siRNA Delivery from an Injectable Scaffold for Wound Therapy

Abstract

Significance: Aberrant overexpression of proinflammatory molecules is believed to be a key mediator in the formation of chronic skin wounds, and the inhibition of these signals may be an effective therapeutic strategy to promote healing. Small interfering RNA (siRNA) can provide gene-specific silencing and may present a safe and effective route for knockdown of inflammatory or other target proteins in chronic skin wounds.

Critical issues: siRNA suffers from delivery barriers in vivo such as susceptibility to degradation, membrane impermeability, and transient activity. Therefore, a delivery strategy that stabilizes siRNA, provides intracellular (cytoplasmic) delivery, and produces temporally sustained activity is needed. The novel approach described combines pH-responsive, siRNA-loaded nanoparticles into a biodegradable polyurethane (PUR) scaffold and presents a promising platform for effective, local silencing of deleterious genes in nonhealing skin wounds.

Recent advances: The siRNA delivery barriers have been overcome using a nanoparticulate carrier that protects siRNA and responds to pH gradients in the endo-lysosomal pathway to mediate cytosolic delivery. Nanoparticle incorporation into a biodegradable PUR scaffold provides a means for controlling the delivery kinetics of siRNA-loaded carriers. Furthermore, the PUR is injectable, making it feasible for clinical use, and provides a porous tissue template for cell in-growth during tissue regeneration and remodeling. This local siRNA delivery platform can be tuned to optimize release kinetics for specific pathologies.

Future directions: siRNA may provide a new class of biologic drugs that will outperform growth factor approaches, which have shown only moderate clinical success. The new platform presented here may provide clinicians with an improved option for wound care.

Craig L. Duvall, PhD

Figure 1.

Schematic illustrating the approach described in this review. (A) Reversible addition fragmentation chain transfer-synthesized diblock co-polymer with the siRNA condensing block shown in gray and the pH responsive block in black. (B) In aqueous solutions, the diblock copolymer self-assembles into micellar nanoparticles with a positive surface charge that can be used to electrostatically condense siRNA. (C) Lyophilized si-SPNs are mixed into the polyol component and then added to the lysine triisocyanate and water to form a porous PUR scaffold containing embedded si-SPNs. (D) The si-SPNs can diffuse out of the PUR scaffold, and, upon release, si-SPNs can be internalized and efficiently delivered in a bioactive form into the cytoplasm of cells. siRNA, small interfering RNA; PUR, polyurethane; si-SPN, smart pH responsive polymeric nanoparticle with complexed siRNA.

Figure 2.

(A) si-SPNs demonstrate finely tuned, pH-dependent membrane disruption in a red blood cell hemolysis assay. Red blood cells incubated without si-SPNs showed no hemolysis at tested pH values. (B) The si-SPNs provide enhanced intracellular uptake relative to naked siRNA as shown by flow cytometry on human cervical carcinoma cells delivered FAM-labeled siRNA. (C, D) Real-time reverse transcriptase–polymerase chain reaction indicates that si-SPNs are capable of delivering siRNA against the model gene GAPDH and efficiently reducing target expression by nearly 90% at a charge ratio of 4:1 and siRNA concentration of 50 nM. These si-SPNs have been subsequently incorporated into PUR scaffolds with (E) interconnected, porous morphology as shown via SEM. Wound healing after application of empty PUR scaffolds (F–H) has been shown to be accelerated when PDGF is incorporated for sustained release into the wound (I–K). GAPDH, glyceraldehyde 3-phosphate dehydrogenase; PDGF, platelet-derived growth factor; SEM, scanning electron microscopy.