Gold nanoparticles and chitosan encapsulation for therapy and sensing

Un sinfín de aplicaciones en el campo de la biomedicina se verían beneficiadas por el futuro uso de nanopartículas de oro (AuNPs). Sin embargo, a pesar de las miles de publicaciones científicas que destacan cada año las fabulosas propiedades de estas nanopartículas en este área, su empleo más allá del ámbito científico es testimonial, especialmente cuando hablamos de su uso con finalidades terapéuticas en el ámbito clínico. El objetivo principal de esta tesis consiste en mejorar la aplicabilidad de las nanopartículas de oro con diferentes geometrías mediante dos líneas de acción: 1) el estudio de la relación entre sus propiedades fisicoquímicas y su rendimiento para las aplicaciones de bio-detección y terapia fototérmica y 2) la búsqueda de una manera complementaria de mejorar sus propiedades intrínsecas para aplicaciones terapéuticas mediante su encapsulación en hidrogeles de quitosano. En la primera parte de esta tesis la inexistencia de una metodología clínica adecuada para la detección de miRNA nos ha llevado a buscar nuevas estrategias para su detección mediante el empleo de nanopartículas de oro como amplificadores de la señal de detección gracias a sus idóneas propiedades: elevada densidad, tamaño adecuado y facilidad para su funcionalización con diferentes proteínas. Además, para descubrir su utilidad en la detección de miRNA en condiciones similares a las necesarias en el ámbito clínico, se ha llevado a cabo un estudio comparativo empleando nanopartículas de oro con diversas geometrías y diferentes estrategias de biofuncionalización para su interacción con los miRNA. En la segunda parte de esta tesis se ha continuado con el estudio de la aplicación de diferentes geometrías de nanopartículas de oro para su uso en terapia fototérmica, centrándonos en sus propiedades ópticas, su interacción celular y eficacia, ya que hay una falta de conocimiento acerca de la importancia que tiene la forma de las nanopartículas en este tema. Gracias a las conclusiones extraídas durante este estudio concernientes a la baja internalización celular de los nanorods, hemos propuesto una estrategia innovadora consiste en su encapsulación en hidrogeles de quitosano motivada por las propiedades de adhesión celular del quitosano que han permitido una mejora remarcable de su eficacia. Finalmente se ha desarrollado una nueva metodología para la encapsulación de nanopartículas de oro en hidrogeles de quitosano empleando la tecnología de eyección automática de tinta o inkjet para mejorar su encapsulación, la cual permite que el proceso sea más fácil de escalar, efectivo y controlable. Esta tecnología ofrece una metodología automática para la producción continua de microcápsulas de quitosano que contengan las nanopartículas de oro en su interior, proporcionando biomateriales con propiedades muy interesante para las aplicaciones biomédicas. Como resultado, la investigación llevada a cabo durante esta tesis doctoral contribuye a ampliar nuestro conocimiento en la importancia del empleo de diferentes geometrías de nanopartículas de oro para las aplicaciones terapéuticas y de detección a la vez que proporciona nuevas herramientas para aumentar su aplicabilidad gracias a la funcionalización de su superficie y a su encapsulación en hidrogeles de quitosano. <br /

Natural Polysaccharides for siRNA Delivery: Nanocarriers Based on Chitosan, Hyaluronic Acid, and Their Derivatives

Natural polysaccharides are frequently used in the design of drug delivery systems due to their biocompatibility, biodegradability, and low toxicity. Moreover, they are diverse in structure, size, and charge, and their chemical functional groups can be easily modified to match the needs of the final application and mode of administration. This review focuses on polysaccharidic nanocarriers based on chitosan and hyaluronic acid for small interfering RNA (siRNA) delivery, which are highly positively and negatively charged, respectively. The key properties, strengths, and drawbacks of each polysaccharide are discussed. In addition, their use as efficient nanodelivery systems for gene silencing applications is put into context using the most recent examples from the literature. The latest advances in this field illustrate effectively how chitosan and hyaluronic acid can be modified or associated with other molecules in order to overcome their limitations to produce optimized siRNA delivery systems with promising in vitro and in vivo result

Surfactant-free synthesis and scalable purification of triangular gold nanoprisms with low non-specific cellular uptake

Gold nanoprisms possess remarkable optical properties that make them useful for medical biotechnology applications such as diagnosis and photothermal therapy. However, shape-selective synthesis of gold nanoprisms is not trivial and typically requires either toxic surfactants or time-consuming purification protocols, which can limit their applicability. Here, we show how triangular gold nanoprisms of different sizes can be purified by precipitation using the non-toxic glutathione ligand, thereby removing the need for toxic surfactants and bottleneck purification techniques. The protocol is amenable for direct scaling up as no instrumentation is required in the critical purification step. The new purification method provides a two-fold increased yield in gold nanoprisms compared to electrophoretic filtration, while providing nanoprisms of similar localized surface plasmon resonance wavelength. Crucially, the gold nanoprisms isolated using this methodology show fewer non-specific interactions with cells and lower cellular internalization, which paves the way for a higher selectivity in therapeutic applications

Obtención y caracterización de proteínas Fur de microorganismos de interés biotecnológico y sanitario

Fur (Ferric uptake regulator) es un regulador global responsable de mantener la homeostasis del hierro en la mayor parte de procariotas. En muchos patógenos, Fur es esencial para la expresión de factores de virulencia y la colonización de los tejidos infectados, por lo que Fur se considera una prometedora futura diana en la búsqueda de nuevos fármacos. En este trabajo se ha llevado a cabo el clonaje de las proteínas Fur de Legionella pneumophila y Staphylococcus aureus en un vector de expresión, se han sobreexpresado en Escherichia coli y se ha llevado a cabo una purificación y una primera caracterización de la proteína Fur de S. aureus, consistente en conocer algunas características básicas de la proteína, así como comprobar que ha mantenido su actividad. Se han elegido estos dos microorganismos ya que ambos son importantes patógenos humanos y pertenecen a grupos distintos, puesto que L. pneumophila se trata de una bacteria Gram negativa, mientras que S. aureus es Gram positiva. Esta primera caracterización tiene como objetivo conseguir toda la información posible que permitirá diseñar una estrategia de búsqueda de potenciales inhibidores mediante cribado de una quimioteca. Por tanto este trabajo se enmarca dentro de un proyecto más amplio de búsqueda de potenciales antimicrobianos basados en la inhibición de la proteína Fur en diferentes microorganismos patógenos

Gold nanoprism-nanorod face off: comparing the heating efficiency, cellular internalization and thermoablation capacity

[Aim]: This work compares the synthesis, heating capability, cellular internalization and thermoablation capacity of two different types of anisotropic gold nanoparticles: gold nanorods (NRs) and nanoprisms (NPrs). [Methods]: Both particles possess surface plasmon resonance absorption bands in the near-IR, and their heating efficiency upon irradiation with a continuous near-IR laser (1064 nm) was evaluated. The cellular internalization, location and toxicity of these PEG-stabilized NPrs and NRs were then assessed in the Vero cell line by transmission electron microscopy and inductively coupled plasma mass spectrometry analysis, and their ability to induce cell death upon laser irradiation was then evaluated and compared. [Results & conclusion]: Although both nanoparticles are highly efficient photothermal converters, NRs possessed a more efficient heating capability, yet the in vitro thermoablation studies clearly demonstrated that NPrs were more effective at inducing cell death through photothermal ablation due to their greater cellular internalization.This work was supported by Shanghai Jiao Tong University, Fondo Social de la DGA (grupos DGA), Ministerio de la Economía y Competitividad del Gobierno de España for the public funding of Proyectos I+D+I – Programa Estatal de Investigación, Desarrollo e Innovación Orientada a los Retos de la Sociedad (project no. SAF2014–54763-C2–2-R) and the ERC-Starting grant 239931-NANOPUZZLE. For financial support SG Mitchell acknowledges the Fundación General CSIC (Programa ComFuturo); A Artiga acknowledges the Ministerio de Educación, Cultura y Deportes for an FPU grant (FPU014/06249); G Alfranca acknowledges the Ministry of Education for a China Scholarship Council (CSC) grant; and M Moros acknowledges the European Commission for an MCSA Fellowship (grant agreement no. 660228). TPeer Reviewe

A clinically compatible drug-screening platform based on organotypic cultures identifies vulnerabilities to prevent and treat brain metastasis

We report a medium-throughput drug-screening platform (METPlatform) based on organotypic cultures that allows to evaluate inhibitors against metastases growing in situ. By applying this approach to the unmet clinical need of brain metastasis, we identified several vulnerabilities. Among them, a blood-brain barrier permeable HSP90 inhibitor showed high potency against mouse and human brain metastases at clinically relevant stages of the disease, including a novel model of local relapse after neurosurgery. Furthermore, in situ proteomic analysis applied to metastases treated with the chaperone inhibitor uncovered a novel molecular program in brain metastasis, which includes biomarkers of poor prognosis and actionable mechanisms of resistance. Our work validates METPlatform as a potent resource for metastasis research integrating drug-screening and unbiased omic approaches that is compatible with human samples. Thus, this clinically relevant strategy is aimed to personalize the management of metastatic disease in the brain and elsewhere.Acknowledgments: This work was supported by MINECO (SAF2017-89643-R, SAF2014-57243-R, SAF2015-62547-ERC) (M.V.), Fundacion FERO (IX FERO Grant for Research in Oncology) (M.V.), Fundacio La Marato de TV3 (141) (M.V.), Melanoma Research Alliance (Bristol-Myers Squibb-Melanoma Research Alliance Young Investigator Award 2017 (https://doi.org/10.48050/pc.gr.75716)) (M.V.), Beug Foundation (Prize for Metastasis Research 2017) (M.V.), Fundacion Ramon Areces (CIVP19S8163) (M.V.) and CIVP20S10662 (E.O.P.), Worldwide Cancer Research (19-0177) (M.V.), H2020-FETOPEN (828972) (M.V.), Cancer Research Institute (Clinic and Laboratory Integration Program CRI Award 2018 (54545)) (M.V.), AECC (Coordinated Translational Groups 2017 (GCTRA16015SEOA) (M.V.), LAB AECC 2019 (LABAE19002-VALI) (M.V.), ERC CoG (864759) (M.V.), Sophien-Stiftung zur Förderung der klinischen Krebsforschung (T.W.), Promedica Stiftung (T.W.), Stiftung f€ur angewandte Krebsforschung (T.W.), Forschungskredit of the University of Zurich (FK-18-054) (T.W.), Betty and David Koetser Foundation for Brain Research (T.W.), Foundation for Applied Cancer Research in Zurich (T.W., M.W.), Comunidad de Madrid (S2017/BMD-3867 RENIM-CM and Y2018/NMT-4949 NanoLiver-CM) and European structural and investment funds (M.D.), ISCIII (PT20/00044) co-funded by FEDER “A way of making Europe” (M.D.), Ministero dell’Istruzione, dell’Universita e della Ricerca-MIUR, “Dipartimenti di Eccellenza 2018-2022”, (D15D18000410001) (L.B. and P.C.), Science Foundation Ireland Frontiers for the Future Award (19/FFP/6443) (L.Y.), Science Foundation Ireland Strategic Partnership Programme, Precision Oncology Ireland (18/SPP/3522) (L.Y.), Breast Cancer Now Fellowship Award/ with the generous support of Walk the Walk (2019AugSF1310) (D.V.), La Caixa-Severo Ochoa International PhD Program Fellowship (LCF/BQ/SO16/52270014) (L.Z.), La Caixa International PhD Program Fellowship-Marie Sklodowska-Curie (LCF/BQ/DI17/11620028) (P.G-G), MINECO-Severo Ochoa PhD Fellowship (BES-2017-081995) (L.A-E.), AECC Postdoctoral Fellowship (POSTD19016PRIE) (N.P.), Boehringer Ingelheim Fonds MD fellowship (L.M.). The contribution of the Experimental Therapeutics Programme was supported by core funding from the Spanish National Cancer Research Center (CNIO). CNIO is supported by the ISCIII, the Ministerio de Ciencia e Innovacion, and is a Severo Ochoa Center of Excellence (SEV-2015-0510). The CNIC is supported by the ISCIII, the Ministerio de Ciencia e Innovacion and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (SEV-2015-0505). M.V. was named Ramon y Cajal Investigator (RYC-2013-13365) and is member of EMBO YIP (4053)

A clinically compatible drug-screening platform based on organotypic cultures identifies vulnerabilities to prevent and treat brain metastasis

We report a medium‐throughput drug‐screening platform (METPlatform) based on organotypic cultures that allows to evaluate inhibitors against metastases growing in situ. By applying this approach to the unmet clinical need of brain metastasis, we identified several vulnerabilities. Among them, a blood–brain barrier permeable HSP90 inhibitor showed high potency against mouse and human brain metastases at clinically relevant stages of the disease, including a novel model of local relapse after neurosurgery. Furthermore, in situ proteomic analysis applied to metastases treated with the chaperone inhibitor uncovered a novel molecular program in brain metastasis, which includes biomarkers of poor prognosis and actionable mechanisms of resistance. Our work validates METPlatform as a potent resource for metastasis research integrating drug‐screening and unbiased omic approaches that is compatible with human samples. Thus, this clinically relevant strategy is aimed to personalize the management of metastatic disease in the brain and elsewhere

Stratification of radiosensitive brain metastases based on an actionable S100A9/RAGE resistance mechanism

© The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.Whole-brain radiotherapy (WBRT) is the treatment backbone for many patients with brain metastasis; however, its efficacy in preventing disease progression and the associated toxicity have questioned the clinical impact of this approach and emphasized the need for alternative treatments. Given the limited therapeutic options available for these patients and the poor understanding of the molecular mechanisms underlying the resistance of metastatic lesions to WBRT, we sought to uncover actionable targets and biomarkers that could help to refine patient selection. Through an unbiased analysis of experimental in vivo models of brain metastasis resistant to WBRT, we identified activation of the S100A9-RAGE-NF-κB-JunB pathway in brain metastases as a potential mediator of resistance in this organ. Targeting this pathway genetically or pharmacologically was sufficient to revert the WBRT resistance and increase therapeutic benefits in vivo at lower doses of radiation. In patients with primary melanoma, lung or breast adenocarcinoma developing brain metastasis, endogenous S100A9 levels in brain lesions correlated with clinical response to WBRT and underscored the potential of S100A9 levels in the blood as a noninvasive biomarker. Collectively, we provide a molecular framework to personalize WBRT and improve its efficacy through combination with a radiosensitizer that balances therapeutic benefit and toxicity.info:eu-repo/semantics/publishedVersio