Monoolein-based lipoplexes (DODAB/MO/DNA) as non-viral vector for transfection- from physicochemical characterization to biological application

Cationic liposomes/DNA (lipoplexes) have been widely used as non-viral vectors for transfection, the role of the neutral lipid in liposome formulation being determinant for the efficiency of this process [1,2]. In this work, we studied the potential of monoolein (MO) as helper lipid for cellular transfection. Lipoplexes composed of pDNA and dioctadecyldimethylammonium bromide (DODAB)/1-monooleoyl-rac-glycerol (MO) at different molar ratios (4:1, 2:1 and 1:1) were investigated, as well as different cationic lipid/DNA ratios. The physicochemical properties of the lipoplexes (size and charge), the formation of the lipoplexes, the effect of MO on pDNA condensation and the effect of heparin on percentage of pDNA release from the lipoplexes were also studied by Ethidium Bromide (EtBr) exclusion assays, Dynamic Light Scattering (DLS), Zeta Potential (æ) and electrophoresis. The cytotoxicity, transfection efficiency, as well as the intracellular localization of labeled DNA were evaluated on 293T cells. It was found that the presence of MO not only increases the efficiency of pDNA compactation, but also affects the physicochemical properties of lipoplexes, which could possibly interfere with lipoplex-cell interactions. The DODAB:MO (2:1) and (4:1) formulations were capable of efficiently mediate in vitro cell transfection. These results were consistent with fluorescence microscopy studies, which illustrated that lipoplexes were able to gain entry into the cytosol and deliver pDNA to the nucleus. Understanding the structure–activity relationship of MO based lipoplexes will give direction toward the development of safe and efficient gene delivery systems.Portuguese Foundation for Science and Technology (FCT) for financial support to Center of Physics and Center of Molecular & Environmental Biology and funding through projects PTDC/QUI/69795/2006 and SFRH/BD/46968/2009

Monoolein as helper lipid for non-viral transfection in mammals

Lipoplexes composed of pDNA and DODAB/MO at different molar ratios (4:1, 2:1 and 1:1) and different cationic lipid/DNA charge ratios were investigated. The physicochemical properties of the lipoplexes (size and charge), the pDNA complexation, and the effect of heparin on pDNA release, were studied by Dynamic Light Scattering, Zeta Potential, and Ethidium Bromide exclusion assays. The cytotoxicity, transfection efficiency and the intracellular localization of DNA were evaluated on 293T cells.The Portuguese Foundation for Science and Technology (FCT) for the financial support to the Center of Physics and Center of Molecular & Environmental Biology and funding through projects PTDC/QUI/69795/2006 and SFRH/BD/46968/2009 are acknowledged

Effects of preparation method on the physicochemical characteristics and cell transfection efficiency of non‐viral nanocarriers

The success of gene therapy is decisively dependent on the development of effective carrier systems, able to compact and thus protect the genetic material as to avoid both in vitro and in vivo barriers for transgene delivery [1]. Given the problems of safety encountered with viral vectors, non-viral carrier systems are a safer alternative for the therapeutic delivery of genes [2]. Cationic liposomes/DNA (lipoplexes) have been widely used as non-viral vectors for cell transfection, with the neutral lipid (helper) of the liposome formulation playing a determinant role for the efficiency of this process. The lipoplex preparation method itself influences the structural properties of the produced lipoplex, affecting its final lipofection capability [3, 4]. In this work, we have studied the potential for cell transfection of a new liposomal formulation containing monoolein (MO) as helper lipid, using various preparation methods. Lipoplexes composed of pSV-β-galactosidase and dioctadecyldimethylammonium bromide (DODAB)/1-monooleoyl-rac-glycerol (MO) at (4:1) molar ratio and at cationic lipid/DNA ratio of 4.0 were tested. Ethidium Bromide (EtBr) exclusion assays, Dynamic Light Scattering (DLS), and Zeta Potential (ζ) were used to study plasmidic DNA complexation and physicochemical properties of the formed lipoplexes (their size and electrical charge). Transfection efficiency was also evaluated on 293T cells by determining the activity of β-galactosidase, the reporter gene. The results indicate that the lipoplexes’ physicochemical properties are strongly dependent on the preparation method (one-step or multi-step complexation, at 25ºC or 50ºC), with resulting mean sizes varying from 350 to 1700 nm and superficial charge densities ranging from +9 mV to +30 mV. No clear correlation was found, however, between lipoplex physicochemical properties and observed cell transfection efficiencies, suggesting that other parameters, such as structure (degree of DNA condensation/compaction) of the complex and the mode of internalization by the cell may also weigh in on cell transfection. Nevertheless, it was possible to determine that lipoplex preparation methods can greatly affect DNA uptake by cells. Optimal nanotherapy requires the therapeutic molecule to be protected from degradation and reach its target cell and intracellular location. This work demonstrates that the MO-based system is a viable nanocarrier for plasmidic DNA and that the conditions to prepare the carriers for gene therapy application can be modulated to achieve maximal delivery efficiency

Photocatalytic thin films coupled with polymeric microcapsules for the controlled-release of volatile agents upon solar activation

This work reports on the application of solar-activated photocatalytic thin films that allow the controlled-release of volatile agents (e.g., insecticides, repellents) from the interior of adsorbed polymeric microcapsules. In order to standardize the tests,a quantification of the inherent controlled-release of a particular volatile agent is determined by gas chromatography coupled to mass spectroscopy, so that an application can be offered to a wide range of supports from various industrial sectors, such as in textiles (clothing, curtains, mosquito nets). This technology takes advantage of the established photocatalytic property of titanium dioxide (TiO2) for the use as an active surface/site to promote the controlled-release of a specific vapor (volatile agentfrom within the aforementioned microcapsules.Portuguese FCT COMPETE scientific programs, with references NANO/NTec-CA/0046/2007 and PTDC/CTM-NAN/119979/201

Propagação de mirtilo do tipo Rabbiteye por estaquia e alporquia.

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Recent advances on antimicrobial wound dressing: A review

Skin and soft tissue infections (SSTIs) have high rates of morbidity and mortality associated. Despite the successful treatment of some SSTIs, those affecting the subcutaneous tissue, fascia, or muscle delay the healing process and can lead to life-threatening conditions. Therefore, more effective treatments are required to deal with such pathological situations. Recently, wound dressings loaded with antimicrobial agents emerged as viable options to reduce wound bacterial colonization and infection, in order to improve the healing process. In this review, an overview of the most prominent antibacterial agents incorporated in wound dressings along with their mode of action is provided. Furthermore, the recent advances in the therapeutic approaches used in the clinic and some future perspectives regarding antibacterial wound dressings are also discussed

Improvement in the molecular diagnosis of Machado-Joseph disease

Abstract BACKGROUND: Direct detection of the gene mutation allows for the confirmation of the clinical diagnosis of Machado-Joseph disease (MJD), the most frequent cause of autosomal dominant spinocerebellar ataxia worldwide. OBJECTIVE: To address the main difficulties in our national MJD predictive testing program. The first was the emergence of intermediate alleles, for which it is not yet possible to determine whether they will cause disease. The second was the issue of homoallelism, ie, homozygosity for 2 normal alleles with exactly the same (CAG)(n) length, which occurs in about 10% of all test results. METHODS: A large pedigree with 1 affected patient carrying a 71 and a 51 CAG repeat and 2 asymptomatic relatives carrying the 51 CAG repeat and normal-size alleles underwent clinical and molecular studies. Intragenic haplotypes for these alleles were determined. A representative sample of the healthy population in the region was obtained to assess the distribution of the normal (CAG)(n) length. We established the genotype for 4 intragenic polymorphisms in the gene for MJD (MJD1) in 21 homoallelic individuals, to distinguish their 2 normal chromosomes. In addition, we developed a new Southern blot method to completely exclude cases of nonamplification of expanded alleles in the homoallelic individuals. RESULTS: The study of the family in which the 51 CAG repeat was found suggests that the allele is apparently not associated with disease. These intermediate alleles were not present in a large sample of the healthy population from the same region. Intragenic polymorphisms allowed distinction of the 2 different normal alleles in all cases of homoallelism. The absence of an expanded allele was also confirmed by Southern blot. CONCLUSIONS: We propose an improved protocol for molecular testing for MJD. These strategies, developed to overcome the practical difficulties mostly in the presymptomatic and prenatal diagnosis of MJD, should prove useful for other polyglutamine-related disorders

Magnetoliposomes based on magnetite nanoparticles

In this work, magnetic nanoparticles of magnetite were prepared by soft chemical methods, using different surfactants as templating media. These nanoparticles were either covered with a lipid bilayer, forming dry magnetoliposomes, or entrapped in liposomes - aqueous magnetoliposomes.FCT, FEDER, COMPETE/QREN/EU for financial support to CFUM (Strategic Project PEst-C/FIS/UI0607/2011) and to the research project PTDC/QUI/81238/2006 (FCOMP-01-0124-FEDER-007467)