Oriented functionalization of magnetic nanoparticles with E-cadherin

The synergy between nanotechnology and biological science is being exploited in the last years, giving rise to a great amount of knowledge generation and applications in a wide variety of fields. This increasing use of this type of nano-sized materials has awak-ened the interest on the interaction between them and biological entities: cells, tissues or organisms. Among all the types of available nanomaterials, iron oxide magnetic nanopar-ticles have shown a huge potential in the biomedical field due to their biocompatibility and other interesting properties, especially their ability to produce heat when exposed to an external alternating magnetic field. A virtually unexplored subject is the interaction of magnetic nanoparticles with cell membranes of living cells. The study of this topic will have impact on fundamental science (i.e. biophysical studies of the cell membrane under localized heating, changes in heating efficiency of nanoparticles when interacting with living cells) and will allow the development of brand new applications (i.e. hyper-thermia treatment, cell transfection). In the work here presented, E-cadherin has been selected as a targeting agent. It is a ubiquitous adhesion protein present in the cell membranes of many cell types, with im-portant physiological functions and a key role in cancer progression. Spherical magnetic nanoparticles have been functionalized with this protein in an oriented manner, so that they are able to interact with the homologue proteins in the cell membrane. In that way, this is proposed as an approach to immobilize magnetic nanoparticles on the cells sur-face. Two different strategies were designed, one of them showing promising results of the oriented functionalization. However, the efficiency of the process needs to be fur-ther optimized for the future applications of this platform

Nanoestructuras híbridas para la activación remota de terapia enzimática dirigida mediada por hipertermia magnética

La terapia enzimática dirigida surge como una estrategia interesante para el empleo de enzimas capaces de metabolizar profármacos en el interior del cuerpo. Sin embargo, cuenta con limitaciones como la respuesta inmune del propio organismo o la temperatura sub-óptima alcanzada por las enzimas en el interior del mismo. Como respuesta a estas limitaciones, en este trabajo se propone la co-encapsulación de la enzima peroxidasa de rábano picante (HRP) con nanopartículas magnéticas (MNPs) por medio de una cubierta de sílica biomimética. De esta manera, el objetivo principal es la activación de estas MNPs mediante la aplicación de un campo magnético alterno externo, para conseguir la temperatura óptima de la HRP. Además, para evaluar la estabilidad y la funcionalidad del nanohíbrido se debe optimizar el protocolo de actividad enzimática. Los resultados obtenidos mostraron que la HRP es funcional en el interior del nanohíbrido. Así mismo, se observó cómo las MNPs son capaces de absorber energía y generar calor al ser expuestas a un campo magnético. Sin embargo, la temperatura alcanzada no se correspondió con la óptima de la enzima. Futuros estudios son necesarios para determinar el efecto de diferentes condiciones del campo magnético, con el objetivo de alcanzar dicha temperatura óptima

High-dose exposure to polymer-coated iron oxide nanoparticles elicits autophagy-dependent ferroptosis in susceptible cancer cells

Ferroptosis, a form of iron-dependent, lipid peroxidation-driven cell death, has been extensively investigated in recent years, and several studies have suggested that the ferroptosis-inducing properties of iron-containing nanomaterials could be harnessed for cancer treatment. Here we evaluated the potential cytotoxicity of iron oxide nanoparticles, with and without cobalt functionalization (Fe2O3 and Fe2O3@Co-PEG), using an established, ferroptosis-sensitive fibrosarcoma cell line (HT1080) and a normal fibroblast cell line (BJ). In addition, we evaluated poly (ethylene glycol) (PEG)-poly(lactic-co-glycolic acid) (PLGA)-coated iron oxide nanoparticles (Fe3O4-PEG-PLGA). Our results showed that all the nanoparticles tested were essentially non-cytotoxic at concentrations up to 100 μg/mL. However, when the cells were exposed to higher concentrations (200–400 μg/mL), cell death with features of ferroptosis was observed, and this was more pronounced for the Co-functionalized nanoparticles. Furthermore, evidence was provided that the cell death triggered by the nanoparticles was autophagy-dependent. Taken together, the exposure to high concentrations of polymer-coated iron oxide nanoparticles triggers ferroptosis in susceptible human cancer cells

Aplicaciones fotónicas de nanomateriales de oro en el desarrollo de biosensores enzimáticos e inmunológicos

El objetivo de esta Tesis Doctoral es la aplicación de las propiedades ópticas de los nanomateriales de oro en el desarrollo de biosensores fotónicos para la determinación de biomarcadores clínicamente relevantes. Durante este trabajo, se han sintetizado, caracterizado e implementado varios nanomateriales de oro en diferentes esquemas de biodetección utilizando tanto enzimas como anticuerpos como elementos de reconocimiento biológico. De hecho, fue posible desarrollar nuevos esquemas de nanobiodetección con mejores características analíticas que las tradicionales, como sensibilidad mejorada y adaptabilidad en el punto de atención. En todos los casos, la gran selectividad alcanzada se basó en la selección de receptores biológicos apropiados para cada analito diana y la optimización de su unión orientada al nanomaterial correspondiente. Esto permitió asegurar la transducción más efectiva entre el reconocimiento biológico y los mecanismos fotónicos involucrados en cada caso.En concreto, en esta Tesis se desarrollaron nuevos nanobiosensores enzimáticos utilizando nanoclusters de oro como etiquetas luminiscentes Vis-NIR, evitando los inconvenientes de los fluoróforos convencionales, como la degradación química y la existencia de interferencias espectrales en muestras biológicas. Para ello, se exploraron dos líneas de investigación; a) la unión covalente de nanoclusters de oro cerca del sitio activo de una enzima redox para promover un fenómeno de transferencia de energía que permita la determinación fluorescente de los analitos objetivo, y b) aprovechar la extinción de la fluorescencia de los nanoclusters de oro por oxígeno para monitorear reacciones enzimáticas de la oxidorreductasa.También se propuso el diseño de nanomateriales de oro mediante síntesis dirigida y específica con ligandos de reconocimiento biológico (enzimas) para explorar dos conceptos sensoriales diferentes impulsados por NP-sintéticos: a) la síntesis in-situ de nanomateriales de oro mediante la reducción de residuos de la enzima yb) la formación de nanopartículas de oro aprovechando las propiedades redox de la enzima desencadenadas por su interacción con el sustrato. En todos los casos, la señal óptica del nanomaterial generado está relacionada con la concentración del analito objetivo.Finalmente, esta Tesis también ha explorado el desarrollo de inmuno-nanobiosensores plasmónicos fototérmicos, basados en el uso de nanoprismas de oro unidos a anticuerpos que pueden actuar como transductores fisicoquímicos del reconocimiento biológico del analito generando una señal térmica cuantificable. El uso de nanoprismas de oro como etiquetas térmicas se implementó en esquemas de biodetección comúnmente utilizados y, por lo tanto, bien aceptados en la clínica (ELISA: Enzyme-Linked ImmunoSorbent Assay y LFIA: lateral flow immunoassay). Esto permitió desarrollar un novedoso concepto térmico de estos esquemas tradicionales de biosensores (Thermo-LISA y Thermo-LFIA) no solo con mejores características analíticas sino también con posibilidades reales de transferencia al mercado en el corto-medio plazo.La optimización y evaluación de la respuesta analítica de los nanobiosensores desarrollados, ha permitido la determinación cuantitativa de analitos relevantes para la detección de cáncer (biomarcadores de cáncer gastrointestinal y de próstata), control de calidad de alimentos (aminas biogénicas) y para otras aplicaciones biomédicas como la determinación de neurotransmisores (acetilcolina) y aminoácidos (L-fenilalanina) relacionados con enfermedades específicas.The aim of this PhD Thesis is the application of the optical properties of gold nanomaterials in the development of photonic biosensors for the determination of clinically relevant biomarkers. During this work, various gold nanomaterials have been synthetized, characterized and implemented in different biosensing schemes using both enzymes and antibodies as biological recognition elements. In fact, it was possible to develop novel nanobiosensing schemes with better analytical characteristics than the traditional ones, such as improved sensitivity and point-of-care adaptability. In all cases, the great selectivity reached was based on the selection of appropriate biological receptors for each target analyte and the optimization of their oriented binding to the corresponding nanomaterial. This allowed ensuring the most effective transduction between the biological recognition and the photonic mechanisms involved in each case. In particular, in this Thesis, new enzymatic nanobiosensors were developed using gold nanoclusters as Vis-NIR luminescent labels, avoiding the drawbacks of conventional fluorophores, such as chemical degradation and the existence of spectral interferences in biological samples. For this, two lines of research were explored; a) the covalent binding of gold nanoclusters close to the active site of a redox enzyme to promote an energy transfer phenomena that allow the fluorescent determination of the target analytes, and b) to take advantage of the quenching of gold nanoclusters fluorescence by oxygen to monitor oxidoreductase enzymatic reactions. The design of gold nanomaterials by means of directed and specific synthesis with biological recognition ligands (enzymes) was also proposed to explore two different NP-synthetic driven sensory concepts: a) the in-situ synthesis of gold nanomaterials by means of the reducing residues of the enzyme and b) the formation of gold nanoparticles taking advantage of the redox properties of the enzyme triggered by its interaction with the substrate. In all cases, the optical signal of the generated nanomaterial is related to the concentration of the target analyte. Finally, this Thesis has also explored the development of photothermal plasmonic immuno-nanobiosensors, based on the use of gold nanoprism bound to antibodies which can act as physicochemical transducers of the biological recognition of the analyte generating a quantifiable thermal signal. The use of gold nanoprism as thermal labels was implemented in biosensing schemes commonly used and therefore well accepted in the clinic (ELISA: Enzyme-Linked ImmunoSorbent Assay, and LFIA: lateral flow immunoassay). This allowed the development of a novel thermal-based concept of these traditional biosensing schemes (Thermo-LISA and Thermo-LFIA) with not only better analytical characteristics but also with real possibilities of transference to the market in the short-medium term. The optimization and the evaluation of the analytical response of the developed nanobiosensors, has allowed the quantitative determination of relevant analytes for cancer detection (gastrointestinal and prostate cancer biomarkers), control food quality (biogenic amines) and for other biomedical applications such as the determination of neurotransmitters (acetylcholine), and amino acids (L-phenylalanine) related to specific diseases.<br /

Hipertermia magnética basada en nanopartículas de óxido de hierro como terapia antitumoral: del cultivo celular tridimensional al modelo in vivo

La hipertermia magnética es una terapia prometedora para el tratamiento localizado del cáncer. Bajo la exposición a un campo magnético alterno externo, las nanopartículas magnéticas actúan como agentes de calentamiento que inducen la muerte celular en la región tratada. La comprensión de los mecanismos moleculares implicados en el daño celular generado por este tratamiento es crucial para la aplicación exitosa de esta terapia. Para esta tesis, se prepararon nanopartículas magnéticas esféricas de 11 nm por el método de descomposición térmica, que posteriormente se recubrieron con PMAO (poli (anhídrido maleico-alt-1-octadeceno) y finalmente se funcionalizaron con glucosa. Con el objetivo de evaluar la influencia de la localización de las nanopartículas en la eficacia del tratamiento térmico, se prepararon diferentes modelos de cultivo celular tridimensional (3D) basados en geles de colágeno, tanto en la línea celular de macrófagos murinos, RAW264.7, como en la línea de células tumorales pancreáticas humanas, MIAPaca-2. En un modelo, todas las partículas se encontraban localizadas dentro de las células (Modelo In), mientras que el otro modelo, contenía partículas tanto dentro como fuera de las células (Modelo In&Out). Además, se desarrolló un modelo murino de xenoinjerto de cáncer pancreático humano basado en las células MIAPaca-2. La internalización de las nanopartículas magnéticas, así como los mecanismos de muerte celular inducidos por diferentes condiciones de tratamiento de hipertermia se evaluaron por microscopía confocal, estudios de citometría de flujo, ensayos de biología molecular, análisis histológicos, medidas magnéticas y otras técnicas de caracterización analítica. Además, se evaluó el efecto del calentamiento intracelular inducido por las nanopartículas bajo acción del campo magnético mediante simulaciones computacionales. En general, los resultados in vitro e in vivo obtenidos en esta tesis han demostrado que la terapia térmica con hipertermia magnética tiene un importante efecto en la modulación de la matriz extracelular, así como en la inducción de mecanismos de inmuno-estimulación. Fenómeno especialmente relevante en la búsqueda de nuevas estrategias terapéuticas para el tratamiento del cáncer de páncreas. Además, las evidencias experimentales de este trabajo demostraron que las vías de muerte celular inducidas por el tratamiento con hipertermia magnética dependen del número de partículas localizadas en el interior de las células. Esto es importante para comprender los mecanismos moleculares que median la respuesta celular a esta terapia térmica. Magnetic hyperthermia is a promising therapy for the localized treatment of cancer. Under the exposure to an external alternating magnetic field, magnetic nanoparticles act as heating agents inducing cell death in the treated region. Understanding the molecular mechanisms involved in the cellular damage generated by this treatment is crucial for the successful application of this therapy. In this thesis, 11 nm spherical magnetic nanoparticles were prepared by thermal decomposition, coated with PMAO (poly (maleic anhydride-alt-1-octadecene) and functionalized with glucose. In order to evaluate the influence of the nanoparticle location in the treatment efficacy, two different three-dimensional (3D) cell culture models, based on collagen gels, were prepared both in the murine macrophage cell line, RAW264.7, as in the human pancreatic tumor cell line, MIAPaca-2. One model kept all the particles inside the cells (In Model) while the other model had particles both inside and outside the cells (In&Out Model). In addition, the xenograft murine model of human pancreatic cancer based in the MIAPaca-2 cells was developed. The magnetic nanoparticle uptake and cell death mechanisms induced by different conditions of the hyperthermia treatment were evaluated by confocal microscopy, flow cytometry studies, molecular biology assays, histological analysis, magnetic measurements and other analytical characterization techniques. In addition, computational simulations to evaluate the intracellular heating effects were also performed. In general, the in vitro and in vivo results obtained in this thesis, showed that magnetic hyperthermia had an important effect in the modulation of the extracellular matrix, as well as in the induction of immune-stimulation mechanisms. Phenomenon especially relevant in the search for new therapeutic strategies for pancreatic cancer. Moreover, the experimental results of this thesis showed that the type of cell death pathways triggered by the magnetic hyperthermia treatment depend on the number of intracellular nanoparticles. This is important in the understanding the molecular mechanisms that mediate the cellular response to this thermal therapy.<br /

Biosensor comprising metal nanoparticles

[ES] La presente invención se refiere a un biosensor donde la detección del analito se realiza de forma visual por el cambio de color en las zonas del soporte en que el analito esté presente producido por las nanopartículas al ser irradiadas con una fuente de luz externa[EN] The present invention discloses a biosensor for visual detection of an analyte, based on the light to heat conversion properties of metal nanoparticles: the analyte is visually detected by the colour change in the support areas (where the analyte is present), produced as a result of the heat generated by the metal nanoparticles where they are irradiated with an external light source. Use of said biosensor in a method for the detection of analytes is also claimed.Peer reviewedUniversidad de Zaragoza, Fundación Agencia Aragonesa para la Investigación y el Desarrollo, Consejo Superior de Investigaciones Científicas (España)B1 Patente sin examen previ

Multifunctional Nanocarriers for diagnostics, drug delivery and targeted treatment across blood-brain barrier: perspectives on tracking and neuroimaging

Nanotechnology has brought a variety of new possibilities into biological discovery and clinical practice. In particular, nano-scaled carriers have revolutionalized drug delivery, allowing for therapeutic agents to be selectively targeted on an organ, tissue and cell specific level, also minimizing exposure of healthy tissue to drugs. In this review we discuss and analyze three issues, which are considered to be at the core of nano-scaled drug delivery systems, namely functionalization of nanocarriers, delivery to target organs and in vivo imaging. The latest developments on highly specific conjugation strategies that are used to attach biomolecules to the surface of nanoparticles (NP) are first reviewed. Besides drug carrying capabilities, the functionalization of nanocarriers also facilitate their transport to primary target organs. We highlight the leading advantage of nanocarriers, i.e. their ability to cross the blood-brain barrier (BBB), a tightly packed layer of endothelial cells surrounding the brain that prevents high-molecular weight molecules from entering the brain. The BBB has several transport molecules such as growth factors, insulin and transferrin that can potentially increase the efficiency and kinetics of brain-targeting nanocarriers. Potential treatments for common neurological disorders, such as stroke, tumours and Alzheimer's, are therefore a much sought-after application of nanomedicine. Likewise any other drug delivery system, a number of parameters need to be registered once functionalized NPs are administered, for instance their efficiency in organ-selective targeting, bioaccumulation and excretion. Finally, direct in vivo imaging of nanomaterials is an exciting recent field that can provide real-time tracking of those nanocarriers. We review a range of systems suitable for in vivo imaging and monitoring of drug delivery, with an emphasis on most recently introduced molecular imaging modalities based on optical and hybrid contrast, such as fluorescent protein tomography and multispectral optoacoustic tomography. Overall, great potential is foreseen for nanocarriers in medical diagnostics, therapeutics and molecular targeting. A proposed roadmap for ongoing and future research directions is therefore discussed in detail with emphasis on the development of novel approaches for functionalization, targeting and imaging of nano-based drug delivery systems, a cutting-edge technology poised to change the ways medicine is administered

From monomeric to multimeric His-tag proteins conjugation to magnetic nanoparticles through NTA-Me2+: shape and size effects

Resumen del póster presentado a la 4th Spanish Conference on Biomedical Applications of Nanomaterials, celebrda online del 2 al 4 de junio de 2021.Peer reviewe

Sterilization matters: Consequences of different sterilization techniques on gold nanoparticles

Nanoparticles (NPs) can offer many advantages over traditional drug design and delivery, as well as toward medical diagnostics. As with any medical device or pharmaceutical drug intended to be used for in vivo biomedical applications, NPs must be sterile. However, very little is known regarding the effect of sterilization methods on the intrinsic properties and stability of NPs. Herein a detailed analysis of physicochemical properties of two types of AuNPs upon sterilization by means of five different techniques is reported. In addition, cell viability and production of reactive oxygen species are studied. The results indicate that sterilization by ethylene oxide seems to be the most appropriate technique for both types of NPs. It is concluded that it is crucial to test several methods in order to establish the specific type of sterilization to be performed for each particular NP.The authors acknowledge financial support from the Xunta de Galicia (PGIDIT06TMT31402PR), SUDOE (IMMUNONET-SOE1/1P1/E014), and Spanish Ministry of Science and Innovation (Consolider Ingenio 2010, CSD2006-12, NANOBIOMED). Ángela França was supported with a Leonardo da Vinci Fellowship. Jesús Martinez de la Fuente thanks ARAID for financial support.Peer reviewe

Dependence of intracellular magnetic nanoparticles amount in the apoptotic death pathway triggered by magnetic hyperthermia treatment

Trabajo presentado a la 4th Spanish Conference on Biomedical Applications of Nanomaterials, celebrada online del 2 al 4 de junio de 2021.Peer reviewe