Biodegradable and biocompatible collagen-based hybrid materials for force sensing applications

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- nc-nd/4.0/). 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 In e na ional Jou nal o Biological Mac omolecules jou nal homepage: www.else ie .com/loca e/ijbiomac 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.