Reducing Agent-Assisted Excessive Galvanic Replacement Mediated

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Reducing Agent-assisted Excessive Galvanic Replacement Mediated Seed-mediated Synthesis of Porous Gold Nanoplates and Highly Efficient Gene-thermo Cancer Therapy Seounghun Kang, Kyunglee Kang, Hyun Huh, Hyungjun Kim, SungJin Chang, Tae Jung Park, Ki Soo Chang, Dal-Hee Min, and Hongje Jang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b13028 • Publication Date (Web): 22 Sep 2017 Downloaded from http://pubs.acs.org on September 25, 2017

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ACS Applied Materials & Interfaces

Reducing Agent-assisted Excessive Galvanic Replacement Mediated Seed-mediated Synthesis of Porous Gold Nanoplates and Highly Efficient Genethermo Cancer Therapy Seounghun Kang2, Kyunglee Kang1, Hyun Huh3, Hyungjun Kim4, Sung-Jin Chang5, Tae Jung Park5, Ki Soo Chang3, Dal-Hee Min2,6,7* and Hongje Jang1* 1

Department of Chemistry, Kwangwoon University, Seoul 01897, Republic of Korea

2

Department of Chemistry, Seoul National University, Seoul 08826, Republic of Korea

3

Optical Instrumentation Development Team, Korea Basic Science Institute, 169-148 Gwahak-ro,

Yuseong-gu, Daejeon 34133, Republic of Korea 4

Department of Chemistry, University of Michigan, Ann Arbor, MI 48109, United States

5

Department of Chemistry, Chung-Ang University, 84 Heukseok-ro, Dongjak-gu, Seoul 06974,

Republic of Korea 6

Center for RNA Research, Institute for Basic Science (IBS), Seoul National University, Seoul 08826,

Republic of Korea 7

Institute of Nanobio Convergence Technology, Lemonex Inc., Seoul 08826, Republic of Korea

Keywords: cancer therapy, galvanic replacement, gene delivery, photothermal therapy, porous nanostructure

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*To whom correspondence should be addressed. Prof. Dal-Hee Min, Tel:+82-2-880-4338, E-mail: [email protected] Prof. Hongje Jang, Tel:+82-2-940-8320, E-mail: [email protected]

Table of Contents

Abstract Porous Au nanoplates (pAuNPs) were manufactured by a reducing agent-assisted galvanic replacement reaction on Ag nanoplates using a seed-mediated synthetic approach. Two core additives, polyvinylpyrrolidone and L-ascorbic acid, prevented fragmentation and proceeded secondary growth. By controlling the concentration of the additives and the amount of replacing ion AuCl4-, various nanostructures including nanoplates with holes, nanoframes, porous nanoplates, and bumpy nanoparticles with unity and homogeneity were synthesized. The present synthetic method is advantageous because it can be used to manufacture porous Au nanoplates (pAuNPs) with ease, robustness, and convenience. The prepared pAuNPs exhibited a highly efficient photothermal conversion effect and cargo loading capacity on exposed surfaces by Au-thiol linkage. By using dual cargo mixed loading -2-


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of the hepatitis C virus (HCV) targeting gene drug DNAzyme and cell-penetrating peptide TAT onto the surface of the pAuNPs and photothermal conversion mediated hyperthermic treatment, successful gene-thermo therapy against HCV genomic human hepatocarcinoma cells were demonstrated.

Introduction Over the past few decades, growth in the design and synthesis of colloidal nanoparticles has increased because they are useful in many fields including biomedicine,1-3 catalysis,4,5 optics,6,7 and even data storage.8,9 The silver (Ag) nanoplate, which is an anisotropic nanoparticle, has been studied for a long time because of its 2-dimensional planar structure and its unique physicochemical properties.10-13 Much effort has been conducted into changing the edge length, thickness, and shape of these nanoplates for collective modulation of their localized surface plasmon resonance (LSPR) properties.14,15 In addition to this relatively simple dimensional control, Ag nanoplates are being manufactured with unique properties through post-synthetic transformation techniques such as chemical etching, light etching, and elemental replacement reactions.16,17 One of the best known post-synthetic transformation methods is the galvanic replacement reaction which was reported for the first time by the Xia group.18 The galvanic replacement reaction is a kind of redox reaction in the solution phase based on the difference in the standard reduction potential between two elements. In general, a sacrificial template such as silver (Ag), copper (Cu), and cobalt (Co) is replaced by added replacing ions such as gold (Au), platinum (Pt), and palladium (Pd) to provide transformed nanostructures including -3-



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hollow nanoshells by stoichiometric redox balance.19,20 Through this attractive redox reaction, Ag nanoplates are widely known to transform into plates with central holes or into nanoframe structures. While previous research trends have looked to obtain more homogeneous and uniform nanostructures through galvanic replacement, recently unprecedented nanostructures have been manufactured with enhanced physicochemical properties using unconventional approaches. For this purpose, modified galvanic replacement techniques have been actively researched and reported including asymmetric diffusion, excessive galvanic replacement, inhibitory galvanic reaction, and competitive galvanic replacement with additives.21-23 One of the most interesting challenge in galvanic replacement can be represented as reducing agent mediated shape-controlled preparation of nanoparticles.24-29 However, above listed extended galvanic replacement approaches were optimized in reaction conditions by stoichiometric balance to accomplish the balancing between replacement and reduction rate for structural maintenance. 24-29 In this work, the reducing agent-assisted excessive galvanic replacement reaction of Ag nanoplates to form porous Au nanoplates (pAuNPs) through seed-mediated synthetic approach was studied. (Figure 1) Unlike the previous reports under optimized replacing reaction conditions,24-29 by using Ag nanoplates as the sacrificial nanotemplate with existence of reducing agent, robust transformation against excessive addition of replacing ion were established here. The excessive addition of replacing ion induced the fluctuation of balance between replacement and reduction rate and led to formation of porous nanoplate structures. The obtained pAuNPs are expected to exhibit an excellent photothermal conversion effect because of their enhanced extinction wavelength in overall regions, and their high volume-tosurface ratio is advantageous for surface loading of delivering cargo. Using the chemical -4-


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bond between the exposed Au surface and thiolated cargo, the hepatitis C virus (HCV) targeting gene drug DNAzyme (Dz) and cell-penetrating peptide TAT was successfully loaded. The combinational treatment of HCV-genomic-encoded human hepatocarcinoma was accomplished by photothermal-conversion-mediated hyperthermic cell ablation under near infrared (NIR) laser irradiation and triggered releasing of loaded Dz into the intracellular environment. To the best of our knowledge, this report is the first to synthesize pAuNPs in excessive one-pot manner and to present their high potential for gene-thermo combinational cancer therapy as a model biomedical application.

Results and discussion First, sacrificial Ag nanoplate was synthesized by a slight modification of the previous report.30 Briefly, Ag nanoplate seed was prepared by shape-controlled reduction of Ag ion by sodium borohydride under the existence of polyvinylpyrrolidone (PVP), trisodium citrate (Na3Cit), hydrogen peroxide. During the seed preparation step, under the combinational shape controlling action of oxidative etchant hydrogen peroxide, Ag (111) facets selective adhesion of Na3Cit and PVP, Ag nanoplate seed was manufactured by reducing and capping agent sodium borohydride.31,32 From the repeated growth of prepared Ag nanoplate seed into larger nanoplate by addition of Ag+ precursor under the existence of L-ascorbic acid (L-AA), the edge lengths of Ag nanoplate could be controlled and we adopted 1st grown Ag nanoplate for following transformation by considering the extinction spectra shift to 800 nm region by galvanic replacement for further applications. (Figure S1) Next, we performed galvanic replacement for further transformation of the Ag -5-



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nanoplates into distinctive nanostructures such as 2-dimensional frames or hollow nanostructures with complex metal distribution, in order to take advantage of their unique physicochemical properties. The amount of AuCl4- added to the 1st grown Ag nanoplates with 2 h of sufficient reaction time was adjusted accordingly to achieve galvanic replacement through the one-pot reaction approach and to form the target nanomaterial by considering that the shift of the extinction spectra from transformation should be located at around 800 nm. Interestingly, the nanostructures obtained up to an addition of 4 v/v% AuCl4- solution followed the transformation step consistent with previously reported cases of central hole formation, internal etching, and nanoframe formation with increasing AuCl4-. However, an excessive addition of AuCl4- over 10 v/v% induced porous nanoplate structures and backfilled bumpy nanostructures. (Figure 2b) The structures and proportions of the main nanoparticles were clearly distinguishable as nanoplates, nanoplates with hole, nanoframe, porous nanoplates, and bumpy nanoparticles depending on the concentration of added AuCl4-. The nanostructures formed under excess AuCl4- were very different from those reported previously in which fragmentation or aggregation of the nanostructures without rigid surface coating or additional treatment such as chemical etching was observed.22,23 The LSPR wavelength from in-plane dipole resonance helped to further analyse the series of structural changes. Addition of 1 v/v% of AuCl4- caused a blue shift of the LSPR wavelength because of rounding of the Ag nanoplate edge, and the addition of 2 and 4 v/v% AuCl4- induced a red shift of the LSPR wavelength and an overall increase in extinction intensity because of structural transformation of the nanomaterial into a holed nanoframe. Addition of 10 v/v% AuCl4- caused the LSPR wavelength to blue shift because of the secondary growth of porous nanostructures and backfilling. The peak at 340 nm (out-of-plane quadrupole resonance) and 470 nm (in-plane quadrupole resonance) disappeared because of the structural -6-


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transformation.12,31 (Figure 2a) The galvanic replacement reaction under the condition in which all additives were removed and 10 v/v% AuCl4- was added caused significant fragmentation and aggregation of the nanostructures as expected. (Figure S2) We obtained similar results when only Na3Cit was present at the same concentration as in the one-pot synthesis, and it was confirmed that Na3Cit, a surface-capping stabilizer, contributes to the dispersion of the nanomaterial but does not affect the structural transformation. By comparing TEM images, it was observed that the Ag nanoplates which were initially 60 nm edge length, fragmented into small debris and that they underwent no significant structural transformation. One interesting observation was that in the absence of Na3Cit, a by-product of galvanic displacement: AgCl, aggregated densely on the surface of the resulting small nanoparticle fragments, as a grey mass. On the other hand, this aggregation was much less in the presence of Na3Cit, which is expected because of the additional stabilizing effect caused by the adsorption behavior of Na3Cit on the surface of the nanomaterial. (Figure S2) In order to investigate the effect of L-AA, the secondary reducing agent, on the transformation of the nanomaterial structure, galvanic replacement was performed with varying concentrations of AuCl4-, in the absence of L-AA. (Figure 3) Without L-AA, the nanostructures formed by galvanic replacement transformed more rapidly by the addition of AuCl4- and more fragmentation occurred when AuCl4- was added at a concentration 4 v/v% or more, compared to the case where L-AA was present. Although fragmentation occurred and the initial structure of the Ag nanoplates was not maintained, cluster formation without aggregation, and a similar behavior as the nanoparticles was confirmed through TEM and UV-Vis spectroscopy. This clustering behavior was likely a result of the surface-adsorptive -7-



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PVP in the additive solution. From this result, some insight can be gained into the effect of LAA on the nanoplate structure transformation and the rate of the galvanic replacement reaction. To obtain additional information on the mechanism, similar reactions were performed by varying the concentration of L-AA with a fixed concentration of other additives to form the pAuNPs. (Figure 4) Nanostructures produced under 0 mM, 1 mM, and up to 10 mM (excess) of L-AA were analyzed by TEM and UV-Vis spectroscopy. It was expected that the remaining L-AA, which was used for the growth of Ag nanoplates, would contribute to the secondary growth process by using the product of the galvanic replacement reaction as a template, which was confirmed by the TEM images of each sample. In the presence of 1 mM L-AA, almost all of the particles transformed into pAuNPs through galvanic replacement, and the proportion of overgrown bumpy nanostructured particles increased as the amount of LAA increased. About 60% and 95% of the particles formed as hollow bumpy AuNPs in presence of 2.5 mM and 5 mM L-AA, respectively. In the presence of excess 10 mM of LAA, the interior vacancy was filled and the swelling formation started by overgrowth. Furthermore, as the concentration of L-AA increased, a spherical hollow nanostructure was selectively formed rather than a nanoplate. The high reaction rate with a large concentration of L-AA had more of an influence on the structure of the nanomaterial than the surfaceadsorbed PVP. The above results suggest that L-AA is a key factor in the reducing agentassisted galvanic replacement of Ag nanoplates, which matches previous related reports. Tests were also performed in which other experimental conditions were fixed in the formation of the pAuNPs and the concentration of PVP was varied to identify the effect of PVP on the transformation. (Figure S3) In the absence of PVP, it was found that the initial -8-


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structure of the nanoplate was not maintained and fragmented into small debris. In the presence of a small amount of PVP (concentration of 0.05 mM), the size and behavior of the nanoparticles were maintained, but shrinking during the replacement reaction resulted in the formation of rugged, tightly packed spherical nanoparticles. The effect of PVP on the structure was observed at higher concentrations over 0.15 mM, and the addition of 0.15 mM PVP lead to the formation of porous nanoplates. More interestingly, when the concentration of PVP was greater than 0.5 mM, hollow spherical nanoshell structures formed, which are generally manufactured from the galvanic replacement of Ag nanospheres, which were not present in the precursor. The proportion of hollow spherical nanoshells increased with increasing PVP concentration. This unconventional structural transformation would be expected to be caused by L-AA with restructuring restricted by PVP. Through these observations, it can be seen that PVP contributes not only to the determination of structure and dispersion in the synthesis of nanoparticles, but also to the structural transformation reaction through its usage as an additive for fine controlling of the experimental conditions. To infer the principle driving the formation of the observed nanostructures, the standard reduction potential was focused on, which is a key element of the galvanic replacement reaction. The standard reduction potentials of AuCl4-(aq) and Ag+(aq) against the aqueous standard hydrogen electrode (SHE) are 0.99 V and 0.80 V, respectively. According to these values, galvanic replacement between the Ag nanoplates and AuCl4- ions is a thermodynamically favorable reaction with a driving force of 0.19 V. By considering the stoichiometric balanced equation, the reduction of Au(III) requires the oxidation of three equivalent Ag: AuCl4-(aq) + 3Ag(s) Au(s) + 4Cl-(aq) + 3Ag+(aq) Au(s) + 3AgCl(s) + Cl-(aq) -9-


(eq.1)


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This stoichiometry means that the silver surface atoms are not enough to reduce gold and core silver atoms are consumed. In addition, the instant precipitation of the AgCl adduct favors oxidation of the silver surface according to Le Chatelier’s principle. Previous reports have pointed out the importance of the Cl- ion, by showing that the galvanic replacement reaction does not take place with different gold precursors such as gold acetate.14 The silver atoms in the form of AgCl(s) can be recovered by single-electron reduction with a potential of 0.22 V. The effects of L-AA in the galvanic replacement and interaction with metal atoms were examined. The oxidized L-AA (denoted as L-AA(ox)) is known to undergo the two-electron, two-proton reduction with a potential of 0.28 ~ 0.35 V.29 The reduction potential of 0.35 V was selected to examine the spontaneity of each redox reaction in this study. L-AA has been previously applied as a reducing agent in galvanic replacement.24-29 Based on standard reduction potential analysis, the participation of L-AA in this galvanic replacement can result in two feasible reduction mechanisms: one-step and two-step reduction. One- and two-step reduction differ in the interaction between the Au(III) precursor and L-AA. One-step reduction represents the direct precipitation of gold from the reduction of AuCl4- by L-AA (Table 1.(i)-2). Simultaneously, L-AA can also reduce net existing silver ions, which are generated by the competition between the galvanic replacement and AgCl adduct formation (Table 1.(i)-3). Equation (i)-1 represents the galvanic replacement between the gold precursor and Ag nanoplate. All redox reactions are exothermic and expected to take place at the same time. In case of two-step reduction, one intermediate oxidation state, Au(I), was additionally considered. Two-electron transfer between AuCl4- and L-AA generates - 10 -


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AuCl2-. AuCl2- has a reduction potential of 1.11 V, which is higher than the corresponding value for AuCl4- by 0.11 V because of the loss of the electron-donating Cl anion.32-34 The conventional galvanic replacement reaction is replaced by equation (ii)-2 during the two-step reduction, and the change of gold precursor from AuCl4- to AuCl2- increases the thermodynamic driving force by 0.12 V (0.31 V for the two-step reduction pathway and 0.19 V for the conventional galvanic replacement). This analysis suggests that galvanic replacement would be facilitated by the pre-reduction of AuCl4- by L-AA in the two-step reduction pathway. Stoichiometry between gold and silver atoms in galvanic replacement is changed to 1:1 in the suggested two-step reduction mechanism, which can explain the observed slower reaction termination in the presence of L-AA compared to the 1:3 ratio in the conventional galvanic replacement by competitive reaction pathways. Furthermore, the 1:1 ratio makes the redox reaction more controllable and can result in a different morphology. Even though there is no decisive evidence to verify this dominant pathway, both mechanisms are considered to operate simultaneously with different contributions. To figure out the present reducing agent assisted excessive galvanic replacement reaction, we observed the structural transformation cascade by TEM observation. From the addition of 10 v/v% replacing AuCl4- ion to L-AA, Na3Cit and PVP containing Ag nanoplate template, reaction mixture was dispensed and quenched by centrifugation mediated purification. The obtained TEM images clearly represented sequential structural transformation through nanoframe (5 min) to pAuNPs (30 min). (Fig 5) At the foremost stage, added excessive AuCl4- could be consumed by both galvanic replacement in one-step reduction pathway (Table 1.(i)-1) and AuCl2- ion formation (Table 1.(ii)-1). Next, galvanic replacement in two- 11 -



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step reduction pathway (Table 1.(ii)-2) became dominant replacement reaction due to generation of sufficient AuCl2- ion and higher reduction potential of 0.31 V compared to 0.19 V from one-step pathway. Galvanic replacement between Ag(0) and AuCl2- should not induce fragmentation due to stoichiometric balance of transferred electron count of one. Moreover, from this stage, reduction of Ag(I), Au(I) and Au(III) were mainly occurred by existing reducing agents. Simultaneous metal cations reduction and additional replacement of newly generated Ag(0) with AuCl2- led to continuous growth of nanoframe as template into pAuNPs. Because of this competed growth pathway, formed pAuNPs exhibited definite porous nanostructure with Ag-Au alloy composition from elemental mapping analysis. (Figure S5) Interestingly, growth of Ag and Au nanostructures were highly regulated to inner direction of nanoframe to accomplish filled-in process and this feature mainly depended on the concentration of PVP which might play a role as structural supporting adsorptive. One of the essential parameter of plasmonic nanomaterials for therapeutic applications, photothermal conversion effect, was originated from the incident light absorption and electron oscillation to kinetic energy transfer through electron-phonon interaction.35-37 The dominant absorption wavelength of pAuNPs is located in the 800-900 nm region where the wavelength coincided with the NIR, which is expected to exhibit an excellent photothermal conversion effect as compared to conventional spherical nanoparticles. In order to investigate the photothermal conversion effect expected from the absorption wavelength, temperature elevation was measured under NIR irradiation of the pAuNPs. In a cuvette assay, they exhibited a significant temperature elevation to 49.8 ℃ (∆T = 26.5 ℃) with exposure to 808 nm NIR laser irradiation for 180 s, whereas the 1X phosphate buffered saline (1X PBS) without pAuNPs exhibited a negligible temperature change, only reaching 24.7 ℃ (∆T = - 12 -


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0.7 ℃). (Figure 6a) For the verification of hyperthermia and the application of the pAuNPs in combinational cancer treatment, loading of cell-penetrating peptide TAT (transactivator of transcription (TAT) of the human immunodeficiency virus, CGGYGRKKRRQRRR, underlined letters represent the essential sequence of the TAT peptide) to the pAuNPs surface and cytotoxicity tests were performed.38 The chemical affinity between the exposed Au surface and the thiol functional group from the side chain of the cysteine end was used to successfully load the TAT peptides onto the surface of the pAuNPs. This was indirectly confirmed by the increase of hydrodynamic radius (from 63.75 ± 0.58 nm (pAuNPs) to 73.04 ± 0.31 nm (TAT-pAuNPs)) by dynamic light scattering (DLS) and the increase of zeta potential (from -18.8 ± 0.18 mV (pAuNPs) to -14.1 ± 0.48 mV (TAT-pAuNPs)) with the loading of the positively charged TAT peptide. (Figure 6d) The enhancement of cell internalization of pAuNPs by the TAT peptide loading was clearly observed through comparing microscopic bright field images. (Figure 6c) Although pAuNPs were rarely introduced into the cells, TAT-pAuNPs were observable as black dots in the bright field image. The cytotoxicity of pAuNPs was investigated by a cell viability assay using MTT(3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazalium bromide)). According to the MTT assay data, both pAuNPs and TAT-pAuNPs exhibited excellent cell viability of over 93% with a nanoparticle optical density (OD) of 0.45.(Figure S4) Based on the enhanced cell internalization ability by TAT peptide conjugation and the low cytotoxicity, cell-based studies for gene delivery and a therapeutic assay at an OD of 0.45 of the TAT-pAuNPs were performed. The mediation of cancer cell hyperthermia by the photothermal conversion of TAT-pAuNPs was confirmed by the treatment of the human hepatocarcinoma-carrying HCV nonstructural protein 3 (NS3) replicon (Luc-Neo NS3 replicon Huh7),39 following 808 nm NIR laser irradiation. After live/dead cell staining with calcein AM and EthiD-1, the - 13 -



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designated area with hyperthermia was observed by fluorescence microscopic imaging. (Figure 6b) Compared to conventional spherical gold nanoparticles which commonly used in the field of drug delivery and therapy, pAuNPs expected to be more efficient due to higher surface-tovolume ratio and significant photothermal conversion efficiency under the NIR irradiation due to their porous nanostructure and NIR wavelength located absorption spectrum.40 Gene delivery using a previously reported DNAzyme (Dz) sequence for binding and cleaving NS3encoded genomic RNA of HCV was performed.41,42 The loading strategy for Dz utilized the same interaction between Au and the end-labeled thiol as in the TAT peptide conjugation. Fluorescein-labelled Dz (FAM-Dz; 5`-FAM-AAT GGG GAG GCT AGC TAC AAC GAG GCT TTG C-thiol-3`, catalytic motif of DNAzyme presented as underlined letters) was used to conveniently quantify and confirm intracellular delivery. To pAuNPs with an extinction value of 1 OD, various concentrations of FAM-Dz were added and incubated to investigate loading capacity and efficiency. By optimization of the FAM-Dz loading process, 150 pmol of FAM-Dz was added to the pAuNPs with an 1 OD and the loading profile was observed for 24 h. Within the first 3 h, about 50 % of FAM-Dz was conjugated to the surface of the pAuNPs and little was loaded thereafter. (Fig 6e) In order to analyze the intracellular releasing of FAM-Dz loaded onto the pAuNPs, the releasing profile of the FAM-Dz in the emulated intracellular environment, which contained a high concentration of glutathione (2 mM GSH, 1X PBS) for ligand exchange with conjugated FAM-Dz, was obtained over 24 h. The control experiment (0 mM GSH, 1X PBS) showed a 2% release of FAM-Dz, whereas about 68% of FAM-Dz was released within 24 h of incubation in the emulated intracellular environment. (Figure 6f) Simultaneous loading of FAM-Dz and the TAT peptide for - 14 -


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therapeutic application was carried out by placing a mixture of them in the pAuNPs. The loading capacity decreased (~11.8%) because of crowding and competitive binding. (Figure 6e) Finally, the therapeutic efficacy of thermo-gene dual-modal treatment by using the FAM-Dz/TAT-pAuNPs against NS3 replicon Huh7 cells was evaluated. In comparing the results to control sets of non-treated cells (100% viable), with only NIR irradiation (93.69%), pAuNPs (101.75%), and TAT-pAuNPs (99.68%), mono-modal treatment exhibited a somewhat enhanced therapeutic efficiency. In the case of mono-treatment of gene (FAM-Dz), FAM-Dz-pAuNPs (81.14%) and FAM-Dz/TAT-pAuNPs (47.45% viable) showed a significant decrease of cell viability compared to the free FAM-Dz treatment (98.65% viable). Furthermore, in a thermal mono-treatment test, TAT-pAuNPs under NIR irradiation showed much higher therapeutic efficacy (45.96% viable) than pAuNPs alone under NIR irradiation (97.53% viable) in cancer cell ablation. These results show the importance of the delivery vehicle in the effectiveness of treatment. In the present evaluation, the 808 nm NIR laser power (or irradiation time) was reduced compared to that used in-vitro heat generation tests for emphasized comparison with the dual-modal approach. The treatment showed an extremely effective cancer cell ablation efficiency (10.98% viable), which successfully confirmed the synergistic effect of combinational therapy. (Figure 7a) To visually show FAM-Dz delivery and the change in cell viability from gene-thermo combination treatment, staining with Hoechst33342 (blue) and EthiD-1 (red) was investigated using fluorescence microscopy. Expressed green fluorescence from the released FAM-Dz and red fluorescence from apoptotic cells showed high agreement with cell viability test results. (Figure 7b)

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Conclusion In conclusion, pAuNPs were conveniently synthesized using seed-mediated reducing agentassisted excessive galvanic replacement of Ag nanoplate with excess of the replacing ion, AuCl4-. Fragmentation of the Ag nanoplate was generally a major problem under this excessive condition, but the nanostructure stabilizer PVP and competitive reductant L-AA in the galvanic replacement helped to overcome the fragmentation and aggregation by producing porous Au nanostructures. As far as we know, this report is the first in-depth analysis of such an unconventional galvanic replacement reaction with respect to the utilized type and concentration of additives. Based on an excellent photothermal conversion effect was expected from the absorption spectra of the nanostructure, it was applied to gene-thermo combinational cancer therapy. The loading of FAM-Dz and TAT peptides using Au-thiol linkage for convenient cargo conjugation on the surface of the pAuNPs was accomplished, and low cytotoxicity without biocompatible coating was observed in the pAuNPs. As a result, a highly efficient gene-thermo combinational therapy was realized by utilizing the selective intracellular releasing strategy of the loaded FAM-Dz and the photothermal conversion effect by NIR laser irradiation. The results showed that the studied reducing agent-assisted excessive galvanic replacement reaction is a viable route for the formation of pAuNPs. Moreover, the resulting porous structures with their excellent physicochemical properties are expected to be highly useful in various fields including bio-sensing and cancer treatment.

Methods Materials. Hydrogen tetrachloroaurate (III) hydrate was purchased from Kojima - 16 -


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Chemicals Co. (Sayama, Saitama, Japan). Silver nitrate, hydrogen peroxide (30%), trisodium citrate dihydrate, sodium borohydride were purchased from Junsei (Tokyo, Japan). 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), L-ascorbic acid, Citric acid, polyvinylpyrrolidone (Mw 29 kD) 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/DEAD viability/cytotoxicity assay kit was purchased from Molecular Probes Invitrogen (Carlsbad, CA, USA). G418 was purchased from A.G. Scientific, Inc. (CA, USA). Fluorescein labelled thiolated DNAzyme (FAM-Dz) which was designed to silence HCV NS3 gene was prepared as 5`-FAM-AAT GGG GAG GCT AGC TAC AAC GAG GCT TTG C-3`-SH and purchased from Genotech (underlined letters: catalytic motif of DNAzyme) (Seoul, Korea). All chemicals were used as received. TAT peptide (CGGYGRKKRRQRRR) was synthesized by solid-phase peptide synthesis (SPPS) method.

Seed-mediated synthesis of porous Au nanoplates (pAuNPs)

Preparation of Ag nanoseeds 250 µL of 10 mM AgNO3, 300 µL of 30 mM trisodium citrate dihydrate Na3Cit, 1.5 mL of 3.5 mM PVP (Mw= 29 kDa), 24.75 mL of DI water were added to 50 mL glass vial, then 60 µL of 30% hydrogen peroxide was added and gently stirred for homogeneous mixing. To the mixture, 250 mL of 100 mM sodium borohydride solution was injected followed by incubation for 3 h at room temperature. The addition of sodium borohydride to the solution - 17 -



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lead to color change to pale yellow, then further changes were occurred to transparent, deep yellow, orange and finally purple during 3 h of reaction time. When no further color change occurs, proceed to the growth reaction step without any purification process.

Synthesis of Ag nanoplates by seed growth method The growth reaction is based on the case that the volume of the seed solution is 10 mL, which is the same as the increase or decrease through proportion. In detail, to the 10 mL of seed solution, 0.125 mL of 75 mM trisodium citrate dihydrate and 0.375 mL of 100 mM Lascorbic acid were added. Next, the growth solution was prepared 20 mL of 1 mM AgNO3, 0.125 mL of 100 mM citric acid and 10 µL of 75 mM trisodium citrate dihydrate. To the seed solution mixture, 5 mL of growth solution was added by 1 mL per 5 sec rates. Through this process, 1st growth Ag nanoplates were formed. For additional growth, 5 mL of growth solution was added per growth step in the same manner to above solution. For the preparation of porous Au nanoplates by reducing-assisted galvanic replacement, proceed to the transformation reaction step without any purification process.

Synthesis of porous Au nanoplates (pAuNPs) by galvanic replacement For reducing-assisted galvanic replacement, prepared Ag nanoplates solution containing additives was diluted by DI water. To 10 mL glass vial, 1 mL of Ag nanoplates were added then 1.5 mL of DI water was added. 1, 2, 4, 10, and 20 v/v% pAuNPs were manufactured by addition of 50, 100, 200, 500, and 1000 µL of 1 mM AuCl4- solution, respectively. After the addition of replacing AuCl4- ion solution, the mixtures were incubated - 18 -


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for 2 h at room temperature without disturbance. Final products were purified by centrifugation at 8000 rpm for 15 min and washed with DI water for 3 times.

Characterization of the prepared Ag nanoplates and pAuNPs Energy-filtering transmission electron microscope LIBRA 120 (Carl Zeiss, Germany), JEM-2100F HR (JEOL Ltd., Japan) were used to obtain images of prepared nanoplates. UVVis spectrophotometer S—3100 (Scinco, Republic of Korea) and SynergyMx (Biotek, UK) were used to obtain UV-Vis absorption spectra. Fluorescence was measured by spectrofluorometer FP-8300 (Jasco Inc., USA). 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) and Ti inverted fluorescence microscope (Nikon Co., Japan) and a CoolSNAP cf charge-coupled device (CCD) camera (photometrics, Tucson, AZ, USA).

Loading of TAT peptide and FAM-Dz onto pAuNPs

TAT peptide conjugation 20 µL of 1 µM TAT paptide in distilled water stock solution was added to the 1 mL of pAuNPs with the extinction value of 1 OD. To achieve the loading of TAT peptide to pAuNPs, the reaction mixture was incubated for 12 h on a horizontal shaker at 180 rpm, room temperature in dark. The unbound TAT peptide was removed by centrifugation at 7,000 rpm for 15 min each and washed with distilled water three times. Finally, TAT peptide loaded pAuNPs was redispersed in 1 mL of 1X PBS. - 19 -



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FAM-Dz conjugation 15 µL of 10 µM FAM-Dz-SH (FDz) in distilled water stock solution was added to the 1mL of pAuNPs with the extinction value of 1 OD. To achieve the loading of FDz to pAuNPs, the reaction mixture was incubated for 12 h on a horizontal shaker at 180 rpm, room temperature in dark. The unbound substrate was removed by centrifugation at 7,000 rpm for 15 min each and washed with distilled water three times. Finally, FDz loaded pAuNPs was redispersed in 1 mL of 1X PBS. Calculation of loaded FDz was based on fluorescence spectra of FAM (λex. = 495 nm, λem. = 520 nm) in centrifuged supernatant.

TAT peptide and FAM-Dz dual conjugation The mixture of 15 µL of 10 µM FDz and 20 µL of 1 µM TAT peptide in distilled water stock solution was sequentially added to the 1 ml of pAuNPs with the extinction value of 1 OD. The reaction mixture was then incubated for 12 h on a horizontal shaker at 180 rpm, room temperature in dark. The unbound substrate was removed by centrifugation at 7,000 rpm for 15 min and washed with distilled water three times. Finally, FDz/TAT peptide coloaded pAuNPs was redisperded in 1 mL of 1 x PBS. Calculation of loaded FDz was based on fluorescence spectra of FAM in centrifuged supernatant.

FAM-Dz releasing profile To monitor the release of loaded FDz under cytoplasm imitated conditions, the FDz-pAuNPs - 20 -


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was dispersed in phosphate buffered saline (pH 7.4) solution with/without 2 mM glutathione at room temperature. At each 0, 1, 2, 3, 6, 9, 12, 18, and 24 h of observation, the solution of FDz-pAuNPs was centrifuged at 7,000 rpm for 15 min by Centrifuge 5418 (Eppendorff, Germany) to pull down the nanocomplex, and the amount of the released FDz was determined by measuring fluorescence spectra of FAM, respectively.

Characterization of photothermal effect

Temperature elevation measurement To assess the temperature elevation by photothermal conversion, 1 mL of pAuNPs with the extinction value of 1 OD, and control 1X PBS were placed in 2 mL tubes. The 808 nm NIR laser was irradiate to each solution with intensity of 4 W/cm2 for 3 min, and the temperature change was measured by a digital thermometer in every 30 s.

Cell based hyperthermia measurement To examine hyperthermia by photothermal conversion, TAT-pAuNPs in 1X PBS was treated to NS3 replicon Huh7 cells in 12-well plate that were seeded with confluency of 80,000 cells/well. After 6 h of incubation in a humidified 5% CO2 incubator at 37 ℃, residual TAT-pAuNPs was removed and washed with 1X PBS two times, followed by replacing with serum-containing media. Next, the cells were irradiated with 808 nm NIR laser with intensity of 4 W/cm2 for 5 min at ambient condition and incubated for additional 12 h. Following incubation, 500 µL of the combined live-dead cell staining solution (2 µM calcein AM and 4

µM EthiD-1 in D-PBS) was added to each well and incubated for 20 min for staining. - 21 -



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Fluorescent images of the cells were obtained using a fluorescence microscopy.

Cell viability assay

Cell culture The human hepatoma cell line Huh7 containing NS3 hepatitis C virus RNA was grown in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 4.5 g/L D-glucose, supplemented with 10% FBS (fetal bovine serum), 100 units/mL penicillin, 100 mg/mL streptomycin and 500 µg/mL of G418. The cells were grown in a humidified 5% CO2 incubator at 37℃.

MTT assay for cell viability measurement MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) powder was dissolved in 1X PBS at 5 mg/mL concentrations and filtered through a 0.2 µm pore sized sterilized syringe filter. The stock solution was stored at 4 ℃. The NS3 replicon Huh7 cells were seeded with a density of 10,000 cells per well of a 96-well culture plate with 100 µL of growth media (about 50-70% confluency). To compare the TAT-pAuNPs and pAuNPs mediated drug delivery efficiency, the cells were treated with free FDz, FDz-pAuNPs, and FDz/TAT-pAuNPs in serum free media and incubated for 6 h at 37 ℃. Then, the cells were rinsed with 1X PBS, 2 times followed by replacing with serum-containing media and incubated for 12 h at 37 ℃. After that, the cells were rinsed with 1 x PBS two times. 100 µL of serum free media at 0.5 mg/mL - 22 -


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concentrations of MTT was added to the cells and incubated for 2 h until a purple color developed to detect the metabolically active cells. The media was discarded, and the cells were rinsed with 1X PBS one time. Then, 100 µL DMSO was added to each well to make water insoluble formazan salt solubilized. The optical densities of each well in the plates were measured at 560 nm. Mean and standard deviation of triplicated were calculated and plotted. To investigate the synergistic effect of NIR irradiation with pAuNPs-based chemotherapy, after cells were treated with free FDz, TAT-pAuNPs, and TAT/FDz-pAuNPs in serum free media and incubated for 6 h at 37 ℃. Then, the cells were rinsed with 1X PBS, 2 times followed by replacing with serum-containing media. Next, the cells were irradiated with 808 nm NIR laser with intensity of 4 W/cm2 for 2 min at ambient condition and incubated for 12 h at 37 ℃.

After that, the cells were rinsed with 1X PBS two times. 100 µL

of serum free media at 0.5 mg/mL concentrations of MTT was added to the cells and incubated for 2 hr until a purple color developed to detect the metabolically active cells. The media was discarded, and the cells were rinsed with 1X PBS one time. Then, 100 µL DMSO was added to each well to make water insoluble formazan salt solubilized. The optical densities of each well in the plates were measured at 560 nm. Mean and standard deviation of triplicated were calculated and plotted.

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Figure 1. Galvanic replacement of Ag nanoplates with L-AA and PVP against AuCl4variables.

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Figure 2. Galvanic replacement mediated nanostructure transformation analysis against various concentrations of [Au3+] with fixed [L-AA], [PVP] and [Na3Cit]. (a) Extinction spectrum of prepared nanostructures and the proportion of each nanostructures. (b) TEM images for 0 to 20 v/v% addition of 1 mM AuCl4- solution to Ag nanoplates. The scale bar is 100 nm. - 25 -



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Figure 3. Galvanic replacement mediated nanostructure transformation analysis without LAA against various concentrations of [Au3+] with fixed [PVP] and [Na3Cit]. (a) Extinction spectrum of prepared nanostructures and the proportion of each nanostructures. (b) TEM images for 0 to 20 v/v% addition of 1 mM AuCl4- solution to Ag nanoplates. The scale bar is 100 nm.

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Figure 4. Galvanic replacement against the concentrations of L-AA with fixed [PVP], [Na3Cit], and [Au3+] as 10 v/v%. (a) Extinction spectrum of formed nanostructures and their proportion variation. (b) TEM observation of nanostructures against the concentrations of LAA. The scale bar is 100 nm.

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Figure 5. Time dependent transformation of Ag nanoplate into pAuNPs under the existence of L-AA, PVP, and Na3Cit. The scale bar is 100 nm.

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Figure 6. Gene-thermo combinational therapeutic feasibility confirmation by using pAuNPs. (a) Photothermal conversion of pAuNPs against control set of 1X PBS under the 808 nm NIR laser. (b) Hyperthermia verification of TAT-pAuNPs to NS3 replicon Huh7 cells. The scale bar is 200 µm. (c) Bright field image of NS3 replicon Huh7 cell after treatment with pAuNPs and TAT-pAuNPs. Introduced pAuNPs can be distinguished by black dots. The scale bar is 100 µm. (d) DLS and zeta potential of pAuNPs and TAT-pAuNPs for the confirmation of TAT conjugation onto pAuNPs surface. (e) loading and (f) releasing profile of FAM-Dz. - 29 -



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Figure 7. Quantitative evaluation of therapeutic efficacy of gene-thermo combinational cancer therapy with FAM-Dz/TAT-pAuNPs against NS3 replicon Huh7 cells. (a) The relative cell viability comparison exhibited therapeutic efficacy of mono- and dual-treatment manner. (b) Fluorescence microscopy images of the cells treated with various experimental sets - 30 -


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clearly exhibited the successful delivery of FAM-Dz and synergistic effect of gene-thermo therapy. The scale bar is 100 µm.

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Table 1. Chemical equations and standard reduction potentials of one- and two-step reduction pathway.

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Supporting Information Transmission electron microscopy (TEM), UV-Vis spectrophotometer, Cell viability assay, Elemental mapping. Acknowledgemet This work was supported by National Research Foundation of Korea (NRF) funded by Korean government (Grant Nos. NRF-2016R1C1B1008090). This work was supported by the Basic Science Research Program (2016R1A4A1010796), the International S&T Cooperation Program (2014K1B1A1073716), and the Research Center Program of IBS (IBS-R008-D1) through the National Research Foundation of Korea (NRF). This research was supported by ‘NST Research Fellowship for Young Scientists’ at KBSI through NST in South Korea. The present research has been conducted by the Research Grant of Kwangwoon University in 2017.

‡Seounghun Kang and Kyunglee Kang were equally contributed.

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Reducing Agent-Assisted Excessive Galvanic Replacement Mediated

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