Cy5.5-Containing matrix metalloproteinase (MMP) activatable peptide conjugated to glycol chitosan (GC)–coated gold nanoparticles (AuNPs)

MMP-GC-AuNPs

Chopra A.

Publication Details

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In vitro Rodents

Background

[PubMed]

The use of gold nanoparticles (AuNPs) for molecular imaging has attracted much attention because these particles are easy to fabricate in different shapes, have a quenching effect, and can be used for computed tomography (CT) (1). Because bare AuNPs are unstable (due to aggregation) and are non-targeted, these particles have to be to be coated with biocompatible materials to improve their stability and application under in vivo conditions. In addition, NPs that are targeted to specific cellular biomarkers are useful for the detection and treatment of diseases such as cancers (for details, see Jiao et al. (2) and Lim et al. (3)). However, the AuNP surface coatings (e.g., thiolated polyethylene glycol) may influence the biological behavior of these particles and prevent their uptake by cells. This can result in reduced clearance of the NPs through the reticuloendothelial system, prolonged blood circulation times, and increased accumulation in tumors (4). Therefore, investigators are devoting much effort to develop AuNPs that are easy to synthesize, physiologically stable, and can be used to detect (e.g., by noninvasive imaging) and treat cancerous tumors (1). In an effort to produce AuNPs that would meet this criteria, the particles were coated with glycol chitosan (GC) and conjugated to a Cy5.5-containing matrix metalloproteinase (MMP) activatable peptide (Cy5.5-Gly-Pro-Leu-Gly-Val-Arg-Gly-Lys-black hole quencher-3 (BHQ-3) dye-Gly-Gly; the MMP cleavable site is in italics) to produce MMP-GC-AuNPs for use as a multimodal computed tomography (CT)/near-infrared fluorescence (NIRF) imaging probe (1). The MMP-GC-AuNPs were then evaluated with CT and NIRF optical imaging techniques for the visualization of HT-29 cell xenograft tumors (these cells express the MMP-2) in nude mice.

Synthesis

[PubMed]

The synthesis, formulation, and storage of the MMP-GC-AuNPs is described by Sun et al. (1). Based on the standard absorption curve of Cy5.5 at 675 nm, 572 molecules of the MMP peptide probe were reported to be conjugated to each GC-AuNP. Transmission electron microscopy showed that the MMP-GC-AuNPs had a spherical shape and a diameter of 20 nm. The GC layer on the AuNPs was uniform with a thickness of 10 nm, and the hydrodynamic diameter of the NPs in an aqueous solution was 99.4 ± 16.8 nm as determined with dynamic light scattering. The ζ potential of the MMP-GC-AuNPs was 34.48 ± 1.00 mV.

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

The MMP-GC-AuNPs were reported to be stable in distilled water and phosphate-buffered saline from pH 3 to pH 11 for at least 80 days, and they maintained their diameter during this period (1). A cell proliferation assay with HeLa cells exposed to MMP-GC-AuNPs for 24 h showed that the NPs were non-toxic to the cells even at a concentration of 1 mg/mL in the growth medium (1).

MicroCt imaging of the MMP-GC-AuNPs showed that the particles had superior X-ray absorption properties compared with eXIA™160, an iodine-based contrast agent used for CT (1).

The investigators reported that 90% of the NIRF signal of MMP-GC-AuNPs was quenched by the fluorescence resonance energy transfer from the BHQ-3 component of the MMP activatable peptide (1). The NIRF of the gold NPs was recovered only in presence of MMP-2, and the NIRF intensity was shown to increase with the concentration of the enzyme.

Exposure of SCC-7 cells (express and secrete the MMP into the growth medium) to MMP-GC-AuNPs for 1 h showed that a strong fluorescence signal was generated with as little as 104 cells (1). This indicated that the MMP-GC-AuNPs had a high sensitivity to the activity of the MMP enzymes.

Animal Studies

Rodents

[PubMed]

Nude mice bearing HT-29 cell tumors (number of animals not reported) were injected with the MMP-GC-AuNP probe (200 μL, 300 mg/kg body weight (BW)) through the tail vein, and the rodents were anesthetized to acquire whole-body CT images after different time periods (1). In the CT images, the tumor was clearly distinguishable from the surrounding tissues at 2 h postinjection (p.i.), and at 24 h p.i. the Hounsfield units value for the lesions was 254, which was approximately five-fold higher than the normal surrounding tissues (Hounsfield units value = ~40). The lesions were not visible when eXIA™160 was used as the contrast agent for the CT imaging of the tumors (1). The investigators attributed this to the lack of tumor specificity and fast clearance of the eXIA™160 contrast agent from the body.

For NIRF imaging, the tumor-bearing animals (number of mice not reported) were injected with 200 μL (5 mg/kg BW) of the MMP-GC-AuNPs as before, and NIRF optical images were acquired from the animals at various time points (1). At 1 h p.i., an increased NIRF signal was apparent only in the tumor compared with the normal surrounding tissues, and the lesions were clearly visible from 4 h p.i. to 48 h p.i. In a blocking study, the animals were injected with the probe 30 min after intratumoral injection of a MMP inhibitor (the inhibitor was not specified), and no NIRF signal was generated in the tumor. At 4 h p.i., the mice (with or without the MMP inhibitor treatment) were euthanized, and organs of interest were removed from the animals. NIRF images of the organs showed that the tumor was clearly distinguishable from rest of the organs. Among all the organs, a low NIRF signal was observed only in the liver. Dark field microscopy of the various tissues confirmed the presence of MMP-GC-AuNPs in the tumors, including those treated with the MMP inhibitor. In addition, generation of the NIRF signal and the presence of the MMP enzymes in the tumor tissues was confirmed with optical fluorescence microscopy and immunostaining (for MMP), respectively (1).

On the basis of these results, the investigators concluded that the MMP-GC-AuNPs accumulated specifically in the tumor tissues and could be used as a dual CT/NIRF probe for the detection of cancerous tumors in rodents (1).

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.

Supplemental Information

[Disclaimers]

No information is currently available.

References

1.
Sun I.C., Eun D.K., Koo H., Ko C.Y., Kim H.S., Yi D.K., Choi K., Kwon I.C., Kim K., Ahn C.H. Tumor-targeting gold particles for dual computed tomography/optical cancer imaging. Angew Chem Int Ed Engl. 2011;50(40):9348–51. [PubMed: 21948430]
2.
Jiao P.F., Zhou H.Y., Chen L.X., Yan B. Cancer-targeting multifunctionalized gold nanoparticles in imaging and therapy. Curr Med Chem. 2011;18(14):2086–102. [PubMed: 21517767]
3.
Lim Z.Z., Li J.E., Ng C.T., Yung L.Y., Bay B.H. Gold nanoparticles in cancer therapy. Acta Pharmacol Sin. 2011;32(8):983–90. [PMC free article: PMC4002534] [PubMed: 21743485]
4.
Arnida M.M. Janat-Amsbury, A. Ray, C.M. Peterson, and H. Ghandehari, Geometry and surface characteristics of gold nanoparticles influence their biodistribution and uptake by macrophages. Eur J Pharm Biopharm. 2011;77(3):417–23. [PMC free article: PMC3379889] [PubMed: 21093587]