Pioneering wound care solutions: triaxial wet-spun fibers with bioactive agents for chronic wounds, part II (controlled release and biological activity of the active agents)

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Ci e his: Ma e . Ad ., 2025,
6, 2029
Pionee ing wound ca e solu ions: iaxial
we -spun ibe s wi h bioac i e agen s o
ch onic wounds, pa II (con olled elease
and biological ac i i y o he ac i e agen s)†
Ca a ina S. Mi anda,
a
A. F ancisca G. Sil a,
b
Camille E enou,
c
Je
´ o
ˆme Lama ine,
c
Be enge e F omy,
c
Sı
´l ia M. M. A. Pe ei a-Lima,
b
A u Ribei o,
de
Susana P. G. Cos a,
b
Na a
´lia C. Homem
and Helena P. Felguei as *
a
The incidence o bac e ial in ec ions associa ed wi h ch onic wounds (CWs) has inc eased in ecen
yea s. Thus, a iaxial we -spun ib ous sys em (con aining h ee laye s) was p oduced o CW healing.
The iaxial ibe s we e loaded wi h cinnamon lea oil (CLO), endowed wi h high an ibac e ial, an ioxidan
and an i-in lamma o y ea u es, and an an imic obial pep ide –alanine–alanine–p oline– aline (AAPV) –
capable o egula ing he ac i i y o human neu ophil elas ase (HNE; highly exp essed du ing in lamma-
o y p ocesses). To o e come he cha ac e is ic high ola ili y o essen ial oils (EOs), CLO was loaded a
he sys em’s co e and blended wi h polycap olac one (PCL) which has excellen elas ici y and ensile
s eng h. The in e media e laye was composed o sodium algina e (SA) which has high hyd a ion capa-
ci y and AAPV. Finally, he shell was made o cellulose ace a e (CA), ensu ing he sys em’s s uc u al
in eg i y and p o iding a po ous ne wo k o he con olled elease o AAPV and CLO. This esea ch was
di ided in o wo pa s, wi h he p esen add essing he biological cha ac e iza ion o he sys em, namely
he con olled elease o bioac i e agen s, hei an ibac e ial, an ioxidan and cy ocompa ibili y p o iles
and he pep ide-loaded ibe abili y o inhibi HNE ac i i y. AAPV-loaded we -spun ibe s a ained a
sus ained elease o up o 55% du ing 24 h o incuba ion in physiological-like media, also p esen ing
e ec i e HNE inhibi ion (E65%). Addi ionally, CLO-loaded ibe s demons a ed a con olled elease o
up o E52% du ing 24 h o incuba ion in PBS, eaching highe an ibac e ial and an ioxidan p o iles in
compa ison wi h he unloaded ibe s. Da a con i med he biological po en ial, sa e y and sui abili y o
he p oposed sys em o u u e applica ions in CW ca e.
1. In oduc ion
Ch onic wounds (CWs) canno heal a he same pace as acu e
wounds.
1
CWs o en s all a in lamma ion, condi ioning he
pa ien s’ heal h and hei quali y o li e, leading o ch onic
pain, loss o unc ion and mobili y, and ul ima ely aising
mo bidi y a es.
2
F equen ly, hese wounds a e he esul o
bac e ial in ec ions, igge ed by S aphylococcus au eus,S aphy-
lococcus epide midis,Esche ichia coli and Pseudomonas ae ugi-
nosa, some o he mos p e alen mic oo ganisms and a majo
conce n o he Wo ld Heal h O ganiza ion.
2,3
Be ween 2005
and 2022, P. ae uginosa was de ec ed in 14.5% o CWs in he
Uni ed S a es o Ame ica (USA), while in he Uni ed Kingdom
(UK) and Po ugal, i s p esence was de ec ed in 8.6% and 11.4%
o he cases, espec i ely.
4
Mo eo e , in CWs, neu ophils
elease p o eases (including human neu ophil elas ase, HNE)
in an uncon olled manne , as soon as bac e ia s a colonizing
he wound si e. Such abno mally high concen a ions o HNE
a
Cen e o Tex ile Science and Technology (2C2T), Uni e si y o Minho, Campus o
Azu e
´m, 4800-058 Guima a
˜es, Po ugal.
E-mail: ca a ina.mi anda@2c2 .uminho.p , helena. elgu[email p o ec ed];
Fax: +351-253-510-293; Tel: +351-253-510-283
b
Cen e o Chemis y (CQ), Uni e si y o Minho, Campus o Gual a , 4710-057
B aga, Po ugal. E-mail: [email protected] , sil [email protected] ,
[email p o ec ed]
c
E
´quipe In e
´g i e
´ onc ionnelle du issu cu ane
´(SKIN), Labo a oi e de biologie
issulai e e d’inge
´nie ie he
´ apeu ique (LBTI), CNRS UMR5305, Uni e si e
´Lyon I,
7 passage du Ve co s, 69367 Lyon Cedex 07, F ance.
E-mail: camille.e enou@ibcp. , [email p o ec ed],
be enge e. omy@cn s.
d
CEB – Cen e o Biological Enginee ing, Uni e si y o Minho, B aga, 4710-057,
Po ugal. E-mail: [email p o ec ed]
e
LABBELS – Associa e Labo a o y, B aga, Guima a
˜es, Po ugal
Simoldes Plas ics S.A., Rua Comendado An o
´nio da Sil a Rod igues, 165, 3720-
193, Oli ei a de Azeme
´is, Po ugal. E-mail: na [email protected]
†Elec onic supplemen a y in o ma ion (ESI) a ailable. See DOI: h ps://doi.o g/
10.1039/d4ma01104j
Recei ed 5 h No embe 2024,
Accep ed 14 h Feb ua y 2025
DOI: 10.1039/d4ma01104j
sc.li/ma e ials-ad ances
Ma e ials
Ad ances
PAPER
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du ing in lamma o y p ocesses lead o he deg ada ion o
endogenous and supplemen al g ow h ho mones, which p e-
en he wounds om p og essing o he ollowing s eps o he
healing p ocess.
1,5,6
The inhibi o y effec o he alanine–alanine–p oline– aline
(AAPV) e apep ide agains HNE has been p o en o be
e ec i e.
6,7
The mechanism o ac ion o he AAPV pep ide is
no ye ully unde s ood. S ill, acco ding o To h e al., he
pep ide i s he P-P1 subsi es o HNE, esul ing in compe i i e
inhibi ion o HNE ac i i y.
6,8
As o o he small pep ides, AAPV
canno be used eely in CW he apies due o i s low s abili y in
physiological media, as well as high sensi i i y o changes in
en i onmen al condi ions. Also, hese pep ides p esen oxici y
when used a high concen a ions du ing sys emic deli e y.
9,10
To p e en such e en s, hese bioac i e agen s can be inco po-
a ed in o polyme ic s uc u es ha o e p o ec ion agains
en i onmen al changes and allow hei con olled and localized
elease, wo king as an al e na i e o he use o an ibio ics.
7
An ibio ics a e a common ool in he ea men o bac e ial
in ec ions in CWs.
10,11
Howe e , hei excessi e use has
esul ed in he need o hink o al e na e solu ions, including
essen ial oils (EOs), he apeu ic agen s o na u al o igin ha a e
mainly composed o mix u es o ola ile and lipophilic com-
pounds ex ac ed om se e al pa s o plan s.
9,11
EOs
con ain hyd ophobic elemen s esponsible o hei inhe en
analgesic, an i-in lamma o y, an ioxidan and an ibac e ial
p ope ies.
12,13
The an ibac e ial effec s o EOs esul om
hei abili y o dis up p ocesses ela ed o ion o solu e
anspo , due o hei lipophilici y, which acili a e hei
pene a ion h ough he bac e ial memb ane and he in e e -
ence wi h in acellula componen s.
9,11,14
Cinnamon lea oil
(CLO) is ex ac ed om cinnamon ba ks and lea es and is
mainly o med om eugenol and cinnamaldehyde, which
endows he EO wi h an ibac e ial abili ies.
12,15
The an ioxidan
and an ibac e ial p ope ies o eugenol ha e been ex ensi ely
add essed.
9,16,17
Addi ionally, his compound has been
epo ed o possess an i-in lamma o y p ope ies, by supp es-
sing he exp ession o he cyclooxygenase II enzyme.
18
Howe e ,
simila ly o small pep ides, he use o EOs in he apeu ic
app oaches comes wi h limi a ions, including cy o oxici y a
high concen a ions, high ola ili y and sensi i i y o ex e nal
ac o s, such as empe a u e, ligh and oxygen.
9,13
S ill, in
ecen yea s, esea ch has been disclosed on he loading o
EOs and he apeu ic pep ides on o polyme ic ibe s ha
inc ease hese bioac i e agen ’s physiological s abili y, while
p ese ing hei chemical ea u es.
9,19
P oducing polyme ic ib ous s uc u es loaded wi h EOs
and/o pep ides can be accomplished using spinning echni-
ques, including we -spinning.
20,21
We -spun ib ous cons uc s
display a wide ange o diame e s and mo phologies, along
wi h high po osi y. Such p ope ies a e a o ed in biomedicine
by enabling cell pene a ion, adhesion and p oli e a ion, and
by esembling physiological mic oen i onmen s.
20,22
The goal
o he cu en esea ch was o enginee a iaxial sys em wi h
bac e ial and enzyme- ela ed inhibi o y ea u es o p omo ing
wound healing. Fo ha , a we -spinning app oach was ca ied
ou o p oduce ibe s wi h h ee laye s, also known as a iaxial
sys em: (1) a co e (inne mos laye ) made o polycap olac one
(PCL), in ended o offe he ibe s high elas ici y, loaded wi h
CLO o inhibi he g ow h o bo h G am-posi i e and G am-
nega i e bac e ia;
7,23,24
(2) an in e media e laye made o a
blend o sodium algina e (SA) and AAPV, o main ain a mois
en i onmen and o egula e he local enzyma ic ac i i y,
espec i ely;
6,7
and (3) a shell (ou e mos laye ) composed o
cellulose ace a e (CA) ha ensu es s uc u al in eg i y o ibe s
and offe s p o ec ion o he inne laye s con aining he bioac-
i e compounds.
25
The po osi y inhe en o he shell allowed a
somewha con olled access o he ac i e agen s p esen in he
in e media e laye and he co e, eleasing AAPV and CLO in a
sus ained manne .
Resea ch was di ided in wo pa s: pa I (unde pee
e ision), chemical, physical, mechanical and he mal cha ac-
e iza ion o he iaxial sys em, along wi h a p oo o concep
consis ing o he p oduc ion o a small d essing; and pa II
(p esen manusc ip ), biological cha ac e iza ion o he iaxial
sys em. The minimum bac e icidal concen a ion (MBC) and
minimum inhibi o y concen a ion (MIC) o CLO, in i s ee
o m, we e examined agains S. au eus,S. epide midis,E. coli
and P. ae uginosa. HNE inhibi ion e alua ions we e pe o med
o de e mine he AAPV’s maximum inhibi o y concen a ion
(IC
M
), also in i s ee o m. Addi ionally, bioac i e agen elease
kine ics s udies, om wi hin he ibe sys ems, we e ca ied
ou . The HNE inhibi ion capaci y o he enginee ed ibe s was
e alua ed, as well as hei an ibac e ial and an ioxidan p ope -
ies. Finally, he sa e y o he iaxial ibe s owa ds skin cell
models we e in es iga ed in i o. Recen ly, his echnique has
d awn a en ion as a p omising ool o de eloping d ug
deli e y sys ems o biomedical pu poses. Howe e , o he
au ho s’ knowledge, he e a e no epo s on he p oduc ion o
Eos and pep ide-loaded iaxial we -spun ibe s o wound
healing applica ions.
2. Ma e ials and me hods
2.1. Ma e ials
CA (M
n
50.000), PCL (M
n
80.000), SA ( om b own algae,
medium iscosi y), 1,1-diphenyl-2-pic ylhyd azyl (DPPH), e hyl
cyanoglyoxyla e-2-oxime (Oxyma), 1,3-diisop opylca bodiimide
(DIC), dichlo ome hane (DCM), chlo oace ic acid, deu e ium
oxide (D
2
O), elas ase om human leukocy es, ypsin inhibi o
om soybean, N-me hoxysuccinyl-Ala-Ala-P o-Val-p-ni oanilide
(N-MeO-Suc-Ala-Ala-P o-Val-p-NA), Dulbecco’s Phospha e Bu -
e ed Saline (DPBS), ypsin–EDTA, T yp an Blue solu ion and
e al bo ine se um (FBS) we e ob ained om Sigma-Ald ich (S
Louis, Missou i, USA). An ibio ic–an imyco ic was ob ained
om G isp (Po o, Po ugal). N,N-dime hyl o mamide (DMF),
ace ic acid (AcOH), ace oni ile (ACN), pipe idine, sodium
hyd oxide, sodium ca bona e, 2,2,2- i luo oe hanol (TFE) and
i luo oace ic acid (TFA) we e acqui ed om Me ck (Da m-
s ad , Ge many). Dime hyl sul oxide (DMSO) and 6-hyd oxy-
2,5,7,8- e ame hylch oman-2-ca boxylic acid (T olox) we e
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ob ained om Fishe (Maha ash a, India). Dulbecco’s Mod-
i ied Eagle’s Medium (DMEM), penicillin–s ep omycin, Alama
Blue and lac a e dehyd ogenase (LDH) ki we e pu chased om
In i ogen (Massachuse s, USA). T i on was acqui ed om Bio-
Rad Labo a o ies (Cali o nia, USA). Anhyd ous calcium chlo -
ide was employed as coagula ion/c osslinking agen du ing
we -spinning and was supplied by Chem-Lab (Zedelgem, Bel-
gium). T is–hyd ochlo ide (T is–HCl) was pu chased om
Roche (Basel, Swi ze land).
AAPV and WAAPV we e syn hesized by us as desc ibed in e .
7. Pu e cinnamon lea oil (CLO, o igin Cinnamomum zeylanicum
Blume, = 1.049) was ob ained om Folha d’A
´gua Company
(San o Ti so, Po ugal).
19
Sodium phospha e dibasic (Sigma-
Ald ich), monosodium phospha e monohyd a e (Me ck), po as-
sium chlo ide (Me ck) and sodium chlo ide (Me ck) we e used
in he p epa a ion o phospha e buffe saline solu ion (PBS a
0.01 M: 1.44 g L
1
o Na
2
HPO
4
, 0.24 g L
1
o KH
2
PO
4
, 0.20 g L
1
KCl and 8.00 g L
1
o NaCl, adjus ed o physiological pH 7.4).
G am-posi i e bac e ia S. au eus (ATCC 6538) and S. epide -
midis (ATCC 35984), along wi h G am-nega i e bac e ia E. coli
(ATCC 25922) and P. ae uginosa (ATCC 25853) we e supplied by
Ame ican Type Cul u e Collec ion (ATCC, Vi ginia, USA). Fo
bac e ia g ow h, yp icase soy b o h (TSB), yp icase soy aga
(TSA), nu ien aga (NA) and nu ien b o h (NB) we e pu -
chased om VWR (Al agide, Po ugal), while he Muelle
Hin on b o h (MHB) was ob ained om CondaLab (Mad id,
Spain). Human ke a inocy es cell line (HaCaT) – DKFZ HaCaT
adhe en cell line (immo alized human ke a inocy es) – was
ob ained om Cy ion (p e iously known as CLS) (Eppelheim,
Ge many), and p o ided by DKFZ (Helm oz, Ge many).
26
Mouse emb yonic ib oblas cell line (NIH 3T3) om Sigma
Ald ich (Da ms ad , Ge many). All eagen s we e used wi hou
u he pu i ica ion.
2.2. We -spun ibe p oduc ion
A PCL solu ion was p epa ed in DMF a 10% w/ and s i ed o
1 h a 50 1C. CLO was hen combined a 16.40 mg mL
1
(amoun es ablished in e . 27), consis ing o 4i s MIC. Bo h
compounds we e le o homogenize o 1 h a 50 1C (PCL–CLO
solu ion).
28
A 2% w/ aqueous SA solu ion was also p epa ed
and s i ed o 3 h a 50 1C. A e wa ds, AAPV was added o he
p e ious solu ion a 50 mgmL
1
, co esponding o i s max-
imum inhibi o y concen a ion (IC
M
) (concen a ion es ab-
lished in e . 7) (SA–AAPV solu ion). Finally, a 10% w/ CA
solu ion was dissol ed in DMF o 3 h a 50 1C. A we -spinning
se up was used, con aining h ee sy inge pumps (NE-300, New
E a Pump Sys ems, No leq, San o Ti so, Po ugal), esponsible
o con olling he a e and ejec ion olume, a iaxial spinne e
(sealed needles o h ee laye s wi h 21, 15 and 11 gauge, om
he inne o he ou e laye ) and a la ge ay con aining 500 mL
o a 2% w/ CaCl
2
coagula ion ba h, a oom empe a u e (RT).
PCL o PCL–CLO solu ions we e loaded on o he sy inge
connec ed o he inne mos po (co e) and ejec ed a
0.11 mL min
1
, whe eas SA o SA–AAPV solu ions we e loaded
on o he in e media e po and ejec ed a 0.13 mL min
1
.
Addi ionally, CA solu ion was ejec ed a 0.15 mL min
1
and
loaded on o he ou e mos po (shell). Fibe s con aining only
one laye (PCL, PCL–CLO, SA, SA–AAPV, CA) we e also p o-
duced, as well as ibe s wi hou one o he laye s (e.g., PCL/SA,
PCL/CA, SA/CA). To each g oup o ibe s, samples wi hou one/
bo h o he ac i e agen s we e also p oduced in o de o es he
in luence o each compound on he p ope ies o he sys em
(e.g., PCL/SA–AAPV, PCL–CLO/SA/CA). The iden i ica ion o he
ibe s used ‘‘/’’ o sepa a ing elemen s belonging o di e en
laye s and ‘‘–‘‘ o componen s wi hin he same laye (Table 1).
2.3. WAAPV and CLO elease kine ics
The moni o ing o AAPV elease by using IV- isible abso p ion
spec oscopy was no possible, since an o e lapping o max-
imum wa eleng hs o abso p ion be ween all ibe compounds
(all biodeg adable polyme s) was obse ed. Due o he absence
o luo escence om AAPV, a modi ied e sion o he pep ide
was syn hesized, by including he luo escen amino acid
yp ophan (WAAPV), used o moni o he elease by luo ime-
y. Al hough he p esence o such amino acid may ha e led o
diffe ences in he pep ide elease a es, such modi ica ion s ill
Table 1 Lis o we -spun ibe ypologies analyzed in his esea ch
Fibe ypology Co e composi ion In e media e laye composi ion Shell composi ion
PCL PCL — —
PCL–CLO PCL blended wi h CLO — —
SA — SA —
SA–AAPV — SA blended wi h AAPV —
CA — — CA
PCL/SA PCL SA —
PCL–CLO/SA PCL blended wi h CLO SA —
PCL/SA–AAPV PCL SA blended wi h AAPV —
PCL–CLO/SA–AAPV PCL blended wi h CLO SA blended wi h AAPV —
SA/CA — SA CA
SA–AAPV/CA — SA blended wi h AAPV CA
PCL/CA PCL — CA
PCL–CLO/CA PCL blended wi h CLO — CA
PCL/SA/CA PCL SA CA
PCL–CLO/SA/CA PCL blended wi h CLO SA CA
PCL/SA–AAPV/CA PCL SA blended wi h AAPV CA
PCL–CLO/SA–AAPV/CA PCL blended wi h CLO SA blended wi h AAPV CA
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allowed he p edic ion o he pep ide’s beha io in physiologi-
cal media, due o hei simila chemical s uc u es. The luo -
escen pep ide was loaded on o he ibe s as desc ibed in e . 7.
A WAAPV calib a ion cu e in PBS a concen a ions anging
om 0.500 and 62.500 mgmL
1
was p epa ed using a spec o-
me e Sa spec (Vila No a de Gaia, Po ugal) and Ligh Scan 2.0
so wa e. WAAPV-loaded ibe s we e imme sed in PBS, pH 7.4,
o 1, 2, 4, 6 and 24 h o incuba ion a 37 1C and 120 pm, and
aliquo s o 150 mL we e collec ed a each ime poin . Fluo es-
cence was hen ead using an LED lamp o 275 nm, in he ange
180–900 nm, wi h a esolu ion o 1 nm and wa eleng h accu acy
o 0.5 nm. Resul s we e epo ed as in ensi y coun s (IC) s.
wa eleng h, based on he WAAPV calib a ion cu e (IC = 32.413
Concen a ion + 4144.7; R
2
= 0.9858).
Rega ding he EO, he amoun o CLO eleased om all we -
spun ibe s we e es ima ed using a UV-1800 UV- isible spec o-
pho ome e (Shimadzu) (measu emen o emaining concen-
a ion a e ibe imme sion). Ini ially, a calib a ion cu e o
CLO a diffe en concen a ions in PBS, anging om 0.02 o
0.20 mg mL
1
, was p epa ed o enable eliable de ec ion o he
spec a’s egion o in e es . The esul s we e plo ed as abso bance
s. wa eleng h. The elease o CLO om he we -spun ibe s was
assessedinPBSa e 1,2,4,6and24h.Theselec iono such
medium was based on p e ious esea ch s udies om ou eam, in
an a emp o app oxima e ha o physiological-like media, also
conside ing he desi ed u u e applica ion o his sys em as a
wound d essing.
7,10
Samples o CLO-con aining ibe s weighing
10 mg each we e le in con ac wi h 1 mL o PBS a 37 1Cand
120 pm, and aliquo s o 150 mL we e collec ed a each ime poin .
Thei abso bances we e hen measu ed in he ange 200–900 nm.
The elease o CLO was de e mined by he di e ences in abso -
bance be ween he i s momen o in e ac ion (0 h) and he
subsequen measu ing pe iods, up o he 24 h ma k.
2.4. AAPV-loaded ibe s’ inhibi o y effec agains HNE
AAPV-loaded and unloaded ibe s we e incuba ed in 1 mL o
PBS, pH 7.4, a 37 1C and 120 pm o 1, 2, 4, 6 and 24 h. A e
each incuba ion pe iod, aliquo s o 150 mL we e collec ed; hese
ep esen ed he es ing solu ions. AAPV-unloaded ibe s we e
also analyzed, in his way sub ac ing he polyme s’ in luence
om he esul s o AAPV-loaded ibe s. HNE inhibi ion expe i-
men s we e conduc ed as desc ibed in e . 7. B ie ly, a subs a e
solu ion (N-MeO-Suc-Ala-Ala-P o-Val-p-NA) (125 mL) was mixed
wi h 0.1 M T is–HCl bu e (405 mL), along wi h he 150 mL
aliquo s o he es ing solu ions in PBS and 20 mL o HNE
solu ion (45 mU). A e 1 h o incuba ion a 37 1C, an inhibi o
solu ion (500 mL) was added o s op he eac ion and abso -
bances we e ead a 405 nm (EZ Read 2000 Mic opla e Reade ,
Bioch om, Camb idge, UK). Da a we e epo ed as maximum
inhibi o y concen a ion (IC
M
). Expe imen s we e conduc ed in
iplica e, wi h h ee abso bance eadings being done pe
eplica e (mean a e aging nine measu emen s).
2.5. An ioxidan ac i i y
F ee adical-sca enging ac i i y was de e mined using he 1,1-
diphenyl-2 pic ylhyd azyl (DPPH) assay. Samples o 10 mg o
each we -spun ibe ypology we e imme sed in 1 mL o
absolu e e hanol and incuba ed a 37 1C and 120 pm o 1,
2, 4, 6 and 24 h. E hanol was selec ed, no only because o he
EO’s high solubili y, bu also due o i s common use in DPPH
assays and he low wa e con en , which could lead o in e -
e ences in he abso bance eadings.
29–31
Aliquo s o 10 mL we e
collec ed a each ime pe iod and mixed, in a 96-well pla e, wi h
140 mL o a DPPH s ock solu ion (400 mM in absolu e e hanol).
DMSO and T olox we e used as nega i e and posi i e con ols,
espec i ely. The abso bances we e moni o ed e e y 5 min o
1 h and measu ed a 515 nm, using an EZ Read 2000 Mic opla e
Reade (Bioch om, Camb idge, UK). Expe imen s we e con-
duc ed in iplica e and da a we e epo ed in e ms o he
pe cen age o educed DPPH a a s eady s a e, which was
calcula ed as ollows (eqn (1)):
DPPH %ðÞ¼
AiA
Ai
100;(1)
in which A
ep esen s he abso bance egis e ed a a s eady
s a e and A
i
co esponds o he ini ial abso bance.
2.6. Bac e ia inhibi ion: ime-kill kine ics
Time-kill kine ics es s we e conduc ed acco ding o s anda d
ASTM E2149-01, adap ed as epo ed p e iously.
7,10
The an i-
bac e ial p o ile o he iaxial we -spun ibe s con aining he
AAPV pep ide and he CLO was e alua ed agains 1 10
5
CFUs
pe mL suspensions o S. au eus,S. epide midis and E. coli in
TSB, and a suspension o P. ae uginosa p epa ed in NB.
Samples o 10 mg we e imme sed in 1 mL o each bac e ium
suspension and incuba ed a 37 1C and 120 pm o 1, 2, 4, 6
and 24 h. A each ime poin , bac e ial suspensions we e
se ially dilu ed in PBS (10
1
o 10
4
), pla ed in TSA and NA,
and incuba ed a 37 1C o 24 h. G own colonies we e coun ed,
and esul s we e exp essed in pe cen age (%) o bac e ia
inhibi ion. All measu emen s we e pe o med in iplica e
( h ee measu emen s we e done pe eplica e, wi h da a a e -
aging nine alues) and da a we e p ocessed using he G aphPad
P ism 8.0 so wa e.
2.7. Bio ilm o ma ion
Bac e ia inoculums o S. au eus,S. epide midis and E. coli we e
p epa ed in TSB and o P. ae uginosa in NB and le o e nigh o
g ow a 37 1C and 120 pm. Fou eplica es om each ibe
ypology (10 mg) we e weighed in o 48-well pla es and co e ed
wi h 5 mL o each inoculum mixed wi h 500 mL o MHB. Con ol
wells con aining he dilu ed inoculums bu wi hou samples
we e also conside ed o his assay. The pla es we e incuba ed
o 48 h a 37 1C o allow bio ilm o ma ion. A e wa ds, ibe s
we e emo ed om each well and he pla e was washed 3wi h
PBS o emo e loosely bound bio ilm cells. The pla e was le a
37 1C o 20 min o ixa ing he bio ilms. Then, a 0.1% w/
c ys al iole (CV) aqueous solu ion was added o each well and
he pla e was le a RT o 15 min. This was ollowed by
3 washes wi h PBS o emo e unbound CV. Finally, 500 mLo
a 30% / ace ic acid solu ion we e added o each well o 15
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min and aliquo s we e ans e ed o a 96 well-pla e o abso -
bance eadings a 595 nm.
2.8. Cy ocompa ibili y es ing
2.8.1. Cell cul u e condi ions. The cy ocompa ibili y and
cell p oli e a ion o all ibe ypologies owa ds mouse emb yo-
nic ib oblas cell line (NIH 3T3) and human ke a inocy e cell
line (HaCaT) we e e alua ed using a di ec con ac assay. Cells
we e hawed and sub-cul u ed in DMEM supplemen ed wi h
10% / inac i a ed FBS (30 min, 56 1C) and 1% / penicillin–
s ep omycin, a 37 1C in a humidi ied a mosphe e o 5% CO
2
.
The ea e , cells we e chemically de ached om cell cul u e
lasks using ypsin–EDTA solu ion. The numbe o cells was
de e mined using a Malassez cell (Ma ien eld, Ge many), wi h
cells being labelled wi h ypan blue (Sigma Ald ich, UK), o
e alua e he p opo ion o dead cells, and coun ed using a Ca l
Zeiss Suzhou Co., L d (Suzhou, China) mic oscope. Cells we e
seeded in 96-well la -bo om issue pla es (G eine , Ge many)
and incuba ed o 24 h a 37 1C in a humidi ied a mosphe e o
5% CO
2
. HaCaT and NIH 3T3 we e seeded a 15 000 and 20 000
cells pe well, espec i ely (concen a ions de e mined in a
p e ious in es iga ion; unpublished da a). The expe imen s
we e pe o med using ib oblas cell line in passages 2 o 12
and ke a inocy e cell line in passages 20 o 30. Fib ous samples
o 2 mg we e used o each es . Expe imen s we e conduc ed in
iplica e. Fibe s we e washed and disin ec ed 3wi h deio-
nized wa e (dH
2
O) and 1e hanol a 70% / , du ing 5 min
each, p io o any es ing.
2.8.2. Cell iabili y assessmen . P io o any ibe es ing,
he de e mina ion o he op imum cell numbe was ca ied ou
by seeding cells in o 96-well la black issue pla es a concen-
a ions anging om 5000 o 25 000 cells pe well. Cells we e
incuba ed o e nigh a 37 1C and 5% CO
2
a mosphe e. A e -
wa ds, he medium was emo ed and 100 mL o a 10% /
Alama Blue
TM
(In i ogen) we e added o each well. Cells we e
again incuba ed o 1 h and he luo escence was ead (l
exc
=
560 and l
em
= 590 nm) using a Tecan luo ime e (Tecan Spa k,
Lyon, F ance) wi h Tecan Spa k Con ol 3.2 so wa e. The
me abolic ac i i y o each cell concen a ion was calcula ed
using eqn (2):
Me abolic ac i i y %ðÞ
¼Flu sample Flu nega i e con ol
Flu posi i e con ol Flu nega i e con ol 100;
(2)
whe e he Flu sample co esponds o he luo escence o
each sample, Flu nega i e con ol co esponds o cells g own
in 5% / DMSO solu ion, and Flu posi i e con ol ep esen s a
seeding o 50 000 cells pe well, supe io o he maximum es ed
densi y in he assay o assu e ha a e y high g ow h o bo h
cell lines was achie ed.
A e de e mining he op imum cell numbe o each cell
line, cells we e seeded and 6 h la e ibe s we e added o he
wells. A e 24 and 48 h o incuba ion a 37 1C and 5% CO
2
a mosphe e, he me abolic ac i i y o he cells was e alua ed
using he Alama Blue assay.
2.8.3. Cell lysis assessmen . The cell lysis es was pe -
o med by ollowing he ins uc ions om LDH ki (In i ogen).
Fi s ly, he op imum cell numbe o pe o ming he LDH assay
was de e mined by seeding cells a concen a ions om 0 o
10 000 cells pe well in 96-well la black issue pla es (6 epli-
ca es o each cell concen a ion). Cells we e incuba ed o e -
nigh a 37 1C and 5% CO
2
a mosphe e. A e wa ds, 10 mLo
s e ile wa e we e added o he i s h ee eplica es o each cell
concen a ion (spon aneous LDH ac i i y), whe eas 10 mLo 2%
/ T i on solu ion in DPBS we e added o he second se o
eplica es o each cell concen a ion (maximum LDH ac i i y).
Cells we e again incuba ed o 45 min, p o ec ed om ligh ,
ollowed by he ans e o 50 mL o each supe na an o a new
pla e, o which 50 mL o a eac ion mix u e was added. Cells
we e once mo e incuba ed a oom empe a u e (RT) du ing
10 min, p o ec ed om ligh . Then, 50 mL o a s op solu ion
we e added o all wells and he luo escence was measu ed
(l
exc
= 560 and l
em
= 590 nm) using Tecan luo ime e . The
maximum LDH elease ac i i y abso bances minus he spon a-
neous LDH elease abso bances e sus he es ed cell numbe s
we e plo ed using he G aphPad P ism 8.0 so wa e.
Cells we e hen seeded a he de e mined op imum cell
numbe and, 6 h la e , ibe s we e added. A e 24 h o
incuba ion, he cell lysis es was epea ed. The pe cen ages
o cell lysis we e hen de e mined using he ollowing eqn (3):
Cell lysis %ðÞ¼ Fluo sample Fluo spon aneous c l
Fluo maximum c l Fluo spon aneous c l
100;(3)
whe e he Fluo sample co esponds o he luo escence o cells
ea ed wi h each ib ous sample, Fluo spon aneous c l ep e-
sen s he cells ea ed wi h s e ile dH
2
O (spon aneous LDH
ac i i y) and Fluo maximum c l co esponds o he cells ea ed
wi h 2% / T i on (maximum LDH ac i i y).
2.8.4. Cell mo phology obse a ions. Cells we e isualized
a b igh ield using a ligh mic oscope (Ca l Zeiss Suzhou Co.,
L d). Ligh mic oscope images we e ob ained a he beginning
o he expe imen ( = 0 h) and a e 24 h o incuba ion in
con ac wi h he samples, o de ec any possible changes in cell
mo phology. The images we e acqui ed a a 10magni ica ion
objec i e using an Axiocam 105 colo came a (Zeiss, Swi ze -
land), using a Zeiss ZEN 3.8 so wa e.
2.9. Cell mig a ion
2.9.1. Cell cul u e. Immo alized HaCaT we e cul u ed in
DMEM, supplemen ed wi h 10% / FBS and 1% / an ibio ic–
an imyco ic solu ion. Cellula subcul u es we e pe o med
when con luence eached alues close o 80–90%. HaCaT cells
we e main ained in 25 cm
2
o 75 cm
2
issue cul u e lasks in an
incuba o a 37 1C in a humidi ied a mosphe e wi h 5% CO
2
.
The cell cul u e medium was enewed 2pe week. Fo sub-
cul u es and pla ing, he adhe en cells we e de ached wi h
ypsin solu ion 0.05% / , and esh medium was added in
o de o neu alize he ypsin. The cell suspension was cen-
i uged 5 min a 160 g. The supe na an was disca ded, and
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esh medium was added o ob ain a new cell suspension. The
cell suspension was injec ed in a Neubaeu chambe , and he
cell concen a ion was de e mined.
2.9.2. Sc a ch assay. The effec o PCL/SA/CA, PCL/SA–
AAPV/CA, PCL–CLO/SA/CA and PCL–CLO/SA–AAPV/CA samples
on he mig a ion p o iles o HaCaT cells was e alua ed by
mic oscopic isualiza ion a e he sc a ch assay. Fibe s we e
washed and disin ec ed 3wi h dH
2
O and 1wi h e hanol a
70% / , du ing 5 min each, p io o he expe imen .
Cells we e seeded a a densi y o 5.0 10
4
cells pe well, on a
24-well issue cul u e pla e (T asadingen, Swi ze land) he day
be o e he expe imen s. A e con i ma ion o cell con luency, a
sc a ch on he cell monolaye was pe o med wi h he help o a
disposable s anda d 200 mL pipe e ip. The wells we e washed
wi h s e ile PBS and esh medium was added o he wells p io
o he isualiza ion and eco ding o he sc a ch in a Leica DMI
3000 B in e ed luo escence mic oscope (Hee b ugg, Swi ze -
land) equipped wi h Leica DFC450 Ccame a (Hee b ugg,
Swi ze land). The cells we e hen exposed o he samples and
we e u he incuba ed a 37 1C in a humidi ied a mosphe e o
5% CO
2
. Cells incuba ed only wi h esh cul u e medium we e
used as con ol o cell mig a ion. A he end o 24 and 48 h o
con ac , he cell mig a ion was e alua ed by mic oscopic isua-
liza ion (phase con as , 10magni ica ion) and de e mined
using he ImageJ
s
so wa e ( e sion 1.53, Na ional Ins i u es o
Heal h, Be hesda, Ma yland, USA), espec i ely. The pe cen-
ages o wound a ea we e de e mined a 24 h and 48 h, using
he ollowing eqn (4):
32
%wound a ea ¼A ðÞ
A0
100;(4)
whe e A( ) co esponds o he a ea o each sample de e mined
a 24 h/48 h and A
0
co esponds o he a ea o each sample a =
0h.
2.10. S a is ical analysis
All measu emen s we e conduc ed in iplica e unless o he -
wise men ioned in he expe imen al sec ions. Nume ical da a
we e epo ed as mean s anda d de ia ion (SD). Da a we e
ea ed using G aphPad P ism 8.0 So wa e (G aphPad So wa e
Inc, USA). No mali y analysis was pe o med, and esul s we e
analyzed using One-way ANOVA and Tukey es s. S a is ically
signi ican diffe ences we e conside ed a po0.05.
3. Resul s and discussion
The iaxial we -spun ibe s we e p oduced ia we -spinning, as
desc ibed in Sec ion 2.2. The p esence o h ee dis inc laye s
on he ibe s was con i med ia b igh ield mic oscopy
and scanning elec on mic oscopy (SEM; Fig. 1). Th ough
Fig. 1 Mic og aphs o iaxial we -spun ibe mo phology ob ained ia b igh ield mic oscopy o (a) he comple e sys em (1: co e composed o PCL–
CLO; 2: in e media e laye composed o SA–AAPV; 3: shell composed o CA); and (b) o he i-axial ibe s wi hou addi i es (1: co e composed o PCL; 2:
in e media e laye composed o SA; 3: shell composed o CA). Visualiza ion o he (c) su ace mo phology (magni ica ion o 500) and (d) c oss-sec ion
(magni ica ion o 70) o he comple e iaxial ibe s ob ained by SEM.
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mo phological, mechanical and he mal p ope y analyses
(da a unde pee e ision), he lexibili y and elas ici y o he
ibe s was demons a ed, eaching alues up o E300% o
maximum elonga ion. Also, he enginee ed iaxial ibe s we e
deemed highly s able when exposed o physiological-like
media, du ing 28 days o incuba ion, losing only 23% in mass
h oughou ha pe iod. In he end, he iaxial we -spun ibe s
we e p o en effec i e o kni ing a wound d essing, in his way
assu ing he sui abili y o his sys em o wound healing
applica ions.
The cu en wo k ocused on he biological p ope ies o he
iaxial sys em, including an imic obial and an ioxidan ac i -
i ies, he elease p o iles o he ac i e agen s (AAPV and CLO),
he egula ion o he local enzyma ic ac i i y, and he cy ocom-
pa ibili y o each ibe ypology (sa e y).
3.1. WAAPV elease kine ics
In o de o map AAPV elease om pep ide-loaded we -spun
ibe s, a modi ied e sion o AAPV, in which a yp ophan was
a ached o he N- e minus, was used (WAAPV). Such modi ica-
ion was necessa y due o an o e lap o maximum abso bance
bands om PCL, SA and CA polyme s, cen e ed a E200 nm,
de ec ed ia UV- isible spec oscopy. As a esul , WAAPV p e-
sen ed a maximum luo escence peak a E275 nm, de ec ed
using luo ime y, hus allowing o eliable de ec ion o his
compound wi hou any in e e ence om he emaining ibe
compounds (con i med by he analysis o he unloaded ibe s).
Cumula i e elease p o iles o WAAPV we e de e mined by
compa ison wi h he calib a ion cu e o he analyzed pep ide
(Fig. 2). SA–WAAPV monolaye ed ibe s a ained he highes
elease p o iles, since no inne o ou e laye s we e p esen o
p o ec he payload, leading o a highe exposu e o he pep ide
o he media. In i s p esence, he inne laye made o PCL o
PCL–CLO condi ioned he pep ide elease om bo h PCL/
SA–WAAPV (di e ence o E3.25% compa ed o SA–AAPV) and
PCL–CLO/SA–WAAPV coaxial ibe s (di e ence o E7.04%
compa ed o SA–AAPV). As obse ed in a p e ious esea ch,
7
PCL ends o pa ially inco po a e he SA o SA–WAAPV in e -
media e laye s, in e e ing wi h he possible mig a ion o he
pep ide owa ds he su ounding media. Howe e , in his
combina ion, he p esence o isible olds and b eakable si es
along he SA–WAAPV shell (Fig. S1 in he ESI†) may ha e
po en ia ed i s deg ada ion, accele a ing pep ide elease. Mo e-
o e , he pa ial dissolu ion o SA while in con ac wi h he
physiological media in esponse o i s highly hyd ophilic na u e
and inc eased a ini y owa ds wa e may also jus i y such
ou come.
33,34
PCL/SA–WAAPV/CA iaxial ibe s epo ed he lowes
WAAPV elease p o iles, since i was condi ioned by bo h he
inne mos laye o PCL and an ou e mos laye made o CA,
which was endowed wi h high mechanical esis ance and
hyd ophobic beha io , limi ing he in e ac ions o WAAPV wi h
wa e molecules om PBS.
35–37
Tha same conclusion could be
e ained o explain he low elease p o iles egis e ed o SA–
WAAPV/CA coaxial ibe s. On he o he hand, PCL–CLO/SA–
WAAPV/CA iaxial ibe s showed a sligh ly supe io elease a e
compa ed o PCL/SA–AAPV/CA iaxial ibe s (E7.70% o inc e-
men ). The p esence o CLO, a compound wi h high a ini y
owa ds PCL, is expec ed o ha e al e ed he con o ma ion o
PCL polyme ic chains, leading o a lowe pa ial inco po a ion
o he SA–WAAPV in e media e laye , he e o e acili a ing
access o he pep ide om he physiological media.
13
O e all,
all WAAPV-loaded ibe s displayed a p olonged and sus ained
elease o he pep ide, assu ing hei sui abili y as e ec i e d ug
deli e y pla o ms. E en hough he mechanism o ac ion o
WAAPV is no ye ully unde s ood, i is plausible o assume a
simila i y wi h AAPV, since bo h pep ides sha e equal amino
acids, apa om yp ophan.
3.2. CLO elease p o ile
CLO’s elease kine ics was assessed a e ibe con ac wi h PBS
up o 24 h o incuba ion (Fig. 3). Da a om unloaded ibe s
we e also collec ed o elimina e in e e ence o polyme s om
he CLO-loaded ibe esul s. As expec ed, he PCL–CLO mono-
laye ed ibe s elease he mos CLO in he sho es pe iod o
ime, because o he easie access o he oil by he su ounding
media.
22,38
In e es ingly, bo h PCL–CLO/SA and PCL–CLO/SA–
AAPV coaxial ibe s also eached simila ou comes. One plau-
sible explana ion o his occu ence may ely on he eeble
mechanical p ope ies and media ins abili y o he SA and SA–
AAPV ou e laye s.
39
I is likely ha he educed s uc u al
in eg i ies along wi h he hyd ophilic cha ac e o he ou e
laye s in bo h PCL–CLO/SA and PCL–CLO/SA–AAPV coaxial
ibe s caused he pa ial dissolu ion o SA and SA–AAPV on o
he physiological media and, hus, p omp ed he elease o
CLO.
33,34
As CA displays high igidi y and s uc u al in eg i y,
con e ing a ba ie e ec on he we -spun ibe s, PCL–CLO/CA
and he iaxial ibe s epo ed a slowe elease kine ics o
CLO.
22,38,40
Gene ally, all ibe ypologies expe ienced a bu s
elease a e 1 h o incuba ion, con inuously inc easing he CLO
libe a ion om ha poin and un il he 6 h ma k. A e ha
pe iod oil elease became mo e sus ained, mos likely because
Fig. 2 Cumula i e elease kine ics o WAAPV-loaded we -spun ibe s.
Da a a e epo ed as mean SD (n= 3). S a is ical signi icance was
de e mined ia he Tukey es , applying mul iple compa isons be ween
he diffe en ibe ypologies (no s a is ical signi icances we e de ec ed).
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access o he emaining molecules was mo e challenging
(s onge in e ac ions wi h he polyme s).
3.3. HNE inhibi ion
The inhibi ion o he HNE ac i i y by AAPV-loaded we -spun
ibe s was assessed by hei incuba ion in PBS a 37 1C o a
pe iod o 24 h, simul aneously con i ming he success ul
inco po a ion o he pep ide (Fig. 4). Da a om unloaded ibe s
was also collec ed and sub ac ed om he esul s om AAPV-
loaded ibe s, hus emo ing he polyme in e e ence. All
ibe s p esen ed HNE inhibi ions supe io o 15% in hei i s
hou o incuba ion, which was associa ed wi h an ini ial bu s
elease o he pep ide (Fig. 2). Howe e , a e 6 h and un il he
24 h ma k, all samples epo ed a smalle inhibi o y p o ile,
since he pep ide was eleased a a slowe pace (Fig. 3).
As expec ed, iaxial ibe s epo ed lowe HNE inhibi o y
capaci ies in compa ison wi h coaxial and monolaye ed ibe s,
due o he p esence o he CA p o ec i e laye , which limi ed he
access o AAPV.
22,38,40
Addi ionally, in he p esence o PCL, SA
is pa ially los , becoming en apped in he PCL polyme
chains, he e o e in e e ing wi h he a ailabili y o AAPV and
i s elease o he media.
7
Tha same heo y explains no only he
supe io HNE inhibi ion om SA–AAPV/CA coaxial ibe s, bu
also he lowe HNE inhibi o y p o iles obse ed o PCL–CLO/
SA–AAPV coaxial ibe s. Since he a ini y be ween CLO and PCL
is g ea ly p omo ed by he in e ac ion be ween he hyd oxyl
g oups o eugenol (one o he main elemen s o CLO) and he
ca bonyl g oups o PCL, leading o he molecula dispe sion o
CLO h oughou he PCL polyme ic ma ix, he inco po a ion o
he SA–AAPV in e media e laye was exace ba ed.
13,41
Howe e ,
a e 6 h, such di e ences we e less p onounced, mos likely
due o he cha ac e is ic high ola ili y o he EOs.
9,13
Despi e
inducing a smalle HNE inhibi o y p o ile (in a e age), he
iaxial ibe s did no epo signi ican di e ences compa ed
o he o he samples, being s ill classi ied as e icien in
egula ing HNE ac i i y, wi h inhibi o y capaci ies up o
E65%, and hus capable o con ibu ing o he healing
p ocess.
6
3.4. An ioxidan ac i i y
The an ioxidan ac i i ies o CLO-loaded ibe s we e e alua ed
by incuba ing he samples o 24 h in absolu e e hanol, p io o
conduc ing he DPPH adical sca enging assay (Fig. 5 and Fig.
S2 in he ESI†). Again, da a om unloaded ibe s we e collec ed
o emo e he polyme in e e ence in he esul s.
CLO’s s ong an ioxidan p ope ies o igina e in eugenol,
one o i s main componen s.
31
B and-Williams e al. showed
ha he sca enging ac i i y o eugenol is achie ed by he ini ial
o ma ion o an an ioxidan adical, ollowed by h ee ypes o
eac ion pa hways, including he dona ion o a second hyd o-
gen a om o he DPPH adical, he o ma ion o a complex
be ween one DPPH adical and one a yl adical, and he
dime iza ion be ween wo phenoxyl adicals ha egene a e
wo hyd oxyl g oups capable o in e ac ing wi h DPPH.
42
Euge-
nol’s abili y o in e e e wi h ee adicals om hyd ogen
pe oxide offe s cell p o ec ion agains oxida i e s ess, which,
consequen ly, accele a es wound healing.
43
PCL–CLO monolaye ed ibe s eached one o he highes
DPPH educ ion a es ( a ying be ween E57.87% a 2 h, he
maximum, and E36.34% a 4 h, he minimum ac i i y). This
occu ed because o he absence o an ou e , p o ec i e ba ie ,
he e o e inc easing he a ailabili y and exposu e o CLO o he
media.
22,38
PCL–CLO/SA and PCL–CLO/SA–AAPV coaxial ibe s
also epo ed high an ioxidan ac i i ies, because o he SA and
SA–AAPV ou e laye s mechanical limi a ions, along wi h hei
pa ial inco po a ion by PCL, which acili a ed access o he oil
and esul ed in highe eleases in compa ison wi h CA-
con aining ibe s (Fig. 3).
7
As expec ed, PCL–CLO/CA and he
iaxial ibe s epo ed he lowes an ioxidan p o iles con i m-
ing he p o ec i e unc ions o he CA shell.
22,38,40
Bo h he
iaxial cons uc s and he PCL–CLO/CA coaxial ibe s
Fig. 3 Cumula i e elease kine ics o CLO-loaded we -spun ibe s. Da a
a e epo ed as mean SD (n= 3). S a is ical signi icance was de e mined
ia he Tukey es , applying mul iple compa isons be ween he diffe en
ibe ypologies (no s a is ical signi icances we e ound).
Fig. 4 HNE inhibi ion p o ile om AAPV-loaded we -spun ibe s. Da a a e
epo ed as mean SD (n= 3). S a is ical signi icance was de e mined ia
he Tukey es , applying mul iple compa isons be ween he diffe en ibe
ypologies (no s a is ical signi icances we e ound).
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demons a ed an ioxidan abili ies o e ime (in a e age 6.89–
56.67%, 1.51–34.30% and 1.87–32.49% o PCL–CLO/CA, PCL–
CLO/SA/CA and PCL–CLO/SA–AAPV/CA ibe s, espec i ely),
making hem sui able o hei in ended applica ion.
3.5. An ibac e ial es ing
S. au eus,S. epide midis,E. coli and P. ae uginosa g ow h
inhibi ion was e alua ed a e 24 h o exposu e o he ibe s
a 37 1C (Fig. 6). Resul s showed ha he an ibac e ial ac i i y
inc eased a e he i s hou o incuba ion, a clea effec o he
CLO ini ial bu s elease (Fig. 2). CLO-loaded ibe s achie ed
highe an ibac e ial efficacy in compa ison wi h unloaded
ibe s. Such an occu ence was p edic ed since many epo s
ha e add essed ha CLO’s s ong an ibac e ial p ope ies as a
esul o one o i s main elemen s, eugenol.
43,44
In ac ,
acco ding o he li e a u e, he CLO mechanism o ac ion is
based on i s accumula ion a he bac e ia cell su aces, dis up -
ing he cy oplasmic memb ane unc ion and in eg i y, conse-
quen ly leading o he leakage o cell con en and cell dea h.
9,11
PCL–CLO monolaye ed ibe s epo ed supe io an ibac e -
ial ac i i ies due o he la ge a ailabili y and exposu e o CLO
o he media as was also e idenced in he elease kine ics and
DPPH educ ion p o iles (Fig. 2 and 4). PCL–CLO/SA and PCL–
CLO/SA–AAPV coaxial ibe s p omo ed one o he la ges inhibi-
ions o bac e ia ac i i y, once mo e due o he eeble s uc u al
in eg i y o he SA laye , in which polyme agmen s could ha e
been eleased and pa ially dissol ed in he media. This way,
he ibe s’ ou e laye no longe es ic ed he access o he EO
loaded in o he ibe s, explaining hei an ibac e ial ac i i y.
45
In con as , he an ibac e ial e ec i eness o PCL–CLO/CA
Fig. 5 DPPH educ ion induced by CLO-loaded we -spun ibe s a e (a) 1, (b) 2, (c) 4, (d) 6 and (e) 24 h o incuba ion. Da a a e epo ed as mean SD
(n= 3). S a is ical signi icance was de e mined ia he Tukey es , applying mul iple compa isons be ween he diffe en ibe ypologies (1 h: po0.030
signi icance be ween PCL and PCL–CLO/SA–AAPV/CA; 2 h: no s a is ical signi icances de ec ed; 4 h: po0.030 signi icance be ween PCL–CLO/SA,
PCL–CLO/SA–AAPV and PCL/SA/CA, PCL–CLO/SA/CA, PCL/SA–AAPV/CA, PCL–CLO/SA–AAPV/CA; 6 h: po0.030 signi icance be ween PCL–CLO
and PCL–CLO/SA–AAPV, PCL–CLO/CA and PCL–CLO/SA/CA; 24 h: po0.002 signi icance be ween PCL–CLO, PCL–CLO/SA, PCL–CLO/SA–AAPV and
PCL/SA/CA, PCL–CLO/SA/CA, PCL/SA–AAPV/CA, PCL–CLO/SA–AAPV/CA).
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