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Bovine serum albumin–stabilized gold nanoclusters conjugated with folate and indocyanine green derivative 02

Au-FA-MPA
, PhD
National Center for Biotechnology Information, NLM, NIH, Bethesda, MD

Created: ; Last Update: January 24, 2013.

Chemical name:Bovine serum albumin–stabilized gold nanoclusters conjugated with folate and indocyanine green derivative 02
Abbreviated name:Au-FA-MPA
Synonym:Au-BSA-FA-MPA, Au-BSA-FA-ICG-Der-02, Au-FA-ICG-Der-02
Agent category:Compound
Target:Folate receptor
Target category:Receptor
Method of detection:Optical, near-infrared fluorescence (NIR) imaging
Source of signal:ICG-Der-02 (MPA)
Activation:No
Studies:
  • Checkbox In vitro
  • Checkbox Rodents
Structure is not available in PubChem.

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 (Km, 1–5 μM). Some cells in the choroid plexus, kidney, lung, thyroid, spleen, placenta, and thymus also possess a higher-affinity receptor (Kd, 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

Rodents

[PubMed]

Chen et al. (9) performed in vivo whole-body NIR fluorescence imaging studies in nude mice (n = 5/group) bearing MDA-MB-231 human breast cancer (high folate receptor expression), HTC116 human colon cancer (moderate to high folate receptor expression) or HepG-2 human lung cancer xenografts (low folate receptor expression) at 0.5–96 h after intravenous injection of Au-FA-MPA (10 mg/kg body weight). Images showed that NIR fluorescence (MPA) spread all over the body at 0.5 h and then concentrated in the liver and kidney. Most NIR fluorescence of the normal tissues was cleared from the body by 10 h. MDA-MB-231 tumors were clearly visualized at 2 h, and the NIR fluorescence peaked at 20 h. The tumor NIR fluorescence was still detectable at 96 h. HTC116 tumors exhibited weaker NIR fluorescence than the MDA-MB-231 tumors at 2–4 h, and fluorescence was almost indistinguishable from the background by 20 h. On the other hand, HepG-2 tumors showed a low fluorescence signal at 3 h, which became undetectable by 10 h. Tumor/background ratios for MDA-MB-231 tumors were 2.9 ± 0.5 at 1 h, 11.4 ± 0.6 at 20 h, and 6.4 ± 0.2 at 72 h. Tumor/background ratios for HTC116 tumors were 2.1 ± 0.4 at 1 h, 6.4 ± 0.6 at 10 h, and 2.2 ± 0.3 at 48 h. Tumor/background ratios for HepG-2 tumors were 3.8 ± 0.5 at 3 h and 1.2 ± 0.1 at 20 h. Ex vivo NIR fluorescence images of the excised tumors and normal organs at 5 h showed strong signals in the MDA-MB-231 tumor, HTC116 tumor, liver, and kidney but weak signal in the HepG-2 tumor. The excised organs from normal mice exhibited little histological change at 7 d after injection of Au-FA-MPA (250 mg/kg). No blocking studies were performed.

Other Non-Primate Mammals

[PubMed]

No publication is currently available.

Non-Human Primates

[PubMed]

No publication is currently available.

Human Studies

[PubMed]

No publication is currently available.

References

1.
Stanger O. Physiology of folic acid in health and disease. Curr Drug Metab. 2002;3(2):211–23. [PubMed: 12003352]
2.
Ke C.Y., Mathias C.J., Green M.A. The folate receptor as a molecular target for tumor-selective radionuclide delivery. Nucl Med Biol. 2003;30(8):811–7. [PubMed: 14698784]
3.
Yannuzzi, L.A., Indocyanine green angiography: a perspective on use in the clinical setting. Am J Ophthalmol, 2011151(5): p. 745-751 e1. [PubMed: 21501704]
4.
Yamamoto M., Sasaguri S., Sato T. Assessing intraoperative blood flow in cardiovascular surgery. Surg Today. 2011;41(11):1467–74. [PubMed: 21969147]
5.
Schaafsma B.E., Mieog J.S., Hutteman M., van der Vorst J.R., Kuppen P.J., Lowik C.W., Frangioni J.V., van de Velde C.J., Vahrmeijer A.L. The clinical use of indocyanine green as a near-infrared fluorescent contrast agent for image-guided oncologic surgery. J Surg Oncol. 2011;104(3):323–32. [PMC free article: PMC3144993] [PubMed: 21495033]
6.
Manizate F., Hiotis S.P., Labow D., Roayaie S., Schwartz M. Liver functional reserve estimation: state of the art and relevance for local treatments: the Western perspective. J Hepatobiliary Pancreat Sci. 2010;17(4):385–8. [PubMed: 19936599]
7.
Huang C.C., Chen C.T., Shiang Y.C., Lin Z.H., Chang H.T. Synthesis of fluorescent carbohydrate-protected Au nanodots for detection of Concanavalin A and Escherichia coli. Anal Chem. 2009;81(3):875–82. [PubMed: 19119843]
8.
Chen H., Li B., Ren X., Li S., Ma Y., Cui S., Gu Y. Multifunctional near-infrared-emitting nano-conjugates based on gold clusters for tumor imaging and therapy. Biomaterials. 2012;33(33):8461–76. [PubMed: 22951103]
9.
Chen H., Li S., Li B., Ren X., Mahounga D.M., Cui S., Gu Y., Achilefu S. Folate-modified gold nanoclusters as near-infrared fluorescent probes for tumor imaging and therapy. Nanoscale. 2012;4(19):6050–64. [PubMed: 22930451]
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