SN-38 loaded polymeric micelles to enhance cancer therapy.

7-Ethyl-10-hydroxycamptothecin (SN-38) loaded poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) (Pluronic F-108) and poly(ethylene glycol)-block-poly(ε-caprolactone) (PEG-b-PCL) nanoparticles were successfully prepared by a modified film hydration method and characterized by scanning electric microscopy (SEM), transmission electron microscopy (TEM), atomic force microscopy (AFM) and dynamic light scattering (DLS). Satisfactory drug loading of 20.73 ± 0.66% and a high encapsulation efficiency of 83.83 ± 1.32% were achieved. The SN-38 nanoparticles (SN-38 NPs) can completely disperse into a phosphate buffered saline (PBS) medium to produce a clear aqueous suspension that remains stable for up to three days. Total drug releases were 67.91% and 91.09% after 24 h in a PBS or fetal bovine serum (FBS) medium. Half maximal inhibitory concentration (IC(50)) tests of SN-38 and SN-38 NPs on A549 lung cells produced results of 200.0 ± 14.9 ng ml(-1) and 80.0 ± 4.6 ng ml(-1), respectively. Similarly, IC(50) tests of SN-38 and SN-38 NPs on MCF-7 breast cells yielded results of 16.0 ± 0.7 ng ml(-1) and 8.0 ± 0.5 ng ml(-1), respectively. These in vitro IC(50) studies show significant (p < 0.01) enhancement of the SN-38 NP drug efficiency in killing cancer cells in comparison to the free drug SN-38 control. All the materials used for this nanoformulation are approved by the US FDA, with the virtue of extremely low toxicity to normal cells.

Pharmacokinetic and toxicological evaluation of multi-functional thiol-6-fluoro-6-deoxy-D-glucose gold nanoparticles in vivo.

We synthesized a novel, multi-functional, radiosensitizing agent by covalently linking 6-fluoro-6-deoxy-D-glucose (6-FDG) to gold nanoparticles (6-FDG-GNPs) via a thiol functional group. We then assessed the bio-distribution and pharmacokinetic properties of 6-FDG-GNPs in vivo using a murine model. At 2 h, following intravenous injection of 6-FDG-GNPs into the murine model, approximately 30% of the 6-FDG-GNPs were distributed to three major organs: the liver, the spleen and the kidney. PEGylation of the 6-FDG-GNPs was found to significantly improve the bio-distribution of 6-FDG-GNPs by avoiding unintentional uptake into these organs, while simultaneously doubling the cellular uptake of GNPs in implanted breast MCF-7 adenocarcinoma. When combined with radiation, PEG-6-FDG-GNPs were found to increase the apoptosis of the MCF-7 breast adenocarinoma cells by radiation both in vitro and in vivo. Pharmacokinetic data indicate that GNPs reach their maximal concentrations at a time window of two to four hours post-injection, during which optimal radiation efficiency can be achieved. PEG-6-FDG-GNPs are thus novel nanoparticles that preferentially accumulate in targeted cancer cells where they act as potent radiosensitizing agents. Future research will aim to substitute the (18)F atom into the 6-FDG molecule so that the PEG-6-FDG-GNPs can also function as radiotracers for use in positron emission tomography scanning to aid cancer diagnosis and image guided radiation therapy planning.

High-throughput quantitative analysis with cell growth kinetic curves for low copy number mutant cells.

The mutation rate in cells induced by environmental genotoxic hazards is very low and difficult to detect using traditional cell counting assays. The established genetic toxicity tests currently recognized by regulatory authorities, such as conventional Ames and hypoxanthine guanine phosphoribosyl-transferase (HPRT) assays, are not well suited for higher-throughput screening as they require large amounts of test compounds and are very time consuming. In this study, we developed a novel cell-based assay for quantitative analysis of low numbers of cell copies with HPRT mutation induced by an environmental mutagen. The HPRT gene mutant cells induced by the mutagen were selected by 6-thioguanine (6-TG) and the cell's kinetic growth curve monitored by a real-time cell electronic sensor (RT-CES) system. When a threshold is set at a certain cell index (CI) level, samples with different initial mutant cell copies take different amounts of time in order for their growth (or CI accumulation) to cross this threshold. The more cells that are initially seeded in the test well, the faster the cell accumulation and therefore the shorter the time required to cross this threshold. Therefore, the culture time period required to cross the threshold of each sample corresponds to the original number of cells in the sample. A mutant cell growth time threshold (MT) value of each sample can be calculated to predict the number of original mutant cells. For mutagenesis determination, the RT-CES assay displayed an equal sensitivity (p > 0.05) and coefficients of variation values with good correlation to conventional HPRT mutagenic assays. Most importantly, the RT-CES mutation assay has a higher throughput than conventional cellular assays.

Sputum microRNA profiling: a novel approach for the early detection of non-small cell lung cancer.

PURPOSE: MicroRNAs (miRNAs) post-transcriptionally regulate hundreds of gene targets involved in tumorigenesis thereby controlling vital biological processes, including cellular proliferation, differentiation and apoptosis. MiRNA profiling is an emerging tool for the potential early detection of a variety of malignancies. This study was conducyed to assess the feasibility and methodological robustness of quantifying sputum miRNAs, employing quantitative real-time polymerase chain reaction (RT-qPCR) and cluster analysis on an optimized miRNA profile as a novel approach for the early detection of non-small cell lung cancer (NSCLC). METHODS: The relative expressions of 11 miRNAs in sputum (miR-21, miR-145, miR-155, miR-205, miR-210, miR-92, miR-17-5p, miR-143, miR-182, miR-372, and let-7a) in addition to U6 were retrospectively assessed in four NSCLC-positive and four negative controls. Subsequently, a set of five miRNAs (miR-21, miR-143, miR-155, miR-210, miR-372) was selected because of degree of relatedness observed in the cluster analysis and tested in the same sputum sample set. The five optimized miRNAs accurately clustered these eight retrospective patients into NSCLC positive cases and negative controls. The five miRNA panel was then prospectively quantified in the sputum of 30 study patients (24 NSCLC cases and six negative controls) in a double-blind fashion to validate a five miRNA panel using hierarchical cluster analysis. RESULTS: The optimized five miRNA panel detected NSCLC (83.3% sensitivity and 100% specificity) in 30 prospectively accrued study patients. CONCLUSION: Sputum miRNA profiling using cluster analysis is a promising approach for the early detection of non-small cell lung cancer. Further investigation using this approach is warranted.

Magnetic gold nanoparticles: synthesis, characterization and its application in the delivery of FITC into KG-1 cells.

In this article, we report a new method-a sonication method to disperse iron oxide nanoparticles into smaller nanoparticles and make gold ions absorb onto the surface or trapped in the micropores of the iron oxide nanoparticles using sonication action. By using quick reduction of ascorbic acid and post-HCI solution treatment, gold covered magnetic nanoparticles (mGNPs) with spherical morphology and uniform size were synthesized in a water solution. The size of the mGNPs was found to be 20-30 nm. Some ideal mGNPs possessed a core-shell structure. The mGNPs were non-cytotoxic and mGNP-fluorescein isothiocyanate (FITC) can enter KG-1 cells when driven by an external magnetic force, which was confirmed by confocal imaging. The confocal image also showed the FITC inside the KG-1 cells was near the nucleus. The fluorescein isothiocyanate (FITC) delivery efficiency is about 100% according to the flow cytometry results.

FITC delivery into plant cells using magnetic single-walled carbon nanotubes.

In this paper, fluorescein isothiocyanate (FITC) was covalently bonded with magnetic single-walled carbon nanotubes (mSWCNTs) that were purified using our previous method. To demonstrate our design, mSWCNT-FITC was delivered into plant cells (canola and carrot cells) driven by external magnetic forces. From FACS results, the FITC delivery efficiency was about 100% for both two canola and carrot protoplasts, which were further confirmed by the confocal and sectional TEM images. Some mSWCNTs were found trapped both inside the endosomes of canola protoplast and outside endosome near the nuclear membrane of carrot protoplast according to the sectional TEM images. All results showed that mSWCNT is a good delivery carrier for biomolecules.

Efficient and rapid uptake of magnetic carbon nanotubes into human monocytic cells: implications for cell-based cancer gene therapy.

Monocyte-based gene therapies in cancer have been hampered by either the resistance of these cells to non-viral molecular delivery methods or their poor trafficking to the tumor site after their ex vivo manipulations. Magnetic nanoparticles (MNP)-loaded genetically engineered monocytes can efficiently delivered to tumor site by external magnetic field, but they are not ideal delivery tools due to their spherical shape. Hence, we have investigated the cellular uptake efficiency and cytotoxicity of fluorescein isothiocyanate (FITC)-labelled magnetic carbon nanotubes (FITC-mCNT) in human monocytic leukemia cell line THP-1 for application in cell-based gene therapy against cancer. Uptake of FITC-mCNT into THP-1 cells reached 100% only 1 h after the delivery. Confocal imaging confirmed that FITC-mCNT entered the cell cytoplasm and even into the nucleus. FITC-mCNT uptake did not compromise cell viability. This delivery system might therefore enhance cell-based cancer gene therapies.

Low-intensity pulsed ultrasound-mediated stimulation of hematopoietic stem/progenitor cell viability, proliferation and differentiation in vitro.

Low-intensity pulsed ultrasound (LIPUS) stimulated the viability, proliferation and differentiation of hematopoietic stem/progenitor cells (HSPC) from fresh and cryopreserved peripheral blood leukapheresis product, as well as cord blood when applied for 10 min each day for 4 days. Cell viability, proliferation and differentiation were assessed on day 5 by viable cell counting, MTS proliferation assay, flow cytometry, and colony-forming unit assay. LIPUS stimulation: (i) enhanced the proliferation of fresh HSPC and maintained the viability of cryopreserved HSPC in vitro; (ii) did not affect the percentage of CD34(+) and CD14(+) cells; and (iii) enhanced burst-forming unit-erythroid colony formation. Hence, we suggest that this novel LIPUS stimulation approach might enhance the efficacy of clinical transplantation and cellular therapies using HSPC.

Impact of carbondiimide crosslinker used for magnetic carbon nanotube mediated GFP plasmid delivery.

1-Ethyl-3-(3-dimethylaminopropyl) carbondiimide hydrochloride (EDC) is commonly used as a crosslinker to help bind biomolecules, such as DNA plasmids, with nanostructures. However, EDC often remains, after a crosslink reaction, in the micro-aperture of the nanostructure, e.g., carbon nanotube. The remaining EDC shows positive green fluorescent signals and makes a nanostructure with a strong cytotoxicity which induces cell death. The toxicity of EDC was confirmed on a breast cancer cell line (MCF-7) and two leukemic cell lines (THP-1 and KG-1). The MCF-7 cells mainly underwent necrosis after treatment with EDC, which was verified by fluorescein isothiocyanate (FITC) annexin V staining, video microscopy and scanning electronic microscopy (SEM). If the EDC was not removed completely, the nanostructures with remaining EDC produced a green fluorescent background that could interfere with flow cytometry (FACS) measurement and result in false information about GFP plasmid delivery. Effective methods to remove residual EDC on macromolecules were also developed.

Thio-glucose bound gold nanoparticles enhance radio-cytotoxic targeting of ovarian cancer.

The treatment of ovarian cancer has traditionally been intractable, and required novel approaches to improve therapeutic efficiency. This paper reports that thio-glucose bound gold nanoparticles (Glu-GNPs) can be used as a sensitizer to enhance ovarian cancer radiotherapy. The human ovarian cancer cells, SK-OV-3, were treated by gold nanoparticles (GNPs) alone, irradiation alone, or GNPs in addition to irradiation. Cell uptake was assayed using inductively coupled plasma atomic emission spectroscopy (ICP-AES), while cytotoxicity induced by radiotherapy was measured using both 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide and clonogenic assays. The presence of reactive oxygen species (ROS) was determined using CM-H2-DCFDA confocal microscopy and cell apoptosis was determined by an Annexin V-FITC/propidium iodide (PI) kit with flow cytometry. The cells treated by Glu-GNPs resulted in an approximate 31% increase in nanoparticle uptake compared to naked GNPs (p < 0.005). Compared to the irradiation alone treatment, the intracellular uptake of Glu-GNPs resulted in increased inhibition of cell proliferation by 30.48% for 90 kVp and 26.88% for 6 MV irradiation. The interaction of x-ray radiation with GNPs induced elevated levels of ROS production, which is one of the mechanisms by which GNPs can enhance radiotherapy on ovarian cancer.

Real-time cell-impedance sensing assay as an alternative to clonogenic assay in evaluating cancer radiotherapy.

Intrinsic radiosensitivity of normal and tumour tissues has been shown to be an independent prognostic factor for patients' response to radiotherapy. This study compares the real-time cell-impedance sensing (RT-CES) assay with the conventional clonogenic assay in terms of in-vitro radiosensitivity. One objective in this study was to predict in-vivo response to gold nanoparticle (GNP) treatment on the basis of in-vitro RT-CES testing results. Four adenocarcinoma cancer cell lines were tested using both the RT-CES and the clonogenic assays. Cell-survival curves were plotted, and the mean SF2 values obtained by these two different assay methods were compared using ANOVA. Radiation sensitivities obtained in-vitro were then compared with the in-vivo results. On the basis of the measurement of cell colonies, the RT-CES assay has similar radiosensitivity to the clonogenic assay, but significantly shortens the testing time from 14-21 days to only 72 h. Intrinsic GNP enhanced radiation sensitivity using tumour volume (mm(3)) in vivo is comparable with that using RT-CES cell survival assay in vitro. Furthermore, the RT-CES system provides real-time information regarding the state of cell radiosensitivity that may give useful information towards personalizing radiotherapy. The RT-CES assay enables more reliable and time-efficient results in the evaluation of radiosensitivity.

Magnetic carbon nanotube labelling for haematopoietic stem/progenitor cell tracking.

Haematopoietic stem and progenitor cell (HSPC) research has significantly contributed to the understanding and harnessing of haematopoiesis for regenerative medicine. However, the methodology for real-time tracking HSPC in vivo is still lacking, which seriously restricts the progress of research. Recently, magnetic carbon nanotubes (mCNT) have generated great excitement because they have been successfully used as vehicles to deliver a lot of biomolecules into various cells. There is, however, no report about mCNT being used for tracking HSPC. In this paper, we investigated the uptake efficiency of fluorescein-isothiocyanate-labelled mCNT (FITC-mCNT) into HSPC and their effect on the cytotoxicity and differentiation of HSPC. We found that cellular uptake of FITC-mCNT was concentration-and time-dependent. The uptake of FITC-mCNT into HSPC reached up to 100% with the highest mean fluorescence (MF). More importantly, efficient FITC-mCNT uptake has no adverse effect on the cell viability, cytotoxicity and differentiation of HSPC as confirmed by colony-forming unit assay (CFU). In conclusion, the results reported here suggest the further tailoring of mCNT for their use in HSPC labelling/tracking in vivo or gene delivery into HSPC.

Identification of a new microRNA expression profile as a potential cancer screening tool.

PURPOSE: Small non-coding microRNAs (miRNAs) are key components of cancer development and are considered as potential biomarkers for cancer diagnosis and treatment monitoring. This study investigated miRNA expression profiles of human cancer cells in order to develop a screening method for lung cancer. METHODS: A series of lung cancer related miRNAs (miR-21, miR-145, miR-155, miR-205, miR-210, miR-92, miR-17-5p, miR-143, miR-182, miR-372, let-7a) were selected as candidates for miRNA expression profiles of human lung cancer cell lines (A549, SK-mes-1). MicroRNA u6 was the endogenous control. Cancer cell lines for positive controls; breast MCF-7, prostate Du-145, and glioblastoma U118. The negative control was normal lung fibroblast cell line MRC-5. RT-PCR was performed on StepOnePlus (Applied Biosystem, USA). MiRNA expressions of malignant cells were compared with normal fibroblast cells as well as endogenous control (u6) using the thermal cycle at threshold. Assessment of miRNA expression profiles were then performed using agglomerative hierarchical cluster analysis software (SPSS13, USA). RESULTS: We demonstrated that miR-21, miR-182 and let7-5a were over-expressed, and miR-145 and miR-155 were under-expressed in all cancer cell lines. Combined with the cluster analysis we were able to clearly distinguish cell lines for normal fibroblasts, breast cancer, prostate cancer, glioblastoma, and lung cancer. CONCLUSION: There is potential utility of screening for lung cancer with miRNA expression profiles. Future work will focus on the sensitivity of such miRNA expression profiles in screening sputum for lung cancer, which can be performed in real time.

Gold nanoparticle sensitize radiotherapy of prostate cancer cells by regulation of the cell cycle.

Glucose-capped gold nanoparticles (Glu-GNPs) have been used to improve cellular targeting and radio-sensitization. In this study, we explored the mechanism of Glu-GNP enhanced radiation sensitivity in radiation-resistant human prostate cancer cells. Cell survival and proliferation were measured using MTT and clonogenic assay. Flow cytometry with staining by propidium iodide (PI) was performed to study the cell cycle changes induced by Glu-GNPs, and western blotting was used to determine the expression of p53 and cyclin proteins that correlated to cell cycle regulation. With 2 Gy of ortho-voltage irradiation, Glu-GNP showed a 1.5-2.0 fold enhancement in growth inhibition when compared to x-rays alone. Comparing the cell cycle change, Glu-GNPs induced acceleration in the G0/G1 phase and accumulation of cells in the G2/M phase at 29.8% versus 18.4% for controls at 24 h. G2/M arrest was accompanied by decreased expression of p53 and cyclin A, and increased expression of cyclin B1 and cyclin E. In conclusion, Glu-GNPs trigger activation of the CDK kinases leading to cell cycle acceleration in the G0/G1 phase and accumulation in the G2/M phase. This activation is accompanied by a striking sensitization to ionizing radiation, which may have clinical implications.

Enhancement of radiation cytotoxicity in breast-cancer cells by localized attachment of gold nanoparticles.

Gold nanoparticles (GNPs) and modified GNPs having two kinds of functional molecules, cysteamine (AET) and thioglucose (Glu), are synthesized. Cell uptake and radiation cytotoxicity enhancement in a breast-cancer cell line (MCF-7) versus a nonmalignant breast-cell line (MCF-10A) are studied. Transmission electron microscopy (TEM) results show that cancer cells take up functional Glu-GNPs significantly more than naked GNPs. The TEM results also indicate that AET-capped GNPs are mostly bound to the MCF-7 cell membrane, while Glu-GNPs enter the cells and are distributed in the cytoplasm. After MCF-7 cell uptake of Glu-GNPs, or binding of AET-GNPs, the in vitro cytotoxicity effects are observed at 24, 48, and 72 hours. The results show that these functional GNPs have little or no toxicity to these cells. To validate the enhanced killing effect on cancer cells, various forms of radiation are applied such as 200 kVp X-rays and gamma-rays, to the cells, both with and without functional GNPs. By comparison with irradiation alone, the results show that GNPs significantly enhance cancer killing.

Enhanced radiation sensitivity in prostate cancer by gold-nanoparticles.

PURPOSE: Nanotechnology is an emerging field with significant translational potential in medicine. In this study, we applied gold nanoparticles (GNP) to enhance radiation sensitivity and growth inhibition in radiation-resistant human prostate cancer cells. METHODS: Gold nanoparticles (GNPs) were synthesized using HAuCl4 as the gold particle source and NaBH4 as the reductant. Either thio-glucose or sodium citrate was then added to the solution separately to bind the GNPs to form thio-glucose-capped gold nanoparticles (Glu-GNP) and neutral gold nanoparticles (TGS-GNPs). Human prostate carcinoma DU-145 cells were exposed to vehicle, irradiation, 15nM TGS-GNPs, or 15nM Glu-GNPs, or GNPs plus irradiation. The uptake assays of GNP were performed using hemocytometer to count cells and the mass spectrometry was applied to calculate gold mass. The cytotoxicity induced by GNPs, irradiation, or GNPs plus irradiation was measured using a standard colorimetric MTT assay. RESULTS: Exposure to Glu-GNPs resulted in a three times increase of nanoparticle uptake compared to that of TGS-GNPs in each target cell (p < 0.005). Cytoplasmic intracellular uptake of both TGS-GNPs and Glu-GNPs resulted in a growth inhibition by 30.57% and 45.97% respectively, comparing to 15.88% induced by irradiation alone, in prostate cancer cells after exposure to the irradiation. Glu-GNPs showed a greater enhancement, 1.5 to 2 fold increases within 72 hours, on irradiation cytotoxicity compared to TGS-GNPs. Tumour killing, however, did not appear to correlate linearly with nanoparticle uptake concentrations. CONCLUSION: These results showed that functional glucose-bound gold nanoparticles enhanced radiation sensitivity and toxicity in prostate cancer cells. In vivo studies will be followed to verify our research findings.

RANKL release from self-assembling nanofiber hydrogels for inducing osteoclastogenesis in vitro.

PURPOSE: To develop a nanofiber hydrogel (NF-hydrogel) for sustained and controlled release of the recombinant receptor activator of NF-kB ligand; (RANKL) and to characterize the release kinetics and bioactivity of the released RANKL. METHODS: Various concentrations of fluorescently-labelled RANKL protein were added to NF-hydrogels, composed of Acetyl-(Arg-Ala-Asp-Ala)4-CONH2 [(RADA)4] of different concentrations, to investigate the resulting in vitro release rates. The nano-structures of NF-hydrogel, with and without RANKL, were determined using atomic force microscopy (AFM). Released RANKL was further analyzed for changes in secondary and tertiary structure using CD spectroscopy and fluorescent emission spectroscopy, respectively. Bioactivity of released RANKL protein was determined using NFATc1 gene expression and tartrate resistant acid phosphatase (TRAP) activity of osteoclast cells as biomarkers. RESULTS: NF-hydrogel concentration dependent sustained release of RANKL protein was measured at concentrations between 0.5 and 2%(w/v). NF-hydrogel at 2%(w/v) concentration exhibited a sustained and slow-release of RANKL protein up to 48h. Secondary and tertiary structure analyses confirmed no changes to the RANKL protein released from NF-hydrogel in comparison to native RANKL. The results of NFATc1 gene mRNA expression and TRAP activities of osteoclast, showed that the release process did not affect the bioactivity of released RANKL. CONCLUSIONS: This novel study is the first of its kind to attempt in vitro characterization of NF-hydrogel based delivery of RANKL protein to induce osteoclastogenesis. We have shown the self-assembling NF-hydrogel peptide system is amenable to the sustained and controlled release of RANKL locally; that could in turn increase local concentration of RANKL to induce osteoclastogenesis, for application to the controlled mobilization of tooth movement in orthodontic procedures. STATEMENT OF SIGNIFICANCE: Orthodontic tooth movement (OTM) occurs through controlled application of light forces to teeth, facilitating the required changes in the surrounding alveolar bone through the process of bone remodelling. The RANKL system regulates alveolar bone remodelling and controls root resorption during OTM. The use of exogenous RANKL to accelerate OTM has not been attempted to date because large quantities of RANKL for systemic therapy may subsequently cause serious systemic loss of skeletal bone. The controlled and sustained local release of RANKL from a carrier matrix could maximize its therapeutic benefit whilst minimizing systemic side effects. In this study a NF-hydrogel was used for sustained and controlled release of RANKL and the release kinetics and biofunctionality of the released RANKL was characterized. Our results provide fundamental insight for further investigating the role of RANKL NF-hydrogel release systems for inducing osteoclastogenesis in vivo.

Increasing vaccine production using pulsed ultrasound waves.

Vaccination is a safe and effective approach to prevent deadly diseases. To increase vaccine production, we propose that a mechanical stimulation can enhance protein production. In order to prove this hypothesis, Sf9 insect cells were used to evaluate the increase in the expression of a fusion protein from hepatitis B virus (HBV S1/S2). We discovered that the ultrasound stimulation at a frequency of 1.5 MHz, intensity of 60 mW/cm2, for a duration of 10 minutes per day increased HBV S1/S2 by 27%. We further derived a model for transport through a cell membrane under the effect of ultrasound waves, tested the key assumptions of the model through a molecular dynamics simulation package, NAMD (Nanoscale Molecular Dynamics program) and utilized CHARMM force field in a steered molecular dynamics environment. The results show that ultrasound waves can increase cell permeability, which, in turn, can enhance nutrient / waste exchange thus leading to enhanced vaccine production. This finding is very meaningful in either shortening vaccine production time, or increasing the yield of proteins for use as vaccines.

Microelectronic cell sensor assay for detection of cytotoxicity and prediction of acute toxicity.

This study reports in-house assessment of a real-time cell electronic sensing (RT-CES) system used as a test platform for both cytotoxicity assay and predicting acute toxicity. For cytotoxicity determination, the RT-CES assay displayed equal sensitivity and coefficients of variation values with good correlation to NRU assay. The IC50 values and the LD50 values for the cytotoxicity reference materials were compared in the context of the proposed prediction model for acute rodent toxicity. The results obtained from RT-CES assay fitted within the acceptance limits of the prediction model and showed that the RT-CES cytotoxicity assay met the qualification guidelines in NIH Publication #01-4500 to accurately predict acute toxicity. In addition to cell viability, the RT-CES assay provided dynamic information that can be used to identify maximum toxicity and reversibility of the toxic effects which are difficult to achieve by the endpoint assays and, therefore, the RT-CES assay is more accurate for assessment of cytotoxicity. The features of the RT-CES assay, such as labeling free, automatic detection, and easy operation, give this assay potential to replace BALB/c 3T3 NRU assay and be used as routine setting for drug monitoring in the toxicological laboratory.

Development of a microelectronic chip array for high-throughput genotyping of Helicobacter species and screening for antimicrobial resistance.

A microelectronic array assay was developed to specifically genotype Helicobacter pylori versus Helicobacter heilmannii and to determine antimicrobial resistance. Helicobacter 16S rRNA and 23S rRNA genes were specifically generated with Helicobacter genus-specific primers, respectively. The single-nucleotide polymorphisms (SNPs) in 16S rRNA, 268T specific in the H. pylori sequence, and 263A specific in H. heilmannii were used as molecular markers for identification of H. pylori and H. heilmannii, respectively. A triple-base-pair resistant mutation, AGA965-967TTC in 16S rRNA, is known to be responsible for H. pylori tetracycline resistance and was detected to identify resistant strains. H. pylori macrolide resistance was determined by the identification of 3 defined mutations in the 23S rRNA gene using the same method. The assay could be directly used to detect H. pylori in feces. The assay performs multiple determinations, including identification of Helicobacter species and antibiotic resistances, on the same microelectronic platform and is highly amenable to the development of other DNA-based assays.