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- Advanced face mask filters based on PCL electrospun meshes dopped with antimicrobial MgO and CuO nanoparticlesPublication . Ferreira, Carolina A. M.; Guerreiro, Sara F.C.; Valente, Joana F. A.; Patrício, Tatiana M.F.; Alves, Nuno; Mateus, Artur; Dias, Juliana R.The pandemic situation caused by coronavirus clearly demonstrated the need for alternatives able to protect the respiratory tract and inactivate the infectious agents. Based on this, antibacterial face-mask filters of polycaprolactone (PCL) dopped with magnesium oxide (MgO) and copper oxide (CuO) nanoparticles (NPs) were produced using an electrospinning technique. A morphological analysis of electrospun meshes evaluated the success of nanoparticles’ incorporation as well as the average fibers’ diameters (481 +- 272 nm). The performance of electrospun nanofibers was also assessed in terms of tensile strength (0.88 +- 0.25 MPa), water vapor permeability (11,178.66 +- 35.78 g.m-2.day-1), stability under wet conditions and antibacterial activity according to the standard guidelines. The filters showed structural stability up to 2 h of washing and improved antibacterial activity against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) for optimized concentrations of MgO and CuO NPs. Overall, electrospun meshes with antibacterial activity were successfully developed for advanced filtering applications.
- A biorefinery approach to the biomass of the seaweed Undaria pinnatifida (Harvey Suringar, 1873): obtaining phlorotannins-enriched extracts for wound healingPublication . Ferreira, Carolina A. M.; Félix, Rafael; Félix, Carina; Januário, Adriana P.; Alves, Nuno; Novais, Sara C.; Dias, Juliana R.; Lemos, Marco F.L.Brown seaweeds are recognized sources of compounds with a wide range of properties and applications. Within these compounds, phlorotannins are known to possess several bioactivities (e.g., antioxidant, anti-inflammatory, and antimicrobial) with potential to improve wound healing. To obtain phlorotannins enriched extracts from Undaria pinnatifida, a biorefinery was set using lowcost industry-friendly methodologies, such as sequential solid–liquid extraction and liquid–liquid extraction. The obtained extracts were screened for their antioxidant and antimicrobial activity against five common wound pathogens and for their anti-inflammatory potential. The ethanolic wash fraction (wE100) had the highest antioxidant activity (114.61 +- 10.04 mmol mg-1 extract by Diphenyl-1-picrylhydrazyl (DPPH) and 6.56 +-1.13mMeq. Fe II mg-1 extract by and Ferric Reducing Antioxidant Power (FRAP)), acting efficiently against Gram-negative (Pseudomonas aeruginosa) and Gram-positive (Staphylococcus aureus) bacteria, and showing a nitric oxide production inhibition over 47% when used at 0.01 ug mL-1. NMR and FTIR chemical characterization suggested that phlorotannins are present. Obtained fraction wE100 proved to be a promising candidate for further inclusion as wound healing agents, while the remaining fractions analyzed are potential sources for other biotechnological applications, giving emphasis to a biorefinery and circular economy framework to add value to this seaweed and the industry.
- Multifunctional Gelatin/Chitosan Electrospun Wound Dressing Dopped with Undaria pinnatifida Phlorotannin-Enriched Extract for Skin RegenerationPublication . Ferreira, Carolina A. M.; Januário, Adriana P.; Félix, Rafael; Alves, Nuno; Lemos, Marco F.L.; Dias, Juliana R.The similarities of electrospun fibers with the skin extracellular matrix (ECM) make them promising structures for advanced wound dressings. Moreover, infection and resistance in wounds are a major health concern that may be reduced with antibacterial wound dressings. In this work, a multifunctional wound dressing was developed based on gelatin/chitosan hybrid fibers dopped with phlorotannin-enrich extract from the seaweed Undaria pinnatifida. The intrinsic electrospun structure properties combined with the antimicrobial and anti-inflammatory properties of phlorotannin-enrich extract will enhance the wound healing process. Electrospun meshes were produced by incorporating 1 or 2 wt% of extract, and the structure without extract was used as a control. Physico-chemical, mechanical, and biological properties were evaluated for all conditions. Results demonstrated that all developed samples presented a homogenous fiber deposition with the average diameters closer to the native ECM fibrils, and high porosities (~90%) that will be crucial to control the wound moist environment. According to the tensile test assays, the incorporation of phlorotannin-enriched extract enhances the elastic performance of the samples. Additionally, the extract incorporation made the structure stable over time since its in vitro degradation rates decreased under enzymatic medium. Extract release profile demonstrated a longstanding delivery (up to 160 days), reaching a maximum value of ~98% over time. Moreover, the preliminary antimicrobial results confirm the mesh’s antimicrobial activity against Pseudomonas aeruginosa and Staphylococcus aureus. In terms of biological characterization, no condition presented cytotoxicity effects on hDNF cells, allowing their adhesion and proliferation over 14 days, except the condition of 2 wt% after 7 days. Overall, the electrospun structure comprising phlorotannins-enriched extract is a promising bioactive structure with potential to be used as a drug delivery system for skin regeneration by reducing the bacterial infection in the wound bed.
- A Biorefinery Approach to the Biomass of the Seaweed Undaria pinnatifida (Harvey Suringar, 1873): Obtaining Phlorotannins-Enriched Extracts for Wound HealingPublication . Ferreira, Carolina A. M.; Félix, Rafael; Félix, Carina; Januário, Adriana P.; Alves, Nuno; Novais, Sara C.; Dias, Juliana R.; Lemos, Marco F.L.Brown seaweeds are recognized sources of compounds with a wide range of properties and applications. Within these compounds, phlorotannins are known to possess several bioactivities (e.g., antioxidant, anti-inflammatory, and antimicrobial) with potential to improve wound healing. To obtain phlorotannins enriched extracts from Undaria pinnatifida, a biorefinery was set using low cost industry-friendly methodologies, such as sequential solid–liquid extraction and liquid–liquid extraction. The obtained extracts were screened for their antioxidant and antimicrobial activity against five common wound pathogens and for their anti-inflammatory potential. The ethanolic wash fraction (wE100) had the highest antioxidant activity (114.61 ± 10.04 mmol·mg−1 extract by Diphenyl-1-picrylhydrazyl (DPPH) and 6.56 ± 1.13 mM eq. Fe II·mg−1 extract by and Ferric Reducing Antioxidant Power (FRAP)), acting efficiently against Gram-negative (Pseudomonas aeruginosa) and Gram-positive (Staphylococcus aureus) bacteria, and showing a nitric oxide production inhibition over 47% when used at 0.01 µg·mL−1 . NMR and FTIR chemical characterization suggested that phlorotannins are present. Obtained fraction wE100 proved to be a promising candidate for further inclusion as wound healing agents, while the remaining fractions analyzed are potential sources for other biotechnological applications, giving emphasis to a biorefinery and circular economy framework to add value to this seaweed and the industry.
- Multifunctional gelatin/chitosan electrospunwound dressing dopped with Undaria pinnatifida phlorotannin-enriched extract for skin regenerationPublication . Ferreira, Carolina A. M.; Januário, Adriana P.; Félix, Rafael; Alves, Nuno; Lemos, Marco F. L.; Dias, Juliana R.The similarities of electrospun fibers with the skin extracellular matrix (ECM) make them promising structures for advanced wound dressings. Moreover, infection and resistance in wounds are a major health concern that may be reduced with antibacterial wound dressings. In this work, a multifunctional wound dressing was developed based on gelatin/chitosan hybrid fibers dopped with phlorotannin-enrich extract from the seaweed Undaria pinnatifida. The intrinsic electrospun structure properties combined with the antimicrobial and anti-inflammatory properties of phlorotannin-enrich extract will enhance the wound healing process. Electrospun meshes were produced by incorporating 1 or 2 wt% of extract, and the structure without extract was used as a control. Physico-chemical, mechanical, and biological properties were evaluated for all conditions. Results demonstrated that all developed samples presented a homogenous fiber deposition with the average diameters closer to the native ECM fibrils, and high porosities (~90%) that will be crucial to control the wound moist environment. According to the tensile test assays, the incorporation of phlorotannin-enriched extract enhances the elastic performance of the samples. Additionally, the extract incorporation made the structure stable over time since its in vitro degradation rates decreased under enzymatic medium. Extract release profile demonstrated a longstanding delivery (up to 160 days), reaching a maximum value of ~98% over time. Moreover, the preliminary antimicrobial results confirm the mesh’s antimicrobial activity against Pseudomonas aeruginosa and Staphylococcus aureus. In terms of biological characterization, no condition presented cytotoxicity effects on hDNF cells, allowing their adhesion and proliferation over 14 days, except the condition of 2 wt% after 7 days. Overall, the electrospun structure comprising phlorotannins-enriched extract is a promising bioactive structure with potential to be used as a drug delivery system for skin regeneration by reducing the bacterial infection in the wound bed.
- Incorporating Undaria pinnatifida ((Harvey) Suringar 1873) bioactive extracts in electrospun wound dressingsPublication . Ferreira, Carolina Alexandra Marzia; Lemos, Marco Filipe Loureiro; Dias, Juliana RosaWounds are a worldwide health concern, as result of resistance to most antibiotics and inappropriate dressings. Therefore, effective treatment against bacterial infections remains a challenge, and new wound healing products with antimicrobial activity are very required. Thus, new natural products would be useful as therapeutic wounds, like, phlorotannins, since have reported antimicrobial, antioxidant, anti-inflammatory, UV-protective activities, among many others. Electrospun fibers are much appreciated in wound healing due to its similarities to the extracellular matrix. In this work, phlorotannins-enriched extracts from invasive Undaria pinnatifida ((Harvey) Suringar 1873) were produced and analyzed to incorporate into electrospun wound dressings. In the first part, a screening was performed, using different solvents and applying liquid-liquid fractionation. After the first outputs results, the extraction pipeline was optimized to produce 6 extracts (H, AQAE, AQs, E50, 100H, and E100s). Analytical procedures such as DPPH (2,2’ – Diphenyl-1-picryldydrazyl radical), FRAP (Ferric Antioxidant Power), TPC (Total Phenolic Content) and antimicrobial assays like microdilution were performed. The results suggest that there are two fractions that possibly have higher content in phlorotannins: AQAE and E100s. However, E100s has the highest antioxidant activity (114.61 mg.mol-1 for DPPH, and 6.56 mM eq. Fe II.mg-1 extract for FRAP), while AQAE shows 59.54 mg.mol-1 for DPPH and 3.49 mM eq. Fe II.mg-1 extract for FRAP. As well as TPC values with 0.47 eq. PGL.mg-1 versus 0.24 eq. PGL.mg-1 for AQAE. Concerning the antimicrobial activity, AQAE inhibits completely the Gram-positive Staphylococcus aureus, while E100s acts efficiently against overall wound Gram-negative and Gram-positives. Cellular effects were also evaluated as a proxy for in vivo safety, with no adverse results recorded. FTIR-ATR (Fourier-Transform Infrared spectroscopy with Attenuated Total Reflectance) and NMR (Nuclear Magnetic Resonance) characterization demonstrate that phlorotannins are present in E100s fraction, over AQAE. Consequently, E100s was selected to be incorporated in electrospun meshes. In the second part of the dissertation, 1 and 2 wt% of E100s extract was incorporated after parameters optimization for a solution with 14 wt% gelatin and 3.6 wt% of chitosan, 70% v/v of glacial Acetic Acid /2% v/v of triethylamine. and 4% v/v of BDDGE (1,4 – Butanonediol diglycydil ether). The ideal parameters achieved were a high tension of 20 kV, 0.2 mL.h-1 of flow rate and 12 cm from tip-to-collector. Physicochemical properties of electrospun meshes, including porosity, density, water contact angle, water absorption, and mechanical properties were analysed. Results showed that increasing the amount of phlorotannins-enriched extract influence the porosity, density, as well water uptake, due to the compact structure and owing to the hydrophilicity of phlorotannins which, resulting in better mechanical properties, delaying the enzymatic degradation and presenting a two-stages extract release effect. Thus, gelatin/chitosan electrospun meshes comprising phlorotannins-enrich extract are promising bioactive structures with potential to be used as a drug delivery system for skin tissue regeneration.
- Producing cyanobacteria to use as biostimulantsPublication . Mouga, Teresa; Simões, Fátima; Moreira, Vitória; Martins, Alice; Ferreira, Carolina; Ramos, Rute; Afonso, CléliaBy 2050, the world’s population will reach about 10 billion people, which will the need for food by about 60% more than is available today. To meet this need, new processes are emerging to allow more efficient and sustainable intensification of agriculture. In this context, the use of biostimulants has received increasing attention, due to their ability to improve plant quality and yield, stress tolerance, and/or improve the availability of nutrients in the soil. When it comes to Cyanobacteria, the preferred ones are N2-fixing species, which increase organic nitrogen in the soil, and produce hygroscopic exopolysaccharides, which alter the distribution of water by reducing soil hydrophobicity. Therefore, they decrease soil evaporation and retain moisture, improving soil stability. Wild strains of filamentous N2 fixing Nostoc 135, Nostoc 136, Nostoc 137, and Anabaena 4, provided by Alga2O Lda., were grown in the laboratory with the aim of selecting those that presented the necessary physiological characteristics to be efficiently grown in the laboratory. Therefore, the growth rate, productivity, and exopolysaccharides (EPS) contents were analysed. The EPS obtained from each strain culture (3% g/L) was applied to lettuce seeds (Lactuca sativa), to determine the germination rate, primary root length and number of primary leaves produced. As confirmed by other authors, Cyanobacteria are interesting biostimulants regarding germination rates. Other effects to be tested in the future are heat and drought tolerance, water, nutrients, essential oil, and pigment content, all of which are positive aspects that have been previously reported for other Nostoc and Anabaena species.