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Optimal fluorescence and photosensitivity properties of dual-functional NaYb1−xF4:Tmx3+ nanoparticles for applications in imaging guided photodynamic therapy

The fluorescence and photosensitivity properties of NaYb1−xF4:Tmx3+ nanoparticles were optimized to develop noninvasive near-infrared fluorescence imaging-guided photodynamic therapy. The emission at 800 nm from Tm3+ presented an exponential increase with an increase in the Tm3+ doping concentration from 0 to 2%. The photosensitivity properties of NaYb1−xF4:Tmx3+ nanoparticles were also studied via the chemoprobe method, which used a reactive oxygen quencher, 1,3-diphenylisobenzofuran (DPBF). With the increase in the doping concentration of Tm3+, the generation rate of reactive oxygen species in NaYb1−xF4:Tmx3+ nanoparticles decreased linearly at a rate of 0.3. The doping concentration of Tm3+ had two opposite effects on the 800 nm emission and generation rates of reactive oxygen species. The competitive relationship was discussed and an optimal value for the Tm3+ doping concentration of approximately 1% was determined. At this concentration, the energy of the Yb3+ excited state can be fully utilized, and the fluorescence and photosensitivity properties are an effective combination.


Introduction
Photodynamic therapy (PDT), due to its highly specic selectivity and minimal invasiveness, has become an intriguing route for treating tumors. [1][2][3] In typical PDT, the photosensitizer (PS) is excited by light and generates reactive oxygen species (ROS), killing tumor cells with minimal damage to surrounding tissues. PSs are key components in the implementation of effective PDT. 4,5 Generally, traditional PSs need to be excited by visible light, which results in poor penetration depth in tissue, due to which their use is limited to the supercial treatment of tumors. 6,7 A tissue is known to have a biological window in the near infrared light (NIR) region from 800 to 1100 nm, as the excitation light in this range exhibits weak scattering and absorption in the tissue, enabling deep penetration. Efforts have been invested to extend the excitation wavelengths of PSs to the NIR range. For instance, two-photon-excited PSs have been developed. [8][9][10] Unfortunately, the absorption cross-section of two-photon-excited PSs is too small, necessitating the use of a high-energy pulse laser, which signicantly exceeds safe limits for clinical applications. Upconversion nanoparticle (UCNP)based PSs, which feature UCNP-loaded traditional PSs (NPs-PSs) to perform PDT, are proposed as alternative candidates. Specically, UCNPs absorb NIR light and emit visible light to trigger traditional PSs to generate ROS. [11][12][13] In the process, severe relaxation losses and low upconversion efficiencies lead to an impractically low ROS quantum yield. Recently, we proposed an NIR-excited inorganic PS, NaYbF 4 NPs, that could achieve NIR-excited PDT with high efficiencies via a simple energy transfer method. 14 In addition, NaYbF 4 NPs serve as PSs directly without drug loading and release, making the therapeutic effect more controllable and stable. However, NaYbF 4 NPs are difficult to guide to the positions of tumor due to the weak emission of Yb 3+ . Therefore, it is imperative to introduce a luminescence element to NaYbF 4 NPs for achieving precise tumor localization and high-efficiency PDT.
In this study, we design a novel nanocomposite NaYb 1Àx -F 4 :Tm x 3+ as the PS for application to imaging-guided PDT. The uorescence characteristics and ROS generation were investigated in detail using NaYb 1Àx F 4 :Tm x 3+ NPs. The energy distribution relationship between the 800 nm uorescence intensity and ROS generation rate was obtained. Next, the energy distribution between imaging and PDT was adjusted by changing the Tm 3+ doping concentration, and an optimal value for the Tm 3+ doping concentration was determined at which uorescence and photosensitivity properties combined effectively.

Results and discussion
To explain the energy transfer mechanism of NaYb 1Àx  Fig. 2a and S1, † respectively. A histogram of the NP size distribution was obtained using the Nano Measurer 1.2.5 soware by calculating the size of NPs in the TEM bright-eld image (Fig. 2b). The mathematical details regarding the calculation of the particle size distribution are presented in Table S1. † The mean NP size is approximately 6.5 nm. The XRD pattern of NaYb 99% F 4 :Tm 1% 3+ NPs was obtained, and the positions of the diffraction peaks were found to closely match the JCPDS-PDF 77-2043 cubic phase of NaYbF 4 NPs (Fig. 2c). Furthermore, the effect of the Tm 3+ doping concentration on the structure of NaYb 1Àx F 4 :Tm x 3+ NPs was investigated through XRD analysis using specimens with Tm 3+ concentrations of 1% and 2%, as shown in Fig. S2. † No distinct difference was observed between the diffraction angles for Tm 3+ concentrations at 1% and 2%. It is probably due to similar radii of 0.0858 nm and 0.087 nm for Yb 3+  is approximately 98%, the XRD pattern is consistent with the JCPDS standard card. 16 Fig. 3a presents the uorescence spectra of NaYb 1Àx F 4 :Tm x NPs. Further, the integrated intensities of the 800 nm emission with different Tm 3+ doping concentrations are shown intuitively in Fig. 3b. The intensity of the 800 nm emission is observed to initially increase. When the doping concentration of Tm 3+ reaches 2%, the uorescence intensity of 800 nm is at its maximum. As the doping concentration of Tm 3+ continues to increase, the uorescence intensity at 800 nm begins to decrease due to Tm 3+ concentration quenching. In this study, we consider the Tm 3+ concentration to be within 2%.
The detailed relationship between the uorescence intensity and Tm 3+ concentration is explored. The typical uorescence spectra of NaYb 1Àx F 4 :Tm x 3+ (x ¼ 0.2, 0.5, 1, 1.5, 2) NPs are shown in Fig. 3c. Fig. 3d shows the 800 nm uorescence intensities of NaYb 1Àx F 4 :Tm x 3+ NPs at different Tm 3+ concentrations. We used the average value and standard deviation of the 800 nm emission for three-repeats of measurement, as shown in Fig. S3, † which was normalized by the intensity for 2% Tm 3+ . The normalized 800 nm uorescence intensities of NaYb 1Àx F 4 :Tm x 3+ NPs were enhanced exponentially with the Tm 3+ doping concentration (x) in the form of y ¼ 1 À exp(Àx/x 0 ), which is shown in Fig. 3d. Although x 0 ¼ 0.6% is the optimized Tm 3+ doping concentration for the uorescence intensity, it is not the optimum concentration for dual functions (imaging and PDT).
To theoretically investigate the effect of the Tm 3+ concentration on the intensity of the 800 nm emission, the rate equations in the steady states based on the energy transitions were studied through eqn (S1)-(S5). † As illustrated in eqn (S5), † the same pump power density was utilized to excite the 800 nm emissions; hence, r is a constant parameter for all the samples. The absorption cross-sections of the Yb 3+ ions are similar. N Yb0 can be regarded as constant because the concentration of Yb 3+ only exhibits a small change. Therefore, eqn (S5) † can be simplied as follows: The intensity of the 800 nm emission in eqn (1) is related to: the energy transfer rate between Yb 3+ and Tm 3+ ions (W i ); the population densities of the ground state (N 0 ) in Tm 3+ ; and the decay times of Yb 3+ in excited states (s Yb1 ) and Tm 3+ in 3 F 4 states (s 1 ). When the Tm 3+ concentration increases, W 0 , W 1 , and N 0 in eqn (1) increase. On the contrary, with increasing Tm 3+ concentration, the distance between the Tm 3+ ions decreases and cross-relaxation is enhanced, leading to a decrease in s Yb1 and s 1 . [16][17][18] At low concentrations of Tm 3+ , the rates of increase for W 0 , W 1 , and N 0 are higher than the rate of decrease for s Yb1 2 s 1 ; hence, the intensity of the 800 nm emission increases with increasing Tm 3+ concentration. As the Tm 3+ concentration continues to increase, the decrease in s Yb1 2 s 1 is faster than the increases in W 0 , W 1 , and N 0 . This is probably because the values of s Yb1 and s 1 are very small and s Yb1 2 s 1 decreases rapidly due to the power function and product form. Therefore, the intensity of the 800 nm emission decreases at a high Tm 3+ concentration. Furthermore, we studied the relationship between singlet oxygen generation and the Tm 3+ doping concentration. The 1,3diphenylisobenzofuran (DPBF) absorbance spectra of NaYb 1Àx F 4 :Tm x 3+ (x ¼ 0.2, 0.5, 1, 1.5, 2%) were measured three times (Fig. S4 †). Fig. 4a displays the normalized DPBF absorbance as a function of the irradiation time in the NaYb 1Àx F 4 :-Tm x 3+ NP solutions. The absorbance shows an exponential decay with the light exposure time. Next, the consumption rate of DPBF as an indicator of ROS generation rate was determined by the slope of linear tting, 19 as shown in the inset of Fig. 4a. Fig. 4b shows the consumption rates of DPBF at different Tm 3+ concentrations, which use the average value and standard deviation for three-repeats of measurement and are normalized   For the application of dual-functional NaYb 1Àx F 4 :Tm x 3+ NPs, the energy competition mechanism between uorescence and ROS generation from Yb 3+ in the excited state should be considered. The Tm 3+ doping concentration has opposite effects on the uorescence and ROS generation (Fig. 5a). On the one hand, an increase in the Tm 3+ doping concentration would facilitate a strong 800 nm upconversion emission. On the other hand, an increased Tm 3+ doping concentration is unfavorable for a high ROS generation rate. Considering the two opposing effects, an optimum Tm 3+ doping concentration is required to balance the energy distribution between uorescence and ROS generation. Here, the variation rate (rate of decrease or increase) of ROS generation and uorescence intensity can be employed to evaluate the relative rate of variations at different Tm 3+ doping concentrations. The variation rate can be determined by the derivation of the curves in Fig. 5a. The variation rates of uorescence intensity and ROS generation are shown in Fig. 5b. The decreasing rate of ROS-generating efficiency is 0.3, which is a constant. Furthermore, the rate of increase of uorescence intensity decreases exponentially. When the Tm 3+ doping concentration is less than 1%, the rate of increase for uorescence intensity is greater than the rate of decrease for ROS generation. Therefore, the improvement in the uorescence intensity is worth the loss of ROS generation. However, when the Tm 3+ concentration exceeds 1%, the increasing rate of uorescence intensity is less than the decreasing rate of ROS generation. Hence, there is an optimal value for the Tm 3+ concentration, which corresponds to approximately 1%. At this concentration, the energy of the Yb 3+ excited state can be fully utilized, allowing for the most efficient manner of energy distribution. of ethanol and cyclohexane to remove impurities. The NPs were then collected by centrifugation. Aer purication, the NPs were stored in cyclohexane.

The generation rate of ROS
The generation rate of reactive oxygen species can be evaluated by consumption rate of DPBF. The time-dependent concentration of DPBF during ROS generation can be described as follows: 21 where [DPBF] and [ROS i ] are the concentrations of DPBF and ROS, respectively; k i is the rate constant of chemical reaction between O 2 and DPBF; and k is the consumption rate of DPBF or the reciprocal of the time constant in the decrease of DPBF. Eqn (2) can be rewritten as: Then, the consumption rate of DPBF (k) can be given by the slope of the eqn (3).
For the concentrations of DPBF, we employ the Beer-Lambert law to calculate. The transmission intensity can be described as: where I t and I 0 are the transmission intensity with and without absorption by samples, s is the absorption cross section of PS, and L is the optical path. Here, Therefore, A can be used to estimate the change of DPBF concentration, and is given by: Absorption spectra measurement DPBF solution (0.02 mmol mL À1 ) with NaYb 1Àx F 4 :Tm x 3+ (12 mmol mL À1 ) was exposed to a 980 nm laser with the power of 1.5 W. The transmission spectra of DPBF in the solutions were measured every 20 minutes. A deuterium lamp served as the light source of transmission spectra, and a miniature QE65000 ber optic spectrometer (Ocean Optics, USA) was used to detect the transmission from UV to visible light. The absorption spectrum of DPBF can be obtained according to the Beer-Lambert law.
Characterization method X-ray diffraction (XRD) was obtained using Rigaku D/MAX-2600/ PC with the Cu Ka radiation (l ¼ 1.5406Å) at the scanning step at 0.02 . Transmission electron microscope (TEM) image was obtained using FEI Tecnai TF20.

Fluorescence spectra measurement
The uorescence spectra were measured at room temperature using 70 mW power-controlled 980 nm laser diode as an excitation source. A monochromator (Zolix Instrument SBP 300) coupled with photomultiplier (Zolix Instrument PMTH-S1-CR131) was used to collect the 800 nm upconversion uorescence. The scanning step is 1 nm. All uorescence spectra were measured using the same measuring system under the same conditions.

Conclusions
The ROS generation and 800 nm emission in NaYb 1Àx F 4 :Tm x 3+ NPs were investigated for application in imaging-guided PDT. The Tm 3+ doping concentration was found to have opposite effects on the ROS generation and 800 nm emission. At low Tm 3+ concentrations, the increasing rate of ROS generation exceeded the decreasing rate of uorescence intensity. Additionally, the former was slower than the latter. When the Tm 3+ concentration reached 1%, the variation rate of ROS generation and uorescence intensity was equal. At this concentration, the energy of Yb 3+ in the excited state could be used efficiently and the combination effect of uorescence imaging and PDT was optimum.

Conflicts of interest
There are no conicts to declare.