Surface Functionalization of PLGA Nanoparticles to Increase Transport across the BBB for Alzheimer’s Disease

applied sciences Re iew Su ace Func ionaliza ion o PLGA Nanopa icles o Inc ease T anspo ac oss he BBB o Alzheime ’s Disease Lau a Del Amo 1, Amanda Cano 1,2,3,4 , Mi en E che o 3,5 , Eliana B. Sou o 6,7, Ma a Espina 1,2 , An oni Camins 3,5 , Ma ia Luísa Ga cía1,2,3 and Elena Sánchez-López 1,2,3,*   Ci a ion: Del Amo, L.; Cano, A.; E che o, M.; Sou o, E.B.; Espina, M.; Camins, A.; Ga cía, M.L.; Sánchez- López, E. Su ace Func ionaliza ion o PLGA Nanopa icles o Inc ease T anspo ac oss he BBB o Alzheime ’s Disease. Appl. Sci. 2021, 11, 4305. h ps://doi.o g/10.3390/ app11094305 Academic Edi o : Ca la Sa do Recei ed: 31 Ma ch 2021 Accep ed: 29 Ap il 2021 Published: 10 May 2021 Publishe ’s No e: MDPI s ays neu al wi h ega d o ju isdic ional claims in published maps and ins i u ional a il- ia ions. Copy igh : © 2021 by he au ho s. Licensee MDPI, Basel, Swi ze land. This a icle is an open access a icle dis ibu ed unde he e ms and condi ions o he C ea i e Commons A ibu ion (CC BY) license (h ps:// c ea i ecommons.o g/licenses/by/ 4.0/). 1 Depa men o Pha macy, Pha maceu ical Technology and Physical Chemis y, Facul y o Pha macy and Food Sciences, Uni e si y o Ba celona, 08028 Ba celona, Spain; [email p o ec ed] (L.D.A.); [email p o ec ed] (A.C.); [email p o ec ed] (M.E.); [email p o ec ed] (M.L.G.) 2Ins i u e o Nanoscience and Nano echnology (IN2UB), Uni e si y o Ba celona, 08028 Ba celona, Spain 3 Cen o de In es igación Biomédica en Red de En e medades Neu odegene a i as (CIBERNED), Uni e si y o Ba celona, 08028 Ba celona, Spain; mi [email p o ec ed] (M.E.); [email p o ec ed] (A.C.) 4Resea ch Cen e and Memo y Clinic, FundacióACE. Ins i u Ca alàde Neu ociències Aplicades—In e na ional Uni e si y o Ca alunya (UIC), 08017 Ba celona, Spain 5Depa men o Pha macology, Toxicology and The apeu ic Chemis y, Facul y o Pha macy and Food Sciences, Uni e si y o Ba celona, 08028 Ba celona, Spain 6CEB—Cen e o Biological Enginee ing, Uni e si y o Minho, Campus de Gual a , 4710-057 B aga, Po ugal; [email p o ec ed] 7Depa men o Pha maceu ical Technology, Facul y o Pha macy, Uni e si y o Coimb a, 3000-458 Coimb a, Po ugal *Co espondence: [email p o ec ed] Abs ac : Alzheime ’s disease (AD) is a ch onic neu odegene a i e diso de ha accoun s o abou 60% o all diagnosed cases o demen ia wo ldwide. Al hough he e a e cu en ly se e al d ugs ma - ke ed o i s ea men , none a e capable o slowing down o s opping he p og ession o AD. The ole o he blood-b ain ba ie (BBB) plays a key ole in he design o a success ul ea men o his neu- odegene a i e disease. Nanosized pa icles ha e been p oposed as sui able d ug deli e y sys ems o o e come BBB wi h he pu pose o inc easing bioa ailabili y o d ugs in he b ain. Biodeg adable poly (lac ic-co-glycolic acid) nanopa icles (PLGA-NPs) ha e been pa icula ly ega ded as p omising d ug deli e y sys ems as hey can be su ace- ailo ed wi h unc ionalized molecules o si e-speci ic a ge ing. In his e iew, a ho ough discussion abou he mos ecen unc ionaliza ion s a egies based on PLGA-NPs o AD and hei mechanisms o ac ion is p o ided, oge he wi h a desc ip ion o AD pa hogenesis and he ole o he BBB in b ain a ge ing. Keywo ds: unc ionalized PLGA nanopa icles; b ain deli e y; blood-b ain ba ie ; Alzheime ’s disease 1. In oduc ion Alzheime ’s disease (AD) is ecognised as a ch onic neu odegene a i e disease cha - ac e ized by amyloid be a accumula ion and b ain in acellula neu o ib illa y angles [ 1 ]. Despi e he se e al d ugs app o ed by he Food and D ug Adminis a ion (FDA) o he ea men o AD, hey a e no ully e ec i e in amelio a ing he symp oms and lose e ec- i eness o e ime. We s ill ace an unme medical need wi h espec o e ec i e ea men and managemen o his neu odegene a i e diso de . To design and de elop a success ul ea men s a egy o AD, he ole o he blood- b ain ba ie (BBB) has o be aken in o accoun [ 2 ]. The BBB consis s o a con inuous laye o di e en ia ed endo helial cells linked oge he by igh junc ions, pe icy es, non enes a ed basal lamina and as ocy ic oo p ocesses [ 3 ]. Due o i s p ope ies, his complex ba ie makes he CNS a challenging mic oen i onmen o be eached by d ug molecules h ough con en ional app oaches [ 2 ]. The BBB is hus a limi ing ba ie o con en ional d ug deli e y [3]. Appl. Sci. 2021,11, 4305. h ps://doi.o g/10.3390/app11094305 h ps://www.mdpi.com/jou nal/applsci Appl. Sci. 2021,11, 4305 2 o 27 The e o e, se e al inno a i e s a egies ha e been p oposed o enhance he ans- po o he apeu ics h ough he BBB [ 3 ]. Among di e en ypes o nanosized pa icles, polyme ic (e.g., PLGA-NPs, PLA-NPs, polyme ic micelles, dend ime s) and lipid (e.g., liposomes, solid lipid nanopa icles) nanopa icles ha e been ex ensi ely s udied o deli e he apeu ic d ugs and mac omolecules o he b ain [4]. In his a ea, polyme ic nanopa icles can be made o biocompa ible copolyme s o poly(D,L-lac ic-co-glycolic) acid ha ha e low solubili y in wa e [ 5 , 6 ]. Mo eo e , cha - ac e is ics such as size, ze a po en ial and hyd ophilici y can be con olled by su ace modi ica ions, such as su ac an coa ing, o enhance b ain up ake [ 7 ]. In addi ion, ligands known o a ge he su ace ecep o s on endo helial cells o he BBB (e.g., ans e in, insulin and lipop o ein ecep o s) can be linked o he su ace o PLGA-NPs o p o ide a ge ed b ain deli e y and imp o e NP up ake [8]. PLGA is, indeed, one o he mos success ul polyme ic ma e ials used o p oduce unc ionalized pa icles, gi en i s biodeg adabili y and biocompa ibili y p ope ies ha ha e con ibu ed o i s app o al by he FDA and EMA (1986) o pa en e al adminis a ion. Besides, he loading o d ugs in o a polyme ic ma ix co e o nanopa icles p omo es d ug p o ec ion agains deg ada ion and o e s he possibili y o modi y he elease p o ile bo h in i o and in i o [9]. The aim o he p esen wo k is o iden i y di e en s a egies o PLGA-NPs su ace unc ionaliza ion o enhance hei anspo h ough he BBB o success ul ea men o neu odegene a i e diseases, in pa icula o AD. In o de o unde ake his objec i e, some seconda y aims ha e been es ablished, such as he ho ough analysis o AD pa hology highligh ing he challenges encoun e ed in he physiological p ope ies o he BBB o d ug deli e y, he ole o PLGA NPs and hei su ace a ge ing o inc ease anspo h ough he BBB o AD ea men . 2. Ma e ials and Me hods Di e en da abases we e used o he li e a u e sea ch: PubMed, Sp inge Link, Sci- enceDi ec , making use o he ollowing key wo ds: “ unc ionalized/ a ge ed PLGA nanopa icles”, “Alzheime ’s disease”, “b ain deli e y” and “blood-b ain ba ie ”. Rele- an s udies we e selec ed based on he yea o publica ion (a e 2010) and he di e en s a egies used o enhance BBB c ossing. A o al o 13 pape s on unc ionalized PLGA NPs o AD ea men we e used. In o ma ion abou NP cha ac e is ics, in i o and in i o models, and he mos ele an indings we e d awn om selec ed pape s, and a e also summa ised. 3. Alzheime ’s Disease 3.1. P e alence and Incidence Alzheime ’s disease (AD) accoun s o abou 60% o all demen ia cases wo ldwide, wi h an es ima ed global incidence o 24.3 million cases, which inc eases wi h he ag- ing popula ion [ 10 , 11 ] wi h an incidence a e ha inc eases exponen ially wi h age un il 85 yea s [12,13]. Age o he onse can hus be used as c i e ion o he ca ego iza ion o ea ly-onse AD (EOAD) and la e-onse AD (LOAD). The o me accoun s o abou 1–6% o all cases, wi h ages anging om 30 o 60–65 yea s old. The la e is he mos common o m o he disease, wi h an onse la e han 60–65 yea s [ 11 ]. Bo h EOAD and LOAD a e cha ac e ized by a p og essi e loss o memo y and o ien a ion oge he wi h o he cogni i e de ici s ha e en ually become incapaci a ing, including impai ed judgmen and decision making, ap axia and language dis u bances. In addi ion, hese a e ypically ollowed by o he neu opsychia ic symp oms such as dep ession, anxie y, apa hy, delusions, agi a ion o hallucina ions [14]. Mo eo e , in pa allel wi h li e ime expec ancy, he inc easing numbe s o pa ien s diagnosed wi h AD cons i u e a global heal h conce n wi h huge implica ions o pa ien s and ca e ake s [ 15 ]. This has led o an eno mous inc ease in esea ch ocused on unde - Appl. Sci. 2021,11, 4305 3 o 27 s anding AD pa hogenesis in o de o disco e d ugs o p e en ion and ea men o he disease [16]. 3.2. Neu opa hology The majo pa hological hallma ks o AD a e he se -up o ex acellula amyloid plaques and in aneu onal neu o ib illa y angles (NFTs) in he b ain. Al hough plaques and angles a e also iden i ied in cogni i ely no mal age-ma ched con ols, hei densi y and dis ibu ion a e signi ican ly mo e se e e in AD pa ien s [17]. Amyloid plaques a e mainly composed o he amyloid- β (A β ) pep ide ha accumula e in he ex acellula co ex [ 18 ]. The 40 amino acids-pep ide is he mos common o m o A β in humans and is called A β 40, whe eas a 42-amino-acid-long agmen , called A β 42, is less abundan . The only di e ence is ha A β 42 has wo addi ional amino acid esidues a he C- e minus [ 19 ]. Howe e , A β 42 has been associa ed wi h AD because i is mo e p one o agg ega ion han A β 40, and hus would be deposi ed be o e A β 40 [ 20 ], leading o he o ma ion o hese amyloid plaques. The gene o he amyloid p ecu so p o ein (APP) ha o igina es he A β pep ide is loca ed in ch omosome 21 in humans wi h h ee majo iso o ms a ising om al e na i e splicing [ 21 ]. Howe e , he physiological unc ion o APP, despi e in ensi e esea ch, is no ye ully disclosed. Th ee dis inc sec e ases ( α , β , o γ ) can induce clea age o ull-leng h APP, which hus unde goes di e en sequen ial p o eoly ic p ocessing. In he nonamyloidogenic pa hway, APP is i s clea ed by α -sec e ase (non-neu o oxic, “no mal” clea age), eleasing a la ge soluble ec odomain o APP (sAPP- α ) in o he ex acellula space. Opposed o A β , sAPP- α plays an impo an ole in su i al and in neu onal plas ici y, showing a p o ec i e e ec agains exci o oxici y. sAPP- α also egula es he p oli e a ion o neu al s em cells, being ins umen al o ea ly neu al de elopmen [ 22 ]. On he con a y, in he amyloidogenic p ocessing, APP is i s clea ed by β -sec e ase (po en ially neu o oxic, “abno mal” clea age), eleasing ano he soluble ec odomain o APP (sAPP- β ) in o he ex acellula space. Upon α o β -clea age, he espec i e ca boxyl e minal agmen s (CTFs) o APP ( α -CTF and β -CTF) a e kep in he cell memb ane and su e γ -sec e ase-media ed clea age. Subsequen o his, γ -clea age, α -CTF and β -CTF gene a e p83 and A β , espec i ely, (Figu e 1). I should be no ed ha γ -sec e ase clea age occu s in he ansmemb ane domain, e en hough he exac si e may a y. Indeed, majo si es o γ -sec e ase clea age a e he A β 40 and 42 posi ions [ 14 ], esul ing in he p oduc ion o A β 40 o A β 42, i.e., wo main o ms o Aβconsis ing, espec i ely, o ei he 40 o 42 amino acid esidues. The p e alen heo y o AD pa hogenesis is cu en ly accep ed o be he amyloid hy- po hesis, sugges ing he accumula ion o insoluble o ms o A β as he p ima y pa hological p ocess, which esul s om he imbalance be ween A β p oduc ion and A β clea ance [ 23 ]. Indeed, he A β 40/A β 42 a io is key in he se -up o his disease. A β 42 is he p edominan o m o A β ound in he b ain pa enchyma o AD pa ien s, whe eas A β 40 is mos ly ound in he ce eb al ascula u e [ 24 ]. Since A β 42 is he mos soluble o m, i has he isk o oligome izing o o m A β - ib ils and p o o ib ils esponsible o he o ma ion o amyloid plaques. Al hough amyloid plaques a e assumed o be non oxic, he o ma ion o amyloid oligome s may be esponsible o neu o oxici y. As a esul , he amyloid cascade would lead o he clinical synd ome o AD [14]. This cascade o e en s includes local oxida ion, in lamma ion, exci o oxici y (due o excessi e glu ama e) and au hype phospho yla ion [ 14 ]. In his con ex , he o ma ion o NFTs is conside ed a downs eam p ocess in which au p o eins agg ega e in a soluble o m, which esul s in neu onal dys unc ion and neu odegene a ion. Mo eo e , his p og essi e neu onal degene a ion would esul in an imbalance and sho age o se e al neu o ansmi e s (e.g., se o onin, dopamine, ace ylcholine), he eby leading o he known cogni i e AD de iciencies [ 25 ]. The b ain changes caused by he disease a e shown in Figu e 2. Appl. Sci. 2021,11, 4305 4 o 27 Figu e 1. Schema ic ep esen a ion o nonamyloidogenic and amyloidogenic pa hways o APP p ocessing. APP clea age akes place ei he by alpha-sec e ase ( he nonamyloidogenic pa hway) gene a ing sAPP-alpha and C83, o by be a-sec e ase ( he amyloidogenic pa hway) gene a ing sAPP-be a and C99 (based on [22]). Figu e 2. Di e ences be ween human b ain. ( A ) Heal hy b ain and ( B ) AD b ain (exce p ed om BioRende ). Al hough a good deal o da a, collec ed o e decades o scien i ic and clinical esea ch o his disease, s ill suppo s he ole o A β as he p ima y ini ia o o he AD complex pa hogenic cascade, an inc easing numbe o indica o s poin ou ha while he igge ing e ec o A β seems necessa y, i does no seem c ucial in la e s ages o he disease [ 26 ]. The e o e, o he hypo heses explaining AD pa hogenesis ha e been de eloped. As al eady men ioned, NFTs (composed o Tau p o ein) cons i u e ano he pa hologi- cal AD hallma k. Tau is a mic o ubule-associa ed p o ein abundan in he neu ons o he CNS ha wo ks as a sca old p o ein, main aining he s abili y o mic o ubules in axons. Unde pa hological condi ions, au hype phospho yla ion is inc eased, which esul s in he Tau emo al om he mic o ubule, which causes he collapse o he mic o ubule and impai s neu onal axons, causing neu odegene a ion [ 27 – 30 ]. In addi ion, his hype phos- pho yla ion gene a es au agg ega es ha e en ually o m neu o ib illa y angles [ 29 – 31 ], leading o loss o neu onal unc ion and esul ing in apop osis [ 32 ]. The e o e, se e al s udies on bioma ke s poin ou ha Tau pa hology is in ima ely ela ed o he p og ession o neu odegene a ion [33]. Appl. Sci. 2021,11, 4305 5 o 27 Mo eo e , esea che s also sugges ha neu oin lamma ion [ 34 ], choline gic neu- onal damage and oxida i e s ess [ 27 ] play an impo an ole in he neu opa hological p og ession o AD. 3.3. T ea men Despi e all he ad ances made since D . Alois Alzheime desc ibed he i s case o AD in 1907, he p ecise mechanisms o amyloid and au pa hology behind AD pa hogenesis ha e s ill no been clea ly iden i ied. As a esul , he e a e s ill no e ec i e pha maco he - apeu ic al e na i es o p ophylaxis, managemen and ea men o AD [ 14 ]. Al hough an idemen ia agen s de eloped o he ea men o AD can be ca ego ized as symp oma ic o disease-modi ying [ 35 ], none o he es ablished ea men s can ully amelio a e AD p og ession [36]. Some o hese cu en symp oma ic ea men s include ace ylcholines e ase (AChE) inhibi o s and N-me hyl-D-aspa a e (NMDA) ecep o an agonis s, such as meman ine. Howe e , all hese d ugs (Table 1) can only alle ia e AD symp oms. Fu he mo e, he e iciency o he d ugs a ies be ween pa ien s and disease s ages. In addi ion, hey possess se e al ad e se e ec s such as nausea, omi ing and dia hoea [37]. Besides, an ipsycho ic and an idep essan ea men s a e also used o amelio a e he beha iou al symp oms [ 38 ]. In addi ion, compounds ha ac on he pa hological subs a e o he disease, namely on ex acellula A β plaques and in acellula NFTs, a e cu en ly unde esea ch [14]. Table 1. FDA-app o ed d ugs o AD (based on [39]). D ug Donepezil (A icep ®, 1996) Ri as igmine (Exelon®, 2000) Galan amine (Razadyne®, 2001) Meman ine (Ebixa®, 2003) Pha maceu ical company P ize , New Yo k, USA No a is, Basel, Swi ze land Janssen, New Je sey, USA Lundbeck, Valby, Denma k Class and indica ion AChE inhibi o p esc ibed o ea symp oms o mild- o-mode a e and mode a e- o-se e e AD AChE inhibi o p esc ibed o ea symp oms o mild- o-mode a e AD AChE inhibi o p esc ibed o ea symp oms o mild- o-mode a e AD NMDA ecep o an agonis p esc ibed o ea symp oms o mode a e- o-se e e AD Mechanism o ac ion P e en s he b eakdown o ACh in he b ain P e en s he b eakdown o Ach and bu y ylcholine in he b ain P e en s he b eakdown o ACh and s imula es nico inic ecep o s o elease mo e ACh in he b ain Blocks he oxic e ec s associa ed wi h excess glu ama e and egula es glu ama e ac i a ion Common ad e se e ec s Nausea (3–19%), omi ing (3–9%), dia hea (5–15%) Nausea (17–47%), omi ing (13–31%), dia hea (5–19%), loss o appe i e (≥17%), weigh loss (3–26%), muscle weakness Nausea (21%), omi ing (11%), dia hea (7%), loss o appe i e (7%), weigh loss Dizziness (5–7%), headache (6%), cons ipa ion (3–5%), con usion I is also wo h no ing ha he limi ed o unsuccess ul de elopmen o new he apeu ic agen s o AD is equen ly a ibu ed o he p esence o he BBB, whose p ope ies make he CNS one o he mos complex mic oen i onmen s o he body, hus comp omising he de elopmen o no el e ec i e compounds [2]. 4. The Blood-B ain-Ba ie On a e age, he adul human b ain accoun s o abou 2% o body weigh . Despi e i s ela i ely small size, i consumes abou 20% o glucose-de i ed ene gy [ 40 ]. Indeed, he mammalian b ain depend upon glucose as i s main sou ce o ene gy. Fu he mo e, neu ons wi hin he CNS ha e a high ene gy demand, equi ing a con inuous supply o ene gy subs a es (mainly glucose) and nu ien s om he blood [41]. Mo eo e , neu ons communica e using se e al chemical (ions, neu o ansmi e s, neu omodula o s and neu opep ides) and elec ical signals (synap ic and ac ion po en ials). The e o e, an accu a e egula ion o he axons and synap ic ionic mic oen i onmen s is Appl. Sci. 2021,11, 4305 6 o 27 c i ical o heal hy b ain physiology [ 42 ]; hus, eliable neu al signaling is go e ned by he ba ie laye s exis ing be ween blood and neu al issue [43]. The adul b ain is composed o wo main in e acial ba ie s, namely: (i) he blood- ce eb ospinal luid ba ie , which is composed o epi helial cells o he cho oid plexus acing he ce eb ospinal luid [ 42 ] and (ii) he a ascula a achnoid epi helium, unde lying du a and enclosing CNS, also o ms a ba ie laye , e en hough i s a ascula na u e and small su ace a ea do no p omo e a signi ican su ace ex ension o exchange be ween he blood and he CNS [ 44 ]. Besides hese wo ba ie s, he BBB ep esen s he la ges in e ace o blood-b ain exchange, and is c ea ed by he endo helial cells ha o m he walls o he capilla ies [ 42 ]. As a esul , he e is a di ec in e ac ion wi h he ci cula ing blood, making he BBB he mos selec i e physical ba ie and allowing i o exe he igh es con ol o e he immedia e mic oen i onmen o b ain cells [ 45 ]. In ac , he BBB is p esen in all o ganisms wi h a well-de eloped CNS [ 46 ], and is esponsible o ensu ing he p ope en i onmen o neu onal ne wo k unc ionali y and b ain homeos asis. The BBB p o ec s he b ain agains pa hogenic agen s and i egula es he in lux and e lux o luids by means o dynamic combina ions o ionic, molecula , ascula and cellula ac o s [47]. 4.1. S uc u e Al hough endo helial cells o he CNS ascula u e o m he main ba ie agains he en y o xenobio ics in he b ain, hese cells do no unc ion independen ly om o he s, bu a he ac as modules wi hin he mul icellula neu o ascula uni . In ac , ci cula ing immune cells, neu ons, mic oglia, pe icy es and as ocy es a e in ima ely linked wi h he endo helium and play suppo ing oles in main aining and unc ioning his ba ie [ 43 , 48 , 49 ] due o an in ica e ne wo k o molecula c oss alk be ween hem [ 45 ] (Figu e 3). Figu e 3. Schema ic ep esen a ion o he cellula ne wo ks o he BBB. The pa acellula pa hway is go e ned by he igh junc ions c ea ed by he b ain endo helial cells. Foo p ocesses om as ocy es o m a complex ne wo k su ounding he capilla ies and p o ide links o neu ons. Pe icy es a e dis ibu ed discon inuously along he leng h o he b ain capilla ies and pa ially su ound he endo helium. Mic oglia a e CNS- esiden immune cells (based on [50]). Appl. Sci. 2021,11, 4305 7 o 27 Indeed, in addi ion o he CNS endo helial cells, se e al unc ional laye s exis be ween he blood and b ain. This is he case o he basemen memb ane, which comple ely co e s he capilla ies and is made o laminin, ib onec in and ype IV collagen, and con ains pe icy es; as ocy es also su ound his basemen memb ane o he BBB. Each o hese laye s may con ibu e o solu e mo emen h ough BBB [51]. Howe e , he endo helial cells lining he ce eb al blood essels s ill ep esen he main ana omical s uc u e o he BBB and can be dis inguished, bo h unc ionally and mo phologically, om he pe iphe al endo helial cells. Fo example, hey ha e a high concen a ion o mi ochond ia, which is an indica ion o a high ene gy expendi u e [52]. Mo eo e , endo helial cells om he CNS composing he igh junc ions, g ea ly limi pa acellula pe meabili y [ 53 – 55 ]. They also display low a es o anscy osis when compa ed o pe iphe al endo helial cells, e ec i ely educing esicle-media ed anscellula anspo [56]. As a esul o his igh pa acellula and anscellula ba ie , endo helial cells a e pola - ized, p esen ing dis inc abluminal and luminal su aces wi h e lux and in lux anspo e s and ecep o s, which oge he con ol he mo emen be ween blood and b ain [52]. In addi ion, hese endo helial cells ha e a ne nega i e su ace cha ge, epelling nega i ely cha ged compounds, and hey also ha e no enes a ions (small anscellula po es ha allow ee di usion), hus p e en ing as exchange o molecules. Endo helial cells also ha e e y low le els o leucocy e adhesion molecules, es ic ing he numbe o immune cells ha can indeed en e he CNS [52]. 4.2. T anspo ac oss he BBB The BBB plays a ole no only as a ba ie o cells and solu es, bu also as a ca ie o selec i e d ug molecules. Po en ial ou es o he anspo and pe mea ion o small molecules and biomac omolecules ac oss he BBB do exis (Figu e 4), p ima ily h ough ei he pa acellula o anscellula anspo [ 57 ]. On he one hand, small hyd ophilic molecules can c oss he BBB and each he b ain h ough an aqueous pa hway ia pa a- cellula anspo . On he o he hand, small lipophilic compounds en e he b ain issue h ough anscellula di usion, a nonsa u able pa hway, which could be op imized by modi ying he physicochemical p ope ies o he d ug. The e is a co ela ion be ween he lipid solubili y o a d ug and he a e a which i en e s he CNS. Fac o s such as molecula weigh (<400–500 Da) and hyd ogen bonding capaci y (<8–10 hyd ogen bonds) comp omise d ug access o he b ain [ 58 ]. Howe e , he e a e se e al o he examples o d ugs en e ing he CNS ha a e no a ec ed by hese p ope ies [50]. Mo eo e , igh junc ions limi ing pa acellula pe meabili y po en ially isola e he b ain om many ionic nu ien s, such as glucose and amino acids which a e needed o b ain me abolism and, he e o e, he BBB endo helium also con ains many speci ic solu e anspo e s o allow ca ie -media ed anspo (CMT) o hese subs ances. Indeed, he endo helial cells p esen in he BBB exp ess anspo p o eins on hei su ace o a wide ange o molecules, he eby media ing hei b ain in lux and e lux [42]. Rega ding la ge molecula weigh solu es such as selec i e pep ides, p o eins and la ge mac omolecules, he mechanisms o c ossing he BBB and en e ing he CNS consis o binding o speci ic ecep o s on he cell su ace o endo helial cells ia endocy osis. This p ocess o endocy osis can be ei he h ough ecep o -media ed anscy osis (RMT), p o iding he main ou e o which hese la ge molecula weigh solu es c oss he BBB and en e he b ain, o h ough adso p i e-media ed endocy osis (AMT) o pinocy osis [57]. In summa y, he mo emen o molecules and d ugs h ough he BBB is ei he passi e o ac i e. The o me is d i en by a concen a ion g adien om plasma o b ain, wi h mo e lipophilic compounds en e ing mo e easily; he la e being acili a ed by ac i e anspo e s in he endo helial cell memb anes [ 42 ]. Howe e , he BBB is an obs acle o d ug deli e y o he b ain. The e o e, inc easing e o s a e cu en ly ongoing o o e come he limi a ions encoun e ed in he BBB o deli e y o he apeu ics [52]. Appl. Sci. 2021,11, 4305 8 o 27 Figu e 4. Schema ic ep esen a ion o po en ial ou es o anspo and pe mea ion ac oss he BBB (based on [47]). 5. S a egies o Enhance he Deli e y o The apeu ic Agen s ac oss he BBB The deli e y o d ugs o he b ain is manda o y o he ea men o b ain-associa ed diseases, bu i is comp omised by he p esence o he BBB [59]. Despi e he mul iple BBB c ossing pa hways, app oxima ely 98% o small molecules, and mos la ge molecules (e.g., an ibodies, ecombinan p o eins and pep ides, i al ec- o s), a e unable o each he b ain h ough he BBB [ 60 , 61 ], esul ing in a e y low bioa ail- abili y in he b ain [62]. All his has slowed down he exploi a ion o immuno he apy in b ain diseases. In ac , in a enously adminis e ed an ibodies apidly en e he b ain and subsequen ly a e quickly expelled om i . Thus, only abou 0.1% o he apeu ic an ibodies each he a ge ing si e, esul ing in a demand o signi ican ly highe an ibody concen a ions in sys emic ci cula ion, which, in u n, is associa ed wi h an inc eased isk o sys emic oxici y [63]. A common nonin asi e s a egy o enhance he BBB pe mea ion o small d ugs is o inc ease he lipophilic cha ac e o he molecule by chemical modi ica ion [ 45 ]. Howe e , his app oach may p omo e a as e clea ance o he d ug om he ci cula o y sys em h ough e lux anspo e s, he eby comp omising he d ug dis ibu ion and e ec i eness. Thus, s uc u al modi ica ion o he d ug o inc ease i s a ini y o endogenous anspo p o eins on he ce ebella endo helium has been p oposed [64]. In addi ion o d ug modi ica ion, inc ease o lipophilici y, o educ ion o he molecula size, may also con ibu e o inc ease BBB pe mea ion by ocusing on he educ ion o e lux anspo , hus enhancing he anscellula di usion pe meabili y o dis up ing he igh junc ion complexes [ 47 ]. Fu he mo e, he use o nanopa icles and o he molecula T ojan ho ses may also be exploi ed o enhance he deli e y o d ug molecules ac oss he BBB [ 65 ]. 5.1. Nano echnological Tools o O e come he BBB Mul idisciplina y e o s a e being made combining chemis y, physics, enginee ing, and biology o c ea e e ec i e deli e y sys ems able o c oss he BBB wi h he aim o diagnose and/o ea b ain diseases [66]. This is especially ele an o o e come he limi a ions encoun e ed wi h cu en ly a ailable s a egies o deli e d ugs in o he CNS h ough he BBB [ 47 ], in pa icula o Appl. Sci. 2021,11, 4305 9 o 27 he deli e y o pep ides, ecombinan p o eins, accines and nucleo ides [ 67 , 68 ]. The mos commonly used deli e y sys ems a e liposomes, micelles, dend ime s and mic o and nanopa icles [ 69 ], which can be designed o educed size, biodeg adabili y and biocompa ibili y, p olonged blood hal -li e and no oxici y, making hese d ug deli e y sys ems e y a ac i e [ 70 ]. These p ope ies ha e been associa ed wi h educed side e ec s, imp o ed si e-speci ic a ge ing capaci y and be e pa ien compliance [71]. 5.2. Polyme ic Nanopa icles Polyme ic nanopa icles (NPs) a e pa icles o nanome ic size (1–1000 nm) composed o a solid co e, and ha e been widely exploi ed o c oss he BBB due o hei p ope ies o d ug deli e y, such as high loading capaci y, high s abili y, con olled d ug elease and a ge ing e iciency [5]. Polyme ic NPs can be p oduced using ei he syn he ic o na u al polyme s (mo e limi ed in e ms o hei syn hesis and p ocessing). Acco ding o hei mo phology, poly- me ic NPs a e classi ied in wo dis inc ca ego ies, namely, nanosphe es and nanocapsules (Figu e 5). In nanosphe es, he d ug is molecula ly dispe sed h oughou he polyme ic ne wo k o is placed on o he su ace o he polyme ic co e, while in he case o nanocap- sules, he d ug molecules a e solubilized in an oil o aqueous co e which is su ounded by a polyme ic laye [59]. Figu e 5. Schema ic ep esen a ion o nanosphe es and nanocapsules wi h he d ug en apped o adso bed on o he su ace o nanopa icles (based on [72]). Mo eo e , he p ope ies o hese NPs can be ailo ed by he in oduc ion o hi d pa y componen s [ 73 ] in o de o inc ease hei hal -li e, o each he BBB mo e easily, and o inc ease d ug bioa ailabili y in o he b ain o he ea men o neu ological diso de s such as AD [69]. The selec ion o he ype o polyme o he p oduc ion o such NPs is based on se e al c i e ia and ac o s, namely, he desi ed size o a pa icula applica ion, physicochemical p ope ies o he d ug o be loaded wi hin he polyme ic co e, su ace cha ac e is ics and equi ed unc ionali y, deg ee o biodeg adabili y and biocompa ibili y, and d ug elease p o ile o he inal o mula ion [ 74 ]. Se e al na u al and syn he ic polyme s ha e been used o p epa e NPs o b ain deli e y [75]. Poly (bu yl cyanoac yla e) (PBCA) NPs we e he i s polyme -based DDS used o deli e d ugs o he CNS [ 76 ]. Un o una ely, despi e many ad an ages hey possess, such as biocompa ibili y and biodeg adabili y, PBCA NPs ha e no ye been launched in clinical use. One o he majo limi a ions o hese pa icles is hei poo d ug loading capaci y, in pa icula o hyd ophobic molecules. Besides, he bu s elease o he d ug om PBCA- NPs has also been poin ed ou as a sho coming in mos o he in i o elease s udies desc ibing he use o PBCA-NPs [ 77 ]. Mo eo e , i has been seen ecen ly ha PBCA NPs could p esen po en ial oxici y, which could be a ibu ed o as e deg ada ion and a mo e apid elease o deg ada ion p oduc s [ 78 ]. Howe e , o he au ho s ha e demons a ed ha hey can induce oxida i e s ess, e op osis and nec osis [79]. Al e na i ely, se e al ypes o polyme ic NPs wi h high posi i e cha ge ha e been epo ed o c oss he BBB due o hei elec os a ic in e ac ion wi h b ain endo helial cells (nega i ely cha ged). Chi osan (CS) is a na u ally occu ing polysaccha ide ha possesses Appl. Sci. 2021,11, 4305 16 o 27 compa ed o o he PLGA NPs, CRT pep ide-modi ied PLGA NPs codeli e ing S1 and cu cumin showed po en ial ad ances o he ea men o AD mice [107]. Following he same line, B6 pep ide (CGHKAKGPRK) is also conside ed as a p omis- ing candida e o enhancing d ug deli e y in o he CNS due o i s abili y o a ge T R as a subs i u e o he ans e in p o ein [ 154 – 156 ]. In ac , i is de i ed om a phage display lib a y, and has p e iously shown high pe meabili y ac oss he BBB [ 157 ] and highe accumula ion in b ain capilla y endo helial cells [ 158 ]. Thus, i is sugges ed ha i would also imp o e cu cumin bioa ailabili y in he b ain. The e o e, Fan e al. [ 106 ] p epa ed PLGA-PEG NPs loaded wi h cu cumin and conjuga ed wi h he B6 pep ide, ob aining NPs wi h a size less han 150 nm. These PLGA-PEG-B6/Cu NPs signi ican ly inc eased cu cumin cellula up ake in i o and could ema kably imp o e cogni i e impai men in APP/PS1 mice, indica ing ha hese NPs can p o oundly imp o e cu cumin deli e y e iciency o he b ain. Mo eo e , ex i o s udies demons a ed ha hey could educe hippocampal β -amyloid o ma ion and deposi s, as well as au hype phospho yla ion. Thus, hese NPs may se e as an in e es ing s a egy o he ea men o AD [106]. Ano he s a egy o o e come he hyd ophobic na u e and nonsolubili y in wa e o cu cumin is conjuga ing PLGA NPs wi h a a ge ing Te -1 pep ide moie y. Te -1 is a 12- amino acid pep ide (HLNILSTLWKYR) iden i ied h ough phage display, which has he binding ea u es o e anus oxin [ 159 ], hus p esen ing a ini y o neu ons, and could a ge ca gos o he b ain, bypassing he BBB [ 160 ]. Fo his eason, Ma hew e al. [ 104 ] syn hesized wa e -soluble PLGA coa ed-cu cumin NPs and coupled hese NPs wi h Te -1, esul ing in NPs wi h a size be ween 150-200 nm. Su ace modi ica ion wi h Te -1 pep ide inc eased neu onal a ge ing e iciency in i o . Indeed, enhanced neu onal up ake o cu cumin-PLGA NPs was obse ed when compa ed o non a ge ed cu cumin-PLGA NPs. The e o e, hese esul s indica e ha Te -1- a ge ed PLGA-coa ed cu cumin NPs can be addi ional ools o he ea men o neu ological diseases, in pa icula AD, wi h espec o i s an iamyloid and an ioxidan ac i i ies [ 104 ]. Howe e , his is a p elimina y in i o s udy ha needs mo e de ailed in i o in es iga ions o d aw u he conclusions. Li e al. [ 101 ] employed ano he phage display pep ide lib a y wi h he pu pose o isola ing pep ides ha may be exploi ed o a ge deli e y sys ems o he BBB and inally selec ed a 12-amino-acid-pep ide (deno ed as Pep TGN). They co alen ly conju- ga ed Pep TGN (TGNYKALHPHNG) on o he su ace o PEG-PLGA based NPs. The su ace-modi ied PLGA NPs wi h Pep TGN had sizes abou 100 nm and esul ed in an imp o ed deli e y o NPs ac oss he BBB and b ain a ge ing. This led o signi ican highe cellula up ake in i o and enhanced in i o b ain accumula ion, a he han li e and spleen accumula ion, showing powe ul b ain selec i i y o Pep TGN. The e o e, Pep TGN modi ied NPs migh be a good s a egy o a ge ed d ug deli e y ac oss he BBB [ 101 ]. Indeed, he abili y o TGN o acili a e he deli e y o NPs o he b ain has been u he demons a ed [ 161 , 162 ], con i ming ha signi ican ly highe cellula up ake and b ain dis ibu ion occu ed wi h TGN-modi ied NPs compa ed o naked NPs. Mo eo e , pep ides such as g7, ha a e simila o syn he ic opioid pep ides [ 163 ], can also be employed o deli e d ugs in o he CNS. In ac , i was p e iously demons a ed ha PLGA-NPs conjuga ed wi h g7 a e able o e icien ly c oss he BBB wi hou damage [ 164 ]. The e o e, Ba ba a e al. [ 83 ] designed and enginee ed cu cumin-encapsula ed PLGA NPs bound o g7 (Cu -NP-g7). The esul ing NPs had a pa icle size a ound 200–250 nm, a ou able o sys emic adminis a ion. The au ho s demons a ed ha Cu -NP-g7 could inc ease he cellula a ini y o he ac i e neu onal cells and be in e nalized by hippocampal neu ons. Besides, hey de e mined he e ec o Cu -NP-g7 on he agg ega ion o A β , which showed an impo an dec ease o A β . Thus, b ain deli e y o cu cumin using BBB-c ossing PLGA-g7 NPs is a p omising al e na i e in he ea men o AD. Howe e , his s udy has been e i ied by an in i o cellula model and would need a comple e in i o s udy in AD animal models so ha u he conclusions could be d awn. Appl. Sci. 2021,11, 4305 17 o 27 6.2.2. Recep o -Media ed T anscy osis CNS endo helial cells exp ess ecep o s, such as T R, low-densi y lipop o ein ecep o (LDLR), insulin-like g ow h ac o ecep o , insulin ecep o , diph he ia oxin ecep o , sca enge ecep o class B ype and nico inic ace ylcholine ecep o (nAChR), on o hei su aces. Thus, deli e y sys ems can be deco a ed wi h a ge ing ligands ha speci ically ecognise hese ecep o s o media e d ug pene a ion o he b ain [165]. Polyso ba e 80 (P80, also known as Tween 80) is pa icula ly in e es ing o b ain deli e y, since PBCA NPs coa ed wi h P80 ha e al eady acili a ed b ain deli e y o se e al d ugs ha we e unable o c oss he BBB in a ee o m [ 166 ]. Poloxame 188 (P188, also known as Plu onic F-68) was also ound o be e ec i e as an NP coa ing ma e ial o b ain a ge ing [ 97 ]. This e ec is a ibu ed o he enhanced adso p ion o ce ain plasma p o eins (especially he apolipop o eins E, A-I and B) on o he NPs. Indeed, a e being exposed o se um o plasma, a ious p o eins quickly adso b on o o eign NPs [ 167 ]. This e ec is known as di e en ial p o ein adso p ion, and was i s desc ibed in 1989 [ 168 ]. The pos ula ed hypo hesis is ha he adso p ion o apolipop o- eins on o P80/P188-coa ed NPs is esponsible o he subsequen in e ac ion wi h hei espec i e ecep o s exp essed by he endo helial cells o ming he BBB, and hus p o- mo es ecep o -media ed endocy osis o NPs, acili a ing hei deli e y in o he b ain [ 169 ]. The e o e, all hese nanopa icle DDS appea o ac like T ojan ho ses ha would anspo he d ugs in o he b ain endo helial cells and, in his way, ep esen a no el pla o m echnology o he ea men o neu odegene a i e diseases [170]. Mo eo e , he coa ing o pa icles wi h P80 causes a ea angemen o he p o eins composing he igh junc ions a he BBB which esul s in he inc ease o pa acellula c ossing o NPs in o he b ain [ 171 , 172 ]. Ano he eason is a ibu ed o he ole o P80 in blocking he e lux sys em, educing he pump-o e ec o he P-glycop o ein, and he eby achie ing a high d ug concen a ion in he b ain [173]. Taha a e al. [ 102 ] s udied di e en su ace-modi ied PLGA NPs o mula ions based on P80, P188 and CS o a ge ing CNS diseases. The esul ing PLGA NPs had sizes anging om 250 o 400 nm depending on he ype o su ace modi ie used, and a e sys emic adminis a ion, NP concen a ion in he b ain inc eased wi h he su ace modi ica ion o he pa icles, in pa icula , CS and P80 PLGA NP. Howe e , CS-PLGA NPs we e only adso bed on endo helial cells and no ans e ed in o b ain issue, whe eas a highe b ain dis ibu ion was seen wi h P80-PLGA NPs, which we e seen in he pa enchyma beyond he ce eb al blood essel endo helial cells. This sugges ed ha P80-PLGA NPs could no only adso b o he endo helial memb ane o ce eb al blood essels bu could also be in e nalized by endo helial cells and c oss he BBB. Mo eo e , P80-PLGA NPs exhibi ed p olonged ci cula ion in he blood compa ed o he o he NPs e alua ed, which migh be a eason o he inc eased b ain dis ibu ion by a oiding up ake by RES. The e o e, P80-PLGA NPs ha e high po en ial as e ec i e d ug ca ie s o CNS deli e y [102]. This was u he con i med by Fo nague a e al. [ 174 ]. Indeed, hey demons a ed he capabili y o galan amine-PLGA NPs o c oss he BBB because o he pe meabilizing and a ge ing e ec o P80, leading o a o mula ion wi h in e es ing p ope ies o be used as ad anced DDS o he symp oma ic ea men o AD. As al eady men ioned, ecep o s ha a e highly exp essed on CNS endo helial cells include T R. Lac o e in (L ) is a na u ally occu ing i on-binding glycop o ein belonging o he ans e in amily. I s ecep o (L R) is highly exp essed in b ain endo helial cells and in neu ons, being especially o e exp essed in capilla ies and neu ons associa ed wi h age- ela ed neu odegene a i e diseases [ 175 , 176 ]. This ac makes L a p omising a ge ing molecule o he ea men o AD. The e o e, Meng e al. [ 108 ] p epa ed L -PLGA NPs loaded wi h Hupe zine A (HupA), a e e sible AChE inhibi o which enhances memo y in animal models [ 177 , 178 ]. The esul ing NPs had an a e age size below 200 nm and L -su ace modi ica ion inc eased cellula up ake o NPs h ough RMT, leading o imp o ed b ain deli e y. The e o e, b ain accumula ion o L -TMC NPs was highe han non a ge ed analogues, especially in Appl. Sci. 2021,11, 4305 18 o 27 he memo y- ela ed hippocampus. This ou come sugges s ha L -PLGA NPs may be a p omising app oach o he deli e y o HupA in AD [ 108 ]. Howe e , u u e s udies a e equi ed o con inue e alua ing hei he apeu ic e icacy in animal models o AD, and i should be no ed ha he use o la ge p o eins such as lac o e in p o ein can esul in p oblems like syn hesis p ocedu e, s abili y and immunological esponse [157]. 6.2.3. Ca ie -Media ed T anspo Besides ecep o s, ac i e anspo in he BBB may also be exploi ed o b ain a ge ing. The glu a hione (GSH) anspo e is highly exp essed a he BBB [ 97 ]. This endogenous ipep ide hiol ac s as an an ioxidan and helps o p o ec cells om ROS [179]. Since he e is a la ge numbe o GSH anspo e s a he BBB, GSH conjuga ed on o he PLGA NP su ace is expec ed o bind o hese anspo e s and inc ease he numbe o NPs a he BBB in e ace [ 179 ]. Howe e , al hough he mechanism o GSH anspo a ion h ough he b ain cells was p o en o be pe o med h ough a speci ic mechanism [ 180 ], he exac molecula mechanism emains o be elucida ed. Paka e al. [ 105 ] de eloped GSH- unc ionalized PLGA-PEG NPs o be loaded wi h cu cumin. The esul ing NPs, o mean size be ween 149 o 180 nm and coa ed wi h bo h PEG and GSH, inc eased imp o ed d ug up ake in i o . Mo eo e , he in e nalized cu cumin was ound localized in almos e e y cell and hei componen s, meaning ha he GSH su ace-modi ica ion allowed be e cellula a icking o he o mula ion. No el insigh s in o he de elopmen o e ec i e deli e y sys ems able o escape lysosomal deg ada ion we e desc ibed, he e o e inc easing he he apeu ic e ec o d ugs use ul o he ea men o AD [ 105 ]. Howe e , in i o expe imen s a e s ill equi ed o d aw mo e gene al conclusions. 6.2.4. Adso p ion-Media ed T anscy osis In addi ion o pa icle size, su ace cha ge (i.e., ze a po en ial) is also expec ed o a ec NP cellula up ake and cy o oxici y. The e o e, nanopa icles wi h a posi i e su ace cha ge a e mo e sui able o cellula up ake. The su ace o CNS endo helial cells shows a nega i e cha ge and hus, a ac s posi i ely cha ged nanopa icles o in e ac wi h he BBB h ough adso p ion-media ed endocy osis [181]. In ac , while ecep o -media ed anscy osis equi es he ini ial binding o a ligand o he memb ane o he BBB endo helial cells, abso p i e-media ed anscy osis elies on nonspeci ic cha ge-based in e ac ions [ 100 ]. Consequen ly, poo selec i i y o abso p i e- media ed anscy osis is p edominan , since i can be easily ini ia ed by polyca ionic compounds binding he nega i e cha ges on o he memb ane. Ca ionic su ac an coa ing is belie ed o ende a posi i e cha ge o NPs, hus im- p o ing hei in e ac ion wi h cells and issues [ 97 ]. Fo ins ance, dioc adecyldime hylam- monium b omide (DODAB), a qua e na y ammonium su ac an , can d as ically al e he nega i e cha ge o PLGA NPs by p e e en ial adso p ion [ 182 ]. In he same way, didode- cyldime hylammonium b omide (DMAB), ano he qua e na y ammonium compound, is also used o nanopa icle s abiliza ion. Pee la e al. [ 183 ] epo ed ha DMAB enabled he in e ac ion o NPs wi h a cell memb ane model in a p opo ional ashion o hei cellula up ake in i o . The e o e, Xu e al. [ 103 ] p epa ed DMAB-PLGA NPs loaded wi h couma in-6 and obse ed ha su ace modi ica ion wi h DMAB no ably imp o ed cellula up ake in i o , which was size-dependen wi h an op imal pa icle size o 100 nm. Indeed, DMAB-modi ied NPs showed smalle sizes and highe ze a po en ials han he PVA-coa ed NPs, meaning ha he DMAB-coa ed nanopa icles could be en apped by he cells mo e easily, inc easing cellula up ake. In addi ion o ca ionic su ac an s, ca ionic polyme s such as chi osan (CS) can be em- ployed. As p e iously men ioned, CS is a na u al ca ionic polyme which has been demon- s a ed o p omo e AMT, he eby enhancing NP cellula up ake. Indeed, Taha a e al. [ 102 ] in es iga ed CS as a su ace-modi ying agen o imp o e PLGA NPs b ain deli e y. NP su ace modi ica ion wi h CS inc eased NP concen a ions in he b ain compa ed o un- Appl. Sci. 2021,11, 4305 19 o 27 modi ied NPs. Mo eo e , CS-PLGA NPs we e abso bed on o he ce eb al blood essel by adhesion o endo helial cells by means o elec os a ic in e ac ion wi h cell memb anes, he eby enhancing cellula up ake o CS-PLGA NPs, whe eas unabso bed pa icles we e elimina ed apidly om blood ci cula ion due o up ake by he RES. The e o e, PLGA NPs su ace-modi ied wi h CS may play an in e es ing ole o CNS d ug deli e y. T ime hyla ed chi osan (TMC) is a qua e nized CS de i a i e ha is posi i ely cha ged unde physiological condi ions [ 184 ]. As a ca ionic ligand, TMC acili a es NP ac i e ans- po ia abso p i e-media ed anscy osis. Thus, TMC-modi ied NPs can be exploi ed o deli e y o he b ain [185,186]. Wang e al. [100] p epa ed PLGA-NPs and hen employed co alen binding o a ach TMC o he su ace o PLGA–NPs and o m TMC/PLGA-NPs. The ob ained pa icles showed a mean diame e o 150 nm and we e dis ibu ed in he pe i en icula egion o he co ex and he hi d en icle ex ensi ely, while no b ain up ake o unmodi ied PLGA–NPs was seen, showing ha posi i ely cha ged TMC con- ibu ed o he elec os a ic in e ac ion wi h he anionic binding si es o he b an capilla ies. This igge ed he abso p i e-media ed anscy osis pa hway, ollowed by he up ake o NPs h ough he BBB, eaching he b ain pa enchyma. The inc eased cellula up ake and anspo in o he b ain a e su ace modi ica ion wi h TMC was u he demons a ed by Meng e al. [ 108 ]. Mo eo e , TMC o med a hyd ophilic su ounding, which also con ibu ed o his enhancemen and a oided up ake by he mononuclea phagocy ic sys em. As a esul , beha iou al es s conduc ed in mice showed ha hese NPs g ea ly educed memo y impai men by es o ing i o a no mal le el. Besides, he senile plaque and biochemical pa ame e es s con i med he b ain- a ge ed e ec s o TMC/PLGA–NPs. Taken oge he , hese esul s indica e ha TMC su ace-modi ied NPs a e able o c oss he BBB and could be a p omising s a egy o b ain a ge ing wi h low oxici y [100]. 6.3. Pos -T anscy osis NP-B ain In e ac ion As PLGA NPs a e able o each he b ain, a ge ed deli e y sys ems a e needed so ha NPs can en e he b ain cells [ 97 ]. As al eady men ioned, CNS endo helial cells o e exp ess se e al ecep o s, including T Rs, and hus, ano he p omising s a egy o imp o e d ug anspo o he b ain is using monoclonal an ibodies (mAbs) o a ge T Rs. Indeed, T Rs a e o e exp essed in he b ain capilla y endo helium and ha e been demon- s a ed o unde go endocy osis ( ecep o media ed anscy osis) h ough he BBB [ 187 ]. Thus, an i ans e in ecep o monoclonal an ibodies such as OX26 a e being used o BBB c ossing. In ac , OX26 abili y o ecognize and bind o cells ha exp ess he T R, such as he BBB endo helial cells, has been well desc ibed [ 188 ]. Lou ei o e al. [ 109 ] p oposed PEG-PLGA NPs unc ionalized wi h wo mAbs o deli e encapsula ed an iamyloid iA β 5 pep ide in o he b ain o AD ea men . On he one hand, PEG-PLGA NPs we e conjuga ed wi h OX26 mAb o bind o he T R and c oss he BBB; hey we e conjuga ed wi h DE2B4 mAb o bind o he A β pep ide, he majo cons i uen o AD plaques, he eby ac ing as a a ge ing ligand. The esul ing PLGA NPs, o mean size o 150–170 nm, we e compa ible wi h he pa en e al ou e. The in i o up ake o PEGyla ed-PLGA NPs (wi hou mAbs a ached) was signi ican ly lowe when compa ed wi h he up ake o he immune NPs, he eby con i ming OX26 abili y o inc ease he cellula up ake o NPs. Fu he mo e, PLGA NP cellula up ake inc eases wi h he densi y o su ace-immobilized an ibody [ 189 ], explaining he inc eased cellula up ake o NPs om 8% (wi h OX26) o 14% (wi h OX26 and DE2B4). Thus, he o mula ion o PLGA NPs conjuga ed wi h hese wo an ibodies is a p omising sys em o p o ec an iamyloid pep ides om p o eoly ic deg ada ion and o inc ease hei up ake in he b ain. Howe e , he mechanism o in e naliza ion o PLGA immune NPs by b ain capilla y endo helial cells needs o be elucida ed, and u u e wo k is equi ed o con i m ha hese NPs a e e icien o he ea men o AD in ansgenic models. Mo eo e , as al eady men ioned, he usage o la ge p o eins such as T R an ibodies can esul in p oblems like syn hesis p ocedu e, s abili y and immunological esponse [157]. Appl. Sci. 2021,11, 4305 20 o 27 7. Conclusions Al hough much p og ess has been made owa ds he unde s anding o AD pa hophys- iology, he e is s ill no clinically accep ed ea men o cu e o hal i s p og ession. Mo eo e , he s uc u e o he BBB is a majo obs acle o he deli e y o d ugs in o he b ain o he ea men o CNS diseases like AD. The e o e, nano echnology-based DDS such as PLGA NPs ha e eme ged and a e unde in es iga ion. Indeed, he use o PLGA NPs appea s o be a p omising di ec ion o he ea men o neu odegene a i e diseases, since se e al s a egies o enhance he anspo o NPs h ough he BBB ha e been de eloped. Indeed, some o he la es app oaches o PLGA NPs su ace modi ica ion we e discussed in his e iew a icle, and in mos cases, a single unc ionaliza ion s a egy was no enough. PLGA NPs composi ion needs o be op imized, choosing app op ia e componen s o ob ain PLGA o mula ions able o achie e BBB c ossing and p ecise a ge ing, so ha hese o mula ions can play a i al ole in AD he apy. Au ho Con ibu ions: Concep ualiza ion, L.D.A., A.C. (Amanda Cano), M.E. (Mi en E che o) and E.S.-L.; w i ing—o iginal d a p epa a ion, L.D.A., A.C. (An oni Camins), and E.B.S.; w i ing— e iew and edi ing, L.D.A., M.E. (Ma a Espina), A.C. (Amanda Cano), A.C. (An oni Camins), E.B.S., M.L.G., and E.S.-L.; isualiza ion, moni o ing and esou ces: L.D.A., M.L.G. and E.S.-L. All au ho s ha e ead and ag eed o he published e sion o he manusc ip . Funding: This esea ch ecei ed no ex e nal unding. Acknowledgmen s: A.C. [Amanda Cano] acknowledges he suppo o he Spanish Minis y o Science, Inno a ion and Uni e si ies unde he g an Juan de la Cie a (FJC2018-036012-I). Au ho s acknowledge he suppo o he Spanish Minis y o Economy and Compe i i eness unde he p ojec SAF2017-84283-R; Biomedical Resea ch Ne wo king Cen e in Neu odegene a i e Diseases (CIBERNED, CB06/05/0024) and Po uguese Science and Technology Founda ion (FCT) o he s a egic und (UIDB/04469/2020). Con lic s o In e es : The au ho s decla e no con lic o in e es . Re e ences 1. Se ano-Pozo, A.; F osch, M.P.; Masliah, E.; Hyman, B.T. Neu opa hological Al e a ions in Alzheime Disease. Cold Sp ing Ha b. Pe spec . Med. 2011,1, a006189. [C ossRe ] 2. Wong, K.H.; Riaz, M.K.; Xie, Y.; Zhang, X.; Liu, Q.; Chen, H.; Bian, Z.; Chen, X.; Lu, A.; Yang, Z. 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