Nanotechnology-based approaches for Imaging and Therapy

Improved therapeutics and diagnostic tools are needed in the clinic, contributing towards better treatment and survivorship. During this presentation, we will discuss how nanoparticle-based tools can support decision-making through imaging, and provide new insights on how nanoparticles interact with their environment. Overall, we will explore how nanoparticles can serve as sensitive probes, as well as translational platforms for personalized medicine

Rapid Nanoparticle-Mediated Monitoring of Bacterial Metabolic Activity and Assessment of Antimicrobial Susceptibility in Blood with Magnetic Relaxation

Considering the increased incidence of bacterial infections and the emergence of multidrug resistant bacteria at the global level, we designed superparamagnetic iron oxide nanoparticles as nanosensors for the assessment of antimicrobial susceptibility through magnetic relaxation. In this report, we demonstrate that iron oxide nanosensors, either dextran-coated supplemented with Con A or silica-coated conjugated directly to Con A, can be used for the fast (1) quantification of polysaccharides, (2) assessment of metabolic activity and (3) determination of antimicrobial susceptibility in blood. The use of these polysaccharide nanosensors in the determination of antimicrobial susceptibility in the clinic or the field, and the utilization of these nanoprobes in pharmaceutical R&D are anticipated

Magnetic Nanosensors For Multiplexed Bacterial Pathogenesis Identification

Developing diagnostic modalities that utilize nanomaterials and miniaturized detectors can have an impact in point-of-care diagnostics. Diagnostic systems that (i) are sensitive, robust, and portable, (ii) allow detection in clinical samples, (iii) require minimal sample preparation yielding results quickly, and (iv) can simultaneously quantify multiple targets, would have a great potential in biomedical research and public healthcare. Bacterial infections still cause pathogenesis throughout the world (Chapter I). The emergence of multi-drug resistant strains, the potential appearance of bacterial pandemics, the increased occurrence of bacterial nosocomial infections, the wide-scale food poisoning incidents and the use of bacteria in biowarfare highlight the need for designing novel bacterial-sensing modalities. Among the most prominent disease-causing bacteria are strains of Escherichia coli, like the E. coli O157:H7 that produces the Shiga-like toxin (Stx). Apart from diarrheagenic E. coli strains, others cause disease varying from hemolytic uremic syndrome and urinary tract infections to septicemia and meningitis. Therefore, the detection of E. coli needs to be performed fast and reliably in diverse environmental and clinical samples. Similarly, Mycobacterium avium spp. paratuberculosis (MAP), a fastidious microorganism that causes Johne\u27s disease in cattle and has been implicated in Crohn\u27s disease (CD) etiology, is found in products from infected animals and clinical samples from CD patients, making MAP an excellent proof-of-principle model. Recently, magnetic relaxation nanosensors (MRnS) provided the first applications of improved diagnostics with high sensitivity and specificity. Nucleic acids, proteins, viruses and enzymatic activity were probed, yet neither large targets (for instance iv bacterial and mammalian cells) nor multiple bacterial disease parameters have been simultaneously monitored, in order to provide thorough information for clinical decision making. Therefore, the goal of this study was to utilize MRnS for the sensitive identification of multiple targets associated with bacterial pathogenesis, while monitoring virulence factors at the microorganism, nucleic acid and virulence factor levels, to facilitate improved diagnosis and optimal treatment regimes. To demonstrate the versatility of MRnS, we used MAP as our model system, as well as several other pathogens and eukaryotic cell lines. In initial studies, we developed MRnS suitable for biomedical applications (Chapter II). The resulting MRnS were composed of an iron oxide core, which was caged within a biodegradable polymeric coating that could be further functionalized for the attachment of molecular probes. We demonstrated that depending on the polymer used the physical and chemical properties of the MRnS can be tailored. Furthermore, we investigated the role of polymer in the enzyme-mimicking activity of MRnS, which may lead to the development of optimized colorimetric in vitro diagnostic systems such as immunoassays and small-molecule-based screening platforms. Additionally, via facile conjugation chemistries, we prepared bacterium-specific MRnS for the detection of nucleic acid signatures (Chapter III). Considering that MAP DNA can be detected in clinical samples and isolates from CD patients via laborious isolation and amplification procedures requiring several days, MRnS detected MAP\u27s IS900 nucleic acid marker up to a single MAP genome copy detection within 30 minutes. Furthermore, these MRnS achieved equally fast IS900 detection even in crude DNA extracts, outperforming the gold standard diagnostic method of nested Polymerase Chain v Reaction (nPCR). Likewise, the MRnS detected IS900 with unprecedented sensitivity and specificity in clinical isolates obtained from blood and biopsies of CD patients, indicating the clinical utility of these nanosensors. Subsequently, we designed MRnS for the detection of MAP via surface-marker recognition in complex matrices (Chapter III). Milk and blood samples containing various concentrations of MAP were screened and quantified without any processing via MRnS, obtaining dynamic concentration-dependent curves within an hour. The MAP MRnS were able not only to identify their target in the presence of interferences from other Gram positive and Gram negative bacteria, but could differentiate MAP among other mycobacteria including Mycobacterium tuberculosis. In addition, detection of MAP was performed in clinical isolates from CD patients and homogenized tissues from Johne\u27s disease cattle, demonstrating for the first time the rapid identification of bacteria in produce, as well as clinical and environmental samples. However, comparing the unique MAP quantification patterns with literatureavailable trends of other targets, we were prompted to elucidate the underlying mechanism of this novel behavior (Chapter IV). We hypothesized that the nanoparticle valency – the amount of probe on the surface of the MRnS – may have modulated the changes in the relaxation times (ΔΤ2) upon MRnS – target association. To address this, we prepared MAP MRnS with high and low anti-MAP antibody levels using the same nanoparticle formulation. Results corroborated our hypothesis, but to further bolster it we investigated if this behavior is target-size-independent. Hence utilizing small-moleculeand antibody-carrying MRnS, we detected cancer cells in blood, observing similar detection patterns that resembled those of the bacterial studies. Notably, a single cancer vi cell was identified via high-valency small-molecule MRnS, having grave importance in cancer diagnostics because a single cancer cell progenitor in circulation can effectively initiate the metastatic process. Apart from cells, we also detected the Cholera Toxin B subunit with valencly-engineered MRnS, observing similar to the cellular targets\u27 diagnostic profiling behavior. Finally, as bacterial drug resistance is of grave healthcare importance, we utilized MRnS for the assessment of bacterial metabolism and drug susceptibility (Chapter V). Contrary to spectophotometric and visual nanosensors, their magnetic counterparts were able to quickly assess bacterial carbohydrate uptake and sensitivity to antibiotics even in blood. Two MRnS-based assay formats were devised relying on either the Concanavalin A (Con A)-induced clustering of polysaccharide-coated nanoparticles or the association between free carbohydrates and Con A-carrying MRnS. Overall, taking together these results, as well as those on pathogen detection and the recent instrumentation advancements, the use of MRnS in the clinic, the lab and the field should be anticipated

Nanoparticle-Mediated Methods for Antimicrobial Susceptibility Testing of Bacteria

A method of testing bacterial cells for antimicrobial susceptibility includes preparing a suspension of the bacterial cells in a non-nutrient medium, mixing with the suspension an antimicrobial, a carbohydrate usable by the bacterial cells, metallic nanoparticles, and a lectin, and incubating the mixture while monitoring a parameter of the nanoparticles responsive to use of the carbohydrate by the bacterial cells. More broadly stated, the invention includes a method of testing an agent for its effect on cell metabolism by preparing a suspension of cells in a non-nutrient medium, mixing the suspension with the agent, adding a carbohydrate usable by the cells, metallic nanoparticles, and a lectin with binding specificity for the added carbohydrate, and monitoring a nanoparticle parameter responsive to the cells

Microbe detection via hybridizing magnetic relaxation nanosensors (US)

Disclosed herein are methods and materials for facilitating the detection of nucleic acid analytes of interest. Specifically exemplified herein are methods for detecting mycobacterial microorganisms, namely Mycobacterium avium spp. paratuberculosis. Also disclosed is new hybridizing magnetic relaxation nanosensor (hMRS) particularly adapted to detect a target nucleic acid analyte of interest

Polymer Coated Ceria Nanoparticles for Selective Cytoprotection (CIP)

Methods, systems and compositions are disclosed wherein normal, nontransformed, healthy biological cells are protected from oxidative stress, radiation therapy and chemotherapy while diseased, transformed cells, such as, cancer cells, are provided no protection by the biocompatible, polymer coated nanoceria composition of the present invention. The polymer-coated nanoceria preparation herein exhibits no toxicity to normal cells and exhibits pH-dependent antioxidant properties at neutral or physiological pH values, between approximately 6.5 to approximately 1 1.0 and is inactive as an antioxidant at acidic pH values between approximately 2.0 to approximately 6.4. Improved therapeutic agents and cytoprotecting devices are based on the newly discovered, pH dependent properties of polymer-coated nanoceria that provide selective cytoprotection

Cerium-Oxide-Nanoparticle-Based Device for the Detection of Reactive Oxygen Species and Monitoring of Chronic Inflammation DIV

polymer-coated cerium oxide based device and system is disclosed for detecting reactive oxygen species and monitoring chronic inflammation. The device and system encapsulate free therapeutic nanoparticle elements not present in a living body in a prosthetic or implantable unit. In one embodiment, the unit is a structure with a reactive oxygen species (ROS) scavenging component on one end and at the opposite end is an imaging agent consisting of at least one of the fluorophore capable of fluorescence emission, a chemiluminescent agent, a magnetic relaxation agent and an X-ray contrast agent. In a second embodiment, a single chamber device consisting of a multifunctional nanocomposite has a ROS-scavenging nanoparticle constituent (nanoceria) and a multimodal reporting nanoparticle component (i.e. Dex-IR-DiR). The device and system is utilized in treatment of diseases with a pro-inflammatory component, including, but not limited to, Crohn\u27s disease, ulcerative colitis, inflammatory bowel disease, cystic fibrosis, arthritis, and cancer chemotherapy

Oxidase Activity of Polymeric Coated Cerium Oxide Nanoparticles

methods, systems, compositions include biocompatible polymer coated nanoceria that function as aqeous redox catalyst with enhanced activity at an acidic to moderaly alkaline pH value between 1 and 8. The compositions are used as oxidizing agents for decompostion, decontamination and inactivation of organic contaminants, such as, pesticides and chemical warfare agents. Another use includes nanoceria as targetable nanocatalyst prepared by conjugating various targeting ligands to the nanoparticle coating to form a colorimetric or fuorescent probe in immunoassays or other molecule binding assays that involve the use of a molecule in solution that changes the color of the solution or emits a fluorescent signal, where localization of the nanoceria to organs or tissue is assessed by treatment with an oxidation sensitive dye or other detection devices. Versatility and uses of the nanoceria compositions are controlled by pH value, choice of dye substrate and thickness of the polymer coating on the ceria nanoparticles

Multiplexed highly sensitive detections of cancer biomarkers in thermal space using encapsulated phase change nanoparticles

We describe a multiplexed highly sensitive method to detect cancer biomarkers using silica encapsulated phase change nanoparticles as thermal barcodes. During phase changes, nanoparticles absorb heat energy without much temperature rise and show sharp melting peaks (0.6 degrees C). A series of phase change nanoparticles of metals or alloys can be synthesized in such a way that they melt between 100 and 700 degrees C, thus the multiplicity could reach 1000. The method has high sensitivity (8 nM) that can be enhanced using materials with large latent heat, nanoparticles with large diameter, or reducing the grafting density of biomolecules on nanoparticles

Rapid and Sensitive Detection of an Intracellular Pathogen in Human Peripheral Leukocytes with Hybridizing Magnetic Relaxation Nanosensors

Bacterial infections are still a major global healthcare problem. The quick and sensitive detection of pathogens responsible for these infections would facilitate correct diagnosis of the disease and expedite treatment. Of major importance are intracellular slow-growing pathogens that reside within peripheral leukocytes, evading recognition by the immune system and detection by traditional culture methods. Herein, we report the use of hybridizing magnetic nanosensors (hMRS) for the detection of an intracellular pathogen, Mycobacterium avium spp. paratuberculosis (MAP). The hMRS are designed to bind to a unique genomic sequence found in the MAP genome, causing significant changes in the sample’s magnetic resonance signal. Clinically relevant samples, including tissue and blood, were screened with hMRS and results were compared with traditional PCR analysis. Within less than an hour, the hMRS identified MAP-positive samples in a library of laboratory cultures, clinical isolates, blood and homogenized tissues. Comparison of the hMRS with culture methods in terms of prediction of disease state revealed that the hMRS outperformed established culture methods, while being significantly faster (1 hour vs 12 weeks). Additionally, using a single instrument and one nanoparticle preparation we were able to detect the intracellular bacterial target in clinical samples at the genomic and epitope levels. Overall, since the nanoparticles are robust in diverse environmental settings and substantially more affordable than PCR enzymes, the potential clinical and field-based use of hMRS in the multiplexed identification of microbial pathogens and other disease-related biomarkers via a single, deployable instrument in clinical and complex environmental samples is foreseen