Application of aptamers for in vivo molecular imaging and theranostics

Abstract

Nucleic acid aptamers are small three-dimensional structures of oligonucleotides selected to bind to a target of interest with high affinity and specificity. In vitro, aptamers already compete with antibodies to serve as imaging probes, e.g. for microscopy or flow cytometry. However, they are also increasingly used for in vivo molecular imaging. Accordingly, aptamers have been evaluated over the last twenty years in almost every imaging modality, including single photon emission computed tomography, positron emission tomography, magnetic resonance imaging, fluorescence imaging, echography, and x-ray computed tomography. This review focuses on the studies that were conducted in vivo with aptamer-based imaging probes. It also presents how aptamers have been recently used to develop new types of probes for multimodal imaging and theranostic applications.

Introduction

Molecular imaging of living subjects allows the study of molecular and cellular events in their native environment, instead of having to remove cells or tissues before analysis. It is now routinely used in clinics to diagnose various diseases, as well as evaluate drug efficacy. There are several imaging modalities available and most require a molecular-imaging probe. Chemistry or biochemistry plays a crucial role in the development of such probes, which are usually constructed by combining three molecular components (Fig. 1): 1) a component to provide a signal that can be detected by an imaging instrument, 2) a ligand that can specifically interact with the molecular target of interest, and 3) a spacer that links the two previous components together. The spacer is not always mandatory and the two first components may sometimes be directly linked together. Once introduced into the living subject, the location of the molecular imaging probes can be monitored by an imaging system. The probe is generally injected intravenously, but other routes of administration can be used. Just after injection, the probe is distributed throughout the body of the subject. Then, part of the probe is expected to bind to its molecular target, whereas the rest is cleared from the tissues. Accordingly, the imaging system should detect a higher signal in tissue expressing the target than the others (Fig. 2).

The first molecular imaging probes were derived from natural molecules that are involved in metabolism. For example, one of the most famous radiotracers used for cancer imaging is 2-deoxy-2-[¹⁸F]fluoro-d-glucose (¹⁸F-FDG), which is a radiolabeled glucose analog. However, there is now an increasing demand to develop new types of molecular imaging probes that can bind to targets for which there are no available or known natural ligands. These new probes are often made from macromolecules that can be tailored to interact with any molecular target of interest. These macromolecules use four forces of interaction (ionic bonds, hydrogen bonds, hydrophobic effects, and van der Waals interactions) to adopt three-dimensional (3D) structures and specifically interact with their target. Such forces are often called “weak bonds” because they require little energy to break. However, the 3D structure of a macromolecule can allow it to form many such weak interactions with a target of interest to, ultimately, bind to it with a very high affinity.

Peptides and proteins can provide a myriad of 3D structures able to generate high affinity ligands. For example, antibodies are well-known ligands that can bind to their target with very high affinity and specificity. This property has been exploited to develop drugs and molecular imaging probes. However, proteins have many disadvantages when used as molecular imaging probes. First, their 3D structures can be unstable and difficult to maintain, not only in vivo but also in vitro during labeling steps. In addition, they are almost impossible to produce chemically, except for small peptides. Thus, they require bio-production processes that are not only costly but can also cause problems of batch-to-batch reproducibility. Finally, they may face problems of immune response, e.g. only humanized antibodies can be injected into humans, but cannot be easily evaluated in immuno-competent animal models, which reject them.

Like amino-acid polymers, nucleic acid sequences can form many 3D structures. This property is used in nature by RNAs to interact with a variety of molecules or to have catalytic activities (e.g. protein synthesis or splicing). Since 1990, several combinatorial approaches have been developed to exploit and analyze these properties of nucleic acids. Tuerk and Gold popularized such methods as SELEX (for systematic evolution of ligands by exponential enrichment) [1] and the nucleic acid-based ligands identified by this technique were baptized “aptamers” by Ellington and Szostak from the Latin “aptus”, meaning “to fit” [2]. Although the selection of aptamers has undergone many improvements and refinements [[3], [4], [5], [6], [7], [8]], they are still based on the same principle (Fig. 3). A population of oligonucleotide sequences known as “candidates” is synthesized. They contain a randomly synthesized region, which is framed by two known sequences that can be amplified by PCR. This population is then subjected to an in vitro selection process, which extracts candidates that bind to a chosen target from the rest. These candidates are then amplified by PCR or RT-PCR and transcribed in vitro before being used in a new in vitro selection round. During the amplification step, mutations may occur, leading to the appearance of candidates that are slightly different from their parents, some of which may have higher affinity for the target. Accordingly, the population is expected to evolve through several rounds of selection and amplification, in a Darwinian fashion, favoring the progressive enrichment of the sequences with the highest affinity. Sequencing a sample of the population can then identify such sequences. Recently, high-throughput sequencing has led to a better understanding of this molecular evolution process and better identification of the aptamers [9]. The main weakness of RNA and DNA sequences is to be rapidly degraded by nucleases. The half-life of the oligonucleotide RNA is a few seconds in the plasma whereas for DNA it is around 30 to 60 min [10]. Several chemical modifications have been introduced in aptamers to protect them from nuclease degradation including modified bases, modified sugar and modifications on the phosphodiester linkage [11]. The most common strategy is to use chemically modified nucleotides that can be directly incorporated by polymerases during SELEX. For example, 2′Fluoro nucleotides are often used and the half-life time of 2’Fluoro-pyrimidine RNA sequences is around 5 to 15 h in plasma [10]. Chemical modifications could also be incorporated post-selection but they can change the structure of aptamers and affect their binding. Since their discovery, aptamers have been selected against a wide variety of targets, from small molecules to proteins to even complexes of targets present on the cell surface [3,12,13]. Aptamers have provided fundamental insights concerning the versatility of RNA, supporting its involvement in the origin of life [14,15]. In parallel, aptamers have been increasingly used for biotechnological applications, such as biosensors [16], bio-purification [17], biomarker discovery [18], and the regulation of gene expression [19]. Furthermore, one aptamer has already been commercialized as a drug for the treatment of age-related macular degeneration and several others are currently in clinical trials [20].

Because of their high specificity and affinity, aptamers are promising tools for molecular imaging [[21], [22], [23], [24]]. Over the last 20 years, several aptamers have been tested as molecular probes in almost every imaging modality, including positron emission tomography (PET), single photon emission computed tomography (SPECT), magnetic resonance imaging (MRI), echography, X-ray computed tomography (X-ray CT), and fluorescence imaging. This review presents an up-to-date summary of the investigations that have been performed in vivo and the recent use of aptamers to develop new types of probes for multimodal imaging and theranostic applications. We have tried to exhaustively list all these studies in tables (see Table 1, Table 2, Table 3), but have chosen not to describe them all in detail, but rather to present general conclusions that can be drawn from these experiments.