Journal of Colloid and Interface Science 455 (2015) 6–15
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Journal of Colloid and Interface Science www.elsevier.com/locate/jcis
Synthesis of ultrastable and multifunctional gold nanoclusters with enhanced fluorescence and potential anticancer drug delivery application Xiaodong Zhang a, Fu-Gen Wu a,⇑, Peidang Liu a,c, Hong-Yin Wang a, Ning Gu a, Zhan Chen b,⇑ a b c
State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing 210096, China Department of Chemistry, University of Michigan, 930 North University Avenue, Ann Arbor, MI 48109, United States School of Medicine, Southeast University, Nanjing 210009, China
g r a p h i c a l a b s t r a c t
a r t i c l e
i n f o
Article history: Received 30 March 2015 Accepted 18 May 2015 Available online 22 May 2015 Keywords: Gold nanoclusters Ultrastable Short-chain PEGs Anticancer
a b s t r a c t The problem of stability hinders the practical applications of nanomaterials. In this research, an innovative and simple synthetic method was developed for preparing ultrastable and multifunctional gold nanoclusters (Au NCs). HS–C11–EG6–X is a class of molecules consisting of four components: a mercapto group (–SH), an alkyl chain (C11), a short chain of polyethylene glycols (EG6) and a functional group (X, X = OH, COOH, NH2, GRGD, etc). The present work demonstrated the importance of using HS–C11–EG6–X to prepare Au NCs with excellent properties and the role each component in this molecule played for synthesizing Au NCs. Au NCs with tunable surface functionalities were successfully synthesized and characterized. It was found that Au NC precursors had a fluorescent quantum yield of 0.4%; in contrast, after capping with HS–C11–EG6–X, the quantum yield significantly increased to 1.3–2.6%. The HS–C11–EG6–X capped Au NCs exhibited superior stability under various solution conditions (including extreme pH, high salt concentration, phosphate buffered saline and cell medium) for at least 6 months, even after conjugation with anticancer drug doxorubicin. Besides, we have also demonstrated that other commonly employed thiol-containing ligands failed to prepare stable fluorescent Au NCs. Moreover, the Au NCs showed negligible toxicity to A549 lung cancer cells up to 100 lM, and the application of the ultrastable Au NCs for anticancer drug delivery has also been demonstrated. Ó 2015 Elsevier Inc. All rights reserved.
⇑ Corresponding authors. E-mail addresses: [email protected] (F.-G. Wu), [email protected] (Z. Chen). http://dx.doi.org/10.1016/j.jcis.2015.05.029 0021-9797/Ó 2015 Elsevier Inc. All rights reserved.
X. Zhang et al. / Journal of Colloid and Interface Science 455 (2015) 6–15
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1. Introduction The instability of nanomaterials is a large obstacle that prevents nanomaterials for practical applications, especially in chemical and biomedical fields [1–4]. Charge stabilization (electrostatic repulsion between nanoparticles) and steric stabilization (such as by using polyethylene glycol ligands) are commonly used to solve the problem. However, the former method is sensitive to salt concentration, and the latter is usually not suitable for ultrasmall nanoparticles especially when using long polyethylene glycol chains. On the other hand, noble metal nanoclusters, consisting of several to several hundred atoms, also have drawn much attention due to their unique physiochemical properties [5–7], catalytic activities [8], ion detection capability [9–13] and potential applications in bio-labeling and sensing [14–30]. At the same time, Au NCs can be synthesized using different capping molecules, such as proteins [31–33], DNA and nucleotides [34,35], peptides [11,36–39], polymers [40–44] and small molecules such as dihydrolipoic acid [10], diphosphine [45], histidine [46,47], purine [48], cytidine [49,50] and alkanethiol [51–56]. Many excellent methods have been developed to prepare Au NCs. Schiffrin and co-workers prepared 1–3 nm Au NCs using two-phase reduction in the presence of alkanethiol [51]. Chang and co-workers produced fluorescent Au NCs by adding various alkanethiol ligands to gold nanoparticles stabilized by tetrakis(hydroxymethyl)phosphonium chloride (THPC) and 11-mercaptoundecanoic acid (MUA), yielding the brightest Au NCs with a quantum yield of 3.1% [9]. Au NCs fabricated by the above-mentioned methods are hydrophobic, which must be undergone a further hydrophilic treatment to become water-soluble for biomedical applications. Recently, Rotello group synthesized a series of water-soluble small gold nanoparticles (2 nm) functionalized with polyethylene glycol (PEG) and quaternary-N groups, and used them in regulating cell behavior, antimicrobial and model nanoparticles in vivo [57–59]. Despite such advances, it is still challenging for the Au NCs to have further modification due to the absence of functional groups on the Au NCs’ surface. Herein, we report two new and simple methods (see Scheme 1) to fabricate water-soluble and multifunctional Au NCs with high stability based on short-chain PEGs ‘‘HS–C11–EG6–X’’. Each HS–C11–EG6–X molecule consists of four parts: a mercapto group (–SH), a methylene group (C11), a short chain of ethylene glycols (EG6) and a terminal functional group (X, X = OH, COOH, NH2 and GRGD) (their molecular structures are shown in Scheme 2). In method A, histidine and HAuCl4 were blended and incubated for more than two hours to synthesize Au NC precursors. After that, HS–C11–EG6–OH was added to the above-prepared Au NC precursors to form the final Au NCs. In the even simpler method B,
Scheme 2. Four thiol-containing short-chain PEGs (HS–Cm–EGn–X, X = OH, NH2, OCH2COOH and GRGD) discussed in this study.
HAuCl4, histidine and HS–C11–EG6–OH were mixed together in one pot to form the final Au NCs. Both products possess ultrahigh stability in aqueous solutions of different pH, high salt concentration and even cell medium. Moreover, the Au NCs were still stable even conjugated with anticancer drug doxorubicin (DOX). Confocal imaging experiments have been carried out to demonstrate that the drug-carrying Au NCs can enter into cells even the cell nucleus.
2. Experimental 2.1. Materials HAuCl43H2O, L-histidine, L-tyrosine, L-cysteine, mercaptosuccinic acid, lipoic acid, quinine sulfate and HS–(CH2)11– (OCH2CH2)6–OH (abbreviated as HS–C11–EG6–OH) were purchased from Sigma–Aldrich. HS–(CH2)11–(OCH2CH2)6–NH2HCl (abbreviated as HS–C11–EG6–NH2), HS–(CH2)11–(OCH2CH2)6–OCH2COOH (abbreviated as HS–C11–EG6–OCH2COOH) and HS–(CH2)11– (OCH2CH2)6–GRGD (abbreviated as HS–C11–EG6–GRGD, where
Scheme 1. Schematic illustration of the formation of Au NCs.
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GRGD refers to glycine–arginine–glycine–aspartic acid) were ordered from Prochimia (Sopot, Poland). Thiol-PEG2000-methoxy (HS-PEG2K) and thiol-PEG5000-methoxy (HS-PEG5K) were obtained from Nanocs Inc. (Boston, MA, USA). The short thiol-containing peptide cysteine–arginine–glycine–aspartic acid– lysine (CRGDK) was acquired from GL Biochem Ltd (Shanghai, China). Doxorubicin (DOX) was from Beijing Huafeng United Technology Co., Ltd (Beijing, China). All solutions were prepared with deionized water purified by a Milli-Q system (Millipore). 2.2. Synthesis of HS–C11–EG6–X capped Au NCs
2.2.1. Two-step synthesis All glassware was cleaned in a bath of freshly prepared aqua regia (HCl:HNO3, 3:1 v/v) and rinsed thoroughly in Milli-Q water prior to use. An aqueous solution of histidine (1.5 mL, 0.1 M) was added to an aqueous solution of HAuCl4 (0.5 mL, 10 mM) in a small vial at room temperature (about 25 °C). The solution mixture was blended gently at the beginning and then was placed at room temperature without stirring in dark. After more than 3 h, an aqueous solution of HS–C11–EG6–OH (2 mL, 3.75 mM) was added to the mixture (pH = 6.88). Afterwards, the final mixture (pH = 6.80) was shaken in dark at room temperature overnight. The residual histidine and HS–C11–EG6–OH were removed by dialysis (2 kDa). Au NCs with different capping agents could be synthesized by replacing HS–C11–EG6–OH with mixture containing HS–C11–EG6– X (X = OCH2COOH, NH2 and GRGD) and HS–C11–EG6–OH. 2.2.2. One pot synthesis An aqueous solution of histidine (1.5 mL, 0.1 M) was added to an aqueous solution of HAuCl4 (0.5 mL, 10 mM) in a small vial at room temperature (about 25 °C). After blending for several times, an aqueous solution of HS–C11–EG6–OH (2 mL, 3.75 mM) was added immediately. Afterwards, the mixture was shaken in dark at room temperature overnight. The residual histidine and HS–C11–EG6–OH were removed by dialysis (2 kDa). 2.3. Synthesis of Au NC-DOX To conjugate DOX to the carboxyl groups on the surface of ‘‘HS–C11–EG6–OCH2COOH:HS–C11–EG6–OH = 1:2’’ capped Au NCs, Au NCs were firstly reacted with EDC/sulfo-NHS dissolved in 0.1 M MES buffer solution (pH = 6.0) at room temperature in the dark for 30 min. The molar ratio of COOH (on the surface of Au NCs): sulfo-NHS:EDC was 1:10:25. Then DOX dissolved in dimethyl sulfoxide (DMSO) and phosphate buffered saline (PBS, pH = 7.4) was added to the above activated Au NC solution (COOH:DOX = 1:1.5, mol/mol). After reaction for 4 h at room temperature in dark, the residual sulfo-NHS, EDC and DOX were removed by dialysis (2 kDa). 2.4. Characterization of Au NCs UV–vis absorption spectra were recorded with a UV-2600 spectrophotometer (Shimadzu). Fluorescence spectra were collected on a RF-5301PC spectrofluorophotometer (Shimadzu) and an F-7000 fluorescence spectrophotometer (Hitachi). Dynamic light scattering (DLS) and zeta potential measurements were carried out on a Malven Nano ZS Zetasizer. Transmission electron microscopic (TEM) experiment was performed on a JEM-2100 transmission electron microscope (JEOL). X-ray photoelectron spectroscopy (XPS) analysis was conducted by a PHI Quantera II X-ray photoelectron spectrometer (Ulvac-Phi).
2.5. Cell culture and cytotoxicity assay To test the cytotoxicity of Au NCs in vitro, A549 cells (cancerous lung cells) were selected in this study. A549 cells were cultured in cell medium (DMEM), supplemented with 10% fetal bovine serum, 100 U of penicillin and 100 lg/mL streptomycin in a humidified incubator at 37 °C and 5% CO2. For the MTT toxicity test of Au NCs, A549 cells were seeded in a 96-well plate in cell medium overnight and subsequently incubated with different concentrations of Au NCs (0, 6.25, 12.5, 25, 50 and 100 lM) in cell medium for 24 h at 37 °C and 5% CO2. Afterwards, cell medium in each well was removed and each well was washed by PBS buffer. 100 lL cell medium containing 0.5 mg/mL MTT was added to each well. After incubation for 4 h at 37 °C and 5% CO2, 150 lL DMSO was added to each well and mixed. The absorbance at 492 and 570 nm was measured with a Multiskan FC microplate photometer (Thermo). The following formula was used to calculate the inhibition of cell growth: cell viability (%) = (mean of absorbance value of Au NC treated group/mean absorbance value of control) 100%. 2.6. Real-time cell analysis The real-time cytotoxicities (cell growth behavior) of free DOX and Au NC-DOX were evaluated using a real-time cell analyzer— the iCELLigence system (ACEA Biosciences Inc.). The iCELLigence system is an impedance-based system, allowing for label-free and real-time monitoring of cell growth/proliferation process. The growth or proliferation of cells leads to an increase of impedance (and an increase in cell index value), while the cell death caused by addition of drugs can decrease the cell index value. The A549 cells were seeded into E-plate L8 (8 wells) at 1.5 104 cells per well in 450 lL culture medium. Proliferating A549 cells were allowed to proceed for 17 h before drug treatment. Subsequently, culture medium was replaced by 450 lL Au NC-DOX or DOX suspension at concentrations of 0.01, 0.1, 1, 3 and 10 lg/mL. 2.7. Cell imaging A549 cells were seeded in a Lab-Tek 8-well chambered coverglass (Nunc) in cell medium in a humidified incubator at 37 °C and 5% CO2. After culturing for 24 h, Au NC-DOX or free DOX was added to the well and incubated for 1 and 3 h, respectively. The final concentration of free DOX and DOX in Au NC-DOX was the same (1 lg/mL). After washing with PBS for three times, the cells were imaged under a confocal laser-scanning microscope (CLSM, Leica TCS-SP8) with an excitation wavelength of 488 nm. 3. Results and discussion 3.1. Synthesis and characterization of Au NCs As we stated above, here the Au NCs were prepared using two different methods (Scheme 1). In method A, to synthesize Au NC precursors, histidine and HAuCl4 were blended and shaken gently for several times, followed by incubation for more than 2 h without stirring. This procedure is similar to that reported by Yang et al. [46]. After addition of HS–C11–EG6–OH to the above-prepared Au NC precursors and incubation at room temperature for more than 8 h, the final HS–C11–EG6–OH capped Au NCs were obtained. In method B, HAuCl4, histidine and HS–C11–EG6–OH were mixed to form HS–C11–EG6–OH capped Au NCs in one step. The TEM image (Fig. S1) reveals that Au NC precursors prepared by method A, step 1, have an average size of 3.06 nm. After capping with 1:4 ‘‘HS–C11–EG6–X (X = OCH2COOH, NH2 and
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Fig. 1. Typical TEM images and corresponding size distribution histograms of Au NCs capped with HS–C11–EG6–X. (A and F) HS–C11–EG6–OH two-step, (B and G) HS–C11– EG6–OCH2COOH:HS–C11–EG6–OH = 1:4, (C and H) HS–C11–EG6–NH2:HS–C11–EG6–OH = 1:4, (D and I) HS–C11–EG6–GRGD:HS–C11–EG6–OH = 1:4 and (E and J) HS–C11–EG6– OH one pot.
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GRGD):HS–C11–EG6–OH’’ mixture (Fig. 1, B&G, C&H, D&I), the Au NCs have a smaller size (1.56–1.85 nm) with better dispersity and uniformity. The Au NCs prepared by ‘‘HS–C11–EG6–OH, one pot’’ (Fig. 1E and J) and ‘‘HS–C11–EG6–OH, two-step’’ (Fig. 1A and F) are similar in size (1.42 vs. 1.65 nm) and morphology. The smaller sizes of the final Au NCs might result from the etching effect of the alkanethiol ligand to the Au NC precursors. The successful binding of the ligands was verified by zeta potential measurements. The HS–C11–EG6–OH capped Au NCs have a surface charge value of 4.5 mV for ‘‘two-step’’ sample and 5.1 mV for ‘‘one pot’’ sample. While the Au NCs capped with ‘‘HS–C11–EG6–OCH2COOH:HS–C11–EG6–OH = 1:4’’ have a surface charge of 9.3 mV, and the Au NC capped with ‘‘HS–C11–EG6– NH2:HS–C11–EG6–OH = 1:4’’ has a surface charge of +9.0 mV. These two latter surface charge values can plausibly be explained by the presence of the respective negatively or positively charged ligands on the surface of Au NCs. The surface charge of Au NC capped with ‘‘HS–C11–EG6–GRGD:HS–C11–EG6–OH = 1:4’’ cannot be obtained from zeta potential measurements (possibly because its surface charge value is too close to zero) and is not given here. All the five products have similar UV–vis spectra (Fig. 2A). There was no absorption for the wavelength region >350 nm. Absorption was observed between 250 and 350 nm, with a small peak residing at 261 nm. The absence of a high wavelength absorption at above 400 nm in the UV–vis spectra suggested that there were no large gold aggregates or gold nanoparticles in the solution. This deduction can also be supported by the following analyses: Our Au NC precursors are the same as those in a previous report [46], in which the Au NCs with a size of around 3 nm include 10 gold atoms. The size of the final Au NCs after capping with
HS–C11–EG6–X decreases to 1.5 nm, much smaller than that of the precursors, which indicates that the final Au NCs consist of less than 10 gold atoms. As a result, no UV absorption above 400 nm can be observed. To investigate the surface ligand changes of the Au NCs after the addition of these thiol PEGs, XPS experiments were carried out. The XPS spectra of the Au NC precursors (Fig. S2A) and HS–C11–EG6– OH-capped Au NCs (Fig. S2B) show the binding energies of all the elements in the product. Specifically, the elements of C, N, O and S are from histidine and HS–C11–EG6–OH in HS–C11–EG6– OH-capped Au NCs, indicating that there are two ligands on the surface of the Au NCs. The decrease in the intensity of the N 1s peak indicates that partial histidine molecules were replaced by the HS–C11–EG6–OH molecules. The presence of S 2p at 162.1 eV peak proves the interaction of Au NCs with HS–C11–EG6–OH through the Au–S bond. The binding energy of Au 4f7/2 shifts from 83.9 to 83.0 eV (Fig. 2D), indicating a reduction of Au(I) to Au(0) in Au NCs by HS–C11–EG6–OH molecules. The fluorescence properties of the Au NCs were also evaluated. Before UV irradiation, the solution of the HS–C11–EG6–OH capped Au NCs was colorless under white light, while it exhibited different colors under the 254 (orange) and 302 nm (yellow) UV excitations (Fig. 2C). Quantitatively, under the 261 nm excitation, all the five samples generated a fluorescence emission peak at about 575 nm, and the only exception was that the emission of the ‘‘HS–C11–EG6–OH one pot’’ sample has a 10 nm red shift compared to those of the other four samples (Fig. 2B). The similar structure of the surface ligands and the small difference in the composition of these ligands can explain the similar UV–vis and fluorescence spectra of these Au NCs. Among the five samples with the same UV–vis
Fig. 2. UV–vis absorption (A) and fluorescence emission (B) spectra of Au NCs capped with ‘‘HS–C11–EG6–OCH2COOH:HS–C11–EG6–OH = 1:4’’, ‘‘HS–C11–EG6–NH2:HS–C11– EG6–OH = 1:4’’, ‘‘HS–C11–EG6–GRGD:HS–C11–EG6–OH = 1:4’’, ‘‘HS–C11–EG6–OH one pot’’ and ‘‘HS–C11–EG6–OH two-step’’. (C) Photographs of HS–C11–EG6–OH capped Au NCs (two-step) in water under irradiations of visible, 254 nm and 302 nm UV light, respectively. (D) XPS spectra of Au 4f for Au NC precursors (black line) and HS–C11–EG6–OH capped Au NCs (red line). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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absorption, Au NCs capped with ‘‘HS–C11–EG6–OCH2COOH: HS–C11–EG6–OH = 1:4’’ is the brightest, followed by ‘‘HS–C11– EG6–OH’’, ‘‘HS–C11–EG6–NH2:HS–C11–EG6–OH = 1:4’’, ‘‘HS–C11– EG6–GRGD:HS–C11–EG6–OH = 1:4’’ and ‘‘HS–C11–EG6–OH one pot’’. Using tyrosine (QY = 14%, excited at 275 nm) [60] as a standard, we calculated quantum yields of the above five products to be 2.6%, 2.3%, 2.0%, 1.3% and 0.6%, respectively. These quantum yield data are all larger than that of Au NC precursors (0.4%, using quinine sulfate as the reference) prepared by mixing HAuCl4 and histidine, suggesting that the surface modification by these thiol-containing short-chain PEGs can significantly increase the fluorescence intensity of the Au NCs. The fluorescence intensity enhancement of AuNCs may be attributed to the ligand to metal charge transfer [61]. Besides, as reported in previous work [46], the Au NC precursors have the maximum excitation and emission peaks of Au NC precursors at 386 and 490 nm, respectively. After capped with HS–C11–EG6–X, the excitation peak has a blue shift to 261 nm, while the emission peak is at 575 nm (314 nm Stokes shift), which is significantly different from the reported value for the Au NC precursors. This also indicates that the thiol-terminated alkyl-containing short-chain PEGs can be tightly bound to the surfaces of the Au NCs. Additionally, although the Au NCs prepared by the ‘‘HS–C11–EG6–OH one pot’’ and ‘‘HS– C11–EG6–OH two-step’’ methods had similar average size and size distribution (Fig. 1), the fluorescence intensity of the latter was three times higher than that of the former. It was indicated that it was better to add HS–C11–EG6–X after forming Au NC precursors than before that. However the ‘‘one pot’’ method is simpler and therefore we believe that it is also a new development. Careful optimization of the experimental conditions of the ‘‘one pot’’ method may lead to stronger emission from Au NCs, which is under the current investigation. 3.2. Stability of ‘‘HS–C11–EG6–X’’ capped Au NCs We also investigated the stability of Au NCs prepared using the ‘‘HS–C11–EG6–OH two-step’’ method. The Au NCs were prepared in different solution conditions, including water, acid and base (pH = 2 and 12), complete DMEM medium (containing 10% fetal bovine serum, 100 U of penicillin, and 100 lg/mL streptomycin), PBS and high salt concentration solution (2 M NaCl). The as-prepared samples were very stable, observed under white light (no precipitates were observed) and 254 nm ultraviolet light (the fluorescence color did not change in different solutions) (Fig. 3A). Since phenol red was present in DMEM, the photo of Au NCs under ultraviolet light is not shown in the figure. The size and morphology of the AuNCs in pure water or cell PBS almost did not change after incubation for more than 6 months (Fig. S3), suggesting that the AuNCs capped with HS–C11–EG6–X have superior stability. We have also investigated the fluorescence stability of the three kinds of Au NCs in different pH solutions (Fig. 3B–D). Although there are some deviations of fluorescence intensities in the ‘‘HS–C11–EG6–OH’’, ‘‘HS–C11–EG6–OCH2COOH:HS–C11–EG6–OH = 1:4’’ and ‘‘HS–C11–EG6–NH2:HS–C11–EG6–OH = 1:4’’ capped Au NCs, the fluorescence emissions of these Au NCs are relatively stable in the pH range of 2–12. The results may indicate that the minor species of HS–C11–EG6–OCH2COOH or HS–C11–EG6–NH2 that accounting for only 20 mol% of the total surface ligands do not influence the fluorescence stability of the Au NCs. We have also carried out zeta potential measurements for Au NCs capped with HS–C11–EG6–OH under various conditions. The zeta potential values of Au NCs in PBS (0.1 M, pH = 7.4), pH = 2, pH = 12 and DMEM cell medium are –4.9, +6.0, –23.3 and –10.1 mV, respectively. The large difference in zeta potential value indicates that the stability of the Au NCs is mainly due to the steric effect of the short-chain PEGs, not the surface charge properties.
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The similar UV–vis and fluorescence emission spectra of Au NCs in water and 2 M NaCl (Fig. 4) also demonstrate that the charge stabilization effect does not play a major role. We believe that the use of HS–C11–EG6–X capping in Au NC synthesis is a key development. Au NC precursors prepared using the first step of the two-step method without HS–C11–EG6–X capping can easily precipitate after standing for 2–5 days, or after stirring the formed Au NC precursors for several hours. Also, Au NC precursors will precipitate if we perform dialysis to remove the unbound histidine molecules. The instability of the Au NC precursors significantly limits their practical applications. As a result, we tried to cap such Au NCs with thiol-containing molecules like L-cysteine, mercaptosuccinic acid, lipoic acid, HS-PEG2K, HS-PEG5K and a short thiol-containing peptide CRGDK. When capped with L-cysteine, the Au NCs were easy to precipitate in water, which is due to the relatively poor water solubility of L-cysteine.
When coated with HS-PEG2K, HS-PEG5K, mercaptosuccinic acid, lipoic acid and CRGDK, the fluorescence intensities of Au NCs significantly decreased and some of these ligands even made the fluorescence diminish. The above experiments demonstrate the critical role the HS–Cm–EGn–X molecule plays toward the successful fabrication of the ultraclean and ultrastable fluorescent Au NCs. As we stated before, there are four parts of the HS–Cm–EGn–X molecule: the mercapto group (–SH), the methylene group (Cm), the short-chain polyethylene glycol (EGn) and the functional group (X) like OH, NH2, OCH2COOH and GRGD. All these components are important for preparing/stabilizing/using the Au NCs. The mercapto group is to bind to gold atoms through the Au-S covalent bond. The alkyl chain (such as (CH2)11) serves as a hydrophobic shield to stabilize nanoparticles and prevent other thiol-containing molecules to replace the bound thoil-ligands [62]. If the Au NCs were first coated with HS-PEG2K or HS-PEG5K, evidence has been reported that some small thiol-terminated molecules (such as cysteine) can replace the surface HS-PEGs, causing possible instability of the nanoclusters [62]. Therefore the methylene group is important. The hydrophilic EGn part is also important. It can increase water solubility of prepared Au NCs, and the introduction of EGn also endows the Au NCs with a superb capability to prevent non-specific adsorption of proteins [63–65], and can therefore ‘‘mask’’ the Au NCs from the host’s immune system (reduced immunogenicity and antigenicity). Finally, the functional group (X) is very critical to make Au NCs multifunctional. For example, when the functional group is an arginine–glycine–aspartic acid (RGD) peptide, the Au NCs can target to cancer cells whose RGD receptors (integrins) are overexpressed on the cell surfaces. Moreover, we can bind carboxyl-containing molecule (e.g., folic acid) or amine-containing molecule (e.g., doxorubicin) to the amine groups or carboxyl groups on the surface of the Au NCs using the EDC/NHS coupling reaction, respectively. This kind of coupling reaction can further extend the functionality of the Au NCs. The ultrasmall Au NCs with carried drugs can possibly pass through the blood brain barrier, enabling the efficient delivery of the drugs to the targeting positions in the brain. If we conjugate antibodies onto the Au NCs’ surfaces (with amine or carboxyl groups) through EDC/NHS coupling reaction, we can use these antibody-conjugated Au NCs to investigate important cellular events via antibody-antigen recognition. Furthermore, with the charged amine (NH+3) or carboxyl (COO–) functional groups on the terminal part of the EGs, we can tune the charge or charge ratio of the Au NCs, and these charged Au NCs can be used in various electrostatic interaction based supramolecular self-assembly systems (such as layer-by-layer assembly and polyion complex micelles) to fabricate functional biomaterials and biofilms for specific purposes. Finally, the synthesized Au NCs are ultraclean
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Fig. 3. (A) HS–C11–EG6–OH capped Au NCs (two-step) at different solution conditions: complete DMEM medium (with phenol red), water, pH = 2, pH = 12, PBS (pH = 7.4) and 2 M NaCl under white light (from left to right, the upper panel) and 254 nm UV (the lower panel) excitations. Dependence of fluorescence intensity on various pH values for (B) HS–C11–EG6–OH capped Au NCs (two-step), (C) ‘‘HS–C11–EG6–OCH2COOH:HS–C11–EG6–OH = 1:4’’ capped Au NCs and (D) ‘‘HS–C11–EG6–NH2:HS–C11–EG6–OH = 1:4’’ capped Au NCs. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
nanoclusters. However, if the density of the functional group is too low, then Au NCs may not have efficient targeting or sensing ability for various purposes. 3.3. Application of the stable Au NCs for anticancer drug carrier
Fig. 4. UV–vis absorption and fluorescence spectra of HS–C11–EG6–OH capped Au NCs in water and 2 M NaCl.
(that is, they can be very stable in pure water, with negligible free molecules and ions), making them excellent models for various physicochemical applications without the need to consider the side effects of free molecules or ions. An optimized surface density of the functional group X on Au NCs is also vital for the preparation of the highly fluorescent and highly stable Au NCs. If HS–C11–EG6–OH is completely changed to HS–C11–EG6–X (X = NH2, OCH2COOH), the obtained Au NCs capped with 100% HS–C11–EG6–NH2 or 100% HS–C11–EG6– OCH2COOH have a much lower fluorescence intensity as compared to those with 1:4 (mol/mol) of HS–C11–EG6–NH2 (or –OCH2COOH) and HS–C11–EG6–OH. The surface density of the above functional groups cannot be too high, since the charged ligands on the surface of Au NCs will repulse each other, causing instability of the
We have also evaluated the stability of the AuNCs after conjugation with drugs, and have investigated the potential ability of these Au NCs for drug delivery into the cells. To this end, the cytotoxicity of the ultraclean Au NCs needs to be investigated first. The MTT assay results indicate that the four products have negligible toxicity to A549 cells (Fig. 5), which indicates that these Au NCs can be safely used in live cells. Since the fluorescence excitation of the above prepared Au NCs resides at around 260 nm, it is difficult to use these Au NCs for cellular imaging. To see if the Au NCs can enter live cells and at the same time to prove that these Au NCs can be used to deliver anticancer drugs, DOX was conjugated to Au NCs capped with ‘‘HS–C11–EG6–OCH2COOH:HS–C11–EG6– OH = 1:2’’ using the EDC/sulfo-NHS coupling reaction. UV–vis absorbance spectra (Fig. S4), fluorescence spectra (Fig. S5) and DLS data (Fig. S6) all demonstrated the successful synthesis of Au NC-DOX complex. Au NCs have no absorbance for the wavelength region >400 nm, while both free DOX and Au NC-DOX complex have a maximum excitation peak at around 475 nm. Meanwhile, Au NC-DOX complex has a similar emission spectrum with free DOX. Besides, the DLS data reveal that the Au NC-DOX complex has a average hydrodynamic diameter of 10.7 nm, which endow the complex to be entered into cell nucleus since the nuclear pore allows that passage of nanoparticles smaller than 20 nm [66]. The DOX-conjugated Au NCs can be used as excellent model systems for drug delivery and cell imaging studies.
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Fig. 5. Cytotoxicity of Au NCs capped with (A) ‘‘HS–C11–EG6–OH two-step’’, (B) ‘‘HS–C11–EG6–OCH2COOH:HS–C11–EG6–OH = 1:4’’, (C) ‘‘HS–C11–EG6–NH2:HS–C11–EG6– OH = 1:4’’ and (D) ‘‘HS–C11–EG6–GRGD:HS–C11–EG6–OH = 1:4’’ on A549 cells determined by MTT assay (after incubation for 24 h).
Fig. 6. Cytotoxicity assays for free DOX (A and B) and Au NC-DOX (C and D). Shown in the left (A and C) are MTT assays after incubation with drugs for 48 h, while shown in the right (B and D) are real-time cell analysis results.
We will show the drug delivery application of Au NC-DOX first. To evaluate the cytotoxicities of Au NC-DOX and free DOX, MTT assay and real-time cell analysis experiments were carried out
(Fig. 6). MTT assay results (Fig. 6A and C) show that after adding 10 and 3 lg/mL Au NC-DOX or free DOX and incubating for 2 days, A549 cells were almost all dead. Addition of 1 lg/mL Au NC-DOX
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X. Zhang et al. / Journal of Colloid and Interface Science 455 (2015) 6–15
Fig. 7. Confocal images of A549 cells 1 h after incubation with free DOX (A–C) and Au NC-DOX (D–F).
Fig. 8. Confocal images of A549 cells after incubation with Au NC-DOX for (A) 1 h and (B) 3 h.
decreases the cell viability value to 50%, while addition of the same concentration of free DOX made almost all the cells dead. The real-time cell analysis results (Fig. 6B and D) show a similar trend at time = 65 h (equal to the 48 h drug incubation time if we plus the 17 h cell incubation time before drug addition). For these high concentration (1, 3 and 10 lg/mL) samples, the real-time cell analysis results show that the Au NC-DOX begins to have a significant cell killing effect at around 30 h, which is about 5 h later than the free DOX which begins to have a significant cell killing effect at around 25 h. Low concentrations (0.1 and 0.01 lg/mL) of Au NC-DOX and DOX have negligible cytotoxicity or even increase the proliferation of the cells, as shown by MTT and real-time cell analysis results. Since the free DOX and Au NC-DOX have a maximum excitation at round 475 nm, we can use confocal imaging technique to study whether the free DOX and Au NC-DOX can enter cells. After incubating A549 cells with 1 lg/mL DOX or Au NC-DOX containing 1 lg/mL DOX for 1 h, a number of red-emission dots can be seen in both groups (Fig. 7A and D), indicating that Au NC-DOX can enter cells as free DOX. However, only a few red fluorescent dots appear in the cell nucleus for Au NC-DOX (Fig. 7F), while a number of red fluorescent dots can be observed for free DOX (Fig. 7C). To demonstrate if Au NC-DOX can enter cell nucleus, A549 cells were incubated with Au NC-DOX for different time periods (1 h and 3 h).
Fig. 8 shows that compared with the case with the incubation time of 1 h, the confocal images taken after an incubation time of 3 h show plenty of red fluorescent dots in both the cytoplasm and nucleus. The above results demonstrated Au NC-DOX complex can enter into the cells and even the nucleus. After conjugation to the surface of Au NCs, the time needed for the drugs to enter into the nucleus was prolonged. The conjugation of DOX onto the surface of Au NCs has several advantages. First, the water solubility of DOX is greatly improved by conjugation of free DOX onto the terminal groups of the short-chain PEGs of Au NCs. The presence of surface PEGs on the Au NCs may prevent protein adsorption and prolong the drug circulation time in vivo. The improved water solubility and the increased drug circulation time make the Au NC-DOX complex be a safer anticancer drug formulation since it has been reported that free DOX molecules may cause severe systemic toxicity [67]. Second, the ultrasmall size of Au NCs endows the conjugated DOX with the ability to enter into the cell nucleus to have cell killing effect, since DOX mainly takes its anticancer effect in the cell nucleus. Besides, the ultrasmall size of the Au NC-DOX complex may have a deeper tumor tissue penetration depth [67]. Moreover, compared with large gold nanoparticles which could be trapped by the reticulo-edothelial system (RES) absorption and cause unavoidable accumulation in liver and spleen, the Au
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NCs with a much smaller size could be efficiently cleared by the kidney [68], making the smaller Au NCs be a better choice for drug delivery system. Third, due to the enhanced permeability and retention (EPR) effect, the nanocluster-conjugated DOX may have passive targeting ability in vivo. Our present work shows that the ultrastable water-soluble Au NCs can be used as an efficient anticancer drug delivery system. 4. Conclusions In summary, based on the thiol-containing short-chain PEGs (HS–C11–EG6–X), we synthesized water-soluble, multifunctional and monodisperse Au NCs using two methods. The two synthetic methods have their respective pros and cons. Although the quantum yield of the Au NCs prepared by method A is higher than that by method B, the preparation of Au NCs by method B is easier. The Au NCs prepared by both methods are ultrastable and have good monodispersity in aqueous media. It is found that the HS–C11– EG6–X capped Au NCs exhibit superior stability under various solution conditions for at least 6 months, even after conjugation with anticancer drug DOX. Besides, HS–C11–EG6–X can significantly increase the quantum yield of the synthesized Au NCs and the Au NCs capped with carboxyl groups have the highest quantum yield. Furthermore, the terminal functional group X can be any functional group, such as hydroxyl, amide, carboxyl and GRGD as demonstrated in this paper. Other functional groups like azido (–N3), biotin, nitrilotriacetic group and acylamino are also commercially available and can be readily used for various purposes. The present work demonstrates the importance of using thiol-terminated, alkyl-containing short-chain PEGs to fabricate and stabilize the Au NCs. Acknowledgments This work was supported by grants from the National Key Basic Research Program of China (973 Program) (No. 2013CB933904), the Natural Science Foundation of China (21303017), the Natural Science Foundation of Jiangsu Province (KB20130601), a project funded by the Priority Academic Program Development (PAPD) of Jiangsu Higher Education Institutions, and the Fundamental Research Funds for the Central Universities (2242015R30016). ZC thanks the University of Michigan to support his sabbatical. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jcis.2015.05.029. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14]
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Contents lists available at ScienceDirect
Journal of Colloid and Interface Science www.elsevier.com/locate/jcis
Synthesis of ultrastable and multifunctional gold nanoclusters with enhanced fluorescence and potential anticancer drug delivery application Xiaodong Zhang a, Fu-Gen Wu a,⇑, Peidang Liu a,c, Hong-Yin Wang a, Ning Gu a, Zhan Chen b,⇑ a b c
State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing 210096, China Department of Chemistry, University of Michigan, 930 North University Avenue, Ann Arbor, MI 48109, United States School of Medicine, Southeast University, Nanjing 210009, China
g r a p h i c a l a b s t r a c t
a r t i c l e
i n f o
Article history: Received 30 March 2015 Accepted 18 May 2015 Available online 22 May 2015 Keywords: Gold nanoclusters Ultrastable Short-chain PEGs Anticancer
a b s t r a c t The problem of stability hinders the practical applications of nanomaterials. In this research, an innovative and simple synthetic method was developed for preparing ultrastable and multifunctional gold nanoclusters (Au NCs). HS–C11–EG6–X is a class of molecules consisting of four components: a mercapto group (–SH), an alkyl chain (C11), a short chain of polyethylene glycols (EG6) and a functional group (X, X = OH, COOH, NH2, GRGD, etc). The present work demonstrated the importance of using HS–C11–EG6–X to prepare Au NCs with excellent properties and the role each component in this molecule played for synthesizing Au NCs. Au NCs with tunable surface functionalities were successfully synthesized and characterized. It was found that Au NC precursors had a fluorescent quantum yield of 0.4%; in contrast, after capping with HS–C11–EG6–X, the quantum yield significantly increased to 1.3–2.6%. The HS–C11–EG6–X capped Au NCs exhibited superior stability under various solution conditions (including extreme pH, high salt concentration, phosphate buffered saline and cell medium) for at least 6 months, even after conjugation with anticancer drug doxorubicin. Besides, we have also demonstrated that other commonly employed thiol-containing ligands failed to prepare stable fluorescent Au NCs. Moreover, the Au NCs showed negligible toxicity to A549 lung cancer cells up to 100 lM, and the application of the ultrastable Au NCs for anticancer drug delivery has also been demonstrated. Ó 2015 Elsevier Inc. All rights reserved.
⇑ Corresponding authors. E-mail addresses: [email protected] (F.-G. Wu), [email protected] (Z. Chen). http://dx.doi.org/10.1016/j.jcis.2015.05.029 0021-9797/Ó 2015 Elsevier Inc. All rights reserved.
X. Zhang et al. / Journal of Colloid and Interface Science 455 (2015) 6–15
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1. Introduction The instability of nanomaterials is a large obstacle that prevents nanomaterials for practical applications, especially in chemical and biomedical fields [1–4]. Charge stabilization (electrostatic repulsion between nanoparticles) and steric stabilization (such as by using polyethylene glycol ligands) are commonly used to solve the problem. However, the former method is sensitive to salt concentration, and the latter is usually not suitable for ultrasmall nanoparticles especially when using long polyethylene glycol chains. On the other hand, noble metal nanoclusters, consisting of several to several hundred atoms, also have drawn much attention due to their unique physiochemical properties [5–7], catalytic activities [8], ion detection capability [9–13] and potential applications in bio-labeling and sensing [14–30]. At the same time, Au NCs can be synthesized using different capping molecules, such as proteins [31–33], DNA and nucleotides [34,35], peptides [11,36–39], polymers [40–44] and small molecules such as dihydrolipoic acid [10], diphosphine [45], histidine [46,47], purine [48], cytidine [49,50] and alkanethiol [51–56]. Many excellent methods have been developed to prepare Au NCs. Schiffrin and co-workers prepared 1–3 nm Au NCs using two-phase reduction in the presence of alkanethiol [51]. Chang and co-workers produced fluorescent Au NCs by adding various alkanethiol ligands to gold nanoparticles stabilized by tetrakis(hydroxymethyl)phosphonium chloride (THPC) and 11-mercaptoundecanoic acid (MUA), yielding the brightest Au NCs with a quantum yield of 3.1% [9]. Au NCs fabricated by the above-mentioned methods are hydrophobic, which must be undergone a further hydrophilic treatment to become water-soluble for biomedical applications. Recently, Rotello group synthesized a series of water-soluble small gold nanoparticles (2 nm) functionalized with polyethylene glycol (PEG) and quaternary-N groups, and used them in regulating cell behavior, antimicrobial and model nanoparticles in vivo [57–59]. Despite such advances, it is still challenging for the Au NCs to have further modification due to the absence of functional groups on the Au NCs’ surface. Herein, we report two new and simple methods (see Scheme 1) to fabricate water-soluble and multifunctional Au NCs with high stability based on short-chain PEGs ‘‘HS–C11–EG6–X’’. Each HS–C11–EG6–X molecule consists of four parts: a mercapto group (–SH), a methylene group (C11), a short chain of ethylene glycols (EG6) and a terminal functional group (X, X = OH, COOH, NH2 and GRGD) (their molecular structures are shown in Scheme 2). In method A, histidine and HAuCl4 were blended and incubated for more than two hours to synthesize Au NC precursors. After that, HS–C11–EG6–OH was added to the above-prepared Au NC precursors to form the final Au NCs. In the even simpler method B,
Scheme 2. Four thiol-containing short-chain PEGs (HS–Cm–EGn–X, X = OH, NH2, OCH2COOH and GRGD) discussed in this study.
HAuCl4, histidine and HS–C11–EG6–OH were mixed together in one pot to form the final Au NCs. Both products possess ultrahigh stability in aqueous solutions of different pH, high salt concentration and even cell medium. Moreover, the Au NCs were still stable even conjugated with anticancer drug doxorubicin (DOX). Confocal imaging experiments have been carried out to demonstrate that the drug-carrying Au NCs can enter into cells even the cell nucleus.
2. Experimental 2.1. Materials HAuCl43H2O, L-histidine, L-tyrosine, L-cysteine, mercaptosuccinic acid, lipoic acid, quinine sulfate and HS–(CH2)11– (OCH2CH2)6–OH (abbreviated as HS–C11–EG6–OH) were purchased from Sigma–Aldrich. HS–(CH2)11–(OCH2CH2)6–NH2HCl (abbreviated as HS–C11–EG6–NH2), HS–(CH2)11–(OCH2CH2)6–OCH2COOH (abbreviated as HS–C11–EG6–OCH2COOH) and HS–(CH2)11– (OCH2CH2)6–GRGD (abbreviated as HS–C11–EG6–GRGD, where
Scheme 1. Schematic illustration of the formation of Au NCs.
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GRGD refers to glycine–arginine–glycine–aspartic acid) were ordered from Prochimia (Sopot, Poland). Thiol-PEG2000-methoxy (HS-PEG2K) and thiol-PEG5000-methoxy (HS-PEG5K) were obtained from Nanocs Inc. (Boston, MA, USA). The short thiol-containing peptide cysteine–arginine–glycine–aspartic acid– lysine (CRGDK) was acquired from GL Biochem Ltd (Shanghai, China). Doxorubicin (DOX) was from Beijing Huafeng United Technology Co., Ltd (Beijing, China). All solutions were prepared with deionized water purified by a Milli-Q system (Millipore). 2.2. Synthesis of HS–C11–EG6–X capped Au NCs
2.2.1. Two-step synthesis All glassware was cleaned in a bath of freshly prepared aqua regia (HCl:HNO3, 3:1 v/v) and rinsed thoroughly in Milli-Q water prior to use. An aqueous solution of histidine (1.5 mL, 0.1 M) was added to an aqueous solution of HAuCl4 (0.5 mL, 10 mM) in a small vial at room temperature (about 25 °C). The solution mixture was blended gently at the beginning and then was placed at room temperature without stirring in dark. After more than 3 h, an aqueous solution of HS–C11–EG6–OH (2 mL, 3.75 mM) was added to the mixture (pH = 6.88). Afterwards, the final mixture (pH = 6.80) was shaken in dark at room temperature overnight. The residual histidine and HS–C11–EG6–OH were removed by dialysis (2 kDa). Au NCs with different capping agents could be synthesized by replacing HS–C11–EG6–OH with mixture containing HS–C11–EG6– X (X = OCH2COOH, NH2 and GRGD) and HS–C11–EG6–OH. 2.2.2. One pot synthesis An aqueous solution of histidine (1.5 mL, 0.1 M) was added to an aqueous solution of HAuCl4 (0.5 mL, 10 mM) in a small vial at room temperature (about 25 °C). After blending for several times, an aqueous solution of HS–C11–EG6–OH (2 mL, 3.75 mM) was added immediately. Afterwards, the mixture was shaken in dark at room temperature overnight. The residual histidine and HS–C11–EG6–OH were removed by dialysis (2 kDa). 2.3. Synthesis of Au NC-DOX To conjugate DOX to the carboxyl groups on the surface of ‘‘HS–C11–EG6–OCH2COOH:HS–C11–EG6–OH = 1:2’’ capped Au NCs, Au NCs were firstly reacted with EDC/sulfo-NHS dissolved in 0.1 M MES buffer solution (pH = 6.0) at room temperature in the dark for 30 min. The molar ratio of COOH (on the surface of Au NCs): sulfo-NHS:EDC was 1:10:25. Then DOX dissolved in dimethyl sulfoxide (DMSO) and phosphate buffered saline (PBS, pH = 7.4) was added to the above activated Au NC solution (COOH:DOX = 1:1.5, mol/mol). After reaction for 4 h at room temperature in dark, the residual sulfo-NHS, EDC and DOX were removed by dialysis (2 kDa). 2.4. Characterization of Au NCs UV–vis absorption spectra were recorded with a UV-2600 spectrophotometer (Shimadzu). Fluorescence spectra were collected on a RF-5301PC spectrofluorophotometer (Shimadzu) and an F-7000 fluorescence spectrophotometer (Hitachi). Dynamic light scattering (DLS) and zeta potential measurements were carried out on a Malven Nano ZS Zetasizer. Transmission electron microscopic (TEM) experiment was performed on a JEM-2100 transmission electron microscope (JEOL). X-ray photoelectron spectroscopy (XPS) analysis was conducted by a PHI Quantera II X-ray photoelectron spectrometer (Ulvac-Phi).
2.5. Cell culture and cytotoxicity assay To test the cytotoxicity of Au NCs in vitro, A549 cells (cancerous lung cells) were selected in this study. A549 cells were cultured in cell medium (DMEM), supplemented with 10% fetal bovine serum, 100 U of penicillin and 100 lg/mL streptomycin in a humidified incubator at 37 °C and 5% CO2. For the MTT toxicity test of Au NCs, A549 cells were seeded in a 96-well plate in cell medium overnight and subsequently incubated with different concentrations of Au NCs (0, 6.25, 12.5, 25, 50 and 100 lM) in cell medium for 24 h at 37 °C and 5% CO2. Afterwards, cell medium in each well was removed and each well was washed by PBS buffer. 100 lL cell medium containing 0.5 mg/mL MTT was added to each well. After incubation for 4 h at 37 °C and 5% CO2, 150 lL DMSO was added to each well and mixed. The absorbance at 492 and 570 nm was measured with a Multiskan FC microplate photometer (Thermo). The following formula was used to calculate the inhibition of cell growth: cell viability (%) = (mean of absorbance value of Au NC treated group/mean absorbance value of control) 100%. 2.6. Real-time cell analysis The real-time cytotoxicities (cell growth behavior) of free DOX and Au NC-DOX were evaluated using a real-time cell analyzer— the iCELLigence system (ACEA Biosciences Inc.). The iCELLigence system is an impedance-based system, allowing for label-free and real-time monitoring of cell growth/proliferation process. The growth or proliferation of cells leads to an increase of impedance (and an increase in cell index value), while the cell death caused by addition of drugs can decrease the cell index value. The A549 cells were seeded into E-plate L8 (8 wells) at 1.5 104 cells per well in 450 lL culture medium. Proliferating A549 cells were allowed to proceed for 17 h before drug treatment. Subsequently, culture medium was replaced by 450 lL Au NC-DOX or DOX suspension at concentrations of 0.01, 0.1, 1, 3 and 10 lg/mL. 2.7. Cell imaging A549 cells were seeded in a Lab-Tek 8-well chambered coverglass (Nunc) in cell medium in a humidified incubator at 37 °C and 5% CO2. After culturing for 24 h, Au NC-DOX or free DOX was added to the well and incubated for 1 and 3 h, respectively. The final concentration of free DOX and DOX in Au NC-DOX was the same (1 lg/mL). After washing with PBS for three times, the cells were imaged under a confocal laser-scanning microscope (CLSM, Leica TCS-SP8) with an excitation wavelength of 488 nm. 3. Results and discussion 3.1. Synthesis and characterization of Au NCs As we stated above, here the Au NCs were prepared using two different methods (Scheme 1). In method A, to synthesize Au NC precursors, histidine and HAuCl4 were blended and shaken gently for several times, followed by incubation for more than 2 h without stirring. This procedure is similar to that reported by Yang et al. [46]. After addition of HS–C11–EG6–OH to the above-prepared Au NC precursors and incubation at room temperature for more than 8 h, the final HS–C11–EG6–OH capped Au NCs were obtained. In method B, HAuCl4, histidine and HS–C11–EG6–OH were mixed to form HS–C11–EG6–OH capped Au NCs in one step. The TEM image (Fig. S1) reveals that Au NC precursors prepared by method A, step 1, have an average size of 3.06 nm. After capping with 1:4 ‘‘HS–C11–EG6–X (X = OCH2COOH, NH2 and
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Fig. 1. Typical TEM images and corresponding size distribution histograms of Au NCs capped with HS–C11–EG6–X. (A and F) HS–C11–EG6–OH two-step, (B and G) HS–C11– EG6–OCH2COOH:HS–C11–EG6–OH = 1:4, (C and H) HS–C11–EG6–NH2:HS–C11–EG6–OH = 1:4, (D and I) HS–C11–EG6–GRGD:HS–C11–EG6–OH = 1:4 and (E and J) HS–C11–EG6– OH one pot.
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GRGD):HS–C11–EG6–OH’’ mixture (Fig. 1, B&G, C&H, D&I), the Au NCs have a smaller size (1.56–1.85 nm) with better dispersity and uniformity. The Au NCs prepared by ‘‘HS–C11–EG6–OH, one pot’’ (Fig. 1E and J) and ‘‘HS–C11–EG6–OH, two-step’’ (Fig. 1A and F) are similar in size (1.42 vs. 1.65 nm) and morphology. The smaller sizes of the final Au NCs might result from the etching effect of the alkanethiol ligand to the Au NC precursors. The successful binding of the ligands was verified by zeta potential measurements. The HS–C11–EG6–OH capped Au NCs have a surface charge value of 4.5 mV for ‘‘two-step’’ sample and 5.1 mV for ‘‘one pot’’ sample. While the Au NCs capped with ‘‘HS–C11–EG6–OCH2COOH:HS–C11–EG6–OH = 1:4’’ have a surface charge of 9.3 mV, and the Au NC capped with ‘‘HS–C11–EG6– NH2:HS–C11–EG6–OH = 1:4’’ has a surface charge of +9.0 mV. These two latter surface charge values can plausibly be explained by the presence of the respective negatively or positively charged ligands on the surface of Au NCs. The surface charge of Au NC capped with ‘‘HS–C11–EG6–GRGD:HS–C11–EG6–OH = 1:4’’ cannot be obtained from zeta potential measurements (possibly because its surface charge value is too close to zero) and is not given here. All the five products have similar UV–vis spectra (Fig. 2A). There was no absorption for the wavelength region >350 nm. Absorption was observed between 250 and 350 nm, with a small peak residing at 261 nm. The absence of a high wavelength absorption at above 400 nm in the UV–vis spectra suggested that there were no large gold aggregates or gold nanoparticles in the solution. This deduction can also be supported by the following analyses: Our Au NC precursors are the same as those in a previous report [46], in which the Au NCs with a size of around 3 nm include 10 gold atoms. The size of the final Au NCs after capping with
HS–C11–EG6–X decreases to 1.5 nm, much smaller than that of the precursors, which indicates that the final Au NCs consist of less than 10 gold atoms. As a result, no UV absorption above 400 nm can be observed. To investigate the surface ligand changes of the Au NCs after the addition of these thiol PEGs, XPS experiments were carried out. The XPS spectra of the Au NC precursors (Fig. S2A) and HS–C11–EG6– OH-capped Au NCs (Fig. S2B) show the binding energies of all the elements in the product. Specifically, the elements of C, N, O and S are from histidine and HS–C11–EG6–OH in HS–C11–EG6– OH-capped Au NCs, indicating that there are two ligands on the surface of the Au NCs. The decrease in the intensity of the N 1s peak indicates that partial histidine molecules were replaced by the HS–C11–EG6–OH molecules. The presence of S 2p at 162.1 eV peak proves the interaction of Au NCs with HS–C11–EG6–OH through the Au–S bond. The binding energy of Au 4f7/2 shifts from 83.9 to 83.0 eV (Fig. 2D), indicating a reduction of Au(I) to Au(0) in Au NCs by HS–C11–EG6–OH molecules. The fluorescence properties of the Au NCs were also evaluated. Before UV irradiation, the solution of the HS–C11–EG6–OH capped Au NCs was colorless under white light, while it exhibited different colors under the 254 (orange) and 302 nm (yellow) UV excitations (Fig. 2C). Quantitatively, under the 261 nm excitation, all the five samples generated a fluorescence emission peak at about 575 nm, and the only exception was that the emission of the ‘‘HS–C11–EG6–OH one pot’’ sample has a 10 nm red shift compared to those of the other four samples (Fig. 2B). The similar structure of the surface ligands and the small difference in the composition of these ligands can explain the similar UV–vis and fluorescence spectra of these Au NCs. Among the five samples with the same UV–vis
Fig. 2. UV–vis absorption (A) and fluorescence emission (B) spectra of Au NCs capped with ‘‘HS–C11–EG6–OCH2COOH:HS–C11–EG6–OH = 1:4’’, ‘‘HS–C11–EG6–NH2:HS–C11– EG6–OH = 1:4’’, ‘‘HS–C11–EG6–GRGD:HS–C11–EG6–OH = 1:4’’, ‘‘HS–C11–EG6–OH one pot’’ and ‘‘HS–C11–EG6–OH two-step’’. (C) Photographs of HS–C11–EG6–OH capped Au NCs (two-step) in water under irradiations of visible, 254 nm and 302 nm UV light, respectively. (D) XPS spectra of Au 4f for Au NC precursors (black line) and HS–C11–EG6–OH capped Au NCs (red line). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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absorption, Au NCs capped with ‘‘HS–C11–EG6–OCH2COOH: HS–C11–EG6–OH = 1:4’’ is the brightest, followed by ‘‘HS–C11– EG6–OH’’, ‘‘HS–C11–EG6–NH2:HS–C11–EG6–OH = 1:4’’, ‘‘HS–C11– EG6–GRGD:HS–C11–EG6–OH = 1:4’’ and ‘‘HS–C11–EG6–OH one pot’’. Using tyrosine (QY = 14%, excited at 275 nm) [60] as a standard, we calculated quantum yields of the above five products to be 2.6%, 2.3%, 2.0%, 1.3% and 0.6%, respectively. These quantum yield data are all larger than that of Au NC precursors (0.4%, using quinine sulfate as the reference) prepared by mixing HAuCl4 and histidine, suggesting that the surface modification by these thiol-containing short-chain PEGs can significantly increase the fluorescence intensity of the Au NCs. The fluorescence intensity enhancement of AuNCs may be attributed to the ligand to metal charge transfer [61]. Besides, as reported in previous work [46], the Au NC precursors have the maximum excitation and emission peaks of Au NC precursors at 386 and 490 nm, respectively. After capped with HS–C11–EG6–X, the excitation peak has a blue shift to 261 nm, while the emission peak is at 575 nm (314 nm Stokes shift), which is significantly different from the reported value for the Au NC precursors. This also indicates that the thiol-terminated alkyl-containing short-chain PEGs can be tightly bound to the surfaces of the Au NCs. Additionally, although the Au NCs prepared by the ‘‘HS–C11–EG6–OH one pot’’ and ‘‘HS– C11–EG6–OH two-step’’ methods had similar average size and size distribution (Fig. 1), the fluorescence intensity of the latter was three times higher than that of the former. It was indicated that it was better to add HS–C11–EG6–X after forming Au NC precursors than before that. However the ‘‘one pot’’ method is simpler and therefore we believe that it is also a new development. Careful optimization of the experimental conditions of the ‘‘one pot’’ method may lead to stronger emission from Au NCs, which is under the current investigation. 3.2. Stability of ‘‘HS–C11–EG6–X’’ capped Au NCs We also investigated the stability of Au NCs prepared using the ‘‘HS–C11–EG6–OH two-step’’ method. The Au NCs were prepared in different solution conditions, including water, acid and base (pH = 2 and 12), complete DMEM medium (containing 10% fetal bovine serum, 100 U of penicillin, and 100 lg/mL streptomycin), PBS and high salt concentration solution (2 M NaCl). The as-prepared samples were very stable, observed under white light (no precipitates were observed) and 254 nm ultraviolet light (the fluorescence color did not change in different solutions) (Fig. 3A). Since phenol red was present in DMEM, the photo of Au NCs under ultraviolet light is not shown in the figure. The size and morphology of the AuNCs in pure water or cell PBS almost did not change after incubation for more than 6 months (Fig. S3), suggesting that the AuNCs capped with HS–C11–EG6–X have superior stability. We have also investigated the fluorescence stability of the three kinds of Au NCs in different pH solutions (Fig. 3B–D). Although there are some deviations of fluorescence intensities in the ‘‘HS–C11–EG6–OH’’, ‘‘HS–C11–EG6–OCH2COOH:HS–C11–EG6–OH = 1:4’’ and ‘‘HS–C11–EG6–NH2:HS–C11–EG6–OH = 1:4’’ capped Au NCs, the fluorescence emissions of these Au NCs are relatively stable in the pH range of 2–12. The results may indicate that the minor species of HS–C11–EG6–OCH2COOH or HS–C11–EG6–NH2 that accounting for only 20 mol% of the total surface ligands do not influence the fluorescence stability of the Au NCs. We have also carried out zeta potential measurements for Au NCs capped with HS–C11–EG6–OH under various conditions. The zeta potential values of Au NCs in PBS (0.1 M, pH = 7.4), pH = 2, pH = 12 and DMEM cell medium are –4.9, +6.0, –23.3 and –10.1 mV, respectively. The large difference in zeta potential value indicates that the stability of the Au NCs is mainly due to the steric effect of the short-chain PEGs, not the surface charge properties.
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The similar UV–vis and fluorescence emission spectra of Au NCs in water and 2 M NaCl (Fig. 4) also demonstrate that the charge stabilization effect does not play a major role. We believe that the use of HS–C11–EG6–X capping in Au NC synthesis is a key development. Au NC precursors prepared using the first step of the two-step method without HS–C11–EG6–X capping can easily precipitate after standing for 2–5 days, or after stirring the formed Au NC precursors for several hours. Also, Au NC precursors will precipitate if we perform dialysis to remove the unbound histidine molecules. The instability of the Au NC precursors significantly limits their practical applications. As a result, we tried to cap such Au NCs with thiol-containing molecules like L-cysteine, mercaptosuccinic acid, lipoic acid, HS-PEG2K, HS-PEG5K and a short thiol-containing peptide CRGDK. When capped with L-cysteine, the Au NCs were easy to precipitate in water, which is due to the relatively poor water solubility of L-cysteine.
When coated with HS-PEG2K, HS-PEG5K, mercaptosuccinic acid, lipoic acid and CRGDK, the fluorescence intensities of Au NCs significantly decreased and some of these ligands even made the fluorescence diminish. The above experiments demonstrate the critical role the HS–Cm–EGn–X molecule plays toward the successful fabrication of the ultraclean and ultrastable fluorescent Au NCs. As we stated before, there are four parts of the HS–Cm–EGn–X molecule: the mercapto group (–SH), the methylene group (Cm), the short-chain polyethylene glycol (EGn) and the functional group (X) like OH, NH2, OCH2COOH and GRGD. All these components are important for preparing/stabilizing/using the Au NCs. The mercapto group is to bind to gold atoms through the Au-S covalent bond. The alkyl chain (such as (CH2)11) serves as a hydrophobic shield to stabilize nanoparticles and prevent other thiol-containing molecules to replace the bound thoil-ligands [62]. If the Au NCs were first coated with HS-PEG2K or HS-PEG5K, evidence has been reported that some small thiol-terminated molecules (such as cysteine) can replace the surface HS-PEGs, causing possible instability of the nanoclusters [62]. Therefore the methylene group is important. The hydrophilic EGn part is also important. It can increase water solubility of prepared Au NCs, and the introduction of EGn also endows the Au NCs with a superb capability to prevent non-specific adsorption of proteins [63–65], and can therefore ‘‘mask’’ the Au NCs from the host’s immune system (reduced immunogenicity and antigenicity). Finally, the functional group (X) is very critical to make Au NCs multifunctional. For example, when the functional group is an arginine–glycine–aspartic acid (RGD) peptide, the Au NCs can target to cancer cells whose RGD receptors (integrins) are overexpressed on the cell surfaces. Moreover, we can bind carboxyl-containing molecule (e.g., folic acid) or amine-containing molecule (e.g., doxorubicin) to the amine groups or carboxyl groups on the surface of the Au NCs using the EDC/NHS coupling reaction, respectively. This kind of coupling reaction can further extend the functionality of the Au NCs. The ultrasmall Au NCs with carried drugs can possibly pass through the blood brain barrier, enabling the efficient delivery of the drugs to the targeting positions in the brain. If we conjugate antibodies onto the Au NCs’ surfaces (with amine or carboxyl groups) through EDC/NHS coupling reaction, we can use these antibody-conjugated Au NCs to investigate important cellular events via antibody-antigen recognition. Furthermore, with the charged amine (NH+3) or carboxyl (COO–) functional groups on the terminal part of the EGs, we can tune the charge or charge ratio of the Au NCs, and these charged Au NCs can be used in various electrostatic interaction based supramolecular self-assembly systems (such as layer-by-layer assembly and polyion complex micelles) to fabricate functional biomaterials and biofilms for specific purposes. Finally, the synthesized Au NCs are ultraclean
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Fig. 3. (A) HS–C11–EG6–OH capped Au NCs (two-step) at different solution conditions: complete DMEM medium (with phenol red), water, pH = 2, pH = 12, PBS (pH = 7.4) and 2 M NaCl under white light (from left to right, the upper panel) and 254 nm UV (the lower panel) excitations. Dependence of fluorescence intensity on various pH values for (B) HS–C11–EG6–OH capped Au NCs (two-step), (C) ‘‘HS–C11–EG6–OCH2COOH:HS–C11–EG6–OH = 1:4’’ capped Au NCs and (D) ‘‘HS–C11–EG6–NH2:HS–C11–EG6–OH = 1:4’’ capped Au NCs. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
nanoclusters. However, if the density of the functional group is too low, then Au NCs may not have efficient targeting or sensing ability for various purposes. 3.3. Application of the stable Au NCs for anticancer drug carrier
Fig. 4. UV–vis absorption and fluorescence spectra of HS–C11–EG6–OH capped Au NCs in water and 2 M NaCl.
(that is, they can be very stable in pure water, with negligible free molecules and ions), making them excellent models for various physicochemical applications without the need to consider the side effects of free molecules or ions. An optimized surface density of the functional group X on Au NCs is also vital for the preparation of the highly fluorescent and highly stable Au NCs. If HS–C11–EG6–OH is completely changed to HS–C11–EG6–X (X = NH2, OCH2COOH), the obtained Au NCs capped with 100% HS–C11–EG6–NH2 or 100% HS–C11–EG6– OCH2COOH have a much lower fluorescence intensity as compared to those with 1:4 (mol/mol) of HS–C11–EG6–NH2 (or –OCH2COOH) and HS–C11–EG6–OH. The surface density of the above functional groups cannot be too high, since the charged ligands on the surface of Au NCs will repulse each other, causing instability of the
We have also evaluated the stability of the AuNCs after conjugation with drugs, and have investigated the potential ability of these Au NCs for drug delivery into the cells. To this end, the cytotoxicity of the ultraclean Au NCs needs to be investigated first. The MTT assay results indicate that the four products have negligible toxicity to A549 cells (Fig. 5), which indicates that these Au NCs can be safely used in live cells. Since the fluorescence excitation of the above prepared Au NCs resides at around 260 nm, it is difficult to use these Au NCs for cellular imaging. To see if the Au NCs can enter live cells and at the same time to prove that these Au NCs can be used to deliver anticancer drugs, DOX was conjugated to Au NCs capped with ‘‘HS–C11–EG6–OCH2COOH:HS–C11–EG6– OH = 1:2’’ using the EDC/sulfo-NHS coupling reaction. UV–vis absorbance spectra (Fig. S4), fluorescence spectra (Fig. S5) and DLS data (Fig. S6) all demonstrated the successful synthesis of Au NC-DOX complex. Au NCs have no absorbance for the wavelength region >400 nm, while both free DOX and Au NC-DOX complex have a maximum excitation peak at around 475 nm. Meanwhile, Au NC-DOX complex has a similar emission spectrum with free DOX. Besides, the DLS data reveal that the Au NC-DOX complex has a average hydrodynamic diameter of 10.7 nm, which endow the complex to be entered into cell nucleus since the nuclear pore allows that passage of nanoparticles smaller than 20 nm [66]. The DOX-conjugated Au NCs can be used as excellent model systems for drug delivery and cell imaging studies.
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Fig. 5. Cytotoxicity of Au NCs capped with (A) ‘‘HS–C11–EG6–OH two-step’’, (B) ‘‘HS–C11–EG6–OCH2COOH:HS–C11–EG6–OH = 1:4’’, (C) ‘‘HS–C11–EG6–NH2:HS–C11–EG6– OH = 1:4’’ and (D) ‘‘HS–C11–EG6–GRGD:HS–C11–EG6–OH = 1:4’’ on A549 cells determined by MTT assay (after incubation for 24 h).
Fig. 6. Cytotoxicity assays for free DOX (A and B) and Au NC-DOX (C and D). Shown in the left (A and C) are MTT assays after incubation with drugs for 48 h, while shown in the right (B and D) are real-time cell analysis results.
We will show the drug delivery application of Au NC-DOX first. To evaluate the cytotoxicities of Au NC-DOX and free DOX, MTT assay and real-time cell analysis experiments were carried out
(Fig. 6). MTT assay results (Fig. 6A and C) show that after adding 10 and 3 lg/mL Au NC-DOX or free DOX and incubating for 2 days, A549 cells were almost all dead. Addition of 1 lg/mL Au NC-DOX
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Fig. 7. Confocal images of A549 cells 1 h after incubation with free DOX (A–C) and Au NC-DOX (D–F).
Fig. 8. Confocal images of A549 cells after incubation with Au NC-DOX for (A) 1 h and (B) 3 h.
decreases the cell viability value to 50%, while addition of the same concentration of free DOX made almost all the cells dead. The real-time cell analysis results (Fig. 6B and D) show a similar trend at time = 65 h (equal to the 48 h drug incubation time if we plus the 17 h cell incubation time before drug addition). For these high concentration (1, 3 and 10 lg/mL) samples, the real-time cell analysis results show that the Au NC-DOX begins to have a significant cell killing effect at around 30 h, which is about 5 h later than the free DOX which begins to have a significant cell killing effect at around 25 h. Low concentrations (0.1 and 0.01 lg/mL) of Au NC-DOX and DOX have negligible cytotoxicity or even increase the proliferation of the cells, as shown by MTT and real-time cell analysis results. Since the free DOX and Au NC-DOX have a maximum excitation at round 475 nm, we can use confocal imaging technique to study whether the free DOX and Au NC-DOX can enter cells. After incubating A549 cells with 1 lg/mL DOX or Au NC-DOX containing 1 lg/mL DOX for 1 h, a number of red-emission dots can be seen in both groups (Fig. 7A and D), indicating that Au NC-DOX can enter cells as free DOX. However, only a few red fluorescent dots appear in the cell nucleus for Au NC-DOX (Fig. 7F), while a number of red fluorescent dots can be observed for free DOX (Fig. 7C). To demonstrate if Au NC-DOX can enter cell nucleus, A549 cells were incubated with Au NC-DOX for different time periods (1 h and 3 h).
Fig. 8 shows that compared with the case with the incubation time of 1 h, the confocal images taken after an incubation time of 3 h show plenty of red fluorescent dots in both the cytoplasm and nucleus. The above results demonstrated Au NC-DOX complex can enter into the cells and even the nucleus. After conjugation to the surface of Au NCs, the time needed for the drugs to enter into the nucleus was prolonged. The conjugation of DOX onto the surface of Au NCs has several advantages. First, the water solubility of DOX is greatly improved by conjugation of free DOX onto the terminal groups of the short-chain PEGs of Au NCs. The presence of surface PEGs on the Au NCs may prevent protein adsorption and prolong the drug circulation time in vivo. The improved water solubility and the increased drug circulation time make the Au NC-DOX complex be a safer anticancer drug formulation since it has been reported that free DOX molecules may cause severe systemic toxicity [67]. Second, the ultrasmall size of Au NCs endows the conjugated DOX with the ability to enter into the cell nucleus to have cell killing effect, since DOX mainly takes its anticancer effect in the cell nucleus. Besides, the ultrasmall size of the Au NC-DOX complex may have a deeper tumor tissue penetration depth [67]. Moreover, compared with large gold nanoparticles which could be trapped by the reticulo-edothelial system (RES) absorption and cause unavoidable accumulation in liver and spleen, the Au
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NCs with a much smaller size could be efficiently cleared by the kidney [68], making the smaller Au NCs be a better choice for drug delivery system. Third, due to the enhanced permeability and retention (EPR) effect, the nanocluster-conjugated DOX may have passive targeting ability in vivo. Our present work shows that the ultrastable water-soluble Au NCs can be used as an efficient anticancer drug delivery system. 4. Conclusions In summary, based on the thiol-containing short-chain PEGs (HS–C11–EG6–X), we synthesized water-soluble, multifunctional and monodisperse Au NCs using two methods. The two synthetic methods have their respective pros and cons. Although the quantum yield of the Au NCs prepared by method A is higher than that by method B, the preparation of Au NCs by method B is easier. The Au NCs prepared by both methods are ultrastable and have good monodispersity in aqueous media. It is found that the HS–C11– EG6–X capped Au NCs exhibit superior stability under various solution conditions for at least 6 months, even after conjugation with anticancer drug DOX. Besides, HS–C11–EG6–X can significantly increase the quantum yield of the synthesized Au NCs and the Au NCs capped with carboxyl groups have the highest quantum yield. Furthermore, the terminal functional group X can be any functional group, such as hydroxyl, amide, carboxyl and GRGD as demonstrated in this paper. Other functional groups like azido (–N3), biotin, nitrilotriacetic group and acylamino are also commercially available and can be readily used for various purposes. The present work demonstrates the importance of using thiol-terminated, alkyl-containing short-chain PEGs to fabricate and stabilize the Au NCs. Acknowledgments This work was supported by grants from the National Key Basic Research Program of China (973 Program) (No. 2013CB933904), the Natural Science Foundation of China (21303017), the Natural Science Foundation of Jiangsu Province (KB20130601), a project funded by the Priority Academic Program Development (PAPD) of Jiangsu Higher Education Institutions, and the Fundamental Research Funds for the Central Universities (2242015R30016). ZC thanks the University of Michigan to support his sabbatical. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jcis.2015.05.029. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14]
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