Introduction:   Small nucleic acids including but not limited to nucleic acid aptamers, small interference RNAs, and anti-sense oligonucleotides have been extensively studied as therapeutic agents. They have demonstrated great potential for the treatment of a wide range of diseases including cancers, diabetes, infections, neurodegenerative diseases, and other generic disorders [1]. Because they are highly potent molecules, it is critical to achieve safe and efficient delivery of these molecules in the body. However, in comparison to the efforts made to design their sequences and to understand their in vivofunctionalities, less attention has been paid to the development of novel polymeric systems for the delivery of small nucleic acids. Therefore, this study was aimed at developing programmable hydrogels for controlled release of therapeutic nucleic acid aptamers using the principle of intermolecular hybridization.Materials and Methods: Anti-thrombin aptamer (ATA) (5'-TGG TTG GTG TGG TTG G-3') was used as a model to develop programmable hydrogels. DNA linked with acrylate was mixed with polyethylene glycol diacrylate to synthesize affinity hydrogels via free radical polymerization. The binding affinity of ATA to the conjugated DNA was measured by electrophoresis and surface plasmon resonance. ATA was loaded into the hydrogels via overnight incubation. The release kinetics of ATA from the hydrogels was measured by Nanodrop 3300. The hydrogels were also imaged in the CRI Maestro EX System. Human plasma and thrombin were used to test the bioactivity of ATA indicated by the thrombin clotting time.Results and Discussion: The overall concept of controlling the release of nucleic acid aptamers is shown in Fig. 1. A set of immobilizing DNA molecules was investigated to determine the effect of binding affinity on aptamer release. In comparison to native hydrogel (i.e., the hydrogel without immobilizing DNAs), the DNA-functionalized hydrogel exhibited smaller burst release. In addition, ATA was slowly released from the hydrogel and the released rate was a function of the hybridizing length. When the hybridizing length reached 10 nucleotides, the ATA release from the hydrogel was very slow after the initial burst release. However, in the presence of triggering molecules, ATA could be rapidly released via the mechanism of intermolecular hybridization. The fast release of ATA was confirmed by the SPR analysis and gel imaging. These results indicate that the optimal ATA release rate can be achieved by varying the hybridization length and the addition of triggering molecules. In addition to the controlled release study, the bioactivity study showed that the released ATA was able to inhibit the functionality of thrombin.  Conclusions:  DNA functionalized hydrogels are a promising polymeric system for controlled release of nucleic acid aptamers. The sustained release rate can be tuned by using different hybridizing length. In addition, the release rate can be programmed and accelerated in the presence of triggering molecules.Acknowledgements:  We greatly acknowledge the support from the United States NSF CAREER Award (DMR-0955358 /1332351).References: [1] Alvarez-Salas LM. Curr Top Med Chem, 2008, 8, 1379.

Programmable hydrogels for the controlled release of therapeutic nucleic acid aptamers via reversible DNA hybridization

247th National Meeting (2014)

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