Cell-free 3D scaffold with two-stage delivery of miRNA-26a to regenerate critical-sized bone defects

Abstract

MicroRNAs (miRNAs) are being developed to enhance tissue regeneration. Here we show that a hyperbranched polymer with high miRNA-binding affinity and negligible cytotoxicity can self-assemble into nano-sized polyplexes with a ‘double-shell’ miRNA distribution and high transfection efficiency. These polyplexes are encapsulated in biodegradable microspheres to enable controllable two-stage (polyplexes and miRNA) delivery. The microspheres are attached to cell-free nanofibrous polymer scaffolds that spatially control the release of miR-26a. This technology is used to regenerate critical-sized bone defects in osteoporotic mice by targeting Gsk-3β to activate the osteoblastic activity of endogenous stem cells, thus addressing a critical challenge in regenerative medicine of achieving cell-free scaffold-based miRNA therapy for tissue engineering.

Scaffold‐Based microRNA Therapies in Regenerative Medicine and Cancer

“…[59] However, this is counterbalanced by their reported cytotoxic and proinflammatory effects, some of which have been associated with their ability to trigger the generation of intracellular vacuoles. [60] Polyethyleneimine (PEI) derivatives have also been extensively studied as miRNA delivery systems, [19,61,62] and specifically PEI-poly(ethylene glycol) (PEG) nanoparticles (NP) have recently demonstrated interesting profiles when encapsulated within poly(lactic-co-glycolic acid) (PLGA) microspheres in a poly(l-lactic acid) (PLLA) scaffold [63] or as part of a calcium chloride cross-linked alginate hydrogel system. [64] PEI is watersoluble and easily attracts negatively charged ions to aid the polyplex cargo escape the endolysosomal compartment in what is known as "the proton sponge effect."…”

“…[102,106] Whether one of the routes may be better suited for clinical translation remains to be elucidated, although in situ transfecting scaffolds hold greater potential to exist as "off-the-shelf" products. [107] Many distinct scaffold types have been tuned to serve the purpose of miRNA delivery, including several hydrogels, [58,92,106,[108][109][110][111][112][113][114] electrospun fibers, [63,[115][116][117][118] and more prolifically porous or spongy scaffolds. [46,47,50,54,55,96,112,[119][120][121][122][123][124][125][126][127][128][129][130] Before reviewing the details of their applications, summarized in Table 2 and described further in the next section, we will discuss the key characteristics making these materials amenable to miRNA delivery.…”

“…Electrospinning is a distinct fabrication method that produces nanofibrous scaffolds typically using material sources such as gelatin, [115] synthetic PLLA, [63] PCL, [92,117] or PEG-PCL copolymers. [116] Interestingly, most of the nonviral, nonlipid vectors tested for 3D-miRNA delivery have utilized biomaterials fabricated using this method: Qureshi's photocleavable silver NPs, [92] the commercial Transit-TKO transfection reagent, [115,117,139] an innovative peptic sequence REDV-trimethyl chitosan-PEG complex, [116] and hyperbranched (HP) PEI-PEG polyplexes.…”

“…[116] Interestingly, most of the nonviral, nonlipid vectors tested for 3D-miRNA delivery have utilized biomaterials fabricated using this method: Qureshi's photocleavable silver NPs, [92] the commercial Transit-TKO transfection reagent, [115,117,139] an innovative peptic sequence REDV-trimethyl chitosan-PEG complex, [116] and hyperbranched (HP) PEI-PEG polyplexes. [63] Moreover, with the exception of the Ag-NPs, all the remaining systems have been developed as in situ "cell-free" miRNA transfecting platforms, underscoring great clinical potential and versatility of this fabrication method. Finally, spongy porous scaffolds account for the largest evidence in miRNA delivery: 50% of these are ceramic-based or ceramic-composite materials, the other half is represented by a mix of compositions.…”

“…[58,63,146] Li et al [58] documented the first report in 2013 using BM-MSCs, HyStem-HP hydrogel, and miR-26a mimic. They demonstrated almost full repair of the defect 12 weeks postimplantation and significantly enhanced vascularization confirming that miR-26a had the capacity to regulate endogenous angiogenic-osteogenic coupling for enhanced bone regeneration.…”