Develpment of electrospun wound-dressings incorporating medicinal plant-extracts

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Development of electrospun wound-dressings incorporating medicinal plant-extracts

Publication . Mouro, Cláudia Filipa Duarte; Gouveia, Isabel Cristina Aguiar de Sousa e Silva; Fangueiro, Raul Manuel Esteves de Sousa

Human skin is a remarkably effective barrier against the invasion of external pathogens. However, when the occurrence of wounds compromises the skin’s integrity, the possibility of pathogenic microorganisms to colonize the wound site increase as well as the risk of acquiring an infection. In particular, the presence and permanence of high levels of pathogenic bacteria in the wound have been identified as the main responsible for the delay or failure in the healing process, especially in patients with a compromised immune system. The skin and soft tissue infections (SSTIs), particularly those caused by bacteria, are among the most common infections that can progress quickly to life-threatening complications. Besides, the aging population, combined with the increased rates of obesity and chronic diseases, like diabetes, have contributed to a higher prevalence of wounds susceptible to bacterial colonization and infection. In this context, to prevent the penetration of bacteria at the wound site and its growth and proliferation, wound dressings have been produced from different materials, with diverse shapes, containing antimicrobial agents into their structure. Among these agents, antibiotics, nanoparticles (NPs), and natural products have been the most used. However, the excessive and indiscriminate use of antibiotics has triggered an alarming rate of multidrug-resistant bacteria. Also, the possible toxicity associated with the use of NPs has limited its application in dressing materials. In this way, we have been witnessing an increasing demand for compounds obtained from natural sources, in particular from medicinal plants, as a more effective and efficient alternative. Medicinal plants are natural sources of bioactive substances that may exert significant effects on the management and treatment of wounds. Besides, the numerous therapeutic properties of the medicinal plants, such as antimicrobial, anti-inflammatory, antioxidant, anesthetic, and analgesic, are helpful in the treatment of injured skin by enhancing fibroblast proliferation, angiogenesis, and collagen biosynthesis. Thus, wound dressing materials containing plant extracts and some compounds obtained from plants, with intrinsic antimicrobial activity and ability to accelerate the healing process, have captured the interest of researchers in recent years in order to avoid or even eliminate undesirable pathogenic infections. Among the different techniques used to produce wound dressing materials, the electrospinning has been highlighted in the development of wound dressings based on bioactive nanofibers due to its simplicity, cost-effectiveness, and versatility. The nanofiber membranes produced by electrospinning have demonstrated properties with remarkable therapeutic potential, such as a 3D architecture that mimics the morphological features of the skin’s extracellular matrix (ECM), a high surface area to volume ratio, and porosity that allow them to control the exudate effectively. These characteristics are also able to maintain a moist environment at the wound site and ensure a continuous supply of nutrients and oxygen that promotes wound healing. Furthermore, the electrospun nanofibrous membranes have been incorporated with different types of bioactive or therapeutic agents, improving the desirable wound healing properties. Therefore, in this doctoral work, new electrospun wound dressing materials containing crude medicinal plant extracts and plant essential oils with remarkable antimicrobial and healing effects were developed from several strategies to protect the wound from both external agents and pathogenic invasion, as well as improve the skin tissue regeneration. In a first approach, Eugenol (EUG), an essential oil extracted from cloves, was incorporated into a polymeric blend composed of Polycaprolactone (PCL), Polyvinyl Alcohol (PVA), and Chitosan (CS) by electrospinning from water-in-oil (W/O) and oil-in-water (O/W) emulsions. From this work, it was achieved better wound healing properties when O/W emulsion was used. However, although emulsion electrospinning shows promising potential for preserving the EUG’s stability and bioactivity, the essential oils require large amounts of raw material, as well as multiple step preparation methods and special laboratory facilities. To overcome the limitations presented by essential oils, two different crude medicinal plant extracts, which are easily obtained from dried and milled plants, were prepared through a simple, easy to perform, and low-cost extraction method, and then incorporated in two different polymeric blends by emulsion electrospinning to corroborate the effectiveness and potential of this technique. Regarding that, a crude extract of Hypericum perforatum L. (HP) was incorporated into a polymeric blend of Poly(L-lactic acid) (PLLA), PVA, and CS, while a crude extract of Chelidonium majus L. (CM) was loaded into a blend of PCL, PVA, and Pectin (PEC). The results revealed that the manufactured nanofiber membranes exhibited suitable properties for use as wound dressing materials. Besides, these membranes have been shown to inhibit the growth of pathogenic bacteria, namely Staphylococcus aureus (S. aureus) and Pseudomonas aeruginosa (P. aeruginosa), and proved to be versatile systems for controlled release of bioactive and/or therapeutic agents. From these studies, the CM extract loaded into electrospun PCL/PVA_PEC nanofibrous membrane achieved a better antibacterial activity, reaching a ~4 Log reduction. Therefore, emulsion electrospinning has demonstrated to exhibit the incomparable ability to produce, in a single step, single-layer wound dressings incorporated with natural products, and the replacement of EUG by crude medicinal plant extracts proved to be an attractive and promising alternative. In a different approach, double-layered electrospun nanofibrous membranes containing crude medicinal plant extracts were produced, aiming to restore the structure and functions of the native skin. Concerning that, two different double-layer materials were developed from electrospinning. PLLA and PCL’s top layers were designed to act as breathable and waterproof protective barriers, capable of preventing bacteria penetration into the wound. In turn, lower layers of Polyethylene oxide (PEO), CS, and HP, as well as Chitosan-Sodium Tripolyphosphate (CS-TPP), combined with PVA and Centella asiatica L. (CA) were produced to improve the biologic performance of these materials. Due to their properties, the lower layers demonstrated to be able to promote the healing process and inhibit the growth of S. aureus and P. aeruginosa without inducing any cytotoxic effect. However, the PVA_CS-TPP_CA revealed a higher bacterial inhibitory effect, reaching a 3 Log reduction. Finally, a cotton gauze bandage, traditionally used to provide support and confer robust protection against external threats, was successfully combined with PVA and CS nanofibers containing Agrimonia eupatoria L. (AG) to produce a nano-coating capable of inhibiting the growth of bacteria at the wound site and support skin regeneration. Overall, the scientific work performed in this thesis has been conducted to encourage the scientific community to give more attention to the potential benefits of bioactive natural products as medicinal plants, which exhibit a low tendency to develop bacterial resistance. Moreover, it has been shown that the use of relatively simple, versatile, and low-cost strategies to produce wound dressing materials displaying antimicrobial properties have an essential impact on the control of bacterial colonization but also prevent bacterial wound infection and consequently accelerate the healing process.

2023-02-03Doctoral thesis

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