Vincenzo Taresco

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.

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.

Antimicrobial polymers for anti-biofilm medical devices: state-of-art and perspectives.

The field of antimicrobial polymers has increasingly grown over the past 10 years, and is expected to have a further rapid expansion in the next few years. The application of these polymers to medical devices has been shown to significantly contribute to the reduction of development of biofilm-based related infections. Antimicrobial polymers can be roughly divided in two classes: antimicrobial agent-releasing polymers and biocidal polymers. Many different antimicrobial agent-releasing medical devices have been so far evaluated in clinical trials and are commercially available. Biocidal polymers, which possess intrinsic antimicrobial properties, represent a new generation of antimicrobial polymers and offer promise for enhancing the efficacy of existing antimicrobial agents, increasing antimicrobial durability and reducing the risk of emergence of resistant pathogens. In this chapter, these two classes of antimicrobial polymers are reviewed and discussed especially in terms of anti-biofilm efficacy.

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.

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.

Rapid quantification of low level polymorph content in a solid dose form using transmission Raman spectroscopy

This proof of concept study demonstrates the application of transmission Raman spectroscopy (TRS) to the non-invasive and non-destructive quantification of low levels (0.62-1.32% w/w) of an active pharmaceutical ingredients polymorphic forms in a pharmaceutical formulation. Partial least squares calibration models were validated with independent validation samples resulting in prediction RMSEP values of 0.03-0.05% w/w and a limit of detection of 0.1-0.2% w/w. The study further demonstrates the ability of TRS to quantify all tablet constituents in one single measurement. By analysis of degraded stability samples, sole transformation between polymorphic forms was observed while excipient levels remained constant. Additionally, a beam enhancer device was used to enhance laser coupling to the sample, which allowed comparable prediction performance at 60 times faster rates (0.2s) than in standard mode.

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.

Combinatorial Biomolecular Nanopatterning for High-Throughput Screening of Stem-Cell Behavior.

A novel combinatorial biomolecular nanopatterning method is reported, in which multiple biomolecular ligands can be patterned in multiple nanoscale dimensions on a single surface. The applicability of the combinatorial platform toward cell-biology applications is demonstrated by screening the adhesion behavior of a population of human dental pulp stem cell (hDPSC) on 64 combinations of nanopatterned extracellular matrix (ECM) proteins in parallel.

Amphiphilic block copolymers from a renewable ε-decalactone monomer: prediction and characterization of micellar core effects on drug encapsulation and release

Here we describe a methoxy poly(ethyleneglycol)-b-poly(ε-decalactone) (mPEG-b-PεDL) copolymer and investigate the potential of the copolymer as a vehicle for solubilisation and sustained release of indomethacin (IND). The indomethacin loading and release from mPEG-b-PεDL micelles (amorphous cores) was compared against methoxy poly(ethyleneglycol)-b-poly(ε-caprolactone)(mPEG-b-PCL) micelles (semicrystalline cores). The drug-polymer compatibility was determined through a theoretical approach to predict drug incorporation into hydrated micelles. Polymer micelles were prepared by solvent evaporation and characterised for size, morphology, indomethacin loading and release. All the formulations generated spherical micelles but significantly larger mPEG-b-PεDL micelles were observed compared to mPEG-b-PCL micelles. A higher compatibility of the drug was predicted for PCL cores based on Flory-Huggins interaction parameters (χsp) using the Hansen solubility parameter (HSP) approach, but higher measured drug loadings were found in micelles with PεDL cores compared to PCL cores. This we attribute to the higher amorphous content in the PεDL-rich regions which generated higher micellar core volumes. Drug release studies showed that the semicrystalline PCL core was able to release IND over a longer period (80% drug release in 110 h) compared to PεDL core micelles (80% drug release in 72 h).