This work of thesis involves various aspects of crystal engineering. Chapter 1 focuses on crystals containing crown ether complexes. Aspects such as the possibility of preparing these materials by non-solution methods, i.e. by direct reaction of the solid components, thermal behavior and also isomorphism and interconversion between hydrates are taken into account. In chapter 2 a study is presented aimed to understanding the relationship between hydrogen bonding capability and shape of the building blocks chosen to construct crystals. The focus is on the control exerted by shape on the organization of sandwich cations such as cobalticinium, decamethylcobalticinium and bisbenzenchromium(I) and on the aggregation of monoanions all containing carboxylic and carboxylate groups, into 0-D, 1-D, 2-D and 3-D networks. Reactions conducted in multi-component molecular assemblies or co-crystals have been recognized as a way to control reactivity in the solid state. The [2+2] photodimerization of olefins is a successful demonstration of how templated solid state synthesis can efficiently synthesize unique materials with remarkable stereoselectivity and under environment-friendly conditions. A demonstration of this synthetic strategy is given in chapter 3. The combination of various types of intermolecular linkages, leading to formation of high order aggregation and crystalline materials or to a random aggregation resulting in an amorphous precipitate, may not go to completeness. In such rare cases an aggregation process intermediate between crystalline and amorphous materials is observed, resulting in the formation of a gel, i.e. a viscoelastic solid-like or liquid-like material. In chapter 4 design of new Low Molecular Weight Gelators is presented. Aspects such as the relationships between molecular structure, crystal packing and gelation properties and the application of this kind of gels as a medium for crystal growth of organic molecules, such as APIs, are also discussed.

The aim of this thesis was to design, synthesize and develop a nanoparticle based system to be used as a chemosensor or as a label in bioanalytical applications. A versatile fluorescent functionalizable nanoarchitecture has been effectively produced based on the hydrolysis and condensation of TEOS in direct micelles of Pluronic® F 127, obtaining highly monodisperse silica - core / PEG - shell nanoparticles with a diameter of about 20 nm. Surface functionalized nanoparticles have been obtained in a one-pot procedure by chemical modification of the hydroxyl terminal groups of the surfactant. To make them fluorescent, a whole library of triethoxysilane fluorophores (mainly BODIPY based), encompassing the whole visible spectrum has been synthesized: this derivatization allows a high degree of doping, but the close proximity of the molecules inside the silica matrix leads to the development of self - quenching processes at high doping levels, with the concomitant fall of the fluorescence signal intensity. In order to bypass this parasite phenomenon, multichromophoric systems have been prepared, where highly efficient FRET processes occur, showing that this energy pathway is faster than self - quenching, recovering the fluorescence signal. The FRET efficiency remains very high even four dye nanoparticles, increasing the pseudo Stokes shift of the system, attractive feature for multiplexing analysis. These optimized nanoparticles have been successfully exploited in molecular imaging applications such as in vitro, in vivo and ex vivo imaging, proving themselves superior to conventional molecular fluorophores as signaling units.

In the scenario of depleting fossil fuels, finding new energy technologies and conserving conventional energy resources have become essential to sustain modern civilization. While renewable energies are on the rise, considerable interest has been turned also to reduce energy consumption of conventional devices and appliances, which are often not yet optimized for this purpose. Modern nanotechnology provides a platform to build such devices by using nanomaterials showing exceptional physico-chemical properties. In particular, carbon materials (fullerenes, carbon nanotubes, graphene etc.), which show high thermal and electrical conductivity, aspect ratio, shear strength and chemical/mechanical resistance, are quite promising for a wide range of applications. However, the problem of solubility often hampers their handling and industrial utilization. These limitations can be mitigated by functionalizing carbon nanostructures, either covalently or non covalently, with organic or inorganic compounds. The exo- and endohedral functionalization of carbon nanotubes (CNTs) with organic/inorganic moieties to produce luminescent materials with desired properties are the main focus of this doctoral work. These hybrids have been thoroughly designed and characterized with chemical, microscopic and photophysical analyses. All the materials based on carbon nanostructures described in this thesis are innovative examples of photoactive and luminescent hybrids, and their morphological and photophysical properties help understanding the nature of interactions between the active units. This may prompt the design and fabrication of new functional materials for applications in the fields of optoelectronics and photovoltaics.

The aim of this thesis was the formulation of new chitosan based delivery systems for transmucosal drug administration. Transmucosal routes, such as buccal, vaginal and nasal routes, allow the circumvention of the hepatic first pass metabolism and avoid the gastrointestinal chemical and enzymatic degradations. Moreover, transmucosal drug administration can allow to avoid pain or discomfort caused by injections, when drugs are administered through parenteral routes, thus increasing patient compliance. On the other side, the major disadvantage of transmucosal drug administration is represented by the presence of biological fluids and mucus that can remove drug systems from the application site, thus reducing the contact time between drug and mucosa and consequently, decreasing drug bioavailability. For this reason, in this study, the investigation of chitosan delivery systems as mucoadhesive formulations able to increase drugs residence time and to improve their bioavailability, was taken into account. In the paper 1, buccal films based on chitosan-gelatin complexes were prepared and loaded with propranolol hydrochloride. The complexes were characterized and studied in order to evaluate their physical- chemical properties and their ability to release the drug and to allow its permeation through buccal mucosa. In the paper 2, vaginal inserts based on chitosan/alginate complexes were formulated for local delivery of chlorhexidine digluconate. Tests to evaluate the interaction between the polymers and to study drug release properties were performed, as well as the determination of antimicrobial activity against the patogens responsible of vaginitis and candidosis. In the project 3, chitosan based nanoparticles containing cyclodextrin and other excipients, with the capacity to modify insulin bioavailabity were formulated for insulin nasal delivery. Nanoparticles were characterized in terms of size, stability and drug release. Moreover, in vivo tests were performed in order to study the hypoglycemic reduction in rats blood samples.

This thesis was focused on the investigation of the linear optical properties of novel two photon absorbers for biomedical applications. Substituted imidazole and imidazopyridine derivatives, and organic dendrimers were studied as potential fluorophores for two photon bioimaging. The results obtained showed superior luminescence properties for sulphonamido imidazole derivatives compared to other substituted imidazoles. Imidazo[1,2-a]pyridines exhibited an important dependence on the substitution pattern of their luminescence properties. Substitution at imidazole ring led to a higher fluorescence yield than the substitution at the pyridine one. Bis-imidazo[1,2-a]pyridines of Donor-Acceptor-Donor type were examined. Bis-imidazo[1,2-a]pyridines dimerized at C3 position had better luminescence properties than those dimerized at C5, displaying high emission yields and important 2PA cross sections. Phosphazene-based dendrimers with fluorene branches and cationic charges on the periphery were also examined. Due to aggregation phenomena in polar solvents, the dendrimers registered a significant loss of luminescence with respect to fluorene chromophore model. An improved design of more rigid chromophores yields enhanced luminescence properties which, connected to large 2PA cross-sections, make this compounds valuable as fluorophores in bioimaging. The photophysical study of several ketocoumarine initiators, designed for the fabrication of small dimension prostheses by two photon polymerization (2PP) was carried out. The compounds showed low emission yields, indicative of a high population of the triplet excited state, which is the active state in producing the reactive species. Their efficiency in 2PP was proved by fabrication of microstructures and their biocompatibility was tested in the collaborator’s laboratory. In the frame of the 2PA photorelease of drugs, three fluorene-based dyads have been investigated. They were designed to release the gamma-aminobutyric acid via two photon induced electron transfer. The experimental data in polar solvents showed a fast electron transfer followed by an almost equally fast back electron transfer process, which indicate a poor optimization of the system.

A series of imidazolium salts of the type [BocNHCH2CH2ImR]X (Boc = t-Bu carbamates; Im = imidazole) (R = Me, X = I, 1a; R = Bn, X = Br, 1b; R = Trityl, X = Cl, 1c) and [BnImR’]X (R’ = Me, X = Br, 1d; R’ = Bn, X = Br, 1e; R’ = Trityl, X = Cl, 1g; R’ = tBu, X = Br, 1h) bearing increasingly bulky substituents were synthetized and characterized. Subsequently, these precursors were employed in the synthesis of silver(I)-N-heterocyclic (NHC) complexes as transmetallating reagents for the preparation of rhodium(I) complexes [RhX(NBD)(NHC)] (NHC = 1-(2-NHBoc-ethyl)-3-R-imidazolin-2-ylidene; X = Cl; R = Me, 4a; R = Bn, 4b; R = Trityl, 4c; X = I, R = Me, 5a; NHC = 1-Bn-3-R’-imidazolin-2-ylidene; X = Cl; R’ = Me, 4d, R’ = Bn, 4e, R’ = Trityl, 4g; R’ = tBu, 4h). VT NMR studies of these complexes revealed a restricted rotation barriers about the metal-carbene bond. While the rotation barriers calculated for the complexes in which R = Me, Bn (4a,b,d,e and 5a) matched the experimental values, this was not true for the complexes 4c,g, bearing a trityl group for which the values are much smaller than the calculated ones. Energy barriers for 4c,g, derived from a line shape simulation, showed a strong dependence on the temperature while for 4h the rotational energy barrier is stopped at room temperature. The catalytic activity of the new rhodium compounds was investigated in the hydrosilylation of terminal alkynes and in the addition of phenylboronic acid to benzaldehyde. The imidazolium salts 1d,e were also employed in the synthesis of new iron(II)-NHC complexes. Finally, during a six-months stay at the University of York a new ligand derived from Norharman was prepared and employed in palladium-mediated cross-coupling.

Microparticelle a base di complessi polielettrolitici di Chitosano/Pectina per il rilascio nasale di Tacrina cloridrato. Lo scopo di questo studio è stata la ricerca di nuove formulazioni solide per la somministrazione nasale di Tacrina cloridrato allo scopo di ridurre l’eccessivo effetto di primo passaggio epatico ed aumentarne la biodisponibilità a livello del Sistema Nervoso Centrale. La Tacrina è stata incapsulata in microparticelle mucoadesive a base di complessi elettrolitici di chitosano e pectina. Le microparticelle sono state preparate mediante due diversi approcci tecnologici (spray-drying e spray-drying/liofilizzazione) e analizzate in termini di caratteristiche dimensionali, morfologiche e chimico-fisiche. Nanoparticelle di Chitosano reticolate con Sodio Cromoglicato per il trattamento della rinite allergica. Il Sodio Cromoglicato è uno dei farmaci utilizzati per il trattamento della rinite allergica. Come noto, la clearance mucociliare provoca una rapida rimozione dei farmaci in soluzione dalla cavità nasale, aumentando così il numero di somministrazioni giornaliere e, di conseguenza, riducendo la compliance del paziente. Per ovviare a tale problema, si è pensato di includere il sodio cromoglicato in nanoparticelle di chitosano, un polimero capace di aderire alla mucosa nasale, prolungare il contatto della formulazione con il sito di applicazione e ridurre il numero di somministrazioni giornaliere. Le nanoparticelle ottenute sono state caratterizzate in termini di dimensioni, resa, efficienza di incapsulazione e caricamento del farmaco, potenziale zeta e caratteristiche mucoadesive. Analisi quantitativa di Budesonide amorfa tramite calorimetria a scansione differenziale. È stato sviluppato un nuovo metodo quantitativo allo stato solido basato sulla Calorimetria a Scansione Differenziale (DSC) in grado di quantificare in modo selettivo e accurato la quantità di Budesonide amorfa presente in una miscela solida. Durante lo sviluppo del metodo sono stati affrontati problemi relativi alla convalida di metodi analitici su campioni solidi quali la miscelazione di polveri solide per la preparazione di miscele standard e il calcolo della precisione. / Microparticles based on chitosan/pectin polyelectrolyte complexes for nasal delivery of tacrine hydrochloride. The purpose of this study was the research of new solid formulations for the nasal administration of tacrine hydrochloride in order to reduce the hepatic first-pass effect and increase its bioavailability in the Central Nervous System. Tacrine was encapsulated in microparticles based on mucoadhesive chitosan/pectin polyelectrolyte complex. The microparticles were prepared using two different technological approaches (spray-drying and spray-drying/lyophilization) and analyzed in terms of dimensional, morphological and chemical-physical characteristics. Nanoparticles of chitosan crosslinked with cromolyn sodium for the treatment of allergic rhinitis. Sodium cromolyn is one of the drugs used for the treatment of allergic rhinitis. As known, the mucociliary clearance causes a rapid removal of a solution of the drug from the nasal cavity, thus increasing the number of daily administrations and, consequently, reducing the patient's compliance. To overcome this problem, the sodium cromoglycate was included in nanoparticles of chitosan, a polymer capable to prolong the contact of the formulation with the nasal mucosa and reduce the number of daily administrations. The obtained nanoparticles were characterized in terms of size, yield, encapsulation efficiency, drug loading, zeta potential and mucoadhesive properties. Quantitative analysis of amorphous Budesonide by Differential Scanning Calorimetry. A solid-state quantitative method, able to selectively and accurately quantify the amount of amorphous Budesonide present in a solid mixture, was developed using the Differential Scanning Calorimetry (DSC). During the development of the method, some problems related to the validation of a solid-state analytical method (such as the mixing of solid powders for the preparation of standard mixtures and the calculation accuracy) have been addressed and solved.

The scope of my research project is to produce and characterize new crystalline forms of organic compounds, focusing the attention on co-crystals and then transferring these notions on APIs to produce co-crystals of potential interest in the pharmaceutical field. In the first part of this work co-crystallization experiments were performed using as building blocks the family of aliphatic dicarboxylic acids HOOC-(CH2)n-COOH, with n= 2-8. This class of compounds has always been an object of study because it is characterized by an interesting phenomenon of alternation of melting points: the acids with an even number of carbon atoms show a melting point higher than those with an odd one. The acids were co-crystallized with four dipyridyl molecules (formed by two pyridine rings with a different number of bridging carbon atoms) through the formation of intermolecular interactions N•••(H)O. The bases used were: 4,4’-bipyridine (BPY), 1,2-bis(4-pyridyl)ethane (BPA), 1,2-(di-4-pyridyl)ethylene (BPE) and 1,2-bis(4-pyridyl)propane (BPP). The co-crystals obtained by solution synthesis were characterized by different solid-state techniques to determine the structure and to see how the melting points in co-crystals change. In the second part of this study we tried to obtain new crystal forms of compounds of pharmaceutical interest. The APIs studied are: O-desmethylvenlafaxine, Lidocaine, Nalidixic Acid and Sulfadiazine. Each API was subjected to Polymorph Screening and Salt/Co-crystal Screening experiments to identify new crystal forms characterized by different properties. In a typical Salt/Co-crystal Screening the sample was made to react with a co-former (solid or liquid) through different methods: crystallization by solution, grinding, kneading and solid-gas reactions. The new crystal forms obtained were characterized by different solid state techniques (X-ray single crystal diffraction, X-ray powder diffraction, Differential Scanning Calorimetry, Thermogravimetric Analysis, Evolved gas analysis, FT-IR – ATR, Solid State N.M.R).

Volatile organic compounds (VOCs) are the key aroma producers in fruits and sensory quality of fruits is widely determined by qualitative and quantitative composition of VOCs. The aroma of grape is a complex of hundreds of VOCs belonging to different chemical classes like alcohols, esters, acids, terpenes, aldehydes, furanones, pyrazines, isoprenoids and many more. VOCs play important role as they determine the flavor of grapes and wine made from it. The objective of this thesis is to study of VOCs through development of different mass spectrometry based analytical methodologies and its applications for the comprehensive investigation and construction of database of the VOCs in grapes. First part of the study was dedicated to generation of the comprehensive database of grape VOCs through the screening of multiple grape varieties (n=124) representing different species, color and origin. The experiment was carried out using headspace solid-phase microextraction (HS-SPME) and gas chromatography mass spectrometry (GC-MS) based approach and according to metabolomics protocols. A customized dataset of reference standards (>350) was generated and, an automated pipeline for data analysis was created in collaboration with data management group of the institute. The results showed annotation of “level 1†of 117 VOCs in grape. The established database in this experiment will represent the significant portion of the future Grape Metabolome database. The second part of the study was dedicated to study the differential behavior of volatile organic compounds and their glycosylated precursors qualitatively and semi quantitatively. Volatile secondary metabolites also exist in the form of nonvolatile and odorless glycosylated precursors in grape and studies have confirmed that concentration of these precursors can be much higher than its free counterparts. The elevated concentrations of volatiles in glycosylated forms can significantly affect the wine aroma because of possible chemical modifications throughout the process of fermentation and wine ageing. In addition, the investigation of the biosynthesis and accumulation of VOCs in the fruit tissues requires the consideration of both the free and bound forms. To study the phenomenon an experiment was carried using solid phase extraction (SPE) of the free and glycosylated precursors; with enzymatic hydrolysis aglycone part of the precursors was released followed by subsequent GC-MS analysis. Over 10 different selected grape varieties were analyzed. Sixty-six significant different aroma compounds in grapes (pre and post hydrolysis) were identified. Identification was done based on several parameters like retention time, retention index and MS spectral database. The multivariate statistical analysis by two-way hierarchical clustering with heat map visualization showed distribution of the compounds within different varieties before and after hydrolysis. In the third part of the study, we performed experiments dedicated to training and applications of atmospheric pressure gas chromatography mass spectrometry (APGC-MS). The experiment was carried out at the Department of Biological Sciences, University of North Texas, under the supervision of Prof. Vladimir Shulaev. We have established the metabolomics protocol for the analysis of fruit volatiles using APGC-MS with an optimized GC and MS conditions and created novel library of the fruit volatile compounds using APGC-MS system. Six different grape varieties were analyzed as a case study and experimental results showed APGC-MS as a valuable solution for metabolomics analysis. The data processing and statistical evaluation was done using XCMS and progenesis QI© software. Moreover, observations based on injections of pure reference standards showed high abundance of molecular ions with minimal fragmentation at low collision energy that is typically missing in traditional vacuum source GC-MS. Moreover, the use of elevated collision energy data resulted in a spectrum similar to the traditional EI data.

A range of different polyphenols can be ingested in a bowl of polyphenol-rich fruit, going from one-phenol hydroxybenzoic acid to more complex polymeric compounds. Epidemiologically, polyphenol consumption has been associated with a reduced risk of cancer and cardiovascular disease and neurological protection against brain ageing. However, only a small proportion of native polyphenols (5-10%) are absorbed and the remainder reach the colon, where they are extensively metabolised by the gut microbiota. The colonic microbiota produces a relatively small number of polyphenol microbial metabolites from a large number of different dietary polyphenols. During subsequent tissue distribution, the target organs and the effective concentration circulating remain largely unreported. This Ph.D. thesis is divided into two parts: chemical analysis of food composition and in vivo bioavailability of polyphenol metabolites. Metabolomics offers an innovative approach that has recently been shown to be effective in both food chemistry and nutritional bioavailability studies. Polyphenol composition in strawberries is studied in the first part of this Ph.D. thesis, with the aim of evaluating nutritionally significant amounts of polyphenols before ingestion. In this context, a targeted method for quantitative analysis of multiple classes of phenols was developed. A high sensitivity MRM-based method for 135 phenolics with a wide dynamic range was obtained, providing valuable insight and assisting with the analysis of complex matrices such as fruit, and more in general food. Application of the method was tested in Fragaria spp., and along with another rapid method for the analysis of anthocyanins and ellagitannins, provided a general overview of polyphenol composition in strawberries. A total of 56 individual compounds were accurately identified and quantified, some of them for the first time, their concentration ranging from 1 ug/100 g for low abundant polyphenols to 40 mg/100 g of fresh fruit. Moreover the isolation of some ellagitannins and definition of their profile in Fragaria spp. was carried out during fruit ripening. Clarification of the main ellagitannin, agrimoniin, was obtained by isolation and it was ambiguously assigned as the main ellagitannin present in the diet. In the second part of this Ph.D. thesis the in vivo bioavailability of a dose of polyphenol microbial metabolites reflecting dietary consumption of fruit was studied. The focus was on the metabolites of polyphenols which can be found in the bloodstream after gut microflora metabolism. Their distribution was explored in rats in different organs, in particular in the brain, considering their possible neuropreventive properties. Development of a specific quantitative method for the quantification of selected polyphenol microbial metabolites made it possible to analyse complex biological samples resulting from in vivo trials with rats treated with a nutritionally significant dose of polyphenol microbial metabolites, intravenously injected. A high-throughput, sensitive and reproducible method for 23 polyphenol metabolites in six different biological matrices was developed. A purification protocol made it possible to obtain cleaner and more concentrated samples, with low limits of quantification. Specific organ-tropism was observed, mainly hepatotropism. Remarkably, in this study the brain was reported to be one of the target organs for these molecules, already being present at basal level or increasing their concentration after treatment. Furthermore, the amount of 10 out of 23 compounds significantly increased with a nutritionally significant dose.