Characterization of Novel Synthetic Polyphenols: Validation of Antioxidant and Vasculoprotective Activities.

Antioxidant compounds, including polyphenols, have therapeutic effects because of their anti-inflammatory, antihypertensive, antithrombotic and antiproliferative properties. They play important roles in protecting the cardiovascular and neurological systems, by having preventive or protective effects against free radicals produced by either normal or pathological metabolism in such systems. For instance, resveratrol, a well-known potent antioxidant, has a counteracting effect on the excess of reactive oxygen species (ROS) and has a number of therapeutic benefits, like anti-inflammatory, anti-cancer and cardioprotective activities. Based on previous work from our group, and on the most frequent OH substitutions of natural polyphenols, we designed two series of synthetically accessible bis-polyhydroxyphenyl derivatives, separated by amide or urea linkers. These compounds exhibit high antioxidant ability (oxygen radical absorbance capacity (ORAC) assay) and interesting radical scavenging activity (RSA) values (2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and α,α-diphenyl-ß-picrylhydrazyl (DPPH) tests). Some of the best polyphenols were evaluated in two biological systems, endothelial cells (in vitro) and whole aorta (ex vivo), highly susceptible for the deleterious effects of prooxidants under different inflammatory conditions, showing protection against oxidative stress induced by inflammatory stimuli relevant in cardiovascular diseases, i.e., Angiotensin II and IL-1ß. Selected compounds also showed strong in vivo antioxidant properties when evaluated in the model organism Saccharomyces cerevisiae.

Thermosensitive macroporous cryogels functionalized with bioactive chitosan/bemiparin nanoparticles.

Thermosensitive macroporous scaffolds of poly(N-isopropylacrylamide) (polyNIPA) loaded with chitosan/bemiparin nanoparticles are prepared by the free radical polymerization in cryogenic conditions. Chitosan/bemiparin nanoparticles of 102 ± 6.5 nm diameter are prepared by complex coacervation and loaded into polyNIPA cryogels. SEM image reveal the highly porous structure of cryogels and the integration of nanoparticles into the macroporous system. Volume phase transition temperature (VPT) and total freezing water content of cryogels are established by differential scanning calorimetry, and their porosity is determined by image-NMR. Swelling of cryogels (above and below the VPT) is highly dependent on nanoparticles concentration. In vitro release profile of bemiparin from cryogel is highly modulated by the presence of chitosan. Bemiparin released from nanoparticles preserves its biological activity, as shown by the BaF32 cell proliferation assay. Cryogels are not cytotoxic for the human fibroblast cells and present excellent properties for application on tissue engineering and controlled release of heparin.

Anti-angiogenic activity of heparin-like polysulfonated polymeric drugs in 3D human cell culture.

The activity of new anti-angiogenic polymeric drugs was tested in a 3D endothelial cell culture system applied as a model of angiogenesis. The assay was performed in a highly reproducible fibrin matrix that supported endothelial cell attachment, proliferation, migration, and formation of capillary-like structures. Active growth factors (FGF and/or VEGF) were added to the medium to induce the formation of blood vessel-like structures, and the effect of the active polymers was then tested by a semi-quantitative immunostaining protocol and visualized by laser-scanning confocal microscopy. The synthetic heparin-like macromolecules that were tested for their anti-angiogenic efficacy were previously characterized in terms of their anti-proliferative activity in 2D tissue culture. Two different anti-angiogenic monomers, a methacrylic derivative of 5-amino-2-naphthalenesulfonic acid (MANSA) and 2-acrylamido-2-methylpropane sulfonic acid (AMPS), were copolymerized with a hydrophilic monomer (vinyl pyrrolidone, VP) or a hydrophobic monomer (butyl acrylate, BA), giving rise to different copolymeric systems with controlled microstructure and supramolecular organization. Both copolymeric systems have demonstrated a composition-dependent anti-mitogenic effect in "2D in vitro" cell culture experiments using aFGF as pro-angiogenic growth factor and BALB/c 3T3 fibroblast as cell model, as was shown in a previous publication. These 3D experiments provide evidence for the strong and specific modulation of angiogenesis by these systems. The 3D experiments constitute an improvement over 2D in vitro experiments and in vivo experiments with angiogenic drugs and may help to reduce the number of animal experiments.

Antimitogenic polymer drugs based on AMPS: monomer distribution-bioactivity relationship of water-soluble macromolecules.

A number of polysulfonated molecules have demonstrated their interaction with fibroblast growth factor (FGF), hampering their binding to its receptors (low affinity heparan sulfate proteoglycans (HSPG) and high affinity tyrosine kinase FGF receptors) and inhibiting the intracellular signaling and mitogenic response in cultured endothelial cells. The aim of this work was the synthesis and characterization of new copolymers based on 2-acrylamido-2-methylpropane sulfonic acid (AMPS) with antiproliferative activity for antitumoral applications. N-Vinylpyrrolidone (VP) or butyl acrylate (BA) was copolymerized with the sulfonated monomer to obtain macromolecules with different hydrophilic/hydrophobic balance and distribution of the sulfonated groups within the macromolecules. In vitro cell culture proliferative assays showed that monomer distribution affected the inhibition of the proliferative action of FGF. Reactivity ratios of the systems were determined following the free radical copolymerization by in situ (1)H NMR, and the correlation of the monomer sequence distribution with the bioactivity is discussed.

Modulation of proteins adsorption onto the surface of chitosan complexed with anionic copolymers. Real time analysis by surface plasmon resonance.

The interpolyelectrolyte complex formation between chitosan and anionic polyacrylic derivatives, bearing sulfonic moieties, as well as the protein adsorption onto the chitosan/polyacrylic complexes were studied by surface plasmon resonance (SPR) optical biosensor. This unique technique allows a real time monitoring of different surface molecular interactions with very high sensitivity. The acrylic macromolecules are two families of copolymers of 2-acrylamido-2-methylpropane sulfonic acid (AMPS) and, respectively, 2-hydroxyethylmethacrylate (HEMA) and N,N'-dimethylacrylamide (DMAA). The complexation process was evaluated through the SPR measurements resulting from the flowing of polyacrylic aqueous solution over the sensor previously coated with chitosan. The SPR was able to differentiate strong ionic bonds from other weak and reversible interactions. By means of the coated sensors (uncomplexed and the whole series of complexed chitosan), SPR cold be used for a simple "in vitro" protein adsorption analysis, by flowing aqueous solutions of albumin and fibrinogen. While both proteins were adsorbed on the uncomplexed chitosan, the complexed coatings exhibited different and very promising behaviors. In particular, they showed no adsorption or only selective adsorption of albumin.