Aaron A. Urbas

Anti-HER2 IgY antibody-functionalized single-walled carbon nanotubes for detection and selective destruction of breast cancer cells.

BackgroundNanocarrier-based antibody targeting is a promising modality in therapeutic and diagnostic oncology. Single-walled carbon nanotubes (SWNTs) exhibit two unique optical properties that can be exploited for these applications, strong Raman signal for cancer cell detection and near-infrared (NIR) absorbance for selective photothermal ablation of tumors. In the present study, we constructed a HER2 IgY-SWNT complex and demonstrated its dual functionality for both detection and selective destruction of cancer cells in an in vitro model consisting of HER2-expressing SK-BR-3 cells and HER2-negative MCF-7 cells.MethodsThe complex was constructed by covalently conjugating carboxylated SWNTs with anti-HER2 chicken IgY antibody, which is more specific and sensitive than mammalian IgGs. Raman signals were recorded on Raman spectrometers with a laser excitation at 785 nm. NIR irradiation was performed using a diode laser system, and cells with or without nanotube treatment were irradiated by 808 nm laser at 5 W/cm2 for 2 min. Cell viability was examined by the calcein AM/ethidium homodimer-1 (EthD-1) staining.ResultsUsing a Raman optical microscope, we found the Raman signal collected at single-cell level from the complex-treated SK-BR-3 cells was significantly greater than that from various control cells. NIR irradiation selectively destroyed the complex-targeted breast cancer cells without harming receptor-free cells. The cell death was effectuated without the need of internalization of SWNTs by the cancer cells, a finding that has not been reported previously.ConclusionWe have demonstrated that the HER2 IgY-SWNT complex specifically targeted HER2-expressing SK-BR-3 cells but not receptor-negative MCF-7 cells. The complex can be potentially used for both detection and selective photothermal ablation of receptor-positive breast cancer cells without the need of internalization by the cells. Thus, the unique intrinsic properties of SWNTs combined with high specificity and sensitivity of IgY antibodies can lead to new strategies for cancer detection and therapy.

Automated Spectral Smoothing with Spatially Adaptive Penalized Least Squares

A variety of data smoothing techniques exist to address the issue of noise in spectroscopic data. The vast majority, however, require parameter specification by a knowledgeable user, which is typically accomplished by trial and error. In most situations, optimized parameters represent a compromise between noise reduction and signal preservation. In this work, we demonstrate a nonparametric regression approach to spectral smoothing using a spatially adaptive penalized least squares (SAPLS) approach. An iterative optimization procedure is employed that permits gradual flexibility in the smooth fit when statistically significant trends based on multiscale statistics assuming white Gaussian noise are detected. With an estimate of the noise level in the spectrum the procedure is fully automatic with a specified confidence level for the statistics. Potential application to the heteroscedastic noise case is also demonstrated. Performance was assessed in simulations conducted on several synthetic spectra using traditional error measures as well as comparisons of local extrema in the resulting smoothed signals to those in the true spectra. For the simulated spectra, a best case comparison with the Savitzky–Golay smoothing via an exhaustive parameter search was performed while the SAPLS method was assessed for automated application. The application to several dissimilar experimentally obtained Raman spectra is also presented.

Probing the intracellular glutathione redox potential by in-cell NMR spectroscopy.

Non-invasive and real-time analysis of cellular redox processes has been greatly hampered by lack of suitable measurement techniques. Here we describe an in-cell nuclear magnetic resonance (NMR) based method for measuring the intracellular glutathione redox potential by direct and quantitative measurement of isotopically labeled glutathione introduced exogenously into living yeast. By using this approach, perturbations in the cellular glutathione redox homeostasis were also monitored as yeast cells were subjected to oxidative stress.

A composition-controlled cross-linking resin network through rapid visible-light photo-copolymerization

An assembly that delivers well-defined functional materials, clinically practical procedures to make these materials in situ, and appropriate analytical tools for chemical structure and kinetic studies is desirable, though currently unavailable. Herein, we introduce a system that addresses this need through the development and characterization of a cross-linking resin network, which is achieved through rapid, visible-light induced polymerization in a solvent-free environment. This resin network is the result of co-polymerization of a distyrenyl-monomer with a dimethacryl-monomer. Ninety percent of vinyl conversion is achieved in seconds. In addition, an azeotropic composition is identified and confirmed through static end-point evaluation, sol–gel experiment, kinetic study, and mathematical modeling of data acquired via FTIR, real-time Raman and 1H NMR spectroscopies. These results yield opportunities for the design and development of new functional materials to be used in various applications.

Quantitative measurements of glutathione in yeast cell lysate using 1H NMR

Methods for quantifying the level of glutathione (GSH) in yeast cell lysate are described using 1H NMR analysis. For quantification purposes, the 1H resonances corresponding to the Cys βCH2 of GSH were identified as having the fewest overlapping spectral interferences from lysate matrix components using GSH spiked yeast lysate samples. Two methods, standard addition based on peak integration and a spectral subtraction approach, were evaluated for quantifying GSH in lysate samples. The peak integration procedure required baseline estimation and a peak fitting step to correct for background interferences while the spectral subtraction procedure was comparatively straightforward. The level of GSH measured by 1H NMR was in good agreement with the concentration measured by the DTNB-GSSG reductase recycling assay. The proposed NMR method can lead to a reliable quantitation of GSH and could be applicable to a variety of other analytes of interest in complex biological matrices.

On the feasibility of determining polymer chemical heterogeneity by SEC with continuous off-line Raman detection

Examined here is the feasibility of employing Raman spectroscopy as a detection method in size-exclusion chromatography (SEC) and related macromolecular separations, for the purposes of determining the chemical heterogeneity of copolymers. To this effect, heart-cutting fractions from the SEC peak of a gradient random copolymer of styrene (S) and methyl methacrylate (MMA) were collected, at concentrations resembling those of eluting slices in an analytical SEC experiment, and subsequently analyzed by off-line Raman. The amount (weight or mole percent) of styrene in each fraction was quantitated by Raman by applying a calibration curve constructed through the analysis of well-characterized block, alternating, and random S-MMA copolymers. The weight percentages of S (w% S) obtained from SEC with off-line Raman analysis of the gradient copolymer were compared to results previously obtained using SEC with on-line multi-angle static light scattering, differential refractometry, and ultraviolet absorption detection. Not only did Raman rank the w% S of the fractions in the correct order but, on average, Raman results differed by 3% or less as compared to the values obtained via multi-detector SEC. The results from this study provide proof of principle of both the feasibility of continuous off-line Raman detection for macromolecular separations and of the possibility of employing for this purpose the same or similar hardware to that currently used for continuous off-line Fourier transform infrared detection.

Efficient electrochemical degradation of multiwall carbon nanotubes

As the production mass of multiwall carbon nanotubes (MWCNT) increases, the potential for human and environmental exposure to MWCNTs may also increase. We have shown that exposing an aqueous suspension of pristine MWCNTs to an intense oxidative treatment in an electrochemical reactor, equipped with an efficient hydroxyl radical generating Boron Doped Diamond (BDD) anode, leads to their almost complete mineralization. Thermal optical transmittance analysis showed a total carbon mass loss of over two orders of magnitude due to the electrochemical treatment, a result consistent with measurements of the degraded MWCNT suspensions using UV-vis absorbance. Liquid chromatography data excludes substantial accumulation of the low molecular weight reaction products. Therefore, up to 99% of the initially suspended MWCNT mass is completely mineralized into gaseous products such as CO2 and volatile organic carbon. Scanning electron microscopy (SEM) images show sporadic opaque carbon clusters suggesting the remaining nanotubes are transformed into structure-less carbon during their electrochemical mineralization. Environmental toxicity of pristine and degraded MWCNTs was assessed using Caenorhabditis elegans nematodes and revealed a major reduction in the MWCNT toxicity after treatment in the electrochemical flow-by reactor.

Abstract 3686: Antibody-functionalized single-walled carbon nanotubes for detection and selective destruction of breast and pancreas cancer cells

Nanocarrier-based antibody targeting is a promising modality in therapeutic and diagnostic oncology. Single-walled carbon nanotubes (SWNTs) exhibit two unique optical properties that can be exploited for such applications, i.e., strong Raman signal for cancer cell detection and near-infrared (NIR) absorbance for selective photothermal ablation of tumors. In the present study, we constructed a HER2 IgY-SWNT complex and a MUC4 IgG-SWNT complex, and demonstrated in vitro their dual functionality for both detection and selective destruction of breast and pancreas cancer cells. The two complexes were constructed by covalently conjugating carboxylated SWNTs with anti-HER2 chicken polyclonal IgY antibody and with anti-MUC4 mouse monoclonal IgG antibody, respectively. Raman signals were recorded on a Raman spectrometer with a laser excitation at 785 nm. NIR irradiation was performed using a diode laser system, and cells with or without nanotube treatment were irradiated by 808 nm laser at 50 W/cm 2 for 5 min. Cell viability was examined by calcein AM/ethidium homodimer-1 (EthD-1) staining. Immunohistochemical studies showed that HER2 IgY-SWNT complex specifically targeted receptor-expressing SK-BR-3 cells but not receptor-negative MCF-7 cells; MUC4 IgG-SWNT complex specifically targeted pancreatic cancer cells but not normal pancreas cells. Using a Raman optical microscope, we found the Raman signal collected at single-cell level from the complex-treated breast or pancreatic cancer cells was significantly greater than that from various control cells. NIR irradiation selectively destroyed the complex-targeted breast and pancreatic cancer cells without harming receptor-free cells. The death rate after NIR irradiation were 94.1% and 96.6% respectively for complex-treated breast and pancreatic cancer cells, but only 1.3% and 1.8% for the untreated controls of the two cancer cell types, respectively. These results demonstrated that the antibody-SWNT complexes have the potential to detect cancer cells at the early stages of tumor development. In addition, the complexes are potentially useful for selectively killing cancer cells while sparing healthy ones. Thus, the unique intrinsic properties of SWNTs combined with high specificity and sensitivity of antibodies can lead to new strategies for cancer detection and therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3686.