Biotechnology and Nanomedicine

UNIMIB researchers have achieved particularly significant advancements in the applications of biotechnology and nanomedicine for theranostic of the diseases that affect the Central Nervous System (CNS). This approach makes the Athenaeum an outstanding reference Centre in Europe for this particular branch. Relevant results have been achieved thanks to several past an currently running grants devoted to design of nanoparticles (NPs) for treatment and diagnosis of CNS diseases.

BIOTECHNOLOGY

1. 1. Electrophysiology. Electrophysiology is crucial in the understanding of the electrical functioning of neurons in the central nervous system (CNS). Both, the single cell- and the slice-level approaches on samples coming from normal tissue and from diseased brains are central for the analysis of the pathophysiological mechanisms responsible for diseases and for the development of new specific drugs. The availability of 64- and 256-electrode multi-site recording apparatus (MEA) allows to test nanomedicine tools and pharmacological agents on living neuronal networks. This offers a way to study in vitro the effects on neuronal cell health, neurotransmission and neurodegenerative processes.
   2. Proteomics. Proteomics is devoted to assess protein pattern in human samples, with the aim to identify molecular markers of disease and to follow the outcome of pharmacological interventions of neurological diseases. Proteomics unit is accustomed with the main proteomics strategies: i) Quantitation by Peptides and Proteome Profiling, either label-free or label-based, using mono and bi-dimensional liquid chromatography-MS/MS; ii) Proteome/Peptidome identification and characterization of their modifications (e.g. phosphorylation, glycosilation, etc) by high-throughputshot-gun (bottom-up) and top-down approaches with a nanoLC-ESI-QqTOF and a nanoLC-ESI-ETD-MS/MS; iii) Proteome /peptidome profiling by MALDI_TOF and in-situ identification by MALDI-TOF/TOF.
   3. Structural analysis of amyloid proteins and aggregates. This topic is aiming to assess the molecular mechanisms of amyloid aggregation and disaggregation to design possible therapeutics. Techniques used are Fourier Transform Infrared Spectroscopy (FTIR), Circular Dichroism (CD),spectrofluorimetry, Atomic Force Microscopy (AFM), Electron Microscopy (EM).

NANOMEDICINE

1. Nanotechnologies for treatment and diagnosis of Alzheimer disease (AD). As concerning AD, the FP7 project “Nanoparticles for therapy and diagnosis of AD” (NAD), leaded by UNIMIB (coordinator: Prof. Masserini), involved 19 partners from 13 European Countries, published 52 scientific articles on the major journals of nanomedicine, was featured in 2 Editorials, and published 4 Patents.Moreover the project has been awarded amongst the best FP7 projects in 2014.
   NAD project was intended to systematically design NPs for AD therapy and diagnosis. The rationale followed for the design of NPs was to provide them with multi-task features, namely the ability: i) to cross the blood-brain barrier (BBB), ii) to bind beta-amyloid peptide (Aβ); iii) to disaggregate brain Aβ assemblies into small soluble species, facilitating their clearance from the brain. During the first part of the project, the Consortium has selected efficient ligands to synthesize multi-functionalized NPs carrying the above said features. Liposomes, polymeric and solid-lipid NPs have been functionalized with ligands for binding Aβ (phosphatidic acid, antibodies, curcumin derivatives) and for crossing the BBB (peptides derived from ApoE and from HIV, anti-Transferrin receptor antibodies). The effectiveness of NPs has subsequently been fine-tuned in vitro on a number of artificial and cellular models, always caring for their biocompatibility and physical stability features. All the in vitro experiments helped the Consortium to keep to a minimum the use of animals, although the effectiveness of NPs had to be eventually evaluated in rodent models of AD. Finally, as a proof of the correctness of NPs design, after a relatively short treatment (about 1 month), NPs injected in transgenic mouse models of AD showed the ability to reduce brain Aβ burden, either plaques or oligomeric aggregates, considered to be the most neurotoxic form. Noteworthy, besides these effects, the treatment with NPs was shown to ameliorate animal impaired memory.
   In conclusion, NAD Consortium has designed and produced multitask therapeutic nano-devices that are encouraging candidates for the therapy of Alzheimer’s disease.
2. Nanotechnology for CNS tumours. NPs can be used to target selective cancer cells resulting in the localization of the therapeutic activity or diagnostic probes. This sector is dedicated to the development of: i) novel hybrid nanoparticles consisting of a magnetic core, useful as MRI contrast agent, and an organic shell responsible for the targeting action. The activities include:NPs synthesis, characterization and functionalization with biomolecules; in vitro studies on cellular models (cells viability and internalization) and/or on tumor bearing mice; ii) gold NPs, both uniform-spherical and anisotropic or in-homogenous, can act as highly localized thermal loaders for direct hypethermia (possibly of tumors) and for indirect temperature assisted chemotherapy. Gold nanoparticle based devices have been used to reach highly sensitive biosensing of tumor markers and to develop constructs for temperature triggered drug release and extremely localized hyperthermic treatments. These can be used for direct hypethermia and for temperature enhanced chemotherapy. iii) The FRRB project “NEVERMIND, New frontiers of engineered nanovectors to improve treatment efficacy and safety in neurological disorders”, in which UNIMIB is involved as project partner (PI: Prof. F. Re), is an ambitious project that strives to couple state-of-art nanotechnologies with cutting edge advancements in neuroscience to develop innovative nanovectors that can reach the brain and selectively halt some detrimental processes occurring during Alzheimer’s disease and Glioblastoma. The involved hospitals have long standing experience in therapy and care of patients with these neurological disorders and their researchers have joined forces with the University partners to identify a common drug delivery strategy.
3. Nanotoxicity. The range of different types of nanoparticles and their biomedical applications is rapidly growing, creating a need to thoroughly examine the effects these particles have on biological entities. This topic has been partially covered by the project “Evaluation of toxicity of nanopraticles designed for therapy of neurodegenerative diseases” funded by
   Fondazione Banca del Monte di Lombardia to Prof. Re F. Moreover, industrial end environmental nanoparticles may enter the body and reach the CNS. For the correct evaluation of cell function and behavior in vivo, any effects of the nanoparticles on the cells in vitro must be completely ruled out. The assays must focus on (i) nanoparticle internalization, (ii) immediate cell toxicity, (iii) cell proliferation, (iv) cell morphology, (v) cell functionality and (vi) cell physiology.
4. Nanoparticles to contrast amyloid toxic effects. The researchers are designing Nanoparticles (NP) engineered to sequester amyloid in plasma. The results achieved until now allow its sequestration when it is present at high concentrations (e.g. exogenous Aβ added). In plasma from patients with natural overexpression of A(Down Syndrome patients) or in cerebrospinal fluid (CSF), multitasks NPs comes from NAD project are able to sequester 10% of total Aβ.
   Moreover, we have designed liposomes able to restore cell energetic activity in cultured fibroblasts after significant reduction of viability induced by exposure to Aβ. NPs are able to modify cell signalling pathways, preventing the toxicity induced by oligomeric Aβ, and to modulate the glutamate transport in an in vitro and ex-vivo cellular models of AD.
5. Tissue engineering. Glycans participate in neural cell migration, neurite outgrowth and synapse formation in both the developing and adult nervous system. Understanding their functions would improve the knowledge in the mechanisms that determine nervous system differentiation and regeneration and could lead to the use of glycans as therapeutic strategies for neurological disorders. For this reason we are studying the effects of native and glycosylated collagen (glc-collagen) on neuronal differentiation.
   In order to assess the effect of the “neoglycosylation” reaction on collagen structure, molecular dynamics simulations, AFM, water contact angle and FTIR have been performed. We used a neuroblastoma, dorsal root ganglion-derived cell line (F11). Sodium current densities showed the tendency to increase from petri dishes to collagen and to glc-collagen and significantly higher potassium current densities were calculated for cells plated on glc-collagen. Mean resting membrane potential was very depolarized in cells from the petri dishes but showed a trend to hyperpolarize in cells plated on collagen and was significantly more negative on glc-collagen. In petri dishes, the majority of the cells showed slow depolarisations which were not able to reach 0 mV, on the contrary, the fraction of cells able to generate action potentials was significantly higher on collagen and reached almost the totality on glc-collagen. Our data show for the first time F11 cells differentiated without the use of chemical and differentiating agents and suggest that glc-collagen is an efficient material to be employed for neuronal differentiation.
6. Biomimicry and bio-inspired nanoparticles. Exploiting biological molecules, organisms and cells, or taking inspiration from them, drug vector performances can be rapidly improved toward the perfect carrier. Following this direction, researchers have refined carrier properties, achieving significant results. For example the design and characterization of lipoproteins-mimicking particles allowed the possibility to better deliver drugs across the blood-brain barriers.
7. Biomodelling. The development of realistic in vitro blood-brain barrier (BBB) models that recapitulate the physiological and/or pathological parameters and molecular aspect of the neurovascular unit (NVU) is of fundamental importance not only in CNS drug discovery but also in translational research. UNIMIB provides a platform of different in vitro BBB models, starting from unicellular models to advanced multicellular models of functional BBB. UNIMIB also working on advanced 3D printing technologies for engineering BBB models, which use is now fast expanding among researchers. These advancements were allowed by the H2020 JPND project “NAB3, Development of novel multicellular in vitro models of Alzheimer disease-like BBB” (coordinator: Prof. F.Re).