In many cases, poor specificity and sensitivity of current optical imaging probes limit their imaging ability and applications. From this point of view, the emergence of activatable molecular imaging probes based on signal amplification strategies are a promising set of imaging tools which can overcome these limitations. They offer several distinct advantages over conventional dye-labeled imaging probes: i) multiple fluorophores are able to be cleaved by a single enzyme resulting in an intrinsic signal amplification, ii) quenching of the probe before activation results in significantly low background fluorescence and iii) the activation efficiency strongly depends on the protease expression of target cells resulting in highly specific detection of target molecules. The combined effect results in high signal-to-background ratios and improved detection sensitivity and specificity.
Over the past decade, activatable fluorescent probes underwent major improvements, but the increased fluorescent signal outputs are not exciting, especially for in vivo application. Besides the high biological tissue absorption of fluorescent signals, fluorescent activatable probes need to prevent additional, equally important, limitations like efficiently decreasing background signal and increasing fluorescent output signal. As mentioned in the previous section, the quencher-fluorophore pairs of peptide probes are generally one-to-one format, limiting improvement in the signal-to-background ratio. In addition, the efficient targeted intracellular delivery of activatable probes limits the development of imaging caspase activity. Combining NPs with activatable probes can ensure high payload delivery of these probes to the target site, penetrating the cytoplasm by a different cellular uptake mechanism. With the incorporation of polymer NPs and novel metals such as gold NPs into one platform, use of complementary imaging modalities like PET, SPECT, MRI and CT is conceivable and is no longer confined to depth penetration concerns of optical imaging techniques.
The largest limitation of BLI is that many clinical trials using viral vectors for transfection have been terminated since the application of these vectors have induced unexpected adverse effects such as toxicity, immunogenicity and oncogenicity. Nanomaterials are gaining more interest as alternatives to viral vectors for gene delivery, but it is hard to achieve a major breakthrough in a short term [79
]. However, it is emerging as a powerful technology to study viral pathogenesis, immune responses to infection, and quantifying effects of therapy in living animals. Although the outlook of activatable probes is clear and exciting, most platforms are in the proof-of-concept stage. Many obstacles, such as pharmacokinetics, non-specific degradation and aggregation, and potential toxicity of the probe, should be considered.
Despite the application of these technologies for optical imaging, “smart probes” for apoptosis imaging is still in its infancy and has a long way to go. The ability to utilize these aforementioned technologies in vivo will surely lead to advancements in early detection, improved diagnosis and therapeutic treatments tailored for individual patients.