Chitosan-Stabilized Self-Assembled Fluorescent Gold Nanoclusters

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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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ACS Biomaterials Science & Engineering

Chitosan-stabilized self-assembled fluorescent gold nanoclusters for cell imaging and bio-distribution in vivo Ying Duana, Ruiping Duanb*, Rui Liub, Man Guanb, Wenjuan Chenb, Jingjing Mab, Mingmao Chena, Bo Dub,Qiqing Zhangab* a Institute of Biomedical and Pharmaceutical Technology, Fuzhou University, Fuzhou 350002, China b Institute of Biomedical Engineering, Chinese Academy of Medical Science and Peking Union Medical College, Tianjin Key Laboratory of Biomedical Material, Tianjin 300192, China * E-mail: [email protected]; [email protected].

Keywords AuNCs@NAC-CS; AuNCs@BSA; Chitosan; Tumor cell imaging

Abstract Biocompatible, near-infrared luminescent gold nanoclusters were synthesized in situ using as-prepared chitosan grafted with N-Acetyl-L-cysteine (NAC-CS). The fluorescent gold nanoclusters coated with chitosan-N-acetyl-L-cysteine (AuNCs@NAC-CS) was aggregated by multiple ultrasmall gold nanoclusters closing with each other, with strong fluorescence emission at 680 nm upon excitation at 360 nm. AuNCs@NAC-CS did not display any appreciable cytotoxicity on cells even at a concentration of 1.0 mg mL-1. AuNCs@NAC-CS were more insensitive to H2O2 and trypsin compared with fluorescent gold nanoclusters coated with Albumin BovineⅤ(AuNCs@BSA), which make them have long time imaging in HeLa cells. Furthermore, the obvious fluorescence signal of AuNCs@NAC-CS appeared in the liver and kidney of the normal mice after 6 h injection. And the fluorescence intensity decreased after that because of the highly efficient clearance characteristics of ultrasmall nanoparticles. These findings demonstrated that AuNCs@NAC-CS possessed good fluorescence, low cytotoxicity, and low sensitivity to some content of cells, allowing imaging of the living cells. ACS Paragon Plus Environment

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Introduction Metal nanoclusters, usually consisting of several to tens of atoms, have received enormous attention in last decade1-3.Compared with bulk metal materials, the metal nanoclusters show lots of special properties such as discrete electronic states4, size-dependent fluorescence5 and intrinsic magnetism6. Therefore, metal nanoclusters have a wide prospects in bio-chemical sensing7, bio-imaging8 and catalysis9. In particular, gold nanoclusters have become more attractive, because gold nanoclusters have exhibited lots of excellent properties quite different from quantum dots and traditional organic dyes such as low toxicity10, good chemical and photo-physical stability11, facile synthesis12, tunable fluorescent emissions13.All of those advantages make gold nanoclusters become more competitive in biomedical application14-16. In the past few years, many coats on the surface of the gold nanoclusters have been studied to reduce the potential toxicity including poly(ethylene glycol)-dithiolane ligands17, bio-surfactant (sodium cholate)18, glutathione19, bovine albumin20 and so on. Especially, AuNCs@BSA have shown great potential application in bio-imaging21 and bio-chemical sensing22 because of simple, rapid, one-pot and green route, bright near-NIR emission, and high efficient and stable fluorescence23. However, AuNCs@BSA have been reported to be oxidative decomposed by reactive oxygen species (ROS) and degraded by proteases or other enzymes in lysosomes which means the fluorescence intensity of AuNCs@BSA could be influenced by H2O2 and proteases or other enzymes24-27. Hence, the further bio-imaging application could be limited because the

existence of reactive oxygen species (ROS) production and the enzyme-containing intracellular milieu could lead to the fluorescence quenching27. Therefore, chitosan was chosen as coating on the surface of the gold nanoclusters to improve resistant ability to ROS and proteases. Chitosan, a cationic biopolymer prepared from alkaline N-deacetylation of

natural chitin, has amino group and hydroxyl group in the repeating every unit28-30. Chitosan has lots of good properties such as biodegradability, non-toxicity, biocompatibility, and environmental friendliness31-33. ACS Paragon Plus Environment

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Thus, chitosan is an ideal material for the gold nanoclusters’ surface coating.

In this work, chitosan-N-acetyl-L-cysteine (NAC) was prepared by immobilizing the carboxylic acid group of NAC on the primary amino groups of CS backbone. And small-sized NAC-CS coated fluorescent gold nanoclusters (AuNCs@NAC-CS) were synthesized and stabilized through thiol groups in thiolated chitosan (NAC-CS) (Fig 1). Biocompatible materials such as chitosan, NAC were used instead of DMF and methanol which were used before. And the synthesized steps were simplified which were simple and green compared with that of before8. The resultant (NAC-CS) was characterized by NMR spectra and the content of thiol groups were determinated. The fluorescent probe (AuNCs@NAC-CS) was characterized by UV-visible spectroscopy, fluorescence spectroscopy, transmission electron microscopy (TEM). Cells uptake, cytotoxicity were evaluated and the metabolism experiment of AuNCs@NAC-CS and AuNCs@BSA were conducted to compare fluorescence stability in vitro with the result that the fluorescence of AuNCs@NAC-CS could be observed longer time compared with AuNCs@BSA. In vivo experiments, the obvious fluorescence signal of AuNCs@NAC-CS appeared in the liver and kidney of the normal mice after 6 h injection. And the fluorescence intensity decreased after that because of the highly efficient clearance characteristics of ultrasmall nanoparticles. Thus, AuNCs@NAC-CS have good potential application in live-cell imaging as a fluorescent probe.

Fig 1. Schematic illustration of the reaction of NAC-CS and AuNCs@NAC-CS

Materials and methods ACS Paragon Plus Environment


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Reagents and chemicals CS (MW=10 kDa, degree of deacetylation=91%) were purchased from Nantong Xingcheng Biological Industry

Limited

Co.

(China).

N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide

N-acetyl-L-cysteine

hydrochloride

(EDC),

(NAC),

N-Hydroxysuccinimide

(NHS), Gold chloride trihydrate (HAuCl4) were obtained from Aladdin Industrial Corporation (Shanghai, China). Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum, penicillin, streptomycin and phosphate buffered saline (PBS) were provided from Gibco (American). Mili-Q purified water was used throughout the experiments. All other chemicals were of analytical grade and used without further modification.

Instruments Fluorescence spectra were recorded with a QuantaMaster40 fluorescence spectrophotometer (PTI, Canada). Absorption spectra were recorded using a Lambda35 UV-Vis spectrophotometer (Perkin Elmer, USA). Transmission electron microscopy (TEM) images were obtained with a Tecnai G2 F20 microscope (FEI, USA). A Nicolet Is10 Fourier transform infrared (FT-IR) spectrophotometer (Thermo, USA) was employed to measure the IR spectra. The cellular fluorescence images were recorded using an LSM710 microscope (Zeiss, Germany). Bio-distribution images were recorded with the CRi Maestro2, MaestroEX-RRO, USA.

Synthesis of NAC-CS and characteristic Chitosan was dispersed in 0.1 M hydrochloric acid solution. NAC was dispersed in the water followed by the addition of a solution of EDC and NHS34-35. After 20 min activation of carboxyl groups in NAC, NAC solution was added into chitosan solution. The reaction mixture was incubated for 8 h at room temperature under stirring in the dark, and then dialyzed for 36 h in the dark. The dialyzed fragment were lyophilized after filtration. Successful conjugation of CS with NAC was confirmed by NMR. The amount of thiol groups attached to the chitosan was determined by a procedure with DTDP36-37. A ACS Paragon Plus Environment

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certain amount of NAC-CS was dissolved in a little 5 mL 0.1 M hydrochloric acid solution and then was dispersed in 20 mL buffer solution (NaH2PO4-EDTA·Na2). Subsequently, 100 µL of DTDP reagent (8.8mg in 5 mL of acetonitrile solution) was added. After 30 min reaction, the absorbance at 320 nm was read. The quantity of thiol groups was calculated using a standard curve which obtained by the thiol group determination of a series of NAC solution with different concentrations.

Synthesis of AuNCs@NAC-CS and AuNCs@BSA An acetate solution containing 2.5 mM of HAuCl4 and 10 mg mL-1 of NAC-CS was incubated on oscillator at 60 ℃ for 5 h in the dark. The resultant mixture (AuNCs@NAC-CS) was dialyzed for 36 h in the dark. The characteristic of AuNCs@NAC-CS was confirmed by UV-Vis, TGA, TEM, fluorescence spectroscopy. AuNCs@BSA were prepared according to the previously reported method14. An aqueous solution containing 5.8 mM of HAuCl4 and 19.2 mg mL-1 of BSA was under vigorous magnetic stirring for 2 min. Then, 38 mM NaOH was added to adjust the pH to 10. This mixture was allowed to incubate at 100 ℃ for 1 h. The resultant mixture was dialyzed for 36 h in the dark. The product could be collected by vacuum freeze drying.

Sensitivity measurements H2O2 stock solution (0.1 M) and trypsin stock solution (0.1 M) were prepared and various concentrations were obtained by serial dilution of the stock solution. A series of 20 µL of standard solution of H2O2 or trypsin were added 20 µL AuNCs@NAC-CS solution respectively and incubated for 30 min before conducting the fluorescence measurements.

Cell culture The HeLa, MCF-7 and 3T3 cells were cultured in DMEM medium with 10% (v/v) fetal bovine serum and 1 % (v/v) penicillin–streptomycin at 37 ℃ in a 5% CO2 incubator.

Cytotoxicity assay ACS Paragon Plus Environment


CCK-8 assays were used to probe cellular viability. The HeLa, MCF-7 and 3T3 cells were cultured overnight in 96-well plates at a density of 5 × 103 cells per well and then exposed to different concentrations of the AuNCs@NAC-CS for 24 h at 37 °C. Subsequently, each well was washed with PBS buffer and the 10% CCK-8 reagent was added. After 1 h incubation, the optical density (OD) of supernate was measured at 450 nm and the viability of cells was calculated. Three replicates were done for each treatment group.

Metabolism assay The HeLa cells were cultured overnight in dishes at a density of 104 cells per dish and then exposed to the AuNCs@NAC-CS for 1 h at 37 °C. Subsequently, each dish was washed with PBS buffer and added new DMEM medium with 10 % (v/v) fetal bovine serum. Then, after 0 h, 1 h, 2 h, 4 h or 8 h incubation, the cells were washed thrice in PBS, fixed with 4 % p-formaldehyde for 30 min. Fluorescence images of cells were recorded using confocal laser scanning microscope.

Bio-distribution of AuNCs@NAC-CS Normal (BALB/C, female, 6-8 weeks) mice were purchased from Institute of Laboratory Animal Sciences, CAMP&PUMC (Beijing, China) for in vivo imaging and bio-distribution investigation of AuNCs@NAC-CS. Normal mice were used for bio-distribution investigation of AuNCs@NAC-CS in vivo. In the experiment, aqueous solution (0.1 mL, 8.3 mg mL-1) was administered into each mouse through tail vein injection. The mice were dissected at the corresponding time points (1 h, 3 h, 6 h, 12 h, 24 h and 36 h) and the fluorescence images of the organs (heart, liver, spleen, lung and kidney) were collected to investigate the bio-distribution of AuNCs@NAC-CS using the imaging system equipped with a 455 nm Laser.

Result and discussion Synthesis and Characterization of NAC-CS ACS Paragon Plus Environment

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Firstly, NAC-CS was prepared by immobilizing the carboxylic acid group of NAC on the primary amino groups of CS backbone. In this reaction, the EDC worked as catalyst and the addition of NHS significantly improved the thiol yield38. The structure of NAC-CS was confirmed by 1H NMR spectra (Fig 2). Compared with spectrum of chitosan, new resonance peaks at δ4.57 (a, 1H, -CH-N-) and δ2.81, 3.14 (b, 2H, -CH2S-) were assigned to the tertiary hydrogen (-CH-NH-) and the side-chain methylene (-CH2SH) of NAC-CS, which was able to confirm that NAC was successfully grafted onto the CS backbone. IR spectra also confirmed that NAC-CS was successfully synthesised (S1).

Fig 2. The 1H NMR spectra of (a) NAC-CS and (b) NAC and (c) CS

As shown in Fig 3, standard curve of the thiol groups determination was obtained by a series of NAC solution with different concentrations, and the thiol groups number in NAC-CS was determinated by standard curve. The results show that the number of thiol group in NAC-CS was closely related with the molar ratio of reagents (NAC and CS). The number of thiol groups in the polymer increase with the NAC: CS ratio. However, the number of thiol groups seemed to have peaked when the NAC: CS ratio was 1:6, and there was no further increase in coupling beyond this level.

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Fig 3. (a) The standard curve of thiol groups and (b) thiol groups concentration in different molar ratio of CS and NAC

Synthesis and Characterization of AuNCs@NAC-CS As can be noticed in Fig 4(a), the UV-vis absorption of AuNCs@NAC-CS increased strongly toward shorter wavelengths from around 400 nm which is similar to AuNCs@DMF39. And there was no obvious

surface plasmon resonance (SPR) band of gold nanoparticles, which suggested that the core diameters of most AuNCs were less than 2 nm8. In addition, Fig 5 clearly showed that AuNCs@NAC-CS had an emission spectra band with a characteristic peak wavelength of 680 nm as shown in Fig 4(b), and a peak wavelength of 360 nm was observed in excitation spectra. The filter was used to avoid the interference by 1/2 fraction frequency scattering peak (shown in Fig S2). The content of Au in AuNCs@NAC-CS is nearly 6.61 % (shown in S5).


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Fig 4. (a) The UV-vis spectra of AuNCs@NAC-CS and (b) The excitation and emission spectra of AuNCs@NAC-CS

As shown in Fig 5, the morphology of AuNCs@NAC-CS were observed as circular or sub circular in TEM images, and had trend to form aggregated nanostructure. There were many thiol groups coated on the surface of gold as the stabilizing agent, which were obtained from NAC. One chain of NAC-CS can be coupled to multiple gold nanoclusters, and the surface of gold can also be coated with several NAC-CS molecules at the same time. Several gold nanoclusters were close to each other to form aggregated structure, because the long-chain of NAC-CS curled in the medium solution.

Fig 5. (a) The TEM image of AuNCs@NAC-CS in 100 nm scale and (b) in 20 nm scale

As shown in Fig 6, the fluorescence emission spectra of AuNCs@NAC-CS was influenced by the ACS Paragon Plus Environment


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reaction temperature and feed ratio of NAC-CS / HAuCl4. At the temperature of 30℃ to 80℃, as the temperature hoisted, the fluorescence intensity increased. Then the fluorescence intensity reduced in the 90 ℃ reaction temperature compared with the 80 ℃. The higher temperature in an appropriate range caused more reaction energy which made fluorescence intensity increase. However, excessively high temperature easily caused nanoparticles aggregation and fluorescence intensity reduction. As shown in Fig 6 (b), the fluorescence intensity of AuNCs@NAC-CS increased, because more AuNCs@NAC-CS were synthesized with more HAuCl4 in reaction solution when the thiol groups were fixed in reaction. However, HAuCl4 couldn’t endlessly increase. Otherwise, AuNCs@NAC-CS subsequently aggregated and formed red-brown precipitation because of less of protected groups. In addition, a slightly redshift could be observed with more HAuCl4 in reaction system, and this phenomenon could be contributed in the different atom number of AuNCs@NAC-CS. As reported, the emission spectra was related with the atom number in AuNCs13. The feed ratio of NAC-CS / HAuCl4 1:2 was chosen in the following experiment. And fluorescence quantum yields (QY) of AuNCs@NAC-CS was estimated to be 3.5 % using Rhodamine B as a standard40.

Fig 6. (a) The fluorescence spectra of AuNCs@NAC-CS which were synthesized by different reaction temperature and (b) different molar ratio of NAC-CS and HAuCl4.


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The fluorescence intensity gradually reduced with higher concentration of acid and basic in the solvent. Meanwhile, the change of fluorescence intensity was partial reversible. The fluorescence intensity was recovered when the pH changed into the neutral, which was shown in Fig S3 and Fig S4. The fluorescence intensity change in different pH solution was accordance with the structure and charge change of NAC-CS which grafted on the surface of gold. At the same time, the peak of the fluorescence intensity showed blue shift in low pH and red shift in high pH, which caused by the number of atoms in the cluster13. Chitosan is a natural alkali polysaccharide containing many amino groups in the molecular chain. And the charge of amino groups was seriously influenced by pH value of solution. The structure and charge change of NAC-CS caused the changes of morphology of AuNCs@NAC-CS which were manifested in fluorescence intensity and max absorption peak. The fluorescence intensity of AuNCs@NAC-CS was much stronger than that of AuNCs@NAC which synthesized with NAC as the ligand (shown in S6). Because of the curling of NAC-CS, the gold nanoclusters were close to each other to restrict their free movement in solution, which in turn might improve their fluorescence efficiency41. Sensitivity measurements As reported, BSA could be easily cleaved into amino acids/peptide fragments by trypsin42, and fluorescence intensity of AuNCs@BSA reduced because of surface coating’s degradation. This fact limited the cell imaging application of AuNCs@BSA owing to kinds of protease existence in cell and culture medium. In addition, the gold nanoclusters have been reported for the possess of intrinsic peroxidase-like activity which could catalyze H2O2 to produce oxygen43, and at that time the fluorescence intensity of AuNCs was reduced. The tumor imaging using AuNCs could be influenced because of H2O2 enrichment in tumor area. As illustrated in Fig 7, the fluorescence intensity of AuNCs@BSA decreased gradually when the concentration of H2O2 was raised from 0 to 100 mM. However, the fluorescence intensity of AuNCs@NAC-CS remained essentially constant when the concentration of H2O2 was raised. And the ACS Paragon Plus Environment


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similar result was showed in Fig 8, when they were treated with trypsin. The fluorescence intensity of AuNCs@BSA reduced gradually when they were treated with different concentration of trypsin. Meanwhile there was no significant change of AuNCs@NAC-CS’s fluorescence intensity. This phenomenon indicated that the fluorescence sensitivity of AuNCs was related with surface coating. The reduction of fluorescence intensity could be attributed to the strong oxidative ability of H2O2 and the catalytic ability of trypsin, which disrupted the BSA protected Au clusters, leading to their aggregation and growth to become larger Au nanoparticles. And CS was stable when they treated with H2O2 and trypsin, so the fluorescence intensity of AuNCs@NAC-CS had more resistibility to H2O2 and trypsin compared with AuNCs@BSA. Thus, there was longer time to evaluate the fluorescence of AuNCs@NAC-CS in cell imaging compared with AuNCs@BSA.

Fig 7. The fluorescence intensity of (a) AuNCs@BSA and (b) AuNCs@NAC-CS treated with different concentration of H2O2.


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Fig 8. The fluorescence intensity of (a) AuNCs@BSA and (b) AuNCs@NAC-CS (b) treated with different concentration of trypsin.

In vitro toxicity Cytotoxicity of AuNCs should be concerned when applied for cell imaging. MCF-7, 3T3 and HeLa cells were used to evaluate the cytotoxicity of AuNCs@NAC-CS through CCK-8 assay. As shown in Fig 9, the viabilities of both MCF-7, 3T3 and HeLa cells maintained above 90% when they had been incubated with AuNCs@NAC-CS in the concentration range of 0.2-1.0 mg/mL. As reported previously8, the cytotoxicity of AuNCs was related with surface coating materials. The high viability demonstrated that CS was suitable for surface coating material because of the good biocompatibility and AuNCs@NAC-CS maybe far more suited for live-cell imaging as a fluorescent probe because of its low cytotoxicity.

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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respectively. All the data were obtained by conducting three parallel experiments.

Cell uptake and metabolism The HeLa cells were incubated with AuNCs@NAC-CS for different time periods to evaluate the cell uptake. It was found in Fig 10 that the Hela cells produced bright fluorescence after incubating with AuNCs@NAC-CS, indicating that AuNCs@NAC-CS entered the cells. And the blank Hela cells had no fluorescence, indicating that there was no auto-fluorescence from the cells themselves. As shown in Supproting Information, there was no obviously difference of fluorescence intensity when cells were incubated with AuNCs for 1 h, 2 h, 4 h, demonstrating that 1 h was enough for AuNCs@NAC-CS to enter into cells. Hence, 1 h incubation time was chosen in cell metabolism assay.

Fig 10. Confocal fluorescent microscopic images of HeLa cells incubated with (a)AuNCs@NAC-CS and (b) the blank HeLa cells. AuNCs@NAC-CS with the concentration of 50 µg/mL were used.

To investigate the temporal evolutions of cellular metabolism of the AuNCs@NAC-CS or AuNCs@BSA, HeLa cells were used as model. As shown in Fig 11, the bright fluorescence of AuNCs@NAC-CS could be observed until 4 h incubation in DMEM medium, and the week ACS Paragon Plus Environment

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fluorescence could be observed even after 8 h incubation in DMED medium. However, the fluorescence intensity of AuNCs@BSA gradually reduced with the prolonged incubation time, and it almost no fluorescence in Hela cells after 8 h incubation. This phenomenon was accordance with the results of sensitivity assay. The content of H2O2 is higher in tumor area compared with normal area, and H2O2 will cause fluorescence intensity reducing. In addition, the proteases in cell or culture medium influence the fluorescence of AuNCs. The AuNCs@NAC-CS had low sensitivity to H2O2 and trypsin compared with AuNCs@BSA, because CS was more stable than BSA in cell and culture environment. Thus, AuNCs@NAC-CS have good potential application in live-cell imaging as a fluorescent probe than BSA-protected AuNCs.

Fig 11. (a-i) Confocal fluorescent microscopic imgaes of showing the metabolism of AuNCs@BSA and AuNCs@NAC-CS 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 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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Bio-distribution of AuNCs@NAC-CS Fig 12 shows the bio-distribution of AuNCs@NAC-CS in different organs, such as heart, liver, spleen, lung and kidney and the dynamic fluorescence intensity change in different organs. The obvious fluorescence signal appeared in the liver and kidney of the normal mice after 6 h injection. The fluorescence intensity in kidney gradually increased until 6 h when achieved the highest intensity. After that, the fluorescence intensity decreased. The ultrasmall nanoparticles were high-efficient cleared to overcome the toxicity by nonspecific accumulation in healthy tissues/organs from renal in vivo44. The AuNCs@NAC-CS exhibited the similar process with gold nanoclusters in bio-distribution and metabolism, which was formed by multiple ultrasmall gold nanoclusters closing with each other. The high level of liver uptake of AuNCs@NAC-CS is owing to mononuclear phagocytic system (MPS) absorption. And the uptake of AuNCs@NAC-CS in kidney can be possibly correlated with renal excretion. Generally the hydrophilic polymer such as polyethylene glycol may extend in vivo circulation time. However, the number of NAC in CS-NAC was limited, and NAC mostly interred in AuNCs@NAC-CS which combined NAC-CS with Au. Therefore, few NAC was on the surface of AuNCs@NAC-CS, and the effect of NAC-chitosan was not very satisfying to extend in vivo circulation time.


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Fig 12. the ex-vivo fluorescence images of isolated organs (heart, liver, spleen, lung, kidney) from (a) the mice at 6 h post-injection, (b) the blank mice; (c) dynamic fluorescence intensity in different organs after injection.

Conclusion A new fluorescent probe (AuNCs@NAC-CS) was successfully synthesized and utilized for imaging of living cells. The as-prepared surface coating (NAC-CS) which worked as reductant and stabilizer is more stable and easily to introduce other groups because of active groups in NAC-CS molecular chain such as amino and hydroxyl groups. Compared with before, the method is simple and green. The probe is demonstrated to possess many advantages in imaging, such as low cytotoxicity, low sensitivity to tumor cells contents (H2O2 and protease) and long-time imaging. The bio-friendly nature and near-infrared fluorescence of AuNCs@NAC-CS can be used as candidates for optical cell imaging, living imaging, targeted drug delivery for cancer treatment or imaging agents for early tumor diagnosis.

Acknowledgements This work was supported by the National Natural Science Foundation of China (Grant Nos. 31271023, 81501578), the Fundamental Research Funds for the Central Universities.

Supporting Information FT-IR spectrum, the fluorescence intensity of AuNCs@NAC and water with filter, the fluorescence intensity of AuNCs@NAC-CS in the different pH of solution, the TGA of AuNCs@NAC-CS, the fluorescence intensity of AuNCs@NAC-CS and AuNCs@NAC, fluorescence images of HeLa cells after different incubation time with AuNCs@BSA and AuNCs@NAC-CS.

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Chitosan-stabilized self-assembled fluorescent gold nanoclusters for cell imaging and bio-distribution in vivo Ying Duana, Ruiping Duanb*, Rui Liub, Man Guanb, Wenjuan Chenb, Jingjing Mab, Mingmao Chena, Bo Dub,Qiqing Zhangab* a Institute of Biomedical and Pharmaceutical Technology, Fuzhou University, Fuzhou 350002, China b Institute of Biomedical Engineering, Chinese Academy of Medical Science and Peking Union Medical College, Tianjin Key Laboratory of Biomedical Material, Tianjin 300192, China * E-mail: [email protected]; [email protected].

For Table of Contents Use Only

For Table of Contents Use Only


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Near-infrared luminescent AuNCs@NAC-CS possessed good fluorescence, low cytotoxicity, and low sensitivity to cells, allowing imaging of the living cells. And AuNCs@NAC-CS could be cleared with nonspecific accumulation in healthy tissues/organs in in vivo experiment.


Chitosan-Stabilized Self-Assembled Fluorescent Gold Nanoclusters

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