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The Future of Chitosans

Nano3Bio Final Event – September 20, 2017


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Contents Contemporary chitosan research by Nano3Bio – The Future of Chitosans ........................................................................................... 2

Abstracts .................................................................................................................... 6Chitosans – High-Tech Functional Biomaterials for the Bio-Economy .......................................................................................................... 7Deciphering chitin biosynthesis: fundamentals and potential applications .............................................................................................. 8The promise of enzymes: natural chitosans through Biotechnology ..................................................................................................... 9Nano3Bio Chitosans – Improved Performance through NanoBioTechnology ...................................................................................... 10The transglycosylation route for synthesis of bio-active chitooligosaccharides ............................................................................... 11Enzyme engineering for the production of chitosans with defined acetylation pattern ............................................................................... 12Expanding the metabolic engineering toolset to develop tailor-made microbial cell factories ......................................................................... 13Electrospun and electrosprayed chitosan nanofibers and particles for biomedical applications .............................................................. 14Chitosan-based nanoparticles and their promising role in tomorrow’s medical therapies ............................................................................... 15Biomineralization of thermo-sensitive chitosan hydrogels for bone tissue regeneration ....................................................................................... 16Intravascular accumulation and retention of chitosan-nanocapsules ............................................................................................. 17Life cycle assessment of chitosans and chitooligosaccharides production ....................................................................................... 18

About Nano3Bio ..................................................................................................... 20Major achievements of Nano3Bio ............................................................................. 21Nano3Bio consortium partners ................................................................................... 22

Acknowledgement .................................................................................................. 23

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Contemporary chitosan research by Nano3Bio –


Chitosans are an amazing class of functional biopolymers, perhaps the most versatile and most promising one. They can be used in medicine, in agriculture, in food industry, in cosmetics, in water and wastewater purification, in paper and textile industries – and in biotechnology.

Traditional chitosan and new requirements

Chitosan can be produced rather easily from chitin, one of the most abundant biopolymers that is wide-spread in nature, e.g. giving strength to insect shells as well as shrimp and crab carapaces. Thus, chitin and chitosans are renewable resources of almost unlimited availability. Waste material from the shrimp fisheries can be transformed into a valuable product of immense potential.

However, there is a catch, as with all things that sound too good to be true. Chitosan has been a ‘promising’ biopolymer for almost fifty years. But initial promises could not be kept. Results on bioactivities reported in the scientific literature did not lead to the development of products be-cause the results were not reliably reproducible. Industry understandably was disappointed and lost interest. Today, two decades of fundamental research on structure-function relationships have led to the development of well-defined chitosans with known physico-chemical properties and reliable biological functionalities. These second generation chitosans are ready for the markets – to be used for the development of reliable appli-cations and successful products. As a consequence, the European Commission recently registered chitosan hydrochloride as a ‘basic sub-stance’ which can now be used e.g. in agricultural products without the need for lengthy and costly toxicity studies and registration processes.


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But even the well-defined second generation chitosans are not perfectly suitable for all applications, in particular not in sensitive fields such as health care, pharmacology, and biomedicine. Here, the animal origin of chitosan is a hindrance to market entry, as is the – real or assumed – danger of allergen or even viral contamination. This is where the Nano3Bio project sets in, aiming at the development of biotechnological production processes for well-defined, third generation chitosans.

New approach, advanced opportunities

Nano3Bio convenes an international team of researchers from universi-ties, research centres and companies around Europe and India, joining forces to make a dream come true: the biotechnological production of third generation chitosans. These chitosans will be even less poly-disperse than hitherto existing chitosans, or even monodisperse in the case of oligomers, with defined, non-random patterns of acetylation, clearly defined biological activities, and known cellular modes of action. These chitosans will create new market opportunities in the future.

The Nano3Bio project significantly improved the capability to deliver chitosans with known and defined, non-random patterns of acetylation. These will then be compared to their conventional counterparts, to benchmark their properties and functionalities against the best per-forming chitosans available today.

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What chitosans can do for you

Some chitosan sponges can stop bleeding, and some chitosan-based dressings can support scar-free healing even of chronic or large-scale wounds.

Some chitosan nanoparticles can transport drugs across cellular barriers, including the blood-brain barrier, and DNA or RNA into cells.

Some chitosans can stimulate the immune system, including that of animals, so that they can be used as a feed additive to reduce the use of antibiotics.

Some chitosans can promote plant growth and induce disease resistance and stress tolerance in plants by strengthening the plant’s own defensive system.

Some chitosans can be used to control postharvest diseases and they can form transparent films to be used e.g. as food packaging, keeping fruits and vegetable fresh and preventing spoilage.

Some chitosans can stabilize creams and shampoos and at the same time, preserve them.

Some chitosans can clean drinking water, filter wine and remove proteins or heavy metals from the waste water of breweries or industry.

Some chitosans can be used as a biocompatible surface to culti-vate human cells, e.g. as a 3D matrix in organ models.

Furthermore there are a multitude of other possible applications of this versatile class of biopolymers. Pure chitosans are completely non-toxic to plants, animals, and humans. They are non-allergenic and easily degraded in the environment.


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The nano aspect

Nano3Bio aims to develop biotechnological approaches to produce chitin and chitosans as well as their nano-formulations, and to analyse their nano-scale solution properties for a wide range of possible applications, e.g. in biomedicine and pharmaceuticals.

Novel chitosans can be used in aqueous solution, as physical hydrogels, or in the form of nanostructures such as nanoparticles, nanocapsules or nanofibre hybrid materials. In addition, Nano3Bio explores molecular nano-imprinting strategies to confer the surface of nanoparticles and nanofibers with high affinity towards biologically relevant molecules that can be exploited in drug delivery and antibacterial activity. While nano-formulations can be bio-compatible carriers for drug, gene and vaccine delivery, nano-structured physical hydrogels are promising bio-materials for tissue engineering, e.g. bio-mineralisation for bone repair.

One example of a promising Nano3Bio achievement are nanocapsules made of a specific novel chitosan which accumulate in tumours. When loaded with a marker, these capsules will allow early diagnosis of even small tumours and metastases. They can also be loaded with anti-tumour drugs which are thus targeted specifically to the tumours, minimizing un-wanted off-site effects. The next step in this development will be to use a biotechnologically produced designer chitosan which will be fully degradable in the human body, preventing the long-term accumulation of the nanocarrier, thus further minimizing adverse effects of targeted cancer therapies.

Learn more about Nano3Bio:

www.nano3bio.eu

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Abstracts

On the following pages you will find the abstracts belonging to the talks on the Nano3Bio event ‘The Future of Chitosans’ on September 20, 2017 in Hyderabad, India. The abstracts are deliberately framed short in this booklet in order to provide easy access to the approaches and outcomes of the consortium’s scientific work.

Visit www.nano3bio.eu/start/final-conference/ in order to find up-to-date information on the event’s agenda.


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Chitosans – High-Tech Functional Biomaterials for the Bio-Economy

Bruno M. Moerschbacher et al. University of Münster, Germany ([email protected])

Nature has all the solutions! Over thousands of millennia, life has evolved fantastic solutions to almost every problem imaginable. One formidable problem faced by humanity is our dependence on fossil oil not only as a source of energy, but also as a raw material for an ever-increasing range of plastics. While these are becoming more and more recyclable and biode-gradable, their quality continuously deteriorates with every cycle and more often than not, final degradation products remain that are accumulating as microplastics in the environment – at a scale that exceeds all imagination.

Nature’s materials – biomaterials – are different. They are multi-purpose materials and fully recycled at the end of their lives, serving as nutrients for micro-organisms which degrade them into their constituent building blocks, to be re-used as new materials or to be burned for energy release, emitting only the CO2 fixed earlier during their biosynthesis. Among the most spec-tacular biomaterials are biopolymers such as cellulose and chitin which are wrapped around plant and fungal cells as re-enforcing fibres in the protecting shields of their cell walls. They are also extremely abundant: cellulose is without doubt the most abundant biopolymer on Earth and arguably, chitin is second. Chitin can be converted into chitosans, a family of highly promising, biologically active biopolymers and smaller oligomers which have the potential to become the basis of a range of sustainable solutions for diverse applications. However, chitosans form a large family of biomolecules differing in their structure and functions.

Some chitosans can protect plants from disease, some chitosans can be used for targeted drug delivery, some chitosans can protect food from spoilage, and some chitosans can support scar-free wound healing. But the chemical processes used today to produce chitosans from natural chitin yield mixtures of chitosans which differ in composition from batch to batch unless strict measures of quality control are employed.

The Nano3Bio project, therefore, aims to design biotechnological ways using nature’s tools to produce well-defined natural chitosans which differ from their conventional, chemically produced counterparts by having better defined and different structures, and more reliable functions.

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Deciphering chitin biosynthesis: fundamentals and potential applications

Elzbieta Rzeszutek, Sara M. Díaz-Moreno, Osei Ampomah, Vincent Bulone Royal Institute of Technology (KTH), Sweden

Chitin is a linear polymer of β-1,4-linked N-acetylglucosamine (GlcNAc) residues and it is one of the most abundant biopolymers in nature. It is present in many diverse organisms including insects, arthropods, fungi, and fungal-like organisms as oomycetes. Chitin is synthesized by membrane-bound proteins called chitin synthases. These enzymes belong to the glycosyltransferase family whose members typically share a conserved catalytic domain flanked by multiple transmembrane regions. The mechanism of chitin biosynthesis is currently not well understood. Gaining insight into the polymerization and assembly of chitin chains into microfibrils is depen-dent on the identification and characterization of the chitin synthase en-zymes. Our work is focused on the characterization of chitin synthase proteins from the oomycete Saprolegnia parasitica, one of the most severe fish pathogens, responsible for important economic losses in aquaculture. Even though the amount of chitin in those organisms is present in very small amounts, the biosynthesis of the polymer hence chitin synthase activity is crucial for hyphal growth. This presentation will describe the full character-ization of a chitin synthase from S. parasitica (CHS5). The enzyme was successfully expressed in a heterologous system and purified to homogeneity as a full-length protein containing all predicted transmembrane domains. The recombinant protein was shown to be catalytically active in vitro. Our data also indicate that CHS5 most likely occurs as a homodimer, the formation of which seems required for activity and translocation of chitin across the membrane to the extracellular space. The product formed in vitro by the recombinant enzyme is organized as crystalline chitin microfibrils with an average chain length of 150. Point mutations of conserved amino acids allowed us to identify the essential residues for activity and processivity of the enzyme. The data shed light on the fundamental mechanism and properties of chitin biosynthesis in oomycetes and other eukaryotic micro-organisms. Protein engineering can be envisaged for the first time to control the structural properties of chitin and derive tailored structural and functional chitin-based products.


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The promise of enzymes: natural chitosans through biotechnology

Lea Hembach, Stefan Cord-Landwehr, Jasper Wattjes, Anna Niehues, Janina Hoßbach, Shoa Naqvi, Nour Eddine El Gueddari, Bruno M. Moerschbacher

University of Münster, Germany ([email protected]) Chitin is an evolutionary ancient molecule found not only in fungal cell walls but also prominently giving strength to insect and crab outer shells and to the skeletons of a many invertebrate animals. Being strikingly absent from higher plants and vertebrate animals, chitin is a tell-tale molecule triggering defense reactions in the immune systems of these organisms. In turn, pathogenic micro-organisms have learned to disguise their presence by masking their chitin, e.g. by slightly modifying it into chitosan. This modified biopolymer - chitosan - is rare in nature, but it can readily be produced by a simple chemical treatment of chitin which is one of the most abundant biopolymers on Earth. However, this chemical process yields a mixture of different chitosans, with poorly defined structural and functional properties. In contrast, organisms use enzymes, called chitin deacetylases, to perform the same process, and these appear to yield different, well-defined chitosans. The task of University of Münster within the Nano3Bio project is to characterize such enzymes from different sources, mostly fungi but also bacteria, viruses, and algae, to use them in a biotechnological process for the conversion of chitin into chitosans. An in depth understanding of the enzymes’ mode of action can then be used to optimize them using bioinformatics-based protein engineering techniques, so that they produce defined chitosans suitable for specific applications. We have hypothesised, and the results are beginning to prove us right, that these biotechnologically produced, “third generation” chitosans are different from today’s chemically produced chitosans, and that they are showing more reliable performance in a range of bioassays. These novel, natural chitosans promise to allow the development of novel, sustainable, environment-friendly and customer-safe solutions e.g. in plant disease protection, food preservation, targeted drug delivery for instance for tumour therapy, and scar-free wound healing even of large, third degree burns.

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Nano3Bio Chitosans – Improved Performance through NanoBioTechnology

Bruno M. Moerschbacher University of Münster, Germany ([email protected])

The Nano3Bio project pursues two different, parallel approaches towards the biotechnological production of novel, natural chitosans. Both approaches make use of nature’s own tools for the biosynthesis of chitosans. Our first approach targets an enzymatic conversion of chitin into chitosans, using chitin deacetylases. The raw material used in this process is natural chitin isolated from shrimp and crab shell wastes of the fishery industries - the same that is used today for the chemical conversion into chitosan. Chitin deacetylases can be found in a range of fungi which are rare natural producers of chitosan, as well as in some bacteria - but we now also discovered them in some viruses and microalgae which we found to also be natural producers of chitosans. However, we had to realise that these enzymes do not perform well in biotechnological processes so that we had to optimise them through bioinformatics-based protein engineering. At the end of the Nano3Bio project, we have achieved the first production of biotechnologically produced, natural chitosans the structure of which differs significantly from all known conventional chitosans, and the biological functionalities of which we are currently investigating. Our second approach for the biotechnological production of chitosans goes one step further by even producing the chitin in an enzymatic process. Nature’s enzymes for this step - chitin synthases - are wide-spread e.g. in fungi and insects, but we again also found them in some viruses, bacteria, and microalgae. These are large, complex enzymes naturally embedded in cell membranes and once isolated, they exhibit extremely poor performance. Therefore, we use these enzymes in their natural environment, i.e. in living cells. Combining different, natural or optimised chitin synthases and chitin deacetylases in bacteria, we achieved the production of a range of fully defined, small chitosan oligomers. Sophisticated metabolic engineering of the production strains led to high purities and yields of the target chitosans, and upscaling of fermentation and down-stream processing led to the production of sufficient amounts of these novel chitosans which are now being studied for their biological activities.


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The transglycosylation route for synthesis of bio-active chitooligosaccharides

Appa Rao Podile, Jogi Madhuprakash, et al., University of Hyderabad, India ([email protected])

Chitinases are a class of family 18 glycosyl hydrolases (GHs) that catalyze hydrolysis of β-1, 4 glycosidic bonds present in the homopolymer chitin which is made of repeating units of N-acetylglucosamine. The degradation products of chitin referred to as chitooligosaccharides are of special interest due to potential biological applications, especially in the food, medical, and agriculture fields. Biological activity was known for chitooligosaccharides with a chain length or degree of polymerization (DP) ≥4. Long-chain chito-oligosaccharides (in particular DP ≥4) could be produced either by using chitin synthases or by using chitinases that have transglycosylation activity. Some chitinases show transglycosylation activity, along with hydrolytic activity, forming new glycosidic bonds between donor and acceptor saccharides. The major goal of University of Hyderabad within the Nano3Bio project is mining of different bacterial isolates for chitinases with potential transglycosylation activity for the production of long-chain chitooligo-saccharides, which could improve disease resistance in plants. But, the transglycosylation efficiency by the wild-type chitinase(s) (and other GHs) is inevitably limited by enzyme-catalyzed (secondary) hydrolysis of the transglycosylation product(s). So, there is a definite need for curtailing the hydrolytic activity while improving transglycosylation activity, to generate longer chain chitooligosaccharides. This problem was addressed by an in-depth characterization of selected chitinases for a clear understanding of the mode of action and further we were able to improve the transglycosylation activity through structure-guided mutational strategies coupled with protein engineering techniques. As it was proposed, we generated hyper-transglyco-sylating mutants of a chitinase and the best mutants were employed for developing a bio-process for the large-scale production of longer chain chito-oligosaccharides. In addition, these chitooligosaccharides were tested for their ability to improve disease resistance in rice plants.

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Enzyme engineering for the production of chitosans with defined acetylation pattern

Antoni Planas, Xevi Biarnés, Hugo Aragunde, Cristina Alsina, Laia Grifoll, Sergi Pascual, Almudena Aranda

University Ramon Llull, Spain ([email protected]) Chitin processing, mainly in the form of depolymerization and de-N-acety-lation reactions by chitin-modifiying enzymes (chitinases and deacetylases), generates a series of derivatives including chitosan and chito-oligosaccha-rides (COS), which play remarkable roles in molecular recognition events, in-cluding the modulation of cell signaling and morphogenesis, the immune res-ponse, and host-pathogen interactions. Chitosans and COS are also attracttive scaffolds for the development of bionanomaterials for drug/gene delivery and tissue engineering applications. Most of the biological activities associated with COS seem to be largely dependent not only on the degree of polymeri-zation but also on the acetylation pattern, which defines the charge density and the distribution of GlcNAc and GlcNH2 moieties in chitosans and COS.

In the Nano3Bio project, the IQS-URL team is addressing strategies to pro-duce sequence-defined chitosan oligomers and polymers by enzymatic pro-cesses starting from simple chitin oligosaccharides (chitobiose to chito-pentaose). The strategy involves the specific deacetylation of a chitin oligo-saccharide by the action of selected and/or engineered chitin deacetylases (defined pattern of acetylation) followed by enzymatic polymerization by the action of engineered glycosynthases to generate new polymers having a regular and well defined pattern of acetylation as single products. Protein engineering of both enzyme families seeks to generate tailor-made biocatalyst for the production of chitosan with defined acetylation pattern.

a) Chitin deacetylases (CDAs): A major challenge is to understand how different CDAs specifically define the distribution of GlcNAc and GlcNH2 moieties in the oligomeric chain. We reported the first 3D structures of a CDA in complex with natural substrates and proposed a structural model to predict the deacetylation pattern exhibited by different CDAs. This model has been used to engineer enzyme specificity towards novel patterns and highly efficient enzymes.

b) Glycosynthases (GS): By engineering the active site of chitinases, the na-tural hydrolytic activity is abolished/decreased, and synthase activity is introduced. Novel mutants have been developed to polymerize activated gly-cosyl donors to give low molecular weight polymers with defined structures.


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Expanding the metabolic engineering toolset to develop tailor-made microbial cell factories: microbial production of

pure and well-defined chitooligosaccharides Marjan De Mey, Jo Maertens, Pieter Coussement, David Bauwens,

Wouter Van Bellegem, Dries Duchi, Chiara Guidi Ghent University, Belgium ([email protected])

Industrial biotechnology, i.e. the use of microorganisms and enzymes to produce chemicals, materials and energy from renewable resources, is a key enabling technology and has the potential to tackle major societal problems such as climate change, the growing human population and the increasing waste mountain. In this context, the growing number of metabolic engine-ering, systems biology and synthetic biology techniques and tool boxes allows researchers to engineer the biotechnological production of an increasingly larger number of complex molecules. The expression of multi-gene pathways is nonetheless a daunting endeavour given that unbalanced enzyme expression in a pathway limits the flux through the biosynthetic pathway. Hence, pathway optimization is being increasingly recognized as a critically important activity. In response, we developed a new combinatorial multi-gene pathway assembly scheme based on Single Strand Assembly (SSA) methods and Golden Gate Assembly (GGA), exploiting the strengths of both assembly techniques. With a minimum of intermediary steps and an accom-panying set of well-characterized and ready-to-use genetic parts, the de-veloped workflow allows effective introduction of various DNA part libraries (sequence variability) and efficient assembly of multi-gene pathways. Here, we have applied these methods to build an efficient microbial-based platform for the production of a portfolio of pure and well-defined chitooligosaccha-rides (COS). Thus far however, the considerable product dispersity in terms of degree of polymerization (DP), degree of acetylation (DA) and pattern of acetylation (PA), obtained with todays’ production technologies severely hampers the exploitation of their application potential in domains ranging from health care to agriculture, as these applications likely rely on stringent structure/function relationships. To this end, E. coli was massively tuned by integrating metabolic engineering and synthetic biology with a view to i) increasing product yield and productivity, ii) improving product purity, and iii) expanding the library of well-defined chitooligosaccharides produced in vivo both by employing engineered chitin synthases and chitin deacetylases and exploiting the natural diversity thereof.

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Electrospun and electrosprayed chitosan nanofibers and particles for biomedical applications

Ana C. Mendes, Ioannis S. Chronakis

Technical University of Denmark (DTU) Chitosan(s), a chitin-derived polysaccharides made of glucosamine and N-actetyl glucosamine, exhibit a set of remarkable biological properties such as biocompatibility, biodegradability, hemostatic activity, antibacterial, anti-mycotic and anticoagulant activity. Subsequently, chitosan’s have been largely explored to develop functional nano-micro structures (particles, fibers, etc.) for biomedical applications.

Nano-microfibers could be used for the encapsulation and release of drugs due to their high surface area, porosity and tunable diameter, as well as scaffolds that mimic the architecture of tissue to seed mammalian cells. Nano-microfibers can be produced by electrospinning, a technique that uses electric field to convert a liquid solution into solid nano-microfiber mats. Parent technique to electrospinning is electrospray that converts liquid solutions into solid nano-microparticles.

In the Nano3Bio project, DTU has developed electrospun nano-microfibers of chitosan and phospholipids as carriers for the controlled delivery of drugs, either orally or via skin (transdermal). These functional fibers were also shown to be mucoadhesive, which will facilitate the release of the drugs in the local areas of the small intestine, where the major absorption of nutrients and pharmaceuticals to the blood circulation occurs. Moreover, chitosan-phospholipid fibers could be used as scaffolds to seed and culture skin cells to construct human tissues (e.g skin). In addition DTU produced electro-sprayed chitosan particles that can be used as a carrier for oral delivery of vaccines, avoiding the discomfort of the patients receiving the vaccines by injection.


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Chitosan-based nanoparticles and their promising role in tomorrow’s medical therapies

Francisco M. Goycoolea et al. University of Leeds, UK / University of Münster, Germany

([email protected]) The main achievements of science and technology in the last century have brought substantial improvements in the quality of life (e.g., improvements of food production, health, energy, transport and communications). However, many gaps in health remain worldwide (e.g. antimicrobial resistance, more effective therapies against cancer, high prevalence of obesity, diabetes and cardiovascular diseases, lack of therapies for rare diseases). To meet these outstanding gaps, new therapeutic paradigms are necessary that lead to rational approaches to influence specific cell responses. Bioinspired nanomaterials not only share similar building blocks, but also the hierarchical organization in the length scale of living systems. Chitosan is a unique biopolymer in this regard, due to its multifunctional material and biological properties. This conference will address the progress achieved in our laboratory, in the frame of the Nano3Bio project, on the development of chitosan-based particles (<~300 nm) produced from fully characterized chito-sans varying in their structure, using different techniques such as ionotropic gelation, covalent crosslinking, electrostatic self-assembly, spontaneous emulsification and layer-by-layer coating. We have addressed the role chitosan’s degree of acetylation and molar mass on the physicchemical and biological characteristics of these systems, including their size distribution, surface charge (zeta potential), capacity to interact with mucin, and their antibacterial activity. We have also studied the formation of nanocomplexes with polynucleotides (pDNA, siRNA and microRNA) and ability to reprogram breast cancer and cystic fibrosis cells using non-viral gene therapy; as well as nanocapsules loaded with natural phytochemicals (e.g. capsaicin) that can influence the absorption of drugs and can disrupt the communication processes (quorum sensing) in Gram negative bacteria, thus potentially reducing their virulence. For some of these systems, we have established benchmark comparisons between the chemically produced and novel chitosans derived from microalgae generated in the Nano3Bio project. The current obstacles for the future translation of these approaches from the laboratory bench to the patient’s bed will also be discussed.

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Biomineralization of thermo-sensitive chitosan hydrogels for bone tissue regeneration

Myriam G. Tardajos, Peter Dubruel Polymer Chemistry and Biomaterials Group, Gent University, Belgium

([email protected]) Regenerative medicine is a growing area constantly in search for new materials where cells proliferate and regenerate damaged tissue. Finding an appropriate technique for bone regeneration still remains a significant clinical challenge. Bone itself consists mainly of organic material and in-organic bone mineral in the form of small crystals. A wide range of synthetic biomaterials have been proposed as bone graft substitutes combining polymers, as the organic part, and calcium phosphates (CaP), as the mineral. The natural biopolymer chitosan is a promissing candidate for bone tissue engineering. Chitosan, apart from being bioresorbable, is biocompatible, non-toxic and a biofunctional polymer.

In terms of scaffolds, one of the most promising materials that resemble the natural environment are hydrogels. Hydrogels are polymeric networks with the ability of imbibing large amounts of water or biological fluids. Chitosan hydrogels are produced in combination with sodium beta-glycerophosphate. These hydrogels are designed to undergo a rapid liquid to solid phase transition in response to temperature changes. The thermo-sensitivity of these hydrogels makes them ideal as injectable materials in the biomedical field due to their gelation at human body temperature (37ºC).

Advantages of using injectable hydrogels as biomaterials for tissue regeneration include exact filling of defects, ease of integration of cells and water-soluble bioactive substances including enzymes. The ability of these hydrogels to imbibe the enzyme ALP accelerates the gelation and promotes the mineralization. ALP is secreted by osteoblasts (bone cells) and assists in liberating phosphates from organic phosphates, facilitating the precipitation of CaP crystals.


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Intravascular accumulation and retention of chitosan-nanocapsules

Christian Gorzelanny et al. Heidelberg University, Medical Faculty Mannheim, Germany

([email protected]) The specific delivery of anti-tumor drugs into tumor tissues is a desired goal of modern cancer therapies. Encapsulation of chemotherapeutic drugs in nano-sized carriers such as nanoparticles or nanocapsules is considered to increase the accumulation of the drug within the tumor due to the enhanced permeability and retention (EPR) effect. However, microthrombi within the tumor vasculature have been speculated to limit the distribution of the nano-carrier within the tumor and thus the therapeutic efficacy of the loaded drug.

Here, we report the trapping of chitosan nanocapsules in blood vessels of primary melanomas in vivo. Tumors were generated through intradermal injection of malignant melanoma cells (ret). Primary skin tumors developed within two weeks and fluorescent labeled nanocapsules were injected intra-venously. Retention of the nanocapsules was detected 24h after particle administration by immune histology in sections of cryconserved tissue. To further support the idea of an intravascular nanoparticle accumulation we performed additional experiments in transgenic animal models with an affected coagulation. Nanocapsule accumulation within the tumor was measured in vivo using intravital imaging (IVIS). In line with our hypothesis, we found a significantly reduced tumoral concentration of nanocarries in animals with a reduced intravascular coagulation due to the lack of von Willebrand factor (vWF). To support our in vivo results we have performed microfluidic experiments mimicking tumor vessels in vitro. In comparison to polystyrene nanoparticle we found that chitosan-based nanocapsules exhibit a significantly increased ability to interact with the pro-coagulant vWF.

In conclusion, we confirm the previously hypothesized limitation of the EPR-effect by tumor-associated thrombosis. We further envision that targeting of intra-tumoral vessel occlusions with chitosan nanocapsules is a novel therapeutic approach in cancer therapy.

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Life cycle assessment of chitosans and chito-oligosaccharides production Ivan Muñoz, 2.-0 LCA consultants, Aalborg, Denmark Franziska Möller, Greendelta GmbH, Berlin, Germany

Life cycle assessment (LCA) is an increasingly used tool for environmental assessment of products and services, involving a comprehensive analysis of the potential environmental impacts associated to product supply chains. We present an LCA applied to production of chitosans based on data from two real producers, located in Europe and India, respectively. We assess in total three chitosan supply chains: from shells of snow crab caught in North America, from shells of shrimp caught in Asia and from squid pen caught in Asia. We also present a detailed analysis of the Nano3bio biotechnological approach for chito-oligosaccharides (COS) production at pilot scale, based on fermentation of glucose, and we compare this novel process to a conven-tional production based on chemically breaking chitin to obtain COS.

The analysed system for each chitosan supply chain included the production of raw materials, their processing to produce chitin and the manufacture of chitosans. Data for biotechnological COS production where obtained first-hand from the pilot-scale tests carried out during the Nano3bio project. We quantified the consumption of nutrients, process chemicals as well as electricity and steam consumed during the different operations, from fermentation to the COS separation. Conventional COS production based on chemical treatment of chitin was included based on a theoretical process derived from patents and industrial know-how by the Nano3bio consortium. In all chitosan and COS production routes we assessed the effects of potential deforestation associated to the demand for land.

The environmental impact assessment was carried out by means of the recommended methods in the International Life Cycle Data (ILCD) handbook, including 15 indicators such as greenhouse-gas emissions, water use and land use. The results show that the assessed chitosans have very different environmental profiles, reflecting their substantially different supply chains in terms of raw material, production locations, as well as the different appli-cations. We also show the potential environmental improvement that biotechnological COS could represent when compared to conventional COS production.


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About Nano3Bio From 2013 to 2017 the international project Nano3Bio pursued the biotechnological production of chitosans as its main overall goal. The European Commission supported the research project with almost nine million Euros. In important fields, the consortium achieved or prepared breakthroughs from basic research to biotechnological applications. The list on page 21 of this booklet contains a selection of Nano3Bio’s major achievements.

Nano3Bio is the highlight of a sequence of international projects, which helped to build knowledge on chitosans for nearly two decades, first laying the path, then paving the way towards today’s successful second generation chitosans. Nano3Bio extended this success story towards third generation chitosans that will be of even higher quality.

Towards this ambitious goal, leading experts from 22 universities, re-search institutes, and companies from Belgium, Denmark, France, Ger-many, India, Spain, and Sweden formed the Nano3Bio consortium, led by Prof. Bruno Moerschbacher (University of Münster, Germany). Page 22 of this booklet provides a list of all Nano3Bio consortium partners.

Nano3Bio generated encouraging results. However, the road ahead is still challenging. For example, it will be important to further determine which biological organisms are best suited to produce exactly the different qualities and quantities of chitosans required for specific applications.

Not least, the consortium is convinced that many other fields of appli-cation can be identified in which a specific chitosan can replace or support other, less sustainable substances. This is desirable, since one of the good qualities of chitosans lies in the fact that they are well-tolerated by the human body and fully biodegradable in the environment.


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Major achievements of Nano3Bio

Major achievements of Nano3Bio

The Nano3Bio project created a host of innovations, such as:

Protocols for the production of chitosans with better defined structures developed

Findings on chitosan binding a red dye in unique and intriguing ways

Self-assembled xanthan-COS nanofibers developed Policy recommendations generated based on regulatory

analysis Electrospun and electrosprayed chitosan nanofibers and

particles developed Thermo-sensitive chitosan hydrogels for cell encapsulation

applications developed New method for chitosan quantification developed A new low-cost protein engineering technology developed Novel chitosan polymers produced biotechnologically in a

bio-refinery process Novel chitosan oligomers produced biotechnologically

in a cell factory approach First natural microalgal chitosans from micro-algal

sources characterized Novel chitosan nanoformulations developed for drug

and gene delivery A chitosan-based hand cream formulation developed First detailed life cycle assessment of chitosan

production processes established

Learn more about Nano3Bio’s promising achievements for winning future chitosans: www.nano3bio.eu

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Nano3Bio consortium partners

Aggregated international capacity – leading experts from 22 universities, research institutes, and companies form the Nano3Bio consortium (in alphabetical order):

Artes Biotechnology GmbH (Germany)

beemo GmbH (Germany)

Bio Base Europe Pilot Plant VZW (Belgium)

Care Sense Consulting (Germany)

Centre National de la Recherche Scientifique (France)

Cosphatec GmbH (Germany)

Danmarks Tekniske Universitet (Denmark)

Enantia SL (Spain)

Gillet Chitosan EURL (France)

Greenaltech SL (Spain)

Greendelta GmbH (Germany)

Heppe Medical Chitosan GmbH (Germany)

Institut Quimic De Sarria (Spain)

Kungliga Tekniska Hoegskolan (Sweden)

Lyon Ingénierie Projets (France)

Perseus BVBA (Belgium)

Ruprecht-Karls-Universität Heidelberg (Germany)

Thermo Fisher Scientific GENEART GmbH (Germany)

Universiteit Gent (Belgium)

University of Hyderabad (India)

University of Münster (Germany)

2.-0 LCA Consultant APS (Denmark)

More information on the consortium: www.nano3bio.eu/about/consortium/


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Acknowledgement The Nano3Bio consortium thanks everybody who contributed to the great success of this project. In terms of the final event ‘The Future of Chitosans’ in September 2017 we particularly thank Prof. Appa Rao Podile and his team from the University of Hyderabad, Department of Plant Sciences, for hosting.

Not least we want to thank the European Union for funding this project. We are convinced that achievements for science, society and the environ-ment generated by Nano3Bio prove that this funding was an efficient investment.

Visit www.nano3bio.eu/start/final-conference/ in order to find up-to-date information on the agenda of the Nano3Bio final public event.

Nano3Bio

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Notes

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the future of chitosans - nano3bio · the future of chitosans ... deciphering chitin biosynthesis:...

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