Iron Oxide Nanoparticles as MRI Contrast Agents: a Physico-chemical Insight

SuperParamagnetic Iron Oxide Nanoparticles (SPIONs) are effective contrast agents for Magnetic Resonance Imaging. The modification of SPION surface has emerged as imperative to induce stability in aqueous media and biocompatibility. On the grounds of the published literature, we optimized a robust strategy for the preparation of SPION aqueous suspension suitable for biocompatible applications. The functionalization strategy was applied to SPIONs synthesized through thermal-decomposition method, but, in principle, it can also be extended to other inorganic nanoparticles. A detailed description of the preparation protocols adopted for the obtainment of functionalized SPIONs is reported together with a detailed characterization of their structure and composition. As a result, monodispersed functionalized SPIONs with high biocompatibility and effective activity as MRI contrast agents were obtained. Nevertheless, reliable information is often required about the behavior of nanoparticles in biological systems. For this purpose, the interaction between the functionalized SPIONs and lipid bilayers mimicking of the plasma cellular membrane was characterized. Finally, as a further step in the development of functionalized SPIONs for biomedical applications, the versatility of the optimized functionalization strategy was exploited to prepare prototypes of theranostic nanoparticles as well as dualmodal imaging contrast agents

Understanding the Nano-bio Interfaces: Lipid-Coatings for Inorganic Nanoparticles as Promising Strategy for Biomedical Applications

Inorganic nanoparticles (NPs) exhibit relevant physical properties for application in biomedicine and specifically for both the diagnosis and therapy (i.e. theranostic) of severe pathologies, such as cancer. The inorganic NP core is often not stable in aqueous suspension and can induce cytotoxic effects. For this reason, over the years, several coating strategies were suggested to improve the NP stability in aqueous solutions as well as the NP biocompatibility. Among the various components which can be used for NP coatings, lipids, and in particular phospholipids emerged as versatile molecular building blocks for the production of NP coatings suitable for biomedical application. The recent synthetic efforts in NP lipid coatings allows today to introduce on the NP surface a large variety of lipid molecules eventually in mixture with amphiphilic or hydrophobic drugs or active molecules for cell targeting. In this review, the most relevant examples of NP lipid-coatings are presented and grouped in two main categories: supported lipid bilayers (SLB) and hybrid lipid bilayers (HLB). The discussed scientific cases take into account the most commonly used inorganic NP for biomedical applications in cancer therapy and diagnosis

PO-485 Low abundance circulating proteins in giant cell tumours of bone

Introduction Circulating low-abundance proteins/fragments generating from tumour cells and tissues, represent the most important source of cancer biomarkers useful for early diagnosis and prognosis. Giant cell tumour of bone (GCT) is a benign neoplasm occurring in the long bone and in the axial skeleton of young adults. Approximately 5% of GCT develop pulmonary metastases. Although many biomarkers have been proposed, identification of circulating low abundance molecules may be useful to predict metastasis with a non invasive method. Material and methods The hydrogel nanoparticles technique followed by mass spectrometry was used to detect low molecular weight serum proteins or protein fragments in serum of 20 GCT patients with different clinical course and in 10 healthy sera used as control. The most representative low-abundant de novo or differentially abundant proteins were submitted to String database in order to define protein-protein interaction network. Cluster analysis was performed to identify prognostic groups of patients with similar abundance of proteins that significantly discriminate between the groups. Results and discussions For the 25 low-abundant de novo or differentially abundant proteins identified, we recognised that the top interconnected pathways included protein activation cascade, wound healing, blood coagulation, cell-substrate adhesion. Proteoma cluster analysis separated metastasis-free from metastatic GCT patients in two well-defined groups where serum levels of signalling transduction mediators and regulators of kinase activity presented a high discriminatory power. Increased expression of proteins STAT5B, GRB2 and OXSR1 was related to a higher probability of metastasis. Conclusion In conclusion, using a no invasive technique, we identified differentially abundant serum biomarkers, also providing prognostic information in patients with GCT of bone. Future studies are ongoing to establish the interplay between these biomarkers in order to fully understand the mechanism involved in tumour development and to focus on the planning of tailored therapies that should be more effective and less toxic

Charge Transport Phenomena in Peptide Molecular Junctions

Inelastic electron tunneling spectroscopy (IETS) is a valuable in situ spectroscopic analysis technique that provides a direct portrait of the electron transport properties of a molecular species. In the past, IETS has been applied to small molecules. Using self-assembled nanoelectronic junctions, IETS was performed for the first time on a large polypeptide protein peptide in the phosphorylated and native form, yielding interpretable spectra. A reproducible 10-fold shift of the I/V characteristics of the peptide was observed upon phosphorylation. Phosphorylation can be utilized as a site-specific modification to alter peptide structure and thereby influence electron transport in peptide molecular junctions. It is envisioned that kinases and phosphatases may be used to create tunable systems for molecular electronics applications, such as biosensors and memory devices

Towards biomimics of cell membranes: Structural effect of phosphatidylinositol triphosphate (PIP3) on a lipid bilayer

Phosphoinositide (PIP) lipids are anionic phospholipids playing a fundamental role for the activity of several transmembrane and soluble proteins. Among all, phosphoinositol-3',4',5'-trisphosphate (PIP3) is a secondary signaling messenger that regulates the function of proteins involved in cell growth and gene transcription. The present study aims to reveal the structure of PIP-containing lipid membranes, which so far has been little explored. For this purpose, supported lipid bilayers (SLBs) containing 1,2-dioleoyl-sn-glycero-3-phospho-(1'-myoinositol-3',4',5'-trisphosphate (DOPIP3) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) were used as mimics of biomembranes. Surface sensitive techniques, i.e. Quartz Crystal Microbalance with Dissipation monitoring (QCM-D), Atomic Force Microscopy (AFM) and Neutron Reflectometry (NR), provided detailed information on the formation of SLB and the location of DOPIP3 in the lipid membrane. Specifically, QCMD and AFM were used to identify the best condition for lipid deposition and to estimate the total bilayer thickness. On the other hand, NR was used to collect experimental structural data on the DOPIP3 location and orientation within the lipid membrane. The two bilayer leaflets showed the same DOPIP3 concentration, thus suggesting the formation of a symmetric bilayer. The headgroup layer thicknesses of the pure POPC and the mixed POPC/DOPIP3 bilayer suggest that the DOPIP3-headgroups have a preferred orientation , which is not perpendicular to the membrane surface, but instead it is close to the surrounding lipid headgroups. These results support the proposed PIP3 tendency to interact with the other lipid headgroups as PC, so far exclusively suggested by MD simulations

Application of Nanotrap technology for high sensitivity measurement of urinary outer surface protein A carboxyl-terminus domain in early stage Lyme borreliosis

Objectives: Prompt antibiotic treatment of early stage Lyme borreliosis (LB) prevents progression to severe multisystem disease. There is a clinical need to improve the diagnostic specificity of early stage Lyme assays in the period prior to the mounting of a robust serology response. Using a novel analyte harvesting nanotechnology, Nanotrap particles, we evaluated urinary Borrelia Outer surface protein A (OspA) C-terminus peptide in early stage LB before and after treatment, and in patients suspected of late stage disseminated LB. Method: We employed Nanotrap particles to concentrate urinary OspA and used a highly specific anti-OspA monoclonal antibody (mAb) as a detector of the C-terminus peptides. We mapped the mAb epitope to a narrow specific OspA C-terminal domain OspA236-239 conserved across infectious Borrelia species but with no homology to human proteins and no cross-reactivity with relevant viral and non-Borrelia bacterial proteins. 268 urine samples from patients being evaluated for all categories of LB were collected in a LB endemic area. The urinary OspA assay, blinded to outcome, utilized Nanotrap particle pre-processing, western blotting to evaluate the OspA molecular size, and OspA peptide competition for confirmation. Results: OspA test characteristics: sensitivity 1.7 pg/mL (lowest limit of detection), % coefficient of variation (CV) = 8 %, dynamic range 1.7-30 pg/mL. Pre-treatment, 24/24 newly diagnosed patients with an erythema migrans (EM) rash were positive for urinary OspA while false positives for asymptomatic patients were 0/117 (Chi squared p < 10-6). For 10 patients who exhibited persistence of the EM rash during the course of antibiotic therapy, 10/10 were positive for urinary OspA. Urinary OspA of 8/8 patients switched from detectable to undetectable following symptom resolution post-treatment. Specificity of the urinary OspA test for the clinical symptoms was 40/40. Specificity of the urinary OspA antigen test for later serology outcome was 87.5 % (21 urinary OspA positive/24 serology positive, Chi squared p = 4.072e-15). 41 of 100 patients under surveillance for persistent LB in an endemic area were positive for urinary OspA protein. Conclusions: OspA urinary shedding was strongly linked to concurrent active symptoms (e.g. EM rash and arthritis), while resolution of these symptoms after therapy correlated with urinary conversion to OspA negative

Use of a Chagas Urine Nanoparticle Test (Chunap) to Correlate with Parasitemia Levels in T. cruzi/HIV Co-infected Patients

BackgroundEarly diagnosis of reactivated Chagas disease in HIV patients could be lifesaving. In Latin America, the diagnosis is made by microscopical detection of the T. cruzi parasite in the blood; a diagnostic test that lacks sensitivity. This study evaluates if levels of T. cruzi antigens in urine, determined by Chunap (Chagas urine nanoparticle test), are correlated with parasitemia levels in T. cruzi/HIV co-infected patients.Methodology/Principal FindingsT. cruzi antigens in urine of HIV patients (N = 55: 31 T. cruzi infected and 24 T. cruzi serology negative) were concentrated using hydrogel particles and quantified by Western Blot and a calibration curve. Reactivation of Chagas disease was defined by the observation of parasites in blood by microscopy. Parasitemia levels in patients with serology positive for Chagas disease were classified as follows: High parasitemia or reactivation of Chagas disease (detectable parasitemia by microscopy), moderate parasitemia (undetectable by microscopy but detectable by qPCR), and negative parasitemia (undetectable by microscopy and qPCR). The percentage of positive results detected by Chunap was: 100% (7/7) in cases of reactivation, 91.7% (11/12) in cases of moderate parasitemia, and 41.7% (5/12) in cases of negative parasitemia. Chunap specificity was found to be 91.7%. Linear regression analysis demonstrated a direct relationship between parasitemia levels and urine T. cruzi antigen concentrations (p 105 pg was chosen to determine patients with reactivation of Chagas disease (7/7). Antigenuria levels were 36.08 times (95% CI: 7.28 to 64.88) higher in patients with CD4+ lymphocyte counts below 200/mL (p = 0.016). No significant differences were found in HIV loads and CD8+ lymphocyte counts.ConclusionChunap shows potential for early detection of Chagas reactivation. With appropriate adaptation, this diagnostic test can be used to monitor Chagas disease status in T. cruzi/HIV co-infected patients.Author SummaryReactivation of Chagas disease in people living with HIV is a serious clinical condition that is associated with high mortality. Hence, early diagnosis and treatment can be lifesaving. Although there are not well accepted criteria to identify patients at risk of reactivation, parasitemia levels are usually considered as the best predictor. Microscopy is used in Latin America for detection of parasitemia levels. However, this has low sensitivity, which usually leads to a delay in diagnosis and treatment. Quantitative PCR is used only for research proposes in endemic areas. Antigens in urine (antigenuria) are correlated with parasitemia levels in animal models, as well as in cases of congenital Chagas disease. We believe that antigenuria can also be used for prediction of parasitemia levels in T. cruzi/HIV co-infected patients. In this study, Chunap (Chagas urine nanoparticle test) was used for concentration and quantification of T. cruzi antigens in urine of T. cruzi/HIV co-infected patients. Values of more than 105 pg of T. cruzi antigens in urine were observed only in patients with reactivation of Chagas disease. This study shows that antigenuria levels are highly correlated to levels of parasitemia and can be used as a non-invasive technique for monitoring parasitemia levels in T. cruzi/HIV co-infected patients

Rapid Detection of Biological and Chemical Threat Agents Using Physical Chemistry, Active Detection, and Computational Analysis

Basic technologies have been successfully developed within this project: rapid collection of aerosols and a rapid ultra-sensitive immunoassay technique. Water-soluble, humidity-resistant polyacrylamide nano-filters were shown to (1) capture aerosol particles as small as 20 nm, (2) work in humid air and (3) completely liberate their captured particles in an aqueous solution compatible with the immunoassay technique. The immunoassay technology developed within this project combines electrophoretic capture with magnetic bead detection. It allows detection of as few as 150-600 analyte molecules or viruses in only three minutes, something no other known method can duplicate. The technology can be used in a variety of applications where speed of analysis and/or extremely low detection limits are of great importance: in rapid analysis of donor blood for hepatitis, HIV and other blood-borne infections in emergency blood transfusions, in trace analysis of pollutants, or in search of biomarkers in biological fluids. Combined in a single device, the water-soluble filter and ultra-sensitive immunoassay technique may solve the problem of early âwarning typeâ detection of aerosolized pathogens. These two technologies are protected with five patent applications and are ready for commercialization

The Italian Rare Pancreatic Exocrine Cancer Initiative

INTRODUCTION: Exocrine pancreatic cancers include common type pancreatic ductal adenocarcinoma and cystic neoplasms, which account for 85% and 10% of cases, respectively. The remaining 5% are rare histotypes, comprising adenosquamous carcinoma, acinar cell carcinoma, signet ring cell carcinoma, medullary carcinoma, pancreatoblastoma, hepatoid carcinoma, undifferentiated carcinoma and its variant with osteoclast-like giant cells, solid pseudopapillary carcinoma, and carcinosarcoma. Due to their low incidence, little knowledge is available on their clinical and molecular features as well as on treatment choices. The national initiative presented here aims at the molecular characterization of series of rare histotypes for which therapeutic and follow-up data are available. METHODS: A nationwide Italian Rare Pancreatic Cancer (IRaPaCa) task force whose first initiative is a multicentric retrospective study involving 21 Italian cancer centers to retrieve histologic material and clinical and treatment data of at least 100 patients with rare exocrine pancreatic cancers has been created. After histologic revision by a panel of expert pathologists, DNA and RNA from paraffin tissues will be investigated by next-generation sequencing using molecular pathway-oriented and immune-oriented mutational and expression profiling panels constructed availing of the information from the International Cancer Genome Consortium. Bioinformatic analysis of data will drive validation studies by immunohistochemistry and in situ hybridization, as well as nanostring assays. CONCLUSIONS: We expect to gather novel data on rare pancreatic cancer types that will be useful to inform the design of therapeutic choices