Hydrogel functionalization with DNA aptamers for sustained PDGF-BB releasew

Hydrogels are one of the most appealing polymeric materials used for preparing sustained-release systems that have a great impact on pharmaceutical development and regenerative medicine. However, hydrogels generally have high permeability, which often leads to fast substance release. Extensively fast release will not only decrease the biological efficacy of the released substances, but also cause severe sideeffects in vivo. Thus, there has been extensive investigation of hydrogel functionalization to improve the sustained-release efficacy. For instance, perlecan domain 1 has been applied to functionalize hyaluronic acid-based hydrogel particles for sustained protein release. Here we aim to explore a novel sustained-release hydrogel system using DNA aptamers. DNA aptamers have recently attracted significant attention in various areas because in principle DNA aptamers can be selected for any molecule of interest with high binding affinity and specificity. In addition, DNA aptamers not only are tolerant of harsh thermal, physical, and chemical conditions in general, but also exhibit tunable stability in biological environments. Importantly, DNA aptamers can be synthesized with a standard chemical procedure and exhibit little immunogenicity due to their small size. These advantages motivated us to explore the feasibility of using aptamers as affinity sites of hydrogels as a framework for sustained protein release. To prove the concept, we used polyacrylamide gel and anti-platelet-derived growth factor-BB (PDGF-BB) aptamer as a model system. The reason for using polyacrylamide was that its synthesis is a well-defined chemical procedure. Although polyacrylamide is not a suitable material for in vivo applications, the concept is expected to be applicable for biocompatible hydrogels made of polymers such as poly(ethylene glycol) and hyaluronic acid. The anti-PDGF-BB aptamer was originally selected from a DNA library. Its binding functionality has been well studied. The aptamer sequence used here is composed of its truncated 36-nucleotide (nt) format and a 10-nt tail attached to the 50 end. The truncated 36-nt aptamer is used to bind to PDGF-BB, whereas the 10-nt tail is used to enhance molecular flexibility. The structures of anti-PDGF-BB aptamers with higher and lower affinity are shown in Fig. 1A and B, respectively. The binding functionality was evaluated with surface plasmon resonance (SPR) analysis, which directly provides information on the binding affinity. The KD values of the higher-affinity and lower-affinity aptamers are 25 nM and 220 nM, respectively (Fig. 1C). To functionalize polyacrylamide gel, the aptamer was conjugated with an acrydite functional group at its 50 end during its chemical synthesis (Fig. 2A). Thus, when ammonium persulfate (APS) and N,N,N0,N0-tetramethylenediamine (TEMED) were added into the mixture of acrydite-modified aptamer, acrylamide, and bis-acrylamide to initiate free-radical polymerization, the unsaturated double bond of the acrydite would then enable the incorporation of aptamer molecules into hydrogel networks (Fig. 2A). To test the feasibility, polyacrylamide gels were subjected to electrophoresis, stained with ethidium bromide (EtBr), and examined by an imaging system. If the gels were functionalized with the aptamers, the aptamers would stay in the gels during the electrophoresis. Thus, EtBr stain would appear on the gels. Fig. 2B demonstrates that the acrydite-modified aptamer molecules were successfully incorporated into the hydrogels during polymerization. Despite the success, we also found that not