4 - Chitosan-Stabilized Self-Assembled Fluorescent Gold

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Article
Chitosan-stabilized self-assembled fluorescent gold
nanoclusters for cell imaging and bio-distribution in vivo
Ying Duan, Ruiping Duan, Rui Liu, Man Guan, Wenjuan
Chen, Jingjing Ma, Mingmao Chen, Bo Du, and Qiqing Zhang
ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.7b00975 • Publication Date (Web): 09 Feb 2018
Downloaded from http://pubs.acs.org on February 12, 2018
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Page 1 of 25 ACS Biomaterials Science & Engineering
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2 Chitosan-stabilized self-assembled fluorescent gold nanoclusters for cell imaging and
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5 bio-distribution in vivo
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7 Ying Duana, Ruiping Duanb*, Rui Liub, Man Guanb, Wenjuan Chenb, Jingjing Mab, Mingmao Chena, Bo
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10 Dub,Qiqing Zhangab*
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a Institute of Biomedical and Pharmaceutical Technology, Fuzhou University, Fuzhou 350002, China
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15 b Institute of Biomedical Engineering, Chinese Academy of Medical Science and Peking Union Medical
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College, Tianjin Key Laboratory of Biomedical Material, Tianjin 300192, China
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20 * E-mail: rpduan@sina.com; zhangqiq@126.com.
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22 Keywords
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26 AuNCs@NAC-CS; AuNCs@BSA; Chitosan; Tumor cell imaging
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28 Abstract
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Biocompatible, near-infrared luminescent gold nanoclusters were synthesized in situ using as-prepared
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34 chitosan grafted with N-Acetyl-L-cysteine (NAC-CS). The fluorescent gold nanoclusters coated with
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chitosan-N-acetyl-L-cysteine (AuNCs@NAC-CS) was aggregated by multiple ultrasmall gold nanoclusters
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39 closing with each other, with strong fluorescence emission at 680 nm upon excitation at 360 nm.
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41 AuNCs@NAC-CS did not display any appreciable cytotoxicity on cells even at a concentration of 1.0 mg
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44 mL-1. AuNCs@NAC-CS were more insensitive to H2O2 and trypsin compared with fluorescent gold
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46 nanoclusters coated with Albumin BovineⅤ(AuNCs@BSA), which make them have long time imaging in
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49 HeLa cells. Furthermore, the obvious fluorescence signal of AuNCs@NAC-CS appeared in the liver and
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51 kidney of the normal mice after 6 h injection. And the fluorescence intensity decreased after that because of
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the highly efficient clearance characteristics of ultrasmall nanoparticles. These findings demonstrated that
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56 AuNCs@NAC-CS possessed good fluorescence, low cytotoxicity, and low sensitivity to some content of
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cells, allowing imaging of the living cells.
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60 ACS Paragon Plus Environment
ACS Biomaterials Science & Engineering Page 2 of 25
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2 Introduction
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6 Metal nanoclusters, usually consisting of several to tens of atoms, have received enormous attention in last
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8 decade1-3.Compared with bulk metal materials, the metal nanoclusters show lots of special properties such as
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11 discrete electronic states4, size-dependent fluorescence5 and intrinsic magnetism6. Therefore, metal
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13 nanoclusters have a wide prospects in bio-chemical sensing7, bio-imaging8 and catalysis9.
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In particular, gold nanoclusters have become more attractive, because gold nanoclusters have exhibited lots
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18 of excellent properties quite different from quantum dots and traditional organic dyes such as low toxicity10,
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20 good chemical and photo-physical stability11, facile synthesis12, tunable fluorescent emissions13.All of those
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23 advantages make gold nanoclusters become more competitive in biomedical application14-16.
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25 In the past few years, many coats on the surface of the gold nanoclusters have been studied to reduce the
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28 potential toxicity including poly(ethylene glycol)-dithiolane ligands17, bio-surfactant (sodium cholate)18,
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30 glutathione19, bovine albumin20 and so on. Especially, AuNCs@BSA have shown great potential application
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in bio-imaging21 and bio-chemical sensing22 because of simple, rapid, one-pot and green route, bright
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35 near-NIR emission, and high efficient and stable fluorescence23. However, AuNCs@BSA have been reported
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to be oxidative decomposed by reactive oxygen species (ROS) and degraded by proteases or other enzymes
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40 in lysosomes which means the fluorescence intensity of AuNCs@BSA could be influenced by H2O2 and
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42 proteases or other enzymes24-27. Hence, the further bio-imaging application could be limited because the
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45 existence of reactive oxygen species (ROS) production and the enzyme-containing intracellular milieu
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47 could lead to the fluorescence quenching27.
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50 Therefore, chitosan was chosen as coating on the surface of the gold nanoclusters to improve resistant
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52 ability to ROS and proteases. Chitosan, a cationic biopolymer prepared from alkaline N-deacetylation of
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55 natural chitin, has amino group and hydroxyl group in the repeating every unit28-30. Chitosan has lots of
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57 good properties such as biodegradability, non-toxicity, biocompatibility, and environmental friendliness31-33.
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2 Thus, chitosan is an ideal material for the gold nanoclusters’ surface coating.
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5 In this work, chitosan-N-acetyl-L-cysteine (NAC) was prepared by immobilizing the carboxylic acid
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7 group of NAC on the primary amino groups of CS backbone. And small-sized NAC-CS coated
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10 fluorescent gold nanoclusters (AuNCs@NAC-CS) were synthesized and stabilized through thiol groups
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12 in thiolated chitosan (NAC-CS) (Fig 1). Biocompatible materials such as chitosan, NAC were used
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instead of DMF and methanol which were used before. And the synthesized steps were simplified which
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17 were simple and green compared with that of before8. The resultant (NAC-CS) was characterized by
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NMR spectra and the content of thiol groups were determinated. The fluorescent probe
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22 (AuNCs@NAC-CS) was characterized by UV-visible spectroscopy, fluorescence spectroscopy,
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24 transmission electron microscopy (TEM). Cells uptake, cytotoxicity were evaluated and the metabolism
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27 experiment of AuNCs@NAC-CS and AuNCs@BSA were conducted to compare fluorescence stability
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29 in vitro with the result that the fluorescence of AuNCs@NAC-CS could be observed longer time
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32 compared with AuNCs@BSA. In vivo experiments, the obvious fluorescence signal of
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34 AuNCs@NAC-CS appeared in the liver and kidney of the normal mice after 6 h injection. And the
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fluorescence intensity decreased after that because of the highly efficient clearance characteristics of
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39 ultrasmall nanoparticles. Thus, AuNCs@NAC-CS have good potential application in live-cell imaging
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as a fluorescent probe.
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56 Fig 1. Schematic illustration of the reaction of NAC-CS and AuNCs@NAC-CS
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59 Materials and methods
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2 Reagents and chemicals
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5 CS (MW=10 kDa, degree of deacetylation=91%) were purchased from Nantong Xingcheng Biological
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7 Industry Limited Co. (China). N-acetyl-L-cysteine (NAC),
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10 N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC), N-Hydroxysuccinimide
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12 (NHS), Gold chloride trihydrate (HAuCl4) were obtained from Aladdin Industrial Corporation (Shanghai,
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China). Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum, penicillin, streptomycin and
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17 phosphate buffered saline (PBS) were provided from Gibco (American). Mili-Q purified water was used
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throughout the experiments. All other chemicals were of analytical grade and used without further
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22 modification.
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24 Instruments
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27 Fluorescence spectra were recorded with a QuantaMaster40 fluorescence spectrophotometer (PTI,
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29 Canada). Absorption spectra were recorded using a Lambda35 UV-Vis spectrophotometer (Perkin Elmer,
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32 USA). Transmission electron microscopy (TEM) images were obtained with a Tecnai G2 F20
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34 microscope (FEI, USA). A Nicolet Is10 Fourier transform infrared (FT-IR) spectrophotometer (Thermo,
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USA) was employed to measure the IR spectra. The cellular fluorescence images were recorded using
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39 an LSM710 microscope (Zeiss, Germany). Bio-distribution images were recorded with the CRi
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Maestro2, MaestroEX-RRO, USA.
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44 Synthesis of NAC-CS and characteristic
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46 Chitosan was dispersed in 0.1 M hydrochloric acid solution. NAC was dispersed in the water followed
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49 by the addition of a solution of EDC and NHS34-35. After 20 min activation of carboxyl groups in NAC,
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51 NAC solution was added into chitosan solution. The reaction mixture was incubated for 8 h at room
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54 temperature under stirring in the dark, and then dialyzed for 36 h in the dark. The dialyzed fragment
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56 were lyophilized after filtration. Successful conjugation of CS with NAC was confirmed by NMR.
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The amount of thiol groups attached to the chitosan was determined by a procedure with DTDP36-37. A
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2 certain amount of NAC-CS was dissolved in a little 5 mL 0.1 M hydrochloric acid solution and then was
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5 dispersed in 20 mL buffer solution (NaH2PO4-EDTA·Na2). Subsequently, 100 µL of DTDP reagent
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7 (8.8mg in 5 mL of acetonitrile solution) was added. After 30 min reaction, the absorbance at 320 nm
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10 was read. The quantity of thiol groups was calculated using a standard curve which obtained by the thiol
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12 group determination of a series of NAC solution with different concentrations.
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Synthesis of AuNCs@NAC-CS and AuNCs@BSA
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17 An acetate solution containing 2.5 mM of HAuCl4 and 10 mg mL-1 of NAC-CS was incubated on
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oscillator at 60 ℃ for 5 h in the dark. The resultant mixture (AuNCs@NAC-CS) was dialyzed for 36 h
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22 in the dark. The characteristic of AuNCs@NAC-CS was confirmed by UV-Vis, TGA, TEM,
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24 fluorescence spectroscopy.
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27 AuNCs@BSA were prepared according to the previously reported method14. An aqueous solution
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29 containing 5.8 mM of HAuCl4 and 19.2 mg mL-1 of BSA was under vigorous magnetic stirring for 2
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32 min. Then, 38 mM NaOH was added to adjust the pH to 10. This mixture was allowed to incubate at
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34 100 ℃ for 1 h. The resultant mixture was dialyzed for 36 h in the dark. The product could be collected
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by vacuum freeze drying.
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39 Sensitivity measurements
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H2O2 stock solution (0.1 M) and trypsin stock solution (0.1 M) were prepared and various
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44 concentrations were obtained by serial dilution of the stock solution. A series of 20 µL of standard
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46 solution of H2O2 or trypsin were added 20 µL AuNCs@NAC-CS solution respectively and incubated for
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49 30 min before conducting the fluorescence measurements.
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51 Cell culture
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54 The HeLa, MCF-7 and 3T3 cells were cultured in DMEM medium with 10% (v/v) fetal bovine serum
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56 and 1 % (v/v) penicillin–streptomycin at 37 ℃ in a 5% CO2 incubator.
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Cytotoxicity assay
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2 CCK-8 assays were used to probe cellular viability. The HeLa, MCF-7 and 3T3 cells were cultured
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5 overnight in 96-well plates at a density of 5 × 103 cells per well and then exposed to different
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7 concentrations of the AuNCs@NAC-CS for 24 h at 37 °C. Subsequently, each well was washed with
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10 PBS buffer and the 10% CCK-8 reagent was added. After 1 h incubation, the optical density (OD) of
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12 supernate was measured at 450 nm and the viability of cells was calculated. Three replicates were done
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for each treatment group.
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17 Metabolism assay
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The HeLa cells were cultured overnight in dishes at a density of 104 cells per dish and then exposed to
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22 the AuNCs@NAC-CS for 1 h at 37 °C. Subsequently, each dish was washed with PBS buffer and added
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24 new DMEM medium with 10 % (v/v) fetal bovine serum. Then, after 0 h, 1 h, 2 h, 4 h or 8 h incubation,
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27 the cells were washed thrice in PBS, fixed with 4 % p-formaldehyde for 30 min. Fluorescence images of
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29 cells were recorded using confocal laser scanning microscope.
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32 Bio-distribution of AuNCs@NAC-CS
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34 Normal (BALB/C, female, 6-8 weeks) mice were purchased from Institute of Laboratory Animal
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Sciences, CAMP&PUMC (Beijing, China) for in vivo imaging and bio-distribution investigation of
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39 AuNCs@NAC-CS. Normal mice were used for bio-distribution investigation of AuNCs@NAC-CS in
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vivo. In the experiment, aqueous solution (0.1 mL, 8.3 mg mL-1) was administered into each mouse
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44 through tail vein injection. The mice were dissected at the corresponding time points (1 h, 3 h, 6 h, 12 h,
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46 24 h and 36 h) and the fluorescence images of the organs (heart, liver, spleen, lung and kidney) were
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49 collected to investigate the bio-distribution of AuNCs@NAC-CS using the imaging system equipped
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51 with a 455 nm Laser.
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54 Result and discussion
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57 Synthesis and Characterization of NAC-CS
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2 Firstly, NAC-CS was prepared by immobilizing the carboxylic acid group of NAC on the primary
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5 amino groups of CS backbone. In this reaction, the EDC worked as catalyst and the addition of NHS
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7 significantly improved the thiol yield38. The structure of NAC-CS was confirmed by 1H NMR spectra
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10 (Fig 2). Compared with spectrum of chitosan, new resonance peaks at δ4.57 (a, 1H, -CH-N-) and δ2.81,
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12 3.14 (b, 2H, -CH2S-) were assigned to the tertiary hydrogen (-CH-NH-) and the side-chain methylene
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(-CH2SH) of NAC-CS, which was able to confirm that NAC was successfully grafted onto the CS
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17 backbone. IR spectra also confirmed that NAC-CS was successfully synthesised (S1).
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41 Fig 2. The 1H NMR spectra of (a) NAC-CS and (b) NAC and (c) CS
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44 As shown in Fig 3, standard curve of the thiol groups determination was obtained by a series of NAC
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46 solution with different concentrations, and the thiol groups number in NAC-CS was determinated by
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49 standard curve. The results show that the number of thiol group in NAC-CS was closely related with the
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51 molar ratio of reagents (NAC and CS). The number of thiol groups in the polymer increase with the
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54 NAC: CS ratio. However, the number of thiol groups seemed to have peaked when the NAC: CS ratio
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56 was 1:6, and there was no further increase in coupling beyond this level.
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21 Fig 3. (a) The standard curve of thiol groups and (b) thiol groups concentration in different molar ratio of CS and NAC
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23 Synthesis and Characterization of AuNCs@NAC-CS
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As can be noticed in Fig 4(a), the UV-vis absorption of AuNCs@NAC-CS increased strongly toward
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28 shorter wavelengths from around 400 nm which is similar to AuNCs@DMF39. And there was no obvious
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surface plasmon resonance (SPR) band of gold nanoparticles, which suggested that the core diameters
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33 of most AuNCs were less than 2 nm8. In addition, Fig 5 clearly showed that AuNCs@NAC-CS had an
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35 emission spectra band with a characteristic peak wavelength of 680 nm as shown in Fig 4(b), and a peak
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38 wavelength of 360 nm was observed in excitation spectra. The filter was used to avoid the interference
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40 by 1/2 fraction frequency scattering peak (shown in Fig S2). The content of Au in AuNCs@NAC-CS is
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43 nearly 6.61 % (shown in S5).
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21 Fig 4. (a) The UV-vis spectra of AuNCs@NAC-CS and (b) The excitation and emission spectra of AuNCs@NAC-CS
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23 As shown in Fig 5, the morphology of AuNCs@NAC-CS were observed as circular or sub circular in
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TEM images, and had trend to form aggregated nanostructure. There were many thiol groups coated on
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28 the surface of gold as the stabilizing agent, which were obtained from NAC. One chain of NAC-CS can
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be coupled to multiple gold nanoclusters, and the surface of gold can also be coated with several
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33 NAC-CS molecules at the same time. Several gold nanoclusters were close to each other to form
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35 aggregated structure, because the long-chain of NAC-CS curled in the medium solution.
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55 Fig 5. (a) The TEM image of AuNCs@NAC-CS in 100 nm scale and (b) in 20 nm scale
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57 As shown in Fig 6, the fluorescence emission spectra of AuNCs@NAC-CS was influenced by the
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2 reaction temperature and feed ratio of NAC-CS / HAuCl4. At the temperature of 30℃ to 80℃, as the
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5 temperature hoisted, the fluorescence intensity increased. Then the fluorescence intensity reduced in the
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7 90 ℃ reaction temperature compared with the 80 ℃. The higher temperature in an appropriate range
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10 caused more reaction energy which made fluorescence intensity increase. However, excessively high
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12 temperature easily caused nanoparticles aggregation and fluorescence intensity reduction. As shown in
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Fig 6 (b), the fluorescence intensity of AuNCs@NAC-CS increased, because more AuNCs@NAC-CS
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17 were synthesized with more HAuCl4 in reaction solution when the thiol groups were fixed in reaction.
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However, HAuCl4 couldn’t endlessly increase. Otherwise, AuNCs@NAC-CS subsequently aggregated
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22 and formed red-brown precipitation because of less of protected groups. In addition, a slightly redshift
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24 could be observed with more HAuCl4 in reaction system, and this phenomenon could be contributed in
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27 the different atom number of AuNCs@NAC-CS. As reported, the emission spectra was related with the
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29 atom number in AuNCs13. The feed ratio of NAC-CS / HAuCl4 1:2 was chosen in the following
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32 experiment. And fluorescence quantum yields (QY) of AuNCs@NAC-CS was estimated to be 3.5 %
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34 using Rhodamine B as a standard40.
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55 Fig 6. (a) The fluorescence spectra of AuNCs@NAC-CS which were synthesized by different reaction temperature and (b)
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57 different molar ratio of NAC-CS and HAuCl4.
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2 The fluorescence intensity gradually reduced with higher concentration of acid and basic in the solvent.
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5 Meanwhile, the change of fluorescence intensity was partial reversible. The fluorescence intensity was
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7 recovered when the pH changed into the neutral, which was shown in Fig S3 and Fig S4. The
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10 fluorescence intensity change in different pH solution was accordance with the structure and charge
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12 change of NAC-CS which grafted on the surface of gold. At the same time, the peak of the fluorescence
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intensity showed blue shift in low pH and red shift in high pH, which caused by the number of atoms in
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17 the cluster13. Chitosan is a natural alkali polysaccharide containing many amino groups in the molecular
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chain. And the charge of amino groups was seriously influenced by pH value of solution. The structure
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22 and charge change of NAC-CS caused the changes of morphology of AuNCs@NAC-CS which were
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24 manifested in fluorescence intensity and max absorption peak. The fluorescence intensity of
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27 AuNCs@NAC-CS was much stronger than that of AuNCs@NAC which synthesized with NAC as the
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29 ligand (shown in S6). Because of the curling of NAC-CS, the gold nanoclusters were close to each other
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32 to restrict their free movement in solution, which in turn might improve their fluorescence efficiency41.
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34 Sensitivity measurements
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As reported, BSA could be easily cleaved into amino acids/peptide fragments by trypsin42, and
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39 fluorescence intensity of AuNCs@BSA reduced because of surface coating’s degradation. This fact
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limited the cell imaging application of AuNCs@BSA owing to kinds of protease existence in cell and
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44 culture medium. In addition, the gold nanoclusters have been reported for the possess of intrinsic
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46 peroxidase-like activity which could catalyze H2O2 to produce oxygen43, and at that time the
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49 fluorescence intensity of AuNCs was reduced. The tumor imaging using AuNCs could be influenced
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51 because of H2O2 enrichment in tumor area.
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54 As illustrated in Fig 7, the fluorescence intensity of AuNCs@BSA decreased gradually when the
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56 concentration of H2O2 was raised from 0 to 100 mM. However, the fluorescence intensity of
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AuNCs@NAC-CS remained essentially constant when the concentration of H2O2 was raised. And the
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2 similar result was showed in Fig 8, when they were treated with trypsin. The fluorescence intensity of
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5 AuNCs@BSA reduced gradually when they were treated with different concentration of trypsin. Meanwhile
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7 there was no significant change of AuNCs@NAC-CS’s fluorescence intensity. This phenomenon indicated
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10 that the fluorescence sensitivity of AuNCs was related with surface coating. The reduction of fluorescence
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12 intensity could be attributed to the strong oxidative ability of H2O2 and the catalytic ability of trypsin, which
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disrupted the BSA protected Au clusters, leading to their aggregation and growth to become larger Au
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17 nanoparticles. And CS was stable when they treated with H2O2 and trypsin, so the fluorescence intensity of
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AuNCs@NAC-CS had more resistibility to H2O2 and trypsin compared with AuNCs@BSA. Thus, there was
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22 longer time to evaluate the fluorescence of AuNCs@NAC-CS in cell imaging compared with
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45 Fig 7. The fluorescence intensity of (a) AuNCs@BSA and (b) AuNCs@NAC-CS treated with different concentration of
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H2O2.
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21 Fig 8. The fluorescence intensity of (a) AuNCs@BSA and (b) AuNCs@NAC-CS (b) treated with different concentration of
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In vitro toxicity
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28 Cytotoxicity of AuNCs should be concerned when applied for cell imaging. MCF-7, 3T3 and HeLa cells
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were used to evaluate the cytotoxicity of AuNCs@NAC-CS through CCK-8 assay. As shown in Fig 9, the
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33 viabilities of both MCF-7, 3T3 and HeLa cells maintained above 90% when they had been incubated with
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35 AuNCs@NAC-CS in the concentration range of 0.2-1.0 mg/mL. As reported previously8, the cytotoxicity of
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38 AuNCs was related with surface coating materials. The high viability demonstrated that CS was suitable for
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40 surface coating material because of the good biocompatibility and AuNCs@NAC-CS maybe far more suited
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43 for live-cell imaging as a fluorescent probe because of its low cytotoxicity.
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Fig 9. The MCF-7 cells, 3T3 cells and HeLa were incubated with different dosages of AuNCs@NAC-CS in vitro for 24 h,
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2 respectively. All the data were obtained by conducting three parallel experiments.
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5 Cell uptake and metabolism
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7 The HeLa cells were incubated with AuNCs@NAC-CS for different time periods to evaluate the cell
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10 uptake. It was found in Fig 10 that the Hela cells produced bright fluorescence after incubating with
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12 AuNCs@NAC-CS, indicating that AuNCs@NAC-CS entered the cells. And the blank Hela cells had no
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fluorescence, indicating that there was no auto-fluorescence from the cells themselves. As shown in
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17 Supproting Information, there was no obviously difference of fluorescence intensity when cells were
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incubated with AuNCs for 1 h, 2 h, 4 h, demonstrating that 1 h was enough for AuNCs@NAC-CS to
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22 enter into cells. Hence, 1 h incubation time was chosen in cell metabolism assay.
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49 Fig 10. Confocal fluorescent microscopic images of HeLa cells incubated with (a)AuNCs@NAC-CS and (b) the blank
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51 HeLa cells. AuNCs@NAC-CS with the concentration of 50 µg/mL were used.
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54 To investigate the temporal evolutions of cellular metabolism of the AuNCs@NAC-CS or
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56 AuNCs@BSA, HeLa cells were used as model. As shown in Fig 11, the bright fluorescence of
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AuNCs@NAC-CS could be observed until 4 h incubation in DMEM medium, and the week
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2 fluorescence could be observed even after 8 h incubation in DMED medium. However, the fluorescence
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5 intensity of AuNCs@BSA gradually reduced with the prolonged incubation time, and it almost no
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7 fluorescence in Hela cells after 8 h incubation. This phenomenon was accordance with the results of
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10 sensitivity assay. The content of H2O2 is higher in tumor area compared with normal area, and H2O2 will
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12 cause fluorescence intensity reducing. In addition, the proteases in cell or culture medium influence the
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fluorescence of AuNCs. The AuNCs@NAC-CS had low sensitivity to H2O2 and trypsin compared with
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17 AuNCs@BSA, because CS was more stable than BSA in cell and culture environment. Thus,
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AuNCs@NAC-CS have good potential application in live-cell imaging as a fluorescent probe than
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22 BSA-protected AuNCs.
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52 Fig 11. (a-i) Confocal fluorescent microscopic imgaes of showing the metabolism of AuNCs@BSA and AuNCs@NAC-CS
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55 in the living Hela cells. The metabolism of AuNCs@BSA and AuNCs@NAC-CS in the living Hela cells at (a and e) 0 h (b
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57 and f) 1 h (c and g) 2 h (d and h) 4 h (e and i) 8h after 1h incubated with diffierent AuNCs.
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1
2 Bio-distribution of AuNCs@NAC-CS
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5 Fig 12 shows the bio-distribution of AuNCs@NAC-CS in different organs, such as heart, liver, spleen,
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7 lung and kidney and the dynamic fluorescence intensity change in different organs. The obvious
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10 fluorescence signal appeared in the liver and kidney of the normal mice after 6 h injection. The
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12 fluorescence intensity in kidney gradually increased until 6 h when achieved the highest intensity. After
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that, the fluorescence intensity decreased. The ultrasmall nanoparticles were high-efficient cleared to
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17 overcome the toxicity by nonspecific accumulation in healthy tissues/organs from renal in vivo44. The
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AuNCs@NAC-CS exhibited the similar process with gold nanoclusters in bio-distribution and
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22 metabolism, which was formed by multiple ultrasmall gold nanoclusters closing with each other. The
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24 high level of liver uptake of AuNCs@NAC-CS is owing to mononuclear phagocytic system (MPS)
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27 absorption. And the uptake of AuNCs@NAC-CS in kidney can be possibly correlated with renal
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29 excretion. Generally the hydrophilic polymer such as polyethylene glycol may extend in vivo
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32 circulation time. However, the number of NAC in CS-NAC was limited, and NAC mostly interred in
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34 AuNCs@NAC-CS which combined NAC-CS with Au. Therefore, few NAC was on the surface of
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AuNCs@NAC-CS, and the effect of NAC-chitosan was not very satisfying to extend in vivo circulation
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39 time.
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2 Fig 12. the ex-vivo fluorescence images of isolated organs (heart, liver, spleen, lung, kidney) from (a) the mice at 6 h
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5 post-injection, (b) the blank mice; (c) dynamic fluorescence intensity in different organs after injection.
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7 Conclusion
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11 A new fluorescent probe (AuNCs@NAC-CS) was successfully synthesized and utilized for imaging of
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13 living cells. The as-prepared surface coating (NAC-CS) which worked as reductant and stabilizer is more
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stable and easily to introduce other groups because of active groups in NAC-CS molecular chain such as
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18 amino and hydroxyl groups. Compared with before, the method is simple and green. The probe is
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20 demonstrated to possess many advantages in imaging, such as low cytotoxicity, low sensitivity to tumor
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23 cells contents (H2O2 and protease) and long-time imaging. The bio-friendly nature and near-infrared
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25 fluorescence of AuNCs@NAC-CS can be used as candidates for optical cell imaging, living imaging,
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28 targeted drug delivery for cancer treatment or imaging agents for early tumor diagnosis.
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31 Acknowledgements
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34
This work was supported by the National Natural Science Foundation of China (Grant Nos. 31271023,
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37 81501578), the Fundamental Research Funds for the Central Universities.
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Supporting Information
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43 FT-IR spectrum, the fluorescence intensity of AuNCs@NAC and water with filter, the fluorescence intensity
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45 of AuNCs@NAC-CS in the different pH of solution, the TGA of AuNCs@NAC-CS, the fluorescence
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48 intensity of AuNCs@NAC-CS and AuNCs@NAC, fluorescence images of HeLa cells after different
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50 incubation time with AuNCs@BSA and AuNCs@NAC-CS.
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2 Chitosan-stabilized self-assembled fluorescent gold nanoclusters for cell imaging and
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5 bio-distribution in vivo
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7 Ying Duana, Ruiping Duanb*, Rui Liub, Man Guanb, Wenjuan Chenb, Jingjing Mab, Mingmao Chena, Bo
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10 Dub,Qiqing Zhangab*
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a Institute of Biomedical and Pharmaceutical Technology, Fuzhou University, Fuzhou 350002, China
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15 b Institute of Biomedical Engineering, Chinese Academy of Medical Science and Peking Union Medical
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College, Tianjin Key Laboratory of Biomedical Material, Tianjin 300192, China
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20 * E-mail: rpduan@sina.com; zhangqiq@126.com.
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