Antioxidant Hydroxytyrosol-Based Polyacrylate with Antimicrobial and Antiadhesive Activity Versus Staphylococcus Epidermidis.

The accumulation of reactive oxygen species (ROS) in microbial biofilms has been recently recognized to play a role in promoting antibiotic resistance in biofilm-growing bacteria. ROS are also over-produced when a medical device is implanted and they can promote device susceptibility to infection or aseptic loosening. High levels of ROS seem also to be responsible for the establishment of chronic wounds.In this study, a novel antioxidant polyacrylate was synthesized and investigated in terms of antimicrobial and antibiofilm activity. The polymer possesses in side-chain hydroxytyrosol (HTy), that is a polyphenolic compound extracted from olive oil wastewaters.The obtained 60 nm in size polymer nanoparticles showed good scavenging and antibacterial activity versus a strain of Staphylococcus epidermidis. Microbial adherence assays evidenced that the hydroxytyrosol-containing polymer was able to significantly reduce bacterial adhesion compared to the control. These findings open novel perspective for a successful use of this antioxidant polymer for the prevention or treatment of biofilm-based infections as those related to medical devices or chronic wounds.

Water soluble usnic acid-polyacrylamide complexes with enhanced antimicrobial activity against Staphylococcus epidermidis.

Usnic acid, a potent antimicrobial and anticancer agent, poorly soluble in water, was complexed to novel antimicrobial polyacrylamides by establishment of strong acidic-base interactions. Thermal and spectroscopic analysis evidenced a molecular dispersion of the drug in the polymers and a complete drug/polymer miscibility for all the tested compositions. The polymer/drug complexes promptly dissolved in water and possessed a greater antimicrobial activity against Staphylococcus epidermidis than both the free drug and the polymer alone. The best results were obtained with the complex based on the lowest molecular weight polymer and containing a low drug content. Such a complex showed a larger inhibition zone of bacterial growth and a lower minimum inhibitory concentration (MIC) with respect to usnic acid alone. This improved killing effect is presumably due to the reduced size of the complexes that allows an efficient cellular uptake of the antimicrobial complexes. The killing effect extent seems to be not significantly dependent on usnic acid content in the samples.

Chemoselective C-4 aerobic oxidation of catechin derivatives catalyzed by the Trametes villosa laccase/1-hydroxybenzotriazole system: synthetic and mechanistic aspects.

Catechin derivatives were oxidized in air in the presence of the Trametes villosa laccase/1-hydroxybenzotriazole (HBT) system in buffered water/1,4-dioxane as reaction medium. The oxidation products, flavan-3,4-diols and the corresponding C-4 ketones, are bioactive compounds and useful intermediates for the hemisynthesis of proanthocyanidins, plant polyphenols which provide beneficial health properties for humans. Determinations of oxidation potentials excluded that catechin derivatives could be directly oxidized by laccase Cu(II), while it resulted in the H-abstraction from benzylic positions being promptly promoted by the enzyme in the presence of the mediator HBT, the parent species producing in situ the reactive intermediate benzotriazole-N-oxyl (BTNO) radical. A remarkable and unexpected result for the laccase/HBT oxidative system has been the chemoselective insertion of the oxygen atom into the C-4-H bond of catechin derivatives. Mechanistic aspects of the oxidation reaction have been investigated in detail for the first time in order to corroborate these results. Since the collected experimental findings could not alone provide information useful to clarify the origin of the observed chemoselectivity, these data were expressly supplemented with information derived by suitable molecular modeling investigations. The integrated evaluation of the dissociation energies of the C-H bonds calculated both by semiempirical and DFT methods and the differential activation energies of the process estimated by a molecular modeling approach suggested that the observed selective oxidation at the C-4 carbon has a kinetic origin.

A convenient and safe O-methylation of flavonoids with dimethyl carbonate (DMC).

Dietary flavonoids exhibit beneficial health effects. Several epidemiological studies have focused on their biological activities, including antioxidant, antibacterial, antiviral, anti-inflammatory and cardiovascular properties. More recently, these compounds have shown to be promising cancer chemopreventive agents in cell culture studies. In particular, O-methylated flavonoids exhibited a superior anticancer activity than the corresponding hydroxylated derivatives being more resistant to the hepatic metabolism and showing a higher intestinal absorption. In this communication we describe a convenient and efficient procedure in order to prepare a large panel of mono- and dimethylated flavonoids by using dimethyl carbonate (DMC), an ecofriendly and non toxic chemical, which plays the role of both solvent and reagent. In order to promote the methylation reaction under mild and practical conditions, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) was added in the solution; methylated flavonoids were isolated in high yields and with a high degree of purity. This methylation protocol avoids the use of hazardous and high toxic reagents (diazomethane, dimethyl sulfate, methyl iodide).

Self-Assembly of Catecholic Moiety-Containing Cationic Random Acrylic Copolymers.

Amphiphilic polyelectrolytes (APEs), exhibiting particular self-association properties in aqueous media, can be used in different industrial applications, including drug delivery systems. Their typical core-shell structure (micelle) depends on the balance of interactions between hydrophobic and ionizable monomer units. In this work, the structure of amphiphilic cationic random copolymers, obtained by employing different molar ratios of two acrylic monomers, one bearing in the side chain a tertiary amine (N,N-diethylethylendiamine, DED) and the other one a hydrophobic catecholic group (hydroxytyrosol, HTy), was investigated by atomistic molecular dynamics (MD) simulation, (1)H NMR analysis, dynamic light scattering (DLS), and zeta potential measurements. The structures of p(AcDED-co-AcHTy) copolymers were compared with that of the cationic homopolymer (pAcDED). MD simulation showed a chain folding in water solution of all polymer materials consistent with the degree of hydrophobicity of the chain, that increases with the number of aromatic residues. This phenomenon was induced by the interaction between the charged amine groups with water and by the associated attraction between aromatic rings inside the molecule. In addition, the p(AcDED-co-AcHTy) 70/30 copolymer had a marked tendency to self-assemble as shown by the radial distribution function among catechol carbon atoms. Electrical conductivity measurements evidenced a micellar arragment for all of the synthesized copolymers, and specially for p(AcDED-co-AcHTy) 70/30, a flower micelle structure seem to be more likely. The stacking interactions among catecholic groups present in the side chain of the copolymers reduced the size and charge density specially for the p(AcDED-co-AcHTy) 70/30 copolymer. Finally, the good antimicrobial activity of all copolymers confirmed the right reached amphiphilic balance. Indeed, a considerable reduction of the minimum inhibitory concentration (from 100 µg/mL to 40 µg/mL for pAcDED and p(AcDED-co-AcHTy) 70/30, respectively) was obtained by introducing a hydrophobic group molar fraction of 0.3.

Antimicrobial and antioxidant amphiphilic random copolymers to address medical device-centered infections.

Microbial biofilms are known to support a number of human infections, including those related to medical devices. This work is focused on the development of novel dual-function amphiphilic random copolymers to be employed as coatings for medical devices. Particularly, copolymers were obtained by polymerization of an antimicrobial cationic monomer (bearing tertiary amine) and an antioxidant and antimicrobial hydrophobic monomer (containing hydroxytyrosol, HTy). To obtain copolymers with various amphiphilic balance, different molar ratios of the two monomers were used. (1)H NMR and DSC analyses evidenced that HTy aromatic rings are able to interact with each other leading to a supra-macromolecular re-arrangement and decrease the copolymer size in water. All copolymers showed good antioxidant activity and Fe(2+) chelating ability. Cytotoxicity and hemolytic tests evidenced that the amphiphilic balance, cationic charge density and polymer size in solution are key determinants for polymer biocompatibility. As for the antimicrobial properties, the lowest minimal inhibitory concentration (MIC = 40 µg/mL) against Staphylococcus epidermidis was shown by the water-soluble copolymer having the highest HTy molar content (0.3). This copolymer layered onto catheter surfaces was also able to prevent staphylococcal adhesion. This approach permits not only prevention of biofilm infections but also reduction of the risk of emergence of drug-resistant bacteria. Indeed, the combination of two active compounds in the same polymer can provide a synergistic action against biofilms and suppress reactive species oxygen (ROS), known to promote the occurrence of antibiotic resistance.

Wet adhesion of buckypaper produced from oxidized multiwalled carbon nanotubes on soft animal tissue.

Buckypaper (BP) is the general definition of a macroscopic assembly of entangled carbon nanotubes. In this paper, a new property of a BP film produced from oxidized multiwalled carbon nanotubes was investigated. In particular, BP shows to be able to promptly and strongly adhere to animal internal soft and wet tissues, as evaluated by peeling and shear tests. BP adhesion strength is higher than that recorded for a commercial prosthetic fabric (sealed to the tissue by fibrin glue) and comparable with that of other reported optimized nanopatterned surfaces. In order to give an interpretation of the observed behavior, the BP composition, morphology, porosity, water wettability, and mechanical properties were analyzed by AFM, X-ray photoelectron spectroscopy, wicking tests, contact angle, and stress-strain measurements. Although further investigations are needed to assess the biocompatibility and safety of the BP film used in this work, the obtained results pave the way for a possible future use of buckypaper as adhesive tape in abdominal prosthetic surgery. This would allow the substitution of conventional sealants or the reduction in the use of perforating fixation.

Synthesis and structure/antioxidant activity relationship of novel catecholic antioxidant structural analogues to hydroxytyrosol and its lipophilic esters.

A large panel of novel catecholic antioxidants and their fatty acid or methyl carbonate esters has been synthesized in satisfactory to good yields through a 2-iodoxybenzoic acid (IBX)-mediated aromatic hydroxylation as the key step. The new catechols are structural analogues of naturally occurring hydroxytyrosol (3,4-DHE). To evaluate structure/activity relationships, the antioxidant properties of all catecholic compounds were evaluated in vitro by ABTS assay and on whole cells by DCF fluorometric assay and compared with that of the corresponding already known hydroxytyrosyl derivatives. Results outline that all of the new catechols show antioxidant capacity in vitro higher than that of the corresponding hydroxytyrosyl derivatives. Less evident positive effects have been detected in whole cells experiments. Cytotoxicity experiments, using MTT assay, on a representative set of compounds evidenced no influence in cell survival.

Efficient synthesis of scutellarein.

Scutellarein is a component of Scutellaria, recently known as a potent cytotoxic agent on human leukaemia cells. The aim of this study was the synthesis of scutellarein and its methylated derivative. The new features are the innovating method to afford flavones from flavanones and the A-ring regioselective bromination step that lead to the target molecule by a facile and high-yielding pathway.

Synthesis of a novel ester of hydroxytyrosol and α-lipoic acid exhibiting an antiproliferative effect on human colon cancer HT-29 cells.

A novel ester of hydroxytyrosol and α-lipoic acid was synthesized in satisfactory yield by original and simple procedures and evaluated about its antiproliferative activity on the human colorectal adenocarcinoma HT-29 cell line. The compound exhibited a cell growth inhibitory activity significantly more potent than the corresponding parent natural compounds, very likely due to the induction of cell cycle arrest in the G2/M phase. These data suggest that the novel ester might exert a more effective antitumour activity than hydroxytyrosol and α-lipoic acid.

Antibiotic delivery polyurethanes containing albumin and polyallylamine nanoparticles.

Nano-structured polymers delivering an antibiotic for the prevention of medical device-related infections were developed. Systems consisted of bovine serum albumin or polyallylamine nanoparticles alone or entrapped in a polyurethane and then loaded with cefamandole nafate, chosen as a drug model. Results showed that nanoparticles alone were able to adsorb high antibiotic amounts due to their high surface/volume ratio. However, they released cefamandole in an uncontrolled fashion, leading to a rapid loss of antibacterial activity. Improvements in the release control were obtained when CEF loaded and non-loaded nanoparticles were entrapped in a carboxylated polyurethane. For these systems the drug delivery was at least of 50% with respect to nanoparticles alone with a prolonged antimicrobial activity up to 9 days.

Convenient synthesis of hydroxytyrosol and its lipophilic derivatives from tyrosol or homovanillyl alcohol.

Hydroxytyrosol, a naturally occurred o-phenolic compound exhibiting antioxidant properties, was synthesized by a three-step high-yielding procedure from natural and low-cost compounds such as tyrosol or homovanillyl alcohol. First, the efficient chemoselective protection of the alcoholic group of these compounds was performed by using dimethyl carbonate (DMC) as reagent/solvent; second, the oxidation with 2-iodoxybenzoic acid (IBX) or Dess-Martin periodinane reagent (DMP) and in situ reduction with sodium dithionite (Na2S2O4) allowed the preparation of carboxymethylated hydroxytyrosol; finally, by a mild hydrolytic step, hydroxytyrosol was obtained in high yield and purity, as confirmed by NMR spectra and HPLC profile. By using a similar methodology, lipophilic hydroxytyrosol derivatives, utilized as additives in pharmaceutical, food, and cosmetic preparations, were prepared. In fact, at first the chemoselective protection of the alcoholic group of tyrosol and homovanillyl alcohol was performed by using acyl chlorides without any catalyst to obtain the corresponding lipophilic derivatives, and then these compounds were converted in good yield and high purity into the hydroxytyrosol derivatives by oxidative/reductive pathway with IBX or DMP and Na2S2O4.