Power absorption measurements during NMR experiments

The heating produced by the absorption of radiofrequency (RF) has been considered a secondary undesirable effect during MRI procedures. In this work, we have measured the power absorbed by distilled water, glycerol and egg-albumin during NMR and non-NMR experiments. The samples are dielectric and examples of different biological materials. The samples were irradiated using the same RF pulse sequence, whilst the magnetic field strength was the variable to be changed in the experiments. The measurements show a smooth increase of the thermal power as the magnetic field grows due to the magnetoresistive effect in the copper antenna, a coil around the probe, which is directly heating the sample. However, in the cases when the magnetic field was the adequate for the NMR to take place, some anomalies in the expected thermal powers were observed: the thermal power was higher in the cases of water and glycerol, and lower in the case of albumin. An ANOVA test demonstrated that the observed differences between the measured power and the expected power are significant

The Use of Silica Microparticles to Improve the Efficiency of Optical Hyperthermia (OH)

Although optical hyperthermia could be a promising anticancer therapy, the need for high concentrations of light-absorbing metal nanoparticles and high-intensity lasers, or large exposure times, could discourage its use due to the toxicity that they could imply. In this article, we explore a possible role of silica microparticles that have high biocompatibility and that scatter light, when used in combination with conventional nanoparticles, to reduce those high concentrations of particles and/or those intense laser beams, in order to improve the biocompatibility of the overall procedure. Our underlying hypothesis is that the scattering of light caused by the microparticles would increase the optical density of the irradiated volume due to the production of multiple reflections of the incident light: the nanoparticles present in the same volume would absorb more energy from the laser than without the presence of silica particles, resulting either in higher heat production or in the need for less laser power or absorbing particles for the same required temperature rise. Testing this new optical hyperthermia procedure, based on the use of a mixture of silica and metallic particles, we have measured cell mortality in vitro experiments with murine glioma (CT-2A) and mouse osteoblastic (MC3T3-E1) cell lines. We have used gold nanorods (GNRs) that absorb light with a wavelength of 808 nm, which are conventional in optical hyperthermia, and silica microparticles spheres (hereinafter referred to as SMSs) with a diameter size to scatter the light of this wavelength. The obtained results confirm our initial hypothesis, because a high mortality rate is achieved with reduced concentrations of GNR. We found a difference in mortality between CT2A cancer cells and cells considered non-cancer MC3T3, maintaining the same conditions, which gives indications that this technique possibly improves the efficiency in the cell survival. This might be related with differences in the proliferation rate. Since the experiments were carried out in the 2D dimensions of the Petri dishes, due to sedimentation of the silica particles at the bottom, whilst light scattering is a 3D phenomenon, a large amount of the energy provided by the laser escapes outside the medium. Therefore, better results might be expected when applying this methodology in tissues, which are 3D structures, where the multiple reflections of light we believe will produce higher optical density in comparison to the conventional case of no using scattering particles. Accordingly, further studies deserve to be carried out in this line of work in order to improve the optical hyperthermia technique.This study was partially supported by CIBER-BBN (Spain) and the NEUROCENTRO-CM (B2017/BMD-3760) Consortium. Characterization of the MNPs was performed by the ICTS ‘NANBIOSIS’, Unit 15, Functional Characterization of Magnetic Nanoparticles of the CIBER in Bioengineering, Biomaterials & Nanomedicine (CIBER-BBN) at the Center for Biomedical Technology (CTB) of the ‘Universidad Politécnica de Madrid’ (UPM). This work was carried out as a part of Project PGC2018-097531-B-I00, funded by the Ministry of Science of Spain

Influence of Medium Viscosity and Intracellular Environment on the Magnetization of Superparamagnetic Nanoparticles in Silk Fibroin Solutions and 3T3 Mouse Fibroblast Cell Cultures

IOP also requests that you include the following statement of provenance: "This is an author-created, un-copyedited versíon of an article published in Nanotechnology. IOP Publishing Ltd is not responsíble for any errors or omissíons in this versíon of the manuscript or any versíon derived from it. The Versíon of Record is available online at https://doi.org/10.1088/1361-6528/aacf4a.[EN] Biomedical applications based on the magnetic properties of superparamagnetic iron oxide nanoparticles (SPIONs) may be altered by the mechanical attachment or cellular uptake of these nanoparticles. When nanoparticles interact with living cells, they are captured and internalized into intracellular compartments. Consequently, the magnetic behavior of the nanoparticles is modified. In this paper, we investigated the change in the magnetic response of 14 nm magnetic nanoparticles (Fe3O4) in different solutions, both as a stable liquid suspension (one of them mimicking the cellular cytoplasm) and when associated with cells. The field-dependent magnetization curves from inert fluids and cell cultures were determined by using an alternating gradient magnetometer, MicroMagTM 2900. The equipment was adapted to measure liquid samples because it was originally designed only for solids. In order to achieve this goal, custom sample holders were manufactured. Likewise, the nuclear magnetic relaxation dispersion profiles for the inert fluid were also measured by fast field cycling nuclear magnetic relaxation relaxometry. The results show that SPION magnetization in inert fluids was affected by the carrier liquid viscosity and the concentration. In cell cultures, the mechanical attachment or confinement of the SPIONs inside the cells accounted for the change in the dynamic magnetic behavior of the nanoparticles. Nevertheless, the magnetization value in the cell cultures was slightly lower than that of the fluid simulating the viscosity of cytoplasm, suggesting that magnetization loss was not only due to medium viscosity but also to a reduction in the mechanical degrees of freedom of SPIONs rotation and translation inside cells. The findings presented here provide information on the loss of magnetic properties when nanoparticles are suspended in viscous fluids or internalized in cells. This information could be exploited to improve biomedical applications based on magnetic properties such as magnetic hyperthermia, contrast agents and drug delivery.The authors are thankful to their supporters: a grant from Universidad Politecnica de Madrid to Ana Lorena Urbano-Bojorge and a grant from Universidad Nacional Experimental del Tachira (UNET)- Venezuela to Oscar Casanova-Carvajal. This study was also financially supported in part by CIBER-BBN (Spain) and Madr.ib-CM (Spain).Urbano-Bojorge, AL.; Casanova-Carvajal, O.; González, N.; Fernández, L.; Madurga, R.; Sánchez-Cabezas, S.; Aznar, E.... (2018). Influence of Medium Viscosity and Intracellular Environment on the Magnetization of Superparamagnetic Nanoparticles in Silk Fibroin Solutions and 3T3 Mouse Fibroblast Cell Cultures. Nanotechnology. 29(38):1-13. https://doi.org/10.1088/1361-6528/aacf4aS113293

Influence of Medium Viscosity and Intracellular Environment on the Magnetization of Superparamagnetic Nanoparticles in Silk Fibroin Solutions and 3T3 Mouse Fibroblast Cell Cultures

IOP also requests that you include the following statement of provenance: "This is an author-created, un-copyedited versíon of an article published in Nanotechnology. IOP Publishing Ltd is not responsíble for any errors or omissíons in this versíon of the manuscript or any versíon derived from it. The Versíon of Record is available online at https://doi.org/10.1088/1361-6528/aacf4a.[EN] Biomedical applications based on the magnetic properties of superparamagnetic iron oxide nanoparticles (SPIONs) may be altered by the mechanical attachment or cellular uptake of these nanoparticles. When nanoparticles interact with living cells, they are captured and internalized into intracellular compartments. Consequently, the magnetic behavior of the nanoparticles is modified. In this paper, we investigated the change in the magnetic response of 14 nm magnetic nanoparticles (Fe3O4) in different solutions, both as a stable liquid suspension (one of them mimicking the cellular cytoplasm) and when associated with cells. The field-dependent magnetization curves from inert fluids and cell cultures were determined by using an alternating gradient magnetometer, MicroMagTM 2900. The equipment was adapted to measure liquid samples because it was originally designed only for solids. In order to achieve this goal, custom sample holders were manufactured. Likewise, the nuclear magnetic relaxation dispersion profiles for the inert fluid were also measured by fast field cycling nuclear magnetic relaxation relaxometry. The results show that SPION magnetization in inert fluids was affected by the carrier liquid viscosity and the concentration. In cell cultures, the mechanical attachment or confinement of the SPIONs inside the cells accounted for the change in the dynamic magnetic behavior of the nanoparticles. Nevertheless, the magnetization value in the cell cultures was slightly lower than that of the fluid simulating the viscosity of cytoplasm, suggesting that magnetization loss was not only due to medium viscosity but also to a reduction in the mechanical degrees of freedom of SPIONs rotation and translation inside cells. The findings presented here provide information on the loss of magnetic properties when nanoparticles are suspended in viscous fluids or internalized in cells. This information could be exploited to improve biomedical applications based on magnetic properties such as magnetic hyperthermia, contrast agents and drug delivery.The authors are thankful to their supporters: a grant from Universidad Politecnica de Madrid to Ana Lorena Urbano-Bojorge and a grant from Universidad Nacional Experimental del Tachira (UNET)- Venezuela to Oscar Casanova-Carvajal. This study was also financially supported in part by CIBER-BBN (Spain) and Madr.ib-CM (Spain).Urbano-Bojorge, AL.; Casanova-Carvajal, O.; González, N.; Fernández, L.; Madurga, R.; Sánchez-Cabezas, S.; Aznar, E.... (2018). Influence of Medium Viscosity and Intracellular Environment on the Magnetization of Superparamagnetic Nanoparticles in Silk Fibroin Solutions and 3T3 Mouse Fibroblast Cell Cultures. Nanotechnology. 29(38):1-13. https://doi.org/10.1088/1361-6528/aacf4aS113293

Contribución al estudio de las propiedades de las nanopartículas magnéticas en aplicaciones biomédicas relacionadas con la detección en tejidos ex-vivo y cultivos celulares

Desde su aparición, la nanotecnología ha irrumpido como área puntera de investigación ofreciendo beneficios significativos a la sociedad a través de aplicaciones y desarrollos tecnológicos que han revolucionado diversas áreas de la ciencia. En el sector salud, el empleo de la nanotecnología (nanomedicina), ha beneficiado el sistema sanitario en áreas de diagnóstico y terapia mediante la introducción de técnicas más eficientes, localizadas y personalizadas. El empleo de Nanopartículas Magnéticas (NPM) con fines biomédicos ha despertado un gran interés debido a que sus propiedades físicas únicas pueden conducir a nuevos métodos terapéuticos. Sin embargo, hay una necesidad de comprender las propiedades magnéticas de las nanopartículas y sus interacciones con los organismos vivos con el objetivo de desarrollar terapias más seguras y eficaces. Así mismo, la carencia de estudios centrados en el análisis de la distribución y destino final de las nanopartículas en el organismo tras una terapia, sumado a los problemas toxicológicos que pueda generar, ha ralentizado la aceptación comercial de muchas terapias que utilizan los nanomateriales. Esta situación se debe tanto a una infravaloración del estudio de la nanotoxicología como a la dificultad de implementar técnicas de seguimiento y detección eficaces. Por consiguiente, este trabajo de tesis doctoral busca dar respuesta directa a estas cuestiones aún sin resolver completamente. Por esta razón, se diseñó un exhaustivo protocolo de medida para llevar a cabo una adecuada caracterización de las NPMs distribuidas en los tejidos biológicos provenientes de ratones de experimentación a los cuales se les administró previamente una dosis de NPMs. Adicionalmente, se realizó la caracterización de NPMs suspendidas en diversos fluidos viscosos (agua desionizada y soluciones de fibroína) e internalizadas en cultivos celulares. A través de este estudio también se pretende demostrar y evaluar la viabilidad de los métodos de medición como una herramienta útil para realizar el estudio del comportamiento magnético de las nanopartículas suspendidas en fluidos viscosos y su internalización en cultivos celulares y tejidos ex-vivo. Para realizar la caracterización magnética de las nanopartículas, el Laboratorio de Bioinstrumentación y Nanomedicina (LBN) del Centro de Tecnología Biomédica (CTB) de la Universidad Politécnica de Madrid (UPM) dispone de una plataforma de Caracterización Funcional de Nanopartículas Magnéticas. Esta plataforma corresponde a la Unidad 15 del sistema NANBIOSIS-ICTS impulsado por el Centro de Investigación Biomédica en Red en Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN) para el fomento de la investigación en biotecnología. La Unidad de Caracterización Funcional de Nanopartículas Magnéticas está formada por dos equipos: un Magnetómetro por Gradiente Alternante MicroMagTM 2900 (AGM System) y un Relajómetro por Ciclado Rápido de Campo (FFCR). El AGM mide la magnetización de saturación de los materiales magnéticos y su extremada sensibilidad y precisión lo hacen especialmente atractivo para caracterizar NPMs. El FFCR mide los tiempos de relajación y los perfiles de dispersión de las nanopartículas magnéticas. Es importante mencionar que fue necesaria una adaptación de la sonda del AGM mediante la fabricación de un portamuestras con características específicas para llevar a cabo el estudio del comportamiento magnético de las nanopartículas suspendidas en fluidos viscosos e internalizadas en cultivos celulares. Los resultados obtenidos mostraron la validez de los procedimientos ya que efectivamente fue posible observar diferencias en el comportamiento magnético de las nanopartículas según el tiempo y el tipo de tejido. En general se apreció que, tras la inyección, las nanopartículas viajan a través del torrente sanguíneo durante las primeras horas y con el tiempo van siendo eliminadas a través de la orina o depositadas en ciertos órganos. Después de un mes de administrada la inyección de las NPMs, se detectó una acumulación de nanopartículas en el bazo e hígado indicando así una respuesta del sistema reticuloendotelial (RES). Sin embargo, en el cerebro no se detectó acumulación de NPMs durante el tiempo que duró el estudio (1 mes) indicando que las NPMs no atravesaron la barrera hematoencefálica (BHE). Por otro lado, se pudo observar una disminución en la magnetización de saturación de las nanopartículas dependiendo de la viscosidad del medio y la concentración de material magnético. Esta alteración es causada por las interacciones dipolo-dipolo, la formación de clústers o cadenas y el impedimento de los momentos magnético para alinearse con el campo magnético por el aumento de la viscosidad del medio y la concentración. También fue posible detectar una disminución en la magnetización de saturación de las nanopartículas cuando se internalizaron en los lisosomas. La variación causada por el encapsulamiento de las NPMs en los lisosomas provoca una disminución en los grados de libertad de las NPMs y la formación de agregados. Por otra parte, se observó un aumento en la relajatividad inducida por el incremento de la viscosidad del fluido debido a que la superficie de las NPMs está protegida contra el agua libre por la matriz de fibroína. Esto nos lleva a concluir que la fibroína podría ser un buen agente de recubrimiento para las NPMs. De manera general, se pudo comprobar la viabilidad de la Unidad 15 como una herramienta eficaz y versátil para llevar a cabo estudios de biodistribución y seguimiento de NPMs a través del estudio de los tejidos ex-vivo. Así mismo, esta herramienta permite realizar investigaciones relacionadas con las propiedades físicas de las NPMs suspendidas en fluidos viscosos e internalizadas en cultivos celulares. ----------ABSTRACT---------- Since its original conception, nanotechnology has emerged as a leading area of research offering significant benefits to society through applications and technological developments that have revolutionized various areas of science. In the health sector, the use of nanotechnology, also known as nanomedicine, has benefited the health system in areas of diagnosis and therapy though the introduction of more efficient, localized, and personalized techniques. The use of Magnetic Nanoparticles (MNPs) for biomedical purposes has generated a great interest due to their unique physical properties, which can lead to new therapeutic methods. However, there is a need for understanding the magnetic nanoparticles properties and their interactions with living organisms to develop safer and more efficient therapies. Moreover, the lack of studies focused on the analysis of nanoparticles distribution and final destination in the body after a therapy, in addition to the toxicological problems that it can generate, has slowed down the commercial acceptance of many therapies that utilize nanomaterials. This situation is due both to an underestimation of the study of nanotoxicology and to the difficulty of implementing effective monitoring and detection techniques. Therefore, this thesis seeks to give a direct answer to these issues still completely unresolved. For this reason, an exhaustive measurement protocol was designed in order to carry out an appropriate characterization of MNPs distributed in biological tissues from experimental mouse models, which a dose of MNPs was previously administered. Additionally, the characterization of MNPs suspended in viscous fluids (deionized water and fibroin solutions) and internalization into cultured cells were performed. Through this study, also intends to demonstrate and to assess the feasibility of the measurement methods as an useful tool to perform the study of the magnetic behavior of the MNPs suspended into viscous fluids and their internalization into cell cultures and ex-vivo tissues. In order to carry out the magnetic characterization of the nanoparticles, the Laboratory of Bioinstrumentation and Nanomedicine (LBN) of the Centre for Biomedical Technology (CTB) of the Technical University of Madrid (UPM), has a platform of functional characterization of MNPs. This platform corresponds to Unit 15 of the system NANBIOSIS-ICTS driven by the Centre for Biomedical Research Network Bioengineering, Biomaterials and Nanomedicine (CIBER- BBN) to promote the biotechnology research. The Unity of functional characterization of MNPs is composed by two devices: an Alternating Gradient Magnetometer MicroMagTM 2900 (AGM System) and a Fast Field Cycling Relaxometry (FFCR). The saturation magnetization of the magnetic materials is measured by an AGM. Their high sensibility and precision make these especially attractive to characterize MNPs. The relaxation times and dispersion profiles of the MNPs are measured with the FFCR. It is important to mention that an adaptation of the AGM’s probe was necessary through the manufacturing of a sample holder with specific characteristics to carry out the study of the magnetic behavior of nanoparticles suspend in viscous fluids and internalized into cell cultures. The results obtained showed the validity of the procedures since it was possible to efficiently observe differences in the magnetic behavior of the nanoparticles according to the time and type of tissue. Overall, it was noticed that after the injection, the nanoparticles travel through the bloodstream during the first hours and over time these are eliminated via urine or deposited in some organs. One month after administrating the injection of the MNPs, an accumulation of nanoparticles was detected in the spleen and liver, thus indicating a response of the reticuloendothelial system (RES). However, in the brain was not detected any accumulation of MNPs during the time the study last (1 month), indicating that MNPs did not cross the blood-brain barrier (BBB). On the other hand, a decrease in the saturation magnetization of the MNPs was observed depending on the viscosity of the medium and the concentration of the magnetic material. This change is caused by the dipole-dipole interactions, the formation of clusters or chains, and the hindrance of magnetic moments to align with the magnetic field by increasing the viscosity of the medium and the concentration. Furthermore, it was possible to detect a decrease in the saturation magnetization of the nanoparticles when they were internalized into the lysosomes. The variation caused by the encapsulation of the MNPs in the lysosomes causes a decrease in the degrees of freedom of the magnetic moments of the MNPs and the formation of aggregates. On the other hand, an increase in the relaxation induced by the increased viscosity of the fluid was observed because the surface of the MNPs is protected against free water by the fibroin matrix. This leads us to conclude that fibroin matrix could be a good coating agent for MNPs. In general, the viability of Unit 15 was verified as an efficient and versatile tool to carry out studies on the biodistribution and monitoring of MNPs through the study of ex-vivo tissues. Likewise, this tool allows research on the physical properties of MNPs suspended in viscous fluids and internalized into cell cultures

Slowdown intracranial glioma progression by optical hyperthermia therapy: study on a CT-2A mouse astrocytoma model

Metallic nanorods are promising agents for a wide range of biomedical applications. We report an optical hyperthermia method capable of inducing slowdown tumor progression of an experimental in vivo CT-2A glioblastoma tumor. The tumor model used in this research is based on the transplantation of mouse astrocytoma CT-2A cells in the striatum of mice by intracranial stereotaxic surgery. Two weeks after cell implant, the resulting tumor is treated by irradiating intratumoral injected gold nanorods, biofunctionalized with CD133 antibody (B-GNRs), using a continuous wave laser. Nanoparticles convert the absorbed light into localized heat (reaching up to 44 °C) due to the effect of surface plasmon resonance. A significant slowdown in CT-2A tumor progression is evident, by histology and magnetic resonance imaging, at one (p = 0.03) and two weeks (p = 0.008) after irradiation treatment. A notable deceleration in tumor size (15%–75%) as compared to the control untreated groups, it is observed. Thus, laser irradiation of B-GNRs is found to be effective for the treatment of CT-2A tumor progression. Similarities between the pre-clinical CT-2A tumor model and the human astrocytoma disease, in terms of anatomy, metastatic behavior and histopathology, suggest that hyperthermic treatment by laser irradiation of B-GNRs administered into high-grade human astrocytoma might constitute a promising alternative treatment to limit the progression of this deadly disease

Research lines in Hyperthermia at the Bioinstrumentation Laboratory of the Centre for Biomedical Technology

The Bioinstrumentation Laboratory belongs to the Centre for Biomedical Technology (CTB) of the Technical University of Madrid and its main objective is to provide the scientific community with devices and techniques for the characterization of micro and nanostructures and consequently finding their best biomedical applications. Hyperthermia (greek word for “overheating”) is defined as the phenomenon that occurs when a body is exposed to an energy generating source that can produce a rise in temperature (42-45ºC) for a given time [1]. Specifically, the aim of the hyperthermia methods used in The Bioinstrumentation Laboratory is the development of thermal therapies, some of these using different kinds of nanoparticles, to kill cancer cells and reduce the damage on healthy tissues. The optical hyperthermia is based on noble metal nanoparticles and laser irradiation. This kind of nanoparticles has an immense potential associated to the development of therapies for cancer on account of their Surface Plasmon Resonance (SPR) enhanced light scattering and absorption. In a short period of time, the absorbed light is converted into localized heat, so we can take advantage of these characteristics to heat up tumor cells in order to obtain the cellular death [2]. In this case, the laboratory has an optical hyperthermia device based on a continuous wave laser used to kill glioblastoma cell lines (1321N1) in the presence of gold nanorods (Figure 1a). The wavelength of the laser light is 808 nm because the penetration of the light in the tissue is deeper in the Near Infrared Region. The first optical hyperthermia results show that the laser irradiation produces cellular death in the experimental samples of glioblastoma cell lines using gold nanorods but is not able to decrease the cellular viability of cancer cells in samples without the suitable nanorods (Figure 1b) [3]. The generation of magnetic hyperthermia is performed through changes of the magnetic induction in magnetic nanoparticles (MNPs) that are embedded in viscous medium. The Figure 2 shows a schematic design of the AC induction hyperthermia device in magnetic fluids. The equipment has been manufactured at The Bioinstrumentation Laboratory. The first block implies two steps: the signal selection with frequency manipulation option from 9 KHz to 2MHz, and a linear output up to 1500W. The second block is where magnetic field is generated ( 5mm, 10 turns). Finally, the third block is a software control where the user can establish initial parameters, and also shows the temperature response of MNPs due to the magnetic field applied [4-8]. The Bioinstrumentation Laboratory in collaboration with the Mexican company MRI-DT have recently implemented a new research line on Nuclear Magnetic Resonance Hyperthermia, which is sustained on the patent US 7,423,429B2 owned by this company. This investigation is based on the use of clinical MRI equipment not only for diagnosis but for therapy [9]. This idea consists of two main facts: Magnetic Resonance Imaging can cause focal heating [10], and the differentiation in resonant frequency between healthy and cancer cells [11]. To produce only heating in cancer cells when the whole body is irradiated, it is necessary to determine the specific resonant frequency of the target, using the information contained in the spectra of the area of interest. Then, special RF pulse sequence is applied to produce fast excitation and relaxation mechanism that generates temperature increase of the tumor, causing cellular death or metabolism malfunction that stops cellular divisio