Injectable nanofibrous spongy microspheres for NR4A1 plasmid DNA transfection to reverse fibrotic degeneration and support disc regeneration

Abstract

Safe and efficient gene therapy is highly desired for controlling pathogenic fibrosis of nucleus pulposus (NP) tissue, which would result in intervertebral disc (IVD) degeneration and disability if left untreated. In this work, a hyperbranched polymer (HP) with high plasmid DNA (pDNA) binding affinity and negligible cytotoxicity is synthesized, which can self-assemble into nano-sized polyplexes with a “double shell” structure that can highly efficiently transfect pDNA into NP cells. These polyplexes are then encapsulated in biodegradable nanospheres (NS) to enable two-stage delivery: 1) temporally-controlled release of pDNA-carrying polyplexes and 2) highly efficient delivery of pDNA into cells by the released polyplexes. These biodegradable NS are co-injected with nanofibrous spongy microspheres (NF-SMS) to localize the cellular transfection of the pDNA encoding orphan nuclear receptor 4A1 (NR4A1), which was recently reported as a therapeutic agent to delay pathogenic fibrosis. It is shown that HP can transfect human NP cells efficiently in vitro with low cytotoxicity. The two-stage delivery system is able to present the polyplexes over a sustained time period (more than 30 days) in the tail of rat. The NR4A1 pDNA carried by the HP polyplexes is found to therapeutically reduce the pathogenic fibrosis of NP tissue in a rat-tail degeneration model. In conclusion, the combination of the two-stage NR4A1 pDNA delivery NS and NF-SMS is able to repress fibrosis and to support IVD regeneration.

Sirtuin 3-dependent mitochondrial redox homeostasis protects against AGEs-induced intervertebral disc degeneration

“…As observed, exogenous NMN administration could ameliorate the NP cell apoptosis and IVD degeneration induced by AGEs, along with the restoration of SIRT3 level. Meanwhile, we cannot neglect the limitations that involved in this animal model using local injection into rat tail discs, although it is widely used [14] , [72] . The rat IVDs experience different biomechanical characteristics with that of human.…”

Bioresorbable Microspheres with Surface‐Loaded Nanosilver and Apatite as Dual‐Functional Injectable Cell Carriers for Bone Regeneration

“…For instance, porous‐structured microspheres could be made by incorporating porogen into polymeric emulsions, followed by removal of the porogen after microsphere solidification . Ma and co‐workers developed nanofibrous microspheres using thermally induced phase separation for cartilage regeneration and knee repairing . Intending to promote regeneration efficiency, growth factors like BMP‐2 and IGF‐1 were incorporated into microspheres, if for bone regeneration, osteoactive inorganic components as calcium phosphates and bioactive glasses were usually introduced .…”

Tooth Regeneration: Insights from Tooth Development and Spatial-Temporal Control of Bioactive Drug Release

“…So far, the use of non-viral gene vectors has been explored in many tissue regeneration approaches, such as bone regeneration [112], cartilage regeneration [115], tendon repair [116], nerve regeneration [117]. For example, Feng et al combined nanofibrous spongy microspheres with biodegradable nanospheres to synthesize a two-stage delivery system for plasmid DNA encoding orphan nuclear receptor 4A1 (NR4A1), and which was proved to be effective in promoting disc regeneration [118].…”