Detection of Glutathione in Vitro and in Cells by the Controlled Self

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Detection of Glutathione in Vitro and in Cells by the Controlled Self-assembly of Nanorings Yue Yuan, Jia Zhang, Mengjing Wang, Bin Mei, Yafeng Guan, and Gaolin Liang Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 07 Jan 2013 Downloaded from http://pubs.acs.org on January 8, 2013

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Analytical Chemistry

Detection of Glutathione in Vitro and in Cells by the Controlled Self-assembly of Nanorings Yue Yuan,† Jia Zhang,§ Mengjing Wang,† Bin Mei,† Yafeng Guan,‡ and Gaolin Liang*†

†

CAS Key Laboratory of Soft Matter Chemistry, Department of Chemistry, University of Science and Technology of China, 96 Jinzhai Road, Hefei, Anhui 230026, China

‡

Key Laboratory of Separation Sciences for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China §

State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, China

*Corresponding author: Gaolin Liang, Fax: (+86)-551-63600730, Tel: (+86)-551-63607935 E-mail: [email protected] Homepage: http://lianglab.ustc.edu.cn

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ABSTRACT Taking advantage of a reduction-controlled biocompatible condensation reaction and selfassembly, we have developed a new method for the determination of glutathione (GSH) concentration in vitro and in HepG2 human liver cancer cells. Upon reduction by GSH under physiological conditions (pH 7.4 in buffer), the small molecule CBT-Cys(SEt) condenses and self-assembles into nanorings, increasing the UV absorbance at 380 nm (with significant linear correlation in the 0 to 87 µM GSH range and a limit of detection of 1 µM). This method is also selective to GSH rather than cysteine in biological samples. Through the use of added internal standards, we successfully determined the concentration of GSH in HepG2 cells to be 14.96 µM (2.99 fmol/cell). To better understand the mechanism of nanoring self-assembly, the condensation product of CBT-Cys(SEt) formed using different concentrations of GSH and different reaction times were characterized by TEM. MANUSCRIPT TEXT Glutathione (GSH) is one of the most important thiolated biomolecules, playing a crucial role in mammalian and eukaryotic cells. It is the main non-protein biothiol found at high concentrations in the intracellular environment to protect cellular components from being damaged by reactive oxygen species (ROS) and toxins.1 When cells are under oxidative stress, there exists an imbalance between GSH and its oxidized form, glutathione disulfide (GSSG). The latter then rapidly converts into GSH under the action of GSH reductase, increasing of the ratio of GSH to GSSG and relieving the oxidative stress of cells. The alteration of the intracellular redox status (ratio of GSH to GSSG) is associated with numerous clinical diseases,2 suggesting that intracellular GSH could be used as an important clinical biomarker. Various methods have been developed previously for in vitro determination or in vivo imaging of GSH, such as fluorometry based on organic fluorophores,3 quantum dots,4 and gold nanoclusters,5 luminescence analysis based on MnO2-modified nanoparticles,6 colorimetry,7 highperformance liquid chromatography (HPLC),8 and electrochemistry.9 The limits of detection (LODs) and linear ranges for the detection of GSH of abovementioned methods are summarized in Supplementary Table S1 (Supporting Information). However, these methods require the addition of a 2 Environment ACS Paragon Plus

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mediator such as dyes, fluorescent substances, quantum dots, or labelled nanoparticles into the specimen before detection, making them complicated, expensive, bioincompatible, and time-consuming. Moreover, the manipulations involved in some of these methods, such us electrode modification and cleaning for electrochemistry, are complex and difficult. Herein we have developed a new spectrophotometric method for specific detection of GSH, in vitro and in HepG2 human liver cancer cells, using a GSH-controlled reduction of a small molecule and self-assembly of the product into nanorings. The LOD of our method is 1 µM with a linear range of 0-87 µM, both are comparable to those reported. The results of our study indicate that this method is simple, lable-free, sensitive, rapid, and biocompatible. Self-assembly is ubiquitous in biological systems, responsible for the generation of a wide range of complex supramolecular structures, including double-stranded DNA chains for the storage and passage of genetic information.10 Compared with those induced by conventional physical perturbations (pH, temperature, light, ionic strength, etc.), self-assemblies initiated by chemical or biochemical triggers have attracted considerable interest due to the possibility of integrating them with biological events.11 Unfortunately, many chemically-induced self-assembly processes cannot take place in aqueous environments because of the incompatibility of the organic monomers with water. Recently, inspired by nature, Rao and co-workers developed a biocompatible condensation reaction between the 1,2aminothiol group of cysteine and the cyano group of 2-cyanobenzothiazole (CBT), with the resulting product self-assembling into a number of supramolecular nanostructures.12 This reaction proceeds under physiological conditions and can be regulated by pH, reduction, or enzymatic cleavage. The corresponding condensation reaction initiated by the reduction of GSH had also been proposed, but was not explored thoroughly. To further investigate the GSH-controlled condensation, and subsequent selfassembly of nanostructures (as illustrated in Scheme 1), we designed a small molecule (CBT-Cys(SEt)) containing both a disulfide-protected cysteine and CBT moiety, This small molecule was also used to create an assay for determining GSH concentrations in vitro and in biological samples.



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Scheme 1. GSH-controlled condensation of CBT-Cys(SEt) to assemble nanorings. N

O S

S NH2

CN S

N H

HS NH2

CBT-Cys(SEt)

HS NH2

N H

CN N H

S

Condensation

CBT-Cys N

O O

N

O GSH

N

N

S

S

CN S

N H

Self-assembly

n

Nanorings

EXPERIMENTAL SECTION Determining the concentration of GSH in vitro. Different concentrations of GSH were added to 140 µM CBT-Cys(SEt) in phosphate buffer (10mM PB, pH 7.4) containing 2% DMF(v/v). The mixture was incubated for 1 hr at room temperature (RT) before taking absorption spectra. Each experiment was performed three times. Determining the concentration of GSH in cell lysate. 1 mL HepG-2 cell lysate (5 × 106 cells) was transferred to an ultrafiltration centrifuge tube (10 kDa) and the large biomolecules were separated from cell lysate by centrifugation at 15,000 rpm for 10 min at RT. For each measurement made, 20 µL of cell lysate supernatant was added to 90 µL of 171 µM CBT-Cys(SEt) in PB (10 mM, pH 7.4) containing 2% DMF(v/v), to give a final CBT-Cys(SEt) concentration of 140 µM. Different concentrations of GSH were then added to each sample and the mixtures incubated for 1 h at RT before taking UV-Vis spectra. Each experiment was performed three times. RESULTS AND DISCUSSION The synthesis of CBT-Cys(SEt) is simple and straightforward, as described in the Supporting Information. We first studied the condensation reaction of CBT-Cys(SEt) (140 µM in 10 mM PB, pH 7.4) in the presence of different reducing agents. As shown in the inset of Figure 1a, after 1 h of reaction the solutions in the presence of GSH, sodium sulfide (Na2S), and tris(2-carboxyethyl)phosphine (TCEP) became turbid monodispersions, in contrast to the transparent blank solution and solution treated with cysteine. UV-Vis spectra of these three turbid monodispersions showed appreciable red-shifts of the absorption peaks from 320 nm to longer wavelengths, suggesting the occurrence of condensation.13 4 Environment ACS Paragon Plus

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Consistent with the photographs, UV-Vis spectra of these monodispersions also showed large increases in the absorbance at 400-600 nm, indicating the formation of nanostructures. Interestingly, the solution of CBT-Cys(SEt) treated with cysteine only exhibited a UV-Vis absorption peak red-shifts from 320 nm to 332 nm but no increase of absorbance at 400-600 nm, suggesting that the condensation products of CBT-Cys(SEt) reduced by cysteine do not self-assemble into nanostructures. To prove this hypothesis, an in vitro model reaction was conducted. A mixture of 70 µM L-Cysteine and 140 µM CBT-Cys(SEt) (in 2% DMF, 10 mM PBS, pH 7.4) was stirred for 1 h at RT, and the reaction product purified and analyzed by HPLC. As shown in Figure S4, two new peaks appeared in the HPLC trace after the reaction (Supporting Information). High resolution matrix-assisted laser desorption /ionization mass spectroscopic (HR-MALDI-MS) analysis indicated that both peak 1 and peak 2 had the same molecular weight, which correspond to the two isomeric condensation products formed between LCysteine and CBT-Cys(SEt) (Figure S5 and S6, Supporting Information). MALDI-MS analysis of the reaction mixture of CBT-Cys(SEt) with GSH revealed that the dominant ionic peak has a m/z value of 612.906 which corresponds to the molecular weight of GSSG (Figure S7, Supporting Information), consistent with oxidation of GSH during its reduction of the disulfide bond of CBT-Cys(SEt). Interestingly, after HPLC purification of the reaction mixture, MALDI mass spectrum of the hump peak on the HPLC trace showed the peaks of linear oligomers of CBT-Cys instead of cyclic ones, different from previous studies (Figure S8, Supporting Information).12,14 This might be ascribed to the rigidity of the condensation products.



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Figure 1. (a) CBT-Cys(SEt) solutions (140 µM) in the presence of different reducing agents at RT for 1 h in PB (pH 7.4, 10 mM). The concentrations of GSH, Na2S and cysteine were 1.75 mM, and the concentration of TCEP was 0.45 mM. The inset is a photograph showing the corresponding change in the appearance of the solutions. (b) The UV-Vis absorption spectra of CBT-Cys(SEt) solutions (140 µM) in the presence of various concentrations of GSH for 1 h at RT in PB (pH 7.4, 10 mM). (c) Correlation of ∆A380 nm in figure (a) with GSH concentrations between 0 and 870 µM. The inset is a fitted calibration line in the linear region of 0 to 87 µM GSH. The error bar represents the standard deviation of three measurements. Figure 1b shows the UV-Vis absorption spectra of CBT-Cys(SEt) (140 µM) incubated with different concentrations of GSH at room temperature (RT) for 1 h in PB. In general, the absorbance at 400-600 nm increased gradually as the GSH concentration was increased, while the absorbance at 320 nm 6 Environment ACS Paragon Plus

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decreased. At 870 µM GSH, an obvious red-shift and widening of the absorption peak at 320 nm was observed, accompanied by an increase of absorbance at longer wavelengths. This is likely due to aggregation of the nanostructures self-assembled from the GSH-controlled condensation products of CBT-Cys(SEt). The concentration of GSH not only determines the final result of the self-assembly, as revealed by the absorption spectra, but also the kinetics of self-assembly (Figure S9, Supporting Information). We recorded the absorbance changes both at 320 nm and at 380 nm to study the timedependence of the condensation reaction. Results indicated that the self-assembly of condensation products into nanostructures had not finished within 1 h for GSH concentrations less than 870 µM. By correlating the absorbance change at 380 nm (∆A380

nm)

with the concentration of GSH, we

obtained a calibration curve for the determination of GSH in vitro. As shown in Figure 1c, a linear relationship between the ∆A380 nm and GSH concentration (Y = 0.00468 * X, R2 = 0.999) was obtained over the range of 0-87 µM. The LOD of GSH of the assay was 1 µM (S/N = 3). In our previous studies,12,14-15 we found that the morphologies of the nanostructures formed after the self-assembly of condensation products depend on both the structural properties of the precursors (i.e., CBT derivatives) and the factors initiating the condensation reaction (e.g., pH change, reduction, or enzymatic cleavage). These nanostructures include three-dimensional fibrous nanostructures, twodimensional snowflake-shaped nanofibres, and uniform nanoparticles. This relationship motivated us to characterize the nanostructures resulting from the self-assembly of the condensation products of GSHreduced CBT-Cys(SEt) by transmission electron microscopy (TEM), as shown in Figure 2a-d. In the absence of GSH, we observed an amorphous deposition of CBT-Cys(SEt) after the evaporation of the solvent (Figure 2a). In the presence of GSH, very different TEM images were observed for CBTCys(SEt) treated with different concentrations of GSH (Figure 2b-d). They showed interconnected nanostructures consisting of individual nanorings, ranging from irregular ones at lower GSH concentrations (Figure 2b and c) to regular, roundish ones at higher GSH concentrations (Figure 2d). At 50 µM GSH and 140 µM CBT-Cys(SEt), the nanorings have an average outer diameter of 148 nm and a ring width of 39 nm. At 256 µM and 870 µM GSH, the nanorings have average outer diameters of 130 7 Environment ACS Paragon Plus


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nm and 148 nm, and ring widths of 25 nm and 30 nm respectively (Figure 2e and f). Interconnections of the nanorings resulting in structures with larger hydrodynamic diameters are proven with the dynamic light scattering (DLS) data (Figure S10, Supporting Information). In addition to the effect of GSH concentration on the morphology of the nanorings, reaction time also has an effect on the final product. After a 20 min incubation of 140 µM CBT-Cys(SEt) with 870 µM GSH, few nanorings were observed in the TEM image (Figure S11a and b, Supporting Information), but when the reaction time was extended to 40 min, many more individual separated nanorings appeared (Figure S11c and d, Supporting Information). This indicates that while the initial GSH concentration controls the structural morphology of condensation products, the reaction time at specific GSH concentrations determines the quantity of the nanorings formed (also corresponding to the absorbance at 380 nm).

Figure 2. TEM images of the nanostructures formed after incubation of CBT-Cys(SEt) (140 µM) with (a) 0 µM, (b) 50 µM, (c) 256 µM, and (d) 870 µM of GSH for 1 h at RT in PB (10mM, pH 7.4). (e) Summary of the outer diameters of the nanorings in figures b to d. (f) Summary of the ring widths of the nanorings in figures b to d. 8 Environment ACS Paragon Plus

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Realizing that GSH is present at millimolar levels within living cells, we further applied this detection method to quantify GSH present in HepG2 human liver cancer cells. Before the assay, any possible interference from cysteine and Na2S was tested to evaluate the selectivity of this method. At the specified concentrations, no interference from these two agents was observed in the optical response induced by GSH (Figure S12, Supporting Information). Healthy HepG2 cells grown in culture medium were trypsinized, incubated with RIPA (radioimmuno- precipitation assay) buffer on ice for 30 min, and then centrifuged. The supernatant after centrifugation was tested using the CBT-Cys(SEt) assay in combination with added internal GSH standards to determine the concentration of GSH in the cell lysate (Figure 3). Figure 3a shows the UV-Vis spectra of CBT-Cys(SEt), CBT-Cys(SEt) with cell lysate, and CBT-Cys(SEt) with cell lysate with additions of different amounts of added GSH. After linearly fitting the absorbance change at 380 nm with the concentration of added GSH (Figure 3b), we determined that the concentration of GSH in HepG2 cell lysate was 14.96 µM (or 2.99 fmol/cell), which matches well with those of previous reports.8, 16 Furthermore, taking advantage of the enhancement of fluorescence emission at 450 nm by CBTCys(SEt) after condensation,12 we were also able to qualitatively detect GSH in living cells using the methods of GSH addition or GSH depletion with H2O2 as described in the Supporting Information (Figure S13-16).



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Figure 3. (a) The UV-Vis absorption spectra of the cell lysate after centrifugation (black), CBTCys(SEt) (0.14 mM, red line), CBT-Cys(SEt) with cell lysate (green), and CBT-Cys(SEt) with cell lysate and the addition of 16.1 µM (blue), 30.9 µM (bluish), and 45.9 µM (purplish) GSH for 1h. (b) Correlation of ∆A380nm with different additions of GSH standards to the solution of CBT-Cys(SEt) with cell lysate. The error bar represents the standard deviation of three measurements.

CONCLUSIONS In conclusion, using reduction by GSH to control the condensation and consequent self-assembly of CBT-Cys(SEt) into nanostructures, we have developed a new method for quantitative GSH detection in vitro and in cells. The structural morphology of the nanorings is dependent upon the initial concentration of GSH while the reaction time determines the final quantity of nanorings formed. Interference tests showed that neither sulfide nor cysteine interferes with the detection of GSH in biological samples, and this method was successfully applied to determine the concentration of GSH in HepG2 human liver cancer cells. Efforts to integrate the excellent plasmonic properties of gold nanoparticles or semiconductor quantum dots with this biocompatible condensation reaction to design more sensitive probes for GSH are currently underway. 10 Environment ACS Paragon Plus

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ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (21175122, 91127036), the Fundamental Research Funds for Central Universities (WK2060190018), and the Anhui Provincial Natural Science Foundation (1108085J17). The authors are grateful to Dr. Lina Cui of Stanford School of Medicine for her help with English editing. SUPPORTING INFORMATION General methods, synthesis and characterization of CBT-Cys(SEt), HPLC conditions, supplementary scheme, tables, and figures (Scheme S1, Supplementary Table S1-S3, Figure S1-S16). REFERENCES (1)

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