Cy o oxici y and In lamma o y E ec s o Chi in Nano ib ils Isola ed
om Fungi
Ai o La anaga, Ca los Bello-Al a ez, and E lan z Lizundia*
Ci e This: Biomac omolecules 2023, 24, 5737−5748
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ABSTRACT: Fungal nanochi in can assis he ansi ion om he linea ossil-based economy o a ci cula biobased economy gi en
i s en i onmen al bene i s o e con en ional c us acean-nanochi in. I s eal-wo ld implemen a ion equi es ca e ully assessing i s
oxici y so ha unwan ed human heal h and en i onmen al issues a e a oided. Acco dingly, he cy o oxici y and in lamma o y e ec s
o chi in nano ib ils (ChNFs) om whi e mush oom is assessed. ChNFs a e ew nanome e s in diame e , wi h a 75.8% N-ace yla ion
deg ee, a c ys allini y o 59.1%, and p esen a 44:56 chi in/glucan weigh a io. S udies a e conduc ed o aqueous colloidal ChNF
dispe sions (0−5 mg·mL−1) and ee-s anding ilms ha ing physically en angled ChNFs. Aqueous dispe sions o chi in nanoc ys als
(ChNCs) isola ed ia hyd ochlo ic acid hyd olysis o α-chi in powde a e also e alua ed o compa ison. Cy o oxici y s udies
conduc ed in human ib oblas s (MRC-5 cells) and mu ine b ain mic oglia (BV-2 cells) e eal a compa a i ely sa e beha io o e
ela ed biobased nanoma e ials. Howe e , a s ong in lamma o y esponse was obse ed when BV-2 cells we e cul u ed in he
p esence o colloidal ChNFs. These no el cy o oxici y and in lamma o y s udies shed ligh on he po en ial o ungal ChNFs o
biomedical applica ions.
■INTRODUCTION
The use o ossil- uel based ma e ials is d i ing ou socie y
owa d an unp eceden ed clima e c isis wi h no able en i on-
men al issues ela ed o aw ma e ial sca ci y,
1
la ge global
oo p in ,
2
declining ossil esou ce a ailabili y,
1
and uncon-
olled accumula ion o plas ic was e in e es ial, i e , o
ma ine ecosys ems.
3
T ansi ioning owa d a biobased economy
could pa ially add ess hese global challenges gi en he
inhe en enewabili y and biodeg adabili y o ma e ials om
biological o igin.
4
In his con ex , a g ea deal o a en ion is
being paid o he exploi a ion o na u al biopolyme s ha a e
mainly composed o a ew building blocks con aining ca bon
and o igina ing om he cells o li ing o ganisms such as plan s
o mic oo ganisms. Gene ally, na u al biopolyme s ul ill he
equisi es o low cos , wide and local a ailabili y, p ocessabili y,
he mal and mechanical pe o mance, and ease o chemical
modi ica ion.
5,6
De eloping (nano) ib illa ed biopolyme s
opens new ho izons owa d new ans o ma i e applica ions
wi h mul iple unc ions, whe e he mechanical, op ical,
he mal, and ionic p ope ies a e abo e he p ope ies shown
by he pa en ma e ial.
7
Di e se biopolyme ibe s can be
sepa a ed in o ib ils o dec easing diame e ( anging om a
ew mic ons o a ew nanome e s) ha a e ul ima ely
composed o o de ed linea molecula chains. Among hose,
cellulosic biocolloids (colloidal en i ies composed o cellulose
and i s de i a i es), in he o m o cellulose nanoc ys als
(CNCs) o cellulose nano ib ils (CNFs), show a dominan
posi ion a bo h undamen al and applied esea ch, wi h ease o
chemical modi ica ion
8
and high echnology eadiness le els
(TRLs).
7,9,10
Cellulose is decomposed in o non oxic glucosidic
chains, enabling i s use in many en i onmen al and biomedical
applica ions.
11
Recei ed: July 19, 2023
Re ised: No embe 4, 2023
Accep ed: No embe 6, 2023
Published: No embe 21, 2023
A iclepubs.acs.o g/Biomac
© 2023 The Au ho s. Published by
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Howe e , he e a e o he po en ial biopolyme s open o
explo a ion gi en he as lib a y o biobased ma e ials ha
na u e o e s. Wi h a simila s uc u e o cellulose, chi in has
also been decons uc ed o ob ain chi in nanoc ys als
(ChNCs) and chi in nano ib ils (ChNFs).
12
Chi in con ains
∼6 w % ni ogen om ace amide g oups and i is ound in
many li ing o ganisms; i.e., as a majo s uc u al componen in
he exoskele ons (shells) o a h opods, insec s, o ungi.
13
Colloidal chi in ex ac ed om c us aceans has shown
impo an applica ions in pho onic de ices,
14
ene gy s o age,
15
o ba ie applica ions in ilms.
16
Howe e , he isola ion o
hese nanopa icles om c us aceans, whe e i appea s oge he
wi h CaCO3, p o eins and mine als, equi es ha sh chemical
and/o mechanical ea men s, o en in ol ing s ong acid
hyd olysis and/o chemical oxida ion s eps o demine aliza-
ion, dep o einiza ion, bleaching, o ib illa ion.
13
These
p ocesses inc ease he en i onmen al impac s o esul ing
biocolloids, jeopa dizing hei implemen a ion as sus ainable
ma e ials. On he con a y, he chi in in ungal esou ces does
no coexis wi h CaCO3. As a esul , colloidal chi in can be
easily isola ed om ungi unde mild condi ions. As a esul ,
ungal ChNF isola ion shows a lowe global wa ming po en ial
(18.5 kg CO2-equi ·kg−1) when compa ed wi h he 543.5 o
906.8 kg CO2-equi ·kg−1gene a ed upon con en ional chi in
nanoc ys al ex ac ion om c ab o sh imp shells, espec-
i ely.
17
These me i s posi ion ungal ChNFs a he o e on o
esea ch owa d en i onmen ally sus ainable ma e ials. In a
pionee ing wo k, Nawawi e al. epo ed in 2019 a e y simple
p ocedu e o isola e chi in nano ibe s om he whi e
mush oom (Aga icus bispo us), whe e solely a sho mechanical
agi a ion in a ki chen blende and a mild alkaline ea men
we e needed.
18
This ma e ial was hen p ocessed in he o m o
nanopape s wi h ensile s eng hs abo e 200 MPa
18
and has
been used o he ul a il a ion o o ganic sol en s and wa e
19
o as ba e y elec oly es in he o m o gels.
20
Howe e , he
sa e y o hese ma e ials emains a ques ion o be answe ed o
pa e hei way in o eal-wo ld applica ions and ensu e ha
hese ma e ials a e no haza dous o bo h human heal h and
he en i onmen .
20
In his sense, al hough CNCs and CNFs,
cellulose analogues o ChNFs, ha e, in gene al, demons a ed
negligible- o-low (cy o) oxici y,
21,22
pulmona y in lamma ion
o CNFs has been seen, while chemical modi ica ion impai s
low cy o oxici y o CNFs.
23−25
The size and mo phology,
c ys allini y deg ee, o su ace chemis y a e key aspec s in
de e mining he nano oxici y o nanocelluloses. Fo example,
CNCs and CNFs show a leng h-dependen mechanisms o
oxici y on li e cells, whe e sho nanopa icles igge ed
signi ican cy o oxici y in Kup e cells.
26
In his con ex , in i o oxici y s udies o ChNFs a e o
pa icula ele ance as nanoma e ials in gene al ha e an
inc eased abili y o mig a e o a ious o gans and issues and
c oss physiological ba ie s.
27
This would help o o esee any
po en ial oxic e ec o chi in biocolloids om ungi induced
by inhala ion, de mal exposu e, o o he ou es o admin-
is a ion. In his con ex , human ib oblas s a e one o he
p e e ed cells o s udy he cy o oxici y aspec s ela ed o
biobased colloids since hey a e well accep ed by he ISO/EN
10993 p ocedu es o he biological e alua ion o biomedical
de ices.
28
In addi ion, mic oglia a e a ele an ype o cells
in ol ed in he egula ion o neu oin lamma o y esponses and
immune su eillance.
29
These cells ha e p o en use ul o
de e mine he nano oxici y o nanoma e ials such as sil e ,
30
o
i anium dioxide.
31
The e o e, s udies on mic oglia could help
o exclude undesi ed p o-in lamma o y e ec s induced by
ChNFs.
Acco dingly, he e we isola ed ChNFs om whi e mush-
ooms o alida e hei non oxici y and open he use o ungal
biocolloids in o eal-wo ld applica ions. ChNFs ha e a
c ys allini y deg ee o 59.1% and a e composed o chi in and
amo phous glucans (44 w % chi in), and he chi in ac ion
has a N-ace yla ion deg ee o 75.8%. S udies a e conduc ed on
colloidally s able ChNF dispe sion and ee-s anding nano-
pape ilms using human ib oblas s (MRC-5 cells) and mu ine
b ain mic oglia (BV-2 cells). No signi ican di e ences on he
me abolic ac i i y o MRC-5 and BV-2 cells we e obse ed,
e en a la ge exposu e doses, indica ing ChNFs om ungal
esou ces a e compa a i ely sa e han CNCs o CNFs. Gi en
he lack o oxici y o hese ma e ials and hei inhe en
p ope ies, we en isage he implemen a ion o chi in nano-
ib ils no only in wa e emedia ion o ene gy s o age, bu also
in biomedical applica ions.
■EXPERIMENTAL SECTION
2.1. Ma e ials. Whi e mush ooms (Aga icus bispo us) pu chased
om a local s o e in Bilbao (Spain) we e used o chi in nano ib il
isola ion. Chi in om sh imp shells (p ac ical g ade powde ) was
pu chased om Sigma-Ald ich. Sodium hyd oxide pelle s (NaOH,
≥97%) and hyd ochlo ic acid (HCl, 37%) we e ob ained om
Honeywell Fluka. Human lung ib oblas s (MRC-5, CCL-171) we e
acqui ed om ATCC (U.S.A.), whe eas mu ine mic oglia (BV-2)
we e acqui ed om AcceGen Bio ech (U.S.A.). Dulbecco’s modi ied
Eagle medium (DMEM), Hanks’ balanced sal solu ion (HBSS),
penicillin−s ep omycin (P/S), e al bo ine se um (FBS), Alama Blue
cell iabili y eagen , and hodamine-phalloidin we e supplied by
Fishe Scien i ic (Spain). Fluo oshield wi h DAPI, T i on X-100,
Tween 20, and lipopolysaccha ides (LPS) we e supplied by Sigma-
Ald ich (Spain), and 16% o maldehyde solu ion and he G iess
Reagen we e supplied by The moFishe Scien i ic (Spain). Mouse
TNF-alpha DuoSe Elisa was pu chased om Bio echne (U.K.).
2.2. Chi in Nano ib il (ChNF) and Chi in Nanoc ys al (ChNC)
Isola ion and Film Fab ica ion. Colloidal chi in nano ib ils
(ChNFs) we e isola ed om whi e mush ooms ollowing a op-
down app oach.
18
Mush ooms we e ozen a −10 °C o 1 week jus
a e pu chase. Fo biocolloid isola ion, 500 g o ozen whi e
mush ooms we e imme sed in 1 L o dis illed wa e du ing 5 min and
we e manually washed o emo e any di . Then, he mush ooms we e
blended o 5 min (Powe Black Ti anium 1800) and he ob ained
slu y was hea ed a 85 °C o 30 min in a h ee-neck ound-bo om
lask. The mix u e was hen washed by il a ion, and he cake was
eco e ed and edispe sed again in dis illed wa e o an addi ional
ea men in 1 M NaOH a 65 °C o 180 min unde magne ic
s i ing. A e being washed by il a ion, he mix u e was blended
again o 1 min. Finally, he biocolloids we e s o ed a 1.0 w %
(aqueous dispe sion) a 4 °C un il use.
Chi in nanoc ys als (ChNCs) we e p epa ed ia acid hyd olysis o
nea α-chi in powde .
32
An amoun o 1:15 (w/ ) comme cial pu e
chi in powde om sh imp was added o a 3 M HCl solu ion and was
magne ically s i ed a 85 °C o 90 min. The eac ion was quenched
by adding a 3- old ice-cold wa e quan i y. The HCl was emo ed by
h ee cen i uga ion s eps (8000 g o 10 min a 25 °C); he
supe na an was disca ded, and he pelle was esuspended in dis illed
wa e . A dispe sion o ∼100 mL was sonica ed using a UP400 S
sonica o (Hielsche ) equipped wi h a S24d14D sono ode a a powe
o 200 W o 8 min while cooled on ice. Subsequen ly, a
cen i uga ion s ep a 8000 g was applied o 10 min a 25 °C, and
he supe na an con aining he ChNCs was collec ed. Fo u he
pu i ica ion, he dispe sion was dialyzed agains dis illed wa e o a
week using egene a ed cellulose dialysis ubing ha ing a molecula
weigh cu o (MWCO) o 12−14 kDa (Medicell Memb anes L d.).
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The ChNC concen a ion was ob ained upon d ying ∼3 g o
suspension and measu ing he d y con en . ChNCs we e s o ed a 4
°C a a concen a ion o 3.5 w %.
ChNF ilms we e p epa ed by sol en cas ing. Ho p essing was no
used o a oid undesi ed su ace pa e ning e ec s by he molding.
B ie ly, 8 mL o 1 w % aqueous ChNF dispe sion was cas ed on o
polys y ene weighing dishes (60 ×60 mm) and was allowed o
e apo a e o 96 h a 20 °C. The esul ing ilm was u he d ied in an
o en a 50 °C o 24 h.
2.3. Physico-Mechanical Cha ac e iza ion. A omic o ce
mic oscopy (AFM) obse a ions we e conduc ed using a Veeco
Ins umen ’s Mul iMode SPM 004-130-000 AFM a oom empe -
a u e. Fo biocolloid obse a ion, a ChNF suspension d ople a a
concen a ion o 0.02 mg·mL−1was coa ed on o a mica subs a e, and
wa e was allowed o e apo a e a oom empe a u e. Fo ilm
cha ac e iza ion, he sol en -cas ed ilm was di ec ly moun ed on o a
s ainless s eel AFM holde , and he ilm was obse ed. The
NanoScope Analysis 1.9 p og am was employed o analyze he
eco ded images. ChNFs and ChNCs we e obse ed by ansmission
elec on mic oscopy (TEM) on a JEOL JEM 1400 Plus appa a us a
an accele a ion ol age o 100 kV. A 3 μL d ople (0.01 w % aqueous
dispe sion) was deposi ed on o a hyd ophilic EMS CF300-Cu g id
(glow discha ge ea men ; 10 mA du ing 30 s in a Leica EM
ACE200) and he biocolloids we e nega i ely s ained wi h 1% u anyl
ace a e (UO2(CH3COO)2) o 20 s ( he u anyl ace a e was hen
emo ed wi h a il e pape ).
A enua ed o al e lec ance Fou ie ans o m in a ed (ATR-
FTIR) spec a we e ob ained by using a Jasco FT/IR-6100
spec ome e (ATR op ics; 2 cm−1 esolu ion). Room empe a u e
X- ay di ac ion (XRD) was conduc ed in a PHILIPS X’PERT PRO
au oma ic di ac ome e in he a− he a con igu a ion, seconda y
monoch oma o wi h Cu−Kα adia ion (λ= 1.5418 Å) and a PIXcel
solid s a e de ec o . Ca bon nuclea magne ic esonance (13C NMR)
spec a we e acqui ed in a B uke A ance DPX 300 (B uke , U.S.A.)
a 75.5 MHz esonance equency. Spec a we e ob ained a oom
empe a u e using 40 mg, in e se ga ed decoupled sequence, 3 s
acquisi ion ime, 4 s delay ime, 5.5 μs pulse, spec al wid h 18800 Hz,
and >10000 scans. Ze a-po en ial o wa e -dispe sed ChNFs and
ChNCs (0.02 mg·mL−1) o pH alues anging om 2 o 10 was
ob ained using a Mal e n Ze asize Nano-ZS. The pH was uned
upon addi ion o 0.1 M NaOH o 0.1 M HCl. The he modeg ada ion
o ChNF ilms was assessed in a TGA METTLER TOLEDO 822e
ins umen using pla inum pans a a hea ing a e o 10 °C·min−1wi h
a 50 mL·min−1N2 low.
The su ace opology o he ChNF ilms was analyzed using a
scanning p obe mic oscope Dimension ICON om B uke wi h
NanoScope Analysis 1.9 so wa e. Expe imen s we e conduc ed in
apping mode wi h an in eg a ed silicon ip/can ile e . Wa e was
used as he p obe liquid o he de e mina ion o he con ac angle.
Measu emen s we e ca ied ou by he sessile d op me hod (5 μL pe
d op) using a K uss D op Shape Analyze DSA100 a oom
empe a u e. The a e age alue was calcula ed by using ou
measu emen s. The ensile p ope ies o he ChNF ilms we e
analyzed using a uni e sal es ing machine (MTC-100 om IDM)
equipped wi h a 500 N load cell. Fi een mm long, 5 mm wide, and 30
±2μm ilms we e used, wi h a de o ma ion a e o 0.5 mm·min−1.
A e age and s anda d de ia ion alues we e de e mined o e h ee
measu emen s. Me cu y in usion po osime y (POREMASTER-60
GT, Quan ach ome Ins umen s, Inc.) was applied o measu e he
po osi y o ilms a a maximum p essu e o 241 MPa
2.4. In Vi o S udies. To s udy he po en ial cy o oxici y o he
isola ed chi in nano ib ils and chi in nanoc ys als, he me abolic
ac i i y o MRC-5 and BV-2 cells was de e mined in he p esence o
inc easing concen a ions (0−5 mg·mL−1) o ChNFs and ChNCs. To
a oid po en ial con amina ion, ChNFs and ChNCs we e ho oughly
washed wi h 70% e hanol p io o hei inco po a ion in he cell
cul u e media. Cells we e seeded in a 96-well pla e a a densi y o
5000 cells pe well. A e 24 h, he medium was aspi a ed and
subs i u ed by comple e medium (DMEM + 10% FBS) con aining
inc easing amoun s o ChNFs and ChNCs. A e 24 h, he me abolic
ac i i y o he cells was de e mined by he Alama Blue assay.
Addi ionally, he po en ial p o-in lamma o y esponse o BV-2 cells o
he p esence o ChNFs and ChNCs was also e alua ed. Acco dingly,
BV-2 cells we e seeded in a 48-well pla e a a densi y o 50,000 cells
pe well. A e 24 h, media was aspi a ed and subs i u ed by comple e
media con aining inc easing amoun s o ChNFs and ChNCs (0, 0.1,
1, and 5 mg·mL−1). LPS a a concen a ion o 20 ng·mL−1was used as
a posi i e con ol. A e 24 h, he media was collec ed, and he
p esence o ni i es was quan i ied by he G iess Reagen . The
p oduc ion o TNF-alpha in he media was de e mined by ELISA
ollowing he p o ocol p o ided by he supplie .
To e alua e he cy o oxici y o ChNF ilms and hei capaci y o
allow cell adhesion and se e as sca olds, ci cula samples o 6 mm
diame e we e i s punched ou om he ee-s anding ilms ob ained
upon sol en cas ing. These ilms we e placed in 24-well pla es,
washed wi h 70% e hanol, and u he s e ilized by exposu e o UV-
ligh (30 min). Then, cells (MRC-5 o BV-2 cells) we e seeded a a
densi y o 10000 cells pe sample. A e 24 and 48 h, he me abolic
ac i i y o cells was de e mined by he Alama Blue assay. A hese
ime-poin s, cells we e ixed wi h 4% pa a o maldehyde solu ion and
hei cy oskele on and nuclei we e espec i ely s ained wi h
hodamine-phalloidin and DAPI, as p e iously desc ibed by us.
33
Cells we e inally obse ed unde an in e ed luo escen mic oscope
(Nikon Eclipse Ts2, Nikon).
2.5. S a is ical Analysis. In he in i o s udies, he esul s a e
p esen ed as mean ±SD. One-way analysis o a iance (ANOVA)
was used o es he s a is ical di e ences be ween g oups, wi h he
Bon e oni pos hoc es and a con idence le el o 95% (p< 0.05).
■RESULTS AND DISCUSSION
In Vi o E alua ion o Dispe sed Chi in Nano ib ils.
The aim o his wo k is o e alua e any po en ial cy o oxic and
in lamma o y esponse ha ChNFs isola ed om ungi may
o igina e. To do so, ou e o s ocus on wa e -dispe sed
ChNFs and ee-s anding ChNF-based ilms (Figu e 1,
highligh ed wi hin he g een box). A op-down app oach is
ollowed o isola e he na i e nano ib ils loca ed a he inne
cell wall o ce ain ungal species. These chi inous nano ib ils
ac as s uc u al polyme embedded wi hin a β-glucan ma ix,
p o iding mechanical s abili y o ungal cell walls.
13,34
Colloidal chi in nano ib ils a e ob ained a e an ini ial
ib illa ion p ocess wi h a con en ional cooking blende , hen
submi ing he slu y o a ho -wa e ea men (85 °C o 30
min, i emo es wa e -soluble componen s) and a inal
dep o einiza ion p ocess (1 M NaOH, 65 °C o 180 min)
o emo e p o eins, lipids, and ce ain polysaccha ides.
18
Buchne il a ion is applied o wash he ex ac soluble
componen s. Finally, a 1 w % colloidal dispe sion o ChNFs in
wa e is ob ained. As indica ed by he isual appea ance o
dispe sions in Figu e S1, ChNFs emain s able in wa e o
days.
35
This colloidal s abili y is o g ea impo ance when
conside ing his nanoma e ial as an injec able ehicle o
biomedical applica ions (e.g., d ug deli e y).
This dispe sion has also been used o p epa e ee-s anding
ilms by a simple sol en -cas ing. Du ing his p ocess, wa e
slowly e apo a es, and he concen a ion o he slu y inc eases
so nano ibe s physically en angle. As wa e con inues o
e apo a e, capilla y o ces p o ide a ac ion be ween indi id-
ual nano ibe s.
36
Wi h u he wa e e apo a ion, hese ibe s
become close o each o he , and seconda y a ac ion o ces
such as hyd ogen bonding occu be ween nano ib ils, which in
u n yields ee-s anding ilms wi h ema kable mechanical
p ope ies.
7
Thanks o he in ica e nanopa icle en anglemen ,
ChNF ilms do no edispe se when imme sed in wa e (Figu e
S2), which is in con as wi h ee-s anding ilms p epa ed
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Biomac omolecules 2023, 24, 5737−5748
5739
upon simple sol en -cas ing o o he biocolloids such as
cellulose nanoc ys als.
37
The mo phological ea u es o ChNFs we e i s cha ac-
e ized by mic oscopy. A ib illa -like ma e ial wi h diame e s
in he ange o ew nanome e s is obse ed in he con ac -
mode AFM heigh and phase images shown in Figu e 2a. Such
mo phology esembles he one p oduced by mechanically o
enzyma ically p ocessing cellulose o ob ain CNFs, which has a
low deg ee o ib illa ion and yields bundles wi h diame e s o
ca. 20 nm.
38
A mo e de ailed mo phological obse a ion by
ansmission elec on mic oscopy (TEM) in Figu e S3 e eals
ha ChNFs each leng hs expanding up o ∼1μm, which is
la ge han he diame e and leng h o e single α-chi in
c ys alli es (a e age alues o 2−5 nm and ∼300 nm,
espec i ely).
13
These obse a ions sugges ha ob ained
ib illa y ma e ial is composed upon he agg ega ion o se e al
α-chi in c ys alli es.
18,39
Fo compa ison, ChNCs we e isola ed
om α-chi in by an acid hyd olysis p ocess assis ed by ip
sonica ion. A 3 M HCl solu ion a 85 °C o 90 min hyd olyzes
he glycosidic bonds o chi in, while a ip-sonica ion s ep
ende s colloidal chi in.
32
This p ocess has been selec ed gi en
i s simplici y, and po en ially lowe en i onmen al oo p in
o e o he chemically in ensi e and ime-consuming p ocesses
(up o 5 M HCl, 104 °C unde e lux o eac ions imes o 18
h).
14,40,41
As demons a ed by he TEM mic og aph in Figu e
2c, od-shaped chi in nanopa icles a e ob ained (50−300 nm
in leng h, 8−15 nm in wid h),
9
which ag ees wi h li e a u e.
14
A enua ed o al e lec ance-Fou ie ans o m in a ed
spec oscopy (ATR-FTIR) and X- ay di ac ion (XRD) in
Figu e 2c and d, espec i ely, p o ide addi ional in o ma ion
abou he isola ed ma e ial. Fo compa ison, da a co espond-
ing o comme cially a ailable pu i ied chi in powde isola ed
om Pandalus bo ealis sh imp a e also shown. ChNFs p esen
he cha ac e is ic abso p ion bands o chi in, wi h he b oad
band a 3650−3200 cm−1due o he −OH s e ching, he
−CH bands a 2911 and 2841 cm−1, he amide I, II, and III
bands a 1628, 1556, and 1315 cm−1, espec i ely, and he
sha p peaks a 1378 and 1029 cm−1due o he CH3
symme ical de o ma ion and C−O−C g oups in chi in,
espec i ely.
42,19
The amide III band con i ms he p esence
o chi in ins ead o chi osan. Howe e , he lowe in ensi y o
he amide bands in compa ison wi h aw chi in sugges he
p esence o an addi ional phase, which acco ding o li e a u e
is iden i ied as β-glucans ha emain co alen ly bonded o
nano ib ils.
19
As β-D-glucans a e polysaccha ides composed o
D-glucose monome s linked by β-glycosidic bonds and do no
con ain ni ogen, he glucosamine con en (o chi in con en )
in he isola ed chi in nano ib il−glucan complexes can be
es ima ed om CHN elemen al analysis (ca bon, hyd ogen,
ni ogen) by mul iplying by 14.199 ni ogen con en .
43
Wi h a
ni ogen con en o 3.07 ±0.09 w % (well below he
heo e ical 6.89 w % ni ogen ound in chi in), a chi in
con en o 43.6% is ob ained, being he emaining ma e ial
mos ly composed by glucans.
19
The XRD pa e n o ChNFs indica ed he occu ence o a
semic ys alline ma e ial, whe e wo well dis inguishable
c ys alline peaks a 2θ= 9.2 and 19.7°co esponding o
(020) and (110) planes o chi in a e seen, co ela ing well wi h
high deg ees o N-ace yla ion.
44
The na owe di ac ion peaks
(no e also he weake 2θ= 20.5 and 26.2°) ma ch wi h he
c ys alline α-chi in o m,
42
while he la ge halo indica es he
p esence o an amo phous ma e ial such as β-glucans.
18
The
c ys allini y index o ChNFs can be ob ained om he (020)
e lec ion as
I I
I
C I 100
am
020
020
020
= ×
(1)
C ys allini y alues o 89.4% and 59.1% a e ob ained o α-
chi in and ChNFs, espec i ely. Such dec eased c ys allini y
does no necessa ily co ela es wi h a lowe N-ace yla ion
(DA) alues o pu e samples,
44
because he con ibu ion o
amo phous glucans should be also conside ed. Besides, he
in e plana spacing (d) be ween adjacen planes ha ing (020)
Mille indices can p o ide aluable in o ma ion on he
con o ma ional ea u es o he chi in. The dcan be ob ained
om he di ac ion pa e ns acco ding o he B agg’s law as
Figu e 1. ChNF isola ion om whi e mush oom (Aga icus bispo us) using mechanical blending and dep o einiza ion. Toxici y s udies a e ca ied
ou on aqueous ChNF dispe sions (a di e en concen a ions) and on ChNF ee-s anding ilms, highligh ed in g een.
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d n2 sin = ×
(2)
whe e θis he di ac ion angle, nis an in ege , and λis he
wa eleng h o he adia ion used. d020 alues o 9.50 and 9.68 Å
a e ob ained o pu e α-chi in and ungal ChNFs, espec i ely.
To gain pe spec i e, d020 alues o 9.70−9.78 Å ha e been
epo ed o chi in mic oc ys als ex ac ed om An a c ic k ill
(Euphausia supe ba), c ab, and sh imp shells upon HCl
hyd olysis.
45
This la ge spacing o ChNCs ob ained a e
HCl ea men esul s om he ha sh demine aliza ion and
dep o eina ion ea men s needed o ex ac chi in om
c us acean exoskele ons.
17
Al hough he inc eased in e plana
spacing sugges s an expansion o he c ys al la ice induced by
a educed DA, one mus conside ha chi osan wi h a DA as
low as 7.2% p esen s a d020 alue o 7.42 Å.
44
The e o e, XRD
esul s sugges ha he mild isola ion p ocess he e applied
(sho eac ion imes, low empe a u es, low basici y) ende s
α-chi in wi h a high DA whe e he hyd ogen bonding wi hin
he chi in c ys alli es is no dis up ed, oge he wi h
amo phous glucans.
As one o he mos accu a e and ep oducible echnique o
he deg ee o DA es ima ion, solid-s a e ca bon-13 nuclea
magne ic esonance (13C NMR) analyses we e conduc ed and
he esul s a e shown in Figu e 2e (enla ged 13C NMR spec a
and he co esponding chemical shi s a e shown in Figu e S4
and Table 1, espec i ely).
46
The DA was es ima ed om he
in eg al o me hyl ca bon di ided by he summa ion in eg als
o ca bon a oms o he D-glucopy anosyl ing as
47
I
I
DA(%) 6 100
CH
C1 C6
3
= × ×
(3)
whe e ICHd
3accoun s o he in eg al o he me hyl peak and
IC1−C6 conside s all he ca bon g oups in he backbone,
espec i ely. Bo h spec a a e domina ed by he ea u es
Figu e 2. Mo phological and con o ma ional cha ac e iza ion: (a) con ac -mode AFM heigh and phase images o isola ed ChNFs om Aga icus
bispo us; (b) ansmission elec on mic oscopy image showing isola ed ChNCs om comme cial α-chi in powde unde HCl ea men ; (c) ATR-
FTIR spec a o ChNFs and α-chi in powde ; (d) XRD pa e ns o ChNFs and α-chi in powde ; (e) 13C NMR spec a o ChNFs and α-chi in
powde ; ( ) Z-po en ial o ChNF and ChNC aqueous dispe sions a di e en pH alues; (g) he mog a ime ic cu es co esponding o ChNF
om Aga icus bispo us, c us acean α-chi in, and c us acean ChNCs.
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asc ibed o chi in, wi h he C�O signal a 174 ppm, he C-2 a
55 ppm, and he CH3a 23 ppm. Besides, C-4 and C-6 ca bon
signals do no show double s, con i ming he occu ence o
chi in a he han chi osan.
47,48
The ma ked in ensi y o he
CH3signals o bo h samples sugges a high DA as opposed o
chi osan, which shows low in ensi ies o his signal. In ac ,
DA alues o 94.1% and 75.8% a e ob ained acco ding o eq 3,
indica ing he success ul isola ion o chi in om ungi. The
sligh asymme y o he C-1 peak o ChNFs (signal occu ing
a 104 and 102 ppm o chi in and β-D-glucan, espec i ely),
49
he new signal a 33 ppm and he shoulde appea ing close o
he C-3 signal (a lowe ppm alues) is indica i e o (1 →3)-
β-D-glucans ha emain co alen ly linked o chi in h ough a
ca bonyl linkage.
47
As nanopa icle su ace cha ge media es cell−ma e ial
in e ac ions,
50
he su ace cha ge o wa e -dispe sed biocolloids
a a ying pH alues was measu ed, and he esul s a e shown
in Figu e 2 . A low pH alues, ChNFs show a posi i e ne
cha ge due o p o ona ion o he N-ace yl g oups o chi in. The
su ace becomes nega i ely cha ged as he media becomes
alkaline o each −20.2 mV a pH 10, wi h an isoelec ic poin
a ound pH 4.5. Ob ained cha ges a e mo e nega i e han he
esul s ob ained o ChNCs (see Figu e S5 o ATR-FTIR and
XRD esul s cha ac e is ic o α-chi in), whe e a ne posi i e
cha ge o +32 mV a pH = 2 wi h an isolelec ic poin a pH
9.1 is seen. The mo e nega i e cha ge o ChNFs o e ChNCs
a a gi en pH sugges s a lowe ac ion o amine g oups
a ailable o unde go dep o ona ion. This lowe cha ge induces
elec os a ic epulsion o ces among nanopa icles o imp o e
hei colloidal s abili y, simila o wha has been obse ed o
CNCs.
51
Finally, Figu e 2g shows he he mog a ime ic
analysis (TGA) cu es o a ChNF ilm, α-chi in powde , and
ChNCs. O e all, a e y simila he mal s abili y o ChNFs and
ChNCs is obse ed. An ini ial weigh loss cen e ed a 80 °C
co esponding o adso bed wa e e apo a ion (blue a ow),
oge he wi h a wide and ma ked he modeg ada ion e en
occu ing in he 280−405 °C ange o igina ing om he
deg ada ion o 2-amino-2-deoxy-D-glucopy anose uni s in
chi in is obse ed.
52
ChNFs adso b mo e wa e (by weigh )
in compa ison wi h chi in powde , and he deg ada ion cu e
becomes wide o ChNFs. Besides, a cha equi alen o ∼25
w % is ob ained a 650 °C o ChNFs. I is wo hy o no e ha
ChNFs show an imp o ed esis ance owa d he modeg ada-
ion o e he ubiqui ous CNCs ex ac ed h ough sul u ic acid
hyd olysis.
53
To e alua e he po en ial cy o oxici y o he isola ed ChNFs,
he me abolic ac i i y o wo di e en cell lines was quan i ied
by means o an Alama Blue assay (Figu e 3). A e 24 h in
con ac wi h inc easing concen a ions o ChNFs ( om 0 o 5
mg·mL−1), no s a is ically signi ican di e ences (p< 0.05)
we e obse ed on he me abolic ac i i y o human lung
ib oblas s (MRC-5) wi h espec o he con ol (i.e., cells in
he absence o ChNFs). Simila ly, he me abolic ac i i y o
mu ine mic oglia (BV-2) was always simila o sligh ly highe
han ha obse ed in he con ol, con i ming again he
cy ocompa ibili y o he ChNFs isola ed om ungi a he
s udied concen a ions. In he p esence o ChNCs, bo h cell
lines showed a simila beha io , and s a is ically signi ican
di e ences we e only obse ed in BV-2 cells exposed o he
highes concen a ions (i.e., 5 mg·mL−1) o ChNCs. I should
be no ed ha he cy o oxici y o no el ma e ials a he
nanoscale based on na u al biopolyme s s ongly depends on
he sou ce, isola ion p o ocol, unc ionaliza ion, dimensions
and s udied cell line.
26,37
Thus, a di ec compa ison be ween
di e en nanoma e ials is di icul due o he lack o well-
es ablished p o ocols. Some e iew pape s ha e ied o
summa ize hose ecen s udies dealing wi h he cy o oxici y o
cellulose-based nanoma e ials, which sha e s uc u al and
chemical simila i ies o he ChNFs p esen ed he ein.
54,55
Hani e al. p epa ed cellulose nanoc ys als o con olled shape
and size and s udied hei po en ial cy o oxici y wi h mu ine
ib oblas s (NIH3T3).
28
The p epa ed CNCs did no ha e any
de imen al e ec on cell iabili y a concen a ions o up o
250 μg·mL−1. Howe e , cell iabili ies below 80% we e
eco ded o highe CNCs concen a ions (i.e., 500 and 1000
μg·mL−1). In a simila s udy, Pe ei a e al. explo ed he
cy o oxici y o CNFs de i ed om co on on bo ine
ib oblas s.
56
As de e mined by low cy ome y, CNFs did
no impac cell iabili y o concen a ions o up o 200 μg·
mL−1. None heless, a dose-dependen cy o oxici y was
obse ed o highe concen a ions wi h cell iabili ies o 72
and 37% o concen a ions o 1 and 5 mg·mL−1, espec i ely.
In iew o ou esul s, i emains easible o a gue ha he
ChNFs isola ed he ein a e compa a i ely sa e han p e iously
epo ed nanoma e ials de i ed om na u ally sou ced
polyme s and show a beha io simila o ha o he ChNCs
Table 1. Chemical Shi s (δ, ppm) o Chi in Samples Ob ained by 13C NMR
signal (ppm)
sample C�O C1 C4 C5 C3 C6 C2 CH3
α-chi in
a
173.8 104.1 83.0 75.7 73.3 60.8 55.2 22.8
α-chi in 173.7 103.6 82.8 74.9 73.3 60.5 54.9 22.6
ChNFs 173.7 103.6 82.9 74.1 74.1 60.5 55.1 22.8
a
α-Chi in om e 47.
Figu e 3. Me abolic ac i i y o MRC-5 and BV-2 cells in he p esence
o inc easing concen a ions o ChNFs (da k o ange and da k g ay) o
ChNCs (ligh o ange and ligh g ay) a e 24 h. As e isks (*) indica e
signi ican di e ences (p< 0.05) wi h espec o he con ol (absence
o ChNFs o ChNCs; n= 5).
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ob ained ia he adi ional acid hyd olysis om chi in powde
om sh imps.
Despi e he posi i e esul s obse ed o dispe sed ChNFs in
e ms o cy o oxici y, whe e he me abolic ac i i y o bo h
MRC-5 and BV-2 cells emained unal e ed o a wide ange o
ChNF concen a ions, deepe biological es s a e equi ed o
ensu e sa e use and explo e u u e applica ions o his
biocolloid. He ein, he sec e ion o in lamma o y media o s
by BV-2 cells a a ying ChNF concen a ions was in es iga ed.
BV-2 is mu ine mic oglia ha play a majo ole in he
egula ion o neu oin lamma o y p ocesses and a e quickly
ac i a ed in esponse o exogenous insul s, including nanoma-
e ials. Thus, hey ha e been p e iously explo ed as a cellula
model o s udy he in lamma o y esponse o a ious
nanopa icles (e.g., sil e and i anium dioxide nanopa -
icles).
30,57
As obse ed in Figu e 4a, he concen a ion o
ni i es in he supe na an o BV-2 cells g adually inc eased
wi h he ChNF concen a ion. A a ChNF concen a ion o 1
mg·mL−1, despi e no s a is ically signi ican di e ences (p<
0.05) being obse ed, he p esence o ni i es was 1.7 imes
highe han o he nega i e con ol (i.e., cells in he absence o
ChNFs), bu s ill signi ican ly lowe han he concen a ion
obse ed in he posi i e con ol (i.e., cells s imula ed wi h 20
ng·mL−1o LPS). When inc easing he ChNF concen a ion
up o 5 mg·mL−1, he concen a ion o ni i es was 18.1 imes
highe han o he nega i e con ol and 2.0 imes highe han
o he posi i e con ol, being hese di e ences s a is ically
signi ican (p< 0.05). In he case o ChNCs, he concen a ion
o ni i es a a ChNC concen a ion o 1 mg·mL−1was
signi ican ly highe (p< 0.05; 8.8 imes highe ) han he one
obse ed in he nega i e con ol, being simila o he le els
obse ed in he posi i e con ol. A he highes concen a ion
o ChNCs (i.e., 5 mg·mL−1), he elease o ni i es was 20.2
imes highe han o he nega i e con ol and 2.0 imes highe
han o he posi i e con ol, being hese di e ences s a is i-
cally signi ican (p< 0.05). The measu emen o ni i es is
egula ly used o es ima e he p oduc ion o ni ic oxide by
cells, which is a p o-in lamma o y media o . As a comple-
men a y assay, he sec e ion o umo nec osis ac o (TNF-α),
which ep esen s an in lamma o y cy okine, by BV-2 cells in
he p esence o ChNFs was u he s udied (Figu e 4b). BV-2
cells sec e ed negligible le els o TNF-αin he absence o
ChNFs (<100 pg·mL−1), whe eas hey sec e ed >4000 pg·
mL−1when s imula ed wi h 20 ng·mL−1(i.e., posi i e con ol),
hus alida ing ou in i o in lamma ion model. BV-2 cells
sec e ed highe le els o TNF-αas he concen a ion o ChNFs
inc eased, being always signi ican ly (p< 0.05) highe han in
he case o he posi i e con ol. In he case o ChNCs, he
sec e ion o TNF-αwas simila o he posi i e con ol a a
concen a ion o 1 mg·mL−1. A a ChNC concen a ion o 5
mg·mL−1, he concen a ion o TNF-αin he cell supe na an
was 6.9×highe han he one obse ed in he posi i e con ol.
Taken oge he , hese esul s sugges a dose-dependen p o-
in lamma o y esponse o mic oglia o bo h ChNFs and
ChNCs.
Ob ained esul s a e in ag eemen wi h p e ious in i o and
in i o obse a ions dealing wi h he in lamma o y esponse
induced by biobased colloids. As o cy o oxici y s udies, he e
exis s a s ong in e play be ween he size, shape, su ace
unc ionali ies, aw ma e ial sou ce, p epa a ion p ocedu e, and
in lamma o y esponse induced by he esul ing nanoma e ials,
making a di ec compa ison be ween di e en s udies
challenging. Fo example, Menas e al. concluded ha CNCs
caused a mo e se e e in lamma o y esponse on human lung
epi helial cells han nano ib illa ed cellulose.
58
In a di e en
s udy, a ca ionic de i a i e o cellulose nanoc ys als also
induced a p o-in lamma o y esponse on mu ine mac ophages,
being he esponse dependen on he su ace unc ionali ies.
59
These in i o obse a ions a e u he suppo ed by in i o
esul s, whe e CNCs induce pulmona y oxici y in mice by
elici ing oxida i e s ess, issue damage and a obus
in lamma o y esponse.
60
Conside ing he g owing use o
eme ging biocolloids and hei po en ial echnological
applica ions,
7,13
i esul s i al o conduc de ailed biological
e alua ions o hese biocolloids o gain mo e insigh s abou he
pa icula in e ac ion o hese nanoma e ials and cells, issues
and o gans. In he p esen p elimina y s udy, ChNFs we e
challenged wi h BV-2 a ela i ely high concen a ions (>100
μg·mL−1) o s udy hei po en ial p o-in lamma o y e ec .
Howe e , we es ima e u he in i o s udies a e equi ed wi h
he aim o e alua ing he e ec o p ocessing, su ace
unc ionali ies, and mo phological aspec s o he ChNFs
isola ed he ein on cell beha io .
Figu e 4. (a) Fold change wi h espec o he nega i e con ol (i.e., cells in he absence o ChNFs o ChNCs) o ni i e concen a ion in he
supe na an o BV-2 cells exposed o di e en concen a ions o ChNFs and ChNCs o 24 h. (b) Fold change wi h espec o he posi i e con ol
(i.e., cells s imula ed wi h 20 ng·mL−1o LPS) o TNF-αconcen a ion in he supe na an o BV-2 cells exposed o di e en concen a ions o
ChNFs and ChNCs o 24 h. “a” and “b” indica e signi ican di e ences (p< 0.05) wi h espec o he nega i e and posi i e con ol, espec i ely (n
= 3).
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Toxici y and Cell P oli e a ion on o Chi in Nano ib il
Films. The su ace cha ac e is ics o ma e ials play a
de e minan ole in he esul ing cell/ma e ial in e ac ions.
61,62
Acco dingly, he su ace mo phology o sol en -cas ed ChNF
ilms has been in es iga ed by apping-mode AFM. Figu e 5a
shows he heigh and phase AFM images o a ee-s anding
ChNF ilm. A ela i ely la su ace composed o o e lapped
nano ib ils wi h a andom in plane o ien a ion is obse ed. The
achie ed ne wo k s uc u e o igina es om he s uc u al
lexibili y and high aspec - a io o ChNFs, which show a
endency owa d physical en anglemen du ing wa e e apo-
a ion. As he nano ib ils emain co e ed by an amo phous
laye (glucans), indi idual nano ib ils a e di icul o obse e in
he images (highe -magni ica ion AFM heigh and phase
images in Figu e S6 sugges a ChNF wid h o app oxima ely 7
o 17 nm). The su ace oughness o he ilms, de e mined by
oo -mean-squa e oughness (Rq) and mean oughness (Ra)
pa ame e s eaches 19.3 and 15.6 nm, espec i ely.
63
Such low
alues deno e he o ma ion o highly smoo h su aces upon
he sol en -cas ing o ChNF aqueous dispe sions. The su ace
we abili y was assessed by wa e con ac angle measu emen s.
A con ac angle o 68.1 ±5°is seen in Figu e 5b o a 5 μL
wa e d op on o a ChNF ilm, indica ing a p edominan ly
hyd ophilic na u e as occu ing wi h nanocellulose ilms.
63
In
spi e o being hyd ophilic, ChNF ilms p esen a good
esis ance o b eak o edispe sion when imme sed in dis illed
wa e (Figu e S2), o e ing an undeniable ad an age owa d
biomedical o packaging applica ions o e hei nanocellulose
analogues, whose wa e s abili y is usually poo .
64
This s abili y
o igina es om he syne gy be ween glucans and nano ib ils,
18
and con as wi h he swelling and subsequen nanopa icle
dispe sion su e ed by CNC-based nanopape s when in con ac
wi h aqueous sys ems.
Besides, he mechanical and mo phological ea u es o
ChNF ilms we e in es iga ed be o e conduc ing biological
es s. The ensile p ope ies o ChNF ilms we e de e mined
acco ding o uniaxial es s and he ep esen a i e s ess−s ain
cu e is shown in Figu e 5c. The ChNF ilm p esen s a b i le
beha io whose Young’s modulus (E) eaches 3415 MPa, a
maximum ensile s ess (σy) o 61.5 MPa and an elonga ion a
b eak (εb) o 4.6%. I should be no ed ha , in spi e o he
p ocesses simplici y do no equi ing il a ion and a
subsequen ho -p essing,
65
he obse ed modulus and ul ima e
s eng h alues emain compa able o he esul s epo ed o
he majo i y o biodeg adable he moplas ic ma e ials.
66,67
This
may be due o he combina ion o he physical en anglemen
and seconda y a ac ion o ces o indi idual nano ibe s,
oge he wi h he na i e amo phous glucans among nano ib ils
imp o ing he binding o he whole ma e ial as na u ally occu s
in he ungal cell walls.
18
Besides, an enhanced duc ili y in
compa ison wi h nanopape s based on CNCs (εb= 1.9 ±
0.2%),
63
ce ain CNF-based ilms (εb= 2.1 o 10.1%,
depending on he cellulose o igin and ilm po osi y),
68
bac e ial cellulose (εb= 2.4 ±0.3%),
69
o c us acean-de i ed
HCl-hyd olyzed chi in nanowhiske ilms (εb= 1.2 ±0.4%)
70
is achie ed. Howe e , a lowe modulus and ensile s eng h
ha e been ob ained in compa ison wi h he ChNF ilm by
Nawawi e al. (6.9 GPa, 204 MPa, espec i ely).
18
The la ges
load- esis ance shown by Nawawi e al. can be explained by a
educed po osi y and enhanced seconda y a ac ion o ces
be ween nano ib ils achie ed upon il a ion and subsequen
p essing in an o en a 120 °C o 3 h unde 5 kg weigh .
18
Fo
Figu e 5. Sol en -cas ed ChNF ilm cha ac e iza ion: (a) su ace mo phology as e ealed by apping-mode AFM heigh and phase images; (b)
ep esen a i e image o a wa e d op a he su ace o a ChNF ilm; (c) ep esen a i e uniaxial ensile s ess−s ain es ; and (d) in usion/ex usion
cu es o me cu y-po osime y.
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u he e i ica ion, we conduc ed a me cu y in usion
po osime y analysis o in es iga e he po e cha ac e is ics o
he ChNF ilms. As seen in Figu e 5d, a po osi y o 62.1% and
ilm su ace a ea o 7.9 m2·g−1was ob ained (simila ly o
nanocellulose ilms ab ica ed by sol en cas ing; po osi y =
56%, su ace a ea = 11.0 m2·g−1).
71
The abundan po es wi hin
he in e io o he ilm can ac as c ack ini ia ion si es and lead
o a ma e ial emb i lemen e ec when subjec ed o ex e nal
ensile s esses.
72
A il a ion app oach could be explo ed in he
u u e o educe ilm po osi y and imp o e he ac u e
p ope ies o he ChNF ilms. This po osi y, despi e ha ing a
de imen al e ec on s eng h- ela ed p ope ies, may be
bene icial o he use o his ma e ial as a sca old o issue
enginee ing applica ions since i acili a es he di usion o
nu ien s and oxygen. I should be no ed ha a sol en -cas ing
app oach was ollowed he e o keep he na i e ilm su ace
mo phology in ac and a oid undesi ed pa e ning o he
ChNF ilm by he molds used du ing ho p essing.
Ob ained ib illa y-like su ace mo phology and mechanical
p ope ies, in combina ion wi h he noncy o oxici y obse ed
o wa e -dispe sed nano ib ils, make ChNF ilms po en ial
candida es o biomedical uses. An addi ional a ac i e
o igina es om i s biobased cha ac e , which enables en i on-
men ally sus ainable biomedical ma e ials as opposed o
cu en con en ional choices elying on pe oleum-de i ed
polyme s, such as poly( inylidene luo ide) o poly(ε-
cap olac one).
73
The e o e, he po en ial o ChNF ilms o
allow cell g ow h and adhesion was also e alua ed wi h MRC-5
and BV-2 cells. As obse ed in Figu e 6a, he me abolic ac i i y
o bo h MRC-5 and BV-2 cells inc eased wi h ime, con i ming
ha cells we e able o g ow in he p esence o he ChNF ilms.
Acco dingly, he calcula ed me abolic ac i i ies a 48 h we e
1.6×and 2.5×highe han hose a 24 h o MRC-5 and BV-2
cells, espec i ely. As concluded om he luo escen mic o-
g aphs in Figu e 6b, e y ew MRC-5 cells we e obse ed on
he ChNF ilms in compa ison o he con ol (i.e., glass slide),
sugges ing a poo in e ac ion be ween he cells and he
bioma e ial. This could be asc ibed o he en isaged s uc u e
o ou ChNFs, ha may con ain hyd ophobins (i.e., cys eine-
ich p o eins ound in ilamen ous ungi and mush ooms),
20
hus limi ing he adhesion o human ib oblas s, as p e iously
epo ed.
74,75
In con as , BV-2 cells we e able o adhe e and
g ow on he ChNF ilms, showing mo phologies compa able o
hose o he cells obse ed on he glass slide. We hypo hesize
ha he ac i a ion o mic oglia by he p esence o ChNFs (as
demons a ed in Figu e 4) may inc ease he exp ession o
in eg ins, hus acili a ing cell adhesion o he bioma e ial.
P e ious s udies ha e demons a ed ha he ac i a ion o
mic oglia h ough he s imula ion wi h p oin lamma o y
cy okines (e.g., TNF-α, IFN-α, e c.) o LPS signi ican ly
inc eases he adhesion o hese cells o o he wise poo ly
adhe en subs a es (e.g., laminin) ia he exp ession o
in eg ins,
76,77
which suppo s ou hypo hesis.
The ela i ely low pu i y (p esence o 56 w % glucans) o
ungi-de i ed ChNFs o e c us acean-de i ed ChNCs o igi-
na es om he combina ion o he inhe en p esence o glucans
polysaccha ide in ungi oge he wi h he mild isola ion p ocess
applied. In spi e o such a lowe pu i y, ChNFs o e clea
ad an ages ega ding en i onmen al sus ainabili y and scale-up
po en ial. In ac , and as opposed o i s c us acean-de i ed
analogues, ungal nanochi in can be ce ainly upscaled
ollowing he 12 p inciples o g een chemis y.
78
Mechanical
blending, il a ion, and alkaline dep o einiza ion a e easily
scalable p ocesses as hey a e cu en ly applied a he indus ial
le el. Besides, he use o enewable eeds ock oge he wi h he
sho and mild dep o einiza ion (low empe a u e, ambien
p essu e) s eps needed ensu e minimal oxici y o human
heal h and he en i onmen wi h low ene gy demand. A eas o
imp o emen in he nea u u e include he op imiza ion o
NaOH concen a ion and yield inc ease, so he use o ma e ials
is maximized (a om economy). As such, we an icipa e ha he
nea u u e could wi ness he indus ial p oduc ion o ungal
nanochi in o i s exploi a ion in a a ie y o applica ions
(whe he biomedical o no ) ha bea he bene i o a
noncy o oxic and a e compa a i ely sa e cha ac e han o he
biocolloid analogues.
Figu e 6. (a) Me abolic ac i i y o MRC-5 and BV-2 cells on o ChNF ilms and hei (b) co esponding luo escen mic og aphs (Blue-Dapi-
Nuclei; Red-Rhodamine phalloidin-ac in ilamen s). Scale ba : 100 μm. As e isks (*) indica e signi ican di e ences (p< 0.05) wi h espec o he
con ol ( luo escen in ensi y o cells a 24 h; n= 6).
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