RESEARCH ARTICLE
Low molecula weigh ε-cap olac one-p-
couma ic acid copolyme s as po en ial
bioma e ials o skin egene a ion
applica ions
Ma co Con a diID
1,2☯
*, Alejand o Al a o-Pulido
1☯
, Pasquale Picone
3
, Susana Guzman-
Puyol
1
, Luca Goldoni
4
, Jose
´J. Benı
´ ez
5
, An onio He edia
6,7
, Ma kus J. Ba hel
8
,
Luca Cese acciu
9
, Gio anni Cusimano
3
, O nella Robe a B anca o
3
, Ma a Di Ca lo
3
,
A hanassia A hanassiou
1
*, Jose
´A. He edia-Gue e o
1
*
1Sma Ma e ials, Is i u o I aliano di Tecnologia, Geno a, I aly, 2DIBRIS, Uni e si y o Genoa, Genoa, I aly,
3Is i u o di Biomedicina ed Immunologia Molecola e "A. Mon oy", CNR, Pale mo, I aly, 4Analy ical
Chemis y Facili y, Is i u o I aliano di Tecnologia, Geno a, I aly, 5Ins i u o de Ciencia de Ma e iales de
Se illa, Cen o mix o CSIC-Uni e sidad de Se illa, Isla de la Ca uja, Se illa, Spain, 6Ins i u o de
Ho o u icul u a Sub opical y Medi e a
´nea (IHSM), La Mayo a Uni e sidad de Ma
´laga-CSIC Alga obo-
Cos a, Ma
´laga, Spain, 7Depa amen o de Biologı
´a Molecula y Bioquı
´mica, Uni e sidad de Ma
´laga, Ma
´laga,
Spain, 8Nanoma e ials o Biomedical Applica ions, Is i u o I aliano di Tecnologia, Geno a, I aly, 9Ma e ials
Cha ac e iza ion Facili y, Is i u o I aliano di Tecnologia, Geno a, I aly
☯These au ho s con ibu ed equally o his wo k.
*ma co.con a di@ii .i (M.C.); a hanassia.a hanassio[email p o ec ed] (A.A.); jose.he edia-gue e o@ii .i (J.A.H.-G.)
Abs ac
ε-cap olac one-p-couma ic acid copolyme s a di e en mole a ios (ε-cap olac one:p-cou-
ma ic acid 1:0, 10:1, 8:1, 6:1, 4:1, and 2:1) we e syn hesized by mel -polycondensa ion and
using 4-dodecylbenzene sul onic acid as ca alys . Chemical analysis by NMR and GPC
showed ha copolyes e s we e o med wi h dec easing molecula weigh as p-couma ic
acid con en was inc eased. Physical cha ac e is ics, such as he mal and mechanical p op-
e ies, as well as wa e up ake and wa e pe meabili y, depended on he mole ac ion o p-
couma ic acid. The p-couma a e epe i i e uni s inc eased he an ioxidan capaci y o he
copolyme s, showing an ibac e ial ac i i y agains he common pa hogen Esche ichia coli.
In addi ion, all he syn hesized copolyes e s, excep he one wi h he highes concen a ion
o he phenolic acid, we e cy ocompa ible and hemocompa ible, hus becoming po en ially
use ul o skin egene a ion applica ions.
In oduc ion
Biodeg adable, bio eso bable, and biocompa ible polyme s a e becoming common
ma e ials o pha maceu ical uses, mainly in he design o no el medical de ices such as
implan s, sca olds, ilms, nanopa icles o nano ibe s ha can con ol he elease o d ugs
and/o ep oduce, mimic o eplace some pa s o he body [1–3]. Among hese polyme s,
poly-ε-cap olac one (PCL), a e a pe iod o unpopula i y espec o o he eso bable
PLOS ONE | h ps://doi.o g/10.1371/jou nal.pone.0214956 Ap il 8, 2019 1 / 18
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OPEN ACCESS
Ci a ion: Con a di M, Al a o-Pulido A, Picone P,
Guzman-Puyol S, Goldoni L, Benı
´ ez JJ, e al.
(2019) Low molecula weigh ε-cap olac one-p-
couma ic acid copolyme s as po en ial bioma e ials
o skin egene a ion applica ions. PLoS ONE 14
(4): e0214956. h ps://doi.o g/10.1371/jou nal.
pone.0214956
Edi o : Tusha Jana, Uni e si y o Hyde abad,
INDIA
Recei ed: Decembe 11, 2018
Accep ed: Ma ch 23, 2019
Published: Ap il 8, 2019
Copy igh : ©2019 Con a di e al. This is an open
access a icle dis ibu ed unde he e ms o he
C ea i e Commons A ibu ion License, which
pe mi s un es ic ed use, dis ibu ion, and
ep oduc ion in any medium, p o ided he o iginal
au ho and sou ce a e c edi ed.
Da a A ailabili y S a emen : All ele an da a a e
wi hin he manusc ip and i s Suppo ing
In o ma ion iles.
Funding: The au ho s ecei ed no speci ic unding
o his wo k.
Compe ing in e es s: The au ho s ha e decla ed
ha no compe ing in e es s exis .
polyme s such as polylac ides and polyglycolides [4], has egained much in e es due o i s
excellen biocompa ibili y, easy and e icien syn hesis, and low deg ada ion a e in wa e
[1,5]. PCL is a pe oleum-based polyes e syn hesized ia high-yield lac one ing-opening
polyme iza ion (ROP) o ε-cap olac one (common name o he hexano-6-lac one) and
whose inal molecula weigh can be s ongly a ec ed by choice o he ca alys used [6,7].
Mo eo e , he molecula weigh , usually be ween 3000 and 80000 g/mol, in luences he deg-
ada ion a e o PCL and can make i sui able o di e en applica ions [5,8]. Thus, high
molecula weigh PCL is equi ed o implan s and sca olds in o de o main ain he s abil-
i y o he ma e ial o mon hs o yea s. Fo ins ance, Pi and Schinde [9] demons a ed
ha PCL capsules (wi h a Mw ~66000 g/mol) emained in ac a e 2-yea s o implan a ion
in a s.
On he o he hand, in he cases o su u es o wound d essings, a well-designed ma e ial
should be able o be eso bed by he skin in 2–3 weeks, in o de o a oid non-p ope healing
o he wound [10]. In his sense, Albe sson e al. [11] epo ed an in i o deg ada ion and
abso p ion o PCL powde (Mn ~3000 g/mol) by mac ophage and gian cells o a pe iod
o 13 days, sugges ing ha low molecula weigh PCL can be e ec i e o skin egene a ion
applica ions. PCL has a mel ing poin a ound 70˚C, which allows i s use as a biopolyme “ink”
o used deposi ion 3D p in ing o sca olds o issue enginee ing [12,13]. In addi ion, Engel-
be g e al. [14] ha e s udied he mechanical p ope ies o pu e PCL, highligh ing i s sui able
pe o mance o se e al biomedical applica ions such as wound d essing, su gical su u es, con-
acep ion, and den is y [5]. Despi e i s e sa ile beha io , PCL, simila ly o o he syn he ic
polyme s such as poly inylpy olidone (PVP), does no show any in insically biological ac i -
i y [15]. Fo his eason, hese mac omolecules a e o en blended wi h bioac i e polyme s [16–
18] and agen s able o p omo e cell a achmen [19], ensu e disin ec ion o educe in lamma-
o y and oxida i e e en s [15,20–22]. Fu he mo e, by exploi ing he p esence o he es e
g oup, a a ie y o block copolyme s be ween PCL and chi osan [23], algina e [24] and hyal-
u onic acid [25] ha e been syn hesized.
To design cu en su u es o wound d essings, a success ul s a egy is he use o na u al
bioac i e agen s o pa ial o comple e eplacemen o common and widely abused an ibio -
ics and an i-in lamma o ies d ugs [26–30]. Among hem, polyphenolic compounds such as
e ulic acid, p-couma ic acid (PCA), and cu cumin, appea ed as he mos p omising ones
[29,31]. In ac , hese molecules a ec se e al componen s o he in lamma o y esponse [32,
33], ensu e a s ong sca enging e ec agains ee adicals [34], and show a ce ain deg ee o
an ibac e ial ac i i y [35]. These molecules, in pa icula p-couma ic acid, ha e been chemi-
cally combined wi h PCL o syn hesize copolyme s ha me ge he p ope ies o polyphenols
and polycap olac one. Fo example, ecen ly, Nguyen e al. [36] copolyme ized ε-cap olac-
one wi h di e en de i a i es o sy ingic, e ulic, and p-couma ic acids by using Sb
2
O
3
as a
ca alys , a ge ing o imp o e he he mal cha ac e is ics o pu e PCL and o eplace non-bio-
deg adable pe oleum-based plas ics. Ins ead, Li e al. [37] syn hesized a poly(ε-cap olac-
one)-co-poly(4-hyd oxycinnamic acid) copolyme by ca alysis wi h s annous oc anoa e o
use in biomedical, d ug-con olled elease and luo escen p obe ields. In e es ingly, in bo h
cases, an impo an educ ion o he molecula weigh was epo ed when PCA pa icipa ed
in he polyme iza ion.
In his wo k, we p esen an al e na i e syn hesis o ε-cap olac one-p-couma ic acid copoly-
me s a di e en mola a ios by mel -polycondensa ion ca alyzed by 4-dodecylbenzenesul o-
nic acid (DBSA) o po en ial biomedical applica ions such as wound d essing and su u es.
The esul an copolyme s we e chemically and physically cha ac e ized and compa ed o pu e
PCL. In addi ion, he an ioxidan and an ibac e ial pe o mance, as well as he biocompa ibil-
i y and hemocompa ibili y, we e de e mined.
ε-cap olac one-p-couma ic acid copolyme s as po en ial bioma e ials o skin egene a ion applica ions
PLOS ONE | h ps://doi.o g/10.1371/jou nal.pone.0214956 Ap il 8, 2019 2 / 18
Ma e ials and me hods
Ma e ials
ε-cap olac one (ε-CL, pu i y: 97%, MW = 114.14 g/mol), p-couma ic acid (PCA, pu i y:
�98.0%, MW = 164.16 g/mol), and 4-dodecylbenzenesul onic acid (DBSA, pu i y: �95%,
MW = 326.49 g/mol) we e pu chased om Sigma-Ald ich and used wi hou u he pu i ica ion.
Polyme syn hesis
The syn hesis o he ε-cap olac one-p-couma ic acid copolyme s a di e en mola a ios
(ε-CL:PCA 1:0, 10:1, 8:1, 6:1, 4:1, and 2:1) was ca ied ou by mel -polycondensa ion ca alyzed
by DBSA, as summa ized in Fig 1A.ε-cap olac one (5 mL) was hea ed a 150˚C o 1 h. Then,
di e en amoun s o p-couma ic acid (0, 0.74, 0.93, 1.23, 1.85, and 3.70 g) and 10 μL o DBSA
we e added and he blends s i ed a 150˚C o 24 h. A e cooling a oom empe a u e, solids
ob ained we e solubilized in 25 mL o chlo o o m and he copolyes e s we e p ecipi a ed wi h
an excess o cold me hanol. Samples we e washed h ee imes wi h wa e and me hanol (50 mL
pe g am o ilm) and d ied in acuum o 24 h a oom empe a u e. To o m homogeneous
ilms, copolyme s we e mel ed a 75˚C o ew minu es in Te lon Pe i dishes and cooled a
oom empe a u e. The appea ance o he ilms is shown in Fig 1B and sample composi ion
and labeling a e summa ized in Table 1.
Table 1. Label, mola a io, mole ac ion, and weigh % o he ε-cap olac one-p-couma ic acid copolyme
p epa ed.
Label Mola a io Mole ac ion Weigh %
ε-CL PCA ε-CL PCA ε-CL PCA
PCL/PCA 1:0 1 0 1.00 0.00 100.0 0.0
PCL/PCA 10:1 10 1 0.91 0.09 87.4 12.6
PCL/PCA 8:1 8 1 0.89 0.11 84.8 15.2
PCL/PCA 6:1 6 1 0.86 0.14 80.7 19.3
PCL/PCA 4:1 4 1 0.80 0.20 73.6 26.4
PCL/PCA 2:1 2 1 0.67 0.33 58.2 41.8
h ps://doi.o g/10.1371/jou nal.pone.0214956. 001
Fig 1. Scheme o syn hesis and pho og aphs o he biopolyme s. (A) Polyme iza ion eac ion be ween ε-cap olac one and p-couma ic acid. ε-CL and
PCA monome s and hei co esponding epe i i e uni s in he polyes e ha e been highligh ed in ligh g een and ligh o ange, espec i ely. (B)
Pho og aphs o he ε-cap olac one-p-couma ic acid copolyme ilms.
h ps://doi.o g/10.1371/jou nal.pone.0214956.g001
ε-cap olac one-p-couma ic acid copolyme s as po en ial bioma e ials o skin egene a ion applica ions
PLOS ONE | h ps://doi.o g/10.1371/jou nal.pone.0214956 Ap il 8, 2019 3 / 18
Nuclea Magne ic Resonance (NMR) spec oscopy
Fo Nuclea Magne ic Resonance (NMR) cha ac e iza ion, ~7–8 mg o compounds we e dis-
sol ed in 1 mL o THF-d8. NMR expe imen s we e pe o med on a B uke A ance III 400
MHz spec ome e equipped wi h a B oad Band In e se (BBI) p obe, wi hou he spinning. 5
mm ubes illed wi h 500 μL o he sample solu ions we e employed. Be o e he acquisi ion,
au oma ic ma ching and uning we e inely egula ed, homogenei y au oma ically adjus ed,
and empe a u e ac i ely con olled a 300K. The
1
H 90˚ pulse was op imized by an au oma ic
pulse calcula ion ou ine [38]. In
1
H-NMR expe imen s, 45 k complex da a poin s o 8 an-
sien s we e accumula ed, a e a 90˚ lip angle, o e a spec al wid h o 18.02 ppm (o se
6.18 ppm), a a ixed ecei e gain (1), using 30 s o elaxa ion delay and no s eady s a e scans.
Spec a we e manually phased and au oma ically baseline co ec ed. An exponen ial line
b oadening equi alen o 0.1 Hz was applied o FIDs be o e Fou ie T ans o m. The
1
H-
1
H
COSY(CO ela ion Spec oscopY) was pe o med wi h 1 FID, 2048 da a poin and 128 inc e-
men , o e a spec al wid h o 8.93 ppm (o se a 4.17 ppm), while he
1
H-
13
C HSQC (mul i-
plici y edi ed He e onuclea Single Quan um Cohe ence) was acqui ed wi h 2 FIDs, 1024 da a
poin s and 256 inc emen s, o e a spec al wid h o 8.93 ppm a he
1
H and 165 ppm a he
13
C
(o se a 4.14 and 75.0 ppm, espec i ely). All spec a we e e e ed o non-deu e a ed THF
esidue peak a 3.58 ppm (
1
H) and a 67.2 ppm (
13
C), acco ding o Fulme e al. [39].
p-couma ic acid:
1
H NMR (THF-d8, 400 MHz): 12.00–7.80 b s, (2H, OH and COOH);
7.55 ppm d, J= 16.0 Hz (1H); 7.42 pseudo d, J= 8.6 Hz (2H, AA’MM’ sys em); 6.75 pseudo d,
J= 8.6 Hz (2H, AA’MM’ sys em), 6.25 ppm d, J= 16.0 Hz (1H).
Gel Pe mea ion Ch oma og aphy (GPC)
GPC measu emen s we e ca ied ou using an Agilen 1260 In ini y qua e na y LC sys em
using wo PLGel 5μm MIXED-C columns a 25 ˚C and a e ac i e index de ec o . Te ahyd o-
u an (THF) was used o dissol e he samples and as eluen a 1 mL/min low a e. The sys em
was calib a ed wi h Agilen EasyVial PS s anda ds.
The mal cha ac e iza ion
The he mal deg ada ion beha io o he samples was in es iga ed by he mog a ime ic anal-
ysis (TGA) me hod, using a TGA Q500 om TA Ins umen s. Measu emen s we e pe o med
using 3–5 mg o sample in an aluminum pan unde ine N
2
low (50 mL/min) in a empe a-
u e ange om 30 o 600 ˚C wi h a hea ing a e o 5 ˚C/min. The weigh loss and i s i s
de i a i e we e eco ded simul aneously as a unc ion o ime/ empe a u e.
Di e en ial Scanning Calo ime y (DSC) he mog ams we e acqui ed wi h a DSC Q20 (TA
Ins umen s) om 30 o 150 ˚C unde ni ogen low (50 mL/min) a 20 ˚C/min by using non-
he me ic aluminum pans. Abou 4 mg (weigh ed wi h ±0.01 mg p ecision) o sample we e
used. Specimens we e i s hea ed om 30 o 150 ˚C o elease mois u e, cooled o 30 ˚C and
inally amped o 150 ˚C.
Mechanical es s
The mechanical p ope ies o he PVP/PCA copolyme s we e de e mined by uniaxial ension
es s on a dual column uni e sal es ing machine (Ins on 3365). Films we e cu in dog bone
specimens (a leas se en o hem o each sample) wi h a wid h o 4 mm and an e ec i e
leng h o 25 mm. Displacemen was applied a a a e o 10 mm/min. The Young’s modulus,
s ess a maximum load, and elonga ion a b eak we e calcula ed om he s ess-s ain cu es.
All he s ess-s ain cu es we e eco ded a 25 ˚C and 44% R.H.
ε-cap olac one-p-couma ic acid copolyme s as po en ial bioma e ials o skin egene a ion applica ions
PLOS ONE | h ps://doi.o g/10.1371/jou nal.pone.0214956 Ap il 8, 2019 4 / 18
Wa e up ake
The wa e up ake capaci y o he samples was ob ained as ollows: d y samples we e weighed
(~80 mg) on a sensi i e elec onic balance and placed in di e en chambe s wi h con olled
humidi y. The humidi y condi ions we e: 0%, 11%, 44%, 84%, 100%. A e condi ioning in di -
e en humidi y chambe s un il equilib ium condi ions (usually 24 h), each ilm was weighed,
and he amoun o adso bed wa e was calcula ed based on he di e ence be ween he weigh
o each ilm and i s ini ial d y weigh .
Wa e apo pe meabili y
The wa e apo ansmission a e (WVTR) and wa e apo pe meabili y (WVP) o he sam-
ples we e de e mined a 25 ˚C and unde 100% ela i e humidi y g adien (ΔRH%) acco ding
o he ASTM E96 s anda d me hod. In his es , pe mea ion chambe s wi h a deposi (7 mm
diame e and 10 mm deep) we e used and illed wi h 400 μL o deionized wa e (which gene -
a es an in e nal 100% RH) [40]. Samples we e cu in ci cles and moun ed by sealing he op o
he wa e deposi . The pe mea ion chambe s we e placed inside a desicca o wi h anhyd ous
silica gel p o iding 0% RH. The mass o wa e ans e ed h ough he ilm was de e mined by
he weigh change o he pe mea ion chambe e e y hou du ing a pe iod o 8 hou s using an
elec onic balance (0.0001 g accu acy). The mass loss was plo ed as a unc ion o ime and he
slope o each cu e was calcula ed by linea eg ession. Then, he wa e apo ansmission
a e (WVTR) was de e mined as below [41]:
WVTR gðm2dÞ1
 �¼Slope
A ea o he sample
The WVP o he samples we e hen calcula ed as below:
WVP gðm d PaÞ1
 �¼WVTR �L�100
ps�DRH
whe e L (m) is he hickness o he sample, measu ed wi h a mic ome e wi h a 0.001 mm
accu acy; ΔRH (%) is he pe cen age ela i e humidi y g adien , and p
s
(Pa) is he sa u a ion
wa e apo p essu e a 25 ˚C [42,43].
WVTR and WVP measu emen s we e eplica ed h ee imes o each ilm.
DPPH
.
ee adical ca ion sca enging assay
The an ioxidan capaci y o he samples was de e mined acco ding o he p ocedu e desc ibed
elsewhe e [44]. The me hod is based on he sca enging o DPPH
.
adical h ough he ac ion o
an an ioxidan ha decolo izes he DPPH
.
solu ion. B ie ly, 5 x 5 mm
2
ilms we e added o 3
mL o 0.1 mM DPPH
.
solu ion in e hanol. The dec ease o he abso bance was de e mined a
515 nm wi h a Ca y 300 Scan UV- isible spec opho ome e a di e en imes. All he mea-
su emen s we e pe o med in iplica es, and he esul s we e a e aged o ob ain a mean alue.
Radical sca enging ac i i y was exp essed as he inhibi ion pe cen age o ee adical by he
sample and calcula ed as ollows:
Radical Sca enging Ac i i y %ð Þ ¼ A0A1
A0
x100
whe e A
0
is he abso bance alue o he con ol (3 mL o 0.1 mM DPPH
.
solu ion in e hanol),
and A
1
is he abso bance alue o he sample a di e en imes.
ε-cap olac one-p-couma ic acid copolyme s as po en ial bioma e ials o skin egene a ion applica ions
PLOS ONE | h ps://doi.o g/10.1371/jou nal.pone.0214956 Ap il 8, 2019 5 / 18

Cell cul u e and ea men s
P ima y human de mal ib oblas s (HDFa) (The mo Fishe Scien i ic, Milan, I aly) we e cul-
u ed wi h DMEM medium (Celbio, Milan, I aly) supplemen ed wi h 10% e al bo ine se um
(FBS) (Gibco-In i ogen, Milan, I aly), 2mM glu amine, 1% penicillin and 1% s ep omycin
(50 mg/mL). Cells we e main ained in a humidi ied 5% CO
2
a mosphe e a 37 ±0.1 ˚C. Fo
cy ocompa ibili y assays, HDFa cells we e seeded on 96-well la -bo om pla e a a densi y o
4x10
3
pe well. A e UV s e iliza ion 2 mg o PCL/PCA ilms a a di e en mola a io (i.e.,
1:0, 10:1, 8:1, 6:1, 4:1, and 2:1) we e pla ed on HDFa monolaye , and he samples we e incu-
ba ed a 37 ˚C o 72 hou s.
De e mina ion o cell iabili y
Cell iabili y was measu ed by MTS assay (P omega I alia, S. .l., Milan, I aly). MTS [3-(4,5-
dime hyl hiazol-2-yl)-5-(3-ca boxyme hoxyphenyl)-2-(4-sulphophenyl)-2H- e azolium]
was u ilized acco ding o he manu ac u e ’s ins uc ions. HDFa cells we e pla ed in a 96-well
pla e, and a e ea men , 20 μL o he MTS solu ion we e added o each well and incuba ed
o 4 h a 37 ˚C in a humidi ied incuba o wi h 5% CO
2
. The abso bance was ead a 490 nm
on he Mic opla e eade Wallac Vic o 2 1420 Mul ilabel Coun e (Pe kinElme Inc., Monza,
I aly). The esul s we e exp essed as he pe cen age o MTS educ ion ela i e o he con ol
and p esen ed as he mean ±s anda d de ia ion (SD) and we e e alua ed acco ding o he
ISO10993-5 s anda d guidelines [45] in which oxici y o di ec -con ac me hod is sco ed
om 0 o 4 (0 = non-cy o oxic, 1 = sligh ly cy o oxic, 2 = mildly cy o oxic, 3 = mode a ely
cy o oxic and 4 = se e ely cy o oxic). The ea ed cul u ed cells and he con ols we e mo pho-
logically analyzed by mic oscopy inspec ion on an Axio Scope 2 mic oscope (Zeiss).
Hemolysis assay
Hemolysis assay was pe o med acco ding o he p o ocol o Picone e al. [46]. B ie ly, 5 mL o
enous blood collec ed ea ly in he mo ning om a heal hy dono was d awn di ec ly in o K2
EDTA coa ed Vacu aine ubes o p e en coagula ion. A e cen i uga ion a 500g o 5 min,
he hema oc i and plasma le el we e ma ked on he ube. Then he plasma was emo ed and
eplaced wi h 150 mM NaCl, and he ube was cen i uged a 500g o 5 min. This s ep was
epea ed h ee imes. A e , he supe na an was eplaced wi h PBS a pH 7.4. 200 μL o dilu ed
(1:50) e y h ocy es we e pipe ed in o a 96-well pla e. The ea e , di e en PCL/PCA samples
(in pa icula , 1:0, 6:1, 4:1, and 2:1) a 10 mg/mL o 10 μL o 20% T i on X-100, as a posi i e
con ol, we e added o he e y h ocy es sample. The pla e was incuba ed a 37 ˚C o 2 h and
hen cen i uged o 5 min a 500g o pelle whole e y h ocy es. Then, 100 μL o he supe na-
an was ans e ed om each well in o a clea , la -bo omed 96-well pla e and abso bance,
due o ee hemoglobin p esence, was ead a 490 nm by using a pla e eade (Wallac Vic o 2
1420 Mul ilabel Coun e (Pe kinElme , Inc. Monza, I aly). A e backg ound sub ac ion, he
a e age abso bance o he posi i e con ol was de e mined. All expe imen al da a poin s we e
no malized wi h his mean abso bance alue, which ep esen s 100% hemolysis.
An ibac e ial es
The an imic obial ac i i y o PCL/PCA samples was es ed agains Esche ichia coli (E.coli) by
using aga -disk di usion me hod. Pieces o PCL/PCA ilms (~250 mg) wi h di e en PCA
a ios (1:0, 6:1, 4:1, and 2:1) we e s e ilized unde UV lamp a 254 nm o 30 minu es. An ali-
quo o E.coli o.n. cul u e, app oxima ely 10
9
CFU/mL, was dilu ed (1:1000) and 10 μL we e
sp ead on o he LB aga pla es. A e 10 minu es, he samples we e placed on he pla es and
ε-cap olac one-p-couma ic acid copolyme s as po en ial bioma e ials o skin egene a ion applica ions
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incuba ed a 37˚C o 24 and 72 h. The inhibi o y e ec o PCL/PCA on bac e ia g ow h was
de e mined by assessing he dimensions o he colonies in he PCA eleased zone. Th ee epli-
ca e pla es we e used o each concen a ion o he ilm. Fo he inhibi o y g ow h es , 100 μL
o E.coli o.n. bac e ial cul u e, app oxima ely 10
9
CFU/mL, was added o esh LB medium
(10 mL) wi h o wi hou PCL/PCA a di e en mola a ios (1:0, 6:1, 4:1, 2:1). The exponen ial
g ow h was de e mined by spec opho ome ic measu emen s (600 nm) e e y 30 minu es up
o 360 minu es. Da a o h ee independen expe imen s we e ob ained.
S a is ical analysis
The signi icance o he di e ences in he mean alues o g oups was e alua ed using he analy-
sis o a iance (one-way ANOVA). The analysis was ollowed by Bon e oni’s pos hoc es .
Di e ences we e conside ed signi ican when he p- alue was �0.05.
Resul s and discussion
Chemical cha ac e iza ion
The chemical cha ac e iza ion o he copolyme s was ca ied ou by
1
H NMR. As ep esen a-
i e examples, he spec a o PCL/PCA 10:1 and 2:1 as well as hose o 1:0 and p-couma ic acid
( o compa ison pu poses) a e shown, Fig 2. PCL/PCA 1:0 showed he ypical chemical shi s
o polycap olac one a 4.01, 2.26, 1.61, and 1.37 ppm, associa ed wi h he di e en local mag-
ne ic en i onmen o p o ons o PCL (a comple e assignmen and cha ac e iza ion can be
ound in Fig 2 and S1 Fig). On he o he hand, o p-couma ic acid, he chemical shi s a 7.55
and 6.24 ppm and 7.41 and 6.74 ppm we e a ibu ed o he p o ons o he double bond and
a oma ic ing g oups, espec i ely (mo e de ails a e p esen in he Ma e ials and me hods sec-
ion, NMR pa ). Ins ead, copolyme s’ spec a showed he inge p in o he alipha ic and a o-
ma ic componen s. Mo eo e , b oad peaks, whose in ensi y depended on he PCA con en ,
we e also obse ed. Such signals a e cha ac e ized by long co ela ion imes (τ
c
) which is ypi-
cal o species ha mo e slowly in solu ion. These b oad peaks ha e been p e iously obse ed
in o he polyes e s whe e phenolic compounds ha e been copolyme ized wi h alipha ic mono-
me s[36].
To u he cha ac e ize he ε-cap olac one-p-couma ic acid copolyme s, hei mola mass
was de e mined by GPC. Table 2 shows M
n
, M
w
, and PDI alues. As obse ed, he pa icipa-
ion o p-couma ic acid as a monome dec eased he mola mass om M
n
= 4500 g/mol and
M
w
= 6850 g/mol o PCL/PCA 1:0 o M
n
= 2800 g/mol and M
w
= 3100 g/mol o PCL/PCA
2:1. This is a educ ion o ~38% and ~55% o M
n
and M
w
, espec i ely. Mos likely, such a
d op o molecula weigh can be a consequence o a lowe eac i i y o PCA in compa ison o
ε-cap olac one du ing he mel -polycondensa ion. On he o he hand, he polydispe si y was
also dec eased om 1.55 o PCL/PCA 1:0 o 1.12 o PCL/PCA 2:1. In a linea s ep- eac ion,
he polydispe si y index can be de ined as PDI = 1 + p, whe e p is he con e sion deg ee [47].
This equa ion p edic s PDI alues anging om 1 o 2 as he con e sion inc eases. Values
lis ed in Table 2 can be in e p e ed in e ms o a compe i ion be ween chain g ow h and chain
condensa ion eac ions. In he case o PCL, he chain condensa ion a e is high and a b oad
dis ibu ion o molecula sizes is ob ained (highe PDI). Howe e , and due o di usion limi a-
ions in he mol en s a e, he con e sion deg ee is low and mode a e PDI and low Mw poly-
me s a e ob ained. When PCA is added, he chain g ow h eac ion is a o ed s he chain
condensa ion p ocess and a mo e uni o m size dis ibu ion is ob ained (lowe PDI alues).
The e o e, a low chain condensa ion deg ee causes he educ ion o he a e age molecula
weigh o PCL-PCA copolyme s.
ε-cap olac one-p-couma ic acid copolyme s as po en ial bioma e ials o skin egene a ion applica ions
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Fig 2. NMR cha ac e iza ion.
1
H-NMR spec a o PCL/PCA 1:0, 10:1, and 2:1 and p-couma ic acid in THF-d
8
. Assignmen s a e included.
h ps://doi.o g/10.1371/jou nal.pone.0214956.g002
Table 2. Numbe a e age molecula mass (M
n
), mass a e age molecula mass (M
w
), and polydispe si y index
(PDI) o PCL/PCA copolyme s.
Sample M
n
(g/mol) M
w
(g/mol) PDI
1:0 4500 6850 1.55
10:1 4200 5800 1.37
8:1 3950 5000 1.27
6:1 4000 5200 1.31
4:1 3650 4400 1.21
2:1 2800 3100 1.12
h ps://doi.o g/10.1371/jou nal.pone.0214956. 002
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The mal analysis
The he mal beha iou o PCL/PCA samples and pu e p-couma ic acid was in es iga ed by
TGA and DSC, Fig 3.Fig 3A shows he he mog a ime ic analysis ( op) and he co espond-
ing de i a i e he mog a ime ic cu es (bo om) o he PCL/PCA 1:0, 10:1, 6:1, and 2:1
samples. PCL/PCA 1:0 p esen ed a single weigh loss a ~404 ˚C, while he deg ada ion o p-
couma ic acid was a ~215˚C. The copolyme s showed wo weigh losses: one a ~404˚C
associa ed wi h he deg ada ion o he alipha ic ac ion in he copolyme and ano he one, a
shoulde in he de i a i e he mog a ime ic cu es, a ~445˚C ela ed o he he mal decom-
posi ion o p-couma a e epe i i e uni s [48]. No deg ada ion o ee p-couma ic acid was
obse ed, indica ing ha i was no p esen in he samples. Fu he mo e, he p esence o PCA
inc eased he cha esidue a e TGA measu emen om ~0.5% o PCL/PCA 1:0 o ~10% o
PCL/PCA 2:1, inse o Fig 3A.
DSC esul s a e epo ed in Fig 3B and 3C. DSC he mog ams o PCL/PCA 1:0, 10:1, 6:1,
and 2:1 samples a e displayed in Fig 3B. A single he mal e en associa ed wi h he mel ing
poin o he copolyme s was obse ed. A dec ease in mel ing empe a u es and lowe en hal-
pies o usion we e de e mined, as PCA con en is aised in Fig 3C. Thus, T
m
and ΔH
anged
om ~62˚C and ~101 J/g, espec i ely, o PCL/PCA 1:0 o ~47˚C and ~25 J/g, espec i ely,
o PCL/PCA 2:1. These dec eases can be a ibu ed o he educ ion o molecula weigh and
Fig 3. The mal s abili y analysis. (A) TGA he mog ams ( op) and de i a i e he mog a ime ic cu es (bo om) o PCL/PCA 1:0, 10:1, 6:1, and 2:1
samples and pu e p-couma ic acid. The inse in he op g aph shows he pe cen age o cha esidue as a unc ion o he mole ac ion o PCA. (B) DSC
cu es o PCL/PCA 1:0, 10:1, 6:1, and 2:1 samples in he ange o empe a u e be ween 20 and 100˚C. (C) Mel ing poin and en halpy o usion o PCL/
PCA copolyme s espec o he mole ac ion o PCA.
h ps://doi.o g/10.1371/jou nal.pone.0214956.g003
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