Interactions of lecithinized superoxide dismutase with serum proteins and cells.

RESEARCH ARTICLE – Pharmaceutical Biotechnology

Interactions of Lecithinized Superoxide Dismutase with Serum Proteins and Cells TSUTOMU ISHIHARA,1 SHUNSUKE NARA,1 TOHRU MIZUSHIMA2 1 2

Department of Chemical Biology and Applied Chemistry, College of Engineering, Nihon University, Fukushima 963-8642, Japan Department of Analytical Chemistry, Faculty of Pharmacy, Keio University, Tokyo 105-8512, Japan

Received 15 April 2014; revised 8 May 2014; accepted 9 May 2014 Published online 27 May 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.24031 ABSTRACT: Superoxide dismutase covalently bound to four lecithin molecules (PC-SOD) is known to be retained in circulating blood for a prolonged period and has a high affinity for cells, resulting in beneficial therapeutic effects in animal disease models. In this study, we evaluated the interaction of PC-SOD with biological components, such as serum proteins and cells, to clarify the mechanism underlying the improved pharmacokinetics of SOD induced by lecithin chemical modification (lecithinization). PC-SOD was distributed in the plasma but not in blood cells after being added to the blood. PC-SOD formed a complex with serum protein(s) such as albumin, whereas unmodified SOD did not. The cellular content of PC-SOD was markedly higher than that of unmodified SOD, and was distributed in lysosomes. The pathway associated with the cellular uptake was found to involve clathrin–/caveolae-independent and cholesterol-sensitive endocytosis. Overall, our data indicated that the increased hydrophobicity of lecithinized SOD enhanced its association to both serum protein(s) and plasma membrane microdomains. The former inhibited SOD excretion and promoted long-term retention in circulating blood, whereas C 2014 Wiley Periodicals, Inc. and the American Pharmacists Association J the latter enhanced internalization into cells via endocytosis. Pharm Sci 103:1987–1994, 2014 Keywords: conjugation; drug delivery systems; macromolecular drug delivery; pharmacokinetics; protein delivery; membrane transport; proteins

INTRODUCTION Since 1980s, the development of genetic engineering has facilitated the production of proteins and peptides, such as cytokines, hormones, and antibodies, as therapeutic pharmaceuticals.1,2 However, intrinsic properties of proteins (e.g., instability, degradation by proteases, rapid excretion, and pleiotropic actions) sometimes limit their clinical applications.2–4 One of the strategies to overcome the limitations is to produce mutant fusion proteins by genetic modification,5 whereas another is chemical conjugation of proteins with various molecules such as synthetic polymers. Oxidative stress induced by elevated levels of reactive oxygen species (ROS) including superoxide anion, results in oxidative damage to DNA, proteins, and lipids, and plays an important role in the mechanism of various diseases, such as hypertension, atherosclerosis, diabetic complications, cancer, and Alzheimer’s disease.6,7 Extensive studies have been carried out for a long-time to develop antioxidants that are capable of scavenging ROS as therapeutic agents. Copper–zinc superoxide dismutase (CuZn-SOD, also known as SOD1, homodimer of 16kDa subunits) is localized in the cytoplasm and catalyzes the dismutation of superoxide anion into hydrogen peroxide and oxygen.8 Genetic overexpression of SOD in animal models of various diseases showed a beneficial effect,9,10 suggesting that SOD is useful as a potential therapeutic antioxidant drug. AlR ) has been clinically used though bovine CuZn-SOD (Orgotein Correspondence to: Tsutomu Ishihara (Telephone: +81-24-956-8805; Fax: +81-24-956-8805; E-mail: [email protected]) This article contains supplementary material available from the authors upon request or via the Internet at http://onlinelibrary.wiley.com/. Journal of Pharmaceutical Sciences, Vol. 103, 1987–1994 (2014)

C 2014 Wiley Periodicals, Inc. and the American Pharmacists Association

in USA and Europe as an anti-inflammatory drug,11 SOD has a two major drawbacks in its therapeutic application, as it has a short life span in blood circulation (half-life: 6–10 min) because of its rapid excretion from the kidney12,13 and low cellular uptake.14 These limitations prompted the development of genetically or chemically modified SOD derivatives to improve its therapeutic characteristics.15 Poly(ethylene glycol) (PEG) has been widely used as a modifying material for proteins, peptides, aptamers, and various types of colloidal carriers.16,17 The modification of proteins with PEG chains leads to altered physicochemical characteristics (e.g., solubility and stability) of proteins, which in turn facilitate the pegylated proteins to remain in the blood circulation for a prolonged duration. As the proteins circulating for prolonged periods have continuous and long-term activity, some pegylated proteins are already being used in clinical settings. Similarly, many researchers have reported the beneficial effect of SOD conjugated with PEG, both in cultured cells and in animal models of diseases.18,19 However, the modification with PEG generally induces reduction in cellular uptake of proteins. Moreover, SOD has been modified with various molecules such as cell penetrating peptides,20,21 antibodies,22 C fragment of tetanus toxin,23 polysaccharides,24 pluronic,25 polyoxazoline,26 and a pyran copolymer27 to alter its pharmacokinetics. Additionally, a carrier for SOD derivatives such as liposomes has also been developed.28,29 However, as far as we know, the therapeutic application of these SOD derivatives in humans has not been approved yet. Copper–zinc superoxide dismutase covalently bound to an average of four molecules of a lecithin derivative (PC-SOD) has also been developed.30,31 PC-SOD exhibits high affinity for cells in vitro,31 prolonged residence in blood circulation, and high accumulation in various tissues in vivo,30,32 compared

Ishihara, Nara, and Mizushima, JOURNAL OF PHARMACEUTICAL SCIENCES 103:1987–1994, 2014

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1988


MATERIALS AND METHODS Materials and Animals

Figure 1. The distribution of FITC-labeled SOD and FITC-labeled PC(H)-SOD in plasma. FITC-labeled SOD or FITC-labeled PC(H)-SOD was added to the blood. After incubation for 10 min, the plasma was collected by centrifugation and the fluorescence intensity of the plasma was measured using fluorescence spectrophotometer.

SOD

PC(L)-SOD

PC(H)-SOD

None

Figure 2. Native polyacrylamide gel electrophoresis of FITC-labeled SOD and FITC-labeled PC-SOD in the presence of excessive amounts of various proteins. The gel was visualized on a transilluminator without staining.

with unmodified SOD. In addition, PC-SOD exhibited beneficial effects in animal models of various diseases, ulcerative colitis,33 bleomycin-induced pulmonary fibrosis,34,35 elastaseinduced emphysema,36 focal cerebral ischemic injury,37 and spinal cord injury-induced motor dysfunction.38 A phase II clinical trial of PC-SOD to treat idiopathic pulmonary fibrosis is actually being carried out in Japan and Republic of Korea. The enhanced therapeutic effects of PC-SOD are proposed to result from its altered biodistribution, but not from its increased enzymatic activity, because PC-SOD exhibited only 83% enzymatic activity in vitro.30 However, the mechanism underlying altered SOD biodistribution induced by lecithin chemical modification remains unclear. Thus, in this study, we attempted to understand the mechanisms by which PC-SOD is retained in the blood circulation and its higher cellular affinity by analyzing the interactions of PC-SOD with biological components, such as serum proteins and cells.

Recombinant human CuZn-SOD (molecular mass: 32 kDa) was supplied by Asahi Kasei Pharma (Tokyo, Japan). 1-Hexadecanoyl-2-(4-hydroxycarbonylbutyroyl)-sn-glycero-3phosphocholine (C3 PC) was supplied by Nippon Fine Chemical Company Ltd. (Osaka, Japan). Fluorescein isothiocyanate isomer I (FITC) was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). C3 PC modified with Nhydroxysuccinimide (NHS-C3 PC, Supplementary Fig. S1) was synthesized according to the literature.30 Male Wistar rats (6-weeks old) were obtained from CLEA Japan, Inc. (Tokyo, Japan), and blood was collected under anesthesia with heparin (Novo-Heparin for injection; Mochida Pharmaceutical Company, Ltd., Tokyo, Japan). The experiments and procedures were carried out in accordance with the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the National Institutes of Health and were approved by the Animal Care and Use Committee of Nihon University. Synthesis of FITC-Labeled PC-SOD Superoxide dismutase (0.606 mM) was mixed with FITC (4.22 mM) in 500-mM sodium carbonate buffer (pH 10.0) for 2 h at room temperature. The solution was dialyzed against distilled water using Spectra/Por7dialysis membrane (MWCO: 1000 Da; Spectrum Lab., Inc., Rancho Dominguez, California) for 2 days, and then it was concentrated using Centriprep Centrifugal Filter Unit (YM-3; MWCO: 3000 Da; EMD Millipore Corporation., Billerica, Massachusetts). FITC-labeled SOD was finally collected by a gel filtration (PD-10 column; GE Healthcare UK Ltd., Buckinghamshire, England) using 5% sucrose aqueous solution as an elution buffer. The molar ratio of FITC bound to SOD was calculated from the amount of FITC determined form the absorbance at 495 nm, and the amount of SOD determined from BCA protein assay (Thermo Fisher Scientific, Inc., Waltham, Massachusetts). Synthesized FITC-labeled SOD was stored at −20◦ C. Four-hundred microliters of 2-propanol was added dropby-drop to 2000 :L of borate buffer (50 mM borate, 50 mM potassium chloride, pH 8.5) containing 4.8 mg of FITClabeled SOD with stirring. To this solution, 1600 :L of 2propanol containing 0.74 or 1.48 mg of NHS-C3 PC was added drop-by-drop. After incubation for 24 h at room temperature, 4 mL of sodium phosphate buffer solution (50 mM, pH 7.2) was added. The solution was concentrated using a Centriprep Centrifugal Filter Unit (YM-3). FITC-labeled PC-SOD was collected by a gel filtration (PD-10 column) using sodium phosphate buffer solution (50 mM, pH 7.2) as an elution buffer. Finally, the solution was centrifuged at 20,000 g for 10 min and the supernatant containing FITC-labeled PC-SOD was stored at −20◦ C. Fluorescein isothiocyanate-labeled PC-SOD were analyzed by HPLC using a TSK gel Phenyl-5PW column (Tosoh Corporation, Tokyo, Japan) with a gradient eluted mobile phase of acetonitrile–water (0.1% trifluoroacetic acid) and elutants were detected by using a UV detector (220 nm) and a fluorescence detector (excitation: 490 nm; emission: 520 nm). In this article, FITC-labeled PC-SOD with lower and higher PC content were shown as PC(L)-SOD and PC(H)-SOD, respectively.


DOI 10.1002/jps.24031


1989

Figure 3. Uptake of FITC-labeled SOD or FITC-labeled PC-SOD in HeLa cells incubated in Opti-MEM or DMEM-containing serum for 2 h at 37◦ C. The nucleus of the cells was stained with Hoechst 33258.

Distribution of FITC-Labeled PC-SOD in the Blood Fifty microgram of FITC-labeled SOD or FITC-labeled PC(H)SOD was added to 500 :L of blood collected from rats. After incubation for 10 min at 37◦ C with gentle shaking, the plasma was collected by centrifugation at 800 g for 10 min. The plasma was diluted 10-fold with Radio-Immunoprecipitation Assay buffer (RIPA bufer; 50 mM Tris–HCl pH 8.0, 150 mM sodium chloride, 1% NP-40, 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate), and the fluorescence intensity of the diluted solution was measured using fluorescence spectrophotometer (excitation: 490 nm; emission: 520 nm; RF5300PC; Shimadzu Corporation, Kyoto, Japan).

Interaction of FITC-Labeled PC-SOD with Serum Proteins Fluorescein isothiocyanate-labeled SOD (100 :g/mL) or FITClabeled PC-SOD (100 :g/mL) was incubated with bovine (globulin (10 mg/mL; Nacalai Tesque, Inc., Kyoto, Japan) or bovine serum albumin (BSA; 10 mg/mL; Sigma–Aldrich, St. Louis, Missouri) in phosphate buffer solution (50 mM sodium phosphate, 200 mM sodium chloride, pH 7.2) for 2 h at 25◦ C. Alternatively, 100 :g/mL of FITC-labeled SOD or FITC-labeled PC-SOD was incubated with the mixture of fetal bovine serum (FBS; Biowest SAS, Nuaill´e, France) and phosphate buffer solution (50 mM sodium phosphate, 200 mM sodium chloride, pH 7.2) (1/1, v/v) for 2 h at 25◦ C. The solutions were analyzed by using 10% polyacrylamide gel electrophoresis in the absence of sodium dodecyl sulfate and reductants (Native-PAGE) with Tris–glycine buffer (25 mM Tris base, 192 mM glycine) as the DOI 10.1002/jps.24031

running buffer. The separated bands were visualized on a UVtransilluminator at 365 nm.

Interaction of FITC-Labeled PC-SOD with Cells HeLa cells (human epithelial carcinoma cell line) and RAW264.7 cells (murine macrophage-like cell line) were provided by the RIKEN BRC through the National Bio-Resource Project of the MEXT Japan, and were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% FBS, streptomycin, and penicillin. HUVEC (normal human umbilical vein endothelial cell) was purchased from Takara Bio Inc. (Shiga, Japan) and was cultured in attached medium (EBM-2). HeLa cells were harvested on the 8-well Nunc Lab-Tek II Chambered Coverglass (Thermo Fisher Scientific) at 5 × 104 cells per well and incubated overnight at 37◦ C in DMEM containing 10% FBS. The cells were washed with phosphate-buffered saline (PBS) and incubated with 50 :g/mL of FITC-labeled SOD or FITC-labeled PC-SOD in Opti-MEM (Opti-MEM I Reduced Serum Medium; Life Technologies Corporation, Carlsbad, California) or DMEM containing 10% FBS. After incubation for a designated period at 37◦ C, the cells were washed with PBS three times and were fixed in 2% paraformaldehyde solution for 30 min at room temperature. In this process, nucleus of cells was also stained with 5 :g/mL of Hoechst 33258 (Dojindo Laboratories, Kumamoto, Japan). The fluorescence images of cells were captured using a DS-Fi1c digital camera connected to Nikon DS-L2 controller (Nikon Corporation, Tokyo, Japan). Lysosomes in cells were costained using Lysotracker-Red DND99 (Life Technologies Corporation). The cells were incubated


1990


fluorescence spectrophotometer (excitation: 490 nm; emission: 520 nm) and the protein concentration of the lysate were determined by BCA protein assay (Thermo Fisher Scientific). The cellular content of FITC-labeled PC(H)-SOD in cells incubated with several endocytosis inhibitors were also determined. HeLa cells were preincubated with endocytosis inhibitors in Opti-MEM for 30 min at 37◦ C. After the addition of FITC-labeled PC(H)-SOD to the medium at 50 :g/mL, the cells were incubated for 1 h at 37◦ C. The cellular content of FITC-labeled PC(H)-SOD was determined according to the method mentioned above. Endocytosis inhibitors were added to the medium at the following concentrations: chlorpromazine hydrochloride (30 :M; Tokyo Chemical Industry Company. Ltd., Tokyo, Japan), dansylcadaverine (150 :M; Sigma– Aldrich), cytochalasin D (30 :M; Wako Pure Chemical Industries, Ltd.), wortmannin (200 nM, Funakoshi Company, Ltd., Tokyo, Japan), methyl-$-cyclodextrin (M$CD; 5 mM; Sigma– Aldrich), filipin III (10 :M; Funakoshi Company, Ltd.), and nystatin (30 :M; Sigma–Aldrich).

RESULTS AND DISCUSSION

Figure 4. The time course of the uptake of FITC-labeled PC(H)-SOD in HeLa cells during incubation in Opti-MEM or DMEM-containing serum. (a) The content of PC(H)-SOD in cells was determined by fluorescence detection, and (b) the cells incubated in Opti-MEM were observed using a fluorescence microscope.

with FITC-labeled PC(H)-SOD and Lysotracker-Red DND-99 in Opti-MEM for 2 h at 37◦ C. The cellular content of FITC-labeled SOD and FITC-labeled PC(H)-SOD was also determined. HeLa cells were harvested on the 96-well plate at 1.5 × 104 cells per well and incubated overnight at 37◦ C in DMEM containing 10% FBS. The cells were washed with PBS and incubated with 50 :g/mL of FITC-labeled SOD or FITC-labeled PC(H)-SOD in Opti-MEM or DMEM containing 10% FBS. After incubation for a designated period at 37◦ C, the cells were washed with PBS three times. After the addition of 100 :L of RIPA buffer to each well, the wells were allowed to stand for 30 min at 37◦ C. After gently pipetting out the contents in each well, the resultant cell lysates were diluted 10-fold with RIPA buffer. The concentration of FITC-labeled PC(H)-SOD in the lysate was determined using

Fluorescein isothiocyanate-labeled PC-SOD was analyzed by HPLC on a hydrophobic interaction chromatography column. The retention time of the peak of SOD detected by absorbance at 220 nm was delayed with the modification of FITC (Supplementary Fig. S2a). Moreover, the peaks were detected at the same retention time by the fluorescence detection (Supplementary Fig. S2b), indicating the modification of SOD with FITC. The molar ratio of FITC to SOD in FITC-labeled SOD was calculated as 1.8. After reacting FITC-labeled SOD with NHS-C3 PC, the retention time of the reactant was further delayed depending on the amount of NHS-C3 PC added, indicating that FITC-labeled PC-SOD with different PC content was synthesized successfully. The photometrical analysis using 2,4,6trinitrobenzenesulfonic acid sodium salt30 indicated that the molecular numbers of lecithin bound to SOD was on an average 2.5 and 3.7, respectively. In this article, FITC-labeled PC-SOD with lower and higher PC content was shown as PC(L)-SOD and PC(H)-SOD, respectively. After incubation of FITC-labeled SOD or FITC-labeled PC(H)-SOD in the blood, its distribution in plasma and blood cells was examined by the analysis of fluorescence detection (Fig. 1), which indicated that most of them were found in plasma, but not in blood cells. Furthermore, blood cells containing fluorescence dye (FITC) were not observed using a fluorescence microscope (data not shown). Next, we examined an interaction of PC-SOD with serum proteins. FITC-labeled SOD and FITC-labeled PC-SOD with excessive amounts of (-globulin, albumin, or FBS were analyzed by Native-PAGE (Fig. 2). The bands of SOD were not shifted in the presence of any serum proteins. On the contrary, the bands of PC-SOD were shifted in the presence of albumin and FBS, but not (-globulin, indicating a complex formation of PC-SOD with serum protein(s) such as albumin. It is well known that a number of drugs with low molecular weight reversibly bind to plasma proteins (e.g., albumin, "1 acid glycoprotein, and globulin), based on various interactions such as hydrogen bond, hydrophobic interaction, ionic interaction, and van der Waals force.39 The drug–serum protein binding is critically involved in determining the pharmacokinetics.


DOI 10.1002/jps.24031


1991

Figure 5. Intercellular distribution of FITC-labeled PC(H)-SOD in HeLa cells. Lysosome and nucleus were costained using Lysotracker Red and Hoechst 33258, respectively.

The binding affinity greatly depends on the properties of both drugs and serum proteins, for example, albumin shows higher binding affinity for lipophilic acidic drugs, and globulin shows higher affinity for cholesterol and lipophilic vitamins, respectively. The band shift of PC(H)-SOD in the presence of albumin is obvious compared with that of PC(L)-SOD, suggesting that lecithin molecules bound to SOD altered the physicochemical characteristics of SOD to facilitate PC-SOD binding to serum protein(s). Although the binding interaction(s) between these proteins was not clarified, increased hydrophobicity and/or decreased isoelectric point (∼4.2)30 of PC-SOD might have influenced the interaction. As reported previously, the biodistribution study of SOD in rats by enzyme-linked immunosorbent assay showed that SOD was immediately accumulated in the kidney.30 As SOD is easily filtrated from glomerulus because of its relatively lower molecular weight (32 kDa), this accumulation occurs. On the contrary, PC-SOD showed longer circulation time in blood, reduced accumulation in the kidney, and enhanced accumulation in other tissues.30 Taken together, our findings suggest that PC-SOD is relatively difficult to filtrate from glomeruli, because of an increase in its apparent molecular weight because of rapid complex formation with serum protein(s) such as albumin. Next, we examined the cellular uptake of PC-SOD. HeLa cells were incubated with FITC-labeled SOD or FITC-labeled PC-SOD in Opti-MEM without serum or DMEM with serum DOI 10.1002/jps.24031

for 2 h at 37◦ C (Fig. 3). PC(H)-SOD showed notable internalization in the cells incubated in Opti-MEM, whereas SOD was not internalized. The PC(L)-SOD content in the cells was lower than that of PC(H)-SOD (data not shown), suggesting that the increased hydrophobicity of SOD by lecithinization enhanced its affinity for the cells. PC(H)-SOD internalization in cells incubated in DMEM-containing serum was lower than that in cells incubated in Opti-MEM (Figs. 3 and 4a). The inhibitory effect of serum on PC-SOD uptake indicated that PC-SOD preferentially formed complexes with serum protein(s). A low level of PC-SOD uptake was observed in the presence of serum, indicating that PC-SOD that had been dissociated from the serum protein(s) could be internalized in the cells. The cellular content of PC(H)-SOD gradually increased when cells were incubated in Opti-MEM (Fig. 4a). PC(H)-SOD was internalized in cells after only 10 min of incubation (Fig. 4b), indicating a short-term trafficking route for PC(H)-SOD internalization. In contrast, the cellular content of SOD was below the detection limit. Distribution of PC(H)-SOD in cells was examined in further detail. Lysosomes and nuclei were stained using Lysotracker Red and Hoechst 33258, respectively, as shown in Figure 5. The merged image of FITC and Hoechst 33258 indicated that PC(H)-SOD was not localized to nuclei. The merged image of FITC and Lysotracker Red, on the other hand, indicated that the majority of PC(H)-SOD was distributed to lysosomes, although it was unclear whether PC-SOD was located inside of


Relative PC(H)-SOD uptake (%)

1992


140 120 100 80 60 40 20

*

0

Figure 6. The effect of various endocytosis inhibitors on internalization of FITC-labeled PC(H)-SOD in HeLa cells. Each datum represents the mean ± SD of three independent wells. Asterisk indicates significance at p < 0.01 versus none.

lysosomes or on the membranes of lysosomes. Thus, these results suggested that PC(H)-SOD was internalized into cells via endocytosis. The intracellular location of SOD as well as its content in cells affects its therapeutic activity. In a previous report, PC-SOD exhibited higher enzymatic activity than unmodified SOD in the cultured cells,31 indicating that PC-SOD internalized via endocytosis also exhibited enzymatic activity. Endocytosis is a fundamental process to internalize extracellular molecules into eukaryotic cells. Current studies indicate that endocytosis occurs by multiple mechanisms, including macropinocytosis, phagocytosis, clathrin-dependent endocytosis, caveolae-dependent endocytosis, and other forms of endocytosis,40,41 although some mechanisms are still poorly characterized. In general, the endocytic pathways are distinguished based on their differential sensitivity to pharmacological/chemical inhibitors, although the use of these inhibitors has potentially poor specificity.42,43 In this study, the pathway of PCSOD uptake in cells was examined using various endocytosis inhibitors. Chlorpromazine and dansylcadaverine are generally used as inhibitors for clathrin-dependent endocytosis.42 Although it is still debatable whether they also inhibit macropinocytosis and phagocytosis, studies have not reported their inhibitory effect on caveolae-dependent pathway.42 On the other hand, wortmannin (inhibitor for phosphatidylinositol-3-kinase) and cytocalacin D (inhibitor for actin polymerization) are well known to block macropinocytosis and phagocytosis, whereas studies also indicated that these inhibitors have the potential for blocking clathrin–/caveolae-dependent endocytosis.42 As shown in Figure 6, the cellular uptake of PC-SOD was not inhibited in the presence of these inhibitors, indicating that the pathway was not involved in clathrin-dependent endocytosis, macropinocytosis, and phagocytosis. Filipin III and nystatin are known to form multimeric globular complexes with cholesterol on the plasma membrane, disrupting the function of the lipid rafts.42,44 Lipid rafts are microdomains of the plasma membrane enriched in cholesterol and sphingolipids, and play many important roles in cell signal transduction and intracellular internalization of endoge-

nous molecules.45,46 Moreover, lipid rafts are essential to form the primary endocytic vehicle in clathrin-independent endocytic pathways such as caveolae-dependent endocytosis.41,47,48 Filipin III and nystatin did not inhibit the cellular uptake of PC-SOD (Fig. 6), suggesting that this pathway is not involved in lipid rafts-mediated endocytosis including caveolae-dependent endocytosis. On the other hand, M$CD is a cyclic oligosaccharide and extract cholesterol from the plasma membrane by binding it with high affinity, inducing disruption of the structure of membrane lipid rafts.42,49 Interestingly, despite their similar inhibitory effect, M$CD significantly inhibited the uptake of PC-SOD. Furthermore, the uptake in the cells washed by PBS after treatment with M$CD was also inhibited (data not shown), indicating that the inhibition was not caused by the direct interaction between PC-SOD and M$CD. The depletion of cholesterol on cell membrane by M$CD has an effect on the diverse cellular functions/structures such as signal transduction and cytoskeleton as well as lipid rafts, resulting in nonspecific inhibition for many endocytic pathways, clathrin– /caveolae-dependent endocytosis, macropinocytosis, and other types of endocytosis,42,43 whereas filipin III and nystatin seem to be selective inhibitors for lipid raft-mediated and caveolaedependent endocytosis.42 In addition, the observation of cells treated with M$CD by fluorescence microscope revealed that PC-SOD was not distributed both in cytoplasm and on surface membrane (data not shown). Thus, the physicochemical characteristics of cell membrane/domain altered by cholesterol depletion probably led to reduced affinity of PC-SOD to the cell surface. Taken together, our data suggested that the increased hydrophobicity of lecithinized SOD induced its association with cell membrane microdomains, thereby allowing PCSOD to internalize via endocytic pathways. Although further studies are necessary to define the precise endocytic pathway involved in PC-SOD internalization, the pathway does not involve clathrin–/caveolae-dependent endocytosis, macropinocytosis, or phagocytosis, but could be related to another type of endocytosis that is sensitive to cholesterol depletion. We further examined the uptake of FITC-labeled PC-SOD in different types of cells, such as HUVEC and RAW264.7 cells. The results were similar to that obtained from HeLa cells. PC(H)-SOD was remarkably internalized in these cells in serum-free medium (Supplementary Fig. S3), whereas the uptake was suppressed in the presence of serum. In contrast, SOD was not internalized in both mediums. Thus, the data suggested that PC-SOD is internalized in diverse types of cells in various tissues in a nonspecific manner. The technique of chemical modification of SOD with lecithin enabled both prolonged blood circulation and higher affinity for cells. Consequently, PC-SOD was more effective than unmodified SOD, PEG-SOD, and pyran copolymer-bound SOD in the suppression of ischemic mouse paw edema as reported previously.31 It is noteworthy that this unique technique of modification of protein with lecithin may enhance the production of various protein-based therapeutics. In this study, PC(H)-SOD exhibited higher affinity for cells and serum protein(s) as compared with PC(L)-SOD. Although the accurate role of lecithin is poorly clarified, the enhanced hydrophobicity of SOD may be crucial. It has also been reported that PC-SOD bound to 10 lecithin molecules exhibited slightly higher affinity for cells and longer plasma retention than PC-SOD bound to four lecithin molecules.30 On the contrary, the therapeutic effect of PC-SOD with 10 lecithin molecules was lower probably because of its


DOI 10.1002/jps.24031


Lecithin

Serum protein

SOD

SOD

PC-SOD

Complex formation

Cholesterol-sensitive endocytosis

SOD

SOD SOD Lysosome

Prolonged residence in blood (inhibition of excretion)

SOD

Cell

SOD

Figure 7. Schematic illustration of PC-SOD interacting with biological components.

reduced enzyme activity. Thus, when applying the technique for other proteins, it may be necessary to optimize the number of lecithin molecules bound to protein to maintain the required enzyme activity and hydrophobicity.

CONCLUSIONS Although PC-SOD reportedly circulates for a prolonged period in the blood and has higher affinity for cells, the underlying mechanisms controlling these properties have not been clarified. In this study, we evaluated the interaction of PC-SOD with biological components, namely, serum proteins and cells. Our data suggest that the increased hydrophobicity of lecithinized SOD enhanced its rapid and reversible complex formation with serum protein(s), resulting in longer retention in circulating blood. Lecithinization also enhanced the association of SOD to plasma membrane microdomains. Thus, dissociated PC-SOD in circulating blood is internalized into the cells of various tissues via cholesterol-sensitive endocytosis (Fig. 7). The altered biodistribution of PC-SOD in vivo results in beneficial therapeutic effects in various diseases.

ACKNOWLEDGMENT This work was supported in part by grants from the New Energy and Industrial Technology Development Organization (NEDO) of Japan.

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DOI 10.1002/jps.24031

catalase mimetics on human breast cancer cells.

19F NMR study of the interactions of fluoride with superoxide dismutase and hemoglobin in erythrocytes.

Quantitative analysis of extracellular-superoxide dismutase in serum and urine by ELISA with monoclonal antibody.

Effect of superoxide dismutase‑entrapped liposomes and protein transduction domain‑superoxide dismutase on human umbilical vein endothelial cells.

Serum Superoxide Dismutase Is Associated with Vascular Structure and Function in Hypertensive and Diabetic Patients.

Superoxide dismutase and Parkinson's disease.

Reconstitution of iron-superoxide dismutase.

Polarographic determination of superoxide dismutase.

Rickettsia rickettsii induces superoxide radical and superoxide dismutase in human endothelial cells.

Assay of superoxide dismutase applicable to whole bacterial cells.

Double-blind controlled trial of lecithinized superoxide dismutase in patients with idiopathic interstitial pneumonia - short term evaluation of safety and tolerability.

Superoxide dismutase activity and superoxide dismutase-1 gene methylation in normal and tumoral human breast tissues.

Radioprotection of bone marrow stem cells by superoxide dismutase.

zinc-superoxide dismutase transgenic mice.

Superoxide dismutase: "positive" spectrophotometric assays.

Lowered superoxide dismutase in highly metastatic B16 melanoma cells.

Superoxide dismutase and pulmonary ozone toxicity.

Superoxide dismutase: pharmacological developments and applications.

Extracellular superoxide dismutase regulates the expression of small gtpase regulatory proteins GEFs, GAPs, and GDI.

Active subunits from superoxide dismutase.

Evolutionary relationships in superoxide dismutase.

Relation of amiodarone hepatic and pulmonary toxicity to serum drug concentrations and superoxide dismutase activity.

Isoelectric focusing of superoxide dismutase isoenzymes.

Radioprotection of mice by superoxide dismutase.