Bioreducible Liposomes for Gene Delivery: From the Formulation to the Mechanism of Action

BACKGROUND: A promising strategy to create stimuli-responsive gene delivery systems is to exploit the redox gradient between the oxidizing extracellular milieu and the reducing cytoplasm in order to disassemble DNA/cationic lipid complexes (lipoplexes). On these premises, we previously described the synthesis of SS14 redox-sensitive gemini surfactant for gene delivery. Although others have attributed the beneficial effects of intracellular reducing environment to reduced glutathione (GSH), these observations cannot rule out the possible implication of the redox milieu in its whole on transfection efficiency of bioreducible transfectants leaving the determinants of DNA release largely undefined. METHODOLOGY/PRINCIPAL FINDINGS: With the aim of addressing this issue, SS14 was here formulated into binary and ternary 100 nm-extruded liposomes and the effects of the helper lipid composition and of the SS14/helper lipids molar ratio on chemical-physical and structural parameters defining transfection effectiveness were investigated. Among all formulations tested, DOPC/DOPE/SS14 at 25:50:25 molar ratio was the most effective in transfection studies owing to the presence of dioleoyl chains and phosphatidylethanolamine head groups in co-lipids. The increase in SS14 content up to 50% along DOPC/DOPE/SS14 liposome series yielded enhanced transfection, up to 2.7-fold higher than that of the benchmark Lipofectamine 2000, without altering cytotoxicity of the corresponding lipoplexes at charge ratio 5. Secondly, we specifically investigated the redox-dependent mechanisms of gene delivery into cells through tailored protocols of transfection in GSH-depleted and repleted vs. increased oxidative stress conditions. Importantly, GSH specifically induced DNA release in batch and in vitro. CONCLUSIONS/SIGNIFICANCE: The presence of helper lipids carrying unsaturated dioleoyl chains and phosphatidylethanolamine head groups significantly improved transfection efficiencies of DOPC/DOPE/SS14 lipoplexes. Most importantly, this study shows that intracellular GSH levels linearly correlated with transfection efficiency while oxidative stress levels did not, highlighting for the first time the pivotal role of GSH rather than oxidative stress in its whole in transfection of bioreducible vectors

Genetic Biomarkers for ALS Disease in Transgenic SOD1G93A Mice

The pathophysiological mechanisms of both familial and sporadic Amyotrophic Lateral Sclerosis (ALS) are unknown, although growing evidence suggests that skeletal muscle tissue is a primary target of ALS toxicity. Skeletal muscle biopsies were performed on transgenic SOD1G93A mice, a mouse model of ALS, to determine genetic biomarkers of disease longevity. Mice were anesthetized with isoflurane, and three biopsy samples were obtained per animal at the three main stages of the disease. Transcriptional expression levels of seventeen genes, Ankrd1, Calm1, Col19a1, Fbxo32, Gsr, Impa1, Mef2c, Mt2, Myf5, Myod1, Myog, Nnt, Nogo A, Pax7, Rrad, Sln and Snx10, were tested in each muscle biopsy sample. Total RNA was extracted using TRIzol Reagent according to the manufacturer's protocol, and variations in gene expression were assayed by real-time PCR for all of the samples. The Pearson correlation coefficient was used to determine the linear correlation between transcriptional expression levels throughout disease progression and longevity. Consistent with the results obtained from total skeletal muscle of transgenic SOD1G93A mice and 74-day-old denervated mice, five genes (Mef2c, Gsr, Col19a1, Calm1 and Snx10) could be considered potential genetic biomarkers of longevity in transgenic SOD1G93A mice. These results are important because they may lead to the exploration of previously unexamined tissues in the search for new disease biomarkers and even to the application of these findings in human studies

Effects of temperature in juvenile seabass (Dicentrarchus labrax L.) biomarker responses and behaviour: implications for environmental monitoring

The effects of temperature on European seabass (Dicentrarchus labrax L.) juveniles were investigated using a 30-day bioassay carried out at 18 and 25 °C in laboratory conditions. A multiparameter approach was applied including fish swimming velocity and several biochemical parameters involved in important physiological functions. Fish exposed for four weeks to 25 °C showed a decreased swimming capacity, concomitant with increased oxidative stress (increased catalase and glutathione peroxidase activities) and damage (increased lipid peroxidation levels), increased activity of an enzyme involved in energy production through the aerobic pathway (isocitrate dehydrogenase) and increased activities of brain and muscle cholinesterases (neurotransmission) compared to fish kept at 18 °C. Globally, these findings indicate that basic functions, essential for juvenile seabass surviving and well performing in the wild, such as predation, predator avoidance, neurofunction and ability to face chemical stress may be compromised with increasing water temperature. This may be of particular concern if D. labrax recruitment phase in northwest European estuaries and coastal areas happens gradually inmore warm environments as a consequence of global warming. Considering that the selected endpoints are generally applied in monitoring studies with different species, these findings also highlight the need of more research, including interdisciplinary and multiparameter approaches, on the impacts of temperature on marine species, and stress the importance of considering scenarios of temperature increase in environmental monitoring and in marine ecological risk assessment

glutathione depletion activates mitogen-activated protein kinase (mapk) pathways that display organ-specific responses and brain protection in mice

Because mitogen-activated protein kinases (MAPK) are downstream effectors of antioxidant responses, changes in GSH levels in an organism might induce organ-specific responses. To test our hypothesis, mice were treated intraperitoneally with L-buthionine-S-R-sulfoximine (BSO) to inhibit GSH synthesis. A time-related GSH depletion in the liver and kidney correlated with p38(MAPK) phosphorylation and induction of thioredoxin ! (Tx-1) transcription. This positive regulation was associated with nuclear translocation of NF-kappa B and ATF-2 and gamma-Jun phosphorylation in the liver, but only c-Jun phosphorylation in the kidney. Increased levels of GSH were observed in the brain together with extracellular regulated kinase 2 (ERK2) activation, Nrf2 nuclear accumulation, and increases in transcription of Nrf2, xCT, T-glutamylcysteine synthetase (gamma GCSr), and Tx-1. Pretreatment with MAPK inhibitors SB203580 and U0126, or addition of the exogenous thiol N-acetylcysteine, abrogated both p38(MAPK) and ERK2 activation as well as downstream effects on gene expression. No effect on gamma GCSr was observed. These results indicate that in mice, GSH depletion is associated with p38(MAPK) phosphorylation in the liver and kidney and with ERK2 activation in the brain, in what could be considered part of the brain's protective response to thiol depletion. (C) 2007 Elsevier Inc. All rights reserved