Carlo Filippeschi

Boron Nitride Nanotube-Mediated Stimulation of Cell Co-Culture on Micro-Engineered Hydrogels

In this paper, we describe the effects of the combination of topographical, mechanical, chemical and intracellular electrical stimuli on a co-culture of fibroblasts and skeletal muscle cells. The co-culture was anisotropically grown onto an engineered micro-grooved (10 µm-wide grooves) polyacrylamide substrate, showing a precisely tuned Young’s modulus (∼ 14 kPa) and a small thickness (∼ 12 µm). We enhanced the co-culture properties through intracellular stimulation produced by piezoelectric nanostructures (i.e., boron nitride nanotubes) activated by ultrasounds, thus exploiting the ability of boron nitride nanotubes to convert outer mechanical waves (such as ultrasounds) in intracellular electrical stimuli, by exploiting the direct piezoelectric effect. We demonstrated that nanotubes were internalized by muscle cells and localized in both early and late endosomes, while they were not internalized by the underneath fibroblast layer. Muscle cell differentiation benefited from the synergic combination of topographical, mechanical, chemical and nanoparticle-based stimuli, showing good myotube development and alignment towards a preferential direction, as well as high expression of genes encoding key proteins for muscle contraction (i.e., actin and myosin). We also clarified the possible role of fibroblasts in this process, highlighting their response to the above mentioned physical stimuli in terms of gene expression and cytokine production. Finally, calcium imaging-based experiments demonstrated a higher functionality of the stimulated co-cultures.

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 core – gold shell nanoparticles for hyperthermia treatments

The development of new tools and devices to aid in treating cancer is a hot topic in biomedical research. The practice of using heat (hyperthermia) to treat cancerous lesions has a long history dating back to ancient Greece. With deeper knowledge of the factors that cause cancer and the transmissive window of cells and tissues in the near-infrared region of the electromagnetic spectrum, hyperthermia applications have been able to incorporate the use of lasers. Photothermal therapy has been introduced as a selective and noninvasive treatment for cancer, in which exogenous photothermal agents are exploited to achieve the selective destruction of cancer cells. In this manuscript, we propose applications of barium titanate core–gold shell nanoparticles for hyperthermia treatment against cancer cells. We explored the effect of increasing concentrations of these nanoshells (0–100 μg/mL) on human neuroblastoma SH-SY5Y cells, testing the internalization and intrinsic toxicity and validating the hyperthermic functionality of the particles through near infrared (NIR) laser-induced thermoablation experiments. No significant changes were observed in cell viability up to nanoparticle concentrations of 50 μg/mL. Experiments upon stimulation with an NIR laser revealed the ability of the nanoshells to destroy human neuroblastoma cells. On the basis of these findings, barium titanate core–gold shell nanoparticles resulted in being suitable for hyperthermia treatment, and our results represent a promising first step for subsequent investigations on their applicability in clinical practice.

Nanostructured ultra-thin patches for ultrasound-modulated delivery of anti-restenotic drug

This work aims to demonstrate the possibility to fabricate ultra-thin polymeric films loaded with an anti-restenotic drug and capable of tunable drug release kinetics for the local treatment of restenosis. Vascular nanopatches are composed of a poly(lactic acid) supporting membrane (thickness: ~250 nm) on which 20 polyelectrolyte bilayers (overall thickness: ~70 nm) are alternatively deposited. The anti-restenotic drug is embedded in the middle of the polyelectrolyte structure, and released by diffusion mechanisms. Nanofilm fabrication procedure and detailed morphological characterization are reported here. Barium titanate nanoparticles (showing piezoelectric properties) are included in the polymeric support and their role is investigated in terms of influence on nanofilm morphology, drug release kinetics, and cell response. Results show an efficient drug release from the polyelectrolyte structure in phosphate-buffered saline, and a clear antiproliferative effect on human smooth muscle cells, which are responsible for restenosis. In addition, preliminary evidences of ultrasound-mediated modulation of drug release kinetics are reported, thus evaluating the influence of barium titanate nanoparticles on the release mechanism. Such data were integrated with quantitative piezoelectric and thermal measurements. These results open new avenues for a fine control of local therapies based on smart responsive materials.

Synthesizing tubular and trapezoidal shaped ZnO nanowires by an aqueous solution method

In this paper we present a novel bottom-up method suitable for developing vertically aligned hollow ZnO nanowires, ZnO nanotubes as well as longitudinally half ZnO nanowires. The procedures used for synthesizing such crystals combine chemical and electrochemical growth processes in aqueous solution at low temperatures (<90 °C), with a growth block process. A thin layer of gold, deposited when the nanowire growth process is at half way, has the crucial role of blocking the growth along the intended directions. The possibility of fabricating highly aligned crystals on a wide range of polymeric substrates, including flexible or transparent ones, is also illustrated. Our proposed methods hold potential for new developments in piezotronics and piezophotonics by allowing fabrication of nanodevices in the inner region of the hollow nanowires and nanotubes.

Three-Dimensional Soft Material Micropatterning via Direct Laser Lithography of Flexible Molds

Three-dimensionally micropatterned surfaces are attracting increasing interest in soft robotics owing to the potential of mimicking natural morphologies at the micro/nanoscale. We employ direct laser lithography to fabricate molds with complex three-dimensional (3D) micrometric features, in a positive photoresist on flexible substrates, to pattern curved macroscopic soft surfaces with shapes not achievable with standard methods (e.g., reentrant angles). We present several 3D intricate microstructures in poly(dimethylsiloxane) (PDMS) and show a soft cylinder patterned with 3D microstructures with one molding process. Finally, we deform PDMS-based 3D architectures and show soft microgripping capability, indicating the potentiality of this approach for future application in soft robotics.

Hierarchical surface patterning for triboelectric nanogenerators and sensors

We develop PDMS and PMMA triboelectric surfaces with micro/nanopatterning via simple and effective methods, and starting from Si substrates anisotropically etched with low KOH concentration solution. A hierarchical structure formed by micro-pyramidal structures with superficial nano-roughness, is developed in a 200 μm PDMS layer. In parallel, by means of a PDMS stamp and nanoimprinting technology, a 2 μm film of PMMA is nanopatterned, showing the feasibility of large scale production of such surfaces using a cost effective and easily repeatable process. We show SEM and AFM characterization of the negative (PDMS) and positive (PMMA) triboelectric surfaces and demonstrate how contact keys, integrating the fabricated soft materials, can be used to investigate the signals generated from the repetitive mechanical stimulation of its taxels.

Artificial System Inspired by Climbing Mechanism of Galium Aparine Fabricated via 3D Laser Lithography.

In this work, we present an artificial dry adhesive system inspired by the leaf-climbing mechanisms in Galium aparine. Among the different species of climbing plants, G. aparine shows a unique capability of adhesion to a wide range of roughness and stiffness objects, mainly via its leaves, using microscopic hooks for the physical interlocking. The adaxial (upper) and abaxial (lower) leaf surfaces differ significantly in attachment properties, which depend on the direction of the applied force (ratchet-like mechanism). In order to mimic this adhesive behavior, we designed artificial abaxial and adaxial leaf hooks by extracting the morphological parameters from the natural structures. We fabricated artificial hooks at different scales (1:1, 1:2, 1:4) using Direct Laser Lithography (DLL), a technique that allows a rapid prototyping of 3D microstructures. The adhesion of the artificial systems was tested on a polyester tissue substrate, obtaining adhesive forces comparable or higher than the natural counterpart. This biomimetic approach can open new opportunities to understand nature through artificial investigations and lead to several applications in the fields of robotics and space technology.

A 3D Real‐Scale, Biomimetic, and Biohybrid Model of the Blood‐Brain Barrier Fabricated through Two‐Photon Lithography

The investigation of the crossing of exogenous substances through the blood-brain barrier (BBB) is object of intensive research in biomedicine, and one of the main obstacles for reliable in vitro evaluations is represented by the difficulties at the base of developing realistic models of the barrier, which could resemble as most accurately as possible the in vivo environment. Here, for the first time, a 1:1 scale, biomimetic, and biohybrid BBB model is proposed. Microtubes inspired to the brain capillaries were fabricated through two-photon lithography and used as scaffolds for the co-culturing of endothelial-like bEnd.3 and U87 glioblastoma cells. The constructs show the maturation of tight junctions, good performances in terms of hindering dextran diffusion through the barrier, and a satisfactory trans-endothelial electrical resistance. Moreover, a mathematical model is developed, which assists in both the design of the 3D microfluidic chip and its characterization. Overall, these results show the effective formation of a bioinspired cellular barrier based on microtubes reproducing brain microcapillaries to scale. This system will be exploited as a realistic in vitro model for the investigation of BBB crossing of nanomaterials and drugs, envisaging therapeutic and diagnostic applications for several brain pathologies, including brain cancer.