Roberto Marotta

Multifunctional nanobeads based on quantum dots and magnetic nanoparticles: synthesis and cancer cell targeting and sorting.

Trifunctional polymer nanobeads are prepared by destabilization of a mixture of magnetic nanoparticles, quantum dots, and an amphiphilic polymer, followed by functionalization of the bead surface with folic acid molecules. The distribution of the nanoparticles within the nanobeads can be tuned using either acetonitrile or water as destabilizing solvent. The luminescence of the resulting beads can be tuned by varying the ratio of quantum dots per magnetic nanoparticles. The application of an external magnetic field (such as a small static magnet of 0.3 T) to the magnetic-fluorescent nanobeads allows the quantitative accumulation of the beads within a few hours depending on the total size of the beads. Furthermore, specific targeting of cancer cells overexpressing folate receptors is achieved thanks to the folic acid decorating the surface of the as-synthesized nanobeads. Folate receptor mediated cellular uptake of the folic acid-functionalized nanobeads is proven via both confocal imaging and transmission electron microscopy characterization. Cell sorting experiments performed with trifunctional nanobeads show quantitative recovering of targeted cells even when they are present at low percentage (up to 1%).

Lipid Droplets: A New Player in Colorectal Cancer Stem Cells Unveiled by Spectroscopic Imaging

The cancer stem cell (CSC) model is describing tumors as a hierarchical organized system and CSCs are suggested to be responsible for cancer recurrence after therapy. The identification of specific markers of CSCs is therefore of paramount importance. Here, we show that high levels of lipid droplets (LDs) are a distinctive mark of CSCs in colorectal (CR) cancer. This increased lipid content was clearly revealed by label‐free Raman spectroscopy and it directly correlates with well‐accepted CR‐CSC markers as CD133 and Wnt pathway activity. By xenotransplantation experiments, we have finally demonstrated that CR‐CSCs overexpressing LDs retain most tumorigenic potential. A relevant conceptual advance in this work is the demonstration that a cellular organelle, the LD, is a signature of CSCs, in addition to molecular markers. A further functional characterization of LDs could lead soon to design new target therapies against CR‐CSCs. Stem Cells 2015;33:35–44

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.

Nanostructured superhydrophobic substrates trigger the development of 3D neuronal networks

The generation of 3D networks of primary neurons is a big challenge in neuroscience. Here, a novel method is presented for a 3D neuronal culture on superhydrophobic (SH) substrates. How nano-patterned SH devices stimulate neurons to build 3D networks is investigated. Scanning electron microscopy and confocal imaging show that soon after plating neurites adhere to the nanopatterned pillar sidewalls and they are subsequently pulled between pillars in a suspended position. These neurons display an enhanced survival rate compared to standard cultures and develop mature networks with physiological excitability. These findings underline the importance of using nanostructured SH surfaces for directing 3D neuronal growth, as well as for the design of biomaterials for neuronal regeneration.

Order versus Disorder: in vivo bone formation within osteoconductive scaffolds

In modern biomaterial design the generation of an environment mimicking some of the extracellular matrix features is envisaged to support molecular cross-talk between cells and scaffolds during tissue formation/remodeling. In bone substitutes chemical biomimesis has been particularly exploited; conversely, the relevance of pre-determined scaffold architecture for regenerated bone outputs is still unclear. Thus we aimed to demonstrate that a different organization of collagen fibers within newly formed bone under unloading conditions can be generated by differently architectured scaffolds. An ordered and confined geometry of hydroxyapatite foams concentrated collagen fibers within the pores, and triggered their self-assembly in a cholesteric-banded pattern, resulting in compact lamellar bone. Conversely, when progenitor cells were loaded onto nanofibrous collagen-based sponges, new collagen fibers were distributed in a nematic phase, resulting mostly in woven isotropic bone. Thus specific biomaterial design relevantly contributes to properly drive collagen fibers assembly to target bone regeneration.

Magnetic nanobeads decorated with silver nanoparticles as cytotoxic agents and photothermal probes.

A versatile method for decorating magnetic nanobeads (being composite materials from polymers and superparamagnetic nanoparticles) with silver nanoparticles of 3-6 nm size is presented. Control over the silver nanoparticle coverage at the nanobead surface is achieved by changing the reaction parameters. Moreover, the silver-decorated magnetic nanobeads (Ag-MNBs) are studied with respect to their in vitro cytotoxicity on two distinct tumour cell lineages under different parameters, i.e., dose, incubation time, magnetic field applied during the culturing, silver ion leakage, and colloidal stability. It is found that enhanced magnetically mediated cellular uptake and silver ion leakage from the Ag-MNBs surface are the main factors which affect the toxicity of the Ag-MNBs and allow the half-maximal inhibitory dose of silver to be reduced to only 32 μg mL(-1) . Furthermore, a synergic cytotoxicity induced by photo-activation of silver nanoparticles was also found.

Laser-assisted synthesis of Staphylococcus aureus protein-capped silicon quantum dots as bio-functional nanoprobes

A novel approach for nanofabricating protein-functionalized luminescent silicon nanoparticles based on infrared ultrafast laser ablation of silicon in an aqueous solution of Staphylococcus aureus protein A is reported. It is demonstrated that 8 nm protein A-capped silicon quantum dots with blue-green photoemissive properties are generated. The conjugation efficiency studies reveal a high percentage of protein A attached to the Si nanoparticle surface through physical adsorption phenomena during the in situ laser process. The biological functionality of laser-generated Staphylococcus aureus protein A-capped Si nanoparticles is investigated. Confocal and electron microscopy together with energy dispersive x-ray spectroscopy analysis show that these Si-based bio-nanostructures selectively bind IgG in the cells. Cell viability studies reveal that these protein A-capped Si nanoparticles are suitable for biological applications, demonstrating their potential as universal secondary biomarkers for in vivo applications such as long-term, real-time cell labeling, cell staining and controlled drug delivery.

Structure of human N-acylphosphatidylethanolamine-hydrolyzing phospholipase D: regulation of fatty acid ethanolamide biosynthesis by bile acids.

The fatty acid ethanolamides (FAEs) are lipid mediators present in all organisms and involved in highly conserved biological functions, such as innate immunity, energy balance, and stress control. They are produced from membrane N-acylphosphatidylethanolamines (NAPEs) and include agonists for G protein-coupled receptors (e.g., cannabinoid receptors) and nuclear receptors (e.g., PPAR-α). Here, we report the crystal structure of human NAPE-hydrolyzing phospholipase D (NAPE-PLD) at 2.65 Å resolution, a membrane enzyme that catalyzes FAE formation in mammals. NAPE-PLD forms homodimers partly separated by an internal ∼ 9-Å-wide channel and uniquely adapted to associate with phospholipids. A hydrophobic cavity provides an entryway for NAPE into the active site, where a binuclear Zn(2+) center orchestrates its hydrolysis. Bile acids bind with high affinity to selective pockets in this cavity, enhancing dimer assembly and enabling catalysis. These elements offer multiple targets for the design of small-molecule NAPE-PLD modulators with potential applications in inflammation and metabolic disorders.

Potent nematicidal activity of phthalaldehyde, salicylaldehyde, and cinnamic aldehyde against Meloidogyne incognita.

The nematicidal activity of selected aromatic aldehydes was tested against the root knot nematode Meloidogyne incognita. The most active aldehyde was phthalaldehyde (1) with an EC(50) value of 11 ± 6 mg/L followed by salicylaldehyde (2) and cinnamic aldehyde (3) with EC(50) values of 11 ± 1 and 12 ± 5 mg/L, respectively. On the other hand, structurally related aldehydes such as 2-methoxybenzaldehyde (21), 3,4-dimethoxybenzaldehyde, and vanillin (23) were not active at the concentration of 1000 mg/L. By liquid chromatography-mass spectrometry the reactivity of tested aldehydes against a synthetic peptide resembling the nematode cuticle was characterized. At the test concentration of 1 mM, the main adduct formation was observed for 3,4-dihydroxybenzaldehyde (22), 2-methoxybenzaldehyde (21), and 3,4-dimethoxybenzaldehyde. Considering that 2-methoxybenzaldehyde (21) and 3,4-dimethoxybenzaldehyde were not active against M. incognita in in vitro experiments led us to hypothesize a different mechanism of action rather than an effect on the external cuticle modification of nematodes. When the toxicity of the V-ATPase inhibitor pyocyanin (10) was tested against M. incognita J2 nematodes, an EC(50) at 24 h of 72 ± 25 mg/L was found. The redox-active compounds such as phthalaldehyde (1) and salicylaldehyde (2) may share a common mode of action inhibiting nematode V-ATPase enzyme. The results of this investigation reveal that aromatic redox-active aldehydes can be considered as potent nematicides, and further investigation is needed to completely clarify their mode of action.

Lamin B1 overexpression increases nuclear rigidity in autosomal dominant leukodystrophy fibroblasts

The architecture and structural mechanics of the cell nucleus are defined by the nuclear lamina, which is formed by A‐ and B‐type lamins. Recently, gene duplication and protein overexpression of lamin B1 (LB1) have been reported in pedigrees with autosomal dominant leukodystrophy (ADLD). However, how the overexpression of LB1 affects nuclear mechanics and function and how it may result in pathology remain unexplored. Here, we report that in primary human skin fibroblasts derived from ADLD patients, LB1, but not other lamins, is overexpressed at the nuclear lamina and specifically enhances nuclear stiffness. Transient transfection of LB1 in HEK293 and neuronal N2a cells mimics the mechanical phenotype of ADLD nuclei. Notably, in ADLD fibroblasts, reducing LB1 protein levels by shRNA knockdown restores elasticity values to those indistinguishable from control fibroblasts. Moreover, isolated nuclei from ADLD fibroblasts display a reduced nuclear ion channel open probability on voltage‐step application, suggesting that biophysical changes induced by LB1 overexpression may alter nuclear signaling cascades in somatic cells. Overall, the overexpression of LB1 in ADLD cells alters nuclear mechanics and is linked to changes in nuclear signaling, which could help explain the pathogenesis of this disease.—Ferrera, D., Canale, C., Marotta, R., Mazzaro, N., Gritti, M., Mazzanti, M., Capellari, S., Cortelli, P., Gasparini, L. Lamin B1 overexpression increases nuclear rigidity in autosomal dominant leukodystrophy fibroblasts. FASEB J. 28, 3906‐3918 (2014). www.fasebj.org