Biofunctionalization of natural fiber-reinforced biocomposites for biomedical applications

In the last ten years, environmental consciousness has increased worldwide, leading to the development of eco-friendly materials to replace synthetic ones. Natural fibers are extracted from renewable resources at low cost. Their combination with synthetic polymers as reinforcement materials has been an important step forward in that direction. The sustainability and excellent physical and biological (e.g., biocompatibility, antimicrobial activity) properties of these biocomposites have extended their application to the biomedical field. This paper offers a detailed overview of the extraction and separation processes applied to natural fibers and their posterior chemical and physical modifications for biocomposite fabrication. Because of the requirements for biomedical device production, specialized biomolecules are currently being incorporated onto these biocomposites. From antibiotics to peptides and plant extracts, to name a few, this review explores their impact on the final biocomposite product, in light of their individual or combined effect, and analyzes the most recurrent strategies for biomolecule immobilization.PTDC/CTM-TEX/28074/2017; Portuguese Foundation for Science and Technology (FCT), FEDER funds by means of Portugal 2020 Competitive Factors Operational Program (POCI) and the Portuguese Government(OE) by means of projects POCI-01-0145-FEDER-028074 and UID/CTM/00264/202

Antimicrobial action and clotting time of thin, hydrated poly(Vinyl Alcohol)/cellulose acetate films functionalized with LL37 for prospective wound-healing applications

Poly(vinyl alcohol)/cellulose acetate (PVA/CA) films were prepared via a new method that combines principles from solvent casting and phase inversion. To guarantee some degree of flexibility, films were produced with a higher percentage of PVA compared to CA, from 90/10 to 50/50. The antimicrobial peptide (AMP) LL37 was then anchored using dopamine as a binding agent. Films were characterized in terms of functional groups, thermal stability, tensile strength, porosity, swelling and degradation (stability in physiological media at different pHs). The antimicrobial performance of LL37 surface-modified films was tested against Staphylococcus aureus, Staphylococcus epidermidis and Escherichia coli in dynamic environment and in the presence and absence of an albumin interface. LL37 treated films demonstrated great antibacterial efficacy against the three kinds of bacteria, ≈ 75% inhibition for S. aureus, ≈ 85% for S. epidermidis and ≈ 60% for E. coli, regardless of PVA/CA ratio. Presence of albumin reduced bacteria inhibition in all tested groups, most likely due to the binding of the protein molecules to the antimicrobial agents, reducing the free fraction available for bacterial killing. Films treated with LL37 accelerated clotting time (≈ 10 min) above vancomycin and bare surfaces, demonstrating great capacity to activate the intrinsic coagulation cascade.Portuguese Foundation for Science and Technology (FCT), FEDER funds by means of Portugal 2020 Competitive Factors Operational Program (POCI) and the Portuguese Government (OE) for funding the project PEPTEX with reference POCI-01-0145-FEDER-028074. Authors also acknowledge project UID/CTM/00264/2019 of Centre for Textile Science and Technology (2C2T), funded by national funds through FCT/MCTE

Functionalization of crosslinked sodium alginate/gelatin wet-spun porous fibers with Nisin Z for the inhibition of Staphylococcus aureus-induced infections

Nisin Z, an amphipathic peptide, with a significant antibacterial activity against Gram-positive bacteria and low toxicity in humans, has been studied for food preservation applications. Thus far, very little research has been done to explore its potential in biomedicine. Here, we report the modification of sodium alginate (SA) and gelatin (GN) blended microfibers, produced via the wet-spinning technique, with Nisin Z, with the purpose of eradicating Staphylococcus aureus-induced infections. Wet-spun SAGN microfibers were successfully produced at a 70/30% v/v of SA (2 wt%)/GN (1 wt%) polymer ratio by extrusion within a calcium chloride (CaCl2) coagulation bath. Modifications to the biodegradable fibers’ chemical stability and structure were then introduced via crosslinking with CaCl2 and glutaraldehyde (SAGNCL). Regardless of the chemical modification employed, all microfibers were labelled as homogeneous both in size (≈246.79 µm) and shape (cylindrical and defect-free). SA-free microfibers, with an increased surface area for peptide immobilization, originated from the action of phosphate buffer saline solution on SAGN fibers, were also produced (GNCL). Their durability in physiological conditions (simulated body fluid) was, however, compromised very early in the experiment (day 1 and 3, with and without Nisin Z, respectively). Only the crosslinked SAGNCL fibers remained intact for the 28 day-testing period. Their thermal resilience in comparison with the unmodified and SA-free fibers was also demonstrated. Nisin Z was functionalized onto the unmodified and chemically altered fibers at an average concentration of 178 µg/mL. Nisin Z did not impact on the fiber’s morphology nor on their chemical/thermal stability. However, the peptide improved the SA fibers (control) structural integrity, guaranteeing its stability for longer, in physiological conditions. Its main effect was detected on the time-kill kinetics of the bacteria S. aureus. SAGNCL and GNCL loaded with Nisin Z were capable of progressively eliminating the bacteria, reaching an inhibition superior to 99% after 24 h of culture. The peptide-modified SA and SAGN were not as effective, losing their antimicrobial action after 6 h of incubation. Bacteria elimination was consistent with the release kinetics of Nisin Z from the fibers. In general, data revealed the increased potential and durable effect of Nisin Z (significantly superior to its free, unloaded form) against S. aureus-induced infections, while loaded onto prospective biomedical wet-spun scaffolds.This research received funding from the Portuguese Foundation for Science and Technology (FCT) under the scope of the projects PTDC/CTM-TEX/28074/2017 (POCI-01-0145-FEDER-028074) and UID/CTM/00264/2021

Combinatory action of chitosan-based blended films and loaded cajeput oil against Staphylococcus aureus and Pseudomonas aeruginosa-mediated infections

Chronic wounds (CW) have numerous entry ways for pathogen invasion and prosperity, damaging host tissue and hindering tissue remodeling. Essential oils exert quick and efficient antimicrobial (AM) action, unlikely to induce bacterial resistance. Cajeput oil (CJO) has strong AM properties, namely against Staphylococcus aureus and Pseudomonas aeruginosa. Chitosan (CS) is a natural and biodegradable cationic polysaccharide, also widely known for its AM features. CS and poly(vinyl alcohol) (PVA) films were prepared (ratio 30/70; 9 wt%) by solvent casting and phase inversion method. Films’ thermal stability and chemical composition data reinforce polymer blending. Films were supplemented with 1 and 10 wt% of CJO in relation to total polymeric mass. Loaded films were 23 and 57% thicker, respectively, than the unloaded films. Degree of swelling and porosity also increased, particularly with 10 wt% CJO. AM testing revealed that CS films alone were effective against both bacteria, eradicating all P. aeruginosa within the hour (*** p < 0.001). Still, loaded CS/PVA films showed improved AM traits, being significantly more efficient than unloaded films right after 2 h of contact. This study is the first proof of concept that CJO can be dispersed into CS/PVA films and show bactericidal effects, particularly against P. aeruginosa, this way opening new avenues for CW therapeutics.Authors acknowledge the Portuguese Foundation for Science and Technology (FCT), FEDER funds by means of Portugal 2020 Competitive Factors Operational Program (POCI) and the Portuguese Government (OE) for funding the project PEPTEX with reference PTDC/CTMTEX/28074/2017 (POCI-01-0145-FEDER-028074). Authors also acknowledge project UID/CTM/00264/2020 of Centre for Textile Science and Technology (2C2T), funded by national funds through FCT/MCTES

The Power of Specialized Biomolecules Against Bacteria

The high resistance of bacteria against conventional pharmaceutical solutions, the antibiotics, has raised serious global public-health concerns. This has stimulated interest in the research of bio-based therapeutics with limited resistance, namely, antimicrobial peptides (AMPs) or essential oils (EOs). This study envisaged the evaluation of the antimicrobial efficacy of selected biomolecules, namely LL37, pexiganan, tea tree oil (TTO), cinnamon leaf oil (CLO) and niaouli oil (NO), against four bacteria commonly associated to nosocomial infections: Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli and Pseudomonas aeruginosa. The antibiotic vancomycin and silver nanoparticles (AgNPs) were used as control for comparison purposes. The biomolecules were initially screened for their antibacterial activity using the agar-diffusion test, followed by the determination of minimal inhibitory concentrations (MICs), kill-time kinetics and the evaluation through scanning electron microscopy (SEM) observations of the cell morphology upon 24 h exposure. All agents were effective against the selected bacteria. Interestingly, the AgNPs required a higher concentration (4000–1250 µg/mL) to induce the same effects as the AMPs (500–7.8 µg/mL). Pexiganan was the most effective biomolecule, requiring lower concentrations to kill both Gram-positive and Gram-negative bacteria (62.5-7.8 µg/mL), within a short period of time (averaging 2 h 15 min for all bacteria). Most biomolecules apparently disrupted the bacteria membrane stability due to the observed cell morphology deformation and by effecting on the intracellular space. AMPs were seen to induce morphological deformations and cellular content release, while EOs were seen to split and completely envelope bacteria. Data unraveled more of the potential of these biomolecules and allowed to take a step forward in the understanding of their mechanisms of action against infection-related bacteria

Antibacterial activity of specialized biomolecules

The increased resistance of bacteria against conventional pharmaceutical solutions, the antibiotics, has raised serious health concerns. This has stimulated interest in the development of bio-based therapeutics with limited resistance, namely, essential oils (EOs) or antimicrobial peptides (AMPs). This study envisaged the evaluation of the antimicrobial efficacy of selected biomolecules, namely LL37, pexiganan, tea tree oil (TTO), cinnamon leaf oil (CLO) and niaouli oil (NO), against four bacteria commonly associated to nosocomial infections: Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli and Pseudomonas aeruginosa. The antibiotic vancomycin and silver nanoparticles (AgNPs) were used as control compounds for comparison purposes. The biomolecules were initially screened for their antibacterial efficacy using the agar-diffusion test, followed by the determination of minimal inhibitory concentrations (MICs), kill-time kinetics and the evaluation of the cell morphology upon 24 h exposure. All agents were effective against the selected bacteria. Interestingly, the AgNPs required a higher concentration (4000–1250 µg/mL) to induce the same effects as the AMPs (500–7.8 µg/mL). Pexiganan was the most effective biomolecule, requiring lower concentrations to kill both Gram-positive and Gram-negative bacteria (62.5-7.8 µg/mL), within a short period of time (averaging 2 h 15 min for all bacteria). Most biomolecules apparently disrupted the bacteria membrane stability due to the observed cell morphology deformation and by effecting on the intracellular space. AMPs were observed to induce morphological deformations and cellular content release, while EOs were seen to split and completely envelope bacteria. Data unraveled more of the potential of these new biomolecules as replacements for the conventional antibiotics and allowed us to take a step forward in the understanding of their mechanisms of action against infection-related bacteria.FEDER funds through COMPETE and by national funds through FCT via the projects POCI-01-0145-FEDER-028074 and UID/CTM/00264/2020. T.D.T also acknowledges FCT for PhD scholarship with reference 2020.06046.B

Eugenol-containing essential oils loaded onto Chitosan/Polyvinyl alcohol blended films and their ability to eradicate Staphylococcus aureus or Pseudomonas aeruginosa from infected microenvironments

Chronic wounds (CW) create numerous entryways for pathogen invasion and prosperity, further damaging host tissue and hindering its remodeling and repair. Essential oils (EOs) exert quick and efficient antimicrobial (AM) action, unlikely to induce bacterial resistance. Cinnamon leaf and clove oils (CLO and CO) display strong AM activity, namely against Staphylococcus aureus and Pseudomonas aeruginosa. Chitosan (CS) is a natural and biodegradable cationic polysaccharide, also widely known for its AM features. CS and poly (vinyl alcohol) (PVA) films were prepared (ratio 30/70 w/w; 9 wt%) by the solvent casting and phase inversion method. The film's thermal stability and chemical composition data reinforced polymer blending and EO entrapment. Films were supplemented with 1 and 10 wt% of EO in relation to total polymeric mass. The film thickness and degree of swelling (DS) tended to increase with EO content, particularly with 10 wt % CLO (* p < 0.05). UV-visible absorbance scans in the 250-320 cm-1 region confirmed the successful uptake of CLO and CO into CS/PVA films, particularly with films loaded with 10 wt% EO that contained 5.30/5.32 times more CLO/CO than films supplemented with 1 wt% EO. AM testing revealed that CS films alone were effective against both bacteria and capable of eradicating all P. aeruginosa within the hour (*** p < 0.001). Still, loaded CS/PVA films showed significantly improved AM traits in relation to unloaded films within 2 h of contact. This study is a first proof of concept that CLO and CO can be dispersed into CS/PVA films and show bactericidal effects, particularly against S. aureus, this way paving the way for efficient CW therapeutics.Portuguese Foundation for Science and Technol-ogy (FCT), FEDER funds by means of Portugal 2020 Competitive Factors Operational Program(POCI), and the Portuguese Government (OE) for funding the project PEPTEX with reference PTDC/CTM-TEX/28074/2017 (POCI-01-0145-FEDER-028074). The authors also acknowledge project UID/CTM/00264/2021 of the Centre for Textile Science and Technology (2C2T), funded by nationalfunds through FCT/MCTES. The authors also thankÂngela Silva for performing STA and DSCmeasurements and assisting with enthalpy calculations and FTIR set-up, Eng. Joaquim Jorge Peixotofor showing us how to use the film thickness meter, as well as to Eng. Paulo Madureira from IsazaScientific for clarifications regarding the employed UV-visible spectroscopy metho

Bacterial nanocellulose films loaded with nisin Z -antibacterial efficacy against Staphylococcus aureus strains

Burn wounds can lead to numerous severe complications including bacterial infections causing patient morbidity and mortality, mostly in low- and middle-income countries. The Grampositive bacteria Staphylococcus aureus is one of the major causes of nosocomial infections in burn patients. Furthermore, the considerable increase of the microbial resistance against traditional antibiotics is leading towards alternative strategies to treat bacterial infections. Nisin Z is an antimicrobial peptide which exhibits a significant antibacterial activity against Gram-positive bacteria. The incorporation of peptide and other biomolecules within a biopolymer matrix provides protection maintaining their antimicrobial potential. Bacterial nanocellulose (BNC) has been widely used as wound dressings. Its impressive water retention capacity (>99%) and porosity are beneficial to manage wounds due to its potential to absorb exudates, providing a breathable and humid environment. In this work, the functionalization of BNC with nisin Z (BNC-NZ) via vacuum filtration is reported. The entrapment of the peptide inside the BNC films was confirmed through morphological characterization using Attenuated Total Reflectance-Fourier Transform Infrared (ATR-FTIR) spectrometry. Typical absorbance peaks of nisin Z are easily identifiable at 1647 cm−1 (amide group) and 1520 cm-1 (bending of primary amines). Thermal Gravimetric Analysis (TGA) suggested that nisin Z did not interfere with the BNC matrix. The antimicrobial activity of nisin Z against S. aureus strains including a multiple drug-resistant, was verified by Minimum Bactericidal Concentration (MBC). Agar Diffusion and Shake Flask methods revealed the potential of BNC-NZ for prospective applications in burn wound dressings

Flexible, biodegradable LL37-anchored poly(vinyl alcohol)/cellulose acetate films for enhanced infection control

Wound care is a growing industry that lately has been facing multiple challenges due to the increasing health care costs, aging of population, appearance of antibiotic-resistant pathogens, and rise in the incidence of chronic diseases. Unlike acute wounds which heal in a predictable amount of time following the stages of healing, chronic wounds (CW) often fail to progress past the inflammatory phase, increasing costs and healing time. Bioactive dressings that incorporate drugs/antibiotics or bioactive molecules in their formulation have been suggested as alternatives to the conventional gauzes and foams. Here, we propose the combination of poly(vinyl alcohol) (PVA) and cellulose acetate (CA), both biodegradable and biocompatible polymers, for the production of films processed via a new method that combines principles from solvent casting and phase inversion, and modified with the antimicrobial peptide (AMP) LL37, as a new active solution. To guarantee some degree of flexibility, films were produced with a higher percentage of PVA compared to CA, from 90/10 to 50/50. LL37 was then anchored using dopamine as a binding agent. Films were characterized in terms of functional groups, thermal stability, tensile strength, porosity, swelling and degradation rate. The antimicrobial performance of LL37 surface-modified films was tested against Staphylococcus aureus, Staphylococcus epidermidis and Escherichia coli in dynamic environment. LL37-modified films demonstrated great antibacterial efficacy against the three bacteria, ≈ 75% inhibition for S. aureus, ≈ 85% for S. epidermidis and ≈ 60% for E. coli, regardless of PVA/CA ratio. Films treated with LL37 accelerated clotting time (≈ 10 min) above vancomycin and bare surfaces, demonstrating great capacity to activate the intrinsic coagulation cascade. In the end, the potential of LL37 functionalized PVA/CA films for prospective wound-healing applications was demonstrated

Antimicrobial activity of a bacterial nanocellulose film functionalized with Nisin Z for prospective burn wounds treatment

Burn wounds can lead to numerous severe complications including bacterial infections causing patient morbidity and mortality, mostly in low- and middle-income countries. The considerable increase of the microbial resistance against traditional antibiotics is leading towards alternative strategies to treat bacterial infections. Nisin Z is an antimicrobial peptide which exhibits a significant antibacterial activity against Gram-positive bacteria. Its efficacy against Gram-negative bacteria is limited, nonetheless it can be improved with the addition of surfactants, such as ethylenediaminetetraacetic acid (EDTA). The incorporation of peptide and other biomolecules within a biopolymer matrix provides protection maintaining their antimicrobial potential. Bacterial nanocellulose (BNC) has been widely used as wound dressings. Its impressive water retention capacity (> 99 %) and porosity are beneficial to manage wounds due to its potential to absorb exudates, providing a breathable and humid environment. In this work, the functionalization of BNC with Nisin Z (BNC-NZ) via vacuum filtration is reported. The entrapment of the peptide inside the BNC films was confirmed through morphological characterization using Attenuated Total Reflectance-Fourier Transform Infrared (ATR-FTIR) spectrometry. Typical absorbance peaks of Nisin Z are easily identifiable at 1647 cm-1 (amide group) and 1520 cm-1 (bending of primary amines). Thermal Gravimetric Analysis (TGA) suggested that Nisin Z did not interfere with the BNC matrix. The antimicrobial activity of Nisin Z against five of the most common bacteria found in burn wounds was verified by Minimum Bactericidal Concentration (MBC) ranging 8.0-256.0 µg/mL. Agar Diffusion and Shake Flask methods revealed the potential of BNC-NZ for prospective applications in burn wound dressings.This work is financed by FEDER funds through COMPETE and by national funds through FCT via the projects POCI-01-0145-FEDER-028074 and UID/CTM/00264/2020. L.M. and T.D.T also acknowledge FCT for their PhD scholarships with references 2020.04919.BD and 2020.06046.BD