Tunable delivery of siRNA from a biodegradable scaffold to promote angiogenesis in vivo

Abstract

A system has been engineered for temporally controlled delivery of siRNA from biodegradable tissue regenerative scaffolds. Therapeutic application of this approach to silence prolyl hydroxylase domain 2 promoted expression of pro-angiogenic genes controlled by HIF1α and enhanced scaffold vascularization in vivo. This technology provides a new standard for efficient and controllable gene silencing to modulate host response within regenerative biomaterials.

Keywords: controlled release; gene knockdown; reversible addition-fragmentation chain transfer (RAFT) polymerization, endosomolytic nanoparticles, injectable scaffolds.

Figure 1

Material synthesis and characterization of the PEUR scaffold si-NP delivery platform. A) The structure of the diblock copolymer developed previously^[30] contains an siRNA condensing block composed of DMAEMA and a pH-responsive block composed of a copolymer of DMAEMA, BMA, and PAA. B) The polyester alcohol (polyol or triol) that was used in the synthesis of polyurethanes were composed of copolymers of poly(ε-caprolactone), poly(glycolide), and poly(D,L-lactide). C) Isocyanate-containing crosslinking components used for PEUR formation included hexamethylene diisocyanate trimer (HDIt) and lysine triisocyanate (LTI). D) The excipient trehalose stabilized the size and ζ-potential of released si-NPs compared to si-NPs prepared in PBS. E) PEUR scaffold-released si-NPs deliver siRNA into the cytoplasm of cells in vitro similar to freshly prepared si-NPs (scale = 30μm). F) Gene silencing activity was similar in PEUR scaffold-released si-NPs compared to freshly-made si-NPs as revealed by RT-PCR IC₅₀ analysis of target gene expression (p=NS). G–J) SEM images of PEUR scaffolds (LTI-based materials shown) containing varying weight% of trehalose (5% by weight is 5T) demonstrate the porous scaffold architecture (Scale = 300μm). K–N) Maximum intensity projections from confocal microscopy showed homogenous loading of si-NPs into the scaffold (note dark areas correspond to pores, scale = 300μm). O–P) Temporal release profile of si-NPs from PEUR scaffolds in vitro demonstrated diffusion controlled release (characterized by Weibull model) that could be modulated through varying the concentration of trehalose or alteration of the isocyanate chemistry. Q–R) The rate of release of si-NPs in vivo was increased relative to the release in vitro but was also tunable based on varying the concentration of the excipient trehalose.

Figure 2

The si-NP-loaded PEUR scaffolds provide a potent and temporally-tunable gene silencing platform. A) PPIB mRNA was significantly silenced by siRNA-NP-PEUR at day 5, 12, and 21 (p<0.002 for all groups, n=4) in subcutaneous implants in mice at a siRNA dose of 200μg/kg. B) A dose response at day 12 demonstrated a low IC₅₀ for siRNA-NP-PEUR of 41.8 μg/kg C) The temporal gene silencing profile was tuned through the use of trehalose to control release kinetics (day 5 p<.0005 0T vs 5T, day 35 p<.0005 0T vs 5T). D) A longitudinal study demonstrated significant luciferase reduction over the time course of wound healing, highlighted in gray, in a COL1A2 luciferase reporter mouse model (p<.01, n=5). E) Western blotting for PPIB at day 12 showed significant protein reduction in PPIB siRNA loaded scaffolds (n=3, p<.05).

Figure 3

Sustained silencing of PHD2 increases angiogenesis within PEUR tissue scaffolds. A) 80% silencing of PHD2 increased VEGF and FGF-2 expression by 200% and 290% respectively (*p<0.01). B) CD31 staining was significantly increased within PHD2 scaffolds at day 14 and day 33 (Scale = 200 μm, vessels appear read, nuclei are counterstained purple with hematoxylin, and white space represents residual PEUR scaffold). C–D) CD31 sections were quantified showing a significant increase in vessel area at day 33 (*p<0.01). E) Micro-CT of explanted PHD2-NP scaffolds showed a significant increase in both vessel number and vessel size for PHD2-NP scaffolds as shown in the histogram. F) Micro-CT images visually demonstrate the increased vasculature within the scaffolds. G) Quantitative analysis of 3D micro-CT vessel images revealed a significant increase in vascular volume and mean vascular thickness within PHD2-NP-loaded scaffolds.