Biochemical and cellular mechanisms regulating Acanthamoeba castellanii adherence to host cells.

Free-living amoebae belonging to the genus Acanthamoeba are the causative agents of infections such as amoebic keratitis (AK), granulomatous amoebic encephalitis (GAE) and cutaneous lesions. The mechanisms involved in the establishment of infection are unknown. However, it is accepted that the initial phase of pathogenesis involves adherence to the host tissue. In this work, we analysed surface molecules with an affinity for epithelial and neuronal cells from the trophozoites of Acanthamoeba castellanii. We also investigated the cellular mechanisms that govern the process of trophozoite adhesion to the host cells. We first used confocal and epifluorescence microscopy to examine the distribution of the A. castellanii actin cytoskeleton during interaction with the host cells. The use of drugs, as cytochalasin B (CB) and latrunculin B (LB), revealed the participation of cytoskeletal filaments in the adhesion process. In addition, to identify the proteins and glycoproteins on the surface of A. castellanii, the trophozoites were labelled with biotin and biotinylated lectins. The results revealed bands of surface proteins, some of which were glycoproteins with mannose and N-acetylglucosamine residues. Interaction assays of biotinylated amoebae proteins with epithelial and neuronal cells showed that some surface proteins had affinity for both cell types. The results of this study provide insight into the biochemical and cellular mechanisms of the Acanthamoeba infection process.

Red fluorescent benzothiadiazole derivative loaded in different nanoformulations: Optical properties and their use in bio-imaging.

Fluorophores with optimized nonlinear optical properties have become prominent as contrast labels in laser scanning microscopy (LSM). The purpose of this work is to report on a novel benzothiadiazole derivative, namely 4,7-bis(5-((9,9-dioctyl-9H-fluoren-2-yl)ethynyl)thiophen-2-yl)benzo[c][1,2,5]thiadiazole (EFBT) and its optical performance when it is loaded into organic nanostructures intended as labels for LSM. Four different nanostructured labels were prepared: i) EFBT-loaded silica nanoparticles (SiNPs); ii) folate-bioconjugated SiNPs (SiNPs-FA); iii) EFBT-loaded PEGylated nanoparticles (NPs-PEG); and iv) EFBT-loaded folate-terminated PEGylated nanoparticles (NPs-PEG-FA). All these nanostructures are reported through a comparative study of their linear and nonlinear optical properties, including their performance as exogenous label agents in the cervical cancer cell line HeLa. This assessment of the performance of a specific fluorophore loaded into different nanostructured matrices (labels), and fairly compared under the same characterization conditions, including the LSM settings, is less common while previous reports had focused in comparing silica and PEGylated nanoparticles but loaded with different fluorophores. The results show that the internal molecular organization into each type of organic nanostructure impacted differently the properties of EFBT, where the silica matrix tend to preserve the optical performance of the fluorophore by preventing intermolecular interactions; in contrast, PEGylated nanoparticles favored molecular interactions and introduced non-radiative decay channels that degrades drastically the optical performance. Nevertheless, the use of functionalized ends entities produced a better cellular label uptake with PEGylated that with silica nanoparticles. In overall, the NPs-PEG-FA label produced the best HeLa imaging.

Fluorescent carbon nanoparticles synthesized from bovine serum albumin nanoparticles.

Fluorescent carbon based-nanoparticles are one of the emerging nanomaterials. Their preparation is relatively simple, rapid and inexpensive, and they are less toxic compared with metal and semiconductor nanoparticles. Here, we report a simple and reliable method to prepare water-soluble fluorescent carbon nanoparticles (FC-NPs) from nanoparticles made from a protein, bovine serum albumin. The obtained mean size of our carbon nanoparticles is between 3.8 and 3.4 nm, and they exhibit its maximum fluorescence emission at 424 and 408 nm respectively (with a reasonable QY of 16.5%) due to the presence of functional groups (NH, NH2, COOH and OH) that contain O and N; the presence of these functional groups was confirmed by FTIR and XPS analysis. The photoluminescent decay lifetime was modeled by a two exponential fit which indicates a contribution from both core and surface states. Also, the preliminary results showed that FC-NPs had a good interaction with HeLa and normal oral epithelial cells; nanoparticles were permeable at the cell membrane and went to the cytosol, and even to the nucleus, in less than 30 min, the fluorescence images of our preliminary results did not show any apparent toxic damage in any of the cell lines.

Biological response of HeLa cells to gold nanoparticles coated with organic molecules.

In this work, gold nanospheres functionalized with low weight organic molecules (4-aminothiphenol and cysteamine) were synthesized in a one-step method for their in vitro cytotoxic evaluation on HeLa cells. To enhance the biocompatibility of the cysteamine-capped GNPs, BSA was used due to its broad PH stability and high binding affinity to gold nanoparticles. Besides, the widely reported silica coated gold nanorods were tested here to contrast their toxic response against our nanoparticles coated with organic molecules. Our results shown, the viability measured at 1.9×10-5M did not show significant differences against negative controls for all the samples; however, the metabolic activity of HeLa cells dropped when they were exposed to silica gold nanorods in the range of concentrations from 2.9×10-7M to 3.0×10-4M, while in the cases of gold nanospheres, we found that only at concentrations below 1.9×10-5M metabolic activity was normal. Our preliminary results did not indicate any perceivable harmful toxicity to cell membrane, cytoskeleton or nucleus due to our nanospheres at 1.9×10-5M. Additional test should be conducted in order to ensure a safe use of them for biological applications, and to determine the extent of possible damage.