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
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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
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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.
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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
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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
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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
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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
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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)
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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