RESUMO
High-resolution electrospray ionization ion mobility mass spectrometry has revealed a gas-phase isomer of the ubiquitous, extremely well-studied Au25(SR)18 cluster both in anionic and cationic form. The relative abundance of the isomeric structures can be controlled by in-source activation. The measured collision cross section of the new isomer agrees extremely well with a recent theoretical prediction (Matus, M. F.; et al. Chem. Commun. 2020, 56, 8087) corresponding to a Au25(SR)18- isomer that is energetically close and topologically connected to the known ground-state structure via a simple rotation of the gold core without breaking any Au-S bonds. The results imply that the structural dynamics leading to isomerization of thiolate-protected gold clusters may play an important role in their gas-phase reactions and that isomerization could be controlled by external stimuli.
RESUMO
Nanodrug delivery systems (NDDSs) based on water-soluble and atomically precise gold nanoclusters (AuNCs) are under the spotlight due to their great potential in cancer theranostics. Gastric cancer (GC) is one of the most aggressive cancers with a low early diagnosis rate, with drug therapy being the primary means to overcome its increasing incidence. In this work, we designed and characterized a set of 28 targeted nanosystems based on Au144(p-MBA)60 (p-MBA = para-mercaptobenzoic acid) nanocluster to be potentially employed as combination therapy in GC treatment. The proposed multifunctional AuNCs are functionalized with cytotoxic drugs (5-fluorouracil and epirubicin) or inhibitors of different signaling pathways (phosphatidylinositol 3-kinases (PI3K)/protein kinase B (Akt)/mammalian target of the rapamycin (mTOR), vascular endothelial growth factor (VEGF), and hypoxia-inducible factor (HIF)) and RGD peptides as targeting ligands, and we studied the role of ligand ratio in their optimal structural conformation using peptide-protein docking and all-atom molecular dynamics (MD) simulations. The results reveal that the peptide/drug ratio is a crucial factor influencing the potential targeting ability of the nanosystem. The most convenient features were observed when the peptide amount was favored over the drug in most cases; however, we demonstrated that the system composition and the intermolecular interactions on the ligand shell are crucial for achieving the desired effect. This approach helps guide the experimental stage, providing essential information on the size and composition of the nanosystem at the atomic level for ligand tuning in order to increase the desired properties.
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The electron binding mechanism in [Ag44(SC6H3F2)30]4- (SC6H3F2 = 3,4-difluorobenzenethiolate) tetra-anion was studied by photoelectron spectroscopy (PES), collision-induced dissociation mass spectrometry (CID-MS), and density functional theory (DFT) computations. PES showed that [Ag44(SC6H3F2)30]4- is energetically metastable with respect to electron autodetachment {[Ag44(SC6H3F2)30]3- + e-} and features a repulsive Coulomb barrier (RCB) with a height of 2.7 eV. However, CID-MS revealed that [Ag44(SC6H3F2)30]4- does not release an electron upon collisional excitation but undergoes dissociation. DFT computations performed on the known structure of [Ag44(SC6H3F2)30]4- confirmed the negative adiabatic electron affinity of [Ag44(SC6H3F2)30]3- and interpreted the experimental PE spectrum by taking into account tunneling electron photodetachment through the RCB.
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Hepatitis E Virus-like particles self-assemble in to noninfectious nanocapsids that are resistant to proteolytic/acidic mucosal delivery conditions. Previously, the nanocapsid was engineered to specifically bind and enter breast cancer cells, where successful tumor targeting was demonstrated in animal models. In the present study, the nanocapsid surface was modified with a solvent-exposed cysteine to conjugate monolayer protected gold nanoclusters (AuNC). Unlike commercially available gold nanoparticles, AuNCs monodisperse in water and are composed of a discrete number of gold atoms, forming a crystalline gold core. Au102 pMBA44 (Au102) was an ideal conjugate given its small 2.5 nm size and detectability in cryoEM. Au102 was bound directly to nanocapsid surface cysteines via direct ligand exchange. In addition, Au102 was functionalized with a maleimide linker (Au102_C6MI) for maleimide-thiol conjugation to nanocapsid cysteines. The AuNC-bound nanocapsid constructs were conjugated in various conditions. We found Au102_C6MI to bind nanocapsid more efficiently, while Au102 remained more soluble over time. Nanocapsids conjugated to Au102_C6MI were imaged in cryoEM for single particle reconstruction to localize AuNC position on the nanocapsid surface. We resolved five unique high intensity volumes that formed a ring-shaped density at the 5-fold symmetry center. This finding was further supported by independent rigid modeling.
Assuntos
Proteínas do Capsídeo/química , Ouro/química , Nanopartículas Metálicas/química , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Microscopia Crioeletrônica , Vírus da Hepatite E/metabolismo , Maleimidas/química , Microscopia Eletrônica de Transmissão , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificaçãoRESUMO
Visualization and tracking of viruses without compromising their functionality is crucial in order to understand virus targeting to cells and tissues, and to understand the subsequent subcellular steps leading to virus uncoating and replication. Enteroviruses are important human pathogens causing a vast number of acute infections, and are also suggested to contribute to the development of chronic diseases like type I diabetes. Here, we demonstrate a novel method to target site-specifically the hydrophobic pocket of enteroviruses. A probe, a derivative of Pleconaril, was developed and conjugated to various labels that enabled the visualization of enteroviruses under light and electron microscopes. The probe mildly stabilized the virus particle by increasing the melting temperature by 1-3 degrees, and caused a delay in the uncoating of the virus in the cellular endosomes, but could not however inhibit the receptor binding, cellular entry or infectivity of the virus. The hydrophobic pocket binding moiety of the probe was shown to bind to echovirus 1 particle by STD and tr-NOESY NMR methods. Furthermore, binding to echovirus 1 and Coxsackievirus A9, and to a lesser extent to Coxsackie virus B3 was verified by using a gold nanocluster labeled probe by TEM analysis. Molecular modelling suggested that the probe fits the hydrophobic pockets of EV1 and CVA9, but not of CVB3 as expected, correlating well with the variations in the infectivity and stability of the virus particles. EV1 conjugated to the fluorescent dye labeled probe was efficiently internalized into the cells. The virus-fluorescent probe conjugate accumulated in the cytoplasmic endosomes and caused infection starting from 6 hours onwards. Remarkably, before and during the time of replication, the fluorescent probe was seen to leak from the virus-positive endosomes and thus separate from the capsid proteins that were left in the endosomes. These results suggest that, like the physiological hydrophobic content, the probe may be released upon virus uncoating. Our results collectively thus show that the gold and fluorescently labeled probes may be used to track and visualize the studied enteroviruses during the early phases of infection opening new avenues to follow virus uncoating in cells.
Assuntos
Enterovirus Humano B/química , Corantes Fluorescentes/química , Oxidiazóis/química , Linhagem Celular Tumoral , Infecções por Coxsackievirus/metabolismo , Infecções por Coxsackievirus/patologia , Enterovirus Humano B/metabolismo , Enterovirus Humano B/ultraestrutura , Ouro/química , Humanos , Interações Hidrofóbicas e Hidrofílicas , OxazóisRESUMO
Development of precise protocols for accurate site-specific conjugation of monodisperse inorganic nanoparticles to biological material is one of the challenges in contemporary bionanoscience and nanomedicine. We report here a successful site-specific covalent conjugation of functionalized atomically monodisperse gold clusters with 1.5-nm metal cores to viral surfaces. Water-soluble Au102(para-mercaptobenzoic acid)44 clusters, functionalized by maleimide linkers to target cysteines of viral capsid proteins, were synthesized and conjugated to enteroviruses echovirus 1 and coxsackievirus B3. Quantitative analysis of transmission electron microscopy images and the known virus structures showed high affinity and mutual ordering of the bound gold clusters on the viral surface and a clear correlation between the clusters and the targeted cysteine sites close to the viral surface. Infectivity of the viruses was not compromised by loading of several tens of gold clusters per virus. These advances allow for future investigations of the structure-function relations of enteroviruses and enterovirus-related virus-like particles, including their entry mechanisms into cells and uncoating in cellular endosomes.