Silver-Assisted Thiolate Ligand Exchange Induced PhotoluminescentBoost of Gold Nanoclusters for Selective Imaging of IntracellularGlutathioneXueqi Hu,§,† Youkun Zheng,§,† Junyu Zhou,‡ Danjun Fang,‡ Hui Jiang,*,† and Xuemei Wang*,†

†State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, National Demonstration Center forExperimental Biomedical Engineering Education (Southeast University), Southeast University, Nanjing 210096, P. R. China‡School of Pharmacy, Nanjing Medical University, Nanjing 210029, P. R. China

*S Supporting Information

ABSTRACT: Metal/ligand exchange is a common strategyfor precise assembly of metal nanoclusters (NCs) in organicphases. However, such a case is still not well studied in theaqueous phase. In this work we have demonstrated the silverions assisted ligand exchange on water-soluble N-acetylcys-teine (NAC) stabilized Au NCs. Silver ions may trigger bothsilver−gold metal exchange and silver addition on Au NCs.Unlike those well-reported silver induced photoluminescent(PL) enhancements, the processes show little changes in PLintensity. The as-obtained AuNAC@Ag NC can furtherpromote the ligand exchange between NAC and glutathione(GSH) and induce a maximum of a 20-fold increase in PLemission at 570 nm. The enhancement was proportional to theconcentration of GSH, with a linear range of 0−0.5 mM. For other thiol compounds such as cysteine, NAC, and cysteamine, nosignificant PL changes were observed. Cytotoxicity evaluation shows that the AuNAC@Ag NCs are biocompatible. Thus, theintracellular GSH can be specially visualized by the formation of stable AuNAC@AgGSH NCs. These results may be helpful toreveal the underlying processes of metal/ligand exchange on NCs in aqueous environment and pave a new avenue for faciledesign and preparation of efficient imaging probe candidates.

■ INTRODUCTION

Intracellular redox balance is a key factor for maintaining cellphysiological stability. The abundant intracellular thiolatecomponents are responsible for the reducing cytoplasmenvironment. Three main types of antioxidant systems havebeen evolved inside cells on the basis of thiol/disulfide redoxpairs, i.e., glutathione pair (glutathione, GSH/glutathionedisulfide, GSSG), cysteine/cystine, and thioredoxin pair.1,2

Among these components, GSH is an important molecule tokeep the redox balance.3 Thus, the determination of intra-cellular GSH levels may be of great importance to evaluatedifferent cell status, especially immunological response,4 aging,5

and tumor metastasis,6 due to the observable GSH levelchanges during their occurrence.Recently photoluminescent (PL) probes for intracellular

thiol detection are well researched, including organic andinorganic candidates.7 Generally, these probes still face greatchallenges. Many thiol-responsive organic dyes have beenreported, but they may face difficulty for long-term observationdue to limited photostability.8−10 On the other hand, some II−VI semiconductor quantum dots show high quantum yields buthave high risks of cytotoxicity.11 In addition to these issues, the

possible influence by intracellular autofluorescence should bewell considered.In the recent decade, metal nanoclusters (NCs), especially

gold NCs, were proposed as alternatives for bioimaging becauseof their stability, biocompatibility, and wide emission spectrumranges.12,13 The NCs have an ultrasmall size of 1−3 nm andtransitional features between molecules and larger nano-particles.14 The PL mechanisms are quite complicated andintegrated both effects from sizes (metal atom numbers) ofNCs and specific ligands. The energy levels interval inside NCsare close to the Fermi wavelengths, resulting in a sizedependent electronic structure.15 Besides, ligands play anessential role in mediating PL emission. Even subtle changes inligands have a dramatic effect on the PL properties. Forexamples, Jin’s group16 showed the special effect ofmethylthiolbenzene ligands. Au130, Au104, and Au40 NCs areobtained by adjusting the methyl group at ortho-, meta-, andpara- sites, rendering different PL emissions. In the meantime,the PL efficiency of NCs is also affected by features of ligands

Received: November 23, 2017Revised: March 10, 2018Published: March 12, 2018

Article

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and valence states of metal (especially Au) cores. It is well-known that the charge transfer from the surface thiolate ligandsto the metal core can determine the PL of Au NCs, and thesurface ligands with electron-rich atoms can effectively enhancethe emission.17 Thus, thiols of different chains are used toassemble versatile NCs with efficient PL.The facile detection of thiols by Au nanostructures is possible

due to the versatile changes in optical and electronic propertiesinduced by gold−sulfur interaction.18,19 We recently becamedevoted to probing the PL enhancement processes bycontrolling the surface coverage of ligands around NCs. Wefound the imaging of thiols inside cells by special surface“unsaturated” GSH coated Au NCs.20 However, it still lackssystematic investigation to develop probes specific for certainanalytes. In this work we have demonstrated the silver-assistedthiolate ligand exchange on N-acetyl cysteine (NAC) coated AuNCs (Scheme 1). The ligand exchange, denoting thesubstitution of weaker ligands on NCs by stronger ligands, isa known strategy for preparation of NCs. However, forthiolated Au NCs containing a large number of strong Au−Sbonds, the ligand exchange is rarely reported. To the best ofour knowledge, only a recent work reported the ligandexchange between thiolated Au NCs by phosphine in organicphase.21 Here we initially observed that silver ions caneffectively combine to AuNAC NCs by d10−d10 metallophilicinteractions to obtain AuNAC@Ag NCs in aqueous phases.This process shows little changes in PL emission at 570 nm,while the further binding of GSH to AuNAC@Ag NCs caninduce a maximum of 20-fold increase in PL. The PLenhancement is specific to GSH, with limited response toother common thiol compounds such as cysteine, NAC, andcysteamine. The AuNAC@Ag NCs are almost noncytotoxiceven at a high concentration and can be used as a candidate tovisualize the intracellular GSH, which show great potential toreveal the relationships of redox balance and physiologicalstatus inside cells.

■ EXPERIMENTAL SECTIONMaterials and Instruments. GSH, NAC, cysteine (Cys),

cysteamine (CyA), and buthionine sulfoximine (BSO) were productsof Sigma-Aldrich (St. Louis, MO, U.S.A.). Hoechst 33342 (beyotime,China) and Lysotracker Red DND-99 (life technology, thermo,U.S.A.) were used for costaining of cell nucleus and lysosomes.Chloroauric acid (HAuCl4) and silver nitrate were purchased fromSinoreagent Co. Ltd. (Shanghai, China). All other reagents are ofanalytical grade. Amicon Ultra-15 ultrafiltration tubes (with a

molecular-weight cutoff (MWCO) of 3 kDa, Millipore, U.S.A.) wereused for separation and concentration of crude Au NCs samples.Phosphate buffer saline (PBS) was purchased from Hyclone (U.S.A.).Deionized water (Milli-Q, Millipore, U.S.A.) was used throughout.

The PL and UV−vis spectra were obtained on an RF-5301PCspectrofluorophotometer (Shimadzu, Japan) and an Evolution 260-BioUV−visible spectrometer (Thermo, U.S.A.), respectively. Themorphology of NCs was observed on a transmission electronmicroscope (JEM-2100, JEOL, Japan). The X-ray photoelectronspectroscopy (XPS) was recorded on a PHI 5000 VersaProbeequipment (ULVAC-PHI, Japan). The mass spectroscopy wasperformed on an ABI-4800 Plus MALDI TOF/TOF analyzer (AppliedBiosystems, U.S.A.). The matrix used was α-cyano-4-hydroxycinnamicacid (CHCA). The CCK-8 assays were recorded on a Multiskan FCmicroplate reader (Thermo, U.S.A.). The cellular imaging was shot ona confocal laser scanning microscopy (Ti-C2, Nikon, Japan).

Synthesis of AuNAC NCs. The previous methods use sodiumborohydride as a reducing agent for preparation of AuNAC NCs.22 Wehere attempted a simple method and found that NAC itself can act asboth an efficient stabilizer and a reducing agent. In a typical method,the chloroauric acid (2.5 mM, 4 mL) and NAC (2.5 mM, 6 mL) wasmixed rapidly with a vortex. The colorless mixture was then stirred inan 80 °C water bath to turn to light yellow gradually in several hours,implying the formation of NCs. The clear solution was cooled to roomtemperature, and its pH was adjusted to neutral by addition of NaOH(1 M, 0.12 mL). The saline in the samples was removed by repeatedultrafiltration. To optimize the synthetic conditions, different molarratios of HAuCl4 and NAC were mixed. For pH selection, the pH ofthe mixture was adjusted to a desired value before heating. In a generaltest, the effect on the PL intensity of AuNAC NCs by silver ions wasinspected in ultrapure water (pH around 6) to avoid the possibleinterference by phosphate saline. Note that the pH changes are notsignificant during the whole experiments in ultrapure water.

Cell Culture and Cytotoxicity Test. The HepG2 liver cancercells and L02 normal liver cells were obtained from KeyGen Biotech.Co. Ltd. (Nanjing, China) and were cultured in Dulbecco’s modifiedeagle medium (DMEM, Hyclone, U.S.A.) containing 10% fetal bovineserum (Gibco, U.S.A.) and penicillin−streptomycin (Hyclone, U.S.A.)at 37 °C and 5% CO2 atmosphere.

The CCK-8 assay kit (Beyotime Co. Ltd., Shanghai, China) wasused to evaluate the cytotoxicity by NCs. All NC samples were firstfiltered by a sterile membrane (0.22 μm, Millipore, U.S.A.) and putunder UV irradiation before use. First, cells of 5 × 103 per well (totalvolume of 100 μL) were suspended and cultured in a 96-well plateovernight to achieve cell adhesion. NCs of a series of concentrationswere then added to the wells and incubated for 24 h. Subsequently, aCCK-8 solution of 10 μL was added to each well. The wells with sameamount of NCs, culture media, and CCK-8 solution but without cellswere used as blank control. After further incubation for 1 h, the

Scheme 1. Silver-Assisted Thiolate Ligands Exchange May Induce Photoluminescent Boost of Au NCs

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absorbance at 450 nm for each well was recorded on a microplate

reader. The cell viability was calculated by

= − − ×A A A AViability % [ ]/[ ] 100%NCs blank 0 blank

where ANCs, A0, and Ablank represent the absorbance for NC treatedcells, nontreated cells, and the blank control, respectively.

Cellular Imaging. Cells of 5 × 105 /mL were seeded into aFluoroDish (FD35-100, WPI, U.S.A.) and cultured for a certain time

Figure 1. (A) PL responses of AuNAC NCs to Ag+ with a concentration ranging from 0.1 to 100 μM. (B) PL response of AuNAC@Ag NCscontaining different amounts of Ag+ to GSH of 200 μM. (C) Dependence of PL intensity of AuNAC@Ag NCs on Ag+ concentrations in thepresence of GSH of 200 μM. Here the PL increasing ratio means the fluorescent ratio after and before addition of GSH to AuNAC@Ag NCs. (D)PL changes of AuNAC NCs (black curve) upon addition of Ag+ and GSH in the dark (green curve) or under exposure of light (red curve).Excitation/emission slit: 5 nm/10 nm.

Figure 2. (A, B) TEM images and (C, D) size distributions of (A, C) AuNAC@Ag and (B, D) AuNAC@Ag-GSH NCs.

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in a 37 °C incubator after the addition of NCs samples. The confocalmicroscopy was used to perform cell imaging processes. Forquantitative comparison, the ImageJ software was used to collect thepixels in a specific region of interest.

■ RESULTS AND DISCUSSIONSynthesis of AuNAC NCs. The general synthetic method

of thiolate-protected Au NCs is performed by formation of

Au(I)−thiolate polymer through reduction of Au(III) to Au(I)by thiol compounds. The Au(I)−thiolate may further bereduced by other reducing agents (commonly borohydride) oretching thiols (for examples, GSH) to produce Au NCs. NAChas been reported as a template for Au NCs for more than adecade.23 In some initial works, special buffers, including aceticacid and ethanol, were required to prepare NCs emitted at 570

nm.24 Recently, Deng et al.25,26 reported the syntheses of redemitted NCs (emission centered at 650 nm) at near neutralpH, with a rather high molar ratio of Au:NAC around 1:16.The two types of NCs should be different according to their PLfeatures. However, as a template, NAC was not as prevalent asGSH, and the properties of NAC coated Au (AuNAC) NCswere still not well studied.Here we demonstrated that AuNAC NCs emitted at 570 nm

can be synthesized just by simply heating a mixture of gold saltsand NAC, without special choice of pH buffer. The as-obtainedNCs have a relatively low PL efficiency. This feature allows thesignificant PL enhancement after attachment of the silver−thiolcomplex.The emission and excitation spectra of Au NCs prepared

with Au and NAC at a feeding molar ratio of 1:1.5 weremeasured after heating the mixture for different times (FigureS1A in the Supporting Information). The PL appears in 0.5 hand gradually increases with the heating time. After 7 h ofheating, the PL tends to be stable. The optimized excitation andemission wavelengths of the NCs are about 420 and 570 nm,respectively. A large Stokes shift (∼150 nm) is beneficial forefficient observation of intracellular targets. The ultrafiltrationexperiments confirm that the AuNAC NCs have a MW largerthan 3 kDa (Figure S2). The UV spectra showed an absorptionpeak at 320 nm for the Au and NAC mixture, while this peakwas moved to 350 nm upon heating, with a significant red shiftin wavelengths (∼30 nm), corresponding to the formation ofAu NCs and changes in solution color (Figure S1B). No furtherchanges were observed at larger heating time.The influence of Au and NAC feeding molar ratios on PL of

NCs was investigated (Figure S1C). The difference in thefeeding ratio does not cause significant changes in excitationand emission peak wavelengths of Au NCs. The sampleprepared with an Au:NAC molar ratio of 1:1.5 shows themaximum PL intensity, while the PL emission reaches almost70% of the maximum at an Au:NAC molar ratio of 1:1 and 1:2.For Au:NAC molar ratio of 0.5:1, only colloidal Au NPs areformed, with a typical surface plasmonic adsorption band. ForAu:NAC molar ratio larger than 2:1, white insoluble aggregatesappear and no PL is observed for the supernatant of thismixture. Besides precursor ratios, pH values also affected thePL performances (Figure S1D). The sample prepared with aAu:NAC molar ratio of 1:1.5 shows an acceptable PL emissionin acidic environments (i.e., pH 2.0 or 2.6), while the PLdecreases with the increasing pH and completely vanishes inbasic solution.The morphologies of as-prepared AuNAC NCs were

inspected by TEM (Figure S3). The size distribution ofAuNAC NCs is 2.1 ± 0.4 nm, according to the statisticalevaluation of 179 individual particles in TEM images. The highresolution TEM clearly shows a group of crystalline latticeswith an equal interdistance of 0.24 nm, corresponding to theAu(111) surface.

Effect of Ag+ and GSH on PL Intensity of AuNAC NCs.Silver ions are well-known for their high affinity toward goldnanostructures. The Au−Ag bimetallic nanocomposites arereadily formed after a simple mix and further reduction.27,28 ForAu NCs, similar conjugation usually induces great PLenhancement. For examples, Le Guevel et al.29 reported thesilver induced a 3−5-fold PL enhancement on GSH coated AuNCs at a dopant percentage of ∼2 wt %. We also observed thesilver dopant (∼5 atom %) can cause a red-shift for cytidinestabilized Au NCs from 490 to 560 nm, accompanied by an

Figure 3. MALDI-TOF MS analysis of (A) AuNAC, (B) AuNAC@Ag, and (C) AuNAC@AgGSH samples. The asterisk (*) indicates thenewly appearing MS peak after silver or GSH binding.

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even larger increase (∼25-fold) in PL efficiency.30 The atomicprecise control of AgxAu25−x NCs even results in a 200-foldquantum yield boost.31 However, in our current case, when Ag+

was added to the AuNAC NCs solution, it only showed anobservable red-shift in wavelength, but with slight PLenhancement. As shown in Figure 1A, the emission spectra atdifferent Ag+ concentrations to AuNAC NCs of 0.03 mg/mLwere measured. The PL intensity hardly shows any changes forAg+ concentration lower than 1 μM. When the concentrationreaches 10 μM, the emission peak wavelength is shifted toabout 590 nm, with a maximum of ∼2-fold in PL intensitycompared with that for Au NCs in the absence of Ag+. Thefurther addition of Ag+ gives no observable PL changes. Whilethe concentration of Ag+ is up to 100 μM, the PL intensity evendecreases, similar to the PL quenching effects reported formany heavy metal ions.12 These results indicate the possiblebinding of Ag+ to NCs, accompanied by slight changes in PL.The PL inhibition by Ag+ at high concentrations may be causedby excess free Ag+.Our previous work reported the attachment of thiol

compounds on the “unsaturated” surface of GSH coated AuNCs induced PL enhancement, which induced a PL enhance-

ment due to the formation of new NCs species.20 Here, the PLemission remains unchanged upon addition of GSH of 200 μMto AuNAC NCs. But interestingly, the PL level completelychanges after the subsequent addition of Ag+. Although Ag+

may weakly affect the PL of AuNAC NCs, this sharp increase(ca. 20-fold) in PL intensity is not only caused by silver ions(Figure 1B). Note that the PL intensity only has a 2−3-foldincrease at a Ag+ concentration of 10 μM. It then shows adramatic increase when the concentration of Ag+ reaches 25μM (Figure 1C). The PL tends to be stable at a Ag+

concentration of 50 μM. The time to reach the PL equilibriumis about 10 min (Figure S4). In the presence of Ag+ of 100 μM,the PL intensity decreased, which is also likely due to the PLinhibition by excess Ag+. In the meantime, the emission peakwavelength is shifted back to ∼570 nm. It can be concludedthat GSH and silver ions have a synergistic effect on increasingthe PL intensity of AuNAC NCs.Since most Ag+ involved reactions are sensitive to light, we

also considered the photosensitivity during assembly ofAuNAC@AgGSH NCs. The influence was inspected bycomparing PL changes after successive addition of Ag+ andGSH to AuNAC NCs in the dark or under exposure to light for

Table 1. Identification of MS m/z Peaks

sample peak m/z calc. m/z formula

AuNAC 914.84 915.29 [Au12NAC8 (−8H)]4−

1273.78 1274.45 [Au12NAC9 (−9H)]3−

1436.75 1436.64 [Au12NAC12 (−12H)]3−

933.9 934.18 [Au14NAC6]4−

1255.77 1255.75 [Au15NAC5]3−

1231.76 1229.64 [Au20NAC6]4−

1394.75 1392.82 [Au20NAC10]4−

1474.73 1474.42 [Au15NAC9]3−

AuNAC@Ag 826.80 826.18 [Au8NAC8Ag4 (−8H)]4−

1185.78 1185.35 [Au9NAC9Ag3 (−9H)]3−

1544.70 1544.51 [Au12NAC12Ag3 (−12H)]3−

AuNAC@AgGSH 968.84 970.32 [Au8NAC4Ag4GSH4 (−8H)]4−

1114.87 1114.45 [Au8Ag4GSH8 (−8H)]4−

1058.87 1059.42 [Au12NAC4GSH4 (−8H)]4−

1202.93 1203.55 [Au12GSH8 (−8H)]4−

1561.84 1562.71 [Au12NAC3GSH6 (−9H)]3−

1706.84 1705.90 [Au12GSH9 (−9H)]3−

Figure 4. Main metal/ligand exchange deduced by mass spectroscopy. The colors of the rectangle frameworks mean AuNAC (red), AuNAC@Ag(blue), and AuNAC@AgGSH (orange) samples, respectively.

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12 h (Figure 1D). No significant differences are observed forthese cases, demonstrating that the PL enhancement is a light-insensitive process. Moreover, the pH influence on the PLenhancement was further investigated. Similar PL enhancementis observed in different buffers with pH ranging from 5 to 9(Figure S5). Thus, the system can possibly work in thephysiological environment.Silver Ion-Assisted Ligand Exchange on Au NCs. We

further probed the possible mechanism for the synergistic PLenhancement by GSH and silver ions. Since the presence ofsilver ions of 10 μM in AuNAC NCs immediately results in thered shift in PL emission peak, it is reasonable to hypothesize

that silver ions are easily bound to NCs. To confirm the role ofsilver ions, we added Ag+ of 50 μM to AuNAC NCs and thentreated the mixture by ultrafiltration (Figure S6A). Aftertreatment, both parts (MW > 3 kDa or < 3 kDa) were dilutedto the original volume to facilitate the comparison. It isnoteworthy that the PL even increases for the component witha MW > 3 kDa, similar to the case without ultrafiltration. Thisresult demonstrates our previous view that excess Ag+ maycause PL inhibition because the excess silver ions can beremoved during the ultrafiltration and the PL recovers. For thefiltrate with a MW < 3 kDa, no PL is observed (blue curve,Figure S5A). This means the formation of silver doped Au

Figure 5. PL response of AuNAC@Ag to thiols including (A) Cys, (B) CyA, or (C) NAC. (D) Comparison of PL responses to different thiols usingAuNAC@Ag with a fixed silver ion concentration of 25 μM. Inset (from Nos. 1 to 6): the picture of AuNAC (1), AuNAC@Ag (2), AuNAC@Ag+GSH (3), AuNAC@Ag+NAC (4), AuNAC@Ag+Cys (5), and AuNAC@Ag+CyA (6) under illumination of 365 nm.

Figure 6. Cytotoxicity of AuNAC@Ag NCs toward HepG2 (left) and L02 (right) cells in 24 h. The error bars are obtained by five duplicateexperiments.

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NCs, namely, AuNAC@Ag NCs, which still have a MW largerthan 3 kDa. The further addition of GSH to AuNAC@Ag NCssolution exhibits a significant PL increase. After ultrafiltration,the PL is still only observed for the component (MW > 3 kDa)(red curve, Figure S6B), indicating that GSH molecules areactually adsorbed on NCs in the presence of silver ions.On the other hand, as mentioned above, the PL emission of

AuNAC NCs was initially not responsive to GSH. This impliesthat the as-prepared AuNAC NCs have a relatively stable thiolcoated structure or the attachment of GSH does happen, butwithout causing PL changes. To reveal the possibility, themixture of GSH and AuNAC solution was treated byultrafiltration (Figure S6C). Only NC components (MW > 3kDa) are luminescent. However, the subsequent addition ofsilver ions of 100 μM to these components gives very slight PLchanges (red curve, Figure S5D), confirming that GSH itselfcannot be bound to AuNAC NCs spontaneously. Therefore, itwas reasonable that the sequential addition of GSH of 200 μMcan cause a typical PL enhancement (green curve, Figure S6D).These results suggested that Ag+ and GSH should be assembledon AuNAC NCs and trigger the great PL enhancement.

Besides the sequential addition of silver ions and GSH, wealso attempted the addition of a silver−GSH premixture(Figure S7). Note that the premixture is usually turbid and cancause an observable background in PL spectra. Aftersubtraction of the background, the complicated silver−GSHstructures actually give no PL enhancement. Thus, theformation of AuNAC@Ag NCs is essential for the followingPL enhancement.We attempted to obtain more details to illustrate the PL

enhancement. Several works have reported the aggregationinduced emission (AIE)-type PL enhancement by Au NCs.32,33

In this case, it shows no evidence of such phenomena from theTEM images (Figure 2A,B). The monodispersed AuNAC@AgNCs and AuNAC@Ag-GSH NCs have similar average sizes,i.e., 2.1 ± 0.4 nm (169 individual particles) and 2.0 ± 0.5 nm(164 individual particles), respectively.The valences of Ag in the AuNAC@AgGSH NCs were

further inspected (Figure S8). The XPS results show that Ag(I)is the main form of silver(I) components, with a 3d5/2 peak at367.5 eV and a 3d3/2 peak at 373.5 eV, respectively (FigureS7B). Therefore, it can be deduced that the Ag(I)-GSHstructure attached to Au NCs is essential for PL enhancement.Moreover, this special structure seems uncommon for otherthiolated Au NCs. For example, after the binding of Ag(I) toGSH coated Au NCs, the additional GSH only causes the PLdecrease (Figure S9).The photophysical properties of the NCs were also

investigated by time-resolved PL spectrum. All the abovespecies have a similar average lifetime between 2.0 and 2.2 μs(Figure S10), corresponding to the ligand metal charge transfermechanism commonly reported in Au−thiol NCs.24 Theadsorption of GSH to NCs shows no typical changes inlifetime, perhaps due to the limited structural changes inclusters during the ligand exchange process.We further probed the NC species during the attachment of

silver ions and GSH by mass spectroscopy (Figure 3). It isnoteworthy that we can only deduce that the newly generatedspecies will cause the PL increase because of the difficulty inseparating the luminescent species effectively at the currentstage. Since all Au species have a MW higher than 3 kDa, theyare normally multiply charged at an m/z of 1000−2000 (Table1). The results show that Au12 species are the main species inAuNAC sample, including the high abundant species[Au12NAC8 (−8H)]4− (m/z 914.84), [Au12NAC9 (−9H)]3−(m/z 1273.78), and [Au12NAC12 (−12H)]3− (m/z 1436.75)(Figure 3A). Note that, according to the evaluation, the Aucores exhibit a mixed valence of Au (0) and Au (I), which are incoincidence with the XPS results. After the treatment of silverions, three AuNAC@Ag species appeared (Figure 3B),corresponding to [Au12NAC12Ag3( −12H)]3− (m/z 1544.7),[Au9NAC9Ag3 (−9H)]3− (m/z 1185.78), and [Au8NAC8Ag4(−8H)]4− (m/z 826.8), respectively. The further addition ofGSH leads to the appearance of new peaks, as well asdiminishment of the forementioned peaks, indicating thetransformation of AuNAC@Ag species (Figure 3C). Themost abundant species contain [Au12NAC4GSH4 (−8H)]4−(m/z 1058.87), [Au12GSH8 (−8H)]4− (m/z 1202.93), and[Au12NAC3GSH6 (−9H)]3− (m/z 1561.84). Considering thatno silver contents are observed in these products, GSH mayactually attack AuNAC@Ag by replacing partial or even allsilver ion−NAC moieties. In the meantime, some reactionintermediates, such as [Au8NAC4Ag4GSH4 (−8H)]4− (m/z968.84) and [Au8Ag4GSH8 (−8H)]4− (m/z 1114.87), can also

Figure 7. PL response of GSH in HepG2 cells by incubation of (A, C)AuNAC NCs or (B, D) AuNAC@Ag NCs before (A, B) and after (C,D) the cells were treated with BSO. Scale bar: 50 μm.

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be observed, reflecting the key roles of silver ions during theligand exchange. The possible transformation processes aresketched in Figure 4.Selectivity of GSH Response to AuNAC@Ag NCs.

Considering the presence of the same thiol groups in Cys,NAC, and CyA, it may form a shell comprised of Ag−thiolcomplexes around AuNAC NCs. After addition of Cys toAuNAC@Ag, the PL emission intensity does not change withthe increase of Cys concentration (Figure 5A). For CyA(Figure 5B) and NAC (Figure 5C), the PL intensity showslimited changes with the increasing concentrations of CyA orNAC and even decreases with the increasing concentration ofsilver ions. Thus, for common biological thiols, the PL onlyresponds to GSH and the PL increment is linear to theconcentration of GSH ranging from 0 to 0.5 mM (R2 = 0.98)(Figure 5D). Although it cannot exclude the ligand exchangeon AuNAC NCs by other thiols, the latter fail to generate PLenhancement except GSH. This offers the opportunity forselective assays of GSH, especially for intracellular use.Cytotoxicity Evaluation and Intracellular GSH Imag-

ing. As a cellular imaging probe candidate, the cytotoxicityshould be carefully evaluated by conventional methods such asCCK-8 assays. Gold NCs are well-known for their excellentbiocompatibility.34 Here the AuNAC NCs of sub-mM levelshow little cytotoxicity (Figure S9). The viability reaches 80%for both HepG2 cancer cells and L02 normal cells after a 24 hincubation, corresponding to a qualitative cytotoxicity evalua-tion of grade 0 (None)−1 (Slight). Usually the involvement ofsilver components brings great concern because of the potenthigh toxicity by released silver ions.35 We observed thatAuNAC@Ag NCs are generally low cytotoxic even at aconcentration of sub-mg/mL level (Figure 6). Even thoughaccurate IC50 values are unavailable, they should be larger than750 μg/mL. Typically, the concentration of AuNAC@Ag forcellular imaging is far less than this value. Therefore, the cellshave a survival rate of 80−90% during the staining processes,allowing the effective in vitro imaging in the following steps.Since the PL of AuNAC@Ag can be selectively enhanced by

GSH, it allows the imaging of this abundant molecule thatregulates the redox intracellular environment. First we havedemonstrated that the selective PL enhancement can beobserved in cell media, indicating the anti-interference capacityof this method (Figure S12). It is noteworthy that GSH cangive observable PL response in cell media at the mM level,much higher than that of 200 μM in the aqueous solution. ThismM level is coincident with intracellular GSH concentrations.For in vitro imaging (Figure 7), the entry rates of NCs aregenerally controlled by the hydrophobicity/hydrophilicity ofligands.36 Normally, for a concentration of AuNAC andAuNAC@Ag of 75 μg/mL, the NCs can efficiently enter theHepG2 cytoplasm after a 4 h incubation. Compared with thecontrol group stained by only Hoechst 33342 dye (Figure S13),the Hoechst 33342 dye/AuNAC@Ag costaining group (FigureS14) showed few colocalized spots are observed in Hoechst33342 channel and NCs channel, demonstrating that the entryof NCs hardly occurs in the nucleus zone during this period.The costaining investigation with lysotracker (Figures S15 andS16) showed that NCs (green channel) are partly distributed inlysosomes (red channel) and partly in the cytosol. Although thecontrast is not high, we can obtain the pixel statistical results inspecific zones collected by ImageJ software. Cells incubatedwith AuNAC NCs show a mean PL grayscale value of 3.66 ±1.19 (Figure 7A), and brighter PL is observed for the

AuNAC@Ag group (grayscale value of 6.49 ± 2.19, Figure7B). For cells pretreated with BSO, a GSH synthase inhibitor,the mean PL grayscale value reduces by ∼43% after furtherincubation with AuNAC@Ag NCs, corresponding to therepression of intracellular GSH levels (Figure 7D,E). As acomparison, few changes are observed for the AuNAC NCgroup after the treatment with BSO (grayscale value of 3.55 ±1.26, Figure 7C), which are in coincidence with the fact thatAuNAC@Ag but not AuNAC can react with GSH. Theseresults strongly support that the prepared NCs can be used aspotential probes for intracellular molecular imaging anddiagnostic studies. Considering that the different cellular uptakeefficiencies, as well as the heterogeneity of single cells, maycause a relatively large error range during the measurement, itwill be more helpful to improve this resolution by uncoveringthe internalization mechanism and modification of the NCsurface for enhanced accessibility.

■ CONCLUSIONS

In summary, we have realized the specific imaging ofintracellular GSH through the GSH induced PL enhancementof AuNAC@Ag NCs. The ligand exchange by GSH and NACon AuNAC@Ag NC triggers a maximum of a 20-fold increasein PL emission at 570 nm. The synergistic effects of GSH andsilver ions on the AuNAC cores have been demonstrated. ThePL changes were proportional to the concentration of GSH. Noother intracellular thiol components can induce similar PLchanges upon addition to AuNAC@Ag NCs. Thus, the almostnoncytotoxic AuNAC@Ag NC can be used as a selectiveimaging probe for intracellular GSH. This work may provide analternative strategy for design of efficient NC based probes.

■ ASSOCIATED CONTENT

*S Supporting InformationThe Supporting Information is available free of charge on theACS Publications website at DOI: 10.1021/acs.chemma-ter.7b04926.

Synthetic optimization, TEM images, PL titration, XPSanalysis, and cytotoxicity of the NCs (PDF)

■ AUTHOR INFORMATION

Corresponding Authors*E-mail: sungi@seu.edu.cn (H.J.).*E-mail: xuewang@seu.edu.cn (X.W.).

ORCIDHui Jiang: 0000-0001-8044-758XXuemei Wang: 0000-0001-6882-7774Author Contributions§(X.H. and Y.Z.) These authors contributed equally.

NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTS

The authors acknowledge the support by the National NaturalScience Foundation of China (21675023, 81325011, and91753106), National Key Research and Development Programof China (2017YFA0205300), Natural Science Foundation ofJiangsu Province (BK20161413), and Southeast University−Nanjing Medical University joint project (2242017K3DN29).

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DOI: 10.1021/acs.chemmater.7b04926Chem. Mater. 2018, 30, 1947−1955

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Chemistry of Materials Article

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