Controlled Release in Hydrogels Using DNA Nanotechnology

Abstract

Gelatin is a biopolymer widely used to synthesize hydrogels for biomedical applications, such as tissue engineering and bioinks for 3D bioprinting. However, as with other biopolymer-based hydrogels, gelatin-hydrogels do not allow precise temporal control of the biomolecule distribution to mimic biological signals involved in biological mechanisms. Leveraging DNA nanotechnology tools to develop a responsive controlled release system via strand displacement has demonstrated the ability to encode logic process, which would enable a more sophisticated design for controlled release. However, this unique and dynamic system has not yet been incorporated within any hydrogels to create a complete release circuit mechanism that closely resembles the sequential distribution of biomolecules observed in the native environment. Here, we designed and synthesized versatile multi-arm DNA motifs that can be easily conjugated within a gelatin hydrogel via click chemistry to incorporate a strand displacement circuit. After validating the incorporation and showing the increased stability of DNA motifs against degradation once embedded in the hydrogel, we demonstrated the ability of our system to release multiple model cargos with temporal specificity by the addition of the trigger strands specific to each cargo. Additionally, we were able to modulate the rate and quantity of cargo release by tuning the sequence of the trigger strands.

Designer DNA Strand Displacement Reaction toward Controlled Release of Cargos

“…The hydrodynamic diameter of the 4WJ motif was measured using dynamic light scattering (DLS) similarly with our previous study (19). The 60 µL of 750 nM 4WJ was prepared with 1X…”

“…The 4-way junction (4WJ) DNA motif is folded as described in our previous work (19). Briefly, the DNA strands were mixed equimolarly at 5 µM in 1X folding buffer (40 mM TRIS-base, 20 mM acetic acid, 2 mM EDTA, 12 mM MgCl2, at pH 8.0) and annealed in a Bio-Rad T100 TM Thermal Cycler using a temperature ramp from 95 to 4℃ over the course of 2h.…”

“…Agarose gel electrophoresis was used to characterize the 4WJ DNA motif to ensure the structure was folded properly using the established method in our previous paper (19).…”

“…Fluorescence of the DNA motif was measured for fluorophore (HEX or TR) samples, quenched (HEX/Iowa Black FQ or TR/Iowa Black RQ) samples, and the unmodified samples using the established method in our previous paper (19). Briefly, the DNA motifs were diluted to 500 nM for all conditions.…”

“…Thus, thanks to the nature of DNA and the variety of DNA motifs available, there are many stimuli that have already been used to generate a dynamic DNA nanotechnology system, which include pH, light, temperature, enzymatic reactions, strand displacement, and aptamer (7)(8)(9)(10)(11)(12). Amongst these stimuli for designing dynamic DNA nanotechnology system, the toeholdmediated strand displacement reaction (TMSDR), which has been used for DNA circuits (13)(14)(15)(16)(17), biosensors (4,5,18), release system (19,20), reconfigurable nanodevices (21)(22)(23) is particularly attractive. Indeed, the TMSDR operates by using a single strand DNA (trigger strand) with higher affinity to displace another strand in a DNA duplex (incumbent) and does not require any other external stimuli (Figure S1).…”