Electrospun polycaprolactone nanofibrous membranes loaded with manuka honey and essential oils by layer-by-layer assembly for antibacterial wound dressings

Chronic wounds cause several discomforts in patients and require huge expenses in terms of time and resources. Estimates indicate that the global market will increase from USD 12.36 billion in 2022 to USD 19.52 billion by 2029. The consequences related to chronic and non-healing wounds are pathological inflammation, impaired angiogenesis, difficult in re-epithelization and formation of drug resistant biofilms. These types of wounds are a global health issue, statistically affecting the 1-2% of the population in developed countries during their lives. Moreover, antimicrobial resistance (AMR) is a correlated issue: this happens when pathogens no longer respond to antimicrobial agents, drugs becomes ineffective and severe infections become threatening. AMR has caused at least 1.27 million deaths worldwide and was associated with ~5 million deaths (2019), costing USD 4.6 billion annually. On account of this, it is fundamental to advance new treatment solutions. Among the emerging therapies are present medicated dressing incorporating bioactive molecules, including natural compounds (e.g. essential oils, Manuka Honey, curcumin, chitosan). These materials exert antimicrobial and antibacterial functions by interacting with bacteria cells causing membrane disruption and impairment in the cell division process. Furthermore, no episode of resistance has been recorded yet; for these reasons, their use is advantageous. Therefore, the aim of this thesis project was to develop antimicrobial dressings for wound treatments and this thesis work was performed at both Newcastle University (UK) and Politecnico di Torino (IT). Particularly, polycaprolactone-membranes were manufactured via electrospinning technique. Subsequently, the membranes were coated with natural polyelectrolytes using the layer-by-layer technique. The natural compounds used were chitosan, as polycation, manuka honey, as polyanion, and essential oils (these latter in form of nanoemulsions). Quartz Crystal Microbalance with Dissipation (QCM-D) analysis was performed simulating the layer-by-layer process onto a sensor in order to monitor the real-time coating formation and growth. Subsequently, morphological, physicochemical and biological characterizations were performed on the membranes. Scansion Electron Microscopy (SEM) imaging was conducted to investigate the thickness of the coating. Fourier Transform Infrared Spectroscopy with Attenuated Total Reflection (FTIR-ATR) analysis was carried out to investigate the functional groups at the surface of the samples; whilst with X-Ray Photoelectron Spectroscopy (XPS) technique, the elemental composition at the surface of the material was quantitatively measured. The effectiveness of the membranes against bacteria was investigated on both gram positive and negative bacterial strains (Staphylococcus aureus and Pseudomonas aeruginosa); moreover, the membranes were tested in presence of fibroblasts cells, to verify biocompatibility. The results obtained from SEM and QCM-D demonstrate the formation of the layers and the growing of the coating, with a final thickness of 160 nm for a 16 layers coating. Moreover, FTIR-ATR and XPS analysis show the distinction among different coating compositions. The membranes (above which bacterial strains were cultured) stained with Crystal Violet and analysed at the microscope, demonstrate the effective antibacterial activity of the coating. At the same time, the cells cultured in presence of the membranes and analysed with Live/Dead assay preserved viability.