Innovative analytical methods for Central Nervous System Drug analysis in biological fluids

During recent years a consistent number of central nervous system (CNS) drugs have been approved and introduced on the market for the treatment of many psychiatric and neurological disorders, including psychosis, depression, Parkinson disease and epilepsy. Despite the great advancements obtained in the treatment of CNS diseases/disorders, partial response to therapy or treatment failure are frequent, at least in part due to poor compliance, but also genetic variability in the metabolism of psychotropic agents or polypharmacy, which may lead to sub-therapeutic or toxic plasma levels of the drugs, and finally inefficacy of the treatment or adverse/toxic effects. With the aim of improving the treatment, reducing toxic/side effects and patient hospitalisation, Therapeutic Drug Monitoring (TDM) is certainly useful, allowing for a personalisation of the therapy. Reliable analytical methods are required to determine the plasma levels of psychotropic drugs, which are often present at low concentrations (tens or hundreds of nanograms per millilitre). The present PhD Thesis has focused on the development of analytical methods for the determination of CNS drugs in biological fluids, including antidepressants (sertraline and duloxetine), antipsychotics (aripiprazole), antiepileptics (vigabatrin and topiramate) and antiparkinsons (pramipexole). Innovative methods based on liquid chromatography or capillary electrophoresis coupled to diode-array or laser-induced fluorescence detectors have been developed, together with the suitable sample pre-treatment for interference removal and fluorescent labelling in case of LIF detection. All methods have been validated according to official guidelines and applied to the analysis of real samples obtained from patients, resulting suitable for the TDM of psychotropic drugs.

Laser driven proton acceleration and beam shaping

In the race to obtain protons with higher energies, using more compact systems at the same time, laser-driven plasma accelerators are becoming an interesting possibility. But for now, only beams with extremely broad energy spectra and high divergence have been produced. The driving line of this PhD thesis was the study and design of a compact system to extract a high quality beam out of the initial bunch of protons produced by the interaction of a laser pulse with a thin solid target, using experimentally reliable technologies in order to be able to test such a system as soon as possible. In this thesis, different transport lines are analyzed. The first is based on a high field pulsed solenoid, some collimators and, for perfect filtering and post-acceleration, a high field high frequency compact linear accelerator, originally designed to accelerate a 30 MeV beam extracted from a cyclotron. The second one is based on a quadruplet of permanent magnetic quadrupoles: thanks to its greater simplicity and reliability, it has great interest for experiments, but the effectiveness is lower than the one based on the solenoid; in fact, the final beam intensity drops by an order of magnitude. An additional sensible decrease in intensity is verified in the third case, where the energy selection is achieved using a chicane, because of its very low efficiency for off-axis protons. The proposed schemes have all been analyzed with 3D simulations and all the significant results are presented. Future experimental work based on the outcome of this thesis can be planned and is being discussed now.

Biomimetic Materials for Biomedical Applications

Objects with complex shape and functions have always attracted attention and interest. The morphological diversity and complexity of naturally occurring forms and patterns have been a motivation for humans to copy and adopt ideas from Nature to achieve functional, aesthetic and social value. Biomimetics is addressed to the design and development of new synthetic materials using strategies adopted by living organisms to produce biological materials. In particular, biomineralized tissues are often sophisticate composite materials, in which the components and the interfaces between them have been defined and optimized, and that present unusual and optimal chemical-physical, morphological and mechanical properties. Moreover, biominerals are generally produced by easily traceable raw materials, in aqueous media and at room pressure and temperature, that is through cheap process and materials. Thus, it is not surprising that the idea to mimic those strategies proper of Nature has been employed in several areas of applied sciences, such as for the preparation of liquid crystals, ceramic thin films computer switches and many other advanced materials. On this basis, this PhD thesis is focused on the investigation of the interaction of biologically active ions and molecules with calcium phosphates with the aim to develop new materials for the substitution and repair of skeletal tissue, according to the following lines: I. Modified calcium phosphates. A relevant part of this PhD thesis has been addressed to study the interaction of Strontium with calcium phosphates. It was demonstrated that strontium ion can substitute for calcium into hydroxyapatite, causing appreciable structural and morphological modifications. The detailed structural analysis carried out on the nanocrystals at different strontium content provided new insight into its interaction with the structure of hydroxyapatite. At variance with the behaviour of Sr towards HA, it was found that this ion inhibits the synthesis of octacalcium phosphate. However, it can substitute for calcium in this structure up to 15 atom %, in agreement with the increase of the cell parameters observed on increasing ion concentration. A similar behaviour was found for Magnesium ion, whereas Manganese inhibits the synthesis of octacalcium phosphate and it promotes the precipitation of dicalcium phosphate dehydrate. It was also found that Strontium affects the kinetics of the reaction of hydrolysis of α-TCP. It inhibits the conversion from α-TCP to hydroxyapatite. However, the resulting apatitic phase contains significant amounts of Sr2+ suggesting that the addition of Sr2+ to the composition of α-TCP bone cements could be successfully exploited for its local delivery in bone defects. The hydrolysis of α-TCP has been investigated also in the presence of increasing amounts of gelatin: the results indicated that this biopolymer accelerates the hydrolysis reaction and promotes the conversion of α-TCP into OCP, suggesting that its addition in the composition of calcium phosphate cements can be employed to modulate the OCP/HA ratio, and as a consequence the solubility, of the set cement. II. Deposition of modified calcium phosphates on metallic substrates. Coating with a thin film of calcium phosphates is frequently applied on the surface of metallic implants in order to combine the high mechanical strength of the metal with the excellent bioactivity of the calcium phosphates surface layers. During this PhD thesis, thank to the collaboration with prof. I.N. Mihailescu, head of the Laser-Surface-Plasma Interactions Laboratory (National Institute for Lasers, Plasma and Radiation Physics – Laser Department, Bucharest) Pulsed Laser Deposition has been successfully applied to deposit thin films of Sr substituted HA on Titanium substrates. The synthesized coatings displayed a uniform Sr distribution, a granular surface and a good degree of crystallinity which slightly decreased on increasing Sr content. The results of in vitro tests carried out on osteoblast-like and osteoclast cells suggested that the presence of Sr in HA thin films can enhance the positive effect of HA coatings on osteointegration and bone regeneration, and prevent undesirable bone resorption. The possibility to introduce an active molecule in the implant site was explored using Matrix Assisted Pulsed Laser Evaporation to deposit hydroxyapatite nanocrystals at different content of alendronate, a bisphosphonate widely employed in the treatments of pathological diseases associated to bone loss. The coatings displayed a good degree of crystallinity, and the results of in vitro tests indicated that alendronate promotes proliferation and differentiation of osteoblasts even when incorporated into hydroxyapatite. III. Synthesis of drug carriers with a delayed release modulated by a calcium phosphate coating. A core-shell system for modulated drug delivery and release has been developed through optimization of the experimental conditions to cover gelatin microspheres with a uniform layer of calcium phosphate. The kinetics of the release from uncoated and coated microspheres was investigated using aspirin as a model drug. It was shown that the presence of the calcium phosphate shell delays the release of aspirin and allows to modulate its action.

Fluid dynamic analysis and modelling of industrial chemical equipment

The research is aimed at contributing to the identification of reliable fully predictive Computational Fluid Dynamics (CFD) methods for the numerical simulation of equipment typically adopted in the chemical and process industries. The apparatuses selected for the investigation, specifically membrane modules, stirred vessels and fluidized beds, were characterized by a different and often complex fluid dynamic behaviour and in some cases the momentum transfer phenomena were coupled with mass transfer or multiphase interactions. Firs of all, a novel modelling approach based on CFD for the prediction of the gas separation process in membrane modules for hydrogen purification is developed. The reliability of the gas velocity field calculated numerically is assessed by comparison of the predictions with experimental velocity data collected by Particle Image Velocimetry, while the applicability of the model to properly predict the separation process under a wide range of operating conditions is assessed through a strict comparison with permeation experimental data. Then, the effect of numerical issues on the RANS-based predictions of single phase stirred tanks is analysed. The homogenisation process of a scalar tracer is also investigated and simulation results are compared to original passive tracer homogenisation curves determined with Planar Laser Induced Fluorescence. The capability of a CFD approach based on the solution of RANS equations is also investigated for describing the fluid dynamic characteristics of the dispersion of organics in water. Finally, an Eulerian-Eulerian fluid-dynamic model is used to simulate mono-disperse suspensions of Geldart A Group particles fluidized by a Newtonian incompressible fluid as well as binary segregating fluidized beds of particles differing in size and density. The results obtained under a number of different operating conditions are compared with literature experimental data and the effect of numerical uncertainties on axial segregation is also discussed.

Synthesis and surface modification of silver and gold nanoparticles. Nanomedicine applications against Glioblastoma Multiforme.

In the last decades noble metal nanoparticles (NPs) arose as one of the most powerful tools for applications in nanomedicine field and cancer treatment. Glioblastoma multiforme (GBM), in particular, is one of the most aggressive malignant brain tumors that nowadays still presents a dramatic scenario concerning median survival. Gold nanorods (GNRs) and silver nanoparticles (AgNPs) could find applications such as diagnostic imaging, hyperthermia and glioblastoma therapy. During these three years, both GNRs and AgNPs were synthesized with the “salt reduction” method and, through a novel double phase transfer process, using specifically designed thiol-based ligands, lipophilic GNRs and AgNPs were obtained and separately entrapped into biocompatible and biodegradable PEG-based polymeric nanoparticles (PNPs) suitable for drug delivery within the body. Moreover, a synergistic effect of AgNPs with the Alisertib drug, were investigated thanks to the simultaneous entrapment of these two moieties into PNPs. In addition, Chlorotoxin (Cltx), a peptide that specifically recognize brain cancer cells, was conjugated onto the external surface of PNPs. The so-obtained novel nanosystems were evaluated for in vitro and in vivo applications against glioblastoma multiforme. In particular, for GNRs-PNPs, their safety, their suitability as optoacoustic contrast agents, their selective laser-induced cells death and finally, a high tumor retention were all demonstrated. Concerning AgNPs-PNPs, promising tumor toxicity and a strong synergistic effect with Alisertib was observed (IC50 10 nM), as well as good in vivo biodistribution, high tumor uptake and significative tumor reduction in tumor bearing mice. Finally, the two nanostructures were linked together, through an organic framework, exploiting the click chemistry azido-alkyne Huisgen cycloaddition, between two ligands previously attached to the NPs surface; this multifunctional complex nanosystem was successfully entrapped into PNPs with nanoparticles’ properties maintenance, obtaining in this way a powerful and promising tool for cancer fight and defeat.

Theoretical and numerical study of the laser-plasma ion acceleration

The laser driven ion acceleration is a burgeoning field of resarch and is attracting a growing number of scientists since the first results reported in 2000 obtained irradiating thin solid foils by high power laser pulses. The growing interest is driven by the peculiar characteristics of the produced bunches, the compactness of the whole accelerating system and the very short accelerating length of this all-optical accelerators. A fervent theoretical and experimental work has been done since then. An important part of the theoretical study is done by means of numerical simulations and the most widely used technique exploits PIC codes (“Particle In Cell'”). In this thesis the PIC code AlaDyn, developed by our research group considering innovative algorithms, is described. My work has been devoted to the developement of the code and the investigation of the laser driven ion acceleration for different target configurations. Two target configurations for the proton acceleration are presented together with the results of the 2D and 3D numerical investigation. One target configuration consists of a solid foil with a low density layer attached on the irradiated side. The nearly critical plasma of the foam layer allows a very high energy absorption by the target and an increase of the proton energy up to a factor 3, when compared to the ``pure'' TNSA configuration. The differences of the regime with respect to the standard TNSA are described The case of nearly critical density targets has been investigated with 3D simulations. In this case the laser travels throughout the plasma and exits on the rear side. During the propagation, the laser drills a channel and induce a magnetic vortex that expanding on the rear side of the targer is source of a very intense electric field. The protons of the plasma are strongly accelerated up to energies of 100 MeV using a 200PW laser.

Development of a method for plasma - induced combustion of intermediate to low-level radioactive waste

This work demonstrates that the plasma - induced combustion of intermediate to low-level radioactive waste is a suitable method for volume reduction and stabilization. Weaknesses of existing facilities can be overcome with novel developments. Plasma treatment of LILW has a high economical advantage by volume reduction for storage in final repositories.

Numerical and Analytical Methods for Laser-Plasma Acceleration Physics

Theories and numerical modeling are fundamental tools for understanding, optimizing and designing present and future laser-plasma accelerators (LPAs). Laser evolution and plasma wave excitation in a LPA driven by a weakly relativistically intense, short-pulse laser propagating in a preformed parabolic plasma channel, is studied analytically in 3D including the effects of pulse steepening and energy depletion. At higher laser intensities, the process of electron self-injection in the nonlinear bubble wake regime is studied by means of fully self-consistent Particle-in-Cell simulations. Considering a non-evolving laser driver propagating with a prescribed velocity, the geometrical properties of the non-evolving bubble wake are studied. For a range of parameters of interest for laser plasma acceleration, The dependence of the threshold for self-injection in the non-evolving wake on laser intensity and wake velocity is characterized. Due to the nonlinear and complex nature of the Physics involved, computationally challenging numerical simulations are required to model laser-plasma accelerators operating at relativistic laser intensities. The numerical and computational optimizations, that combined in the codes INF&RNO and INF&RNO/quasi-static give the possibility to accurately model multi-GeV laser wakefield acceleration stages with present supercomputing architectures, are discussed. The PIC code jasmine, capable of efficiently running laser-plasma simulations on Graphics Processing Units (GPUs) clusters, is presented. GPUs deliver exceptional performance to PIC codes, but the core algorithms had to be redesigned for satisfying the constraints imposed by the intrinsic parallelism of the architecture. The simulation campaigns, run with the code jasmine for modeling the recent LPA experiments with the INFN-FLAME and CNR-ILIL laser systems, are also presented.