Smart dressings based on nanostructured fibers containing natural origin antimicrobial and anti-inflammatory compounds

Resumen

The current Doctoral Thesis, named “Smart dressings based on nanostructured fibers containing natural origin antimicrobial and anti-inflammatory compounds” has been developed in the Department of Chemical Engineering and Environmental Technology (University of Zaragoza, Spain) in the group of Nanostructured Films and Particles (NFP), which is a member of the Aragon Institute of Nanoscience (INA). This research has been developed in this institute and also in the Biomedical Research Center of Aragon (CIBA). A predoctoral stay of 6 months was performed in the Methodist Hospital Research Institute in Houston (Texas, US) supervised by Professor Ennio Tasciotti. This research was supported by a FPI predoctoral fellowship, funded by the Spanish Ministry of Economy and Competitiveness (CTQ2014-52384-R). The short stay was also funded by the Network of Biomedical Research Center in the field of Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN). Due to the development of bacterial resistances, the search of new compounds to treat infections is in the spotlight of researchers. In this scenario, this thesis is focused on the design of a bioactive dressing based on electrospun polycaprolactone (PCL) nanofibers loading natural compounds obtained from essential oils (EOs) acting as anti-inflammatory and antimicrobial compounds for wound healing treatment. For this aim, a deep research on the bactericidal and anti-inflammatory EOs compounds properties was carried out. The first purpose of the thesis was to tackle wound infection with plant-derived natural compounds. For this goal, the bactericidal properties and mechanisms of different free EOs in planktonic bacteria cultures using a Gram-positive strain, Staphylococcus aureus (ATCC 25923) and a Gram-negative strain, Escherichia coli S17 were studied. In addition, since biofilm formation is a challenge in wound infections, the effect of natural compounds was studied on S. aureus biofilm. The high volatility character of EOs and the fact that they can be oxidized in contact with air or ultraviolet light, make difficult their application. Encapsulation of EOs is a good technique to protect their properties. Thus, thymol (THY), the compound that demonstrated the best antimicrobial activity against planktonic bacteria and biofilm, was encapsulated into PCL nanofibers. Nanofibers synthesis and THY encapsulation were carried out using the electrospinning technique. This method allows the fabrication of patches with high load of those volatile compounds such as THY. The bactericidal properties of these patches were studied using a cGFP-expressing S. aureus strain through quantitative assays and confocal microscopy. In order to use these natural compounds for wound healing applications, free-compound cytotoxicity assays were carried out in three different cell types: fibroblast, keratinocytes and macrophages. Moreover, a co-culture model using J774 macrophages and cGFP-expressing S. aureus was developed to study and monitoring the effect of THY loaded PCL patches in intracellular bacteria. The second goal in the thesis was to find an anti-inflammatory natural compound effective for wound treatment. For these assays, an in vitro inflammatory model was optimized in lipopolysaccharide (LPS) -activated J774 macrophages. These experiments were developed in the Methodist Hospital Research Institute in Houston (Texas, US). The study of the increase or reduction levels of inflammatory cytokines allowed us to determine the best anti-inflammatory natural compounds evaluated in our studies. Among them, PCL-THY patches demonstrated to be the wound dressings that better reduced inflammation in an in vitro inflammatory model. After concluding that THY had superior antimicrobial and anti-inflammatory properties when loaded in PCL nanofibers, an in vivo experiment to determine the effectiveness of the designed patches in an infected skin wound model was developed. SKH1 hairless mice were used to analyze the in vivo bactericidal and anti-inflammatory properties of PCL-THY patches for their potential clinical application. The document is structured in different chapters that address each of the goals of this doctoral thesis: Chapter I corresponds with the introductory part where general concepts are exposed. The chapter includes a description of the wound healing processes, pointing the factors affecting the procedure such as infection and inflammation. Different types of wound healing treatments such as antibiotics are described, highlighting antibiotic resistance as the reason to choose plant-derived compounds as a wound healing treatment. Electrospinning technique is explained to understand the synthesis of PCL nanofibers and the encapsulation of the active principle. Chapter II focuses on antimicrobial activity of free EOs to tackle wound infection. Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) were measured for different plant-derived natural compounds in S. aureus (ATCC 25923) and E. coli S17 strains. In addition, a S. aureus biofilm model was optimized to determine the effect of EOs in these bacterial formations. The mechanism of action of EOs against bacteria was studied by scanning electron microscopy (SEM) and flow cytometry. Cytotoxicity of EOs treatment was also evaluated in skin-related cell types such as fibroblasts, keratinocytes and macrophages. Carvacrol (CAR), cinnamaldehyde (CIN) and THY exhibited the highest in vitro antimicrobial activities against E. coli and S. aureus by disrupting the bacteria membrane. These results are included in the published article entitled “Evaluation of the antimicrobial activity and cytotoxicity of different components of natural origin present in essential oils”. García-Salinas, S.; Elizondo-Castillo, H.; Arruebo, M.; Mendoza, G.; Irusta, S. Molecules 2018, 23 (6), 1–18. https://doi.org/10.3390/molecules23061399. Chapter III considering the results obtained in Chapter II, THY was chosen as the compound to be loaded into electrospun PCL nanofibers due to its antimicrobial activity and low cytotoxicity compared with the other EOs tested. PCL-THY patches were synthetized, and their bactericidal properties were measured against both, planktonic culture and biofilm of cGFP-expressing S. aureus. A co-culture model using cGFP-expressing S. aureus and J774 macrophages was developed to study the effect of PCL-THY patches on infected cells. Compared to non-loaded dressings, PCL-THY dressings were able to eliminate the pathogenic bacteria in coculture models using infected murine macrophages. In addition, it was corroborated the successful ability of the developed patch in preventing biofilm formation. These results are included in the recently submitted article entitled “Antimicrobial Wound Dressings Against Fluorescent and Methicillin-Sensitive Intracellular Pathogenic Bacteria” Garcia-Salinas, S.; Gamez-Herrera, E.; Landa, G.; Arruebo, M.; Irusta, S.; Mendoza, G. ACS Applied Materials & Interfaces Manuscript ID: am-2020-05668q. Chapter IV describes the anti-inflammatory activity of EOs with the aim of reducing and controlling the inflammatory process during wound healing. Excessive inflammation can cause the activation of unnecessary cells or cytokines, generating deleterious effects, limiting healing. To develop this goal, an inflammatory model of J774 macrophages activated with LPS was carried out. Different free natural compounds reported as anti-inflammatory molecules were assessed. Thus, infected macrophages were treated and analyzed at the genetic level, measuring interleukin (Il) 1b, iNos (inducible Nitric oxide synthase) and Il10 cytokines by RT-PCR (Reverse Transcription-Polymerase Chain Reaction). PCL patches loaded with EOs were tested using the same in vitro model, confirming previous results. In addition, it was assessed cell morphology and average cell area comparing treated cells with positive (LPS-activated cells without treatment) and negative (non LPS-activated cells) controls. This experiment demonstrated the similarity among treated cells and non-activated cells, confirming the anti-inflammatory effect of THY loaded PCL patches. These results are included in the published article entitled “Electrospun Anti-Inflammatory Patch Loaded with Essential Oils for Wound Healing”. García-Salinas, S.; Evangelopoulos, M.; Gámez-Herrera, E.; Arruebo, M.; Irusta, S.; Taraballi, F.; Mendoza, G.; Tasciotti, E. Int. J. Pharm. 2020, 577 (January), 119067. https://doi.org/10.1016/j.ijpharm.2020.119067. Chapter V joins all the research performed during the thesis related to an S. aureus (ATCC 25923) infected wound in vivo model using SHK1 hairless mice. Thus, it was analyzed the in vivo bactericidal and inflammatory properties of the designed dressings. In addition, controls of free EO and a clinical compound (chlorhexidine) were added to assess the advantages of our patch against other treatments. Starting by infection, quantitative and qualitative measurements of bacteria present in the wound were collected at different dpi (days post infection). Moreover, the histological analysis of skin wounds was carried out to evaluate the inflammatory reaction and new vessel formation. Studies demonstrated that PCL-THY can prevent infection, promote wound healing and prompt regeneration. These results are included in the recently accepted article entitled “Efficiency of Antimicrobial Electrospun Thymol-Loaded Polycaprolactone Mats in vivo” Garcia-Salinas, S.; Gamez-Herrera, E.; Asin, J.; de Miguel, R.; Andreu, V.; Sancho-Albero, M.; Mendoza, G.; Irusta, S.; Arruebo, M. ACS Applied Biomaterials. ACS Appl. Bio Mater. 2020, 3 (5), 3430–3439. https://doi.org/10.1021/acsabm.0c00419 Chapter VI summarizes the main conclusions obtained during this doctoral thesis. Appendix I describes the main characterization techniques and biological methods in order to evaluate the antimicrobial and anti-inflammatory ability of free and loaded compounds. Appendix II compiles the references used to write this work. Appendix III points out the published scientific papers and the participation in conferences during this thesis.