Unveiling in Vivo Subcutaneous Thermal ... - ACS Publications

Letter pubs.acs.org/NanoLett

Unveiling in Vivo Subcutaneous Thermal Dynamics by Infrared Luminescent Nanothermometers Erving Clayton Ximendes,†,‡ Weslley Queiroz Santos,† Uéslen Rocha,‡ Upendra Kumar Kagola,† Francisco Sanz-Rodríguez,‡,⊥ Nuria Fernández,‡ Artur da Silva Gouveia-Neto,† David Bravo,‡ Agustín Martín Domingo,‡ Blanca del Rosal,‡ Carlos D. S. Brites,§ Luís Dias Carlos,§ Daniel Jaque,*,‡,⊥ and Carlos Jacinto*,† †

Grupo de Fotônica e Fluidos Complexos, Instituto de Física, Universidade Federal de Alagoas, 57072-900, Maceió-AL, Brazil Fluorescence Imaging Group, Departamento de Física de Materiales, Faculdad de Ciencias, Universidad Autónoma de Madrid, 28049, Madrid, Spain § Departamento de Física and CICECOAveiro Institute of Materials, University of Aveiro, 3810-193, Aveiro, Portugal ⊥ Instituto Ramón y Cajal de Investigación Sanitaria. Ctra. Colmenar Viejo, km. 9100 28034 Madrid, Spain ‡

S Supporting Information *

ABSTRACT: The recent development of core/shell engineering of rare earth doped luminescent nanoparticles has ushered a new era in fluorescence thermal biosensing, allowing for the performance of minimally invasive experiments, not only in living cells but also in more challenging small animal models. Here, the potential use of active-core/active-shell Nd3+- and Yb3+-doped nanoparticles as subcutaneous thermal probes has been evaluated. These temperature nanoprobes operate in the infrared transparency window of biological tissues, enabling deep temperature sensing into animal bodies thanks to the temperature dependence of their emission spectra that leads to a ratiometric temperature readout. The ability of active-core/ active-shell Nd3+- and Yb3+-doped nanoparticles for unveiling fundamental tissue properties in in vivo conditions was demonstrated by subcutaneous thermal relaxation monitoring through the injected core/shell nanoparticles. The reported results evidence the potential of infrared luminescence nanothermometry as a diagnosis tool at the small animal level. KEYWORDS: Nanothermometry, rare earth nanoparticles, second biological window, subcutaneous thermal sensing

T

experiments. Such studies would, in turn, provide access to the basic properties of tissues from which possible alterations related to incipient diseases could be detected.6,7 Contactless subcutaneous thermal sensing is not an easy task, and it has remained an unsolved challenge for many years. Where the widely used infrared thermometry has failed, luminescent nanothermometers (LNTHs) have emerged as a reliable solution. LNTHs are luminescent NPs whose spectral properties experience significant changes under temperature variations within the physiological range (37−50 °C).8,9 In this case, contactless thermal reading comes from the analysis of their spectroscopic properties. It is nowadays possible to find in the literature numerous LNTHs based on a great variety of luminescent NPs, including quantum dots, polymeric NPs, metallic NPs, nanodiamonds, and rare earth-doped NPs.8,9 Despite this large list of systems, only a few of them show real potential for subcutaneous thermal sensing. This is so because

he fast development that nanotechnology has experienced during the last years has made possible the emergence of new nanosized materials with pretailored properties and functionalities. The contributions of nanotechnology have been especially remarkable in the field of nanomedicine, in which these nanosized systems now offer new perspectives for imaging, diagnosis, and therapy.1 Nanotechnology has indeed provided clinicians with new materials and tools constituting straightforward solutions to challenging problems that remained unsolved. For instance, nanotechnology has made possible the treatment of brain tumors with magnetic nanoparticles (NPs), selective and remotely controlled drug delivery, and real deep tissue optical imaging.2−5 Moreover, nanotechnology has also played a fundamental role in the renewed interest that contactless thermal sensing procedures and techniques are currently receiving. Noninvasive thermal sensing has been widely proposed as an early diagnosis tool capable of early detection of several diseases, including cancer, as well as a control technique to be used during hyperthermia treatments. Furthermore, real-time subcutaneous thermal sensing has also been proposed as a powerful tool for the study of the thermal dynamics of tissues during in vivo © 2016 American Chemical Society

Received: November 11, 2015 Revised: January 28, 2016 Published: February 4, 2016 1695

DOI: 10.1021/acs.nanolett.5b04611 Nano Lett. 2016, 16, 1695−1703

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Figure 1. Scaled model of (a) Nd:Yb LaF3 (single-core), (b) Yb@Nd LaF3 (core/shell) and (c) Nd@Yb LaF3 (core/shell) NPs. Upon 790 nm excitation, all of the NPs present the emission bands ascribed to Nd3+ (900, 1060, 1350 nm) and to Yb3+ (1000 nm).

small animal tissues during in vivo experiments has also been explored. Figure 1 depicts a schematic representation of the samples investigated in this work, corresponding to one single-core and two active-core/active-shell structures. In the single-core structure (hereafter Nd:Yb LaF3 NPs) both Nd3+ and Yb3+ ions coexist in the whole volume of the particle, whereas in the core/shell structures they have been spatially separated by selective doping during the synthesis procedure. Two different core/shell structures were synthesized, one of them having the core doped with Nd3+ ions and the shell with Yb3+ ions (hereafter Nd@Yb LaF3 NPs) and the other constituted by a Yb3+-doped core and a Nd3+-doped shell (Yb@Nd LaF3 NPs). The Nd3+ and Yb3+ concentrations were both fixed to 10 mol % in all structures. These concentrations were chosen to optimize the overall luminescence brightness of the NPs and to get similar emission intensities from both Nd3+ and Yb3+ ions (for more details see Section S2 of Supporting Information). Figure 2a and b shows the characteristic TEM images of single-core and core/shell LaF3 NPs, respectively. Size histograms have been obtained for each case and are included in Figure 2c. Nd@Yb and Yb@Nd LaF3 core/shell NPs presented identical size histograms. For the single-core sample, the average size was found to be close to 15 nm, in good agreement with previous works.29 In the case of the core/shell NPs, the average size was found to be close to 24 nm, so the thickness of the shell layer was estimated to be ∼4.5 nm. The selective incorporation of Nd3+ and Yb3+ ions in the core/shell structures was further evidenced by high resolution TEM and EDX studies. Figure 2d shows a high resolution TEM image of a characteristic Yb@Nd LaF3 NP. The EDX concentration profiles corresponding to both Nd3+ and Yb3+ ions have been superimposed so that the location of the specific elements within the NP are identified. As it can be seen, the Yb3+ ions are well-localized at the central part of the profile, indicating their sole presence at the core. On the other hand, the presence of Nd3+ ions has been found to be maximum at the edges of the NP, in accordance with the existence of a Nd3+-doped shell. Note that, as expected, the EDX scan also reveals the existence of Nd3+ ions at the center of the profile due to the tridimensional nature of the sample. To reinforce the core/shell structure of our samples, EPR experiments were also carried out. Figure 2e shows typical spectra for single core and core/shell NPs. The g values reported for both Nd3+ and Yb3+ ions in LaF3 crystals have also been included, allowing for the identification of the different contributions to the net EPR spectra.32 In the case of singlecore samples, where there is a spatial coexistence between Nd3+ and Yb3+ ions, an additional contribution to the line width of

most of them operate in the visible spectrum domain, where optical penetration into tissues is minimal. Avoiding this limitation implies the shift of their operation spectral range from the visible to either the first (I-BW, 700−950 nm) or second (II-BW, 1000−1400 nm) biological windows, where both tissue absorption and scattering are minimized.10−12 In particular, the use of LNTHs working in the II-BW would open the possibility of not only deep tissue imaging but also of high spatial resolution in vivo thermal sensing, as it has already been demonstrated in imaging applications.13−16 It is also possible to find recent works demonstrating the possibility of developing LNTHs that can be simultaneously used as multimodal bioimaging probes.17 As an additional requirement, LNTHs to be used for subcutaneous thermal sensing should operate under infrared radiation with wavelengths around 800 nm. This responds to the fact that this particular wavelength, provided by cost-effective laser diodes, has demonstrated to be minimally harmful when compared, for example, with other widely excitation wavelengths used in biophotonics (such as 765 or 980 nm).18−21 Two different strategies could be followed for the synthesis of 800 nm excited LNTHs operating in the II-BW. The first consists in the use of infrared-emitting QDs providing thermal reading based on either their marked thermal quenching or on their temperature-induced spectral shift.22,23 Alternatively, single or multiple rare earth-doped NPs can be used. In this second case, thermal reading could be achieved by the so-called ratiometric approach, in which temperature is determined from the analysis of the relative intensities of their different emission bands/lines.24 Subcutaneous thermal sensing with rare earthdoped LNTHs in the infrared has already been proposed by using ytterbium and neodymium codoped NPs25 and fully demonstrated by using single Nd3+-doped NPs.26−31 In both cases, thermal sensing was performed by analyzing emission lines lying within the I-BW so that the potential advantages of working in the II-BW (including larger penetration depths, improved resolution and autofluorescence free measurements) were not fully exploited. In this work, we have designed and synthesized a new type of LNTH capable of subcutaneous luminescence thermal sensing in the II-BW under single beam 790 nm excitation. The LNTH is based on a Nd3+/Yb3+ double-doped core/shell structure in which both ions are spatially separated by selective doping during synthesis process. The interaction between Nd3+ and Yb3+ ions at the core/shell interface gives rise to their infrared thermal sensitivity, as demonstrated by time-resolved spectroscopy. The ability of the active-core/active-shell structures as ratiometric thermal sensors for unveiling basic properties of 1696


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Figure 3. (a) Room temperature emission spectra of Nd@Yb LaF3 (red), Yb@Nd LaF3 (purple), and Nd:Yb LaF3 (blue) NPs under 790 nm laser excitation (97 mW). The transitions ascribed to Nd3+ and Yb3+ are identified in green and blue, respectively. Spectra are normalized to their intensity at 1350 nm. (b) Fluorescence lifetime of the 4F3/2 Nd3+ emitting level of Nd@Yb (red), Yb@Nd (purple), and Nd:Yb LaF3 (blue) NPs. The decay curve obtained for Nd LaF3 NPs (black) is included for comparison. (c) Simplified energy scheme of the Nd3+ and Yb3+ emitting centers representing excitation and radiative decays (full lines), nonradiative decays (curved line), and possible ion−ion energy transfer paths (dashed lines).

Figure 2. Characteristic TEM images of (a) single-core and (b) core/ shell LaF3 NPs. Scale bars are 90 nm. (c) Size histogram corresponding to both single-core and core/shell NPs as obtained from the statistical analysis of the TEM images. The solid lines are the best fit to the data using log-normal distributions (r2 > 0.952) with center half-width-at-half-maximum values of 15 ± 4.6 nm and 24 ± 5.6 nm, for single-core and core/shell NPs, respectively. (d) Highresolution TEM image of an illustrative core/shell Yb@Nd LaF3 NP. The EDX concentration profiles corresponding to Nd (green) and Yb (blue) have been superimposed so that the elements localization within the NP have been identified. (e) Typical EPR spectra for single core (top spectrum) and core/shell NPs (middle and bottom spectra). The g values reported for Nd3+ (green) and Yb3+ (blue) ions in LaF3 crystals have been also included as vertical dashed lines.

band that corresponds to the 2F5/2 → 2F7/2 transition. The energy transfer processes taking place under 790 nm excitation are summarized in Figure 3c. The absorption of 790 nm laser photons promotes the excitation of Nd3+ ions from their ground state to the 4F5/2 state. Then, a rapid phonon-assisted relaxation to the metastable 4F3/2 state takes place. Once at the metastable state, energy diffusion between Nd3+ ions could take place until nonradiative energy transfer processes, involving phonon emission, are produced. Energy transfer leads to a relaxation of Nd3+ ions down to their ground state and to a simultaneous excitation of Yb3+ ions from their ground state up to the 2F5/2 state, from which infrared emission is produced. Although both Nd3+ and Yb3+ bands are present in the 850− 1500 nm emission range for all of the structures, their relative contribution varies from single-core to core/shell NPs. The emission spectrum obtained from Nd:Yb LaF3 single-core NPs shows dominant contribution of Yb3+ emission, revealing an expected large Nd3+ → Yb3+ energy transfer. In single-core double-doped structures, Nd3+ ions are in close proximity to Yb3+ ions in all the NP volume, leading to reduced Nd3+-toYb3+ distances and, hence, to large energy transfer efficiencies.33 Such large energy transfer efficiency is also evidenced in the lifetime measurements included in Figure 3b, where the fluorescence lifetimes from the 4F3/2 Nd3+ emitting level are represented for all of the samples. The decay curve obtained for single Nd3+-doped single-core LaF3 NPs (hereafter Nd:LaF3 NPs) is also included for the sake of comparison. Single-doped Nd:LaF3 NPs show the longest lifetime (τNd = 33 μs), as well as

EPR lines is observed due to the spin−spin interaction between dissimilar ions. In the case of core/shell structures, EPR lines appear better defined and narrower due to the reduction of spin−spin interactions as a consequence of the spatial separation between Nd3+ and Yb3+ ions. In this case, the EPR line width is mostly due to spin−spin interactions between similar ions at either the core or the shell. Thus, both EDX and EPR measurements included in Figure 2 demonstrate the successful synthesis of core/shell structures with selective Yb3+ and Nd3+ doping. The room temperature emission spectra generated from the single-core and core/shell NPs under infrared 790 nm laser excitation are included in Figure 3a. Due to the negligible absorption of Yb3+ ions at this particular wavelength, Nd3+ ions act as the only sensitizer units. The emission spectra included in Figure 3a have been normalized to the emission intensity at 1350 nm generated by Nd3+ ions. In all cases, the emission spectra include the characteristic emission bands of Nd3+ ions centered at 900, 1060, and 1350 nm, assigned to the 4F3/2 → 4 I9/2, 4F3/2 → 4I11/2, and 4F3/2 → 4I13/2 transitions, respectively. The Nd3+ → Yb3+ energy transfer is evidenced by the appearance of the Yb3+ characteristic 970−1030 nm emission 1697


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Nano Letters nonpure single-exponential decay that very likely reveals the presence of self-quenching due to Nd3+-Nd3+ interactions. For the Nd:Yb LaF3 single-core sample, the fluorescence lifetime is strongly reduced down to τNd:Yb = 3 μs, indicating an overall Nd Nd3+ → Yb3+ energy transfer with an efficiency of ηNd:Yb (Nd → Yb) = 1 − τNd:Yb Nd /τNd = 0.9. This large energy transfer efficiency is in accordance with the dominant contribution of Yb3+ ions to the emission spectrum of Nd:Yb single-core LaF3 NPs. The contribution of Yb3+ ions to the infrared fluorescence is reduced in the core/shell structures indicating a less efficient Nd3+ → Yb3+ energy transfer. This was indeed expected, since in this case only Nd3+ ions at the core/shell interface find Yb3+ ions in proximity, so that only a reduced fraction of Nd3+ ions is transferring their energy to the Yb3+ ones. This is in agreement with the fluorescence decay curves included in Figure 3b from which it is evident that the fluorescence lifetime of the (4F3/2) state of Nd3+ ions is substantially longer for the core/shell structures than for the Nd:Yb single-core LaF3 NPs. The fluorescence lifetimes in Nd@Yb and Yb@Nd core/shell NPs have been found to be τNd@Yb = 24 μs and τYb:Nd = 25 μs, Nd Nd respectively. These values yield virtually the same overall Nd3+ → Yb3+ energy transfer efficiency, i.e., ηNd@Yb (Nd → Yb) ≅ /τNd ≅ 0.24. Figure S1 ηNd@Yb (Nd → Yb) = 1 − τNd@Yb Nd includes the comparison of the emission spectra for single-core and core/shell NPs, taken under the very same excitation and emission conditions, revealing a larger emitted intensity for core/shell NPs with respect to single-core ones. This fact is attributed to the self-quenching effect originating from nonradiative mechanisms such as cross-relaxation, energy migration, and even to energy trap processes between emitting ions and OH− surface radicals appearing in single core structures. Figure S1 also reveals similar emission intensities for both Nd@Yb and Yb@Nd core/shell LaF3 NPs in agreement with the similar lifetimes. The ability of the Nd3+/Yb3+double-doped LaF3 NPs for luminescence nanothermometry in the II-BW has been investigated by analyzing their emission spectra under 790 nm excitation as a function of temperature in the physiological (10−50 °C) temperature range. Results are summarized in Figure 4, in which the 790 nm excited emission spectra of Nd@ Yb, Yb@Nd, and Nd:Yb LaF3 NPs (as obtained at 10 and 50 °C) are displayed (Figure 4a, b, and c, respectively). Whereas for core/shell NPs the contribution of ytterbium ions to the overall emission spectra decreases with increasing temperature, single-core NPs show an opposite behavior. For Nd:Yb LaF3 NPs the temperature increase leads to an increment in the contribution of Yb3+ ions to the overall emission spectra. The observed variations open the avenue to use the ratio between the emitted intensity of Nd3+ ions at ≈1.3 μm (4F3/2 → 4I13/2, hereafter INd) and that of Yb3+ ions at around 1000 nm (2F5/2 → 2F7/2, hereafter IYb). Figure 4d shows the temperature variation of the intensity ratio Δ = INd/IYb as obtained for the three samples investigated in this work (INd and IYb represent the peak intensities). A linear calibration relation between Δ and T was obtained in all three cases with a positive (core/shell NPs), or a negative (single-core NPs) slope, respectively. Presently, the explanation for this opposite behavior between core/shell and single-core NPs is not clear. Previous works dealing with the temperature dependence of the net Nd3+ → Yb3+ energy transfer efficiency evidenced that, even in the same system, temperature could enhance or decrease the net Nd3+ → Yb3+ energy transfer efficiency depending on the precise balance between direct transfer and phonon-assisted Nd3+ ←

Figure 4. Emission spectra of (a) Nd@Yb LaF3, (b) Yb@Nd LaF3, and (c) Nd:Yb LaF3 NPs obtained at 10 and 50 °C under 790 nm excitation. (d) Calibration curves of Nd@Yb LaF3 (red), Yb@Nd LaF3 (purple), and Nd:Yb LaF3 (blue) NPs. Dots are experimental Δ values, and the lines represent the best fit to the experimental data using straight lines (r2 > 0.913). (e) Sensitivity curves of Nd@Yb LaF3 (red), Yb@Nd LaF3 (purple), and Nd:Yb LaF3 (blue) NPs. The sensitivity of the two other luminescent thermometers reported until now emitting in the II-BW are also included for comparison, Cerón et al. (green) and Marciniak et al. (orange).21,23

Yb3+ back transfer. When Nd3+ → Yb3+ energy transfer dominates, the Δ ratio is expected to increase with temperature.34 On the other hand, when phonon assisted Nd3+ ← Yb3+ back transfer is the dominant mechanism then Δ is expected to decrease with temperature. According to this argument, the temperature-induced decrease of Δ in single-core NPs suggests Nd3+ ← Yb3+ back transfer as the dominant mechanism in these structures. A more detailed discussion about the mechanism leading to the linear increment of Δ with temperature can be made based on Figure S3 that shows the temperature dependence of the fluorescence lifetime of both 2 F5/2 (Yb3+) and 4F3/2 (Nd3+) states. The 4F3/2 (Nd3+) fluorescence lifetime remains almost constant in the 10−60 °C range, indicating a temperature-independent Nd3+ → Yb3+ energy transfer efficiency. On the other hand, the 2F5/2 (Yb3+) lifetime was found to decrease with increasing temperature. This suggests a temperature-induced increment in the Nd3+ ← Yb3+ back transfer efficiency. In this scenario (temperatureindependent Nd3+ → Yb3+ energy transfer efficiency and temperature-enhanced Nd3+ ← Yb3+ back transfer efficiency), the net contribution of the Yb3+ emission band to the overall emission of core/shell NPs is expected to decrease. This is in accordance with the experimentally observed increase of Δ with temperature. A linear fit to the experimental data included in Figure 4d allowed us to estimate the relative thermal sensitivity Sr = (1/ Δ) ∂Δ/∂T (presented as a figure of merit to compare the performance of different thermometers operating in II-BW).8,35 We have obtained thermal sensitivities of 0.41 ± 0.01%·°C1−, 0.36 ± 0.02%·°C1−, and 0.1 ± 0.02%·°C1− for Nd@Yb, Yb@ Nd, and Nd:Yb LaF3 NPs, respectively, at 10 °C. At this point it 1698


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conductive (for a more detailed discussion on this aspect, see Section S6 of Supporting Information). For the former process (skin thermal relaxation), the Newton’s law of cooling could be applied.40 In the absence of a heating source the skin temperature, Tskin, during thermal relaxation is given by41,42

should be highlighted that the same thermal sensitivities were found when the intensity ratio Δ was computed by using the Nd3+ emission intensity at 890 nm (4F3/2 → 4I9/2 transition) or at 1060 nm (4F3/2 → 4I11/2 transition). This fact is clearly demonstrated in Figure S4 in which the temperature dependence of the intensity ratio Δ as obtained by using the three emission lines of Nd3+ ions is included. The possibility of using different emission lines for thermal sensing with identical thermal sensitivities is, indeed, an outstanding property of Nd@ Yb core/shell NPs as they can operate in a broad spectral range, depending on the particular application. Note that, in addition, core/shell engineering has allowed for a 4-fold improvement in the thermal sensitivity of Nd3+ /Yb3+ double-doped LaF3 NPs. Different works regarding the synthesis and application of core/ shell structures clearly demonstrated how they could be used to enhance the luminescence brightness of the NPs and to tailor their spectral properties by activating or deactivating energy transfer processes between rare earth ions at core and/or shell.36−38 In addition to these benefits of core/shell engineering, the results included in this work demonstrate that it can also be used to enhance the thermal sensitivity of rare earthdoped NPs. In order to give a comprehensive comparison of the highest thermal sensitivity obtained for Nd@Yb core/shell NPs (0.44%·°C−1) with respect to other rare earth-based LNTHs, Figure S5 constitutes a figure of merit including the thermal sensitivities and spectral operating ranges of other rare earth-based LNTHs. As it can be observed, there are few rareearth-based LNTHs operating in the II-BW. Restricting our comparison to this particular spectral range, the thermal sensitivities achieved by our core/shell engineered LaF3 NPs are slightly larger than the recently reported for Er3+ and Yb3+ codoped LiLaP4O12 NPs, although in that case they used for reference the 4F3/2 → 4I9/2 transition of Nd3+ that, strictly speaking, does not lie within the II-BW.10,25 Although the core/ shell structures here reported showed lower thermal sensitivities than that of hybrid-nanostructures based on QDs and Nd3+-doped NPs,23 they offer additional advantages such as a reduced nanoparticle size as well as a more direct and robust synthesis procedure. The potential use of our Nd@Yb LaF3 NPs (those showing the largest thermal sensitivity) as infrared luminescent nanothermometers for real-time subcutaneous in vivo thermometry has also been evaluated in this work. In particular, we explored their ability for accurate measurement of subcutaneous thermal transients as a potential future theranostics tool. It should be noted that in many thermal therapies the relevant parameter usually monitored is the steady state temperature achieved at the end of the heating procedure. Its magnitude is what really determines the efficacy of the treatment. Nevertheless, in this work we focused on the possible use of subcutaneous thermal reading not as a control tool during photothermal treatments but, instead, as an alternative diagnosis tool. The basic idea behind our work is that when a tissue is undergoing a thermal relaxation (cooling in the absence of any heating source), the cooling dynamics strongly depends on the tissue properties.39 Thus, accurate measurements of the cooling relaxation profiles provides information about the “tissue status” and, hence, could be used to detect anomalies caused by incipient diseases. The temperature relaxation process depends on the physical mechanisms responsible for the heat dissipation: whereas for the skin the dissipation may be essentially convective, for the subcutaneous tissues it could be assumed to be mainly

⎛ t ⎞ Tskin(t ) = T∞ + ΔTskin exp⎜ − ⎟ ⎝ τconv ⎠

(1)

where T∞ is the steady tissue’s surrounding temperature (achieved in the limit t ≫ τconv), ΔTskin = Tmax skin − T∞ is the temperature difference between the initial temperature (Tmax skin) and T∞, and τconv is the convective relaxation time. According to basic theory of Newton’s cooling, this convective relaxation time depends on several factors, including the thermal contact area and the convective heat transfer coefficient of the surroundings.41,42 On the other hand, as explained in Section S6 of Supporting Information, the thermal relaxation of the subcutaneous tissue can be described by the Fourier’s law. For a unidimensional medium at constant temperature at a boundary, Ts, the time evolution of subcutaneous temperature, Tscut(t), is given by41,43 ⎛ τ ⎞ Tscut(t ) = Ts + ΔTscut erf⎜ cond ⎟ ⎝ t ⎠

(2)

where erf denotes the Gaussian error function, and τcond is the characteristic tissue relaxation time. We note that τcond is determined by the tissue’s thermal properties, so that it is given by39,43 τcond =

L2 4αtissue

(3)

where L and αtissue are the characteristic length and thermal diffusivity of the tissue undergoing the thermal relaxation, respectively. The thermal diffusivity of a given tissue depends on its thermal conductivity (ktissue), density (ρtissue), and specific heat capacity (ctissue) according to44,45 αtissue =

k tissue ρtissue ctissue

(4)

Thus, as we will address next, a proper analysis of subcutaneous thermal relaxation dynamics would allow us to access the characteristic thermal relaxation time of a given subcutaneous tissue that can be unequivocally related to its fundamental properties. In addition, detection of small variations in the subcutaneous tissue relaxation times would allow for the identification of possible alterations of its thermal diffusivity, specific heat, thermal conductivity and density associated, for instance, to the presence of cancer tumors and other diseases. Moreover, subcutaneous thermal monitoring would also make possible the quantification of the local thermal dose administrated in a hyperthermia treatment that is nowadays considered as one of the most important factors which influence the efficacy of hyperthermia treatments.46,47 In order to demonstrate the potential use of our Nd@Yb LaF3 NPs for the measurement of subcutaneous thermal relaxation dynamics, a simple in vivo experiment was designed. It is schematically described in Figure 5. A total volume of 200 μL of a PBS (phosphate-buffered saline) dispersion of Nd@Yb LaF3 NPs (1% in mass) was subcutaneously injected on a CD1 mouse. The injected mouse was anesthetized and placed in a small animal imaging chamber equipped with a body 1699


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contribution. Once thermal stabilization was achieved and the subcutaneous presence of the Nd@Yb LaF3 NPs was confirmed, we proceeded to induce a moderate temperature increment at the injection site. Such local heating was induced by taking advantage of the ability of 808 nm laser radiation for CD1 mouse heating mainly due to Nd3+ absorption and also to residual skin absorption at this wavelength.49 The mouse was illuminated with an 808 nm heating laser beam providing an A808 nm = 5.0 ± 0.5 cm2 illumination area over the skin. An infrared thermal camera was added to the setup to monitor the skin temperature. Safe heating was then performed by setting the power density of the 808 nm laser beam at 0.7 W/cm2 and extending the thermal treatment during 4 min. Under these irradiation conditions the mouse surface temperature increased from 34.2 up to 40.5 °C as it can be observed in the thermal images included in Figure 5d. Note that such heating did not produce any damage to the skin. This is in agreement with the fact that the power density used for heating was set to be close to the conservative limit set for human skin exposure (0.7 W/cm2 at 980 nm).50,51 During the heating procedure, the strong background created by the high intensity 808 nm laser beam precludes accurate measurement of the luminescence generated by the subcutaneously injected Nd@Yb LaF3 NPs, so that they could not be used as thermal sensors during the heating procedure. Once the heating cycle is finished (808 nm laser is turned off), the thermal relaxation starts, and the mouse tissues gradually recover their initial temperature. In absence of the heating 808 nm radiation, the luminescence of Nd@Yb LaF3 NPs was used to monitor the dynamics of the thermal relaxation. For this purpose, a low power 790 nm, 30 mW laser diode was focused into the Nd@Yb LaF3 subcutaneous injection by using an infrared long working distance objective (see Figure 5b). The subcutaneous luminescence generated by Nd@Yb LaF3 NPs was collected by the same microscope objective and its subsequent spectral analysis provided us the time evolution of the subcutaneous temperature through the Δ = INd/IYb intensity ratio that, in this case, is computed from the Nd3+ emitted intensity at 1060 nm. For in vivo experiments, this particular line was selected since, as it is explained in detail in Section S7 of Supporting Information, it leads to a minimum uncertainty in the determination of the subcutaneous temperature. Typical in vivo emission spectra obtained at different times after the heating procedure are included in Figure S6. At this point we should note that for subcutaneous thermal measurements the calibration curve obtained for Nd@Yb LaF3 NPs in aqueous medium has been used. This is a valid approach as the fluorescence properties of rare earth doped LaF3 NPs are not modified when incorporated into a tissue (see section S10 in Supporting Information).26 Data included in Figure 5f constitutes, to the best of our knowledge, the first time that thermal relaxation dynamics are measured in a living animal by luminescence nanothermometry. Just after the end of heating cycle, the subcutaneously injected Nd@Yb LaF3 NPs provide a thermal reading of 39 ± 1 °C that is in good agreement with the skin temperature value provided by infrared thermometry (38 °C as obtained from Figure 5d and e). It should be noted at this point that the 790 nm “reading” beam could also heat both tissue and Nd@Yb LaF3 NPs. In order to elucidate this possible additional contribution, thermal images obtained after complete tissue relaxation but with the 790 nm reading laser still on were measured. Figure S7 includes a thermal image acquired 220 s after the 808 nm laser-induced heating cycle in which it is clear

Figure 5. (a) Fluorescence top-image of the CD1 mouse (delimited by the dashed line) where the subcutaneous injection of Nd@Yb core/ shell LaF3 NPs is evidenced by the bright fluorescence spot. (b) Digital picture of the CD1 mouse during in vivo thermal relaxation experiments. (c) Schematic representation of the subcutaneous thermal relaxation experiments. Thermal infrared images of the CD1 mouse before (d) and at the end (e) of the heating stimulus. (f) Time evolution of the temperatures measured by the subcutaneous luminescent thermometer (gray) and the IR thermal camera (orange). Dots are experimental subcutaneous (circles) and skin (squares) temperatures, whereas the solid line is the best fit to eq 2.

temperature controller so that the mouse temperature was kept at 34 ± 1 °C, accordingly to the control infrared camera measurement (see thermal image included as Figure 5b). The efficient subcutaneous incorporation of the Nd@Yb LaF3 NPs was then confirmed by infrared (1100−1700 nm) fluorescence in vivo imaging. The obtained fluorescence image included in Figure 5a was obtained by illuminating the whole mouse with an 808 nm laser beam at 0.35 W·cm−2 and collecting the generated fluorescence in the 1100−1700 nm range by using a set of bandpass filters and an InGaAs camera. Experimental details about the small animal imaging setup can be found in the Supporting Information. The fluorescence image included in Figure 5a reveals the presence of infrared autofluorescence (it is possible to intuit the shape of the CD1 mouse). The intensity of this autofluorescence background is remarkably lower than that generated by the subcutaneously injected core/ shell nanoparticles, as expected when measuring in the 1100− 1700 nm range.48 Further experimental evidence of the minimum presence of autofluorescence is provided in the inset of Figure S6, in which it is clear that subcutaneous fluorescence spectra are not affected by any background 1700


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of the 560 J of the deposited laser energy. This yields to a tissue absorbance at 808 nm equal to 10 ± 2%. The absorption coefficient of the tissue (αabs tissue) measured in in vivo conditions can be estimated applying the Lambert−Beer law:55

that no local heating is produced at the location of the 790 nm laser spot on the mouse skin. When the thermal relaxation curves corresponding to the subcutaneous and skin temperatures (provided by Nd@Yb LaF3 NPs and IR camera, respectively) are compared, clear differences are observed. Whereas the skin temperature decay follows a single exponential profile, accordingly eq 1, the time evolution of the subcutaneous temperature deviates from a simple single exponential decay. It, indeed, can be well fitted to an erf decay as indicated in eq 2. Figure 5f includes as solid lines the best fit of experimental data to eq 2 with τcond = 39 ± 8 s (r2 > 0.909). We may use the characteristic tissue relaxation time τcond to compute the thermal diffusivity of the mouse tissue by setting the characteristic length/dimension of the tissue undergoing the thermal relaxation, L. Previous works dealing with laserinduced tissue heating experiments have concluded that the characteristic length of the tissue corresponds to the optical penetration depth of the heating radiation into the tissue.52 In our experiments, heating was produced by an 808 nm laser beam so that L can be assumed to be the penetration depth of the 808 nm radiation into tissues, l808nm, so that L ≈ l808nm.52 A penetration length at 808 nm close to L = 4.5 ± 0.5 mm has already been estimated in the literature.11 Thus, according to eq 3, a tissue thermal diffusivity is obtained with the value of αtissue = 0.13 ± 0.04 mm2·s−1. This value is in very good agreement with the one calculated through eq 3, α = 0.135 mm2·s−1, using published data for the thermal conductivity (ktissue = 0.5 W· m−1·°C−1), the density (ρtissue = 103 kg·m−3) and the specific heat (ctissue = 3.7 × 103 J·kg−1·°C−1) of animal tissues.53 Moreover, the calculated thermal diffusivity agrees with that reported for breast tissue at 810 nm,54 α = 0.142 mm2·s−1. The fitting of the subcutaneous temperature to eq 2, results in a surrounding tissue temperature of Ts = 28 ± 1 °C, which is below the normal body temperature of an anesthetized mouse (34 °C). Besides using the skin and subcutaneous thermal relaxation curves to elucidate the thermal diffusivity of tissues, they were also used to estimate the total thermal energy dose absorbed by the tissue. According to the definition of heat capacity, the thermal energy dose, Qtissue, required to cause a temperature increment ΔT in a tissue of mass mtissue is given by Q tissue = mtissue × ctissue × ΔT

abs (808 nm) = αtissue

⎞ Q 808nm 1 ⎛ ⎟⎟ ln⎜⎜ L ⎝ Q 808nm − Q tissue ⎠

(6)

αabs tissue

With this expression, a tissue absorption coefficient of = 0.23 ± 0.05 cm−1 is obtained. This value is found to be in good agreement with previous works published by S. L. Jacques, who estimated a tissue absorption coefficient ranging from 0.1 to 0.4 cm−1 depending on fat, water, and melanin content.56 The in vivo tissue absorption coefficient obtained through subcutaneous thermal measurements has also been found to be close to the absorption coefficient at 808 nm of human skin, as measured under ex vivo conditions, by A. N. Bashkatov et al.,11 who reported an absorption coefficient close to 0.35 cm−1. The good agreement between the values obtained by subcutaneous luminescence nanothermometry for both tissue absorption coefficient and tissue thermal diffusivity with those previously reported in the literature supports the validity of using our Nd@Yb core/shell LaF3 NPs as accurate and reliable subcutaneous thermal sensors for in vivo applications. In summary, we have shown that infrared-emitting activecore/active-shell Nd3+- and Yb3+-codoped LaF3 NPs can be successfully used as subcutaneous thermal probes operating in the second biological window. Selective doping during the core/shell growth has been demonstrated to be a promising approach for tailoring luminescence and thermal sensitivity of the nanostructure. In particular, it has been found that if Yb3+ and Nd3+ emitting ions are spatially separated in the core and shell (or vice versa), a 4-fold improvement in the thermal sensitivity is reached. The ratio between the Yb3+ and Nd3+ infrared emissions in core/shell structures provide a full optical method to measure subcutaneous temperatures in a minimally invasive way. Moreover, the codoped Yb3+/Nd3+ core/shell NPs have been successfully used to measure subcutaneous thermal transients in small animal models from which accurate information about basic properties of tissues (e.g., absorption coefficient and thermal diffusivity) have been obtained. Results have been found to be in excellent agreement with published data obtained by completely different methods. The results reported here demonstrate the bright potential of NIR emitting nanothermometers at the small animal level in the study of heat transfer, noninvasive detection of subcutaneous anomalies, and as a diagnosis tool.

(5)

In our conditions, the mass of tissue subjected to the 808 nm laser-induced heating can be estimated assuming a tissue volume equal to the irradiated area (Atissue = 5.0 ± 0.5 cm2) multiplied by the penetration length, l808nm, so that mtissue = ρtissue × Atissue × L ≅ (2.2 ± 0.3) × 10−3 kg. From the skin and subcutaneous thermal transients included in Figure 5f, we can estimate, in a first order approximation, total temperature increment after heating procedure close to ΔT ∼ 6 °C. Assuming this temperature increment we estimated a total thermal dose of Qtissue = 54 ± 8 J. This can be compared to the total 808 nm laser energy deposited in the tissue that can be calculated as Qdeposited = I808nm × Δt × A808nm = 560 ± 63 J, where I808nm = 0.7 W·cm−2 is the 808 nm laser intensity, Δt = 160 s is the effective irradiation time, and A808nm = 5.0 ± 0.5 cm2 is the 808 nm laser spot. The effective irradiation time is the time required for thermal stabilization as estimated from the thermal transient of skin temperature measured during heating cycles (more details about the definition and estimation of this effective irradiation time can be found in Section 9 of Supporting Information). Thus, the tissue absorbs only 54 J

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.nanolett.5b04611. Section S1, Luminescence brightness of single-core and core/shell nanoparticles. Section S2, Fluorescence intensity ratio and doping levels of Nd3+ and Yb3+. Section S3, Temperature dependence of fluorescence lifetimes. Section S4, Emission wavelength dependence of thermal sensitivity. Section S5, Figure of merit of rare earth based nanothermometers. Section S6, Cooling dynamics of skin and subcutaneous tissue. Section S7, Estimation of temperature uncertainty during in vivo 1701


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(3) Jiang, S.; Gnanasammandhan, M. K.; Zhang, Y. J. R. Soc., Interface 2010, 7, 3−18. (4) Liu, Y.; Miyoshi, H.; Nakamura, M. Int. J. Cancer 2007, 120, 2527−2537. (5) Zhang, X. Q.; Lam, R.; Xu, X.; Chow, E. K.; Kim, H. J.; Ho, D. Adv. Mater. 2011, 23, 4770−4775. (6) Wisaksana, A. In Measurement of mechanical properties of soft limb tissue as a diagnostic tool. Proceedings of the 1993 IEEE Nineteenth Annual Northeast, V.A. Med. Center, Castle Point, NY, USA, 18−19 Mar 1993, Ostrander, L. E., Cagir, B., Lee, B. Y.; 1993; pp 54−55. (7) Ozerdem, U.; Hargens, A. R. Microvasc. Res. 2005, 70, 116−120. (8) Brites, C. D. S.; Lima, P. P.; Silva, N. J. O.; Millan, A.; Amaral, V. S.; Palacio, F.; Carlos, L. D. Nanoscale 2012, 4, 4799−4829. (9) Jaque, D.; Vetrone, F. Nanoscale 2012, 4, 4301−4326. (10) Smith, A. M.; Mancini, M. C.; Nie, S. Nat. Nanotechnol. 2009, 4, 710−1. (11) Bashkatov, A.; Genina, E.; Kochubey, V.; Tuchin, V. J. Phys. D: Appl. Phys. 2005, 38, 2543. (12) Quek, C.-H.; Leong, K. W. Nanomaterials 2012, 2, 92. (13) Robinson, J. T.; Hong, G.; Liang, Y.; Zhang, B.; Yaghi, O. K.; Dai, H. J. Am. Chem. Soc. 2012, 134, 10664−10669. (14) Welsher, K.; Sherlock, S. P.; Dai, H. Proc. Natl. Acad. Sci. U. S. A. 2011, 108, 8943−8948. (15) Hong, G.; Robinson, J. T.; Zhang, Y.; Diao, S.; Antaris, A. L.; Wang, Q.; Dai, H. Angew. Chem., Int. Ed. 2012, 51, 9818−9821. (16) Zhang, Y.; Hong, G.; Zhang, Y.; Chen, G.; Li, F.; Dai, H.; Wang, Q. ACS Nano 2012, 6, 3695−702. (17) Wang, Z.; Zhang, P.; Yuan, Q.; Xu, X.; Lei, P.; Liu, X.; Su, Y.; Dong, L.; Feng, J.; Zhang, H. Nanoscale 2015, 7, 17861−17870. (18) Liang, H.; Vu, K. T.; Krishnan, P.; Trang, T. C.; Shin, D.; Kimel, S.; Berns, M. W. Biophys. J. 1996, 70, 1529−1533. (19) Haro-González, P.; Ramsay, W. T.; Maestro, L. M.; del Rosal, B.; Santacruz-Gomez, K.; del Carmen Iglesias-de la Cruz, M.; SanzRodríguez, F.; Chooi, J. Y.; Sevilla, P. R.; Bettinelli, M.; Choudhury, D.; Kar, A. K.; Solé, J. G.; Jaque, D.; Paterson, L. Small 2013, 9, 2162− 2170. (20) Wang, Y.-F.; Liu, G.-Y.; Sun, L.-D.; Xiao, J.-W.; Zhou, J.-C.; Yan, C.-H. ACS Nano 2013, 7, 7200−7206. (21) Bregnhøj, M.; Blázquez-Castro, A.; Westberg, M.; Breitenbach, T.; Ogilby, P. R. J. Phys. Chem. B 2015, 119, 5422−5429. (22) Li, S.; Zhang, K.; Yang, J. M.; Lin, L. W.; Yang, H. Nano Lett. 2007, 7, 3102−3105. (23) Cerón, E. N.; Ortgies, D. H.; del Rosal, B.; Ren, F.; Benayas, A.; Vetrone, F.; Ma, D.; Sanz-Rodríguez, F.; Solé, J. G.; Jaque, D.; Rodríguez, E. M. Adv. Mater. 2015, 27, 4781−4787. (24) Brites, C. D.; Lima, P. P.; Silva, N. J.; Millán, A.; Amaral, V. S.; Palacio, F.; Carlos, L. D. Adv. Mater. 2010, 22, 4499−4504. (25) Marciniak, L.; Bednarkiewicz, A.; Stefanski, M.; Tomala, R.; Hreniak, D.; Strek, W. Phys. Chem. Chem. Phys. 2015, 17, 24315− 24321. (26) Carrasco, E.; del Rosal, B.; Sanz-Rodríguez, F.; de la Fuente, Á . J.; Gonzalez, P. H.; Rocha, U.; Kumar, K. U.; Jacinto, C.; Solé, J. G.; Jaque, D. Adv. Funct. Mater. 2015, 25, 615−626. (27) Rocha, U.; Jacinto da Silva, C.; Ferreira Silva, W.; Guedes, I.; Benayas, A.; Martinez Maestro, L.; Acosta Elias, M.; Bovero, E.; van Veggel, F.; Garcia Sole, J. A.; Jaque, D. ACS Nano 2013, 7, 1188− 1199. (28) Rocha, U.; Kumar, K. U.; Jacinto, C.; Villa, I.; Sanz-Rodríguez, F.; del Carmen Iglesias de la Cruz, M.; Juarranz, A.; Carrasco, E.; van Veggel, F. C. J. M.; Bovero, E.; Solé, J. G.; Jaque, D. Small 2014, 10, 1141−1154. (29) Rocha, U.; Upendra Kumar, K.; Jacinto, C.; Ramiro, J.; Caamaño, A. J.; García Solé, J.; Jaque, D. Appl. Phys. Lett. 2014, 104, 053703. (30) Wawrzynczyk, D.; Bednarkiewicz, A.; Nyk, M.; Strek, W.; Samoc, M. Nanoscale 2012, 4, 6959−6961. (31) Benayas, A.; del Rosal, B.; Pérez-Delgado, A.; Santacruz-Gómez, K.; Jaque, D.; Hirata, G. A.; Vetrone, F. Adv. Opt. Mater. 2015, 3, 687− 694.

measurements. Section S8, Thermal loading caused by the 790 nm probe beam. Section S9, Thermal dose and energy deposited in the tissue. Section S10, Luminescence spectrum of the NPs in tissue and aqueous environment. Section S11, Experimental Details (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: cjacinto@fis.ufal.br. Author Contributions

L.D.C., D.J., A.d.S.G.N., and C.J. conceived the study and designed the experiments. E.C.X., W.Q.S., U.R., K.U.K., and C.D.S.B. performed the experiments on optical spectroscopy of nanoparticles. F.S.R., N.F., B.d.R., E.C.X., W.Q.S., and U.R. performed the in vivo experiments. D.B. and A.M.D. performed the EPR experiments. All the authors contributed to the analysis of the data and to the writing process of the manuscript. E.C.X. and W.Q.S. contributed equally. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Spanish Ministerio de Educacion y Ciencia (MAT2013-47395-C4-1-R), by EU Framework Programme (COST-CM1403 action) by Brazilian Agencies: FINEP (Financiadora de Estudos e Projetos) through the grants INFRAPESQ-11 and INFRAPESQ-12; CNPq (Conselho Nacional de Desenvolvimento Cientıfico e Tecnologico) Grants INCT NANO(BIO)SIMES and Project Universal nr. 483238/2013-9; CAPES (Coordenadoria de Aperfeiçoamento de Pessoal de Ensino Superior) by means of the Project PVE nr. A077/2013. E.C.X. is supported by a PhD scholarship from CNPq and currently by the PVE A077/ 2013 project by means of a PhD sandwich program developed at the Universidad Autonoma de Madrid, Spain. D.J. is the PVE (Pesquisador Visitante Especial) of the Project A077/2013. K.U.K. is a postdoctoral fellow of this Project. W.Q.S. is a postdoctoral fellow of the PNPD/CAPES program. U.R. is supported by a Post Doctoral Fellowship grant PDE/CAPES at the Universidad Autonoma de Madrid-Spain through the Project No. 2108-14-3. B.d.R. aknowledges support from Universidad Autonoma de Madrid through an FPI grant. This work was developed in the scope of the project CICECO Aveiro Institute of Materials (ref. FCT UID/CTM/50011/ 2013), financed by national funds through the FCT/MEC and when applicable cofinanced by FEDER under the PT2020 Partnership Agreement. C.D.S.B. (SFRH/BPD/89003/2012) thanks Fundaçaõ para a Ciência e Tecnologia (Portugal) for the postdoctoral grant.

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ABBREVIATIONS NP, nanoparticle; LNTH, luminescent nanothermometer; BW, biological window; TEM, transmission electron microscopy; EDX, energy-dispersive X-ray; EPR, electron paramagnetic resonance

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