In vivo evaluation of riboflavin receptor targeted fluorescent USPIO in mice with prostate cancer xenografts.

Riboflavin (Rf) receptors bind and translocate Rf and its phosphorylated forms (e.g. flavin mononucleotide, FMN) into cells where they mediate various cellular metabolic pathways. Previously, we showed that FMN-coated ultrasmall superparamagnetic iron oxide (FLUSPIO) nanoparticles are suitable for labeling metabolically active cancer and endothelial cells in vitro. In this study, we focused on the in vivo application of FLUSPIO using prostate cancer xenografts. Size, charge, and chemical composition of FLUSPIO were evaluated. We explored the in vitro specificity of FLUSPIO for its cellular receptors using magnetic resonance imaging (MRI) and Prussian blue staining. Competitive binding experiments were performed in vivo by injecting free FMN in excess. Bio-distribution of FLUSPIO was determined by estimating iron content in organs and tumors using a colorimetric assay. AFM analysis and zeta potential measurements revealed a particulate morphology approximately 20-40 nm in size and a negative zeta potential (-24.23 ± 0.15 mV) in water. X-ray photoelectron spectroscopy and time-of-flight secondary ion mass spectrometry data confirmed FMN present on the USPIO nanoparticle surface. FLUSPIO uptake in prostate cancer cells and human umbilical vein endothelial cells was significantly higher than that of control USPIO, while addition of excess of free FMN reduced accumulation. Similarly, in vivo MRI and histology showed specific FLUSPIO uptake by prostate cancer cells, tumor endothelial cells, and tumor-associated macrophages. Besides prominent tumor accumulation, FLUSPIO accumulated in the liver, spleen, lung, and skin. Hence, our data strengthen our hypothesis that targeting riboflavin receptors is an efficient approach to accumulate nanomedicines in tumors opening perspectives for the development of diagnostic and therapeutic systems. ELECTRONIC SUPPLEMENTARY MATERIAL: Supplementary material is available for this article at 10.1007/s12274-016-1028-7 and is accessible for authorized users.

Imaging dehydration kinetics of a channel hydrate form of the HIV-1 attachment inhibitor prodrug BMS-663068.

An analysis of the free acid form of the HIV-1 attachment inhibitor prodrug BMS-663068-01 revealed a reversible moisture sorption event in the 42%-46% relative humidity (RH) range. An existing single-crystal analysis indicated that these observations were due to the formation of a nonstoichiometric channel hydrate. This effect was reproducible on repeated cycles, suggesting that the material's structural integrity was not compromised because of the interconversion process. Small, reversible, and predictable changes in the atomic structure were observed by solid-state nuclear magnetic resonance (ssNMR). Atomic force microscopy (AFM) and environmental scanning electron microscopy (ESEM) could discern changes in surface topography as a function of RH. Surface cracks were visible at 25% RH, most of which disappeared at 60% RH. This change was reversible on reducing the RH, with cracks reappearing in the same locations. A reduction in surface roughness was seen at high humidity, which was consistent with the uptake of moisture causing surface swelling. The observations by AFM/ESEM were consistent with the atomic alterations seen with ssNMR. Changes in unit cell dimensions are not uncommon with channel hydrates as the crystal lattice expands or contracts when the crystal structure absorbs/desorbs water, but concomitant, reversible surface morphology property changes have not been widely reported.

Elucidating the molecular mechanism of PAMAM-siRNA dendriplex self-assembly: effect of dendrimer charge density.

Dendrimers are attractive vehicles for nucleic acid delivery due to monodispersity and ease of chemical design. The purpose of this study was to elucidate the self-assembly process between small interfering RNA (siRNA) and different generation poly(amidoamine) dendrimers and to characterize the resulting structures. The generation 4 (G4) and G7 displayed equal efficiencies for dendriplex aggregate formation, whereas G1 lacked this ability. Nanoparticle tracking analysis and dynamic light scattering showed reduced average size and increased polydispersity at higher dendrimer concentration. The nanoparticle tracking analysis indicated that electrostatic complexation results in an equilibrium between differently sized complex aggregates, where the centre of mass depends on the siRNA:dendrimer ratio. Isothermal titration calorimetric data suggested a simple binding for G1, whereas a biphasic binding was evident for G4 and G7 with an initial exothermic binding and a secondary endothermic formation of larger dendriplex aggregates, followed by agglomeration. The initial binding became increasingly exothermic as the generation increased, and the values were closely predicted by molecular dynamics simulations, which also demonstrated a generation dependent differences in the entropy of binding. The flexible G1 displayed the highest entropic penalty followed by the rigid G7, making the intermediate G4 the most suitable for dendriplex formation, showing favorable charge density for siRNA binding.

Scanning electron microscopy of nuclear structure.

Accessing internal structure and retaining relative three dimensional (3D) organization within the nucleus has always proved difficult in the electron microscope. This is due to the overall size and largely fibrous nature of the contents, making large scale 3D reconstructions difficult from thin sections using transmission electron microscopy. This chapter brings together a number of methods developed for visualization of nuclear structure by scanning electron microscopy (SEM). These methods utilize the easily accessed high resolution available in field emission instruments. Surface imaging has proved particularly useful to date in studies of the nuclear envelope and pore complexes, and has also shown promise for internal nuclear organization, including the dynamic and radical reorganization of structure during cell division. Consequently, surface imaging in the SEM has the potential to make a significant contribution to our understanding of nuclear structure.

A protocol for isolation and visualization of yeast nuclei by scanning electron microscopy (SEM).

This protocol details methods for the isolation of yeast nuclei from budding yeast (Saccharomyces cerevisiae) and fission yeast (Schizosaccharomyces pombe), immuno-gold labeling of proteins and visualization by field emission scanning electron microscopy (FESEM). This involves the removal of the yeast cell wall and isolation of the nucleus from within, followed by subsequent processing for high-resolution microscopy. The nuclear isolation step can be performed in two ways: enzymatic treatment of yeast cells to rupture the cell wall and generate spheroplasts (cells that have partially lost their cell wall and their characteristic shape), followed by isolation of the nuclei by centrifugation or homogenization; and whole cell freezing followed by manual cell rupture and centrifugation. This protocol has been optimized for the visualization of the yeast nuclear envelope (NE), nuclear pore complexes (NPCs) and associated cyto-skeletal structures. Samples once processed for FESEM can be stored under vacuum for weeks, allowing considerable time for image acquisition.

HIV-1 DNA Flap formation promotes uncoating of the pre-integration complex at the nuclear pore.

The HIV-1 central DNA Flap acts as a cis-acting determinant of HIV-1 genome nuclear import. Indeed, DNA-Flap re-insertion within lentiviral-derived gene transfer vectors strongly stimulates gene transfer efficiencies. In this study, we sought to understand the mechanisms by which the central DNA Flap mediates HIV-1 nuclear import. Here, we show that reverse transcription (RT degrees) occurs within an intact capsid (CA) shell, independently of the routing process towards the nuclear membrane, and that uncoating is not an immediate post-fusion event, but rather occurs at the nuclear pore upon RT degrees completion. We provide the first observation with ultrastructural resolution of intact intracellular HIV-1 CA shells by scanning electron microscopy. In the absence of central DNA Flap formation, uncoating is impaired and linear DNA remains trapped within an integral CA shell precluding translocation through the nuclear pore. These data show that DNA Flap formation, the very last event of HIV-1 RT degrees, acts as a viral promoting element for the uncoating of HIV-1 at the nuclear pore.

High resolution analysis of mammalian nuclear structure throughout the cell cycle: implications for nuclear pore complex assembly during interphase and mitosis.

Nuclear pore complexes (NPCs) are the gateways for both active and passive bidirectional molecular transport between the nucleoplasm and cytoplasm. These mega-dalton assemblies are composed of multiple copies of approximately 30 distinct proteins termed nucleoporins. Higher eukaryotes display an "open" mitosis in which the NPCs, nuclear envelope, and lamina disassemble. During mitosis several nucleoporins are redistributed to kinetochores until they are recruited back to the periphery of chromatin as the NPCs are reassembled. Within this study we have developed and optimized the visualization of mammalian cells and their chromosome profiles throughout the cell-cycle. Close attention has been paid to the preservation of chromatin, membranes, and NPC structure to investigate the ultrastructural locations of specific proteins in both interphase and mitosis.

Actin- and protein-4.1-containing filaments link nuclear pore complexes to subnuclear organelles in Xenopus oocyte nuclei.

We imaged the interiors of relatively intact Xenopus oocyte nuclei by field emission scanning electron microscopy (feSEM) and visualized a network of filaments that attach to nuclear pore complexes and extend throughout the nucleus. Within the nucleus, these 'pore-linked filaments' (PLFs) were embedded into spherical structures 100 nm to approximately 5 microm in diameter. A subset of spheres was identified as Cajal bodies by immuno-gold labeling; the rest were inferred to be nucleoli and snurposomes both of which are abundant in Xenopus oocyte nuclei. Most PLFs were independent of chromatin. The thickness of a typical PLF was 40 nm (range, approximately 12-100 nm), including the 4 nm chromium coat. PLFs located inside the nucleus merged, bundled and forked, suggesting architectural adaptability. The PLF network collapsed upon treatment with latrunculin A, which depolymerizes actin filaments. Jasplakinolide, which stabilizes actin filaments, produced PLFs with more open substructure including individual filaments with evenly-spaced rows of radially projecting short filaments. Immuno-gold labeling of untreated oocyte nuclei showed that actin and protein 4.1 each localized on PLFs. Protein 4.1-gold epitopes were spaced at approximately 120 nm intervals along filaments, and were often paired ( approximately 70 nm apart) at filament junctions. We suggest that protein 4.1 and actin contribute to the structure of a network of heterogeneous filaments that link nuclear pore complexes to subnuclear organelles, and discuss possible functions for PLFs in nuclear assembly and intranuclear traffic.

Yeast nuclear pore complexes have a cytoplasmic ring and internal filaments.

The nuclear pore complex (NPC) controls transport of macromolecules across the nuclear envelope. It is large and complex but appears to consist of only approximately 30 different proteins despite its mass of > 60MDa. Vertebrate NPC structure has been analyzed by several methods giving a comprehensive architectural model. Despite our knowledge of yeast nucleoporins, structural data is more limited and suggests the basic organization is similar to vertebrates, but may lack some peripheral and other components. Using field emission scanning electron microscopy to probe NPC structure we found that the yeast, like higher eukaryotic, NPCs contain similar peripheral components. We can detect cytoplasmic rings and evidence of nucleoplasmic rings in yeasts. A filamentous basket is present on the nucleoplasmic face and evidence for cytoplasmic filaments is shown. We observed a central structure, possibly the transporter, that which may be linked to the cytoplasmic ring by internal filaments. Immuno-gold labeling suggested that Nup159p may be attached to the cytoplasmic ring, whereas Nup116p may be associated, partly, with the cytoplasmic filaments. Analysis of a Nup57p mutant suggested a role in maintaining the stability of cytoplasmic components of the NPC. We conclude that peripheral NPC components appear similar in yeasts compared to higher organisms and present a revised model for yeast NPC structural composition.