Inhalable Microparticles Embedding Biocompatible Magnetic Iron-Doped Hydroxyapatite Nanoparticles.

Recently, there has been increasing interest in developing biocompatible inhalable nanoparticle formulations, as they have enormous potential for treating and diagnosing lung disease. In this respect, here, we have studied superparamagnetic iron-doped calcium phosphate (in the form of hydroxyapatite) nanoparticles (FeCaP NPs) which were previously proved to be excellent materials for magnetic resonance imaging, drug delivery and hyperthermia-related applications. We have established that FeCaP NPs are not cytotoxic towards human lung alveolar epithelial type 1 (AT1) cells even at high doses, thus proving their safety for inhalation administration. Then, D-mannitol spray-dried microparticles embedding FeCaP NPs have been formulated, obtaining respirable dry powders. These microparticles were designed to achieve the best aerodynamic particle size distribution which is a critical condition for successful inhalation and deposition. The nanoparticle-in-microparticle approach resulted in the protection of FeCaP NPs, allowing their release upon microparticle dissolution, with dimensions and surface charge close to the original values. This work demonstrates the use of spray drying to provide an inhalable dry powder platform for the lung delivery of safe FeCaP NPs for magnetically driven applications.

Cryo-electron tomography of intact cardiac muscle reveals myosin binding protein-C linking myosin and actin filaments.

Myosin binding protein C (MyBP-C) is an accessory protein of the thick filament in vertebrate cardiac muscle arranged over 9 stripes of intervals of 430 Å in each half of the A-band in the region called the C-zone. Mutations in cardiac MyBP-C are a leading cause of hypertrophic cardiomyopathy the mechanism of which is unknown. It is a rod-shaped protein composed of 10 or 11 immunoglobulin- or fibronectin-like domains labelled C0 to C10 which binds to the thick filament via its C-terminal region. MyBP-C regulates contraction in a phosphorylation dependent fashion that may be through binding of its N-terminal domains with myosin or actin. Understanding the 3D organisation of MyBP-C in the sarcomere environment may provide new light on its function. We report here the fine structure of MyBP-C in relaxed rat cardiac muscle by cryo-electron tomography and subtomogram averaging of refrozen Tokuyasu cryosections. We find that on average MyBP-C connects via its distal end to actin across a disc perpendicular to the thick filament. The path of MyBP-C suggests that the central domains may interact with myosin heads. Surprisingly MyBP-C at Stripe 4 is different; it has weaker density than the other stripes which could result from a mainly axial or wavy path. Given that the same feature at Stripe 4 can also be found in several mammalian cardiac muscles and in some skeletal muscles, our finding may have broader implication and significance. In the D-zone, we show the first demonstration of myosin crowns arranged on a uniform 143 Å repeat.

Multimetallic Microparticles Increase the Potency of Rifampicin against Intracellular Mycobacterium tuberculosis.

Mycobacterium tuberculosis ( M.tb) has the extraordinary ability to adapt to the administration of antibiotics through the development of resistance mechanisms. By rapidly exporting drugs from within the cytosol, these pathogenic bacteria diminish antibiotic potency and drive the presentation of drug-tolerant tuberculosis (TB). The membrane integrity of M.tb is pivotal in retaining these drug-resistant traits. Silver (Ag) and zinc oxide (ZnO) nanoparticles (NPs) are established antimicrobial agents that effectively compromise membrane stability, giving rise to increased bacterial permeability to antibiotics. In this work, biodegradable multimetallic microparticles (MMPs), containing Ag NPs and ZnO NPs, were developed for use in pulmonary delivery of antituberculous drugs to the endosomal system of M.tb-infected macrophages. Efficient uptake of MMPs by M.tb-infected THP1 cells was demonstrated using an in vitro macrophage infection model, with direct interaction between MMPs and M.tb visualized with the use of electron FIB-SEM tomography. The release of Ag NPs and ZnO NPs within the macrophage endosomal system increased the potency of the model antibiotic rifampicin by as much as 76%, realized through an increase in membrane disorder of intracellular M.tb. MMPs were effective at independently driving membrane destruction of extracellular bacilli located at the exterior face of THP1 macrophages. This MMP system presents as an effective drug delivery vehicle that could be used for the transport of antituberculous drugs such as rifampicin to infected alveolar macrophages, while increasing drug potency. By increasing M.tb membrane permeability, such a system may prove effectual in improving treatment of drug-susceptible TB in addition to M.tb strains considered drug-resistant.

Intracellular nanoparticles mass quantification by near-edge absorption soft X-ray nanotomography.

We used soft X-ray three-dimensional imaging to quantify the mass of superparamagnetic iron oxide nanoparticles (SPION) within whole cells, by exploiting the iron oxide differential absorption contrast. Near-edge absorption soft X-ray nanotomography (NEASXT) combines whole-cell 3D structure determination at 50 nm resolution, with 3D elemental mapping and high throughput. We detected three-dimensional distribution of SPIONs within cells with 0.3 g/cm(3) sensitivity, sufficient for detecting the density corresponding to a single nanoparticle.

Cryo-soft X-ray tomography as a quantitative three-dimensional tool to model nanoparticle:cell interaction.

BACKGROUND: Recent advances in nanoparticle design have generated new possibilities for nano-biotechnology and nano-medicine. Here we used cryo-soft X-ray tomography (cryo-SXT) to collect comprehensive three-dimensional (3D) data to characterise the interaction of superparamagnetic iron oxide nanoparticles (SPION) with a breast cancer cell line. RESULTS: We incubated MCF-7 (a human breast cancer cell line) from 0 to 24 h with SPION (15 nm average diameter, coated with dimercaptosuccinic acid), a system that has been studied previously using various microscopy and bulk techniques. This system facilitates the validation and contextualization of the new 3D data acquired using the cryo-SXT-based approach. After vitrification, samples tested by correlative cryo-epifluorescent microscopy showed SPION accumulation in acidic vesicles related to the endocytic pathway. Microscopy grids bearing MCF-7 cells were then analysed by cryo-SXT to generate whole cell volume 3D maps. Cryo-SXT is an emerging technique that benefits from high X-ray penetration into the biological material to image close-to-native vitrified cells at nanometric resolution with no chemical fixation or staining agents. This unique possibility of obtaining 3D information from whole cells allows quantitative statistical analysis of SPION-containing vesicle (SCV) accumulation inside cells, including vesicle number and size, distances between vesicles, and their distance from the nucleus. CONCLUSIONS: Correlation between fluorescent microscopy, cryo-SXT and transmission electron microscopy allowed us to identify SCV and to generate 3D data for statistical analysis of SPION:cell interaction. This study supports continuous transfer of the internalized SPION from the plasma membrane to an accumulation area near the cell nucleus. Statistical analysis showed SCV increase in number and size concomitant with longer incubation times, and therefore an increase in their accumulated volume within the cell. This cumulative effect expands the accumulation area and cell organelles such as mitochondria are consequently displaced to the periphery. Our 3D cryo-SXT approach demonstrates that a comprehensive quantitative description of SPION:cell interaction is possible, which will serve as a basis for metal-based nanoparticle design and for selection of those best suited for hyperthermia treatment, drug delivery and image diagnosis in nanobiomedicine.

Cholesterol Flux Is Required for Endosomal Progression of African Swine Fever Virions during the Initial Establishment of Infection.

UNLABELLED: African swine fever virus (ASFV) is a major threat for porcine production that has been slowly spreading in Eastern Europe since its first appearance in the Caucasus in 2007. ASFV enters the cell by endocytosis and gains access to the cytosol to start replication from late endosomes and multivesicular bodies. Cholesterol associated with low-density lipoproteins entering the cell by endocytosis also follows a trafficking pathway similar to that of ASFV. Here we show that cholesterol plays an essential role in the establishment of infection as the virus traffics through the endocytic pathway. In contrast to the case for other DNA viruses, such as vaccinia virus or adenovirus 5, cholesterol efflux from endosomes is required for ASFV release/entry to the cytosol. Accumulation of cholesterol in endosomes impairs fusion, resulting in retention of virions inside endosomes. ASFV also remodels intracellular cholesterol by increasing its cellular uptake and redistributes free cholesterol to viral replication sites. Our analysis reveals that ASFV manipulates cholesterol dynamics to ensure an appropriate lipid flux to establish productive infection. IMPORTANCE: Since its appearance in the Caucasus in 2007, African swine fever (ASF) has been spreading westwards to neighboring European countries, threatening porcine production. Due to the lack of an effective vaccine, ASF control relies on early diagnosis and widespread culling of infected animals. We investigated early stages of ASFV infection to identify potential cellular targets for therapeutic intervention against ASF. The virus enters the cell by endocytosis, and soon thereafter, viral decapsidation occurs in the acid pH of late endosomes. We found that ASFV infection requires and reorganizes the cellular lipid cholesterol. ASFV requires cholesterol to exit the endosome to gain access to the cytoplasm to establish productive replication. Our results indicate that there is a differential requirement for cholesterol efflux for vaccinia virus or adenovirus 5 compared to ASFV.

Characterization of interaction of magnetic nanoparticles with breast cancer cells.

BACKGROUND: Different superparamagnetic iron oxide nanoparticles have been tested for their potential use in cancer treatment, as they enter into cells with high effectiveness, do not induce cytotoxicity, and are retained for relatively long periods of time inside the cells. We have analyzed the interaction, internalization and biocompatibility of dimercaptosuccinic acid-coated superparamagnetic iron oxide nanoparticles with an average diameter of 15 nm and negative surface charge in MCF-7 breast cancer cells. RESULTS: Cells were incubated with dimercaptosuccinic acid-coated superparamagnetic iron oxide nanoparticles for different time intervals, ranging from 0.5 to 72 h. These nanoparticles showed efficient internalization and relatively slow clearance. Time-dependent uptake studies demonstrated the maximum accumulation of dimercaptosuccinic acid-coated superparamagnetic iron oxide nanoparticles after 24 h of incubation, and afterwards they were slowly removed from cells. Superparamagnetic iron oxide nanoparticles were internalized by energy dependent endocytosis and localized in endosomes. Transmission electron microscopy studies showed macropinocytosis uptake and clathrin-mediated internalization depending on the nanoparticles aggregate size. MCF-7 cells accumulated these nanoparticles without any significant effect on cell morphology, cytoskeleton organization, cell cycle distribution, reactive oxygen species generation and cell viability, showing a similar behavior to untreated control cells. CONCLUSIONS: All these findings indicate that dimercaptosuccinic acid-coated superparamagnetic iron oxide nanoparticles have excellent properties in terms of efficiency and biocompatibility for application to target breast cancer cells.

T cells kill bacteria captured by transinfection from dendritic cells and confer protection in mice.

Dendritic cells (DCs) phagocytose, process, and present bacterial antigens to T lymphocytes to trigger adaptive immunity. In vivo, bacteria can also be found inside T lymphocytes. However, T cells are refractory to direct bacterial infection, leaving the mechanisms by which bacteria invade T cells unclear. We show that T cells take up bacteria from infected DCs by the process of transinfection, which requires direct contact between the two cells and is enhanced by antigen recognition. Prior to transfer, bacteria localize to the immunological synapse, an intimate DC/T cell contact structure that activates T cells. Strikingly, T cells efficiently eliminate the transinfecting bacteria within the first hours after infection. Transinfected T cells produced high levels of proinflammatory cytokines and were able to protect mice from bacterial challenge following adoptive transfer. Thus, T lymphocytes can capture and kill bacteria in a manner reminiscent of innate immunity.

Molecular characterization of HIV-1 subtype C gp-120 regions potentially involved in virus adaptive mechanisms.

The role of variable regions of HIV-1 gp120 in immune escape of HIV has been investigated. However, there is scant information on how conserved gp120 regions contribute to virus escaping. Here we have studied how molecular sequence characteristics of conserved C3, C4 and V3 regions of clade C HIV-1 gp120 that are involved in HIV entry and are target of the immune response, are modulated during the disease course. We found an increase of "shifting" putative N-glycosylation sites (PNGSs) in the α2 helix (in C3) and in C4 and an increase of sites under positive selection pressure in the α2 helix during the chronic stage of disease. These sites are close to CD4 and to co-receptor binding sites. We also found a negative correlation between electric charges of C3 and V4 during the late stage of disease counteracted by a positive correlation of electric charges of α2 helix and V5 during the same stage. These data allow us to hypothesize possible mechanisms of virus escape involving constant and variable regions of gp120. In particular, new mutations, including new PNGSs occurring near the CD4 and CCR5 binding sites could potentially affect receptor binding affinity and shield the virus from the immune response.

Cryo X-ray nano-tomography of vaccinia virus infected cells.

We have performed full-field cryo X-ray microscopy in the water window photon energy range on vaccinia virus (VACV) infected cells to produce tomographic reconstructions. PtK2 cells were infected with a GFP-expressing VACV strain and frozen by plunge fast freezing. The infected cells were selected by light fluorescence microscopy of the GFP marker and subsequently imaged in the X-ray microscope under cryogenic conditions. Tomographic tilt series of X-ray images were used to yield three-dimensional reconstructions showing different cell organelles (nuclei, mitochondria, filaments), together with other structures derived from the virus infection. Among them, it was possible to detect viral factories and two types of viral particles related to different maturation steps of VACV (immature and mature particles), which were compared to images obtained by standard electron microscopy of the same type of cells. In addition, the effect of radiation damage during X-ray tomographic acquisition was analyzed. Thin sections studied by electron microscopy revealed that the morphological features of the cells do not present noticeable changes after irradiation. Our findings show that cryo X-ray nano-tomography is a powerful tool for collecting three-dimensional structural information from frozen, unfixed, unstained whole cells with sufficient resolution to detect different virus particles exhibiting distinct maturation levels.

Performance of V3-based HIV-1 sero subtyping in HIV endemic areas.

HIV-1 serosubtyping based on reactivity to peptides from the V3 region of gp120 is a low-cost and easy to perform procedure often used in geographical areas with high prevalence and incidence of HIV infection. We evaluated the performance of V3-based serotyping on 148 sera from 118 HIV-1-infected individuals living in Uganda, with estimated dates of seroconversion. Of the 148 tested samples, 68 (46.0%) specifically reacted with only one of the V3 peptides included in the test (SP), 64 (43.2%) did not react with any peptide (NR) and 16 (10.8%) reacted with two or more peptides (CR). According to the estimated seroconversion date, the large majority of samples collected early after infection belonged to the NR group. These samples had also a low Avidity Index. In contrast, samples collected later after infection belonged mainly to CR and SP groups and had also a higher avidity index. These results indicate that the performance of V3-based assays depends on maturation of HIV-specific immune response and can be significantly lowered when these tests are carried out on specimens collected from recently infected individuals.

Detection of recent HIV infections in African individuals infected by HIV-1 non-B subtypes using HIV antibody avidity.

BACKGROUND: To estimate HIV incidence several methods have been used to discriminate recent HIV infections from long-standing infections using a single serum sample. OBJECTIVE: To evaluate the performance of the anti-HIV avidity index (AI) for identifying recent HIV infections in individuals with a known date of seroconversion from Uganda, where the predominant HIV subtypes are A and D. STUDY DESIGN: We selected 149 repository serum samples from Ugandan HIV-positive individuals and evaluated the AI. Specimens collected < or =6 months after seroconversion were considered as recent infections, and those collected >6 months as long-standing infections. All specimens were serotyped using a V3 peptide enzyme immunoassay. RESULTS: The mean AI was 0.55+/-0.21 among the 108 patients with recent infections and 0.93+/-0.14 among the 41 samples from long-standing infections (p<0.0001). The AI test showed a sensitivity of 85.2% and a specificity of 85.4% at a cutoff of 0.80. No significant association was observed between serotype and the misclassification of samples by AI. CONCLUSIONS: The AI, which is inexpensive and easy-to-perform, can be useful in identifying recent HIV infections in countries where HIV-1 non-B subtypes are prevalent.