MATERIALS AND METHODS
ChemicalsProtoporphyrin IX (PP) was purchased from Sigma Chemical Co. (St. Louis, MO, U.S.A.). The succinimidyl derivative of PEG (ME-020CS), with an average molecular weight of 2000, was from NOF Co. (Tokyo, Japan). N-tert-butoxycarbonyl (Boc)-ethylenediamine was purchased from Tokyo Chemical Industry Co. Ltd. (Tokyo, Japan). Tetrahydrofuran (THF), triethylamine (TEA), ethyl chloroformate, N,N-dimethylformamide (DMF), diethyl ether, and trifluoroacetic acid (TFA) were from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Manganese (II) acetate tetrahydrate was from Nacalai Tesque Inc. (Kyoto, Japan). 6-Hydroxydopamine hydrochloride (6-OHDA, H4381) and 2′,7′-dichlorofluorescein diacetate (DCFH-DA, D6883) were purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.). APAP was from Terumo Corporation (Tokyo, Japan) and from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). MnTBAP was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, U.S.A.). MitoSOX™ Red mitochondrial Superoxide Indicator (M36008) was from Thermo Fisher Scientific (Waltham, MA, U.S.A.).
Synthesis of PEG-MnPPPEG-MnPP was synthesized according to a protocol similar to that reported previously12–14) with some modifications. Figure 1 illustrates the overall scheme, which contains three major steps: (i) introduction of amino groups into the protoporphyrin ring by reacting ethylenediamine with the intrinsic carboxyl groups of the ring; (ii) PEG conjugation to the amino groups; and (iii) chelation of Mn2+ to the PEG-porphyrin ring. Detailed information for each step is presented below.
Synthesis of Bis(ethylenediamino)-Protoporphyrin (PPED)PP (200 mg, 0.36 mmol) was suspended in 40 mL of THF and cooled to 0°C on ice. With stirring, TEA (0.5 mL, 3.6 mmol) was added, followed by drop-wise addition of ethyl chloroformate (0.68 mL, 7.2 mmol). The reaction was carried out for 20 min and continued at room temperature for 15 min. The resultant suspension was filtered to remove the TEA–HCl and was then subjected to evaporation in vacuo to remove THF and ethyl chloroformate, which thereby generated activated PP. Then, 0.25 mL of Boc-ethylenediamine (1.6 mmol, 2.2 M excess to carboxyl groups in PP) was dissolved in 2 mL of DMF, to which the activated PP (described above), which had been dissolved in 10 mL of DMF, was added in drop-wise fashion. The reaction was continued at room temperature with stirring for 2 h. The product was then precipitated with 10× volumes of chilled diethyl ether by using centrifugation (4000 × g, 5 min, 4°C). The precipitate was washed 3 times with ice-cold diethyl ether, and a solid product (PPED-Boc) was obtained after removing diethyl ether by evaporation. The purity of PPED-Boc was 99.5% according to HPLC, as described below. Finally, the Boc group was removed from PPED-Boc by dissolving PPED-Boc in 3 mL of TFA and keeping it on ice for 2 h. The product (PPED-TFA) was precipitated and washed with diethyl ether as described above to yield 190 mg of product with a purity of 98.9% as determined by HPLC.
Conjugation of PPED with PEGThe PPED-TFA obtained (190 mg), which consisted of about 80% PPED (0.23 mmol), was dissolved in 20 mL of DMF. To this solution were added succinimidyl PEG, 2.0 g (1 mmol, about a 2× molar excess to the amino groups in PPED), and 95 µL of TEA (0.69 mmol). The reaction proceeded at room temperature for 24 h with stirring. The product was then precipitated with 20× volumes of chilled diethyl ether by using centrifugation (4000 × g, 5 min, 4°C), after which the sample was washed 3 times with ice-cold diethyl ether. Finally, 2.1 g of PEG-conjugated PP (PEG-PP) was obtained with a purity of 97.6%, as determined with HPLC. The PP content (loading) was quantified as 7.6%, on the basis of an analysis with UV absorption at 406 nm and using a standard curve for free PP in dimethyl sulfoxide (DMSO) as a reference.
Chelation of Manganese to PEG-PPPEG-PP (745 mg, 0.1 mmol porphyrin equivalent) was dissolved in 75 mL of methanol, to which 2.89 g (10.8 mmol) of manganese (II) acetate tetrahydrate was added. The solution was stirred at room temperature for 22 h to complete the chelation of Mn2+ to the porphyrin ring. After the reaction, methanol was removed by evaporation to yield the crude PEG-MnPP. PEG-MnPP was then purified by precipitating unreacted manganese (II) acetate tetrahydrate with chloroform and then washing 3 times with chloroform. The final product, after evaporation of chloroform, was dissolved in 100 mL of deionized water followed by lyophilization. The yield of PEG-MnPP after lyophilization was 645 mg.
HPLCThe formation and purity of products were confirmed by using the LC-2000Plus series HPLC system (JASCO, Tokyo, Japan) equipped with a PU-2080 pump, UV-2075 UV/Visible detector (integrator output scale was 1 V/1.0 AU), and 807-IT integrator. The Asahipak GF-310 HQ column (7.5 × 300 mm) (Showa Denko, Tokyo, Japan) was used. The mobile phase consisted of 70% methanol, 30% DMSO, and 0.001% TFA at a flow rate of 0.8 mL/min; eluate was monitored at 415 nm for PP and at 470 nm for MnPP.
Dynamic Light ScatteringPEG-MnPP was dissolved in deionized water at 7 mg/mL and filtered through a 0.45-µm filter. Particle size was measured by using dynamic light scattering (ELS-Z2; Photal Otsuka Electronics, Osaka, Japan) and was calculated by using the cumulant and histogram methods.
O2– Decomposition AssaySOD activity was measured by means of the SOD Assay Kit-WST (Dojindo Laboratories, Kumamoto, Japan) according to the manufacturer’s instructions. This assay is based on the reduction of WST-1 by O2− to formazan. In brief, 200 µL of WST working solution was added to 20 µL of PEG-MnPP (100 µM) or MnTBAP (100 µM); the samples were mixed by pipetting them into a 96-well plate, after which the samples were mixed with 20 µL of enzyme working solution. After incubation for 20 min at 37°C, absorbance was measured at 490 nm via a microplate reader (Bio-Rad, Hercules, CA, U.S.A.). The rate of WST-1 reduction was calculated by using an SOD standard curve as described in the manufacturer’s instructions.
H2O2 Decomposition AssayTo determine the H2O2 decomposition activity of test compounds, 50 µM solutions of the test compounds prepared in Dulbecco’s Modified Eagle’s Medium (DMEM; Wako Pure Chemical Industries, Ltd.) and supplemented with 10% heat-inactivated fetal bovine serum were reacted with 2 mM H2O2, as described in the figure legends. After the reaction, H2O2 decomposition was evaluated by means of the Hydrogen Peroxide Colorimetric Detection Kit (Enzo Sciences, Plymouth Meeting, PA, U.S.A.) according to the manufacturer’s protocol. In brief, 2-µL samples of the reaction mixtures were added to 18 µL of water, and the diluted samples were pipetted into blank wells, to which was added 40 µL of detection reagent. After incubation for 15 min at 37°C, absorbance was measured at 595 nm with a microplate reader (Bio-Rad).
Cell Lines and Culture ConditionsHeLa cells, the human cervical cancer cell line, were cultured in Eagle’s Minimum Essential Medium (Wako Pure Chemical Industries, Ltd.) supplemented with 10% heat-inactivated fetal bovine serum (MP Biomedicals, Santa Ana, CA, U.S.A.) and 1% penicillin–streptomycin (Nacalai Tesque). SH-SY5Y cells, the human neuroblastoma cell line, and human hepatocellular carcinoma cell line, HuH-7 cells were cultured in DMEM supplemented with 10% heat-inactivated fetal bovine serum and 1% penicillin-streptomycin. THP-1 cells were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum and 1% penicillin–streptomycin. All cells were cultured in a 5% CO2 humidified incubator at 37°C.
Cell TreatmentCell viability was determined by using two different methods—the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Dojindo Laboratories) or the trypan blue exclusion assay (Automated Cell Counter Countess, Invitrogen, Eugene, OR, U.S.A.) depending on the cell type. For adhesive cells such as HeLa and SH-SY5Y cells, the MTT assay was used. For floating cells such as THP-1 cells, the trypan blue exclusion assay was utilized. In our first experiments, we determined the cytotoxicity of PEG-MnPP and related substances. HeLa cells or THP-1 cells were seeded in a 96-well plate at a density of 1 × 104 cells/well. After overnight incubation, cells were treated with PEG-MnPP, PEG-PP, or MnTBAP for 24 h or cells were untreated. Cell viability was then determined via the MTT assay and the trypan blue exclusion assay for HeLa cells and THP-1 cells, respectively, according to the manufacturer’s instructions.
We determined the effects of PEG-MnPP on ROS-induced cell death by using two different models. HeLa cells were seeded in a 96-well plate at a density of 1 × 104 cells per well and were cultured overnight. Cells were then exposed to 1 mM H2O2 in the absence or the presence of additives such as PEG-MnPP for 5 h. SH-SY5Y cells were seeded in a 96-well plate at a density of 5 × 104 cells per well and were cultured for 8 h. Cells were treated with 6-OHDA at specific concentrations (12.5–100 µM) in the absence or presence of 30 µM PEG-MnPP for 24 h. Cell viability after each treatment was determined by means of the MTT assay.
Measurement of Intracellular ROS ProductionSH-SY5Y cells were seeded in a 96-well plate at a density of 5 × 104 cells per well and were treated with 6-OHDA at indicated concentrations (12.5–50 µM) in the absence or presence of 30 µM PEG-MnPP or PEG-PP. After incubation for 3 h, cells were rinsed and then loaded with 5 µM DCFH-DA (Sigma-Aldrich) in Modified Krebs–Ringer solution (44 mM NaHCO3, 0.9 mM NaH2PO4, 110 mM NaCl, 5 mM KCl, 0.8 mM MgSO4, 10 mM glucose, 2 mM CaCl2, pH 7.4) for 15 min. HuH-7 cells were seeded in a 96-well plate at a density of 1 × 104 cells per well and were treated with APAP at indicated concentrations (0.2–20 mM) in the absence or presence of 30 µM PEG-MnPP or PEG-PP. After incubation for 24 h, cells were rinsed and then loaded with 5 µM MitoSOX™ Red mitochondrial Superoxide Indicator (M36008, Thermo Fisher Scientific) for 15 min. Fluorescent images were observed by means of a fluorescence microscope (BZ-X700, Keyence, Osaka, Japan). Three wells for each treatment were analyzed and the relative fluorescent intensity was expressed as a quantitative data.
Animals and Experimental ProtocolsMale ICR mice (6–8 weeks old) were purchased from Kyudo Co. (Saga, Japan). The animals were allowed free access to food and water at all times and were maintained on a 12-h light/dark cycle in an environment of controlled temperature (20–25°C) and humidity (50 ± 5%) for 1 week before use. All animal experiments were carried out according to the Guidelines of the Laboratory Protocol of Animal Handling, Sojo University and Kumamoto University.
ICR mice were fasted overnight, after which liver injury was induced via intraperitoneal (i.p.) injections of APAP (300 mg/kg).15) In the group receiving the PEG-PP or PEG-MnPP treatment, mice received one intravenous injection of PEG-PP or PEG-MnPP (0.5 mg PP or MnPP equivalent/kg dissolved in physiological saline, 0.1 mL) via the tail vein at 2 h after APAP administration. In the control group, mice were injected intravenously with physiological saline. Mice were killed at 24 h after the APAP injection. The body weights and liver weights were measured, and serum samples were collected for measurement of alanine aminotransferase (ALT).
Statistical AnalysesAll data are expressed as means ± standard deviation (S.D.) Data for each experiment were obtained from at least three independent experiments. Statistical analyses were performed by using Student’s t-test, with the level of significance set at p < 0.05.
RESULTS AND DISCUSSION
Synthesis of PEG-MnPPAs Fig. 1 shows, PEG-MnPP was synthesized by incorporating manganese into the PEG-PP structure. Chelation of manganese was carried out by incubating PEG-PP and manganese acetate in methanol, with success of the chelation confirmed by the appearance of the characteristic UV/VIS absorption spectrum for manganese porphyrin (Fig. 2). In fact, PEG-MnPP in methanol has a distinctive split Soret band, which is characteristic for manganese porphyrins,16) with one component appearing at 377 nm and a weaker one appearing at 466 nm (Fig. 2(b)). The purity of PEG-MnPP was determined to be higher than 98% by means of HPLC.
Physicochemical Properties of PEG-MnPPConjugation of PEG chains to manganese porphyrin greatly increased water solubility of PEG-MnPP. For instance, PEG-MnPP could be dissolved in neutral aqueous buffer at a concentration of approximately 30 mM without heating. We previously found that PEG-ZnPP formed micelle-like molecular assemblies in aqueous media, with a hydrophobic core consisting of ZnPP moieties surrounded by hydrophilic PEG chains.11–13) In this study, we investigated whether PEG-MnPP formed similar molecular assemblies in water. Dynamic light scattering analysis suggested that PEG-MnPP formed nanoparticles having an average diameter of 73.5 ± 17.9 nm in water (Fig. 3). Porphyrins are known to exhibit intense fluorescence when they are well dispersed in solution, whereas after aggregation the fluorescence is strongly quenched.17) As Fig. 4 shows, the fluorescence intensity of PEG-MnPP was smaller in water than in methanol and was increased by increasing the methanol content. These data suggest that PEG-MnPP forms micelle-like molecular assemblies in aqueous media.
ROS-Scavenging Activities of PEG-MnPPMnPP derivatives reportedly mimic both SOD and catalase activities.9) We first investigated the SOD mimic activity of PEG-MnPP. MnTBAP is one of a class of manganese porphyrins and mimics both SOD and catalase activities.18) We thus compared MnTBAP with PEG-MnPP. As Fig. 5 illustrates, PEG-MnPP did demonstrate SOD mimic activity but its activity was 5.5 times weaker than that of MnTBAP.
We next studied whether PEG-MnPP could exhibit catalase mimic activity. Figure 6(a) clearly shows that a catalytic concentration (50 µM) of PEG-MnPP completely decomposed excess H2O2 (2 mM) in aqueous media. Metal-free PEG-PP failed to decompose H2O2. Glutathione (GSH) is a peptidyl antioxidant that is involved in the elimination of H2O2 in cells. Because glutathione can eliminate H2O2 by reacting directly with H2O2, only a marginal decrease in H2O2 was observed for it under current conditions (Fig. 6(a)). It is interesting that the potency of PEG-MnPP as a catalase mimic was higher than that of MnTBAP. During 30 min of incubation, PEG-MnPP at 25 µM completely decomposed 2 mM H2O2, whereas MnTBAP required a concentration higher than 50 µM for complete decomposition of H2O2 (Fig. 6(b)). PEG-MnPP also decomposed H2O2 more rapidly than did MnTBAP (Fig. 6(c)). These data suggest that PEG-MnPP is a potent catalase mimic.
Cytoprotective Effects of PEG-MnPP against ROS-Mediated Cell DeathWe first investigated the cytotoxicity of PEG-MnPP alone to human cell lines to determine the biocompatibility of PEG-MnPP with cells. As seen in Fig. 7, PEG-MnPP treatment did not affect the viability of the epithelial cell line (HeLa cells) as well as the monocyte cell line (THP-1 cells) under the present experimental conditions. We observed no morphological changes of cells treated with PEG-MnPP under these conditions (data not shown).
We then studied whether PEG-MnPP could protect cells from oxidative stress-associated cell death. Treatment of HeLa cells with authentic H2O2 at concentrations higher than 0.125 mM reduced cell viability to approximately 20% of untreated controls (Fig. 8(a)). Addition of PEG-MnPP resulted in a marked increase in cell viability, whereas PEG-PP did not show such a cytoprotective effect (Fig. 8(b)). Consistent with H2O2 removal activities determined in vitro (Fig. 6), the cytoprotective effect of PEG-MnPP was greater than that of MnTBAP (Fig. 8(b)). We next examined the cytoprotective effect of PEG-MnPP in a more biologically relevant model. 6-OHDA is a dopaminergic neurotoxin that can cause neuronal cell death.19) 6-OHDA-induced cell death was reportedly mediated via extracellular auto-oxidation of 6-OHDA to form H2O2.20) Consistent with previous report,21) 6-OHDA treatment significantly increased intracellular ROS levels as indicated by the marked increase of fluorescent intensity derived from ROS sensitive probe DCFH-DA (Supplementary Fig. S1 (a,b)). Co-treatment with PEG-PP did not affect ROS production induced by 6-OHDA treatment, whereas PEG-MnPP treatment significantly reduced ROS production in cells (Fig. 8(c), Supplementary Fig. S1 (c,d)). In human neuroblastoma SH-SY5Y cells, 6-OHDA treatment caused cell death in a dose-dependent manner (Fig. 8(d)). PEG-MnPP at 30 µM resulted in a marked increase in cell viability, i.e., protection against 6-OHDA-induced cell death (Fig. 8(d)). Under the present conditions, PEG-PP did not have any cytoprotective effects (data not shown). These data clearly indicate that PEG-MnPP effectively eliminated cytotoxic ROS under these cell culture conditions and protected cells from cell death associated with oxidative stress. It should also be noted that although PEG-MnPP eliminated ROS in cells treated with 50 µM 6-OHDA, cell viability at this dose of 6-OHDA did not recovered by PEG-MnPP treatment (Fig. 8). This may suggest that, at higher doses of 6-OHDA, ROS independent cell damages may dominate for 6-OHDA-induced cell death.
Therapeutic Effects of PEG-MnPP in APAP-Induced Acute Liver FailureAPAP is one of the drugs used most often for its analgesic and antipyretic properties.22,23) At the recommended doses APAP is safe and effective; overdose may result in hepatotoxicity and acute liver failure.22,23) APAP overdose leads to the saturation of the main elimination pathways of APAP in the liver, e.g., the glucuronidation and sulfation pathways; thus, the highly reactive metabolite N-acetyl-p-benzoquinone imine (NAPQI) accumulates.22,23) NAPQI so formed can damage mitochondria in hepatocytes and result in excess production of ROS.22,23) In this study, we examined the therapeutic effects of PEG-MnPP on acute liver failure caused by APAP overdose in a mouse model. Under the current experimental conditions, APAP at 300 mg/kg induced acute liver failure, as indicated by the marked increase in liver-to-body weight ratio and serum ALT levels (Fig. 9). Administration of PEG-MnPP (0.5 mg MnPP equivalent/kg) at 2 h after the APAP injection significantly reduced both the liver-to-body weight ratio and the serum ALT levels (Fig. 9). On the other hand, treatment with PEG-PP failed to reduce liver damages caused by APAP overdose (Supplementary Fig. S2). As mentioned above, APAP treatment induce mitochondria damages and associated ROS production,24,25) we examined mitochondria-derivaed ROS production by using MitoSOX probe. We found that APAP treatment increased mitochondrial ROS production (Supplementary Fig. S3). Importantly, APAP-induced mitochondrial ROS production was significantly reduced by PEG-MnPP (Supplementary Fig. S3).
Ferret et al. reported the protective effect of MnTBAP against APAP-induced liver failure.26) In their experiments, MnTBAP was administered via the intraperitoneal route at 10 and 20 mg/kg doses to APAP-treated mice.26) The present study demonstrated that a single injection of PEG-MnPP at a much lower dose was sufficient to produce a therapeutic effect against severe liver damage caused by APAP in vivo.
To our knowledge, this is the first demonstration of potent catalase-like activity of a manganese porphyrin after pegylation. PEG-MnPP behaves as nanoparticles in aqueous media by forming a molecular assembly. As reported for other nanoparticles,27) PEG-MnPP may circulate in vivo for longer time periods because it may escape from renal clearance. At a site of inflammation such as a damaged liver caused by APAP, increased vascular permeability may facilitate leakage of PEG-MnPP and subsequent accumulation in inflamed tissues.
Naruta and Maruyama reported that catalase-like activity of manganese porphyrin could be significantly enhanced when two manganese porphyrin rings were connected by an anthracene linkage to form manganese porphyrin dimers.28) These dimers act synergistically to decompose H2O2.28) Kubota et al. developed water-soluble manganese porphyrin dimers that act as a potent catalase mimic, similar to the anthracene-linked manganese porphyrins.8) In the present study, we found that PEG-MnPP more effectively catalyzed decomposition of H2O2 than did the monomeric MnTBAP (Fig. 6). As just discussed, the manganese porphyrin rings of PEG-MnPP may assemble in aqueous media, which may facilitate intermolecular interactions of manganese porphyrin rings and hence promote H2O2 decomposition.
The therapeutic potential of manganese porphyrin derivatives has been reported for oxidative stress-associated diseases such as paraquat-induced lung injury and carrageenan-induced lung injury.29) Li et al. found that the manganese porphyrin derivative HSJ-0017 possesses both SOD and catalase mimic activities; it exerted potent antitumor effects in a tumor-bearing mouse model.30) Additional study is needed to clarify the accumulation in tumors and the antitumor effects of PEG-MnPP.
In summary, we demonstrated here that PEG-MnPP is a water-soluble and biocompatible synthetic catalase mimic that can effectively eliminate toxic H2O2. The in vivo mouse model revealed that PEG-MnPP may be able to treat APAP-induced acute liver failure. Continued study is warranted to determine the therapeutic effects of PEG-MnPP on inflammatory disorders that are associated with extensive production of ROS.
