Suwimon Boonrungsiman

Human primary erythroid cells as a more sensitive alternative in vitro hematological model for nanotoxicity studies: Toxicological effects of silver nanoparticles.

Although immortalized cells established from cancerous cells have been widely used for studies in nanotoxicology studies, the reliability of the results derived from immortalized cells has been questioned because of their different characteristics from normal cells. In the present study, human primary erythroid cells in liquid culture were used as an in vitro hematological cell model for investigation of the nanotoxicity of silver nanoparticles (AgNPs) and comparing the results to the immortalized hematological cell lines HL60 and K562. The AgNPs caused significant cytotoxic effects in the primary erythroid cells, as shown by the decreased cell viability and induction of intracellular ROS generation and apoptosis, whereas they showed much lower cytotoxic and apoptotic effects in HL60 and K562 cells and did not induced ROS generation in these cell lines. Scanning electron microcopy revealed an interaction of AgNPs to the cell membrane in both primary erythroid and immortalized cells. In addition, AgNPs induced hemolysis in the primary erythroid cells in a dose-dependent manner, and transmission electron microcopy analysis revealed that AgNPs damaged the erythroid cell membrane. Taken together, these results suggest that human primary erythroid cells in liquid culture are a more sensitive alternative in vitro hematological model for nanotoxicology studies.

Chitosan-based DNA delivery vector targeted to gonadotropin-releasing hormone (GnRH) receptor

The main purpose of this study was to investigate the application of modified chitosan as a potential vector for gene delivery to gonadotropin-releasing hormone receptor (GnRHR)-expressing cells. Such design of gene carrier could be useful in particular for gene therapy for cancers related to the reproductive system, gene disorders of sexual development, and contraception and fertility control. In this study, a decapeptide GnRH was successfully conjugated to chitosan (CS) as confirmed by proton nuclear magnetic resonance spectroscopy (1H NMR) and Attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR). The synthesized GnRH-conjugated chitosan (GnRH-CS) was able to condense DNA to form positively charged nanoparticles and specifically deliver plasmid DNA to targeted cells in both two-dimensional (2D) and three-dimensional (3D) cell cultures systems. Importantly, GnRH-CS exhibited higher transfection activity compared to unmodified CS. In conclusion, GnRH-conjugated chitosan can be a promising carrier for targeted DNA delivery to GnRHR-expressing cells.

Shape and surface properties of titanate nanomaterials influence differential cellular uptake behavior and biological responses in THP-1 cells

We investigated cellular uptake behavior and biological responses of spherical and fibrous titanate nanomaterials in human monocyte THP-1 cells. Two titanate nanofibers (TiNFs), namely TF-1 and TF-2, were synthesized from anatase TiO2 nanoparticles (TNPs) via hydrothermal treatment. The synthesized TiNFs and TNPs were thoroughly characterized for their size, crystallinity, surface area and surface pH. TF-1 (∼2 µm in length) was amorphous with an acidic surface, while TF-2 (∼7 µm in length) was brookite with a basic surface. The results demonstrated that none of these titanate nanomaterials resulted in significant cytotoxicity, even at the highest doses tested (50 µg/ml), consistent with an absence of ROS generation and lack of change of mitochondrial membrane potential. While no cytotoxic effect was found in the titanate nanomaterials, TF-2 tended to decrease the proliferation of THP-1 cells. Furthermore, TF-2 resulted in an inflammatory cytokine response, as evidenced by dramatic induction of IL-8 and TNF-α release in TF2 but not TF-1 nor TNPs. These results suggest that shape of titanate nanomaterials plays an important role in cellular internalization, while surface pH may play a prominent role in inflammatory response in THP-1 cells.

Alterations of mineralized matrix by lead exposure in osteoblast (MC3T3-E1) culture

The present study investigated the effect of lead (Pb) on bone ultrastructure and chemistry using an in vitro bone model. MC3T3-E1 preosteoblasts were differentiated and treated with lead acetate at 0.4, 2, 10, and 50 μM. No abnormalities in either cell growth or bone nodule formation were observed with the treated dose of lead acetate. However, Pb treatments could significantly increase Pb accumulation in differentiated osteoblast cultures and upregulate expression of Divalent metal transporter 1 (Dmt1) in a dose dependent manner. Pb treatments also altered the expression of osteogenic genes, including secreted phosphoprotein 1, osteocalcin, type I collagen, and osteoprotegerin. Moreover, in mineralized osteoblast cultures, Pb was found to be mainly deposited as Pb salts and oxides, respectively. Ultrastructure analysis revealed Pb localizing with calcium and phosphorus in the mineralized matrix. In mineralizing osteoblast cells, Pb was found in the intracellular calcified vesicles which is one of the bone mineralization mechanisms. Pb was also present in mineral deposits with various shapes and sizes, such as small and large globular or needle-like mineral deposits representing early to mature stages of mineral deposits. Furthermore, Pb was found more in the globular deposits than the needle shaped mineral crystals. Taken together, our observations revealed how Pb incorporates into bone tissue, and showed a close association with bone apatite.

Thermoresponsive Bacteriophage Nanocarrier as a Gene Delivery Vector Targeted to the Gastrointestinal Tract

The use of the gastrointestinal tract as a site for the local delivery of DNA is an exciting prospect. In order to obtain an effective vector capable of delivering a gene of interest to target cells to achieve sufficient and sustained transgene expression, with minimal toxicity, we developed a new generation of filamentous bacteriophage. This particular bacteriophage was genetically engineered to display an arginine-glycine-aspartic acid (RGD) motif (an integrin-binding peptide) on the major coat protein pVIII and carry a mammalian DNA cassette. One unanticipated observation is the thermoresponsive behavior of engineered bacteriophage. This finding has led us to simplify the isolation method to purify bacteriophage particles from cell culture supernatant by low-temperature precipitation. Our results showed that, in contrast to non-surface modified, the RGD-modified bacteriophage was successfully used to deliver a transgene to mammalian cells. Our in vitro model of the human intestinal follicle-associated epithelium also demonstrated that bacteriophage particles were stable in simulated gastrointestinal fluids and able to cross the human intestinal barrier. In addition, we confirmed an adjuvant property of the engineered bacteriophage to induce nitric oxide production by macrophages. In conclusion, our study demonstrated the possibility of using bacteriophage for gene transfer in the gastrointestinal tract.

Comparison of suitable protein measurement for Thalassiosira weissflogii (Bacillariophyta) and Tetraselmis chuii (Chlorophyta)

To perform effective measurement of the protein contents, it is necessary to apply the most suitable extraction technique. We compared protein extraction techniques for two microalgal species with entirely different cell wall structures, such as diatom Thalassiosira weissflogii and green flagellate Tetraselmis chuii, using mechanical, physical, and chemical extraction methods. Our results showed that in Th. weissflogii, the control treatment without extraction and mechanical disruption using ultrasonication was sufficient to disrupt its brittle cell wall, resulting in the protein yield of 19.03 and 19.46% of dry weight (DW), respectively. In case of Te. chuii, the chemical extraction using 6% trichloroacetic acid at 95°C was the most suitable extraction method, resulting in the protein yield of 23.78% of DW. Therefore, the most suitable extraction technique must be individually selected for each species depending on their cell wall properties.