Smart design for a flexible, functionalized and electroresponsive hybrid platform based on poly(3,4-ethylenedioxythiophene) derivatives to improve cell viability

1 Sma design o lexible, unc ionalized and elec o esponsi e hyb id pla o m based on poly(3,4-e hylenedioxy hiophene) de i a i es o imp o e cell iabili y B enda G. Molina,1,2,* Anca D. Bend ea,3 Sonia Lanzalaco,1,2 Lou des F anco,1,2 Lumini a Cianga,3 Luis J. del Valle,1,2 Jo di Puiggali,1,2,4 Pau Tu on,5 Elaine A melin,1,2 Ioan Cianga3,* and Ca los Aleman1,2,4,* 1 Depa amen d’Enginye ia Química, EEBE, Uni e si a Poli cnica de Ca alunya, C/ Edua d Ma is any, 10-14, Ed. I2, 08019, Ba celona, Spain 2 Ba celona Resea ch Cen e o Mul iscale Science and Enginee ing, Uni e si a Poli cnica de Ca alunya, C/ Edua d Ma is any, 10-14, Ed. C, 08019, Ba celona, Spain 3 Pe u Poni” Ins i u e o Mac omolecula Chemis y, 41A, G igo e–Ghica Voda Alley, 700487, Iasi, Romania 4 Ins i u e o Bioenginee ing o Ca alonia (IBEC), The Ba celona Ins i u e o Science and Technology, Baldi i Reixac 10-12, 08028 Ba celona Spain 5 B. B aun Su gical, S.A. Ca e e a de Te asa 121, 08191 Rubí (Ba celona), Spain Co espondence o: [email p o ec ed], [email p o ec ed] and [email p o ec ed]du  Romanian au ho s dedica e his pape as a ibu e o la e P o esso C is o o I. Simionescu, o his li e-long ema kable achie emen in polyme science esea ch and educa ion and o he cen ena y commemo a i e celeb a ion o his bi h (1920-2007). 2 ABSTRACT De elopmen o sma unc ionalized ma e ials o issue enginee ing has a ac ed signi ican a en ion in ecen yea s. In his wo k we ha e unc ionalized a ee-s anding ilm iso ac ic polyp opylene (i-PP), a syn he ic polyme ha is ypically used o biomedical applica ions (e.g. ab ica ion o implan s), o enginee ing a 3D all-polyme lexible in e ace ha enhances cell p oli e a ion by a ac o o ca. h ee. A hie a chical cons uc ion p ocess consis ing o h ee s eps was enginee ed as ollows: 1) unc ionaliza ion o i-PP by applying a plasma ea men , esul ing in i-PP ; 2) i-PP su ace coa ing wi h a laye o polyhyd oxyme hy-3,4-e hylenedioxy hiophene nanopa icles (PHMeEDOT NPs) by in si u chemical oxida i e polyme iza ion o HMeEDOT; and 3) deposi ion on he p e iously ac i a ed and PHMeEDOT NPs coa ed i-PP ilm (i-PP /NPs) o a g a conjuga ed copolyme , ha ing poly(3,4- e hylenedioxy hiophene) (PEDOT) backbone, and andomly dis ibu ed sho poly(- cap olac one) (PCL) side chains (PEDOT-g-PCL), as a coa ing laye o 9 m in hickness. The p ope ies o he esul ing biopla o m, which can be de ined as a obus mac oscopic composi e coa ed wi h a "molecula composi e", we e in es iga ed in de ail, and bo h adhesion and p oli e a ion o wo human cell lines ha e been e alua ed, as well. Resul s demons a e ha he inco po a ion o he PEDOT-g-PCL laye signi ican ly imp o es cell a achmen and cell g ow h no only when compa ed o i-PP bu also wi h espec o he same pla o m coa ed wi h only PEDOT, cons uc ed in a simila manne , as con ol. 3 INTRODUCTION Conduc ing polyme s (CPs) as polyme ic bio-in e aces ha e gained signi ican a en ion due hei biocompa ibili y combined wi h hei unique elec ochemical and elec ical p ope ies.1-6 Thus, hei abili y o accommoda e and anspo elec onic and ionic cha ge ha e made CPs a ac i e candida es as bioma e ials in applica ions ha equi e communica ion ac oss in e aces by exchanging cha ge, as o example bioac i e in e aces. Thus, CPs in e change cha ge a he in e ace wi h cell memb anes, s imula ing cell adhesion and p oli e a ion.7-12 Fu he mo e, CPs a e esponsi e o elec ical, elec ochemical and mechanical s imuli, in luencing he beha io o cells o issue cul u ed on o hem.9-12 Func ionaliza ion wi h biomolecules has been used o op imize hei cell binding abili y. Fo example, conjuga es in ol ing CPs unc ionalized wi h he A g-Gly-Asp (RGD) pep ide ha e shown imp o emen s in cell a achmen , u he coloniza ion and cell su i al.13,14 In he las decade, g a copolyme s wi h a CP backbone ha e become a p omising amily o ma e ials o di e se biomedical applica ions. These ma e ials ake ad an age o he unique elec ochemical and elec ical p ope ies o CPs and he chemical and/o physical p ope ies o he g a ed side chains. Fo example, conduc ing g a ed copolyme s ha e been used o ab ica e elec ochemical senso s o bioanaly ics (e,g quan i a i e de ec ion o NADH, se o onin, dopamine, cocaine and non-Hodgkin lymphoma gene);15-19 o cons uc su aces wi h an imic obial16 o imaging ac i i y;20 o p e en he non-speci ic adhesion o cells and p o eins;21,22 o p epa e nanoca ie sys ems ha enable size-selec i e encapsula ion o d ug molecules and hei sus ained elease;23 and o p omo e cell ac i i y o issue enginee ing.24-26 G a copolyme s wi h CP backbones and lexible, s imuli-sensi i e side chains a e in e es ing polyme ic a chi ec u es ha , in o me epo s, we e also named "molecula 4 composi es" based on hei mac oscopic p ope ies.27,28 This ype o polyme s can be p epa ed using h ee di e en app oaches:29 1) “g a ing o” in which end- unc ionalized side chains (al eady polyme ized) a e connec ed o eac i e si es o he CP backbone; 2) “g a ing om” ha consis s on he g owing o he side chains om he ini ia ion si es o he CP; and 3) “g a ing h ough”, which in ol es he syn hesis o elec oac i e mac omonome s ha can be homopolyme ized22 o copolyme ized wi h an app op ia e low-molecula weigh compound16,17,20 o wi h ano he mac omonome .15 The “g a ing h ough” s a egy allows o he uning o side chains g a ing densi y and b anching poin s dis ance adjus men , gi ing he possibili y o modula e he ma e ials p ope ies o a a ge ed applica ion.30,31 In ecen yea s we ha e used such an app oach o p epa e g a copolyme s ha ing poly hiophene backbone and polye hylene glycol (PTh-g-PEG) as side chains.15,22,25,26 Also, we ob ained a conduc ing g a copolyme by elec ochemical copolyme iza ion o hyd oxyme hy-3,4-e hylenedioxy hiophene (HMeEDOT) and a hiophene-ended poly(-cap olac one) (PCL) mac omonome (PTh-g-PCL).17 In spi e o he p omising biomedical applica ions o PTh-g-PEG and PTh-g-PCL, he applicabili y o hose ma e ials is se e ely limi ed because o he elec odes (i.e. ypically s eel o indium in oxide (ITO)) used in he elec o(co)polyme iza ion p ocesses. The e o e, achie emen o sel -s anding and lexible g a -copolyme s based on CPs is highly desi able o p o ide applicabili y. In o de o o e come he abo e men ioned d awbacks, in his wo k we enginee ed a new 3D all-polyme s lexible sys em, which was cons uc ed in a hie a chical manne , combining using di e en polyme iza ion echniques and based on a combina ion o a mac oscopic composi e wi h a "molecula composi e" as an ou e laye . This op laye is ep esen ed by a g a CP, ha ing poly(3,4-e hylenedioxy hiophene) (PEDOT) 5 backbone and andomly dis ibu ed sho PCL side chains (PEDOT-g-PCL). This ma e ial has been ob ained using, in he las s ep, he g a ing h ough app oach. Mo e speci ically, a new syn hesized EDOT-con aining mac omonome has been elec ocopolyme ized wi h 3,4-e hylenedioxy hiophene (EDOT) as coa ing laye on iso ac ic polyp opylene (i-PP) used o biomedical implan s, which has been p e iously unc ionalized and elec oac i a ed by applying a plasma ea men and deposi ing poly(hyd oxyme hyl-3,4-e hylenedioxy hiophene) nanopa icles (PHMeEDOT NPs), espec i ely. A e chemical, elec ochemical, he mal and mo phological cha ac e iza ion o esul ing bioac i e pla o m, which is lexible and ee-s anding, he adhesion and p oli e a ion o wo human cell lines we e examined. Resul s demons a e he p omising po en ial o he biopla o m laye ed wi h PEDOT-g-PCL o issue enginee ing applica ions, imp o ing by a ac o o ca. h ee he capaci y o p oli e a e cells wi h espec o he biomedical i-PP used as suppo . RESULTS AND DISCUSSION Syn hesis o he mac omonome The EDOT-PCL mac omonome (Scheme S1), one o he ew epo ed o da e,32-36 was ob ained by ing-opening polyme iza ion o ε-cap olac one (ε-CL) using HMeEDOT as ini ia o and s annous oc anoa e, Sn(Oc )2, as ca alys . De ails abou in bulk ε-CL polyme iza ion a e p o ided in he Supplemen a y In o ma ion. 1H and 13C NMR spec a o he mac omonome in CDCl3 a e shown in Figu es S1 and S2, espec i ely, while he FTIR spec um is displayed in Figu e S3. The molecula weigh o he EDOT-PCL mac omonome , as es ima ed om 1H-NMR da a (Mn H- NMR), is 2055 Da, indica ing ha he polyme iza ion deg ee (PD) is 16.5. The esul s o GPC measu emen (Figu e S4) and an explana o y discussion on he s uc u al 6 cha ac e iza ion o EDOT-PCL mac omonome and o i s p ope ies in bulk can be ound in he Suppo ing In o ma ion. 1H NMR (CDCl3, δ in ppm): 6.37 (a,b); 4.42-4.36 (c); 4.33-4.23 (d,e), 4.10-4.05 (j), 3,69-3.66 (k); 2.42-2.29 ( ); 1.73-1.58 (i,g); 1.44-1.36 (h); (Figu e S1) 13C NMR (CDCl3, δ in ppm): (173.46, 172.9 (h); 141 (c,d); 99.9(a,b); 71.9, 71.48 (e); 65.56 ( ); 64.08(g,o); 62.6, 62.36 ( ); 34, 33.76 (i), 32.2 (p); 28.28 (m); 25.47 (j), 24.53 (k); (Figu e S2). IR (KB ), cm−1: 3539.32; 3439.55;(PCL); 3114.58; ( hiophene ing -Th- o EDOT); 2946.39 - 2866.58; (-CH2, -CH alipha ic in EDOT and PCL); 1725.13; (PCL); 1487.3; (Th); 1471.63; (PCL); 1420.36 (Th); 1399; (PCL) 1369.09; (EDOT); 1295.04; 1245.19; (PCL); 1192.5; (PCL and EDOT); 1107.05; 1045.81; (EDOT); 962; (PCL); 933.3; 860.67; 840.73; (Th); (Figu e S3 and de ailed discussion in Suppo ing In o ma ion). P epa a ion o bioac i e pla o m The bioac i e pla o m was p epa ed using a h ee-s ep p ocess (Figu e 1a). Fi s ly, he su ace o i-PP ilms (10  10 cm2) commonly used o biomedical applica ions (e.g. su gical su u es and meshes), which we e kindly supplied by B aun Su gical S.A. (Rubí, Ba celona, Spain), was unc ionalized wi h oxygen plasma (0.30 mba ) du ing 180 seconds and using a powe discha ge o 250 W. Secondly, he unc ionalized i-PP (i- PP ) ilms we e cu in 0.5  1.5 cm2 samples, which we e coa ed wi h PHMeEDOT NPs by imme sing each one in 5 mL o 0.2 M HCl wi h 50 mM MHeEDOT monome du ing 30 min and unde agi a ion (250 pm). The oxida i e chemical polyme iza ion o he monome was conduc ed by slowly d opping 1 mL o 0.2 M HCl solu ion con aining 60 mM o ammonium pe sul a e (APS). The eac ion was kep a 37 °C and 80 pm o 24 h. A e his pe iod, he unc ionalized and elec oac i a ed ma e ial, 7 he ea e named i-PP /NPs, was washed wi h a 0.2 M NaOH solu ion o balance he cha ge, and hen d ied. These wo cons uc ion s eps p o ided he unique oppo uni y o enginee he biopla o ms su ace a he molecula le el. Thus, he i s o med PHMeEDOT polyme chains, due o he ee hyd oxyl unc ional g oups, could be o ien ed and s ongly ancho ed o i-PP su ace ia he hyd ogen bonding wi h exis ing pola g oups ((e.g. C=O and C–O) o med du ing he plasma discha ge. This o ien a ion o he i s polyme chains will d i e he o ganiza ion o all o he g own polyme chains inside o NPs go e ned also by o he ypes o physical in e molecua in e ac ions (hyd ophobic, - s acking). In he las s ep, he EDOT-PCL mac omonone and he EDOT monome we e elec ocopolyme ized on i-PP /NPs ilms. Fo his pu pose, each i-PP /NPs ilm was in oduced in a h ee-elec ode cell illed wi h 20 mL o an ace oni ile solu ion con aining 0.1 M LiClO4 as suppo ing elec oly e and bo h EDOT and EDOT-PCL in a millimola a io o 7/3. Elec ocopolyme iza ion was conduc ed a a cons an po en ial o 1.40 V and adjus ing he polyme iza ion cha ge o 1.0 C. I should be no ed ha , in his p ocess PHMeEDOT NPs ac ed as polyme iza ion nuclei o he g owing o PEDOT-g-PCL chains. Fo he sake o compa ison, biopla o ms wi hou PCL side g oups we e also p epa ed using only EDOT monome (10 mM) in he elec opolyme iza ion s ep, which was conduc ed using iden ical expe imen al condi ions. I is wo h no ing ha he used elec o(co)polyme iza ion echnique enables a p ecise con ol o he coa ing hickness (see below) h ough he polyme iza ion cha ge, as was p o ed in ea ly kine ic s udies on PEDOT and o he ICPs.37 Though elec ochemically silen ( ide in a), he PHMeEDOT NPs laye seems ha did no block he cha ge anspo h ough he in e ace. This laye , o e ing su ace- 8 con ined elec ochemically eac ion si es in he o m o adical ca ions, allow o co alen connec ion o he new g owing copolyme ic/polyme ic chains o he suppo su ace.38,39 Thus, subsequen PEDOT-g-PCL copolyme o PEDOT homopolyme o ma ion con inue due o nuclea ion om exis ing PHMeEDOT,40 in he o m o su ace e he ed chains. Howe e , he exis ence o he physically adso bed PEDOT-g- PCL copolyme o PEDOT chains, as non-co alen coa ing on he PHMeEDOT NPs ilm su ace, especially o med in he la es phase o he elec opolyme iza ion, can' be excluded. These chains could o m s ong - s acks wi h he NPs su ace in a simila manne as he p e iously epo ed PCL-deco a ed poly-p-phenylene o med wi h he ca bon nano ubes su ace.41 He ea e , lexible and ee-s anding biopla o ms o med by i-PP /NPs and coa ed wi h a PEDOT-g-PCL o PEDOT (con ol) laye a e deno ed i- PP /PEDOT-g-PCL o i-PP /PEDOT, espec i ely. Figu e 1b shows pho og aphs o i-PP, i-PP /NPs, i-PP /PEDOT and i-PP /PEDOT-g- PCL. The anslucen i-PP ilm becomes opaque and blueish a e he inco po a ion o PHMeEDOT NPs, which in u n con e s in da k blue when PEDOT and PEDOT-g- PCL laye s a e deposi ed by elec o(co)polyme iza ion. These isual ans o ma ions sugges ha bo h he oxida i e polyme iza ion and he elec o(co)polyme iza ion occu ed success ully. Chemical cha ac e iza ion and we abili y The FTIR and Raman spec a o i-PP /PEDOT and i-PP /PEDOT-g-PCL a e compa ed in Figu e 2a-b. On he o he hand, he spec a o biomedical i-PP and i-PP we e epo ed in ecen wo k42,43 and ha e no been epea ed he e, while Figu es S5 and S6 show he FTIR and Raman spec a, espec i ely, o i-PP /NPs wi h he co esponding discussion. 9 The FTIR spec a o bo h biopla o ms a e domina ed by he abso p ion bands o i- PP subs a e and PEDOT backbone. The cha ac e is ic abso p ion peaks o i-PP co espond o he de o ma ion ib a ion o he CH2 g oup a 1461 cm-1, he me hyl g oup ib a ions a 1385 cm-1, and he cha ac e is ic ib a ions o CH2 g oups a abso p ion peaks a 998 and 1164 cm-1.42-44 Besides, he in ense b oad signals appea ing a a ound 1600 cm-1 a e a ibu ed o he C=O s e ching o he unc ional g oups c ea ed by he oxygen plasma ea men .42,43 The PEDOT bands e ealed in he spec a a e he C–S and C–S–C ib a ions in he hiophene ing a a ound 869, 757 and 628 cm- 1. Un o una ely, he dis inc i e peak o PCL side chains, which co esponds o he C=O s e ching a 1723 cm-1,45 o e laps wi h he b oad peak o i-PP . Al hough he de ec ion by FTIR spec oscopy o PCL side chains is almos hinde ed by hei sho leng h and low g a ing densi y, as compa ed o PEDOT chains, as well as by he in e e ences wi h i-PP signals, hei iden i ica ion ha e achie ed by high esolu ion Raman spec oscopy. Al hough he cha ac e is ics peaks o PEDOT backbone p edomina e in he 785 nm lase Raman spec a eco ded o wo coa ed biopla o ms (Figu e 2b), some clea di e ences allowed us o iden i y he p esence o PCL side chains in i-PP /PEDOT-g- PCL. Mo e speci ically, he ollowing peaks we e collec ed in he spec a o he wo sys ems: he ib a ion mode o he hiophene C–S bond a 988 cm-1; he s e ching o he e hylendioxy g oup a 1085 cm-1; he C–C in e - ing s e ching a 1258 cm-1; he C– C s e ching a 1365 cm-1; and he C=C s e ching a 1420 cm-1. The C=O and C–C s e ching peaks (1575 and 1137 cm-1, espec i ely), which can be a ibu ed o bo h i- PP and PCL, a e consis en ly mo e in ense o i-PP /PEDOT-g-PCL han o i- PP /PEDOT. E idence o he success ul EDOT-PCL mac omolecula inco po a ion in he i- PP /PEDOT-g-PCL biopla o m is also p o ided by he su ace we abili y. The con ac 16 Figu e S10 displays ep esen a i e scanning elec on mic oscopy (SEM) mic og aphs and bo h 2D heigh and phase con as a omic o ce mic oscopy (AFM) images o biomedical i-PP. The su ace o his subs a e is homogeneous, smoo h and ela i ely la , exhibi ing a oo -mean-squa e oughness (Rq) o 30  6 nm only. The e ec s o he unc ionaliza ion and elec oac i a ion on he mo phology and opog aphy o i-PP and i-PP /NPs a e shown in Figu e 6. The su ace becomes mo e complex a e unc ionaliza ion. Thus, he plasma ea men causes he appa i ion o a supe icial and homogeneous nano-pa e ning on he whole su ace (Figu e 6a). This mo phological change a ec s signi ican ly he opog aphy, showing a sligh inc ease o he su ace oughness (Rq = 38  6 nm) due o he appa i ion o small and sha p peaks abundan ly and homogeneously dis ibu ed. Abundan PHMeEDOT NPs wi h cap icious mo phology a e clea ly de ec ed in he su ace o i-PP /NPs (Figu e 6b), which expe iences a d as ic inc emen o he oughness (Rq = 312  12 nm) in compa ison o i- PP . Those NPs, which o ganize in a po ous laye o 1.1  0.1 m in hickness, we e andomly dis ibu ed on he i-PP su ace, o ming a andom con ac ne wo k s uc u e. Howe e , he con ac be ween such NPs was no la ge enough o ensu e pe cola ion and o o m conduc ion pa hs, which explains he inexis en elec ochemical ac i i y o i- PP /NPs ilms (Figu e 2c). Figu e 7 shows signi ican mo phological di e ences be ween i-PP /PEDOT and i- PP /PEDOT-g-PCL. In he o me , he PEDOT homopolyme comple ely co e s he PHMeEDOT NPs, in eg a ing hem in o a single conduc ing laye wi h con inuous and well-de ined conduc ion pa hs. This laye exhibi s he ypical he e ogeneous mo phology o elec opolyme ized PEDOT ilms,64,65 which consis s on he agg ega ion o dense clus e s o packed molecules. Thus, he linea g owing o PEDOT chains, which a e exclusi ely o med by - linkages (i.e. he -posi ions o he hiophene ing 17 a e occupied by he dioxane ing), a o s he o ma ion o compac sphe oidal clus e s ha agg ega e lea ing a la ge numbe o submic ome ic po es among hem. This unique s uc u e a ou s he access and escape o dopan ions du ing edox p ocesses, explaining he excellen elec ochemical beha io epo ed o PEDOT.64-67 Mo eo e , his mo phological o ganiza ion esul s in a ough su ace wi h Rq = 611  57 nm, as is shown in he AFM images included in Figu e 7a. The mic oscopic ex u e o i-PP /PEDOT-g-PCL is smoo he han in i-PP /PEDOT, which has been a ibu ed o he supe icial loca ion o he PCL side chains. This assump ion is con i med by he con as in he AFM phase image included in Figu e 7b, which allows dis inguishing be ween wo phases in i-PP /PEDOT-g-PCL. Phase imaging is a powe ul ool ha is sensi i e o su ace s i ness/so ness and adhesion be ween he ip and su ace. The image eco ded o i-PP /PEDOT-g-PCL shows b igh and da k domains, which co espond o chemical-dependen phase shi s o up o 174º. The b igh a eas, which co espond o la ge phase angles, a e associa ed wi h he PEDOT phase, whe eas he da k a eas ep esen he PCL phase. I is wo h no ing ha he PCL phase occupies he main pa o he su ace, whe eas he PEDOT phase only appea s in egions la gely domina ed by he inco po a ion o EDOT monome s wi h espec o EDOT-PCL mac omonome . These esul s can be explained i he expe imen al de ails a e aken in o accoun . Fo elec opolyme iza ion, i was used he ace oni ile as eac ion medium, which is a ma ginal sol en o PCL. Du ing he sol en e apo a ion and ilm o ming, he PCL side chains o PEDOT-g-PCL na u ally mig a e owa d he su ace, mos p obable exposing he iches alipha ic pa o hem. On he o he hand, he oughness o i-PP /PEDOT-g-PCL (Rq = 448  34 nm) is signi ican ly lowe han ha o i-PP /PEDOT, e en hough he hickness o he PEDOT- g-PCL and PEDOT laye s a e e y simila (9.0  2.5 and 8.2  2.4 m, espec i ely). 18 Ano he impo an di e ence be ween i-PP /PEDOT-g-PCL and i-PP /PEDOT e e s o he s uc u e o he po es. Al hough bo h biopla o ms display a po ous su ace wi h a la ge numbe o submic ome ic po es, i-PP /PEDOT-g-PCL shows a unique dis ibu ion o nanome ic po es wi h sizes comp ised wi hin 50 and 100 nm. These addi ional nanopo es, which ha e been a ibu ed o he sel -o ganiza ion o he PCL side chains, a e consis en wi h bo h he high elec ochemical ac i i y and he low elec ochemical s abili y o i-PP /PEDOT-g-PCL in compa ison o i-PP /PEDOT. When cycled in he ange om -0.3 V o 0.9 V, PCL side chains a e expec ed o be e y sensi i e o he elec ochemical deg ada ion, especially due o hei low molecula weigh . Thus, nanome ic po es p obably collapsed a e a ew consecu i e edox cycles, educing d as ically he elec ochemical ac i i y o he sys em when he numbe o edox cycles inc eases. Moni o ing he in luence o PEDOT-g-PCL in cellula adhesion and p oli e a ion The e ec o a g a copolyme laye in cell adhesion and p oli e a ion was e alua ed by conside ing wo cell lines, HeLa and IMR-90, bo h ex ensi ely used in scien i ic esea ch because o hei as g ow h. HeLa is a human immo al ca cinogenic cell line wi h epi helial mo phology, while IMR-90 p ima y cells a e human Caucasian e al lung ib oblas s (no-ca cinogenic). Quan i a i e esul s o cell adhesion and p oli e a ion assays (24 h and 7 days o cell cul u e, espec i ely) on issue cul u e poly s y ene (TCPS, con ol), i-PP, i-PP , i-PP /NPs, i-PP /PEDOT and i-PP /PEDOT-g-PCL a e displayed in Figu e 8. Resul s, which co espond o he a e age o h ee independen eplicas o each sys em, a e exp essed in e ms o cell iabili y (c ) ela i e o he TCPS con ol ma e ial. 19 The amoun o cells adhe ed o he su ace o biomedical i-PP is simila (HeLa) o sligh ly highe (IMR-90) han ha o TCPS con ol, which is a well-known biocompa ible ma e ial used o me al implan s. This beha io is app oxima ely main ained a e plasma ea men o i-PP , indica ing ha he unc ionaliza ion does no ha e a majo impac on he in e ac ion and a achmen o he cells o he su ace. This has been a ibu ed o he ac ha , al hough he plasma ea men causes he appa i ion o a homogeneous nano-pa e ning (Figu e 6a), he inc emen in he su ace oughness ( om Rq = 30  6 nm o i-PP o Rq = 38  6 nm o i-PP ) is no la ge enough o a ec he adhesion o he cells. Thus, he oughness o bo h i-PP and i-PP su aces is signi ican ly lowe han he diame e o ilopodia ilamen s in cells (ca. 100-200 nm68), which a e hin, ac in- ich s uc u es p o uding om he lamellipodial ac in ne wo k ha play a c ucial ole in cell adhesion.69,70 The inco po a ion o he i s CP laye in oduces impo an changes ha , in addi ion, depend on he cell line. Mo e speci ically, PHMeEDOT NPs p omo es IMR-90 cells adhesion by a ound 80% wi h espec o he con ol, while educes he a achmen o HeLa cells by app oxima ely 50%. Conside ing he sub-mic ome ic oughness o i- PP /NPs, his a ia ion is consis en wi h he ilopodia ecogni ion capaci y o submic ome ic opog aphies, which was ound o be dependen on he cell shape and ex u e.69 IMR-90 p esen s he ypical usi o m-shape wi h no mal ex u e while HeLa a e so i egula ly-shaped cells, as shown in con ocal mic og aphs displayed in Figu e 9, which makes di icul he ecogni ion o he ex u es. Howe e , he addi ion o he second CP laye esul s in a signi ican inc emen o cell adhesion wi h espec o i- PP /NPs, his e ec being especially ema kable o IMR-90 cells. Thus, he a achmen o ib oblas cells is highe o i-PP /PEDOT and i-PP /PEDOT-g-PCL han o i-PP by 20 70% and 90%, espec i ely, while o HeLa cells he adhesion inc emen is negligible o i-PP /PEDOT and o 60% o i-PP /PEDOT-g-PCL. The di e en esponse o HeLa and IMR-90 cells owa ds CPs can be in e p e ed on he basis o hei con as ed ib onec in con en . Mo e speci ically, PEDOT and, in gene al CPs, ha e a g ea a ini y owa ds ib onec in, i s adso p ion being an essen ial s ep o p omo ing cell-adhesion.71,72 In cell lines de i ed om non-ca cinogenic issues ib onec in was ound o be p edominan ly in he ex acellula ma ix, whe eas ca cinogenic cell lines display e y li le o no ib onec in.73 The absence o ex acellula ib onec in was speci ically demons a ed o umo HeLa cells ha , ins ead, con ain such a p o ein in he cell nucleus.74 This ea u e explains he signi ican ly lowe adhesion o HeLa cells o i-PP /PEDOT-g-PCL and, especially o i- PP /PEDOT, wi h espec o ha ound o IMPR-90 cells. Co alen g a ing o biocompa ible side chains o CPs is a sma s a egy o imp o e he biocompa ibili y o s uc u es based on such class o polyme s, he chemical and physical p ope ies o which mimics he ea u es o a na u al ex acellula ma ix (e.g. CP s uc u es enable ion di usion and ion exchange a he in e ace).75 Analysis o cell iabili y h ough simple p oli e a ion assays is a use ul ool o assessing he impac o sca olds in cell me abolism and, he e o e, de e mine i s po en ial use o issue enginee ing applica ions. Resul s om cell p oli e a ion o he examined ma e ials (and he TCPS con ol) a e shown in Figu e 8b, while ep esen a i e con ocal images a e displayed in Figu e 9 (mic og aphs o con ol TCPS a e shown in Figu e S12). No unexpec edly, esul s depend on he cell line. P oli e a ion o HeLa cells on i-PP and i-PP (c = 116%  2% and 129%  19%, espec i ely) was sligh ly be e han o he con ol TCPS, whe eas ha o i-PP /NPs was clea ly wo se (c = 60%  24%). Ins ead, i-PP /PEDOT (c = 144%  19%) shows a signi ican inc ease in cell numbe 21 due o cell di ision (cy okinesis) wi h espec o bo h p is ine and plasma- unc ionalized i-PP subs a es. These esul s indica e ha he doping le el o chemically polyme ized PHMeEDOT NPs is no high enough o acili a e he exchange o ions wi h adhe ed HeLa cells, which in u n is a ec ed by he low cell adhesion due o he lack o ib onec in in he ex acellula ma ix (as discussed abo e). In con as , PEDOT p epa ed using he expe imen al condi ions desc ibed in his wo k is known o achie e a e y high doping le el.37 I gene ally occu s o CPs syn hesized by elec opolyme iza ion wi h espec o chemical oxida i e polyme iza ion,76 and he e o e, a e y high concen a ion o coun e ions is con ained inside he polyme ic ma ix. These unique elec onic and chemical s uc u es acili a e he exchange o ions a he PEDOT···cell in e ace, p omo ing cell di ision. Indeed, his beha io is so a o able ha i o e comes he limi a ions associa ed o he lack o ex acellula ib onec in in HeLa cells, imp o ing he esponse o elec ochemically ine su aces (i.e. s eel, i-PP, i-PP and i-PP /NPs). I is wo h no ing ha hese esul s, which a e suppo ed by con ocal mic og aphs displayed in Figu e 9, a e ully consis en wi h he cyclic ol ammog ams displayed in Figu e 2c. i-PP /PEDOT-g-PCL exhibi s an ex ao dina y, unp eceden ed high capaci y o p omo ing HeLa cells g ow h wi h c = 268%  15% (Figu e 9d). The supe io ea u es o his biopla o m has been a ibu ed o he syne gy among he elec ochemical ac i i y o he PEDOT, he high biocompa ibili y o PCL, which is equen ly used o biomedical applica ions, and he ionic conduc i i y due o he dopan anions o PEDOT ha , in u n, is enhanced by ion sol a ing polyme p ope y o PCL chains.49,50 The addi ion o he g a copolyme laye o biomedical i-PP imp o es he cell iabili y by a ac o o 2.3. 22 Resul s a e mo e imp essi e o IMR-90 cells. In his case he iabili y o i-PP and i-PP su aces (c = 68%  9% and 72%  4%, espec i ely) is lowe han o he con ol, which has been a ibu ed o he poo we abili y o hese subs a es (i.e. g ow h o ib in-con aining cells is p omo ed on hyd ophilic su aces in compa ison o hyd ophobic ones). This si ua ion is e e sed o i-PP /NPs (c = 98%  9%), e en hough i s wa e con ac angle is simila o ha o i-PP . Again, his beha io has been associa ed o he abili y o CPs o exchange ions wi h cells, as is suppo ed by he esul s ob ained when a ela i ely hick laye o PEDOT is deposi ed on PHMeEDOT NPs. Thus, he c o i-PP /PEDOT (c = 137%  11%) inc eased by wice when compa ed o ha o he i-PP subs a e o biomedical applica ions. Fo i-PP /PEDOT-g-PCL (c = 196%  22%), he imp o emen ep esen s an inc ease o almos a ac o o 3 and 1.5 wi h espec o i-PP and i-PP /PEDOT, e idencing he bene i s o he PCL g a ing. These esul s p o e he syne gy be ween PEDOT and PCL e ec s a he biopla o m in e ace. Thus, he g a ed copolyme app oach p esen ed in his s udy ep esen s a sma s a egy o unc ionalize cu en ly used biomedical plas ics, as i-PP, o ma ching o lexibili y and cha ge anspo be ween he bioin e ace ma e ial and li ing issues, p omo ing conside ably cell adhesion and g owing. CONCLUSIONS Imp o emen o well-s ablished polyme ic ma e ials o biomedical applica ions is a cu en challenge. In his s udy, a conjuga ed g a copolyme is used as a molecula composi e o coa p e iously unc ionalized and elec oac i a ed i-PP in ended o biomedical applica ions. Ou idea was o op imize he p ope ies o a new ma e ial used a abio ic/bio ic in e ace and, by add essing he chemical design owa d side chain 23 unc ionaliza ion o a CP wi h PCL pola side chains (biocompa ible, wi h high ionic conduc i i y, ion-sol a ing capabili y and b oad elec ochemical s abili y window), we succeeded o signi ican ly imp o e he ion anspo and ion- o elec on ansduc ion wi h an ion- ich, li ing, wa e -laden, dynamic biological en i onmen . The su ace o i-PP unc ionalized wi h oxygen plasma ea men has been coa ed wi h PHMeEDOT NPs p epa ed by chemical oxida i e polyme iza ion, which unc ioned as polyme iza ion nuclei o in si u elec ocopolyme iza ion o EDOT-PCL mac omonome and EDOT monome . The esul ing biopla o m is a lexible subs a e coa ed wi h a g a copolyme ha p esen s, syne gis ically, he ad an ages o PEDOT backbone and hose o PCL side chains. This s udy shows ha i-PEDOT/PEDOT-g- PCL has many a o able p ope ies wi h espec o i-PP and i-PP/PEDOT o issue enginee ing applica ions. This has been con i med by cell adhesion and p oli e a ion assays using wo human cell lines wi h e y di e en cha ac e is ics (HeLa and IMR- 90), i-PEDOT/PEDOT-g-PCL imp o ing cell iabili y o i-PP by a ac o o almos h ee. O e all, his s udy has wide implica ions in polyme ic medical de ices cu en ly used as implan s, which can be upg aded enhancing hei issue in eg a ion pe o mance. Fu u e in es iga ions will be o ien ed owa ds his ield, s udying he impac o PEDOT-g-PCL in cell di e en ia ion and he in eg a ion o i- PEDOT/PEDOT-g-PCL. CONFLICTS OF INTEREST The e a e no con lic s o decla e. ACKNOWLEDGEMENTS 24 This wo k was suppo ed by Spanish and Romanian unding agencies. 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Human HeLa and IMR-90 cells we e cul u ed du ing (a) 24 h and (b) 7 days. As e isk ma ks (*) ep esen a signi ican di e ence wi h he con ol when he S uden ’s T- es was applied (p < 0.05). Figu e 9. Con ocal mic oscopy mic og aphs (10×) displaying he mo phology o HeLa (le ) and IMR-90 ( igh ) cells a e incuba ion o 7 days on (a) i-PP, (b) i-PP (c) i-PP /NPs (d) i-PP /PEDOT and (e) i-PP /PEDOT-g-PCL. 33 Figu e 1 34 Figu e 2 35 Figu e 3 36 Figu e 4 37 Figu e 5 38 Figu e 6 39 Figu e 7 40 Figu e 8 41 Figu e 9