Inco po a ion o Tb and Gd imp o es he diagnos ic unc ionali y o
magne o ac ic bac e ia
Lucía Ganda ias
a
,
1
,
*
, Elizabe h M. Je emo as
b
,
c
, Da id Gandia
d
, Lou des Ma cano
e
,
,
g
,
Vi ginia Ma ínez-Ma ínez
h
, Ped o Ramos-Cab e
i
,
j
, Daniel M. Che ie
k
, Se gio Valencia
,
Luis Fe n
andez Ba quín
b
, M. Luisa Fdez-Gubieda
d
,
e
, Ja ie Alonso
b
, Ana Ga cía-P ie o
l
,
**
,
Alicia Muela
a
a
Dp o. Inmunología, Mic obiología y Pa asi ología, Uni e sidad del País Vasco (UPV/EHU), Leioa, 48940, Spain
b
CITIMAC, Uni e sidad de Can ab ia, San ande , 39005, Spain
c
Ins i u ü Physik, Johannes Gu enbe g Uni e si €
a , Mainz, 55128, Ge many
d
BCMa e ials, Bld. Ma ina Casiano 3 d Floo , Leioa, 48940, Spain
e
Dp o. Elec icidad y Elec 
onica, Uni e sidad del País Vasco (UPV/EHU), Leioa, 48940, Spain
Helmhol z-Zen um Be lin ü Ma e ialien und Ene gie, Albe -Eins ein-s . 15, Be lin, 12489, Ge many
g
Depa men o Physics, Facul y o Science, Uni e si y o O iedo, O iedo, 33007, Spain
h
Dp o. Química Física, Uni e sidad del País Vasco (UPV/EHU), Leioa, 48940, Spain
i
Cen e o Coope a i e Resea ch in Bioma e ials (CIC BiomaGUNE), Basque Resea ch and Technology Alliance (BRTA), Donos ia-San Sebas i
an, 20014, Spain
j
IKERBASQUE, Basque Founda ion o Science, Bilbao, 48009, Spain
k
Aix-Ma seille Uni e si 
e, Cen e na ional de la eche che scien ifique (CNRS), Commissa ia 
al'

ene gie a omique e aux 
ene gies al e na i es (CEA), UMR7265,
Bioscience and bio echnology ins i u e o Aix-Ma seille (BIAM), Sain -Paul-lez-Du ance, 13108, F ance
l
Dp o. Física Aplicada, Uni e sidad del País Vasco (UPV/EHU), Bilbao, 48013, Spain
ARTICLE INFO
Keywo ds:
Magne o ac ic bac e ia
The anos ic agen s
Luminescen ma ke s
Magne ic esonance imaging
X- ay abso p ion spec oscopy
Magne ic hype he mia
ABSTRACT
Magne o ac ic bac e ia a e en isaged as po en ial he anos ic agen s. Thei in e nal magne ic compass, chemical
en i onmen specifici y and na u al mo ili y enable hese mic oo ganisms o beha e as nano obo s, as hey can be
acked and guided owa ds specific egions in he body and ac i a ed o gene a e a he apeu ic esponse. He e we
p o ide addi ional diagnos ic unc ionali ies o magne o ac ic bac e ia Magne ospi illum g yphiswaldense MSR-1
while e aining hei in insic capabili ies. These addi ional unc ionali ies a e achie ed by inco po a ing Tb o
Gd in he bac e ia by cul u ing hem in Tb/Gd supplemen ed media. The inco po a ion o Tb p o ides lumi-
nescence p ope ies, enabling po en ial applica ions o bac e ia as bioma ke s. The inco po a ion o Gd u ns
bac e ia in o dual con as agen s o magne ic esonance imaging, since Gd adds T
1
con as o he exis ing T
2
con as o unmodified bac e ia. Gi en hei po en ial clinical applica ions, he diagnos ic abili y o he modified
MSR-1 has been success ully es ed in i o in wo cell models, confi ming hei sui abili y as fluo escen ma ke s
(Tb-MSR-1) and dual con as agen s o MRI (Gd-MSR-1).
1. In oduc ion
Magne o ac ic bac e ia (MTB) a e aqua ic mic oo ganisms ha syn-
hesize in e nally chains o memb ane-enclosed magne ic nanopa icles,
called magne osomes [1–4]. These magne osome chains beha e as a
compass needle, allowing bac e ia o o ien a e and na iga e along he
geomagne ic field lines.
MTB a e en isaged as p omising biomedical nano obo s (nanobio s)
because hey combine he guidance, con ol, and he anos ic capabili ies
o magne ic nanopa icles wi h he mo ili y, chemical specifici y, and he
capaci y o being gene ically modified o bac e ia [5–7]. Mo eo e , MTB
a e non-pa hogenic bu can be modified o deli e and/o exp ess ce ain
* Co esponding au ho .
** Co esponding au ho .
E-mail add esses: lucia.ga[email p o ec ed] (L. Ganda ias), [email p o ec ed] (A. Ga cía-P ie o).
1
Cu en add ess: Bioscience and Bio echnology Ins i u e o Aix-Ma seille (BIAM), UMR7265, Aix-Ma seille Uni e si 
e, CNRS, CEA Cada ache, 13108 Sain -Paul-
lez-Du ance, F ance.
Con en s lis s a ailable a ScienceDi ec
Ma e ials Today Bio
jou nal homepage: www.jou nals.else ie .com/ma e ials- oday-bio
h ps://doi.o g/10.1016/j.m bio.2023.100680
Recei ed 18 Janua y 2023; Recei ed in e ised o m 11 May 2023; Accep ed 19 May 2023
A ailable online 24 May 2023
2590-0064/©2023 The Au ho s. Published by Else ie L d. This is an open access a icle unde he CC BY-NC-ND license (h p://c ea i ecommons.o g/licenses/by-
nc-nd/4.0/).
Ma e ials Today Bio 20 (2023) 100680
cy o oxic molecules. Se e al wo ks ha e demons a ed he po en ial o
magne o ac ic bac e ia as hea ing agen s o magne ic hype he mia
[8–12], as d ug deli e y ca ie s [13–15], and as con as agen s o
magne ic esonance imaging (MRI) [16–18] o magne ic pa icle imaging
(MPI) [19].
Beyond he ou s anding cha ac e is ics o MTB, a numbe o s a egies
ha e been de eloped o p o ide MTB wi h addi ional unc ionali ies and
imp o e hei pe o mance as biomedical agen s o applica ion. One
app oach comes om uning hei magne ic esponse, which elies on he
magne osome chains. In his line, e en hough he syn hesis o magne-
osomes is a p ocess ha is conduc ed unde a s ic gene ic con ol, i has
been shown ha me allic elemen s can be inco po a ed in o he
magne i e mine al co e o he magne osomes, which esul s in significan
changes o he bac e ial magne ic p ope ies. The inco po a ion o hese
elemen s can be achie ed h ough a simple ou e ha consis s o g owing
he MTB in a cul u e medium supplemen ed wi h he me allic elemen
[20–26].
Following his line, in a p e ious wo k we in es iga ed he magne ic
p ope ies o he magne o ac ic bac e ium Magne ospi illum g y-
phiswaldense MSR-1 cul u ed in Tb o Gd supplemen ed media [27]. We
showed ha Tb
3þ
and Gd
3þ
we e inco po a ed as dopan s in o he
magne i e (Fe
3
O
4
) c ys al s uc u e o he magne osome co es,
subs i u ing Fe
3þ
ions. Despi e he mino inco po a ion, i was shown
ha he magne ic p ope ies o he magne osomes changed d as ically a
low empe a u es, bu emained unchanged a he empe a u e window
o in e es o biomedical applica ions. This means ha no changes a e
expec ed in hose unc ionali ies o he modified bac e ia which ely on
he magne ic p ope ies o he magne osomes (i.e. guiding, acking, o
hea ing).
In his wo k, we ocus on he po en ial biomedical applica ions o
MSR-1 g own in he p esence o Tb and Gd. We show ha Tb and Gd a e
no only inco po a ed in o he magne i e c ys al co es o he magne o-
somes, bu a e also ound in o he bac e ial compa men s. As a esul o
he p esence o Tb and Gd in hese o he compa men s, he modified
bac e ia achie e new unc ionali ies ha expand hei po en ial as
biomedical agen s while e aining o he in insic capabili ies, such as he
hea ing e ficiency. Bac e ia ha inco po a e Tb (and hei isola ed
magne osomes) a e pho oluminescen unde UV i adia ion, which p o-
ides hem wi h enhanced diagnos ic capabili ies o in i o s udies.
Al e na i ely, bac e ia ha inco po a e Gd p o ide T
1
con as o mag-
ne ic esonance imaging (MRI) in addi ion o he T
2
con as o unmod-
ified bac e ia, hus se ing as dual con as agen s, which ecen ly ha e
gained inc eased a en ion o hei use in clinical MRI [28,29]. Finally,
gi en hei po en ial biomedical applica ions, he diagnos ic abili y o he
modified MSR-1 has been success ully es ed in i o in wo cell models,
confi ming hei sui abili y as fluo escen ma ke s (Tb-MSR-1) and dual
con as agen s o MRI (Gd-MSR-1).
2. Ma e ials and Me hods
2.1. Bac e ial g ow h condi ions and magne osome pu ifica ion
Magne ospi illum g yphiswaldense s ain MSR-1 (DMSZ 6631) was
cul u ed a 28 C o 48 h in Flask S anda d Medium (FSM) [30] con-
aining (pe li e o deionized wa e ) 0.1 g KH
2
PO
4
, 0.15 g MgSO
4
⋅7H
2
O,
2.38 g HEPES, 0.34 g NaNO
3
, 0.1 g yeas ex ac , 3 g soybean pep one,
0.3% (w / ol) o sodium py u a e as ca bon sou ce and 100
μ
Mo
Fe(III)-ci a e. Tb-MSR-1 and Gd-MSR-1 we e g own by supplemen ing
he FSM media wi h 100
μ
M o Tb(III)-quina e and 100
μ
Mo
Gd(III)-quina e, espec i ely. This concen a ion was chosen a e pe -
o ming sensi i i y cu es o MSR-1 o inc easing concen a ions o
Tb(III)-quina e and Gd(III)-quina e (see Fig. S1 in he Supplemen a y
In o ma ion). Magne osomes we e isola ed ollowing he p o ocol
desc ibed by G ünbe g e al. [31] wi h mino modifica ions. Cells we e
collec ed by cen i uga ion, suspended in 20 mM HEPES/4 mM EDTA
bu e (pH 7.4) and dis up ed using a F ench P ess (1250 psig). The lysed
cells we e hen sonica ed o dispe se cell deb is om magne osomes and
hese we e collec ed and washed 5 imes using magne ic sepa a ion and
10 mM HEPES/200 mM NaCl bu e (pH 7.4). Finally, he isola ed
magne osomes we e suspended in ul apu e wa e .
2.2. T ansmission elec on mic oscopy (TEM)
T ansmission elec on mic oscopy (TEM) images we e ob ained o
bac e ia adso bed on o 300 mesh ca bon-coa ed coppe g ids, using a
JEOL JEM-1400 Plus elec on mic oscope a an accele a ing ol age o
120 kV.
2.3. X- ay abso p ion nea -edge s uc u e (XANES)
Bac e ia and isola ed magne osomes we e measu ed a he Fe K-edge
(7112 eV), and a Tb L
3
(7514 eV) and Gd L
3
(7243 eV) edges. Bac e ia
we e collec ed by cen i uga ion a e 48 h o cul u e. The ob ained
pelle s we e eeze-d ied and compac ed in o 5 mm pills con aining ~2
10
9
bac e ia. Isola ed magne osomes we e eeze-d ied and 2 mg o he
ob ained powde we e mixed wi h 20 mg o bo on ni ide. The mix u e
was compac ed in o 5 mm pills.
X- ay abso p ion nea -edge s uc u e (XANES) spec a o he samples
we e collec ed a oom empe a u e a he BL22-CLÆSS beamline o he
ALBA synch o on acili y (Spain) using a double Si c ys al o ien ed in
he (111) di ec ion as he monoch oma o . Measu emen s we e pe -
o med in ansmission configu a ion excep o he Gd L
3
-edge spec a o
isola ed magne osomes, which we e collec ed in fluo escence yield
mode. Gd e e ences (GdCl
3
(Sigma-Ald ich, 203289) and Gd
2
O
3
(Alpha-
Aesa , 11289)) we e measu ed in ansmission configu a ion, and he Tb
e e ence (Tb(NO
3
)
3
) spec um was kindly p o ided by D . Aida Se ano.
The expe imen al spec a we e no malized using s anda d p ocedu es
o backg ound sub ac ion and da a no maliza ion as implemen ed in
he ee so wa e A hena o he IFEFFIT package [32,33].
2.4. Ha d X- ay nanop obe measu emen s
Scanning X- ay fluo escence mic oscopy (SXFM) measu emen s we e
ca ied ou a he I-14 ha d X- ay nanop obe beamline (Diamond Ligh
Sou ce L d., Didco , UK) [34]. Bac e ia we e deposi ed on o 300 mesh
ca bon-coa ed coppe g ids and moun ed o measu emen using cus om
holde s designed and supplied by he beamline. Measu emen s we e
pe o med unde ambien condi ions using an inciden pho on ene gy o
8 (Gd) o 9 keV (Tb) wi h a ocused X- ay beam o 50–60 nm (FWHM) in
diame e . X- ay fluo escence (XRF) was collec ed in on o he sample
using a ou -elemen silicon d i de ec o (RaySpec, UK) wi h sample
s age as e scanning s ep o 50 nm and a dwell ime o 15 ms. A
pho on-coun ing Me lin de ec o (Quan um De ec o s, UK) was used in
ansmission configu a ion o gene a e no malized phase-g adien (PG)
images [35]. The da a analysis was pe o med using PyMca [36] so wa e
o fi he fluo escence spec a aking in o accoun he emission peaks o
Fe (K
α
1
6.405 keV), Tb (L
α
1
6.273 keV), and Gd (L
α
1
6.053 keV) o o m
he XRF images.
2.5. Magne ic hype he mia
Magne ic hype he mia s udies we e pe o med by AC magne ome y
in a cus om-made se up [37] using a equency ¼133 kHz and a
magne ic field ampli ude (
μ
0
H) om 0 o 90 mT. The dynamic magne-
iza ion was measu ed o bac e ia dispe sed in Milli-Q wa e and a a
concen a ion o 10
11
bac e ia mL
1
. The SAR alues we e ob ained om
he a ea (A) o he AC hys e esis loops acco ding o Equa ion (1) [38]:
SARW
g¼
cA¼
cI
μ
0M dH (1)
whe e M
is he ins an aneous magne iza ion a ime ,H
is he sinusoidal
L. Ganda ias e al. Ma e ials Today Bio 20 (2023) 100680
2
magne ic field o equency a ime , and cis he magne i e mass
concen a ion in he dispe sing medium. The in eg a ion is done o e a
pe iod o he oscilla ing magne ic field, T¼2
π
/ . Gi en he low doping
con en o he magne osomes, he magne i e mass concen a ions (c)
we e calcula ed expe imen ally using he sa u a ion magne ic momen o
he DC hys e esis loops measu ed in a ib a ing sample magne ome e
and conside ing as a e e ence he sa u a ion magne iza ion alue o
magne i e (92 Am
2
kg
1
), esul ing in c¼0.24 mg mL
1
o MSR-1, c¼
0.17 mg mL
1
o Tb-MSR-1 and c¼0.22 mg mL
1
o Gd-MSR-1.
2.6. Luminescence measu emen s
Luminescence measu emen s o Tb-MSR-1 and Tb-magne osomes
we e pe o med in a spec ofluo ime e Edinbu gh Ins umen s (FLSP
920 model) wi h a Xenon flash lamp 450 W as he exci a ion sou ce. The
samples, in 1 cm op ical pa h cu e es in igh -angle configu a ion, we e
exci ed wi h a λ
ex
¼285 nm and he spec a we e eco ded a a λ
em
¼
520–570 nm. Fo he cul u e medium no o in e e e, i was emo ed by
cen i uga ion and he bac e ia we e esuspended in Milli-Q wa e . The
concen a ion o Tb-MSR-1 measu ed was 10
9
bac e ia mL
-1
and he
concen a ion o Tb-magne osomes was 50 µgmL
-1
.
2.7. Fluo escence mic oscopy
Fluo escence mic oscopy images o Tb-MSR-1 and Tb-MSR-1-loaded
euka yo ic cells we e eco ded wi h an op ical in e ed mic oscope
wi h epi configu a ion (Olympus BX51) equipped wi h a X-CITE se ies
120Q fluo escen ligh sou ce and a DP72 colo CCD came a. Samples
we e exci ed wi h UV ligh by espec i e Ch oma band-pass (350/50)
and dich oic (380DCLP) fil e s and emission was collec ed wi h a Ch oma
cu -o fil e om 500 nm (E515LP 2).
2.8. Magne ic esonance imaging (MRI)
MRI s udies o MSR-1, magne osomes, and euka yo ic cells, we e
conduc ed a 11.7 T using a B uke Biospec 117/16 USR MRI sys em
(B uke Biospin GmbH, E lingen, Ge many), in e aced o an ad ance III
console and a BGA12S g adien inse o 660 mT m
1
. Images we e ac-
qui ed using a 4 cm inne diame e olume ic coil o adio equency
ansmission and ecep ion (RX/TX) om B uke and Pa aVision 6.1
so wa e om B uke .
Fo bac e ia and magne osomes, T
2
elaxa ion was measu ed using a
mul i-slice mul i-echo pulse (MSME) sequence acqui ing a field o iew
o 32 32 mm (320 320 image ma ix, gi ing an in-plane esolu ion o
100 100
μ
m) and acqui ing 3 slices o 1 mm hickness wi h a 1 mm
in e slice gap. The main imaging pa ame e s we e: echo ime TE ¼32
consecu i e echoes wi h alues anging be ween 5 and 135 ms (del a TE
¼5 ms); epe i ion ime TR ¼4500 ms; Na ¼3 a e ages; SW ¼163 kHz.
The T
1
elaxa ion was measu ed using a a iable TR apid acquisi ion
wi h elaxa ion enhancemen (VTR-RARE) pulse sequence acqui ing wi h
he same field o iew and ma ix as o T
2
. The main imaging pa ame e s
we e: echo ime TE ¼6.87 ms (Ra e ac o ¼2); 12 epe i ion imes TR ¼
150, 386.5, 647.7, 939.4, 1269.8, 1650.5, 2099.8, 2647.9, 3350.9,
4332.6, 5972.0, 12500 ms; Na ¼3 a e ages; SW ¼85 kHz.
In he case o euka yo ic cells, he T
2
maps we e cons uc ed by pixel-
by-pixel fi ing o MSME images o he aga /cells-filled Eppendo ubes,
acqui ed wi h he ollowing pa ame e s: field o iew o 64 48 mm
(213 160 image ma ix, gi ing an in-plane esolu ion o 300 300
μ
m)
acqui ing 12 consecu i e co onal slices o 1 mm hickness. Main imaging
pa ame e s we e: echo ime TE ¼48 consecu i e echoes wi h alues
anging be ween 5.5 and 264 ms (del a TE ¼5.5 ms); epe i ion ime TR
¼7.5 s; Na ¼1 a e ages; SW ¼65 kHz. The T
1
maps we e cons uc ed
by pixel-by-pixel fi ing o RARE images o he aga /cells-filled Eppen-
do ubes, acqui ed wi h same field o iew and ma ix as o T
2
. The
main imaging pa ame e s we e: echo ime TE ¼6.75 ms; 9 epe i ion
imes TR ¼300, 700, 1200, 1800, 2400, 3000, 4500, 12500 ms; Na ¼1
a e ages; SW ¼65 kHz.
T
1
and T
2
pa ame ic maps we e calcula ed pixel-by-pixel using sel -
de eloped ou ines o he NIH so wa e Image-J, adjus ing da a o
mono-exponen ial cu es.
2.9. In i o assays
A549 human lung ca cinoma cells (DSMZ, ACC 107) and RAW 264.7
mu ine mac ophages (ATCC, TIB-71) we e cul u ed in RPMI medium
supplemen ed wi h 10% e al bo ine se um, 2 mM L-glu amine and a
mix u e o an ibio ics (100 U mL
1
penicillin and 100
μ
gmL
1
s ep o-
mycin) and 0.25
μ
gmL
1
ampho e icin B (an imyco ic) a 37 Cina
humidified a mosphe e (95% ela i e humidi y) and 5% CO
2
. Fo
cellula up ake o bac e ia, 5 10
9
bac e ia mL
1
we e added o cell
cul u es con aining 2 10
5
cell mL
1
in RPMI medium.
Fo he cy o oxici y assessmen , he iabili y o cells cul u ed wi h
bac e ia was es ed a e 24 h and 48 h using flow cy ome y (Beckman
Coul e Gallios). In o de o dis inguish be ween li ing and dead cells,
cells we e s ained wi h p opidium iodide, a ed fluo escen s ain ha
links o DNA bu can only pene a e he cells when hei memb anes a e
damaged, he e o e s aining only dead cells. I was exci ed wi h a blue
lase (488 nm) and eco ded in he FL3 channel (620/20 nm). To es i-
ma e cell concen a ion, comme cial fluo escen beads om a known
concen a ion we e used (BD T ucoun ™Tubes, 340334).
In o de o measu e he T
1
and T
2
con as o euka yo ic cells (wi h o
wi hou bac e ia), he samples we e p epa ed by mixing 1:1 o 10
6
cells
mL
1
and 1% aga in Eppendo ubes whe e he measu emen s we e
ca ied ou .
3. Resul s and discussion
3.1. Bac e ial inco po a ion o Tb and Gd
The inco po a ion o Tb/Gd in MSR-1 was in es iga ed by means o X-
ay abso p ion nea -edge s uc u e (XANES) spec oscopy measu ed a
he BL22-CLÆSS beamline a he ALBA synch o on (Spain). This is an
elemen -specific echnique ha allows de e mina ion o he oxida ion
s a e and coo dina ion chemis y a ound he elemen o in e es , he e Fe
and Tb o Gd, by selec ing he ene gy o he inciden X- ay ene gy a ound
he Fe K- o Tb/Gd L
3
-abso p ion edges.
XANES spec oscopy was p e iously used o analyze magne osomes
isola ed om bac e ia g own in p esence o Tb o Gd [27]. These mea-
su emen s indica ed ha Tb/Gd a oms a e in oduced in he c ys al
s uc u e o he magne i e co e o magne osomes subs i u ing app oxi-
ma ely 3–4% o Fe
3þ
ions by Tb
3þ
o Gd
3þ
. Now, by measu ing whole
bac e ia, we aim o disce n whe he Tb/Gd a e also s o ed in o he
bac e ial compa men s apa om he magne osomes.
Fe K-edge XANES measu emen s o bac e ia g own in he p esence o
Tb o Gd ( om now on designa ed Tb-MSR-1 and Gd-MSR-1, espec-
i ely) confi m ha , as in MSR-1, he Fe is o ming magne i e om he
magne osome co es, and is also ound in a e ihyd i e-like phase as he
one o bac e io e i in co es (see he Supplemen a y In o ma ion,
Fig. S2). Al e na i ely, Fig. 1 shows he Tb/Gd L
3
-edges XANES spec a o
Tb-MSR-1 and Gd-MSR-1 and hei co esponding isola ed magne o-
somes (Tb-mag and Gd-mag) oge he wi h Tb
3þ
and Gd
3þ
e e ences
(Tb(NO
3
)
3
and GdCl
3
, espec i ely). The posi ion o he edge ene gy o
he bac e ia and isola ed magne osomes is coinciden wi h he e e -
ences, which indica es ha he inco po a ed Tb/Gd ions a e in he þ3
oxida ion s a e. We no e he XANES spec a o whole bac e ia and hei
co esponding isola ed magne osomes show dis inc di e ences in he
whi e line in ensi y ( ea u e a ound 7520 and 7250 eV o Tb and Gd,
espec i ely; a ibu ed o he exci a ion o 2p co e-le el elec ons o
alence-le el 5d o bi al acancies). The in ensi y o he whi e line is
ela ed o elec onic s uc u al changes a ound he abso bing a om,
which depend on he na u e o he ligands, coo dina ion geome y and/
o chemical en i onmen . Fo bo h Tb and Gd XANES, he whi e line is
L. Ganda ias e al. Ma e ials Today Bio 20 (2023) 100680
3
Fig. 1. Tb L
3
-edge (le ) and Gd L
3
-edge ( igh ) XANES spec a o Tb-MSR-1 and Gd-MSR-1 and co esponding isola ed magne osomes (Tb-mag and Gd-mag). Spec a
o e e ences o Tb
3þ
(Tb(NO
3
)
3
) and Gd
3þ
(GdCl
3
) a e displayed as well. Inse : de ail o he pos -edge oscilla ions.
Fig. 2. TEM, phase-g adien (PG), and scanning X- ay fluo escence mic oscopy (SXFM) images o Tb-MSR-1 and Gd-MSR-1. Fo SXFM maps, he Tb/Gd L
α
1
XRF maps,
Fe K
α
1
XRF maps, and he composi e maps o indi idual bac e ia a e displayed.
L. Ganda ias e al. Ma e ials Today Bio 20 (2023) 100680
4
mo e in ense o he isola ed magne osomes han o he bac e ia, while
he pos -edge oscilla ions also di e be ween hem (see inse s o Fig. 1).
This sugges s ha he Tb
3þ
and Gd
3þ
ions inco po a ed by he bac e ia
a e no only wi hin he magne osomes, bu can also be ound in addi-
ional cellula compa men s whe e hey a e likely coo dina ed wi h
biomolecules.
The p esence o Tb and Gd in o he bac e ial compa men s was
confi med by scanning X- ay fluo escence mic oscopy pe o med a he
X- ay nanop obe beamline (I-14) a Diamond Ligh Sou ce (UK). This is a
mic oscopy echnique based on he cha ac e is ic fluo escence emission
ene gies o elemen s ha allows he localiza ion o specific elemen s wi h
sub-100 nm esolu ion. By disce ning he di e en fluo escence peaks
(Supplemen a y In o ma ion, Fig. S3), we could de e mine he spa ial
dis ibu ion o each elemen inside he bac e ia. Mo eo e , he X- ays
sca e ed by he bac e ia we e used o gene a e no malized phase-
g adien (PG) images o obse e he shape o he bac e ia. As displayed
in Fig. 2 he Fe is loca ed o ming a line in he middle o each bac e ium
ha co esponds o he magne osome chain on he TEM images. How-
e e , bo h Tb and Gd a e dis ibu ed along he whole bac e ia, o ming
clus e s in some occasions. This confi ms he da a ob ained by XANES
ha Tb and Gd a e dis ibu ed h oughou he bac e ia and no only
inco po a ed in o he magne osomes as we obse ed in ou p e ious
publica ion [27].
Finally, ansmission elec on mic oscopy (TEM) images o Tb-MSR-1
and Gd-MSR-1 (Fig. 3) show ha he inco po a ion o Tb and Gd does no
induce significan changes in he mo phology o he bac e ia and mag-
ne osome chain a angemen s wi h espec o he con ol MSR-1 g own
in s anda d condi ions. De ails abou he mo phological cha ac e iza ion
o he magne osome chains can be ound in Je emo as e al. [27].
3.2. The anos ic po en ial o Tb-MSR-1 and Gd-MSR-1
In his sec ion we will analyze he po en ial o Tb-MSR-1 and Gd-
MSR-1 o he anos ic applica ions. A e confi ming ha he inco po-
a ion o Tb/Gd does no al e he hea ing e ficiency o he modified
bac e ia o magne ic hype he mia applica ions, we will analyze sepa-
a ely he addi ional diagnos ic unc ionali ies p o ided o MSR-1 ia he
inco po a ion o Tb (luminescence) and Gd (dual con as o MRI).
Finally, we will es he pe o mance o Tb-MSR-1 and Gd-MSR-1 in i o
in wo cell models.
3.2.1. Magne ic hype he mia o Tb-MSR-1 and Gd-MSR-1
In magne ic hype he mia, nanopa icles exci ed by al e na ing
magne ic fields o ampli ude Hand equency gene a e hea ha in-
c eases he empe a u e o he su ounding medium. The hea ing ca-
paci y is quan ified h ough he specific abso p ion a e (SAR), which
measu es he capaci y o he sys em o ans o m he abso bed magne ic
ene gy in o hea .
He e, he SAR alues o Tb-MSR-1 and Gd-MSR-1 dispe sed in Milli-Q
wa e we e measu ed ia an AC magne ome y me hod and compa ed o
ha o MSR-1 [10]. Measu emen s we e ca ied ou using an AC mag-
ne ic field o a fixed equency ¼133 kHz and a magne ic field
ampli ude (
μ
0
H) o up o 90 mT in a cus om-made equipmen [37].
Fig. 4 shows he SAR alues as a unc ion o he magne ic field
ampli ude o Tb-MSR-1 and Gd-MSR-1 oge he wi h MSR-1. The SAR
s. H cu es a e simila o he h ee samples and ollow he end e-
po ed in o he s udies [10,39,40], so ha o low applied magne ic fields
he SAR alue is negligible bu inc eases significan ly abo e 20 mT,
eaching sa u a ion a a ound 40 mT. The e a e no essen ial di e ences
be ween he sa u a ion alues eached by MSR-1 and Gd-MSR-1 (1120
Wg
1
o 8.4 Wg
1
kHz
1
i no malized by he equency) and Tb-MSR-1,
which is only 7% lowe . These a e among he highes alues epo ed a
clinically sa e alues o equency and field ampli ude [5], su passing
hose ypically ob ained o i on oxide-based nanopa icles, which ange
be ween 2 and 5 Wg
1
kHz
1
[40–43]. In sum, hese esul s confi m ha
inco po a ing Tb/Gd in o MSR-1 does no a ec he ou s anding hea ing
e ficiency o he MSR-1 bac e ia.
3.2.2. Luminescence o Tb-MSR-1
Lan hanides such as Tb ha e been p oposed as e sa ile molecula
p obes o biological sys ems due o hei pho ophysical p ope ies,
including long luminescence li e imes, na ow bandwid hs, and insen-
si i i y o chemical en i onmen [44]. I is known ha Tb
3þ
can eplace
Fig. 3. Rep esen a i e TEM images o MSR-1, Tb-MSR-1, and Gd-MSR-1.
Fig. 4. Specific abso p ion a e (SAR) measu ed a ¼133 kHz co esponding
o MSR-1, Tb-MSR-1, and Gd-MSR-1 dispe sed in Milli-Q wa e .
L. Ganda ias e al. Ma e ials Today Bio 20 (2023) 100680
5

Ca
2þ
in biological sys ems as hey ha e a simila ionic adius (0.92 Å o
Tb
3þ
and 0.99 Å o Ca
2þ
)[45]. As a esul , Tb can o m s able complexes
wi h polypep ides eplacing Ca
2þ
, and con e s pho oluminescence unde
UV exci a ion in he ange o maximum p o ein abso p ion (λ
ex
¼285
nm) showing a cha ac e is ic maximum emission peak a 545 nm [44,
46]. Unde his p emise, he pho oluminescence p o ided by he inco -
po a ion o Tb was es ed wi h fluo ime y measu emen s and fluo es-
cence mic oscopy.
The emission band was measu ed a 285 nm exci a ion in he ange o
520–570 nm o ou samples: i) Tb-MSR-1, ii) MSR-1, iii) magne osomes
isola ed om Tb-MSR-1 (Tb-mag), and i ) magne osomes isola ed om
MSR-1 (mag). As shown in Fig. 5A, bo h he Tb-MSR-1 and Tb-mag
display he Tb-specific emission peak a 545 nm, whe eas hei co e-
sponding con ol coun e pa s, MSR-1 and mag, do no show any fluo-
escence signal, confi ming ha he de ec ed emission comes om Tb. In
he case o he Tb-mag, he fluo escence emission occu ing upon exci-
a ion wi h he maximum p o ein abso p ion wa eleng h sugges s ha Tb
is linked o he p o eins o he magne osome memb ane in addi ion o
hei p esence in he mine al co e, as in e ed by XANES measu emen s.
The dis ibu ion o Tb in he bac e ia is u he e idenced by means o
fluo escence mic oscopy imaging o Tb-MSR-1. Due o echnical limi a-
ions, he sample could no be exci ed a he op imum wa eleng h o 285
nm. Al e na i ely, images we e collec ed exci ing Tb-MSR-1 wi h UV
ligh using a band-pass fil e (350/50 nm) and he emission was collec ed
wi h a cu -o fil e om 500 nm. Unde hese condi ions, Fig. 5B shows
b igh field and fluo escence images o Tb-MSR-1.
E en hough he spa ial esolu ion and he measu emen condi ions
we e no op imal, he good ag eemen be ween b igh field and fluo-
escence images o bac e ial bodies demons a es ha he inco po a ion
o Tb is a success ul ou e owa ds he p oduc ion o luminescen bac e ia
wi h applica ions, o example, as bioma ke agen s.
3.2.3. Gd-MSR-1 as dual con as agen s o magne ic esonance imaging
Magne osomes a e p omising candida es as magne ic esonance
imaging (MRI) con as agen s since magne i e nanopa icles p o ide
nega i e con as by sho ening he T
2
ans e se elaxa ion ime
[47–49]. On he o he hand, pa amagne ic subs ances, such as Gd- and
Mn-based ma e ials, ac as posi i e con as agen s by sho ening he T
1
longi udinal elaxa ion ime.
MRI s udies ha e been ca ied ou in ou samples, o compa ison: i)
Gd-MSR-1, ii) MSR-1, iii) magne osomes isola ed om Gd-MSR-1 (Gd-
mag), and i ) magne osomes isola ed om MSR-1 (mag). In all cases,
samples we e suspended in wa e wi h Fe concen a ions anging om 0
mM o 0.36 mM and we e measu ed in an 11.7 T MRI scanne .
Fig. 6A shows he T
1
and T
2
pa ame ic maps ob ained wi h
inc easing Fe con en . These maps ha e been econs uc ed om he T
1
images acqui ed wi h 12 inc easing epe i ion ime (TR) alues
(150–12500 ms) and om he T
2
images acqui ed wi h 32 inc easing
ime- o-echo (TE) alues (5–135 ms, del a ¼5 ms) (see Figs. S4 and S5 in
he Supplemen a y In o ma ion). The T
2
maps o all he samples e eal a
clea sho ening o he T
2
elaxa ion ime when inc easing he Fe con en ,
indica ing ha hey can wo k as T
2
-sho ening con as agen s, as ex-
pec ed o magne i e nanopa icles. In e es ingly, while he T
1
maps o
MSR-1, Gd-mag, and mag a e essen ially independen o he concen a-
ion o Fe, Gd-MSR-1 shows a con as change wi h he Fe concen a ion,
enabling a po en ial use o hese bac e ia as T
1
-sho ening con as
agen s.
F om T
1
and T
2
maps he con as e ficiency o he samples can be
quan ified. The con as e ficiency, o signal enhancemen p oduced by a
con as agen , is measu ed by he longi udinal and ans e se elaxi -
i ies,
1
and
2
, espec i ely, which a e ob ained om he slope o he
linea fi s o he co esponding elaxa ion a es (R
i¼1,2
¼1/T
i
) s. Fe
concen a ion (Fig. 6B). The esul s o he elaxi i ies a e shown in
Table 1.
The
2
alues o all samples a e simila , anging be ween 315 and 370
mM
1
s
1
. This indica es ha he inco po a ion o Gd
3þ
ions does no
a ec he T
2
con as e ficiency. Such an obse a ion is easonable
conside ing he low Gd doping con en o hese magne osome co es
Fig. 5. A) Pho oluminescence o Tb-MSR-1 and MSR-1 and hei isola ed magne osomes (Tb-mag and mag) exci ed a λ
ex
¼285 nm. B) Mic oscopy images o Tb-MSR-
1. B igh field (le ) and fluo escence ( igh ) (λ
ex
¼350/50 nm, λ
em
500 nm).
L. Ganda ias e al. Ma e ials Today Bio 20 (2023) 100680
6
(34%, as es ima ed om XANES [27]), and he nea absence o
di e ences in he magne ic esponse o he MSR-1 and Gd-MSR-1 a ound
oom empe a u e [27]. The alues ob ained o
2
a e compa able o
o he alues epo ed o magne osomes in he li e a u e (be ween 200
and 526 mM
1
s
1
o fields anging be ween 4.7 and 17.2 T) [47,50,51].
On he o he hand, MSR-1, Gd-mag, and mag show negligible
1
alues, acco dingly o he T
1
maps shown ea lie , bu Gd-MSR-1 s ands
ou wi h a clea linea inc ease o he elaxa ion a e wi h he
concen a ion, esul ing in a
1
alue o 4.9 mM
1
s
1
. Thus, he inco -
po a ion o Gd adds T
1
con as ing capabili ies o Gd-MSR-1.
In sum, hese esul s showcase ha Gd-MSR-1 a e p omising dual
con as agen s o magne ic esonance imaging since, depending on he
pulse sequence used o imaging, hey will appea as posi i e in T
1
weigh ed images and as nega i e in T
2
weigh ed images. Dual con as
agen s ha e ecen ly gained inc eased a en ion o hei use in clinical
MRI [28,29]. A dual T
1
/T
2
con as agen p oduces a dis inguishable
con as in bo h T
1
-weigh ed and T
2
-weigh ed images, acili a ing hei
dis inc ion om po en ial endogenous subs ances o he body ha may
p o ide con as only in T
1
o T
2
modes [52].
3.2.4. In i o assessmen o he diagnos ic po en ial o Tb-MSR-1 and Gd-
MSR-1
The diagnos ic unc ionali ies acqui ed by MSR-1 h ough inco po-
a ion o Tb and Gd ha e been es ed in i o in wo cell models: A549
lung ca cinoma cells and RAW 264.7 mac ophages.
Fi s , we ha e analyzed he cy o oxici y o he bac e ia. As epo ed
p e iously [10], MSR-1 do no cause cy o oxici y in A549 lung ca cinoma
Fig. 6. A) T
1
(le ) and T
2
( igh ) pa ame ic maps ob ained a inc easing Fe concen a ions a 11.7 T. B) Longi udinal (R
1
¼1/T
1
) and ans e se (R
2
¼1/T
2
)
elaxa ion a es s. Fe concen a ion. The slopes o he linea fi s gi e he elaxi i ies (
1
and
2
) displayed in Table 1.
Table 1
Longi udinal (
1
) and ans e se (
2
) elaxi i ies a 11.7 T ob ained om he
linea fi ings in Fig. 6B.
1
(mM
Fe
)
1
s
1
2
(mM
Fe
)
1
s
1
Gd-MSR-1 4.87 315.3
MSR-1 0.05 354.1
Gd-mag 0.00 375.9
mag 0.00 349.1
L. Ganda ias e al. Ma e ials Today Bio 20 (2023) 100680
7
cells as hey do no a ec hei iabili y no hei g owing abili y. He e
we aim o e i y whe he Tb/Gd inco po a ion causes any change in
MSR-1 cy o oxici y.
As obse ed in Fig. 7, cells con inue o p oli e a e when incuba ed
wi h MSR-1 (wi hou o wi h Tb/Gd). Howe e , i mus be no ed ha in
he case o RAW 264.7, he e is a significan di e ence in cell p oli e -
a ion a e 48 h when he cells a e incuba ed wi h Tb/Gd-MSR-1 in
compa ison o MSR-1.
To u he in es iga e he cy o oxici y o he bac e ia, we measu ed
cell iabili y as he a io be ween li ing and o al numbe o cells. As
obse ed in Fig. 7, he e a e no significan changes in cell iabili y a e
24 h and 48 h o cell incuba ion wi h MSR-1, Tb-MSR-1, o Gd-MSR-1 as
he mean alue o iabili y in bo h cell ypes anges be ween 87% and
96% a e 48 h, indica ing ha he inco po a ion o Tb/Gd does no in-
c ease he cy o oxici y o MSR-1.
Then, aiming o es he diagnos ic po en ial o Tb-MSR-1 and Gd-
MSR-1 in i o, bo h es ed cell lines we e incuba ed o e nigh wi h
bac e ia as explained in he Ma e ials and Me hods sec ion.
Fig. 8A shows b igh field and fluo escence images o con ol A549
cells and Tb-MSR-1-loaded A549 cells ob ained in he same condi ions as
in Fig. 5B. As obse ed in he images, he con ol cells do no show any
fluo escence when exci ed wi h UV ligh whe eas cells incuba ed wi h
Tb-MSR-1 do show fluo escence. Ano he conclusion ha can be in e ed
om hese images is ha Tb-MSR-1 bac e ia a e inside he cells as hey
can be obse ed in he cy oplasm su ounding he nuclei ha appea as
non-fluo escen .
Rega ding he con as ing pe o mance o Gd-MSR-1, Fig. 8B shows
he T
1
and T
2
pa ame ic maps o con ol and Gd-MSR-1-loaded A549
and RAW 264.7 cells. These maps ha e been econs uc ed om he T
1
and T
2
images p esen ed in Figs. S6 and S7. While no con as is obse ed
in he con ol cells, he inco po a ion o Gd-MSR-1 p oduces a sho ening
o bo h T
1
and T
2
in bo h cell lines, p o ing he po en ial o Gd-MSR-1 as
dual con as agen s.
4. Conclusions
The inco po a ion o Tb and Gd in o Magne ospi illum g yphiswaldense
MSR-1 imp o es hei po en ial as biomedical agen s because he bac-
e ia e ain he cha ac e is ics o he unmodified MSR-1 in e ms o
hea ing e ficiency o magne ic hype he mia and T
2
con as o MRI,
while adding supplemen a y unc ionali ies. Tb-MSR-1 gain lumines-
cence p ope ies unde UV exci a ion and Gd-MSR-1 u n in o dual
con as agen s o magne ic esonance imaging because Gd
3þ
adds T
1
con as o he T
2
con as p o ided by he magne i e co es o magne-
osomes. Finally, we ha e e ified ha Tb-MSR-1 and Gd-MSR-1 a e no
cy o oxic and ha e demons a ed hei new diagnos ic unc ionali ies in
i o in wo cell models. Thus, we conclude ha hese lan hanide-
modified MSR-1 could be po en ial biomedical agen s wi h imp o ed
Fig. 7. Le : Time e olu ion o he numbe o li ing A549 and RAW 264.7 cells no malized o ¼0 h. Righ : Viabili y pe cen ages a e 24 h and 48 h o cul u ing
MSR-1/Tb-MSR-1/Gd-MSR-1-loaded A549 and RAW 264.7 cells. The esul s ep esen he mean s anda d de ia ion alues, n ¼3.
L. Ganda ias e al. Ma e ials Today Bio 20 (2023) 100680
8
diagnos ics capaci ies, u he ad ancing magne o ac ic bac e ia as
p omising nano obo s.
C edi au ho s a emen
Lucía Ganda ias: Da a cu a ion, Fo mal analysis, In es iga ion,
Me hodology, W i ing –o iginal d a , W i ing – e iew &edi ing.
Elizabe h M. Je emo as: In es iga ion, Me hodology, W i ing – e iew
&edi ing. Da id Gandia: In es iga ion, W i ing – e iew &edi ing.
Lou des Ma cano: In es iga ion, W i ing – e iew &edi ing. Vi ginia
Ma ínez-Ma ínez: In es iga ion, W i ing – e iew &edi ing. Ped o
Ramos-Cab e : In es iga ion, Fo mal analysis, W i ing – e iew &
edi ing. Daniel M. Che ie : In es iga ion, Fo mal analysis, W i ing –
e iew &edi ing. Se gio Valencia: In es iga ion, W i ing – e iew &
edi ing. Luis Fe n
andez Ba quín: Concep ualiza ion, P ojec adminis-
a ion, W i ing – e iew &edi ing. M. Luisa Fdez-Gubieda: Concep-
ualiza ion, Me hodology, Supe ision, P ojec adminis a ion, W i ing –
e iew &edi ing. Ja ie Alonso: Concep ualiza ion, Me hodology,
P ojec adminis a ion, W i ing – e iew &edi ing. Ana Ga cía-P ie o:
In es iga ion, Fo mal analysis, P ojec adminis a ion, Me hodology,
Supe ision, W i ing –o iginal d a , W i ing – e iew &edi ing. Alicia
Muela: Concep ualiza ion, Me hodology, Supe ision, W i ing –o iginal
d a , W i ing – e iew &edi ing.
Funding
This wo k has been unded by he Spanish Go e nmen (g an s
PID2020-115704RB-C31, PID2020-114347RB-C32, PID2020-
115704RB-C33 and RED 2018–102626–T (HIPERNANO) unded by
MCIN/AEI/10.13039/501100011033) and by he Basque Go e nmen
(p ojec s IT1479-22 and IT1639-22). LG acknowledges he Spanish
Go e nmen o he PhD/Pos doc o al ellowship (PRE2018-083255
unded by MCIN/AEI/10.13039/501100011033 and by Eu opean Union
Nex Gene a ionEU/PRTR). EMJ acknowledges he Beca Concepci
on
A enal (Gobie no de Can ab ia –G an n. 406333) and Alexande on
Humbold Pos doc o al Fellowship. LM acknowledges he BBVA Foun-
da ion o he Leona do Fellowships o Resea che s and Cul u al C ea-
o s 2022.
Decla a ion o compe ing in e es
The au ho s decla e ha hey ha e no known compe ing financial
in e es s o pe sonal ela ionships ha could ha e appea ed o influence
he wo k epo ed in his pape .
Da a a ailabili y
Da a will be made a ailable on eques .
Acknowledgemen s
We acknowledge he BL22-CLÆSS beamline s a o he ALBA syn-
ch o on o assis ance du ing he expe imen and he s a o he I-14
beamline a he Diamond Ligh Sou ce, especially D . Miguel G
omez-
Gonz
alez o his help du ing he expe imen and he da a analysis. We
acknowledge he human and echnical suppo p o ided by he mic o-
scopy se ices o SGIke (UPV/EHU). We hank P o . Jos
e
Angel Ga cía,
D . I~
naki O ue, and Danny Yosma Villanue a-
Al a o o he hype -
he mia measu emen s, and Se gio de la Vega o echnical suppo .
Appendix A. Supplemen a y da a
Supplemen a y da a o his a icle can be ound online a h ps://
doi.o g/10.1016/j.m bio.2023.100680.
Fig. 8. A) Mic oscopy images o con ol and Tb-MSR-1-loaded A549 cells. B igh field (uppe panel) and fluo escence (lowe panel) (λ
ex
¼350/50 nm, λ
em
500 nm).
B) T
1
and T
2
pa ame ic maps o con ol and Gd-MSR-1-loaded A549 and RAW 264.7 cells ob ained a 11.7 T.
L. Ganda ias e al. Ma e ials Today Bio 20 (2023) 100680
9