PEGyla ed Te bium-Based Nano ods as Mul imodal Bioimaging
Con as Agen s
Ca los Ca o, Jose M. Paez-Munoz, Ana M. Bel án, Manuel Pe nia Leal,*
and Ma ía Luisa Ga cía-Ma ín*
Ci e This: ACS Appl. Nano Ma e . 2021, 4, 4199−4207
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ABSTRACT: Diagnos ic imaging s ongly elies on he use o
con as agen s (CAs). In gene al e ms, cu en CAs p esen
undesi able side effec s ha encou age esea che s and pha ma-
ceu ical companies o con inually sea ch o sa e and mo e
e sa ile al e na i es. He e, we desc ibe he syn hesis and
cha ac e iza ion o e bium-based nano ods (TbNRs) as a
po en ial al e na i e o adi ional CAs o magne ic esonance
imaging (MRI) and X- ay compu ed omog aphy (CT). The
pa amagne ism and high a omic numbe o Tb p o ide TbNRs
wi h bo h magne ic elaxi i y and X- ay a enua ion capabili ies.
A e su ace unc ionaliza ion wi h a polye hylene glycol (PEG)-
de i ed ligand, TbNRs showed high colloidal s abili y in
physiological media. Addi ionally, oxici y s udies conduc ed in
cell cul u es and zeb afish emb yos demons a ed he sa e y o he as-syn hesized TbNRs, hus suppo ing hei po en ial use as CAs.
Las ly, in i o imaging expe imen s in mice demons a ed ha TbNRs p oduce ema kable con as enhancemen on bo h MRI and
CT.
KEYWORDS: Ra e-ea h nanopa icles, e bium, MRI, CT, con as agen s, mul imodal imaging
■INTRODUCTION
Imaging-based diagnosis has long since become an essen ial
ool o daily clinical p ac ice. Among he diffe en in i o
imaging modali ies, MRI and CT s and ou o hei abili y o
ende de ailed 3D ana omical in o ma ion along wi h
unc ional in o ma ion, especially in he case o MRI.
1
Al hough he use o con as agen s (CAs) is o en
unnecessa y, in ce ain si ua ions, hey a e essen ial o he
p ope isualiza ion o a pa icula egion o in e es o o ge
ele an unc ional in o ma ion, such as pe usion o diffusion
pa ame e s.
2
The mos commonly used in a enous CAs a e
gadolinium- and iodine-based compounds o MRI and CT,
espec i ely.
3,4
Gd chela es a e ou inely used as MRI CAs o
gene a e posi i e con as in T1-weigh ed images as a esul o
sho ening he longi udinal elaxa ion ime (T1) p oduced by
he Gd ca ions on he p o ons o he nea by wa e molecules.
As o iodine de i a i es, hey a e used as CT CAs owing o he
high a omic numbe o iodine (Z= 53), which s ongly
co ela es wi h he X- ay a enua ion coefficien . Image
con as in CT is p oduced by diffe ences in he X- ay
a enua ion o issues and, he e o e, he p esence o iodine
de i a i es will po en ia e image con as by inc easing he
a enua ion coefficien o he issues whe e hey accumula e.
5
Al hough Gd chela es and iodina ed compounds a e s ill
widely used a p esen , hey ha e some impo an limi a ions.
On he one hand, Gd chela es can p oduce oxic effec s caused
by he unexpec ed elease o Gd3+ and subsequen accumu-
la ion in diffe en issues,
6−8
whe eas iodina ed compounds
can cause alle gic eac ions, con as -induced neph opa hy, o
hype hy oidism.
9−11
On he o he hand, bo h ypes o CAs
exhibi sho blood hal -li es since hey a e apidly exc e ed by
enal fil a ion due o hei small size and, he e o e, hei use is
limi ed o applica ions in which long ci cula ion imes a e no
equi ed.
Cu en ad ances in medical imaging aim o inc ease
diagnos ic accu acy h ough diffe en s a egies, such as
mul imodal imaging e alua ions. In his scena io, nano-
medicine plays a key ole, hanks o he eno mous possibili ies
i offe s o p oduce new nanoscale ma e ials wi h imp o ed
p ope ies and unc ionali ies.
12
Thus, nume ous new nano-
scale CAs wi h g ea po en ial o imp o e he diagnos ic
accu acy o MRI and CT ha e been p oduced o e he pas ew
yea s.
13,14
Among hem, nanos uc u es based on a e ea h
Recei ed: Feb ua y 24, 2021
Accep ed: Ma ch 10, 2021
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(RE) elemen s ha e a ac ed a g ea deal o a en ion because
o hei unique physicochemical p ope ies, which p o ide
hem wi h g ea po en ial o bioimaging applica ions.
15
Thus,
he ou s anding magne ic p ope ies o se e al RE elemen s,
such as Gd, Ho, Dy, o Tb, ha e been exploi ed o he
de elopmen o a new gene a ion o nanos uc u ed CAs o
MRI.
16−24
Likewise, lan hanide-based nanos uc u es a e also
sui able o CT imaging, hanks o he high a omic numbe (Z)
o hese elemen s, which allows hem o efficien ly abso b X-
ays.
25,26
The as majo i y o hese s udies use he lan hanide
as a dopan agen and no as he main componen o he
nanos uc u ed ma e ial. Ye , o he RE-based s uc u es,
mos ly lan hanide oxide nanopa icles (NPs), ha e also been
explo ed as CAs o in i o imaging. Among hem, Gd-oxide
NPs as MRI CAs a e he mos widely s udied.
27−30
Rega ding
Tb-oxide NPs, Ma asini e al.
31
ecen ly epo ed he syn hesis
o ul asmall pa amagne ic NPs based on se e al lan hanide
oxides, including Tb oxide. This wo k shows he po en ial o
hese NPs as MRI CAs, al hough no in i o e idence has been
epo ed so a . Indeed, i is wo h no ing he e ha a key aspec
lacking in many o hese s udies is de ailed in i o
cha ac e iza ion ha demons a es he eal po en ial o hese
nanos uc u es as CAs. In addi ion o high biocompa ibili y,
NP-based CAs mus be able o escape he mononuclea
phagocy e sys em and emain in ci cula ion long enough o
efficien ly each hei a ge , especially in umo imaging.
Howe e , many o he RE-based CAs, pa icula ly hose ha
use RE elemen s as he doping agen , exhibi la ge hyd o-
dynamic diame e s (HDs), usually abo e 100 nm, which in i o
lead o apid li e up ake, limi ing hei applicabili y as
CAs.
23,29,32
On he o he hand, long ci cula ion imes a e
de e mined no only by he size o he NPs bu also by hei
coa ing. In his ega d, s eal h polyme s such as polye hylene
glycol (PEG) ha e been shown o efficien ly p olong blood
ci cula ion imes.
33
He e, we epo he syn hesis o e bium-
based nano ods (TbNRs) wi h sizes o 2 ×9 nm wi h high
colloidal s abili y and excellen luminescen , magne ic, and X-
ay a enua ion p ope ies. These TbNRs we e well dispe sed
in physiological medium by a simple ligand exchange wi h a
ca echol-de i ed PEG ligand. The cy o oxici y o he esul ing
PEGyla ed TbNRs (PEG−TbNRs) was assessed in i o using
diffe en assays, including MTT, li e-dead, and flow cy ome y,
and in i o using zeb afish emb yos. Then, in i o imaging
s udies we e conduc ed in mice o de e mine he sui abili y o
PEG−TbNRs as CT and MRI CAs.
■EXPERIMENTAL SECTION
Ma e ials. Comme cial eagen s and sol en s we e pu chased om
acc edi ed supplie s (Sigma-Ald ich, Fishe Scien ific, and Ac os
O ganics): e bium(III) chlo ide, oleic acid 99%, sodium olea e, 1-
oc adecene, PEG, gallic acid, 4-dime hylaminopy idine, ie hylamine,
dicyclohexylca bodiimide (DCC), ni ic acid (HNO3), sodium sul a e
(Na2SO4), hyd ochlo ic acid (HCl), 3-[4,5-dime hyl hiazol-2yl]-2,5-
diphenyl e azolium b omide (MTT), T i on X-100, TO-PRO-3
iodine, DAPI (4′,6-diamidino-2-phenylindole), Roswell Pa k Memo-
ial Ins i u e (RPMI) medium, and phospha e buffe ed saline (PBS).
The sol en s: e hanol, oluene, chlo o o m, hexane, ace one,
dime hylsulphoxide (DMSO), e ahyd o u an, and dichlo ome hane
we e used in he anhyd ous o m and wi h HPLC g ade; Milli-Q
wa e was used (Millipo e, fil e po e size 0.22 μM, 18.2 MΩ).
Syn hesis o TbNRs. Syn hesis o Tb Olea e. Fo he syn hesis o
Tb olea e, we used an adap ed e sion o he p o ocol epo ed by Liu
e al.
24
In b ie , a mix u e o 1.87 g o e bium chlo ide (7.05 mmol)
and 4.75 g o sodium olea e (15.60 mmol) was dissol ed in 7 mL o
dis illed wa e , 10 mL o e hanol, and 17 mL o hexane. This solu ion
was efluxed o 4 h unde an ine a mosphe e, cooled o oom
empe a u e and ans e ed in o a sepa a ion unnel. The o ganic
laye , which con ains he desi ed e bium olea e, was washed se e al
imes using dis illed wa e . The hexane was e apo a ed using a
o a apo .
Syn hesis o TbNRs. A o al o 0.511 g (0.51 mmol) o e bium
olea e, along wi h 0.250 g (0.89 mmol) o oleic acid and 3.5 mL o 1-
oc adecene was hea ed a 320 °C o 1 h in an ine a mosphe e
(Scheme 1). Then, he mix u e was cooled down o oom
empe a u e and washed se e al imes wi h a mix u e o e hanol
and ace one (1:1) as p ecipi a ing agen s, and subsequen ly
cen i uged (10 min a 5.000 pm). TbNRs we e hen esuspended
in oluene.
Func ionaliza ion o TbNRs. The de ailed p ocedu e conce ning
he ligand syn hesis is desc ibed in he Suppo ing In o ma ion.In
b ie , a solu ion con aining 1.0 mL o TbNRs (10 g/L), 1.0 mL o he
ligand GA-PEG3000-OH (0.1 M in CHCl3), and 50 μLo
ie hylamine was pu in a sepa a ing unnel. This mix u e was
shaken gen ly and hen dilu ed by adding 5 mL o oluene, 10 mL o
ace one, and 5 mL o Milli-Q wa e . Subsequen ly, his mix u e was
shaken, he aqueous laye collec ed in a ound-bo om flask, and he
emaining o ganic sol en s o ae apo a ed. The esul ing wa e -
soluble PEG−TbNRs we e pu ified using 100 kDa cu -offcen i uge
fil e s a 450 c un il he ee ligand was comple ely emo ed. Then,
he PEG−TbNRs we e dilu ed in he co esponding media and
cen i uged du ing 5 min a 150 c .
Cha ac e iza ion. T ansmission Elec on Mic oscopy. T ans-
mission elec on mic oscopy (TEM) and scanning-TEM (STEM)
images we e ob ained on a FEI TALOS F200 (FEI, Hillsbo o, OR,
USA) ope a ing a 200 kV accele a ing ol age and equipped wi h
Supe -X ene gy-dispe si e X- ay spec ome y (EDX). Composi ional
analyses o he samples we e pe o med by he combina ion o high-
angle annula da k-field images (HAADF) and EDX acquisi ions in
TEM and STEM modes (0.2 s dwell ime and spa ial d i co ec ion).
Sample p epa a ion consis ed o d opwise adding TbNR solu ion (∼1
g/L o Tb) on o a ca bon-coa ed coppe g id and le ing i d y. The
leng h and diame e o TbNRs we e calcula ed as he a e age o a
hund ed TbNRs measu ed.
Scheme 1. Syn hesis P ocedu e o TbNRs
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UV− is Spec oscopy. The UV− is spec a we e egis e ed on a
Ca y 100 (Agilen , Wal ham, MA, U.SA) spec ome e using a qua z
ay wi h 1 cm ligh pa h.
Fluo escence Spec oscopy. The fluo escence spec a we e
eco ded wi h an Edinbu gh Ins umen s FLS920 (Edinbu gh, U.K)
spec ome e wi h a qua z ay (ligh pa h = 1 cm).
Vib a ing Sample Magne ome e . The analysis o he magne -
iza ion beha io was pe o med on a ib a ing sample magne ome e
(VSM) (Mic oMag 3900, P ince on Measu emen s Co p., U.S.A)
using magne ic fields anging om −1 o 1 T a 5 and 300 K.
Dynamic Ligh Sca e ing. PEG−TbNR size dis ibu ion was
analyzed using a Ze asize Nano ZS90 (Mal e n Ins umen s,
Mal e n, Wo ces e shi e, U.K). PEG−TbNRs we e dispe sed in
PBS o cul u e medium a a final concen a ion o 50 mg/L(Tb).
ZEN0118-low- olume disposable sizing cu e es we e used o hese
measu emen s; 2.420 was se as he e ac i e index wi h an angle o
de ec ion o 173°backsca e (NIBS de aul ). The measu emen ime
was de e mined au oma ically. Th ee measu emen s we e pe o med
pe sample. The size dis ibu ion was de e mined using he numbe
mean. The analysis was done acco ding o he gene al-pu pose
(no mal esolu ion) model.
Fou ie T ans o m In a ed Spec oscopy. Fou ie ans o m
in a ed spec oscopy (FTIR) spec oscopy was pe o med on a
JASCO FTIR-4100 equipped wi h an ATR accesso y (MIRacle ATR,
PIKE Technologies) coupled o a me cu y cadmium ellu ide (MCT)
de ec o cooled wi h liquid ni ogen. Spec a acquisi ion was
pe o med om 4000 o 800 cm−1(4 cm−1 esolu ion, 50 scans).
Nuclea Magne ic Resonance Spec oscopy. The 1HNMR
spec um o he ligand p epa ed in CDCl3was acqui ed on a B uke
400 MHz nuclea magne ic esonance (NMR) sys em (B uke
BioSpin, Rheins e en, Ge many).
T ans e sal Relaxi i ies ( 2). 2was measu ed in PBS a 1.44 and
9.4 T using concen a ions o PEG−TbNRs anging om 2 o 0.2
mM Tb. T2 alues a 1.44 T we e de e mined on a B uke Minispec
MQ-60 (B uke BioSpin, Rheins e en, Ge many) using a Ca l−
Pu cell−Meiboom−Gill (CPMG) sequence. T2 alues a 9.4 T we e
measu ed on a B uke Biospec MRI spec ome e (B uke Biospec,
B uke BioSpin, E lingen, Ge many) equipped wi h a 40 mm
quad a u e esona o and 400 mT m−1field g adien s. T2 alues we e
measu ed using a 64-echo CPMG imaging sequence (TE alues
be ween 7.5 and 640 ms).
T ans e se elaxi i y, 2, was de e mined as he slope o he linea
fi o 1/T2o e [Tb].
Induc i ely Coupled Plasma High-Resolu ion Mass Spec osco-
py. Tb concen a ion was de e mined on a NexION ICP−HRMS
(Pe kinElme , Wal ham, MA, U.SA). In b ie , 25 μL o he TbNR
solu ion was dilu ed wi h 2.5 mM aqua egia. This mix u e was le
o e nigh and hen dilu ed up o 25 mL wi h wa e .
Cell Cul u e. N13 mouse mic oglial cells we e g own in RPMI
medium supplemen ed wi h 10% FBS, 2 mM L-glu amine, and 1%
penicillin/s ep omycin a 37 °C and 5% CO2.
Cy o oxici y. The cy o oxici y o PEG−TbNRs was assessed in
N13 cells by he MTT assay (de ails a e gi en in he Suppo ing
In o ma ion).
Cell Mo phology S udies. These s udies we e pe o med using a
Pe kinElme ope e a high con en imaging sys em, which allowed us
o e alua e cell mo phology and calcula e he pe cen age o cell dea h.
Fu he de ails a e gi en in he Suppo ing In o ma ion.
Te a ogenici y Assay. Wild- ype zeb afish we e used o hese
expe imen s. A day 0, hey we e ou c ossed, and hen, he emb yos
we e collec ed and incuba ed in E3 medium o 4 h a 28 °C.
Un e ilized eggs we e disca ded. Those emb yos ha de eloped
p ope ly we e exposed o diffe en concen a ions o PEG−TbNRs in
E3 medium. Eggs we e deposi ed in eigh -well Pe i dishes (∼30 eggs
in 4 mL). The ha ching a e, mal o ma ions, and su i al we e
analyzed a 24, 48, and 96 h o PEG−TbNRs exposu e.
Animals. BALB/c mice (male, ca. 22 g weigh , n= 3), p o ided by
Jan ie Labs (Le Genes -Sain -Isle, F ance), we e used in all in i o
imaging expe imen s. The expe imen s we e ca ied ou in acco dance
wi h ou local e hics commi ee guidelines and we e consis en wi h
he na ional egula ions o he use and ca e o expe imen al animals
(R.D. 53/2013). P io o imaging expe imen s, mice we e
Figu e 1. TEM (a) and EDX (b) images o TbNRs be o e unc ionaliza ion. TEM (c) and STEM (d) images o PEGyla ed TbNRs. Inse shows a
highe -magnifica ion image (scale ba co esponds o 10 nm).
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anes he ized (1% isoflu ane), and a ca he e was in oduced in o hei
ail ein o in a enous adminis a ion o he PEG−TbNRs. Then,
he animals we e ans e ed o he imaging sys em (CT o MRI). The
PEG−TbNRs we e adminis e ed in a enously (10 mg o Tb pe kg).
In Vi o and In Vi o CT. CT images we e ob ained on a B uke
Albi a sys em (B uke Biospec, B uke BioSpin, E lingen, Ge many)
using he ollowing pa ame e s: 35 μm X- ay ocal spo size
(nominal), 45 kVp ene gy, wo king a 400 μA. A enua ion alues
we e exp essed in Hounsfield uni s, wi h he scale adjus ed o be e
isualize so issues, o e exposing he bones. Li e and kidney
pha macokine ics we e calcula ed om he a e age alues wi hin he
same egions o in e es (ROIs) a diffe en expe imen al imes.
In Vi o MRI. MRI s udies we e pe o med on he 9.4 T B uke
Biospec sys em desc ibed in he T ans e sal elaxi i ies sec ion by
ollowing he p o ocol desc ibed by us elsewhe e.
34
In b ie , high-
esolu ion T2-weigh ed images and a sequence o as T2-weigh ed
images we e acqui ed and analyzed o cha ac e ize he sho - e m
pha macokine ics (fi s 30 min). Then, quan i a i e T2maps we e
acqui ed a 0, 1, 24, 48, and 168 h o e alua e long- e m
biodis ibu ion and pha macokine ics.
His ology. A 168 h pos injec ion o he PEG−TbNRs, mice we e
eu hanized and he kidneys, spleen, and li e we e collec ed. Tissue
a chi ec u e was assessed by ligh mic oscopy o hema oxylin and
eosin (H&E)-s ained issue sec ions. De ails a e p o ided in he
Suppo ing In o ma ion.
S a is ical Analysis. The SPSS package (SPSS Inc., Chicago,
Illinois) was used o he s a is ical analysis. Diffe ences be ween
diffe en expe imen al condi ions we e de e mined by s uden ’s - es
o one-way analysis o a iance (ANOVA), wi h a significance le el o
0.05.
■RESULTS AND DISCUSSION
Syn hesis, Cha ac e iza ion, and Func ionaliza ion o
TbNRs. TbNRs we e syn hesized by he mal decomposi ion o
e bium−olea e in he p esence o oleic acid in 1-oc adecene.
The mo phology and size o he syn hesized TbNRs we e
examined by TEM and EDX. The as-p epa ed pa icles showed
an elonga ed shape o a ound 9.0 nm in leng h and 2.1 nm in
wid h. In e es ingly, hese elonga ed pa icles we e o ganized
in elonga ed supe s uc u es o a ound 80.2 nm in leng h and
9.2 nm in wid h (Figu es 1a and S1). The o ma ion o hese
assemblies o NPs occu ed du ing he syn hesis p ocess, likely
due o he diffe en eac i i y along he ods. These assemblies
o oleic acid-capped NPs we e ligand exchanged by ca echol-
de i ed PEG ligands, esul ing in wa e -soluble assemblies wi h
a s uc u e simila o ha o he as-syn hesized TbNRs, as
shown in he TEM images. The e o e, he gen le ligand
exchange p ocedu e was no sufficien o disassemble he NPs,
p obably due o he high ene gy o in e ac ion be ween hem.
The as-p epa ed TbNRs we e capped by oleic acid
su ac an s, gi ing a hyd ophobic cha ac e o he nanoma e i-
al. Thus, o make hem biocompa ible, TbNRs we e ans-
e ed o aqueous media by ollowing a p e iously epo ed
ligand exchange me hod,
35
using a ca echol-de i ed PEG−OH
ligand. As p e iously shown, ca echol ancho s s ongly in e ac
wi h he i on oxide NP su ace ia hei phenolic hyd oxyl
g oups.
36
These ancho s o m a s able 5-membe ed ing
complex wi h he Fe ca ion. Simila ly, lan hanide ca echola e
complexes a e likely o med om i alen lan hanide ca ions
and ca echol-de i ed ligands. Then, he bi-den a e ca echol
ancho o he ligand would eac wi h he Tb a he su ace o
he NRs o ming a s able complex simila o he e bium−
ca echola e complex [Tb2(ca )3] p oduced in solu ion.
37,38
Cha ac e iza ion o PEGyla ed TbNRs. PEG−TbNRs
we e ho oughly cha ac e ized by diffe en physicochemical
echniques o de e mine hei mo phology, size, op ical
p ope ies, magne ic p ope ies, and colloidal s abili y. The
o iginal mo phology and sizes o he p is ine NRs we e
p ese ed a e ligand exchange, as confi med by TEM and
STEM (Figu es 1c,d and S1). Mo eo e , he a achmen o he
PEGyla ed ligand o he su ace o he TbNRs was confi med
by FTIR spec oscopy (Figu e S2). Addi ionally, hese
unc ionalized TbNRs we e e y s able in wa e wi hou any
sign o p ecipi a ion o e ime, u he confi ming he success
in inco po a ing he PEGyla ed ligands on he su ace o he
TbNRs. Fu he analysis by dynamic ligh sca e ing (DLS)
showed he high s abili y o PEG−TbNRs in physiological
media, specifically in PBS and cell cul u e medium. PEG−
TbNRs showed HDs a ound 75 nm in PBS, and sligh ly lowe
(∼60 nm) in cell cul u e medium. A e 24 h, he HDs o
PEG−TbNRs inc eased by a ound 30% and hen emained
s able h oughou he measu emen ime (1 week). This
beha io demons a es he s abili y o PEG−TbNRs in
physiological media and also ha no agg ega es we e o med
du ing ligand exchange (Figu e S3).
REs also show in e es ing op ical p ope ies, which a e
go e ned by he elec on configu a ion and can be
cha ac e ized by UV− is and pho oluminescence (PL) spec-
oscopy. Thus, he op ical abso p ion in he UV egion o REs
can o igina e om ansi ions 4 →5d. The e o e, he peak
obse ed a 277 nm in he UV− is spec um could be assigned
o he 4 8→4 75d ansi ion, which no mally appea s a mo e
han 200 nm in he case o Tb ions (Figu e 2).
39
The PL
emission spec um o he TbNRs dispe sed in oluene a oom
empe a u e is shown (Figu e 2). The emission spec um o
TbNRs unde 325 nm exci a ion displayed ou emission
peaks, which could be assigned o he elec on ansi ions om
5D4→7F6(490 nm), 5D4→7F5(546 nm), 5D4→7F4(583
nm), and 5D4→7F3(620 nm). Among hese ansi ions, he
g een emission (546 nm) appea ed as he s onges , which is in
ag eemen wi h he Judd−O el heo y.
24
Addi ionally, PEG−TbNRs we e e alua ed as po en ial CAs
o in i o imaging, specifically o CT and MRI. The abili y o
Figu e 2. (a) UV− is spec um o PEG−TbNRs. (b) Fluo escence emission spec a o PEG−TbNRs.
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PEG−TbNRs o be used as CT CA was de e mined by
measu ing he X- ay a enua ion. In e es ingly, he slope o he
linea fi o he X- ay a enua ion e sus he PEG−TbNRs
concen a ion was 21.4 HU mM−1[Tb], which is 10- old
highe han ha ob ained o he comme cial CA iohexol (2.69
HU mM−1)(Figu e 3b).
40
On he o he hand, he magne ic
p ope ies o PEG−TbNRs we e s udied o e alua e hei
po en ial as MRI CAs. Fi s , he magne ic hys e esis loop
measu emen s by he VSM showed ha PEG−TbNRs exhibi
supe pa amagne ic beha io a 5 K and pa amagne ic a 300 K
(Figu e 3a). Then, hei magne ic ans e se elaxi i y, 2, was
de e mined a 1.44 and 9.4 T. PEG−TbNRs showed 2 alues
o 10.4 mM−1•s−1and 48.5 mM−1•s−1(Figu e 3c) a low and
high magne ic fields, espec i ely. The e o e, we can conclude
ha ou PEG−TbNRsbeha easmul imodalimaging
nanosys ems o op ical imaging, CT, and MRI. Howe e ,
he emission wa eleng h o hese PEG−TbNRs, a ound 540
nm, limi s hei applicabili y in i o in op ical imaging due o
he high issue au ofluo escence a ha wa eleng h,
41
oge he
wi h weak issue pene a ion. On he con a y, hei excellen
X- ay a enua ion and magne ic elaxa ion p ope ies make
hem e y p omising CT and MRI CAs o bioimaging.
In Vi o Cy o oxici y Assessmen . The e alua ion o he
cy o oxici y o new nanoma e ials designed o biological
applica ions is a c ucial s ep ha equi es an exhaus i e
analysis. The e o e, we ca ied ou a de ailed analysis including
mul iple assays, namely, cell mo phology, li e-dead, and MTT
assays. These assays we e pe o med on he mouse mic oglial
cell line, N13. A e exposu e o g owing concen a ions o
PEG−TbNRs, om 0.1 o 100 μg/mL o Tb, N13 cells did no
show e idence o mo phological changes, e en o he highes
concen a ion e alua ed (Figu es 4a−c and S4). Fu he mo e,
he o al numbe o cells pe well emained unal e ed o all he
concen a ions es ed (Figu es 4dandS5). Indeed, he
pe cen age o dead cells emained a ound 0%, wi hou a
s a is ically significan inc ease in all cases (Figu es 4e and S5).
Finally, no s a is ically significan effec on mi ochond ial
ac i i y was obse ed wi h he MTT assay (Figu e 4 ). These
esul s demons a e ha PEG−TbNRs exhibi excellen
cellula biocompa ibili y, suppo ing hei po en ial o in i o
applica ions. Howe e , in i o oxici y depends on many
diffe en ac o s ha canno be e alua ed in cell cul u es.
The e o e, be o e p oceeding wi h in i o expe imen s on mice,
oxici y was also assessed in i o using zeb afish emb yos.
Figu e 3. (a) hys e esis loop o PEG-TbNRs measu ed a 5 K (black) and 300 K (blue); (b) X- ay a enua ion measu ed as he linea ela ionship
be ween he in ensi y o he CT images and he concen a ion o Tb in he PEG−TbNR solu ions; (b); (c) elaxa ion a e (1/T2) s he
concen a ion o Tb in he PEG−TbNR solu ions measu ed a 1.44 T (black do s) and 9.4 T ( ed do s), and he linea fi s (black and ed lines)
whose slopes co espond o he ans e se elaxi i ies ( 2) o PEG−TbNRs a bo h magne ic fields.
Figu e 4. Op ical mic oscopy images o N13 cells: (a) nega i e con ol, (b) posi i e con ol, (c) cells exposed PEG−TbNRs (100 μg/mL). Images
co espond o he me ging o TO-PRO-3 iodine ( ed), DAPI (blue), and b igh field (g ay). Scale ba s co espond o 100 μm. Ba cha s
co espond o (d) o al numbe o cells/well, (e) pe cen age o dead cells, and ( ) cell iabili y (MTT assay) a e exposu e o inc easing
concen a ion o PEG−TbNRs o 24 h.
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Te a ogenici y Assessmen on Zeb afish Emb yos.
Te a ogenici y o PEG−TbNRs was e alua ed in zeb afish
emb yos, as p e iously desc ibed.
42
The su i al and ha ching
a es o zeb afish emb yos exposed o g owing concen a ions
o PEG−TbNRs (0.1, 1, 10, 50, and 100 μg/mL o Tb) we e
assessed a diffe en ime poin s a e e iliza ion. A e 24 h
pos exposu e, PEG−TbNRs we e clea ly obse ed o be
in e ac ing wi h he cho ion o he emb yos (Figu e S6a).
Rega ding he ha ching p ocess, bo h con ol emb yos and
hose exposed o 48 h o PEG−TbNRs showed 100%
ha ching (Figu e S6b). These esul s ag ee wi h ha ching a es
epo ed o no mal zeb afish emb yos.
43
In addi ion, nei he
mo ali y no mal o ma ions we e obse ed a any ime poin
in he emb yos exposed o PEG−TbNRs (Figu es 5 and S7).
In Vi o CT and MRI S udies. CT imaging s udies showed
an inc emen o 5.5 ±2.1 HU in he li e 1 h
pos adminis a ion o PEG−TbNRs (10 mg o Tb pe kg),
which inc eased up o 12.1 ±3.1 a e 24 h (Figu e 6). Then,
he ΔHU alues dec eased, being 10.4 a 24 h and 6.1 a 168 h.
The e o e, he maximal accumula ion was de ec ed a 24 h, and
hen, PEG−TbNRs we e slowly clea ed om he li e .
Impo an ly, hese esul s we e ob ained wi h an injec ion
dose app oxima ely 50 imes lowe han ha ecommended o
iohexol, which is 430 mg pe kilog am o body imaging
applica ions.
44
In i o biodis ibu ion and pha macokine ics o he PEG−
TbNRs we e also e alua ed by MRI. Sho - e m pha macoki-
ne ics by dynamic T2imaging showed ha he PEG−TbNRs
we e apidly aken up by he li e , eaching a alue o 50% o
ela i e enhancemen du ing he fi s 30 min a e he
in a enous adminis a ion o PEG−TbNRs (10 mg o Tb)
(Figu e 7a,b).
No significan accumula ion was obse ed in he es o he
o gans. Mo eo e , long- e m pha macokine ics showed a
maximal T2dec ease (ΔT2o 7.1 ms) a 24 h a e
adminis a ion, in ag eemen wi h he CT esul s shown
abo e. A simila end was obse ed in he kidneys, whe e he
maximum T2dec ease (ΔT2o 5.9 ms) was eached a 24 h.
A e 48 h, he kidneys eco e ed hei ini ial T2 alues,
whe eas he li e only showed a sligh T2 eco e y, in
ag eemen wi h he slow li e clea ance desc ibed in he CT
s udies (Figu e 7c).
Quan i a i e E alua ion o Ci cula ing Li e ime o
PEG−TbNRs in he Bloods eam. Tb con en in blood and
he li e a 1 and 24 h was quan ified by ICP−MS (Figu e 8).
The concen a ion o Tb in blood was 2.3 and 1.4 mg/L a 1
and 24 h pos adminis a ion, which co esponds o 10 and 6%
o he injec ed dose, espec i ely. These esul s confi med he
long blood hal -li e o he PEG−TbNRs. As expec ed, an
opposi e end was obse ed in he li e , showing 16.2 mg/kg
o issue a 1 h and 34.5 mg/kg o issue a 24 h, espec i ely.
Thus, he ICP esul s a e in e y good ag eemen wi h he CT
and MRI esul s, demons a ing ha PEG−TbNRs ha e long
blood esidence ime, being s ill p esen a 24 h, and ha hey
unde go slow hepa ic clea ance. Blood esidence ime is o
u mos impo ance o some in i o applica ions, in pa icula
o umo a ge ing. Efficien umo a ge ing, whe he passi e,
ia enhanced pe meabili y and e en ion (EPR) effec , o
ac i e, has been shown o equi e long ci cula ion imes.
45
The e o e, he ICP−MS esul s, which a e in good ag eemen
wi h he MRI findings and wi h he long-ci cula ing na u e o
ou PEG−TbNRs, u he suppo he g ea po en ial o hese
nanosys ems o in i o applica ions, specifically o cance
imaging.
In Vi o Toxici y Assessmen . Finally, po en ial ha m
caused by he in a enous adminis a ion o PEG−TbNRs was
assessed his ologically and also by ollowing he weigh p ofile
o he animals. H&E s aining was ca ied ou on issue samples
o he li e , kidneys, and spleen a 168 h pos adminis a ion o
PEG−TbNRs. The li e sec ions o he animals injec ed wi h
PEG−TbNRs esembled hose injec ed wi h PBS. No e idence
o acu e and subacu e li e inju y, such as acuola ed swelling
o he hepa ocy es’cy oplasm, was obse ed in he li e
sec ions (Figu e S8). Likewise, no subs an ial changes we e
obse ed in he kidneys o he animals injec ed wi h PEG−
TbNRs when compa ed o con ol animals. Kidneys exhibi ed
egula in ac b ush bo de s and glome uli a ound he
Bowman’s capsule (Figu e S8).
46
Finally, he spleen also
p esen ed no mal his ological ea u es wi h unal e ed ed and
whi e pulp, which is cha ac e is ic o hei no mal a chi ec u e.
Rega ding he weigh p ofile, a ansien dec ease in body
weigh was obse ed, wi h a maximum diffe ence be ween
g oups a 24 h a e he adminis a ion o PEG−TbNRs.
Howe e , a e 168 h, he animals egained hei no mal
weigh , indica ing he absence o oxic effec s (Figu e S9).
Figu e 5. Mo phological analysis o zeb afish emb yos exposed o
g owing concen a ions o PEG−TbNRs a diffe en ime poin s (24,
48, and 96 h).
Figu e 6. Rep esen a i e in i o CT images a diffe en ime poin s (0,
1, 24, 48, and 168 h) a e he injec ion o PEG−TbNRs. The li e
has been highligh ed o be e isualiza ion. Values a e in Hounsfield
uni s and he scale adjus ed o be e isualiza ion o so issues,
o e exposing he bones.
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■CONCLUSIONS
We epo he syn hesis o PEGyla ed TbNRs wi h po en ial
applica ion as mul imodal imaging CAs. TbNRs we e
syn hesized by he mal decomposi ion o he as-p epa ed
Tb−olea e. Then, TbNRs we e success ully unc ionalized
using a ca echol-de i ed PEG ligand o ende wa e -s able
TbNRs. These PEG−TbNRs showed high colloidal s abili y in
diffe en media, absence o oxici y in i o in cell cul u es and
in i o in zeb afish emb yos, and excellen p ope ies as CT
and MRI CAs. On he o he hand, a e in a enous injec ion
in mice, he PEG−TbNRs showed excellen in i o imaging
con as bo h in CT and MRI, long blood esidence ime,
being s ill p esen a e 24 h, and effec i e hepa ic clea ance.
Al oge he , hese esul s demons a e he high po en ial o
PEG−TbNRs as in i o mul imodal CAs.
■ASSOCIATED CONTENT
*
sıSuppo 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.1c00569.
De ails on he expe imen al sec ion, TEM his og ams,
FTIR, DLS, UV− is, PL, VSM, in i o CT e alua ion,
magne ic elaxi i y, in i o cy o oxici y, in i o oxici y,
and in i o side effec s in oden s (PDF)
■AUTHOR INFORMATION
Co esponding Au ho s
Manuel Pe nia Leal −Depa amen o de Química O gánica y
Fa macéu ica, Facul ad de Fa macia, Uni e sidad de Se illa,
Se ille 41012, Spain; o cid.o g/0000-0001-8160-0574;
Email: [email p o ec ed]
Ma ía Luisa Ga cía-Ma ín−BIONAND−Cen o Andaluz
de Nanomedicina y Bio ecnología (Jun a de Andalucía-
Figu e 7. In i o pha macokine ics o in a enously injec ed PEG−TbNRs in BALB/c mice: (a) sho - e m pha macokine ics in he li e ; (b)
sho - e m pha macokine ics in he kidneys; (c) T2-weigh ed MR images a diffe en imes pos adminis a ion o PEG-TbNRs; (d) T2 a ia ion in
diffe en issues de e mined om in i o T2maps ( alues co espond o a e age T2and ΔT2 om 3 animals).
Figu e 8. ICP−MS quan ifica ion o Tb con en a 1 and 24 h pos in a enous injec ion o PEG−TbNRs. (a) Absolu e Tb concen a ion, (b)
ela i e Tb concen a ion (% o he injec ed dose). E o ba s co espond o s anda d de ia ion (n= 3).
ACS Applied Nano Ma e ials www.acsanm.o g A icle
h ps://doi.o g/10.1021/acsanm.1c00569
ACS Appl. Nano Ma e . 2021, 4, 4199−4207
4205
Uni e sidad de Málaga), Málaga 29590, Spain; Biomedical
Resea ch Ne wo king Cen e in Bioenginee ing, Bioma e ials
&Nanomedicine (CIBER-BBN), Málaga 29590, Spain;
o cid.o g/0000-0002-2257-7682; Email: mlga cia@
bionand.es
Au ho s
Ca los Ca o −BIONAND−Cen o Andaluz de
Nanomedicina y Bio ecnología (Jun a de Andalucía-
Uni e sidad de Málaga), Málaga 29590, Spain;
o cid.o g/0000-0003-4758-3816
Jose M. Paez-Munoz −BIONAND−Cen o Andaluz de
Nanomedicina y Bio ecnología (Jun a de Andalucía-
Uni e sidad de Málaga), Málaga 29590, Spain
Ana M. Bel án−Depa amen o de Ingenie ía y Ciencia de
Los Ma e iales y Del T anspo e, Escuela Poli écnica
Supe io , Uni e sidad de Se illa, Se illa 41011, Spain;
o cid.o g/0000-0003-2599-5908
Comple e con ac in o ma ion is a ailable a :
h ps://pubs.acs.o g/10.1021/acsanm.1c00569
Au ho Con ibu ions
All au ho s con ibu ed o he w i ing o he manusc ip . The
final e sion o he manusc ip has been app o ed by all
au ho s. C.C. and J.M.P.-M. con ibu ed equally o his wo k.
Funding
Financial suppo was p o ided by he Minis y o Economy,
Indus y and Compe i i eness o he Spanish Go e nmen ,
g an CTQ2017-86655-R o MLGM and MPL; and he
Minis y o Heal h o he Andalusian Regional Go e nmen ,
g an OH-0026-2018 o MLGM and g an PI2013-0559 o
MPL.
No es
The au ho s decla e no compe ing financial in e es .
The aw/p ocessed da a equi ed o ep oduce hese findings
canno be sha ed a his ime as he da a also o ms pa o an
ongoing s udy.
■ACKNOWLEDGMENTS
The au ho s wan o exp ess special hanks o Alejand o
Domínguez o his in aluable help wi h he in i o oxici y
expe imen s. The au ho s also hank D . Juan F. López o his
suppo wi h he TEM expe imen s, D . Inaki O ue o VSM
measu emen , D . John Pea son and Luisa Macías o
assis ance wi h he cell expe imen s and help ul discussion,
Reyes Molina o assis ance wi h animal expe imen s, and
Ma ia Somoza o helping wi h he MRI expe imen s. Au ho s
hank BIONAND’s Nanoimaging Uni . Op ical mic oscopy,
TEM, CT, and MRI expe imen s ha e been pe o med in he
ICTS “NANBIOSIS”, mo e specifically in he U28 Uni a
BIONAND.
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