Facile one-pot synthesis of Au(0)@Au(I)–NAC core–shell nanoclusters with orange-yellow luminescence for cancer cell imaging

Abstract

We report a facile strategy to synthesize ultrabright core–shell gold nanoclusters in one pot by using N-acetyl-L-cysteine (NAC) as both a reducing and protecting agent, in which the core is Au(0) atoms and the shell is oligomeric Au(I)–NAC complexes. The Au(0)@Au(I)–NAC core–shell nanoclusters (NCs) displayed excitation and emission bands at 340 and 590 nm, respectively. It showed a strong orange-yellow photoluminescence with a quantum yield of 14%. The thermogravimetric analysis and mass spectrometry data suggest that the as-synthesized NCs comprise mainly Au₂₇NAC₃₂, in which a uniquely high thiolate-to-Au ratio (1.2 : 1) endows the gold clusters better biocompatibility. The Au(0)@Au(I)–NAC core–shell NCs offered ultra-small size, excellent stability, large Stokes shift and microsecond-scale lifetime, and exhibited negligible cytotoxicity to cancer cells. Based on the excellent properties of the gold nanoclusters (AuNCs), cell experiments were conducted. Cytotoxicity studies showed that the AuNCs exhibited negligible effects in altering cell proliferation or triggering apoptosis. The as-synthesized AuNCs have been successfully applied as a photoluminescent probe for human bladder cancer cellular imaging.

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Introduction

Gold nanoclusters (AuNCs), possessing numerous peculiar features, have become the subject of intensive research in biological, catalysis, environmental, and pharmaceutical fields.^1–5 These widespread applications primarily arise from their unique size- and morphology-dependent optical and electrical properties that are different from their bulk counterparts. Owing to the electronic properties of AuNCs transitioning from bulk-like continuum electronic states to molecule-like and discrete electronic orbital levels, the size regime between 1 and 5 nm is of striking concern.⁶ Therefore, ultrasmall gold NCs are expected as promising candidates for the design of novel probes. The most interesting feature of AuNCs is their luminescence properties for advanced diagnostics and in vivo imaging of cellular processes. Luminescence AuNCs, which combine strong luminescence with low toxicity, ultrafine size, and good biocompatibility, are ideal bioimaging and theranostic probes^7–9 only if they can be synthesized by general and convenient methods.

In the past decade, the synthesis of gold clusters have been described extensively since the pioneering work of Brust and coworkers in 1994,¹⁰ especially their applications arising from their unique luminescence characteristics. Several approaches have been developed for the synthesis of luminescence AuNCs on the subnanometer scale. For example, AuNCs are commonly prepared by the reaction between Au(I)–thiolate complexes and a strong reducing agent, such as sodium borohydride.^11,12 Moreover, mild reductant such as tetrakis(hydroxymethyl)phosphonium chloride has been applied to synthesize AuNCs.¹³ A one-step synthesis of luminescence gold clusters receives much concern recently, in which gold clusters can be obtained directly by reducing gold salt with suitable stabilizing reagents.^14–16 4-(2-Hydroxyethyl)-piperazine-1-ethanesulfonic acid and 2-(N-morpholino)ethanesulfonic acid have been used to assist the synthesis of green-emitting AuNCs due to their inherent reducing properties.¹⁷ Gold clusters with blue luminescence stabilized by poly(amidoamine) dendrimers and N,N′-dimethylformamide were achieved.^15,18 Blue light-emitting AuNCs were synthesized in a simple one-pot process via reflux of Au ions with amino-terminated poly(1,2-butadiene) in toluene.¹⁹ Importantly, the introduction of biomolecules in the synthesis of AuNCs can make the procedure greener and the biocompatibility of AuNCs could be enhanced. For instance, biomaterials such as L-3,4-dihydroxyphenylalanine, histidine and L-amino acid oxidase were employed as the reducing/capping agents for the synthesis of AuNCs with green and red luminescence.^20–22 But the drawbacks of these existing synthetic methods are that they require ether multiple/complex steps or long reaction times. And some synthesis strategy used extra assistant chemical reagent such as NaOH, or the as-synthesized gold clusters tended to aggregation under high temperature.

In this work, N-acetyl-L-cysteine (NAC) was used as a green reducing and stabilizing agent to prepare aqueous Au(0)@Au(I)–NAC core–shell nanoclusters (Au(0)@Au(I)–NAC NCs or NCs for short). The proposed method was really convenient and environmental friendly, exempted from pressuring, special treatment and media and these reactions are green and atom-economic processes, only involving the reactants of HAuCl₄ and NAC possessing biocompatibility without extra catalysts, templates or other chemical reagents. Herein, we report a facile satisfying one-pot synthesis procedure for an orange-yellow emitting core–shell AuNCs. The thermogravimetric analysis and mass spectrometric data suggest that the as-synthesized NCs comprises mainly Au₂₇NAC₃₂, in which uniquely high thiolate-to-Au ratio (1.2 : 1) endow the gold clusters better biocompatibility. The NCs displayed excellent stability, including storage in water at room temperature (25 °C) and elevated temperature (70 °C), in solutions of high salt concentration (e.g., 500 mM NaCl), and in common buffer solutions over the pH range 2–12. The good biocompatibility, excellent stability, large Stokes shift (250 nm), microsecond-scale lifetime and strong emission of the core–shell AuNCs give them potential as a probe for cellular imaging applications.

Experimental section

Reagents and materials

N-Acetyl-L-cysteine (NAC) was obtained from International Laboratory (San Bruno, CA, USA). Hydrogen tetrachloroaurate trihydrate (HAuCl₄·3H₂O) was provided by Aldrich (Milwaukee, WI, USA). Glacial acetic acid (CH₃COOH) and absolute ethyl alcohol (CH₃OH) were purchased from Tianjin Chemical Reagent Company (Tianjin, China). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetra-zolium bromide (MTT) was obtained from Solarbio (Beijing, China). 10 mM phosphate buffer solutions (PBS) were prepared by mixing appropriate volumes of standard solutions of 10 mM Na₂HPO₄ and 10 mM NaH₂PO₄. Cellulose ester membrane tube (500 Da cutoff) was purchased from Spectrum Laboratories (Rancho Dominguez, CA, USA). Ultrapure water (Milli-Q) with a resistivity of 18.2 MΩ was used as the general solvent throughout the study. All reagents of analytical reagent grade or above were used as received.

Synthesis of Au(0)@Au(I)–NAC NCs

Au(0)@Au(I)–NAC NCs was synthesized based on a reported Au–thiolate NCs method with some modifications.²³ All glassware was thoroughly cleaned with aqua regia (HNO₃/HCl, 1 : 3) and rinsed extensively with Milli-Q water prior to use. Freshly prepared solutions of HAuCl₄ (0.10 M, 0.4 mL) and NAC (0.10 M, 0.6 mL) were mixed with a 1.6 mL MeOH/glacial acetic acid (6 : 1, v/v) solution under magnetic stirring at 25 °C for 30 min, which turned from bright yellow to orange with some white suspensions. 8.7 mL Milli-Q water was then added to the mixture, and the precipitate was dissolved within seconds. Subsequently, the solution was heated to 70 °C with reflux under magnetic stirring for 24 h. An aqueous solution of strongly orange-yellow emitting Au(0)@Au(I)–NAC NCs was formed. A cellulose ester dialysis membrane (MWCO: 500 Da) was then used to separate the Au(0)@Au(I)–NAC NCs from any residual unreacted species. After dialysis for 3 days, the solvent (H₂O) was removed by a freeze-dryer. The orange-yellow emitting Au(0)@Au(I)–NAC NCs solid could be stored at room temperature for 6 months or longer with negligible changes in their optical properties.

Materials characterization

UV-vis absorption spectra were recorded on a Shimadzu UV-2450 absorption spectrophotometer (Tokyo, Japan). Photoluminescence (PL) spectra and lifetimes were measured using time-resolved/steady state fluorescence spectrometers (Edinburgh Instrument FLS-920, Livingston, UK). pH measurements were taken on a FE20 pH-meter (Mettler Toledo Instrument Inc, Shanghai, China). Transmission electron microscopic (TEM) measurements were acquired on an FEI Tecnai G220 S-TWIN TEM (Portland, OR, USA) operated at an accelerating voltage of 300 kV. The dynamic light scattering analysis was performed with a Malvern Instruments Nano-ZS90 Zetasizer (Malvern, UK). The matrix-assisted laser desorption/ionization-time-of-flight mass spectra (MALDI-TOF MS) of Au(0)@Au(I)–NAC NCs were acquired on an Autoflex MALDI-TOF mass spectrometer (Bruker, Bremen, Germany) equipped with a nitrogen (N₂) laser (337 nm). Fourier transform infrared (FTIR) spectra were performed on a Perkin-Elmer Paragon 1000 FTIR spectrometer (Waltham, MA, USA). Thermogravimetric analysis (TGA) was conducted on a Perkin-Elmer TGA 6 thermogravimetric analyzer (TGA) (Waltham, MA, USA). X-ray photoelectron spectrum (XPS) was acquired on a Leybold Heraeus SKL-12 X-ray photoelectron spectrometer (Shenyang, China).

Luminescence quantum yield of Au(0)@Au(I)–NAC NCs

The quantum yield of the Au(0)@Au(I)–NAC NCs was calculated using the following equation:

Φ_x = Φ_s × (A_s/A_x) × (I_x/I_s) × (n_x/n_s)²

where the subscripts s and x refer to photoluminescence standard and Au(0)@Au(I)–NAC NCs, respectively, Φ is the quantum yield, A is the absorbance, I is the integrated luminescence intensity, and n is the refractive index of solvent. Quinine sulfate in 0.10 M H₂SO₄ with a quantum yield of 0.54 was used as the luminescence standard. The excitation wavelength of the standard and Au(0)@Au(I)–NAC NCs was 340 nm.

MTT assay

For the cell cytotoxicity text, BIU-87 cells were first plated on a Costar 96-well tissue-culture cluster and cultured at 37 °C with 5% CO₂ in air for 3 h to adhere cells onto the surface. The well without cells and treatment with Au(0)@Au(I)–NAC NCs was taken as a zero set. The medium was then changed with 100 μL of fresh DMEM supplemented with 10% FBS containing Au(0)@Au(I)–NAC NCs, and the cells were allowed to grow for another 24 h. At least five parallel samples were performed in each group. Cells without treatment with Au(0)@Au(I)–NAC NCs were taken as a control. After adding 20 μL of 5.0 mg mL⁻¹ MTT reagent into every well, the cells were further incubated for 4 h, followed by removing the culture medium with MTT, and then 150 μL of DMSO was added. The resulting mixture was shaken for ca. 10 min at room temperature. The absorbance value of the mixture was measured at 490 nm with a SunRise microplate reader (Tecan Austria GmbH, Grödig, Austria). The cell viability was estimated using the equation of cell viability (%) = [Σ(A_i/A_control) × 100]/n, where A_i is the absorbance of different concentrations of the probe, A_control is the average absorbance of the control well in which the probe was absent, and n (6) is the number of the data point.

Cellular imaging

BIU-87 cells were cultured in DMEM supplemented with 10% FBS and incubated at 37 °C in a 5% CO₂ atmosphere. The Au(0)@Au(I)–NAC NCs aqueous solution (100 μL, 2.2 mg mL⁻¹) was added to the culture medium (1.0 mL) at 0.20 mg mL⁻¹ final concentration. After incubation for 2 h, the BIU-87 cells were harvested using 0.25% trypsin/0.020% EDTA, washed three times (1.0 mL each) with pH 7.4 PBS (comprising 137 mM NaCl, 2.7 mM KCl, 8.0 mM Na₂HPO₄, and 2.0 mM KH₂PO₄), and kept in PBS for optical imaging by a Olympus FV1000 confocal microscope (Tokyo, Japan) with 40× objective.

Results and discussion

Synthesis of Au(0)@Au(I)–NAC NCs

In this study, we prepared photoluminescence Au(0)@Au(I)–NAC core–shell NCs in aqueous solutions incorporating NAC as both a reducing and protecting agent. The NCs were formed by mixing organic solutions of NAC and aqueous solutions of HAuCl₄ at 25 °C for 30 min, followed by ultrapure water addition dissolving the white insoluble solid and heating to 70 °C with reflux under magnetic stirring for 24 h. Formation of the Au(0)@Au(I)–NAC NCs involved three steps as shown in Scheme 1(A). The first step was the reduction of Au(III) to Au(I) by the thiol group of NAC, followed by the coordination of Au(I) to the thiol group to form insoluble white aggregates of Au(I)–thiolate complexes, or to the carboxyl groups of NAC or Cl⁻ to form Au(I)–carboxyl/Cl complexes. The second step, plentiful H₂O was added, the pH value of the solution increased from 1.42 to 1.96. NAC in the Au(I)–thiolate complexes was negatively charged at pH 1.96, which is consistent with the reported dissociation constant (pK_a = 1.71) of the carboxyl group in the cysteine residue.²⁴ The negative charge imparts the Au(I)–thiolate complexes good water solubility to form the oligomer of Au(I)–NAC complexes. Due to the high affinity between Au(0) atoms and Au(I), the third step was the selective reduction the Au(I)–carboxyl/Cl complexes to Au(0) atoms and subsequent sequestration of the Au(0) species by the oligomeric Au(I)–NAC complexes at the elevated temperature of 70 °C. Scheme 1(B) illustrates the schematic structure of Au(0)@Au(I)–NAC NCs end-product, in which the core is Au(0) atoms and the shell is oligomeric Au(I)–NAC complexes. NAC was the reducing-cum-protecting agent in the synthesis, and the reaction temperature was used to vary its dual functionality. The effect of reaction temperature on luminescence NCs was studied, and control experiments were carried out at 50, 60, 80, and 90 °C. The results (Fig. 1) show the luminescence intensity of 70 °C is higher than that of other temperatures. We speculate that Au(I)–carboxyl/Cl complexes could not be effectively reduced to form Au(0) core when the reaction temperature was less than 60 °C. When the reaction temperature was higher than 80 °C, the oligomer of Au(I)–NAC complexes started to be reduced, resulting in the decrease in the luminescence of NCs.

Scheme 1 Schematic of synthesis of luminescence Au(0)@Au(I)–NAC NCs.

Fig. 1 The normalized photoemission spectra of as-synthesized NCs at different reaction temperatures.

As such, we chose 70 °C as the optimal reaction temperature in the synthesis process, where sufficient Au(I)–carboxyl/Cl complexes could be reduced to Au(0) atoms while the oligomeric Au(I)–NAC complexes were left mainly unaffected. Protection of oligomeric Au(I)–NAC complexes from reduction in the process and control of their aggregation on the Au(0) surface were crucial to the formation of highly luminescence Au(0)@Au(I)–NAC core–shell NCs.

Optical properties of Au(0)@Au(I)–NAC NCs

Fig. 2A depicts the UV-vis absorption spectrum and the photoluminescence spectrum of Au(0)@Au(I)–NAC core–shell NCs. The absence of the typical surface plasmon resonance band at 520 nm (blue line) indicates that the as-synthesized NCs product has a dimension <2 nm.²¹ The UV-vis spectrum of the NCs does not show a molecular-like absorption that is the typical characteristic of conventional thiolate-protected AuNCs with more than 15 Au atoms.²⁵ Strong emission of the NCs appears at 590 nm (black line), with an excitation maximum at 340 nm (red line). Orange-yellow photoluminescence can be observed under UV light irradiation (inset of Fig. 2A). In contrast to organic fluorophores, the NCs exhibited a large Stokes shift (250 nm), suggesting that the emission was mainly phosphorescence. This was also supported by the photoluminescence lifetime measurement.

Fig. 2 (A) UV-vis absorption (blue line), normalized photoemission (black line, λ_ex = 340 nm), and photoexcitation (red line, λ_em = 590 nm) spectra of the luminescent Au(0)@Au(I)–NAC NCs. Inset: photographs of the NCs under room light (left) and a hand-held UV lamp with excitation at 365 nm (right). (B) Photoluminescence decay as a function of lifetime for Au(0)@Au(I)–NAC NCs.

Photoluminescence lifetime measurement (Fig. 2B) reveals that the luminescence decay of the NCs can be fitted with a biexponential curve, suggesting the possible existence of two components with life times of τ₁ = 2.04 μs (55.06%) and τ₂ = 8.54 μs (44.94%). The emission of NCs was assigned to a triplet metal-centered state via the relatively long lifetime (microsecond-scale). Through comparison with quinine sulfate, the quantum yield of the NCs was determined to be 14%, which is higher than that of most previously reported luminescent Au–thiolate NCs (typically 0.001–0.1%).^25–28 The luminescent properties of gold complexes are known to be affected by gold–gold interactions, and these interactions depend on the nature of the ligands, that is, electron donating power, size, and energy level of the frontier orbital.^29–31 The origin of such strong emission of the as-synthesized NCs could be ascribed to the ligand-to-metal charge transfer (LMCT) which is mainly affected by the types of ligands as well as the ligand-to-metal–metal charge transfer (LMMCT) from the sulfur atom in the NAC ligands of Au(I)–NAC oligomer to the Au atoms.

The Au–Au interaction and the associated luminescent properties can be perturbed through different environmental stimuli, such as pH, ionic strength of media, and temperature. The luminescence stability of the NCs was investigated under various experimental conditions including pH, temperature, salt concentration and storage. As shown in Fig. 3, the luminescent properties of the as-synthesized NCs are quite stable. The luminescence intensity of NCs could be maintained above 90% in the pH range of 2.0–12.0 (Fig. 3A). Even under elevated temperature (70 °C), the luminescence intensity of NCs almost did not change (Fig. 3B). The change of luminescence intensity was less than 3% even after 6 months storage at room temperature (25 °C, Fig. 3C). It is obvious that the intensity did not change appreciably by subjecting the clusters to 0.0–500 mM NaCl (Fig. 3D).

Fig. 3 Normalized photoluminescent intensity of Au(0)@Au(I)–NAC NCs in PBS at different pH (A), temperature (B), time (C) and concentrations of NaCl (D). The error bars represent variations among six separate measurements.

Characterization of the Au(0)@Au(I)–NAC NCs

Various spectroscopic techniques were employed for characterization of the as-synthesized NCs product. The morphology and hydrodynamic size of the NCs were assessed by HRTEM and dynamic light scattering (DLS) analysis as depicted in Fig. 4. The HRTEM image (Fig. 4A) shows that the NCs display nearly spherical shape and have an average diameter of below 2 nm, which is in agreement with that of the UV-vis data. Fig. 4B shows the size distribution histogram generated from analyzing several TEM images. 100 particles in the images were used to assess the size of the NCs sample. The average diameter is calculated to be 1.41 ± 0.11 nm. Fig. 4C shows the average hydrodynamic size of the Au(0)@Au(I)–NAC NCs to be ∼3.5 nm from the DLS measurement. The hydrodynamic diameter of nanoparticle obtained by DLS was larger than those by TEM due to the presence of solvation layer around the NCs in aqueous solution.³²

Fig. 4 (A) HRTEM image. (B) The particle size histogram from analyzing several TEM images. (C) Particle size distribution from DLS measurement of the Au(0)@Au(I)–NAC NCs.

IR spectrometry can be employed to identify the types of functionality of ligands attached to the nanoclusters. Fig. 5A displays the IR spectra of the free NAC (black line) and the NCs samples (red line). When the two FTIR spectra are compared, the NAC molecule in the form of thiolate protects the Au core and this is confirmed by the absence of the band at 2550 cm⁻¹ assigned to the S–H stretching vibration. Amide I belt V_C=O and amide II belt δ_NH of the NCs occur at ∼1641 and ∼1535 cm⁻¹. An obvious difference is observed at ∼3376 with a red shift of 18 cm⁻¹ and a wide loose peak for amide group of NCs as compared with the free NAC, which might be ascribed to the hydrogen bonding interactions. The lower energy of the NCs amide II band as compared to the free ligand (1541–1585 cm⁻¹) provides another evidence that the hydrogen bonding in antiparallel, intercluster, which could transpire through interdigitation of ligands or ligand bundles on adjacent cluster molecules as postulated previously for alkane thiolate protected AuNCs.^33–35 The presence of the carboxyl group on the Au(0)@Au(I)–NAC NCs is confirmed by the band at the carboxyl stretching at ∼1728 cm⁻¹.³⁴ In addition, the fine structure at <1500 cm⁻¹ is broadened as compared to that of the free ligand due to the effect of the conduction electrons on the Au shell. In brief, the IR spectra demonstrate the attachment of NAC in the form of thiolate to the Au of the Au(0)@Au(I)–NAC NCs.

Fig. 5 (A) FTIR spectra of (a) pure NAC, (b) Au(0)@Au(I)–NAC NCs with dialysis; (B) Au 4f XPS spectra of Au(I)–NAC complexes (red line), as-synthesized luminescent Au(0)@Au(I)–NAC NCs (black line) and Au(0) nanoclusters (blue line).

The XPS technique was employed to investigate the valence state of Au in the Au(0)@Au(I)–NAC NCs and intermediate product white Au(I)–NAC complexes. For comparison, NAC protecting Au(0) NCs was prepared according to our previous report.¹¹ As shown in Fig. 5B, the binding energies of Au 4f_7/2 peaks of these three Au materials is 84.2 (black line), 84.4 (red line), and 84.0 eV (blue line). The Au 4f spectrum of the luminescence Au(0)@Au(I)–NAC NCs was further deconvoluted into Au(I) and Au(0) components. The integrated areas of these two bands illustrate that the Au(I) content constitutes ∼60% of all Au atoms in the NCs.

TGA is commonly employed to determine the relative compositions of the organic ligand and the ligand-to-metal ratio of gold nanoparticles. The NAC-to-Au ratio in Au(0)@Au(I)–NAC NCs was estimated by TGA at a heating rate of 10 °C min⁻¹ as shown in Fig. 6A. The results show that there was nearly no weight loss up to 170 °C. The weight loss mainly occurred in the temperature range from 170 to 500 °C. As shown in Fig. 6A, it was clear that there is an onset at 260 °C. The probable explanation is the thermal desorption of NAC as volatile disulfide before 260 °C. The weight loss from 260 to 500 °C is attributed to the combustion of NAC. The TGA data also determined the organic fractions to be 50 wt% of the nanoclusters. This can be translated into a NAC-to-Au mole ratio of 1.2 : 1, which is slightly different with the synthesis ratio (1.5 : 1). There is still some unreacted NAC in the crude product which can be removed by the dialysis. This value is significantly higher than that of the reported conventional Au–thiolate NCs with more than 15 Au atoms.^36–38 It is consistent with 1 : 1 thiolate-to-Au ratio of many oligomeric or polymeric Au(I)–thiolate complexes.^39–41 The high Au(I) content and high NAC-to-Au ratio are indications of a high content of oligomeric Au(I)–thiolate complexes in the AuNCs, which is consistent with the reported AuNCs.²³

Fig. 6 (A) Thermogravimetric analysis of Au(0)@Au(I)–NAC NCs; (B) MALDI-TOF mass spectra of Au(0)@Au(I)–NAC NCs.

Fig. 6B displays the MALDI-TOF MS spectrum of Au(0)@Au(I)–NAC NCs. A broad mass peak is obtained at ∼10.5 kDa in the largest abundance (tag with star). Since NAC-to-Au mole ratio is 1.2 : 1 that obtained in TGA data, according to the largest abundance of MS spectrum, we can estimate the numbers of Au and NAC approximately as 27 and 32, respectively. Namely, the average mass of the as-prepared Au(0)@Au(I)–NAC NCs is assigned as Au₂₇NAC₃₂.

Cellular imaging applications

Since Au(0)@Au(I)–NAC NCs possess a set of features including their biocompatibility, ultrasmall size, good stability and long Stokes shift that make them attractive as intracellular imaging probes.⁴² After incubation with Au(0)@Au(I)–NAC NCs for 2 h, the BIU-87 cells were imaged by using a spinning disc laser microscope, where the bright luminescence from Au(0)@Au(I)–NAC NCs can easily be seen (Fig. 7B). The overlay of luminescence (Fig. 7C) and bright field (Fig. 7A) images revealed that Au(0)@Au(I)–NAC NCs were localized inside the cells (Fig. 7C), demonstrating the capability of our Au(0)@Au(I)–NAC NCs to stain the interior of the living cells. With respect to the detailed uptake mechanism of ultra-small NCs and their accurate intracellular behavior, much work still requires to be done. Recently, most researchers have proposed that the internalization rate and mechanism of gold nanoparticles is closely related to cell type, nanoparticle size, charge and other surface properties.⁴³ Moreover, it was reported that, the nanoparticles of size less than 50 nm entered cells mainly by endocytosis through a clathrin-mediated process, and intracellular trafficking through the endosomal pathway.^44–46 We therefore speculate that Au(0)@Au(I)–NAC NCs may also enter cells through clathrin-mediated endocytosis. Certainly, further study on the mechanism of the entrance of Au(0)@Au(I)–NAC NCs into cells is needed. The successful trial in the present work to apply Au(0)@Au(I)–NAC NCs for real-time monitoring of BIU-87 cells in living subjects confirmed the remarkable application potential of Au(0)@Au(I)–NAC NCs in the biomedical imaging and related fields.

Fig. 7 The luminescence microscopy images of the optical sections of BIU-87 bladder cancer cells after incubation with Au(0)@Au(I)–NAC NCs for 2 h (A) bright-field and (B) 405 nm excitation. (C) The merged image of (A) and (B).

Cell cytotoxicity assay

Cytotoxicity is an indispensable parameter for future application of Au(0)@Au(I)–NAC NCs especially in live cell-labelling. In vitro toxicity experiments of Au(0)@Au(I)–NAC NCs were evaluated using the human bladder cancer (BIU-87) cell lines through the MTT assay. As shown in Fig. 8, the results manifested that BIU-87 cells remained above 85% viability after 24 h of treatment with 0.1–0.3 mg mL⁻¹ NCs. Even up to relatively high concentration, BIU-87 cells still had a viable percentage of more than 75% after having been incubated with 0.5 mg mL⁻¹ NCs for 24 h. In the previous studies, evidence indicates that the cytotoxicity of metal NCs is mostly from the release of toxic ions, such as Ag⁺, Au^1+/3+, and Cd²⁺, in the acidic environment of lysosomes.^47,48 Although BIU-87 cells did not show any major toxicity, the decreased cellular viability with the increase in the treatment dose of NCs is attributed to the increase of the NCs inside the cells, especially in the lysosomes. The low toxicity toward human bladder cancer cells further strengthens our view that Au(0)@Au(I)–NAC NCs are promising luminescent probes for advanced diagnostics and in vivo imaging of cellular processes.

Fig. 8 The viability (%) of BIU-87 cells after 24 h treatment with NCs calculated from MTT assay.

Conclusions

We have successfully developed a “green” method for the preparation of core–shell AuNCs using NAC as both a reducing and protecting agent in a facile one-pot strategy. The as-prepared Au(0)@Au(I)–NAC core–shell NCs showed a strong orange-yellow photoluminescence with an improved quantum yield of 14%. Besides, the Au(0)@Au(I)–NAC NCs has several other attractive features, e.g., ultra-small size (core diameter: 1.4 nm), long luminescence lifetime (τ₁ = 2.04 μs, τ₂ = 8.54 μs), large Stokes shift (250 nm), excellent biocompatibility and high stability. All these make the Au(0)@Au(I)–NAC NCs highly attractive for bioimaging. The developed facile synthesis method is scalable and may provide a meaningful reference for the further design and application of NCs in the fields of analytical detection and chemical sensing. This work is in progress in our research group.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (No. 21305082 and 21403135), Program for the Outstanding Innovative Teams of Higher Learning Institutions of Shanxi, Research Project Supported by Shanxi Scholarship Council of China (No. 2014-017), Fund Program for the Scientific Activities of Selected Returned Overseas Professionals in Shanxi Province, 131 Leading Talents Project of Higher Learning Institutions of Shanxi, and Youth Foundation of Shanxi Province (No. 2011021005-1 and 2013021008-5).

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