Polymer-based nanocarriers to transport therapeutic biomacromolecules across the blood-brain barrier

Re iew a icle Polyme -based nanoca ie s o anspo he apeu ic biomac omolecules ac oss he blood-b ain ba ie Elena Rome o-Ben a , Upashi Goswami a,b , Jackeline So o-C uz a , Ami eza Mansoo i-Ke mani b,c,d , Dhi aj Mish a a,e , Se gio Ma in-Salda˜ na a , Jone Mu˜ noz-Uga emendia b , Alejand o Sosnik e , Ma celo Calde ´ on a, , Ana Beloqui a, , Ai o La a˜ naga b,* a POLYMAT, Applied Chemis y Depa men , Facul y o Chemis y, Uni e si y o he Basque Coun y UPV/EHU, Paseo Manuel de La dizabal 3, Donos ia-San Sebas i´ an 20018, Spain b Depa men o Mining-Me allu gy Enginee ing and Ma e ials Science, POLYMAT, Bilbao School o Enginee ing, Uni e si y o he Basque Coun y (UPV/EHU), Plaza To es Que edo 1, Bilbao 48013, Spain c Is i u o I aliano di Tecnologia, Sma Bio-In e aces, Viale Rinaldo Piaggio 34, Pon ede a 56025, I aly d Scuola Supe io e San ’Anna, The Bio obo ics Ins i u e, Viale Rinaldo PIaggio 34, Pon ede a 56025, I aly e Labo a o y o Pha maceu ical Nanoma e ials Science, Depa men o Ma e ials Science and Enginee ing, Technion - Is ael Ins i u e o Technology, Hai a 3200003, Is ael IKERBASQUE, Basque Founda ion o Science, Plaza Euskadi 5, Bilbao 48009, Spain ARTICLE INFO Keywo ds: Biomac omolecules Blood-b ain ba ie anspo Polyme ic nanopa icles Neu ological diso de s ABSTRACT The apeu ic biomac omolecules such as gene ic ma e ial, an ibodies, g ow h ac o s and enzymes ep esen a no el he apeu ic al e na i e o neu ological diseases and diso de s. In compa ison o adi ional he apeu ics, which a e mainly based on small molecula weigh d ugs ha add ess he symp oms o hese diso de s, he a- peu ic biomac omolecules can educe undesi ed side e ec s and a ge speci ic pa hological pa hways, hus pa ing he way owa ds pe sonalized medicine. Howe e , hese biomac omolecules unde go deg ada ion/ dena u a ion p ocesses in he physiological en i onmen and show poo capaci y o c oss he blood-b ain ba ie (BBB). Consequen ly, hey a ely each he cen al ne ous sys em (CNS) in hei ac i e o m. He ein, we c i - ically o e iew se e al polyme ic nanoca ie s ha can p o ec and deli e he apeu ic biomac omolecules ac oss he BBB. Polyme ic nanoca ie s a e i s ca ego ized based on hei a chi ec u e (biodeg adable solid nanopa icles, nanogels, dend ime s, sel -assembled nanopa icles) ha ul ima ely de e mines hei physico- chemical p ope ies and unc ion. The a ailable polyme ic o mula ions a e hen ho oughly analyzed, placing pa icula a en ion on hose s a egies ha ensu e he s abili y o he biomac omolecules du ing hei encap- sula ion p ocess and p omo e hei passage ac oss he BBB by con olling hei physical (e.g., mechanical p ope ies, size, su ace cha ge) and chemical (e.g., su ace unc ional g oups, a ge ing mo i s) p ope ies. Acco dingly, his e iew gi es a unique pe spec i e on polyme ic nanoca ie s o he deli e y o he apeu ic biomac omolecules ac oss he BBB, ep esen ing a concise, comple e and easy- o- ollow guide, which will be o high in e es o chemis s, ma e ial scien is s, pha macologis s, and biologis s. Besides, i also p o ides a c i ical pe spec i e abou he limi ed clinical ansla ion o hese sys ems. S a emen o signi icance: The inc easing incidence o cen al ne ous sys em diso de s is a majo heal h conce n. The use o he apeu ic biomac omolecules has been placed in he spo ligh o many in es iga ions. Howe e , eaching he apeu ic concen a ion le els o biomac omolecules in he cen al ne ous sys em is es ic ed by he blood-b ain ba ie and, hus, his ep esen s he main clinical challenge when de eloping e icien he apies. He ein, we p o ide a c i ical discussion abou he use o polyme ic nanoca ie s o deli e he apeu ic bio- mac omolecules in o he cen al ne ous sys em, highligh ing po en ial u u e di ec ions o o e come he cu en challenges. * Co esponding au ho . E-mail add ess: [email p o ec ed] (A. La a˜ naga). Con en s lis s a ailable a ScienceDi ec Ac a Bioma e ialia jou nal homepage: www.else ie .com/loca e/ac bio h ps://doi.o g/10.1016/j.ac bio.2025.02.065 Recei ed 16 Sep embe 2024; Recei ed in e ised o m 20 Feb ua y 2025; Accep ed 28 Feb ua y 2025 Ac a Bioma e ialia 196 (2025) 17–49 A ailable online 1 Ma ch 2025 1742-7061/© 2025 The Au ho s. Published by Else ie Inc. on behal o Ac a Ma e ialia Inc. This is an open access a icle unde he CC BY-NC-ND license ( h p://c ea i ecommons.o g/licenses/by-nc-nd/4.0/ ). 1. In oduc ion In he las hi y yea s, he e has been a subs an ial ise in he global occu ence and dea h a es associa ed o neu ological diso de s, cu en ly ep esen ing a majo cause o mo bidi y and mo ali y wo ldwide. This phenomenon can be asc ibed o he expansion and ma u a ion o he wo ld popula ion, along wi h inc easing ulne abili y o en i onmen al, me abolic, and li es yle haza ds. Acco ding o he “Global Bu den o Disease, Inju ies, and Risk Fac o s S udy (GBD) 2021 ″ , a ound 3.4 billion people wo ldwide expe ienced a ne ous sys em diso de in 2021 [1]. These include, among o he s: (i) neu odegene a- i e diseases, namely Alzheime ’s disease (AD), Pa kinson’s disease (PD), and Hun ing on’s disease (HD); (ii) au oimmune diseases like mul iple scle osis (MS); (iii) b ain cance , pa icula ly glioblas oma mul i o me (GBM); (i ) ce eb o ascula acciden s (e.g., ischemic s oke); ( ) auma ic b ain inju y (TBI); and ( i) o he diseases (e.g., epilepsy, ansien ischemic a ack, and ce eb al palsy) a ec ing he cen al ne ous sys em (CNS). In he u u e, i is an icipa ed ha b ain diso de s will eme ge as a signi ican global heal h conce n, posing subs an ial mo ali y a es and inancial bu dens [2]. To ace his challenge, g ea e o s a e being cu en ly in es ed in de eloping ea men s ha no only alle ia e he symp oms o hese diseases bu also ea he unde lying pa hology. Se e al ac o s con ibu e o he ailu e o cu en medica ion ea men s (mainly based on small molecula weigh (MW) d ugs) o b ain diseases, including he equi emen o la ge he apeu ic doses and p olonged and highly in asi e in a en icula injec ions [3]. These a iables s ongly impac he medioc e esul s e ealed by s anda d ea men op ions in he clinic [4,5]. The use o biomac omolecules such as oligopep ides, monoclonal an ibodies, g ow h ac o s, an ioxidan enzymes o nucleic acids, ep- esen s a no el he apeu ic al e na i e and has ga ne ed conside able a en ion om esea che s, p omp ing ex ensi e endea o s o acili a e hei ansla ion in o clinical applica ions. This new class o he apeu ics o e s highe speci ici y and can g ea ly educe o - a ge e ec s (Table 1). Al hough signi ican ad ances in molecula biology pe mi s now he la ge-scale syn hesis o such delica e biomac omolecules, hei use as a he apy s ill aces unme challenges. The cell memb ane has e ol ed o e billions o yea s o accu a ely con ol he mo emen o molecules in o and ou o he cy osol, p o ec ing he in acellula en i onmen om ex acellula dis u bance. Hence, he high MW and he hyd ophilic na u e o mos o he biomac omolecules esul s in a limi ed pe me- abili y h ough biological ba ie s like cell memb anes [29]. Besides, mac omolecula s uc u es can unde go con o ma ional changes due o he dis up ion o na i e non-co alen in e ac ions and he clea age o pep ide bonds, esul ing in impai ed he apeu ic agen s. Fu he mo e, deli e ing biomac omolecules o he b ain sys emically aces di e se challenges such as: insu icien d ug adminis a ion, deg ada- ion/dena u a ion in he bloods eam, i s -pass clea ance, immune esponse, oxici y o no mal issues, and es ic ions imposed by he blood-b ain ba ie (BBB); mos o he biomac omolecules a e unable o nonin asi ely c oss he BBB and en e he b ain pa enchyma [30]. The seek o s a egies o he e icien espassing o bio- mac omolecules h ough he BBB has opened new esea ch oppo u- ni ies in which s a e-o - he-a echnologies a e me ged wi h enginee ed deli e y sys ems. Thus, new app oaches encompass he dis up ion o he BBB ia he u iliza ion o magne ic esonance-guided ocused ul asound [31] o he lipidiza ion o wa e -soluble d ugs [32]. In his ega d, nano echnology could p o ide sophis ica ed s a egies o design and o mula e nanoma e ials loaded wi h biomolecules, signi ican ly enhancing pa ien p ospec s and achie ing posi i e pha maco-economic esul s [33,34]. A clea example is he applica ion o an ioxidan s [35] in he managemen o b ain inju ies and neu odegene a ion, whe ein an ioxidan enzymes such as supe oxide dismu ase (SOD1) migh Table 1 Cu en medica ions and biomac omolecules unde esea ch o he ea men o CNS diso de s o b ain cance [3]. Neu ological diso de Cha ac e is ic Cu en medica ions Common side e ec s S udies wi h biomac omolecules Alzheime ’s disease (AD) G adually ad ancing demen ia accompanied by memo y impai men . Cholines e ase inhibi o s, NMDA an agonis s. Nausea, omi ing, dia hea, dec eased appe i e, dizziness, headache, con usion. Monoclonal an ibody agains Aβ agg ega es [6] (Aducanumab [7], Lecanemab [8] and Donanemab [9]). Pa kinson´s disease (PD) Pa hological degene a ion o nig os ia al dopamine neu ons cha ac e ized by mo o igidi y, es ing emo , and b adykinesia. Le odopa, dopamine agonis , Monoamine oxidase-B inhibi o s, ca echol o-me hyl ans e ase (COMT) inhibi o s, su gical he apies. Dyskinesias, nausea, omi ing, o hos a ic hype ension, hallucina ions, li e damage, in ec ions. Neu o ophic ac o s (glial cell-de i ed neu o ophic ac o (GDNF) and b ain- de i ed neu o ophic ac o (BDNF)) [10,11], monoclonal an ibodies agains α -synuclein (Snca) (P asinezumab) [12,13], shRNA a ge ing Snca [14]. Hun ing on’s disease (HD) Au osomal neu odegene a i e diso de . Pa ien s exhibi a ange o symp oms, including cho ea, men al dis u bances, and cogni i e de e io a ion. Cho ea and an ipsycho ic medica ion, an idep essan s (Selec i e se o onin eup ake inhibi o s), mood s abilize s. D owsiness, pa kinsonism, dep ession, insomnia, anxie y, aka hisia, excess weigh gain and dyslipidemia. Di alen small in e e ing RNA (siRNA) silencing he hun ing in gene [15]. Chape one p o eins o comba he neu odegene a i e e ec [16]. Mul iple scle osis (MS) Demyelina ing disease cha ac e ized by he immune sys em’s a ack on he myelin shea h su ounding ne e ibe s, esul ing in communica ion impai men s be ween he b ain and he body. gamma-aminobu y ic acid analogs, ac i a ion o α 2-ad ene gic ecep o s, mi ochond ial dihyd o-o o a e dehyd ogenase (DHODH) inhibi o s, immunomodula o s (Gla i ame ace a e). Dia hea, nausea, li e p oblems, weakness, hallucina ions, hypo ension, inc eased isk o in ec ions. Neu o ophic ac o s (BDNF) [17]. Remyelina ion-p omo ing p o eins (Insulin-like g ow h ac o 1 (IGF-1)) [18]. Monoclonal an ibodies (Na alizumab) [19]. S oke Neu ological de iciency a ibu ed o a se e e ocal lesion o he cen al ne ous sys em (CNS) due o a ascula disease, including ce eb al in a c ion, suba achnoid hemo hage (SAH), and in ace eb al hemo hage (ICH). Se ine p o ease issue- ype plasminogen ac i a o . Nausea, omi ing, hypo ension, dizziness, alle gic eac ions. Neu o ophic ac o s (NGF) [20]. RNAi a ge ing umo nec osis ac o (TNF)- α o in e leukin (IL)-1β o educe pos -ischemic in lamma ion [21]. miR-124, which p omo es human neu ogenesis [22]. siRNAs a ge ing caspase [23]. Glioblas oma Mos common and agg essi e umo among glial neoplasms. Radia ion he apy, emozolomide adminis a ion (chemo he apy DNA base alkyla ion), and su ge y. Ce eb al edema, con ulsions, a igue, omi ing, skin damage, alopecia, bone ma ow supp ession. Monoclonal an ibodies: Be acizumab ha selec i ely binds o ascula endo helial g ow h ac o (VEGF) [24], Ce uximab and Pani umumab ha a ge he mu a ed epide mal g ow h ac o ecep o (EGFR) III a ian , common in glioblas oma [25,26], siRNA agains Bcl-2 [27] o miR-124 [28]. E. Rome o-Ben e al. Ac a Bioma e ialia 196 (2025) 17–49 18 assume a pi o al ole. None heless, due o hei limi ed s abili y and adminis a ion, he li e a u e lacks ele an s udies. The po en ial o clinical ans e a ises solely when u ilized in conjunc ion wi h nano- ca ie s [36–38]. O e he yea s, signi ican ad ancemen s ha e been made in he de elopmen o ailo ed nanoca ie s, wi h special a en ion o lipid nanopa icles as deli e y sys ems o enhance he e ec i eness o hese he apeu ics [39–41]. Howe e , he body ecognizes hese nanopa icles as o eign ma e ials and ac i a es he inna e immuni y, he eby in lu- encing adap i e immuni y [42]. In such si ua ions, polyme nano- pa icles may o e supe io cha ac e is ics such as educed immunogenici y, inc eased s abili y, and enhanced ep oducibili y [43–45]. In e es ingly, hese p omising nanoca ie s, when combined wi h cell-pene a ing echniques, can add ess he challenges associa ed wi h he deli e y o biomac omolecules o he ea men o CNS dis- o de s [46,47]. In his e iew, we emphasize he sophis ica ed a chi ec u es a ail- able in he ecen li e a u e (i.e., ~60% o he e e ences used in his e iew belong o s udies pe o med in he las 5 yea s) o he ab ica ion o polyme ic nanoca ie s capable o c ossing he BBB om he blood- s eam, oge he wi h he cu en app oaches o encapsula ing he a- peu ic biomac omolecules (Fig. 1). This e iew does no aim solely o p o ide examples o polyme ic nanoca ie s ha deli e bio- mac omolecules ac oss he BBB, which a e summa ized in Table 2. Ins ead, ou goal is o o e a c i ical pe spec i e on he ea u es ha make hese polyme ic nanoca ie s ideal o his pu pose, as well as o Fig. 1. Gene al illus a ion ha summa izes he main polyme ic nanoca ie s desc ibed in his e iew o anspo he apeu ic biomac omolecules in o he b ain. (A) Biodeg adable solid nanopa icles encompass nanopa icles ha p esen (i) a b oad o syn he ic al e na i es o deco a e hei su ace wi h speci ic ligands o enhance an ac i e a ge deli e y, and (ii) allow wo main encapsula ion app oaches, such as double wa e in-oil-wa e o nanop ecipi a ion echniques o encapsula e biomac omolecules. (B) Nanogels a e so polyme ic nanopa icles, ha (i) pe mi he use o a b oad mul i- esponsi e c osslinke s o lead di e en s imuli- esponsi e nanoma e ials and also (ii) allow he encapsula ion o biomac omolecules media ed by di e en echniques such as he o ma ion o sel -assembled complex, UV polyme iza ion in liposomes, a om ans e adical polyme iza ions (ATRP) in in e se micelles and in si u adical polyme iza ion leading single enzyme nanogels. (C) Dend i ic polyme s ha e a con olled syn hesis in mul i-b anched a chi ec u es, ha opens he possibili y o expose mul i alen ly speci ic g oups on hei su ace o enhance ligand- ecep o s in e ac ions, and, a he same ime, modula e he su ace cha ge o encapsula e biomac omolecules as nucleic acids by elec os a ic in e ac ion inside hei in e nal po es. (D) Sel -assembled polyme ic nanopa icles can be syn he ized h ough spon aneous in e ac ion o molecules (physic o ces) o o m o ganized s uc u es ha acili a e he implemen a ion o encapsula ion app oaches. Bio ende has been used o c ea ing he desc ip i e image. E. Rome o-Ben e al. Ac a Bioma e ialia 196 (2025) 17–49 19 highligh he ad an ages o using ce ain ypes o polyme ic sys ems o e o he s. Addi ionally, i highligh s clinical ansla ion, explo es cu en challenges, and o e s insigh s in o u u e p ospec s in his ield. This app oach allows he eade o d aw hei own conclusions, conside ing ha each nanosys em possesses impo an ea u es o di e en po en ial applica ions. 2. The blood-b ain ba ie issue o he apy design 2.1. Physiology o he blood-b ain ba ie The BBB se es as a p o ec i e, semi-pe meable memb ane ha in ica ely egula es he exchange o essen ial nu ien s, oxygen, and ions om he bloods eam in o he CNS, while i p e en s he in ac ion by exogenous subs ances, oxins, and pa hogens [69,70]. Ana omically, he BBB is a complex and in eg a ed ne wo k o endo helial, neu al, and immune cells, known as he neu o ascula uni [71–73]. The endo helial cells, ep esen ing he main componen o he BBB, o m he inne line o b ain capilla y walls wi h la ges su ace a ea. They a e pola ized wi h nega i e su ace cha ge and no enes a ions, p e en ing he apid di usion and exchange o nega i ely-cha ged molecules be ween he blood and he b ain [69,70]. P ima ily, endo helial cells a e allied wi h one ano he by igh junc ions (TJs), which is dis inc i e o endo helial cells o CNS along wi h adhe ens junc ion, which oge he o m a obus physical ba ie [72]. Unde he endo helial cells lies he basemen memb ane. I consis s o ex acellula ma ix p o eins like collagen, hepa in, laminin, nidogen, and pe lecan [69,70,72,73]. Wi hin he basemen memb ane, pe icy es (a ype o mu al cells) g ow abluminal o endo helial cells, and co e 90% o capilla y beds [73]. Pe icy es a e esponsible o main aining he diame e o he essels and egula e he speci ic gene exp ession o endo helial cells om BBB, essen ial o main aining he TJs [71,74]. Due o hei close associa ion wi h endo- helial cells, hey play a pi o al ole in egula ing BBB in eg i y, ho- meos asis, and in con olling ce eb al blood low in capilla ies along wi h exchange o ions and me aboli es be ween hem [69,75,76]. Addi ionally, hey a e also impo an in ascula de elopmen , egula- ion o in lamma o y esponse by con olling leucocy e in il a ion, sec e ion o in lamma o y media o s, and neu o oxin emo al [75,76]. A he in e ace be ween neu ons and endo helial cells a e he as o- cy es, a ype o glial cells connec ed wi h he end walls o endo helium. They co e mos o he CNS and gi e in eg i y by s eng hening he TJs, while p o iding biological suppo o he cells [69,71]. They play a majo ole in egula ion o ascula unc ion, exp ession o anspo e s such as P-glycop o ein (P-gp) and glucose anspo e -1 (GLUT-1), wa e and ion homeos asis, pH egula ion, neu o ascula coupling (as as o- cy es a e he cen al link be ween mic o ascula u e and neu al ne wo k), and p o ide ene gy- ich subs a es and insula ion o he neu ons [70,71,76]. Acco dingly, close associa ion o as ocy es wi h endo helial cells and pe icy es holds con ol o e pe meabili y ac oss he BBB [69,77]. Mic oglia ac s as he mac ophages o he CNS, mig a ing o pa hologically a ec ed egions o phagocy ose ne ous issue [77]. Mic oglia a e igilan , and ensu e p ope esponse o any Table 2 Polyme -based nanoca ie s ha anspo he apeu ic biomac omolecules ac oss he BBB o ea CNS diso de s o b ain cance . Classi ica ion D ug deli e y sys em Ca go Disease BBB Pene a ion Re e ence Biodeg adable solid nanopa icles (NPs) Poly(lac ic-co-glycolic acid) PLGA NPs Enzyme galac osylce amidase K abbe disease Angiopep-2 (Ang2), g7 and ans e in (T ). [48] PLGA NPs Small in e e ing RNA (siRNA) T auma ic b ain inju y Polyso ba e 80, poloxame 188, DSPE- PEG-glu a hione, and DSPE-PEG-T . [49] PLGA NPs Vi amin D-binding p o ein Alzheime ´s Disease (AD) - [50] PLGA NPs Epide mal g ow h ac o ecep o siRNA Gliomas Ang2. [51] Nanogels PLA-PEG-PLA NGs Ri uximab (RTX) Non-Hodgkin lymphoma 2-me hac yloyloxye hyl phospho ylcholine (MPC). [52] PLA-PEG-PLA NGs Ne e g ow h ac o and RTX Cen al ne ous sys em (CNS) diseases MPC. [53] Poly(N-(3-aminop opyl) me hac ylamide- co-me hac yloyloxy e hyl phospho ylcholine) and MMP sensi i e pep ide sequence as c osslinke Nimo uzumab and as uzumab B ain umo s MPC. [54,55] Poly(e hylene glycol)-b-poly(me hac ylic acid) (PEG-b-PMAA) diblock copolyme Cispla in T ea men o glioma Monoclonal an ibodies agains Cx43 and BSAT1. [56] Dend i ic polyme s Polyamidoamine dend ime Heme oxygenase-1 (HO-1) plasmid In lamma o y diseases including ischemic s oke - [57] Dend i ic polyglyce ol sul a e. Dend i ic polyglyce olamine. Pep idomime ic o he an i- angiogenic p o ein h ombospondin-1 (TSP-1 PM) mic oRNA-34a (miR-34a) Glioblas oma Glioma T . [58,59] Ca bosilane dend ime . Lac o e in bea ing polyp opylenimine dend iplex siRNA. 2G-(SNMe3I)11-FITC. Neu ological diso de s Lac o e in. [60,61] Sel -assembled pa icle Ca ionic micelles assembled om i amin E succina e g a ed ε -polylysine polyme s TRAIL plasmids (pDNA) Gliomas Apolipop o ein E (ApoE). [62] Glucosyla ed-polyion complex polyme ic micelle An isense oligonucleo ide (ASO) Cen al ne ous sys em (CNS) diso de s Glucose coa ing (GLUT-1- media ed anspo s a egy). [63] Chime ic polyme somes siRNA Glioblas oma Ang2. [64] Chime ic polyme somes Sapo in Glioblas oma (ApoE) pep ide. [65] O he polyme ic ca ie s Chi osan-PMMA-PAA NPs Topoisome ase I inhibi o SN-38 B ain umo s Re oenan io pep ide shu le H- pw pswmpp h -NH2. [66] Chi osan NPs G een Fluo escen P o ein (GFP)- agged plasmid in HEK293–293 B ain cance - [67] Hyalu ona e Nanopa icles Neu oglobin S oke - [68] E. Rome o-Ben e al. Ac a Bioma e ialia 196 (2025) 17–49 20 inju y, in ec ion, and in lamma ion o main aining CNS homeos asis [76]. These highly complex, dynamic, and selec i e ea u es o he BBB a e o u mos impo ance o he co ec homeos asis o he CNS. Un- de s anding he ole o BBB anspo e s o main aining homeos asis is e y c ucial, since hey go e n he ansloca ion o a ious molecula en i ies ac oss he BBB (Fig. 2a). 2.2. Blood-b ain ba ie dis up ion unde pa hological condi ions The BBB unde goes dis inc i e changes in a ious neu ological dis- o de s and diseases [70,71,77,78]. The pa hology o BBB in ol es s uc u al, molecula , and unc ional modi ica ions o BBB cons i uen s, ha impac he o e all physiological in eg i y and unc ion o he ba - ie . E en hough he p ime cause o di e en diseases impac ing BBB di e s, he p ima y ocus o BBB pa hology cen e s on i s pe meabili y. In no mal condi ions, BBB is selec i ely pe meable (Fig. 2b), bu mos o hese diso de s esul in inc eased pe meabili y due o al e a ions o endo helial cells (sh inkage), loss o TJs p o eins, augmen ed anspo o molecules ( anscy osis), and al e a ion in anspo (pa acellula and anscellula ) sys ems [69,78]. Addi ionally, BBB b eakdown is e iden by he loss o basemen memb ane, enhanced leucocy e ac i i y, de e- io a ion o pe icy es esul ing in impai men o ascula ne wo k, and de ached as ocy es, as desc ibed in Fig. 2c [71,76]. The o he hallma k o BBB pa hogenesis is he elease o in lamma o y media o s such as in e leukin (IL)-1β, in e e on (IFN)-γ, umou nec osis ac o (TNF)- α , oxida i e compounds (e.g., NO, H 2 O 2 ), lipid media o s (p os aglandin E2 and F2a), asogenic agen s (e.g., his amine), and enzymes (e.g., ma ix me allop o einases) by bo h local and in il a ing immune cells [77,78]. 2.3. S a egies o blood-b ain ba ie c ossing 2.3.1. Physical and chemical dis up ion o he blood-b ain ba ie Ex e nal s imuli-media ed me hods ha can egula e BBB pe me- abili y o deli e y o he apeu ic agen s ha e been widely explo ed, and hey showed high e ec i eness. They in ol e physical and biochemical app oaches (Fig. 3). Physical dis up ion: Ex e nal physical app oaches, like ocused ul a- sound, magne ic esonance imaging, high pulse elec ic ields, lase s, and elec o acupunc u e, ha e been used o p omo e he pe meabili y o he BBB and he ollowing deli e y o he apeu ic agen s and (bio) mac omolecules in he b ain issue [69,79–82]. Focused ul asound, along wi h mic obubbles (gas- illed), in ol es high- equency sound wa es ha a e p ecisely a ge ed o a speci ic a ea wi hin he b ain o igge mic obubbles in he blood o ca i a ion [77]. This mechanical in e ac ion esul s in empo a y and e e sible dis up ion o he BBB, allowing he deli e y o he apeu ic agen s in CNS. The e ha e been ecen ad ancemen s in using his app oach, some o which a e unde - going clinical ials [83]. Addi ionally, ocused ul asound has been coupled wi h magne ic esonance imaging and mic obubbles o p ecise a ge ing o d ugs [78,80,83]. Low- and high-pulsed elec ical ields ha e also been used o anspo small molecules by dis up ing he TJs o he BBB [81]. Recen ly, lase s imula ion has been explo ed o modu- la ing BBB pe meabili y. Fo his, plasmonic gold nanopa icles (AuNPs) we e conjuga ed wi h an ibody BV11 (AuNPBV11) o a ge ing junc- ional adhesion molecule A (JAM-A) and we e injec ed o mice Fig. 2. (A) Schema ic ep esen a ion o a ious anscy osis pa hways o blood-b ain ba ie (BBB) ha egula es he anspo a ion o di e en molecules, bio- mac omolecules and ions. Small lipophilic molecules passi ely di use h ough BBB ia anscellula lipophilic pa hway whe eas hyd ophilic molecules pass ia a pa acellula pa hway. Essen ial nu ien s and ions along wi h glucose, i amins, elec oly es, amino acids, and nucleosides c oss he BBB h ough ca ie media ed anscy osis (CMT) ia glucose anspo e iso o m 1 (GLUT-1) and la ge neu al amino acid anspo e 1 (LAT1). La ge biomac omolecules like p o eins, lipo- p o eins, o pep ides, c oss he BBB h ough anscy osis mechanisms ia in e ac ion wi h speci ic ecep o s and is known as ecep o media ed anscy osis (RMT). This in ol es ans e in ecep o (T R), insulin ecep o (IR), lipop o ein ecep o s, and lac o e in (L ) ecep o . Adso p i e media ed anscy osis (AMT) allows anscy osis o la ge biomac omolecules (e.g., pep ides) and is based on elec os a ic in e ac ions be ween he posi i ely-cha ged subs a es and he nega i ely- cha ged plasma memb ane. (B) Schema ic ep esen a ion o ans e se sec ion o BBB wi h i s componen s and unc ion du ing heal hy s a e, and (C) a e BBB b eakdown. Bio ende has been used o c ea ing he desc ip i e image. E. Rome o-Ben e al. Ac a Bioma e ialia 196 (2025) 17–49 21 in a enously. One-hou pos -adminis a ion, 532 nm picosecond lase was applied ansc anially, leading o ac i a ion o AuNPBV11. This esul ed in he enhancemen o BBB pe meabili y, acili a ing he de- li e y o immunoglobulins, adeno-associa ed i al ec o s, and lipo- somes [82]. The e ec o i adia ion on he b ain is in ica ely associa ed wi h powe and i adia ion ime, along wi h he dis ance be ween he sou ce and he a ge ed a ea. No ably, nea in a ed ligh wa eleng h in he ange om 700 o 1600 nm has ga ne ed p o ound in e es due o i s abili y o pene a e deep issues and has been used o egula e BBB pe meabili y. Recen s udies ha e applied nea in a ed i adia ion o imp o e he abili y o ligh -sensi i e d ug deli e y sys ems wi h pho- o he mal e ec s o pene a e BBB, o e ec i e ea men o dep ession [69]. The use o elec oacupunc u e o imp o ing he pe meabili y o BBB has also gained in e es . One o he s udies highligh s he augmen a ion o BBB pe meabili y h ough he applica ion o elec o- acupunc u e a ge ing he GV20 and GV26 acupoin s in a models [69]. Chemical dis up ion: The mos commonly used chemical eagen s o dis up he in eg i y o he BBB, oge he wi h hei wo king condi ions, applica ions, ad an ages and disad an ages a e summa ized in Table 3. [69,77,78,80,84–92] Some o he pha maceu ical biological compounds like zonula occludens oxins, Ce epo (a syn he ic pep ide analog o b adykinin), LipoB idge (a nonimmunogenic o mula ion con aining sho -chain oligoglyce olipids), ascula endo helial g ow h ac o (VEGF), and chemical compounds such as oleic acid, lysophospha idic acid, cyclodex ins, and sodium dodecyl sul a e a e also in ol ed in ansien opening o he BBB [77,78,86]. The i uses also se e as s imula ing biological agen s ha help in he opening o TJs by up egula ing chemokines. As example, s udies on human b ain mic o ascula endo helial cells highligh ed he in ol e- men o he human immunode iciency i us (HIV) ype 1 gp 120 i us in enhancing BBB pe meabili y by deg ading TJs [78]. Addi ionally, adeno-associa ed i us and Wes Nile i us also exhibi ed p omising abili y o a ge CNS, wi hou dis up ing he BBB [78,95]. 2.3.2. In asi e s a egies Di ec injec ion/implan a ion: Di ec injec ions in ol e localized de- li e y o he apeu ics o hei sus ained elease ia implan s in o he b ain. This is used o ea condi ions such as cance s, s oke, neu o- logical and men al diso de s [96]. Su gically placed implan s can p o- ide sus ained elease o d ugs by opening he skull. The ca mus ine-loaded biodeg adable implan s (Gliadel® wa e ), app o ed by he Food and D ug Adminis a ion (FDA), placed du ing umou esec ion, ha e shown imp o ed su i al a es in pa ien s acing GBM. Also, nica dipine- eleasing solid implan s ha e been success ully used o p e en neu ological diso de s ollowing s okes [96]. A he same ime, long- e m (5 mon hs) deli e y sys ems ha e been explo ed o schizo- ph enia o add ess ea men adhe ence issues associa ed wi h men al diso de s [96]. In ace eb o en icula adminis a ion: In his app oach, he he a- peu ics a e deli e ed in o he ce eb ospinal luid, speci ically in he la e al en icle sys em o he b ain, ia an ou le ca he e o in a- ce eb o en icula po implan ed unde he scalp o by pump [77,88]. Ou o his, he use o pumps is mo e p e alen , as i main ains con in- uous and ele a ed d ug concen a ions in ce eb ospinal luid [77]. In ace eb o en icula implan s ha e been used o deli e chemo he - apeu ics o b ain cance , opioids o pain managemen , and d ugs o he ea men o neu ological diso de s like lysosomal s o age diso de s (LSD), mucopolysaccha idosis, and o diso de s associa ed wi h ce e- b al palsy (e.g., baclo en) [90]. Addi ionally, clinical ials a e going on o amyo ophic la e al scle osis (ALS) and PD by adminis e ing VEGF and pla ele -de i ed g ow h ac o s, espec i ely [88,90]. In a hecal adminis a ion: He e, he he apeu ics a e di ec ly injec ed in o he space su ounding he spinal co d by lumba punc u e and a e deli e ed o CNS pa enchyma ia ce eb ospinal luid. This app oach has been app o ed o he deli e y o an isense oligonucleo ide o he ea men o spinal muscula a ophy. Clinical ials a e going on o ALS and HD [77,90]. 2.3.3. Non-in asi e s a egies Non-in asi e s a egies o he deli e y o he apeu ics ely on pha macological app oaches ha exploi he endogenous anspo mechanisms o enhanced BBB pe meabili y. These s a egies, ha include chemical modi ica ion o he he apeu ic d ugs, anspo ca - ie s, chime ic pep ides, nanoca ie s, T ojan ho se, e c., ha e Fig. 3. Schema ic ep esen a ion o he mos common s a egies o blood-b ain ba ie (BBB) c ossing. Physical/chemical dis up ion includes ansien opening o he BBB o acili a e he deli e y o he apeu ics. In asi e s a egies in ol e di ec implan a ion/injec ion o he apeu ics ei he di ec ly in he b ain o h ough he ce eb ospinal luid (CSF). Non-in asi e s a egies equi e he use o ca ie s capable o deli e ing he apeu ics ac oss he BBB. Bio ende has been used o c ea ing he desc ip i e image. E. Rome o-Ben e al. Ac a Bioma e ialia 196 (2025) 17–49 22 Table 3 Summa y o a ious chemical eagen s used o BBB dis up ion. Agen s Examples Mechanism Wo king Condi ion Applica ion Ad an ages/Disad an ages Hype osmo ic Agen s Manni ol [77,78,80, 84,87–89] Osmo ic p essu e g adien c ea ed, which leads o sh inkage o endo helial cells and dis up ion o igh junc ion, and esul ing in empo a y opening o he blood- b ain ba ie (BBB) A e ial Injec ion/in usion (25%) D ug Deli e y, (chemo he apeu ics, imaging agen s) Ad an ages: Clinically app o ed, enhanced pe meabili y, non-in asi e, apid ac i i y, b oad ange o agen s, cos e ec i e Disad an ages: Non selec i e, inconsis en ba ie dis up ion, neu ological oxici y Glyce ol [80,85]Neu ological S udy, d ug deli e y Ad an ages: A ailabili y, low cos Disad an ages: Lowe e icacy, Immunogenic isks A abinose [77,78,80, 87,89,93] In a enous adminis a ion D ug Deli e y Ad an ages: Biodeg adable, e ec i e a low concen a ion, used o hyd ophilic and lipophilic d ugs Disad an ages: Limi ed in o ma ion, isk o me abolic imbalances Saline [89,90,93] In a enous adminis a ion (23.4%) Used in p eclinical models o d ug deli e y and con as agen s o CNS Ad an ages: Cos e ec i e, a ailabili y, easy adminis a ion Disad an age: Edema, issue damage, hype na emia (high sodium le els) U ea [85,89,90,93]Used in p eclinical s udies o d ug deli e y Ad an ages: Cos e ec i e, dis up s BBB a con olled doses Disad an ages: Sho li ed dis up ion, elec oly e imbalance F uc ose [85] In a enous adminis a ion Used in p eclinical s udies o d ug deli e y and con as agen s Ad an ages: Less oxic, biocompa ible Disad an ages: Limi ed In o ma ion, me abolic imbalances Vasoac i e Compounds His amine [77,80,89, 90] Binds wi h B2 ecep o s o endo helial cells leading o dis up ion o igh junc ion and enhanced d ug pe meabili y In a enous (10 o 100 µM) Used in p eclinical models o d ug deli e y Ad an ages: Minimal oxici y Disad an ages: sho du a ion o ac ion, non-speci ic B adykinine [77,80, 87,88,88,90] In a enous o in ace eb al adminis a ion D ug deli e y (enhanced deli e y o chemo he apeu ics, small molecules) Ad an ages: Selec i e, minimal oxici y Disad an age: Sho du a ion o ac ion, limi ed e icacy o la ge molecules, cos RMP-7 (analogues o b andykinin [88,89] Ad an ages: Mo e po en and speci ic o B2 ecep o s han b adykinin, highe hal -li e as esis an o deg ada ion Alkylglyce ols (e.g., 1- O-pen ylglyce ol [80, 88,89] In aca o id injec ion (200 mM) D ug Deli e y (Chemo he apeu ics) Ad an ages: Re e sible, inc ease in BBB pe meabili y Disad an ages: Limi ed e ec i eness, side e ec s Leuco ienes (e.g., cys einyl leuko ienes) [88,89] Binds o he G-p o ein coupled ecep o s CysLTR1 and CysLTR2, igge ing he con ac ion o endo helial cells and dis up ing igh junc ion esul ing in enhanced pe meabili y In ace eb o e–n icula injec ion (6pmol) D ug Deli e y Ad an ages: Immune esponse acili a ion Disad an ages: Tissue damage, long e m oxici y Tumo nec osis ac o A (TNF-A)/in e e on c (INF-c) [94] Tigh junc ion p o eins a e al e ed ia in lamma o y cy okines Sys emic o local adminis a ion D ug Deli e y, neu oin lamma ion and CNS esea ch Ad an ages: Ta ge speci ic, mechanis ic insigh s Disad an ages: Sys emic side e ec s, isk o neu oin lamma ion Chemical compounds Oleic Acid [77,78] I changes p o ein kinase C-induced p o ein phospho yla ion, esul ing in e e sible opening o BBB In aca o id in usion Used in p eclinical s udies o d ug deli e y Ad an ages: Mild and e e sible e ec Disad an ages: Low speci ici y, limi ed in o ma ion Lysophospha idic acid [77,78] In a enous Injec ion Ad an ages: Re e sible e ec Disad an ages: Limi ed in o ma ion, isk o neu oin lamma ion Sodium lau yl sulpha e [77,78] Dis up s endo helial cell memb ane by al e ing lipid bilaye Di ec In usion on BBB D ug Deli e y and BBB s udies Ad an ages: Cos , Dose dependen (con inued on nex page) E. Rome o-Ben e al. Ac a Bioma e ialia 196 (2025) 17–49 23 demons a ed p omising esul s wi h minimal ad e se e ec s. This e- iew ocusses on he T ojan ho se app oach, and highligh s hei mode o ac ion and he po en ial applica ion in he deli e y o bio- mac omolecules ac oss he BBB. Molecula T ojan ho ses a e designed o deli e he apeu ic mole- cules (e.g., small-molecule d ugs, ecombinan p o eins, and genes) ac oss he BBB by mimicking na u al biological p ocesses and s uc u es. This s a egy in ol es non-in asi e app oaches, using an ibodies, pep- ides, cells, and nanopa icles as T ojan ho ses, a ge ing endogenous BBB ecep o s such as ecep o -media ed anscy osis (RMT) and adso p i e-media ed anscy osis (AMT) o anscy osis o he apeu ic biomac omolecules [97–99]. Examples o some o he T ojan ho se ap- p oaches o BBB pene a ion a e desc ibed below (Fig. 3). An ibody T ojan ho ses: This echnique uses gene ic enginee ing o he apeu ics (neu o ophins, he apeu ic an ibodies, enzymes and decoy ecep o s) wi h monoclonal an ibodies (mAb) (IgG domain) ha ing speci ici y owa ds ans e in ecep o (T R) and human insulin e- cep o (HIR) o gi e T RmAb and HIRmAb. These ac as a T ojan ho se o deli e y o a ached d ugs ia RMT [100,101]. Fo example, T ojan ho se Pabina usp al a, enginee ed by usion o idu ona e 2-sul a ase (lysosomal enzyme) and hea y chain o T RmAb, has been used o he Hun e synd ome and ecei ed ma ke app o al o he ea men o b ain diso de s in Japan [100,102]. Pep ide T ojan ho ses: Angiopep-2 pep ide (Ang2) is a low-densi y lipop o ein ecep o (LPR1)- a ge ing pep ide, ha enhances he BBB pene a ion o an i-human epide mal g ow h ac o -2 mAb [103]. Simila ly, a no el de i a i e o pacli axel wi h Ang2 (ANG1005) e ealed imp o ed d ug up ake in gliomas and is unde clinical ial [99, 104]. TAT is a posi i ely-cha ged cell pene a ing pep ide (CPP) de i ed om he ansac i a o o ansc ip ion p o ein o he HIV. I has been ound ha many ca ionic pep ides a e aken up by he cells h ough AMT in i o. Howe e , poo b ain pe meabili y was obse ed in i o [98, 103]. Con a ily, ano he CPP, SynB pep ides, de i ed om a na u al mammalian an imic obial pep ide, ha e shown be e deli e y o pola biomolecules such as mo phine-6-glucu onide o he b ain in a clinical ial, bo h in i o and in i o [78]. Addi ionally, SynB3 conjuga ed wi h a ious low b ain-pene a ing chemo he apeu ic d ugs like doxo ubicin, benzylpenicillin, pacli axel, and dala gin has demons a ed signi ican b ain pene a ion and can be a po en ial s a egy o b ain cance ea men [78]. Func ionaliza ion o nanopa icles: The apies based on app o ed nanoca ie s (e.g., liposomes and polyme ic nanopa icles) and associ- a ed echnologies (in asi e and non-in asi e) a e cu en ly being in es iga ed o ea CNS diseases [99]. In his con ex , liposomes wi h high biocompa ibili y, biodeg adabili y, and in insic compe ence o BBB ha e been ex ensi ely explo ed as T ojan ho ses. Liposomes unc- ionalised wi h polye hylene glycol (PEG) and biomolecules (T and insulin) ha e been used o he deli e y o DNA encoding lysosomal enzyme o he b ain o he ea men o mucopolysaccha idosis [105]. PEGyla ed liposomes u he unc ionalised wi h glu a hione (GSH) o a ge ing GSH anspo e s o deli e y o doxo ubicin, me hylp ednis- olone, and iba i in a e in p eclinical s udies [99]. Fo example, PEGyla ed doxo ubicin-loaded liposomes a ge ing GSH anspo e s ha e shown posi i e ou comes in e ms o an i umou ac i i y, and ha e eached phase I/II clinical ials. The ea men was ound o be sa e and well- ole a ed [105]. Simila ly, ea men wi h GSH PEGyla ed lipo- somal me hylp ednisolone o phase I ials on 42 pa ien s, was deemed sa e and well ole a ed a he apeu ic doses o MS [105]. In he case o in a enous adminis a ion o iba i in encapsula ed GSH PEGyla ed liposome, a ou old inc emen o he d ug in b ain mic odialysa es was obse ed [99]. Two o he liposome-based T ojan ho se sys ems, SGT-53 (wi h T Ab and plasmid p53), and doxo ubicin-loaded PEGyla ed li- posomes wi h ce uximab a e in phase I and phase II clinical ials o GBM [105]. Cellula ca ie s: Inno a i e pa adigms, whe ein he immune cells (monocy es, neu ophils, and mac ophages) a e ha nessed o BBB he apeu ic in e en ions, ha e gained in e es in ecen imes. In his app oach, cells a e used as T ojan ho ses. Cells a e ei he gene ically modi ied o p oduce he apeu ically ac i e pep ides and RNA mole- cules/p o eins o can ca y he apeu ics a ge ing BBB [99]. Immune cells ha e inhe en p ope ies o ansmig a e ac oss BBB by changing hei shape (diapedesis) and ha e been used o b ain deli e y in dis- eases in ol ing in lamma ion. Enginee ed mac ophages exp essing glial cell line-de i ed neu o ophic ac o (GDNF) in a PD model demon- s a ed long- e m neu op o ec i e e ec s a he onse o disease p o- g ession and educed b ain in lamma ion [99]. Fu he , neu al p ecu so s em cells and hema opoie ic s em cells can also in e ac wi h BBB cells unde in lamma ion. In ecen s udies, neu al p ecu so s em cells and hema opoie ic s em cells we e enginee ed o exp ess he an i umou biomolecule umou -selec i e p oapop o ic dea h ecep o ligand (TRAIL), esul ing in ea ing me as a ic b ain umou [99]. In ano he se o cell-media ed d ug deli e y examples, magne ic lipo- somes loaded wi h he d ug diclo enac and unc ionalised wi h he pep ide mo i RGD (a ginylglycylaspa ic acid) we e able o a ge monocy es and neu ophils in an IL-1β induced b ain in lamma ion a model. In ha s udy, RGD ac ed as a a ge ing ligand o in eg in Table 3 (con inued) Agen s Examples Mechanism Wo king Condi ion Applica ion Ad an ages/Disad an ages Disad an ages: Nonspeci ic, Side e ec s Sodium dodecyl sul a e [77,78] In e ac s wi h lipid and p o ein o cell memb ane   Ad an ages: Dose dependen , e e sible Sodium cap a e (C10) [89,91] In eg a es in o he lipid bilaye o endo helial cells which inc eases memb ane luidi y and loosening he igh junc ion a o ing pa acellula anspo s In aca o id adminis a ion (5–25 mM) D ug Deli e y Ad an ages: Re e sible, e ec i e o bo h small and la ge molecules Disad an ages: Limi ed in o ma ion, non-selec i e, in lamma ion Cyclodex ins [78] BBB dis up ion by ex ac ing choles e ols om endo helial cells In a enous (1mM-10 mM) D ug Deli e y (hyd ophobic d ugs) Ad an ages: Biodeg adable, selec i e ac ion on cell memb ane Disad an ages: Limi ed e icacy o hyd ophilic d ugs, side e ec s A oma ic subs ances Bo neol [84] Re e sibly disassembles he igh junc ion p o eins (claudins and occludins), ansien ly dis up ing he BBB in eg i y D ug Deli e y, neu op o ec ion Ad an ages: Re e sible e ec , na u al o igin, b oad applicabili y Disad an ages: Concen a ion dependen oxici y, nonspeci ic, limi ed ac i i y E. Rome o-Ben e al. Ac a Bioma e ialia 196 (2025) 17–49 24 ecep o s exp essed on monocy es and neu ophils, which enhanced he up ake o diclo enac-loaded liposomes by he cells. As a esul , i was obse ed 9.1 old inc emen o d ug eaching he b ain. Table 4 sum- ma izes he di e en s a egies o BBB c ossing desc ibed in his sec ion, highligh ing he ad an ages and disad an ages o each app oach [69,77, 84,87,88,90,93,94,106–109]. Among he s a egies explained abo e, we will conside he ein he use o polyme ic nanoca ie s o deli e he apeu ic biomac omolecules (e.g., enzymes, g ow h ac o s, genes) ac oss he BBB om he blood- s eam. The molecula s uc u e o polyme s can be inely adjus ed du ing he syn he ic app oach o ia pos -polyme iza ion app oaches, esul ing in polyme ic nanoca ie s wi h uneable mechanical, physi- cochemical, and su ace p ope ies. The wide a ie y o a ailable poly- me s (e.g., syn he ic s. na u al, biodeg adable s. non-biodeg adable, s imulus- esponsi e) allows he ab ica ion o a ple ho a o nano- o mula ions ha ha e shown p omising esul s ega ding he p o ec- ion o sensi i e biomac omolecules om ex e nal condi ions (e.g., p esence o p o einases, changes in pH and ionic s eng h) and BBB c ossing. 3. Polyme -based nanoca ie s o c oss he blood-b ain ba ie In he p esen e iew, he a ious polyme ic nanoca ie s a e o ga- nized acco ding o hei a chi ec u e, which ul ima ely dic a es hei physicochemical p ope ies and unc ionali y. In his con ex , biode- g adable solid nanopa icles a e no able o hei compac ness, hyd o- phobici y, and unable biodeg adabili y; nanogels a e dis inguished by hei so , h ee-dimensional s uc u e and en i onmen al adap abili y; dend ime s a e ecognized o hei well-de ined, monodispe se, highly b anched, and symme ical a chi ec u e; and sel -assembled nano- pa icles/nanocapsules a e cha ac e ized by hei e sa ili y in e ms o syn he ic app oaches, enabling he encapsula ion o sensi i e biomac omolecules. 3.1. Biodeg adable solid nanopa icles Biodeg adable solid nanopa icles a e mainly o mula ed by using alipha ic polyes e s. In his con ex , biocompa ible and biodeg adable nanopa icles based on FDA-app o ed poly( ε -cap olac one) (PCL), poly (lac ide) (PLA), and i s copolyme s wi h glycolide (PLGA) ha e ecei ed pa icula a en ion as ma e ials o he ab ica ion o b ain- a ge ed d ug deli e y sys ems. A wide a ie y o ca gos, mainly based on small MW d ugs, ha e been success ully encapsula ed in o hese nanopa icles o add ess neu odegene a i e diseases such as AD, PD, and o he diseases and diso de s o he CNS [110]. Mo eo e , hese nano- pa icles ha e been p o ed o be p omising nanoca ie s o deli e (bio) mac omolecules ac oss he BBB. Hence, hey a e pos ula ed as po en ial en i ies o enzyme eplacemen he apies (ERT) [48], small in e e ing RNA (siRNA) he apies [49,51], b ain-de i ed neu o opic ac o he - apies (BDNF) [111] and i amin D-binding p o ein (DBP) [50] o LSDs, GBM, o TBI, among o he s. As (bio)mac omolecule nanoca ie s, PCL, PLA, and PLGA nano- pa icles p esen ce ain ad an ages o e o he deli e y sys ems. They a e comme cially a ailable, easy o manu ac u e, and o e e sa ile nanopa icle o mula ions wi h adjus ed physical, chemical, and me- chanical p ope ies. The MW, as well as he co-monome cons i uen s and a io, di ec ly a ec he iscosi y, solubili y, glass ansi ion em- pe a u e (T g ), c ys allini y deg ee, and phase beha io o he polyme [112,113]. Amo phous ma ices acili a e a mo e uni o m dis ibu ion o he ac i e/ he apeu ic compounds and endow he polyme wi h highe deg ada ion a es. In his sense, he PLA componen (i.e., lac ide) plays an impo an ole. The c ys alline phase can be supp essed by modi ying he s e eochemis y o lac ide: while s e eo egula PLA (pu e D- o L-lac ide) is c ys allizable, he s e eo-i egula PLA (D,L-lac ide) is amo phous [114]. The e o e, (co)polyme s based on D,L-lac ide, such as poly(D,L-lac ide) (PDLLA), poly(D,L-lac ide-co- ε -cap olac one) (PDLCL), and poly(D,L-lac ide-co-glycolide) PDLLGA (usually epo ed in bibliog aphy and comme cialized as PLGA) a e he mos sui able candida es o his d ug deli e y applica ions. Among hese polyme s, PLGAs a e he mos epo ed and used as s anda d nanopa icles. In gene al, he biodeg ada ion kine ics o hese nanopa icles, which is essen ial o ca go elease, is easily p edic ed by adjus ing he MW and he composi ion o he copolyme s. The inc ease in MW and D,L-lac ide uni s gene ally educes he biodeg ada ion a e in PLGA copolyme s. This ea u e allows he design o nanopa icles wi h con olled and ailo able elease p o iles o ac i e agen s anging om mon hs o yea s o deg ada ion imes. Mo eo e , MW and he co-monome a io play an impo an ole in uning nanopa icle elas ici y. This pa ame e has been epo ed o be an impo an condi ion o con ol he biological in- e ac ions o nanopa icles wi h he b ain endo helium, he eby ha ing a di ec impac on hei anspo ac oss he BBB. In his sense, high MW and lac ide- ich polyme s will exhibi highe s i ness, which acco ding o epo ed s udies, migh p omo e highe associa ion wi h endo helial cells and anspo h ough hem [115,116]. The ab ica ion echnique is essen ial o design speci ic nanopa icles wi h he abili y o ca y hyd ophilic (bio)mac omolecules o he b ain Table 4 Compa a i e analysis o di e en s a egies o BBB c ossing, highligh ing he ad an ages and disad an ages o each s a egy. S a egies Examples Ad an ages Disad an ages Physical Dis up ion Focused Ul asound (FUS) [77,84,87,88, 93] P ecise a ge ing, non-in asi e, empo a y opening, con olled and adjus able pa ame e s. Tissue damage, equi emen o high-end equipmen , ope a ional expe ise, cos . High Pulse Elec ic Field [94] Pe meabili y h ough elec opo a ion, e e sible. Cellula damage, p ecise deli e y, limi ed esea ch. Lase s and Elec opunc u e [69,94] Non-in asi e, adjus able pa ame e s. Cellula damage, side e ec s, limi ed clinical da a, echnical challenges. Chemical Dis up ion Hype osmo ic Agen s [84,90] Clinically app o ed, enhanced pe meabili y, non- in asi e, apid ac i i y, b oad ange o agen s, cos e ec i e. Non- a ge ing, cellula damage, ansien and inconsis en he apeu ic e ec , sys emic oxici y, egula o y and sa e y conce n. In asi e S a egies Di ec Injec ion/Implan a ion [84,88,90, 106] Localized deli e y, ci cum en ing BBB, highe d ug concen a ion, educed sys emic oxici y. In asi e, limi ed d ug dis ibu ion, ad e se local eac ions, echnical complexi y, cos . In ace eb o en icula and In a hecal Adminis a ion [77,88,90,93,106,109] Di ec CNS deli e y, ci cum en ing BBB, uni o m d ug dis ibu ion wi h enhanced he apeu ic e iciency. In asi e, p ocedu e ela ed complica ions, echnical complexi y, side e ec s. Non-In asi e S a egies An ibody and Pep ide T ojan Ho se [103, 108,109] Ta ge ed deli e y, high selec i i y and speci ici y, educed sys emic oxici y, high anscy osis, e sa ili y in payload. Complex enginee ing and op imiza ion, immune esponse, a iable BBB pene a ion, p oduc ion and scalabili y, s abili y issue, cos . Func ionaliza ion o Nanopa icles [69,77, 88,90,107,109] Ta ge ed deli e y, speci ici y, imp o ed pene a ion, con ol-led elease, e sa ili y, mul i unc ional. Ba ch o ba ch a iabili y, oxici y and clea ance, s abili y issues. Cellula Ca ie s [77,88,90,107,109] Ta ge ed deli e y, educed immunogenici y, biodeg adable, mul i unc ional, e sa ili y, e icien gene deli e y. Pa hogenici y, s abili y, cul u ing and main enance, scalabili y, limi ed payload, egula o y and e hical issues. E. Rome o-Ben e al. Ac a Bioma e ialia 196 (2025) 17–49 25 3.4. Sel -assembled polyme ic nanopa icles Inspi ed by biology and combining sup amolecula chemis y wi h nano echnology, sel -assembled nanoca ie s can be p oduced h ough spon aneous in e ac ion o molecules o o m o ganized s uc u es [181–183]. The e a e a ious sel -assembled nanoca ie s ha ha e been s udied o c oss he BBB, such as liposomes [62,184], niosomes (using solu e ca ie anspo e s) [185], lipid nanoca ie s [186], polyme ic micelles [187], polyme somes (polyme esicles) [188], and laye -by-laye (LbL) capsules, among o he s. As he ocus o he p esen e iew is on polyme -based nanoca ie s, he ein we elabo a e upon h ee sel -assembled polyme ic nanoca ie s: polyme ic nanopa icles based on amphiphilic block and g a copolyme s, polyme somes, and LbL capsules. Amphiphilic block and g a copolyme s ep esen a no able ca ego y o polyme ic nanopa icles, ea u ing dis inc hyd ophilic and hyd o- phobic polyme blocks chemically linked o c ea e s uc u es wi h excep ional sel -assembly capabili ies. These blocks can ei he be neu al polyme s (hyd ophilic o hyd ophobic) o polyelec oly es (anionic, ca ionic, o zwi e ionic) [189–191]. In an aqueous en i on- men , hese amphiphilic block copolyme s sel -assemble o o m poly- me ic micelles abo e hei c i ical micelle concen a ion (CMC). Abo e his concen a ion, he hyd ophobic inne co e o he copolyme comes close o agg ega e and dis ances i sel om wa e molecules [192]. These a e gene ally made o biocompa ible, non-immunogenic, biode- g adable blocks such as polyes e s, poly(p opylene oxide), o poly (amino acids) linked o biologically compa ible co ona- o ming blocks like PEG [193,194] and display a co e-shell nanos uc u e. In addi ion, g a copolyme s p oduced by he hyd ophobiza ion o hyd ophilic polyme ic backbones (e.g., polysaccha ides, polyols) can gi e place o mo e complex nanos uc u es such as mul imicella sys ems ha enable g ea e EE [195]. Thei ease o unc ionaliza ion makes hem an e icien nanosys em o b ain a ge ing by bo h in a enous and in anasal adminis a ion [196,197]. Howe e , depending on he adminis a ion s a egy, nanoca ie ea u es such as size and su ace mus be p ope ly enginee ed. Fo example, he g oup o Sosnik de eloped chi osan-based sel -assembled nanoca ie s ha we e su ace-modi ied wi h shu le pep ides and inc eased he deli e y o small-molecule an icance d ugs o he CNS [66,198]. Thei in es iga ion in he CNS deli e y o (bio) mac omolecules is cu en ly being in es iga ed. Va ious app oaches used o he unc ionaliza ion o polyme micelles o b ain a ge ing a e shown in (Fig. 8) [199]. Plu onic® block copolyme s, also known as poloxame s, exempli y his phenomenon and ha e ga ne ed signi ican in e es due o hei abili y o impede d ug e lux anspo e s, such as he inhibi ion o P-gp exp essed on he BBB, hus enhancing d ug de- li e y o he CNS [200,201]. Plu onic® a e poly(e hylene oxide)-poly (p opylene oxide)-poly(e hylene oxide) (PEO-PPO-PEO) iblocks, o ming an amphiphilic copolyme . The manipula ion o he numbe o hyd ophilic EO and hyd ophobic PO uni s p o ides a e sa ile means o ailo ing hese copolyme s o speci ic applica ions [200,202]. Wi h o e 50 Plu onic® molecules comme cially a ailable om BASF Co p. (Pa sippany, NJ, USA), each cha ac e ized by di e en hyd ophilic-lipophilic balance (HLB) and CMC, hese copolyme s o e a spec um o possibili ies o ailo ing pa icle size, d ug loading, s a- bili y, and d ug elease p o iles in he pu sui o op imized he apeu ic ou comes. The classi ica ion o Plu onic® copolyme s in o ou ca ego ies based on di e ences in HLB and hyd ophobic PPO chain leng h u he elu- cida es hei di e si y [203]. The i s class includes hyd ophilic Plu - onic® wi h an HLB o 20–29, such as F68, F108, and F127, whe e F means ha he p oduc is supplied in he o m o lakes [190,204,205]. These no only exhibi good hyd ophilici y bu also boas excellen biocompa ibili y [206], con ibu ing o d ug accumula ion and p o- longed blood ci cula ion ime [207]. The second class comp ises Fig. 8. Schema ic ep esen a ion o polyme ic micelles o d ug deli e y. The image illus a es mul i unc ional polyme ic micelles wi h a ious modi ica ions o enhanced d ug deli e y. The co e is hyd ophobic, consis ing o polyme s like PCL (polycap olac one), PLA (polylac ic acid), and PPO (polyp opylene oxide), while he co ona is hyd ophilic, o med by PEG (polye hylene glycol), PVP (poly inylpy olidone), o PEO (polye hylene oxide). Modi ica ions include co e c oss-linking o s abili y, ligand a ge ing ecep o s (e.g., ans e in, ola e) o ecep o -media ed a ge ing, and s imuli- esponsi e elemen s o con olled d ug elease. Addi ional ea u es such as luo opho es and imaging agen s allow in i o acking, cell-pene a ing pep ides enhance cellula up ake, and c oss-linked co onas imp o e s uc u al in eg i y. Ap ame s a e used o speci ic a ge ing, and su ace cha ge modi ica ions op imize in e ac ions wi h biological memb anes. These micelles encapsula e d ugs, siRNA, p o eins, and o he agen s o a ge ed and e icien he apeu ic deli e y. Adap ed wi h pe mission om [199]. Copy igh © 2022 Else ie . E. Rome o-Ben e al. Ac a Bioma e ialia 196 (2025) 17–49 32 Plu onic® wi h a lowe HLB and sho e PPO chain, ep esen ed by liquid L64 [208], L44 [209], and L35 [210], which impa s nano- s uc u es wi h he abili y o sel -assemble in aqueous media, o e ing a mo e sui able size (10–100 nm) and s able s uc u e [211]. The hi d class ea u es Plu onic® wi h a lowe HLB and a PPO chain leng h anging om 30 o 60 and in pas e o m a oom empe a u e, including P85 [212], P105 [213], and L61 [214], which exhibi dissolu ion and sel -assembly p ope ies in wa e , making hem sui able o loading hyd ophobic d ugs. Addi ionally, hese copolyme s consume adenosine iphospha e (ATP) in mul id ug- esis an cance cells, inhibi ing P-gp and p olonging d ug ci cula ion ime [215,216]. The ou h class o Plu onic® wi h a lowe HLB and a PPO chain exceeding 60, ea u ing P123 and L121, wi h he longes PO chain, is widely used o loading d ugs due o i s g ea e hyd ophobici y [217,218]. The encapsula ion o (bio)mac omolecules wi hin sel -assembled nanoca ie s is a challenge ha can be o e come by ha nessing he ad an ages o sel -assembled nanopa icles and s a egically binding biomac omolecules h ough en apmen , adso p ion, o co alen a achmen , ans o ming hese copolyme s in o T ojan ho ses, acili- a ing he enhanced anspo and deli e y o he apeu ic payloads o he b ain. Ce ain amphiphilic nanopa icles modi ied wi h p o eins, pep ides, RNA, and d ugs ha e shown he abili y o c oss he BBB. Fo ins ance, Poly(β-L-malic acid- i leucine)-copolyme has been used o anspo miRNA ac oss he BBB. This sys em u ilizes he LRP-1 ans- cy osis pa hway and amyloid be a (Aβ) pa hway by conjuga ing a D- con igu ed (D3)-pep ide as a ec o o speci ic a ge ing, achie ing neu on-selec i e deli e y o miRNA in an AD mouse model [219]. Ano he no able example in ol es he use o Lep in-Plu onic® 85 (P85) conjuga es. This amphiphilic copolyme a ge s he lep in anspo e and has shown p omising esul s as an an i-obesi y d ug. I s bene i s include enhanced pe iphe al bioa ailabili y, inc eased b ain up ake, and he abili y o c oss he BBB independen ly o he lep in anspo e [220]. Fu he mo e, he T R-T12-PEG-PLGA and TATH7-PEG-PLGA sys ems u ilize T R o anspo he T R-T12 pep ide, TATH7 pep ide, and pacli axel ac oss he BBB. The s a egy in ol es conjuga ion wi h he T R-T12 and TATH7 pep ide shu le, and in i o pha macodynamic e alua ion demons a ed a po en an i- umo e ec in subcu aneous and no mo opic glioma models, signi ican ly ex ending he li e cycle o umo -bea ing mice [221]. On he o he hand, while amphiphilic copolyme s can c oss he BBB, speci ic a ge ing o desi ed b ain egions emains challenging. S a e- gies like conjuga ion wi h b ain-speci ic ligands o an ibodies a e c ucial o maximizing e icacy and minimizing o - a ge e ec s. E icien encapsula ion and con olled elease o (bio)mac omolecules wi hin amphiphilic copolyme s emain key challenges. Op imizing polyme a ios, inco po a ing s imuli- esponsi e elease mechanisms, and explo ing co-assembly wi h o he polyme s a e p omising s a egies. Ex ensi e p eclinical and clinical s udies a e necessa y o es ablish sa e y and e icacy o egula o y app o al. Despi e hese di icul ies, amphiphilic copolyme s hold immense po en ial o anspo ing bio- mac omolecules ac oss he BBB. Con inued esea ch e o s ocused on add essing hese limi a ions and explo ing inno a i e design s a egies can help o mo e close he echnology o ea ing a ious neu ological diso de s. Polyme somes a e syn he ic esicles composed o amphiphilic block copolyme s, wi h a hyd ophobic bilaye memb ane and g ea e deg ee o complexi y in compa ison o he a o emen ioned co e-shell polyme ic micelles (Fig. 9a). Polyme somes a e usually ab ica ed using diblock, iblock o mul iblock copolyme s by ollowing di e en me hods o induce hei sel -assembly including sol en -swi ch (o mic o luidic me hod), pH- uning, polyme ehyd a ion, polyion complex esicles (PICsomes) ab ica ion app oach, a combina ion o sel -assembly/ polyme iza ion (i.e., polyme iza ion-induced sel -assembly), and cen i uga ion me hod. A e hei sel -assembly, sonica ion, memb ane ex usion, o al e na i e me hods can be used o pu i y and/o adjus hei size [222]. Some ypical polyme s o p oduce polyme somes o nanomedicine a e PEG [223], diblock copolyme s o dex an and PLGA (DEX–PLGA), poly(bu adiene)(PBd)-PEG [224], and diblock copolyme s o PEG and PCL [225,226]. Compa ed o liposomes, polyme somes ha e a hicke bilaye s uc u e, which imp o es hei physical s abili y and inc ease hei blood ci cula ion [224]. Con a ily o polyme ic micelles, ha a e usually limi ed o he encapsula ion o hyd ophobic d ugs, polyme somes can accommoda e bo h hyd ophilic and hyd ophobic ca gos in hei s uc u e. Polyme somes ha e gained a en ion in cance nanomedicine and d ug deli e y sys ems due o hei ad an ageous p ope ies, such as g ea s abili y, e sa ili y in design, and unable ca- pabili ies o e ec i e ca go encapsula ion and con olled elease o an icance he apeu ics [222]. Rega ding he use o polyme somes o deli e he apeu ic agen s in he b ain en i onmen , se e al a emp s ha e been desc ibed in li e a- u e. Yu e al. unc ionalized he su ace o 100 nm PEG-PLGA poly- me somes wi h lac o e in o c oss he BBB and imp o e he deli e y o luo escen 6-couma in and S14G-humanin (a neu op o ec i e pep ide) o he b ain issue. Pha macokine ic s udies ollowing in a enous in- jec ion sugges ed ha 101 lac o e in ligands pe polyme some was he op imized numbe o p omo e he pe meabili y o he polyme somes ac oss he BBB and ensu e b ain- a ge ed deli e y [223]. Geo gie a e al. p esen ed a o mula ion o PBd-b-PEG polyme somes, which we e deco a ed wi h pep ide G23 o inc ease hei capaci y o a ge gangli- oside GM1 o ca eolae-media ed endo helial anscy osis. P elimina y in i o s udies o hese polyme somes in an endo helial T answell® model showed ha he polyme somes we e capable o pene a ing he BBB h ough e icien endo helial cells anscy osis [224]. In addi ion, in i o biodis ibu ion s udies a e in a enous injec ion in mice showed ha G23- unc ionalized polyme somes accumula e in he b ain pa en- chyma [227]. This is an ad an age o G23- unc ionalized polyme somes o e some epo ed T - a ge ed sys ems, which mainly accumula e in he b ain capilla y and do no each o he pa enchyma egion. Ac i e a ge ing s a egies enable he nanoca ie s o c oss he BBB by means o an in e ac ion h ough he speci ic ligands and hey ha e been applied o bo h polyme ic micelles and polyme somes, aiming o imp o e he up ake o he nanoca ie s by endo helial cells. One ac i e a ge ing s a egy is o a e se he nanoca ie s in he BBB using ca ie p o eins, such as GLUT-1, which is speci ically and highly exp essed on endo helial cells o he BBB. Su Min e al. de eloped glucosyl-poly (e hylene glycol)-b-poly(L-lysine) modi ied wi h 3-me cap op opyl amidine and 2- hiolaneimine (Glu-PEG-PLL(MPA/IM)) polyme ic mi- celles. As explained be o e, polyme ic micelles a e also ob ained by sel - assembly o amphiphilic polyme s [228]. These glucosyla ed-polyion complex polyme ic micelles we e loaded wi h an isense oligonucleo- ide (ASO) ha we e unc ionalized wi h di e en quan i ies o glucose o s udy he e ec o he ligand densi y and he accumula ion o nano- pa icles in b ain. A g oup o nanoca ie s ha was coa ed wi h 52 glucose ligands pe nanopa icle showed 17 imes highe accumula ion in he b ain compa ed o he g oup wi hou glucose coa ing, indica ing he g ea po en ial o GLUT-1-media ed anspo s a egy [63]. Shi Yanan e al. disco e ed a new me hod o enhance he clinical use o RNAi in glioblas oma he apy. This me hod in ol es he de elopmen o an Ang2 pep ide-deco a ed chime ic polyme some (ANG-CP), which e ec i ely packs and p o ec s an i-PLK1 siRNA (siPLK1). In i o s udies indica e ha ANG-CP may e icien ly a e se he immo alized mouse b ain endo helial cell line (bEnd.3) monolaye , acili a e he anspo o siRNA in o he cy oplasm o U-87 MG glioma umo cells h ough he LRP-1-media ed pa hway, and ma kedly silence PLK1 mRNA and i s associa ed oncop o ein in U-87 MG cells [64]. In his way, Jiang Yu e al. sugges using a chime ic polyme some (CP) deco a ed wi h ApoE ins ead o Ang2 as a a ge ed p o ein he apy o ea ing glioblas oma. This showed be e pene a ion h ough he bEnd.3 monolaye in in i o BBB models [65]. Among sel -assembled polyme ic nanoca ie s, mul ilaye polyme capsules ha e a ac ed inc easing a en ion in ecen yea s. Mul ilaye polyme capsules a e usually ab ica ed ia he LbL app oach, which E. Rome o-Ben e al. Ac a Bioma e ialia 196 (2025) 17–49 33 Fig. 9. (A) Schema ic illus a ion o polyme some s uc u es, which a e ob ained by sel -assembly o amphiphilic block copolyme s. (B) A schema ic illus a ion o laye -by-laye (LbL) deposi ion o opposi ely cha ged polyelec oly es on a nega i e co e o sac i icial empla e. Final LbL nanopa icle wi h blood-b ain ba ie (BBB)- a ge ed ex e nal laye o acili a e he passage o nanopa icles h ough he BBB and deli e i s ca go in o he b ain. Bio ende has been used o c ea ing he desc ip i e image. (C) Up ake o LbL nanopa icles wi h h ee co es (lipo: liposomes, PLGA: poly (lac ic-co-glycolic acid), PS: ca boxyla ed polys y ene), which we e deposi ed by one laye o poly-L-a ginine (PLR) polyca ion and inal laye o di e en polyanions (CMDex: ca boxyme hyldex an, HA: hyalu onic acid, PLD: polyL- aspa ic acid, PLE: poly-L-glu amic acid, and PSialA: polysialic acid). The up ake by he monolaye s ha we e g own in T answell shows supe io i y o liposome co e o e o he s i e co es. (D) Pe meabili y o he LbL nanopa icles wi h di e en co es bu wi h he same las laye o PLD in he mice BBB demons a es mo e pe meabili y o LbL nanopa icles wi h liposomal co e. (E) BBB pe meabili y o ba e liposomes and liposome-based LbL nanopa icles wi h di e en su ace unc- ionaliza ion in mice showed ha he pe meabili y o LbL nanopa icles wi h hyalu onic acid (HA) ex e nal laye ac oss mice BBB was he g ea es . Adap ed wi h pe mission om [243]. Copy igh © 2023 John Wiley & Sons. Bio ende has been used o c ea ing he desc ip i e image. E. Rome o-Ben e al. Ac a Bioma e ialia 196 (2025) 17–49 34 elies on he al e na e deposi ion o opposi ely-cha ged polyme s on o a empla e (Fig. 9b) [229]. The s epwise p ocess in he LbL app oach al- lows he p ecise con ol o e he su ace p ope ies (i.e., cha ge and unc ionali ies) and he size o he capsule [181,230]. LbL capsules ha e been acco dingly conside ed as po en ial d ug deli e y and he anos ic nanosys ems o a wide a ie y o biomedical applica ions [229, 231–233]. Beyond d ugs o small MW, LbL (nano)capsules ha e demons a ed hei capaci y o deli e he apeu ic biomac omolecules, such as genes and enzymes [234–237]. As an illus a ion o his e sa- ili y and mul i- unc ionali y associa ed o LbL sys ems, Boehnke e al. in oduced click chemis y in o LbL nanopa icles, which enables hem o combine a biosensing pep ide and a a ge ing pep ide wi hin he same ca ie . This mul i unc ional sys em demons a ed a sensi i e de ec ion o h ee di e en ypes o cance s and simul aneous gene silencing as a esul o success ul siRNA deli e y [232]. This s udy highligh s he po- en ial o LbL nanopa icles o load high amoun s o a ious la ge bio- mac omolecules, such as siRNA and pep ides, in a single o mula ion. One o he impo an aspec s o a ge ed d ug deli e y o b ain issue is ha nanopa icles a e no clea ed ou om he blood s eam ia phagocy osis and he ecogni ion o immune sys em. The e a e some s a egies ha ha e been implemen ed o a oid he up ake by mac o- phages, such as cell hi chhiking, su ace modi ica ion o nanopa icles, and he modula ion o he physiological en i onmen [238]. Łukasie- wicz e al. de eloped 100 nm LbL nanocapsules loaded wi h clozapine and unc ionalized wi h PEG o in es iga e hei po en ial o c oss he BBB [239]. Resul s demons a ed ha he p oposed su ace modi ica ion s a egy dec eased hei phagocy osis and up ake by mac ophages, bu did no dec ease he endo helial cells up ake. Mo eo e , all he g oups we e able o c oss he BBB in an in i o BBB T answell® model, and PEG unc ionaliza ion imp o ed he anscy osis and educed he cy o ox- ici y o clozapine-encapsula ed nanocapsules. In addi ion, hei inhibi- ion s udies indica ed ha ca eolae-dependen anscy osis played a ole in he in e naliza ion o nanocapsules by endo helial cells [239]. Ano he s a egy o BBB anspo elies on acili a ing anscy osis in BBB endo helial cells h ough a mechanism media ed by low-densi y lipop o ein ecep o - ela ed p o eins, such as LRP1 [188,240]. S aehla e al. ab ica ed LbL nanocapsules using liposomes as a empla e and p opa gyl-modi ied poly(L-aspa ic acid) as he ex e nal laye , which was u he modi ied wi h Ang2 pep ide [241]. They used hese nano- pa icles o cispla in deli e y in a mic o luidic BBB-GBM. In compa i- son wi h ba e liposomes and non- unc ionalized LbL nanopa icles, he LbL nanocapsules unc ionalized wi h Ang2 we e p e e en ially accu- mula ed in mic o ascula ne wo k nea GBM sphe oids. Thus, Ang2- unc ionalized LbL nanopa icles showed mo e e ec i eness in GBM umo cell killing wi h espec o ba e liposomes and ee cispla in. These ou comes demons a ed he supe io i y o Ang2- unc ionalized nanopa icles, pene a ing he BBB by LRP1-media ed anscy osis. This wo k also demons a ed he inc eased exp ession o LRP1 in he ascula ne wo k in p esence o GBM sphe oids. Addi ionally, he in insic mechanical p ope ies o he LbL pa i- cles/capsules can also de e mine hei capaci y o c oss he BBB. I was shown ha he s i ness and de o mabili y o LbL nanopa icles is mainly dependen on he co e a he han on su ace unc ionali ies. Kong e al. we e able o une he s i ness o LbL nanopa icles by al e ing he choles e ol con en in liposome syn hesis, which was used as a co e. This s udy epo s ha mo e de o mable, luid, and complian LbL nano- pa icles we e ob ained by choles e ol addi ion o liposomal co e. Compa ed o s i LbL nanopa icles, complian LbL ones can pene a e il e memb anes (wi h 100 nm po e size) mo e easily, which could also ha e be e BBB pene a ion [242]. In a simila s udy, Lamson e al. ocused on he e ec o co e s i ness and su ace unc ionaliza ion o LbL nanopa icles on hei anspo h ough he BBB. To his end, hey in es iga ed h ee di e en nega i ely-cha ged co es, i.e., liposomes, PLGA, and ca boxyla ed polys y ene, and modi ied he las laye o he nanopa icles by using di e en polyanions such as ca boxyme hyldex- an, hyalu onic acid (HA), poly(L-aspa ic acid), poly(L-glu amic acid,) and poly(sialic acid). Thei esul s e ealed ha liposome-based LbL nanopa icles showed he highes o simila anspo in T answell® assay and he highes up ake in b ain mic o ascula endo helial cell line (hCMEC/D3) monolaye associa ion (Fig. 9c) compa ed o he nano- pa icles wi h s i e co e. I was also e ealed ha he su ace chemis y o he LbL nanopa icles de e mines he mechanism by which nano- pa icles a e anspo ed in acellula ly. A e e alua ing he anspo in he BBB o mice, liposomal nanopa icles exhibi ed he highes pe meabili y compa ed o o he LbL nanopa icles wi h he same ou e laye (Fig. 9d). Finally, liposome-based nanopa icles wi h he las laye o HA showed he highes pe meabili y in mice BBB compa ed o o he su ace modi ica ions (Fig. 9e) [243]. Al hough his wo k p o ides insigh ul esea ch on he e ec o co e s i ness and su ace chemis y on he capaci y o LbL nanopa icles o c oss he BBB, i is no exac ly clea why HA unc ionaliza ion imp o ed pe meabili y in i o. Despi e p e ious a emp s, he e a e ew esea ch wo ks on sel - assembled nanopa icles ocusing on ca ying biomac omolecules and c ossing he BBB. The e o e, many mo e s udies a e needed o in es i- ga e di e en p ospec i e o mula ions using a ious empla es and su ace chemis ies, de e mine hei e ec i eness, and unco e he eal po en ial o sel -assembled polyme ic nanoca ie s. Co e emo al a e laye deposi ion could esul in nanopla o ms wi h mo e elas ic and luid memb ane ha can pene a e h ough he ba ie [244]. All he men ioned examples show he e sa ili y and po en ial o sel -assembled nanoca ie s, including polyme ic micelles, polyme somes, and LbL nanopa icles/nanocapsules, o combine a ious unc ionali ies in one sys em o e ec i e deli e y o he apeu ic molecules ac oss he bio- logical ba ie s, such as he BBB. 3.5. O he polyme ic ca ie s Nanopa icles based on na u al polyme s like polysaccha ides o p o eins ha e been explo ed o hei po en ial use in a ge ing BBB and deli e he apeu ic biomac omolecules [95,245–247]. The p esence o la ge su ace a eas con aining unc ional g oups (e.g., hyd oxyl, ca boxyl, amino) allows ailo ing he physicochemical p ope ies o polyme ic nanoca ie s o enhancing biomolecule conjuga ion and a ge ing ecep o s o c ossing he BBB. Along wi h his, he size, aspec a io (shape), su ace cha ge and ligands o he pa icles can also be modula ed, which also ha e an impo an ole in BBB pene a ion [248–250]. In his sec ion, we discuss examples o some o he nano- ca ie s de i ed om na u al polyme s o a ge ed deli e y o d ugs and s a egies o imp o e hei abili y o c oss he BBB. Chi osan: Chi osan (CS) is a na u al linea ca ionic copolyme de i ed om chi in and is a widely used ma e ial o nanoca ie ab ica ion ha ing p o ound applica ion in many CNS diso de s [251,252]. CS consis s o β-(1,4)-linked d-glucosamine and N-ace yl-d-glucosamine g oups. Owing o i s good biocompa ibili y, i is classi ied as ‘gene ally ecognized as sa e’ (GRAS) by he FDA. CS nanopa icles can be ab i- ca ed by a ious me hods including ionic gela ion, emulsi ica ion, and chemical c osslinking [253]. The ionic gela ion me hod was ollowed o encapsula e me ho exa e, dopamine, and si aglip in d ug o he ea men o GBM, PD, and AD, espec i ely [251,254,255]. Recen ly, p oline-loaded CS nanopa icles, syn hesized by ion c osslinking me hod, ha e been used o deli e p oline, esul ing in neu op o ec ion a e ischemic inju y [256]. Addi ionally, due o poo pene a ion o mos o he CNS associa ed d ugs, CS was used o deco a e PLGA and PCL nanopa icles o a ge ing he d ugs ac oss BBB [251]. The posi i e su ace-cha ge o he nanopa icles shows high a ini y owa ds he endo helial cells o enhanced b ain deli e y and cellula adso p ion, ia AMT. Fo his, CS is modi ied wi h me hyl iodide and glycidyl e he s o gene a e N- ime hyl CS (TMC) and alkylglyce yl-modi ied CS, espec- i ely [249]. TMC imp o es mucoadhesion, solubili y (in a pH-independen manne ), enhances d ug loading and abso p ion e i- ciency wi h espec o na i e CS. Fo example, nanopa icles o mula ed wi h TMC loaded wi h an an i-neu oexci a ion pep ide displayed E. Rome o-Ben e al. Ac a Bioma e ialia 196 (2025) 17–49 35 e ec i e BBB pene a ion, and ha e been used o epilepsy ea men [251,257]. In case o alkylglyce yl CS, ini ially he p ima y hyd oxyl unc ionali ies o he low MW CS we e modi ied h ough selec i e g a ing wi h alkylglyce yl g oups [249,257]. Following his, he nanopa icles we e syn hesized using ionic gela ion me hod and he esul ing nanopa icles displayed enhanced pe meabili y o he apeu ics ac oss BBB [257]. Fu he mo e, he su ace modi ica ion o CS nano- pa icles wi h PEG has also eme ged as a p omising s a egy o enhance he b ain a ge ing e iciency o he he apeu ics [257]. To u he op imize his app oach, he PEG-modi ied nanopa icles we e unc- ionalized wi h a ge ing an ibodies like OX26 mAb o an i-T R [257]. This a ge ed deli e y s a egy has been used o deli e Z-DEVD-FMK pep ide and an isense oligonucleo ides, showing imp o ed he apeu ic e icacy ia RMT [252]. Fo a a ie y o he apeu ic applica ions, CS nanopa icles ha e also shown p omise o ca y biomac omolecules ac oss BBB. As an illus a ion, dual an ibody a ge ing siRNA-loaded CS nanopa icles we e enginee ed o block HIV eplica ion in he CNS [95]. The wo an ibodies T and b adykinin B2 speci ically bind wi h he T R and b adykinin B2 ecep o , espec i ely, acili a ing siRNA deli e y in o as ocy e a ge cells. This esul ed in inhibi ion o exp ession o p o eins such as SART3 and hCycT1 ha a e associa ed wi h HIV eplica ion [258]. In a simila wo k, CS nanopa icles we e used o in i o ans ec ion o GFP- agged plasmids on HEK293 (human emb y- onic kidney cells) and MG-U87 (b ain cance ) cells on mu ine models [67]. The wo k highligh s he e ec i e BBB pene a ion o CS nanopa icles. Algina e: Algina e is a linea unb anched anionic and hyd ophilic na u al polysaccha ide ob ained om b own seaweed. S uc u ally, i is composed o a copolyme o α -1-gulu onic acid and β-D-mannu onic acid linked by 1,4-glycosidic moie ies. FDA classi ied algina e as GRAS due o i s e y good biocompa ibili y and hus, i has widesp ead ap- plica ions in he ield o d ug and gene deli e y, wound healing, and issue enginee ing [249]. Repo s ha e shown ha pH- esponsi e algi- na e nanopa icle o ma ion in ol es me hods like ionic gela ion, emulsi ica ion-sol en e apo a ion, emulsi ica ion-gela ion, wa e /oil emulsion me hod, in e -chain c osslinking, o by mixing wi h o he polyme s [259]. Fo example, algina e nanopa icles ha e been ecen ly p epa ed along wi h CS o deli e small pep ide SpBMP-9 de i ed om neu al g ow h ac o BMP-9. This p omo es he di e en ia ion o choline gic neu ons and inac i a e GSK3be a which a e p omising ou - comes in he ea men o b ain degene a i e diso de s, such as AD [260]. The widely used ionic gela ion me hod has been applied o p e- pa e enla axine-loaded algina e nanopa icles, which ha e been u i- lized as an i-dep essan s [261]. Following his app oach, an in anasal ca ie sys em was de eloped, exploi ing elec os a ic in e ac ion o algina e and doxo ubicin o gi e algina e-doxo ubicin nanocomplex, which was inco po a ed in o CS nanopa icles. The wo k highligh s he e icien deli e y o doxo ubicin o b ain issue wi h a a ge ing e i- ciency o 480%, signi ying a p omising s a egy o in anasal a ge ed deli e y o he b ain [262]. In ano he example, sodium algina e along wi h doxo ubicin and hodamine we e emulsi ied in o dioc yl sodium sul osuccina e [Ae osol OT (AOT)]–algina e nanopa icles and we e able o o e come P-gp media ed d ug esis ance in umou cells [263]. In ecen p og essions wi hin egene a i e medicine, he ea men o PD encompasses he deli e y o he apeu ic cells and neu o ophic ac- o s capable o eleasing dopamine o he b ain. The app oach o deli e ing cells aims o eplace los neu ons and acili a e unc ional einne a ion by eleasing dopamine ha sec e es neu o ophic ac o s. Howe e , clinical ials e ealed high a es o cell dea h pos implan- a ion. To o e come his, algina e has been used o make dual laye beads along wi h HA o slow and sus ained elease o he apeu ic cells SH-SY5Y (neu oblas oma cell line) and immunosupp essan FK506 compound showing p olonged cell su i al and unc ionali y o PD ea men [264]. Hyalu onic Acid: HA is a wa e soluble, biocompa ible linea poly- saccha ide composed o D-glucu onic acid and N-ace yl-D-glucosamine uni s ound in he ex acellula ma ix o a ious issues. The p esence o hyd oxyl g oups makes i hyd ophilic, whe eas ca boxyl, hyd oxyl, and ace amido g oups in i s s uc u e can be exploi ed o chemical modi- ica ion [265]. HA has been ex ensi ely u ilized in d ug deli e y sys ems due o i s a ini y owa ds CD44 ( ans-memb ane glycop o ein) e- cep o s ha a e o e exp essed in b eas , lung and me as a ic b ain cance [266,267]. The syn hesis o nanopa icles in ol es elec os a ic in e ac ions be ween anionic HA and ca ionic CS, which we e used o deli e cu cumin and neu oglobin in he b ain o GBM and s oke ea men [68,268]. Va ious HA-based modi ied nanopla o ms, like PEGyla ed HA, HA wi h lipoic acid- lysine, HA wi h i amin E succina e copolyme s, HA wi h glycy he inic acid, doxo ubicin, and sodium i- polyphospha e nanopa icles ha e been used o doxo ubicin deli e y o an icance he apy and o e coming chemo esis ance [269]. Fo enhanced a ge ed he apy o GBM, a hyb id HA nanoca ie sys em was unc ionalized wi h pep ide HRK-19, con aining RGD and NGR o bind α β3 and aminopep idase-N (CD13) ecep o s o e exp essed in glioma cells and/o angiogenic essels. Wi h limi ed in es iga ion on he po en ial use o algina e nanoca ie s, he e is plen y o oom o imp o e hese nanoca ie s o biomac omolecule deli e y ac oss BBB. Bo h algina e and HA nanopa icles, being easily designable, and wi h long ci cula ion ime in he sys emic bloods eam, can be easily modula ed o achie e enhanced BBB pe mea ion [249–270]. Besides, na u al p o eins a e in ended o se e as ca ie s o a ge ing moie ies o enhance he anspo o d ugs and he apeu ic agen s o he CNS [271,272]. P o eins ha ing high s abili y and ac i i y wi h low enzyma ic deg ada ion, immunogenici y, phagocy osis, enal clea ance, leading o inc ease in hal -li e o he d ug, has shown p omising ou - comes [271]. Gela in: Among all na u al polyme s, gela in has been ex ensi ely used o he deli e y o biomac omolecules o he b ain. The syn hesis o gela in nanopa icles in ol es emulsi ica ion sol en e apo a ion, des- ol a ion, nanop ecipi a ion, and mic o luidic de ices me hods [245]. Ideally, gela in needs c osslinking agen s o o e come i s limi a ions o low mechanical in eg i y and apid decomposi ion. Fo CNS diso de s, s a egies like ca ionic cell pene a ion based on he pep ide-media ed endocy osis a e conduc ed o ans e se he BBB. Fo example, a b ain-pene a ing pep ide conjuga ed wi h PEG and TAT pep ide has been used o modi ica ion o gela in–siloxane nanopa icles o enhancing hei e iciency in c ossing BBB [245–252]. Fu he , gela in nanopa icles ha e demons a ed hei e ec i eness as a eliable ca ie o RNA deli e y in he b ain en i onmen , along wi h anspo o g ow h ac o s, mic oRNAs, and siRNA o di e en ia e s em cells and silence genes [245]. The in anasal deli e y o inducible ni ic oxide syn hase siRNA and os eopon in pep ide inc eased i s e icacy o he ea men o ischemic s oke by inducing s ong neu op o ec i e e ec [245–252]. Recen ly, gela in nanopa icles loaded wi h neu opep ide Subs ance P deli e ed in CNS enhanced he dopamine gic neu on e- co e y in hemipa kinsonian a s and can be an e ec i e he apy o PD [273]. Human Se um Albumin: Human se um albumin (HSA) is a globula p o ein commonly used o imp o e he solubili y and anspo o hy- d ophobic d ugs ac oss he BBB. D ug-albumin complexes can inc ease d ug s abili y and p olong ci cula ion ime, enhancing hei po en ial o each he CNS. The syn hesis in ol es chemical (emulsion and complex coace a ion), physical (elec osp ay and nano sp ay), and sel -assembly (desol a ion) me hods. HSA has eac i e amino, hiol and ca boxylic g oups on i s su ace, which a ou co alen ligand and su ace modi- ica ions whe eas hey also a ou non-co alen in e ac ions o a ious d ugs and pep ides. Albumin nanopa icles unc ionalised wi h he p o ein ApoE could a ge endo helial cells by ecep o media ed endocy osis, whe eas nanopa icles wi hou ApoE we e incapable o eaching b ain issues. Fu he , albumin pa icles can be modi ied wi h cell pene a ing pep ides like low MW p o amine (LMWP) and b ain pene a ion enhance s based on adi ional Chinese medicines like bo neol, muscone o men hol o imp o e BBB pene a ion [274,275]. As E. Rome o-Ben e al. Ac a Bioma e ialia 196 (2025) 17–49 36 an illus a ion, BSA nanopa icles and LMWP-modi ied BSA nano- pa icles we e ab ica ed based on sel -assembly o LMWP and BSA wi h hyd ophobic d ugs pacli axel and en e inide (Fig. 10a). Bo h ypes o nanopa icles esul ed in a es ing umou g ow h in bo h subcu aneous and o ho opic glioma models. In he o ho opic glioma model, he whole-body imaging da a e ealed up ake o bo h ypes o nanopa icles (Fig. 10b). No ably, LMWP-modi ied BSA nanopa icles demons a ed apid and subs an ial accumula ion in b ain wi h highe luo escence signals (Fig. 10c). In compa ison, he up ake o BSA nanopa icles was no ably lowe han ha o he LMWP (Figs. 10c and e). The esul s a e u he co obo a ed wi h ex i o imaging o he dissec ed o gans om mice (Fig. 10d). A majo disad an age o his model is he lack o BBB. Cu en ly, o GBM, clinical ials a e going on wi h FDA app o ed Ab axane (albumin bound pacli axel) along wi h ca bopla in and implan ed ul asound emi e s de ice [246]. Fu he , albumin is used o modi y he su ace o polyme ic nanopa icles o enhancing BBB ansloca ion [246]. Fu he , modi ica ions o nanopa icles wi h na u al ligands/ Fig. 10. Blood-b ain ba ie (BBB) pene a ing low molecula weigh p o amine (LMWP) bo ine se um albumin (BSA) nanopa icles (L-BSA-NPs) and BSA-NPs de eloped wi h sel -assembly o LMWP and BSA along wi h hyd ophobic d ugs pacli axel (PTX) and en e inide(4-HPR). Cha ac e iza ion o he syn hesized BSA- NPs and L-BSA-NPs. (A) T ansmission elec on mic og aphy and he pa icle size dis ibu ion o he BSA-NPs and L-BSA-NPs. (B) In i o imaging o mice bea ing U87 xenog a umou s showing in e umo al pene a ion abili y o BSA-NPs and L-BSA-NPs. Full body imaging showing biodis ibu ion o BSA-NPs and L-BSA-NPs, wi h p og essi e ise in luo escen in ensi y om 2 o 8 h, wi h L-BSA-NPs displaying enhanced umo accumula ion compa ed o BSA-NPs. (C) Quan i ica ion o he a e age luo escence e iciency in i o a he umo si e. (D) Ex i o imaging o he majo dissec ed o gans, whe e umo shows highe luo escence in ensi y. (E) Quan i ica ion o luo escence in ensi y o only umou s om D. Adap ed wi h pe mission om [274]. Copy igh © 2016 Ame ican Chemical Socie y. E. Rome o-Ben e al. Ac a Bioma e ialia 196 (2025) 17–49 37 p o eins like T , lac o e in, apolipop o ein, and melanoT ha e been success ully used o a ge he exp essed ecep o s on endo helial cells and imp o e BBB anscy osis [249,252]. T is a glycop o ein ha in- e ac s wi h T R o e exp essed on he su ace o BBB h ough ligand- ecep o ecogni ion. Addi ionally, nanoca ie made om na - u al i on s o age p o ein, e i in, exhibi s BBB- a e sing and he glioma- a ge ing p ope ies. Fe i in nanoca ie s along wi h hei modi ica ion wi h in eg in α 2β1 a ge ing ligand (DGEAGGDGEA) ha e been used o deli e doxo ubicin o enhanced o ho opic glioma he - apy [276]. Ad ances in ma e ial chemis y ha e sugges i ely augmen ed he polyme ic nanoca ie s o mula ions and i s usage in CNS deli e y. The na u ally occu ing polyme s owing o i s less oxici y, biocompa ibili y, a ge ed d ug deli e y, e sa ili y, biodeg adabili y, a ailabili y, and low cos o e g ea p omise o imp o ing d ug deli e y ac oss he BBB. Howe e , due o hei low mechanical s eng h, polyme ic nanoca ie s ha e poo s abili y and s uc u al in eg i y du ing ci cula ion and d ug leakage compa ed o syn he ic polyme s. Fu he , challenges like apid clea ance, ba ch o ba ch a iabili y, lowe d ug encapsula ion and po en ial immunogenici y should be ca e ully conside ed in he a ional designed o s a egies o acili a e d ug anspo a ion ac oss he BBB o po en ial long- e m and clinical applica ion. 4. Clinical ansla ion Basic and applied neu oscience has b ough o ligh new echnolo- gies and he disco e y o new he apeu ic in e en ions wi h po en ial o o e come some o he majo CNS diso de s. Fu he mo e, an inc easing in-dep h unde s anding and desc ip ion o new molecula a ge s o CNS diso de s is also enhancing he oolbox o he design o new he apies. Hence, bo h academia and indus y e o s a e ocused on he wo ldwide bu den o he neu ological diso de s in he las decades, which ep esen he leading cause o disabili y and he second leading cause o dea h [277]. Cases o b ain cance as well as he p e alence o CNS diso de s a e inc easing due o he longe li e-expec ancy acco ding o Wo ld Heal h O ganiza ion. Howe e , some p ac ical challenges emain in es ablishing no el d ugs and biologicals when mo ing om he bench o in i o s udies o in o he clinical use. Eme ging app oaches, such as in anasal deli e y, a e being implemen ed as a non-in asi e op ion o he d ug deli e y o he CNS while educing pe iphe al exposu e, mos ly a a basic esea ch le el. Thus, de eloping new s a egies o each he CNS, while limi ing sys emic e ec s is s ill one o he main clinical challenges due o he inhe en high complexi y o he BBB. Decades o neu oscience esea ch ha e led o he de elopmen o biologic d ugs ha ha e gi en clinicians and pa ien s enewed hope o imp o ed ea men op ions o complex diseases like MS, AD, PD, o b ain can- ce s, among o he s. The de elopmen and use o biomac omolecules a e blossoming in he las yea s due o hei abili y o a ge speci ic pa h- ways, which educes undesi ed side e ec s, also allowing o pe sonal- ized ea men ha add esses he pa ien ’s pa icula condi ion. Al hough he a ailabili y o biologic ea men s o CNS diso de s is limi ed, he e is no denying ha he exis ing adi ional d ugs unda- men ally change symp oms o hese diseases bu do no change he ch onic onse o he diso de . Mos o he a ailable he apeu ic bio- mac omolecules in he ma ke a e an ibodies, such as LEMTRADA® (alem uzumab), TYSABRI® (na alizumab), o OCREVUS® (oc elizu- mab) o he ea men o MS, Aduhelm® (aducanumab) o AD, and Be acizumab o Naxi amab-gqgk o b ain cance . Rega dless o he pa hology, clinical ials ha e ailed o a mul i ude o easons, such as se e e ad e se e ec s, minimal o no change in disease onse o poo bioa ailabili y due o he inhe en complexi y o he diso de s and he BBB c ossing abili y [278]. Howe e , he inc easing numbe o people a ec ed by CNS diso de s and b ain umo s poin ou he need o new de elopmen s o ackle his global bu den, especially in low and mid-income coun ies. Fu he mo e, abou one hi d o he FDA ap- p o als in 2022 we e biologics, pushing hem ahead o small molecules o he i s ime, wi h new app oaches such as an ibody–d ug conju- ga es, bispeci ic p o eins, and cell and gene he apies [279]. This end is e idenced wi h mo e han 300 en ies o “Alzheime ’s Disease and enzymes”, 34 en ies o “mul iple scle osis and g ow h ac o s”, 127 en ies o “neu oblas oma and an ibody”, o 63 en ies o “GBM and an ibodies”, in he websi e clinical ials.go among o he s. When sea ching o ials in ol ing “nanopa icles”, he numbe s dec ease d ama ically, being mos ly ocused on he use o me allic nanopa icles and liposomes (Table 5). The i s liposomal based d ug, Doxil, was app o ed by he FDA in 1995, while he i s polyme ic nanopa icle, Ab axane, go he app o al in 2005 bo h using small molecules as hei ca goes [280]. Two decades la e , he FDA app o ed a nanome ic ca ie based on Lipid and Nucleic acids, called ONPATTRO®. In addi- ion, PEGyla ion echnology has been widely applied o e he las 30 yea s o imp o e he pha macokine ics and pha macodynamics o di e en he apeu ic modali ies such as small molecules, pep ides, o p o eins, which leads o o e 30 PEGyla ed d ugs cu en ly used in he clinic [281]. Fu he mo e, some clinical ials a e ocused on he use o Ab axane o Doxil on d ug epu posing app oaches nowadays (Table 5). O e all, i is expec ed ha he endency o using biomac omolecules ollow up o he use o polyme ic nanoca ie s loading hem o o e come hei inhe en ly poo physicochemical p ope ies in he upcoming yea s. 5. Pi alls and u u e pe spec i es As explained along his e iew, a ge ed nanopa icles ha e shown p omise o o e come he BBB. Howe e , hey also accumula e and elease he ca go in o - a ge body si es, which o en esul s in sys emic side e ec s. Nasal adminis a ion was ini ially u ilized o local d ug deli e y in he ea men o alle gic condi ions and sys emic adminis- a ion in he so-called ansnasal ou e [282]. Fo example, calci onin salmon nasal sp ay is used o ea pos menopausal os eopo osis. The ansnasal deli e y o o he biologicals such as he human g ow h ac o and oxy ocin has been also in es iga ed [283,284]. The ansnasal ou e bypasses hepa ic i s -pass me abolism and could lead o sys emic bioa ailabili y compa able o he in a enous and he in amuscula injec ions. An adminis a ion s a egy ha capi alizes on di ec ans- po mechanisms be ween he ol ac o y nasal epi helium and he CNS, namely he in anasal o nose- o-b ain ou e, was in oduced by P o . W. H. F ey II in he 1990s o he deli e y o pep ides and o he he apeu ic molecules [285–288]. Majo ad an ages a e he quick onse o ac ion, no hepa ic i s -pass me abolism, and pa ien compliance. Daun less e o s ha e been de o ed o un a el he molecula and cellula pa hways in ol ed in he nose- o-b ain anspo , which a e s ill unde scien i ic deba e [289]. The anspo is an in e play among di e en in acellula and ex acellula mechanisms wi h a le el o con ibu ion ha depends on he p ope ies o he d ug (in solu ion o as a nanopa icle) and he o mula ion. O e all, he e is b oad ag eemen ha he ol ac o y egion in gene al and he ol ac o y neu ons in pa icula play a key ole [290, 291]. Also b anches o he igeminal ne e a e ele an playe s as neu ons om hese b anches connec he nasal mucosa wi h he ol ac- o y bulb. In addi ion, he apeu ic compound can access he b ain ia he blood ascula u e and he lympha ic sys em. The possible ole o mic oglia in he nose- o-b ain anspo o di e en ypes o nano- pa icles has been ecen ly epo ed [292,293]. I is wo h s essing ha he egion o he CNS in which he nanopa icles accumula e depends on ea u es such as size and su ace chemis y [197]. Thus, a mo e comp ehensi e in es iga ion o his pa hway has o be ca ied ou o which he de elopmen o clinically ele an in i o and ex i o models ha will enable he sc eening o he mos p omising p o o ypes is a c ucial s age [197–294]. The de elopmen o in i o models ha ai h ully ecapi ula e he complexi y o he BBB and allow a apid sc eening o he apeu ics has been also placed in he spo ligh o many in es iga ions. These models a e belie ed o ci cum en , o a leas minimize, he need o in i o models in he nea u u e, being in line wi h he 3R p inciples ( eplace, E. Rome o-Ben e al. Ac a Bioma e ialia 196 (2025) 17–49 38 Table 5 Ongoing clinical ials using nanopa icles o ea CNS diso de s o b ain cance . S udy Ti le Phase Sys em Disease G oups Pa icipan s P ima y Ou come S a us Iden i ie High-Field MRI I on- Based Con as - Enhanced Cha ac e iza ion o Mul iple Scle osis and Demyelina ing Diseases NA Fe aheme ( e umoxy ol) RRMS In e en ions: D ug: Fe aheme D ug: Gadolinium-based con as NA Numbe and loca ion o enhancing b ain lesions seen on 7 esla MRI ollowing Fe aheme adminis a ion. Wi hd awn NCT01973517 In Vi o Cha ac e iza ion o In lamma ion Wi h Fe umoxy ol, an Ul asmall Supe pa amagne ic I on Oxide Nanopa icle, on 7 Tesla Magne ic Resonance Imaging 1 Fe umoxy ol, an Ul asmall Supe pa amagne ic I on Oxide Nanopa icle MS Expe imen al: Fe umoxy ol A 510 mg dose (17 mL) o e umoxy ol dilu ed in 50 mL o 0.9% no mal saline will be in a enous 14 To de e mine he change in g adien -echo T2*-weigh ed signal in an i on- ich b ain s uc u e, he globus pallidus [Time F ame: 6 mon hs ollowing e umoxy ol adminis a ion] de e mine i e umoxy ol induces long-las ing b ain signal in ensi y changes in HV and MS Comple ed NCT02511028 No el Imaging Ma ke s in SPMS 1 Fe umoxy ol SPMS D ug: Fe umoxy ol in usion D ug: Gado e idol Diagnos ic Tes : MRI B ain and Ce ical Spine 10 To de e mine a signal change on T1-weigh ed and 3D UTE MRI b ain (and uppe ce ical co d) be o e and 96 hou s (±24 hou s) a e e umoxy ol adminis a ion [Time F ame: 96 hou s ±24 hou s] Rec ui ing NCT05357833 31P-MRS Imaging o Assess he E ec s o CNM-Au8 on Impai ed Neu onal Redox S a e in Mul iple Scle osis. (REPAIR-MS) 2 CNM-Au8 (Gold Nanoc ys als) MS PD In e en ions: D ug: gold nanoc ys als Expe imen al: 60mg CNM-Au8 60mg suspension o clean-su aced, ace ed, gold nanoc ys als in 120ml o sodium bica bona e bu e ed wa e In e en ions: D ug: gold nanoc ys als NA The change om baseline o week 12 in CNS me abolic changes, based on 31P-MRS Redox Ra io. [Time F ame: A 12 Weeks] Rec ui ing Comple ed NCT03993171 NCT03815916 The apeu ic Nanoca alysis o Slow Disease P og ession o Amyo ophic La e al Scle osis (ALS) (RESCUE-ALS) 2 CNM-Au8 ALS D ug: CNM-Au8 D ug: Placebo 45 Elec omyog aphy measu es o disease p og ession. [Time F ame: 36 weeks] Comple ed NCT04098406 S udy o APH-1105 in Pa ien s Wi h Mild o Mode a e Alzheime ’s Disease 2 APH-1105 (Alpha Sec e ase Modula o ) AD D ug: APH-1105 O he : Placebo NA Sa e y: Incidence o T ea men -eme gen Ad e se E en s [Time F ame: Baseline h ough 30 days pos inal ea men dose up o day 60] E icacy: Cogni ion Change [Time F ame: Baseline - day 60] Change in Alzheime ’s Disease Assessmen Scale-Cog (ADAS-COG) o al sco e om baseline o pos inal ea men dose. No ye ec ui ing NCT03806478 Radiosensi iza ion o Mul iple B ain Me as ases Using AGuIX Gadolinium Based Nanopa icles (NANO-RAD) 1 AGuIX® Gadolinium chela ed polysiloxane based nanopa icles wi h Magne ic Resonance B ain Cance In e en ions: D ug: AGuIX Radia ion: whole b ain adia ion he apy 15 Maximum- ole a ed dose (MTD) o polysiloxane gadolinium-chela es based nanopa icles (AGuIX) gi en concu en ly o he whole b ain adia ion he apy o he Comple ed NCT02820454 (con inued on nex page) E. Rome o-Ben e al. Ac a Bioma e ialia 196 (2025) 17–49 39 Table 5 (con inued) S udy Ti le Phase Sys em Disease G oups Pa icipan s P ima y Ou come S a us Iden i ie ea men o mul iple b ain me as ases [Time F ame: 18 mon hs] E alua ing AGuIX® Nanopa icles in Combina ion Wi h S e eo ac ic Radia ion o B ain Me as ases (NANOSTEREO) 2 AGuIX®B ain Cance D ug: AGuIX 2 IV injec ions (100 mg/ Kg/injec ion) a day 4 and day 8 +S e eo ac ic Radia ion om day 8 o day 15 as pe s anda d p ac ice. 1 Ra e o local con ol [Time F ame: 1 yea ] The p ima y endpoin is he a e o local con ol de ined as he p opo ion o pa ien s wi h a comple e esponse, a pa ial esponse o a s able disease. Te mina ed NCT04094077 S e eo ac ic B ain- di ec ed Radia ion Wi h o Wi hou Aguix Gadolinium- Based Nanopa icles in B ain Me as ases 2 AGuIX®B ain Cance Radia ion: S e eo ac ic Radia ion D ug: AGuIX gadolinium-based nanopa icles Radia ion: S e eo ac ic Radia ion O he : Placebo 134 Local Recu ence [Time F ame: F om en ollmen o 6 mon hs] Assessed wi h Response Assessmen in Neu o- Oncology (RANO) - B ain Me as asis Guidelines Time o local ailu e on a pe me as asis basis will be pe o med using he log- ank es . Rec ui ing NCT04899908 Radio he apy o Mul iple B ain Me as ases Using AGuIX® (NANORAD2) 2 AGuIX®B ain Cance In e en ions: D ug: AGuIX® Radia ion: Whole B ain Radia ion The apy In e en ions: Radia ion: Whole B ain Radia ion The apy NA E alua ion o b ain me as ases esponse, acco ding o RECIST 1.1 c i e ia (o modi ied RECIST) by MRI, wi h MRI cen alized eading Rec ui ing NCT03818386 Pemb o+Chemo in B ain Me s 2 Nab-Pacli axel B ain Cance Pemb olizumab wi h s anda d o ca e chemo he apy ea men : Pa ien s will ecei e 200mg o 400mg o Pemb olizumab (s anda d o ca e dosing a he disc e ion o ea ing physician) e e y h ee o six weeks wi h s anda d o ca e chemo he apy ea men (ca bopla in, peme exed, pacli axel, nab-pacli axel). In e en ions: D ug: Pemb olizumab D ug: Nab pacli axel D ug: Pacli axel D ug: Peme exed D ug: Ca bopla in NA Disease con ol a e [Time F ame: 6 mon hs (baseline o 6 mon hs)] In ac anial bene i de ined as s able disease, pa ial esponse, and comple e esponse Rec ui ing NCT04964960 Lapa inib and Pacli axel in T ea ing Pa ien s Wi h Ad anced Solid Tumo s 1 Ab axane B ain Cance D ug: lapa inib D ug: pacli axel albumin-s abilized nanopa icle o mula ion Ab axane 28 Maximum ole a ed dose (MTD) o lapa inib in cou se 1 [Time F ame: es ima ed o be 12 weeks] Comple ed NCT00313599 MTX110 by Con ec ion- Enhanced Deli e y in T ea ing Pa icipan s Wi h Newly- Diagnosed Di use In insic Pon ine Glioma (PNOC015) 1 and 2 MTX110 (panobinos a nanopa icle o mula ion) B ain Cance In e en ions: D ug: Panobinos a Nanopa icle Fo mula ion MTX110 D ug: Con ec ion- Enhanced Deli e y (CED) 7 Ad e se e en s and clinically signi ican labo a o y abno mali ies which mee G ade 3, 4, o 5 c i e ia acco ding o Common Te minology C i e ia o Ad e se E en s (CTCAE) Comple ed NCT03566199 CED o MTX110 Newly Diagnosed Di use Midline Gliomas 1 MTX110 B ain Cance In e en ions: D ug: In usa e wi h MTX110 and gadolinium De ice: Con ec ion- Enhanced Deli e y (CED) NA Sa e y o epea ed con ec ion-enhanced deli e y (CED) o MTX110 will be epo ed by summa izing he incidence a e o ad e se e en s obse ed o epo ed. Rec ui ing NCT04264143 (con inued on nex page) E. Rome o-Ben e al. Ac a Bioma e ialia 196 (2025) 17–49 40 educe, e ine) and acili a ing he p e-clinical selec ion o BBB- pe meable he apeu ics [295]. As explained along his e iew, he BBB is a he e ocellula ba ie , in which he c oss alk be ween he di e en cell ypes and he cha ac e is ics o he ex acellula ma ix play an impo an ole. Thus, esembling his complex scena io in i o is challenging. Mos o he s udies epo ed in li e a u e ely on he use o wo-dimensional models. The complexi y o hese wo-dimensional models can be u he expanded by including a ious cell ypes (e.g., pe icy es, as ocy es, neu ons) ei he p ima y, es ablished cell lines o de i ed om induced plu ipo en s em cells (iPSCs) [296,297]. These models ha e gained popula i y hanks o hei simplici y and possibili y o ob ain p elimina y esul s in a ela i ely sho pe iod o ime. How- e e , hey poo ly ep esen he mo phological ea u es o b ain capil- la ies in e ms o geome y and s uc u al o ganiza ion. Besides, he semipo ous memb ane is made ou o igid polyme s (e.g., poly- ca bona e o polye hylene e eph hala e) wi h mechanical s i ness a abo e he ones obse ed in he ex acellula ma ix. The e o e, e- sea che s a e ying o combine he simplici y o hese wo-dimensional models wi h mo e biomime ic memb anes o achie e pe meabili y alues close o he in i o scena io [298]. The use o mic o luidics has eme ged as a po en ool o de elop mo e clinically ele an BBB models. Mic o luidic de ices usually con ain in e connec ed compa men s ha allow he cocul u e o di e en cell ypes in indi idual chambe s. Mo eo e , hese compa men s can be illed wi h cell-laden gels, hus c ea ing a 3-dimensonal en i onmen ha be e ep esen he physio- logical cell o ganiza ion [299,300]. The use o mic o luidic de ices, oge he wi h he es ablishmen o imp o ed in i o cul u e p o ocols, he use o human iPSCs and he possibili y o mimic physiological condi ions (e.g., luid shea s ess, blood-like iscosi y) a e allowing he de elopmen o obus in i o pla o ms ha ep esen a eal al e na i e o in i o models. As an illus a ion, Pa k e al. epo ed a mic o luidic chip ha main ained human BBB unc ion (i.e., low ba ie pe me- abili y, exp ession o e lux pumps, high le els o TJs) o up o one week in i o, and showed in i o-like selec i e anscy osis o pep ides and an ibodies [301]. Al e na i e models exploi he capaci y o cells o sel -assemble in o h ee-dimensional essel a chi ec u es. Fo example, he cocul u e o human endo helial cells, as ocy es and pe icy es wi hin ib in gels esul ed in he o ma ion o a mic o ascula ne wo k ha shows gene exp ession p o iles and pe meabili y alues simila o hose obse ed in i o [302,303]. As explained be o e, his e iew examines a ious polyme ca ie s sui able o deli e ing biomac omolecules o he b ain. These ca ie s ha e been de eloped using a ial-and-e o -based app oach, which is cu en ly a signi ican bo leneck in he de elopmen o polyme ic Table 5 (con inued) S udy Ti le Phase Sys em Disease G oups Pa icipan s P ima y Ou come S a us Iden i ie Maximum Tole a ed Dose (MTD) o MTX110 [Time F ame: 14 days] A S udy o E alua e he Sa e y, Tole abili y and Immunogenici y o EGFR(V)-EDV-Dox in Subjec s Wi h Recu en GBM (Ce eb alEDV) 1 EGFR(V)-EDV-Dox ((V)EDVDox con ains doxo ubicin (Dox) wi hin he minicells and a ge s EGFR h ough Vec ibix) B ain Cance GBM In e en ions: D ug: EGFR(V)-EDV-Dox NA Sa e y measu es will be conduc ed om S udy Day 1 as pe s udy schedule o sa e y ollow-up isi 30 (+5 days) pos las dose. Unknown NCT02766699 AGuIX Nanopa icles Wi h Radio he apy Plus Concomi an Temozolomide in he T ea men o Newly Diagnosed GBM (NANO-GBM) 1 and 2 AGuIX®GBM Expe imen al: AGuIX + chemo adio he apy ( adio he apy + emozolomide) Sham Compa a o : chemo adio he apy ( adio he apy + emozolomide) NA The ecommended dose (phase I) o AGuIX in combina ion wi h TMZ and and adio he apy du ing he adio- chemo he apy pe iod [Time F ame: du ing 6 weeks a e he i s injec ion o AGuIX] 6-mon h P og ession F ee Su i al (PFS) a e (phase II) [Time F ame: 6 mon hs om he s a o ea men ] Rec ui ing NCT04881032 Phase II S udy o Combined Temozolomide and SGT-53 o T ea men o Recu en GBM 2 SGT-53 (a complex o ca ionic liposome encapsula ing a no mal human wild ype p53 DNA sequence in a plasmid backbone) GBM In e en ions: Gene ic: SGT-53 D ug: Temozolomide 1 Tumo Response [Time F ame: 6 mon hs] The 6-mon h p og ession- ee su i al (PFS) was e alua ed using RANO Response C i e ia. Te mina ed NCT02340156 NU-0129 in T ea ing Pa ien s Wi h Recu en GBM o Gliosa coma Unde going Su ge y 1 NU-0129 (gold base sphe ical nucleic acid (SNA) nanoconjuga e a ge ing BCL2L12) GBM Expe imen al: T ea men (NU-0129) 8 To e alua e he sa e y o in a enous NU-0129 in pa ien s wi h ecu en GBM o GS. Comple ed NCT03020017 A Phase I T ial o Nanoliposomal CPT- 11 (NL CPT-11) in Pa ien s Wi h Recu en High-G ade Gliomas 1 NL CPT-11 GBM In e en ions: D ug: Nanoliposomal CPT-11 34 To assess he sa e y and pha macokine ics o NL CPT-11 in pa ien s wi h ecu en malignan glioma s a i ied based on UGT1A1 geno yping. [Time F ame: 1–2 yea s] Comple ed NCT00734682 (Da a om: h ps://clinical ials.go ). T ials wi h jus expec ed numbe o pa icipan s we e conside ed as no applicable (NA). Abb e ia ions: MS, Mul iple Scle osis; RRMS, Relapsing Remi ing Mul iple Scle osis; SPMS, Seconda y p og essi e Mul iple Scle osis; PD, Pa kinson’s Disease; ALS, Amyo ophic La e al Scle osis; AD, Alzheime ’s Disease. E. 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