Elisabetta Casarin

In Vivo Fate of Avidin-Nucleic Acid Nanoassemblies as Multifunctional Diagnostic Tools

This study describes the formulation optimization and body-cell distribution and clearance in mice of a dually fluorescent biodegradable poly avidin nanoassembly based on the novel Avidin-Nucleic-Acid-Nano-ASsembly (ANANAS) platform as a potential advancement of classic avidin/biotin-based targeted delivery. The nanoformulation circulates freely in the bloodstream; it is slowly captured by filter organs; it is efficiently cleared within 24-48 h, and it is poorly immunogenic. The system displays more favorable properties than its parent monomeric avidin and it is a promising tool for diagnostic purposes for future translational aims, for which free circulation in the bloodstream, safety, multifunctionality and high composition definition are all necessary requirements. In addition, the assembly shows a time-dependent cell penetration capability, suggesting it may also function as a NP-dependent drug delivery tool. The ease of preparation together with the possibility to fine-tune the surface composition makes it also an ideal candidate to understand if and how nanoparticle composition affects its localization.

Evaluation of cytotoxic activity of Schisandra chinensis lignans

Using exhaustive chromatographic separation we have isolated (-)-tigloyl-deangeloyl-gomisin F as a novel dibenzocyclooctadiene lignan from schisandra chinensis. With the help of HPLC, we further isolated (+)-schisandrin, (+)-deoxyschisandrin, (+)-γ-schisandrin, (-)-gomisin J, (+)-gomisin A, (-)-gomisin N, (-)-tigloyl-gomisin P, (-)-wuweizisu C, (-)-gomisin D, rubrisandrin A, (-)-gomisin G, (+)-gomisin K (3) and (-)-schisantherin C. A full NMR description of (-)-schisantherin C was carried out with the aim to confirm previous reports of its structure. Compounds isolated were identified on the basis of UV, IR, (1)H- and (13)C-NMR and MS. The cytotoxicity of lignans was tested for the BY-2 cell line alone and as a synergistic effect with the cytotoxic agent camptothecin. Lignans showed various toxicity and synergistic and antagonistic effects on camptothecin-induced cytotoxicity. Cytotoxicity against colon cancer cell line LoVo was also tested.

Molecular mechanisms of antiproliferative effects induced by Schisandra-derived dibenzocyclooctadiene lignans (+)-deoxyschisandrin and (-)-gomisin N in human tumour cell lines.

A different behavior of the two dibenzocyclooctadiene lignans (+)-deoxyschisandrin (1) and (-)-gomisin N (2), from Schisandra chinensis fruits, was observed against two human tumour cell lines, (2008 and LoVo). These lignans inhibited cell growth in a dose-dependent manner on both cell lines, but inducing different types of cell death. In particular, (+)-deoxyschisandrin (1) caused apoptosis in colon adenocarcinoma cells (LoVo) but not in ovarian adenocarcinoma cells (2008), while (-)-gomisin N (2) induced apoptosis on both the cell lines used. Mitochondrial-mediated pathway was not involved in apoptotic stimuli. Both compounds caused G2/M phase cell growth arrest correlated with tubulin polymerization.

A peptide nucleic acid label-free biosensor for Mycobacterium tuberculosis DNA detection via azimuthally controlled grating-coupled SPR

Grating coupled surface plasmon resonance phenomena under azimuthal control of incident light (φ ≠ 0° GC-SPR) have recently been exploited for the development of biosensing solutions with a sensitivity similar to that of classic prism-coupled SPR sensors, with the advantage of higher miniaturization potential. Here we combined the use of φ ≠ 0° GC-SPR with the use of peptide nucleic acid (PNA) probes and a strategy for maximizing the signal-to-noise ratio for the sensitive detection of Mycobacterium tuberculosis (MT) DNA. We focused our attention on the optimization of the PNA-based sensing layer by controlling the sensing surface composition with the PNA-based probe and a poly(ethylene oxide) (PEO)-based antifouling layer. We tested the sensor response first in the presence of complementary and non-complementary oligonucleotides, and then we applied our strategy for the detection of PCR amplified samples, using the fluorescence-based microarray technology as the control. With the φ ≠ 0° GC-SPR set-up adopted, a limit of detection (LOD 0.26 pM) more than one order of magnitude lower than that obtained by the fluorescence method (LOD 8.9 pM) was observed using a complementary oligonucleotide target. Also when PCR amplicons were analysed on SPR grating surfaces, lower DNA concentrations were detectable with the SPR readout as compared to the fluorescence one, and with an experimental protocol that does not include the need to use expensive fluorophore molecules. The whole approach, involving the sensor fabrication, the sensing surface control and DNA detection, has demonstrated that φ ≠ 0° GC-SPR is a good starting point for a sensitive, versatile and scalable biosensing technique that will be further investigated in future experiments.

Detection of a fluorescent-labeled avidin-nucleic acid nanoassembly by confocal laser endomicroscopy in the microvasculature of chronically inflamed intestinal mucosa

Inflammatory bowel diseases are chronic gastrointestinal pathologies causing great discomfort in both children and adults. The pathogenesis of inflammatory bowel diseases is not yet fully understood and their diagnosis and treatment are often challenging. Nanoparticle-based strategies have been tested in local drug delivery to the inflamed colon. Here, we have investigated the use of the novel avidin-nucleic acid nanoassembly (ANANAS) platform as a potential diagnostic carrier in an experimental model of inflammatory bowel diseases. Fluorescent- labeled ANANAS nanoparticles were administered to mice with chemically induced chronic inflammation of the large intestine. Localization of mucosal nanoparticles was assessed in vivo by dual-band confocal laser endomicroscopy. This technique enables characterization of the mucosal microvasculature and crypt architecture at subcellular resolution. Intravascular nanoparticle distribution was observed in the inflamed mucosa but not in healthy controls, demonstrating the utility of the combination of ANANAS and confocal laser endomicroscopy for highlighting intestinal inflammatory conditions. The specific localization of ANANAS in inflamed tissues supports the potential of this platform as a targeted carrier for bioactive moieties in the treatment of inflammatory bowel disease.

A New ELISA Using the ANANAS Technology Showing High Sensitivity to diagnose the Bovine Rhinotracheitis from Individual Sera to Pooled Milk

Diagnostic tests for veterinary surveillance programs should be efficient, easy to use and, possibly, economical. In this context, classic Enzyme linked ImmunoSorbent Assay (ELISA) remains the most common analytical platform employed for serological analyses. The analysis of pooled samples instead of individual ones is a common procedure that permits to certify, with one single test, entire herds as “disease-free”. However, diagnostic tests for pooled samples need to be particularly sensitive, especially when the levels of disease markers are low, as in the case of anti-BoHV1 antibodies in milk as markers of Infectious Bovine Rhinotracheitis (IBR) disease. The avidin-nucleic-acid-nanoassembly (ANANAS) is a novel kind of signal amplification platform for immunodiagnostics based on colloidal poly-avidin nanoparticles that, using model analytes, was shown to strongly increase ELISA test performance as compared to monomeric avidin. Here, for the first time, we applied the ANANAS reagent integration in a real diagnostic context. The monoclonal 1G10 anti-bovine IgG1 antibody was biotinylated and integrated with the ANANAS reagents for indirect IBR diagnosis from pooled milk mimicking tank samples from herds with IBR prevalence between 1 to 8%. The sensitivity and specificity of the ANANAS integrated method was compared to that of a classic test based on the same 1G10 antibody directly linked to horseradish peroxidase, and a commercial IDEXX kit recently introduced in the market. ANANAS integration increased by 5-fold the sensitivity of the 1G10 mAb-based conventional ELISA without loosing specificity. When compared to the commercial kit, the 1G10-ANANAS integrated method was capable to detect the presence of anti-BHV1 antibodies from bulk milk of gE antibody positive animals with 2-fold higher sensitivity and similar specificity. The results demonstrate the potentials of this new amplification technology, which permits improving current classic ELISA sensitivity limits without the need for new hardware investments.

Discrimination between Ulcerative Colitis and Crohn's Disease using Phage Display identified Peptides and Virus-mimicking Synthetic Nanoparticles

Abstract Discrimination between Crohn disease (CD) and ulcerative colitis (UC) is an unsolved clinical issue in up to 10–15% of inflammatory bowel disease (IBD) patients. We developed a method capable to discriminate between the two clinical conditions by combining random peptide phage display screenings and nanoparticle (NP) based assays. We identified two peptides able to recognize selectively biopsies of inflamed mucosa of either CD or UC patients. The peptides were integrated into synthetic virus-mimicking nanoassemblies using a poly-avidin NP platform. Peptide functionalized nanoassemblies carrying about 400 peptides/NP were used to optimize a highly selective and specific microplate colorimetric test which allowed us to distinguish CD from UC in inflamed IBD epithelia tissues. The method could complement and expand the diagnostic armamentarium in IBDs, especially when discrimination between these CD and UC is not straightforward.