INTRODUCTION AND LITERATURE REVIEW
1.3 Aptamers
1.3.9 Applications of Aptamer
1.3.9.1 Molecular imaging
An accurate and specific diagnosis of carcinoma to identify its type or even subtype is especially crucial and directly determines the treatment design from doctors. In recent years, as a rapidly emerging field, aptamers as an imaging probe has received much attention in biomedical research and clinical diagnoses. Small oligonucleotide aptamers have clear superiority over antibodies for in vitro or in vivo bioimaging applications. The antibody-based bioimaging agents suffer from poor tissue penetration, high immunogenicity, and long blood residence time that can result in unwanted side effects (Kobayashi and Choyke, 2011). Using aptamers as imaging agents has the advantage of their being non-toxic, because oligonucleotide moieties are present in the human body.
Additionally, as aptamers have high specificity for their target, accurate targeting and rapid diffusion through the blood circulation, use of these molecules can increase the certainty of the results obtained during diagnosis or clinical analysis. Aptamers can be chemically synthesized and easily modified, thus they can be modified with fluorescent
groups or quantum dots differential dyes or colorimetric reporter molecules, and used for imaging. Based on these advantages, aptamers have been considered as an imaging agents for cell imaging as well as single-protein imaging (Song et al., 2012). Shi et al were the first group to report the imaging of tumors in mice with high specificity using Ramos lymphoma cells specific Cy5-labeled TD05 aptamer.(Shi et al., 2010).
Fluorescence imaging of cancerous cell or tumors with aptamers has been well documented in the literature. The aptamer probes were labelled with fluorescent molecules like Cy5, FITC, 6-FAM etc and are used for imaging as well as tracking cancerous cells in vitro and in vivo. In one study, an RNA aptamer that binds to recombinant integrin αvβ3 was labeled with Cy5 and tested for its ability to bind to endogenous integrin αvβ3 on cell surface, as well as to subsequently affect cellular responses (Mi et al., 2005). In another study, aptamers generated against Epithelial cell adhesion molecule (EpCAM) were labelled with FITC and used for imaging MDA-MB- 231, Kato III, and HEK 293T cells (Fig. 1.11) (Song et al., 2013). These are one step detection method where aptamers were directly conjugated to fluorescent molecule.
Another approach involves two step detection strategy where capture aptamers were biotinylated and the detection molecules were fluorescent tagged. For instance Bayrac et al labelled GMT 8 aptamers (specific to A172 cell line) with biotin group and streptavidin-PE were used for signal generation. Sometimes nanorods were used as a scaffold for multivalent binding of multiple aptamers to enhance both the signal intensity and binding affinity for cancer cell recognition. In one example aptamer, KK1HO8 with relatively weak binding affinities to its targets on conjugation with nanorod showed a 300 fold increase in fluorescence signal on binding to K-562 cells (Huang et al., 2008).
Quantum dots (QDs) are inorganic fluorescent semiconductor nanoparticles with many desirable properties for imaging applications such as high quantum yields, high molar extinction coefficients, strong resistance to photobleaching and chemical degradation, narrow emission spectra and large effective Stokes shifts (Cai et al., 2007; Li et al., 2007). A biotin labeled ssDNA aptamer TLS9a specific to mouse liver hepatoma cell line BNL 1ME A.7R.1 (MEAR) was fabricated on the streptavidin-modified quantum dots (SA-QDs) to obtain a biocompatible probe for live cell imaging. In another example, multifunctional Quantum dot aptamer approach was devised where quantum dot (QD)-
aptamer(Apt)-doxorubicin (Dox) conjugate [QD-Apt(Dox)] was used for cancer imaging as well as targeted delivery vehicle (Bagalkot et al., 2007).
Figure 1.11: Confocal images of cultured HEK-293T, MDA-MB-231, and Kato III cells stained with the FITC-labelled initial library and SYL3C aptamer. "Reprinted with permission from Song et al., 2013. Copyright 2013 American Chemical Society."
Gold nanoparticles possess unusual optical and electronic properties, high stability and biological compatibility, controllable morphology and size dispersion, and easy surface functionalization (Daniel and Astruc, 2004; Grzelczak et al., 2008; Rosi et al., 2006;
Sperling et al., 2008; Storhoff et al., 2004). Aptamer‑conjugated gold NPs (Apt‑AuNPs) provide a new platform to facilitate targeted recognition and detection. . The Mirkin group pioneered the use of AuNP-DNA conjugates, that is, AuNPs modified with thiolated oligonucleotide probe, which led to development of a series of novel assay methods for the ultrasensitive detection of DNA and proteins (Mirkin et al., 1996; Rosi and Mirkin, 2005). Apt-AuNP conjugates were also used to develop colorimetric assays for the detection of cancerous cells (Medley CD et al., 2008) , biomolecules such as
platelet-derived growth factors (PDGFs) (Huang et al., 2005) and small molecules like k+ ions (L. Wang et al., 2006), cocaine (Zhang et al., 2008), ATP (Wang et al., 2007) etc. Nanospheres were also used for fabricating aptamers. A labeled anti-human epidermal growth factor receptor antibody or aptamer was conjugated with hollow gold nanospheres for in vivo imaging of head and neck cancer. Interestingly, the tumor uptake of aptamer-guided imaging probes was much higher than that of the antibody-guided imaging probes (Melancon et al., 2014).