Attilio Marino

Piezoelectric Nanoparticle-Assisted Wireless Neuronal Stimulation.

Tetragonal barium titanate nanoparticles (BTNPs) have been exploited as nanotransducers owing to their piezoelectric properties, in order to provide indirect electrical stimulation to SH-SY5Y neuron-like cells. Following application of ultrasounds to cells treated with BTNPs, fluorescence imaging of ion dynamics revealed that the synergic stimulation is able to elicit a significant cellular response in terms of calcium and sodium fluxes; moreover, tests with appropriate blockers demonstrated that voltage-gated membrane channels are activated. The hypothesis of piezoelectric stimulation of neuron-like cells was supported by lack of cellular response in the presence of cubic nonpiezoelectric BTNPs, and further corroborated by a simple electroelastic model of a BTNP subjected to ultrasounds, according to which the generated voltage is compatible with the values required for the activation of voltage-sensitive channels.

Two-photon polymerization of sub-micrometric patterned surfaces: investigation of cell-substrate interactions and improved differentiation of neuron-like cells

Direct Laser Writing (DLW) is an innovative tool that allows the photofabrication of high resolution 3D structures, which can be successfully exploited for the study of the physical interactions between cells and substrates. In this work, we focused our attention on the topographical effects of submicrometric patterned surfaces fabricated via DLW on neuronal cell behavior. In particular, we designed, prepared, and characterized substrates based on aligned ridges for the promotion of axonal outgrowth and guidance. We demonstrated that both rat PC12 neuron-like cells and human SH-SY5Y derived neurons differentiate on parallel 2.5 μm spaced submicrometric ridges, being characterized by strongly aligned and significantly longer neurites with respect to those differentiated on flat control substrates, or on more spaced (5 and 10 μm) ridges. Furthermore, we detected an increased molecular differentiation toward neurons of the SH-SY5Y cells when grown on the submicrometric patterned substrates. Finally, we observed that the axons can exert forces able of bending the ridges, and we indirectly estimated the order of magnitude of these forces thanks to scanning probe techniques. Collectively, we showed as submicrometric structures fabricated by DLW can be used as a useful tool for the study of the axon mechanobiology.

Two-Photon Lithography of 3D Nanocomposite Piezoelectric Scaffolds for Cell Stimulation

In this letter, we report on the fabrication, the characterization, and the in vitro testing of structures suitable for cell culturing, prepared through two-photon polymerization of a nanocomposite resist. More in details, commercially available Ormocomp has been doped with piezoelectric barium titanate nanoparticles, and bioinspired 3D structures resembling trabeculae of sponge bone have been fabricated. After an extensive characterization, preliminary in vitro testing demonstrated that both the topographical and the piezoelectric cues of these scaffolds are able to enhance the differentiation process of human SaOS-2 cells.

Barium titanate nanoparticles and hypergravity stimulation improve differentiation of mesenchymal stem cells into osteoblasts

Background Enhancement of the osteogenic potential of mesenchymal stem cells (MSCs) is highly desirable in the field of bone regeneration. This paper proposes a new approach for the improvement of osteogenesis combining hypergravity with osteoinductive nanoparticles (NPs). Materials and methods In this study, we aimed to investigate the combined effects of hypergravity and barium titanate NPs (BTNPs) on the osteogenic differentiation of rat MSCs, and the hypergravity effects on NP internalization. To obtain the hypergravity condition, we used a large-diameter centrifuge in the presence of a BTNP-doped culture medium. We analyzed cell morphology and NP internalization with immunofluorescent staining and coherent anti-Stokes Raman scattering, respectively. Moreover, cell differentiation was evaluated both at the gene level with quantitative real-time reverse-transcription polymerase chain reaction and at the protein level with Western blotting. Results Following a 20 g treatment, we found alterations in cytoskeleton conformation, cellular shape and morphology, as well as a significant increment of expression of osteoblastic markers both at the gene and protein levels, jointly pointing to a substantial increment of NP uptake. Taken together, our findings suggest a synergistic effect of hypergravity and BTNPs in the enhancement of the osteogenic differentiation of MSCs. Conclusion The obtained results could become useful in the design of new approaches in bone-tissue engineering, as well as for in vitro drug-delivery strategies where an increment of nanocarrier internalization could result in a higher drug uptake by cell and/or tissue constructs.

P(VDF-TrFE)/BaTiO3 Nanoparticle Composite Films Mediate Piezoelectric Stimulation and Promote Differentiation of SH-SY5Y Neuroblastoma Cells.

Poly(vinylidene fluoride-trifluoroethylene, P(VDF-TrFE)) and P(VDF-TrFE)/barium titanate nanoparticle (BTNP) films are prepared and tested as substrates for neuronal stimulation through direct piezoelectric effect. Films are characterized in terms of surface, mechanical, and piezoelectric features before in vitro testing on SH-SY5Y cells. In particular, BTNPs significantly improve piezoelectric properties of the films (4.5-fold increased d31 ). Both kinds of films support good SH-SY5Y viability and differentiation. Ultrasound (US) stimulation is proven to elicit Ca(2+) transients and to enhance differentiation in cells grown on the piezoelectric substrates. For the first time in the literature, this study demonstrates the suitability of polymer/ceramic composite films and US for neuronal stimulation through direct piezoelectric effect.

Folate-grafted boron nitride nanotubes: Possible exploitation in cancer therapy

Boron nitride nanotubes (BNNTs) have generated considerable interest among the scientific community because of their unique physical and chemical properties. They present good chemical inertness, high thermal stability, and optimal resistance to oxidation, that make them ideal candidates for biomedical applications, in particular as nanovectors for drug, gene and protein delivery into the cells. In this study, BNNTs were prepared through a synthesis based on a chemical vapor deposition (CVD) method, and thereafter chemically functionalized with folic acid. The obtained nanostructures have been characterized by Fourier transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), thermogravimetric analysis (TGA), scanning electron microscopy (SEM), and transmission electron microscopy (TEM). The characterization showed efficiently functionalized BNNTs of length of about 1 μm. Furthermore, confocal laser microscopy demonstrated that our nanotubes can be fluorescently-traced under appropriate excitation. Thanks to this property, it has been possible to investigate their internalization by HeLa cells through confocal microscopy, demonstrating that the BNNT up-take clearly increases after the functionalization with folate, a result confirmed by inductively coupled plasma (ICP) assessment of boron content inside the treated cell cultures.

Gold Nanoshell-Mediated Remote Myotube Activation

Mild heat stimulation of muscle cells within the physiological range represents an intriguing approach for the modulation of their functions. In this work, photothermal conversion was exploited to remotely stimulate striated muscle cells by using gold nanoshells (NSs) in combination with near-infrared (NIR) radiation. Temperature increments of approximately 5 °C were recorded by using an intracellular fluorescent molecular thermometer and were demonstrated to efficiently induce myotube contraction. The mechanism at the base of this phenomenon was thoroughly investigated and was observed to be a Ca2+-independent event directly involving actin-myosin interactions. Finally, chronic remote photothermal stimulations significantly increased the mRNA transcription of genes encoding heat shock proteins and sirtuin 1, a protein which in turn can induce mitochondrial biogenesis. Overall, we provide evidence that remote NIR + NS muscle excitation represents an effective wireless stimulation technique with great potential in the fields of muscle tissue engineering, regenerative medicine, and bionics.

Gelatin/nanoceria nanocomposite fibers as antioxidant scaffolds for neuronal regeneration

BACKGROUND The design of efficient nerve conduits able to sustain the axonal outgrowth and its guidance towards appropriate targets is of paramount importance in nerve tissue engineering. METHODS In this work, we propose the preparation of highly aligned nanocomposite fibers of gelatin/cerium oxide nanoparticles (nanoceria), prepared by electrospinning. Nanoceria are powerful self-regenerative antioxidant nanomaterials, that behave as strong reactive oxygen species scavengers, and among various beneficial effects, they have been proven to inhibit the cell senescence and to promote the neurite sprouting. RESULTS After a detailed characterization of the developed substrates, they have been tested on neuron-like SH-SY5Y cells, demonstrating strong antioxidant properties and beneficial multi-cue effects in terms of neurite development and alignment. CONCLUSIONS Obtained findings suggest efficiency of the proposed substrates in providing combined topographical stimuli and antioxidant effects to cultured cells. GENERAL SIGNIFICANCE Proposed nanocomposite scaffolds represent a promising approach for nerve tissue engineering and regenerative medicine.

Evaluation of the effects of boron nitride nanotubes functionalized with gum arabic on the differentiation of rat mesenchymal stem cells

The use of nanostructured materials in biomedical applications has been widely investigated, in particular with the aim of using these materials to selectively carry drugs or molecules of interest to a target tissue or organ. Boron nitride nanotubes (BNNTs) functionalized with specific moieties have been demonstrated to be useful candidates for the targeted delivery of proteins, drugs and nucleic acids. We evaluated the influence of BNNTs functionalized with gum arabic on the differentiation of mesenchymal stem cells. The nanotubes were characterized by transmission electron microscopy, scanning electron microscopy, Fourier transform infrared spectrometry, Raman spectrometry and dynamic light scattering. In vitro assays were performed on mesenchymal stem cells to evaluate the cytocompatibility of the functionalized BNNTs in terms of cell viability and metabolic activity. The interactions of the cells with the nanoparticles were further investigated by analyzing the conformation of the cytoskeleton. The differentiation of the mesenchymal stem cells into adipocytes and osteocytes after treatments with safe doses of BNNTs was assessed at both the gene and phenotype levels.

Piezoelectric Effects of Materials on Bio-Interfaces

Electrical stimulation of cells and tissues is an important approach of interaction with living matter, which has been traditionally exploited in the clinical practice for a wide range of pathological conditions, in particular, related to excitable tissues. Standard methods of stimulation are, however, often invasive, being based on electrodes and wires used to carry current to the intended site. The possibility to achieve an indirect electrical stimulation, by means of piezoelectric materials, is therefore of outstanding interest for all the biomedical research, and it emerged in the latest decade as a most promising tool in many bioapplications. In this paper, we summarize the most recent achievements obtained by our group and by others in the exploitation of piezoelectric nanoparticles and nanocomposites for cell stimulation, describing the important implications that these studies present in nanomedicine and tissue engineering. A particular attention will be also dedicated to the physical modeling, which can be extremely useful in the description of the complex mechanisms involved in the mechanical/electrical transduction, yet also to gain new insights at the base of the observed phenomena.