To confirm the conjugation of siGFP-SH to DEX-MAES macromer via a Michael-addition reaction, polyacrylamide gel analysis was performed. A solution of 10 μg of annealed siGFP-SH was mixed with 10% (w/w) DEX-MAES solution in nuclease-free PBS (pH 8.0) containing 0.05% (w/w) Irgacure D-2959 photoinitiator (PI) (Sigma-Aldrich) to a total volume of 50 μl, and the mixture was allowed to react under ambient conditions in darkness. After 0 min (right after mixing) and 2, 6, 12, or 24 hours, the samples (10 μl) were collected and frozen in −80°C at the desired time points to halt the reaction and to be thawed immediately before running acrylamide gels (described in the previous section). A standard of 1 μl of antisense strand siRNA, which did not contain a thiol group to bind to the DEX, and a standard of RNA-free DEX were both added as controls. The antisense RNA was also used to standardize the densitometric calculations, on which the estimates of reaction completion were based.

To examine the release of Michael-addition tethered siRNA from DEX hydrogels, 50 μl of each DEX-siRNA conjugate solution was added to wells of a clear plastic 96-well plate. The wells were irradiated with UV light (OmniCure S1000 UV Spot Cure System, Lumen Dynamics Group, Mississauga, Ontario, Canada) for 150.0 s at the intensity of 2.5 mW/cm2 to cross-link the hydrogels. To fabricate the hydrogels with different macromer concentrations [6, 8, and 10% (w/w)] while keeping the total RNA amount constant, the same amount of RNA (10 μg) was conjugated with the same volume of macromer solutions with different DEX-MAES concentrations before cross-linking. The cross-linked gels were then gently moved to nuclease-free Eppendorf tubes containing 1.0 ml of nuclease-free PBS (pH 7.4). The tubes were placed in a 37°C incubator for subsequent siRNA release. The PBS was periodically carefully removed from the Eppendorf tubes and subsequently frozen. One milliliter of fresh nuclease-free PBS (pH 7.4) was then added, and the gels were allowed to continue degrading. In this manner, the amount of siRNA released from the gels could be detected within the PBS at each time point to monitor the degradation of the hydrogels. Once complete degradation occurred, the frozen aliquots were thawed and their siRNA contents were determined by a RiboGreen (Thermo Fisher Scientific) assay, as per the manufacturer’s procedure. The assay was performed using a plate reader (VersaMax, Molecular Devices, Sunnyvale, CA) with an excitation of 485 nm and an emission of 520 nm. Standards were made of fresh corresponding annealed double-stranded siRNA to establish standard curves to calculate the concentration of released RNAs. Assayed values from degraded RNA-free hydrogels were then subtracted from the experimental samples to take into account any potential assay interference from DEX (n = 3). To examine the bioactivity of released siRNA, the releasates containing released siGFP from days 1 to 10 were pooled and used to treat monolayer cultured HeLa cells for 2 days, followed by quantification of cellular GFP expression via flow cytometry (fig. S4B).

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