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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).
- Amino Acid-Based Material for the Complementary Therapy of Decubitus UlcersPublication . Nogueira, Frederico; Gouveia, Isabel C.Chronic wounds, pressure sores, lesions, and infections of microbial origin in bedridden, paralyzed, or malnutrition patients remain the object of study of many researchers. A variety of factors behind the development of these disorders are related to the patient's immune system, making it unable to respond effectively to the treatment of the wound. These factors can be properly controlled, giving particular importance to the ethiology and stage of the wound, as well as the time periods corresponding to the replacement of the dressings. The present research reports a novel foam/soft material, L-Cys-g-PCL, with an application for decubitus/pressure ulcers, especially for wounds with a difficult healing process due to infections and constant oxidation of the soft tissues. During this work, the interactions between S. aureus and L-Cys-g-PCL foam were studied under conditions that simulate decubitus ulcers; namely, pH and exudate. The effects of duration of grafting (1 or 8 h) and pH (7.0 and 8.9) on wettability, surface energy, swelling, and porosity were also evaluated. Results showed an effective microbicidal activity exhibiting an inhibition ratio of 99.73% against S. aureus. This new L-Cys-g-PCL soft material showed saftey to contact skin, ability to be shaped to fill in sunken holes (craters) - pressure ulcers stage III - and to act as a smart material responsive to pH, which can be tailored to develop better swelling properties at alkaline pH where exudates are normally higher, so as to address exudate self-cleaning and prevention of desiccation..
- Electrospinning polypropylene with an amino acid as a strategy to bind the antimicrobial peptide Cys-LC-LL-37Publication . Nogueira, Frederico; Teixeira, Pilar; Gouveia, Isabel C.Hospital isolation gowns are increasingly competitive, with brands and manufacturers contesting consumer preferences. The textile materials in contact with the skin can acquire secretions and multiresistant microorganisms, causing discomfort and health risks, respectively. A new nanofibrous substrate— polypropylene grafted with L-Cys—was developed with an increased crystallinity, providing its surface with –SH hooks necessary to efficiently cross-link the antimicrobial peptide Cys-LC-LL-37 in order to protect against nosocomial pathogens and their spread to community. Furthermore, this application does not require environmental control of humidity, and it is not susceptible to enzyme and microorganism degradation.
- 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.
- Immobilization of bacteriophage in wound-dressing nanostructurePublication . Nogueira, Frederico; Karumidze, Natia; Kusradze, Ia; Goderdzishvili, Marina; Teixeira, Pilar; Gouveia, Isabel C.Opportunistic bacteria that cause life-threatening infections are still a central problem associated with a healthcare setting. Bacteriophage capsid immobilization on nanostructured polymers maximizes its tail exposure and looks promising in applications toward skin-infections as alternative to antibiotics standardly used. The main goal of this work was to investigate the covalent immobilization of vB_Pae_Kakheti25 bacteriophage capsid on polycaprolactone (PCL) nanofibers (non-woven textile), as a potential effective antimicrobial, laundry resistant and non-toxic dressing for biomedical use. Surface analyses showed that the immobilization of vB_Pae_Kakheti25 bacteriophage capsid on PCL nanofibres oriented bacteriophage tails to interact with bacteria. Furthermore, antimicrobial assays showed a very effective 6 log bacterial reduction, which was equivalent to 99.9999%, after immediate and 2 hours of contact, even following 25 washing cycles (due to covalent bond). The activity of PCL-vB_Pae_Kakheti25 against P. aeruginosa was immediate and its reduction was complete.
- Synergistically enhanced stability of laccase immobilized on synthesized silver nanoparticles with water-soluble polymersPublication . Cunha, M. N. M.; Felgueiras, Helena Prado; Gouveia, Isabel C.; Andrea, ZilleSilver nanoparticles (AgNPs) were synthesized by citrate reduction method in the presence of polymers, poly(ethylene glycol) (PEG), poly(vinyl alcohol) (PVA) and chitosan, used as stabilizing agents, and an oxidoreductase enzyme, laccase (Lac), with the goal of expanding the NPs antimicrobial action. AgNPs were characterized by UV-vis spectrometry, dynamic light scattering and transmission electron microscopy. As protecting agents, PEG and PVA promoted the formation of spherical uniformly-shaped, small-sized, monodispersed AgNPs (≈20nm). High Mw polymers were established as most effective in producing small-sized NPs. Chitosan's viscosity led to the formation of aggregates. Despite the decrease in Lac activity registered for the hybrid formulation, AgNPs-polymer-Lac, a significant augment in stability over time (up to 13days, at 50°C) was observed. This novel formulation displays improved synergistic performance over AgNPs-Lac or polymer-Lac conjugates, since in the former the Lac activity becomes residual at the end of 3days. By enabling many ionic interactions, chitosan restricted the mass transfer between Lac and substrate and, thus, inhibited the enzymatic activity. These hybrid nanocomposites made up of inorganic NPs, organic polymers and immobilized antimicrobial oxidoreductive enzymes represent a new class of materials with improved synergistic performance. Moreover, the Lac and the AgNPs different antimicrobial action, both in time and mechanism, may also constitute a new alternative to reduce the probability of developing resistance-associated mutations.
- Layer-by-Layer Deposition of Antibacterial Polyelectrolytes on Cotton FibresPublication . Gomes, Ana; Mano, João F.; Queiroz, João; Gouveia, Isabel C.The introduction of molecules with biological properties on textile materials is essential for a number of biotechnological applications. With the purpose of testing new processes applied to textiles, in this study, we present the first results on the feasibility of using the Layerby-Layer (LbL) deposition process in natural fibers such as cotton, with natural polyelectrolytes like chitosan (CH) and alginic acid sodium salt (ALG), the durability of CH/ALG multilayer on cotton were evaluated. The increase of negative charges to the substrate cotton was made with NaBr and TEMPO, to ensure the success of the process of LbL. Three characterization methods to assess electrostatic LbL deposition were performed: the contact angle between a liquid (water) and the sample surface, in order to characterize the wettability of the samples with the different layers of CH and ALG; dyeing of the CH/ALG assembled cotton fabric with cationic methylene blue that shows regular changes in terms of color depth (K/S value), which indicate that the surface were alternately deposited with CH and ALG layers and, finally, the analysis by infrared spectroscopy using Fourier Transform with Attenuated Total Reflection (ATR-FTIR), to assess the changes in the interaction between CH and ALG deposited on cotton samples.
- New garment proposal for prevention of spreading Gram-negative bacteria resistant to carbapenem antibiotic class under hospital settingsPublication . Nogueira, Frederico; Gomes, Ana; Gouveia, Isabel C.Sensitive skin diseases, including atopic dermatitis, skin inflammation and bedsores, leave patients vulnerable under hospital setting. It is important for the development of a hospital gown with ‘‘soft hand’’ properties and at the same time as a protector against nosocomial infections. Klebsiella pneumoniae has developed resistance to antibiotics in the carbapenem antibiotic class, known as carbapenem-resistant K. pneumoniae (CRKP). CRKP is resistant to nearly all antibiotics and can kill up to 50% of infected patients. This work consisted in the development of a washable recycled silk fibroin-based gown covalently linked with an amino acid L-Cysteine(L-Cys), focused on prevention of K. pneumoniae establishment, proliferation and spreading to community, for use under hospital settings. With the growing problem of resistance to antibiotics and few new therapies on the horizon, gowns adsorbed with L-Cys show to function as a barrier to the establishment and proliferation of microorganisms, providing user protection from infectious disease. This gown was knitted at a rectilinear needle loom with a Jersey knit structure. Then it was cross-linked with L-Cys, subjected to laundry, and subsequently characterized by energy-dispersive X-ray spectroscopy, Fourier transform infrared spectroscopy, contact angle, free energy of adhesion, scanning electron microscopy and transmission electron microscopy. Results presented a bactericidal effect against K. pneumoniae of 94.92% after three rinses and 88.88% after five washing cycles, with the few adhered bacteria with an altered and compromised morphology.
- Layer-by-layer deposition of antimicrobial polymers on cellulosic fibers: a new strategy to develop bioactive textilesPublication . Gomes, Ana; Mano, João F.; Queiroz, João; Gouveia, Isabel C.In recent years, there has been an increase of infectious diseases caused by different microorganisms and the development of antibiotic resistance. In this way, the search for new and efficient antibacterial materials is imperative. The main polysaccharides currently used in the biomedical and pharmaceutical domains are chitin and its derivative chitosan (CH) and alginates (ALG). In this study, a simple technique of Layer by Layer (LbL) of applying polycation CH and polyanion ALG was used to prepare CH/ALG multilayers on cotton samples via the electrostatic assembly with success. The CH/ALG cotton samples (functionalized) were investigated for their antibacterial properties towards Staphylococcus aureus and Klebsiella pneumonia using the international standard method JIS L 1902:2002. The antibacterial activity of the functionalized samples was tested in terms of bacteriostatic and bactericidal activity, and results showed that the samples exhibited a bacteriostatic effect on the two bacteria tested, as expected. In addition, samples with five layers (CH/ALG/CH/ALG/CH) were more effective in inhibiting bacterial growth. This new coating for cellulosic fibers is a new strategy and may open new avenues for the development of antimicrobial polymers with potential application in health-care field.
- Incorporation of antimicrobial peptides on functionalized cotton gauzes for medical applicationsPublication . Gomes, Ana; Mano, João F.; Queiroz, João; Gouveia, Isabel C.A large group of low molecular weight natural compounds that exhibit antimicrobial activity has been isolated from animals and plants during the past two decades. Among them, peptides are the most widespread resulting in a new generation of antimicrobial agents with higher specific activity. In the present study we have developed a new strategy to obtain antimicrobial wound-dressings based on the incorporation of antimicrobial peptides into polyelectrolyte multilayer films built by the alternate deposition of polycation (chitosan) and polyanion (alginic acid sodium salt) over cotton gauzes. Energy dispersive X ray microanalysis technique was used to determine if antimicrobial peptides penetrated within the films. FTIR analysis was performed to assess the chemical linkages, and antimicrobial assays were performed with two strains: Staphylococcus aureus (Gram-positive bacterium) and Klebsiella pneumonia (Gram-negative bacterium). Results showed that all antimicrobial peptides used in this work have provided a higher antimicrobial effect (in the range of 4 log-6 log reduction) for both microorganisms, in comparison with the controls, and are non-cytotoxic to normal human dermal fibroblasts at the concentrations tested.