Enhanced Antibiotic Tolerance of an In Vitro Multispecies Uropathogen Biofilm Model, Useful for Studies of Catheter-Associated Urinary Tract Infections.

Catheter-associated urinary tract infections (CAUTI) are a common clinical concern as they can lead to severe, persistent infections or bacteremia in long-term catheterized patients. This type of CAUTI is difficult to eradicate, as they are caused by multispecies biofilms that may have reduced susceptibility to antibiotics. Many new strategies to tackle CAUTI have been proposed in the past decade, including antibiotic combination treatments, surface modification and probiotic usage. However, those strategies were mainly assessed on mono- or dual-species biofilms that hardly represent the long-term CAUTI cases where, normally, 2-4 or even more species can be involved. We developed a four-species in vitro biofilm model on catheters involving clinical strains of Escherichia coli, Pseudomonas aeruginosa, Klebsiella oxytoca and Proteus mirabilis isolated from indwelling catheters. Interspecies interactions and responses to antibiotics were quantitatively assessed. Collaborative as well as competitive interactions were found among members in our model biofilm and those interactions affected the individual species' abundances upon exposure to antibiotics as mono-, dual- or multispecies biofilms. Our study shows complex interactions between species during the assessment of CAUTI control strategies for biofilms and highlights the necessity of evaluating treatment and control regimes in a multispecies setting.

A new catheter-integrated drug-delivery system for controlled intravesical mitomycin C release.

BACKGROUND: Intravesical treatment of bladder cancer is preferred over systemic administration. However, the efficacy of intravesical instillations is challenged by the periodic voiding that flushes out the instilled drug and ultimately reduces drug exposure to the bladder epithelium. Here, we demonstrate a new catheter-integrated drug-delivery concept that utilizes a silicone-based interpenetrating polymer network (IPN) as material for the catheter balloon, to facilitate continuous release of the bladder cancer adjuvant, Mitomycin C, from a balloon-reservoir to the urinary bladder. METHODS: Long-term release properties and anti-carcinoma cell efficacy of released drug was investigated in vitro. Short-term release experiments were performed in live pigs to evaluate the IPN prototype catheter in a physiological relevant environment in vivo. RESULTS: Sustained zero-order release of Mitomycin C was achieved for 12 days in vitro without refilling the balloon. Mitomycin C was released from the IPN-balloons into the urinary bladder of live pigs in concentrations adequate to inhibit carcinoma cell growth. CONCLUSION: The IPN catheter represents a new drug-delivery concept for prolonged Mitomycin C delivery to the urinary bladder.

A Novel Device-Integrated Drug Delivery System for Local Inhibition of Urinary Tract Infection.

Background: Catheter-associated urinary tract infection (CAUTI) is a frequent community-acquired infection and the most common nosocomial infection. Here, we developed a novel antimicrobial catheter concept that utilizes a silicone-based interpenetrating polymer network (IPN) as balloon material to facilitate a topical slow-release prophylaxis of antibacterial agents across the balloon to the urinary bladder. Methods: The balloon material was achieved by modifying low shore hardness silicone tubes with a hydrogel interpenetrating polymer in supercritical CO2 using the sequential method. Release properties and antibacterial efficacy of the IPN balloon treatment concept was investigated in vitro and in a porcine CAUTI model developed for the study. In the latter, Bactiguard Infection Protection (BIP) Foley catheters were also assessed to enable benchmark with the traditional antimicrobial coating principle. Results: Uropathogenic Escherichia coli was undetectable in urinary bladders and on retrieved catheters in the IPN treatment group as compared to control that revealed significant bacteriuria (>105 colony forming units/ml) as well as catheter-associated biofilm. The BIP catheters failed to prevent E. coli colonization of the bladder but significantly reduced catheter biofilm formation compared to the control. Conclusion: The IPN-catheter concept provides a novel, promising delivery route for local treatment in the urinary tract.

On-Demand Reversible UV-Triggered Interpenetrating Polymer Network-Based Drug Delivery System Using the Spiropyran-Merocyanine Hydrophobicity Switch.

A reversible switchable on-demand UV-triggered drug delivery system (DDS) based on interpenetrating polymer networks (IPNs) with silicone as the host polymer and spiropyran (SP)-functionalized guest polymer is designed and demonstrated. The photo-responsive IPNs provide a new triggered drug delivery concept as they exploit the change in intermolecular interactions (work of adhesion) among the drug, matrix, and solvent when the incorporated hydrophobic SP moieties transform into the hydrophilic merocyanine form upon light irradiation without degradation and disruption of the DDS. The change in how the copolymer composition (hydrophilicity and content) and the lipophilicity of the drug (log P) affect the release profile was investigated. A thermodynamic model, based on Hansen solubility parameters, was developed to design and optimize the polymer composition of the IPNs to obtain the most efficient light-triggered drug release and suppression of the premature release. The developed IPNs showed excellent result for dopamine, l-dopa, and prednisone with around 90-95% light-triggered release. The model was applied to study the release behavior of drugs with different log P and to estimate if the light-induced hydrophobic-to-hydrophilic switch can overcome the work of adhesion between polymers and drugs and hence the desorption and release of the drugs. To the best of our knowledge, this is the first time that work of adhesion is used for this aim. Comparing the result obtained from the model and experiment shows that the model is useful for evaluating and estimating the release behavior of specific drugs merocyanine, IPN, DDS, and spiropyran.

Molecular-Gated Drug Delivery Systems Using Light-Triggered Hydrophobic-to-Hydrophilic Switches.

A photoresponsive molecular-gated drug delivery system (DDS) based on silicone-hydrogel (poly(HEMA-co-PEGMEA)) interpenetrating polymer networks (IPNs) functionalized with carboxylated spiropyran (SPCOOH) was designed and demonstrated as an on-demand DDS. The triggered-release mechanism relies on controlling the wetting behavior of the surface by light, exploiting different hydrophobicities between the "closed" and "open" isomers of spiropyran as a photoswitchable molecular gate on the surface of IPN (SP-photogated IPN). Light-triggered release of doxycycline (DOX) as a model drug indicated that the spiropyran (SP) molecules provide a hydrophobic layer around the drug carrier and have a good gate-closing efficiency for IPNs with 20-30% hydrogel content. Upon UV light irradiation, SP converts into an open hydrophilic merocyanine state, which triggers the release of DOX. These results were compared with a previously developed SP-bulk modified IPN using the same hydrogel as a control, proving the efficiency of the gated IPN system. The covalent attachment of SPCOOH to the alcohol groups of the hydrogel and the structural change caused by UV light was indicated with FTIR analysis. XPS results also confirm the presence of SP by indicating the atomic percentage of nitrogen with respect to the hydrogel content.

Evaluating Efficacy of Antimicrobial and Antifouling Materials for Urinary Tract Medical Devices: Challenges and Recommendations.

In Europe, the mean incidence of urinary tract infections in intensive care units is 1.1 per 1000 patient-days. Of these cases, catheter-associated urinary tract infections (CAUTI) account for 98%. In total, CAUTI in hospitals is estimated to give additional health-care costs of £1-2.5 billion in the United Kingdom alone. This is in sharp contrast to the low cost of urinary catheters and emphasizes the need for innovative products that reduce the incidence rate of CAUTI. Ureteral stents and other urinary-tract devices suffer similar problems. Antimicrobial strategies are being developed, however, the evaluation of their efficacy is very challenging. This review aims to provide considerations and recommendations covering all relevant aspects of antimicrobial material testing, including surface characterization, biocompatibility, cytotoxicity, in vitro and in vivo tests, microbial strain selection, and hydrodynamic conditions, all in the perspective of complying to the complex pathology of device-associated urinary tract infection. The recommendations should be on the basis of standard assays to be developed which would enable comparisons of results obtained in different research labs both in industry and in academia, as well as provide industry and academia with tools to assess the antimicrobial properties for urinary tract devices in a reliable way.

Controlled Release of Plectasin NZ2114 from a Hybrid Silicone-Hydrogel Material for Inhibition of Staphylococcus aureus Biofilm.

Staphylococcus aureus is a major human pathogen in catheter-related infections. Modifying catheter material with interpenetrating polymer networks is a novel material technology that allows for impregnation with drugs and subsequent controlled release. Here, we evaluated the potential for combining this system with plectasin derivate NZ2114 in an attempt to design an S. aureus biofilm-resistant catheter. The material demonstrated promising antibiofilm properties, including properties against methicillin-resistant S. aureus, thus suggesting a novel application of this antimicrobial peptide.

Enhanced plasmid loss in bacterial populations exposed to the antimicrobial compound irgasan delivered from interpenetrating polymer network silicone hydrogels.

The spread of antimicrobial resistance, usually mediated by horizontal transfer of plasmids, limits the options of treating bacterial infections and thereby poses a crucial human health problem. The disturbance of plasmid stability within bacterial species in clinical environments serves as a novel strategy to reduce the development and dissemination of antibiotic resistance. We tested the ability of irgasan to destabilize plasmids from Escherichia coli K-12 cells when added directly into liquid growth medium at concentrations below levels of marked bacterial growth inhibition, or when released into liquid growth medium from irgasan-impregnated Interpenetrating Polymer Network (IPN) silicone hydrogel objects, a novel technology developed as drug-delivery platform. IPN-mediated irgasan-release was indirectly monitored as the extent of plasmid loss from bacterial cells during a 24-hour period or during repeated exposure to new irgasan-loaded IPN devices every 24h for a total of 10days. The cells were genetically modified so that plasmid loss could be quantified by applying a combination of fluorescence-based reporter gene technology and flow cytometry. When exposing bacterial cells to the irgasan-impregnated IPNs for 24h, we observed a modest (2.8-4.7%), but significant (P<0.05), plasmid loss as well as an inhibition of bacterial growth, both gradually increasing with increasing impregnation concentration. Repeated exposure to irgasan-impregnated IPNs drastically increased the plasmid loss of up to 83%, but cells adapted over time, which indicated the limitations of this specific drug for future medical applications. This study, however, illustrates the ability of IPNs to release an impregnated compound into a liquid suspension to induce a significant biological impact on growing bacterial cells.

Co-release of dicloxacillin and thioridazine from catheter material containing an interpenetrating polymer network for inhibiting device-associated Staphylococcus aureus infection.

Approximately half of all nosocomial bloodstream infections are caused by bacterial colonization of vascular catheters. Attempts have been made to improve devices using anti-adhesive or antimicrobial coatings; however, it is often difficult to bind coatings stably to catheter materials, and the low amounts of drug in thin-film coatings limit effective long-term release. Interpenetrating polymer networks (IPNs) are polymer hybrid materials with unique drug release properties. While IPNs have been extensively investigated for use in tablet- or capsule-based drug delivery systems, the potential for use of IPNs in drug release medical devices remains largely unexplored. Here, we investigated the use of silicone-hydrogel IPNs as a catheter material to provide slow anti-bacterial drug-release functionality. IPN catheters were produced by the sequential method, using supercritical CO2 as a solvent to polymerize and crosslink poly(2-hydroxyethyl methacrylate) (PHEMA) in silicone elastomer. The design was tested against Staphylococcus aureus colonization after loading with dicloxacillin (DCX) alone or in combination with thioridazine (TDZ), the latter of which is known to synergistically potentiate the antibacterial effect of DCX against both methicillin-sensitive and methicillin-resistant S. aureus. The hydrophilic PHEMA component allowed for drug loading in the catheters by passive diffusion and provided controlled release properties. The drug-loaded IPN material inhibited bacterial growth on agar plates for up to two weeks and in blood cultures for up to five days, and it withstood 24h of seeding with resilient biofilm aggregates. The combined loading of DCX+TDZ enhanced the antibacterial efficiency in static in vitro experiments, although release analyses revealed that this effect was due to an enhanced loading capacity of DCX when co-loaded with TDZ. Lastly, the IPN catheters were tested in a novel porcine model of central venous catheter-related infection, in which drug-loaded IPN catheters were found to significantly decrease the frequency of infection.

Investigation of pharmaceuticals in processed animal by-products by liquid chromatography coupled to high-resolution mass spectrometry.

There is an on-going trend for developing more sustainable salmon feed in which traditionally applied marine feed ingredients are replaced with alternatives. Processed animal products (PAPs) have been re-authorized as novel high quality protein ingredients in 2013. These PAPs may harbor undesirable substances such as pharmaceuticals and metabolites which are not previously associated with salmon farming, but might cause a potential risk for feed and food safety. To control these contaminants, an analytical strategy based on a generic extraction followed by ultra-high performance liquid chromatography coupled to high resolution mass spectrometry (UHPLC-HRMS) using quadrupole time-of-flight mass analyzer (QTOF MS) was applied for wide scope screening. Quality control samples, consisting of PAP commodities spiked at 0.02, 0.1 and 0.2 mg/kg with 150 analytes, were injected in every sample batch to verify the overall method performance. The methodology was applied to 19 commercially available PAP samples from six different types of matrices from the EU animal rendering industry. This strategy allows assessing possible emergent risk exposition of the salmon farming industry to 1005 undesirables, including pharmaceuticals, several dyes and relevant metabolites.

3D Printed Silicone-Hydrogel Scaffold with Enhanced Physicochemical Properties.

Scaffolds with multiple functionalities have attracted widespread attention in the field of tissue engineering due to their ability to control cell behavior through various cues, including mechanical, chemical, and electrical. Fabrication of such scaffolds from clinically approved materials is currently a huge challenge. The goal of this work was to fabricate a tissue engineering scaffold from clinically approved materials with the capability of delivering biomolecules and direct cell fate. We have used a simple 3D printing approach, that combines polymer casting with supercritical fluid technology to produce 3D interpenetrating polymer network (IPN) scaffold of silicone-poly(2-hydroxyethyl methacrylate)-co-poly(ethylene glycol) methyl ether acrylate (pHEMA-co-PEGMEA). The pHEMA-co-PEGMEA IPN materials were employed to support growth of human mesenchymal stem cells (hMSC), resulting in high cell viability and metabolic activity over a 3 weeks period. In addition, the IPN scaffolds support 3D tissue formation inside the porous scaffold with well spread cell morphology on the surface of the scaffold. As a proof of concept, sustained doxycycline (DOX) release from pHEMA-co-PEGMEA IPN was demonstrated and the biological activity of released drug from IPN was confirmed using a DOX regulated green fluorescent reporter (GFP) gene expression assay with HeLa cells. Given its unique mechanical and drug releasing characteristics, IPN scaffolds may be used for directing stem cell differentiation by releasing various chemicals from its hydrogel network.

Soft hydrogels interpenetrating silicone--A polymer network for drug-releasing medical devices.

Materials for the next generation of medical devices will require not only the mechanical stability of current devices, but must also possess other properties such as sustained release of drugs in a controlled manner over a prolonged period of time. This work focuses on creating such a sophisticated material by forming an interpenetrating polymer network (IPN) material through modification of silicone elastomers with a poly(2-hydroxyethyl methacrylate) (PHEMA)-based hydrogel. IPN materials with a PHEMA content in the range of 13%-38% (w/w) were synthesized by using carbon dioxide-based solvent mixtures under high pressure. These IPNs were characterized with regard to microstructure as well as ability of the hydrogel to form a surface-connected hydrophilic carrier network inside the silicone. A critical limit for hydrogel connectivity was found both via simulation and by visualization of water uptake in approximately 25% (w/w) PHEMA, indicating that entrapment of gel occurs at low gel concentrations. The optimized IPN material was loaded with the antibiotic ciprofloxacin, and the resulting drug release was shown to inhibit bacterial growth when placed on agar, thus demonstrating the potential of this IPN material for future applications in drug-releasing medical devices.

Sustained prevention of biofilm formation on a novel silicone matrix suitable for medical devices.

Bacterial colonization and biofilm formation on medical devices constitute major challenges in clinical long-term use of e.g. catheters due to the risk of (re)infection of patients, which would result in additional use of antibiotics risking bacterial resistance development. The aim of the present project was to introduce a novel antibacterial approach involving an advanced composite material applicable for medical devices. The polymeric composites investigated consisted of a hydrogel network of cross-linked poly(2-hydroxyethyl methacrylate) (PHEMA) embedded in a poly(dimethylsiloxane) (PDMS) silicone elastomer produced using supercritical carbon dioxide (scCO2). In these materials, the hydrogel may contain an active pharmaceutical ingredient while the silicone elastomer provides the sufficient mechanical stability of the material. In these conceptual studies, the antimicrobial agent ciprofloxacin was loaded into the polymer matrix by a post-polymerization loading procedure. Sustained release of ciprofloxacin was demonstrated, and the release could be controlled by varying the hydrogel content in the range 13-38% (w/w) and by changing the concentration of ciprofloxacin during loading in the range of 1-20mg/mL. Devices containing 25% (w/w) hydrogel and loaded with ciprofloxacin displayed a strong antibacterial effect against Staphylococcus aureus bacterial colonization and subsequent biofilm formation on the device material was inhibited for 29days. In conclusion, the hydrogel/silicone composite represents a promising candidate material for medical devices that prevent bacterial colonization during long-term use.

Safety evaluation for a biodiesel process using prion-contaminated animal fat as a source.

BACKGROUND: Due to the bovine spongiform encephalopathy (BSE), specified risk material (SRM) as well as animal meat and bone meal (MBM) are banned from the food and feed chain because of a possible infection with pathogenic prions (PrP(Sc)). Nowadays, prions are widely accepted to be responsible for TSE(transmissible spongiform encephalopathies)-caused illnesses like BSE and scrapie, and especially for the occurrence of the new variant of CJD in humans. Presently, SRM and MBM are burnt under high temperatures to avoid any hazards for humans, animals or the environment. The aim of this study was to evaluate a method using animal fat separated from Category I material which includes SRM and the carcasses of TSE-infected animals, or animals suspected of being infected with TSE, as a source for producing biodiesel by transesterification, analogous to the biodiesel process using vegetable oil. METHODS: For this purpose, animal fat was spiked with scrapie-infected hamster brain equivalents--as representative for a TSE-infected animal--and the biodiesel manufacturing process was downscaled and performed under lab-scale conditions. RESULTS AND DISCUSSION: The results analysed by Western blotting showed clearly that almost each single step of the process leads to a significant reduction of the concentration of the pathogenic prion protein (PrP(Sc)) in the main and side-products. CONCLUSION: The data revealed that the biodiesel production, even from material with a high concentration of pathogenic prions, can be considered as safe. RECOMMENDATIONS AND OUTLOOK: The obtained results indicated that biodiesel produced from prion-contaminated fat was safe under the tested process conditions. However, it has to be pointed out that the results cannot be generalized because a different process control using other conditions may lead to different results and then has to be analysed independently. It is clear that the production of biodiesel from high risk material represents a more economic usage than the combustion of such material.