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- Antimicrobial functionalization of wool: assessment of the effect of Cecropin-B and [Ala5]-Tritrp7 antimicrobial peptidesPublication . Mouro, Cláudia; Gouveia, Isabel C.This investigation provides a new strategy to impart antimicrobial properties into wool-based materials using Cecropin-B and [Ala5]-Tritrp7 antimicrobial peptides (AMPs). The process was conducted using exhaustion method at 40 °C for 1–3 h. The presence of the AMPs in the modified-wool samples was confirmed by colorimetric assay of Bradford reagent and possible changes in the morphology of the fibers and damage to its surface were analyzed by scanning electron microscopy. Results showed that 1 h were long enough for the functionalization to occur effectively and that the morphology of the fibers was not influenced by the functionalization process. Furthermore, the antimicrobial activity of the AMPs applied on wool was assessment by JIS L 1902-2002 against Staphylococcus aureus (ATCC 6538) and Klebsiella pneumoniae (ATCC 4352). The results showed that both AMPs have a high reduction in bacterial growth (Cecropin-B resulting in 71.67% reduction against S. aureus and 85.95% against K. pneumoniae. While [Ala5]-Tritrp7 resulting in 66.74% reduction against S. aureus and 88.65% against K. pneumoniae).
- Development of electrospun wound-dressings incorporating medicinal plant-extractsPublication . Mouro, Cláudia Filipa Duarte; Gouveia, Isabel Cristina Aguiar de Sousa e Silva; Fangueiro, Raul Manuel Esteves de SousaHuman 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.
- Emulsion Electrospun Fiber Mats of PCL/PVA/Chitosan and Eugenol for Wound Dressing ApplicationsPublication . Mouro, Cláudia; Simões, Manuel; Gouveia, Isabel C.In recent years, the damaging e ects of antimicrobial resistance relating to wound management and infections have driven the ongoing development of composite wound dressing mats containing natural compounds, such as plant extracts and their derivatives. e present research reports the fabrication of novel electrospun Polycaprolactone (PCL)/Polyvinyl Alcohol (PVA)/Chitosan (CS) ber mats loaded with Eugenol (EUG), an essential oil, known for its therapeutic properties. e electrospun ber mats were prepared via electrospinning from either water-in-oil (W/O) or oil-in-water (O/W) emulsions and characterized using scanning electron microscopy (SEM), Fourier-transform infrared spectroscopy (FT-IR), total porosity measurements, and water contact angle. e in vitro EUG release pro le and antibacterial activity against Staphylococcus aureus and Pseudomonas aeruginosa were also evaluated. e obtained results proved that the EUG was loaded e ciently into electrospun PCL/PVA/CS ber mats and the two W/O and O/W emulsions prepared from the PCL/PVA/CS (7 : 3 : 1) and PCL/PVA/CS (3 : 7 : 1) revealed porosity within the ideal range of 60–90%, even when EUG was loaded. e measured contact angle values showed that the O/W emulsion exhibited a more hydrophilic character and the wettability noticeably decreased a¢er adding EUG in both emulsion blends. Furthermore, the electrospun PCL/PVA/CS ber mats demonstrated a rapid release of EUG during the rst 8 hours, which enhanced gradually a¢erward (up to 120 hours). Moreover, an e cient antibacterial activity against S. aureus (inhibition ratios of 92.43% and 83.08%) and P. aeruginosa (inhibition ratios of 94.68% and 87.85%) was displayed and the in vitro cytotoxic assay demonstrated that the normal human dermal broblasts (NHDF) remained viable for at least 7 days, a¢er direct contact with the produced electrospun ber mats. erefore, such ndings support the biocompatibility and suitability of using these EUG-loaded electrospun PCL/PVA/CS ber mats as a new innovative wound dressing material with potential for preventing and treating microbial wound infections.
- Péptidos antimicrobianos: uma nova estratégia na funcionalização bioativa de fibras de lã e poliamidaPublication . Mouro, Cláudia Filipa Duarte; Gouveia, Isabel Cristina Aguiar de Sousa e SilvaOs materiais têxteis em condições de humidade e temperatura adequadas são um excelente substrato para a proliferação de microrganismos, particularmente bactérias e fungos podendo, em ambiente hospitalar, contaminar doentes e profissionais de saúde. Assim, o uso de materiais têxteis antimicrobianos pode reduzir significativamente o risco de infeções especialmente quando usados em contacto direto com a pele. Para isso, várias substâncias com características antimicrobianas têm sido utilizadas, no entanto, preocupações devido aos seus efeitos colaterais têm levado a uma procura crescente de agentes antimicrobianos naturais, como soluções eficazes, ecológicas e não tóxicas. Consequentemente, neste trabalho, uma nova estratégia prevê o uso de péptidos antimicrobianos (AMPs) como agentes bioativos para a potencial biofuncionalização de materiais têxteis. A cecropina-B, o [Ala5]-Tritrp7 e o Cys-LC-LL-37 foram os AMPs selecionados para conferir propriedades antimicrobianas aos materiais têxteis. Neste trabalho, os AMPs foram usados no tratamento da lã (fibra natural) e da poliamida 6.6 (PA 6.6) (fibra sintética) e a sua ligação às fibras foi confirmada pelo teste do reagente de Bradford em solução e na amostra. Nesse sentido, pelos resultados obtidos, processos de funcionalização de apenas 1 hora foram estabelecidos para a biofuncionalização ocorrer de forma eficaz. Além disso, possíveis alterações nas características estruturais da lã e da PA 6.6 foram analisadas por difração de raio-x (DRX) e comprovou-se que a composição macromolecular das fibras não foi influenciada pelo processo de biofuncionalização, de forma prejudicial. Foram também obtidas imagens de microscopia eletrónica de varrimento (SEM) que comprovaram que a ligação dos AMPs nas fibras não alterou nem provocou danos na sua superfície. Entre os resultados obtidos, é de realçar que as amostras de lã e de PA 6.6 funcionalizadas com os AMPs apresentaram percentagens de redução bacterianas elevadas. No caso da lã, as amostras funcionalizadas apresentaram uma redução no crescimento bacteriano para o Staphylococcus aureus de 71,67% para a cecropina-B e de 66,74% para o [Ala5]-Tritrp7, enquanto para Klebsiella pneumoniae houve uma redução no crescimento bacteriano de 85,95% para a cecropina-B e de 88,65% para o [Ala5]-Tritrp7. Relativamente à PA 6.6, a redução de crescimento para o Staphylococcus aureus, das amostras de PA 6.6 funcionalizadas, foi de 68,81% para a cecropina-B e de 73,81% para o Cys-LC-LL-37. Por outro lado, para a Klebsiella pneumoniae reduções de crescimento de 85,25% para a cecropina-B e de 87,31% para o Cys-LC-LL-37 foram alcançadas. Apesar do efeito antibacteriano dos AMPs ser promissor, procedeu-se também à avaliação da sua citotoxicidade. Os resultados demonstraram que os AMPs não são tóxicos nas condições testadas e que os materiais funcionalizados podem ser utilizados com segurança. Assim, os resultados revelaram que os novos processos conferem propriedades antimicrobianas inequívocas às fibras tratadas. Esse efeito foi preferencialmente visível para as bactérias em estudo e foi avaliado pela norma JIS L 1902-2002.
- Advances in the Production of Biomaterials through Kombucha Using Food Waste: Concepts, Challenges, and PotentialPublication . Cubas, Anelise Leal Vieira; Provin, A. P.; Dutra, Ana; Mouro, Cláudia; Gouveia, I.C.In recent years, several researchers have focused their studies on the development of sustainable biomaterials using renewable sources, including the incorporation of living biological systems. One of the best biomaterials is bacterial cellulose (BC). There are several ways to produce BC, from using a pure strain to producing the fermented drink kombucha, which has a symbiotic culture of bacteria and yeasts (SCOBY). Studies have shown that the use of agricultural waste can be a low-cost and sustainable way to create BC. This article conducts a literature review to analyze issues related to the creation of BC through kombucha production. The databases used were ScienceDirect, Scopus, Web of Science, and SpringerLink. A total of 42 articles, dated from 2018 to 2022, were referenced to write this review. The findings contributed to the discussion of three topics: (1) The production of BC through food waste (including patents in addition to the scientific literature); (2) Areas of research, sectors, and products that use BC (including research that did not use the kombucha drink, but used food waste as a source of carbon and nitrogen); and (3) Production, sustainability, and circular economy: perspectives, challenges, and trends in the use of BC (including some advantages and disadvantages of BC production through the kombucha drink).
- Antimicrobial and antioxidant surface modification toward a new silk-fibroin (SF)-l-Cysteine material for skin disease managementPublication . Nogueira, Frederico; Granadeiro, Luiza Breitenfeld ; Mouro, Cláudia; Gouveia, Isabel C.A novel dressing material – silk fibroin fabric (SF)-l-Cysteine (l-Cys) – is here developed to be used asstandard treatment for atopic dermatitis (AD), which combines comfort, thermic, and tensile strengthproperties of silk materials with antioxidant and antimicrobial effects of l-Cys. A careful understand-ing about the linking strategies is needed in order not to compromise the bioavailability of l-Cys anddeplenish its bioactivity. Durability was also addressed through washing cycles and compared with hos-pital requirements, according to international Standard EN ISO 105-C06:2010. The present research alsoanalyze the interactions between Staphylococcus aureus and SF-l-Cys under simulating conditions of ADand demonstrated the effectiveness of a double covalent grafting, with the importance of SF tyrosine(Tyr) covalent linkage with l-Cys (SF-g-l-Cys/Tyr-g-l-Cys) even after several washing cycles, twenty five,whereas for a disposable application a single covalent mechanism of grafting l-Cys proved to be suffi-cient (SF-g-l-Cys). Results showed effective antimicrobial activities exhibiting higher inhibition ratios of98.65% for SF-g-l-Cys after 5 washing cycles, whereas 97.55% for SF-g-l-Cys/Tyr-g-l-Cys after 25 washingcycles, both at pH 9.5 grafting strategy. Furthermore, it is also reported a non-protumoral effect of l-Cys. Anew advance is herein achieved at the world of medical antimicrobial textiles tailored to address woundmoisture environment and exudate self-cleaning, which may open novel applications as complementarytherapy for AD disease.