(170g) Localized and Sustained Delivery of siRNA from Hydrogels Expedites Fracture Healing

Introduction: Impaired fracture healing, which stems from reduced mesenchymal stem cell (MSC) osteogenesis, is a major clinical challenge ¹. To augment MSC-mediated fracture healing, known inhibitors of bone formation can be downregulated using siRNA-mediated gene knockdown. For example, mouse knockouts of WW domain containing E3 ubiquitin protein ligase 1 (WWP1) exhibit robust fracture healing ². However, siRNA delivery suffers from inefficiencies that preclude fracture-targeted and sustained delivery of protected siRNA ³. Thus, we developed a hybrid nanoparticle (NP)/hydrogel system where NPs protect and increase siRNA delivery efficiency and hydrogels provide localized and sustained delivery by controlling release of embedded siRNA/NPs.

Materials and Methods: Diblock copolymers composed of cationic dimethylaminoethyl methacrylate (DMAEMA) blocks (m~140) and endosomolytic blocks of DMAEMA, propylacrylic acid (PAA), and butyl methacrylate (BMA) (n~80) were synthesized by reversible addition-fragmentation chain-transfer (RAFT) polymerization ³. Self-assembled siRNA/NPs embedded in hydrolytically degradable hydrogels prepared using 10 wt% poly(ethylene glycol)-b-poly(lactide)-b-dimethacrylate, 1 μM siRNA/NPs, and 0.05 wt% lithium arylphosphonate (LAP) photoinitiator, following exposure to ~5 mW/cm² 365 nm light for 10 min. Hydrogels with embedded siRNA/NPs were characterized via scanned electron microscope (SEM). Characterization of localization of both Cy5-siRNA and Cy7-PEG hydrogels at Mid-diaphyseal femur fractures (Balb/c mice) was accomplished using XENOGEN/IVIS imaging. Fractures were treated with WWP1 siRNA/NPs hydrogels, followed by RT-PCR, histology (Alcian blue & Orange G) and microcomputed tomography (µCT) to assess fracture healing.

Results and Discussion: siRNA/NPs were loaded into degradable hydrogels for treatment of femur fractures. SEM imaging showed uniform distribution of NPs within hydrogels. According to IVIS imaging, siRNA and hydrogels were co-localized at fractures for ~21 days, whereas free siRNA/NP dispersed from injection sites rapidly. 14 days after WWP1 siRNA treatment, we observed significantly decreased WWP1 expression compared with untreated controls. Histological analysis showed enhanced bridging bone formation in siRNA/NPs hydrogel treated fractures, whereas extensive unmineralized cartilage and undifferentiated mesenchyme tissue were still present in non-treated and hydrogel only controls. µCT showed 1.3-fold increase in bone callus volume at week 3 in siRNA/NPs hydrogel treated fractures compared with untreated fractures, indicating accelerated bone formation and mineralization.

Conclusions: PEG hydrogels were designed for siRNA/NPs delivery to fracture sites in a sustained and localized manner. Target knockdown of Wwp1 using siRNA/NPs hydrogels showed significantly increased bone formation and accelerated healing. In sum, the development of the hydrogel system has outstanding therapeutic promise.

Acknowledgements: Funding: NSF-DMR1206219; NSF-CBET1450987 NYSTEM N11G-035; NIH-R01AR056696; NIH-R01 AR064200; NIHS10 RR027340.

References: [1] Hofmann, A., et al., Bone, 2008. 42(5): p. 894-906 [2] Shu, L., et al., J Bone Miner Res, 2013. 28(9): p. 1925-35 [3] Benoit, D.S., et al., Biomacromolecules, 2012. 13(11): p. 3841-9