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Understanding the chemical events following trauma to the central nervous system could assist in identifying causative mechanisms and potential interventions to protect neural tissue. Here, we apply a partial optic nerve transection model of injury in rats and use synchrotron X-ray fluorescence microscopy (XFM) to perform elemental mapping of metals (K, Ca, Fe, Cu, Zn) and other related elements (P, S, Cl) in white matter tracts. The partial optic nerve injury model and spatial precision of microscopy allow us to obtain previously unattained resolution in mapping elemental changes in response to a primary injury and subsequent secondary effects. We observed significant elevation of Cu levels at multiple time points following the injury, both at the primary injury site and in neural tissue near the injury site vulnerable to secondary damage, as well as significant changes in Cl, K, P, S, and Ca. Our results suggest widespread metal dyshomeostasis in response to central nervous system trauma and that altered Cu homeostasis may be a specific secondary event in response to white matter injury. The findings highlight metal homeostasis as a potential point of intervention in limiting damage following nervous system injury.
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Traumatismos del Sistema Nervioso , Sustancia Blanca , Animales , Ratas , Cobre , Homeostasis , Modelos AnimalesRESUMEN
Recurrences frequently occur following surgical removal of primary tumors. In many cancers, adjuvant therapies have limited efficacy. Surgery provides access to the tumor microenvironment, creating an opportunity for local therapy, in particular immunotherapy, which can induce local and systemic anti-cancer effects. Here, we develop a surgically optimized biodegradable hyaluronic acid-based hydrogel for sustained intraoperative delivery of Toll-like receptor 3 agonist poly(I:C) and demonstrate that it significantly reduces tumor recurrence after surgery in multiple mouse models. Mechanistically, poly(I:C) induces a transient interferon alpha (IFNα) response, reshaping the tumor/wound microenvironment by attracting inflammatory monocytes and depleting regulatory T cells. We demonstrate that a pre-existing IFN signature predicts response to the poly(I:C) hydrogel, which sensitizes tumors to immune checkpoint therapy. The safety, immunogenicity, and surgical feasibility are confirmed in a veterinary trial in canine soft tissue tumors. The surgically optimized poly(I:C)-loaded hydrogel provides a safe and effective approach to prevent cancer recurrence.
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Hidrogeles , Recurrencia Local de Neoplasia , Ratones , Animales , Perros , Hidrogeles/uso terapéutico , Recurrencia Local de Neoplasia/prevención & control , Inmunoterapia , Modelos Animales de Enfermedad , Microambiente TumoralRESUMEN
Non-viral delivery is an important strategy for selective and efficient gene therapy, immunization, and RNA interference, which overcomes problems of genotoxicity and inherent immunogenicity associated with viral vectors. Liposomes and polymers are compelling candidates as carriers for intracellular, non-viral delivery, but maximal efficiencies of around 1% have been reported for the most advanced non-viral carriers. Here, we develop a library of dendronized bottlebrush polymers with controlled defects, displaying a level of precision surpassed only by biological molecules like DNA, RNA, and proteins. We test concurrent and competitive delivery of DNA and show for the first time that, while intracellular communication is thought to be an exclusively biomolecular phenomenon, such communication between synthetic macromolecular complexes can also take place. Our findings challenge the assumption that delivery agents behave as bystanders that enable transfection by passive intracellular release of genetic cargo and improve upon coarse strategies in intracellular carrier design lacking control over polymer sequence, architecture, and composition, leading to a hit-or-miss outcome. Understanding the communication that takes place between macromolecules will help improve the design of non-viral delivery agents and facilitate translation of genome engineering, vaccines, and nucleic acid-based therapies.
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Liposomas , Polímeros , Comunicación Celular , ADN/metabolismo , Técnicas de Transferencia de Gen , Liposomas/metabolismo , TransfecciónRESUMEN
We present a series of synthetic polymer hydrogels which break the traditional correlation between pore size and mechanical properties. The hydrogels are prepared from a dendronised polymer architecture based on a methacrylate copolymer to which poly(amido amine) dendrons are attached. Our approach will be useful in tailoring hydrogels for tissue engineering, controlled drug release, and flexible electronics.
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Despite decades of study, the molecular mechanisms and selectivity of the biomolecular components of honeybee (Apis mellifera) venom as anticancer agents remain largely unknown. Here, we demonstrate that honeybee venom and its major component melittin potently induce cell death, particularly in the aggressive triple-negative and HER2-enriched breast cancer subtypes. Honeybee venom and melittin suppress the activation of EGFR and HER2 by interfering with the phosphorylation of these receptors in the plasma membrane of breast carcinoma cells. Mutational studies reveal that a positively charged C-terminal melittin sequence mediates plasma membrane interaction and anticancer activity. Engineering of an RGD motif further enhances targeting of melittin to malignant cells with minimal toxicity to normal cells. Lastly, administration of melittin enhances the effect of docetaxel in suppressing breast tumor growth in an allograft model. Our work unveils a molecular mechanism underpinning the anticancer selectivity of melittin, and outlines treatment strategies to target aggressive breast cancers.
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Scar formation after wound healing is a major medical problem. A better understanding of the dynamic nuclear architecture of the genome during wound healing could provide insights into the underlying pathophysiology and enable novel therapeutic strategies. Here, we demonstrate that TGF-ß-induced fibrotic stress increases formation of the dynamic secondary DNA structures called G-quadruplexes in skin fibroblasts, which is coincident with increased expression of collagen 1. This G-quadruplex formation is attenuated by a small molecule inhibitor of intracellular Ca2+ influx and an anti-fibrotic compound. In addition, we identify G-quadruplex-forming sequences in the promoter region of COL1A1, which encodes collagen 1, and confirm their ability to form G-quadruplex structures under physiologically relevant conditions. Our findings reveal a link between G-quadruplexes and scar formation that may lead to novel therapeutic interventions.
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Colágeno Tipo I/química , Colágeno Tipo I/genética , Fibroblastos/efectos de los fármacos , Factor de Crecimiento Transformador beta/farmacología , Calcio/metabolismo , Células Cultivadas , Colágeno Tipo I/metabolismo , Cadena alfa 1 del Colágeno Tipo I , Fibroblastos/citología , Fibroblastos/metabolismo , Fibrosis , G-Cuádruplex , Humanos , Regiones Promotoras Genéticas , Sitio de Iniciación de la Transcripción , Regulación hacia Arriba , Cicatrización de HeridasRESUMEN
Polymers are an attractive anchoring platform for the synthesis of radioimmunoconjugates. They enable independent control over the amount of radioisotope loading and antibody attachment, which is pivotal in developing tailorable formulations for personalised medicine. Herein, we report the synthesis of p(HEMA-ran-GMA) for the conjugation of lutetium ions and rituximab as a functional platform for radioimmunotherapy. We demonstrate the suitability of this platform using non-Hodgkin's lymphoma cells.
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Inmunoconjugados/química , Linfoma no Hodgkin/radioterapia , Radioinmunoterapia , Antineoplásicos Inmunológicos/química , Antineoplásicos Inmunológicos/farmacología , Antineoplásicos Inmunológicos/uso terapéutico , Supervivencia Celular/efectos de los fármacos , Química Clic , Compuestos Epoxi/química , Humanos , Inmunoconjugados/farmacología , Inmunoconjugados/uso terapéutico , Lutecio/química , Metacrilatos/química , Polímeros/química , Rituximab/química , Rituximab/farmacología , Rituximab/uso terapéuticoRESUMEN
Aberrant gene expression is a hallmark of cancer. Although transcription is traditionally considered 'undruggable', the development of CRISPR-associated protein 9 (Cas9) systems offers enormous potential to rectify cancer-associated transcriptional abnormalities in malignant cells. However delivery of this technology presents a critical challenge to overcome in order to realize clinical translation for cancer therapy. In this article we demonstrate for the first time, a fully synthetic strategy to enable CRISPR-mediated activation (CRISPRa) of tumour suppressor genes in vivo using a targeted intravenous approach. We show this via highly efficient transcriptional activation of two model tumour suppressor genes, Mammary Serine Protease Inhibitor (MASPIN, SERPINB5) and cysteine-rich 61/connective tissue growth factor/nephroblastoma-overexpressed 6 (CCN6, WISP3), in a mouse model of breast cancer. In particular, we demonstrate that targeted intravenous delivery of can be achieved using a novel nanoscale dendritic macromolecular delivery agent, with negligible toxicity and long lasting therapeutic effects, outlining a targeted effective formulation with potential to treat aggressive malignancies.
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The adsorption of serum proteins on the surface of nanoparticles (NPs) delivered into a biological environment has been known to alter NP surface properties and consequently their targeting efficiency. In this paper, we use random copolymer (p(HEMA- ran-GMA))-based NPs synthesized using 2-hydroxyethyl methacrylate (HEMA) and glycidyl methacrylate (GMA). We show that serum proteins bind to the NP and that functionalization with antibodies and peptides designed to facilitate NP passage across the blood-brain barrier (BBB) to bind specific cell types is ineffective. In particular, we use systematic in vitro and in vivo analyses to demonstrate that p(HEMA- ran-GMA) NPs functionalized with HIV-1 trans-activating transcriptor peptide (known to cross the BBB) and α neural/glial antigen 2 (NG2) (known for targeting oligodendrocyte precursor cells (OPCs)), individually and in combination, do not specifically target OPCs and are unable to cross the BBB, likely due to the serum protein binding to the NPs.
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Barrera Hematoencefálica/metabolismo , Nanopartículas/química , Nanopartículas/metabolismo , Animales , Transporte Biológico/fisiología , Compuestos Epoxi/química , Femenino , Masculino , Metacrilatos/química , Microscopía Confocal , Células Precursoras de Oligodendrocitos/metabolismo , Polímeros/química , RatasRESUMEN
Advances in the field of genome engineering demand the development of efficient non-viral transfection agents capable of delivering multiple distinct nucleic acids efficiently to cells (co-transfection). However, current delivery methods result in lower co-transfection efficiency than single plasmid transfections, and the efficiency decreases further with increasing numbers of plasmids. The development of a high-throughput methodology is required for the validation of co-transfection platforms to facilitate independent tracking of not only the multiple DNA plasmids during transfection but also the localisation of transfection agents. This is pivotal to determine the bottlenecks in achieving high transfection efficiencies at various stages of the cell internalisation and plasmid expression process. Herein we demonstrate that this can be achieved using a facile methodology in which quantum dots (QDs) are used to label two different plasmid DNA assemblies that are delivered to cells simultaneously using a dendronised polymer system. Multispectral confocal imaging can be used to separate signals from each polyplex as well as the expressed fluorescent reporter proteins to determine whether co-transfection difficulties result from poor internalisation or the inability of DNA to escape from polyplexes. The results demonstrate the utility of this facile approach to label polyplexes without interfering with gene expression, and enable high-throughput screening of transfection reagents for achieving optimal co-transfection.
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Gold nanorods are one of the most widely explored inorganic materials in nanomedicine for diagnostics, therapeutics and sensing1. It has been shown that gold nanorods are not cytotoxic and localize within cytoplasmic vesicles following endocytosis, with no nuclear localization2,3, but other studies have reported alterations in gene expression profiles in cells following exposure to gold nanorods, via unknown mechanisms4. In this work we describe a pathway that can contribute to this phenomenon. By mapping the intracellular chemical speciation process of gold nanorods, we show that the commonly used Au-thiol conjugation, which is important for maintaining the noble (inert) properties of gold nanostructures, is altered following endocytosis, resulting in the formation of Au(I)-thiolates that localize in the nucleus5. Furthermore, we show that nuclear localization of the gold species perturbs the dynamic microenvironment within the nucleus and triggers alteration of gene expression in human cells. We demonstrate this using quantitative visualization of ubiquitous DNA G-quadruplex structures, which are sensitive to ionic imbalances, as an indicator of the formation of structural alterations in genomic DNA.
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Núcleo Celular/genética , ADN/química , G-Cuádruplex , Oro/metabolismo , Nanotubos , Compuestos de Sulfhidrilo/metabolismo , Núcleo Celular/metabolismo , ADN/genética , Endocitosis , Regulación de la Expresión Génica , Oro/análisis , Células HEK293 , Humanos , Células MCF-7 , Nanotubos/análisis , Nanotubos/ultraestructura , Compuestos de Sulfhidrilo/análisisRESUMEN
Multimodal polymeric nanoparticles have many exciting diagnostic and therapeutic applications, yet their uptake and passage by the placenta, and applications in the treatment of pregnancy complications have not been thoroughly investigated. In this work, the maternal-fetal-placental biodistribution of anionic and cationic multimodal poly(glycidyl methacrylate) (PGMA) nanoparticles in pregnant rats at mid (ED10) and late (ED20) gestation was examined. Fluorescently-labelled and superparamagnetic PGMA nanoparticles functionalized with/without poly(ethyleneimine) (PEI) were administered to pregnant rats at a clinically-relevant dose and biodistribution and tissue uptake assessed. Quantitative measurement of fluorescence intensity or magnetic resonance relaxometry in tissue homogenates lacked the sensitivity to quantify tissue uptake. Confocal microscopy, however, identified uptake by maternal organs and the decidua (ectoplacental cone) and trophoblast giant cells of conceptuses at ED10. At ED20, preferential accumulation of cationic vs. anionic nanoparticles was observed in the placenta, with PGMA-PEI nanoparticles localised mainly within the chorionic plate. These findings highlight the significant impact of surface charge and gestational age in the biodistribution of nanoparticles in pregnancy, and demonstrate the importance of using highly sensitive measurement techniques to evaluate nanomaterial biodistribution and maternal-fetal exposure.
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Intercambio Materno-Fetal , Nanopartículas , Polímeros/metabolismo , Animales , Femenino , Edad Gestacional , Metilmetacrilatos/química , Metilmetacrilatos/metabolismo , Microscopía Confocal , Sistema Mononuclear Fagocítico/metabolismo , Imagen Óptica/métodos , Polímeros/química , Embarazo , Ratas , Distribución TisularRESUMEN
Tools for editing the genome and epigenome have revolutionised the field of molecular biology and represent a new frontier in targeted therapeutic intervention. Although efficiencies and specificities of genome editing technologies have improved with the development of TALEs and CRISPR platforms, intracellular delivery of these larger constructs still remains a challenge using existing delivery agents. Viral vectors, including lentiviruses and adeno-associated viruses, as well as some non-viral strategies, such as cationic polymers and liposomes, are limited by packaging capacity, poor delivery, toxicity, and immunogenicity. We report a highly controlled synthetic strategy to engineer a flexible dendritic polymer using click chemistry to overcome the aforementioned delivery challenges associated with genome engineering technologies. Using a systematic approach, we demonstrate that high transfection efficiencies and packaging capacity can be achieved using this non-viral delivery methodology to deliver zinc fingers, TALEs and CRISPR/dCas9 platforms.
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The highly restrictive blood-brain barrier (BBB) plays a critically important role in maintaining brain homeostasis and is pivotal for proper neuronal function. The BBB is currently considered the main limiting factor restricting the passage of large (up to 200 nm) intravenously administered nanoparticles to the brain. Breakdown of the barrier occurs as a consequence of cerebrovascular diseases and traumatic brain injury. In this article, we report that remote injuries in the CNS are also associated with BBB dysfunction. In particular, we show that a focal partial transection of the optic nerve triggers a previously unknown transient opening of the mammalian BBB that occurs in the visual centres. Importantly, we demonstrate that this transient BBB breakdown results in a dramatic change in the biodistribution of intravenously administered large polymeric nanoparticles which were previously deemed as BBB-impermeable.
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Barrera Hematoencefálica/metabolismo , Encefalopatías/fisiopatología , Nanopartículas/metabolismo , Traumatismos del Nervio Óptico , Nervio Óptico/patología , Polímeros/farmacocinética , Administración Intravenosa , Animales , Transporte Biológico , Encefalopatías/cirugía , Modelos Animales de Enfermedad , Femenino , Humanos , Nervio Óptico/cirugía , Ratas , Ratas Endogámicas , Distribución TisularRESUMEN
Synthetic multifunctional electrospun composites are a new class of hybrid materials with many potential applications. However, the lack of an efficient, reactive large-area substrate has been one of the major limitations in the development of these materials as advanced functional platforms. Herein, we demonstrate the utility of electrospun poly(glycidyl methacrylate) films as a highly versatile platform for the development of functional nanostructured materials anchored to a surface. The utility of this platform as a reactive substrate is demonstrated by grafting poly(N-isopropylacrylamide) to incorporate stimuli-responsive properties. Additionally, we demonstrate that functional nanocomposites can be fabricated using this platform with properties for sensing, fluorescence imaging, and magneto-responsiveness.
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The paradigm of using nanoparticle-based formulations for drug delivery relies on their enhanced passive accumulation in the tumor interstitium. Nanoparticles with active targeting capabilities attempt to further enhance specific delivery of drugs to the tumors via interaction with overexpressed cellular receptors. Consequently, it is widely accepted that drug delivery using actively targeted nanoparticles maximizes the therapeutic benefit and minimizes the off-target effects. However, the process of nanoparticle mediated active targeting initially relies on their passive accumulation in tumors. In this article, it is demonstrated that these two tumor-targeted drug delivery mechanisms are interrelated and dosage dependent. It is reported that at lower doses, actively targeted nanoparticles have distinctly higher efficacy in tumor inhibition than their passively targeted counterparts. However, the enhanced permeability and retention effect of the tumor tissue becomes the dominant factor influencing the efficacy of both passively and actively targeted nanoparticles when they are administered at higher doses. Importantly, it is demonstrated that dosage is a pivotal parameter that needs to be taken into account in the assessment of nanoparticle mediated targeted drug delivery.
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Nanopartículas/química , Ácidos Polimetacrílicos/química , Taxoides/farmacología , Transferrina/química , Animales , Línea Celular Tumoral , Docetaxel , Relación Dosis-Respuesta a Droga , Endocitosis , Humanos , Hígado/efectos de los fármacos , Hígado/metabolismo , Ratones Desnudos , Nanopartículas/ultraestructura , Bazo/efectos de los fármacos , Bazo/metabolismo , Taxoides/uso terapéuticoRESUMEN
Colloidal poly(glycidyl methacrylate) nanoparticles (NPs) are demonstrated to be platforms facilitating the "click" chemistry approach of surface functionalization for receptor targeting. Folate receptor-targeted NPs were synthesized, physicochemically characterized, confirmed for their biocompatibility, and validated for their selective targeting capabilities for ovarian cancer cells in vitro.
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Prion-like domains (PLDs) are low complexity sequences found in RNA binding proteins associated with the neurodegenerative disorder amyotrophic lateral sclerosis. Recently, PLDs have been implicated in mediating gene regulation via liquid-phase transitions that drive ribonucleoprotein granule assembly. In this paper, we report many PLDs in proteins associated with paraspeckles, subnuclear bodies that form around long noncoding RNA. We mapped the interactome network of paraspeckle proteins, finding enrichment of PLDs. We show that one protein, RBM14, connects key paraspeckle subcomplexes via interactions mediated by its PLD. We further show that the RBM14 PLD, as well as the PLD of another essential paraspeckle protein, FUS, is required to rescue paraspeckle formation in cells in which their endogenous counterpart has been knocked down. Similar to FUS, the RBM14 PLD also forms hydrogels with amyloid-like properties. These results suggest a role for PLD-mediated liquid-phase transitions in paraspeckle formation, highlighting this nuclear body as an excellent model system for understanding the perturbation of such processes in neurodegeneration.
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Núcleo Celular/metabolismo , Péptidos y Proteínas de Señalización Intracelular/fisiología , Priones/química , Proteínas de Unión al ARN/química , Proteínas Amiloidogénicas/química , Células HeLa , Humanos , Hidrogeles/química , Péptidos y Proteínas de Señalización Intracelular/química , Priones/metabolismo , Unión Proteica , Mapas de Interacción de Proteínas , Proteínas de Unión al ARN/metabolismoRESUMEN
Advances in nanotechnology have resulted in the design of effective, safe and tissue-selective nanocarriers for delivering therapeutics to treat malignancies, infections and other diseases. In pregnancy, nanoparticle-based drug formulations could have the potential to selectively target either the placenta and/or fetus, enabling 'fetal-friendly' drugs to be administered in pregnancy with minimal risk of off-target effects. A considerable amount of research has been carried out on maternal-placental-fetal nanoparticle uptake, transfer and toxicity using rodent and ex vivo models. However, the development of placental targeting strategies and the therapeutic evaluation of nanoformulations in pregnancy remains in its infancy. While some promising avenues are currently under investigation, much work is needed to bring the advantages of nanoparticle-based drug therapy in pregnancy to clinical reality.
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Sistemas de Liberación de Medicamentos/efectos adversos , Sistemas de Liberación de Medicamentos/métodos , Nanopartículas/administración & dosificación , Nanopartículas/efectos adversos , Nanotecnología/métodos , Femenino , Humanos , EmbarazoRESUMEN
SFPQ, (a.k.a. PSF), is a human tumor suppressor protein that regulates many important functions in the cell nucleus including coordination of long non-coding RNA molecules into nuclear bodies. Here we describe the first crystal structures of Splicing Factor Proline and Glutamine Rich (SFPQ), revealing structural similarity to the related PSPC1/NONO heterodimer and a strikingly extended structure (over 265 Å long) formed by an unusual anti-parallel coiled-coil that results in an infinite linear polymer of SFPQ dimers within the crystals. Small-angle X-ray scattering and transmission electron microscopy experiments show that polymerization is reversible in solution and can be templated by DNA. We demonstrate that the ability to polymerize is essential for the cellular functions of SFPQ: disruptive mutation of the coiled-coil interaction motif results in SFPQ mislocalization, reduced formation of nuclear bodies, abrogated molecular interactions and deficient transcriptional regulation. The coiled-coil interaction motif thus provides a molecular explanation for the functional aggregation of SFPQ that directs its role in regulating many aspects of cellular nucleic acid metabolism.