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Active wound dressings based on biological mimicry

Final Report Summary - BACTERIOSAFE (Active wound dressings based on biological mimicry)

Executive Summary:
The Bacteriosafe project developed responsive biocomposite materials and integrated them into sophisticated engineering processes for the manufacture of biologically active devices to potentially revolutionise burn treatment. We utilised the natural behaviour of bacterial pathogens to rupture biomimetic polymer nanocapsules to release antimicrobials and colourimetric signalling molecules. The polymeric nanocapsules were surface immobilized on non-woven and polymeric materials and processes to do this were evaluated. The surface immobilized nanocapsules provide both a simple optical indicator of bacterial infection in a burn wounds and will also kill any infection at its source. The objective was to develop different types of nanocapsules and nanoparticles including: Phospholipid – fatty acid vesicles, amphiphilic block copolymer systems, mini-emulsion polymerised nanocapsules and hybrid nanocapsules. During the course of the project these have been evaluated and only the most promising have been carried forward for further development and implementation into engineering processes for wound dressing development. In order to bring the nanocapsules onto the modified non-woven, we have investigated different methodologies such as: (i) activation of the individual non-woven fibres using plasma assisted surface modification or the deposition of “adhesive” thin films, (ii) spray droplets containing the nanocapsules onto the textile, (iii) gentle plasma assisted modification to bind nanocapsules to the fibre surface, and (iv) immobilisation of the nanocapsules within surface attached hydrogel materials. The biological performance of the nanocapsules, both in suspension and immobilized on the surface of non-wovens have been evaluated by the biology-clinical team of the project. This has been done in vitro as well as with some mimics in vivo studies using cell tissue cultures. The Bacteriosafe project has succeeded in developing a large contingent of biomimetic nanocapsules and nanoparticles (NC/NPs) with well over 400 variations of biomimetic NC/NPs, differing in chemical composition, capsule wall thickness and cargo. Characterization, evaluation and selection of the most promising have been based on stability, ease of synthesis, processability and suitability for upscaling, bio-functionality and cytocompatibility. The microbiological and cell biological investigations form the basis of decisions taken during validation of the different materials. Selected microbiological insights and tests include (i) Minimum Inhibitory Concentrations of different antiseptics (2) hyaluronidase activity of clinical isolates of S. aureus strains, determination of hyaluronidase activity compared to a hyaluronidase standard, and (3) tests to detect the cleavage of a fluorescence labeled peptide sequence integrated in a capsule shell. Toxicity tests investigated the effect on inflammatory marker expression in endothelial cells and determined endotoxin residues. In addition and to enable knowledge based decisions within the program bacterial virulence factors, molecular genetic and quorum sensing mechanisms were studied both qualitatively and quantitatively. For all processes and procedures the consortium has established Standard Operating Conditions (SOPs) to ensure reproducibility and consistency. Extensive efforts went into optimising the stability of the nanocapsules. Several optimized NC/NPs systems that have been validated in simulated wound environment and that have been shown to be compatible with selected engineering solutions are now available. Immobilisation processes include aerosol spraying, ink jet printing, dip coating, electrostatic attachment and embedding NC/NPs in (responsive) hydrogel layers. The final prototype dressing, using a lab based dip coating approach, already shows the potential of a wound dressing that will release a fluorescent indicator and antimicrobial components in response to interaction with bacterial cytolytic virulence factors.
Project Context and Objectives:
The idea of utilizing natures’ own mechanisms to combat bacterial infection is yet to be matched and has provided a basis for high-level research and development on a European level. The Bacteriosafe project is inspired by the nanoscale life science principles used by pathogenic bacteria to kill healthy cells. Pathogenic bacteria secrete an array of virulence factors, which rupture the outer membrane of eukaryotic cells leading to cell death and infection. Biodegradable and bioresponsive polymers were to be implemented for the construction of nanocapsules using: (i) Phospholipid – fatty acid vesicles, (ii) amphiphilic block copolymer systems, (iii) mini-emulsion polymerised nanocapsules and (iv) hybrid nanocapsules. These were to be surface immobilized using plasma assisted processes, ink jet printing, spray coating, dip coating and gel encapsulation. The project aimed to cover research into the basic mechanisms of device operation, the development of a prototype device and the process engineering and validation, which is necessary to prepare it for large scale industrial production based on plasma and aerosol processing of polymeric films and non-wovens. Thus, the project aimed to develop the materials and validate the processes necessary to industrially produce wound dressings of great novelty that will open new pathways for future manufacturing processes and improve European health care. The scientific and technological objectives of the Bacteriosafe proposal were:
• Development of novel nanocapsules that can be controllably lysed by toxins released by pathogenic bacteria. The developed nanocapsules were to be rated according to their stability, functionality, response and sensitivity towards pathogenic bacteria.
• Improve our understanding of the nano-technological mechanisms involved and the factors influencing them.
• Integrated engineering solutions that allow for the stable immobilisation of the nanocapsules on non-woven fabrics. This involved research into process design and engineering solutions with the aim to develop new ways for future industrial processing.
• Validation was to be a major component within the project and involved (i) validation of all materials developed in terms of functionality, control, stability and sensitivity, (ii) validation of methods for the immobilization of nanocapsules on non-wovens and polymer films in terms of longevity/stability, cost reduction and energy efficiency (iii) validation of the biomimetic concepts and the engineering solutions developed in order to prove industrial viability of the proposed technological solution.
The overall program objectives can be schematically described as shown in Figure 1 below, indicating the synthesis of responsive nanocapsule/ nanoparticle systems as the main objective of the program, immediately followed by their evaluation in terms of infection diagnostic, i.e. their ability to recognise infection and display a suitable optical signal, and infection therapy, i.e. their ability to recognize infection and rapidly deliver a payload of antimicrobials to combat the infection. Selected systems that showed a significant diagnostic and/ or therapeutic potential were subsequently to be studied in terms of their cytocompatibility as well as their functionality in vitro using lab scale environments to simulate different stages of infection and wound healing. Again selected systems were then to be tested for their mechanical stability and feasibility for applications in engineering solutions for prototyping lab scale demonstrator wound bandages.

Figure 1 Concept behind the Bacteriosafe project and project goals 
The program was divided into 9 work packages (WPs) of which 6 WPs involved scientific development and 3 involved dissemination, financial and scientific management.
The objectives of WP 1-3 were the synthesis of nanocapsules, nanoparticles and lipid vesicles of suitable composition, stability and functionality for both detection (bacterial sensing) and infection control (infection therapy), whereby WP1 involved block copolymer systems and polymerised nanocapsules / nanoparticles (NC/NP) ) while WP2 focussed entirely on phospholipid vesicles. WP1 and 2 both concentrated on NC/NP systems exhibiting a single function of either sensing or release of an antimicrobial. WP3 focussed on hybrid capsules that combined the functions of detection and therapy in a single system.
WP4 involved the lab scale development of methodologies for the surface attachment of the nanocapsules on materials specified by the industrial partners. The work was done in collaboration with a textile testing institute, the industrial partners and a third country partner (Partner 12 UniSA, Adelaide, Australia).
Bio-validation constitutes the work in WP5 and was carried out by biologists and clinicians with guidance from a specialized paediatric burns unit in Bristol, UK. WP5 was aimed to enable detailed biological characterisation mostly based on standard procedures and protocols with the objective to understand and optimise the biological response of the nanocapsules containing antimicrobials and colourimetric dyes or fluorescent reporter molecules both in suspension and surface immobilised.
The most promising processes developed during the project were validated for industrial scale up in WP6. In order to bring the nanocapsules onto a typical non-woven, the Bacteriosafe engineering team set out to investigate different methodologies such as:
• Plasma assisted surface modification or the deposition of “adhesive” thin films,
• Spray droplets containing the nanocapsules onto the textile, and
• Attachment of the nanocapsules to the dressing surface by electrostatic forces.
• Attachment of patterned gel-nanocapsule composites to polymer film wound closure devices
WP7 and WP8 were concerned with scientific and administrative project management respectively, while WP9 was concerned with the dissemination of results emanating from the project.
There were 60 deliverables distributed over 8 interim activity periods (8x 6 months) and 3 reporting periods (18, 18 and 12 months) over the project duration of 48 months. At the end of each activity period a number of deliverables had to be met and the results and progress was to be assessed by the Project Coordination Committee (PCC) at the “ordinary meetings”, which were to be held every 6 months. Each interim activity period was terminated with a meeting and an interim activity report, which was submitted to the commission.
The main risks originally identified within the project involved (i) finding suitable formulations for bioresponsive capsule/ nanoparticle systems, (ii) incorporation of these systems in suitable engineering processes for the manufacture of burn wound dressings and (iii) their bio-compatibility, long term stability and functionality.

Project Results:
4.1.3. Description of the main S & T results/foregrounds.
The Bacteriosafe project ran as anticipated over the entire 48 months and the project partners succeeded to fulfill all deliverables. The last four years have seen an overall increase in the use of nanoparticles and nanocapsules in an ever increasing range of application. In particular the use of nanoparticles and nanocarriers is today an import area of research and development in modern medicine enabling new diagnostic tools and therapeutic solutions. As such the research in this project has been very timely.
Novel Nanocapsules and Nanoparticles
During the course of the project the different methodologies were established and evaluated by the consortium members and only the most promising systems were carried forward for further development and implementation into engineering processes for wound dressing development. Over the course of the project the Bacteriosafe project studied over 400 different NC/NP formulations dividing then into 3 subgroups: 1. Lipid vesicle systems, 2. Block-copolymer systems and 3. Miniemulsion polymerized systems.
More than 400 different capsule systems have been designed, synthesized, characterized and evaluated in terms of stability, functionality, efficacy and processability. At the end of the project 3 classes of nanocarrier systems have been identified as suitable candidates: lipid vesicles with improved stability, amphiphilic block copolymer and hybrid polymeric systems, of which 10 candidates have been taken forward into final tests exploring demonstrator and prototype development.
Lipid-based systems (P2-BATH)
Conceptually, the lipid nanocapsules (vesicles) pursued by P2-BATH were designed to mimic the eukaryotic cell membrane and be lysed by bacterial virulence factors, releasing their encapsulated payload: either an antimicrobial or a signalling dye. Later development of the project involved the dispersion of vesicles into a patterned gel matrix, cytotoxicity testing and the development of prototype dressings ready for initial animal studies. Particular emphasis was placed on the development of stable and responsive lipid vesicles and their application in a prototype wound dressing. In addition to this efforts were directed towards understanding the virulence factors secreted by key bacterial pathogens and their genetic regulation to aid in the design of the nanocarrier system. The work funneled into a final prototype design with a combined detection and therapeutic release system, going into first considerations of packaging with final post program development.
Development of stable and responsive lipid vesicles ,
A new methodology for detecting the microbiological state of a wound dressing in terms of its colonization with pathogenic bacteria such as Staphylococcus aureus or Pseudomonas aeruginosa has been developed. Here we reported how stabilized lipid vesicles containing self-quenched carboxyfluorescein dye are sensitive to lysis only by toxins/virulence factors from P. aeruginosa and S. aureus but not by a non-toxic E.coli species. The development of the stabilized vesicles is discussed and their response to detergent (triton), bacterial toxin (α-hemolysin) and lipases phospholipase A2). Finally, fabrics with stabilized vesicles attached via plasma deposited maleic anhydride coupling are shown visibly responding to S. aureus (MSSA 476) and P. aeruginosa (PAO1) but not E. coli DH5 in a prototype dressing.
The concept was to study how the mol% of the photo-polymerizable cross-linker would affect vesicle stability (as measured by very brief part dehydration and rehydration) and sensitivity, measured by the fluorescence increase on adding either Triton detergent or alpha haemolysin. Experiments using a variable mixture of mol % TCDA mixed with cholesterol and DMPC and photo-polymerized showed that the stability of the vesicles increased with increased TCDA concentration, while the sensitivity decreased with increased TCDA concentration. The conclusion therefore was to utilise between 20 – 30 mol% TCDA to optimise both stability and sensitivity. The 30 mol% vesicles responded well to virulence factors from S. aureus and P. aeruginosa, as shown in figure 2, which shows increase in fluorescence following vesicle lysis as these bacteria grow.

Figure 2.
Time-resolved fluorescence data showing the release of a reporter dye from 30% TCDA vesicle inoculated with 1000 CFU ml−1 P. aeruginosa PAO1, S. aureus, MSSA 476, E. coli DH5 α and a non-inoculated. control over 18 h.

Although these vesicles were stable for short periods, they were not stable over prolonged periods, especially at elevated temperatures and more than 24h. For this reason longer fatty acid chains from DPPC and DSPC (C16 and C18) respectively, together with different lipid head groups and variation in cholesterol concentration were studied, with over 100 vesicle types being made and measured. The twelve most promising combinations were subjected to lengthier testing and the three systems with the best stability – response parameter were eventually tested against bacterial supernatants.
One of the outcomes from the stability – sensitivity experiments discussed above was the realization that vesicles could be designed to respond differently to virulence factors from P. aeruginosa or S. aureus. At this time we were uncertain of the precise nature of the cytolytic virulence factors being produced, though this is now understood: S. aureus secretes 21 amino acid peptides: phenol soluble modulins, which assemble in the membrane and form (we believe) pores. P. aeruginosa secretes detergent like rhamnolipids which rapidly solubilize the lipid membrane. By changing the lipid composition, it was possible to tune the response to virulence factors from either or both species of bacteria, and this was consistent over many strain types. This data is part of the patent application now at EC and US national phase.
To be effective, the vesicles must not dry out and need to be dispersed in a hydrogel matrix. To this end a number of gels were evaluated, with the osmotic balance and pH all affecting stability. Agarose, a polysaccharide came out top in these tests, which is convenient as it is cheap, no-toxic and easy to work with.

Amphiphilic block copolymer systems (P3-USiegen):
P3-USiegen developed two large families of very stable, yet bacterial enzyme-labile vesicles made of amphiphilic block copolymers (polymersomes), comprising each a hydrophilic and a hydrophobic block, that respond selectively to proteinase (of pseudomonas) as well as hyaluronidase and lipase (both of S. aureus). Within each family the hydrophobic polyester block and the block lengths were varied to achieve optimized self-assembly, efficient encapsulation of reporter dyes or antimicrobials, adequate stability regarding shelf-life and contact with biological media and rapid response. In addition to the establishment of processing, which led to control over vesicles size, the degradation mechanisms were investigated and unravelled.
The synthesis of poly(ethylene glycol)-block-polyester systems (family 1), in which both poly(lactic acid) (PLA) and poly(-caprolactone) (PCL) were utilized, comprises exclusively FDA approved polymer blocks and synthetic protocols. The successful adaptation of these known block copolymer systems to generate hollow capsules with selective lability and fast response against enzymes of relevant pathogenic bacteria was aided by the detailed studies that identified the degradation mechanisms.
Likewise the hyaluronic acid based block copolymer systems (family 2) contain exclusively FDA approved polymeric blocks. The novel block copolymer materials hyaluronic acid-block-PLA and hyaluronic acid-block-PCL were designed, synthesized and fully characterized for the first time. Their relevance due to the sensitivity to bacterial hyaluronidase, a highly conserved enzyme, who’s climaxing production before the exponential growth phase of S. aureus, as was established by partner P2-BATH makes these hyaluronic acid systems prime candidates for reporter capsule materials.
All amphiphilic block copolymers synthesized within the two families mentioned were assembled by selective solvent treatments and thoroughly characterized by dynamic light scattering (DLS) and fluorescence optical, atomic force microscopy (AFM), as well as scanning and transmission electron microscopy (SEM and TEM). Among the various morphologies found, vesicular polymer aggregates with external diameters between ca. 100 - 200 nm were identified and the corresponding preparation conditions were optimized. A large range of reporter dye molecules (incl. calcein, carboxyfluorescein, sulforhodamine B, and nile red) and antimicrobials (incl. sodium azide, Gentamicin, Chlorhexidine, Polyhexanide) were encapsulated successfully during the assembly step and after purification by dialysis could be released upon shell degradation. The degradation halftime could be adjusted, depending on the enzyme concentration and the wall thickness to < 60 minutes for all systems. Pronounced signalling action following enzymatic attack was confirmed for the following light-up and light down systems: sulforhodamine B, calcein, fluoresceine, alpha chymotrypsin-sensitive fluorogenic substrates (light up) and nile red ( as light off system), respectively.
In close cooperation with the consortium partners the degradation of the P3-USiegen capsules with bacterial supernatants (P. aeruginosa and S.aureus provided by Partner P2-Bath), the absence of cytotoxicity (with partners P5-UMC and P6-DCU), sterilization, immobilization (also with Partner P1-MPIP), and finally after upscaling of the synthesis and vesicles production for the PEG-based systems to the > 100 g scale (see section “Activities towards industrial manufacture and validation” for this additional task) the compatibility with large scale coating processes developed by partners P7-Centexbel and P4-LUT were ensured.
P3-USiegen also developed hybrid metal nanoparticle-polymer vesicles, hybrid films of reporter polymersomes in enzyme labile films prepared by the layer-by-layer assembly technique and investigated the generation of much larger polymersomes (500 - 800 nm external diameter) by electroformation processes. A spin-off activity led to the development of infection-signalling reporter hydrogels comprising a chitosan hydrogel with conjugated fluorogenic and chromogenic substrates (Figure 3). The most recent data based on bacterial supernatants from partner Bath (P. aeruginosa and S.aureus) as well as tests with a safety level S-3 strain of E. coli (EHEC) by partner UMC confirm the potential of this approach, which will be further refined.

Figure 3. Detection of pseudomonas: a) UV-Vis spectra of elastase sensitive hydrogel prior to and after exposure to supernatant of pseudomonas provided by Partner P2-BATH; b) corresponding photograph of hydrogel in buffer (left) and enzyme containing solution (right).

Biodegradable polymer capsules (P1-MPIP)
P1-MPIP’s contribution to the project was the synthesis of different hyaluronic acid based nanocapsules, biodegradable poly (L lactic acid) (PLLA) nanoparticles and peptide based nanocapsules. The formation of polymeric hyaluronic acid based nanocapsules was achieved through a crosslinking reaction at the interface of miniemulsion droplets. The SEM and TEM images show clearly a capsule morphology and the chemical composition of nanocapsules was confirmed by FTIR spectroscopy. For the enzyme-triggered release (hyaluronidase) from the nanocapsules, the fluorescent dye SR101 dye was encapsulated with a high yield in the range of 89 and 94%. The nanocapsules were treated with different concentrations of the enzyme hyaluronidase (32, 16, 8 and 1 mg per mL) and after given time periods (0 h, 1 h, 2 h, 4 h, 7 h and 23 h) the fluorescence intensities in the supernatant were determined. The kinetic studies showed that within 23 h the amount of released material increases by a factor 3 to 5. From the hyaluronic acid based nanocapsules, a release of the antimicrobial polyhexanide was obtained in the case of > 8 mg mL-1; a concentration of 1 mg mL-1 hyaluronidase was too small to trigger a significant release. The cleavage of the nanocapsules was confirmed using UV spectroscopy. The average size, size distribution and morphology of the different nanocapsules before and after enzyme treatment were measured using DLS and SEM. SEM images of the crosslinked nanocapsules, taken before and after 8 h and 24 h of hyaluronidase exposure, show clear disintegration of the nanocarrier system. Antimicrobial tests were performed against S. aureus ATCC 29213, S. aureus ATCC 43300 and E. coli ATCC 25922 with different concentrations of nanocapsules ranging from 1000 µg mL-1 to 1.9 µg mL-1. The minimum inhibitory concentration (MIC) study showed that the polyhexanide based nanocapsules exhibited the same antibacterial activity as the hyaluronic acid/polyhexanide based nanocapsules against both strains of S. aureus, preventing detectable growth at 62.5 µg mL-1 in vitro. E. coli ATCC 25922 exhibited a higher resistance to the nanocapsules having a MIC of 250 µg mL-1. The stability of nanocapsules in different biological media was validated as well.
Biocompatible and biodegradable PLLA nanoparticles were created by the combination of miniemulsion and the solvent evaporation method. The size of PLLA nanoparticles, stabilized with an ionic surfactant (SDS, CTACl) is in the diameter range between 90 and 160 nm, whereas the diameter of nanoparticles increases up to 240 nm when a non-ionic stabilizer (PVA, HES and HSA) was employed. The values of the zeta potential are slightly more positive when using higher amounts of octenidine. The observed data from HPLC measurements show that the antimicrobial agent octenidine is released out of PLLA-NPs which are stabilized with PVA, HES or HSA and from the pH studies it can be concluded that proteinase K accelerates the degradation of these nanoparticles, whereas esterase did not influence the degradation of PLLA-NPs. No degradation was observed for the SDS und CTACl stabilized NPs. In bacteria tests the PLLA nanoparticles showed a greater ability to inhibit the growth of S. aureus compared to E. coli. In order to immobilize fluorescent labeled PLLA-NPs in fibres, the dispersion was electrospun using PVA as a fibre template. In the SEM smooth fibres are detected, the cLSM image clearly shows the successful incorporation of the fluorescently labelled PLLA-NPs inside the fibres. The whole nanocarrier setup was provided for toxicity test, bacteria test, cell tests and immobilization experiments to the project partners.
Activities towards industrial manufacture and validation
Since the Bacteriosafe program focussed strongly on the development of NC/NP systems suitable for diagnostic and/or therapeutic applications in wound dressing development, all materials were synthesised on the research lab scale in typical batch processes which showed a general yield in milligram quantities. These quantities were sufficient for most of the biological evaluation and for initial studies concerned with the immobilisation on substrate materials for analytic tools such IR-Spectroscopy and XPS. For the immobilisation on substrates such as non-wovens and other textiles the program soon ran into considerable challenges for producing sufficient quantities of NC/NPs for immobilisation on larger samples. In order to address this, the partner P3-USiegen has during the final year of the program developed prototype lab equipment that enables an upscaling to synthesize and assemble hundreds of grams of approved PEG-b-PLA and PEG-b-PCl materials.
The initial goal of the Bacteriosafe program aimed to immobilize NC/NP systems to non-wovens via electrostatic interactions or via specific chemical bonds directly to the textile fibres. While it was possible to show that this indeed works very well using spray coating, ink jet printing, dip- or drop coating, the prototypes generally showed insufficient longevity that would be required for a future mass product. Criteria that were considered to assess the stability of a dressing prototype included surface attachment of the NC/NPs under different biologically relevant (wound) environments, including pH range from pH4 to pH9, shear forces, ionic concentrations, hydrolysis reactions, bond cleavage as a result of “other” (yet unknown) enzyme activity in the wound.

Biological evaluation
The Bacteriosafe final prototype is based on NC/NP materials that will release a signalling molecule (dye) or a therapeutic molecule (antimicrobial) upon a biological stimulus. The success of this strategy is dependent on:
(i) Fast response by the NC/NP system involved
(ii) Availability of a sufficient quantity of the biological trigger molecules at the site of infection to rapidly “open” the NC/NPs.
(iii) Delivery of sufficient quantities of antimicrobial released by the NC/NP systems to enable the “kill”.
The microbiological groups at P2-BATH, P5-UMC and P6-DCU examined the antimicrobial efficacy of the NC/NP dispersions from P1-MPIP, P2-Bath and P3-USiegen as part of the evaluation of the different systems. S. aureus, a typical pathogen responsible for wound and burn wound infections is known to produce hyaluronidase as a constitutive extracellular enzyme. P2-Bath and P5-UMC collected up to 250 different strains of S. aureus (of which approximately 85% were MSSA, 15% were MRSA) for the project work and carried out qualitative and quantitative screening tests for hyaluronidase activity. The groups were able to show that 94% of clinical S. aureus isolates produce this enzyme. Quantitative determination of hyaluronidase activity on 54 strains of S. aureus showed an average value of 8.6 U/ml (5.4 U/ml) of hyaluronidase activity, which should be sufficient to degrade the hyaluronic acid based nanocapsules and to inhibit 50% (90%) S. aureus strains after antiseptic release. MIC90 measurements using the antiseptics polyhexanide and chlorhexidine on 102 clinical isolates of S. aureus and 49 P. aeruginosa strains showed MIC90s for S. aureus of approximately 1.56 µg/ml for polyhexanide and 0.781 µg/ml for chlorhexidine. Similarly for P. aeruginosa MIC90s were shown to be 6.25 µg/ml for polyhexanide and 12.5 µg/ml for chlorhexidine. Systematic protocols were established to test the selected NC/NP systems first in suspension and later after immobilization on solid surfaces and textiles (non-wovens from P11-Freudenberg).

Cytotoxicity, cell uptake and angiogenesis (P5-UMC, P6-DCU, P2-BATH)
The final dressing prototype should be designed to firmly hold the NC/NPs within the dressing and NOT allow for the release of the NC/NPs into the wound. Even after lysis of the NC/NP no material should elude into the open wound. Beside the antimicrobial response it was therefore also necessary to establish a solid background knowledge on the cytocompatibility of the nanocapsules/ nanoparticles in order to evaluate their suitability for wound healing applications. Here we addressed the following issues:
• Sterility, endotoxin testing protocol for all nanocapsules received for cell culture testing.
• Toxicity testings in different primary dermal cells and macrophages
• IC50 determinations have been made for the NC/NPs.
• Impact of NC/NPs in 2D and 3D wound models has been determined, cellular uptake. Development of angiogenesis assay, optimization of three different models.
• Impact of NC/NPs on altering gene expression of selected markers of the 3 basic phases of wound healing. Investigation of the effect on inflammatory marker expression in endothelial cells.
• Establishment of the isolation and cultivation of primary human dermal and immune cells. NC/NP impact on growth with various cell types and models. Analyses of the uptake of NC into the cytoplasm.
Neovascularization plays a key role during wound healing. Therefore, analysis of the cellular wound response to the biodegradable, antimicrobial NC/NP systems has included investigations into their effect on angiogenesis. In order to enable this different standard angiogenesis models were optimized and adapted for the specific NC/NP systems and appropriate assays to check for and quantify the influence of nanovesicles was established. Wound healing consists of a number of complex and sensitive processes aimed at the regeneration of injured tissue. To validate the compatibility of responsive NC/NP prototypes with these processes, cellular response(s) after exposure to the materials was investigated. Primary human microvascular endothelial cells (HDMEC), monocytic cell line THP-1 (P5-UMC) and HaCaT (P6-DCU) cells were used as test system since they play a key role in various wound healing processes such as inflammation and angiogenesis. Besides cytotoxicity studies, the investigations included an analysis for the cellular uptake of the materials as well as their effects on inflammatory response and the potential to form capillary-like structures in vitro. Results of cytotoxicity investigations obtained to date always showed a moderate toxic effect of suspended polymer nanosystems on primary dermal cells. This was partially attributed to the medium in which the nanocapsules are stored as well as the inherent toxicity of the encapsulated dye used during the evaluation of the NC/NPs. Later work showed a sufficient decrease in this effect after immobilization of the NC/NPs on dressing materials. A wound assay of human epithelial cells was used to show that the final formulations of NC/NPs after removal of any carrier media and any residual chemicals from manufacture do not negatively affect the repair of an epithelial wound.
In order to establish robust data, standard operating procedures (SOP) for assessing the effect of the different nanocapsules on mammalian cell growth and viability were established. This standardisation involved the selection of test cell types (both cell lines and primary cultures). In addition the duration of growth and viability assays was also standardised. Finally, the selection of a reporter method to assess growth and viability that would not be subject to interference from test components (e.g. fluorescent compounds etc.) was also standardised. The standardised protocol developed exposed mammalian cells to nanocapsules and test components for 7 days and used acid phosphatase measurement as a reporter to fully assess any effects on growth rate as well as viability. The final nanocapsule designs as assessed in accordance with the SOP recorded minimal impact on growth and viability in skin cells
More advanced methods to assess interactions that would be important between mammalian cells and prototypes were developed. These in-depth studies provided early information on any potential off target effects prior to any animal testing. The methods developed assessed the crucial wound biocompatibility aspects of innate immune response, wound re-epithelisation and remodelling. These methods involved qRT-PCR for specific targets, neutrophil assays, zymograms and repair of in vitro wound models. The also included the development of advanced multi-layered skin models to assess nano-capsule and prototype interaction in vitro. These in vitro studies were beyond the standard short term toxicity assays normally conducted. These studies indicated that earlier formulations nanocapsule in particular residual amounts of carrier chemicals could generate off target effects. In particular provoke a strong immune reaction. In later formulations which addressed these problems, off targets effects were minimum or non-existent.
Tests with human umbilical cells (HUVECs), red blood cells, neutrophils and T cells show lipid vesicles (P2-Bath) to be not cytotoxic. Pure toxins (PSMs and rhamnolipids) were purchased and the response of vesicles to pure toxins quantified, as well as the toxicity of these virulence factors to T cells and neutrophils. The vesicles were found to be 100x more sensitive to these toxins than T cells or neutrophils, higlighting the potential for detection before significant tissue damage can occur. Finally, neutrophils in human blood were activated with lipopolysaccharide in order to induce an inflammatory response and vesicles added. No lysis of vesicles was observed.
Collectively these proof-of-principle studies indicate that our 'smart dressing' technology could detect infection at an early stage before major damage to host tissues occurs, or clinical symptoms become overt, and inform descision making directly at point-of-care.
Hyaluronidase screening and nanocapsule lysis (P5-UMC)
The microbiological group of UMC examined bacteria-wound dressing interaction especially focusing on hyaluronic acid based nanocapsules. The choice for the encapsulated antiseptic was based on MIC measurements with the antiseptics polyhexanide and chlorhexidine on 101 clinical isolates of S. aureus (85 MSSA and 16 MRSA). 90% of the S. aureus strains were inhibited at a concentration of 1.56 µg/ml polyhexanide. In contrast the lower concentration of only 0.781 µg/ml led to the same result by using chlorhexidine. These results point to a higher antimicrobial effect of chlorhexidine than for polyhexanide.
As opening mechanism for the hyaluronic acid based nanocapsules we considered extracellular hyaluronidase, which production is described in the literature as a typical pathogenicity factor of S. aureus. For the project it was important to find out a capsule opening mechanism, which reacts with a large number of relevant bacterial strains. Of 101 clinical S. aureus isolates screened for extracellular hyaluronidase activity, a positive result was detected in 93% of cases. A quantitative determination assay was developed to compare the hyaluronidase activity of S. aureus to a commercial testes hyaluronidase standard. 54 S. aureus strains from our collection were investigated and the three highest determined values were 149, 83 and 41 U/ml. The measurements pointed to a minimum value of 5.4 U/ml of hyaluronidase activity exhibited by 90% of the investigated strains. For an inhibition of 90% S. aureus strains the nanocapsules must be degraded by this concentration.
The Bacteriosafe partner P1-MPIP provided nanocapsule dispersions which consist of hyaluronic acid (HYA) in the shell and antiseptic polyhexanide (PHMB) as well as a fluorescent dye in the core. Our main tasks were to examine the opening mechanism and the antimicrobial effect of these capsule samples after hyaluronidase attack. According to the manufacturer’s specification, a pH value of 5.3 was chosen for testes hyaluronidase pre-incubation in our test settings. In these experiments we could never show an antimicrobial effect of the HYA/PHMB nanocapsules under a range of different conditions and performances. Finally, we could demonstrate that the hyaluronidase activity was stronger at a pH of 7 and was regarded as the best possible pH condition for the next experiments. The release of the encapsulated dye after incubation with 32, 16, 8, 4 and 1 mg/ml as well as with 150 and 50 U/ml (0.187 and 0.062 mg/ml) applied hyaluronidase concentration indicated capsule degradation in any cases. Increasing fluorescence values of pre-incubated nanocapsules in buffer and subsequent incubation with nutrient broth containing samples indicated that addition of nutrient broth initiates a continuing HYA/PHMB nanocapsule degradation which was further analyzed. The incubation of HYA/PHMB nanocapsules in two different nutrient broths resulted in strongly increased fluorescence signals compared to the control with pure buffer. A common component of the nutrient broths is peptone which was used for another fluorescence measurement resulting in similar values for the pure peptone solution and the nutrient broth. OD measurements of bacterial growth with pre-incubated HYA/PHMB nanocapsules showed a complete inhibition of the strains, even without any hyaluronidase influence so that no release of the antiseptic polyhexanide should be induced. The degradation and release of polyhexanide from the HYA nanocapsules core could be demonstrated by addition of the enzyme hyaluronidase, nutrient broth and even with pure peptone. Test with some selected nanocapsule systems have suggested interfering reactions with some proteins present in wound exudate.
Evaluation of wound exudate (P5-UMC, P10-Frenchay, P6-DCU)
In order to further develop an advanced understanding and modelling of the burn wound environment initial proteomic studies were conducted on the collected wound exudate. This is an area where there is currently no data available. The study validated that wound exudate samples could be collected at multiple sites (Frenchay Hospital, NHS, Bristol, UK and Universitätsmedizin der Johannes Gutenberg-Universität Mainz) stored and transported for proteomics analysis without any protein degradation occurring. Using state of the art LC-MS/MS, the peptide samples were separated using 1 hour RPLC gradients. A novel protein profile of wound exudate was identified following interrogation of mass spec raw files using ProteomeDiscoverer searched against the human protein database (Uniprot). This preliminary protein profile of burn wound exudate consisted of approximately 200 proteins. To further refine and analyse deeper this novel profile immuno-depletion columns were used to remove highly abundant proteins in the exudate samples. This resulted in the identification of lower abundant proteins in the exudate sample, and increased proteome coverage. Overall we have identified approximately 450 different proteins in unfractionated wound exudate from these very first experiments. In order to carry out a deeper analysis of the wound exudate proteome it will be recommended to use immuno-depletion columns to remove highly abundant proteins in the exudate samples, thus allowing the identification of lower abundant proteins in the exudate sample, and increasing proteome coverage. From this very preliminary analysis it looks like we can easily transfer wound exudate patient samples from sites in different EU locations that are suitable for use for proteomic studies. The result of this study has been the completion of a first ever protein profile of burn wound exudate.
Stability and functionality of wound dressing prototypes in wound exudate
In order validate the developed systems within the Bacteriosafe program the partners P5-UMC and P10- Frenchay applied for and received ethics permission to retrieve wound exudate from patients to be used for (i) first evaluation of constitutents of wound exudate and, (ii) stability testing of selected nancapsule systems. This was run as a separate, but parallel program to Bacteriosafe.
The three ‘lead’ vesicle types from P2-Bath were all tested in wound exudate obtained from paediatric patients at Frenchay hospital (Partner north Bristol NHS trust), following full ethical and R&D approvals. Similar tests were performed with selected nanocapsules from P1-MPIP. The data for the vesicles is shown below (figure 4). All vesicles were broadly stable, with the only system showing a small significant loss of stability was the DOPC-DS system. This data was very encouraging and encouraged utilization of the TCDA-DS vesicle system in prototype development.
Combined detection and therapeutic release
The primary focus of the Bath contribution to Bacteriosafe has been on the development of the vesicle carrier vehicle, which we have demonstrated and tested efficacy using the fluorescent dye assay. This approach is equally effective at delivering via a virulence factor trigger, antimicrobials including silver nitrate, gentamicin sulphate and sodium azide. The more complex regulatory pathway and difficulty of getting a working system ready for first human use has meant these approaches have not received the time and effort of the detection system, however a parallel approach based on the work of partner P3-USiegen, where the breakdown of hyaluronic acid by secreted hyaluronidase was used as a trigger for the release of novel bacteriophage.
Bacteriophage research
The Bath team have prospected for, purified and characterised novel bacteriophage (viruses) which specifically kill S. aureus or P. aeruginosa strains. These have been isolated from flood and waste water. Bacteriophages are obligate parasites of bacteria. They multiply intracellularly and lyse their bacterial host releasing their progeny. We isolated a novel phage, DRA88 that has a broad host range amongst S. aureus. Morphologically the phage belongs to the Myoviridae family and comprises a large dsDNA genome of 141,907 bp. DRA88 was mixed with phage K to produce a high titre mixture that showed strong lytic activity against a wide range of isolates including representatives of the major international MRSA clones. Its efficacy was assessed when treating established biofilms produced by three different biofilm producing S. aureus isolates. A significant reduction of biofilm biomass over 48 hours of treatment was recorded in all cases. The phage mixture may form the basis of an effective treatment for infectious caused by S. aureus biofilms.
Phage was dispersed in an agarose matrix and coated in cross-linked hyaluronic acid film which is selectively degraded by hyaluronidase secreted by most S. aureus, Streptococcus and other bacterial species. The result from demonstrated that hyaluronidase positive S. aureus release phage (measured as a titre) whilst hyaluronidase negative S. aureus do not release phage.

Immobilisation protocols, final prototype design and packaging
Immobilisation of particles on lab scale
The NC/NP immobilisation procedures studied in the project were based on either electrostatic interactions, or on chemical reactions between specific chemical groups to form covalent bonds. Processes were adapted to work with the non wovens supplied by P11-Freudenberg as well as for other surfaces. For electrostatic interactions we have concentrated efforts on terminal amino-, alcohol and carboxylic acid groups on either the substrate surface or on the particles/ capsules to be immobilised. The immobilisation protocol involved a dry plasma process and a wet chemical immobilisation.
A particular emphasis in the work was to determine the functional group density per square nanometer and correlate this to the efficacy of attachment. Functional group density was evaluated using X-ray Photoelectron spectroscopy, carried out at P11-UniSA and at other partner sites. The efficiency of particle attachment was evaluated by SEM and AFM before and after washing steps using different solvents, body fluids and wound exudate by visual inspection of the images. The success of this was highly dependent on the nature of the surface and the particle. The efficacy of attachment of nanocapsules was sensitive to the stabilizers used during the NC/NP synthesis which in many cases prevented stable immobilisation of the NC/NP to model surfaces, Figure 5. Some improvement was observed for selected particles embedded within several layers of plasma polymers. For single layer immobilisation of NP/NCs oxygenated surfaces, such as those bearing –COOH (acid) or C-O-O (hydroperoxide) were found to demonstrate the most reliable and reproducible results, yet were still prone to unspecific attachment of biomolecules present in the wound exudate and broth. This could be improved by the additional deposition of an “antifouling” layer deposited from triglyme, Figure 6.
Figure 5. AFM surface-images of different PLLA-NPs immobilized on either pp-AA, pp-AlAl or pp-Diglyme/He-plasma-treatment surfaces.
A final demonstrator was built based on a multiple step process involving plasma/ or ozone activation of the fibre surface followed by wet chemical grafting and subsequent wet chemical NC/NP immobilisation, see Figure 7.

Figure 6 Summary of antimicrobial efficacy of PLLA (containg calcein or octenidine) nanoparticles embedded within different plasma polymers. A study for S. aureus and E. Coli.
Fig.7: Proof of functionality, UV-irradiated nonwoven, left: as received, middle: equipped with surface immobilized PLLA nanoparticles right: nonwoven after release of the dye from the capsules with detergent.

Equipment for continuous manufacture of wound dressing
Equipment was built at P7-Centexbel to enable tests for spray coating of NC/NP systems onto non wovens and foils. In this setup, a spray device was developed allowing continuous and homogeneous deposition of the Bacteriosafe particles on flat surfaces. State of the art spray nozzles, available to industrial application where used and further tuned to meet the Bacteriosafe requirements for NC/NP deposition. In particular for batch processes requiring a minimum quantity of NC/NPs. Homogenous deposition of the NC/NP was achieved at high enough densities.
Using a DBD discharge with an atomisation unit, particle solutions were injected into the plasma field while a nonwoven passed through the discharge. In addition and as an alternative, a gel coating was developed enabling immobilisation of moisture sensitive NC onto cotton mesh fabric, Figure 8. The process allows for roll to roll coating deposition with standard coating equipment used in textiles. For both, the spray and the gel coating, an engineering plan was set-up for straightforward implementation at industrial scale using application methods commonly used in industry. Alternatively, separate compound can be offered to hospitals for custom made gel casting. This will significantly increase shelf life of the products and add freedom to the physicians to design custom shaped dressings.

Figure 8 Equipment for gel encapsulation
Partner P4-LUT focused on a process involving printing of different types of nanoparticles on non-woven polymers polypropylene (PP) and polyethylene terephthalate (PET) surfaces by using the roll-to-roll inkjet printer EPSON photo stylus R2880 (Figure 9). For proof of principle the PLLA (source P1) and a PEG/PCL (source P3) nanocarrier systems were used. In order to improve the adhesion and immobilization of the nanoparticles on the non-woven fabrics the Diffuse Coplanar Surface Barrier Discharge (DCSBD) was applied. DCSBD is the dielectric barrier discharge plasma source, which is able to generate the low-temperature and non-isothermal plasma at the atmospheric pressure for activation, modification and improvement of surface properties of various types of materials. DCSBD system was integrated to the printing line to perform pre-treatment of non-woven polymers before nanoparticles printing.

Figure 9. Prototype of the roll-to-roll system with the integrated DCSBD plasma pre-treatment for nanoparticles printing. Visual appearance of the DCSBD plasma burning in ambient air.

Lab scale roll-to-roll integrated engineering solutions were successfully applied for the immobilization of different types of nanoparticles on the hydrophilic surface of non-woven fabrics. A beneficial effect of plasma treatment is its ability to destroy pathogens and to sterilize materials. In the case of the large scale application an industrial textile printer should be used for higher efficiency.

Alternative gel-based solutions for immobilisation and packaging
As a result of the foreground knowledge created, the Bacteriosafe program has included considerable activities into immobilization of the NC/NP systems into gels, which enable (i) embedding of the NC/NP ensuring a “firm” immobilisation, (ii) careful formulation allows for chemical binding between gel and NC/NP which reduces the release of NC/NP fragments after lysis, and (iii) a “moist” environment which improved the storage time of several of the final NC/NP systems. Gel encapsulation of NC/NPs on non wovens from the partner P11-Freudenberg was established at P2-BATH, P3-USiegen and P7-Centexbel (Figure 10) and showed promising results.

Figure 10 lipid vesicles (P2-BATH) sprayed on wet (left) and dry (right) cotton mesh) at partner P7-Centexbel. Yellow halo in left image is due to triton positive control
A prototype dressing prepared by P2-Bath during the final year has enabled a validation of this. The preparative steps towards the lab scale prototype includes capsule synthesis, immobilisation in a gel spotted onto a non-woven and suitable packaging. All steps were performed under sterile conditions, but are not yet GMP/GLP compliant.
A final design prototype has evolved from insights of the previous research and consists of a regular array of embossed wells in a high viscosity agarose gel. Inside these wells are added the vesicles in a low viscosity agarose gel, with a proprietary backing sheet / closure device being added (Figure 11). The dressing itself was packaged in a vacuum packing / sealing system designed for food preparation (cost €40), and shelf life studies suggest dressings are stable at room temperature for at least 6 months.

Figure 11. Packaged, stable dressing prototype at P2-BATH (b) Response to S. aureus USA 300 supernatant 15 min (8 wells on right, control (8 wells on left)
The great advantage of this approach is that fewer vesicles are required, reducing costs, and that the vesicles are stabilized, but best of all, the brightness of the response is enhanced as the dye diffuses into the surrounding hydrogel matrix (figure 11 (b)).
A system diagram for the up-scale manufacture of this dressing system has been drawn (figure 12) which anticipates post-project development. The current cost of a single dressing unit is currently calculated at €7 in materials / consumables costs. It is anticipated that unit cost could be reduced to < €1 when mass-produced.

Figure 12. Process diagram for manufacture of dressings, potentially transferable to continuous manufacture on a ‘line’.(P2-BATH)
Testing of prototypes: biofilm wound model and Limit of Detection and Cytotoxicity of Vesicles
Much of the previous research was carried out using planktonic bacterial cultures. Whilst these are the ‘traditional method’ of doing microbiology, they arguably have little to do with how bacteria actually behave in a wound matrix. In a real wound matrix, bacteria will rapidly colonize both the dressing and wound surface and form confluent layers leading to biofilms. A simple wound biofilm model (figure 13) was thus developed at P2-Bath to model this environment and permit quantification of start bacterial inoculum vs dressing response.

Figure 13: In-vitro biofilm model (P2-BATH). Polycarbonate membrane inoculated with bacteria then covered with dressing. Response as a function of time measured.
The in-vitro biofilm model was inoculated with 10-10,000 bacterial cells/cm2, before the prototype dressing placed on top. It was found that the dressings have a limit of detection of ~100 cells at 4 hours following initial inoculation to both S. aureus and P. aeruginosa: that being a clear colour, visible by eye.

Scientific achievements beyond the original Bacteriosafe goals
After completion of the Bacteriosafe program, the original schematic for the project achievements can be modified to the following, Figure 14. The additional steps taken (khaki-brown boxes) are described below.

Figure 14 Actually achieved goals and specific details of achievements

Qualitative and quantitative investigation of bacterial virulence factors secreted by key bacterial pathogens and their genetic regulation (P2-BATH). ,
When Bacteriosafe was conceived, the proof of principle data had led us to assume that S. aureus secreted a-haemolysin and P. aeruginosa secreted phospholipases, and that these were the key virulence factors responsible for vesicle lysis. However, on study of the virulence action of a range of S. aureus and P. aeruginosa clinical isolates, supplemented by utilisation of gene knock out mutants, where for example the gene for phospholipase in P. aeruginosa was suppressed or removed it was found that the situation was more complex.
Staphylococcus aureus
The staphylococcal accessory gene regulatory (agr) operon is a well-characterised global regulatory element that is important in the control of virulence gene expression for Staphylococcus aureus, a major human pathogen. Hence, accurate and sensitive measurement of Agr activity is central in understanding the virulence potential of Staphylococcus aureus, especially in the context of Agr dysfunction, which has been linked with persistent bacteraemia and reduced susceptibility to glycopeptide antibiotics. Agr function is typically measured using a synergistic haemolysis CAMP assay, which is believe to report on the level of expression of one of the translated products of the agr locus, delta toxin. In this study we develop a vesicle lysis test (VLT) that is specific to small amphipathic peptides, most notably delta and Phenol Soluble Modulin (PSM) toxins. To determine the accuracy of this VLT method in assaying Agr activity, we compared it to the CAMP assay using 89 clinical Staphylococcus aureus isolates. Of the 89 isolates, 16 were designated as having dysfunctional Agr systems by the CAMP assay, whereas only three were designated as such by VLT. Molecular analysis demonstrated that of these 16 isolates, the 13 designated as having a functional Agr system by VLT transcribed rnaIII and secreted delta toxin, demonstrating they have a functional Agr system despite the results of the CAMP assay. The agr locus of all 16 isolates was sequenced, and only the 3 designated as having a dysfunctional Agr system contained mutations, explaining their Agr dysfunction. Given the potentially important link between Agr dysfunction and clinical outcome, we have developed an assay that determines this more accurately than the conventional CAMP assay.
It was observed that the lysis of vesicles temporally correlated with bacteria reaching their post-exponential (stationary) growth phase. This suggested that the regulation of toxin secretion required a critical density of bacteria via a quorum sensing effect. The Accessory gene regulator is a genetic ‘super-regulator’ which might provide the mechanism for such control. Vesicle lysis was observed to occur during early stationary phase growth. Different starting inocula (104, 105, and 106 cfu/ml) of MSSA 476 were used to assess vesicle lysis during bacterial growth. By monitoring optical density and fluorescence it was evident that vesicle lysis occurs at early stationary phase. Bacteria were grown for 18 h with lipid vesicles. Supernatants of wild-type (RN6390B) and the isogenic agr knockout (RN6911) strains, highlight the central role that the agr operon plays in vesicle lysis. 0.01% Triton X-100 and HEPES buffer was used as positive and negative controls respectively. Understanding exactly what the agr controlled lytic agents were required the measurement of vesicle response to isogenic S. aureus mutants. The deletion of a-or b-toxin had no measurable effect on vesicle lysis. A 5-fold dilution of MW2 and MW2Dpvl mutant illustrated the lack of involvement of the Panton-Valentine leukocidin and leukocidin AB, leukocidin DE and the gamma-haemolysin in lysis of vesicles. The Phospholipase plate assay show phospholipase activity as an orange halo around un-treated supernatant filled wells in contrast to no activity with heat-inactivated supernatants. 95 ˚C Heat-treatment of supernatants retains vesicle lysis ability, suggesting no involvement of either phospholipases or gamma haemolysin in vesicle lysis.
Since none of the pre-assumed toxins were involved in vesicle lysis a screen of 89 S. aureus clinical isolates was performed to see if any of the strains did not lyse the vesicles (figure 15). Of the 89 strains, only three did not lyse the vesicles and all were found to be agr negative pointing to the crucial role of the accessory gene regulator in regulating toxin secretion – and in the control via RNAIII of the Phenol Soluble Modulin toxins which include haemolytic toxin.
Figure 15. Differences in agr activity observed using two methods. A) Delta haemolysin plate assay of agr positive (RN6390B) and negative (RN6911) strains, 17 S. aureus clinical isolates, 16 which are designated as agr – negative, one agr positive isolate and agr – positive LAC and its corresponding isogenic hld mutant strain, signifying the effects of delta toxin and PSM on the haemolysin plate assay. B) Normalized fluorescence measurements of 89 clinical S. aureus strains using the vesicle–supernatant method, highlighting the three strains causing no vesicle lysis.

Scale up of polymermersome production
To be able to address the need for large area coating by printing and spraying approaches developed by Partners P7-Centexbel and P4-LUT, the fabrication of proteinase labile polymersome was upscaled by Partner P3-USiegen in an additional deliverable (see report M 42). For this purpose, ring-opening polymerization was expanded from the conventional small scale synthesis to larger scale batch polymerizations of ≥ 20 g. Pure block copolymers, such as PEG114-b-PCL250 and related PEG114-b-PLA systems, were obtained with yields of > 90 %. The synthesis scale can without problems be further increased in batch polymerizations. In parallel, continuous polymersome production using continuous flow afforded polymersome solutions on the liter scale (typical scale: 2 L; one line with a single pump was shown to possess a capacity of 6L in 24 h continuous operation). By using parallel lines and pumps the production is further upscalable. Excess reporter dye (nile red) was removed by dialysis, which is not a scalable technique and should be replaced with advanced ultra- / microfiltration in future work. Characterization of the polymersomes (neat and nile red filled) by dynamic light scattering showed virtually identical size distributions to smaller batches under comparable conditions, excellent reproducibility and the established selective degradation by lipase and proteinase K for PEG-b-PCL and PEG-b-PLA, respectively. Samples were provided to partners P7-Centexbel and P4-LUT for coating experiments.
From the trial scaled up production the approximate cost for a 100g batch of dry capsules is estimated at around €100 - 200, depending on the particular polymer and including chemical & technical supplies. The overall time for production (including drying) was approximately 2 days.

Interaction with non cost industrial partners
Throughout the program the academic partners have received a tremendous support from the industrial non cost partners. The non cost industrial partners in the project were present at all meetings and participated actively in discussions concerning relevant details to the work in the project. They were vital participants in discussions on issues concerning regulatory aspects, sterilization, packaging, marketing and innovative development towards a realistic product and helped to focus the development within the project. Several visits to the site of the industrial partners were organized. Throughout the four years of the program the non cost industrial partners were also always available for telephone discussions and finding rapid solutions concerning sample supplies and evaluating the significance of the collected data. Their interest in the project work continues and discussions are underway to pursue further collaborations in this area.

Interaction with the partner (P12-UniSA) from a 3rd country
In 2010 a group of 7 consortium members, including the partner from a 3rd country, applied for an International Research Staff Exchange Program under IRSES (acronym KOALA, grant 295155). This was granted at the end of 2011, it started in 2/2012 and finished officially on 31.7.2014. In the frame of this program it has been possible to enhance substantially the contribution of P12-UniSA towards the Bacteriosafe project. P12-UniSA was taken into the Bacteriosafe consortium because of their long standing expertise in wound healing, their experience in product formation, regulatory aspects and spin offs. The KOALA program enabled us to actively work together with P12-UniSA to advance our knowledge on responsive materials and nanocapsules for sensing bacterial infection in wounds. The two programs Bacteriosafe and KOALA have enabled a continuous exchange of personnel, and thus the exchange of ideas and concepts towards improved wound healing strategies and have nourished discussions between the groups across the continents. The joint work has led to several publications and joint follow up projects. Results from the KOALA project are being reported separately for grant 295155.

Potential Impact:
Socio economic impact
The societal impact of the project and its future development are immense. The project results set the foundation for hugely improved burn wound care, and enables technological advances towards other types of wounds that affect the ever increasing aging population. The developed materials may enable faster and more reliable evaluation of wounds, indicate infection and deliver on demand antimicrobials to combat infection. It will improve bedside care, minimize hospital stays, and contribute as a central strategy to reduce the risk of spreading antibiotic resistance. The final anticipated prototype dressing is compatible with existing semi-biological wound dressings.
Socio-economic benefits that accompany the outcome of Bacteriosafe are:
(A) Increased knowledge
The immense knowledge gained during the last 4 years of the Bacteriosafe project has been successfully disseminated at a large number of conferences, workshops and symposia worldwide. A total of 22 peer reviewed publications are published at the end of the project, with at least 10 more being prepared for publication in the upcoming months.
The results of the work have fed into the higher education system and have been integrated into undergraduate teaching program at Universities and research institutions in the UK, Ireland and Germany. It has hugely impacted the post graduate training at the academic partner sites in Germany, UK, and Ireland and has led to highly educated European graduates with an international, interdisciplinary training necessary for the innovative future developments in Europe.
(B) Access to new markets
The knowledge gained during the program has opened new concepts and ideas for applications of the basic technology in a variety of markets.
The development of a diagnostic tool that signals the presence of pathogens has a huge field of application in the medical field, not only in burn wound treatment, but in all aspects of wound healing associated with surgery, trauma and bedside diagnostic. The encapsulation of drugs and their targeted/triggered release was still at a very academic level four years ago, but has today advanced into a new aura of industrial interest. With patents expiring on numerous “blockbuster” drugs, pharmaceutical companies are searching for new competitive business strategies. Drug revenues approaching $70–$80 billion were lost in 2011 as various drugs went off-patent. The potential for improving product quality and accompanying advantages of continuous nanopharmaceutical manufacturing has been recognized by both pharmaceutical industries and regulatory authorities. The output from Bacteriosafe feeds into these new and competitive business strategies opening doors towards a variety of pharmaceutical applications. This in turn will impact equipment builders to provide the tools and machines for the manufacture of the different products, the chemical industry in Europe to mass produce and market the chemicals, biological reagents and pharmaceuticals.
With the evolution of resistance reported in ever increasing numbers of pathogenic bacterial strains there is a trend towards delivery on demand in antimicrobial dressings and therapies. This is possible using the concepts established in the Bacteriosafe program and by “outsmarting” bacteria. One step towards this is the triggered release utilizing toxins expressed by the bacteria themselves as established in this project.
(C) Regulatory aspects
From the start of the project a pronounced effort was made to concentrate on materials, precursors, solvents and reagents which are preferably already FDA approved or which are considered “biocompatible”. The final materials used and developed within the Bacteriosafe project have been initially selected on the basis of their biocompatibility (proven non cytotoxic effects). As such we anticipate that future innovation utilizing the Bacteriosafe technology will enable a faster way to market. During the second part of the project the scientific project committee of the Bacteriosafe project approached the Irish regulatory board to obtain advice on the development of a medical device. Their advice has fed into the selection and validation of materials within the project.

Use and Dissemination of foreground
Main dissemination activities
• Jan 2012: Int. Symposium, Hirschegg, Austria
• 2/2012-7/2014: KOALA staff exchange program, collaboration with Wound CRC of Australia
• Jan 2013: Int. Symposium “Responsive materials in wound healing”,: NTU, Singapore
• June 2013: EuroNanoForum Exhibition & Fair: Dublin, Irland
• April 2014: Int. Symposium (“Antimicrobial surfaces”) at the Int. Conference,: Australasian Society for Biomaterials and Tissue Engineering (ASBTE), Lorne (Melbourne), Australia
• Fliers, several articles in public press
• Press releases, U-Tube videos, radio/ TV interviews throughout program
• Conference contributions (oral and poster), invited talks worldwide

Exploitation of results
The Bacteriosafe program has set the ground for the development of an innovative burn wound dressing which will diagnose infection AND deliver the therapeutic antimicrobials to treat the wound. The availability of diagnostic and treatment within a single wound dressing is not yet available on the market and wound represent a significant improvement in patient care in Europe.
At the end of the program the exploitable foreground in Bacteriosafe includes the following:
1) DIAGNOSTIC SYSTEM 1: Stable lipid based nano carrier systems with proven optical response to hyaluronidase (from S. aureus, Steptococcus), phenol soluble Modulins (S.aureaus) and Rhamnolipids (P. aeruginossa) with validation in relevant lab environment, TRL 4-5
2) THERAPEUTIC SYSTEM 1: Stable lipid based nano carrier systems with proven therapeutic response to hyaluronidase (from S. aureus, Steptococcus), phenol soluble Modulins (S.aureaus) and Rhamnolipids (P. aeruginossa) with validation in relevant lab environment, TRL 4-5
3) DETECTION SYSTEM 2: Stable amphiphilic block copolymer vesicles with proven response to prote(in)ase, lipase and in particular hyaluronidase with validation in the lab environment, TRL 4
4) THERAPEUTIC SYSTEM 2: Stable amphiphilic block copolymer vesicles with proven response to prote(in)ase, lipase and in particular hyaluronidase with validation in the lab environment, TRL 4
5) THERAPEUTIC SYSTEM 4: Stable nanocapsules consisting of a hyaluronic shell with proven response to lipase and in particular hyaluronidase (S.aureus) with validation in the lab environment, TRL 4.
6) THERAPEUTIC SYSTEM 5: stable nanocapsules consisting of a polylactic acid with proven response to lipase and in particular hyaluronidase (S.aureus) with validation in the lab environment, TRL 4.
7) Upscaling the production of the nanocarrier systems by by 2 - 3 orders of magnitude.
8) Engineering processes validated for the immobilization of the nanocarriers with retention of morphology and functionality. Validation for selected systems in relevant lab environments, TRL 4-5
Validation of the developed materials will continue in the groups at laboratory level.

Anticipated 5 year plan
The anticipated short and long term goals of the Bacteriosafe project are schematically shown in figure 16. We anticipate the short term impact (<1 year) of the Bacteriosafe project to become apparent as a number of smaller projects by individual Bacteriosafe partners and smaller groups of Bacteriosafe partners aimed to address some of the challenges that were not part of the original Bacteriosafe plan. Figure 18 below indicates a predicative time plan for bringing a functional responsive wound dressing, such as developed in Bacteriosafe, to market. The Bacteriosafe project aimed to develop on the lab scale and up to the beginning of pre-clinical trials, but not including animal trials. This goal was successfully accomplished by the consortium. The next most critical steps include:
(i) The scale up of the nanocapsules under GMP/GLP conditions
(ii) Prototyping the wound dressing in larger quantities, and
(iii) Pre-clinical trials of first prototypes.
We anticipate this to take at least 2 years, depending on funding. At the same time the partners are actively seeking to spread the technology evolving from Bacteriosafe towards other applications and other industrial sectors such as hygiene, food packaging, nanopharmaceuticals, sensors etc.

Figure 16 Predicted short term and long term plan and impact of the Bacteriosafe outcome.

Activities towards the production of validated prototypes
During year 3 of the Bacteriosafe program (2013) the consortium submitted a proposal (SMARTWOUND proposal#604067-2) for the production of prototypes and preclinical trials. This was unfortunately not granted.
Activities into upscaled production of nanopharmaceuticals
Large quantities of nanopharmaceuticals (drugs encapsulated in nanosized matrices or containers) are required to enable sufficient reproducible prototypes for preclinical and clinical trials. We continue to pursue solutions to enable sufficient amounts of nanoscale material. A proposal for upscaling engineering processes for the encapsulation of enzymes and proteins in biocompatible polymeric nanocapsules up to a GMP compliant pilot plant (SEPA proposal# 646279) has been submitted and is waiting for a decision by the commission. The anticipated plan within this project will enable upscaled encapsulation of different types of “cargo” in a variety of “nanocontainers” and can be applied to Bacteriosafe technology.
Activities into Prototyping & Pre-clinical development of lipid based dressing
A post-project development plan has been submitted by P2-Bath for funding by the UK Medical Research Council in July 2014. Briefly, the plan is:
The research programme consists of three distinct phases, shown in figure 19. Phase one contains three interlinked work packages which will drive iterative development (WP1) informed by WP2&3. This will include the in-vitro testing of prototypes against a broad range of bacteria obtained from burn wounds, and performance in ex-vivo wound infection models with porcine skin. Satisfactory performance in the extensive in-vitro and ex-vivo tests conducted in WP2&3 will constitute milestone 1 and a critical stop/go point for progression to phase 2. Phase 2 is an outsourced toxicology study (WP4) which will deploy ISO10993 tests including cytotoxicity, genotoxicity & carcinogenicity of dressing components and dressing ensemble. A parallel development and planning WP5 runs along side. Satisfactory outcomes from this study (milestone 2, stop / go gate) will lead to phase 3, an in-vivo pig study of dressing performance on an infected burn model. (WP6) Milestone 3.
Activities towards novel biosensors evolving from Bacteriosafe technology
A post program development project has been applied for by P3-USiegen under AiF (ZIM-cooperation projects) with an SME: „Entwicklung eines Indikatorstreifens zum Point of care Nachweis von Verderbniserregern in Lebensmitteln“
Another program that has been granted and will enable post program development between 2014-2015 includes the ATN - DAAD project between P3-USiegen with P11-UniSA “Optical Biosensors for Wound-Colonising Bacteria”.
In 2014 we received a 6 Month DAAD Research Grant for PhD Student: Jessica Bean (P2-Bath) to P3-USiegen “Formation of photo-crosslinked hyaluronic acid systems by nano-imprint lithography, for the creation of stimuli-responsive hydrogel burn wound dressings”
A grant application is in preparation involving partners P2-Bath and P3-USiegen: EU Horizon 2020 consortium for an upcoming 2014 call, Working title “Fluorescence Imaging Assisted Infection Diagnostics in Diabetic Foot Care“
A EU Horizon 2020 consortium involving partner P3-USiegen focusing on “Encapsulation of active for functional textiles” (working title) is currently shaping up for upcoming 2015 calls.
Industrial support from different sectors to enable innovation
Pharma industry has been approached for the encapsulation of selected enzymes and have entered into partnership in the above mentioned proposals.
European SMEs specializing in the production of (i) specific and selected enzymes and (ii) chemicals for the production of nanopharmaceuticals.
Wound dressing companies have been addressed and are partners in the above described proposal.
SME’s in food safety diagnostics have shown interest in the approaches developed.

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