Methotrexate (MTX), a stoichiometric inhibitor of dihydrofolate reductase, is a chemotherapeutic agent for treating a variety of neoplasms. Impairment of drug import into cells and increase in drug export from cells may render cells resistant to MTX. MTX, when locally administered in a soluble form, is rapidly absorbed through capillaries into the circulatory system, which may also account for therapeutic failure in patients. To retain MTX within tumor cells for longer duration and alter its pharmacokinetic behavior, we proposed a new formulation of MTX bound to the gold nanoparticle (AuNP) that serves as drug carriers. In this study, we developed the MTX-AuNP conjugate and examined its cytotoxic effect in vitro and antitumor effect in vivo. Spectroscopic examinations revealed that MTX can be directly bound onto AuNP via the carboxyl group (-COOH) to form the MTX-AuNP complex and kinetically released from the nanoparticles. The accumulation of MTX is faster and higher in tumor cells treated with MTX-AuNP than that treated with free MTX. Notably, MTX-AuNP shows higher cytotoxic effects on several tumor cell lines compared with an equal dose of free MTX. This can be attributed to the "concentrated effect" of MTX-AuNP. Administration of MTX-AuNP suppresses tumor growth in a mouse ascites model of Lewis lung carcinoma (LL2), whereas an equal dose of free MTX had no antitumor effect. In conclusion, these results suggest that by combining nanomaterials with anticancer drugs MTX-AuNP may be more effective than free MTX for cancer treatment.

Objective. Angiogenesis plays a part in the pathogenesis of rheumatoid arthritis (RA), and nanogold inhibits the activity of an angiogenic factor, vascular endothelial growth factor (VEGF). We therefore investigated whether intraarticular delivery of nanogold ameliorates collagen-induced arthritis (CIA) in rats.Methods. Binding of 13-nm nanogold to VEGF in human RA synovial fluid (SF) and its effects on RA SF-induced endothelial cell proliferation and migration were assessed. Nanogold was administered intraarticularly to rats with CIA before the onset of arthritis. Progression of CIA was monitored by measures of clinical, radiologic, and histologic changes. In addition, the microvessel density and extent of infiltrating macrophages as well as levels of tumor necrosis factor ␣ (TNF␣) and interleukin-1␤ (IL-1␤) in the ankle joints were determined.Results. Nanogold bound to VEGF in RA SF, resulting in inhibition of RA SF-induced endothelial cell proliferation and migration. Significant reductions in ankle circumference, articular index scores, and radiographic scores were observed in the nanogoldtreated rats with CIA compared with their control counterparts. In addition, the histologic score (of synovial hyperplasia, cartilage erosion, and leukocyte infiltration), microvessel density, macrophage infiltration, and levels of TNF␣ and IL-1␤ were also significantly reduced in the ankle joints of nanogold-treated rats.Conclusion. Our results are the first to demonstrate that intraarticular administration of nanogold ameliorates the clinical course of CIA in rats. Nanogold exerted antiangiogenic activities and subsequently reduced macrophage infiltration and inflammation, which resulted in attenuation of arthritis. These results demonstrate proof of principle for the use of nanogold as a novel therapeutic agent for the treatment of

We developed an integrated proteomics approach using a chemically functionalized gold nanoparticle (AuNP) as a novel probe for affinity purification to analyze a large protein complex in vivo. We then applied this approach to globally map the transcriptional activation complex of the estrogen response element (ERE). This approach was designated as quantitative nanoproteomics for protein complexes (QNanoPX). In this approach, the positive AuNP-ERE probes were functionalized with polyethylene glycol (PEG), and the consensus sequence of ERE and negative AuNP-PEG probes were functionalized with PEG without the ERE via a thiolated self-assembly monolayer technique. The AuNP-ERE probe had substantially low nonspecific binding and high solubility, which resulted in a 20-fold enrichment of the factor compared with gel beads. In addition, the surface-only binding allows the probe to capture a large protein complex without any restrictions due to pore size. The affinity purification method was combined with MS-based quantitative proteomics and statistical methods to reveal the components of the ERE complex in MCF-7 cells and to identify those components within the complex that were altered by the presence of 17␤-estradiol (E2).

Gold nanoparticles (AuNPs) are widely used as carriers or therapeutic agents due to their great biocompatibility and unique physical properties. Transforming growth factor-beta 1 (TGF-β1), a member of the cysteine-knot structural superfamily, plays a pivotal role in many diseases and is known as an immunosuppressive agent that attenuates immune response resulting in tumor growth. The results reported herein reflect strong interactions between TGF-β1 and the surface of AuNPs when incubated with serum-containing medium, and demonstrate a time- and dose-dependent pattern. Compared with other serum proteins that can also bind to the AuNP surface, AuNP-TGFβ1 conjugate is a thermodynamically favored compound. Epithelial cells undergo epithelial-mesenchymal transition (EMT) upon treatment with TGF-β1; however, treatment with AuNPs reverses this effect, as detected by cell morphology and expression levels of EMT markers. TGF-β1 is found to bind to AuNPs through S-Au bonds by X-ray photoelectron spectroscopy. Fourier transform infrared spectroscopy is employed to analyze the conformational changes of TGF-β1 on the surface of AuNPs. The results indicate that TGF-β1 undergoes significant conformational changes at both secondary and tertiary structural levels after conjugation to the AuNP surface, which results in the deactivation of TGF-β1 protein. An in vivo experiment also shows that addition of AuNPs attenuates the growth of TGF-β1-secreting murine bladder tumor 2 cells in syngeneic C3H/HeN mice, but not in immunocompromised NOD-SCID mice, and this is associated with an increase in the number of tumor-infiltrating CD4⁺ and CD8⁺ T lymphocytes and a decrease in the number of intrasplenic Foxp3(+) lymphocytes. The findings demonstrate that AuNPs may be a promising agent for modulating tumor immunity through inhibiting immunosuppressive TGF-β1 signaling.

Recently, it has been found that thiolated oligonucleotides can be used to direct the assembly of gold colloids into ordered functional nanostructures. However, approaches for fabricating Au/DNA conjugates by using a linear, functional, double-stranded (ds) DNA fragment that directly binds to a Au NP surface have not been reported. In this paper, we used a ligase-dependent strategy to synthesize a 1.7 kb, thiolated dsDNA fragment and generated novel nanocrystals by coupling it to 13 nm Au NPs. The conjugates contained Au/EGFP with EGFP/Au molar ratios of 1:1, 2:1, and 3:1, as determined by their electrophoretic mobility and AFM imaging. The Au/EGFP nanoconjugates could still be digested by restriction endonuclease (RE) and expressed as functional proteins in mammalian cells, indicating the biological activities of DNA fragments were retained after conjugation with Au NPs. This biological strategy for fabrication of Au/DNA conjugates may be applied to molecular imaging, nanomedicine, and nanobiosensor technology.