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In this research activity, we studied the nano-biointeractions occurring between cell culture media and differently sized AuNPs, to determine how media composition influences the formation of protein/NPs complexes that, in turn, may affect the cellular response. To this purpose, we performed an accurate investigation of the bio-physicochemical properties of model spherical AuNPs of different sizes upon incubation with DMEM and RPMI, supplemented with the protein source FBS. We analyzed protein-NP interactions in solution over time by several spectroscopic techniques, finding that, while DMEM elicited the formation of a large time-dependent protein corona, RPMI showed different dynamics with a reduced protein coating. As a consequence, we detected remarkable differences in the cellular uptake of the NPs as well as in the cell viability, as a function of the nano-bioentities formed in the two media. These results may provide important information for the development of reliable protocols in in vitro studies.

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The interactions between biological systems and nanostructured materials are attracting increasing interest, due to the possibility to open up novel concepts for the design of smart nano-biomaterials that actively play a functional biological role. In this frame, the research activity focused on the response of neurons to gold surfaces with different levels of nanoroughness, finding out that they are capable to sense and actively respond to these nanotopography features, with a surprising sensitivity to variations of few nanometers. Moreover, by seeding SH-SY5Y cells onto micropatterned flat and nanorough gold surfaces, we demonstrated the possibility to realize substrates with cytophilic or cytophobic behavior, simply by fine tuning their surface topography at nanometer scale, inducing a clear self-alignment of neurons. These nanostructured substrates were also investigated to explore their use as suitable materials which can prevent bacterial colonization.

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The growing use of nanomaterials in commercial goods and as novel carriers for drug delivery is generating increasing questions about possible risks for human health and environment, due to the lack of an in-depth assessment of their potential toxicity. In this frame, we focused on the study of metrology of nanoparticles (characterization of their size, shape, surface chemistry, aggregation/agglomeration, formation of protein/NP complexes) in order to design standardized in vitro protocols to assess dose-dependence toxicity. Moreover, we investigated the in vivo effects of AuNPs and QDs with different sizes, surface coatings, and nanostructuration, on the model system Drosophila melanogaster upon ingestion. We observed that nanoparticles induce clear adverse effects (such as reduction of life span and genotoxicity) in treated organisms. The toxic effects were found to be dependent on size, surface coatings and nanoscale surface features. These results open up important questions related to the safe use of nanoparticles and suggest some experimental routes for the development of safe nanocarriers.

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We reviewed recent findings about the toxicity of gold nanoparticles (AuNPs) in in vitro and in vivo experiments. We showed that, beyond the wide variability of the experimental conditions (including physical/chemical AuNPs characteristics) and a slight discrepancy of some published results, as-synthesized (naked) AuNPs are significantly toxic both in vitro and in vivo, while appropriate coating may partially prevent their harmful effects. Image reports the quantitative analysis of in vitro and in vivo toxicity outcomes reported in reviewed articles, upon treatment with AuNPs (polymers or proteins conjugated AuNPs were referred to as coated NPs, while NPs stabilized with weakly bound or small ligands were named uncoated AuNPs).

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We are developing photochemical method to synthezie good quality metal nanoparticles of different sizes with potential for largescale synthesis. The approach is completely green chemical using simple natural molecules as stabilizing and photoreducing agents in an aqueous medium. Metal nanoparticles ranging from very small fluorescent nanodots to few tens of nanometers can be conveniently synthesized in large quantity and made into redispersible powders. Surface modification of metal nanoparticles in order to control their surface charge and thereby tailor their invasion into cells is also being carried out with a motive to study cellular interaction and possible bio imaging applications.

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This research activity deals with the synthesis and characterization of several NPs composed of different materials, shapes and surface coatings. The easy functionalization of the NPs surface with different biomolecules (proteins, enzymes, etc.) allows to a site-specific cellular uptake and targeting. Furthermore, we investigate the toxicity of such NPs in order to develop novel biomedical applications of the NPs as diagnostic and therapeutic agents.

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This research activity deals with the synthesis and characterization of SiO₂ NPs showing different sizes and surface properties. We study the in vitro biocompatibility of the SiO₂NPs through the evaluation of different parameters, such as: viability, membrane integrity and the generation of ROS. We evaluate the ability of SiO₂NPs to bind, transport and release DNA, so to act as gene delivery vectors. In the picture it is reported the tGFP silencing in green fluorescent HeLa by SiO₂ NPs

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A novel seed-mediated synthetic route to produce multibranched gold nanoparticles has been developed in the EHS laboratory, in which it is possible to precisely tune both their size and nanostructuration, while maintaining an accurate level of monodispersion. The nanoscale control of surface nanoroughness/branching, ranging from small bud-like features to elongated spikes, allows to obtain fine tuning of the nanoparticle optical properties, up to the red and near-IR region of the spectrum. Such anisotropic nanostructures were demonstrated to be excellent candidates for SERS applications, showing significantly higher signals with respect to the standard spherical nanoparticles Nanoscale, 3, 2227 (2011).

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The research activity deals with the synthesis and characterization of protein nanoparticles. Currently, we are producing BSA NPs which are: i) water soluble and stable over time (more than 6 months) ii) lyophilizable, iii) monodispersed in the range of 200 nm, iv) negatively charged and v) biocompatible. By applying simple functionalization chemistry such as click chemistry, several molecules are linked on the surface of BSA NPs and/or loaded as cargo (siRNS, labile molecules, etc.). Applications: cancer therapy. In the picture, we show the silencing of tGFP expression in fluorescent HeLa cell line upon transfection with pDNA/BSA NPs.

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Development of a lab-on-chip device for diagnostic and proteomic analyses (implementation of amplification reactions of nucleic acids, on-chip microarray technology, and development of miniaturized chips based on plastics and/or soft and biocompatible materials. In this area, we are also interested in developing soluble nanosensors based on NPs for diagnostics in biological fluids.

Ongoing Projects:
AIRC 2010-2011 (Associazione Italiana Ricerca Cancro) Translating innovation into colorectal cancer control; Unit Coordinator; Funded

References:
J. Anal Chem, 2011, 66 (5):528; Anal Biochem 2010, 397(1):53; Applied Physics Letters 2010, 96:113702; Microelectronic Engineering 2010, 87:747; Materials Letters 2010, 64:41; Biomedical microdevices 2009, 11(6):1289; Nanoscale Research Letters 2009, 4(10):1222; Langmuir 2008, 24 (23):13266.

Patents:
U.S. Pat. Appl. Publ., US 2010028898 (2010);

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We have developed different methods for fabricating patterns of metals on various planar substrates. We fabricate metallic and bi-metallic highly spatially controlled nanostructured substrates. We fabricated, by electron beam lithography (EBL), highly ordered gold nanopatterns on planar substrates with different shapes and dimensions. Such substrates were used for MEF applications. Furthermore, we use other approaches to precisely control the pattern of different metals at the micro- and nanoscale, along with their topology by combining lithographic techniques and wet chemistry. We show the ability to fabricate micro- and nanoscale patterns of different metals, with highly controlled surface roughness, onto a number of suitable substrates. Such substrates exhibit very efficient metal enhanced fluorescence in patterned regions and patterned hydrophobicity and hydrophilicity. Owing to their diverse features, these substrates can serve as good substrates for immunosensing, tissue engineering and single molecule detection based on SERS effect.