Super-sensitive time-resolved fluoroimmunoassay for thyroid-stimulating hormone utilizing europium(III) nanoparticle labels achieved by protein corona stabilization, short binding time, and serum preprocessing.

Thyrotropin or thyroid-stimulating hormone (TSH) is used as a marker for thyroid function. More precise and more sensitive immunoassays are needed to facilitate continuous monitoring of thyroid dysfunctions and to assess the efficacy of the selected therapy and dosage of medication. Moreover, most thyroid diseases are autoimmune diseases making TSH assays very prone to immunoassay interferences due to autoantibodies in the sample matrix. We have developed a super-sensitive TSH immunoassay utilizing nanoparticle labels with a detection limit of 60 nU L(-1) in preprocessed serum samples by reducing nonspecific binding. The developed preprocessing step by affinity purification removed interfering compounds and improved the recovery of spiked TSH from serum. The sensitivity enhancement was achieved by stabilization of the protein corona of the nanoparticle bioconjugates and a spot-coated configuration of the active solid-phase that reduced sedimentation of the nanoparticle bioconjugates and their contact time with antibody-coated solid phase, thus making use of the higher association rate of specific binding due to high avidity nanoparticle bioconjugates. Graphical Abstract We were able to decrease the lowest limit of detection and increase sensitivity of TSH immunoassay using Eu(III)-nanoparticles. The improvement was achieved by decreasing binding time of nanoparticle bioconjugates by small capture area and fast circular rotation. Also, we applied a step to stabilize protein corona of the nanoparticles and a serum-preprocessing step with a structurally related antibody

Diversity of actin architecture in human osteoclasts: Network of curved and branched actin supporting cell shape and intercellular micrometer level tubes

Osteoclasts are multinucleated bone-resorbing cells with a dynamic actin cytoskeleton. Osteoclasts are derived from circulating mononuclear precursors. Confocal and stimulated emission depletion (STED) super-resolution microscopy was used to investigate peripheral blood-derived human osteoclasts cultured on glass surfaces. STED and confocal microscopy demonstrated that the actin was curved and branched, for instance, in the vicinity of membrane ruffles. The overall architecture of the curved actin network extended from the podosomes to the top of the cell. The other novel finding was that a micrometer-level tube containing actin bridged the osteoclasts well above the level of the culture glass. The actin filaments of the tubes originated from the network of curved actin often surrounding a group of nuclei. Furthermore, nuclei were occasionally located inside the tubes. Our findings demonstrated the accumulation of c-Src, cortactin, cofilin, and actin around nuclei suggesting their role in nuclear processes such as the locomotion of nuclei. ARP2/3 labeling was abundant at the substratum level of osteoclasts and in the branched actin network, where it localized to the branching points. We speculate that the actin-containing tubes of osteoclasts may provide a means of transportation of nuclei, e.g., during the fusion of osteoclasts. These novel findings can pave the way for future studies aiming at the elucidation of the differentiation of multinucleated osteoclasts.</p

Factors affecting intracellular delivery and release of hydrophilic versus hydrophobic cargo from mesoporous silica nanoparticles on 2D and 3D cell cultures

Intracellular drug delivery by mesoporous silica nanoparticles (MSNs) carrying hydrophilic and hydrophobic fluorophores as model drug cargo is demonstrated on 2D cellular and 3D tumor organoid level. Two different MSN designs, chosen on the basis of the characteristics of the loaded cargo, were used: MSNs with a surface-grown poly(ethylene imine), PEI, coating only for hydrophobic cargo and MSNs with lipid bilayers covalently coupled to the PEI layer as a diffusion barrier for hydrophilic cargo. First, the effect of hydrophobicity corresponding to loading degree (hydrophobic cargo) as well as surface charge (hydrophilic cargo) on intracellular drug release was studied on the cellular level. All incorporated agents were able to release to varying degrees from the endosomes into the cytoplasm in a loading degree (hydrophobic) or surface charge (hydrophilic) dependent manner as detected by live cell imaging. When administered to organotypic 3D tumor models, the hydrophilic versus hydrophobic cargo-carrying MSNs showed remarkable differences in labeling efficiency, which in this case also corresponds to drug delivery efficacy in 3D. The obtained results could thus indicate design aspects to be taken into account for the development of efficacious intracellular drug delivery systems, especially in the translation from standard 2D culture to more biologically relevant organotypic 3D cultures.</p

Stimuli-responsive hybrid nanocarriers developed by controllable integration of hyperbranched PEI with mesoporous silica nanoparticles for sustained intracellular siRNA delivery

Neeraj Prabhakar,1,2 Jixi Zhang,3 Diti Desai,1 Eudald Casals,1 Tina Gulin-Sarfraz,1 Tuomas N&auml;reoja,2,4 Jukka Westermarck,5,6 Jessica M Rosenholm1 1Pharmaceutical Sciences Laboratory, Faculty of Science and Engineering, &Aring;bo Akademi University, 2Laboratory of Biophysics, Faculty of Medicine, University of Turku, Turku, Finland; 3College of Bioengineering, Chongqing&nbsp;University, Chongqing, People&rsquo;s Republic of China; 4Department of Neuroscience, Karolinska Institute, Stockholm, Sweden; 5Centre for Biotechnology, University of Turku and &Aring;bo Akademi, 6Department of Pathology, University of Turku, Turku, Finland Abstract: Small interfering RNA (siRNA) is a highly potent drug in gene-based therapy with the challenge being to deliver it in a sustained manner. The combination of mesoporous silica nanoparticles (MSNs) and polycations in the confined pore space allows for incorporation and controlled release of therapeutic siRNA payloads. We hereby constructed MSNs with expanded mesopores and pore-surface-hyperbranched poly(ethyleneimine) (PEI) tethered with redox-cleavable linkers that could carry a high payload of siRNA (120&nbsp;mg&middot;g-1). The developed nanocarriers were efficiently taken up by cancer cells and were subsequently able to escape to the cytoplasm from the endosomes, most likely owing to the integrated PEI. Triggered by the intracellular redox conditions, the siRNA was sustainably released inside the cells over a period of several days. Functionality of siRNAs was demonstrated by using cell-killing siRNA as cargo. Despite not being the aim of the developed system, in vitro experiments using cell-killing siRNAs showed that the efficacy of siRNA transfection was comparable to the commercial in vitro transfection agent Lipofectamine. Consequently, the developed MSN-based delivery system offers a potential approach to hybrid nanocarriers for more efficient and long-term siRNA delivery and, in a longer perspective, in vivo gene silencing for RNA interference (RNAi) therapy. Keywords: mesoporous silica nanoparticles, RNAi therapy, siRNA delivery, stimuli-responsive drug release, hybrid nanocarrier

Prolonged dye release from mesoporous silica-based imaging probes facilitates long-term optical tracking of cell populations in vivo

Nanomedicine is gaining ground worldwide in therapy and diagnostics. Novel nanoscopic imaging probes serve as imaging tools for studying dynamic biological processes in vitro and in vivo. To allow detectability in the physiological environment, the nanostructure-based probes need to be either inherently detectable by biomedical imaging techniques, or serve as carriers for existing imaging agents. In this study, the potential of mesoporous silica nanoparticles carrying commercially available fluorochromes as self-regenerating cell labels for long-term cellular tracking is investigated. The particle surface is organically modified for enhanced cellular uptake, the fluorescence intensity of labeled cells is followed over time both in vitro and in vivo. The particles are not exocytosed and particles which escaped cells due to cell injury or death are degraded and no labeling of nontargeted cell populations are observed. The labeling efficiency is significantly improved as compared to that of quantum dots of similar emission wavelength. Labeled human breast cancer cells are xenotransplanted in nude mice, and the fluorescent cells can be detected in vivo for a period of 1 month. Moreover, ex vivo analysis reveals fluorescently labeled metastatic colonies in lymph node and rib, highlighting the capability of the developed probes for tracking of metastasis. Mesoporous silica nanoparticles (MSNs) as carriers for optical imaging agents facilitate long-term monitoring of tumor progression and tracking of metastatic cells. MSNs are surface modified for efficient cellular labeling and incorporated with commercially available, affordable fluorescent dyes via different means. The optimized MSN-dye formulation preserves fluorescence intensity signal retention in vivo for a period of 1 month

Discovery of Compounds Inhibiting the ADP-Ribosyltransferase Activity of Pertussis Toxin

The targeted pathogen-selective approach to drug development holds promise to minimize collateral damage to the beneficial microbiome. The AB$_{5}$-topology pertussis toxin (PtxS1-S5) is a major virulence factor of Bordetella pertussis, the causative agent of the highly contagious respiratory disease whooping cough. Once internalized into the host cell, PtxS1 ADP-ribosylates α-subunits of the heterotrimeric Gαi-superfamily, thereby disrupting G-protein-coupled receptor signaling. Here, we report the discovery of the first small molecules inhibiting the ADP-ribosyltransferase activity of pertussis toxin. We developed protocols to purify milligram-levels of active recombinant B. pertussis PtxS1 from Escherichia coli and an in vitro high throughput-compatible assay to quantify NAD$^{+}$ consumption during PtxS1-catalyzed ADP-ribosylation of Gαi. Two inhibitory compounds (NSC228155 and NSC29193) with low micromolar IC$_{50}$-values (3.0 μM and 6.8 μM) were identified in the in vitro NAD$^{+}$ consumption assay that also were potent in an independent in vitro assay monitoring conjugation of ADP-ribose to Gαi. Docking and molecular dynamics simulations identified plausible binding poses of NSC228155 and in particular of NSC29193, most likely owing to the rigidity of the latter ligand, at the NAD$^{+}$-binding pocket of PtxS1. NSC228155 inhibited the pertussis AB$_{5}$ holotoxin-catalyzed ADP-ribosylation of Gαi in living human cells with a low micromolar IC$_{50}$-value (2.4 μM). NSC228155 and NSC29193 might prove to be useful hit compounds in targeted B. pertussis-selective drug development

Core-shell designs of photoluminescent nanodiamonds with porous silica coatings for bioimaging and drug delivery, II : Application

Recent advances within materials science and its interdisciplinary applications in biomedicine have emphasized the potential of using a single multifunctional composite material for concurrent drug delivery and biomedical imaging. Here we present a novel composite material consisting of a photoluminescent nanodiamond (ND) core with a porous silica (SiO2) shell. This novel multifunctional probe serves as an alternative nanomaterial to address the existing problems with delivery and subsequent tracing of the particles. Whereas the unique optical properties of ND allows for long-term live cell imaging and tracking of cellular processes, mesoporous silica nanoparticles (MSNs) have proven to be efficient drug carriers. The advantages of both ND and MSNs were hereby integrated in the new composite material, ND@MSN. The optical properties provided by the ND core rendered the nanocomposite suitable for microscopy imaging in fluorescence and reflectance mode, as well as super-resolution microscopy as a STED label; whereas the porous silica coating provided efficient intracellular delivery capacity, especially in surface-functionalized form. This study serves as a demonstration how this novel nanomaterial can be exploited for both bioimaging and drug delivery for future theranostic application

Discovery of compounds inhibiting the ADP-ribosyltransferase activity of pertussis toxin

Abstract The targeted pathogen-selective approach to drug development holds promise to minimize collateral damage to the beneficial microbiome. The AB₅-topology pertussis toxin (PtxS1-S5) is a major virulence factor of Bordetella pertussis, the causative agent of the highly contagious respiratory disease whooping cough. Once internalized into the host cell, PtxS1 ADP-ribosylates α-subunits of the heterotrimeric Gαi-superfamily, thereby disrupting G-protein-coupled receptor signaling. Here, we report the discovery of the first small molecules inhibiting the ADP-ribosyltransferase activity of pertussis toxin. We developed protocols to purify milligram-levels of active recombinant B. pertussis PtxS1 from Escherichia coli and an in vitro high throughput-compatible assay to quantify NAD⁺ consumption during PtxS1-catalyzed ADP-ribosylation of Gαi. Two inhibitory compounds (NSC228155 and NSC29193) with low micromolar IC₅₀-values (3.0 μM and 6.8 μM) were identified in the in vitro NAD⁺ consumption assay that also were potent in an independent in vitro assay monitoring conjugation of ADP-ribose to Gαi. Docking and molecular dynamics simulations identified plausible binding poses of NSC228155 and in particular of NSC29193, most likely owing to the rigidity of the latter ligand, at the NAD⁺-binding pocket of PtxS1. NSC228155 inhibited the pertussis AB₅ holotoxin-catalyzed ADP-ribosylation of Gαi in living human cells with a low micromolar IC₅₀-value (2.4 μM). NSC228155 and NSC29193 might prove to be useful hit compounds in targeted B. pertussis-selective drug development