Tetrafenilciclopentadienon-derivati ed analoghi: sintesi, "self-assembly" e caratterizzazione reologica

Gels are materials that are easier to recognize than to define. For all practical purpose, a material is termed a gel if the whole volume of liquid is completely immobilized as usually tested by the ‘tube inversion’ method. Recently, supramolecular gels obtained from low molecular weight gelators (LMWGs) have attracted considerable attention in materials science since they represent a new class of smart materials sensitive to external stimuli, such as temperature, ultrasounds, light, chemical species and so on. Accordingly, during the past years a large variety of potentialities and applications of these soft materials in optoelectronics, as electronic devices, light harvesting systems and sensors, in bio-materials and in drug delivery have been reported. Spontaneous self-assembly of low molecular weight molecules is a powerful tool that allows complex supramolecular nanoscale structures to be built. The weak and non-covalent interactions such as hydrogen bonding, π–π stacking, coordination, electrostatic and van der Waals interactions are usually considered as the most important features for promoting sol-gel equilibria. However, the occurrence of gelation processes is ruled by further “external” factors, among which the temperature and the nature of the solvents that are employed are of crucial importance. For example, some gelators prefer aromatic or halogenated solvents and in some cases both the gelation temperature and the type of the solvent affect the morphologies of the final aggregation. Functionalized cyclopentadienones are fascinating systems largely employed as building blocks for the synthesis of polyphenylene derivatives. In addition, it is worth noting that structures containing π-extended conjugated chromophores with enhanced absorption properties are of current interest in the field of materials science since they can be used as “organic metals”, as semiconductors, and as emissive or absorbing layers for OLEDs or photovoltaics. The possibility to decorate the framework of such structures prompted us to study the synthesis of new hydroxy propargyl arylcyclopentadienone derivatives. Considering the ability of such systems to give π–π stacking interactions, the introduction on a polyaromatic structure of polar substituents able to generate hydrogen bonding could open the possibility to form gels, although any gelation properties has been never observed for these extensively studied systems. we have synthesized a new class of 3,4-bis (4-(3-hydroxy- propynyl) phenyl) -2, 5-diphenylcyclopentadienone derivatives, one of which (1a) proved to be, for the first time, a powerful organogelator. The experimental results indicated that the hydroxydimethylalkynyl substituents are fundamental to guarantee the gelation properties of the tetraarylcyclopentadienone unit. Combining the results of FT-IR, 1H NMR, UV-vis and fluorescence emission spectra, we believe that H-bonding and π–π interactions are the driving forces played for the gel formation. The importance of soft materials lies on their ability to respond to external stimuli, that can be also of chemical nature. In particular, high attention has been recently devoted to anion responsive properties of gels. Therefore the behaviour of organogels of 1a in toluene, ACN and MeNO2 towards the addition of 1 equivalent of various tetrabutylammonium salts were investigated. The rheological properties of gels in toluene, ACN and MeNO2 with and without the addition of Bu4N+X- salts were measured. In addition a qualitative analysis on cation recognition was performed. Finally the nature of the cyclic core of the gelator was changed in order to verify how the carbonyl group was essential to gel solvents. Until now, 4,5-diarylimidazoles have been synthesized.

Functional dna nanostructures for in vitro biosensing in live cells

DNA is a fascinating biomolecule that is well known for its genetic role in living systems. The emerging area of DNA nanotechnology provides an alternative view that exploits unparallel self-assembly ability of DNA molecules for material use of DNA. Although many reports exist on the results of DNA self-assembling systems, still few of them focus on the in vitro study about the function of such DNA nanostructures in live cells. Due to this, there are still a limited research about the in vitro functionality of such designs. To address an aspect of this issue, we have designed, synthesized and characterized two multifunctional fluorescencent nanobiosensors by DNA self-assembling. Each structure was designed and implemented to be introduced in live cells in order to give information on their functioning in real-time. Computational tools were used in order to design a graphic model of two new DNA motifs and also to obtain the specific sequences to all the ssDNA molecules. By thermal self-assembly techniques we have successfully synthesized the structure and corroborate their formation by the PAGE technique. In addition, we have established the conditions to characterize their structural conformation change when they perform their sensor response. The sensing behavior was also accomplished by fluorescence spectroscopy techniques; FRET evaluation and fluorescence microscopy imaging. Providing the evidence about their adequate sensing performance outside and inside the cells detected in real-time. In a preliminary evaluation we have tried to show the in vitro functionality of our structures in different cancer cell lines with the ability to perform local sensing responses. Our findings suggest that DNA sensor nanostructures could serve as a platform to exploit further therapeutic achievements in live cells.

Simulating the aggregation of DNA oligonucleotides

In this work we have studied, by means of Molecular Dynamics simulations, the process of denaturation and self-assembly of short oligonucleotides. Supramolecular ordering of DNA short strands is a promising field which is constantly enriched with new findings. Examples are provided by micellar and fibrils formations and due to the selectivity of DNA bindings, "intelligent" devices have been developed to perform simple logic operations. It is worth to notice that computer simulations of these DNA nanosystems would complement experiments with detailed insight into processes involved in self-assembly. In order to obtain an accurate description of the interactions involved in the complex structure of DNA we used oxDNA, a coarse-grained model developed by Ouldridge. We simulated the melting transition of 4, 6, and 8 base pair sequences. Sequence and length dependence were analyzed, specifically we compared thermodynamic parameters DeltaH, DeltaS and the melting temperature with literature results. Moreover, we have attempted to reproduce liquid crystal ordering of the ultrashort sequence GCCG at relatively high saline concentration, until now only experimentally observed in Bellini's works. We found that our simple model successfully reproduces the experimental phase sequence (isotropic, nematic, columnar) at T= 5 °C as a function of oligonucleotide concentration, and we fully characterized the microscopic structure of the three phases.

Design, preparazione e caratterizzazione di nanostrutture di DNA come candidati farmaci per terapia a RNA

Le terapie a RNA stanno attraendo interesse crescente vista la loro capacità di colpire target che venivano dapprima considerati undruggable. Uno degli ambiti di applicazione suggeriti della terapia a RNA è la neuroinfiammazione, una condizione patologica che accompagna e agisce da concausa nelle malattie neurodegenerative. In particolare, si è verificato che nei processi neuroinfiammatori, alcuni microRNA risultano sovra-regolati e tra questi miR-34a. Si è quindi proposto di sviluppare metodi atti a ridurre il contenuto cellulare di miR-34a soprattutto nelle cellule la cui attivazione causa maggiormente la neuroinfiammazione: la microglia. L’obiettivo del lavoro di tesi è stato di sviluppare una nanostruttura di DNA in grado di veicolare una sequenza catalitica (DNAzima) che porti al taglio del miR-34a, una volta internalizzata nelle cellule. Durante il lavoro di tesi si sono sviluppati 2 diversi dendrimeri di DNA pensati per ridurre il contenuto di miR-34a. I sistemi sono stati progettati con l’ausilio di strumenti bioinformatici e poi realizzati in laboratorio e caratterizzati con tecniche biochimiche. Il sistema più promettente è stato caratterizzato per quanto riguarda la sua attività enzimatica di taglio di miR-34a e l’efficienza di internalizzazione da parte di cellule vive di microglia. I risultati ottenuti confermano la solidità del metodo utilizzato per il design del sistema progettato. Le prove condotte sul dendrimero finale, contenente la sequenza attiva, dimostrano il mantenimento dell’attività catalitica del DNAzima e l’internalizzazione della nanostruttura nelle cellule bersaglio.

Double-stimulus sensitive (temperature and pH) self-assemblable polymers

The rheological properties of block co-polymers in water solution at different pH have been investigated. The block copolymers are based on different architectures containing poly(ethylene glycol), poly(propylene glycol) and different blocks of polymer that change their hydrophobic/hydrophilic behavior as a function of pH. The polymer chains of the starting material were extended at their functional ends with the pH-sensitive units using ATRP; this mechanism of controlled radical polymerization was chosen because of the need to minimize polydispersity and avoid transfer reactions possibly leading to homopolymeric inpurities. The starting material were modified in order to use them as macroinitiator for ATRP. The kinetic of each ATRP reaction has been investigated, in order to be able to synthesize polymers with different degree of polymerization, stopping the reaction when the desired polymers chain length has been reached. We will use polymer chains with different basicity and degree of polymerization to link any possible effect of their presence to the conditions under which they become hydrophobic. It has been shown that the rate of polymerization changes changing the type of macroinitiator and the type of monomer synthesized. The slowest rate of polymerization is the one with the most hindered monomer synthesized using the macroinitiator with the highest molecular weight. The water solubility of the synthesized polymers changes depending on the pH of the solution and on the structure of the polymers. It has been shown using 1H-NMR that some of the synthesized polymers are capable to self-aggregation in water solution. The self-aggregation and the type of aggregation is influenced from the structure of the polymer and from the pH of the solution. Changing the structure of the polymers and the pH it is possible to obtain different type of aggregates in solution. This aggregates differ for the volume occupied from them, and for their hardness. Rheological measurements have been demonstrated that the synthesized polymers are capable to form gel phases. The gelation temperature changes changing the structure of the aggregates in solution and it is possible to correlate the changing in the gelation temperature with the changing in the structure of the polymer.

The effect of poly(lactide)-poly(carbonate) based block copolymers on the morphology and crystallization of double crystalline poly(lactide)/poly(ε-caprolactone) blends

Block copolymers of poly(lactide) and poly(carbonate) were synthetized in three different compositions and characterized by 1H-NMR and ATR analyses. The compatibilization effect of this copolymers on 80/20 (w/w%) PLA/PCL blend was evaluated. SEM micrographs show that all the blends exhibit the typical sea-island morphology characteristic of immiscible blends with PCL finely dispersed in droplets on a PLA matrix. Upon the addiction of the copolymers a reduction on PCL droplets size is observable. At the same time, a Tg depression of the PLA phase is detected when the copolymers are added in the blend. These results indicate that these copolymers are effective as compatibilizers. The copolymer that acts as the best compatibilizer is the one characterized by the same amount of PLA and PC as repeating units. As result, in the blend containing this copolymer PLA phase exhibits the highest spherulitic growth rate. An analyses on PLA phase crystallization behaviour from the glassy state within the blends was evaluated by DSC experiments. Isothermal cold crystallization of the PLA phase is enhanced up an order of magnitude upon the blending with PCL. Annealing experiments demonstrated that the crystallization of the PCL phase induces the formation of active nuclei in PLA when cooled above cooled below Tg. When the crystallization rate of PCL is retarded, a reduction on PLA nucleation is observed.