Hydrogel microspheres for spatiotemporally controlled delivery of RNA and silencing gene expression within scaffold-free tissue engineered constructs

Abstract

Delivery systems for controlled release of RNA interference (RNAi) molecules, including small interfering (siRNA) and microRNA (miRNA), have the potential to direct stem cell differentiation for regenerative musculoskeletal applications. To date, localized RNA delivery platforms in this area have focused predominantly on bulk scaffold-based approaches, which can interfere with cell-cell interactions important for recapitulating some native musculoskeletal developmental and healing processes in tissue regeneration strategies. In contrast, scaffold-free, high density human mesenchymal stem cell (hMSC) aggregates may provide an avenue for creating a more biomimetic microenvironment. Here, photocrosslinkable dextran microspheres (MS) encapsulating siRNA-micelles were prepared via an aqueous emulsion method and incorporated within hMSC aggregates for localized and sustained delivery of bioactive siRNA. siRNA-micelles released from MS in a sustained fashion over the course of 28 days, and the released siRNA retained its ability to transfect cells for gene silencing. Incorporation of fluorescently labeled siRNA (siGLO)-laden MS within hMSC aggregates exhibited tunable siGLO delivery and uptake by stem cells. Incorporation of MS loaded with siRNA targeting green fluorescent protein (siGFP) within GFP-hMSC aggregates provided sustained presentation of siGFP within the constructs and prolonged GFP silencing for up to 15 days. This platform system enables sustained gene silencing within stem cell aggregates and thus shows great potential in tissue regeneration applications. STATEMENT OF SIGNIFICANCE: This work presents a new strategy to deliver RNA-nanocomplexes from photocrosslinked dextran microspheres for tunable presentation of bioactive RNA. These microspheres were embedded within scaffold-free, human mesenchymal stem cell (hMSC) aggregates for sustained gene silencing within three-dimensional cell constructs while maintaining cell viability. Unlike exogenous delivery of RNA within culture medium that suffers from diffusion limitations and potential need for repeated transfections, this strategy provides local and sustained RNA presentation from the microspheres to cells in the constructs. This system has the potential to inhibit translation of hMSC differentiation antagonists and drive hMSC differentiation toward desired specific lineages, and is an important step in the engineering of high-density stem cell systems with incorporated instructive genetic cues for application in tissue regeneration.

Keywords: Aggregates; Gene therapy; Mesenchymal stem cells; RNA interference; Sustained delivery.

Figure 1.

A) Chemical structure of DEX-HEMA. B) Schematic showing the fabrication of DEX-based MS encapsulating siRNA-micelles. C) Schematic depicting the incorporation of siRNA-MS into an hMSC aggregate for localized and sustained siRNA presentation and subsequent sustained gene silencing within a stem cell aggregate. Graphics are not to scale.

Figure 2.

DLS characterization of micelle size shows the influence of siRNA loading lyophilization (lyo), pre- and post-lyo, on particle size in the presence of trehalose. * p<0.05.

Figure 3.

Light microscopy images of siGLO-MS6 and siGLO-MS10. Scale bars indicate 20 μm.

Figure 4.

siGLO release profiles from A) MS6 and B) MS10 over the course of 28 days. Cumulative siRNA (left) and % cumulative siRNA (right) release data from 3 different MS batches are plotted separately. The dashed lines (left) represent the actual incorporated siRNA content in 2.0 mg MS for each batch. MS6 release 1 was significantly different from the other two MS6 release profiles, p<0.05.

Figure 5.

GFP expression of monolayer deGFP-Hela cells treated with MS releasates. & p<0.05 compared to all other groups, # p<0.05 compared to None and (−) ctrl, * p<0.05. None = untreated cells.

Figure 6.

Distribution of incorporated MS in hMSC aggregates for sustained siRNA presentation. A) Fluorescence photomicrographs of rhodamine-labeled MS (red) incorporated into hMSC aggregates (0.15 and 0.30 mg MS per aggregate) and DAPI-stained cell nuclei (blue) after 3 days in vitro culture. Scale bars indicate 200 μm. B) Fluorescence confocal photomicrographs of rhodamine-labeled (red) siGLO-MS incorporated into hMSC aggregates to visualize siGLO uptake (green) and DAPI-stained hMSC nuclei (blue) in 3D aggregates after different culture periods. Scale bars indicate 30 μm.

Figure 7.

siGFP-mediated knockdown of GFP mRNA expression within GFP-hMSC aggregates. Analysis of fold changes in GFP mRNA expression of GFP-hMSC aggregates embedded with different MS formulations compared to cells only aggregates at A) 5, B) 10, C) 15 and D) 20 days in vitro culture. * p<0.05.

Figure 8.

siGFP-mediated silencing of GFP expression within GFP-hMSC aggregates. Tissue sections of aggregates show GFP signal (green) of GFP-hMSC aggregates incorporated with cells only, and empty-, siNC- and siGFP-MS6 at different culture times. Scale bar indicates 100 μm.