SINTHESES AND FUNCTIONALIZATION OF POLY(L-LACTIDE) BASED NANOCOMPOSITES SYSTEMS FOR SELECTIVE AND CONTROLLED DRUG RELEASE IN BIOMEDICAL USES

In this thesis, the research activity, focused on two investigations topics, both addressed to the preparation and characterization of poly(L-lactide) (PLLA) based nano-materials for biomedical applications, is reported. In particular, the research deals with the preparation of a new polymeric substrate for vaccine (line 1) and superparamagnetic nanoparticles-PLLA core-shell nanocomposites for targeted and controlled release of anti-tumor drugs (line 2).The polymer we used in both the researches, the poly (L-lactide) (PLLA), is one of the most investigated synthetic product in the biomedical field because of its good mechanical properties, biocompatibility and biodegradability. In this work the PLLA has been used in: - Line 1, as a possible biodegradable carrier for the development of a vaccine. We employed the polymer in guise of lamellar single crystals (PLLAsc) growth from dilute solution, characterized by a 1D-nano-2D-micro morphology and by a high specific area. - Line 2, as the outer coating of micro-nano-superparamagnetic particles (MNPs) for the realization of nano-composites with core-shell morphology to be used for the preparation of drug-delivery systems. Line 1. In the last years, the research on innovative immunization systems took into account the use of polymer micro or nano particles as substrate, vehicle or/and adjuvant for development of new vaccines. In traditional vaccination systems, the conventional antigens are highly immunogenic and induce a strong and durable protective response in the individual immune system. At the same time, the antigen may be potentially harmful or induce dangerous side effects. In contrast, vaccines based on antigens composed of sub-molecular units, obtained by recombinant DNA techniques, usually has negligible side effects. Unfortunately, with rare exceptions, this type of antigen induces low antibody responses and need an "adjuvant" that can enhance the immunogenicity. The research in this field is boosted by the possibility to use immunotherapy for the tumors prevention and eradication. In fact, the cancer, during the malignant progression, is able to evade the body immune response by developing mechanisms that hide its presence to the host. If the immune system is activated by a proper vaccine against these tumours, the body could be able to kill the tumoral cells In this research, the E7 protein of human papillomavirus (HPV16), related to the formation of cervical cancer, has been used for the preparation of a polymer/antigen vaccine. PLLA single crystals (PLLAsc), characterized by an lamellar hexagonal shape with lateral dimensions of about 15 micron and thickness of 10 nm, were used as biodegradable antigene carrier. To increase the hydrophilicity and provide the PLLAsc of active sites useful to the subsequent protein adsorption, a controlled reaction of aminolysis with tetraethylenepentamine (TEPA) was carried out on the surface ester bond of PLLAsc (APLLAsc).The pristine and functionalized single crystals were characterized and the effectiveness of the APLLAsc-E7 vaccine was evaluated by tumour protection experiments on mice. It has been observed that, unlike the behaviour of non-functionalized single crystals, the lamellae have a strong adjuvant effect. The results opens interesting perspectives for a possible use of the APLLAsc-E7 system as a single dose vaccine. Line 2. Superparamagnetic nano-particles (MNPs) represent the object of an extensive research in the material science and medicine fields. In fact, by a proper selection of the composition, it is possible to prepare hybrid inorganic core-polymer shell nanocomposites able to fulfill diagnostic and therapeutic tasks. The MNPs are magnetized only in presence of an external magnetic fields and show no residual magnetism when the field is removed, that is no hysteresis in the magnetization curve is observed. In this class of nanoparticles are included superparamagnetic iron oxides and iron oxides doped with +2 cations (M: Mn, Co, Fe, Ni) to form MFe2O4 spinel structures. The coating of MNPs by organic shell is mandatory to overcome their agglomeration driven by their high surface-to-volume ratio. Moreover, the outermost sheath can increase the nanoparticles circulation time in vivo before their clearance by macrophages in the reticulo-endothelial system. The MNPs can be directed toward a target organ or tissue by external magnetic field. Once they reach the target they can perform their therapeutic task by means physical or chemical process. As far as the former is concerned, when subject to alternating external magnetic field, magnetic nanoparticles absorb energy and convert it into heat through a mechanism called magnetic hyperthermia. The generated local temperature increase, up to 41-47 °C, can kills the surrounding tissue cells, such as cancer cells. As far the chemical action, MNPs can be loaded with a drug and used as drug delivery vehicles. The possibility to direct them on a specific target has the advantage to increase the potency of many therapeutics and reduce their possible systemic toxicity. Moreover, the drug release can be trigged by external stimuli. The local magnetic hyperthermia, for instance, can activate polymer transition, like volume collapse of a thermo-sensitive polymer shell, and induce an accelerative drug release. In this research, superparamagnetic nanoparticles (MNPs), composed of manganese and iron oxides (MnFe2O4) and synthesized by the technique of water-in-oil microemulsion, have been used.The obtained MNPs have sizes in the order of 10 nm and present superparamagnetic behaviour. Three different methods were used to coat the nanoparticles with PLLA and to obtain a core-shell nanocomposite: - Grafting from: -OH groups on the surface of nanoparticles have been used as initiators for catalyzed ring opening polymerization (ROP) of L-lactide. - Grafting to: a series of carboxylated-poly(L-lactide) with different molecular weight (CPLLAs), based on 2,2-bis(hydroxymethyl) propionic acid (DMPA) as initiator, have been prepared by ROP. Then, CPLLAs were adsorbed onto nanoparticles surface by exploiting the specific interaction of the carboxylic acid with the MNPs surface. - One step: the monomer (L-lactide), the initiator (DMPA), MNPs and the catalyst were reacted simultaneously. All the CPLLAs and the three types of nano-composites were chemical and physico-chemical characterized. Even if the grafting from and one step methods are more attractive from the scale-up point of view, they have provided low yields. The grafting to method was observed to be more controllable in terms of molecular weights and thickness of the shell. The nanocomposites were then loaded with an anti-tumor model drug (Usnic acid) and the static and dynamic release of the drug in PBS was evaluated.