Nanoengineering tools for biomedical applications: sustained drug-delivery and vascular biophysics

Nanotechnology is a rapidly-expanding field dealing with the control, manipulation and understanding of matter at scale ranging approximately between 1 and 1000 nanometers. Nanomaterial is any object with a characteristic size falling within the above range, either naturally-occurring or man-made, presenting features that are uniquely related to its size. Nanotechnology has led to the development of a wide ensemble of structures and devices with relevant applications in a variety of fields including automotive, robotics, manufacturing, electronics, communication and medicine. Nanomedicine represents the branch of nanotechnology with immediate application to the biomedical sciences and healthcare system. It permits to finely tune the interaction between a man-created device and a biological system, for applications such as imaging, drug delivery and thermal ablation. Particularly interesting are the nanochannel systems and engineered nanoparticles, because of their versatility and tunability. The nanochannel systems are micro-fabricated molecular sieves bearing nano-sized channels with adjustable geometry and can be used for drug delivery and discovery, clinical diagnostics, biomolecules separation and analysis, cell immunoisolation and analysis. The engineered nanoparticles are nano-sized particles with adjustable composing material, particle shape and size and surface conjugation and can be used for therapeutic, diagnostic and imaging purposes. This thesis presents two engineering tools falling in the realm of nanomedicine. The first tool is a nanochannel membrane for the long-term controlled release of drug molecules; the second tool is the atomic force microscopy here used for mechano-biological cell characterization. During the first year of my visiting scholarship in the Texas Medical Center, working in the laboratory of Dr. Mauro Ferrari, my research activity has focused on the in-vivo and in-lab analysis of drug and small molecules release through a nanochanneled system. After that, in the last one year of activity, I turned my attention to the mechano-biological characterization of endothelial cells exposed to different external stimuli, such as pro-inflammatory cytokines and gold nanoparticles. In this case, the cell properties have been assessed using several techniques, including conventional epifluorescent, confocal, darkfield and atomic force microscopy. In the first project, the release of different molecules –drugs (interferon alpha, IFN-alpha;) and a molecular probe (dextran)– was measured through a nano-channeled delivery system (nDS), obtained by photolithographic microfabrication. The IFN-alpha; diffusion was studied both in-vivo (healthy rats) as well as in-lab (diffusion devices) condition; the IFN concentration was measured, in blood and in proper solvent respectively, by ELISA immunoassay. The in-vivo studies have shown a linear and sustained release, which would be of interest from the clinical point of view. The release of a fluorescent dextran, evaluated only in-lab condition and measured by spectrophotometer, demonstrated a constant release for about 60 days, thus confirming literature already demonstrating such diffusive behavior for similar nDS membranes with other molecules under certain conditions. This activity has led to a manuscript on the IFN diffusion that will be shortly submitted. In the second project, the mechano-biological response of endothelial cells to external stimuli was evaluated. Two different stimuli were considered: a well-established pro-inflammatory cytokine (TNF-alpha;); and two differently-sized spherical gold nano-particles (30 and 100 nm diameters). Upon stimulation, the mechanical properties such as cell stiffness and adhesion, cytoskeletal organization; and biological properties, such as cell inflammation and viability were assessed by atomic force microscopy (AFM), confocal microcopy, epifluorescent microscopy, darkfield microscopy, ELISA immunoassay, viability and proliferation testing. In particular, an AFM bearing a colloidal micrometric particle (colloidal probe) was used, which permits to probe the biomechanics of the cells with high sensitivity and specificity. Both external stimuli were shown to significantly affect cell response in terms of cell stiffness, adhesion and cytoskeletal organization. Stimulation with TNF-α led to cell stiffening, overall adhesion decreasing and actin cytoskeleton thickening; whereas stimulation with gold nanoparticles led to an overall decrease in cell stiffness and increase in non-functional actin filaments formation. These findings would suggest a bio-active role for these nano-particles when internalized by endothelial cells in sufficient amount. This activity has led to two manuscripts: one, already published, focusing on the TNF-alpha; stimulation; and one, in preparation, focusing on the gold nanoparticles stimulation.