion
mos a ec ed by he lack o blood low and is i e e sibly inju ed in a ew minu es. The
penumb a is a ulne able, hypo-pe used, and me abolically comp omised egion ha can
be eco e ed i he blood low supply is e-es ablished in he i s ew hou s a e a s oke.
The penumb a is also he a ge o mul iple p o ec i e d ugs aimed a educing o blocking
he p og ession o cell dea h [4,5].
Pha maceu ics 2021, 13, x FOR PEER REVIEW 2 o 27
due o he p esence o a clo , and cons i u es app oxima ely 85% o s oke e en s [2] (Fig-
u e 1)
Figu e 1. Rep esen a ion o ischemic s oke and hemo hagic s oke. In an ischemic s oke, he clo
blocks blood low o an a ea o he b ain; meanwhile, hemo hagic s oke happens when a blood
essel b eaks and bleeds in o he b ain. Figu e c ea ed wi h BioRende .com.
The ischemic in a c is a dynamic egion in which mul iple molecula and cellula
p ocesses a e in ol ed (de ined as an ischemic cascade), which s a s immedia ely a e
he ischemic insul and changes e en weeks and mon hs la e . Based on he blood pe u-
sion le el and me abolic ac i i y in he in a c b ain, wo egions ha e been ex ensi ely
desc ibed: he ischemic co e and he penumb a [3] (Figu e 2). The co e is he egion mos
a ec ed by he lack o blood low and is i e e sibly inju ed in a ew minu es. The penum-
b a is a ulne able, hypo-pe used, and me abolically comp omised egion ha can be
eco e ed i he blood low supply is e-es ablished in he i s ew hou s a e a s oke.
The penumb a is also he a ge o mul iple p o ec i e d ugs aimed a educing o block-
ing he p og ession o cell dea h [4,5].
Figu e 2. Illus a ion o he penumb a concep . Ischemic co e ep esen s he in a c ed issue and
he penumb a egion he sal ageable issue a isk o in a c ion in case o pe sis ence essel occlu-
sion. While neu ons in he ischemic co e a e conside ed beyond escue, neu ons in he penumb a
a e po en ial a ge s o he apeu ic in e en ion. Figu e c ea ed wi h BioRende .com.
The apeu ic app oaches a e s oke onse may be classi ied in o h ee main g oups:
epe usion, p o ec i e, and eco e y s a egies (Figu e 3).
Figu e 2.
Illus a ion o he penumb a concep . Ischemic co e ep esen s he in a c ed issue and he penumb a egion
he sal ageable issue a isk o in a c ion in case o pe sis ence essel occlusion. While neu ons in he ischemic co e a e
conside ed beyond escue, neu ons in he penumb a a e po en ial a ge s o he apeu ic in e en ion. Figu e c ea ed wi h
BioRende .com.
The apeu ic app oaches a e s oke onse may be classi ied in o h ee main g oups:
epe usion, p o ec i e, and eco e y s a egies (Figu e 3).
Repe usion he apies, based on he use o pha macological h ombolysis and/o me-
chanical ecanaliza ion, a e ocused on es o ing CBF in he i s hou s pos s oke onse [
6
].
Th ombolysis wi h in a enous ecombinan issue plasminogen ac i a o ( PA) is he only
Food and D ug Adminis a ion/Eu opean Medicines Agency (FDA/EMA)-app o ed d ug
ea men o pa ien s wi h acu e ischemic s oke, bu i s use is limi ed by a na ow he a-
peu ic window, selec i e e icacy, and hemo hagic complica ions [
7
]. Nowadays, ad anced
endo ascula ecanaliza ion echnology based on he use o mechanical ca he e s has sig-
ni ican ly imp o ed he ecanaliza ion a es, showing a be e o e all ou come in ea ed
pa ien s. Howe e , his mechanical he apy is e ec i e when s oke e en s a e caused
by a la ge essel occlusion, which ep esen s less han 30% o he pa ien s. Mo eo e , in
occlusions loca ed in small essels, whe e he PA has a success ul ecanaliza ion a e, he
e icacy o mechanical h ombec omy is also limi ed. In addi ion, some clinical ials ha e
Pha maceu ics 2021,13, 757 3 o 26
shown ha p e ea men wi h in a enous PA p o ides addi ional bene i s o pa ien s
unde going mechanical h ombec omy. These e idences e lec ha PA h ombolysis
emains he gold s anda d ea men o acu e s oke [7].
Pha maceu ics 2021, 13, x FOR PEER REVIEW 3 o 27
Figu e 3. Scheme o he di e en he apeu ic s a egies o he ischemic s oke. Figu e c ea ed wi h
BioRende .com.
Repe usion he apies, based on he use o pha macological h ombolysis and/o me-
chanical ecanaliza ion, a e ocused on es o ing CBF in he i s hou s pos s oke onse
[6]. Th ombolysis wi h in a enous ecombinan issue plasminogen ac i a o ( PA) is
he only Food and D ug Adminis a ion/Eu opean Medicines Agency (FDA/EMA)-ap-
p o ed d ug ea men o pa ien s wi h acu e ischemic s oke, bu i s use is limi ed by a
na ow he apeu ic window, selec i e e icacy, and hemo hagic complica ions [7]. Now-
adays, ad anced endo ascula ecanaliza ion echnology based on he use o mechanical
ca he e s has signi ican ly imp o ed he ecanaliza ion a es, showing a be e o e all ou -
come in ea ed pa ien s. Howe e , his mechanical he apy is e ec i e when s oke
e en s a e caused by a la ge essel occlusion, which ep esen s less han 30% o he pa-
ien s. Mo eo e , in occlusions loca ed in small essels, whe e he PA has a success ul
ecanaliza ion a e, he e icacy o mechanical h ombec omy is also limi ed. In addi ion,
some clinical ials ha e shown ha p e ea men wi h in a enous PA p o ides addi-
ional bene i s o pa ien s unde going mechanical h ombec omy. These e idences e lec
ha PA h ombolysis emains he gold s anda d ea men o acu e s oke [7].
Neu onal p o ec ion is a e m ha conglome a es a a ie y o s a egies ocused on
educing cell dea h a e an ischemic e en wi hou a ec ing issue epe usion. To da e,
se e al compounds ha e been p oposed o block he pa hway leading o ischemia-in-
duced cell dea h a di e en s eps o he ischemic cascade. These in e en ions ange om
physical app oaches such as hypo he mia o he use o pha macological d ugs [8]. Despi e
he posi i e esul s ob ained in p eclinical s udies, he e is no p o ec i e compound o
s oke pa ien s ye , due o he na ow he apeu ic window, abili y o he d ug o each
b ain a ge s, side e ec s, o sho hal -li e a e d ug adminis a ion [8].
Figu e 3.
Scheme o he di e en he apeu ic s a egies o he ischemic s oke. Figu e c ea ed wi h
BioRende .com.
Neu onal p o ec ion is a e m ha conglome a es a a ie y o s a egies ocused on
educing cell dea h a e an ischemic e en wi hou a ec ing issue epe usion. To da e,
se e al compounds ha e been p oposed o block he pa hway leading o ischemia-induced
cell dea h a di e en s eps o he ischemic cascade. These in e en ions ange om
physical app oaches such as hypo he mia o he use o pha macological d ugs [
8
]. Despi e
he posi i e esul s ob ained in p eclinical s udies, he e is no p o ec i e compound o
s oke pa ien s ye , due o he na ow he apeu ic window, abili y o he d ug o each
b ain a ge s, side e ec s, o sho hal -li e a e d ug adminis a ion [8].
Neu o epai s a egies, usually based on cell he apies o g ow h ac o adminis a ion,
in ol e he es o a ion o b ain unc ion, ei he by egene a ion o he damaged ce eb al
issue o by he es ablishmen o al e na i e neu al pa hways o synapses ( e e ed o as
b ain plas ici y). The he apeu ic window o hese he apies is wide han he one in
h omboly ic o neu op o ec i e app oaches. The aim o he ea men s o neu ological
unc ion eco e y a e s oke is no es ic ed o he neu ons; i is mo e ocused on he
neu o ascula uni , including p ocedu es ha enhance synap ogenesis and angiogenesis.
Thus, neu o epai ea men s may use s em cells and p o-neu ogenic, p o-angiogenic,
and/o p o-synap ogenic d ug deli e y, among o he s [9].
Pha maceu ics 2021,13, 757 4 o 26
1.2. Nanomedicine
Nano echnology eme ged in 1959 as a new a ea o s udy ha in ol ed he c ea ion
o ma e ials o sys ems a he nanome e scale, de ined as nanopa icles (NPs). La e ,
his echnology was applied o he medical ield, opening a new e a in he diagnosis and
ea men o many diseases, cu en ly known as nanomedicine [
10
]. The de elopmen
o NPs o clinical use has allowed, o ins ance, he encapsula ion o hyd ophilic d ugs,
a dec ease in he he apeu ic doses, a educ ion in side e ec s, an imp o emen in he
biocompa ibili y and he apeu ic e ec , o a ge ing and deli e y o he d ug o he desi ed
egion [11].
O e he las ew yea s, he complexi y o nanosys ems has inc eased exponen ially.
Ini ially, he i s o mula ions we e mainly ocused on he encapsula ion o d ugs o
inc easing hei sa e y and e icacy; howe e , he new ad ances in he ield o nanomedicine
ha e allowed he de elopmen o “sma ” deli e y nanopla o ms ha , a e adminis a ion,
can speci ically ecognize he pa hological egion and induce a con olled d ug deli e y
ha can be induced by in e nal o ex e nal s imuli. In e nal s imuli in ol e chemical and
biochemical changes in he a ge ed egion, such as pH, empe a u e, edox, and ionic
changes, o e en enzyma ic ac i i ies, whe eas ex e nal s imuli comp ise, o ins ance, ligh ,
magne ic ield, o ul asound [11].
Owing o he e sa ili y o nanomedicine, as obse ed in o he pa hologies, his
echnology has been widely applied in he ield o s oke, no only o diagnosis bu also o
imp o e he e icacy o h omboly ic he apy and p o ec i e d ugs, as well as o help in he
de elopmen o eco e y he apies.
2. Nanomedicine o S oke Diagnosis
Compu ed omog aphy (CT) and magne ic esonance imaging (MRI) ha e e olu ion-
ized ischemic s oke diagnosis and managemen . Ini ially es ic ed o s uc u al imaging
o exclude bleeding, hese imaging modali ies can now de ec in ac anial essel occlusion,
e alua e he ischemic penumb a o selec candida es o h ombec omy, and play a key
ole in iden i ying s oke e iology. Molecula imaging has he po en ial o u he expand
he in o ma ion p o ided by CT and MRI by e ealing he biological p ocesses ha cons i-
u e po en ial diagnos ic o he apeu ic a ge s [
12
]. The use o nano echnology o design
con as agen s has led o signi ican ad ances in he ield [13].
The ecen de elopmen o a new amily o con as agen s o MRI based on mic o-
sized pa icles o i on oxide (MPIOs) has allowed a la ge inc ease in he sensi i i y and
speci ici y o his imaging modali y, pa ing he way o clinical applica ion [
14
]. The use o
la ge pa icles wi h a diame e o a leas 1
µ
m has se e al ad an ages compa ed o he
mo e classically used ul asmall supe pa amagne ic i on oxide pa icles
(20–50 nm)
. In-
deed, i p e en s he passi e leakage o pa icles in he b ain pa enchyma and inc eases he
payload o he con as ma e ial pe pa icle [
15
]. Using MPIOs coupled wi h monoclonal
an ibodies, non-in asi e de ec ion o speci ic p o eins exp essed by he ce eb o ascula u e
has been demons a ed in neu o ascula diso de s in he las ew yea s [16].
The combina ion o
in i o
MRI wi h NP con as agen s has allowed he cla i ica ion
o many aspec s o pos -s oke in lamma ion, which is one o he main pa hogenic p ocesses
occu ing in he acu e and subacu e phases o s oke [
17
]. Moni o ing he empo o-spa ial
egula ion o he in lamma o y eac ion in he a ec ed b ain pa enchyma could ha e sig-
ni ican clinical applica ions, such as iden i ying pa ien subse s ha could bene i om
immunomodula o y ea men s. In his ega d, he endo helial cells o he blood–b ain
ba ie (BBB) a e key playe s in pos -s oke in lamma ion by media ing he diapedesis o
leukocy es om he blood o he b ain h ough he exp ession o adhesion molecules. Since
hese adhesion molecules a e easily accessible by la ge con as -ca ying pa icles, hey con-
s i u e in e es ing a ge s o molecula imaging. Using MPIOs coupled wi h monoclonal
an ibodies a ge ing ascula cell adhesion molecule-1 (VCAM-1), molecula MRI has
p o ided e idence o he exis ence o a new concep de ined as “in lamma o y penumb a”
in ischemic s oke [
18
]. In mice, 24 h a e ischemic s oke, his echnique un eiled an
Pha maceu ics 2021,13, 757 5 o 26
in lamma o y a ea a isk, su ounding he ini ial ischemic lesion, which was seconda ily
in il a ed by lymphocy es and ul ima ely ec ui ed by he ischemic co e [
18
]. By analogy
wi h he ischemic penumb a, he misma ch be ween he VCAM-1-o e exp essing egion
( e ealed by molecula MRI) and he ischemic co e ( e ealed by di usion-weigh ed imag-
ing 24 h pos onse ) was called he “in lamma o y penumb a”. In e es ingly, he size o
his “in lamma o y penumb a” a ied acco ding o he du a ion and sub ypes o ischemic
s oke in expe imen al models, sugges ing ha he e could be a wide a iabili y in he
in ensi y and spa ial ex en o he in lamma o y eac ion be ween pa ien s.
Besides VCAM-1, o he adhesion molecules a e in ol ed in he diapedesis o leuko-
cy es om he blood o he b ain. Fo ins ance, Deddens and co-wo ke s pe o med
molecula MRI o in e cellula adhesion molecule-1 (ICAM-1) in an expe imen al model o
ischemic s oke in mice. They used MPIOs a ge ing ICAM-1 o e eal he o e exp ession
o ICAM-1 by ac i a ed endo helial cells a di e en ime poin s a e ischemic onse [
19
].
They ound ha ICAM-1 exp ession was maximal a 48 h and ex ended in he pe i-in a c
a ea, in line wi h he “in lamma o y penumb a” concep . Whe he VCAM-1 and ICAM-1
imaging me hods p o ide di e en ial in o ma ion on s oke, pa hophysiology emains o
be explo ed. Mo eo e , Quenaul e al. demons a ed ha molecula MRI o P-selec in
using MPIOs could be employed o diagnose a ansien ischemic a ack (TIA). In a TIA,
he du a ion o ischemia is oo sho o induce changes on an unenhanced MRI scan. In e -
es ingly, expe imen al s udies ha e e ealed ha P-selec in is o e exp essed o a leas
24 h in he a ec ed ascula e i o y. Using molecula MRI o P-selec in, i is he e o e
possible o de ec he endo helial ac i a ion igge ed by he TIA and dis inguish TIA om
s oke mimics, such as epilepsy o mig aine [20].
Molecula MRI can also aid in he e iologic assessmen o ischemic s oke. Indeed, i
has been p e iously demons a ed ha up u ed a he oscle o ic plaques o e exp ess en-
do helial ac i a ion ma ke s. Using MPIOs a ge ing P-selec in and VCAM-1, McA ee e al.
demons a ed ha i is possible o de ec he ac i a ed endo helium inside a he oscle o ic
plaques using MRI [
21
]. Molecula MRI o endo helial ac i a ion in s oke pa ien s could
hus be u ilized o iden i y he culp i ascula lesion and he e o e be e cha ac e ize
s oke e iology.
E en i i s sensi i i y o de ec con as ma e ial is lowe han ha o MRI, CT can also
be used o molecula imaging using NPs. This has been demons a ed in expe imen al
models o ca o id a e y h ombosis and embolic ischemic s oke [
22
]. Kim e al. showed
ha ib in- a ge ed gold NPs can e eal ce eb al h ombus as a high-densi y endo ascula
ma e ial on CT images. In acu e s oke se ings, his would allow he assessmen o
h ombus bu den and moni o ing o h omboly ic he apy in a non-in asi e manne .
Gi en he demons a ion o he high sensi i i y and speci ici y o molecula imaging
o in lamma ion o e ed by MPIO-enhanced molecula MRI in expe imen al models, e o s
a e ongoing o ansla e his me hod o clinical imaging. Indeed, he MPIOs used in
p eclinical s udies a e non-biodeg adable because o hei coa ing and inne s uc u e. In
his con ex , he de elopmen o biocompa ible MPIOs is manda o y. To o e come his
limi a ion, Pe ez-Balde as e al. ecen ly epo ed he p oduc ion o mul ime ic magne i e
pa icles ha o m la ge MPIO-like pa icles ha a e biodeg adable [
23
]. In an expe imen al
model o neu oin lamma ion, hey demons a ed ha his me hod allowed non-in asi e
imaging o ac i a ed endo helial cells.
3. Nanopa icles o Recanaliza ion The apies
App oxima ely 85% o all s oke cases a e ischemic s okes ha occu due o he
obs uc ion by blood clo s wi hin a blood–b ain essel. To da e, PA emains he only
h omboly ic d ug o he ea men o acu e ischemic s oke [
24
]. PA is a ib in-speci ic
se ine p o ease ha ac i a es he endogenous p oenzyme plasminogen and con e s i o
he ac i e o m plasmin, hus deg ading he h ombus ib in ne wo k. In addi ion o he
isk o hemo hagic ans o ma ion (HT), sys emic deli e y o PA has o be adminis e ed
in a enously, 10% as bolus, and 90% as in usion due o i s sho hal -li e (3.5 min, wi h a

Pha maceu ics 2021,13, 757 6 o 26
clea ance o 75% in he i s 8 min). This sho hal -li e is caused by he e icien inac i a ion
o esiden blood enzymes, such as plasminogen ac i a o inhibi o s (PAI-1 and PAI-2) [
25
].
Adminis a ion o PA is also ela ed o neu o oxic side e ec s due o he ac i a ion o
mic oglia, N-me hyl-d-aspa a e (NMDA) ecep o s, and me allop o einases [26].
Owing o he isk o HT, PA in usion is es ic ed o a na ow he apeu ic window
(4.5 h o s oke symp om onse when injec ed alone and 6 h when combined wi h mechani-
cal h ombec omy). Un o una ely, only app oxima ely 30% o pa ien s ea ed wi h PA
achie e ull o pa ial ecanaliza ion a e in a enous adminis a ion. In addi ion, PA
adminis a ion has o be done by expe ienced neu ologis s a e exclusion o in ac anial
hemo hage by CT o MRI, which inc eases he “doo o needle ime.” All hese limi a ions
mean ha less han 10% o s oke pa ien s can bene i om his ea men [27,28].
In he pas wo decades, s udies ha e ocused on ex ending he PA he apeu ic ime
window beyond 4.5 h o explo ing al e na i e h omboly ic agen s, while mos d ugs
ha e ailed in clinical ials [
29
]. Cu en ly, endo ascula ecanaliza ion he apy (ERT) by
mechanical h ombec omy has eme ged as a new ea men o acu e ischemic s oke. ERT
has he ad an age o a highe ecanaliza ion a e o p oximal in ac anial a e y occlusions
ha a e usually esis an o in a enous PA. Howe e , despi e he clea bene i s o ERT,
his no el ecanaliza ion s a egy s ill has impo an limi a ions. Fo ins ance, i is no
e ec i e when he occlusion is in he dis al ce eb al a e ies (small a e ies), which occu s
in mo e han 30% o ischemic s okes. The use o ERT may also elease mic oclo s wi h
he isk o subsequen dis al s okes; he e o e, i is usually employed in combina ion wi h
PA o imp o e he a e o epe usion as well as o dissol e he mic oclo s p oduced
du ing mechanical h ombec omy. In addi ion, ERT equi es he a ailabili y o pe sonnel
o imaging, su ge y, and endo ascula acili ies, which limi s i s use o he main ci ies
wi h la ge hospi als [30].
This shows ha PA pe usion emains he gold s anda d o acu e ischemic s oke
and is a well-es ablished p ocedu e widely used in all hospi als [31].
The las ew yea s ha e seen a new clo -bus ing d ug, named enec eplase, ha is
al eady accep ed in he ea men o hea a ack. Based on i s chemical p ope ies, i could
be a sa e and e ec i e d ug o ea ing s oke, becoming a se ious PA compe i o .
In addi ion o he new ad ances in he chemical cus omiza ion o PA, nanomedical
app oaches o he a ge ed deli e y o h omboly ic agen s ha e been in ensi ely in es i-
ga ed. These nanomedical app oaches a e ocused on a ge ing he d ug o he a e ial clo
egion o inc ease he h omboly ic e icacy and induce con olled d ug elease o educe
he isk o HT associa ed wi h delayed adminis a ion [32] (Figu e 4).
3.1. Nanoca ie s o PA
To da e, many nanomedical ca ie s ha e been de eloped o encapsula e and op imize
he e icacy and sa e y o PA, such as liposomes, polyme ic NPs, magne ic NPs (MNPs),
mic obubbles, and echogenic liposomes, wi h di e en le els o success [32,33] (Figu e 5).
Liposomes, comp ising an amphiphilic phospholipid bilaye and a hyd ophilic aque-
ous co e, can encapsula e bo h wa e -soluble and wa e -insoluble compounds. Lipo-
somes we e one o he i s nanosys ems employed as d ug deli e y sys ems o PA.
Hee emans e al.
showed, o example, ha PA-loaded liposomes ha e a be e h om-
boly ic e ec han ee PA [
34
]. In compa ison wi h o he nanoca ie s, liposomes also
ha e many ad an ages such as biodeg adabili y, biocompa ibili y, low immunogenici y,
and lexibili y in coupling wi h si e-speci ic ligands.
Bo h syn he ic and na u al polyme s ha e been used o ab ica e NPs o h omboly ic
he apy because o hei biocompa ibili y and biodeg adabili y. Syn he ic polyme s can be
easily unc ionalized, and he esul ing NPs can be cus omized in e ms o size, po osi y,
and hyd ophobici y. Poly(lac ic-co-glycolic acid) (PLGA), an FDA-app o ed polyme , ep-
esen s one o he mos common polyme s used o PA deli e y. Na u al polyme s such
as chi osan and gela in ha e also been in es iga ed as al e na i e ma e ials o PA deli -
Pha maceu ics 2021,13, 757 7 o 26
e y [
33
]. Al hough hese na u al polyme s a e biocompa ible and biodeg adable, hey ha e
a iable composi ions depending on he na u al sou ce and hey need o be s anda dized.
Pha maceu ics 2021, 13, x FOR PEER REVIEW 7 o 27
Figu e 4. To inc ease he ecanaliza ion e ec i eness o ecombinan issue plasminogen ac i a o
( PA) and dec ease he side e ec s, some app oaches ha e been ca ied ou . Fo example, combi-
na ion wi h o he d ugs, he chemical cus omiza ion o he molecule, in a-a e ial adminis a ion,
combina ion wi h sono h ombolysis, and he encapsula ion o he d ug in deli e y sys ems. Figu e
c ea ed wi h BioRende .com.
3.1. Nanoca ie s o PA
To da e, many nanomedical ca ie s ha e been de eloped o encapsula e and op i-
mize he e icacy and sa e y o PA, such as liposomes, polyme ic NPs, magne ic NPs
(MNPs), mic obubbles, and echogenic liposomes, wi h di e en le els o success [32,33]
(Figu e 5).
Figu e 4.
To inc ease he ecanaliza ion e ec i eness o ecombinan issue plasminogen ac i a o
( PA) and dec ease he side e ec s, some app oaches ha e been ca ied ou . Fo example, combi-
na ion wi h o he d ugs, he chemical cus omiza ion o he molecule, in a-a e ial adminis a ion,
combina ion wi h sono h ombolysis, and he encapsula ion o he d ug in deli e y sys ems. Figu e
c ea ed wi h BioRende .com.
Pha maceu ics 2021, 13, x FOR PEER REVIEW 7 o 27
Figu e 4. To inc ease he ecanaliza ion e ec i eness o ecombinan issue plasminogen ac i a o
( PA) and dec ease he side e ec s, some app oaches ha e been ca ied ou . Fo example, combi-
na ion wi h o he d ugs, he chemical cus omiza ion o he molecule, in a-a e ial adminis a ion,
combina ion wi h sono h ombolysis, and he encapsula ion o he d ug in deli e y sys ems. Figu e
c ea ed wi h BioRende .com.
3.1. Nanoca ie s o PA
To da e, many nanomedical ca ie s ha e been de eloped o encapsula e and op i-
mize he e icacy and sa e y o PA, such as liposomes, polyme ic NPs, magne ic NPs
(MNPs), mic obubbles, and echogenic liposomes, wi h di e en le els o success [32,33]
(Figu e 5).
Figu e 5.
Examples o nanodeli e y sys ems o PA o ex end he hal -li e o he he apeu ic agen ,
o educe seconda y e ec s, imp o e he ecanaliza ion, a ge he he apy, and imp o e he e icacy.
Figu e c ea ed wi h BioRende .com.
Conjuga ion o PA wi h MNPs has been equen ly employed in h omboly ic he apy.
This s a egy allows he e alua ion o he a e o he ea men based on he capabili y o he
Pha maceu ics 2021,13, 757 8 o 26
pa icles o ac as a magne ic esonance con as agen . In addi ion, unde he applica ion
o a local magne ic ield, he h omboly ic e ec o he d ugs ends o accumula e a a
speci ic si e, which is a a o able p ope y o a ge ed h ombolysis [
35
]. Cu en ly, i on
oxide NPs a e he mos explo ed MNPs because o hei biodeg adabili y and known
me abolic pa hways. Among i on oxides, magne i e (Fe
3
O
4
) and maghemi e (
γ
-Fe
2
O
3
)
a e e y popula candida es ha possess sui able magne ic p ope ies o biomedical
applica ions [33].
Mic obubbles (MBs) a e mic osphe es illed wi h gas o ai used as con as agen s
o eco-Dopple imaging owing o hei acous ic cha ac e is ics. The combina ion wi h
PA o e alua e epe usion success showed ha hese MBs we e able o accele a e he
h omboly ic ac i i y, which was a ibu ed o s able and ine ial ca i a ion, esul ing
in mic os eaming and e osion o he clo su ace, which enhanced he pene a ion o
h omboly ic agen s in o he clo s. These esul s led o he ab ica ion o MBs loaded wi h
PA o op imize he h omboly ic e icacy and po en ially educe he hemo hagic isk
unde ul asound exposu e [36,37].
Simila o MBs, echogenic liposomes a e mul i unc ional phospholipid bilaye -encapsula ed
esicles, which can be used as con as agen s o sonog aphy and d ug deli e y, including
PA. The exposu e o PA-loaded echogenic liposomes o ul asound can dis up he lipid
shell and hence igge d ug elease. As a esul , unde ul asound s imuli, PA can be eleased
locally, hus inc easing he concen a ion o PA in he a ea o h ombus, educing he equi ed
he apeu ic dose o PA and consequen ly he isk o hemo hage. Mo eo e , he gas encapsu-
la ed in echogenic liposomes can exe a ca i a ion- ela ed mechanism, leading o enhanced
h omboly ic e ec s [38].
3.2. Clo Ta ge s o PA Nanoca ie Vec o iza ion
Al hough passi e a ge ing o PA nanosys ems has shown p omising h omboly ic
e icacy, ecen ac i ely a ge ed nanoca ie s ha e been designed. Ac i e a ge ing pe mi s
d ug accumula ion speci ically a he h ombus si e and has he po en ial o enhance
enzyme pene a ion in o deeply localized h ombi. Many o he a ge s a e ela ed o clo
componen s, such as ac i a ed pla ele ecep o s, h ombin, ib in, ed blood cells, o on
Willeb and ac o ( WF) [32] (Figu e 6).
A e h ombus o ma ion, h ombin and collagen included in he clo cause he
ac i a ion o he pla ele glycop o ein (GP) IIb/IIIa ecep o , which leads o changes in
he pla ele cells om an inac i e o an ac i e s a e. The c i ical ole o hese ecep o s
in h ombus o ma ion has p o ided an excellen a ge o nanosys ems designed o
h omboly ic he apy. Fo example, he pep ides a ginine–glycine–aspa ic acid (RGD)
o a ginine–glycine–aspa ic acid–se ine ha e been used o a ge NPs o he GP IIb/IIIa
ecep o s o he clo egion. Thus, deco a ing he liposome su ace wi h RGD pep ide
esul ed in an inc ease in ib inoly ic ac i i y and a ge ing o pla ele s [
39
]. Simila esul s
on clo dissolu ion we e also obse ed in o he independen s udies employing PLGA
and chi osan NPs cus omized wi h he RGD pep ide [
40
]. These pep ide sequences ha e
been used o a ge magne ic NPs wi h PA, allowing he moni o ing o h ombolysis by
MRI [
41
]. P-selec in, a ansmemb ane glycop o ein o ac i a ed pla ele s, is ano he clo
a ge o PA ec o iza ion using he polyme ucoidan [42].
Th ombin is o he an abundan componen o he clo ha has been widely used o
a ge h omboly ic NPs, as shown by Absa e al. [43].
Fib in (also called Fac o Ia), which is in ol ed in blood clo ing, is o med by he
ac ion o he p o ease h ombin on ib inogen, which causes i o polyme ize. The poly-
me ized ib in, oge he wi h pla ele s, o ms a hemos a ic plug o clo o e a wound si e.
Fib in appea s only in he clo egion, bu is no p esen in blood ci cula ion and in no mal
issue, which makes i a sui able goal o ib inoly ic nanosys ems [
43
]. Cus omiza ion o
h omboly ic NPs wi h an ibodies agains di e en agmen s o he ib in, such as B-chain
o D-dime , con i med he highe e icacy and sa e y o hese o mula ions wi h espec o
ee PA adminis a ion [44,45]. Recen s udies epo ed by Huai-An e al. desc ibed ha
Pha maceu ics 2021,13, 757 9 o 26
he associa ion be ween magne ic NPs coa ed wi h PLGA, PA, and a pep ide agains he
ib in clo egion was linked wi h highe h ombolysis a e
in i o
and educ ion o clo
lysis ime in i o [46].
Pha maceu ics 2021, 13, x FOR PEER REVIEW 9 o 27
Figu e 6. S a egies o a ge he nanosys ems o he h ombus. Figu e c ea ed wi h BioRen-
de .com.
A e h ombus o ma ion, h ombin and collagen included in he clo cause he ac i-
a ion o he pla ele glycop o ein (GP) IIb/IIIa ecep o , which leads o changes in he
pla ele cells om an inac i e o an ac i e s a e. The c i ical ole o hese ecep o s in
h ombus o ma ion has p o ided an excellen a ge o nanosys ems designed o
h omboly ic he apy. Fo example, he pep ides a ginine–glycine–aspa ic acid (RGD) o
a ginine–glycine–aspa ic acid–se ine ha e been used o a ge NPs o he GP IIb/IIIa e-
cep o s o he clo egion. Thus, deco a ing he liposome su ace wi h RGD pep ide e-
sul ed in an inc ease in ib inoly ic ac i i y and a ge ing o pla ele s [39]. Simila esul s
on clo dissolu ion we e also obse ed in o he independen s udies employing PLGA and
chi osan NPs cus omized wi h he RGD pep ide [40]. These pep ide sequences ha e been
used o a ge magne ic NPs wi h PA, allowing he moni o ing o h ombolysis by MRI
[41]. P-selec in, a ansmemb ane glycop o ein o ac i a ed pla ele s, is ano he clo a ge
o PA ec o iza ion using he polyme ucoidan [42].
Th ombin is o he an abundan componen o he clo ha has been widely used o
a ge h omboly ic NPs, as shown by Absa e al. [43].
Fib in (also called Fac o Ia), which is in ol ed in blood clo ing, is o med by he
ac ion o he p o ease h ombin on ib inogen, which causes i o polyme ize. The pol-
yme ized ib in, oge he wi h pla ele s, o ms a hemos a ic plug o clo o e a wound si e.
Fib in appea s only in he clo egion, bu is no p esen in blood ci cula ion and in no mal
issue, which makes i a sui able goal o ib inoly ic nanosys ems [43]. Cus omiza ion o
h omboly ic NPs wi h an ibodies agains di e en agmen s o he ib in, such as B-chain
o D-dime , con i med he highe e icacy and sa e y o hese o mula ions wi h espec o
ee PA adminis a ion [44,45]. Recen s udies epo ed by Huai-An e al. desc ibed ha
he associa ion be ween magne ic NPs coa ed wi h PLGA, PA, and a pep ide agains he
ib in clo egion was linked wi h highe h ombolysis a e in i o and educ ion o clo
lysis ime in i o [46].
Figu e 6.
S a egies o a ge he nanosys ems o he h ombus. Figu e c ea ed wi h BioRende .com.
Gela in nanos uc u es we e also designed o encapsula e PA o con olled d ug
deli e y. Gela in is used because o i s biocompa ibili y, and i s abili y o bind o he WF, an
endo helium ac o ha pa icipa es ac i ely in clo o ma ion. Uesugi and cowo ke s used
his polyme o encapsula e PA and gene a e ul asound- esponsi e NPs. These assays
showed ha PA ac i i y was supp essed a ound 50% in hese gela in complexes, and
he ac i i y was comple ely eco e ed on applica ion o ul asound (2 MHz, 0.72 W/cm
2
).
In i o
analysis in swine con i med he endency o NPs o bind o he clo s, and inc ease
he ecanaliza ion e ec i eness unde ul asound adia ion [47,48].
The in e es in biomime ic NPs o encapsula e PA has inc eased signi ican ly in
ecen yea s. Biomime ic NPs, mainly based on he use o cell memb anes o bo h pla ele s
and ed blood cells, a e in isible o he phagocy ic sys em, and aim a inc easing he
hal -li e o he NPs and imp o ing he a ge ing e icacy o he h ombus egion. P omising
esul s we e ob ained o he ib inoly ic ac i i y wi h PA conjuga ed on he su ace
o ed blood cell memb anes [
49
]. One o he i s success ul s udies using pla ele s o
encapsula e PA was ca ied ou by Hu e al., who published a biomime ic NP based on
pla ele memb ane coa ing. This no el design showed an imp o emen in he e icacy and
sa e y o he h omboly ic ac i i y o PA in s oke models. The e icacy o his “sma ”
s a egy was also enhanced by he a ini y binding o he pla ele memb ane o he clo
componen s [50].
3.3. T igge ing Con olled Release
The con olled elease o an encapsula ed d ug (such as PA), has been one o he
mos impo an ad an ages in he design o new nanosys ems. This d ug-con olled elease
allows o ocus on he e ec o he d ug on a speci ic a ge , inc eases he e icacy o he
d ug, and impo an ly, educes he side e ec s.
Pha maceu ics 2021,13, 757 16 o 26
syn hesized and es ed in a oden s oke model wi h signi ican in a c olume educ-
ion and neu ological de ici imp o emen [
91
]. In a ecen expe imen al s udy, a new
bioenginee ed nanopla ele was de eloped o sequen ial si e-speci ic deli e y o PA and
neu op o ec an ZL006e, esul ing in a dec eased ischemic olume and ROS le els in a a
model o b ain ischemia [50].
5. Nanomedicine o S oke Reco e y The apies
S em cell-based he apies ha e eme ged as a p omising ool o eco e y ea men
in delayed phases o s oke owing o hei mul ipo en iali y, abili y o elease g ow h
ac o s, and immunomodula o y capaci ies. Thus, his ansdi e en ia ion can p oduce
cells wi h a neu al lineage, induce neu ogenesis, angiogenesis, synap ogenesis, and ac i a e
endogenous es o a i e p ocesses h ough he p oduc ion o di e en cy okines and ophic
ac o s. Mo eo e , he egula ion o CBF, he BBB, and o he neu op o ec i e mechanisms,
such as he educ ion o apop osis, in lamma ion, demyelina ion, and he inc ease in
as ocy e su i al ha e been shown o be bene icial mechanisms a e s oke [92].
Despi e special a en ion o s em cells as a p omising he apeu ic candida e o s oke,
pa ame e s such as adminis a ion ou e o cell dosage a e s ill unde discussion. MRI in
combina ion wi h con as agen s o NPs o
in i o
cell acking is widely used o e alua e
hese pa ame e s [3,93].
5.1. MRI Con as Cell Agen s
MRI con as agen s o cell agging equi e NPs wi h special cha ac e is ics, such as
high magne iza ion alues and small and na ow size dis ibu ions. The mal decomposi-
ion and chemical cop ecipi a ion a e he mos widely used p ocedu es. The cop ecipi a ion
echnique is p obably he simples and mos e icien chemical ou e o ob ain magne ic
NPs. The main ad an age o his me hod is ha a la ge numbe o pa icles can be syn-
hesized easily and economically [
94
]. Howe e , aside om he magne ic p ope ies, he
a ge ing su ace coa ing o he magne ic pa icles mus be non- oxic and biocompa ible o
be use ul o localized deli e y [95].
Among all he coa ing possibili ies, dex an is one o he mos widely used polyme s
o MNP coa ing because o i s biocompa ibili y. This polysaccha ide polyme is composed
exclusi ely o
α
-D-glucopy anosyl uni s wi h a ying deg ees o chain leng h and b anch-
ing. In 1982, Molday and Mackenzie we e he i s o epo he o ma ion o magne i e
in he p esence o dex an 40,000 [
96
]. The syn hesis o dex an-coa ed NPs is an in si u
p ocedu e, and he e ec o educing he e minal glucose o dex an on he o ma ion and
s abili y o dex an-coa ed supe pa amagne ic NPs has been demons a ed o be signi ican
o pa icle size, coa ing s abili y, and magne ic p ope ies [94].
Fo cell labeling applica ions, in addi ion o he selec ion o op imal ma e ial and
biocompa ible coa ing, ans ec ion agen s (TAs) o elec opo a ion p ocesses a e equi ed
o allow NPs o en e he cells, which is a c i ical s ep [
97
,
98
]. Fo ins ance, Tas, such
as poly-L-lysine (PLL) a e oxic o cells; he e o e, in expe imen al s udies based on cell
acking, i is necessa y o de e mine he app op ia e a io o NPs/TAs o e icien ly label
cells and minimize oxic side e ec s. In addi ion, s em cells a e sensi i e o cul u e medium
and can exhibi di e en beha io s when hei composi ion is changed; he e o e, cellula
pheno ype and unc ionali y ( o example, g ow h ac o elease) a e labeling mus be
e i ied o gua an ee he biocompa ible na u e o he ma e ial [
97
]. O he pa ame e s ha
a e impo an o p ope ly cha ac e ize he cell labeling a e he amoun o NP up ake, ROS
gene a ion, and in acellula NP localiza ion [99].
O he me hods o syn hesize coa ed magne ic NP cell labeling a e much mo e complex
and equi e esol ing o solubili y issues. Fo ins ance, Jain e al. [
100
] de eloped a no el
oleic acid (OA)–Plu onic-s abilized i on oxide MNP o mula ion, in which he OA shell
su ounds he i on oxide NPs, and hen he polyme (Plu onic) is ancho ed a he in e ace
o he OA shell o con e an aqueous dispe si y o he o mula ion. Plu onic coa ing also has
he ad an age o no equi ing ans ec ion agen s o elec opo a ion o cell labeling [
101
].

Pha maceu ics 2021,13, 757 17 o 26
5.2. MNPs o MRI Cell T acking in S oke
Rela i ely ew s udies ha e compa ed he di e en possible ou es o s em cell ad-
minis a ion o s oke eco e y. The i s s udies ha used s em cells o ce eb o ascula
diseases in ol ed looking o a neu onal eplacemen , so an in apa enchymal injec ion
was chosen as he mos di ec ou e o cell eng a men . These s udies showed ha s em
cells no only su i ed bu mig a ed o he a ec ed zone [
102
,
103
]. Howe e , his choice
is no he mos sui able because o he need o open a c anial window and also because i
damages he b ain pa enchyma, which is no con enien o s oke pa ien s.
The main al e na i e o his ou e o adminis a ion is he ascula ou e, ei he in a-
a e ial (i.a.) o in a enous (i. .), which a e cu en ly he mos used ou es o cell deli e y.
I. . injec ions a e minimally in asi e bu cell acking s udies ollowing his ou e ha e
shown ha mos adminis e ed cells emain apped in he lungs [
104
,
105
], li e [
106
], and
spleen [
107
], indica ing ha a educed numbe o cells each he b ain. On he con a y, i.a.
adminis a ion is a p omising s a egy o di ec he majo i y o injec ed cells o he b ain,
howe e , his is a isky adminis a ion ou e and he a e o injec ed cells ollowing his
ou e emains unknown due o high a ia ion in he epo ed esul s [97].
Whe he one ou e is mo e e icien han he o he is no clea and depends on he
cell ype used. Thus, in some s udies, i was ound ha he injec ion o neu al p ogeni o
cells by an i.a. ou e h ough he ca o id a e y p esen ed a highe mig a ion a e and a
wide dis ibu ion pa e n han i. . adminis a ion. Ne e heless, he mo ali y a e o i.a.
deli e y was signi ican ly highe (41%) han ha o i. . deli e y (8%) [
108
]. Howe e , in
o he s udies wi h bone ma ow s em cells and bone ma ow mononuclea cells, he e was
no g ea e mo ali y o g ea e eco e y o in a c olume o one ou e compa ed o he
o he [109,110].
The size o s em cells is also a c i ical pa ame e when passing h ough he lungs,
and should be aken in o conside a ion when deciding he bes ou e o adminis a ion.
Fo example, when using mesenchymal s em cells (MSCs), he majo i y o hem become
apped in he lungs, while he neu onal s em cells (NSCs) ha e a wo- old highe pass-
h ough a e [105].
To cla i y he disc epancies in he bes ou e o cell adminis a ion in s oke and
using syn hesized dex an-coa ed supe pa amagne ic NPs o MRI cell acking, MSCs
we e in es iga ed o de e mine i hey a e able o each he b ain ollowing i.a. o i. .
adminis a ion. They we e adminis e ed a e ansien ce eb al ischemia in a s, and hei
he apeu ic e ec s we e e alua ed o bo h ou es o adminis a ion [
97
]. The indings
om hese s udies show ha MSCs we e inside he b ain issue ollowing i.a. bu no i. .
adminis a ion in ischemic a s (Figu e 11). Howe e , he i.a. ou e inc eased he isk o
ce eb al lesions (mic os okes) (Figu e 12) and did no imp o e unc ional eco e y, while
in a enous deli e y p oduced unc ional eco e y and was sa e. This ac implies ha
he cell ea men bene i s a e no a ibu able o b ain MCS eng a ing a e s oke [
97
].
P esumably, MSCs apped in he lung would a ec immune sys em unc ion sys emically,
suppo ing he hypo hesis ha bene icial MSC e ec s a e s oke can be achie ed in
he absence o signi ican MSC ec ui men o he in a c egion, and e en o e come he
associa ed isks o i.a. adminis a ion. The bene icial e ec o i. . MSC adminis a ion is
mainly associa ed wi h he sec e ion o a la ge numbe o neu op o ec i e and neu o ophic
ac o s ha p omo e epai and eco e y h ough nume ous pa hways [97].
5.3. Magne ic Vec o iza ion
Owing o he ele ance o he ou e o adminis a ion and cell/dose on he e icacy
o s em cell he apy, di e en s a egies ha e eme ged o imp o e cell deli e y in a ge
a eas. In his ega d, in o de o inc ease he numbe o injec ed cells close o he ischemic
lesion, magne ic ec o iza ion o cells agged wi h supe pa amagne ic NPs has been es ed
wi h success ul esul s, showing ha magne ic cell loading ep esen s a sa e and e ec i e
s a egy o p ecise cell guidance in o speci ic b ain a eas [111].
Pha maceu ics 2021,13, 757 18 o 26
Pha maceu ics 2021, 13, x FOR PEER REVIEW 18 o 27
The size o s em cells is also a c i ical pa ame e when passing h ough he lungs, and
should be aken in o conside a ion when deciding he bes ou e o adminis a ion. Fo
example, when using mesenchymal s em cells (MSCs), he majo i y o hem become
apped in he lungs, while he neu onal s em cells (NSCs) ha e a wo- old highe pass-
h ough a e [105].
To cla i y he disc epancies in he bes ou e o cell adminis a ion in s oke and us-
ing syn hesized dex an-coa ed supe pa amagne ic NPs o MRI cell acking, MSCs we e
in es iga ed o de e mine i hey a e able o each he b ain ollowing i.a. o i. . admin-
is a ion. They we e adminis e ed a e ansien ce eb al ischemia in a s, and hei he -
apeu ic e ec s we e e alua ed o bo h ou es o adminis a ion [97]. The indings om
hese s udies show ha MSCs we e inside he b ain issue ollowing i.a. bu no i. . ad-
minis a ion in ischemic a s (Figu e 11). Howe e , he i.a. ou e inc eased he isk o ce -
eb al lesions (mic os okes) (Figu e 12) and did no imp o e unc ional eco e y, while
in a enous deli e y p oduced unc ional eco e y and was sa e. This ac implies ha
he cell ea men bene i s a e no a ibu able o b ain MCS eng a ing a e s oke [97].
P esumably, MSCs apped in he lung would a ec immune sys em unc ion sys emi-
cally, suppo ing he hypo hesis ha bene icial MSC e ec s a e s oke can be achie ed
in he absence o signi ican MSC ec ui men o he in a c egion, and e en o e come he
associa ed isks o i.a. adminis a ion. The bene icial e ec o i. . MSC adminis a ion is
mainly associa ed wi h he sec e ion o a la ge numbe o neu op o ec i e and neu-
o ophic ac o s ha p omo e epai and eco e y h ough nume ous pa hways [97].
Figu e 11. Magne ic esonance image (MRI) o a a b ain slice, 4 h a e in aa e ial adminis a-
ion o mesenchymal s em cells (MSCs) labeled wi h magne ic pa icles. Labeled cells can be ob-
se ed as black punc a e pa e ns in he igh b ain hemisphe e. Rep oduced wi h pe mission
om [97], Sp inge Na u e, 2017.
Figu e 11.
Magne ic esonance image (MRI) o a a b ain slice, 4 h a e in aa e ial adminis a ion
o mesenchymal s em cells (MSCs) labeled wi h magne ic pa icles. Labeled cells can be obse ed
as black punc a e pa e ns in he igh b ain hemisphe e. Rep oduced wi h pe mission om [
97
],
Sp inge Na u e, 2017.
Pha maceu ics 2021, 13, x FOR PEER REVIEW 19 o 27
Figu e 12. Uppe igu e: Mul i ocal ischemia is obse ed ac oss he b ain a e adminis a ion o 1 ×
10
6
MSCs (indica ed wi h yellow a ows). Lowe igu e. Elec on ansmission mic og aph o he a
b ain co ex 4 h a e in a-a e ial (i.a.) deli e y o MSCs wi h magne ic pa icles, showing he a -
e ial occlusion caused by he cell adminis a ion. (A) Dila ed b ain essel su ounded by neu opils
(scale ba 5 µm). (B) Magni ica ion o essel dila ion in A. Red blood (RB) co puscles a e on he
igh , and pla ele s (PTs) and MSCs can be obse ed inside he essel (scale ba 2 µm). (C) Magni i-
ca ion o he uppe essel expansion in B (scale ba 1 µm). (D) Longi udinal sec ion o a b ain essel
in which wo labeled cells can be obse ed (scale ba 2 µm). Rep oduced wi h pe mission om [97],
Sp inge Na u e, 2017.
5.3. Magne ic Vec o iza ion
Owing o he ele ance o he ou e o adminis a ion and cell/dose on he e icacy o
s em cell he apy, di e en s a egies ha e eme ged o imp o e cell deli e y in a ge a -
eas. In his ega d, in o de o inc ease he numbe o injec ed cells close o he ischemic
lesion, magne ic ec o iza ion o cells agged wi h supe pa amagne ic NPs has been
es ed wi h success ul esul s, showing ha magne ic cell loading ep esen s a sa e and
e ec i e s a egy o p ecise cell guidance in o speci ic b ain a eas [111].
6. Discussion
S oke is a disease ha occu s unexpec edly and has a disas ous ou come. App oxi-
ma ely 15 million people expe ience a s oke episode e e y yea wo ldwide, o which 33%
a e le wi h a pe manen disabili y, whe eas app oxima ely 40% o cases esul in dea h.
Due o he high impac o he disease a ound he wo ld, s oke is anked as he second
deadlies disease o indi iduals o e 60 yea s o age. Despi e he bene i s o mechanical
ecanaliza ion, PA h ombolysis emains he mains ay o acu e s oke he apy in many
hospi als, bu i s use is limi ed o a educed numbe o pa ien s, mainly due o he na ow
he apeu ic window and hemo hagic complica ions. Many e o s ha e been made o in-
es iga e new p o ec i e d ugs wi h success ul esul s du ing p eclinical analysis, bu
Figu e 12.
Uppe igu e: Mul i ocal ischemia is obse ed ac oss he b ain a e adminis a ion o
1×106
MSCs (indica ed wi h yellow a ows). Lowe igu e. Elec on ansmission mic og aph o he
a b ain co ex 4 h a e in a-a e ial (i.a.) deli e y o MSCs wi h magne ic pa icles, showing he
a e ial occlusion caused by he cell adminis a ion. (
A
) Dila ed b ain essel su ounded by neu opils
(scale ba 5
µ
m). (
B
) Magni ica ion o essel dila ion in A. Red blood (RB) co puscles a e on he igh ,
and pla ele s (PTs) and MSCs can be obse ed inside he essel (scale ba 2
µ
m). (
C
) Magni ica ion
o he uppe essel expansion in B (scale ba 1
µ
m). (
D
) Longi udinal sec ion o a b ain essel in
which wo labeled cells can be obse ed (scale ba 2
µ
m). Rep oduced wi h pe mission om [
97
],
Sp inge Na u e, 2017.
Pha maceu ics 2021,13, 757 19 o 26
6. Discussion
S oke is a disease ha occu s unexpec edly and has a disas ous ou come. App oxi-
ma ely 15 million people expe ience a s oke episode e e y yea wo ldwide, o which 33%
a e le wi h a pe manen disabili y, whe eas app oxima ely 40% o cases esul in dea h.
Due o he high impac o he disease a ound he wo ld, s oke is anked as he second
deadlies disease o indi iduals o e 60 yea s o age. Despi e he bene i s o mechanical
ecanaliza ion, PA h ombolysis emains he mains ay o acu e s oke he apy in many
hospi als, bu i s use is limi ed o a educed numbe o pa ien s, mainly due o he na ow
he apeu ic window and hemo hagic complica ions. Many e o s ha e been made o
in es iga e new p o ec i e d ugs wi h success ul esul s du ing p eclinical analysis, bu
none o hem ha e demons a ed bene i s in clinical p ac ice, and eco e y cell he apies
equi e addi ional clinical analysis and s anda dized p o ocols ( o example, cell ype, dose,
o ou e o adminis a ion) be o e becoming a he apeu ic esou ce o s oke pa ien s.
In he las decade, he de elopmen o nanomedicine has opened new and p omising
oppo uni ies o he diagnosis and ea men o neu ological pa hologies, including s oke
(Table 1). Fo ins ance, he use o NP con as agen s o s oke diagnosis o
in i o
cell
acking has shown o be a powe ul ool o s udy new aspec s ela ed o he imaging
diagnosis o o e alua e he isks o cell he apies a e adminis a ion; howe e , bioac-
cumula ion o deg ada ion a e p ocesses ha a e s ill no well known o many NPs and
o en depend on he ma e ial used; he e o e, he use o NP con as agen s o clinical use
equi es u he sa e y analysis.
Table 1. Summa y o he mos ele an nanosys ems de eloped in he ischemia ield.
Diagnosis
Imaging Tool Type o Nanopa icle Ta ge T igge ing Re e ence
MRI
MPIOs
VCAM-1 [18]
ICAM-1 [19]
P-selec in [20]
VCAM-1 and P-selec in [21]
Mul ime ic magne i e pa icles Pep ide o in acellula mac ophage
p o eases [23]
CT Gold NPs Fib in [22]
Th omboly ic T ea men
Type o Nanopa icle Ta ge T igge ing Re e ence
Liposomes Liposomes Plasminogen [33,34]
Magne oliposomes
The mosensi i e and magne ic guidance
[60]
Polyme ic NPs
PLGA NPs coa ed wi h chi osan Fib in [39,40]
Polysaccha ide-poly(isobu ylcyanoac yla e) nanopa icles P-selec in [42]
Laye -by-laye capsules Glycop o ein (GP) IIb/IIIa
Th ombin esponsi e [43]
Poly(N-isop opylac ylamide) nanogels Fib in [44]
Nea -in a ed luo escen dye-conjuga ed bo ona ed
mal odex in
Fib in
H2O2 esponsi e [51]
NIR luo escen dye-conjuga ed bo ona e an ioxidan
polyme s
( BAP) and ib in- a ge ing lipopep ides
Fib in
H2O2 esponsi e [52]
P od ug nanoca ie s based on PEG and diosgenin
de i a i es pH esponsi e [53]
Agg ega es o mul iple smalle NPs o PLGA Shea s ess esponsi e [57]
Laye -by-laye sub-mic ome ic nanocapsules wi h gela in Von Willeb and ac o
Ul asounds [65]
Nea -in a ed luo escen dye-conjuga ed bo ona ed
mal odex in
Fib in
H2O2 esponsi e [51]
Pha maceu ics 2021,13, 757 20 o 26
Table 1. Con .
Diagnosis
Imaging Tool Type o Nanopa icle Ta ge T igge ing Re e ence
Me al NPs
Magne ic ods Magne ic guidance [35]
Fe3O4-based PLGA Fib in [41]
PLGA magne ic NPs Fib in
Magne ic guidance [46]
Me al–o ganic amewo k-de i ed ca bon nanos uc u es
GP IIb/IIIa
Hype he mia and ROS sensi i e unde
NIR lase
[59]
Chi osan nanocomposi es wi h Fe3O4Magne ic guidance [61]
Magne ic nanocubes wi hin a deoxychi osan co e Magne ic guidance [62]
Magne ic Fe3O4mic o ods Magne ic guidance [63]
Mic obubbles
Fib in
Ul asounds [36]
Fib in
Ul asounds [37]
Echogenic liposomes Ul asounds [38]
Ul asounds [64]
Biomime ic NPs
Red blood cells doped wi h NIR imaging agen [49]
Nanopla ele wi h PA and a neu op o ec an (ZL006e) Th ombin [50]
Pla ele mic opa icle inspi ed GP IIb/IIIa and P-selec in [54]
Neu op o ec ion
App oach Type o Nanopa icle Ta ge T igge ing Re e ence
In lamma ion
Exosomes loading cu cumin In eg in α β3 [71]
Neu ophil-like cell memb ane-coa ed mesopo ous
nanozyme In lamed endo helium [72]
Sphe es and ellip ical disks ICAM-1 [79]
Gold NPs wi h couma in-PEG- hiol [80]
Sphe e and od NPs VCAM-1 [81]
Oxida i e s ess
NPs o es e a ol [85]
Ce ium oxide NPs [86]
Glu ama e
exci o oxici y Gold NPs wi h meman ine [88]
Combina ion wi h PA
Liposomes loading PA and asudil [90]
Sel -assembled an ioxidan NPs wi h PA [91]
Neu o eco e y
App oach Type o Nanopa icle Ta ge T igge ing Re e ence
MRI cell acking Supe pa amagne ic i on oxide nanopa icles (SPIONs)
Cell acking o mesenchymal s em cells
(MSCs) [97]
Magne ic ec o iza ion Supe pa amagne ic i on oxide nanopa icles (SPIONs)
Cell acking o endo helial p ogeni o
cells (EPCs)
Magne ic guidance
[111]
In e cellula adhesion molecule 1 (ICAM-1); magne ic esonance imaging (MRI); nea -in a ed (NIR); nanopa icles (NPs); polye hylene
glycol (PEG); poly lac ic-co-glycolic acid (PLGA); eac i e oxygen species (ROS); ecombinan issue plasminogen ac i a o ( PA); ascula
cell adhesion molecule 1 (VCAM-1).
New ad ances in he ield o nano echnology ha e allowed he de elopmen o “sma ”
deli e y nanopla o ms ha ha e signi ican ly imp o ed he e icacy o h omboly ic ea -
men s and p o ec i e d ugs by educing he side e ec s. The sea ch o no el he apeu ic
s a egies o s oke has inc eased signi ican ly, and he combina ion o hese he apies
wi h e icien solu ions p o ided by he ield o nano echnology would de ini ely help o
ind new e ec i e he apeu ic o mula ions o imp o e he ou comes o s oke pa ien s.
Pha maceu ics 2021,13, 757 21 o 26
One o he majo limi a ions o NPs as medicinal p oduc s is ha a e adminis a ion,
hese ex e nal “nanobodies” may ac i a e he coagula ion p ocess wi h he isk o clo ing.
Mo eo e , NPs a e apidly seques e ed om he bloods eam by ci cula ing phagocy ic
cells and mac ophages. This apid seques e ing ep esen s one o he majo challenges o
nano o mula ions o e ec i e
in i o
a ge ing o speci ic cells and issues. The de elop-
men o biomime ic nanosys ems de i ed om cellula memb anes has o e ed a no el
solu ion o sol e hese limi a ions as hey emain “in isible” o he phagocy ic sys em,
he eby inc easing hei ee-li e in blood ci cula ion and imp o ing he a ge ing e iciency.
In he ield o s oke, biomime ic nanosys ems de i ed om cellula memb anes, mo e
speci ically om pla ele s, ha e eme ged as a p omising s a egy o imp o e he e icacy
and sa e y o PA, wi h speci ic a ge ing o blood clo s.
Finally, success ul p eclinical esul s wi h “sma ” deli e y nanopla o ms o s oke o
o he pa hologies usually equi e scaled-up p oduc ion o subsequen clinical alida ion.
Risk analysis o scaled-up p oduc ion should be aken in o conside a ion o an icipa e
limi a ions such as syn hesis issues, ime consump ion o poo ba ch consis ency, and
ep oducibili y, which could limi he ansla ionali y o he he apy.
Au ho Con ibu ions:
C.C.-P. and A.d.S.-C.: w i ing—o iginal d a p epa a ion, E.P., J.P., D.M.,
D.V., B.P., and F.C.: w i ing— e iew and edi ing, F.C.: supe ision, F.C.: p ojec adminis a ion. All
au ho s ha e ead and ag eed o he published e sion o he manusc ip .
Funding:
This p ojec was suppo ed by he FRQS, ISCIII (AC19/00031 and AC20/00041), and
ANR unde he amewo k o Eu oNanoMed III_2020 (PLATMED_p ojec ); he Eu opean Union
p og am FEDER and he Eu opean Regional De elopmen Fund–ERDF; and he Xun a de Galicia
(IN607D2020/03 and ED431G2019/03). E.P. and B.P acknowledge he AEI g an s (PID2019-111218RB-
I00 and RyC-2017-23457). Finally, F.C. hanks he ISCIII and Miguel Se e p og am (CPII19/00020).
Ins i u ional Re iew Boa d S a emen : No applicable.
In o med Consen S a emen : No applicable.
Da a A ailabili y S a emen : No applicable.
Con lic s o In e es :
The au ho s decla e no con lic o in e es . J.P., Gene al Manage o he company
Op2Lysis (Caen, F ance). Op2Lysis had no ole in he design o he s udy; in he collec ion, analyses,
o in e p e a ion o da a; in he w i ing o he manusc ip , o in he de-cision o publish he esul s.
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