Biocompa ibili y and
inflamma o y esponse o sil e
ungs a e, sil e molybda e, and
sil e anada e mic oc ys als
B una Na ália Al es da Sil a Pimen el
1
,
Sa ah Raquel De Annunzio
1
, Ma celo Assis
2
,
Paula Aboud Ba bugli
1
, Elson Longo
3
and
Ca los Edua do Ve gani
1
*
1
School o Den is y, São Paulo S a e Uni e si y (Unesp), A a aqua a, B azil,
2
Depa amen o Physical and
Analy ical Chemis y, Uni e si y Jaume I (UJI), Cas elló, Spain,
3
CDMF, Fede al Uni e si y o São Ca los
(UFSCa ), São Ca los, B azil
Sil e ungs a e (α-Ag
2
WO
4
), sil e molybda e (β-Ag
2
MoO
4
), and sil e anada e
(α-AgVO
3
) mic oc ys als ha e shown in e es ing an imic obial p ope ies.
Howe e , hei biocompa ibili y is no ye ully unde s ood. Cy o oxici y and
he inflamma o y esponse o sil e -con aining mic oc ys als we e analyzed in
THP-1 and THP-1 di e en ia ed as mac ophage-like cells, wi h he alama Blue™
assay, flow cy ome y, con ocal mic oscopy, and ELISA. The p esen in es iga ion
also e alua ed edox signaling and he p oduc ion o cy okines (TNFα, IL-1β, IL-6,
and IL-8) and ma ix me allop o einases (MMP-8 and -9). The esul s showed ha
α-AgVO
3
(3.9 μg/mL) did no a ec cell iabili y (p>0.05). α-Ag
2
WO
4
(7.81 μg/mL),
β-Ag
2
MoO
4
(15.62 μg/mL), and α-AgVO
3
(15.62 μg/mL) sligh ly dec eased cell
iabili y (p≤0.003). All sil e -con aining mic oc ys als induced he p oduc ion o
O
2
−
and his e ec was mi iga ed by Reac i e Oxygen Species (ROS) sca enge and
N-ace ylcys eine (NAC). TNFα, IL-6 and IL-1βwe e no de ec ed in THP-1 cells,
while hei p oduc ion was ei he lowe (p≤0.0321) o simila o he con ol g oup
(p≥0.1048) o mac ophage-like cells. The p oduc ion o IL-8 by bo h cellula
pheno ypes was simila o he con ol g oup (p≥0.3570). The elease o MMP-8
was no de ec ed in any condi ion in THP-1 cells. Al hough MMP-9 was eleased
by THP-1 cells exposed o α-AgVO
3
(3.9 μg/mL), no significan di e ence was
ound wi h con ol (p= 0.7). Rega ding mac ophage-like cells, he elease o
MMP-8 and -9 dec eased in he p esence o all mic oc ys als (p≤0.010). O e all,
he p esen wo k shows a p omising biocompa ibili y p ofile o , α-Ag
2
WO
4
,β-
Ag
2
MoO
4
, and α-AgVO
3
mic oc ys als.
KEYWORDS
sil e -based me al oxides, cy okines, ma ix me allop o einases, eac i e oxygen species,
monocy es, mac ophages
1 In oduc ion
In ecen yea s, se e al s udies ha e e alua ed he an imic obial p ope ies o medical
ma e ials unc ionalized wi h nanopa icles o an ibio ics o imp o e hei p ope ies and
p e en in ec ions (T an and Webs e , 2013;Cas o e al., 2014;Zhu e al., 2014;Cas o e al.,
2016a;Cas o e al., 2016b;Hogan e al., 2019;Rangel e al., 2020;Ve za e al., 2021). Sil e
OPEN ACCESS
EDITED BY
Nagend a Kuma Kaushik,
Kwangwoon Uni e si y, Republic o
Ko ea
REVIEWED BY
Juliana Sil a Ribei o de And ade,
Fede al Uni e si y o San a Ca a ina,
B azil
Angel León-Bui imea,
Mon e ey Ins i u e o Technology and
Highe Educa ion (ITESM), Mexico
*CORRESPONDENCE
Ca los Edua do Ve gani,
[email p o ec ed]
RECEIVED 02 May 2023
ACCEPTED 12 July 2023
PUBLISHED 20 July 2023
CITATION
Pimen el BNAS, De Annunzio SR, Assis M,
Ba bugli PA, Longo E and Ve gani CE
(2023), Biocompa ibili y and
inflamma o y esponse o sil e
ungs a e, sil e molybda e, and sil e
anada e mic oc ys als.
F on . Bioeng. Bio echnol. 11:1215438.
doi: 10.3389/ bioe.2023.1215438
COPYRIGHT
© 2023 Pimen el, De Annunzio, Assis,
Ba bugli, Longo and Ve gani. This is an
open-access a icle dis ibu ed unde he
e ms o he C ea i e Commons
A ibu ion License (CC BY). The use,
dis ibu ion o ep oduc ion in o he
o ums is pe mi ed, p o ided he o iginal
au ho (s) and he copy igh owne (s) a e
c edi ed and ha he o iginal publica ion
in his jou nal is ci ed, in acco dance wi h
accep ed academic p ac ice. No use,
dis ibu ion o ep oduc ion is pe mi ed
which does no comply wi h hese e ms.
F on ie s in Bioenginee ing and Bio echnology on ie sin.o g01
TYPE O iginal Resea ch
PUBLISHED 20 July 2023
DOI 10.3389/ bioe.2023.1215438
has been used o cen u ies o ea in ec ions and he use o sil e
and sil e -con aining pa icles has inc eased in he pas ew yea s
(Poli ano e al., 2013). The li e a u e shows ha his me al has
an imic obial p ope ies agains a a ie y o mic oo ganisms, such
as S aphylococcus au eus,Esche ichia coli,Pseudomonas ae uginosa
and Candida albicans (Kim e al., 2009;Panáček e al., 2009;
Ma ínez-Gu ie ez e al., 2012;Dizaj e al., 2014;Shang e al.,
2019). Howe e , a limi ed numbe o s udies ha e in es iga ed i s
biocompa ibili y (Zhu e al., 2014;Rangel e al., 2020;Ve za e al.,
2021).
Acco ding o he li e a u e, depending on he size and
concen a ion o he pa icle, sil e can dec ease cell me abolism,
inc ease ROS p oduc ion, cy okine elease and e en induce
p og ammed cell dea h (Foldbje g e al., 2009;Liu e al., 2010;
Foldbje g e al., 2011;Pa k e al., 2011;Ma ínez-Gu ie ez e al.,
2012;Mu phy e al., 2016). To op imize he an imic obial p ope ies
and imp o e he biocompa ibili y o sil e , some in es iga o s ha e
combined his me al wi h di e en me al oxides, such as anada e
(VO
3
), ungs a e (WO
4
) and molybda e (MoO
4
)(Fabb o e al., 2016;
Foggi e al., 2017a;Foggi e al., 2017b;Pimen el e al., 2020;Pimen el
e al., 2022). P e ious s udies ha e shown ha sil e ungs a e (α-
Ag
2
WO
4
), sil e molybda e (β-Ag
2
MoO
4
) and sil e anada e (α-
AgVO
3
) showed no cy o oxic e ec on no mal o al ke a inocy es
(NOK-si) and gingi al fib oblas s (FGH) (Ha o Chá ez e al., 2018;
Assis e al., 2019;Pimen el e al., 2020). Fu he mo e, s udies ha e
shown ha α-Ag
2
WO
4
and α-AgVO
3
do no p omo e DNA
deg ada ion (Ha o Chá ez e al., 2018;Pimen el e al., 2020).
Howe e , despi e hese p omising findings, he biocompa ibili y
o hese ma e ials could no be gua an eed un il specific s udies
add essing oxida i e s ess, inflamma o y esponses, and
ex acellula ma ix pa hways we e conduc ed.
The inflamma o y esponse is a complex, mul i-s ep p ocess ha
occu s du ing inju y and in ec ion (Tu ne e al., 2014;Tu e al.,
2022). Inflamma ion is pa o he immune esponse and aims o
elimina e he o ending agen and ini ia e he healing p ocess leading
o issue and unc ional es o a ion (Tu e al., 2022). The li e a u e
epo s ha sil e pa icles, especially nanopa icles, ha e unique
chemical and physical p ope ies esponsible o hei an imic obial
ac i i y. I is al eady known ha me allic pa icles can indi ec ly
induce he p oduc ion o ROS due o he p esence o me allic ions
(Ha o Chá ez e al., 2018;Assis e al., 2019). Acco ding o he
li e a u e, ROS, including he supe oxide anion (O2•-), ac i a es he
NF-κB (nuclea ac o kappa B) and MAPK (mi ogen-ac i a ed
p o ein kinase) pa hways, which a e esponsible o s imula ing
IL-1β, TNFαand IL—6 genes (Ndengele e al., 2005;Ma ínez-
Gu ie ez e al., 2012;Mu phy e al., 2016;Yu e al., 2020;Canapa o
e al., 2021). Thus, he p esence o ROS can ac i a e he immune
esponse (Ak e e al., 2018) and s imula e he immune sys em o
p oduce a ious cy okines and o he inflamma o y media o s (Pa ks
e al., 2004;Abdulkhaleq e al., 2018).
Gi en he po en ial applica ion o α-Ag
2
WO
4
,β-Ag
2
MoO
4
and
α-AgVO
3
in den al ma e ials and medical de ices o p e en o al
in ec ions, i is impe a i e o es ablish hei abili y o mi iga e any
excessi e inflamma o y esponses. Fu he mo e, he ole o ma ix
me allop o einases (MMPs), which a e esponsible o issue
emodeling and healing (A aújo e al., 2011), mus be
unde s ood. P e ious s udies ha e shown ha MMPs a e s ongly
associa ed wi h pe iodon al disease, leading o he loss o
pe iodon al a achmen and bone des uc ion (F anco e al., 2017;
Al-Majid e al., 2018). Among he 23 ypes o MMPs al eady
iden ified, he up egula ion o MMP-8 and -9 has been ela ed o
pe iodon i is and pe i-implan i is (F anco e al., 2017;Checchi e al.,
2020) and i is associa ed wi h disease p og ession and bone loss
(A akawa e al., 2012;Al-Majid e al., 2018). High le els o MMP-8
and -9 a e ound in pe iodon al issues whe e he disease is
es ablished, possibly indica ing se e i y and p og ession o he
pa hology (F anco e al., 2017;Al-Majid e al., 2018;Checchi
e al., 2020). Addi ionally, MMP p oduc ion can con ibu e o
he ailu e o den al es o a ions (Hashimo o e al., 2016).
The e o e, he apies aimed a con olling MMP p oduc ion, while
a oiding cy o oxic and geno oxic e ec s and educing hei le els,
ha e he po en ial o e ec i ely p e en pe iodon al disease and
pe i-implan i is.
In his con ex , he p esen s udy e alua ed he cy o oxici y
p ofile, and he p oduc ion o eac i e oxygen species (ROS), p o-
inflamma o y cy okines (IL-1β, TNFα, IL-6, and IL-8), and MMPs
(−8 and −9), by THP-1 cells (human monocy es) and THP-1
mac ophage-like cells ollowing exposu e o sil e -con aining
mic oc ys als (α-Ag
2
WO
4
,β-Ag
2
MoO
4
and α-AgVO
3
).
2 Ma e ials and me hods
2.1 P epa a ion o mic oc ys als
Sil e ungs a e, sil e molybda e, and sil e anada e we e
p epa ed as p e iously desc ibed (Fabb o e al., 2016;Foggi e al.,
2017a;Oli ei a e al., 2017). B iefly, 1 × 10
−3
mol o sil e ni a e
(AgNO
3
; 99.98% pu i y; Cennab as, Gua ulhos, SP, B azil) was
dilu ed in 50 mL o dis illed wa e . Simul aneously, 5 × 10
−4
mol
o sodium ungs a e dihyd a e (Na
2
WO
4
•2H
2
O; 99.99% pu i y;
Sigma-Ald ich, S . Louis, MO, Uni ed S a es) o sodium
molybda e dihyd a e (Na
2
MoO
4
•2H
2
O; 99.98% pu i y; Al a
Aesa , Ha e hill, MA, Uni ed S a es) o 1 × 10
−3
mol o
ammonium me a anada e (NH
4
VO
3
; 99.99% pu i y; Sigma-
Ald ich, S . Louis, MI, Uni ed S a es) we e dilu ed in 50 mL o
dis illed wa e . Tempe a u es o 70°C o α-Ag
2
WO
4
and β-
Ag
2
MoO
4
and 10°C o α-AgVO
3
we e used. A e eaching he
empe a u es equi ed, he solu ions we e mixed, ins an ly o ming a
p ecipi a e. These p ecipi a es we e washed wi h dis illed wa e o a
pH o 7 and o en-d ied a 60°C o 12 h. A e syn hesis, all
mic oc ys als we e dilu ed in PBS o 2 mg/mL (s ock solu ion),
and he samples we e main ained in he da k and a oom
empe a u e un il u he use.
2.2 Physicochemical assessmen and sil e
concen a ion
The s uc u al cha ac e iza ion o he ma e ials was pe o med
a long- ange, a D/Max-2500 PC di ac ome e (Rigaku, Japan)
wi h Cu Kα adia ion (λ= 1.54056 Å) in he 2θ ange o 10°–80°
a a scan a e o 0.01°min
−1
. To analyze he mo phologies, a scanning
elec on mic oscope wi h a field emission gun (FEG-SEM) FEI
Model Inspec F50, ope a ing a 5 kV was used. Pa icle coun
analysis was pe o med using ImageJ so wa e, wi h a minimum
F on ie s in Bioenginee ing and Bio echnology on ie sin.o g02
Pimen el e al. 10.3389/ bioe.2023.1215438
coun o 100 pa icles. The sil e con en p esen in he
mic oc ys als su e s oxida ion du ing he syn hesis p ocess. To
calcula e he amoun o oxidized sil e [Ag
+
] in he mic oc ys als
s uc u e, fi s , he mic oc ys al concen a ion was con e ed om
µg/mL o µmol/mL using he ollowing equa ion: sil e con en in
each mic oc ys al concen a ion = mic oc ys al concen a ion
(µmol/mL) × 10
–6
/Molecula Weigh o he mic oc ys al. Then,
he amoun o sil e was calcula ed based on he numbe o mols
eleased by each mic oc ys al.
2.3 Mic oc ys als concen a ion agains
Candida albicans
The expe imen al g oups we e defined based on he minimal
inhibi o y concen a ion (MIC) and minimal ungicidal
concen a ion (MFC) om C. albicans ATCC 90028 pe o med
p e iously by Fabb o e al. (2016);Foggi e al. (2017a),; Pimen el
e al. (2020). Bo h α-Ag
2
WO
4
and β-Ag
2
MoO
4
p esen ed he same
MIC and MFC alues (7.81 μg/mL and 15.62 μg/mL, espec i ely o
each mic oc ys al). Fo α-AgVO
3
, he MIC and MFC alues we e
3.9 μg/mL and 15.62 μg/mL, espec i ely. Wo king solu ions we e
p epa ed immedia ely be o e use by dilu ing each mic oc ys al s ock
solu ion in Dulbecco’s modified Eagle’s medium (DMEM).
2.4 In i o THP-1 and mac ophages-like cell
cul u e and g ow h condi ions
The cell line THP-1 (human monocy es om pe iphe al blood)
was ob ained om he Rio de Janei o Cell Bank (BCRJ; cell line code
0234) and ou inely cul u ed a 37°C in a 5% CO
2
-humidified
en i onmen in Roswell Pa k Memo ial Ins i u e medium
(RPMI-1640; Sigma-Ald ich, S . Louis, MO, Uni ed S a es),
supplemen ed wi h 2 mM o glu amine (LONZA, Basel,
Swi ze land), 10 mM o HEPES (Sigma-Ald ich, S . Louis, MO,
Uni ed S a es), 1 mM o sodium py u a e (Sigma-Ald ich, S .
Louis, MO, Uni ed S a es), 4.5 g/L o glucose (Syn h, Diadema,
SP, B azil), 1.5 g/L o sodium bica bona e (Syn h, Diadema, SP,
B azil), 1% o an ibio ic/an imyco ic solu ion (Sigma-Ald ich, S .
Louis, MO, Uni ed S a es), 10% o e al bo ine se um (FBS; Gibco,
G and Island, NY, Uni ed S a es), and 0.09% o β-me cap oe hanol
(Gibco, G and Island, NY, Uni ed S a es). To ob ain he
mac ophages-like om THP-1 cells, be o e each expe imen , he
THP-1 cells we e seeded and s imula ed wi h 100 ng/mL o pho bol
12-my is a e 13-ace a e (PMA; Sigma-Ald ich, S . Louis, MO,
Uni ed S a es) (Pa k e al., 2007), which was added o he cell
cul u e medium and main ained a 37°C in a 5% CO
2
-humidified
en i onmen o achie ed he mac ophage pheno ype. A e 48 h, he
supe na an was disca ded and he mac ophages cells we e washed
wice wi h PBS. Subsequen ly, a esh medium was added and
main ained o e nigh be o e he assays.
2.5 Cell iabili y assay
Cell iabili y was pe o med a e 24 h o con ac wi h sil e -
con aining mic oc ys als, and i was assessed by alama Blue™
assay. THP-1 and mac ophages-like cells (1 × 10
6
/well) we e
seeded on 12-well pla es a a final olume o 3 mL o RPMI
medium wi h 5% FBS and main ained a 37°Cin5%CO
2
.A e
16 h, he cells we e washed wi h PBS, and he cell cul u e medium
wi hou FBS was added wi h sil e -con aining mic oc ys als (α-
Ag
2
WO
4
:7.81μg/mL; β-Ag
2
MoO
4
:15.62μg/mL; α-AgVO
3
:
3.9 μg/mL and 15.62 μg/mL). The pla es we e main ained a
37°Cin5%CO
2
, and a e 4 h and 24 h an aliquo o 100 µL
o he supe na an s om each well we e collec ed and s o ed
a −20°C un il he cy okine p oduc ion assay. A e 24 h o
con ac wi h he mic oc ys als, he cells we e incuba ed o 4 h
in a esh cell cul u e medium con aining 10% o alama Blue™
eagen (In i ogen, Ca lsbad, CA, Uni ed S a es). Then, 200 µL
o each well was ans e ed in quad uplica e o a black 96-well
pla e, and he fluo escence emission was measu ed (exci a ion:
544 nm; emission: 590 nm; Fluo oskan Ascen II, The moFishe
Scien ific, Wal ham, MA, Uni ed S a es). S anda d cell cul u e
condi ions we e used as li e cell con ol (CT) and cells incuba ed
wi h 10 µL o lysis bu e solu ion (LB; T i on-x 100 9%; Sigma-
Ald ich, S . Louis, MO, Uni ed S a es) we e used as dead cell
con ol. This assay was pe o med in quad uplica e and on h ee
di e en occasions.
2.6 In acellula ROS (O
2
−
) quan ifica ion
The p oduc ion o supe oxide (O
2
−
) induced by sil e -
con aining mic oc ys als on THP-1 and mac ophages-like cells
was conduc ed wi h dihyd oe hidium eagen (DHE; D23107;
In i ogen, Ca lsbad, CA, Uni ed S a es), a selec i ely p obe o
O
2
−
de ec ion. Cells we e seeded in a 96-well pla e a 2 × 10
4
cells/well
in K ebs-Henselei bu e (pH 7.0 ± 0.2). Then, 200 µL o DHE (1:
1000) was added o each well and he pla es we e main ained a 37°C
o 1 h. Fu he , he α-Ag
2
WO
4
(7.81 μg/mL), β-Ag
2
MoO
4
(15.62 μg/mL), and α-AgVO
3
(3.9 μg/mL and 15.62 μg/mL)
sil e -con aining mic oc ys als we e added o he co esponding
wells and he pla es we e main ained a 37°C o 1 h. The ea e , he
cells we e washed and, 100 µL o esh K ebs-Henselei bu e was
added o each well. The in acellula supe oxide p oduc ion was
measu ed by fluo escence emission in a fluo escence eade
(FLUOs a Omega, BMG Lab ech, Ca y, NC, Uni ed S a es; Ex.:
540-10nm; Em.: 620–10 nm). Cells unde s anda d cul u e
condi ions we e used as nega i e O
2
−
con ol, hyd ogen pe oxide
(H
2
O
2
[0.125 mM]; Sigma-Ald ich, S , Louis, MO, Uni ed S a es)
was used as posi i e O
2
−
con ol, and N-Ace yl-L-cys eine (NAC)
[0.01 mM] (Sigma-Ald ich, S . Louis, MO, Uni ed S a es) as a
sca enge . This assay was pe o med in quad uplica e and on
h ee di e en occasions.
2.7 In acellula ROS (O
2
−
) de ec ion by
con ocal lase scanning mic oscopy (CLSM)
Fo he CLSM assay, THP-1 and mac ophage-like cells we e
seeded in a 48-well pla e a 3 × 10
4
cells/well and incuba ed wi h
DHE p obe o 1 h a 37°Cin5%CO
2
. Then, he α-Ag
2
WO
4
(7.81 μg/mL), β-Ag
2
MoO
4
(15.62 μg/mL), and α-AgVO
3
(3.9 μg/
mL and 15.62 μg/mL) mic oc ys als we e added o he
F on ie s in Bioenginee ing and Bio echnology on ie sin.o g03
Pimen el e al. 10.3389/ bioe.2023.1215438
co esponding wells and he pla e we e incuba ed o ano he hou
a 37°Cin5%CO
2
. The p obe excess was emo ed and he CLSM
images we e ob ained wi h an LSM 800 mic oscope (Ca l Zeiss,
Obe kochen, Ge many) using a 561-nm lase , de ec ion o
b igh field and fluo escence spec a up o 700 nm,
wi h ×20 objec i e. Cells unde s anda d cul u e condi ions we e
used as nega i e O
2
−
con ol, hyd ogen pe oxide (H
2
O
2
[0.125 mM];
Sigma-Ald ich, S . Louis, MO, Uni ed S a es) as posi i e O
2
−
con ol,
and NAC [0.01 mM] (Sigma-Ald ich, S . Louis, MO, Uni ed S a es)
as sca enge con ol.
2.8 P oduc ion o p o-inflamma o y
cy okines
The IL-1β,TNFα, IL-6, and IL-8 cy okines p oduc ion was
assessed a e THP-1 and mac ophage-like cells we e exposed o
α-Ag
2
WO
4
(7.81 μg/mL), β-Ag
2
MoO
4
(15.62 μg/mL), and α-
AgVO
3
(3.9 μg/mL and 15.62 μg/mL) mic oc ys als, a 4 and
24 h o exposu e. The samples we e ob ained as desc ibed in
sec ion 2.5 and main ained a —20°C un il he analysis. The
Human Inflamma o y Cy okine Ki (Ca . No. 551811; BD
Biosciences, San Jose, CA, Uni ed S a es) was used acco ding
o he manu ac u e ’s ins uc ions. B iefly, while he samples
hawed a oom empe a u e, he lyophilized Human
Inflamma o y Cy okines S anda ds we e econs i u ed wi h
2 mL o Assay diluen , and hen a se ial dilu ion was
pe o med om 1:2 un il 1:256. The nega i e con ol (0 pg/
mL) was p epa ed only wi h Assay Diluen . Nex , a mix o
cap u e beads was p epa ed and 50 µL was added in each ube
(s anda d cu e and samples). Then, 50 µL o s anda d cy okines
o samples we e added o each co esponding ube, and finally
50 µL o Human Inflamma o y PE De ec ion Reagen we e added
o all ubes. A e 3 h o da k oom incuba ion, 1 mL o Wash
Bu e wasadded oall ubesandcen i ugeda 200g o 5 min.
The supe na an s we e ca e ully disca ded, and he pelle s we e
esuspended in 300 µL o Wash Bu e . The samples we e
analyzed using a BD FACSA ia™Fusion Flow Cy ome e (BD
Biosciences, San Jose, CA, Uni ed S a es), and all da a ob ained
we e e alua ed wi h he FCAP A ay so wa e 3 (BD
Biosciences, San Jose, CA, Uni ed S a es).
2.9 MMPs signaling
To e alua e he p oduc ion o MMP-8 and -9, THP-1 and
mac ophage-like cells we e seeded in 25-cm
2
flasks a a
concen a ion o 5 × 10
5
cells/flask in RPMI cul u e medium
con aining 5% FBS and 5% CO
2
a 37°C. A e 16 h, he cells
we e washed wi h PBS, and esh cul u e medium, wi hou FBS,
con aining α-Ag
2
WO
4
(7.81 μg/mL), β-Ag
2
MoO
4
(15.62 μg/mL),
and α-AgVO
3
(3.9 μg/mL and 15.62 μg/mL) mic oc ys als we e
added o he co esponden ea men flask. The cells we e
main ained a 37°Cin5%CO
2
o 24 h. Nega i e con ol cells
(CT) we e main ained unde s anda d cell cul u e condi ions, and
he posi i e con ol o MMP p oduc ion was assessed wi h cells
incuba ed wi h 1 μg/mL o lipopolysaccha ide om Esche ichia coli
(LPS; Sigma-Ald ich, S . Louis, MO, Uni ed S a es). Subsequen ly,
he supe na an s we e collec ed and s o ed a −20°C un il analysis.
This assay was pe o med in duplica e on wo independen
occasions. Be o e he ELISA assay, he amoun o o al p o ein in
each sample was measu ed wi h he B ad o d p o ein assay
(B ad o d, 1976) (Sigma-Ald ich, S Louis, MO, Uni ed S a es)
using bo ine se um albumin (BSA; Sigma-Ald ich, S . Louis, MO,
Uni ed S a es) as he s anda d. Spec opho ome ic measu emen s
we e pe o med a 595 nm (EZ Read 400 Mic opla e Reade ;
BioCh om, Cambou ne, CAM, Uni ed Kingdom). The MMPs
(−8 and −9) p oduc ion was de ec ed wi h he MMP-8 Human
ELISA Ki (ab100609, Abcam, Camb idge, CBE, Uni ed Kingdom)
and MMP-9 SimpleS ep ELISA Ki (ab246539; Abcam, Camb idge,
CBE, Uni ed Kingdom), acco ding o he manu ac u e ’s
ins uc ions. The OD was immedia ely ead a 600 nm using a
mic opla e eade (EZ Read 400 Mic opla e Reade ; BioCh om,
Cambou ne, CAM, Uni ed Kingdom). The final da a we e
no malized by he amoun o p o ein pe sample. This assay was
pe o med in iplica e in a single occasion.
2.10 S a is ical analysis
All da a ob ained we e analyzed o no mali y (Shapi o-Wilk’s
es ) and homoscedas ici y (Le ene es ). The s a is ical analysis o cell
iabili y and O
2
−
p oduc ion was pe o med wi h one-way ANOVA,
ollowed by Tukey’spos hoc on he IBM SPSS S a is ics so wa e
( e sion 23). Fo cy okine and MMP p oduc ion, a 95% confidence
in e al (CI) was defined o compa e he esul s among g oups. A
significance le el o 5% was adop ed.
3 Resul s
3.1 Mic oc ys als’cha ac e iza ion and sil e
concen a ion
The XRD and FEG-SEM analyses a e shown in Figu e 1. Fo he
α-Ag
2
WO
4
sample, he o ho hombic phase was ob ained, wi h a
Pn2n space g oup (PDF 34–61) (Figu e 1A). This phase has a
complex s uc u e, o med by se e al clus e s o [AgO
x
](x=2,
4, 6, and 7) and dis o ed oc ahed al clus e s o [WO
6
](Assis e al.,
2020). I s mo phology is composed o hexagonal mic o ods
(Figu e 1B) o a e age leng h and wid h o 0.95 ± 0.35 and
0.15 ± 0.06 µm, espec i ely. The β-Ag
2
MoO
4
phase was also
ob ained, wi h cubic s uc u e and Fd-3m space g oup (PDF
8–473) (Figu e 1C). This s uc u e has a lowe complexi y in
e ms o cons i uen clus e s, being o med by dis o ed
oc ahed al and e ahed al clus e s o [AgO
6
] and [MoO
4
],
espec i ely (Foggi e al., 2020). I s mo phology does no ha e a
polyhed al mic os uc u e, known as bean-like mo phology
(Figu e 1D). These pa icles ha e a high deg ee o agg ega ion,
coalescing in many cases. The a e age leng h and wid h ob ained o
his sample was 3.80 ± 0.80 and 1.40 ± 0.31 µm, espec i ely. Fo α-
AgVO
3
, i is obse ed ha he pu e phase is ob ained, wi hou any
addi ional peak, e e ing o he monoclinic phase wi h C2/c space
g oup (PDF 89–4396) (Figu e 1E). This phase is o med by dis o ed
oc ahed al clus e s o [AgO
6
] and dis o ed clus e s o [VO
4
](Sil a
e al., 2019). I s mo phology is homogeneous, wi h he shape o 4-
F on ie s in Bioenginee ing and Bio echnology on ie sin.o g04
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sided mic o ods (Figu e 1F). I s a e age leng h and wid h a e 9.17 ±
4.98 and 0.52 ± 0.18 µm, espec i ely, showing high sample size
dispe sibili y. The esul s ob ained o he h ee samples a e in
ag eemen wi h hose published in p e ious wo ks (Oli ei a e al.,
2017;Assis e al., 2021;Teodo o e al., 2022).
To calcula e he o al o sil e ions [Ag
+
] concen a ion ound in
he mic oc ys als, he a ge concen a ion o each one was
con e ed om µg/mL o µmol/mL, as ollowing:
(7.81 μg/mL)/(463.57 g/mol) o α-Ag
2
WO
4
= 0.0168 μmol/mL
o α-Ag
2
WO
4
;
(15.62 μg/mL)/(375.68 g/mol) o β-Ag
2
MoO
4
= 0.0416 μmol/
mL o β-Ag
2
MoO
4
;
(3.9 μg/mL)/(206.81 g/mol) o α-AgVO
3
= 0.0188 μmol/mL o
α-AgVO
3
;
(15.62 μg/mL)/(206.81 g/mol) o α-AgVO
3
= 0.0755 μmol/mL
o α-AgVO
3
.
Since, acco ding o he chemical defini ion, 1 mol o α-Ag
2
WO
4
and β-Ag
2
MoO
4
eleases 2 mol o Ag
+
each, he mola concen a ion
o Ag
+
in hese wo mic oc ys als is wice he concen a ion o α-
Ag
2
WO
4
and β-Ag
2
MoO
4
, 0.0168 μmol/mL and 0.0416 μmol/mL,
espec i ely. Thus, he o al [Ag
+
] was 0.0336 μmol/mL o
0.0156 μg/mL o α-Ag
2
WO
4
and 0.0832 μmol/mL o 0.0313 μg/
mL o β-Ag
2
MoO
4
. In he same way, 1 mol o α-AgVO
3
eleases
1 mol o Ag
+
, so he o al [Ag
+
] was 0.0188 μmol/mL o 0.0039 μg/
mL o α-AgVO
3
a 3.9 μg/mL, and 0.0755 μmol/mL o 0.0156 μg/
mL o α-AgVO
3
a 15.62 μg/mL.
3.2 Cell iabili y
The cell iabili y was e alua ed by alama Blue™assay (Figu e 2).
Fi s , when THP-1 cells we e main ained in con ac wi h α-Ag
2
WO
4
(7.81 μg/mL) and α-AgVO
3
(3.9 μg/mL), cell iabili y was
s a is ically simila o he con ol g oup (CT) (p>0.05)
(Figu e 2A). Howe e , he con ac o THP-1 cells wi h β-
Ag
2
MoO
4
(15.62 μg/mL) and α-AgVO
3
(15.62 μg/mL) p omo ed
a dec ease in cell iabili y as compa ed o CT (p= 0.0003 and p=
0.017, espec i ely) (Figu e 2A). Simila ly, he cell iabili y o
mac ophage-like cells was dec eased a e exposu e o α-Ag
2
WO
4
(7.81 μg/mL) and α-AgVO
3
(15.62 μg/mL), when compa ed o CT
(p= 0.0009 and p<0.0001, espec i ely) (Figu e 2B). The con ac o
α-AgVO
3
(3.9 μg/mL) and β-Ag
2
MoO
4
(15.62 μg/mL) mic oc ys als
wi h mac ophage-like cells p omo ed cell iabili y simila o CT (p=
0.05 and p= 0.991, espec i ely) (Figu e 2B).
Despi e he obse ed changes in cell iabili y, based on he
cy o oxici y classifica ion p oposed by Lonn o h and Dahl (2001),
Lönn o h and Dahl (2003) and Sle en and Dahl (1999),no
cy o oxici y was no ed when THP-1 cells we e ea ed wi h α-
FIGURE 1
X- ay di ac ion (XDR) pa e ns and FE-SEM images. (A,B) α-Ag2WO4; (C,D) β-Ag2MoO4; (E,F) α-AgVO3.
F on ie s in Bioenginee ing and Bio echnology on ie sin.o g05
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AgVO
3
(3.9 μg/mL; educ ion o 8.31% in cell iabili y), whe eas α-
AgVO
3
(15.62 μg/mL), α-Ag
2
WO
4
(7.81 μg/mL), and β-Ag
2
MoO
4
(15.62 μg/mL) we e sligh ly cy o oxic ( educ ion o 26.04%, 22.7%,
and 30.71% in cell iabili y, espec i ely). Fo mac ophage-like cells,
α-AgVO
3
(3.9 μg/mL), α-Ag
2
WO
4
(7.81 μg/mL), and β-Ag
2
MoO
4
(15.62 μg/mL) we e non-cy o oxic ( educ ion o 8.27%, 9.42%,
and −2.84% in cell iabili y, espec i ely), whe eas α-AgVO
3
(15.62 μg/mL) p esen ed sligh cy o oxici y ( educ ion o 29.64%
in cell iabili y).
3.3 In acellula O
2
−
quan ifica ion and
imaging
The gene a ion o O
2
−
by he cells a e he con ac wi h sil e -
con aining mic oc ys als was e alua ed wi h fluo escence emission
using a DHE p obe. The THP-1 cells exposed o sil e -con aining
mic oc ys als inc eased he p oduc ion o O
2
−
. As expec ed, when
cells we e incuba ed wi h mic oc ys als and he NAC sca enge was
added (+NAC), he e was a d op in he O
2
−
p oduc ion (Figu e 3A).
FIGURE 2
Mean alues o cell iabili y (%) o (A) THP-1 cells and (B)
mac ophage-like cells a e con ac wi h α-Ag2WO4 (7.81 µg/mL),
β-Ag2MoO4 (15.62 µg/mL), and α-AgVO3 (3.9 µg/mL and 15.62 µg/
mL) mic oc ys als o 24 hou s. E o ba s: s anda d de ia ion.
CT: li e cell con ol; LB: lysis bu e , dead cell con ol. Do ed line: 70%
o cell iabili y. G oups wi h as e isks a e s a is ically di e en om
con ol. *: p = 0.017; **: p <0.009; ***: p <0.0001. α= .05.
FIGURE 3
Mean alues o fluo escence in ensi y o THP-1 cells (A) and
mac ophage-like cells (B) using DHE p obe a e con ac wi h NAC
[0.01 mM], H2O2 [0.125 mM], and α-Ag2WO4 (7.81 µg/mL), β
Ag2MoO4 (15.62 µg/mL), and α-AgVO3 (3.9 µg/mL and
15.62 µg/mL) mic oc ys als (alone o wi h NAC). E o ba s: s anda d
de ia ion. NAC: N-Ace yl-L-cys eine; H2O2: hyd ogen pe oxide.
G oups wi h as e isks a e s a is ically di e en om con ol *: p =
0.026; **: p = 0.005; ***: p = 0.001; ****: p <0.0001. α= .05.
F on ie s in Bioenginee ing and Bio echnology on ie sin.o g06
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The highes dec ease ega ding O
2
−
p oduc ion was obse ed when
THP-1 cells we e exposed o α-Ag
2
WO
4
(7.81 μg/mL) + NAC and α-
AgVO
3
(3.9 μg/mL) + NAC, bu ye hey we e simila o con ol
g oup (CT; p>0.170) (Figu e 3A).
Mac ophage-like cells showed lowe p oduc ion o O
2
−
compa ed o THP-1 cells, which was simila o con ol g oup
(p>0.05) (Figu e 3B). Ne e heless, simila o THP-1, he e was
a dec ease in he O
2
−
p oduc ion in he p esence o NAC when
mac ophage-like cells we e exposed o all mic oc ys als, pa icula ly
o α-Ag
2
WO
4
(7.81 μg/mL; p<0.0001), α-AgVO
3
(3.9 μg/mL;
p<0.0001) and α-AgVO
3
(15.62 μg/mL; p<0.001) (Figu e 3B).
The CLSM images confi med he da a ob ained wi h he
in acellula fluo escence emission quan ifica ion. The THP-1
(Figu e 4) and mac ophage-like cells (Figu e 5) ea ed wi h
H
2
O
2
showed highe fluo escence han con ol (CT) (Figu es 4A,
B;Figu es 5A, B, espec i ely). The ea men wi h mic oc ys als also
p esen ed high fluo escence, which was dec eased when
mic oc ys als we e associa ed wi h NAC (Figu es 4C–J;
Figu es 5C–J).
3.4 P oduc ion o p o-inflamma o y
cy okines
The flow cy ome y analysis showed ha THP-1 cells p oduced only
IL-8 a bo h 4 and 24 h (Figu es 6A, B, espec i ely). Fo his cell line, only
α-Ag
2
WO
4
(7.81 μg/mL) was able o inc ease IL-8 p oduc ion a e 4 h o
con ac (p= 0.0136), when compa ed o he con ol g oup (CT).
Howe e , a e 24 h o con ac , he e we e no significan di e ences
(p≥0.7161) in he p oduc ion o IL-8 be ween all expe imen al g oups
and he con ol g oup. The o he cy okines e alua ed (IL-1β,TNFα,and
IL-6) we e no de ec ed in his cell line a any condi ions (da a no shown).
FIGURE 4
CLSM o THP-1 cells. (A) s anda d cul u e condi ions; (B)
H2O2 [0.125 mM]; C and D: αAg2WO4 (7.81 µg/mL) wi hou NAC (C)
and wi h NAC [0.01 mM] (D); E and F: β-Ag2MoO4 (15.62 µg/mL)
wi hou NAC (E) and wi h NAC [0.01 mM] (F); G and H: α-AgVO3
(3.9 µg/mL) wi hou NAC (G) and wi h NAC [0.01 mM] (H); I and J: α-
AgVO3 (15.62 µg/mL) wi hou NAC (I) and wi h NAC [0.01 mM] (J).
Red fluo escence: O2—p oduc ion.
FIGURE 5
CLSM o mac ophage-like cells. (A) s anda d cul u e condi ions;
(B) H2O2 [0.125 mM]; C and D: α-Ag2WO4 (7.81 µg/mL) wi hou NAC
(C) and wi h NAC [0.01 mM] (D); E and F: β-Ag2MoO4 (15.62 µg/mL)
wi hou NAC (E) and wi h NAC [0.01 mM] (F); G and H: α-AgVO3
(3.9 µg/mL) wi hou NAC (G) and wi h NAC [0.01 mM] (H); I and J: α-
AgVO3 (15.62 µg/mL) wi hou NAC (I) and wi h NAC [0.01 mM] (J).
Red fluo escence: O2—p oduc ion.
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Fo mac ophage-like cells, all cy okines we e de ec ed (Figu e 7).
The p oduc ion o TNFα, IL-6 and IL-1βwas lowe han CT a e 4 h
o con ac wi h all sil e -con aining mic oc ys als (p≤0.0321;
Figu es 7A, C, E). In con as , when compa ed o con ol, no
significan changes in he p oduc ion o IL-8 we e obse ed a e
4 h o exposu e o all expe imen al mic oc ys al (p≥0.1789;
Figu e 7G). A e 24 h o exposu e o α-AgVO
3
(15.62 μg/mL),
mac ophage-like cells showed a educ ion in TNFαp oduc ion
(p= 0.0035) (Figu e 7B). A his ime, no significan changes in
IL-6 p oduc ion we e obse ed, ega dless he expe imen al
mic oc ys als (p≥0.1549; Figu e 7D). Simila esul s we e
obse ed o IL-1βand IL-8, excep o α-AgVO
3
(15.62 μg/mL)
and β-Ag
2
MoO
4
(15.62 μg/mL), whe e he e was an inc eased
p oduc ion o IL-1β(p= 0.0006) and IL-8 (p= 0.0039),
espec i ely, a e 24 h o exposu e (Figu es 7F, H).
3.5 P oduc ion o MMP-8 and -9
The elease o MMPs by THP-1 and mac ophage-like cells, a e 24 h
o exposu e o sil e -con aining mic oc ys als, was measu ed by he
ELISA. I was no possible o de ec he p oduc ion o MMP-8 by THP-1
cells, e en unde s anda d cell cul u e condi ions o in he p esence o LPS
(da a no shown). The elease o MMP-9 was no de ec ed when hese
FIGURE 6
Mean alues o pg/mL o IL-8 p oduced by THP-1 cells a e
4 hou s (A) and 24 hou s (B) o con ac wi h α-Ag2WO4 (7.81 µg/mL),
β-Ag2MoO4 (15.62 µg/mL), and α-AgVO3 (3.9 µg/mL and 15.62 µg/
mL) mic oc ys als. E o ba s: s anda d de ia ion; CT: s anda d
cul u e con ol; LPS: lipopolysaccha ide, con ol. G oups wi h
as e isks a e s a is ically di e en om con ol. *: p <0.0463; ***: p <
0.0001. α= .05.
FIGURE 7
Mean alues o pg/mL o IL-1β, TNFα, IL-6, and IL-8 p oduced by
mac ophage-like cells a e 4 hou s (A,C,E,G) and 24 hou s (B,D,F,H)
o con ac α-Ag2WO4 (7.81 µg/mL), β-Ag2MoO4 (15.62 µg/mL), and
α-AgVO3 (3.9 µg/mL and 15.62 µg/mL) mic oc ys als. E o ba s:
s anda d de ia ion; CT: s anda d cul u e con ol; LPS:
lipopolysaccha ide, con ol. G oups wi h as e isks a e s a is ically
di e en om con ol. *: p <0.0321; **: p = 0.0035; ***: p <
0.0039. α= .05.
F on ie s in Bioenginee ing and Bio echnology on ie sin.o g08
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cells we e s imula ed wi h α-Ag
2
WO
4
(7.81 μg/mL), β-Ag
2
MoO
4
(15.62 μg/mL), and α-AgVO
3
(15.62 μg/mL) (Figu e 8A). The small
amoun o MMP-9 eleased a e 24 h o con ac wi h α-AgVO
3
(3.9 μg/mL) was no s a is ically di e en om he con ol g oup (p=0.7).
When e alua ing he elease o MMP-8 and -9 by mac ophage-like
cells, i was no ed ha all mic oc ys als p omo ed a dec ease in he
amoun o MMP-8 eleased (p<0.0001). Also, MMP-8 was no
de ec ed when hese cells we e main ained in con ac wi h α-AgVO
3
(15.62 μg/mL) o 24 h (Figu e 8B). The exposu e o mac ophage-like
cells o α-Ag
2
WO
4
(7.81 μg/mL), α-AgVO
3
(3.9 μg/mL), and α-AgVO
3
(15.62 μg/mL) esul ed in MMP-9 elease s a is ically simila o con ol
g oup (p≥0.216; Figu e 8C). Only mac ophage-like cells exposed o β-
Ag
2
MoO
4
(15.62 μg/mL) o 24 h showed a dec ease in MMP-9
eleased (p= 0.010).
In Figu e 9 we ha e a summa y o he main findings o his wo k.
4 Discussion
The use o sil e as an an imic obial agen has been ex ensi ely
s udied in ecen yea s. A he nanoscale, sil e has demons a ed
excellen an imic obial p ope ies by inducing he p oduc ion o
eac i e oxygen species (ROS) (Ca lson e al., 2008;Liu e al., 2010;
Pa k e al., 2011;Ak e e al., 2018;Canapa o e al., 2021;Liu e al.,
2021). Howe e , he gene a ion o ROS can also be esponsible o
cy o oxic e ec s on mammalian cells (Ca lson e al., 2008;Foldbje g
e al., 2009;Foldbje g e al., 2011;Nishan h e al., 2011;Canapa o
e al., 2021). Fu he mo e, sil e concen a ion has been epo ed as
a oxic ac o wi hin he ange o 10–100 μg/mL (Che nouso a and
Epple, 2013). To enhance he an imic obial p ope ies o sil e and
imp o e i s biocompa ibili y, esea che s ha e combined sil e wi h
di e en me al oxides, such as anada e (VO
3
), ungs a e (WO
4
),
and molybda e (MoO
4
). The compounds α-AgVO
3
,α-Ag
2
WO
4
, and
β-Ag
2
MoO
4
ha e shown an imic obial ac i i y agains C. albicans
(Fabb o e al., 2016;Foggi e al., 2017a;Foggi e al., 2017b;Assis
e al., 2019;Pimen el e al., 2020;Pimen el e al., 2022), me hicillin-
esis an S. au eus (MRSA) (Longo e al., 2014;Oli ei a e al., 2017;
Assis e al., 2018;Foggi e al., 2020) and E. coli (Canapa o e al.,
2021). They ha e been e ec i e in educing 3 log
10
(CFU/mL) o
6 log
10
(CFU/mL) (Longo e al., 2014;Fabb o e al., 2016;Foggi e al.,
2017a;Foggi e al., 2017b;Oli ei a e al., 2017;Pimen el e al., 2020;
Pimen el e al., 2022), wi hou causing damage o bo h human
ke a inocy es and fib oblas s cells (Ha o Chá ez e al., 2018;Assis
e al., 2019;Pimen el e al., 2020). The esul s epo ed he e indica e
a sligh dec ease in cell iabili y a e 24 h o con ac wi h α-α-
Ag
2
WO
4
(7.81 μg/mL), β-Ag
2
MoO
4
(15.62 μg/mL), and AgVO
3
(3.9 μg/mL and 15.62 μg/mL) mic oc ys als. This dec ease in
iabili y may be pa ly a ibu ed o he sil e con en and i s
abili y o gene a e eac i e oxygen species. P e ious heo e ical
s udies ha e sugges ed ha hese sil e -con aining mic oc ys als
a e o med by complex clus e s connec ed by weak in e ac ions and,
when in an aqueous en i onmen , hese clus e s can b eak wa e
molecules in o hyd oxyl adicals and p o ons (OH* and H
•
).
Simul aneously, he e is an elec on ans e o oxygen molecules
(O
2
), esul ing in he o ma ion o Oʹ
2
, which in e ac s wi h he
p o on (H
•
) o o m he adical HO
2
*(Fabb o e al., 2016;Foggi
e al., 2017a;Oli ei a e al., 2017).
The calcula ed sil e con en in he mic oc ys als used in his
s udy was app oxima ely 0.0156 μg/mL o α-Ag
2
WO
4
(a 7.81 μg/
mL), 0.0313 μg/mL o β-Ag
2
MoO
4
(a 15.62 μg/mL), 0.0039 μg/mL
o α-AgVO
3
(a 3.9 μg/mL), and 0.0156 μg/mL o α-AgVO
3
(a
15.62 μg/mL). A hese concen a ions, he sil e -con aining
mic oc ys als we e conside ed ei he non-cy o oxic o sligh ly
cy o oxic. Such concen a ions a e significan ly lowe when
compa ed o hose ound in sil e nanopa icles desc ibed in
p e ious s udies (Ma ínez-Gu ie ez e al., 2012;Ma inez-
Gu ie ez e al., 2013).
I is al eady known ha me al pa icles can indi ec ly induce
ROS p oduc ion due o he p esence o me al ions (Ha o Chá ez
e al., 2018;Assis e al., 2019). This oxida i e s ess can be
FIGURE 8
Mean alues o pg/µg o MMP-9 eleased by THP-1 cells (A) and
MMP-8 (B) and -9 (C) eleased by mac ophage-like cells a e
24 hou s o con ac wi h α-Ag2WO
4
(7.81 µg/mL), β-Ag2MoO
4
(15.62 µg/mL), and α-AgVO
3
(3.9 µg/mL and 15.62 µg/mL)
mic oc ys als. CT: s anda d cul u e condi ions; LPS:
lipopolysaccha ide, posi i e con ol; ND: non-de ec ed. G oups wi h
as e isks a e s a is ically di e en om con ol. *: p= 0.010; ***: p<
0.0001. α= .05.
F on ie s in Bioenginee ing and Bio echnology on ie sin.o g09
Pimen el e al. 10.3389/ bioe.2023.1215438