ARTICLE

Joining Time-Resolved Thermometry

and Magnetic-Induced Heating in a
Single Nanoparticle Unveils Intriguing
Thermal Properties
Rafael Pin~ ol,† Carlos D. S. Brites,‡ Rodney Bustamante,† Abelardo Martı́nez,§ Nuno J. O. Silva,‡
José L. Murillo,† Rafael Cases,† Julian Carrey, Carlos Estepa,† Cecilia Sosa,^ Fernando Palacio,† Luı́s D. Carlos,*,‡

)
and Angel Millán*,†
†
Departamento de Fisica de la Materia Condensada, Facultad de Ciencias and Instituto de Ciencia de Materiales de Aragón, CSIC
Universidad de Zaragoza, 50009
See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

Zaragoza, Spain, ‡Departamento de Física and CICECO Aveiro Institute of Materials, Universidade de Aveiro, 3810
193 Aveiro, Portugal, §Departamento de Electrónica de
)

Potencia. I3A, Universidad de Zaragoza, 50018 Zaragoza, Spain, Laboratoire de Physique et Chimie des Nano-Objets (LPCNO)Université de Toulouse, INSA, UPS,
CNRS (UMR 5215), F-31077, Toulouse, France, and ^Departamento de Toxicología, Facultad de Veterinaria, Universidad de Zaragoza, 50013 Zaragoza, Spain
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ABSTRACT Whereas efficient and sensitive nanoheaters and nanothermometers are

demanding tools in modern bio- and nanomedicine, joining both features in a single
nanoparticle still remains a real challenge, despite the recent progress achieved, most of it
within the last year. Here we demonstrate a successful realization of this challenge. The
heating is magnetically induced, the temperature readout is optical, and the ratiometric
thermometric probes are dual-emissive Eu3þ/Tb3þ lanthanide complexes. The low
thermometer heat capacitance (0.021 3 K
1) and heater/thermometer resistance
(1 K 3 W
1), the high temperature sensitivity (5.8% 3 K
1 at 296 K) and uncertainty
(0.5 K), the physiological working temperature range (295
315 K), the readout
reproducibility (>99.5%), and the fast time response (0.250 s) make the heater/thermometer nanoplatform proposed here unique. Cells were incubated
with the nanoparticles, and fluorescence microscopy permits the mapping of the intracellular local temperature using the pixel-by-pixel ratio of the
Eu3þ/Tb3þ intensities. Time-resolved thermometry under an ac magnetic field evidences the failure of using macroscopic thermal parameters to describe
heat diffusion at the nanoscale.

KEYWORDS: nanothermometers . nanoheaters . magnetic hyperthermia . intracellular temperature . heat diffusion

N
anothermometers, nanoheaters, nanoheater's local temperature. Notwith-
and nanoscale heat transfer are standing great activity in the past decade,
hot topics in nanotechnology.1
3 sensitive and efficient nanoparticles em-
These three fields are interrelated and bedding both heaters and thermometers
depend on each other for their parallel have not yet been realized, despite several
development. A detailed understanding of intriguing reports, particularly in the last
heat propagation processes at the nano- year.13
20
scale requires the development of thermo- This work develops heating and thermo-
metric and heating tools with nanometric metry in a single nanoparticle with unpre-
resolution.3
5 Magnetic-, plasmonic-, and cedented thermal contact (the thermom- * Address correspondence to
phonon-induced thermal heating of nano- eter is located just on the surface of the [email protected],
[email protected].
particles are powerful noninvasive techni- heater) and explores a glimpse of the
ques for bio- and nanotechnology appli- possibilities opened by such nanoparticles, Received for review January 5, 2015
cations, such as drug release,6,7 remote a tool that could give a definitive impulse to and accepted February 18, 2015.
control of single-cell functions,8,9 plasmonic an effective use of local heat generation
Published online February 18, 2015
devices,10 and hyperthermia therapy of at the nanoscale. Simple experiments 10.1021/acsnano.5b00059
cancer11,12 and other diseases.8 To be effec- performed with these nanoparticles with
tive, local heating requires measuring the high time (up to 0.250 s) and temperature C 2015 American Chemical Society

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 ARTICLE
resolutions (0.5 K) show a quasi-instantaneous onset of In another (ii) work, Gd2O3:Yb,Er nanorods (thermom-
temperature gradient from the nanoheater to the eters) are decorated with Au nanoparticles (heaters).16
medium that implies a huge heat resistance that This single integrating nanoplatform has the advan-
cannot be explained from the macroscopic thermal tage of measuring the absolute local temperature of
properties of the system. Moreover, the nanoparticles the sample volume under irradiation (rather than an
offer additional advantages, such as an optical thermo- average temperature, as in (i)), over a wide range (300
metric readout, allowing an easy implementation in a to 2000 K). It has, however, two important disadvan-
large variety of systems, a high stability in aqueous tages: the thermometric probe is overdimensioned
medium, and a capacity for biological multifunctiona- relative to the heater, and the plasmonic-induced
lization and targeting (Supplementary Section 1). heating power is low because the excitation wave-
The heater/thermometer nanoplatform here pro- length required to excite the thermometer (980 nm) is
posed can be beneficial in many technological applica- off resonance with the localized surface plasmonic
tions from microelectronics to bio- and nanomedicine, band of the Au NPs (maximum at ∼550 nm).
namely, in hyperthermia. Current strategies for hyper- An early example following the single-particle strat-
thermia include heating the whole tumor tissue above egy was performed on Au nanoparticle suspensions
315
316 K by radio frequency electromagnetic fields, using intense pulse lasers (15  10
6 J/pulse) for
UV
visible
IR radiation, and ac magnetic fields, using, heating and pump
probe spectroscopy for thermom-
respectively, dielectric, photonic, and magnetic micro/ etry.22 This system has an important limitation since
nanomaterials acting as heat sources. While light- heating and temperature measurements are discon-
induced approaches are suitable for near-surface ap- tinuous, occurring in a very short time scale (∼10
10 s).
plications, the use of magnetic fields allows in-depth In a second example, a fluorophore was attached to the
applications, now in phase II clinical applications.11 shell of the magnetic heater by a bond that breaks at a
However, to achieve 315
316 K at the tumor mass, a certain temperature.13 An analysis of the fluorophore
high load of magnetic nanoparticle is needed. The content in the supernatant after heating gives the
efficacy of this technique will be much improved when moment at which the bond-breaking temperature is
the heat is applied at localized targeted sites in the reached, but this is still far from a real time and
cells, causing a similar death effect with a much smaller continuous temperature readout. This system was
number of nanoparticles.12 The question then is how soon improved by using the denaturalization of DNA
high the local temperature can rise before the heat strands as the temperature probe14 instead of bond
generated by the nanoparticles is dissipated to the breaking, but still the temperature reading was neither
medium. This is where the incorporation of local continuous nor instantaneous. A third example uses
thermometry to the nanoheater will provide the an- LaF3 nanoparticles doped with a high Nd3þ concentra-
swer. The accurate measurement of the nanoheater's tion (25 at. %) behaving as heaters/thermometers with
surface temperature by a nanothermometer is crucial 0.2% 3 K
1 sensitivity and a heating performance from
for regulating the heat released to the surroundings, room temperature to ∼320 K with a high laser power
allowing the adjustment of the irradiation parameters density (up to 3 W 3 cm
2).19 The same group reported a
and thus assisting the therapy. step forward toward nanoparticle-based photothermal
Progress in the measurement of the nanoheater's therapy at clinical level, applying the LaF3:Nd3þ (5.6 at. %)
temperature followed two directions: (i) using a second nanoparticles to temperature-controlled photothermal
nanoparticle for thermometry (dual-particle approach) therapy of cancer tumors in mice.20
and (ii) anchoring a molecular thermometric probe at
the surface or outer shell of the nanoheater (single- RESULTS AND DISCUSSION
particle approach). The heater/thermometer nanoplatform was pre-
In contrast with strategy (ii), the dual-particle ap- pared from iron oxide cores functionalized with Eu3þ
proach has the inherent limitation of the uncontrolled and Tb3þ complexes, coated with a P4VP-b-P(PMEGA-
spatial distribution of nanoheaters and nanothermom- co-PEGA) copolymer and dispersed in water to obtain
eters,15
18,21 with the concomitant large distribution of an aqueous ferrofluid suspension (Supplementary
the nanoheater/nanothermometer distances (thermal Scheme 1, Table 1, Figures 1
8). At neutral pH, the
sensing is not achieved at the same heating volume). copolymer has a hydrophobic part (P4VP) and a hydro-
These restrictions were partially mitigated recently via philic part (PEG) that is well solvated by water mol-
strategy (ii) by encapsulating magnetic nanoparticles ecules, thus restraining the agglomeration of the
and thermometric luminescent NaYF4:Yb,Er nano- nanoparticles. Consequently, the ferrofluid suspension
particles in a larger mesoporous silica nanoparticle.18 is stable for months, as described in the Supporting
Still, the determination of the heater's temperature was Information. P4VP-PEGA copolymer coatings have also
indirect, as the thermometer was 8
9 nm from the shown to be advantageous in terms of blood and cell
heater, and the low temporal resolution (30 s) pre- toxicity, as explained in the Supporting Information
cluded a fine screening of the heat transfer process. (Section 1.3). Before use, the ferrofluid was filtered

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 ARTICLE
Figure 1. Structural properties of the maghemite multiparticle core
shell beads. (A) Schematic scaled representation of a
typical bead. In the central region, the Eu3þ/Tb3þ complexes (reddish layer) for temperature measurement cover the
maghemite NPs (orange). The P4VP (green) forms a first shell encapsulating the magnetic NPs, and the P(PMEGA-co-PEGA)
chains (blue) occupy the outer part of the bead. (B) Hydrodynamic size distribution of the maghemite multiparticle core (blue
bars) and of the whole bead (red bars). (C) TEM/STEM image of the beads showing the multiparticle iron oxide core and
chemical image of the sample by STEM showing that all the heavy elements are located in the core. (D) EEL spectrum and (E)
EDX spectrum at a point inside the multiparticle core.

(through a 2.2  10
7 m filter, so it can be easily as Db = 48.0 nm (PDI = 0.2, Figure 1B). A key point in this
sterilized for biological use) and purified by magnetic nanoplatform is the location of the thermometric Eu3þ
separation to ensure that all the heater/thermometer and Tb3þ ions. A first indication is given by STEM
nanoplatforms (also called beads throughout the images (Figure 1C, Supplementary Figure 12), showing
article) contain both the thermometric and magnetic that bright heavy elements (Fe from the magnetic
components. multiparticle core, Eu and Tb from the complexes) are
Chemical analysis, scanning transmission electron concentrated in the same regions. Furthermore, EELS
microscopy (STEM) and cryoTEM (Figure 1C, Supple- and EDX analyses confirmed a co-localization of Fe and
mentary Figures 9, 10), electron diffraction (Supple- Tb (Eu is too scarce to be detected, Supplementary
mentary Figure 9), electron energy loss spectroscopy Figure 11) in the bead multiparticle core. EDX analyses
(EELS) (Figure 1D), energy-dispersive spectroscopy at points outside the bead multiparticle core con-
(EDX) (Figure 1E, Supplementary Figures 11 and 12), firm the absence of these elements (Supplementary
and dynamic light scattering (DLS) (Figure 1B, Supple- Figure 12). Therefore, the thermometric Eu3þ and Tb3þ
mentary Figure 13) permit building up a structural ions are located on the surface of the iron oxide
model of the heater/thermometer bead (Figure 1A). nanoparticles.
Accordingly, TEM images (Supplementary Figures 9 The absolute local temperature is inferred through
and 10) show the magnetic multiparticle core has an the dependence on temperature of the emission spec-
average diameter Dc = 23 ( 9 nm and is formed by an tra of the nanoplatform (Figure 2), as reported pre-
aggregate of 1 to 7 iron oxide magnetic nanoparticles viously for Eu3þ/Tb3þ-containing organic
inorganic
with a spinel crystal structure and a nanoparticle hybrids.3,23
25 The thermometric response of such
diameter Dp = 10 ( 2 nm. The DLS results for the size systems results from thermally activated energy trans-
of the magnetic multiparticle core, Dc = 23.2 nm (with a fer between Eu3þ- and Tb3þ-emitting levels and trip-
polydispersity index, PDI, of 0.3) are very similar to let energy states of the ligands and of the host
those observed in cryoTEM images (Figure 1B). The matrix,25 resulting in both the temperature range of
polymer shell did not yield any contrast in TEM and maximum sensitivity and excitation wavelength
cryo-TEM images (Figure 1C); thus the outer diameter capable of being tuned by a proper selection of
of the beads was established from DLS measurements organic ligands and host matrix.23,25 In this case the

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Figure 2. Emission spectra and thermometric performance of the multicore beads. (A) Emission spectra of the water
suspension multicore beads (6.2 g Fe2O3 3 L
1) in the temperature range 295
315 K. The excitation wavelength is 365 nm. The
5
D0 f 7F2
4 (Eu3þ) and the 5D4 f 7F5 (Tb3þ) transitions are identified, and the asterisk marks the spectral region where an
overlap is observed between the 5D0 f 7F0,1 and the 5D4 f 7F4 lines. The inset shows a simplified energy scheme of the Eu3þ
and Tb3þ ions and btfa ligand, where the most intense Eu3þ and Tb3þ transitions are presented. The orange arrows represent
the thermally driven 5D4 f host energy transfer and the corresponding back transfer. (B) Relative sensitivity Sr of the water
suspension multicore beads in the temperature range 295
315 K. (C) Temperature cycling of the water suspension multicore
beads between 297 and 310 K, with a repeatability better than 99.5% in the 10 consecutive cycles. The solid and interrupted
lines are guides for the eyes. The error bars result from the standard deviation of each histogram (60 s acquisition).

nanothermometer was designed using Eu3þ and Tb3þ sensitivity of 5.8% 3 K
1 at 296 K, Figure 2B). No hyster-
ions, btfa (4,4,4-trifluoro-1-phenyl-1,3-butanedione) esis and/or photobleaching distorting temperature
ligands, and P4VP, aiming for an optimal sensitivity at measurements are discernible in the time scale of the
the physiological temperature range and a cost-effec- experiments (Figure 2). Upon 10 consecutive tempera-
tive 365 nm excitation wavelength (Supplementary ture cycles between 297 and 310 K, the thermometer
Figures 14
20). The emission spectra of the water reproducibility is as high as 99.5% (Figure 2C), demon-
suspension of the beads shows that the 5D0 f 7F2 strating the robustness of the temperature readout. In
(Eu3þ) integrated intensity (IEu) is insensitive to tem- our experimental conditions, the time fluctuations of
perature, whereas the intensity of the 5D4 f 7F5 (Tb3þ) the thermometric parameter Δ are always below 0.7%,
transition (ITb) decreases with the temperature increase decreasing when the integration time is increased. Con-
(Figure 2A). This temperature dependence is rationa- verting these fluctuations into temperature allows one to
lized assuming that the first excited triplet state of the calculate the uncertainty of the temperature measure-
btfa ligand with energy above that of the 5D4 emitting ment, which is 0.5 K (Supplementary Figure 21B).
state is populated through thermally driven Tb3þ- The heater/thermometer nanoplatform is applied
to-ligand energy transfer (diminishing, therefore, the to monitor local temperature changes under ac mag-
5
D4 f 7F5 intensity). The energy difference between that netic fields (Figure 3). A scheme of the experimental
triplet state and the 5D0 emitting level is too large to setup used is depicted in Figure 3A (Supplementary
permit the thermally driven depopulation of the Eu3þ Scheme 2). Two on
off field protocols are described,
emitting state in the 295
315 K interval, and the illustrating a classic one-pulse protocol (Figure 3B)
thermometric parameter Δ = ITb/IEu guarantees the and a multiple-pulse protocol where the fine heating

absolute measurement of temperature.25 The ratio- thermometry ability of the beads is highlighted
metric thermometric parameter Δ allows the emission (Figure 3C,D, Supplementary Figure 22). For compar-
spectrum/temperature conversion through the calibra- ison purposes, temperature readings through a semi-
tion curve generated for the particular experimental conductor optical reference thermometer immersed in
conditions used (Supplementary Table 2, Figure 21A). the fluid and an infrared thermal camera focused on
As discussed elsewhere,3 the ratiometric (or self- the wall of the container are also used. As expected,
referencing) intensity measurements are not compro- before the field was turned on, temperature values
mised by the well-known disadvantages of experi- in all thermometers were coincident and constant
ments based on the intensity of only one transition. (Figure 3B
D).
The thermometric performance of the beads is eval- When the field was switched on, the molecular
uated using the relative sensitivity Sr = (∂Δ/∂T)/Δ,3,23 thermometer responded immediately and with a quite
ranging from 0.5 to 5.8% 3 K
1, 295
315 K (maximum sharp slope, while the semiconductor thermometer

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Figure 3. Temperature dynamics during the on
off switching of an ac magnetic field. (A) Schematic representation of the
experimental setup showing the cuvette filled with the water-suspended multicore beads and the optical fiber bundle,
composed of 1 core fiber for emission recovery and 6 lateral fibers for excitation (fiber diameter of 400  10
6 m).
(B) Temperature measurement of a 0.75 mL ferrofluid suspension during a 600 s one-pulse experiment using the molecular
(blue circles) and semiconductor (read circles) thermometers. The shadowed area marks the time interval when the magnetic
field is turned on (60
220 s). (C) Temperature measurement of the same suspension during a 300 s multipulse experiment
using the molecular (blue circles) and semiconductor (red circles) thermometers. The shadowed areas mark the time intervals
when the magnetic field is turned on (15 s). The solid lines correspond to the fit of experimental data with the lumped
elements model (Schemes S3
S5). (D) Zoom of the first 90 s of the measurements (marked area in part C). For comparison, the
infrared thermal camera (black squares) temperature readout is included.

had a delay of about 10 s and a smoother slope. e.g., 2 orders of magnitude higher than the only value
Similarly, when the field is switched off, the response reported so far, 30 s for magnetically heated nano-
of the molecular thermometer is again faster and particles.18 However, there are examples of nano-
sharper than that of the semiconductor thermometer thermometers demonstrating faster response, e.g.,
(Figure 3B
D), converging then for longer times the in vitro nonratiometric temperature measurements
(Supplementary Figure 22A). Clearly, the most inter- with a temporal resolution of 10
2 s.26
esting thermal events occur during 15 s after switching The temperature variation at the nanoheater and
the field on and off, and they can be discerned only its temperature gap with the surrounding media
when the time response of the thermometer is fast, as presented here are in consonance with previous
is the case here (characteristic time, at least, on the findings9,13,18 and deserve a critical look. With this
order of the detector integration time, 0.250 s). Besides purpose, we focus our attention on one single bead
the necessary high temporal resolution, the observa- (Figure 1A) surrounded by water. The expected power
tion of the sharp features for t < 10 s is expected only if dissipated by each heater, P ≈ (7
10)  10
16 W/bead,
the system has a good thermal conductivity and a is estimated based on the out-of-phase component
small heat capacity between the heat source and the of the ac magnetic susceptibility (Supplementary
thermometer. This is a signature of a thermometer truly Figures 23
25). This value corresponds to a specific
coupled to a heat source, and, to the best of our power loss of 33 to 50 W/g (Fe2O3), which is within the
knowledge, it is the first time that such a signature is range of those usually found for Fe2O3 nanoparticles.27
found for heat sources of such small size. The time Naturally, this power results in a heat flux and in a
response of the heater/thermometer nanoplatform temperature gradient, whose values depend on the
reported here is much faster when compared with thermal properties of the polymer coating, water, and
the thermometers that are coupled with heat sources, the container. On the basis of the P4VP and P(PEGA)

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bulk thermal conductance (and for a coating with a (lumped) polymer coating, the water and the wall of
thickness of ∼13 nm), the temperature gradient across the container, and thermal resistances between each
the polymers is expected to be on the order of 10
8 K. element and between water and air and the wall and
Then, no temperature gap between the nanoparticles air (Supplementary Scheme 5). The best fit is shown in
and the water should be observed (Supplementary Figure 3C,D and in Supplementary Figure 26, using the
Tables 3 and 4 for dimensions and macroscopic ther- effective lumped resistances presented in Supplemen-
mal parameters of the materials). tary Table 5. As expected, given the relation between
Currently, it is not yet clear whether an increasing the capacitance of the polymer and water, the tem-
number of experimental results9,13,15,18,28 (including perature at the polymer (where the molecular thermo-
those reported here) are meaningless or whether the meter is placed) follows the jumps of the heating
macroscopic models/parameters applied to the nano- source. The measurement of the semiconductor thermo-
heaters are not valid. Evidence for the existence of meter is close to the measurement of the infrared
large temperature gaps between nanoheaters and sur- camera at the surface of the wall of the container
rounding media comes from different heating sources (Figure 3D), which is well reproduced by the simulation.
(i.e., magnetothermia,9,13,18 absorption/dissipation by While the heat capacitances of the ∼1017 beads
Au nanoparticles,21 and nonradiative decay in LaF3: (present in the ferrofluid suspension) are close to those
Nd3þ nanoparticles19) and different thermometric sys- expected from bulk, the equivalent thermal resistances
tems (i.e., Au nanoparticles, quantum dots, proteins, are 10 orders of magnitude higher than those pre-
nanodiamonds, and trivalent lanthanide ions). These dicted using the bulk thermal conductivity and the
systems have so far passed the most obvious reliability bead dimensions (Supplementary Section 7), stressing
tests under different chemical environments and the profound contradiction between models and
external fields,28 and they not contradict the thermo- experimental results aforementioned.
dynamic arguments presented by Baffou et al.29 Opossum kidney (OK) cells were incubated with the
demonstrating the impossibility of having tempera- heater/thermometer nanoparticles. Fluorescence
ture heterogeneities of up to a few Kelvins inside a images under UV excitation (340
380 nm) were taken
single cell resulting from internal energy dissipation simultaneously after half-splitting the emission in two
mechanisms (endogenous thermogenesis). We also separate wavelength ranges covering the main emis-
notice that the application of an ac magnetic field to sion bands of Tb3þ (529
555 nm) and Eu3þ (597

cells containing a small number of magnetic nanopar- 620 nm) (Figure 4). Observation (through the ocular
ticles induces cell death without increasing the global lenses) of cells stained with DAPI showed the inter-
cell temperature.30 This may be taken as indirect nalization of the nanoparticles, mostly around the
evidence of local temperature gaps like the one mea- nucleus, although it seems they have not penetrated
sured in the present experiments. Concerning the inside (Supplementary Section 8). A comparison of
models/parameters applied to the nanoheaters, it has Figure 5A,B unequivocally shows the co-localization
been pointed out that nanoparticles behave very of both Tb3þ and Eu3þ ions in the OK cells over a region
differently when compared to bulk systems.1 For in- near the nucleus. This enables the thermometric
stance, the emergence of a resistance for the heat mapping at subcellular scale by simply taking the
transport at the nanointerfaces induces the appear- pixel-by-pixel ratio of the Tb3þ and Eu3þ intensities
ance of temperature gradients.31,32 Interphase heat and using the calibration curve of Supplementary
resistances are usually given as the equivalent thick- Figure 21A. Figure 5C shows a temperature mapping
ness of an extra layer of material producing the same of the regions of interest (where both emissions are
effect (Kapitza length, lK). In our case lK is on the order of discernible), permitting estimating the temperature
20 nm, which means that the overall thermal resistance near the cell's nucleus. The histogram of Figure 5D
would increase by an order of magnitude at most with indicates that the temperature is constant over the
respect to heat transfer across the polymer shell region mapped (299.3 ( 0.2 K) within the uncertainty
(Supplementary Section 7). of the molecular thermometer (0.5 K). The maps of two
An estimation of the equivalent thermal parameters other imaged nuclei (squares 2 and 3 in Figure 4C,D)
needed to reproduce the experimental results under reveal a temperature distribution similar to that of
the classical heat laws can be obtained by considering square 1 (Supplementary Figure 27), despite the
a lumped thermal capacity model, based on a simpli- changes on the intensity values of the Tb3þ and Eu3þ
fication of the structural model of Figure 1 (Sup- emissions. The lateral resolution of such mapping is
plementary Schemes 4, 5). This approach is justified defined by the pixel image and corresponds to
since a uniform temperature within each element of ∼220 nm. The penetration depth of the UV-excited
the bead is expected based on the estimated Biot heater/thermometer nanoplatforms reported here is
number (Supplementary Section 7.6). The model con- estimated as ∼0.2  10
3 m.33 Although the penetra-
sists of a heat source simulating the heat dissipated by tion depth could be improved by shifting the excita-
the iron oxide nanoparticles, the heat capacitances of a tion to the NIR spectral range,34,35 the thermal

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Figure 4. Imaging of Tb3þ and Eu3þ emissions from cell-internalized multicore beads. Fluorescence images of OK cells treated
with the nanoparticles showing the Tb3þ (545 nm) (A and C) and Eu3þ (610 nm) (B and D) emissions. The brightness and
colors have been modified for better visualization and do not correspond to the actual intensity ratio. All scale bars
correspond to 40 μm.

Figure 5. Temperature mapping of OK cells. Magnification of the areas in Figure 4C,D delimited by the squares showing the
(A) Eu3þ (5D0 f 7F2, 610 nm) and (B) Tb3þ (5D4 f 7F5, 545 nm) emissions. The pseudocolored maps were chosen to illustrate
the co-localization of the Eu3þ and Tb3þ emissions. For better visualization, the maximum of the Tb3þ colormap was scaled by
a factor of 3. The interrupted lines delimitate the nucleus of the OK cell, marking the region of interest where the temperature
map presented in part C was computed. (D) The histogram of the temperature distribution near the OK nucleus follows a
Gaussian distribution of mean value ((standard deviation) 299.3 ( 0.2 K (r2 > 0.997), in accord with the cell culture
temperature. All scale bars correspond to 10 μm.

sensitivity is, in general, poorer. For instance, in Nd3þ- possibilities in studies of the heat flow at the nanoscale,
doped luminescent thermometers with NIR excitation including thermal capacitance and conductivity across
and emission (within the first biological optical window) nanostructured media,36 as for instance detailed stud-
the relative sensitivity is almost 40 times lower that that ies in cellular thermal processes.37,38 In addition, we
reported here, despite the much larger penetration have established the local temperature mapping near
depth, ∼(1
2)  10
3 m.34,35 However, NIR excitation the nucleus of OK cells incubated with the heater/
is less damaging to cells compared with UV excitation thermometer beads simply using the pixel-by-pixel
(especially when high-power densities are used), and ratio of the Tb3þ and Eu3þ intensities. Furthermore,
the signal measurements are not disturbed as much by the heater/thermometer single nanoplatform reported
tissue autofluorescence as UV-excited signals. here shows great potential for the magnetically trig-
gered gene expression control that resulted in tumor
CONCLUSIONS growth inhibition39 and for impact on the design of
In summary, the unprecedented relatively small heat hyperthermia therapies based on localized manipula-
capacitance of the molecular thermometer and its tion of heat flows and short application times. In this
physical contact with the nanoheaters reveal the way, local energy supply that is not immediately
existence of an unexpected temperature gradient dissipated to the surrounding media could be enough
between nanoheaters and surrounding media for re- to induce irreversible intracellular damage in tumor
latively long time intervals (t > 10 s) and relatively low cells within a short time period, while maintaining the
heat powers (10
16 W/heater). Moreover, the contin- temperature of the neighboring tissue.40 Together
uous temperature monitoring with high time resolu- with an adequate vectorization of the particles, unpre-
tion allows the observation of previously undisclosed cedented specificity would be achieved. The use of
changes during the first few seconds (t < 10 s) of the system presented here can help to settle these
heating by an ac magnetic field. This opens intriguing questions and to give a fair account of the real

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potential of local hyperthermia. In a more extended enable thermal conductivity studies in cellular orga-
vision, accurately controlled local heating and precise nelles and across membranes, as well as detailed
temperature determination in the cellular media will studies in cell physiology related to thermal processes.

METHODS modified Eagle's medium-Ham's F12 (Gibco-Life Technologies),

supplemented with 10% fetal calf serum (FCS), glutamine, and
Synthesis. All the synthetic procedures are described in
antibiotics. The cells were made quiescent for 24 h previous to
detail in Supplementary Section 1. P4VP-Cl and the final diblock
the treatments with ferrofluids by incubating them in the same
copolymer P4VP-b-P(PMEGA-co-PEGA) were prepared by atom
medium containing only 0.5% FCS. For fluorescence observa-
transfer radical polymerization. [Ln(btfa)3(H2O)2] (Ln = Eu, Tb)
tions, quiescent cells grown in chamber slides (Millipore) were
complexes were prepared by mixing the reactants in ethanol
treated with thermometric nanoparticles for an additional 24 h.
solutions. Iron oxide multiparticle cores were prepared by
Then, the supernatants were aspirated out, and the cells were
precipitation of iron salts in ammonia solutions. Core
shell
washed three times with cold PBS, fixed with 3% (w/v) para-
Eu3þ/Tb3þ-containing Fe2O3@P4VP-b-P(MPEGA-co-PEGA) nano-
formaldehyde for 10 min, washed again three times, and
particles were prepared by addition of the polymer to an acidic
mounted for microscopy. Control cell cultures were also grown
suspension of iron oxide nanoparticles, increasing the pH to 7.4,
in the absence of the ferrofluid. A series of samples were stained
and addition of ethanol solutions of the two lanthanide com-
with DAPI for the localization of cell nuclei. Fluorescence
plexes. The suspension was filtered (0.22 μm), and the nanopar-
microscope observations were carried out in a Leica DMI3000B
ticles were collected by magnetic separation and resuspended in
inverted microscope under 340
380 nm excitation light. The
water to obtain the final ferrofluid.
images were taken in an Orca 4.0 camera coupled to a Gemini
Composition and Structure Characterization. The Fe, Tb, and Eu
beam splitter, both from Hamamatsu.
contents in the Eu3þ/Tb3þ-containing Fe2O3@P4VP-b-P-
(MPEGA-co-PEGA) nanoparticles were determined by coupled Conflict of Interest: The authors declare no competing
plasma atomic emission spectrometry (ICP-AES). TEM observa- financial interest.
tions were carried in a JEOL 2000-FXII microscope equipped Supporting Information Available: Detailed materials and
with EDX analyzer, and STEM and cryo-TEM observations were methods, as well as additional experimental data (Figures
performed in a FEI Tecnai F30 microscope equipped with both S1
S27). This material is available free of charge via the Internet
EELS and EDX analyzers. Dynamic light scattering measure- at http://pubs.acs.org.
ments were performed in a Zetasizer Nano ZS from Malvern
Laser (see Supplementary Section II).
Magnetic Heating Equipment. A homemade magnetic heating Acknowledgment. This work was partially supported by the
source was used consisting of a signal generator, a high-power Spanish Ministry of Science and Innovation (MAT2011-259911)
amplifier, and a matching transformer connected to an RCL and in part developed in the scope of the project CICECO-Aveiro
circuit. The magnetic field produced at the L element consists of Institute of Materials (ref. FCT UID/CTM/50011/2013), financed
low-inductance Litz wires around a ferrite nucleus with a section by Portuguese funds through the FCT/MEC (RECI-CTM-CER-
of 3  2.7 cm and a gap of 1 cm where the sample is placed. The 0336-2012, EXPL-CTM-NAN-0295-2012) and when applicable
field intensity and frequency during the measurements were cofinanced by FEDER under the PT2020 Partnership Agreement.
23 mT and 97.771 kHz, respectively (Supplementary Section 6.1). C.D.S.B. (SFRH/BPD/89003/2012) and N.J.O.S. acknowledge
Optical Characterization. Photoluminescence spectra of Eu3þ FCT for a postdoctoral grant and an IF 2013 contract, respec-
and Tb3þ complexes encapsulated in the block copolymer were tively. R.B. thanks ICMA-CSIC for a JAE-predoc grant. The authors
obtained at room temperature by exciting the samples with a would like to acknowledge the use of Servicio General de Apoyo
1000 W ORIEL 66187 tungsten halogen lamp and a double a la Investigación-SAI, Universidad de Zaragoza.
0.22 m SPEX 168OB monochromator. Fluorescence emission
was detected using a 0.5 JAREL-ASH monochromator with a
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