Surface nano-structured materials to control bacterial contamination

Surface nano-structured materials to control bacterial contamination Petya Stoyanova Petkova A thesis submitted in fulfilment of the requirements for degree of International Doctor of Philosophy at the Universitat Politècnica de Catalunya Supervised by Dr. Tzanko Tzanov Group of Molecular and Industrial Biotechnology (GBMI) Department of Chemical Engineering Universitat Politècnica de Catalunya Terrassa (Barcelona) 2016 ii iii This thesis was financially supported by: Ayudas para la formación de profesorado universitario (FPU) Ref beca: FPU 12/06258 A pilot line of antibacterial and antifungal medical textiles based on a sonochemical process, FP7-228730 Group of Molecular and Industrial Biotechnology iv v На моето семейство To my family vi vii Abstract The spread of bacteria and infections, initially associated with an increased number of hospital-acquired infections, has now extended into the community causing severe and difficult to diagnose and treat diseases. Additionally, many of those diseases are evoked by bacteria that have become resistant to antibiotics. Overcoming the ability of bacteria to develop resistance will potentially reduce the burden of these infections on the healthcare systems worldwide and prevent thousands of deaths each year. At present, there is a great interest in manufacturing antimicrobial materials that would provide bacteria-free environment. The nano-scale particles are promising candidates to fight bacterial pathogens, since developing of resistance to their action is less likely to occur. Nanoparticles (NPs) can be incorporated into polymeric matrices to design a wide variety of nanocomposites. Such nano-structures consisting of inorganic and inorganic/organic NPs represent a novel class of materials with a broad range of applications. This thesis is about the development of antibacterial nano-structured materials aimed at preventing the spread of bacteria. To achieve this, two versatile physicochemical and biotechnological tools, namely sonochemistry and biocatalysis were innovatively combined. Ultrasound irradiation used for the generation of various nano-structures and its combination with biocatalysts (enzymes) opens new perspectives in materials processing, here illustrated by the production of NPs coated medical textiles, water treatment membranes and chronic wound dressings. The first part of the thesis aims at the development of antibacterial medical textiles to prevent the bacteria transmission and proliferation using two single step approaches for antibacterial NPs coating of textiles. In the first approach antibacterial zinc oxide NPs (ZnO NPs) and chitosan (CS) were deposited simultaneously on cotton fabric by ultrasound irradiation. The obtained hybrid NPs coatings demonstrated durable antibacterial properties after multiple washing cycles. Moreover, the presence of biopolymer in the NP hybrids improved the biocompatibility of the material in comparison with ZnO NPs coating alone. In the second approach, a simultaneous sonochemical/enzymatic process for durable antibacterial coating of cotton with ZnO NPs was carried out. The enzymatic treatment provides better adhesion of the ZnO NPs and, as a consequence, enhanced coating stability during exploitation. Likewise to the antibacterial coatings obtained in the first approach, the antibacterial efficiency of these textiles was maintained after multiple intensive laundry viii regimes used in hospitals. The NPs-coated cotton fabrics inhibited the growth of the most medically relevant bacteria species. In the second part of the thesis, hybrid antibacterial biopolymer/silver NPs and cork matrices, were enzymatically assembled into an antimicrobial material with potential for water remediation. Intrinsically antibacterial amino-functional biopolymers, namely CS and aminocellulose were used as doping agents to stabilize colloidal dispersions of silver NPs (AgNPs), additionally providing the particles with functionalities for covalent immobilization on cork to impart durable antibacterial effect. The biopolymers promoted the antibacterial performance of the obtained nanocomposites in conditions simulating a real situation in constructed wetlands. In the last, third part of the thesis, a bioactive nanocomposite hydrogel for wound treatment was developed. Sonochemically synthesized epigallocatechin gallate nanospheres (EGCG NSs) were incorporated and simultaneously crosslinked enzymatically into a thiolated chitosan hydrogel. The potential of the generated EGCG NSs for chronic wound treatment was evaluated by assessing its antibacterial properties and inhibitory effect on myeloperoxidase and collagenase biomarkers of chronic wound infection. Sustained release of the EGCG NSs from the biopolymer matrix was achieved. The latter, coupled with the good biocompatibility of the hydrogel suggested its potential for chronic wound management. Keywords: antibacterial nanoparticles, chitosan, antibacterial coatings, medical textiles, ultrasound, water disinfection, nanocomposites, natural phenolics, enzymatic crosslinking, hydrogels, wound healing ix Table of Contents Abstract ............................................................................................................................ vii Abbreviation list ............................................................................................................... xiii Figure, table and scheme captions ................................................................................... xv 1 Introduction ................................................................................................................. 1 2 State-of-the-art ........................................................................................................... 5 2.1 The global threat of antimicrobial resistance. Hospital-associated infections ....... 7 2.2 Strategies for prevention and control of hospital-associated infections .............. 11 2.3 Antibacterial nanoparticles ................................................................................. 13 2.3.1 Synthesis of nanoparticles. Sonochemistry - a versatile tool for nanoparticles synthesis and antimicrobial nano-functionalization of surfaces ................................. 15 2.3.2 Metal and metal oxide nanoparticles ........................................................... 21 2.3.3 Hybrid polymer/metal nanoparticles ............................................................ 24 2.3.4 Nanoparticles as drug delivery system........................................................ 26 2.4 Antibacterial nanocomposites to prevent bacterial contamination and transmission ................................................................................................................. 27 2.4.1 Antibacterial nanoparticles-coated medical textiles ..................................... 27 2.4.2 Potential of sonochemical processing in manufacturing of functional materials ................................................................................................................... 31 2.4.3 Antimicrobial nanocomposites for water disinfection ................................... 32 2.4.4 Antimicrobial hydrogels for wound treatment .............................................. 34 3 Objectives of the thesis ............................................................................................. 38 4 Materials and Methods .............................................................................................. 42 4.1 Reagents ........................................................................................................... 44 4.2 Enzymes ............................................................................................................ 44 4.3 Bacteria ............................................................................................................. 45 4.4 Experimental methods ....................................................................................... 45 4.4.1 Simultaneous sonochemical coating of textiles with antibacterial ZnO/chitosan nanoparticles ...................................................................................... 45 4.4.1.1 Ultrasound-assisted coating of cotton .................................................. 45 4.4.1.2 Quantification of deposited ZnO .......................................................... 46 xvi Figure 5.3 HRSEM micrographs of cotton fabrics coated with ZnO (A, B), ZnO/CS (C, D), and CS (E, F). Composition of the starting solution: 2 mM ZnO NPs and 0.3 % (w/v) CS. The images on the left were taken with 10 000x, whereas the right-side images were taken with 200 000x magnification. ............................................................................................ 65 Figure 5.4 STEM images taken to evaluate: (A) the NPs presence in the remaining solutions after the sonochemical coating of cotton fabrics with 2 mM ZnO in absence (top image), and presence (bottom image) of 0.3 % (w/v) CS; and (B) the NPs leaching into a solution incubated with the fabrics coated with ZnO (top image), and ZnO/CS (bottom image). .... 67 Figure 5.5 Antibacterial activity of ZnO (light grey bars) and Zn2+/CS (dark grey bars) coated cotton fabrics towards S. aureus and E. coli after 15 min of contact. ................................ 68 Figure 5.6 (A) Percentage of remaining antibacterial activity of the coatings after 10 washing cycles at 75 °C against S. aureus (light grey bars) and E. coli (dark grey bars), after 15 min of contact. (B) Images of antibacterial activities of the Zn2+/CS fabrics before and after washing. .......................................................................................................................... 70 Figure 5.7 Human skin fibroblasts viability after 24 h (light grey bars) and 1 week (dark grey bars) contact with the coated cotton fabrics. .................................................................... 72 Figure 5.8 HRSEM image of cotton fabrics after 30 min of US irradiation (amplitude 35 %, intensity 17.30 W/cm2, 55ᵒC) in absence (A) and presence (B) of enzyme. ..................... 77 Figure 5.9 Temperature profile of cellulase (Kappacell ETU 39) activity (A) and the residual cellulase activity after 30 min of US irradiation (amplitude 35 %, intensity 17.30 W/cm2) as a function of temperature (B). The residual activity was calculated as a percentage of the activity of the enzyme not exposed to US at 55 °C for 1 h in presence of Whatman No. 1 filter paper as a substrate................................................................................................. 78 Figure 5.10 Reducing sugars released under different US amplitudes after 30 min of US irradiation at 55 °C in presence of Whatman No. 1 filter paper as a substrate. The US amplitudes of 20, 25, 30, 35 and 40 % correspond to the US intensities of 6.90, 10.40, 13.10, 17.30 and 30.80 W/cm2, respectively. .............................................................................. 79 Figure 5.11 Intrinsic fluorescence spectra of untreated (black chart line) and ultrasound treated for 30 min cellulase (gray chart line). ................................................................... 80 Figure 5.12 Representative HRSEM micrographs of cotton fabrics coated with 1 mM of ZnO NPs at 55 °C in presence of denatured enzyme before (a, b) and after 10 washing cycles (c, d) and in presence of cellulase before (e, f) and after10 washing cycles (g, h). The images on the left were taken with 15000x, whereas the right-side images were taken with 50000x magnification. .................................................................................................................. 82 Figure 5.13 Size histograms (fitted by Gaussian curves (solid line)) of the ZnO NPs deposited on the fabric in presence of denatured enzyme (a) and cellulase (b) associated with Fig. 5.12b and 5.12f, respectively. ............................................................................ 83 xvii Figure 5.14 Antibacterial activity of the fabrics coated with 1 mM of ZnONPs: light gray bars represent non-washed fabrics, whereas dark gray bars show the antibacterial activity of the fabrics after 10 washing cycles at 75°C. The assays were performed with S. aureus (a) and E. coli (b), after 1 h of contact with the fabrics. ................................................................. 84 Figure 5.15 STEM images of the AgNPs dispersions synthesized in absence and presence of CS and AC. The images were taken to evaluate the presence of NPs in the dispersions immediately after the synthesis of the particles (images A, B and C) and after 3 h of their preparation (images D, E and F). The inset photographs represent the analyzed dispersions. ........................................................................................................................................ 91 Figure 5.16 UV-Vis spectra obtained for the pure AgNPs dispersion and the dispersions doped with CS and AC after 72 h of their synthesis. ........................................................ 93 Figure 5.17 FTIR-ATR spectra of untreated and enzymatically functionalized cork with AgNPs, AgNPs-CS and AgNPs-AC dispersions. .............................................................. 96 Figure 5.18 SEM images of cork surface enzymatically treated in absence (A) and presence of NPs: (B) pure AgNPs, (C) AgNPs-CS, and (D) AgNPs-AC. ......................................... 97 Figure 5.19 Antimicrobial activity of untreated and enzymatically functionalized cork with AgNPs, AgNPs-CS and AgNPs-AC dispersions. The materials were subjected to the washing treatment in water (stirring 100 rpm) for up to 5 days, during which the antibacterial activity is evaluated at different time points. ..................................................................... 98 Figure 5.20 SEM images of E. coli accumulated on the surface of unmodified cork (A, B) and on the CS/AgNPs-cork composite (C, D) after 16 h of contact with E. coli suspension. The images on the left were taken with 10000×, whereas the right-side images were taken with 100000× magnification. .......................................................................................... 101 Figure 5.21 Images of natural cork granules after incubation in sterile distilled water (A) and water inoculated with E. coli (B); and CS/AgNPs-cork composite after incubation in sterile distilled water (C) and water inoculated with E. coli (D) for 16 h at room temperature. The images B and D are related to Figure 5.20 A, B and Figure 5 C, D, respectively. ........... 102 Figure 5.22 Data of EGCG NSs hydrodynamic radius (at 25 ° C) (n=3) (A), and fluorescence microscopy image of the spheres filled with Nile red (B). Scale bar 2 µm....................... 108 Figure 5.23 Functional properties of EGCG NSs (light grey bars) and free solution (dark grey bars). Inhibition of the activity of MPO (A) and collagenase (B). All results are reported as mean values ± standard deviation (n=3). ................................................................... 109 Figure 5.24 The antioxidant activity of EGCG NSs (light grey bars) and free solution (dark grey bars). All results are reported as mean values ± standard deviation (n=3). ............ 111 Figure 5.25 The antibacterial activity of EGCG NSs (light grey bars) and free solution (dark grey bars) towards Staphylococcus aureus. All results are reported as mean values ± standard deviation (n=3). ............................................................................................... 112 xviii Figure 5.26 In situ gel formation of TC/EGCG hydrogels in the presence of HRP (9.65 U/mL) and different concentrations of H2O2. Data of the mean time of gelation ± standard deviation (n=3) are reported. .......................................................................................... 114 Figure 5.27 TC hydrogels swelling ratio (A) and degradation (B). The results show the mean hydrogel swelling and degradation of each experimental group ± standard deviation (n=6 for each time point). ............................................................................................................ 115 Figure 5.28 Hydrogels morphology (A) TC and (B) TC hydrogels loaded with EGCG NSs. ...................................................................................................................................... 116 Figure 5.29 Time-dependent release of EGCG NSs from TC hydrogels. Three samples were evaluated for each time point and data are reported as mean EGCG NSs release (%) ± standard deviation. ...................................................................................................... 117 Figure 5.30 Cytotoxicity of EGCG NSs, TC and their mixture to human cell fibroblasts. The results show the mean cytotoxicity values of 3 independent assays ± standard deviation (n=6). ............................................................................................................................. 118 Table 5.1 Antibacterial activity of the coated fabrics against S. aureus and E. coli after different incubation time. .................................................................................................. 69 Table 5.2 Amount of ZnO deposited on the cotton fabrics in a simultaneous sono-enzymatic process (experimental error ± 10 %). ............................................................................... 81 Table 5.3 ζ potential and mean hydrodynamic radius of AgNPs synthesized in absence and presence of CS and AC. .................................................................................................. 89 Table 5.4 Antibacterial activity of enzymatically modified cork against Escherichia coli and Staphylococcus aureus after 5 days washing in water. The results are expressed in % of bacteria reduction compared to the untreated cork. ......................................................... 99 Table 5.5 Hydrogels time of gelation at room temperature using horseradish peroxidase (9.65 U/mL) and different concentrations of hydrogen peroxide and EGCG NSs:TC ratios. ...................................................................................................................................... 116 Scheme 5.1 Mechanism of immobilization of the hybrid AgNPs-biopolymers onto cork. Mechanisms above the scheme, show two possibilities of reaction: (1) Michael addition, (2) Schiff base formation. ...................................................................................................... 94 Scheme 5.2 Sonochemical synthesis of EGCG NSs preparation and their purification. . 107 Scheme 5.3 EGCG NSs loading into TC hydrogels. ...................................................... 113 1 1 Introduction 2 3 Antibiotic resistance is a growing public health concern worldwide. Each year the number of people who acquire infections caused by antibiotic resistant bacteria increases, resulting in increased number of deaths. Antibiotics are the most commonly prescribed drugs to treat infections caused by bacteria. However, the emergence of new resistant microorganisms has accelerated alongside with their misuse and overuse, which contribute to the antibiotic resistance around the globe. Direct or indirect contact with a source of infectious pathogens e.g. environmental surfaces, healthcare settings, food and water supplies brings about the spread and transmission of pathogens. Therefore, an efficient strategy to address the threat of antimicrobial resistance should be aimed at: i) the prevention and control of the spread of bacteria and infections, and ii) the development of new bactericides to which bacteria cannot easily acquire resistance. Recent approaches addressing this challenge explore non-traditional antibiotic agents such as antimicrobial nanoparticles (NPs) and nanosized carriers for target drug delivery. Antimicrobial NPs are found to be effective in inactivating various microorganisms. A variety of chemical and physical synthetic routes have been exploited for the NPs synthesis. Among them, the use of ultrasound (US) is currently positioned as one of the most powerful tools in nano-structured materials synthesis. Moreover, the US irradiation has been successfully applied to homogeneously coat the surface of variety of solid substrates, including textiles, glass and nylon. It has been demonstrated that the microjets and shock waves produced after cavitation collapse are able to drive the NPs at such high velocities towards the solid substrate that fusion and strong adherence by physical or chemical interactions with the surface occurs during collision. Another promising approach exploiting US is the combination of ultrasonic energy and enzymatic treatments as a strategy to boost the enzyme activity e.g. to accelerate the mass transfer during some of the textiles’ manufacturing steps and thus, shorten the processing time. Both innovative tools (US and biocatalysts) could be also employed, alone or in combination, to create antibacterial surfaces and devices. This thesis aims at designing of nano-functionalized materials able to prevent/reduce the bacterial spread. The rationale of the work is the integration of nano- and bio-technological approaches with sonochemistry. The innovative combination of antimicrobial NPs, biopolymers, enzymes, and sonochemical irradiation is exploited to develop the following nano-structured surfaces and devices: i) medical textiles coated with antibacterial metal oxide NPs and biopolymer/metal oxide nano-hybrids, ii) antibacterial biopolymer/metal NPs- 4 cork composites for water disinfection, and iii) bioactive nanocomposite hydrogels for wound treatment. 5 2 State-of-the-art 6 7 2.1 The global threat of antimicrobial resistance. Hospital-associated infections “Antimicrobial resistance is resistance of a microorganism to an antimicrobial drug that was originally effective for treatment of infections caused by it.”1 The antimicrobial resistance threatens the effective prevention and treatment of an ever-increasing range of infections caused by resistant microorganisms, including bacteria, fungi, viruses and parasites. Those microorganisms are able to withstand the attack of antibacterial drugs, antifungals and antivirals, so that conventional treatments become ineffective and infections persist, increasing the risk of transmission. The development of resistant strains is a natural occurrence that happen when resistant markers are exchanged between them or when microorganisms replicate themselves erroneously. The use and misuse of antimicrobial drugs accelerates the emergence of drugresistant strains. Poor infection control practices, inadequate sanitary conditions and inappropriate food-handling encourage the further spread of antimicrobial resistance. The World Health Organization (WHO) warns that at present the so called “post-antibiotic era”, in which common infections and minor injuries can kill, is a very real possibility due to the fact that resistance to common bacteria has reached alarming levels worldwide.1 The mechanisms by which antimicrobials act involve interference with proteins,2 nucleic acid synthesis,3 and inhibition of cell-wall synthesis i.e. the peptidoglycan biosynthesis.4,5 However, bacteria have evolved genetic and biochemical ways of resisting these antimicrobial actions and to develop a tolerance to the antimicrobial drugs by several ways:6  enzymatic inactivation of the drugs by beta-lactamases, acetylases, adenylases, and phosphorylases,  limiting the drug access by virtue of membrane characteristics,  altering the drug target so that the antimicrobial no longer binds to it,  by passing the drug's metabolism,  increasing the expression of efflux pumps. 14 metal oxides (MeO) NPs etc. Some of these nano-scaled particles act with multiple simultaneous mechanisms against microbes (Figure 2.4) which makes the development of resistance to these NPs unlikely, because multiple simultaneous gene mutations in the same bacterial cell would be required for that resistance to develop.32 The antimicrobial NPs can be used effectively by coating them on the surfaces that require antimicrobial functions, for instance, on medical devices, textiles and water treatment filters.33 Figure 2.4 Multiple mechanisms of antimicrobial action of, chitosan-containing NPs (chitosan NPs), silver-containing NPs (AgNPs), zinc oxide-containing NPs (ZnO NPs), copper-containing NPs, and magnesium-containing NPs; ROS refers to reactive oxygen species (adapted from32). 15 2.3.1 Synthesis of nanoparticles. Sonochemistry - a versatile tool for nanoparticles synthesis and antimicrobial nano-functionalization of surfaces Different physical and chemical processes are currently used to synthesize metal NPs, with desired size, size distribution and morphology/shape. While some methods are bottoms up (synthesis), some are called top down ("milling"). Top down methods involve braking the larger materials (bulk or micro structures) into NPs, while bottom up methods start form homogeneous solution or gas to build up the nano-structure (particle, layer, wire etc.). The synthesis methods include liquid and gas phase processes such as micro-emulsion, reverse micelle process, novel spray methods and sol-gel assisted in-situ techniques. Some of the most common techniques used to prepare biodegradable NPs (nanospheres (NSs) and nanocapsules (NCs)) include coacervation and phase separation, emulsification diffusion, solvent displacement and solvent evaporation, sol-gel encapsulation, supercritical fluid technology, spray drying and spray congealing.34 However, the majority of the synthesis routes for generation of polymeric nanoscale biomedical materials are based on emulsification techniques, and are achieved either directly from a macromolecule, or through a polymerization reaction during the preparation.35,36 Selection of an appropriate route depends on different factors including the particle size, their thermal, chemical and physiological stability, and the requirement of an organic solvent and surfactants in the reaction. The latter two are being directly associated with the biocompatibility of the final product. However, whenever referring to the industry, the main requirements to the NPs synthesis process are to be low-cost, environmentally friendly and to be suitable for both continuous operation and a high production rate. Sonochemistry is a research field in which due to the application of powerful ultrasound (US) radiation molecules undergo a chemical reaction. The US irradiation of liquids (20 KHz–10 MHz) creates conditions important for the improvement of the stoichiometric and catalytic chemical reactions by increasing the reactivity, in some cases, nearly by a million-fold. The greatest advances in sonochemistry, however, reside in the production of new materials with unusual properties. The synthesis capacity of US comes from the acoustic cavitation phenomenon, which consists of the formation, growth and the implosive collapse of bubbles in a liquid.37 The dynamics of i) cavity growth, through the diffusion of solute vapor into the volume of bubble, and ii) collapse, which occurs when the bubble size reaches its maximum, are highly dependent on the local environment and are different in homogeneous liquids and near the liquid-solid interface. The implosions of cavities inside which gases and vapors are 16 compressed, generate intense heat that raises the temperature of the liquid surrounding the cavity and creates local hot spots.38 It is claimed that upon the collapse of bubbles, temperatures of nearly 5000 °C and local pressures as high as 500 atmospheres are obtained (Figure 2.5A). Since the collapse of bubbles occurs in less than nanoseconds, the heating and cooling rates during cavitation are greater than 109 K/s.39 In general, no chemical reactions happen when US irradiation is applied to solid and solidgas systems as the cavitation phenomenon takes place only in liquids. Thus, the chemical effects of US fall into three areas: i) homogeneous sonochemistry of liquids, ii) heterogeneous sonochemistry of liquid-liquid or liquid-solid systems, and iii) sonocatalysis (initiation of a catalytic reaction by irradiation with US).39 The source of homogeneous sonochemistry is the localized hot spot, while related phenomena occur with cavitation in liquid-solid systems. There are two important aspects - microjets and shock waves - derived from cavitation observed nearby surfaces. The cavitation and the shock waves, created by US can accelerate solid particles to very high velocities, as the non-spherical collapse of the cavities drives high speed microjets of liquid directed towards the surface (Figure 2.5B). Figure 2.5 A) Bubble formation, growth and subsequent collapse over several acoustic cycles. A bubble oscillates in phase with the applied sound wave, contracting during compression and expanding during rarefactions,40 B) Collapsing bubbles near a surface experience non-uniformities in their surroundings that results in the formation of high-velocity microjets.41 These extreme conditions cause the rupture of chemical bonds and enhance mass transport and thus, cause changes in the surface morphology, reactivity and composition. In the process of formation of nanomaterials, their solid state is determined by the high cooling A B 17 rates coupled with the type of precursors used. In the case, when volatile precursors are used, gas phase reactions are predominant and the formed NPs are amorphous.42 On the other hand, when for the synthesis of NPs the precursor is a non-volatile compound, the reaction occurs in the liquid phase and in this case the products formed could be nanoamorphous or nanocrystalline.38 The latter depends upon the temperature of the ring region, where the reactions take place. In the former case, the volatile precursor (e.g. a volatile organometallic compound) will produce free metal atoms generated by bond dissociation due to the high temperatures created during bubble collapse. These atoms can be injected into the liquid phase and nucleate to form NPs or other nano-structured materials if appropriate templates or stabilizers are present in the solution. Nonvolatile precursors may still undergo sonochemical reactions, even outside of the collapsing bubbles by undergoing reactions with radicals or other high energy species produced from the sonolysis of vapor molecules inside the collapsing bubbles that then diffuse into the liquid phase to initiate a series of reactions (e.g. reduction of metal cations, Figure 2.6).43 Figure 2.6 Primary sonochemistry and secondary sonochemistry for preparation of nanomaterials. An example of primary sonochemistry is the generation of metal atoms from sonolysis of weak metal– carbon bonds from volatile organometallic compounds inside the collapsing bubble that then diffuse into the bulk liquid to form functional nanomaterials. Secondary sonochemical products may arise from chemically active species (e.g., organic radicals from sonolysis of vapour) that are formed inside the bubble, but then diffuse into the liquid phase and subsequently react with solution precursors to form a variety of nano-structured materials.43 The importance of sonochemistry in the fields of material science and nanotechnology rises up through its advantages for i) preparation of amorphous NPs, ii) formation of 18 proteinaceous and polymer micro- and nano-particles, iii) insertion of nanomaterials into mesoporous materials, and iv) deposition of NPs on metal, ceramic and polymeric surfaces. The advantages of US over the conventional methods for NPs preparation include shorter reaction times, milder reaction conditions, higher yields, improved selectivity and clean reactions.44–47 Moreover, sonochemical routes often do not employ any binding agents or stabilizers. For instance, Gedanken and coworkers have readily synthesized by US irradiation different metallic NPs and core-shells with encapsulated metal without using coupling agents.48–51 The extreme conditions attained during bubble collapse (localized hotspot through adiabatic compression or shock wave formation within the gas phase) have been exploited to decompose chemical bonds and generate a variety of nano-structured materials such as metal,52,53 MeO/metal hydroxides54–62 metal sulfides,63 metal nitrides,64 metal carbide NPs65,66 bimetallic NPs,67–71 semiconductor NPs,72,73 metal-polymer composites,74,75 ceramic materials69,76 and others.62,77 For the generation of these structures acetate,53,58,60,73,78 aqueous metal salts solution,61,72,79 ammonia complexes57,63 and organometallic precursors55,65,77,80,81 have been used. The sonochemical synthesis of nanostructured materials is also extremely versatile as various forms of nanophase materials can be generated simply by changing the reaction conditions, e.g. temperature, concentration of precursors, sonication power and time.44,57,60,69,82–84 Nanowires,85–87 nanorings,52,88,89 nanotubes,72,88 nanorods,88,90–92 nanoplates,52,74 nested inorganic fullerenes93, spheres,63,94 nanocylinders95 and many others structures, such as whiskers-like,72,96 flower-like94,97 and wormhole-like98 are among the shapes of sonochemical products. For instance, by controlling the process parameters zinc oxide (ZnO) nanostructures with different morphology were synthesized by Jung et al.94 ZnO nanorods and nanocups were obtained by changing the concentration of the precursors (zinc nitrate hexahydrate and hexa-methylenetetramine) and sonication time, while triethyl citrate was used as an additional chemical to synthesize ZnO nanodisks. For the synthesis of ZnO nanoflowers and NSs, zinc acetate dihydrate and ammonia–water were used as zinc cation and hydroxide anion precursors, respectively. In this case, for the synthesis of ZnO NSs triethyl citrate was added to the above mixture. Recently, US-assisted anionic biosurfactants (rhamnolipids and surfactin)-template route was developed to synthesise α- HH nano-CaSO4.44 Control over the crystal polymorph, size and shape of nano-CaSO4 was achieved by adjusting the molar ratio of (NH4)2SO4/CaCl2 and the mass ratios of 19 rhamnolipid/H2O, surfactin/H2O and rhamnolipid/surfactin. It was observed that with increasing the biosurfactants/H2O ratios, crystal length and aspect ratio decreased whereas by increase the surfactin ratio, the crystal morphology gradually changes from long rods to hexagonal plate and then to plate-like structures. The preferential adsorption of rhamnolipid on the side facets and surfactin on the top facets contributes to the morphology control. In this study, the US approach was found to be superior to conventional in situ deposition technique used for the synthesis of nano-CaSO4 in terms of energy efficiency, processing time, yield and crystallization habit (crystal shape, morphology and l/w aspect ratio). Surface modification has been recognized as one of the most advanced and intriguing methods to build tailored nanomaterials. One of the benefits of coating NPs on 2D and 3D surfaces is the avoidance of the release of the NPs to the environment. The coated nanomaterials provide a very high surface area and possess chemical and physical properties that are distinct from those of the individual components. Moreover, the sonochemistry is a method that enables to simultaneously produce NPs and deposit them on the substrates in one single step. Gedanken and co-workers have defined two routes for the NPs deposition on substrates under US: i) ‘‘throwing stones’’ mode of deposition99 and ii) in situ mode of deposition.100,101 In the “throwing stones” mode of deposition, the substrates are coated with preliminarily synthesized NPs or commercial NPs, while in the in situ mode the substrates are coated with NPs that were directly synthesized during the sonochemical reaction. An additional route for the deposition of NPs very similar to the in situ coating method was found for the sonication of aqueous solutions of either inorganic102 or organic compounds.103 For instance, it was found that the sonication of aqueous solutions of inorganic compounds (NaCl, CuSO4, KI) resulted in the formation of salt NPs, but not metal or MeO NPs (as in the case of in situ method).102 The sonochemical coating is performed by the “bombardment” of 2D and 3D solid surfaces (ceramic, polymeric, glass, metal, textiles and paper) by liquid microjets carrying NPs. These microjets are formed when the acoustic bubble collapses near a solid surface. In the onestep sono-process the newly formed NPs (or commercial one) are embedded physically into the solid surface by the microjets moving at speeds >200 m/sec. This process produces a uniform homogenous and stable coating of NPs on surfaces with different functional groups. Various metal NPs were deposited with the aid of US on different organic and inorganic supports, such as polystyrene104, poly(methyl methacrylate),105,106 silica,107–111 and alumina.112 For example, a nanocomposite from silver/nylon 6,6 has been produced from an 20 aqueous solution of silver nitrate (AgNO3) in the presence of ammonia and ethylene glycol by an US-assisted reduction method. Larger NPs (50 – 100 nm) were finely dispersed onto the polymer surface, while smaller NPs (20 nm) have penetrated the surface and distributed inside the polymer grains. The obtained nanocomposites were stable to washing and were used as a master batch for the production of nylon yarns by melting and spinning processes. The produced nylon fibers have demonstrated good antibacterial performance towards Gram-positive S. aureus and Gram-negative Pseudomonas aeruginosa (P. aeruginosa) bacteria.113 Zinc oxide NPs (ZnO NPs) were deposited on a glass slides by sonochemical irradiation of the Zn2+ ions precursor in a water–ethanol mixture.114 The leaching of NPs was tested in water, or ethanol and/or acetone solutions. The results demonstrated that the sonochemically deposited ZnO NPs are strongly anchored to the glass, as no NPs were detected in the leaching solutions after 7 days of incubation. Recently, Kiel et al. reported on sonochemical preparation of salt NPs from an aqueous solution of NaCl, CuSO4 and KI and simultaneous deposition on glass microscope slide, a parylene-coated glass slide, or a silicon wafer.102 It was found that the increasing of the initial ions concentration and US treatment time, lead to enlarged NPs and appearance of aggregates on the substrate surfaces. The application of US to produce high performance nanocomposite coatings on steel was also demonstrated by several research groups. For example, Zn–Ni–Al2O3 nanocomposite coatings on low carbon steel were fabricated using eletrodeposition technique with the aid of US by Zheng and co-workers.115 It was shown that the US vibration significantly improved the uniform dispersion of nano-alumina particles in the matrix and increased the nanoalumina content incorporated in the Zn–Ni–Al2O3 nanocomposite coating. Further, the anticorrosion properties and the hardness of Zn–Ni–Al2O3 composite coatings were improved significantly as compared to the Zn–Ni alloy, which was related to the nanoaluminia content and dispersion in the matrix. Soloviev and co-workers have generated silver NPs (AgNPs) by reduction of AgNO3 by ethylene glycol and simultaneously coated them on stainless steel plate.116 They found that the adherence of the particles on the steel substrate is not as good as in the case when soft substrates are used. Moreover, by increasing the distance between the substrate and the sonochemical horn the coating was found to be unstable and easily washed. The metal flat surfaces usually provide fewer possibilities for direct functionalization especially without any additives as compared to polymeric materials. This leads to lower coating stability and thus, decreased life-time at use of the product. 21 The sonochemical process enables also to coat the inner walls of hollow tubing e.g. AgNPs were deposited in a one-step US process on tubing made of rubber, PVC, Teflon and polyethylene.117 In another approach enzyme NPs of α-amylase were also in situ generated and immobilized onto polyethylene films via high-intensity US.118 Sonochemical methods have also been applied to solve the stability problem of magnetic NPs119,120 through their sonochemical coating with polymers or deposition on surfaces. The US irradiation was used as a way to prevent the agglomeration of the magnetite particles and accelerate the hydrolysis and condensation of tetraethyl orthosilicate.121,122 The method resulted in homogeneously coated NPs due to the improved mass transfer of silica sols to NPs surface. It was found that the thickness of the silica layer over the NPs could be easily controlled by the sonication time.121 The challenges in synthesis of iron air-stable structures were also overcome by sonochemistry. Air-stable iron NPs were synthesized and deposited on the surface of carbon spherules by US.49 The process consist of sonochemical decomposition of Fe(CO)5 in diphenylmethane. This approach was previously applied by Nikitenko and co-workers, who synthesized highly magnetic, air-stable iron-iron carbide NPs by using power US.123 Their finding showed that during the sonolysis of neat diphenylmethane a polymer-like solid product is formed, which coats the surface of iron NPs formed simultaneously from Fe(CO)5. Recently, the synthesis of magnetically recyclable thin-layer MnO2 nanosheet-coated Fe3O4 nanocomposites with photocatalytic properties by combined hydrothermal/US approach was also reported.124 2.3.2 Metal and metal oxide nanoparticles Because of their biocidal activity, metals have been widely used for centuries as antimicrobial agents. Nano-metal and metal based compounds such oxides or salts are claimed to be more biocidal (at very low concentrations) than many conventional antibiotics, which utilization at high concentrations can induce adverse effects and toxicity to human cells.125,126 Moreover, the use of antibacterial NPs is also among the most promising strategies to overcome the microbial drug resistance.127 The generally accepted mechanism of metallic NPs antibacterial action involves generation of reactive oxygen species (ROS) including hydrogen peroxide (H2O2), OH¯ (hydroxyl radicals), and O−2 2 (peroxide), followed by the disruption of the bacterial cell membrane (Figure 2.4).126,128–131 These killing mechanisms are usually less prone for the development of antimicrobial resistance.33 22 However, while for some of the metallic NPs the mechanism of antibacterial activity is widely investigated and clarified, for others still remains a subject of debate. The bactericidal effect of those nanosystems depends on several parameters such as NP’s size, stability and concentration. Many studies indicate that smaller NPs have better antibacterial performance than the bigger ones. Morones et al. have reported on the AgNPs size effect against Gram-negative bacteria.132 They investigated particles in the range of 1–100 nm and had found that the NPs in a direct interaction with the bacteria preferentially had a diameter of ~1–10 nm. Another factor that could play a role for the antibacterial properties of the NPs is their shape. For instance, the shape dependent antibacterial performance against Gram-positive Streptococcus iniae and Streptococcus parauberis and Gram-negative E. coli and Vibrio anguillarum bacteria was observed for the copper oxide NPs (CuO NPs).133 The tested NPs shapes included surface morphologies such as rice grain-like, needle-like and platelike. Among these differently shaped CuO nanomaterials, the plate-like CuO displayed better antibacterial activity than grain or needle shaped nano-CuO. Recently, Perelshtein and co-authors have reported that the antibacterial properties of metal oxide NPs (MeO NPs), namely ZnO and CuO, depends on their crystalline structure, whereas the presence of more defects and less organized structure leads to higher toxicity.134 Their study compared the activity of commercial and sonochemically produced NPs. A significant increase in the production of ROS was found in the more defective, sonochemically prepared metal nanooxides, as compared to the commercial ones with the same particle size range. The bactericidal efficiency of the metallic NPs may be also affected by the type of microorganism i.e. to be strain-specific. For instance, E. coli, S. aureus and Bacillus subtilis (B. subtilis) bacteria strains susceptibility to silver and copper NPs (CuNPs) was studied.135,136 The E. coli and S. aureus were more susceptible to the effect of the AgNPs compared to the CuNPs, while B. subtilis depicted the highest sensitivity to NPs compared to the other strains and was more adversely affected by the CuNPs.135 The greater biocidal efficiency of AgNPs for E.coli (Gram-negative) than to S. aureus (Gram-positive) was attributed to the difference in cell wall structure between Gram-negative and Gram-positive microorganisms.137 The antibacterial properties of metal NPs were largely studied against human pathogenic bacteria such as P. aeruginosa, E. coli, S. aureus, Klebsiella pneumoniae and the antibiotic resistant bacteria as methicillin-resistant Staphylococcus aureus (MRSA). For instance, 23 copper,138 CuO,131,139 zinc140,141 and ZnO,142 titanium143 and TiO2,144 MgO,145 gold,146 alginate147 and silver140,143,146 are among the NPs that antibacterial effectiveness against bacteria, viruses and other eukaryotic micro-organisms have been proved. Silver and zinc NPs supported on various suitable materials, such as carbon, polyurethane foam,148 polymers and sepiolite149 have been effectively used for biological applications due to their bactericidal properties. In the medicine, materials comprising nano-silver are used also to prevent bacteria colonization on catheters,150 contact lenses,151 dental materials,152 medical instruments,153 and human skin.154 The application of AgNPs in the medical and pharmaceutical fields is widely exploited due to their high thermal stability, low volatility and low toxicity to human cells. The AgNPs are among the approved by the U.S. Food and Drug Administration nano-devices for antibacterial wound dressings. Silver-containing materials can be also employed to eliminate microorganisms on textile fabrics,155,156 or they can be used for water treatment purposes.157 In fact, by far silver is the most widely used antimicrobial material incorporated in the form of nano-silver in functional textiles. For example, powdered AgNPs were prepared using alkali dissolved starch as reducing and stabilizing agent followed by applying these particles to cotton fabrics. The developed material showed potential as wound dressing material as it demonstrated good antibacterial and anti-inflammatory properties.158 In another approach, AgNPs impregnated paper as point-of-use device for microbial purification of drinking water was designed. The embedded in the blotter paper NPs release was minimal, with values under the WHO limit of 0.1 ppm for silver in drinking water.159 Composite metal-metal NPs have been also applied in biomedical areas as antibacterial agents as well as for bioseparations, targeted drug delivery and magnetic resonance imaging. The combined use of silica NPs with the other biocidal metals such as silver has been extensively studied in the recent years. For example, the production of novel antimicrobial silver–silica nanocomposite material was reported.160 The results revealed that the nanocomposite have better antimicrobial effect against a wide range of microorganisms compared to conventional materials, such as silver nitrate and silver zeolite. In another study, silica nanowire substrates decorated with Ag- or CuNPs have demonstrated strong antibacterial activity against E. coli. However, while silver decorated silica nanowires were found to be biocompatible with human lung adenocarcinoma epithelial cell line A549, the copper decorated silica nanowires showed high cytotoxicity.161 In another study, the antibacterial activity of nanostructured ZnMgO was compared to its pure nano-ZnO and 30 Moreover, the fabrics coated with NPs after preliminary treatment with enzyme showed uniform NPs dispersion along the fibres, and particle sizes in lower nanometric range in comparison with non-treated fabric. Perelshtein et al. have applied one-step sonochemical process to obtain antimicrobial coatings of titanium dioxide NPs (TiO2 NPs) on cotton. The produced nano coatings displayed antimicrobial effect (when light illuminated) against common antimicrobial pathogens.100 In another study, ZnO NPs were synthesized and simultaneous deposited on the surface of cotton bandages using US irradiation. The formed ZnO NPs were homogeneously dispersed on the cotton surface. In line with the concerns for the NPs toxicity, the authors performed leaching test for evaluating the stability of the coatings. It was found that the concentration of zinc ions in the leaching solution increases by increasing the temperature, however, no NPs presence in the solution was detected by transmission electron microscope (TEM) and dynamic light scattering (DLS) analysis. These findings indicated that during the sonochemical process the NPs were strongly anchored to the textile substrate. The coating of fabrics with polymer NPs by US was also demonstrated. Tannic acid NPs were synthesized from an aqueous solution without the use of stabilizers and embedded on to cotton textiles in a one-step sonochemical process.103 The obtained materials possessed antimicrobial and anti-inflammatory properties. The anti-inflammatory properties were assessed in terms of inhibition of inflammatory enzymes (myeloperoxidase (MPO) and collagenase) activities. It was found that the inhibitory effect of the tested specimens was directly proportional to the concentration of organic compound used for bandage coating. The authors assumed that tannic acid NPs exhibited their functions from the surface of the cotton bandages. In recent years, Morsali and coworkers have demonstrated in several studies the NPs coating of polyester and silk fibers through sequential dipping steps in alternating bath of metal salts under US.156,210,211 For example, silver bromide NPs were coated onto polyester fabrics by sequential dipping steps in alternating bath of potassium bromide and AgNO3 under US irradiation.156 It was demonstrated that the NPs size and morphology depend on the US power, sequential dipping steps and precursor concentration. The results suggested that an increasing of the sequential dipping steps and salts concentration lead to an increasing of the particles size, while an increase in the sonication power is accompanying with decreasing the size of the particles. 31 2.4.2 Potential of sonochemical processing in manufacturing of functional materials The use of US in laboratory reactions is becoming commonplace, and the extension of the technology to industrial-scale reactions is likely to follow. Underlying these developing technologies are the recent advances in our understanding of the nature of cavitation and the chemical effects of US. The sonochemical coating route could be easily scaled-up to meet the requirements of large-scale textile coatings. Recently, the European project SONO (FP7-228730, http://www.fp7-sono.eu/), where our group was a key partner, demonstrated the development of a pilot line for the production of medical antibacterial textiles based on sonochemistry. The antibacterial efficiency of the produced medical textiles have been reported in several research papers30,204,208 and in validated in a clinical study.209 Pilot scale installation for sonochemically assisted coating of textile fabrics with various kinds of NPs was designed by Abramov et al. (Figure 2.8).30 The principle of the developed machine (operating in a roll-to-roll mode) involved feeding the fabric through a gap between two flat vibrating plates driven by magnetostrictive transducers. The speed of the moving fabric could be regulated between 0.004 and 0.050 m/s. Up to 50 m of fabric can be continuously coat with CuO and ZnO NPs using this pilot machine. The obtained textiles were characterized in terms of coating morphology, structure and stability to washing. The antibacterial assay of the coatings demonstrated that the loading of the nano-oxide appear to be decisive for their biocide performance. The obtained cotton fabrics displayed good antibacterial activity towards E. coli, K. pneumoniae, S. aureus and MRSA.212 32 Figure 2.8 Photo of the roll-to-roll sonochemical pilot installation. In the inset the CuO-coated and uncoated spools are shown. The front panel is open to show the change in the color of bandage after coating with CuO NPs. In the inset, one can observe that with the deposition of CuO NPs the color of the bandage changed from white to brown. When the ZnO is deposited on the bandage, the white color is retained.30 2.4.3 Antimicrobial nanocomposites for water disinfection Constructed wetlands in various designs are the most versatile systems for removal of pesticides, heavy metals, polycyclic aromatic compounds and pharmaceutical residues.214– 217 Currently, a major issue with these systems is related to the additional microbial contamination, especially the growth of undesired pathogen bacteria. Microbiologically unsafe water is a global problem and among major causes of preventable morbidity and mortality. The WHO reports that by 2025 half of the world’s population will be living in water-stressed areas with access only to inadequate drinking water.218 One such example is microbiologically contaminated water associated with transmission of so-called bacteria waterborne diseases such as diarrhea, cholera, dysentery and typhoid fever.219 Annually 2.2 million diarrheal disease deaths are being linked with the consumption of contaminated water.220 The concerns about these diseases have even worsened during the last years due to the effects of global warming, namely extreme rainfall and flooding that contribute to the inordinate microbial proliferation in surface and groundwater.221,222 33 Strategies to efficiently recover wastewater acceptable for domestic or recreational purposes are therefore considered of global importance. Some conventional methods for treating microbiologically unsafe water, e.g. boiling, reduce most waterborne pathogens and it is nowadays widely used to disinfect household water.223 However, these methods are only efficient for small volumes. Whenever the purification of larger volumes is required, the disinfection process involves the use of chemicals, such as chlorine, chloramines, iodine or ozone.224,225 Among them, the most accepted is the chlorination due to its efficiency and low price. Nevertheless, chlorine disinfection leads also to the formation of by-products as trihalomethanes,223,226,227 which are related with developing certain cancers or adverse reproductive outcomes.228,229 Many researchers are therefore investigating new sustainable techniques to disinfect large volumes of water without endangering the environment and the human health.230,231 Recently, considerable interest has arisen in the application of nanotechnology for water purification. In particular, functional antibacterial NPs and their potential for disinfection or microbial control in system level water treatment. Nanomaterials are providing novel opportunities to develop more efficient and cost effective nano-structured and reactive membranes for water purification. Several nanomaterials, such as silver, CS, and TiO2, have found applications as antimicrobial agents in diverse products and industrial processes, including for water treatment.224 Among all, nano-silver have been widely used for water filtration applications to inactivate bacteria232 and viruses.233,234 Moreover, AgNPs have been demonstrated to possess remarkable antimicrobial properties when employed in very low concentrations without harmful side effects.235–238 Another NPs that have great potential as water purification catalysts and redox active media are the TiO2 NPs. TiO2 NPs can be employed as both oxidative and reductive catalysts to degrade organic and inorganic pollutants; to reduce toxic metal ions under UV and as antibacterial agents for water purification.239240 However, for disinfection purposes the NPs immobilization on preferably low-cost materials is necessary, where the permanent NPs binding ensures the disinfection efficiency during long-term exploitation.241 For example, cellulose acetate hollow fiber membrane loaded with AgNPs for water treatment were prepared via dry-jet wet-spinning technique. The fibers were active against E. coli and S. aureus after being immersed in water bath for 180 days, despite that 60 % of the initially loaded silver remained. However, during filtration the silver content in the hollow fiber was depleted quickly within 5 days and the material lost it antibacterial activity against both tested bacteria strains, meaning that silver content must 34 be periodically replenished after permeation.232 In another approach, a two-step process was used to design composite membranes on a glass support consisting of 50 nm thick chitosan layer on a poly(acrylic acid)/poly(ethylene glycol) diacrylate layer. The obtained material possess antibacterial activity towards S. aureus and E. coli bacteria, and the antibacterial activity of the membrane improved with increasing chitosan content.242 In a similar way, nano-composite membranes incorporating other functional (e.g. catalytic, photocatalytic and antimicrobial) NPs into water treatment membranes can be developed. The strategies for the NPs immobilization of onto various 2D or 3D macroscopic surface mainly involve the NPs modification with polymers or molecules containing functional groups allowing their further anchoring on a target surface. However, for achieving this goal the use of facile and environmentally friendly method is an important issue. The potential application of bio-catalysts (enzymes) could results into direct immobilization of various functionalized NPs onto different substrates under mild conditions. 2.4.4 Antimicrobial hydrogels for wound treatment Wide range of new functional dressing materials are frequently introduced to target different aspects of the wound healing process as a result of the existing variety of wound types (acute or chronic). The ideal wound dressings are defined as those capable to achieve rapid healing at reasonable cost coupled with minimal inconvenience to the patients. The wound treatment should be based on the controlled-release delivery of active substances directly into wound sites which promotes the wound healing process such as grow factors,243,244 antimicrobials245 or inhibitors of deleterious enzymes found in wounds.246 Many of the new topical dressings are based on novel polymers and are employed for drugs’ disposal due to their physical characteristics (e.g. fluid affinity, water uptake, rheological properties) but mainly because of their suitability for delivery of therapeutic agents when maintaining the wound surfaces moist. Hydrogels are three-dimensional structures with high water content often comprising covalently cross-linked hydrophilic polymers. The extent of cross-linking allows for tuning of physical, chemical and biological properties, which in the case of biopolymers are usually coupled with the intrinsic biocompatibility and similarity with the ECM of various tissues. Hence, biopolymer hydrogels are used for a wide range of biomedical applications such as drug carriers or composite matrices for tissue engineering.247,248 For instance, they appear attractive alternatives to conventional wound dressings to overcome their common 35 disadvantages such as low flexibility, lack of porosity and high adherence to wound surface that provokes painful removal.249 Additionally, the hydrogel-based wound materials provide and maintain beneficial for wound healing moisture environment. Compounds with intrinsic antimicrobial properties, e.g. polyphenolic molecules or metal oxides, could be added to polymer mixtures prior to gelation, targeting their sustained or controlled release from the hydrogel platform.250,251 In one such approach, plant polyphenols were encapsulated for protection and delivered from the biopolymeric NPs or matrices to improve the resistance of cells to oxidative stress, and to inhibit bacteria growth and deleterious chronic wound enzyme activities.246,252 Their processing into nanoscale particles is yet another attempt to improve their bioavailability and increase the efficiency in order to reduce the administration doses.253 Further integration of the NPs within a hydrogel platform aiming their protection and controlled/sustained release is expected to aid in the development of more efficient drug delivery systems for e.g. medical applications.251 Polysaccharides, such as cellulose and starch, are the most abundant natural polymers in the biosphere. Naturally occurring alginate and chitosan are well known wound management materials due to their biological and haemostatic properties, which also facilitate the wound healing process.254 These are often applied in combination with pharmaceutical agents as bioactive dressings capable for sustained, and thus prolonged delivery of actives to the wound site.255 CS and its derivatives are potentially exploitable in the surface modification of colloidal carriers to modulate the bio-distribution of drugs and/or control their absorption.256–258 CS materials have been widely used as a topical dressing to treat wound and burn infections not only because of its intrinsic antimicrobial properties, but also by virtue of its ability to deliver extrinsic antimicrobial agents to wounds and burns. Another, promising approach for designing of hydrogel wound dressings are based on collagen or glycosaminoglycans (GAG) such as hyaluronan and chondroitin sulfate, which are found in the ECM.259,260 The GAG molecules can be chemically modified, creating new bio-materials with important bio-medical applications e.g. delivery of growth factors to the wound or as scaffolding material for tissue regeneration.261,262 Dressings prepared from chemicallycrosslinked GAG hydrogel films acts as bio-interactive wound dressings participating actively in the wound healing process rather than simply being passive barriers to desiccation. To achieve the polymer gelation different physical and chemical cross-linking strategies have been employed. The physical cross-linking can be achieved by changing the physical entanglements or by non-covalent physical associations, such as secondary forces 36 (hydrogen, ionic, or hydrophobic bonding).263,264 For example, the physical characteristics of the hydrogels could be modulated to the desired ones by changing pH, ionic strength and temperature of gelation.265,266 It was demonstrated that the mechanical properties of collagen gels are easily tuned by varying those parameters.267 In another study, it was shown that the ionotropic gelation method for formation of crosslinked CS can be modified from ionic crosslinking to deprotonation by adjusting the pH of tripolyphosphate. In such conditions the CS ionic cross-linking takes place at lower pH, while the deprotonation mechanism occur when higher pH is used.268 A powerful approach for the generation of new materials with desired properties are the supramolecular interactions. Such hydrogel materials based on supramolecular self-assembly synthesis attracted special interest in the biological and electronics fields. The supramolecular self-assembly of cyclodextrins and polymers has led to the development of novel supramolecular hydrogels for drug delivery applications. For example, supramolecular hydrogel suitable for relatively long-term sustained controlled release were formulated based on the complexation α-cyclodextrin with various triblock copolymers bearing a hydrophobic block.263,269 The general advantage of physically crosslinked hydrogels lies on a forming a gel without the need for chemical modification or the addition of cross-linking entities which make them suitable for in vivo practices. However, such networks held together by physical interactions only form mechanically weak scaffolds, resulting in fast erosion or degradation which limits their applications.270 Because of these it is difficult to decouple hydrogels variables such as gelation time, network pore size, chemical functionalization, and degradation time, restricting the design flexibility.271 The presence of non-homogeneities, free chain ends or chain loops and defects due to the formation of clusters of molecular entanglements, or hydrophobically- or ionically-associated domains can be also observed. A low viscosity liquid phase or syneresis of the hydrogel after the gel formation is another drawback of solely physical networks.272 For these reasons, chemically cross-linkable systems allowing the formation of covalent bonds in situ have been developed. The chemical cross-linking process requires the addition of small molecules or conjugated directly to reactive pre-polymers. Chemical cross-linking of biopolymers to produce hydrogels results in higher mechanical properties compared to the physically assembled materials. However, the residual toxic chemicals and organic solvents employed often make them unsuitable for biomedical applications due to the toxic effects on human cells.273,274 The formation of free radicals or the nonselective reactivity with nucleophiles found in biomolecules such as proteins can cause undesired side reactions such as inflammation.275,276 As an alternative, covalently cross-linked gels could be obtain in situ via 37 enzyme-catalyzed reactions applying mild conditions and avoiding the use of harsh chemicals.277,278 Such enzyme cross-linking systems are exploitable for encapsulation of various bio-entities (e.g. active agents and/or cells) and for further application in the pharmaceutical industry.279 For example, peroxidase-catalyzed oxidation of CS derivatives was used for the preparation of biocompatible hydrogels for cartilage tissue engineering and drug delivery.280,281 The sustained release of therapeutic proteins produced by genetically engineered to secrete an anticancer drug (interleukin-2) cells through 4 % gelatin hydrogels enzymatically cross-linked by microbial transglutaminase was recently reported. The findings in this study have demonstrated that the enzymatically cross-linked hydrogels were not only biocompatible, but also have suitable transport properties that would facilitate the design of sustained drug release devices.282 38 3 Objectives of the thesis 39 46 remove the loosely fixed particles and chitosan. Control samples were prepared by deposition of ZnO NPs alone. 4.4.1.2 Quantification of deposited ZnO The amount of ZnO embedded on the fabrics was determined after extraction with 0.5 M nitric acid. The concentration of solubilized Zn2+ ions was probed by inductive coupled plasma atomic emission spectroscopy (ICP – AES) analysis using ULTIMA JY2501 (France). The obtained results are reported as mean values ± standard deviation (n = 3). 4.4.1.3 Characterization of the coatings and nanoparticles The surface morphology of the coatings and size of the deposited NPs were studied with high resolution scanning electron microscope (HRSEM) model Quanta 200 FEG from FEI (USA). Additionally, the presence of zinc and chitosan on the coated fabric was detected by energy dispersive X – ray spectroscopy (EDS). ZnO/CS mass and molar ratios were determined by X-ray photoelectron spectroscopy (XPS) on a SPECS system equipped with an Al anode XR50 source operating at 150 mW and a Phoibos 150 MCD-9 detector (Germany). The morphology of the ZnO NPs in the remaining after the sonochemical process suspensions was investigated by a Ziess Neon FIb microscope (Carl Zeiss, Germany) in scanning transmission electron microscopy (STEM) mode operating at 30 kV acceleration voltage. The samples for observation were drop-casted on a TEM holey carbon grid. Additionally, STEM images were taken to evaluate the leaching of the NPs from the fabrics. Prior to STEM analysis the coated fabrics were incubated for 1 h in 0.3 mM KH2PO4 solution at 37 °C and 230 rpm. 4.4.1.4 Antibacterial tests Antimicrobial activity of the coated samples was assessed according to the standard shake flask method, recommended by the American Society for Testing and Materials (ASTM) for permanently immobilized active agents on fabrics (ASTM-E2149-01). The method provides quantitative data for measuring the reduction rate in number of colonies formed, converted to the average colony forming units per milliliter of buffer solution in the flask (CFU/mL). For preparation of E. coli and S. aureus suspensions, a single colony from the corresponding 47 stock bacterial cultures was used. The culture was then inoculated overnight in 20 mL sterile nutrient broth (NB, Sharlab, Spain) in a 100 mL Erlenmeyer flask and incubated at 37 ºC and 110 rpm. The inoculated bacterial culture was diluted with sterile buffer (0.3 mM KH2PO4) until solution absorbance of 0.28 ± 0.01 at 475 nm was reached, which corresponds to 1.5 ÷ 3.0 x 108 CFU/mL. This bacterial culture was then diluted appropriately into sterile buffer solution to obtain a final concentration of 1.5 ÷ 3.0 x 105 CFU/mL (working bacterial dilution). Thereafter, the cotton fabrics (0.035 g) were incubated with 5 mL of bacterial suspension at 37 ºC and 230 rpm. For determination of the inoculum cell density the suspensions were withdrawn before introducing the textile sample and after 15, 30 and 60 min in contact with the fabrics. These suspensions were serially diluted in sterile buffer solution, plated on a plate count agar and further incubated at 37 ºC for 24 h to determine the number of surviving bacteria. Antimicrobial activity is reported in terms of percentage of bacteria reduction calculated as the ratio between the number of surviving bacteria before and after the contact with the coated textiles using the following formula: Bacteria reduction (%) = ((A-B) / A) x 100 (Equation 4.1) where A and B are the average number of bacteria before and after the contact with the coated textiles, respectively. The durability of the antibacterial effect was evaluated after performing 10 washing cycles in a laboratory dyeing machine (Ahiba Nuance, Datacolor ) at 75 °C, 30 rpm, for 15 min with 0.1 g/L non-ionic surfactant Cotemol NI (Colorcenter, Spain), in liquor to good ratio 30:1. 4.4.1.5 Cell culture The BJ-5ta cells (human foreskin fibroblasts, ATCC-CRL-4001) were maintained in 4 parts Dulbecco’s Modified Eagle’s Medium (DMEM, ATCC) containing 4 mM of L-glutamine (ATCC), 4500 mg/L glucose, 1500 mg/L sodium bicarbonate and 1 mM sodium pyruvate, and 1 part of Medium 199 supplemented with 10 % (v/v) of fetal bovine serum (FBS) and 10 g/mL Hygromycin B, at 37 ºC in a humidified atmosphere with 5 % CO2. The culture medium was replaced every 2 days. At pre-confluence, cells were harvested using trypsin-EDTA (ATCC-30-2101, 0.25 % (w/v) trypsin / 0.53 mM EDTA solution in Hank’s BSS without calcium or magnesium) and seeded at a density of 4.5 x 104 cells/well in a 96-well tissue culture-treated polystyrene plate (Nunc, Spain). 48 4.4.1.6 Cytotoxicity evaluation by indirect contact Coated fabric samples (25 mg) were first sterilized under UV light for 1 h. The samples were then placed in contact with 3 mL of complete growth medium (DMEM) in a CO2 incubator at 37 °C for 1 and 7 days. At the end of these periods, the samples were removed and the growth media withdrawn. Medium without the contact with the cotton was used as a negative control, whereas a 30 % dimethyl sulfoxide (DMSO) prepared in fresh culture medium was used as a toxicity positive control. The previously seeded cells were put into contact with the withdrawn culture media and incubated at 37 °C in a humidified atmosphere of 5 % CO2 for 24 h. The cells were further examined for signs of toxicity using Alamar Blue assay kit (AlamarBlue®, Invitrogen). Resazurin, the active blue ingredient of the kit, is a nontoxic, cell permeable compound that, once in a viable cell, is reduced to red colored resorufin. AlamarBlue® was diluted in culture medium and added to each well after aspirating the culture medium containing the samples. After 4 h incubation at 37 ºC the absorbance at 570 nm was measured, using 600 nm as a reference wavelength, in a microplate reader Infinite M200, Tecan (Austria). The quantity of resorufin formed is directly proportional to the number of viable cells. The error bars for each data are the standard deviation of three independent measurements. No cell viability was detected after the contact with the positive control regardless of the incubation time (data not reported). 4.4.2 Simultaneous sonochemical enzymatic coating of textiles with antibacterial ZnO nanoparticles 4.4.2.1 Cellulase activity measurements The cellulase activity was measured using the filter paper (FPU) assay.284 Reactions were carried out in 50 mL test tubes with Whatman No. 1 filter paper strip (1 x 6 cm, 50 mg) in 1.0 mL distilled water and 0.5 mL enzyme formulation. The mixtures were incubated at temperatures ranging from 20 to 65 °C for 1 h. Thereafter, a colorimetric reagent (DNSA) was added to quantify the amount of reducing sugars. The DNSA solution was prepared by dissolving 120 g sodium potassium tartrate in 80 mL of previously heated (60 °C) 0.2 M 49 NaOH, prior to addition of 200 mL of 96 mM DNSA and volume completion to 400 mL with distilled water. The reaction mixtures were placed in a boiling water bath for 5 min, cooled to room temperature and diluted with 20 mL distilled water prior to measuring the absorbance at 540 nm with Infinite M200 (Tecan, Austria) multiplate reader. All experiments were performed in triplicate. 4.4.2.2 Enzyme stability in US field One mL of cellulase product diluted to 50 mL with water was subjected to US irradiation (20 kHz, 21.5 W, 17.30 W/cm2, 0.43 W/cm3 and 35 % of amplitude) for 30 min at temperatures ranging from 20 to 60 °C. After the treatment, enzyme aliquots (0.5 mL) were incubated for 1 h at 55 °C with Whatman No. 1 filter paper strip (1 x 6 cm) and the reducing sugars released were measured using the FPU assay as previously described. The residual enzyme activity was calculated as a percentage of the activity of the enzyme not exposed to US. The possible effect of the sonochemical treatment on the tertiary and secondary structure of cellulase was assessed by measuring the intrinsic fluorescence of the enzyme as a result of protein unfolding and denaturing. For the purpose of the assay, cellulase water solutions not exposed (control) and exposed to US treatment (30 min) were evaluated. The fluorescence was measured at room temperature (25 ± 1 °C) with Quanta Master 4 spectrofluorometer (PTI, USA) at 280 nm excitation wavelength (slit = 2 nm), 300 - 450 nm emission wavelength (slit = 2 nm) and 1200 nm/s of scanning speed. 4.4.2.3 Effect of the US parameters on the enzyme performance To study the hydrolytic potential of the cellulase toward cellulose substrate under sonication, the US amplitude of vibration (and thus intensity) was varied to determine its effect on the yield of the enzyme catalyzed reaction expressed in reducing sugars concentration. For this aim, 1 mL of cellulase solution (diluted to 50 mL with water) was subjected to ultrasonic irradiation at different US amplitudes (range of 20 – 40 %) for 30 min at 55 °C and in presence of 0.5 g of Whatman No. 1 filter paper as a substrate. Thereafter, the liberated reducing sugars were determined using the aforementioned method (section 2.4.2.1., FPU assay). Control treatments without enzyme were carried out in parallel. All results are reported as mean values ± standard deviation (n = 3). 50 4.4.2.4 Ultrasound-enzyme assisted coating of cotton with ZnO NPs The sonochemical coating was carried out using an ultrasonic transducer Ti-horn (20 kHz, Sonics and Materials VC750, USA). The US intensity (17.30 W/cm2), density (0.43 W/cm3) and power (21.5 W) used for the textile treatment were calorimetrically determined as described in 2.4.1.1. The cotton samples (5 x 10 cm, approx. 0.7 g) were immersed in the ultrasonic pot containing 50 mL ZnO NPs aqueous solution (1 mM) and 2 % of weight of fabric (owf) cellulase formulation and the coating of the cotton samples was carried out during 30 min at 55 ± 2 °C and amplitude of 35 %. To maintain the fabric at the bottom of the US pot without using any additional accessories, the fabric sample was cut bigger than the diameter of the pot (4 cm). Thereafter, the sample was folded in a way that its diameter is slightly wider than the diameter of the pot, thereby once placed at the bottom of the pot the contact/friction of its edges with the walls prevents it from moving during the sonochemical process. The sample area exposed to the US irradiation was used for the further experiments. The ultrasonic horn was dippen 1 cm in the ZnO dispersion at a distance from the fabric (at the bottom of the ultrasonic pot without any additional support) of approximately 3 cm. Thereafter the samples were thoroughly washed with distilled water to remove the loosely fixed particles. Controls were prepared by sonochemical deposition of ZnO NPs in presence of temperature-denatured enzyme. 4.4.2.5 Characterization of the coatings The quantification of the amount of ZnO deposited on the fabrics (by ICP – AES) and the surface morphology studies of the coatings (by HRSEM) were carried out as described previously in section 4.4.1.2. For both washed and unwashed samples, the HRSEM images (section 4.4.1.2) were obtained from fabrics treated in three independent experiments. ImageJ software was used to build three size histograms of the ZnO NPs deposited on to the fabrics. 4.4.2.6 Antibacterial test The antibacterial performance of the coatings were assessed as previously described in Section 4.4.1.4 51 4.4.3 Enzyme-assisted functionalization of cork surface with antibacterial biopolymer/silver hybrid nanoparticles 4.4.3.1 Preparation of biopolymer-doped silver nanoparticles Biopolymers, CS and aminocellulose (AC) were dissolved to reach 0.8 % (w/v) in 1 % CH3COOH (CS) and distilled water (AC). The pH of the solutions were adjusted to 5 by adding 2 M NaOH in case of CS and 1 M HCl in case of AC. AgNPs synthesis was carried out by chemical reduction of Ag+ to elemental silver (Ag) using NaBH4 (17.5 mg/mL in dH2O), starting from aqueous solutions of AgNO3 (5 mg/mL) in presence of CS or AC. First, 2 mL of NaBH4 was added to 30 mL of a biopolymer solution under vigorous stirring, and then AgNO3 was slowly added to reach the final volume of 50 mL. 4.4.3.2 Characterization of biopolymer-doped silver nanoparticles The hybrid structures were characterized using ζ potential for the particles charge, DLS for the mean particle size, UV-Vis spectrophotometry to check for the presence and intensity of the AgNPs surface plasmon resonance (SPR) band, and STEM to visualize the particles and evaluate their stability in the obtained dispersions. ζ potential was measured using Malvern ®Zetasizer Nano ZS, DLS measurements were performed using DL135 Particle Size Analyzer (Cordouan Technologies, France). Three samples of each AgNPs dispersion (in absence and presence of CS of AC) were processed acquiring 5 measurement cycles with 1 % signal-to-noise ratio. The data were analyzed using NanoQ 1.2.1.1 software. UV- Visible spectra were recorded between 300-600 nm for the dispersions diluted 100 times in distilled water using a Cary 100 Bio spectrophotometer (Varian). The AgNPs in the dispersions were visualized by a Zeiss Neon FIB microscope (Carl Zeiss, Germany) in STEM mode operating at 30 kV acceleration voltage. The samples for observation were drop-casted on a TEM holey carbon grid. 4.4.3.3 Immobilization of the hybrid biopolymer-doped AgNPs onto cork Prior to the treatment with biopolymer-doped AgNPs, the cork granules were cleaned with aqueous solution of HCl (pH 2), distilled H2O, NaOH solution (pH 10) and finally with 96 % EtOH, and dried at 60 ºC for 12 h. Thereafter, 1 g of the material was placed in a mixture containing 18 mL of AgNPs dispersion (pure, doped with CS or doped with AC – immediately after their preparation), 18 mL of 0.1 M succinic acid/succinate buffer (pH 5), and 0.5 mL of 52 laccase (final concentration 0.1 U/mL). The reaction was allowed to proceed for 24 h at 50 °C and 30 rpm in a laboratory dying machine Ahiba (Datacolor). Control samples were also prepared using the same treatment conditions without laccase (in presence of hybrid biopolymer-AgNPs) and with laccase and CS or AC alone (without AgNPs). The treated cork was washed thoroughly with water to remove the loosely fixed particles and NaBH4 from the material, and finally dried at 50 ºC for 12 h. Fourier transform infrared spectroscopy (FTIR) was used to analyze the surface of the treated cork. IR spectra of the samples were collected in the range of 4000-600 cm-1 using Perkin-Elmer Spectrum 100 (USA) equipped with universal ATR sampling accessory, performing 64 scans for each spectrum. Scanning electron microscopy (SEM) was performed to examine the morphology of the AgNPs-biopolymer hybrids immobilized onto cork and to examine the presence of bacteria on the non-modified cork (control) and on the cork-AgNPs-CS composite. The micrographs with magnification ×30K were obtained using a Zeiss Neon FIB microscope (Carl Zeiss, Germany) operating in SEM mode. 4.4.3.4 Antimicrobial activity of AgNPs embedded cork matrices The antimicrobial performance of treated cork matrices was assessed against E. coli and S. aureus using the dynamic shake ASTM E 2149-01 test, as described in section 2.4.1.4. The results were expressed as log10 of CFU/mL of buffer solution in the flask. Besides the unwashed matrices (only rinsed with water after the treatment), the antibacterial performance of the untreated and treated cork was evaluated after immersing those in water for 1, 2 and 5 days at room temperature while stirring (100 rpm) to mimic the environment in which the materials are intended to be used, i.e. water flow in constructed wetlands. Finally, the antimicrobial activity after 5 days washing is also expressed as a reduction percentage of the survived bacteria colonies. The results were expressed as log reduction in CFU counts. To study the viability of the bacteria accumulated on the cork surface, unmodified and functionalized with AgNPs-CS cork pieces were incubated with E. coli contaminated water for 16 h. Thereafter, the cork samples were washed with sterile distilled water. To grow the alive bacteria, five of the washed cork granules were placed on Coliform agar and incubated at 37 °C for 24 h, while another five granules were used for SEM measurements in order to detect the presence of bacteria on the materials’ surface. Before the SEM analysis the 53 bacteria accumulated on the cork were subjected to fixation procedure preserving their initial structure. Briefly, the samples were placed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 30 min, then washed twice with sterile PBS, and placed in 25, 50, 75 and 96% ethanol for 10 min. The fixation was conducted at room temperature. 4.4.4 Enzyme-assisted formation of nanospheres-embedded hybrid biopolymer hydrogels for wound healing 4.4.4.1 Nanospheres preparation and characterization NSs were generated by applying 20 kHz ultrasonic irradiation (0.43 W/cm3 intensity and 35 % amplitude) on the interface of water/oil mixture (15:6 (v/v)) containing 0.5 mM EGCG for 3 min at 4 ºC. The NSs suspended in the aqueous phase were thereafter subjected to several cleaning steps after centrifugation (2000 rpm for 15 min) to remove the oil phase. Three samples of each NSs suspension were assayed by DLS at room temperature to determine the mean sphere size of the obtained phenolic NSs as described in section 2.4.3.2. The NSs morphology was evaluated using a NIKON Eclipse Ti−S fluorescence microscope (Nikon Instruments, Inc., The Netherlands). The phenol groups of EGCG NSs and free solution were estimated according to the Folin-Ciocalteu method for determination of total phenol content 285 by monitoring the reaction at 760 nm in a microplate reader (Infinite M200, Tecan, Austria) and the results were expressed in gallic acid equivalents (GAE). Prior measuring the absorbance, the samples were filtered through Millex® GP 0.22 µm PES filters (Merck Millipore Ltd., Spain) after which 200 µL of the filtrates were transferred to a 96-well microplate. 4.4.4.2 Myeloperoxidase inhibition The MPO inhibition with the EGCG NSs and free solution was measured using guaiacol as a substrate. The samples were serially diluted in 50 mM phosphate buffer, pH 6.5 (in a concentration range of 0.0025 to 0.001 mM) and further 450 µL of each dilution was incubated for 1 h with 20 µL of 0.60 U MPO and 30 µL of guaiacol (167 mM) at 37 °C. The mixtures were then filtered through Millex® GP 0.22 µm PES filters (Merck Millipore Ltd., Spain) and 200 µL of the filtrates were transferred to a 96-well microplate. The reaction was 54 started by adding 22 µL of 1 mM H2O2 and the absorbance increase was followed at 470 nm for 90 s. 4.4.4.3 Collagenase inhibition The collagenase activity was measured using EnzChek® kit. Briefly, 80 µL of the EGCG free solution or NSs suspension, 100 µL of collagenase (0.5 U/mL) and 20 µL gelatinfluorescein conjugate (to a final substrate concentration of 100 µg/mL) were incubated at room temperature protected from light. After 6 h the fluorescence was measured (493/528 nm) using a microplate reader Infinite M200, Tecan (Austria). For each sample, the background fluorescence values derived from the controls without the enzyme were subtracted. The measurements were performed in triplicate and the results are expressed as the percentage of collagenase inhibition compared to the control without EGCG. 4.4.4.4 Antioxidant activity Radical scavenging activity of the EGCG NSs and free solution were determined by measuring the decrease in absorbance of 1,1-diphenyl-2-picrylhydrazyl radical (DPPH•) at 517 nm. Different dilutions of the samples (100 µL) were incubated with 2.5 mL of DPPH• solution (9.84 mM) in methanol at room temperature in dark for 30 min. All experiments were carried out in triplicate. The antioxidant activity was calculated using the following formula: DPPH inhibition (%) = [1 – (A/Ao)] x 100 where A0 is the absorbance of the negative control (DPPH solution alone) and A is the absorbance in presence of the EGCG solution/NSs suspension. 4.4.4.5 Antibacterial tests S. aureus growth inhibition assay was performed in presence of the EGCG NSs or free solution. For the preparation of S. aureus suspension, a single colony from the corresponding stock bacterial culture was used. The culture was then inoculated overnight in 20 mL sterile NB (Sharlab, Spain) in a 100 mL Erlenmeyer flask and incubated at 37 °C and 110 rpm. In order to obtain the bacteria stock solutions the inoculated bacterial culture 55 was diluted with NB until reaching the absorbance of 0.01, monitored at 600 nm. Thereafter, 250 µL undiluted (0.5 mM) and diluted (0.25; 0.1; 0.05; 0.025 mM) in NB EGCG NSs/solution were incubated with 250 µL of bacteria stock solution for 24 h at 37 °C and 230 rpm. After, the suspensions were serially diluted in NB, plated on a Baird-Parker agar, and further incubated at 37 °C for 24 h to determine the number of surviving bacteria colonies. Antimicrobial activity is reported in terms of percentage of bacteria reduction calculated as the ratio between the number of surviving colonies before and after the contact with the samples using Equation 4.1 (Section 4.4.1.4), where A and B are the average number of bacteria colonies at 24 h for bacteria incubated without (control sample) and with EGCG NSs/solution, respectively. 4.4.4.6 Thiolation of chitosan The incorporation of thiol groups into chitosan was carried out following the procedure of Bernkop-Schnürch et al.286 First, chitosan was dissolved in CH3COOH (1% v/v) and TBA was added at the chitosan:TBA ratio 5:2 (w/w). The pH was then adjusted to 6 with 5 M NaOH. In order to avoid the oxidation of free thiols to -S-S- during the coupling reaction, dithiotreitol was added in a final concentration of 100 mM. The reaction mixture was stirred for 24 h at room temperature. The obtained thiolated chitosan (TC) conjugate was purified by 24 h lasting different dialysis steps: first three times against 5 mM HCl containing 1 % NaCl, then two times against 5 mM HCl. Thereafter, TC and the control (non-thiolated chitosan) were freeze-dried and stored at 4°C until further use. The free thiol content in the TC conjugate was determined spectrophotometrically using Ellman’s reagent287 and found to be 878±14 µmol/g of conjugate. 4.4.4.7 Hydrogels preparation and characterization Hydrogels were obtained by enzymatic cross-linking between 46 to 660 sec at room temperature, using horseradish peroxidase (9.65 U/mL) and hydrogen peroxide as an oxidizing agent. For the hydrogels containing EGCG NSs, the spheres were dispersed in 2 % TC solution in a spheres:TC 10/90 (v/v) ratio prior to cross-linking. The gelation time (the time which it takes for the formation of gel) was determined using a vial tilting method by regarding the gel state when the solution does not flow for 1min after inverting a vial.288 The 62 5.1.1 Introduction The properties of hybrid polymer-metal nanocomposites are influenced by the components interactions which affect the composite shape, size distribution and stability. Chitosan (CS) strongly complexes, unlike other natural compounds, with metal ions due to its free amine groups.289,290 Two models are proposed for hybrid CS and metal ions structures: “pendant model” where ion is bound to only one amino group of CS,291 and “bridge model” where ion is bound to several nitrogen atoms and hydroxyl groups of one or bridging more CS chains.292 This ability of CS was explored in our previous study293 to sonochemically synthesize zinc oxide (ZnO)/CS NPs (ZnO/CS NPs) and simultaneously deposit them on cotton fabrics. Here, the study is extended to the optimization of the sonochemical process in terms of metal oxide (MeO) concentration and reaction time in order to obtain efficient and durable antimicrobial textiles that do not cause toxic effects to human cells. Such development is in the scope of a general strategy to decrease the occurrence of nosocomial infections in clinical settings through sustainable manufacturing of efficient antimicrobial hospital textiles. The sonochemical process is carried out in aqueous solutions, in line with the current focus on development of environmentally friendly manufacturing technologies. The durability of the antimicrobial effect is also evaluated after multiple hightemperature washing cycles used in hospitals against two medically relevant bacterial species – S. aureus and E. coli. 63 5.1.2 Characterization of the coated fabrics ICP measurements were performed for determination of zinc on the cotton treated with different ZnO NPs concentrations in the presence and absence of CS. As expected, increased ZnO NPs concentration in the coating process led to higher amount of zinc deposited on the fabric (Figure 5.1A). On the contrary, considerably less amount of zinc was detected on the fabrics treated in presence of CS. Moreover, the EDS analysis of the hybrid coatings detected the presence of N and Zn on the fabric, thereby confirming the deposition of both CS and ZnO (Figure 5.1B). Figure 5.1 (A) Amount of zinc sonochemically deposited on cotton fabrics from solutions of ZnO NPs, in absence (light grey bars) and presence (dark grey bars) of 0.3 % (w/v) CS, and (B) EDS spectrum of cotton coated with 2 mM ZnO in presence of CS. XPS analysis was conducted to determine the molar and mass ratios of ZnO/CS on the cotton fiber surface. Due to the large experimental groups, XPS results presented herein are for the hybrid 2 mM ZnO NPs/CS coating. The energy spectrum of the photoelectrons, produced by the x-rays photoelectronic effect allows the determination of the sample composition (up to 10 nm sampling depth). The position of the peaks (binding energies of electron orbitals) in the spectrum and their relative areas are used to quantitatively identify the composition of the sample surface (Figure 5.2A). Figure 5.2B shows high - energy resolution carbon C 1s spectrum obtained for the hybrid coating. Three peaks are assigned to the different chemical bonds of carbon atoms, namely C-O (286.21 eV), C=O / O-C-O (287.29 eV) and C-H / C-C (284.80eV). The N 1s spectrum peaks (Figure 5.2C) with binding energies of 399.35 eV and 401.9 eV were assigned to the chitosan amino and protonated amino groups, respectively. Zn 2p 3/2 spectrum is presented with only one peak with binding energy at 1021.97 eV (Figure 5.2D).293 Atomic concentrations obtained from XPS analysis A B 64 for Zn (14.87 %) and N (85.13 %) were used for calculation the molar (14.19) and mass (0.08) ratios of ZnO/CS within the hybrid coating. Figure 5.2 N and Zn XPS spectra of cotton sonicated in the presence of 2mM ZnO NPs and CS. The HRSEM surface analysis of the sample coated only with ZnO showed a dense layer of NPs on the fibers (Figure 5.3A, B). Comparatively lower amount of NPs with bigger average size was found on the fabric treated with ZnO/CS system (Figure 5.3C, D), in good agreement with the ICP data. It is hard to distinguish between the individual and hybrid ZnO/CS particles, because chitosan itself is able to form nanospheres under sonochemical irradiation that are similar in shape to the ZnO NPs.293 Indeed, the pure CS coating (Figure 5.3E, F) appeared quite similar morphologically to the hybrid coating. Nevertheless, the NPs within the hybrid coating were bigger in size than the pure ZnO or CS particles, indicating that these probably were comprised of both components. It was reported that the mean diameter of CS NPs increased when metal ions were loaded to the polymer.294 On the other hand, the effect of CS on the metal NPs is resumed in its action as a controller of the nucleation or stabilizer.295,296 A B D C 65 Figure 5.3 HRSEM micrographs of cotton fabrics coated with ZnO (A, B), ZnO/CS (C, D), and CS (E, F). Composition of the starting solution: 2 mM ZnO NPs and 0.3 % (w/v) CS. The images on the left were taken with 10 000x, whereas the right-side images were taken with 200 000x magnification. 66 To explain the differences in the amounts of ZnO deposited on the fibers, the remaining after the sonochemical process solutions were evaluated for the presence of ZnO NPs using STEM. Given the lower amount of Zn detected on the fabrics treated in presence of chitosan, it was expected that higher amount of MeO particles would be found in the corresponding remaining solution compared to the one remained after coating in absence of chitosan. Surprisingly, the ZnO NPs density was also lower in the solution left after the hybrid coating (Figure 5.4A). The lower amount of NPs both on the fabric and in the remaining solution could be explained only if the effect of pH on the solubility of both ZnO and CS is considered. ZnO dissolution occurs over a wide pH range,297 whereas the oxide is stable at pH 7.2 due to a minimal interference of the dissolution products. On the other hand, due to intermolecular and intra-molecular hydrogen bonding, chitosan can be dissolved only in aqueous solutions of organic or mineral acids below pH 7. At these conditions the stability of ZnO rapidly decreases due to reaction with acidic substances. It is thus expected that the addition of CS dissolved in CH3COOH to ZnO aqueous suspension will decrease the pH of the system and affect the ZnO stability. After CS addition the pH of the ZnO NPs suspension indeed dropped from 8.2 to 6.9. Zn2+ complexation could also contribute to the lowering of the system pH. Similar behavior was already reported for various metal ion systems containing CS.298–300 The complex reaction could be described according to the Lewis acidbase theory, where the acid (Zn2+) is an acceptor of a pair of electrons, provided by the base (CS).298 Thus, the physicochemical properties of the individual composite components most probably dictate the formation of Zn2+/CS complex within the hybrid coatings. On the other hand, no NPs leaching was detected from the coated fabrics, revealing high stability of both ZnO and the hybrid coatings (Figure 5.4B). 67 Figure 5.4 STEM images taken to evaluate: (A) the NPs presence in the remaining solutions after the sonochemical coating of cotton fabrics with 2 mM ZnO in absence (top image), and presence (bottom image) of 0.3 % (w/v) CS; and (B) the NPs leaching into a solution incubated with the fabrics coated with ZnO (top image), and ZnO/CS (bottom image). 5.1.3 Antibacterial activity Both ZnO and CS are largely reported as efficient antibacterial agents. The most widely accepted mechanism of ZnO antibacterial action involves the oxide dissolution to Zn2+ further associated with oxidative stress in bacteria cells and generation of ROS.129,301 ROS cause inhibition of cell enzymes, lysosomal and mitochondrial damage, and consequently cell death.302 The antimicrobial effect of CS depends on its molecular weight and varies among different microorganisms.303,304 Among the proposed mechanisms of CS antimicrobial action are: i) binding to the cell DNA to inhibit the protein synthesis, and ii) interaction with negatively charged microbial membranes to alter the cell permeability.305,306 Recently, hybrid complexes of CS and metal ions showed several-fold enhancement of their A B 68 antibacterial activity as compared to the individual components.182,298 In addition, the antibacterial activity was directly proportional to the amount of metal ions in these complexes. In a next step, the antibacterial efficiency of the sonochemically generated on cotton fabrics hybrid Zn2+/CS was evaluated against two medically relevant bacterial species and further compared to the effect of pure ZnO coatings. The Zn2+/CS coatings reduced the viabilities of both S. aureus and E. coli, though to a different extent (Figure 5.5). As expected, the presence of chitosan enhanced the antibacterial effect of the coatings against both strains regardless of zinc concentration in the complex. However, the fabrics coated with the lowest NPs concentration (0.2 mM) in presence of CS brought about only 30 % reduction of bacteria viabilities. Increasing the amount of zinc in the coatings resulted in enhanced antibacterial effect. The coatings with 2 and 20 mM ZnO showed comparable antibacterial efficiency for both bacteria. Thus, all further experiments were carried out with the fabrics treated with 2 mM NPs, as this concentration was considered appropriate to produce efficient antimicrobial textiles. Figure 5.5 Antibacterial activity of ZnO (light grey bars) and Zn2+/CS (dark grey bars) coated cotton fabrics towards S. aureus and E. coli after 15 min of contact. In Table 5.1 the antibacterial activity of fabrics coated with 0.3 % (w/v) CS, 2 mM ZnO, and the corresponding hybrid is shown as a function of the incubation time. The hybrid coating led to 98 % reduction of bacteria growth for S. aureus after 60 min, whereas ZnO and CS alone reduced the bacteria growth by 61 and 31 %, respectively. It is worthy to mention that the CS coating reached its maximum activity against S. aureus already after 30 min, while 69 the longer incubation time resulted in improved antimicrobial effect for the coatings containing ZnO. In the case of E. coli a progressive improvement in antibacterial effect was observed for both CS and pure ZnO coatings. The different efficiency of the CS-containing coatings against E. coli (78 % after 60 min incubation) in comparison to S. aureus (31 %) could be due by the low Mw of the biopolymer (15 kDa) used in the study. Low Mw chitosans are reported to be more active against Gram-negative than against Gram-positive bacteria.304 On the other hand, the hybrid coating reduced the bacterial viability by more than 96 % even after 15 min of incubation. Nearly 100 % reduction was observed for both fabrics treated with ZnO and the hybrid coating after 60 min incubation. Table 5.1 Antibacterial activity of the coated fabrics against S. aureus and E. coli after different incubation time. Staphylococcus aureus Reduction in viability, % Coating 15 min 30 min 60 min CS 26.84 ± 4.07 33.00 ± 1.94 30.99 ± 1.81 2 mM ZnO NPs 15.39 ± 1.93 41.50 ± 2.12 60.77 ± 0.16 2 mM ZnO NPs/CS 63.15 ± 0.23 81.62 ± 3.58 98.48 ± 0.22 Escherichia coli Reduction in viability, % Coating 15 min 30 min 60 min CS 25.18 ± 1.02 72.66 ± 1.02 78.06 ± 0.51 2 mM ZnO NPs 79.88 ± 0.60 89.81 ± 1.31 99.86 ± 0.02 2 mM ZnO NPs/CS 96.60 ± 0.12 96.72 ± 0.18 99.88 0.04 5.1.4 Durability of antibacterial effect Despite the advances in antimicrobial finishing, the hospital textiles still become contaminated at use.307 Frequent laundering is therefore necessary in order to entirely prevent the transfer of pathogens. In order to evaluate the durability of the antimicrobial coatings, the fabrics were subjected to 10 washing cycles at 75 ºC and their antibacterial performance was assessed afterwards against the selected bacterial strains. The results 70 are expressed in percentage of remaining biocide activity compared to non-washed fabrics (Figure 5.6A). The ZnO coating retained about 50 % of its initial activity against both strains, whereas the hybrid coating preserved about 70 % and 85 % of the efficacy against S. aureus and E. coli, respectively (illustrated at Figure 5.6B). It is the presence of CS that improved the washing stability of the hybrid coating, as the individual CS coating was showed to maintain between 85 and 90 % of its initial activity. The sonochemical deposition of polymers onto solid surfaces usually results in their stable embedding on the substrate.308 Under ultrasound irradiation of liquids, the microjets and shock waves produced after cavitation collapse are able to drive the biopolymers at such high velocities towards a solid surface that fusion and strong adherence by physical or chemical interactions with the fabric occurs during collision.309 Conversely, sonochemically deposited metal oxides are not particularly stable and additional pre-treatment of the solid substrate are required for improved coating stability.141 Figure 5.6 (A) Percentage of remaining antibacterial activity of the coatings after 10 washing cycles at 75 °C against S. aureus (light grey bars) and E. coli (dark grey bars), after 15 min of contact. (B) Images of antibacterial activities of the Zn2+/CS fabrics before and after washing. A B 71 5.1.5 Cell viability Although zinc and copper oxides are safer alternatives to more toxic silver, the studies continue to highlight their biological toxicity using soil and aquatic organisms310 and mammalian cell lines.311,312 The toxic action of metal and MeO NPs is mainly a consequence of the release of cytotoxic metal ions from such systems.301,313 Since ZnO NPs partially dissolve in water, their dissolution in aqueous systems is expected to involve both ionic and particulate species. Solubilized Zn2+ proved to contribute substantially to the cytotoxicity of ZnO NPs.165,310 Another assumption claims that ZnO produces toxic species associated with its photocatalytic property. ROS generated under environmentally relevant UV radiation increase significantly the toxicity of these systems to human cells.301 Some studies even report the generation of ROS in absence of photo-chemical energy.314,315 It is therefore crucial to evaluate the potential toxicity to humans of the systems comprising ZnO. After optimizing the ZnO concentration in the antimicrobials-coated textiles, the potential cytotoxicity of these systems was evaluated for medical application requiring contact with human skin. Indirect contact method was used to determine the potential toxicity that CS, ZnO or hybrid nano-coatings might induce to fibroblasts cell culture. During the first 24 h the cultured cells were metabolically active with no difference in cell viability observed among the experimental groups. However, after one week of contact with the ZnO coated fabric cell viability dropped to less than 5 %. In contrast, the CS and the hybrid coating did not induce considerable cell toxicity even after one week. As in the case of bacteria killing, ZnO NPs induce oxidative stress in human cells through the generation of free radicals and ROS.316 The reason for the lower cytotoxicity value in the case of the hybrid coating could be the lower amount of ZnO impregnated on the fabric compared to the fabric treated with ZnO alone (Figure 5.1A). It could be also hypothesized that the CS inhibits the generation of ROS below the threshold of oxidative stress due to its antioxidant capacity.317 78 During US irradiation free hydroxyl and hydrogen radicals created by the high localized pressure and temperature could inactivate the enzyme.325 To verify whether the cellulase activity would be affected by the US applied in the sonochemical coating process, an aqueous cellulase solution without substrate was exposed for 30 min to US irradiation at different temperatures. Thereafter, filter paper samples were incubated with aliquots of this solution, and the amount of reducing sugars was determined. The residual enzyme activity was calculated as a percentage of the activity of the enzyme not exposed to US irradiation. Only 10 to 20 % activity loss was detected after the US treatment regardless of the temperature in the cell (Figure 5.9A), suggesting that the simultaneous sono-enzymatic process would be feasible without compromising significantly the activity of the enzyme. Figure 5.9 Temperature profile of cellulase (Kappacell ETU 39) activity (A) and the residual cellulase activity after 30 min of US irradiation (amplitude 35 %, intensity 17.30 W/cm2) as a function of temperature (B). The residual activity was calculated as a percentage of the activity of the enzyme not exposed to US at 55 °C for 1 h in presence of Whatman No. 1 filter paper as a substrate. The hydrolytic potential of the cellulase used in our experiments was studies under ultrasonic irradiation with different amplitudes/intensities. Figure 4.10 compares the final reducing sugar concentrations after 30 min of enzymatic hydrolysis on a model cellulose substrate at various amplitudes. The highest amount of reducing sugars released during the enzymatic process was obtained when the US amplitude and intensity were 35 % and 17.30 W/cm2, respectively. Thus, all further experiments were carried out at these conditions, considered as the optimal for the enzyme performance in presence of US irradiation. 79 Figure 5.10 Reducing sugars released under different US amplitudes after 30 min of US irradiation at 55 °C in presence of Whatman No. 1 filter paper as a substrate. The US amplitudes of 20, 25, 30, 35 and 40 % correspond to the US intensities of 6.90, 10.40, 13.10, 17.30 and 30.80 W/cm2, respectively. Thereafter, the enzyme activity at the selected processing conditions (amplitude of 35 % and intensity of 17.30 W/cm2) in terms of reducing sugars released during the 30 min sonication process in the presence of filter paper substrate (0.5 g) was determined and compared to the activity of the enzyme upon mechanical stirring (110 rpm). The assay was performed at the temperature of maximum enzyme activity (55 °C), used further for the enzyme/US coating of cotton with ZnO NPs. A 3-fold increase of the released reducing sugars, and thus, cellulase activity, was observed upon sonication (0.311 ± 0.01 mg/mL) in comparison to mechanical stirring (0.096 ± 0.02 mg/mL), due to the intensified mass transport.324 In addition, changes in the molecular structure of the enzyme reported by others 326 to explain its increased activity were not observed in the fluorescence spectra of cellulase before and after sonication (Figure 5.11). 80 Figure 5.11 Intrinsic fluorescence spectra of untreated (black chart line) and ultrasound treated for 30 min cellulase (gray chart line). 5.2.3 Simultaneous sonochemical/enzymatic coating of cotton with ZnO NPs Cotton fabrics were coated with antibacterial ZnO NPs in a simultaneous enzyme/US process carried out with both active and denatured enzyme as a control. ICP - AES measurements were further performed for quantifying the amount of ZnO on the treated fabrics (Table 5.2). Similar amount of ZnO was deposited sonochemically in the processes using either active or denatured cellulase. In addition to determining the amount of ZnO NPs embedded onto the fabrics, we also examined the durability of the coatings subjected to 10 washing cycles at 75 °C in presence of non-ionic surfactant. The fabrics treated with the active enzyme resisted better the applied washing cycles, though ~67 % of the initially deposited ZnO were removed after the washings. At the same time, 95 % of the ZnO on the fabrics coated in presence of denatured enzyme was washed-off. Greater resistance to washings of the NPs on the samples treated with active enzyme demonstrates the significance of the enzymatic activation of the fibers surface for improved NPs adhesion and more durable treatment. The hydrolytic bio-treatment of the cotton fibers generates free hydroxyl groups on their surface, serving as anchoring points for NPs deposition.141 81 Table 5.2 Amount of ZnO deposited on the cotton fabrics in a simultaneous sono-enzymatic process (experimental error ± 10 %). Sample ZnO, % wt (per 100 g of fabric) after coating ZnO on the fabrics after 10 washing cycles (%) Non-washed Washed Enzyme 0.8048 0.2690 33.4 Denatured enzyme 0.8533 0.0428 5.0 The HRSEM surface analysis of the coated samples showed that NPs agglomeration was observed regardless of the enzyme used in the coating process (active or denatured). However, the tendency of the particles to form aggregates was more pronounced for the fabric coated in presence of denatured protein (Figure 5.12A, B). More uniformly distributed NPs were observed on the cotton surface coated in presence of active cellulase (Figure 5.12E, F). This was additionally confirmed from the size histograms of the NPs deposited on both fabrics (Figure 5.13A, B), showing more narrow distribution of the deposited NPs when the active enzyme was used. These observations corroborate our previous findings that the enzymatic activation of cotton surface leads to uniformly dispersed along the fibers NPs.141 In the current study a similar effect was achieved using a single-step sono-enzymatic process. The morphology of the fabrics treated in presence of denatured and active cellulase after subjecting to 10 washing cycles is shown in Figure 5.12C, D and Figure 5.12G, H, respectively. On the surface of the fabric coated in presence of denatured enzyme few NPs were found after the washings, while still a dense NPs layer is observed on to the surface of the enzyme treated samples. These observations were in a good agreement with the ICP – AES findings (Table 5.2), namely NPs improved stability when deposited in a simultaneous sono-enzymatic process. 82 Figure 5.12 Representative HRSEM micrographs of cotton fabrics coated with 1 mM of ZnO NPs at 55 °C in presence of denatured enzyme before (a, b) and after 10 washing cycles (c, d) and in presence of cellulase before (e, f) and after10 washing cycles (g, h). The images on the left were taken with 15000x, whereas the right-side images were taken with 50000x magnification. A) D) C) E) F) G) B) H) 83 Figure 5.13 Size histograms (fitted by Gaussian curves (solid line)) of the ZnO NPs deposited on the fabric in presence of denatured enzyme (a) and cellulase (b) associated with Fig. 5.12b and 5.12f, respectively. 5.2.4 Antibacterial activity ZnO NPs are largely reported as efficient antibacterial agents against both Gram-positive and Gram-negative bacteria. The antibacterial efficiency of the fabrics coated with ZnONPs was evaluated against S. aureus and E. coli. The non-washed fabrics treated with active or denatured cellulase showed comparable antibacterial performances (~70 % reduction in bacteria viability) toward S. aureus (Figure 5.14a). However, after 10 washings the coatings obtained in presence of denatured enzyme lost entirely their antibacterial efficacy toward this bacterial strain. On the other hand, the coating deposited in presence of the active enzyme retained ~50 % of its initial antibacterial activity, which is in good agreement with the amount of remaining ZnO on this fabric. All non-washed fabrics were more efficient against E. coli, and the samples obtained in presence of active or denatured protein reduced the bacterial viability by 98 % and 90 %, respectively. After washing, however, the antibacterial performance of the coating obtained in presence of active cellulase was maintained. The sample coated in presence of denatured enzyme lost more than 30 % of its initial antibacterial efficiency (Figure 5.14b), though the ZnONPs amount for this sample was reduced by 95 % after the washings. A possible explanation for these results could be that the amount of the ZnONPs needed to inhibit the E. coli growth is much lower than the one initially deposited on to the fabric. On the other hand, the ZnO concentration on the fabrics after the washings is considerably reduced and it is below the concentration needed for full kill of this bacterium but still enough to inhibit 50 84 % of its growth. These findings reconfirmed that the cellulase treatment improved the durability of the coating effect. The higher resistance of S. aureus to ZnONPs in comparison with E. coli is due to the thicker peptidoglycan layer in the cell wall of Gram-positive bacteria, which makes them less susceptible to MeO NPs.137,327 The results also showed that the durability of the antibacterial effect of the ZnO NP coating obtained in the one step sonoenzymatic coating approach against E. coli was higher compared to the already reported durability of the coating obtained in the two step-process (consisting of enzymatic pretreatment of the cotton textiles followed by sonochemical deposition of ZnO NPs).141 Figure 5.14 Antibacterial activity of the fabrics coated with 1 mM of ZnONPs: light gray bars represent non-washed fabrics, whereas dark gray bars show the antibacterial activity of the fabrics after 10 washing cycles at 75°C. The assays were performed with S. aureus (a) and E. coli (b), after 1 h of contact with the fabrics. 85 5.2.5 Conclusions Novel industry-attractive technologies are required for the manufacturing of antibacterial medical textiles used to reduce the risk of hospital-acquired infections. In this study we assessed the feasibility of a single-step sono-enzymatic process for durable coating of cotton fabrics with antibacterial ZnO NPs. The coating procedure resulted in the uniform multilayer deposition of ZnO NPs onto the fibers. As a consequence of the boosted cellulase activation of the fabric surface during sonication, the adhesion of the coated NPs was improved and thus, the antibacterial performance was ensured. Although the outer NP layers were not firmly fixed on the textiles, 33 % of the initially deposited ZnO NPs remained on the surface after multiple intensive washing cycles. The remaining after washings NPs still exhibited nearly 100 % reduction of the viability of E. coli, whereas the efficiency toward S. aureus decreased by 50 %. In summary, the one-step sono-enzymatic process is an attractive alternative to the currently used technologies for antibacterial textile functionalization that rely on aggressive and time-consuming chemical pre-treatments to achieve durable antimicrobial fabrics. 86 5.3 Enzyme-assisted functionalization of cork surface with antibacterial biopolymer/silver hybrid nanoparticles This section is based on the following publication: Francesko A, Blanón L, Vázquez M, Petkova P, Morató J, Pfeifer A, Heinze T, Mendoza E, Tzanov T, Enzymatic functionalization of cork surface with antimicrobial hybrid biopolymer/silver nanoparticles. Applied Materials & Interfaces, 2015, 7(18), pp 9792-9799 87 94 Scheme 5.1 Mechanism of immobilization of the hybrid AgNPs-biopolymers onto cork. Mechanisms above the scheme, show two possibilities of reaction: (1) Michael addition, (2) Schiff base formation. 95 FTIR spectra were recorded to verify whether the functionalization of the cork surface was successful. Prior to analysis, unmodified cork, cork enzymatically treated with pure AgNPs (immediately after their preparation) and cork treated with the hybrid biopolymer-AgNPs were subjected to thorough washing during which the samples were immersed in distilled water and vigorously stirred for 5 days. The water was changed each 24 h and the remaining solutions were checked for the presence of NPs by optical microscopy using a Nikon Eclipse Ti microscope, with a 100x oil-immersion objective. After 4 days in water, no particles were detected in any of the remaining solution meaning that after this time there was no AgNPs leaching from the samples. The unmodified cork and cork treated with pure AgNPs displayed characteristic for cork spectra with some of the most prominent bands as markers of: i) suberin at 1462 cm-1, 1236 cm-1 and 1157 cm-1, its aliphatic chains at 2918 cm-1 and 2852 cm-1, and its ester groups at 1735 cm-1 and 721 cm-1, ii) lignin aromatics at 1600 cm-1, 1510 cm-1 and 849 cm-1, and iii) cork polysaccharides at 1096 cm-1 and 1035 cm-1 (Figure 5.17).333 Clear and comparable differences were found in the spectra of the cork treated with AgNPs-biopolymer hybrids. All peaks attributed to the cork suberin including the bands constituting the typical suberin fingerprint (1462 cm-1, 1236 cm-1 and 1157 cm-1)334 decreased considerably after the enzymatic functionalization. Also the bands representing the lignin aromatics decreased or completely disappeared. Thus, both suberin and lignin from cork were involved in the functionalization reaction. At the same time, the differences in the spectra in polysaccharide regions between 1532 – 1681 cm-1 and 900 – 1120 cm-1 suggest a successful biopolymer grafting, but this regions are rather complicated for deeper analysis. Instead, a shoulder peak was noticed at 1260 cm−1 in the spectra of the treated cork (especially pronounced for the cork treated with AgNPs-CS), which could be attributed to C-N stretching of aryl amides formed via Michael addition.332 Therefore, the FTIR spectra confirmed that the phenolic moieties from suberin and lignin in cork were covalently linked with CS or AC, where the reaction occurred predominantly via Michael addition. 96 Figure 5.17 FTIR-ATR spectra of untreated and enzymatically functionalized cork with AgNPs, AgNPs-CS and AgNPs-AC dispersions. SEM analysis was carried out in order to confirm the presence of AgNPs on the cork surface after the enzymatic treatment. On average, cork cells rarely exceed 50 µm height, with a hexagonal face of 15-20 µm and a thickness of 1-2 µm.17 SEM images were thus taken inside the cells, first for the untreated cork without the NPs (Figure 5.18A). The cell surface was not smooth and appeared granulated, where the remaining deposits, not removed during the cleaning procedure, were noticed. The same deposits were observed on the cork treated in presence of laccase with the AgNPs immediately after their synthesis (Figure 5.18B). The AgNPs were thus not fixed on the cork surface or were removed during the immersion in water for 5 days. In contrast, particles and macromolecular structures were observed on the cork enzymatically embedded with the hybrid AgNPs-biopolymer systems even after extensive washing with water (Figure 5.18C and D). This was especially the case of AgNPs-CS treated cork, which displayed a large amount of NPs on the surface. Both individual submicron particles and larger agglomerates could be visualized. A lower number of AgNPs was observed on the cork surface treated with AgNPs-AC system (marked with red arrows on Figure 5.18D), but these were still fixed strong enough to resist the removal 97 by thorough washing. A larger number of permanently deposited AgNPs was achieved via the laccase-assisted grafting of CS on cork. Figure 5.18 SEM images of cork surface enzymatically treated in absence (A) and presence of NPs: (B) pure AgNPs, (C) AgNPs-CS, and (D) AgNPs-AC. 5.3.4 Antimicrobial activity of AgNPs-embedded cork matrices The antibacterial activity of cork matrices functionalized with biopolymer-doped AgNPs was evaluated at different time points after immersing the materials in water up to 5 days. The results of the time-kill experiments showed that the cork matrices treated with pure Ag-NPs (immediately upon their preparation) fully inhibited the bacterial growth for both E. coli and S. aureus if no washing procedure was performed (Figure 5.19). Although not expected in such extent, the antibacterial effect of cork-AgNPs sample could be explained by a moderate 98 adsorption capacity of cork towards silver.335 In contrast, other materials such as nanoclay have been associated with AgNPs in absence of auxiliary molecules to impart diffusioncontrolled antimicrobial activity, with long term impact.336 Adsorption of metals onto different sorbents may occur by physiosorption and chemisorption.337,338 Considering that the chemisorption involves the permanent modification through generation of new chemical bonds at the surface of a material and the progressive loss of the antibacterial effect of cork- AgNPs during 5 days in water, it is logical to conclude that this material maintained the activity whereas the physically adsorbed particles persisted on its surface. On the other hand, the cork matrices functionalized with AgNPs-CS and AgNPs-AC hybrids preserved most of the antibacterial effect (against E. coli) or fully retained the effect (against S. aureus) during the extensive washing procedure. Although it is probable that some of the hybrid structures were also physically adsorbed on surface (reflected in somewhat lower effect of the cork-AgNPs-CS and AgNPs-AC in case of E. coli after 5 days of washing), these results indirectly confirm the FTIR and SEM findings for the permanent functionalization of cork with AgNPs-biopolymer hybrids using the laccase-assisted approach. Figure 5.19 Antimicrobial activity of untreated and enzymatically functionalized cork with AgNPs, AgNPs-CS and AgNPs-AC dispersions. The materials were subjected to the washing treatment in water (stirring 100 rpm) for up to 5 days, during which the antibacterial activity is evaluated at different time points. The antimicrobial effect of cork-AgNPs-CS and cork-AgNPs-AC after 5-day washing was also compared to the matrices enzymatically functionalized with only CS and AC (without AgNPs), in order to reveal if the effect of the biopolymers was restricted only to the material functionalization. Calculated in percentage of bacterial inhibition compared to the effect of 99 untreated cork, the matrices with enzymatically embedded hybrid AgNPs-CS and AgNPs- AC reduced respectively by 99.6 % and 85.3 % the growth of E. coli, whereas showing the full kill potential in the case of S. aureus (Table 5.4). Although the SEM images showed more AgNPs particles immobilized on cork in the case of AgNPs-CS, the antimicrobial efficiency of these two materials were comparable for the used bacteria inoculums. Such scenario did not allow to clearly distinguish the individual effects of AgNPs on one side and intrinsically antimicrobial CS or AC on another. Indeed, CS is a known while AC (a more economical substitute of CS) is an emerging antimicrobial macromolecule in different forms.339,340 Cork treated with CS and AC in absence of AgNPs did not show activity against E. coli, whereas the efficiency for S. aureus reduction was of 10.2 % and 4.3 %, respectively. The antibacterial potential of these matrices is measured in the assay optimized to distinguish the effect of the cork embedded with AgNPs (small amount of the material and high bacteria count), which certainly possess higher antibacterial activity than the biopolymers. Thus, the absence or the low extent of the antibacterial potential of the matrices treated only with biopolymers is explained with the assay conditions applied. Moreover, a slightly higher antibacterial potential of the CS- treated compared to the matrices containing AC is attributed to better grafting of CS macromolecule compared to AC (concluded from the SEM images). Overall, the antibacterial efficiency of the cork grafted with hybrid AgNPsbiopolymers is largely dependent on the presence of silver. Nevertheless, some synergistic antibacterial effect of the AgNPs and the intrinsically antibacterial amino-functional biopolymers could still be envisaged, though the enhancement of antimicrobial activity in hybrid nanoscale architectures based on silver for long-term effect is not a new concept.341 Table 5.4 Antibacterial activity of enzymatically modified cork against Escherichia coli and Staphylococcus aureus after 5 days washing in water. The results are expressed in % of bacteria reduction compared to the untreated cork. Sample E. coli reduction (%) S. aureus reduction (%) Cork-AgNPs- CS 99.6 100 Cork-AgNPs-AC 85.3 100 Cork-CS 0 10.2 Cork-AC 0 4.3 100 In order to better understand the effect of the hybrid nano-coating on the bacteria, we further studied (by SEM) the surface morphology of the cork granules (natural cork or functionalized with AgNPs-CS complex) after incubation in water inoculated with E. coli. The SEM analysis revealed the presence of accumulated E. coli on the cork surface (Figure 5.20). However, the morphology of the bacteria found on the untreated cork and on the cork-AgNPs-CS composite was very different. Proliferating bacteria with well-formed flagella were observed on the surface of the untreated cork (Figure 5.20 A, B), whereas the E. coli found on the functionalized with AgNPs-CS cork granules were not flagellated (Figure 5.20 C, D). Apparently, the hybrid nanocomposite affected the bacteria structure and could prevent their attachment and proliferation on the surface. More bacteria were attached on the surface of the unmodified cork than on the surface of the cork-AgNPs-CS composite. The loss of flagella in E. coli after water disinfection with TiO2-based photocatalyst was previously reported.342 The presence or absence of flagella is important to bacterial survival and growth, since these appendages are not only driving cell locomotion, but also allow the bacteria to attach to surfaces or to epithelial cells.343 It is well established that flagellar-mediated motility and the ability to produce a number of pili are essential for biofilm formation 344 and contribute to the virulence of pathogenic bacteria.345 101 Figure 5.20 SEM images of E. coli accumulated on the surface of unmodified cork (A, B) and on the CS/AgNPs-cork composite (C, D) after 16 h of contact with E. coli suspension. The images on the left were taken with 10000×, whereas the right-side images were taken with 100000× magnification. As a next step, the viability of the bacteria accumulated on the cork surface was tested by placing the cork pieces on Coliform agar plates specific for E. coli. The results showed that the flagellated bacteria observed on Figure 5.20A and B on the surface of the unmodified cork samples were viable, while bacteria growth was not observed for the cork-AgNPs–CS samples on which surface the bacteria were without flagella (Figure 5.21D). Therefore, the bacteria found observed on Figure 5.20 C and D were not viable, demonstrating the disinfection potential of the designed material. 102 Figure 5.21 Images of natural cork granules after incubation in sterile distilled water (A) and water inoculated with E. coli (B); and cork-AgNPs-CS composite after incubation in sterile distilled water (C) and water inoculated with E. coli (D) for 16 h at room temperature. The images B and D are related to Figure 5.20 A, B and Figure 5.20 C, D, respectively. 103 5.3.5 Conclusions In this study, the laccase oxidation of the phenol moieties in cork into reactive quinones and their further reaction with nucleophilic amino groups in CS- or AC-doped AgNPs was exploited to impart durable antibacterial activity onto cork matrices. The role of CS and AC biopolymers in the hybrid NPs structures was three-fold in order to: (i) stabilize the AgNPs and prevent their aggregation, (ii) serve as an interface for permanent grafting of AgNPs on cork, and iii) synergistically improve the AgNPs antibacterial effect. The biocatalytically functionalized cork matrices with AgNPs-CS and AgNPs-AC efficiently reduced the Escherichia coli and Staphylococcus aureus growth during the course of 5 days. Such longterm stability and durability of the antimicrobial effect in conditions of continuous water flow, suggest the potential application of the functionalized cork matrices in constructed wetlands as an adsorbent for removal of wastewater impurities at the same time avoiding microbial contaminations. 110 control over MPO and MMPs activities (total inhibition is not required), in order to provide favorable for the healing and extracellular matrix reconstruction conditions. The current therapies aiming to modulate the activity of major chronic wound enzymes involve the application of anti-inflammatory agents as persistent inflammation is often a key to impaired wound healing. Alkaloids,352 plants polyphenol extracts,353 synthetic polymers with hydroactive properties,354 oleic acid355 and peptides356 are therapeutic agents that through different mechanisms stimulate healing of chronic wounds. In the most recent attempts, chitosan and collagen based dressings were previously upgraded with bioactive phenolic compounds to simultaneously control MPO and collagenase activities in vitro.246,278 5.4.4 Radical scavenging activity The DPPH assay was used to study the antioxidant capacity of EGCG NSs and EGCG in solution. In contrast to the inhibition of enzymes, no differences in the radical scavenging activities between NSs and free solution were observed (Figure 5.24). The antioxidant properties of phenols are linked with their chemical structure as their efficiency increases with increasing the number of OH groups. In the case of nano-scaled materials radical scavenging activity depends on a surface reaction since only the surface comes in contact with the free radicals.357 Thus, our results suggest that the amount of phenolic groups is similar in the NSs and free solution, meaning that these are not involved in the sonochemical stabilization of the nanospheres. The latter was confirmed by evaluating the total phenol content, which was 0.176 ± 0.006 and 0.173 ± 0.004 mM GAE, respectively for the EGCG NSs and free solution. In addition, the surface contact with the functional groups is probably not crucial in the case of DPPH scavenging, as in the case of the enzyme inhibition. 111 Figure 5.24 The antioxidant activity of EGCG NSs (light grey bars) and free solution (dark grey bars). All results are reported as mean values ± standard deviation (n=3). 5.4.5 Antibacterial activity The loss of skin integrity and the exposure of the cutaneous tissue in chronic wounds provide a favorable environment for bacteria to colonize and proliferate. The antimicrobial activity of the NSs suspension/solution was therefore evaluated against the Gram-positive S. aureus - being one of the most commonly found pathogen in chronic wounds associated with changes to wound healing times.358 The results demonstrated that the EGCG NSs have better antibacterial performance than free solution, for all tested dilutions (Figure 5.25). Whereas the highest concentrations of NSs and free solution showed comparable antibacterial properties, their dilution led to different behavior in terms of inhibition of the S. aureus growth. The NSs displayed no changes in their performance down to 0.1 mM, whereas the EGCG solution did not show any antibacterial capacity at this concentration. The better antibacterial performance of the EGCG NSs could be attributed to the improved biophysical interactions between the NSs and bacteria including the NSs biosorption and bacterial uptake.359 It has been also reported that the size and shape of the NPs could affect their antibacterial performance, usually following the rule that smaller structures of spherical shape are better antibacterials than bigger NPs with elongated shapes.350 The small size of the NPs and the high surface to volume ratio aid to their interaction with the bacteria and the enhanced antimicrobial potential. 112 Figure 5.25 The antibacterial activity of EGCG NSs (light grey bars) and free solution (dark grey bars) towards Staphylococcus aureus. All results are reported as mean values ± standard deviation (n=3). 5.4.6 Enzyme-assisted hydrogel formation The hydrogel preparation was achieved via HRP-mediated coupling reaction of thiol groups in TC (serving as a nucleophile) in presence and absence of phenolic NSs (Scheme 5.3). The ability to control the gelation time by varying the concentration of H2O2 used in the reaction was demonstrated regardless of the EGCG NSs presence (Figure 5.26). As expected, the gelation times decrease from 660 to 46 sec and 420 to 23 sec in presence and absence of NSs respectively, as the H2O2 concentrations increase from 0.05 wt % to 1.0 wt %. After the addition of HRP and H2O2, the thiols are susceptible to oxidization and are coupled to each other to give disulfide (-S-S-) bonds. The system consists of two interdependent reactions: the HRP/H2O2 mediated oxidation of thiols to thiyl radicals and later non-enzymatic transformation of these radicals to disulfides.360 113 Scheme 5.3 EGCG NSs loading into TC hydrogels. Oil core 114 Figure 5.26 In situ gel formation of TC/EGCG hydrogels in the presence of HRP (9.65 U/mL) and different concentrations of H2O2. Data of the mean time of gelation ± standard deviation (n=3) are reported. To study the behavior of the thiolated chitosan hydrogels when placed in aqueous environment, their swelling and degradation properties prior the NSs inclusion were evaluated. The swelling capacity of the TC hydrogels revealed that half of the total water uptake was absorbed within the first hour (34 %), while the swelling progressively continued reaching 66 % after 24 h, and then increased slowly to its maximum of 74 % at day 3 (Figure 5.27A). The results for hydrogel degradation indicated that after 3 weeks the hydrogels only lost ~25 % of their initial weight (Figure 5.27B). The weight loss was more pronounced during the first week as the weight of the samples decreased by 15 %, suggesting that during this period the non-cross linked polymer is released into the aqueous medium. When a hydrogel material is placed in an aqueous solution, water molecules penetrate into the polymer network. The water molecules are expected to occupy the free space in the network meshes, which will start to expand, allowing other water molecules to enter. The most important parameters influencing the biopolymer constructs swelling and stability are the hydrophilicity of its constituents and the extent of cross-linking in the matrix. 115 Figure 5.27 TC hydrogels swelling ratio (A) and degradation (B). The results show the mean hydrogel swelling and degradation of each experimental group ± standard deviation (n=6 for each time point). The oxidative enzymes, such as the HRP, can also convert phenol groups in the EGCG to reactive quinones, which subsequently undergo non-enzymatic reactions with a variety of nucleophiles including the available free thiols and/or amino groups. It is generally accepted that the phenolic compounds are promoters of the peroxidase-catalyzed thiol oxidation by reducing the rate-limiting HRP intermediate and/or by oxidation of thiols by free radicals of phenols.360,361 It was therefore expected that in presence of phenolic NSs, the enzymemediated gelation will be faster in comparison with the control (absence of phenolic NSs). Instead, the time of gelation increased with the nanospheres inclusion (Figure 5.26). The conventional peroxidase cycle involves reaction with H2O2, which is reduced to water while phenols are oxidized to phenoxy radicals that dimerize. On the other hand, when the thiols are oxidized by peroxidases (reactions characterized with the oxygen consumption) the process is yielding H2O2. The higher the concentration of H2O2 is, the more decomposition of H2O2 and more thiyl radicals are produced. The additional production of the oxidizing agent (H2O2) during the -S-S- bonds formation could be the reason why the gelation time in absence of phenols (which will reduce the H2O2 concentration) is faster.360 Similarly to the findings reported by Sakai et al. (2014), namely decreased gelation time with decreasing of phenol groups content of chitosan/phenol system, was observed in our case by addition of the phenol NSs formulations of lower concentrations (Table 5.5).362 116 Table 5.5 Hydrogels time of gelation at room temperature using horseradish peroxidase (9.65 U/mL) and different concentrations of hydrogen peroxide and EGCG NSs:TC ratios. EGCG NSs:TC ratio (v/v) H2O2 concentration (%) Time of gelation (sec) 1/99 0.3 83 10/90 0.3 150 1/99 0.5 60 10/90 0.5 90 5.4.7 Hydrogel morphology The SEM of the inner section of the hydrogels loaded with the EGCG NSs confirmed the spheres incorporation into the material. The NSs were also intact after their incorporation into the gel matrix (Figure 5.28). The presence of a few bigger than 1 µm NSs is probably due to the nanospheres aggregation (Figure 5.28B, inset image). Figure 5.28 Hydrogels morphology (A) TC and (B) TC hydrogels loaded with EGCG NSs. 5.4.8 In vitro release study After being loaded into the TC hydrogels, the EGCG NSs release profile from the biopolymer matrices was studied. The results (obtained after measuring the turbidity of the incubation solution) showed a potential of the obtained hydrogels for the sustained delivery of the intact A B 117 EGCG NSs. Nearly 50 % of the incorporated EGCG NSs were released during the course of 6 days in close to linear regime (Figure 5.29). The evaluation period of 6 days was chosen as a maximum and desirable period for the wound dressing change. Beyond this time the cells at the wound bed strongly adhere and proliferate within the material, which would impede the dressing removal.363 On the other hand, the lower frequency of dressing change is costly while the strategy to maintain the environment moist in order to facilitate the woundhealing process through prevention of tissue dehydration and cell death is compromised. Moreover, the extent of the enzyme inhibition could be easily controlled by applying the hydrogel dressing on the wound site for different time periods. Figure 5.29 Time-dependent release of EGCG NSs from TC hydrogels. Three samples were evaluated for each time point and data are reported as mean EGCG NSs release (%) ± standard deviation. 5.4.9 Cytotoxicity There is a notable rise in the number of publications discussing the toxicity of nanomaterials, based on their size, safe dose administration, and material constituents. Hence, the evaluation of their biocompatibility properties is highly recommendable, especially when such materials are aimed for medical applications. Thus, the toxicity of TC, EGCG NSs and their mixture to human foreskin fibroblasts cell line was assessed. All materials displayed good biocompatibility (above 90 % compared with the control), indicating that the application of the generated hydrogels as drug delivery devices would be safe (Figure 5.30). 118 Figure 5.30 Cytotoxicity of EGCG NSs, TC and their mixture to human cell fibroblasts. The results show the mean cytotoxicity values of 3 independent assays ± standard deviation (n=6). CS is a nontoxic polymer and widely applied as a drug delivery material. In addition, EGCG has been reported to have high antitumor activity,364 and also ability to protected human normal bronchial epithelial BEAS-2B cells from Cr(VI)-induced cell death and apoptosis.365 However, it was also reported that low concentrations of EGCG have inhibited the DNA damage induced by reactive oxygen and nitrogen species, while higher concentrations of the compound may itself result in damage to cellular DNA.366 The EGCG conjugation with other materials (as in our case) is also a way to reduce its harmful effect.367 119 5.4.10 Conclusions EGCG nanospheres were successfully generated via a one-step sonochemical process and further incorporated into the TC hydrogel platforms prepared by enzyme-mediated gelation without compromising the particles integrity. By optimizing the H2O2 concentration, the enzymatic gelation can be controlled to occur within less than 1 min. The potential of the phenolic NSs as chronic wound healing promoters was demonstrated via the improved antibacterial and chronicity factors inhibition compared to the EGCG in solution. Moreover, the intact EGCG NSs were released from the highly biocompatible hybrid hydrogel in a sustained manner over 6 days, suggesting potential for prolonged usage and low frequency of dressing change for wound healing applications. 126 Imparting antibacterial properties to the materials and surfaces is a key for achieving of bacteria-free environment in health care facilities and public areas. The results of this thesis have demonstrated that a wide variety of surface nanostructured 2D and 3D polymer-based materials for medical and industrial applications can be developed by applying an innovative combination of versatile sonochemical and enzymatic nano-coating technologies. The development of the antibacterial medical textiles (sections 5.1 and 5.2) was carried out under the scope of the large scale European project SONO - A pilot line of antibacterial and antifungal medical textiles based on a sonochemical process, FP7-228730, which was acknowledged as a “Success Story” by the European Commission. Within this project a pilot line for the production of medical antibacterial textiles based on the scale-up of the sonochemical coating process was developed. The antibacterial efficiency of the produced textiles, e.g. hospital sheets, medical coats and bandages was proven in a clinical trial carried out in the Multi-profile Hospital for Active Medical Treatment and Emergency Medicine "N.I.Pirogov" in Sofia, Bulgaria. In a further step, the sonochemical pilot will be brought to a pre-commercial level and demonstrated in relevant industrial environment for coating of antibacterial substrates such as medical textiles, upholstery, and water/air membranes among others. The disinfection efficacy of the developed at biopolymer/silver nanoparticles cork composites (section 5.3) will be evaluated at the level of a point-of-use water filtration device (water volume up to 2 L/day) prior constructing of larger disinfection installations e.g. point- of-entry systems (disinfection capacity of 100 - 150 L water per day). In addition, the hybrid biopolymer/silver nanoparticles cork material can be used as disinfection element in already existing water purification systems as it is facile to manipulate. The efficiency of developed nanocomposite hydrogels (section 5.4) will be further tested ex vivo with real wound exudates and biofilms, prior to in vivo evaluation. In the production of nano-functionalized materials, nanotoxicology studies will ensure the safety of the workers, end users and the environment. A large number of applications can be foreseen based on the outcomes and the knowledge derived from this thesis. A variety of nano-sized actives can be generated via ultrasound irradiation e.g. nano-antibiotics, enzyme nano-particles, and hybrid nano-antibiotics, among others, and further incorporated, by employing different biotechnological approaches in polymer matrices to obtain antibacterial and antibiofilm materials. 127 Acknowledgements At the end of my thesis I would like to thank all people who made this thesis possible and an unforgettable experience for me. First, I would like to sincere gratitude my supervisor Dr. Tzanko Tzanov for his patient guidance, encouragement and useful critiques during my research. I know there is a lot more to learn. Thanks for giving me the opportunity to participate in European projects and international congresses, to gain experience and know people from all parts of the world. My sincere thanks also goes to Dr. Antonio Francesko who was helping me with his advices and knowledge during my studies and I would say was sharing (more than anyone else) with me my “scientific ups and downs” almost to the end of my PhD. Хвала пуно, Тони! I acknowledge my gratitude to prof. Aharon Gedanken (Bar-Ilan University) and prof. Georg Gubitz (University of Natural Resources and Life Sciences) and the members of their groups (Betina, Anjani, Gregor, Kartin and Katrin, Barbara, the “container team” (Ale, Dani, Sara and Martin)) and all the rest gorgeous people I met in Tel-Aviv and Vienna for helping me to spent unforgivable time during my stay there. Special thanks to Dr. Ilana Perelshtein for her advices and cooperation whenever I needed during my PhD. I cannot miss to say thanks to Dr. Jamie Beddow (Coventry University) who introduce me to the field of microbiology. Thanks to all my colleagues Carlos, Guillem, Margarida, Sonia, Kamelia, Teresa, Rosa, Elisabetta, Eva, Ivaylo, Silvia, Diana and Javi for the nice moments we shared the last years, without you and your support and help this journey won’t be the same. Specially Moltes, moltes gràcies to Carlos and Eva per seva ajuda! You were always willing to help me in the lab and especially in arranging, translating and fixing the documents (in catalan and castellano) knowing better than me all the administrative steps I needed to do. Thanks and to Krisi (I will not forget you) for being my friend (before all) and colleague. I still remember when we met (and Stanimir next to you) ….almost 11 years ago in this big auditorium and „Ти коя група си? “. Криси и Мири, винаги е хубаво когато имаш някой от „вкъщи“ когато си в чужбина, а когато този някой са някои и са твои приятели прави всичко много по-леко. Благодаря ви за подкрепата, вечерите, разходките, „обикалянето по магазините“, приятелството (мога да изреждам още много). Без вас нищо не би било същото! I would like to thank and to other part of our group members Dong, Lupita, Merce, Sundar, 128 and Miguel, supervised by Prof. Pere Garriga. I spent countless hours doing antimicrobial experiments in “your lab”. You tuned easily from my colleagues to my friends, sharing nice moments and talks which made me more familiar with your countries and cultures. Thanks and to all my friends outside the laboratory and those I met in GAIA. Lina, Ricard, Ignacio and Giulio I will always remember our first dinner when you read a book in Catalan and spoke for some diodes (anyway I still do not like the physics). Shubham, thank you for being here after all the rest have left (good you got your grant after me). Kostas, it is always nice to have coffee and croissant with you. Alessandra, Alexia and Weiyi you were one of the best flat-mates/friends I could wish for. Finally, I would like to thank to my family. My parents and sister, you are always there for me. Не бих могла да си пожелая по-прекрасни родители и сестра. Винаги сте ме подкрепяли безусловно във всяко мое начинание и това, което научих през годините е, че винаги, винаги трябва да се вслушвам в съветите, които ми давате. Мамо и тате, ние (аз и кака) сме това, което сме благодарение на вас и на всичко, което сте направили за нас, за да ни осигурите най-доброто каквото и да ви е струвало. Благодаря ви! 129 Bibliography: (1) World Health Organization. Antimicrobial resistance http://www.who.int/mediacentre/factsheets/fs194/en/. (2) Poehlsgaard Jacob; Douthwaite Stephen. Nat. Rev. Microbiol. 2005, 3, 870–881. (3) Gale, E. F.; Folkes, J. 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