Bioactividad intrínseca de nanopartículas magnéticas recubiertas con poli-etilénimina sobre células tumorales pancreáticas y del sistema fagocítico mononuclear

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 12-06-2015La nanotecnología ha significado un considerable salto para aplicaciones biomédicas, debido a las propiedades químico‐ físicas intrínsecas de los biomateriales, y a la posibilidad de modificarlos de acuerdo a los intereses terapéuticos y diagnósticos específicos. Particularmente, las nanopartículas superparamagnéticas de óxido de hierro (SPION) son ampliamente explotadas por sus propiedades magnéticas, no solo en estrategias diagnósticos como agentes de contraste, sino también, como nanotransportadores de drogas. La posibilidad de concentrar estas nanopartículas en el sitio de interés mediante la aplicación de campo magnético externo aporta otra vía para disminuir los efectos secundarios sistémicos de muchas de las actuales drogas anti‐tumorales. Entre las terapias anti-tumorales en las que se han utilizado las nanopartículas está la terapia génica. Para funcionalizar las nanopartículas para transportar ADN/ARN, se han desarrollado diferentes polímeros catiónicos con la capacidad de conjugar ADN y/o ARN, garantizando su estabilidad química y funcional ante las diferentes condiciones fisiológicas (torrente sanguíneo, microambiente tumoral, compartimento endosomal, etc.). Sin embargo, un aspecto importante es la interacción de las propias nanopartículas magnéticas con diferentes sistemas celular como el sistema fagocítico mononuclear, células endoteliales, fibroblastos, y las propias células tumorales, que eventualmente podría definir la eficacia del tratamiento. En este trabajo, estudiamos la interacción de SPION recubiertas con polietilenimina (PEI) desnudas con macrófagos, fibroblastos, células endoteliales, y células tumorales murinas de páncreas. Demostramos que, además de poseer la capacidad de transfectar eficientemente (células HEK293), las SPIONs recubiertas con PEI inhiben la migración e invasión de las células tumorales murinas pancreáticas a través de la disminución de invadosomas y metaloproteinasas, MT1-MMP y MMP2, así como, probablemente, mediante la modulación de la ruta dependiente de microARN-21. Igualmente, estudiamos el efecto biológico que esta nanopartículas tienen sobre células endoteliales y fibroblastos, en los que induce la expresión de genes vinculados con procesos biológicos como la angiogénesis y respuesta inmune, así como inhiben la migración de las células endoteliales. Finalmente, demostramos que las SPIONs activan los macrófagos hacia un fenotipo M1 y modulan la dinámica de formación de podosomas. Además, inhiben la degradación de matriz extracelular por los macrófagos a través de la expresión de inhibidores de metaloproteinasas y la modulación de MMP2. En resumen, estos resultados demuestran que las SPIONs recubiertas con PEI no solo son un agente transfectante, sino también, poseen en sí mismas propiedades anti-metastásica y adyuvantes independientes de la capacidad de transfección, lo que sugiere su uso en estrategias anti-tumorales e inmunoterapéuticasNanotechnology has meant a considerable jump for biomedical applications, due to the intrinsic chemical and physical properties of biomaterials, and the possibility to modify them for specific diagnostic and therapeutic interest. Particularly, the superparamagnetic iron oxide nanoparticles (SPION) are widely exploited for their magnetic properties, not only in diagnostic strategies as contrast agents, but also as drug nanocarriers. The ability to concentrate these nanoparticles at the site of interest by applying external magnetic field provides another way to reduce systemic side effects of current anti-­tumor drugs. Gene therapy is among the anti-­tumor therapies in which the nanoparticles have been used. In this sense, different cationic polymers have been developed with the ability to package DNA and / or RNA, thereby preserving their chemical and functional stability in different physiological conditions (bloodstream, tumor microenvironment, endosomal compartment, etc.). However, an important consideration is the interaction of the magnetic nanoparticles themselves with cellular systems, e.g. the mononuclear phagocyte system, endothelial cells, fibroblasts and the tumor cells themselves, which could eventually influence the efficacy of treatment. In this study, we analyzed the interaction of naked polyethyleneimine (PEI)-­coated SPIONs with macrophages, fibroblasts, endothelial cells, and murine pancreatic tumor cells. We showed that, besides having the ability to efficiently transfect (HEK293 cells), PEI-­coated SPIONs inhibit migration and invasion of murine pancreatic tumor cells by decreasing invadosomas and metalloproteinases MT1-­MMP and MMP2 and, likely by modulating the microRNA-­21-­dependent pathways. Also we studied the biological effects on endothelial cells and fibroblasts, where they induce the expression of genes linked to biological processes, e.g. angiogenesis and immune response, and inhibit migration of endothelial cells. Finally, we showed that PEI-­coated SPIONs activate macrophages (M1 like phenotype) and modulate podosome dynamics. Also, they inhibited the degradation of extracellular matrix by macrophages through the metalloproteinase expression inhibition (MMP2) and induction of metalloproteinase inhibitors. In summary, these results demonstrate that the PEI-­coated SPIONs are not only are able to efficiently transfect cells, but also exert potential anti-­metastatic and adjuvant properties themselves, independently of transfection capacity, suggesting their use in anti-­tumor and immunotherapeutic strategie

The Intrinsic Biological Identities of Iron Oxide Nanoparticles and Their Coatings: Unexplored Territory for Combinatorial Therapies

Over the last 20 years, iron oxide nanoparticles (IONPs) have been the subject of increasing investigation due to their potential use as theranostic agents. Their unique physical properties (physical identity), ample possibilities for surface modifications (synthetic identity), and the complex dynamics of their interaction with biological systems (biological identity) make IONPs a unique and fruitful resource for developing magnetic field-based therapeutic and diagnostic approaches to the treatment of diseases such as cancer. Like all nanomaterials, IONPs also interact with different cell types in vivo, a characteristic that ultimately determines their activity over the short and long term. Cells of the mononuclear phagocytic system (macrophages), dendritic cells (DCs), and endothelial cells (ECs) are engaged in the bulk of IONP encounters in the organism, and also determine IONP biodistribution. Therefore, the biological effects that IONPs trigger in these cells (biological identity) are of utmost importance to better understand and refine the efficacy of IONP-based theranostics. In the present review, which is focused on anti-cancer therapy, we discuss recent findings on the biological identities of IONPs, particularly as concerns their interactions with myeloid, endothelial, and tumor cells. Furthermore, we thoroughly discuss current understandings of the basic molecular mechanisms and complex interactions that govern IONP biological identity, and how these traits could be used as a stepping stone for future research.Peer reviewe

Multiphoton imaging of melanoma 3D models with plasmonic nanocapsules

We report the synthesis of plasmonic nanocapsules and the cellular responses they induce in 3D melanoma models for their perspective use as a photothermal therapeutic agent. The wall of the nanocapsules is composed of polyelectrolytes. The inner part is functionalized with discrete gold nanoislands. The cavity of the nanocapsules contains a fluorescent payload to show their ability for loading a cargo. The nanocapsules exhibit simultaneous two-photon luminescent, fluorescent properties and X-ray contrasting ability. The average fluorescence lifetime (τ) of the nanocapsules measured with FLIM (0.3 ns) is maintained regardless of the intracellular environment, thus proving their abilities for bioimaging of models such as 3D spheroids with a complex architecture. Their multimodal imaging properties are exploited for the first time to study tumorspheres cellular responses exposed to the nanocapsules. Specifically, we studied cellular uptake, toxicity, intracellular fate, generation of reactive oxygen species, and effect on the levels of hypoxia by using multi-photon and confocal laser scanning microscopy. Because of the high X-ray attenuation and atomic number of the gold nanostructure, we imaged the nanocapsule-cell interactions without processing the sample. We confirmed maintenance of the nanocapsules’ geometry in the intracellular milieu with no impairment of the cellular ultrastructure. Furthermore, we observed the lack of cellular toxicity and no alteration in oxygen or reactive oxygen species levels. These results in 3D melanoma models contribute to the development of these nanocapsules for their exploitation in future applications as agents for imaging-guided photothermal therapy. Statement of Significance: The novelty of the work is that our plasmonic nanocapsules are multimodal. They are responsive to X-ray and to multiphoton and single-photon excitation. This allowed us to study their interaction with 2D and 3D cellular structures and specifically to obtain information on tumor cell parameters such as hypoxia, reactive oxygen species, and toxicity. These nanocapsules will be further validated as imaging-guided photothermal probe

Iron oxide and iron oxyhydroxide nanoparticles impair SARS-CoV-2 infection of cultured cells

Background Coronaviruses usually cause mild respiratory disease in humans but as seen recently, some human coronaviruses can cause more severe diseases, such as the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the global spread of which has resulted in the ongoing coronavirus pandemic. Results In this study we analyzed the potential of using iron oxide nanoparticles (IONPs) coated with biocompatible molecules like dimercaptosuccinic acid (DMSA), 3-aminopropyl triethoxysilane (APS) or carboxydextran (FeraSpin™ R), as well as iron oxyhydroxide nanoparticles (IOHNPs) coated with sucrose (Venofer®), or iron salts (ferric ammonium citrate -FAC), to treat and/or prevent SARS-CoV-2 infection. At non-cytotoxic doses, IONPs and IOHNPs impaired virus replication and transcription, and the production of infectious viruses in vitro, either when the cells were treated prior to or after infection, although with different efficiencies. Moreover, our data suggest that SARS-CoV-2 infection affects the expression of genes involved in cellular iron metabolism. Furthermore, the treatment of cells with IONPs and IOHNPs affects oxidative stress and iron metabolism to different extents, likely influencing virus replication and production. Interestingly, some of the nanoparticles used in this work have already been approved for their use in humans as anti-anemic treatments, such as the IOHNP Venofer®, and as contrast agents for magnetic resonance imaging in small animals like mice, such as the FeraSpin™ R IONP. Conclusions Therefore, our results suggest that IONPs and IOHNPs may be repurposed to be used as prophylactic or therapeutic treatments in order to combat SARS-CoV-2 infection.This work was supported by the following Grants: CSIC-COV19-012/012202020E154 funded by the Spanish National Research Council Interdisciplinary Thematic Platform (PTI) Global Health (PTI Salud Global), SGL2103021 funded by the European Commission-NextGenerationEU (Regulation EU2020/2094) through CSIC’s Global Health Platform (PTI Salud Global); PDC2021-120759-100 funded by MCIN/AEI/10. 13039/50110 00110 33 and by the “European Union NextGenerationEU/PRTR”, PID2020-112685RB-100 funded by MCIN/AEI/10. 13039/50110 00110 33, and the “Atracción de Talento Investigador” programme (2017-T1/BMD-5155) funded by the “Comunidad de Madrid”. Y. Portilla was first a predoctoral FPU scholar (FPU15/06170) funded by MCIN/AEI/10. 13039/50110 00110 33 and by “ESF Investing in your future”, then a predoctoral scholar funded by CSIC-COV19-012/012202020E154 and is now a postdoctoral scholar funded by the European Commission-NextGenerationEU (Regulation EU2020/2094) through the CSIC’s Global Health Platform (PTI Salud Global, SGL2103021). D. López-García received a JAE-INTRO 2020 Fellowship from the Spanish National Research Council (CSIC, JAEINT-20-01805). V. Mulens-Arias was a postdoctoral scholar working under a Juan de La Cierva-Incorporación Contract (IJCI-2017-31447) funded by MCIN/AEI/10. 13039/50110 00110 33. N. Daviu is a predoctoral scholar (FPU18/04828) funded by MCIN/AEI/10. 13039/50110 00110 33 and by “ESF Investing in your future”. This research work was performed in the framework of the Nanomedicine CSIC HUB (ref. 202180E048).Peer reviewe

Unravelling the mechanisms that determine the uptake and metabolism of magnetic single and multicore nanoparticles in a Xenopus laevis model.

Multicore superparamagnetic nanoparticles have been proposed as ideal tools for some biomedical applications because of their high magnetic moment per particle, high specific surface area and long term colloidal stability. Through controlled aggregation and packing of magnetic cores it is possible to obtain not only single-core but also multicore and hollow spheres with internal voids. In this work, we compare toxicological properties of single and multicore nanoparticles. Both types of particles showed moderate in vitro toxicity (MTT assay) tested in Hep G2 (human hepatocellular carcinoma) and Caco-2 (human colorectal adenocarcinoma) cells. The influence of surface chemistry in their biological behavior was also studied after functionalization with O,O′-bis(2-aminoethyl) PEG (2000 Da). For the first time, these nanoparticles were evaluated in a Xenopus laevis model studying their whole organism toxicity and their impact upon iron metabolism. The degree of activation of the metabolic pathway depends on the size and surface charge of the nanoparticles which determine their uptake. The results also highlight the potential of Xenopus laevis model bridging the gap between in vitro cell-based assays and rodent models for toxicity assessment to develop effective nanoparticles for biomedical applications

Dissecting the Inorganic Nanoparticle-Driven Interferences on Adhesome Dynamics

Inorganic nanoparticles have emerged as an attractive theranostic tool applied to different pathologies such as cancer. However, the increment in inorganic nanoparticle application in biomedicine has prompted the scientific community to assess their potential toxicities, often preventing them from entering clinical settings. Cytoskeleton network and the related adhesomes nest are present in most cellular processes such as proliferation, migration, and cell death. The nanoparticle treatment can interfere with the cytoskeleton and adhesome dynamics, thus inflicting cellular damage. Therefore, it is crucial dissecting the molecular mechanisms involved in nanoparticle cytotoxicity. This review will briefly address the main characteristics of different adhesion structures and focus on the most relevant effects of inorganic nanoparticles with biomedical potential on cellular adhesome dynamics. Besides, the review put into perspective the use of inorganic nanoparticles for cytoskeleton targeting or study as a versatile tool. The dissection of the molecular mechanisms involved in the nanoparticle-driven interference of adhesome dynamics will facilitate the future development of nanotheranostics targeting cytoskeleton and adhesomes to tackle several diseases, such as cancer

The Use of Iron Oxide Nanoparticles to Reprogram Macrophage Responses and the Immunological Tumor Microenvironment

The synthesis and functionalization of iron oxide nanoparticles (IONPs) is versatile, which has enhanced the interest in studying them as theranostic agents over recent years. As IONPs begin to be used for different biomedical applications, it is important to know how they affect the immune system and its different cell types, especially their interaction with the macrophages that are involved in their clearance. How immune cells respond to therapeutic interventions can condition the systemic and local tissue response, and hence, the final therapeutic outcome. Thus, it is fundamental to understand the effects that IONPs have on the immune response, especially in cancer immunotherapy. The biological effects of IONPs may be the result of intrinsic features of their iron oxide core, inducing reactive oxygen species (ROS) and modulating intracellular redox and iron metabolism. Alternatively, their effects are driven by the nanoparticle coating, for example, through cell membrane receptor engagement. Indeed, exploiting these properties of IONPs could lead to the development of innovative therapies. In this review, after a presentation of the elements that make up the tumor immunological microenvironment, we will review and discuss what is currently known about the immunomodulatory mechanisms triggered by IONPs, mainly focusing on macrophage polarization and reprogramming. Consequently, we will discuss the implications of these findings in the context of plausible therapeutic scenarios for cancer immunotherapy.VM-A is a post-doctoral scholar working under a Juan de la Cierva-Incorporación Contract (IJCI-2017-31447) from the Spanish Ministry of Science and Iion. The European Commission-funded VetBioNet INFRAIA-731014 project supports JR. This work was supported in part by grants from the Spanish Ministry of Science and Innovation (SAF-2017-82223-R and PID-2020-112685RB-100 to DB). DFB group is part of the Network “Nanotechnology in Translational Hyperthermia” (HIPERNANO, RED2018-102626-T) supported by the Spanish Ministry of Science and Innovation.Peer reviewe

Rational Design of Fractal Gold Nanosphere Assemblies with Optimized Photothermal Conversion using Quantitative Structure Property Relationship (QSPR) Approach

Assemblies of plasmonic nanoparticles have been proposed for various applications, including photothermal therapy, exploiting surface plasmon coupling phenomena. However, the rational design of fractal nanoparticle assembly remains challenging due to the lack of structural characterizations and modelization of real systems. Here we used the quantitative structure property relationship (QSPR) approach, driven by experimental data and statistical analysis, to establish a relationship between structural descriptors of fractal gold nanoparticle (GNP) aggregates and their light-to-heat conversion. A total of 160 assemblies of various size spherical GNPs with different polyelectrolyte chains were synthesized, which differ in their global charge, size, mass fractal dimension, and plasmonic properties. Fifteen independent descriptors of structure and properties were extracted and further analyzed by QSPR. Principal component analysis and multilinear regression reveal that light-to-heat conversion is mainly governed by the structure of the aggregates and not by the characteristics of the building blocks. This highlights the key role of the fractal dimension of the aggregate and of the ratio of GNP/polyelectrolyte mass to optimize photothermal effects. Rational criteria to optimize light-to-heat conversion within nonideal fractal assemblies of GNP were identified, relaxing on the choice of other parameters, such as GNP or aggregate size, that can be adapted to the desired biomedical applications.A.B. received a PhD fellowship from the doctoral school Physique en Ile de France (EDPIF). V.M.-A. received a postdoc fellowship from the Association pour le Recherche contre le Cancer (ARC, Aides Individuelles, postdoctorant, dossier 20150603405). The authors thank the ANR CarGold16-CE09-026, ANR Coligomere-18-CE06-0006, and the European Union’s Horizon 2020 research and innovation program under Grant Agreement No. 801305 for funding

Endocytosis-driven gold nanoparticle fractal rearrangement in cells and its influence on photothermal conversion

Cellular endocytosis and intracellular trafficking of nanoparticles induce dynamic rearrangements that profoundly modify the physical properties of nanoparticle and govern their biological outcomes when activated by external fields. The precise structure, organization, distribution, and density of gold nanoparticles (AuNPs) confined within intracellular compartments such as lysosomes have not been studied comprehensively, hampering the derivation of predictive models of their therapeutic activity within the cells of interest. By using transmission electron microscopy and small-angle X-ray scattering, we have determined that canonical spherical citrate-coated AuNPs in the 3–30 nm size range form fractal clusters in endolysosomes of macrophages, endothelial cells, and colon cancer cells. Statistical analysis revealed that the cluster size and endolysosome size are correlated but do not depend on the size of AuNPs unless larger preformed aggregates of AuNPs are internalized. Smaller AuNPs are confined in greater numbers in loose aggregates covering a higher fraction of the endolysosomes compared to the largest AuNPs. The fractal dimensions of intracellular clusters increased with the particle size, regardless of the cell type. We thus analyzed how these intracellular structure parameters of AuNPs affect their optical absorption and photothermal properties. We observed that a 2nd plasmon resonance band was shifted to the near-infrared region when the nanoparticle size and fractal dimensions of the intracellular cluster increased. This phenomenon of intracellular plasmon coupling is not directly correlated to the size of the intralysosomal cluster or the number of AuNPs per cluster but rather to the compacity of the cluster and the size of the individual AuNPs. The intracellular plasmon-coupling phenomenon translates to an efficient heating efficiency with the excitation of the three cell types at 808 nm, transforming the NIR-transparent canonical AuNPs with sizes below 30 nm into NIR-absorbing clusters in the tumor microenvironment. Harnessing the spontaneous clustering of spherical AuNPs by cells might be a more valuable strategy for theranostic purposes than deploying complex engineering to derive NIR-absorbent nanostructures out of their environment. Our paper sheds light on AuNP intracellular reorganization and proposes a general method to link their intracellular fates to their in situ physical properties exploited in medical applications.This work has received funding from the European Union's Horizon 2020 research and innovation program under grant agreement No 685795 and 801305. The authors thank the ANR CarGold-16-CE09-026, ANR Coligomere-18-CE06-0006 and ANR CycLys-18-CE09-0015-01. VMA received a Post-doc fellowship from Association pour le Recherche contre le Cancer (ARC, Aides Individuelles, post-doctorant, dossier 20150603405). ANB received a PhD fellowship by the Institute thematique multi-organismes (ITMO) Cancer and the doctoral school Frontières du Vivant (FdV) – Programme Bettencourt. AB received a PhD fellowship by the doctoral school Physique en Ile de France (EDPIF). This work was supported by the ITMO-Inserm Plan Cancer 2014-2019

An electrostatically conjugated-functional MNK1 aptamer reverts the intrinsic antitumor effect of polyethyleneimine-coated iron oxide nanoparticles in vivo in a human triple-negative cancer xenograft

The authors acknowledge the Scientific and Technical Assistance of the Transmission Electron Microscopy, Flow cytometry, Histology and Animal services at the Centro Nacional de Biotecnología (CNB, CSIC). ICP-OES analysis was carried out in the support laboratories at the Instituto de Ciencia de Materiales de Madrid (ICMM, CSIC). The authors are also grateful to Dr M. Sefton for author editing of the manuscript.Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature. This study was supported by the DTS18/00029 Grant (to VM Gonzalez and DF Barber) from the Instituto de Salud Carlos III (Plan Estatal de I + D + I 2017-2020), co-financed by the European Development Regional Fund ''A way to achieve Europe'' (ERDF), and by the PID2020-112685RB-100 Grant (to DF Barber) funded by MCIN/AEI/10.13039/501100011033. V. Mulens-Arias was a postdoctoral scholar working under a Juan de La Cierva-Incorporación Contract (IJCI-2017-31447, funded by MCIN/AEI/10.13039/501100011033) and Y. Portilla received a predoctoral FPU Grant (FPU15/06170 funded by MCIN/AEI/10.13039/501100011033 and the ESF Investing in your future). This research work was performed in the framework of the Nanomedicine CSIC HUB and Cancer CSIC HUB.Background: Triple-negative breast cancer (TNBC) remains a difficult breast cancer subtype to treat as it exhibits a particularly aggressive behavior. The dysregulation of distinct signaling pathways underlies this aggressive behavior, with an overactivation of MAP kinase interacting kinases (MNKs) promoting tumor cell behavior, and driving proliferation and migration. Therefore, MNK1 is an excellent target to impair the progression of TNBC and indeed, an MNK1-specific aptamer has proved to be efficient in inhibiting TBNC cell proliferation in vitro. Although polyethyleneimine-coated iron oxide nanoparticles (PEI-IONPs) have been used as transfection and immunomodulating agents, no study has yet addressed the benefits of using these nanoparticles as a magnetic carrier for the delivery of a functional aptamer. Results: Here, we tested the antitumor effect of a PEI-IONP complexed to the functional MNK1b-specific aptamer in vitro and in vivo. We demonstrated that these apMNKQ2@PEI-IONP nanoconjugates delivered three times more apMNKQ2 to MDA-MB-231 cells than the aptamer alone, and that this enhanced intracellular delivery of the aptamer had consequences for MNK1 signaling, reducing the amount of MNK1 and its target the phospho(Ser209)-eukaryotic initiation factor 4E (eIF4E). As a result, a synergistic effect of the apMNKQ2 and PEI-IONPs was observed that inhibited MDA-MB-231 cell migration, probably in association with an increase in the serum and glucocorticoid-regulated kinase-1 (SGK1) and the phospho(Thr346)-N-myc down-regulated gene 1 (NDRG1). However, intravenous administration of the apMNKQ2 alone did not significantly impair tumor growth in vivo, whereas the PEI-IONP alone did significantly inhibit tumor growth. Significantly, tumor growth was not inhibited when the apMNKQ2@PEI-IONP nanocomplex was administered, possibly due to fewer IONPs accumulating in the tumor. This apMNKQ2-induced reversion of the intrinsic antitumor effect of the PEI-IONPs was abolished when an external magnetic field was applied at the tumor site, promoting IONP accumulation. Conclusions: Electrostatic conjugation of the apMNKQ2 aptamer with PEI-IONPs impedes the accumulation of the latter in tumors, which appears to be necessary for PEI-IONPs to exert their antitumor activity. Graphical Abstract: [Figure not available: see fulltext.