In e na ional Jou nal o Biological Mac omolecules 227 (2023) 1070–1077
A ailable online 1 Decembe 2022
0141-8130/© 2022 The Au ho s. Published by Else ie B.V. This is an open access a icle unde he CC BY-NC-ND license (h p://c ea i ecommons.o g/licenses/by-
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Magne ically esponsi e chi osan-pec in ilms inco po a ing Fe
3
O
4
nanopa icles wi h enhanced an imic obial ac i i y
I a xe Za andona
a
, Daniela M. Co eia
b
, Joana Mo ei a
c
, Ca los M. Cos a
c
,
d
,
e
,
Senen xu Lance os-Mendez
,
g
,
*
, Ped o Gue e o
a
,
,
h
,
**
, Ko o de la Caba
a
,
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
Cen e o Chemis y, Uni e si y o Minho, 4710-053 B aga, Po ugal
c
Physics Cen e o Minho and Po o Uni e si ies (CF-UM-UP), Uni e si y o Minho, 4710-057 B aga, Po ugal
d
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
e
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
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
g
Ike basque, Basque Founda ion o Science, 48009 Bilbao, Spain
h
P o einma ma e ials SL, A enida de Tolosa 72, 20018 Donos ia-San Sebas i´
an, Spain
ARTICLE INFO
Keywo ds:
Chi osan-pec in ilms
Fe
3
O
4
nanopa icles
An imic obial capaci y
Magne ic p ope ies
ABSTRACT
Chi osan-pec in ilms wi h i on oxide (Fe
3
O
4
) magne ic nanopa icles we e p epa ed by solu ion cas ing in o de
o p oduce biopolyme based magne ically ac i e ma e ials. In a ed (FTIR) spec a indica ed physical in-
e ac ions be ween he ma ix and nanopa icles, co obo a ed by di e en ial scanning calo ime y (DSC) esul s.
In addi ion, he mal cha ac e iza ion sugges ed ha he in e ac ions be ween chi osan, pec in and he nano-
pa icles esul ed in a less compac s uc u e, in luencing he ilm mechanical p ope ies. Rega ding ib a ing-
sample magne ome y (VSM) and elec ical analysis, chi osan-pec in ilms wi h Fe
3
O
4
nanopa icles showed
e imagne ic beha io , wi h an inc ease o he dielec ic cons an as he nanopa icle concen a ion inc eased.
Fu he mo e, ilms displayed enhanced an imic obial ac i i y agains Esche ichia coli (G am-nega i e) and
S aphylococcus epide midis (G am-posi i e) bac e ia. The e o e, chi osan-pec in ilms wi h Fe
3
O
4
magne ic
nanopa icles p o ide p omising esul s o ac i e and in elligen ood packaging applica ions.
1. In oduc ion
Food packaging plays an essen ial ole in he ood supply chain,
gua an eeing ood quali y and sa e y om ex e nal ac o s, such as mi-
c oo ganisms, empe a u e, odo s and ligh exposu e, du ing ood
anspo and s o age, and suppo ing he p e en ion o ood was e [1].
In his ega d, packaging echnologies a e in con inuous e olu ion
seeking o imp o e he quali y and eshness o ood and p olonging i s
shel li e [2]. Addi ionally, wi h he goal o a enua e en i onmen al
issues and o implemen ci cula economy models, sus ainable ma e ials
a e gaining ele ance as po en ial candida es o ood packaging [3].
Pa icula ly, biopolyme s could be a sus ainable al e na i e o pack-
aging ma e ials due o hei biodeg adable cha ac e and non- oxici y
[4–6]. Among he di e en biopolyme s, and due o hei speci ic
p ope ies, chi osan and pec in could be sui able ma e ials o in elligen
packaging ilms, since bo h polysaccha ides a e pH dependen . Unde
low pH condi ions, posi i ely cha ged chi osan and nega i ely cha ged
pec in bind ia ionic in e ac ions o ming a polyelec oly e complex,
esul ing in he enhancemen o he mechanical p ope ies and hyd o-
philici y o e chi osan and pec in ma ices sepa a ely [7,8]. Few wo ks
ha e been epo ed on he use o chi osan-pec in ma ix o ood pack-
aging. Among hem, [9] p epa ed chi osan-pec in ilms wi h an hocy-
anin as a pH indica o de ice o in elligen ood packaging, and [10]
added S ep omyces coelicolo , which imp o ed CO
2
ba ie p ope ies o
he ilm.
Rega ding in elligen packaging, he aim is o moni o s physical,
chemical o biological s a us o ood i ems by de ec o s and senso s,
om he beginning o he ood supply chain un il i eaches he
* Co espondence o: S. Lance os-Mendez, 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.
** Co espondence o: P. Gue e o, 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.
E-mail add esses: [email p o ec ed] (S. Lance os-Mendez), [email p o ec ed] (P. Gue e o).
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.2022.11.286
Recei ed 13 July 2022; Recei ed in e ised o m 21 No embe 2022; Accep ed 28 No embe 2022
In e na ional Jou nal o Biological Mac omolecules 227 (2023) 1070–1077
1071
consume , p o iding in o ma ion abou he ood quali y and in e nal
en i onmen condi ions o he package [11,12]. Thus, in elligen pack-
aging can p o ide in o ma ion o he in si u condi ions o he ood quali y
and eshness, leading o a loss o dependency on ood expi y da es. In
his ega d, nanopa icles o me al oxides ha e a g ea po en ial o
applica ions in ood indus y because o hei an ibac e ial capaci y,
non- oxici y, oxygen and e hylene sca enging capabili y, and he mal
s abili y p ope ies [13]. Indeed, me al oxides show sensing p ope ies
by he mechanism o adso p ion and deso p ion wi h di e en gaseous
compounds on he su ace o he ma e ial, leading o changes in he
elec ical conduc ance [14]. In his ega d, Fe
3
O
4
nanopa icles (NP)
ha e been applied in a a ie y o including biomedicine, cosme ics and
ood p ese a ion, due o hei an imic obial ac i i y, magne ic
esponse, biocompa ibili y and non- oxic p ope ies [15,16]. Fo ood
applica ions, Fe
3
O
4
NPs ha e been used as senso s o de ec ing di e en
compounds, such as hea y me als [17], ca eic acid (Depa men o Food
Science and Technology, Aya ollah Amoli B anch, Islamic Azad Uni-
e si y, Amol 46311-39631, Mazanda an, I an; & Abdi, [18]), o ood-
bo ne spoilage bac e ia [19].
In his con ex , he aim o he p esen wo k was o p epa e chi osan-
pec in ilms wi h Fe
3
O
4
nanopa icles by solu ion cas ing, in o de o
assess he e ec o Fe
3
O
4
NPs concen a ions on he chi osan-pec in
ma ix, physicochemical, he mal, s uc u al, magne ic, elec ic and
an imic obial p ope ies.
2. Ma e ials and me hods
2.1. Ma e ials
Chi osan wi h a molecula weigh o 375 kDa and a deace yla ion
deg ee ≥75 % was supplied by Sigma-Ald ich, Spain. High me hoxy-
la ed pec in, wi h a molecula weigh o 472 kDa and an es e i ica ion
deg ee o 58 %, was kindly supplied by CEAMSA, Spain. I on oxide
(Fe
3
O
4
) powde , wi h pa icle size o 50–100 nm and a pu i ica ion
deg ee o 97 %, was supplied by Nanos uc u ed & Amo phous Ma e-
ials, Inc., USA. Ace ic acid solu ion (1 N), used as sol en , was supplied
by Pan eac, Spain.
2.2. Film p epa a ion
Chi osan-pec in ilms wi h Fe
3
O
4
nanopa icles we e p ocessed by
solu ion cas ing. The polyme s we e dissol ed sepa a ely. On he one
hand, he equi ed amoun o chi osan was dissol ed in 1 w % ace ic
acid solu ion by s i ing o 30 min. On he o he hand, Fe
3
O
4
nano-
pa icles we e dispe sed in a 0.1 w % aqueous solu ion o T i on 100-X
by sonica ion o 3 h. Then, he equi ed amoun o pec in was added o
he NP aqueous solu ion and s i ed a 67 ◦C. Bo h solu ions we e mixed
a 8000 pm o 10 min (Ul a u ax UT25, IKA, Ge many), and ai
bobbles we e emo ed by acuum. The mix u e was placed in a pe i
dish and le o d y a oom empe a u e. Mix u e composi ions inco -
po a ing Fe
3
O
4
NP concen a ions be ween 0.1 and 10 w % a e shown in
Table 1.
2.3. Film cha ac e iza ion
2.3.1. Fou ie ans o m in a ed (FTIR) spec oscopy
An Alpha II spec ome e (B uke , Mad id, Spain), wi h a Pla inum
ATR accesso y, was used o collec FTIR spec a o chi osan-pec in ilms
wi h Fe
3
O
4
nanopa icles. A o al o 32 scans we e pe o med wi h a
esolu ion o 4 cm
−1
in he wa eleng h be ween 4000 and 800 cm
−1
.
2.3.2. The mo-g a ime ic analysis (TGA)
A Me le Toledo TGA/SDTA 851 he mo-balance was used o mea-
su e he he mal s abili y o he samples. Dynamic scans om 25 o
900 ◦C we e ca ied ou a a cons an a e o 10 ◦C/min unde ni ogen
a mosphe e o a oid he mo-oxida i e eac ions.
2.3.3. Di e en ial scanning calo ime y (DSC)
A Me le Toledo DSC 822 was used o pe o m di e en ial scanning
calo ime y. Samples o a ound 3 mg we e hea ed om −50 ◦C o 300 ◦C
a a hea ing a e o 10 ◦C/min unde ni ogen a mosphe e o a oid
oxida i e eac ions.
2.3.4. Scanning elec on mic oscopy (SEM)
Mo phology was examined by using a Hi achi S-4800 scanning
elec on mic oscope a an accele a ing ol age o 15 kV. Be o e analysis,
ac u ed su aces we e coa ed wi h a gold laye by spu e ing wi h a
Pola on SC502 appa a us.
Addi ionally, scanning elec on mic oscopy/ene gy-dispe si e X- ay
spec oscopy (SEM/EDX) was used o analyze he pa icle dis ibu ion
wi hin he samples wi h a Hi achi TM3000 Table op Mic oscope.
2.3.5. Mechanical p ope ies
Mechanical p ope ies we e measu ed wi h an Ins on 5967 elec-
omechanical es ing sys em (Ins on, Spain). Acco ding o ASTM
D638-14 [20], es s we e ca ied ou wi h a load cell o 500 N and a
c osshead a e o 1 mm/min. Films we e cu in o bone shaped samples o
4.75 mm ×22.25 mm. Fi e samples we e measu ed o each sys em.
Tensile s eng h (TS), elonga ion a b eak (EAB) and elas ic modulus (E)
we e e alua ed om he measu emen s.
2.3.6. Vib a ing-sample magne ome y (VSM)
The magne ic p ope ies o he ilms we e analyzed wi h a Mic o-
Sense EZ7 VSM om −1.8 o 1.8 T a oom empe a u e. The hys e esis
loops o he samples we e measu ed and emanence (M
), magne iza ion
sa u a ion (M
s
) and coe ci e ield (H
c
) pa ame e s we e ob ained.
2.3.7. Elec ical cha ac e iza ion
Fo he elec ic measu emen s, ci cula gold elec odes o 5 mm
diame e we e deposi ed by magne on spu e ing wi h a Pola on Coa e
SC502 on o bo h sides o each sample. The elec ical conduc i i y o he
ilms was measu ed h ough a Kei hley 487 picoamme e / ol age sou ce
wi h a ±10 V ol age, and he olume conduc i i y o he ilms (
σ
) was
calcula ed by:
σ
=d
R.A(1)
whe e R is he esis ance o he ilm ob ained om he slope o he I–V
cu es, d is hickness, and A is he elec ode a ea.
Dielec ic measu emen s we e pe o med using a Quad ech 1920
LCR p ecision me e . The capaci ance (C) and he dielec ic losses ( an δ)
we e ob ained a oom empe a u e in he equency ange om 20 Hz o
1 MHz wi h an applied ol age o 0.5 V.
The eal (
ε
´) pa o he dielec ic unc ion was calcula ed as:
ε
′=C⋅d
ε
0⋅A(2)
whe e C is he indi idual sample capaci y,
ε
0
is he pe mi i i y o
Table 1
Composi ion o chi osan-pec in ilms wi h di e en con en s o Fe
3
O
4
nanopa icles.
Sys em
designa ion
Chi osan
concen a ion (w %)
Pec in
concen a ion (w
%)
NP concen a ion
(w %)
Con ol 50.00 50.00 0
0.1NP 49.95 49.95 0.1
0.5NP 49.75 49.75 0.5
1NP 49.50 49.50 1
5NP 47.50 47.50 5
10NP 45.00 45.00 10
I. Za andona e al.
In e na ional Jou nal o Biological Mac omolecules 227 (2023) 1070–1077
1072
acuum (8.85 ×10
−12
F⋅m
−1
), A is he elec ode a ea and d is he ilm
hickness.
The eal pa o he conduc i i y o he dielec ic ma e ial can be
calcula ed om he dielec ic measu emen s as ollows:
σ
′(
ω
) =
ε
0
ωε
′′(
ω
)(3)
whe e
ε
0
is he pe mi i i y o ee space,
ω
=2
π
is he angula e-
quency and
ε
′′(
ω
) =
ε
′ an δ is he equency dependen imagina y pa o
he dielec ic pe mi i i y.
2.3.8. An imic obial analysis
To de e mine he inhibi ion capaci y o ilms, wo ood pa hogens
we e es ed: a G am nega i e E. coli K12 and a G am-posi i e S aphylo-
coccus epide midis NCTC 11,047 pu chased om Ame ican Type Cul u e
Collec ion (LGC S anda ds S.L.U.) The bac e ial p e-inoculum was p e-
pa ed by using a single colony om he co esponding s ock and
esuspended in nu ien b o h (NB). A e incuba ing o e nigh a 37 ◦C
and 200 pm, he p e-inoculum was cen i uged (5000 pm) and he
pelle was esuspended in 0.9 % solu ion o NaCl a pH 6.5. The op ical
densi y, OD, o E. coli and S. epide midis was measu ed a 600 nm and
adjus ed o 0.28 ±0.01 and 0.36 ±0.01 o E. coli and S. epide midis,
espec i ely.
Samples we e cu in ci cula pieces o 10 mm diame e , placed in o
alcons, and s e ilized wi h ul a iole ligh o 30 min on each side.
Then, 2 mL o he inal bac e ial suspension was added and came in o
con ac wi h he ilms o 2 h a 37 ◦C and 200 pm. Falcons wi hou any
ma e ial we e used as con ols o bac e ial g ow h.
The iabili y o bac e ial cells in suspension a e con ac ing he
ma e ial was e alua ed using colony- o ming uni s (CFUs) assay. Ten-
old se ial dilu ions o he bac e ial cul u es o he alcons we e ca -
ied ou in 0.9 % NaCl aqueous solu ion. A olume o 10
μ
L was placed
on sp ead pla es o NB and colony- o ming uni s pe millili e (CFU ⋅
mL
−1
) coun was ca ied ou a e incuba ing he pla es a 37 ◦C o 24 h.
An imic obial ac i i y was de e mined by compa ing iable bac e ia o
each sys em wi h ha incuba ed wi hou any ilm. The bac e ial g ow h
inhibi ion was calcula ed based on he ollowing equa ion:
Bac e ial g ow h inhibi ion (%) = 100 −(CFU sample
CFU con ol)×100% (4)
2.4. S a is ical analysis
Wi h he pu pose o de e mining he signi ican di e ences be ween
measu emen s, analysis o a iance (ANOVA) was ca ied ou by means
o SPSS so wa e (SPSS S a ic ic 25.0). Tukey's mul iple ange es was
used o mul iple compa isons among di e en sys ems wi h a s a is ical
signi icance a he p <0.05 le el.
3. Resul s and discussion
3.1. FTIR analysis and he mal cha ac e iza ion
In o de o e alua e he in e ac ions among he componen s o he
ilms, FTIR analysis was ca ied ou and he spec a a e shown in
Fig. 1a). The cha ac e is ic bands o chi osan and pec in we e obse ed
in con ol ilms: he band a 3247 cm
−1
, associa ed o O
–
H bonds in
bo h polyme s and o N
–
H bonds in chi osan; he bands be ween 2925
and 2850 cm
−1
, a ibu ed o C
–
H s e ching ib a ions; he band a
1742 cm
−1
, associa ed o C
–
–
O in he es e bonds o pec in; he band a
1633 cm
−1
, assigned o he s e ching o C
–
–
O bond in chi osan and o
he asymme ic s e ching o COO
−
in pec in; and he band be ween
1150 and 890 cm
−1
, a ibu ed o he C-O-C o he saccha ide ing o
chi osan and pec in. As shown in a p e ious s udy, he bands ela ed o
C
–
–
O s e ching o chi osan and o he es e bond o pec in showed
displacemen s owa ds lowe wa enumbe s, indica ing ha he in-
e ac ions be ween chi osan and pec in we e physical [21].
Addi ionally, some bands displacemen s we e obse ed when Fe
3
O
4
nanopa icles we e added in o he chi osan-pec in sys em. In pa icula ,
4000 3500 3000 2500 2000 1500 1000
a)
10 NP
5 NP
1 NP
0.5 NP
0.1 NP
.u.a/ecna imsna T
Wa enumbe / cm
-1
Con ol
100 200 300 400 500 600 700 800
-0.008
-0.006
-0.004
-0.002
0.000
b)
Cº%
/GTD -1
Tempe a u e / ºC
Con ol
Chi osan
Pec in
100 200 300 400 500 600 700 800
-0.005
-0.004
-0.003
-0.002
-0.001
0.000 c)
Cº%GTD
-1
Tempe a u e / ºC
Con ol
0.1NP
0.5NP
1NP
5NP
10NP
0 50 100 150 200 250 300
g
.
W/wolF aeH
-1
Tempe a u e / ºC
10NP
5NP
1NP
0.5NP
0.1NP
Con ol
odnE
d)
Fig. 1. a) FTIR spec a o chi osan-pec in ilms, b) DTG o nea chi osan, nea pec in and con ol ilm, c) DTG o chi osan-pec in ilms and d) DSC o chi osan-
pec in ilms.
I. Za andona e al.
In e na ional Jou nal o Biological Mac omolecules 227 (2023) 1070–1077
1073
O
–
H and N
–
H ib a ion bands shi ed o highe wa enumbe s, om
3247 cm
−1
o con ol ilms up o 3263 cm
−1
o 10NP, as he nano-
pa icle concen a ion inc eased. Mo eo e , he band associa ed o C
–
H
s e ching ib a ions a 2853 cm
−1
shi ed o highe wa enumbe s,
becoming a shoulde o he band a 2923 cm
−1
. All hese band dis-
placemen s indica ed ha Fe
3
O
4
nanopa icles in e ac ed physically
wi h chi osan-pec in ma ix.
TGA and DSC analyses we e ca ied ou in o de o de e mine he
he mal s abili y o he ma e ial. Conce ning TGA, de i a i e he mo-
g a ime ic cu es a e p esen ed in Fig. 1b and c. Rega ding nea chi-
osan and pec in samples (Fig. 1b), he mal deg ada ion was obse ed a
300 ◦C and 240 ◦C, espec i ely. Fo con ol ilms, 4 in lec ion poin s
we e p esen ed. The i s one was obse ed a ound 68 ◦C, ela ed o he
wa e e apo a ion due o mois u e. The second in lec ion poin , a ound
230 ◦C, was he g ea es one and i was ela ed o he he mal deg a-
da ion o chi osan and pec in polyme s. I should be no ed ha he
he mal deg ada ion o chi osan-pec in ilm happened a lowe em-
pe a u e han ha o pu e pec in and pu e chi osan (Fig. 1b). This e en
could indica e ha he ionic bonding be ween chi osan and pec in led o
s uc u e changes in he ma e ial [9]. The hi d in lec ion peak was a
shoulde a 283 ◦C, ela ed o he chi osan ha was no bonded o pec in.
Finally, a sligh in lec ion poin was obse ed a 447 ◦C, ela ed o he
decomposi ion o by-p oduc s. The addi ion o Fe
3
O
4
nanopa icles
(Fig. 1c) caused he p esence o a new in lec ion peak a ound 680 ◦C,
which became mo e in ense as he concen a ion o NP inc eased and
can be a ibu ed o he ansi ion om Fe
3
O
4
o FeO [22].
The endo he mic peaks o he DSC he mog am o chi osan-pec in
sys ems wi h Fe
3
O
4
NP a e shown in Fig. 1d. Two endo he mic peaks
we e obse ed o con ol ilms: he i s , a 94 ◦C, was a ibu ed o he
ilm mois u e; he second, a 211 ◦C, was ela ed o he en apped wa e
linked by hyd ogen bonding wi h he pola g oups o he biopolyme s
[23]. Rega ding he ilms wi h nanopa icles, he same endo he mic
peaks we e obse ed as o he con ol sample, al hough o he samples
wi h highe concen a ion o nanopa icles, 5NP and 10NP, he peak a
94 ◦C o he con ol sample was shi ed o lowe empe a u es, 82 ◦C
and 76 ◦C, espec i ely. This displacemen indica ed ha he e we e
in e ac ions be ween he ma ix and he NP, as obse ed by FTIR. In
addi ion, he shi o lowe empe a u es would indica e ha he
Fig. 2. SEM images o chi osan-pec in ilm c oss-sec ion: a) con ol, b) 0.1NP, c) 0.5NP, d) 1NP, e) 5NP, and ) 10NP. Yellow dashed ci cles indica e nanopa icle
agg ega ions.
I. Za andona e al.
In e na ional Jou nal o Biological Mac omolecules 227 (2023) 1070–1077
1074
s uc u e o med was less compac and, he e o e, would equi e less
ene gy o elease he mois u e.
3.2. S uc u e and mechanical p ope ies
The mo phology o he ilms was analyzed by SEM and he c oss-
sec ion images o he samples a e shown in Fig. 2. Con ol ilms p e-
sen ed a homogeneous s uc u e, indica ing he compa ibili y be ween
chi osan and pec in. When Fe
3
O
4
nanopa icles we e added a low
concen a ions, he s uc u e o he ilms emained homogeneous bu , as
NP concen a ion inc eased, especially o 5NP and 10NP samples,
nanopa icle agg ega ions we e obse ed. In pa icula , he nanopa icle
clus e ing in 10NP ilms was bigge han in 5NP ilms. These esul s
explain he empe a u e dec ease obse ed by DSC analysis, since he
nanopa icle agg ega ions led o a less compac polyme s uc u e.
The dispe sion o i on on 1NP, 5NP and 10NP ilms su ace was
analyzed by SEM/EDX. As can be obse ed in Fig. 3, he Fe signal in blue
showed ha he nanopa icles we e homogeneously dispe sed h ough
he su ace o he ilms, hough la ge NP agg ega es a e shown o he
samples wi h la ge ille con en s.
Tensile s eng h (TS), elonga ion a b eak (EAB), and elas ic modulus
(E) o he ilms, shown in Table 2, we e measu ed in o de o assess he
in luence o he magne ic nanopa icles on he chi osan-pec in ma ix.
Rega ding o con ol ilms, high alues o TS we e ob ained, due o he
s ong in e molecula bonds be ween chi osan and pec in, which
de i ed in o a compac s uc u e. These alues a e highe han hose
Fig. 3. SEM/EDX images o he su ace o chi osan-pec in ilms wi h 1 % Fe
3
O
4
(1NP), 5 % Fe
3
O
4
(5NP) and 10 % Fe
3
O
4
(10NP).
Table 2
Tensile s eng h (TS), elonga ion a b eak (EAB) and elas ic modulus (E) o
chi osan-pec in ilms wi hou Fe
3
O
4
nanopa icles (Con ol) and wi h di e en
con en s o Fe
3
O
4
nanopa icles.
Films TS (MPa) EAB (%) E (MPa)
Con ol 47.0 ±1.1
a
6.0 ±0.5
a
2395 ±52
a
0.1NP 43.7 ±1.2
b
5.9 ±0.2
a
2397 ±18
a
0.5NP 43.9 ±1.2
b
6.0 ±0.6
a
2443 ±27
a
1NP 44.0 ±0.5
b
6.3 ±0.8
a
2417 ±40
a
5NP 40.7 ±0.8
c
7.0 ±0.5
a
2076 ±58
b
10NP 40.1 ±1.0
c
7.0 ±0.7
a
2053 ±46
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 acco ding o he Tukey's mul iple ange es .
-16000 -8000 0 8000 16000
-200
-100
0
100
200
g.ume/noi azi engaM -1
Applied ield / Oe
0.1NP
0.5NP
1NP
5NP
10NP
a)
0246810
0
50
100
150
200 M
M
s
H
c
NP con en / %
g.ume/noi azi engaM -1
30
35
40
45
50
55
60
Coe ci e ield / Oe
b)
Fig. 4. a) Hys e esis loops and b) magne ic p ope ies (M , emanence; magne iza ion sa u a ion, Ms.; and Hc, coe ci e ield) o chi osan-pec in ilms wi h di e en
con en s o Fe
3
O
4
nanopa icles.
I. Za andona e al.
In e na ional Jou nal o Biological Mac omolecules 227 (2023) 1070–1077
1075
ound by o he au ho s o chi osan-pec in ilms [24]. When he nano-
pa icles we e added, no signi ican di e ences (p >0.05) we e
obse ed in EAB esul s. Howe e , TS alues dec eased as he nano-
pa icles concen a ion inc eased, as well as E alues o 5NP and 10NP,
indica ing ha he NP ac s as de ec i e si es o mechanical p ope ies,
in pa icula o concen a ions >5 %. These esul s indica e ha Fe
3
O
4
nanopa icles a ec ed he s uc u e o he polyme ic ma ix, as obse ed
in SEM images, hinde ing he in e ac ions be ween chi osan and pec in
chains [25].
3.3. Magne ic p ope ies
Rega ding magne ic p ope ies, VSM analysis was ca ied ou o
ob ain he hys e esis loops and calcula e he emanence, he magne i-
za ion sa u a ion, and he coe ci e ield (Fig. 4). The sys ems exhibi ed a
e imagne ic beha io , since he alues o emanence and coe ci e ield
we e unequal o ze o [26]. As obse ed in Fig. 4b, he magne ic beha io
o he sys ems was dependen o he nanopa icle concen a ion. As he
nanopa icle concen a ion inc eased, M
alues inc eased, om 0.23
emu/g (0.1NP) o 19.70 emu/g (10NP), as well as M
s
alues om 2.38
emu/g (0.1NP) o 206.11 emu/g (10NP). Howe e , coe ci e ield
inc eased as nanopa icle concen a ion inc eased up o 0.5 % (50 Oe)
bu s a ed o dec ease un il 41 Oe (10NP) as nanopa icle concen a ion
inc eased. The coe ci e ield is dependen on he nanopa icle size: i he
size inc eases, he coe ci i y inc eases un il a c i ical pa icle size is
eached, a e which he coe ci i y dec eases. Ne e heless, since he
size o he nanopa icles in his s udy a e he same o all samples, esul s
may be ela ed o he agglome a ion o he nanopa icles, as desc ibed in
p e ious wo ks [27].
Fig. 5. a) Cu en - ol age (I-V) cu es and b) elec ical conduc i i y alue o ilms as a unc ion o Fe
3
O
4
con en in he 1s egime (black) and in he 2nd
egime ( ed).
Fig. 6. a) Real pa o dielec ic cons an (
ε
´), b) dielec ic losses ( an δ), and c) a.c. conduc i i y (
σ
´) o chi osan-pec in ilms wi h di e en con en s o Fe
3
O
4
nanopa icles.
I. Za andona e al.
In e na ional Jou nal o Biological Mac omolecules 227 (2023) 1070–1077
1076
3.4. Elec ic cha ac e iza ion
Elec ical conduc i i y o he ilms wi h Fe
3
O
4
nanopa icles was
e alua ed by pe o ming I-V cu es (Fig. 5a). I can be obse ed ha he
I-V cu es depend on he Fe
3
O
4
con en and does no p esen he
s aigh -line dependence cha ac e is ics o Ohm's law. Ins ead, wo e-
gimes a e obse ed a low (−10 V o −3 V) and high applied ol ages (2
V o 10 V). These egimes a e ela ed o he wa e linked by hyd ogen
bonding o he pola g oups and he mo emen o ee ions by he
applica ion o he elec ic ield. As can be obse ed in Fig. 5b, he
elec ical conduc i i y inc eases s ongly as a unc ion o Fe
3
O
4
con en
o bo h egimes. The elec ical conduc i i y o con ol ilms and 10NP
ilms was 3.2 ×10
−10
S⋅cm
−1
and 9.6 ×10
−10
S⋅cm
−1
, espec i ely in
he 2nd egime. This beha io is due o he ac ha he addi ion o
Fe
3
O
4
nanopa icles inc eases he cha ge ca ie s and he conduc ion is
assigned o he elec on hopping.
Addi ionally, he dielec ic analysis was e alua ed and esul s a e
shown in Fig. 6. The dielec ic cons an (Fig. 6a) and an δ (Fig. 6b)
depend on he equency due o dipole elaxa ion. Rega dless o e-
quency ange, he dielec ic cons an and an δ inc eases as a unc ion o
NP concen a ion due o pola iza ion con ibu ions de i ed om he
addi ion o nanopa icles, mainly domina ed by in e acial and spa ial
cha ge pola iza ion [28]. Conce ning a.c. conduc i i y (Fig. 6c), alues
inc eased wi h equency, indica ing he local con ibu ion o he elec-
ical conduc i i y. Rega dless o equency ange, he addi ion o Fe
3
O
4
nanopa icles inc eased he a.c. conduc i i y due o he cha ge ca ie
hopping [29].
3.5. An imic obial capaci y
All ilms showed an imic obial ac i i y agains E. coli and S. epide mis
as can be seen in he ep esen a i e pho og aphs p esen ed in Fig. 7a,
al hough di e en esponse was obse ed o bo h bac e ia (Fig. 7b). In
he case o S. epide midis, 10 NP ilms eached 43 % o inhibi ion, while
98 % was eached o E. coli. The an imic obial capaci y o he ilms was
d i en by bo h chi osan and magne ic nanopa icles. The an imic obial
mechanism o chi osan was caused by he posi i e cha ge o he amino
g oup o chi osan, which in e ac ed wi h he nega i e cha ges o cell
memb anes, a ec ing he loss o p o ein and o he in acellula com-
ponen s [30]. On he o he hand, he an imic obial e ec o i on
nanopa icles was ela ed o he capaci y o he nanopa icles o in e ac
wi h he cell memb ane and o pene a e inside he cells, causing
memb ane damage and inac i a ion o he bac e ia [31]. The e o e,
chi osan-pec in ilms wi h Fe
3
O
4
nanopa icles showed inc eased an i-
bac e ial ac i i y due o he coope a i e ac ion o chi osan and Fe
3
O
4
nanopa icles.
I is wo h no ing ha Fe
3
O
4
nanopa icles imp o ed an imic obial
capaci y when compa ed o con ol ilms. Addi ionally, i should be
no ed he di e ence in he inhibi o y capaci y o he ilms wi h espec
o he wo bac e ia unde s udy. This di e ence migh be due o he
di e en cell walls o G am-posi i e and G am-nega i e bac e ia. The
cell wall o G am-posi i e bac e ia consis s o an ou e hick laye o
pep idoglycan ha ac s as a laye o esis ance agains mos inhibi o y
molecules, whe eas he cell wall o G am-nega i e bac e ia has a hinne
pep idoglycan laye [32].
4. Conclusions
The p epa a ion o chi osan-pec in ilms wi h Fe
3
O
4
magne ic
nanopa icles by solu ion cas ing led o homogeneous ilms. FTIR esul s
indica ed ha he physical in e ac ions be ween chi osan and pec in
we e no a ec ed by he addi ion o he nanopa icles. Howe e , he
mechanical p ope ies we e in luenced by Fe
3
O
4
nanopa icles, due o
he agg ega ion o he nanopa icles, as shown by SEM. The inco po-
a ion o nanopa icles also in luenced he elec ical, magne ic and
an imic obial p ope ies. As he nanopa icle concen a ion inc eased,
he dielec ic cons an , he emanence and he magne iza ion sa u a ion
inc eased. Fo he an imic obial p ope ies, he addi ion o he nano-
pa icles inc eased he an imic obial capaci y o he ilms o pa hogenic
Esche ichia coli (G am-nega i e) and S aphylococcus epide midis (G am-
posi i e) bac e ia. The e o e, Fe
3
O
4
nanopa icles inco po a ed in o
chi osan-pec in ilms o ming solu ions could ex end ood shel -li e,
besides unc ioning as a po en ial senso o ood packaging due o
hei elec ic and magne ic p ope ies.
CRediT au ho ship con ibu ion s a emen
I a xe Za andona: Da a cu a ion, Fo mal analysis, In es iga ion,
W i ing – o iginal d a . Daniela M. Co eia: Da a cu a ion, Fo mal
analysis, In es iga ion, Me hodology, W i ing – o iginal d a . Joana
Mo ei a: Da a cu a ion, Fo mal analysis, In es iga ion, Me hodology,
W i ing – o iginal d a . Ca los M. Cos a: Concep ualiza ion, In es i-
ga ion, Me hodology, Resou ces, Funding acquisi ion, Supe ision,
Valida ion, W i ing – e iew & edi ing. Senen xu Lance os-Mendez:
Concep ualiza ion, In es iga ion, Resou ces, Funding acquisi ion, Su-
pe ision, Valida ion, W i ing – e iew & edi ing. Ped o Gue e o:
Concep ualiza ion, In es iga ion, Me hodology, Supe ision, Valida-
ion, W i ing – e iew & edi ing. Ko o de la Caba: Concep ualiza ion,
In es iga ion, Resou ces, Funding acquisi ion, Supe ision, Valida ion,
W i ing – e iew & edi ing.
Fig. 7. a) Pho og aphs o he an imic obial ac i i y agains E. coli and S. epide mis o chi osan-pec in ilms wi h 10 w % o Fe
3
O
4
nanopa icles, and b) inhibi ion
capaci y o chi osan-pec in ilms, wi hou Fe
3
O
4
nanopa icles (Con ol) and wi h di e en con en s o Fe
3
O
4
nanopa icles, agains E. coli and S. epide midis s ains in
solu ion a e 2 h in con ac wi h ma e ial. The esul s a e he a e age o 3 independen assays.
I. Za andona e al.
In e na ional Jou nal o Biological Mac omolecules 227 (2023) 1070–1077
1077
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 .
Acknowledgmen s
G an PID2021-124294OB-C22 unded by MCI/AEI10.13039/
501100011033 and by “ERDF A way o making Eu ope”. Bioma g oup
hanks he Basque Go e nmen o unding (IT1658-22) and I.Z. hanks
he Basque Go e nmen o he ellowship (22-2018-00078). This wo k
was also suppo ed by he Po uguese 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, and In es iga o
FCT Con ac s 2020.02915.CEECIND (D.M.C) and 2020.04028.CEE-
CIND (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). Thanks a e also due o he Ad anced
Resea ch Facili ies (SGIke ) om he UPV/EHU.
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