Diana Paiva

The effect of a fluorinated cholesterol derivative on the stability and physical properties of cationic DNA vectors

Liposomes of the cationic lipid DOTAP (1,2-dioleoyl-3-trimethylammonium-propane) and the fluorinated cholesterol derivative heptafluorocholesterol (F7-CHOL) were tested at the molar ratio of 1:1 for DNA compaction and transfection. Their properties were correlated with the characteristics of the well-known system, DOTAP and cholesterol (CHOL). The mass lipid–DNA (L/D) ratios at the isoelectric point were within the ranges 3–4 for DOTAP:CHOL and 4–5 for DOTAP:F7-CHOL, as determined by electrophoretic mobility measurements. These results and the ethidium bromide fluorescence intercalation assays confirmed that more DOTAP:F7-CHOL liposomes are needed to compact the same amount of DNA as DOTAP:CHOL. The phase diagrams of aggregation and re-entrant condensation phenomena obtained by phenomenological theory support this conclusion and also establish that the liposome–DNA binding is stronger in the case of the DOTAP:F7-CHOL–DNA system. The stability rates of both liposomes in the presence of DNA and their transfection efficiencies were similar, which support the application of heptafluorocholesterol as a helper lipid in cationic liposomes for DNA delivery.

Langmuir Monolayers of Monocationic Lipid Mixed with Cholesterol or Fluorocholesterol: DNA Adsorption Studies

Monolayers of the cationic lipid DOTAP (1,2-dioleoyl-3-trimethylammonium-propane) and cholesterol or heptafluorocholesterol were prepared, and their interaction with DNA was characterized. The mixture of DOTAP with each of the sterols at 1:1 molar ratios leads to monolayers in a liquid expanded state, similarly to that of DOTAP alone. The area per molecule of the mixtures was smaller than that expected according to the additivity rule applicable if the two components are either completely miscible or immiscible within the monolayer. The observed negative deviation from the additivity indicates the existence of additional attractive interactions between the components. The surface potential of DOTAP monolayer is positive (+560 mV). It decreases only slightly after the addition of cholesterol (+540 mV) but drastically after the addition of heptafluorocholesterol (+20 mV) in the 1:1 mixtures at a surface pressure of 35 mN/m. This difference is attributed to the negative dipole moment of the fluorinated component. The adsorption of DNA is similar for both systems, which supports the possibility of using fluorinated cholesterol as helper lipid in DNA transfection vectors.

Chitosan conjugates for DNA delivery

Graft copolymers of chitosan and maltodextrin were synthesized by reductive amination of a low molecular weight chitosan. The degree of substitution is 70% or above, as quantified by nuclear magnetic resonance spectroscopy, at molar ratios of chitosan glucosamine units and maltodextrin of 1 : 1 and 1 : 5. The high substitution degree generates a water-soluble compound at pH 7.4 by reducing the amino groups of chitosan. In addition, the copolymer self-assembles into spherical nanoparticles with diameters of about 300 nm. The mass polymer/DNA ratios at the isoelectric point are within the range of 3-3.5 for chitosan-maltodextrin nanoparticles as determined by electrophoretic mobility measurements. These results confirm that DNA interacts efficiently with the copolymer nanoparticles indicating a potential application of the system for DNA delivery.

Oxidized Xanthan Gum and Chitosan as Natural Adhesives for Cork

Natural cork stopper manufacturing produces a significant amount of cork waste, which is granulated and combined with synthetic glues for use in a wide range of applications. There is a high demand for using biosourced polymers in these composite materials. In this study, xanthan gum (XG) and chitosan (CS) were investigated as possible natural binders for cork. Xanthan gum was oxidized at two different aldehyde contents as a strategy to improve its water resistance. This modification was studied in detail by 1H and 13C nuclear magnetic resonance (NMR), and the degree of oxidation was determined by the hydroxylamine hydrochloride titration method. The performance of the adhesives was studied by tensile tests and total soluble matter (TSM) determinations. Xanthan gum showed no water resistance, contrary to oxidized xanthan gum and chitosan. It is hypothesized that the good performance of oxidized xanthan gum is due to the reaction of aldehyde groups—formed in the oxidation process—with hydroxyl groups on the cork surface during the high temperature drying. Combining oxidized xanthan gum with chitosan did not yield significant improvements.

Synthesis and study of the complex formation of a cationic alkyl-chain bola amino alcohol with DNA: in vitro transfection efficiency

Recent studies point out that bolaamphiphiles can be used in nonviral gene therapy. Due to their bipolar character, they may span a membrane and thus stabilize or destabilize it which could be relevant for DNA transfer across a biological membrane. Since there are only very few studies on bolaamphiphile application in DNA transfection, it is difficult to assess whether they will bring additional advantages to the class of nonviral vectors. A bolaamphiphile with a hydrophobic chain of 22 carbon atoms with trimethylammonium and hydroxyl groups at each end was synthesized (22-hydroxydocos-1-yl-N,N,N-trimethylammonium bromide). The bolaamphiphile conferred stability and lowered the zeta potential of vesicles of a cationic lipid. Confocal laser scanning microscopy and flow cytometry experiments have shown that this bolaamphiphile can transfect the green fluorescent protein plasmid in cells if mixed with a cationic lipid. It can compete with standard cationic lipids in terms of transfection but is at the same time less toxic. The potential of this class of molecules in gene delivery results from the fact that they will confer high stability to the DNA vector.

Reinforcement of Thermoplastic Corn Starch with Crosslinked Starch/Chitosan Microparticles

Microparticles of corn starch and chitosan crosslinked with glutaraldehyde, produced by the solvent exchange technique, are studied as reinforcement fillers for thermoplastic corn starch plasticized with glycerol. The presence of 10% w/w chitosan in the microparticles is shown to be essential to guaranteeing effective crosslinking, as demonstrated by water solubility assays. Crosslinked chitosan forms an interpenetrating polymer network with starch chains, producing microparticles with a very low solubility. The thermal stability of the microparticles is in agreement with their polysaccharide composition. An XRD analysis showed that they have crystalline fraction of 32% with Va-type structure, and have no tendency to undergo retrogradation. The tensile strength, Young’s modulus, and toughness of thermoplastic starch increased by the incorporation of the crosslinked starch/chitosan microparticles by melt-mixing. Toughness increased 360% in relation to unfilled thermoplastic starch.

Dynamic mechanical analysis and creep-recovery behavior of agglomerated cork

The mechanical behavior of agglomerated cork, made of cork granules bound with polyurethane moisture-cured adhesive is investigated and compared to natural cork in the small strain regime (strain <5%). Dynamic mechanical analysis (DMA) of the agglomerated material revealed two distinct thermal transitions, one at −45 °C, related to the glass transition of polyurethane, and one at 3 °C, associated with melting of suberin, a natural polyester that is the main component of cork’s cell walls. Natural cork showed the latter transition to occur at a higher temperature range, between 10 and 25 °C, probably due to a different crystalline arrangement being formed upon cooling the cork granules under pressure in the mold. The storage modulus of agglomerated cork was found to be similar to that of natural cork. Creep and recovery experiments were well described by Burgers model and Weibull distribution function, respectively. Agglomerated cork showed higher instantaneous creep strain and viscous flow than natural cork, probably due to relative displacement and slippage of the granules being allowed by the binder. In all cork materials, not all the instantaneous creep strain was instantaneously recovered. A fraction underwent delayed recovery and another turned into permanent strain. This behavior was associated with the deformation of corrugations in the cork cell walls. Cyclic creep-recovery tests showed for all cork materials recoveries above 90%, except for the first cycle.