Venlafaxine-PLGA Nanoparticles Provide a Fast Onset of Action in an Animal Model of Depression Via Nose-to-brain

Venla axine-PLGA nanopa icles p o ide a as onse o ac ion in an animal model o dep ession ia nose- o-b ain M. Dolo es Caye o-O e o a , Lau a Pe ez-Caballe o b , I ene Sua ez-Pe ei a c,d , Ma ía Hidalgo-Figue oa c,d,e , Alejand a Delgado-Seque a d,e , Juan Manuel Mon esinos g , Es he Be ocoso c,d,e,1 , Lucía Ma ín-Bande as a, ,1,* a Depa men o Pha macy and Pha maceu ical Technology, Facul y o Pha macy, Uni e sidad de Se illa, Se illa, Spain b Depa men o Psychobiology, Uni e si y o G anada, Campus de Ca uja, G anada, Spain c Cen o de In es igaci´ on Biom´ edica en Red en Salud Men al (CIBERSAM), Mad id, Spain d Neu opsychopha macology & Psychobiology Resea ch G oup, Depa men o Psychology, Uni e si y o Cadiz, C´ adiz, Spain e Biomedical Resea ch and Inno a ion Ins i u e o C´ adiz (INiBICA) Resea ch Uni , Pue a del Ma Uni e si y Hospi al, Uni e si y o C´ adiz, C´ adiz, Spain Ins i u o de Biomedicina de Se illa (IBIS)-Campus Hospi al Uni e si a io Vi gen del Rocío, Se illa, Spain g Hospi al de Pue o Real, C´ adiz, Spain ARTICLE INFO Keywo ds: Venla axine Nose- o-b ain PLGA nanopa icles Dep ession ABSTRACT Backg ound: Cu en ea men o dep ession is hinde ed by he delayed onse o he ac ion o an idep essan d ugs, o en esul ing in ea men ailu e. The e o e, new he apeu ic solu ions a e impe a i e. Me hodology: Venla axine-loaded poly(lac ic-co-glycolic acid) nanopa icles we e p oduced by a double emulsion-sol en e apo a ion me hod. Cellula sa e y assessmen and in e naliza ion assays we e ca ied ou in i o in human ol ac o y neu oepi helium cells. The an idep essan e ec o in anasal (nose- o-b ain) nano- pa icle adminis a ion was assessed in animals submi ed o an animal model o dep ession by beha io al es s, including open- ield, suc ose p e e ence es and ail suspension es . Resul s: The d ug en apmen e iciency (55–65 %), pa icle size (190–210 nm), polydispe si y index (<0.2), and ze a po en ial (−20 mV) o Venla axine-loaded poly(lac ic-co-glycolic acid) nanopa icles we e de e mined o be adequa e. Nanopa icles did no show cy o oxic e ec s. Cell iabili y was mo e han 90 % o all o mula ions and concen a ions assayed. The esul s o he quan i a i e and quali a i e cell up ake assays we e consis en , showing an e iden in e naliza ion o he nanopa icles in o he cells. Fu he mo e, enla axine-loaded nano- pa icles adminis e ed o jus 7 days we e able o e e se he pheno ype induced by a dep essi e-like model, showing a signi ican an idep essan -like e ec compa ed o hose ea ed wi h ee enla axine. Conclusions: These indings indica ed ha in anasal enla axine-loaded poly(lac ic-coglycolic acid) nano- pa icles could become a iable echnique o imp o ing enla axine b ain up ake ia nose- o-b ain. I could also be a p omising nanopla o m o enhancing he ea men o dep ession. 1. In oduc ion One o he majo hu dles in de eloping e ec i e ea men s o cen al ne ous sys em (CNS) diso de s is he accessing he b ain due o he p esence o he blood–b ain ba ie (BBB)(Dong, 2018). The BBB is mainly composed o endo helial cells igh ly sealed by igh junc ions which p e en he en y o ha m ul subs ances in he blood o he CNS (Wang e al., 2019). The es ic i e s uc u e o his ba ie esul s in a he apeu ic ailu e o many compounds because 100 % o la ge and hyd ophilic molecules and 98 % o small compounds a e unable o c oss i (Pi es and San os, 2018). Fo una ely, a ecen eliable ou e has been in es iga ed. This ou e, called nose- o-b ain can bypass he BBB and he neu o he apeu ics could each he b ain di ec ly. Al hough he ou e is no comple ely desc ibed, i is known ha he access o nanopa icles o he b ain could be h ough ex acellula and in acellula pa hways (Fo mica e al., 2022; Huang * Co esponding au ho a : Depa men o Pha macy and Pha maceu ical Technology, Facul y o Pha macy, Uni e sidad de Se illa, Se illa, Spain. E-mail add ess: [email p o ec ed] (L. Ma ín-Bande as). 1 Join senio au ho s. Con en s lis s a ailable a ScienceDi ec In e na ional Jou nal o Pha maceu ics jou nal homepage: www.else ie .com/loca e/ijpha m h ps://doi.o g/10.1016/j.ijpha m.2025.125692 Recei ed 3 Sep embe 2024; Recei ed in e ised o m 17 Ap il 2025; Accep ed 5 May 2025 In e na ional Jou nal o Pha maceu ics 678 (2025) 125692 A ailable online 6 May 2025 0378-5173/© 2025 The Au ho (s). Published by Else ie B.V. This is an open access a icle unde he CC BY license ( h p://c ea i ecommons.o g/licenses/by/4.0/ ). e al., 2024) using igeminal and ol ac o y ne es (C owe e al., 2018; Giunchedi e al., 2020). A e in anasal adminis a ion, ac i es a e anspo ed o ol ac o y egion by di e en pa hways (Mi al e al., 2014). This a ea is he only exposed sec ion o he CNS o he ex e nal en i onmen , and i is esponsible o d ug deli e y o di e en loca ions o he b ain (Shukla e al., 2021). Mo eo e , he ou e o e s po en ial p ope ies compa ed o o al o in a enous adminis a ion. This non- in asi e and painless echnique enhances pa ien compliance by allowing d ugs o pass di ec ly o he b ain. This bypasses hepa ic i s - pass me abolism, esul ing in a ela i ely as onse o ac ion (E d˝ o e al., 2018; Szab´ o-R´ e ´ esz e al., 2022). The use o nanomedicine o in anasal adminis a ion is being widely in es iga ed due o he ad an ages o hese adminis a ion sys- ems can o e o he nose- o-b ain ou e. Among he main ad an ages, i can be men ioned: access o emo e places; educ ion o oxici y and side e ec s; g ea e dose– esponse; enhanced solubili y; imp o emen o pha macokine ics p o iles compa ed wi h con en ional medicines o he con ol o d ug elease (Caban e al., 2014; Choi and Han, 2018; G¨ ange e al., 2018; Pa a e al., 2018). Then, a g ea a ie y o d ugs ha e been encapsula ed and es ed o nume ous b ain pa hological si ua ions such as glioblas oma (Ahmad e al., 2022; B uinsmann e al., 2022; Fe ei a e al., 2021; De F aga e al., 2021), b ain inju y (Li e al., 2022; Wang e al., 2021), epilepsy (Se alhei o e al., 2015; Shah e al., 2021; Yous an e al., 2021), schizoph enia (Pandey e al., 2022; Ruby and Pandey, 2016), Pa kinson’s disease (Dimiou e al., 2022; T apani e al., 2022), mig aine (Jha and Mish a, 2022; Yada e al., 2022), acqui ed immunode iciency synd ome (AIDS) (Kakad and Kshi saga , 2021), Alzheime s disease (Sunena and Mish a, 2018), a en ion de ici hy- pe ac i i y diso de (ADHD) (Sha ma e al., 2023), amyo ophic la e al scle osis (ALS) (Lu e al., 2023) o dep ession (Liu e al., 2023; So en- ino e al., 2020; Vi o ino e al., 2020). Mo eo e , ecen s udies ha e demons a ed ha in anasal admin- is a ion o nanopa icle-based o mula ions can p oduce an idep essan -like e ec s in animal models, sugges ing po en ial e i- cacy in humans. Nanopa icles can be enginee ed o deli e an ide- p essan agen s di ec ly o speci ic b ain egions in ol ed in mood egula ion, po en ially enhancing he apeu ic ou comes (Xinchen e al., 2023). Focusing on dep ession disease, Albe o e al., 2022 compiled 11 s udies ( h ee o hem using enla axine-nanosys ems) ca ied ou using lipid and polyme ic nanosys ems o he in anasal adminis a ion o an idep essan subs ances, among hem, a p e ious s udy om he au- ho s Caye o-O e o e al., 2019 which indings we e p o ec ed by a pa en applica ion (WO2020193838A1) (Ma ín-Bande as e al., 2020) . The e ision by Albe o e al. 2022 desc ibed he ideal cha ac e is ics o nanopa icle o mula ion o in anasal adminis a ion and summa- ized he main s udies ca ied ou wi h a ious ac i e an idep essan ing edien s adminis e ed in anasally, including enla axine. Mo e ecen , Upadhyay e al. 2024 collec ed he mos ele an pape s ela ed o he adminis a ion o an idep essan d ugs adminis e ed ia he nose- o-b ain. Au ho s analyzed he o al numbe o 18 published a icles. Six o hem desc ibed he use o enla axine encapsula ed in o di e en nanosys ems (Upadhyay e al., 2024). These ecen e iews clea ly highligh he need o no el he apeu ic s a egies o ea men o dep ession. Mo eo e , au ho s poin ou he in e es in he nose- o-b ain ou e as an al e na i e o d ug deli e y o he cen al ne ous sys em and emphasize he abili y o a ious nanosys ems o each he b ain a e in anasal adminis a ion. In all he s udies included in bo h e- iews, encapsula ion o an idep essan ac i es led o imp o ed deli e y o he d ug o he b ain, ega dless o he s udy design. This ansla es in o a g ea e an idep essan e ec and as e onse o ac ion o encap- sula ed an idep essan s, whe he compa ing di e en ou es o admin- is a ion o di e en o mula ions ( ee o encapsula ed d ug) a e in anasal adminis a ion. As i is well known, he design o nanoca ie s is a key poin . Ou g oup ca ied ou an in i o biodis ibu ion s udy in mice o di e en ypes o nanopa icles (NPs): plain NPs, ans e in su ace-modi ied poly(lac ic-co-glycolic) acid (PLGA) NPs (T -PLGA NPs) and speci ic pep ide o ans e in ecep o su ace-modi ied PLGA NPs (T Rp-PLGA NPs). Ou s udy concluded ha he simples app oach, plain NPs, showed he highes abili y o each he b ain a e in anasal adminis- a ion (Caye o-O e o e al., 2019), a oiding ecep o -media ed endocy osis. Acco ding o WHO, majo dep ession will be he i s global cause o bu den in 2030 and mo e han 300 million people wo ldwide a e a ec ed (Malhi and Mann, 2018). Dep ession is a s a e o low mood cha ac e ized by hopelessness and sadness, which can in luence emo- ions, eelings, ac s o ela ionships leading, in many cases, o suicide (Jani e al., 2019). Many ypes o an idep essan s a e cu en ly a ail- able, such as selec i e se o onin eup ake inhibi o s (SSRIs), se o onin- no epineph ine eup ake inhibi o s (SNRIs), monoamine oxidase in- hibi o s (MAOIs), o icyclic an idep essan s (TCAs), howe e he lack o a comple e esponse in some pa ien s emains a signi ican challenge o global heal hca e (Ja e e al., 2019). The limi ed e ec i eness o an idep essan d ugs, combined wi h hei delayed onse o ac ion, con ibu es o unsa is ac o y he apeu ic esul s in pa ien s wi h dep ession, highligh ing he impo ance o explo ing new medica ions o al e na i e app oaches o add ess hese challenges (Na ional Ins i u e o Heal h and Ca e Excellence, 2022; Oli ei a-Maia e al., 2023; Pos - e nak and Zimme man, 2005). In ou s udy, enla axine (VLF), a widely used d ug, has been used as a model d ug. VLF is a SNRI has dual- monoamine gic ac ion can cause se e al side e ec s, including nausea, insomnia, d owsiness, cons ipa ion, achyca dia, and d y mou h, among o he s (Lipid e al., 2022). Fu he mo e, like o he monoamine eup ake inhibi o s, i has a slow onse o he apeu ic ac ion ollowing o al adminis a ion, ypically aking a ound 2–4 weeks o pa ien s o expe ience mood imp o emen (En suah e al., 1998). Al hough he unde lying pha macodynamic o pha macokine ic eason o his delay a e no well unde s ood, i is clinically signi ican because i p olongs pa ien s associa ed disabili ies and inc eases he likelihood o ea men discon inua ion (Na ional Ins i u e o Heal h and Ca e Excellence, 2022). Mo eo e , o al bioa ailabili y o VLF is low (40–45 %) and i shows a sho hal -li e (4–5 h), which esul s in equen adminis a ion o main ain he apeu ic le els (Xu e al., 2020). Al hough he e a e com- me cial con olled- elease able s and capsules, VLF is a hyd ophilic compound and p esen s a limi ed BBB pe meabili y. Fo all his, VLF becomes a sui able candida e o be encapsula ed in o NPs. Mo eo e , VLF NPs adminis a ed ia nose- o-b ain ou e could bols e in anasal VLF deli e y o he b ain. The pu pose o he p esen s udy was o in es iga e he b ain- a ge ing po en ial o VLF-loaded PLGA NPs ia a non-in asi e in a- nasal nose- o-b ain ou e. Thus, he s udy explo es he use o nano- pa icles o an idep essan d ug deli e y, wi h he goal o enhancing he e ec i eness, a ge ing, and sa e y o ea men s such as enla axine. A majo challenge in an idep essan he apy is he di icul y many d ugs ace in c ossing he blood–b ain ba ie . By le e aging he nose- o-b ain ou e, nanopa icles can signi ican ly imp o e d ug deli e y o he b ain, inc easing bioa ailabili y and educing he ime equi ed o each he apeu ic concen a ions. As a esul , he slow neu oadap i e changes—such as ecep o egula ion and neu oplas ici y— ha ypi- cally ake weeks wi h monoamine gic an idep essan s could po en ially be accele a ed. Addi ionally, nanopa icles o e con olled and sus- ained d ug elease, minimizing dosing equency and side e ec s. By enhancing b ain up ake, hey may also lowe he equi ed dosage, u he educing he isk o ad e se e ec s associa ed wi h con en ional o al an idep essan s. Thus, he e we explo ed i VLF-loaded PLGA NPs p oduced a as and obus an idep essan -like esponses a e in anasal adminis a ion in mice submi ed o an animal model o dep ession. M.D. Caye o-O e o e al. In e na ional Jou nal o Pha maceu ics 678 (2025) 125692 2 2. Ma e ials and me hods 2.1. Ma e ials Resome ® RG 504H, PLGA e mina ed was ob ained om E onik (Ge many). VLF (Venla axine hyd ochlo ide) and NHS (N-hyd ox- ysuccinimide) we e ob ained om Sigma-Ald ich (USA), e hyl ace a e (HPLC g ade); dime hyl sul oxide (DMSO) (HPLC g ade) and poly inyl alcohol (PVA) (MW =72000 g/mol) we e pu chased om Pan eac (Spain). Fo in i o expe imen s, DMEM-F12 medium om Biowes (F ance) was supplemen ed wi h inac i a ed e al bo ine se um (FBS) om Bio- wes (F ance), Glu aMAX om GibcoBRL (USA) and P imocin om In i oGen (USA). Penicillin-s ep omycin, FITC (Fluo escein 5-iso hio- cyana e), ypsin-EDTA, 3-(4,5-dime hyl hiazol-2-yl)-2,5-diphenyl e- azolium b omide (MTT), PBS (pH =7.4 ±0.1), nile ed (NR), and pa a o maldehyde (PFA) we e p o ided by Sigma-Ald ich. Collagen I a ail was pu chased om Gibco (The mo Fishe , USA). Hoechs ® 22,242 om In i ogen (USA) and WGA (Whea Ge m Agglu inin) Alexa Fluo ™ 555 Conjuga e was ob ained om The mo Scien i ic (USA). 2.2. VLF-PLGA NPs p epa a ion Fo he p epa a ion o he NPs, a double emulsion-sol en e apo a- ion me hod (DE-SEV) was used wi h sligh modi ica ions (Ma ín-Ban- de as e al., 2013). B ie ly, 80 mg o PLGA we e dissol ed in e hyl ace a e (4 % w/ ) and we e emulsi ied by addi ion d op o d op o 100 uL o an aqueous solu ion o PVA (0.5 % w/ ) con aining 25 % w/w o d ug (VLF) unde ul asonica ion o 1 min a RT in an ul asonic ba h (JP Selec a, Ba celona, Spain. 50 W). The esul ing emulsion (W 1 /O) was quickly d opped (wi h a pipe e) in o 10 mL o aqueous PVA solu- ion a 0.5 % w/ (p e iously placed in he homogenize on ice) employing a high-speed homogenize (Ul a u ax® T25 wi h S25N-8G dispe sing ool, IKA, Ge many) o 1 min o ob ain a double W 1 /O/W 2 emulsion a 24.000 pm. This emulsion was ob ained using ice du ing he emulsi ica ion p ocess o p e en he o e hea ing o he emulsion. A e wa ds, he emulsion was le unde magne ic s i ing employing a magne ic s i e (IKA RT-15, IKA Ge many) un il comple e e apo a ion o he o ganic sol en a oom empe a u e (6 pm). Then, NPs sus- pension was collec ed ( i s cen i uga ion) and washed wice wi h 45 ml o Milli-Q wa e by ul acen i uga ion a 14.610 RCF o 30 min a 4 ◦C (Eppendo 5804R cen i uge, Eppendo AG). Finally, NPs we e esus- pended in ehalose solu ion (5 % w/ ) ( inal olume =1 ml) used as c yop o ec an and eeze-d ied o ob ain a ine and s able powde ( ozen and lyophilized a −80 ◦C and 0.078 mba ; Tels a C yodos, Spain). 2.3. FITC-PLGA NPs p oduc ion Fo cell up ake s udies, FITC was used as a luo opho e and co a- len ly bonded o PLGA o e alua e he in e naliza ion o NPs in cells. The luo escen polyme was syn he ized h ough ca bodiimide me hod employing he p o ocol used in p e ious expe imen s (Caye o-O e o e al., 2019). Then, luo escen NPs we e p epa ed by DE-SEV ollowing he same p o ocol ollowed o p epa e VLF-PLGA NPs. 2.4. Nps cha ac e iza ion NPs we e cha ac e ized o mean hyd odynamic diame e , size dis- ibu ion (PdI) and su ace cha ge (ZP). The mean pa icle size and polydispe si y index o he blank o bo h VLF-loaded and FITC-PLGA NPs was measu ed by pho on co ela ion spec oscopy and he ZP was analysed by Lase Dopple using a Ze asize ZS90 (Mal e n Ins umen s L d, Mal e n, UK). Measu emen s we e ca ied ou in iplica e a e dilu ing an aliquo o ecen ly p epa ed NPs suspension wi h dis illed wa e a oom empe a u e. Fo he encapsula ion capabili ies s udies, he d ug con en o he NPs was e alua ed by he p e iously alida ed HPLC me hod (Haque e al., 2012) wi h sligh modi ica ions. Ch oma og aphic sepa a ion was achie ed wi h a LiCh oCART® 250–4 HPLC Ca idge LiCh osphe ® 100 5 μ m column. Fi s , aqueous supe na an was collec ed and il e ed (Millex GV sy inge il e , 0.22 μ m) and, hen 20 μ L o he supe na an was injec ed in o he HPLC sys em o d ug de ec ion and quan i ica ion using a Hi achi LaCh om (D-7000) se ies HPLC equipped wi h a L-7200 au oma ic injec o and a qua e na y pump (model L-7100). Column was kep a 25 ◦C (L2350 column o en, Eli e LaCh om) and he measu e- men s we e pe o med a 225 nm. The amoun o d ug encapsula ed in he de elopmen nanosys ems was calcula ed indi ec ly (Jain and Da a, 2014), measu ing he amoun o ac i e in he supe na an a e NPs collec ion by ul acen i uga ion and compa ed wi h an ini ial amoun o d ug used o p epa e he NPs. The d ug con en in o NPs was exp essed as encapsula ion e iciency (EE %) and d ug loading (DL%) acco ding o he equa ions (1) and (2) espec i ely: EE% =(( o al d ug amoun −unencapsula ed d ug amoun )/ o al d ug amoun ) x 100 (1). DL% =(( o al d ug amoun −unencapsula ed d ug amoun )/ o al mass o NPs) x 100 (2). 2.5. In i o cell s udies 2.5.1. Cell cul u e I is well known ha he s uc u e o he ol ac o y bulb comp ises di e en ypes o cells, including he cells used in his s udy de i ed om he ol ac o y neu oepi helium (ONE) (La oie e al., 2017). ONE cells we e used o p elimina y in i o s udies as hey a e one o he cell ypes ha he pa icles may ini ially encoun e on hei jou ney h ough he in anasal ou e. ONE cells we e ob ained om a heal hy olun ee by nasal b ushing as desc ibed p e iously (Delgado-Seque a e al., 2021). This heal hy subjec p o ided w i en, in o med consen , app o ed by he E hics Commi ee (PI-151019). Cells ob ained by he nasal b ushing included highly p oli e a i e neu al p ecu so s ha we e g own a 37 ◦C wi h 5 % CO 2 , in Dulbecco’s Modi ied Eagle Medium/Ham F-12 (DMEM/F12) supplemen ed wi h 10 % FBS, 2 % Glu aMAX and 0.2 % P imocin. When he con luence was eached, cells we e de ached wi h 0.25 % ypsin-EDTA and epla ed in DMEM/F12 medium supple- men ed wi h FBS (10 % / ), an ibio ic–an imyco ic (1 % / ) and Glu amax® (2 % / ) in humidi ied en i onmen (Au oFlow NU-4750 Wa e Jacke CO 2 incuba o ; NUAIRE, USA). The medium was eplaced e e y day, and he in i o expe imen s we e ca ied ou in passage 5. 2.5.2. In i o cell cy o oxici y assay The cy o oxici y o he NPs was e alua ed by he MTT p oli e a ion assay, de e mining mi ochond ial dehyd ogenase ac i i y (Mosmann, 1983). The MTT (yellow) is clea ed o o mazan ( iole ) by a sys em o he mi ochond ial espi a o y chain o li ing cells. This change o colou can be quan i ied by spec opho ome y. Cells om he ONE we e pla ed a a densi y o 4 x 10 4 cells/well in a 96-well pla e (Nunclon®) and incuba ed o 24 h a 37 ◦C o allow cell a achmen . Then, he medium was comple ely emo ed, and cells we e washed wice wi h 200 µL o PBS be o e he incuba ion wi h di e en concen a ions o NPs ( om 0.1 o 1000 µg/mL, app oxima ely equi - alen o 0.0155–––155 µg/ml o VLF) o 24 h. NPs suspension was emo ed a e incuba ion ime and cells we e washed wice wi h PBS. 50 µL o MTT solu ion (1 mg/ml in medium) was added o each well o 2.5 h in da k. Finally, MTT solu ion was disca ded and MTT o mazan c ys als we e dissol ed wi h 100 µL o isop opanol, shaking in da k o 10 min a oom empe a u e. Abso bance was measu ed in a mic opla e eade (Syne gy HT, BioTech, USA) a 570 nm and cell iabili y was calcula ed ollowing equa ion (Caye o-O e o e al., 2019): Viabili y (%) =((expe imen al alue – nega i e con ol) / (posi i e M.D. Caye o-O e o e al. In e na ional Jou nal o Pha maceu ics 678 (2025) 125692 3 con ol – nega i e con ol)) x 100 (Eq. 3). Cells incuba ed only wi h DMEM/F12 medium we e used as nega i e con ol and DMSO as posi i e con ol. 2.5.3. In i o in e naliza ion s udies The up ake o NPs in o he cells was de e mined by quali a i e and quan i a i e analysis and FITC-PLGA NPs we e used o hose pu poses. Analysis by con ocal lase mic oscopy. Fo quali a i e s udies o NPs in e naliza ion, ONE cells we e seeded (4 x 10 4 cells/well) in a µ-Slide 8 well (iBidi, Ge many) and we e allowed o a ach o e nigh . A e ha , cells we e washed wice wi h PBS (pH 7.4) and incuba ed wi h FITC-PLGA NPs suspension (250 µg/ mL in cul u e medium wi hou FBS) o 1 and 2 h a 37 ◦C. A he end o incuba ion ime, cells we e washed wice wi h PBS o emo e he e- mains o NPs suspension and he nuclei we e coun e -s ained wi h Hoechs ® 33,342 o 10 min. Then, cells we e washed wice wi h PBS and he cy oplasms we e s ained wi h WGA-Alexa Fluo 555. Finally, cells we e washed wice wi h PBS and ixed wi h PFA (4 % w/ in PBS) Fig. 1. (A) Expe imen al design o he in i o expe imen s. Mice unde wen a baseline suc ose aining es ou days p io o he s a o he CORT egimen. Animals we e exposed o he ap wa e (con ol animals) o CORT ia d inking wa e o 28 days in dec easing doses equi alen o a daily dose o 6.6 mg/kg/day o 15 days, ollowed by 2.7 mg/kg o 3 days and 1.1 mg/kg o 10 days. T ea men s (VLF-loaded NPs, Blank NPs, VLF ee d ug o PBS) we e adminis e ed in anasally daily du ing he las 7 days o he CORT egimen. Beha iou al expe imen s including he open- ield es (OF) he suc ose p e e ence es (SPT) and he ail suspension es (FST) we e pe o med. (B) E ec s o CORT adminis e ed o ally ia he d inking wa e upon luid and ood consump ion. The in ake was acked h ee imes a week o e he 28 days o he CORT egimen. Es ima ion o daily in ake o luid pe mice de e mined by weighing bo les and es ima ion o ood in ake pe mice measu ed in g ams o pelle consumed. All alues a e p esen ed as mean ±SEM. S a is ical signi icances displayed a e esul s o dep ession model ac o ob ained by a epea ed measu e ANOVA analysis (n =9–10 pe g oup). (C) E ec o ea men s on locomo o ac i i y in he OF. G aph ep esen s he mean o he o al dis ance a elled exp essed in a bi a y uni s (A.U.) ±S.E.M. Two-way ANOVA es (n =13–17 pe g oup). (D) E ec o ea men s on he SPT. G aph ep esen s he mean o he pe cen age o suc ose p e e ence ±S.E.M. Two-way ANOVA ollowed by Bon e oni pos -hoc es (n =12–21 pe g oup). (E) E ec o ea men s on he ail sus- pension es . G aph ep esen s he mean o he immobili y ime exp essed in seconds ±S.E.M. Two-way ANOVA ollowed by Bon e oni pos -hoc es (n =5–10 pe g oup). * p <0.05 s espec i e con ol g oup, ## p <0.01 s PBS- ea ed g oup, & p <0.05 s VLF- ea ed g oup, +p <0.05 s Blank NPs- ea ed g oup. M.D. Caye o-O e o e al. In e na ional Jou nal o Pha maceu ics 678 (2025) 125692 4 o 20 min and main ained in PBS o be obse ed by con ocal lase scanning mic oscope (Leica S ella is 8 Falcon; Leica; Ge many) wi h imaging so wa e (Las X). Cells wi hou NPs we e used as con ol. Quan i a i e s udy by low cy ome y. Fo his pu pose, cells we e pla ed in a 6-well pla e (9.6 cm 2 /well) a a densi y o 1 x 10 6 cells pe well (2 ml) and we e allowed o a ach o e nigh . The nex day, medium was e i ed, and cells we e washed wice wi h PBS and incuba ed wi h FITC-PLGA NPs suspension (500 µg/ ml in cul u e medium wi hou FBS) o 2 h. A e incuba ion, he sus- pension was emo ed, and he wells we e washed wice wi h PBS o elimina e he pa icle suspension esidues. The cells we e de ached wi h ypsin-EDTA (1 mL) o 5 min. Finally, he cell suspension was washed and esuspended wi h PBS and placed di ec ly in cy ome e ubes. The luo escen NPs inside o cells we e measu ed using a low cy ome e (MACSQuan VYB; Mil enyi Bio ec; Ge many) and he da a we e analyzed wi h MACS Quan i y so wa e). Cells non-incuba ed wi h luo escen pa icles we e used as a con ol o basal au o luo escence. 2.6. In i o s udies 2.6.1. Animals Adul male C57BL/6J mice (10 weeks old a he beginning o he expe imen s) we e housed unde con olled condi ions (22 ±1 ◦C; 12-h ligh /da k cycle) wi h ood and wa e a ailable ad libi um. All p oced- u es we e pe o med in acco dance wi h he Eu opean Guidelines (2010/63/EU) and Spanish Law (RD 53/2013) egula ing animal esea ch. All he expe imen al p o ocols we e app o ed by he E hical Commi ee o Animal Expe imen a ion o he Uni e si y o C´ adiz. 2.6.2. Expe imen al design In b ie , he dep ession-like model was es ablished by o al co ico- s e one (CORT) ch onic adminis a ion. Animals we e p esen ed wi h wa e (con ol g oup) o CORT (CORT g oup) dilu ed in he d inking wa e o 28 days. In o de o assess he baseline beha iou , a suc ose p e e ence es (SPT) was pe o med 3 days be o e he CORT egimen. D inking and ood consump ion was ollowed h ee imes a week du ing he 4-weeks o CORT adminis a ion in each cage. Addi ionally, mice we e adminis e ed daily om he 21s o he 28 h day o he CORT egimen wi h i.n. adminis a ion o VLF-loaded NPs, Blank NPs, VLF ee d ug o PBS. A e ha , o examine he possible an idep essan -like e - ec o hese ea men s animals we e es ed in he open- ield es (OF), he suc ose p e e ence es (SPT) and ail suspension es (TST) wi h an in e al o 2 days be ween es s (Fig. 1). 2.6.3. Mouse model o dep ession The animal model o dep ession used is he one induced by ch onic CORT adminis a ion. This model induces a dep essi e-like pheno ype ha e ec i ely mimics bo h he beha io al symp oma ology and he neu obiological mechanisms ypical o dep ession (Gou ley and Taylo , 2009; Zhao e al., 2008). The mice model o dep ession was induced as desc ibed p e iously (Fe ´ es-Coy e al., 2016). CORT (Sigma-Ald ich, Spain) was dissol ed in egula ap wa e and b ough o a pH o 7.4. Mice we e exposed o CORT o 28 days in dec easing doses equi - alen o a daily dose o 6.6 mg/kg/day o 15 days, ollowed by 2.7 mg/ kg o 3 days and 1.1 mg/kg o he las 10 days un il he s a o he beha iou al e alua ion. CORT was p epa ed eshly e e y 72 h and kep ligh p o ec ed in opaque bo les. Animals in he con ol g oup ecei ed wa e only. 2.6.4. T ea men T ea men was ini ia ed on day 21 pos -exposu e o CORT and was main ained o 7 consecu i e days. All ea men s we e applied daily in anasally in bo h nos ils, 5 µL d op in each nos il ( o al olume 10 µL/day). Fo i.n. adminis a ion, animals we e ligh ly anaes he ized wi h 2 % iso lu ane (Fa ma e , Spain) and placed in a supine posi ion. VLF-PGLA NPs a a dose o 0.06075 mg/day o an equi alen amoun o blank NPs we e adminis e ed. F ee VLF was dilu ed in PBS and admin- is e ed daily a 0.06075 mg/day. Con ol mice ecei ed PBS. 2.6.5. Beha iou al assessmen Open- ield es (OF). Locomo o ac i i y was measu ed using an open- ield appa a us o assess he spon aneous mo emen and explo a o y beha io o he mice. The appa a us consis ed o a squa e plexiglass box (45 ×45 cm) wi h 35 cm high walls. Mice we e placed in one co ne o he box and allowed o oam eely o a pe iod o 15 min. Du ing his ime, he mo emen o he animals was eco ded and acked using a came a connec ed o he S. M.A.R.T sys em (Spon aneous Mo o Ac i i y Reco ding and T acking: Panlab, S.L.), which measu es he o al dis ance a elled in a bi a y uni s. This es allows o he quan i ica ion o locomo o ac i i y, p o iding aluable insigh s in o he e ec s o he ea men s on gene al mo o unc ion and explo a o y beha io . Suc ose p e e ence es (SPT). The SPT is a ewa d-based es , used as an indica o o anhedonia, a co e symp om o dep ession. SPT was assessed in a wo-bo le pa adigm o 8 h (9:00–17:00 h), whe e he mice ecei ed wo bo les, one illed wi h 1 % suc ose solu ion and one wi h ap wa e . A e hal o he ime (4 h), he places o he bo les we e changed o a oid side bias. No p e ious ood o wa e dep i a ion was applied be o e he es . The weigh o each bo le was de e mined be o e and a e he es o accu a ely de e mine he olume o liquid consumed. The suc ose p e e ence was calcula ed in pe cen age as he amoun o consumed suc ose solu ion di ided by he o al liquid in ake (Liu e al., 2018). This measu emen p o ides an index o he animal’s mo i a ion o a swee ewa d, wi h a dec ease in suc ose p e e ence compa ed o wa e indi- ca ing an anhedonic-like beha io , a hallma k o dep essi e s a es (Liu e al., 2018). The SPT is pe o med bo h be o e (as baseline) and a e he CORT egimen o assess any changes in ewa d sensi i i y and beha io ollowing he expe imen al ea men . Tail suspension es (TST). The TST is one o he mos widely used p eclinical ools o s udy an i- dep essan ac i i y in mice (S e u e al., 1985). This es is based on he beha io al esponse o mice o an inescapable si ua ion, speci ically assessing he du a ion o immobili y as a p oxy o dep essi e-like beha io . In he TST, mice we e indi idually suspended by he ail 20 cm abo e he loo using adhesi e ape placed 1 cm om he ip o he ail. This posi ion o ces he animal o emain suspended in a helpless s a e, leading o a na u al endency o ei he a emp o escape o emain immobile. The es session las ed o 6 min, and hey we e ideo aped and subsequen ly sco ed by a ained obse e . The o al du a ion o immobili y du ing he es was measu ed. A educ ion in immobili y in his es was conside ed o indica e an idep essan ac i i y (Be ocoso e al., 2013; Can e al., 2012). 2.7. S a is ical analysis Fo he in i o expe imen s, da a we e exp essed as he mean ±S.D. o he pa ame e measu ed. Fo he in i o expe imen s, da a we e analysed by wo-way analysis o a iance (ANOVA) ollowed by he Bon e oni pos -hoc es . The ac o s e alua ed (be ween subjec s) we e he dep ession model and ea men . Da a we e analysed using P ism 7.0 G aphPad and S a is ica 10.0 so wa e. All p- alues <0.05 we e conside ed s a is ically signi ican . 3. Resul s 3.1. Nps p oduc ion and physicochemical cha ac e iza ion The p oduc ion o blank and VLF-loaded NPs (VLF-PLGA NPs) was ca ied ou by DE-SEV me hod and he mean pa icle size, polydispe si y index (PdI) and ze a po en ial (ZP) we e measu ed as p e iously we e desc ibed (subsec ion 2.4). The da a a e exp essed as he mean alue ± M.D. Caye o-O e o e al. In e na ional Jou nal o Pha maceu ics 678 (2025) 125692 5 SD. The mean hyd odynamic diame e o blank PLGA and VLF-PLGA NPs was 192.1 ±6.7 nm and 208 ±2.9 nm, espec i ely. The PdI was less han 0.2 indica ing a high homogenei y and na ow size dis ibu ion (S e e eld e al., 2016). The su ace cha ge was measu ed a e washing NPs wice by ul acen i uga ion o emo e he PVA esidues (Zambaux e al., 1998). ZP we e e y simila o blank and loaded NPs, –21.4 ±0.3 mV and –22.4 ±1.3 mV, espec i ely. Rega ding FITC-PLGA NPs used o in e naliza ion s udies, simila esul s we e ob ained. The a e age size o hese NPs was 277 ±7 nm wi h a PdI =0.102 ±0.065 and ZP alues we e –21.7 ±0.2 mV. The encapsula ion p ocess was measu ed indi ec ly, calcula ing he amoun o d ug in he supe na an as desc ibed in he Me hod sec ion (subsec ion 2.4). The EE% and DL% o PLGA NPs we e 55–65 % and 10–12 %, espec i ely. 3.2. In i o cell s udies 3.2.1. In i o cell cy o oxici y assay VLF was app o ed in 1993 by FDA (Wa anabe e al., 2018) and is known o ha e a a ou able sa e y p o ile. Howe e , oxici y cell s udies a e ex emely impo an in he de elopmen o NPs. Thus, an MTT assay was ca ied ou in o de o e alua e he me abolic ac i i y o iable ONE cells a e incuba ion wi h hese o mula ions a di e en concen a ions. Fo his assay, we used ONE cells incuba ed wi h a ange o con- cen a ions (0.1–––1000 ug/mL) o blank and VLF-loaded PLGA NPs. Cells incuba ed wi h medium (wi hou PLGA NPs) we e used as nega i e and DMSO as posi i e con ol. A e 24 h o incuba ion, he me abolic ac i i y o li ing cells was supe io o 85 %, main aining cell iabili y in he ange o 90––100 % o all he o mula ions (blank and VLF-loaded NPs) and concen a ions assayed. Resul s ob ained a e summa ized in Fig. 2. The ob ained esul s shown less han 15 % iabili y loss a e incuba ion, sugges ing ha unloaded and VLF-loaded NPs a e compa - ible wi h hese cells acco ding o he ISO 10993–5 guidelines (Biological e alua ion o medical de ices. Pa 5: Tes o in i o cy o oxici y). Addi ionally, he cy o oxici y o he cells was also e alua ed a e in- cuba ion wi h he ee d ug and he iabili y was supe io o 90 % o all concen a ions (Fig. 2). 3.2.2. In i o in e naliza ion s udy To e alua e he associa ion o NPs o ONE cells, lase con ocal mi- c oscopy was used. B ie ly, FITC-PLGA NPs we e incuba ed (250 µg/mL) o 1 h o 2 h wi h ONE cells and hei beha iou was isually e alua ed. Resul s (Fig. 3) showed ha FITC-PLGA NPs can in e ac wi h ONE cells (60 and 120 min) wi h a g ea e accumula ion a e 120 min, Addi ionally, in o de o check he localiza ion o NPs, a Z-s ack (op ical sec ion: 2.5 µm) was pe o med o con i m ha NPs a e inside o cells. Images ob ained con i m NPs in e naliza ion and hei loca ion in cy oplasm and nucleus (supplemen a y da a). To suppo he quali a i e assays, a quan i a i e s udy was ca ied ou o con i m he in e naliza ion o luo escen NPs (FITC-PLGA NPs) in ONE cells by low cy ome y. Fo his pu pose, cells and luo escen nanopa icles we e incuba ed unde same condi ions o isual assay (250 µg/mL, 60 and 120 min, 37 ◦C) and a e incuba ion, samples we e analysed by low cy ome y. As i shown in Fig. 4, compa ed wi h nega i e con ol (cells incu- ba ed wi hou NPs), he in ensi y o luo escence inc eased a ound 33 % (46.24 % s 78.91 %) a e 60 min and a ound 44 % a e 120 min (46.24 % s 90.55 %) con i ming he p esence o luo escen NPS inside o he cells. These esul s sugges ha NPs in e naliza ion is a ime- dependen p ocess. Beyond he use ul in o ma ion abou he ole able dose, he associ- a ion s udies (NPs cell up ake) could p o ide an insigh in o he access mechanism o he b ain. In his sense, and due o he physicochemical p ope ies o he nanosys ems assayed in his wo k (diame e >100 nm and sligh ly nega i e ze a po en ial), he main hypo heses suppo ed by he scien i ic communi y o unde s and he nose- o-b ain access o nano o mula ions o he b ain is he in a-cellula pa hway (Chen e al., 2024; Clemen ino e al., 2021). 4. In i o s udies 4.1. Fluid and ood consump ion The luid and ood consump ion we e moni o ed o e he CORT egimen o check he induc ion o he dep essi e-like beha io . As ex- pec ed, CORT adminis a ion p oduced a signi ican inc ease o bo h luid and ood in ake (Fig. 1B). 4.2. Locomo o ac i i y The OF was pe o med o e alua e he e ec o CORT and he adminis a ion o each i.n. ea men on he spon aneous locomo ion o he animals. The esul s showed ha none o he ea men s signi ican ly modi ied he animal’s mo o ac i i y in he OF (Fig. 1C). 4.3. Suc ose p e e ence es No di e ences be ween g oups in suc ose p e e ence be o e he CORT egimen (a baseline measu emen ) we e obse ed (da a no shown). Howe e , CORT signi ican ly dec eases he suc ose p e e ence in bo h PBS and Blank NPs- ea ed animals demons a ing ha he dep ession model p oduced anhedonia. Rega ding he e ec o i.n. ea men s, none o he ea men s induced any e ec on he suc ose p e e ence in con ol animals. Howe e , in animals submi ed o CORT while he VLF- ee d ug was no able o modi y he suc ose p e e ence, he VLF-loaded NPs es o ed he de ici induced by he animal model, showing a signi ican inc ease in suc ose p e e ence compa ed o all PBS-, VLF- and Blank NPs- ea ed animals. Thus, he dose employed o VLF ee d ug was no able o no malize he suc ose p e e ence, bu he same dose adminis e ed h ough he NPs was able o block he anhe- donic e ec induced by he dep ession model (Fig. 1D). 4.4. Tail suspension es Rega ding he ail suspension es , he esul s showed ha he CORT egimen induced a dep essi e-like beha iou due o inc eases in he immobili y ime in PBS- ea ed animals. Rega ding he e ec o i.n. ea men s, none o hem al e ed he immobili y ime in con ol animals, howe e , in CORT animals VLF-loaded NPs signi ican ly dec eased he immobili y ime and es o ed he dep essi e-like beha io induced by he model. As in SPT, he dose employed o VLF ee d ug was no able o es o e he dep essi e-like beha iou . Fig. 2. Viabili y o ONE cells a e 24 h o incuba ion wi h di e en concen- a ions (0.1–––1000 ug/mL) o blank NPs, VLF-loaded NPs and ee d ug (VLF). G aph ep esen s he mean ±S.D (n =6). M.D. Caye o-O e o e al. In e na ional Jou nal o Pha maceu ics 678 (2025) 125692 6 5. Discussion 5.1. Nps p oduc ion and physicochemical cha ac e iza ion VLF is a dual ac ion an idep essan which p esen s a sho hal -li e, na ow abso p ion window and ema kable hepa ic i s -pass me a- bolism, which leads o poo o al bioa ailabili y (Li e al., 2021). In he con en ional o al he apy, hese disad an ages lead o equen adminis a ion o main ain he he apeu ic le els and es ic ed access o he b ain due o he p esence o he BBB. The e o e, he encapsula ion o he an idep essan in polyme ic NPs could imp o e he cu en o al he apy compa ed o adi ional dosage o ms becoming a p omising o mula ion o dep ession ea men . Taking in o accoun he solubili y o he polyme (hyd ophobic) and he ac i e (wa e -soluble d ug), NPs we e p epa ed by double emulsion- e apo a ion me hod (Ding e al., 2019; Iqbal e al., 2015) using PLGA, a polyme ha is app o ed o human use by FDA and EMA. The ype o nanopa icle o employ in he in i o s udies was based on he esul s ob ained in p e ious wo k, as men ioned abo e (Caye o-O e o e al., 2019). Fo VLF NPS, esul s ob ained by Mal e n Ze asize showed a low PdI (<0.4) indica ed he homogenei y o he popula ion and he Fig. 3. Me ged con ocal mic oscopy images o ONE cells a e 60 min (A) and 120 min (B) o incuba ion wi h FITC-PLGA NPs (250 µg/mL). Rep esen a i e immuno luo escence images in ONE cell o FITC-PLGA NPs in g een (A1, B1), cy oplasm ma ked in ed wi h WGA-Alexa Fluo ® (A2, B2), nuclei s ained in blue wi h Hoechs ® (A3, B3) and me ged (A and B) in ONE cells using 20x objec i e o A and 60x objec i e o B. M.D. Caye o-O e o e al. In e na ional Jou nal o Pha maceu ics 678 (2025) 125692 7 ae odynamic diame e no exceeding 200 nm. This pa icle size, ac- co ding o he li e a u e, is he limi o an e icien b ain a ge ing e - iciency h ough ol ac o y egion (Tong e al., 2017). Then, a low PdI is essen ial o ensu e a monodispe se popula ion because size is a c i ical ac o o access o he CNS by he in anasal ou e, de e mining he mechanism o en y in o he b ain (Bonacco so e al., 2017). FITC-PLGA NPs exhibi ed a sligh ly la ge bu compa ible size (a ound 270 nm) o hei use in in e naliza ion s udies wi h a highly homogeneous popula ion. Rega ding he ze a po en ial, nega i e alues o all o mula ions indica ed a s able sys em wi hou agg ega ion and easy edispe sion and he absence o PVA on he su ace o nanosys ems (Zambaux e al., 1998). I is essen ial o emo e he PVA esidues o ensu e a su icien nega i e cha ge and p e en agg ega ion. Ze a po en ial also in luences bo h nanopa icle s abili y in biological luids and he isk o immune clea ance and longe ci cula ion imes in he bloods eam. This is mainly due o hei educed in e ac ion wi h plasma p o eins and dec eased up ake by he mononuclea phagocy e sys em (MPS) (Blanco e al., 2015). Au ho s p e iously demons a ed an accumula ion o less han 5 % o nanopa icles in spleen o li e , in an in i o biodis ibu ion assays, indica ing minimal clea ance. Finally, EE% and DL% alues ob ained by DE-SEV me hod we e ema kably ep oducible, wi h mo e han 50 % o ini ial amoun encapsula ed in o he NPs sugges ing he pa icles p oduc ion me hod was op imal. 5.2. In i o MTT and in e naliza ion s udies As men ioned abo e, al hough he mechanism o nose- o-b ain ou e is no comple ely elucida ed (Yokel, 2022), mos s udies sugges ha he e a e se e al possible ou es may be implica ed (Bo ajo e al., 2022) in he nose- o-b ain pa hway and i is well-known ha igeminal and ol ac o y ne es a e in ol ed (in acellula and ex acellula pa hway) (Giunchedi e al., 2020)(Kuma e al., 2014). The e o e, he s udy o cellula up ake o PLGA NPs can be a use ul ool o de e mine he abili y o hem o each he b ain ac oss he ol ac o y cells in u u e in i o expe imen s. In he in i o expe imen s, cell up ake assays (quali a i e and quan i a i e) o luo escen NPs (FITC-PLGA) and cy o oxici y es o o mula ions we e e alua ed a e he incuba ion wi h ol ac o y cells. On he one hand, cell iabili y analysis indica ed ha incuba ion wi h he NPs assayed did no esul in signi ican cy o oxici y, as cell iabili y emained abo e 85 %. Acco ding o he ISO 10993–5:2009 guidelines, hese esul s sugges ha he es ed o mula ions a e non- oxic wi hin he s udied concen a ion ange. On he o he hand, in o de o es ablish a co ela ion be ween in i o and in i o assessmen s and elucida e he access o he luo escen NPs o he b ain, we s udied he in e naliza ion o NPs in ONE cells o 60 and 120 min. Ou esul s showed ha luo escen NPs can in e ac wi h ol ac o y cells, sugges ing ha NPs could c oss he cells in nose- o-b ain ou e in case o in acel- lula pa hway. The quan i a i e up ake s udies o NPs, con i m he e- sul s ob ained o quali a i e analysis. These esul s a e in conco dance wi h ecen li e a u e. I is belie ed ha pa icles wi h sizes g ea e han 100 nm could access he b ain h ough he in acellula ou e, which would ein o ce he need o e icien cellula in e naliza ion (Clemen ino e al., 2021)(Chen e al., 2024). 5.3. An idep essan -like e ec In he p esen s udy, we use a dep essi e model in oden s based on he ch onic adminis a ion o CORT. The exposu e o exogenous CORT p oduces a pe sis en dep ession-like s a e sensi i e o an idep essan ea men in oden s. This beha io al pheno ype is accompanied by b ain changes inhe en o dep essi e-like s a es, which o e all makes his model an app op ia e ool o assess he an idep essan -like e ec o no el d ugs (Gou ley and Taylo , 2009)(Zhao e al., 2008). Fi s , we con olled he p ope induc ion o his animal model by he e alua ion o he ood and d inking consump ion du ing he en i e p o ocol. In ag eemen wi h p e ious indings, he ch onic adminis a ion o CORT induces an inc ease in liquid and ood consump ion (Da id e al., 2009; Fe ´ es-Coy e al., 2015). Addi ionally, a he end o he p o ocol we demons a ed he dep essi e-like s a e in CORT– ea ed animals as measu ed by he dec ease in he suc ose p e e ence as well as wi h he inc ease in he immobili y ime in he TST. To e alua e he beha io al e ec o he di e en in anasal ea - men s, we e alua e he animals in he OF, he suc ose p e e ence es Fig. 4. Flow cy ome y analysis o in e naliza ion e alua ion o FITC-PLGA NPs a e 60 min (g een) and 120 min (blue) a 250 µg/mL o concen a ion. Nega i e con ol is illus a ed in ed. M.D. Caye o-O e o e al. In e na ional Jou nal o Pha maceu ics 678 (2025) 125692 8 and he TST. Spon aneous locomo o ac i i y assessed in he OF is a sensi i e measu e in oden s o seda i e and s imulan e ec s induced by d ugs. Ou esul s showed ha nei he VLF-loaded NPs no VLF- ee d ug p oduced any e ec on he spon aneous ac i i y. Indeed, p e i- ous s udies demons a ed ha acu e high doses o VLF p oduced an in- c ease in locomo o ac i i y while ch onic adminis a ion did no a ec o e en induce a seda i e e ec measu ed in he OF (Red obe e al., 1998) (Ka lsson e al., 2011), (Zhang e al., 2022). The low po en ial o s imulan o seda i e e ec s a e VLF-loaded NPs adminis a ion sug- ges s a po en ially a o able side-e ec p o ile o his compound. In addi ion, hese esul s ule ou he possibili y ha basal ac i i y le els a e con ounding he esul s ob ained in o he es s, such as TST. Anhedonia is one o he co e symp oms o dep ession. In he p esen s udy, we use he SPT, which is one o he mos common es s o assessing anhedonia in oden s. Ou esul s showed ha VLF-loaded NPs es o ed he de ici ound in CORT– ea ed animals, while VLF- ee d ug was no able o modi y he suc ose p e e ence. Simila esul s we e ob ained in he o al ime spen immobile sco ed as a measu e o beha io al despai . In he TST, only he adminis a ion o VLF-loaded NPs was able o signi ican ly educe he immobili y ime in he ani- mals submi ed o he dep essi e model. The absence o an idep essan -like esponse ound a e VLF ee d ug adminis a ion in he beha io al assessmen was expec ed. Based on p e ious s udies, he dose selec ed in ou s udy is oo low o p oduce a solid an idep essan e ec (Cou ens e al., 2022). As obse ed in clinical se ings, i ypically akes o e wo weeks o an idep essan adminis- a ion o achie e a signi ican e ec capable o e e sing he dep essi e pheno ype induced by an animal model (Da id e al., 2009; Cou ens e al., 2022) . Howe e , ou esul s show ha he same dose and egimen o adminis a ion (1 week) o in anasal VLF-loaded NPs can p oduce a clea an idep essan -like esponse. Thus, ou esul s sugges ha he capsula ion o VLF in PLGA NPs po en ia es he an idep essan -like e - ec o his d ug and may also be a p omising s a egy o educe he onse o ac ion o his d ug, al hough u he esea ch is needed. Al hough we canno de e mine he p ecise mechanism by which NPs imp o e he pha macological p o ile o VLF in i o, one possibili y is ha his o mula ion o e s a long-ac ing con olled elease. We p opose ha NPs-based d ug deli e y sys ems can po en ially o e come he e ec s o P-glycop o ein (P-gP) in he BBB by achie ing a slow elease o VLF. P- gP, an ATP dependen d ug anspo p o ein, ac s as an ac i e e lux pump ha emo es subs ances om he b ain. VLF has been shown o induce P-gP a ce ain concen a ions (1–50 µM) (Bachmeie e al., 2011). In a p e ious s udy conduc ed by ou eam, we compa ed he e ec s o inc easing concen a ions o ee VLF e sus VLF-NPs a same dose on he basola e al side in an in i o BBB model. Ou esul s indi- ca ed ha he g adual elease o VLF om NPs o he basola e al side (d ug concen a ion <0.25 µM) did no each le els su icien o induce P-gP (Caye o-O e o e al., 2019). This obse a ion aligns wi h p e ious epo s sugges ing ha b ain concen a ions and he beha io al e ec s induced by ch onic VLF adminis a ion a e in luenced by he ac i i y o P-gP (Ka lsson e al., 2011). The e o e, hese da a highligh he po en ial o NPs-based d ug deli e y sys ems o mi iga e he impac o P-gP on VLF pha macokine ics a he BBB. By achie ing a con olled and g adual elease o VLF, hese NPs e ec i ely main ain d ug concen a ion below he h eshold known o induce P-gP ac i i y, as demons a ed in ou in i o BBB model. Addi ionally, nanopa icles cause no only ex ension in he e en ion ime i also inc eases he cellula up ake. Fu he mo e, in addi ion o i s e ec s a he BBB le el, i is concei able ha VLF encapsula ed in PLGA NPs al e s i s pha macological ac i i y a he si e o ac ion. Recen s udies sugges ha he onse o he an idep essan e ec o monoamine gic eup ake inhibi o s is media ed by hei a ini y o he ansmemb ane domains o T kB ecep o s (Casa o o e al., 2021). T kB is a ecep o y osine kinase o b ain-de i ed neu o ophic ac o (BDNF), and he ole o BDNF in media ing an idep essan -like beha io al esponses has been well-es ablished (Cas ´ en and Mon- eggia, 2021; Ne o e al., 2011). Recen e idence indica es ha di ec binding o an idep essan d ugs o T kB ecep o s p omo es BDNF signaling, po en ially media ing he onse o an idep essan esponses o hese compounds (Enka i e al., 2024). This concep may explain why ypical an idep essan s ac slowly; hey g adually accumula e in he b ain, achie ing a pla eau a e se e al weeks o ea men (Ka son e al., 2006; Ko nhube e al., 1995). This sugges s ha clinical esponse occu s only when d ug concen a ions in he b ain a e su icien ly high o in e ac wi h low-a ini y binding a ge s such as T kB. The e o e, he enhanced cellula up ake o VLF acili a ed by NPs could be c ucial in inc easing i s binding o T kB ansmemb ane domains, po en ially explaining he enhanced po ency o he an idep essan e ec obse ed wi h VLF-loaded NPs in i o. O e all, we hypo hesized ha he non-in asi e nose- o-b ain de- li e y o ou o mula ion would imp o e he an idep essan ac ion o VLF and be as e ec i e as a ch onic ea men o a highe dose o VLF. 6. Conclusions PLGA NPs con aining VLF could be a po en ial pla o m o he ea men o neu ological diso de s, speci ically o he dep ession. A p e ious biodis ibu ion s udy de eloped by au ho s, es ablished he p esence o his ype o plain-PLGA NPs in he b ain a e in anasal adminis a ion and, in his wo k, in i o beha iou al assays ha e con i med he access o NPs o b ain. The NPs p epa ed in he expe i- men s showed a size which no exceeding 200 nm, an essen ial equi emen o each he b ain by ol ac o y egion acco ding o he li e a u e. Mo eo e , in acco dance wi h ISO guidelines, he p epa ed PLGA-NPs nanopa icles did no exhibi cy o oxici y a he concen a- ions es ed in ONE cells. F om he in i o s udies, we can conclude ha VLF-loaded NPs e oke e y as (1 week) and obus an idep essan -like e ec s in CORT- ea ed mice. Thus, he use o nanopa icle o mula ion could o e come some o he limi a ions o an idep essan ea men s such as he induc ion o ad e se e ec s o high doses o VLF o he slow onse o ac ion, imp o ing compliance and he ou come. Fu he in i o s udies in animal models a e necessa y o alida e hese indings and con i m hei eal he apeu ic po en ial, as well as he mechanisms o he NPs ha enabled a success ul access o he b ain, a e wa an ed o p o ide addi ional in o ma ion o u he po en ial op i- miza ion o he o mula ion, as well as o he de elopmen o al e na- i e ela ed neu ological he apies. Also, p eclinical es ing o assess he sa e y o hese NPs, including hei long- e m e ec s and cy o oxici y, immunogenici y, and po en ial o bioaccumula ion will be necessa y. O e all, ou esul s highligh he po en ial use o plain PLGA NPs as a no el he apy and, pa icula ly, poin o nose- o-b ain adminis a ion o plain PLGA NPs o mula ions as easible and p omising an idep essan non-in asi e he apy. Funding. This s udy was suppo ed by Jun a de Andalucía (2021/CTS-480; 2019/CTS-480). CRediT au ho ship con ibu ion s a emen M. Dolo es Caye o-O e o: W i ing – e iew & edi ing, W i ing – o iginal d a , Valida ion, Me hodology, In es iga ion, Fo mal analysis, Da a cu a ion. Lau a Pe ez-Caballe o: W i ing – e iew & edi ing, W i ing – o iginal d a , Me hodology, In es iga ion, Fo mal analysis, Da a cu a ion. I ene Sua ez-Pe ei a: W i ing – o iginal d a , In es i- ga ion, Fo mal analysis, Da a cu a ion. Ma ía Hidalgo-Figue oa: Me hodology, In es iga ion, Fo mal analysis, Da a cu a ion. Alejand a Delgado-Seque a: In es iga ion, Fo mal analysis, Da a cu a ion. Juan Manuel Mon esinos: Me hodology, In es iga ion. Es he Be ocoso: W i ing – e iew & edi ing, Visualiza ion, Valida ion, Supe ision, Me hodology, In es iga ion, Funding acquisi ion, Fo mal analysis, Da a cu a ion, Concep ualiza ion. Lucía Ma ín-Bande as: W i ing – e iew & edi ing, W i ing – o iginal d a , Visualiza ion, Valida ion, M.D. Caye o-O e o e al. In e na ional Jou nal o Pha maceu ics 678 (2025) 125692 9