pha maceu ics
Re iew
B eaking Ba ie s: Bioinspi ed S a egies o Ta ge ed
Neu onal Deli e y o he Cen al Ne ous Sys em
Ana P. Spence 1,2,3 , Ma ília To ado 1,2,4,†, Bea iz Cus ódio 1,2,4,†, Sa a C. Sil a-Reis 1,2,
So ia D. San os 1,2 , Vic o ia Lei o 1,2 and Ana P. Pêgo 1,2,3,4,*
1i3S—Ins i u o de In es igação e Ino ação em Saúde, Uni e sidade do Po o, Rua Al edo Allen 208,
4200-135 Po o, Po ugal; [email p o ec ed] (A.P.S.); [email p o ec ed] (M.T.);
[email p o ec ed] (B.C.); [email p o ec ed] (S.C.S.-R.); [email p o ec ed] (S.D.S.);
[email p o ec ed] (V.L.)
2INEB—Ins i u o de Engenha ia Biomédica, Uni e sidade do Po o, Rua Al edo Allen 208,
4200-135 Po o, Po ugal
3FEUP—Faculdade de Engenha ia, Uni e sidade do Po o, 4200-465 Po o, Po ugal
4ICBAS—Ins i u o de Ciências Biomédicas Abel Salaza , Uni e sidade do Po o, 4050-313 Po o, Po ugal
*Co espondence: [email p o ec ed]
†These au ho s con ibu ed equal o his wo k.
Recei ed: 4 No embe 2019; Accep ed: 19 Feb ua y 2020; Published: 23 Feb ua y 2020


Abs ac :
Cen al ne ous sys em (CNS) diso de s encompass a as spec um o pa hological
condi ions and ep esen a g owing conce n wo ldwide. Despi e he high social and clinical in e es in
ying o sol e hese pa hologies, he e a e many challenges o b idge in o de o achie e an e ec i e
he apy. One o he main obs acles o ad ancemen s in his ield ha has hampe ed many o he
he apeu ic s a egies p oposed o da e is he p esence o he CNS ba ie s ha es ic he access o
he b ain. Howe e , adequa e b ain biodis ibu ion and neu onal cells speci ic accumula ion in he
a ge ed si e also ep esen majo hu dles o he a ainmen o a success ul CNS ea men . O e he
las ew yea s, nano echnology has aken a s ep o wa d owa ds he de elopmen o he apeu ics
in neu ologic diseases and di e en app oaches ha e been de eloped o su pass hese obs acles.
The e sa ili y o he designed nanoca ie s in e ms o physical and chemical p ope ies, and he
possibili y o unc ionalize hem wi h speci ic moie ies, ha e esul ed in imp o ed neu o a ge ed
deli e y p o iles. Wi h he concomi an p og ess in biology esea ch, many o hese s a egies ha e
been inspi ed by na u e and ha e aken ad an age o physiological p ocesses o achie e b ain deli e y.
He e, he di e en nanosys ems and a ge ing moie ies used o achie e a neu onal deli e y epo ed
in he open li e a u e a e comp ehensi ely e iewed and c i ically discussed, wi h emphasis on
he mos ecen bioinspi ed ad ances in he ield. Finally, we exp ess ou iew on he pa amoun
challenges in a ge ed neu onal deli e y ha need o be o e come o hese p omising he apeu ics o
mo e om he bench o he bedside.
Keywo ds: CNS diso de s; d ug deli e y; nanomedicine; nanoca ie s; bioinspi ed; neu o a ge ing
1. The Need o New Neu o a ge ed The apies
Neu ological diso de s a e one o he majo causes o dea h (9 million in 2016) and he leading cause
o disabili y-adjus ed li e yea s (276 million in 2016) wo ldwide [
1
]. The numbe o people su e ing om
cen al ne ous sys em (CNS) diso de s has inc eased d ama ically in he las ew decades due o he
popula ion g owing and aging, placing a emendous bu den on amilies and social economies [
1
]. CNS
diso de s encompass a huge numbe o pa hological condi ions induced by inju ies o diseases, such as
s oke, p ima y and me as a ic umo s o he CNS, CNS in ec ions, auma ic b ain and spinal co d inju ies,
Alzheime ’s and Pa kinson’s diseases, and mul iple scle osis, among o he s [2,3].
Pha maceu ics 2020,12, 192; doi:10.3390/pha maceu ics12020192 www.mdpi.com/jou nal/pha maceu ics
Pha maceu ics 2020,12, 192 2 o 38
Despi e his ala ming scena io, he e is a lack o e ec i e he apies o he g ea majo i y
o CNS diso de s. Many e o s ha e been made in explo ing app oaches aimed a minimizing
he clinical ou come caused by neu onal degene a ion and dea h; howe e , he po en ial bene i s
obse ed in p e-clinical esea ch con as s wi h he ailu e when ansla ed o he clinics. In he as
majo i y o hese s a egies, his has been mainly a ibu ed o poo blood s abili y, limi ed issue
pe mea ion be o e eaching he neu onal cells and, ul ima ely, low bioa ailabili y a he CNS issue
o he in es iga ed he apeu ic agen s, as well as hei inadequa e biocompa ibili y o pe iphe al
side-e ec s [
4
,
5
]. The e o e, he e is an impe a i e necessi y o he de elopmen o new app oaches
ha comply wi h clinical equi emen s.
Wi h he p og ess o he nano echnology ield, one expec s o obse e g ea ad ances in CNS
he apeu ics, since nanoscaled bioma e ials a e an icipa ed o se e as p omising deli e y pla o ms
(nanoca ie s) o he apeu ic (bio)molecules, being able o o e come he e e ed ca ea s. Besides
he unable cha ac e is ics (size, shape, su ace p ope ies, hyd ophobici y, d ug encapsula ion o
conjuga ion capaci y and eleasing), mos o he known nanoca ie s a e able o be u he unc ionalized
wi h a ge ing moie ies o imp o e hei e icacy in eaching he issue and/o cells o in e es , inc easing
he bioa ailabili y and deli e y e iciency a he a ge si e [6].
Indeed, he de elopmen o e ec i e CNS he apies has been hampe ed by he lack o he ‘magic
bulle ’, able o speci ically a ge he pa hological a ea. Consequen ly, majo e o s ha e been aken in
he de elopmen o nano echnology s a egies wi h a ge ing p ope ies o inc ease he bioa ailabili y
and deli e y e iciency a he speci ic CNS o neu onal si e.
In any adminis a ion o a he apeu ic agen aimed a he CNS, he e is he challenge in eaching
he issue i s and neu ons o o he cell ypes la e , as e y ew molecules ha e he abili y o
c oss he CNS ba ie s. Among hese ba ie s, we can enume a e he blood–b ain ba ie (BBB),
he blood–ce eb ospinal luid ba ie (BCSFB), he blood–spinal co d ba ie (BSCB) and he a ascula
a achnoid ba ie (AAB) [
7
,
8
], which will be u he desc ibed in Sec ion 4.2. Limi a ions in CNS ba ie
pe mea ion inspi ed esea che s in applying biological-inspi ed molecules o a ge CNS issue o cells.
In his e iew, we will p esen some o he de eloped nanobiomedical app oaches, wi h pa icula
ocus on he bioinspi ed neu onal- a ge ed ones, as well as he main challenges along he deli e y pa h
ha hese he apeu ic s a egies may ind.
2. The Nano echnological Answe o an E icien The apy
Nanomedicineis heapplica iono nano echnology obiomedicalapplica ions, includingdiagnosis
and d ug deli e y pu poses, in a li ing o ganism. D ugs wi h e y poo wa e solubili y p esen se e al
limi a ions, including limi ed anspo and bioaccessibili y a e adminis a ion, and hus a highe
quan i y is o en equi ed so ha he clinical e ec can be a ained, o en a he cos o undesi able side
e ec s. O he he apeu ic agen s, while soluble, may be apidly deg aded when adminis e ed, apidly
exc e ed and/o p e en ed by he di e en biological ba ie s o come in o con ac wi h he a ge si e.
In ecen yea s, one has seen ha many o hese limi a ions could be ci cum en ed by he in oduc ion
o nano echnological-based deli e y sys ems [
9
]. O e he pas ew decades, a a ie y o i al and
non- i al nanoca ie s ha e been ex ensi ely in es iga ed o imp o e he clinical pe o mance o
se e al ea men s. By explo ing di e en deli e y sys ems, he encapsula ion/immobiliza ion o d ugs
has imp o ed hei bioa ailabili y and subsequen he apeu ic e icacy, by inc easing hei solubili y,
p o ec ion om deg ada ion in biological luids and ci cula ion imes.
2.1. Nanosys ems o The apeu ics Deli e y
2.1.1. Vi al Deli e y Sys ems
Vi al ec o s a e cons uc ed based on i uses, since hey possess he na u al abili y o e ade
he hos cells, deli e ing and exploi ing he cellula machine y o exp ess i s own gene ic ma e ial.
The ideal i al ca ie would be ob ained by main aining all he in e naliza ion, opism and deli e y
Pha maceu ics 2020,12, 192 3 o 38
capabili ies o he wild ype i us, as well as i s nanome ic size ( ypically bellow 100 nm) bu no
ca ying i al payloads, which could esul in oxici y and an immunogenic esponse. This can be
achie ed by gene ic enginee ing h ough he emo al o he i al componen s esponsible o he
pa hogenici y, while lea ing in ac all he necessa y componen s o he payload deli e y [10,11].
Se e al ypes o i us, including e o i us, adeno i us, adeno-associa ed i us, and he pes
simplex i us, ha e been explo ed [
12
,
13
]. Inspi ed by hese i uses, a ious d ug deli e y sys ems,
such as i us-like pa icles and i osomes ha e been p oposed [
11
,
14
]. Howe e , o e he yea s, he use
o hese ec o s has e ealed some d awbacks, such as hei di icul and expensi e p oduc ion in
la ge quan i ies and he p esence o esidual i al elemen s ha can po en ially cause an in lamma o y
esponse, cy o oxici y, immunogenici y and inse ional mu agenesis [
12
,
14
]. Thus, se e al e o s a e
being made o o e come hese sho comings. Fo u he eading on i us-inspi ed nanoca ie s o
d ug deli e y, please e e o he e iew by Sabu e al. [14].
The disad an ages associa ed wi h i al ec o s ha e led scien is s o look o sa e al e na i es [
5
].
The de elopmen o non- i al nanosys ems has gla ing sa e y ad an ages as hey p esen limi ed oxic
and immunological p oblems. Mo eo e , non- i al deli e y sys ems a e easie o p oduce on a la ge
scale h ough inno a i e syn hesis schemes and hey a e no limi ed by he payload capaci y o he
i us capsule and, he e o e, can ans e highe payloads. Howe e , despi e he g ea ea u es o his
class o ec o s, when applied o gene deli e y, he esul s o ans ec ion a e s ill a om he deli e y
e iciency ob ained wi h i al ec o s. The e o e, se e al esea ch eams a e wo king ha d owa ds he
imp o emen o non- i al ec o design o enhance ans ec ion e iciencies.
The ou main ypes o non- i al deli e y sys ems ha ha e been explo ed o he deli e y o
he apeu ics a e discussed in he ollowing sec ions: ino ganic-, lipid-, polyme -, and dend ime -based
ec o s (Figu e 1).
Pha maceu ics 2020, 12, x FOR PEER REVIEW 3 o 38
ideal i al ca ie would be ob ained by main aining all he in e naliza ion, opism and deli e y
capabili ies o he wild ype i us, as well as i s nanome ic size ( ypically bellow 100 nm) bu no
ca ying i al payloads, which could esul in oxici y and an immunogenic esponse. This can be
achie ed by gene ic enginee ing h ough he emo al o he i al componen s esponsible o he
pa hogenici y, while lea ing in ac all he necessa y componen s o he payload deli e y [10,11].
Se e al ypes o i us, including e o i us, adeno i us, adeno-associa ed i us, and he pes
simplex i us, ha e been explo ed [12,13]. Inspi ed by hese i uses, a ious d ug deli e y sys ems,
such as i us-like pa icles and i osomes ha e been p oposed [11,14]. Howe e , o e he yea s, he
use o hese ec o s has e ealed some d awbacks, such as hei di icul and expensi e p oduc ion
in la ge quan i ies and he p esence o esidual i al elemen s ha can po en ially cause an
in lamma o y esponse, cy o oxici y, immunogenici y and inse ional mu agenesis [12,14]. Thus,
se e al e o s a e being made o o e come hese sho comings. Fo u he eading on i us-inspi ed
nanoca ie s o d ug deli e y, please e e o he e iew by Sabu e al. [14].
The disad an ages associa ed wi h i al ec o s ha e led scien is s o look o sa e al e na i es
[5]. The de elopmen o non- i al nanosys ems has gla ing sa e y ad an ages as hey p esen limi ed
oxic and immunological p oblems. Mo eo e , non- i al deli e y sys ems a e easie o p oduce on a
la ge scale h ough inno a i e syn hesis schemes and hey a e no limi ed by he payload capaci y o
he i us capsule and, he e o e, can ans e highe payloads. Howe e , despi e he g ea ea u es o
his class o ec o s, when applied o gene deli e y, he esul s o ans ec ion a e s ill a om he
deli e y e iciency ob ained wi h i al ec o s. The e o e, se e al esea ch eams a e wo king ha d
owa ds he imp o emen o non- i al ec o design o enhance ans ec ion e iciencies.
The ou main ypes o non- i al deli e y sys ems ha ha e been explo ed o he deli e y o
he apeu ics a e discussed in he ollowing sec ions: ino ganic-, lipid-, polyme -, and dend ime -
based ec o s (Figu e 1).
Figu e 1. Schema ic ep esen a ion o nanopa icles based on ino ganic, lipid, polyme and dend ime
nanos uc u es applied as he apeu ics deli e y ec o s. Pa s o he igu e we e made wi h
BioRende .
2.1.2. Non-Vi al Deli e y Sys ems
Ino ganic Nanosys ems
The ield o ino ganic-based nanosys ems has exploded wi hin ecen decades. Silica, gold, sil e
and i on oxide nanopa icles (NPs) a e among he ino ganic nanosys ems being explo ed o d ug
deli e y. Cu en ly, he po osi y, shape and size o ino ganic NPs can be p ecisely uned.
Addi ionally, al hough no exclusi ely cha ac e is ic o his ype o NP, hei su ace can be easily
modi ied.
Figu e 1.
Schema ic ep esen a ion o nanopa icles based on ino ganic, lipid, polyme and dend ime
nanos uc u es applied as he apeu ics deli e y ec o s. Pa s o he igu e we e made wi h BioRende .
2.1.2. Non-Vi al Deli e y Sys ems
Ino ganic Nanosys ems
The ield o ino ganic-based nanosys ems has exploded wi hin ecen decades. Silica, gold, sil e
and i on oxide nanopa icles (NPs) a e among he ino ganic nanosys ems being explo ed o d ug
deli e y. Cu en ly, he po osi y, shape and size o ino ganic NPs can be p ecisely uned. Addi ionally,
al hough no exclusi ely cha ac e is ic o his ype o NP, hei su ace can be easily modi ied.
Gold (Au) NPs p esen cha ac e is ics ha ha e a oused he in e es o he scien i ic communi y,
such as small sizes (a ound 2 nm) and sphe ical mo phologies [
15
]. The mos common me hod o
AuNP syn hesis is he ci a e educ ion o e achlo oau ic (III) acid. A e ob aining hese NPs, hey can
easily be unc ionalized wi h d ugs and/o a ge ing moie ies by physical abso p ion o by chemical
bonding (ionic o co alen ) due o he s ong binding a ini y o hiols, ca boxylic acids, disulphides
Pha maceu ics 2020,12, 192 4 o 38
and p o eins o Au [
16
]. In he nine een h cen u y, Michael Fa aday epo ed he i s scien i ic no e
on AuNP syn hesis. Since hen, se e al syn he ic ou es and d ug conjuga ions ha e been p oposed.
Xiao e al. p oposed a conjuga ed nanosys em aimed a he p omo ion o human neu oblas oma
(SH-SY5Y) cell p oli e a ion and neu i e g ow h. They de eloped AuNPs wi h 6-me cap opu ine,
an an i-in lamma o y d ug, exposed on he pa icle su ace h ough an Au-sulphu bond, and a
neu on-pene a ing pep ide (RDP) [
17
]. The unc ionaliza ion wi h RDP allowed an inc eased up ake
e iciency in he neu al cells. These ligands can b ing nume ous bene icial p ope ies o nanosys ems
ha ha e al eady shown e y p omising esul s; such is he case o he gold nanoclus e s (AuNCs),
de eloped by Gao e al. and composed o AuNPs, which ha e been epo ed o ha e a a e di ec
medicinal ac i i y [
18
]. AuNCs inhibi ed he agg ega ion and ib illa ion o
α
-Syn. The uncommon
agg ega ion and ib illa ion o his synuclein p o ein, p edominan ly exp essed in neu ons’ p esynap ic
e minals, leads o he o ma ion o Lewy bodies, a ema kable pa hological hallma k o Pa kinson’s
disease (PD) [
18
]. The p omising esul s p o ided a p oo o p inciple o he p ospec o AuNCs in
new d ug disco e y agains PD and he unc ionaliza ion o hese nanoclus e s wi h a a ge ing moie y
(see Sec ion 3) can u he con e neu ospeci ici y, imp o ing he e ec i eness o his sys em.
Recen ly, ano he ype o ino ganic nanosys em has been eme ging in he d ug deli e y ield— he
supe pa amagne ic i on oxide NPs (SPIONs) o i on oxide NPs (IONPs). These pa icles a e made o
small c ys als o i on oxide, commonly magne i e (Fe
3
O
4
) o maghemi e (
γ
-Fe
2
O
3
), wi h a ac i e
nanome ic sizes (usually in he ange o 20 o 150 nm) [
19
,
20
]. These NPs a e usually syn hesized
by cop ecipi a ion and can be coa ed wi h di e en polyme s. This s a egy, besides imp o ing
biocompa ibili y, allows o an easy unc ionaliza ion wi h a ge ing moie ies.
Many IONPs ha e been e alua ed in p eclinical and clinical ials, and se e al o hem a e clinically
a ailable [
19
]. Despi e he e s ill being a low numbe o app o ed i on oxide-based sys ems o d ug
deli e y o he CNS, in e es ing nanosys ems ha e been p oposed by a ious esea ch eams and, in he
u u e, could be clinically es ed and hope ully app o ed. Al hough no being an example o IONPs
being used as a d ug deli e y sys em, Nano he m
®
is a an as ic i s app o ed applica ion in he CNS.
In 2010, his i on oxide nanosys em was app o ed by he Food and D ug Adminis a ion (FDA) o he
ea men o glioblas omas [
21
]. Nano he m
®
is an inno a i e he apeu ic op ion based on magne ic
hype he mia whe e i on oxide pa icles modi ied wi h an aminosilane coa ing a e di ec ly injec ed
in o he umo . Ne e heless, his is an impo an esul ha con ibu es o b inging he use o IONPs
close o he clinic o he apeu ics deli e y pu poses.
Also, ocusing on ea ing diseases in he CNS, Naja abadi e al. de eloped p omising SPION
nanosys ems conjuga ed wi h que ce in, known o p o ec neu onal cells agains oxida i e s ess and
apop osis [
22
]. The epo ed esul s indica ed ha he que ce in conjuga ed o he dex an-coa ed Fe
3
O
4
NPs,
enhancing he bioa ailabili y o que ce in in he b ain and, he e o e, could be conside ed as ea men o
neu odegene a i e diso de s. Besides hei size, he magne ic p ope ies o SPIONs can be ad an ageously
applied. Due o hei i on oxide co e, one app oach o a ge he CNS can be h ough hei magne ic
p ope ies. In ac , he e a e some s udies demons a ing ha he applica ion o an ex ac anial magne ic
ield can media e he BBB c ossing o hese NPs, allowing o hei accumula ion in he b ain [23,24].
Silica-based o sil e -based NPs a e o he ino ganic NPs ha ha e ecei ed a en ion. Silica-based
NPs a e widely used because hei p oduc ion is easy and inexpensi e [
25
], hey p esen in low
nanosizes (less han 50 nm), hey ha e a po ous na u e ( ha can g ea ly inc ease hei su ace/ olume
a io and, consequen ly, pe mi highe su ace unc ionaliza ioncapaci y) and hey dono showswelling
o a ia ions in po osi y wi h pH o empe a u e. The e o e, hese pa icles can success ully p o ec
dis inc molecules agains dena u a ion induced by hese wo la e pa ame e s [
26
]. Jampilek e al.
deli e ed h ee noo opics loaded in silica nanosys ems. Noo opics a e neu oenhance s used a e
a auma o as a ea men o neu odegene a i e diso de s, o enhance memo y o o he cogni i e
unc ions [
25
]. The si e o ac ion o hese d ugs is he b ain and, he e o e, he BBB is a se ious
obs acle when conside ing sys emic injec ion. Howe e , in his s udy, i was ound ha he d ug,
when ca ied by silica-based ec o s, pe mea ed h ough he ba ie o he b ain issue, inc easing he
Pha maceu ics 2020,12, 192 5 o 38
amoun o d ug in he b ain issue, app oxima ely 200- old. The combina ion o wo o mo e ypes
o deli e y sys em has been a e y p ominen p oposal and could be an in e es ing op ion o he
u u e. Tu an e al. combined silica NPs wi h an i on oxide co e as a ea men o a b ain umo .
This ino ganic-combina ion nanosys em allowed o he igge ed d ug elease, by an inno a i e
swi chable mechanism, which can be ac i a ed by ex e nal adio equency ields nea a b ain umo
si e [27].
Rega ding sil e NPs (AgNPs), hese ha e many in e es ing ea u es, especially hei excep ional
an imic obial ac i i y. In ac , CNS ea men s o in ec ious diseases caused by b ain-ea ing amoebae
(Acan hamoeba species,Balamu hia mand illa is, and Naegle ia owle i) ha e been p oposed using hese
NPs [
28
]. Howe e , e y ew s udies ha e been pe o med using AgNPs as d ug deli e y sys ems
as a ea men o CNS neu ons, especially due o se e al sa e y- ela ed aspec s [
29
–
31
]. Bu hese
sa e y issues a e no only associa ed wi h AgNPs; in ac , in spi e o all o he men ioned ad an ageous
p ope ies o ino ganic NPs, he majo i y s ill ha e majo d awbacks and ha e been shown o be
pa icula ly c i ical o he b ain, as hey can induce neu o oxici y, neu oin lamma ion and can
pe manen ly al e he BBB pe meabili y [23,32,33].
Lipid-Based Nanosys ems
Liposomes, i s epo ed by Alec Bangham in 1961, a e he mos common and well-in es iga ed
nanoca ie s o d ug deli e y [
9
,
34
,
35
]. These sphe ical esicles, based on phospholipids, amphiphilic
molecules and wo a y acid hyd ophobic chains, can enclose aqueous co e spaces su ounded
by a hyd ophobic memb ane, and can be p epa ed wi h sizes anging om 30 nm o se e al
mic ome e s [34–36]
. Liposomes’ basic p ope ies, such as size, luidi y, igidi y and su ace
beha io , can be changed by using di e en lipids and p epa a ion me hods. Due o hei size,
amphipa hic cha ac e and biocompa ibili y, liposomes a e p omising candida es o d ug deli e y [
35
].
An al e na i e o adi ional liposomes may be o he lipid-based nanoca ie s, such as solid lipid
NPs (SLNs), nanos uc u ed lipid ca ie s (NLCs) and lipidic nanobubbles. SLNs a e biocolloidal
dispe sions composed o biocompa ible solid lipids, as a pa icle ma ix, p epa ed by high-p essu e
homogeniza ion and using mainly polyhyd oxy su ac an s (suc ose, glucose, o glyce ol) as s abilize s.
On he o he hand, NLCs, which we e de eloped o o e come he limi ed loading capaci y o SLNs,
ha e a ma ix composed o solid and liquid lipid blends. Lipidic nanobubbles a e a di e en app oach,
consis ing o gaseous ca i ies, usually wi h diame e s o <100 nm, ha ha e shown p omising esul s
in he neu onal deli e y ield. Se e al esea ch g oups applied ocused ul asound combined wi h
nanobubbles, accomplishing a eliable BBB opening [
37
,
38
]. Howe e , some sa e y issues we e aised
due o he collapse o nanobubbles, which can damage memb ane p o eins, such as p o eins in he BBB
ha play an impo an ole in p o ec ing b ain issue [
39
]. Because o his, u he s udies using his
ype o NP a e needed o con i m hei sa e sui abili y o neu onal deli e y. The amphiphilici y o all
hese lipidic nanosys ems allows he encapsula ion o lipophilic and wa e -soluble d ugs, ha can be
loaded by ac i e (d ug encapsula ed a e liposome/SLN/NLC assembly) o passi e (d ug encapsula ed
du ing assembly) app oaches [
9
]. In addi ion o his encapsula ion capabili y, lipidic nanosys ems can
be uned o elease he d ug molecules in a con olled and p ecise way a e hei unc ionaliza ion
wi h a ge ing ligands [
34
]. The conjuga ion wi h hese a ge ing moie ies is usually ca ied ou by
eac ion be ween he ca boxyl g oup linkage o deli e y ec o and he unc ional g oups, such as
hyd oxyl, amine, and hiol g oup, among o he s, p esen in d ugs.
O e he yea s, ad ances in he design o he mos used lipidic nanoca ie s, liposomes, ha e led
o new he apeu ic op ions in he ea men o di e en condi ions, and a numbe o liposome-based
nanosys ems ha e been app o ed by he FDA and Eu opean D ug Agency (EMA) [
34
,
40
]. Al hough
mos o hem a e applied as cance ea men s, some o hem a e indica ed o he ea men o
meningi is, a pa hology cha ac e ized by he acu e in lamma ion o he b ain and spinal co d p o ec i e
memb anes. Depocy
®
is a liposome-based d ug app o ed in U.S. and Eu ope o he ea men o
lymphoma ous meningi is and neoplas ic meningi is. This may ha e been he i s s ep in in oducing

Pha maceu ics 2020,12, 192 6 o 38
lipidic-based nanosys ems as a ea men o CNS diseases. O no e is he ac he i s e e app o ed
RNA in e e ence d ug by he FDA (Pa isi an) is o mula ed wi h hepa o opic lipid nanopa icles o
he ea men o amilial amyloid polyneu opa hy [41].
The ea esomeo he liposomed ugconjuga es o he ea men o CNSdiso de sindi e en le els
o p eclinical de elopmen o e alua e hei pha macokine ics and biodis ibu ion p o iles [
34
]. Some
o hese nanosys ems ha e been explo ed as glioblas oma he apeu ics. Se e al s udies ha e epo ed
he use o liposomal o mula ions o deli e an i-cance d ugs, such as me ho exa e, 5- luo ou acil,
pacli axel, e lo inib and doxo ubicin [
42
–
47
]. Liposomes wi h sizes smalle han ~200 nm ha e he
capaci y o c oss he BBB [
48
–
51
]. Wen e al. p epa ed quan um-do -loaded liposomes (size <150 nm)
ha we e capable o o e coming he ba ie mo e e icien ly han ee quan um do s [51].
Howe e , o enhance he e iciency o he passage, hey ha e be unc ionalized wi h
ecep o - a ge ing molecules o enhance he deli e y a he a ge ed si e [
42
,
43
,
52
]. Rod igues e al.
modi ied liposomes wi h TAT, a ca ionic pep ide de i ed om he ansac i a ing p o ein o HIV-1.
TAT, known as an enhance o NP passage h ough BBB, is one o many cell-pene a ing pep ides ha
can be used o unc ionalize he nanosys em’s su ace o imp o e i s in e naliza ion capabili ies [
53
].
Fo u he eading on cell pene a ing pep ides, please e e o he e iew by Guido i e al. [54].
Despi e all he g ea ad an ages ela ed o hese ec o s, he e a e some ea u es limi ing hei
applicabili y o some ex en , such as low solubili y, poo s abili y and low encapsula ion e iciency,
which s ill need o be o e come [34,55].
Polyme ic Nanosys ems
Polyme ic nanosys ems could be conside ed a iendly ma e ial o nanomedical applica ions due
o hei simplis ic syn hesis, a o able ea u es, such as biocompa ibili y and unable physicochemical
p ope ies, and widesp ead applicabili y [
21
]. Inno a i e polyme ic deli e y sys ems ha e
been de eloped and op imized o diminished oxici y, enhanced bioa ailabili y, biodeg adabili y,
and desi able pha macokine ics. These op imized nanosys ems can en ap and p o ec d ug molecules
om enzyma ic and hyd oly ic deg ada ion, media e hei anspo and, when he d ug is linked ia a
s imuli-sensi i e (e.g., pH, empe a u e, edox) bond, sus ain he d ug elease wi hin he a ge si e
o e a pe iod o days o e en weeks [26].
Polyme s canbe syn hesizedby a iousmechanisms, such as adical polyme iza ion, condensa ion
polyme iza ion, g a copolyme iza ion, pho opolyme iza ion, and ing-opening polyme iza ion [
56
].
The mos explo ed biocompa ible syn he ic polyme ic sys ems ha e been poly(glycolic acid) (PGA),
poly(e hylene glycol) (PEG), poly(lac ic acid) (PLA), poly(
ε
-cap olac one) (PCL), poly(lac ic-co-glycolic
acid)(PLGA), poly(amino acids)and N-(2-hyd oxyp opyl)me hac ylamidecopolyme s(HPMA) [
26
,
57
].
In addi ion o hese syn he ic polyme s, ca bohyd a e based polyme s, such as chi osan and de i a i es,
as well as poly(cyclodex ins), ha e also been s udied o neu onal deli e y [58–62].
The biodeg ada ion is a p ope y ha can be assu ed by he exis ence o labile bonds in hei
backbone o c osslinke (in he case o e icula ed sys ems). The mos commonly explo ed a e he
hyd olysable (enzyma ic media ed o hyd olysis) es e bonds, such as in he case o he commonly
used PLGA, o which he deg ada ion p oduc s (lac ic acid and glycolic acid) a e na u ally p esen
in he body and, he e o e, i is ully biocompa ible [
42
,
43
]. PCL is ano he biodeg adable polyes e
which has a slow deg ada ion p ocess [44].
I is no ewo hy ha PEG is a s anda d syn he ic polyme o en used in he de elopmen o
deli e y nanosys ems. This polyme can be easily modi ied a i s e minal hyd oxyl g oup wi h
di e en eac i e g oups, enabling c osslinking and conjuga ion chemis ies; consequen ly, i is used in
se e al biomedical applica ions [
63
]. Mo eo e , PEGyla ion o nanosys ems is an app oach commonly
adop ed o inc ease hei wa e solubili y, biocompa ibili y and ci cula ion ime in he bloods eam (by
he educ ion in opsoniza ion). The use o PEG has also been ex ensi ely explo ed as a linke be ween
a ge ing moie ies and NPs in o de o expose he ligand on he NP su ace, o coa he NP su ace o
o bo h o hese pu poses a he same ime [64].
Pha maceu ics 2020,12, 192 7 o 38
Recen s udies ha e ocused on he use o PLGA o he p epa a ion o NPs o encapsula e
he apeu ic agen s o he ea men o CNS diso de s, such as spinal co d inju y (SCI), AD o
b ain cance [
42
,
65
]. Low y e al. used biodeg adable PLGA mic osphe es o encapsula e sonic
hedgehog (Shh), a mul i unc ional g ow h ac o in ol ed in he p oli e a ion and di e en ia ion o
oligodend ocy es and neu ons du ing spinal co d de elopmen . They e alua ed he e icacy o hese
NPs as d ug deli e y sys ems o Shh a he si e o lesion and he consequen unc ional eco e y in he
case o SCI [
66
]. Howe e , he injec ion o mic osphe es a he inju y si e may no be an easy me hod o
adminis a ion. Thus, he p epa a ion o pa icles wi h smalle sizes and he unc ionaliza ion wi h
a ge ing moie ies can be bene icial, as i enables adminis a ion o be pe o med in a less in asi e
way and each he si e o ac ion equally. Fo ins ance, Li e al. op ed o unc ionalize PLGA NPs
wi h lac o e in (L ) o ac as a ca ie o shikonin, a po en ial an iglioma agen , and hese we e
able o pass he BBB and accumula e in he b ain a e in a enous (IV) injec ion in he a ail ein.
The accumula ion in he b ain inc eased h ee- old, compa ed o ee shikonin [67].
In 2013, one o he op 10 bes -selling d ugs in he U.S. was Copaxone
®
, a polyme ic-based d ug
in which he polyme is gla i ame ace a e (a la ge polypep ide composed o l-glu amic acid, l-alanine,
l-lysine and l- y osine) [
68
]. Ini ially app o ed in 1996, Copaxone
®
, an immunomodula o ha is a
esul o many yea s o esea ch, was used in he ea men o mul iple scle osis.
Rega ding na u al polyme s, chi osan has p obably been he mos ex ensi ely explo ed. Chi osan
is a polyme o d-glucosamine and
β
-1-4 N-ace ylglucosamine esidues de i ed by he pa ial
deace yla ion o chi in [
69
]. Bha na e al. de eloped chi osan NPs loaded wi h donepezil, a d ug
commonly used o ea AD. The in anasal adminis a ion o
14
C-labeled donepezil-loaded NPs in
a s esul ed in a high pe cen age o adioac i i y pe g am in he b ain issue when compa ed o he
d ug solu ion [
59
,
70
]. To enhance NP deli e y a he neu ons, chi osan has also been unc ionalized
wi h di e en neu o a ge ing ligands. Fo ins ance, o a ge he pe iphe al ne ous sys em neu ons
(PNS), Lopes e al. selec ed he non- oxic ca boxylic e minal agmen om he e anus neu o oxin
( u he discussed in Sec ion 3) as he NP- a ge ing moie y. These chi osan NPs had g ea esul s in
escuing pe iphe al neu ons in i o om degene a ion a e in amuscula adminis a ion [71].
Despi e hese and o he e y p omising success s o ies, se e al d awbacks may limi he easibili y
o his ype o NP, such as he oxici y and/o he non-deg adabili y o some cases, whils he syn he ic
p ocess can be e y complica ed, wi h high cos s [60].
Dend ime -Based Nanosys ems
Ini ially, he ield o polyme chemis y was mainly ocused on linea -shaped molecules. Howe e ,
inno a i e s uc u es ha e eme ged which di e om his ini ial idea. One o he ypes o compound
ha e olu ionized he polyme chemis y was a g oup o e y b anched and globula mac omolecules
called ‘dend ime s’ [72–74].
Dend ime -like s uc u es we e i s syn hesized in 1978 and, du ing he ollowing decade,
Denkewal e , Tomalia, Newkome, and F eche inc eased he complexi y o hese hype b anched
molecules and named hem ‘dend ime s’ [74,75].
A ypical dend ime is composed by h ee main pa s: he cen al co e, which may be based on
one o se e al unc ional g oups, epea ing uni s o monome s co alen ly linked o he co e o ganized
in b anching laye s (gene a ions), and he su ace wi h mul iple unc ional g oups (o pe iphe al
g oups), which play a key ole in hei physicochemical p ope ies [73,76,77].
Donald Tomalia de ined he di e gen syn hesis by he o ma ion o he dend ime om he
inne mul i unc ional co e ex ending ou wa d h ough se e al epea ed coupling eac ions. On he
o he hand, he con e gen syn hesis is ini ia ed on he pe ec b anched dend ons (dend ime
wedges), which a e hen linked o a mul i unc ional co e a e he ac i a ion/dep o ec ion o hei ocal
poin [
77
]. As a esul , he g ea numbe o unc ional g oups in he pe iphe y o dend ime s (like
hiols, amines, hyd oxyl, ca boxylic acids and azide g oups, among o he s) allows o he e he ing o
Pha maceu ics 2020,12, 192 8 o 38
di e en bioac i e ligands in a speci ic and con ollable manne acco ding o he desi ed p ope ies
and applica ions, mimicking he mul i alency p esen in se e al biological sys ems [78].
Rega ding he d ug immobiliza ion/load o hese ec o s, he e a e wo main app oaches:
by con aining he d ug encapsula ed inside he dend ime (“dend i ic box” model o complexa ion)
o by conjuga ion h ough ionic coo dina ion o co alen linkage o he e minal g oups o he
dend ime [
77
]. Fo a ge ing, he la e mus be he me hod o choice o link a ge ing moie ies while
keeping hem exposed o he dend ime su ace.
Thanks o hei unable chemical s uc u e, dend ime s ha e shown many ad an ages, such as
hei high solubili y in di e en media, hyd ophobic o hyd ophilic ca i ies in he in e io ,
high biocompa ibili y, and low immunogenici y [
73
,
76
,
79
,
80
]. Rela ed o his, and also due o
hei a ac i e chemical and physical cha ac e is ics, se e al applica ions ha e been p esen ed in he
biomedical and ma e ials ields [73,77,79,81].
The e a e a wide ange o di e en dend ime amilies wi h g ea po en ial o gene deli e y, d ug
deli e y, and magne ic esonance and compu ed omog aphy imaging [
73
,
74
,
82
–
84
]. Among hem,
he mos explo ed dend ime s as deli e y ec o s o CNS applica ions ha e been he poly(amido
amine) (PAMAM) dend ime s [
80
,
85
,
86
], bu o he s, like poly(p opylene imine) (PPI) [
80
,
87
–
90
],
poly(e he )-copoly(es e ) (PEPE) [
91
], poly(l-lysine) based (PLL) [
92
], ca bosilane [
93
], phospho us [
94
],
and poly(e he imine) (PETIM) [77,95], ha e also been epo ed.
In 2013, Ce quei a e al. p oposed a dend i ic nanosys em based on a PAMAM co e g a ed wi h
ca boxyme hylchi osan o in acellula ly deli e he loaded me hylp ednisolone, a neu op o ec i e
d ug used o p e en seconda y inju y damage ollowing SCI, o glial cells. They assessed he abili y
o une he in lamma o y eac ion a e he auma ic inju y, speci ically by mi iga ing he esponse
o mic oglial cells. The NP- ea ed animals had a educ ion in he numbe o seconda y inju ies [
96
].
Mo eo e , Dhanikula e al. explo ed PEPE dend ime s as me ho exa e ca ie s o he ea men o
gliomas. Thei dend i ic nanosys ems we e conjuga ed wi h glucosamine, which in e ac s wi h he
p edominan anspo e a he mammalian BBB (GLUT1), o enhance passage h ough he ba ie ,
as well as o umo a ge ing. The quan i y o me ho exa e anspo ed ac oss BBB was h ee o
i e imes mo e a e loading in he dend ime . This inno a i e nanosys em had p omising esul s
agains glioma cells and a ascula human glioma umo sphe oids [
91
]. Using a di e en app oach,
Iezzi e al. conjuga ed luocinolone ace onide (a d ug o diminish e ina neu oin lamma ion) o a
hyd oxyl- e mina ed ou h-gene a ion PAMAM. In his case, sus ained d ug elease was obse ed o
mo e han 90 days [97].
Despi e he g ea po en ial shown o biological applica ions, pa icula ly as d ug deli e y ec o s,
some limi a ions ha e been associa ed wi h dend ime s, such as hei high cos and complica ed
syn hesis p ocess, and he non-deg adable cha ac e o he mos -used dend ime s, which can lead o
bioaccumula ion and cy o oxici y, aising sa e y issues.
2.2. The Impac o he Physical and Chemical P ope ies o he Nanosys ems on Thei Biological Pe o mance
The physicochemical p ope ies o a nanosys em, such as he size, mo phology,
hyd ophobici y/hyd ophilici y, su ace unc ional g oups, cha ge densi y, and su ace p ope ies
(Figu es 2and 3), among o he s, a e e y impo an in assu ing he e icien anspo and elease o
a he apeu ic agen and o ensu e he speci ici y o neu onal a ge ing. Figu e 2B summa izes some
o he mos ele an physicochemical p ope ies o he nanosys ems when en isaging biomedical
applica ions. Di e en app oaches, ei he du ing syn hesis o pos -syn hesis, can be ollowed o
une hese p ope ies, allowing a ine design o he deli e y ec o . Fo ins ance, one can con e o
imp o e ea u es such biological s abili y, which can be c ucial o each neu ons a e a pe iphe al
adminis a ion. Biological s abili y is a e y desi able ea u e; howe e , high s abili y can cause
excessi e ci cula ion ime and undesi able e ec s. Thus, his p ope y should be p ope ly uned,
depending on he biological applica ion.
Pha maceu ics 2020,12, 192 9 o 38
Pha maceu ics 2020, 12, x FOR PEER REVIEW 9 o 38
Figu e 2. (A). Main ea u es o he nanosys ems in luencing deli e y e iciency. Nanopa icles (NPs) can be classi ied in o ino ganic (e.g., gold, silica, sil e ), lipidic,
polyme ic and dend i ic. NPs can be uned ega ding hei size (p e e ably unde 100 nm o CNS applica ions) and a e able o bind d ugs by he es ablishmen o
di e en bonds/in e ac ions be ween he unc ional g oups o he d ugs and he ec o ( he a oms we e ep esen ed acco ding o he s anda d CPK colo ing ules).
The NP su ace cha ge can be posi i e, nega i e o neu al. Addi ionally, NPs can be unc ionalized wi h di e en ypes o a ge ing ligand, such as ap ame s,
an ibodies, pep ides and p o eins. The impac o hese ea u es on NP pe o mance a e u he explo ed in his e iew. Adap ed wi h pe mission om: Sa ai a e
al., Jou nal o Con olled Release; published by Else ie , 4773020068925 [98]. Pa s o he igu e we e made wi h BioRende . (B). The in luence among he main
physicochemical p ope ies o nanosys ems and p incipal biological ac o s. The colo ba s ep esen he in e ela ionship be ween he di e en p ope ies.
Figu e 2. (A). Main ea u es o he nanosys ems in luencing deli e y e iciency. Nanopa icles (NPs) can be classi ied in o ino ganic (e.g., gold, silica, sil e ), lipidic,
polyme ic and dend i ic. NPs can be uned ega ding hei size (p e e ably unde 100 nm o CNS applica ions) and a e able o bind d ugs by he es ablishmen
o di e en bonds/in e ac ions be ween he unc ional g oups o he d ugs and he ec o ( he a oms we e ep esen ed acco ding o he s anda d CPK colo ing
ules). The NP su ace cha ge can be posi i e, nega i e o neu al. Addi ionally, NPs can be unc ionalized wi h di e en ypes o a ge ing ligand, such as ap ame s,
an ibodies, pep ides and p o eins. The impac o hese ea u es on NP pe o mance a e u he explo ed in his e iew. Adap ed wi h pe mission om: Sa ai a e al.,
Jou nal o Con olled Release; published by Else ie , 4773020068925 [
98
]. Pa s o he igu e we e made wi h BioRende . (
B
). The in luence among he main
physicochemical p ope ies o nanosys ems and p incipal biological ac o s. The colo ba s ep esen he in e ela ionship be ween he di e en p ope ies.
Pha maceu ics 2020,12, 192 16 o 38
su aces had signi ican neu on- a ge ed deli e y
in i o
and
in i o
, wi h imp o ed deli e y and
enhanced he apeu ic e ec in glioblas oma o he encapsula ed doxo ubicin [
106
]. Also de i ed om
a neu o oxin, Te -1 (a 12 amino acid pep ide), which is an analog o e anus oxin non- i ulen HC
agmen , has been ecognized by phage display [
137
–
139
]. A Te 1-modi ied PEI was syn hesized and
hese NPs showed speci ic and enhanced binding o PC12 and do sal oo ganglion cells [
140
]. In he
pos e io wo k, a simila polyme ic sys em, based on biodeg adable ca ionic chi osan, was employed
o in es iga e he deli e y o a neu o ophic ac o in a ne e c ush inju y in i o model [71,138].
Chlo o oxin (CTX), a 4-kDa pep ide isola ed om he enom o Is aeli sco pion Leiu us
quinques ia us, binds speci ically o b ain glioma cells, making i a p omising op ion as a ea men ,
especially because his pep ide inhibi s he mig a ion and he in asion o umo cells [
141
]. This e ec
esul s in he binding o glioma-speci ic chlo ide channels and ma ix me allop o einase-2 (MMP2).
Recen ly, Zhao e al. p epa ed PEI dend ime s conjuga ed wi h PEG, CTX, wi h a
131
I adiolabeling,
and hen en apped AuNPs. This nanosys em was used o he a ge ed SPECT/CT imaging and
adionuclide he apy o glioma cells
in i o
and
in i o
using a subcu aneous glioma umo model.
The de eloped nanosys em demons a ed po en ial o be applied o glioma a ge ed diagnosis and
he apy [
142
]. Mo eo e , Sun e al. de eloped a nanosys em based on IONPs conjuga ed o CTX and
me ho exa e. I demons a ed imp o ed speci ici y, ex ended NP e en ion and inc eased cy o oxici y
owa d umo cells, sugges ing ha i also possesses po en ial o applica ions in cance diagnosis and
ea men [
143
]. Se e al pep ides ha e been de i ed om he p e iously men ioned RVG using phage
display [
119
,
144
]. These can bind speci ically o he nico inic ace ylcholine ecep o , allowing access
h ough he BBB and he sp ead h oughou he b ain un il eaching he neu ons [
121
]. The RVG29 (29
amino acid pep ide) was conjuga ed o PAMAM- and PEI-based NPs o pDNA deli e y. In he case
o RVG29-modi ied PEI NPs, a 1.3- old inc ease in gene exp ession was ob ained
in i o
ega ding
non-modi ied NPs, a e IV adminis a ion [
145
]. Using he RVG29-PAMAM NPs, he gene exp ession
assays showed a 2- old inc ease in he b ain [
146
]. Mo eo e , PEGyla ed TMC modi ied wi h RVG
pep ide ca ying Cy5-labeled siRNA o mouse b ains led o an inc eased accumula ion when compa ed
o he non a ge ed coun e pa [
147
]. These s udies ha e shown he excellen abili y RVG bes ows on
NPs, allowing hem o each he b ain, and, in he u u e, he ac ion on neu ons, ha also ha e RVG
ecep o s, can be achie ed.
Ano he pep ide is he BDNF-de i ed pep ide (IKRG), a ou -amino acid pep ide sequence which
has been epo ed o a ge T kB ecep o s abundan ly p esen in neu ons. Xu e al. used his ligand o
unc ionalize hei PEG-PCL NPs, signi ican ly imp o ing hei selec i e in e naliza ion in o neu ons
o he do sal oo ganglion [
148
]. The possibili y o eaching he CNS neu ons makes his pep ide a
po en ial op ion o he de elopmen o a ge ed NPs as a ea men o neu ological diso de s. Also
based on BDNF, LM22A, a mime ic o BDNF, has been s udied. Simila ly o BDNF, i has he abili y
o bind o he same highly speci ic ecep o s (T kB); i can also ac i a e hem and p omo e neu onal
su i al, di e en ia ion, and synap ic unc ion [
149
]. P esen ly, he use o LM22A conjuga ed o
deli e y ec o s has no ye been published.
Neu o ensin (NT) is ano he p omising 13 amino acid pep ide ha was o iginally isola ed om he
bo ine hypo halamic neu ons, and a e wa ds i has been iden i ied in neu ons o o he species. NT was
ound o unde go quick in e naliza ion a e ecep o binding [
150
,
151
]. This pep ide was al eady
conjuga ed o PLL and applied as a deli e y sys em in hemipa kinsonism-induced a s [
152
,
153
].
The p omising esul s ob ained in his s udy opened he doo o he use o NT as a neu o a ge ing
moie y and, he e o e, o he eams a e e alua ing i [154].
Selle s e al. iden i ied a di e en pep ide, he a ge ed axonal impo (TAxI) pep ide,
which en iched ecombinan bac e iophage accumula ion and deli e ed an ac i e enzyme in o
spinal co d mo o neu ons in he a e ma h o an in amuscula injec ion in mice [
155
]. Acco ding o
he au ho s, he TAxI pep ide showed g ea po en ial o clinics, since i can also in e ac wi h human
spinal co d mo o neu ons. Howe e , o he bes o ou knowledge, he use o his pep ide conjuga ed
o deli e y ehicles has no ye been explo ed.

Pha maceu ics 2020,12, 192 17 o 38
Fo se e al o he CNS pa hologies, di e en p omising pep ide conjuga es ha e also been
in oduced, like dend ig a PLl-PEG-Angiopep o he ea men o PD [
156
,
157
]. Angiopep-2, a 19
amino acid pep ide, was conjuga ed o PLL using PEG as linke . Huang and co-wo ke s chose
Angiopep-2 as a ligand o lipop o ein ecep o - ela ed p o ein 1 (LRP1), which is highly exp essed in a
a ie y o cell ypes in he b ain, including neu ons, ascula cells and glial cells [
157
]. The esul s
ob ained in a ch onic pa kinsonian model upon IV adminis a ion we e e y p omising. La e , PAMAM
was unc ionalized, also ia a PEG linke wi h his pep ide, which is also o e exp essed in b ain
umo cells, o ob ain a new deli e y nanosys em o glial umo s [
158
]. Mo eo e , Xin e al. assessed
he po en ial o Angiopep conjuga ed o PEG-PCL NPs as a b ain- a ge ed d ug deli e y sys em.
A e injec ion in he mouse caudal ein, he b ain co onal sec ion showed a highe accumula ion
o Angiopep-PEG-PCL NPs in he co ical laye , la e al en icle, hi d en icles and hippocampus
han ha o non- a ge ed NPs [
159
]. Fo a di e en pu pose, Kada i e al. de eloped a no el
glioblas oma- a ge ing app oach based on SLN unc ionalized wi h angiopep-2 unc ionalized and
loaded wi h doce axel. The
in i o
a ge ing capabili y was assessed in a glioblas oma-induced
syngeneic mouse model and he esul s showed he huge po en ial o deli e he apeu ic molecules
o glioma cells [
160
]. Del G osso e al. also de eloped angiopep-2- unc ionalized NPs, bu his ime
o he b ain- a ge ed deli e y o enzymes as a eplacemen he apy in K abbe disease. The
in i o
deli e y o enzymes by unc ionalized PLGA NPs was assessed 4 h a e in ape i oneal injec ion in
di e en o gans o CNS and PNS. Mo eo e , his same eam used he PLGA NPs unc ionalized wi h a
di e en pep ide, he T pep ide (T 2). The enzyma ic ac i i y was measu ed in he ne ous sys em
demons a ing ac i i y eco e y in he b ain up o he una ec ed mice le el. San i e al. designed
T binding pep ides and T 2 was he mos p omising pep ide, in e ms o binding e iciency and
in e naliza ion capaci y [
161
]. T 2 was conjuga ed o AuNPs and he in e naliza ion was e ec i ely
p omo ed. This eam is now ocused on he u he e alua ion o his ligand o ac as a a ge ing moie y
o he deli e y o he apeu ics
in i o
and
in i o
. In a di e en s udy, he pep ide lep in30 (30 amino
acids), which was de i ed om he polypep ide lep in (146 amino acids), which is sec e ed in o he
bloods eam by adipocy es, was linked o dend ig a PLL h ough a PEG [
92
]. Lep in is an endogenic
ho mone ha ac s cen ally on cells exp essing he lep in ecep o si ua ed in he hypo halamus
and o he pa s o he b ain. Au ho s p o ed ha lep in30-PLL NPs c ossed an
in i o
BBB model
e ec i ely and accumula ed mo e in he b ain a e IV adminis a ion [
92
]. Mo eo e , block copolyme s
consis ing o PEG and PCL we e used o link h ombin- o MMP-9-clea able pep ides p esen in he
ischemic b ain issue [162].
Recen ly, Pep-TGN, which is a new 12 amino acid pep ide selec ed using he phage display
pep ide lib a y, was iden i ied. When conjuga ed wi h PEG-PLGA NPs, i enabled he c ossing o he
BBB [
163
]. These NPs we e ca ying Couma in-6, which is a luo escen p obe, o show he ma ke ’s
accumula ion in he b ain. The pep ide’s p omising ea u es will be u he explo ed in ega d o CNS
he apeu ics deli e y [163].
3.3. An ibodies
An ibodies a e conside ed g ea a ge ing ligands, especially due o hei speci ic beha io
in i o
.
Consequen ly, he apeu ic monoclonal an ibody (mAb) de elopmen o he pu pose o ansla ion
o he clinic con inues an ac i e ield and, he e o e, mAbs p e ail as one o he mos explo ed NP
a ge ing ligands.
Se e alpolyme icnanosys ems ha ebeen su ace unc ionalized wi hmAb. Chi osannanosphe es
we e unc ionalized wi h PEG bea ing he OX26 mAb, which has g ea a ini y o he T R. Ano he
high a ini y mAb o T R is 8D3, which has al eady been added o AuNPs [
164
]. Mo eo e , Shi e al.
linked o liposomes a mAb a ge ing he T R, which is p esen in he BBB and aided he passage o his
ba ie and a i al o he b ain [
165
]. These new NPs p esen ed good physicochemical p ope ies and
g ea in e naliza ion capaci y [
166
]. Al hough no speci ic o neu ons, his mAb can be e y use ul o
inc easing he a i al o nanosys ems o he b ain.
Pha maceu ics 2020,12, 192 18 o 38
Speci ic moie ies a e also being explo ed; o ins ance, an inno a i e PLl-based nanosys em
coupled o a mAb (MC192) agains he neu o ophin ecep o p75
NTR
was de eloped [
167
].
Seebu gue e al. epo ed ha he exp ession o p75
NTR
is up egula ed in spinal mo o neu ons in
pa ien s su e ing om amyo ophic la e al scle osis [
168
]. Mo eo e , Ba a i e al. showed ha his
deli e y sys em, injec ed in amuscula ly, is in e nalized ollowing he binding o he ecep o and is
anspo ed in o he neu ons o he b ain and spinal co d [167].
Zhang e al. de eloped a new p omising liposome-based nanosys em a ge ed wi h he 83-14
mu ine mAb o he human insulin ecep o a he human glioma cells. The inhibi ion o cance
cell g ow h was achie ed a e IV adminis a ion [
169
]. In ano he s udy, 83-14 mu ine mAb was
conjuga ed o SLN o imp o e he b ain deli e y o saquina i , a p o ease inhibi o able o hampe he
HIV-1 and HIV-2 duplica ion in hei la e ma u e s age. An inc ease in he concen a ion o mAb in
he NP su ace enhanced he pe cen age pe meabili y ac oss he BBB, and in he
in i o
up ake [
170
].
3.4. Ap ame s
Ap ame s a e single s anded oligonucleo ide sequences wi h a s able, de ined and easily adap ed
h ee-dimensional con igu a ion [
171
,
172
]. Simila o an ibodies, ap ame s ha e a speci ic binding
capabili y o
in i o
and ex i o ligands and ha e a ac ed a en ion in he pas ew yea s o a ge ed
d ug deli e y app oaches [
171
–
175
]. Cu en ly, many de eloped ap ame s can in e ac wi h a ious
a ge s, om simple ino ganic molecules o la ge p o ein complexes, and e en en i e cells [
176
]. When
compa ed o mAbs, ap ame s a e signi ican ly easie and cheape o p oduce and ha e less non-speci ic
biodis ibu ion and o - a ge e ec s [
172
]. Due o hese cha ac e is ics, ap ame s ha e na u ally been
a ousing a en ion in he ield o CNS deli e y.
Pegap anib (Macu agen
®
) is an ap ame -based FDA- and Eu opean Medicines Agency-app o ed
d ug ha has been de eloped o ea macula degene a ion using an an iangiogenic s a egy by
inhibi ing he in e ac ion o ascula endo helial g ow h ac o wi h i s ecep o [
177
,
178
]. This d ug
was al eady discon inued, bu his is an impo an o no e as, al hough no neu on a ge ing, his was
he i s app o ed ap ame -based d ug ha eached he ma ke .
An inno a i e bioinspi ed d ug deli e y nanosys em was de eloped by conjuga ing PLGA and
he AS1411 ap ame , which is a DNA ap ame capable o speci ically binding o nucleolin. Bo h cance
and endo helial cells highly exp ess his p o ein in hei plasma memb ane. The unc ionaliza ion
wi h he a ge ing ligand acili a ed he deli e y o pacli axel [
179
]. The mice ea ed wi h hese
loaded-NPs had signi ican ly highe umo inhibi ion. These esul s demons a ed he po en ial e icacy
o AS1411- unc ionalized NPs o he ea men o gliomas. Also wi h high a ini y o glioma cells,
GMT8 has been applied o enhance in acellula d ug deli e y and sphe oid umo pene a ion in
combina ion wi h NPs and doce axel [180].
Ap ame 17 is ano he de eloped ap ame ha has al eady shown i s abili y o dis inguish
di e en ia ed om undi e en ia ed PC12 cells [
181
]. Al hough he e is no ye any ansla ion
o a ge ed deli e y sys ems, his can be explo ed as a p omising a ge ing moie y speci ic o he
ne ous sys em.
O he ap ame s ha ha e shown p omising ea u es ha e been de eloped o a ge mis olded o
agg ega ed p o eins, such as amyloid-ß, au,
α
-synuclein, hun ing in and p ion p o ein, which a e
o en associa ed wi h mos CNS pa hologies [
182
]. Fo u he eading on ap ame s a ge ing hese
neu opa hies, please e e o he e iew by Bou ie e al. [182].
4. The In icacies and Challenges o CNS Ta ge ing
As sugges ed by he p e ious sec ions, a chi ec ing a NP o each he CNS and neu ons in pa icula
is no a i ial ask. Despi e he p omising de elopmen s ob ained so a , and he cu en knowledge o
di e en neu o a ge ing moie ies ha can be explo ed o encompass a speci ic deli e y o di e en
CNS a eas, many challenges ha e o be aced e e y s ep o he way o achie e an e ec i e he apy.
The di icul y in c ossing he CNS ba ie s, he choice o an adequa e and e icien adminis a ion
Pha maceu ics 2020,12, 192 19 o 38
ou e and he demand o achie ing an op imal neu ospeci ici y a e some o he majo conce ns in NP
design. These opics a e u he app aised in he nex sec ions, whe e he pi alls o each deli e y s ep
a e desc ibed.
4.1. Ba ie s
The CNS ba ie s a e one o he majo obs acles in neu onal deli e y app oaches; he e o e, in his
sec ion, hese challenges will be desc ibed in de ail and many possibili ies o o e come hem will be
discussed (Figu e 3).
4.1.1. Blood-B ain Ba ie
The BBB is a dynamic and highly selec i e in e ace be ween he blood and he b ain issue.
I cons i u es he majo obs acle in sys emic b ain he apies, due o he composing endo helial cells
s ongly linked o each o he by igh junc ion p o eins, whe e his endo helium is also egula ed
by as ocy es, pe icy es and neu ons [
183
,
184
]. The e o e, CNS he apeu ic esea ch has been mo e
ocused on su passing his ba ie .
Many deli e y sys ems o CNS he apies a e designed o ake ad an age o BBB anspo a ion
pa hways in o de o imp o e hei e ec i eness [
185
]. The anspo o NPs ac oss he BBB can
happen h ough di e en pa hways: (a) pa acellula di usion; (b) adso p i e media ed anspo ; (c)
ca ie -media ed anspo ; and (d) ecep o -media ed anspo . The pa acellula pa hway plays an
impo an ole in blood–b ain exchanges and is media ed by he igh junc ions’ cohesi eness ha
con ols he passage o lipophilic o soluble low molecula weigh molecules [
186
]. Ne e heless, a e a
sys emic adminis a ion, he BBB pe mea ion h ough he e e ed ou e may be enhanced by ex e nally
inducing a BBB dis up ion o uning he NPs’ cha ac e is ics. The BBB-induced dis up ion o enhance
b ain deli e y h ough he pa acellula pa hway c ea es a empo a y pe u ba ion o he ba ie , which
can be achie ed by osmo ic, chemical (manni ol, b adykinin analogue) o ul asound dis up ions,
depending on he echnique used [
187
–
189
]. Also, enginee ed NPs coa ed wi h su ac an s a e able o
s imula e a ansien opening o he BBB and inc ease he di usion o he apeu ic compounds o he
b ain pa enchyma [
190
]. Howe e , he limi ed selec i i y challenges he e icacy o hese app oaches
( he BBB is comp omised so besides he he apeu ics o he compounds can pe mea e, including
pa hogens). Unde pa hological condi ions, as, o example, in s oke, auma ic b ain inju y, mul iple
scle osis, AD and b ain umo s, he BBB unde goes signi ican changes [
184
,
191
,
192
]. As he BBB
su e s a dis up ion caused by igh junc ion disassembly, his can be seen as a window o oppo uni y
o he apeu ic NP deli e y h ough pa acellula di usion [86,193].
The in e ac ion o he NPs wi h BBB endo helial cells can also be explo ed as a way o inc ease hei
e en ion in he b ain issue, which can po en ia e hei pe mea ion o he b ain pa enchyma. In his case,
he he apeu ic app oach akes ad an ageo headso p ion-media ed anspo . NPsa ep ope ly uned
o in e ac wi h he b ain capilla y and p omo e hei pe mea ion o he b ain pa enchyma by igge ing
endocy osis o inc easing he d ug concen a ion g adien [
190
,
194
]. The adso p ion capaci y o he
b ain endo helial cells elies on he chemical and biological su ace cha ac e is ics o he nanoca ie s.
This phenomenon is acili a ed by a lipophilic and/o posi i ely cha ged su ace, which p omo e
he non-speci ic in e ac ion wi h he cell memb anes. Rega ding mo e speci ic in e ac ions wi h
mic o essel endo helial cells, a ge ing moie ies can be linked o he NPs, such as molecules o a ge
low-densi y lipop o ein (LDL) [
195
], insulin [
196
] and T ecep o s [
110
]. The ecep o -media ed
anspo shu les a ange o p o eins di ec ly o he b ain pa enchyma by using he esicula a icking
o he b ain endo helial cells (endocy osis, anscy osis and exocy osis) [
190
,
194
]. The conjuga ion
o ligands na i ely ecognized by b ain endo helial cells, such as T [
111
,
112
] and L [
115
–
117
], is a
p omising al e na i e o b ain d ug deli e y. Fo u he eading on hese and o he po en ial ligands,
please e e o he e iew by Lajoie e al. [
194
]. This desc ibed mechanism seems o be one o he mos
secu e and mos e icien me hods o a ge ing d ugs o he CNS, and i is pe cei ed o be one o he
s a egies mos likely o succeed [197], as demons a ed in p e ious sec ions.
Pha maceu ics 2020,12, 192 20 o 38
Also, he BBB ca ie -media ed anspo e s con ol he changes o small hyd ophilic molecules
induced by he linkage o he espec i e ligands, such as he glucose anspo e GLUT1 and he amino
acid ca ie LAT1 [
185
,
194
]. He eupon, enginee ed NPs wi h moie ies able o in e ac wi h hese ligands
a e p one o pe mea e he BBB. Glu a hione (GSH) has been used as a po en ial BBB a ge ing s a egy,
since BBB endo helial cells exp ess anspo e s o his molecule. GSH- agged PEGyla ed liposomal
loaded wi h doxo ubicin has al eady en e ed clinical ials o he ea men o b ain umo s [198].
Al hough he c ea ion o a CNS he apy able o c oss he BBB is a challenging ask, nano echnology
has oom o imp o ing and enginee ing NPs o de y ou main b ain gua d.
4.1.2. Blood-Ce eb ospinal Fluid Ba ie
The BCSFB consis s o he cho oid plexus epi helium be ween he blood and ce eb ospinal luid
(CSF) whe e cells a e also linked by igh junc ions and he issue is highly ascula ized. Besides he
mechanical p o ec ion coming om he igh s uc u e, hese cells ha e specialized anspo e s o
main ain he homeos asis and p o ec he CNS om ex e nal pa hogen p oduc s [
199
–
201
]. Howe e ,
when compa ed o he BBB, his ba ie is leakie and NP p esen in ci cula ion wi h app op ia e sizes
can mo e easily c oss i and en e in o he CSF. The highe pe missi eness o his ba ie does no mean
a signi ican inc ease in subs ances’ bioa ailabili y in he deep b ain pa enchyma; he e o e, o inc ease
he b ain pene a ion, he ec o can be conjuga ed o b ain anspo e s and enzymes [185,202].
4.1.3. Blood-Spinal Co d Ba ie
Addi ionally, he BSCB is a majo playe in egula ing he spinal co d en i onmen by in e acing
his issue and he blood. The BSCB and he BBB ha e no iceable di e ences, e en hough hey sha e
he cellula building blocks. One o he majo di e ences is he dec eased exp ession o he igh
junc ion complex, which explains he sligh ly inc eased pe meabili y o he BSCB compa ed o he
BBB [203].
Simila ly o he BBB, he ansien dis up ion o he cha ac e is ic igh junc ions occu ing unde
pa hological condi ions, o example in SCI, con ibu es o imp o ing he deli e y o NPs o he
issue [
204
–
207
]. Despi e he enhanced pe mea ion unde pa hological condi ions, he e is a lack o
esea ch in su passing he BSCB using nano echnology [
208
]. Howe e , wi h i s analogy o he BBB,
i is in ui i e ha he same p inciples can be applied o d ug deli e y sys ems.
4.1.4. A ascula A achnoid Ba ie
Las ly, he AAB, pa o he meningeal co e ing, enw aps he b ain unde he du a in e acing
he enes a ed du a blood essels and he CSF [
209
]. Howe e , i does no ep esen a signi ican
blood-b ain exchange ba ie due o i s educed su ace a ea [8].
4.2. How o Reach he B ain?
As discussed abo e, despi e all he e o s made so a , only a small pe cen age o d ugs can
pe mea e he BBB [
105
], and e en i a ca ie is success ully designed wi h speci ic a ge ing o
pene a e his ba ie , side e ec s in o - a ge ed a eas o he b ain may eme ge. Also, pe iphe al
o gan accumula ion and as elimina ion a e e i ied in ce ain adminis a ion ou es and may esul
in ine icien deli e y and a educ ion in he he apeu ic e ec in he CNS. I is no ewo hy ha he
biodis ibu ion o NPs is highly dependen on hei in insic cha ac e is ics, such as he size, shape,
hyd ophobici y/hyd ophilici y and su ace p ope ies [210].
The e o e, adminis a ion me hods should be chosen conside ing he physicochemical p ope ies
o each ype o NP in o de o achie e highe accumula ions in he a ge si e, a oiding some o
he po en ial ba ie s and possible side e ec s. In his way, he choice o he deli e y me hod is o
g ea impo ance o he success o he he apy and many aspec s should be aken in conside a ion.
An o e iew o he mos con en ionally used me hods and some al e na i e ou es o adminis a ion
o CNS d ug deli e y a e desc ibed below.
Pha maceu ics 2020,12, 192 21 o 38
4.2.1. ‘Con en ional’ Adminis a ion Rou es
When hinking abou he apeu ics deli e y, one idealizes a ‘magic pill’ ha is capable o eaching
ou a ge success ully a e non-in asi e adminis a ion. O al adminis a ion is a well-known ou e bu ,
despi e o e ing he g ea es compliance o pa ien s, his ou e has majo d awbacks owa ds an e ec i e
d ug deli e y o he CNS. The ha sh en i onmen o he gas oin es inal ac , he i s -pass e ec ,
and he ac ha he apeu ics ha e o c oss he in es inal wall o each sys emic ci cula ion, make he
mission o b ain deli e y almos impossible [
211
]. S udies wi h NP o mula ions, specially using lipidic
NPs, ha e been de eloped o imp o e d ug deli e y o he CNS a e o al adminis a ion [
212
,
213
].
In spi e o hese imp o emen s, d ug adso p ion and bioa ailabili y a e s ill e y poo [214].
Con a ily o o al deli e y, in ace eb al, in ace eb o en icula (ICV) o in a hecal
adminis a ions (injec ions in o he CSF), as well as implan s, a e he mos commonly used me hods
when e e ing o CNS d ug deli e y, bu also ep esen he mos in asi e s a egies. While hese
adminis a ion ou es ha e he ad an ages o bypassing he BBB, as hey can di ec ly and speci ically
access he a ge si e, hey p esen a se e e isk o damaging he su ounding issues [
215
]. Fu he mo e,
he low di usion o he d ug in o he b ain pa enchyma limi s he a ea o ac ion and also may esul
in neu o oxic e ec s a he adminis a ion si e due o an exace ba ed local d ug concen a ion [
216
].
I is impo an o emphasize ha he e is some con o e sy abou he use o CSF injec ions as a
di ec me hod o d ug deli e y o he b ain pa enchyma. Some s udies sugges ha hese ou es o
adminis a ion a e mo e adequa e o b ain ependyma deli e y, as only a small pe cen age o he CSF
d ug concen a ion pe mea es he b ain and only a ew millime e s om he su ace [
202
,
217
]. Howe e ,
o he au ho s ha e claimed ha in a hecal adminis a ion is able o success ully deli e he apeu ics o
widesp ead b ain a eas due o he close communica ion be ween he CSF and pe i ascula spaces [
218
].
This discussion may be explained by he use o NPs o di e en na u es (e.g., lipid- o polyme -based)
and cha ac e is ics, which will in luence he desi ed dis ibu ion o he he apy by hese me hods [
219
].
IV adminis a ion is conside ed he mos clinically p e e able me hod o he apeu ics
adminis a ion and is highly employed o NP deli e y, as i allows o as single o epea ed
adminis a ion o e e y ascula ized issue in a sa e way han he me hods desc ibed be o e. This ou e
bypasses he abso p ion ba ie s o o al adminis a ion, meaning ha he e ec a e IV is as e , and in
he case o CNS diseases, i can ake ad an age o he comp omised BBB obse ed in many b ain
pa hologies o each he b ain pa enchyma [
118
]. On he o he hand, he ubiqui ous dis ibu ion o
NPs in he blood by IV injec ion may esul in diminished accumula ion in he desi ed ac ion si e a he
b ain and undesi ed accumula ion in pe iphe al o gans [
104
]. Because o his, designing NPs o a oid
his o - a ge ed deli e y is e y impo an . As p e iously discussed in Sec ion 3, he unc ionaliza ion
o he deli e y sys ems wi h a ge ing moie ies is one o he mos ele an ea u es being explo ed o
o e come his p oblem, bu o he aspec s, such as NPs’ biocompa ibili y, cha ge and hemocompa ibili y,
should also be aken in o conside a ion in IV deli e y. I is well es ablished ha ca ionic NPs can be e
in e ac wi h he nega i ely cha ged memb anes, a he han neu al o anionic NPs, con ibu ing o
imp o ed in e naliza ion p o iles. Howe e , posi i ely cha ged NPs ha e sho plasma hal -li es due
o he apid clea ance, which makes he selec i e deli e y o NPs in o he b ain di icul [
103
]. Also,
wi h he blood being he i s poin o con ac wi hin he body, many pa hophysiological e en s can
be elici ed due o he in e ac ion o he NPs wi h he di e en blood componen s. This could esul
in he ac i a ion o he immune sys em, coagula ion o ib inolysis cascades, hemolysis and p o ein
adso p ion [
220
]. As p e iously commen ed, he PEGyla ion o he deli e y agen s can inc ease he
ci cula ion ime a e IV and also help o a oid he immune sys em and imp o e biocompa ibili y [
221
],
bu pe iphe al accumula ion and non-selec i e deli e y may s ill be e i ied.
4.2.2. ‘Al e na i e’ Adminis a ion Rou es
An e ec i e b ain deli e y echnique, capable o su passing he BBB and he di usion issue
o in ace eb al injec ions discussed in he p e ious sec ion, is he con ec ion-enhanced deli e y
(CED). This ou e is based on a con ec i e bulk- low p ocess, whe e d ugs a e di ec ly in used in o

Pha maceu ics 2020,12, 192 22 o 38
he ex acellula space wi h a ca he e , aking ad an age o he in e s i ial pa hway o a ain an
homogeneous pe usion h oughou he b ain [
222
]. This app oach was al eady success ully used,
especially o pe o m CNS in a umo al in usions o di e en ypes o deli e y nanosys em, such as
IONPs [
223
] and polyme ic NPs [
224
,
225
], among o he s. Howe e , besides being an in asi e ou e o
adminis a ion, some a eas o he b ain ep esen an ana omic ba ie o his echnique, such as he
ependymal o he pial su aces and, addi ionally, pa hological issue s uc u al changes may also al e
he in usa e di usion. Mo eo e , d ugs ha can leak h ough he BBB o ha a e apidly me abolized
in he CNS may no be adequa e o his ype o adminis a ion, as hey will impai e icien d ug
deli e y [
222
]. Fu he mo e, his p ocess equi es long pe iods o ime (hou s o days) o be e icien
and esul s ha e shown high a iabili y be ween expe imen s [226].
The adminis a ion o NPs ia in aa e ial injec ions has also been explo ed as an al e na i e o
IV deli e y. As he ca o id a e y is he majo blood supplie o he b ain, his deli e y me hod ac s
like a ‘ eeway’, allowing o a highe exposu e o he NPs o he a ea o in e es , owing o he i s pass
h ough ci cula ion. This me hod delays he na iga ion o he deli e y ec o s h oughou he sys emic
ci cula ion, acili a ing an inc eased accumula ion o NPs a he a ge si e and diminishing he apid
sys emic clea ance e i ied in IV adminis a ion. In aca o id d ug deli e y has been widely s udied o
he adminis a ion o chemo he apeu ic agen s, namely o magne ic NPs, which a e known o p esen
sho plasma hal -li es [
227
,
228
]. Addi ionally, Che ok e al. e i ied a 30- old highe accumula ion o
PEI-modi ied IONPs in b ain umo s wi h in aca o id adminis a ion, when compa ed o IV [
229
].
Ne e heless, he in aa e ial injec ion me hod poses signi ican isks o embolism o hemo hages
du ing he in e en ion [
230
], and, he e o e, he isk- o-bene i a io needs o be ca e ully conside ed.
In anasal adminis a ion o he CNS is gaining g owing in e es o e he o al o sys emic ou es,
as d ugs can be deli e ed in a sel and non-in asi e ashion di ec ly o he b ain. This me hod allows
he deli e y o molecules h ough he igeminal and ol ac o y pa hways, bypassing he BBB and he
BCSFB. The di ec abso p ion o d ugs by he nasal epi helial mucosa esul s in highe bioa ailabili y,
be e pha macokine ic p o iles and he apid onse o he he apy [
231
]. The success o in anasal
adminis a ion in NP deli e y o he b ain was al eady shown in se e al s udies, whe e he e icacy
o his s a egy o e he IV ou e was shown [
232
,
233
]. Ne e heless, simila ly o he o he ou es o
adminis a ion, in anasal deli e y also has some associa ed pi alls. The small adminis a ion olume
allowed by he nose- o-b ain ou e could be a limi ing ac o in assu ing ha su icien he apeu ic d ug
doses each he b ain and he e o e, i is mo e app op ia e o po en d ugs. Also, he small epi helial
a ea, sho e en ion ime and he mucocilia y clea ance may educe he apeu ics’ pe mea ion h ough
he nasal mucosa [
231
]. I should also be highligh ed ha he majo i y o s udies o CNS deli e y
ound in he open li e a u e a e pe o med using oden models, and he su ace epi helial a ea o he
ol ac o y egions o oden s is 10 imes highe han humans [
226
], which can hampe he ansla ion o
his me hod o he clinic.
4.3. Ta ge ing Ligands Dilemmas in Neu ospeci ic Deli e y
I bypassing he ba ie s is a challenge o CNS he apeu ics, a ge ing a speci ic pa hological
neu onal egion o ype o neu on cell popula ion is also an undeniable s uggle in his a ea.
Neu ospeci ic deli e y is pa icula ly challenging once neu ons a e embedded by glial cells in he b ain,
which ha e a mo e phagocy ic na u e and, he e o e, a e mo e p one o in e nalize he apeu ics [
188
].
Howe e , he inc easing knowledge abou CNS pa hologies a he cellula and molecula le el has
acili a ed he de elopmen o ac i e a ge ed nanosys ems, and success ul app oaches ha e al eady
been de eloped o a ge ing speci ic CNS cell popula ions, as p e iously discussed. Ne e heless,
some conside a ions ega ding he choice o he neu ospeci ic s a egy ha e o be ca e ully ponde ed.
Fi s o all, inding a ecep o wi h exclusi e neu ospeci ic exp ession is a ha d ask. Fo example,
he al eady men ioned T kB, he ecep o s o BDNF o i s mime ics, a e ex ensi ely exp essed in he
CNS, including he ce eb al co ex, hippocampus and spinal co d. Howe e , hese ecep o s a e also
p esen in non-neu onal cells like he oligodend ocy es [
234
], o in cells o he pe iphe al ne ous
Pha maceu ics 2020,12, 192 23 o 38
sys em, which may hampe a neu onal- a ge ed deli e y. Mo eo e , T kB is p esen in a a ie y o
pe iphe al issues, like he kidneys and he panc eas, which may lead o unspeci ic biodis ibu ion
p o iles [235].
Ano he impo an conce n is he e alua ion o he endogenous ligands o he a ge ecep o and
he possibili y o compe i ion be ween hem and he chosen a ge ing moie y. The LRP-1, a ecep o
ha is o e exp essed in gliomas and, he e o e, has been as ly explo ed o a ge b ain umo cells,
has mo e han 30 di e en ligands o di e se na u es (lipop o eins, p o eases, i us, oxins) [
236
]. Also,
gangliosides a e ex ensi ely explo ed o b ain a ge ing s a egies as ecep o s o oxins and pep ide
de i a i es, like Te 1. Howe e , hese ecep o s, in pa icula he GT1b ganglioside, a e in ol ed in a
a ie y o neu ological p ocesses and he e o e ha e a a ie y o endogenous ligands, such as lec ins,
g ow h ac o s and in eg ins [
237
]. The e o e, besides ha ing success ul applica ions, he use o ce ain
a ge ing moie ies mus be ca e ully equa ed due o he high compe i ion wi h endogenous ligands,
which may hampe he p ocess o he desi ed a ge ligand- ecep o in e ac ion. A solu ion could be
he de elopmen o al e na i e a ge ing ligands wi h an a ini y o sligh ly di e en epi opes o he
ecep o s, such as he ones de eloped in he case o T R (OX26 and 8D3, o example) [238].
The e icacy o he in e ac ion be ween ligands and ecep o s and ecep o hal -li e a e also
impo an ac o s o ac i e a ge ing deli e y [
236
]. The educ ion in ligand a ini y o i s ecep o
can be obse ed when a ached o NPs. This limi a ion is usually o e come by he unc ionaliza ion
o NPs wi h mul iple ligand molecules o inc ease a idi y. Howe e , high a idi y can also impede
NP deli e y, as he s eng h o he in e ac ion is oo s ong and hus NPs will emain a ached o he
plasma memb ane [
98
]. One s a egy o su pass his d awback is o unc ionalize NPs wi h a ge ing
ligands h ough acid- esponsi e clea able linke s, which can dissocia e in endosomes and p omo e he
elease o NPs om memb ane p o eins [239].
Also, he de e mina ion o he op imal ligand densi y, allowing maximal cell-speci ic in e ac ion,
is a c i ical issue in he ailo ing o a ge ed sys ems. We ha e p e iously epo ed he use o a omic
o ce mic oscopy (AFM) as a sc eening ool o he e icien design o a ge ed NP sys ems [
240
].
Explo ing o ce spec oscopy, we we e able o ob ain new insigh s in o he ligand– ecep o mechanism
and o de e mine he op imal a ge ed NP o mula ion ega ding neu onal cell in e naliza ion o PEI
complexes wi h plasmid DNA unc ionalized wi h he HC o he e anus oxin. In line wi h ou
indings, Chu e al. in es iga ed he e ec o Te 1 ligand densi y in neu onal cell up ake and concluded
ha lowe densi ies o ligand esul ed in imp o ed in e naliza ion o HPMA–oligolysine copolyme
NPs [
241
]. This could be explained by he ac ha he excessi e a ge ing ligand densi y can esul in
educed selec i i y, as s e ic hind ances can be o med due o he o e p oximi y be ween ligands,
dec easing hei binding abili y o neu onal cells and enhancing he binding o non- a ge ed cells [
242
].
Hence, i is impo an o op imize he NPs’ ligand densi y o enhance a ge ing e iciency.
Besides he amoun o ligand moie ies, one aspec ha is c ucial o he a ge ing po en ial
o nanosys ems is he co ec exposu e o he a ge ing ligands. As p e iously commen ed,
PEGyla ion is he mos popula mul i unc ional app oach o su ace coa ing o bo h imp o e
physicochemical p ope ies and ensu e he co ec su ace exposu e o he co esponding ligands [
243
].
Howe e , he impac o PEG space densi y and chain leng h in ligands op imal exposu e has been
dis ega ded. The e o e, ecen ly ou g oup s udied his issue, showing ha highe densi ies o PEG
chains esul ed in imp o ed neu onal in e naliza ion pe o mances [
244
]. This is p obably due o he
“b ush-like” con o ma ion ha PEG molecules adop when g a ed o a su ace a highe densi ies,
allowing a be e exposu e o he ligands. Also, in he same s udy, we showed be e in e naliza ion
and deli e y e iciencies wi h NPs g a ed wi h a mix u e o longe (5 kDa) and sho e (2 kDa) PEG
chains. These sho e spaced chains esul ed in imp o ed ligand mobili y, in his way enhancing
in e ac ions wi h he hos ecep o [
244
]. Thus, he ine- uning o PEG unc ionaliza ion is also o g ea
impo ance o neu ospeci ic deli e y.
O he aspec s ha ha e also been neglec ed when designing ac i e a ge ed NPs o CNS
he apeu ics a e disease p og ession and aging [
105
]. The o e exp ession o selec ed a ge s, ei he on
Pha maceu ics 2020,12, 192 24 o 38
he BBB o in neu onal cells in he case o neu onal diseases, is he mos commonly used s a egy o
a ge ing deli e y. Howe e , he exp ession o ecep o s can be modula ed h oughou he pa hological
e en s o wi h aging. Osgood e al. con i med ha LRP-1 exp ession was signi ican ly educed in a
a model o aging [
245
]. Thus, nanosys ems designed o a ge his BBB- and glioma cell-associa ed
ecep o migh no be e icien when adminis e ed o senio pa ien s. Mo eo e , ecep o egula ion is
disease speci ic and is a om being well known, posing an e en oughe challenge o d ug deli e y
s a egies. The e o e, me hods o e alua e and alida e he e ec i e NPs’ neu ospeci ici y in biological
models ha can mimic CNS diseases a e equi ed. An inno a i e s a egy using AFM was p oposed
by Gomes e al. o ace his p oblem, whe e NPs we e a ached o he AFM ip and p obed agains
models o di e en complexi ies [
246
]. This molecula ecogni ion o ce spec oscopy model is a as
sc eening ool ha can be used o iden i y and s udy po en ial a ge ing agen s. Bu , all in all, u he
in es iga ions ega ding neu ospeci ic nano echnologies o CNS he apeu ics and hei dynamic
beha io a e needed o be e unde s and he apeu ics oppo uni ies.
5. Concluding Rema ks and Fu u e Pe spec i es
Fo decades, esea che s ha e been s uggling o ind sa e, con ollable and e icien deli e y
ec o s o ackle he ala ming and p e ailing p oblem o CNS diseases. The de elopmen o ‘sma ’
s a egies ha can inco po a e and deli e he apeu ics in an e ec i e way and, a he same ime,
can display he ligand moie ies so ha hey exe hei a ge ing unc ions in a speci ic manne , is a
easible answe . The nano echnology ield has made conside able p og ess and non- i al nanosys ems
ha e been p oposed and in ensi ely explo ed o his pu pose as an al e na i e o he haza dous i al
ec o s. Inno a i e and mul i unc ional enginee ed NPs ha e al eady p o en hei e icacy in eaching
he b ain by he inco po a ion o ac i e a ge ing moie ies. Some o he de eloped nanos a egies
a e, in ac , inspi ed by he in insic neu ospeci ici y o some i uses, and o he s we e ins iga ed in
physiological cues o a ain a sa e and e icien CNS deli e y. Despi e he abundance o di e en
ec o s o neu onal a ge ed d ug deli e y, polyme s and dend ime s ha e been among he mos
explo ed, due o he possibili y o e sa ile chemical design and unc ionaliza ion.
When de eloping s a egies o neu o a ge ing, he ec o o choice needs o o e come mul iple
cellula ba ie s, wi h he BBB ep esen ing he main hu dle. Besides his, a ge ing he exac si e
o ac ion a he b ain ep esen s a c ucial ea u e o a oid he common oxici y and low he apeu ic
esponse obse ed in unselec i e dis ibu ions. Dual- a ge ed sys ems a e an a ac i e s a egy o
su pass he di e en ba ie s sequen ially and achie e a speci ic deli e y. Se e al s udies ha e al eady
p o ed he po en ial o his app oach o di e en CNS diseases [
247
,
248
]. Ne e heless, a p oblem
ha has been neglec ed in NP design is b ain di usion. Once in he b ain, NPs ha e o di use
h ough he nega i ely cha ged and na ow ex acellula spaces o each hei a ge , simul aneously
a oiding b ain clea ance mechanisms. I is no ewo hy ha in he con ex o some neu ologic diseases,
he in lamma o y p ocess makes he di usion p ocess mo e di icul , as mic oglia cells a e ac i a ed and
inc ease in numbe , diminishing he al eady educed ex acellula spaces [
249
]. The e o e, nanoca ie s
need o display s abili y in he biological milieu and many ac o s such as size, molecula weigh ,
mo phology, hyd ophobici y, su ace composi ion and p ope ies can al e NPs’ a es [
103
]. Mo eo e ,
he sui able choice o he chemical bonds o link he a ge ing moie y and he ca ied d ug may also
in luence he success o d ug deli e y (Figu e 2A).
Rega ding NP enginee ing, special a en ion mus also be paid o he possible al e a ions o
hei physicochemical p ope ies upon in e ac ion wi h biological luids and e en a e a i al in
he b ain. Depending on NPs’ composi ion, size, shape, hyd ophobici y/hyd ophilici y balance and
su ace p ope ies (specially cha ge), hund eds o ypes o se um p o ein can apidly adso b o he
NPs when in con ac wi h he bloods eam, o ming he known ‘p o ein co ona’ [
250
]. This p o ein
coa ing changes he su ace p ope ies and may induce NP agg ega ion, inducing a as e clea ance
by phagocy ic and immune cells and acili a ing he non-speci ic up ake o he NPs by andom cell
ypes [
221
]. Also, his shell could in luence ligand– ecep o in e ac ions, educing NPs’ a ge ing
Pha maceu ics 2020,12, 192 25 o 38
skills [
250
]. Impo an ly, he o ma ion o p o ein co ona can induce he al e a ion o he pa icle
ze a po en ial o sligh ly nega i e alues, which can also al e NP biodis ibu ion, in e ac ion wi h
BBB and hampe he up ake and he in acellula a icking in he a ge cells, impeding a success ul
he apy [
104
]. In addi ion o he al eady di icul ask o p edic ing he p o ein co ona e ec s in NPs’
a es, his biomolecula shell is dynamic and i s composi ion is modula ed upon passage h ough he
BBB [
251
]. Mo eo e , he composi ion o p o ein co ona can be in luenced by he adminis a ion ou e
and, also impo an , i will be disease-speci ic, which makes he p edic ion o NPs’ beha io e en mo e
challenging [
252
]. The e o e, ca e ully designed models o s udy he in luences o p o ein co ona in
NP-cell in e ac ions a e u gen ly needed.
In ac , imp o ed and mo e ealis ic models a e needed o encompass he s udy o NPs’ CNS
a ge ing o mula ions in all s ages. Fi s o all, ele an models o mimic BBB dynamics would be
essen ial o p edic he op imal ime o adminis a ion in b ain diseases ha induce BBB opening.
Fo ins ance, in he case o s oke, a discussion has been aised abou he BBB dis up ion: mono- o
biphasic, and o how long and when i occu s. Knowing he exac mechanisms o BBB opening and i s
pa hological changes h ough disease p og ession would be c ucial in deciding he bes he apeu ic
window o oppo uni y. Also, ca e ul a en ion has o be paid o he choice o he speci ic disease model.
Di e en ou comes we e obse ed be ween he ansien and he pe manen ischemic models [
253
],
showing he possible bias o he esul s o he same disease. Fu he mo e, a ia ions be ween he
in i o
and
in i o
en i onmen s may lead o con adic i e esul s and he ex apola ion o hese
esul s o humans is isky. In ac , his is one o he ac o s ha mos con ibu es o he gap be ween
esea ch and clinical NP applica ion. Wi h he aim o o e coming his gap, 3D models ha be e
mimic he
in i o
mic oen i onmen and he in e ac ions be ween di e en issues o cells ha e
been de eloped in ecen yea s, such as o ganoids o o gan-on-a-chip de ices. These models can be
manipula ed o simula e di e en CNS diseases and, in his way, hey can help o be e p edic NPs’
e ec i eness and biocompa ibili y [
254
,
255
]. Mo eo e , hese models can be used o s udy disease
p og ession and p edic , o example, o e exp essed ecep o s o be explo ed as a ge s, o he bes
ime o he success o he s a egy.
In addi ion o his, he unce ain neu o oxici y o nanoma e ials induced by he signi ican
accumula ion in issues has hampe ed nano echnologies’ egula o y app o al and comme cializa ion.
Fo example, magne ic NPs ha e al eady shown success ul esul s, ei he o d ug deli e y, cance
he apy o imaging pu poses. Howe e , hei ansla ion o he clinic has been hampe ed by he
immense con o e sies ega ding hei biological sa e y [
256
]. The e o e, he use o biocompa ible and
biodeg adable ma e ials could be an answe o a oid sa e y conce ns and exace ba ed bioaccumula ion,
and many g oups a e now ocused in he de elopmen o his ype o ca ie s. Recen ly, we epo ed
a new amily o biodeg adable PEG-GATGE (gallic acid ie hylene glycol es e ) dend i ic block
copolyme s, as well as hei unc ion as siRNA ec o s, which showed an adequa e deg ada ion
a e [
78
]. Addi ionally, hese dend i ic s uc u es allow an easy and e sa ile unc ionaliza ion by ‘click’
chemis y, opening a b oad ange o unc ionaliza ion possibili ies o hese ec o s. In ac , ega ding
he ca ie s’ de elopmen , new syn he ic ‘g een’ and o hogonal s a egies, such as he men ioned
‘click’ chemis y, a e being explo ed.
Finally, i is no ewo hy ha despi e he success and e sa ili y o PEG, some li e a u e epo s
ha e highligh ed he p esence o an ibodies ha speci ically ecognize his polyme (an i-PEG Abs).
This has been co ela ed wi h he he apeu ic e icacy loss and he inc ease in he apeu ics’ side
e ec s [
63
]. The e o e, di e en an i-bio ouling polyme s a e being explo ed, such as poly(2-oxazoline)s
and poly(glyce ol) [257].
Despi e all hese hu dles, CNS- a ge ed nano he apeu ics hold emendous po en ial o be used
as ea men s o hese complex diseases. So, u he e o s should be combined o achie e e ec i e
and pa ien - iendly he apies. Based on he discussed hu dles, i can also be expec ed ha he “ideal”
ca ie o he apeu ic agen s o he CNS will ha e o combine, in one en i y, se e al bio-physicochemical
Pha maceu ics 2020,12, 192 32 o 38
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