Both in vitro and in vivo glutathione (GSH)-triggered anticancer drug releases were monitored in real time from the PEGylated graphene oxide (PEG-GO) platform. The assembly of the anticancer drug doxorubicin (DOX) on PEG-GO was verified by UV-Vis absorption and infrared spectroscopic tools. The fluorescence of DOX appeared to be quenched significantly by PEG-GO. A part of the initial DOX (10 À4 M) in PEG-GO was found to be released by $23.5% after treatment with 2 mM glutathione (GSH) within 15 min. Our fluorescence colocalization experiments indicated that PEG-GO-DOX was endocytosed and localized in either lysosomes or endosomes of intracellular compartments. Using fluorescence imaging techniques in real time, we were able to observe an approximately 2.5 times higher in vitro drug release in the live cells by externally triggering glutathione ethyl ester (GSH-OEt) rather than endogeneous GSH. In vivo fluorescence images of DOX were obtained with an order of magnitude larger intensity from the subcutaneous site in living mice after treatment with 0.3 mg of GSH. A realtime release of DOX on PEG-GO at the intended locus can be achieved in vivo after an external triggering of GSH.
Gold nanorod-attached PEGylated graphene-oxide (AuNR-PEG-GO) nanocomposites were tested for a photothermal platform both in vitro and in vivo. Cytotoxicity of AuNR was reduced after encapsulation with PEG-GO along with the removal of cetyltrimethylammonium bromide (CTAB) from AuNR by HCl treatment. Cellular internalization of the CTAB-eliminated AuNR-PEG-GO nanocomposites was examined using dark-field microscopy (DFM), confocal Raman microscopy and transmission electron microscopy (TEM). To determine the photothermal effect of the AuNR-PEG-GO nanocomposites, A431 epidermoid carcinoma cells were irradiated with Xe-lamp light (60 W cm(-2)) for 5 min after treatment with the AuNR-PEG-GO nanocomposites for 24 h. Cell viability significantly decreased by ~40% when the AuNR-PEG-GO-encapsulated nanocomposites were irradiated with light as compared with the cells treated with only the AuNR-PEG-GO nanocomposites without any illumination. In vivo tumor experiments also indicated that HCl-treated AuNR-PEG-GO nanocomposites might efficiently reduce tumor volumes via photothermal processes. Our graphene and AuNR nanocomposites will be useful for an effective photothermal therapy.
We study the adsorption behaviors of rhodamine dyes on gold nanoparticles (Au NPs) depending on their surface charges. Rhodamine 6G (Rh6G) dye is tested comparatively for positively and negatively charged Au NPs prepared by the reduction of chitosan and citric acid, respectively. The adsorption of Rh6G is found to be weaker on the positively charged Au NPs, whereas more substantial aggregation is found on negatively charged Au NPs. An increase in the concentration of Au NPs enhances the surface-enhanced Raman scattering (SERS) intensities only for the Au(−) NPs, whereas the Au(+) NPs do not exhibit any strong SERS signals. Our findings suggest that SERS and reciprocal fluorescence measurements of Rh6G can be used to estimate the surface charges and atomic percentages of Au NPs less than ∼5 ppm.
We investigated the cellular uptake behavior of non-fluorescent metal nanoparticles (NPs) by use of surface-enhanced Raman scattering (SERS) combined with dark-field microscopy (DFM). The uptake of Au NPs inside a single cell could also be identified by DFM first and then confirmed by z-depth-dependent SERS at micrometer resolution. Guided by DFM for the location of Au NPs, an intracellular distribution assay was possible using Raman dyes with unique vibrational marker bands in order to identify the three-dimensional location inside the single cell by obtaining specific spectral features. Au NPs modified by 4-mercaptobenzoic acid (MBA) bearing its -COOH surface functional group were used to conjugate transferrin (Tf) protein using the 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) reaction. The protein conjugation reaction on Au surfaces was examined by means of color change, absorption spectroscopy, and SERS. Our results demonstrate that DFM techniques combined with SERS may have great potential for monitoring biological processes with protein conjugation at the single-cell level.
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