Mitochondrial Gene Therapy: Advances in Mitochondrial Gene Cloning, Plasmid Production, and Nanosystems Targeted to Mitochondria.

Mitochondrial gene therapy seems to be a valuable and promising strategy to treat mitochondrial disorders. The use of a therapeutic vector based on mitochondrial DNA, along with its affinity to the site of mitochondria, can be considered a powerful tool in the reestablishment of normal mitochondrial function. In line with this and for the first time, we successfully cloned the mitochondrial gene ND1 that was stably maintained in multicopy pCAG-GFP plasmid, which is used to transform E. coli. This mitochondrial-gene-based plasmid was encapsulated into nanoparticles. Furthermore, the functionalization of nanoparticles with polymers, such as cellulose or gelatin, enhances their overall properties and performance for gene therapy. The fluorescence arising from rhodamine nanoparticles in mitochondria and a fluorescence microscopy study show pCAG-GFP-ND1-based nanoparticles' cell internalization and mitochondria targeting. The quantification of GFP expression strongly supports this finding. This work highlights the viability of gene therapy based on mitochondrial DNA instigating further in vitro research and clinical translation.

Development, characterization, and skin delivery studies of related ultradeformable vesicles: transfersomes, ethosomes, and transethosomes.

Ultradeformable vesicles (UDV) have recently become a promising tool for the development of improved and innovative dermal and transdermal therapies. The aim of this work was to study three related UDV: transfersomes, ethosomes, and transethosomes for the incorporation of actives of distinct polarities, namely, vitamin E and caffeine, and to evaluate the effect of the carrier on skin permeation and penetration. These actives were incorporated in UDV formulations further characterized for vesicles imaging by transmission electron microscopy; mean vesicle size and polydispersity index by photon correlation spectroscopy; zeta potential by laser-Doppler anemometry; deformability by pressure-driven transport; and incorporation efficiency (IE) after actives quantification by high-performance liquid chromatography. Topical delivery studies were performed in order to compare UDV formulations regarding the release, skin permeation, and penetration profiles. All UDV formulations showed size values within the expected range, except transethosomes prepared by "transfersomal method", for which size was smaller than 100 nm in contrast to that obtained for vesicles prepared by "ethosomal method". Zeta potential was negative and higher for formulations containing sodium cholate. The IE was much higher for vitamin E- than caffeine-loaded UDV as expected. For flux measurements, the following order was obtained: transethosomes (TE) > ethosomes (E) ≥ transfersomes (T). This result was consistent with the release and skin penetration profiles for Vitamin E-loaded UDV. However, the releasing results were totally the opposite for caffeine-loaded UDV, which might be explained by the solubility and thermodynamic activity of this active in each formulation instead of the UDV deformability attending to the higher non-incorporated fraction of caffeine. Anyway, a high skin penetration and permeation for all caffeine-loaded UDV were obtained. Transethosomes were more deformable than ethosomes and transfersomes due to the presence of both ethanol and surfactant in their composition. All these UDV were suitable for a deeper skin penetration, especially transethosomes.

Nutritional and nutraceutical potential of rape (Brassica napus L. var. napus) and "tronchuda" cabbage (Brassica oleraceae L. var. costata) inflorescences.

Two traditional cultivated vegetables highly consumed among Northern Portuguese regions were tested for their chemical composition, nutritional profile and in vitro antioxidant properties using four assays: 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals scavenging activity, reducing power, inhibition of ß-carotene bleaching and inhibition of lipid peroxidation by thiobarbituric acid reactive substances (TBARS) assay. The studied varieties of two Brassica species, locally known as "grelos" (rape) and "espigos" ("tronchuda" cabbage) are nutritionally well-balanced vegetables; particularly "tronchuda" cabbage revealed the highest levels of moisture, proteins, fat, energy, ß-carotene and vitamin C; rape gave the highest contents of ash, carbohydrates, sugars (including fructose, glucose, sucrose and raffinose), essential n-3 fatty acid α-linolenic acid, and the best ratios of PUFA/SFA and n-6/n-3 fatty acids, tocopherols, lycopene, chlorophylls, phenolics, flavonoids, and also the highest antioxidant properties. The health benefits associated to the antioxidant properties reinforce their contribution to a healthy and balanced diet, highlight the interest of their consumption, validate the empirical use and add new values to traditional/regional products which have been used for a long time.