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. In Spain
au ho s acknowledge MINECO/FEDER (RTI2018-098951-B-I00 and RTI2018-
101827-B-I00) and he Agència de Ges ió d'Aju s Uni e si a is i de Rece ca
(2017SGR359, 2017SGR373 and 2017SGR128). Suppo o he esea ch o C.A. is
g a e ul o ICREA Academia p og am o excellence in esea ch. In Romania his wo k
was suppo ed by a g an o Minis y o Resea ch and Inno a ion, CNCS–UEFISCDI
(PN-III-P4-ID-PCCF-2016-0050, wi hin PNCDI III), which he Romanian au ho s
g ea ly acknowledge. All au ho s a e indeb ed o B B aun Su gical S.A. o suppo and
help ul discussion.
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subs a e. 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