Synthesis of Fluorescent Au NanocrystalsâSilica Hybrid

Subscriber access provided by READING UNIV

Article

Synthesis of Fluorescent Au Nanocrystals-Silica Hybrid Nanocomposite (FLASH) with Enhanced Optical Features for Bio-imaging and Photodynamic Activity Taeshik Kim, Hongje Jang, Seongchan Kim, Jong-Hwan Lee, Sung-Yon Kim, Noo Li Jeon, Joon Myong Song, and Dal-Hee Min Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b02562 • Publication Date (Web): 03 Dec 2017 Downloaded from http://pubs.acs.org on December 4, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Langmuir is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 23 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Synthesis of Fluorescent Au Nanocrystals-Silica Hybrid Nanocomposite (FLASH) with Enhanced Optical Features for Bio-imaging and Photodynamic Activity

Taeshik Kim1,2‡, Hongje Jang3‡, Seongchan Kim1,2, Jong-Hwan Lee1,2, Sung-Yon Kim2, Noo Li Jeon4, Joon Myong Song5 and Dal-Hee Min1,2,6*

1

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

2

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

3

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

Republic of Korea 4

Department of Mechanical and Aerospace Engineering, Seoul National University, Seoul 08826,

Republic of Korea 5

College of Pharmacy, Seoul National University, Seoul 08826, Republic of Korea

6

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

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

KEYWORDS: gold nanocrystal, hybrid nanocomposite, bio-imaging, photodynamic therapy, cancer therapy

ACS Paragon Plus Environment

1

Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 23

Abstract

Fluorescent Au nanocrystals-silica hybrid nanocomposite (FLASH) was synthesized by co-condensation of surface modified Au nanocrystals (AuNCs). Present FLASH nanocomposite exhibited 4 times enhanced photoluminescence and photocatalytic activity compared to single nanocrystals. Based on these enhanced optical features, we successfully demonstrated in vitro fluorescence bio-imaging of introduced FLASH to human cervical cancer cell line HeLa. Beyond the confirmation of photocatalytic activity from the photodegradation of methylene blue as model compound, regional selective photodynamic therapy of HeLa cells under the UV irradiation was also presented. Taken together the enhanced optical features and further potential in theranostic applications, we expect the present FLASH can be promising tool for nanobiotechnology field.

Introduction

Gold nanocrystals (AuNCs) were defined as 2-5 nm sized nanostructures consisting of tens to hundreds of Au atoms.1 Since the birth of AuNCs, numerous approaches have been reported for facile synthesis of AuNCs by using thiolated organic or biomolecules including alkyl chains, glutathione (GSH), and bovine serum albumin (BSA).2,3 Generally, AuNCs exhibited quite different physical and optical properties from conventional gold nanoparticles (AuNPs).4 Although AuNCs does not exhibit a distinctive localized surface plasmon resonance (LSPR) peaks due to their ultra-small size, AuNCs are characterized by its photoluminescence (PL) under the irradiation of ultraviolet (UV) or short wavelength visible light (VIS) by quantum confinement effect.5 Based on the small size and PL characteristics, AuNCs are favoured as a type of quantum dots in various application fields


2

Page 3 of 23 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

including

fluorescence-based

sensing,6

bio-imaging,7-9

drug

delivery,10

and

nanocatalyst.11,12 Beyond these properties, another interesting feature of AuNCs is the generation of reactive oxygen species (ROS) under the light irradiation.13,14 ROS, referred also as oxidative radicals, is generated from incomplete reduction of oxygen.15 The production of ROS in living organisms induces oxidation of various essential biomolecules including DNA, amino acids, and organelles, thereby triggering aging or apoptosis.16 However, controlled production of ROS under regulated conditions enables its therapeutic application, so called photodynamic therapy (PDT).17 Researches on the efficient ROS generation using nanocrystals have been extensively studied mainly focusing on

the

development

of

new

materials

including

copper

chalcogenides,18-21

semiconductors,22-24 and plasmonic metal nanocrystals.25,26 Herein, we synthesized Fluorescent Au nanocrystals-silica hybrid nanocomposite (FLASH) by co-condensation method for enhanced bio-imaging and cancer cell therapy via photodynamic activity. Our study revealed that FLASH possessed enhanced photoluminescence compared to general stand-alone AuNCs by higher population from encapsulated structure. The four times enhanced quantum yield of FLASH was sufficient to perform bio-imaging for monitoring the internalization of nanocomposite in vitro. In addition, despite the surface coverage with silica nanoshell, FLASH efficiently represented enhanced photocatalytic activity than naked AuNCs as verified by degradation of methylene blue (MB) as a model. Based on these enhanced optical features, we successfully demonstrated bio-imaging and PDT in vitro.

Results and discussion


3

Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 23

First, we synthesized light emitting AuNCs through previously reported method with slight modification.27 (Figure 1) Briefly, appropriate amount of AuCl4- was added to pre-heated GSH which plays a key role as reducing and surface stabilizing agent at the same time, followed by further incubation of 2 hr to ensure the formation of monodisperse AuNCs. After the purification, manufactured AuNCs were re-dispersed in phosphate buffered

saline

hydroxysuccinimde

(PBS)

and

(EDC/NHS)

1-ethyl-3-(3-dimethylaminopropyl)carbodiimide/Ncoupling

reaction

was

performed

with

(3-

aminopropyl)triethyoxysilane (APTES) for following AuNCs embedded silica shell formation by sol-gel method. The prepared APTES-AuNCs complex was mixed with tetraethyl orthosilicate (TEOS) in ethanol, and addition of ammonium hydroxide triggered the assembling and coating process at room temperature. The finally obtained FLASH was purified and re-dispersed in deionized water for further characterizations. The AuNCs coreclustered formation mechanism of FLASH can be explained by essential role of propyl ammonium group from ATPES conjugation onto AuNCs during sol-gel process. The balance between hydrophilic ammonium head for maintaining water dispersity and hydrophobic propyl chain for directing silica precursor enabled clustered nanostructure in single silica nanospheres.28,29 We next performed characterizations for the prepared AuNCs, APTES-AuNCs and FLASH mainly on their diameter, spatial distribution and photoluminescence properties. According to the UV-Vis spectrophotometer analysis, AuNCs exhibited strong absorption at 200 nm, but exponentially diminished to show no optical density over 500 nm wavelength. (Figure S1a) Observed extinction spectra were identical to those in the previously reported AuNCs.[16] In case of APTES-AuNCs, overall extinction spectrum was


4

Page 5 of 23 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

coincided except newly appeared small shoulder around 260 nm from conjugation with silane.28(Figure S1b) On the other hand, FLASH exhibited relatively gentle decrease of absorption up to 800 nm wavelength might be originated from superposition and population of clustered AuNCs in the nanostructure core. (Figure 2b) From the Fourier transformed infrared (FT-IR) measurement of synthesized AuNCs and APTES-AuNCs, successful preparation was confirmed by the existence of bands at 1,533 cm-1 (amide II, C-N stretching in combination with N-H bending) and 1,637 cm-1 (Amide I, C=O stretching of peptide bonds) from GSH, and newly emerged strong peak at 950-1,250 cm-1 (Si-O-Si stretching) from APTES. (Figure S2a) The silica shell formation for FLASH was also verified by FT-IR measurement in comparison with silica nanoparticles (SNs) without APTES-AuNCs core. The amide I & II bands were observed only in FLASH due to the APTES-AuNCs existence, and stronger shoulder band around 1,250 cm-1 might imply superposition of Si-O-Si stretching and Si-O-M stretching peak compared to SNs. (Figure S2b) According

to

the

transmission

electron

microscope

(TEM)

and

photoluminescence(PL) analysis, synthesized AuNCs exhibited diameter distribution of 3 ± 0.5 nm and distinctive emission spectrum with 610 nm absorption maxima with 3% quantum yield(QY) under the 394 nm excitation. (Figure 3) Interestingly, synthesized FLASH from the co-condensation of APTES-AuNCs with TEOS, 100 nm of central domain AuNCs clustered core and 29.6 nm thickness shell nanostructure was formed, which are different from the general core-shell nanostructures consisting of individual AuNCs and coating material. (Figure 2a) Present core domain clustered nanostructure of FLASH is different from previously reported carbon dots (CDots)-silica nanocomposite.29


5

Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 23

This difference may be originated from the greater affinity of AuNCs than CDots towards amine functional group of APTES which can affect instant crosslinking during cocondensation process. We also expect that relatively homogeneous dispersion of AuNCs in core domain might be originated from preventing of AuNCs aggregation by existence of AuNCs surface stabilizing ligand, GSH. Because of the core clustered AuNCs distribution, FLASH represented relatively weak 467 nm and strong 610 nm PL peaks under the 394 nm excitation with four times enhanced QY of 12.8%. (Figure 2b) The enhanced photoluminescence was also verified by brightness of light emitting colloidal solution under the UV irradiation. (Figure S1) High angle annular dark field scanning TEM (HAADF-STEM) image and energy dispersive spectrometer (EDS) elemental mapping image additionally supported the central domain clustered core-shell structure of FLASH. (Figure 2c) MB solution was next applied to verify the photocatalytic activity of FLASH, which was visualized by photodegradation of MB under the 48 W ultraviolet(UV) irradiation from blue to colourless. 86.2% of MB was degraded by photocatalytic reaction of 100 µg/mL FLASH for 90 min, which was calculated by extrapolation followed by relative absorption maxima (λ=664 nm) comparison. In case of AuNCs, 40.2% of MB was degraded by theoretically same concentration of AuNCs as in FLASH. In contrast, MB without nanomaterial and silica nanoparticle (SN) exhibited degradation of just 11.0 and 4.1% of MB during 90 min of irradiation, respectively. Observed photocatalytic enhancement of FLASH could be explained by the increased absorbance at UV-Vis region due to interparticle electromagnetic coupling by close AuNCs proximity locations. (Figure 3 and Figure S5, S6) To consider the adsorption of MB onto nanomaterial surface and inevitable


6

Page 7 of 23 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

auto-degradation, we further performed the MB degradation experiment with same concentrations in dark condition for 90 min of incubation time. Absorbance at 664 nm of MB only, with SN, with AuNCs, and with FLASH represented 3%, 0%, 7%, and 5% of intensity decrease, respectively. (Figure S7) Comprehensively, the photocatalytic activity of FLASH was higher than 80% even considering the decrease of the absorbance through the nanomaterial surface adsorption and auto-degradation. To reveal the origin of photocatalytic activity of FLASH, we further synthesized and characterized the FLASH derivatives by using the different amount of TEOS for nanocomposite formation. (Figure S8) Interestingly, the photocatalytic activity of FLASH was determined not solely by the amount of embedded AuNCs, but combinational feature with the formed SiO2 shells environment. (Figure S9) The observed enhanced QY and significant photocatalytic activity of FLASH suggested further applications for bio-imaging agent and photosensitizer (PS). Prior to cell based bio-imaging and photodynamic therapeutic applications, we studied cytotoxicity of manufactured FLASH against human cervical cancer cell line (HeLa) by using cell counting kit-8 (CCK-8) assay. Up to the concentration of 500 µg/mL FLASH treatment, over 91% of cell viabilities were observed without UV irradiation. (Figure 4a) Compared to the low cytotoxicity of FLASH, UV irradiation mediated photodynamic treatment clearly exhibited dose-dependent decrease of cell viability. According to the 0.2 W/cm2 of 365 nm UV irradiation for 10 min followed by CCK-8 assay, drastic cell viability decreases were observed as 53.67% (125 µg/mL), 27.08% (250 µg/mL) and 6.32% (500 µg/mL). (Figure 4a) Photodynamic therapy was also confirmed by Calcein AM/EthiD-1 live/dead staining followed by fluorescent microscopic observation. The HeLa cell ablation


7

Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 23

was selectively accomplished only in designated region where UV treated. Compared to FLASH, AuNCs did not show sufficient photodynamic therapeutic efficiency from live/dead staining image. (Figure 4b) Next, we performed fluorescence live cell bio-imaging by using FLASH against HeLa cell. After the treatment of FLASH and following 4 hr of incubation, cell nuclei were stained by Hoechst 33342 for clear observation of the intracellular FLASH distribution. According to the Hoechst 33342 (λex/λem= 365/461 nm) and FLASH (λex/λem= 492/575 nm) signal, intracellular uptake and bio-imaging of the intracellular distribution via FLASH fluorescence were successfully demonstrated. (Figure 5)

Conclusion

In conclusion, we synthesized fluorescent AuNCs through facile route by using GSH as reducing and surface stabilizing agent. Further modification with APTES and following sol-gel silica shell formation resulted silica@clustered-AuNCs shell@core FLASH nanocomposite. FLASH exhibited enhanced QY and photocatalytic activity under the UV irradiation. Taken together, FLASH showed excellent biocompatibility, water dispersibility, enhanced photoluminescence and photocatalytic activity, so we expect that the present FLASH can be a promising nanocomposite for various biomedical applications.

Methods Materials: Hydrogen tetrachloroaurate (III) hydrate was purchased from Kojima Chemicals Co.


8

Page 9 of 23 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

(Sayama, Saitama, Japan). L-Glutathione reduced (GSH), (3-aminopropyl)triethoxysilane (APTES) and tetraethyl orthosilicate (TEOS) were purchased from Sigma (St. Louis, MO, USA). Ammonium hydroxide (25%), ethyl alcohol (95%) and hydrochloric acid (35%) were purchased from DAEJUNG chemical & metals (Siheung, Kyungki-do, Republic of Korea). 10X phosphatebuffered 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). 10K Amicon filter was purchased from Millipore (Billerica, MA, USA).

Synthesis of AuNCs: 50 mL of 3.6 mM L-glutathione solution was heated at 95 ºC. Then, 605 μL of 0.248 M HAuCl4 solution was added under vigorous stirring for 2hr. The synthesized AuNCs were purified by centrifuging at 17,000g to remove large aggregates after reaction. The supernatant was further purified with using 20 mM HCl to adjust the solution pH to 3~ 4, and then ethanol was added to the solution to make precipitation. The product was purified with using ethanol 3 times at 6500 rpm for 10 min by centrifuge 5810R (Eppendorf, Germany). The precipitates were dried in a vacuum oven and re-dispersed in 50 mL of 1X PBS buffer.

Preparation of APTES-AuNCs: Both 11 mg of EDC and 8 mg of NHS added 20 mL of AuNCs solution with stirring for 15 min to activate carboxylic group. After pre-activation, 25 μL of APTES was added in the solution and reacted for 4 hr with stirring. The product was purified with distilled water using 15 mL of 10K Amicon filter 3 times at 6500 rpm for 10 min. Finally, APTES modified AuNCs were re-dispersed in 5 mL of distilled water.


9

Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 23

Synthesis of FLASH: 200 μL of APTES-AuNCs and 5 μL of TEOS were mixed in 1 mL of ethanol. After that, 1 mL of ethanol, 300 μL of distilled water and 30 μL of 25 % NH4OH solution were added. Co-condensation was reacted for 3 hr at room temperature. After reaction, the product was purified 3 times with distilled water by centrifugation at 8000 rpm for 10 min until supernatant did not emit fluorescence of unreacted AuNCs under UV irradiation.

Measurement of photocatalytic activity: 1 mL of prepared 20 μM MB without nanomaterials, 20 μM MB with 100 μg/mL FLASH, 20 μM MB with 10 μL of 187.7 nM AuNCs that are theoretically same concentration with those in FLASH, 20 μM MB with 100 μg/ mL silica nanoparticles (SNs) were irradiated by using 48W UV-transilluminator (ATTO, Korea) during 90 min. Photodegradation of MB was measured by UV-Vis spectroscopy in every 10 min in 1hr.

Characterization of AuNC, APTES-AuNC, FLASH: Energy-filtering transmission electron microscope LIBRA 120 (Carl Zeiss, Germany) and high-resolution TEM (JEM-2100F, USA) were used to obtain images of nanoparticles. Energy dispersive spectroscopy was carried out by HADDF-STEM. UV-Vis spectrophotometer S-3100 (Scinco, Korea) were used to obtain UV-Vis absorption spectra. Fluorescence was measured by spectrofluorometer FP-8300 (Jasco Inc., U.S.A.). 365 nm irradiation was performed by 365-nm LED (Shanghai Yulitech Co., Ltd., China, 0.2 W/cm2). Quantum yield was measured by Quantum Efficiency Measurement System QE-1200. Photoluminescence (PL) was measured by ACTON spectrometer (Princeton instruments). Fourier transform Infrared (FT-IR) was measured by VERTEX 70 (Bruker, USA) with HYPERION


10

Page 11 of 23 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

microscope.

Cell culture: Human cervical cancer cell line (HeLa) was grown in DMEM containing 10% FBS, 100 units/mL penicillin, 100 mg/mL streptomycin at 5% CO2, 37°C.

Cell viability test: To quantitatively investigate cell viability related to the photodynamic effect of FLASH, HeLa cells (1 × 104 cells/well) were plated in a 96-well plate, incubated for 24 hr, and incubated with various concentrations of FLASH. After 12 hr incubation, each well was irradiated with a 365-nm LED (Shanghai Yulitech Co., Ltd., China, 0.2 W/cm2) for 10 min, and then, the medium was replaced with a serum-containing medium. After 12 hr incubation, the cells were then carefully washed with 1x PBS and CCK-8 assay solution was added for 1 hr with serum-free medium, followed by measuring absorbance at 450 and 670 nm wavelength by using a microplate reader (Molecular Devices, Inc., USA). All experiments were carried out in triplicates.

Photodynamic therapy in vitro: To investigate the therapeutic effect of photodynamic therapy in vitro, FLASH (200 μg/mL) and AuNC in serum-free medium were treated to HeLa cells (1.2 × 105 cells/well), which had been pre-incubated in a 12-well plate for 24 hr. After the medium exchange to a serum-containing cell culture medium, the cells were irradiated with a 365 nm LED (0.2 W/cm2) for 10 min. After further 12 hr incubation, each well was treated with Live/Dead assay reagent based on the manufacturer’s protocol. The bright field and fluorescence images of the cells were obtained by using an inverted fluorescence microscope, IX70 (Olympus, Japan) with a 4x objective. The acquisition images were operated for pseudo-coloring, fluorescent intensity and


11

Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 23

background elimination process using internal supplied software.

Cellular imaging of FLASH: HeLa cells (5.0 × 104 cells/well) were seeded and grown in an 8-well glass chamber for 24 hr and FLASH (200 μg/mL) was added to each well in a serum-free medium for 4 hr. After incubation, the cells were carefully rinsed with 1x PBS, and the medium was replaced with a serum-containing fresh medium. After nucleus was stained with Hoechst 33342 staining kit by manufacturer’s protocol, the bright field and fluorescence images were obtained by using a Delta Vision Elite Microscopy System with a 60x objective. The acquisition images were operated for pseudo-coloring, fluorescent intensity and background elimination process using internal supplied software.


12

Page 13 of 23 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Figure 1. Schematic illustration of FLASH synthetic procedure.


13

Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 23

Figure 2. Characterization of synthesized FLASH. (a) TEM, (b) UV-Vis and PL spectrum (left) with digital image of light emission under UV irradiation (right), and (c) elemental mapping of Au Lα, Si Kα, Si Kα with dark-field image. The scale bars are 50 nm in (c).


14

Page 15 of 23 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Figure 3. photocatalytic degradation of MB under the 48 W UV light irradiation. (a) MB only and (b) MB with SNs did not showed enough photodegradation property. (c) MB with AuNCs exhibited decrease of absorption spectra and (d) MB with FLASH clearly represented significantly enhanced photocatalytic activity.


15

Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 23

Figure 4. FLASH cytotoxicity assay against HeLa cell line to verify photodynamic therapy feasibility. (a) CCK-8 assay exhibited UV irradiation triggered FLASH mediated photodynamic cell ablation and (b) live/dead staining visually supported selective photodynamic therapy in designated region. The scale bar is 100 µm.


16

Page 17 of 23 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Figure 5. Fluorescence images of the HeLa cells treated with FLASH. The cell nuclei were stained by Hoechst 33342 (blue) and introduced FLASH was observed as red signal in perinuclear region. The scale bar is 25 µm.


17

Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 23

Acknowledgment This work was supported by the Basic Science Research Program (2016R1E1A1A01941202, 2016R1A4A1010796), International S&T Cooperation Program (2014K1B1A1073716) and the Research Center Program (IBS-R008-D1) of IBS (Institute for Basic Science) through the National Research Foundation of Korea (NRF).

Author Contributions ‡ Taeshik Kim and Hongje Jang equally contributed to this manuscript.

Notes The authors declare no competing financial interest.

References 1. M. Sakamoto, T. Tachikawa, M. Fujitsuka, T. Majima, Photoreactivity of As-Fabricated Au Clusters at the Single-Cluster Level. J. Am. Chem. Soc. 2009, 131(1), 6-7. 2. J. Xie, Y. Zheng, J. Y. Ying, Protein-Directed Synthesis of Highly Fluorescent Gold Nanoclusters. J. Am. Chem. Soc. 2009, 131(3), 888-889. 3. J. Liu, M. Yu, C. Zhou, S. Yang, X. Ning, J. Zheng, Passive Tumor Targeting of RenalClearable Luminescent Gold Nanoparticles: Long Tumor Retention and Fast Normal Tissue Clearance. J. Am. Chem. Soc. 2013, 135(13), 4978-4981. 4. G. Ramakrishna, O. Varnavski, J. Kim, D. Lee, T. Goodson, Quantum-Sized Gold Clusters as Efficient Two-Photon Absorbers. J. Am. Chem. Soc. 2008, 130(15), 5032.


18

Page 19 of 23 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

5. L. Hu, L. Deng, S. Alsaiari, D. Zhang, N. Khashab, “Light-on” Sensing of Antioxidants Using Gold Nanoclusters. Anal. Chem. 2014, 86(10), 4989-4994. 6. Y. T. Wu, C. Chanmugam, W. B. Tseng, M. M. Hiseh, W. L. Tseng, A Gold Nanoclusterbased Fluorescent Probe for Simultaneous pH and Temperature Sensing and Its Application to Cellular Imaging and Logic Gates. Nanoscale, 2016, 8, 11210-11216. 7.

F. Bi, H. Yin, S. Zheng, Q. Zhu, H. Yang, M. Kang, F. Gan, X. Chen, One-step Synthesis of Peptide Conjugated Gold Nanoclusters for the High Expression of FGRF2 Tumor Targeting and Imaging. RSC Adv. 2016, 6, 4627-4633.

8. L. Shang, R. M. Dorlich, S. Brandholt, R. Schneider, V. Trouillet, M. Bruns, D. Gerthsen, G. U. Nienhaus, Facile Preparation of Water-soluble Fluorescent Gold Nanoclusters for Cellular Imaging Applications. Nanoscale, 2011, 3, 2009-2014. 9. X. Wu, X. He, K. Wang, C. Xie, B. Zhou, Z. Qing, Ultrasmall Near-infrared Gold Nanoclusters for Tumor Fluorescence Imaging in vivo. Nanoscale, 2010, 2, 2244-2249. 10. Y. Lei, L. Tang, Y. Xie, Y. Xianyu, L. Zhang, P. Wang, Y. Hamada, K. Jiang, W. Zheng, X. Jiang, Gold Nanoclusters-assisted Delivery of NGF siRNA for Effective Treatment of Pancreatic Cancer. Nat. Commun. 2017, 8, 15130. 11. C. Yu, G. Li, S. Kumar, H. Kawasaki, R. Jin, Stable Au25 (SR) 18/TiO2 Composite Nanostructure with Enhanced Visible Light Photocatalytic Activity. J. Phys. Chem. Lett. 2013, 4(17), 2847-2852. 12. L. Polavarapu, M. Manna, Q. H. Xu, Biocompatible Glutathione Capped Gold Clusters as One-and Two-photon Excitation Fluorescence Contrast Agents for Live Cells Imaging. Nanoscale, 2011, 3, 429-434.


19

Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 23

13. K. Zheng, M. I. Setyawati, D. T. Leong, J. Xie, Anticancer Luminescent Gold Quantum Clusters for in situ Cancer-selective Marking-imaging-targeting. ACS Nano, 2017, DOI:10.1039/C7NR02229H 14. F. Gao, W. Zheng, L. Gao, P. Cai, R. Liu, Y. Wang, Q. Yuan, Y. Zhao, X. Gao, Au Nanoclusters and Photosensitizer Dual Loaded Spatiotemporal Controllable Liposomal Nanocomposites Enhance Tumor Photodynamic Therapy Effect by Inhibiting Thioredoxin Reductase. Adv. Healthc. Mater. 2017, 6, 1601453. 15. C. S. Foote, Photosensitized Oxygenations and the Role of Singlet Oxygen. Acc. Chem. Res. 1968, 1, 104-110. 16. T. M. Buttke, P. A. Sandstrom, Oxidative Stress as a Mediator of Apoptosis. Immunol. Today, 1994, 15(1), 7-10. 17. G. Palumbo, Photodynamic Therapy and Cancer: a Brief Sightseeing Tour. Expert Opin. Drug Delivery, 2007, 4(2), 131-148. 18. Q. W. Tian, F. R. Jiang, R. J. Zou, Q. Liu, Z. G. Chen, M. F. Zhu, S. P. Yang, J. L. Wang, J. H. Wang, J. Q. Hu, Hydrophilic Cu9S5 Nanocrystals: A Photothermal Agent with a 25.7% Heat Conversion Efficiency for Photothermal Ablation of Cancer Cell in vivo. ACS Nano, 2011, 5, 9761-9771. 19. C. M. Hessel, V. P. Pattani, M. Rasch, M. G. Panthani, B. Koo, J. W. Tunnell, B. A. Korgel, Copper Selenide Nanocrystals for Photothermal Therapy. Nano Lett. 2011, 11, 2560-2566. 20. M. Zhou, R. Zhang, M. A. Huang, W. Lu, S. L. Song, M. P. Melancon, M. Tian, D. Liang, C. A. Li, A Chelator-free Multifunctional [64Cu] CuS Nanoparticle Platform for


20

Page 21 of 23 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Simultaneous Micro-PET/CT Imaging and Photothermal Ablation Therapy. J. Am. Chem. Soc. 2010, 132, 15351-15358. 21. S. Wang, A. Riedinger, H. Li, C. Fu, H. Liu, L. Li, T. Liu, L. Tan, M. J. Barthel, G. Pugliese, F. De Donato, M. S. D’Abbusco, X. Meng, L. Manna, H. Meng, T. Pellegrino, Plasmonic Copper Sulfide Nanocrystals Exhibiting Near-infrared Photothermal and Photodynamic Therapeutic Effects. ACS Nano, 2015, 9, 1788-1800. 22. R. Bakalova, H. Ohba, Z. Zhelev, M. Ishikawa, Y. Baba, Nat. Biotechnol. Quantum Dots as Photosensitizers? 2004, 22, 1360-1361. 23. A. C. Samia, X. Chen, C. Burda, Semiconductor Quantum Dots for Photodynamic Therapy. J. Am. Chem. Soc. 2003, 125, 15736-15737. 24. B. I. Ipe, M. Lehnig, C. M. Niemeyer, On the Generation of Free Radical Species from Quantum Dots. Small, 2005, 1, 706-709. 25. L. V. Nair, S. S. Nazeer, R. S. Jayasree, A. Ajayaghosh, Fluorescence Imaging Assisted Photodynamic Therapy using Photosensitizer-linked Gold Quantum Clusters. ACS Nano, 2015, 9, 5825-5832. 26. Y. -Z. Chen, Z. U. Wang, H. Wang, J. Lu, S. -H. Yu, H. -L. Jiang, Singlet Oxygen-engaged Selective Photo-oxidation over Pt Nanocrystals/Porphyrinic MOF: The Roles of Photothermal Effect and Pt Electronic State. J. Am. Chem. Soc. 2017, 139, 2035-2044. 27. J. Liu, P. N. Duchesne, M. Yu, X. Jiang, X. Ning, R. D. Vinluan III, P. Zhang, J. Zheng, Luminescent Gold Nanoparticles with Size-independent Emission. Angew. Chem. Int. Ed. 2016, 55(31), 8894-8898; Angew. Chem. 2016, 128(31), 9040-9044.


21

Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 23

28. A. Samanta, T. Rajesh, R. N. Devi, Confined Space Synthesis of Fully Alloyed and SinterResistant AuPd Nanoparticles Encapsulated in Porous Silica. J. Mater. Chem. A, 2014, 2, 4398-4405. 29. A. Samanta, B. B. Dhar, R. N. Devi, Ultrasmall Gold Cluster Arrays Encapsulated in Silica Nanospheres: Applications in Fluorescence Imaging and Catalysis. J. Phys. Chem. C, 2012, 116, 1748-1754. 30. T. Höfler, A. M. Track, P. Pacher, Q. shen, H. -G. Flesch, G. Glawacek, G. Koller, M. G. Ramsey, R. Schennach, R. Resel, C. Teichert, W. Kern, Photoreactive Molecular Layers Containing Aryl Ester Units: Preparation, UV Patterning and Post-exposure Modification. Mater. Chem. Phys. 2010, 119, 287-293. 31. C. Liu, L. Bao, B. Tang, J. -Y. Zhao, Z. -L. Zhang, L. -H. Xiong, J. Hu, L. -L. Wu, D. W. Pang, Fluorescence-converging Carbon Nanodots-hybridized Silica Nanosphere. Small, 2016, 12(34), 4702-4706.


22

Page 23 of 23 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir


Synthesis of Fluorescent Au NanocrystalsâSilica Hybrid

Recommend Documents

Synthesis of Fluorescent Au NanocrystalsâSilica Hybrid