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Despite the relevance of carbohydrates as cues in eliciting specific biological responses, glycans have been rarely exploited in the study of neuronal physiology. We report thereby the study of the effect of neoglucosylated collagen matrices on neuroblastoma F11 cell line behavior. Morphological and functional analysis clearly showed that neoglucosylated collagen matrices were able to drive cells to differentiate. These data show for the first time that F11 cells can be driven from proliferation to differentiation without the use of chemical differentiating agents. Our work may offer to cell biologists new opportunities to study neuronal cell differentiation mechanisms in a cell environment closer to physiological conditions.

A new approach for tethering of bioactive molecules via arginine is proposed and validated on collagen 2D matrices. The method involves the introduction of a methyl ketone on arginine side-chains, followed by reaction with model alkoxyamino derivatives. This journal is © the Partner Organisations 2014.

Osteogenic Growth Peptide (OGP) motifs were tethered to SULFHYDRYL-BINDTM polystyrene plates tuning spatial presentation of the different functional domains. The effects of the different surface-tethered OGP motifs have been evaluated in mesenchymal stem cells (MSCs) osteogenesis. A culture medium without any osteogenic supplements was used in order to evaluate the OGP effect, avoiding any interference by additional factors. The motif YGFGG resulted to be the most interesting, strongly enhancing the expression of BMP2 that plays a fundamental role in bone regeneration. With this study we present new evidence of the role of OGP in osteogenesis and new insights into different OGP motifs able to act as best ligands for the design of innovative bioactive materials for osteogenesis promotion and regenerative medicine applications.

Despite the relevance of carbohydrates as cues in eliciting specific biological responses, the covalent surface modification of collagen-based matrices with small carbohydrate epitopes has been scarcely investigated. We report thereby the development of an efficient procedure for the chemoselective neoglycosylation of collagen matrices (patches) via a thiol-ene approach, between alkene-derived monosaccharides and the thiol-functionalized material surface. SR-XPS, FTIR and ELLA assays confirmed the effectiveness of the collagen neoglycosylation. Preliminary biological evaluation in osteoarthritic models is reported. The proposed methodology can be extended to any thiolated surface for the development of smart biomaterials for innovative approaches in regenerative medicine.

Hybrid materials with interpenetrating networks of silica and degradable polymers have the potential to outperform current biomaterials as their mechanical properties and degradation rates can be tightly controlled. GPTMS is one of the most widely used precursors for sol–gel hybrid fabrication. It can be used as the silica precursor or as a coupling agent to create hybrids with covalent bonds between the silica and organic components. Understanding its reactivity with nucleophilic groups on organic molecules in aqueous conditions is of key importance to hybrid synthesis. NMR spectrometry assisted by mass spectrometry was successfully used for the investigation of the reaction system of (3-glycidoxypropyl)trimethoxysilane (GPTMS) in the presence of different simple nucleophiles. Inorganic condensation and silica network formation is accelerated in the presence of propylamine; propanoic acid gives nucleophilic attack on the epoxy ring; propanol and propanthiol do not seem to participate in the reaction.

A set of new metabolically stable arabinose 5-phosphate analogues possessing phosphate mimetic groups at position 5 was synthesised. Their ability to interact with arabinose 5-phosphate isomerase from Pseudomonas aeruginosa was evaluated by STD-NMR studies. The synthesised compounds were also characterised for their activity in vivo on P. aeruginosa and E. coli strains. Unfortunately, none of the synthesised compounds was able neither to bind API nor to inhibit bacterial growth.

My PhD project has been focused on the design, synthesis and biological activity studies of glicomimetics. During these three years I have been mainly interested in three different arguments (three proteins: API, Akt, CIM6Pr) that I will elucidate in detail in this thesis. Even if the target proteins (enzymes and receptors) are very different, these three arguments have a common element. In all cases, starting from the known mechanistic, structural and biological information of the single protein, I have designed glycomimetics that are potentially able to interact with the target protein. All the studied proteins are biologically relevant, so the final aim of the synthetic effort is always a biological test, in order to check the hypothesis done during the design step and to get new useful information about the biological system. The synthesized compounds are always glycomimetics or, more generally speaking, glycans: they can be arabinose-based mimetic of A5P, glucose-based mimetic of an inisitol structure, or synthetic glycoproteins. The most used analytical instrument for the characterization of all the synthesized compounds and for the study of their interaction with enzymes is NMR spectroscopy. The wide use of this analytical technique led me to study topics that were not related to carbohydrates themselves, but related to NMR techniques. Therefore, I increased my knowledge and applied NMR spectroscopy to the biomaterial field and in particular I have studied the reactivity of reaction systems widely used for biomaterial production using NMR. I will briefly discuss these works in the last part of my thesis.

Glycans participate in neural cell migration, neurite outgrowth and synapse formation in both the developing and adult nervous system. Understanding their functions would improve the knowledge in the mechanisms that determine nervous system differentiation and regeneration and could lead to the use of glycans as therapeutic strategies for neurological disorders. For this reason we studied the effects of native and glycosylated collagen (glc-collagen) on neuronal differentiation by using a neuroblastoma, dorsal root ganglion-derived cell line (F11). The choice of cells was motivated by their capability to differentiate into neurons when exposed to differentiating agents. We compared the behaviour of cells plated on native and glc-collagen with cells seeded on petri dishes. After 7 days, the electrophysiological properties of cells were analysed by the patch-clamp technique in the whole-cell configuration. Sodium current densities showed the tendency to increase from petri dishes to collagen and to glc-collagen and significantly higher potassium current densities were calculated for cells plated on glc-collagen. Mean resting membrane potential was very depolarized in cells from the petri dishes but showed a trend to hyperpolarize in cells plated on collagen and was significantly more negative on glc-collagen. The electrical activity was tested by applying depolarizing current pulses by the patch-electrode; in petri dishes, the majority of the cells showed slow depolarisations which were not able to reach 0 mV, on the contrary, the fraction of cells able to generate action potentials was significantly higher on collagen and reached almost the totality on glc-collagen. Our data show for the first time F11 cells differentiated without the use of chemical and differentiating agents and suggest that glc-collagen is an efficient material to be employed for neuronal differentiation.

Recent efforts in the design and synthesis of potential anti-cancer vaccines based on carbohydrates are outlined, covering literature up to 2012. This approach capitalises on the fact that cancer cells often display alterations in their glycan repertoire if compared to normal cells, resulting in the accumulation of new structures, usually referred as to tumour-associated carbohydrate antigens (TACA). Several preclinical and clinical studies demonstrate that naturally acquired, passively administered, or actively induced antibodies against TACAs are able to eliminate circulating tumour cells and micro-metastases in cancer patients. TACAs can be ideal candidates to elicit an antitumor immune response, however they are often not immunogenic enough to induce protection, since many of them are of embryonic origin or expressed in normal tissue, even if at low levels, and consequently tolerated by the immune system. Approaches toward breaking self-tolerance in the design of novel glycocnjugates structures, together with new conjugation methods appeared in the last years will be considered.

Type I collagen films have been functionalized on their surfaces by plasma treatment with carboxyl and amino groups to improve their potential for grafting bioactive molecules. The physico-chemical properties of the plasma-treated films were evaluated and compared to the untreated materials by water contact angle, SEM and AFM. The presence of new functional groups on the film surfaces has been assessed by ATR-FTIR spectra after chemical derivatization. Moreover, the biocompatibility of the plasma-treated films was studied with MG-63 human osteoblast-like cells, evaluating cell proliferation, viability and morphology at 1, 3 and 7 days.

Collagen matrices have been neoglycosylated with lactose by reductive amination at lysine side chains. AFM analysis highlights that the chemical neoglycosylation does not affect molecular assembly into fibrils. Moreover, ELLA biochemical assays show that the glycan moiety is efficiently exposed on the matrix surface for receptor recognition.

Biomaterials are needed for tissue regeneration applications that provide control of mechanical properties and enhanced toughness compared to conventional bioceramics. New sol-gel hybrids were developed with interpenetrating networks of silica and bis(3-aminopropyl) polyethylene glycol. Covalent coupling between the organic and the inorganic components was used to control mechanical properties of the hybrids. The objective was to synthesise and characterise a bis(3-aminopropyl) polyethylene glycol silica hybrid material with 35 wt% organic and 65 wt% inorganic and covalent coupling between the components. A coupling agent, 3-glycidopropyltrimethoxysilane (GPTMS) was used to form the covalent links. The hypothesis was that the epoxy ring of the GPTMS would react with the polymer, leaving a polymer functionalised with siloxane groups. In a sol of hydrolysed tetraethylorthosilicate (TEOS) the siloxanes from the GPTMS form –Si–O–Si– bonds between the functionalised polymer and the silica network. Bis(3-aminopropyl) polyethylene glycol contains two terminal amino groups available for the covalent functionalisation with the epoxy group of GPTMS. Hybrids with 35 wt% organic and 65 wt% inorganic with a ratio of GPTMS:PEG of 1:4 were proven to have an excellent balance between strain to failure (10 %) and compressive strength (20 MPa). However, the functionalisation of the polymer was followed by liquid NMR as a function of the aging time and temperature and the expected reaction of nucleophilic attack of the epoxy ring by the amino group of the polymer did not happen until the water was removed from the system during drying

Different methods for the functionalization of carbonate hydroxyapatite granules with free amine groups by reaction with (3-aminopropyl)triethoxysilane (APTES) have been compared in order to improve the potential for tethering of bioactive molecules to bioceramics. The combined use of tetraethoxyorthosilicate (TEOS) and APTES with acid catalysis resulted in an evident increase of amine surface grafting.

Metabolically stable arabinose 5-phosphate analogues possessing phosphate mimetic groups at the 5-position were synthesized and evaluated by saturation-transfer-difference (STD) NMR studies for their ability to interact with arabinose 5-phosphate isomerase. Isosteric methylphosphonate 1 and (difluoromethyl)phosphonate 3 were recognised and bound by the catalytic pocket of the enzyme. The synthesized compounds were tested in vivo on E. coli and P. aeruginosa strains in order to evaluate their antibacterial activity.

Hybrid organic–inorganic solids represent an important class of engineering materials, usually prepared by sol–gel processes by cross-reaction between organic and inorganic precursors. The choice of the two components and control of the reaction conditions (especially pH value) allow the synthesis of hybrid materials with novel properties and functionalities. 3-Glycidoxypropyltrimethoxysilane (GPTMS) is one of the most commonly used organic silanes for hybrid-material fabrication. Herein, the reactivity of GPTMS in water at different pH values (pH 2– 11) was deeply investigated for the first time by solution-state multinuclear NMR spectroscopic and mass spectrometric analysis. The extent of the different and competing reactions that take place as a function of the pH value was elucidated. The NMR spectroscopic and mass spectrometric data clearly indicate that the pH value determines the kinetics of epoxide hydrolysis versus silicon condensation. Under slighly acidic conditions, the epoxy-ring hydrolysis is kinetically more favourable than the formation of the silica network. In contrast, under basic conditions, silicon condensation is the main reaction that takes place. Full characterisation of the formed intermediates was carried out by using NMR spectroscopic and mass spectrometric analysis. These results indicate that strict control of the pH values allows tuning of the reactivity of the organic and inorganic moities, thus laying the foundations for the design and synthesis of sol–gel hybrid biomaterials with tuneable properties

The general aim of the project is the development of innovative materials for bone and cartilage tissue engineering, focusing on the design of smart biomimetic materials, fully integrating principles from cell and molecular biology able to directly regulate cell differentiation and metabolism. Materials equipped with molecular cues mimicking the structure or function of natural extracellular microenvironments are able to interact with surrounding tissues by biomolecular recognition (Lutolf, MP 2005). Biomimetic materials should be capable of eliciting specific cellular responses and directing new tissue formation mediated by specific interactions, which can be manipulated by altering design parameters. Smart materials should provide the opportunity for chemically linking biological signalling molecules such as peptides or small proteins, or carbohydrates, eliciting cell responses to help the healing process. For tissue repair, biomaterials should have several properties to support viable repair. Firstly, the material must act as a support structure for cells and possess enough mechanical strength to protect the cells contained within it. Secondly, some level of bioactivity should be provided to accommodate cellular attachment and migration. Furthermore, the materials should be biodegradable and remodel as the new tissue forms and replaces the original construct. In this regard, the matrix should be non-toxic, non-attractive and non-stimulatory of inflammatory cells, and also non-immunogenic, which would be detrimental to tissue regeneration. Finally, the scaffolds should provide easy handling under clinical conditions, enabling fixation of the materials into the implant site [Stoop R. 2008]. In the present work, we are studying and applying functionalization strategies of different materials with signaling biomolecules. New smart materials were designed and synthetized to obtain innovative scaffolds useful in forming tissues with oriented mechanical and cellular pro

Lipopolysaccharides (LPSs), which constitute the lipid portion of the outer leaflet of Gram-negative bacteria, are essential for growth, and are responsible for a variety of biological effects associated with Gram-negative sepsis. LPSs are amphiphilic molecules comprising three regions: lipid A, the core region, and a polysaccharide portion; the lipid A was proven to represent the toxic principle of endotoxic active lipopolysaccharides. In addition, it is known that the minimal conserved structure of LPS is the lipophylic oligoasaccharidic structure containing Kdo residues linked to the-LipA moiety. Thus, the design and development of novel antibacterial drugs can focus on different aspects, related to the biosynthesis and chemical features of LPS: 1) Inhibitors of lipid A biosynthesis 2) Inhibitors of Kdo biosynthesis. Both Kdo and Lipid A are needed for the construction of the minimum structural element Kdo2-LipidA, needed for bacterial survival. Any inhibitors acting on the biogenetic pathway of this molecule can act as antibacterial. 3) Antagonists of the interaction between endotoxins and the host receptors: LPS is recognised by the CD14 and the Toll-like receptor (TLR)- 4/MD2 complex, where Lipid A is the crucial moiety in the interaction. Any drug acting as an antagonist of this process can have antisepsis potential. Considerable efforts have been made in this direction to identify natural or synthetic molecules able to interfere with the interaction between LPS and inflammatory cells. This review will highlight recent efforts in the design and biological activity of enzyme inhibitors and antagonist acting on the 3 key aspects outlined above