The role of surface functionalization on the pulmonary inflammogenicity and translocation into mediastinal lymph nodes of graphene nanoplatelets in rats.

Graphene, a two-dimensional monocrystalline layer of carbon atoms, has potential in many applications not only in material sciences, but also in the biomedical fields, but there is little information about the role of surface modification on the toxicity of graphene-based nanomaterials. Here, we evaluated the role of surface functionalization of the graphene nanoplatelets (GNPs) on the pulmonary inflammogenicity and translocation into mediastinal lymph nodes using a rat intratracheal instillation model. Six types of GNPs were used: All types of GNPs were based on the pristine GNPs (GNPdot), and different functional groups were conjugated onto them including a COOH (GNPCOOH), COH [Formula: see text], N-H [Formula: see text], F x (GNPF), and N=H [Formula: see text]. All types of GNPs showed very high potential for the generation of reactive oxygen species (ROS) in a dose-dependent manner when measured by a 2'7'-dichlorofluorescin diacetate assay. GNPs were instilled into the lungs of rats at 0.3 and 1 mg/rat for the evaluation of acute (24 h) inflammation and at 3 mg/rat for chronic (1 and 4 weeks) inflammation. At 24 h after instillation, all types of GNPs showed good dose-dependent increases in polymorphonuclear leukocytes with a clear dose-dependency although significant increases compared to vehicle control were found only in positively charged GNPs [Formula: see text]. While the acute inflammation in all treatment groups was returned to control levels at 1 and 4 weeks after instillation, GNPs showed similar patterns of translocation into the mediastinal lymph nodes with a higher degree over time. This study implies that the main factors of GNPs for producing lung inflammation are the potential for ROS generation and surface charge. In addition, functional groups on the GNPs might not play an important role in the extrapulmonary translocation into the mediastinal lymph nodes.

Evaluation of Haemagglutinin Content by RP-HPLC to Generate Pandemic Influenza Vaccine.

The potency of influenza vaccine is determined based on its hemagglutinin (HA) content. In general, single radial immunodiffusion (SRID) assay has been utilized as the standard method to measure HA content. However, preparation of reagents for SRID such as antigen and antibody takes approximately 2~3 months, which causes delays in the development of influenza vaccine. Therefore, quantification of HA content by other alternative methods is required. In this study, we measured HA contents of H1N1 antigen and H1N1 influenza vaccine by reverse phase-high performance liquid chromatography (RP-HPLC) methods. The presence of HA1 and HA2 was investigated by silver staining and Western blot assay. In addition, accuracy and repeatability of HA measurement by RP-HPLC were evaluated. Comparison of HA concentration by SRID and RP-HPLC revealed a precise correlation between the two methods. Our results suggest that RP-HPLC assay can replace SRID in the event of a pandemic flu outbreak for rapid vaccine development.

Retinopathy Induced by Zinc Oxide Nanoparticles in Rats Assessed by Micro-computed Tomography and Histopathology.

Nanotechnology has advanced at an extremely rapid pace over the past several years in numerous fields of research. However, the uptake of nanoparticles (NPs) into the body after administration through various routes may pose a risk to human health. In this study, we investigated the potential ocular toxicity of 20-nm, negatively- charged zinc oxide (ZnO) NPs in rats using micro-computed tomography (micro-CT) and histopathological assessment. Animals were divided into four groups as control group, ZnO NPs treatment group (500 mg/kg/day), control recovery group, and ZnO NPs treatment and recovery group. Ocular samples were prepared from animals treated for 90 days (10 males and 10 females, respectively) and from recovery animals (5 males and 5 females, respectively) sacrificed at 14 days after final treatment and were compared to age-matched control animals. Micro-CT analyses represented the deposition and distribution of foreign materials in the eyes of rats treated with ZnO NPs, whereas control animals showed no such findings. X-ray fluorescence spectrometry and energy dispersive spectrometry showed the intraocular foreign materials as zinc in treated rats, whereas control animals showed no zinc signal. Histopathological examination revealed the retinopathy in the eyes of rats treated with ZnO NPs. Neuronal nuclei expression was decreased in neurons of the ganglion cell layer of animals treated with ZnO NPs compared to the control group. Taken together, treatment with 20-nm, negatively-charged ZnO NPs increased retinopathy, associated with local distribution of them in ocular lesions.

Organ-specific distribution of gold nanoparticles by their surface functionalization.

The behavior and fate of intravenously (i.v.) injected nanoparticles (NPs) can be controlled by several physicochemical factors including size, shape and surface charge. To evaluate the role of surface charge on distribution of NPs, we used neutral-charged 15-nm-sized polyethylene glycol-coated gold nanoparticles (AuNP(PEG)) as a core NP and carboxyl or amine groups were conjugated to AuNP(PEG) to generate negative (AuNP(COOH)) or positive AuNP (AuNP(NH2)), respectively. Each type of AuNP was i.v. injected into mice (1 mg kg(-1)) and the concentration of Au was measured in different organs at 30 min, 4, 24 h, 7, 14 days, 1, 3 and 6 months post-injection. The organ distribution also showed the higher deposition rate depending on their functional groups: AuNP(PEG) for mesenteric lymph node, kidney, brain and testis; AuNP(COOH) for liver; AuNP(NH2) for spleen, lung and heart. The blood circulation time and the major excretion route were different depending on their functional groups. In conclusion, functional groups conjugated on the surface of AuNPs produce differences in blood kinetics, organ distribution and elimination pattern which can be important information for directing NPs to specific organs or improving the kinetic properties.

A hemagglutinin quantification method for development of an influenza pandemic vaccine using size exclusion high performance liquid chromatography.

Single radial immunodiffusion (SRID) assay requires a reference antigen and an antibody to the hemagglutinin (HA) of an influenza vaccine. As it takes 2­3 months to develop the reference antigen, vaccine development is delayed in cases of an influenza pandemic. In the present study, the measurement of the HA content of influenza vaccines was assessed using size exclusion high performance liquid chromatography (SE­HPLC) for the rapid development of a pandemic vaccine. When the 2009 H1N1 reference antigen, pandemic 2009 H1N1 vaccine and 2010 seasonal influenza vaccines were analyzed by SE­HPLC, the HA of the reference antigen and vaccines was specifically separated. The presence and specificity of HA were evidenced with immunoprecipitation and ELISA assays. For the influenza vaccines, the chromatogram pattern and retention time of HA were similar among the antigen types (2009 H1N1, 2010 H3N2 and 2010 B). In addition, when SE­HPLC was applied, the ratio of HA chromatogram to peak area revealed a significant correlation with HA concentration for the reference antigen and vaccine. The result of the HA content calculation based on SE­HPLC exhibited 99.91­100% similarity, compared with that of SRID. These findings suggest that the measurement of peak area ratio/HA content using SE­HPLC may be a substitute for SRID and rapidly measure HA content to enable faster development of a vaccine during an influenza pandemic.

Potent acetylcholinesterase inhibitory compounds from Myristica fragrans.

The anti-cholinesterase activity was evaluated of the ethyl acetate fraction of the methanol extract of Myristica fragrans Houtt (Myristicaceae) seeds and of compounds isolated from it by various chromatographic techniques. The chemical structures of the compounds were determined from spectroscopic analyses (NMR data). Thirteen compounds (1-13) were isolated and identified. Compound 8 { [(7S)-8'-(4'-hydroxy-3'-methoxyphenyl)-7-hydroxypropyl]benzene-2,4-diol) showed the most effective activity with an IC50 value of 35.1 microM, followed by compounds 2 [(8R,8'S)-7'-(3',4'-methylenedioxyphenyl)-8,8'-dimethyl-7-(3,4-dihydroxyphenyl)-butane] and 11 (malabaricone C) with IC50 values of 42.1 and 44.0 pM, respectively. This is the first report of significant anticholinesterase properties of M. fragrans seeds. The findings demonstrate that M. fragrans could be used beneficially in the treatment of Alzheimer's disease.

Toxicity assessment of Leonuri Herba aqueous extract orally administered to rats for 13 consecutive weeks.

ETHNOPHARMACOLOGICAL RELEVANCE: The Leonuri Herba has been traditionally used for women's disease in Asian countries. AIM OF THE STUDY: The objective of the present study was to evaluate the subchronic toxicity of Leonuri Herba aqueous extract in male and female F344 rats. MATERIAL AND METHODS: Leonuri Herba aqueous extract was administered orally once daily at dose levels of 0, 125, 250, 500, 1000 and 2000 mg/kg/day for 13 weeks. Toxicological assessment was performed including mortality, clinical signs, body and organ weights, food consumption, ophthalmology, urinalysis, hematology, serum chemistry, gross findings and histopathologic examination. RESULTS: There were no treatment related differences in clinical signs, urinalysis, hematology and serum chemistry, except for a histopathologic examination. The squamous cell hyperplasia in the forestomach was observed in both sexes of rats given 2000 mg/kg/day of Leonuri Herba aqueous extract. CONCLUSION: In conclusion, the NOAEL (No Observed Adverse Effect Level) for Leonuri Herba aqueous extract was determined as 1000 mg/kg/day in both sexes of rats under the present experimental conditions. And the acceptable daily intake value for Leonuri Herba aqueous extract was calculated to be 10mg/kg body weight per day using a safety factor of 100 to the NOAEL.