Modifications of Parylene by Microstructures and Selenium Nanoparticles: Evaluation of Bacterial and Mesenchymal Stem Cell Viability

Modifica ions o Pa ylene by Mic os uc u es and Selenium Nanopa icles: E alua ion o Bac e ial and Mesenchymal S em Cell Viabili y Jana Peka ko a 1 , 2 , Im ich Gablech 1 , 2 , Ta iana Fialo a 3 , Ond ej Bilek 4 and Zdenka Fohle o a 1 , 2 , 5 * 1 Cen al Eu opean Ins i u e o Technology, B no Uni e si y o Technology, B no, Czechia, 2 Depa men o Mic oelec onics, Facul y o Elec ical Enginee ing and Communica ion, B no, Uni e si y o Technology, B no, Czechia, 3 Depa men o Chemis y and Biochemis y, Mendel Uni e si y in B no, B no, Czechia, 4 Ins i u e o En i onmen al Technology, VŠB—Technical Uni e si y o Os a a, Os a a, Czechia, 5 Depa men o Biochemis y, Facul y o Medicine, Masa yk Uni e si y, B no, Czechia Pa ylene-based implan s o coa ings in oduce su aces su e ing om bac e ia coloniza ion. He e, we syn hesized poly inylpy olidone-s abilized selenium nanopa icles (SeNPs) as he an ibac e ial agen , and a ious app oaches a e s udied o hei ep oducible adso p ion, and hus he modifica ion o pa ylene-C–coa ed glass subs a e. The nanopa icle deposi ion p ocess is op imized in he nanopa icle concen a ion o ob ain e enly dis ibu ed NPs on he fla pa ylene-C su ace. Mo eo e , he a ay o pa ylene-C mic opilla s is ab ica ed by he plasma e ching o pa ylene-C on a silicon wa e , and he su ace is modified wi h SeNPs. All designed su aces a e es ed agains wo bac e ial pa hogens, Esche ichia coli (G am-nega i e) and S aphylococcus au eus (G am-posi i e). The esul s show no an ibac e ial e ec owa d S. au eus, while some bac e ios a ic e ec is obse ed o E. coli on he fla and mic os uc u ed pa ylene. Howe e , SeNPs did no enhance he an ibac e ial e ec agains bo h bac e ia. Addi ionally, all designed su aces show cy o oxic e ec s owa d mesenchymal s em cells a high SeNP deposi ion. These esul s p o ide aluable in o ma ion abou he po en ial an ibac e ial ea men o widely used pa ylene-C in biomedicine. Keywo ds: pa ylene-C, mic opilla s, selenium nanopa icles, biocompa ibili y, an imic obial INTRODUCTION Pa ylene is a commonly used polyme ic p o ec i eness o a wide ange o de ices in he o m o hin-film coa ing. Due o i s excellen an ico osion p ope ies, i is o en used as an insula o o elec onic componen s (Ma szalek e al., 2017). I s long- e m s abili y and biocompa ibili y in con ac wi h biofluids and issues make his polyme excellen o biomedical applica ions (Golda-Cepa e al., 2020). To da e, pa ylene has been used o coa implan able senso s and ansduce s (Zeniieh e al., 2014), and o hopedic o den al implan s, such as ca he e s and s en s (S au e e al., 2018). Due o i s na u al hyd ophobic cha ac e , he su ace has o be chemically ea ed (e.g., oxygen plasma o silaniza ion) o modified by p o eins o suppo , o example, cell g ow h (Chang, 2007;Song e al., 2009). Mo eo e , bac e ial coloniza ion and subsequen in ec ions emain one o he pos -complica ions o he many ha d and polyme ic implan s so Edi ed by: Magdalena M. S e ano ić, Ins i u e o Technical Sciences (SASA), Se bia Re iewed by: Hi ak K Pa a, Uni e si y College London, Uni ed Kingdom Manuel Ahumada, Uni e sidad Mayo , Chile *Co espondence: Zdenka Fohle o a [email p o ec ed].cz Special y sec ion: This a icle was submi ed o Bioma e ials, a sec ion o he jou nal F on ie s in Bioenginee ing and Bio echnology Recei ed: 24 Sep embe 2021 Accep ed: 10 No embe 2021 Published: 03 Decembe 2021 Ci a ion: Peka ko a J, Gablech I, Fialo a T, Bilek O and Fohle o a Z (2021) Modifica ions o Pa ylene by Mic os uc u es and Selenium Nanopa icles: E alua ion o Bac e ial and Mesenchymal S em Cell Viabili y. F on . Bioeng. Bio echnol. 9:782799. doi: 10.3389/ bioe.2021.782799 F on ie s in Bioenginee ing and Bio echnology | www. on ie sin.o g Decembe 2021 | Volume 9 | A icle 7827991 ORIGINAL RESEARCH published: 03 Decembe 2021 doi: 10.3389/ bioe.2021.782799 a , and pa ylene is no excep ion (Filipo iće al., 2020). Ne e heless, con en ional sys ema ic an ibio ic he apy o he in ec ed pa ien s has been ound o ha e low e ec i eness in some cases, and ad e se side e ec s, and inc eases he numbe o d ug- esis an bac e ia. D ug esis ance en o ces a high dose o an ibio ic (ATB) applica ion, o en gene a ing in ole able oxici y o he mammalian cells, de eloping new an ibio ics, and sea ching o he ad anced an imic obial p o ec ions and coa ings (Huh e al., 2011). Cu en ly, he u iliza ion o non-d ug an imic obial ma e ials and coa ings has been widely s udied. This in oduces nanoma e ials, including sil e (A ya e al., 2019), gold (Pik el e al., 2021), coppe (Usman e al., 2013), selenium (Huang e al., 2019), i anium oxide (Yousse e al., 2020), and zinc oxide (Janaki e al., 2015) nanopa icles; ca bon nanoma e ials (Azizi-Lalabadi e al., 2020); an imic obial pep ides (Raheem and S aus, 2019); o chi osan (Qi e al., 2004) which shows mo e o less an imic obial e ec i eness by hemsel es owa d a specific ype o bac e ia. The e o e, he di e en s a egies and combina ions o a ious ma e ials, geome ies, and chemis y a e eme ging oday o find enhanced an ibac e ial e ficiency. Fo ins ance, he modifica ion wi h an imic obial agen s (nanopa icles, pep ides, e c.), oxida ion, o su ace unc ionaliza ion has been widely s udied agains G am-posi i e and G am-nega i e bac e ia (Sha ma and Con ad, 2014;Bilek e al., 2020). Besides, he mechanical o physicochemical mic o-/nanos uc u ing o he su ace has been p o en o enhance he an ibac e ial p ope ies o some bioma e ials such as i anium, an alum, ha nium, and zi conium and i s oxides (Bilek e al., 2019;Fohle o a e al., 2021;2019). Since he polyme ic ma e ials used in medicine such as poly inylchlo ide (PVC), polyu e hanes (PU), silicone, and pa ylene also su e om bac e ial coloniza ion and in ec ion, he an imic obial modifica ion o hese polyme s and hei biocompa ibili y in i o and in i o ha e been s udied (Lee e al., 2013;Kupka e al., 2016). Fo example, h ee deposi ion modes o ZnO nanopa icle deco a ion o pa ylene-coa ed glass ha e been es ed o an ibac e ial ac ions (Apple o e al., 2010). The ZnO–pa ylene–glass composi es di e ed in hei pa icle sizes, coa ing hickness, and dep h o pene a ion, and demons a ed significan an ibac e ial ac i i y agains E. coli and S. au eus. The an imic obial coa ing has been de eloped o inhibi bac e ial g ow h on PVC, PU, and silicone by a coa ing o polyme ic subs a es wi h SeNPs in si u (T an and Webs e , 2013). The cy o oxici y o po ous silicon ma e ials wi h immobilized Au, Ag, and Cu nanopa icles has been s udied on L929 fib oblas s wi h he s a is ical di e ence o he CuNP subs a e (Solano-Umaña and Vega-Baud i , 2017). Te acycline nanopa icles embedded in o pa ylene film o p e en bac e ia- associa ed in ec ions ha e been s udied, and sel -s e ilizing he p ope ies o he su ace has been sugges ed (G inbe g e al., 2015). Se e al s udies showed he biocompa ibili y o deposi ed o pa e ned pa ylene-C o he mammalian cells (Gołda e al., 2013;Deli opoulos e al., 2015;Tu e al., 2017) wi h sugges ed good pe o mance a e plasma and p o ein modifica ion. To ou knowledge, he mic o-/nanos uc u ed pa ylene-C has no been s udied as he su ace wi h po en ial an ibac e ial ac i i y. Also, he syne ge ic e ec o such su ace wi h an ibac e ial SeNPs has no been e alua ed and compa ed wi h he fla pa ylene-C film so a . In his wo k, we p esen he basic expe imen al s udy o he syne gis ic e ec o SeNPs and mic os uc u ed pa ylene-C su ace in he o m o mic opilla s on he g ow h o G am- posi i e S. au eus and G am-nega i e E. coli bac e ia. Mic os uc u ed su ace has been ab ica ed by he op-down pa ylene-C e ching echnique. The adso p ion o poly inylpy olidone (PVP)-s abilized SeNPs was es ed on na u ally hyd ophobic, plasma- ea ed, and posi i ely cha ged silanized pa ylene-C su ace. Besides, due o he impo ance o cellula compa ibili y wi h he po en ially designed an ibac e ial su ace, we used mesenchymal s em cells o le hem in e ac wi h bo h fla and mic os uc u ed pa ylene-C su ace o e alua e he cellula iabili y and mo phology. Resul s showed he e con ibu e o he knowledge o he design and de elopmen o an ibac e ial biomedical coa ings based on pa ylene-C. MATERIALS AND METHODS P epa a ion o Fla Pa ylene-C Film Pa ylene-C laye wi h a hickness o ≈5 µm was deposi ed on Si wa e acco ding o he ollowing p ocedu e. 5.5 g o pa ylene-C was deposi ed on ≈100 mm Si (100) wa e by chemical apo deposi ion (CVD). In he nex s ep, wa e s wi h pa ylene-C laye we e cu in o (2 ×2) cm 2 using a semiau oma ic dicing saw ESEC 8003. Fab ica ion o Pa ylene-C Mic opilla s The a ay o mic opilla s was ab ica ed ia a“ op-down”p ocess in which 10 µm hick laye o pa ylene-C was deposi ed on he Si (100) wa e using he CVD me hod (Figu e 1A). The deposi ion o an ≈300-nm hick Ti laye using he elec on beam e apo a ion echnique was ollowed by s anda d UV pho oli hog aphy ia a mask aligne in he acuum con ac mode using he pho o esis (PR) AZ 5214E (Figu e 1B). The hickness o PR was 1.4 µm. PR de elopmen c ea ed he pa e n o hexagonally a anged ea u es wi h a diame e o 2 µm and a cen e - o-cen e dis ance o 4 µm. Then, i anium was e ched using eac i e-ion e ching (RIE) in Cl 2 plasma by means o chlo ine-based RIE. Finally, he wa e was placed in o he ion beam e ching (IBE) ins umen employing a Kau man ion-beam sou ce wi h collima ed g ids using pu e O 2 plasma o e ching he pa ylene-C om he a eas unco e ed by Ti. The PR was comple ely emo ed du ing he IBE p ocess, and he Ti esidue was addi ionally emo ed using chlo ine-based RIE (Figu e 1C). The las s ep was wa e cu ing in o single chips wi h dimensions o (2 ×2) cm 2 using em osecond lase , which elimina es he gene a ion o impu i ies in compa ison o o he cu ing echniques. Syn hesis o Selenium Nanopa icles Colloidal selenium nanopa icles we e p epa ed by we -chemical eac ion (Li e al., 2010). The aqueous solu ion o 25 mM sodium seleni e (Na 2 SeO 3 ) was mixed wi h aqueous solu ions o 28 mM L-cys ein (C 3 H 7 NO 2 S) and 25 μM PVP unde igo ous s i ing. When he uby ed colo o he mix u e de eloped, i F on ie s in Bioenginee ing and Bio echnology | www. on ie sin.o g Decembe 2021 | Volume 9 | A icle 7827992 Peka ko a e al. Biocompa ibili y o Mic os uc u ed Pa ylene-C indica ed he end o he eac ion, and s i ing was s opped. A e wa d, he colloidal solu ion was pu ified h ee imes wi h deionized wa e by ul acen i uga ion. SeNP solu ion wi h final concen a ion o 0.2 mg·ml −1 was subsequen ly dilu ed as ollows: no dilu ion (0×), 5×,10×,20×, and 50×. All chemicals we e pu chased om Sigma-Ald ich wi h a chemical pu i y g ade o pe analysis (p.a.). Sodium seleni e was used as a p ecu so o Se ions, L-cys ein as a educing agen , and poly inylpy olidone wi h an a e age molecula weigh o ≈40,000 g·mol −1 as a s abilizing agen . Nanopa icle mo phology and size we e cha ac e ized by he scanning ansmission elec on mic oscope (STEM). Nanopa icle ze a po en ial (Ze asize Nano ZS ins umen ; Mal e n Ins umen L d., Uni ed Kingdom) was measu ed a he condi ion o 25°C and equilib a ing ime 0 s. Calcula ions conside ing he diminishing o pa icle concen a ion we e based on he Smoluchowski model, wi h pa ame e s F (κa) o 1.50. Fo measu emen , disposable cu e es o ype DTS1070 we e used. The measu emen s we e pe o med unde he au oma ic se ing o a enua ion and ol age selec ion. All measu emen s we e done in iplica es. Deco a ion o Pa ylene-C Wi h Nanopa icles Si squa es wi h pa ylene film we e ea ed wi h O 2 plasma o 1 min o induce pa ylene film hyd ophilici y. Then, a sel -assembled monolaye (SAM) o 3-aminop opyl ime hoxysilane (APTES) was deposi ed using CVD. A 30 µl o APTES was pu on he glass slide nex o he wa e and hea ed o 120°C o 30 min in an SAM chambe (cus om-made). The adso p ion o SeNPs wi h a ious dilu ions (0×,5×,10×,20×, and 50×) o he pa ylene film was es ed and op imized o ba e, plasma- ea ed, and silanized su ace. Si wa e wi h pa ylene-C film was imme sed in o he pe i dish wi h a ious dilu ions o SeNP solu ion o 90 min. A e wa d, he Si wa e s deco a ed wi h SeNPs we e ho oughly insed wi h DI wa e and ai -d ied. We named ou samples Fc and Mc o con ol expe imen s on he fla (F) and mic opilla (M) subs a e, espec i ely. SeNP deco a ed samples we e named as F0-F50 and M0-M50 o a ious dilu ions (0×,5×, 10×,20×,and50×). Su ace Cha ac e iza ion The pa ylene-C modifica ion was cha ac e ized by measu ing he con ac angle (CA) by applying a wa e d op on he su ace. The CA was e alua ed using Su acewa e 8 so wa e, and he s a is ical analysis was pe o med on 5 d ops om each s ep o he modified su ace. The dis ibu ion o SeNPs on pa ylene-C was cha ac e ized wi h he scanning elec on mic oscope (SEM), and X- ay pho oelec on spec oscopy has been used o analyze su ace chemis y (XPS). XPS spec a we e analyzed by a peak fi ing so wa e (CasaXPS e sion 2.3.18PR1.0). An ibac e ial Tes An ibac e ial p ope ies o SeNP deco a ed pa ylene-C films (fla and mic opilla s) we e u he e alua ed ia he colony coun ing me hod using G am-nega i e bac e ia such as E. coli and G am- posi i e bac e ia such as S. au eus. Bac e ial s ains we e cul u ed on Columbia blood aga 5% (Labmediase is) a 37°C o e nigh . Bac e ial inoculum was p epa ed by dilu ing he bac e ial colonies in Muelle Hin on b o h (Sigma-Ald ich) o cell densi y 1–2·10 6 CFU·mL −1 . The samples we e insed in s e ile wa e , he bac e ial inoculum was sp ead on o he su ace, and he olume o he inoculum was 25 μm·cm 2 . Samples wi h inoculum we e co e ed by a s e ile plas ic oil and incuba ed o 24 h a 37°C. Subsequen ly, he samples we e insed in PBS, and adhe ed bac e ia we e de-a ached by o exing and sonica ion. Collec ed bac e ial suspensions we e dilu ed in PBS by decimal dilu ion, and he colony coun ing me hod was used o de e mine he bac e ial coun . The suspensions we e inocula ed on PCA aga pla es and incuba ed o 24 h a 37°C. Bac e ial colonies g owing on he pla es we e hen coun ed, and he colony o ming uni was calcula ed (CFU·cm −2 ). The expe imen was pe o med in iplica es. The samples wi hou nanopa icles bu plasma- ea ed we e aken as he con ol. The dimension o each sample was 2 ×2cm 2 . Viabili y o Mesenchymal S em Cells Mesenchymal s em cells (MSCs) isola ed du ing he plas ic su ge y we e cul u ed in he MSC g ow h medium (Sigma- Ald ich) supplemen ed wi h 5% FBS, 1% penicillin/ s ep omycin, and 1% L-glu amine. The cells we e ha es ed by ypsiniza ion in 0.25% solu ion o ypsin/EDTA a 80% confluence. The iabili y o cells was s udied by he e azolium- based assay. The p oli e a ion o MSCs on he samples was measu ed wi h 2,3-bis-(2-me hoxy-4-ni o-5-sul ophenyl)-2H- e azolium-5-ca boxanilide (XTT) and e alua ed on days 1 and 3 a e seeding; he ini ial cell densi y was 1·10 4 cells pe a ea. B iefly, he cells we e cul u ed o a defini e pe iod and hen FIGURE 1 | Scheme o mic opilla a ay ab ica ion by plasma e ching o pa ylene-C. Deposi ion o pa ylene-C, i anium, and pho o esis on Si wa e (A). T ans e o mask pa e n by pho oli hog aphy and i anium e ching (B). E ching o pa ylene-C o de elop mic opilla a ay (C). F on ie s in Bioenginee ing and Bio echnology | www. on ie sin.o g Decembe 2021 | Volume 9 | A icle 7827993 Peka ko a e al. Biocompa ibili y o Mic os uc u ed Pa ylene-C gen ly washed wice wi h p ehea ed PBS. The mix u e o 150 ml cul u e medium and 50 ml e azolium dye (XTT, 1 mg·mL −1 in PBS, pH 7.4) was added o he samples. A e 4-h incuba ion, he abso bance o he solu ion was measu ed a 450 nm. Fu he , op ical mic oscopy was used o obse e he cellula mo phology on he designed su aces. MSCs on he mic opilla s we e fixed wi h 4% pa a o maldehyde in PBS o 15 min, washed wice in bu e solu ion, and pe meabilized in 4% T i on-X100 solu ion o 1 h. Then, he cells we e washed in bu e , and immunos aining o cells was pe o med. Acco ding o he manu ac u e , he eady p obe Ac inG een (The mo Fishe ) hasbeenapplied os ainac in filamen s, and he cells we e imaged a 480 nm. MSCs on a fla su ace ha e been imaged by he DIC mode o he op ical mic oscope. The cell su ace a ea was measu ed using ImageJ so wa e. The expe imen was pe o med in iplica es. The samples wi hou nanopa icles bu plasma- ea ed we e aken as he con ol. The dimension o each sample was (2 ×2) cm 2 . S a is ical Analysis The da a a e p esen ed as mean ±s anda d e o . The s a is ical one-way analysis o a iance (ANOVA) wi h a confidence le el o 95% was used. RESULTS Cha ac e iza ion o Syn he ized Selenium Nanopa icles The mo phology and size o SeNPs we e de e mined by a high- esolu ion STEM wi h an accele a ing ol age o 30 kV. A 10 μlo SeNP colloidal solu ion was d opped on o a ca bon memb ane o STEM analysis and d ied a an ambien empe a u e o 22°C. STEM image in Figu e 2 p esen s SeNPs ha indica e a uni o m sphe ical shape o nanopa icles wi h no agglome a ions. The a e age pa icle size o SeNPs has been es ima ed (42 ±7) nm. The s abili y o SeNPs was analyzed by measu ing hei elec okine ic ze a (ζ)po en ial.The measu ed ζpo en ial o PVP-s abilized SeNPs syn hesized in his wo k was (−31.6 ±1.9) mV a pH 6.3 and 25°C. P epa a ion and Cha ac e iza ion o Selenium Nanopa icles/Pa ylene-C Films The deco a ion o pa ylene-C films was ini ially es ed by he adso p ion o PVP–SeNPs on un ea ed and plasma- ea ed fla pa ylene-C su aces. The SEM imaging did no show any nanopa icle deposi ion on such su aces (da a no shown). The su ace unc ionaliza ion o fla pa ylene-C wi h amino- e mina ed silane APTES p o ided ep oducible adso p ion o SeNPs as ollows: ≈12 SeNPs·µm −2 (no dilu ion), ≈9 SeNPs·µm −2 (5×dilu ion), ≈5 SeNPs·µm −2 (10×dilu ion), ≈3 SeNPs·µm −2 (20×dilu ion), and ≈1 SeNPs·µm −2 (50×dilu ion) (Figu e 3). The same modifica ion p ocedu e has been pe o med o mic os uc u ed pa ylene-C. Pa ylene-C mic opilla s ab ica ed by he e ching echnique o pa ylene-C p o ided a mic opilla a ay o he hexagonally o de ed pa ylene-C pilla s wi h ≈2µm diame e , ≈4 µm cen e - o-cen e dis ance, ≈8 µm leng h, and cylind ical mo phology, as confi med by a scanning elec on mic oscopy (Figu es 4A,B, he ep esen a i e MSCs a e shown on Figu e 4C). The pa ylene-C unc ionaliza ion was cha ac e ized by con ac angle measu emen (Figu e 5). A e oxygen plasma ea men , he fla pa ylene-C film wi h a con ac angle (CA)≈90°d opped o ≈25°.TheCA alue o he highly hyd ophobic mic os uc u ed pa ylene-C film d opped down om CA ≈110° o 90°. Silaniza ion o he su aces wi h APTES inc eased he CA alue o ≈65° o he fla pa ylene-C while emaining unchanged (≈90°) o he mic os uc u ed su ace. The p ocess o APTES and PVP–SeNP deposi ion on pa ylene- Cfilm was confi med by XPS analysis (Figu e 6). The wide spec um o he fla pa ylene-C film shows he main peak cha ac e is ics o un ea ed pa ylene-C (C1s, Cl 2s, and Cl 2p). The spec um o he APTES/SeNPs/pa ylene-C sample showed N1s peak om he APTES and PVP s abilizing agen , Si 2s and Si 2p peaks a ising om he silane deposi ion, and Se 3d; Se LMM peaks a e cha ac e is ic o he selenium nanopa icles. Table 1 summa izes he a omic pe cen age alues o he selec ed. An ibac e ial P ope ies o Pa ylene-C Films The an ibac e ial p ope ies o SeNP deco a ed pa ylene-C films agains G am-posi i e and G am-nega i e bac e ia ha e been e alua ed quan i a i ely. Figu e 7 shows esul s om he colony coun ing me hod o bo h bac e ia a e 24 h’incuba ion wi h he designed pa ylene-C su aces. The alues a e ela ed o he ini ial bac e ial densi y o 2.5·10 4 CFU·cm −2 (blue line) o easie e alua ion o an ibac e ial e ficiency. Da a showed ha he g ow h o S. au eus was no inhibi ed on any su aces, he fla (F; g ay colo ) and mic os uc u ed (M; pink colo ). Unexpec edly, selenium nanopa icles did no enhance he an ibac e ial e ficiency o designed su aces agains S. au eus as was expec ed. Mo e posi i e esul s ha e been ob ained o G am-nega i e E. coli. By compa ing bo h g oups (F and M), we can obse e he inhibi ion e ec o bac e ia g ow h on fla pa ylene-C and he mo e e iden bac e ios a ic e ec on mic os uc u ed pa ylene-C. Howe e , no significan di e ence has been obse ed o SeNP-deco a ed su aces (F0-F50 and M0-M50). FIGURE 2 | STEM image o selenium nanopa icles. F on ie s in Bioenginee ing and Bio echnology | www. on ie sin.o g Decembe 2021 | Volume 9 | A icle 7827994 Peka ko a e al. Biocompa ibili y o Mic os uc u ed Pa ylene-C Viabili y o Mesenchymal S em Cells on Pa ylene-C Films We pe o med he XTT es wi h mesenchymal s em cells o s udy he iabili y and p oli e a ion o MSCs on all designed su aces a day 1 and day 3 (Figu e 8). Figu e 8A showed a significan dec ease in cell adhesion and he p oli e a ion a e on SeNP- deco a ed fla pa ylene-C films compa ed o he undeco a ed pa ylene-C “con ol.”The samples wi h he lowes nanopa icle dilu ions (5×and 10×) exhibi ed almos 100% killing e ficiency o he mesenchymal s em cells e en a e 24 h. The cy o oxici y/ iabili y o cells was u he confi med by DIC imaging o MSCs by op ical mic oscopy (Figu e 9). By looking a he da a ob ained o he mic opilla su ace (Figu e 8B) and compa ing hem wi h he mic oscopic obse a ion (Figu e 10), one can no ice ha he MSCs adhe ed and p oli e a ed slowly on mic os uc u es compa ed o he fla su ace. Mo eo e , 5×dilu ion was s ill a o able o he cell su i al, and well-sp ead cells we e clea ly obse ed on he cap u ed images. We also measu ed he cell su ace a ea on each sample (Figu e 11). The cell su ace a ea co esponded well wi h he iabili y s age o cells. DISCUSSION In his wo k, mic os uc u ed and fla pa ylene-C film we e deco a ed wi h SeNPs o e alua e an ibac e ial e ec i eness o such designed su aces and hei biocompa ibili y o mesenchymal s em cells. SeNPs ha e been chosen as he widely p esen ed an ibac e ial agen (Geo ion e al., 2020;Filipo iće al., 2021). The syn hesized nanopa icles we e cha ac e ized by ζpo en ial and STEM mic oscopy. ζpo en ialisusedasapa ame e o helong- e m s abili y p edic ion o he colloidal solu ions (Low y e al., 2016). In gene al, he solu ion wi h he absolu e alue o ζpo en ial abo e 30 mV can be conside ed s able. The ζpo en ial is also dependen on se e al ac o s such as concen a ion, cha ge, empe a u e, and pH. The measu ed ζpo en ial o PVP-s abilized SeNPs syn hesized in ou FIGURE 3 | SEM images o SeNP deco a ed fla pa ylene-C wi h he di e en nanopa icle dilu ions shown o wo di e en scale ba s. FIGURE 4 | SEM images o he hexagonally o de ed mic opilla s ab ica ed ia e ching o pa ylene-C (A,B). The ep esen a i e SEM image o fixed mesenchymal s em cells laying on he mic opilla subs a e (C). F on ie s in Bioenginee ing and Bio echnology | www. on ie sin.o g Decembe 2021 | Volume 9 | A icle 7827995 Peka ko a e al. Biocompa ibili y o Mic os uc u ed Pa ylene-C wo k has been es ima ed a abou (−31.6 ±1.9) mV a pH 6.3 and 25°C, which co ela es wi h simila esul s o PVP-s abilized nanopa icles (V andečiće al., 2020). STEM mic oscopy showed nanopa icles wi h sphe ical shapeandana e agepa iclesizeo (42 ±7) nm (Figu e 2). The nanopa icle deposi ion on pa ylene-C films by simple adso p ion was fi s es ed o di e en ly ea ed fla pa ylene-C films. The s eng h o adso p ion o nanopa icles on he su ace depends mainly on he su ace chemical p ope ies and he opog aphy o bo h ma e ials. Ini ially, we s a ed wi h he adso p ion o PVP–SeNPs on un ea ed and plasma- ea ed fla pa ylene-C su aces ha , o ou su p ise, did no show any nanopa icle deposi ion. The e o e, we decided o pe o m he su ace unc ionaliza ion o pa ylene-C wi h amino- e mina ed silane APTES, and hus p o ide he posi i ely cha ged su ace o nega i ely cha ged PVP–SeNPs a pH 7. This unc ionaliza ion p o ided e enly dis ibu ed nanopa icle deposi ion on he fla pa ylene-C film (Figu e 3). The same modifica ion p ocedu e has been pe o med o he mic os uc u ed pa ylene-C film. Howe e , he SEM images o nanopa icle deco a ed mic os uc u es did no show any nanopa icles due o he bad con as e en a e su ace me alliza ion (da a no shown). The unc ionaliza ion o pa ylene-C films was cha ac e ized by con ac angle measu emen s in each s ep o he su ace modifica ion (Figu e 5). The fla pa ylene-C wi h a con ac angle (CA)≈90° d ama ically d opped o ≈25°a e oxygen plasma ea men , making he su ace hyd ophilic. Howe e , he CA dec ease o he highly hyd ophobic mic os uc u ed pa ylene-C was no ob ious, d opping down om CA ≈110° o ≈90°, making he su ace sligh ly hyd ophobic. Silaniza ion o plasma- ea ed su aces wi h APTES exposed he amino g oup on he pa ylene-C and he CA inc eased o ≈65°, and emained almos unchanged (≈90°) o hefla and mic os uc u ed su ace, espec i ely. The minimal change o CA o he mic os uc u ed su ace could co espond wi h he ac ha jus he uppe pa o pilla s has been exposed o oxygen plasma, and hus APTES. Fu he mo e, he XPS analysis was pe o med o bo h films o confi m he p ocess o APTES and PVP–SeNP deposi ion (Figu e 6 and Table 1). Pa ylene-C is poly (pa a-xylylene), in which chlo ine a om subs i u es one hyd ogen a om. The wide spec um o he fla pa ylene-C film shows he main peak cha ac e is ics o un ea ed pa ylene-C, such as C 1s, Cl 2s, and Cl 2p (Bi e al., 2014). The spec um o he APTES/SeNPs/pa ylene-C sample showed ha N 1s peak om he APTES and PVP-s abilizing agen , Si 2s and Si 2p peaks coming om he silane deposi ion and Se 3d, and Se LMM peaks a e he cha ac e is ics o he selenium nanopa icles. The pe cen age elemen al analysis o he fla and mic os uc u ed pa ylene-C film is summa ized in Table 1.Thelowe alues o he mic os uc u ed film could co espond wi h he p esence o gaps be ween pilla s. Ne e heless, he XPS analysis confi med he success ul unc ionaliza ion and deco a ion o pa ylene-C su aces wi h APTES and SeNPs, espec i ely. By finding he success ul adso p ion o nanopa icles by elec os a ic in e ac ion on he silanized su ace, we p oceeded o p epa e ep esen a i e samples di e ing in he numbe o nanopa icles pe a ea. Thus, we made a se ial dilu ion o nanopa icle s ock solu ion om 0× o 50×,andwele heSeNPs in e ac wi h he silanized su ace o 90 min. The densi y o nanopa icle co e age was con olled by SEM mic oscopy (Figu e 3). A e he op imiza ion o he nanopa icle adso p ion, we we e able o ep oducibly ob ain samples wi h ≈12 SeNPs·µm −2 (no dilu ion), ≈9SeNPs·µm −2 (5×dilu ion) ≈5SeNPs·µm −2 (10× dilu ion), ≈3SeNPs·µm −2 (20×dilu ion), and ≈1SeNPs·µm −2 (50× dilu ion). The SEM images o SeNP deco a ed pa ylene-C films also showed ha he nanopa icle size di e s om ha ob ained by STEM mic oscopy. SEM images in Figu e 3 es ima ed he size o FIGURE 5 | Con ac angle measu emen o he pa ylene-C–coa ed glass slide and mic opilla a ay unde he di e en su ace ea men (PC  pa ylene-C, O 2 plasma ea men , and APTES silane). FIGURE 6 | Rep esen a i e XPS wide spec um o he fla pa ylene- C–coa ed glass slide (blue) and pa ylene-C su ace wi h immobilized SeNPs ia APTES silaniza ion. F on ie s in Bioenginee ing and Bio echnology | www. on ie sin.o g Decembe 2021 | Volume 9 | A icle 7827996 Peka ko a e al. Biocompa ibili y o Mic os uc u ed Pa ylene-C nanopa icles abou (88 ±8) nm, which may be due o he sample coa ing by ≈10 nm laye o gold and a low- esolu ion SEM imaging. Ne e heless, we managed o ep oducibly deco a e he nanopa icles on amino- unc ionalized pa ylene-C o ob ain he ep esen a i e samples o u he an ibac e ial and cy o oxici y assays. Pa ylene-C, as many o he medical polyme s, does no exhibi any significan an ibac e ial p ope ies. On he o he hand, some ino ganic nanopa icles (Ag, Au, Se, e c.) possess an ibac e ial ac ion hemsel es (Gue e o Co ea e al., 2020). Deco a ion o such polyme ic ma e ials wi h hese nanopa icles has been ound as one o he s a egies o imp o ing he an ibac e ial p ope ies o polyme s. Selenium is he ace elemen o human heal h, and hus, SeNPs ha e become eme ging nanoma e ials in biomedicine. SeNPs ha e been shown in many s udies as p omising an imic obial agen s (Nguyen e al., 2017;Menon e al., 2020). Besides, he deco a ion o PU,PVC,andsilicon(T an and Webs e , 2013), as well as non- polyme ic bioma e ials (Bilek e al., 2019) wi h SeNPs, has inc eased he an ibac e ial p ope ies o ma e ials owa d G am-nega i e and G am-posi i e bac e ia in a ela i ely posi i e manne . The sugges ed co ela ion be ween SeNP size and an ibac e ial ac ion has been s udied o he nanopa icle size ange be ween 40 and 205 nm (Huang e al., 2019). The au ho s showed ha he bes an ibac e ial e ficiency was ound o 81 nm SeNPs. The an imic obial ac i i y o SeNPs wi h di e en su ace chemis y and s uc u e has also been s udied o se e al common bac e ial s ains (Filipo iće al., 2021). Mo eo e , mic o- and nanos uc u ing o ha d bioma e ials ha e also been ound o ha e significan impac agains he bac e ia compa ed o he non-s uc u ing ones (Li e al., 2019;Liu e al., 2020). He e, we s udied he syne gis ic an ibac e ial e ec o he modified pa ylene-C polyme wi h bo h, selenium nanopa icles and mic os uc u es in he o m o mic opilla s. As o he model o bac e ial cells, we chose G am-posi i e S. au eus and G am-nega i e E. coli.Bo hbac e ia di e om hememb anecomponen s,and hus, heymigh ha ea di e en esponse o nano-/mic o ma e ials (Epand and Epand, 2009). The p esen ed da a in Figu e 7 a e ela ed o he ini ial bac e ial densi y o 2.5·10 4 CFU·cm −2 (blue line). I enables one o es ima e whe he he su ace is a o able o bac e ia adhesion and g ow h o i exhibi s bac e ios a ic o bac e icide e ec . The g ow h o S. au eus was no inhibi ed on any su aces, he fla (F; g ay colo ) and mic os uc u ed (M; pink colo ), espec i ely, as i is shown in Figu e 7. Unexpec edly, selenium nanopa icles did no enhance he an ibac e ial e ficiency o bo h su aces, and mo e impo an ly, he significan di e ence has no been obse ed e en o he se ies o nanopa icle dilu ions. The mechanism o an ibac e ial ac ion o SeNPs is supposed o damage he cellula memb ane and p oduce eac i e oxygen species (ROS). (Huang e al., 2019). The possible explana ion o his non-e ec i i y o SeNPs o ac as an ibac e ial agen s can be due o he unsui able nanopa icle size, memb ane composi ion o G am-posi i e bac e ia, as well as he posi i e cha ge on he un-deco a ed pa ylene-C esidues, which can a ac he bac e ia and suppo hem in he g ow h. Mo eo e , he ab ica ed TABLE 1 | A omic pe cen age alues o he main elemen s p esen ing he fla and mic os uc u ed pa ylene-C be o e and a e he modifica ions as ex ac ed om he XPS spec um. Fla pa ylene-C O 1s (%) Se 3d (%) N 1s (%) Si 2p (%) Un ea ed 0.7 0 0.2 0 APTES and SeNP modifica ion 8.4 0.7 1.8 1.9 Mic os uc u ed pa ylene-C O 1s (%) Se 3d (%) N 1s (%) Si 2p (%) Un ea ed 0.4 0 0.2 0 APTES and SeNP modifica ion 2.9 0.3 0.8 0.8 FIGURE 7 | Colony coun ing me hod o G am-posi i e S. au eus and G am-nega i e E. coli a e 24 h incuba ion wi h he es ed samples. The blue line indica es he ini ial amoun o bac e ia deposi ed on he su ace a ea. Fc and Mc means fla and mic opilla su ace wi hou nanopa icles (con ol), espec i ely. F0-F50 and M0-M50 means nanopa icle dilu ions on he fla and mic opilla su ace, espec i ely. The ** indica es s a is ical significance o be ween g oups a p≤0.05. F on ie s in Bioenginee ing and Bio echnology | www. on ie sin.o g Decembe 2021 | Volume 9 | A icle 7827997 Peka ko a e al. Biocompa ibili y o Mic os uc u ed Pa ylene-C FIGURE 9 | DIC images o adhesion and iabili y o MSCs on SeNP deco a ed fla pa ylene-C film. FIGURE 10 | Recolo ed fluo escence images o adhesion and iabili y o MSCs on SeNP deco a ed mic opilla s. FIGURE 8 | P oli e a ion assay o MSCs on he SeNP deco a ed pa ylene-C fla subs a e (A) and SeNP deco a ed pa ylene-C mic opilla s (B). The * indica es s a is ical significance be ween each dilu ion a p≤0.05 and ** indica es significance be ween he con ol and each dilu ion a p≤0.05. F on ie s in Bioenginee ing and Bio echnology | www. on ie sin.o g Decembe 2021 | Volume 9 | A icle 7827998 Peka ko a e al. Biocompa ibili y o Mic os uc u ed Pa ylene-C mic opilla s did no influence S. au eus g ow h, p obably due o he “bigge ”mic os uc u es and pi ches ha made he su ace a o able o bac e ia coloniza ion e en in he in e pilla space. Conside ing he S. au eus and E. coli size o 0.5–1μmand1–2μm, espec i ely, bo h bac e ia can easily pene a e he ee space be ween pilla s, and hey may colonize he bo om o he subs a e. Ano he eason o he ine ec i eness o he su ace agains S. au eus may be he s onge cell wall s uc u e o G am-posi i e bac e ia and he sphe ical shape o he cocci, which allows hem o esis su ace i egula i ies mo e e ec i ely. Howe e , mos o hese easons esul ing in he g ow h o S. au eus on hese su aces a e p obably caused by he combina ion o wo o mo e ac o s. A li le bi di e en esul was ob ained o G am-nega i e E. coli. Compa ing bo h g oups (F and M), one can obse e an inhibi ion e ec o bac e ia g ow h on fla pa ylene-C and a mo e isible bac e ios a ic e ec on he mic os uc u ed pa ylene-C. Howe e , wi hin he wo g oups (F0-F50 and M0- 50), no significan di e ence has been obse ed. I means ha he SeNPs did no ac as an an ibac e ial agen , and mo e likely, he pa ylene i sel and mic os uc u ing played a mo e impo an ole in an ibac e ial ac ion owa d G am-nega i e bac e ia. Howe e , he e ec o he posi i ely cha ged su ace om he APTES unc ionaliza ion o pa ylene which has been men ioned abo e has been obse ed o a ac and suppo he g ow h o E. coli (Sha ma and Con ad, 2014). He e, i could con ibu e o he E. coli adhesion bu no o he g ow h, as shown in he g aph. Mo e likely, heposi i echa gehadbe e impac onG am-posi i eS. au eus adhesion and g ow h. Ou esul s om he SeNPs’ine ec i eness o ac as an an ibac e ial agen ha e also been obse ed p e iously (Nguyen e al., 2017). We belie e ha e en i he e can be he a ac ion o nega i ely cha ged bac e ial memb ane o he posi i ely cha ged su ace, he SeNPs, due o he size and epulsi e nega i e cha ge, canno pene a e he cell memb ane success ully and cause damage. Also, he di e en memb ane composi ions o G am- posi i e and G am-nega i e bac e ia can be mo e o less sensi i e o hese designed su aces. I has been published p e iously ha bo h ypes o bac e ia adhe ed apidly on he posi i ely cha ged su aces, bu wi h no ob ious g ow h o he G am-nega i e bac e ia, which can also co ela e wi h ou esul s (Go enbos e al., 2001). In conclusion, ou esul s highligh he di ficul y in unde s anding he oleo bac e ialcellsu acecha ac e is ics o henanopa icle esis ance and bioma e ial su ace p ope ies, and i mus be s udied in a mo e comp ehensi e way. The in e ac ion o he specific medical su ace wi h mammalian cells is he key ac o in de e mining ma e ial biocompa ibili y. A a ie y o bioma e ials a e designed, o example, o ha e high e ficiency o an ibac e ial ac ion and do no necessa ily exhibi cy ocompa ibili y o li ing cells. Since he nanopa icles like an ibio ics can kill bac e ia in a ce ain lowe o highe dose, no mal mammalian cells exposed o he same concen a ion o nanopa icles can also be killed. The e o e, i is impo an o find some balance be ween e ec i e an ibac e ial ac ion and none o low cy o oxici y o no mal cells and/o good e ec i eness agains cance cells. Mo eo e , mechanical o physicochemical mic o-/ nanos uc u ing o he bioma e ial su ace has been sugges ed as one o he key ac o s de e mining he cell adhesion, p oli e a ion, di e en ia ion, e c (Bilek e al., 2019;Fohle o a e al., 2021). In his wo k, XTT cy o oxici y and p oli e a ion assay ha e been pe o med wi h mesenchymal s em cells (Figu es 8A,B)and confi med by mic oscopic imaging o cells on es ed su aces (Figu e 9,10). Quan i a i e da a om he mic opilla -based su aces show mo e cy o oxic e ec owa d MSCs han he fla - based samples. Howe e , he image analysis confi med ha MSCs on he mic os uc u ed su aces we e jus less adhe ed and slowly p oli e a ed. Fu he mo e, he mo phology o MSCs in e ac ing wi h he fla pa ylene-C su aces o lowe SeNP dilu ions (5×and 10×) was mos ly o ounded shape due o he ob ious apop osis. MSC iabili y on mo e SeNP dilu ed fla samples (20×-50×) looked p olonged in shape, compa ed o he con ol sample, no fla ened, ec angula , and widesp ead on he su ace, which is shown by he DIC con as mode (Figu e 9). This could be caused by he su ace chemical and mo phological p ope ies inducing some s ess in cells. On he o he hand, he 5×dilu ion o nanopa icles on he mic os uc u ed su aces has s ill been a o able o he MSC iabili y. In he 10×dilu ed sample, he cells we e well sp ead wi h a p olonged and ec angula shape. MSCs on a con ol sample a e ound, widesp ead cells ha can be done by he highe cell densi y, hus leading o spa ial inhibi ion (Figu e 10). FIGURE 11 | Cell su ace a ea o MSCs cul u ed on selenium deco a ed he pa ylene-C fla subs a e (A) and SeNP deco a ed pa ylene-C mic opilla s (B).The su ace a ea was measu ed a day 1. The * indica es s a is ical significance be ween each dilu ion a p≤0.05, and ** indica es significance be ween he con ol and each dilu ion a p≤0.05. F on ie s in Bioenginee ing and Bio echnology | www. on ie sin.o g Decembe 2021 | Volume 9 | A icle 7827999 Peka ko a e al. Biocompa ibili y o Mic os uc u ed Pa ylene-C