Antonella Piozzi

Usnic Acid, a Natural Antimicrobial Agent Able To Inhibit Bacterial Biofilm Formation on Polymer Surfaces

ABSTRACT In modern medicine, artificial devices are used for repair or replacement of damaged parts of the body, delivery of drugs, and monitoring the status of critically ill patients. However, artificial surfaces are often susceptible to colonization by bacteria and fungi. Once microorganisms have adhered to the surface, they can form biofilms, resulting in highly resistant local or systemic infections. At this time, the evidence suggests that (+)-usnic acid, a secondary lichen metabolite, possesses antimicrobial activity against a number of planktonic gram-positive bacteria, including Staphylococcus aureus, Enterococcus faecalis, and Enterococcus faecium. Since lichens are surface-attached communities that produce antibiotics, including usnic acid, to protect themselves from colonization by other bacteria, we hypothesized that the mode of action of usnic acid may be utilized in the control of medical biofilms. We loaded (+)-usnic acid into modified polyurethane and quantitatively assessed the capacity of (+)-usnic acid to control biofilm formation by either S. aureus or Pseudomonas aeruginosa under laminar flow conditions by using image analysis. (+)-Usnic acid-loaded polymers did not inhibit the initial attachment of S. aureus cells, but killing the attached cells resulted in the inhibition of biofilm. Interestingly, although P. aeruginosa biofilms did form on the surface of (+)-usnic acid-loaded polymer, the morphology of the biofilm was altered, possibly indicating that (+)-usnic acid interfered with signaling pathways.

Synergistic Activity of Dispersin B and Cefamandole Nafate in Inhibition of Staphylococcal Biofilm Growth on Polyurethanes

ABSTRACT Antibiotic therapies to eradicate medical device-associated infections often fail because of the ability of sessile bacteria, encased in their exopolysaccharide matrix, to be more drug resistant than planktonic organisms. In the last two decades, several strategies to prevent microbial adhesion and biofilm formation on the surfaces of medical devices, based mainly on the use of antiadhesive, antiseptic, and antibiotic coatings on polymer surfaces, have been developed. More recent alternative approaches are based on molecules able to interfere with quorum-sensing phenomena or to dissolve biofilms. Interestingly, a newly purified β-N-acetylglucosaminidase, dispersin B, produced by the gram-negative periodontal pathogen Actinobacillus actinomycetemcomitans, is able to dissolve mature biofilms produced by Staphylococcus epidermidis as well as some other bacterial species. Therefore, in this study, we developed new polymeric matrices able to bind dispersin B either alone or in combination with an antibiotic molecule, cefamandole nafate (CEF). We showed that our functionalized polyurethanes could adsorb a significant amount of dispersin B, which was able to exert its hydrolytic activity against the exopolysaccharide matrix produced by staphylococcal strains. When microbial biofilms were exposed to both dispersin B and CEF, a synergistic action became evident, thus characterizing these polymer-dispersin B-antibiotic systems as promising, highly effective tools for preventing bacterial colonization of medical devices.

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.

Polyurethane anionomers containing metal ions with antimicrobial properties: Thermal, mechanical and biological characterization

In recent years the employment of implantable medical devices has increased remarkably, notwithstanding that microbial infections are a frequent complication associated with their use. Different strategies have been attempted to overcome this problem, including the incorporation of antimicrobial agents into the device itself. In this study a new approach to obtain intrinsically antimicrobial materials was developed. Polymer anionomers containing Ag(I), Cu(II), Zn(II), Al(III) and Fe(III) were prepared by neutralization of a carboxylated polyurethane. In the case of the PEUA-Ag, PEUA-Fe and PEUA-Cu ionomers the ion aggregates behaved as reinforcing filler particles, increasing the mechanical properties of the systems in terms of hardness and strength at break over the pristine carboxylated polymer. With the exception of the Al-containing polymer, all the other experimented ionomers showed satisfactory antimicrobial properties. The best antibacterial effect was obtained with the silver ion-containing polymer, which inhibited Staphylococcus epidermidis growth for up to 16days. Ciprofloxacin was also adsorbed onto the above mentioned ionomers. A synergistic effect of the antibiotic and silver ions on bacterial growth inhibition was observed for at least 25days.

New polyurethane compositions able to bond high amounts of both albumin and heparin: Part I

In order to prepare polymers provided with better haemocompatibility with respect both to the coagulative cascade and to platelet aggregation and activation, we have synthesized new polyurethanes containing in the chain-extender [di(2-hydroxyethyl)hexadecylamine] both a long chain alkyl group (able to bond albumin) and a tertiary ammonium group able, after suitable quaternization reaction, to bind ionically significant amounts of heparin. The amounts of heparin and albumin bonded to the polymer films were determined spectrophotometrically. A biological in vitro evaluation of the heparinized and albuminized films was also carried out with respect to blood coagulation factors (by activated partial thromboplastin time measurements) and to platelet adhesion and activation (by platelet count and scanning electron microscopy examination). It was seen that the type of adsorption sequence for albumin and heparin, respectively, onto the various homo- and copolymer films, plays an important role on their biological properties; the possible mechanisms involved are also discussed on the basis of X-ray photoelectron spectroscopy and attenuated transmission reflectance evaluation of the polymer surfaces.

Polyurethanes Loaded with Antibiotics: Influence of Polymer-Antibiotic Interactions on In Vitro Activity Against Staphylococcus epidermidis

Abstract Acidic or basic polyurethanes were loaded with antibiotics to develop materials to prevent medical device-related infections. A correlation between polymer-antibiotic interactions and amount of drug absorbed by polymers and released over time was found. Since the employed antibiotics, i.e. amoxicillin, cefamandole nafate, rifampin and vancomycin, possessed at least an acidic group in their structural formula, the introduction of basic tertiary amines in the polyurethane side-chain resulted in an increased polymer ability to adsorb antibiotics. However, a stronger ionic interaction between this polymer and the antibiotics caused a release of lower amount of drug over time. Antibiotics released from polymers inhibited Staphylococcus epidermidis growth on agar. Antibiotic-loaded polyurethanes kept in water for increasing times were still able to show inhibition zones of bacterial growth. The antibacterial activity lasted up to 3 hours for amoxicillin, 24 hours for vancomycin, 8 days for cefamandole nafate and 8 months for rifampin.

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.

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.

Pore formers promoted release of an antifungal drug from functionalized polyurethanes to inhibit Candida colonization.

Aims:  As a preventive strategy to inhibit fungal biofilm formation on medical devices, we planned experiments based on polyurethane loading with fluconazole plus pore‐former agents in order to obtain a promoted release of the antifungal drug.

Synthesis of biomimetic segmented polyurethanes as antifouling biomaterials

Controlling the non-specific adsorption of proteins, cells and bacteria onto biomaterial surfaces is of crucial importance for the development of medical devices with specific levels of performance. Among the strategies pursued to control the interactions between material surfaces and biological tissues, the immobilization of non-fouling polymers on biomaterial surfaces as well as the synthesis of the so-called biomimetic polymers are considered promising approaches to elicit specific cellular responses. In this study, in order to obtain materials able to prevent infectious and thrombotic complications related to the use of blood-contacting medical devices, heparin-mimetic segmented polyurethanes were synthesized and fully characterized. Specifically, sulfate or sulfamate groups, known to be responsible for the biological activity of heparin, were introduced into the side chain of a carboxylated polyurethane. Due to the introduction of these groups, the obtained polymers possessed a higher hard/soft phase segregation (lower glass transition temperatures) and a greater hydrophilicity than the pristine polymer. In addition, the synthesized polymers were able to significantly delay the activated partial thromboplastin time, this increased hemocompatibility being related both to polymer hydrophilicity and to the presence of the -SO3H groups. This last feature was also responsible for the ability of these biomimetic polymers to prevent the adhesion of a strain of Staphylococcus epidermidis.