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Digital archive of theses discussed at the University of Pisa


Thesis etd-03072008-171802

Thesis type
Tesi di dottorato di ricerca
Thesis title
Academic discipline
Course of study
Relatore Prof. Chiellini, Emo
Relatore Dott. Chiellini, Federica
  • biomaterials
  • drug delivery
  • nanoparticles
  • nanotechnology
Graduation session start date
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.