Biocompatible Heat-Shock Protein Inhibitor-Delivered Flowerlike Short

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Biological and Medical Applications of Materials and Interfaces

Biocompatible HSP Inhibitor-Delivered Flower-Like SWIR Nanoprobe for Mild Temperature-Driven Highly Efficient Tumor Ablation Anqi Jiang, Yuxin Liu, Liyi Ma, Fang Mao, Lidong Liu, Xuejiao Zhai, and Jing Zhou ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b21483 • Publication Date (Web): 24 Jan 2019 Downloaded from http://pubs.acs.org on January 29, 2019

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ACS Applied Materials & Interfaces

Biocompatible HSP Inhibitor-Delivered Flower-Like SWIR Nanoprobe for Mild Temperature-Driven Highly Efficient Tumor Ablation Anqi Jiang ,Yuxin Liu , Liyi Ma , Fang Mao , Lidong Liu , Xuejiao Zhai , Jing Zhou* Department of Chemistry, Capital Normal University, Beijing, 100048, P. R. China E-mail: [email protected]

Abstract: Multifunctional nanomaterials for dual-mode imaging guided cancer therapy are highly desirable in clinical applications. Herein, the flower-like NiS2 coated-NaLuF4:Nd nanoparticle (Lu:Nd@NiS2) was synthesized as a novel therapeutic agent for short-wave infrared light imaging and magnetic resonance imaging to guide photothermal therapy. The material was then loaded with phenolic epigallocatechin 3-gallate (EGCG), which is a natural heat shock protein 90 (HSP90) inhibitor. Upon NIR irradiation, EGCG was released from the Lu:Nd@NiS2-EGCG, which bound heat HSP90 and reduced cell tolerance to heat, resulting in a better therapeutic effect at the same elevated temperature. Therefore, with minimal side effects and remarkable antitumor efficacy in vivo, Lu:Nd@NiS2-EGCG appeared to be a promising photothermal agent for enhanced photothermal therapy.

Keywords:

rare earth; multifunctional imaging; heat shock protein; natural resources;

photothermal; semiconductor

Introduction: 1

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Noninvasive photothermal therapy (PTT) is a critically important method in cancer treatment that involves irradiation of the tumor area after injection of photothermal conversion agents in order to induce locally high temperatures and thereby destroy cancer cells 1. Commonly used photothermal conversion nanomaterials are noble metal nanomaterials polymers

6-7,

and semiconductor materials

8-10.

2-4,

graphene 5, conjugated

However, cancer cells in deeper tissues are not

significantly treated as limited near-infrared light could penetrate tissue. Therefore, most PTTs were only available for the treatment of superficial tumors. Heat shock proteins (HSP) are functionally related proteins that assist other proteins to fold, maintain their structure, and be disposed of properly

11.

They are upregulated by heat shock to

protect cells at high temperatures. The presence of the HSP inhibitor reduces the tolerance of cancer cells to heat, which means that the tumor in deeper tissue can be ablated by PTT

12-13.

In

our early work, small interfering RNA (siRNA) was used to inhibit the synthesis of HSP70 14 and the small molecule inhibitor PES was used to inhibit the action of HSP70

15

to improve the

efficacy of PTT. However, there are certain challenges in these studies, such as the easy inactivation of siRNA, and the relatively high toxicity of some small molecule inhibitors. Epigallocatechin gallate (EGCG), which belongs to the class of phenols, is a natural HSP90 inhibitor extracted from green tea that has the advantages of low cytotoxicity, stability, selective interaction with HSP90, and capacity to disrupt HSP90 activity via multicellular signaling 16. In order to meet the requirements of real-time diagnosis, luminescence imaging has become a hot topic in recent research

17-20.

Short-wave infrared (SWIR) light has the advantages of deep

tissue penetration, low light scattering, high signal, and reduced photodamage effects

21-28.

Rare

earth nanomaterials have unique properties, causing many researches to focus on developing

2


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high-efficiency rare earth-based SWIR imaging agents

29-31.

Due to the long excitation (808 nm)

and emission wavelengths (850 nm–1400 nm), Nd-doped nanomaterials have potential applications in accurate real-time cancer diagnosis

32.

However, luminescence imaging does not

provide detailed anatomical and physiological images in vivo. The combination of magnetic resonance imaging (MRI) and luminescence imaging combines the advantages of both imaging modalities to provide precise positioning of the tumor

33-38.

Previous work has shown that

semiconductor nickel sulfide could be used for effective T2-weighted MRI in vitro and in vivo. In three-dimentional flower-like structures, incident photons are scattered and reflected on the surface of the materials with a longer optical path and actuation time, resulting in improved NIR absorption and enhanced photothermal conversion capacity compared to normal nanoparticles. In addition, the pleated state of the flower-like nanoparticle makes the specific surface area larger, resulting in excellent drug load capacity. Therefore, the flower-like semiconductor has advantages regarding both drug loading properties and photothermal conversion 39. In this work, Nd-doped NaLuF4 nanoagents (Lu:Nd) for SWIR imaging was first reported. The flower-like NiS2 was then applied to Lu:Nd through a series of steps for T2-MRI and PTT, and their imaging and theranostic properties in vitro were further discussed. The toxicity of Lu:Nd@NiS2 nanoparticles was also evaluated. Finally, HSP90 inhibitor EGCG was loaded by electrostatic adsorption. Further multifunctional imaging and enhanced therapeutic effects were demonstrated by experiments with tumor-bearing mice.

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Results and Discussions Synthesis and characterization Multifunctional flower-like nanoparticles were synthesized in several steps (Figure 1a). First, we used a modified solvothermal method to synthesize NaLuF4:Nd nanoparticles (Lu:Nd) 40. The size, morphologies, and crystal structure of the obtained samples were investigated by transmission electron microscope images (TEM) and high-resolution TEM (HRTEM). The TEM images showed that the Lu:Nd nanoparticles were dispersed with a uniform spherical shape on the copper grid, and the size of the nanoparticles was about 75 nm (Figure S1a). The X-ray power diffraction (XRD) pattern showed good crystallinity and conformed to the NaLuF4 being in the hexagonal phase according to the standard card of β-phase of NaLuF4 (JCPDS: 027-0726) (Figure S3a). The typical HRTEM and the selective area electronic diffraction (SAED) also supported this observation (Figure S2a, S4a). The energy-dispersive X-ray (EDX) analysis spectrum of Lu:Nd confirmed the presence of Na, Lu, Nd, and F elements in the nanoparticles (Figure S5). For biological applications, water solubility of the materials needs to be improved. Silica dioxide (SiO2) coating is widely used for hydrophobic and hydrophilic materials 41. After coating the Lu:Nd nanoparticles with mesoporous SiO2 (mSiO2), a Lu:Nd@mSiO2 core-shell structure was obtained (Figure S1b). The thickness of the mesoporous silica shell was around 20 nm, as shown in Figure S2b. Subsequently, we transformed the mSiO2 shell into a nickel silicate shell by the hydrothermal method

42.

Under alkaline conditions, the Si-O bond of the mesoporous silica

layer broke to form silicate ions, which then continuously reacted with the nickel-ammonia complex at high temperature. Due to the mesoporous structure of silica, the flower-like Lu:Nd@NiSiO3 nanoparticles were finally formed. The TEM and HRTEM images demonstrated

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that the average diameter of the flower-like surface was approximately 150 nm (Figure S1c, S2c). The NiS2 coated-nanoparticles (Lu:Nd@NiS2) were then synthesized by a further hydrothermal method

43.

The color of the Lu:Nd@NiSiO3 suspension was light green, while that of the

Lu:Nd@NiS2 suspension was black (Figure S6). The Lu:Nd@NiS2 adopted a flowerlike architecture and had a mean size of 150 ± 5 nm (Figure 1b,c). The EDX analysis spectrum of Lu:Nd@NiS2 confirmed the presence of Na, Lu, Nd, F, Ni, and S elements in Lu:Nd@NiS2 nanoparticles (Figure 1d). To further investigate their microstructure, elemental mapping was used to study the elemental distributions in the core-shell structure. Energy-dispersive X-ray spectroscopy (EDS) mapping images corresponded to the elemental distribution of Lu, S, and Ni (Figure 1e). The Lu element remained in the core region, while the Ni and S elements could be detected in the shell region. This result further confirmed the core-shell structure of Lu:Nd@NiS2 microspheres. N2 adsorption and desorption were then analyzed, which revealed the specific surface area and porosity of the core-shell structure of Lu:Nd@NiS2. The Brunauer-Emmett-Teller (BET) surface area was about 13.66 m2 g-1, and the pore volume was 0.119 cm3 g-1, which were calculated from N2 isotherms (Figure 1f). The pore-size distribution plot showed a rough surface and inside pores of the flowerlike structure of Lu:Nd@NiS2. The XRD pattern conformed to the hexagonal NaLuF4 and the NiS2 according to the standard card of β-phase of NaLuF4 (JCPDS: 027-0726) and NiS2 (JCPDS: 11-009), respectively (Figure S3b). We then tested the stability of Lu:Nd@NiS2 in water, 0.9% NaCl, 5% glucose and PBS solution by Ultraviolet–visible–near infrared (UV-vis-NIR) spectra, as the absorbance of Lu:Nd@NiS2 was directly related to their morphology and dispersity. It was found that there was no obvious difference between the absorbance of Lu:Nd@NiS2 in those solutions, which suggested that the Lu:Nd@NiS2 can retain

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its morphology in various biological solutions (Figure S7a). Furthermore, there was no observable change in the absorbance of Lu:Nd@NiS2 in water within 30 days storage period, which confirmed that Lu:Nd@NiS2 was stable in various biological solutions (Figure S7b). Finally, the optical properties of Lu:Nd@NiS2 nanoparticles were analyzed. The luminescence peaks at 860 nm, 1064 nm, and 1332 nm originated from the transition of Nd3+ from the excited state 4F3/2 to ground state 4I9/2, 4I11/2, and 4I13/2, respectively (Figure 1g). The luminescence intensity of Lu:Nd@NiS2 increased with concentration (Figure S8). SWIR has ideal laser penetration depth 44, thus, we then chose luminescence at 1332 nm for further study and imaging application. Therefore, Lu:Nd@NiS2 have potential applications in accurate real-time cancer diagnosis. However, luminescence imaging did not provide detailed anatomical and physiological results in vivo. Magnetic resonance imaging (MRI) has unique advantages, such as high sensitivity to soft tissue, theoretically no imaging depth limitations, and no ionizing radiation damage

45.

T2-weighted MRI is widely used in clinical cancer diagnosis which can provide high resolution three-dimentional images of pathological phenomena such as tumors and inflammation. In this study, we used a 0.5 T human clinical scanner to obtain the T2-weighted MR images of Lu:Nd@NiS2 samples. The relaxivities of Lu:Nd@NiS2 were calculated from the slope of the plot of the relaxation rate 1/T2 in relation to the concentration of nanoparticles. Relaxation rates were strongly correlated with the concentration, as evidenced by an R value of 0.9957 (Figure 1h). The intensity of the T2-weighted MRI signal increased as the molarity increased, making the image darker (Figure 1h inset). Typically, an r2/r1 ratio more than 10 is an essential factor for T2 contrast agents. Therefore, we also obtained the value of r1 and calculated the ratio of r2/r1 to be 129.56. These results proved that Lu:Nd@NiS2 is a potential T2 MR contrast agent for T2-weighted MRI.

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Photothermal properties of Lu:Nd@NiS2 We found that the UV-vis-NIR spectra of Lu:Nd@NiS2 (0, 0.175, 0.35, and 0.7 mg mL-1) exhibited broad absorbance in the 210–900 nm region, which suggested photothermal properties of our material (Figure 2c). To detect the photothermal properties of the Lu:Nd@NiS2 solution, the different concentrations solution was irradiated with the 808 nm NIR laser (1.6 W cm-2). The color of the photothermal images of the solutions changed continuously as a function of time (Figure 2a). The temperature elevation values of deionized (DI) water and Lu:Nd@NiS2 solutions at concentrations of 0.25, 0.5, 0.75, and 1.5 mg mL-1 were 1.5, 9, 14.5, 19.7, and 24.7 °C, respectively (Figure 2b). The results revealed that the temperature rise in the solution was strongly related to the concentration. Photothermal ability was dependent not only on the solution concentration but also on the laser power. The Lu:Nd@NiS2 solutions were irradiated with an 808 nm laser at different laser intensities (0.008, 0.58, 1, 1.6 W) for 8 min, corresponding to temperature rises of 1.3, 9.1, 15.1, and 24.6 ℃, respectively (Figure S9). As shown in Figure 2d and 2e, when heating and cooling processes reached equilibrium, the photothermal conversion efficiency (η) of Lu:Nd@NiS2 was 39.38%, which was comparable to the previously reported photothermal coupling agents

46.

In addition, photothermal stability of Lu:Nd@NiS2

nanocomposites was examined upon laser irradiation (Figure 2f). For each cycle, the dispersion was irradiated for 120 s and then cooled to room temperature. After six cycles, no loss in temperature rise was observed. Moreover, the absorbance curves of Lu:Nd@NiS2 substantially coincided before and after laser irradiation (Figure 2g), which indicated excellent photostability of Lu:Nd@NiS2. In conclusion, the flower-like Lu:Nd@NiS2 could be used as an excellent photothermal agent for thermal ablation in cancer treatment .

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EGCG loading and release Phenolic epigallocatechin gallate (EGCG) is an ester of epigallocatechin and gallic acid. For improved therapeutic applications, we loaded EGCG on Lu:Nd@NiS2 and studied its loading and release properties. First, EGCG and Lu:Nd@NiS2 were simply stirred in DI water for 24 h. The negative charge of EGCG formed static adsorption with positive charge on the Lu:Nd@NiS2 surface, thus, EGCG loaded on Lu:Nd@NiS2. As shown in Figure 3a, the absorbance of EGCG was positively correlated with its concentration. The ultraviolet-visible (UV-vis) absorbance of EGCG, Lu:Nd@NiS2 and Lu:Nd@NiS2-EGCG revealed that EGCG was successfully absorbed on Lu:Nd@NiS2 (Figure 3c). We measured the absorbance at the EGCG characteristic absorption (273 nm) in the supernatant, and by linear fitting, we obtained that when the mass ratio of EGCG to Lu:Nd @NiS2 is 2:1, and the loading efficiency of EGCG reached maximum at 343.1 mg g-1 (Figure 3b, d). Considering the utilization rate of comprehensive drugs and environmental protection issues, we choose this ratio to carry out drug loading. EGCG release from Lu:Nd@NiS2 was detected in buffer solutions (pH 6.4 and 7.4) by UV-vis absorption. EGCG release was found to be time- and pH-dependent. The release rate at pH 6.4 was higher than that at pH 7.4, probably due to protonation of the phenolic hydroxyl (Figure 3e). In detail, EGCG possess various dissociable OH groups in its structure. When Lu:Nd@NiS2-EGCG was dissolved in an acidic environment, the hydrogen ions protonated the phenolic hydroxyl moiety of the EGCG, which caused it to be released from Lu:Nd@NiS2-EGCG. According to previous reports, the encapsulated EGCG, after release, can serve as a heat shock protein 90 (HSP90) inhibitor by binding at the C-terminal ATP binding region on HSP90 to inhibit dimerization, promote conformation, and interfere with chaperone function, in the same manner as the aryl hydrocarbon

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receptor (AhR) or immunophilin-like protein XAP2 and p23

47-48.

Because HSP90 is required for

luciferase refolding, HSP90 activity can be studied by luminescence measurements. Diluted rabbit reticulocyte lysate (RRL) 49, which is rich in HSP90, was incubated with EGCG or Lu:Nd@NiS2 for 30 min before the addition of luciferase. The luminescence showed that active HSP90 was only reduced in the presence of EGCG, and the results suggested that Lu:Nd@NiS2-EGCG had an obvious inhibitory effect on HSP90 (Figure 3f). Moreover, the recovery study also demonstrated that neither Lu:Nd@NiS2 nor EGCG facilitated luciferase refolding (Figure 3f). Therefore, the EGCG loaded on nanoparticles was suitable for the enhanced photothermal therapy (PTT) study as the HSP90 inhibitor. Toxicity study The toxicity of Lu:Nd@NiS2 was studied to determine the safety in bioapplications. We first measured cytotoxicity in human colorectal adenocarcinoma HCT116 cells by performing a methyl thiazolyl tetrazolium (MTT) assay. The MTT assay showed no significant difference in the proliferation of HCT116 cells with and without the application of Lu:Nd@NiS2 within 12 and 24 h (Figure S10a). When substituting human non-small lung cancer cells (NCI-H1975) in the above experiments, similar results were obtained (Figure S10b). The results of MTT assay demonstrated low cellular cytotoxicity of Lu:Nd@NiS2. To determine the potential toxicity of Lu:Nd@NiS2 in vivo, healthy mice were sacrificed at 1 h, 24 h, 7 d, 15 d, 30 d, and 60 d after administration of Lu:Nd@NiS2 (20 mg kg-1 body-1) for blood analysis and histological examination (n = 3). In addition, mice of the same age injected with PBS were used as a control group (n = 3). Comparatively, the liver function indices, including total protein (TP), aspartate aminotransferase (AST), albumin (ALB), alanine aminotransferase (ALT),

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creatinine (CREA), and total bilirubin (TBIL), showed similar levels in the test and control group (Figure S11 and Table S1). Histological examination using hematoxylin-eosin (H&E) staining revealed that there was no significant adverse reaction in main organs of mice at 1 h, 24 h, 7 d, 15 d, 30 d, and 60 d after the injection of Lu:Nd@NiS2 (Figure S12). Thus, the final results demonstrated that Lu:Nd@NiS2 exhibited little toxicity as our tested dose in mice after administration. Moreover, the blood half-life time of the Lu:Nd@NiS2-EGCG nanoparticles reaches 4 h, which was sufficient to achieve benefit from material after intravenous injection (Figure S13). In vivo multifunctional imaging Primarily, SWIR imaging in vivo was measured. Lu:Nd@NiS2 (100 μL, 2 mg mL-1) was injected into tumor-bearing mice. Luminescence imaging in vivo was performed at the tumor region post-injection (Figure 4a). Numerical analysis obtained by ImageJ software revealed high sensitivity in luminescence imaging in vivo (Figure 4b). Consequently, the result confirmed that Lu:Nd@NiS2-EGCG can be a luminescence probe in the diagnosis of tumor optical imaging. In the previous section, negative MR enhancement of T2 magnetic resonance was obtained (Figure 1h inset). An MRI scanner with a spin echo sequence was used to further examine the suitability of Lu:Nd@NiS2 as a T2-weighted MR contrast agent for in vivo applications. We then injected Lu:Nd@NiS2 (100 μL, 2 mg mL-1) into tumor-bearing mice. Negative significantly enhanced MR contrast images were found in the tumor areas pre- and post-injection (Figure 4c). Image analysis also showed significant MRI signal differences between tumor areas and normal tissue areas after injection (Figure 4d). The experiments demonstrated that Lu:Nd@NiS2 has potential to serve as an imaging probe for multi-modality image-guided cancer therapy.

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As Lu:Nd@NiS2-EGCG has excellent photothermal conversion properties, we subsequently performed photothermal imaging in tumor-bearing mice and monitored local temperature changes by thermal imaging. The color of corresponding photothermal images of the solutions changed continuously with time (Figure 4e, S14b). The surface temperature of the blank control group increased to 38 ℃ within 7 min under irradiation by an 808 nm laser at a power density of 0.97 W cm-2 (Figure S14a). The surface temperature of the tumor in mice, which were injected with Lu:Nd@NiS2-EGCG and irradiated at a power density of 0.97 W cm-2, increased rapidly to 42 ℃ within 7 min (Figure 5a), while that in mice operating at a power density of 1.5 W cm-2 within 7 min increased to 45 ℃ (Figure 5b). Comparably, while irradiating with a 2.5 W cm-2 power density, the surface temperature of the tumor increased rapidly to 55 ℃ within 7 min (Figure 5c). The photothermal performance was strongly related to the duration and power density of the laser. The results demonstrated that Lu:Nd@NiS2-EGCG has potential for in vivo PTT application within reasonable conditions. In vivo photothermal therapy Before studying the effect of photothermal therapy on the Lu:Nd@NiS2-EGCG nanoparticles in vivo, we first studied the viability of HCT116 cells treated with Lu:Nd@NiS2-EGCG under laser irradiation. Figure S10c shows the significant difference in HCT116 cell proliferations with and without laser irradiation after adding Lu:Nd@NiS2-EGCG. The result indicated that reduced cell viability was found in the Lu:Nd@NiS2-EGCG after irradiation and cell viability in Lu:Nd@NiS2-EGCG samples (1.2 mg mL-1) even decreased to below 20% which indicated the Lu:Nd@NiS2-EGCG is an excellent photothermal agent. Tumor-bearing mice were then divided into nine groups to further study photothermal therapy

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with the Lu:Nd@NiS2-EGCG nanoparticles in vivo. First, the control groups (i.e., PBS only, laser only, EGCG + laser groups) were designed for comparison. Within the treatment period, tumors in these groups grew continuously, reaching a relative tumor volume (V/V0) as high as 3–4 (Figure S15). The results indicated that PBS injected alone, NIR laser irradiation alone or EGCG injection under irradiation had almost no effect on tumor development. The Lu:Nd@NiS2 + low laser group and Lu:Nd@NiS2-EGCG + low laser group consisted of tumor-bearing mice that were respectively intravenously injected with a PBS suspension of Lu:Nd@NiS2 (100 μL, 2 mg mL-1) and Lu:Nd@NiS2-EGCG (100 μL, 2 mg mL-1), then exposed to an 808 nm laser operating at a power density of 0.97 W cm-2. During the treatment period, tumors in these two groups grew continuously, while tumors in the Lu:Nd@NiS2-EGCG + low laser group grew slower than the Lu:Nd@NiS2 + low laser group (Figure 5d). Mice in the Lu:Nd@NiS2 + middle laser and the Lu:Nd@NiS2-EGCG + middle laser groups were irradiated by a 1.5 W cm-2 laser. Tumors in the Lu:Nd@NiS2 + middle laser group did not significantly change within the treatment period, while that in the Lu:Nd@NiS2-EGCG + middle laser group were partially ablated (Figure 5e). Tumor volumes in the Lu:Nd@NiS2 + high laser group and the Lu:Nd@NiS2-EGCG + high laser group were studied at laser of 2.5 W cm-2 (Figure 5f). The V/V0 of the Lu:Nd@NiS2-EGCG + high laser group decreased to zero after the first treatment, while the tumor ablation rate in the Lu:Nd@NiS2 + high laser group was slower, nevertheless tumor ablation was quick, and over-treatment of tumor sites in mice was observed. This result was due to the combination of released EGCG from the nanoparticles and HSP90 in the tumor cells which made photothermal ablation more effective. A similar conclusion was obtained following hematoxylin and eosin (H&E) staining. H&E histological sections indicated that tumor cells and adipocytes in normal tissue of the mice

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irradiated with low power were compact and showed no conspicuous necrosis (Figure 5g). Following medium-power irradiation, the tumor area became loose and fragile, and the tumor site showed obvious shrinkage (Figure 5h). The adipocytes in normal tissue were intact with minimal damage (Figure 5i). However, the tumor region and adipocytes both suffered extreme damage under high-power irradiation. These results indicated that injection of Lu:Nd@NiS2 by the laser irradiation could inhibit tumor growth, with a not entirely obvious effect, while the effect of the Lu:Nd@NiS2-EGCG group was significant under the same irradiation conditions. We also observed body weights of the mice. Compared with other groups (recorded without significant change), body weight in the Lu:Nd@NiS2-EGCG + high laser group was lighter during treatment, implying that some of the normal tissues in mice in this group were likewise damaged and these mice were overtreated (Figure S16). The results in tumor volume and mice weight indicated that Lu:Nd@NiS2-EGCG can ablate tumors in mice after laser irradiation at 808 nm with medium intensity with minimal side effects. These results demonstrated that Lu:Nd@NiS2-EGCG was an excellent photothermal agent in vivo for PTT of cancer within reasonable conditions.

Conclusion: In this work, we first reported flowerlike NiS2 modified-NaLuF4:Nd (Lu:Nd@NiS2) core-shell nanoparticles. Multifunctional Lu:Nd@NiS2 was successfully applied in T2-weighted MRI and SWIR luminescence imaging guided photothermal therapy. Lu:Nd@NiS2 treated cells and animals exhibited unobservable toxicity. Considering its theranostic properties, natural EGCG was loaded by electrostatic adsorption, enhancing the PTT effect by inhibiting the action of HSP90. Our work provided a novel optimization strategy for development of multifunctional nanoagents and a potentially better method for enhancing the efficacy of cancer photothermal therapy.

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Acknowledgements The authors thank the funding of Beijing Talent Foundation Outstanding Young Individual Project (2015000026833ZK02), The Joint Foundation Program of Beijing Municipal Natural Science Foundation and Beijing Municipal Education Commission (KZ201810028045), Capacity Building for Sci-Tech Innovation-Fundamental Scientific Research Funds (025185305000/195), Project of High-level Teachers in Beijing Municipal Universities in the Period of 13th Five-year Plan (IDHT20180517), Project of Construction of Scientific Research Base by the Beijing Municipal Education Commission, Yanjing Young Scholar Development Program of Capital Normal University, and Youth innovative research team of Capital Normal University.

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Figure

1.

a)

Schematic

illustration

of

synthesis

of

multifunctional

nanoflower

Lu:Nd@NiS2-EGCG. TEM image b), and HRTEM image c), EDX analysis (* represents the Si element from the silicon plate used in the preparation of the sample) d), EDS mapping e), and the N2 adsorption-desorption isotherms f) of Lu:Nd@NiS2. g) The luminescence spectrum of Lu:Nd@NiS2 excited by 808 nm laser. h) Relaxation rate (1/T2) vs various concentrations of Lu:Nd@NiS2.

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Figure 2. Photothermal images a) and heating curves b) of Lu:Nd@NiS2 at the concentration of 0.25, 0.5, 0.75, and 1.5 mg mL-1 under laser (808 nm, 1.6 W cm-2) respectively. c) UV-vis-NIR absorbance spectra of Lu:Nd@NiS2 at different concentrations, and DI water. d) Heating and cooling curves of Lu:Nd@NiS2 under laser of 808nm and corresponding e) linear time data versus ln(θ) of Lu:Nd@NiS2. f) Temperature variations of Lu:Nd@NiS2 (1.5 mg mL-1) under the continuous irradiations of 808 nm laser for six cycles. (g) UV-vis-NIR spectra and visual appearance (inset) of the Lu:Nd@NiS2 before and after radiation of 808 nm laser.

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Figure 3. a) UV-vis absorbance spectra of EGCG solutions at the concentrations of 5, 10, 20, 40, and 80 mg L-1. b) Liner relation between absorbance value at peak 273 nm vs the concentrations of EGCG. c) UV-vis absorbance spectra of EGCG, Lu:Nd@NiS2 and Lu:Nd@NiS2-EGCG. d) The loading efficiency of the EGCG in Lu:Nd@NiS2-EGCG. e) The release ratio of the EGCG released from Lu:Nd@NiS2 in buffer solutions (pH 6.4 and 7.4). f) The relative units studied by luminescence determination of luciferase in various situations.

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Figure 4. Luminescence imaging collecting 1332 nm signal excited by 808 nm laser a) and T2-weighted MRI c) in vivo and corresponding numerical analysis b,d) after injection of Lu:Nd@NiS2-EGCG. e) Photothermal images of mice after injection of Lu:Nd@NiS2-EGCG at different conditions. 1) Lu:Nd@NiS2-EGCG + low laser group (0.97 W cm-2), 2) Lu:Nd@NiS-EGCG2 + middle laser group (1.5 W cm-2), 3) Lu:Nd@NiS2-EGCG + high laser group (2.5 W cm-2). Statistical significance was determined by one-way t tests. *p < 0.05, **p < 0.01, and ***p < 0.001.

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Figure 5. Heating curves of mice which injected with Lu:Nd@NiS2-EGCG under laser (808 nm) irradiation at the power density of 0.97 W cm-2 a), 1.5 W cm-2 b), and 2.5 W cm-2 c), respectively. Tumor growth rates of Lu:Nd@NiS2-EGCG + low laser d), Lu:Nd@NiS2-EGCG + middle laser e), and Lu:Nd@NiS2-EGCG + high laser f). H&E histologic sections of the border of tumor and adipocytes of Lu:Nd@NiS2-EGCG + low laser group g), Lu:Nd@NiS2-EGCG + middle laser group h), and Lu:Nd@NiS2-EGCG + high laser group i). Statistical significance was determined by one-way t tests. *p < 0.05, **p < 0.01, and ***p < 0.001.

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