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Chitosan and its derivatives are interesting biopolymers for different field of analytical chemistry, especially in separation techniques. The present study was aimed at testing chitosan water soluble derivatives as dynamic coating agents for application to capillary electrophoresis. In particular, chitosan was modified following three different chemical reactions (nucleophilic substitution, reductive amination, and condensation) to introduce differences in charge and steric hindrance, and to assess the effect of these physico-chemical properties in capillary electrophoresis. The effects were tested on the capillary electrophoretic separation of the glycoforms of human transferrin, an important iron-transporting serum protein, one of which, namely disialo-transferrin (CDT), is a biomarker of alcohol abuse. Chitosan derivatives were characterized by using NMR and 1H NMR, HP-SEC-TDA, DLS, and rheology. The use of these compounds as dynamic coatings in the electrolyte running buffer in capillary electrophoresis was tested assessing the peak resolution of the main glycoforms of human transferrin and particularly of disialo-transferrin. The results showed distinct changes of the peak resolution produced by the different derivatives. The best results in terms of peak resolution were achieved using polyethylene glycol (PEG)-modified chitosan, which, in comparison to a reference analytical approach, provided an almost baseline resolution of disialo-transferrin from the adjacent peaks.

Biomaterials used for biomedical applications are composed by natural, synthetic or hybrid crosslinked polymers. Furthermore, they can also be functionalized with bioactive molecules, to obtain suitable biological and morphological properties, to provide in vitro tissue models to study cell fate [1,2]. Gelatin is a biopolymer obtained from hydrolysis of collagen, it is biodegradable, biocompatible, and relatively inexpensive [3]. All these features make gelatin ideal for the design and development of new functional materials for biomedical applications. In addition, gelatin-based materials are excellent bioactive compounds to support cell adhesion and proliferation [2]. Heparin is a sulphated linear polysaccharide belonging to the family of glycosaminoglycans (GAG); it is widely used in pharmaceutical field as an anticoagulant and it’s able to interact with many cell surface receptors and extracellular (ECM) proteins [5]. In this work, gelatin was functionalized, by a reductive amination reaction, with deaminated heparin to generate composite bioactive polymers useful for medical device coating or for tissue regeneration strategies. The structural characterizations of starting materials and final products were investigated with NMR spectroscopy, High-Performance-Size Exclusion Chromatography with Triplo Detector Array, Zeta Potential, Photo Correlation Spectroscopy and finally studies of the rheological properties were performed. To test the activity of heparin linked to gelatin, the interaction of the conjugate Hep-Gelatin product with Platelet factor 4 protein (PF4), a protein involved in thrombocytopenia, a side effect of the administration of heparin, was investigated. The Hep-Gelatin product is characterized by a molecular weight (Mw) around 230KDa, higher than the started gelatin (Mw of 150KDa), a molar ratio Heparin-gelatin of 0.14, determined by colorimetric assay and different physicochemical properties than the gelatin, such for example a lower viscosity.

The substitution of perfluorinated substances (PFAS) as chemical applied in anti-adhesive and hydrophobic applications has become an urgent need, due to raising concerns for their potential hazard. In this framework, chitosan-based coatings represent a promising alternative because of their properties of non-toxicity, biodegradability, and sustainability as raw materials. Our work developed modified chitosan-based coatings, with a fine-tuning of transparency and superhydrophobicity (Figure 1). Chitosan-based fluorine-free superhydrophobic coatings were obtained via functionality modification through esterification and amidation reaction with stearoyl chloride, followed by freeze-drying and coating deposition using a novel solvent-free approach followed by thermal treatment. A full characterization of the modified-chitosan was performed to assess its chemico-physical properties and confirmed the functionality modification. The application of an innovative and sustainable deposition method demonstrates to be effective in tuning the coating properties of transparency and superhydrophobicity, combining durability under physical abrasion.

Protein analysis by capillary electrophoresis is an established benchmark technique, where electrophoretic separation performances are frequently affected by protein adsorption on capillary walls. The protein-wall interaction can be suppressed or mitigated by the application of the so-called dynamic coating agents. In this study, the potential of chitosan derivatives as dynamic coating was investigated, targeting the separation of the carbohydrate deficient transferrin (CDT), a relevant biomarker in forensic analyses to detect chronic alcohol abuse. To this aim, chitosan was derivatized through different chemical reactions, including nucleophilic substitution, reductive amination, and condensation reaction, to provide different characteristics to the dynamic coatings. After chemical and physical characterization, chitosan derivatives were tested as dynamic coating additives in the buffer separation solution of CDT. Evidence of the improvement in the CDT separation efficiency were found applying PEGmodified chitosan via nucleophilic substitution.

In cancer microenvironment, aberrant glycosylation events of ECM proteins and cell surface receptors occur. We developed a protocol to generate 3D bioprinted models of colorectal cancer (CRC) crosslinking hyaluronic acid and gelatin functionalized with three signalling glycans characterized in CRC, 3′-Sialylgalactose, 6′-Sialylgalactose and 2′-Fucosylgalactose. The crosslinking, performed exploiting azide functionalized gelatin and hyaluronic acid and 4arm-PEG-dibenzocyclooctyne, resulted in biocompatible hydrogels that were 3D bioprinted with commercial CRC cells HT-29 and patient derived CRC tumoroids. The glycosylated hydrogels showed good 3D printability, biocompatibility and stability over the time. SEM and synchrotron radiation SAXS/WAXS analysis revealed the influence of glycosylation in the construct morphology, whereas MALDI-MS imaging showed that protein profiles of tumoroid cells vary with glycosylation, indicating that sialylation and fucosylation of ECM proteins induce diverse alterations to the proteome of the tumoroid and surrounding cells.

One of the current greatest challenges in materials science and technology is the development of safe- and sustainable-by-design coatings with enhanced functionalities, e.g. to substitute fluorinated substances raising concerns for their potential hazard on human health. Bio-based polymeric coatings represent a promising route with a high potential. In this study, we propose an innovative sustainable method for fabricating coatings based on chitosan with modified functionality, with a fine-tuning of coating properties, namely transparency and superhydrophobicity. The process consists in two main steps: i) fluorine-free modification of chitosan functional groups with stearoyl chloride and freeze-drying to obtain a superhydrophobic powder, ii) coating deposition using a novel solvent-free approach through a thermal treatment. The modified chitosan is characterized to assess its chemico-physical properties and confirm the functionality modification with fatty acid tails. The deposition method enables tuning the coating properties of transparency and superhydrophobicity, maintaining good durability.

Non‐covalent interactions in supramolecular chemistry provide useful systems to understand biological processes, and self‐assembly systems are suitable assets to build‐up innovative products for biomedical applications. In this field, polyelectrolyte complexes are interesting, especially when polysaccharides are involved, due to their non‐toxicity and bio-absorbability. In this work, we investigated a polyelectrolyte formed by hyaluronic acid (HA), a negatively charged linear polysaccharide, with Chitlac (Ch), a positively charged lactose‐modified chitosan. The aim of the study was the investigation of a novel Ch–HA polyelectrolyte complex, to understand the interaction between the two polysaccharides and the stability towards enzymatic activity. By means of gel permeation chromatography–triple detector array (GPC–TDA), nuclear magnetic resonance (NMR), dynamic viscosity, Zeta Potential and scanning electron microscopy (SEM), the polyelectrolyte complex properties were identified and compared to individual polysaccharides. The complex showed monodisperse molecular weight distribution, high viscosity, negative charge, and could be degraded by specific enzymes, such as hyaluronidase and lysozyme. The results suggest a close interaction between the two polysaccharides in the complex, which could be considered a self‐assembly system.

A protocol based on the combination of different analytical methodologies is proposed to standardize the experimental conditions for reproducible formulations of hybrid hydrogels. The final hybrid material, based on the combination of gelatin and chitosan functionalized with methylfuran and cross-linked with 4-arm-PEG-maleimide, is able to mimic role, dynamism, and structural complexity of the extracellular matrix. Physical–chemical properties of starting polymers and finals constructs were characterized exploiting the combination of HP-SEC-TDA, UV, FT-IR, NMR, and TGA.

The development of 3D printable hydrogels based on the crosslinking between chitosan and gelatin is proposed. Chitosan and gelatin were both functionalized with methyl furan groups. Chemical modification was performed by reductive amination with methyl furfural involving the lysine residues of gelatin and the amino groups of chitosan to generate hydrogels with tailored properties. The methyl furan residues present in both polymers were exploited for efficient crosslinking via Diels-Alder ligation with PEG-Star-maleimide under cell-compatible conditions. The obtained chitosan-gelatin hybrid was employed to formulate hydrogels and 3D printable biopolymers and its processability and biocompatibility were preliminarily investigated.