Ferric Hydroxide-Modified Upconversion Nanoparticles for 808 nm

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Biological and Medical Applications of Materials and Interfaces

Ferric Hydroxide-Modified Upconversion Nanoparticles for 808 nmNIR-Triggered Synergetic Tumor Therapy with Hypoxia Modulation Xiao Wu, Peijian Yan, Zhaohui Ren, Yifan Wang, Xiujun Cai, Xiang Li, Renren Deng, and Gaorong Han ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b18427 • Publication Date (Web): 17 Dec 2018 Downloaded from http://pubs.acs.org on December 17, 2018

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ACS Applied Materials & Interfaces

Ferric Hydroxide‐Modified Upconversion Nanoparticles for 808 nm‐NIR‐ Triggered Synergetic Tumor Therapy with Hypoxia Modulation Xiao Wu,† Peijian Yan,‡ Zhaohui Ren,† Yifan Wang,‡ Xiujun Cai,‡ Xiang Li,*,† Renren Deng,*,† and † Gaorong Han †

State Key Laboratory of Silicon Materials, School of Materials Science and Engineering, Zhejiang University, Hangzhou 310027, P. R. China ‡Key Laboratory of Endoscopic Technique Research of Zhejiang Province, Sir Run Run Shaw Hospital, Zhejiang University, Hangzhou 310016, P. R. China ABSTRACT: The efficacy of dynamic therapy for solid tumors suffers daunting challenges induced by tumor hypoxia. Herein, we report a biocompatible nanosystem containing Fe(OH)3-modified upconversion nanoparticles (UCNPs) for promoted synergetic chemo- and photodynamic therapy with the modulation of tumor hypoxia. In the system, UCNPs convert 808 nm-NIR excitation to visible photon energy which stimulates Ce6 photosensitizers to generate toxic reactive oxygen species (ROS) by consumption of dissolved oxygen in cancer cells. Importantly, we employ Fe(OH)3 compounds to enable continuous oxygen generation in cancer cells, and meanwhile induce extra ROS formation through the Fenton-like reaction. The system consequently improves the tumor treatment efficacy in vitro and in vivo. This study puts forwards a novel combinatorial therapeutic platform for tumor microenvironment modulation and enhanced cancer therapy. KEYWORDS: Fenton-like reaction • Ferric hydroxide nanocolloid • Photodynamic therapy • Tumor hypoxia • Upconversion nanoparticles

1. INRODUCTION Photodynamic therapy (PDT) has been extensively recognized as a highly promising approach for cancer treatment. It conventionally consists of three main elements: photosensitizers, excitation light, and oxygen supply.1-4 During a PDT treatment, photosensitizers stimulate the conversion of oxygen into toxic reactive oxygen species (ROS) under an incident beam of light excitation. The generated ROS may disrupt the homeostasis of tumor metastasis leading to severe cell apoptosis and then inhibit tumor progression.5,6 However, solid tumors present typical hypoxia characteristics owing to its local recurrence and aggressive metastasis. It severely compromises the efficacy of PDT because of the shortage in oxygen supply.7-10 The approaches, enabling continuous supply of oxygen locally within tumors, are therefore of particular interest for overcoming the hypoxia challenge. In view of the fact that malignant proliferation of cancer cells produces high content of hydrogen peroxide (H2O2, up to 0.5 nmol/104 cells/h, 50-100 μM in cytoplasm), one strategy to achieve localized oxygen production is to employing exogenous agents which can react with the overexpressed H2O2 in tumor microenvironment.11,12 In this regard, materials such as catalase, and MnO2 have been applied for selectively catalyzing O2 production.13-19 Despite the usefulness, these materials may easily deactivate by intracellular protease or glutathione which limits their abilities for continuous in situ oxygen production. Alternatively, ferrite-based nanomaterials have recently emerged as more durable oxygen generators and attracted

growing attention.20-23 During a course of our study, we noticed that Fe(OH)3 nanocolloidal can induce effective oxygen production in H2O2 solution via the Fenton-like reaction. Intriguingly, the production of oxygen is accompanied by the formation of hydroxyl radical --- a more toxic ROS through a Fe3+/Fe2+ catalytic cycle. Accordingly, we hypothesized that Fe(OH)3 can be used to relieve tumor hypoxia, meanwhile, induce chemodynamic cytotoxicity to cancer cells. Herein, we propose a composite system, by conjugating Fe(OH)3 with lanthanide-doped upconversion nanoparticles (UCNPs), for synergetic chemo- and photodynamic therapy of malignant tumors in living mice (Scheme 1). Notably, UCNPs, which are featured by the ability of converting near-infrared (NIR) radiation to visible/UV emission, have been exploited as promising platforms for deep tissue PDT.24-31 Conventional photosensitizers, based on triplet-sensitized organic fluorophores or inorganic photocatalysts, are not suitable for treating deep tumors because of the low tissue penetration offered by ultraviolet (UV) or visible excitation light sources. By coupling conventional photosensitizers with UCNPs, NIR excitation with a substantially higher depth of tissue penetration can be utilized for sensitized ROS generation.32-35

2. MATERIALS AND METHODS 2.1. Materials. Yttrium(III) acetate hydrate (99.9%), ytterbium(III) acetate hydrate (99.9%), neodymium(III) acetate hydrate (99.9%), erbium(III) acetate hydrate (99.9%), sodium hydroxide (NaOH; >98%), ammonium fluoride (NH4F; >98%), 1-octadecene (90%), oleic acid (90%), tetraethylorthosilicate (TEOS, >99%), Iron(III) chloride hexahydrate (≥98%),

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Chlorin-e6 (Ce6) were purchased from Sigma-Aldrich. Ammonia solution (AR), Hydrogen peroxide (H2O2, 30 wt%) were purchased from Sinopharm Chemical Reagent Co., Ltd. Cell Counting Kit-8 (CCK8) were purchased from Beyotime. All chemicals were used as received without further purification. 2.2. Synthesis of NaYF4:Yb,Er@NaYF4:Nd@NaYF4 core-shell-shell UCNPs. The UCNPs were syntheiszied according to a previousy reported method.36-38 Futher details are described in the Supporting Information. 2.3. Synthesis of silica coated UCNPs (UCS). In a typical experiment, the as-prepared oleic acid capped NaYF4:Yb,Er@NaYF4:Nd@NaYF4 nanoparticles (30 mg mL-1, 0.5 mL in cyclohexane) were precipitated and collected by centrifugation followed by dispersed in 1 mL ethanol solution. After that, 1 mL of HCl (0.2 M in ethanol) was added, and the mixture was sonicated for 30 min to remove the oleic ligand from the surface of UCNPs. The resulting product was washed with ethanol for several times and finally dispersed in 1 mL deionized water. The as-prepared ligand-free nanoparticles were then added to 5 mL aqueous solution containing 200 mg of PVP-K10 before it was sonicated for 30 min. Subsequently, 20 mL of ethanol was added to the solution. The mixture was then dropwise added with 800 μL of ammonia solution and 100 μL of TEOS under vigorous stirring before it was allowed to react at room temperature for 12 h. The resulting UCS nanoparticles were washed with ethanol several times and redispersed in 6 mL ethanol. 2.4. Synthesis of Fe(OH)3 nanocolloidals. The Fe(OH)3 nanoparticles were synthesized by dropwise adding 200 μL solution of FeCl3 (1.068 g mL-1) into 25 mL boiling deionized water. The solution was kept boiling until the color became reddish brown. The as-synthesized Fe(OH)3 nanocolloidal was reserved at room temperature for further use without any purification. 2.5. Synthesis of UCS-Ce6-Fe(OH)3 nanocomposites. An ethanol solution of UCS (0.2 mL) was added to a 2 mL microcentrifuge tube containing 0.9 mL acetone solution of Ce6 (0.6 mg mL-1). After sonication for 30 min, the Ce6 loaded nanoparticles were collected by centrifugation (18000 rpm, 10 min) followed by dispersing in 1 mL ethanol. Thereafter, 20 μL of the as-prepared Fe(OH)3 nanocolloidal solution was added. The resulting mixture was sonicated for 10 min. Subsequently, the Fe(OH)3-modified UCS-Ce6 nanoparticles were collected by centrifugation and washed with ethanol for three times, and finally dispersed in 1 mL of deionized water. 2.6. In vitro toxicity assessment of UCS-Ce6-Fe(OH)3. Murine 4T1 breast cancer cells seeded in several 96-well plates at a density of 2×104 cells per well were cultured for 12 h at 37 o C under 5% CO2. Various concentrations of UCS-Ce6Fe(OH)3 (0.01, 0.025, 0.05, 0.1, 0.2 and 0.5 mg mL-1) were added into the culture media and co-incubated with the cells for 12 h and 24 h, followed by removing the medium containing non-internalized nanoparticles. The Cell Counting Kit-8 (CCK8) solutions were then added, and the cells were incubated in the cell incubator for another 2 h. Thereafter, the absorbance at 450 nm of each well was monitored by a microplate reader. The cytotoxicity of the nanoparticles was determined by comparing the relative cell viabilities to untreated cells. 2.7. In vitro therapy evaluation. Murine breast cancer 4T1 cells were seeded in 96-well plates at a density of 2×104 cells per well. After being incubated for 12h at 37 oC in normoxia environment (5 % CO2, 21 % O2) or hypoxia environment (5 %

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Scheme 1. Schematic illustration for the therapeutic mechanism of UCS-Ce6-Fe(OH)3 nanocomposites under NIR irradiation at 808 nm.

CO2, 2 % O2), the cells were treated with UCS-Ce6 or UCSCe6-Fe(OH)3 at concentrations of 0.2 mg mL-1 or 0.5 mg mL-1 for 5 h. Then the cells were exposed to 808 nm laser irradiation at a power density of 1 W cm-2 for 10 min. After the irradiation, the cells were transferred into fresh medium and further incubated for another 24 h before the cell viability was tested by the CCK-8 assay. 2.8. In vivo PDT experiments on 4T1 solid tumor models. Male BALB/c mice were subcutaneously implanted with 4T1 cells. When the tumor reached approximately 100 mm3, in vivo PDT experiment were performed. Each group of experiments carried out in five mice. Six groups of mice were treated with (1) PBS, (2) PBS+808 nm laser, (3) UCS-Ce6, (4) UCS-Ce6Fe(OH)3, (5) UCS-Ce6+808 nm laser, and (6) UCS-Ce6Fe(OH)3+808 nm laser, respectively. The solution of PBS, UCS-Ce6 or UCS-Ce6-Fe(OH)3 (50 μL, 20 mg mL-1) was intratumorally injected into each group. The irradiation power and irradiation time are 1 W and 30 min (1 min interval after each 5 minutes of irradiation)

3. RESULTS AND DISCUSSION In a typical experiment, we prepared hexagonal-phased NaYF4 UCNPs (~35 nm) with a refined core-shell-shell structure (NaYF4:Yb,Er@NaYF4:Nd@NaYF4) by a well-documented coprecipitation method (Figure 1a, b, and Figure S1).33-35 In the core-shell-shell nanoparticles, Nd3+ was doped as sensitizers to enable efficient excitation at 808 nm. In comparison with conventional 980 nm NIR-to-visible upconversion using Yb3+ ions as sensitizers, the use of ~800 nm excitation induces less overheating for the aqueous environment and maximizes depth of tissue penetration.39-42 A ~10 nm thick silica layer was then coated on the UCNPs by a modified literature procedure to render the particles with desired hydrophilicity and surface functionality (Figure 1a, c).43 In the meantime, Fe(OH)3 nanocolloidal was synthesized by dropwise addition of FeCl3 solution into boiling deionized water (Figure S2).44,45 Thereafter, photosensitizer Ce6 was loaded on silica surface of the UCS followed by conjugating the UCS-Ce6 with the Fe(OH)3 (Figure 1a, d). The as-prepared Fe(OH)3 was found to be amorphous without any sharp diffraction peaks in the corresponding X-ray diffraction (XRD) pattern (Figure S2). Transmission electron microscopy (TEM) showed irregular particle shapes of the Fe(OH)3 having an average diameter of 4-5 nm (Figure S2). The formation of UCS/Fe(OH)3 assemblies, dominated by


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Figure 1. a) Schematic illustration for the synthesis of UCS-Ce6-Fe(OH)3 nanocomposites. b-d) TEM images of the as-synthesized NaYF4:Yb,Er@NaYF4:Nd@NaYF4 core-shell-shell UCNPs; UCNP@SiO2 nanoparticles (UCS), and UCS-Ce6-Fe(OH)3 nanocomposites, respectively. All of the scale bars are 100 nm. e) High resolution Fe(2p) XPS spectrum of UCS-Ce6-Fe(OH)3. f) STEM and corresponding element mapping images of a typical UCS-Ce6-Fe(OH)3 particle. g) FTIR spectra of Ce6, UCS, and UCS-Ce6, respectively.

electrostatic interactions, was confirmed by TEM and Zeta potential measurements (Figure 1d, and Figure S3). The electrostatic bounding between Ce6/Fe(OH)3 and UCS was found to be strong enough to maintain the composite assembly in aqueous solution. After treating the nanocomposites with several centrifugation-washing-redispersion cycles, no obvious detachment was observed (Figure S4). Note that free Fe ions and Ce6 molecules should be easily removed through the centrifugation-washing steps. X-ray photoelectron spectroscopy (XPS) of the as-synthesized UCS-Ce6-Fe(OH)3 revealed the presence of Fe in trivalent state (Figure 1e).46 Elemental mapping of the composite material by energydispersive X-ray spectroscopy (EDS) at a single particle level under scanning transmission electron microscopy (STEM) further confirmed the chemical composition of UCS-Ce6Fe(OH)3 (Figure 1f). Furthermore, the modification of silica and Ce6 on the UCNPs was verified by Fourier-transform infrared spectroscopy (FTIR) analysis (Figure 1g). We next employed the Dynamic Light Scattering (DLS) analysis to evaluate the stability of the nanocomposites in physiological media. Basically, UCS, UCS-Ce6, and UCS-Ce6Fe(OH)3 showed good stability in deionized water having average hydrodynamic diameters of 108.7, 110.2, and 142.2 nm, respectively (Figure S5 and Table S1). However, UCS-Ce6Fe(OH)3 alone was found not stable in pure phosphate buffered saline (PBS) over 12 h of dispersion. It resulted in large aggregates to a diameter >300 nm probably due to the phosphate electrolyte induced colloidal aggregation (Figure S5). Nevertheless, we found that blood plasma proteins such as serum albumin could stabilize UCS-Ce6-Fe(OH)3 in complex biological media. When supplemented with 10% v/v fetal bovine serum in PBS, the UCS-Ce6-Fe(OH)3 dispersion caused stable colloidal with a hydrodynamic diameter of 175.8 nm (Table S1). We attributed the improved stability to the formation of a protein layer on nanocomposite surface which act as a physical barrier against direct particle-particle interaction.47-49 As an added benefit, we found that the protein

Figure 2. a) UV-VIS absorption spectra of UCS-Ce6-Fe(OH)3 nanocomposites at various of Ce6:UCS loading ratios. (Inset) Plot of real loading capacities in the final products against the Ce6:UCS loading ratios. b) Upconversion emission spectra of UCS before and after loaded with Ce6Fe(OH)3. c) Time-dependent evolution of dissolved oxygen concentration measured in solutions with UCS-Ce6-Fe(OH)3 (2.5 mg/mL) + H2O2 (500 μM), H2O2, and UCS-Ce6-Fe(OH)3, respectively. d) Plot of relative absorbance change of DPBF as a function of time indicating the rate of ROS generation at various conditions.

modification may also prevent detachment of Ce6/Fe(OH)3 from UCS in complex media with strong ionic strength. As a proof, we monitored the leakage of Ce6 from the nanocomposite dispersion in Dulbecco's modified eagle's medium (DMEM, containing electrolyte buffer and 10% fetal calf serum) by UV-vis absorption spectroscopy. Upon 24h of incubation, over 80% of Ce6 was found still attached to nanoparticles indicating good stability of the composite structure in biological media (Figure S6). The loading efficiency of Ce6 on the UCS-Ce6-Fe(OH)3 was optimized by monitoring the changes in featured Ce6 absorbance at 660 nm with various feeding concentration of Ce6. As shown in Figure 2a, the final absorbance of Ce6 reaches maximum when the weight ratio of Ce6 to UCS in the reactants increases to 1:0.3. It indicates a maximum loading capacity of ~0.9 wt% under our investigation (Figure S7). We then examined the optical properties of the UCS-Ce6Fe(OH)3 nanocomposites at the optimized Ce6 loading condition (1:0.3 w/w). Upon excitation with a continuous-wave (CW) diode laser at 808 nm, the nanoparticles exhibit three main upconversion luminescence peaks centered at around 408 nm, 540 nm, and 660 nm, respectively (Figure S8 and S9). These emissions can be ascribed to 4f-4f electron transitions of Er3+ through 2H9/2→4I15/2, 2H11/2/4S3/2 →4I15/2, and 4F9/2 →4I15/2, respectively (Figure S10).50 The emission at 660 nm overlaps with the absorption of Ce6, thereby enabling energy transfer from UCS to Ce6. This was confirmed by the observation of PL quenching and lifetime decreasing in UCS after loading with Ce6 (Figure 2b and Figure S15). The energy transfer efficiency was determined to be 29.6% by calculating the percentage change of the upconversion emission intensity at 662 nm. It is important to note that assembly of Fe(OH)3 does not affect the energy transfer process as Fe(OH)3 has no obvious absorption



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Figure 3. a) In vitro cell viability of 4T1 cells incubated with UCS-Ce6-Fe(OH)3 nanocomposites under dark condition at 37 oC for 12h and 24 h at different concentrations. b, c) CCK-8 assay of 4T1 cells after incubating at normoxia/hypoxia environment and treated by different methods. d) Fluorescence images of 4T1 cells indicating intracellular ROS generation. The cells were stained with DAPI (blue, nuclei) and DCFH-DA (green, ROS species) after hypoxia incubation and different treatments. The scale bar is 200 μm. e) Fluorescence images of 4T1 cells staining with calcein-AM (green, live cells) and propidium iodide (red, dead cells) after different treatments at hypoxia incubation. The scale bar is 100 μm. ***p < 0.001, **p < 0.01, or *p < 0.05.

in red wavelength region (600-700 nm; Figure S2). Besides, the UCS-Ce6-Fe(OH)3 nanocomposites do not afford significant overheating at 808 nm irradiation. In comparison with that in deionized water, the temperature rise of an aqueous dispersion containing 0.5 mg/mL UCS-Ce6-Fe(OH)3 was observed to increase by only ~1 oC after 10 min continuous laser irradiation at a relatively high photon flux of 4 W/cm2 (Figure S11). To demonstrate the feasibility of the Fe(OH)3-modifed UCS for therapeutics, we further determined the oxygen and ROS generation by adding the UCS-Ce6-Fe(OH)3 to H2O2 solution. As anticipated, the nanocomposites promoted the disproportionation of H2O2. The amount of dissolved oxygen in solution was found to gradually increase within 300 s, while it remained unaltered in control samples with only H2O2 or UCSCe6-Fe(OH)3 in the same period (Figure 2c). These results suggest the possibility of using Fe(OH)3-modified UCS to relieve tumor hypoxia. We next employed 1,3-diphenylisobenzofuran (DPBF) to verify the production of ROS.51-54 Without irradiation, we observed a gentle decline in DPBF absorbance at around 400 nm in solution containing H2O2 and UCS-Ce6-Fe(OH)3, which evidences the generation of ROS by the Fenton-like reaction (Figure S12). Under 808 nm-NIR irradiation (1 W), we observed that the reduction rate of DPBF drastically increased by about six times in comparison with the same sample under dark condition, due to efficient conversion of oxygen to reactive singlet oxygen through upconverted photo-sensitization (Figure 2d). By contrast, in the control samples without H2O2 or Fe(OH)3, the decay of DPBF was found negligible in the dark, and two times slower under the same irradiation (Figure 2d). We then investigated which types of ROS species were generated in the systems. We applied singlet oxygen sensor green (SOSG) to determine the generation of single oxygen.55 The SOSG assay confirm that 1O2 is one of the major ROS products for UCS-Ce6-Fe(OH)3 upon NIR irradiation (Figure S16a). We also conducted electron spin resonance (ESR)

spectra to identify the production of free radicals. 5,5-dimethyl1-pyrroline N-oxide (DMPO) was applied to capture short-lived free radicals, forming radical-adducts which could be detected by ESR.56,57 The ESR spectra indicate that Fenton-like reaction by UCS-Ce6-Fe(OH)3 produces only hydroperoxyl radicals (·OOH) under dark condition, while both hydroperoxyl and hydroxyl radicals (·OH) were yielded upon NIR irradiation (Figure S16b). Taken together, these results ambiguously validate the cooperative effect of Fe(OH)3 and UCS-Ce6 on light-triggered ROS production at H2O2 rich conditions. The cytotoxicity of the as-prepared UCS-Ce6-Fe(OH)3 nanocomposites to both 4T1 breast cancer cells and 293T human embryonic kidney cells was subsequently examined using a cell counting kit 8 (CCK-8). It was found that the asprepared nanocomposites do not induce clear negative effect to the cellular viabilities within 24 h at a concentration range of 0.01-0.5 mg/mL (Figure 3a and Figure S14). Besides, UCSCe6-Fe(OH)3 also show negligible hemolytic effect against red blood cells. These results suggest good biocompatibility of the nanocomposites for therapeutic applications (Figure S17). The in vitro anticancer effect of the nanocomposites was tested by the CCK-8 cell viability assay. When cultured at normoxia atmosphere, in the absence of H2O2, the phototoxicity of UCS-Ce6-Fe(OH)3 particles presented at a similar magnitude to that of UCS-Ce6 samples, which is attributed solely to the singlet oxygen sensitization of Ce6 molecules agitated by emission from the UCNPs. In the presence of exogenous H2O2 (100 μM), the UCS-Ce6-Fe(OH)3 particles was found to induce much stronger cell apoptosis because of enhanced ROS production by the Fenton-like reaction (Figure 3b). We further evaluated the therapeutic outcome in a hypoxia environment during cell culture. Notably, only the H2O2-incubated UCSCe6-Fe(OH)3 group maintained appreciable therapy efficacy post to laser exposure (Figure 3c). Without laser irradiation, the cell-killing efficacy was less than 25% for the same material even at a high H2O2 level (100 μM) and high particle


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Figure 4. a) Changes in body weight of mice with 4T1 breast tumor after treating with Control (PBS), NIR (PBS + 808 nm NIR irradiation), UCS-Ce6, UCSCe6-Fe(OH)3, UCS-Ce6 + 808 nm NIR irradiation, and UCS-Ce6-Fe(OH)3 + 808 nm NIR irradiation. b) Changes in relative tumor volume of mice with 4T1 breast tumor in response to various treatments. c) Corresponding tumor weights of mice in 14 days post the different treatments. d) Corresponding photos of treated mice and tumors in 14 days post the different treatments. e) H&E-stained tumor slices collected 24 h after laser treatment. The scale bars are 100 μm. (***p < 0.001, **p < 0.01, or *p < 0.05).

concentration (0.5 mg/mL). This finding suggests that the Fenton-like reaction by Fe(OH)3 does not induce considerable non-specific cytotoxicity in dark condition, therefore possibly resulting in less side effects during the therapy process. In addition, the in situ ROS generation was also identified by cell imaging using 2,7-dichlorofluorescin diacetate (DCFH-DA) as a fluorescence label. As shown in Figure 3d, when UCS-Ce6Fe(OH)3 particles were cultured with 4T1 cells, sole NIR irradiation or H2O2 presence may induce a certain degree of ROS content within cells. In contrast, when the cells were cultured with the particles and H2O2, after NIR irradiation a dramatically enhanced green fluorescence presented within all cells, indicating the promoted intracellular ROS production. This is induced by the combined effects from Fenton-like reaction of Fe(OH)3 compound and agitated photosensitizer. In consequence, UCS-Ce6-Fe(OH)3 particles showed the strongest in-vitro anticancer effect in all sample groups. The fluorescence staining examination of living/dead cells further verified the enhancement of in vitro therapeutic efficacy of UCS-Ce6Fe(OH)3 nanocomposites due to the promoted ROS induction, as expected (Figure 3e). Given the promising in vitro results, a series of examinations using 4T1 mouse tumor model were proceeded to validate the in vivo pharmacodynamics of UCS-Ce6-Fe(OH)3. In a typical experiment, the mice were intratumorally injected with PBS, UCS-Ce6 and UCS-Ce6-Fe(OH)3 particles, respectively, followed by 808 nm laser irradiation (1 W). The mice body weight, tumor volume were monitored in the following 14 days (Figure 4a and b). All of the mice were sacrificed and tumors were collected and weighted on day 14 (Figure 4c-d，Figure S13). It was found that all groups of mice remained at a similar body weight, indicating no clear acute toxicity of those nanoparticles. Compared with the tumors injected with pure PBS solution, those on mice treated with NIR alone or samples without NIR irradiation presented no clear delay in their growth.

For the tumor-bearing mice injected with UCS-Ce6 after NIR irradiation, the tumor growth was inhibited by certain degree. Significantly, for those treated with UCS-Ce6-Fe(OH)3 particles, the volume of tumor shrunk rapidly and remained at low dimensions within 14 days. H&E-stained microscopy images of tissue slices also revealed that the mice treated with UCS-Ce6-Fe(OH)3 nanoparticles suffered dramatically severe damages in tumor while tumor tissues from the other treatment schemes basically retained their regular morphology (Figure 4e).

4. CONCLUSIONS In summary, we have developed a combinatorial approach through the use of Fe(OH)3-modified UCNPs for enhanced photo- and chemodynamic therapy against hypoxia tumors. In our design, Fe(OH)3 nanoparticles not only act as an oxygen generator, but also produce hydroxyl radicals for promoting chemo- therapy effect through Fenton-like reaction. Taking advantage of the robust oxygen production by the Fe(OH)3/H2O2 reaction and the high penetration depth of 808 nm excitation offered by the UCNPs, we demonstrate NIRtriggered in vivo inhibition of tumor growth in living animal models with significantly improved efficacy. This study establishes a smart material platform suited not only for theranostic treatment of cancers with unfavorable microenvironment, but also for targeted diagnosis or energy conversion applications when one considers the potential magnetic and catalytic properties of ferrite nanomaterials.58

ASSOCIATED CONTENT Supporting Information



The Supporting Information is available free of charge on the ACS Publications website. Experimental procedures, Figures S1−S17. (PDF)

AUTHOR INFORMATION Corresponding Author [email protected] [email protected]

Notes The authors declare no competing financial interests.

ACKNOWLEDGMENT This work was financially supported by the National Nature Science Foundation of China (51672247, 51872256), the ‘111’ Program funded by Education Ministry of China and State Administration of Foreign Experts Affairs (B16043), the Major State Research Program of China (2016YFC1101900), the National Key Research and Development Program of China (2018YFB0703803), and the Fundamental Research Funds for the Central Universities (2017QNA4010).

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Ferric Hydroxide-Modified Upconversion Nanoparticles for 808 nm

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