Polypeptide-based multilayer capsules with anti-inflammatory properties: exploring different strategies to incorporate hydrophobic drugs

Ma e ials o biology and medicine
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Ma celo Calde ón, Ai o La añaga e al .
Polypep ide-based mul ilaye capsules wi h
an i-in lamma o y p ope ies: explo ing di e en
s a egies o inco po a e hyd ophobic d ugs
ISSN 2050-750X
Volume 13
Numbe 18
14 May 2025
Pages 5193–5484
This jou nal is © The Royal Socie y o Chemis y 2025 J. Ma e . Chem. B, 2025, 13, 5297–5314 | 5297
Ci e his: J. Ma e . Chem. B, 2025,
13, 5297
Polypep ide-based mul ilaye capsules wi h
an i-in lamma o y p ope ies: explo ing diffe en
s a egies o inco po a e hyd ophobic d ugs†
Ma ia Angela Mo a,
ab
Se gio Ma in-Saldan
˜a,
a
Ana Beloqui,
ac
Ma celo Calde o
´n *
ac
and Ai o La an
˜aga *
b
Mo e han 90% o d ug candida es used in he d ug de elopmen pipeline and abou 40% o d ugs on
he ma ke a e poo ly soluble in wa e based on he de ini ion o he biopha maceu ical classi ica ion
sys em. The ad en o d ug deli e y app oaches has ep esen ed a s iking ool o o e come he
challenges associa ed wi h he use o hyd ophobic d ugs, such as hei low bioa ailabili y and off- a ge
effec s. D ug ca ie o mula ions composed o biodeg adable and biocompa ible polyme s, such as
polypep ides, ha e been explo ed as pla o ms o hos poo ly wa e -soluble d ugs o p olong d ug
ci cula ion, enhance hei sa e y, educe hei immunogenici y, and p omo e hei con olled elease.
In his wo k, we e alua ed h ee s a egies—co-p ecipi a ion, pos -encapsula ion, and conjuga ion— o
inco po a e a hyd ophobic model d ug, i.e., cu cumin (CUR), in o biodeg adable mul ilaye capsules
ab ica ed ia a laye -by-laye (LbL) app oach. Poly(L-lysine) (PLys) and poly(L-glu amic acid) (PGlu) we e
adop ed as building blocks and al e na ely assembled on o calcium ca bona e (CaCO
3
) mic opa icles o
build a polypep ide-mul ilaye memb ane, which ac ed as a ba ie o con ol he elease o he d ug.
The applica ion o ou h ee o mula ions in in i o in lamma o y models o THP-1 de i ed human
mac ophages and mu ine mic oglia showed a educ ion o he in lamma ion wi h he supp ession o
h ee pi o al p o-in lamma o y cy okines (i.e., in e leukin (IL)-1b, IL-6, and umo nec osis ac o (TNF)-
a). Mo eo e , he in acellula elease o CUR de ec ed upon up ake s udies on ac i a ed mic oglia
sugges ed ha ou sys ems could ep esen a po en ial he apeu ic app oach o educe acu e
neu oin lamma ion and modula e mic oglia pheno ype.
1. In oduc ion
D ug deli e y sys ems based on biodeg adable polyme s ha e
ound di e se applica ions in o al, nasal, pa en e al, and ans-
de mal adminis a ion o small d ug molecules, p o eins, and
nucleic acids. Biodeg adable polyme s o syn he ic (e.g., poly-
es e s, polyanhyd ides, and polyamides) o na u al o igin (e.g.,
p o eins and polysaccha ides) ha e se e al ad an ages when
used o d ug deli e y applica ions due o hei biocompa ibil-
i y and deg ada ion in o ha mless by-p oduc s unde speci ic
condi ions.
1
Fo ins ance, poly(lac ide-co-glycolide) (PLGA),
which is app o ed by he Food and D ug Adminis a ion
(FDA), is widely used as a polyme ic ca ie because i deg ades
in o CO
2
and H
2
O, which a e non- oxic o he human body and
a e emo ed h ough he K ebs cycle.
2
Syn he ic polypep ides
a e ano he class o polyme s ha ha e been in es iga ed as
componen s o d ug deli e y sys ems due o hei simila i y
wi h p o eins, including sa e y, biocompa ibili y, biodeg ada-
ion, and low immunogenici y.
3
The co ne s one o polyme ic ca ie s is o imp o e he
pha macological p ope ies o he apeu ics, by ei he encapsu-
la ion o conjuga ion echniques. The encapsula ion s a egy is
e y simple and scalable, bu i usually suffe s om low loading
efficiencies and leakage o he payload om he ca ie s. As an
al e na i e, he co alen conjuga ion o a d ug o a polyme
ca ie ep esen s an effec i e s a egy o imp o e he solubili y
and he bioa ailabili y o he d ug, p olong he d ug ci cula ion,
enhance he sa e y, educe he immunogenici y, and p omo e he
con olled elease o he apeu ics.
4
Pa icula ly, syn he ic polypep-
ides, such as poly(L-lysine) (PLys), poly(L-glu amic acid) (PGlu),
a
POLYMAT, Applied Chemis y Depa men , Facul y o Chemis y, Uni e si y o he
Basque Coun y UPV/EHU, Paseo Manuel de La dizabal 3,
20018 Donos ia-San Sebas ia
´n, Spain. E-mail: [email p o ec ed]
b
Depa men o Mining-Me allu gy Enginee ing and Ma e ials Science, POLYMAT,
Bilbao School o Enginee ing, Uni e si y o he Basque Coun y (UPV/EHU),
Plaza To es Que edo 1, 48013 Bilbao, Spain. E-mail: [email p o ec ed]
c
IKERBASQUE, Basque Founda ion o Science, Plaza Euskadi 5, 48009 Bilbao,
Spain
†Elec onic supplemen a y in o ma ion (ESI) a ailable. See DOI: h ps://doi.o g/
10.1039/d4 b01906g
Recei ed 23 d Augus 2024,
Accep ed 27 h Ma ch 2025
DOI: 10.1039/d4 b01906g
sc.li/ma e ials-b
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and poly(L-aspa ic acid) (PAsp), which ha e al eady eached he
p e-clinical o clinical s age, ha e been widely used as polypep-
ide–d ug conjuga es hanks o hei con o ma ions (i.e.,a-helix,
b-shee , and andom coil), abili y o sel -assemble in e sa ile
a chi ec u es, high unc ionali y due o hei side chains, and
possibili y o ailo he esponsi eness o biological applica ions.
3
As an illus a ion, Vi agel
s
—a PLys-dend ime used as an an i-
bac e ial and an an i i al agen —and Copaxone
s
—a copolyme
o L-glu amic, L-alanine, L-lysine, and L- y osine agains mul iple
scle osis—ha e al eady been app o ed and eached he ma ke .
Addi ionally, PEG-block-copolyme s o PLys, PGlu, and PAsp a e
among he mos ad anced o mula ions used in clinical se ings.
3
Despi e he ou s anding ea u es o polypep ides, academic
esea ch is o ien ed owa ds he de elopmen o mo e ad anced
d ug deli e y sys ems o o e come he biological issues associa ed
wi h se e al diseases and educe he off- a ge effec s. Wi h
his pu pose, he laye -by-laye (LbL) echnique has been used
o ab ica e mul i unc ional d ug deli e y sys ems, which ha e
he unique p ope y o hos one o mul iple d ugs, s imuli-
esponsi e moie ies, and addi ional unc ionali ies o cell a -
ge ing in one single en i y.
5–11
The LbL app oach is a s a egy
ha has gained g ea in e es in he ield o d ug deli e y o he
ab ica ion o highly unc ionalized mul ilaye capsules by
assembling ei he syn he ic o na u al polyme s as building
blocks on o a empla e. The assembled polyme s can ac as a
ba ie memb ane, con olling he di usion o he payload and
limi ing acco dingly he ini ial bu s elease ha would cause he
elease o he d ug be o e eaching he a ge cells.
12,13
Polypep-
ides a e a ac i e LbL building blocks due o hei biocompa -
ibili y, biodeg adabili y, high unc ionali y, and high endency o
sel -assemble in o o ganized mul ilaye nanos uc u es.
14
One
o he mos commonly used empla es o pha maceu ical
applica ions is calcium ca bona e (CaCO
3
) in bo h he mic o-
and nano-me e scales, hanks o i s acile syn hesis, high
po osi y, biocompa ibili y, easy dissolu ion unde mild condi ions
(i.e., e hylenediamine e aace ic acid (EDTA) solu ion, pH o7.0),
and capaci y o hos molecules o di e se na u e, including
p o eins, g ow h ac o s, and small d ug molecules.
15–17
Fo
ins ance, lipid-coa ed CaCO
3
nanopa icles, syn hesized by a
e e sed mic oemulsion me hod,
18
ha e been epo ed o e i-
cien ly encapsula e a he apeu ic pep ide o lung cance ea -
men .
19
CaCO
3
a e i e pa icles ep esen a p omising al e na i e
o hyd ogels,
20
i al ec o s,
21
liposomes,
22
and micelles.
23
Speci-
ically, hey can be conside ed o sol e he challenges associa ed
wi h he deli e y o hyd ophobic d ugs, such as he sho esi-
dence ime in he body due o hei low solubili y in physio-
logical luids, and he low bioa ailabili y and apid clea ance
ha esul in educed e icacy.
24
The encapsula ion in o CaCO
3
pa icles can be pe o med using h ee main app oaches:
(i) adso p ion, (ii) in il a ion, and (iii) co-p ecipi a ion.
25,26
The adso p ion app oach elies on he di usion o he payload
o in e es in o he p e-syn hesized po ous pa icles. Depend-
ing on he size o he molecules, his ype o encapsula ion can
esul in e y low loading capaci ies and in an inhomogeneous
dis ibu ion o he ca go molecules due o he s e ic limi a-
ions wi h espec o he po e size (a e age o 20–60 nm).
27
In he in il a ion app oach, he loading o he molecules
occu s by dec easing hei solubili y o by sol en e apo a ion.
None heless, he co-p ecipi a ion is he mos elegan me hod
o load molecules in o CaCO
3
pa icles, allowing e y high
encapsula ion e iciencies and e y good dis ibu ion o
he loaded molecules.
28,29
This me hod in ol es he simul a-
neous encapsula ion o he payload and he assembly o he
pa icles.
In his wo k, we compa ed diffe en me hodologies o
inco po a e cu cumin (CUR) as a hyd ophobic model d ug in o
biodeg adable LbL mic ocapsules. In wo sys ems, we encapsu-
la ed CUR in o CaCO
3
mic opa icles by co-p ecipi a ion o
pos -encapsula ion. In a diffe en app oach, we conjuga ed
he d ug o one o he laye s in o he mul ilaye memb ane.
The polypep ides, PLys and PGlu, we e chosen o hei bio-
compa ibili y and biodeg adabili y as posi i ely and nega i ely
cha ged building blocks, espec i ely, o ab ica e a mul ilaye
memb ane on o he CaCO
3
mic opa icles ia he LbL app oach.
The LbL sys ems con aining CUR in he co e we e compa ed in
e ms o encapsula ion efficiency, d ug loading, and d ug elease
unde neu al (i.e., pH 7.4) and acidic (i.e.,pH5.0)condi ionsand
in he p esence o p o eoly ic enzymes. Howe e , hese sys ems
may cause some conce ns abou he e en ion o he d ug du ing
he nume ous s eps o he LbL p ocess and he dissolu ion o he
CaCO
3
mic opa icles. Indeed, She e al. epo ed ha encapsu-
la ing a basic ib oblas g ow h ac o in o CaCO
3
mic opa icles
led o a o al loss o 57% o he encapsula ed p o ein a e he
deposi ion o six-laye s and he co e dissolu ion.
17
As an al e -
na i e o he encapsula ion o CUR, we de eloped a new LbL
sys em whe e CUR was included in he mul ilaye memb ane
a e being conjuga ed o PGlu (CUR–PGlu). This app oach was
expec ed o (i) enhance he solubili y o he d ug, (ii) con ol he
d ug loading quan i y in o he capsules, and (iii) p omo e he
elease o he d ug concomi an ly wi h he disassembly o
he mul ilaye nanos uc u e. Fu he mo e, aking ad an age o
he epo ed pleio opic p ope ies o CUR (e.g., an i- umo al,
an i-oxidan , an i-bac e ial, an i-in lamma o y, e c.),
30
ou LbL-
capsules, ob ained a e co e emo al, we e es ed in wo in i o
in lamma o y models o mac ophages de i ed om human mono-
cy es (THP-1) and mu ine mic oglia (BV-2) o highligh hei
cy ocompa ibili y and he in lamma o y esponse o he cells in
he p esence o he CUR-loaded mic ocapsules.
2. Ma e ials and me hods
2.1 Ma e ials
Calcium chlo ide anhyd ous (CaCl
2
), e hylenediamine e aace-
ic acid disodium sal dehyd a e (EDTA), poly(sodium 4-s y ene
sul ona e) (PSS, molecula weigh MWB70 kDa), sodium
ca bona e (Na
2
CO
3
), sodium chlo ide (NaCl), po assium chlo ide
(KCl), calcium chlo ide dihyd a e (CaCl
2
2H
2
O), magnesium
chlo ide hexahyd a e (MgCl
2
6H
2
O), sodium phospha e mono-
basic monohyd a e (NaH
2
PO
4
H
2
O), sodium phospha e dibasic
hep ahyd a e (Na
2
HPO
4
7H
2
O), phospha e buffe saline (PBS),
1-e hyl-3-(3-dime hylaminop opyl)ca bodiimide hyd ochlo ide
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(EDCHCl), is(hyd oxyme hyl)aminome hane hyd ochlo ide
(TRIS), pho bol 12-my is a e 13-ace a e (PMA), lipopolysaccha -
ide (LPS), Hank’s Balanced sal solu ion (HBSS), T i on X-100,
Tween 20, bo ine se um albumin (BSA), Flu oshield wi h 40,6-
diamidino-2-phenylindole (DAPI), and cu cumin (CUR) we e
pu chased om Sigma-Ald ich (Spain). 4-(2-Hyd oxye hyl)-1-
pipe azinee hanesul onic acid buffe 1 M (HEPES) a pH 7.3
was pu chased om Al a Aesa . 4-Dime hylaminopy idine (DMAP)
and d y N,N-dime hyl o mamide (DMF) we e pu chased om
Ac os O ganics. N-Bu yl-poly(L-lysine hyd ob omide) (PLys, MW=
39.9 kDa) and n-bu yl-poly(L-glu ama e sodium sal ) (PGlu, MW=
30.6 kDa) we e pu chased om Polypep ide The apeu ic Solu ions
S. L. (Spain). Dichlo ome hane (DCM), die hyl e he , sodium dode-
cyl sul a e (SDS), Dulbecco’s modi ied Eagle’s medium (DMEM),
RPMI 1640 medium, e al bo ine se um (FBS), penicillin–
s ep omycin solu ion (P/S), alama Blue
s
cell iabili y eagen ,
Quan -iT PicoG een dsDNA assay ki , e ame hyl hodamine
iso hiocyana e (TRITC)-phalloidin, 16% o maldehyde solu ion
(w/ ), he G iess eagen ki , and Human ma ix me allop o einase
(MMP)-2 Recombinan P o ein (Pep oTech
s
) we e pu chased
om The moFishe Scien i ic (Spain). DuoSe ELISA ki s we e
pu chased om R&D Sys ems (UK). Human monocy es isola ed
om an acu e monocy ic leukemia pa ien we e acqui ed
om ATCC, whe eas mu ine mic oglia (BV-2) om AcceGen
Bio ech (USA).
2.2 Encapsula ion o cu cumin in o he CaCO
3
mic opa icles
2.2.1 Encapsula ion o cu cumin by adso p ion. The encap-
sula ion o CUR by adso p ion, named he e pos -encapsula ion,
was pe o med a e he syn hesis o he unloaded CaCO
3
mic o-
pa icles (i.e., empla e) o ob ain POST-T. B ie ly, 1.5 mL o 0.2 M
CaCl
2
solu ion was pou ed in o 1.5 mL o 0.2 M Na
2
CO
3
solu ion
con aining 4 mg mL
1
PSS. The mix u e was s i ed o 1 min
a high speed, and he empla e was allowed o p ecipi a e o
30 min. A e wa ds, he empla e was collec ed by cen i uga ion
(18800 g o 2 min) and washed h ee imes wi h a 5 mM NaCl
solu ionanddis illedwa e .TheCUR solu ion was added ei he
d opwise o di ec ly o he empla e. In b ie , CUR (1 mg) was
dissol ed in 0.5 mL o e hanol, and he solu ion was added o
he empla e(30mg)suspendedin0.5mLo dis illedwa e .
Then, he empla e was incuba ed o 30 min shel e ed om ligh .
A e cen i uga ion (18800 g o 2 min), he supe na an was
collec ed o de e mine he encapsula ion efficiency (EE%)
(eqn (1)) and he loading capaci y (LC%) (eqn (2)) by measu ing
he abso bance (Pe kinElme PDA Lambda 265 spec opho -
ome e , l= 427 nm). A e wa ds, he empla e was washed once
wi h a 5 mM NaCl solu ion o dis illed wa e and s o ed o
u he cha ac e iza ion. All he s eps o syn hesis we e ca ied ou
a oom empe a u e.
EE %ðÞ¼ CUR en apped ðweigh Þ
Ini ial amoun o CUR ðweigh Þ100%(1)
LC %ðÞ¼ CUR en apped ðweigh Þ
Amoun o mic opa icles ðweigh Þ100%(2)
2.2.2 Encapsula ion o cu cumin by co-p ecipi a ion. The
CUR-loaded CaCO
3
mic opa icles by co-p ecipi a ion (i.e.,
COPRE-T) we e syn hesized by using wo app oaches. B ie ly,
CUR was dissol ed in e hanol and hen d opwise o di ec ly
added o a 0.2 M CaCl
2
solu ion wi h diffe en e hanol- o-wa e
a ios. In he op imized app oach, CUR (1 mg) was dissol ed in
0.3 mL o e hanol and di ec ly added o 1.5 mL o a 0.2 M CaCl
2
solu ion. This mix u e was pou ed o 1.5 mL o a 0.2 M Na
2
CO
3
solu ion con aining 4 mg mL
1
PSS a high s i ing speed o
1 min. The o med mic opa icles we e allowed o p ecipi a e
o 30 min shel e ed om ligh . COPRE-T we e cen i uged
and he supe na an was collec ed o calcula e he alues o
EE% (eqn (1)) and LC% (eqn (2)) by UV-Vis measu emen s
(Pe kinElme PDA Lambda 265 spec opho ome e , l=
427 nm). Then, COPRE-T we e washed once wi h a 5 mM NaCl
solu ion o dis illed wa e and s o ed o u he cha ac e i-
za ion. All he s eps o syn hesis we e ca ied ou a oom
empe a u e.
2.3 Conjuga ion o cu cumin o poly(L-glu amic acid)
PGlu (20 mg) was dissol ed in 1 mL o d y DMF unde a N
2
a mosphe e. EDCHCl (5.3 mg, 0.20 eq.) and DMAP (2.4 mg,
0.14 eq.) dissol ed in DMF we e added, and he mix u e was
s i ed o 20 min o ac i a e he ca boxyla e g oups. A e -
wa ds, CUR (3.5 mg, 0.07 eq., dissol ed in d y DMF) was added
o he mix u e. The mix u e was le s i ing o e nigh shel e ed
om ligh . Then, 10 mL o dis illed wa e was added o he
mix u e, and he un eac ed eagen s we e emo ed by ex ac-
ion wi h DCM (3) and die hyl e he (2). CUR–PGlu in he
aqueous phase was pu i ied using a Sephadex column, lyophi-
lized and s o ed a 20 1C o u he use (yield = 45%).
2.4 Fab ica ion o LbL mic ocapsule o mula ions
2.4.1 Syn hesis o unloaded and CUR-loaded (PLys/PGlu)
3
mic ocapsules. PLys and PGlu solu ions (1 mg mL
1
) we e
p epa ed in 25 mM HEPES/20 mM NaCl buffe solu ion a pH
6.5. S a ing om PLys, he polypep ide solu ions we e al e -
na ely adso bed on he su ace o he unloaded empla e, o
POST-T, o COPRE-T o 15 min o o m he bilaye PLys/PGlu.
A e each deposi ion cycle, he mic opa icles we e cen i uged
(18800 g o 2 min) and he non-adso bed polypep ide was
emo ed by washing once wi h a 5 mM NaCl solu ion. The
z-po en ial was measu ed o de e mine he su ace cha ge o
he coa ed mic opa icles. Following his p ocedu e, he mic o-
pa icles we e coa ed by h ee (PLys/PGlu) bilaye s. Finally,
he co e was dissol ed by incuba ing each o he coa ed mic o-
pa icles wi h a 0.1 M EDTA solu ion o 5 min o ob ain
unloaded mic ocapsules (i.e., CPS) and CUR-loaded mic o-
capsules (i.e., COPRE-cps and POST-cps). Then, he capsules
we e washed once wi h a 5 mM NaCl solu ion o dis illed wa e .
2.4.2 Syn hesis o (PGlu/PLys)(CUR–PGlu/PLys)(PGlu/PLys)
mic ocapsules. The LbL mic ocapsules named CONJ-cps we e
ab ica ed s a ing om he unloaded empla e acco ding o
he LbL p o ocol epo ed abo e, whe e he PGlu o he ou h
laye was subs i u ed by CUR–PGlu.
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2.5 S abili y s udies o LbL mic ocapsules
The s abili y o CPS was es ed in diffe en media a pH 7.4 and
37 1C a wo diffe en ime poin s (i.e., 6 and 24 h). B ie ly,
mic ocapsules (abou 3 mg) we e incuba ed wi h 0.2 mL o (i)
0.1 M TRIS, (ii) 0.1 M TRIS + 0.1% o 0.5% SDS, (iii) 0.1 M TRIS +
0.1% o 0.5% SDS + 2 mg mL
1
p onase buffe solu ions, and
(i ) a i icial ce eb ospinal luid (ACSF) in he p esence o
absence o 0.1% BSA + MMP-2 a diffe en concen a ions
(i.e., 0, 200, 500, and 1000 ng mL
1
). A he selec ed ime
poin s, he capsules we e collec ed by cen i uga ion (18800 g
o 2 min) and washed once wi h dis illed wa e be o e u he
cha ac e iza ion.
2.6 In i o elease o cu cumin om LbL mic ocapsule
o mula ions
The in i o elease o CUR om each o mula ion was de e -
mined unde acidic and neu al en i onmen s and in he
p esence o a mix u e o p o eoly ic enzymes (i.e., p onase) a
37 1C. In b ie , he CUR o mula ions we e edispe sed in 0.2
mL o (i) 0.1 M ace a e buffe con aining 0.1% SDS a pH 5.0 o
(ii) 0.1 M TRIS con aining 0.1% SDS a pH 7.4 o (iii) 0.1 M TRIS
con aining 0.1% SDS and 2 mg mL
1
p onase a pH 7.4. The
elease o CUR unde he espec i e condi ions was assessed
e e y 15 min o he i s 6 h and hen e e y 1 h o 6 h o each
18 h. A he es ablished ime poin s, he mic ocapsules we e
cen i uged (18800 g o 3 min), and he supe na an was
collec ed o measu e he abso bance o he CUR eleased (l=
430 nm o COPRE-cps and POST-cps, and l= 427 nm o CONJ-
cps) using a mic opla e eade (BioTek Syne gy H1M). Then,
esh medium was added o he pelle o u he incuba ion
imes. The elease s udies we e conduc ed in iplica e o each
condi ion, and a cumula i e elease o CUR was de e mined
o e 18 h (eqn (3)).
Cumula i e elease %ðÞ¼
Amoun o CUR eleased ðweigh Þ
Amoun o CUR loaded ðweigh Þ100
(3)
2.7 Physico-chemical and mo phological cha ac e iza ion
The size dis ibu ion o he unloaded empla e, COPRE-T, and
POST-T was de e mined using a lase sca e ing pa icle size
dis ibu ion analyze (HORIBA LA-350) a oom empe a u e.
The c ys allog aphic s uc u e o he unloaded empla e,
COPRE-T and POST-T was de e mined by X- ay diff ac ion
analysis (XRD). The analysis was pe o med by means o a
PHILIPS X’PERT PRO au oma ic diff ac ome e ope a ing a
40 kV and 40 mA, in a he a– he a con igu a ion, a seconda y
monoch oma o wi h Cu-Ka(l= 1.5418 Å) and a PIXcel solid
s a e de ec o .
The su ace cha ge o he unloaded empla e, COPRE-T,
POST-T, and he co esponding mul ilaye mic opa icles a e
each deposi ion s ep was measu ed using a Mal e n Ins u-
men Ze asize (ZEN 3690) o a minimum o en uns. The
z-po en ial was de e mined o mic opa icles dispe sed in a
5 mM NaCl solu ion a oom empe a u e. The mean alue and
s anda d de ia ion (mean SD) o he da a we e calcula ed
om h ee measu emen s.
The unloaded empla e and unloaded LbL mic opa icles/
capsules we e cha ac e ized by a enua ed o al e lec ion-
Fou ie ans o m in a ed spec oscopy (ATR-FTIR) using a
Nicole AVATAR 370 spec ome e . Spec a we e aken wi h a
esolu ion o 2 cm
1
and a e aged o e 64 scans.
The mo phological analysis o he unloaded empla e,
COPRE-T, POST-T, he co esponding LbL mic opa icles/cap-
sules, and he CPS moni o ed du ing he s abili y s udies was
pe o med by means o scanning elec on mic oscopy (SEM,
Hi achi FEG-SEM S-4800) wi h an accele a ion ol age o 5.0 kV.
Be o e aking he images, he samples we e coa ed wi h a 10 nm
laye o gold using an Emi ech K550X ion spu e .
Nuclea magne ic esonance (NMR) spec oscopy and high-
pe o mance liquid ch oma og aphy (HPLC) we e used o cha -
ac e ize he conjuga ed compound CUR–PGlu. In he case o
NMR spec oscopy, CUR–PGlu was dissol ed in deu e a ed
wa e (D
2
O), and
1
H-NMR spec a we e acqui ed (128 scans,
64 s o elaxa ion ime) a oom empe a u e using a B uke
Ad ance DPX 300 spec ome e wi h 300 MHz o esonance
equency. HLPC measu emen s we e ca ied ou using a HPLC
Nexe a Li e sys em (Shimadzu), equipped wi h a SIL-40C au o-
sample (Shimadzu), an OHpak SB-803 HQ column (8.0 
300 mm), and a SPD-M40 pho o diode a ay de ec o (Shimadzu).
The samples analyzed by HPLC we e dissol ed in PBS ( low a e
0.5 mL min
1
,251C, l= 220 and 427 nm).
2.8 In i o cellula s udy
2.8.1 Cell cul u e. THP-1 monocy es and BV-2 cell lines
we e cul u ed in RPMI 1640 medium and DMEM, espec i ely,
supplemen ed wi h 10% FBS and 1% P/S. The cells we e
incuba ed a 37 1C unde a 5% CO
2
a mosphe e in a humidi ied
incuba o .
2.8.2 Me abolic ac i i y, cell iabili y, and in lamma o y
esponse o THP-1 de i ed mac ophages in he p esence o
LbL mic ocapsule o mula ions. THP-1 cells (2.5 10
4
cells pe
well) we e seeded in 24-well pla es wi h 100 ng mL
1
PMA
condi ioned media o 24 h o induce he diffe en ia ion o
THP-1 monocy es in o mac ophages. Then, he condi ioned
medium was e eshed wi h RPMI medium o 24 h o allow
he cul u e o es . The cells we e subsequen ly ea ed wi h
100 ng mL
1
LPS and/o diffe en concen a ions (i.e., 10, 100,
1000 capsules pe cell) o CPS and each CUR o mula ion in
RPMI medium o 24 h o de e mine he effec on he acu e
phase o in lamma ion. A e wa ds, he supe na an s we e
collec ed and s o ed a 20 1C o u he analysis. The me a-
bolic ac i i y was assessed using an alama Blue
s
assay.
In b ie , 10% alama Blue
s
in RPMI medium was added o
he cul u e and incuba ed o 4 h a 37 1C (5% CO
2
, humidi y),
and i s educ ion was de e mined using a mic opla e eade
(BioTek Syne gy H1M, l
ex
= 560 nm, l
em
= 590 nm, oom
empe a u e). Fu he mo e, he dsDNA con en was quan i ied
by using he Quan -iT PicoG een dsDNA assay ki . B ie ly,
a known olume o MilliQ wa e was added o he cell cul u e
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a e he alama Blue
s
assessmen . The samples we e hen sub-
jec ed o a leas h ee cycles o eeze– haw and he PicoG een
assay was pe o med ollowing he manu ac u e ’s p o ocol. Bo h
analyses we e pe o med a leas in iplica e (NZ3) and he a io
o me abolic ac i i y pe dsDNA con en was calcula ed.
The esponse o THP-1 diffe en ia ed mac ophages o wo
diffe en concen a ions o CPS, COPRE-cps, POST-cps, and
CONJ-cps (i.e., 100 and 1000 capsules pe cell) was measu ed
using an ELISA acco ding o he manu ac u e ’s speci ica ion.
In b ie , h ee pi o al p o-in lamma o y cy okines (i.e., umo
nec osis ac o (TNF)-a, in e leukin (IL)-1b, and IL-6) we e
quan i ied in he eco e ed cul u e media a e he ea men .
The elease o he cy okines was assessed a leas in iplica e
(NZ3).
2.8.3 Me abolic ac i i y and in lamma o y esponse o BV-2
cells in he p esence o LbL mic ocapsule o mula ions. BV-2
cells (5 10
4
cells pe well) we e seeded in a 48-well pla e, and
any po en ial de imen al effec o CPS, COPRE-cps, POST-cps,
and CONJ-cps was assessed in he p esence o absence o LPS
ea men , ollowing a p e iously epo ed p o ocol.
31
B ie ly,
he cells we e incuba ed o e nigh wi h eshly p epa ed LbL
mic ocapsules a h ee diffe en concen a ions (i.e., 10, 100,
and 1000 capsules pe cell in 250 mL o DMEM). A e wa ds,
250 mL o DMEM con aining LPS was added o he cells ( inal
LPS concen a ions o 20 ng mL
1
o ac i a ed mic oglia and
0ngmL
1
o es ing mic oglia) and incuba ed o 24 h a 37 1C
(5% CO
2
, humidi y). Cells cul u ed in he absence o LbL
mic ocapsules and LPS we e used as he nega i e con ol
(i.e., heal hy), whe eas he ones cul u ed in he absence o
LbL mic ocapsules bu in he p esence o LPS we e conside ed
as he LPS con ol. A e 24 h, he condi ioned media we e
collec ed and used o de e mine he concen a ion o ni i es
and TNF-asec e ed by he cells. The me abolic ac i i y was
de e mined by incuba ing he cells wi h 10% alama Blue
s
in
DMEM a 37 1C (5% CO
2
, humidi y) o 3 h shel e ed om ligh .
The luo escence in ensi y (l
ex
= 560 nm and l
em
= 590 nm) o
each well was measu ed using a mic opla e eade (BioTek
Syne gy H1M) a oom empe a u e. Fo he con ol g oups
and each concen a ion o LbL mic ocapsules, ou echnical
eplica es we e assessed.
The con en o ni i es in he supe na an s was measu ed
using a G iess eagen ki , ollowing he p o ocol p o ided by
he supplie (abso bance 548 nm, oom empe a u e). The TNF-a
eleased by cells was quan i ied by means o an ELISA, ollowing
he p o ocol p o ided by he supplie . Bo h analyses we e pe -
o med in iplica e.
2.8.4 Up ake o cu cumin-loaded LbL mic ocapsules by
BV-2 cells. Theup akeo COPRE-cps,POST-cps,andCONJ-cps
was ca ied ou on es ing and LPS-ac i a ed mic oglia. B ie ly,
BV-2 cells (2 10
4
cells pe well) we e seeded in a 24-well pla e
andallowed oadhe e o 6h.Toac i a e he mic oglia, BV-2 cells
we e ea ed wi h 20 ng mL
1
LPS o 18 h. Then, bo h es ing and
ac i a ed BV-2 cells we e incuba ed a 37 1C(5%CO
2
,humidi y)
wi h COPRE-cps, POST-cps, and CONJ-cps (100 capsules pe cell)
o 24 h. A e wa ds, he medium was aspi a ed, and he cells
we e washed wi h HBSS and ixed wi h 4% pa a o maldehyde o
5 min. Fo immunos aining, he cells we e washed wi h HBSS and
pe meabilized wi h 0.5% T i on X-100 in PBS o 10 min. The cells
we e washed wice wi h PBS and incuba ed wi h TRITC-Phalloidin
solu ion in 1% BSA o 15 min. Finally, he cells we e washed
wice wi h PBS-T (i.e., 0.1% Tween 20 in PBS) and once wi h PBS
by incuba ing 5 min unde sligh agi a ion. Images o he up ake
o he LbL mic ocapsules by BV-2 cells we e aken using a con ocal
mic oscope (Zeiss LSM800, 63) om h ee echnical eplica es.
The nuclei we e s ained wi h DAPI by applying a d op o
Flu oshield moun ing medium on a glass slide.
2.9 S a is ical analysis
All quan i a i e da a ela ed o he ab ica ion and he cha ac-
e iza ion o LbL mic ocapsule o mula ions a e ep esen ed as
he mean SD. Fo he in i o s udies, a leas h ee echnical
eplica es (NZ3) we e used, and he esul s a e indica ed as
he mean SD. To p ocess he da a o he cell expe imen s,
one-way analysis o a iance (ANOVA) ollowed by he Dunne
pos hoc es was used o compa ison wi h he con ol g oups
(po0.05; po0.01, po0.00005; po0.00001).
3. Resul s and discussion
3.1 Inco po a ion o cu cumin in o he calcium ca bona e
mic opa icles
CaCO
3
a e i e pa icles ep esen p omising ino ganic bioma-
e ials o applica ions in d ug deli e y hanks o hei biocom-
pa ibili y, po osi y, high d ug loading capaci y, and p ese a-
ion o he p ope ies o he payload.
26,32,33
Molecules o di e se
na u e can be inco po a ed in o he pa icles, whose s abili y
may be enhanced by he p esence o addi i es. Fo ins ance, he
s abili y o he CaCO
3
a e i e pa icles in non-pola media has
made hem good candida es o he inclusion o hyd ophobic
d ugs.
15,32,34
In his wo k, CUR, used as a hyd ophobic model d ug, was
inco po a ed in o CaCO
3
mic opa icles h ough wo diffe en
app oaches: co-p ecipi a ion and pos -encapsula ion. In he
co-p ecipi a ion s a egy, CUR was encapsula ed du ing he
o ma ion o he CaCO
3
mic opa icles, whe eas in he pos -
encapsula ion app oach he loading was ca ied ou a e hei
syn hesis by adso p ion.
As CUR is poo ly soluble in wa e and he syn hesis o he
mic opa icles is pe o med in aqueous solu ion, an op imiza-
ion o he encapsula ion p ocedu e was ca ied ou o achie e a
high loading efficiency and a con ol on he mo phology o he
mic opa icles. In he case o he co-p ecipi a ion app oach,
wo diffe en pa ame e s, i.e., he olume o e hanol used as co-
sol en o dissol e CUR and he speed o i s addi ion o he
CaCl
2
solu ion, we e e alua ed o he syn hesis o he mic o-
pa icles. Speci ically, when 1 mL o CUR in e hanol was mixed
wi h 0.5 mL o 0.2 M CaCl
2
ia di ec o d opwise addi ion, he
inal CaCO
3
mic opa icles we e cha ac e ized by mo e o
less de ined lowe -like shapes, espec i ely (Fig. 1(A) ad (B)).
Fu he mo e, no signi ican changes we e obse ed in he encap-
sula ion efficiency ha was abou 70% in bo h cases. Con a ily,
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bydec easing he olumeo e hanolin hemix u eo CUR/CaCl
2
,
he SEM images showed pa icles wi h sphe ical mo phology
(Fig. 1(C) and (D)). Speci ically, when CUR was dissol ed in
0.5 mL o e hanol and di ec ly added o 1 mL o 0.2 M CaCl
2
,
inal CaCO
3
mic opa icles wi h a spongy su ace we e obse ed in
SEM (Fig. 1(C)). When CUR in 0.3 mL o e hanol was di ec ly
added o 1.5 mL o 0.2 M CaCl
2
, he inal CaCO
3
mic opa icles
appea ed po ous bu smoo he han he p e ious ones (Fig. 1(D)).
Despi e he diffe en mo phologies o he syn hesized pa icles,
he XRD peaks e ealed he o ma iono pu e a e i epa icles
(Fig. S1, ESI†). The e o e, we hypo hesized ha he olume o
e hanol could al e he mo phology o he mic opa icles wi hou
affec ing hei c ys allini y s a e. T adi ionally, equimola concen-
a ions o CaCl
2
and Na
2
CO
3
in equal olumes lead o he
o ma ion o iso opic CaCO
3
pa icles. The syn hesis is d i en
by nuclea ion, g ow h, and agg ega ion o calcium ca bona e
g ains a he mic oscale.
35
In a epo ed s udy, i was co obo a ed
ha he ini ial sal concen a ion had a s ong impac on he
nuclea ion p ocess o he CaCO
3
pa icles, allowing he gene a ion
o sphe ical pa icles when an equilib ium be ween ions was
eached.
36
Howe e , he addi ion o diffe en addi i es, such as
mac omolecules, di alen ca ions, o o ganic sol en s, affec s he
mo phology o he CaCO
3
mic opa icles.
33
In pa icula , in a
epo ed s udy, i was s a ed ha e hanol, isop opanol, and
die hylene glycol could affec he c ys al g ow h a e and s abilize
he a e i e c ys als p e en ing he ans o ma ion in o calci e
c ys als.
37
The diffe en mo phologies (non-sphe ical) o he
pa icles a e epo ed o be caused by he inhomogenei y o he
sol en alcohol–wa e mix u e and he p e e en ial sol a ion o
he ions.
38
In ac , as CO
32
ions a e p e e en ially sol a ed by
wa e and Ca
2+
ions bo h by wa e and e hanol, a compa men a-
lized wa e - ich mic ophase is o med in luencing he shape and
size o he p ima y CaCO
3
pa icles in he i s phase o he
eac ion.
38
In ou wo k, we could obse e ha he educ ion o he
olume o e hanol o he minimum amoun o dissol e he
hyd ophobic molecules o CUR (i.e., 0.3 mL) allowed o ob ain
sphe ical-like and s able CaCO
3
mic opa icles wi h high encap-
sula ion e iciency (Table 1). The speed o addi ion o CUR did no
ha e a ele an impac on he mo phology o he pa icles and on
he encapsula ion e iciency. Howe e , i is no excluded ha he
c ys als o CUR o med du ing he syn hesis could in luence he
p ocess o he o ma ion o he pa icles.
In he case o he pos -encapsula ion app oach, he p e-
syn hesized mic opa icles we e esuspended in dis illed wa e
and incuba ed wi h CUR dissol ed in e hanol in a inal / a io
o 1:1. The d opwise o di ec addi ion o CUR o he mic o-
pa icles led o a diffe ence o 20% in he encapsula ion
efficiency, esul ing in a highe alue in he case o he d op-
wise addi ion (Table 1). The p ese a ion o he sphe ical-like
shape o he o iginal mic opa icles a e he incuba ion wi h
CUR in e hanol was co obo a ed by he SEM images (Fig. 1(E)
and (F)), and he s abili y o he a e i e c ys als was u he
con i med by he XRD peaks (Fig. S1, ESI†).
O e all, by compa ing he co-p ecipi a ion and pos -
encapsula ion s a egies, he diffe ences in he encapsula ion
efficiencies shown in Table 1 may be due o he diffe en
mechanisms o inclusion o CUR in o he CaCO
3
mic opa i-
cles. Indeed, he en apmen o CUR du ing he o ma ion o
he mic opa icles in he case o he co-p ecipi a ion app oach
de e mined a highe encapsula ion efficiency compa ed o he
pos -encapsula ion app oach, whe e he CUR molecules migh
simply diffuse in and ou h ough he po es o he pa icles.
The e o e, his aspec migh in luence he successi e elease
s udies o CUR.
3.2 Conjuga ion o cu cumin o poly(L-glu amic acid)
The poo wa e -solubili y o CUR is one o he d awbacks ha
limi i s use as a d ug, despi e i s excellen p ope ies, including
Fig. 1 SEM images showing he mo phology o he cu cumin (CUR)-loaded calcium ca bona e (CaCO
3
) mic opa icles syn hesized du ing he
op imiza ion o he p ocedu e. (A) and (B) Flowe -like CaCO
3
mic opa icles ob ained by he co-p ecipi a ion app oach a e di ec ly (A) o d opwise
(B) adding CUR (1 mg) dissol ed in he mix u e e hanol/0.2 M calcium chlo ide (CaCl
2
) (1 mL/0.5 mL) o 1.5 mL o a 0.2 M sodium ca bona e (Na
2
CO
3
)
solu ion. (C) and (D) Sphe ical-like CaCO
3
mic opa icles ob ained by pou ing he mix u e composed o CUR (1 mg) in 0.5 mL (C) o in 0.3 mL (D) o
e hanol and 1 mL o 1.5 mL o a 0.2 M CaCl
2
solu ion, espec i ely, o 1.5 mL o a 0.2 M Na
2
CO
3
solu ion. (E) and (F) Sphe ical-like CaCO
3
mic opa icles
syn hesized by he pos -encapsula ion app oach, esuspending he p e-syn hesized CaCO
3
mic opa icles (30 mg) in dis illed wa e and adding di ec ly
(E) o d opwise (F) CUR (1 mg) dissol ed in 0.5 mL e hanol. The scale ba is 3 mm.
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an i-cance , an i-oxidan , and an i-in lamma o y effec s.
39
The
conjuga ion o polyme s o o m polyme –d ug conjuga es
o e s he ad an age o enhance he solubili y, bioa ailabili y,
and plasma hal -li e o hyd ophobic d ugs. P io wo ks ha e
epo ed he conjuga ion o CUR o PGlu o amino acids.
40–43
Fo ins ance, s a -shaped polyglu ama e conjuga es o bisde-
me hoxycu cumin ha e shown ex ended blood ci cula ion
imes, exhibi ing a po en ial neph op o ec ion agains acu e
kidney inju y
42
and neu op o ec ion agains Alzheime ’s
diseases.
43
In his wo k, we conjuga ed CUR o PGlu o enhance
i s solubili y, included i in a d ug deli e y o mula ion, and
explo e i s po en ial an i-in lamma o y p ope ies. Speci ically,
he conjuga ion o CUR o PGlu was ca ied ou ia an es e
bond by means o an es e i ica ion eac ion o ob ain CUR–
PGlu wi h an o e all yield a e pu i ica ion o 45% (Fig. 2).
A e he pu i ica ion s ep, CUR–PGlu was cha ac e ized by
1
H-NMR spec oscopy, whe e, in addi ion o he ypical peaks o
PGlu aken as a e e ence, he peaks in he ange o 2.8–3.3 ppm
and 6.8–7.9 ppm we e a ibu ed o CUR (Fig. S2A, ESI†). The
UV-Vis spec a o CUR–PGlu we e cha ac e ized by a sligh shi
o he abso p ion band (420 nm) wi h espec o he abso bance
o CUR (l= 427 nm) p obably due o he conjuga ion o PGlu
(Fig. S2B, ESI†). Addi ionally, in he ch oma og am ob ained
by HPLC analysis, a main peak appea ed in he case o he
conjuga ed compound a a e en ion ime o 6.7 min (l=
220 nm and l= 427 nm) wi h espec o PGlu, which was
cha ac e ized by a e en ion ime o 14.1 min (l= 220 nm)
(Fig. S2C, ESI†). The sho e e en ion ime o CUR–PGlu wi h
espec o he one o PGlu highligh ed he success ul eac ion.
Howe e , a small and b oad peak a a ound 15 min may be
a ibu ed o a a ie y o combina ions o CUR–PGlu (Fig. S2C,
ESI†). Indeed, due o he lack o selec i i y o he eac ion, all
he ca boxylic g oups could be ac i a ed du ing he i s s ep o
he eac ion; he e o e a di e en numbe o CUR molecules
could be conjuga ed a he end o he p ocess. Mo eo e , i is
no excluded ha , being CUR a symme ic molecule bea ing
wo OH g oups, i can eac oge he wi h wo ac i a ed ca -
boxylic g oups in he same polypep ide chain o be ween wo
polypep ide chains, gi ing di e en conjuga ed compounds.
The z-po en ial o he inal compound was 11.0 2.2 mV
agains 20.3 1.7 mV o PGlu. This highligh ed ha mos o
he ca boxylic g oups o he PGlu chains we e ee om he
conjuga ion. Hence, due o he nega i e cha ge, CUR–PGlu
could be u he used as one o he building blocks in he
mul ilaye ne wo k o he capsules.
3.3 Fab ica ion o he cu cumin-loaded LbL mic ocapsule
o mula ions
The LbL echnique was subsequen ly used o ab ica e mul i-
laye biodeg adable mic ocapsules con aining CUR. Th ee di -
e en sys ems we e syn hesized: COPRE-cps, POST-cps, and
CONJ-cps (Fig. 3).
To ab ica e he biodeg adable mic ocapsules, wo poly-
pep ides we e used as building blocks, i.e., PLys and PGlu.
Gene ally, due o hei side chains and pK
a
alues, polypep ides
a e cha ac e ized by a pH-dependen cha ge, which can be
Table 1 Encapsula ion efficiency (EE%) and loading capaci y (LC%) o each s a egy o cu cumin loading in o sphe ical-like calcium ca bona e (CaCO
3
)
mic opa icles
Me hod o encapsula ion Type o addi ion Shape Encapsula ion efficiency (EE%) Loading capaci y (LC%)
Co-p ecipi a ion
a
Di ec Sphe ical 87.3 1.0
e
2.9 0.0
e
Pos -encapsula ion
b
Di ec
c
Sphe ical 51.8 1.4
e
1.7 0.1
e
D opwise
d
Sphe ical 71.7 4.7
e
2.4 0.2
e
a
The co-p ecipi a ion app oach was pe o med a oom empe a u e by di ec ly adding cu cumin (CUR, 1 mg) dissol ed in 0.3 mL o e hanol o 1
.5 mL o a 0.2 M calcium chlo ide (CaCl
2
) solu ion. The mix u e CUR/CaCl
2
was hen added o 1.5 mL o a 0.2 M sodium ca bona e (Na
2
CO
3
)
solu ion con aining 4 mg mL
1
poly(sodium 4-s y ene sul ona e) (PSS) a high s i ing speed o 1 min. The pa icles we e allowed o p ecipi a e o
30 min.
b
The pos -encapsula ion app oach was pe o med a oom empe a u e by di ec ly o d opwise adding CUR (1 mg) dissol ed in 0.5 mL o
e hanol o he p e-syn hesized CaCO
3
mic opa icles (30 mg) e-dispe sed in 0.5 mL o dis illed wa e . The CaCO
3
mic opa icles we e incuba ed o
30 min.
c
Di ec addi ion o CUR.
d
D opwise addi ion o CUR.
e
The pelle o he syn hesized CUR-loaded CaCO
3
mic opa icles was collec ed o
measu e he abso bance o he supe na an (l= 427 nm) and calcula e he encapsula ion efficiency (EE%) and he loading capaci y (LC%).
Fig. 2 Scheme o he syn hesis o cu cumin–conjuga e poly(L-glu amic acid) (CUR–PGlu) ia an es e i ica ion eac ion be ween cu cumin (CUR) and
poly(L-glu amic acid) (PGlu). The eac ion was pe o med o e nigh a oom empe a u e (RT) a e ac i a ing he side chains o PGlu wi h 1-e hyl-3-(3-
dime hylaminop opyl)ca bodiimide hyd ochlo ide (EDCHCl) and dime hylaminopy idine (DMAP).
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exploi ed o use hem as polyelec oly es in he LbL me hodo-
logy. Speci ically, unde ou wo king pH condi ions (i.e.,pH
6.5), PLys (pK
a
= 9.0) and PGlu (pK
a
= 4.8) bea posi i e and
nega i e cha ges, espec i ely. The e o e, by al e na ing he
posi i ely cha ged PLys and he nega i ely cha ged PGlu on o
he empla e, we could build a mul ilaye memb ane made o
h ee (PLys/PGlu) bilaye s mainly in e ac ing each o he by
elec os a ic in e ac ions (Fig. 3(B)-i–iii).
The op imiza ion o he me hodology o encapsula e CUR
in o CaCO
3
mic opa icles by he co-p ecipi a ion app oach
desc ibed in Sec ion 3.1 allowed explo a ion o he op imal
condi ions o ob ain sphe ical-like CaCO
3
mic opa icles.
Impo an ly, he mo phology o he empla e affec s he shape
o he pa icles a e applying he LbL app oach. We could
con i m his phenomenon h ough he SEM mic og aphs a e
he deposi ion o he mul ilaye memb ane on o he sphe ical-like
and lowe -like CaCO
3
empla es. The ob ained LbL-pa icles kep
he mo phology o he co esponding uncoa ed mic opa icles,
ei he lowe -like (Fig. S3A and B, ESI†) o sphe ical-like (Fig. 4(B))
shape. Mo eo e , a e he dissolu ion o he empla e, he LbL-
capsules looked collapsed, bu no ele an changes we e obse ed
o bo h mo phologies (Fig. S3C and D, ESI,†and Fig. 4(B),
espec i ely). As he mo phology o he LbL sys ems may also
de e mine he kine ics o he elease o he payload, and/o he
in e ac ions wi h cells (e.g., up ake), in his wo k, we pe o med
a compa a i e s udy be ween he h ee LbL sys ems wi h he
sphe ical-like shape (Fig. 3(A)-i–iii).
To p oceed wi h he ab ica ion o he CUR-loaded LbL
sys ems, we selec ed he CUR-loaded CaCO
3
mic opa icles ha
showed he highes encapsula ion efficiency in he case o
co-p ecipi a ion and pos -encapsula ion (Table 1).
The mean sizes o 5.8 1.6 mm, 5.4 2.5 mm, and 3.9 
1.6 mm o he COPRE-T, POST-T, and he CaCO
3
-T, espec-
i ely, we e obse ed by measu ing he pa icle size dis ibu ion
Fig. 3 (A) and (B) Schema ic illus a ion o he laye -by-laye (LbL) p ocess o syn hesize cu cumin (CUR)-loaded pa icles (i.e., COPRE-pa icles, POST-
pa icles, and CONJ-pa icles) ia co-p ecipi a ion (i), pos -encapsula ion (ii), and conjuga ion (iii) app oaches. COPRE-T was syn hesized by di ec ly
adding 0.3 mL o CUR in e hanol o 1.5 mL o 0.2 M CaCl
2
solu ion. POST-T was syn hesized by d opwise addi ion o CUR in e hanol o p e-syn hesized
CaCO
3
-T. (C) Co e emo al o ob ain he co esponding capsules: COPRE-cps (i), POST-cps (ii), and CONJ-cps (iii). Poly(L-lysine) (PLys) was used as he
posi i ely cha ged building block. Poly(L-glu amic acid) (PGlu) and CUR–PGlu we e used as he nega i ely cha ged building blocks.
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iew showed ha he CUR-loaded capsules we e engul ed by cells
(Fig. 8). This phenomenon could be associa ed wi h he inc eased
phagocy ic ac i i y o he ac i a ed cells in o M1 pheno ype, which
has been demons a ed in he li e a u e.
61,62
Al hough he s udy
o he pola iza ion o mic oglia is beyond he scope o ou wo k,
he demons a ed p e e en ial up ake by classically ac i a ed
mic oglia migh be e y in e es ing o se e al neu oin lamma o y
diseases (e.g., Alzheime ’s diseases, Pa kinson’s diseases, au-
ma ic b ain inju y, and s oke). In ac , al hough he M1-like
mic oglia a e c ucial in elici ing he inna e immune esponse
agains pa hogens, he pe sis ence o he in lamma o y s imulus
may induce ch onic neu oin lamma ion, whe e he M1 pheno ype
exe s i s neu o oxic na u e.
71
The e o e, modula o s o he mic o-
glia pheno ype may ep esen a po en ial he apeu ic app oach o
he ea men o se e al diseases and diso de s occu ing in he
CNS. In e es ingly, we obse ed ha when POST-cps we e aken
up by he LPS-ac i a ed cells, an in acellula elease was e iden
(Fig. 8(C) and (F)). Con a ily, in he case o COPRE-cps and CONJ-
cps, e y low in acellula elease was app ecia ed, and CUR
appea ed homogeneously localized in he capsules (Fig. 8(B), (E)
and (D), (G)). Zoomed con ocal images a e epo ed in Fig. S11 o
he ESI.†This inding co ela es wi h he elease s udies showed
in Fig. 5, and migh jus i y he di e se in i o elease p o iles o
CUR be ween he h ee di e en o mula ions.
The e o e, we belie e ha he acili a ed elease o CUR in o
he cells and he down egula ion o p o-in lamma o y media o s
could be e y p omising o ea pa hological neu oin lamma ion.
4. Conclusions
In his wo k, we de eloped biodeg adable mul ilaye mic o-
capsules by using he LbL app oach. PLys and PGlu we e used
as posi i ely and nega i ely cha ged building blocks due o
hei biocompa ibili y and biodeg adabili y. In pa icula , we
buil high enginee ed ca ie s, es ablishing h ee me hodolo-
gies o include a poo wa e -soluble d ug (i.e., CUR) in he co e
by co-p ecipi a ion o pos -encapsula ion o in he mul ilaye
memb ane as polyme -conjuga e.
An ini ial op imiza ion p ocedu e was ca ied ou o he
encapsula ion o he ee CUR in o he CaCO
3
mic opa icles
o achie e high encapsula ion efficiencies and sys ems wi h a
sphe ical-like shape. In he case o he co-p ecipi a ion s a egy,
he e hanol played a c ucial ole in he mo phology o he
esul ing pa icles, p omo ing he o ma ion o a lowe -like
shape a high olume and a sphe ical-like shape when he
olume used o dissol e CUR was minimized compa ed o he
sal solu ions. Fu he mo e, in hecaseo hepos -encapsula ion
s a egy, he d opwise addi ion o CUR o he p e-syn hesized
CaCO
3
mic opa icles pu sued high encapsula ion efficiencies
compa ed o he di ec addi ion. In e es ingly, he wo selec ed
app oaches p o ed o be decisi e ei he in e ms o he loca ion
o he d ug in he LbL-capsules o in he subsequen elease
o CUR, which was accele a ed in POST-cps compa ed o he
COPRE-cps.
We also p esen ed an al e na i e manne o include CUR in
ou LbL sys ems, which consis ed o inc easing he solubili y o
he d ug by syn hesizing d ug-conjuga es and hen inco po a -
ing hem in o he mul ilaye memb ane. This app oach is e y
p omising in he ield o d ug deli e y as i would allow o
include esponsi e en i ies in he mul ilaye memb ane, con-
ol he amoun o he d ug loaded in he nanos uc u e, and
ha e an ideal sys em o he con olled elease o he d ug.
Howe e , he lack o selec i i y in he eac ion we pe o med
did no allow us o con ol he conjuga ion o CUR, which
u he exhibi s wo po en ial OH g oups ha could eac wi h
Fig. 8 Con ocal images showing he up ake o COPRE (B), POST (C), and CONJ (D) capsules by ac i a ed BV-2 cells by 20 ng mL
1
LPS a e 24 h o
incuba ion wi h he capsules wi h espec o he con ol (CTRL) (A). An o hogonal iew o he up ake o COPRE (E), POST (F), and CONJ (G) capsules by
ac i a ed BV-2 cells a e 24 h o incuba ion. The nuclei a e s ained in blue (DAPI), F-ac in is s ained in ed ( e ame hyl hodamine iso hiocyana e (TRITC)-
phalloidin), and he g een is a ibu ed o he p esence o cu cumin (CUR). The scale ba is 5 mm.
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PGlu. The e o e, a u he imp o emen o his me hodology
migh be needed o u u e s udies.
Mo eo e , he s udy o he elease p o ile o CUR om he
h ee o mula ions unde acidic and neu al condi ions and in
he p esence o p o eoly ic enzymes e ealed ha se e al pa a-
me e s may in luence he elease o he d ug (e.g., he pH, he
ionic s eng h, he na u e o he laye s and he d ug, o he
ypology o loading). No ewo hily, he LbL echnique offe ed a
alid ool o ab ica e d ug deli e y sys ems ha allowed a
con olled and sus ained elease o he d ug, p e en ing i s
ini ial bu s elease.
Due o he documen ed pleio opic p ope ies o CUR as
he apeu ics, in i o s udies on wo diffe en models o
in lamed cell lines (i.e., THP-1 de i ed mac ophages and BV-2
cells) we e pe o med o e alua e whe he CUR was s ill bio-
ac i e a e he loading p ocess, he conjuga ion, and in he
p esence o he mul ilaye memb ane. Ou CUR-loaded cap-
sules did no induce any de imen al effec ega dless o he
explo ed concen a ion. No ably, al hough he capsule compo-
si ion showed an an i-in lamma o y effec on THP-1 de i ed
mac ophages, he highes concen a ions o CUR loaded in o
he capsules could amelio a e he in lamma ion by supp essing
he elease o p o-in lamma o y cy okines, such as TNF-aand
IL-6. This sugges ed ha he capsules and he conjuga ion
p ese ed he bioac i i y o he d ug, which esul ed clea e
in he ea men o in lamed mic oglia. Fu he mo e, he
up ake s udies o he capsules by LPS-ac i a ed mic oglia
compa ed o he co esponding es ing cells highligh ed he
endency o he M1-like pheno ype o phagocy ize he capsules
when an in lamma ion occu s. In pa icula , he in acellula
elease o CUR con i med he diffe en elease p o iles o CUR
ob ained in i o. In ac , he up ake s udies ia con ocal
mic oscopy indica ed ha he elease o CUR was acili a ed
in POST-cps, p obably due o a mechanism o diffusion,
whe eas, in he o he wo sys ems, CUR looked mo e localized
inside he capsules.
O e all, ega dless o he me hodology applied o include
he hyd ophobic d ug in o he biodeg adable mul ilaye ca -
ie s, ou LbL o mula ions could ha e a g ea po en ial as
modula o s o he mic oglia pheno ype owa ds he educ ion
o he acu e neu oin lamma ion. Fu he mo e, he possibili y
o apply he LbL echnique on nanopa icles (e.g., CaCO
3
nanopa icles, silica nanopa icles, and nanoc ys als) and unc-
ionalize he ex e nal laye o he mul ilaye memb ane may
lead o explo a ion o al e na i e adminis a ion ou es o
speci ic neu onal diseases. Hence, we belie e ha his s udy
could pa e he way o ex ended in es iga ions on no el d ug
deli e y sys ems in bo h he mic o- and nano-me e scales o
ea he in lamma ion associa ed wi h diffe en diseases.
Au ho con ibu ions
M.A.M.:concep ualiza ion,in es iga ion,me hodology, o mal
analysis, and w i ing – o iginal d a .S.M.-S.:in es iga ion,
o mal analysis, w i ing – o iginal d a , and w i ing – e iew
and edi ing. A. B.: w i ing – e iew and edi ing, supe ision, and
unding acquisi ion. M. C.: concep ualiza ion, w i ing – e iew
and edi ing, supe ision, and undingacquisi ion.A.L.:concep-
ualiza ion, w i ing – e iew and edi ing, supe ision, and unding
acquisi ion.
Da a a ailabili y
The da a suppo ing his a icle ha e been included as pa o
he ESI.†
Con lic s o in e es
The au ho s decla e ha hey ha e no compe ing in e es s.
Acknowledgemen s
The au ho s acknowledge he inancial suppo om he Basque
Go e nmen (p ojec s 2023333010, 2023333023, PIBA2023-1-0043,
IT-1766-22, IKUR S a egy), he Uni e si y o he Basque Coun y
(p ojec s COLLAB22/05 and GIU21/033), he IKERBASQUE-Basque
Founda ion o Science, he Minis y o Science and Inno a ion o
he Go e nmen o Spain (g an PID2022-142739OB-I00 unded
by MICIU/AEI/10.13039/501100011033 and by FEDER, UE;
’Ma ı
´a de Maez u’ P og amme o Cen e o Excellence in R&D,
g an CEX2023-001303-M unded by MICIU/AEI/10.13039/
501100011033; PID2022-142128NB-I00 unded by MCIN/AEI/
10.13039/501100011033/and by he ‘‘Eu opean Union Nex Gene -
a ionEU/PRTR’’; RYC2018-025923-I om RyC p og am – MCIN/
AEI/10.13039/501100011033 and FSE ‘‘in ie e en u u u o’’).
SGIke echnical se ices (UPV/EHU) a e g a e ully acknowledged
o he suppo in ATR-FTIR, con ocal mic oscopy, XRD, and SEM.
Open Access unding p o ided by Uni e si y o he Basque
Coun y.
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