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Cy5.5-Conjugated matrix metalloproteinase cleavable peptide nanoprobe

Cy5.5-MMP probe
, PhD
National Center for Biotechnology Information, NLM, Bethesda, MD 20894

Created: ; Last Update: April 12, 2012.

Chemical name:Cy5.5-Conjugated matrix metalloproteinase cleavable peptide nanoprobe
Abbreviated name:Cy5.5-MMP probe
Synonym:
Agent Category:Peptide
Target:Matrix metalloproteinase
Target Category:Enzyme
Method of detection:Optical imaging (near-infrared fluorescence (NIRF) imaging)
Source of signal / contrast:Cy5.5
Activation:Yes
Studies:
  • Checkbox In vitro
  • Checkbox Rodents
Structure not available in PubChem.

Background

[PubMed]

Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidase enzymes that are known to regulate diverse physiological processes such as cell growth, differentiation, and migration, and they play an important role in the development and progression of osteoarthritis (1) and cancer (2, 3). The classification, structural characteristics, induction, regulation, and biological functions of >25 MMPs have been reviewed by Klein and Bischoff (3). Because MMPs are overexpressed in malignant tumors and support the establishment of neoplasms, these enzymes are the target of many anti-cancer drugs (4). In addition, researchers are interested to develop and evaluate imaging probes that target the MMPs so as to detect cancers at an early stage, track progression of the disease, and monitor the response to cancer therapy (5). A strategy using activatable cell-penetrating peptides (ACPPs) labeled with a radionuclide (6) or an optical imaging dye (7) has been utilized by investigators for the successful detection of tumors that overexpress MMPs in mice. However, it was shown that the ACPP probe was probably not suitable for the detection of these lesions because the probe was most likely activated in the vasculature and the tumors showed nonspecific uptake of the label (6).

Investigators have used fluorogenic probes that consist of a fluorophore and a quencher attached to a targeting peptide for in vivo detection of the MMP activity (1). A characteristic feature of these probes is that, in the normal state, fluorescence emitted by the fluorophore is absorbed by the quencher through resonance energy transfer and no signal is detected from the peptide (for a schematic diagram, see Ryu et al. (1)). The fluorescence generated by the probe is detectable only when the targeting peptide is cleaved by a suitable protease such as the MMP and the quencher is released from the complex. A limitation of using these probes in vivo is that they exhibit low stability and the constituent peptides may be cleaved nonspecifically by different proteases (1). Therefore, it was apparent that a probe containing a substrate peptide that is specific for an MMP would be most suitable for the detection of these enzymes. On the basis of this information, a fluorogenic probe containing an MMP-13–specific peptide substrate (Gly-Pro-Leu--Gly-Met-Arg-Gly-Leu-Gly-Lys; the substrate amino acid sequence is italicized, and the MMP cleavage site is indicated with a double hyphen) sandwiched between the Cy5.5 fluorescence dye and the black hole quencher-3 (BHQ-3) was developed (1). The intact probe (Cy5.5-MMP probe) exhibited low background fluorescence that increased significantly only after the peptide substrate was cleaved by the MMP. The biodistribution of the Cy5.5-MMP probe and its ability to detect neoplastic lesions in mice bearing murine squamous cell carcinoma SCC-7 cell tumors were investigated by Yhee et al. (8).

Synthesis

[PubMed]

The MMP substrate peptide was synthesized with standard solid-phase Fmoc chemistry, and the Cy5.5-MMP probe was prepared as described by Ryu et al. (1). The final Cy5.5-labeled probe was characterized with ultraviolet-visible spectroscopy and matrix-assisted laser desorption/ionization-time of flight mass spectroscopy. Purity of the final preparation of Cy5.5-MMP was reported to be >95% as determined with reverse-phase high-performance liquid chromatography (8). The formulation, stability, storage conditions, and the ratio of Cy5.5 to the MMP peptide in the probe were not reported.

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

Generation of fluorescence by Cy5.5-MMP was investigated by incubating 20 nM of the probe with increasing concentrations of MMP-13 (2 nM, 4 nM, and 8 nM) in a 96-well microplate for 30 min at 37°C as described by Yhee et al. (8). Control wells either contained no MMP-13 or had the protease in the presence of an inhibitor (4 nM; the nature of the inhibitor was not reported). Measurement of the fluorescence intensity of the wells showed that wells with 2 nM, 4 nM, and 8 nM of the protease had 13.4-, 24.7-, and 48.4-fold higher fluorescence intensity, respectively, compared with the wells without any MMP-13 or with the protease inhibitor. This indicated that the MMP probe was functional.

To confirm that the Cy5.5-MMP probe is suitable for in vitro detection of the MMP activity, different concentrations of the fluorescence-labeled peptide (ranging from 3.125 nM to 50 nM) were incubated as before with serum-free growth medium harvested from SCC-7 cells (these cells express and secrete the MMP into the growth medium) in a 96-well microplate (8). For use as controls, similar concentrations of the probe were exposed to distilled water. Fluorescence imaging of the plates showed that approximately 400 units, 600 units, and 900 units of fluorescence were generated with 12.5 nM, 25 nM, and 50 nM of the probe, respectively. The control wells exhibited constant fluorescence intensity at all concentrations of the optical probe (approximately 200 units).

Animal Studies

Rodents

[PubMed]

Biodistribution of the Cy5.5-MMP probe was investigated in nude mice bearing SCC-7 cell tumors (8). The animals (n = 5 mice/group) were injected with 40 nM of the probe through the tail vein, and fluorescence images were acquired from the animals at different time periods from 1 h to 12 h postinjection (p.i.). At 1 h p.i. the tumor was visible in the images, and the fluorescence intensity from the lesion peaked at approximately 6.25 × 104 counts/sec at 3 h p.i. By 12 h p.i., the intensity decreased to approximately 4.75 × 104 counts/sec. Ex vivo fluorescence imaging of various organs obtained from the animals at 3 h p.i. showed that maximum fluorescence intensity was generated in the tumors (approximately 200 counts/sec), followed by the kidneys (approximately 75 counts/sec) and the liver (approximately 50 counts/sec). Fluorescence microscopy and immunostaining of the tumor and liver tissues showed that the protease activity was present mainly in the tumor tissue and only low levels of enzyme activity were evident in the liver. No blocking studies were reported.

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.
Ryu J.H., Lee A., Na J.H., Lee S., Ahn H.J., Park J.W., Ahn C.H., Kim B.S., Kwon I.C., Choi K., Youn I., Kim K. Optimization of matrix metalloproteinase fluorogenic probes for osteoarthritis imaging. Amino Acids. 2011;41(5):1113–22. [PubMed: 20953646]
2.
Hua H., Li M., Luo T., Yin Y., Jiang Y. Matrix metalloproteinases in tumorigenesis: an evolving paradigm. Cell Mol Life Sci. 2011;68(23):3853–68. [PubMed: 21744247]
3.
Klein T., Bischoff R. Physiology and pathophysiology of matrix metalloproteases. Amino Acids. 2011;41(2):271–90. [PMC free article: PMC3102199] [PubMed: 20640864]
4.
Gialeli C., Theocharis A.D., Karamanos N.K. Roles of matrix metalloproteinases in cancer progression and their pharmacological targeting. FEBS J. 2011;278(1):16–27. [PubMed: 21087457]
5.
Scherer R.L., McIntyre J.O., Matrisian L.M. Imaging matrix metalloproteinases in cancer. Cancer Metastasis Rev. 2008;27(4):679–90. [PubMed: 18465089]
6.
van Duijnhoven S.M.J., Robillard M.S., Nicolay K., Grull H. Tumor Targeting of MMP-2/9 Activatable Cell-Penetrating Imaging Probes Is Caused by Tumor-Independent Activation. Journal of Nuclear Medicine. 2011;52(2):279–286. [PubMed: 21233187]
7.
Olson E.S., Jiang T., Aguilera T.A., Nguyen Q.T., Ellies L.G., Scadeng M., Tsien R.Y. Activatable cell penetrating peptides linked to nanoparticles as dual probes for in vivo fluorescence and MR imaging of proteases. Proc Natl Acad Sci U S A. 2010;107(9):4311–6. [PMC free article: PMC2840175] [PubMed: 20160077]
8.
Yhee J.Y., Kim S.A., Koo H., Son S., Ryu J.H., Youn I.C., Choi K., Kwon I.C., Kim K. Optical imaging of cancer-related proteases using near-infrared fluorescence matrix metalloproteinase-sensitive and cathepsin B-sensitive probes. Theranostics. 2012;2(2):179–89. [PMC free article: PMC3287424] [PubMed: 22375156]

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