Aptamers for In Vivo Imaging

Sandra Borkowski and Ludger M. Dinkelborg


In Vivo Imaging: Modalities and Requirements

Imaging Modalities

In clinical in vivo diagnostics today, several imaging modalities are used that provide different types of information (Table 16.1). Conventional imaging modalities in radiology such as computed tomography (CT), magnetic resonance imaging (MRI), and ultrasound mainly provide anatomical information about the human body with a high spatial resolution (morphological imaging). Because of their significantly higher sensitivities, single photon emission computer tomography (SPECT), positron emission tomography (PET), or optical imaging (OI) devices allow the generation of pathophysiological information on biological processes at the cellular or even subcellular level, making use of molecular targeting principles (molecular imaging). (Fig. 16.1). PET in particular is a growing technology and new tracers and molecule classes addressing a plethora of interesting preclinical and clinical questions are currently evolving (Rohren et al., 2004). The intrinsic spatial resolution of these technologies is inferior when compared with the above mentioned morphological imaging modalities. However, the recent combi-

Signal moieTy

SPECT isotope le.j. Te-Sari. 1-123, ln-111J PET isMspe (Bfl. F-18. 0-11, Ga-6&> Di/iiof optital imaging [e.g. N|Rt

Fig. 16.1 Molecular targeting in imaging by single photon emission computer tomography (SPECT), positron emission tomography (PET), and optical approaches.

Fig. 16.2 Computed tomography (CT), 18F- CT image of a lung cancer (non-small cell lung fluorodeoxyglucose (18F-FDG) positron emis- cancer) patient with lymph node metastasis. sion tomography (PET) image and fused PET- From von Schulthess (2003), with permission.
Table 16.1 Comparison of imaging modalities

Modality mode


Spatial resolution

Blood concentration



300 mm

>50 mmol/L



800 mm

>50 mmol/L



500 mm



Functional and molecular

5-10 mm



Functional and molecular

2-8 mm



Functional and molecular

1-5 mm


Modified after von Schulthess (2003).

Modified after von Schulthess (2003).

nation of the high spatial resolution of morphological imaging (CT) and the high sensitivity of molecular imaging (PET and SPECT) in one instrument (PET/CT and SPECT/CT) combines the strength of both procedures synergistically in one device. For example, in a lung cancer patient the pathophysiological information of enhanced metabolism in lymph node metastases with high localized precision can be clearly demonstrated by fusing a 18F-fluorodeoxyglucose (FDG) PET image with a CT image (Fig. 16.2) (von Schulthess, 2003).

In anatomical imaging the morphological information gained by the use of imaging technologies such as CT, MRI, and ultrasound is enhanced by the application of contrast agents. The kinetics of these contrast agents is mainly determined by their physicochemical characteristics (size, charge, etc.) and the biodistribution is heavily dependent on the blood supply to the targeted organ (e.g. a tumor). The typical blood concentrations of contrast agents lie within the micromolar range. Molecular imaging approaches need a signal-generating moiety such as an radioisotope or a near infrared dye attached to a targeting agent, allowing the localization of the injected tracer by the dedicated imaging device (PET, SPECT, OI). The injected amounts of molecular imaging agents are far below those of contrast agents. Provided a high specific activity can be achieved, injected amounts below 100 mg are typically sufficient to obtain diagnostically effective images. Therefore, toxic side-effects caused by molecular targeting agents, especially in the SPECT and PET field, are not expected.

In this review we will focus on the use of aptamers for SPECTand PET. We will mainly concentrate on cancer indications, but also touch upon other diseases such as neurological disorders, infection, and inflammation.

Requirements for Imaging

Along with small molecules, peptides, and antibody fragments, aptamers are promising tools in molecular imaging. In research, aptamers have been successfully applied for molecular targeting of nucleotides and proteins. As well as high target specificity and affinity, appropriate chemical stability, rapid pharmacokinetics, and the exclusion of immunogenicity and toxic side-effects are prerequisites for effective imaging agents. In order to achieve an early and high signal-to-noise ratio, rapid tissue penetration and a high target binding affinity are the dominant requirements as described already in the concept of "escort" aptamers (Hicke et al., 2000).

In contrast to therapeutic interventions, in imaging, direct interference with the disease process such as the activation or inhibition of enzymes or other signaling pathways is not necessary. The accumulation of the radiolabeled targeting agent in the diseased organ or tissue can occur merely by binding and elimination of the unbound material via renal or hepatobiliary excretion. In general, rapid tissue penetration and target accumulation combined with fast excretion is preferred for SPECTand PET applications, which mainly use isotopes with a short half-life, like 99mTc (t/2 = 6h) for SPECT and 18F (t/2=2h) for PET. Therefore, high signal-to-noise ratios at early time points are needed in routine nuclear medicine diagnosis. Important parameters in this regard are high tumor-to-blood ratios, tumor-to-normal tissue ratios, and urinary excretion that occurs more rapidly than hepatobili-ary clearance (see Table 16.2 for optimal properties of diagnostic tracers in oncology). In contrast to therapeutic applications, in diagnostic imaging a high concen-

Table 16.2 Optimal properties of in vivo targeting agents in radiodiagnostics of cancer


Parameters (examples)

In vitro

High affinity to the target

K or Kd in nanomolar range


High specificity to the target

Specific blocking by >70%


Rapid uptake in target tissue

Maximum tumor uptake within 15 min

in mice

Accumulation and retention

Wash-out from tumor < 50%

in the target tissue

within 1 h

Low non-target tissue retention

Tumor-to-tissue ratios >5

Rapid blood clearance

Tumor-to-blood ratios >10,

High urinary excretion

>70% ID in the urine

Table 16.3 Tumor and blood uptake (in % ID/g) and tumor-to-organ ratios of 99mTc-and 125I-labeled TTA1 (5 h post injection) in comparison to 125I-anti-TN-C-IgG1 (4 h post injection) in U251 tumor-bearing mice (n = 3; ± SD)

99mTc-TTA1 (5 h p.i.) 125I-TTA1 (5 h p.i.) 125I-anti-TN-C-IgG1 (4 h p.i.)

99mTc-TTA1 (5 h p.i.) 125I-TTA1 (5 h p.i.) 125I-anti-TN-C-IgG1 (4 h p.i.)

Table 16.3 Tumor and blood uptake (in % ID/g) and tumor-to-organ ratios of 99mTc-and 125I-labeled TTA1 (5 h post injection) in comparison to 125I-anti-TN-C-IgG1 (4 h post injection) in U251 tumor-bearing mice (n = 3; ± SD)

Tumor (% ID/g)

0.45 ±


1.18 ±


9.12 ±


Blood (% ID/g)

0.05 ±


0.46 ±



± 1.00


5.40 ±


4.95 ±


2.11 ±



2.36 ±


1.53 ±


1.84 ±



2.81 ±


2.10 ±


2.14 ±



7.14 ±


3.52 ±


0.74 ±




± 10.75

2.69 ±


0.51 ±




± 15.88


± 8.10


± 7.66


2.14 ±


0.63 ±


1.29 ±



0.19 ±


0.42 ±


5.08 ±


TTA1, aptamer targeting human tenascin-C (TN-C).

TTA1, aptamer targeting human tenascin-C (TN-C).

tration of a radiolabeled aptamer at the target site is less important than a high signal-to-noise ratio which can even be achieved by a low tracer uptake at the diseased organ if the elimination from healthy organs and tissues occurs rapidly.

Table 16.3 compares the biodistribution of 99mTc and 125I-labeled TTA1, an ap-tamer targeting the human matrix protein tenascin-C, with an iodinated full-size antibody against the same target in mice. Although the antibody exhibits a much higher tumor uptake, the tumor-to-tissue ratios and especially the tumor-to-blood ratio is significantly lower than those achieved with the TTA1 aptamer. However, a high number of target molecules and the efficient and rapid delivery of labeled aptamers to these targets are remaining critical factors for generating sufficient signal-to-noise ratios in imaging.


Aptamers for In Vivo Imaging

Oligonucleotide Properties for In Vivo Applications

Aptamers against several interesting targets, and with promising in vitro characteristics, have been generated making use of the SELEX process. However, only a few aptamers have been reported so far to be successful in in vivo imaging experiments. Since an extrapolation of in vitro data of oligonucleotides and other targeting agents to the in vivo situation is often not possible, studying the biodistribu-

tion and imaging characteristics of oligonucleotides after radiolabeling is a valuable method to characterize their in vivo behavior. This relates to both the use of oligonucleotides as tools for drug discovery as well as for development of oligonucleotides themselves as drugs.

Two key properties - stability in biological fluids and systemic elimination -determine the bioavailability of aptamers. A main obstacle to the development of this compound class as drugs is still their instability against plasma endo-and exonucleases. A rapid degradation of aptamers in the blood prevents target binding, leading to insufficient signal-to-noise ratios for imaging. In this respect, RNA-based aptamers were proven to be more stable than DNA-based aptamers. Post-SELEX modifications of the oligonucleotide backbone (such as phosphodi-ester, phosphothioate, methyldiester) or the introduction of a 2'-amino, -fluoro or -O-methyl groups on the ribose can improve the stability of oligonucleotides significantly. Appropriate positions within the aptamer sequence for these post-SELEX modifications have to be identified because they can significantly influence binding affinity or the in vivo properties of the aptamer (Schmidt et al., 2004). Phosphothioate oligonucleotides exhibit high plasma protein binding and persistent liver and kidney uptake (Tavitian et al., 1998), and their metabolites were found to be excreted into the urine. In contrast 2'-O-methyl RNA or 2'-fluoropyrimidine aptamers are mainly excreted intact via the kidneys. Substitutions of nucleotides or other modifications such as increasing the molecular size by PEGylation also heavily influence the systemic clearance rate. Furthermore, the choice of the radioisotope (see also Table 16.3) as well as its chelate have an important influence on the biodistribution of radiolabeled oligonucleotides (Zhang et al., 2000 and Kuhnast et al., 2000).

The commonly used capping of the 3' end to prevent degradation by 3' exonucleases in the plasma (Dougan et al., 1997) increases the blood stability of aptamers significantly. Other strategies to gain higher in vivo stability resulted in the synthesis of Spiegelmers, locked nucleic acids (LNAs), and peptide nucleic acids (PNAs). Due to their higher stability and rapid body clearance, PNAs are especially interesting for imaging. In addition, they can be designed to cross cell membranes (Mier et al., 2000), opening the possibility that they could be used to image intracellular targets (for review see Tung, 2000).

Although stabilization of aptamers by one or more of the aforementioned strategies is a prerequisite for their use in vivo, it has to be considered that higher stability might also lead to a higher background activity leading to a low signal-to-noise ratio in imaging. For example, LNA derivatives of the aptamer TTA1 have been investigated for in vivo tumor targeting after labeling with 99mTc (Schmidt et al., 2004). Although the higher plasma stability led to an increased tumor uptake in mice, the observed increased tissue background and slower renal and hepatobiliary excretion resulted in a lower signal-to-noise ratio (Table 16.4). Additionally, 2'-O-methyl and LNA backbone modifications of TTA1 shifted the murine biodistribution towards higher urinary clearance and kidney uptake, whereas the unmodified TTA1 exhibited higher intestinal uptake and more rapid fecal excretion. In conclusion, the balance between the two key properties

Table 16.4 Tumor and blood uptake and tumor-to-organ ratios of 99mTc-labeled TTA1 and its stabilized 2'-O-methyl and locked nucleic acid (LNA) analogs in U251 tumor-bearing mice 1 hour post injection (n = 3; ± SD)


99mTc-TTA1.1 (2'-O-methyl analog)

99mTc-TTA1.2 (LNA analog)

Tumor (% ID/g)

0.87 ± 0.25

1.28 ± 0.06

2.89 ± 0.55

Blood (% ID/g)

0.21 ± 0.01

0.24 ± 0.04

1.03 ± 0.09


2.09 ± 0.718

0.89 ± 0.34

0.78 ± 0.30


0.78 ± 0.20

0.13 ± 0.02

0.23 ± 0.05


1.47 ± 0.49

0.06 ± 0.01

0.14 ± 0.02


4.42 ± 1.49

3.69 ± 0.82

1.95 ± 0.78


4.23 ± 1.42

5.38 ± 1.02

2.81 ± 0.55


13.91 ± 2.82

8.70 ± 3.50

8.56 ± 4.09


4.17 ± 1.26

1.52 ± 0.04

1.14 ± 0.13


0.06 ± 0.02

1.16 ± 0.28

4.44 ± 0.78

of aptamers - stability, allowing for a sufficient targeting and body clearance, and low background activity in non-targeted organs and tissues - must be optimized for every individual aptamer. Rapid renal clearance can result in very high signal-to-noise ratios early after administration, provided a specific target retention can be achieved.

It has been observed in animals that aptamers have a tendency to non-specifi-cally stick to serum proteins or cells and that they are retained in non-target tissues such as excretory organs after systemic administration. The high negative charge of the aptamer's backbone seems to be responsible for this phenomenon, which increases the background activity during imaging.

Since efficient delivery of aptamers to their target is another main obstacle of their in vivo use, several attempts have been made to improve perfusion across endothelial cell layers and enhance cellular uptake and internalization. Vectors such as cationic lipids, polyamines, or synthetic vehicles have been used to overcome the highly charged backbone of most oligonucleotides, which prevents them from binding to intracellular targets. However, successful vector applications for aptamers in imaging have not been reported.

Comparison of Different Classes of Targeting Agents

With approximately 10-15 kDa, aptamers have a molecular weight between peptides and antibody fragments. Compared with antibodies and antibody fragments (scFv, Fab) aptamers have several characteristics that are advantageous for imaging:

• Because of their smaller size, aptamers can diffuse more rapidly into tissues and organs, leading to faster targeting (and imaging) when compared with antibodies.

• The lower molecular weight of aptamers can result in a shorter circulation time and faster body clearance, leading to a low background noise during imaging.

• The fast body clearance decreases the radiation dose (total body dose) to the patient.

• Aptamers do not induce immune responses.

• Because they are fully synthetic, the production of aptamers is less expensive.

Peptides are much smaller than aptamers and have been used for targeting cell surface proteins such as G-protein-coupled receptors. Together with small molecules they represent useful tools, especially in the PET imaging field where rapid pharmacokinetics are needed due to the short half-lives of most PET isotopes. Because of the fact that high-throughput screening of aptamer libraries is feasible by use of the SELEX process, their high affinity and specificity as well as their automated synthesis, aptamers combine the advantages of antibodies and peptides for in vivo imaging (Hicke and Stephens, 2000). Based on their molecular weight they still seem to be small enough and attractive for use in PET imaging. Although oligonucleotides seem to be excellent tools for rational drug design, their systemic therapeutic use has rarely been transferred to in vivo applications. Reasons for this situation are their inherent fragility, low bioavailability, and tendency to non-specific interactions (Younes et al., 2002). Opposite to the limitations in systemic therapeutic use, the general suitability of oligonucleotides has been proven in imaging and a growing use especially of radiolabeled aptamers in imaging is foreseen.

Aptamer Targets for Imaging

Medical needs in targeted imaging arise from the localization and staging of a disease, patient selection for an individualized treatment, and early therapy monitoring. Due to the negative charge of the oligonucleotide backbone, aptamers lack the ability to efficiently cross cell membranes. This inherent characteristic limits their in vivo application to extracellular targets. Therefore, aptamers for imaging applications are best suited for targeting of spatially confined extracellular disease targets. Vascular targets seem to be well suited and are often selected in indications like thrombosis, restenosis/intimal hyperplasia and angiogenesis (White et al., 2000). Also with therapy as the goal, both DNA and RNA aptamers have been raised against thrombin (Bock et al., 1992), platelet-derived growth factor (PDGF) (Leppanen et al., 2000), and vascular endothelial growth factor (VEGF) (Ruckman et al., 1998).

Making use of the SELEX process, RNA aptamers were generated binding to human L-selectin for use in imaging of inflammation (Ringquist and Parma, 1998). In vitro tests demonstrated that these aptamers preferentially recognize intracellular L-selectin with a high specificity and sensitivity. However, in vivo application have not been reported yet.

Besides cardiovascular diseases, several questions in oncology (e.g. determining the aggressiveness of a tumor, predicting the best therapeutic intervention, and the early identification of those patients responding to a given treatment) can be addressed with molecular (targeted) imaging. 18F-Fluorodeoxyglucose (FDG) is a prominent tracer for PET imaging in oncological indications. Tumors are diagnosed with 18F-FDG, making use of the well-known "Warburg effect" whereby tumors have a higher glucose consumption than other tissues. However, because FDG uptake is also enhanced in inflammation, more specific tracers that are able to differentiate between tumor growth and inflammation are desired. Therefore, there is a clear need for alternative tracers from different molecular classes to overcome these limitations of FDG in tumor diagnosis and also to allow for the combination of imaging and therapy approaches with the same compound.

Targets that are overexpressed in a variety of tumors such as the extracellular matrix protein tenascin-C (TN-C) are well suited for radiolabeled aptamers. The post-SELEX modified RNA aptamer TTA1, generated against human TN-C, has been successfully applied in tumor imaging in mice using SPECT (Hicke et al., in press).

Another example for oncological indications are stabilized RNA aptamers that have been generated by SELEX against the extracellular domain of prostate-specific membrane antigen (PSMA), which is overexpressed in prostate cancer (Lupoldt et al., 2002). Although high in vitro binding affinities in the nanomolar range have been reported, no in vivo applications have been carried out so far.

Several examples of in vivo applications of oligonucleotides have been reported for both antisense DNAs and RNAs. A 99mTc-labeled antisense oligonucleotide (20-mer) was raised against CAPL, a cancer-related gene (Hjelstuen et al., 1998). Biodistribution experiments in normal mice showed a higher stability and a slower blood and organ clearance when the 3' end was capped by the MAG3-aminohexyl chelator. Another 18-mer antisense DNA was synthesized against RIa, a subunit of protein kinase A (PKA) (Zhang et al., 2000). The biodistribution of this antisense oligonucleotide in mice was compared after labeling via HYNIC, MAG3, or DTPA chelators with 99mTc. The oligonucleotide labeled via the HYNIC approach led to significantly higher liver, spleen, and kidney uptake, as well as a high intestinal uptake compared with the MAG3-labeled oligonucleotide (Table 16.5). It has also been demonstrated that an increased sequence length of antisense oligonu-cleotides not only reduces the achieved labeling yields and specific activities but also strongly influences their pharmacokinetics (Wu et al., 2000). It has to be stated that the lack of well-controlled and convincing results questions the usefulness of antisense approaches for imaging applications (for review see Duatti, 2004). Compared with antisense oligonucleotides, the use of globular extracellular binding aptamers are much more promising for in vivo imaging.

Table 16.5 Biodistribution (% ID/g, mean ± SD) in normal mice 4h following the injection of mTc labeled to DNA via three different chelators HYNIC, MAG3, or DTPA (n = 5)






24.0 ± 4.5

3.3 ± 0.7

14.0 ± 2.5


0.84 ± 0.06

0.37 ± 0.09

0.74 ± 0.16


30.1 ± 3.0

2.5 ± 0.2

6.0 ± 1.0


1.5 ± 0.3

0.51 ± 0.07

1.0 ± 0.30


9.4 ± 1.4

1.4 ± 0.14

6.1 ± 1.6


0.46 ± 0.08

1.2 ± 1.4

0.49 ± 0.04

Small intestine

1.5 ± 0.3

1.1 ± 0.8

0.52 ± 0.09

Large intestine

1.9 ± 0.4

22.2 ± 9.4

0.70 ± 0.06


0.17 ± 0.09

0.13 ± 0.01

0.12 ± 0.00


0.21 ± 0.02

0.65 ± 0.22

1.8 ± 0.6

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