Introduction: Impaired fracture healing, which commonly stems from reduced mesenchymal stem cell (MSC) osteogenic capacity, is a major clinical challenge[1]-[3]. To augment MSC function and subsequent fracture healing, known inhibitors of bone formation can be downregulated. For example, mouse knockouts of WW domain containing E3 ubiquitin protein ligase 1 (WWP1) exhibit robust fracture healing[4]. To realize clinically-relevant approaches to enhance fracture healing motivated by gene knockout studies, siRNA delivery can be exploited. However, siRNA delivery has many challenges including inefficient delivery vehicles that are incapable of local and sustained delivery of protected siRNA to achieve tissue regeneration[5]. Thus, we developed and tested a hybrid nanoparticle (NP)/hydrogel delivery system where NPs protect siRNA and increase siRNA delivery efficiency, while PEG hydrogels provide localized and sustained siRNA delivery by controlling release of embedded siRNA/NPs.
Materials and Methods: Diblock copolymers used to form NPs were synthesized by reversible addition-fragmentation chain-transfer (RAFT) polymerization as detailed previously (Fig. 1A)[6]. siRNA/NP complexes were formed via electrostatic interaction[6] and characterized in vitro for size, MSC uptake, cytocompatibility, and silencing efficiency. siRNA/NP-loaded hydrogels (Fig.1B) were prepared using 10 wt% 10 kDa PEG-b-poly(lactide)-dimethacrylate macromers, 1 μM siRNA/NP complexes, and 0.05 wt% lithium arylphosphonate (LAP) photoinitiator in phosphate buffered saline (PBS), following exposure to ~ 5 mW/cm2 365 nm UV light for 10 min. The release of siRNA/NP from hydrogel were quantified using fluorescently-labeled siRNA. Localization of hydrogels at murine mid-diaphyseal femur fractures was evaluated qualitatively using Cy7-labeled hydrogels and XENOGEN/IVIS imaging. Fractures were treated with WWP1 siRNA/NP-releasing hydrogels (Fig. 1D), followed by RNA extraction and RT-PCR 14 days after fracture to evaluate in vivo silencing of released siRNA.
Results and Discussion: Scanning electron microscope (SEM) imaging (Fig. 1C) showed uniform distribution of NPs with size of 23 ± 3 nm within hydrogels. Robust MSC uptake of FAM-labeled siRNA/NPs was observed 24 hr after treatment (Fig. 2A). NP-mediated WWP1 silencing was observed 48 hrs after treatment, which was comprable to a commercially available transfection reagent Lipofectamine 2000 (Fig. 2B). No significant difference in cell viability was observed (Fig. 2C). According to IVIS imaging, hydrogels were localized at the fracture site for ~31 days (Fig. 2D). Fourteen days after WWP1 siRNA treatment via varying delivery vehicles, we observed that WWP1 was upregulated for ~4.3-fold at fractures in non-treated fractures, and siRNA/NP hydrogels was able to reduce WWP1 expression to no fracture control levels (Fig. 2E). We also observed increased levels of runt-related transcription factor 2 (RUNX2), an osteogenic transcription factor, corresponding to decreased WWP1 expression (Fig. 2F).
Conclusions: Tunable PEG hydrogels were designed to deliver bioactive siRNA/NPs to fracture sites in a sustained and localized manner. Current experiments are focused on evaluating fracture healing rates after treatment with siRNA/NP hydrogel via histology, X-ray, and microcomputed tomography, with comparisons to WWP1 knockout healing.
National Science Foundation (NSF) DMR1206219; New York State Stem Cell Science (NYSTEM) Foundation (N11G-035)
References:
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