POLYMERIC NANOSTRUCTURED FORMULATIONS FOR TARGETED ADMINISTRATION OF BIOACTIVE AGENTS

Chemotherapy is an effective treatment for cancer and other serious diseases such as cardiovascular restenosis and AIDS. It is a complicated procedure in which many factors are involved in determining its success or failure. Furthermore it carries a high risk due to drug toxicity, with the more effective drugs tending to be more toxic. Patients have to tolerate severe side effects and sacrifice their quality of life. The inefficiency and side effects of chemotherapy are caused mainly by the formulation, pharmacokinetics and toxicity of chemotherapeutic agents and the drug resistance. Hence, the development of effective carriers for both existing and newly developed drugs may be as important as the discovery of new anticancer drugs. All these concerns lead to the concept of sustained, controlled and targeted release of drugs. The ideal goal of a drug delivery system is to deliver high efficacy drug(s) at the right time to the desired location, at high concentration but safe enough, over a sufficiently long period. Effective drug delivery has also important implications from a market viewpoint. More effective drug delivery can provide drug companies with a means of expanding market share or of revitalizing therapeutics with previously unrealized potential because of poor pharmacokinetic profiles. Nanomedicine is not only important from the social and welfare aspects, but also for its economic potential. In the framework of a long–standing research activity ongoing in the Bioactive Polymeric Materials Group at the Department of Chemistry and Industrial Chemistry of the University of Pisa, the present work was aimed at the development of polymeric nanoparticles for the site specific and controlled delivery of drugs for cancer treatment and tissue engineering application. The primary objective of the present PhD was the advancement of knowledge and the theoretical understanding of the relations among variables that play important roles in the development and optimization of procedures for the preparation of polymeric nanoparticle suspensions. The work was conducted keeping in mind the practical aspects of preparing nanodelivery structures for bioactive agents administration in cancer therapy and tissue engineering applications. A variety of polymers including PLGA, PHB, VAM41, multi block copolymers of PCL-PLU, and Chitosan, were investigated. Retinoic Acid and Chondroitin Sulphate, were loaded into nanoparticles as hydrophobic and hydrophilic model drugs respectively. Proteins have been also co-encapsulated as stabilizing agent or as model proteic drugs. For the preparation of RA loaded nanoparticles several methods were investigated and set up: dialysis, nanoprecipitation, co-precipitation and colloidal-coating. Colloidal-coating is an original solvent-free method developed in the framework of the research activity carried out in the present PhD thesis. The first two methods were employed for the preparation of formulations based on PLGA, PHB and the multi block copolymer PCL-co-PLU, while the last two for formulations based on VAM41. The resulting nanoparticle suspensions were analyzed macroscopically in terms of homogeneity and formation of precipitates, and characterized from a dimensional and morphological point of view by means of light scattering (LS) and scanning electron microscopy (SEM). Particles were purified and storage modalities were set up. Drug release studies were carried out by an original method that might better reflect bioavailability data. Since HSA is one of the most abundant plasma protein it was added to the releasing medium overcoming problems related to the poor solubility of RA in water. Solubility problems often hamper the evaluation of drugs release kinetics of hydrophobic drugs in phosphate buffer solution (PBS) at 37°C, which is commonly used in in vitro environment and that should match the in vivo condition. The obtained results showed that HSA affected the stability and life of nanoparticle formulations. The activity of RA encapsulated by colloidal-coating method was evaluated on SK-N-SH human neuroblastoma cell line that is known to undergo inhibition of proliferation and neuronal differentiation upon treatment with RA. The data obtained suggested that the anticancer activity of RA was not impaired by incorporation and purification processes. A careful in vitro investigation of PHB and the relative nanoparticles cytotoxicity was carried out using two type of assays aimed at the evaluation of the interactions of the materials with cell metabolism (WST-1 tetrazolium salts) and cell endocytosis functions (Neutral Red Uptake). In vitro cytotoxicity of PHB resulted fairly low as highlighted by the high IC50 values obtained both with Tetrazolium Salts and Neutral Red assays. PHB based nanoparticles prepared by dialysis method exhibited as well a very good cytocompatibility and can be considered fully biocompatible It was also investigated the preparation of suitable vehicles for intra-articular injection in regenerated cartilages tissue, in form of nanoparticles, to allow Chondroitin Sulphate sustained release. Synthetic bioerodible (VAM41), biodegradable (PLGA), natural (Chitosan) polymers and nanoprecipitation or co-precipitation methods were used to develop the nanostructured formulations. The limit of nanoprecipitation method mainly concerns the possibility to achieve an efficiently loading of hydrophilic drug substances inside the nanoparticles. Drug is easily lost in water during the preparation and incompatibilities between the polymer and the drug could worse the situation. Due to the wide differences among the utilized polymers the nanoprecipitation and co-precipitation methods were adjusted and modified in relation to the physical-chemical characteristics of the utilized material. PLGA, VAM 41 and Chitosan nanoparticles were prepared. No detectable amounts of CS were loaded onto the PLGA nanoparticles indicating the not suitability of the method for the encapsulation of this drug or a strong incompatibility between the hydrophobic polymer and hydrophilic drug. CS was detected into VAM41 and Chitosan NPs. A quantitative determination of the drug encapsulation was not possible due to several occurrences. Spectrophotometric UV assays have been based on changes in the absorption spectrum of the dye 1,9-dimethylmethylene blue (DMMB) when bound to Glycosaminoglycans (GAGs). CS interacted somehow with VAM41 and the characteristic UV band of absorbance of DMMB was shifted to higher wavelengths. Another attempt to evaluate encapsulation was done by IR spectrophotometry. The ratio between the intensity of the peak characteristic for CS and the intensity of the peak characteristic for VAM41 was evaluated and related to the percentage of CS in the samples. Although the developed method was effective for evaluating the encapsulation of CS it was not possible to verify whether the observed interaction affected these results or not. Chitosan nanoparticles were insoluble in all the tested solvents. Hence, an approximate evaluation of CS content was carried out by XPS analysis. The presence of sulphur in the sample qualitatively confirmed the encapsulation of CS into the NPs. The reported results and relevant discussions, which give exhaustive theoretical understanding of the phenomena occurring during colloidal formations, contribute to both basic and applied research in biomedical science and pharmaceutical technology

Program and abstracts

We are pleased that the program in 2022 will be more interesting than ever and it will include the following topics: Mathematical Modeling in Cancer Therapy, Gene Therapy, Archaeological Genetics, New perspectives in Human Forensic Molecular Biology, Genomics in Medicine, Pharmacogenomics and Drug Development, Stem Cells in Medicine, Regenerative Medicine, Ribosomes in Medicine, Epigenomics, Crime Scene Investigation, Forensic Genetics, and Mass Catastrophes Managements. This year, the third "Nobel Spirit" will provide a forum to the three Nobel laureates to stimulate public discussion on the role of science in solving global health issues, acute regional problems such as brain drain, demographic decline, as well as cultural and social change. In addition, we are organizing a very stimulating Session on Bioanthropology and global health in the times of crisis, as well as Joint Event ISABS and Ministry of the Interior - Crime Scene Investigation Training Course: Mystery on the ship —Investigation of the water-related crime scene

Study of Liver Surface Imaging Marker to Monitor Chronic Liver Disease Progression

Ph.DDOCTOR OF PHILOSOPH

Biocomposite Inks for 3D Printing

Three-dimensional (3D) printing has evolved massively during the last years. The 3D printing technologies offer various advantages, including: i) tailor-made design, ii) rapid prototyping, and iii) manufacturing of complex structures. Importantly, 3D printing is currently finding its potential in tissue engineering, wound dressings, tissue models for drug testing, prosthesis, and biosensors, to name a few. One important factor is the optimized composition of inks that can facilitate the deposition of cells, fabrication of vascularized tissue and the structuring of complex constructs that are similar to functional organs. Biocomposite inks can include synthetic and natural polymers, such as poly (ε-caprolactone), polylactic acid, collagen, hyaluronic acid, alginate, nanocellulose, and may be complemented with cross-linkers to stabilize the constructs and with bioactive molecules to add functionality. Inks that contain living cells are referred to as bioinks and the process as 3D bioprinting. Some of the key aspects of the formulation of bioinks are, e.g., the tailoring of mechanical properties, biocompatibility and the rheological behavior of the ink which may affect the cell viability, proliferation, and cell differentiation.The current Special Issue emphasizes the bio-technological engineering of novel biocomposite inks for various 3D printing technologies, also considering important aspects in the production and use of bioinks

Advanced Therapy Medicinal Products for Eye Diseases: Goals and Challenges

The concept of advanced therapy medicinal products (ATMPs) encompasses novel kinds of medicines for human use that are based on genes, cells or tissues. These intend to offer not only regeneration, but complete functional recovery of diseased tissues and organs using different strategies. Gene therapy, cell therapy and tissue engineering are the main areas in which promising advanced therapies are emerging. The eye is a very complex organ whose main structures, the cornea and the retina, play a pivotal role in maintaining normal vision, as severe alterations in these tissues can lead to blindness. Ocular tissues are starting to benefit from ATMPs by fighting against the enormous complexity and devastating potential of many ocular diseases. However, developments arising from this field of work face important challenges related to vectors to deliver drugs and genetic material to target tissues, suitable biomaterials to prepare cell scaffolds and cell stemness, among others—not to mention the complicated legislation around ATMPs, the complexity in production and quality control and the absence of standardized protocols.The purpose of this Special Issue is to serve as an overview of the current progress in the application of cell and gene therapies, as well as tissue engineering to restore functionality in diseased ocular structures, and the challenges linked to reaching patients

Proteomics

Biomedical research has entered a new era of characterizing a disease or a protein on a global scale. In the post-genomic era, Proteomics now plays an increasingly important role in dissecting molecular functions of proteins and discovering biomarkers in human diseases. Mass spectrometry, two-dimensional gel electrophoresis, and high-density antibody and protein arrays are some of the most commonly used methods in the Proteomics field. This book covers four important and diverse areas of current proteomic research: Proteomic Discovery of Disease Biomarkers, Proteomic Analysis of Protein Functions, Proteomic Approaches to Dissecting Disease Processes, and Organelles and Secretome Proteomics. We believe that clinicians, students and laboratory researchers who are interested in Proteomics and its applications in the biomedical field will find this book useful and enlightening. The use of proteomic methods in studying proteins in various human diseases has become an essential part of biomedical research