The role of the helper lipid on the DNA transfection efficiency of lipopolyplex formulations.

Multifunctional, lipopolyplex formulations comprising a mixture of cationic liposomes and cationic, receptor-targeting peptides have potential use in gene therapy applications. Lipopolyplex formulations described here are typically far more efficient transfection agents than binary lipoplex or polyplex formulations. It has been shown previously that the peptide component mediates both DNA packaging and targeting of the nanoparticle while in this report we investigate the contribution of the lipid component. We hypothesised that the lipid components synergise with the peptides in the transfection process by promoting endosomal escape after lipid bilayer fusion. Lipopolyplexes were prepared with cationic liposomes comprising DOTAP with either neutral lipid DOPE or DOPC. DOPE promotes fusogenic, inverted hexagonal lipid structures while DOPC promotes more stable laminar structures. Lipopolyplexes containing DOPE showed substantially higher transfection efficiency than those formulated with DOPC, both in vitro and in vivo. DOPE-containing lipopolyplexes showed rapid endosomal trafficking and nuclear accumulation of DNA while DOPC-containing formulations remained within the late endo-lysosomal compartments. These findings are consistent with previous finding for the role of DOPE in lipoplexes and support the hypothesis regarding the function of the lipid components in lipopolyplexes. These findings will help to inform future lipopolyplex design, strategies and clinical development processes

Dioctadecyldimethylammonium:monoolein nanocarriers for efficient in vitro gene silencing

This study describes a novel liposomal formulation for siRNA delivery, based on the mixture of the neutral lipid monoolein (MO) and cationic lipids of the dioctadecyldimethylammonium (DODA) family. The cationic lipids dioctadecyldimethylammonium bromide (DODAB) and chloride (DODAC) were compared in order to identify which one will most efficiently induce gene silencing. MO has a fluidizing effect on DODAC and DODAB liposomes, although it was more homogeneously distributed in DODAC bilayers. All MO-based liposomal formulations were able to efficiently encapsulate siRNA. Stable lipoplexes of small size (100-160 nm) with a positive surface charge (>+45 mV) were formed. A more uniform MO incorporation in DODAC:MO may explain an increase of the fusogenic potential of these liposomes. The siRNA-lipoplexes were readily internalized by human nonsmall cell lung carcinoma (H1299) cells, in an energy dependent process. DODAB:MO nanocarriers showed a higher internalization efficiency in comparison to DODAC:MO lipoplexes, and were also more efficient in promoting gene silencing. MO had a similar gene silencing ability as the commonly used helper lipid 1,2-dioleyl-3-phosphatidylethanolamine (DOPE), but with much lower cytotoxicity. Taking in consideration all the results presented, DODAB:MO liposomes are the most promising tested formulation for systemic siRNA delivery.This work was supported by FEDER through POFC - COMPETE and by national funds from FCT through the projects PEst-C/BIA/UI4050/2011 (CBM.A), PEst-C/FIS/UI0607/2011 (CFUM), and PTDC/QUI/69795/2006, while Ana Oliveira holds scholarship SFRH/BD/68588/2010. Eloi Feitosa thanks FAPESP (2011/03566-0) and CNPq (303030/2012-7), and Renata D. Adati thanks FAPESP for scholarship (2011/07414-0). K. Raemdonck is a postdoctoral fellow of the Research Foundation - Flanders (FWO-Vlaanderen). We acknowledge NanoDelivery-I&D em Bionanotecnologia, Lda. for access to their equipment

In vitro and in vivo mRNA delivery using lipid-enveloped pHresponsive polymer nanoparticles

Biodegradable core−shell structured nanoparticles with a poly(β-amino ester) (PBAE) core enveloped by a phospholipid bilayer shell were developed for in vivo mRNA delivery with a view toward delivery of mRNA-based vaccines. The pH-responsive PBAE component was chosen to promote endosome disruption, while the lipid surface layer was selected to minimize toxicity of the polycation core. Messenger RNA was efficiently adsorbed via electrostatic interactions onto the surface of these net positively charged nanoparticles. In vitro, mRNA-loaded particle uptake by dendritic cells led to mRNA delivery into the cytosol with low cytotoxicity, followed by translation of the encoded protein in these difficult-to-transfect cells at a frequency of 30%. Particles loaded with mRNA administered intranasally (i.n.) in mice led to the expression of the reporter protein luciferase in vivo as soon as 6 h after administration, a time point when naked mRNA given i.n. showed no expression. At later time points, luciferase expression was detected in naked mRNA-treated mice, but this group showed a wide variation in levels of transfection, compared to particle-treated mice. This system may thus be promising for noninvasive delivery of mRNA-based vaccines.United States. Dept. of Defense (Institute for Soldier Nanotechnology, contract W911NF-07-D-0004)Ragon Institute of MGH, MIT and HarvardSingapore. Agency for Science, Technology and ResearchHoward Hughes Medical Institute (Investigator

Nonviral Approaches for Neuronal Delivery of Nucleic Acids

The delivery of therapeutic nucleic acids to neurons has the potential to treat neurological disease and spinal cord injury. While select viral vectors have shown promise as gene carriers to neurons, their potential as therapeutic agents is limited by their toxicity and immunogenicity, their broad tropism, and the cost of large-scale formulation. Nonviral vectors are an attractive alternative in that they offer improved safety profiles compared to viruses, are less expensive to produce, and can be targeted to specific neuronal subpopulations. However, most nonviral vectors suffer from significantly lower transfection efficiencies than neurotropic viruses, severely limiting their utility in neuron-targeted delivery applications. To realize the potential of nonviral delivery technology in neurons, vectors must be designed to overcome a series of extra- and intracellular barriers. In this article, we describe the challenges preventing successful nonviral delivery of nucleic acids to neurons and review strategies aimed at overcoming these challenges

Gene delivery by cationic lipids: in and out of an endosome

Cationic lipids are exploited as vectors ('lipoplexes') for delivering nucleic acids, including genes, into cells for both therapeutic and cell biological purposes. However, to meet therapeutic requirements, their efficacy needs major improvement, and better defining the mechanism of entry in relation to eventual transfection efficiency could be part of such a strategy. Endocytosis is the major pathway of entry, but the relative contribution of distinct endocytic pathways, including clathrin- and caveolae-mediated endocytosis and/or macropinocytosis is as yet poorly defined. Escape of DNA/RNA from endosomal compartments is thought to represent a major obstacle. Evidence is accumulating that non-lamellar phase changes of the lipoplexes, facilitated by intracellular lipids, which allow DNA to dissociate from the vector and destabilize endosomal membranes, are instrumental in plasmid translocation into the cytosol, a prerequisite for nuclear delivery. To further clarify molecular mechanisms and to appreciate and overcome intracellular hurdles in lipoplex-mediated gene delivery, quantification of distinct steps in overall transfection and proper model systems are required

Aspects sanitaires du travail des enfants dans les carrieres d’exploitation de gravier et de sable dans la Region Maritime au Togo

Introduction : Le travail des enfants (TDE) est un grave problème de santé publique en raison de ses multiples conséquences. L’Afrique subsaharienne y est particulièrement exposée en raison de la pauvreté.Objectifs : Décrire le profil sociodémographique de ces enfants travailleurs et répertorier les affections liées au travail de ces enfants dans les carrières.Méthodologie : Etude transversale descriptive portant sur les aspects sanitaires du TDE conduite d’août à novembre 2014, sur 18 sites d’extraction de graviers et de sable des préfectures de la région maritime du Togo.Résultats : Nous avons recensé 99 sujets de moins de 18 ans sur une population totale de 345 travailleurs. Les enfants avaient été retrouvés sur tous les sites d’extraction de gravier dans la région maritime.Les enfants étaient dans 77,7% de sexe masculin (77 enfants) avec un sex-ratio 3,3. Tous les enfants avaient au moins 7 ans avec une moyenne de 12 ans et dans 49,5% (49 enfants) leur âge était compris entre 11 et 14 ans.Les accidents de travail ont été signalés dans 81,8% des cas (81 enfants). Les types de lésions rapportées ont été essentiellement des plaies dans 79,8% (79enfants) et des entorses dans 36,4% des cas (36 enfants).Les symptômes respiratoires comme rhume et toux ont été retrouvé dans respectivement 43,4% (43 enfants) et 42,4% (42 enfants) ; des douleurs musculaires étaient retrouvées dans 92,9% des cas.Sur tous les sites visités aucun moyen de protection adapté n’a été observé. La prise en charge des accidents du travail a été effectuée par les enfants eux-mêmes et par leurs parents dans respectivement 46,5% (46 enfants) et 53,5% des cas (53 enfants).Conclusion : Les enfants présents sur tous les sites d’extraction étaient le plus souvent de sexe masculin et avaient un âge moyen de 12 ans. Les pathologies rencontrées sont dominées par les atteintes respiratoires avec les douleurs comme plaintes chez les enfants. Des accidents de travail fréquents sont pris en charge par les enfants ou leurs parents. Mots-clés : Travail des enfants, maladies professionnelles, accidents du travail, carrières, Togo.   English Title: Aspects sanitaires du travail des enfants dans les carrières d’exploitation de gravier et de sable dans la Région Maritime au TogoIntroduction: Child labor (CL) is a serious public health problem because of its multiple consequences. Sub-Saharan Africa is particularly vulnerable because of poverty. Objectives: To describe the socio-demographic profile of these child workers and to list the conditions related to the work of these children in careers. Methodology: Cross-sectional descriptive study on the health aspects of the CL conducted from August to November 2014, on 18 gravel and sand extraction sites in the prefectures of the maritime region of Togo. Results: We identified 99 subjects under 18 years old out of a total population of 345 workers. The children had been found at all gravel extraction sites in the Maritime Region. Children were 77.7% male (77 children) with a sex ratio of 3. 3. All children were at least 7 years old with an average of 12 years old and in 49.5% (49 children) their age was between 11 and 14 years old. Work-related accidents were reported in 81.8% of cases (81 children). The types of lesions reported were mainly wounds in 79.8% (79 children) and sprains in 36.4% of the cases (36 children). Respiratory  symptoms such as colds and coughs were found in respectively 43.4% (43 children) and 42.4% (42 children); muscle pain was found in 92.9% of cases. At all the sites visited no suitable means of protection were observed. The management of accidents related to work was carried out by the children themselves and their parents in respectively 46.5% (46 children) and 53.5% of the cases (53 children). Conclusion: The children present at all extraction sites were mostly male and had an average age of 12 years. The pathologies encountered are dominated by respiratory disorders with pains as complaints in children. Frequent work accidents are dealt with by children or their parents. Keywords: Child labor, occupational diseases, accidents at work, careers, Togo

Randomised comparison of a bioresorbable everolimus-eluting scaffold with a metallic everolimus-eluting stent for ischaemic heart disease caused by de novo native coronary artery lesions: the 2-year clinical outcomes of the ABSORB II trial

The one-year randomised data of the ABSORB II trial showed that the everolimus-eluting bioresorbable scaffold and the everolimus-eluting metallic stent were comparable for the composite secondary clinical outcomes of patient-oriented composite endpoint (PoCE) and device-oriented composite endpoint (DoCE)/target lesion failure (TLF), MACE and TVF. This report describes the two-year clinical outcomes of the ABSORB II trial. Patients were randomly assigned in a 2:1 ratio to receive treatment with an everolimus-eluting bioresorbable scaffold (Absorb; Abbott Vascular, Santa Clara, CA, USA) or treatment with an everolimus-eluting metallic stent (XIENCE; Abbott Vascular). The trial enrolled 501 patients. Clinical follow-up at two years was available in 320 patients in the Absorb BVS arm and 160 patients in the XIENCE arm. At two years, the PoCE for the Absorb and XIENCE arms was 11.6% and 12.8% (p=0.70) and the DoCE/TLF was 7.0% and 3.0% (p=0.07), respectively. The hierarchical ID-MACE rate was 7.6% vs. 4.3% (p=0.16) and the rate of TVF was 8.5% vs. 6.7% (p=0.48). The definite/probable thrombosis rate was 1.5% in the Absorb arm vs. 0% in the XIENCE arm (p=0.17). Thirty-six percent and 34% of patients remained on DAPT at two years, respectively. Ninety-two percent of patients in both arms remained on aspirin. Two-year clinical results demonstrate sustained low rates of PoCE, MACE, DoCE and TVF with the Absorb BVS as compared to the XIENCE sten

Towards positional cloning of the RN gene in the pig

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