Functionalization and Characterization of Magnetic Nanoparticles for the Detection of Ferritin Accumulation in Alzheimer's Disease

Early diagnosis in Alzheimer's disease (AD), prior to the appearance of marked clinical symptoms, is critical to prevent irreversible neuronal damage and neural malfunction that lead to dementia and death. Therefore, there is an urgent need to generate new contrast agents which reveal by a noninvasive method the presence of some of the pathological signs of AD. In the present study, we demonstrate for the first time a new nanoconjugate composed of magnetic nanoparticles bound to an antiferritin antibody, which has been developed based on the existence of iron deposits and high levels of the ferritin protein present in areas with a high accumulation of amyloid plaques (particularly the subiculum in the hippocampal area) in the brain of a transgenic mouse model with five familial AD mutations. Both in vitro and after intravenous injection, functionalized magnetic nanoparticles were able to recognize and bind specifically to the ferritin protein accumulated in the subiculum area of the AD transgenic mice.Fil: Fernández Cabada, Tamara. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Politécnica de Madrid; EspañaFil: Martínez Serrano, Alberto. Consejo Superior de Investigaciones Científicas; España. Universidad Autónoma de Madrid; EspañaFil: Cussó, Lorena. Instituto de Investigacion Sanitaria Gregorio Marañón; España. Universidad Carlos III de Madrid; España. Centro de Investigación Biomédica en Red de Salud Mental; EspañaFil: Desco, Manuel. Instituto de Investigacion Sanitaria Gregorio Marañón; España. Centro de Investigación Biomédica en Red de Salud Mental; España. Universidad Carlos III de Madrid; EspañaFil: Ramos Gómez, Milagros. Universidad Politécnica de Madrid; Españ

Caracterización de nanopartículas magnéticas y de oro para posibles aplicaciones biomédicas en diagnóstico y terapia

Los nanomateriales han adquirido recientemente un gran interés debido a la gran variedad de aplicaciones que pueden llegar a tener en el ámbito de la biomedicina. Este trabajo recoge las posibilidades tanto diagnósticas como terapéuticas que presentan dos modalidades de nanomateriales: nanopartículas de óxido de hierro y nanopartículas de oro. Para ello, en una primera aproximación se ha llevado a cabo la caracterización de las nanopartículas desde el punto de vista de la biocompatibilidad asociada a su tamaño y al tiempo de contacto o circulación en células y tejidos, ensayada tanto in vitro como in vivo así como la cinética de acumulación de dichas nanopartículas en el organismo vivo. Posteriormente se ha realizado la biofuncionalización de los dos tipos de nanopartículas para reconocer dianas moleculares específicas y poder ser utilizadas en el futuro en dos aplicaciones biomédicas diferentes: diagnóstico de enfermedad de Alzheimer mediante imagen de resonancia magnética y destrucción selectiva de células tumorales mediante hipertermia óptica. ABSTRACT Nanomaterials have recently gained a great interest due to the variety of applications that can have in the field of biomedicine. This work covers both diagnostic and therapeutic possibilities that present two types of nanomaterials: iron oxide nanoparticles and gold nanoparticles. Therefore, in a first approximation it has performed the characterizing of nanoparticles from the standpoint of biocompatibility associated with their size and time of contact or movement in cells and tissues, tested both in vitro and in vivo as well as the kinetics of accumulation of the nanoparticles into the living organism. Subsequently the biofunctionalization of two types of nanoparticles was made to recognize specific molecular targets and can be used in the future in two different biomedical applications: diagnosis of Alzheimer's disease by magnetic resonance imaging and selective destruction of tumor cells by optical hyperthermia

A Novel Contrast Agent Based on Magnetic Nanoparticles for Cholesterol Detection as Alzheimer’s Disease Biomarker

Abstract Background Considering the high incidence of Alzheimer’s disease among the world population over the years, and the costs that the disease poses in sanitary and social terms to countries, it is necessary to develop non-invasive diagnostic tests that allow to detect early biomarkers of the disease. Within the early diagnosis methods, the development of contrast agents for magnetic resonance imaging becomes especially useful. Accumulating evidence suggests that cholesterol may play a role in the pathogenesis of Alzheimer’s disease since abnormal deposits of cholesterol surrounding senile plaques have been described in animal transgenic models and patients with Alzheimer’s disease. In vivo experiments have also shown that diet-induced hypercholesterolemia enhances intraneuronal accumulation of β-amyloid protein accompanied by microgliosis and accelerates β-amyloid deposition in brains. Presentation of the Hypothesis In the present study, we propose for the first time the synthesis of a new nanoconjugate composed of magnetic nanoparticles bound to an anti-cholesterol antibody, to detect the abnormal deposits of cholesterol observed in senile plaques in Alzheimer’s disease by magnetic resonance imaging. The nanoplatform could also reveal the decrease of cholesterol observed in neuronal plasmatic membranes associated with this pathology. Testing the Hypothesis Experimental design to test the hypothesis will be done first in vitro and then in ex vivo and in vivo studies in a second stage. Implications of the Hypothesis The designed nanoplatform could therefore detect cholesterol deposits at the cerebral level. The detection of this biomarker in areas coinciding with senile plaque accumulations could provide early information on the onset and progression of Alzheimer’s disease

Evaluation of the bioaccumulation kinetics of gold nanorods in vital mammalian organs by mean of TXRF spectrometry

This work presents the first application of total-reflection X-ray fluorescence (TXRF) spectrometry, a new and powerful alternative analytical method, to evaluation of the bioaccumulation kinetics of gold nanorods (GNRs) in various tissues upon intravenous administration in mice. The analytical parameters for developed methodology by TXRF were evaluated by means of the parallel analysis of bovine liver certified reference material samples (BCR-185R) doped with 10 μg/g gold. The average values (n = 5) achieved for gold measurements in lyophilized tissue weight were as follows: recovery 99.7%, expanded uncertainty (k = 2) 7%, repeatability 1.7%, detection limit 112 ng/g, and quantification limit 370 ng/g. The GNR bioaccumulation kinetics was analyzed in several vital mammalian organs such as liver, spleen, brain, and lung at different times. Additionally, urine samples were analyzed to study the kinetics of elimination of the GNRs by this excretion route. The main achievement was clearly differentiating two kinds of behaviors. GNRs were quickly bioaccumulated by highly vascular filtration organs such as liver and spleen, while GNRs do not show a bioaccumulation rates in brain and lung for the period of time investigated. In parallel, urine also shows a lack of GNR accumulation. TXRF has proven to be a powerful, versatile, and precise analytical technique for the evaluation of GNRs content in biological systems and, in a more general way, for any kind of metallic nanoparticles

Optical Hyperthermia Using Anti-Epidermal Growth Factor Receptor-Conjugated Gold Nanorods to Induce Cell Death in Glioblastoma Cell Lines

Gold nanorods (GNRs) are able to efficiently convert absorbed light into localized heat within a short period of time due to the surface plasmon resonance effect. This property, along with their easy bioconjugation, allows the use of GNRs in photothermal therapy as selective photothermal agents to target cancer cells. In this study, GNRs were combined with an antibody against anti-epidermal growth factor receptor (EGFR), a receptor that is frequently overexpressed in brain tumors, and the potential of the nanoconjugate (EGFR-GNRs) to eliminate tumor cells was assessed in vitro. Two human glioblastoma cell lines (U373-MG and 1321N1) expressing EGFR at different levels were incubated with unfunctionalized GNRs and EGFR-GNRs, and then exposed to irradiation with a continuous-wave laser at 808 nm. The pretreatment with the EGFR-GNR nanoconjugate significantly increased the cell death rate after laser irradiation compared to unconjugated GNRs. No photothermal cell destruction was observed in the absence of GNRs. Our data suggest that the EGFR modification improves GNR-mediated cell death after laser irradiation, even when EGFR is present at low doses in cancer cells, and may have the potential to be used clinically as a tool to help complete resection of brain tumors during surgery.Fil: Fernández Cabada, Tamara. Universidad Politécnica de Madrid; España. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Pablo, Cristina Sánchez López de. Universidad Politécnica de Madrid; España. Centro de Investigación Biomédica en Red; EspañaFil: Pisarchyk, Liudmila. Universidad Politécnica de Madrid; EspañaFil: Serrano Olmedo, José Javier. Universidad Politécnica de Madrid; España. Centro de Investigación Biomédica en Red; EspañaFil: Ramos Gómez, Milagros. Universidad Politécnica de Madrid; España. Centro de Investigación Biomédica en Red; Españ

Magnetic nanoparticles and clusters for magnetic hyperthermia : Optimizing their heat performance and developing combinatorial therapies to tackle cancer

Magnetic hyperthermia (MHT) is a therapeutic modality for the treatment of solid tumors that has now accumulated more than 30 years of experience. In the ongoing MHT clinical trials for the treatment of brain and prostate tumors, iron oxide nanoparticles are employed as intra-Tumoral MHT agents under a patient-safe 100 kHz alternating magnetic field (AMF) applicator. Although iron oxide nanoparticles are currently approved by FDA for imaging purposes and for the treatment of anemia, magnetic nanoparticles (MNPs) designed for the efficient treatment of MHT must respond to specific physical-chemical properties in terms of magneto-energy conversion, heat dose production, surface chemistry and aggregation state. Accordingly, in the past few decades, these requirements have boosted the development of a new generation of MNPs specifically aimed for MHT. In this review, we present an overview on MNPs and their assemblies produced via different synthetic routes, focusing on which MNP features have allowed unprecedented heating efficiency levels to be achieved in MHT and highlighting nanoplatforms that prevent magnetic heat loss in the intracellular environment. Moreover, we review the advances on MNP-based nanoplatforms that embrace the concept of multimodal therapy, which aims to combine MHT with chemotherapy, radiotherapy, immunotherapy, photodynamic or phototherapy. Next, for a better control of the therapeutic temperature at the tumor, we focus on the studies that have optimized MNPs to maintain gold-standard MHT performance and are also tackling MNP imaging with the aim to quantitatively assess the amount of nanoparticles accumulated at the tumor site and regulate the MHT field conditions. To conclude, future perspectives with guidance on how to advance MHT therapy will be provided. This journal i

Development of image analysis software for quantification of viable cells in microchips.

Over the past few years, image analysis has emerged as a powerful tool for analyzing various cell biology parameters in an unprecedented and highly specific manner. The amount of data that is generated requires automated methods for the processing and analysis of all the resulting information. The software available so far are suitable for the processing of fluorescence and phase contrast images, but often do not provide good results from transmission light microscopy images, due to the intrinsic variation of the acquisition of images technique itself (adjustment of brightness / contrast, for instance) and the variability between image acquisition introduced by operators / equipment. In this contribution, it has been presented an image processing software, Python based image analysis for cell growth (PIACG), that is able to calculate the total area of the well occupied by cells with fusiform and rounded morphology in response to different concentrations of fetal bovine serum in microfluidic chips, from microscopy images in transmission light, in a highly efficient way

Analysis of tumoral spheres growing in a multichamber microfluidic device

Lab on a Chip (LOC) farming systems have emerged as a powerful tool for single cell studies combined with a non‐adherent cell culture substrate and single cell capture chips for the study of single cell derived tumor spheres. Cancer is characterized by its cellular heterogeneity where only a small population of cancer stem cells (CSCs) are responsible for tumor metastases and recurrences. Thus, the in vitro strategy to the formation of a single cell‐derived sphere is an attractive alternative to identify CSCs. In this study, we test the effectiveness of microdevices for analysis of heterogeneity within CSC populations and its interaction with different components of the extracellular matrix. CSC could be identify using specific markers related to its pluripotency and self‐renewal characteristics such as the transcription factor Oct‐4 or the surface protein CD44. The results confirm the usefulness of LOC as an effective method for quantification of CSC, through the formation of spheres under conditions of low adhesion or growing on components of the extracellular matrix. The device used is also a good alternative for evaluating the individual growth of each sphere and further identification of these CSC markers by immunofluorescence. In conclusion, LOC devices have not only the already known advantages, but they are also a promising tool since they use small amounts of reagents and are under specific culture parameters. LOC devices could be considered as a novel technology to be used as a complement or replacement of traditional studies on culture plates.Fil: Belgorosky, Denise. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Fernández Cabada, Tamara. Universidad Tecnológica Nacional. Facultad Regional Haedo; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Peñaherrera Pazmiño, Ana Belén. Universidad Tecnológica Nacional. Facultad Regional Haedo; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Langle, Yanina Verónica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Booth, Ross. Millipore Sigma Corporation; Estados UnidosFil: Bhansali, Shekhar. University of Florida; Estados UnidosFil: Perez, Maximiliano Sebastian. Universidad Tecnológica Nacional. Facultad Regional Haedo; Argentina. University of Florida; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Eijan, Ana Maria. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Lerner, Betiana. Universidad Tecnologica Nacional. Facultad Regional Haedo. Centro de Ingenieria de Recubrimientos Especiales y Nanoestructuras.; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Hybrid microchannel-solid state micropore device for fast and optical cell detection

This paper presents a methodology for cell detection and counting using a device that combines PDMS (polydimethylsiloxane) microfluidic multilayer channels with a single solid state micropore. Optimal conditions of solid-state micropore fabrication from crystalline silicon wafers are presented. Micropores of varying size can be obtained by directly etching using an etchant agent concentration of 50 wt% KOH, at varying temperatures (40, 60, 80 °C) and voltages (100, 500, 1000 mV). Scanning Electron Microscopy (SEM), and profilometry techniques have been used for the micropore characterization. In order to find optimal conditions for cell detection a COMSOL Multiphysics simulation was performed. Pressure drop, shear stress, fluid viscosities and flow rates parameters were evaluated. The potential viability of the device for cell detection and counting, avoiding cellular damage, is demonstrated.Fil: Olmos Carreno, Carol Maritza. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Tecnológica Nacional. Facultad Regional Haedo; ArgentinaFil: Rosero Yánez, Gustavo Ivan. Universidad Tecnológica Nacional. Facultad Regional Haedo; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Fernández Cabada, Tamara. Universidad Tecnológica Nacional. Facultad Regional Haedo; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Booth, Ross. MilliporeSigma Corporation; Estados UnidosFil: Der, Manuel. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Física; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Cabaleiro, Juan Martin. Universidad de Buenos Aires. Facultad de Ingeniería. Departamento de Ingeniería Mecánica. Laboratorio de Fluidodinámica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Debut, Alexis. Universidad de Las Fuerzas Armadas; EcuadorFil: Cumbal Flores, Luis. Universidad de Las Fuerzas Armadas; EcuadorFil: Perez, Maximiliano Sebastian. Universidad Tecnológica Nacional. Facultad Regional Haedo; Argentina. Universidad de Buenos Aires. Facultad de Ingeniería. Instituto de Ingeniería Biomédica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Lerner, Betiana. Universidad Tecnológica Nacional. Facultad Regional Haedo; Argentina. Florida International University; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Determination of percentage cell area.

<p>From the determination of mean cell size, PIACG software estimates the mean height and cell area to calculate the percentage of total area occupied by the cells in the well.</p