In e na ional Jou nal o Biological Mac omolecules 256 (2024) 128486
A ailable online 1 Decembe 2023
0141-8130/© 2023 The Au ho s. Published by Else ie B.V. This is an open access a icle unde he CC BY-NC-ND license (h p://c ea i ecommons.o g/licenses/by-
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Biodeg adable and biocompa ible collagen-based hyb id ma e ials o o ce
sensing applica ions
Mi eia Andonegi
a
,
b
, Ra aela M. Mei a
b
, Daniela M. Co eia
c
, Nelson Pe ei a
b
,
Ca los M. Cos a
b
,
e
, Senen xu Lance os-Mendez
b
,
d
,
,
*
, Ko o de la Caba
a
,
d
,
*
, Ped o Gue e o
a
,
d
,
g
a
BIOMAT Resea ch G oup, Uni e si y o he Basque Coun y (UPV/EHU), Escuela de Ingenie ía de Gipuzkoa, Plaza de Eu opa 1, 20018 Donos ia-San Sebas i´
an, Spain
b
Physics Cen e o Minho and Po o Uni e si ies (CF-UM-UP) and Labo a o y o Physics o Ma e ials and Eme gen Technologies, LapMET, Uni e si y o Minho 4710-
057, B aga, Po ugal
c
Cen e o Chemis y, Uni e si y o Minho, 4710-057 B aga, Po ugal
d
BCMa e ials, Basque Cen e o Ma e ials, Applica ions and Nanos uc u es, UPV/EHU Science Pa k, 48940 Leioa, Spain
e
Ins i u e o Science and Inno a ion o Bio-Sus ainabili y (IB-S), Uni e si y o Minho, 4710-053 B aga, Po ugal
Ike basque, Basque Founda ion o Science, 48009 Bilbao, Spain
g
P o einma Ma e ials SL, A enida de Tolosa 72, 20018 Donos ia-San Sebas i´
an, Spain
ARTICLE INFO
Keywo ds:
Collagen
Elec ical esponse
P essu e senso
ABSTRACT
Wi h he aim o eplacing syn he ic mac omolecules by biological mac omolecules o ad anced applica ions,
collagen ilms we e p oduced wi h wo di e en ionic liquids (ILs), choline dihyd ogen phospha e ([Ch][DHP])
and choline se ina e ([Ch][Se i]), added in o de o modula e he elec ical esponses. The ilms we e p epa ed
by cas ing, a ying IL con en be ween 0 and 6 w %. The mo phology and he mal p ope ies o he esul ing
ilms we e ound o be independen o bo h IL ype and con en . Howe e , he highes di ec cu e (d.c.)
elec ical conduc i i y (1.4 ×10
−8
S⋅cm
−1
) was achie ed o collagen ilms con aining 3 w % [Ch][DHP].
Fu he mo e, i was demons a ed ha IL/collagen ilms we e non-cy o oxic, wi h cell ac i i y alues exceeding
70 %. These collagen ilms we e p o en o be sui able o o ce sensing applica ions, displaying excellen
sensi i i y and s abili y upon epea ed es ing.
1. In oduc ion
Collagen ep esen s he mos abundan p o ein wi hin he ex acel-
lula ma ix (ECM) o e eb a es, cons i u ing oughly 30 % o he o al
p o ein mass in mammals. Speci ically, ype I collagen, he p ima y
ocus o his s udy, cons i u es abou 90 % o he collagen ound in skin
and bones [1,2]. Thus, he main ole a ibu ed o collagen is o p o ide
s uc u al suppo , con ibu ing o he mechanical p ope ies o issues,
such as s eng h and oughness. Beyond i s s uc u al ole, collagen has
been demons a ed o play a signi ican pa in a ious unc ional oles
[3]. I plays an impo an ole in issue healing, p o iding he biological
mic oen i onmen o cell g ow h, aiding cell a achmen , mig a ion,
and p oli e a ion [4]. Mo eo e , collagen exhibi s low immunogenici y
and an igenici y, ende ing i biocompa ible [5,6]. Al hough collagen is
esis an o common p o eases showing a long li e ime, unde goes
biodeg ada ion wi hin he body h ough ce ain enzymes om he ma-
ix me allop o einase (MMP) amily, a c ucial ac o o designing
biodeg adable collagen-based ma e ials [7]. Consequen ly, modi ica ion
o c oss-linking is necessa y o con ol he deg ada ion a e o issue
enginee ing applica ions [8,9]. The physical and biological p ope ies o
collagen, coupled wi h i s e sa ili y in o ming gels, ilms, meshes,
sca olds and ibe s make collagen an a ac i e candida e o a ious
applica ions.
Cu en ly, he majo i y o indus ial-g ade collagen ype I is sou ced
om ca le. Ca le skin collagen is used o o i y endons and p omo e
wound healing, while neona al bo ine de mis is employed in he nia
epai , plas ic and econs uc i e su ge y, among o he applica ions
[10]. Ne e heless, he u iliza ion o his collagen is limi ed because o
i s high manu ac u ing cos s. Fo una ely, an un apped and mo e cos -
e ec i e sou ce o collagen exis s: was e gene a ed by he lea he in-
dus y. App oxima ely 80 % o his was e comp ises non- anned esi-
dues, which con ain abou 70–80 % o ib ous collagen [11]. Thus,
eco e ing collagen-con aining was e has he po en ial o be a p o i able
and en i onmen ally sus ainable app oach, con ibu ing o he Uni ed
* Co esponding au ho s a : BCMa e ials, Basque Cen e o Ma e ials, Applica ions and Nanos uc u es, UPV/EHU Science Pa k, 48940 Leioa, Spain.
E-mail add ess: [email p o ec ed] (K. de la Caba).
Con en s lis s a ailable a ScienceDi ec
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h ps://doi.o g/10.1016/j.ijbiomac.2023.128486
Recei ed 3 Oc obe 2023; Recei ed in e ised o m 13 No embe 2023; Accep ed 27 No embe 2023
In e na ional Jou nal o Biological Mac omolecules 256 (2024) 128486
2
Na ions Sus ainable De elopmen Goals [12].
Collagen is a p omising ma e ial o elec onic de ices, including
elec ochemical applica ions [13], and biomem is i e memo y de ices,
which exhibi a high- esis ance s a e/low- esis ance s a e a io o ~100
[14]. In his con ex , a lexible s ain senso based on collagen ibe s was
de eloped, boas ing a ensile s eng h o 59.9 MPa, elec ical conduc-
i i y o 6.5 S⋅m
−1
and supe -amphiphobic p ope ies, making i sui able
o wea able elec onic de ices [15]. Fu he mo e, polyme -based
hyb id ma e ials ea u ing collagen, polyaniline as conduc i e ma ix,
and i on oxide nanopa icles as magne ic ille s we e de eloped o
achie e ailo ed dielec ic, magne ic and conduc ing p ope ies. This
composi e ma e ial exhibi ed an elec ical conduc i i y o 2.44 ×10
−2
S⋅cm
−1
and a sa u a ion magne iza ion o 8.32 emu/g [16].
Wi hin his con ex , polyme -based hyb id ma e ials inco po a ing
ionic liquids (ILs) o e a no el pla o m o c ea ing mul i unc ional
hyb id ma e ials wi h educed en i onmen al oo p in when compa ed
o nanopa icle-con aining composi es [17]. Building upon p e ious
wo k in ol ing collagen wi h a ious ILs, including choline dihyd ogen
phospha e ([Ch][DHP]), choline de ina e ([Ch][Se i]), choline bis( i-
luo ome hylsul onyl)imide ([Ch][TFSI]), and 1-e hyl-3-me hylimida-
zolium bis( i luo ome hylsul onyl)imide ([EMIM][TFSI]), which
demons a ed hei sui abili y o esis i e ouch senso s [18], his s udy
aims o de elop sus ainable blends o collagen wi h low IL con en (up o
6 w %) u ilizing wo IL ypes: [Ch][DHP] and [Ch][Se i]. These ILs we e
selec ed o hei biocompa ibili y and wa e miscibili y. The e alua ion
encompasses an assessmen o mo phology, he mal, mechanical, elec-
ical and biological p ope ies, in addi ion o he unc ional sensing
esponse.
2. Ma e ials and me hods
2.1. Ma e ials
Bo ine collagen was supplied by P o einma S.L. (Donos ia, Spain),
choline dihyd ogen phospha e [Ch][DHP] (>98 %) and choline de ina e
[Ch][Se i] (>95 %, 60 % in H
2
O) by Ionic Liquids Technologies GmbH
(Ge many), and ace ic acid by Pan eac Quimica S.L.U. (Ba celona,
Spain).
2.2. Film p epa a ion
Cas ing was employed in he p epa a ion o collagen ilms wi h
a ying con en s (0, 1, 3, and 6 w %) o [Ch][DHP] and [Ch][Se i]. Fi s ,
bo ine collagen and he co esponding amoun o IL o each o mula-
ion we e mixed in a solu ion o 0.5 M ace ic acid (1:50 collagen/ace ic
acid). To ob ain he ilms, he mix u es we e le a oom empe a u e o
2 h unde con inuous s i ing a 400 pm be o e being pou ed in o Pe i
dishes and allowed o d y a oom empe a u e. Films wi h an a e age
hickness o 125
μ
m we e ob ained, i espec i e o he ille con en .
These ilms we e iden i ied as 1[Ch][DHP], 1[Ch][Se i], 3[Ch][DHP], 3
[Ch][Se i], 6[Ch][DHP], and 6[Ch][Se i], as a unc ion o IL ype and
con en . Con ol ilms we e hose wi hou ILs. Be o e unde going ilm
cha ac e iza ion, all ilms we e condi ioned o 48 h in an ACS Sun ise
700 V biochambe (Ala a Ingenie os, Mad id, Spain) a 25 ◦C and 50 %
ela i e humidi y.
2.3. Film cha ac e iza ion
To assess he he mal ansi ions o he samples, di e en ial scanning
calo ime y (DSC) measu emen s we e pe o med wi h a Me le Toledo
DSC 822 (Mad id, Spain) equipmen . Abou 3.0 ±0.2 mg o each sample
we e enclosed in 50
μ
L aluminum pans, o a oid mass loss du ing he
expe imen , and subjec ed o a hea ing amp om 25 o 300 ◦C a a a e
o 10 ◦C/min unde an ine a mosphe e (10 mL N
2
/min) o a oid
he mo-oxida i e eac ions.
Fou ie T ans o med In a ed (FTIR) measu emen s we e pe o med
using an Alpha II Compac FTIR spec ome e coupled wi h an a enu-
a ed o al e lec ance (ATR) c ys al (ZnSe). The FTIR spec a o he
composi es we e eco ded a oom empe a u e om 4000 o 800 cm
−1
and collec ed a e 32 scans wi h a spec al esolu ion o 4 cm
−1
.
X- ay di ac ion (XRD) was pe o med a 40 kV and 40 mA wi h a
PANaly ic Xpe P o (PANaly ical, Almelo, The Ne he lands) appa a us
using Cu
–
K (λ =1.5418) as he adia ion sou ce. The da a we e
collec ed be ween 2 and 50◦(s ep size =0.026, ime pe s ep =118 s).
The mo phology o he ilms was examined by SEM, using an Hi achi
S-4800 scanning elec on mic oscope (Hi achi, Mad id, Spain) a 15 kV
accele a ing ol age. P io o SEM measu emen s, ilms we e placed on a
me al s ub and coa ed wi h gold using a JEOL ine-coa ion spu e JFC-
1100 and a gon a mosphe e.
Mechanical ensile es s o he samples we e pe o med using an
Ins on 5967 mechanical es ing sys em (Ins on, Ba celona, Spain).
Tensile es s we e ca ied ou a a a e o 1 mm/min on bone-shaped
samples (4.75 mm ×22.25 mm) in acco dance wi h he ASTM D
638–03 s anda d. To de e mine signi ican di e ences be ween samples,
an analysis o a iance (ANOVA) was pe o med using SPSS so wa e
(SPSS S a is ic 25). Fo compa ison ac oss a ious sys ems, Tukey's es
wi h s a is ical signi icance a he P <0.05 le el was conside ed.
The d.c. elec ical conduc i i y (
σ
, S/cm) o he ilms was ob ained a
oom empe a u e h ough a Kei hley 287 picoamme e / ol age sou ce,
applying ol age be ween ±10 V. Measu emen s we e pe o med in he
pa allel pla e con igu a ion and he elec ical conduc i i y was ob ained
om he I-V cu es h ough Eq. (1):
σ
=d
R.A(1)
whe e d (cm) is he sample hickness, R (Ω) is he esis ance alue, and A
(cm
2
) is he elec ode a ea.
2.4. Film deg ada ion and cy o oxici y
Fo he deg ada ion assay, ci cula samples wi h a 13 mm diame e
we e cu om di e en collagen ilms, weighed (wi) and placed in a 24-
well cul u e pla e wi h 500
μ
L o phospha e-bu e ed saline solu ion
(PBS, pH 7.4) and cell cul u e medium (DMEM). The samples we e
subsequen ly incuba ed in an o en a 37 ◦C o 1, 2, 4 and 7 days. A each
ime poin , he samples we e emo ed om he cul u e pla e and le o
d y a oom empe a u e be o e being weighed (w ). The weigh loss o
h ee specimens o each sample was calcula ed using Eq. (2):
Weigh loss (%) = wi −w
wi ⨉100 (2)
To assess he cy o oxici y o he samples, MC3T3-E1 p e-os eoblas
cells we e cul i a ed in Dulbecco's modi ied Eagle's medium (DMEM,
Gibco) con aining 1 g⋅L
−1
glucose, 10 % e al bo ine se um (FBS, Bio-
ch om) and 1 % penicilin/s ep omycin (P/S, Bioch om) unde s anda d
cul u e condi ions (a 37 ◦C in a 95 % humidi ied ai con aining 5 %
CO
2
). The cul u e medium was e esehed e e y 2 days and he cells
we e ha es ed be o e eaching ~70 % con luence. The ci o oxici y o
he IL/collagen composi e ilms was e alua ed using he 3-(4,5-dime-
hyl hiazol-2-yl)-2,5-diphenyl e azolium b omide (MTT, P omega)
assay. Ci cula samples wi h a 13 mm diame e we e cu om collagen
ilms wi h a ying [Ch][DHP] and [Ch][Se i] con en s (1, 3 and 6 w %).
Fi s , he samples we e exposed o ul a iole ligh (UV) o 40 min (20
min on each side), washed wice wi h s e ile phospha e-bu e ed saline
solu ion (PBS, pH 7.4) o 30 min pe wash on an o bi al shake o
emo e any esidual sol en . Subsequen ly, hey we e exposed again o
UV ligh o an addi ional hou (30 min on each side) o ensu e comple e
s e iliza ion. Following s e iliza ion, he samples we e placed in a 24-
well issue cul u e polys y ene pla e con aining cell cul u e medium
and incuba ed a 37 ◦C in 95 % humidi ied a mosphe e con aining 5 %
CO
2
o 72 h. MC3T3-E1 cells (cell densi y =3 ×10
4
cells⋅mL
−1
) we e
cul i a ed in 96-well issue cul u e polys y ene pla es o 24 h o
M. Andonegi e al.
In e na ional Jou nal o Biological Mac omolecules 256 (2024) 128486
3
acili a e cell a achmen . A e his pe iod, he cell cul u e medium in
he 96-well pla e was eplaced and he MC3T3-E1 cells we e exposed o
a cul u e medium ha had been in con ac wi h a ious samples. A e
72 h, he medium om each well was emo ed and esh medium
con aining MTT solu ion in a 1:10 a io was added. A e 2 h o incu-
ba ion, 100
μ
L om each well was ans e ed in quad uplica e o a 96-
well pla e and he op ical densi y was measu ed a 490 nm. MC3T3-E1
cell iabili y was calcula ed using Eq. (3):
Cell iabili y (%) = abso bance o sample
abso bance o nega i e con ol ⨉100 (3)
2.5. Fo ce senso de elopmen
An in e digi ma ix o 9 ci cula digi s (digi wid h and spacing o
500
μ
m and a channel leng h o 95 mm) was ab ica ed wi h conduc i e
sil e ink om No acen ix (Me alon HPS-021LV). A manual sc een-
p in ing machine was used wi h a mesh o 100 h eads by cen ime e
and he conduc i e pa e ns we e deposi ed on op o a PET subs a e
(Melinex 506, 100
μ
m hick PET ilm), as shown in Fig. 1a. Fo he
sensing assays, he ilms we e cu in ci cles o 12 mm in diame e and
placed on op o a pape sepa a o wi h a hole o 10 mm in diame e ,
allowing an ai gap be ween he in e digi and he collagen ilm
(Fig. 1b), and he ma ix was encapsula ed wi h ano he PET ilm. The
elec ical connec ion o he ma ix wi h he elec onic sys em was ach-
ie ed wi h a la lexible cable wi h a pi ch o 1 mm, glued wi h Z-axis
conduc i e ape om 3 M (9703).
A eensy 4.0 mic ocon olle was used o acqui e he esis ance
a ia ion o he ma ix h ough a ol age di ide wi h a esis ance (R) o
10 MΩ. The mic ocon olle analog- o-digi al con e e (ADC) acqui es
he ol age alue o he nine senso s and sends he con e ed da a o a
G aphical use in e ace (GUI) ia USB, as shown in Fig. 2a). Fig. 2b
shows he de eloped ic- ac- oe game, aking ad an age o he nine-
senso ma ix.
3. Resul s and discussion
3.1. The mal and physicochemical p ope ies
DSC analysis was used o de e mine he he mal ansi ions and
s abili y o collagen ilms con aining ILs. The in luence o IL inclusion on
he glass ansi ion empe a u e (T
g
), dena u a ion empe a u e (T
d
),
and en halpy (ΔH) o he ilms is shown in Table 1. All samples showed
an endo he mic peak be ween 45 ◦C and 185 ◦C, associa ed wi h he
dehyd a ion and he mal dena u a ion o he amo phous egion o
collagen [19]. I was obse ed ha T
d
inc eased om 84.1 ◦C o 91.3 ◦C
o 6[Ch][DHP] and 98.4 ◦C o 6[Ch][Se i] samples, indica ing ha a
sligh imp o emen in he he mal s abili y o collagen upon he inclu-
sion o bo h ILs, pa icula ly in he case o [Ch][Se i]-con aining sam-
ples [20]. ΔH alues, which ep esen he ene gy equi ed o elease ee
and bound wa e as well as o collagen dena u a ion [21], dec eased
om 324.5 J/g in con ol ilms o 223.5 J/g o 6[Ch][DHP] and 193.6
J/g o 6[Ch][Se i], in acco dance wi h he dec ease in he s uc u al
o de obse ed in he XRD analysis (Fig. 5). Rega ding he glass an-
si ion empe a u e, T
g
dec eased om 38.5 ◦C in con ol ilms o 36.2 ◦C
o 6[Ch][DHP] and 32.53 ◦C o 6 [Ch][Se i]. This educ ion in Tg can
be a ibu ed o he in e ac ions be ween collagen and ILs ha dis up
hyd ogen bond in collagen, esul ing in changes in he collagen s uc u e
[22].
The in luence o inco po a ing di e en IL ypes and con en s was
s udied by ATR-FTIR measu emen s. As can be seen in Fig. 3a), he main
abso p ion bands o collagen a e obse ed in he FTIR spec a o all
samples, i espec i e o IL concen a ion o ype. The abso p ion band a
3500–3000 cm
−1
is a ibu ed o N
–
H s e ching ib a ion o amide A
and he band a 1632 cm
−1
is associa ed wi h he C
–
–
O s e ching i-
b a ion o amide I. The band a 1547 cm
−1
co esponds o he N
–
H
bending ib a ion o amide II, and he band a 1238 cm
−1
ep esen s he
C
–
N s e ching ib a ion o amide III [23,24]. Fu he mo e, he ab-
so p ion bands a 1080 and 1032 cm
−1
a e associa ed o C
–
O and C-O-C
s e ching ib a ions, espec i ely [23,25]. Rega ding ILs, he sligh
abso p ion band de ec ed a 960 cm
−1
in [Ch][DHP]/collagen and [Ch]
[Se i]/collagen ilms is a ibu ed o he C
–
N s e ching o he [Ch]
+
ca ion [18,26,27]. Addi ionally, o he 6[Ch][DHP] sample, wo
shoulde s a 946 cm
−1
and 1080 cm
−1
a e no iceable, co esponding o
he P-OH g oup and P
–
–
O g oups o [DHP]
−
, espec i ely [18,28,29].
When ILs we e added, sligh changes in he ela i e in ensi y be-
ween he amide I and amide II bands we e obse ed (Fig. 3b). These
changes may be a ibu ed o con o ma ional changes in he collagen
s uc u e induced by he p esence o ILs. Speci ically, he in ensi y o he
amide I band was lowe han ha o he amide II band o he con ol
ilms. Howe e , o he 6[Ch][Se i] sample, he in ensi y o amide I band
became smalle han ha o amide II. This shi is a ibu ed o he
physical in e ac ions, speci ically ionic in e ac ions be ween collagen
chains and ILs. I is wo h no ing ha hese in e ac ions can change he
na i e s uc u e o collagen [20]. S ill, a low concen a ions, ionic liq-
uids p ima ily a ec he sup amolecula s uc u e o collagen wi hou
signi ican ly impac ing he seconda y s uc u e o collagen ibe s a
molecula le el [30]. This obse a ion aligns wi h he SEM images o he
ilm c oss-sec ion (Fig. 4). In ac , [Ch][DHP] was ound o p omo e he
o ma ion o in a- and in e molecula o ces wi hin he helices o
collagen due o elec os a ic in e ac ions be ween collagen and [Ch]
[DHP] [31].
3.2. Mo phological analyses
SEM and XRD es s we e conduc ed o s ablish a connec ion be ween
he p e iously men ioned p ope ies and he s uc u al cha ac e is ics o
he collagen ilm. This in es iga ion is essen ial because ILs ha e he
po en ial o dis u b he iple helix s uc u e o collagen. The c oss-
Fig. 1. Schema ic ep esen a ion o he de eloped p essu e senso sys em.
M. Andonegi e al.
In e na ional Jou nal o Biological Mac omolecules 256 (2024) 128486
4
sec ion mo phology o he IL/collagen composi es was e alua ed by
SEM and ep esen a i e images a e shown in Fig. 4, which includes
p is ine collagen (con ol), 6[Ch][DHP] and 6[Ch][Se i]. I is no e-
wo hy ha all ilms showed he compac and uni o mly dense ib illa
s uc u e consis en wi h collagen. These indings con i m ha he
ib illa s uc u e o he samples emained in ac i espec i e o he
addi ion o ILs [32,33].
Addi ionally, all samples showed he semic ys alline XRD pa e n
cha ac e is ic o collagen, ea u ing di ac ion peaks a 2ɵ =7.80◦and
20.50◦(Fig. 5). On he one hand, he dis inc peak deno ed as A co e-
sponds o he c ys alline s uc u e o collagen and ep esen s he dis-
ance be ween he collagen chains. On he o he hand, he b oade band
deno ed as B is associa ed wi h he sca e ing caused by componen s
Fig. 2. Schema ic ep esen a ion o he elec onic ci cui o da a acquisi ion (a) and image o he applica ion de eloped (b).
Table 1
Glass ansi ion empe a u e (T
g
), dena u a ion empe a u e (T
d
), and en halpy
(ΔH) alues ob ained by DSC analysis o collagen ilms con aining [Ch][DHP]
and [Ch][Se i].
Films T
g
(◦C) ±1 ◦C T
d
(◦C) ±1 ◦C ΔH (J/g) ±1 %
Con ol 38.5 84.1 324.5
1[Ch][DHP] 36.9 86.4 275.5
3[Ch][DHP] 36.3 88.6 266.3
6[Ch][DHP] 36.2 91.3 223.5
1[Ch][Se i] 34.9 91.1 213.7
3[Ch][Se i] 32.8 92.4 210.5
6[Ch][Se i] 32.5 98.4 193.6
Fig. 3. – FTIR-ATR spec a o collagen ilms wi h di e en con en s o [Ch][DHP] and [Ch][Se i]: a) om 4000 o 800 cm
−1
and b) om 1800 o 800 cm
−1
.
Fig. 4. SEM c oss-sec ion images o a) p is ine collagen (con ol), b) 6[Ch][DHP], and c) 6[Ch][Se i] ilms.
M. Andonegi e al.
In e na ional Jou nal o Biological Mac omolecules 256 (2024) 128486
5
wi hin collagen ibe s, ep esen ing he amo phous phase [31,34]. In he
case o samples con aining [Ch][DHP] (Fig. 5a), a sligh dec ease in he
in ensi y o peak A was obse ed upon he inco po a ion o 1 w % IL.
This educ ion became mo e p onounced in samples wi h 3 and 6 w %
[Ch][DHP], indica ing a dec ease o he s uc u al o de in collagen as IL
con en s inc eased. This obse a ion may be a ibu ed o he abili y o
ILs o dis up hyd ogen bonds in collagen and modi y i s s uc u e
[22,35,36]. The shi in his peak posi ion o 7.35◦ o 6[Ch][DHP]
samples indica es an inc ease in he dis ance be ween molecula chains
om 1.14 nm o 1.20 nm. Addi ionally, an inc ease in he in ensi y o he
band B was obse ed wi h he addi ion o [Ch][DHP], ega dless o he
IL con en , and a shoulde a app oxima ely 40◦appea ed in he 6[Ch]
[DHP]samples.
A simila e ec was obse ed wi h he inco po a ion o [Ch][Se i]
(Fig. 5b), esul ing in a mo e signi ican dec ease in he in ensi y o peak
A o he samples wi h 3 and 6 w % [Ch][Se i] han o he samples wi h
he same [Ch][DHP] con en s. This obse a ion poin s o a mo e sub-
s an ial educ ion in he collagen s uc u al o de o [Ch][Se i] con-
aining samples, al hough no peak shi was obse ed. I mus be no ed
ha he absence o he peak a a ound 30◦, cha ac e is ic o he iple
helical s uc u e o collagen [37], sugges s ha he iple helical s uc-
u e o collagen is pa ially dis up ed in all samples [38], an e ec
a ibu ed o he p ocessing condi ions.
3.3. Mechanical and d.c. elec ical p ope ies
Tensile es s we e used o assess he e ec o ILs on he mechanical
p ope ies o he ilms, and he esul s a e shown in Fig. 6a) and sum-
ma ized in Table 2. I was obse ed ha he e was no signi ican di -
e ence in ensile s eng h bu a sligh inc ease in elonga ion a b eak
was no iced o [Ch][DHP] con aining samples as he IL con en
inc eased. This phenomenon can be a ibu ed o he hyg oscopic na u e
o his IL, which enhanced he ilm wa e e en ion capaci y [39,40]. In
con as , o samples con aining [Ch][Se i], a signi ican dec ease in
ensile s eng h and an inc ease in elonga ion a b eak we e obse ed in
he case o 3[Ch][DHP] and 6[Ch][DHP] ilms. This beha io is asc ibed
o he plas icizing e ec o he IL. Fu he mo e, he dec ease in he
s uc u al o de in he samples con aining 3 and 6 w % [Ch][Se i], as
obse ed by XRD analysis (Fig. 5b), could also con ibu e o he dec ease
in ensile s eng h.
The d.c. elec ical conduc i i y alue o collagen ilms as a unc ion
o IL con en is shown in Fig. 6b. The inse shows he cha ac e is ic I-V
cu es o he ilms con aining 6 w % IL, demons a ing nea ly linea
Ohmic beha io , wi h sligh non-linea con ibu ions. Rega dless o IL
ype, he elec ic conduc i i y (
σ
) inc eased wi h ising IL con en up o
6 w %, when a sligh dec ease is obse ed. This imp o emen in elec-
ical esponse can be a ibu ed o he inc eased p esence o mobile
anions and ca ions acili a ed by ILs [41]. A highe IL concen a ions (6
w %) IL-IL in e ac ions in ensi y, limi ing ion mobili y and leading o a
Fig. 5. XRD pa e ns o a) [Ch][DHP] and b) [Ch][Se i] con aining collagen ilms.
Fig. 6. a) S ess/s ain cu es ob ained by ensile es s and b) d.c. elec ical conduc i i y alues o collagen ilms con aining [Ch][DHP] and [Ch][Se i] ILs. Inse : I-V
cu es o 6[Ch][DHP] and 6[Ch][Se i] collagen ilms.
Table 2
Tensile s eng h (TS) and elonga ion a b eak (EB) o collagen ilms p epa ed
wi h di e en [Ch][DPH] and [Ch][Se i] con en s.
Films TS (MPa) EAB (%)
Con ol 25.16 ±4.29
b,c
10.41 ±0.66
a
1[Ch][DHP] 26.92 ±1.20
c
16.59 ±1.45
b
3[Ch][DHP] 27.08 ±1.97
c
17.71 ±1.45
b
6[Ch][DHP] 26.03 ±2.59
b,c
16.88 ±0.83
b
1[Ch][Se i] 27.40 ±3.78
c
12.97 ±0.54
a
3[Ch][Se i] 21.05 ±3.94
a,b
15.70 ±2.43
b
6[Ch][Se i] 18.09 ±2.57
a
16.09 ±1.56
b
a-c: Two means ollowed by he same le e in he same column a e no signi -
ican ly (P >0.05) di e en h ough he Tukey's mul iple ange es .
M. Andonegi e al.
In e na ional Jou nal o Biological Mac omolecules 256 (2024) 128486
6
sligh dec ease o he conduc i i y [42]. Fu he mo e, he ype o IL
sligh ly a ec ed elec ic conduc i i y due o di e ences in size and in-
e ac ions s ablished wi hin he collagen ma ix, a ec ing ion mobili y.
Collagen ilms con aining 3 w % o [Ch][DHP] exhibi ed he highes
elec ical conduc i i y wi h a alue o 1.4 ×10
−8
S⋅cm
−1
.
3.4. Deg ada ion and cy o oxici y
Deg ada ion assays we e pe o med o e alua e he beha io o
collagen ilms con aining IL when exposed o PBS and DMEM. As can be
seen in Fig. 7a, all samples comple ely deg aded a e 4 days in PBS
despi e hei di e en p o ile. Collagen and [Ch][DHP]/collagen com-
posi es displayed a simila deg ada ion p o ile. A e 1 day, collagen
ilms e ealed a weigh loss o ~25 %, which inc eased o ~30 % on he
second day, and eached 100 % by he ou h day. In pa icula , he
weigh loss inc eased om 19 % on he i s day o 28 % on he second
day o 1[Ch][DHP] ilms, om 18 % on he i s day o 24 % on he
second day o 3[Ch][DHP] ilms, and om 17 % on he i s day o 27 %
on he second day o 6[Ch][DHP] ilms. These esul s indica ed ha he
inco po a ion o [Ch][DHP] did no a ec he deg ada ion p o ile o he
polyme . In con as , he addi ion o IL [Ch][Se i] induced a as e
weigh loss when compa ed o p is ine collagen. An inc ease in [Ch]
[Se i] con en led o a signi ican inc ease in he weigh loss o he
composi e ilms. In ela ion o he deg ada ion assays in DMEM
(Fig. 7b), i was obse ed ha he deg ada ion p o iles o he samples
we e qui e simila , wi h he excep ion o 6[Ch][Se i] ilms, which p e-
sen ed a as e deg ada ion a e. A e 1 day in DMEM, 6[Ch][Se i] ilms
e ealed a weigh loss o 25 %, which inc eased app oxima ely o 43 %
on he second day, 83 % on he ou h day, and 88 % on he se en h day.
Addi ionally, he cy o oxici y o he samples was e alua ed and he
ob ained esul s a e p esen ed in Fig. 8. None o he IL/collagen ilms
exhibi ed ci o oxixiy, as all samples p esen ed MC3T3-E1 cell iabili ies
highe han 70 %. This demons a es he sui abili y o bo h [Ch][DHP]/
collagen and [Ch][Se i]/collagen composi es o biomedical
applica ions.
3.5. Senso esponse
Taking he elec ical p ope ies in o accoun , 3[Ch][DHP] ilms we e
selec ed o de elop a o ce senso . In Fig. 9a, he esis ance a ia ion
du ing100 comp ession cycles o e ime is depic ed using a Shimadzu
AG-IS wi h a 500 N load cell. A mo e de ailed ep esen a ion o hese
a ia ions o e 3 cycles is p esen ed in Fig. 9b, illus a ing he co ela-
ion be ween he esis ance a ia ion and he o ce on he senso . The
esis ance a ia ion inc eased p opo ionally wi h he o ce applied o
he ilm and ice e sa. Va ia ions in he ai gap be ween he in e digi
and he collagen ilm allowed a ia ions in he con ac a ea wi h he
in e digi , changing he elec ical beha io and esul ing in he dec ease
o he esis ance alue (inc ease o ΔR) wi h he applied o ce (Fig. 9b),
measu ed as an elec ical signal. Fig. 9c) shows he esis ance a ia ion
as a unc ion o he applied o ce o a speci ic senso , which is ep e-
sen a i e o he emaining senso s. The dec ease in esis ance was only
de ec ed when he applied o ce eached app oxima ely 1.2 N, he
minimum o ce equi ed o place he collagen ilm in con ac wi h he
in e digi . The senso exhibi ed a sensi i i y o app oxima ely 8 MΩ/N,
and i s esponse emained s able h oughou cycling wi h a good
epea abili y and no hys e esis.
Fig. 9 d) shows he a ia ion ecei ed by he applica ion when he
di e en senso s we e p essed sequen ially wi h a inge , om senso S1
o S9, showing an iden ical a ia ion in each senso and allowing a
eliable de ec ion o he p essu e. The GUI buil in Q Modeling Lan-
guage (QML) ecei ed he alue o he 9 senso s and de ec ed i he
esis ance alue was abo e a h eshold, based on he applied p essu e. I
he esis ance alue was less han he h eshold, he algo i hm assumed
ha he bu on was p essed, changing he s a e o each squa e.
Fig. 7. Weigh loss ela i e o he o iginal mass a e deg ada ion o 7 days in a) PBS and b) DMEM media o IL/collagen ilms.
Fig. 8. Cy o oxici y assays wi h MC3T3-E1 cells in con ac wi h he as-p epa ed
ex ac ion media exposed o he IL/collagen composi es wi h di e en [Ch]
[DHP] and [Ch][Se i] con en s o 72 h ( ela i e cell iabili y was p esen ed as
he pe cen age o he nega i e con ol (DMEM, n =3 ±s anda d e o o
mean (SEM)).
M. Andonegi e al.
In e na ional Jou nal o Biological Mac omolecules 256 (2024) 128486
7
4. Conclusions
Biocompa ible ionic liquid (IL)/collagen ilms we e de eloped o
o ce senso applica ions. These collagen ilms we e p epa ed using he
cas ing me hod inco po a ing wo di e en ILs, choline dihyd ogen
phospha e ([Ch][DHP]) and choline de ina e ([Ch][Se i]), wi h IL
con en a ia ions (1, 3, 6 w %). These ilms we e cha ac e ized by a
compac and homogeneous ib illa s uc u e, which p o ided sui able
he mal and mechanical p ope ies. Addi ionally, IL/collagen ilms we e
p o ed o be non-cy o oxic wi h a cell iabili y >70 %. No ably, he d.c.
elec ical conduc i i y o he composi es inc eased pa icula ly o
collagen ilms wi h 3 w % o [Ch][DHP], eaching 1.4 ×10
−8
S.cm
−1
.
Consequen ly, his pa icula o mula ion was selec ed o he de el-
opmen o o ce esis i e senso s, which showed excellen esponse and
sus ained s abili y o e ime.
Au ho s a emen
We con i m ha he manusc ip has been ead and app o ed by all
named au ho s and ha he e a e no o he pe sons who sa is ied he
c i e ia o au ho ship bu a e no lis ed. We u he con i m ha he
o de o au ho s lis ed in he manusc ip has been app o ed by all o us.
Decla a ion o compe ing in e es
The au ho s decla e ha hey ha e no known compe ing inancial
in e es s o pe sonal ela ionships ha could ha e appea ed o in luence
he wo k epo ed in his pape .
Acknowledgemen s
G an PID2021-124294OB-C22 unded by MCI/AEI10.13039/
501100011033 and by “ERDF A way o making Eu ope”. This wo k was
also suppo ed by he Basque Go e nmen (IT1658-22) and he Po u-
guese Founda ion o Science and Technology (FCT) unde s a egic
unding UIDB/04650/2020, UID/FIS/04650/2021, p ojec PTDC/FIS-
MAC/28157/2017, 2022.05932.PTDC and In es iga o FCT Con ac
2020.02915.CEECIND (D.M.C) and 2020.04028.CEECIND (C.M.C.)
unded by na ional unds h ough FCT and by he ERDF h ough he
COMPETE2020-P og ama Ope acional Compe i i idade e In e -
nacionalizaç˜
ao (POCI). The au ho s also acknowledge unding om he
Basque Go e nmen Indus y and Educa ion Depa men unde he
ELKARTEK p og am. M.A hanks he Basque Go e nmen 1 o he
ellowship (POS_2022_1_0007).
Re e ences
[1] C. Ding, e al., The esponse o collagen molecules in acid solu ion o empe a u e,
Polyme 55 (22) (2014) 5751–5759.
[2] P.C. Balau e, e al., In i o and in i o s udies o no el ab ica ed bioac i e
d essings based on collagen and zinc oxide 3D sca olds, In . J. Pha m. 557 (2019)
199–207.
[3] S.A. Ghodbane, M.G. Dunn, Physical and mechanical p ope ies o c oss-linked
ype I collagen sca olds de i ed om bo ine, po cine, and o ine endons,
J. Biomed. Ma e . Res. A 104 (11) (2016) 2685–2692.
[4] A. I as o za, e al., The e sa ili y o collagen and chi osan: om ood o biomedical
applica ions, Food Hyd ocoll. 116 (2021), 106633.
[5] A.K. Lynn, I.V. Yannas, W. Bon ield, An igenici y and immunogenici y o collagen,
J. Biomed. Ma e . Res. B Appl. Bioma e . 71B (2) (2004) 343–354.
Fig. 9. a) 100 cycles o loading and unloading o ce applied o he senso and he co esponding esis ance a ia ion o e ime. b) Magni ica ion o some o he o ce
applica ion cycles and c) esis ance a ia ion co ela ion wi h he o ce applied o he senso . d) Digi al ou pu ecei ed in he so wa e o he 9 senso s p essed
sequen ially.
M. Andonegi e al.
In e na ional Jou nal o Biological Mac omolecules 256 (2024) 128486
8
[6] M. Andonegi, e al., Physicochemical and biological pe o mance o aloe Ve a-
inco po a ed na i e collagen ilms, Pha maceu ics 12 (2020), h ps://doi.o g/
10.3390/pha maceu ics12121173.
[7] J.L. Laue -Fields, D. Juska, G.B. Fields, Ma ix me allop o einases and collagen
ca abolism, Pep . Sci. 66 (1) (2002) 19–32.
[8] D. Benayahu, e al., Unique collagen ibe s o biomedical applica ions, Ma . D ugs
16 (2018), h ps://doi.o g/10.3390/md16040102.
[9] M. Andonegi, e al., S uc u e-p ope ies ela ionship o chi osan/collagen ilms
wi h po en ial o biomedical applica ions, Ca bohyd . Polym. 237 (2020),
116159.
[10] A. Le´
on-L´
opez, e al., Hyd olyzed collagen-sou ces and applica ions, Molecules 24
(2019) 22.
[11] L. Mais enko, e al., Collagen ob ained om lea he p oduc ion was e p o ides
sui able gels o biomedical applica ions, Polyme s 14 (2022), h ps://doi.o g/
10.3390/polym14214749.
[12] J. Bebbing on, J. Une man, Achie ing he Uni ed Na ions sus ainable de elopmen
goals, Accoun . Audi . Accoun . J. 31 (1) (2018) 2–24.
[13] X. Zhang, e al., Collagen-based lexible elec onic de ices o elec ochemical
ene gy s o age and sensing, Mac omol. Rapid Commun. 44 (10) (2023), p.
2200977.
[14] Y. Zeng, e al., A sus ainable biomem is i e memo y de ice based on na u al
collagen, Ma e ials Today Chemis y 13 (2019) 18–24.
[15] Z. Bai, e al., Ve sa ile nano–mic o collagen ibe -based wea able elec onics o
heal h moni o ing and he mal managemen , J. Ma e . Chem. A 11 (2) (2023)
726–741.
[16] B.T. Mekonnen, M. Rago haman, T. Palanisamy, Bi unc ional hyb id composi es
om collagen biowas es o he e ogeneous applica ions, ACS Omega 2 (8) (2017)
5260–5270.
[17] D.M. Co eia, e al., Ionic liquid–polyme composi es: a new pla o m o
mul i unc ional applica ions, Ad . Func . Ma e . 30 (24) (2020) 1909736.
[18] M. Andonegi, e al., Sus ainable collagen blends wi h di e en ionic liquids o
esis i e ouch sensing applica ions, ACS Sus ainable Chemis y & Enginee ing 11
(15) (2023) 5986–5998.
[19] J. Gonz´
alez-Masís, e al., Sel -assembly s udy o ype I collagen ex ac ed om male
Wis a Hanno e a ail endons, Bioma e ials Resea ch 24 (1) (2020) 19.
[20] A. Ta annum, e al., A e aqueous solu ions o choline-based ionic liquid
biocompa ible c oss-linke s o collagen? J. Mol. Liq. 303 (2020), 112654.
[21] V. Samouillan, e al., The use o he mal echniques o he cha ac e iza ion and
selec ion o na u al bioma e ials, Jou nal o Func ional Bioma e ials 2 (2011)
230–248, h ps://doi.o g/10.3390/j b2030230.
[22] A. Ta annum, J.R. Rao, N.N. Fa hima, Choline-based amino acid ILs–collagen
in e ac ion: enuncia ing i s ole in s abiliza ion/des abiliza ion phenomena,
J. Phys. Chem. B. 122 (3) (2018) 1145–1151.
[23] K. Belbachi , e al., Collagen ypes analysis and di e en ia ion by FTIR
spec oscopy, Anal. Bioanal. Chem. 395 (3) (2009) 829–837.
[24] T. Riaz, e al., FTIR analysis o na u al and syn he ic collagen, Appl. Spec osc. Re .
53 (9) (2018) 703–746.
[25] X. Bi, e al., A no el me hod o de e mina ion o collagen o ien a ion in ca ilage
by Fou ie ans o m in a ed imaging spec oscopy (FT-IRIS), Os eoa h . Ca il.
13 (12) (2005) 1050–1058.
[26] R.M. Mei a, e al., Ionic liquid-based elec oac i e ma e ials: a no el app oach o
ca diac issue enginee ing s a egies, J. Ma e . Chem. B 10 (34) (2022) 6472–6482.
[27] V. Rigual, e al., P o ic, ap o ic, and choline-de i ed ionic liquids: owa d
enhancing he accessibili y o ha dwood and so wood, ACS Sus ain. Chem. Eng. 8
(3) (2020) 1362–1370.
[28] S.M. Kamal Mohamed, e al., Facile p epa a ion o biocompa ible and anspa en
silica ae ogels as Ionogels using choline dihyd ogen phospha e ionic liquid, Appl.
Sci. 11 (2021), h ps://doi.o g/10.3390/app11010206.
[29] A. Reizabal, e al., Silk ib oin bending ac ua o s as an app oach owa d na u al
polyme based ac i e ma e ials, ACS Appl. Ma e . In e aces 11 (33) (2019)
30197–30206.
[30] A. Meh a, J.R. Rao, N.N. Fa hima, E ec o ionic liquids on he di e en
hie a chical o de o ype I collagen, Colloids Su . B Bioin e aces 117 (2014)
376–382.
[31] S.Y. Bak, e al., Assessmen o he in luence o ace ic acid esidue on ype I collagen
du ing isola ion and cha ac e iza ion, Ma e ials 11 (2018), h ps://doi.o g/
10.3390/ma11122518.
[32] P. Ji, e al., Collagen ilm wi h bionic laye ed s uc u e and high ligh
ansmi ance o pe sonalized co neal epai ab ica ed by con olled sol en
e apo a ion echnique, Jou nal o Func ional Bioma e ials (2022) 13, h ps://doi.
o g/10.3390/j b13020052.
[33] Y. Ma, e al., A op-down app oach o imp o e collagen ilm’s pe o mance: he
compa isons o mac o, mic o and nano sized ibe s, Food Chem. 309 (2020),
125624.
[34] N. Re´
a egui-Pinedo, e al., Cha ac e iza ion o collagen om h ee gene ic lines
(g ay, ed and F1) o O eoch omis nilo icus (Tilapia) skin in young and old adul s,
Molecules (2022) 27, h ps://doi.o g/10.3390/molecules27031123.
[35] Y. Hu, e al., E alua ion o 1-e hyl-3-me hylimidazolium ace a e based ionic liquid
sys ems as a sui able sol en o collagen, J. Appl. Polym. Sci. 130 (4) (2013)
2245–2256.
[36] M.M. Gi aud-Guille, e al., S uc u al aspec s o ish skin collagen which o ms
o de ed a ays ia liquid c ys alline s a es, Bioma e ials 21 (9) (2000) 899–906.
[37] C. Li, e al., S uc u al p ope ies o pepsin-solubilized collagen acyla ed by lau oyl
chlo ide along wi h succinic anhyd ide, Ma e . Sci. Eng. C 55 (2015) 327–334.
[38] Z. Meng, e al., Dissolu ion and egene a ion o collagen ibe s using ionic liquid,
In . J. Biol. Mac omol. 51 (4) (2012) 440–448.
[39] M. Rahman, C.S. B azel, Ionic liquids: new gene a ion s able plas icize s o poly
( inyl chlo ide), Polym. Deg ad. S ab. 91 (12) (2006) 3371–3382.
[40] E. Ve ou is, e al., In a- and in e -molecula in e ac ions in choline-based ionic
liquids s udied by 1D and 2D NMR, J. Mol. Liq. 322 (2021), 114934.
[41] M. Qu, e al., Ion anspo in ionic liquid/poly( inylidene luo ide) sys em unde
elec ic ields: a molecula dynamics simula ion, Colloids Su . A Physicochem.
Eng. Asp. 642 (2022), 128328.
[42] F.C.A. Sil a, e al., Polyme -ion in e ac ions in PVDF@ionic liquid polyme
elec oly es: a combined expe imen al and compu a ional s udy, Elec ochim. Ac a
427 (2022), 140831.
M. Andonegi e al.
