Solid lipid nanopa icles loaded wi h
a no el indole-based mul i a ge ligand:
p epa a ion, cha ac e iza ion, and
neu op o ec i e e alua ion in an H₂O₂-
induced SH-SY5Y cell model
Ch is ina Voyche a1, Teodo a Popo a1, Ma a Sla ko a1, Bo isla Tzanko 1,
Bo is S oyano 1, Deni sa S a ano a1, Violina Angelo а1
1 Facul y o Pha macy, Medical Uni e si y o So ia, So ia, Bulga ia
Co esponding au ho :
Ch is ina Voyche a (h o[email p o ec ed]u-so ia.bg)
Recei ed
29 Sep embe 2025♦
Accep ed
23 Oc obe 2025♦
Published
18 No embe 2025
Ci a ion:
Voyche a C, Popo a T, Sla ko a M, Tzanko B, S oyano B, S a ano a D, Angelo а V (2025) Solid lipid nanopa icles
loaded wi h a no el indole-based mul i a ge ligand: p epa a ion, cha ac e iza ion, and neu op o ec i e e alua ion in an H₂O₂-in-
duced SH-SY5Y cell model. Pha macia 72: 1–13. h ps://doi.o g/10.3897/pha macia.72.e173489
Abs ac
A newly syn hesized indole-based N-benzylpipe idine de i a i e (3a), ac ing as a mul i a ge -di ec ed ligand wi h ace ylcholines e ase
inhibi o y ac i i y, was encapsula ed in o solid lipid nanopa icles (SLNs) o imp o e neu op o ec i e po en ial and enable in anasal
b ain deli e y in u u e in i o s udies. Lipid p esc eening was pe o med, and a spec opho ome ic me hod o he quan i ica ion
o 3a was de eloped and alida ed. SLNs we e p epa ed using he nano- empla e enginee ing echnique and e alua ed o pa icle
size, ze a po en ial, d ug en apmen e iciency, in i o elease, and s abili y. The op imized o mula ion (3a-SLNs) was u he
cha ac e ized by ansmission elec on mic oscopy (TEM), X- ay powde di ac ion (XRD), and di e en ial scanning calo ime y
(DSC). The nanopa icles exhibi ed a o able physicochemical p ope ies, eleasing 52% o he d ug o e 24 h. Pe meabili y s udies
indica ed imp o ed cellula up ake o 3a upon SLN encapsula ion. The o mula ion emained s able o 6 mon hs unde e ige a ed
(4 ± 2 °C) and oom empe a u e (25 ± 2 °C; 60 ± 5% RH) condi ions. The neu op o ec i e po en ial o 3a-SLNs was e alua ed
in an H₂O₂-induced oxida i e s ess model in SH-SY5Y neu onal cells using he MTT assay. Cells we e p e ea ed wi h ee 3a
(0.1–50 µM) o 90 min be o e H₂O₂ exposu e (1 mM). Hyd ogen pe oxide induces oxida i e damage ia eac i e hyd oxyl adicals,
a ec ing cellula lipids, p o eins, and DNA. 3a-SLNs exhibi ed supe io p o ec i e e ec s compa ed wi h ee 3a, indica ing ha
nanopa icle encapsula ion enhances in acellula a ailabili y and an ioxidan de ense. These indings highligh SLN-encapsula ed
3a as a p omising in anasal nanoca ie sys em o Alzheime ’s disease he apy, which will be u he in es iga ed in in i o models.
By imp o ing b ain deli e y po en ial and educing oxida i e s ess–induced neu onal inju y, 3a-SLNs o e a s a egy o o e come
pha macokine ic limi a ions o con en ional compounds and enhance neu op o ec i e e icacy.
Keywo ds
Alzheime ’s disease, mul i a ge ligand 3a, neu op o ec ion, nose- o-b ain deli e y, oxida i e s ess, SH-SY5Y cells, solid lipid
nanopa icles
Copy igh Voyche a C e al. This is an open access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion License
(CC-BY 4.0), which pe mi s un es ic ed use, dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal au ho and sou ce
a e c edi ed.
Pha macia 72: 1–13
DOI 10.3897/pha macia.72.e173489
Resea ch A icle
Voyche a C e al.: SLNs loaded wi h a no el indole-based mul i a ge ligand2
In oduc ion
Alzheime ’s disease (AD) is a p og essi e neu odegene a-
i e diso de cha ac e ized by cogni i e decline, memo y
loss, and neu onal dea h (Ca dinali e al. 2010). Among
he majo pa hological mechanisms unde lying AD a e
choline gic dys unc ion, oxida i e s ess, and accumula-
ion o amyloid-be a (Aβ) pep ides. Choline gic de ici s
a e associa ed wi h dec eased ace ylcholine (ACh) sig-
naling, p ima ily due o al e ed ac i i y o ace ylcholines-
e ase (AChE) and bu y ylcholines e ase (BChE), which
hyd olyze ACh. In AD, AChE ac i i y is o en unchanged
o dec eased, while BChE ac i i y may inc ease, con-
ibu ing o impai ed choline gic ansmission (Fe nán-
dez-Bolaños and López 2022). Concu en ly, Aβ pep ides
accumula e due o imbalances in p oduc ion and clea -
ance, o ming neu o oxic oligome s ha igge oxida i e
s ess, lipid pe oxida ion, and neu onal apop osis (Li e
al. 2020). Donepezil (DNPZ) is a selec i e, noncompe -
i i e, and apidly e e sible ace ylcholines e ase inhib-
i o . Mela onin (MEL) modula es he Aβ p oduc ion/
clea ance balance and educes Aβ neu o oxici y (Ramos
e al. 2017; Li e al. 2020; Ve ma e al. 2023). Molecules
con aining a mela onin ame and a donepezil emnan
show p ominen inhibi o y ac i i y agains ace ylcholin-
es e ase (Luo e al. 2013; Gulcan and Kosa 2022; P a in
and Jozwiak 2022; Eissa e al. 2023). A ecen ly de eloped
indole-based N-benzylpipe idine de i a i e, 3a (N’-[(E)-
(1-benzylpipe idin-4-yl)me hylidene]-1H-indole-3-ca bo-
hyd azide), has shown po en AChE and BChE inhibi-
ion, s ong an ioxidan ac i i y, and low cy o oxici y in
SH-SY5Y and Neu o-2a cells. Molecula docking s udies
sugges ha 3a in e ac s wi h AChE, BChE, and mela-
onin MT1/MT2 ecep o s, suppo ing i s po en ial as a
neu op o ec i e agen . Angelo a e al. (2023) explo ed
wo se ies o hyb id molecules combining mela onin
and donepezil h ough hyd azone o sul onyl hyd a-
zone linkages. The lead indole-based hyb id compound
3a (N’-[(E)-(1-benzylpipe idin-4-yl)me hylidene]-1H-in-
dole-3-ca bohyd azide) exhibi ed subs an ial AChE inhi-
bi ion (76.51 ± 3.04 µM) and BChE inhibi ion (56.50 ±
0.20 µM), along wi h s ong an ioxidan and lipid pe -
oxida ion inhibi ion ac i i ies. In an ioxidan assays, 3a
showed obus 1,1-diphenyl-2-pic ylhyd azyl (DPPH)
ac i i y and excelled in bo h e ic educing an ioxidan
powe (FRAP) and e ic hiocyana e (FTC) assays. No-
ably, 3a displayed low cy o oxici y in Neu o-2a (IC₅₀ >
300 µM) and SH-SY5Y cells (129 ± 7.5 µM), ou pe o m-
ing donepezil, and demons a ed a o able blood–b ain
ba ie pe meabili y (PAMPA BBB–logPe = 4.420). Mo-
lecula docking s udies sugges ed ha 3a a ge s AChE,
BChE, and MT1 and MT2 ecep o s, ma king i as a
p omising candida e o Alzheime ’s disease ea men
(Angelo a e al. 2023). In ollow-up esea ch (Mihaylo a
e al. 2024), he e ec s o 3a on Aβ-induced neu o oxici y
and memo y impai men we e e alua ed in mouse mod-
els. The N-benzylpipe idine de i a i e (3a) signi ican ly
a ec ed Aβ₄₂ p oduc ion in SH-SY5Y neu onal cul u es
a e 24 h and showed supe io an i-amyloid ac i i y
compa ed wi h he e e ence d ugs DNPZ and galan h-
amine (GAL). Addi ionally, compound 3a adminis e ed
alone educed he amoun o malondialdehyde (MDA)
in mouse b ain homogena e s a is ically signi ican ly by
21.4%, while scopolamine (SC) inc eased i by 76% com-
pa ed wi h he con ol g oup in he SC-induced b ain
oxici y es (Mihaylo a e al. 2024). In animals ea ed
wi h bo h SC and he es compound 3a, he e was a s a-
is ically signi ican dec ease in he MDA le el by 19.3%
compa ed wi h he g oup ea ed wi h SC alone. In con-
as o DNPZ, compound 3a was able o p o ec cells in
he do sal hippocampus agains SC-induced neu o ox-
ici y (Mihaylo a e al. 2024). Fu he mo e, 3a e ec i e-
ly in luenced b ain inju y and AD-like pa hology in he
SC-induced demen ia model in mice by a enua ing cho-
line gic damage, lipid pe oxida ion (LP), and neu onal
loss in he hippocampus (Mihaylo a e al. 2024). Acu e
oxici y s udies in mice (Mihaylo a e al. 2024) showed
ha he compound is mildly oxic (500–5000 mg/kg)
when adminis e ed o ally and mode a ely oxic (50–500
mg/kg) when gi en in ape i oneally, based on he Hodge
and S e ne scale (Go e nmen o Canada 2024). The low
o al oxici y migh be linked o poo o al bioa ailabili-
y, as he compound may ha e limi ed abso p ion in he
gas oin es inal ac o unde go signi ican hepa ic and
in es inal me abolism, yielding non oxic me aboli es.
Fu he pha macokine ic s udies a e needed o con i m
his by measu ing me aboli e and pa en compound le -
els in plasma, u ine, and eces. Biochemical analysis o 3a
e ealed only sligh di e ences om he e e ence d ug
donepezil, speci ically in o al p o ein and ASAT ac i i y,
indica ing no associa ed hepa o oxic o neph o oxic e -
ec s (Mihaylo a e al. 2024).
In anasal d ug deli e y p o ides a unique oppo uni-
y o nonin asi e access o he cen al ne ous sys em.
I s ad an ages include as abso p ion, highe pa ien ac-
cep abili y, and ci cum en ion o me abolic deg ada ion
(Gandhi e al. 2024; Koo e al. 2024). Inco po a ion o
ac i e molecules in o nanoca ie sys ems, pa icula ly
solid lipid nanopa icles, enhances hei s abili y and
bioa ailabili y while o e ing con olled elease and p o-
longed esidence ime in he nasal ca i y (Cos a e al.
2021; Zheng e al. 2024). These combined bene i s make
in anasal nanoca ie s a ac i e candida es o he
managemen o Alzheime ’s disease and ela ed condi-
ions (Nguyen and Duong 2025).
Among he di e en nanoca ie pla o ms explo ed
o nose- o-b ain deli e y, solid lipid nanopa icles
(SLNs) ha e gained special a en ion due o hei biocom-
pa ibili y and po en ial o imp o e d ug pe mea ion. SLNs
a e colloidal sys ems ha can deli e bo h hyd ophilic and
lipophilic ac i e subs ances. Thei co e is a composi e o
solid lipids s abilized by a monolaye coa ing o su ac an
on he su ace (Schwa z e al. 1994). Thei small pa icle
size, as well as he p esence o emulsi ie s, de e mines
hei abili y o pass h ough biological memb anes and
enables he pa i ioning o nanosized d ople s in he nasal
issues, esul ing in enhanced CNS deli e y ia in anasal
applica ion (Riz i e al. 2019; Mehne and Mäde 2001).
Pha macia 72: 1–13 3
The physiological lipids in hei composi ion enhance he
o al bioa ailabili y o poo ly wa e -soluble ac i e sub-
s ances (Mehne and Mäde 2001), whe eas hei occlu-
si e e ec and excellen adhesion o mucous memb anes
inc ease nasal e en ion ime (Kau e al. 2008). Mo e-
o e , SLNs p o ide excellen d ug loading, p o ec ion o
he loaded ac i e subs ance om deg ada ion, imp o ed
s abili y, and supe io con olled d ug elease. They a e
also biodeg adable and ha e good ole abili y, as he e
a e no epo s o acu e o ch onic oxici y, which makes
hem sui able o p oduc ion scalabili y (Kau e al. 2008;
Helgason e al. 2009; Kuma e al. 2013).
Conside ing hese ad an ages, encapsula ion o 3a in o
SLNs is expec ed o enhance s abili y, cellula up ake, and
e en ual b ain deli e y ia he in anasal ou e. The p esen
s udy aimed o p epa e and cha ac e ize 3a-loaded SLNs,
op imize hei physicochemical p ope ies, and e alua e
hei neu op o ec i e e ec s agains H₂O₂-induced oxida-
i e s ess in SH-SY5Y neu onal cells. The in i o model
allows assessmen o an ioxidan and neu op o ec i e e i-
cacy, p o iding insigh in o he po en ial o SLN-encapsu-
la ed 3a as a no el he apeu ic s a egy o AD.
Ma e ials and me hods
S ea ic acid, palmi ic acid, glyce yl monos ea a e, and
glyce yl palmi os ea a e we e pu chased om Sasol Ge -
many GmbH (Wi en, Ge many). Tween 80 was pu -
chased om Sigma-Ald ich (S . Louis, USA). E hanol, di-
me hyl sul oxide (DMSO), disodium hyd ogen phospha e
dihyd a e, and po assium dihyd ogen phospha e we e all
pu chased om Me ck (Da ms ad , Ge many). Deionized
wa e was p epa ed in he labo a o y. All sol en s, chem-
icals, and eagen s we e ob ained comme cially and used
wi hou u he pu i ica ion.
Syn hesis and cha ac e iza ion o 3a
A solu ion o 20 mmol o he co esponding ca bonyl
compound in 10 mL o absolu e e hanol was mixed wi h
a ho solu ion o 20 mmol a oylhyd azide in 10 mL o ab-
solu e e hanol a 60 °C and s i ed o 1–8 h. The ob ained
c ys alline p ecipi a es we e il e ed, washed wi h an e ha-
nol–e he mix u e, and ec ys allized om e hanol. A e
he chemical eac ions, he c ude compounds we e pu i-
ied h ough s anda d me hods such as column ch oma-
og aphy and ec ys alliza ion o isola e he pu e hyb id
molecules. The inal syn hesized compounds we e cha ac-
e ized by echniques such as nuclea magne ic esonance
(NMR) spec oscopy, mass spec ome y, and elemen al
analysis o con i m hei s uc u es and pu i y.
De elopmen o an analy ical me hod
o 3a
The assay o 3a was de e mined using UV– is spec opho-
ome y. The me hod was alida ed wi h espec o selec-
i i y, linea i y, and p ecision. E alua ion o selec i i y was
pe o med by compa ing he UV– is spec a o he pu e
ac i e pha maceu ical ing edien (API) and he placebo
mix u e in he ange o 200–600 nm. Linea i y o he an-
aly ical me hod was es ed in wo media: dis illed wa e
and pH 6.5 bu e . 3a (2 mg) was quan i a i ely ans e ed
in o 50 mL olume ic lasks and solubilized in 5 mL o
DMSO, ollowed by dilu ion up o 50 mL wi h he espec-
i e medium. Se en s anda d s ock solu ions we e dilu ed
o co e he concen a ion ange o 0.002–0.04 mg/mL.
The es s we e pe o med in iplica e. Calib a ion cu es
o he UV– is spec opho ome ically measu ed s ock
solu ions we e plo ed based on concen a ion e sus
abso bance. The co ela ion coe icien (R²) se ed as an
indica o o linea i y. Based on he calib a ion cu e equa-
ion, he co ela ion coe icien , y-in e cep , slope o he
eg ession line, and esidual sum o squa es we e de e -
mined and used in he o mula o calcula ing he amoun
o API eleased om he dosage o m. The p ecision was
assessed by es ing h ee concen a ion le els co e ing
he speci ied ange (50%, 100%, and 125% o he API
concen a ion used in he s anda d expe imen s), p e-
pa ing iplica es a each le el. The es s we e pe o med
in bo h dis illed wa e and pH 6.5 bu e media. Based
on he ob ained esul s, he ela i e s anda d de ia ion
(RSD%) was calcula ed.
De e mina ion o solubili y o 3a in solid
lipids
The solubili y o 3a in s ea ic acid, palmi ic acid, glyce yl
monos ea a e, and glyce yl palmi os ea a e was e alua ed
semi-quan i a i ely. 3a was added in s eps o 1 mg o 100
mg o mel ed lipid and s i ed wi h a magne ic s i e . The
expe imen con inued un il incomple e dissolu ion o he
las added po ion was obse ed. The es was epea ed
wi h he de e mined amoun o 3a ha showed comple e
dissolu ion. Solubili y was e alua ed isually in he mel ed
lipid and mic oscopically in a hin sec ion o he cooled
solu ion (Shah aeini e al. 2020; Wolska and B ach 2022).
P epa a ion o SLNs and loading o 3a
The SLNs we e p epa ed using he nano- empla e enginee -
ing echnique wi h some modi ica ions (Sohail e al. 2023).
Glyce yl monos ea a e was mel ed a 70 °C. Tween 80
was homogenized in 10 mL o dis illed wa e and hen
hea ed o 70 °C. The su ac an solu ion was added d op-
wise o he mel ed lipid unde con inuous s i ing on a
magne ic s i e . A e adding he las po ion, s i ing
was con inued a 800 pm o 30 min. The ob ained emul-
sion was sonica ed o 4 min a 75% ampli ude in a 20/10 s
on/o cycle using a p obe sonica o (Bandelin Sonopuls)
o ob ain a nanoemulsion. The sonica ed nanoemulsion
was placed in o cold wa e (4 °C) o o m he SLN dispe -
sion. To p epa e loaded SLNs, 10 mg o 3a was p e iously
dissol ed in he lipid. The amoun s o lipid and su ac an
o he di e en samples a e p esen ed in Table 2. The e-
sul ing dispe sion was passed h ough a 0.45 µm sy inge
il e o elimina e unen apped 3a and la ge agg ega es.
Voyche a C e al.: SLNs loaded wi h a no el indole-based mul i a ge ligand4
SLNs we e ob ained a e lyophiliza ion a −80 °C o
24 h unde a 1 × 10–4 mba acuum using an Alpha 3–4
LSCbasic semi-indus ial eeze-d ye (Ma in Ch is Ge-
ie ocknungsanlagen GmbH, Os e ode, Ge many). The
lyophilized SLNs we e econs i u ed wi h deionized wa e
o bu e be o e use in u he s udies.
De e mina ion o SLN encapsula ion
e iciency (EE)
The SLN (10 mL) suspension was cen i uged a 1300
pm o 20 min. The supe na an , con aining ee non-
encapsula ed 3a, was sui ably dilu ed wi h 10% DMSO
and analyzed by UV spec opho ome y a λ = 258 nm,
as desc ibed in he De elopmen o an analy ical me h-
od o 3a sec ion. EE was calcula ed using he ollowing
equa ion:
Dynamic ligh sca e ing (DLS) analysis
The nanopa icle size, polydispe si y index, and ze a po-
en ial we e de e mined using a Ze asize (Ze asize Nano
ZS, Mal e n Panaly ical, Wo ces e shi e, UK). The sam-
ples (0.1% w/ ) we e dispe sed in dis illed wa e , sonica -
ed o 20 min, and measu ed a a sca e ing angle o 90°
and a 25 °C.
T ansmission elec on mic oscopy
(TEM) analysis
The size and s uc u e o 3a-loaded SLNs we e cha ac e ized
using ansmission elec on mic oscopy (JEOL JEM 2100 h
STEM; 200 kV; poin esolu ion = 0.23 nm). Samples we e
p epa ed by placing he aqueous suspension o nanopa icles
on a polyme mic og id suppo ed on a Cu g id. The wa e
was subsequen ly e apo a ed unde acuum.
X- ay di ac ion analysis (XRD)
X- ay powde di ac ion pa e ns o he d ug, lipid, and
op imized 3a-SLNs we e measu ed using an X- ay di ac-
ome e (B uke D8 ADVANCE, Ge many). The samples
we e analyzed using Cu Kα adia ion (45 kV, 40 mA) and
scanned om 5° o 80° (2θ) wi h a s ep size o 0.03° (2θ)
and a coun ing ime o 17.5 s/s ep.
Di e en ial scanning calo ime y (DSC)
DSC cu es o 3a, SLNs, and 3a-SLNs we e eco ded using
a di e en ial scanning calo ime e (Pe kinElme DSC-
8500) equipped wi h an In acoole 3 cooling sys em. The
samples we e scanned in he empe a u e ange o −50 °C
o 180 °C a a hea ing a e o 10 °C/min. Cooling was pe -
o med a 20 °C/min om 180 °C o 20 °C. Ins umen
con ol, da a collec ion, and p ocessing we e conduc ed
using Py is so wa e e sion 10.1.0.0412.
In i o d ug elease s udy
The d ug elease om 3a-SLNs was e alua ed in simula ed
nasal luid (SNF; pH 6.5) using he dialysis bag me hod.
The SNF medium con ained 7.45 mg/mL NaCl, 1.29 mg/
mL KCl, and 0.32 mg/mL CaCl₂·2H₂O (Fa id e al. 2013).
To main ain sink condi ions o 3a, 10% DMSO was add-
ed o he elease medium. The dialysis memb ane (po e
size 2.4 nm; molecula weigh cu o 12,000–14,000; Sig-
ma-Ald ich–Millipo eSigma) was soaked in dis illed wa-
e o 12 h be o e use. The 3a-SLN dispe sion, equi alen
o 1.8 mg o 3a, was pou ed in o he bag, and bo h ends
we e secu ed wi h clamps. The dialysis bag was placed in
a 25 mL conical lask con aining he dissolu ion medium.
The elease s udy was pe o med using an incuba o shak-
e a 37 °C. Aliquo s o he dissolu ion medium we e wi h-
d awn a p ede e mined ime in e als, and an equal ol-
ume o esh medium was added o main ain a cons an
olume. The samples we e analyzed spec opho ome i-
cally a 258 nm agains a sol en blank. All expe imen s
we e pe o med in iplica e.
In i o pe meabili y o 3a
Pe meabili y s udies we e pe o med using a F anz
di usion cell sys em (Logan Ins umen s Co p. 913-6
au oma ed ansde mal di usion cell sampling sys em,
Some se , NJ, USA). The uppe chambe (dono com-
pa men ) was illed wi h 2 mL o 3a-SLN dispe sion
equi alen o 1.8 mg o pu e 3a (Zhang e al. 2020). The
Pe meaPad® memb ane was placed be ween he dono
and ecep o compa men s, wi h an e ec i e su ace
a ea o 1.54 cm². The ecep o compa men con ained
12 mL o SNF (pH 6.5) wi h 10% DMSO added o main-
ain sink condi ions. The s i ing a e was main ained
a 100 pm, and he empe a u e was kep a 34 ± 0.5 °C
using a ci cula ing wa e ba h h oughou he 12 h pe -
mea ion s udy (Nai e al. 2022). A p ede e mined in-
e als (1, 2, 3, 4, 5, 6, and 12 h), 1 mL o he sample was
wi hd awn om he ecep o phase and eplaced wi h
esh medium. The amoun o pe mea ed d ug was an-
alyzed spec opho ome ically a λ = 258 nm. All sam-
ples we e es ed in iplica e. The cumula i e amoun
o he d ug ha pene a ed h ough he memb ane
su ace (Q , µg/cm2) was calcula ed using he ollowing
equa ion (Liu e al. 2018):
Q = (Cn·VA + ∑i
n-1 Ci ·Vs)/A
whe e Q is he cumula i e amoun o 3a ha pene a -
ed pe uni memb ane su ace a ea; Cn is he 3a concen-
a ion in he n- h sample; Ci is he 3a concen a ion in
he i- h sample; VA is he olume o he ecep o phase
(12 mL); Vs. is he olume o he wi hd awn sample
(2 mL); and A is he e ec i e pe mea ion a ea o he
memb ane (1.54 cm²). The cumula i e Q was plo ed
agains ime, and he s eady-s a e lux (Jss, µg/cm²/h)
was calcula ed om he slope o he linea po ion o
he cu e (Liu e al. 2018).
Pha macia 72: 1–13 5
S abili y s udies
The op imized 3a-SLN dispe sion was subjec ed o s a-
bili y es ing in iplica e. S o age condi ions we e as
ollows: 4 ± 2 °C ( e ige a ed), 25 ± 2 °C and 60 ± 5% RH,
and 40 ± 2 °C and 75 ± 5% RH o 6 mon hs. The a e age
pa icle size, polydispe si y index, and ze a po en ial we e
measu ed unde hese condi ions (Luo e al. 2006).
Cell line and cul u e condi ions
The human neu oblas oma cell line SH-SY5Y (ECACC
No. 94030304) was ob ained om he Eu opean Collec-
ion o Au hen ica ed Cell Cul u es (ECACC, UK). This
cell line is widely used as an in i o model o neu onal
di e en ia ion, neu o oxici y, and neu op o ec ion due
o i s ca echolamine gic p ope ies and abili y o acqui e
neu on-like cha ac e is ics unde speci ic condi ions.
Cells we e cul u ed in RPMI 1640 medium supplemen ed
wi h 10% hea -inac i a ed e al bo ine se um (FBS), 2 mM
L-glu amine, and 1% penicillin–s ep omycin. Cul u es we e
main ained a 37 °C in a humidi ied incuba o wi h 5% CO₂.
The medium was eplaced e e y 2–3 days, and cells we e
subcul u ed a 70–80% con luence using ypsin–EDTA and
eseeded a sui able densi ies o expe imen s.
H₂O₂-induced oxida i e s ess model in
SH-SY5Y neu onal cells
To e alua e he cy op o ec i e po en ial o he es com-
pound, bo h ee 3a (0.1, 1, 5, 10, 25, and 50 µM) and
nanopa icle-loaded 3a (3a-SLNs) a he same concen a-
ions we e es ed. Unloaded nanopa icles we e also assessed
sepa a ely o e a concen a ion ange o 0.208–104 µg/mL
o de e mine any e ec s o he ca ie sys em alone. SH-
SY5Y cells we e seeded in 96-well pla es a 3.5 × 10⁴ cells
pe well and allowed o a ach o 24 h unde s anda d cul-
u e condi ions (37 °C, 5% CO₂). Cells we e hen p e ea ed
wi h he espec i e o mula ions o 90 min o allow cellu-
la up ake. Following p e ea men , cells we e washed wi h
s e ile PBS and exposed o 1 mM H₂O₂ o 10 min o induce
oxida i e s ess. A e he H₂O₂ challenge, esh comple e
RPMI 1640 medium was added, and cells we e incuba ed
o an addi ional 24 h. Cell iabili y was subsequen ly mea-
su ed using he MTT assay, wi h un ea ed cells se ing as
he nega i e con ol (100% iabili y) and H₂O₂- ea ed cells
wi hou compound o nanopa icles se ing as he posi i e
con ol o maximal oxida i e damage. All da a we e ana-
lyzed using G aphPad P ism 8, applying one-way ANOVA
ollowed by Dunne ’s pos hoc es o de e mine s a is ical-
ly signi ican di e ences ela i e o he con ol g oups.
Resul s and discussion
Syn hesis and cha ac e iza ion o 3a
The syn hesis o 3a began wi h comme cially a ail-
able s a ing ma e ials, such as indole de i a i es and
donepezil analogues, as p esen ed in ou p e iously pub-
lished a icle (Angelo a e al. 2023). A s epwise syn he ic
app oach was used o inco po a e indole and donepe-
zil-like pha macopho es in o a single hyb id molecule.
Va ious chemical eac ions, including nucleophilic sub-
s i u ion and condensa ion, we e employed o o m he
desi ed hyb id s uc u e. The syn hesis aimed o op i-
mize he balance be ween he wo pha macopho es (in-
dole and donepezil) o ensu e ha he inal compound
exhibi ed bo h ace ylcholines e ase (AChE) inhibi o y
ac i i y (donepezil-like) and a ini y o o he po en ial
Alzheime ’s- ela ed a ge s. A e he chemical eac ions,
he c ude compounds we e pu i ied h ough s anda d
me hods such as column ch oma og aphy and ec ys al-
liza ion o isola e he pu e hyb id molecules. The inal
syn hesized compounds we e cha ac e ized by nuclea
magne ic esonance (NMR) spec oscopy, mass spec-
ome y, and elemen al analysis o con i m hei s uc-
u es and pu i y. The ob ained compound was N’-[(E)-
(1-benzylpipe idin-4-yl)me hylidene]-2-(1H-indol-3-yl)
ace ohyd azide, and i s s uc u e is shown in Fig. 1.
De elopmen o an analy ical me hod
o 3a
Valida ion o analy ical me hods is an essen ial p ocess
because i demons a es ha he analy ical p ocedu e used
o a speci ic de e mina ion is sui able o i s in ended
pu pose and ensu es i s eliabili y by e alua ing accu a-
cy, p ecision, speci ici y, linea i y, ange, and obus ness.
Conside ing he assay o 3a om solid lipid nanopa icles,
he me hod was alida ed wi h espec o selec i i y, lin-
ea i y, and p ecision.
Selec i i y e e s o he ex en o which a me hod can
de e mine a pa icula analy e in a complex mix u e
wi hou in e e ence om o he componen s (Valcá cel
e al. 2001). To demons a e selec i i y, he UV– is spec-
a o pu e 3a and a placebo mix u e con aining glyce yl
monos ea a e (GMS) and Tween 80 we e eco ded. The
ac i e pha maceu ical ing edien (API) exhibi ed a max-
imum abso p ion peak a 258 nm (λmax), whe eas he
placebo solu ion did no show any abso p ion a he same
wa eleng h, con i ming he selec i i y o he assay me hod.
Figu e 1. S uc u e o N’-[(E)-(1-benzylpipe idin-4-yl)me-
hylidene]-1H-indole-3-ca bohyd azide (3a).
Voyche a C e al.: SLNs loaded wi h a no el indole-based mul i a ge ligand6
Acco ding o ICH guideline Q2, he linea i y o an ana-
ly ical p ocedu e is i s abili y (wi hin a gi en ange) o p o-
ide analy ical es esul s ha a e di ec ly p opo ional o
he concen a ion (amoun ) o he API in he sample. Based
on he UV– is abso p ion de e mina ions a 258 nm o
he p epa ed s anda d s ock solu ions, calib a ion cu es
we e cons uc ed in dis illed wa e and bu e medium (pH
6.5) (Figs 2, 3), and he co esponding equa ions we e plo -
ed. The high R² alues con i med he linea dependence
be ween he concen a ion o 3a and i s abso bance in he
gi en concen a ion ange in bo h media.
The p ecision o an analy ical p ocedu e exp esses he
closeness o he esul s (deg ee o sca e ) among a se ies o
measu emen s ob ained om mul iple samplings o he same
sou ce unde iden ical condi ions. P ecision da a a e gene -
ally exp essed as s anda d de ia ion, ela i e s anda d de ia-
ion (RSD), and con idence in e al. The RSD% esul s o 3a
a e shown in Fig. 4. RSD% alues below 2% con i med he
p ecision o he analy ical me hod o he assay o 3a.
Solubili y o 3a in lipids
Ini ially, he solubili y o 3a was de e mined in a ious lip-
ids, and he esul s a e p esen ed in Table 1.
Solubili y s udies indica ed ha 3a showed he highes
solubili y in glyce yl monos ea a e (GMS). I s selec ion
o he p esen s udy was based on i s solubilizing po en-
ial, biocompa ibili y, and sui abili y o nose- o-b ain de-
li e y (Joshi e al. 2012; Singh e al. 2012).
The choice o su ac an was based on he hyd ophilic–
lipophilic balance (HLB) alue. Tween 80, wi h an HLB
o 15, can emulsi y GMS o o m a s able nanoemulsion
a an accep able concen a ion. Polyso ba es a e a sui able
choice as coa ing ma e ials in he p epa a ion o ca ie s
o b ain- a ge ed deli e y o ac i e subs ances, as hey
acili a e anspo ac oss he blood–b ain ba ie (BBB)
(K eu e e al. 2003). Thei mechanism o ac ion is as-
socia ed wi h endocy osis by b ain capilla y endo helial
cells (Oli ie 2005; Yada e al. 2018). Se e al epo s ha e
shown ha Tween 80 p oduces ine-sized SLNs sui able
o nose- o-b ain d ug deli e y (Gau e al. 2014).
A e he selec ion o componen s, op imiza ion was
pe o med. The app op ia e a io o lipid and su ac an
was chosen based on pa icle size, polydispe si y index,
ze a po en ial, and loading e iciency, as hese pa ame-
e s can in luence in i o pe meabili y, oxici y, and b ain
a ge ing. Pa icle size has a signi ican e ec on s abili y,
d ug loading, and elease beha io , whe eas pa icle size
dis ibu ion a ec s ci cula ion ime, abso p ion, and bio-
dis ibu ion. Thus, small pa icle sizes and la ge su ace
a eas lead o imp o ed d ug solubili y, be e mucoadhe-
sion, and enhanced in i o pe mea ion compa ed wi h a
pu e d ug solu ion o suspension.
The esul s o he dynamic ligh sca e ing (DLS) anal-
ysis and encapsula ion e iciency (EE%) calcula ions a e
p esen ed in Table 2.
In ag eemen wi h li e a u e da a, an inc ease in lip-
id concen a ion om 200 o 400 mg led o an inc ease
in he pa icle size and PDI o SLNs (Shah aeini e al.
2020; Vi o ino e al. 2011). The insu icien amoun o
Table 1. Solubili y o 3a in lipids.
Lipid Amoun o lipid equi ed* (mg)
s ea ic acid 369.3 ± 1.52
palmi ic acid 486.6 ± 2.64
glyce yl monos ea a e 219.7 ± 0.56
glyce yl palmi os ea a e 306.6 ± 1.14
*Da a a e exp essed as mean ± SD, n = 3.
Figu e 2. S anda d cu e o 3a in dis illed wa e wi h 10%
DMSO (mean ± SD; n = 3).
Figu e 3. S anda d cu e o 3a in bu e (pH 6.5) wi h 10%
DMSO (mean ± SD; n = 3).
Figu e 4. RSD% alues o he i e samples om h ee di e en
concen a ions o 3a, co esponding o 100%, 50%, and 125% o
he es sample amoun in dis illed wa e and bu e (pH 6.5).
Pha macia 72: 1–13 7
su ac an equi ed o emulsi y GMS was he p obable
eason o his p opo ional dependency. The a e age
pa icle size and PDI dec eased wi h inc easing Tween
80 con en om 100 o 300 mg. This obse a ion ma ches
he indings o o he au ho s who epo ed ha he size
o SLNs dec eases wi h inc easing su ac an concen a-
ion (Kuma e al. 2018). Du ing sonica ion, he size o
he d ople s is educed and a new su ace is gene a ed.
An inc ease in su ac an concen a ion ensu es s abili-
za ion o his newly o med su ace. The polydispe si y
index (PDI) indica es he pa icle size dis ibu ion, wi h
alues below 0.3 con i ming homogenei y and uni o mi-
y (Abousam a and Mohsen 2016). In he p esen s udy,
PDI alues below 0.3 we e obse ed in samples wi h he
lowes lipid and highes su ac an concen a ions o
bo h emp y and loaded pa icles. The ze a po en ials o
he emp y and loaded SLNs we e compa able, likely due
o hei simila su ace s uc u es and he o ma ion o
a su ac an laye a ound he lipid ma ix. The alues
anged om −16.30 o −25.30 mV. The nega i e cha ge
was p obably induced by he adso p ion o OH⁻ ions
om wa e on o he SLN su ace. Simila obse a ions
ha e been epo ed by o he esea che s (Ho and Ah-
mad 1999; Hsu and Nacu 2003; Abousam a and Mohsen
2016). Samples wi h a ζ-po en ial g ea e han 25 mV,
pa icula ly hose wi h highe su ac an concen a ions,
exhibi ed be e long- e m colloidal s abili y o bo h
emp y and loaded SLNs. The highes EE% was obse ed
in samples con aining he highes lipid concen a ion.
Since a lowe lipid concen a ion leads o smalle pa icle
size, d ug molecules end o escape om he lipid ma ix,
esul ing in lowe EE% (Singh e al. 2012). Pa ial expul-
sion o he ac i e subs ance can occu a lowe lipid con-
cen a ions du ing c ys alliza ion o he lipid phase. EE%
also dec eased wi h dec easing su ac an concen a ion,
likely due o pa icle coalescence caused by insu icien
Tween 80. This was con i med by he inc eased PDI al-
ues obse ed when su ac an concen a ion was educed
in loaded pa icles. SLNs wi h smalle mean pa icle sizes
can e ec i ely c oss he BBB, whe eas hose wi h a PDI
below 0.3 and a ze a po en ial abo e −25 mV a e ex-
pec ed o exhibi he g ea es long- e m s abili y. Based
on hese indings, samples 3a/1 (emp y) and 3a-SLN/1
(loaded) we e selec ed o u he in es iga ion.
T ansmission elec on mic oscopy
(TEM) in es iga ion
The TEM image p esen ed in Fig. 5 shows he sphe ical
shape and homogeneous shading o he nanopa icles
loaded wi h 3a. The pa icle size, anging om app oxi-
ma ely 50 o 60 nm, as con i med by TEM, is consis en
wi h he Ze asize measu emen s.
Di e en ial scanning calo ime y (DSC)
The DSC he mog am o 3a showed a sha p endo he mic
mel ing peak in he empe a u e ange o 150–160 °C,
wi h a maximum a 158 °C (Fig. 6). The peak o SLNs
appea ed be ween 50 °C and 80 °C, wi h a maximum a
75 °C. The 3a-SLNs peak exhibi ed highe in ensi y and
a b oade ange han ha o he emp y SLNs due o he
inco po a ion o 3a wi hin he lipid c ys al la ice. The
he mog am o 3a-SLNs did no show he mel ing peak o
3a, sugges ing ha 3a was dissol ed in he lipid ma ix and
exis ed in an amo phous s a e.
X- ay di ac ome ic analysis
In he X- ay di ac ion (XRD) pa e n o 3a, sha p peaks
a 2θ-sca e ing angles o 24.2°, 33.6°, 34.8°, and 35.9°
we e dis inc ly no iceable, indica ing i s high c ys al-
lini y (Fig. 7). The c ys alline na u e o he emp y SLNs
was e iden om peaks a app oxima ely 20.6°, 24.0°,
Table 2. Samples, o mula ion, and physicochemical p ope ies o emp y and 3a-loaded SLNs (mean ± SD; n = 3).
S № 3a (mg) GMS (mg) Tween 80 (mg) PS (nm) PDI Zp (mV) EE %
3a/1 – 200 300 47.96 ± 2.02 0.288 –25.30 ± 4.06 –
3a/2 – 300 300 63.67 ± 4.03 0.368 –23.23 ± 2.02 –
3a/3 – 400 300 99.17 ± 1.36 0.513 –21.18 ± 3.03 –
3a/4 – 200 100 78.16 ± 4.12 0.415 –16.30 ± 0.32 –
3a/5 – 200 200 59.87 ± 4.01 0.313 –20.23 ± 3.21 –
3a-SLN/1 10 200 300 56.23 ± 4.36 0.268 –25.16 ± 0.17 89.5 ± 3.65
3a-SLN/2 10 300 300 80.44 ± 0.02 0.467 –22.12 ± 2.11 92.6 ± 1.73
3a-SLN/3 10 400 300 103.24 ± 3.51 0.543 –21.11 ± 0.42 95.1 ± 2.34
3a-SLN/4 10 200 100 98.10 ± 6.87 0.473 –17.89 ± 4.27 83.6 ± 0.45
3a-SLN/5 10 200 200 78.56 ± 2.09 0.389 –21.25 ± 1.16 86.4 ± 3.21
S – Sample, GMS – glyce yl monos ea a e, PS – pa icle size, PDI – polydispe si y index, Zp – ze a po en ial, EE% – en apmen e iciency.
Figu e 5. TEM image o 3a-SLNs (sample 3a-SLN/1).
Voyche a C e al.: SLNs loaded wi h a no el indole-based mul i a ge ligand8
25.3°, and 25.6°. In he spec a o 3a-SLNs, he cha ac e -
is ic peaks o 3a we e absen , and he lipid peaks displayed
lowe in ensi y. This obse a ion sugges s ha 3a was suc-
cess ully inco po a ed in o he SLNs. The educed peak
in ensi y o 3a-SLNs was p obably due o he in e cala ion
o 3a wi hin he lipid c ys al la ice, which al e ed he c ys-
allini y o he loaded SLNs.
In i o d ug elease s udy
In i o elease o 3a om 3a-SLNs was compa ed wi h he
di usion o ee 3a ac oss he dialysis memb ane (Fig. 8).
The s udy was conduc ed in simula ed nasal luid (SNF;
pH 6.5). Nea ly 99% o ee 3a c ossed he dialysis ba -
ie wi hin 3 h. In con as , abou 38% o 3a was eleased
om 3a-SLNs du ing he i s 6 h o he dissolu ion es ,
ollowed by sus ained elease eaching 52% a 24 h. The
ini ial bu s elease was likely due o he p esence o 3a
nea he SLN su ace, whe eas he subsequen sus ained
elease esul ed om he ex ended di usion pa h h ough
he lipid ma ix. Simila elease p o iles ha e been epo -
ed o SLNs p epa ed by he nano- empla e enginee ing
echnique (Qu eshi e al. 2016, 2017; Riz i e al. 2019;
Sohail e al. 2023).
In i o pe meabili y assay
In i o pe meabili y assays a e essen ial in d ug esea ch
and de elopmen . Mos pe meabili y s udies simula ing
in anasal deli e y employ ex i o animal memb ane
models, such as sheep nasal mucosa, due o hei s uc-
u al simila i y o human mucosa (Sha ma e al. 2014).
Howe e , hese s anda d ex i o models o en exhibi
poo ep oducibili y and a iabili y in esul s ac oss is-
sue sou ces, labo a o ies, and esea che s (Hayeshi e al.
2008). Mo eo e , he use o animal memb anes aises
e hical conce ns and is ime-consuming, as issues mus
be incuba ed unde speci ic condi ions be o e es ing (Di
Cagno e al. 2015S). To ob ain mo e ep oducible esul s,
he p esen s udy employed Pe meaPad®, a non-cellula ,
phospholipid esicle-based pe mea ion assay (PVPA)
me hod, ins ead o ex i o models (Ma and Wu 2017; Ra-
hamim and Azagu y 2021). Pe meaPad® is a biomime ic
memb ane ha simula es passi e di usion ac oss a ious
biological ba ie s (gas oin es inal, buccal, and nasal) and
p o ides a mo e eliable assay because i s ba ie consis s
o phospha idylcholine liposomes (Leci hin S-100) im-
mobilized be ween ni ocellulose il e suppo s, mimick-
ing he s uc u e and geome y o biological memb anes
(Cagno and Baue -B andl 2019). Due o his unique de-
sign, he Pe meaPad® ba ie is obus , cos -e ec i e, easy
o use (wi hou p e ea men ), and esis an o a wide pH
ange (1–10) as well as o su ac an s and co-sol en s such
as DMSO (Bibi e al. 2015).
The F anz cell di usion sys em was used o analyze he
pe mea ion o 3a om 3a-SLNs (sample 3a-SLN/1), and
he di usion was compa ed wi h ha o ee 3a (3a dis-
pe sion). The s udy was conduc ed in SNF (pH 6.5), co -
esponding o he nasal en i onmen , o 12 h. The pe -
mea ion p o iles shown in Fig. 9 illus a e he di e ences
in 3a beha io be ween he wo o mula ions. The esul s
Figu e 6. DSC analysis o 3a, SLNs, and 3a-SLNs.
Figu e 7. X- ay di ac ome ic analysis o 3a, SLNs, and
3a-SLNs.
Figu e 8. In i o elease o 3a om 3a dispe sion and 3a-SLNs
(sample 3a-SLN/1) in pH 6.5 a 34 °C (mean ± SD; n = 3).
Pha macia 72: 1–13 9
clea ly demons a ed ha d ug anspo om 3a-SLNs
was signi ican ly highe (1.27- old) han om ee 3a.
This enhancemen could be a ibu ed o he in insic lipid
s uc u e o he SLNs ( esembling biological lipids), hei
small size, and he p esence o emulsi ie s, which acili a e
pene a ion h ough he Pe meaPad® memb ane due o i s
lipid composi ion simila o nasal mucosa. The indings
sugges ha he p epa ed 3a-SLNs could also p omo e he
pa i ioning o nanosized d ople s wi hin he nasal mu-
cosa, he eby inc easing esidence ime and enhancing
b ain d ug deli e y ollowing in anasal adminis a ion o
3a-SLNs.
The slowe elease o compound 3a om solid lipid
nanopa icles (SLNs) h ough a dialysis memb ane com-
pa ed wi h i s as e passage h ough Pe meaPad® can be
a ibu ed o se e al ac o s ela ed o he ma e ial p ope -
ies o he ba ie s and he SLN o mula ion.
The dialysis memb ane used o elease s udies is
ypically composed o a semipe meable ma e ial wi h a
de ined po e size (2.4 nm in his s udy) and molecula
weigh cu o (12,000–14,000 Da). This memb ane p i-
ma ily allows passi e di usion o ee d ug molecules
bu can es ic he passage o d ug-loaded nanopa icles
o la ge agg ega es, esul ing in slowe d ug elease. In
con as , Pe meaPad® is a phospholipid-based biomime ic
memb ane designed o simula e biological memb anes.
I s lipidic composi ion closely esembles ha o cellula
memb anes, enhancing he in e ac ion and di usion o
lipid-based ca ie s such as SLNs.
The SLN s uc u e also plays a signi ican ole in he
pe mea ion p ocess. In SLNs, he d ug (3a) is encapsu-
la ed wi hin a lipid ma ix. D ug elease occu s in wo
s ages—an ini ial bu s elease o su ace-associa ed d ug
ollowed by sus ained di usion h ough he lipid co e.
The lipid ma ix p o ides a con olled elease en i on-
men , educing he a e a which 3a becomes a ailable o
di usion h ough he dialysis memb ane. The sus ained
elease beha io o 3a-SLNs obse ed h ough he dialysis
memb ane is consis en wi h he ex ended di usion pa h
ha he d ug mus a e se wi hin he lipid ma ix be o e
eaching he elease medium.
In addi ion, Pe meaPad®, due o i s lipidic na u e,
may acili a e in e ac ion wi h SLNs, enhancing he pa -
i ioning and anspo o d ug-loaded nanopa icles o
eleased d ug. Con e sely, he dialysis memb ane lacks
his lipid compa ibili y, which may educe in e ac ion and
pe mea ion o bo h SLNs and he eleased d ug.
Pa icle size and su ace cha ac e is ics a e addi ional
ac o s con ibu ing o highe pe meabili y. The small size
(50–60 nm) and lipid-based composi ion o SLNs imp o e
hei in e ac ion wi h lipidic memb anes such as Pe mea-
Pad®. These cha ac e is ics may p omo e be e pa i ion-
ing in o he Pe meaPad® memb ane, enhancing he a e o
d ug elease and anspo .
In conclusion, he di e ences in he elease p o iles can
be explained by he con as ing mechanisms o in e ac-
ion and anspo h ough he espec i e memb anes,
wi h Pe meaPad® o e ing a mo e e icien model o sim-
ula ing biological memb ane in e ac ions.
S abili y s udies
No signi ican changes we e obse ed in pa icle size
du ing s o age a 4 ± 2 °C ( e ige a ed) o a oom em-
pe a u e (25 ± 2 °C, 60 ± 5% RH). Howe e , pa icle size
inc eased signi ican ly (p < 0.05) unde accele a ed condi-
ions (40 ± 2 °C, 75 ± 5% RH) due o agg ega ion (Table 3).
Ze a po en ial and polydispe si y index did no show
signi ican changes when he nanopa icle dispe sion was
s o ed a 4 ± 2 °C and 25 ± 2 °C, 60 ± 5% RH. A 40 ± 2 °C
and 75 ± 5% RH, he PDI inc eased. A possible explana-
ion o his obse a ion is he change in he ze a po en ial
alue om −25 mV in he ini ial mon h o −15 mV in
he hi d mon h and −6 mV in he six h mon h o s o -
age unde hese condi ions. This a ia ion migh be due
o pa ial dissolu ion o he lipid coa ing, which leads o
pa icle agg ega ion.
H₂O₂-induced oxida i e s ess model in
SH-SY5Y neu onal cells
To e alua e he neu op o ec i e po en ial o he newly
syn hesized compound 3a, SH-SY5Y cells we e p e ea -
ed wi h a ious concen a ions (0.1–50 µM) o ee 3a o
3a-SLNs (sample 3a-SLN/1) o 90 min, ollowed by ex-
posu e o 1 mM H₂O₂ o induce oxida i e s ess. Expo-
su e o H₂O₂ o 15 min signi ican ly educed cell iabili y
compa ed wi h un ea ed con ols (Fig. 10). P e ea men
wi h ee 3a p o ided only modes p o ec ion, signi ican
a 25 and 50 µM, p ese ing 9% and 16% o cell iabili y,
espec i ely (p < 0.05; p < 0.001 s. H₂O₂).
In con as , 3a-SLNs exhibi ed a concen a ion-dependen
neu op o ec i e e ec o e a b oade ange (1–50 µM), p e-
se ing cell iabili y by 7%, 9%, 17%, 26%, and 36%, espec-
i ely (p < 0.05; p < 0.01; p < 0.001 s. H₂O₂), and demons a -
ing signi ican ly s onge p o ec ion han he ee compound
Figu e 9. In i o pe meabili y o 3a om 3a dispe sion and
3a-SLNs (sample 3a-SLN/1) ac oss Pe meaPad® memb ane in
SNF (pH 6.5) o 12 h (mean ± SD; n = 3).
