pha maceu icals
A icle
Highly E icien T2 Cobal Fe i e Nanopa icles Vec o ized o
In e naliza ion in Cance Cells
E a Maza ío1,* , Magdalena Cañe e 2, Fe nando He anz 3, Jo ge Sánchez-Ma cos 1, Jesús M. de la Fuen e 4,5 ,
Pila He as i 1and Nie es Menéndez 1


Ci a ion: Maza ío, E.; Cañe e, M.;
He anz, F.; Sánchez-Ma cos, J.;
de la Fuen e, J.M.; He as i, P.;
Menéndez, N. Highly E icien T2
Cobal Fe i e Nanopa icles
Vec o ized o In e naliza ion in
Cance Cells. Pha maceu icals 2021,14,
124. h ps://doi.o g/10.3390/
ph14020124
Academic Edi o : Okhil Kuma Nag
Recei ed: 4 Janua y 2021
Accep ed: 2 Feb ua y 2021
Published: 5 Feb ua y 2021
Publishe ’s No e: MDPI s ays neu al
wi h ega d o ju isdic ional claims in
published maps and ins i u ional a il-
ia ions.
Copy igh : © 2021 by he au ho s.
Licensee MDPI, Basel, Swi ze land.
This a icle is an open access a icle
dis ibu ed unde he e ms and
condi ions o he C ea i e Commons
A ibu ion (CC BY) license (h ps://
c ea i ecommons.o g/licenses/by/
4.0/).
1Depa amen o de Química Física Aplicada, Facul ad de Ciencias, Uni e sidad Au ónoma de Mad id,
F ancisco Tomás y Valien e 7, Can oblanco, 28049 Mad id, Spain; [email p o ec ed] (J.S.-M.);
pila [email p o ec ed] (P.H.); [email p o ec ed] (N.M.)
2Depa amen o de Biología, Facul ad de Ciencias, Uni e sidad Au ónoma de Mad id, C/Da win 2,
Can oblanco, 28049 Mad id, Spain; [email p o ec ed]
3Ins i u o de Química Médica (IQM-CSIC) and CIBER de En e medades Respi a o ias (CIBERES),
Juan de la Cie a 3, 28006 Mad id, Spain; [email p o ec ed]
4Ins i u o de Nanociencia y Ma e iales de A agón, CSIC, Uni e sidad de Za agoza, C/Ped o Ce buna 12,
50009 Za agoza, Spain; jm uen e@uniza .es
5Ne wo king Biomedical Resea ch Cen e o Bioenginee ing, Bioma e ials and Nanomedicine (CIBER-BBN),
28029 Mad id, Spain
*Co espondence: [email p o ec ed]
Abs ac :
Uni o m cobal e i e nanopa icles ha e been syn hesized using an elec ochemical
syn hesis me hod in aqueous media. Thei colloidal, magne ic, and elaxome ic p ope ies ha e been
analyzed. The no el y o his syn hesis elies on he use o i on and cobal oils as p ecu so s, which
assu es he ep oducibili y o he i on and cobal a io in he s uc u e. A s able and biocompa ible
a ge ing conjuga e nanopa icle- olic acid (NP-FA) was de eloped ha was capable o a ge ing FA
ecep o posi i i y in HeLa (human ce ical cance ) cance cells. The biocompa ibili y o NP-FA was
assessed
in i o
in HeLa cells using he MTT assay, and mo phological analysis o he cy oskele on
was pe o med. A high le el o NP-FA binding o HeLa cells was con i med h ough quali a i e
in i o
a ge ing s udies. A alue o 479 Fe+Co mM
−1
s
−1
o ans e se elaxi i y (
2
) was ob ained
in colloidal suspension. In addi ion,
in i o
analysis in HeLa cells also showed an impo an e ec
in nega i e T2 con as . The e o e, he esul s show ha NP-FA can be a po en ial bioma e ial o use
in bio medical ials, especially as a con as agen in magne ic esonance imaging (MRI).
Keywo ds: a ge ing; con as agen ; cobal e i e; nanopa icles; olic acid; in e naliza ion
1. In oduc ion
Magne ic nanopa icles (NPs) in gene al ha e ecei ed subs an ial a en ion o hei
he anos ic po en ial ac i i y (abili y o combine he apeu ic and diagnos ic agen s wi hin
he same de ice). Some o he mos impo an cha ac e is ics in he nanome e egime ha
can be unable o op imize he magne ic p ope ies needed o an e ec i e imaging and
he mal ac i a ion a e he sa u a ion o magne iza ion, coe ci i y, and magne oc ys alline
aniso opy [
1
]. Among o he s, e i e nanopa icles ha e a ac ed special a en ion in he
las decades because hey a e easily syn hesizable. The e is a g ea a ie y o syn hesis
me hods, ha a e inexpensi e and easy o pe o m, esul ing in e i es wi h excellen
c ys allini y and magne ic p ope ies [2–4].
The supe pa amagne ic (SPM) cha ac e and la ge magne ic momen s o e i e mag-
ne ic nanopa icles con e hem in o good candida es o be used as magne ic esonance
imaging (MRI) con as agen s, which esul s om he coope a i i y o he indi idual spins
when aligned in he p esence o an ex e nal magne ic ield. SPM nanopa icles pe u b
he ex e nal magne ic ield, dec easing he elaxa ion imes (T
1
o T
2
) by de ocusing he
magne iza ion ec o (M) ensemble in he p ecession axis, being T
1
he elaxa ion ime o
Pha maceu icals 2021,14, 124. h ps://doi.o g/10.3390/ph14020124 h ps://www.mdpi.com/jou nal/pha maceu icals
Pha maceu icals 2021,14, 124 2 o 13
M
y
pa allel o he p ecession axis and T
2
he elaxa ion ime o he magne iza ion ec o
(M
x,y
) in he pe pendicula plane o he p ecession axis. The e o e, hese magne ic ield
pe u ba ions a e esponsible o he changed elaxi i ies o he wa e molecules measu ed.
This p oduces a da ke in ensi y signal in he case o pe pendicula o T
2
elaxa ion and
a b igh e signal o T
1
o pa allel elaxa ion. The g ea e he elaxa ion o he ma e ial,
he be e i s beha io as a con as agen should be, wi hou aking in o conside a ion
biological ac o s ha can al e i s beha io , such as due o issues pe aining o hei s a-
bili y, agg ega ion, low cell in e naliza ion dose, o incomple e clea ance. To imp o e he
e iciency o T
2
con as agen s, i is necessa y o con ol he magne ic p ope ies o he
NPs h ough he design o he physical chemical p ope ies, such as composi ion, c ys-
alline s uc u e, shape, hyd odynamic size, su ace cha ge, and he na u e o he coa ing.
These cha ac e is ics also a ec he biodis ibu ion, s abili y, opsoniza ion, and me abolism.
Among all hese physicochemical pa ame e s, only h ee magne ic cha ac e is ics di ec ly
de e mine he T
2
elaxi i y: (1) sa u a ion o magne iza ion alue, (2) magne ic ma e ial
olume ac ion, (3) he in a-agg ega e olume ac ion occupied by he magne ic ma e ials
ela i e o he whole (hyd odynamic) sphe e [5].
P e ious epo s highligh he ela ionship be ween he size o he NPs, he sa u a ion
magne iza ion (M
s
), and he
2
alues. The la ge he size and he M
s
alue, he highe
he
2
elaxi i y o he NPs [
6
]. Changes in he magne ic p ope ies o he NPs a e also
obse ed depending on he syn hesis me hod used [
7
,
8
]. These di e ences a e mainly
a ibu ed o changes in he c ys allog aphic o de o he ma e ial o he p esence o
impu i ies. Ano he app oach widely desc ibed in he li e a u e is he modi ica ion o he
con as e iciency by uning he composi ion and s uc u e o he o ganic coa ing
[9–11]
.
Al hough mos comme cial con as agen s a e Gd-based, some o he compounds based
on i on oxides ha e come o be comme cialized, such as Reso is , Lumi em e c., bu
hei p oduc ion was abandoned a e a ew yea s, due o oxici y and ad e se e ec
p oblems [
12
]. Ano he s a egy, based on he doping o i on oxide NPs wi h ca ions wi h
a omic adii simila o Fe
2+
, such as Co, Ni, Mn, and Zn, has been ca ied ou [
13
–
15
].
Taking in o accoun an in e sion deg ee o 100% in he spinel s uc u e, AB
2
O
4
, his means
ha all he doped elemen s a e placed in he B posi ion, and he magne ic momen pe
o mula uni co esponds o he
µB
o he doped ca ion. Conside ing he spin quan um
numbe s o Mn
2+
, Ni
2+
, and Co
2+
ca ions, which co espond o S = 5/2, 3/2, and 1,
espec i ely, he equi alen numbe o
µB
o pa amagne ic ansi ion me al ca ions can
be es ima ed as ollows:
µB=
2
·pS·(S+1)
. Those magne ic momen s co espond o
he maximum heo e ical alues o 5.9
µB
, 3.9
µB
, and 2.8
µB
o MnFe
2
O
4
, NiFe
2
O
4
, and
CoFe
2
O
4
e i e s uc u es. Howe e , in he nanome ic scale, he sa u a ion magne iza ion
is always lowe han he bulk magne iza ion due o magne ic diso de o he su ace. In
any case, hese e i es show good pe o mance as T
2
con as agen s. Some examples
om he li e a u e show
2
alues o app oxima ely 358 mM
−1
s
−1
o manganese e i es
wi h an M
s
alue o 110 emug
−1
(Mn+Fe). An
2
alue o app oxima ely 394 mM
−1
s
−1
has al eady been published by ou g oup o elec ochemically syn he ized Mn
0.5
Fe
2.5
O
4
,
wi h an M
s
alue o 108 emug
−1
(Fe + Mn) [
16
]. Values o
2
= 152 mM
−1
s
−1
wi h
Ms= 85 emug−1(Fe + Ni)
we e ound o nickel e i e [
17
,
18
]. Finally, in he case o cobal
e i e NPs, se e al wo ks ha e been published conce ning se e al ac o s. Fo example, he
ela ionship be ween sizes, M
s
, and ans e se elaxi i y (
2
) alues has been e alua ed o
cobal e i e nanocubes (NCs) in he ange o 6–25 nm [
15
]. Albino e al. ecen ly published
he e ec o he Zn inclusion in he cobal e i e s uc u e, in he
2
elaxome ic alue.
They es ablished an op imal Zn subs i u ion o 0.4 in he spinel uni o mula o ob ain an
2
alue abou 500 mM
−1
s
−1
o 8 nm nanopa icles [
19
]. An imp o ed modi ica ion o cobal
e i e con as agen has been ca ied ou , doping wi h eu opium and encapsula ed in o
mesopo ous silica [
20
]. No el T
2
con as agen ma e ials ecen ly published a e ocused
on ca bon-coa ed pa amagne ic dysp osium oxide (DYO@C) nanopa icles [21].
Op imiza ion o he magne ic p ope ies is no he only poin o be ul illed. The
s abili y and
in i o
biocompa ibili y a e also a o e on in he design o ou nanopa icle
Pha maceu icals 2021,14, 124 3 o 13
MRI con as agen s. The long- e m s abili y o hese pa icles wi hou agglome a ion o
p ecipi a ion is an impo an equi emen o almos any applica ion o magne ic nanopa -
icles. Mo eo e , ce ain ypes o coa ings could a o a ge ing o speci ic o gans and,
he e o e, an inc ease in he accumula ion o he con as agen in he desi ed a ea [
22
,
23
].
Among o he small molecules, olic acid is well known as a a ge molecule ha ecognizes
ola e ecep o s o e exp essed in ce ain umo cell lines [24].
Unde physiological condi ions, mos NPs pene a e he cells by endocy osis. Mos
endocy ic pa hways include lysosomes, whe e he deg ada ion o nanopa icles akes place
and educes he diame e depending on he na u e o he coa ing. This size educ ion di-
minishes he
in i o
signal and he e o e educes he inal T
2
con as signal [
25
,
26
]. Finally,
he cy o oxici y and cellula up ake o NPs also depend on mo phological pa ame e s o he
pa icles, such as size, shape, coa ing, and su ace cha ge, as well as biological pa ame e s,
such as cell line ype, pa icle concen a ion, medium composi ion, and empe a u e.
He e, we p opose he elec osyn hesis o cobal e i e nanopa icles conjuga ed wi h
olic acid o he speci ic ecogni ion o he ola e ecep o exp essed a he su ace o cance
cells (HeLa). We cha ac e ized he nanocomposi e (NP-FA) using Fou ie ans o m in-
a ed spec oscopy (FTIR), dynamic ligh sca e ing (DLS), and he mog a ime ic analysis
(TGA). The sa u a ion magne iza ion alues o cobal e i e nanos uc u es we e measu ed
by a supe conduc ing quan um in e e ence de ice (SQUID). A e wa ds, he cy o oxic-
i y and cy oskele on mo phology we e e alua ed a e nanopa icle incuba ion. Finally,
in i o
MRI elaxi i y measu emen s o HeLa cells in e nalized wi h e i e nanos uc u es
we e in es iga ed o applica ion in biomedicine.
2. Resul s and Discussion
2.1. Nanopa icle Physicochemical P ope ies
Figu e 1a shows he TGA da a o ba e NP and NP-FA. The non- unc ionalized sample
exhibi s a o al weigh loss o app oxima ely 6.5% a 700
◦
C, and he weigh dec ease
ha appea ed in he ange o 100–400
◦
C was due o mois u e emo al and e en ual
e abu ylammonium b omide e apo a ion. The NP-FA sample shows a o al weigh loss
o 20% a 700
◦
C. Simila conside a ions as o ba e NPs can be made; he s eepes dec ease
be ween 200 and 500
◦
C could be due o he elimina ion o he co alen ly conjuga ed FA
molecule. By conside ing he di e ence in mois u e con en be ween ba e NP and NP-FA,
we can es ima e an expe imen al loss o olic acid o app oxima ely 12%.
Figu e 1.
(
a
) The mog a ime ic analysis o ba e nanopa icles (NP) (black line) and nanopa icle-
olic acid (NP-FA) (blue line) unde ai condi ions. The s iped squa e is a guide o eyes o highligh
he di e ence in weigh loss be ween samples. (
b
) FTIR spec um o ba e NPs, NPs unc ionalized
wi h olic acid, and he FA molecule.
The su ace g oups o olic acid-modi ied CoFe2O4nanopa icles we e cha ac e ized
by FTIR, as shown in Figu e 1b. The abso p ion peak a 596 cm
−1
is a ibu ed o Fe-O
bond abso p ion. Abso p ion a 1604 cm
−1
co esponds o he ca bonyl and a oma ic ing
Pha maceu icals 2021,14, 124 4 o 13
s e ching ib a ions in he olic acid molecule, and abso p ion a 1396 cm
−1
co esponds
o he benzoic ib a ions in he olic acid molecule. The e o e, in a ed da a p o ed ha
olic acid molecules success ully modi ied he su ace o CoFe2O4nanopa icles.
A selec ed TEM image o NP-FA is shown in Figu e 2a. P ocessing o hese TEM
images by ImageJ so wa e p o ided NP diame e alues o 17(4) nm (N = 180). Acco ding
o TEM, he unc ionalized nanopa icles p esen some agg ega ion.
Figu e 2.
(
a
) T ansmission elec on mic oscopy (TEM) images o nanopa icles unc ionalized wi h olic acid. (
b
) Compa ison
o he nanopa icle size and hyd odynamic size dis ibu ion.
The hyd odynamic diame e (HD) o dispe sed NP-FA ( he e ec i e diame e o he
NP-FA when di using in wa e , ypically unde s ood as he sum o he co e diame e and
wice he shell hickness) can be assessed wi h sca e ing echniques, e.g., dynamic ligh
sca e ing (DLS). In Figu e 2b, a compa ison o he HD and he pa icle size is p esen ed.
The HD o he colloidal NP-FA sample was highe han he pa icle size, wi h a mean
alue o 78 nm (measu ed in in ensi y mode) and a polydispe si y index (PDI) o 0.16.
A PDI alue < 0.2 e lec s he size homogenei y o he sample [27].
One essen ial pa ame e conce ning he beha io o magne ic nanopa icles o ac as a
T
2
con as agen is hei sa u a ion o magne iza ion (M
s
). A high M
s
o supe pa amagne ic
NPs locally induces highe magne ic ield g adien s i dispe sed in solu ions and subjec ed
o an ex e nal homogeneous magne ic ield. Thus, he highe he M
s
o NPs wi h e e y hing
else being equal, he mo e e ec i e hey a e as con as agen s.
Figu e 3a shows he hys e esis loops, M(H), measu ed up o 50 kOe a 300 K. A
his empe a u e, an S- ype cu e wi h a hys e e ic cu e appea s wi h H
c
= 60 Oe,
M = 11 emug−1
, and an M
s
alue o 80.6(2) emug
−1
. This las alue is close o he bulk
alue p e iously epo ed (80 emug
−1
) and highe han o he alues epo ed o cobal
e i e nanopa icles wi h simila sizes and composi ions [28,29].
The ze o- ield-cooling (ZFC) and ield-cooling (FC) magne iza ion cu es, om 5 o
395 K
, a se e al ex e nal magne ic ields a e depic ed in Figu e 3b. The FC cu es show
a nea ly cons an alue o magne iza ion ha dec eases a high empe a u e and high
magne ic ields, whe eas he ZFC cu es show a high i e e sibili y ha dec eases wi h
he ield. This beha io is ela ed o e o- o e imagne ic ma e ials a he nanoscale wi h
magne ic o de empe a u es abo e oom empe a u e. All cu es show simila beha io
wi h a maximum in he ZFC cu e a T
B
, he empe a u e ela ed o he eezing o blocking
p ocess o he nanopa icle magne ic momen . As shown, he blocking empe a u e de-
c eases wi h he ex e nal magne ic ield, and a maximum is only p esen a app oxima ely
335 K when wo king a 1000 Oe. The e olu ion o T
B
wi h he magne ic ield is ela ed
Pha maceu icals 2021,14, 124 5 o 13
o he s ong exchange in e ac ion be ween he pa icles ha allows a low- empe a u e
o de [30].
Figu e 3.
(
a
) Magne ic hys e esis loop a 300 K, (
b
) ze o- ield-cooling/ ield-cooling (ZFC/FC) cu es, bo h o hem o he
sample NP-FA. Magne iza ion (M) was e lec ed as emu pe g am o nanopa icle.
This inding indica es ha below his empe a u e, NP-FA exhibi s e omagne ic
beha io wi h emanence and coe ci i y and ha abo e 300 K, he hys e esis ea u e
anishes and is expec ed o exhibi supe pa amagne ic beha io .
2.2. Relaxi i y o NP-FA
We hen measu ed he MRI elaxa ion imes (T
1
and T
2
) om 0 o 0.8 mM i on and
cobal o ob ain he ans e se elaxi i y (
2
) and longi udinal elaxi i y alues (
1
) o NP-
FA, as shown in Figu e 4a. The
1
alue o NP-FA was app oxima ely
10.3 Fe+Co mM−1s−1(2)
,
whe eas he
2
alue was app oxima ely 479(5)
Fe+Co mM−1s−1
, p o iding an
2
/
1
a-
io o 48.3. The la ge
2
and
2
/
1
a ios indica e ha hese nanopa icles a e p omising
candida es o high-e iciency T
2
con as agen s. The elaxa ion a e o ze o me al concen-
a ion we e ex ac ed om he linea i ing o he expe imen al da a, esul ing in alues
o
0.2(1) s−1
and 4(2) s
−1
o (1/T
1
)
0
and (1/T
2
)
0
, espec i ely. Such a signi ican imp o e-
men in he T
2
MR signal a ises om he high alue o sa u a ion o magne iza ion o hese
NPs, which is able o dis o he local magne ic ield in an e ec i e way.
2
is ela ed o he
M
s
, which, in u n, is a ec ed by he size o he NP [
11
]. T
2
-weigh ed phan om images
o a colloidal solu ion o NP-FA wi h di e en Fe and Co me al concen a ions a e shown
in Figu e 4b. Wi h inc easing concen a ion, he signal in ensi y o T
2
-weigh ed phan om
images ob iously dec eases, as shown in Figu e 4b, indica ing ha he T
2
elaxa ion ime
dec eases wi h inc easing me al concen a ion. The e o e, he sample has he po en ial o
gene a e MRI con as enhancemen on T2-weigh ed sequences.
Table 1depic s he elaxi i y alues o mos ypical comme cial agen s based on
i on oxide nanopa icles. Fo a simple compa ison, se e al
2
alues o cobal e i e
nanopa icles ound in he li e a u e ha e also been summa ized. The
2
alue ob ained in
his wo k o cobal e i e nanopa icles unc ionalized wi h olic acid is he highes alue
ound in he li e a u e o nanopa icles wi h sphe ical shapes.

Pha maceu icals 2021,14, 124 6 o 13
Figu e 4.
(
a
) T
1
and T
2
in e se measu emen s s. cobal and i on millimola concen a ion. The
slope o hese cu es co esponds o
1
and
2
elaxi i ies. (
b
) T
2
-weig hed magne ic esonance
(MR) images o NP-FA in aqueous solu ion a a ious me al concen a ions using a Va ian 7T mic o
magne ic esonance imaging (MRI) scanne .
Table 1. Relaxi i y collec ion alues ound in he li e a u e as a unc ion o nanopa icle diame e and coa ing.
Con as Agen s [31] Ma e ial Coa ing D(TEM) nm 1 2 2/ 1
This wo k
CoFe2O4
Folic acid 17(4) 10.3 479 46
Lee e al. [17] 2,3-Dime cap osuccinic acid 172
Schul z-Sikma e al. [32] Silica 7 142
Joshi e al. [15] 11-aminoundecanoic acid 15 301
Kim e al. [18] DMSA 8 6 392 62
Venka esha e al. [33] Chi osan 6 32
Liu e al. [34] Ci a e 37.89 27.8 75.1 2.7
Sine em®
γ-Fe2O3
Dex an 4–15 9.9 65 7
Reso is ®Ca boxydex an 4–15 9.7 189 19
VSOP_C184®Ci ic acid 5 14 33.4 2
Endo em®Dex an 4–15 10.1 120 12
2.3. Cell Viabili y and In e naliza ion
The esul s ob ained wi h he MTT assay do no exhibi any oxic e ec o he concen-
a ions used in his s udy. All iabili y pe cen ages we e simila o he con ols, i.e., nea
100% a e 6 o 24 h o incuba ion, as shown in Figu e 5.
Pha maceu icals 2021,14, 124 7 o 13
Figu e 5.
Ba s diag am o biocompa ibili y o NPs unc ionalized wi h olic acid in HeLa cells
measu ed by MTT assay. Blue columns, cells incuba ed 6 h wi h di e en concen a ions o NPs
and hei iabili y measu ed 24 h la e . G een columns, cells incuba ed o 24 h and hei iabili y
measu ed 24 h la e . The esul s ep esen he a e age o 6 independen expe imen s. Op ical
mic og aphies o con ol cells, NP-FA 0.6 mM in e naliza ion in HeLa cells incuba ed o 6 and 24 h.
Scale ba 10 µm.
A e bo h incuba ion imes (6 o 24 h), he cells we e ixed and s ained wi h oluidine
blue (TB) and obse ed by op ical mic oscopy, as shown in Figu e 5. A e 6 h o incuba ion
a 0.6 mM, he NPs we e clea ly localized inside he cells and we e de ec ed as small
agg ega es, a con ol cell mic og aph was shown o compa ison. Mo eo e , he quan i y
o NPs inside he cells was highe i he incuba ion ime was inc eased o 24 h. In addi ion,
he NPs we e kep inside he cells 24 h a e he incuba ion ime (da a no shown). Repo ed
oxici y in es iga ions ha e e ealed ha he CoFe
2
O
4
nanopa icles wi h app op ia e
su ace modi ica ion show ela i ely low cy o oxici y [35,36].
2.4. Analysis o Cy oskele on Componen s
Two cy oskele al componen s, mic o ubules (MTs) and ac in mic o ilamen s, ha e also
been analyzed o s udy he cy o oxic e ec o NPs. Mic o ubules a e highly dynamic ibe s
o he cy oskele on o ganized as a adial a ay a ound he cen osome wi h c i ical unc ions
in euka yo ic cells (e.g., mo ili y, di ision, esicle anspo ). Ins ead, ac in mic o ilamen s
a e in ol ed in a wide a ie y o cell unc ions, such as he es ablishmen o cell mo phology,
anspo o esicles and o ganelles, posi ioning o cellula componen s, cy okinesis, cell
locomo ion, cell–cell and cell–subs a e in e ac ions, and signal ansduc ion.
We analyzed he e ec o nanopa icle in e naliza ion on MTs and ac in by indi ec
immuno luo escence analysis o
α
- ubulin and TRITC- hodamine, espec i ely, and DNA
coun e s aining wi h Hoechs 33258. The in e phase mic o ubule ne wo k appea ed well
o ganized in cells incuba ed o 24 h a a me al concen a ion o 0.6 mM compa ed wi h
HeLa con ol cells, as shown in Figu e 6a. Figu e 6a also shows ha he amoun o NPs
inside he cells (me ged image) did no al e he mic o ubule mo phology and dis ibu ion.
To ob ain mo e insigh in o possible indi ec cell damage by hese NPs, we also in es iga ed
hei e ec s on ac in mic o ilamen s. As shown in Figu e 6b, con ol cells showed s ess
ibe s ( ed) c ossing he cy oplasm, and unde he cell co ex, hey we e ancho ed o
he plasma memb ane a ocal con ac s. In compa ison, mo phologic analysis o ac in
mic o ilamen s a e incuba ion wi h NPs did no show any al e a ion o his componen
unde ou expe imen al condi ions, as shown in he me ged image in Figu e 6b.
Pha maceu icals 2021,14, 124 8 o 13
Figu e 6.
Analysis o cy oskele on in HeLa cells incuba ed 24 h wi h 0.6 mM me al concen a ion
and obse ed unde luo escence and b igh - ield mic oscopy immedia ely a e incuba ions. (
a
)
Immuno luo escence s aining o
α
- ubulin obse ed unde blue ligh exci a ion. (
b
) TRITC-phalloidin
isualiza ion o ac in mic o ilamen s obse ed unde g een ligh exci a ion.
In conclusion, he beha io o NPs in HeLa cells demons a ed easy NP pene a ion
in o cells unde ou expe imen al condi ions, as well as he absence o e ec s on cell
su i al. As shown in se e al igu es o his wo k, he mo phology o cells incuba ed wi h
NPs did no di e om ha o con ol cells. The esul s ob ained in he mo phological
analysis o he cy oskele al componen s con i m he biocompa ibili y o he NPs.
2.5. In Vi o Phan om Image Con as
The MR images om
in i o
phan oms o NP-FA wi h di e en me al concen a ions
a e shown in Figu e 7. T
2
-weigh ed phan om MRI images we e ob ained om a se ies o
colloidal suspensions o cells incuba ed wi h NP-FA in he ans e se and longi udinal
posi ions, as shown in Figu e 7a,b, espec i ely. Wi h inc easing concen a ions o NP-FA,
he signal in ensi y d opped, esul ing in a signi ican inc ease in T
2
elaxa ion compa ed
wi h phospha e-bu e ed saline (PBS) and con ol cells [
11
]. This sugges s ha a minimal
concen a ion o NP-FA o 0.6 mM would be su icien o de ec an app eciably in ense
signal on MRI. In conclusion, he T
2
-weigh ed phan om images exhibi ed nega i e dose-
dependen con as imp o emen , sugges ing ha cobal e i e unc ionalized wi h FA as
a a ge molecule is e y p omising o imaging pu poses.
Pha maceu icals 2021,14, 124 9 o 13
Figu e 7.
MRI o phan oms we e pe o med o measu e he T
2
elaxi i y o he SPION complex-labeled cells. (
a
,
b
)
T ans e sal and longi udinal T2-weigh ed images o HeLa cells incuba ed wi h a ied concen a ion o NP-FA.
3. Ma e ials and Me hods
3.1. Syn hesis and Func ionaliza ion o Cobal Fe i e Nanopa icles
Cobal e i e nanopa icles we e p epa ed in one s ep by an elec ochemical
me hod
[37,38]
. The g a ing o he NPs wi h olic acid (FA) was pe o med by adding
he as-syn he ized NPs in an aqueous solu ion o 0.02 gmL
−1
FA. The pH o his solu ion
was inc eased by means o KOH un il pH 8.0, and his solu ion was hea ed and s i ed o
1 h a 80 ◦C
. The NPs we e sepa a ed by means o a magne o 0.6 T and washed se e al
imes wi h dis illed wa e o emo e he excess FA, a e which he pH o he aqueous
solu ion was dec eased o 7.2 wi h HNO
3
, and colloidal suspensions o pa icles we e
di ec ly ob ained by simple ul asonic ea men . Finally, he suspension was dialyzed o
24 h o emo e he FA no physi-abso bed o he nanopa icle su ace.
3.2. Cha ac e iza ion Techniques
Elec on ansmission mic oscopy images we e ob ained a e placing a single d op
(10
µ
L) o he aqueous solu ion o NP-FA on o a coppe g id coa ed wi h a ca bon ilm.
The g id was le o d y in ai o se e al hou s a oom empe a u e. TEM analysis was
ca ied ou using a Tecnai T20 (FEI, Ne he lands) elec on mic oscope wo king a
200 kV
.
Magne ic cha ac e iza ion was ca ied ou using a SQUID magne ome e (Quan um De-
sign MPMS XL-5). The magne iza ion cu es we e measu ed a oom empe a u e a e
applying a maximum magne ic ield o 50 kOe. F om he magne iza ion cu es, alues o
pa ame e s such as coe ci i y (H
c
) and sa u a ion magne iza ion (M
s
) we e ob ained. The
sa u a ion magne iza ion was calcula ed by in ini e ield ex apola ion o he expe imen al
da a ob ained in he high ield ange whe e he magne iza ion linea ly dec eases wi h
1/H. The magne iza ion e sus empe a u e cu es we e measu ed in ze o- ield-cooling
(ZFC) and ield-cooling (FC) p ocedu es unde di e en ex e nal applied magne ic ields,
H = 100, 200, and 1000 Oe and be ween 5 and 395 K.
The mog a ime ic analysis (TGA) was conduc ed using a Me le Toledo Ins umen
(TG/SDTA851e model) unde ai and wi h a DSC/821e model wi h a hea ing a e o
10 ◦C min−1
om oom empe a u e o 800
◦
C. The p esence o o ganic molecules a ached
o he nanopa icle su ace was s udied by in a ed spec oscopy in a Nicole 20 SXC FTIR.
CoFe
2
O
4
–FA NPs we e dispe sed in KB a 2 w % and p essed in a pelle . The IR spec a
we e egis e ed be ween 4000 and 300 cm−1.
Dynamic ligh sca e ing (DLS) measu emen s we e ca ied ou a 25
◦
C wi h a Nano
ZS (Mal e n Ins umen s) equipped wi h a solid-s a e He-Ne lase (
λ
= 633 nm) o de e -
mine he hyd odynamic diame e o he wa e dispe sible NPs. The e ac ion index was
2.42 and he abso p ion 0.8. Finally, Fe and Co me al concen a ions we e measu ed wi h a
Pe kin-Elme Op ima 2100 DV induc i ely coupled plasma op ical emission spec ome e
(ICP-OES). Fo his pu pose, samples we e diges ed wi h ni ic acid o oxidize he o ganic
coa ing and hen wi h hyd ochlo ic acid o dissol e he pa icles.