nanoma e ials
A icle
Role o Su ace Chemis y in he In Vi o Lung Response o
Nano ib illa ed Cellulose
Kukka Aimonen 1, Sa u Suhonen 1, Mi a Ha ikainen 1, Vi iana R. Lopes 2,† , Hannu No ppa 1,
Na alia Fe az 2and Julia Ca alán1,3,*


Ci a ion: Aimonen, K.; Suhonen, S.;
Ha ikainen, M.; Lopes, V.R.; No ppa,
H.; Fe az, N.; Ca alán, J. Role o
Su ace Chemis y in he In Vi o
Lung Response o Nano ib illa ed
Cellulose. Nanoma e ials 2021,11, 389.
h ps://doi.o g/10.3390/nano
11020389
Academic Edi o : Takuya Ki aoka
Recei ed: 8 Janua y 2021
Accep ed: 30 Janua y 2021
Published: 3 Feb ua y 2021
Publishe ’s No e: MDPI s ays neu al
wi h ega d o ju isdic ional claims in
published maps and ins i u ional a il-
ia ions.
Copy igh : © 2021 by he au ho s.
Licensee MDPI, Basel, Swi ze land.
This a icle is an open access a icle
dis ibu ed unde he e ms and
condi ions o he C ea i e Commons
A ibu ion (CC BY) license (h ps://
c ea i ecommons.o g/licenses/by/
4.0/).
1Finnish Ins i u e o Occupa ional Heal h, Box 40, Työ e eyslai os, 00032 Helsinki, Finland;
[email p o ec ed] (K.A.); [email p o ec ed] (S.S.); [email p o ec ed] (M.H.);
[email p o ec ed] (H.N.)
2Nano echnology and Func ional Ma e ials, Depa men o Ma e ials Science and Enginee ing,
Uppsala Uni e si y, Box 35, 751 03 Uppsala, Sweden; [email p o ec ed] (V.R.L.);
[email p o ec ed] (N.F.)
3Depa men o Ana omy, Emb yology and Gene ics, Uni e si y o Za agoza, 50013 Za agoza, Spain
*Co espondence: [email p o ec ed]
† P esen a ilia ion: Depa men o Medicinal Chemis y, T ansla ional PET Imaging, Uppsala Uni e si y,
751 83 Uppsala, Sweden.
Abs ac :
Wood-de i ed nano ib illa ed cellulose (NFC) has eme ged as a sus ainable ma e ial wi h
a wide ange o applica ions and inc easing p esence in he ma ke . Su ace cha ges a e in oduced
du ing he p epa a ion o NFC o acili a e he de ib illa ion p ocess, which may also al e he
oxicological p ope ies o NFC. In he p esen s udy, we examined he
in i o
oxici y o NFCs wi h
i e su ace chemis ies: non unc ionalized, ca boxyme hyla ed, phospho yla ed, sul oe hyla ed, and
hyd oxyp opyl ime hylammonium-subs i u ed. The NFC samples we e cha ac e ized o su ace
unc ional g oup densi y, su ace cha ge, and ibe mo phology. Fib il agg ega es p edomina ed
in he non unc ionalized NFC, while indi idual nano ib ils we e obse ed in he unc ionalized
NFCs. Di e ences in su ace g oup densi y among he unc ionalized NFCs we e e lec ed in he
ibe hickness o hese samples. In human b onchial epi helial (BEAS-2B) cells, all NFCs showed
low cy o oxici y (CellTi e -GloVR luminescen cell iabili y assay) which ne e exceeded 10% a
any exposu e ime. None o he NFCs induced geno oxic e ec s, as e alua ed by he alkaline come
assay and he cy okinesis-block mic onucleus assay. The non unc ionalized and ca boxyme hyla ed
NFCs we e able o inc ease in acellula eac i e oxygen species (ROS) o ma ion (chlo ome hyl
de i a i e o 2
0
,7
0
-dichlo odihyd o luo escein diace a e assay). Howe e , ROS induc ion did no
esul in inc eased DNA o ch omosome damage.
Keywo ds:
nano ib illa ed cellulose; su ace chemis y; geno oxici y; nano oxici y; nanocellulose;
eac i e oxygen species; human b onchial epi helial cells
1. In oduc ion
Cellulose nano ibe s ha e eme ged as sus ainable and en i onmen ally iendly ma e-
ials wi h a wide ange o indus ial and medical applica ions [
1
]. The e a e se e al ypes
o nanocelluloses ha can be ob ained om di e en aw ma e ials, he mos common
sou ces being ha d- and so wood chemical pulp ibe s [
2
]. Wood pulp ibe s a e p ocessed
wi h chemical and enzyma ic p e ea men s o acili a e he s uc u al decons uc ion o he
ibe s in o wo main ypes o nanocelluloses: nano ib illa ed cellulose (NFC) and cellulose
nanoc ys al [
2
,
3
]. Wood NFC is composed o amo phous and c ys alline domains [
4
] and
is cha ac e ized by ha ing a high-aspec a io, wi h a ypical diame e o 2–10 nm and
leng h in he mic ome e -scale (>1
µ
m) [
5
,
6
]. NCFs a e ob ained om wood pulp ibe s by
mechanical ib illa ion, which can be pe o med by using, e.g., homogenize s, luidize s
and g inde s [
7
]. P io o he mechanical ib illa ion, a ious chemical and enzyma ic
p e ea men s can be applied o ease he ib illa ion o ibe s in o homogeneous nano ib il
Nanoma e ials 2021,11, 389. h ps://doi.o g/10.3390/nano11020389 h ps://www.mdpi.com/jou nal/nanoma e ials
Nanoma e ials 2021,11, 389 2 o 17
dispe sions. Chemical p e ea men s in oduce o example ca boxyl, ca boxyme hyl, and
aldehyde g oups o phospho yl side g oups on he su ace o NFC [
8
]. Such p e ea -
men s a ec no only he su ace chemis y o he nano ib ils bu also p ope ies like ibe
dimensions, speci ic su ace a ea, and deg ee o b anching o he nano ib ils [9].
The inc easing use o nanocelluloses in mul iple applica ions should be accompanied
by an adequa e assessmen o hei sa e y—especially in occupa ional se ings, whe e
inhala ion is conside ed he p ima y ou e o exposu e [
10
]. A li e cycle isk assessmen o
nanocelluloses iden i ied inhala ion o d y nanocellulose powde s o , in he case o we
slu y, ai bo ne nanocellulose-con aining d ople s, as he mos ele an exposu e scena ios
du ing he p oduc ion and manu ac u ing o nanocelluloses [11].
Due o hei na u al o igin, cellulosic ma e ials a e o en assumed no o be oxic [
10
].
Howe e , he long pulmona y biope sis ence o hese ma e ials [
12
–
15
], oge he wi h
he high aspec a io, aise conce ns abou po en ial e ec s on human heal h, especially i
inhaled [
10
]. Fu he mo e, some ea u es o NFCs, such as nanome e size, la ge su ace
a ea, and modi ied su ace chemis y, may impa no el ma e ial p ope ies and biological
beha io , as compa ed wi h mac o-scale ma e ials [
16
]. The e o e, i is necessa y o add ess
he human heal h and en i onmen al sa e y aspec s o nanocelluloses be o e scaling up
hei p oduc ion.
The e is s ill sca ce knowledge on he po en ial ad e se heal h e ec s o NFCs, despi e
he inc easing numbe o s udies pe o med du ing he las ew yea s. As summa ized in
di e en e iews on his opic [
17
–
21
], oxicological s udies show con adic o y indings.
The con lic ing esul s may pa ly be due o a ia ion among NFCs because o di e en ac-
o s, e.g., cellulose sou ce, mechanical ib illa ion p ocedu e, o p e ea men s [
21
], which
can modi y he ma e ial p ope ies. I is well- ecognized ha he physicochemical ea u es
o nanoma e ials may a ec hei oxici y [
16
,
22
,
23
], su ace chemis y being one o he mos
ele an ones [
18
]. Su ace modi ica ions can impa new bene icial p ope ies o nanocel-
luloses, inc easing hei applicabili y in, e.g., heal hca e p oduc s and ood packaging [
24
].
Howe e , di e en unc ionaliza ion will de e mine di e ences in he agglome a ion a e,
hyd ophobici y, su ace cha ge, and su ace chemis y o nanocelluloses, which may a ec
hei cellula up ake, in e ac ion wi h subcellula o ganelles, and downs eam biological
esponses [
19
]. Su ace chemis y was epo ed o d i e
in i o
in lamma o y esponse o
NFC [
25
], while no di e ences in cell me abolic ac i i y o cell memb ane in eg i y we e
obse ed when di e se
in i o
cell models whe e exposed o di e en ly unc ionalized
NFC ma e ials [14,24,25].
S udies add essing he geno oxic po en ial o NCF a e oo sca ce o allow clea con-
clusions [
2
,
19
]. Geno oxic e ec s we e obse ed in mouse 3T3 ib oblas s exposed o
cu auá- and b own co on-de i ed NFC [
26
]. Ven u a e al. [
27
] also epo ed an inc ease in
ch omosome damage, bu no DNA damage, in human lung epi helial al eola A549 cells
cocul u ed wi h THP-1 mac ophages a e being ea ed wi h a TEMPO (2,2,6,6- e ame hyl-
pipe idin-1-oxyl) oxidized NFC. Fu he mo e, geno oxic e ec s we e obse ed in he lung
issue o mice exposed o an enzyma ically p e ea ed NFC [
13
,
15
] and o a TEMPO ox-
idized NFC [
28
]. On he o he hand, no DNA o ch omosome damage was induced by
unmodi ied and enzyma ically o chemically p e ea ed NFC on human b onchial epi helial
BEAS-2B cells [
13
]. Howe e , none o hese s udies compa ed NFC de i ed om he same
sou ce wi h di e en su ace chemis y.
In he p esen s udy, we in es iga ed he ole o su ace chemis y in modula ing he
in i o
oxic po en ial o NFC, by analyzing he abili y o he ma e ials o induce cy o oxic
e ec s, he o ma ion o eac i e oxygen species (ROS), and DNA and ch omosomal dam-
age in human b onchial epi helial BEAS-2B cells. Addi ionally, luo escen s aining o he
nano ib ils was used o assess he cellula up ake o NFC. Fou NFC ma e ials wi h a i-
ous su ace unc ionaliza ion (ca boxyme hyla ion, hyd oxyp opyl ime hylammonium
subs i u ion, phospho yla ion, and sul oe hyla ion), oge he wi h non unc ionalized NFC,
we e included in his s udy. While o he s udies ha e in es iga ed he in luence o su ace
modi ied NFC on se e al oxicological endpoin s using di e en cell models, o he bes o
Nanoma e ials 2021,11, 389 3 o 17
ou knowledge, his is he i s s udy in es iga ing he geno oxic esponse o NFCs ha
only di e in hei su ace chemis y.
2. Ma e ials and Me hods
2.1. Syn hesis and Su ace Modi ica ion o he NFC Ma e ials
The NFC ma e ials we e p o ided by RISE Bioeconomy (S ockholm, Sweden) and
we e p oduced om comme cial ne e -d ied bleached sul i e so wood dissol ing pulp
(Domsjö Fab ike AB, Sweden). Non unc ionalized NFC, he ein e e ed as unmodi-
ied (U-NFC), was p oduced by enzyma ic p e ea men o he wood pulp ollowing he
p o ocol p esen ed by Pääkoo e al. [
29
]. Ca boxyme hyla ed NFC (C-NFC) and hyd ox-
yp opyl ime hylammonium NFC (H-NFC) we e p epa ed as desc ibed by Hua e al. [
30
].
Phospho yla ed NFC (P-NFC) was p oduced ollowing he me hod desc ibed by Nade i
e al. [
31
], using a 4:1 phospho ous:glucopy anose mola a io, ollowed by 5 mic o luidize
passes a 1700 ba . To p epa e sul oe hyla ed NFC (S-NFC), he p o ocol desc ibed by
Nade i e al. [
32
] was ollowed. The su ace unc ional g oup densi y o all NFC samples
was de e mined as desc ibed in Lopes e al. [24].
The chemical s uc u es o he NFC ma e ials a e shown in Figu e 1.
Nanoma e ials 2021, 11, x FOR PEER REVIEW 3 o 18
NFC, we e included in his s udy. While o he s udies ha e in es iga ed he in luence o
su ace modi ied NFC on se e al oxicological endpoin s using di e en cell models, o
he bes o ou knowledge, his is he i s s udy in es iga ing he geno oxic esponse o
NFCs ha only di e in hei su ace chemis y.
2. Ma e ials and Me hods
2.1. Syn hesis and Su ace Modi ica ion o he NFC Ma e ials
The NFC ma e ials we e p o ided by RISE Bioeconomy (S ockholm, Sweden) and
we e p oduced om comme cial ne e -d ied bleached sul i e so wood dissol ing pulp
(Domsjö Fab ike AB, Sweden). Non unc ionalized NFC, he ein e e ed as unmodi ied
(U-NFC), was p oduced by enzyma ic p e ea men o he wood pulp ollowing he p o-
ocol p esen ed by Pääkoo e al. [29]. Ca boxyme hyla ed NFC (C-NFC) and hyd oxyp o-
pyl ime hylammonium NFC (H-NFC) we e p epa ed as desc ibed by Hua e al. [30].
Phospho yla ed NFC (P-NFC) was p oduced ollowing he me hod desc ibed by Nade i
e al. [31], using a 4:1 phospho ous:glucopy anose mola a io, ollowed by 5 mic o luid-
ize passes a 1700 ba . To p epa e sul oe hyla ed NFC (S-NFC), he p o ocol desc ibed by
Nade i e al. [32] was ollowed. The su ace unc ional g oup densi y o all NFC samples
was de e mined as desc ibed in Lopes e al. [24].
The chemical s uc u es o he NFC ma e ials a e shown in Figu e 1.
Figu e 1. Chemical s uc u es o he nano ib illa ed cellulose (NFC) ma e ials unde s udy.
2.2. P epa a ion o he NFC Exposu e Suspensions
The s ock suspensions o he NFCs we e p epa ed in phospha e bu e (PBS) a 5
mg/mL and dispe sed as p e iously desc ibed [25]. B ie ly, he s ock suspensions we e
dispe sed using a B anson Soni ie 450D (400 W, 60 Hz; Danbu y, CT, USA) wi h a 13-mm
ho n o 12 min. The suspensions we e hen s e ilized by au ocla ing, excep o H-NFC
which was subjec o ul a iole adia ion (UV) ea men du ing wo cycles o 45 min
each. Then, he s ock suspensions we e dilu ed in cell cul u e medium and sonica ed o
30 min in a 37 kHz Elmasonic S15H ul asonic ba h cleane (Elma Schmidbaue GmbH,
Singen, Ge many) be o e being added o he cells.
2.3. Cha ac e iza ion o he NFC Ma e ials
2.3.1. Bac e ial Con amina ion, Endo oxin and (1,3)-β-D-Glucan Le els
Bac e ial con amina ion was es ed using he 3M™ Pe i ilm™ Ae obic Coun Pla es,
whe eas bac e ial lipopolysaccha ide con en (endo oxin le el) was measu ed using he
Pie ceTM LAL Ch omogenic Endo oxin Quan i a ion Ki (The mo Fishe Scien i ic, Wal-
ham, MA, USA), ollowing he ins uc ions p o ided by he manu ac u e . P io o he
Figu e 1. Chemical s uc u es o he nano ib illa ed cellulose (NFC) ma e ials unde s udy.
2.2. P epa a ion o he NFC Exposu e Suspensions
The s ock suspensions o he NFCs we e p epa ed in phospha e bu e (PBS) a
5 mg/mL and dispe sed as p e iously desc ibed [
25
]. B ie ly, he s ock suspensions we e
dispe sed using a B anson Soni ie 450D (400 W, 60 Hz; Danbu y, CT, USA) wi h a 13-mm
ho n o 12 min. The suspensions we e hen s e ilized by au ocla ing, excep o H-NFC
which was subjec o ul a iole adia ion (UV) ea men du ing wo cycles o 45 min each.
Then, he s ock suspensions we e dilu ed in cell cul u e medium and sonica ed o 30 min
in a 37 kHz Elmasonic S15H ul asonic ba h cleane (Elma Schmidbaue GmbH, Singen,
Ge many) be o e being added o he cells.
2.3. Cha ac e iza ion o he NFC Ma e ials
2.3.1. Bac e ial Con amina ion, Endo oxin and (1,3)-β-D-Glucan Le els
Bac e ial con amina ion was es ed using he 3M
™
Pe i ilm
™
Ae obic Coun Pla es,
whe eas bac e ial lipopolysaccha ide con en (endo oxin le el) was measu ed using he
Pie ce
TM
LAL Ch omogenic Endo oxin Quan i a ion Ki (The mo Fishe Scien i ic, Wal ham,
MA, USA), ollowing he ins uc ions p o ided by he manu ac u e . P io o he es , he
samples we e hea ed a 75
◦
C o 15 min, o p omo e he elease o endo oxins om
he ma e ial.
Nanoma e ials 2021,11, 389 4 o 17
The le el o (1,3)-
β
-D-glucans in he NFC samples was in es iga ed using he (1,3)-
β
-
D-glucan de ec ion ki Glucan ell wi h diazo- eagen s o endpoin assay (Associa es o
Cape Cod Inc., Li e pool, UK), acco ding o he p o ocol desc ibed by he manu ac u e .
The analyzed samples we e ex ac s ob ained a e incuba ing he NFC suspensions a
75 ◦C o 15 min.
2.3.2. Fibe Mo phology
T ansmission elec on mic oscopy (TEM) imaging was used o in es iga e he mo -
phology o he ibe s. Samples we e p epa ed as desc ibed by Uso e al. [
33
]. B ie ly,
5
µ
L o NFC suspension (0.1% in deionized wa e , dispe sed h ough ul asonica ion as
desc ibed in Sec ion 2.2) we e deposi ed on o coppe TEM g ids wi h o m a ca bon
suppo ilm o 1 min. The ea e , he sample g ids we e s ained by adding 5
µ
L o 2%
u anyl ace a e o 1 s and again 5
µ
L o 5% u anyl ace a e o 15 s. A e each s ep, he
excess o mois u e was d ained along he pe iphe y using il e pape . The g ids we e
examined using a FEI Ti an Themis TEM (The mo ishe Scien i ic, Wal ham, MA, USA)
ope a ed a 200 kV.
2.3.3. Ze a-Po en ial
The z-po en ial o he NFC ma e ials was de e mined in 10 mM NaCl and in cell
cul u e medium (se um- ee LHC-9 medium). The measu emen s in 10 mM NaCl we e
done ollowing he p o ocol desc ibed by Lopes e al. [
24
]. Fo he measu emen s in cell
cul u e medium, z-po en ial was de e mined as an a e age o h ee sepa a e measu emen s
wi h Ze asize Nano ZS (Mal e n Ins umen s L d., Mal e n, UK). Dispe sions o 0.001%
(w/w) o he NFC we e p epa ed in cell cul u e medium, ollowing he same dispe sion
p o ocol as used o he
in i o
exposu es. The measu emen s we e conduc ed a 37
◦
C in
olded capilla y cell di ec ly a e dispe sing he ibe s.
2.4. Cell Cul u e
T ans o med human b onchial epi helial BEAS-2B cells, exhibi ing an epi helial phe-
no ype [
34
], we e ob ained om he Ame ican Type Cul u e Collec ion h ough LGC
P omochem AB (Bo ås, Sweden). The BEAS-2B cells we e g own in se um- ee LHC-9
medium (Gibco, Li e Technologies Co po a ion, G and Island, NY, USA) a 37
◦
C in a
humidi ied a mosphe e o 5% CO
2
. Log-phase BEAS-2B cells we e pla ed on 48-well pla es
(come assay), 96-well pla es (cy o oxici y and ROS assays) and 2-well chambe slides
(mic onucleus assay) om one o h ee days p io o exposu e o he NFC ma e ials.
2.5. Cellula In e naliza ion o NFC
To assess he po en ial in e naliza ion o NFC by BEAS-2B cells, NFC was s ained
wi h he Calco luo Whi e S ain (Me ck KGaA, Da ms ad , Ge many). The s aining was
pe o med on he same slides ha we e used o he sco ing o he mic onucleus equency
( o slide p epa a ion, see below). A ha es , he cells we e ea ed o 15 min a 25
◦
C
wi h cellulase (8.7
µ
L/mL, Cellic CTec2, No ozymes, Bages ae d, Denma k) o emo e
excess NFC ou side he cells. The p ocedu e o he calco luo s aining has p e iously been
desc ibed [
35
]. In b ie , a d op o calco luo was applied on o ac idine o ange-s ained slides
and incuba ed unde a co e slip o 1 min. The sample was he ea e examined wi h a
luo escence mic oscope (ZEISS Axio Image Z1, Ca l Zeiss AG, Obe kochen, Ge many),
using DAPI/FITC/TRITC iple il e and 40x objec i e lens.
2.6. Cy o oxici y Assessmen
BEAS-2B cells we e seeded in whi e clea la bo om 96-well pla es (Co ning, NY,
USA) a a densi y o 2
×
10
4
cells/well (200
µ
L/well; cul u e a ea 0.32 cm
2
/well) and
g own o semicon luency, a e which hey we e exposed o NFC dispe sions o 24 and
48 h a eigh doses: 3.9, 7.8, 15.6, 31.2, 62.5, 125, 250, and 500
µ
g/mL (equi alen o 2.4, 4.9,
9.8, 19.5, 39.1, 78.1, 156.2, and 312.5
µ
g/cm
2
). 0.1% T i on X-100 (AppliChem, Da ms ad ,
Nanoma e ials 2021,11, 389 5 o 17
Ge many) was used as posi i e con ol, while un ea ed cells se ed as nega i e con ol
a each ime poin . All ea men s we e pe o med in quad uplica e, and he expe imen s
we e epea ed h ee imes.
Cy o oxici y was measu ed using he CellTi e -GloVR Luminescen Cell Viabili y As-
say (P omega, Madison, WI, USA) acco ding o ins uc ions p o ided by he manu ac u e
(Technical bulle in, P omega Co po a ion, e ised 6/09, pa # TB288). This assay es ima es
he numbe o me abolically ac i e iable cells in he cul u e, based on he quan iza ion o
ATP, using Ul a-Glo
TM
Recombinan Luci e ase and measu emen o ela i e luminescence
by Fluo oskan Ascen FL (The mo Elec on Co po a ion, Van aa, Finland). Because some
nanoma e ials ha e been desc ibed o ha e op ical in e e ence [
36
], he measu emen s also
included he cellulose ma e ials wi hou cells. Howe e , he NFCs showed no in e e ence
wi h he luminome ic de e mina ions.
Cy o oxici y was exp essed as ela i e luminescence in he ea ed cul u es in compa -
ison wi h he un ea ed cul u es. This assay e lec s all ea men - ela ed e ec s (nec osis,
cell cycle delay and apop osis) ha educe he numbe o iable o li ing cells. In addi ion,
as eques ed by he OECD TG 487 [
37
], cy os asis was always measu ed when pe o ming
he mic onucleus assay, as a way o assessing cy o oxici y in he same cul u es ha we e
used o he mic onucleus assay. In his way, he adequacy o he chosen dose ange based
on he luminome ic assay could be con i med.
2.7. Fo ma ion o In acellula Reac i e Oxygen Species (ROS)
The le els o in acellula ROS we e measu ed using he chlo ome hyl de i a i e
o 2
0
,7
0
-dichlo odihyd o luo escein diace a e (CM-H
2
DCFDA) (In i ogen, Eugene, OR,
USA), acco ding o he manu ac u e ’s guidelines. DCFDA is a lipophilic cell pe meable
compound ha is deace yla ed in he cy oplasm by cellula es e ases and la e oxidized by
ROS o a highly luo escen molecule [
25
]. De i a i es wi h a hiol- eac i e chlo ome hyl
g oup allow o co alen binding o in acellula componen s, pe mi ing e en longe
e en ion wi hin he cell.
BEAS-2B cells we e pla ed on black clea la bo om 96-well pla es (Co ning, NY,
USA) a a densi y o 20,000 cells/well (200
µ
L/well; cul u e a ea 0.32 cm
2
/well) and
g own o semicon luency o wo days. A e being washed wi h Gibco
™
Dulbecco’s
Phospha e-Bu e ed Saline (DPBS, The mo Fishe Scien i ic, Paisley, Sco land), he cells
we e loaded wi h 2.5
µ
M CM-DCFDA in PBS o 30 min a 37
◦
C. The ea e , he loading
bu e was emo ed, and he cells we e washed wi h PBS. Then he cells we e ea ed
wi h he NFC dispe sions a eigh doses: 3.9, 7.8, 15.6, 31.2, 62.5, 125, 250 and 500
µ
g/mL
(equi alen o 2.4, 4.9, 9.8, 19.5, 39.1, 78.1, 156.2 and 312.5
µ
g/cm
2
). 2 mM H
2
O
2
(Sigma-
Ald ich Chemie, S einheim, Ge many) was used as a posi i e con ol, while un ea ed
cells se ed as a nega i e con ol a each ime poin . Fluo escence was eco ded a 3, 6,
and 24 h (exci a ion 485 nm, emission 538 nm) using a pla e eade (Fluo oskan Ascen
FL, Van aa, Finland). The a e age luo escen in ensi y was calcula ed by sub ac ing
backg ound alues. All ea men s we e pe o med in quad uplica es, and he expe imen s
we e epea ed h ee imes.
2.8. Geno oxici y Assessmen
2.8.1. Come Assay
The come (single cell gel elec opho esis) assay was used o s udy DNA s and b eaks
and alkaline labile si es in BEAS-2B cells a e exposu e o he NFC ma e ials. BEAS-2B
cells in log phase we e pla ed in 48-well pla es (cul u e a ea 0.95 cm
2
/well, cul u e medium
olume 250
µ
L/well; Co ning, NY, USA) wo days p io o exposu e. Exposu e ime
was 24 h, and he cells we e exposed o 6, 19, 56, 167, and 500
µ
g/mL (co esponding
o 1.6, 5.0, 14.7, 43.9, and 131.6
µ
g/cm
2
) o each NFC. Un ea ed con ols and posi i e
con ols ea ed wi h hyd ogen pe oxide (20 mM, Riedel-de Haen, Seelze, Ge many) we e
included in all se ies. All ea men s we e pe o med in duplica e, and he expe imen s
we e epea ed wice.

Nanoma e ials 2021,11, 389 6 o 17
The come assay was pe o med in alkaline condi ions (pH > 13) as desc ibed p e i-
ously [
38
]. The slides we e coded, and one sco e pe o med he come analysis using a
luo escence mic oscope (Axioplan 2, Zeiss, Jena, Ge many) and an in e ac i e au oma ed
come coun e (Kome 5.5, Kine ic Imaging L d., Li e pool, UK). The pe cen age o DNA in
he come ail om 200 cells pe dose and expe imen ( wo eplica es pe dose, wo slides
pe eplica e, 50 cells/slide) was used as a measu e o he amoun o DNA damage.
2.8.2. Cy okinesis-Block Mic onucleus Assay
The cy okinesis-block mic onucleus assay was applied o s udy ch omosomal damage
in BEAS-2B cells a e exposu e o he NFCs. The cells we e pla ed on 2-well chambe slides
(cul u e a ea 4.2 cm
2
/well, cul u e medium olume 2 mL/well; Nunc, Roskilde, Denma k)
a a densi y o 300,000 cells pe well and incuba ed o 24 h, o each semicon luency, p io
o he ea men .
Based on he cy o oxici y assay, he cells we e exposed o 48 h o i e doses o he
dispe sed ma e ials: 6, 19, 56, 167, and 500
µ
g/mL ( he co esponding doses we e 2.8,
9.0, 26.7, 79.5, and 238.1
µ
g/cm
2
). Cy ochalasin B (9
µ
g/mL; Sigma-Ald ich Chemie,
S einheim, Ge many) was added o he cell cul u es 6 h a e s a ing he ea men , o
induce binuclea ion o di iding cells. Un ea ed cul u es and cul u es ea ed wi h he
posi i e con ol mi omycin C (MMC; Sigma-Ald ich, S einheim, Ge many; 150 ng/mL)
we e included in all expe imen s. All cul u es we e p epa ed in duplica e.
A e he exposu e, he cells we e b ie ly insed in PBS, ea ed o 15 min a 25
◦
C
wi h cellulase enzyme blend (8.7
µ
L/mL), and insed again wi h PBS. Cellulase ea men
was pe o med o ge id o he nonin e nalized nano ib ils ha could in e e e wi h he
mic onucleus sco ing. The slides we e ai -d ied o 2 h, ixed o 10 min in absolu e
me hanol a 25
◦
C, ai -d ied, and kep a
−
20
◦
C un il s aining. The cells we e s ained
wi h ac idine o ange (32 mg/mL) o 1 min a e which he slides we e insed h ee imes
in Sö ensen bu e (pH 6.8), s ained wi h 4,6-diamidino-2-phenylindole (DAPI, 1
µ
g/mL)
o 5 min, insed in ap wa e , and allowed o d y. The slides we e kep a 4
◦
C p o ec ed
om ligh . Immedia ely be o e analysis, he slides we e moun ed in Sö ensen bu e and
co e ed wi h a co e slip.
The slides we e coded o a blinded analysis. All analyses we e pe o med by one
sco e . Cell p oli e a ion was i s measu ed o assess he possible e ec o he ea men s on
cell cycle delay (cy os a ic e ec ), as a means o ensu e ha he ea men s we e conduc ed
a app op ia e le els o cy o oxici y. To his end, cy os asis, based on he cy okinesis-
blocked p oli e a ion index (CBPI; OECD 2016), was calcula ed om 100 cells pe eplica e
cul u e (200 cells pe dose) as ollows:
%Cy os asis = 100 −100[(CBPI ea ed −1)/(CBPIcon ol −1)]
whe e: CBPI = [(No. mononuclea e cells) + 2(No. binuclea e cells) + 3(No. mul inuclea e
cells)]/( o al No. cells).
To e alua e he equency o mic onuclea ed cells, mic onuclei we e sco ed in 4000 bin-
uclea e cells pe ea men (2000 binuclea e cells pe eplica e; wo eplica es pe ea men )
using ZEISS Axio Image Z1 mic oscope (Ca l Zeiss AG, Obe kochen, Ge many). Binu-
clea e cells and mic onuclei in hem we e iden i ied using a 40
×
objec i e lens using a
FITC/TRITC double il e o ac idine o ange, and he mic onuclei we e e i ied wi h DAPI.
2.9. S a is ical Analyses
One-way analysis o a iance (ANOVA), ollowed by Dunne ’s mul iple compa ison
pos hoc es , was used o assess he e ec o he dose in all he assays. Dose- esponse in
he o ma ion o ROS and in he come and mic onucleus assays was e alua ed by linea
eg ession analysis. Compa isons be ween he posi i e and nega i e con ol g oups we e
pe o med by unpai ed one- ailed - es o all he assays. All analyses we e pe o med
using G aphPad P ism 8, e sion 8.3.1 (G aphPad So wa e, San Diego, CA, USA). The
e ec s we e conside ed signi ican i p< 0.05.
Nanoma e ials 2021,11, 389 7 o 17
3. Resul s
3.1. Cha ac e iza ion o he NFCs
Table 1summa izes he p ope ies o he NFC samples unde s udy. P-NFC had he
highes su ace g oup densi y among he unc ionalized ma e ials, ollowed by H-NFC,
S-NFC, and C-NFC. Low le els o ca boxyl g oups we e quan i ied in U-NFC, which can
be a ibu ed o he p esence o esidual hemicellulose.
Table 1. P ope ies o he nano ib illa ed cellulose (NFC) ma e ials unde s udy.
z-Po en ial (mV)
NFC
Sample Su ace Modi ica ion
Func ional G oup
Con en
(µmol/g)
Fibe Diame e
(Aqueous Suspension) 1
NaCl (10 mM,
pH 7.5) 2
Cell Cul u e Medium
LHC-9
(pH 6–8) 3
U-NFC None 30 410–30 nm agg ega es −10 ±2.5 5−14.1 ±5.2
C-NFC Ca boxyme hyla ion 371 Some indi idual ib ils,
ibe agg ega es (10–15
nm)
−20.9 ±1.8 −20.8 ±0.6
H-NFC Hyd oxyp opyl-
ime hylammonium
subs i u ion 634 4–5 nm indi idual ib ils 17.4 ±2.2 518.7 ±1.0
P-NFC Phospho yla ion 1109 4–5 nm indi idual ib ils −31.1 ±1.2 5−29.6 ±1.1
S-NFC Sul oe hyla ion 444 Some indi idual ib ils,
ibe agg ega es (10–12
nm)
−23.8 ±1.6 5−17.8 ±0.7
1
Es ima ed by TEM images;
2
measu emen s done a 25
◦
C,
3
measu emen s done a 37
◦
C;
4
esidual ca boxyl g oups;
5
F om Lopes e al. [
24
].
Z-po en ial measu emen s o he NFC suspensions in 10 mM NaCl e lec ed he
p esence o he su ace cha ged g oups, wi h posi i e alues o he NFC ma e ial wi h
he hyd op opyl ime hylammonium subs i u ion (H-NFC) and nega i e alues o he C-
NFC, P-NFC, and S-NFC samples. The di e ences in su ace g oup densi y we e e lec ed
in he z-po en ial absolu e alues, wi h he highes deg ee o unc ionaliza ion (P-NFC)
co esponding o he highes z-po en ial alue. The z-po en ial alues ob ained o he
suspensions in cell cul u e medium did no signi ican ly di e om he alues ob ained
wi h he NaCl suspensions. The absence o p o eins in he cell cul u e media and he simila
pH compa ed wi h he NaCl suspensions explain his simila i y.
The mo phology o he NFC ma e ials can be obse ed in he TEM images displayed
in Figu e 2. As expec ed, due o he lack o su ace cha ged g oups in U-NFC (only low
le els o esidual ca boxy g oups we e de ec ed), he ibe s o med agg ega es (10–30 nm
in diame e ; Figu e 2a), while he unc ionaliza ion o he NFC ma e ials esul ed in be e
dispe sion o he indi idual ibe s. Howe e , some di e ences we e ound be ween he
unc ionalized ma e ials, whe e he densi y o he su ace g oups seemed o in luence he
ibe mo phology. H-NFC and P-NFC (Figu e 2c,d, espec i ely) p esen ed indi idual
ib ils (4–5 nm in diame e ), while C-NFC wi h he lowes su ace g oup densi y o all
unc ionalized ma e ials showed ibe agg ega es wi h hickness up o 15 nm (Figu e 2b).
Sligh ibe agg ega ion was obse ed in he S-NFC sample (Figu e 2e), which may also be
ela ed o i s sligh ly lowe su ace g oup densi y compa ed wi h he highly de ib illa ed
H-NFC and P-NFC.
No bac e ial con amina ion was ound in he dispe sed NFC suspensions a e being
s e ilized by au ocla ing o UV ea men .
The esul s om he LAL assay showed ha he NFC ma e ials, excep H-NFC, had
a high le el o endo oxins, which was abo e he uppe de ec ion limi (>1.2 EU/mL) o
he assay. H-NFC showed a le el o 0.12 EU/mL which was below he 0.5 EU/mL limi
alue es ablished by he US Food D ug Agency o inhala ion s udies [
39
]. No es ki
in e e ence was de ec ed wi h any o he ma e ials. Howe e , he po en ial con ibu ion o
β
-glucan con aminan s in he NFC samples o he obse ed endo oxin le els could no be
dismissed, since he endo oxin ki employed did no inhibi he eac ion wi h β-glucans.
Nanoma e ials 2021,11, 389 8 o 17
When he p esence o (1,3)-
β
-D-glucans in ex ac s o NFC was measu ed, alues in
he concen a ion ange 250–20 pg/mL we e ound (Figu e S1), hus indica ing low le els
o (1,3)-β-D-glucan con aminan s in he NFC ma e ials [40].
Nanoma e ials 2021, 11, x FOR PEER REVIEW 8 o 18
Figu e 2. Rep esen a i e TEM images o nano ib illa ed cellulose (NFC) aqueous suspensions showing he mo phology
o he nano ibe s. (a) U-NFC, (b) C-NFC, (c) H-NFC, (d) P-NFC and (e) S-NFC.
No bac e ial con amina ion was ound in he dispe sed NFC suspensions a e being
s e ilized by au ocla ing o UV ea men .
The esul s om he LAL assay showed ha he NFC ma e ials, excep H-NFC, had
a high le el o endo oxins, which was abo e he uppe de ec ion limi (>1.2 EU/mL) o he
assay. H-NFC showed a le el o 0.12 EU/mL which was below he 0.5 EU/mL limi alue
es ablished by he US Food D ug Agency o inhala ion s udies [39]. No es ki in e e -
ence was de ec ed wi h any o he ma e ials. Howe e , he po en ial con ibu ion o β-
glucan con aminan s in he NFC samples o he obse ed endo oxin le els could no be
dismissed, since he endo oxin ki employed did no inhibi he eac ion wi h β-glucans.
When he p esence o (1,3)-β-D-glucans in ex ac s o NFC was measu ed, alues in
he concen a ion ange 250–20 pg/mL we e ound (Figu e S1), hus indica ing low le els
o (1,3)-β-D-glucan con aminan s in he NFC ma e ials [40].
3.2. In e naliza ion o NFCs
As calco luo s aining was pe o med on cellulase p e ea ed slides, nonin e nalized
NFC was no expec ed o be p esen , and hence de ec ed by he s aining, in hese p epa-
a ions. The e o e, s ained NFC ha appea ed associa ed wi h some cells (Figu e 3a,b)
may ha e e lec ed cellula in e naliza ion. Howe e , i may also ha e been due o NFC
ma e ial a ached o he cell memb ane ha was no e icien ly elimina ed by he cellulase
ea men . Ne e heless, mos cells did no show calco luo -s ained ma e ial, sugges ing
ha he possible NFC in e naliza ion conce ned a mino i y o he cells. No calco luo -
s ained ma e ial could be ound in he un ea ed cul u es (Figu e 3c). As NFC on he cells
could no eliably be dis inguished om in e nalized NFC, i emains unclea how much
he NFCs we e ac ually aken up.
Figu e 2.
Rep esen a i e TEM images o nano ib illa ed cellulose (NFC) aqueous suspensions showing he mo phology o
he nano ibe s. (a) U-NFC, (b) C-NFC, (c) H-NFC, (d) P-NFC and (e) S-NFC.
3.2. In e naliza ion o NFCs
As calco luo s aining was pe o med on cellulase p e ea ed slides, nonin e nalized
NFC was no expec ed o be p esen , and hence de ec ed by he s aining, in hese p epa a-
ions. The e o e, s ained NFC ha appea ed associa ed wi h some cells (Figu e 3a,b) may
ha e e lec ed cellula in e naliza ion. Howe e , i may also ha e been due o NFC ma e ial
a ached o he cell memb ane ha was no e icien ly elimina ed by he cellulase ea -
men . Ne e heless, mos cells did no show calco luo -s ained ma e ial, sugges ing ha
he possible NFC in e naliza ion conce ned a mino i y o he cells. No calco luo -s ained
ma e ial could be ound in he un ea ed cul u es (Figu e 3c). As NFC on he cells could
no eliably be dis inguished om in e nalized NFC, i emains unclea how much he
NFCs we e ac ually aken up.
Nanoma e ials 2021, 11, x FOR PEER REVIEW 9 o 18
Figu e 3. Examples o calco luo s aining (blue) in cells ea ed wi h U-NFC (167 µg/mL) (a), H-NFC (56 µg/mL) (b) and
un ea ed cells (c). Coun e s aining by ac idine o ange.
3.3. Cy o oxici y
None o he es ed NFCs signi ican ly educed he numbe o li ing cells p esen in
he cul u e as compa ed wi h he nega i e con ol (Figu e 4).
Figu e 4. Cy o oxici y o he nano ib illa ed cellulose (NFC) ma e ials. The o al numbe o li ing cells was coun ed a 24
and 48 h exposu e o (a) U-NFC, (b) C-NFC, (c) H-NFC, (d) P-NFC and (e) S-NFC, and he esul s we e no malized wi h
he unexposed con ol. Resul s a e p esen ed as he mean ± s anda d e o o he mean. G aphs a e plo ed wi h 95%
con idence in e al (colo ed a eas). The ange o 45 ± 5% li ing cells (co esponding o he 55 ± 5% cy o oxici y) is indica ed
by he g ey a ea in each g aph.
Figu e 3.
Examples o calco luo s aining (blue) in cells ea ed wi h U-NFC (167
µ
g/mL) (
a
), H-NFC (56
µ
g/mL) (
b
) and
un ea ed cells (c). Coun e s aining by ac idine o ange.
Nanoma e ials 2021,11, 389 9 o 17
3.3. Cy o oxici y
None o he es ed NFCs signi ican ly educed he numbe o li ing cells p esen in
he cul u e as compa ed wi h he nega i e con ol (Figu e 4).
Nanoma e ials 2021, 11, x FOR PEER REVIEW 9 o 18
Figu e 3. Examples o calco luo s aining (blue) in cells ea ed wi h U-NFC (167 µg/mL) (a), H-NFC (56 µg/mL) (b) and
un ea ed cells (c). Coun e s aining by ac idine o ange.
3.3. Cy o oxici y
None o he es ed NFCs signi ican ly educed he numbe o li ing cells p esen in
he cul u e as compa ed wi h he nega i e con ol (Figu e 4).
Figu e 4. Cy o oxici y o he nano ib illa ed cellulose (NFC) ma e ials. The o al numbe o li ing cells was coun ed a 24
and 48 h exposu e o (a) U-NFC, (b) C-NFC, (c) H-NFC, (d) P-NFC and (e) S-NFC, and he esul s we e no malized wi h
he unexposed con ol. Resul s a e p esen ed as he mean ± s anda d e o o he mean. G aphs a e plo ed wi h 95%
con idence in e al (colo ed a eas). The ange o 45 ± 5% li ing cells (co esponding o he 55 ± 5% cy o oxici y) is indica ed
by he g ey a ea in each g aph.
Figu e 4.
Cy o oxici y o he nano ib illa ed cellulose (NFC) ma e ials. The o al numbe o li ing
cells was coun ed a 24 and 48 h exposu e o (
a
) U-NFC, (
b
) C-NFC, (
c
) H-NFC, (
d
) P-NFC and
(
e
) S-NFC, and he esul s we e no malized wi h he unexposed con ol. Resul s a e p esen ed as he
mean
±
s anda d e o o he mean. G aphs a e plo ed wi h 95% con idence in e al (colo ed a eas).
The ange o 45
±
5% li ing cells (co esponding o he 55
±
5% cy o oxici y) is indica ed by he g ey
a ea in each g aph.
The pu pose o he cy o oxici y assay was also o choose he ange o doses o each
NFC o be es ed in he geno oxici y assays. Acco ding o OECD guidelines on he
in i o
mic onucleus es [
37
], he highes es subs ance concen a ion o be included in he
assay (in he absence o mic onucleus induc ion a ea lie doses) should p oduce 55 ±5%
cy o oxici y. Highe le els may induce ch omosome damage as a seconda y e ec o
cy o oxici y [
41
] and should, he e o e, be a oided. In he p esen s udy, he highes es ed
dose (500
µ
g/mL) did no each he cy o oxici y limi o any o he NFCs (Figu e 4). On
he o he hand, mo e concen a ed dispe sions we e no s able enough. Hence, 500
µ
g/mL
was chosen as he highes dose o be es ed in he o he assays.
The posi i e con ol, 0.1% T i on X-100, induced a s a is ically signi ican dec ease
in he numbe o li ing cells in all he expe imen s a bo h 24 h (3.31
±
0.14% li ing cells
Nanoma e ials 2021,11, 389 16 o 17
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