Abstract
Background: Metastasis plays a crucial role in cancer-related mortality. Patients under different metastasis status usually have varies prognosis and survival. Hence early detection of metastasis is of vital importance in cancer diagnosis and treatment. Promisingly, several specific metastasis biomarkers have been identified as their expression in cancer sites are unique, thus providing a potential strategy in cancer diagnosis. Previous studies have demonstrated Matrix Metalloproteinase 9 (MMP-9) is a favorable metastasis marker since its expression was shown to be correlated with metastasis level. Therefore, in this study, a MMP-9-stimuli peptide that could be cleaved by MMP-9 specifically was utilized to develop nanodiamond (ND)-peptide complex as MMP-9 specific biosensor for metastasis detection.
Methods: The MMP-9 substrate peptide was chemically conjugated onto NDs to obtain ND-peptide biosensor complex, ND-MMP9. The peptide was also labelled with fluorescent dyes so as to better monitor the cleavage signal, which represented the protease activity of MMP-9. Characterization of ND-MMP9, as well as sensitivity and accuracy of MMP-9 detection, were then investigated in vitro.
Results: ND-MMP9 could be rapidly synthesized and had favorable drug delivery properties as well. In addition, ND-MMP9 could be cleaved by MMP-9 and its cleavage signal could be quantified as well as visualized using confocal microscopy. Furthermore, in human hepatocellular carcinoma cell lines, the cleavage was correlative with MMP-9 expression level. Importantly, ND-MMP9 exhibited stronger in-tube serum stability compared to the naïve substrate peptide, revealing an enhanced ability to protect the base sensor peptide from nonspecific serum protease cleavage, which further expands the applications of such substrate peptide.
Conclusions: This enhanced peptide stability, combined with a quantitative stimuli-responsive output function, provides strong evidence for the further development of a ND-MMP9 biosensor for metastasis site detection. More importantly, this work provides the foundation for use of NDs as a platform for stimuli-responsive biosensors and theranostic complexes that can be implemented across a wide range of biomedical applications.
