Seleni e-Inco po a ed Amo phous Calcium−Magnesium Ca bona e
Nanopa icles Reduce Bac e ial G ow h
Yagmu Göc u, Caga ay M. O al, and Ba u E can*
Ci e This: ACS Appl. Nano Ma e . 2023, 6, 16286−16296
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ABSTRACT: Amo phous calcium ca bona e (ACC) is a non oxic and
deg adable nanoma e ial. ACC can be syn hesized using he cop ecipi a ion
echnique, which enables he inco po a ion o ions in o i s amo phous
s uc u e. Al hough ACC has been in es iga ed o a ious applica ions, such
as was ewa e ea men , in i o imaging, and d ug deli e y, i s an ibac e ial
p ope ies ha e no been explo ed. Conside ing he ex ao dina y capabili y o
bac e ia o adap an imic obial s a egies, as well as he ex ensi e bu den o
bac e ia-induced p oblems on heal hca e sys ems and he wo ld economy, he
need o e ec i e an ibac e ial agen s is becoming a p essing issue. He ein, we
in oduced seleni e-inco po a ed magnesium-s abilized amo phous calcium
ca bona e (ACMC) nanopa icles as a sus ainable an ibac e ial ma e ial. Fo
he i s ime, we demons a ed ha seleni e ions could be inco po a ed in o
ACMC nanopa icles while p ese ing he amo phous s uc u e. An ibac e ial
ac i i y analysis showed ha seleni e-inco po a ed ACMC (Se-ACMC)
nanopa icles a 1 g/L concen a ion could signi ican ly educe he g ow h o G am-posi i e (S aphylococcus au eus and
S aphylococcus epide midis) and G am-nega i e (Esche ichia coli and Pseudomonas ae uginosa) bac e ia s ains wi hin 24 h o
in e ac ion. As an impo an obse a ion, e en he lowes seleni e inco po a ion (4.38 ±0.19 mg selenium pe g o nanopa icles)
led o a mo e han 3-log educ ion in he numbe o S. epide midis colonies. Addi ionally, he an ibac e ial ac i i y was enhanced wi h
an inc ease in he amoun o inco po a ed seleni e. These esul s indica ed ha ion-inco po a ed ACMC nanopa icles can pa e he
way o applica ions as an ibac e ial agen s.
KEYWORDS: CaCO3, amo phous, ion inco po a ion, s abiliza ion, an ibac e ial
1. INTRODUCTION
Calcium ca bona e (CaCO3) is a well-known mine al used in
nume ous indus ial applica ions.
1
CaCO3has h ee anhyd ous
c ys alline polymo phs ( a e i e, a agoni e, and calci e) and
wo hyd ous o ms (monohyd ocalci e and ikai e).
2
Aside om
i s c ys alline polymo phs, CaCO3is also p esen in a
nonc ys alline o m as amo phous calcium ca bona e (ACC).
ACC is he ansien p ecu so o he c ys alline polymo phs,
3
and due o i s me as able na u e, i ans o ms in o he mo e
s able o ms o CaCO3in aqueous en i onmen s ia
dissolu ion and ec ys alliza ion eac ions.
4
Despi e he me as able na u e o ACC, se e al o ganisms in
na u e, such as sea u chins and mollusks, embody bo h he
amo phous and c ys alline CaCO3in hei hie a chical
s uc u es along wi h a ious o ganic molecules.
5
Hence,
inspi ed by na u e, he s abiliza ion o ACC using mac o-
molecules and ions was in es iga ed.
6,7
In he li e a u e, a ious
s udies u ilized Mg2+ ions o s abilize ACC and demons a ed
he p ecipi a ion o amo phous calcium−magnesium ca bona e
(ACMC) nanopa icles. I is known ha ACC has wo o ms:
hyd ous ACC and anhyd ous ACC. Hyd ous ACC ans o ms
o anhyd ous ACC h ough dehyd a ion and hen, ollowing an
ene ge ically downhill pa h, c ys allizes o calci e apidly.
8
Al hough he e ec s o Mg2+ on he s abiliza ion mechanism o
ACC ha e no been clea , i was mainly a ibu ed o he
c ea ion o a high dehyd a ion- ee ene gy ba ie , which could
decele a e he c ys alliza ion o calci e.
9
ACC is a non oxic, biodeg adable, and biocompa ible
nanoma e ial, and, consequen ly, i is a po en ial candida e
o nume ous enginee ing applica ions. Fo ins ance, in he
biomedical ield, polyme -modi ied ACC nanopa icles we e
p oposed o cance ea men by u ilizing hei pH-dependen
deg ada ion and high d ug-loading capaci y.
10
Likewise,
sodium algina e and phospha e-s abilized ACC nanoca ie s
loaded wi h cu cumin we e also in es iga ed o igh agains
cance .
7
Gadolinium-doped poly(ac ylic acid)-s abilized ACC
nanopa icles we e in es iga ed as a magne ic esonance
Recei ed: May 29, 2023
Accep ed: Augus 29, 2023
Published: Sep embe 12, 2023
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imaging con as agen due o he ex ensi e wa e con en o
ACC.
11
Addi ionally, ACC nanopa icles we e in es iga ed o
elimina e phospha e ions om was ewa e due o hei high
adso p ion capaci y.
12
These nanopa icles we e also p oposed
as a cemen addi i e o he sel -healing o mic oc acks,
13
and
upon s abiliza ion wi h magnesium, hey could be used as an
ink in 3D p in ing.
14
In spi e o he po en ial use o ACC
nanopa icles in a ious indus ies, hei e icacy as an i-
bac e ial agen s has no been explo ed.
Bac e ial coloniza ion and bac e ia-d i en in ec ion pose a
g owing h ea o he heal h o indi iduals. Bac e ia a e usually
ap o ga he ins ead o exis ing as sepa a e cells. Once bac e ia
colonize a su ace, hey syn hesize an o ganic ex acellula
ma ix laye . This s uc u e is called bio ilm and makes i much
ha de o igh agains bac e ia.
15,16
Fu he mo e, excessi e
an ibio ic use along wi h he ex ao dina y gene ic adap ion
capabili y o bac e ia led o an uncon ollable inc ease in
an ibio ic- esis an s ains. In ac , i is es ima ed ha he
cumula i e loss o economic ou pu due o an imic obial
esis ance in he OECD coun ies will amoun o app ox-
ima ely $20−35 illion by 2050.
17
Al hough he e a e a ious
s a egies o limi bac e ial coloniza ion, gene a ion o
haza dous byp oduc s, cos , oxici y, and de elopmen o
an ibio ic esis ance a e among he p oblema ic issues, which
g adually led o dec ease in e icacy o mos o he u ilized
an ibac e ial s a egies.
18,19
Consequen ly, he e is a emen-
dous need o edesign he cu en ly used disin ec ion me hods
and an ibac e ial ma e ials. To add ess he a o emen ioned
challenges, he u iliza ion o se e al al e na i e an ibac e ial
chemis ies, such as ca bon-based nanoma e ials, sil e ions,
ca ionic polyme s, and selenium (Se) nanopa icles, has
cap u ed g owing in e es in he pas decade.
20
Se is an essen ial elemen o li e wi h i s in ol emen in he
egula ion o cellula me abolism o many o ganisms.
21
I is
p esen in he amino acid selenocys eine (Sec), which
e en ually o ms selenop o eins o a ious enzymes.
22
In
addi ion, Se can neu alize he oxic e ec o hea y me als,
such as a senic and lead.
23
I is also known o ha e an ioxidan
and an icance p ope ies.
24
Among he oxyanions o Se,
seleni e (SeO32−) has he po en ial o be inco po a ed in o he
ACC s uc u e due o ha ing a simila geome y and cha ge
wi h ca bona e. In he li e a u e, he dose-dependen
an ibac e ial e ec o SeO32−ions agains se e al bac e ia
s ains, including S aphylococcus au eus,Esche ichia coli, and
Pseudomonas ae uginosa, was e i ied.
25,26
In his s udy, in an a emp o u ilize he deg adable na u e
o Mg2+-s abilized ACC (ACMC) nanopa icles, we in o-
duced SeO32−ions, and hus p o ided an ibac e ial cha ac e -
is ics. This is he i s s udy in he li e a u e in es iga ing
SeO32−-inco po a ed ACMC (Se-ACMC) nanopa icles
agains se e al bac e ia s ains (Scheme 1).
2. RESULTS AND DISCUSSION
2.1. Syn hesis and Cha ac e iza ion o Se-ACMC
Nanopa icles. To syn hesize ACMC nanopa icles, aqueous
solu ions o calcium ace a e monohyd a e (solu ion A),
sodium bica bona e, and magnesium chlo ide hexahyd a e
(solu ion B) we e p epa ed sepa a ely. Ha ing a high iscosi y,
e hylene glycol was added o each solu ion o dec ease he
di usion a e o he ions and, consequen ly, decele a e he
c ys alliza ion.
27
A e wa d, solu ion A was pou ed in o
solu ion B o ini ia e ACMC p ecipi a ion. SeO32−-ion-
inco po a ed nanopa icles we e syn hesized using he same
p o ocol excep o he addi ion o sodium seleni e (0.001,
0.005, o 0.01 M) in o solu ion B p io o mixing wi h solu ion
A (Figu e 1a). Acco ding o induc i ely coupled plasma mass
spec ome y (ICP-MS) analysis (Figu e 1b), he quan i y o
Se inco po a ed in o he nanopa icles inc eased when a highe
amoun o sodium seleni e was used du ing hei syn hesis. Fo
Se(I)-ACMC, 4.38 ±0.19 mg o Se was p esen o 1 g o he
nanopa icles, whe eas his alue inc eases o 21.83 ±0.19 and
40.98 ±0.14 mg o Se(II)-ACMC and Se(III)-ACMC
nanopa icles, espec i ely.
Scanning elec on mic oscopy (SEM) images showed ha
ACMC nanopa icles had an i egula pa icle mo phology
(Figu e 1c,e,g,i). I is impo an o no e ha low-magni ica ion
SEM images did no e eal any sign o a seconda y pa icle
mo phology independen o he amoun o inco po a ed
seleni e (Supplemen a y Figu e 1). The pa icle size
dis ibu ion his og ams we e d awn o each nanopa icle
specimen, and D50 alues we e ound as 32.0 ±0.4, 31.1 ±0.4,
31.9 ±0.3, and 32.5 ±0.4 nm o ACMC, Se(I)-ACMC,
Se(II)-ACMC, and Se(III)-ACMC nanopa icles, espec i ely
(Figu e 1d, ,h,j). T ansmission elec on mic oscopy (TEM)
cha ac e iza ions (Figu es 1k,l) u he con i med ha pa icles
had an i egula mo phology. The size measu emen o he
nanopa icles indica ed ha seleni e ion inco po a ion did no
lead o any changes in he pa icle size. The s uc u es o he
nanopa icles we e in es iga ed wi h X- ay di ac ion (XRD;
Figu e 1m), which exp essed he cha ac e is ic amo phous
humps o he ACMC nanopa icles
2
wi hou indica ing any
seconda y c ys alline phase (Supplemen a y Figu e 2). Addi-
ionally, he selec ed-a ea elec on di ac ion (SAED) pa e ns
o he nanopa icles u he alida ed he amo phous na u e o
he pa icles p io o (ACMC) and a e he highes seleni e
inco po a ion [Se(III)-ACMC]. These esul s con i med ha
he inco po a ion o seleni e in o he ACMC nanopa icles did
no al e he amo phous s uc u e o he nanopa icles.
Scheme 1. Se-ACMC Nanopa icles owa d An ibac e ial Applica ions
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Fou ie ans o m in a ed spec oscopy (FTIR) was used o
in es iga e he chemical bonding o he syn hesized nano-
pa icles. In Figu e 2a, he cha ac e is ic ca bona e bands o
ACMC a e designa ed by g ay sec ions. The symme ic
s e ching band a ∼1084 cm−1(ν1), he ou -o -plane band
a ∼859 cm−1(ν2), he asymme ic s e ching bands a 1412
and 1477 cm−1(ν3), and in-plane de o ma ion bands a a ound
688 and 728 cm−1(ν4) a e associa ed wi h he ca bona e ions
in ACMC.
28
Fu he mo e, he lack o calci e peak a 718 cm−1,
a e i e peak a 745 cm−1, and a agoni e peaks a 700 and 712
cm−1indica ed ha c ys alline polymo phs did no o m.
29
Compa ed o he FTIR spec um o ACMC, Se-ACMC
nanopa icles exp essed a band a ∼770 cm−1, which could be
asc ibed o he Se−O bending ib a ions o seleni e.
30
The
in ensi y o his band inc eased wi h an inc ease in he seleni e
concen a ion. The wide band a 3400 cm−1and he small
band a 1640 cm−1we e a ibu ed o O−H bonding:
s e ching and bending ib a ions o wa e , espec i ely,
which could s em om he physically and chemically bonded
wa e and hyd oxide ions in he molecule.
31
Wide-scan X- ay pho oelec on spec oscopy (XPS) esul s
show he elemen al composi ion o he nanopa icles (Figu e
2b). The p esence o calcium, magnesium, and oxygen
elemen s in all o he samples and Se in Se-ACMC
Figu e 1. (a) Ske ch showing he syn hesis p o ocol o he nanopa icles. (b) ICP-MS esul s o Se(I)-ACMC, Se(II)-ACMC, and Se(III)-ACMC
nanopa icles showing millig ams o Se pe g am o nanopa icles. SEM images o (c) ACMC, (e) Se(I)-ACMC, (g) Se(II)-ACMC, and (i)
Se(III)-ACMC nanopa icles ( he scale ba s a e 500 nm). Pa icle size his og ams o (d) ACMC, ( ) Se(I)-ACMC, (h) Se(II)-ACMC, and (j)
Se(III)-ACMC nanopa icles. TEM images and SAED pa e ns o (k) ACMC and (l) Se(III)-ACMC nanopa icles ( he scale ba s a e 100 nm).
(m) XRD pa e ns o he nanopa icles.
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nanopa icles was con i med wi h he analysis. The p esence o
hese elemen s was also in line wi h ene gy-dispe si e X- ay
spec oscopy (EDS) analysis o he nanopa icles (Supple-
men a y Figu e 1). Fu he mo e, a high- esolu ion scan o he
3d co e le el o Se was pe o med o de e mine i s oxida ion
s a e (Figu e 2c,d). Since Se(I)-ACMC nanopa icles had he
lowes Se inco po a ion, hey did no p o ide an in ense signal
su icien enough o accu a ely pe o m he decon olu ion
p ocess. On he o he hand, he binding ene gy o Se 3d5/2 was
ound o be 58.97 eV o Se(II)-ACMC (Figu e 2c) and 59.10
eV o Se(III)-ACMC (Figu e 2d), which could indica e he
oxida ion s a e as Se4+, highligh ing he p esence o seleni e.
32
The he mal p ope ies and wa e ac ion o he nano-
pa icles we e de e mined by he mog a ime ic analysis
(TGA; Figu e 3). The TGA cu e o ACMC nanopa icles
exhibi ed h ee majo mass loss e en s upon hea ing o 800 °C
(Figu e 3a). These mass loss e en s could be a ibu ed o he
emo al o physically and chemically bound wa e om he
nanopa icles, decomposi ion o MgO, and decomposi ion o
CaO, espec i ely.
33
XRD analysis o he hea - ea ed nano-
pa icles e ealed ha he amo phous s uc u e o he ACMC
nanopa icles could be main ained o a la ge ex en un il
app oxima ely 300 °C (Supplemen a y Figu e 3). Once
chemically bound wa e was emo ed om he sys em,
ACMC nanopa icles c ys allized as magnesian calci e. In
addi ion o his, in he second e en , magnesian calci e c ys als
decomposed in o MgO, and he emo al o CO2 om he
sys em led o he second mass loss. In he hi d e en ,
comple e decomposi ion o CaO and MgO occu ed wi h he
emo al o he emaining CO2 om he sys em. Fo he Se-
ACMC nanopa icles (Figu e 3b−d), simila o ACMC, he
ini ial elimina ion o physically and chemically bound wa e
and decomposi ion e en s we e also obse ed. I is wo h
men ioning ha , depending on he seleni e concen a ion, he
wa e con en o he ACMC nanopa icles ini ially inc eased
up o Se(II)-ACMC and hen dec eased o he Se(III)-ACMC
nanopa icles. While ACMC nanopa icles had ∼17% wa e ,
Se(I)-ACMC, Se(II)-ACMC, and Se(III)-ACMC nanopa -
icles had app oxima ely 28, 34, and 20% wa e , espec i ely.
This indica ed ha seleni e inco po a ion inc eased he
hyd a ion le el o ACMC. Fo he second mass loss e en ,
unlike ACMC, he de i a i e weigh loss cu es o Se-ACMC
nanopa icles exp essed double peaks. The exp ession o wo
peaks wi h o e lapping empe a u es indica ed wo sepa a e
decomposi ion e en s occu ing o he Se-ACMC nano-
pa icles. We belie e ha a seleni e phase o med du ing
c ys alliza ion o he Se-ACMC nanopa icles, which u he
decomposed in o a Se compound.
We can specula e ha he eason o he o ma ion o a
seconda y seleni e phase, a he han seleni e being inco po-
a ed in o he magnesian calci e c ys al, could be he size o
seleni e. In he li e a u e, i was shown ha , du ing he
c ys alliza ion o phospha e-inco po a ed ACC, calci e and
hyd oxyapa i e phases o med sepa a ely since phospha e ions
Figu e 2. (a) FTIR and (b) wide-scan XPS spec a o he nanopa icles. High- esolu ion XPS spec a o (c) Se(II)-ACMC and (d) Se(III)-ACMC
nanopa icles o Se 3d.
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could no be inco po a ed in o he calci e c ys al.
34
Conside ing ha seleni e (0.239 nm) has a size simila o
ha o phospha e (0.238 nm),
35
seleni e ions migh no be
inco po a ed in o he c ys al la ice and o med a seconda y
phase du ing c ys alliza ion. Hence, we deduced ha
amo phous CaCO3could be mo e a o able o seleni e
inco po a ion han c ys alline CaCO3.
Deg ada ion es s we e pe o med by measu ing he pH
alues o he samples in 1x phospha e-bu e ed saline (PBS) a
pH 7.4 (Supplemen a y Figu e 4a). The dissocia ion o ACMC
nanopa icles led o an inc ease in he pH alues, and his
could be associa ed wi h he elease o seleni e and ca bona e
o he aqueous solu ion. Fo he ACMC nanopa icles, he e
was a con inuous inc ease in he pH alue up o 24 h, and
a e wa d, he pH alue s abilized. Howe e , o Se-ACMC
nanopa icles, he e was a swi inc ease in he pH, which
indica ed a sudden dissocia ion and elease o ions wi hin
minu es. In ac , he pH alue was s able wi hin app oxima ely
he ange o 9.0 ±0.05 a all ime poin s. The ea lie inc ease
in he pH alue o Se-ACMC nanopa icles indica ed as e
dissolu ion o he nanopa icles and as e elease o he
seleni e ions in o he aqueous solu ion. To demons a e he
ime-dependen elease p o ile o seleni e om he nano-
pa icles, we conduc ed ICP-MS analysis using he ex ac s o
Se(III)-ACMC nanopa icles (Supplemen a y Figu e 4b). The
amoun o eleased Se in he solu ions was measu ed o be
0.267 ±0.008, 0.251 ±0.002, and 0.155 ±0.002 mg a 5 min,
1 h, and 72 h ime poin s, espec i ely. This analysis was in line
wi h he pH change esul s and con i med he sudden elease
o seleni e ions in o he solu ion. I is in e es ing o no e ha
he amoun o Se in he ex ac s dec eased wi h ime. One o
he easons o his beha io migh be he c ys alliza ion o
seleni e compounds in he ex ac s.
Pa icle size is one o he ac o s ha a ec s he solubili y o
ACMC pa icles. In gene al, he smalle he pa icle size, he
highe he solubili y.
36
In his s udy, we syn hesized highly
small pa icles wi h sizes less han 40 nm, which migh
con ibu e o he solubili y o he nanopa icles and he elease
o an ibac e ial seleni e ions. In addi ion, he TGA cu es
exhibi ed ha seleni e ions enhanced he hyd a ion le el o
ACMC. Speci ically, wa e molecules hea ily a ec he ion
mobili y and s uc u al al e a ions du ing c ys alliza ion,
37
consequen ly expedi ing he dissolu ion o ACC.
4
The
deg ada ion esul s showed ha Se-ACMC nanopa icles
deg aded as e han ACMC nanopa icles. When he
deg ada ion esul and TGA esul s a e in e p e ed oge he ,
Figu e 3. TGA showing he mass loss and de i a i e o he mass loss cu es o (a) ACMC, (b) Se(I)-ACMC, (c) Se(II)-ACMC, and (d) Se(III)-
ACMC nanopa icles.
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i could be specula ed ha seleni e ions in he ACMC s uc u e
could lead o an inc ease in he ion mobili y by inc easing he
wa e con en o he nanopa icles and, consequen ly, lead o
highe solubili y o he nanopa icles in an aqueous medium.
These esul s we e in line wi h he li e a u e, whe e Mg2+ ions
we e shown o b ing ex ensi e amoun s o wa e molecules
in o he ACC s uc u e, which impai ed he s eng h o he
ionic s uc u e in an aqueous medium.
37
In his s udy, Mg2+
con en s we e simila o all nanopa icles, and seleni e ions
u he con ibu ed o b inging mo e wa e o he nanopa icle
s uc u e, and hus inc easing he solubili y o he nano-
pa icles.
2.2. An ibac e ial Ac i i y o Se-ACMC Nanopa icles.
To e alua e he an ibac e ial ac i i y o he nanopa icles,
Figu e 4. An ibac e ial ac i i y o he nanopa icles agains (a and c) S. au eus, (b and d) S. epide midis, (e and g) E. coli, and ( and h) P. ae uginosa.
(c, d, g, and h) Pho og aphs o he aga pla es showing he di e ences in he CFUs be ween he con ol and Se(II)-ACMC o di e en bac e ia
s ains (***,p< 0.001; ns = no signi ican ).
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G am-posi i e S. au eus and S aphylococcus epide midis and
G am-nega i e E. coli and P. ae uginosa colonies we e cul u ed
wi h he nanopa icle ex ac s a 1 g/L concen a ion, and
colony- o ming uni s (CFUs) we e coun ed (Figu e 4a,b,e, ).
To isually demons a e he dec ease in CFUs, S. au eus,S.
epide midis,E. coli, and P. ae uginosa colonies g own in he
p esence o Se(II)-ACMC nanopa icle ex ac s a e displayed
in Figu e 4c,d,g,h, espec i ely. The esul s indica ed ha , a e
24 h o cul u e, ACMC nanopa icles did no signi ican ly
educe bac e ial g ow h, excep o S. epide midis. On he o he
hand, he seleni e-inco po a ed pa icles signi ican ly dec eased
he g ow h o all ou bac e ia s ains o all in es iga ed
composi ions (p< 0.001). Fo S. au eus (Figu e 4a,c), 81, 77,
and 87% dec eases in he CFUs we e obse ed o Se(I)-
ACMC, Se(II)-ACMC, and Se(III)-ACMC nanopa icles
compa ed o he con ol g oup wi hou nanopa icles (p<
0.001). Fo S. epide midis, ACMC nanopa icles educed he
CFUs by 85%, and Se-ACMC nanopa icles educed he CFUs
by 99.9% compa ed o he con ol g oup (p< 0.001). Fo he
case o E. coli, Se(I)-ACMC, Se(II)-ACMC, and Se(III)-
ACMC nanopa icles educed he CFUs by 30, 30, and 60%,
espec i ely, compa ed o he con ol g oup (p< 0.001). Las ,
o P. ae uginosa, he e we e 27, 38, and 80% educ ions in
CFUs upon cul u ing wi h Se(I)-ACMC, Se(II)-ACMC, and
Se(III)-ACMC nanopa icle ex ac s, espec i ely, compa ed o
he con ol g oup (p< 0.001). Among he es ed bac e ia
s ains, Se-ACMC nanopa icles we e he mos e ec i e
owa d S. epide midis, dec easing he CFUs by mo e han 3-log.
Acco ding o he CFU assay esul s, he e was no s a is ically
signi ican di e ence in he CFUs be ween he Se(I)-ACMC,
Se(II)-ACMC, and Se(III)-ACMC nanopa icles o he
G am-posi i e bac e ia s ains. In con as , Se(III)-ACMC
nanopa icles we e signi ican ly mo e e ec i e han ei he
Se(I)-ACMC o Se(II)-ACMC nanopa icles owa d G am-
nega i e bac e ia s ains. The hal -maximal inhibi o y concen-
a ion (IC50) alues o Se(I)-ACMC nanopa icles we e
calcula ed o be 0.64 ±0.09, 0.35 ±0.14, 1.69 ±0.10, and
1.52 ±0.08 g/L agains S. au eus,S. epide midis,E. coli, and P.
ae uginosa, espec i ely (Table 1). As he IC50 alue o he
nanopa icles, e en a he lowes concen a ion o seleni e
inco po a ion [Se(I)-ACMC], was conside ably highe han
he concen a ion u ilized in he CFU assay (1 g/L), i is
plausible ha he selec ed nanopa icle concen a ion did no
esul in a s a is ically signi ican di e ence in he CFUs o he
G am-posi i e s ains. Howe e , when he inco po a ed
an ibac e ial seleni e concen a ion inc eased 10- old, an-
si ioning om Se(I)-ACMC o Se(III)-ACMC nanopa icles, a
nanopa icle concen a ion o 1 g/L could elucida e he
di e ences in he an ibac e ial ac i i y o he nanopa icles.
CFU assay esul s clea ly showed ha Se-ACMC nano-
pa icles could signi ican ly educe bo h G am-posi i e and
G am-nega i e bac e ia g ow h. When he esul s we e
in es iga ed in mo e de ail, i was clea ha he an ibac e ial
ac i i y o seleni e ions owa d G am-posi i e bac e ia (S.
au eus and S. epide midis) was mo e p ominen han ha
owa d G am-nega i e bac e ia (E. coli and P. ae uginosa).
Al hough he p ecise an ibac e ial mechanism o ac ion o Se
and i s compounds emains unclea , i is unde s ood ha
ce ain bac e ial s ains, when capable o ole a ing hei
concen a ion, ha e he abili y o educe seleni e ions o
elemen al Se.
38
Wang e al. obse ed he p esence o biogenic
Se nanopa icles bo h wi hin and ou side cells along wi h
cellula emnan s as a esul o he in e ac ion be ween
seleni e- esis an bac e ia and seleni e. They hypo hesized ha
he an ibac e ial e ec migh s em om he elease o Se
nanopa icles wi hin he cells.
39
Based on his no ion, i can be
specula ed ha he bac e ial cell wall could po en ially hold
signi icance in e ms o p o iding p o ec ion agains seleni e.
The di e ence be ween he an ibac e ial e ec s on G am-
posi i e and G am-nega i e bac e ia could be explained by he
dis inc s uc u al p ope ies o he bac e ial cells. The cell wall
o G am-posi i e bac e ia consis s o a hick pep idoglycan
laye , while G am-nega i e bac e ia ha e a hinne pep idogly-
can laye enclosed by an ou e memb ane
40
and possess an
e lux pump sys em.
41
The lack o an ou e memb ane and
e lux sys em can make G am-posi i e bac e ia mo e
suscep ible o en i onmen al h ea s.
As shown in Supplemen a y Figu e 5, a e a 24 h incuba ion
o Se-ACMC nanopa icles wi h S. au eus,E. coli, and P.
ae uginosa, he colo o he T yp ic Soy B o h (TSB) medium
was obse ed o change o ed-o ange. I was epo ed ha his
colo change was an indica o o he in ol emen o bac e ia in
he educ ion o seleni e o Se,
42
and hose elemen al Se
nanopa icles educed by he bac e ial enzymes ha e a less
oxici y han seleni e ions.
43
Fo ins ance, biogenic Se
nanopa icles p oduced by S. au eus,E. coli, and P. ae uginosa
s ains a 25−175 g/L concen a ion did no exe any
cy o oxic e ec on he p ima y human de mal ib oblas cell
line.
44
Conside ing he oxic and alle gen na u e o
an ibac e ial agen s, such as qua e na y ammonium sal s, he
u iliza ion o seleni e anions o dope ACMC nanopa icles
agains bac e ia could be a sus ainable al e na i e. I is
in e es ing o no e ha , upon 24 h cul u e o S. epide midis
wi h Se-ACMC nanopa icle ex ac s, an appa en colo change
was no de ec ed (Supplemen a y Figu e 5). The lack o o
limi ed occu ence o seleni e o Se educ ion in S. epide midis
migh explain he signi ican decline in he g ow h o S.
epide midis compa ed wi h he o he s ains. The high
concen a ions o seleni e emaining inside he TSB solu ion,
a he han being educed o biogenic Se, migh ha e led o
highe an ibac e ial ac i i y and led o a mo e han 3-log
dec ease in he S. epide midis colony coun s. I should be no ed
ha , o S. epide midis, Se-ACMC and ACMC nanopa icles a
1 g/L concen a ion signi ican ly educed bac e ia g ow h.
Al hough he eason behind his end is no clea , i could be
possible ha he eleased Mg2+ ions migh ha e con ibu ed o
he an ibac e ial ac i i y agains S. epide midis.
45
None heless,
u he esea ch is equi ed o explain why seleni e- ee ACMC
nanopa icles we e e ec i e in he inhibi ion o S. epide midis
g ow h.
Al hough he syn hesized nanopa icles exhibi ed an ibac e-
ial p ope ies, he concen a ion-dependen oxici y o Se and
i s compounds is a challenge. Se is ad an ageous when
expe ienced in small amoun s bu can become ha m ul in
la ge quan i ies. In his ega d, he spec um be ween
insu iciency and abundance is qui e na ow.
38
To assess he
Table 1. IC50 Values o Se(I)-ACMC Nanopa icles agains
S. au eus,S. epide midis,E. coli, and P. ae uginosa
G am-posi i e G am-nega i e
S. au eus S. epide midis E. coli P. ae uginosa
Se(I)-
ACMC
(g/L)
0.64 ±0.09 0.35 ±0.14 1.69 ±0.10 1.52 ±0.08
ACS Applied Nano Ma e ials www.acsanm.o g A icle
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16292
cy o oxici y o he nanopa icles, a cell iabili y assay was
conduc ed (Supplemen a y Figu e 6). While Se(II)-ACMC
and Se(III)-ACMC nanopa icles did no suppo ib oblas
p oli e a ion a ei he 0.01 o 0.1 g/L concen a ion, Se(I)-
ACMC nanopa icles p omo ed ib oblas iabili y and
p oli e a ion up o 5 days in i o. Howe e , Se-ACMC
nanopa icles showed oxici y on ib oblas s a 1 g/L
concen a ion independen o he amoun o inco po a ed
seleni e. Since oxici y is a big conce n o biomedical
applica ions, we belie e ha he aqueous s abili y o he Se-
ACMC nanopa icles equi es u he imp o emen o
ansla ion in o comme cial applica ions.
3. CONCLUSIONS
In his s udy, we syn hesized Se-ACMC nanopa icles o
an ibac e ial applica ions. SEM, TEM, and XRD esul s
con i med ha ACMC nanopa icles could main ain hei
pa icle size on he nanome e scale upon seleni e inco po-
a ion, and XPS analysis e i ied he p esence o seleni e ions
in he nanopa icles. Ou esul s also e ealed ha he soluble
na u e o he nanopa icles led o he elease o an ibac e ial
seleni e ions, which inhibi ed bac e ial g ow h o bo h G am-
posi i e (S. au eus and S. epide midis) and G am-nega i e (E.
coli and P. ae uginosa) s ains. The an ibac e ial ac i i y o he
ACMC nanopa icles inc eased wi h an inc ease in he
concen a ion o he inco po a ed seleni e ions. In summa y,
he esul s indica ed ha Se-ACMC nanopa icles ha e he
po en ial o be used as an e ec i e an ibac e ial agen .
4. MATERIALS AND METHODS
4.1. Ma e ials. Calcium ace a e monohyd a e [Ca(CH3CO2)2·
H2O], sodium bica bona e (NaHCO3), magnesium chlo ide hexahy-
d a e (MgCl2·6H2O), and sodium seleni e (Na2SeO3) we e pu chased
om Sigma-Ald ich. E hylene glycol [CH2(OH)2] and e hanol
(C2H5OH) we e ob ained om Isolab Chemicals. Ul apu e wa e
ob ained om a Millipo e Milli-Q pu i ica ion sys em was used in he
expe imen s. All chemicals we e used as ecei ed wi hou u he
pu i ica ion.
4.2. Nanopa icle Syn hesis. ACMC nanopa icles we e
syn hesized using he cop ecipi a ion me hod. 5 mL o an aqueous
calcium ace a e monohyd a e solu ion (0.5 M) and 5 mL o an
aqueous sodium bica bona e (0.5 M)/magnesium chlo ide hexahy-
d a e solu ion (0.5 M) we e p epa ed sepa a ely in wo di e en
beake s. Then, he o al olume o each beake was adjus ed o 25 mL
wi h he addi ion o e hylene glycol. The calcium ace a e
monohyd a e solu ion was pou ed in o he sodium bica bona e/
magnesium chlo ide solu ion. The eac ion ook place o 15 min
unde magne ic s i ing, and a e wa d, he solu ion was kep
s a iona y o 1 h a oom empe a u e. The p ecipi a e was isola ed
by cen i uga ion, ollowed by washing wi h e hanol and ul apu e
wa e , espec i ely. Las , he powde was lyophilized o s o age. Fo
he syn hesis o Se-ACMC nanopa icles, he same p o ocol was
ollowed, excep he addi ion o Na2SeO3a 0.001, 0.005, and 0.01 M
concen a ions [ e e ed o as Se(I)-ACMC, Se(II)-ACMC, and
Se(III)-ACMC, espec i ely] in o he solu ion o sodium bica bona e.
In Table 2, he mola i ies o he p ecu so solu ions a e shown.
4.3. Ma e ials Cha ac e iza ion. The mo phology o ACMC
nanopa icles was obse ed using a FEI Ve ios 450L scanning elec on
mic oscope (B no, Czechia) a 5 kV. The size o he nanopa icles was
measu ed om 200 pa icles o each syn hesized nanopa icle g oup
using ImageJ so wa e. Gaussian o log-no mal dis ibu ion was used
o cu e- i he nanopa icle size dis ibu ion his og ams. TEM
analysis was pe o med using an FEI Tecnai G2 F30 ansmission
elec on mic oscope (Hillsbo o, O egon) o in es iga e he nano-
pa icle mo phology and amo phous s uc u e. Specimens we e
p epa ed o TEM cha ac e iza ion by dispe sion in e hanol. 10 μL
o he mix u e was d opped on o a holey ca bon-coa ed coppe g id
and d ied o 10 min. To u he demons a e he amo phous na u e
o he syn hesized nanopa icles, XRD analyses we e pe o med using
a Rigaku D/Max-2200 X- ay di ac ome e (Tokyo, Japan) wi h
monoch oma ic Cu Kα adia ion (λ= 1.54 Å) a 2°/min scanning
a e be ween 20 and 60°. Fo analysis o he chemical bonding, FTIR
spec a we e ob ained in he 4000−400 cm−1 ange using a
Pe kinElme Spec um 100 (Wal ham, MA) in a enua ed- o al-
e lec ance (ATR) mode. To analyze he chemical composi ion o he
nanopa icles and he chemical s a e o Se, XPS analysis was
pe o med using a K a os Axis Sup a (Manches e , U.K.), which has
a monoch oma ic Al Kαexci a ion sou ce. The Na ional Ins i u e o
S anda ds and Technology XPS online da abase was used o in e p e
he XPS da a. All spec a we e calib a ed o he ad en i ious ca bon
peak a 284.8 eV. The binding ene gies o Se 3d5/2 and Se 3d3/2 we e
ob ained by he decon olu ion o peaks using CasaXPS so wa e. Peak
i ing was done ia Shi ley’s backg ound using he combina ion o
Gaussian and Lo en zian unc ions and pu ing cons ain s on he
peak a ea a io and peak posi ions. The peak sepa a ion be ween Se
3d5/2 and Se 3d3/2 was es ic ed o 0.86 eV. TGA was pe o med
using a TA Ins umen s SDT650 analyze (New Cas le, DE) in a
ni ogen a mosphe e be ween 25 and 800 °C wi h a hea ing a e o 10
°C/min. ICP-MS analyses we e conduc ed o e eal he amoun o Se
inco po a ed in o he nanopa icles and o show he Se elease p o ile
o he nanopa icles using an X Se ies II ICP-MS (The mo Fishe
Scien i ic Inc., Wal ham, MA). P io o he ICP-MS analysis, he
nanopa icles we e mic owa e-diges ed in a HCl/HNO3solu ion o
quan i y he amoun o inco po a ed Se. In ano he se o
expe imen s, he nanopa icles we e ex ac ed in 1x PBS a 10 g/L
concen a ion o 5 min, 1 h, and 72 h o analyze he Se elease p o ile
om he nanopa icles. Deg ada ion es s we e conduc ed in 1x PBS.
Fo hese es s, nanopa icles we e added a a concen a ion o 1 g/L
in 1x PBS and kep a 37 °C h ough he deg ada ion expe imen s.
The pH alues o he solu ions we e measu ed a 1 min, 5 min, 15
min, 1 h, 24 h, 48 h, and 72 h ime poin s.
4.4. Cell Viabili y Assay. L929 ib oblas s (ATCC CCL-1) we e
used o assess he cellula iabili y. Dulbecco’s modi ied Eagle
medium (Sigma-Ald ich) supplemen ed wi h 1% penicillin/s ep o-
mycin and 10% e al bo ine se um was used as a g ow h medium. The
ib oblas s we e main ained in a humidi ied 5% CO2incuba o a 37
°C and subcul u ed a 90% con luency.
P io o he iabili y assay, he nanopa icles we e s e ilized by UV
i adia ion o 1 h. Subsequen ly, he s e ilized nanopa icles we e
ex ac ed using g ow h media a 0.1 and 0.01 g/L concen a ions a 37
°C o 72 h. The ex ac ed media we e used o he cell iabili y assay.
Fo he iabili y expe imen s, ib oblas s we e seeded in o he wells o
a 96-well pla e a a concen a ion o 1 ×104cells/well. A e a 24 h
incuba ion pe iod, he g ow h media we e eplaced wi h 200 μL o he
nanopa icle ex ac s. On he i s , hi d, and i h days o incuba ion,
3-(4,5-dime hyl-2- hiazolyl)-2,5-diphenyl-2H- e azolium b omide
(MTT) assay was conduc ed. Cells incuba ed wi h he nanopa icle
ex ac s we e insed wi h 1x PBS, and a e wa d, 125 μL o a s e ilized
MTT solu ion (1 g/L) was added o each well. Cells we e incuba ed
wi h MTT solu ions o 4 h o o m o mazan c ys als. Then, he
c ys als we e dissol ed wi h a 0.77% HCl solu ion p epa ed in
isop opyl alcohol (1:1 olume a io). The abso bance alues we e
measu ed a 570 nm by using a The mo Scien i ic Mul iskan Go
Table 2. Mola i ies o he P ecu so Solu ions P epa ed o
ACMC and Se-ACMC Nanopa icles
solu ion A solu ion B
sample
designa ions Ca(CH3CO2)2·
H2O (M) NaHCO3
(M) MgCl2·6H2O
(M) Na2SeO3
(M)
ACMC 0.5 0.5 0.5 -
Se(I)-ACMC 0.5 0.5 0.5 0.001
Se(II)-ACMC 0.5 0.5 0.5 0.005
Se(III)-
ACMC 0.5 0.5 0.5 0.01
ACS Applied Nano Ma e ials www.acsanm.o g A icle
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ACS Appl. Nano Ma e . 2023, 6, 16286−16296
16293
mic opla e spec opho ome e . Cells ha we e no ea ed wi h
nanopa icle ex ac s we e used as he con ol g oup.
4.5. An ibac e ial Ac i i y Tes s. 4.5.1. Pa icle S e iliza ion
and P epa a ion. ACMC nanopa icles we e s e ilized by UV-ligh
exposu e o 1 h. S e ilized nanopa icles we e p epa ed a 1 g/L
concen a ion using 0.3% TSB. A e he nanopa icles we e ex ac ed
in he TSB solu ion o 72 h a 37 °C, he supe na an s we e sepa a ed
by cen i uga ion and he ex ac ed media we e used in he biological
expe imen s.
4.5.2. CFU Assay. ACMC nanopa icles we e es ed agains wo
di e en G am-posi i e bac e ia s ains, S. au eus (ATCC 25923) and
S. epide midis (ATCC 35984), and wo di e en G am-nega i e
bac e ia s ains, E. coli (ATCC 10536) and P. ae uginosa (ATCC
27853). Bac e ia om he s ock cul u e we e inocula ed on o Lu ia−
Be ani aga pla es and incuba ed o e nigh . Once he colonies g ew, a
single CFU was isola ed and inocula ed in 3% TSB. A e wa d, he
cells we e ans e ed o a shaking incuba o and cul u ed a 37 °C
and 200 pm o 18 h. A he end o 18 h, bac e ia we e dilu ed o an
op ical densi y o 0.01 a 625 nm (OD625), which was u he dilu ed
by 1:100 using 1x PBS. A o al o 100 μL o he suspended bac e ia
was seeded in o 96-well pla es a a densi y o a×105/mL (a= 4, 9, 8,
and 5 o S. au eus,S. epide midis,E. coli, and P. ae uginosa,
espec i ely). A o al o 100 μL o he nanopa icle ex ac s we e also
added in o each well o inc easing he o al olume o 200 μL/well.
A e 24 h o incuba ion, he bac e ia in he wells we e se ially dilu ed
wi h 1x PBS, and 20 μL o he dilu ions we e seeded on o aga pla es
o coun he CFUs.
4.5.3. IC50 De e mina ion. Nanopa icles we e ex ac ed a a 10 g/
L concen a ion in 0.3% TSB a 37 °C o 72 h. The ex ac s we e
se ially dilu ed by hal in o he wells o a 96-well pla e o a olume o
100 μL o each well. The concen a ion o each bac e ia solu ion was
adjus ed o be 1 ×106CFU/mL. Then, 100 μL o a bac e ia solu ion
was added o he nanopa icle ex ac s o yield a bac e ia
concen a ion o 5 ×105CFU/mL. A e 18 h o incuba ion,
abso bance alues we e measu ed a 600 nm using a The mo
Scien i ic Mul iskan Go mic opla e spec opho ome e . Dose-depend-
en inhibi ion da a we e analyzed using G aphPad P ism so wa e.
Ex ac concen a ions ha educed he bac e ia densi y o hal we e
epo ed as IC50 alues.
4.6. S a is ical Analysis. All o he biological expe imen s we e
epea ed h ee imes, and h ee samples we e used o each
expe imen . The esul s we e epo ed as mean ±s anda d de ia ion.
The one-way ANOVA me hod wi h Tukey’s pos hoc es was used o
de e mine whe he he da a showed a meaning ul s a is ical di e ence.
The s a is ical signi icance was ixed a p< 0.05.
■ASSOCIATED CONTENT
*
sı Suppo ing In o ma ion
The Suppo ing In o ma ion is a ailable ee o cha ge a
h ps://pubs.acs.o g/doi/10.1021/acsanm.3c02415.
Low-magni ica ion SEM images and EDS spec a o
nanopa icles, SEM image o sodium seleni e sal , XRD
spec um o a mix u e o ACMC and sodium seleni e
sal , XRD esul s o hea - ea ed nanopa icles, deg ada-
ion es esul s and Se elease analysis, images o he
media colo change, and he esul s o cell iabili y
expe imen s (PDF)
■AUTHOR INFORMATION
Co esponding Au ho
Ba u E can −Depa men o Me allu gical and Ma e ials
Enginee ing, Middle Eas Technical Uni e si y, Cankaya,
Anka a 06800, Tu key; BIOMATEN, Cen e o Excellence in
Bioma e ials and Tissue Enginee ing and Biomedical
Enginee ing P og am, Middle Eas Technical Uni e si y,
Cankaya, Anka a 06800, Tu key; o cid.o g/0000-0003-
1657-1142; Phone: +90 (312) 210-2513;
Email: [email p o ec ed]
Au ho s
Yagmu Göc u−Depa men o Me allu gical and Ma e ials
Enginee ing, Middle Eas Technical Uni e si y, Cankaya,
Anka a 06800, Tu key; o cid.o g/0000-0001-8312-3679
Caga ay M. O al −Fu u e Ene gy and Inno a ion Labo a o y,
Cen al Eu opean Ins i u e o Technology, B no Uni e si y o
Technology, B no 61200, Czech Republic; o cid.o g/
0000-0001-5220-2104
Comple e con ac in o ma ion is a ailable a :
h ps://pubs.acs.o g/10.1021/acsanm.3c02415
Au ho Con ibu ions
Y.G. syn hesized and cha ac e ized he nanopa icles and
conduc ed in i o expe imen s. C.M.O. ob ained SEM images
and XPS spec a o he nanopa icles. B.E. supe ised he
p ojec . All au ho s con ibu ed o he manusc ip p epa a ion.
No es
The au ho s decla e no compe ing inancial in e es .
■ACKNOWLEDGMENTS
This esea ch was unded by he Tu kish Scien i ic Resea ch
Council unde G an s 117M187, 118M652, and 219M480,
and The Young Scien is Awa d P og am o he Tu kish
Academy o Sciences. We acknowledge he Middle Eas
Technical Uni e si y (METU) Cen al Labo a o y o TEM
and TGA cha ac e iza ions and he BIOMATEN-METU
Cen e o Excellence in Bioma e ials and Tissue Enginee ing
(BIOMATEN) o FTIR cha ac e iza ion. CzechNanoLab
P ojec LM2023051 unded by MEYS CR is g a e ully
acknowledged o inancial suppo o he measu emen s a
CEITEC Nano Resea ch In as uc u e.
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