ABSTRACT
Aptamers are short single-stranded oligonucleotides selected to bind with high affinity and specificity to a target. In contrast to antibodies, aptamers can be produced in large-scale in vitro systems without the need for any biological agents, making them highly attractive as targeting ligands for bioimaging and drug delivery. For in vivo applications it is often desirable to multimerize the aptamers in order to increase their binding strength and overall specificity. Additional functionalities, such as imaging and therapeutic agents, as well as pharmacokinetic modifiers, need to be attached in a stoichiometric fashion. Herein, we present a robust method for assembly of up to three aptamers and a fluorophore in a single well-defined nanostructure. The process is entirely modular and can be applied to any aptamer requiring only a single reactive "click handle." Multimerization of two aptamers, A9g and GL21.T, previously shown to target cancer cells, led to a strong increase in cell uptake. A similar effect was observed for the prostate-specific membrane antigen (PSMA)-targeting A9g aptamer in mice where multivalent aptamer binding led to increased tumor specificity. Altogether, this method provides a platform for multimerization of aptamers with advantages in terms of combinatorial screening capacity and multifunctional design of nanomedicine.
ABSTRACT
Rationale: Within the field of personalized medicine there is an increasing focus on designing flexible, multifunctional drug delivery systems that combine high efficacy with minimal side effects, by tailoring treatment to the individual. Methods: We synthesized a chemically stabilized ~4 nm nucleic acid nanoscaffold, and characterized its assembly, stability and functional properties in vitro and in vivo. We tested its flexibility towards multifunctionalization by conjugating various biomolecules to the four modules of the system. The pharmacokinetics, targeting capability and bioimaging properties of the structure were investigated in mice. The role of avidity in targeted liver cell internalization was investigated by flow cytometry, confocal microscopy and in vivo by fluorescent scanning of the blood and organs of the animals. Results: We have developed a nanoscaffold that rapidly and with high efficiency can self-assemble four chemically conjugated functionalities into a stable, in vivo-applicable system with complete control of stoichiometry and site specificity. The circulation time of the nanoscaffold could be tuned by functionalization with various numbers of polyethylene glycol polymers or with albumin-binding fatty acids. Highly effective hepatocyte-specific internalization was achieved with increasing valencies of tri-antennary galactosamine (triGalNAc) in vitro and in vivo. Conclusion: With its facile functionalization, stoichiometric control, small size and high serum- and thermostability, the nanoscaffold presented here constitutes a novel and flexible platform technology for theranostics.
