Enabling low molecular weight electrospinning through binary solutions of polymer blends

Resea ch a icle Enabling low molecula weigh elec ospinning h ough bina y solu ions o polyme blends Vic o M. Pe ez-Puyana a , Albe o Rome o a , An onio Gue e o a , Lo enzo Mo oni b , Paul A. Wie inga b , * a Depa amen o de Ingenie ía Química, Uni e sidad de Se illa, Facul ad de Química, Se illa 41012, Spain b Depa men o Complex Tissue Regene a ion, MERLN Ins i u e o Technology-Inspi ed Regene a i e Medicine, Maas ich Uni e si y, Maas ich 6200 MD, he Ne he lands ARTICLE INFO Keywo ds: PCL Gela in Elec ospinning Bina y solu ion, Nano ibe s ABSTRACT The o ma ion o nano ib ous memb anes ia elec ospinning is ypically es ic ed o high molecula weigh polyme s in an app op ia e sol en , co ela ed wi h he necessa y o ma ion o polyme chain en anglemen s ha a e needed o achie e success ul p oduc ion o elec ospun ibe s. The p esen wo k ex ends he elec ospinning o low molecula weigh polyme s by in es iga ing he elec ospinning o a bina y solu ion sys em consis ing o wo di e en low molecula weigh polyme s, using as a model sys em polycap olac one (PCL) and gela in in di e en a ios. The iscosi ies o he polyme solu ions we e cha ac e ized as a p oxy o polyme chain en anglemen and he esul ing ibe s we e mo phologically cha ac e ized by SEM imaging and u he assessed wa e con ac angle and molecula composi ion o de e mine he impac and homogenei y o he bina y mix u es. We ound ha uni a y solu ions o ei he PCL o gela in ailed o gene a e p ope ibe s despi e indica ions o chain en anglemen . In con as , bina y solu ions o low molecula weigh PCL and gela in gene a ed di e en ibe quali y and size dis ibu ions, depending on he a io used, wi h di ec co ela ions be ween ibe p ope ies and he PCL:Gela in a io. I was disco e ed ha he a io o PCL o gela ion was mos p edic i e o success ul ibe gene a ion, wi h e ec i e elec ospinning occu ing only o a de ine in e media e ange o high blend a ios while bo h low and high blended bina y solu ions esul ed in poo ibe p oduc ion. Ou s udy con i med ha his beha io was independen om absolu e polyme concen a ion, indica ing a unique in e ac ion be- ween hese bina y species which exis s only unde speci ic a io concen a ions and indica es p omising new a enues o p ocess low molecula weigh polyme s solu ions. 1. In oduc ion The elec ospinning echnique is a p ocess ha can p oduce nonwo en ib ous memb anes comp ised o ibe s wi h diame e s anging om nanome e s o mic ome e s [1]. Thus, hese memb anes p esen in e es ing p ope ies, such as mode a e po osi y and high spe- ci ic a ea. Du ing elec ospinning, a polyme ic solu ion is pumped h ough a me allic spinne e and a high ol age is applied be ween he spinne e and a su ace, called collec o , ha is held a ixed dis ance way. The esul ing elec ic ield elec os a ically ex udes he polyme solu ion om he spinne e , o ming a je o polyme solu ion ha a els om he spinne e o he collec o . Unde op imum condi ions, he je unde goes high speed chao ic whipping which inc eases e apo- a ion a e o he sol en , o ming solid ibe s ha a e e en ually deposi ed on o he collec o o c ea e a ib ous memb ane [2]. The ibe o ma ion and he p ope ies o he memb anes depend on he co ec e olu ion o he polyme je du ing he elec ospinning p ocess. Fibe o ma ion is an in e play be ween p ocessing pa ame e s, en i onmen al condi ions and polyme solu ion p ope ies. P ocessing pa ame e s can be adjus ed o ensu e s able je o ma ion and o une he ibe diame e [3]. In his sense, se e al au ho s ha e e alua ed he in luence o he di e en p ocessing pa ame e s ( ol age, humidi y, wo king dis ance, e c.) on he elec ospinning p ocess [4–7]. En i on- men al condi ions ha e been s udied in e ms o hei in luence on he e apo a ion a e o he sol en and he subsequen e ec s on ibe o - ma ion [8,9]. Bu polyme solu ion is a c i ical aspec in e ms o su ace ension, conduc i i y o iscosi y. The la e is used as an indica ion o he deg ee o chain en anglemen . I chain en anglemen is insu icien , * Co esponding au ho . E-mail add ess: [email p o ec ed] (P.A. Wie inga). Con en s lis s a ailable a ScienceDi ec Nex Ma e ials jou nal homepage: www.sciencedi ec .com/jou nal/nex -ma e ials h ps://doi.o g/10.1016/j.nxma e.2024.100306 Recei ed 1 May 2024; Recei ed in e ised o m 18 June 2024; Accep ed 4 July 2024 Nex Ma e ials 6 (2025) 100306 A ailable online 30 July 2024 2949-8228/© 2024 The Au ho s. Published by Else ie L d. This is an open access a icle unde he CC BY-NC license ( h p://c ea i ecommons.o g/licenses/by- nc/4.0/ ). he je canno be con inuously main ained and he p ocess ansi ions om elec ospinning o elec osp aying whe e he je b eaks up in o d ople s ha quickly dispe se as a esul o columbic o ces and deposi on o he collec o as polyme mic osphe es [10]. As desc ibed by Colby e al. [11], a polyme solu ion alls in o “ ou di e en concen a ion egimes: dilu e, semidilu e unen angled, semi- dilu e en angled, and concen a ed”. Fo elec ospinning, i is he bounda y be ween he semidilu e unen angled and semidilu e en angled egimes, commonly known as he en anglemen concen a ion (C e ), which is o mos in e es ; his is he poin ha he numbe o polyme chains in solu ion is su icien ly high o cons ain each o he , hus p o- ducing en anglemen couplings [12] ha esis he b eakup o he e ol ing polyme je and p omo e consis en ibe o ma ion [13]. Al hough he en anglemen concen a ion di e s depending on he wo king sys em, McKee e al. ound ha a concen a ion be ween 2 and 2.5 imes he en anglemen concen a ion is necessa y o achie e de ec - ee nano ib ous ma s [14]. This has been a gene ally accep ed guideline and is ollowed by he elec ospinning- esea ch communi y. The C e o a polyme /sol en sys em is o en de e mined by measu ing he solu ion iscosi y, since his depends on he in e molecula in- e ac ions in hese polyme solu ions and can se e as a p oxy o he deg ee o chain en anglemen [15]. The sol en sys em used has a g ea in luence on iscosi y, due o hei e ec on he dissolu ion and ho- mogeniza ion o he polyme in solu ion [16]. Simila ly, he polyme molecula weigh and polyme concen a ion in solu ion ha e a signi - ican e ec on chain en anglemen wi hin he solu ion, wi h an inc ease in molecula weigh o concen a ion causing an inc ease in iscosi y [17]. The e o e, he limi ing concen a ion om which homogeneous ibe s a e ob ained may di e depending on he polyme o he sol en used. In he cu en s udy, we in es iga ed a bina y polyme sys em in a single sol en , unde he p emise ha in e molecula in e ac ions o wo dis inc polyme ypes in solu ion can be op imized and co ela ed o an imp o ed elec ospinning p ocess compa ed o espec i e uni a y polyme solu ions. F om a wide a ie y o polyme s cu en ly a ailable o elec ospinning, we selec ed a bina y sys em o PCL and Gela in as wo dis inc polyme ypes o syn he ic and na u al o igin, espec i ely, wi h good solubili y in simila sol en s and wi h epo ed applica ions in issue enginee ing applica ions. Gela in, in pa icula , has been used ex ensi ely in he elec ospinning o issue enginee ed ib ous cons uc s as a na u al p o ein polyme ha has high biocompa ibili y compa ed o syn he ic polyme s [18–20]. Howe e , he elec ospinning o gela in alone is di icul o ca y ou p ope ly and i o en equi es he use o o ganic sol en s o achie e app op ia e solu ion iscosi y and he use o a blended solu ion o syn he ic and na u al polyme s o achie e homo- geneous ibe s [21–23]. Polycap olac one (PCL) is simila ly a common syn he ic polyme o elec ospun meshes due o i s sui able mechanical p ope ies and easy p ocessabili y [24], hough molecula weigh and polyme concen a ion emain c i ical o nano ibe o ma ion. Consis- en elec ospinning o PCL has been epo ed when using an a e age molecula weigh o 45,000 o 80,000 and concen a ions anging om 5 o 20 w % [18,25–28] while no success ul epo s could be ound o elec ospinning low molecula weigh polycap olac one (LW-PCL, M w =14,000 g/mol). Poo elec ospinning pe o mance is ypically indica ed by he o ma ion o beads, which is a ibu ed o he low su ace ension o he polyme solu ion o low molecula weigh o he dissol ed polyme , which p e en s a con inuous polyme je du ing he elec ospinning p ocess. Fo low molecula weigh s, in pa icula , i is sugges ed ha small polyme s chains make i di icul o achie e su i- cien chain en anglemen o ibe o ma ion e en a high concen a- ions in solu ion. Ins ead o ibe s, polyme solu ions o his ype o en esul in he consis en p oduc ion o elec osp ayed mic osphe es, whe e LW-PCL a e epo ed o p oduce mic osphe es o e a ange o concen a ions up o 30 % w/ (21.4 mM) [29]. Ou main hypo hesis is ha gela in and LW-PCL, wo polyme s which canno be success ully elec ospun alone, can be combined o c ea e a bina y polyme solu ion wi h op imized chain en anglemen and, hus, success ul elec ospinning. This would lead o nano ibe p oduc ion using lowe cos polyme s o lowe molecula weigh . The iscosi y o polyme solu ions wi h a ying concen a ions o LW-PCL and gela in was measu ed and he elec ospinning ou come o he same polyme solu ions we e e alua ed using HFIP as sol en , wi h special ocus on how he addi ion o gela in al e ed ib ous memb ane p oduc ion. To his end, PCL and PCL:Gela in nano ib ous sca olds we e ab ica ed and a physicochemical (wa e con ac angle and ene gy dispe si e analysis) and mic os uc u al cha ac e iza ion (scanning elec on mic oscopy) we e pe o med. The main no el y o his wo k is he iden i ica ion o an op imal egime o concen a ions wi hin a bina y sys em, as de ined by he iscosi y measu emen s and ela i e polyme a ios. This s udy was ocused on he ole o change he solu ion p ope ies, so all o he pa- ame e s, such as he p ocessing condi ions, we e main ained cons an . This s udy p o ides a p oo -o -p inciple o u he explo a ion o p o- duce ib es wi h lowe cos p ocessing condi ions. 2. Ma e ials and me hods 2.1. Ma e ials Gela in p o ein (gela in ype B, 80–120 g Bloom) was supplied by Henan Boom Gela in Co. L d (China). Gela in is mainly composed o p o ein (ca 98 w %) and mino componen s, such as ash, lipids and mois u e. Ve y low molecula weigh poly( ε -cap olac one) (LW PCL, M w =14,000 g/mol) and 1,1,1,3,3,3-hexa luo o-2-p opanol (HFIP) we e pu chased om Sigma Ald ich (Ge many). 2.2. Elec ospinning p ocess PCL and gela in in di e en concen a ions we e mixed oge he di ec ly in HFIP a oom empe a u e by s i ing o ca 24 h on a mag- ne ic s i e ollowing he di e en a ios e alua ed. The selec ion o he sol en was based on he op imiza ion o he elec ospinning p ocess since HFIP dissol e bo h PCL and gela in p o ein. Once he solu ions we e p epa ed, he elec ospinning p ocess was ca ied ou using a Fluidna ek LE-100 equipmen (Bioinicia). Memb anes wi h PCL and gela in in di e en a ios we e p ocessed ollowing he same p ocessing condi ions used in p e ious s udies [30]: 14 kV o ol age, 0.4 mL/h o low a e and 14 cm as he needle-collec o dis ance. The sys ems p o- cessed wi h di e en p opo ions o PCL and gela in a e shown in Table 1. The ma s ob ained we e cha ac e ized by analysing he mean ibe size and he ibe size dis ibu ion measu ed om SEM imaging. In addi ion, he con ac angle and he N con en (wi h EDAX) we e also e alua ed. The p e ious polyme ic solu ions we e cha ac e ised by means o hei iscosi y. Table 1 Sys ems p ocessed wi h di e en PCL:Gela in a ios modi ying he PCL o gela in concen a ion. Ra io PCL Concen a ion Gela in Concen a ion To al polyme Concen a ion Nomencla u e 3:1 12 w/ % 4 w/ % 16 w/ % 12/4 4:1 16 w/ % 20 w/ % 16/4 5:1 20 w/ % 24 w/ % 20/4 6:1 24 w/ % 28 w/ % 24/4 8:1 32 w/ % 36 w/ % 32/4 6:1* 6 w/ % 38 w/ % 32/6 5:1* 6.4 w/ % 38.4 w/ % 32/6.4 4:1* 8 w/ % 40 w/ % 32/8 3:1* 10.7 w/ % 42.7 w/ % 32/10.7 V.M. Pe ez-Puyana e al. Nex Ma e ials 6 (2025) 100306 2 2.3. Cha ac e iza ion s udies 2.3.1. Fibe mo phology cha ac e iza ion 2.3.1.1. Scanning elec on mic oscopy (SEM). Mic oscopy examina ion o sca olds was assessed wi h a XL 30 (Philips XL Se ies) a an accel- e a ion ol age o 10 kV, a spo size o 3 and a wo king dis ance o 10 mm. The samples we e co e ed wi h an Au ilm in a high- esolu ion spu e coa e . ImageJ (a digi al p ocessing so wa e) was used o analyze he mean ibe size and he po osi y. The ibe size dis ibu ion was also ob ained o he di e en sys ems p oduced. Fu he mo e, he a io o hei s anda d de ia ion o hei mean was calcula ed o de e - mine he uni o mi y o he sys ems (Eq. 1): Uni o mi y(%) = (1−s anda d de ia ion mean ibe diame e )⋅100 (1) The pa ame e “uni o mi y”has been de ined o e alua e he ho- mogenei y o he memb anes since i allows compa ing he s anda d de ia ion and mean ibe diame e o he ibe size dis ibu ion ob- ained. In his sense, an inc ease in uni o mi y implies a dec ease in he s anda d de ia ion, which ep esen s a lowe a iabili y o ibe size ob ained. 2.3.1.2. Ene gy dispe si e x- ays spec oscopy (EDAX). The composi ion o he elec ospun memb anes was e alua ed using he ene gy dispe si e spec oscopy capabili y o he SEM equipmen (EDAX Si(Li) de ec o ). An accele a ion ol age o 20 kV, a spo size o 3 and a wo king dis ance o 10 mm we e he pa ame e s selec ed. 2.3.2. Wa e con ac angle (WCA) The hyd ophobici y and we abili y o he sca olds we e analyzed by measu ing he wa e con ac angle (WCA) wi h he sessile d op me hod (d ople s wi h an app oxima e olume o 5 μ L). WCA alues we e ob- ained as he a e age be ween he igh /le sides alues o he deionized wa e d ople s. The equipmen used was a D op Shape Analyze (K üss). 2.3.3. Viscosi y A glass capilla y Ubbelohde iscosime e (VWR, The Ne he lands) was used o measu e he iscosi y o he PCL:Gela in solu ions. The empe a u e was main ained a 20±2ºC o all he measu emen s. The speci ic iscosi y was ob ained using he ollowing equa ion [31] (Eq. 2): Speci ic Viscosi y = η − η 0 η 0 (2) Whe e η is he iscosi y o he solu ion (Pa⋅s) and η 0is he iscosi y o he sol en used (1.65 mPa⋅s a 20 ◦C o HFIP). 2.4. S a is ical analysis Each measu emen was pe o med, a leas , in iplica e. S a is ical analyses we e ca ied ou wi h - es s and one way analysis o a iance (p <0.05) using PASW S a is ics o Windows (Ve sion 18: SPSS, Chi- cago, IL). S anda d de ia ions we e ob ained o selec ed pa ame e s. 3. Resul s and discussion 3.1. Uni a y PCL and gela in solu ions: elec ospinning and iscosi y As men ioned abo e, p e ious epo s ha e co ela ed he ou comes o he elec ospinning p ocess o he polyme solu ion p ope ies, spe- ci ically solu ion iscosi y and polyme concen a ion. These wo pa- ame e s a e in e connec ed, ob aining a co ela ion wi h he minimum concen a ion needed o achie e a sui able solu ion iscosi y o ca y ou he elec ospinning p ocess p ope ly. This is achie ed hanks o he en anglemen be ween he polyme chains, which a ou s he elec ospinning p ocess. I has been con i med ha ibe s canno be consis en ly gene a ed om uni a y solu ions p oduced wi h PCL (supplemen a y ma e ial) o gela in [30] by elec ospinning. To co ela e his o iscosi y, uni a y solu ions o PCL and gela in we e i s s udied in a speci ic sol en (HFIP in his case). Fig. 1A shows he e olu ion o he iscosi y o PCL solu- ions by inc easing i s concen a ion. Two di e en egions can be highligh ed by a change in he slope ( om 4.95 o 10.35). The i s e- gion ends when he o al polyme concen a ion is 20 w/ %, s a ing a new egion in which he in luence o he concen a ion o e he iscosi y exhibi s a highe slope. Conside ing he s udy conduc ed by Colby e al. [11], hese wo egions obse ed co espond o he semidilu e unen- angled and en angled egimes, wi h he c osso e poin a 20 w/ % as he en anglemen concen a ion (Ce). As expec ed, he iscosi y o uni a y solu ions o gela in was also obse ed o inc ease wi h inc easing p o ein concen a ions (Fig. 1B). Simila ly, he esul s show an ini ial linea egion up o a concen a ion o 8 % w/ (wi h a slope o 0.061), wi h a sha p inc ease in iscosi y a 10.7 % w/ (wi h a slope o 1.39). This sudden inc ease sugges s gela in molecules s a o inc ease hei in e ac ion wi h each o he , indica i e o inc eased en anglemen . Despi e hese indica ions o an inc ease in en anglemen , he elec- ospinning ou come o bo h solu ions emains poo . The elec o- spinning o LW-PCL esul s in mic osphe es a all concen a ions, bo h abo e and below he 20 %w/ ansi ion (Figu e S1). Simila ly, ou p e ious inding o gela in-only s udies ound ha he elec ospinning p ocess could no be pe o med p ope ly [30]. This beha io can be explained by he low en anglemen o he polyme chains despi e he inc easing gela in concen a ion and subsequen inc ease in iscosi y (Fig. 1B). This low en anglemen is a possible consequence o a low molecula weigh , which is known o con ibu e o bead o ma ion since he elec ospinning p ocess canno be ca ied ou con inuously. Howe e , in con as o bo h uni a y solu ions, which ailed o gene a e ibe s, we obse ed ha combina ions o LW-PCL and gela in in di e en a ios we e success ully elec ospun unde ce ain condi ions. 3.2. In luence o he PCL: gela in a io wi h inc easing PCL concen a ion 3.2.1. Viscosi y measu emen s To be e unde s and his phenomenon, we u he s udied his bi- na y sys em whe eby he gela in concen a ion was held cons an and he amoun o LW-PCL was sys ema ically a ied. The e o e, di e en PCL concen a ions (12, 16, 20, 24 and 32 w/ %) we e combined wi h a cons an amoun o gela in (4 w/ %) and he measu ed iscosi y o hese mix u es measu ed, in o de o co ela e and p edic possible elec ospinning ou comes o hese solu ions (labelled as a io o PCL/ Gela in, speci ically 12/4, 16/4, 20/4, 24/4 and 32/4). Fig. 2 shows he e olu ion o he speci ic iscosi y o he sys ems p oduced wi h a 4 % w/ o gela in and inc easing concen a ions o PCL ( om 12 % o 32 % w/ ), wi h an ini ial slope o 2.49 om 12 % o 16 % w/ ha inc eases o 13.57 om 16 % o 32 % w/ . This de ines wo well-di e en ia ed egions, whe e he i s egion ( om 12 % o 16 %) co esponds o he semidilu e unen angled egime and he second egion co ela es o a semidilu e en angled egime; abo e he concen- a ion o 16 %w/ PCL, chains s a o en angle and, as a consequence, he iscosi y o he solu ions ma kedly inc eases. The e o e, he com- bina ion o 16 % o PCL and 4 % o gela in (12/4 PCL:Gela in a io) can be conside ed he en anglemen concen a ion (Ce, wi h a 20 % o o al polyme concen a ion). Acco ding o Kong and Ziegle [32], he spin- ning o good ibe s equi es ha he solu ion concen a ion be 1.2–2.0 imes he en anglemen concen a ion. O he di e en alues s udied, a PCL:Gela in a io be ween 20/4 and 24/4, o a o al polyme concen- a ion o 24 % and 28 % w/ , espec i ely, should ep esen he s a o good ibe o ma ion since he polyme concen a ion is 1.25 and 1.50 imes he en anglemen concen a ion. V.M. Pe ez-Puyana e al. Nex Ma e ials 6 (2025) 100306 3 3.2.2. Mic os uc u al e alua ion SEM imaging o he e olu ion o he PCL-based memb anes gene - a ed wi h a cons an amoun o gela in (4 %w/ ) and inc easing con- cen a ions o LW PCL (12, 16, 20, 24 and 32 %w/ ) is shown in Fig. 3. These s a ing solu ions ha e ela i e a ios o PCL o gela in o 3:1, 4:1, 5:1, 6:1, and 8:1, labelled espec i ely as 12/4, 16/4, 20/4, 24/4 and 32/4 o cla i y he polyme con en pe condi ion. The lowes a io (12/ 4) showed a combina ion o ibe s and beads on i s s uc u e. Howe e , he inc ease in he a io lead o he homogeniza ion o he sys em wi h he disappea ance o he beads and he o ma ion o consis en ibe s. This imp o emen is mainly obse ed o he 16/4 and 20/4 a io, which co espond wi h he op imal ange ound in he iscosi y measu emen s shown in he p e ious sec ion. In e es ingly, when he a io is highe han 20/4 (24/4 and 32/4), he SEM images showed again he o ma ion o sphe oids in he s uc u e. F om he SEM images, he dis ibu ion o sizes o he elemen s (ei he ibe s o beads) p esen in he s uc u e we e analysed and plo ed in Fig. 3. A shi in he p o ile can be seen, om a wide dis ibu ion obse ed o he sys em wi h a 12/4 (due o he p esence o bo h ibe s and beads) o a na ow dis ibu ion owa ds a cen al alue (a ound 0.5 µm) o he 20/4 sys em and a e u n o a wide dis ibu ion when he a io is highe a 24/4 and 32/4, which gi es ise o a sys em wi h mo e he e ogeneous ibe sizes and he e-eme gence o sphe oid o ma ion. To be e assess he e ec o he PCL:Gela in a io, we quan i ied ibe uni o mi y (Fig. 3) as unc ion o ibe and bead size (Table 2) o each o he mixed PCL:Gela in sys ems. This clea ly shows an op imal egion, whe e uni o mi y inc eases o a maximum be ween PCL:Gela in a ios o 16/4 and 24/4, e lec ing an inc ease in he sys em homoge- nei y and a educ ion in bead o ma ion. Howe e , mo ing away om ha op imal egion encou ages he appea ance o beads esul s in a dec ease in he mean ibe size. The p oli e ollows a end o an in e ed second polynomial, eaching he maximum alue a a a io o 20/4 ha coincides wi h he disappea ance o he beads in i s s uc u e and a na owed dis ibu ion o ibe diame e s. O e all, wi h he ansi ion om a semidilu e unen angled o a semidilu e en angled polyme solu ion we obse ed he expec ed shi om poo quali y elec ospun ibe gene a ion, deno ed by bead o - ma ion and a b oad ange o ibe diame e s, o he p oduc ion o consis en , homogeneous ibe s. In e es ingly, as he polyme concen- a ion con inued o inc ease, we obse ed a e u n o poo ibe o - ma ion. This inding is inconsis en wi h cu en heo y which apply o elec ospinning o uni a y polyme solu ions and sugges s o he unde - lying mechanisms a e in ol ed when conside ing a semidilu e en angled bina y polyme sys em. 3.3. In luence o he PCL: gela in a io wi h inc easing gela in concen a ion We wan ed o explo e he impo ance o main aining a speci ic PCL: Gela in a io, i espec i e o he absolu e concen a ions o iscosi y. We s a ed om an ini ial PCL and gela in concen a ion o 32 % and 4 %, Fig. 1. E olu ion o he speci ic iscosi y o uni a y sys ems as a unc ion o he (A) PCL concen a ion and (B) Gela in concen a ion. Fig. 2. (A) E olu ion o he speci ic iscosi y o bina y sys ems PCL:Gela in as a unc ion o he PCL concen a ion. (B) E olu ion o he speci ic iscosi y o bina y sys ems PCL:Gela in as a unc ion o he PCL concen a ion wi h a dashed egion in which he spinning p ocess canno be ca ied ou p ope ly. SEM images o he di e en bina y sys ems ha e also been included (scale ba : 10 µm). V.M. Pe ez-Puyana e al. Nex Ma e ials 6 (2025) 100306 4 espec i ely, o a PCL:Gela in a io o 32/4 in an HFIP sol en ; in ela i e e ms, his has a a io o 8:1. This cons i u ed semidilu e en angled polyme solu ion ha , despi e su icien ly high iscosi y, was al eady shown o ha e poo ibe o ma ion and ex ensi e mic osphe e p oduc ion. To be e unde s and he ansi ion om beaded ibe s o consis en ibe s, we held he PCL concen a ion cons an his highe alue o 32 % w/ and sys ema ically inc eased he quan i y o gela in wi h concen a ion o 4.0, 6.0, 6.4, 8.0 and 10.7 w/ %, he eby achie ing he same ela i e a ios o 8:1, 6:1, 5:1, 4:1, and 3:1 (in e e se o de ); o dis inguish hese solu ions om he p e ious condi ions, he ela i e a ios include an as e isk ‘*’when men ioned and he condi ions a e labelled wi h he absolu e a io o PCL o gela in, speci ically 32/ 10.7, 32/8, 32/6.4, 32/6 and 32/4. In addi ion, a polyme solu ion o 32 % PCL wi h no gela in was also included o assess he impac o adding gela in. SEM images and ibe /bead size dis ibu ions o hese sys ems a e shown in Fig. 4. The sys ems composed o only PCL p esen ed a s uc u e mainly o med by beads. In e es ingly, he addi ion o gela in o a cons an concen a ion o PCL (32 %) ollowed he same end as was p e iously obse ed. The mo phology o elec ospun deposi ion e ol ed om he e ogeneous ibe s wi h bead o ma ion (sys em 32/4) o homoge- neous ibe s o in e media e a ios (32/6, 32/6.4 and 32/8), ollowed again by beads and small ibe s (sys em 32/10.7). Bead o ma ion was la ges o he 32/4 a io, while bo h highes and lowes a ios also displayed a dec ease in he size o he ibe s gene a ed. An analysis o ibe uni o mi y shows he same in e ed second o de polynomial end o his 32 % PCL-based bina y sys em (Fig. 5), whe e he 32/10.7 and 32/4 PCL:Gela in a ios led o less han 40 % uni o mi y ha e lec s he eme gence o sphe oids. Con e sely, uni o mi y and ibe diame e in- c ease o he in e media e a ios (shown in Fig. 5). Fig. 3. SEM images (scale ba : 10 µm) and ibe /bead size dis ibu ions o he memb anes gene a ed wi h mixed PCL:Gela in sys ems wi h di e en a ios: 12 %+4 % (12/4, a io 3:1), 16 %+4 % (16/4, a io 4:1), 20 %+4 % (20/4, a io 5:1), 24 %+4 % (24/4, a io 6:1) and 32 %+4 % (32/4, a io 8:1). Table 2 Mean ibe and beads diame e s o memb anes p ocessed wi h di e en PCL and gela in pe cen ages and PCL:Gela in a ios: 12 %+4 % (12/4), 16 %+4 % (16/ 4), 20 %+4 % (20/4), 24 %+4 % (24/4), 32 %+4 % (32/4), 32 %+6 % (32/6), 32 %+6.4 % (32/6.4), 32 %+8 % (32/8) and 32 %+10.7 % (32/10.7). SYSTEM Nomencla u e Ra io Fibe Size (nm) Beads Size (nm) PCL (12 %) +Gela in (4 %) 12/4 3:1 288 ±86 807 ±223 PCL (16 %) +Gela in (4 %) 16/4 4:1 485 ±170 - PCL (20 %) +Gela in (4 %) 20/4 5:1 415 ±91 - PCL (24 %) +Gela in (4 %) 24/4 6:1 443 ±95 874 ±176 PCL (32 %) +Gela in (4 %) 32/4 8:1 308 ±100 1.160 ±450 PCL (32 %) +Gela in (6 %) 32/6 6:1* 590 ±218 - PCL (32 %) +Gela in (6.4 %) 32/6.4 5:1* 357 ±93 - PCL (32 %) +Gela in (8 %) 32/8 4:1* 724 ±376 - PCL (32 %) +Gela in (10.7 %) 32/10.7 3:1* 290 ±107 1.299 ±328 V.M. Pe ez-Puyana e al. Nex Ma e ials 6 (2025) 100306 5 Fig. 4. SEM images (scale ba : 10 µm) and ibe /bead size dis ibu ions o he memb anes gene a ed wi h mixed PCL:Gela in sys ems wi h di e en a ios: 32 %+4 % (32/4, a io 8:1), 32 %+6 % (32/6, a io 6:1*), 32 %+6.4 % (32/6.4, a io 5:1*), 32 %+8 % (32/8, a io 4:1*) and 32 %+10.7 % (32/10.7, a io 3:1*). Fig. 5. SEM images and ibe /bead size dis ibu ions o he memb anes gene a ed wi h mixed PCL:Gela in sys ems wi h di e en PCL:Gela in a io: (A) 12 %+4 % (12/4), 16 %+4 % (16/4), 20 %+4 % (20/4), 24 %+4 % (24/4), 32 %+4 % (32/4); (B) 32 %+10.7 % (32/10.7), 32 %+8 % (32/8), 32 %+6.4 % (32/6.4), 32 %+ 6 % (32/6) and 32 %+4 % (32/4). V.M. Pe ez-Puyana e al. Nex Ma e ials 6 (2025) 100306 6 Simila o he PCL:Gela in (4 %) sys ems, hese esul s o he PCL (32 %):Gela in sys ems a e pa ly consis en wi h he p oposed expla- na ion ega ding polyme concen a ion and chain en anglemen as i pe ains o he elec ospinning p ocess. As he polyme solu ion ansi- ions om a lowe concen a ion (e.g. 4 % gela in) o highe concen- a ion (wi h gela in concen a ions be ween 6 % and 8 %), he e ogeneous ibe s gi e way o he p oduc ion o homogeneous ibe s wi h no beads. Howe e , om his concen a ion on, homogenei y is los again, and beaded memb anes a e ob ained, wi h he subsequen dec ease in uni o mi y. One possibili y is ha he o e all high iscosi y o he solu ion, when bo h PCL and gela in concen a ions a e high, p e en s a consis en elec ospinning je and, he e o e, inc easing he he e ogenei y o he esul ing memb anes. Al e na i ely, ano he possible eason is a change in miscibili y o he wo polyme s as he ela i e concen a ions inc ease; his has been p e iously epo ed o o he na u al-syn he ic polyme solu ion blends [33,34]. Wi h such a dec ease in polyme miscibili y and subsequen polyme phase sepa a- ion, i would be expec ed ha less in e -species polyme chain in e - ac ion would occu , consequen ly esul ing in poo elec ospinning ou comes, and ha he e would be a consis en inhomogeneous polyme species dis ibu ion wi hin he deposi ed ibe s. 3.4. Physicochemical e alua ion Di ec ly measu ing he beha iou o a bina y polyme solu ion du - ing elec ospinning is a challenge gi en he dynamic na u e o he p o- cess, including he con inual e apo a ion o sol en , subsequen apid changes in polyme concen a ion, and high-speed e olu ion o luidic je du ing p ocessing. To look o e idence o polyme phase sepa a ion in he esul ing ibe , wa e con ac angle (WCA) and EDAX measu e- men s we e used o assess ibe p ope ies as indica o s o inhomoge- neous ibe composi ion. These wo echniques p o ide an indica ion o he su ace composi ion o he ibe s, whe ein we expec ed homoge- neous ibe s o ha e a change in WCA and EDAX eadou ha is di ec ly p opo ional o he changes in polyme concen a ion. In con as , we an icipa ed any inhomogenei y in he ibe su ace composi ion, esul ing om phase sepa a ion and a subsequen unde - o o e - ep esen a ion o one polyme species e sus he o he on he ibe su ace, would p esen as a nonlinea ela ionship wi h espec o polyme composi ion. As can be obse ed in Fig. 6A and 6B, he alue o he con ac angle inc eased when he a io be ween he polyme s was highe , due o he p esence o he hyd ophobic polyme (PCL) in a highe concen a ion compa ed o he concen a ion o gela in, which is he hyd ophilic polyme o he mix u e. Fo ha eason, he con ac angle a ied (almos doubled) compa ing he alues ob ained o he sys ems wi h he lowes (a ound 40º, a io 12/4) and he highes (a ound 75º, a io 32/4) a io be ween PCL and gela in. Mo eo e , an e olu ion owa ds a mo e hy- d ophilic sys em ook place when he concen a ion o gela in was inc eased, swi ching om ca 70º(32/4 sys em) o ca 40º(32/10.7 sys- em). Fu he mo e, simila con ac angle alues we e ob ained o he sys ems p ocessed wi h a simila PCL:Gela in a io. Pe o ming a linea eg ession o he con ac angle measu emen s wi h espec o he PCL:Gela in a ios om 12/4–32/4 and om 32/ 4–32/10.7, i was possible o de e mine a clea linea end (Fig. 5A and 5B) ha esul ed in he ollowing equa ion (Eqs. 3 and 4, espec i ely): WCA =6.68⋅[PCL :Gela in Ra io] + 20.28 (3) WCA =6.91⋅[PCL :Gela in Ra io] + 36.03 (4) This con i ms expec a ions ha an inc ease in he a io be ween PCL and gela in p oduces an inc ease in he con ac angle, since he sys em ob ained is mo e hyd ophobic due o he g ea e p esence o PCL. Fu he , acco ding o i pa ame e s o di e en co ela ions shown in Table 3, a linea end was obse ed, which achie ed an imp o ed i (an inc ease in R 2 alue) when he ou e mos a ios (12/4 and 32/4) we e excluded. Since he o e all dis ibu ion o ma e ial deposi ion is he same o all samples, his imp o ed i can e lec he change in ibe mo phology (beads o ibe s) bu can also indica e an imp o ed homo- genei y in ibe composi ion o in e media e PCL:Gela in a ios. To u he in es iga e he co ela ion be ween he homogenei y o ibe composi ion and ini ial polyme solu ion a io, he ela ion be- ween PCL and gela in was assessed ia EDAX analysis o he elec ospun ibe s, whe e he % amoun o ni ogen pe uni a ea was used as a me ic o p o ein dis ibu ion wi hin he ibe s uc u e (Table S1). The ni ogen p esen in he su ace ob ained om he EDAX p o iles, shown in Fig. 5A and 5B, shows an ob ious co ela ion be ween N con en and he changing a ios be ween he wo polyme s. The esul s shown Fig. 6. (A) Co ela ion be ween he WCA and N con en wi h he PCL:Gela in a io and (B) ela ion be ween he uni o mi y and mean ibe size o he memb anes p ocessed wi h di e en PCL:Gela in a io: 12 %+4 % (12/4), 16 %+4 % (16/4), 20 %+4 % (20/4), 24 %+4 % (24/4), 32 %+4 % (32/4), 32 %+6 % (32/6), 32 %+ 6.4 % (32/6.4), 32 %+8 % (32/8) and 32 %+10.7 % (32/10.7). Table 3 R 2 alues o he di e en co ela ions o Con ac Angle and Ni ogen con en o memb anes p ocessed wi h di e en PCL and gela in pe cen ages and PCL: Gela in a ios: 12 %+4 % (12/4), 16 %+4 % (16/4), 20 %+4 % (20/4), 24 %+ 4 % (24/4), 32 %+4 % (32/4), 32 %+6 % (32/6), 32 %+6.4 % (32/6.4), 32 %+8 % (32/8) and 32 %+10.7 % (32/10.7). CORRELATIONS Sys ems R 2 R 2 (wi hou 12/4 sys em) R 2 (wi hou 12/4 and 32/4 sys ems) Con ac Angle 12/4–32/4 0.9607 0.9768 0.9839 Ni ogen con en 12/4–32/4 0.8908 0.9809 0.9998 Con ac Angle 32/ 10.7–32/4 0.9511 0.9082 0.7992 Ni ogen con en 32/ 10.7–32/4 0.9774 0.9871 0.9886 V.M. Pe ez-Puyana e al. Nex Ma e ials 6 (2025) 100306 7 conclude ha he e is a simila dec easing linea end (Eqs. 5 and 6): Ncon en = − 0.63⋅[PCL :Gela in Ra io] + 5.26 (5) Ncon en = − 0.70⋅[PCL :Gela in Ra io] + 5.94 (6) This con i ms ha inc easing he PCL:Gela in a io esul s in a lowe amoun o p o ein-associa ed ni ogen. Fu he mo e, he analysis o he di e en co ela ions pe o med (Table 3) showed he simila beha iou exhibi ed by he con ac angle measu emen s, whe e an inc ease in he R 2 i ing alue is achie ed when excluding he ou e mos a ios o 12/4 and 32/4. While is i cu en ly no possible o di ec ly con i m he esponse o he wo polyme species as hey a e subjec ed o a apid inc ease in concen a ion and simul aneous mechanical o ces du ing he e apo a ion and whipping phases o he elec ospun polyme je , he obse ed changes in ibe mo phology and bo h he con ac angle and e alua ion o ni ogen dis ibu ion p o ide indi ec suppo wi h he hypo hesis o inhomogenei y in he dis ibu ion o gela in and, hus, phase sepa a ion wi hin he esul ing ibe s [35]. 4. Conclusions Sui able PCL/Gela in nano ibe memb anes can be ob ained using low molecula weigh (LW) PCL. The ypical o ma ion o beads du ing he elec ospinning p ocess using LW PCL can be a oided wi h he addi ion o gela in, which is homogeneously dis ibu ed in he mem- b ane. The g ea e hyd ophilici y, he lowe su ace ension and he inc eased iscosi y o he polyme sys ems leads o imp o e he elec- ospinning p ocess. Howe e , a u he inc ease in gela in p o ein concen a ion leads o an inc ease in iscosi y, which could cause g ea e di icul ies in he elec ospinning p ocess, o could igge phase sepa a ion wi hin he bina y solu ion. Consequen ly, i would be con enien o cha ac e ize he solu ion e olu ion du ing he elec o- spinning p ocess in e ms o iscosi y, su ace ension, and composi ion, al hough his cu en ly p esen s a challenge due o he dynamic na u e o his manu ac u ing p ocess. Di e en co ela ions be ween he PCL:Gela in a io and he di e en p ope ies measu ed (N con en and con ac angle) we e es ablished. Linea co ela ions we e ob ained, al hough wi h opposi e slopes, since he hyd ophobici y o he sys ems inc eases wi h he PCL: Gela in a io whe eas he N con en dec eases as he a io inc eases. On he o he hand, he homogenei y o he sys ems was analyzed calcu- la ing he uni o mi y o he nano ib ous memb anes om he SEM im- aging. A maximum was ound o he a io wi h he lowes mean ibe size ( a io 20/4). In ac , highe mean sizes lead o an inc ease in he he e ogenei y o he sys ems. The p esen wo k demons a es ha no only he o al polyme concen a ion a ec s he ibe o ma ion, bu also he a io be ween he wo polyme s when using a bina y sys em; ini ial indica ions sugges ha phase sepa a ion may occu o some polyme sys ems as hey become mo e concen a ed du ing he elec ospinning p ocess, esul ing in poo en anglemen and ibe uni o mi y. Fo PCL and gela in, he mos sui able a ios we e ound o be 16/4, 20/4 and 24/4. The use o bina y solu ions p esen s a new a enue o p ocess low molecula weigh polyme s ia elec ospinning, whe e low molecula weigh polyme s a e also ypically mo e economic o p oduce. While a ca ea is he use o HFIP as a necessa y uni e sal sol en o bo h species o his bina y sys em, he eby no in line wi h eme ging obliga ions o g een manu ac u ing p ocesses, i p o ides a case s udy o he de elopmen o low cos , low molecula weigh polyme species ha a e compa ible wi h sui able sol en sys ems. In any case, memb anes wi h con ollable mo phology, s uc u e and p ope ies could be achie ed by adjus ing he PCL:Gela in a io. Funding sou ces This wo k is pa o a esea ch p ojec sponso ed by he “Minis e io de Economía y Compe i i idad”(MCI/AEI/FEDER, EU) om he Span- ish Go e nmen (Re . RTI2018-097100-B-C21). The au ho s g a e ully acknowledge hei inancial suppo . The au ho s also acknowledge he Uni e si y o Se ille o he VPPI-US p edoc o al g an and Jun a de Andalucía (Eu opean Social Fund, PAIDI DOCTOR –Con oca o ia 2019–2020, DOC_00586) o he pos doc o al con ac o Vic o M. Pe ez-Puyana. Pa o his wo k was ca ied ou a he Depa men o Complex Tissue Regene a ion (Maas ich Uni e si y, MERLN-Ins i u e o Technology-Inspi ed Regene a i e Medicine) and by inancial sup- po om he p og am “Es ancias b e es en Espa˜ na y en el ex anje o pa a bene icia ios de Becas p edoc o ales o PIF de la US y de Becas de la Fundaci´ on C´ ama a” om he Uni e si y o Se ille. 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