Development of PEGylated Cysteine-Modified Lysine Dendrimers with

Jun 23, 2016 - To inhibit hepatic ischemia/reperfusion injury, we developed polyethylene glycol (PEG) conjugated (PEGylated) cysteine-modified lysine ...
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Development of PEGylated Cysteine-Modified Lysine Dendrimers with Multiple Reduced Thiols To Prevent Hepatic Ischemia/ Reperfusion Injury Hidemasa Katsumi,*,† Makiya Nishikawa,‡ Rikiya Hirosaki,† Tatsuya Okuda,§,∥ Shigeru Kawakami,§,⊥ Fumiyoshi Yamashita,§ Mitsuru Hashida,§ Toshiyasu Sakane,† and Akira Yamamoto† †

Department of Biopharmaceutics, Kyoto Pharmaceutical University, Yamashina-ku, Kyoto 607-8414, Japan Department of Biopharmaceutics and Drug Metabolism, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan § Department of Drug Delivery Research, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan ∥ Department of Chemistry, Dokkyo Medical University, Shimotsuga-gun, Tochigi 321-0293, Japan ⊥ Department of Pharmaceutical Informatics, Graduate School of Biomedical Sciences, Nagasaki University, Sakamoto, Nagasaki 852-8523, Japan ‡

ABSTRACT: To inhibit hepatic ischemia/reperfusion injury, we developed polyethylene glycol (PEG) conjugated (PEGylated) cysteine-modified lysine dendrimers with multiple reduced thiols, which function as scavengers of reactive oxygen species (ROS). Second, third, and fourth generation (K2, K3, and K4) highly branched amino acid spherical lysine dendrimers were synthesized, and cysteine (C) was conjugated to the outer layer of these lysine dendrimers to obtain K2C, K3C, and K4C dendrimers. Subsequently, PEG was reacted with the C residues of the dendrimers to obtain PEGylated dendrimers with multiple reduced thiols (K2C−PEG, K3C− PEG, and K4C−PEG). Radiolabeled K4C−PEG (111In-K4C− PEG) exhibited prolonged retention in the plasma, whereas 111In-K2C−PEG and 111In-K3C−PEG rapidly disappeared from the plasma. K4C−PEG significantly prevented the elevation of plasma alanine aminotransferase (ALT) activity, an index of hepatocyte injury, in a mouse model of hepatic ischemia/reperfusion injury. In contrast, K2C−PEG, K3C−PEG, L-cysteine, and glutathione, the latter two of which are classical reduced thiols, hardly affected the plasma ALT activity. These findings indicate that K4C−PEG with prolonged circulation time is a promising compound to inhibit hepatic ischemia/reperfusion injury. KEYWORDS: drug delivery, dendrimer, PEGylation, cysteine, thiol, reactive oxygen species, hepatic ischemia/reperfusion injury

1. INTRODUCTION

To date, the representative reduced thiols N-acetylcysteine (a prodrug of L-cysteine) and glutathione have been examined for the prevention of hepatic ischemia/reperfusion injury,13−15 because reduced thiols are ROS scavengers. However, reduced thiols are eliminated rapidly from the bloodstream after being intravenously injected, limiting their therapeutic effect. Although thiolated polyamidoamine (PAMAM) dendrimers, which are macromolecular reduced thiols, have been reported,16,17 the tissue distribution and controlled delivery of thiols have hardly been examined so far. We recently developed polyethylene glycol (PEG) conjugated thiolated bovine serum albumin, a novel macromolecular reduced thiol, in which there are approximately 18 SH functional groups.18 PEG−BSA-SH

Oxidative stress contributes to the etiology of numerous liver disorders,1−4 including the onset and progression of hepatic ischemia/reperfusion injury.3,4 Hepatic ischemia/reperfusion injury is associated with hepatic failure after shock and liver transplantation.3−5 During reperfusion after hepatic ischemia, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation in Kupffer cells generates reactive oxygen species (ROS). Subsequently, ROS activate inflammatory factor expression via the nuclear factor-κB (NF-κB) mediated pathway.6−10 Therefore, it is likely that ROS play a critical role in hepatic ischemia/reperfusion injury, and scavenging ROS may be a suitable approach to inhibit hepatic injury. Therefore, antioxidants that scavenge ROS have been considered therapeutic candidates for hepatic ischemia/ reperfusion injury.5,11,12 © XXXX American Chemical Society

Received: June 21, 2016 Accepted: June 23, 2016

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DOI: 10.1021/acs.molpharmaceut.6b00557 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Molecular Pharmaceutics

Figure 1. Structures of PEGylated cysteine-modified lysine dendrimers (K2C−PEG, K3C−PEG, and K4C−PEG). Molecular weight was calculated based on the average number of PEG chains/dendrimers and the theoretical molecular weight of cysteine-modified lysine dendrimers.

2. MATERIALS AND METHODS 2.1. Animals. Male ddY mice (weighing 25−27 g) were purchased from Japan SLC Inc. (Shizuoka, Japan) and maintained under conventional housing conditions. All animal experiments were conducted according to the principles and procedures outlined in the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The Animal Experimentation Committee of the Kyoto Pharmaceutical University approved all animal experiment protocols. 2.2. Chemicals. 1-Hydroxy-1-benzotriazole monohydrate (HOBt), ethylenediamine, 1,4-dioxane, N,N-dimethylformamide dehydrate (DMF), trifluoroacetic acid (TFA), ethyl acetate, dithiothreitol (DTT), 2,2′-dipyridyl disulfide (PDS), 2,4,6-trinitrobenzenesulfonate sodium salt dehydrate (TNBS), petroleum ether, L-cysteine, glutathione, and triethylamine were purchased from Wako Pure Chemical Industries (Osaka, Japan). Diethylenetriaminepentaacetic acid (DTPA) anhydride and ethylenediaminetetraacetic acid tetrasodium salt (EDTA 4Na) were purchased from Dojindo Laboratory (Kumamoto, Japan). 1-[Bis(dimethylamino)methylene]-1H-benzotriazolium 3-oxide hexafluorophosphate (HBTU) and N-(tert-butoxycarbonyl)-S-trityl-L-cysteine (Boc-Cys(Trt)-OH) were purchased from Novabiochem (La Jolla, CA). Methoxypolyethylene glycol N-succinimidyl succinate (activated PEG; average molecular weight 2,000 kDa) was purchased from NOF Co. (Tokyo, Japan). N-Boc-protected lysine (Boc-Lys(Boc)-OH) was prepared as previously reported.20 All other chemicals were commercial products of reagent grade. 2.3. Synthesis and Characteristics of PEGylated Cysteine-Modified Lysine Dendrimers. Structures and synthetic routes of K2C−PEG, K3C−PEG, and K4C−PEG are shown in Figure 1. K2, K3, and K4 were synthesized according to a previously published method,20 and then coupled with Boc-Cys(Trt)-OH by the HBTU-HOBt method.

had a prolonged retention time in the systemic circulation after being intravenously injected into mice. Moreover, the ROSmediated hepatitis induced by D-galactosamine and lipopolysaccharide was effectively prevented by an intravenous injection of PEG−BSA-SH in mice. These results suggest that conjugation of reduced thiols to a macromolecular carrier prolongs retention in the systemic circulation after delivery and is a promising approach to treat ROS-mediated diseases. Lysine dendrimers, which are highly branched unique spherical lysine polymers, possess some advantages as macromolecular carriers such as biodegradability, high water solubility, and low toxicity.19 It was reported that changing the constituent amino acids of lysine dendrimers could alter their physicochemical properties, including size and charge. Furthermore, tissue distribution of lysine dendrimers can be controlled by various chemical modifications.20,21 Therefore, we used lysine dendrimers for prolonged delivery of reduced thiols. The purpose of this study was to develop an amino acid dendrimer with multiple reduced thiols for the efficient prevention of hepatic ischemia/reperfusion. We selected second, third, and fourth generations of lysine dendrimers (K2, K3, and K4) as bioinert dendrimer backbones. We then conjugated the K2, K3, and K4 backbones with cysteine, a reduced thiol, to obtain amino acid dendrimers with multiple reduced thiols (K2C, K3C, and K4C). Then, to increase plasma retention, PEG was covalently bound to K2C, K3C, and K4C to obtain PEGylated cysteine-modified lysine dendrimers (K2C−PEG, K3C−PEG, and K4C−PEG). Tissue distribution was investigated after iv injection in mice. Finally, the efficacy of these PEGylated cysteine-modified lysine dendrimers in the inhibiting hepatic ischemia/reperfusion injury was examined. B

DOI: 10.1021/acs.molpharmaceut.6b00557 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Molecular Pharmaceutics Table 1. Physicochemical Characteristics of PEGylated Cysteine-Modified Lysine Dendrimers dendrimers

no. of SH groups

av no. of PEG chains

theor mol wt

particle diam Dn (nm)

polydispersity index (PDI)

zeta-potential (mV)

K2C−PEG K3C−PEG K4C−PEG

8 16 32

2.0 ± 0.16 8.3 ± 0.14 14.2 ± 0.44

5,700 20,100 35,600

6.6 7.0 8.2

0.682 0.371 0.219

0.04 0.64 −1.14

2.5. Tissue Distribution of 111In-PEGylated CysteineModified Lysine Dendrimers in Mice. K2C−PEG, K3C− PEG, and K4C−PEG were radiolabeled with 111In (Nihon Medi-Physics, Takarazuka, Japan) using DTPA anhydride as previously described.18,24,30,31 111In-K2C−PEG, 111In-K3C− PEG, and 111In-K4C−PEG were intravenously injected in mice at a dose of 1 mg/kg (8.0 × 106 cpm/kg), which is almost equivalent to the dose administered in the hepatic ischemia/ reperfusion study. Blood and major organs were collected under ether anesthesia at predetermined times after injection. The radioactivity in each sample was measured in a gamma counter (1480 Wizard, PerkinElmer, Boston, MA, USA). 2.6. Hepatic Ischemia/Reperfusion. Hepatic ischemia was induced by occluding the portal vein and hepatic artery with a vascular clamp for 15 min under ether anesthesia.8,11,12 Blood reflow to the liver (reperfusion) occurred. Just before the start of reperfusion, saline, K2C−PEG, K3C−PEG, K4C−PEG, L-cysteine, and glutathione were intravenously injected at an equivalent thiol dose (4.5 μmol of SH/kg). After 6 h of reperfusion, blood was taken under ether anesthesia, and plasma ALT activity (an indicator of hepatic injury) was assayed as reported previously.12 The oxidized glutathione (GSSG) and reduced glutathione (GSH) levels in the liver were determined as reported previously.12 2.7. Statistical Analysis. Statistical significance was assessed by one-way analysis of variance followed by the Student−Newman−Keuls multiple comparison test for multiple groups at a significance level of p < 0.05.

Briefly, K4C was prepared by coupling between K4 (111 mg, 0.028 mmol (0.90 mmol of amine functional groups)) and BocCys(Trt)-OH (632 mg, 1.36 mmol) in DMF containing HBTU (517 mg, 1.36 mmol), HOBt (209 mg, 1.36 mmol), and triethylamine (190 μL, 1.36 mmol) for 4 h at room temperature. The reaction mixture was evaporated, and the product was dissolved with chloroform. The organic phase was washed with 5% NaHCO3 five times, 10% citric acid three times, and saturated sodium chloride three times. The organic phase was dried with anhydrous MgSO4. The products were recrystallized from petroleum ether, filtered by vacuum filtration, and vacuum dried. The products were then deprotected in TFA for 1 h at room temperature. The mixture was evaporated and crystallized from diethyl ether to obtain cysteine-modified K4 (K4C). K2C and K3C were synthesized with the same method as described above. The lysine dendrimers and cysteine-modified lysine dendrimers could be identified by MALDI TOF-MS (Voyager-DE STR, Applied Biosystems, Inc., Foster City, CA, USA). The lysine dendrimers showed clear single peak values corresponding to the calculated ones. Although the ion peaks of cysteine-modified lysine dendrimers were broad, the peak values corresponded to the calculated ones. The thiol groups derived from cysteine were detected by Ellman’s method.22 In order to protect the K2C, K3C, and K4C thiols, these compounds were coupled with PDS (molar ratio of PDS to thiol functional groups of 1.5:1) in methanol for 0.5 h at room temperature. The reaction mixture was evaporated, the product was recrystallized from diethyl ether, and the products were filtered by vacuum filtration and vacuum dried. Then, the PDS-coupled K2C, K3C, and K4C (K2C−PDS, K3C−PDS, and K4C−PDS) were reacted with activated PEG (molar ratio of activated PEG to amine functional groups of 5:1) in methanol adjusted to pH 8.0 or above by the addition of triethylamine for 24 h at room temperature.20,23,24 The reaction mixture was then evaporated, washed, and concentrated by ultrafiltration. For the deprotection of thiols, the obtained compounds were treated with DTT (molar ratio of DTT to PDS of 20:1) in sodium phosphate buffer (1 mM EDTA, pH 7.5). After incubation for 1 h at room temperature, the products (K2C−PEG, K3C−PEG, and K4C−PEG) were washed and concentrated by ultrafiltration. The thiol groups derived from cysteine were detected by Ellman’s method.22 The average number of PEG chains/ dendrimers was estimated by measuring the number of unreacted free amino groups as reported previously.25,26 The mean diameters and zeta-potentials were measured by Zetasizer Nano (Malvern Instruments Ltd., Worcestershire, U.K.) at 25 °C. 2.4. Scavenging of ROS by PEGylated CysteineModified Lysine Dendrimers. The scavenging potential of K2C−PEG, K3C−PEG, K4C−PEG, L-cysteine, and glutathione on the superoxide anion that was generated by xanthine− xanthine oxidase was evaluated according to previous reports.12,27 The hydrogen peroxide and hydroxyl radical scavenging potential of these thiols was assessed using fluorescent probe methods, as reported previously.12,18,28,29

3. RESULTS 3.1. Physicochemical Characteristics of PEGylated Cysteine-Modified Lysine Dendrimers. Table 1 shows the physicochemical characteristics of K2C−PEG, K3C−PEG, and K4C−PEG. K2C−PEG, K3C−PEG, and K4C−PEG had an average of 2, 8.3, and 14.2 PEG chains per dendrimer, respectively. The particle diameters of the dendrimers increased depending on the generation of dendrimers. The zetapotentials of the dendrimers ranged from 0.04 to −1.14. 3.2. Scavenging of ROS by PEGylated CysteineModified Lysine Dendrimers. Figure 2A−C shows the effects of K2C−PEG, K3C−PEG, K4C−PEG, L-cysteine, and glutathione on the concentrations of ROS, including superoxide anion, hydrogen peroxide, and hydroxyl radicals in sodium phosphate buffer. K2C−PEG, K3C−PEG, K4C−PEG, Lcysteine, and glutathione significantly reduced the levels of superoxide anion, hydrogen peroxide, and hydroxyl radical. Furthermore, treatment with K2C−PEG, K3C−PEG, and K4C−PEG markedly reduced the levels of all three ROS compared with treatment with L-cysteine or glutathione. There were no significant differences in the reduction of hydrogen peroxide between K2C−PEG, K3C−PEG, and K4C−PEG, although treatment with K2C−PEG reduced the levels of superoxide anion and hydroxyl radical slightly more than K3C− PEG or K4C−PEG treatment. 3.3. Distribution of PEGylated Cysteine-Modified Lysine Dendrimers after Intravenous Injection in Mice. C

DOI: 10.1021/acs.molpharmaceut.6b00557 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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contrast, 111In-K2C−PEG and 111In-K3C−PEG quickly disappeared from plasma with accumulation of approximately 60% and 48% of the dose at 6 h in the liver, respectively. Although 111 In-K4C−PEG slightly accumulated in the kidneys, distribution to the spleen, heart, and lungs was hardly observed for all dendrimers. 3.4. Inhibition of Hepatic Ischemia/Reperfusion Injury by PEGylated Cysteine-Modified Lysine Dendrimers. Figure 4 shows the plasma ALT levels 6 h after hepatic

Figure 4. Effect of PEGylated cysteine-modified lysine dendrimers and various thiols on plasma ALT level in mice with hepatic ischemia/ reperfusion. K2C−PEG, K3C−PEG, K4C−PEG, L-cysteine, and glutathione were administered at an equivalent SH dose (4.5 μmol of SH/kg). Results are expressed as the mean ± standard error of at least three mice. *p < 0.05, significantly different from the salinetreated group. ALT: alanine aminotransferase. L-Cys: L-cysteine. GSH: reduced glutathione.

Figure 2. Effects of PEGylated cysteine-modified lysine dendrimers on superoxide anion (A), hydrogen peroxide (B), and hydroxyl radicals (C) in sodium phosphate buffer. The results are expressed as the mean ± standard error of three samples. *p < 0.05 and **p < 0.01, significantly different from the control group. ††p < 0.01, significantly different from the L-cysteine group. ##p < 0.01, significantly different from the glutathione group. L-Cys: L-cysteine. GSH: reduced glutathione.

ischemia/reperfusion. The ALT activity increased and reached a level of approximately 199 ± 48 at 6 h after reperfusion, indicating the induction of hepatic injury. K4C−PEG significantly suppressed the increase in ALT level, whereas K2C−PEG, K3C−PEG, L-cysteine, and glutathione did not significantly affect the plasma ALT levels. K4C−PEG prevented increases in the ALT level by about 35, 47, and 50% compared to levels in mice treated with saline, L-cysteine, and glutathione, respectively. Furthermore, in comparison to L-cysteine and glutathione, K4C−PEG significantly attenuated the decrease in

Figure 3A−C shows the plasma concentrations and tissue distribution of 111In-K2C−PEG, 111In-K3C−PEG, and 111InK4C−PEG after iv in mice. 111In-K4C−PEG exhibited a prolonged retention in the plasma and gradually distributed to the liver to a level of approximately 17% of the dose at 6 h. In

Figure 3. Time courses of plasma concentration and tissue accumulation of 111In-labeled K2C−PEG(A), K3C−PEG(B), and K4C−PEG(C) in mice after intravenous injection at a dose of 1 mg/kg. Results are expressed as the mean ± standard error for three mice. ●, plasma; ■, liver; ▲, kidney; ○, lung; □, spleen; △, heart. D

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K2C, K3C, and K4C a greater opportunity to interact with ROS and increases the ROS scavenging potential of the compounds.18 We demonstrated that the plasma retention of PEGylated cysteine-modified lysine dendrimers was dependent on dendrimer generation and the degree of PEG modification, which is in agreement with previous reports.20,21 The high plasma retention of K4C−PEG could be attributed to fewer interactions with macrophages, other types of cells, and extracellular matrices.23,35 It has been reported that an increase in the number of attached PEG residues increases the plasma retention of lysine dendrimers.21 These results suggest that the lower plasma retention of K2C−PEG and K3C−PEG was because the plasma retention of PEGylated macromolecules is generally proportional to the number of PEG chains. It was reported that chemically modified macromolecules are easily taken up by the mononuclear phagocyte system in the liver (i.e., Kupffer cells).36 Therefore, we propose that the hepatic distribution of PEGylated cysteine-modified lysine dendrimers is caused by Kupffer cells. It was reported that the oxidative stress generated from Kupffer cells and neutrophils is associated with vascular and parenchymal cell injury in hepatic ischemia/reperfusion injury. In hepatic ischemia/reperfusion, activated NADPH oxidase generates ROS in Kupffer cells, leading to recruitment and activation of neutrophils. ROS and several proteases generated from the activated neutrophils may exacerbate tissue injury.10,37,38 It has been reported that targeting ROS scavenging enzymes (superoxide dismutase and catalase) to liver nonparenchymal cells (i.e., Kupffer cells and endothelial cells) is an effective approach for inhibiting hepatic ischemia/ reperfusion injury.10,11 ROS are generated mainly from Kupffer cells during the early stages of reperfusion. However, the preventive effect of PEGylated cysteine-modified lysine dendrimers in hepatic ischemia/reperfusion injury was proportional to their retention in the plasma, and not proportional to their liver accumulation. Therefore, it is reasonable to speculate that PEGylated cysteine-modified lysine dendrimers remaining in the systemic circulation have a greater chance of continuously scavenging ROS. We previously reported that ROS-mediated hepatitis induced by lipopolysaccharide and Dgalactosamine was effectively prevented by an intravenous injection of PEG−BSA-SH, which also remained in the bloodstream for a long period of time.18 These results suggest that prolonged delivery of reduced thiols to hepatic sinusoid (where ROS are generated by Kupffer cells and neutrophils) is an effective approach for the inhibition of hepatic ischemia/ reperfusion injury. Glutathione redox status has been used as an index of oxidative stress in cells.39 Therefore, the recovery of glutathione redox status after treatment with K4C−PEG indicates that K4C−PEG prevented hepatic injury by reducing ischemia/ reperfusion-induced oxidative stress. It was reported that NFκB, a transcription factor, modulated the expression of various inflammatory factors in ischemia/reperfusion injury.40−42 Because ROS are associated with activation of NF-κB,41 we propose that K4C−PEG suppresses the expression of inflammatory factors by scavenging ROS, thereby preventing hepatic ischemia/reperfusion injury. However, additional studies are required to elucidate how K4C−PEG prevents hepatic injury. In conclusion, we successfully developed PEGylated cysteinemodified lysine dendrimers as amino acid dendrimers with

reduced glutathione levels (Figure 5A) and the increase in oxidized glutathione levels (Figure 5B) in the liver, which are indices of oxidative stress.

Figure 5. Effect of various thiols on reduced glutathione (A) and oxidized glutathione (B) levels in the livers of mice 6 h after reperfusion. Each of the thiols was administered at an equivalent SH dose (4.5 μmol of SH/kg). Results are expressed as the mean ± standard error of at least three mice. *p < 0.05 and **p < 0.01, significantly different from the saline-treated group. L-Cys: L-cysteine. GSH: reduced glutathione. GSSG: oxidized glutathione.

4. DISCUSSION In this study, we successfully developed PEGylated cysteinemodified lysine dendrimers as amino acid dendrimers with multiple reduced thiols and improved ROS scavenging activity. The diameters and zeta-potentials of PEGylated cysteinemodified lysine dendrimers are in agreement with previous reports of PEGylated lysine dendrimers.21 Cysteine-modified lysine dendrimers consist of amino acids. Thus, these dendrimers can be considered biocompatible compounds, although the biocompatibility of the dendrimers needs to be examined for future clinical applications. Low molecular weight reduced thiols, including L-cysteine and glutathione, easily form intermolecular disulfide linkages that prevent them from directly reacting with and therefore scavenging ROS.32−34 However, the addition of PEG to K2C, K3C, and K4C compounds may result in steric hindrance, preventing the formation of intermolecular disulfide linkages. This affords E

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protects the rat liver against reperfusion injury after prolonged warm ischemia. Ann. Surg. 2004, 239, 220−231. (16) Yandrapu, S. K.; Kanujia, P.; Chalasani, K. B.; Mangamoori, L.; Kolapalli, R. V.; Chauhan, A. Development and optimization of thiolated dendrimer as a viable mucoadhesive excipient for the controlled drug delivery: an acyclovir model formulation. Nanomedicine 2013, 9, 514−522. (17) Day, B. S.; Fiegland, L. R.; Vint, E. S.; Shen, W.; Morris, J. R.; Norton, M. L. Thiolated dendrimers as multi-point binding headgroups for DNA immobilization on gold. Langmuir 2011, 27, 12434− 12442. (18) Katsumi, H.; Nishikawa, M.; Nishiyama, K.; Hirosaki, R.; Nagamine, N.; Okamoto, H.; Mizuguchi, H.; Kusamori, K.; Yasui, H.; Yamashita, F.; Hashida, M.; Sakane, T.; Yamamoto, A. Development of PEGylated serum albumin with multiple reduced thiols as a longcirculating scavenger of reactive oxygen species for the treatment of fulminant hepatic failure in mice. Free Radical Biol. Med. 2014, 69, 318−323. (19) Ohsaki, M.; Okuda, T.; Wada, A.; Hirayama, T.; Niidome, T.; Aoyagi, H. In vitro gene transfection using dendritic poly(L-lysine). Bioconjugate Chem. 2002, 13, 510−517. (20) Okuda, T.; Kawakami, S.; Maeie, T.; Niidome, T.; Yamashita, F.; Hashida, M. Biodistribution characteristics of amino acid dendrimers and their PEGylated derivatives after intravenous administration. J. Controlled Release 2006, 114, 69−77. (21) Okuda, T.; Kawakami, S.; Akimoto, N.; Niidome, T.; Yamashita, F.; Hashida, M. PEGylated lysine dendrimers for tumor-selective targeting after intravenous injection in tumor-bearing mice. J. Controlled Release 2006, 116, 330−336. (22) Ellman, G. L. Tissue sulfhydryl groups. Arch. Biochem. Biophys. 1959, 82, 70−77. (23) Abuchowski, A.; van Es, T.; Palczuk, N. C.; Davis, F. F. Alteration of immunological properties of bovine serum albumin by covalent attachment of polyethylene glycol. J. Biol. Chem. 1977, 252, 3578−3581. (24) Katsumi, H.; Nishikawa, M.; Yamashita, F.; Hashida, M. Development of polyethylene glycol-conjugated poly-S-nitrosated serum albumin, a novel S-Nitrosothiol for prolonged delivery of nitric oxide in the blood circulation in vivo. J. Pharmacol. Exp. Ther. 2005, 314, 1117−1124. (25) Habeeb, A. F. Determination of free amino groups in proteins by trinitrobenzenesulfonic acid. Anal. Biochem. 1966, 14, 328−336. (26) Hu, T.; Prabhakaran, M.; Acharya, S. A.; Manjula, B. N. Influence of the chemistry of conjugation of poly(ethylene glycol) to Hb on the oxygen-binding and solution properties of the PEG-Hb conjugate. Biochem. J. 2005, 392, 555−564. (27) Hakozaki, T.; Date, A.; Yoshii, T.; Toyokuni, S.; Yasui, H.; Sakurai, H. Visualization and characterization of UVB-induced reactive oxygen species in a human skin equivalent model. Arch. Dermatol. Res. 2008, 300 (Suppl. 1), S51. (28) Maeda, H.; Fukuyasu, Y.; Yoshida, S.; Fukuda, M.; Saeki, K.; Matsuno, H.; Yamauchi, Y.; Yoshida, K.; Hirata, K.; Miyamoto, K. Fluorescent probes for hydrogen peroxide based on a non-oxidative mechanism. Angew. Chem., Int. Ed. 2004, 43, 2389−2391. (29) Setsukinai, K.; Urano, Y.; Kakinuma, K.; Majima, H. J.; Nagano, T. Development of novel fluorescence probes that can reliably detect reactive oxygen species and distinguish specific species. J. Biol. Chem. 2003, 278, 3170−3175. (30) Hnatowich, D. J.; Layne, W. W.; Childs, R. L. The preparation and labeling of DTPA-coupled albumin. Int. J. Appl. Radiat. Isot. 1982, 33 (5), 327−332. (31) Nishikawa, M.; Staud, F.; Takemura, S.; Takakura, Y.; Hashida, M. Pharmacokinetic evaluation of biodistribution data obtained with radiolabeled proteins in mice. Biol. Pharm. Bull. 1999, 22, 214−218. (32) Sokolowska, I.; Ngounou Wetie, A. G.; Woods, A. G.; Darie, C. C. Automatic determination of disulfide bridges in proteins. J. Lab. Autom. 2012, 17, 408−416. (33) Carballal, S.; Radi, R.; Kirk, M. C.; Barnes, S.; Freeman, B. A.; Alvarez, B. Sulfenic acid formation in human serum albumin by

multiple reduced thiols and improved ROS scavenging activity. The therapeutic potential of PEGylated cysteine-modified lysine dendrimers for hepatic injury was proportional to the plasma retention of dendrimers. These findings indicate that PEGylated cysteine-modified lysine dendrimers with prolonged circulation time are promising compounds to inhibit hepatic ischemia/reperfusion injury.

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Corresponding Author

*Phone: +81-75-595-4662. Fax: +81-75-595-4761. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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REFERENCES

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DOI: 10.1021/acs.molpharmaceut.6b00557 Mol. Pharmaceutics XXXX, XXX, XXX−XXX