Bioma e ials
Science
PAPER
Ci e his: Bioma e . Sci., 2015, 3,
1395
Recei ed 26 h May 2015,
Accep ed 22nd June 2015
DOI: 10.1039/c5bm00160a
www. sc.o g/bioma e ialsscience
Elec oac i e polyme –pep ide conjuga es o
adhesi e bioin e aces†
Sil ana Maione,
a,b
Ana M. Gil,
c
Geo gina Fab ega ,
a,b
Luis J. del Valle,
a,b
Jo di T igue o,
a
Adele Lau en ,
d,e
Denis Jacquemin,
d,e
F ancesc Es any,
b,
Ana I. Jiménez,
c
Da id Zanuy,
a
Ca los Ca i iela*
c
and Ca los Alemán*
a,b
Elec oac i e polyme –pep ide conjuga es ha e been syn hesized by combining poly(3,4-e hylenedioxy-
hiophene), a poly hiophene de i a i e wi h ou s anding p ope ies, and an A g-Gly-Asp (RGD)-based
pep ide in which Gly has been eplaced by an exo ic amino acid bea ing a 3,4-e hylenedioxy hiophene
ing in he side chain. The inco po a ion o he pep ide a he ends o p e o med PEDOT chains has been
co obo a ed by bo h FTIR and X- ay pho oelec on spec oscopy. Al hough he mo phology and opo-
logy a e no influenced by he inco po a ion o he pep ide a he ends o PEDOT chains, his p ocess
la gely affec s o he su ace p ope ies. Thus, he we abili y o he conjuga es is conside ably highe han
ha o PEDOT, independen ly o he syn he ic s a egy, whe eas he su ace oughness only inc eases
when he conjuga e is ob ained using a compe ing s a egy (i.e. g ow h o he polyme chains agains e -
mina ion by end capping). The elec ochemical ac i i y o he conjuga es has been ound o be highe
han ha o PEDOT, e idencing he success o he polyme –pep ide links designed by chemical simila i y.
Densi y unc ional heo y calcula ions ha e been used no only o asce ain he con o ma ional p e e -
ences o he pep ide bu also o in e p e he elec onic ansi ions de ec ed by UV- is spec oscopy.
Elec oac i e su aces p epa ed using he conjuga es displayed he highe bioac i i ies in e ms o cell
adhesion, wi h he ela i e iabili ies being dependen on he oughness, we abili y and elec ochemical
ac i i y o he conjuga e. In addi ion o he influence o he pep ide agmen in he ini ial cell a achmen
and subsequen cell sp eading and su i al, he esul s indica e ha PEDOT p omo es he exchange o
ions a he conjuga e–cell in e ace.
In oduc ion
The RGD (A g-Gly-Asp) amino acid sequence is he uni o a
cell adhesi e ac i i y domain in adhe en p o eins (e.g. ib o-
nec in, ib in and i onec in).
1–3
This cell adhesion mo i has
been widely used in he de elopmen o syn he ic ma e ials o
bioenginee ing, diffe en app oaches being applied o such
pu poses (e.g. in eg a ion in la ge pep ide sequences,
4–7
su ace unc ionalized subs a es
8–12
and polyme –pep ide
conjuga es
12–15
). In all cases RGD-con aining bioma e ials
inc eased he cellula adhesion wi h espec o he con ol
when he adhesion mo i was immobilized main aining i s
biological ac i i y.
Among ad anced o ganic bioma e ials, polyme –pep ide
conjuga es, which esul om he co alen in eg a ion o a
pep ide wi h a syn he ic polyme block, a e especially a ac i e
because his kind o hyb id mac omolecule combines unique
p ope ies ha a e de i ed om he p ecise chemical s uc u e
and unc ionali y o pep ides and he s abili y, unc ions and
p ocessabili y o syn he ic polyme s.
16–19
In o de o ake
ad an age o he elec ochemical p ope ies o elec oac i e
conduc ing polyme s (ECPs), diffe en ECP–pep ide conjuga es
ha e been p epa ed du ing he las decade. Fo example, he
i s oligo hiophene di ec ly conjuga ed o a β-shee pen a-
pep ide was epo ed in 2004,
20
while he modi ica ion o he
†Elec onic supplemen a y in o ma ion (ESI) a ailable: Syn he ic in e media es,
cha ac e iza ion me hods, XPS spec a, SEM and AFM mic og aphs, cyclic ol-
ammog ams and plo s o he elec on densi y diffe ence. See DOI: 10.1039/
c5bm00160a
a
Depa amen d’Enginye ia Química, E. T. S. d’Enginye ia Indus ial de Ba celona,
Uni e si a Poli ècnica de Ca alunya, Diagonal 647, 08028 Ba celona, Spain.
E-mail: [email p o ec ed]u
b
Cen e o Resea ch in Nano-Enginee ing, Uni e si a Poli ècnica de Ca alunya,
Campus Sud, Edi ici C’, C/Pasqual i Vila s/n, Ba celona E-08028, Spain
c
Depa men o de Química O gánica, Ins i u o de Sín esis Química y Ca álisis
Homogénea (ISQCH), Uni e si y o Za agoza –CSIC, 50009 Za agoza, Spain.
E-mail: [email p o ec ed]
d
CEISAM, UMR CNRS 6230, Facul é des Sciences e des Techniques, BP 92208,
Uni e si é de Nan es, 2, ue de la Houssiniè e, 44322 Nan es Cedex, F ance
e
Ins i u Uni e si ai e de F ance, 103 bd S Michel, 75005 Pa is Cedex 5, F ance
Depa amen d’Enginye ia Química, Escola Uni e si a ia d’Enginye ia Tècnica
Indus ial de Ba celona, Uni e si a Poli ècnica de Ca alunya, Com e d’U gell 187,
08036 Ba celona, Spain
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β-posi ion on polypy ole (PPy) o c ea e s ong disul ide bonds
wi h he Cys o he RGDS sequence (A g–Gly–Asp–Cys) was
desc ibed in ea lie s udies.
21,22
Wi hin his con ex , we ha e
ecen ly epo ed a new s a egy o he p epa a ion o ECP–
pep ide conjuga es ha is based on he chemical simila i y o
hei wo componen s.
23,24
This app oach is based on he
design o exo ic syn he ic amino acids bea ing he main
chemical g oups o he polyme o acili a e he co alen conju-
ga ion be ween he componen s. Such a me hodology a oids
he p esence o long alipha ic linke s, which a e ypically used
o join he wo componen s o he polyme –pep ide conjuga e.
The absence o linke s is expec ed o p omo e he ex ension o
he polyme p ope ies owa ds he pep ide su ace egion,
which is an impo an limi a ion ypically ound in ECP–
pep ide conjuga es.
In his wo k, we ha e used a s a egy based on chemical
simila i y o design an elec oac i e RGD-based ECP–pep ide
conjuga e. The polyme selec ed o his pu pose is poly(3,4-
e hylenedioxy hiophene), he ea e abb e ia ed as PEDOT,
which is among he mos success ul ECPs due o i s excellen
elec ochemical and he mal p ope ies, high conduc i i y,
good en i onmen al s abili y in i s doped s a e, mechanical
lexibili y, ela i e ease o p epa a ion, and as doping–undop-
ing p ocess.
25–27
Rega ding he pep ide, he Gly esidue o he
RGD sequence has been eplaced by GlE, which consis s o an
amino acid bea ing a 3,4-e hylenedioxy hiophene as he side
g oup a ached o an addi ional me hylene g oup. The esul -
ing sequence, he ea e deno ed as RG
E
D, has been a ached
o he ends o PEDOT chains o ming he PEDOT–RG
E
D conju-
ga e (Scheme 1). A e chemical cha ac e iza ion, he physical
p ope ies o he conjuga e ha e been in es iga ed a diffe en
leng h scales. Finally, he bene i s induced by conjuga ion in
issue egene a ion ha e been in es iga ed by compa ing he
beha io o PEDOT and PEDOT–RG
E
D as so bioelec oac i e
suppo s o cell a achmen .
Resul s and discussion
Syn hesis o he p o ec ed RG
E
D pep ide
The syn he ic ou e used o ob ain he RG
E
D sequence, which
was p o ec ed a he eac i e posi ions by e -bu yl es e
g oups (
Bu-COO) o a oid in e e ence du ing he coupling o
PEDOT chains, is p o ided in Fig. 1.
The p epa a ion and chemical cha ac e iza ion o 1–5in e -
media es is epo ed in he ESI.†The p ocedu e used o
p epa e he p o ec ed RG
E
D pep ide was as ollows: o a solu-
ion o Boc-L-A g(Boc)
2
-OH (500 mg, 1.05 mmol) in dichlo o-
me hane (60 mL) cooled o 0 °C in an ice ba h, we e added
1-hyd oxybenzo iazole hyd a e (HOB ) (161 mg, 1.05 mmol)
and N-[3-(dime hylamino)-p opyl]-N′-e hylca bodiimide hyd o-
chlo ide (EDC·HCl) (201 mg, 1.05 mmol) ollowed by a solu-
ion o H-D,L-GlE-Asp(O
Bu)-O
Bu (1 mmol) [ob ained by
dep o ec ion o 5(678 mg, 1 mmol) wi h die hylamine (DEA;
5 mL, 50 mmol)] in dichlo ome hane (10 mL) and inally
N-me hylmo pholine (NMM; 0.11 mL, 1 mmol). The eac ion
mix u e was s i ed a oom empe a u e o 24 h. Then, he
solu ion was washed wi h 5% NaHCO
3
(3 × 30 mL) ollowed by
5% KHSO
4
(3 × 30 mL). The o ganic phase was d ied o e
magnesium sul a e and e apo a ed o d yness. The c ude
p oduc was pu i ied by column ch oma og aphy (eluen :
hexane/e hyl ace a e 6/4) o p o ide he p o ec ed RG
E
D
(a mix u e o dias e eoisome s) as a whi e solid (822 mg,
0.90 mmol, 90% yield).
IR (KB ) ν: 3381, 1715, 1610 cm
−1
.
1
H NMR (CDCl
3
,
400 MHz): δ1.37–1.85 (m, 4H), 1.43, 1.50, 1.51 (3 s, 45H),
2.59–2.72 (m, 1H), 2.73–2.87 (m, 1H), 3.02–3.22 (m, 2H),
3.72–3.89 (m, 2H), 4.08–4.27 (m, 5H), 4.55–4.71 (m, 2H),
5.49–5.66 (m, 1H), 6.11, 6.12 (2 s, 1H), 6.83–7.13 (m, 2H),
9.08–9.52 (m, 2H).
13
C NMR (CDCl
3
, 125 MHz): δ24.8, 24.9,
28.0, 28.18, 28.21, 28.4, 28.5, 28.8, 29.0, 29.8, 37.56, 37.59,
44.25, 44.29, 49.3, 49.5, 53.9, 54.0, 54.5, 54.7, 64.70, 64.72,
64.9, 79.1, 79.2, 81.5, 81.6, 82.29, 82.33, 84.0, 84.1, 97.3, 97.4,
111.26, 11.32, 139.3, 141.52, 141.54, 155.05, 155.08, 155.9,
160.7, 160.8, 163.71, 163.73, 169.4, 169.5, 169.92, 169.98,
170.1, 171.97, 172.22. HRMS (ESI) C
42
H
69
N
6
O
14
S[M+H]
+
:
calcd 913.4587, ound 913.4609.
Scheme 1 Chemical s uc u e o he PEDOT–RG
E
D conjuga e.
Fig. 1 Reagen s and condi ions o he syn hesis o p o ec ed RG
E
D: (a)
KOH 2 N/MeOH, 2 h, 99%; (b) HCl (3N)/AcOE , 30 min, 100%; (c)
FmocOsu, K
2
CO
3
,CH
3
CN/H
2
O 3/1, 80%; (d) HOB , EDC·HCl, NMM, H-L-
Asp(O
Bu)-O
Bu, CH
2
Cl
2
, 0 °C 30 min, 24 h, 95%; (e) DEA, CH
2
Cl
2
,
4 h, 100%; ( ) HOB , EDC·HCl, NMM, Boc-L-A g(Boc)
2
-OH, CH
2
Cl
2
,0°C
30 min, 24 h, 90%.
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Syn hesis o he PEDOT–RG
E
D conjuga e
The PEDOT–RG
E
D conjuga e was p epa ed using wo al e na-
i e s a egies (A and B) o h ee s eps each, which a e known
o p o ide ma e ials wi h diffe en su ace p ope ies.
28
In he
i s s ep, which is he same o he wo s a egies (Fig. 2),
PEDOT ilms we e deposi ed on o s eel shee s by ch ono-
ampe ome y (CA) unde a cons an po en ial o 1.40 V and
using a 10 mM 3,4-e hylenedioxy hiophene (EDOT) solu ion in
ace oni ile con aining 100 mM LiClO
4
as he suppo ing elec-
oly e. The polyme iza ion ime (θ
1
) was only 10 s. A e elimi-
na ing he excess o he monome and dopan om he
p epa ed PEDOT ilms and washing wi h ace oni ile, he
second s ep o each s a egy was add essed (Fig. 2).
In s a egy A, he elec ode coa ed wi h PEDOT was in o-
duced in a cell con aining an ace oni ile solu ion wi h 1 mM
o he p o ec ed RG
E
D and 0.1 M LiClO
4
, a cons an po en ial
o 1.37 V being applied o θ
2
= 30 s. In s a egy B, he PEDOT
ilm was in oduced in a cell illed wi h a 1 mM RG
E
D, 0.5 mM
EDOT and 0.1 M LiClO
4
ace oni ile solu ion. In his case, a
cons an po en ial o 1.37 V was applied o θ
2
= 10 s only.
Acco dingly, he main diffe ences be ween s a egies A and B
can be summa ized as ollows: (i) he gene a ion medium
used in B o he p epa a ion o he conjuga e con ains bo h
pep ide and monome in he second s ep, while ha employed
in A con ains only pep ides; and (ii) he polyme iza ion ime,
θ
2
, is signi ican ly la ge in A han in B. These modi ica ions
a e expec ed o affec conside ably he oughness and we abil-
i y o he su ace.
28
Finally, in he las s ep, which was iden ical
o s a egies A and B (Fig. 2), he desi ed PEDOT–RG
E
D conju-
ga es we e ob ained by imme sing elec odes coa ed wi h he
PEDOT–(p o ec ed RG
E
D) sys ems in o a 1 : 1 i luo oace ic
acid : dichlo ome hane mix u e (TFA : DCM) o 2 hou s and,
subsequen ly, washing wi h DCM. Thus, his s ep was exclu-
si ely ocused on he dep o ec ion o he pep ide agmen s
once hey ha e been a ached o he ECP chains. He ea e , he
conjuga es de i ed om s a egies A and B ha e been labelled
as PEDOT–RG
E
D/A and PEDOT–RG
E
D/B, espec i ely.
The a e age hickness, as de e mined by con ac p o ilo-
me y, o PEDOT–RG
E
D/A and PEDOT–RG
E
D/B ilms was 296 ±
21 and 472 ± 15 nm, espec i ely. On he o he hand, PEDOT
ilms used as a con ol in he whole wo k we e p epa ed by
imme sing he ilms achie ed in he i s s ep in o a cell con-
aining an ace oni ile solu ion wi h 0.1 M LiClO
4
(i.e. wi hou
pep ides and monome s) and applying a cons an po en ial o
1.37 V du ing θ
2
= 10 s. This me hodology allowed us o ob ain
ECP ilms wi h doping le els simila o ha achie ed o
PEDOT–RG
E
D/B. The a e age hickness o ilms de i ed om
his p ocess was 280 ± 44 nm.
Compa ison o he FTIR spec a o PEDOT, PEDOT–RG
E
D/A
and PEDOT–RG
E
D/B (Fig. 3) co obo a es he inco po a ion o
he RG
E
D pep ide. Thus, he amide II and amide III bands,
which a ise om he coupling be ween he N–H in-plane
bending and C–N s e ching modes, a e clea ly ecognizable a
app oxima ely 1520 and 1320 cm
−1
, espec i ely, o he wo
conjuga es. Un o una ely, he A g and Asp side chain bands
Fig. 2 Scheme displaying he wo syn he ic s a egies (A and B) used o p epa e PEDOT–RG
E
D. Each s a egy in ol ed a h ee-s ep p ocess, e en
hough A and B only diffe in he condi ions used o he in e media e s ep. RE, WE and CE e e o he e e ence elec ode, wo king elec ode and
coun e elec ode, espec i ely.
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a a ound 1620 and 1708 cm
−1
,
29
espec i ely, a e no iden i-
ied because o he o e lapping associa ed wi h he hiophene
ing (C C and C–C s e ching) o PEDOT.
23,30
The chemical s uc u e o PEDOT–RG
E
D ilms was u he
cha ac e ized by X- ay pho oelec on spec oscopy (XPS).
Table 1 compa es he su ace a omic composi ions. The pene-
a ion o X- ay adia ion using he condi ions desc ibed in he
Expe imen al sec ion is expec ed o be ∼10 nm, e en hough
in his case he pene a ion is unknown because o bo h he
nanome ic hickness and po osi y (nex sub-sec ion) o he
ilms. This ea u e explains he de ec ion o a small pe cen age
o ni ogen in PEDOT samples, which canno be a ibu ed o
he aces o ace oni ile since he samples we e s o ed o
almos wo weeks be o e XPS analyses. Thus, de ailed
in e p e a ion o he N 1s (see below) allowed us o conclude
ha he ni ogen de ec ed in he composi ion o PEDOT
comes om he AISI 316 s eel subs a e. Howe e , he N 1s
inc eases om 0.43% in PEDOT o 0.81% and 1.58% in
PEDOT–RG
E
D/A and PEDOT–RG
E
D/B, espec i ely, suppo ing
he success ul inco po a ion o he pep ide. Fo PEDOT, he
C/S a io, 6.22, is close o he heo e ical alue o 6.00, while
such a a io inc eases o 6.90 and 7.20 o PEDOT–RG
E
D/A and
PEDOT–RG
E
D/B, espec i ely. This obse a ion is consis en
wi h he ac ha he inc emen o he con en o C in he wo
conjuga es is due o he inco po a ion o he RG
E
D pep ide.
Fig. 4a–d display he high- esolu ion XPS spec um in he
C 1s, N 1s, O 1s and S 2p egions o PEDOT–RG
E
D/B, he
spec a o PEDOT–RG
E
D/A being epo ed in Fig. S1.†Be o e
discussing he analysis, i should be men ioned ha he peaks
o he pep ide may be in luenced by hose o PEDOT and ice
e sa, explaining he small de ia ions ound in some peaks
wi h espec o he alues epo ed in he li e a u e.
Decon olu ion o he C 1s peak led o i e Gaussian cu es
ha ha e been a ibu ed o he sa u a ed and conjuga ed C–C
(284.5 eV) and C C–O (286.4 eV) bonds o PEDOT chains,
30,31
he C O o he amide (288.3 eV),
32,33
he C–N bonds o he gua-
nidinium g oup in A g (289.6 eV)
33
and he C–F bonds o he
esidual TFA molecules (292.2 eV)
33
a ising om he dep o ec ion
s ep in he syn hesis (Fig. 2). The high esolu ion N 1s spec um
shows a peak cen e ed a 400.3 eV, which seems o include bo h
he guanidinium (400.1 eV) and he backbone amide (400.6 eV)
signals iden i ied o he RGD pep ide sequence.
33
The peak a
402.0 eV, which was also de ec ed in he PEDOT samples, has
been a ibu ed o impu i ies o he subs a e.
23
The O 1s signal consis s o h ee componen s, wi h he
mos in ense one a 533.3 eV co esponding o he C–O–C
bond in he e hylene b idge o PEDOT.
30
The componen a
531.4 eV is assigned o he ca boxyla e g oups in he conju-
ga ed RG
E
D pep ide,
33
while he peak a 535.2 eV co esponds
o he C O o he con amina ion p oduc s (e.g. CO
2
adso bed
om he a mosphe e).
23,34
The la e is ypically ound in
PEDOT p oduced by anodic polyme iza ion and doped wi h
li hium pe chlo a e.
23,34
The high esolu ion XPS o he S 2p
egion o he conjuga e shows he spin-spli sul u coupling
S2p
3/2
and S 2p
1/2
, wi h a sepa a ion o 1.2 eV, o he C–S–C
bond o he hiophene ing (163.9 and 165.1 eV, espec i ely)
in PEDOT, i s homologues wi h posi i ely cha ged sul u (i.e.
C–S
+
–C 165.5 and 166.7 eV, espec i ely), and he C–S–C bond
o he side chain o he GlE esidue in he pep ide (a 167.8
and 169.0 eV, espec i ely).
35,36
The spec a in he Cl 2p egions o PEDOT–RG
E
D/B and
PEDOT–RG
E
D/A show peaks a 207.6 and 209.2 eV (Fig. S2†),
which co espond o he spin-spli chlo ide coupling Cl 2p
3/2
and Cl 2p
1/2
, wi h a sepa a ion o 1.6 eV, o he Cl–O bond o
he pe chlo a e dopan agen . The Cl/S a ios ob ained using
he a omic pe cen composi ions displayed in Table 1 ha e
been di ec ly associa ed wi h he doping le el (DL), which co -
esponds o he numbe o posi i e cha ges pe hiophene
ing. The DL is e y high o PEDOT, DL = +0.49, indica ing
ha he o mula o he ECP is: [(EDOT
0.49+
)
n
(ClO
4−
)
0.49n
]
solid
.
Ve y simila esul s (DL = +0.54) ha e been ob ained by de e -
mining he amoun o chlo ides wi h s anda d ion ch oma o-
g aphy. In con as , he DL o he wo conjuga es dec eases o
∼+0.05, indica ing a no able educ ion o he sal s uc u e be-
ha io . In o de o asce ain one o he possible easons o
such d as ic educ ion, PEDOT ilms we e imme sed in o a
Fig. 3 FTIR spec a o PEDOT, PEDOT–RG
E
D/A and PEDOT–RG
E
D/B.
Table 1 A omic pe cen composi ion (C 1s, O 1s, N 1s, S 2p and Cl 2p)
ob ained by XPS o PEDOT, PEDOT–RG
E
D/A and PEDOT–RG
E
D/B
samples
C 1s N 1s O 1s S 2p Cl 2p
PEDOT 50.56 0.43 36.90 8.12 3.99
PEDOT + TFA 57.74 0.47 31.62 8.80 1.17
PEDOT–RG
E
D/A 62.01 0.81 27.85 8.98 0.35
PEDOT–RG
E
D/B 61.54 1.58 27.78 8.55 0.55
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1 : 1 TFA : DCM o 2 hou s and, subsequen ly, washed wi h
DCM, emula ing he s ep used o elimina e he p o ec ion o
pep ide agmen s in PEDOT–(p o ec ed RG
E
D) sys ems. XPS
analyses o he esul ing ilms (PEDOT + TFA in Table 1) indi-
ca e ha he DL o PEDOT dec eases om +0.49 o +0.13 upon
ea men wi h TFA.
Su ace cha ac e iza ion
The su ace mo phology and opog aphy o PEDOT–RG
E
D con-
juga es we e in es iga ed using scanning elec on mic oscopy
(SEM) and a omic o ce mic oscopy (AFM), espec i ely. SEM
mic og aphs o PEDOT–RG
E
D/B (Fig. 5a) indica e a s uc u e
o med by he agg ega ion o s icks wi h a ibe -like mo -
phology, which acili a e he o ma ion o na ow and o uous
po es. Compa ison wi h mic og aphs ob ained o PEDOT–
RG
E
D/A, as p epa ed PEDOT, and PEDOT ea ed wi h 1 : 1
TFA : DCM o 2 hou s (Fig. S3†) indica es ha he impac on
he su ace mo phology o bo h end capping pep ides and he
applied dep o ec ion ea men is p ac ically negligible. Simi-
la ly, he su ace opog aphy o PEDOT–RG
E
D/B (Fig. 5b), ha
can be desc ibed as a dense and homogeneous dis ibu ion o
sha p peaks g ouped in clus e s, is appa en ly indis inguish-
able om hose o PEDOT and PEDOT–RG
E
D/A (Fig. S4†). This
opology, which is a consequence o he linea g ow h o
polyme chains (i.e. molecules a e exclusi ely o med by α–α
linkages because he β-posi ions o he hiophene ing a e
occupied by he dioxane ing),
37
is no al e ed by he inco po -
a ion o he RG
E
D pep ide a he ends.
In spi e o hei mo phological simila i y, he su ace
oughness o PEDOT–RG
E
D conjuga es has been ound o
Fig. 4 High- esolu ion XPS spec a o PEDOT–RG
E
D/B: C 1s (a), N 1s
(b), O 1s (c) and S 2p (d) egions. Peaks om decon olu ion a e also dis-
played. The ed line co esponds o he expe imen al p ofile while he
black line is he sum o he decon olu ed peaks.
Fig. 5 (a) SEM mic og aphs, high and low (inse ) magnifica ions, and (b)
2D and 3D opog aphic images o PEDOT–RG
E
D/B. (c) Con ac angles
o wa e (a e age and s anda d de ia ion o 14 independen measu es)
o as p epa ed PEDOT, PEDOT ea ed wi h 1 : 1 TFA : DCM o 2 hou s
and, subsequen ly, washed wi h DCM (PEDOT + TFA), emula ing he
s ep used o elimina e he p o ec ion o pep ide agmen s in PEDOT–
(p o ec ed RG
E
D) sys ems, and he wo p epa ed conjuga es.
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depend on he app oach used in he in e media e s ep o he
syn he ic p ocess. Thus, he oo -mean-squa e oughness (R
q
)
o PEDOT and PEDOT–RG
E
D/A (R
q
= 117 ± 28 and 114 ±
14 nm, espec i ely) is ∼25% lowe han ha o PEDOT–RG
E
D/
B(R
q
= 149 ± 22 nm). This effec has been a ibu ed o he
coexis ence and compe i ion o wo chemical p ocesses in he
in e media e s ep o s a egy B: g ow h o polyme chains by
inco po a ing EDOT monome s and e mina ion o polyme
chains by inco po a ing RG
E
D. In con as , he e mina ion
was he only p ocess p esen in s a egy A. On he o he hand,
he hickness de e mined by AFM sc a ching es s using he
ip in con ac mode (Fig. S5†) was 350 ± 10, 361 ± 15 and 443 ±
7 nm o PEDOT, PEDOT–RG
E
D/A and PEDOT–RG
E
D/B,
espec i ely, hese alues being ully consis en wi h hose
ob ained by con ac p o ilome y.
The con ac angles (Θ) measu ed in wa e (Fig. 5c) indica e
ha PEDOT is a hyd ophilic ma e ial (i.e. Θ< 90°), which
should be a ibu ed o he oxygen a oms o he used dioxane
ing. The we abili y inc eases signi ican ly upon he inco po -
a ion o RG
E
D a he ends o polyme chains, independen ly o
he s a egy used in he syn he ic p ocess (i.e. ΔΘ> 40° in all
cases). Howe e , he effec o he syn he ic s a egy is no negli-
gible, he con ac angle o PEDOT–RG
E
D/B being 18° lowe
han ha o PEDOT–RG
E
D/A. The inc emen o bo h we a-
bili y and R
q
sugges s ha he conjuga e de i ed om syn-
he ic s a egy B is mo e app op ia e o issue enginee ing
applica ions han ha p oduced using A. On he o he hand,
he con ac angle de e mined o PEDOT ea ed wi h 1 : 1
TFA : DCM o 2 hou s (Fig. 5c) indica es ha he end capping
pep ide is esponsible o he ema kable we abili y o he wo
conjuga es, he impac o he changes p oduced by he acid
ea men being p ac ically null (ΔΘ≈4°).
Elec oac i i y and elec os abili y
Cyclic ol amme y (CV) s udies we e conduc ed o de e mine
he in luence o he end capping pep ide on he elec ochemi-
cal p ope ies o he ECP. Con ol ol ammog ams eco ded in
phospha e buffe ed saline solu ion (PBS), which ep esen s a
physiological medium, a e displayed in Fig. 6a. The abili y o
exchange cha ges e e sibly, he ea e deno ed as elec oac i -
i y, inc eases wi h he simila i y be ween he anodic and ca ho-
dic a eas o he con ol ol ammog am. The elec oac i i y o
PEDOT–RG
E
D/A and PEDOT–RG
E
D/B is, espec i ely, 57% and
75% highe han ha o PEDOT. This ea u e poin s ou he
ema kable success o he s a egy based on he design o
ECP–pep ide conjuga es by chemical simila i y. Thus, conju-
ga ion be ween he wo componen s h ough a syn he ic
amino acid bea ing he epea uni o PEDOT as he side chain
a he han using long and lexible linke s, as is equen ly
done, acili a es he mo emen o cha ge om he bulk o he
ilm–solu ion in e ace du ing he oxida ion p ocess (o om
he in e ace o he bulk in he educ ion p ocess). Fu he -
mo e, cha ges loca ed a ionized A g and Asp esidues, which
a e placed a he su ace o ilms, also con ibu e o acili a e
he access o escape o dopan ions du ing he edox p o-
cesses. Consequen ly, he low o ions is highe o PEDOT–
RG
E
D han ha o PEDOT. On he o he hand, he abili y o
s o e cha ge is 11% highe o PEDOT–RG
E
D/B han ha o
PEDOT–RG
E
D/A. This has been a ibu ed o he accessibili y
o bo h PEDOT agmen s and RG
E
D pep ides a he su ace o
ilms, which is p omo ed by syn he ic s a egy B (Fig. 6b).
These su ace cha ac e is ics affec no only he elec oac i i y,
oughness and we abili y bu also he elec ochemical s abili y
(elec os abili y).
The elec os abili y was e alua ed by applying en consecu-
i e oxida ion– educ ion cycles in PBS. Fig. 6c ep esen s he
loss o elec oac i i y (LEA) ela i e o he i s cycle agains he
Fig. 6 (a) Con ol ol ammog ams o PEDOT, PEDOT–RG
E
D/A and
PEDOT–RG
E
D/B in PBS. Ini ial and final po en ials, −0.40 V; e e sal
po en ial, 0.80 V; scan a e, 50 mV s
−1
. (b) Scheme eflec ing su ace
s uc u al diffe ences be ween PEDOT–RG
E
D/A and PEDOT–RG
E
D/B
conjuga es. (c) Loss o elec oac i i y (LEA) agains he numbe o con-
secu i e oxida ion and educ ion cycles in 0.1 M PBS o PEDOT,
PEDOT–RG
E
D/A and PEDOT–RG
E
D/B.
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numbe o cycles. Fo PEDOT, he LEA s abilizes a 17% in he
ou h cycle, eaching a pla eau ha e lec s he high elec o-
chemical s abili y o his ECP. In con as , he LEA g ows p o-
g essi ely wi h he numbe o cycles o PEDOT–RG
E
D/A, he
obse ed alue a e 10 cycles being LEA = 29%. PEDOT–RG
E
D/B
exhibi s an in e media e beha io . Thus, he LEA slowly
inc eases wi h he numbe o edox cycles (i.e. LEA = 22% a e
10 cycles) and, simul aneously, ends o s abilize a ound a
sligh ly highe alue. Acco ding o hese esul s, he conju-
ga ion o RG
E
D has a nega i e effec on he elec ochemical
s abili y o PEDOT, which should be a ibu ed o he deg a-
da ion o he pep ide du ing he oxida ion and educ ion p o-
cesses. This undesi able effec is pa ially mi iga ed by he
syn he ic s a egy B, which akes ad an age o he excellen
elec ochemical p ope ies o PEDOT (Fig. 6b). Thus, he nega-
i e effec s on he elec os abili y caused by he pep ide
de e io a ion dec ease wi h he inc easing su ace eachabili y
o PEDOT chains.
Molecula con o ma ion and op ical p ope ies
The con o ma ional po en ial ene gy su ace o he RG
E
D
pep ide was sys ema ically explo ed using a p ocedu e inspi ed
in he build-up me hod, ea lie de eloped by Sche aga and co-
wo ke s
38
(see he ESI†). This me hod uses he geome ies o
smalle pep ide segmen s o build he s uc u es o la ge seg-
men s. Acco dingly, 559 s a ing con o ma ions we e con-
s uc ed by combining he mos s able minima iden i ied o
each o he in ol ed esidues. The geome y o all hese s uc-
u es was op imized a he B3LYP/6-31+G(d,p) le el o heo y in
aqueous solu ion, sol en effec s being modeled by he Pola iz-
able Con inuum Model (PCM). The esul ing 552 minima we e
g ouped in a lis o 26 unique minimum ene gy con o -
ma ions, which was cons uc ed by assessing he simila i ies
among he diffe en s uc u es (clus e ing o s uc u es
desc ibed in he Expe imen al sec ion). Fig. 7a displays he
dis ibu ion o such unique minima in he maps cons uc ed
using he backbone dihed al angles o A g (ω,ψ), GlE (φ,ψ) and
Asp (ω,φ) esidues. As i can be seen, A g and Asp end o
adop an ex ended a angemen wi h ψ≈180° and φ≈180°,
espec i ely, e en hough he la e also shows h ee minima
wi h φ≈60°. Despi e his appa en backbone homogenei y,
bo h A g and Asp p esen signi ican con o ma ional a iabil-
i y a hei side chains gi ing place o diffe en ne wo ks o
side chain⋯backbone in e ac ions (Fig. 7b). In con as , he
GlE esidue exhibi s ema kable con o ma ional a iabili y a
he backbone displaying minima in he α
R
(φ,ψ≈−70°,−20°),
C
7eq
(φ,ψ≈−80°,80°) and β(φ,≈−150°, 60°) egions o he
φ,ψ-map.
Fig. 7b depic s he i e unique minimum ene gy con o -
ma ions wi h ela i e ene gy (ΔE) lowe han 5 kcal mol
−1
,
which ha e been labeled wi h le e s A–E in inc easing o de o
ene gy. The lowes ene gy minimum (A) is s abilized by
a complex ne wo k o hyd ogen bonds ha include: (i) wo
backbone⋯backbone (b–b) in e ac ions in ol ing he ionized
N- and C- e minal g oups and he C O and N–H moie ies o
he adjacen amide g oups (i.e. known as C
5
in e ac ions
because a oms in ol ed in such in e ac ions de ine a i e-
membe ed ing); (ii) one side chain⋯backbone (sc–b) sal
b idge be ween he ca boxyla e side g oups o Asp and he
ionized N- e minus; and (iii) one side chain⋯side chain
(sc–sc) in e ac ion be ween he guanidinium g oup o A g and
he dioxane ing o GlE. In addi ion, his s uc u e is s abilized
by a N–H⋯πin e ac ion be ween he guanidinium g oup o
A g and he hiophene ing o GlE. Such an in e ac ion has no
been iden i ied in any o he unique minimum wi h ΔE≤
5 kcal mol
−1
. The nex minimum (B), which is p ac ically
isoene ge ic o A (ΔE< 0.1 kcal mol
−1
), exhibi s he same b–b
and sc–b in e ac ions. Thus, he only diffe ence be ween A and
B e e s o he sc–sc in e ac ion ha , in he la e , consis s o
Fig. 7 (a) Con o ma ional maps cons uc ed by conside ing he back-
bone dihed al angles o he maps A g (ω,ψ), GlE (φ,ψ) and Asp (ω,φ) esi-
dues, using he 25 unique minimum ene gy con o ma ions iden ified in
he clus e ing analysis o RG
E
D. (b) Rep esen a ion o he fi e low-
ene gy s uc u es (labeled om A o E in he inc easing o de o ene gy)
ound in he clus e ing analysis o RG
E
D/B in PBS. Sal b idges and
hyd ogen bonds a e indica ed by ed and iole dashed lines, espec i-
ely. The ed ci cle in A indica es ha he N–H g oup is in ol ed in he
N–H⋯πin e ac ion wi h he hiophene g oup. Dis ances and angles
associa ed wi h each o hese in e ac ions a e gi en in Å and deg ees,
espec i ely. (c) UV- is spec um eco ded o dilu e ace oni ile RG
E
D
solu ion (37 μM). (d) Elec onic densi y diffe ence (exci ed–g ound)
co esponding o he fi s elec onic ansi ion (λ
(1)
max
). Blue ( ed) egions
indica e an inc ease (dec ease) o elec on densi y upon elec onic
ansi ion.
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a hyd ogen bond be ween he guanidinium g oup and he
C O o A g.
The mos cha ac e is ic in e ac ion in he nex minimum
(C), wi h ΔE= 1.5 kcal mol
−1
, co esponds o a double sc–sc
sal b idge be ween he guanidinium and ca boxyla e g oups
o A g and Asp, espec i ely. As a consequence o such in e -
ac ion, he cha ged side g oups a e less exposed o he sol en
in C han in A and B. This con o me also p esen s b–b in e -
ac ions be ween he cha ged ammonium-end g oup and he
amide o A g as well as be ween he ca boxyla e-end g oup and
he amide o Asp. In minimum D (ΔE= 3.9 kcal mol
−1
) he A g
side chain adop s a ully ex ended a angemen ha p ecludes
he pa icipa ion o he guanidinium g oup in he o ma ion
o sc–sc and sc–b in amolecula in e ac ions. Thus, in e -
ac ions obse ed in D a e simila o hose desc ibed o A–C.
Finally, minimum E, wi h ΔE= 4.7 kcal mol
−1
, exhibi s a
double sc–sc sal b idge be ween A g and Asp as well as wo
sc–b in e ac ions in ol ing he cha ged end g oups. These a e
simila o he in e ac ions iden i ied o C.
The esul s displayed in Fig. 7b indica e ha , in gene al,
he o e all shape o he unique minimum ene gy con o -
ma ions wi h ΔE< 5 kcal mol
−1
is app op ia e o o m in e -
molecula in e ac ions wi h he in eg in ecep o . Thus, he
gene al shape adop ed by hese con o ma ions can be simply
desc ibed as a small and de o med hai pin wi h he cha ged
g oups poin ing ou wa ds. As PEDOT chains a e connec ed o
RG
E
D h ough he side g oup o he GlE esidue, all hese con-
o ma ions a e no expec ed o be affec ed by he o ma ion o
he conjuga e. Acco dingly, he unc ionali y o he RGD
adhesi e sequence is expec ed o be p ese ed in he pep ide
o PEDOT–RG
E
D conjuga es.
While he EDOT abso p ion spec um exhibi s a b oad peak
cha ac e ized by a double hump (264 nm and 255 nm), he
spec um o RG
E
D unde simila condi ions (dilu ed in ace o-
ni ile solu ion) exhibi s a clea ansi ion maximum which
appea s a 234 nm wi h a small shoulde a 264 nm (see
Fig. 7c). In a p e ious wo k
24
on EDOT, we showed he impo -
ance o accoun ing o se e al con o ma ions o such a lex-
ible s uc u e o ep oduce he expe imen al da a wi h
compu a ional means. In he same ein, he op ical p ope ies
we e he ein simula ed in ace oni ile o A–E con o ma ions
s a ing om he i e abo e-men ioned s uc u es. All con o -
ma ions p esen simila spec a wi h wo close maximum
abso p ion wa eleng hs (λ
(1)
max
and λ
(2)
max
) a 234 nm and 232 nm
(associa ed oscilla o s eng hs >0.1). No signi ican a ia ion
was obse ed while selec ing one con o ma ion o ano he . By
looking a he elec onic densi y diffe ence o hese ansi ions
o A con o ma ion, i is c ys al clea ha he elec onic an-
si ion occu s on he G
E
moie y and is he e o e no affec ed by
A g and Asp lexibili y (Fig. 7d, see Fig. S6† o simila plo s
o B–D). By compa ing he compu a ional esul s wi h he
expe imen , he compu ed peak a 234 nm ep oduces he
expe imen al main peak while he obse ed expe imen al
shoulde a 264 nm could no be esol ed using such me hodo-
logy. The shoulde is he e o e no he esul o con o ma ional
effec s bu is ela ed o he ine s uc u e o G
E
.
Moni o ing he in luence o RG
E
D in cell adhesion
The effec o he pep ide in cell adhesion was e alua ed by con-
side ing wo epi helial (Ve o and Saos-2) and wo ib oblas
(Cos-7 and MRC-5) cell lines. Ve o and Cos-7 a e cell lines
de i ed om monkey kidney, while he o igin o Saos-2 and
MRC-5 was human bone and human lung, espec i ely. The
quan i a i e esul s o cell adhesion assays a e displayed in
Fig. 8a. These a e exp essed as pe a ea o subs a e ma e ial
and ela i e o hose o he issue cul u e polys y ene (TCPS),
which was used as a con ol ma e ial. The numbe o cells
adhe ed o he su ace o PEDOT–RG
E
D is signi ican ly highe
han ha o PEDOT and TCPS o all cell lines, e idencing he
cell-speci ic adhesion p omo ed ia he RG
E
D ligands conju-
ga ed o he PEDOT chains. The RGD pep ide has been ecog-
nized as a molecula ac i a o o he in eg in cell su ace
ecep o , which can induce in acellula signaling
pa hways.
39–41
This abili y is p ese ed in RG
E
D, whe e he Gly
o RGD has been eplaced by GlE. In pa icula , he ac i a ion
o he in eg in ecep o h ough he in e ac ions wi h he
RG
E
D pep ide in he ini ial cell a achmen s ep seems o
affec subsequen cell sp eading and su i al h ough cell sig-
naling molecules, such as ocal adhesion kinase (FAK), as p o-
posed o RGD.
40
Al hough his beha io is ema kable o he
Fig. 8 (a) Cellula adhesion on he su ace o PEDOT, PEDOT–RG
E
D/A
and PEDOT–RG
E
D/B. TCPS was used as a con ol subs a e. Ve o, Saos-
2, Cos-7 and MRC-5 cells we e cul u ed o 24 h. The expe imen s we e
pe o med using six samples o each subs a e. (b) Adhesion o Ve o,
Saos-2, Cos-7 and MRC-5 cells (b1–b4) on o PEDOT–RG
E
D/B. SEM
mic og aphs eflec he homogeneous cell sp eading. (c) High and low
(inse ) magnifica ion SEM mic og aphs o Saos-2 and Cos-7 cells (c1 and
c2, espec i ely) a ached o PEDOT–RG
E
D/B. (d) Con ol ol ammo-
g ams in PBS o PEDOT–RG
E
D/B uncoa ed and coa ed wi h Saos-2 and
MRC-5 cells.
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wo conjuga es, i is conside ably mo e e iden o PEDOT–
RG
E
D/B han o PEDOT–RG
E
D/A. This has been a ibu ed o
he bene i s p oduced by he syn he ic s a egy B in he p o-
pe ies o he esul ing conjuga e (i.e. enhancemen o he
oughness, we abili y and elec oac i i y). In gene al, he ela-
i e iabili y o Saos-2 and MRC-5 human cell lines is highe
han ha o Ve o and Cos-7 monkey kidney cell lines, indepen-
den ly o he subs a e su ace.
Fig. 8b, which displays low magni ica ion SEM mic og aphs
o cells cul u ed on o bo h PEDOT–RG
E
D/A and PEDOT–RG
E
D/
B, e eals no only he high densi y o adhe ed cells bu also
hei homogeneous sp eading on o he su ace o he wo con-
juga e ilms. De ails abou he adhesion o MRC-5 and Saos-2
cells on o he su ace o PEDOT–RG
E
D/B a e p o ided in
Fig. 8c. As i can be seen, he cellula mechanism ope a ing o
he adhesion o he cells on o he conjuga e ilms is he con-
nec ion o he su ace wi h ilopodia. Thus, sp eading o he
cells is achie ed h ough an in ima e con ac be ween he cells
and he su ace o he ilms.
On he o he hand, he pa icula elec ochemical p o-
pe ies o PEDOT, which exhibi highe cell iabili y han ine
TCPS, also con ibu e o he supe io cell a achmen o
PEDOT–RG
E
D. Thus, he abili y o his ECP o exchange ions
wi h he cells h ough he cellula memb ane is known o
a o he o ma ion o cell-su ace in e ac ions.
42
In o de o
e alua e he in luence o cul u ed cells in he elec ochemical
ac i i y o he conjuga es, PEDOT–RG
E
D/A and PEDOT–RG
E
D/
B ilms coa ed wi h cell monolaye s we e in es iga ed by CV in
PBS. Fig. 8d compa es he con ol ol ammog ams eco ded
o PEDOT–RG
E
D/B coa ed wi h Saos-2 and MRC-5 cells
wi h he ol ammog am ob ained o he unco e ed ma e ial
( esul s ob ained o PEDOT–RG
E
D/A samples coa ed wi h a e
displayed in Fig. S7†). The cells do no affec he p o ile o he
ol ammog ams, e en hough hey p o oke an enhancemen
o bo h he anodic cu en densi y a he e e sal po en ial and
o he elec oac i i y, which e lec he elec obioac i i y o he
p epa ed conjuga es. Thus, cell monolaye s inc ease he mobi-
li y o ions a he in e aces, he elec oac i i y being inc eased
by abou 10–25% wi h espec o he uncoa ed conjuga es.
Acco ding o hese esul s, he adhe ed cells p omo e he
exchange o ions a he conjuga e–cell in e ace a he han
blocking he channels ha allow he access and escape o ions.
Conclusions
The RG
E
D pep ide, which is an analogue o he adhesi e RGD
sequence, has been designed by chemical simila i y wi h
PEDOT and, subsequen ly, p epa ed h ough chemical syn-
hesis. A e his, PEDOT–RG
E
D has been ob ained by anodic
polyme iza ion, he EDOT side g oup o he GlE esidue ac ing
as a linke be ween he pep ide and he polyme . Two diffe en
s a egies ha e been used o p oduce ma e ials wi h diffe en
su ace oughness, we abili y and elec ochemical ac i i y.
Con ol on o hese p ope ies has been ound o modula e he
beha io o PEDOT–RG
E
D conjuga es as so bioelec oac i e
suppo s o cell a achmen .
Because he cell ecogni ion abili ies o he RGD mo i
emain in he RG
E
D sequence, cell a achmen and sp eading
on PEDOT–RG
E
D ha e been signi ican ly p omo ed wi h
espec o PEDOT. Fu he mo e, he elec ochemical ac i i y o
he ECP is p ese ed in he conjuga e because bo h he small
size o he pep ide and he u iliza ion o an EDOT ing as a
linke be ween he ECP and he pep ide, do no affec he
anspo o cha ge. Indeed, he elec ochemical ac i i y o he
conjuga es inc eases upon he adhesion o cell monolaye s.
The success ul design and con olled p epa a ion o effec i e
ECP–pep ide conjuga es open new and a ied possibili ies
wi hin he biomedical ield. Fo example, he ab ica ion o
mul i unc ional biomedical pla o ms o egene a i e medi-
cine, combining he p esence o he RG
E
D adhesi e sequence
wi h o he unc ional pep ides in he same pla o m, and he
de elopmen o a i icial skin based on ECPs, which in
addi ion, p omo e he egene a ion o na u al skin.
43
Expe imen al sec ion
Chemical cha ac e iza ion o RG
E
D
Mel ing poin s we e de e mined using a Gallenkamp appa -
a us and a e unco ec ed. IR spec a we e eco ded on a
Nicole A a a 360 FTIR spec opho ome e ; ν
max
is gi en o
he main abso p ion bands.
1
H and
13
C NMR spec a we e
eco ded on a B uke AV-500, AV-400, ARX-300 o Va ian
Gemini 300 ins umen a oom empe a u e unless o he wise
indica ed, using he esidual sol en signal as he in e nal
s anda d, chemical shi s (δ) a e exp essed in ppm and coup-
ling cons an s (J) in he z. High- esolu ion mass spec a we e
ob ained on a B uke Mic o o -Q spec ome e .
Syn hesis o PEDOT–RG
E
D
S eps 1 and 2 o he syn he ic p ocess desc ibed in he ex
we e ca ied ou in a s anda d h ee elec ode cell o 50 mL
using s eel AISI 316 shee s o 4 cm
2
a ea (su ace oughness
de e mined by a omic o ce mic oscopy: 11.4 nm) as he
wo king and coun e elec odes. The e e ence elec ode was
an Ag|AgCl elec ode con aining a KCl sa u a ed aqueous solu-
ion (E° = 0.222 V a 25 °C). The p epa a ion o PEDOT and
PEDOT–(p o ec ed RG
E
D) ilms was pe o med by CA using a
cons an po en ial o 1.40 and 1.37 V, espec i ely. In all cases,
he cells we e illed wi h 50 mL o he co esponding ace o-
ni ile solu ion. All elec opolyme iza ions and cyclic ol am-
me y assays we e conduc ed on a PGSTAT302N AUTOLAB
po en ios a –gal anos a (Ecochimie, The Ne he lands)
equipped wi h he ECD module, which was connec ed o a PC
compu e con olled h ough he NOVA 1.6 so wa e.
Cha ac e iza ion
The ESI†p o ides de ails o he equipmen and condi ions
used o cha ac e iza ion by con ac p o ilome y, FTIR
spec oscopy, X- ay pho oelec on spec oscopy (XPS), s anda d
Bioma e ials Science Pape
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