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Research Project
COVALENT AND NON-COVALENT STRATEGIES FOR SURFACE MODIFICATION OF DIFFERENT TEXTILE MATERIALS WITH ANTIMICROBIAL PROPERTIES
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Antimicrobial and antioxidant surface modification toward a new silk-fibroin (SF)-l-Cysteine material for skin disease management
Publication . 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.
Immobilization of bacteriophage in wound-dressing nanostructure
Publication . 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.
Covalent and non-covalent strategies for surface modification of different textile materials with antimicrobial properties
Publication . Nogueira, Frederico Álvaro Sequeira; Gouveia, Isabel Cristina Aguiar de Sousa e Silva a; Teixeira, Erhan Piskin e Doutora Pilar
Nowadays, millions of people become infected with bacteria that cause hospital infections,
which is a major cause of mortality in hospitals, killing 700,000 people per year in the world.
It is even projected that the number of deaths in hospitals will grow to 10 million by 2050.
The use of antimicrobial textiles, especially in close contact with the patients and in the
immediate and non-immediate surroundings, may significantly reduce the risk of infections.
However, they should possess broad spectrum biocidal properties, be safe for use and highly
effective against antibiotic resistant microorganisms, including those that are commonly
involved in hospital-acquired infections.
Most nosocomial infections are primarily by opportunistic microorganisms, i. e., they rarely
cause diseases in a healthy immune system, but seek to exploit any weaknesses in the body of
immunocompromised patients, such as victims of burns, cancer patients or beddriden with
open wounds, in order to cause infections. These strains have the ability to grow in any
environment, present important virulence factors, and have resistance to a large variety of
antibiotics.
Several antimicrobial agents have been tested in textiles. Quaternary ammonium compounds,
silver, polyhexamethylene biguanides and triclosan have been used, with limited success.
They have powerful bactericidal activity, however, the majority have a reduced spectrum of
microbial inhibition and may cause skin irritation, citotoxicity, ecotoxicity and bacterial
resistance. In addition, its incorporation in the textiles reduces their activity substantially and
limits availability. Moreover, the biocide can gradually lose activity during the use and textile
repeated laundering.
To overcome these disadvantages, natural compounds such as L-Cysteine (L-Cys),
bacteriophages and antimicrobial peptides (AMPs), were tested in this work as antimicrobial
agents for fibrous materials.
As such, in a first approach we carried out studies in order to confer antimicrobial properties
on textile and polymeric surfaces in such a way that they could irreversibly attract, bind and
eliminate microorganisms, paving the way to a dynamic protective barrier. For this purpose,
the amino acid L-Cys and the AMPs Magainin I, LL-37, and Cys-LC-LL-37 were used in order to
provide antimicrobial properties to cotton fibrous materials . L-Cys was selected due to its
proven antimicrobial properties granted by its thiol group and also proved its capacity to
ensure antioxidant activity by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) reagent. Covalent and
non-covalent immobilization strategies were tested on different fibrous materials and
subjected to intensive washing cycles, such as cotton, silk, polycaprolactone, and
polypropylene, in order to immobilize L-Cys in a durable manner. For a better understanding
of the interactions material-L-Cys-bacteria, cotton textile substrates were chemically modified with N, N-carbonyldiimidazole (CDI) and subsequently functionalized with different
concentrations of L-Cys. These studies revealed that there was a specific amount of CDI
activator (4%) which would be ideal to more efficiently bind L-Cys (5%). These results
revealed a higher antimicrobial efficiency, when compared to another study, in which the
cotton substrate was non-covalently immobilized with Magainin I and LL-37. Cotton-L-Cys
caused most death among bacteria, after washing cycles, due exclusively to its covalent
bound that was able to immobilize L-Cys more permanently. In support of this hypothesis, a
polymer difficult to modify - polypropylene - was grafted with L-Cys, which strengthened its
nanostructure and endowed it with thiol groups that allowed to bind the peptide Cys-LC-LL-37
via disulfide bond (covalent). It was found that Cys-LC-LL-37 resisted to successive wash
cycles, and the flexibility of this peptide was unique to the elimination of the
microorganisms.
Subsequently, the knowledge acquired when using cotton and polypropylene were transferred
to silk and polycaprolactone, in order to test the applicability of this developed concept to
other fibrous structures potentially to be used as antimicrobial textiles. Different percentages
of L-Cys were immobilized, by different chemical reactions, on samples of aforementioned
polymers with biomedical potential, and X-ray spectroscopy (EDS), Fourier transform infrared
spectroscopy (FT-IR), calorimetry (DSC), Ellman's reagent, and contact angle were used to
chemically check L-Cys immobilization, as well as antimicrobial and cytotoxicity assays, so as
to ensure that the applications would not be toxic to humans. Also, silk and polycaprolactone
samples covalently bound by 1 and 5% L-Cys, respectively, eliminated very well the
microorganisms. In addition, these samples retained L-Cys during several wash cycles.
At this stage, after the work developed and the knowledge acquired, enabled us to move into
a new strategy of immobilization of bacteriophages in fibrous materials. The covalent
coupling of the vB-Pae-Kakheti phage capsid to the surface of polycaprolactone nanofibers
produced by electrospinning was performed, so that the phage had its tail facing outwards,
maintaining its infectivity. The results again confirm that not only the presence of an
antimicrobial, but also the way it is immobilized, makes all the difference in the development
strategy of antimicrobial textiles.
It was concluded, therefore, that an optimized amount of "new" antimicrobial compounds
alternative to antibiotics and synthetic biocides, as well as their specific orientation,
consisted of a better performance upon contact and elimination of bacteria, being crucial for
the development of biomaterials for contact with skin and mucosa.
Amino Acid-Based Material for the Complementary Therapy of Decubitus Ulcers
Publication . 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..
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Funding agency
Fundação para a Ciência e a Tecnologia
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Funding Award Number
SFRH/BD/91444/2012