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
