Controlled surface nanotopography and oxygen plasma treatment of PEEK to improve cellular response

Poly(aryl-ether-ether-ketone) (PEEK) is a semi crystalline polymer which exhibits properties that make it an attractive choice for use as an implant material. It displays natural radiolucency, and MRI compatibility, as well as good chemical and sterilization resistance, both of which make it of particular interest in orthopaedic implants. However, PEEK has demonstrated poor cellular adhesion both in vitro and in vivo. This is problematic as implant surfaces that do not develop a layer of adhesive cells are at risk of undergoing fibrous encapsulation, which in turn leads to lack of a strong interface between the implant device and the patient tissue, which can in turn lead to failure of the implant and revision surgery . As incorporating nanotopography into a polymer surface has been demonstrated to be able to direct the differentiation behaviour of stem cells, a possible solution to PEEKs underlying issues with poor cellular response would be to incorporate specific nanoscale topography into the material surface through injection moulding, and then analysing if this is a viable method for addressing PEEKs issues with cellular response. In addition to nanoscale topography, the experimental PEEK surfaces were treated with oxygen plasma to address the underlying cytophobicity of the material. As this type of treatment has been documented to be capable of etching the PEEK surface, experiments were carried out to quantify the effect of this treatment, both on the ability of cells to adhere to the PEEK surface, as well as the effect it has upon the nanotopography present at the PEEK surface. The results demonstrated that there were a range of plasma treatments which would significantly improve the ability of cells to adhere to the PEEK surface without causing unacceptable damage to the nanotopography. Three different types of cells with osteogenic capacity were tested with the PEEK surfaces to gauge the ability of the topography to alter their behaviour: SAOS-2, osteoprogenitors and 271+ MSCs. Due to PEEKs material properties (it is non transparent, exhibits birefringence and is strongly autofluorescent) a number of histological techniques were used to investigate a number of different stages that take place in osteogenesis. The different cell types did display slightly different responses to the topographies. The SAOS-2 cells cultured on surfaces that had been plasma treated for 2 minutes at 200W had statistically significantly higher levels of von Kossa staining on the NSQ surface compared to the planar surface, and the same experiment employing alizarin red staining, showed a statistically significantly lower level of staining on the SQ surface compared to the planar surface. Using primary osteoprogenitor cells designed to look into if whether or not the presence of nanotopography effected the osteogenic response of these cells, we saw a lack of statistically significant difference produced by the surfaces investigated. By utilising HRP based immunostaining, we were able to investigate, in a quantitative fashion, the production of the two osteogenic markers osteopontin and osteocalcin by cells. When stained for osteocalcin, the SQ nanotopography had total percentage of the surface with stained material, average area and average perimeter all statistically significantly lower than the planar surface. For the cells that were stained for osteopontin, the SQ nanotopgraphy had a total percentage of the surface with stained material, average area and average perimeter all highly statistically significantly lower than those of the planar surface. Additionally, for this marker the NSQ nanotopography had average areas and average perimeters that were highly significantly higher than those of the planar surface. There were no significant differences for any of the values investigated for the 271+ MSC’s When plasma treatment was varied, the SAOS-2 cells demonstrated an overall trend i.e. increasing the energy of plasma treatment in turn leads to an increase in the overall percentage of staining. A similar experiment employing stem cells isolated from human bone marrow instead of SAOS-2 cells showed that for polycarbonate surfaces , used as a control, mineralization is statistically significantly higher on the NSQ nanopattern compared to the planar surface, whereas on the PEEK surfaces we observe the opposite trend i.e. the NSQ nanotopography having a statistically significantly lower amount of mineralization compared to the planar surface at the 200W 2min and 30W 1min plasma treatments. The standout trend from the PEEK results in this experiment was that the statistically significant differences on the PEEK substrates were clustered around the lower energy plasma treatments, which could suggest that the plasma treatment disrupted a function of the nanotopograhy which is why, as the energy increases, there are less statistically significant differences between the NSQ nanotopography and the Planar surface This thesis documents the response of a number of different types of cells to specific nanoscale topographies incorporated into the PEEK surface which had been treated with oxygen plasma. It outlines the development of a number of histological methods which measure different aspects of osteogenesis, and were selected to both work with PEEK, and produce quantitative results through the use of Cell Profiler. The methods that have been employed in this body of work would be of interest to other researchers working with this material, as well as those working with similarly autofluorescent materials.

Sintese de derivados 3 - substituídos de coumarinas

Este trabalho descreve a síntese de novos derivados de coumarinas 3-substituídas por grupos arilo, etenilarilo e etenil-organometálicos, através de novas metodologias via reacções de Heck e de metátese (Grubbs), com controlo da regioquímica e com significativos rendimentos reaccionais. A aplicação destas metodologias permitiu a síntese dos derivados, 3-fenilcoumarina (131), 3-(4-bromofenil)coumarina, (132), 3-(4-iodofenil)­coumarina (134), 3-(4-nitrofenil)coumarina (136), 3-(4-etilfenil)coumarina (133), 4-(coumarin-3-il)benzaldeído (135), 3-(4-metoxifenil)coumarina (137), (E)-3-acrilato-[4-(coumarin-3-il)fenil] de metilo (138), 6,7-metileno­dioxi-[3-(E)-2'-feniletenil]coumarina (145), 6,7-dimetoxi-[-(E)-2'-fenil­etenil]coumarina (146), 6,7-dimetoxi-[3-(E)-2'-(6'-nitrofenil)etenil]coumarina (147), 4-[2-(E)-(6,7-dimetoxicoumarin-3-il)etenil]benzaldeído (148) e 6,7-dimetoxi-[3-(E)-2'-ferroceniletenil]coumarina (149), dos quais os últimos nove, são compostos novos, identificados e caracterizados pela primeira vez. A deslocalização do sistema de electrões  conjugados, induzida pelos diversos substituintes das coumarinas, foi igualmente avaliada através da espectroscopia de UV/Vis. De referir que parte deste trabalho foi publicado como: "New Methodology for the Synthesis of 3-Substituted Coumarins via Pd-Catalyzed Site-Se/ective Cross-Coupling Reactions”, Sérgio Martins, Paula S. Branco, María C. de la Torre, Miguel A. Sierra e António Pereira, Synlett, 2010 (https://www.thieme-connect.com/ejournals/abstract/ synlett/doi/1 O.1 OS5/s-0030-1259014). ABSTRACT: This work describes the synthesis of new 3-aryl, ethenylaryl and ethenyl-organometallics coumarin derivatives, using a new methodology via Heck and metathesis (Grubbs) reactions, with regiochemistry control and significant reaction yields. The application of these methodologies allowed the synthesis of derivatives, 3-phenylcoumarin (131), 3-(4-bromophenyl)coumarin (132), 3-(4-iodophenyl)coumarin (134), 3-(4-nitrophenyl)coumarin (136), 3-(4-ethylphenyl)coumarin {133), 4-(coumarin-3-yl)benzaldehyde {135), 3-(4-methoxiphenyl)coumarin (137), (E)-ethyl 3-[4(coumarin-3-yl)phenyl]­acrylate (138), 6,7-methylenedioxy-[3-(E)-2'-phenylethenyl]coumarin (145), 6,7-dimethoxy-[-(E)-2'-phenylethenyl]coumarin (146), 6,7-dimethoxy-[3-(E)­-2'-(6'-nitrophenyl)ethenyl]coumarin (147), 4-[2-(E)-(6,7-dimethoxy­coumarin-3-yl)ethenyl]benzaldehyde {148) e 6,7-dimethoxy-[3-(E)-2'-(ferro­ cene)ethenyl]coumarin (149), the last nine of these are new compounds, identified and characterized for the first time. The delocalization of conjugated -electron system, induced by different substituents of coumarins, was also assessed by spectroscopy UV/Vis. Part of this work was published at: "New Methodology for the Synthesis of 3-Substituted Coumarins via Pd-Catalyzed Site-Selective Cross-Coupling Reactions", Sérgio Martins, Paula S. Branco, María C. de la Torre, Miguel A. Sierra e António Pereira, Synlett, 2010 (https://www.thieme­connect.com/ejournaIs/abstract/synlett/doi/1O.1 055/s-0030-1259014).

Bis-Alkoxycarbonylation And Difunctionalization Of Alkenes Employing Palladium(II) Or Nickel(II) Complexes As Catalysts

Section 1 is focused on the bis-alkoxycarbonylation reaction of olefins, catalyzed by aryl α-diimine/Pd(II) complexes, for the synthesis of succinic acid ester derivatives, important compounds in many industrial fields. The opening chapter (Chapter 1) of this thesis presents an overview of the basic chemistry of organopalladium compounds and carbonylation reactions, focusing on oxidative bis-alkoxycarbonylation processes. In Chapter 2 the results obtained in the bis-alkoxycarbonylation of 1,2-disubstituted olefins are reported. The reaction proceeds under very mild reaction conditions, using an aryl α-diimine/Pd(II) catalyst and p-benzoquinone as oxidant, in the presence of a suitable alcohol. This process proved to be very efficient, selective and diastereospecific and various 2,3-disubstituted succinic esters have been obtained in high yields. In Chapter 3 the first bis-alkoxycarbonylation reaction of acrylic esters and acrylic amides, leading to the synthesis of 2-alkoxycarbonyl and 2-carbamoyl succinates respectively, is reported. Remarkably, the utilized aryl α-diimine/Pd(II) catalyst is able to promote the carbonylation of both the β- and the generally non-reactive α- positions of these alkenes. The proposed catalytic cycle is supported by DFT calculations. Section 2 is mainly focused on the Ni-catalyzed difunctionalization of unactivated alkenes tethered to unstabilized ketones. This reaction allows for a wide range of pharmaceutically useful cyclic architectures to be obtained. Chapter 4 consists of an introduction to the difunctionalization reactions of unactivated olefins. In particular, intramolecular reactions will be discussed in detail. In Chapter 5 the results obtained from the Ni-catalyzed difunctionalization of unactivated alkenes tethered to unstabilized ketones are reported. The reaction proceeds through the formation of a zinc-enolate compound, followed by a cyclization/cross-coupling reaction, which takes place in the presence of a phosphine/Ni(II) complex and an (hetero)aryl electrophile, leading to different cyclic and bicyilc architectures. In Chapter 6, preliminary results concerning the anionic cyclization of zinc enolates tethered to unactivated alkenes are presented.

Novel dual D3R/GSK-3β modulators: an innovative multitarget strategy for bipolar disorder

Among the psychiatric diseases, bipolar disorder (BD) is the sixth leading cause of disability with a prevalence up to 4 % worldwide. BD is a complex neuropsychiatric condition which alternates episodes of mania with symptoms of depression. Although the neurobiological pathways are not completely clarified, the dopamine (DA) hypothesis, recognized as the leading theory explaining the pathophysiology of the malady, states that the dramatically compromised homeostatic regulation of dopaminergic circuits leads to alternated changes in DA neurotransmission. Modulation of D2 and D3 receptors (D2/3R) through partial agonists represents the first-line therapeutic strategy for psychiatric diseases. Moreover, a deregulation of the enzyme glycogen synthase kinase-3β (GSK-3β) has been reported as peculiar feature of BD. In this scenario, the concomitant modulation of D3R and GSK-3β, by employing multitarget compounds, could offer promises to achieve an effective cure of this illness. In the light of these findings, we rationally envisaged the pharmacophoric model at the basis of the design of several D3R partial agonists, suitable to be exploited for the dual D3R/GSK-3β ligand design. Thus, synthetic efforts were addressed to develop a first set of hybrid molecules able to concurrently modulate the selected targets. For a chemical structure point of view, we employed different spacers to combine a substituted aryl-piperazine moiety, reported in previously discovered D3R modulators, with a pyrazole-based fragment, already identified in GSK-3β inhibitors. A fluorescent and a cellular functional assays were carried out to assess the activity of all synthetized compounds against GSK-3β and on D3R, respectively. Most of the derivatives proved to effectively modulate both GSK-3β and D3R with potencies in the low-µM and low-nM range, respectively. The consistent biological data allowed us to identify some lead candidates worth to be further modified with the aim to optimize their biological profile and to perform a structure-activity relationship (SAR) study.

New Metal Tetrazolate Complexes: Design, Synthesis, Characterization and Applications

This final thesis is aimed at summarizing the research program I have carried out during my PhD studies, that has been dealing with the design, the preparation, characterization and applications of new Re(I), Ru(II), and Ir(III) metal complexes containing anionic ligands such as 5-aryl tetrazolates [R-CN4]- or their neutral analogues, N-alkyltetrazoles [R-CN4-R1]. Chapter 1 consists of a brief introduction on tetrazoles and metal-tetrazolato complexes, and on the photophysical properties of d6 transition metal complexes. In chapter 2, the synthesis, characterization and study of the photophysical properties of new luminescent Ir(III)-tetrazolate complexes are discussed. Moreover, the application of one of the new Ir(III)-CN complexes as emissive core in the fabrication of an OLED device is reported. In chapter 3, the study of the antimicrobial activity of new Ru(II)-alkyltetrazole complexes is reported. When the pentatomic ring was substituted with a long alkyl residue, antimicrobial activity toward Deinococcus radiodurans was observed. In chapter 4, a new family of luminescent Re(I)-tetrazolate complexes is reported. In this study, different N-alkyl tetrazoles play the role of diimine (diim) ligands in the preparation of new Re(I) tricarbonyl complexes. In addition, absorption and emission titration experiments were performed to study their interaction with Bovine Serum Albumin (BSA). In chapter 5, the synthesis and characterization of new luminescent Re(I)-tetrazolate complexes are discussed. The use of sulfonated diimine ligands in the preparation of new Re(I) tricarbonyl complexes led to the first example Re(I) complexes for the luminescent staining of proteins. In chapter 6, the synthesis, a new family of Ir(III)-NO2 tetrazole complexes displaying unexpected photophysical properties are discussed. Moreover, the possibility to tune the luminescent output of such systems upon chemical modification of the pending nitro group was verified by performing reduction tests with sodium dithionite; this represents encouraging evidence for their possible application as hypoxia-responsive luminescent probes in bioimaging.

Molecular modelling of organic functional materials

The investigation of the mechanisms lying behind the (photo-)chemical processes is fundamental to address and improve the design of new organic functional materials. In many cases, dynamics simulations represent the only tool to capture the system properties emerging from complex interactions between many molecules. Despite the outstanding progresses in calculation power, the only way to carry out such computational studies is to introduce several approximations with respect to a fully quantum mechanical (QM) description. This thesis presents an approach that combines QM calculations with a classical Molecular Dynamics (MD) approach by means of accurate QM-derived force fields. It is based on a careful selection of the most relevant molecular degrees of freedom, whose potential energy surface is calculated at QM level and reproduced by the analytic functions of the force field, as well as by an accurate tuning of the approximations introduced in the model of the process to be simulated. This is made possible by some tools developed purposely, that allow to obtain and test the FF parameters through comparison with the QM frequencies and normal modes. These tools were applied in the modelling of three processes: the npi* photoisomerisation of azobenzene, where the FF description was extended to the excited state too and the non-adiabatic events were treated stochastically with Tully fewest switching algorithm; the charge separation in donors-acceptors bulk heterojunction organic solar cells, where a tight-binding Hamiltonian was carefully parametrised and solved by means of a code, also written specifically; the effect of the protonation state on the photoisomerisation quantum yield of the aryl-azoimidazolium unit of the axle molecule of a rotaxane molecular shuttle. In each case, the QM-based MD models that were specifically developed gave noteworthy information about the investigated phenomena, proving to be a fundamental key for a deeper comprehension of several experimental evidences.

Diazene photoswitches in artificial molecular machines and light-effected materials

The research developed in this thesis focused on the spectroscopic and photochemical characterization of molecular diazene photoswitches, both as individual species and as functional components of mechanically interlocked molecules, molecular-based materials and artificial molecular machines and motors. Among the plethora of photochromes reported so far, azobenzene is the most versatile photoswitch due to its reproducible and well-established photochemical properties. Part I of this thesis work focuses on the characterization of light-responsive supramolecular systems based on azobenzene: a photochemically-driven rotary motor, a light-responsive supramolecular polymeric material and a supramolecular system capable of photoinduced entantiodiscrimination. Despite the wide success of azobenzene photoswitches, the tunability of their photochemical properties as a function of the diversified substitution pattern on its aryl ring presents intrinsic limitations. To overcome this issue, in the last decade heteroaryl azoswitches (i.e., azobenzene having heterocyclic rings in place of one or both phenyl groups) have attracted a great deal of attention. Hence, Part II of this thesis work treats the photochemical characterization of two different families of azoheteroarenes embedding imidazolium and thienyl functionalities in their structures. Their potential implementation in water-soluble artificial molecular machines and light-effected semiconductor materials is also assessed.

Green approach to Palladium Cross-Coupling reactions and development of new methodologies based on highly abundant metals

Transition metal catalyzed cross-coupling reactions represent among the most versatile and useful tools in organic synthesis for the carbon-carbon (C-C) bond formation and have a prominent role in both the academic and pharmaceutical segments. Among them, palladium catalyzed cross-coupling reactions are currently the most versatile. In this thesis, the applications, impact and development of green palladium cross-coupling reactions are discussed. Specifically, we discuss the translation of the Twelve Principles of Green Chemistry and their applications in pharmaceutical organometallic chemistry to stimulate the development of cost-effective and sustainable catalytic processes for the synthesis of active pharmaceutical ingredients (API). The Heck-Cassar-Sonogashira (HCS) and the Suzuki-Miyaura (SM) protocols, using HEP/H2O as green mixture and sulfonated phosphine ligands, allowed to recycle and recover the catalyst, always guaranteeing high yields and fast conversion under mild conditions, with aryl iodides, bromides, triflates and chlorides. No catalyst leakage or metal contamination of the final product were observed during the HCS and SM reactions, respecting the very low limits for metal impurities in medicines established by the International Conference of Harmonization Guidelines Q3D (ICH Q3D). In addition, a deep understanding of the reaction mechanism is very important if the final target is to develop efficient protocols that can be applied at industrial level. Experimental and theoretical studies pointed out the presence of two catalytic cycles depending on the counterion, shedding light on the role of base in catalyst reduction and acetylene coordination in the HCS coupling. Finally, the development of a cross-coupling reaction to form aryldifluoronitriles in the presence of copper is discussed, highlighting the importance of inserting fluorine atoms within biological structures and the use of readily available metals such as copper as an alternative to palladium.