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140. Effect of Intracellular Glutathione Levels on Cytotoxicity of Bioreducible Polycations
PHYSICAL METHODS OF DELIVERY the drug administered, only cells exposed to quinazolines showed significant increase in overall mean fluorescence.
A second transfection applied with increasing concentrations of quinazoline showed consistent increase in total mean fluorescence across the ranges tested.
amine)s were synthesized by Michael addition copolymerization of equal molar ratio of N,N’-dimethylamino dipropylenetriamine (DMADPTA) to N,N’-cystaminebisacrylamide (CBA) and N,N’hexamethylenebisacrylamide (HMBA). Through controlling the ratio of CBA and HMBA, the percentage of reducible component was adjusted at 100%, 75%, 50%, 25% and 0%. Cytotoxicity was determined by MTS assay. The results indicate that the IC50 values of the series of polycations in GSH-rich human pancreatic adenocarcinoma cells Panc-28 were 98.3 ± 1.35, 75.6 ± 4.11, 47.5 ± 2.62, 26.9 ± 1.28 and 4.09 ± 0.147 µg/mL, respectively. By contrast, the results in human ductal pancreatic adenocarcinoma cells Panc-1, which have relatively low GSH levels, the IC50 value were 44.1 ± 3.77, 26.4 ± 1.99, 24.5 ± 1.69, 18.9 ± 1.78 and 3.64 ± 0.124 µg/mL, respectively. Treatment with 2.5 mM diethyl maleate (DEM) which inhibits intracellular GSH biosynthesis resulted in the decrease of IC50 value for the 100% reducible polymer to 51.9 ± 3.48 and 13.9 ± 2.42 µg/mL in Panc-28 and Panc-1, respectively. Furthermore, treatment of cells with 1 mM 5,5’-Dithiobis(2-nitrobenzoic acid) (DTNB) which blocks thiol groups on the cell surface, resulted in the increase of IC50 for the 100% reducible polymer to 188 ± 17.2 and 112 ± 9.26 µg/mL in Panc-28 and Panc-1 cells, respectively. In contrast, the IC50 values of the non-reducible polymer were not affected by the changes in intracellular GSH levels. The results confirm that introduction of disulfide bonds into the structure of polycations significantly decreases their cytotoxicity and that both intracellular GSH levels and reducing capacity of cellular plasma membrane are involved in the cytotoxicity decrease.
Physical Methods of Delivery 141. Systematic Examination and Molecular Analysis of DNA-Nanoparticles-Mediated Toxicity Leads to Organ Site Identification and Methods To Overcome Toxicity In Vivo: Translation from Laboratory to the Clinic
Gopalan Began,1 Cynthia D. Branch,1 Jack A. Roth,1 Rajagopal Ramesh.1 1 Department of Thoracic and Cardiovascular Surgery, M.D. Anderson Cancer Center, Houston, TX.
These data suggest a possible involvement of the NF-κB pathway in attenuating transgene expression. However, the exact triggering receptor/ligand within the pathway would require further investigation.
140. Effect of Intracellular Glutathione Levels on Cytotoxicity of Bioreducible Polycations Chao Wu,1 Jun Chen,1 Jing Li,1 David Oupicky.1 1 Department of Pharmaceutical Sciences, Wayne State University, Detroit, MI.
The toxicity of polycations such as poly(ethylenimine) has been recognized since their use in gene delivery. Redox-sensitive polymers attract attention because of their intracellular reducibility and significantly lower toxicity. Common method employed in the design of reducible polymer is introducing disulfide bonds into the structures of the polycations. Bioreducible polycations easily form polyplexes with plasmid DNA, mRNA or siRNA that were already proven to show significantly improved efficacy of the delivery process. Bioreducible polycations are expected to be degraded rapidly and with a good intracellular selectivity by glutathione (GSH). GSH is able to participate in disulfide exchange reactions with polymers containing disulfide group. In our study, hyperbranched poly(amido Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy
Preclinical studies have demonstrated systemic gene delivery encapsulated in nanoparticles of various compositions has limited applicability due to toxicity. Therefore, treatment of metastatic cancers via systemic treatment has remained a challenge. We have previously reported effective systemic tumor suppressor gene delivery to experimental lung metastasis using a cationic-lipidbased nanoparticle. However, to translate our preclinical findings to the clinic for treatment of lung cancer we focused our efforts on examining the nanoparticle-mediated toxicity; the primary organ where the toxicity is initiated; and methods to overcome toxicity. For this purpose immunocompetent C3H mice were treated intravenously with Fus1 tumor suppressor gene (100 ug of plasmid) encapsulated in a cationic DOTAP:Cholesterol lipid-nanoparticle (Fus1-nanoparticles). The DNA concentration used was previously determined to be lethal in C3H mice with 100% death occurring within 24h after treatment. Animals that did not receive any treatment or were treated with empty nanoparticles or naked plasmid DNA alone served as controls. Time course (2h, 4h, 6h, 12h, and 24h) studies after treatment were performed that involved collection of blood and organs (lung, liver, spleen) for analysis of inflammatory cytokines; inflammation associated molecular signaling; and for histopathology. Induction of acute inflammatory cytokines (TNF, IL-1, IL-6, IFN) at 2h after treatment was observed in the serum of mice treated with Fus1-nanoparticles. The cytokine levels were very high at 2h compared to the cytokine levels in other treatment groups. However, the cytokine levels decreased by 12h after treatment and returned to S55
A second transfection applied with increasing concentrations of quinazoline showed consistent increase in total mean fluorescence across the ranges tested.
amine)s were synthesized by Michael addition copolymerization of equal molar ratio of N,N’-dimethylamino dipropylenetriamine (DMADPTA) to N,N’-cystaminebisacrylamide (CBA) and N,N’hexamethylenebisacrylamide (HMBA). Through controlling the ratio of CBA and HMBA, the percentage of reducible component was adjusted at 100%, 75%, 50%, 25% and 0%. Cytotoxicity was determined by MTS assay. The results indicate that the IC50 values of the series of polycations in GSH-rich human pancreatic adenocarcinoma cells Panc-28 were 98.3 ± 1.35, 75.6 ± 4.11, 47.5 ± 2.62, 26.9 ± 1.28 and 4.09 ± 0.147 µg/mL, respectively. By contrast, the results in human ductal pancreatic adenocarcinoma cells Panc-1, which have relatively low GSH levels, the IC50 value were 44.1 ± 3.77, 26.4 ± 1.99, 24.5 ± 1.69, 18.9 ± 1.78 and 3.64 ± 0.124 µg/mL, respectively. Treatment with 2.5 mM diethyl maleate (DEM) which inhibits intracellular GSH biosynthesis resulted in the decrease of IC50 value for the 100% reducible polymer to 51.9 ± 3.48 and 13.9 ± 2.42 µg/mL in Panc-28 and Panc-1, respectively. Furthermore, treatment of cells with 1 mM 5,5’-Dithiobis(2-nitrobenzoic acid) (DTNB) which blocks thiol groups on the cell surface, resulted in the increase of IC50 for the 100% reducible polymer to 188 ± 17.2 and 112 ± 9.26 µg/mL in Panc-28 and Panc-1 cells, respectively. In contrast, the IC50 values of the non-reducible polymer were not affected by the changes in intracellular GSH levels. The results confirm that introduction of disulfide bonds into the structure of polycations significantly decreases their cytotoxicity and that both intracellular GSH levels and reducing capacity of cellular plasma membrane are involved in the cytotoxicity decrease.
Physical Methods of Delivery 141. Systematic Examination and Molecular Analysis of DNA-Nanoparticles-Mediated Toxicity Leads to Organ Site Identification and Methods To Overcome Toxicity In Vivo: Translation from Laboratory to the Clinic
Gopalan Began,1 Cynthia D. Branch,1 Jack A. Roth,1 Rajagopal Ramesh.1 1 Department of Thoracic and Cardiovascular Surgery, M.D. Anderson Cancer Center, Houston, TX.
These data suggest a possible involvement of the NF-κB pathway in attenuating transgene expression. However, the exact triggering receptor/ligand within the pathway would require further investigation.
140. Effect of Intracellular Glutathione Levels on Cytotoxicity of Bioreducible Polycations Chao Wu,1 Jun Chen,1 Jing Li,1 David Oupicky.1 1 Department of Pharmaceutical Sciences, Wayne State University, Detroit, MI.
The toxicity of polycations such as poly(ethylenimine) has been recognized since their use in gene delivery. Redox-sensitive polymers attract attention because of their intracellular reducibility and significantly lower toxicity. Common method employed in the design of reducible polymer is introducing disulfide bonds into the structures of the polycations. Bioreducible polycations easily form polyplexes with plasmid DNA, mRNA or siRNA that were already proven to show significantly improved efficacy of the delivery process. Bioreducible polycations are expected to be degraded rapidly and with a good intracellular selectivity by glutathione (GSH). GSH is able to participate in disulfide exchange reactions with polymers containing disulfide group. In our study, hyperbranched poly(amido Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy
Preclinical studies have demonstrated systemic gene delivery encapsulated in nanoparticles of various compositions has limited applicability due to toxicity. Therefore, treatment of metastatic cancers via systemic treatment has remained a challenge. We have previously reported effective systemic tumor suppressor gene delivery to experimental lung metastasis using a cationic-lipidbased nanoparticle. However, to translate our preclinical findings to the clinic for treatment of lung cancer we focused our efforts on examining the nanoparticle-mediated toxicity; the primary organ where the toxicity is initiated; and methods to overcome toxicity. For this purpose immunocompetent C3H mice were treated intravenously with Fus1 tumor suppressor gene (100 ug of plasmid) encapsulated in a cationic DOTAP:Cholesterol lipid-nanoparticle (Fus1-nanoparticles). The DNA concentration used was previously determined to be lethal in C3H mice with 100% death occurring within 24h after treatment. Animals that did not receive any treatment or were treated with empty nanoparticles or naked plasmid DNA alone served as controls. Time course (2h, 4h, 6h, 12h, and 24h) studies after treatment were performed that involved collection of blood and organs (lung, liver, spleen) for analysis of inflammatory cytokines; inflammation associated molecular signaling; and for histopathology. Induction of acute inflammatory cytokines (TNF, IL-1, IL-6, IFN) at 2h after treatment was observed in the serum of mice treated with Fus1-nanoparticles. The cytokine levels were very high at 2h compared to the cytokine levels in other treatment groups. However, the cytokine levels decreased by 12h after treatment and returned to S55