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Reversal of oxidative stress in endothelial cells by controlled release of adiponectin
Journal of Controlled Release 130 (2008) 234–237
Contents lists available at ScienceDirect
Journal of Controlled Release j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j c o n r e l
Reversal of oxidative stress in endothelial cells by controlled release of adiponectin Mohanad Mossalam a, Ji-Hoon Jeong a,b, E. Dale Abel c, Sung Wan Kim a,d, Yong-Hee Kim a,d,⁎ a
Center for Controlled Chemical Delivery, Department of Pharmaceutics and Pharmaceutical Chemistry, University of Utah, Salt Lake City, UT 84112, USA College of Pharmacy, Sungkyun Kwan University, Republic of Korea c Program in Human Molecular Biology and Genetics, Department of Internal Medicine, University of Utah, USA d Department of Bioengineering, Hanyang University, Republic of Korea b
A R T I C L E
I N F O
Article history: Received 26 May 2008 Accepted 12 June 2008 Available online 19 June 2008 Keywords: Controlled release Adiponectin Oxidative stress Endothelial cells
A B S T R A C T Hyperglycemia causes endothelial dysfunction due to its effect on increasing reactive oxygen species (ROS). Adiponectin (Adp) has been reported to suppress hyperglycemia-associated ROS generation. It was hypothesized that administering globular adiponectin (gAdp) via injectable biodegradable thermosensitive triblock copolymer might effectively reduce ROS generation in endothelial cells. In this study, gAdp was incorporated into and released from the polymer gel. The released gAdp was further investigated by comparing it with the intact gAdp with regard to the efficiency in reducing ROS and activating cAMP. The released gAdp effectively suppressed excess ROS production in the in vitro endothelial cell culture model under high-glucose condition via cAMP/PKA pathway. These data provide a rationale for developing controlled release dosage form of gAdp as a therapeutic tool for oxidative stress-related pathology in patients with diabetes. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Hyperglycemia is one of the main causes of vascular complications in diabetic patients due to its effect on increasing ROS production, which causes endothelial dysfunction [1–3]. In blood vessels, ROS have been implicated in the pathogenesis of hypertension, vascular lesion formation, atherosclerosis, and diabetic vascular disease [4–8]. Consequently, a novel approach to restore vascular function in diabetes might be to reduce mitochondrial ROS-overproduction. Adiponectin (Adp) is a circulating hormone secreted by adipose tissue that plays a key role in glucose homeostasis and fatty acid metabolism [9]. Adp levels are reduced in patients with obesity and cardiovascular disease [10,11]. Studies have shown the potential role of Adp in coronary artery disease patients with diabetes by suppressing ROS generation [12–15]. Adp has also been very recently recognized to suppress hyperglycemia-associated ROS generation via a cAMP-dependent protein kinase (PKA) pathway [16]. Adiponectin could be administered using an injectable biodegradable thermosensitive triblock copolymer delivery system. The copolymer, which is composed of hydrophilic poly (ethylene glycol) (PEG) B-block and hydrophobic poly (lactide-co-glycolide) (PLGA) A block, allows the delivery of insoluble or unstable substances by generating a hydrogel depot at body temperature [17,18]. It has also been used to enhance gene delivery efficiency [19]. Potentially, these copolymers
⁎ Corresponding author. Department of Bioengineering, Hanyang University, 17 Haengdang-dong, Seongdong-gu, Seoul, 133-791, Republic of Korea. Tel.: +82 2 2220 2345; fax: +82 2 2220 1998. E-mail address: [email protected] (Y.-H. Kim). 0168-3659/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.jconrel.2008.06.009
were proved to be valuable components of controlled delivery systems or site-specific administration of therapeutics [20,21]. The aim of this study was to develop and characterize a controlled drug delivery system containing gAdp, and test the hypothesis that administering gAdp via a polymer gel might effectively reduce ROS generation in endothelial cells and therefore lead to recovery of endothelial dysfunction. We investigated the antioxidant effect and mechanism of action of released gAdp by the copolymer on reducing ROS in Bovine Aortic Endothelial Cells (BAEC). This study investigates a new therapeutical advantage in gAdp delivery and also provides the framework for further work involving in vivo studies. 2. Materials and methods BAECs were obtained from Cambrex Bio Science Walkersville, Inc. Cell culture materials were from Invitrogen (Carlsbad, CA). The copolymer (ReGel® R40/60) was kindly provided by MacroMed, Inc (SLC, UT), Chen S. et al. has have detailed synthesis and characterization of the copolymer [22]. The mouse recombinant gAdp (16.6 kDa in E. coli host) and fAdp (35 kDa in HEK 293 cells host) were from Kamiya Biomedical Company (Seattle, WA). All other chemicals and reagents, unless otherwise noted, were obtained from Sigma (St. Louis, MO). 2.1. Cell culture BAECs were cultured in Dulbecco's modified eagle medium (DMEM) containing 10% fetal bovine serum (FBS), and 1% penicillin– streptomycin. Cells were routinely studied before the tenth passage and at 80–90% confluence for ROS and cAMP measurements.
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2.2. In vitro ROS measurements BAECs at a density of 2×105 cells/well in 6 well plates were treated under different glucose concentrations: 0 mM (as a negative control), 15 mM, and 25 mM (as a positive control). The 25 mM glucose treated cells were then treated with 2 µg/ml, 3 µg/ml, 4.5 µg/ml, 7 µg/ml and 9 µg/ml globular adiponectin (gAdp), and 4.5 µg/ml released globular adiponectin from the polymer gel (rAdp) to measure the effect of adiponectin on the hyperglycemia-induced ROS levels. The cells were cultured for 36 h, then treated with 10 µM 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA) for 30 min. They were then suspended in Phosphate-Buffered Saline (PBS). The fluorescence was then measured using flow cytometry. The higher the fluorescence, the greater the ROS production [23]. 2.3. Measurements of cellular cAMP BAECs at a density of 2 × 105 cells/well were cultured under the same conditions as in the in vitro ROS studies. cAMP was then measured by following the manufacturer's instruction using a direct immunoassay kit (BioVision) [24]. 2.4. In vitro release study of gAdp from the copolymer Firstly, 25 µg gAdp was dissolved in 0.5 ml the copolymer and incubated in a 37 °C water bath. Once gel is formed, 1 ml of PBS was added to and removed from the vial containing the polymer gel every 24 h. The released protein was quantified using Adiponectin ELISA kit (B-Bridge International) and then concentrated at different time intervals using centriprep (Amicon) to further investigate its activity. SDS was performed on the concentrated protein [25]. 2.5. Statistical analyses Quantitative data are presented as means ± SD as indicated from the number of experiments. Statistical analysis was based on Student's t-test for comparison of two groups. A p value less than 0.05 was used to determine statistical significance. 3. Results and discussion The method used to measure ROS-overproduction was confirmed by testing the effect of gAdp on reducing ROS. BAECs were incubated in different concentrations of glucose. There was an increase in the ROS production with the glucose treated cells in a dose dependent manner up to 7.5 fold (P b 0.05) (Fig. 1). However, when using mannitol at the same concentrations, there was no significant difference in the
Fig. 1. Glucose effect on ROS production. After treatment with glucose, ROS was measured via FACSCAN. Mannitol had no effect on ROS. Data are expressed as the percent of basal (0 mM glucose) and represent the means ± SD of n = 3. ⁎P b 0.05 vs. basal.
Fig. 2. Dose response of gAdp in reducing ROS induced by high glucose in endothelial cells. After treatment, ROS was measured via FACSCAN. The gAdp showed no effect on glucose untreated cells. Data are expressed as the percent of basal (0 mM glucose) and represent the means ± SD of n = 3. ⁎P b 0.05 vs. glucose treated cells.
ROS level ruling out any osmotic effect. Hyperglycemia induces ROSoverproduction through several pathways. A major mechanism is through the overproduction of the superoxide anion (O− 2) by the mitochondrial electron transport chain [26]. Incubating the cells in 25 mM glucose medium puts the cells in hyperglycemic condition. The high-glucose treated cells followed by the incubation with gAdp showed reduction in ROS production in a dose dependent manner with optimum effect at 4.5 µg/ml concentration (Fig. 2). The gAdp had no effect on the glucose untreated cells showing its activity merely on the ROS induced by the glucose treatment. The controlled delivery system containing gAdp was developed by solubilizing the protein in the polymer gel. In vitro release of gAdp from the polymer gel and its activity on ROS and cAMP were tested to characterize and prove the efficacy of the delivery model. The release profile showed almost 100% release of the 25 µg gAdp (Fig. 3) over 27 days without initial burst release. The protein was released in a first order manner in the first 5 days, then in a zero-order manner in the following 10 days, and then in a zero-order manner with a different release rate in the final 12 days. The protein is released from the gel upon degradation of the copolymer. The released protein was then concentrated using centriprep (Amicon) to further investigate its activity. The SDS PAGE performed on the released Adp at different time intervals did not show any degradation or aggregation, which shows the stability of the released protein from the polymer gel (Fig. 4). The released gAdp (rAdp) from the copolymer was compared to the activity of the intact gAdp. It showed that the released gAdp had the same effect as the intact gAdp on reducing ROS in endothelial cells with no significant difference (Fig. 5). These results represent the
Fig. 3. In vitro release study of 25 µg gAdp from injectable, biodegradable, and thermosensitive triblock copolymer. The gAdp is fully released over the 27 days. The data represent means ± SD of n = 5.
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Fig. 6. Effect of the released gAdp (rAdp) from the polymer gel on activating cAMP compared to the intact gAdp. This confirms the preservation of gAdp activity. The data represent means ± SD of n = 3. ⁎P b 0.05 vs. high glucose.
Fig. 4. SDS PAGE for the released adiponectin compared to the intact adiponectin. It shows no degradation of the protein upon release from the polymer gel.
potential of the triblock copolymer as a safe delivery carrier for the gAdp. cAMP was measured in high high-glucose induced cells treated with either gAdp or rAdp. The gAdp and rAdp showed the same effect on increasing the cellular content of cAMP with no significant difference (Fig. 6) proving the pathway in which gAdp reduces ROS and the preservation of the activity of the released gAdp. The rAdp had no change in its characteristics in relation to its activity on ROS and cAMP. The polymer gel was not added directly to the cells due to its viscosity. However, the protein release could be achieved in animal models. This was performed in our laboratory using other proteins. The characterization of the gAdp delivery system raises the possibility to be used therapeutically in animal models. 4. Conclusions The gAdp effectively suppressed excess ROS production in the in vitro BAECs under high-glucose condition via cAMP pathway. The
Fig. 5. Effect of the released gAdp (rAdp) from the polymer gel on reducing ROS compared to the intact gAdp. Data are expressed as the percent of basal (0 mM glucose) and represent the means ± SD of n = 3. ⁎P b 0.05 vs. high glucose.
controlled release profile of gAdp from the polymer gel was achieved showing almost 100% sustained release over 27 days with no initial burst release. The released gAdp preserved its activity in reducing ROS and activating cAMP. These data raise the possibility that injectable gels containing gAdp could be used therapeutically to reduce ROS in the blood vessels of animals with diabetes. This released adiponectin might compensate for the reduced adiponectin concentrations in the blood stream of diabetic or obese animals. This study provides a new direction for developing controlled release dosage form of gAdp as a therapeutic tool for oxidative stress stress-related pathology in patients with diabetes. The results provide a framework for developing scientific foundation for further work involving in vivo studies. Acknowledgments This work was partially supported by the Korea Science and Engineering Foundation (KOSEF) grant funded by the Korea government (MEST) (F104AA010005-07A0101-00510, R01-2007-00020602-0(2007)) and Seoul R&BD program (CR070027) in Korea.
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M. Mossalam et al. / Journal of Controlled Release 130 (2008) 234–237 [12] H. Motoshima, X. Wu, K. Mahadev, B.J. Goldstein, Adiponectin suppresses proliferation and superoxide generation and enhances eNOS activity in endothelial cells treated with oxidized LDL, Biochem. Biophys. Res. Commun. 315 (2004) 264–271. [13] T. Costacou, J.C. Zgibor, R.W. Evans, J. Otvos, M.F. Lopes-Virella, R.P. Tracy, T.J. Orchard, The prospective association between adiponectin and coronary artery disease among individuals with type 1 diabetes. The Pittsburgh Epidemiology of Diabetes Complications Study, Diabetologia 48 (2005) 41–48. [14] S. Furukawa, T. Fujita, M. Shimabukuro, M. Iwaki, Y. Yamada, Y. Nakajima, O. Nakayama, M. Makishima, M. Matsuda, I. Shimomura, Increased oxidative stress in obesity and its impact on metabolic syndrome, J. Clin. Invest. 114 (2004) 1752–1761. [15] S. Nakanishi, K. Yamane, N. Kamei, H. Nojima, M. Okubo, N. Kohno, A protective effect of adiponectin against oxidative stress in Japanese Americans: the association between adiponectin or leptin and urinary isoprostane, Metabolism 54 (2005) 194–199. [16] R. Ouedraogo, X. Wu, S.Q. Xu, L. Fuchsel, H. Motoshima, K. Mahadev, K. Hough, R. Scalia, B.J. Goldstein, Adiponectin suppression of high-glucose-induced reactive oxygen species in vascular endothelial cells: evidence for involvement of a cAMP signaling pathway, Diabetes 55 (2006) 1840–1846. [17] T. Kissel, Y. Li, S. Gorich, C. Volland, R. Koneberg, Parenteral protein delivery systems using biodegradable ABA-block-copolymers, J. Control. Release 39 (1996) 315–326. [18] S. Duvvuri, K.G. Janoria, A.K. Mitra, Development of a novel formulation containing poly(D,L-lactide-co-glycolide) microspheres dispersed in PLGA-PEG-PLGA gel for sustained delivery of ganciclovir, J. Control. Release 108 (2005) 282–293. [19] C.W. Chang, D. Choi, W.J. Kim, J.W. Yockman, L.V. Christensen, Y.H. Kim, S.W. Kim, Non-ionic amphiphilic biodegradable PEG-PLGA-PEG copolymer enhances gene delivery efficiency in rat skeletal muscle, J. Control. Release 118 (2007) 245–253.
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Contents lists available at ScienceDirect
Journal of Controlled Release j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j c o n r e l
Reversal of oxidative stress in endothelial cells by controlled release of adiponectin Mohanad Mossalam a, Ji-Hoon Jeong a,b, E. Dale Abel c, Sung Wan Kim a,d, Yong-Hee Kim a,d,⁎ a
Center for Controlled Chemical Delivery, Department of Pharmaceutics and Pharmaceutical Chemistry, University of Utah, Salt Lake City, UT 84112, USA College of Pharmacy, Sungkyun Kwan University, Republic of Korea c Program in Human Molecular Biology and Genetics, Department of Internal Medicine, University of Utah, USA d Department of Bioengineering, Hanyang University, Republic of Korea b
A R T I C L E
I N F O
Article history: Received 26 May 2008 Accepted 12 June 2008 Available online 19 June 2008 Keywords: Controlled release Adiponectin Oxidative stress Endothelial cells
A B S T R A C T Hyperglycemia causes endothelial dysfunction due to its effect on increasing reactive oxygen species (ROS). Adiponectin (Adp) has been reported to suppress hyperglycemia-associated ROS generation. It was hypothesized that administering globular adiponectin (gAdp) via injectable biodegradable thermosensitive triblock copolymer might effectively reduce ROS generation in endothelial cells. In this study, gAdp was incorporated into and released from the polymer gel. The released gAdp was further investigated by comparing it with the intact gAdp with regard to the efficiency in reducing ROS and activating cAMP. The released gAdp effectively suppressed excess ROS production in the in vitro endothelial cell culture model under high-glucose condition via cAMP/PKA pathway. These data provide a rationale for developing controlled release dosage form of gAdp as a therapeutic tool for oxidative stress-related pathology in patients with diabetes. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Hyperglycemia is one of the main causes of vascular complications in diabetic patients due to its effect on increasing ROS production, which causes endothelial dysfunction [1–3]. In blood vessels, ROS have been implicated in the pathogenesis of hypertension, vascular lesion formation, atherosclerosis, and diabetic vascular disease [4–8]. Consequently, a novel approach to restore vascular function in diabetes might be to reduce mitochondrial ROS-overproduction. Adiponectin (Adp) is a circulating hormone secreted by adipose tissue that plays a key role in glucose homeostasis and fatty acid metabolism [9]. Adp levels are reduced in patients with obesity and cardiovascular disease [10,11]. Studies have shown the potential role of Adp in coronary artery disease patients with diabetes by suppressing ROS generation [12–15]. Adp has also been very recently recognized to suppress hyperglycemia-associated ROS generation via a cAMP-dependent protein kinase (PKA) pathway [16]. Adiponectin could be administered using an injectable biodegradable thermosensitive triblock copolymer delivery system. The copolymer, which is composed of hydrophilic poly (ethylene glycol) (PEG) B-block and hydrophobic poly (lactide-co-glycolide) (PLGA) A block, allows the delivery of insoluble or unstable substances by generating a hydrogel depot at body temperature [17,18]. It has also been used to enhance gene delivery efficiency [19]. Potentially, these copolymers
⁎ Corresponding author. Department of Bioengineering, Hanyang University, 17 Haengdang-dong, Seongdong-gu, Seoul, 133-791, Republic of Korea. Tel.: +82 2 2220 2345; fax: +82 2 2220 1998. E-mail address: [email protected] (Y.-H. Kim). 0168-3659/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.jconrel.2008.06.009
were proved to be valuable components of controlled delivery systems or site-specific administration of therapeutics [20,21]. The aim of this study was to develop and characterize a controlled drug delivery system containing gAdp, and test the hypothesis that administering gAdp via a polymer gel might effectively reduce ROS generation in endothelial cells and therefore lead to recovery of endothelial dysfunction. We investigated the antioxidant effect and mechanism of action of released gAdp by the copolymer on reducing ROS in Bovine Aortic Endothelial Cells (BAEC). This study investigates a new therapeutical advantage in gAdp delivery and also provides the framework for further work involving in vivo studies. 2. Materials and methods BAECs were obtained from Cambrex Bio Science Walkersville, Inc. Cell culture materials were from Invitrogen (Carlsbad, CA). The copolymer (ReGel® R40/60) was kindly provided by MacroMed, Inc (SLC, UT), Chen S. et al. has have detailed synthesis and characterization of the copolymer [22]. The mouse recombinant gAdp (16.6 kDa in E. coli host) and fAdp (35 kDa in HEK 293 cells host) were from Kamiya Biomedical Company (Seattle, WA). All other chemicals and reagents, unless otherwise noted, were obtained from Sigma (St. Louis, MO). 2.1. Cell culture BAECs were cultured in Dulbecco's modified eagle medium (DMEM) containing 10% fetal bovine serum (FBS), and 1% penicillin– streptomycin. Cells were routinely studied before the tenth passage and at 80–90% confluence for ROS and cAMP measurements.
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2.2. In vitro ROS measurements BAECs at a density of 2×105 cells/well in 6 well plates were treated under different glucose concentrations: 0 mM (as a negative control), 15 mM, and 25 mM (as a positive control). The 25 mM glucose treated cells were then treated with 2 µg/ml, 3 µg/ml, 4.5 µg/ml, 7 µg/ml and 9 µg/ml globular adiponectin (gAdp), and 4.5 µg/ml released globular adiponectin from the polymer gel (rAdp) to measure the effect of adiponectin on the hyperglycemia-induced ROS levels. The cells were cultured for 36 h, then treated with 10 µM 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA) for 30 min. They were then suspended in Phosphate-Buffered Saline (PBS). The fluorescence was then measured using flow cytometry. The higher the fluorescence, the greater the ROS production [23]. 2.3. Measurements of cellular cAMP BAECs at a density of 2 × 105 cells/well were cultured under the same conditions as in the in vitro ROS studies. cAMP was then measured by following the manufacturer's instruction using a direct immunoassay kit (BioVision) [24]. 2.4. In vitro release study of gAdp from the copolymer Firstly, 25 µg gAdp was dissolved in 0.5 ml the copolymer and incubated in a 37 °C water bath. Once gel is formed, 1 ml of PBS was added to and removed from the vial containing the polymer gel every 24 h. The released protein was quantified using Adiponectin ELISA kit (B-Bridge International) and then concentrated at different time intervals using centriprep (Amicon) to further investigate its activity. SDS was performed on the concentrated protein [25]. 2.5. Statistical analyses Quantitative data are presented as means ± SD as indicated from the number of experiments. Statistical analysis was based on Student's t-test for comparison of two groups. A p value less than 0.05 was used to determine statistical significance. 3. Results and discussion The method used to measure ROS-overproduction was confirmed by testing the effect of gAdp on reducing ROS. BAECs were incubated in different concentrations of glucose. There was an increase in the ROS production with the glucose treated cells in a dose dependent manner up to 7.5 fold (P b 0.05) (Fig. 1). However, when using mannitol at the same concentrations, there was no significant difference in the
Fig. 1. Glucose effect on ROS production. After treatment with glucose, ROS was measured via FACSCAN. Mannitol had no effect on ROS. Data are expressed as the percent of basal (0 mM glucose) and represent the means ± SD of n = 3. ⁎P b 0.05 vs. basal.
Fig. 2. Dose response of gAdp in reducing ROS induced by high glucose in endothelial cells. After treatment, ROS was measured via FACSCAN. The gAdp showed no effect on glucose untreated cells. Data are expressed as the percent of basal (0 mM glucose) and represent the means ± SD of n = 3. ⁎P b 0.05 vs. glucose treated cells.
ROS level ruling out any osmotic effect. Hyperglycemia induces ROSoverproduction through several pathways. A major mechanism is through the overproduction of the superoxide anion (O− 2) by the mitochondrial electron transport chain [26]. Incubating the cells in 25 mM glucose medium puts the cells in hyperglycemic condition. The high-glucose treated cells followed by the incubation with gAdp showed reduction in ROS production in a dose dependent manner with optimum effect at 4.5 µg/ml concentration (Fig. 2). The gAdp had no effect on the glucose untreated cells showing its activity merely on the ROS induced by the glucose treatment. The controlled delivery system containing gAdp was developed by solubilizing the protein in the polymer gel. In vitro release of gAdp from the polymer gel and its activity on ROS and cAMP were tested to characterize and prove the efficacy of the delivery model. The release profile showed almost 100% release of the 25 µg gAdp (Fig. 3) over 27 days without initial burst release. The protein was released in a first order manner in the first 5 days, then in a zero-order manner in the following 10 days, and then in a zero-order manner with a different release rate in the final 12 days. The protein is released from the gel upon degradation of the copolymer. The released protein was then concentrated using centriprep (Amicon) to further investigate its activity. The SDS PAGE performed on the released Adp at different time intervals did not show any degradation or aggregation, which shows the stability of the released protein from the polymer gel (Fig. 4). The released gAdp (rAdp) from the copolymer was compared to the activity of the intact gAdp. It showed that the released gAdp had the same effect as the intact gAdp on reducing ROS in endothelial cells with no significant difference (Fig. 5). These results represent the
Fig. 3. In vitro release study of 25 µg gAdp from injectable, biodegradable, and thermosensitive triblock copolymer. The gAdp is fully released over the 27 days. The data represent means ± SD of n = 5.
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Fig. 6. Effect of the released gAdp (rAdp) from the polymer gel on activating cAMP compared to the intact gAdp. This confirms the preservation of gAdp activity. The data represent means ± SD of n = 3. ⁎P b 0.05 vs. high glucose.
Fig. 4. SDS PAGE for the released adiponectin compared to the intact adiponectin. It shows no degradation of the protein upon release from the polymer gel.
potential of the triblock copolymer as a safe delivery carrier for the gAdp. cAMP was measured in high high-glucose induced cells treated with either gAdp or rAdp. The gAdp and rAdp showed the same effect on increasing the cellular content of cAMP with no significant difference (Fig. 6) proving the pathway in which gAdp reduces ROS and the preservation of the activity of the released gAdp. The rAdp had no change in its characteristics in relation to its activity on ROS and cAMP. The polymer gel was not added directly to the cells due to its viscosity. However, the protein release could be achieved in animal models. This was performed in our laboratory using other proteins. The characterization of the gAdp delivery system raises the possibility to be used therapeutically in animal models. 4. Conclusions The gAdp effectively suppressed excess ROS production in the in vitro BAECs under high-glucose condition via cAMP pathway. The
Fig. 5. Effect of the released gAdp (rAdp) from the polymer gel on reducing ROS compared to the intact gAdp. Data are expressed as the percent of basal (0 mM glucose) and represent the means ± SD of n = 3. ⁎P b 0.05 vs. high glucose.
controlled release profile of gAdp from the polymer gel was achieved showing almost 100% sustained release over 27 days with no initial burst release. The released gAdp preserved its activity in reducing ROS and activating cAMP. These data raise the possibility that injectable gels containing gAdp could be used therapeutically to reduce ROS in the blood vessels of animals with diabetes. This released adiponectin might compensate for the reduced adiponectin concentrations in the blood stream of diabetic or obese animals. This study provides a new direction for developing controlled release dosage form of gAdp as a therapeutic tool for oxidative stress stress-related pathology in patients with diabetes. The results provide a framework for developing scientific foundation for further work involving in vivo studies. Acknowledgments This work was partially supported by the Korea Science and Engineering Foundation (KOSEF) grant funded by the Korea government (MEST) (F104AA010005-07A0101-00510, R01-2007-00020602-0(2007)) and Seoul R&BD program (CR070027) in Korea.
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