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693. Non-Viral Polymers with Bioreducible Bonds Modulate Oxidative Stress Mechanisms in Mesenchymal Stem Cells
Mechanistic Studies and Carrier Design 693. Non-Viral Polymers with Bioreducible Bonds Modulate Oxidative Stress Mechanisms in Mesenchymal Stem Cells
James W. Yockman,1 Jonathan H. Brumbach,1 Mei Ou,1 SungWan Kim.1 1 Pharmaceutics and Pharmaceutical Chemistry, University of Utah, Salt Lake City, UT. Improvements in polymeric gene delivery efficiency have incorporated molecules that distinguish between differences in biological microenvironments. We designed a new reducible polymer that exploits physiological redox potentials within the cytoplasm. Reducible poly(amido triethlenetetramine) (SS-PATETA) is a novel cationic carrier that is comprised of the polyamine triethlenetetramine (TETA) and cystamine bisacrylamide (CBA) to contain multiple disulfide bonds. The disulfide bonds allow for responsive degradation during reductive challenges found intracellularly. SS-PATETA complex size and zeta potential remains stable at under 200nm and +32mV while protecting pDNA from DNase I degradation unless in the presence of reducing agents. However, using cellular redox mechanisms for polymer degradation may upset cellular reductive homeostasis and was investigated. Human mesenchymal stem cells were chosen for their known sensitivity to oxidative stress. Transfection with increasing w/w ratios of SS-PATETA demonstrated decreasing luciferase expression with increasing toxicity beginning at 6:1. Toxicity was observed within 4hr following transfection. Cellular glutathione (GST) concentrations were measured following transfection as glutathione is a primary regulator of redox status within cells. GST concentrations within SS-PATETA transfected cells 3 hrs after transfection were significantly lowered from 220 nmoles/ml in non-transfected and naked DNA controls, and bPEI25k positive control to 73 nmoles/ml. Reactive oxygen species (ROS) levels were measured using the fluorescent dye CM-H2DCFDA (Invitrogen). SS-PATETA transfected cells demonstrated a 200% increase in fluorescence over non-transfected controls and 140% increase over bPEI25k controls. To determine if the loss of GST alone is responsible for cell death, the hMSCs were incubated in increasing amounts of buthionine sulfoximine (BSO). Only high levels of BSO (20mM) were able to demonstrate any toxicity. Antioxidants known to effect GST levels or function, N-acetyl cysteine (NAC) and SeO3 respectively, were compared to superoxide dismutase (SOD) to determine specific ROS mechanisms of toxicity. NAC and SeO3 were both able to reverse toxicity associated with 12:1 w/w SS-PATETA transfection at low concentrations (0.125mM and 25mM) while SOD could only partially restore viability to 45% at 100U/ml. Interestingly, the molecules not only reversed toxicity but completely eliminated transfection efficiency in SS-PATETA treated cells. No effect on bPEI25k controls was observed at lower concentrations. Enzymatic reactions responsible for oxidative/reductive homeostasis utilized in this type of polymeric degradation may be perturbed and result in unusually high levels of reactive oxygen species in certain cell types and possibly under certain disease conditions. Increases in glutathione concentrations or enzymatic activity inhibit DNA transfection using these types of bioreducible polymers. The use of these bioreducible polymers must be further investigated to elucidate the mechanism(s) responsible for induction of oxidative stress pathways and how GST concentrations can play such a divisive role in DNA transfection efficiency.
Molecular Therapy Volume 16, Supplement 1, May 2008 Copyright © The American Society of Gene Therapy
694. Influence of the Chemical Composition of Amphiphilic Triblock Copolymers on Muscle Gene Transfer
Debborah Alimi,1 Blandine Brissault,2 Christian Leborgne,1 Christine Guis,2 Daniel Scherman,1 Antoine Kichler.1 1 Exploratory Research, Genethon-FRE3087 CNRS, Evry, France; 2 Laboratoire Materiaux Polymeres aux Interfaces, Universite Evry - UMR7581 CNRS, Evry, France. Amphiphilic triblock copolymers such as the polyethylene glycolpolypropylene glycol-polyethylene glycol L64 (PEG13-PPG30-PEG13) have been recently shown to promote muscle gene transfer. In the present study, we investigated the relation between structure of the copolymers and their capacity to enhance muscle transfection. To this end, we evaluated the in vivo activity of 6 copolymers that differ either by their molecular weight or by their hydrophilic/ hydrophobic balance. Our results indicate that there is not a unique compound able to improve muscle gene transfer but there is a large flexibility in terms of molecular weight and EO/PO ratio. Further, we investigated the effect of a chemical change of the PEG moiety on the transfection activity. Therefore, we synthesized and characterized new amphiphilic copolymers in which the PEG end blocks were replaced by more hydrophilic poly(2-methyl-2-oxazoline) (PMeOXZ) chains of various lengths. The PMeOXZ-PPG-PMeOXZ compounds were assayed for in vivo muscle gene transfer and the results indicate that they increased by 20-fold the transfection efficiency as compared to naked DNA. Thus, the capacity to enhance DNA transfection in skeletal muscles is not restricted to PEG-PPG-PEG arrangements. Finally, we studied the membrane permeabilizing activity of these compounds by performing different membrane leakage assays in vitro and in vivo.
695. Design and Characterization of Polymeric Nanoparticles Containing Conjugated Phospholipase A2 for Non-Viral Gene Delivery Huong T. Le,1 Gururaj A. Rao,1 Jeffrey A. Hughes.1 1 Pharmaceutics, University of Florida, Gainesville, FL.
Progress is being made in the development of non-viral gene delivery vectors, although they remain less efficient than the viral counterparts. Their major limitations reside in the fact that they must be tailored to overcome intracellular barriers to DNA delivery that viruses already master. Improvement of non-viral vectors has focused on cationic lipids and polymers, which possess properties required to address the gene delivery problems. Research in the field of cationic polymer mediated gene delivery hinges upon exploring new polymers or modifying existing ones to provide sufficient safety and transfection efficiency. Our hypothesis is based on the fact that many viral coat proteins have phopholipase activity, for example VP1 of AAV. Thus, conjugating phospholipase A2 (PLA2) to the cationic polymer polyethylenimine (PEI) could improve gene transfer. Here we present the biological characterization of PEI-PLA2/ DNA polyplexes in terms of PLA2 activity, cytotoxicity and gene expression. Method. PLA2 was conjugated via its carboxyl end to the amine groups of PEI (branched; 25 kDa) was, using EDC as a coupling agent. Conjugates synthesized in PEI to PLA2 molar ratio of 50:1 were used for the present set of experiments. The PLA2 activity in the conjugates was determined by measuring hydrolysis rates using 1,2-dithio analog of diheptanoyl phosphatidylcholine as a substrate. Transfection studies were done at N/P ratios of 5, 7, 10, 15, 20, and 40 in human embryonic kidney (HEK 293) and human hepatoma (HepG2) cell lines using a luciferase plasmid DNA and a green fluorescent protein (GFP) plasmid. Cytotoxicity of the conjugates and PEI was compared using the MTT assay. Results. Conjugation of PEI to PLA2 at molar ratios 50:1 does not adversely affect PLA2 enzyme activity. The cytotoxicity of conjugates and PEI was similar S259
James W. Yockman,1 Jonathan H. Brumbach,1 Mei Ou,1 SungWan Kim.1 1 Pharmaceutics and Pharmaceutical Chemistry, University of Utah, Salt Lake City, UT. Improvements in polymeric gene delivery efficiency have incorporated molecules that distinguish between differences in biological microenvironments. We designed a new reducible polymer that exploits physiological redox potentials within the cytoplasm. Reducible poly(amido triethlenetetramine) (SS-PATETA) is a novel cationic carrier that is comprised of the polyamine triethlenetetramine (TETA) and cystamine bisacrylamide (CBA) to contain multiple disulfide bonds. The disulfide bonds allow for responsive degradation during reductive challenges found intracellularly. SS-PATETA complex size and zeta potential remains stable at under 200nm and +32mV while protecting pDNA from DNase I degradation unless in the presence of reducing agents. However, using cellular redox mechanisms for polymer degradation may upset cellular reductive homeostasis and was investigated. Human mesenchymal stem cells were chosen for their known sensitivity to oxidative stress. Transfection with increasing w/w ratios of SS-PATETA demonstrated decreasing luciferase expression with increasing toxicity beginning at 6:1. Toxicity was observed within 4hr following transfection. Cellular glutathione (GST) concentrations were measured following transfection as glutathione is a primary regulator of redox status within cells. GST concentrations within SS-PATETA transfected cells 3 hrs after transfection were significantly lowered from 220 nmoles/ml in non-transfected and naked DNA controls, and bPEI25k positive control to 73 nmoles/ml. Reactive oxygen species (ROS) levels were measured using the fluorescent dye CM-H2DCFDA (Invitrogen). SS-PATETA transfected cells demonstrated a 200% increase in fluorescence over non-transfected controls and 140% increase over bPEI25k controls. To determine if the loss of GST alone is responsible for cell death, the hMSCs were incubated in increasing amounts of buthionine sulfoximine (BSO). Only high levels of BSO (20mM) were able to demonstrate any toxicity. Antioxidants known to effect GST levels or function, N-acetyl cysteine (NAC) and SeO3 respectively, were compared to superoxide dismutase (SOD) to determine specific ROS mechanisms of toxicity. NAC and SeO3 were both able to reverse toxicity associated with 12:1 w/w SS-PATETA transfection at low concentrations (0.125mM and 25mM) while SOD could only partially restore viability to 45% at 100U/ml. Interestingly, the molecules not only reversed toxicity but completely eliminated transfection efficiency in SS-PATETA treated cells. No effect on bPEI25k controls was observed at lower concentrations. Enzymatic reactions responsible for oxidative/reductive homeostasis utilized in this type of polymeric degradation may be perturbed and result in unusually high levels of reactive oxygen species in certain cell types and possibly under certain disease conditions. Increases in glutathione concentrations or enzymatic activity inhibit DNA transfection using these types of bioreducible polymers. The use of these bioreducible polymers must be further investigated to elucidate the mechanism(s) responsible for induction of oxidative stress pathways and how GST concentrations can play such a divisive role in DNA transfection efficiency.
Molecular Therapy Volume 16, Supplement 1, May 2008 Copyright © The American Society of Gene Therapy
694. Influence of the Chemical Composition of Amphiphilic Triblock Copolymers on Muscle Gene Transfer
Debborah Alimi,1 Blandine Brissault,2 Christian Leborgne,1 Christine Guis,2 Daniel Scherman,1 Antoine Kichler.1 1 Exploratory Research, Genethon-FRE3087 CNRS, Evry, France; 2 Laboratoire Materiaux Polymeres aux Interfaces, Universite Evry - UMR7581 CNRS, Evry, France. Amphiphilic triblock copolymers such as the polyethylene glycolpolypropylene glycol-polyethylene glycol L64 (PEG13-PPG30-PEG13) have been recently shown to promote muscle gene transfer. In the present study, we investigated the relation between structure of the copolymers and their capacity to enhance muscle transfection. To this end, we evaluated the in vivo activity of 6 copolymers that differ either by their molecular weight or by their hydrophilic/ hydrophobic balance. Our results indicate that there is not a unique compound able to improve muscle gene transfer but there is a large flexibility in terms of molecular weight and EO/PO ratio. Further, we investigated the effect of a chemical change of the PEG moiety on the transfection activity. Therefore, we synthesized and characterized new amphiphilic copolymers in which the PEG end blocks were replaced by more hydrophilic poly(2-methyl-2-oxazoline) (PMeOXZ) chains of various lengths. The PMeOXZ-PPG-PMeOXZ compounds were assayed for in vivo muscle gene transfer and the results indicate that they increased by 20-fold the transfection efficiency as compared to naked DNA. Thus, the capacity to enhance DNA transfection in skeletal muscles is not restricted to PEG-PPG-PEG arrangements. Finally, we studied the membrane permeabilizing activity of these compounds by performing different membrane leakage assays in vitro and in vivo.
695. Design and Characterization of Polymeric Nanoparticles Containing Conjugated Phospholipase A2 for Non-Viral Gene Delivery Huong T. Le,1 Gururaj A. Rao,1 Jeffrey A. Hughes.1 1 Pharmaceutics, University of Florida, Gainesville, FL.
Progress is being made in the development of non-viral gene delivery vectors, although they remain less efficient than the viral counterparts. Their major limitations reside in the fact that they must be tailored to overcome intracellular barriers to DNA delivery that viruses already master. Improvement of non-viral vectors has focused on cationic lipids and polymers, which possess properties required to address the gene delivery problems. Research in the field of cationic polymer mediated gene delivery hinges upon exploring new polymers or modifying existing ones to provide sufficient safety and transfection efficiency. Our hypothesis is based on the fact that many viral coat proteins have phopholipase activity, for example VP1 of AAV. Thus, conjugating phospholipase A2 (PLA2) to the cationic polymer polyethylenimine (PEI) could improve gene transfer. Here we present the biological characterization of PEI-PLA2/ DNA polyplexes in terms of PLA2 activity, cytotoxicity and gene expression. Method. PLA2 was conjugated via its carboxyl end to the amine groups of PEI (branched; 25 kDa) was, using EDC as a coupling agent. Conjugates synthesized in PEI to PLA2 molar ratio of 50:1 were used for the present set of experiments. The PLA2 activity in the conjugates was determined by measuring hydrolysis rates using 1,2-dithio analog of diheptanoyl phosphatidylcholine as a substrate. Transfection studies were done at N/P ratios of 5, 7, 10, 15, 20, and 40 in human embryonic kidney (HEK 293) and human hepatoma (HepG2) cell lines using a luciferase plasmid DNA and a green fluorescent protein (GFP) plasmid. Cytotoxicity of the conjugates and PEI was compared using the MTT assay. Results. Conjugation of PEI to PLA2 at molar ratios 50:1 does not adversely affect PLA2 enzyme activity. The cytotoxicity of conjugates and PEI was similar S259