Preparation and characterization of electrospun double-layered nanocomposites membranes as a carrier for centella asiatica (L.)

polyme s A icle P epa a ion and Cha ac e iza ion o Elec ospun Double-laye ed Nanocomposi es Memb anes as a Ca ie o Cen ella asia ica (L.) Cláudia Mou o 1, Raul Fanguei o 2and Isabel C. Gou eia 1,* 1FibEnTech Resea ch Uni , Facul y o Enginee ing, Uni e si y o Bei a In e io , 6201-001 Co ilhã, Po ugal; [email p o ec ed] 2Cen e o Tex ile Science and Technology (2C2T), Uni e si y o Minho, 4710-057 Guima ães, Po ugal; [email p o ec ed] *Co espondence: [email p o ec ed] Recei ed: 16 Oc obe 2020; Accep ed: 9 No embe 2020; Published: 11 No embe 2020   Abs ac : A wide ange o na u ally de i ed and syn he ic biodeg adable and biocompa ible polyme s a e oday ega ded as p omising ma e ials o imp o ing skin egene a ion. Alongside his, hese ma e ials ha e been explo ed in conjunc ion wi h di e en ypes o an imic obial and bioac i e agen s, especially na u al-de i ed compounds, o enhance hei biological p ope ies. He ein, a double-laye ed nanocomposi e d essing memb ane was ab ica ed wi h wo dis inc laye s. A bo om laye om Chi osan-Sodium ipolyphospha e (CS-TPP) and Poly( inyl alcohol) (PVA) con aining Cen ella asia ica (L.) (CA) was elec ospun di ec ly o e a Polycap olac one (PCL) laye o imp o e he biologic pe o mance o he elec ospun nano ibe s. In u n, he PCL laye was designed o p o ide mechanical suppo o he damaged issue. The esul s e ealed ha he p oduced double-laye ed nanocomposi e memb ane closely esembles he mechanical, po osi y, and we abili y ea u es equi ed o skin issue enginee ing. On he o he hand, he in i o d ug elease p o ile o he PCL/PVA_CS-TPP con aining CA exhibi ed a con olled elease o 10 days . Mo eo e , he PVA_CS-TPP_CA’s bo om laye displayed he highes an ibac e ial ac i i y agains S aphylococcus au eus (S. au eus) (99.96 ± 6.04%) and Pseudomonas ae uginosa (P. ae uginosa) (99.94 ± 0.67%), which is esponsible o a oiding bac e ial pene a ion while endowing bioac i e p ope ies. Finally, he 3-(4,5-Dime hyl-2- hiazolyl)-2,5-diphenyl-2H- e azolium b omide (MTT) assay showed ha his nanocomposi e memb ane was no cy o oxic o no mal human de mal ib oblas s (NHDF) cells. The e o e, hese indings sugges he po en ial use o he double-laye ed PCL/PVA_CS-TPP_CA as an e icien bionanocomposi e d essing ma e ial. Keywo ds: polycap olac one; chi osan; sodium ipolyphospha e; poly( inyl alcohol); Cen ella asia ica; double-laye ed nanocomposi e memb anes 1. In oduc ion Wound healing is a dynamic and complex p ocess ha equi es cellula in e ac ions be ween a wide a ie y o cell ypes [ 1 – 3 ]. These in e ac ions a e media ed h ough a coo dina ed cascade o biological e en s syne gis ically con olled by nume ous bioac i e molecules, such as g ow h ac o s, an i-in lamma o y agen s, and i amins. Howe e , he physiology o he healing p ocess may be impai ed by se e al ac o s [ 1 – 3 ]. Among hem, bac e ial coloniza ion and subsequen in ec ions emain one o he mos se ious complica ions a e skin s uc u e is comp omised [1,4,5]. Gene ally, when pa hogenic mic oo ganisms con amina e skin wounds, he immune sys em mobilizes i s ene gy ying o supp ess he in asion o hese pa hogens ins ead o ocusing on he e-es ablishmen o he na i e skin’s s uc u al and unc ional ea u es [ 1 , 4 – 6 ]. I in ec ion occu s, Polyme s 2020,12, 2653; doi:10.3390/polym12112653 www.mdpi.com/jou nal/polyme s Polyme s 2020,12, 2653 2 o 18 mic oo ganisms, pa icula ly bac e ia, can p oduce endo oxins ha s imula e he exp ession o p o-in lamma o y cy okines and encou age an ex ended in lamma o y esponse. In his case, wounds exhibi inc eased le els o me allop o einases (MMPs), which p o ide an unsui able en i onmen o he p oduc ion o new skin’s ex acellula ma ix (ECM) componen s, delaying o e en in e up ing he healing p ocess [1,5,6]. Conce ning his, se e alwoundd essingsdisplayingan imic obial p ope ies ha ebeende eloped o p o ec he wound om in ec ion and enhance he healing p ocess. None heless, i is essen ial o imp o e hei pe o mance o supp ess his heal h p oblem and educe he occu ence o li e- h ea ening complica ions [ 1 , 5 – 7 ]. Fo his pu pose, di e en combina ions o bo h syn he ic and na u al biocompa ible and biodeg adable polyme s ha e been explo ed o p oduce 3D nano ib ous memb anes ha mimic he a chi ec u e o he skin’s ECM [1,6–8]. So a , o success ully p oduce nano ibe s as po en ial wound d essings, se e al echniques ha e been used, namely sel -assembly, phase sepa a ion, d awing, empla e syn hesis, and elec ospinning. Elec ospinning has been conside ed as one o he mos e icien , e sa ile, and cos -e ec i e me hods o p oduce nanocomposi e d essing ma e ials wi h he desi able ea u es [1,7]. The unique s uc u al and mo phological p ope ies o he elec ospun nano ibe s, like he high speci ic a ea o olume a io, in e connec ed po es, and he smalle ibe diame e s, closely esemble he s uc u e o collagen ibe s ound in he no mal skin’s ECM. Alongside his, hei po ous s uc u e can suppo cell adhesion, mig a ion, and p oli e a ion, and egula e he elease o bo h g ow h ac o s and signaling molecules, which a e equi ed o achie e wound healing [ 1 , 7 – 11 ]. The elec ospun nano ibe s can also p o ide quick hemos asis, enhance exuda e abso p ion, nu ien s, and gas pe meabili y, as well as p e en ing bac e ial pene a ion and coloniza ion. Mo eo e , elec ospinning p o ides he ope a ional abili y o inco po a ing an imic obial and/o bioac i e agen s, which enable he imp o emen o he biologic beha io o hese wound d essing ma e ials [1,7–9]. To accomplish ha , se e al di e en app oaches ha e been used as an al e na i e o adi ional single- luid elec ospinning [ 12 ]. Among hem, double- luid and mul iple- luid elec ospinning me hods ha e been ex ensi ely s udied o p oduce co e–shell nano ibe s and p o ide speci ic elease p o iles [ 13 , 14 ]. In he coaxial elec ospinning sys em, namely in modi ied coaxial elec ospinning p ocesses, di e en coaxial spinne e needles ha e been designed [ 15 ]. Fu he mo e, wo needles side-by-side ha e been applied o p oduce nano ibe s wi h Janus s uc u es [ 14 , 16 , 17 ]. On he o he hand, mul iple- luid sys ems wi h dis inc spinne e a angemen s like adi ional and modi ied iaxial spinne e s and elec ospun nano ibe s wi h a common shell and wo sepa a e co es ha e been de eloped o ensu e a sus ained elease o he inco po a ed agen s [ 14 , 18 – 21 ]. Howe e , hose new me hods o manipula ing he inne chambe s uc u e a e complica ed. Thus, adi ional single- luid blending elec ospinning is s ill he mains eam me hod due o i s s aigh o wa d ope a ion, easy scale-up, and ema kable powe o ailo ing he componen s and composi ions o esul an composi e nano ibe s. Addi ionally, manipula ion o deposi ion on he collec o wi h di e en elec ospun nano ibe s has been s udied in ecen wo ks and is being u he explo ed o imp o e he unc ional pe o mances o he elec ospun wound d essing ma e ials con aining an imic obial agen s [11,22–24]. Among he di e en an imic obial agen s inco po a ed so a in elec ospun wound d essings, an ibio ics and nanopa icles ha e been widely explo ed due o hei capabili y o a oid bac e ia pene a ion and coloniza ion in o he wound si e [ 1 , 5 , 7 , 9 ]. None heless, he g owing h ea o an ibio ic esis ance and hei oxici y ha e encou aged he use o na u al p oduc s o a oid bac e ial con amina ion. Rega ding ha , compounds ob ained om na u al sou ces, like medicinal plan s, ha e been ega ded as a powe ul na u al supplemen o he managemen and ea men o wounds [ 1 , 7 , 9 , 25 , 26 ]: mainly c ude plan ex ac s, which a e ecologically sus ainable mix u es ich in in e es ing bioac i e phy ochemicals, such as annins, alkaloids, ca bohyd a es and glycosides, e penoids, s e oids, la onoids, and couma ins wi h mul iple healing bene i s [7,9,26,27]. He ein, we p oduced a no el bionanocomposi e d essing memb ane wi h a double-laye ed s uc u e h ough elec ospinning. Polycap olac one (PCL), a hyd ophobic syn he ic polyme , Polyme s 2020,12, 2653 3 o 18 was used as he main componen o he i s laye , due o i s biocompa ibili y, desi able mechanical s eng h, and abili y o ac as a p o ec i e ba ie [ 10 , 28 – 30 ]. On he o he hand, he second laye o Poly( inyl alcohol) (PVA) and Chi osan-Sodium ipolyphospha e (CS-TPP) con aining Cen ella asia ica (L.) (CA) was designed o be in di ec con ac wi h he inju ed skin and enhance he healing p ocess [30,31]. CA is a membe o he Apiaceae amily, and i has been widely used o he ea men o de ma oses, skin lesions such as bu ns, exco ia ions, hype ophic sca s o eczema, and o he skin diseases, like lep osy and pso iasis, as well as in non-de ma ologic condi ions. CA displays di e en e penoids, known as cen elloids, including asia icoside, madecassoside, cen elloside, cen ellose, b ahminoside, hankunizide, sce oleoside, b ahmoside, and asia ic, cen ellic, b ahmic, and madecassic acids which a e esponsible o con e ing se e al he apeu ic p ope ies o CA [ 32 – 34 ]. Mo eo e , he ex ac s ob ained om his medicinal plan a e known o hei capabili y o s imula e ib oblas p oli e a ion, collagen syn hesis, and angiogenesis [ 32 ]. In u n, Chi osan (CS), one o he mos abundan na u al polysaccha ides, is known o i s abili y o s imula e collagen syn hesis, as well as bac e icidal and hemos a ic p ope ies. Alongside his, CS possesses amine unc ional g oups on i s backbone chains, which in acidic aqueous media ensu e a high densi y o posi i e cha ges [ 31 , 35 – 37 ]. Thus, unde hese condi ions, CS can be ionically c oss-linked wi h biodeg adable and biocompa ible polyanions, as he Sodium ipolyphospha e (TPP), o ming polyelec oly e complexes as an imic obial and/o bioac i e agen s deli e y ca ie s [ 31 , 35 ]. Ne e heless, he CS-TPP solu ions a e di icul and uns able o elec ospun in o a ib ous s uc u e, due o he high iscosi y o he CS a low pH alues [ 31 , 37 – 39 ]. To o e come his limi a ion, PVA, one o he mos commonly used wa e -soluble syn he ic polyme s, was added o he CS-TPP blend o enhance ibe - o ming abili y [31,36,38,39]. Hence, in his wo k, we aimed o ake ad an age o he capabili y o e ed by double-laye ed PCL/PVA_CS-TPP_CA o imp o e he wound healing p ocess, namely he bene i s ha he ionically c oss-linked elec ospun PVA_CS-TPP nano ibe s display o con ol he elease o he c ude CA ex ac acco ding o he demands o an imic obial wound ca e p oduc s. 2. Ma e ials and Me hods 2.1. Ma e ials Cen ella asia ica (CA) was supplied om a Po uguese bo anic shop (CH Á HUNOS, Lda., Po ugal) wi hou any addi i es. No mal human de mal ib oblas s (NHDF) cells we e pu chased om ATCC—Ame ican Type Cul u e Collec ion. Polycap olac one (PCL) (MW 80.000 g/mol), Chi osan (CS) (low molecula weigh ) we e acqui ed om Sigma-Ald ich. Poly inyl Alcohol (PVA) (MW 115.000 g/mol) was pu chased om VWR Chemicals. E hanol absolu e, Chlo o o m, Dime hyl o mamide (DMF), and Glacial ace ic acid we e pu chased om Fishe Chemical. Nu ien aga (NA), Nu ien b o h (NB), and Aga o mic obiology we e p o ided om Fluka. B ain Hea In usion (BHI) b o h was ob ained om Pan eac. Muelle Hin on b o h (MHB), Tween 80, Sodium Hyd oxide (NaOH), Sodium Chlo ide (NaCl), T ypsin, and 3-(4,5-Dime hyl-2- hiazolyl)-2,5-diphenyl-2H- e azolium b omide (MTT) we e bough om Sigma-Ald ich. Phospha e-bu e ed saline (PBS) and Sodium ipolyphospha e (TPP) we e pu chased om Al a Aesa . All sol en s we e used as ecei ed om he manu ac u e . 2.2. E hanol Ex ac ion o C ude Cen ella Asia ica (CA) Plan The d ied and powde ed ae ial plan pa s (4 g) we e mace a ed using 40 mL o 95% e hanol as sol en a oom empe a u e o 24 h. Then, he supe na an om he e hanol ex ac ion was di ec ly il e ed h ough Wha man il e pape , and a e ha , he acqui ed il a e was d ied unde educed p essu e o ob ain d y CA ex ac . The yield o he esh plan was 18.81% (d y weigh o he ex ac ob ained a e sol en emo al pe weigh o plan ) (w/w). Finally, he c ude CA ex ac was s o ed ollowing good s o age p ac ices and nex e-suspended in 45% ( / ) e hanol o u he expe imen s. Polyme s 2020,12, 2653 4 o 18 2.3. Minimum Inhibi o y Concen a ion (MIC) o he C ude CA ex ac Minimum inhibi o y concen a ion (MIC) o he c ude CA ex ac was assessed agains S aphylococcus au eus (ATTC 6538) (S. au eus) and Pseudomonas ae uginosa (PA25) (P. ae uginosa) using he b o h mic odilu ion me hod on 96 mul i-well polys y ene pla es (Sigma-Ald ich), acco ding o he CLSI M07-A6 documen . B ie ly, se ial dilu ions o c ude CA ex ac we e p epa ed in s e ile Muelle Hin on B o h (MHB) o ob ain he desi ed ex ac concen a ions (be ween 50 and 0.15 mg/mL). Then, 50 µ L o each CA dilu ion con aining 50 µ L o a bac e ial suspension (adjus ed o ~10 7 CFU/mL in MHB) was applied o he mic opla e wells in iplica e. The pla es we e a e ha incuba ed a 37 ◦C o 18–24 h. The MIC was de ined as he lowes concen a ion o he c ude CA ex ac a which he e was no isible g ow h o S. au eus and P. ae uginosa (no solu ion u bidi y on naked eyes). MHB wi h bac e ial suspensions was added as a posi i e con ol, whe eas only MHB was used as a nega i e con ol. 2.4. Fab ica ion o he Double-Laye ed Nanocomposi es Memb anes The double-laye ed nanocomposi es memb anes we e ab ica ed using he Nanospide echnology (Nanospide labo a o y machine NS LAB 500S om Elma co S.R.O., Czech Republic, h p://www. elma co.com), as a modi ied elec ospinning me hod. Top laye : Ini ially, a PCL solu ion (8% PCL (w/ )) was p epa ed in chlo o o m/DMF a 30:20 olume a io. The esul an solu ion was elec ospun a 75.0 kV, using a wo king dis ance o 15 cm and an elec ode o a ion a e o 55 Hz (elec ode spin =8.8 /min). Bo om laye : CS-TPP blend was p epa ed acco ding o a p e iously epo ed me hod by Nguyen e al. [ 40 ] using sodium ipolyphospha e (TPP) as a c osslinking agen . B ie ly, 0.2% (w/ ) chi osan was dissol ed in 0.35% ace ic acid and kep o e nigh a oom empe a u e. The pH o he esul ing chi osan solu ion was hen adjus ed o pH 5.5 using a 0.5 M sodium hyd oxide (NaOH) solu ion. In u n, a TPP solu ion was p epa ed in dis illed wa e a a concen a ion o 0.25% (w/ ). The CS-TPP was p oduced by d opping he TPP solu ion in o he CS solu ion unde igo ous s i ing in a olume a io o 6:1 o 60 min a oom empe a u e. A e wa d, he CS-TPP was blended wi h 10% (w/ ) o Poly inyl Alcohol (PVA) dissol ed in dis illed wa e a 90 ◦ C wi h a olume a io o 70:30, espec i ely. Addi ionally, he c ude CA ex ac inco po a ion in o CS-TPP was achie ed by adding 3 mg/mL o he CA ex ac in he TPP solu ion. The CS-TPP_CA was p oduced ollowing he same p ocedu e as o he CS-TPP. A e polyme solu ions we e ob ained, hey we e placed in a con aine wi h a o a ing spinning elec ode, and elec ospun on op o he ecen ly p epa ed PCL’s op laye a an elec ode spin o 45 Hz (elec ode spin =7.2 /min), using a wo king dis ance o 15 cm and an applied ol age o 75 kV. Finally, he ab ica ed double-laye ed nanocomposi es memb anes (PCL/PVA_CS-TPP and PCL/PVA_CS-TPP_CA) we e cha ac e ized h ough in i o assays o assess hei app op ia eness as a wound d essing ma e ial. 2.5. Cha ac e iza ion o he P oduced Double-Laye ed Nanocomposi es Memb anes 2.5.1. Scanning Elec on Mic oscopy (SEM) Imaging and Analysis The su ace mo phology o he elec ospun nano ibe s o he op laye (PCL) and bo om laye s (PVA_CS-TPP and PVA_CS-TPP_CA) o he de eloped double-laye ed nanocomposi e memb anes was obse ed using scanning elec on mic oscopy (SEM) (S2700, Hi achi, Tokyo, Japan) a an accele a ing ol age o 20 kV. Fi s , he samples we e moun ed on aluminum s ubs and spu e -coa ed wi h a hin gold laye in an Emi ech K550 spu e coa e (Quo um Technologies L d., Laugh on, Eas Sussex, UK) o be e conduc i i y du ing imaging. The ibe diame e s we e measu ed om he ob ained SEM images using ImageJ so wa e (Na ional Ins i u es o Heal h, MD, USA) and he size- equency dis ibu ions cons uc ed wi h G aphPad P ism 6 so wa e (G aphPad So wa e, La Jolla, CA, USA). Polyme s 2020,12, 2653 5 o 18 2.5.2. A enua ed To al Re lec ance–Fou ie T ans o m In a ed Spec oscopy S udy The chemical composi ion o he op laye (PCL), he bo om laye s (PVA_CS-TPP and PVA_CS-TPP_CA), and hei aw ma e ials was analyzed using a enua ed o al e lec ance–Fou ie ans o m in a ed spec oscopy (ATR–FTIR, The mo-Nicole is10 FT-IR Spec opho ome e , Wal ham, MA, USA). The spec a o he samples we e eco ded in a spec al wid h anging om 400–4000 cm −1 wi h an a e age o 32 scans min−1and a spec al esolu ion o 4 cm−1. 2.5.3. Di e en ial Scanning Calo ime y (DSC) The he mal beha io o he PCL’s op laye and he bo om laye s o PVA_CS-TPP wi h and wi hou CA ex ac was e alua ed by di e en ial scanning calo ime y (DSC) (DSC 204 Phoenix Ne zsch, Ge many). B ie ly, abou 5 mg o each sample was illed in small aluminum con aine s, and he non-iso he mal scans pe o med om 30 ◦ C o 200 ◦ C a a hea ing a e o 5 ◦ C/min, wi h a ni ogen- eplacing a mosphe e. 2.5.4. Assessmen o he Mechanical Cha ac e is ics o he P oduced Double-Laye ed Nanocomposi es Memb anes The ensile es was ca ied ou in d y condi ions acco ding o he ASTM s anda d D3039/D3039M o e alua e he mechanical cha ac e is ics o he p oduced double-laye ed nanocomposi es memb anes. B ie ly, samples o he PCL/PVA_CS-TPP and PCL/PVA_CS-TPP con aining CA samples (n =5) we e cu in o ec angula s ips o 1 cm × 4 cm, and hen he hickness was measu ed wi h a mic ome e (Adamel Lhoma gy MI20, F ance). The ensile es was pe o med using a dynamome e (DY-35 Adamel Lhoma gy, F ance) by using a load cell o 10-N. The samples we e moun ed e ically be ween he clamps o he ensile es e , and a speed o 2 mm/min used un il he memb anes we e up u ed. Finally, he ensile s eng h, Young’s modulus, and elonga ion a b eak we e de e mined. 2.5.5. Measu emen o he To al Po osi y The o al po osi y o he d ied PCL’s op laye and bo om laye s o PVA_CS-TPP and PVA_CS-TPP_CA was measu ed using a luid displacemen me hod and conduc ed as p e iously desc ibed by Yeh e al. [ 41 ]. Absolu e e hanol wi h densi y ρε was used as displacemen liquid because i can easily pene a e he po ous s uc u e wi hou inducing negligible sh inking o swelling as a non-sol en o bo h laye s. B ie ly, a g adua ed cylinde wi h e hanol was weighed (W 1 ), hen a d ied sample wi h a known weigh (W s ) was imme sed in o he cylinde con aining he displacemen liquid. A e ha , his assembly was placed in an ul asonic ba h (Ul asons-H, P-Selec a) o 40 min a 30 ◦ C. A e his pe iod, he olume o e hanol in he g adua ed cylinde was e illed and weighed as W 2 . The sample sa u a ed wi h e hanol was aken ou om he cylinde , and i s weigh de e mined as W 3 . The po osi y (ε) o bo h laye s was es ima ed h ough he ollowing (Equa ions (1)–(3)): Vs=(W1−W2+Ws) ρε (1) Vp=(W2−W3−Ws) ρε (2) ε(%)=Vp (Vp+Vs)×100 ⇔ε(%)=(W2−W3−Ws) (W1−W3)×100 (3) whe e Vs is he olume o he sample, and Vp is he olume o he sample po es. Fo each sample, he po osi y measu emen s we e pe o med in iplica e, and he a e age ± s anda d de ia ion (S.D.) shown o each sample. Polyme s 2020,12, 2653 6 o 18 2.5.6. E alua ion o We abili y P ope ies The wa e con ac angles (WCA) a he su ace o bo h laye s (PCL, PVA_CS-TPP, and PVA_CS-TPP_CA) we e de e mined using a Da a Physics Con ac Angle Goniome e (OCAH-200) o su ace-we ing cha ac e iza ion. B ie ly, each sample was placed on he measu ing s age, hen wa e d ops (4 µ L) we e sea ed on o he su ace o he samples a di e en loca ions a 25 ◦ C. The epo ed WCA alues we e he a e age o a leas h ee independen measu emen s (n =3). 2.5.7. Analysis o he In Vi o Swelling Beha io The swelling deg ee o he p oduced double-laye ed nanocomposi es memb anes (PCL/PVA_CS-TPP and PCL/PVA_CS-TPP_CA) was in es iga ed in a phospha e bu e solu ion (PBS) a a pH o 5.5 by using a g a ime ic me hod. B ie ly, he p e-weigh ed d y samples (W 0 ) we e imme sed in he PBS a 37 ◦ C. A speci ic ime poin s, he swollen samples we e emo ed om he PBS bu e solu ion and eweigh ed a e kindly wiping he excess bu e o he samples (W ). All measu emen s we e pe o med in iplica e (n =3) and he amoun o wa e up ake de e mined acco ding o he ollowing Equa ion (4): Swelling Ra io (%)=(W −W0) W0 ×100 (4) 2.5.8. S udy o he In Vi o Biodeg ada ion P o ile The physical in eg i y beha io s we e analyzed om he weigh loss o he p oduced double-laye ed nanocomposi es memb anes. B ie ly, he d ied samples wi h he ini ial weigh o (W 0 ) we e imme sed in o PBS solu ion (pH =5.5) a 37 ◦ C. A p ede e mined ime in e als (1, 4, 7, and 10 days), he samples (n =3) we e emo ed om he PBS solu ion, insed wi h dis illed wa e o emo e esidual bu e sal s, o en-d ied, and eweigh ed (W d ). Finally, he weigh loss (%) o each sample was de e mined based on Equa ion (5): Weigh loss (%)=(W0−Wd) Wd ×100 (5) 2.5.9. Wa e Vapo T ansmission Ra e (WVTR) Analysis The g a ime ic assay based on he ASTM E96/E96M-15 s anda d was used o e alua e he wa e apo ansmission a e (WVTR) o he p oduced double-laye ed nanocomposi es memb anes. B ie ly, sample ci cles (1.2 cm diame e ) we e cu and ca e ully a ached o he mou hs o es ubes con aining 10 mL o deionized wa e . The ci cula opening o he es ubes was sealed using pa a ilm, and he samples–glass ubes assembly placed in an incuba o a 37 ◦ C. A p ede e mined in e als, he amoun o wa e e apo a ion was es ima ed by he changes in hei weigh o e ime. The WVTR was calcula ed acco ding o Equa ion (6): Wa e apo ansmission a e (WVTR)=Wloss Ag/m2/day(6) whe e Wloss is he daily weigh loss o wa e and A is he es a ea in m2. 2.6. Analysis o he In Vi o CA Release om Double-Laye ed Nanocomposi es Memb anes The in i o elease p o ile o he double-laye ed nanocomposi es memb anes con aining c ude CA ex ac was in es iga ed in PBS (pH =5.5) con aining 10% ( / ) o e hanol. The amoun o eleased CA in PBS was moni o ed by a UV–Vis spec opho ome e a a wa eleng h o 370 nm [ 42 ]. B ie ly, he double-laye ed PCL/PVA_CS-TPP_CA memb anes we e kep imme sed in PBS bu e a 37 ◦ C and 100 pm o 10 days. A speci ic ime poin s, a ixed olume o eleased medium was aken ou om he incuba ion medium, and an equal amoun o esh bu e solu ion e illed o main ain he Polyme s 2020,12, 2653 7 o 18 sink condi ion. The amoun o c ude CA ex ac eleased was measu ed by con e ing i s de ec ed UV abso bance o i s concen a ion acco ding o he calib a ion cu e cons uc ed om a se ies o CA s anda d solu ions ( om 0.00 mg/mL o 5.00 mg/mL). A e ha , he da a ob ained we e e alua ed o de e mine he cumula i e pe cen age o he eleased CA om he samples a each imme sion ime poin . The expe imen s we e conduc ed in iplica e (n =3). 2.7. Assessmen o he An ibac e ial P ope ies o he P oduced Double-Laye ed Nanocomposi es Memb anes Thean ibac e ialac i i y o bo hlaye s(PCL,PVA_CS-TPP,andPVA_CS-TPP_CA)wasexanima ed agains S. au eus and P. ae uginosa ollowing he guidelines es ablished by he S anda d Tes Me hod o De e mining he Ac i i y o Inco po a ed An imic obial Agen (s) in Polyme ic o Hyd ophobic Ma e ials (ASTM E2180-07 s anda d). Fi s ly, S. au eus and P. ae uginosa we e cul i a ed in nu ien b o h (NB) and b ain–hea in usion b o h (BHI) in a shaking incuba o a 37 ◦ C and 110 pm o 18–24 h, espec i ely. A e ha , he bac e ial suspensions we e dilu ed un il he bac e ial concen a ion eached ~10 8 CFU/mL, hen added o he p e iously p epa ed aga slu y o acili a e su ace in e ac ion. A hin laye o inocula ed aga slu y was pipe ed on o he samples and hen le o gel a oom empe a u e be o e incuba ion a 37 ◦ C o 18–24 h. The su i ing bac e ia we e analyzed immedia ely (T 0h ) and a e incuba ion (T 24h ) by elu ion o he aga slu y inoculum om he es samples. A e bac e ia elu ion, se ial dilu ions we e made in NaCl and pipe ed on aga pla es, and incuba ed a 37 ◦ C o 18–24 h. Finally, he numbe o su i ing colonies ollowing incuba ion was coun ed, and he coun s used o es ablish he log (CFU/mL). 2.8. Analysis o he In Vi o Cell Viabili y The cy o oxici y o he double-laye ed nanocomposi es memb anes (PCL/PVA_CS-TPP and PCL/PVA_CS-TPP_CA) was e alua ed h ough colo ime ic 3-(4,5-Dime hyl-2- hiazolyl)-2,5-diphenyl- 2H- e azolium b omide (MTT) assay acco ding o ISO 10993–5 (Biological e alua ion o medical de ices–Pa 5: Tes s o in i o cy o oxici y). Fi s ly, he no mal human de mal ib oblas s (NHDF) cells we e cul u ed in a medium supplemen ed wi h e al bo ine se um (FBS) in a humidi ied incuba o a 37 ◦ C unde a 5% CO 2 a mosphe e. A e wa d, he samples cu in o ound disks (wi h a diame e o ~6 mm) we e placed a he cen e o each well in 24-well pla es, hen s e ilized by UV i adia ion o 1 h be o e cell seeding. A e ha , 1 × 10 4 cells/well we e seeded in each well con aining he s e ilized memb anes and incuba ed wi h 5% CO 2 a 37 ◦ C o 1, 3, and 7 days. Du ing hese in e als o ime, he medium was emo ed, and a mix u e o esh cul u e medium wi h he MTT eagen added o each well. A e being incuba ed o 4 h unde he same condi ions, he con en o each well was again emo ed and eplaced by DMSO o dissol e he o mazan c ys als. Finally, he abso bance o each memb ane was measu ed a 570 nm using a spec opho ome ic pla e eade (Bio ad xMa k mic opla e spec opho ome e ). Cells incuba ed wi hou samples (K − ) and cells wi h E OH (96%) (K + ) we e chosen as con ol g oups. The posi i e con ol (K + ) was added in sepa a e 24 well pla es o a oid alse esul s caused by E OH (96%). 2.9. S a is ical Analysis S a is ical analysis was pe o med om he one-way ANOVA, ollowed by mul iple compa ison es Tu key using G aphPad P ism 6 so wa e (G aphPad So wa e, La Jolla, CA, USA) wi h a s a is ical signi icance o p<0.05. 3. Resul s and Discussion 3.1. Minimal Inhibi o y Concen a ion (MIC) o he C ude CA Ex ac The an imic obial suscep ibili y o he c ude CA ex ac was de e mined by he MIC. The MIC alue agains S. au eus was ound o be 1.40 mg/mL, while he alue o P. ae uginosa was 2.80 mg/mL. These alues we e lowe han hose ob ained by Yao e al. [ 43 ], who e ealed MIC alues o he Polyme s 2020,12, 2653 8 o 18 e hanolic ex ac o CA o 6.25 mg/mL and 25 mg/mL agains S. au eus and P. ae uginosa, espec i ely. These esul s p o ed ha he an ibac e ial ac i i y o he medicinal plan s depends on he speci ic ac i e compounds p esen in he ex ac . 3.2. Cha ac e iza ion o he P oduced Double-Laye ed Nanocomposi es Memb anes 3.2.1. Scanning Elec on Mic oscopy (SEM) Imaging and Analysis In his s udy, he su ace mo phologies and diame e dis ibu ions o he elec ospun nano ibe s om PCL’s op laye and he bo om laye s o PVA_CS-TPP and PVA_CS-TPP inco po a ed wi h he c ude CA ex ac , espec i ely, a e displayed in Figu e 1a. The SEM images show ha bo h laye s exhibi a andom dis ibu ion o nano ibe s wi h in e connec ed po es. The a e age diame e s o he smoo h PCL s uc u e we e de e mined o be 277.63 ± 85.19 nm, which is in ag eemen wi h o he s udies pe o med wi h PCL [ 44 ]. In u n, he a e age ibe diame e o he smoo h and bead- ee s uc u es o PVA_CS-TPP was dec eased om 323.85 ± 91.07 nm o 284.34 ± 75.79 nm when he c ude CA ex ac was inco po a ed, as a esul o educ ion o he iscosi y o he elec ospinning solu ion. In his way, hese esul s sugges ha he p oduced double-laye ed nanocomposi es memb anes esemble he ib ous mo phology and a chi ec u e o he na u al ex acellula ma ix (ECM) since he nano ibe s exhibi diame e s wi hin he size ange o he collagen ibe s o ECM (50–400 nm), being able o p omo e cell adhesion and p oli e a ion [45,46]. Polyme s 2020, 12, x FOR PEER REVIEW 8 o 19 3.2. Cha ac e iza ion o he P oduced Double-Laye ed Nanocomposi es Memb anes 3.2.1. Scanning Elec on Mic oscopy (SEM) Imaging and Analysis In his s udy, he su ace mo phologies and diame e dis ibu ions o he elec ospun nano ibe s om PCL’s op laye and he bo om laye s o PVA_CS-TPP and PVA_CS-TPP inco po a ed wi h he c ude CA ex ac , espec i ely, a e displayed in Figu e 1a. The SEM images show ha bo h laye s exhibi a andom dis ibu ion o nano ibe s wi h in e connec ed po es. The a e age diame e s o he smoo h PCL s uc u e we e de e mined o be 277.63 ± 85.19 nm, which is in ag eemen wi h o he s udies pe o med wi h PCL [44]. In u n, he a e age ibe diame e o he smoo h and bead- ee s uc u es o PVA_CS-TPP was dec eased om 323.85 ± 91.07 nm o 284.34 ± 75.79 nm when he c ude CA ex ac was inco po a ed, as a esul o educ ion o he iscosi y o he elec ospinning solu ion. In his way, hese esul s sugges ha he p oduced double-laye ed nanocomposi es memb anes esemble he ib ous mo phology and a chi ec u e o he na u al ex acellula ma ix (ECM) since he nano ibe s exhibi diame e s wi hin he size ange o he collagen ibe s o ECM (50–400 nm), being able o p omo e cell adhesion and p oli e a ion [45,46]. Acco ding o he c oss-sec ional image, Figu e 1b, i is possible o obse e he wo di e en laye s o he p oduced double-laye ed nanocomposi es memb anes. Figu e 1. Mo phology and ibe diame e dis ibu ion o he Polycap olac one (PCL)’s op laye and he Poly( inyl alcohol) (PVA) and Chi osan-Sodium ipolyphospha e (CS-TPP) (PVA_CS-TPP) and PVA_CS-TPP con aining Cen ella asia ica (L.) (CA)’s bo om laye s (a); and c oss-sec ional SEM images Figu e 1. Mo phology and ibe diame e dis ibu ion o he Polycap olac one (PCL)’s op laye and he Poly( inyl alcohol) (PVA) and Chi osan-Sodium ipolyphospha e (CS-TPP) (PVA_CS-TPP) and PVA_CS-TPP con aining Cen ella asia ica (L.) (CA)’s bo om laye s ( a ); and c oss-sec ional SEM images o he double-laye ed memb anes; ( b ) PCL/PVA_CS-TPP wi h c ude CA ex ac (on he igh ) and wi hou (on he le ). Polyme s 2020,12, 2653 9 o 18 Acco ding o he c oss-sec ional image, Figu e 1b, i is possible o obse e he wo di e en laye s o he p oduced double-laye ed nanocomposi es memb anes. 3.2.2. A enua ed To al Re lec ance-Fou ie T ans o m In a ed Spec oscopy S udy The acqui ed ATR-FTIR spec a o he p oduced double-laye ed nanocomposi es memb anes a e p esen ed in Figu e 2. The spec um o he PCL’s op laye displays i s cha ac e is ic bands, Figu e 2a. The peaks a 2865.22 and 2943.10 cm −1 belongs o he symme ic and asymme ic CH 2 s e ching ib a ion, while he band a 1722.95 cm−1co esponds o he C=O s e ching ib a ion [47]. Polyme s 2020, 12, x FOR PEER REVIEW 9 o 19 o he double-laye ed memb anes; (b) PCL/PVA_CS-TPP wi h c ude CA ex ac (on he igh ) and wi hou (on he le ). 3.2.2. A enua ed To al Re lec ance-Fou ie T ans o m In a ed Spec oscopy S udy The acqui ed ATR-FTIR spec a o he p oduced double-laye ed nanocomposi es memb anes a e p esen ed in Figu e 2. The spec um o he PCL’s op laye displays i s cha ac e is ic bands, Figu e 2a. The peaks a 2865.22 and 2943.10 cm −1 belongs o he symme ic and asymme ic CH 2 s e ching ib a ion, while he band a 1722.95 cm −1 co esponds o he C=O s e ching ib a ion [47]. In u n, he spec um o he PVA_CS-TPP’s bo om laye shows he cha ac e is ic peaks o he PVA and CS a 3316.14 and 2936.47 cm −1 , a ibu ed o he O-H and CH 2 s e ching ib a ion, espec i ely, and a peak a 1642.57 cm −1 assigned o C=O s e ching o a p ima y amide, Figu e 2b. These bands e ealed ha he PVA and CS-TPP we e success ully dispe sed in he nano ibe s [31]. Mo eo e , when he CA ex ac was inco po a ed in o he PVA_CS-TPP nano ibe s, a highe in ensi y o he peaks was obse ed once he cha ac e is ic peaks o he c ude CA ex ac o e lapped wi h he bands o PVA_CS-TPP, Figu e 2b. A simila e ec was p e iously epo ed by Amina e al. [48], who showed ha he cha ac e is ic peaks o PU nano ibe s o e lapped wi h he bands o aqueous ex ac o G ewia mollis (G. mollis), leading o a highe in ensi y in he PU/G. mollis nano ibe s’ spec um. Howe e , he spec um o aw CA con i ms i s cha ac e is ics peaks a 3322.12 cm −1 (O-H s e ching ib a ion o ca boxylic acid g oup), 1656.07 cm −1 (C-O s e ching ib a ion), 1451.17 cm −1 (C-H in- plane bending ib a ion), 1375.48 cm −1 (C-N s e ching ib a ion, a oma ic amide), and 1024.98 cm −1 (C-O s e ching) [49]. Figu e 2. ATR-FTIR analysis o he p oduced double-laye ed memb anes. FTIR spec a o he PCL’s op laye (a), he bo om laye s o PVA_CS-TPP and PVA_CS-TPP_CA, and hei aw ma e ials (b). 3.2.3. Di e en ial Scanning Calo ime y (DSC) The he mal p ope ies o bo h laye s (PCL’s op laye and he bo om laye s o PVA_CS-TPP and PVA_CS-TPP con aining c ude CA ex ac ) we e e alua ed by DSC, as demons a ed in Figu e 3. In PCL’s op laye , he endo he mic peak a 62.96 °C co esponds o he mel ing empe a u e (T m ) o PCL. This esul is in ag eemen wi h he da a a ailable in he li e a u e o elec ospun PCL memb anes (T m (PCL) = 60.10 °C) [50]. On he o he hand, he aw PVA_CS wi h he PVA_CS-TPP nano ibe s display an endo he mic peak a 62.24 °C co esponding o he e apo a ion o wa e and ace ic acid sol en s [51,52]. In addi ion, a weak endo he mic peak was ound a 188.43 °C, which is due o he mel ing o PVA c ys als [51]. Addi ionally, he DSC he mog ams sugges ha he p esence o CS-TPP shi ed he endo he mic peaks o a highe empe a u e, con i ming he he mal s abili y o he ionically c oss- Figu e 2. ATR-FTIR analysis o he p oduced double-laye ed memb anes. FTIR spec a o he PCL’s op laye (a), he bo om laye s o PVA_CS-TPP and PVA_CS-TPP_CA, and hei aw ma e ials (b). In u n, he spec um o he PVA_CS-TPP’s bo om laye shows he cha ac e is ic peaks o he PVA and CS a 3316.14 and 2936.47 cm −1 , a ibu ed o he O-H and CH 2 s e ching ib a ion, espec i ely, and a peak a 1642.57 cm −1 assigned o C=O s e ching o a p ima y amide, Figu e 2b. These bands e ealed ha he PVA and CS-TPP we e success ully dispe sed in he nano ibe s [ 31 ]. Mo eo e , when he CA ex ac was inco po a ed in o he PVA_CS-TPP nano ibe s, a highe in ensi y o he peaks was obse ed once he cha ac e is ic peaks o he c ude CA ex ac o e lapped wi h he bands o PVA_CS-TPP, Figu e 2b. A simila e ec was p e iously epo ed by Amina e al. [ 48 ], who showed ha he cha ac e is ic peaks o PU nano ibe s o e lapped wi h he bands o aqueous ex ac o G ewia mollis (G. mollis), leading o a highe in ensi y in he PU/G. mollis nano ibe s’ spec um. Howe e , he spec um o aw CA con i ms i s cha ac e is ics peaks a 3322.12 cm −1 (O-H s e ching ib a ion o ca boxylic acid g oup), 1656.07 cm −1 (C-O s e ching ib a ion), 1451.17 cm −1 (C-H in-plane bending ib a ion), 1375.48 cm−1(C-N s e ching ib a ion, a oma ic amide), and 1024.98 cm−1(C-O s e ching) [49]. 3.2.3. Di e en ial Scanning Calo ime y (DSC) The he mal p ope ies o bo h laye s (PCL’s op laye and he bo om laye s o PVA_CS-TPP and PVA_CS-TPP con aining c ude CA ex ac ) we e e alua ed by DSC, as demons a ed in Figu e 3. In PCL’s op laye , he endo he mic peak a 62.96 ◦ C co esponds o he mel ing empe a u e (T m ) o PCL. This esul is in ag eemen wi h he da a a ailable in he li e a u e o elec ospun PCL memb anes (Tm(PCL) =60.10 ◦C) [50]. On he o he hand, he aw PVA_CS wi h he PVA_CS-TPP nano ibe s display an endo he mic peak a 62.24 ◦ C co esponding o he e apo a ion o wa e and ace ic acid sol en s [ 51 , 52 ]. In addi ion, a weak endo he mic peak was ound a 188.43 ◦ C, which is due o he mel ing o PVA c ys als [ 51 ]. Addi ionally, he DSC he mog ams sugges ha he p esence o CS-TPP shi ed he endo he mic peaks o a highe empe a u e, con i ming he he mal s abili y o he ionically c oss-linked elec ospun Polyme s 2020,12, 2653 16 o 18 9. Zhang, W.; Ronca, S.; Mele, E. Elec ospun nano ib es con aining an imic obial plan ex ac s. Nanoma e ials 2017,7, 42. [C ossRe ] 10. dos San os, D.M.; Lei e, I.S.; de Lace da Bukzem, A.; de Oli ei a San os, R.P.; F ollini, E.; Inada, N.M.; Campana-Filho, S.P. 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[C ossRe ] Publishe ’s No e: MDPI s ays neu al wi h ega d o ju isdic ional claims in published maps and ins i u ional a ilia ions. © 2020 by he au ho s. Licensee MDPI, Basel, Swi ze land. This a icle is an open access a icle dis ibu ed unde he e ms and condi ions o he C ea i e Commons A ibu ion (CC BY) license (h p://c ea i ecommons.o g/licenses/by/4.0/).