pha maceu ics
A icle
Endogenous An ioxidan Cock ail Loaded Hyd ogel o Topical
Wound Healing o Bu ns
JoséL. So iano 1, Ana C. Calpena 2,3,* , Ma ía J. Rod íguez-Lagunas 4,5 ,Òsca Domènech 2,3 ,
Nu ia Bozal-de Feb e 6, Ma ía L. Ga duño-Ramí ez 7and Bea iz Cla es 1,3,8,*


Ci a ion: So iano, J.L.; Calpena, A.C.;
Rod íguez-Lagunas, M.J.; Domènech,
Ò.; Bozal-de Feb e , N.;
Ga duño-Ramí ez, M.L.; Cla es, B.
Endogenous An ioxidan Cock ail
Loaded Hyd ogel o Topical Wound
Healing o Bu ns. Pha maceu ics 2021,
13, 8. h ps://dx.doi.o g/10.3390/
pha maceu ics13010008
Recei ed: 26 No embe 2020
Accep ed: 17 Decembe 2020
Published: 22 Decembe 2020
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1Depa men o Pha macy and Pha maceu ical Technology, Facul y o Pha macy, Uni e si y o G anada,
18071 G anada, Spain; [email p o ec ed].es
2
Depa men o Pha macy and Pha maceu ical Technology and Physical Chemis y, Facul y o Pha macy and
Food Sciences, Uni e si y o Ba celona, 08028 Ba celona, Spain; [email p o ec ed]
3Nanoscience & Nano echnology Ins i u e (IN2UB), Uni e si y o Ba celona, 08028 Ba celona, Spain
4Depa men o Biochemis y and Physiology, Facul y o Pha macy and Food Sciences,
Uni e si y o Ba celona, 08028 Ba celona, Spain; mj [email p o ec ed]
5Nu i ion and Food Sa e y Resea ch Ins i u e (INSA-UB), 08921 San a Coloma de G amene , Spain
6Depa men o Biology, Heal hca e and he En i onmen , Facul y o Pha macy and Food Sciences,
Uni e si y o Ba celona, 27-31 Joan XXIII A e., 08028 Ba celona, Spain; nu iabozalde eb [email p o ec ed]
7Cen o de In es igaciones Químicas, Uni e sidad Au ónoma del Es ado de Mo elos,
A . Uni e sidad No. 1001, Col Chamilpa, 62209 Cue na aca, Mexico; [email p o ec ed]
8Biosani a y Ins i u e o G anada (ibs.GRANADA), 18012 G anada, Spain
*Co espondence: [email p o ec ed] (A.C.C.); bea izcla es@ug .es (B.C.); Tel.: +34-934-024-560 (A.C.C.);
+34-958-246-664 (B.C.)
Abs ac :
The main goal o his wo k is he s udy o he skin wound healing e icacy o an an ioxidan
cock ail consis ing o i amins A, D, E and he endogenous pineal ho mone mela onin (MLT), wi h
all o hese loaded in o a he mosensi i e hyd ogel deli e y sys em. The esul ing o mula ion
was cha ac e ized by scanning elec on mic oscopy. The an ioxidan e icacy and mic obiological
ac i i y agains G am posi i e and G am nega i e s ains we e also assayed. The skin healing
e icacy was es ed using an
in i o
model which included his ological e alua ion. Fu he mo e,
a omic o ce mic oscopy was employed o e alua e he wound healing e icacy o a skin bu ns
h ough he de e mina ion o i s elas ici y a he nanoscale using o ce spec oscopy analysis. The
esul ing hyd ogel exhibi ed sol s a e a low empe a u e and u ned in o a gel a 30
±
0.2
◦
C. The
hyd ogel con aining he an ioxidan cock ail showed highe sca enging ac i i y han he hyd ogel
con aining i amins o MLT, sepa a ely. The o mula ion showed op imal an imic obial ac i i y. I
was compa able o a comme cial e e ence. I was also e idenced ha he hyd ogel con aining he
an ioxidan cock ail exhibi ed he s onges healing p ocess in he skin bu ns o a s, simila o he
assayed comme cial e e ence con aining sil e sul adiazine. His ological s udies con i med he
obse ed esul s. Finally, a omic o ce mic oscopy demons a ed a simila dis ibu ion o Young’s
modulus alues be ween bu ned skin ea ed wi h he comme cial e e ence and bu ned skin ea ed
wi h hyd ogel con aining he an ioxidan cock ail, and all hese wi h heal hy skin. The use o an
an ioxidan cock ail o i amins and MLT migh be a p omising ea men o skin wounds o u u e
clinical s udies.
Keywo ds: i amins; mela onin; an ioxidan ; skin; healing; ATF mic oscopy
1. In oduc ion
The skin is a mic obiological, chemical, immunological and physical ba ie ha
con e s p o ec ion o he o ganism agains ex e nal ha ms [
1
]. This ba ie can be dis up ed,
allowing ex e nal pa hogens o en e he body, which causes in lamma ion and in ec ion.
Among skin wounds, bu ns a e he mos equen skin inju ies. This kind o inju y can
be caused by he mal, elec ical, chemical, o elec omagne ic ene gy. The se e i y o
Pha maceu ics 2021,13, 8. h ps://dx.doi.o g/10.3390/pha maceu ics13010008 h ps://www.mdpi.com/jou nal/pha maceu ics
Pha maceu ics 2021,13, 8 2 o 17
he bu n co ela es wi h he laye s o he epide mis, de mis and hypode mis a ec ed,
inc easing he mo bidi y and mo ali y when he su ace a ea o he bu n inc eases [
2
].
The induced damage is accompanied by he ac i a ion o in lamma o y and coagula ion
p ocesses, as well as an excess o cy o oxic eac i e oxygen and ni ogen species (ROS,
RNS), in ol ing seconda y issue damage [
3
]. In his ega d, he de elopmen o e ec i e
an ioxidan ools o cu aneous wound healing would help o educe issue damage and
wound in ec ion, as well as o he associa ed complica ions [
4
]. Recen ly, his an ioxidan
s a egy has been also in es iga ed [
5
,
6
]. Classical ea men s o opical wound bu ns
include he gold s anda d sil e sul adiazine. Howe e , some de imen al e ec s such as
cy o oxic ac i i y and wound healing delay ha e been desc ibed [
7
]. Thus, he esea ch o
new app oaches o con olling in ec ion and a oiding undesi ed cy o oxic e ec s, a he
same ime, ep esen s a challenge o pa amoun impo ance nowadays. Among an ioxidan
agen s o wound healing, se e al ypes o i amins ha e been p oposed, such as i amins
E (alpha ocophe ol) and D. Alpha ocophe ol p e en s he oxida ion damage o collagen
and glycosaminoglycans du ing wound healing, being able o accele a e he wound closu e
and amelio a e bu n in ec ion [
8
,
9
]. Rega ding i amin D, his i amin has been epo ed
o modula e immune esponses [
10
], and i has also been p o ed o p esen a bene icial
e ec in sun bu ns [
11
]. Mo eo e , i amin D igge s he o ma ion o an imic obial
pep ides ha may con ibu e o he wound healing p ocess [
12
]. Vi amin A, despi e no
being a common an ioxidan , is able o educe oxida i e s ess [
13
] and e en p e en he
cance isks o oxida i e damage [
14
]. Recen s udies ha e shown ha i amin A a o s
supe oxide dismu ase and glu a hione ans e ase ac i i ies [
15
]. Fu he mo e, i has also
been associa ed wi h pa icipa ion in wound healing, speci ically in mac ophage-media ed
in lamma o y p ocesses as well as angiogenesis [
16
]. Taking all hese i amins oge he , we
ha e p e iously de eloped a he mosensi i e hyd ogel loaded wi h i amin A, D and E o
he ea men o skin bu ns [17].
O he an ioxidan molecules, such as he endogenous ho mone mela onin (MLT; 5-
me hoxy-N-ace yl- yp amine), has also been assayed in his ield [
18
]. MLT is sec e ed
by he pineal gland and plays an impo an ole as a po en adical sca enge o eac i e
oxygen species (ROS) and eac i e ni ogen species (RNS). In a p e ious pape , we e iewed
he ole o mela onin (MLT) as a powe ul an ioxidan [
19
]. The an ioxidan e ec s o MLT
occu h ough di ec and indi ec mechanisms. On one hand, MLT sca enges ee adicals
in all compa men s o he body due o i s amphiphilia and i s dis ibu ion capaci y [
20
].
MLT has been shown o de oxi y up o 10 adicals [
21
]. On he o he hand, MLT inc eases
he ac i i y o an ioxidan enzymes. This causes an inc ease in he endogenous an ioxidan
de ense capaci y and induces he up egula ion o gene exp ession, inc easing he i s line
o de ense agains oxida i e damage o cells [
22
]. Due o hese p ope ies, i has been
p oposed as a po en ial ea men o wound healing [
23
,
24
], being mo e e ec i e han
i amins C and E [
25
,
26
]. In addi ion o he an i-in lamma o y e ec o MLT, i has some
ad an ages wi h espec o o he an ioxidan molecules, such as i s endogenous na u e
and i s acili y o en e in o subcellula compa men s, among o he s [
19
,
27
]. Among
he bene icial e ec s o MLT,
in i o
and
in i o
an imic obial p ope ies ha e been also
epo ed [
28
,
29
]. In his sense, MLT has shown ac i i y agains ungi such as Saccho amyces
ce e isae o Candida albicans [
30
], some i uses [
31
] and bo h G am posi i e and G am
nega i e bac e ia. Some au ho s ha e concluded ha i s an imic obial ac i i y could be
due o he educ ion in in acellula subs a es, which leads o a p olonga ion o he lag
phase o bac e ial g ow h [32].
Thus, he possibili y o using i amins and MLT oge he migh be a sui able com-
bina ion o ea ing skin bu ns and imp o ing he p oli e a ion and di e en ia ion o
skin cells. Fo his ask, a d ug-deli e y sys em o skin wound healing able o abso b
wound exuda es and p o ide a mois en i onmen wi h in insic an imic obial p ope ies
should be desi able. Di e en d ug-deli e y sys ems o loading hese an ioxidan d ugs
ha e been de eloped so a o ea ing wounds, such as mic osphe es [
33
], nanopa i-
cles [
34
], nanosphe es [
35
], e c. I is also ema kable o ake in o conside a ion he biological
Pha maceu ics 2021,13, 8 3 o 17
p ope ies o bioma e ials o ming he d ug ca ie . In p e ious s udies, ou g oup has
demons a ed he po en ial o a hyd ogel loading an ioxidan ac i es in wound healing, con-
sis ing o poloxame 407 (PLX), chi osan (CS) and hyalu onic acid (HA) [
17
,
36
]. Hyd ogel
migh abso b exuda es and p o ide a mois en i onmen , p e en ing wound dehyd a-
ion [
37
]. In his con ex , in si u gel- o ming hyd ogel is e en mo e app op ia e due o i s
abili y o ill he wound a ea as sol s a e jus a e opical adminis a ion and adhe e when
con e ed in o gel [38].
As a nex s ep, i is ou in en ion o e alua e he e ec o an ioxidan ac i es, i amins
and MLT oge he , as well as he use o echniques ha help o eco d he healing ac ion o
hese an ioxidan d ugs on he skin.
A omic Fo ce Mic oscopy (AFM) has eme ged as a use ul echnique no only o
isualize samples a a nanome ic le el, bu also as a powe ul echnique o quan i y
unc ional and s uc u al cha ac e is ics om he nanoscale o he mic oscale, om single
molecules o whole cells and issues. AFM has been widely used o in es iga e indi idual
cells [
39
] o he ex acellula ma ix (ECM) o so issues [
40
,
41
]. In ecen yea s, he
in es iga ion o he whole issue [
42
,
43
] is an eme ging ield o unde s and, in a bo om-up
way, he classical biological expe imen s on issues.
In he p esen wo k, we p oposed a s a egy o he wound healing o bu ns, which
combined an ioxidan i amins A, E, D and he endogenous ho mone MLT. The an ioxidan
cock ail was loaded in a PLX/CS/HA hyd ogel. Fo his ask, he wound healing e icacy
was assessed
in i o
and his ologically. Fu he mo e, we e alua ed he elas ic modulus,
Young’s modulus, by means o o ce spec oscopy AFM (FS-AFM) o bu ned a skin
samples a e di e en ea men s o in es iga e he he apeu ic goodness on a cu aneous
wound model. In o de o suppo he p e ious es s, he an ibac e ial and an ioxidan
ac i i y was also de e mined.
2. Ma e ials and Me hods
2.1. Ma e ials and Animals
MLT and sodium hyalu ona e (1.46 MDa) we e pu chased om Aco a (Mad id,
Spain). Re inol palmi a e ( i amin A) 200,000 IU/mL, cholecalci e ol ( i amin D3)
100,000 IU/g, and D,L alpha ocophe ol ace a e ( i amin E) we e supplied by Fag on
Ibe ica (Ba celona, Spain). PLX was ob ained om BASF (Ba celona, Spain). Sigma-Ald ich
(Mad id, Spain) supplied 75–85% deace yla ed CS (190–310 KDa).
The comme cial opical c eam sil ede ma
®
con aining sil e sul adiazine 10 mg/g
(Aldo-Union Co., Ba celona, Spain) expi y da e 01/2021, ba ch 0012M003, was pu chased
om a local pha macy. Double dis illed wa e was ob ained om a de ice Milli-Q
®
G adien A10 (Millipo e Ibe ica, Mad id, Spain). All o he ma e ials used in expe imen al
sec ions we e o analy ical g ade and supplied by Sigma-Ald ich (Mad id, Spain) unless
o he wise speci ied.
Wis a a s, 3–5 mon hs old and weighing 300–500 g, we e pu chased om he Labo a-
o y Animal Cen e o Ba celona Uni e si y. The animals we e main ained unde s anda d
condi ions wi h ee access o ood and wa e in he labo a o y animal acili ies o one
week be o e he beginning o s udies. All a s we e ea ed humanely and unde e e ina y
supe ision h oughou he expe imen al pe iod.
2.2. P epa a ion and Cha ac e iza ion o Hyd ogel
The PLX/CS/HA based hyd ogel was p epa ed ia we con en ional syn hesis by
di ec dispe sion in wa e . B ie ly, 0.2% HA solu ion (w/ ) was p epa ed by adding he
equi ed amoun o HA o double dis illed wa e and hen s i ed o 1 h, subsequen ly
he solu ion was il e ed. Then, a 0.5 % (w/ ) CS/ace ic acid solu ion was ob ained by
dispe sing he co ec amoun o CS in 0.5% ace ic acid. Equally, 1.8% PLX (w/ ) was
dissol ed in double dis illed wa e wi h con inuous s i ing. The inclusion o ac i es was
ca ied ou as ollows.
Pha maceu ics 2021,13, 8 4 o 17
A p e-selec ed amoun o i amin D was pou ed in o he equi ed olumes o i amins
A and E and dispe sed. This mix u e, oge he wi h he amoun o MLT equi ed o each
1% (w/ ), was added o he p e ious PLX solu ion un il solubiliza ion a 4
◦
C. A e wa ds,
his solu ion con aining ac i es and he CS solu ion we e added o he HA solu ion a 4
◦
C
and s i ed con inuously o 24 h. Finally, he equi ed amoun o PLX o each 18% (w/ )
was added, and his was le o s and o 24 h. The inal composi ion o he o mula ion
labeled as PLX/CS/HA-VM was: PLX 18% (w/ ), CS 0.5% (w/ ), HA 0.2% (w/ ), i amin
A 6000 IU/g, i amin D 400 IU/g, i amin E 2% (w/ ) and MLT 1% (w/ ). Fu he mo e,
PLX/CS/HA hyd ogel loading i amins (PLX/CS/HA-V) o MLT (PLX/CS/HA-M) we e
also p epa ed o an ioxidan , his ological and AFM s udies compa isons.
The sol/gel ansi ion empe a u e and ime was assessed in iplica e by he measu e-
men o ime and empe a u e a which a magne ic ba s opped mo ing owing o gela ion.
Fo his ask, a 10 mL sample was pu in o a anspa en ial con aining he magne ic ba
a cons an o a ion speed o 400 pm in a magne ic wa e ba h om 4 o 37 ±0.1 ◦C.
Scanning elec on mic oscopy (FEI Quan a
®
FEG 650, The mo Fishe Scien i ic-FEI,
Hillsbo o, CA, USA) was u ilized o in es iga e he mo phology o PLX/CS/HA-VM.
Samples we e p ocessed using he c i ical poin d ying echnique. PLX/CS/HA-VM was
spu e ed wi h ca bon be o e obse a ion.
The pH alue o hyd ogel was eco ded using a digi al pH me e GLP 22 (C ison
Ins umen s, Alella, Spain) a oom empe a u e. The measu emen s we e conduc ed by
di ec imme sion o he de ice elec ode in he sample. Ob ained da a a e exp essed as he
mean ±SD o h ee eplica es.
2.3. An ioxidan E iciency
The adical sca enging ac i i y o blank PLX/CS/HA, PLX/CS/HA-V, PLC/CS/HA-
M and PLX/CS/HA-VM was es ed by measu ing hei capaci y o sca enge he s able 1,
1-diphenyl-2-pic ylhyd azyl (DPPH, Sigma-Ald ich Chemie, S einheim, Ge many). Fo
his ask, each hyd ogel was p epa ed by aking 500
µ
L o he o mula ion and 1500
µ
L
o he DPPH e hanolic solu ion. The eac ion mix u es we e shaken igo ously and hen
kep a 30
◦
C. Thei abso bances we e eco ded in iplica e spec opho ome ically wi h
he me hod o a sligh ly modi ied es o B and-Williams [
44
] a 515 nm on a Spec onic
Genesys 8 UV/Vis spec opho ome e (Fishe Scien i ic, Roches e , NJ, USA).
The pe cen age o adical sca enging ac i i y (RSA%) was calcula ed acco ding o he
ollowing equa ion:
RSA% =
A0−As
A0×100 (1)
whe e A
0
is he abso bance o he con ol and A
s
is he abso bance o he samples a 515 nm.
2.4. An imic obial Ac i i y
The an imic obial ac i i y es was ca ied ou by he Ki by-Baue Disk Di usion
Suscep ibili y Tes [
45
]. G am nega i e bac e ia such as Acine obac e baumanii ATCC 19606,
A. baumanii ABAU clinically isola ed, Esche ichia coli ATCC 25922, Pseudomonas ae uginosa
ATCC 27823 and P. ae uginosa PAO-1 clinically isola ed, as well as G am posi i e bac e ia
such as S aphylococcus au eus ATCC 29213 and S aphylococcus au eus MARSA ATCC 43300)
and ungi Candida albicans ATCC10231 we e assayed. MacFa land s anda d suspensions
(1.5
×
108 colony- o ming uni s/mL) we e used o each s ain and hen added on Muelle -
Hin on aga pla es. Then, s e ile il e -pape discs (6 mm diame e ) we e placed on he
su ace and 25
µ
L o a sample was placed on o he discs. The aga pla es we e incuba ed a
37 ◦C o 48 h.
2.5. In Vi o Wound Healing S udy
This s udy was p e iously app o ed by he animal esea ch e hical commi ee 387/18
o he Uni e si y o Ba celona acco ding o Spanish law (Royal Dec ee 53/1 Feb ua y 2013)
based on he Eu opean di ec i e 2010/63/UE.
Pha maceu ics 2021,13, 8 5 o 17
The animals we e anes he ized by iso lu ane 0.5% a 5 L/min adminis a ion ollowed
by an in ade mal injec ion o Bup ex
®
0.05 mg/kg analgesic. The animals we e supe ised
by a e e ina ian h oughou he s udy.
Fi s ly, he backs o he animals we e sha ed and cleaned wi h 70% e hanol. The a s
we e di ided in o g oups o h ee indi iduals (n= 3). Skin bu ns we e caused by a 1.0 cm
2
supe icial con ac a ea o cylind ical de ices a 100
◦
C. The ea men s we e applied
once daily o wo weeks in he ollowing way: G oup I posi i e con ol, he animals
in his g oup ecei ed no ea men ; G oup II, he animals we e ea ed wi h 100
µ
L o
PLX/CS/HA wi hou an ioxidan ac i es; G oup III, he animals we e ea ed wi h 100
µ
L
o PLX/CS/HA-VM; G oup IV, he animals we e ea ed wi h he comme cial e e ence
sil ede ma®. The wound healing p ocess (WH) was eco ded by he measu emen o he
wound size and applying he ollowing equa ion:
WH(%) = (ini ial wound size- inal wound size)/(ini ial wound size) ×100 (2)
2.6. His ological Analysis
A e he bu ns we e induced, he s udy animals we e eu hanized by ce ical dis-
loca ion. The skin o he bu ned a eas was excised. The issues we e ixed o 24 h a
25
◦
C in o maldehyde and hen washed wi h a phospha e bu e ed solu ion (PBS). The
samples we e hen dehyd a ed by imme sion in ascending g ades o e hanol solu ions
and clea ed wi h xylene. Once he samples we e dehyd a ed, hey we e embedded in
pa a in. To p oceed wi h isualiza ion, he samples we e cu in o 5
µ
m sec ions, s ained
wi h hema oxylin/eosin and moun ed in a ligh mic oscope Olympus BX41 equipped
wi h an Olympus XC50 came a (Olympus Co., Tokyo, Japan). A neu ophil quan i ica ion
was ca ied ou , which in ol ed di iding samples in o h ee pa s and coun ing in h ee
andom oci o he de mis.
2.7. AFM Fo ce Spec oscopy Expe imen s
Fo he AFM expe imen s, each skin sample was de os ed a oom empe a u e and
cu by de ma ome (Model GA 630, Aesculap, Tu lingen, Ge many) in o 400
µ
m hick pieces.
A e ha , he 0.5
×
0.5 cm
2
pieces o a skin we e immedia ely glued on o a s eel disc and
insed gen ly wi h bu e and wi h deionized wa e o elimina e any su ace con aminan .
Finally, he sample su ace was d ied unde a ni ogen s eam. Each sample was moun ed
di ec ly on o he AFM scanne loca ed in a chambe wi h a con olled empe a u e and
humidi y o p e en wa e loss om he skin a 24 ◦C and 70%, espec i ely.
The AFM de ice consis ed o an AFM Mul imode IV con olled by Nanoscope V elec-
onics (B uke AXS Co po a ion; San a Ba ba a, CA) equipped wi h a 15
µ
m piezoelec ic
scanne . Silicon AFM ips wi h a nominal sp ing cons an o 42 nN nm
−1
we e used. The
samples we e s udied in con ac mode and in ai , wi h a scan a e o 1.5 Hz and a scan
angle o 0◦.
Fo AFM Fo ce Spec oscopy measu emen s, he sp ing cons an o each can ile e
used was de e mined using he he mal noise me hod. Hund eds o o ces e sus ip–
sample dis ance cu es we e acqui ed in a leas 10 di e en spo s o each sample. The
ip–sample app oaching eloci y was se o all o ce cu es a 1000 nm s
−1
. Applied o ces
F a e gi en by F = kc
×∆
, whe e kc is he sp ing cons an o he can ile e and
∆
s ands
o he can ile e de lec ion. The su ace de o ma ion is gi en as pene a ion (
δ
), e alua ed
as δ= z −∆, whe e z ep esen s he piezo-scanne displacemen .
On he o he hand, he a skin elas ic modulus (Young’s modulus) was calcula ed
using he He z model by con e ing o ce e sus ip–sample dis ance cu es in o o ce
e sus inden a ion cu es. The He z model is a good app oxima ion o ou sys em as
i assumes inden a ion is negligible in compa ison o he sample hickness, so ha he
subs a e does no in luence he calcula ions. Thus, expe imen al o ce e sus
δ
da a we e
adjus ed o Equa ion (3) o ob ain he Young0s modulus [46].
F = E/(1 −υ2)× anα/√2×δ2 (3)

Pha maceu ics 2021,13, 8 6 o 17
whe e E is he Young’s modulus,
α
is he hal angle o he AFM ip and
υ
is he so-called
Poisson’s a io. In ou expe imen s,
α
was 22.5
◦
and
υ
was assumed o be 0.4 o a sligh ly
comp essi e su ace such as skin.
2.8. S a is ical Analysis
The esul s we e analyzed wi h one-way analysis o a iance (ANOVA) o e alua e di -
e ences among mean alues. P ism
®
so wa e, . 3.0 (G aphPad So wa e, Inc., San Diego,
CA, USA) was used. A p- alue < 0.05 was conside ed s a is ically signi ican .
3. Resul s
3.1. Cha ac e iza ion
The de eloped hyd ogel showed a whi ish hue and a homogeneous and luid appea -
ance, wi h a empe a u e-dependen sol-gel ansi ion a 30
±
0.2
◦
C a e
1.7 ±0.1 min.
The hyd ogel was a ee- lowing sol a low empe a u e (measu emen s s a ed om
18
◦
C) and u ned in o a non- lowing gel abo e 30
◦
C (Figu e 1). The ob ained pH o
PLX/CS/HA-VM was 5.0 ±0.1.
Figu e 1. (A) PLX/CS/HA-VM a 18 ◦C; (B) PLX/CS/HA-VM a 32 ◦C.
The in e nal mo phology o PLX/CS/HA-VM was obse ed by SEM. Figu e 2shows
he in e nal po ous h ee-dimension s uc u e wi h app oxima ely sphe ical and homoge-
neous micella size.
Figu e 2.
Scanning Elec on Mic oscopy image ob ained om PLX/CS/HA-VM, 30,000
×
magni ica ion.
Pha maceu ics 2021,13, 8 7 o 17
3.2. An ioxidan Ac i i y
Figu e 3depic s he an ioxidan e icacy esul s o he hyd ogel loading i amins, MLT
and he an ioxidan cock ail ( i amins A, D, E and MLT). As expec ed, blank hyd ogel
PLX/CS/HA showed no adical sca enging ac i i y. Rega ding MLT and i amins, s a is i-
cally signi ican di e ences we e obse ed be ween bo h o mula ions, wi h RSA alues
~18% and ~52%, espec i ely. Howe e , he alues o RSA o PLX/CS/HA-VM we e
signi ican ly highe , in he icini y o 70%.
Figu e 3. An ioxidan ac i i y o hyd ogels.
3.3. Mic obiological S udies
Table 1depic s he mic obiological esul s o PLX/CS/HA-VM and he comme cial e -
e ence agains G am posi i e, G am nega i e and ungi s ains. I is clea ha PLX/CS/HA
and PLX/CS/HA-VM p oduced signi ican inhibi ion o bac e ial g ow h; hey we e
oughly simila o he comme cial e e ence, excep in he case o E. coli ATCC 25922, o
which bo h he blank hyd ogel and he loaded hyd ogel did no show an imic obial ac ion.
Simila esul s we e ob ained in he case o g ow h inhibi ion assay.
Table 1.
Inhibi o y halos and g ow h educ ion p oduced by PLX/CS/HA, PLX/CS/HA-VM and he e e ence o mula ion
sil ede ma®agains di e en pa hogen mic oo ganisms.
Mic oo ganisms Inhibi ion Halos (mm) G ow h Reduc ion
Re e ence PLX/CS/HA PLX/CS/HA-
VM Re e ence PLX/CS/HA PLX/CS/HA-
VM
Acine obac e baumanii ATCC 19606 7 * 8 8 + * + +
Acine obac e baumanii ABAU 15 10 9 + + +
Esche ichia coli ATCC 25922 7 0 0 + − −
Pseudomonas ae uginosa ATCC 27823 7 10 7 + + +
Pseudomonas ae uginosa PAO-1 7 8 7 + + +
S aphylococcus au eus ATCC 29213 7 9 9 + + +
S aphylococcus au eus MARSA ATCC
43300 7 11 11 + + +
Candida albicans ATCC 10231 7 9 9 + + +
(*) Obse ed esis an colonies; (−) no g ow h inhibi ion; (+) g ow h inhibi ion.
Pha maceu ics 2021,13, 8 8 o 17
3.4. Wound Healing E ec on Ra Skin
The wound healing e icacy induced by PLX/CS/HA-VM on a skin bu ns is shown
in Figu e 4. Wounds ea ed wi h PLX/CS/HA-VM exhibi ed simila healing p og ess
o hose ea ed wi h he comme cial e e ence sil ede ma
®
. A e 14 days, a signi ican
accele a ion in wound healing was obse ed as compa ed wi h animals ea ed wi h he
unloaded PLX/CS/HA hyd ogel and he un ea ed g oup.
Figu e 4.
Wound healing e olu ion o 14 days in animals wi h no ea men (G oup I), ea ed wi h
PLX/CS/HA (G oup II), ea ed wi h PLX/CS/HA-VM (G oup III), and animals ea ed wi h he
comme cial e e ence sil ede ma®(G oup IV).
On he o he hand, wound closu e was analyzed in each g oup as a pe cen age o he
educ ion in he wounded a ea a e 14 days (Equa ion (2)), ob aining he ollowing a es:
54.23% in he case o he un ea ed g oup, 63.52% in he case o animals ea ed wi h blank
hyd ogel, 96.12% o he g oup ea ed wi h PLX/CS/HA-VM, and 98.53% o he g oup
ea ed wi h he comme cial e e ence.
3.5. His ological Obse a ion
His ological images o bu n si es 14 days pos -bu ning a e shown in Figu e 5. These
obse a ions we e used o e alua e he healing po en ial o assayed o mula ions. In he
case o un ea ed animals, an ulce and he p esence o in lamma o y cells co e ed by a
scab could be obse ed (Figu e 5B). The issue also showed inc eased epide mal hickness
and a loss o epide mal appendices such as hai ollicles. A simila pa e n was obse ed
in skin samples ea ed wi h blank hyd ogel (Figu e 5B), al hough he scabs we e smalle .
In specimens ea ed wi h he comme cial e e ence sil ede ma
®
(Figu e 5F), epide mis
g ow h showing wound con ac ion could be obse ed. The g oups ea ed wi h i amin
loaded hyd ogel and MLT loaded hyd ogel (Figu e 5E,D, espec i ely) also showed a good
eco e y, simila o he comme cial e e ence g oup. Howe e , i seems ha in conjunc ion
hey could amelio a e he egene a ion o he epide mis and de mis, as i can be obse ed
om he his ological e alua ion o samples ea ed wi h PLX/CS/HA-VM, in which bo h
Pha maceu ics 2021,13, 8 9 o 17
g oups o an ioxidan ac i es we e included (Figu e 5G). In his las case, skin showed
less in il a ion o in lamma o y cells, smalle epide mal hickness and mo e epide mal
appendices when compa ed o he comme cial e e ence ea men .
Figu e 5.
His ology o skin; (
A
) heal hy skin; (
B
) bu ned skin wi hou ea men ; (
C
) skin ea ed wi h PLX/CS/HA; (
D
)
skin ea ed wi h PLX/CS/HA-V; (
E
) skin ea ed wi h PLX/CS/HA-M; (
F
) skin ea ed wi h comme cial e e ence; (
G
) skin
ea ed wi h PLX/CS/HA-VM. Hema oxylin and eosin s ains nuclei blue/black while ke a in and cy oplasm a e s ained
ed. The as e isk indica es loss o s a um co neum and he a ow indica es in il a ion o in lamma o y cells, s = sca . Scale
ba = 200 µm.
3.6. A omic Fo ce Mic oscopy
Figu e 6shows ep esen a i e images o he heal h o a skin unde s udy. Figu e 6A
shows an op ical image o clean egions wi hou any de ached scales o hai . B igh e zones
we e p e e ed o e da ke ones (in e cellula gaps) o a oid any bo de e ec . Figu e 6A
shows he h ee-dimensional (3D) opog aphic iew o a skin su ace co esponding o
one o he b igh e a eas displayed in Figu e 6A. Some andom s uc u es ~500 nm wide
wi h mean heigh alues o 50–100 nm can be obse ed, bu no ke a in ib ils o o ganized
s uc u es we e de ec ed. As he scan size o his image is ~15
×
15
µ
m
2
, he pic u e is
equi alen o one ac ion o he su ace o a co neocy e. Fu he mo e, Figu e 6A shows he
co esponding de lec ion AFM image de i ed om Figu e 6B. This image highligh s he
bo de o he scanned s uc u es and e eals he oughness due o co neocy e disposi ion
ypical o heal hy skin [
47
]. Fu he mo e, he ic ion AFM image o skin is shown in
Figu e 6D o assu e he cleanliness o he e alua ed sample, om which i can be obse ed
ha no changes in colo a e eco ded. This is indica i e ha no lipids o o he con aminan s
a e p esen in he su ace o he skin unde s udy.
Pha maceu ics 2021,13, 8 16 o 17
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