By definition, aptamers are synthetic, highly structured, small, typically <100 mer, ssDNA or RNA ligands. The term ‘aptamer’ means ‘to fit’ (aptus) in Latin (6), which indicates two important properties of aptamers: i) their ability to fold into complex tertiary structures and recognize their targets with high affinity (low nM to high pM equilibrium dissociation constants); and ii) their specificity, somewhat analogous to antigen-antibody interactions. Using this technique, a number of aptamers that specifically recognize targets, such as metal ions (11–13), organic dyes and amino acids (14–17), antibiotics (18,19) and peptides (20,21), as well as proteins of various sizes and functions (6,22,23), whole cells (24–27), whole organisms (28), viruses (29) and bacteria (30), have been obtained (31–33). The SELEX process is based on the ability of these small oligonucleotides to fold into unique 3D structures that interact with a target with high specificity and affinity through such interactions as van der Waals surface contacts, hydrogen bonding and base stacking. Starting with a library containing random RNA sequences with ∼10 (34–36) varieties of RNA molecules, in vitro binding, elution and reverse polymerase chain reaction (PCR) amplification techniques allow for the selection of RNA molecules that efficiently bind to a specific receptor or ligand with high affinity (37,38). Conceptually, the marked specificity and high affinity of aptamers to a wide variety of targets, coupled with the ease of design and molecular engineering, as mentioned above, have made them broadly popular and highly suitable for development as clinical diagnostic and therapeutic agents in cancer research (39–41). Pegaptanib (Pfizer/Eyetech), an aptamer targeting VEGF, has already been approved by the FDA for clinical use in treating AMD (42). A variety of aptamers against other molecular targets are currently under clinical investigation (10), with several more in the pipeline.

Aptamers are identified through an in vitro process, which specifically isolates aptamers for a target of interest involving iterative rounds, termed as SELEX. Briefly, for the SELEX process, as shown in Fig. 1, a random aptamer or oligonucleotide library pool (DNA or RNA) is incubated with the target of interest, with heating and cooling to promote formation of stable structures. After washing, the bound sequences are eluted and incubated with a control target (if negative selection is required) to remove sequences that exhibit recognition with the control as well. The protein-bound aptamers are then recovered. These sequences are amplified with PCR or reverse transcription-PCR. ssRNA or DNA sequences representing the recovered sequences are then generated from these PCR products and used in the subsequent selection round. The process is repeated until the pool is enriched for sequences that specifically recognize the target. The enriched pool is cloned and then sequenced to obtain the individual sequences known as aptamers (7). As this is an in vitro process, the selection conditions may be manipulated to obtain aptamers with properties desirable for a particular purpose, i.e., aptamers that bind to a target at different temperatures and buffer compositions may be used for different purposes, or modified oligonucleotide bases which may be introduced to enhance stability.

The cancer cell surface represents a complex environment with a vast array of potential targets of diagnostic and therapeutic interest; therefore, the aptamers selected by SELEX may exhibit low or no affinity towards these targets, due to shielding of the aptamer binding domain. To overcome this hindrance, a process called Cell-SELEX (cell-based selection of aptamers specific to cancer cells) has been reported to isolate aptamers from living cells as targets, such as cancer cells (24). In Cell-SELEX, instead of using purified protein targets, whole living cells are used as the targets. To generate aptamers that specifically target cancer cells, a library of ssDNA is used (24). This library, which has a random sequence of 30–40 bases and a region flanked by primer sequences of 18–20 bases, is incubated with the target cells. The library and forward primers are labeled with a fluorophore, so that the sense strand of the PCR product for each round, which serves as the the library for the next round, is also fluorescently labeled, enabling the entire process to be monitored by flow cytometry. After washing, the DNA sequences bound to the target cell surface are collected and then incubated with the negative control cells. All the DNA sequences that bind to the negative control cells are removed. To avoid recognition of normal cells, the aptamers bound to these non-specific proteins are removed. The remaining sequences are amplified for the next round of selection. Generally, ∼20 rounds of Cell-SELEX are required to isolate aptamers with the highest selective affinity to the target cells.

A panel of aptamer probes has been successfully selected by Cell-SELEX (Fig. 1) for several types of cancer cells, including lymphocytic leukemia, myeloid leukemia, liver cancer, small- and non-small-cell lung cancer cells (25–27). All these aptamers were found to exhibit high affinity and specific selectivity for their targets. Those results illustrated how aptamers may be selected without specific knowledge of the molecular signature of the cell or the number or type of proteins on the cell surface. Moreover, it is possible to profile the molecular characteristics of the target cancer type, which has been a main objective in the development of cell-based aptamers for use in cancer diagnosis and treatment. The applications of aptamers for cancer diagnosis and therapy through cancer cell detection, cancer cell imaging and targeted drug delivery are discussed below.

The binding affinity of aptamers to their targets enables the detection of cancer cells at low levels, resulting in an early and sensitive diagnosis. In addition, DNA molecules have the advantage of predictable structure and easy site-specific chemical modification. Therefore, aptamers may be quickly and reproducibly synthesized with fluorescent molecules and nanoparticles (NPs) conjugated for targeted cells and tissue detection and imaging.

It is well known that gold nanomaterials possess unusual optical and electronic properties, high stability and biological compatibility, controllable morphology and size dispersion, and easy surface functionalization (47–51) from the standpoints of engineering and application. Aptamer-conjugated gold NPs (Apt-AuNPs) provide a powerful platform to facilitate targeted recognition and detection. By using Apt-AuNPs, a colorimetric assay for the direct detection of cancer cells has been developed (52). Aptamers specific to their targets on the cell membrane surface were conjugated to gold NPs. These Apt-AuNPs were then targeted to assemble on the cell surface membrane through the recognition of the aptamers to their targets. The assembly of the Apt-AuNPs around the cell surface causes a shift in the extinction spectra of the particles when the AuNPs are in sufficient proximity for their surface plasmon resonances to overlap. This assay exhibits a significantly high sensitivity, with a detection limit for simple absorbance of just 90 cells. When the number of target cells increases to 1,000, the color change reaction may be observed by the naked eye. This method demonstrates the potential of aptamer-based analysis for rapid, simple, direct and sensitive detection of cancer cells in clinical diagnosis. In addition, using the two-photon scattering (TPS) technique, Lu et al (53) captured the signal from Apt-AuNP-based cell detection, resulting in the highly selective and sensitive detection of the SK-BR-3 breast cancer cell line at a level of 100 cells/ml, using multifunctional (monoclonal anti-HER2/c-erb-2 antibody and S6 RNA aptamer) AuNP conjugates. Oval-shaped AuNPs were introduced instead of spherical AuNPs. As the particle aspect ratio increased, the TPS intensity change became higher, resulting in improved sensitivity (53).

The research on the applications of aptamer-conjugated fluorophores has advanced rapidly over the last few years (54), including the use of quantum-dot (QD)-conjugated aptamers to perform multiplex detection of cancer cells (44). In that study, three aptamers (TTA1, AS1411 and Muc1) were conjugated to QDs with emission wavelengths of 605, 655 and 705 nm, respectively; for the confocal microscopic analysis of multiplex imaging of cancer, healthy and disease cell lines were incubated with each QD-conjugate. As expected, fluorophore-labeled aptamers were able to detect and differentiate between different types of cancer cells and produced a visible fluorescence signal in the presence of target cells, which indicated that aptamers have high potential as a clinical diagnostic tool.

Gold nanorods (NRs) have been utilized as a platform for multiple aptamer immobilization to detect cancer cells (55). Through covalent linkages of fluorophore-labeled aptamers on the NR surface, ≤80 fluorophore-labeled aptamers may be attached on a single NR, causing a 26-fold higher affinity and >300-fold higher fluorescence signal. In this case, the use of NRs as a nanoplatform for multivalent binding of aptamers increases the signal as well as and binding strength of these aptamers in cancer cell recognition. As a consequence of the selective recognition properties of aptamers, this technique may be used in clinical detection to enhance binding affinity and signaling strength when the concentration of target cells is relatively low.

With increasing detection sensitivity, a dye-doped silica NPs-based method for sensitive and rapid detection of cancer cells was constructed (56). To achieve specific recognition, the biotinylated aptamers are immobilized onto the surfaces of neutravidin-coated fluorescent NPs (FNPs) to form aptamer-conjugated FNPs (Apt-FNPs), which have demonstrated low background signal, high signal enhancement and efficient functionalization. The FNPs are then functionalized with polyethylene glycol (PEG) to prevent non-specific interactions with neutravidin and allow universal binding with biotinylated molecules. Multiplexed cancer cells may also be detected by employing a combination of fluorescence resonance energy transfer of FNPs and the conjugation of different aptamers (57). Furthermore, single-, dual- and triple-dye-doped silica NPs were prepared according to this technology (58). This method provided an additional potential candidate for cancer cell imaging.

In addition to cancer cell detection, improved molecular imaging techniques together with novel near-infrared fluorescence probes, which may also be attached to aptamers, allow in vivo multiparameter 3D exploration of tumors, even deep inside the tissue (59). Aptamer-mediated technology is likely to gain applications in clinical cancer diagnosis, with cancer cell detection as well as cancer cell and tissue visualization. Fluorescein isothiocyanate (FITC)-labeled aptamers were used for tissue staining and imaging under confocal microscopy (60). Compared to the negative control (FITC-labeled unselected library), targeted tissue was recognized with fluorescence signal following incubation with FITC-labeled aptamers, which indicated aptamers as potential molecular probes for cancer visualized diagnosis. Other methods were also developed for labeling aptamers for flow cytometry applications (23,61). Flow cytometric analysis was employed to compare binding of a FITC-labeled DNA aptamer with a complex of mouse anti-human neutrophil elastase (HNE) antibody and FITC-labeled rat antimouse antibody to HNE-labeled beads. The results revealed that aptamers and antibodies were equally efficient in HNE detection (61). Further studies demonstrated that, based on aptamers' high specificity of target recognition, fluorescein-labeled aptamers were selective in flow cytometric experiments for binding to human recombinant CD4-stained mouse T cells expressing human CD4, but did not detect control mouse T cells lacking human CD4 (23).

For cancer cell imaging, the antiprostate-specific membrane antigen (PSMA) aptamer was used. Due to its high affinity and specificity, this aptamer is able to mediate cell-specific endocytosis of the NPs upon molecular recognition between the aptamer and its receptors. The synthesized aptamer-functionalized QDs and super-paramagnetic iron oxide NPs were used for the recognition of cell receptors as well as optical imaging or MRI (62,63). The same concept was also applied to functionalize gold NPs with anti-PSMA aptamer for CT. The results demonstrated that the functionalized gold NPs may enhance the CT image by 4-fold (64). For further study in vivo, molecular imaging of tumors inside mice was performed. TD05, an aptamer that specifically binds Ramos cells, was modified with fluorophores, successfully exhibiting aptamer fluorescence overlapping the tumor site in a mouse cancer model under a whole-animal imaging system.

Overall, the studies discussed in this section helped expedite the process of using aptamers in the clinical setting for cancer diagnosis.

Several aptamers, including pegaptanib and REG1, have already been used for clinical treatment (42).

From a therapeutic point of view, a useful therapeutic agent may stop being used when a more cost-effective, safer or simply generic product is released. Aptamers, targeting a broad range of receptors, with numerous ideal properties, such as high specificity and affinity, easy modification, easy synthesis, stability, low immunogenicity and toxicity, have emerged as promising therapeutic reagents.

PSMA, a transmembrane protein that is highly expressed in human prostate cancer cells and the vascular endothelium, is an attractive target, as it is abundantly expressed on the surface of prostate cancer cells, is known to internalize certain ligands, possesses enzymatic properties as a glutamate carboxypeptidase and is a verified target of a clinically applied imaging agent for prostate cancer (65). In 2002, a 2′-fluorpyrimidine RNA aptamer screen was performed and two stabilized RNA aptamers, A9 and A10, which were capable of binding PSMA with low nanomolar affinity, were first identified (66). These aptamers have been applied to several anticancer preclinical targeting studies of NPs, toxins, therapeutics and siRNAs. siRNAs are double-stranded oligonucleotides (21–23 bp) that bind specifically to cognate mRNA and promote its degradation, effectively decreasing the expression of the target gene product. To develop its ability, a siRNA has to be efficiently delivered to the intracellular space in vivo. For this purpose, an aptamer-siRNA chimera construct was generated to combine the RNA aptamer A10, specific for PSMA, with a siRNA against the survival factors polo-like kinase 1 (Plk-1) and B-cell lymphoma 2 (Bcl-2), which are two antiapoptotic genes involved in cell survival and are commonly overexpressed in human tumors. After being injected at the site of a tumor, aptamer-Plk-1 or Bcl-2 siRNA chimeras were shown to deliver the siRNAs effectively to PSMA-expressing cells, inducing apoptosis in targeted cancer cells and tumor regression in animal models of prostate cancer (67).

An improved treatment of prostate cancer was aptamer-short hairpin RNA (shRNA) chimera, a PSMA-targeted aptamer-conjugated shRNA, which was used to attenuate DNA-repair pathways, resulting in tissue-selective sensitization to ionizing radiation (IR) therapy (68). A10-3-shRNAs targeting the catalytic subunit of the DNA protein kinase (DNAPK) caused a selective reduction of DNAPK RNA and protein in PSMA-positive cells, xenografts and human prostatic tissues. Intratumorally, the administration of aptamer-targeted DNAPK shRNAs combined with IR significantly and specifically enhanced PSMA-positive tumor response to IR, leading to decreased DNA-repair and cancer cell survival following chemotherapy or radiation treatment.

The application of antitumor agents, such as docetaxel, dextran, cisplatin, daunorubicin and doxorubicin, is limited due to their side-effects and toxicity. The first report of targeted drug delivery with NP-aptamer bioconjugates was described by Farokhzad et al (69). The anti-PSMA A10 aptamer, or its truncated version A10-3, was used to target NPs, demonstrating that the A10-3 aptamer may be used to target poly(lactic acid)-block PEG copolymer NPs to PSMA-positive prostate cancer cells (69). Furthermore, the A10-3 aptamer was modified by poly(D,L-lactic-co-glycolic acid; PLGA) NPs to deliver docetaxel (Dtxl); an antimitotic chemotherapy drug (70) to prostate tumors in vivo. When compared with non-targeted NPs that lacked the anti-PSMA aptamer, the Dtxl-NP-Apt conjugates mediated targeted uptake and controlled release of drugs, resulting in potent efficacy and reduced toxicity after intratumoral injection (71). In an extensive study, following systemic administration, A10-3 aptamer induced tumor accumulation of PLGA NPs by 3.7-fold when compared to the non-targeted particle (72). Further investigations demonstrated that the NP-aptamer bioconjugates may be precisely engineered and formulated by controlling size, composition, polydispersity, aptamer density and drug loading, thereby resulting in the desired functions and biodistribution required for further clinical development (72–74). Another polymeric formulation has been used to deliver cisplatin to PSMA expressing tumors, whereas a dosage of 0.3 mg/kg of aptamer-targeted cisplatin NPs was more efficacious compared to a 1 mg/kg of free cisplatin (75). These preclinical studies demonstrated the potential of aptamers as therapeutic reagents to efficiently deliver drugs to targeted cancers. In addition, these results also indicated aptamers as targeting ligands to alter the biodistribution, tumor uptake and tumor retention of therapeutic NP reagents.

The AS1411 aptamer against nucleolin, a Bcl-2 mRNA-binding protein expressed on the surface of several cancer cell types, including those associated with acute myeloid leukemia (AML), has demonstrated promising antineoplastic effects, which may be attributed to interference with the multitude of nucleolin-mediated cell processes. AS1411, a DNA aptamer, was shown to interfere with DNA replication, causing S-phase arrest (76), as well as with the stabilization of Bcl-2 mRNA, a known inhibitor of apoptosis, suggesting another possible mechanism of action (77). The use of AS1411-conjugated NPs for drug delivery was performed on MCF-7 breast cancer cells which overexpress nucleolin, exhibiting increased cell death compared to that of cancer cells that do not overexpress nucleolin. Moreover, the extent of delivery and dosage may be controlled using complementary DNA of the aptamer as an antidote (78). Furthermore, in vitro and in vivo, AS1411 has demonstrated growth inhibitory effects and antitumor activity against multiple cancer cell lines and tumor tissues, respectively. The subsequent toxicity studies in rats and dogs exhibited a favorable safety profile, prompting the initiation of human clinical trials. Notably, internalization of the aptamer results in a decrease in the expression of several cancer-associated mRNAs and, therefore, reduced cell proliferation.

Completed phase I trials demonstrated that AS1411 exhibited minimal toxicity in 17 patients with advanced solid tumors. Moreover, there was evidence of promising clinical activity, with 7 patients achieving disease stabilization and 1 patient with renal cell cancer achieving a complete response by 11 months (79). Phase II studies on metastatic renal cell cancer and AML are currently underway. The development and clinical trial data were reviewed by Mongelard and Bouvet (80).

Mucin 1 (Muc1) is a glycoprotein found on the surface of normal epithelial cells and functions as a barrier against microorganisms and degradative enzymes (81). Muc1 is released into the bloodstream and has been successfully used as a tumor marker for bladder cancer. In addition, Muc1 is an attractive target, as its overexpression has been associated with various types of cancer and its distribution pattern changes on the cell surface (82,83). DNA aptamers directed against Muc1 (AptA and AptB) were identified in 2006 (84) and have since been implemented in ex vivo imaging and radiotherapy (85). In subsequent studies, when such an DNA aptamer was coupled to the light-activated photodynamic therapy agent chlorine e6, it specifically delivered chlorine e6 into epithelial cancer cells, with a significant enhancement in efficiency (>500-fold increase) compared to that of the drug alone, and achieved cancer-specific cytotoxicity (86). These results suggested the anti-Muc1 aptamers may function as drug carriers to specific epithelial cancer cells. As target QD scaffolds allow for labelling of a single QD with multiple aptamers, the efficiency of binding anti-Muc1 aptamers with QDs may be enhanced by avidity (87). In a mouse model bearing A2780/AD human ovarian cancer xenografts, an increased amount of QD-Muc1 accumulated in the tumors compared to non-modified QD. This data support that anti-Muc1 aptamer targeting may be a therapeutic candidate for drug delivery when improved by the avidity associated with NP decoration.

NOX-A12 is a 45-nt L-RNA aptamer, based on L-ribose, which is clinically developed by NOXXON Pharma AG (Berlin, Germany) (88). NOX-A12 was selected against stromal cell-derived factor-1 (SDF-1), which binds to chemokine (C-X-C motif) ligand 12, a chemokine that is involved in tumor metastasis, angiogenesis, cell homing and tissue regeneration (89). Inhibition of the SDF-1 by NOX-A12 may prove useful in the treatment of several types of cancer. NOX-A12 is currently in phase I clinical trials for the treatment of lymphoma, multiple myeloma and hematopoietic stem cell transplantation.

The ultimate aim in cancer therapy remains the development of treatment modalities that effectively eliminate cancer cells without affecting normal cells. Due to their uniqueness and high specificity, aptamers and aptamer-based drug delivery systems are promising tools for targeted cancer therapy. Several more aptamers may soon be joining pegaptanib in the field of cancer diagnosis and treatment.