Delivery of RNAi-Based Therapeutics for Bone Regeneration.

PURPOSE OF REVIEW: The clinical significance, target pathways, recent successes, and challenges that preclude translation of RNAi bone regenerative approaches are overviewed. RECENT FINDINGS: RNA interference (RNAi) is a promising new therapeutic approach for bone regeneration by stimulating or inhibiting critical signaling pathways. However, RNAi suffers from significant delivery challenges. These challenges include avoiding nuclease degradation, achieving bone tissue targeting, and reaching the cytoplasm for mRNA inhibition. Many drug delivery systems have overcome stability and intracellular localization challenges but suffer from protein adsorption that results in clearance of up to 99% of injected dosages, thus severely limiting drug delivery efficacy. While RNAi has myriad promising attributes for use in bone regenerative applications, delivery challenges continue to plague translation. Thus, a focus on drug delivery system development is critical to provide greater delivery efficiency and bone targeting to reap the promise of RNAi.

Serpine1 Knockdown Enhances MMP Activity after Flexor Tendon Injury in Mice: Implications for Adhesions Therapy.

Injuries to flexor tendons can be complicated by fibrotic adhesions, which severely impair the function of the hand. Adhesions have been associated with TGF-ß1, which causes upregulation of PAI-1, a master suppressor of protease activity, including matrix metalloproteinases (MMP). In the present study, the effects of inhibiting PAI-1 in murine zone II flexor tendon injury were evaluated utilizing knockout (KO) mice and local nanoparticle-mediated siRNA delivery. In the PAI-1 KO murine model, reduced adherence of injured tendon to surrounding subcutaneous tissue and accelerated recovery of normal biomechanical properties compared to wild type controls were observed. Furthermore, MMP activity was significantly increased in the injured tendons of the PAI-1 KO mice, which could explain their reduced adhesions and accelerated remodeling. These data demonstrate that PAI-1 mediates fibrotic adhesions in injured flexor tendons by suppressing MMP activity. In vitro siRNA delivery to silence Serpine1 expression after treatment with TGF-ß1 increased MMP activity. Nanoparticle-mediated delivery of siRNA targeting Serpine1 in injured flexor tendons significantly reduced target gene expression and subsequently increased MMP activity. Collectively, the data demonstrate that PAI-1 can be a druggable target for treating adhesions and accelerating the remodeling of flexor tendon injuries.

The Effects of Biological Fluids on Colloidal Stability and siRNA Delivery of a pH-Responsive Micellar Nanoparticle Delivery System.

Nanoparticles (NPs) interact with complex protein milieus in biological fluids, and these interactions have profound effects on NP physicochemical properties and function. Surprisingly, most studies neglect the impact of these interactions, especially with respect to NP-mediated siRNA delivery. Here, the effects of serum on colloidal stability and siRNA delivery of a pH-responsive micellar NP delivery system were characterized. Results show cationic NP-siRNA complexes aggregate in ≥2% serum in buffer, but are stable in serum-free media. Furthermore, nonaggregated NP-siRNA delivered in serum-free media result in 4-fold greater siRNA uptake in vitro, compared to aggregated NP-siRNA. Interestingly, pH-responsive membrane lysis behavior, which is required for endosomal escape, and NP-siRNA dissociation, necessary for gene knockdown, are significantly reduced in serum. Consistent with these data, nonaggregated NP-siRNA in serum-free conditions result in highly efficient gene silencing, even at doses as low as 5 nM siRNA. NP-siRNA diameter was measured at albumin and IgG levels mimicking biological fluids. Neither albumin nor IgG alone induces NP-siRNA aggregation, implicating other serum proteins in NP colloidal instability. Finally, as a proof-of-principle that stability is maintained in established in vivo models, transmission electron microscopy reveals NP-siRNA are taken up by ductal epithelial cells in a nonaggregated state when injected retroductally into mouse salivary glands in vivo. Overall, this study shows serum-induced NP-siRNA aggregation significantly diminishes efficiency of siRNA delivery by reducing uptake, pH-responsive membrane lysis activity, and NP-siRNA dissociation. Moreover, these results highlight the importance of local NP-mediated drug delivery and are broadly applicable to other drug delivery systems.

Diblock Copolymer Hydrophobicity Facilitates Efficient Gene Silencing and Cytocompatible Nanoparticle-Mediated siRNA Delivery to Musculoskeletal Cell Types.

pH-responsive diblock copolymers provide tailorable nanoparticle (NP) architecture and chemistry critical for siRNA delivery. Here, diblock polymers varying in first (corona) and second (core) block molecular weight (Mn), corona/core ratio, and core hydrophobicity (%BMA) were synthesized to determine their effect on siRNA delivery in murine tenocytes (mTenocyte) and murine and human mesenchymal stem cells (mMSC and hMSCs, respectively). NP-mediated siRNA uptake, gene silencing, and cytocompatibility were quantified. Uptake is positively correlated with first block Mn in mTenocytes and hMSCs (p ≤ 0.0005). All NP resulted in significant gene silencing that was positively correlated with %BMA (p < 0.05) in all cell types. Cytocompatibility was reduced in mTenocytes compared to MSCs (p < 0.0001). %BMA was positively correlated with cytocompatibility in MSCs (p < 0.05), suggesting stable NP are more cytocompatible. Overall, this study shows that NP-siRNA cytocompatibility is cell type dependent, and hydrophobicity (%BMA) is the critical diblock copolymer property for efficient gene silencing in musculoskeletal cell types.

Controlled and sustained delivery of siRNA/NPs from hydrogels expedites bone fracture healing.

Despite great potential, delivery remains as the most significant barrier to the widespread use of siRNA therapeutics. siRNA has delivery limitations due to susceptibility to RNase degradation, low cellular uptake, and poor tissue-specific localization. Here, we report the development of a hybrid nanoparticle (NP)/hydrogel system that overcomes these challenges. Hydrogels provide localized and sustained delivery via controlled release of entrapped siRNA/NP complexes while NPs protect and enable efficient cytosolic accumulation of siRNA. To demonstrate therapeutic efficacy, regenerative siRNA against WW domain-containing E3 ubiquitin protein ligase 1 (Wwp1) complexed with NP were entrapped within poly(ethylene glycol) (PEG)-based hydrogels and implanted at sites of murine mid-diaphyseal femur fractures. Results showed localization of hydrogels and controlled release of siRNA/NPs at fractures for 28 days, a timeframe over which fracture healing occurs. siRNA/NP sustained delivery from hydrogels resulted in significant Wwp1 silencing at fracture callus compared to untreated controls. Fractures treated with siRNA/NP hydrogels exhibited accelerated bone formation and significantly increased biomechanical strength. This NP/hydrogel siRNA delivery system has outstanding therapeutic promise to augment fracture healing. Owing to the structural similarities of siRNA, the development of the hydrogel platform for in vivo siRNA delivery has myriad therapeutic possibilities in orthopaedics and beyond.

Evaluating side effects of nanoparticle-mediated siRNA delivery to mesenchymal stem cells using next generation sequencing and enrichment analysis.

RNA interference has immense potential to modulate cell functions. However, effective delivery of small interfering RNA (siRNA) while avoiding deleterious side effects has proven challenging. This study investigates both intended and unintended effects of diblock copolymer nanoparticle (NP) delivery of siRNA delivery to human mesenchymal stem cells (hMSC). Specifically, siRNA delivery was investigated at a range of NP-siRNA:hMSC ratios with a focus on the effects of NP-siRNA treatment on hMSC functions. Additionally, next generation RNA sequencing (RNAseq) was used with enrichment analysis to observe side effects in hMSC gene expression. Results show NP-siRNA delivery is negatively correlated with hMSC density. However, higher NP-siRNA:hMSC ratios increased cytotoxicity and decreased metabolic activity. hMSC proliferation was largely unaffected by NP-siRNA treatment, except for a threefold reduction in hMSCs seeded at 4,000 cells/cm2. Flow cytometry reveals that apoptosis is a function of NP-siRNA treatment time and seeding density; ∼14% of the treated hMSCs seeded at 8,000 cells/cm2 were annexin V+-siRNA+ 24 hr after treatment, while 11% of the treated population was annexin V+-siRNA-. RNAseq shows that NP-siRNA treatment results in transcriptomic changes in hMSCs, while pathway analysis shows upregulation of apoptosis signaling and downregulation of metabolism, cell cycle, and DNA replication pathways, as corroborated by apoptosis, metabolism, and proliferation assays. Additionally, multiple innate immune signaling pathways such as toll-like receptor, RIG-I-like receptor, and nuclear factor-κB signaling pathways are upregulated. Furthermore, and consistent with traditional siRNA immune activation, cytokine-cytokine receptor signaling was also upregulated. Overall, this study provides insight into NP-siRNA:hMSC ratios that are favorable for siRNA delivery. Moreover, NP-siRNA delivery results in side effects across the hMSC transcriptome that suggest activation of the innate immunity that could alter MSC functions associated with their therapeutic potential.

RPA-mediated unfolding of systematically varying G-quadruplex structures.

G-quadruplex (GQ) is a noncanonical nucleic acid structure that is formed by guanine rich sequences. Unless it is destabilized by proteins such as replication protein A (RPA), GQ could interfere with DNA metabolic functions, such as replication or repair. We studied RPA-mediated GQ unfolding using single-molecule FRET on two groups of GQ structures that have different loop lengths and different numbers of G-tetrad layers. We observed a linear increase in the steady-state stability of the GQ against RPA-mediated unfolding with increasing number of layers or decreasing loop length. The stability demonstrated by different GQ structures varied by at least three orders of magnitude. Those with shorter loops (less than three nucleotides long) or a greater number of layers (more than three layers) maintained a significant folded population even at physiological RPA concentration (≈1 µM), raising the possibility of physiological viability of such GQ structures. Finally, we measured the transition time between the start and end of the RPA-mediated GQ unfolding process to be 0.35 ± 0.10 s for all GQ constructs we studied, despite significant differences in their steady-state stabilities. We propose a two-step RPA-mediated GQ unfolding mechanism that is consistent with our observations.