Identificazione di bersagli terapeutici e realizzazione di tecnologie innovative in oncologia ortopedica

Controlled delivery of anticancer drugs through osteotropic nanoparticles (NP) is a novel approach for the adjuvant therapy of osteolytic bone metastases. Doxorubicin (DXR) is widely used in chemotherapy, although its activity is restricted by dose-dependent cardiotoxicity and marrow toxicity. However, its efficacy can be improved when specific targeting at the tumor site is obtained. The aim of this study was to obtain osteotropic biodegradable NP by nanoprecipitation of a copolymer between poly(D,L-lactide-co-glycolide) (PLGA) and an osteotropic bisphosphonate, sodium alendronate (ALE). NP were subsequently characterised for their chemical-physical properties, biocompatibility, and the ability to inhibit osteoclast-mediated bone resorption, and then loaded with DXR. The effectiveness of NP-loaded DXR was investigated through in vitro and in vivo experiments, and compared to that of free DXR. For the in vitro analysis, six human cell lines were used as a representative panel of bone tumors, including breast and renal adenocarcinoma, osteosarcoma and neuroblastoma. The in vitro uptake and the inhibition of tumor cell proliferation were verified. To analyse the in vivo activity of NP-loaded DXR, osteolytic bone metastases were induced through the intratibial inoculation in BALB/c-nu/nu mice of a human breast cancer cell line, followed by the intraperitoneal administration of the free or NP-loaded DXR. In vitro, aAll of the cell lines were able to uptake both free and NP-loaded drug, and their proliferation was inhibited up to 80% after incubation either with free or NP-loaded DXR. In addition, in vivo experiments showed that NP-loaded DXR were also able to reduce the incidence of bone metastases, not only in comparison with untreated mice, but also with free DXR-treated mice. In conclusion, this research demonstrated an improvement in the therapeutic effect of the antineoplastic drug DXR, when loaded to bone-targeted NP conjugated with ALE. Osteotropic PLGA-ALE NP are suitable to be loaded with DXR and offer as a valuable tool for a tissue specific treatment of skeletal metastases.

Biomimetic Scaffolds for the Controlled Release of Bioactive Molecules for Tissue Engineering Applications

The temporospatial controlled delivery of growth factors is crucial to trigger the desired healing mechanisms in target tissues. The uncontrolled release of growth factors has been demonstrated to cause severe side effects in its surrounding tissues. Thus, the first working hypothesis was to tune and optimize a newly developed multiscale delivery platform based on a nanostructured silicon particle core (pSi) and a poly (dl-lactide-co-glycolide) acid (PLGA) outer shell. In a murine subcutaneous model, the platform was demonstrated to be fully tunable for the temporal and spatial control release of the payload. Secondly, a multiscale approach was followed in a multicompartment collagen scaffold, to selectively integrate different sets of PLGA-pSi loaded with different reporter proteins. The spatial confinement of the microspheres allowed the release of the reporter proteins in each of the layers of the scaffold. Finally, the staged and zero-order release kinetics enabled the temporal biochemical patterning of the scaffold. The last step of this PhD project was to test if by fully embedding PLGA microspheres in a highly structured and fibrous collagen-based scaffold (camouflaging), it was possible to prevent their early detection and clearance by macrophages. It was further studied whether such a camouflaging strategy was efficient in reducing the production of key inflammatory molecules, while preserving the release kinetics of the payload of the PLGA microspheres. Results demonstrated that the camouflaging allowed for a 10-fold decrease in the number of PLGA microspheres internalized by macrophages, suggesting that the 3D scaffold operated by cloaking the PLGA microspheres. When the production of key inflammatory cytokines induced by the scaffold was assessed, macrophages' response to the PLGA microspheres-integrated scaffolds resulted in a response similar to that observed in the control (not functionalized scaffold) and the release kinetic of a reporter protein was preserved.

3D nanostructured microcarriers for cell therapy in regenerative medicine

Supercritical Emulsion Extraction technology (SEE-C) was proposed for the production of poly-lactic-co-glycolic acid microcarriers. SEE-C operating parameters as pressure, temperature and flow rate ratios were analyzed and the process performance was optimized in terms of size distribution and encapsulation efficiency. Microdevices loaded with bovine serum insulin were produced with different sizes (2 and 3 µm) or insulin charges (3 and 6 mg/g) and with an encapsulation efficiency of 60%. The microcarriers were characterized in terms of insulin release profile in two different media (PBS and DMEM) and the diffusion and degradation constants were also estimated by using a mathematical model. PLGA microdevices were also used in a cultivation of embryonic ventricular myoblasts (cell line H9c2 obtained from rat) in a FBS serum free medium to monitor cell viability and growth in dependence of insulin released. Good cell viability and growth were observed on 3 µm microdevices loaded with 3 mg/g of insulin. PLGA microspheres loaded with growth factors (GFs) were charged into alginate scaffold with human Mesenchimal Steam Cells (hMSC) for bone tissue engineering with the aim of monitoring the effect of the local release of these signals on cells differentiation. These “living” 3D scaffolds were incubated in a direct perfusion tubular bioreactor to enhance nutrient transport and exposing the cells to a given shear stress. Different GFs such as, h-VEGF, h-BMP2 and a mix of two (ratio 1:1) were loaded and alginate beads were recovered from dynamic (tubular perfusion system bioreactor) and static culture at different time points (1st, 7th, 21st days) for the analytical assays such as, live/dead; alkaline phosphatase; osteocalcin; osteopontin and Van Kossa Immunoassay. The immunoassay confirmed always a better cells differentiation in the bioreactor with respect to the static culture and revealed a great influence of the BMP-2 released in the scaffold on cell differentiation.

Computational insight into materials properties

The aim of the work was to explore the practical applicability of molecular dynamics at different length and time scales. From nanoparticles system over colloids and polymers to biological systems like membranes and finally living cells, a broad range of materials was considered from a theoretical standpoint. In this dissertation five chemistry-related problem are addressed by means of theoretical and computational methods. The main results can be outlined as follows. (1) A systematic study of the effect of the concentration, chain length, and charge of surfactants on fullerene aggregation is presented. The long-discussed problem of the location of C60 in micelles was addressed and fullerenes were found in the hydrophobic region of the micelles. (2) The interactions between graphene sheet of increasing size and phospholipid membrane are quantitatively investigated. (3) A model was proposed to study structure, stability, and dynamics of MoS2, a material well-known for its tribological properties. The telescopic movement of nested nanotubes and the sliding of MoS2 layers is simulated. (4) A mathematical model to gain understaning of the coupled diffusion-swelling process in poly(lactic-co-glycolic acid), PLGA, was proposed. (5) A soft matter cell model is developed to explore the interaction of living cell with artificial surfaces. The effect of the surface properties on the adhesion dynamics of cells are discussed.

Porous Polymeric Bioresorbable Scaffolds for Tissue Engineering

Tissue engineering is a discipline that aims at regenerating damaged biological tissues by using a cell-construct engineered in vitro made of cells grown into a porous 3D scaffold. The role of the scaffold is to guide cell growth and differentiation by acting as a bioresorbable temporary substrate that will be eventually replaced by new tissue produced by cells. As a matter or fact, the obtainment of a successful engineered tissue requires a multidisciplinary approach that must integrate the basic principles of biology, engineering and material science. The present Ph.D. thesis aimed at developing and characterizing innovative polymeric bioresorbable scaffolds made of hydrolysable polyesters. The potentialities of both commercial polyesters (i.e. poly-e-caprolactone, polylactide and some lactide copolymers) and of non-commercial polyesters (i.e. poly-w-pentadecalactone and some of its copolymers) were explored and discussed. Two techniques were employed to fabricate scaffolds: supercritical carbon dioxide (scCO2) foaming and electrospinning (ES). The former is a powerful technology that enables to produce 3D microporous foams by avoiding the use of solvents that can be toxic to mammalian cells. The scCO2 process, which is commonly applied to amorphous polymers, was successfully modified to foam a highly crystalline poly(w-pentadecalactone-co-e-caprolactone) copolymer and the effect of process parameters on scaffold morphology and thermo-mechanical properties was investigated. In the course of the present research activity, sub-micrometric fibrous non-woven meshes were produced using ES technology. Electrospun materials are considered highly promising scaffolds because they resemble the 3D organization of native extra cellular matrix. A careful control of process parameters allowed to fabricate defect-free fibres with diameters ranging from hundreds of nanometers to several microns, having either smooth or porous surface. Moreover, versatility of ES technology enabled to produce electrospun scaffolds from different polyesters as well as “composite” non-woven meshes by concomitantly electrospinning different fibres in terms of both fibre morphology and polymer material. The 3D-architecture of the electrospun scaffolds fabricated in this research was controlled in terms of mutual fibre orientation by properly modifying the instrumental apparatus. This aspect is particularly interesting since the micro/nano-architecture of the scaffold is known to affect cell behaviour. Since last generation scaffolds are expected to induce specific cell response, the present research activity also explored the possibility to produce electrospun scaffolds bioactive towards cells. Bio-functionalized substrates were obtained by loading polymer fibres with growth factors (i.e. biomolecules that elicit specific cell behaviour) and it was demonstrated that, despite the high voltages applied during electrospinning, the growth factor retains its biological activity once released from the fibres upon contact with cell culture medium. A second fuctionalization approach aiming, at a final stage, at controlling cell adhesion on electrospun scaffolds, consisted in covering fibre surface with highly hydrophilic polymer brushes of glycerol monomethacrylate synthesized by Atom Transfer Radical Polymerization. Future investigations are going to exploit the hydroxyl groups of the polymer brushes for functionalizing the fibre surface with desired biomolecules. Electrospun scaffolds were employed in cell culture experiments performed in collaboration with biochemical laboratories aimed at evaluating the biocompatibility of new electrospun polymers and at investigating the effect of fibre orientation on cell behaviour. Moreover, at a preliminary stage, electrospun scaffolds were also cultured with tumour mammalian cells for developing in vitro tumour models aimed at better understanding the role of natural ECM on tumour malignity in vivo.