Background

[PubMed]

Folic acid (FA) is a water-soluble B vitamin (1). It is essential for methylation and DNA synthesis. The primary pathway for entry of folate into cells is via a facilitated transporter, which has a low affinity for folate (K_m, 1–5 μM). Some cells in the choroid plexus, kidney, lung, thyroid, spleen, placenta, and thymus also possess a higher-affinity receptor (K_d, 0.5 nM) that allows folate uptake via receptor-mediated endocytosis. Some human epithelial tumor cells were found to overexpress folate receptors (2). More than 90% of human ovarian and endometrial cancers express the high-affinity receptor, which is absent in normal tissues. Breast, colorectal, renal, and lung carcinomas also overexpress the high-affinity folate receptor but to a lesser frequency (20%–50%).

Among the various optical imaging agents, only indocyanine green (ICG), with NIR fluorescence absorption at 780 nm and emission at 820 nm, is approved by the United States Food and Drug Administration for clinical applications in angiography, blood flow evaluation, and liver function assessment (3-6). It is also under evaluation in several clinical trials for other applications, such as optical imaging and mapping of both the lymphatic vessels and lymph nodes in cancer patients for surgical dissection of tumor cells and endoscopic imaging of the pancreas and colon. Gold (Au) nanoclusters (NCs) are comprised of a few tens of Au atoms that possess the ability to emit strong fluorescence, of which the wavelength is tunable in the range of the visible to near-infrared spectral region (7). Chen et al. (8) prepared bovine serum albumin–stabilized Au NCs (Au-BSA) for conjugation with biomolecules for specific targeting and with near-infrared dye for optical imaging. Folate was conjugated to the amino groups of BSA. The ICG derivative 02 (ICG-Der-02, also known as MPA) contains one carboxyl functional group for covalent conjugation to the amino groups of BSA on the surface of Au-BSA NCs. MPA is a hydrophilic dye. Chen et al. (9) evaluated Au-BSA-FA-MPA (Au-FA-MPA) for in vivo NIR optical imaging of folate receptor expression in tumor-bearing mice.

Synthesis

[PubMed]

Au-BSA NCs were prepared by addition of HAuCl₄ solution (10 mM) to BSA solution (50 mg/ml) under vigorous stirring for 30 min at 37°C (9). The mixture was stirred for an additional 12 h at 37°C. FA was activated with N,N'-dicyclohexylcarbodiimide (DDC) and N-hydroxysuccinimide (NHS) (molar ratio of FA:DCC:NHS was 1:1.5:2) in dimethyl sulfoxide for 12 h at 50°C. The activated FA was extracted with acetone. MPA (1.0 mg) was activated with DDC and NHS (molar ratio of MPA:DCC:NHS was 1:1.5:2) for 3 h at room temperature. The activated FA and Au-BSA were mixed in Tris buffer (pH 8) for 12 h at 4°C. Au-BSA-FA NCs were isolated with column chromatography. The activated MPA was incubated with AU-BSA-FA NCs for 12 h at room temperature. The final product, Au--FA-MPA, was isolated with column chromatography. The mean hydrodynamic diameter of Au-BSA (measured with laser particle size analysis) was 3.74 nm (polydispersity = 0.373), whereas Au-FA-MPA exhibited a relatively larger diameter of 4.82 nm (polydispersity = 0.453), indicating the successful addition of FA and MPA molecules. The absorption spectra of FA, Au-BSA, MPA, Au-BSA-FA, and Au-FA-MPA exhibited absorbance peaks at 365, 232, and 780 nm, which corresponded to FA, Au, and MPA, respectively. The number of Au, BSA, FA, and MPA moieties per Au-FA-MPA was not reported. Au-FA-MPA displayed spectral properties similar to those of MPA, with maximum absorption at 780 nm and maximum emission at 810 nm.

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

The cytotoxic effects of Au-BSA and Au-FA-MPA (0.01–1,000 µM) were assessed in human cell lines HepG-2 (liver cancer) and MDA-MB-231 (breast cancer) for 24 h at 37°C (9). Approximately 95% viability was observed with 0.01–1 µM, with 85% at 1 mM. Confocal fluorescence microscopy analysis showed little binding of Au-BSA and Au-FA-MPA to HepG-2 cells (low folate receptor expression), whereas strong fluorescence signal (MPA) was observed around the nuclei of MCF-7 cells (high folate receptor expression) with Au-FA-MPA but not with Au-BSA-MPA. No blocking studies were performed with excess FA.

Animal Studies

Human Studies

[PubMed]

No publication is currently available.

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