Hydrothermal Galvanic-Replacement-Tethered Synthesis of Ir–Ag

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Hydrothermal Galvanic-Replacement Tethered Synthesis of Ir-Ag-IrO Nanoplates for Computed Tomography-Guided Multi-Wavelength Potent Thermodynamic Cancer Therapy 2

Gyeonghye Yim, Seounghun Kang, Young-Jin Kim, Young-Kwan Kim, Dal-Hee Min, and Hongje Jang ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.8b09516 • Publication Date (Web): 12 Mar 2019 Downloaded from http://pubs.acs.org on March 13, 2019

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Hydrothermal

Galvanic-Replacement

Tethered

Synthesis of Ir-Ag-IrO2 Nanoplates for Computed Tomography-Guided

Multi-Wavelength

Potent

Thermodynamic Cancer Therapy Gyeonghye Yim1‡, Seounghun Kang2‡, Young-Jin Kim4, Young-Kwan Kim4, Dal-Hee Min2,3* and Hongje Jang1*

1Department of Chemistry, Kwangwoon University, 20, Gwangwoon-ro, Nowon-gu, Seoul 01897,

Republic of Korea 2Center

for RNA Research, Institute for Basic Science (IBS), Seoul, 08826, Republic of Korea.

3Institute

of Biotherapeutics Convergence Technology, Lemonex Inc., Seoul 08826, Republic of

Korea 4Carbon

Composite Materials Research Center, Institute of Advanced Composite Materials, Korea

Institute of Science and Technology, San 101, Eunha-ri, Bongdong-eup, Wanju-gun, Jeollabuk-do 565-905, Republic of Korea

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KEYWORDS. cancer therapy, galvanic replacement, photothermal conversion, photodynamic therapy, iridium

ABSTRACT

Beyond the synthesis of typical nanocrystals, various breakthrough approaches have been developed to provide more useful structural features and functionalities. Among them, galvanic replacement, a structural transformation reaction accompanied by constituent element substitution, has been applied to various areas. However, the innovative improvement for galvanic replacement needs to be considered because of the limitation of applicable element pairs to maintain structural stability. To expand the boundary of galvanic-replacement-mediated synthesis, we have become interested in the Group 9 metallic element Ir, which is considered a fascinating element in the field of catalysis, but whose size and shapes regulation has been conventionally regarded as difficult. To overcome the current limitations, we developed a hydrothermal galvanic-replacement tethered synthetic route to prepare Ir-Ag-IrO2 nanoplates (IrNPs) with a transverse length of tens of nanometers and a rough surface morphology. A very interesting photoreactivity was observed from the prepared IrNPs, with Ag and IrO2 coexisting partially, which showed photothermal conversion and photocatalytic activity at different ratios against extinction wavelengths of 473, 660, and 808 nm. The present IrNPs platform showed excellent photothermal conversion efficiency under nearinfrared laser irradiation at 808 nm and also represented an effective cancer treatment in vitro and in vivo through a synergistic effect with reactive oxygen species (ROS) generation. In addition,

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computed tomography (CT) imaging contrast effects from Ir and IrO2 composition were also clearly observed.

Noble metal nanoparticles, including gold, silver, platinum, and others, have been extensively applied to various fields owing to their attractive physico-chemical properties and characteristics derived from the limiting dimensions in the nanometer scale.1-3 In addition to such applications as chemical4,5 and electrochemical catalysts,6-8 environmental applications,9,10 and signal amplification for sensors,11,12 they have also been widely applied in the biomedical field, including diagnosis13-15 and therapy,16-18 owing to their excellent resistance to corrosion, biocompatibility,19 and fascinating optical properties such as localized surface plasmon resonance (LSPR).20,21 In recent years, numerous challenges for interdisciplinary potential have continued beyond the conventional research areas where each noble metal nanoparticle has been applied, such as the following representative examples, namely, Pd,22,23 Pt,24-26 and Rh nanoparticles,27 which are used for cancer treatment. One of the least abundant noble metal elements, iridium (Ir) has been used in the international prototype meter and kilogram measurement units since it was first reported in 1804 until the middle of the 20th century owing to its extremely superior anticorrosion properties.28,29 Despite its high price and processing difficulties, Ir alloy electrodes30,31 and organoiridium compounds32,33 have attracted great interest in the academic and industrial fields owing to their potential for oxygen-evolution reaction and organometallic catalysis.34-36 In addition, an Ir oxide (IrO2) with semiconducting behavior has been known to exhibit excellent photocatalytic efficiency.37,38 Recently, numerous attempts have been made to achieve a high surface-to-volume

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ratio by synthesizing Ir and IrO2 nanoparticles. Although synthetic methods for Ir and IrO2 nanoparticles have been continuously studied, their size and morphology have been restricted to 660 nm > 808 nm, and the photodegradation was also found to follow the same order of efficiency. (Figures 3c,d) Highly efficient photocatalytic activity at 473 nm and 660 nm was originated from the electron-hole pair photogeneration from light irradiation

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exceed the band gap energy of semiconducting IrO2 portion. On the other hand, in case of 808 nm NIR irradiation with the relatively lower energy, photocatalytic activity was induced by injection of hot plasmonic electrons generated from metallic Ir-Ag into the conduction band of IrO2. Observed less efficient photocatalytic activity under NIR than that of the visible-light irradiation condition should be originated from the charge recombination by rapid back transfer by Ir-Ag-IrO2 alloy composition.47 Similar to the case of general photocatalyst, not only 1O2 but also various radicals such as hydroperoxyl (·HO2), superoxide anion (·O2-), and hydroxyl (·OH), were found. Interestingly, a simple comparison based on the temperature-elevation ratio (473 nm:660 nm:808 nm = 0.286:0.322:1.000) and MB decomposition ratio (473 nm:660 nm:808 nm = 1.000:0.893:0.252) at 480-J irradiation demonstrated an inverse relationship between the efficiencies obtained at the respective wavelengths, which proved that the total amount of irradiated energy was distributed to each relaxation pathway. Prior to the confirmation of the photothermal therapy (PTT) and photodynamic therapy (PDT) efficiency at the cellular level, a cell-viability assay was performed to optimize the treatment concentration in which no IrNPs cytotoxicity was solely observed. Human cervical cancer cells HeLa were treated with various concentrations of IrNPs and TAT functionalized IrNPs (TAT-IrNPs, TAT: cell penetrating peptide transactivator of transcription from human immunodeficiency virus), followed by short (1 day)- and long (14 days)- term incubation. Viability tests were performed by using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). According to relative cell viability result, both IrNPs and TAT–IrNPs showed negligible cytotoxicity less than 1.25 mg Ir/L concentration. (Figures S12 and S13) Several additional experiments have confirmed that TAT peptide modification was essential for intracellular

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nanoparticle delivery and that colloidal stability, photothermal conversion, and photocatalytic activity were equally observed from TAT-IrNPs. (Figures S14–S17) For the visual confirmation of the cytotoxicity of the IrNPs derivatives and the effect of PTT and/or PDT under the laser wavelength-dependent irradiation, calcein AM/ethidium homodimer-1 live/dead staining and fluorescent microscopic observation were performed after various experimental treatments of HeLa cells. In the PTT- and PDT-alone cases and the combination treatment of active cells using TAT–IrNPs, significant cell ablation was observed from the designated region compared with that in the case where dead cells were absent under conditions of untreated cells (control), IrNPs-derivative-only treatment, laser-only irradiation, and cellular-internalization unfavorable IrNPs with laser irradiation. (Figures 3e and S18) Similar to the trends observed in the temperature-elevation and photocatalytic-activity verification, the PTTonly condition using L-histidine as a singlet-oxygen scavenger demonstrated efficiency on the order of 473 nm < 660 nm < 808 nm, and the PDT-only condition at 4 C temperature equilibrium followed the efficiency order 473 nm > 660 nm > 808 nm. In the combined treatment that simultaneously used PTT and PDT, effective cancer-cell ablation was confirmed in all three wavelengths. (Figures 3e and S19) These results demonstrated that TAT–IrNPs, which can be applied to PTT and PDT with split efficiency, provided a more convenient and effective process in combinational cancer phototherapy than the conventional methods in which additional photocatalysts or different wavelengths of light must be combined to achieve PTT and PDT effects. Since the dominance of type I (ROS-mediated PDT) and type II (singlet-oxygen-mediated PDT) remains controversial, we further verified the PDT efficacy of TAT–IrNPs on HeLa cells using various scavengers, including L-histidine (singlet-oxygen scavenger), sodium pyruvate (H2O2 scavenger), dimethyl sulfoxide: hydroxyl radical scavenger), and manganese (III)

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tetrakis(4-benzoic acid)porphyrin chloride): superoxide anion radical scavenger) for each mechanism, in combination with PTT. In the 473- and 660-nm irradiation where PDT was relatively dominant compared with PTT, the type-II pathway blockage effectively inhibited cell apoptosis, and the type-I pathway was less effective. At 808 nm where PTT was relatively dominant, the overall cell viability was reduced by hyperthermic apoptosis, but a tendency for scavenger use was equally observed. In summary, PDT that used the TAT–IrNPs appeared to more effectively work with the type-II pathway in the proposed platform. (Figure 4) The sufficient intracellular generation of ROS was characterized by using indicator carboxy-H2DFFDA staining after laser irradiation. According to the subsequent fluorescent microscopic observation, the TAT–IrNPs demonstrated singlet oxygen and ROS in all three wavelengths but the IrNPs did not because of the unfavorable internalization. (Figure S20) To identify the intracellular responses of PTT and PDT, we additionally tested the combined therapeutic-efficiency against various intractable cancer-cell lines, including MDAMB-231 (triple-negative breast cancer cell), HepG2 (human hepatocarcinoma), A375P (melanoma), and HeLa. After the treatment using TAT–IrNPs and following laser irradiation at 473-, 660-, and 808-nm wavelengths, the cell viability and intracellular mRNA expression levels of the heat-stress resistance factors (HSP 70 and HSP 90)48,49 and oxidative stress resistance factors (HIF-1 and NQO-1)50,51 were measured by using the reverse transcription polymerase chain reaction (RT-PCR). (Figure 5) According to the RT-PCR data, the relative expression level change in HSP 70 and 90 significantly increased in MDA-MB-231 because of the PTT-dominant 808-nm irradiation, but the oxidative-stress resistance factors exhibited a negligible increase by the PDT-dominant 473- and 660-nm laser irradiation compared with all the other cell lines. On the

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other hand, A375P showed sufficient increase in HIF-1 and NQO-1 at 473 and 660 nm but with lesser increase in the PTT factors at 808 nm. The overall tendency to increase the PTT and PTT resistance factors by heat and oxidative stress in these cell lines was consistent with that of the MTT assay results. (Figure S21) The results indicated that a cancer cell with a higher expression of heat-stress resistance and oxidative-stress factors were directly correlated to the resistance to the actual PTT and PDT therapeutic effects, which depended on the cell lines. One of the limitations in the current cancer therapy could be considered as the difficulty of finding an optimal treatment method because of the unclear resistance factors of each cancer type. Since the present platform can provide simultaneous but fractionally dominant PTT and PDT, we can possibly achieve the most suitable cancer-treatment method by simple modulation of the laser-wavelength irradiation. Because the liver and kidney are generally regarded as the most important toxicological organs against substances from outside the body, including harmful compounds or nanomaterials, we performed toxicology profiling using blood samples of TAT–IrNP-injected mice at time points of 0, 10, and 20 days. A standard liver-toxicology test against hepatocyte enzyme alanine aminotransferase and liver-tissue enzyme aspartate aminotransferase (AST) revealed that no significant increase in the error range occurred. (Figures 6a,b) The absence of toxicity or renal damage to the kidney was confirmed by blood urea nitrogen and creatine concentration measurements as a standard kidney toxicology test. (Figures 6c,d) The total serum protein levels and lactate dehydrogenase data, which were similar or slightly lower than those of the 1× PBSinjected control-mouse test, also indicated that the TAT–IrNPs are non-cytotoxic. (Figures 6e–f) The red-blood cell, white-blood cell, and platelet counts represented the short-term and spleen toxicity, immune and inflammatory responses, and internal bleeding, respectively. According to

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the blood-cell count results, no significant difference was observed between the control and TAT– IrNPs injected group in all targets. (Figures 6g–i) Both the IrNPs and TAT–IrNPs did not induce hemolysis under all tested concentrations, and no significant adverse effect on the mouse body weight was observed. (Figures 6j,k) For in vivo CT imaging and cancer therapy, HeLa cells (6 × 106 cells/100 L 1× PBS) were subcutaneously injected into five-week old BALB/c nude male mice to prepare for tumor xenograft. After the tumor xenograft volume reached ~100 mm3, 1× PBS (control: 100 L) and TAT–IrNPs dispersed in 1× PBS (experimental: 2 mg Ir/L; 100 L) were separately injected by using tail-vein intravenous injection. CT imaging was performed after 24 h from the TAT–IrNPs injection for sufficient blood circulation and accumulation in the tumor tissue. An increase in the contrast from the CT image of the cross section (Hounsfield unit (HU) = 10.6) was observed. When compared with the HU trend line of the TAT–IrNPs, approximately 0.13 mg Ir/L particles, which are suitable for cancer thermo-dynamic therapy without cytotoxicity, were successfully delivered. (Figures 7a and S22) The functionality of IrNPs as a CT contrast agent resulted from the IrO2 and Ir composition with high atomic number (Z = 77) and K-edge (71 keV). The three-dimensional CT image also clearly showed the tumor accumulation of TAT–IrNPs and the feasibility of imaging-guided therapy. To confirm the feasibility of the combined PTT/PDT in vivo cancer treatment, in addition to the CT imaging, 808-nm diode laser (2 W/cm2 for 3 min) irradiation was performed. (Figure 7b) Typical experiments were performed using the 808-nm laser only to confirm the effective in vivo therapeutic effect by considering the excellent skin penetration of NIR wavelength. In the control (untreated), NIR-irradiation only treatment, and TAT–IrNPs injection, the mice were

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observed to grow to approximately 10 times larger volume without any therapeutic effect. On the other hand, in the PDT- and PTT-only treatment cases, the treated mice showed a partial inhibition of six and two times volume growth, respectively, in the measurement after the 20-day incubation. The combined PTT and PDT conditions showed excellent therapeutic efficiency as the growth of the tumor tissue was completely suppressed, and no further growth was observed during the whole period. (Figures 7c,d) After confirming the trend by directly comparing the size of the extracted tumor tissue, sucrose-infiltrated sectioning and subsequent hematoxylin and eosin (H&E) staining were performed to verify the tumor apoptosis and toxic side effect on major organs. (Figures 7e,f) Terminal deoxynucleotidyl transferase dUTP nick and labeling (TUNEL) and Ki-67 assay on the major organs and tumor tissues harvested from the mice 20 days after treatment provided detailed information of cancer treatment feature. According to the fluorescent microscope images, the major organs (heart, lung, liver, spleen, and kidney) did not show signs and implied rack of apoptotic cells due to cytotoxicity of the TAT–IrNPs (Figure S23). In contrast, the variously treated tumor tissues exhibited exactly the same therapeutic tendency in terms of the tumor-volume measurement. (Figure 7e) Ki-67, which is regarded as the reference for cell proliferation, exhibited a generally reported trend in the major organs from the 1× PBS and TAT–IrNPs treatment due to their non-cytotoxicity. (Figure S24) In the case of the tumor tissue, strong fluorescence was observed from the control-mouse tissue, and it decreased in the PTT- and/or PDT-treated tissues. (Figure 7e) The current TAT–IrNPs were believed to have accumulated in the cancer tissue through passive targeting due to the enhanced permeability and retention (EPR) effect because no targeting ligand was present for the specific organs or tissues.52 For further verification of the biodistribution of the TAT–IrNPs, we fluorescently labeled the TAT peptide using Cy5, and the organs were

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extracted after 24 h from the Cy5–TAT–IrNPs injection to obtain a fluorescent image. Most fluorescent signals were observed from the liver and tumor tissue, and signals were also detected from the lung, kidney, and spleen. The overall tendency was the same as that of the passive targeting due to the EPR effect (which is commonly known), and we confirmed that the required amounts of nanoparticles for cancer treatment can be delivered to the tumor tissue. (Figure S25) Inductively coupled plasma mass spectrometry (ICP-MS) analysis also supported the biodistribution of TAT-IrNPs against major organs and tumor tissue. (Figure S26) Taken together, TAT–IrNPs can be delivered to a tumor tissue via a passive targeting manner, and subsequent combined PTT/PDT treatment can guarantee successful in vivo cancer therapy.

CONCLUSION The formation of Ir nanostructures, which is difficult to accomplish with AgNPs template, were achieved by hydrothermal galvanic-replacement tethered synthesis, which resulted in reproducible manufacturing of IrNPs with rough surface morphology. The synthesized IrNPs exhibited biocompatibility comparable to biocompatible AuNPs, which are widely used in biomedical applications, and also showed PTT and/or PDT effects at various wavelengths. In particular, the PTT and PDT efficiencies had different ratios, depending on the irradiated wavelength—PDT was more effective in shorter wavelengths (473 and 660 nm), and PTT was dominant with extremely high efficiency (81.7%) in longer wavelength (808 nm). As a result, excellent in vitro and in vivo cancer-cell ablation was successfully accomplished through the combined effect of PTT and PDT. We believe that the present study presents a brilliant idea in both material chemistry and biomedical application areas, and it is a versatile method of

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synthesizing conventionally unfavorable nanoparticles by controlling the size and shapes. Moreover, as an example of a proposed application for cancer treatment, we are confident that this method can be applied to the fields that have not yet been considered before.

EXPERIMENTAL Materials. Silver nitrate, hydrogen peroxide (30%), trisodium citrate dihydrate, and sodium borohydride were purchased from Junsei (Tokyo, Japan). 3-(4,5-Dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT), L-ascorbic acid, citric acid, poly(vinylpyrrolidone) (Mw 29 kDa), ethylene glycol (EG), dimethylsulfoxide (DMSO), In situ cell death detection kit, fluorescein, and iridium (III) chloride hydrate were purchased from Sigma (St. Louis, MO, USA). 10X phosphate buffered saline (PBS), Dulbecco’s modified eagle’s medium (DMEM), fetal bovine serum (FBS), Roswell Park Memorial Institue 1640 medium (RPMI 1640), PenicillinStreptomycin, and 0.05% Trypsin-EDTA were purchased from WelGene (Seoul, Korea). LIVE/DEAE viability/cytotoxicity assay kit, Singlet oxygen sensor green (SOSG), methylene blue, ROS sensing reagent, and cell lysis solution Trizol were purchased from Molecular Probes Invitrogen (Carlsbad, CA, USA). Carboxyfluorescein (FAM) labeled thiolated-oligonucleotide (5`-FAM-AATGGGGAGGCTAGCTACAACGAGGCTTTG C-3`-SH) was purchased from Genotech (Seoul, Korea). DeadEndTM fluorometric TUNEL assay kit was purchased from Promega (Madison, WI, USA). All PCR reagents were purchased from TaKaRa Bio Inc. (Shiga, Japan). All PCR primers were purchased from Cosmogenetech (Seoul, Korea). All chemicals were used as received.

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TAT peptide (CGGYGRKKRRQRRR) was synthesized by solid-phase peptide synthesis (SPPS) method.

Preparation of AgNPs AgNPs were prepared by previously reported seed-mediated growth method. Briefly, 1.5 mL of 3.5 mM PVP (Mw 29 kDa), 250 L of 10 mM AgNO3, 300 L of 30 mM Cit, and 24.75 mL of DI water were added into 50 mL glass vial. Then, 60 L of 30% H2O2 and 250 L of 100 mM NaBH4 were added in order under magnetic stirring at ambient condition. The mixture was allowed to be incubated for 3 h for the formation of Ag nanoseeds by pale blue color emerging and for the depletion of NaBH4. To the prepared Ag nanoseeds, 1 mL of 100 mM L-AA and 330 L of 75 mM Cit were sequentially added without any purification steps for the following growth. To the mixture, 13 mL of separately prepared growth solution which containing 13 mL of 1 mM AgNO3 and 80 L of 100 mM citric acid was added by 0.2 mL/sec. Along with the addition of growth solution, the solution color changed into deep-blue by AgNPs formation. Manufactured

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AgNPs should be used as a template for following galvanic replacement reaction freshly without any purification.

Hydrothermal galvanic replacement tethered synthesis of IrNPs The preparation of IrNPs were accomplished by hydrothermal reaction by using Teflon vessel stainless autoclave. To the Teflon vessel, 20 mL of as-synthesized AgNPs and 3 mL of 10 mM Ir3+ solution were added and mixed with gently pipetting for several times to ensure the formation of homogeneous solution. Then, the autoclave was placed on the pre-heated at 200 oC hot-plate and incubated for 1 h without magnetic stirring. When the reaction finished, naturally cooled IrNPs solution was gathered and purified by centrifugation at 9000 rpm for 10 min with DI water washing for more than three times. Finally, synthesized IrNPs were re-dispersed in 10 mL of DI water, as concentration of 1 mg Ir/L characterized by ICP-MS.

Characterizations of IrNPs Size and morphology of IrNPs were characterized by energy-filtering transmission electron microscope LIBRA 120 (Carl Zeiss, Germany), field-emission scanning electron miscroscope AURIGA (Carl Zeiss, Germany), and Tecnai F20 TEM (FEI, USA). X-ray diffraction was measured by D8-Advance (Bruker Miller Co., USA), and X-ray photoelectron spectroscopy was measured by AXIS-His (KRATOS, UK). UV-Vis spectrophotometer Lambda 465 (PerkinElmer, USA) and SynergyMx (Biotek, UK) were used to obtain UV-Vis-NIR extinction spectra. Dynamic light scattering and -potential were measured by Zetasizer Nano ZS (Malvern, UK). 473 nm and

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660 nm laser irradiation were performed by SOLC laser (Shanghai Laser & Optics Century Co., Ltd., China) and 808 nm irradiation was performed by OCLA surgical laser accessories (Soodogroup Co., Korea), respectively. Cell images were taken using an In-cell analyzer 2000 (GE healthcare, USA), Ti-inverted fluorescence microscope (Nikon Co., Japan), and a CoolSNAP cf charge-coupled device (CCD) camera (Photometrics, USA) with Metamorph image analysis software (Molecular Devices, USA). The Hounsfield units in test tubes and tumor were measured by IVIS SpectrumCT In Vivo Imaging System (PerkinElmer, USA). In vivo thermography images were taken with a FLIR one pro (FLIR System, USA). PCR was performed by T100 thermal cyclers (BIO-RAD, USA).

TAT peptide loading on IrNPs (TAT-IrNPs) 20 L of 1 M TAT peptide in DI water stock solution was added to 1 mL of 0.5 mg Ir/L IrNPs at room temperature. The mixture was allowed to be incubated for 12 h on a horizontal shaker at 180 rpm at ambient condition. The unbound TAT peptides were removed by centrifugation at 7000 rpm for 15 min and washed with DI water at least three times to ensure purification. Finally, TAT-IrNPs were re-dispersed in 1 mL of 1x PBS and characterized by DLS and zeta-potential for further applications.

Characterization of photothermal conversion effect in cuvette Photothermal conversion efficiency was confirmed by temperature elevation under the 473, 660, and 808 nm laser irradiation for various concentrations of IrNPs and TAT-IrNPs. 1 mL of

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0.125, 0.063, and 0.031 mg Ir/L concentration IrNPs and TAT-IrNPs were placed and 473 nm (100 mW/cm2), 660 nm (200 mW/cm2), and 808 nm (2 W/cm2) diode laser was irradiated to each sample with total energy of 480 J. Temperature change was observed by digital thermometer in every 120 J irradiation points.

Characterization of photocatalytic activity in cuvette For type one ROS sense, one microliters of 5 mM SOSG in methanol was added to 1 mL of IrNPs (0.125 mg Ir/L) in 1xPBS. The solution was irradiated with 473, 660, and 808 nm laser separately at 4 oC with sealing for preventing the solution form evaporating. The SOSG fluorescence intensity of the solution was measured every 120 J of each laser output and measured until it reaches 480 J. Fluorescence was measured on Ex: 504 nm and Em: 525 nm using a plate reader. For type two ROS, one microliters of 10 mM methylene blue in DI water was added to 1 mL of IrNPs (0.125 mg Ir/L) in 1xPBS. The laser was irradiated to the solution under the same conditions as for type one ROS sensing. The absorbance of methylene blue was measured every 120 J of each laser output and measured until it reaches 720 J. The absorbance was measure on 665 nm using UV-Vis spectrometer.

Cytotoxicity measurement MTT powder was dissolved in 1x PBS at 5 mg/mL concentrations, then filtered through sterilized syringe filter (0.2 m pore diameter). Prepared MTT stock solution was

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stored at 4 oC. HeLa Cells were seeded with a density of 10,000 cells per well of a 96-well culture plate with 100 L of growth media (50-70% confluency). The cells were treated with appropriate concentrations of IrNPs and TAT-IrNPs in serum-containing culture media and incubated for 24 h at 37 oC incubator. After the incubation, the cells were rinsed with 1x PBS twice, then 100 L of serum free media with 0.5 mg/mL concentrations of MTT was added followed by additional incubation of 2 h until purple color developed to detect the metabolically active cells. The media was discarded and the cells were rinsed with 1xPBS once to remove remaining MTT. Finally, 100 L of DMSO was added to each well to dissolve water insoluble formazan salt. The optical densities of each well in the culture plate were measured at 560 nm wavelength. Mean and standard deviation of triplicated were calculated and plotted.

Cell based PDT and/or PTT ablation To demonstrate the PDT effect of IrNPs in vitro, 100 L of TAT-IrNPs (0.125 mg Ir/L) in serum-free cell media was treated with HeLa cells in 96-well plates that were seeded with confluency of 10,000 cells/well. After 6 h of incubation in a humidified 5% CO2 incubator at 37 oC,

the serum-free cell media was removed and washed with 1xPBS twice, followed by replacing

the serum-containing cell media. Then, the HeLa cells were treated by 240 J of 473, 660, and 808 nm laser at 4 oC to prevent the cells from death by photothermal therapy and incubated for an additional 12 h. After then, the cells were rinsed with 1xPBS twice and incubated for 2 h with 100 L of 0.5 mg/mL concentrations of MTT in serum-free cell media. Finally, 100 microliters of DMSO was added to each well to dissolve water insoluble formazan salt.

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To confirm the photothermal therapeutic effect of IrNPs, 100 L of TAT-loaded IrNPs (0.125 mg Ir/L) in serum-free cell media was treated with HeLa cells in 96-well plates that were seeded with confluency of 10,000 cells/well. After 6 h of incubation in a humidified 5% CO2 incubator at 37 oC, the particle containing cell media was removed and washed with 1xPBS twice, followed by replacing 100 L of L-histidine (20 mM), sodium pyruvate (10 mM), DMSO (1%), and MnTBAP (100 M) (L-histidine is singlet oxygen(1O2) scavenger, Sodium pyruvate is hydrogen peroxide (H2O2) scavenger, DMSO is hydroxyl radical (HO·) scavenger, and MnTBAP is superoxide anion (·O2-) scavenger) in serum-containing cell media and incubated for 1 h. Then, the HeLa cells were treated by 240 J of 473, 660, and 808 nm laser at room temperature. Subsequently, the ROS scavenger containing cell media was removed and washed 1xPBS twice, followed by replacing the serum-containing cell media. The subsequent procedure was the same as the photodynamic therapeutic effect confirmation experiment. To confirm the photodynamic and photothermal combination therapeutic effect, 100 L of 0.125 mg Ir/L TAT-IrNPs in serum-free cell media was treated with the cells that were seeded with confluency of 10,000 cells/well. After 6 h of incubation in a humidified 5% CO2 incubator at 37 oC, the serum-free cell media was removed and washed with 1xPBS twice, followed by replacing the serum-containing cell media. Then, the HeLa cells were treated by 240 J of 473, 660, and 808 nm laser at room temperature. The subsequent procedure was the same as the photodynamic therapeutic effect confirmation experiment. The optical densities of each well in the plate was measured at 560 nm. Mean and standard deviation of triplicates were calculated and plotted.

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Reactive oxygen species (ROS) detection in cells The nanoparticles in serum free culture media were treated to HeLa cells in 12-well plate that were seeded with confluency of 80,000 cells/well. After 6h incubation in humidified 5% CO2 incubator at 37 oC to ensure nanoplate internalization, remaining nanoplates were removed and washed with 1x PBS twice, followed by media replacing with serum-containing culture media. Then, HeLa cells were treated by 808 nm NIR diode laser irradiation with 4 W/cm2 for 5 min and allowed to be incubated for additional 1 h. For ROS assay, 500 L of 10 M 5-(and-6)-carboxy2’, 7’-dihydrofluorescein diacetate (Carboxy-H2DFFDA) in 1x PBS was added to each well and incubated for 30 min. Fluorescence images of the cells were obtained by using a fluorescence microscope.

HSP 70, HSP 90α, HIF-1α, and NQO-1 mRNA expression level (PCR) 500 L of 0.125 mg Ir/L TAT-IrNPs in serum-free cell media was treated with the MDAMB-231, HeLa, HepG2, and A375P cells in 24-well plates that were seeded with confluency of 50,000 cells/well. After 6 h of incubation in a humidified 5% CO2 incubator at 37 oC, the serumfree cell media was removed and washed with 1xPBS twice, followed by replacing the serumcontaining cell media. Then, the cells were treated by 180 J of 473, 660, and 808 nm laser at room temperature. After 6 h again, the total RNA was extracted using Trizol. The RT-PCR was performed according to the thermo cyclic condition below. cDNA synthesis: 1 cycle 65 oC for 5 min, 42 oC for 2 min, 42 oC for 50 min, inactivation at 70 oC for 15 min. Amplification: 28 cycles at 95 oC for 30 sec, at 54 oC for 60 sec, and at 72 oC for 30 sec.

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Hemolysis assay 1 mL of mouse whole blood was added to 14 mL 1x PBS and centrifuged 9,500 rpm for 5 min. Washing process was repeated by 5 times. The washed red blood cells were dispersed in 15 mL 1x PBS. Add 0.2 mL of red blood cell solution to 0.8 mL of TAT-IrNPs in 1x PBS and the nanoparticles-red blood cell mixture was incubated on horizontal shaker at 90 rpm (room temperature in dark place). Hemolysis assay positive control and negative control were accomplished by DI water, and 1x PBS, respectively. After 4 h, the mixture was centrifuged at 9,500 rpm for 3 min. 0.2 mL of supernatant from the mixture was transferred to a 96 well plates then characterized by absorbance measurement of hemoglobin at 577 nm and reference at 655 nm.

Toxicology profile Mouse blood was collected during the sacrifice process. Whole blood was collected using EDTA tube and serum was separated using serum-separating tube. Toxicity profile (AST, ALT, BUN, Creatinine, Total protein, Lactic acid dehydrogenase, red blood cells, white blood cells, and platelets) was measured using a FUJI DRI-CEHM FDC3500 and Hemavet 950.

In vivo PTT/PDT combinational therapeutic efficiency test All animal experiments were carried out in compliance with the Institutional Animal Care and Use Committees (IACUC) of Seoul National University. Balb/c nude male mice (5-week old)

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were purchased from ORIENT BIO (Sungnam-si, Korea). Mouse tumor model was prepared by subcutaneous injection of HeLa cells (6 x 106 cells) in 100 L sterilized 1xPBS solution (n = 4). When the size of the tumor is ~100 mm3, 100 L of TAT-IrNPs in 1xPBS (4 eq.) and 1xPBS (as control) were injected into the tail veins of the mouse tumor model. After 24 hr of injection, the mouse tumor were treated by 808 nm NIR diode laser irradiation with 2 W/cm2 for 3 min for demonstrate of IrNPs’ anti-cancer phototherapeutic effect. For PTT only, 100 microliters of Lhistidine (20 mM) in 1xPBS was injected into the tumor before laser irradiation. For PDT only, the laser was irradiated for 20 sec and the resting for 40 sec was repeated to prevent the tumor from raising the temperature and causing apoptosis. The tumor sizes were measured every two days by using the equation of 1/2 x longest diameters x (shortest diameters)2.

Histological evaluation Histological samples were obtained after 24 days of NIR irradiation. Harvested samples from heart, lung, liver, spleen, kidney, and tumor were placed in 4% PFA solution. After sucrose infiltration, the samples were embedded in optimal cutting temperature (OCT) compound and sectioned of 10 micrometers. For H&E staining, the sectioned samples were processed by using H&E staining (BBC Biochemical, Mt Vernon, WA, USA). For TUNEL assay, the sectioned samples were washed with 1xPBS for 30 min and incubated in permeabilization solution (0.1% Triton X-100, and 0.1% Sodium citrate) for 2 min at 4℃. 50 microliters of tunel mixture solution was added on the sectioned samples and incubated in humidified chamber for 60 min at 37℃ in dark place. The sectioned was washed three times with 1xPBS and stained with DAPI. For Ki-67 staining, the sectioned samples were washed with 1xPBS and incubated in the permeabilization

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solution (0.2% tween 20 in 1xPBS) twice for 10 min each. The samples were incubated with blocking solution (5% normal goat serum, 0.2% tween 20 in 1xPBS) for 45 min in humidified chamber. The sectioned samples were incubated with primary antibody diluted solution (2% normal goat serum, 0.2% tween 20 in 1xPBS) (dilution 1:100, anti-human is Santa Cruz, and antimouse is Abcam) in humidified chamber for 3 h at room temperature. The samples were washed 3 times for 10 min in a permeabilization solution and incubated with secondary antibody diluted solution (2% normal goat serum, 0.2% tween 20 in 1xPBS) for 2 h at room temperature. The samples were washed with permeabilization solution and stained with DAPI. The samples were observed under a BX71 microscope with 20x objective lens (Olympus, Tokyo, Japan)

AUTHOR INFORMATION Corresponding Author *Dal-Hee Min Phone: +82-2-880-4338. E-mail: [email protected] *Hongje Jang Phone: +82-2-940-8320. E-mail: [email protected]

ACKNOWLEDGMENT This work was supported by National Research Foundation of Korea (NRF) funded by Korean government (Grant No. NRF-2016R1C1B1008090 and NRF-2019R1C1C1002305). This work was supported by the Basic Science Research Program (2016R1E1A1A01941202 and

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2016R1A4A1010796), the International S&T Cooperation Program (2014K1B1A1073716), and the Research Center Program of IBS (IBS-R008-D1) through the NRF. The present research has been conducted by the Research Grant of Kwangwoon University in 2019.

AUTHOR CONTRIBUTION ‡ G. Yim and S. Kang contributed equally.

SUPPORTING INFORMATION Supporting information contains the characterizations of detailed optimization steps including Ag nano-template, EF-SEM with EDS mapping, HR-TEM image, XPS survey spectrum, photothermal temperature elevation with different concentration of IrNPs and TAT-IrNPs, colloidal stability, cytotoxicity, singlet oxygen generation, control in vitro cellular images, Hounsfield unit plotting in cuvette system, organ fluorescence images and ICP-MS data. Supporting information is available online.

COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests.

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Figure 1. Preparation of IrNPs by hydrothermal galvanic-replacement tethered synthetic method. (a) Schematic illustration of the IrNPs synthesis and application to cancer treatment. (b) UV–Vis spectrum against increase of added Ir3+ amount and (c) detailed feathers. The notation i to x implies

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the Ir/Ag molar ratio of 1.2, 2.3, 3.5, 4.7, 5.8, 7.0, 8.1, 9.3, 10.5, and 11.6, respectively. (d) TEM images of the above-listed synthetic conditions. The scale bar is 100 nm.

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Figure 2. Characterization of the synthesized IrNPs. (a) The XPS and (b) XRD spectra of the IrNPs exhibit co-existence of alloyed Ag from the template composition, Ir0, and Ir4+ due to replacement reaction. (c) The HR-TEM with FFT image and (d) HAADF-STEM/EDS image of single IrNPs clearly exhibit a crystalline structure with overall alloyed characteristics. The scale bar is 50 nm.

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Figure 3. Photothermal conversion and photocatalytic activity of the IrNPs in 1× PBS. (a) Temperature elevation under different wavelength laser irradiation values at the same supplied energy. (b) Repeated heating–cooling trend. (c) SOSG indicator-mediated singlet-oxygen generation. (d) Methylene blue degradation against different wavelength laser irradiation values. (e) In vitro cell-based PTT and/or PDT fluorescent images showing the correlated result using cuvette assay. The scale bar is 50 m.

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Figure 4. Quantitative in vitro efficiency test of PTT and/or PDT against HeLa cells with irradiation of (a) 473 nm, (b) 660 nm, and (c) 808 nm diode laser. (Left) The overall tendency of the therapeutic efficiency is correlated to the PTT and/or PDT feature in the cuvette assay. (Right) Calcein AM/EthiD-1 mediated live/dead staining of mono- or combined cancer treatment showing reliable images using the viability assay. The scale bar is 50 m.

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Figure 5. PTT- and PDT-related mRNA expression-level characterization using PCR. (a) PCR band and (b) relative expression level plot represented that PTT (HSP 70 and HSP 90) and PDT (HIF-1 and NQO-1) show an increasing expression against irradiation of a longer wavelength (473 < 660 < 808 nm) and shorter (473 > 660 > 808 nm) wavelength with deviation from the cell lines.

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Figure 6. Toxicology profile of the TAT–IrNPs (0.125 mg Ir/L) compared with the 1× PBS injection from (a)–(i) mouse blood samples with (j) hemolysis and (k) body-weight changes. In all test references, the TAT–IrNPs do not exhibit significant toxicity.

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Figure 7. In vivo CT-guided therapeutic efficiency test against a xenograft-mouse model. (a) The CT contrast is clearly enhanced with the injection of TAT–IrNPs following 24 h of incubation to accumulate in the tumor tissue. (b) Thermographic-camera images of the 808-nm laser irradiationmediated PTT effect. (c) Relative tumor volume of the PTT and/or PDT treatment and (d) extracted tumor tissues. (e) Histological evaluation of the extracted tumor tissue using H&E staining, Ki-67, and TUNEL assay. (f) H&E staining of the major organs to evaluate the organ toxicity. The scale bar is 50 m.

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