Catarina Madeira

Preparation and characterisation of new oxovanadium(IV) Schiff base complexes derived from amino acids and aromatic o-hydroxyaldehydes

Abstract A range of mostly new oxovanadium(IV) complexes is described. They contain coordinated Schiff bases, made from natural amino acids (glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, threonine, aspartic acid, and histidine) and salicylaldehyde or such derivatives as 3-, 4-, or 5-methoxy-salicylaldehyde. The coordination sphere is completed by simple ligands like water, 2,2′-bipyridyl or pyridine. The compounds are characterised and the nature of their coordination spheres shown by analysis, TLC, and by appropriate spectroscopy (EPR, IR, electronic and circular dichroism of solution and solids). In a few cases, magnetic properties are described to establish oxidation state. In several cases, the solubility of the compounds from racemic amino acids differs markedly from those containing the single enantiomer. The crystal and molecular structure of the related (and novel) compound with N-pyridoxylidene- d , l -isoleucinate, [VO(pyr- d , l -Ile)(bipy)]·H2O is described. It contains two diastereomers. Denoting the chiral vanadium centres as A or C, these are and [A(pyr- l -Ile)(bipy)] [C(pyr- d -Ile)(bipy)].

Advanced cell therapies for articular cartilage regeneration

Advanced cell-based therapies are promising approaches for stimulating full regeneration of cartilage lesions. In addition to a few commercially available medicinal products, several clinical and preclinical studies are ongoing worldwide. In preclinical settings, high-quality cartilage tissue has been produced using combination strategies involving stem or progenitor cells, biomaterials, and biomolecules to generate a construct for implantation at the lesion site. Cell numbers and mechanical stimulation of the constructs are not commonly considered, but are important parameters to be evaluated in forthcoming clinical studies. We review current clinical and preclinical studies for advanced therapy cartilage regeneration and evaluate the progress of the field.

Nonviral Gene Delivery to Mesenchymal Stem Cells Using Cationic Liposomes for Gene and Cell Therapy

Mesenchymal stem cells (MSCs) hold a great promise for application in several therapies due to their unique biological characteristics. In order to harness their full potential in cell-or gene-based therapies it might be advantageous to enhance some of their features through gene delivery strategies. Accordingly, we are interested in developing an efficient and safe methodology to genetically engineer human bone marrow MSC (BM MSC), enhancing their therapeutic efficacy in Regenerative Medicine. The plasmid DNA delivery was optimized using a cationic liposome-based reagent. Transfection efficiencies ranged from ~2% to ~35%, resulting from using a Lipid/DNA ratio of 1.25 with a transgene expression of 7 days. Importantly, the number of plasmid copies in different cell passages was quantified for the first time and ~20,000 plasmid copies/cell were obtained independently of cell passage. As transfected MSC have shown high viabilities (>90%) and recoveries (>52%) while maintaining their multipotency, this might be an advantageous transfection strategy when the goal is to express a therapeutic gene in a safe and transient way.

Characterization of DNA/lipid complexes by fluorescence resonance energy transfer.

Fluorescence resonance energy transfer (FRET) is a potential method for the characterization of DNA-cationic lipid complexes (lipoplexes). In this work, we used FRET models assuming a multilamellar lipoplex arrangement. The application of these models allows the determination of the distance between the fluorescent intercalator on the DNA and a membrane dye on the lipid, and/or the evaluation of encapsulation efficiencies of this liposomal vehicle. The experiments were carried out in 1,2-dioleoyl-3-trimethylammonium-propane/pUC19 complexes with different charge ratios. We used 2-(3-(diphenylhexatrienyl)propanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine (DPH-PC) and 2-(4,4-difluoro-5-octyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine (BODIPY-PC) as membrane dyes, and ethidium bromide (EtBr) and BOBO-1 as DNA intercalators. In cationic complexes (charge ratios (+/-) >or= 2), we verified that BOBO-1 remains bound to DNA, and FRET occurs to the membrane dye. This was also confirmed by anisotropy and lifetime measurements. In complexes with all DNA bound to the lipid (charge ratio (+/-) = 4), we determined 27 A as the distance between the donor and acceptor planes (half the repeat distance for a multilamellar arrangement). In complexes with DNA unbound to the lipids (charge ratio (+/-) = 0.5 and 2), we calculated the encapsulation efficiencies. The presented FRET methodology is, to our knowledge, the first procedure allowing quantification of lipid-DNA contact.

Gene delivery to human bone marrow mesenchymal stem cells by microporation

Electroporation has been considered one of the most efficient non-viral based methods to deliver genes regardless of frequently observed high cell mortality. In this study we used a microporation technique to optimise the delivery of plasmid DNA encoding green fluorescence protein (GFP) to human bone marrow mesenchymal stem cells (BM-MSC). Using resuspension buffer (RB) and as low as 1.5 x 10⁵ cells and 1 μg of DNA, we achieved 40% of cells expressing the transgene, with cell recovery and cell viabilities of 85% and 90%, respectively. An increase in DNA amount did not significantly increase the number of transfected cells but clearly reduced cell recovery. A face-centered composite design was used to unveil the conditions giving rise to optimal plasmid delivery efficiencies when using a sucrose based microporation buffer (SBB). The BM-MSC proliferation kinetics were mainly affected by the presence of plasmid and not due to the microporation process itself although no effect was observed on their immunophenotypic characteristics and differentiative potential. Based on the data shown herein microporation demonstrated to be a reliable and efficient method to genetically modify hard-to-transfect cells giving rise to the highest levels of cell survival reported so far along with superior gene delivery efficiencies.

Plasmid DNA Size Does Affect Nonviral Gene Delivery Efficiency in Stem Cells

Genetic modification of stem cells, prior to transplantation, can enhance their survival and can improve their function in cell therapy settings. Mesenchymal stem cells (MSC) are considered one of the most promising tools for cell-based gene therapy, due to their multipotency, ease of isolation, as well as their high ex vivo expansion potential. Neural stem cells (NSC) may also present an ideal route for gene therapy and have been considered for use in cell replacement therapies in various neurodegenerative diseases. Gene therapy-based applications require the transfer of genetic material, either by viral or nonviral gene delivery methods, although the latter has been associated with low efficiencies, especially within hard to transfect cells as stem cells. Herein, we present results on the influence of plasmid size in gene delivery to human MSC and mouse NSC. We used minimized plasmids encoding a fluorescent protein but lacking the antibiotic resistance gene. This work shows that (1) for smaller plasmids the intracellular plasmid copy number can be up to 2.6-fold higher, and (2) the number of cells presenting fluorescence can be twice the number obtained for larger plasmids. Furthermore, by using plasmid constructs containing different polyA signals, we also demonstrated that differences between the plasmids depend largely on the transgene mRNA level. Based on our data we demonstrate that plasmid size severely affects the efficiency of nuclear uptake and we propose that it can also affect the rate of heterochromatin associated gene silencing in stem cells.

Direct head-to-head comparison of cationic liposome-mediated gene delivery to mesenchymal stem/stromal cells of different human sources: a comprehensive study.

Nonviral gene delivery to human mesenchymal stem/stromal cells (MSC) can be considered a very promising strategy to improve their intrinsic features, amplifying the therapeutic potential of these cells for clinical applications. In this work, we performed a comprehensive comparison of liposome-mediated gene transfer efficiencies to MSC derived from different human sources-bone marrow (BM MSC), adipose tissue-derived cells (ASC), and umbilical cord matrix (UCM MSC). The results obtained using a green fluorescent protein (GFP)-encoding plasmid indicated that MSC isolated from BM and UCM are more amenable to genetic modification when compared to ASC as they exhibited superior levels of viable, GFP(+) cells 48 hr post-transfection, 58 ± 7.1% and 54 ± 3.8%, respectively, versus 33 ± 4.7%. For all cell sources, high cell recoveries (≈50%) and viabilities (>85%) were achieved, and the transgene expression was maintained for 10 days. Levels of plasmid DNA uptake, as well as kinetics of transgene expression and cellular division, were also determined. Importantly, modified cells were found to retain their characteristic immunophenotypic profile and multilineage differentiation capacity. By using the lipofection protocol optimized herein, we were able to maximize transfection efficiencies to human MSC (maximum of 74% total GFP(+) cells) and show that lipofection is a promising transfection strategy for MSC genetic modification, especially when a transient expression of a therapeutic gene is required. Importantly, we also clearly demonstrated that intrinsic features of MSC from different sources should be taken into consideration when developing and optimizing strategies for MSC engineering with a therapeutic gene.

Nonviral gene delivery to neural stem cells with minicircles by microporation.

The main purpose of this work was to evaluate the transfection of novel DNA vectors, minicircles (mC), on embryonic stem cell-derived neural stem cells (NSC). We demonstrated that by combining microporation with mC, 75% of NSC expressing a transgene is achieved without compromising cell survival, morphology, and differentiation potential. When comparing mC with their plasmid DNA (pDNA) counterparts, both gave rise to similar transfection levels but cells harboring mC showed 10% higher cell viability, maintaining 90% of survival at least for 10 days. Long-term analysis showed that NSC harbor a higher number of mC copies and consequently exhibit higher transgene expression when compared to their pDNA counterpart. Taken together, our results offer the first insights on the use of mC as a novel and safe strategy to genetically engineer NSC envisaging their use as biopharmaceuticals in clinical settings for the treatment of neurodegenerative or neurological diseases.

Effect of ionic strength and presence of serum on lipoplexes structure monitorized by FRET

BackgroundSerum and high ionic strength solutions constitute important barriers to cationic lipid-mediated intravenous gene transfer. Preparation or incubation of lipoplexes in these media results in alteration of their biophysical properties, generally leading to a decrease in transfection efficiency. Accurate quantification of these changes is of paramount importance for the success of lipoplex-mediated gene transfer in vivo.ResultsIn this work, a novel time-resolved fluorescence resonance energy transfer (FRET) methodology was used to monitor lipoplex structural changes in the presence of phosphate-buffered saline solution (PBS) and fetal bovine serum. 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP)/pDNA lipoplexes, prepared in high and low ionic strength solutions, are compared in terms of complexation efficiency. Lipoplexes prepared in PBS show lower complexation efficiencies when compared to lipoplexes prepared in low ionic strength buffer followed by addition of PBS. Moreover, when serum is added to the referred formulation no significant effect on the complexation efficiency was observed. In physiological saline solutions and serum, a multilamellar arrangement of the lipoplexes is maintained, with reduced spacing distances between the FRET probes, relative to those in low ionic strength medium.ConclusionThe time-resolved FRET methodology described in this work allowed us to monitor stability and characterize quantitatively the structural changes (variations in interchromophore spacing distances and complexation efficiencies) undergone by DOTAP/DNA complexes in high ionic strength solutions and in presence of serum, as well as to determine the minimum amount of potentially cytotoxic cationic lipid necessary for complete coverage of DNA. This constitutes essential information regarding thoughtful design of future in vivo applications.

Effect of cationic liposomes/DNA charge ratio on gene expression and antibody response of a candidate DNA vaccine against Maedi Visna virus.

Maedi Visna virus (MVV) is an ovine lentivirus with high prevalence all over the world. Since conventional vaccines had failed in protecting animals against the infection, the development of a DNA vaccine can be an alternative. The candidate vaccine was constructed by cloning the sequence encoding MVV p25 protein and was tested both in vitro and in vivo experiments associated with cationic liposomes. The lipoplexes (plasmid DNA-liposome complexes) with charge ratios ranging from 0 to 18 were prepared in physiological saline solution and characterized at a physical-chemistry level. Agarose gel electrophoresis was used as a first approach to evaluate qualitatively the amount of unbounded DNA by the liposomes. Dynamic light scattering measurements revealed that under the studied conditions lipoplexes with theoretical charge ratios (+/-) from 3 to 6 are unstable and prone to aggregation displaying sizes higher than 1 microm. At lower and higher charge ratios lipoplex size range from 200 to 500 nm. Using a Foster Resonance Energy Transfer methodology previously reported by us, complexation efficiency of the same complexes was related to in vitro and in vivo results. Higher transfection efficiencies were obtained in vitro with lipoplexes with charge ratio (+/-)=10, where 97% of the DNA were protected by the liposomes. However, the subcutaneous immunization of mice induced higher antibody titers with lipoplexes at charge ratio (+/-)=1, in which only 23% DNA is protected by the liposomes. Moreover, use of cationic liposomes has shown an increased antibody response when compared with a naked DNA immunization.