Bottom-up Layer-by-Layer Assembling of Antibacterial Freestanding Nanobiocomposite Films.

In this study, freestanding nanobiocomposite films were obtained by the sequential deposition of biopolymer-capped silver nanoparticles (AgNPs) and hyaluronic acid (HA). At first, dispersions of AgNPs decorated with chitosan (CS) or aminocellulose (AC) were synthesized by applying high intensity ultrasound. These polycationic nanoentities were layer-by-layer assembled with the HA polyanion to generate stable 3D supramolecular constructs, where the biopolymer-capped AgNPs play the dual role of active agent and structural element. SEM images of the assemblies revealed gradual increase of thickness with the number of deposited bilayers. The composites of ≥50 bilayers were safe to human cells and demonstrated 100% antibacterial activity against Staphylococcus aureus and Escherichia coli. Moreover, the films containing CSAgNPs brought about the total prevention of biofilm formation reducing the cells surface adherence by up to 6 logs. Such nanobiocomposites could serve as an effective barrier to control bacterial growth on injured skin, burns, and chronic wounds.

Hydrogel Dressings for Advanced Wound Management.

BACKGROUND: Composed in a large extent of water and due to their nonadhesiveness, hydrogels found their way to the wound dressing market as materials that provide a moisture environment for healing while being comfortable to the patient. Hydrogels' exploitation is constantly increasing after evidences of their even broader therapeutic potential due to resemblance to dermal tissue and ability to induce partial skin regeneration. The innovation in advanced wound care is further directed to the development of so-called active dressings, where hydrogels are combined with components that enhance the primary purpose of providing a beneficial environment for wound healing. OBJECTIVE: The objective of this review is to concisely describe the relevance of hydrogel dressings as platforms for delivery of active molecules for improved management of difficult- to-treat wounds. The emphasis is on the most recent advances in development of stimuli- responsive hydrogels, which allow for control over wound healing efficiency in response to different external modalities. Novel strategies for monitoring of the wound status and healing progress based on incorporation of sensor molecules into the hydrogel platforms are also discussed.

Advances in Magnetic Nanoparticles for Biomedical Applications.

Magnetic nanoparticles (NPs) are emerging as an important class of biomedical functional nanomaterials in areas such as hyperthermia, drug release, tissue engineering, theranostic, and lab-on-a-chip, due to their exclusive chemical and physical properties. Although some works can be found reviewing the main application of magnetic NPs in the area of biomedical engineering, recent and intense progress on magnetic nanoparticle research, from synthesis to surface functionalization strategies, demands for a work that includes, summarizes, and debates current directions and ongoing advancements in this research field. Thus, the present work addresses the structure, synthesis, properties, and the incorporation of magnetic NPs in nanocomposites, highlighting the most relevant effects of the synthesis on the magnetic and structural properties of the magnetic NPs and how these effects limit their utilization in the biomedical area. Furthermore, this review next focuses on the application of magnetic NPs on the biomedical field. Finally, a discussion of the main challenges and an outlook of the future developments in the use of magnetic NPs for advanced biomedical applications are critically provided.

Nanotransformation of Vancomycin Overcomes the Intrinsic Resistance of Gram-Negative Bacteria.

The increased emergence of antibiotic-resistant bacteria is a growing public health concern, and although new drugs are constantly being sought, the pace of development is slow compared with the evolution and spread of multidrug-resistant species. In this study, we developed a novel broad-spectrum antimicrobial agent by simply transforming vancomycin into nanoform using sonochemistry. Vancomycin is a glycopeptide antibiotic largely used for the treatment of infections caused by Gram-positive bacteria but inefficient against Gram-negative species. The nanospherization extended its effect toward Gram-negative Escherichia coli and Pseudomonas aeruginosa, making these bacteria up to 10 and 100 times more sensitive to the antibiotic, respectively. The spheres were able to disrupt the outer membranes of these bacteria, overcoming their intrinsic resistance toward glycopeptides. The penetration of nanospheres into a Langmuir monolayer of bacterial membrane phospholipids confirmed the interaction of the nanoantibiotic with the membrane of E. coli cells, affecting their physical integrity, as further visualized by scanning electron microscopy. Such mechanism of antibacterial action is unlikely to induce mutations in the evolutionary conserved bacterial membrane, therefore reducing the possibility of acquiring resistance. Our results indicated that the nanotransformation of vancomycin could overcome the inherent resistance of Gram-negative bacteria toward this antibiotic and disrupt mature biofilms at antibacterial-effective concentrations.

Escherichia coli and Pseudomonas aeruginosa eradication by nano-penicillin G.

The transformation of penicillin G into nano/micro-sized spheres (nanopenicillin) using sonochemical technology was explored as a novel tool for the eradication of Gram-negative bacteria and their biofilms. Known by its effectiveness only against Gram-positive microorganisms, the penicillin G spherization boosted the inhibition of the Gram-negative Pseudomonas aeruginosa 10-fold (from 0.3 to 3.0 log-reduction) and additionally induced 1.2 log-reduction of Escherichia coli growth. The efficient penetration of the spheres within a Langmuir monolayer sustained the theory that nanopenicillin is able to cross the membrane and reach the periplasmic space in Gram-negative bacteria where they inhibit the ß-lactam targets: the transferases that build the bacteria cell wall. Moreover, it considerably suppressed the growth of both bacterial biofilms on a medically relevant polystyrene surface, leaving majority of the adhered cells dead compared to the treatment with the non-processed penicillin G. Importantly, nanopenicillin was found innocuous towards human fibroblasts at the antibacterial-effective concentrations.

Bacteria-responsive multilayer coatings comprising polycationic nanospheres for bacteria biofilm prevention on urinary catheters.

This work reports on the development of infection-preventive coatings on silicone urinary catheters that contain in their structure and release on demand antibacterial polycationic nanospheres. Polycationic aminocellulose conjugate was first sonochemically processed into nanospheres to improve its antibacterial potential compared to the bulk conjugate in solution (ACSol). Afterward the processed aminocellulose nanospheres (ACNSs) were combined with the hyaluronic acid (HA) polyanion to build a layer-by-layer construct on silicone surfaces. Although the coating deposition was more effective when HA was coupled with ACSol than with ACNSs, the ACNSs-based coatings were thicker and displayed smoother surfaces due to the embedment of intact nanospheres. The antibacterial effect of ACNSs multilayers was 40% higher compared to ACSol coatings. This fact was further translated into more effective prevention of Pseudomonas aeruginosa biofilm formation. The coatings were stable in the absence of bacteria, whereas their disassembling occurred gradually during incubation with P. aeruginosa, and thus eradicate the biofilm upon release of antibacterial agents. Only 5 bilayers of HA/ACNSs were sufficient to prevent the biofilm formation, in contrast to the 10 bilayers of ACSol required to achieve the same effect. The antibiofilm efficiency of (HA/ACNSs)10 multilayer construct built on a Foley catheter was additionally validated under dynamic conditions using a model of the catheterized bladder in which the biofilm was grown during seven days. STATEMENT OF SIGNIFICANCE: Antibacterial layer-by-layer coatings were fabricated on silicone that efficiently prevents Pseudomonas aeruginosa biofilm formation during time beyond the useful lifetime of the currently employed urinary catheters in medical practice. The coatings are composed of intact, highly antibacterial polycationic nanospheres processed from aminated cellulose and bacteria-degrading glycosaminoglycan hyaluronic acid. The importance of incorporating nanoscale structures within bacteria-responsive surface coatings to impart durable antibacterial and self-defensive properties to the medical indwelling devices is highlighted.

Simultaneous sonochemical-enzymatic coating of medical textiles with antibacterial ZnO nanoparticles.

The antimicrobial finishing is a must for production of medical textiles, aiming at reducing the bioburden in clinical wards and consequently decreasing the risk of hospital-acquired infections. This work reports for the first time on a simultaneous sonochemical/enzymatic process for durable antibacterial coating of cotton with zinc oxide nanoparticles (ZnO NPs). The novel technology goes beyond the "stepwise" concept we proposed recently for enzymatic pre-activation of the fabrics and subsequent sonochemical nano-coating, and is designed to produce "ready-to-use" antibacterial medical textiles in a single step. A multilayer coating of uniformly dispersed NPs was obtained in the process. The enzymatic treatment provides better adhesion of the ZnO NPs and, as a consequence, enhanced coating stability during exploitation. The NPs-coated cotton fabrics inhibited the growth of the medically relevant Staphylococcus aureus and Escherichia coli respectively by 67% and 100%. The antibacterial efficiency of these textile materials resisted the intensive laundry regimes used in hospitals, though only 33% of the initially deposited NPs remained firmly fixed onto the fabrics after multiple washings.

Quorum-Quenching and Matrix-Degrading Enzymes in Multilayer Coatings Synergistically Prevent Bacterial Biofilm Formation on Urinary Catheters.

Bacteria often colonize in-dwelling medical devices and grow as complex biofilm communities of cells embedded in a self-produced extracellular polymeric matrix, which increases their resistance to antibiotics and the host immune system. During biofilm growth, bacterial cells cooperate through specific quorum-sensing (QS) signals. Taking advantage of this mechanism of biofilm formation, we hypothesized that interrupting the communication among bacteria and simultaneously degrading the extracellular matrix would inhibit biofilm growth. To this end, coatings composed of the enzymes acylase and α-amylase, able to degrade bacterial QS molecules and polysaccharides, respectively, were built on silicone urinary catheters using a layer-by-layer deposition technique. Multilayer coatings of either acylase or amylase alone suppressed the biofilm formation of corresponding Gram-negative Pseudomonas aeruginosa and Gram-positive Staphylococcus aureus. Further assembly of both enzymes in hybrid nanocoatings resulted in stronger biofilm inhibition as a function of acylase or amylase position in the layers. Hybrid coatings, with the QS-signal-degrading acylase as outermost layer, demonstrated 30% higher antibiofilm efficiency against medically relevant Gram-negative bacteria compared to that of the other assemblies. These nanocoatings significantly reduced the occurrence of single-species (P. aeruginosa) and mixed-species (P. aeruginosa and Escherichia coli) biofilms on silicone catheters under both static and dynamic conditions. Moreover, in an in vivo animal model, the quorum quenching and matrix degrading enzyme assemblies delayed the biofilm growth up to 7 days.

Size and Aging Effects on Antimicrobial Efficiency of Silver Nanoparticles Coated on Polyamide Fabrics Activated by Atmospheric DBD Plasma.

This work studies the surface characteristics, antimicrobial activity, and aging effect of plasma-pretreated polyamide 6,6 (PA66) fabrics coated with silver nanoparticles (AgNPs), aiming to identify the optimum size of nanosilver exhibiting antibacterial properties suitable for the manufacture of hospital textiles. The release of bactericidal Ag(+) ions from a 10, 20, 40, 60, and 100 nm AgNPs-coated PA66 surface was a function of the particles' size, number, and aging. Plasma pretreatment promoted both ionic and covalent interactions between AgNPs and the formed oxygen species on the fibers, favoring the deposition of smaller-diameter AgNPs that consequently showed better immediate and durable antimicrobial effects against Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus bacteria. Surprisingly, after 30 days of aging, a comparable bacterial growth inhibition was achieved for all of the fibers treated with AgNPs <100 nm in size. The Ag(+) in the coatings also favored the electrostatic stabilization of the plasma-induced functional groups on the PA66 surface, thereby retarding the aging process. At the same time, the size-related ratio (Ag(+)/Ag(0)) of the AgNPs between 40 and 60 nm allowed for the controlled release of Ag(+) rather than bulk silver. Overall, the results suggest that instead of reducing the size of the AgNPs, which is associated with higher toxicity, similar long-term effects can be achieved with larger NPs (40-60 nm), even in lower concentrations. Because the antimicrobial efficiency of AgNPs larger than 30 nm is mainly ruled by the release of Ag(+) over time and not by the size and number of the AgNPs, this parameter is crucial for the development of efficient antimicrobial coatings on plasma-treated surfaces and contributes to the safety and durability of clothing used in clinical settings.

Enzymatic functionalization of cork surface with antimicrobial hybrid biopolymer/silver nanoparticles.

Laccase-assisted assembling of hybrid biopolymer-silver nanoparticles and cork matrices into an antimicrobial material with potential for water remediation is herein described. Amino-functional biopolymers were first used as doping agents to stabilize concentrated colloidal dispersions of silver nanoparticles (AgNP), additionally providing the particles with functionalities for covalent immobilization onto cork to impart a durable antibacterial effect. The solvent-free AgNP synthesis by chemical reduction was carried out in the presence of chitosan (CS) or 6-deoxy-6-(ω-aminoethyl) aminocellulose (AC), leading to simultaneous AgNP biofunctionalization. This approach resulted in concentrated hybrid NP dispersion stable to aggregation and with hydrodynamic radius of particles of about 250 nm. Moreover, laccase enabled coupling between the phenolic groups in cork and amino moieties in the biopolymer-doped AgNP for permanent modification of the material. The antibacterial efficiency of the functionalized cork matrices, aimed as adsorbents for wastewater treatment, was evaluated against Escherichia coli and Staphylococcus aureus during 5 days in conditions mimicking those in constructed wetlands. Both intrinsically antimicrobial CS and AC contributed to the bactericidal effect of the enzymatically grafted on cork AgNP. In contrast, unmodified AgNP were easily washed off from the material, confirming that the biopolymers potentiated a durable antibacterial functionalization of the cork matrices.

Sonochemically processed cationic nanocapsules: efficient antimicrobials with membrane disturbing capacity.

Bacterial-mediated diseases are a major healthcare concern worldwide due to the rapid spread of antibiotic-resistant bacteria. One strategy to manage the bacterial infections while avoiding the emergence of resistant strains implies specific targeting and disruption of bacteria membranes. This work evaluates the potential of nanostructured biopolymer derivatives, nanocapsules (NCs), to disrupt the bacteria cell walls and effectively kill planktonic microorganisms. Two biopolymers, chitosan and cellulose, were chemically modified to synthesize derivatives with improved cationic character (thiolated chitosan and aminocellulose) prior to their processing into nanocapsules via a one-step sonochemical process. The interactions of NCs, displaying an average size of around 250 nm, with bacteria membrane were evaluated using two membrane models: Langmuir monolayers and liposome bilayers composed of a l-α-phosphatidylglycerol phospholipid extracted from Escherichia coli. NCs possessed improved membrane disturbing capacity in comparison to the nonprocessed biopolymer derivatives, by drastically increasing the monolayer fluidity and inducing more than 50% leakage of a dye inserted in the bilayered liposomes. In addition, membrane disturbance was directly proportional to the NCs cationic charge. Whereas evidence showed that thiolated chitosan and aminocellulose interacted with the bacteria membrane through a "carpet model", the NCs were found to induce larger surface defects and high local perturbance through a "detergent model". Importantly, the degree of disruption caused by the biopolymer derivatives and NCs correlated well with the antimicrobial capacity against Escherichia coli, selectively killing bacteria cells without imparting toxicity to human fibroblasts.

Sonochemical coating of textiles with hybrid ZnO/chitosan antimicrobial nanoparticles.

Textiles are good substrates for growth of microorganisms especially under moisture and temperature conditions found in hospitals. Microbial shedding from the body occurs continuously at contact of the patient with textile materials used in medical practices, contributing to the occurrence of hospital acquired infections. Thus, the use of efficient antimicrobial textiles is necessary to prevent the transfer of pathogens and the infection incidence. In this work, hybrid antimicrobial coatings were generated on cotton fabrics by means of a one-step simultaneous sonochemical deposition of ZnO nanoparticles (NPs) and chitosan. The process was further optimized in terms of reagents concentration and processing time in order to improve the antibacterial properties of the fabric and ensure their biocompatibility. The highest antibacterial activity of the fabrics against two medically relevant bacterial species was achieved in a 30 min sonochemical coating process using 2 mM ZnO NPs suspension. When chitosan was simultaneously deposited with the same amount of ZnO, the obtained hybrid NPs coating displayed higher by 48 and 17% antibacterial activity against Staphylococcus aureus and Escherichia coli, respectively. The presence of biopolymer also improved the durability of the antimicrobial effect of the coatings by 21% for Staphylococcus aureus and 40% for Escherichia coli, evaluated after applying multiple washing cycles at hospital laundering regimes. Finally, 87% biocompatibility improvement supported by fibroblast viability was observed for the hybrid ZnO/chitosan coating compared to the steady decrease of cells viability over one week in contact with the fabrics coated with ZnO alone.

Tannic acid NPs - synthesis and immobilization onto a solid surface in a one-step process and their antibacterial and anti-inflammatory properties.

Tannic acid nanoparticles were synthesized from an aqueous solution without the use of stabilizers via a sonochemical process. In order to avoid the dissolution of the formed nanoparticles, the sonochemical reaction was performed in the presence of a cotton fabric: following their formation, the tannic acid nanoparticles were embedded into the cotton substrate in a one-step process. The bioactive properties of the tannic acid coated surface were examined towards the inhibition of myeloperoxidase and collagenase, two major enzymes related with inflammatory processes. In addition, the antibacterial activity of the tannic acid nanoparticles coated textiles was evaluated against Staphylococcus aureus and Pseudomonas aeruginosa.

One-step sonochemical preparation of redox-responsive nanocapsules for glutathione mediated RNA release.

Efficient RNA delivery to targeted cells requires the use of stable interactive carriers that provide RNA protection during the extracellular transit and trigger release once internalised. One strategy to avoid the premature extracellular RNA drain coupled to sufficient intracellular release is the use of stimuli-responsive delivery materials exploiting as a triggering mechanism the redox gradient between the extra- and intracellular compartments. This work describes a facile route for the preparation of redox-active nanocarriers containing disulphides that combine RNA protection and delivery on demand based on intracellular glutathione (GSH) levels. A one-step sonochemical technology was employed to generate thiolated chitosan (TC) nanocapsules with a diameter between 250 and 570 nm and simultaneously load them with RNA. Their size and physiological stability were directly proportional to the extent of disulphide cross-linking, which in turn could be ruled by adjusting the processing pH and degree of chitosan thiolation. TC processing into nanocapsules showed to be advantageous in terms of RNA condensation and protection compared to the typically employed nanocomplexation. Fluorescence microscopy imaging revealed that: (i) the nanocapsules enter the human fibroblasts and migrate to the perinuclear regions within 1 h, and (ii) the cargo release may occur after the internalisation. These redox-responsive and biocompatible drug carriers demonstrated an effective (∼60%) and sustained (up to 72 h) RNA release at intracellular GSH concentrations (10 mM) in vitro, based on disulphide reduction and consequent capsule disassembly.

Laccase-assisted formation of bioactive chitosan/gelatin hydrogel stabilized with plant polyphenols.

Laccase-assisted simultaneous cross-linking and functionalization of chitosan/gelatin blends with phenolic compounds from Hamamelis virginiana was investigated for the development of bioactive hydrogel dressings. The potential of these hydrogels for chronic wound treatment was evaluated in vitro, assessing their antibacterial and inhibitory effect on myeloperoxidase and collagenase. Rheological studies revealed that the mechanical properties of the hydrogels were a function of the enzymatic reaction time. Stable hydrogels and resistant to lysozyme degradation were achieved after 2 h laccase reaction. The inhibitory capacity of the hydrogel for myeloperoxidase and collagenase was 32% and 79% respectively after 24 h incubation. Collagenase activity was additionally suppressed by adsorption (20%) of the enzyme onto the hydrogel. Therefore, the bioactive properties of the hydrogels were due to the effect of both released phenolic compounds and the permanently functionalized platform itself. The hydrogels showed antibacterial activity against Pseudomonas aeruginosa and Staphylococcus aureus.

Functional biopolymer-based matrices for modulation of chronic wound enzyme activities.

Collagen, collagen/hyaluronic acid (HA) and collagen/HA/chitosan (CS) sponges loaded with epigallocatechin gallate (EGCG), catechin (CAT) and gallic acid (GA) were developed and evaluated as active chronic wound dressings. Their physico-mechanical properties, biostability, biocompatibility and ability to inhibit in vitro myeloperoxidase (MPO) and collagenase--major enzymes related with the persistent inflammation in chronic wounds--were investigated as a function of the biopolymer composition and the polyphenolic compound used. The results demonstrated that the molecular weight of HA influences significantly the bulk properties of the obtained materials: higher elastic modulus, swelling ability and biostability against collagenase were measured when HA with higher molecular weights (830 and 2000 kDa) were added to the collagen matrices. The addition of CS and the polyphenols increased further the biostability of the sponges. Preliminary in vitro tests with fibroblasts revealed that the cells were able to adhere to all sponges. Cell viability was not affected significantly by the addition of the polyphenols; however, the presence of CS or high molecular weight HA in the sponge composition was associated with lower cellular viability. Finally, all specimens containing polyphenols efficiently inhibited the MPO activity. The highest inhibition capacity was observed for EGCG (IC50=15±1µM) and it was coupled to the highest extent of binding to the biopolymers (>80%) and optimal release profile from the sponges that allowed for prolonged (up to 3-5 days) effects.

Sensor materials for the detection of human neutrophil elastase and cathepsin G activity in wound fluid.

Human neutrophil elastase (HNE) and cathepsin G (CatG) are involved in the pathogenesis of a number of inflammatory disorders. These serine proteinases are released by neutrophils and monocytes in case of infection. Wound infection is a severe complication regarding wound healing causing diagnostic and therapeutic problems. In this study we have shown the potential of HNE and CatG to be used as markers for early detection of infection. Significant differences in HNE and CatG levels in infected and non-infected wound fluids were observed. Peptide substrates for these two enzymes were successfully immobilised on different surfaces, including collagen, modified collagen, polyamide polyesters and silica gel. HNE and CatG activities were monitored directly in wound fluid via hydrolysis of the chromogenic substrates. Infected wound fluids led to significant higher substrate hydrolysis compared with non-infected ones. These different approaches could be used for the development of devices which are able to detect elevated enzyme activities before manifestation of infection directly on bandages. This would allow a timely intervention by medical doctors thus preventing severe infections.

Chitin, chitosan and derivatives for wound healing and tissue engineering.

Naturally derived polymers possess a number of properties beneficial to wound healing and tissue engineering. The polysaccharides chitin and chitosan appear to be suitable candidates for the preparation of dressing materials and scaffolds for tissue regeneration due to their unique structural, physico-chemical and functional properties. Functionalization of these biopolymers for improvement of properties such as solubility or introduction of active functions and blending with other intrinsically bioactive polymers has attracted considerable attention in recent years. Such modifications would allow going beyond traditional approaches for treatments of dermal injuries. This chapter is a critical review of the advances in chitin and chitosan functionalization for wound-healing and tissue-engineering applications.