In The October Issue
- Determining Aptamer Affinity
- Coming Soon! Next Generation Sequencing and Bioinformatics Services
- Aptamers in Drug Delivery, Pharmacokinetics, and Dosing
Did you miss the latest article from Base Pair? There are several popular methods for measuring biomolecular interactions and evaluating aptamer affinity. This article gives an overview of SPR, ITC, MST, and BLI and describes some of the advantages of each method.
Coming Soon! Next Generation Sequencing and Bioinformatics Services for Aptamer Selection
Aptamer discovery is a complex process. Once selection design and SELEX rounds are complete, the best aptamer candidates must be chosen. Base Pair can help! Send us your libraries for sequencing and bioinformatics analysis or simply send us your sequencing results. Base Pair will apply our years of experience and proprietary algorithms to choose the best aptamer candidates. Contact us to learn more!
Aptamers in Drug Delivery, Pharmacokinetics, and Dosing
Aptamers for Targted Drug Delivery
In many therapeutic areas, current treatments involve significant side effects. Combining drug compounds with affinity agents that target specific cell surface receptors can dramatically reduce off-target effects and improve treatment effectiveness. While antibodies are popular affinity agents, antibody-drug conjugates have some limitations due to large antibody size and immunogenicity. Development of antibody-drug conjugates is expensive and requires a long lead time and batch-to-batch reproducibility of antibodies is sometimes a concern. Aptamers are small, single strands of DNA or RNA that can selectively bind a target. Due to their small size, aptamers can better penetrate tissues and cells and have low immunogenicity in vivo. They can be easily conjugated without affecting affinity or selectivity and are chemically synthesized. Aptamers can also be selected for binding to non-immunogenic small molecules (9).
Researchers in Germany selected aptamers that bind the hepatocyte growth factor receptor cMet for targeted delivery of the chemotherapy drug doxorubicin to tumor cells. They combined lipidated aptamers for cell targeting and drug loading and incorporated a photoswitch to trigger drug release. The lipidized aptamers showed increased resistance to nuclease degradation and improved cellular uptake in vitro. The brief application of low-intensity UV irradiation led to 80% cell mortality in just 8 hours, while the non-irradiated control reached 57% mortality after 24 hours and 80% cell mortality after 48 hours. The team confirmed selective targeting, effective cell uptake and triggered release of drug through imaging and flow cytometry (7).
Researchers at Duke University selected for aptamers that are internalized into prostate cancer cells without affecting normal prostate cells. Anti-prostate cancer cell aptamer E6 was then conjugated to the highly toxic auristatin drugs MMAE and MMAF. In vitro, the aptamer-drug conjugates selectively killed prostate cancer cells. Addition of a complementary oligonucleotide to the aptamer prevented aptamer-drug targeting and internalization, providing an effective fail-safe in the event of drug accumulation or toxicity. The E3 aptamer-MMAF drug conjugate had a half-life of 18 hours in mouse serum. In vivo administration in a mouse prostate cancer model inhibited tumor growth and increased survival (6).
Combining drug compounds with selective aptamers for targeted delivery can improve drug function and reduce off-target effects.
Aptamer Labeling for Biodistribution and Pharmacokinetics Studies
Aptamers have proven to be particularly effective as affinity agents for imaging, both in vitro and in vivo. Aptamers are non-immunogenic and retain selectivity and affinity after labelling. At only 3nm, DNA and RNA aptamers have been shown to find a greater number of targets and achieve higher labeling densities than traditional antibody affinity agents (3,11). This enhanced degree of labeling can be applied to increase fluorescence signal strength and uniformity in the study of both cell surface markers and intracellular targets. While there are an increasing number of studies showing the advantages of aptamers in diagnostic imaging, the same techniques can be used to evaluate the biodistribution of aptamer-drug compounds and small molecule drug targets.
The research team in Germany used a fluorescence label and confocal microscopy, combined with flow cytometry, to demonstrate selective internalization of lipidized aptamer micelles into NCI-HI838 lung cancer cells. The researchers at Yale used a fluorescence label and confocal microscopy to verify selective accumulation of aptamer-drug conjugates in prostate cancer cells in vitro. Small animal imaging with a near-IR dye was used to visualize selective accumulation of aptamer-drug within prostate tumors in vivo. Selective aptamers have also been used to image HIF-1-positive cancer stem cells via MRI (12), a technique that could be applied to imaging of aptamer-drug conjugates or circulating small molecule targets to assess biodistribution.
Imaging of labeled aptamers selected for binding to small molecule drugs or drug metabolites could be used to study biodistribution and pharmacokinetics.
Aptamer-Based Biosensors for Pharmacokinetics and Dosing
Biosensors produce a change in detectable signal upon reversible interaction with a target of interest in a biological sample. Several unique features of aptamers make them ideal affinity reagents for biosensor development. The small size of capture aptamers enables binding events to take place close to the biosensor surface for enhanced detection. Aptamers have been successfully immobilized on a wide range of materials and labeled with a wide range of signaling molecules without loss of selectivity or affinity. Aptamer stability and re-folding enables biosensor regeneration. The ability to select aptamers that bind non-immunogenic small molecule drugs and drug metabolites in complex samples makes aptamer-based biosensors a particularly useful platform for the study of pharmacokinetics and drug monitoring. (1,5,10).
Several groups have developed biosensors using the Base Pair aptamer to tenofovir (1,8). Researchers at the University of California utilized an aptamer-based electrochemical sensor for real-time measurement of cocaine, doxorubicin, and kanamycin in flowing whole blood (4). More recently, they combined aptamer-based electrochemical sensors and chronoamperometry to detect levels of the small molecule drug tobramycin in vivo in rats. The sensors achieved accurate, reproducible measurement over a range of 1μM to 1mM of tobramycin in whole blood. The rapid, calibration-free aptamer-based sensor technology can be applied to pre-clinical pharmacokinetic and dosing studies and downstream monitoring of circulating drug levels for personalized treatment (2).
Selective aptamers can be used to measure the circulating concentration of small molecule drugs.
Custom Aptamer Discovery
Whether you’re looking to target specific cells for drug delivery, study biodistribution of aptamer-drug conjugates/drug compounds, or measure circulating drug levels, the process starts with discovery of a selective aptamer to your target(s) of interest. Base Pair has discovered aptamers to many cell surface markers, small molecule drugs and drug metabolites. Base Pair’s patented multiplex selection enables simultaneous discovery of selective aptamers to a panel of positive and negative targets, reducing the overall time and cost of aptamer discovery and improving aptamer selectivity.
Contact Base Pair today for more information on custom aptamer development.
1. Aliakbarinodeni, N. et al. Aptamer-based field-effect biosensor for tenofovir detection. Scientific Reports. 2017. 7:44409.
2. Arroyo-Currás, N., Subsecond-resolved molecular measurements in the living body using chronoamperometrically interrogated aptamer-based sensors. ACS Sensors. 2018. 3(2):360-366.
3. Jing, Y., et al. Aptamer-recognized carbohydrates on the cell membrane revealed by super-resolution microscopy. Nanoscale. 2018. 10:7457-7464.
4. Li, H., et al. Calibration-free electrochemical biosensors supporting accurate molecular measurements directly in undiluted whole blood. Journal of the American Chemical Society. 2017. 139:11207-11213.
5. Pfeiffer, F. et al. Selection and biosensor application of aptamers for small molecules. Frontiers in Chemistry. 2016. 4:25.
6. Powell Gray, B., et al. Tunable cytotoxic aptamer-drug conjugates for the treatment of prostate cancer. PNAS. 2018. https://doi.org/10.1073/pnas.1717705115
7. Prusty, D.K., et al. Supramolecular aptamer constructs for receptor-mediated targeting and light-triggered release of chemotherapeutics into cancer cells. Nature Communications. 2018. 9:535.
8. Vishnubhotla, R., et al. Scalable graphene aptasensors for drug quantification. AIP Advances. 2017. 7:115111
9. Wen, J., et al. A unique aptamer-drug conjugate for targeted therapy of multiple myeloma. Leukemia. 2016. 30: 987–991.
10. Wiedman, G.R. et al. An aptamer-based biosensor for the azole class of antifungal drugs. mSphere. 2017. 2(4):e00274-17.
11. Yan, Q., et al. Using an RNA aptamer probe for super-resolution imaging of native EGFR. Nanoscale Advances. 2018. DOI: 10.1039/c8na00143j.
12. Zhu, H., et al. Aptamer-PEG-modified Fe3O4@Mn as a novel T1- and T2- dual-model MRI contrast agent targeting hypoxia-induced cancer stem cells. Nature: Scientific Reports. 2016. 6:39245.