Morphology-Controlled Synthesis of Rhodium Nanoparticles for

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Morphology-Controlled Synthesis of Rhodium Nanoparticles for Cancer Phototherapy Seounghun Kang, Woojun Shin, Myung-Ho Choi, Minchul Ahn, Young-Kwan Kim, Seongchan Kim, Dal-Hee Min, and Hongje Jang ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.8b02698 • Publication Date (Web): 14 Jun 2018 Downloaded from http://pubs.acs.org on June 14, 2018

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Morphology-Controlled

Synthesis

of

Rhodium

Nanoparticles for Cancer Phototherapy Seounghun Kang2‡, Woojun Shin1‡, Myung-Ho Choi2, Minchul Ahn2, Young-Kwan Kim4, Seongchan Kim2,5, Dal-Hee Min2,3* and Hongje Jang1*

1

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

01897, Republic of Korea 2

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

3

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

Korea 4

Carbon 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 5

Division of Chemistry & Molecular Engineering, Seoul National University, Seoul 08826,

Republic of Korea

ACS Paragon Plus Environment

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KEYWORDS. cancer therapy, galvanic replacement, morphology control, photothermal conversion, rhodium

ABSTRACT

Rhodium nanoparticles are promising transition metal nanocatalysts for electrochemical and synthetic organic chemistry applications. However, notwithstanding their potential, to date, Rh nanoparticles have not been utilized for biological applications; there has been no cytotoxicity study of Rh reported in literature. In this regard, the absence of a facile and controllable synthetic strategy of Rh nanostructures with various sizes and morphologies might be responsible for the lack of progress in this field. Herein, we have developed a synthetic strategy for Rh nanostructures with controllable morphology through an inverse-directional galvanic replacement reaction. Three types of Rh-based nanostructures – nanoshells, nanoframes, and porous nanoplates – were successfully synthesized. A plausible synthetic mechanism based on thermodynamic considerations has also been proposed. The cytotoxicity, surface functionalization, and photothermal therapeutic effect of manufactured Rh nanostructures were systematically investigated to reveal their potential for in vitro and in vivo biological applications. Considering the comparable behavior of porous Rh nanoplates to that of gold nanostructures that are widely used in nanomedicine, present study introduces Rh-based nanostructures into the field biological research.


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Although rhodium was discovered in the early 19th century,1 its practical applications were limited for a long time because of the difficulties in handling and rarity.2-4 This situation changed dramatically when it was reported that Rh could be used in three-way catalytic convertors and exhibited superior efficiency compared to conventional Pt or Pd catalysts.5 Ever since, attempts have been made to utilize Rh more practically as a catalyst, and with the development of nanomaterials and the emergence of nanochemistry, research on the catalytic activity and recycling of metallic Rh nanoparticles has been actively pursued.6-10 The catalytic properties of Rh nanoparticles depend on two major factors, i.e., their size and shape.11-16 To date, these properties have been comparatively well-studied with possible factors against the size effect.17,18 On the other hand, because of the lack of a reproducible synthetic strategy, the morphological characteristics have not been studied extensively and demand further investigation. In addition, as conventional Rh nanoparticles are generally limited to a size of 42 °C), this temperature elevation was considered to be at a sufficient level, and further feasibility for photothermal therapy was verified in vitro for porous nanoplates, which demonstrated the highest photothermal conversion efficiency. Prior to cell-based therapeutic applications, we checked the cytotoxicity of the Rh nanoshells, nanoframes, and porous nanoplates against the human cervical cancer cell line (HeLa cells). According to the results of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay against half-dilution treatments of the Rh nanoparticles followed by 24 h incubation, all Rh based nanoparticles exhibited cytotoxicity at concentrations higher than 1 equivalent. In addition, we performed a comparison test with the porous Au nanoplates that we previously reported as promising nanoparticles for cancer therapy.27,28 Interestingly, none of the four particles exhibited cytotoxicity at concentrations below 0.5 equiv. and the decreasing order of biocompatibility was Rh nanoframes > porous Rh nanoplates > Rh nanoshells> porous Au nanoplates at concentrations above 0.5 equiv. (Figure 4a). These results suggest that the Rh


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nanoparticles were comparable to those of Au, which have been previously used for various biological applications because of their high biocompatibility. We next scheduled hyperthermic cancer cell (HeLa) ablation by the photothermal conversion effect of the different Rh nanoparticles, based on the highest concentration of 0.4 equiv., which did not exhibit any notable cytotoxicity but instead showed high photothermal conversion efficiency. For more efficient cell internalization, we mixed the as-synthesized Rh nanostructures with

a cell-penetrating peptide transactivator of transcription

(TAT) from

human

immunodeficiency virus CGGYGRKKKRRQRRR (underlined letters represent the essential sequence of the TAT peptide, and bold letters represent the thiol-containing sequence for conjugation) based on potential metal-thiol affinity and electrostatic interaction (Figure S13).41 At the present stage, it was impossible to determine the reason for the difference in the TAT peptide loading efficiency, but it is presumed that the surface area difference and morphological factors (Figure S15). Cellular uptake of nanoparticles against TAT peptide conjugation was further

confirmed

by

fluorescence

microscopy with

additional

fluorescein

labelled

oligonucleotide loaded nanoparticles. (Figure S16) To verify the requirement for biological applications, we performed the colloidal stability evaluation of nanoparticles against 10% fetal bovine serum

(FBS)

containing complete

cell

media. According to

the UV-Vis

spectrophotometer and digital photo image observation up to 24 h of incubation, colloidal stability of nanoparticles were relatively well maintained. (Figure S17 and S18) From these observations, it was supposed that the cellular internalization of the nanoframe was inefficient because of the difficulty of surface modification. Furthermore, hyperthermic cell death by NIR irradiation was hard to observe even though the photothermal conversion efficiency of the nanoframe was excellent on the cuvette scale. Results from in vitro


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hyperthermic ablation and subsequent Calcein AM/Ethidium homodimer-1 live/dead staining fluorescence were consistent with the present hypothesis (Figure 4d and Figure S21-23). Annexin V-FITC / propidium iodide staining and reactive oxygen species (ROS) indicator carboxy-H2DFFDA staining of photothermal therapy treated cells additionally supported the apoptosis pathway mediated cancer cell ablation and chemically induced cell death by synergistic effect with ROS generation during photothermal therapy. (Figure S24 and S25) In vitro quantitative photothermal therapeutic efficiency of the three distinctively structured Rh nanoparticles and porous Au nanoplate as a positive control was evaluated against HeLa cell line through MTT assay. Compared with the cell viability of untreated control condition (Cont, 100%), simple treatment with Rh nanoshells (98.1%), Rh nanoframes (101.7%), porous Rh nanoplates (102.1%), and porous Au nanoplates (98.6%) did not exhibit any significant toxicity. Even with enhanced cell permeability through surface modification with TAT peptide, all Rh nanoparticles (nanoshells (100.5%), nanoframes (101.0%), and porous nanoplates (101.2%)) were observed to have excellent biocompatibility that was comparable to Au (101.1%), which is often used for biological applications. The acceptability of the 808 nm NIR diode laser irradiation used in the PTT test (4 W/cm2, 5 min) could be verified from the cell viability determined when the cells were treated with only NIR treatment condition (100.1% viable). Finally, after the introduction of each nanoparticle, the samples were irradiated with NIR and the photothermal therapeutic efficiencies were compared. In the case of treatment of the nanoparticles with a base, slightly enhanced PTT efficiencies were observed for Rh nanoshells (97.8%), porous Rh nanoplates (95.3%), and porous Au nanoplates (96%), but none for Rh nanoframes (103.0%). PTT efficiency was significantly enhanced with TAT peptide modification followed by NIR irradiation for Rh nanoshells (26.9%), porous Rh nanoplates


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(8.46%), and porous Au nanoplates (9.1%), but no effect was observed for Rh nanoframes (101.0%) again (Figure 5a and Figure S27). The absence of the PTT feature in the Rh nanoframes was the same as that observed in the cell ablation test according to the area limited NIR irradiation test, and it was confirmed that cell permeability plays a key role in this process when compared to the photothermal conversion of the nanoparticle itself. By conducting several comparisons against the nanoparticles and surface modification, we concluded that the porous Rh nanoplates exhibited the best PTT feasibility. Live/dead staining followed by fluorescence microscopy supported the photothermal therapeutic features (Figure 5b and Figure S28, S29) The tumor bearing mice were prepared by subcutaneous injection of the HeLa cells (6 × 106 cells) in 100 µL of 1x PBS into BALB/c nude male mice (5-week old). When the size of the tumor reached ~100 mm3, TAT peptide modified porous Rh nanoplates (0.5 equiv., 100 µL) and 1x PBS (100 µL) were injected by intravenous injection. After 12 h, the tumor was irradiated with 808 nm diode laser (3 W/cm2) for 3 min. The change in tumor size in each group was measured at intervals of two days for 24 days. As shown in Figure 6a, the group of porous Rh nanoplates treated and irradiated with 808 nm laser showed significant inhibition of tumor growth, which had decreased in size. But in other groups, i.e., 1x PBS with or without laser irradiation, and porous Rh nanoplates without laser irradiation, the tumor growth inhibition was not observed. This demonstrated that the porous Rh nanoplates mediated PTT had an excellent effect on cancer cell death and tumor growth inhibition in vivo. Based on these tumor growth inhibition results, sucrose infiltrated tumor sectioning and H&E staining were carried out for 24 days after 808 nm laser irradiation to compare the tumor necrosis of porous Rh nanoplates treated with 808 nm irradiation and other groups. As shown in Figure 6e, the group of porous Rh nanoplates treated with 808 nm laser irradiation showed severe apoptosis of the tumor. In


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contrast, porous Rh nanoplates treated without laser irradiation and 1x PBS with or without laser irradiation groups did not show the apoptosis tendency. This showed that apoptosis was selectively induced by porous Rh nanoplates-mediated PTT only when irradiated with 808 nm laser; otherwise, porous Rh nanoplates exhibited low cell cytotoxicity and the histological sectioning images of the major organs (heart, lung, liver, spleen, and kidney) were free of systematic toxicity when treated with porous Rh nanoplates (Figure 6f). Organ cytotoxicity and photothermal therapeutic effect were also verified by terminal deoxynucletidyl transferase dUTP nick end labeling (TUNEL) assay and Ki-67 immunohistochemistry assay (Figure S34-39). It is well known that the enhanced permeability and retention (EPR) effect, which is a passive targeting phenomenon in which nanoparticles are delivered to the tumor via gaps junction between the blood vessel epithelial cells around the tumor when the nanoparticles are to be delivered to the tumor intravenously in vivo42-45. However, the nanoparticles circulating the body blood system are not efficiently delivered to the tumor as they are cleared by the immune cells present in the liver or lungs46. The porous Rh nanoplates we used did not show any systematic toxicity (Figure 6b and Figure S32) as they circulated in the blood in an amount sufficient to induce tumor growth inhibition and cancer cell apoptosis through PTT via the EPR effect because of their excellent biocompatibility (Figure 6c, d). To summarize, the porous Rh nanoplates were delivered to the tumor through the EPR effect and effectively inducing tumor growth inhibition and cancer cell death through PTT under 808 nm laser irradiation in vivo.

CONCLUSION


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In the present study, we successfully achieved the inverse-directional galvanic replacement by thermodynamic control and demonstrated practical examples of morphologycontrolled synthesis of Rh nanoplate derivatives. The synthesis of Rh nanostructures, including nanoshells, nanoframes, and porous nanoplates, was achieved by analyzing the key factors such as reaction time, concentration, temperature, and the role of additives. Among the various Rh derivatives, the porous Rh nanoplates showed excellent biocompatibility, photothermal conversion, and surface functionalization. Consequently, they were evaluated for cancer phototherapy, which is the new potential for the biological applicability of Rh-based compounds. Both in vitro and in vivo examination revealed impeccable efficiency without any adverse effect, which was comparable to that of Au, commonly used in the field of nanomedicine. We expect that the present study will broaden the boundaries of nanomaterials science, which has not been addressed to date because of the limitations of existing synthetic methods, and the biological applications that are limited by preconceived notions of component.

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), and rhodium(III) chloride were purchased from Sigma (St. Louis, MO, USA). 10X phosphate buffered saline (PBS), Dulbecco’s modified eagle’s medium (DMEM), and fetal bovine serum (FBS) were purchased from WelGene (Seoul, Korea). LIVE/DEAE viability/cytotoxicity assay kit was purchased from


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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 chemicals were used as received. TAT peptide (CGGYGRKKRRQRRR) was synthesized by solid-phase peptide synthesis (SPPS) method.

Preparation of template Ag nanoplates by seed-mediated method Ag nanoseeds were synthesized by reduction of Ag(I) under the existence of shapecontrolling additives. Briefly, 250 µL of 10 mM AgNO3, 300 µL of 30 mM trisodium citrate dihydrate, 1.5 mL of 3.5 mm PVP, and 24.75 mL of deionized (EI) water were added into a 50 mL clear glass vial; then, 60 µL of 30% hydrogen peroxide was added under the magnetic stirring at 300 rpm with incubation for 3 min to ensure homogeneous mixing. To the above mixture, freshly prepared 250 µL of 100 mM sodium borohydride solution was rapidly injected followed by additional incubation for 3 h. The addition of sodium borohydride led to the color change from transparent to pale yellow; then, further color changes occurred to transparent again, deep yellow, orange, and finally blue in the course of 3 h of reaction time. The prepared Ag nanoseeds were used without purification. To the as-prepared Ag nanoseeds, 333 µL of 75 mM trisodium citrate dihydrate solution and 1 mL of 100 mM L-ascorbic acid were sequentially added under the magnetic stirring. Meanwhile, Ag growth solution composed of 20 mL of 1 mM AgNO3, 0.125 mL of 100 mM


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citric acid, and 10 µL of 75 mM trisodium citrate dihydrate. To the seed solution, 13 mL of asprepared Ag growth solution was added by 0.2 mL/s rates. During the growth reaction, solution color became deep-blue. After the additional reaction time of 10 min at ambient condition, proceed to the inverse-directional galvanic replacement to manufacture Rh nanoplates without any purifications.

Inverse-directional galvanic replacement of Ag nanoplates with Rh(III) To the clear 25 mL glass vial, 4 mL of as-synthesized Ag nanoplates were placed, then 10 mL of EG was added with several times of pipetting to accomplish homogeneous mixing. To the Ag nanoplates in DI water/EG mixed solvent, appropriate volume of 4.2 mM Rh(III) aqueous stock solution was added. For the successful synthesis of nanoshells, nanoframes, and porous nanoplates, 500, 600, and 900 µL of 4.2 mM Rh(III) should be added to each vial, respectively. IGR reaction was accomplished by heating at 190 oC for 4 h without capping to evaporate the DI water content. During the reaction, the color changed into blue indigo, blue-brown, and dark brown for nanoshells, nanoframes, and porous nanoplates, respectively. After the 4 h from heating, remove the heat source and 10 mL of DI water was directly added to each vial for quench the reaction. Synthesized Rh nanostructures were purified by centrifugation at 9000 rpm (Eppendorf E5803) for 10 min, and washed with DI water for 3 times. The final products were re-dispersed in 4 mL of DI water and denoted as 1 eq. concentration. The manufactured Rh nanostructures could be used without any colloidal stability problems even when stored at room temperature for over 3 months.


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Characterization of the prepared Rh nanostructures Energy-filtering transmission electron microscope LIBRA 120 (Carl Zeiss, Germany) and field-emission scanning electron microscope AURIGA (Carl Zeiss, Germany) were used to obtain images of synthesized Rh nanostructures. HAADF-STEM/EDS was measured by Tecnai F20 TEM (FEI, USA). The surface area was measured by nitrogen-adsorption experiment using a Tristar-II (Micromeritics, 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-VisNIR extinction spectra. Dynamic light scattering and zeta-potential were measured by Zetasizer Nano ZS (Malvern, UK). 808 nm NIR irradiation was performed by surgical laser accessories OCLA (Soodogroup Co., Republic of Korea). 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).

TAT peptide loading on Rh nanostructures 20 µL of 1 µM TAT peptide in DI water stock solution was added to 1 mL of 1 eq. Rh nanostructures (nanoshells, nanoframes, and porous nanoplates) 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 peptide loaded Rh nanostructures were re-dispersed in 1 mL of 1x PBS for further applications.


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Characterization of photothermal conversion in cuvette Photothermal conversion efficiency was confirmed by temperature elevation under the 808 nm NIR irradiation for same concentrations of nanoplates. 1 mL of 0.5 eq. concentration Rh nanoshells, Rh nanoframes, porous Rh nanoplates, and porous Au nanoplates were placed and 808 nm NIR diode laser was irradiated to each sample with intensity of 4 W/cm2 for 120 s of measurement time. Temperature change was observed by digital thermometer in every 30 s.

Hyperthermic cancer cell ablation The human cervical cancer cell line HeLa was grown in DMEM containing 4.5 g/L Dglucose, supplemented with 10% FBS, 100 units/mL penicillin, and 100 mg/mL streptomycin. The cells were grown in a humidified 5% CO2 incubator at 37 oC. For the in vitro hyperthermic cancer cell ablation test, 1 mL of 0.5 eq. TAT loaded nanoplates 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 6 h or 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 4W/cm2 for 5 min, and allowed to be incubated for additional 12 h. For live/dead fluorescent image obtaining, 500 uL of combined staining solution composed of 2 µM Calcein AM and 4 µM EthiD-1 in D-PBS


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was added to each well and incubated for min. Fluorescence images of the cells were obtained by using a fluorescence microscope.

Annexin V-FITC/propidium iodide (PI) staining 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 6 h of incubation in humidified 5% CO2 incubator at 37 oC to ensure nanoplate internalization, remaining nanoparticles were discarded 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 4 h. For Annexin V-FITC/PI staining (Annexin V-FITC Apoptosis Detection Kit, Sigma, St. Louis, MO, USA), the cells were collected to 1.5 mL ep tube and washed 1xPBS, 2 times and resuspension 1x annexin binding buffer 500 µL. Add 5 µL of Annexin V-FITC conjugate and 10 µL of PI solution to the 1.5 tube. The mixture was incubated at room temperature for 10 min and protect from light. Fluorescence images of the cells were obtained by using a fluorescence microscope.

Reactive oxygen species (ROS) detection 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


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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 µL of 10 µM 5-(and-6)carboxy-2’, 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.

Bio-TEM sample preparation Bio-TEM samples were obtained after 12 h of intravenous injection. The tumor samples were placed in 4% paraformaldehyde solution and incubated at 4 oC, overnight. The samples were cut to 1 mm3 size and washed with 0.05 M sodium cacodylate buffer for 10 min, 3 times. For post-fixation, add 2 % Osmium tetroxide with 0.1 M cacodylate buffer (1:1) solution to the samples and incubated at room temperature for 2 h. The samples were briefly washed twice with DI water. and 0.5 % Uranyl acetate for 30 min. For dehydration process, the samples were incubated in ethanol 30 %, 50 %, 70 %. 80 %, and 90 % solution for 10 minutes in sequence. Finally, incubated three times for 10 min in 100 % ethanol solution. The samples were incubated in propylene oxide twice for 10 min and propylene oxide + Spurr’s resin (1:1) for 2 hr. The solution was exchanged to 100 % spurr’s resin and incubated overnight. The resin solution was replaced with a clean resin solution and incubated for 2 h. The resin solution was incubated in 70 o

C oven for overnight.

Cytotoxicity measurement


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MTT powder was dissolved in 1x PBS at 5 mg/mL concentrations, then filtered through sterilized syringe filter (0.2 µm pore diameter). Prepared MTT stock solution was 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 µL of growth media (50-70% confluency). The cells were treated with appropriate concentrations of Rh nanoplates 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 µL 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 1x PBS once to remove remaining MTT. Finally, 100 µL 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.

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 MNPs (M = pAu, sRh, fRh, and pRh) 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


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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 and serum was separated using serum-separating tube. Toxicity profile (AST, ALT, BUN, Creatinine, Total protein, and Lactic acid dehydrogenase) was measured using a FUJI DRI-CEHM FDC3500.

In vitro photothermal therapeutic efficiency test For in vitro photothermal therapeutic efficiency test, HeLa cells were seeded with a density of 10,000 cells per well of a 96-well culture plate with 100 µL of growth media (50-70% confluency). The cells were treated with 0.5 eq. of bare and TAT conjugated Rh nanoplates in serum free culture media and incubated for 6 h at 37 ℃ incubator. After the incubation, the cells were rinsed with 1x PBS twice, then media replacing with serum-containing culture media. Then, HeLa cells were treated by 808 nm NIR diode laser irradiation with 4W/cm2 for 5 min and allowed to be incubated for additional 12 h. For live/dead fluorescent image obtaining, 500 uL of combined staining solution composed of 2 µM Calcein AM and 4 µM EthiD-1 in D-PBS was added to each well and incubated for min. Fluorescence images of the cells were obtained by using a fluorescence microscope.


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In vivo photothermal 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) 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 µL sterilized 1xPBS solution (n = 4). When the size of the tumor is ~100 mm3, 100 µL of porous Rh nanoplates in 1xPBS (0.5 eq.) and 1xPBS (as control) were injected into the tail veins of the mouse tumor model. After 12 hr of injection, the mouse tumor were treated by 808 nm NIR diode laser irradiation with 3 W/cm2 for 3 min for demonstrate of porous Rh nanoplates’ anti-cancer phototherapeutic effect. 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. The sectioned samples were processed by using H&E staining (BBC Biochemical, Mt Vernon, WA, USA). The samples were observed under a BX71 microscope with 20x objective lens (Olympus, Tokyo, Japan)

TUNEL assay and Ki-67 immunohistochemistry


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Mouse organ and tumor samples were placed in 4 % PFA solution and incubated at 4 oC, overnight. After sucrose infiltration, the samples were embedded in optimal cutting temperature (OCT) compound and sectioned of 10 µm. The sectioned samples were processed by using in situ cell death detection kit, fluorescein (Roche, Mannheim, Germany). The samples were washed with 1x PBS for 30 min. For the permeable process, incubated in permeabilization solution (0.1 % Triton X-100, and 0.1 % Sodium citrate) for 2 min at 4 ℃. 50 mL of TUNEL reaction mixture was added on the samples and incubated in humidified chamber for 60 min at 37 oC in dark place. The samples were washed 3 times with 1x PBS and mounted in antifade mounting medium with DAPI. The samples were observed under a BX71 microscope with 20x objective lens (Olympus, Tokyo, Japan). The OCT embedded and sectioned samples were washed once with 1x PBS and twice with permeabilization solution (0.2 % tween 20 in 1x PBS) for 10 min. For blocking process, the samples were incubated with blocking solution (5 % normal goat serum, 0.2 % tween 20 in 1x PBS) for 45 min in humidified chamber. The samples were incubated with primary antibody diluted solution (0.2 % tween 20, 2 % normal goat serum in 1x PBS) (dilution 1:100, anti-human from Santa Cruz, and anti-mouse from Abcam) in humidified chamber for overnight at 4 oC. The samples were washed 3 times for 10 min with permeabilization solution and incubated with secondary antibody (anti-Ki67) diluted solution (0.2 % tween 20, 2 % normal goat serum in 1x PBS) for 2 hr at 37 oC in humidified chamber, dark place. The samples were washed with permeabilization solution and mounted in antifade mounting medium with DAPI. The samples were observed under a BX71 microscope with 20x objective lens (Olympus, Tokyo, Japan)


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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). This work was supported by the Basic Science Research Program (2016R1E1A1A01941202), the International S&T Cooperation Program (2014K1B1A1073716), and the Research Center Program of IBS (IBS-R008-D1) through the NRF. This work was supported by Nano Material Technology Development Program through the NRF funded by the Ministry of science, ICT and Future Planning (2016M3A7B4027223) and (2016M3A7B4905609).


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AUTHOR CONTRIBUTION ‡ S. Kang and W. Shin contributed equally.

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Figure 1. Schematic illustration of inverse-directional galvanic replacement of Ag nanoplate template with Rh(III). Due to the positive net cell voltage from the contribution of concentration and temperature, Rh based nanostructures of various morphologies are formed.


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Figure 2. IGR mediated nanostructure transformation. (a) concentration dependent UV-Vis spectrum change and (b) normalized extinction spectra were displayed by matching with their (c) TEM and SEM images. (d) All observed nanostructures and reaction conditions were demonstrated in table. Based on the identified 1st and 2nd threshold conditions (red lined in (d)),


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we selected distinctive three Rh based nanostructures including nanoshells, nanoframes, and porous nanoplates. The scale bar is 100 nm.


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Figure 3. Time dependent observation of IGR and secondary growth of (a) nanoshells, (b) nanoframes, and (c) porous nanoplates by TEM. Skeletal nanostructures were formed within 1 h of reaction by replacement, then evolved into distinctive nanostructures by one-pot secondary growth process. HAADF-STEM/EDS line profile and elemental mapping images clearly exhibited abundant Rh in formed nanostructures. The scale bar is 100 nm.


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Figure 4. According to the (a) MTT cell viability assay, all three Rh nanostructures started to represent cytotoxicity over 0.5 eq. of concentrations. (b) Extinction spectra from UV-Vis spectrophotometer measurement exhibited overall absorption in the UV-Vis-NIR region, and it was stronger for nanostructures containing higher amount of Rh was involved in IGR reaction.


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(c) Compare to 1x PBS control experiment condition, all three nanostructures clearly showed photothermal conversion mediated temperature elevation in cuvette assay. (d) Based on the identified characteristics, NIR mediated hyperthermic HeLa cell ablation feasibility was confirmed by TAT surface attached nanoshells, nanoframes, and porous nanoplates. In case of nanoframes, no hyperthermic cell death was observed and it might be originated from poor TAT conjugation efficiency. The scale bar is 250 µm.


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Figure 5. In vitro cancer phototherapeutic efficiency measurement against HeLa cells. (a) MTT cell viability assay represented that TAT peptide loaded Rh nanoshells and porous Rh nanoplates induced significant cancer cell ablation through NIR irradiation, whereas other control groups did not exhibit phototherapeutic efficiency. In the case of Rh nanoshells In the case of Rh


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nanoframes, no such therapeutic efficiency was observed. RhNPs implies all three Rh nanostructures. (b) Hoechst 33342 (blue, nuclei), Calcein-AM (green, live cells), and EthiD-1 (red, dead cells) staining followed by fluorescent microscope observation supported the result from MTT assay against Rh nanoshells (sRhNPs), Rh nanoframes (fRhNPs), and porous Rh nanoplates (pRhNPs). The scale bar is 50 µm.


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Figure 6. In vivo cancer phototherapy and ex vivo histologic examination by using porous Rh nanoplates against tumor bearing mice. (a) relative tumor volume and (b) body weight change with time for treatment groups. (c) In vivo and (d) ex vivo comparison of tumor size. (e) H&E stained image of tumor and (f) major organs (heart, lung, liver, spleen, and kidney) for 24 d treatment. pRhNPs implies porous Rh nanoplates. The scale bar is 50 µm.


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TOC Figure


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Morphology-Controlled Synthesis of Rhodium Nanoparticles for

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