RESUMO
Increasing evidence suggests that amyloid polymorphism gives rise to different strains of amyloids with distinct toxicities and pathology-spreading properties. Validating this hypothesis is challenging due to a lack of tools and methods that allow for the direct characterization of amyloid polymorphism in hydrated and complex biological samples. Here, we report on the development of 11-mercapto-1-undecanesulfonate-coated gold nanoparticles (NPs) that efficiently label the edges of synthetic, recombinant, and native amyloid fibrils derived from different amyloidogenic proteins. We demonstrate that these NPs represent powerful tools for assessing amyloid morphological polymorphism, using cryogenic transmission electron microscopy (cryo-EM). The NPs allowed for the visualization of morphological features that are not directly observed using standard imaging techniques, including transmission electron microscopy with use of the negative stain or cryo-EM imaging. The use of these NPs to label native paired helical filaments (PHFs) from the postmortem brain of a patient with Alzheimer's disease, as well as amyloid fibrils extracted from the heart tissue of a patient suffering from systemic amyloid light-chain amyloidosis, revealed a high degree of homogeneity across the fibrils derived from human tissue in comparison with fibrils aggregated in vitro. These findings are consistent with, and strongly support, the emerging view that the physiologic milieu is a key determinant of amyloid fibril strains. Together, these advances should not only facilitate the profiling and characterization of amyloids for structural studies by cryo-EM, but also pave the way to elucidate the structural basis of amyloid strains and toxicity, and possibly the correlation between the pathological and clinical heterogeneity of amyloid diseases.
Assuntos
Amiloide/genética , Amiloide/metabolismo , Encéfalo/metabolismo , Microscopia Crioeletrônica/métodos , Ouro/química , Nanopartículas Metálicas/química , Polimorfismo Genético , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Amiloide/química , Humanos , Amiloidose de Cadeia Leve de Imunoglobulina/genética , Amiloidose de Cadeia Leve de Imunoglobulina/metabolismo , Amiloidose de Cadeia Leve de Imunoglobulina/patologia , Emaranhados NeurofibrilaresRESUMO
The deposition of amyloid-ß (Aß) plaques in the brain is a significant pathological signature of Alzheimer's disease, correlating with synaptic dysfunction and neurodegeneration. Several compounds, peptides, or drugs have been designed to redirect or stop Aß aggregation. Among them, the trideca-peptide CWG-LRKLRKRLLR (mApoE), which is derived from the receptor binding sequence of apolipoprotein E, is effectively able to inhibit Aß aggregation and to promote fibril disaggregation. Taking advantage of Atomic Force Microscopy (AFM) imaging and fluorescence techniques, we investigate if the clustering of mApoE on gold nanoparticles (AuNP) surface may affect its performance in controlling Aß aggregation/disaggregation processes. The results showed that the ability of free mApoE to destroy preformed Aß fibrils or to hinder the Aß aggregation process is preserved after its clustering on AuNP. This allows the possibility to design multifunctional drug delivery systems with clustering of anti-amyloidogenic molecules on any NP surface without affecting their performance in controlling Aß aggregation processes.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Apolipoproteínas E/metabolismo , Nanopartículas Metálicas/química , Fragmentos de Peptídeos/metabolismo , Doença de Alzheimer/metabolismo , Amiloide/metabolismo , Encéfalo/metabolismo , Análise por Conglomerados , Ouro/química , Humanos , Placa Amiloide/metabolismo , Ligação Proteica/fisiologiaRESUMO
We have learned from the recent COVID-19 pandemic that the emergence of a new virus can quickly become a global health burden and kill millions of lives. Antiviral drugs are essential in our fight against viral diseases, but most of them are virus-specific and are prone to viral mutations. We have developed broad-spectrum antivirals based on multivalent nanoparticles grafted with ligands that mimic the target of viral attachment ligands (VALs). We have shown that when the ligand has a sufficiently long hydrophobic tail, the inhibition mechanism switches from reversible (virustatic) to irreversible (virucidal). Here, we investigate further how ligand density and particle size affect antiviral efficacy, both in terms of half-inhibitory concentration (IC50) and of reversible vs irreversible mechanism. We designed antiviral silica nanoparticles modified with 11-mercaptoundecane-1-sulfonic acid (MUS), a ligand that mimics heparan sulfate proteoglycans (HSPG) and we showed that these nanoparticles can be synthesized with different sizes (4-200 nm) and ligand grafting densities (0.59-10.70 /nm2). By testing these particles against herpes simplex virus type 2 (HSV-2), we show that within the size and density ranges studied, the antiviral IC50 is determined solely by equivalent ligand concentration. The nanoparticles are found to be virucidal at all sizes and densities studied.
Assuntos
Antivirais , Nanopartículas , Humanos , Antivirais/farmacologia , Ligantes , Pandemias , Herpesvirus Humano 2 , Nanopartículas/químicaRESUMO
The arrangement of nanoscale building blocks into patterns with microscale periodicity is challenging to achieve via self-assembly processes. Here, we report on the phase-transition-driven collective assembly of gold nanoparticles in a thermotropic liquid crystal. A temperature-induced transition from the isotropic to the nematic phase under anchoring-driven planar alignment leads to the assembly of individual nanometer-sized particles into arrays of micrometer-sized agglomerates, whose size and characteristic spacing can be tuned by varying the cooling rate. Phase field simulations coupling the conserved and nonconserved order parameters exhibit a similar evolution of the morphology as the experimental observations. This fully reversible process offers control over structural order on the microscopic level and is an interesting model system for the programmable and reconfigurable patterning of nanocomposites with access to micrometer-sized periodicities.
RESUMO
One of the biggest threats for bacteria-based bioreactors in the biotechnology industry is infections caused by bacterial viruses called bacteriophages. More than 70% of companies admitted to encountering this problem. Despite phage infections being such a dangerous and widespread risk, to date, there are no effective methods to avoid them. Here we present a peptide-grafted compounds that irreversibly deactivate bacteriophages and remain safe for bacteria and mammalian cells. The active compounds consist of a core (cyclodextrin or gold nanoparticle) coated with a hydrophobic chain terminated with a peptide selective for bacteriophages. Such peptides were selected via a phage display technique. This approach enables irreversible deactivation of the wide range of T-like phages (including the most dangerous in phage infections, phage T1) at 37 °C in 1 h. We show that our compounds can be used directly inside the environment of the bioreactor, but they are also a safe additive to stocks of antibiotics and expression inducers (such as isopropyl ß-d-1-thiogalactopyranoside, i.e., IPTG) that cannot be autoclaved and are a common source of phage infections.
Assuntos
Infecções Bacterianas , Bacteriófagos , Ciclodextrinas , Nanopartículas Metálicas , Animais , Ciclodextrinas/farmacologia , Ouro/farmacologia , Bactérias , Peptídeos/farmacologia , MamíferosRESUMO
Viral infections caused by bacteriophages, i.e., viruses that kill bacteria are one of the most dangerous and common threats for bacteria-based bioreactors. More than 70% of biotechnology companies have admitted to encountering this problem. Despite phage infections being such a dangerous and widespread risk, there are no effective methods to avoid them to date. Herein, we present a novel technology based on nanoparticles that irreversibly deactivates bacteriophages and is safe for bacteria. Our method allows for the unsupervised protection of bacterial processes in the biotechnology industry. Gold nanoparticles coated with a mixture of negatively charged 11-mercapto 1-undecanesulfonic acid (MUS) and hydrophobic 1-octanethiol (OT) ligands are effective at deactivating various types of Escherichia coli-selective phages: T1, T4, and T7. The nanoparticles can lower the titer of phages up to 2 and 5 logs in 6 and 24 h at 50 °C, respectively. A comparative analysis of nanoparticles with different ligand shells illustrates the importance of the combination of negatively charged and hydrophobic ligands that is the key to achieving a good inhibitory concentration (EC50 ≤ 1 µg mL-1) for all tested phages. We show that the nanoparticles are harmless for the commonly used bacteria in industry Escherichia coli and are effective under conditions simulating the environment of bioreactors.
Assuntos
Bacteriófagos , Nanopartículas Metálicas , Bactérias , Escherichia coli , OuroRESUMO
Influenza viruses are a leading cause of morbidity and mortality worldwide. These air-borne pathogens are able to cross the species barrier, leading to regular seasonal epidemics and sporadic pandemics. Influenza viruses also possess a high genetic variability, which allows for the acquisition of resistance mutations to antivirals. Combination therapies with two or more drugs targeting different mechanisms of viral replication have been considered an advantageous option to not only enhance the effectiveness of the individual treatments, but also reduce the likelihood of resistance emergence. Using an in vitro infection model, we assessed the barrier to viral resistance of a combination therapy with the neuraminidase inhibitor oseltamivir and human interferon lambda against the pandemic H1N1 A/Netherlands/602/2009 (H1N1pdm09) virus. We serially passaged the virus in a cell line derived from human bronchial epithelial cells in the presence or absence of increasing concentrations of oseltamivir alone or oseltamivir plus interferon lambda. While the treatment with oseltamivir alone quickly induced the emergence of antiviral resistance through a single mutation in the neuraminidase gene, the co-administration of interferon lambda delayed the emergence of drug-resistant influenza virus variants. Our results suggest a possible clinical application of interferon lambda in combination with oseltamivir to treat influenza.
RESUMO
Influenza is one of the most widespread viral infections worldwide and represents a major public health problem. The risk that one of the next pandemics is caused by an influenza strain is high. It is important to develop broad-spectrum influenza antivirals to be ready for any possible vaccine shortcomings. Anti-influenza drugs are available but they are far from ideal. Arguably, an ideal antiviral should target conserved viral domains and be virucidal, that is, irreversibly inhibit viral infectivity. Here, a new class of broad-spectrum anti-influenza macromolecules is described that meets these criteria and display exceedingly low toxicity. These compounds are based on a cyclodextrin core modified on its primary face with long hydrophobic linkers terminated either in 6'sialyl-N-acetyllactosamine (6'SLN) or in 3'SLN. SLN enables nanomolar inhibition of the viruses while the hydrophobic linkers confer irreversibility to the inhibition. The combination of these two properties allows for efficacy in vitro against several human or avian influenza strains, as well as against a 2009 pandemic influenza strain ex vivo. Importantly, it is shown that, in mice, one of the compounds provides therapeutic efficacy when administered 24 h post-infection allowing 90% survival as opposed to no survival for the placebo and oseltamivir.
RESUMO
Diagnosis and treatment of brain disorders, such as epilepsy, neurodegenerative diseases and tumors, would benefit from innovative approaches to deliver therapeutic or diagnostic compounds into the brain parenchyma, with either a homogeneous or a targeted localized distribution pattern. To assess the mechanistic aspect of penetration of nanoparticles (NPs) into the brain parenchyma, a complex, yet controlled and facilitated environment was used: the isolated guinea pig brain maintained in vitro by arterial perfusion. In this unique preparation the blood-brain barrier and the interactions between vascular and neuronal compartments are morphologically and functionally preserved. In this study, superparamagnetic Au/Fe nanoparticles (MUS:OT Au/Fe NPs), recently studied as a promising magnetic resonance T2 contrast agent with high cellular penetration, were arterially perfused into the in vitro isolated brain and showed high and homogeneous penetration through transcytosis into the brain parenchyma. Ultramicroscopy investigation of the in vitro isolated brain sections by TEM analysis of the electron-dense core of the MUS:OT Au/Fe NPs was conducted to understand NPs' brain penetration through the BBB after in vitro arterial perfusion and their distribution in the parenchyma. Our data suggest that MUS:OT Au/Fe NPs enter the brain utilizing a physiological route and therefore can be exploited as brain penetrating nanomaterials with potential contrast agent and theranostics capabilities.
Assuntos
Encéfalo/metabolismo , Meios de Contraste/química , Ouro/química , Ferro/química , Nanopartículas de Magnetita/química , Nanopartículas Metálicas/química , Animais , Transporte Biológico , Barreira Hematoencefálica , Difusão , Sistemas de Liberação de Medicamentos , Cobaias , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Neurônios/metabolismo , Perfusão , Ratos , Ratos Sprague-Dawley , Nanomedicina TeranósticaRESUMO
Gold nanoparticles protected by a binary mixture of thiolate molecules have a ligand shell that can spontaneously separate into nanoscale domains. Complex morphologies arise in such ligand shells, including striped, patchy, and Janus domains. Characterization of these morphologies remains a challenge. Scanning tunneling microscopy (STM) imaging has been one of the key approaches to determine these structures, yet the imaging of nanoparticles' surfaces faces difficulty stemming from steep surface curvature, complex molecular structures, and the possibility of imaging artifacts in the same size range. Images obtained to date have lacked molecular resolution, and only domains have been resolved. There is a clear need for images that resolve the molecular arrangement that leads to domain formation on the ligand shell of these particles. Herein we report an advance in the STM imaging of gold nanoparticles, revealing some of the molecules that constitute the domains in striped and Janus gold nanoparticles. We analyze the images to determine molecular arrangements on parts of the particles, highlight molecular "defects" present in the ligand shell, show persistence of the features across subsequent images, and observe the transition from quasi-molecular to domain resolution. The ability to resolve single molecules in the ligand shell of nanoparticles could lead to a more comprehensive understanding of the role of the ligand structure in determining the properties of mixed-monolayer-protected gold nanoparticles.
RESUMO
The ligand shell that coats, protects, and imparts a large number of properties to gold nanoparticles is a 2-D self-assembled monolayer wrapped around a 3-D metallic core. Here we present a study of the molecular packing of ligand shells on gold nanoparticles based on the analysis of scanning tunneling microscopy (STM) images. We discuss methods for optimal nanoparticle sample preparation in relation to STM imaging conditions. We show that the packing of a self-assembled monolayer composed solely of octanethiols on gold nanoparticles depends on the particle's diameter with an average headgroup spacing of 5.4 A, which is different from that of similar monolayers formed on flat Au(111) surfaces (5.0 A). In the case of nanoparticles coated with mixtures of ligands-known to phase separate into randomly shaped and ordered domains on flat surfaces-we find that phase separation leads to the formation of concentric, ribbonlike domains of alternating composition. The spacing of these domains depends on the ligand shell composition. We find that, for a given composition, the spacing increases with diameter in a manner characterized by discontinuous transitions at "critical" particle sizes. We discuss possible interpretations for the observed trends in our data.