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.

Lipase immobilization on differently functionalized vinyl-based amphiphilic polymers: influence of phase segregation on the enzyme hydrolytic activity.

Microbial lipase from Candida rugosa was immobilized by physical adsorption onto an ethylene-vinyl alcohol polymer (EVAL) functionalized with acyl chlorides. To evaluate the influence of the reagent chain-length on the amount and activity of immobilized lipase, three differently long aliphatic fatty acids were employed (C8, C12, C18), obtaining EVAL functionalization degrees ranging from 5% to 65%. The enzyme-polymer affinity increased with both the length of the alkyl chain and the matrix hydrophobicity. In particular, the esterified polymers showed a tendency to give segregated hydrophilic and hydrophobic domains. It was observed the formation of an enzyme multilayer at both low and high protein concentrations. Desorption experiments showed that Candida rugosa lipase may be adsorbed in a closed form on the polymer hydrophilic domains and in an open, active structure on the hydrophobic ones. The best results were found for the EVAL-C18 13% matrix that showed hyperactivation with both the soluble and unsoluble substrate after enzyme desorption. In addition, this supported biocatalyst retained its activity for repetitive cycles.

Supramolecular organization of toluidine blue dye in solid amorphous phases.

Toluidine blue (TB) dye molecules are intensively utilized for large-area photophysics applications such as carcinoma detection, photoinactivation of bacteria, biosensors, and photovoltaic cells. Understanding the nature of the TB aggregation state becomes an essential point of the research process in order to know the structure-function relationship and to foresee technological applications of this class of metachromatic-dye molecules. However, no structural information on toluidine blue is available in the literature, maybe because of the poor crystalline character of the aggregate. Here, we present the first structural determination of TB organic molecules using the energy dispersive X-ray diffraction technique. The investigation highlights dimeric arrangements of stacked molecules in antiparallel fashion, forming a superstructure of two dimers in a transverse arrangement. The behavior of the TB higher aggregates indicates that these dye molecules, in spite of repulsion due to similar charge (cationic dyes), undergo self-aggregation to form helical conformations.

Design and characterization of antimicrobial usnic acid loaded-core/shell magnetic nanoparticles.

The application of magnetic nanoparticles (MNPs) in medicine is considered much promising especially because they can be handled and directed to specific body sites by external magnetic fields. MNPs have been investigated in magnetic resonance imaging, hyperthermia and drug targeting. In this study, properly functionalized core/shell MNPs with antimicrobial properties were developed to be used for the prevention and treatment of medical device-related infections. Particularly, surface-engineered manganese iron oxide MNPs, produced by a micro-emulsion method, were coated with two different polymers and loaded with usnic acid (UA), a dibenzofuran natural extract possessing antimicrobial activity. Between the two polymer coatings, the one based on an intrinsically antimicrobial cationic polyacrylamide (pAcDED) resulted to be able to provide MNPs with proper magnetic properties and basic groups for UA loading. Thanks to the establishment of acid-base interactions, pAcDED-coated MNPs were able to load and release significant drug amounts resulting in good antimicrobial properties versus Staphylococcus epidermidis (MIC = 0.1 mg/mL). The use of pAcDED having intrinsic antimicrobial activity as MNP coating in combination with UA likely contributed to obtain an enhanced antimicrobial effect. The developed drug-loaded MNPs could be injected in the patient soon after device implantation to prevent biofilm formation, or, later, in presence of signs of infection to treat the biofilm grown on the device surfaces.

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.

Amino-functionalized poly(L-lactide) lamellar single crystals as a valuable substrate for delivery of HPV16-E7 tumor antigen in vaccine development.

BACKGROUND: Poly(L-lactide) (PLLA) is a biodegradable polymer currently used in many biomedical applications, including the production of resorbable surgical devices, porous scaffolds for tissue engineering, nanoparticles and microparticles for the controlled release of drugs or antigens. The surfaces of lamellar PLLA single crystals (PLLAsc) were provided with amino groups by reaction with a multifunctional amine and used to adsorb an Escherichia coli-produced human papillomavirus (HPV)16-E7 protein to evaluate its possible use in antigen delivery for vaccine development. METHODS: PLLA single crystals were made to react with tetraethylenepentamine to obtain amino-functionalized PLLA single crystals (APLLAsc). Pristine and amino-functionalized PLLAsc showed a two-dimensional microsized and one-dimensional nanosized lamellar morphology, with a lateral dimension of about 15-20 µm, a thickness of about 12 nm, and a surface specific area of about 130 m(2)/g. Both particles were characterized and loaded with HPV16-E7 before being administered to C57BL/6 mice for immunogenicity studies. The E7-specific humoral-mediated and cell-mediated immune response as well as tumor protective immunity were analyzed in mice challenged with TC-1 cancer cells. RESULTS: Pristine and amino-functionalized PLLAsc adsorbed similar amounts of E7 protein, but in protein-release experiments E7-PLLAsc released a higher amount of protein than E7-APLLAsc. When the complexes were dried for observation by scanning electron microscopy, both samples showed a compact layer, but E7-APLLAsc showed greater roughness than E7-PLLAsc. Immunization experiments in mice showed that E7-APLLAsc induced a stronger E7-specific immune response when compared with E7-PLLAsc. Immunoglobulin G isotyping and interferon gamma analysis suggested a mixed Th1/Th2 immune response in both E7-PLLAsc-immunized and E7-APLLAsc-immunized mice. However, only the mice receiving E7-APLLAsc were fully protected from TC-1 tumor growth after three doses of vaccine. CONCLUSION: Our results show that APLLA single crystals improve the immunogenicity of HPV16-E7 and indicate that E7-APLLAsc could be used for development of an HPV16 therapeutic vaccine against HPV16-related tumors.

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.

Release behavior and antibiofilm activity of usnic acid-loaded carboxylated poly(L-lactide) microparticles.

The use of controlled drug delivery systems could give a significant contribution to the improvement of therapies against biofilm-based infections. The aim of this study was to develop polymer microparticles, based on carboxylated poly(L-lactide)s, to be employed as carriers for usnic acid (UA), a poorly soluble drug possessing antiviral, antiproliferative and wide spectrum antimicrobial activity. Thanks to polymer surfactant-like structure, 2.4 µm-in-size microparticles were obtained by a surfactant-free oil-in-water emulsion/evaporation method. UA was encapsulated into these microparticles with a high loading efficiency (80%). The drug release kinetics was found to be temperature dependent (the released dose increasing with temperature) and showed bimodal release behavior. By polarized optical microscopy observations and the application of kinetics models, the initial burst effect was attributed to the delivery of the drug amorphous fraction while the slower release occurring for longer times to the crystalline one, both entrapped in the polymer amorphous phase. UA-loaded microparticles were able to promote the killing of a 24h-old Staphylococcus epidermidis biofilm more efficaciously than free UA.

Antifouling polyurethanes to fight device-related staphylococcal infections: synthesis, characterization, and antibiofilm efficacy.

In hospital settings, biofilm-based medical device-related infections are considered a threat to patients, the sessile growing bacteria playing a key role in the spreading of healthcare-associated infections. In recent decades, the design of antifouling coatings for medical devices able to prevent microbial adhesiveness has emerged as one of the most promising strategies to face this important issue. In order to obtain suitable antifouling materials, segmented polyurethanes characterized by a hard/soft domain structure, having the same hard domain but a variable soft domain, have been synthesized. The soft domain was constituted by one of the following macrodiols: polypropylenoxide (PPO), polycaprolactide (PCL), and poly-l-lactide (PLA). The effects of the polymer hydrophilicity and the degree of hard/soft domain separation on antifouling properties of the synthesized polyurethanes were investigated. Microbial adherence assays evidenced as the polymers containing PCL or PLA were able to significantly reduce the adhesion of Staphylococcus epidermidis with respect to the PPO-containing polymer.

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.

Water state effect on drug release from an antibiotic loaded polyurethane matrix containing albumin nanoparticles.

Water mobility plays a crucial role in determining transport properties of small molecules in polymer matrices. In particular, in drug delivery systems, water state affects the pharmacokinetics, especially drug absorption, diffusion and release. In the present study, the state of water in an antibiotic-loaded composite consisting of albumin nanoparticles (BSA(np)) dispersed into a carboxylated polyurethane (PEUA) has been investigated and compared with that of the single drug-loaded components. The antibiotic cefamandole nafate was used as a model drug. DSC analysis, used to evaluate the freezing and non-freezing water fractions in the hydrated samples, showed that in BSA(np) water can adsorb both in the inter-particles regions and inside the particles. With increasing of total adsorbed water amount, the contribution of the freezing water fraction was higher than the non-freezing one. As for PEUA, the majority of water molecules absorbed is in a mobile freezing state (about 60% of the W(tot)). As for the PEUA/BSA(np) composite, the higher polyurethane phase segregation induced by the nanoparticles as well as the higher non-freezing water fraction significantly enhanced drug uptake with respect to PEUA. Moreover, the greater non-freezing water fraction allowed the drug to penetrate within BSA nanoparticles and to give rise then to a controlled drug release. Indeed, the diffusion barrier exerted by nanoparticles and the matrix prolonged the antimicrobial activity from 4 to 9 days. Finally, the higher polyurethane phase segregation also improved composite mechanical properties, as evidenced in stress-strain experiments and dynamic mechanical analysis.

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.