Lorena Tedeschi

Aptamer-Mediated Codelivery of Doxorubicin and NF-κB Decoy Enhances Chemosensitivity of Pancreatic Tumor Cells

Aptamers able to bind efficiently cell-surface receptors differentially expressed in tumor and in healthy cells are emerging as powerful tools to perform targeted anticancer therapy. Here, we present a novel oligonucleotide chimera, composed by an RNA aptamer and a DNA decoy. Our assembly is able to (i) target tumor cells via an antitransferrin receptor RNA aptamer and (ii) perform selective codelivery of a chemotherapeutic drug (Doxorubicin) and of an inhibitor of a cell-survival factor, the nuclear factor κB decoy oligonucleotide. Both payloads are released under conditions found in endolysosomal compartments (low pH and reductive environment). Targeting and cytotoxicity of the oligonucleotidic chimera were assessed by confocal microscopy, cell viability, and Western blot analysis. These data indicated that the nuclear factor κB decoy does inhibit nuclear factor κB activity and ultimately leads to an increased therapeutic efficacy of Doxorubicin selectively in tumor cells.

A gel-free approach in vascular smooth muscle cell proteome: perspectives for a better insight into activation

BackgroundThe use of chromatography coupled with mass spectrometry (MS) analysis is a powerful approach to identify proteins, owing to its capacity to fractionate molecules according to different chemical features. The first protein expression map of vascular smooth muscle cells (VSMC) was published in 2001 and since then other papers have been produced. The most detailed two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) map was presented by Mayr et al who identified 235 proteins, corresponding to the 154 most abundant unique proteins in mouse aortic VSMC. A chromatographic approach aimed at fractionating the VSMC proteome has never been used before.ResultsThis paper describes a strategy for the study of the VSMC proteome. Our approach was based on pre-fractionation with ion exchange chromatography coupled with matrix assisted laser desorption-time of flight mass spectrometry analysis assisted by a liquid chromatography (LC-MALDI-TOF/TOF). Ion exchange chromatography resulted in a good strategy designed to simplify the complexity of the cellular extract and to identify a large number of proteins. Selectivity based on the ion-exchange chemical features was adequate if evaluated on the basis of protein pI. The LC-MALDI approach proved to be highly reproducible and sensitive since we were able to identify up to 815 proteins with a concentration dynamic range of 7 orders of magnitude.ConclusionsIn our opinion, the large number of identified proteins and the promising quantitative reproducibility made this approach a powerful method to analyze complex protein mixtures in a high throughput way and to obtain statistical data for the discovery of key factors involved in VSMC activation and to analyze a label-free differential protein expression.

Innovative Erythrocyte-based Carriers for Gene Delivery in Porcine Vascular Smooth Muscle Cells: Basis for Local Therapy to Prevent Restenosis

Vascular restenosis is affecting 30-40% of patients treated by percutaneous coronary angioplasty (PTCA). The advent of stenting reduced but not abolished restenosis. The introduction of drug eluting stent (DES) further reduced restenosis, but impaired endothelization exposed to intracoronary thrombosis as late adverse event. It is widely accepted that the endothelial denudation and coronary wall damages expose Vascular Smooth Muscle Cells (VSMC) to multiple growth factors and plasma circulating agents thus activating migration and proliferative pathways leading to restenosis. Among the major players of this processes, phosphorylated Elk-1, forming the Elk-1/SRF transcription complex, controls the expression of a different set of genes responsible for cell proliferation. Therefore, it is feasible that gene-specific oligonucleotide therapy targeting VSMC migration and proliferation genes can be a promising therapeutic approach. While a plethora of vehicles is suitably working in static in vitro cultures, methods for in vivo delivery of oligonucleotides are still under investigation. Recently, we have patented a novel erythrocyte-based drug delivery system with high capability to fuse with targeted cells thus improving drug bioavailability at the site of action. Here, the potential of these engineered porcine erythrocytes to deliver a synthetic DNA Elk-1 decoy inside syngenic porcine VSMC was tested. The results of this study indicate that Elk-1 decoy is actually able to inhibit cell proliferation and migration of VSMC. Our data also suggest that erythrocyte-based carriers are more efficient in delivering these oligonucleotides in comparison to conventional vehicles. As a consequence, a lower dose of Elk-1 decoy, delivered by engineered erythrocytes, was sufficient to inhibit cell growth and migration. This approach represents the translational step to reach in vivo experiments in pigs after PTCA and/or stent implantation where oligonucleotide drugs will be site-specific administered by using erythrocyte-based carriers to prevent restenosis.

Optical PMMA Chip Suitable for Multianalyte Detection

A new optical platform for the interrogation of a polymethylmethacrylate (PMMA) multichannel array for chemical and biochemical parameters is described. It consists of a plastic chip formed by two pieces of PMMA properly shaped in order to obtain different microchannels, 500 mum in width and 400 mum in height. A fluorescent sensing layer is immobilized on the internal wall of the microchannel. The emitted light travels along the thickness of the upper PMMA piece and it is detected with an optical fiber connected with a spectrum analyzer. The anisotropy of the fluorescence emitted by the fluorophore immobilized at the liquid/plastic interface gives rise to preferential directions of the emitted fluorescence. The collection of the fluorescence at an angle of 50deg implies an amplification of roughly one order of magnitude in the ratio between the fluorescence signal and the scattered light. The optical platform was further characterized as a pH sensor and as potential chip for immunoassay. In the first case, the fluorescein dye is directly immobilized onto the internal wall of the channel and the fluorescence changes are measured as a function of the pH of the flowing sample. In the second case, a direct antigen-antibody interaction is carried out. The mouse-IgG is covalently immobilized onto the internal wall of the channel and the Cy5-labeled anti-mouse IgG is used for the specific interaction.

Ribozyme-mediated gene knock down strategy to dissect the consequences of PDGF stimulation in vascular smooth muscle cells

BackgroundVascular Smooth Muscle Cells (VSMCs), due to their plasticity and ability to shift from a physiological contractile-quiescent phenotype to a pathological proliferating-activated status, play a central role in the onset and progression of atherosclerosis and cardiovascular diseases. PDGF-BB, among a series of cytokines and growth factors, has been identified as the critical factor in this phenotypic switch. In order to obtain new insights on the molecular effects triggered by PDGF-BB, a hammerhead ribozyme targeting the membrane receptor PDGFR-β was applied to inhibit PDGF pathway in porcine VSMCs.FindingsRibozymes, loaded on a cationic polymer-based vehicle, were delivered into cultured VSMCs. A significant impairment of the activation mechanisms triggered by PDGF-BB was demonstrated since cell migration decreased after treatments. In order to functionally validate the effects of PDGFR-β partial knock down we focused on the phosphorylation status of two proteins, protein disulfide isomerase-A3 (PDI-A3) and heat shock protein-60 (HSP-60), previously identified as indicative of VSMC phenotypic switch after PDGF-BB stimulation. Interestingly, while PDI-A3 phosphorylation was counteracted by the ribozyme administration indicating that PDI-A3 is a factor downstream the receptor signalling cascade, the HSP-60 phosphorylation status was greatly increased by the ribozyme administration.ConclusionThese contradictory observations suggested that PDGF-BB might trigger different parallel pathways that could be modulated by alternative isoforms of the receptors for the growth factor. In conclusion the knock down strategy here described enables to discriminate between two tightly intermingled pathways. Moreover it opens new attractive perspectives in functional investigations where combined gene knock down and proteomic technologies would allow the identification of key factors and pathways involved in VSMC-linked pathological disorders.

Label-Free Detection of Specific RNA Sequences by a DNA-Based CMOS BioMEMS

In this work we propose a resonant mass sensor based on a CMOS-compatible MEMS bulk technology, targeted at the label-free, selective detection of biomolecules. Both the MEMS fabrication phase and the bioactivation protocol were designed to ensure functionality of on-chip test electronic circuitry. Specifically, the bioactivation steps were performed with single drops of the reagents on the active part of the chip to minimize impact on the electronics and package. The CMOS compatibility of the final device is demonstrated by simultaneous operation of the MEMS resonator and the test electronics. The resonator mass sensitivity, determined by resonator loading with gold nanoparticles, compares favorably with those of QCMs and other MEMS resonant mass sensors. To validate the device operation as a biosensor, synthetic oligonucleotide sequences designed to bind to a specific human mRNA (involved in the synthesis of human methylguanine-DNA methyltransferase, a DNA repair protein) were used as probes and covalently bound to the resonator surface. The resonance frequency shift of different sensors at the same concentration of the analyte confirms the inverse dependence of the sensitivity on the mass of the resonator.

FRET based biosensor for detection of active NF-kB

The Nuclear Factor kB is a transcription factor, ubiquitously expressed, involved in the regulation of a large number of genes and in a variety of human disease including inflammation, asthma, atherosclerosis, AIDS, septic shock, arthritis and cancer. The critical need for a simple and direct method to evaluate the quantity of active NF-kB in a biological sample can be addressed using a suitable and reusable biosensor. For this purpose, a novel method, using fluorescence resonance energy transfer (FRET), to detect the active form of NF-kB binding a specific DNA sequence has been developed. A single-stranded DNA (ssDNA) with auto-complementary sequence has been properly designed and synthesized. In order to evaluate FRET due to the DNA/protein binding interaction taking place between double-stranded DNA (dsDNA) immobilized in a capillary wall and NF-kB proteins, a highly sensitive FRET-based biosensor system developed in our laboratory was used. Preliminary results show that our system was capable of detecting the active form of NF-kB protein with a detection efficiency of about 90% and that the system has a good regenerability.

Lab-on-glass system for DNA treatments

This paper presents the fabrication and testing of a lab-on-chip system suitable for treatment of DNA. It includes two main modules: a system-on-glass (SoG) and a disposable microfuidic chip. The SoG integrates, on the same glass substrate, thin film metal heaters and amorphous silicon temperature sensors to achieve a uniform temperature distribution (within ±1°C) in the heated area. Two polydimethylsiloxane microfluidic chips have been developed: a PCR-Chip for DNA amplification and a dsDNA-Chip for separation and selective isolation of a ssDNA from a dsDNA. The proposed system aims therefore to develop compact, low-cost devices that can implement multiple functions in biochemical procedures. In particular, the tested bioanalytical procedures are well suited for carrying-out an on-chip SELEX process, a combinatorial chemistry technique for the selection of aptamers.

An optical platform based on fluorescence anisotropy for C — reactive protein assay

A novel optical platform for point of care testing application was developed. The core of the platform is a miniaturised polymethylmetacrylate (PMMA) chip through which the analysed sample flows. Thanks to the fluorescence anisotropy exhibited by any dipole emitting at a distance from a medium interface of the order of the emitted wavelength, a large fraction of the fluorescence emitted by the sensing layer immobilized onto the internal flow-channel travels along the thickness of the PMMA up to its end-face where it is collected by a plastic optical fibre connected to a spectrum analyser. The potentiality of the optical chip was investigated with a sandwich assay for the C-reactive protein, one of the markers for inflammatory diseases.

A compact optical system for the interrogation of microcantilevers

The present paper is concerned with the development of an optical system capable of interrogating an array of silicon microcantilevers. The use of a laser line-generator and of a CCD camera allows the simultaneous interrogation of the eight cantilevers of the chip without movable parts. The resolution achieved in terms of displacement of each cantilever end is better than 7.5 nm. As an example of the operation, the system has been characterized as a refractometer by filling the cell with liquids having different index of refraction.