Toward a Novel Cellular Copper Detoxifying Drug for Wilson's Disease

Nov 12, 2013 - A layer-by-layer self-assembly method leads to the formation of Au@ZnMoS4 core–shell nanoparticles (NPs). The PEGylated Au@ZnMoS4 ...
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Cell-Permeable Au@ZnMoS4 Core−Shell Nanoparticles: Toward a Novel Cellular Copper Detoxifying Drug for Wilson’s Disease Vindya S. Perera, Haijian Liu, Zhi-Qiang Wang,* and Songping D. Huang* Department of Chemistry and Biochemistry, Kent State University, Kent, Ohio 44240, United States S Supporting Information *

ABSTRACT: A layer-by-layer self-assembly method leads to the formation of Au@ ZnMoS4 core−shell nanoparticles (NPs). The PEGylated Au@ZnMoS4 NPs are highly water-dispersible, exhibit no cytotoxicity, and can penetrate the cell membrane to selectively remove copper(I) ions from HepG2 cells in the presence of other endogenous and biologically essential metal ions, including Mg2+, Ca2+, Mn2+, and Fe2+, demonstrating their potential as a novel intracellular copper detoxifying agent.

KEYWORDS: core/shell nanoparticles, drug delivery, functional coatings, gold nanoparticles, biomedical applications, layer-by-layer self-assembly

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would never recover.12 Despite these undesirable and severe side effects, D-pen has remained the treatment of choice for WD because a safer and more effective drug has not been developed.13 Recently, an investigational oral drug, the ammonium salt of tetrathiomolybdate (TTM, i.e., (NH4)2MoS4) for WD has completed several clinical trials with promising results.14 TTM forms a nonbioabsorbable form of certain ternary complexes with copper and food proteins in the gastrointestinal (GI) tract to block the intestinal absorption of copper from the diet.15 Such a slow-acting mechanism renders improvement of treatment efficacy by TTM for WD incremental. On the other hand, TTM is known to be susceptible to hydrolysis that releases hydrogen sulfide (H2S) under the acidic conditions of the stomach.16,17Although a small amount of H2S is constantly produced in the human digestive tract from the anaerobic digestion of food and can be detoxified by several enzymes, this gas is considered more toxic than hydrogen cyanide (HCN) to the neural and circulating system.18 These facts suggest that the manufacture, storage, and clinical use of TTM will be a safety concern. In 1997, the U.S. Food and Drug Administration (FDA) approved the use of zinc acetate as a clinical drug for WD.19 Zn ions from this drug can stimulate the production of metallothionein in gut cells. This protein, in turn, can bind Cu ions and prevent their absorption and transport to the liver. However, zinc acetate is only effective as a maintenance therapy

INTRODUCTION Progressive hepatolenticular degeneration, which is also known as Wilson’s disease (WD), is a genetic disorder characterized by excess copper accumulation in the liver and other vital organs.1,2 There are wide clinically presenting symptoms of WD including a variety of hepatic, neurological, ophthalmic, and psychiatric symptoms.3,4 If untreated, WD can lead to severe disability, a need for liver transplantation, and death.5 The current treatment of WD is based on the use of copper chelating agents to block the adsorption of this ion in the stomach and to promote its urinary excretion from the body.6 Before chelation therapy was introduced by John Walshe in 1956, WD had been almost invariably progressive and fatal.7 Dpenicillamine or (2S)-2-amino-3-methyl-3-sulfanyl-butanoic acid (D-pen), which is a metabolite of penicillin, has been used as an orally active drug for WD in the clinic for over five decades.3,8 However, D-pen is an immunosuppressant that is also used to treat rheumatoid arthritis. Its use in WD causes a score of severe side effects, with symptoms ranging from bone marrow and immune suppression, to skin rash, and to deterioration of various neurological functions, to name but a few.3,8,9 For patients who exhibit an intolerance to D-pen, another oral drugtriethylenetetraamine (trientine)may be used, although this drug is less effective as a copper chelator.10 There is clear evidence to suggest that both D-pen and trientine can mobilize Cu ions stored in the body tissues and reroute them into circulation, thus increasing the concentrations of copper in the brain.11 It has been estimated that 50% of the WD patients treated with these drugs can suffer neurologic deterioration, and half of them or 25% of the original patients © 2013 American Chemical Society

Received: July 1, 2013 Revised: November 12, 2013 Published: November 12, 2013 4703

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Figure 1. Schematic of layer-by-layer self-assembly of Au@ZnMoS4 nanoparticles (NPs).

for WD.20 It should be noted that, as small molecules, none of the above drugs can penetrate cells to remove free Cu ions in the vital organs for WD patients, particularly those who have progressed to a stage characterized by a significant amount of hepatic copper deposition. In the latter case, the only option for the patient is liver transplantation.21 Clearly, there is an unmet clinical need for a safer and more effective treatment for WD, especially one that can function at the cellular level. Pujol and co-workers recently developed a strategy of using a cluster of carbohydrates conjugated to a Cu(I)-selective chelator to target the asialoglycoprotein receptor (ASGP-R) expressed on the surface of hepatocytes. They demonstrated that the cysteinebased peptide chelators delivered as such conjugates can effectively reduce the intracellular Cu(I) concentration.22,23 The work here is aimed to develop a novel drug delivery system that can penetrate the cell membrane to selectively remove excess free Cu ions from the cell. Gold nanoparticles (Au NPs) are chosen as the drug carrier for the investigation, because of its many desirable properties, such as the high surface-area-to-volume ratio, and multivalent surface architecture that enables the incorporation of multiple therapeutic agents and targeting molecules on the surface to improve the delivery efficiency of therapeutic payloads.24−28 In addition, the thiophilic properties of gold allows for a facile functionalization of the ligand molecule [MoS4]2− to the Au NP surface. We prepared the citrate-coated Au NP cores with an average size of 16 ± 1 nm and treated the NPs with the [MoS4]2− and Zn2+ ions alternately to form a ZnMoS4 shell, which was then PEGylated to impart hydrophilicity to the core−shell NPs. We examined the kinetics, capacity, and selectivity of ion-exchange using such NPs toward copper in aqueous solution. We also studied the cellular uptake, cytotoxicity, and intracellular copper removal of these NPs to demonstrate their potential as a novel cell-permeable copper detoxifying agent. To the best of our knowledge, this study is the first example to rely on the receptor-independent endocytosis to deliver nanoparticles to remove intracellular Cu ions via ion exchange rather than chelation. This work provides a new design paradigm for the development of the next-generation therapy for WD.

ions anchored on the Au surface via the thiophilic interaction of gold.32,33 Such an anchoring process was shown to produce robust attachment of the [MoS4]2− ions on the surface of Au NPs as the immediate dialysis against distilled water did not cause the loss of the [MoS4]2− anions from the Au NPs. While still remaining in the dialysis bag, the [MoS4]2−-plated Au NPs were then treated with an aqueous solution of zinc acetate, followed by another dialysis against distilled water. The coordination of the Zn2+ ions to the S atoms from the [MoS4]2− anions displaced the noncoordinating NH4+ counterions. This was confirmed by the detection of the latter from the solution outside the dialysis bag, thus completing the assembly of the first layer of ZnMoS4 on the Au NP surface.34,35 This process was repeated for the assembly of the subsequent layers of ZnMoS4. The maximum number of cycles we could carry out for the deposition without triggering the core−shell NPs to aggregate was 11. We noticed that, by now, the original citrate coating on the Au NPs was completely displaced. To increase the surface stability and impart water dispersibility, we coated the core−shell NPs with polyethylene glycol (PEG; MW = 8000) after 11 cycles of deposition were completed. We had noticed that the uncoated core−shell NPs would agglomerate and separate from solution if the aqueous dispersion of such NPs was left to stand at room temperature overnight. After PEGylation, the core−shell NPs were dialyzed using regenerated cellulose tubular membrane (MWCO is 12000−14000) against distilled water for two days. Figure 2 shows the TEM images of both the citrate-coated Au NPs and the Au@ZnMoS4 NPs formed from 11 cycles of layer-by-layer deposition followed by PEGylation. The particle size and size distribution for each batch of samples were obtained from measuring and averaging the size of 120 NPs. The average size of the Au@ ZnMoS4 NPs is 22 ± 3 nm, while the average size of the Au cores is 16 ± 1 nm. The TEM images also show that the shape is almost spherical for both the citrate-coated Au NPs and the PEGylated Au@ZnMoS4 NPs. Furthermore, the energydispersive X-ray spectroscopy (EDS) analysis on individual PEGylated Au@ZnMoS4 NPs clearly showed the presence of Zn, Mo, and S, in addition to Au, suggesting that the sample consists of surface-coating ZnMoS4 shell on the Au core rather than the presence of a mechanical mixture of Au and ZnMoS4 NPs (see Figure S1 in the Supporting Information). As shown in Figure 3, the UV−vis spectra of the sample aliquots taken from the reaction mixture after each cycle showed a red-shift of the localized surface plasmon resonance (LSPR) of Au NPs, indicating the altered surface characteristics of the Au NPs after the layer-by-layer assembly of the ZnMoS4 shell.36 This red-

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RESULTS AND DISCUSSION The ZnMoS4 shell was grown on the surface of Au NPs using a layer-by-layer self-assembly technique, as shown in Figure 1.29,30 The as-synthesized citrate-coated Au NPs in aqueous solution31 was sealed in a dialysis bag and immersed in a waterformamide (FM) solution of (NH4)2MoS4 (the ratio of H2O/ FM was 1/5 in volume) to form the first layer of the [MoS4]2− 4704

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we carried out all of ion-exchange studies of Au@ZnMoS4 NPs using Cu(I) ions in aqueous solution. Because Cu(I) disproportionates in aqueous solution to give Cu(II) and Cu(0), copper(I) chloride was dissolved in 80% acetonitrile solution to form a stable Cu(I)-acetonitrile complex.39 The kinetics of the on-exchange between ZnMoS4 NPs and Cu+ ions were determined by placing a dialysis bag containing NPs into an acetonitrile−water solution containing Cu(I) ions. The decrease of the copper concentration in this solution was monitored by elemental analysis using atomic adsorption spectrometry (AA). The results showed that the ion-exchange reaction followed a pseudo-first-order reaction up to the time point of ∼60 min with a rate constant of k1 = 3.0 × 10−4 s−1 or the half-life of t1/2 = 38 min (see Figure 5 and the Supporting

Figure 2. Transmission electron microscopy (TEM) images of asprepared Au NPs (upper left) and Au@ZnMoS4 NPs (lower left) and histograms of the size distribution for NPs corresponding to each panel on the left.

Figure 3. Ultraviolet−visible (UV-vis) spectra of pure Au NPs and the Au@ZnMoS4 NPs at the different stages of layer-by-layer assembling process. Figure 5. Kinetics of copper removal from the aqueous solution by Au@ZnMoS4 NPs.

shift in LSPR is accompanied with a color change of the solution from purple to burgundy, indicative of nanoparticles approaching each other closer than their mean particle diameter.37 The FT-IR spectrum of the Au@ZnMoS4 NPs is shown in Figure 4 in comparison to that of PEG. This IR spectrum exhibits bands attributable to PEG, clearly indicating the presence of a PEG coating on the surface of the Au@ ZnMoS4 assembly. Although Cu(II) is more stable than Cu(I) in aqueous solution where oxygen is present, in the biological system, Cu(II) is first reduced to Cu(I) by various metalloreductases and then transported by the copper transport protein (Ctr1) into cells where this oxidation state is maintained.38 Therefore,

Information for the detailed kinetic data analysis). After this time point, the ion exchange suddenly switched to a slower and second-order reaction with a rate constant of k2 = 1.5 × 10−1 M−1 s−1, indicating the need for Cu+ ions now to penetrate into the inner layers of ZnMoS4 to react with Zn2+ ions after the surface Zn2+ ions are consumed. Overall, these rate measurements suggested that such NPs are kinetically suitable for depleting intracellular Cu ions (vide infra). The ability of a substance to remove metal ions from a solution is often expressed in terms of metal removal capacity. This parameter specifies the maximum amount of metal in milligrams that can be removed by one gram of the given substance. Metal removal capacity (q, mg/g) can be determined using the mass balance equation, q=

Ci − C V /W

where Ci is the initial metal concentration (mg/L), C (mg/L) the metal concentration in solution after time t (min), V the volume of metal solution (L), and W is the weight of NPs (g). To evaluate the metal removal capacity, we performed the metal removal experiments for our NPs using a batch reaction method. The copper removal capacity was found to be 107 mg/

Figure 4. Fourier transform infrared (FT-IR) spectra of PEG, PEGcoated Au@ZnMoS4 NPs, and Au@ZnMoS4 NPs without PEG. 4705

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metabolism, toxicity, and drug targeting in human hepatocytes.42 For live cell imaging, cell cultures were incubated with the dye-labeled Au@ZnMoS4 NPs, washed with PBS, and then directly imaged using a laser scanning confocal microscope without fixation. The images of confocal microscopy showed the presence of bright green fluorescent signals inside the cells that were incubated with the dye-labeled Au@ZnMoS4 NPs for 3 h, while the untreated HepG2 cells were used as the negative control. Figure 8 shows the typical confocal fluorescent images

g, as shown in Figure 6. In a comparison study, we showed that bulk ZnMoS4 reacting with excess CuCl gave Cu2MoS4 and

Figure 6. Copper removal capacity of Au@ZnMoS4 NPs.

ZnCl2 by ion exchange. The structure of Cu2MoS4 was previously determined by the powder X-ray data to be a layered compound.40,41 It is tempting to conjecture that the ion exchange between Cu+ ions and the surface-bound Cu2MoS4 layers proceeds via a similar mechanism. To quantitatively evaluate the selectivity of the Cu(I) removal by Au@ZnMoS4 NPs in the presence of other biologically relevant divalent metal ions, including Mg2+, Ca2+, Fe2+, and Mn2+, we studied the ion-exchange competition among all of these ions. In the typical experiment, 5-mL NPs (10 mM) were sealed in a dialysis bag and placed in a solution (20 mL) containing the Cu+, Mg2+, Ca2+, Fe2+, and Mn2+ ions, each at the 100-ppm level. The solution concentration for each of the above ions was analyzed after 24 h of incubation at 22 °C. The selectivity for each ion was normalized against the removal of Cu+ ions being set as 100%, and expressed as the percent removal for each ion. Figure 7 clearly shows that Au@

Figure 8. Confocal microscopic images of HepG2 cells: (upper left) fluorescence image of cells incubated with dye-conjugated NPs for 3 h; (upper right) bright-field image of cells incubated with dye-conjugated NPs for 3 h; (lower left) fluorescence image of the untreated cells; and (lower right) bright-field image of the untreated cells.

of HepG2 cells treated with the dye-labeled Au@ZnMoS4 NPs and the control cells. The uniform fluorescent emission in the perinuclear region of the cell indicates an untargeted cytoplasmic distribution of NPs without specific binding to any small organelle in the region, which is consistent with cellular uptake via endocytosis. It should be noted that the nuclear uptake of NPs is negligible, as can be seen from the much weaker fluorescent signals inside the nuclei. To assess the cytotoxicity, we performed cell viability assays in HepG2 cells using the MTT method. The cells were incubated for 12 or 24 h at 37 °C under 5% CO2 with varying concentrations of Au@ZnMoS4 NPs suspended in PBS and Dulbecco’s Modified Eagle Medium (DMEM) media. Three independent trials were conducted, and the averages and standard deviations were reported. The reported percent cell survival values are relative to the control cells. Figure 9 shows the viability of HepG2 cells treated with Au@ZnMoS4 NPs. The results clearly indicate that the NPs are nontoxic to cells. More than 92% of the cells were viable even after incubation of NPs with concentration of 100 μM for 12 h. More than 89% of the cells were viable after incubating with 100 μM nanoparticle solution for 24 h. To determine whether the selectivity toward the Cu+ ion by Au@ZnMoS4 NPs is retained in living cells, we studied the in vitro ion-exchange competition in cells that were first incubated with Cu2+, Mg2+, Ca2+, Fe2+, and Mn2+ ions, each at the 100-μM level for 14 h. The use of Cu2+ rather than Cu+ in this study was

Figure 7. Selectivity of several divalent metal ions by Au@ZnMoS4 NPs in aqueous solution.

ZnMoS4 NPs are most selective toward the Cu+ ion in the presence of all the other divalent metal ions tested. The observed selectivity toward the Cu+ ion is also consistent with the expected outcomes based on the HSAB principle. Copper(I) is a soft Lewis acid and should strongly bind to a soft Lewis base, i.e., the S atoms from the [MoS4]2− ligand in this case. We studied cellular uptake of Au@ZnMoS4 NPs in HepG2 cells using the fluorescent confocal microscopic imaging technique. Because of their morphological and functional differentiation, HepG2 cells are often employed as a model for the study of intracellular trafficking and dynamics, liver 4706

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Figure 9. Effect of Au@ZnMoS4 NPs on viability of HepG2 cells after 12 h and 24 h of incubation.

Figure 11. Kinetics of copper uptake in HepG2 cells.

justified by the fact that prior to its transport into cells, Cu2+ is inevitably reduced to Cu+ by metalloreductases and maintained in cells at this oxidation state.38 After the above incubating treatment, cells were found to be alive and viable with the concentrations of the metal ions elevated to this level. The cells were then washed with PBS to remove uninternalized metal ions and incubated with a serum-free fresh medium containing 0.2 mg/mL Au@ZnMoS4 NPs for another 3 h. The cell lysates were analyzed by atomic absorption (AA), and the analysis results were checked for accuracy by inductively coupled plasma−atomic emission spectroscopy (ICP-AES; see the Supporting Information for details). As shown in Figure 10,

The concentration of copper in each cell lysate was determined using the AA technique. As the control experiment, HepG2 cells with elevated copper concentrations were incubated with fresh culture medium, but not treated with Au@ZnMoS4 NPs. The concentrations of copper in these cell lysates were determined side-by-side with those treated with Au@ZnMoS4 NPs. The results were normalized by the number of cells in order to determine the intracellular amount of copper per cell. As shown in Figure 12, the cells treated with Au@ZnMoS4 NPs showed a substantial decrease in the cellular copper level.

Figure 12. Kinetics of copper removal from HepG2 cells.

Specifically, the intracellular concentration of copper dropped from the highly elevated level of 1.05 ± 4 pg/cell to 0.58 ± 5 pg/cell after 1 h of incubation with Au@ZnMoS4 NPs, and to 0.35 ± 6 pg/cell after 6 h of incubation with NPs. The latter is essentially at the same level of endogenous copper concentrations (i.e., 0.21 ± 6 pg/cell) in HepG2 cells if the standard deviations of metal analysis are taken into consideration. We noticed that the cells remained alive and viable during the entire duration of the copper detoxifying studies (i.e., 6 h). In comparison, the cells incubated with the regular medium (i.e., the control cells) had a small efflux of copper ions from 1.05 ± 4 pg/cell to 0.79 ± 4 pg/cell within the first two hours, and then the copper concentration remained steady at this level. Therefore, it is tempting to conjecture that the ion-exchange led to the formation of Au@Cu2MoS4 NPs and that the faster and continuous decrease of copper concentrations in these cells might have occurred via exocytosis of such NPs.

Figure 10. In vitro selectivity of several divalent metal ions by Au@ ZnMoS4 NPs.

the in vitro selectivity of Au@ZnMoS4 NPs toward the Cu+ ion is essentially unchanged when compared to that found in aqueous solution when the standard deviations of metal analysis are considered (see Figure 7). The elevated copper concentrations inside the HepG2 cells were artificially induced by incubating cells with the medium supplemented with a Cu(II) salt (i.e., CuSO4), because Cu(II) is more stable in the aqueous culture medium, but can be reduced to Cu(I) upon being taken up by cells.38 The intracellular copper concentrations were determined from the cell lysates as a function of incubation time using AA. As can be seen from the curve in Figure 11, it took ∼12 h of incubation for the cells to become saturated with Cu ions, while the cells remained healthy and thriving. In order to evaluate the Au@ ZnMoS4 NPs as a potential copper detoxifying agent, we incubated the HepG2 cells containing elevated copper concentrations with Au@ZnMoS4 NPs. After 4 h of incubation, cells were washed twice with PBS and further incubated with fresh culture medium. Cells in each flask were then washed and lysed with concentrated nitric acid at different time intervals.

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CONCLUSION We have developed a stable and biocompatible nanoplatform for delivering ZnMoS4 as an intracellular copper detoxifying agent. Currently, the treatment of choice for Wilson’s Disease (WD) relies on the use of D-pen, which is a slow-acting oral chelating agent with numerous adverse side effects. This clinical 4707

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Nanoparticle-Surface Conjugation of Fluorescence Dye Molecules. To prepare dye-labeled Au@ZnMoS4 nanoparticles, 45 μL of 0.25 mM ethylenediamine solution was added to a 200-μL Au@ ZnMoS4 nanoparticle solution (250 μM) under vigorous stirring. The resulting reaction mixture was continuously stirred for 24 h. The product was purified by dialysis to remove unbound ethylene diamine molecules. Next, 10 mL of carboxyfluorescene dye (0.5 mM) was allowed to react with 1.2 equiv of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) (1.15 mg) for 24 h in a separate container. Finally, ethylene diamine coated Au@ZnMoS4 nanoparticle solution was added to 100 μL of the above-mentioned dye solution and stirred for another 24 h. To remove the unconjugated dye molecules, the resulting product was dialyzed against distilled water for two days and analyzed by fluorescence spectroscopy. Cell Viability Assays. Cytotoxicity studies were performed using an MTT viability assay. HepG2 cells were seeded in a 96-well plate at a density of 2 × 104 cells per well with the DMEM low-glucose medium and incubated for 5 h at 37 °C in an atmosphere of 5% CO2 and 95% air to allow cells to attach to the surface. Cells in each well were then treated with 100 μL of fresh medium containing varying concentrations of the nanoparticles and then incubated for 12 or 24 h. Control wells contained the same medium without nanoparticles. The cells were incubated in media containing 0.1 mg/mL of 3-[4,5dimethylthialzol-2-yl]-2,5-diphenyltetrazolium bromide (i.e., the MTT dye) for 3 h. After the MTT solution was removed, the precipitated violet crystals were dissolved in 200 μL of DMSO. The absorbance was measured at 560 and 630 nm using a microplate reader. The assay results were presented as percent viable cells. In Vitro Selectivity Studies. Cells grown to 80% confluence in DMEM low-glucose media were first incubated with a solution containing Cu2+, Fe2+, Mn2+, Mg2+, and Ca2+ ions (each at 100 μM) to elevate the cellular concentration of these metal ions. After 14 h of incubation, cells were washed with PBS to remove uninternalized ions and incubated with serum-free fresh medium containing 0.2 mg/mL nanoparticles for 3 h. The culture medium was then replaced by fresh complete medium and incubated for another 3 h to allow the exocytosis of NPs to occur. Treated and untreated control cells were then rinsed once with media and twice with PBS. The cells were then trypsinized and centrifuged. The cell pellet was washed once more in PBS. The cells were resuspended in 3 mL of PBS and were counted using a hemocytometer. The cells were then centrifuged and digested using 0.5 mL concentrated nitric acid in order to destroy all organics. The residual matrix was diluted with deionized water to 2 mL and analyzed by atomic absorption (AA) spectrometry. The results of the metal concentrations by AA were checked by the use of ICP-AES to confirm that metal analysis results given by the two different methods are consistent with each other (see the Supporting Information for the validation of our analytical procedures for cellular copper, iron, manganese, magnesium, and calcium concentrations based on AA and ICP-AES). Cellular Uptake of Au@ZnMoS4 Nanoparticles. Confocal fluorescence microscopy technique was used to study the cellular uptake of nanoparticles. First, 1.2 × 105 HepG2 cells per well were seeded in an 8-well chamber slide and incubated for 24 h. The culture medium was then replaced with a medium containing dye-labeled nanoparticles at the concentration of 150 μM. After 3 h of incubation, cells were washed three times with PBS to remove free nanoparticles. Following this, the fresh medium was added to the cells before imaging. Cellular Copper Detoxification by Au@ZnMoS4 Nanoparticles. First, the elevated copper level in HepG2 cells was induced by incubating the cells with DMEM medium supplemented with 400 μM of copper(II) sulfate solution. Copper uptake kinetics by HepG2 cells were determined for different time intervals by measuring the copper levels of the cells that were washed three times with PBS, resuspended in serum-free medium, and counted using a hemocytometer. When the copper concentration became saturated after 12 h of incubation, cells were washed three times with PBS and then incubated with the culture medium containing nanoparticles (200 μg/ mL) for 1, 2, 4, and 6 h, respectively, at 37 °C. The cells were washed

drug also lacks the ability to cross the cell membrane in order to remove excess Cu ions deposited in the cells. Our studies have shown that Au@ZnMoS4 NPs are readily internalized by cells via endocytosis and can selectively remove Cu ions in the presence of other biologically essential ions from the cell. This novel approach may provide a real possibility of developing a safer and more effective treatment to reverse the progression in the late onset of WD, which is often characterized by liver cirrhosis, psychosis, and organ failure. Research is now underway in our laboratory to test the safety and efficacy of this novel drug in small animal models with elevated copper levels in the bloodstream and the other vital organs.

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MATERIALS AND METHODS

Materials. Human hepatocellular carcinoma cell line (HepG2) was obtained from the American Type Culture Collection (Rockville, MD, USA) and cryopreserved at −200 °C prior to use. Dulbecco’s Modified Eagle’s Medium (DMEM, M0643, Sigma−Aldrich, USA) supplemented with 10% fetal bovine serum, 2.2 g/L NaHCO3, 1 mM sodium pyruvate, 100 U/mL penicillin, 100 μg/mL streptomycin, 0.25 μg/mL amphotericin, and 1% penicillin-streptomycin was used at 37 °C in an atmosphere of 5% CO2 for culture and other cellular experiments with HepG2 cells. Synthesis of Au@ZnMoS4 Nanoparticles. The citrate-coated Au NP cores were synthesized using the modified Turkevich method. Specifically, an aqueous solution of HAuCl4 (0.25 mM, 50 mL) was first heated to boiling point with rigorous stirring, to which an aliquot of sodium citrate solution (1%, 0.40−1.75 mL) was added. In less than a minute, the solution turned from pale yellow to wine red, indicating the formation of Au NPs. After boiling and stirring for another 30 min, the resulting solution was cooled to room temperature. The assynthesized Au NP solution was first placed in a dialysis bag soaked in a solution of 0.025% (wt %) (NH4)2MoS4 for 15 min, followed by dialyzing against distilled water three times. The dialysis bag was then transferred to an aqueous solution of 0.025% (wt %) Zn(O2CCH3)2 for 30 min, followed by dialyzing against distilled water three times. This process was repeated 11 times. During each step, the unbound Mo42− or Zn2+ ions were removed by dialysis in distilled water. Finally, the NP solution was coated with polyethylene glycol (PEG, MW = 8000) followed by dialysis using a regenerated cellulose tubular membrane (MWCO = 12000−14000) against distilled water for two days. The solid product was collected by lyophilization. TEM Imaging and EDX Measurements. The samples were first suspended in water, and then placed as the droplet onto a carboncoated copper TEM grid (400-mesh). Specimens were allowed to airdry and analyzed at 200 KV using a FEI Tecnai F20 transmission electron microscope (TEM) equipped with a field-emission gun. The energy-dispersive X-ray spectroscopy (EDX) results were obtained with the integrated scanning TEM (STEM) unit and attached EDAX spectrometer (see Figure S1 in the Supporting Information). The spatial resolution is