Lisa Vaccari

Sharp beveled tip hollow microneedle arrays fabricated by LIGA and 3D soft lithography with polyvinyl alcohol

This paper describes a fabrication process of hollow microneedle arrays with a sharp beveled tip for transdermal drug delivery. A master is fabricated through a double deep x-ray lithography process. First, a polymethylmethacrylate (PMMA) sheet is exposed to produce single PMMA parts with a sawtooth profile. The tip angle of each tooth determines the final tip angle of the microneedles. The PMMA parts are assembled and glued on a conductive substrate and then exposed through a second x-ray mask containing an array of hollow triangles as absorbing structures. A metal layer is then electrodeposited around the needles in order to form the future base of the array. A polyvinyl alcohol (PVA) solution is cast on top of the master to form a negative mold of the microneedle array after a low temperature curing and peel-off steps. A liquid PMMA solution is cast on top of the PVA negative mold and after the full PMMA polymerization the PVA is dissolved in water. This fabrication method can be performed in a non-clean room environment and requires little instrumentation. It is therefore compatible with a low-cost mass-fabrication scheme.

Nanoporous surfaces as harvesting agents for mass spectrometric analysis of peptides in human plasma.

Silica-based nanoporous surfaces have been developed in order to capture low molecular weight peptides from human plasma. Harvested peptides were subjected to mass spectrometric analysis by using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) as a means of detecting and assessing the bound molecules. Peptide profiles consisting of about 70 peaks in the range 800-10,000 m/z were generated. The method could allow detection of small peptides at ng/mL concentration levels, either in standard solutions or in plasma. The same molecular cutoff effect was observed for mixtures of standard proteins and peptides incubated with silicon-based nanoporous surfaces.

Fabrication of Advanced Functional Devices Combining Soft Chemistry with X‐ray Lithography in One Step

Deep X-ray lithography combined with sol-gel techniques offers facile fabrication of controlled patterned films. Using sol-gel, different functional properties can be induced; deep X-ray lithography alters the functionality in the exposed regions. Miniaturized devices based on local property changes are easily fabricated: this technique requires no resist, enabling direct patterning of films in a one-step lithographic process.

Probiotics Can Induce Follicle Maturational Competence: The Danio rerio Case

ABSTRACT In the present study, the effects of the probiotic Lactobacillus rhamnosus IMC 501 on the acquisition of oocyte maturational competence was examined in zebrafish (Danio rerio). L. rhamnosus administration induced the responsiveness of incompetent follicles (stage IIIa) to 17,20-dihydroxy-4-pregnen-3-one and their in vitro maturation. Acquisition of competence by the stage IIIa follicles was further validated by changes of lhr, mprb, inhbaa (activin betaA1), tgfb1, and gdf9 gene expression, which have recently emerged as key regulators of oocyte acquisition of maturational competence, and pou5f1 gene expression, which in other models has been shown to govern the establishment of developmental competence of oocytes. In addition, a DNA microarray experiment was conducted using the same follicles, and with relative gene ontology (GO) data analysis, the molecular effects of probiotic administration emerged. Molecular analysis using PCR-DGGE (denaturing gradient gel electrophoresis) approach, providing information about only the most abundant bacterial members of the microbial community, revealed that the probiotic was able to populate the gastrointestinal tract and modulate the microbial communities, causing a clear shift in them and specifically enhancing the presence of the lactic acid bacteria Streptococcus thermophilus. At the same time, PCR-DGGE analysis revealed that the probiotic was not directly associated with the ovaries. Finally, the effects of probiotic treatment on zebrafish follicle development were also analyzed by FPA (focal plane array) Fourier transform-infrared (FT-IR) imaging, a technique that provides the overall biochemical composition of samples. Changes were found above all in stage IIIa follicles from probiotic-exposed females; the modifications, observed in protein secondary structures as well as in hydration and in bands related to phosphate moieties, allowed us to hypothesize that probiotics act at this follicle stage, affecting the maturation phase.

Infrared microspectroscopy of live cells in microfluidic devices (MD-IRMS): toward a powerful label-free cell-based assay.

Until nowadays most infrared microspectroscopy (IRMS) experiments on biological specimens (i.e., tissues or cells) have been routinely carried out on fixed or dried samples in order to circumvent water absorption problems. In this paper, we demonstrate the possibility to widen the range of in-vitro IRMS experiments to vibrational analysis of live cellular samples, thanks to the development of novel biocompatible IR-visible transparent microfluidic devices (MD). In order to highlight the biological relevance of IRMS in MD (MD-IRMS), we performed a systematic exploration of the biochemical alterations induced by different fixation protocols, ethanol 70% and formaldehyde solution 4%, as well as air-drying on U937 leukemic monocytes by comparing their IR vibrational features with the live U937 counterpart. Both fixation and air-drying procedures affected lipid composition and order as well as protein structure at a different extent while they both induced structural alterations in nucleic acids. Therefore, only IRMS of live cells can provide reliable information on both DNA and RNA structure and on their cellular dynamic. In summary, we show that MD-IRMS of live cells is feasible, reliable, and biologically relevant to be recognized as a label-free cell-based assay.

Primate cathelicidin orthologues display different structures and membrane interactions

The human cathelicidin LL-37 displays both direct antibacterial activities and the capacity to modulate host-cell activities. These depend on structural characteristics that are subject to positive selection for variation, as observed in a previous analysis of the CAMP gene (encoding LL-37) in primates. The altered balance between cationic and anionic residues in different primate orthologues affects intramolecular salt-bridging and influences the stability of the helical conformation and tendency to aggregate in solution of the peptide. In the present study, we have analysed the effects of these structural variations on membrane interactions for human LL-37, rhesus RL-37 and orang-utan LL-37, using several complementary biophysical and biochemical methods. CD and ATR (attenuated total reflection)-FTIR (Fourier-transform IR) spectroscopy on model membranes indicate that RL-37, which is monomeric and unstructured in bulk solution [F-form (free form)], and human LL-37, which is partly structured and probably aggregated [A-form (aggregated form)], bind biological membranes in different manners. RL-37 may insert more deeply into the lipid bilayer than LL-37, which remains aggregated. AFM (atomic force microscopy) performed on the same supported bilayer as used for ATR-FTIR measurements suggests a carpet-like mode of permeabilization for RL37 and formation of more defined worm-holes for LL-37. Comparison of data from the biological activity on bacterial cells with permeabilization of model membranes indicates that the structure/aggregation state also affects the trajectory of the peptides from bulk solution through the outer cell-wall layers to the membrane. The results of the present study suggest that F-form cathelicidin orthologues may have evolved to have primarily a direct antimicrobial defensive capacity, whereas the A-forms have somewhat sacrificed this to gain host-cell modulating functions.

Fabrication of 3D metallic photonic crystals by X-ray lithography

Photonic crystals (3D) represent one of the most important building blocks towards the achievement of a full optics communication technology. So far the largest interest has been attracted by two-dimensional photonic crystals because they are potentially more amenable to fabrication and much closer to application. Straightforward application of the photonic band gap concept is generally thought to require three-dimensional (3D) photonic crystals that, however, represent a challenge from a fabrication point of view. Recent works have shown that 3D metallic PC can be fabricated and that they can be advantageous in the low frequency region where the metals become almost completely reflectors. In this work we show the possibility to fabricate 3D PC structures by X-ray lithography. Gold and nickel 3D photonic crystals with threefold (Yablonovite) and fourfold rotation symmetry have been fabricated with a lattice parameter ranging from 1 µm down to 300 nm. The total thickness of the 3D PC is of the order of 10 µm, a value which should allow to achieve a complete bulk behavior. This is supported by variable angle reflectance measurements, which have shown clear indications for true 3D dimensionality of our samples.

LILIT beamline for soft and deep X-ray lithography at Elettra

In this paper we present the beamline for X-ray lithography installed at ELETTRA (Trieste, Italy). The peculiarity of the beamline design consists mainly in its wide lithographic window. This is achieved by combining high-pass filters (Beryllium windows or other suitable material filters) with low-pass filters (mirrors at increasing angles of incidence). The design allows to change the spectral range of interest continuously, from the soft (around 1.5 keV) where we can achieve the highest lithographic resolution, to hard X-ray region (higher than 10 keV) where sensitive materials of thickness of tens of microns can be exposed.

Artificial β-defensin based on a minimal defensin template

We have designed and chemically synthesized an artificial beta-defensin based on a minimal template derived from the comparative analysis of over 80 naturally occurring sequences. This molecule has the disulfide-bridged beta-sheet core structure of natural beta-defensins and shows a robust salt-sensitive antimicrobial activity against bacteria and yeast, as well as a chemotactic activity against immature dendritic cells. An SAR (structure-activity relationship) study using two truncated fragments or a Cys-->Ser point-mutated analogue, from which one or two of the three disulfide bridges were absent, indicated that altering the structure resulted in a different type of membrane interaction and a switch to different modes of action towards both microbial and host cells, and that covalent dimerization could favour antimicrobial activity. Comparison of the structural, aggregational and biological activities of the artificial defensin with those of three human beta-defensins and their primate orthologues provided useful information on how their mode of action may relate to specific structural features.

Glucose is a key driver for GLUT1-mediated nanoparticles internalization in breast cancer cells

The mesenchymal state in cancer is usually associated with poor prognosis due to the metastatic predisposition and the hyper-activated metabolism. Exploiting cell glucose metabolism we propose a new method to detect mesenchymal-like cancer cells. We demonstrate that the uptake of glucose-coated magnetic nanoparticles (MNPs) by mesenchymal-like cells remains constant when the glucose in the medium is increased from low (5.5 mM) to high (25 mM) concentration, while the MNPs uptake by epithelial-like cells is significantly reduced. These findings reveal that the glucose-shell of MNPs plays a major role in recognition of cells with high-metabolic activity. By selectively blocking the glucose transporter 1 channels we showed its involvement in the internalization process of glucose-coated MNPs. Our results suggest that glucose-coated MNPs can be used for metabolic-based assays aimed at detecting cancer cells and that can be used to selectively target cancer cells taking advantage, for instance, of the magnetic-thermotherapy.