Deny Hartono

Autophagy and oxidative stress associated with gold nanoparticles.

Elemental metal nanoparticles like cadmium and silver are known to cause oxidative stress and are also highly toxic. Yet for gold nanoparticles (AuNPs), it is not well established whether these particles are biologically toxic. Here we show that AuNPs, which were taken up by MRC-5 human lung fibroblasts in vitro, induce autophagy concomitant with oxidative stress. We also observed formation of autophagosomes together with the uptake of AuNPs in the lung fibroblasts as well as upregulation of autophagy proteins, microtubule-associated protein 1 light chain 3 (MAP-LC3) and autophagy gene 7 (ATG 7) in treated samples. AuNP treated cells also generated significantly more lipid hydroperoxides (p-value<0.05), a positive indication of lipid peroxidation. Verification with western blot analysis for malondialdehyde (MDA) protein adducts confirmed the presence of oxidative damage. In addition, AuNP treatment also induced upregulation of antioxidants, stress response genes and protein expression. Exposure to AuNPs is a potential source of oxidative stress in human lung fibroblasts and autophagy may be a cellular defence mechanism against oxidative stress toxicity.

Translocation and effects of gold nanoparticles after inhalation exposure in rats

This study was carried out to test the hypothesis that nanogold particles can accumulate in the olfactory bulb, and translocate from the lung to other organs after inhalation exposure. Gold nanoparticles were aerosolized and introduced through an exposure chamber. The number concentration of airborne nano-sized particles was 2×106 #NSPs/cm3 with >75% of particulates between 30 and 110 nm. Exposure for 5 days resulted in significant increase of Au in the lung and olfactory bulb as detected by ICP-MS, but after 15 days, significant accumulation of gold was detected in the lung, esophagus, tongue, kidney, aorta, spleen, septum, heart and blood. Microarray analysis showed downregulation of many genes related to muscle in the nanogold-exposed lung. Lipidomic analysis of the lung showed a specific decrease in phosphatidylserine 36:1 species. We conclude that nanogold is able to translocate from the lung to other organs with time, and causes significant effects in exposed tissues.

Genomic instability of gold nanoparticle treated human lung fibroblast cells

Gold nanoparticles (AuNPs) are one of the most versatile and widely researched materials for novel biomedical applications. However, the current knowledge in their toxicological profile is still incomplete and many on-going investigations aim to understand the potential adverse effects in human body. Here, we employed two dimensional gel electrophoresis to perform a comparative proteomic analysis of AuNP treated MRC-5 lung fibroblast cells. In our findings, we identified 16 proteins that were differentially expressed in MRC-5 lung fibroblasts following exposure to AuNPs. Their expression levels were also verified by western blotting and real time RT-PCR analysis. Of interest was the difference in the oxidative stress related proteins (NADH ubiquinone oxidoreductase (NDUFS1), protein disulfide isomerase associate 3 (PDIA3), heterogeneous nuclear ribonucleus protein C1/C2 (hnRNP C1/C2) and thioredoxin-like protein 1 (TXNL1)) as well as proteins associated with cell cycle regulation, cytoskeleton and DNA repair (heterogeneous nuclear ribonucleus protein C1/C2 (hnRNP C1/C2) and Secernin-1 (SCN1)). This finding is consistent with the genotoxicity observed in the AuNP treated lung fibroblasts. These results suggest that AuNP treatment can induce oxidative stress-mediated genomic instability.

A liquid crystal-based sensor for real-time and label-free identification of phospholipase-like toxins and their inhibitors.

We report a liquid crystal (LC)-based sensor for real-time and label-free identification of phospholipase-like toxins. Beta-bungarotoxin exhibits Ca(2+)-dependent phospholipase A(2) activity whereas alpha-bungarotoxin and myotoxin II do not exhibit any phospholipase activity. The sensor can selectively identify beta-bungarotoxin, when it hydrolyzes a phospholipid monolayer self-assembled at aqueous-LC interface, through orientational responses of LCs. As a result, optical signals that reflect the spatial and temporal distribution of phospholipids during the hydrolysis can therefore be generated in a real-time manner. The sensor is very sensitive and requires less than 5pg of beta-bungarotoxin for the detection. When phospholipase A(2) inhibitors are introduced together with beta-bungarotoxin, no orientational response of LCs can be observed. In addition, the regeneration of the sensor can be done without affecting the sensing performance. This work demonstrates a simple and cost-effective LC-based sensor for identifying phospholipase-like toxins and for screening compound libraries to find potential toxin inhibitors.

Imaging the disruption of phospholipid monolayer by protein-coated nanoparticles using ordering transitions of liquid crystals.

We report an easily visualized liquid crystal (LC)-based system to study the molecular interactions between protein-coated gold nanoparticles (AuNPs) and supported phospholipid monolayer self-assembled at the aqueous-LC interface. Protein-coated AuNPs were found to disrupt the phospholipid monolayer and resulted in the orientational transitions of LCs that support the phospholipid layer. The disruption of the phospholipid monolayer depends on the type of protein (albumin, neutravidin, and fibrinogen) adsorbing onto nanoparticles. Furthermore, our results suggest that hydrophobic interaction plays a major role in the disruption of the phospholipid layer by protein-coated AuNPs. Results obtained from this study may offer new understanding in the potential cytotoxicity of nanomaterials, where the interaction between nanoparticles and cell membrane is an important step.

The effect of cholesterol on protein-coated gold nanoparticle binding to liquid crystal-supported models of cell membranes

We report an easily visualized liquid crystal (LC)-based system to study the biophysical interactions between protein-coated gold nanoparticles (AuNPs) and LC-supported cell membrane model. The model consists of mixed phospholipid/cholesterol monolayer self-assembled at aqueous-LC interface. Protein-coated AuNPs were found to disrupt the mixed phospholipid/cholesterol monolayer. As a result, orientational transitions of LCs were triggered and optical responses of LCs from dark to bright were observed. The mixed monolayers with higher cholesterol contents were found to be more susceptible to the disruption by protein-coated AuNPs, and hydrophobic interaction played a major role in the monolayer disruption. We also found that the time for non-specific binding of fibrinogen-coated AuNPs to the mixed phospholipid/cholesterol monolayer was similar to that of specific binding of neutravidin-coated AuNPs to the mixed phospholipid/biotin-capped phospholipid monolayer. Results obtained from this study may offer new understanding in the potential nanotoxicity pathway, where the biophysical interaction between nanomaterials and cell membrane is an important step.

Characterization and separation of Cryptosporidium and Giardia cells using on-chip dielectrophoresis

Dielectrophoresis (DEP) has been shown to have significant potential for the characterization of cells and could become an efficient tool for rapid identification and assessment of microorganisms. The present work is focused on the trapping, characterization, and separation of two species of Cryptosporidium (C. parvum and C. muris) and Giardia lambia (G. lambia) using a microfluidic experimental setup. Cryptosporidium oocysts, which are 2-4 μm in size and nearly spherical in shape, are used for the preliminary stage of prototype development and testing. G. lambia cysts are 8-12 μm in size. In order to facilitate effective trapping, simulations were performed to study the effects of buffer conductivity and applied voltage on the flow and cell transport inside the DEP chip. Microscopic experiments were performed using the fabricated device and the real part of Clausius-Mossotti factor of the cells was estimated from critical voltages for particle trapping at the electrodes under steady fluid flow. The dielectric properties of the cell compartments (cytoplasm and membrane) were calculated based on a single shell model of the cells. The separation of C. muris and G. lambia is achieved successfully at a frequency of 10 MHz and a voltage of 3 Vpp (peak to peak voltage).

Self-assembly of cholesterol DNA at liquid crystal/aqueous interface and its application for DNA detection

In this letter, we report a strategy of detecting the DNA targets by using a thin layer of self-assembled cholesterol-labeled DNA probes at the liquid crystal (LC)/aqueous interface. When the system is exposed to 51 μg/ml of complementary DNA targets, the optical appearance of LC shows a continuous change from dark to bright under the crossed polars within 15 min. No obvious change can be observed when the system is exposed to one or two base-pair mismatch DNA targets. This system provides a principle for label-free and real-time detection of DNA targets without any fluorescent labeling.

Detecting and differentiating Escherichia coli strain TOP10 using optical textures of liquid crystals

We report a method for detecting Escherichia coli using a nematic liquid crystal (LC), 4-cyano-4′-pentylbiphenyl (5CB). Among three E. coli strains tested, TOP10 strain grown on agar plates induces a homeotropic orientation of LCs whereas DH5α and JM109 strains do not. This results in a clear distinction in the optical appearance of LCs as either uniformly dark or bright under polarised light. The LC-based method provides a simple, rapid and low-cost method of identifying E. coli strains.

Cell separation and transportation between two miscible fluid streams using ultrasound

This paper presents a two-stream microfluidic system for transporting cells or micro-sized particles from one fluid stream to another by acoustophoresis. The two fluid streams, one being the original suspension and the other being the destination fluid, flow parallel to each other in a microchannel. Using a half-wave acoustic standing wave across the channel width, cells or particles with positive acoustic contrast factors are moved to the destination fluid where the pressure nodal line lies. By controlling the relative flow rate of the two fluid streams, the pressure nodal line can be maintained at a specific offset from the fluid interface within the destination fluid. Using this transportation method, particles or cells of different sizes and mechanical properties can be separated. The cells experiencing a larger acoustic radiation force are separated and transported from the original suspension to the destination fluid stream. The other particles or cells experiencing a smaller acoustic radiation force continue flowing in the original solution. Experiments were conducted to demonstrate the effective separation of polystyrene microbeads of different sizes (3 μm and 10 μm) and waterborne parasites (Giardia lamblia and Cryptosporidium parvum). Diffusion occurs between the two miscible fluids, but it was found to have little effects on the transport and separation process, even when the two fluids have different density and speed of sound.