Efficient nanoheater operated in a biological window for photo-hyperthermia therapy.

Remotely monitoring and regulating temperature in a small area are of vital importance for hyperthermia therapy. Herein, we report ~11 nm NaErF4 nanocrystal as the ultra-small nanoheater, which is highly safe for biological applications. Under 1530 nm photon excitation, upconversion intensity of NaErF4 is significantly enhanced as compared to the conventionally used 980 nm pumping source. Upconversion mechanisms are discussed on the basis of power dependence measurements. Importantly, light-to-heat transformation efficiency of NaErF4 through 1530 nm pumping is determined as high as 75%. Efficient NIR emission, centered at ~800 nm and thus within the biological window, is used for the temperature feedback. The potential applications of this highly efficient nanoheater for controlled photo-hyperthermia treatments are also demonstrated.

To make practically valuable nanoheaters for hyperthermia therapy, two factors should be focused. The first factor is the efficient light-to-heat conversion ability, which lowers the operating laser power corresponding to the lethal temperature of the nanoheater and thus suppresses the side effect of laser, that is, overheating effect. Second factor is the efficient photoluminescence signal of the nanoheater, specifically, detectable light signal dependent on the surrounding temperature. This signal should be strong enough to validate the non-invasive temperature sensing by monitoring the spectra of the nanoheater passing through the human tissue.
However, above mentioned factors are actually contradictory since the light-to-heat transformation and photoluminescence of rare earth are two ways consuming the absorbed energy and compete with each other. To simultaneously achieve effective heating and detectable photoluminescence, one feasible way is to enhance the absorption of incident light energy, including enlarging the absorption cross section of rare earth at the incident wavelength, as well as increasing the doping concentration of the nanoheater. Thus, one should choose rare earth with strong absorption at the incident wavelength and should increase the doping concentration, by adopting this method, the absorbed energy can be greatly enhanced [28].
Unfortunately, the effects of concentration quenching result in extremely weak emission under high doping situation and thus prevents heavily doping of rare earth [29][30][31]. In another word, although possessing efficient light-to-heat conversion, it remains formidable challenge to achieve detectable signal, falling in the BW, for temperature feedback in high doping nanoheaters.
Er 3+ shows efficient energy absorption at 1530 nm (Ш-BW), as Er 3+ possesses larger absorption cross section here. Most importantly, we found that the upconversion intensity of Er 3+ heavily doped fluoride nanocrystals is highly enhanced under 1530 nm pumping source as compared to 980 nm excitation, especially for the NIR emission centered at ~800 nm (І-BW). This means one can simultaneously obtain efficient heat conversion and strong light signal within BW. Therefore, 1530 nm excited Er 3+ heavily doped fluoride nanocrystals should be promising candidate for self-monitoring photothermal probe.
In this paper, we synthesized small NaErF 4 nanocrystals as the photothermal nonoheater. Comparison of upconversion properties were carried out under 980 nm and 1530 nm excitation, and upconversion mechanisms were also investigated. Light-to-heat conversion efficiency was evaluated through monitoring one heating/cooling cycle of the nanoheater in liquid. In addition, FIR technique was utilized for the temperature feedback of the nanoheater and pork tissue was used to demonstrate the feasibility of the proposed nanoheater.  Fig. 1. TEM photograph of as-prepared NaErF 4 nanocrystals, scale bar is 50 nm. Distribution of the particle size is given by measuring 100 particles, and the mean size is calculated to be ~11.3 nm.

Results and discussion
NaErF 4 (namely, 100 mol% Er 3+ doping) nanocrystals are synthesized by a modified thermal decomposition method [32]. The TEM image in Fig. 1 shows the morphology of NaErF 4 nanocrystals, which exhibit a mono-dispersed spherical shape with an average diameter of ~11 nm. Such small size is highly useful for biological applications, due to small nanoparticles can efficiently penetrate subcellular membranes, can be easily cleared from the human body, and would be more suitable for ex vivo diagnostics [33]. Erbium (Ш) is the most commonly used upconversion activator and Yb 3+ is usually added as the sensitizer to utilize its strong absorption at around 980 nm. Herein, strong absorption of Er 3+ appears at 1.5 μm [ Fig. 2(a)], providing solid foundation of efficient upconversion emissions upon 1.5 μm excitation by using Er 3+ singly doped system. As expected, 1530 nm excitation leads to significant improvement on the upconversion intensity of NaErF 4 as compared to 980 nm pumping source by using identical output power [ Fig. 2(b)]. Importantly, the efficient NIR emission at around 800 nm validates the subcutaneous imaging and treatment as demonstrated later. As shown in our recent work [34], severe concentration quenching still occurs under 1530 nm excitation, indicating that the efficient energy harvest (due to 1530 nm pumping combined Er 3+ heavily doping) is mainly responsible for the highly improved upconversion emission. Upconversion mechanisms of Er 3+ excited by 1530 nm photon are investigated by power dependence measurements. It can be seen from Fig. 2(c) that green and red emissions are 3-photon processes, and NIR emission at 800 nm is 2-photon process, as detailed depicted in Fig. 2(d).
As shown in Fig. 3(a), when the sample cell (a 10 × 10 mm 2 quartz cuvette containing NaErF 4 /cyclohexane dispersed solution) is irradiated by 1530 nm laser, temperature of the solution raises gradually. The maximum temperature is achieved after 8-min heating and then another 10-min is needed to cool down to the ambient temperature without heating. In addition, 5 heating/cooling cycles are performed to show the good reproducibility of our nanoheater. Light-to-heat conversion efficiency is one of the most important parameter of the photoheater. This efficiency can be calculated through monitoring one heating/cooling cycle of sample cell, using the following equation [18].
where T max = 34.3 °C is the equilibrium temperature; T 0 = 19.6 °C is the initial ambient temperature [ Fig. 3(a)]. Q b , expressing heat dissipated from 1530 nm light absorbed by the sample, was measured independently to be 97 mW by using a quartz cuvette containing pure cyclohexane without NaErF 4 nanocrystals; I = 1 W is the incident laser power; A = 0.043 is the absorbance of Er 3+ in the cuvette cell. To calculate hS, the heat transfer coefficient from quartz cuvette to the surroundings, time constant τ s is introduced as follows [18].
From Eq. (2), the obtained value of the time constant τ s = 128 s is given, as shown in Fig.  3(b) by the linear fitting. According to the expression τ s = mC/hS, where m = 0.80 g and C = 1.82 J/K·kg are the mass and heat capacity of cyclohexane in the cuvette, respectively, we obtained hS = 11.40 mW/K. Hence, on the basis of the above mentioned parameters, the light-to-heat efficiency is calculated to be η = 75% by using Eq. (1). Due to the plenty of energy levels and thus multiple emission bands of Er 3+ , besides heat energy the residual of absorbed energy should be the summation of the light energy, including mainly the visible emissions and NIR emissions centered at around 800 nm, 980 nm, and 1550 nm, if one neglect the higher-order ultraviolet emissions. This conversion efficiency is much higher than the previously reported nanoheaters such as Au nanorods (21%) and nanoshells (13%), Cu 9 S 5 (25.7%), and Carbon coating nanocrystals (38.1%) [15][16][17], and slightly higher than the NaNdF 4 @NaYF 4 @1Nd:NaYF 4 core-shell nanoheater (72.7%) [28]. Although still lower than some gold nanorods with 100% heating efficiency [35,36], the presented nanoheater exhibits potential of self-monitoring due to its efficient NIR emission.
In addition, it is found that light-to-heat conversion efficiency drastically decreases by using 980 nm pumping source compared to 1530 nm excitation, due to the slump of light harvest. The estimated efficiencies of NaErF 4 under 1530 nm and 980 nm excitation are 75% and 39%, respectively, evidencing the strong dependence of heat conversion on pumping wavelength. Similarly, decreasing the doping concentration of Er 3+ also leads to evident decrease of the heat conversion efficiency (conversion efficiency of 80 mol% Er 3+ doped NaGdF 4 under 1530 nm excitation is evaluated to be 42%), due to the same reason as mentioned above.
To validate the temperature feedback, NIR emission band corresponding to the 4 I 9/2 → 4 I 15/2 transition is utilized for the temperature sensing. FIR (ratio of the luminescence intensities at 804 nm and 825 nm) increases with the ambient temperature and can be well fitted by a linear line [Figs. 4(a) and 4(b)] as follows.
where k = 0.00116 and b = 1.50843 are obtained by the fitting. At the end, pork skin is used to demonstrate the ex vivo hyperthermia treatment. Experimental setup is depicted in Fig. 4(c), NaErF 4 nanocrystals dispersed in cyclohexane are sprayed onto the back side of the pork skin (~2 mm of thickness). 1530 nm laser outputting various powers penetrate the pork skin and illuminate the nanocrystals. Upconversion signal, passing through the pork skin, is transmitted into the spectrometer to deduce the eigen temperature of NaErF 4 , using the calibration in Eq. (3) with determined parameters. Surface temperature of the skin is monitored by a thermal camera. The relative intensity at 804 nm and 825 nm evidently increases by increasing the pumping power [ Fig. 4(d)], indicating the remarkable heating effect. As shown in Fig. 4(e), the heating temperature (increased temperature of NaErF 4 ) approximately increases linearly with the incident laser power. The maximum temperature value goes above 100 °C and the surface heating of the pork skin is still weak when the power density is ~1 W/cm 2 , evidencing the high heat conversion efficiency of our nanoheater. The heating temperature is much higher than that reported in Nd 3+ /Yb 3+ doped LaF 3 core-shell nanocrystals [6], which can be attributed to the efficient light-to-heat transformation of NaErF 4 (smaller size and core-only nature both benefit the heat accumulation), as well as the more concentrated nanocrystal used in the demonstration. showing the heating effect of the nanoheater through 2 mm thickness pork tissue; (d) Normalized spectra of NIR emission under different laser power excitation; (e) Heating effect under different laser powers, surface temperatures are monitored by the thermal camera and eigen temperatures are calculated from FIR sensing. The inset is the digital photograph of the scattered laser beam after passing through the pork tissue, the radius of the scattered beam is evaluated to be 0.4 cm, which is used for estimating the incident power density.

Conclusions
In summary, we have demonstrated a novel rare earth based nanoheater. Using NaErF 4 optically excited with a single 1530 nm infrared beam, this nanoheater simultaneously achieved compact structure (core only nanocrystals), ultra-small particle size (~11 nm), efficient light-to-heat conversion (efficiency of ~75%) and temperature feedback (monitoring the heating). Luminescence mechanisms of the nanoheater were investigated on the basis of the power dependence variations. In addition, the application of NaErF 4 nanoparticles for the achievement of fully controlled photo-hyperthermia processes under single infrared beam excitation through pork tissue has been demonstrated. The obtained results are going to open a new way for the development of biological nanoheater with enhanced efficiency and reduced risk.

Disclosures
The authors declare that there are no conflicts of interest related to this article.