Low molecular weight ϵ-caprolactone-pcoumaric acid copolymers as potential biomaterials for skin regeneration applications

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 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 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 PLOS ONE | h ps://doi.o g/10.1371/jou nal.pone.0214956 Ap il 8, 2019 6 / 18 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 PLOS ONE | h ps://doi.o g/10.1371/jou nal.pone.0214956 Ap il 8, 2019 7 / 18 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 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 8 / 18 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 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 9 / 18 9. Pi C, Schindle A. Cap ono —a biodeg adable deli e y sys em o le ono ges el. 1984. 10. Cla k RAF. Chap e 76—Wound Repai : Basic Biology o Tissue Enginee ing A2—Lanza, Robe . In: Lange R, Vacan i J, edi o s. 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