Development of In Vitro and Ex Vivo Biofilm Models for the Assessment of Antibacterial Fibrous Electrospun Wound Dressings.

Increasing evidence suggests that the chronicity of wounds is associated with the presence of bacterial biofilms. Therefore, novel wound care products are being developed, which can inhibit biofilm formation and/or treat already formed biofilms. A lack of standardized assays for the analysis of such novel antibacterial drug delivery systems enhances the need for appropriate tools and models for their characterization. Herein, we demonstrate that optimized and biorelevant in vitro and ex vivo wound infection and biofilm models offer a convenient approach for the testing of novel antibacterial wound dressings for their antibacterial and antibiofilm properties, allowing one to obtain qualitative and quantitative results. The in vitro model was developed using an electrospun (ES) thermally crosslinked gelatin-glucose (GEL-Glu) matrix and an ex vivo wound infection model using pig ear skin. Wound pathogens were used for colonization and biofilm development on the GEL-Glu matrix or pig skin with superficial burn wounds. The in vitro model allowed us to obtain more reproducible results compared with the ex vivo model, whereas the ex vivo model had the advantage that several pathogens preferred to form a biofilm on pig skin compared with the GEL-Glu matrix. The in vitro model functioned poorly for Staphylococcus epidermidis biofilm formation, but it worked well for Escherichia coli and Staphylococcus aureus, which were able to use the GEL-Glu matrix as a nutrient source and not only as a surface for biofilm growth. On the other hand, all tested pathogens were equally able to produce a biofilm on the surface of pig skin. The developed biofilm models enabled us to compare different ES dressings [pristine and chloramphenicol-loaded polycaprolactone (PCL) and PCL-poly(ethylene oxide) (PEO) (PCL/PEO) dressings] and understand their biofilm inhibition and treatment properties on various pathogens. Furthermore, we show that biofilms were formed on the wound surface as well as on a wound dressing, indicating that the demonstrated methods mimic well the in vivo situation. Colony forming unit (CFU) counting and live biofilm matrix as well as bacterial DNA staining together with microscopic imaging were performed for biofilm quantification and visualization, respectively. The results showed that both wound biofilm models (in vitro and ex vivo) enabled the evaluation of the desired antibiofilm properties, thus facilitating the design and development of more effective wound care products and screening of various formulations and active substances.

Antibacterial Porous Electrospun Fibers as Skin Scaffolds for Wound Healing Applications.

Electrospun fiber scaffolds have a huge potential for the successful treatment of infected wounds based on their unique properties. Although several studies report novel drug-loaded electrospun fiber-based biomaterials, many of these do not provide information on their interactions with eukaryotic and bacterial cells. The main aim of this study was to develop antibacterial drug-loaded porous biocompatible polycaprolactone (PCL) fiber scaffolds mimicking the native extracellular matrix for wound healing purposes. Mechanical property evaluation and different biorelevant tests were conducted in order to understand the structure-activity relationships and reveal how the surface porosity of fibers and the fiber diameter affect the scaffold interactions with the living bacterial and eukaryotic fibroblast cells. Cell migration and proliferation assays and antibiofilm assays enabled us to enlighten the biocompatibility and safety of fiber scaffolds and their suitability to be used as scaffolds for the treatment of infected wounds. Here, we report that porous PCL microfiber scaffolds obtained using electrospinning at high relative humidity served as the best surfaces for fibroblast attachment and growth compared to the nonporous microfiber or nonporous nanofiber PCL scaffolds. Porous chloramphenicol-loaded microfiber scaffolds were more elastic compared to nonporous scaffolds and had the highest antibiofilm activity. The results indicate that in addition to the fiber diameter and fiber scaffold porosity, the single-fiber surface porosity and its effect on drug release, mechanical properties, cell viability, and antibiofilm activity need to be understood when developing antibacterial biocompatible scaffolds for wound healing applications. We show that pores on single fibers within an electrospun scaffold, in addition to nano- and microscale diameter of the fibers, change the living cell-fiber interactions affecting the antibiofilm efficacy and biocompatibility of the scaffolds for the local treatment of wounds.

Monitoring of Antimicrobial Drug Chloramphenicol Release from Electrospun Nano- and Microfiber Mats Using UV Imaging and Bacterial Bioreporters.

New strategies are continuously sought for the treatment of skin and wound infections due to increased problems with non-healing wounds. Electrospun nanofiber mats with antibacterial agents as drug delivery systems provide opportunities for the eradication of bacterial infections as well as wound healing. Antibacterial activities of such mats are directly linked with their drug release behavior. Traditional pharmacopoeial drug release testing settings are not always suitable for analyzing the release behavior of fiber mats intended for the local drug delivery. We tested and compared different drug release model systems for the previously characterized electrospun chloramphenicol (CAM)-loaded nanofiber (polycaprolactone (PCL)) and microfiber (PCL in combination with polyethylene oxide) mats with different drug release profiles. Drug release into buffer solution and hydrogel was investigated and drug concentration was determined using either high-performance liquid chromatography, ultraviolet-visible spectrophotometry, or ultraviolet (UV) imaging. The CAM release and its antibacterial effects in disc diffusion assay were assessed by bacterial bioreporters. All tested model systems enabled to study the drug release from electrospun mats. It was found that the release into buffer solution showed larger differences in the drug release rate between differently designed mats compared to the hydrogel release tests. The UV imaging method provided an insight into the interactions with an agarose hydrogel mimicking wound tissue, thus giving us information about early drug release from the mat. Bacterial bioreporters showed clear correlations between the drug release into gel and antibacterial activity of the electrospun CAM-loaded mats.

Effects and efficacy of different sterilization and disinfection methods on electrospun drug delivery systems.

Microbiological quality of a pharmaceutical product is an essential requirement ensuring patient safety, thus effective sterilization/disinfection methods need to be found. The aim of this study was to evaluate the efficacy of different sterilization/disinfection methods on drug-loaded electrospun matrices and the impact of these treatments on the functionality related characteristics of these matrices. The sterilization efficacy of gamma-irradiation, ultraviolet-irradiation, in situ generated chlorine gas and low-pressure argon plasma treatment were evaluated on two different chloramphenicol-loaded electrospun matrices using pristine polycaprolactone (PCL) as a carrier polymer or PCL in combination with polyethylene oxide. Drug stability, solid state properties, morphology, mechanical properties, swelling, biodegradation and drug release kinetics were studied before and after the treatments. It was shown that all tested methods help to reduce bioburden and only plasma treated matrices were not sterile. At the same time drug degradation after the treatment can be considerable and depends not only on the susceptibility of the drug to degradation, but also on matrix properties (e.g. the nature of carrier polymers). Even though no morphological changes were observed, gamma sterilization increased the hardness and elasticity of PCL matrices as a result of increased crystallinity of the polymer. Plasma treatment was able to significantly enhance water absorption to otherwise hydrophobic PCL/CAM matrix and had tremendous impact on its drug release kinetics as the drug was instantly released from otherwise prolonged release formulation.

Impact of PCL nanofiber mat structural properties on hydrophilic drug release and antibacterial activity on periodontal pathogens.

Electrospinning enables to design and manufacture novel drug delivery systems capable of advancing the local antibacterial therapy. In this study, two hydrophilic drugs - metronidazole and ciprofloxacin hydrochloride - were loaded both individually and in combination into hydrophobic poly(ε-caprolactone) (PCL) matrix using electrospinning. We aimed to develop prolonged release drug delivery systems suitable for the treatment of periodontal diseases and understand how different rarely studied structural features, such as nanofiber mat thickness, surface area, wettability, together with intrinsic properties, like solid state and localization of incorporated drugs in nanofibers, affect the drug release. Furthermore, the safety of nanofiber mats was assessed in vitro on fibroblasts, and their antibacterial activity was tested on selected strains of periodontopathogenic bacteria. The results showed that the structural properties of nanofiber mat are crucial in particular drug-polymer combinations, affecting the drug release and consequently the antibacterial activity. The hydrophobicity of a PCL nanofiber mat and its thickness are the key characteristics in prolonged hydrophilic drug release, but only when wetting is the rate-limiting step for the drug release. Combination of drugs showed beneficial effects by inhibiting the growth of all tested pathogenic bacterial strains important in periodontal diseases.

Interactions between Chloramphenicol, Carrier Polymers, and Bacteria-Implications for Designing Electrospun Drug Delivery Systems Countering Wound Infection.

Antibacterial drug-loaded electrospun nano- and microfibrous dressings are of major interest as novel topical drug delivery systems in wound care. In this study, chloramphenicol (CAM)-loaded polycaprolactone (PCL) and PCL/poly(ethylene oxide) (PEO) fiber mats were electrospun and characterized in terms of morphology, drug distribution, physicochemical properties, drug release, swelling, cytotoxicity, and antibacterial activity. Computational modeling together with physicochemical analysis helped to elucidate possible interactions between the drug and carrier polymers. Strong interactions between PCL and CAM together with hydrophobicity of the system resulted in much slower drug release compared to the hydrophilic ternary system of PCL/PEO/CAM. Cytotoxicity studies confirmed safety of the fiber mats to murine NIH 3T3 cells. Disc diffusion assay demonstrated that both fast and slow release fiber mats reached effective concentrations and had similar antibacterial activity. A biofilm formation assay revealed that both blank matrices are good substrates for the bacterial attachment and formation of biofilm. Importantly, prolonged release of CAM from drug-loaded fibers helps to avoid biofilm formation onto the dressing and hence avoids the treatment failure.