Programmably switching the NIR upconversion for orthogonal activation of photoacoustic imaging and on-demand phototherapy

Upconversion nanoparticles (UCNPs) based phototheranostics offer signicant expectations for the personalized cancer medicine via integrating both modalities of imaging diagnostics and phototherapeutics. However, programmably controlling the photoactivation of imaging and therapy towards the accurate diagnosis with minimum side effects for on-demand therapy has remained challenging due to the lack of ideal switchable UCNPs agents. Herein, we demonstrate a facile strategy to simply switch the near infrared emission at 800 nm from rationally designed UCNPs by modulating the irradiation laser into pulse output. Through synthesis of the theranostic UCNPs-DI agent combining with a photosensitizer and a photoabsorbing agent assembled on the UCNPs, the orthogonal activation of in vivo photoacoustic imaging and photodynamic therapy was further achieved by simply altering the excitation modes from pulse to continuous-wave output upon a single 980-nm laser. Importantly, no obvious harmful effects during photoexcitation caused by reactive oxygen species (ROS) photooxidation and photohyperthermia were generated under imaging modality, which facilitates the long-term and real-time imaging-guidance for the subsequent phototherapy. This work provides a new facile approach for the orthogonal activation of imaging diagnostics and photodynamic therapeutics towards the target cancers.


Introduction
Cancer phototheranostics, an emerging combinatorial modality of light-diagnostics and -therapeutics, has been developed as personalized precision medicine to enhance the treatment accuracy and e ciency of conventional phototherapies particularly the representative photodynamic therapy (PDT). [1][2][3][4][5][6][7] The clinical-approved PDT methodology refers to the photoactivation of a photosensitizer in the presence of molecular oxygen to generate reactive oxygen species (ROS) for destruction of pathological tissues. [8][9][10] To date, efforts have been devoted to establish the photodynamic theranostics by developing functional photosensitizers to serve as both an imaging agent and a therapeutic agent. [10][11][12][13] However, in light of the limitations of currently available photosensitizers with synchronous activation of both diagnostic and therapeutic signals, the long-term and real-time diagnostic imaging may also induce unnecessary phototoxicity (including photooxidation by ROS and the photo-induced hyperthermia effect) in the normal tissues. [14][15][16] To meet the demand for precision medicine against cancers, programmable photoactivation of the photosensitizers for the safety imaging and e cient therapy in an orthogonal manner is highly desirable but still challenging due to lack of ideal photoswitchable agents.
Among the reported orthogonal theranostic agents, the lanthanide-doped upconversion nanoparticles (UCNPs) with multiple wavelength-tunable emissions in the visible or near-infrared (NIR) region have been demonstrated to be a remarkable candidate for the development of photoswitchable agents. [17][18][19][20][21][22] Some orthogonal UCNPs-based platforms were constructed for the programmable activation of the cancer diagnosis and therapy. [23][24][25][26][27] However, in previous studies, the two (or more) excitation laser wavelengths (e.g., 980 and 808 nm) and exquisite agents with core-multi-shells structure designs or different components are indispensable to achieve the switchable emissions, 28-30 which makes the manufacturing process quite complicated and time-consuming as well as requires complex instruments.
Moreover, the orthogonal emissions mainly locate in the UV-visible region such as UV/blue, 31,32 green/red light. [33][34][35][36][37] The switchable NIR emission is still not achieved, which is believed to be more signi cant in the phototheranostics.
Interestingly, photon upconversion is a multi-step anti-Stokes process and each step may have a temporal characteristic. This means that some upconversion processes would be sensitive to the excitation duration, consequently generating tunable emission pro les as evident in the full-color emissive nanoparticles upon pulse laser excitation. 38 However, it should be noted that such orthogonal upconversion needs complex core-multi-shell nanoparticles which produce great di culties in the experimental synthesis. By contrast, we recently constructed a simple migratory core-shell nanostructure toward red/green color switchable output by temporal manipulation of the Er 3+ -Yb 3+ interactions under 980 nm single-wavelength excitation. 38, 39 Moreover, the tunable NIR upconversion luminescence can be obtained by constructing suitable transitions in speci c lanthanide ions. This result provides new chances for the orthogonal NIR upconversion in a much simpler nanostructure through tuning the pulse widths of a single wavelength excitation, showing a remarkable advantage for bioimaging and phototherapy.
In this study, we report a facile strategy to programmably switch the 800 nm NIR emission in Yb/Tm/Er codoped UCNPs by precisely tuning the pulse width of 980 nm excitation laser to achieve the orthogonal real-time safe photoacoustic imaging and effective photodynamic therapy. We discovered for the rst time that the NIR emission of Tm 3+ at 800 nm in such UCNPs is very sensitive to the excitation laser pulse width. This design markedly reduces the complex core-multi-shell nanostructure and the multiple components as well as the synthetic procedure. We further constructed the upconversion nanocomposite (UCNPs-DI) by surface anchoring the photosensitizer indocyanine green (ICG) dye 40,41 with response to the switchable upconverting NIR emission and the semiconductor (DPP) 42,43 with absorption covering the visible emission of the UCNPs. Under the excitation of short-pulse 980 nm laser, the ICG is not activated but the visible emissions of UCNPs can be captured by DPP to generate intense photoacoustic signals with no photodynamic and photothermal effect, which facilitates the long-term and real-time imaging of the in vivo tumors without signi cant phototoxicity observed. Under the continuous-wave (CW) 980 nm excitation, the switch-on NIR upconversion emission could e ciently activate the PDT effect to afford accurate and effective inhibition of the in vivo tumor growth. Moreover, such UCNPs-DI nanoagent can avoid the harmful effect during photoactivated imaging diagnosis and achieve e cient programmable and orthogonal phototherapy by regulating the laser pulse output upon the single 980-nm irradiation, further highlighting the bioapplications of non-steady-state mediated upconversion nanomaterials.

Mechanistic design of UCNPs with switchable NIR emission
In general, the energy transfer rate in a given sensitizer-activator system depends closely on the energy matching between the emission of sensitizer and the absorption of the activator, which shows a decline following an exponential function of the energy gap (Fig. 1a). The upconversion system with an energy mismatch should result in a relatively slow upconversion process than that of the resonantly coupled system (Fig. 1b), allowing to ltrate such upconversion luminescence by reducing the pulse duration time of the excitation laser. Therefore, the dynamic control of switchable upconversion performance in a much simpler structure nanoparticles becomes highly desirable.
Intriguingly, upon the pulse 980 nm laser, the UCNPs show a gradually decreased NIR emission with reducing the pulse width to 10 ns, and the emission light intensity ratio of UCL 650 nm to UCL 800 nm dramatically increases from 0.89 to 8.25, as illustrated in Fig. 1d,e and Figure S3. This observation quanti es the UCNPs an ideal NIR photoswitchable candidate to be used to construct the orthogonal theranostic agents.
To further examine the non-steady-state upconversion mechanism, we measured the time-dependent pro les at 650 nm and 800 nm, respectively. As displayed in Fig. 2a, the red upconversion emission exhibits a faster rise time than that of the NIR emission before reaching the steady-state. This observation suggests that the upconversion of the NIR emission of Tm 3+ indeed needs more time by contrast to the red emission originating from the non-resonant energy transfer from Yb 3+ ( 2 F 5/2 ) to Tm 3+ ( 3 H 5 ). Consequently, the red to NIR emission intensity ratio shows a monotonous decline with the time during the excitation pulse. Hence, reducing the pulse width of the excitation laser become a facial but effective way to switch off the NIR emission. Another merit of this upconversion system lies in the stable color output during a large range of pump power densities (Fig. 2b) and different excitation frequencies ( Figure S4), which lays a solid foundation for the subsequent biological application. It should be noted that a co-doping of Er 3+ and Tm 3+ into NaYF 4 lattice imposes no impact on their rise time feature (Fig. 2c), which con rms the dynamic manipulation of the NIR switchable output. The total energy transfer processes during the dynamic control of the excitation are schematically illustrated in Fig. 2d. In fact, the presence of Tm 3+ can promote the red upconversion of Er 3+ slightly through the energy circling of Er 3+ ( 4 I 11/2 ) → Tm 3+ ( 3 H 5 ) → Er 3+ ( 4 I 11/2 ) ( Figure S5). This is in agreement with the faster rise time of the red upconversion emission of the UCNPs with codoping of Tm 3+ (Fig. 2e). Taken together, the results clearly con rm the validity of using the non-steady-state excitation towards the remarkable NIRswitchable effect of UCNPs, which has never previously been reported involving the lanthanide-based nanoparticles. In the next, the typical pulse laser with width of 10 ns and 20 Hz was selected for the following phototheranostics investigation.

Construction and characterization of orthogonal phototheranostic nanoagent
To demonstrate the dynamic control of the orthogonal photodiagnostics and phototherapeutics for the safe imaging-guided on-demand phototherapy, a nanoagent (UCNPs-DI) was synthesized via assembly with a visible-absorbing semiconductor DPP and an NIR-activatable photosensitizer ICG on the surface of the UCNPs (Fig. 3a and Figure S6). The as-prepared DPP possesses high optical absorption extinction coe cient in the visible region of 550-700 nm, ensuring the effective energy transfer from UCNPs to DPP to generate photoacoustic signal via pulsed photoirradiation of the nanoagent. In view of the typical NIR absorption of ICG around 800 nm and the NIR-switchable emission of UCNPs, the photodynamic effect is non-responsive to the short-pulse irradiation of UCNPs-DI, facilitating the safe long-term and real-time photoacoustic imaging. Upon CW 980 nm irradiation, the switching-on NIR emission could activate the ICG to e ciently produce the cytotoxic reactive oxygen species (ROS) for phototherapy. Therefore, the orthogonal control of the designed UCNPs-DI under programmable photoactivation guarantees the safety of imaging and the precision of therapy.  Figure 3c presents the absorption spectra of UCNPs, DPP, ICG, and UCNPs-DI. According to the absorption peaks recorded at 650 and 800 nm and the Fourier-transform infrared (FTIR) spectroscopy ( Figure S7) of UCNPs-DI, the DPP and ICG were loaded on the nanoagent successfully. The luminescence spectra of UCNPs-DI together with the starting UCNPs were then recorded under the PW/CW excitation of 980 nm laser. As illustrated in Fig. 3d and S8, compared with the starting UCNPs, the visible and NIR emission of UCNPs-DI under CW irradiation was suppressed dramatically due to the energy transfer from UCNPs to DPP and ICG. This was also characterized by the lifetime decline of the 800 nm emission (from 463 to 293 µs, Figure S9). 44,45 These results con rmed the successful construction of the aimed phototheranostic nanoagent UCNPs-DI.
To verify the stability of the nanoagent in aqueous solution and physiological uids, the absorption of UCNPs-DI in PBS solution and blood serum with different concentrations and times were recorded ( Figure   S10). 46 The absorption of nanoagent does not show an obvious change under these conditions.
Adjusting the pH scope of the PBS solution from 8.5 to 5.0 also did not affect its absorption and UCL emission under both 980 nm PW and CW excitations ( Figure S11 and S12). 47 The results show that UCNPs-DI has an excellent consistency in a physiological environment, revealing its high stability under the physiological conditions.
To study the theranostic nanoagent for orthogonal photoacoustic imaging and photodynamic therapy with programmable excitations of CW/PW 980 nm laser, we then evaluated its performance in the photoinduced generation of acoustic effect and singlet oxygen ( 1 O 2 ) in aqueous solutions. Compared with the starting UCNPs, as depicted in Fig. 3e and S13, we observed a strong and concentrationdependent photoacoustic signal (R 2 = 0.99359) when the UCNPs-DI irradiated with 980 nm pulsed laser (10 ns, 0.5 W/cm 2 ). 48 The PW irradiation of UCNPs-DI had no obvious photothermal effect, while the temperature of UCNPs-DI solution increased dramatically after exposing to the CW irradiation (0.5 W/cm 2 ), which suggests that the UCNPs-DI does not induce hyperthermia damage during the photoacoustic diagnosis ( Figure S14a). The high photostability was con rmed by the measurement of the 10-min photoirradiation of the sample with 4 cycles (Fig. 3f and S14b). 49 The 1 O 2 photogeneration of UCNPs-DI was then evaluated using the dye singlet oxygen sensor green (SOSG) as an indicator. 50 Fig. 3g and S15a demonstrated that almost no change was observed for the uorescence of SOSG at 525 nm in the UCNPs-DI solution with PW 980 nm irradiation, revealing no photodynamic toxicity during the photoacoustic diagnosis. On the contrary, 980-nm CW irradiation of UCNPs-DI can generate abundant 1 O 2 , in which the uorescence of SOSG increased rapidly within 18 min ( Figure S15b).
The above results clearly indicate that the UCNPs-DI produced intense photoacoustic signals with negligible photothermal and photodynamic effect upon 980 nm PW irradiation, providing robust ex-vitro evidence for the feasibility of UCNPs-DI as a safe PA imaging candidate for long-time and real-time diagnose or monitoring the therapeutic treatments. On the other side, switching the 980 nm laser into CW modulation activated the signi cant photodynamic effect to kill cells in lesions through the generation of adequate reactive oxygen species. The overall results provide substantial supports for us to achieve photoacoustic imaging and photodynamic therapy in an orthogonal manner through programmable excitations of UCNPs-DI by CW/PW 980 nm laser.

Orthogonally regulated target recognition and photodynamic effect in vitro
The cytotoxicity of the UCNPs-DI with PW (10 ns, 20 Hz, 0.5 W/cm 2 ) and CW 980 nm (0.5 W/cm 2 ) irradiation was rstly investigated in detail. The standard MTT assay was performed on human nonsmall cell human breast adenocarcinoma cancer cells (MCF-7). Figure 4a presents the viability of MCF-7 cancer cells incubated with different concentrations (25, 50, 100, 150, and 200 µg/mL) of PBS (gray), UCNPs-DI (pink), UCNPs-DI + PW (blue), and UCNPs-DI + CW (red). The cell viability was found still over 90% when the concentration of UCNPs-DI up to 200 µg/mL. Thus, the nanoagent should not in uence MCF-7 cells viability when their concentration is less than 200 µg/mL. Signi cantly, PW irradiation of the cells treated with UCNPs-DI did not induce obvious decrease in the cell viability compared with control cells. By contrast, it shows an evident drop in the cell viability under 980 nm CW excitation. When the MCF-7 cancer cells were incubated with 200 µg/mL UCNPs-DI, the cell viability was found dropping to 28 % in the UCNPs-DI + CW group. At this concentration, as shown in Fig. 4b, light dose-dependent cell-killing effect of UCNPs-DI after incubated for 4 h was recorded when exposure to 980 nm CW irradiation. Remarkably, the cell viability of MCF-7 incubated with UCNPs-DI inhibited dramatically to 27.2% with 0.5 W/cm 2 980 nm continuous lasers for 3 min.
The intracellular ROS generation in the UCNPs-DI + CW group and ROS non-generation in the UCNPs-DI + PW group were also con rmed using confocal laser scanning microscope (CLSM) as shown in Fig. 4c, which is consistent with the ROS detection results in Fig. 3g. In addition, the cell viability results were also veri ed by calcein AM (live cells staining; green) and propidium iodide (PI, dead cells staining; red) double-staining, respectively (Fig. 4d). The uorescence imaging results showed that the UCNPs-DI has negligible cytotoxicity under PW irradiation but high photodynamic cytotoxicity under CW irradiation. We then further evaluated the cell damage mechanism of UCNPs-DI + PW/CW using ow cytometry with Annexin V-uorescein isothiocyanate (FITC)/propidium iodide (PI) staining. The data revealed that in the cells treated with UCNPs-DI and pulsed laser irradiation, 2.67% of cells were stained with Annexin V-FITC and 0.23% cells were stained with PI, suggesting that most cells was undamaged under this condition (Fig. 4e). The measurement taken at the group with UCNPs-DI after 980 nm CW irradiation showed that 33.87% of the cells were stained with FITC and 30.70% of the cells were stained with PI, suggesting that the UCNPs-DI + CW treatment caused obvious membrane damage and massive cell death. In addition, the UCNPs-DI showed negligible inhibitory effects to the normal LO 2 cells with or without the 980 nm PW irradiation ( Figure S16). The above results con rmed the evidential potentials of UCNPs-DI as safe photoacoustic imaging candidate. Moreover, the results of UCNPs-DI under the 980 nm CW irradiation have validated the nanoagent as a photoswitchable agent for the effective phototherapy.

In vivo real-time photoacoustic imaging of tumor
With the intense photoacoustic effect and non-photocytotoxicity of UCNPs-DI validated in vitro and in living cells, we then investigated how to use the agent to overcome certain biological drug delivery barriers for the long-term and real-time photoacoustic imaging of speci c tumors (Fig. 5a). The PA imaging is a promising diagnostic imaging technique to monitor the molecular distribution and tumor size/morphology for therapeutical guidance due to its high resolution and noninvasive visualization of tissue structures. 51 After intravenous injection of UCNPs-DI into the MCF-7 tumor-bearing nude mice which were pretreated with either UCNPs or UCNPs-DI, the PA images of tumor region were recorded at times of post-injection in 0, 1, 4, 8, 12 and 24 h, respectively. As depicted in Fig. 5b,d, only very slight PA signal increase was observed over time in the UCNPs cohort. In contrast, the PA signal in the tumor region of the UCNPs-DI pretreated cohort started to be detectable at 4 h post-injection, and the PA signal intensity gradually increased and reached the maximum at 12 h post-injection (Fig. 5c,d). The PA signal in the UCNPs-DI cohort at 12 h post-injection was determined as 10.8 times greater than that in the UCNPs controls (Fig. 5e). Moreover, the PA signal strength still maintained a relative high level after 24 h post-injection of UCNPs-DI, which further proves UCNPs-DI as an excellent PA diagnosing agent. Taken together, these results indicate that UCNPs-DI provide outstanding real-time PA imaging for de nition of the tumor region and precise guidance for the subsequent laser irradiation.

Orthogonally regulated tumor targeting and therapy in vivo
We further evaluated the photoacoustic imaging-guided "on-demand" PDT e cacy in vivo by using the MCF-7 tumor-bearing mice as the animal model (Fig. 6a). 52 The mice were randomly divided into six groups. The control group was injected with PBS; three groups received laser (PW/CW, 980 nm, 0.5 W/cm 2 for 10 min) or UCNPs-DI i.v. injected alone; the other two groups received UCNPs-DI and then went through PW/CW corresponding irradiation (980 nm, 0.5 W/cm 2 for 10 min). The changes in tumor volume and body weight were then monitored and recorded over the next 21 days. As shown in Fig. 6b, c, after injection of the nanoagent for 12 h and laser exposure to the pulsed laser, no obvious tumor growth inhibition effect was observed on during the whole treatment. By contrast, the tumor received the nanoagent and CW 980 nm laser treatment, the volume shrunk persistently, and the tumor growth was almost completely inhibited after treatment for 21 days, as also depicted in Fig. 6d. Figure 6e shows few changes in the body weight of the mice during feeding time, indicating that UCNPs-DI possesses high therapeutic e ciency and the systemic toxicity of the UCNPs-DI nanoagent with PW/CW irradiation was insigni cant.
In order to further con rm the anti-tumor effect of UCNPs-DI, we used immunohistochemical staining for histological analysis to evaluate the pathological changes of the tumor tissue collected after the above treatment (Fig. 6f). Tumor sections revealed almost no cell necrosis and apoptosis in the nanoagent cohort with PW irradiation treatment, while the highest level of tumor cell damage was observed in the nanoagent cohort with CW irradiation treatment. In addition, we also performed hematoxylin and eosin (H&E) staining assays to study tumor cell death and organ damage after the phototherapy on the 21st day (Fig. 6g). The images of H&E staining demonstrated the tumor in the CW laser PDT group revealed signi cantly inhibited proliferation capability of the tumor cells, which is in agreement with the therapeutic results of the above-mentioned immunohistochemical staining in vivo. The photographs of the pathomorphological analysis of heart, liver, spleen, lung, and kidney are provided in Figure S17, which shows no obvious organ damage in all cases, indicating that the prepared UCNPs-DI nanocomposite have very good biocompatibility in vivo. Taken together, these results reveal that we can achieve the in vivo orthogonal safe PA imaging and highly effective phototherapy in the UCNPs-approved nanoagent through programmablly switching the on-duty ratio of the excitation laser with the same wavelength.
In summary, we have successfully demonstrated a facile but e cient strategy to switch the NIR 800 nm emission of the UCNPs by programmablly regulating the on-duty ratio of the excitation laser to achieve the orthogonal real-time safe photoacoustic imaging and effective "switching on-demand" photodynamic therapy with a rational design nanoagent. The nanoagent UCNPs-DI is composed of a highly visibleabsorbing semiconductor DPP and an NIR photosensitizer ICG loaded on the surface of the typical Er 3+ /Tm 3+ -doped UCNPs. The nanoagent with programmablly controlling upconversion process can e ciently transduce the 980 nm excitation to the steady-state visible light and dynamic NIR light. The spectral overlap between the absorption of DPP and the upconverted visible emission makes better use of the highly emissive upconversion for photoacoustic imaging for determining the lesion area under 980 nm pulsed laser, under which conditions, no photothermal and photodynamic effect can be activated. By switching the excitation light into CW mode to lighten the 800 nm emission, the ICG can be activated to generates cytotoxic ROS for antitumor therapy. In short, with UCNPs-DI, we can avoid the harmful effects caused by the ROS photooxidation and photohyperthermia during the long-term and real-time PA imaging diagnosis and achieve effective PA imaging-guided phototherapeutics. This work provides a new facile approach for the orthogonal activation of imaging diagnostics and photodynamic therapeutics towards the target cancers.

Materials
The chemicals mentioned in this article were all reagent grade and can be used immediately without further puri cation and ltration. Rare earth chlorides X(CH 3 COO) 3

Characterizations
The absorbance values of UCNPs and UCNPs-DI were measured by an ultraviolet/visible absorption spectrometer (Lambda-35 UV/visible spectrophotometer, Perkin-Elmer, MA, USA). Upconversion uorescence emission of UCNPs and UCNPs-DI were obtained using 500 mW/cm2 980 nm laser was surveying. Obtain the uorescence spectra of UCNPs and UCNPs-DI on the spetrasuite software.The TEM image of UCNPs and UCNPs-DI is a high-resolution 2100F eld emission transmission electron microscope (JEOL, Japan) operating at a capture acceleration voltage of 200 kV. ZEN3690 zeta sizer (Malvern, USA) was used to measure the size of nanoagent. At room temperature, in the Bio-Rad FTS 6000 spectrometer (Bio-Rad Company, Hercules, California, USA) recorded in FT-IR spectrum in the form of KBr particles. UCNPs and UCNPs-DI stability by getting in different pH (from pH 5.0 to 8.5) the absorption and uorescence spectra assessed UCNPs-DI.

Photoacoustic imaging in tumor in vivo
In addition, Balb/c mice carrying MCF-7 cells were divided into 6 groups. The blank group was injected with PBS only; the 980 nm group was focused with980 nm pulsed light and continuous light laser (0.5 W/cm 2 , 10 minutes); the mice in the UCNPs-DI group were injected intravenously with 100 µL UCNPs-DI (200 µg/mL). The treatment group used 100 µL UCNPs-DI (200 µg/mL) and 980 nm continuous and pulsed laser (0.5 W/cm 2 , 10 minutes) to focus on the tumor area for 10 minutes, respectively. Finally, the 6 groups of mice were injected with UCNPs-DI through the tail vein every 2 days, and the tumor area was treated with laser 12 hours later. During the treatment, the mice were anesthetized with oxygen containing 2.5% iso urane. The treatment effects of the 6 cohorts were appraised by detecting and recording the relative tumor volume and weight changes of mice.

Detection of singlet oxygen in vivo.
Mice with MCF-7 tumors were intravenously injected with 200 µg/mL UCNPS-DI (100 µL). At the time point 12 hours after the injection, 50 µM SOSG (30 µL) was injected into the tumor tissue of the mouse. Then, 980 nm continuous light and pulsed light were used to irradiate the tumor area at a power density of 0.5 W/cm 2 (10 minutes). After the mice were euthanized, the tumor tissues of the mice were dissected, and the tumor tissues were xed in 4% paraformaldehyde. Frozen sections are 10 µm thick. Finally, the tumor slices were blurred with SOSG, then recorded and observed under the Olympus FV3000 confocal laser scanning microscope.

Histology examination
After the treatment, the mice were euthanized. The tumor tissues and major organs (heart, liver, spleen, lung and kidney) were removed from 6 groups of mice and cut into sections with thickness of 4 µm. The tumor sections were xed in 10% paraformaldehyde solution for 12 h, then dehydrated with ethanol and processed into para n. Finally, the tissue section was stained with H&E and recorded and imaged by a uorescence microscope system (Nikon E 200). The H&E staining method is processed according to the method provided by the supplier (BBC Biochemical)

Declarations Con icts of interest
The authors declare no competing nancial interests.

Data availability statement
The raw/processed data required to reproduce these ndings are available from the authors.

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