Communication pubs.acs.org/IC
Selective Ion Exchange Governed by the Irving−Williams Series in K2Zn3[Fe(CN)6]2 Nanoparticles: Toward a Designer Prodrug for Wilson’s Disease Murthi S. Kandanapitiye, Fan Jennifer Wang, Benjamin Valley, Chamila Gunathilake, Mietek Jaroniec, and Songping D. Huang* Department of Chemistry and Biochemistry, Kent State University, Kent, Ohio 44240, United States S Supporting Information *
Among the numerous side effects, the most detrimental and deplorable one is the irreversible neurological damage caused by the mobilization of copper ions stored in the body tissues and rerouting them into circulation by D-PEN, causing the concentrations of copper in the brain to increase.13 Subsequently, two new oral drugs, i.e., triethylenetetramine (Trientine)13,14 and zinc acetate (Galzin),15 were introduced to the clinical treatment of WD. Compared to D-PEN, Trientine is a less effective copper chelator but has proven to be beneficial to the patients who show intolerance to D-PEN. Although not a chelating agent, zinc ions in Galzin act as an antagonist to copper by stimulating the production of metallothionein in cells.15,16 An investigational drug, tetrathiomolybdate (TTM), has recently emerged as a potential alternative oral drug for WD.17,18 Despite its many adverse side effects, D-PEN remains the treatment of choice for WD because of its proven efficacy. It seems clear that there is an unmet clinical need for a novel WD drug with improved organ specificity and/or reduced systemic toxicity.19 Our approach to tackling these problems focuses on the development of cell-permeable copper-depleting nanoparticles (NPs) that can be surface-engineered to be potentially organspecific when targeting agents are used to form new-generation drugs for WD.20,21 In this Communication, we report on the synthesis, characterization, and intracellular copper detoxification of NPs formed from a zinc analogue of Prussian Blue, K2Zn3[Fe(CN)6]2 (thereafter abbreviated as ZnPB). NPs of Prussian Blue and its many analogues have recently attracted a lot of attention.22−29 As a novel strategy for ensuring exclusive selectivity of these NPs toward copper, we have explored a predetermined ion-exchange reaction rather than relying on the normal metal-chelation process. Specifically, we notice that the relative stability exhibited by homologous divalent 3d metal complexes would follow the trend Cr2+ < Mn2+ < Fe2+ < Co2+ < Ni2+ < Cu2+ > Zn2+, regardless of the nature of the ligand. The properties governing the stability of these homologous complexes are atomic radii and ligand-field stabilization energy.30,31 To the best of our knowledge, this is the first example showing that NPs of Prussian Blue analogues can be tailor-made as a designer intracellular copper-detoxifying prodrug. We prepared poly(vinylpyrrolidone) (PVP)-coated ZnPB NPs by a simple one-step procedure in aqueous solution (see the SI for details). Powder X-ray diffraction (XRD) studies showed that the
ABSTRACT: The principle of the Irving−Williams series is applied to the design of a novel prodrug based on K2Zn3[Fe(CN)6]2 nanoparticles (ZnPB NPs) for Wilson’s disease (WD), a rare but fatal genetic disorder characterized by the accumulation of excess copper in the liver and other vital organs. The predetermined ionexchange reaction rather than chelation between ZnPB NPs and copper ions leads to high selectivity of such NPs for copper in the presence of the other endogenous metal ions. Furthermore, ZnPB NPs are highly water-dispersible and noncytotoxic and can be readily internalized by cells to target intracellular copper ions for selective copper detoxification, suggesting their potential application as a new-generation treatment for WD.
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ellular copper plays a key role as an essential cofactor in numerous enzymes and proteins because of its ability to switch between the 1+ and 2+ oxidation states.1−3 Although an essential element, copper in the form of free ions is a potent cytotoxin.4,5 When accumulated inside the cell at a level higher than that required by cellular needs, free copper ions can catalyze the production of reactive oxygen species (ROS) including hydroxyl radicals.2 The latter are known to cause deleterious cellular damage such as lipid peroxidation in membranes, oxidation of proteins, and cleavage of DNA and RNA molecules.6 In the case of Wilson’s disease (WD), accumulation of excess copper in the liver and other vital organs damages these organs because of excessive ROS, which renders the patient severely disabled with a variety of hepatic, neurological, ophthalmic, and psychiatric symptoms. If untreated, the disease can rapidly progress to liver cirrhosis and then a need for liver transplantation or death.7,8 Before chelation therapy using D-penicillamine (D-PEN) was introduced in 1956, WD had always been progressive and fatal.9 DPEN or (2S)-2-amino-3-methyl-3-sulfanylbutanoic acid is a cysteine-like chelating agent originally discovered as a metabolite of penicillin. It contains oxygen, nitrogen, and sulfur donors and exhibits a preferential binding but not sufficient selectivity toward either the CuI or CuII ion.7,10,11 Furthermore, D-PEN is an oral drug with systemic toxicity but no organ specificity. Therefore, its use causes severe side effects including bone marrow and immune suppression, deterioration of various neurological functions, and drug-induced systemic lupus erythematosus, to name a few.12 © XXXX American Chemical Society
Received: December 10, 2014
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DOI: 10.1021/ic502957d Inorg. Chem. XXXX, XXX, XXX−XXX
Inorganic Chemistry
Communication
XRD patterns of the bulk materials and NPs match those calculated from the previously reported X-ray crystallographic data for K2Zn3[FeII(CN)6]2, confirming the phase identity and purity of both materials prepared in this work (see Figure S1 in the Supporting Information, SI). This compound crystallizes in the rhombohedral space group R3̅c with a three-dimensional framework structure consisting of octahedral [Fe(CN)6]4− units linked to tetrahedral Zn2+ ions to create large hexagonalbipyramidal cages encapsulating K+ ions and water molecules.32 Transmission electron microscopy (TEM) imaging revealed that the NPs have near-spherical shape with an average diameter of 44 ± 8 nm, as determined by counting and averaging the size of 51 particles in this TEM picture frame (see Figure 1). Furthermore,
Figure 2. Kinetics of ion exchange between the ZnPB NPs and the CuII ion in aqueous solution.
details). It should be noted that 1+ is the predominate oxidation state for intracellular copper ions.1 We therefore examined the ion exchange between the Cu+ ion and ZnPB NPs in aqueous solution in the presence of air and found that such ion exchange would still take place with the concomitant oxidation of the Cu+ ion to the Cu2+ ion in the reaction product (see Figure S9 in the SI for details). On the other hand, the copper removal capacity, a parameter that specifies the maximum amount of copper in milligrams that can be removed from aqueous solution by 1 g of ZnPB NPs, was measured to be 88 mg g−1 using a batch-reaction method (see the SI for details). The selectivity of the metal removal was evaluated through a competition study involving the ZnPB NPs equilibrating with the Cu2+, Fe2+, Mn2+, Mg2+, and Ca2+ ions in a single aqueous solution with each ion at 100 ppm concentration over a time period of 24 h. The results were normalized against the percent removal of the Cu2+ ion from the solution. In a separate batch reaction, the ZnPB NPs were brought in contact with the Zn2+ ions at the same concentration for 24 h and showed no ion exchange. As predicted by the Irving− Williams series, the ZnPB NPs showed a high selectivity toward Cu2+ ions over all of the divalent ions included in the competition study, with the Ca2+ ion being an exception (see Figure 3).
Figure 1. TEM image of the as-synthesized ZnPB NPs (left) and a histogram of the size distribution (right).
the energy-dispersive X-ray spectroscopy (EDX) measurements showed the characteristic signals of zinc, iron, and potassium from several individual NPs randomly selected from the TEM grid (see Figure S2 in the SI), while the results from the metal and the C, H, and N elemental analysis showed an empirical formula of K2Zn3[FeII(CN)6]2·3.8H2O (see the SI for details) for the bulk material. (see the SI for details). The presence of zeolitic water, ranging from 4 to 12 per formula, was further confirmed by thermogravimetric analysis (TGA) of both the bulk and NP samples. The TGA data also showed that the average surface loading of PVP is 31 wt % (see Figure S3 in the SI). The Fourier transform infrared (FTIR) spectra of the PVP-coated ZnPB NPs are fully consistent with this structural assignment (see Figure S4 in the SI). The kinetics of copper removal was followed by measuring the concentration change of both Cu2+ and Zn2+ ions in the aqueous solution inside which a dialysis bag containing the PVP-coated ZnPB NPs was immersed. The simultaneous decrease in the copper concentration coincided with an increase in the zinc concentration in solution, as determined quantitatively by AA, signifying an ion-exchange reaction between the ZnPB NPs and Cu2+ ions to give also NPs of both copper and zinc (vide infrra). The kinetics data can be fitted to two separate rate laws, i.e., a pseudo-first-order reaction up to the time point of ∼7 h with a rate constant of k1 = 5.7 × 10−5 s−1 and a half-life of t1/2 = 202 min and a second-order reaction with a rate constant of k2 = 4.02 × 10−2 M−1 s−1 (see Figures 2 and S5 and S6 in the SI for details). When we examined the NPs isolated at this point and studied by TEM, EDX, and metal elemental analysis, we found that the NPs now consisted of a solid K2CuxZn3−x[FeII(CN)6]2 solution. The x value ranges from 0.07 to 0.11 depending on the extent of ion exchange (see Figure S7 in the SI). The solubility product constants for K2Zn3[Fe(CN)6]2 and K2Cu3[Fe(CN)6]2, as measured by a static method, are 1.1 ± 2 × 10−38 and 3.8 ± 1 × 10−46 mol7 dm−21, respectively, indicating that such an ionexchange reaction is driven by the difference in the thermodynamic stability of the two analogous compounds (see the SI for
Figure 3. Selectivity of metal-ion removal by the ZnPB NPs from aqueous solution.
Subsequently, we showed that such unwanted ion exchange can be suppressed by pretreating our ZnPB NPs with a solution containing CaCl2 (see the SI for details). Additionally, we demonstrated that the ZnPB NPs can effectively compete against D-PEN for copper ions from aqueous solution (see Figure S11 in the SI). Our studies of cytotoxicity of PVP-coated ZnPVP NPs using an MTT viability assay clearly showed that such NPs exhibit no significant cytotoxicity (see Figure S12 in the SI). We also determined the release of free cyanide ions from the NPs into solutions by a fluorometric method using the Konig reaction33 and found that the cyanide concentration detected after 24 h of incubation with ZnPB NPs was below ∼0.2 ± 2 ppm at pH = 7.0 (see the SI for details). This concentration of CN− ions is at the same level allowed in drinking water as that stipulated by the U.S. Environmental Protection Agency (i.e., 0.2 ppm).34 B
DOI: 10.1021/ic502957d Inorg. Chem. XXXX, XXX, XXX−XXX
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We observed that PVP-coated ZnPB NPs can be readily internalized by HeLa cells. The cellular fluorescent signals showed an even and noncompartmentalized cytoplasmic distribution of the dye-labeled ZnPB NPs in the peripheral region of the nucleus, as shown in Figure 4, suggesting that cellular
uptake of such NPs is most likely through endocytosis. We also confirmed that the internalized ZnPB NPs can remove Cu2+ ions that were previously introduced into HeLa cells. In essence, after incubation with NPs, the copper concentration in the cells decreased from the saturated level of 1093 ± 54 fg cell−1 to 907 ± 45 fg cell−1 in 2 h to 533 ± 45 fg cell−1 in 4 h and to 369 ± 18 fg cell−1 in 8 h. The latter is comparable to the copper concentration of 262 ± 18 fg cell−1 in the control cells (see Figure 5).
Figure 5. Kinetics of copper removal from HeLa cells.
In summary, WD is essentially a liver disease caused by copper accumulation. The ability to target liver and other vital organs using NPs such as the one demonstrated in this work may prove to be clinically more desirable than the current systemic delivery of drug for treating WD, which causes too many adverse side effects.
ASSOCIATED CONTENT
* Supporting Information S
Experimental details including the synthesis and characterization of ZnPB NPs. This material is available free of charge via the Internet at http://pubs.acs.org.
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Figure 4. Confocal microscopic image of HeLa cells treated with the dyelabeled ZnPB NPs.
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Communication
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank the NIH-NCI for financial support (Grant 1R21CA143408-01A1). C
DOI: 10.1021/ic502957d Inorg. Chem. XXXX, XXX, XXX−XXX
