Silver-Assisted Thiolate Ligand Exchange Induced Photoluminescent

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Silver-assisted Thiolate Ligand Exchange Induced Photoluminescent Boost of Gold Nanoclusters for Selective Imaging of Intracellular Glutathione Xueqi Hu, Youkun Zheng, Junyu Zhou, Danjun Fang, Hui Jiang, and Xuemei Wang Chem. Mater., Just Accepted Manuscript • DOI: 10.1021/acs.chemmater.7b04926 • Publication Date (Web): 12 Mar 2018 Downloaded from http://pubs.acs.org on March 12, 2018

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Chemistry of Materials

Silver-assisted Thiolate Ligand Exchange Induced Photoluminescent Boost of Gold Nanoclusters for Selective Imaging of Intracellular Glutathione Xueqi Hu, ‡1 Youkun Zheng, ‡1 Junyu Zhou,2 Danjun Fang,2 Hui Jiang,1* and Xuemei Wang 1* 1 State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, National Demonstration Center for Experimental Biomedical Engineering Education (Southeast University), Southeast University, Nanjing 210096, P. R. China 2 School of Pharmacy, Nanjing Medical University 210029, P. R. China ABSTRACT: Metal/ligand exchange is a common strategy for precise assembly of metal nanoclusters (NCs) in organic phases. However, such case is still not well studied in aqueous phase. In this work we have demonstrated the silver ions assisted ligand exchange on water soluble N-acetylcysteine (NAC) stabilized Au NCs. Silver ions may trigger both silvergold metal exchange and silver addition on Au NCs. Unlike those well-reported silver induced photoluminescent (PL) enhancement, the processes show little changes in PL intensity. The as-obtained AuNAC@Ag NC can further promote the ligand exchange between NAC and glutathione (GSH) and induce a maximum of 20-fold increase in PL emission at 570 nm. The enhancement was proportional to the concentration of GSH, with a linear range of 0-0.5 mM. For other thiol compounds such as cysteine, NAC, and cysteamine, no significant PL changes were observed. Cytotoxicity evaluation shows that the AuNAC@Ag NC are biocompatible. Thus the intracellular GSH can be specially visualized by the formation of stable AuNAC@AgGSH NCs. These results may be helpful to reveal the underlying processes of metal/ligand exchange on NCs in aqueous environment and pave a new avenue for facile design and preparation of efficient imaging probe candidates.

INTRODUCTION Intracellular redox balance is a key factor for maintaining cell physiological stability. The abundant intracellular thiolate components are responsible for the reducing cytoplasm environment. Three main types of antioxidant systems have been evolved inside cells on basis of thiol/disulfide redox pairs, i.e., glutathione pair (glutathione, GSH/ glutathione disulfide, GSSG), cysteine/cystine, and thioredoxin pair.1, 2 Among these components, GSH is an important molecule to keep the redox balance. 3 Thus the determination of intracellular GSH levels may be of great importance to evaluate different cell status, especially immunological response, 4 aging, 5 and tumor metastasis, 6 due to the observable GSH level changes during their occurrence. Recently photoluminescent (PL) probes for intracellular thiol detection are well concerned, including organic and inorganic candidates. 7 Generally, these probes still face great challenges. Many thiol-responsive organic dyes have been reported, but they may face the difficulty for long term observation due to limited photostability. 8-10 On the other hand, some II-VI semiconductor quantum dots show high quantum yields, but have high risks of cytotoxicity. 11 In addition to these issues, the possible influence by intracellular autofluorescence should be well considered.

In the near decade, metal nanoclusters (NCs), especially gold NCs, were proposed as alternatives for bioimaging because of their stability, biocompatibility and wide emission spectrum ranges. 12, 13 The NCs have an ultrasmall size of 1-3 nm and transitional features between molecules and larger nanoparticles. 14 The PL mechanisms are quite complicated and integrated both effects from sizes (metal atom numbers) of NCs and specific ligands. The energy level interval inside NCs are close to the Fermi wavelengths, resulting in a size dependent electronic structure. 15 Besides, ligands play an essential role in mediating PL emission. Even subtle changes in ligands have a dramatic effect on the PL properties. For examples, Jin’s group 16 showed the special effect of methylthiolbenzene ligands. Au130, Au104 and Au40 NCs are obtained by adjusting the methyl group at ortho-, meta-, and para- site, rendering different PL emissions. In the meantime, the PL efficiency of NCs is also affected by features of ligands and valence states of metal (especially Au) cores. It’s well known that the charge transfer from the surface thiolate ligands to the metal core can determine the PL of Au NCs and the surface ligands with electron-rich atoms can effectively enhance the emission.17 Thus thiols of different chains are used to assemble versatile NCs with efficient PL.

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The facile detection of thiols by Au nanostructures is possible due to the versatile changes in optical and electronic properties induced by gold-sulfur interaction.18, 19 We recently devoted to probing the PL enhancement processes by controlling the surface coverage of ligands around NCs. We found the imaging of thiols inside cells by special surface “unsaturated” GSH coated Au NCs. 20 However, it still lacks systematic investigation to develop probes specific for certain analyte. In this work we have demonstrated the silver-assisted thiolate ligand exchange on N-acetyl cysteine (NAC) coated Au NCs. The ligand exchange, denoting the substitution of weaker ligands on NCs by stronger ligands, is a known strategy for preparation of NCs. However, for thiolated Au NCs containing a large number of strong Au-S bonds, the ligand exchange is rarely reported. To the best of our knowledge, only a recent work reported the ligand exchange between thiolated Au NCs by phosphine in organic phase. 21 Here we initially observed that silver ions can effectively combine to AuNAC NCs by d10-d10 metallophilic interactions 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 can induce a maximum of 20-fold increase in PL. The PL enhancement is specific to GSH, with limited response to other common thiol compounds such as cysteine, NAC and cysteamine. The AuNAC@Ag NCs are almost non-cytotoxic even at a high concentration and can be used as a candidate to visualize the intracellular GSH, which show great potentials to reveal the relationships of redox balance and physiological status inside cells.

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recorded on a PHI 5000 VersaProbe equipment (ULVACPHI, Japan). The mass spectroscopy was performed on an ABI-4800 Plus MALDI TOF/TOF Analyzer (Applied Biosystems, USA). The matrix used was α-cyano-4hydroxycinnamic acid (CHCA). The CCK-8 assays were recorded on a Multiskan FC microplate reader (Thermo, USA). The cellular imaging was shot on a confocal laser scanning microscopy (Ti-C2, Nikon, Japan). Synthesis of AuNAC NCs The previous methods use sodium borohydride as a reducing agent for preparation of AuNAC NCs. 22 We here attempted a simple method and found that NAC itself can act as both 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) was mixed rapidly with a vortex. The colorless mixture was then stirred in 80 °C water bath to turn to light yellow gradually in several hours, implying the formation of NCs. The clear solution was cooled to room temperature and its pH was adjusted to neutral by addition of NaOH (1 M, 0.12 mL). The saline in the samples were removed by repeated ultrafiltration. To optimize the synthetic conditions, different molar ratios of HAuCl4 and NAC were mixed. For pH selection, the pH of mixture was adjusted to a desired value before heating. In a general test, the effect on the PL intensity of AuNAC NCs by silver ions was inspected in ultrapure water (pH around 6) to avoid the possible interference by phosphate saline. Note that the pH changes are not significant during the whole experiments in ultrapure water. Cell Culture and Cytotoxicity Test

Scheme 1. Silver-assisted thiolate ligands exchange may induce photoluminescent boost of Au NCs. EXPERIMENTAL SECTION Materials and Instruments GSH, NAC, cysteine (Cys), cysteamine (CyA), and buthionine sulfoximine (BSO) were products of Sigma Aldrich (St. Louis, MO, USA). Hoechst 33342 (beyotime, China) and Lysotracker Red DND-99 (life technology, thermo, USA) were used for co-staining of cell nucleus and lysosomes. Chloroauric acid (HAuCl4) and silver nitrate were purchased from Sinoreagent Co. Ltd. (Shanghai, China). All other reagents are of analytical grade. Amicon Ultra-15 ultrafiltration tubes (with a molecular-weight cut-off (MWCO) of 3 kDa, Millipore, USA) were used for separation and concentration of crude Au NCs samples. Phosphate buffer saline (PBS) was purchased from Hyclone (USA). Deionized water (MilliQ, Millipore, USA) was used throughout. The PL and UV-vis spectra were obtained on an RF5301PC spectrofluorophotometer (Shimadzu, Japan) and an Evolution 260-Bio UV-Visible spectrometer (Thermo, USA), respectively. The morphology of NCs was observed on a transmission electron microscope (JEM-2100, JEOL, Japan). The X-ray photoelectron spectroscopy (XPS) was

The HepG2 liver cancer cells and L02 normal liver cells were obtained from KeyGen Biotech. Co. Ltd. (Nanjing, China) and were cultured in Dulbecco’s modified eagle medium (DMEM, Hyclone, USA) containing 10% fetal bovine serum (Gibco, USA) and penicillin-streptomycin (Hyclone, USA) at 37 °C and 5% CO2 atmosphere. The CCK-8 assay kit (Beyotime Co. Ltd., Shanghai, China) were used to evaluate the cytotoxicity by NCs. All NCs samples were firstly filtered by a sterile membrane (0.22 µm, Millipore, USA) and put under UV irradiation before use. Firstly, cells of 5×103 per well (total volume of 100 µL) were suspended and cultured in a 96-well plate overnight to achieve cell adhesion. NCs of a series of concentrations were then added to the wells and incubated for 24 h. Subsequently, CCK-8 solution of 10 μL was added to each well. The wells with same amount of NCs, culture media and CCK-8 solution but without cells were used as blank control. After further incubation for 1 h, the absorbance at 450 nm for each well was recorded on a microplate reader. The cell viability was calculated by: Viability%= [ANCs− Ablank]/[ A0− Ablank] × 100% where ANCs, A0, and Ablank represents the absorbance for NCs treated cells, non-treated cells, and the blank control, respectively.


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Cellular Imaging Cells of 5×105 /mL were seeded into a FluoroDish (FD35-100, WPI, USA) and cultured for a certain time in a 37 °C incubator after the addition of NCs samples. The confocal microscopy was used to perform cell imaging processes. For quantitative comparison, the Image J software was used to collect the pixels in a specific region of interest. RESULTS AND DISCUSSION Synthesis 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 be reduced by other reducing agent (commonly borohydride) or etching thiols (for examples, GSH) to produce Au NCs. NAC has been reported as a template for Au NCs for more than a decade. 23 In some initial works, special buffers, including acetic acid and ethanol, were required to prepare NCs emitted at 570 nm. 24 Recently, Deng et al. 25, 26 reported the syntheses of red emitted NCs (emission centered at 650 nm) at near neutral pH, with a rather high molar ratio of Au:NAC around 1:16. The two types of NCs should be different according to their PL features. However, as a template, NAC was not as prevalent as GSH and the properties of NAC coated Au (AuNAC) NCs were 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 salts and NAC, without special choice of pH buffer. The as-obtained NCs have a relatively low PL efficiency. This feature allows the significant PL enhancement after attachment of silver-thiol complex. The emission and excitation spectra of Au NCs prepared with Au and NAC at a feeding molar ratio of 1:1.5 were measured after heating the mixture for different time (Figure S1A in the Supplementary Information). The PL appears in 0.5 h and gradually increases with the heating time. After 7-h heating, the PL tends to stable. The optimized excitation and emission wavelength of the NCs is about 420 nm and 570 nm, respectively. A large Stokes shift (~150 nm) is beneficial for efficient observation of intracellular targets. The ultrafiltration experiments confirm that the AuNAC NCs have a MW larger than 3 kDa (Figure S2). The UV spectra showed an absorption peak at 320 nm for the Au and NAC mixture, while this peak was moved to 350 nm upon heating, with a significant red shift in wavelengths (~30 nm), corresponding to the formation of Au NCs and changes in solution color (Figure S1B). No further changes were observed at larger heating time. The influence of Au and NAC feeding molar ratios on PL of NCs were investigated (Figure S1C). The difference

in the feeding ratio does not cause significant changes in excitation and emission peak wavelengths of Au NCs. The sample prepared with an Au:NAC molar ratio of 1:1.5 shows the maximum PL intensity, while the PL emission reaches almost 70% 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 are formed, with a typical surface plasmonic adsorption band. For Au:NAC molar ratio larger than 2:1, white insoluble aggregates appear and no PL is observed for the supernatant of this mixture. Besides precursor ratios, pH values also affected the PL performances (Figure S1D). The sample prepared with an Au:NAC molar ratio of 1:1.5 shows an acceptable PL emission in acidic environments (i.e., pH 2.0 or 2.6), while the PL decreases with the increasing pH and completely vanishes in basic solution. The morphology of as-prepared AuNAC NCs were inspected by TEM (Figure S3). The size distribution of AuNAC NCs is 2.1 ± 0.4 nm, according to the statistical evaluation of 179 individual particles in TEM images. The high resolution TEM clearly shows a group of crystalline lattices with an equal inter-distance of 0.24 nm, corresponding to Au (111) surface. Effect of Ag+ and GSH on PL Intensity of AuNAC NCs Silver ions are well-known for their high affinity towards gold nanostructures. The Au-Ag bimetallic nanocomposites are readily formed after a simple mix and further reduction. 27, 28 For Au NCs, similar conjugation usually induces great PL enhancement. For examples, Le Guevel et al. 29 reported the silver induced a 3~5-fold PL enhancement on GSH coated Au NCs at a dopant percentage of ~2 wt%. We also observed the silver dopant (~5 atom%) can cause a red-shift for cytidine stabilized Au NCs from 490 nm to 560 nm, accompanied with an even larger increase (~ 25-fold) in PL efficiency. 30 The atomic precise control of AgxAu25-x NCs even results in a 200-fold quantum yield boost. 31 However, in our current case, when Ag+ was added to the AuNAC NCs solution, it only showed an observable red-shift in wavelength, but with slight PL enhancement. As shown in Figure 1A, the emission spectra at different Ag+ concentrations to AuNAC NCs of 0.03 mg/mL were measured. The PL intensity hardly shows any changes for Ag+ concentration lower than 1 μM. When the concentration reaches 10 μM, the emission peak wavelength is shifted to about 590 nm, with a maximum of ~2 fold in PL intensity compared with that for Au NCs in the absence of Ag+. The further addition of Ag+ gives no observable PL changes. While the concentration of Ag+ is up to 100 μM, the PL intensity even decreases, similar to the PL quenching effects reported for many heavy metal ions. 12 These results indicate the possible binding of Ag+ to NCs, accompanied with slight changes in PL. The PL inhibition by Ag+ at high concentrations may be caused by excess free Ag+.



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+

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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 NCs containing different amount of Ag to GSH of 200 µM. (C) Dependence of PL intensity of AuNAC@Ag NCs on + Ag concentrations in the presence 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) The 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.

Our previous work reported the attachment of thiol compounds on “unsaturated” surface of GSH coated Au NCs induced PL enhancement, which induced a PL enhancement due to the formation of new NCs species. 20 Here, the PL emission keeps unchanged upon addition of GSH of 200 µM to AuNAC NCs. But interestingly, the PL level completely changes 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 fold increase at Ag+ concentration of 10 μM. It then shows a dramatic increase when the concentration of Ag+ reaches 25 μM (Figure 1C). The PL tends to be stable at Ag+ concentration of 50 μM. The time to reach the PL equilibrium is about 10 min (Figure S4). In the presence of Ag+ of 100 µM, the PL intensity decreased, which is also likely due to the PL inhibition by excess Ag+. In the meantime, the emission peak wavelength is shifted back to ~570 nm. It can be concluded that GSH and silver ions have synergistic effect on increasing the PL intensity of AuNAC NCs. Since most Ag+ involved reactions are sensitive to light, we also considered the photosensitivity during assembly of AuNAC@AgGSH NCs. The influence was inspected by comparing PL changes after successive addition of Ag+ and GSH to AuNAC NCs in the dark or under exposure of light for 12 h (Figure 1D). No significant differences are observed for these cases, demonstrating that the PL enhancement is a light-insensitive process. Moreover, the pH influence on the PL enhancement were further investigated. Similar PL enhancement is observed in different buffers with pH range from 5 to 9 (Figure S5). Thus the system can possibly work in the physiological environment. Silver Ions-Assisted Ligand Exchange on Au NCs We further probed the possible mechanism for the synergistic PL enhancement by GSH and silver ions. Since the presence of silver ions of 10 µM in AuNAC NCs immediately results in the red shift in PL emission peak, it is reasonable to hypothesize that silver ions are easily bound to NCs. To confirm the role of silver ions, we added Ag+ of 50 µM to AuNAC NCs and then treated the mixture by ultrafiltration (Figure S6A). After treatment, both parts (MW > 3 kDa or < 3 kDa) were diluted to the original volume to facilitate the comparison. It is noteworthy that the PL even increases for the component with a MW > 3 kDa, similar with the case without ultrafiltration. This result demonstrates our previous view that excess Ag+ may cause PL inhibition because the excess silver ions can be removed during the ultrafiltration and the PL recovers. For the filtrate with a

MW < 3 kDa, no PL is observed (blue curve, Figure S5A). This means the formation of silver doped Au NCs, namely, AuNAC@Ag NCs, which still have a MW larger than 3 kDa. The further addition of GSH to AuNAC@Ag NCs solution 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 are actually 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 implies that the as-prepared AuNAC NCs have a relatively stable thiol coated structure, or the attachment of GSH does happen, but without causing PL changes. To reveal the possibility, the mixture of GSH and AuNAC solution was treated by ultrafiltration (Figure S6C). Only NCs components (MW > 3 kDa) are luminescent. However, the subsequent addition of silver ions of 100 µM to these components give very slight PL changes (red curve, Figure S5D), confirming that GSH itself cannot be bound to AuNAC NCs spontaneously. Therefore it was reasonable that the sequent addition of GSH of 200 µM can cause a typical PL enhancement (green curve, Figure S6D). These results suggested that Ag+ and GSH should be assembled on AuNAC NCs and trigger the great PL enhancement. Besides the sequential addition of silver ions and GSH, we also attempted the addition of silver-GSH premix (Figure S7). Note that the premix is usually turbid and can cause an observable background in PL spectra. After subtraction of the background, the complicated silverGSH structures actually give no PL enhancement. Thus the formation of AuNAC@Ag NCs is essential for the following PL enhancement. We attempted to obtain more details to illustrate the PL enhancement. Several works have reported the aggregation induced emission (AIE)-type PL enhancement by Au NCs. 32, 33 In this case, it shows no evidences of such phenomena from the TEM images (Figure 2A, B). The monodispersed AuNAC@Ag NCs 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.

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

The valence 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 at 367.5 eV and a 3d3/2 peak at 373.5 eV, respectively (Figure S7B). Therefore it can be deduced that the Ag (I)-GSH structure attached to Au NCs are


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essential for PL enhancement. Moreover, this special structure seems uncommon for other thiolated Au NCs. For examples, after the binding Ag (I) to GSH coated Au NCs, the additional GSH only causes the PL decrease (Figure S9). The photophysical properties of the NCs was also investigated by time-resolved PL spectrum. All above species have a similar average lifetime between 2.0 and 2.2 µs (Figure S10), corresponding to the ligand metal charge transfer mechanism commonly reported in Au-thiol NCs.24 The adsorption of GSH to NCs shows no typical changes in lifetime, perhaps due to the limited structural changes in clusters during ligand exchange process.

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

We further probed the NCs species during the attachment of silver ions and GSH by mass spectroscopy (Figure 3). It is noteworthy that we can only deduce that the new-generated species will cause the PL increase because of the difficulty to separate the luminescent species effectively at current stage. Since all Au species have a MW higher than 3 kDa, they are normally multiply charged at an m/z of 1000-2000 (Table 1). The results show that Au12 species are main species in AuNAC 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 Au cores exhibit a mixed valence of Au (0) and Au (I), which are in coincidence with the XPS results. After the treatment of silver ions, 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 of GSH leads to the appearance of new peaks, as well as diminishment of the forementioned peaks, indicating the transformation of AuNAC@Ag species (Figure 3C). The most 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 that no silver contents are observed in these products, GSH may actually attack AuNAC@Ag by replacing partial or even all silver ions-NAC moiety. In the meantime, some reaction intermediates, such as [Au8NAC4Ag4GSH4(8H)]4- (m/z 968.84) and [Au8Ag4GSH8(-8H)]4- (m/z 1114.87), can also be observed, reflecting the key roles of silver ions during the ligand exchange. The possible transformation processes are sketched in Figure 4. Table 1. Identification of MS m/z Peaks Sample

Peak m/z

Calc. m/z

AuNAC

914.84

915.29

Formula [Au12NAC8(-8H)]

4-

3-

1273.78

1274.45

[Au12NAC9(-9H)]

1436.75

1436.64

[Au12NAC12(-12H)]

933.9

934.18

[Au14NAC6]

1255.77

1255.75

[Au15NAC5]

1231.76

1229.64

[Au20NAC6]

1394.75

1392.82

[Au20NAC10]

1474.73

1474.42

[Au15NAC9]

826.80

826.18

[Au8NAC8Ag4(-8H)]

1185.78

1185.35

[Au9NAC9Ag3(-9H)]

1544.70

1544.51

[Au12NAC12Ag3(-12H)]

968.84

970.32

[Au8NAC4Ag4GSH4(48H)]

1114.87

1114.45

[Au8Ag4GSH8(-8H)]

1058.87

1059.42

[Au12NAC4GSH4 (-8H)]

1202.93

1203.55

[Au12GSH8(-8H)]

1561.84

1562.71

[Au12NAC3GSH6(-9H)]

1706.84

1705.90

[Au12GSH9(-9H)]

AuNAC@ Ag

AuNAC@ AgGSH

3-

4344-

3433-

44-

43-

3-

Figure 4. The main metal/ligand exchange deduced by mass spectroscopy. The color of the rectangle frameworks means AuNAC (red), AuNAC@Ag (blue) and AuNAC@AgGSH (orange) sample, respectively.

Selectivity of GSH Response to AuNAC@Ag NCs Considering the presence of same thiol groups in Cys, NAC and CyA, it may form a shell comprised of Ag-thiol complexes around AuNAC NCs. After addition of Cys to AuNAC @ Ag, the PL emission intensity does not change with the increase of Cys concentration (Figure 5A). For CyA (Figure 5B) and NAC (Figure 5C), the PL intensity shows limited changes with the increasing concentrations of CyA or NAC, and even decreases with the increasing concentration of silver ions. Thus for common biological thiols, the PL only responses to GSH and the PL increment is linear to the concentration of GSH ranging from 0 to 0.5 mM (R2=0.98) (Figure 5D). Although it cannot exclude the ligand exchange on AuNAC NCs by other thiols, the latter fail to generate PL enhancement except GSH. This offers the opportunity for selective assays of GSH, especially for intracellular use. Cytotoxicity Imaging

Evaluation

and

Intracellular

GSH

As a cellular imaging probe candidate, the cytotoxicity should be carefully evaluated by conventional methods such as CCK-8 assays. Gold NCs are well-known for their excellent biocompatibility. 34 Here the AuNAC NCs of sub-mM shows little cytotoxicity (Figure S9). The viability reaches 80% for both HepG2 cancer cells and L02 normal cells after a 24 h incubation, corresponding to a qualitative cytotoxicity evaluation of Grade 0 (None)-1 (Slight). Usually the involvement of silver components brings great concern because of the potent high toxicity



by released silver ions. 35 We observed that AuNAC@Ag NCs are generally low cytotoxic even at a concentration of sub mg/mL level (Figure 6). Despite the accurate IC50 values are unavailable, they should be larger than 750 µg/mL. Typically, the concentration of AuNAC@Ag for

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cellular imaging is far less than this value. Therefore the cells have a survival rate of 80-90% during the staining processes, allowing the effective in vitro imaging in the following steps.

Figure 5. PL response of AuNAC@Ag to thiols including (A) Cys, (B) CyA, or (C) NAC. (D) The comparison of PL responses to different thiols using AuNAC@Ag with a fixed silver ion concentration of 25 µM. Inset (from No. 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 towards HepG2 (left) and L02 (right) cells in 24-h. The error bars are obtained by 5 duplicate experiments.

Since the PL of AuNAC@Ag can be selectively enhanced by GSH, it allows the imaging of this abundant molecule that regulates the redox intracellular environment. Firstly we have demonstrated that the selective PL enhancement can be observed in cell media, indicating the anti-interference capacity of this method (Figure S12). It is noteworthy that GSH can give observable PL response in cell media at mM level, much higher than that of 200 µM in the aqueous solution. This mM level is coincident with intracellular GSH concentrations. For in vitro imaging (Figure 7), the entry rates of NCs are generally controlled by the hydrophobicity/hydrophilicity of ligands. 36 Normally, for a concentration of AuNAC and AuNAC@Ag of 75 μg/mL, the NCs can efficiently enter the HepG2 cytoplasm after a 4 h-incubation. Compared with the control group stained by only Hoechst 33342 dye (Figure S13), the Hoechst 33342 dye/ AuNAC@Ag co-staining group (Figure S14) showed few co-localized spots are observed in Hoechst 33342 channel and NCs channel, demonstrating that the entry of NCs hardly occurs in the nucleus zone during this period. The co-staining investigation with lysotracker (Figure S15, S16) showed that NCs (green channel) are partly distributed in lysosomes (red channel) and partly in the cytosol. Although the contrast is not high, we can obtain the pixels statistical results in specific zones collected by Image J software. Cells incubated with AuNAC NCs show a mean PL grayscale value of 3.66±1.19 (Figure 7A) and brighter PL is observed for AuNAC@Ag group (grayscale value of 6.49±2.19, Figure 7B). For cells pretreated with BSO, a GSH synthase inhibitor, the mean PL grayscale value reduces by ~43% after further incubation with AuNAC@Ag NCs, corresponding to the repression of intracellular GSH levels (Figure 7D and 7E). As a comparison, little changes are observed for AuNAC NCs group after the treatment with BSO (grayscale value of 3.55±1.26, Figure 7C), which are in coincidence with the fact that AuNAC@Ag, but not AuNAC can react with GSH. These results strongly support that the prepared NCs can be used as potential probes for intracellular molecular imaging and diagnostic studies. Considering that the different cellular uptake efficiencies, as well as the heterogeneity of single cells may cause a relative large

error range during the measurement, it will be more helpful to improve this resolution by uncovering the internalization mechanism and modification of NCs surface for enhanced accessibility.

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.

CONCLUSIONS In summary, we have realized the specific imaging of intracellular GSH through the GSH induced PL enhancement of AuNAC@Ag NCs. The ligand exchange by GSH and NAC on AuNAC@Ag NC trigger a maximum of 20-fold increase in PL emission at 570 nm. The synergistic effect of GSH and silver ions on the AuNAC cores have been demonstrated. The PL changes were proportional to the concentration of GSH. No other intracellular thiol components can induce similar PL changes upon addition to AuNAC@Ag NCs. Thus the almost non-cytotoxic AuNAC@Ag NC can be used as a selective imaging probe for intracellular GSH. This work may provide an alternative strategy for design of efficient NCs based probes.

ASSOCIATED CONTENT Supporting Information. Synthetic optimization, TEM images, PL titration, XPS analysis, cytotoxicity of the NCs. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected] (H. J.) or [email protected] (X. W.) ORCID: 0000-0001-8044-758X (H. J.); 0000-0001-6882-7774 (X. W.)

Author Contributions ‡These authors contributed equally.

ACKNOWLEDGMENT


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The authors acknowledge the support by the National Natural Science Foundation of China (21675023, 81325011, and 91753106), National Key Research and Development Program of China (2017YFA0205300), Natural Science Foundation of Jiangsu Province (BK20161413), and Southeast UniversityNanjing Medical University joint project (2242017K3DN29).

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SYNOPSIS TOC


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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 NCs containing different amount of Ag+ to GSH of 200 µM. (C) Dependence of PL intensity of AuNAC@Ag NCs on Ag+ concentrations in the presence 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) The 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. 199x151mm (300 x 300 DPI)



Figure 2. (A, B) TEM images and (C, D) size distribution of (A, C) AuNAC@Ag and (B, D) AuNAC@Ag-GSH NCs. 199x143mm (300 x 300 DPI)


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Figure 3. MALDI-TOF MS analysis of (A) AuNAC, (B) AuNAC@Ag, and (C) AuNAC@AgGSH samples. The asterisk (*) indicates the new appeared MS peak after silver or GSH binding. 99x195mm (300 x 300 DPI)



Figure 4. The main metal/ligand exchange deduced by mass spectroscopy. The color of the rectangle frameworks means AuNAC (red), AuNAC@Ag (blue) and AuNAC@AgGSH (orange) sample, respectively. 107x46mm (300 x 300 DPI)


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Figure 5. PL response of AuNAC@Ag to thiols including (A) Cys, (B) CyA, or (C) NAC. (D) The comparison of PL responses to different thiols using AuNAC@Ag with a fixed silver ion concentration of 25 µM. Inset (from No. 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. 199x146mm (300 x 300 DPI)



Figure 6. Cytotoxicity of AuNAC@Ag NCs towards HepG2 (left) and L02 (right) cells in 24-h. The error bars are obtained by 5 duplicate experiments. 199x75mm (300 x 300 DPI)


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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. 175x313mm (300 x 300 DPI)



Scheme 1. Silver-assisted thiolate ligands exchange may induce photoluminescent boost of Au NCs. 73x39mm (300 x 300 DPI)


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Silver-Assisted Thiolate Ligand Exchange Induced Photoluminescent

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