A novel biointerface that suppresses cell morphological changes by scavenging excess reactive oxygen species Yutaka Ikeda,1 Tomoki Yoshinari,1 Yukio Nagasaki1,2,3 1
Department of Materials Science, Graduate School of Pure and Applied Sciences, University of Tsukuba, Tennodai 1-1-1, Tsukuba, Ibaraki 305-8573, Japan 2 Master’s School of Medical Sciences, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tennodai 1-1-1, Tsukuba, Ibaraki 305-8573, Japan 3 Satellite Laboratory, International Center for Materials Nanoarchitectonics (WPI-MANA), National Institute of Materials Science (NIMS), Tennodai 1-1-1, Tsukuba, Ibaraki 305-8573, Japan Received 11 December 2014; revised 4 February 2015; accepted 5 February 2015 Published online 26 February 2015 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.a.35419 Abstract: During cell cultivation on conventional culture dishes, various events results in strong stresses that lead to the production of bioactive species such as reactive oxygen species (ROS) and nitric oxide. These reactive species cause variable damage to cells and stimulate cellular responses. Here, we report the design of a novel biocompatible surface that decreases stress by not only morphologically modifying the dish surface by using poly(ethylene glycol) tethered chains, but also actively scavenging oxidative stress by using our novel nitroxide radical-containing polymer. A block copolymer, poly(ethylene glycol)-b-poly[(2,2,6,6-tetramethylpiperidine-N-oxyl)aminomethylstyrene] (PEG-b-PMNT) was used to coat the surface of a dish. Differentiation of undifferentiated human leukemia (HL-60) cells was found to be suppressed on the polymer-coated dish. Notably, HL-60 cell cultivation caused
apoptosis under high-density conditions, while spontaneous apoptosis was suppressed in cells plated on the PEG-b-PMNTmodified surface, because a healthy mitochondrial membrane potential was maintained. In contrast, low molecular weight antioxidants did not have apparent effects on the maintenance of mitochondria. We attribute this to the lack of cellular internalization of our immobilized polymer and selective scavenging of excessive ROS generated outside of cells. These results demonstrate the utility of our novel biocompatible material for actively scavenging ROS and thus maintaining cellular morC 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part A: phology. V 103A: 2815–2822, 2015.
Key Words: oxidative stress, cell morphology, suppression of cell activation, scaffold, PEG
How to cite this article: Ikeda Y, Yoshinari T, Nagasaki Y. 2015. A novel biointerface that suppresses cell morphological changes by scavenging excess reactive oxygen species. J Biomed Mater Res Part A 2015:103A:2815–2822.
INTRODUCTION
Reactive oxygen species (ROS) play important roles in regulating cellular morphology.1 In particular, mitochondrial ROS are involved in the regulation of cellular physiology including differentiation, autophagy, metabolic adaptation, apoptosis, and immunity.1,2 The balance between generation and elimination of ROS is essential for signaling pathway maintenance. Redox balance disruptions lead to changes in cellular morphology and disturb cellular homeostasis. For example, ROS cause chromosome instability, which eventually leads to immortalization and transformation of mouse mesenchymal stem cells.3,4 Moreover, excessive ROS levels cause senescence or apoptosis in hematopoietic stem cells.5 Therefore, suppression of excessively generated ROS is essential in order to maintain cellular homeostasis.6 Low molecular weight antioxidants (LMWAs) such as ascorbic acid and N-acetyl cysteine (NAC) have been used to reduce oxidative damage. However,
LMWAs are not always appropriate for use in cell culture because several of these antioxidants have been reported to cause cellular damage, morphological changes, and prooxidant effects.7,8 Thus, alternative approaches for scavenging excess ROS should be explored. Excessive ROS are generated in response to cell contact with biomaterials,9 cell–cell contact, or shear stress that occurs during cell culture.10 Contact with biomaterials causes complex reactions at cell surfaces, generating a variety of ROS that stimulate cellular responses including immune cell activation.11 These findings indicate that biomaterial design, particularly the surface design of biodevices, has significant effects on their compatibility with biological substances. Several polymers such as poly (ethylene glycol) (PEG),12 zwitterionic polymers,13–15 microphase-separated polymers,16,17 and poly(2-methoxyethyl acrylate)18 have been used as coating agents to
Additional Supporting Information may be found in the online version of this article. Correspondence to: Y. Nagasaki; e-mail: [email protected] Contract grant sponsor: Grant-in-Aid for Scientific Research S; contract grant number: 25220203
C 2015 WILEY PERIODICALS, INC. V
2815
reduce adhesion of cells to biodevice surfaces. These polymers are thought to prevent protein adsorption because of their physicochemical characteristics such as repulsive entropic force of the polymer brush and surface water structures. However, none of these polymers completely suppresses contact activation of cells in culture experiments. Recently, we reported a novel surface composed of polymers containing nitroxide radicals, 2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO), which catalytically reacts with ROS and reduces ROS levels.19 This surface was found to strongly suppress blood cell activation, particularly that of platelets and leucocytes, and resulted in almost no coagulation on the modified surface. Thus, ROS scavenging might be an effective strategy for actively suppressing activation of cells and cell–surface biomolecules. In this work, we report the design of a new biocompatible surface with ROSscavenging properties as well as a physicochemically bioinert surface formed from a densely packed PEG-tethered chain surface (PEG brush). Furthermore, we assess the utility of our ROS-scavenging polymer as a novel biocompatible material for maintaining cell morphology in culture by comparing it with LMWAs. MATERIAL AND METHODS
Materials Carbodiimide hydrochloride, amino acids, ascorbic acid, tocopherol, 4-amino-TEMPO, NAC, nitroblue tetrazolium, 12O-tetradecanoylphorbol 13-acetate, and JC-1 (5,50 ,6,60 -tetrachloro-1,10 ,3,30 tetraethylbenzimidazolylcarbocyanine iodide) were used without further purification. Water was purified using the Milli-Q system (Millipore, Molsheim, France). Measurements Electron paramagnetic resonance (ESR) spectra were recorded at room temperature by using a Bruker BioSpin EMXPlus 9.5/2.7 spectrometer operating at 2.0 GHz with a 100-kHz magnetic field modulation. Spectra were collected using the following parameters: sweep width, 2 G; microwave power, 0.200 mW; receiver gain, 1.00 3 103; time constant, 20.48 ms; and conversion time, 40.0 ms. Flow cytometric data were collected for 2,000 events by using a Guava EasyCyte Mini System (Guava Technologies) and analyzed using CytoSoft software (version 4.1). Polymer preparation and coating of cell culture dishes Nitroxide radical-containing poly(ethylene glycol)-bpoly[(2,2,6,6-tetramethylpiperidine-N-oxyl)aminomethylstyrene] (PEG-b-PMNT) polymers were prepared according to previously reported methods.20 As a control polymer, poly(ethylene glycol)-b-poly[(cyclohexyl)aminomethylstyrene] PEG-b-PCHAMS was prepared using the same procedure, except that cyclohexylamine was used in place of 4-aminoTEMPO. The synthesis and analytical data for PEG-bPCHAMS are described in the Supporting Information (Fig. S1). To polymer-coat 24-well cell culture dishes (PureCoat carboxyl dish, BD Biosciences, Franklin Lakes, NJ), carboxyl groups were activated with 20 mg of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) in metha-
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nol (300 lL), and was then reacted with polymer (30 mg) in methanol (300 lL) overnight. The culture dish was washed 25 times with 1M NaCl solution (200 lL) to remove excess polymers that interacted electrostatically with the dish surface. This was followed by two washes with phosphate-buffered saline (PBS). The amount of immobilized polymer on the surface was quantified by measuring recovered polymer that had been released from the dish in response to dimethyl sulfoxide (DMSO) treatment. Preparation of LMWA-free cell culture medium Cell culture medium lacking LMWAs was prepared as follows: L-(1)-arginine (200 mg), L-asparagine (50 mg), DL-asparaginic acid (20 mg), L-cystine dihydrochloride (65.2 mg), L-glutamic acid (20 mg), glycine (10 mg), L-histidine (15 mg), Lleucine (50 mg), L-isoleucine (50 mg), L-(1) lysine hydrochloride (40 mg), L-methionine (15 mg), L-phenylalanine (15 mg), L-proline (20 mg), hydroxyl-L-proline (20 mg), L-(-)-threonine (20 mg), L-tryptophan (5 mg), L-(-)-tyrosine (20 mg), L-valine (10 mg), L-serine (30 mg), D-(1)-glucose (2.0 g), calcium nitrate tetrahydrate (100 mg), magnesium sulfate (49 mg), KCl (400 mg), NaCl (6.0 g), and disodium hydrogen phosphate dodecahydrate (400 mg) were dissolved in 950 mL of ultrapure water. The pH was adjusted to 4 by using 1M and 0.1M HCl, and ultrapure water was added to obtain a final volume of 1.0 L. The resulting solution was sterilized in an autoclave. After autoclaving, NaHCO3 (2.0 g) and 200 mM L-glutamine (10 mL) were added to adjust the pH to 7.4. Cell culture Undifferentiated human leukemia cells (HL-60) from the Riken Cell Bank (Ibaraki, Japan) were maintained in LMWAfree culture medium supplemented with 10% heatinactivated fetal bovine serum. Cell number and viability were assessed using the trypan blue dye-exclusion method. Cellular differentiation in the presence of butyric acid HL-60 cells (1 3 105 cells mL21) were cultured in LMWAfree medium in a 5% CO2 atmosphere in a 24-well plate (BD PureCoat carboxyl dish) in the presence and absence of the polymer coating. Cells were incubated with butyric acid at a final concentration of 0.4 mM. After 48 h of incubation, cells were transferred into another dish that was not coated with PEG-b-PMNT, because immobilized TEMPO might have interfered with subsequent cell differentiation analyses. HL-60 cell differentiation was assessed using nitroblue tetrazolium (NBT) dye reduction as described previously.21,22 In brief, cells were incubated with NBT (4 mg mL21) and freshly prepared 12-O-Tetradecanoylphorbol 13-acetate (TPA) (5 lg mL21), which induces phagocytosis-associated oxidative metabolism, generating superoxide anions, and reduces NBT dye. Differential counts were then performed using light microscopy. A minimum of 300 cells were assessed. Induction of cellular differentiation by using LMWAs HL-60 cells (1 3 105 cells/well) were cultured in LMWAfree medium in a 24-well plate (BD PureCoat carboxyl dish)
SUPPRESSION OF CELL MORPHOLOGICAL CHANGES BY SCAVENGING EXCESS REACTIVE OXYGEN SPECIES
ORIGINAL ARTICLE
in the absence of polymer coating. To the medium, one of four types of LMWAs—ascorbic acid (final concentration 50 lM), tocopherol (final concentration 50 lM), 4-aminoTEMPO (final concentration 50 lM), or NAC (final concentration 5 mM)—was added. Mitochondrial membrane potential measurements Mitochondrial membrane potential was quantified by performing flow cytometric analysis on cells stained with JC-1. HL-60 cells (5 3 105 cells mL21) were cultured in LMWAfree medium in a 24-well plate in the presence or absence of polymer coating. To evaluate the effects of LMWAs on mitochondrial membrane potential, HL-60 cells (5 3 105 cells mL21) were also cultured in the presence of LMWAs under the described conditions for 48 h. After 48 h of incubation, cells were stained using JC-1 and then analyzed using flow cytometry, according to the manufacturer’s instructions. Statistical analysis Statistical analyses were performed using the Student’s t test. A p values of
Department of Materials Science, Graduate School of Pure and Applied Sciences, University of Tsukuba, Tennodai 1-1-1, Tsukuba, Ibaraki 305-8573, Japan 2 Master’s School of Medical Sciences, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tennodai 1-1-1, Tsukuba, Ibaraki 305-8573, Japan 3 Satellite Laboratory, International Center for Materials Nanoarchitectonics (WPI-MANA), National Institute of Materials Science (NIMS), Tennodai 1-1-1, Tsukuba, Ibaraki 305-8573, Japan Received 11 December 2014; revised 4 February 2015; accepted 5 February 2015 Published online 26 February 2015 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.a.35419 Abstract: During cell cultivation on conventional culture dishes, various events results in strong stresses that lead to the production of bioactive species such as reactive oxygen species (ROS) and nitric oxide. These reactive species cause variable damage to cells and stimulate cellular responses. Here, we report the design of a novel biocompatible surface that decreases stress by not only morphologically modifying the dish surface by using poly(ethylene glycol) tethered chains, but also actively scavenging oxidative stress by using our novel nitroxide radical-containing polymer. A block copolymer, poly(ethylene glycol)-b-poly[(2,2,6,6-tetramethylpiperidine-N-oxyl)aminomethylstyrene] (PEG-b-PMNT) was used to coat the surface of a dish. Differentiation of undifferentiated human leukemia (HL-60) cells was found to be suppressed on the polymer-coated dish. Notably, HL-60 cell cultivation caused
apoptosis under high-density conditions, while spontaneous apoptosis was suppressed in cells plated on the PEG-b-PMNTmodified surface, because a healthy mitochondrial membrane potential was maintained. In contrast, low molecular weight antioxidants did not have apparent effects on the maintenance of mitochondria. We attribute this to the lack of cellular internalization of our immobilized polymer and selective scavenging of excessive ROS generated outside of cells. These results demonstrate the utility of our novel biocompatible material for actively scavenging ROS and thus maintaining cellular morC 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part A: phology. V 103A: 2815–2822, 2015.
Key Words: oxidative stress, cell morphology, suppression of cell activation, scaffold, PEG
How to cite this article: Ikeda Y, Yoshinari T, Nagasaki Y. 2015. A novel biointerface that suppresses cell morphological changes by scavenging excess reactive oxygen species. J Biomed Mater Res Part A 2015:103A:2815–2822.
INTRODUCTION
Reactive oxygen species (ROS) play important roles in regulating cellular morphology.1 In particular, mitochondrial ROS are involved in the regulation of cellular physiology including differentiation, autophagy, metabolic adaptation, apoptosis, and immunity.1,2 The balance between generation and elimination of ROS is essential for signaling pathway maintenance. Redox balance disruptions lead to changes in cellular morphology and disturb cellular homeostasis. For example, ROS cause chromosome instability, which eventually leads to immortalization and transformation of mouse mesenchymal stem cells.3,4 Moreover, excessive ROS levels cause senescence or apoptosis in hematopoietic stem cells.5 Therefore, suppression of excessively generated ROS is essential in order to maintain cellular homeostasis.6 Low molecular weight antioxidants (LMWAs) such as ascorbic acid and N-acetyl cysteine (NAC) have been used to reduce oxidative damage. However,
LMWAs are not always appropriate for use in cell culture because several of these antioxidants have been reported to cause cellular damage, morphological changes, and prooxidant effects.7,8 Thus, alternative approaches for scavenging excess ROS should be explored. Excessive ROS are generated in response to cell contact with biomaterials,9 cell–cell contact, or shear stress that occurs during cell culture.10 Contact with biomaterials causes complex reactions at cell surfaces, generating a variety of ROS that stimulate cellular responses including immune cell activation.11 These findings indicate that biomaterial design, particularly the surface design of biodevices, has significant effects on their compatibility with biological substances. Several polymers such as poly (ethylene glycol) (PEG),12 zwitterionic polymers,13–15 microphase-separated polymers,16,17 and poly(2-methoxyethyl acrylate)18 have been used as coating agents to
Additional Supporting Information may be found in the online version of this article. Correspondence to: Y. Nagasaki; e-mail: [email protected] Contract grant sponsor: Grant-in-Aid for Scientific Research S; contract grant number: 25220203
C 2015 WILEY PERIODICALS, INC. V
2815
reduce adhesion of cells to biodevice surfaces. These polymers are thought to prevent protein adsorption because of their physicochemical characteristics such as repulsive entropic force of the polymer brush and surface water structures. However, none of these polymers completely suppresses contact activation of cells in culture experiments. Recently, we reported a novel surface composed of polymers containing nitroxide radicals, 2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO), which catalytically reacts with ROS and reduces ROS levels.19 This surface was found to strongly suppress blood cell activation, particularly that of platelets and leucocytes, and resulted in almost no coagulation on the modified surface. Thus, ROS scavenging might be an effective strategy for actively suppressing activation of cells and cell–surface biomolecules. In this work, we report the design of a new biocompatible surface with ROSscavenging properties as well as a physicochemically bioinert surface formed from a densely packed PEG-tethered chain surface (PEG brush). Furthermore, we assess the utility of our ROS-scavenging polymer as a novel biocompatible material for maintaining cell morphology in culture by comparing it with LMWAs. MATERIAL AND METHODS
Materials Carbodiimide hydrochloride, amino acids, ascorbic acid, tocopherol, 4-amino-TEMPO, NAC, nitroblue tetrazolium, 12O-tetradecanoylphorbol 13-acetate, and JC-1 (5,50 ,6,60 -tetrachloro-1,10 ,3,30 tetraethylbenzimidazolylcarbocyanine iodide) were used without further purification. Water was purified using the Milli-Q system (Millipore, Molsheim, France). Measurements Electron paramagnetic resonance (ESR) spectra were recorded at room temperature by using a Bruker BioSpin EMXPlus 9.5/2.7 spectrometer operating at 2.0 GHz with a 100-kHz magnetic field modulation. Spectra were collected using the following parameters: sweep width, 2 G; microwave power, 0.200 mW; receiver gain, 1.00 3 103; time constant, 20.48 ms; and conversion time, 40.0 ms. Flow cytometric data were collected for 2,000 events by using a Guava EasyCyte Mini System (Guava Technologies) and analyzed using CytoSoft software (version 4.1). Polymer preparation and coating of cell culture dishes Nitroxide radical-containing poly(ethylene glycol)-bpoly[(2,2,6,6-tetramethylpiperidine-N-oxyl)aminomethylstyrene] (PEG-b-PMNT) polymers were prepared according to previously reported methods.20 As a control polymer, poly(ethylene glycol)-b-poly[(cyclohexyl)aminomethylstyrene] PEG-b-PCHAMS was prepared using the same procedure, except that cyclohexylamine was used in place of 4-aminoTEMPO. The synthesis and analytical data for PEG-bPCHAMS are described in the Supporting Information (Fig. S1). To polymer-coat 24-well cell culture dishes (PureCoat carboxyl dish, BD Biosciences, Franklin Lakes, NJ), carboxyl groups were activated with 20 mg of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) in metha-
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IKEDA, YOSHINARI, AND NAGASAKI
nol (300 lL), and was then reacted with polymer (30 mg) in methanol (300 lL) overnight. The culture dish was washed 25 times with 1M NaCl solution (200 lL) to remove excess polymers that interacted electrostatically with the dish surface. This was followed by two washes with phosphate-buffered saline (PBS). The amount of immobilized polymer on the surface was quantified by measuring recovered polymer that had been released from the dish in response to dimethyl sulfoxide (DMSO) treatment. Preparation of LMWA-free cell culture medium Cell culture medium lacking LMWAs was prepared as follows: L-(1)-arginine (200 mg), L-asparagine (50 mg), DL-asparaginic acid (20 mg), L-cystine dihydrochloride (65.2 mg), L-glutamic acid (20 mg), glycine (10 mg), L-histidine (15 mg), Lleucine (50 mg), L-isoleucine (50 mg), L-(1) lysine hydrochloride (40 mg), L-methionine (15 mg), L-phenylalanine (15 mg), L-proline (20 mg), hydroxyl-L-proline (20 mg), L-(-)-threonine (20 mg), L-tryptophan (5 mg), L-(-)-tyrosine (20 mg), L-valine (10 mg), L-serine (30 mg), D-(1)-glucose (2.0 g), calcium nitrate tetrahydrate (100 mg), magnesium sulfate (49 mg), KCl (400 mg), NaCl (6.0 g), and disodium hydrogen phosphate dodecahydrate (400 mg) were dissolved in 950 mL of ultrapure water. The pH was adjusted to 4 by using 1M and 0.1M HCl, and ultrapure water was added to obtain a final volume of 1.0 L. The resulting solution was sterilized in an autoclave. After autoclaving, NaHCO3 (2.0 g) and 200 mM L-glutamine (10 mL) were added to adjust the pH to 7.4. Cell culture Undifferentiated human leukemia cells (HL-60) from the Riken Cell Bank (Ibaraki, Japan) were maintained in LMWAfree culture medium supplemented with 10% heatinactivated fetal bovine serum. Cell number and viability were assessed using the trypan blue dye-exclusion method. Cellular differentiation in the presence of butyric acid HL-60 cells (1 3 105 cells mL21) were cultured in LMWAfree medium in a 5% CO2 atmosphere in a 24-well plate (BD PureCoat carboxyl dish) in the presence and absence of the polymer coating. Cells were incubated with butyric acid at a final concentration of 0.4 mM. After 48 h of incubation, cells were transferred into another dish that was not coated with PEG-b-PMNT, because immobilized TEMPO might have interfered with subsequent cell differentiation analyses. HL-60 cell differentiation was assessed using nitroblue tetrazolium (NBT) dye reduction as described previously.21,22 In brief, cells were incubated with NBT (4 mg mL21) and freshly prepared 12-O-Tetradecanoylphorbol 13-acetate (TPA) (5 lg mL21), which induces phagocytosis-associated oxidative metabolism, generating superoxide anions, and reduces NBT dye. Differential counts were then performed using light microscopy. A minimum of 300 cells were assessed. Induction of cellular differentiation by using LMWAs HL-60 cells (1 3 105 cells/well) were cultured in LMWAfree medium in a 24-well plate (BD PureCoat carboxyl dish)
SUPPRESSION OF CELL MORPHOLOGICAL CHANGES BY SCAVENGING EXCESS REACTIVE OXYGEN SPECIES
ORIGINAL ARTICLE
in the absence of polymer coating. To the medium, one of four types of LMWAs—ascorbic acid (final concentration 50 lM), tocopherol (final concentration 50 lM), 4-aminoTEMPO (final concentration 50 lM), or NAC (final concentration 5 mM)—was added. Mitochondrial membrane potential measurements Mitochondrial membrane potential was quantified by performing flow cytometric analysis on cells stained with JC-1. HL-60 cells (5 3 105 cells mL21) were cultured in LMWAfree medium in a 24-well plate in the presence or absence of polymer coating. To evaluate the effects of LMWAs on mitochondrial membrane potential, HL-60 cells (5 3 105 cells mL21) were also cultured in the presence of LMWAs under the described conditions for 48 h. After 48 h of incubation, cells were stained using JC-1 and then analyzed using flow cytometry, according to the manufacturer’s instructions. Statistical analysis Statistical analyses were performed using the Student’s t test. A p values of
