Role of surface chemistry in the in vitro lung response to nanofibrillated cellulose

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. 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