1. Introduction
Zinc ion (Zn2+) is known to be a novel intracellular second messenger related to various biological functions [1] . As well, Zn2+ is involved in the structure of all enzyme classes, a micro nutrient, and necessary for growth and development [2] . Zinc transporter proteins, divided into two major families, ZIPs to increase and ZnTs to decrease Zn2+ concentration in the cytosol, are responsible for keeping zinc at certain concentrations [3] . Intracellular free Zn2+ is considered to be in the nM to pM range because many zinc metalloproteins have metal binding affinities in that range [4] [5] [6] . The imidazole group of histidine in a zinc metalloprotein is recognized as an excellent ligand for the tetrahedral coordination of the Zn2+ transition metal ion [7] [8] . The carboxyl group of aspartic or glutamic acid also coordinates Zn2+ in the protein [9] . These studies have inspired us to control the intracellular free Zn2+ concentration, without zinc transporter, by using delivery carrier containing imidazole and carboxyl groups for exploring unknown biological functions.
We have previously reported the design of carboxymethyl poly(1-vinylimidazole) (CM-PVIm) for biocompatibility [10] . The CM-PVIm is a net nonionic polyampholyte under physiological pH conditions, because the CM-PVIm possesses both imidazole and carboxyl groups, exhibiting the suppression of the nonspecific interaction with serum proteins. The imidazole groups are considered to possess a buffering capacity in the endosome, resulting in the endosomal escape by endosome membrane destabilization after protonation of imidazole groups for efficient delivery to cytoplasm [11] [12] . Furthermore, we have also reported Zn2+-chelated PVIm derivatives, not polyampholyte but polycation, for efficient gene delivery [13] [14] [15] [16] .
In this study, we have focused on the preparation of Zn2+-chelated CM-PVIm for intracellular Zn2+ delivery. The resulting intracellular Zn2+ delivery is expected to control various biological functions.
2. Materials and Methods
2.1. Materials
Zinc chloride (ZnCl2) and zinc acetate (Zn(OAc)2) were purchased from Kanto Chemical Co., Inc. (Tokyo, Japan). All other chemicals were of a special grade and were used without further purification.
2.2. Preparation of Zn2+-Chelated CM-PVIm
As CM-PVIm, 18 mol% carboxymethyl PVIm were synthesized according to our previous paper [10] . 1-Vinylimidazole (VIm) (500 mg) and 2,2’-Azobis (isobutyronitrile) (AIBN) as an initiator were dissolved in 8 mL N,N-dimethylformamide (DMF). Radical polymerization reaction was carried out for 120 h at 60˚C. After the reaction, the content was poured into a large excess of diethylether, and the precipitate was dried. The resulting polymer (PVIm) and iodoacetic acid (180 mg) were dissolved in 8.5 mL of DMF. The reaction mixture was incubated at 40˚C for 24 h, followed by dialysis against distilled water using a Spectra/Por 7 membrane (molecular weight cutoff = 103) to remove unreacted iodoacetic acid. The resulting polymer (CM-PVIm) was obtained by freeze-drying.
Each resulting CM-PVIm was mixed with ZnCl2 or Zn(OAc)2 (Zn2+/imidazole = 0.5) and incubated at room temperature for 24 h, followed by dialysis against distilled water using a Spectra/Por 7 membrane (103 molecular weight cut-off) to remove free Zn2+. The control sample of ZnCl2 or Zn(OAc)2 in the absence of CM-PVIm was also dialyzed under the same conditions.
2.3. Atomic Absorption Spectrometry
Under acidic conditions, which were achieved by the addition of 1 M HCl to the resulting dialysis fluid, the Zn2+ content in the CM-PVIm was determined by atomic absorption spectrometry at 213.9 nm using an atomic absorption spectrophotometer AA-6200 (Shimadzu Co., Kyoto, Japan).
2.4. Size and ζ-Potential Measurement
The size and ζ-potential of the resulting Zn2+/CM-PVIm complexes were measured in the resulting dialysis fluid. The size of each sample was measured by a dynamic light scattering (DLS) method using an electrophoresis light scattering spectrophotometer (ELS-Z2, Otsuka Electronics Co., Ltd., Tokyo, Japan) and the zeta potential was measured by ELS with electrodes.
2.5. Cell Viability Assay
HepG2 cells (from Riken Bioresource Center Cell Bank), human hepatoblastoma cell line, were cultured in tissue culture flasks containing Dulbecco’s modified Eagle’s medium supplemented with 10% heat-inactivated of fetal bovine serum (FBS). The cells were seeded at 1 × 104 cells/well (100 μL/well) in a 96-well plate and incubated at 37˚C in a 5% CO2 incubator, overnight. After the addition of 20 μL of the sample containing the Zn2+/CM-PVIm complexes or the control sample containing ZnCl2 or Zn(OAc)2 alone, as well as CM-PVIm alone, the cells were incubated at 37˚C for 24 h. After washing the medium with PBS (+), by further incubated for 4 h, the cell viability was measured using the Alamar Blue assay [17] in triplicate.
2.6. Determination of Intracellular Zn2+ Ions
To HepG2 cells seeded at 1 × 104 cells/well in a 96-well plate, 20 μL of the sample containing the Zn2+/CM-PVIm complexes or the control sample containing ZnCl2 or Zn(OAc)2 alone was added according to the cell viability assay. After incubation for 24 h, the cells were washed with PBS (+) twice, followed by the addition of 20 μL of cell culture lysis reagent (25 mM Tris-phosphate (pH 7.8), 2 mM DTT, 2 mM 1,2-diaminocyclohexane-N,N,N’,N’-tetraacetic acid, 10% glycerol, 1% Triton X-100) (Promega, Madison, WI). After 5 min incubation, 100 μL H2O was added. Subsequently, 400 μL of 0.4 M HCl was added to 120 μL of the resulting cell lysate diluted to 1.5 mL with H2O. The concentration of Zn2+ in the resulting sample was determined by atomic absorption spectrometry at 213.9 nm using an atomic absorption spectrophotometer AA-6200 (Shimadzu Co., Kyoto, Japan).
3. Results and Discussion
![]()
Figure 1. Design of Zn2+-chelated CM-PVIm, that is, Zn2+/CM-PVIm complex.
sites, that is, imidazole and carboxyl groups. To examine the effect of counter anions on the chelation to CM-PVIm, we mixed CM-PVIm with ZnCl2 or Zn(OAc)2, as shown in Figure 2, followed by the removal of free Zn2+. The interaction between Zn2+ ions and CM-PVIm is considered to form a cross-linking structure with measurable particle size and ζ-potential. After mixing CM-PVIm with ZnCl2, as shown in Figure 3, the particle size was 6.6 nm and the ζ-potential was +16.3 mV. On the other hand, larger particle size (79.6 nm) and lower ζ-potential (+8.74 mV) were exhibited by mixing with Zn(OAc)2. These results suggest that the Zn2+ ions chelated to the imidazole and/or carboxyl groups of CM-PVIm and that counter anions affected the resulting cross-linking structure by the difference in their coordination interaction with CM-PVIm polyampholytes.
We used atomic absorption spectrometry to determine the amount of Zn2+ ions chelated to the CM-PVIm. The concentration of Zn2+ ions chelated to CM-PVIm by use of ZnCl2 and Zn(OAc)2 was 1.3, and 0.51 mol%, respectively, based on all the imidazole groups of the CM-PVIm (Table 1).
The calculated amount of the Zn2+ chelated to the CM-PVIm is in proportion to the ζ-potential. Conversely, that of the Zn2+ is in inverse proportion to the particle size, presumably due to higher condensation of CM-PVIm polyampholyte chains by larger amount of the Zn2+ ions. Namely, taking these results into account, free Zn2+ ions are considered to be easy of access to imidazole and/or carboxyl groups of CM-PVIm without steric hindrance by use of ZnCl2, because ZnCl2 released 3-fold higher concentration than Zn(OAc)2 [18] .
As shown in Figure 4, the cell viability in the presence of CM-PVIm alone and the resulting Zn2+/CM-PVIm complex for use of intracellular Zn2+ delivery. We confirmed almost 100% cell viability in the presence of CM-PVIm alone, up to the concentration of 16 mg/mL, which was higher than Zn2+ delivery experimental conditions (Figure 4(a)). Furthermore, the Zn2+/CM-PVIm complexes by use of both ZnCl2 and Zn(OAc)2 exhibited no significant cytotoxicity, as well as ZnCl2 and Zn(OAc)2 alone, under the experimental conditions (Figure 4(b)).
Based on the lack of significant cytotoxicity of the Zn2+/CM-PVIm complexes, as shown in Figure 5, the intracellular Zn2+ delivery by the Zn2+/CM-PVIm complexes was examined. When ZnCl2 or Zn(OAc)2 was added to the cells in the absence of CM-PVIm, intracellular Zn2+ hardly increased. On the other hand, the Zn2+/CM-PVIm complex led to increase in intracellular Zn2+, succeeding in
![]()
Figure 2. Typical images just after mixing CM-PVIm with ZnCl2 or Zn(OAc)2.
![]()
Figure 3. Particle size and ζ-potential of the Zn2+/CM-PVIm complexes.
![]()
Figure 4. Effect of CM-PVIm (a) and Zn2+/CM-PVIm (b) on the viability of HepG2 cells. Symbols and error bars represent the mean and standard deviation of the measurements made in paired wells ((a) n = 3 or (b) n = 4). * means that the highest value was dismissed (n = 3).
![]()
Table 1. Amount of Zn2+ chelated to CM-PVIm.
*CM-PVIm (−) as blank.
![]()
Figure 5. Cellular uptake of Zn2+ by the Zn2+/CM-PVIm complexes. Symbols and error bars represent the mean and standard deviation of the measurements made in paired wells (n = 3). Statistical significance (p < 0.05) is indicated when compared to the absence of CM-PVIm.
intracellular Zn2+ delivery. The resulting amount of intracellular Zn2+ treated with the Zn2+/CM-PVIm complex by use of ZnCl2 was higher than that by use of Zn(OAc)2. These results suggest that the Zn2+/CM-PVIm complex with larger amount of Zn2+ as well as smaller particle size is more suitable for intracellular Zn2+ delivery.
From an applied point of view, because zinc transporter-8 (ZnT8), to deliver Zn2+ from the cytosol to insulin granules, is known to regulate hepatic insulin clearance [19] , the optimization of our intracellulr Zn2+ delivery system has the possibility of use for type 2 diabetes therapy.
4. Conclusion
We have chelated Zn2+ ions to CM-PVIm by mixing ZnCl2 or Zn(OAc)2. The resulting Zn2+/CM-PVIm complex by mixing ZnCl2 exhibited smaller particle size and possessed larger amount of Zn2+ ions, as compared to the Zn2+/CM-PVIm by mixing Zn(OAc)2, delivering larger amount of intracellular Zn2+ ions without cytotoxicity. Collectively, from these results, the optimal Zn2+/CM-PVIm complex is considered to be a useful tool for intracellular Zn2+ delivery to control various biological functions.
Acknowledgements
The experimental apparatus was partially supported by Prof. Hiroyoshi Kawakami’s Laboratory at Tokyo Metropolitan University.