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