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 nanobiomedicin

Characterization of variable regions of the Gp120 protein from HIV-1 subtype C virus variants obtained from individuals at different disease stages in Sub-Saharan Africa

Background: The development of a vaccine against HIV/AIDS capable of preventing virus infection has been hampered by the HIV envelope (Env) heterogeneity that makes it difficult to induce neutralizing antibodies against Env proteins from different HIV clades. Several studies have indicated that gp120 Env protein sequence tends to change considerably during the course of HIV disease which allows the virus to escape the immune responses. In order to define gp120 sequence changes, we have characterized the V1, V2, V4 and V5 variable regions of gp120 variants from 72 HIV-1-clade-C-infected subjects from South Africa and Swaziland, which were naïve to antiretroviral (ARV) therapy and at different disease stages. Sequence characteristics, such as aminoacid sequence length, presence of Putative N- Glycosylation Sites (PNGSs) and electric charge were investigated. Methods: According to the Avidity Index value and CD4+ T cell count, patients were classified for disease stage in three groups: recent, chronic and late stage, each one comprised of 24 patients. The V1 to V5 Env variable regions were directly PCR amplified from plasma virus RNA and sequenced. Results: A significant increase in the amino acid sequence length of V1 and V4 domains, and a corresponding increase of the “shifting” PNGSs were observed in the HIV variants obtained from individuals at chronic stage of disease, as compared to the recent infection group. Finally, a significant increase of the net electric positive charge of the V5 loop was found in the HIV variants from the group of subjects with late disease, as compared to the chronic disease group. Conclusion: We conclude that changes in sequence length, glycosylation pattern and net electrical charge in the variable V1, V4 and V5 regions of gp120 occur in the course of HIV infection, possibly in response to the pressure of the host immune response

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

Characterization of variable regions of the Gp120 protein from HIV-1 subtype c virus variants obtained from individuals at different disease stages in Sub-Saharan Africa

Background: The development of a vaccine against HIV/AIDS capable of preventing virus infection has been hampered by the HIV envelope (Env) heterogeneity that makes it difficult to induce neutralizing antibodies against Env proteins from different HIV clades. Several studies have indicated that gp120 Env protein sequence tends to change considerably during the course of HIV disease which allows the virus to escape the immune responses. In order to define gp120 sequence changes, we have characterized the V1, V2, V4 and V5 variable regions of gp120 variants from 72 HIV-1-clade-C-infected subjects from South Africa and Swaziland, which were naïve to antiretroviral (ARV) therapy and at different disease stages. Sequence characteristics, such as aminoacid sequence length, presence of Putative N-Glycosylation Sites (PNGSs) and electric charge were investigated. Methods: According to the Avidity Index value and CD4+ T cell count, patients were classified for disease stage in three groups: recent, chronic and late stage, each one comprised of 24 patients. The V1 to V5 Env variable regions were directly PCR amplified from plasma virus RNA and sequenced. Results: A significant increase in the amino acid sequence length of V1 and V4 domains, and a corresponding increase of the "shifting" PNGSs were observed in the HIV variants obtained from individuals at chronic stage of disease, as compared to the recent infection group. Finally, a significant increase of the net electric positive charge of the V5 loop was found in the HIV variants from the group of subjects with late disease, as compared to the chronic disease group. Conclusion: We conclude that changes in sequence length, glycosylation pattern and net electrical charge in the variable V1, V4 and V5 regions of gp120 occur in the course of HIV infection, possibly in response to the pressure of the host immune response. © 2012 Cenci A