NANOBIOTECHNOLOGY

I. Introductory lessons that summarize the general features of nanoassembled systems; these lessons are meant to go over the main contents of the course "Nanosystems", for the benefit of those students who followed it; at the same time, they are meant to provide a basis for those student who do not have it. Outline of the essential features of nano-structured systems. The ideal nanostructure: components. Modified "natural" nanostructures (bacterial Outer Membrane Vescicles, viruses). Engineered nanoparticles: inorganic (silica, gold), organic (nanoformulations, polymers), liposomes and lipidic nanoparticles, quantum dots. Derivatization with small organic molecules (conjugation, orthogonal bioconjugation), with proteins or antibodies for specific cell targeting.

II. Lectures on nano-biomedicine and nanotoxicology. Physio-structural features of living organisms that come primarily into play in the interaction with nanomaterials.
Blood circulation, endothelial cells, renal filter. Reticuloendothelial system (RES): tissue-resident macrophages. Professional phagocytes: PMN, monocytes-macrophages, APCs. Accessibility to tissues and systems: physiological and pathological endothelial permeability (in chronic inflammation, and neoplasms); Permeabilisation Retention Effect (lymphatic system); "Shrines": blood-brain barrier: structure and its alteration. Cellular and humoral responses to nano-materials, toxicology and pharmacokinetic aspects. The chemical basis of the interaction between nanomaterials and biomolecules: multivalency and cooperativity. Acute cytotoxic cell damage. Toxic mechanisms, principles, measurements. Current knowledge on the toxicity of inorganic (silica, gold) and organic (microgels, liposomes, nanotubes, polymers) nanostructures. Uptake-clearance, endocytosis and phagocytosis. Opsonization: plasma opsonins. Complement. Concept of protein crown. Concept of stealth property (or "invisibility") of a nano-structure. PEGylation. Proinflammatory, pro-immune, pro-coagulant activities: cytokines induction, radicals production, leukocyte and endothelial activation. Complement and coagulation cascades induced by macroscopic or nanoscopic bio-materials. Immune reaction. In vitro measurements. Biodegradation and elimination from the body (kidney, bile).

III. Bio-active (transported) portion and applications: drugs, immunostimulants, DNA. Direct action of the nanomaterial, photoactivation, magnetic field activation. Applications: fluorescent biomarking of tissues and cells, in vivo imaging, diagnosis. Drug and gene delivery. Vaccines. Immunological adjuvants. Detection of pathogens. Detection of proteins. Probing the structure of DNA. Tissue engineering.
Hyperthermal therapies. Separation and purification of biological molecules and cells. Contrast agents in magnetic resonance imaging (MRI). Phagokinetic studies.

IV. Laboratory. The practical part will be introduced by preparatory lectures. It will consist of the synthesis of nanosystems, among which will be nanoparticles (both organic and inorganic/metallic) coated with organic (charged) ligands; liposomes (some fluorophoric molecules will be incapsulated and released by appropriate stimuli); hydrogels based on amino acids and peptides. These nanosystems will be characterized using spectroscopic techniques, such as UV-vis, fluorescence, and dinamic light scattering. Next, the student will test the biocompatibility of the nanosystems produced in biological a-cellular (plasma) or cellular (stabilized human cell lines) models. Examples of possible characterization are: blood coagulation tests, complement activation, citotoxicity, cellular uptake.