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

Dextran-Coated Magnetic Supports Modified with a Biomimetic Ligand for IgG Purification

The authors thank the financial support from Fundacao para a Ciencia e a Tecnologia through Grant PEst-C/EQB/LA0006/2011 and contracts no. PTDC/EBB-BIO/102163/2008, PTDC/EBB-BIO/098961/2008, PTDC/EBB-BIO/118317/2010, SFRH/BD/72650/2010 for V.L.D, and Santander Totta Bank - Universidade Nova de Lisboa for the Scientific Award 2009/2010. The authors are grateful to Dr. Abid Hussain and M. Telma Barroso (REQUIMTE, FCT-UNL, Portugal) for the preparation of the synthetic affinity ligands, to Lonza Biologics, U.K. (Dr. Richard Alldread), and the Animal Cell Technology Unit of ITQB-UNL/IBET (Dr. Paula M Alves and Dr. Ana Teixeira) for providing the cells and the culture bulks and to Mr. Filipe Cardoso and Prof. Paulo Freitas (INESC-MN, Lisbon, Portugal) for the help with the VSM measurements.Dextran-coated iron oxide magnetic particles modified with ligand 22/8, a protein A mimetic ligand, were prepared and assessed for IgG purification. Dextran was chosen as the agent to modify the surface of magnetic particles by presenting a negligible level of nonspecific adsorption. For the functionalization of the particles with the affinity ligand toward antibodies, three methods have been explored. The optimum coupling method yielded a theoretical maximum capacity for human IgG calculated as 568 ± 33 mg/g and a binding affinity constant of 7.7 × 10⁴ M⁻¹. Regeneration, recycle and reuse of particles was also highly successful for five cycles with minor loss of capacity. Moreover, this support presented specificity and effectiveness for IgG adsorption and elution at pH 11 directly from crude extracts with a final purity of 95% in the eluted fraction.proofpublishe

Evaluation of RNAi Therapeutics VIR-2218 and ALN-HBV for Chronic Hepatitis B: Results From Randomized Clinical Trials.

BACKGROUND & AIMS: Current treatment for chronic hepatitis B virus (cHBV) infection requires lifelong treatment. New therapy aimed towards HBV functional cure would represent a clinically meaningful treatment advancement. ALN-HBV and VIR-2218 (modified from ALN-HBV by Enhanced Stabilization Chemistry Plus technology reducing off-target, seed-mediated binding while maintaining on-target antiviral activity) are investigational RNAi therapeutics that target all major HBV transcripts. METHODS: We report the safety of single doses of VIR-2218 and ALN-HBV in humanized mice, a cross-study comparison of single doses of VIR-2218 and ALN-HBV safety in human heathy volunteers (n=24 and n=49, respectively), and the antiviral activity of two monthly doses of 20, 50, 100, 200 mg of VIR-2218 (total n=24) vs. placebo (n=8) in participants with cHBV infection. RESULTS: In humanized mice, alanine aminotransferase (ALT) levels were markedly lower following administration with VIR-2218 compared with ALN-HBV. In healthy volunteers, posttreatment ALT elevations occurred in 28% of participants receiving ALN-HBV compared with none in those receiving VIR-2218. In participants with cHBV infection, VIR-2218 was associated with dose-dependent reductions in hepatitis B surface antigen (HBsAg). The greatest mean reduction of HBsAg at Week 20 in participants receiving 200 mg was 1.65 log IU/mL. The HBsAg reduction was maintained at 0.87 log IU/mL at Week 48. No participants had serum HBsAg loss or hepatitis B surface antibody seroconversion. CONCLUSIONS: VIR-2218 demonstrated an encouraging hepatic safety profile in preclinical and clinical studies as well as dose-dependent HBsAg reductions in patients with cHBV infection. These data support future studies with VIR-2218 as part of combination regimens with a goal of HBV functional cure. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT02826018 and NCT03672188

Identification Of Bacterial Drug Resistance In Blood Using Iron Oxide Nanoparticles

Development of antibiotic resistance by microorganisms has been on the rise in recent years. Therefore, identifying drug resistant strains and effective antibiotic agents is critical. Traditional microbiological methods assess antimicrobial susceptibility within 24 to 48 hours, whereas optical nanoparticle-based methods cannot be used in opaque media. Hence, we have developed a nanoparticle- based antimicrobial susceptibility assay, utilizing Concanavalin A-conjugated iron oxide nanoparticles. When the bacteria do not grow, the changes in the solution\u27s T2 relaxation times are proximal to those of the sterile medium. On the other hand, when the bacteria grow, the levels of free carbohydrates decrease, thus the changes in the T2 times are significantly higher than those of the sterile medium. The iron-oxide-nanoparticle-based antimicrobial susceptibility assay (i) monitors bacterial metabolism, (ii) provides results within 2 hours, (iii) determines the minimum effective antibiotic concentration with sensitivity comparable to those of the gold standard methods, and (iv) determines antimicrobial susceptibility in biological fluids, such as blood

From Rational Design To Probe Optimization: The Role Of Nanoparticle Valency In Single Cancer Cell Detection In Blood Via Magnetic Relaxation

The conjugation of targeting ligands to iron oxide nanoparticles has achieved the development of specific assays for the detection of biomedical targets through magnetic relaxation. However, limited studies investigated how the nanoparticle valency modulates the detection response and the assays\u27 sensitivity. Through nanoparticle valency grafting, we were able to achieve single cancer cell detection in blood, with a small-molecule-carrying multivalent nanoparticle in just 15 minutes. Although exhibiting a similar diagnostic trend, a corresponding high-valency antibody-carrying nanosensor did not achieve the same detection threshold, indicating that small molecules might be attractive probes for cancer diagnostics. Overall, these studies indicate that nanoparticle multivalency is a critical parameter in the design of robust and sensitive nanoprobes for diverse clinical and field-based applications

Identification Of Bacterial Susceptibility To Antibiotics Using Gold Nanoparticles

Inexpensive and rapid bacterial detection and antimicrobial susceptibility assays are desperately needed, as the determination of which antibiotic to use and its administration at effective dosages is critical. Currently, the assessment of antimicrobial susceptibility takes 24 to 48 hours. Hence, we have developed a nanoparticle-based antimicrobial susceptibility assay, utilizing the Concanavalin A-induced clustering of dextran-coated gold nanoparticles. When the bacteria do not grow, addition of Concanavalin A promotes the formation of extensive dextran gold nanoassemblies. The induction of these nanoassemblies is mediated by the presence of free carbohydrates, causing large shifts in the nanoparticles\u27 surface plasmon band. In contrast, when the bacteria grow, the levels of free carbohydrates decrease, thus the size of the gold nanoparticle clusters and the plasmonic shifts are smaller. This gold nanoparticle-based assay provides results within 3 hours and can be used for the high-throughput screening of samples during epidemics

Synthesis Of Dextran-Coated Nanoceria With Ph-Dependent Antioxidant Properties

Cerium oxide nanoparticles (nanoceria) are potent free radical scavengers with neuroprotective, radioprotective, and anti-inflammatory properties 1-4. However, most synthetic procedures for nanoceria results in nanoparticles with poor water solubility or synthesized by procedures involving toxic solvents, therefore hindering their potential clinical use. To advance these studies, we have developed a facile synthetic procedure for monodisperse, water soluble and highly crystalline dextran-coated nanoceria. These nanoparticles are composed of a cerium oxide core of 4 nm surrounded by a dextran coating for a total nanoparticle size of 10nm. One of the most interesting properties of nanoceria is its autocatalytic behavior. The ability of these nanoparticles to reversibly switch from Ce+3 to Ce +4 is a key factor for their catalytic and biological applications as antioxidants. Our results show that the dextran coating does not affect the autocatalytic properties of nanoceria, as hydrogen peroxide and peroxyl radicals can diffuse through the hydrophilic dextran coating and oxidize Ce+3 to Ce+4. Most importantly, we made the surprising discovery that the antioxidant activity of dextran nanoceria is significantly reduced at acidic pH. This could have important applications in the design of improved therapeutics in the radioprotection of healthy tissue during radiation therapy of acidic tumors

Intrinsic Oxidase Activity Of Cerium Oxide Nanoparticles Facilitate The Detection Of Cancer Biomarkers And Cancer Cells

Nanomaterials exhibit unique size-dependent physical properties. Thus, identifying novel properties of these materials at the nanoscale, and their applications in energy, catalysis and medicine, among others, is an area of current and intense research. Cerium oxide has been widely used in catalytic converters for many years, but only recently its interesting nanoscale-derived properties have been elucidated. Among the most interesting properties that nanoceria (cerium oxide nanoparticles) possesses is its ability to behave as a potent antioxidant at pH 7, due to the reversible switching from two oxidation states (Ce+3 and Ce+4). Recently, we reported that the antioxidant activity of nanoceria is pH-dependent, and this property can be used for the selective protection of normal tissue during cancer chemotherapy and radiotherapy. In the present study, we investigated the oxidase activity of nanoceria, which is also pH-dependent. In addition to this, we found that (i) at pH 4 nanoceria oxidized various organic substrates without the need of an oxidizing agent, (ii) nanoceria\u27s oxidase kinetics can be modulated via the thickness of the polymer coating, and (iii) nanoceria can be used in immunoassays via the oxidation of sensitive dyes that facilitate detection of biological targets