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

Cholangiocyte Apoptosis Is an Early Event During Induced Metamorphosis in the Sea Lamprey, Petromyzon Marinus L

Background: Research in biliary atresia has been hindered by lack of a suitable animal model. Lampreys are primitive vertebrates with distinct larval and adult life cycle stages. During metamorphosis the biliary system of the larval lamprey disappears. Lamprey metamorphosis has been proposed as a model for biliary atresia. We have begun to explore cellular events during lamprey metamorphosis by assessing for cholangiocyte apoptosis.Materials and Methods: Sea lamprey larvae were housed under controlled environmental conditions. Premetamorphic larvae were induced to undergo metamorphosis by exposure to 0.01% KClO(4). Animals were photographed weekly, and the stage of metamorphosis was assigned based upon external features. Livers were harvested and processed for routine histology and immunohistochemistry. DNA fragmentation was detected using deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assays and cholangiocytes were identified with antibodies to cytokeratin-19. Percent TUNEL+ cholangiocytes at different stages of metamorphosis was determined.Results: The percentage of TUNEL+ cholangiocytes was 10% in premetamorphic (stage 0) lamprey (n = 6), 51% at stage 1 (n = 6), 40% at stage 2 (n = 5), 18% at stage 3 (n = 5), and 9% stage 4 (n = 4). Routine hemotoxylin and eosin stained paraffin-embedded tissue sections revealed frequent apoptotic bodies at stages 3 and 4 of metamorphosis without histologic evidence of necrosis.Conclusions: DNA fragmentation is identified at the earliest stages of metamorphosis during induced metamorphosis in lampreys. Additional studies are necessary to validate this potentially valuable animal model. (C) 2010 Elsevier Inc. All rights reserved

Timing and volume of crystalloid and blood products in pediatric trauma: An Eastern Association for the Surgery of Trauma multicenter prospective observational study

Copyright © 2020 Wolters Kluwer Health, Inc. All rights reserved. BACKGROUND The purpose of this study was to determine the relationship between timing and volume of crystalloid before blood products and mortality, hypothesizing that earlier transfusion and decreased crystalloid before transfusion would be associated with improved outcomes. METHODS A multi-institutional prospective observational study of pediatric trauma patients younger than 18 years, transported from the scene of injury with elevated age-adjusted shock index on arrival, was performed from April 2018 to September 2019. Volume and timing of prehospital, emergency department, and initial admission resuscitation were assessed including calculation of 20 ± 10 mL/kg crystalloid boluses overall and before transfusion. Multivariable Cox proportional hazards and logistic regression models identified factors associated with mortality and extended intensive care, ventilator, and hospital days. RESULTS In 712 children at 24 trauma centers, mean age was 7.6 years, median (interquartile range) Injury Severity Score was 9 (2-20), and in-hospital mortality was 5.3% (n = 38). There were 311 patients(43.7%) who received at least one crystalloid bolus and 149 (20.9%) who received blood including 65 (9.6%) with massive transfusion activation. Half (53.3%) of patients who received greater than one crystalloid bolus required transfusion. Patients who received blood first (n = 41) had shorter median time to transfusion (19.8 vs. 78.0 minutes, p = 0.005) and less total fluid volume (50.4 vs. 86.6 mL/kg, p = 0.033) than those who received crystalloid first despite similar Injury Severity Score (median, 22 vs. 27, p = 0.40). On multivariable analysis, there was no association with mortality (p = 0.51); however, each crystalloid bolus after the first was incrementally associated with increased odds of extended ventilator, intensive care unit, and hospital days (all p \u3c 0.05). Longer time to transfusion was associated with extended ventilator duration (odds ratio, 1.11; p = 0.04). CONCLUSION Resuscitation with greater than one crystalloid bolus was associated with increased need for transfusion and worse outcomes including extended duration of mechanical ventilation and hospitalization in this prospective study. These data support a crystalloid-sparing, early transfusion approach for resuscitation of injured children. LEVEL OF EVIDENCE Therapeutic, level IV