Drug-loaded PCL electrospun nanofibers as anti-pancreatic cancer drug delivery systems

Vol.:(0123456789) Polyme Bulle in h ps://doi.o g/10.1007/s00289-022-04425-6 1 3 ORIGINAL PAPER D ug‑loaded PCL elec ospun nano ibe s asan i‑panc ea ic cance d ug deli e y sys ems OihaneMi xelena‑I iba en1,2 · Ma cRie a‑Pons1,2· SheilaPe ei a3,4· F anciscoJoséCale o‑Cas o3,4· JuanManuelCas illoTuñón5· Ja ie Padillo‑Ruiz3,4,6· Mai eMujika1,2· Se gioA ana1,2 Recei ed: 14 Ma ch 2022 / Re ised: 24 July 2022 / Accep ed: 3 Augus 2022 © The Au ho (s) 2022 Abs ac Cance is one o he main causes o dea h wo ldwide, being panc ea ic cance he second deadlies cance in Wes e n coun ies. Su ge y, chemo he apy and adio he - apy o m he basis o panc ea ic cance ’s cu en ea men . Howe e , hese ech- niques ha e se e al disad an ages, such as su ge y complica ions, chemo he apy sys emic side e ec s and cance ecu ence. D ug deli e y sys ems can educe side e ec s, inc easing he e ec i i y o he ea men by a con olled elease a he a - ge ed umo cells. In his con ex , coaxial elec ospun ibe s can inc ease he con- ol on he elease p o ile o he d ug. The aim o his s udy was o encapsula e and elease di e en an icance d ugs (5-Fluo ou acil and Me ho exa e) om a poly- me ic ibe ma . Di e en lows and a ios we e used o es hei e ec on ibe mo - phology, FTIR spec um, d ug encapsula ion and elease. Good in eg a ion o he an icance d ugs was obse ed and he use o a desicca o o 24h showed o be a key s ep o emo e sol en emanence. Mo eo e , he esul s o his s udy demon- s a ed ha he polyme ic solu ion could be used o encapsula e and elease di e en d ugs o ea cance s. This makes coaxial elec ospinning a p omising al e na i e o deli e complex chemo he apies ha in ol e mo e han one d ug, such as FOL- FIRINOX, used in panc ea ic cance ea men . Keywo ds D ug deli e y· Elec ospinning· An icance · 5- luo ou acil· Me ho exa e Oihane Mi xelena-I iba enand Ma c Rie a-Pons ha e con ibu ed equally o his wo k. * Oihane Mi xelena-I iba en omi x[email p o ec ed] Ex ended au ho in o ma ion a ailable on he las page o he a icle Polyme Bulle in 1 3 In oduc ion Cance is one o he deadlies diseases wo ldwide. One o he wo s p ognoses o his disease is o he panc ea ic cance , as only he 4% o he pa ien s su i e a e 5yea s o he diagnosis da e[1, 2]. I is also he second mos deadly can- ce - ela ed cause in Wes e n coun ies[3]. Su ge y, chemo he apy such as FOL- FIRINOX o Gemci abine, and adio he apy a e he mos common ea men s used o ea his illness [1, 4–6]. Howe e , hey ha e s ong disad an ages such as chemo he apy sys emic side e ec s, limi a ions o echniques and ecu ence due o es s o cance no emo ed du ing he ea men , among o he s [7, 8] These disad an ages and limi a ions can be educed i d ug deli e y sys ems (DDSs) a e used du ing he healing p ocess[9]. The main ad an age o hese ech- nologies is a ocalized and mo e e icien deli e y, which de i es on a educ ion in he side e ec s and umo ecu ence[10]. Polyme s a e he main ehicles used o DDS because o he wide ange o mac omolecules ha can be used. Each o hem has a speci ic biodeg ada ion p o ile[11], which can p o ide a con olled deli e y as he deg ada ion kine ics will desc ibe he d ug elease p o ile[12]. Fo example, polycap olac one (PCL) is a biodeg adable polyme widely used as DDS, e en wi h encapsula ed an icance d ugs [13–16]. DDS can be di ided in o wo g oups, depending on he libe a ion p o ile hey ha e: hey can do i sys ema ically (such as he case o nanopa icles) o locally (as in he case o elec ospun ibe s, ilms o hyd ogels)[17]. Sys emic deli e y sys ems can be injec ed o in oduced o ally[18]. The main ad an ages o hese sys ems a e hei nanome ic size, speci ici y and low in asi eness, as he ehi- cles can each smalle essels and can be enginee ed o ecognize ce ain ecep- o s and no su ge y is equi ed o he ea men [19–21]. On he o he hand, some imes hese pa icles a e ha d o unc ionalize o can lose hei speci ici y due o he lack o molecules ha can iden i y he a ge and he in e ac ions in he deli e y p ocess[22]. Local d ug deli e y sys ems elease he d ug in a speci ic si e, loco- egionally, by di usion o deg ada ion mainly[23]. These sys ems can ake se e al o ms, such as oams, hyd ogels o ibe s among o he s[10, 24, 25]. This echnology has a s ong backg ound, as i has been in he ma ke since 1995 when GLIA- DEL® wa e s we e comme cialized o ea b ain cance s. The main disad an age o hyd ogels o oams is ha he d ug is deli e ed by di usion[10], which is a as occu ing phenomenon ha eleases he d ug in a sho pe iod o ime[26]. E en mo e, he a ge o local DDS is no accessible wi hou su ge y. Tha is why hese de ices a e mean o be complemen a y o con en ional su ge y, in o de o ensu e ha he en i e umo is emo ed. Elec ospinning is a ab ica ion p ocess o ob ain a local DDS. This me hod is easy o use and e sa ile, i gene a es high su ace- o- olume a io meshes, and i usually is a low-cos p ocess[27]. The gene a ed sca olds a e o med by ibe s, which ha e a mixed deli e y p o ile, as bo h di usion and deg ada ion ela ed elease occu [19, 25]. The shape and size o he ibe s play an impo an ole in he d ug eleasing p o ile. The e o e, he e a e se e al pa ame e s ha can be 1 3 Polyme Bulle in al e ed o modi y he size o hose elec ospun ibe s in o de o con ol he d ug elease, such as po en ial di e ence, nozzle–collec o dis ance, polyme ic solu- ion low, polyme concen a ion and conduc i i y[28–31]. One limi a ion o his echnique is ha he ibe has he same p ope ies ac oss i s whole s uc u e and a bu s d ug elease can occu , as he molecule is o ally blended wi h he ibe [16]. This can be sol ed i wo solu ions a e injec ed a he same ime by coaxial elec- ospinning, inc easing he e sa ili y and he con ol on he coaxial ibe p op- e ies, composi ion and d ug loading and elease[32, 33]. B ie ly, coaxial elec- ospinning is a modi ica ion o he con en ional elec ospinning p ocess, whe e mo e han one solu ion is elec ospun [34]. The spinne e s con aining he di e - en solu ions sha e an axis. This allows he injec ion o one solu ion in o he o he a he needle ip: he co e luid is d awn wi hin he ou e one o p oduce con inu- ous illed nano ibe s. Ac ual chemo he apy ea men s use a ious d ugs, such as 5-Fluo ou acil (5F), i ino ecan, oxalipla in and olinic acid a he same ime o ea panc ea ic cance cells[35–37]. In addi ion, including o he d ugs such as painkille s in he ea men could imp o e he pa ien ’s li e quali y. The e o e, es ing PCL o encapsula e d ugs ela ed o gene al cance ea men , such as Me ho exa e (MTX), is a equi ed s ep o keep DDS on he same oad ha chemo he apy is unde going. Fo his eason, in o de o ob ain an al e na i e ea men o panc ea ic cance , PCL was used o encapsula e and elease di e en an ineoplas ic molecules by coaxial elec ospin- ning. PCL-blended nano ibe s loaded ei he wi h 5F o MTX we e in es iga ed in his esea ch. Va ious PCL p opo ions and ab ica ion pa ame e s we e used o p o- duce di e en elec ospun nano ibe s. The e ec o hose speci ica ions in nano ibe cha ac e is ics and d ug elease was s udied in his wo k. Me hods Ma e ials Polycap olac one (PCL, Mw = 100 000 g/mol), 99% chlo o o m, 100% me hanol, 98–100% o mic acid and 99% ace ic acid we e pu chased om Sigma Li e Sci- ences, USA. 5-Fluo ou acil (5F) was pu chased om AK Scien i ic Inc, USA, and Me ho exa e (MTX) was kindly p o ided by D . A. Aldaz, om he Depa men o Pha macy o Clínica Uni e sidad de Na a a, Spain. Elec ospinning o PCL nano ibe s Elec ospinning solu ions we e p epa ed by dissol ing 14 w .% o PCL in o mic acid/ace ic acid/chlo o o m wi h a a io o 47.5: 47.5: 5 ( / / ), espec i ely. The solu ion was s i ed a oom empe a u e o e nigh and used wi hin he i s 72h. A second solu ion was p epa ed o each d ug, 5F and MTX, by adding 28mg/mL o 8.4mg/mL, espec i ely, o he o iginal polyme ic solu ion. Polyme Bulle in 1 3 Once he solu ions we e eady, coaxial elec ospinning was used o elec ospun he nano ibe s (Fig.1a). The se up consis ed o a coaxial nozzle wi h an inne gauge o 26G and an ou e gauge o 18G (Ramé-Ha Ins umen Co, USA; Fig. 1b), wo sy inge pumps (KD Scien i ic Inc, USA) and a high- ol age DC powe sup- ply (Heinzinge , Ge many). A schema ic o he coaxial elec ospinning is shown in Fig.1c Th ee di e en con igu a ions we e used o ab ica e he ibe s. The pa ame e s used o each con igu a ion es ed a e shown in Table 1. These pa ame e s we e based on he s udy o Iqbal e al. (2017). The sca olds ab ica ed only wi h he poly- me ic solu ion we e used as con ol and iden i ied wi h he code CoPP. The d ug- loaded ibe s we e ab ica ed using ei he o he d ug-solu ions in he inne ube and he polyme ic solu ion in he ou e one. The ibe s loaded wi h 5F we e iden i ied as P5F and he ones loaded wi h MTX as PMTX. Cha ac e iza ion o nano ibe s Su ace mo phology To assess he mo phology o he nano ibe s, scanning elec on mic oscopy (SEM, Phenom G2 P o, USA) was used. The diame e o he ibe s was measu ed by using ImageJ so wa e (Na ional Ins i u e o Heal h). A o al o 4 images pe sca old we e analyzed by measu ing he diame e o 25 ibe s pe ame. The e o e, he diam- e e o 100 ibe s was measu ed andomly in each sample and he a e age diame e Fig. 1 a) Coaxial elec ospinning se -up; b) coaxial nozzle o Ramé-Ha Ins umen Co; c) schema ic o he coaxial elec ospinning p ocess Table 1 Elec ospinning ab ica ion pa ame e s o he h ee di e en con igu a ions, wi h and wi hou d ug Sample code Collec o -nozzle dis- ance (cm) Powe supply (kV) Ou e low (mL/h) Inne low (mL/h) CoPP1/P5F1/PMTX1 13.5 21 0.20 0.05 CoPP2/P5F2/PMTX2 13.5 21 0.15 0.05 CoPP3/P5F3/PMTX3 13.5 21 0.10 0.05 1 3 Polyme Bulle in was epo ed. Nano ibe ma s po osi y and po e size we e measu ed also by image analysis, using Diame e J, an ImageJ package. The ob ained alues a e exp essed as mean alue ± s anda d de ia ion. Sol en emanence Fou ie ans o m in a ed spec oscopy (FTIR) was unde aken on pu e PCL pel- le s and he h ee sol en s ( o mic acid, ace ic acid and chlo o o m), as well as on all he elec ospun ma samples by he FTIR Spec um 100 wi h he ATR (Pe ki- nElme , USA). The scanning ange es ed goes om 650 o 4000 cm−1 wi h a o al amoun o 8 accumula ions. In o de o pe o m he FTIR analysis, solid samples we e comp essed whe eas liquid samples we e no . Ma samples we e analyzed jus a e being ab ica ed o a e 24h. In he case o he la e , he elec ospun ma s we e ei he s o ed o e nigh a oom empe a u e o desicca ed in a desicca o o he 24h. D ug loading e iciency Di e en ial Scanning Calo ime y (DSC) Di e en ial scanning calo ime y was used o e alua e he d ug in eg a ion inside he ibe s (DSC, 4000 Pe kinElme ). Samples wi h a mass be ween 5 and 20mg we e used o he analyses. PCL pelle s we e used wi hou any u he modi ica ion, whe eas sca old pieces we e cu and weigh ed in o de o no malize he esul s. The analyzed empe a u e anged om 20 o 100°C, wi h a empe a u e inc ease o 10°C/min. FTIR analysis D ug loading was semiquan i a i ely assessed by FTIR analysis o he d ug-loaded sca olds. A comp essed sample o each d ug-loaded ma was analyzed once he sol- en was ully e apo a ed om he ibe s. The same equipmen , ange, and num- be o accumula ions as in sol en pe du abili y cha ac e iza ion we e used in hese analyses. In i o d ug elease The d ug elease o he di e en ma ypes was de e mined by imme sing he loaded nano ibe s in PBS. The p ocedu e was as ollows: h ee samples om each con igu- a ion o bo h 5F and MTX ma s we e cu in o 2.25 cm2 squa es and subme ged o 2h in MiliQ wa e , in o de o emo e he ma e ials ha could be ou side he ibe s. The ma s we e hen d ied and weigh ed, be o e being in oduced in 15mL o PBS (pH = 7.4) o wo weeks a 37°C on an o bi al shake (Polymax 1040 om Hei- dolph, Ge many). Du ing his pe iod, spec opho ome y was used o cha ac e ize d ug elease: 100µl o he imme sing PBS we e aken o abso bance eading e e y Polyme Bulle in 1 3 24 h. In o de o de e mine he eleased d ug amoun , he espec i e calib a ion cu e was ob ained o each d ug. Bee -Lambe equa ions we e used o calcula e d ug concen a ion a λ = 270nm and λ = 350nm o 5F and MTX espec i ely. The i ed line o calib a ion o 5F in PBS is acco ding o Eq.1 and he amoun o R2 is 0.9411. On he o he hand, MTX calib a ion ollowed Eq.2, wi h a R2 o 0.9164. S a is ical analysis To end up wi h he s a is ical analysis, independen S uden ’s es was pe o med o compa e he e ec o d ugs and di e en elec ospinning con igu a ions on ibe diame e . The s a is ical signi icance c i e ion was he same o all he analyses. S a- is ical di e ence was de e mined as no signi ican (p- alue > 0.05), signi ican (0.01 < p- alue < 0.05), e y signi ican (0.001 < p- alue < 0.01) o ex emely sig- ni ican (p- alue < 0.001). RESULTS ANDDISCUSSION Su ace mo phology SEM was used o obse e he nano ibe s mo phology and diame e o he di e - en con igu a ions es ed wi h and wi hou he d ugs. The images ob ained by SEM (Fig.2) showed ha all con igu a ions p oduced nano ibe s, which had a g ea su - ace o olume a io. No d ug c ys als o o he de o ma ions such as beads we e obse ed in any o he images ob ained, as all polyme ic ma s p esen ed homogene- ous ibe s o di e en diame e s. All ibe s we e in he nano ibe ange as he maxi- mum a e age diame e s we e less han 400 mic ons. The ma s wi h g ea e diam- e e s we e he ones wi hou d ug encapsula ion (Table2), as all h ee samples had hicke ibe s han any o he ma s blended wi h d ug. These di e ences we e p ob- ably caused due o he conduc i i y di e ences be ween PCL and he d ugs[38]. In addi ion, he ibe dis ibu ions o he sca olds we e o e all a Gaussian dis ibu- ion posi i ely skewed. Howe e , he his og ams o he con igu a ions CoPP2 and PMTX1 looked like an in e se amped dis ibu ion a he han a Gaussian. Signi ican di e ences we e obse ed in he ibe diame e when ou e low was dec eased om 0.20mL/h o 0.15mL/h in he case o con ol sca olds, as CoPP1 ma s s CoPP2 ones showed a p- alue = 2.40 × 10–20. In addi ion, educing he ou e low om 0.15 o 0.10 mL/h, signi ican ly educed he ibe diame e , be ween CoPP2 and CoPP3 (p- alue = 8.07 × 10–14). When he polyme ic solu ions we e blended wi h 5F, no signi ican di e ences we e obse ed i he ou e low was educed om 0.20 o 0.15mL/h no om 0.15 o 0.10mL/h. On he o he hand, in oducing 5F in o he ma s signi ican ly educed he ibe diame e wi h espec (1) Y=3.5564x0.0086 (2) Y=6.9286x+3.8287 1 3 Polyme Bulle in o he con ol sca olds: when he homologous samples we e compa ed, ex emely signi ican di e ence was obse ed in he h ee cases wi h p- alues o 6.84 × 10–13, 4.76 × 10–20 and 4.17 × 10–4 o CoPP1 e sus P5F1, CoPP2 e sus P5F2 and CoPP3 e sus P5F3, espec i ely. Simila beha io s we e obse ed in he case o he MTX sca olds, as all he MTX-blended ibe s had ex emely signi ican smalle diam- e e han he pu e polyme ic ones (p- ales < 0.001). Mo eo e , as o he con ol Fig. 2 SEM images wi h hei co esponding his og am o he di e en elec ospun con igu a ions: a CoPP1; b CoPP2; c CoPP3; d PMTX1; e PMTX2; PMTX3; g P5F1; h P5F2; i P5F3 Table 2 Resul s o SEM images analysis on mo phology (U = uni o m, B = beaded), ibe diame e and po osi y, acco ding o con igu a ion and blended d ug Code Mo phology Diame e (µm) Po osi y (%) Con ol 5F MTX Con ol 5F MTX Con ol 5F MTX 1 U U U 0.26 ± 0.11 0.16 ± 0.06 0.20 ± 0.09 68.87 63.18 50.75 2 U U U 0.37 ± 0.18 0.16 ± 0.05 0.17 ± 0.06 56.25 50.27 60.24 3 U U U 0.20 ± 0.08 0.17 ± 0.04 0.15 ± 0.05 75.33 64.10 56.52 Polyme Bulle in 1 3 polyme ic sca olds, a ela ionship be ween he o al low a e and he ibe diame e was obse ed in MTX-loaded sca olds. In his case, educing he ou e low om 0.20 o 0.10mL/h signi ican ly educed he ibe diame e (p- alue = 1.27 × 10–6). This e ec , which was no obse ed only in he case o 5F, could be due o solu ion conduc i i y. MTX is less pola han 5F [39, 40]. The e o e, i was mo e likely o beha e as PCL, ending up in a ibe dec easing e ec o he polyme ic low. Po osi y was also e alua ed, ob aining po osi ies in he ange o 50–75%. The d ug loaded ma s had o e all less po ous sca olds, as wo o he highes po osi- ies ob ained belonged o he con ol g oup (Table2). This oge he wi h he su - ace- o- olume a io imp o ed he d ug exposi ion and enhanced he in e ac ion wi h he en i onmen by an easy luid low h ough he sca old. These esul s oge he wi h he ibe uni o mi y obse ed in his esea ch ma ched wi h he esul s ha Iqbal e al.[16] ob ained in hei s udy. Sol en emanence The non-desicca ed ma s did no show exac ly he same spec um as he pelle , as hey showed wo peaks a 1509 and 1545 cm−1, which co espond o he ca boxyl g oup o he acids p esen in he sol en s (Fig.3a). This mean ha he sol en was no comple ely emo ed du ing he ab ica ion. The e o e, in oducing he sca old in o a body igh a e being elec ospun could cause se e al p oblems, as he sol en s we e no ully e apo a ed. Fig. 3 FTIR spec um o : a) coaxial polyme ic ma s wi hou d ug analyzed igh a e being elec ospun (Co 0h) o le a oom condi ions o 24h be o e pe o ming he FTIR (Co 24h), ace ic acid and o mic acid; b) he PCL pelle and a coaxial polyme ic ma desicca ed o 24h (Co 24h Desicca ed) 1 3 Polyme Bulle in Inc easing he wai ing ime o 24h educed no o iously he sol en p esence, as he dep h o he peak was mo e p onounced in he ma analyzed jus a e he elec- ospinning p ocess han in he ma le 24h unde oom condi ions be o e pe o m- ing he FTIR. Ne e heless, he sol en - ela ed peaks we e s ill no iceable. Howe e , he sol en p esence was comple ely e ased a e he ma was desic- ca ed o 24h, as he cu e o e lapped exac ly he same peaks o he cu e o he pu e polyme pelle (Fig. 3b). This is possible because o he low-p essu e en i- onmen gene a ed in he desicca o . The e o e, i was p o en ha his widely used me hod was comple ely necessa y o ge id o he sol en s used in he elec ospin- ning p ocess[16, 41, 42]. D ug loading The di e en ial scanning calo ime y pe o med on PCL showed he mel ing ansi- ion o he polyme . No majo di e ences we e obse ed be ween a PCL ma agains a pelle (Fig. 4a), as bo h samples unde wen a solid o liquid ansi ion a ound 65 °C. The only obse able di e ence was he mel ing empe a u e and he hea low, as he mel ing empe a u e was g ea e o he pelle han he ma : 66.1°C and 63.7°C, espec i ely. The hea low o he pelle was o e all highe . These alues concu wi h he ones obse ed in he li e a u e[43–45]. On he o he hand, bo h pu e MTX and 5F did no show any ansi ion in he analyzed ange (Fig.4b and c), as he cu es we e comple ely la . The sca olds blended wi h hese molecules showed a peak ha was no p esen in he DSC pe - o med on PCL, a ound 5°C. This peak was mo e no able in he ma s blended wi h MTX. The obse ed mel ing poin o he ma s blended wi h MTX and 5F was be ween 62.9–66.0°C and 62.4–64.1, espec i ely. The obse ed di e ence be ween he con ol and he loaded sca olds could sugges he p esence o he d ug in he ma s. In addi ion o he DSC analyses, o u he con i m he p esence, he in a ed spec um was used o obse e he d ug loading o MTX and 5F in he ibe s (Fig.5), as shown in he li e a u e[12]. Acco ding o hese FTIR esul s, i was concluded ha a highe a io o inne /ou e low in bo h MTX and 5F-loaded sca olds was ela ed o a g ea e d ug concen a ion. As he ou e solu ion did no con ain he an icance d ug, a educ ion o he ou e low ela ed o he o al solu ion low induced a g ea e d ug low, which mean a highe d ug encapsula ion. This end was clea e in he ibe s blended wi h 5F a 1661 cm−1 and be ween 3000 and 3300 cm−1, belonging ha peak o he C = O s e ching and he band o he N–H s e ching, espec i ely. In he case o MTX-loaded sca olds, he wo anges in which his d ug p esence was obse able we e om 1525 o 1617 cm−1 and om 3000 o 3500 cm−1, also due o he C = O s e ching and he N–H s e ching. Conside ing ha no d ug c ys als no de o ma ions on he ibe s we e obse ed in he SEM images, and he d ug p esence obse ed wi h bo h he DSC and FTIR anal- yses, i was depic ed ha he d ug was success ully encapsula ed du ing he coaxial elec ospinning p ocess pe o med in his s udy. Polyme Bulle in 1 3 Au ho s and A ilia ions OihaneMi xelena‑I iba en1,2 · Ma cRie a‑Pons1,2· SheilaPe ei a3,4· F anciscoJoséCale o‑Cas o3,4· JuanManuelCas illoTuñón5· Ja ie Padillo‑Ruiz3,4,6· Mai eMujika1,2· Se gioA ana1,2 1 CEIT-Basque Resea ch andTechnology Alliance (BRTA), Manuel La dizábal 15, 20018Donos ia-SanSebas ián, Spain 2 Uni e sidad de Na a a, Manuel La dizábal 13, 20018TecnunDonos ia-SanSebas ián, Spain 3 Depa men o Gene al Su ge y, Hospi al Uni e si y Vi gen del Rocio, CSIC, IBiS, Uni e si y o Se ille, Se ille, Spain 4 Oncology Su ge y, Cell The apy, andO gan T ansplan a ion G oup, Ins i u e o Biomedicine o Se ille (IBiS), Vi gen del Rocio Uni e si y Hospi al, Uni e si y o Se ille, Se ille, Spain 5 Depa men o Hepa obilia y andPanc ea ic Su ge y andLi e T ansplan a ion, Uni e si y Hospi al o Badajoz, Badajoz, Spain 6 Su ge y Depa men , Uni e si y o Se ille, Se ille, Spain