Liquid Chromatography-Mass Spectrometry, Biomaterials and 3D Printing in Analytical Applications

Liquid chromatography-mass spectrometry (LC-MS) is a powerful and versatile analytical technique widely used in life sciences. This thesis work aims to group different analytical applications, where LC-MS is the technique of choice to investigate biomarkers of biomedical interest. In this context targeted and untargeted LC-MS/MS approaches were adopted using different analytical instruments such as HPLC, nano-HPLC, electrospray, triple quadrupole, orbitrap and trapped ion mobility spectrometry. The other technology investigated and involved in some of the analytical applications reported in this work is three-dimensional (3D) printing, used for the fabrication of 3D hydrogels based on polysaccharide biomaterials, particularly alginate and hyaluronic acid. In detail, an extrusion 3D printer was used, which, by depositing the biomaterial-based ink, layer upon layer, enabled the construction of porous polymeric scaffolds with customized and tuneable properties. These 3D polymeric networks, sometimes functionalized with drugs, bacterial lysates, nanoparticles, have been used in applications of regenerative medicine, tissue regeneration, treatment of infected wounds and drug delivery. This thesis paper has therefore been divided into 10 chapters. The first chapter is a general introduction on the technologies used in the analytical applications reported; therefore, there are two sections: the first deals with LC-MS in proteomics and metabolomics, respectively; the second deals with the use of 3D printing extrusion-based technique for the manufacturing of natural polysaccharide hydrogels for applications in the biomedical field. From Chapter II to Chapter IX the analytical applications with biomedical interest, developed during the PhD period, are described. Chapter II focuses on the European Horizon 2020 SCRENEED project (#825745) that aim to develop 3D in vitro assays for screening the effect of low doses of Endocrine Disruptors on thyroid cell function in a sex-specific manner. Chapter III reports the use of untargeted and targeted LC-MS for extracellular matrix proteins investigation on porcine tissues decellularized with different detergents. Chapters IV and V concern the development of simple and reliable analytical methods based on targeted LC-MS, for the quantification of the protein insulin like growth factor-1 (IGF-1) in bovine milk samples and, for the quantification of cholesterol metabolites 24-, 25- and 27-hydroxycholesterol in mouse brain and sera, respectively. Chapters VI, VII, VIII, IX, share both the use of targeted LC-MS for the analysis of proteins or lipid biomarkers, and the use of 3D printing to fabricate alginate-based hydrogels crosslinked by ionotropic external (chapters VI and VII) or internal (chapter VIII and IX) gelation. Chapter VI focuses on the development and characterization of 3D printed hydrogels based on alginate, nanocrystalline cellulose and silver nanoparticles to promote antimicrobial and cytotoxic effects. Chapter VII reports the study on the adaptation of lipid profile of human fibroblasts to 2D alginate films or 3D printed alginate scaffolds. In chapter VIII, the development of new self-crosslinking alginate-based inks for 3D printing is described; this method, by exploiting the internal gelation of alginate, avoids the post-printing crosslinking process and allows the loading of water-soluble drugs, such as epirubicin-HCl. Chapter IX reports the development of 3D printed hydrogels based on alginate and hyaluronic acid, functionalized with two strains of Lactobacillus delbrueckii subs. bulgaricus derivatives that have been investigated for their potential in wound healing as advanced therapeutic solutions. In chapter X both conclusions and future perspectives of the various analytical applications discussed in this thesis have been summarized.