RESUMO
These studies are focused on antagonizing organophosphorous (OP) intoxications by a new conceptual approach using recombinant enzymes encapsulated within sterically stabilized liposomes to enhance diisopropylfluorophosphate (DFP) degradation. The OP hydrolyzing enzyme, organophosphorous acid anhydrolase (OPAA), encapsulated within the liposomes, was employed either alone or in combination with pralidoxime (2-PAM) and/or atropine. The recombinant OPAA enzyme, from the ALTEROMONAS: strain JD6, has high substrate specificity toward a wide range of OP compounds, e.g., DFP, soman, and sarin. The rate of DFP hydrolysis by liposomes containing OPAA (SL)* was measured by determining the changes in fluoride-ion concentration using a fluoride ion-selective electrode. This enzyme carrier system serves as a biodegradable protective environment for the OP-metabolizing enzyme (OPAA), resulting in an enhanced antidotal protection against the lethal effects of DFP. Free OPAA alone showed some antidotal protection; however, the protection with 2-PAM and/or atropine was greatly enhanced when combined with (SL)*.
Assuntos
Inibidores da Colinesterase/toxicidade , Esterases/farmacologia , Isoflurofato/antagonistas & inibidores , Isoflurofato/toxicidade , Lipossomos , Animais , Arildialquilfosfatase , Portadores de Fármacos , Isoflurofato/metabolismo , Dose Letal Mediana , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Sarina/metabolismo , Soman/metabolismo , Especificidade por SubstratoRESUMO
The objective of this study was to determine the potential toxicity generated by the interaction of the Nd:YAG laser and Alcon IOGEL intraocular hydrogel lens material. The IOGEL lens is composed of poly 2-hydroxyethylmethacrylate, containing 38% water, previously shown to be highly biocompatible in a wide range of tissue culture and implantation experiments. In this study, intraocular lenses (IOLs) immersed in serum-free cell culture medium were purposely exposed to exaggerated doses of laser energy to cause extensive damage. An IOLAB polymethylmethacrylate (PMMA) lens served as a control lens material. The resultant solutions were assayed for cytotoxicity in a bioassay system using fourth passage human corneal endothelial cells. No cytotoxicity was seen in the bioassay for the IOGEL hydrogel IOLs or the PMMA control IOL at any laser range/dosage tested over a 72-hour incubation period. Hydrogel lenses exhibited decreasing yellowing with decreasing energy levels, and no lens discoloration was apparent at the lowest level of irradiation, 5 mJ/50 laser bursts; the PMMA control lens exhibited moderate yellowing at 15 mJ/50 bursts. Lens marking was moderate for all IOGEL IOLs; the PMMA lens marking was severe at the power level tested.
Assuntos
Extração de Catarata , Endotélio Corneano/patologia , Terapia a Laser , Lentes Intraoculares , Polietilenoglicóis , Complicações Pós-Operatórias/patologia , Sobrevivência Celular , Células Cultivadas , Humanos , Hidrogel de Polietilenoglicol-Dimetacrilato , Metilmetacrilatos , Desenho de Prótese , Falha de PróteseRESUMO
This investigation effort is focused on increasing organophosphate (OP) degradation by phosphotriesterase to antagonize OP intoxication. For these studies, sterically stabilized liposomes encapsulating recombinant phosphotriesterase were employed. This enzyme was obtained from Flavobacterium sp. and was expressed in Escherichia coli. It has a broad substrate specificity, which includes parathion, paraoxon, soman, sarin, diisopropylfluorophosphate, and other organophosphorous compounds. Paraoxon is rapidly hydrolyzed by phosphotriesterase to the less toxic 4-nitrophenol and diethylphosphate. This enzyme was isolated and purified over 1600-fold and subsequently encapsulated within sterically stabilized liposomes (SL). The properties of this encapsulated phosphotriesterase were investigated. When these liposomes containing phosphotriesterase were incubated with paraoxon, it readily degraded the paraoxon. Hydrolysis of paraoxon did not occur when these sterically stabilized liposomes contained no phosphotriesterase. These sterically stabilized liposomes (SL) containing phosphotriesterases (SL)* were employed as a carrier model to antagonize the toxic effects of paraoxon by hydrolyzing it to the less toxic 4-nitrophenol and diethylphosphate. This enzyme-SL complex (SL)* was administered intravenously to mice either alone or in combination with pralidoxime (2-PAM) and/or atropine intraperitoneally. These results indicate that this carrier model system provides a striking enhanced protective effects against the lethal effects of paraoxon. Moreover when these carrier liposomes were administered with 2-PAM and/or atropine, a dramatic enhanced protection was observed.