Abstract
BackgroundTraumatic spinal cord injury (SCI) includes the primary insult as well as a sequela of biochemical and cellular cascades that amplifies the initial injury. This degenerative process, known as secondary injury, is often mediated by both reactive oxygen and nitrogen species released from damaged cells. Previous data suggests that dysregulated production of nitric oxide via inducible nitric oxide synthase (iNOS) is detrimental to spinal cord recovery. M1 macrophages have been implicated to overexpress iNOS post-SCI. In this work, we propose to inhibit iNOS expression through small interfering RNA (siRNA) complexed chitosan nanoparticles (NPs) that primarily target M1 macrophages.MethodssiRNA conjugated chitosan complexes were fabricated with and without an antibody (Ab) targeting moiety and screened for efficiency to reduce iNOS expression in vitro. Best formulations were subsequently applied in vivo following acute SCI in a rodent model. iNOS expression as well as Bax and Bcl-2 biomarkers were used to assess cell apoptosis within the lesion at 24 h post-injury.ResultsAb-siRNA conjugated chitosan NPs significantly reduced iNOS expression in vitro in M1 polarized macrophages. Results show high transfection efficiency with low cytotoxicity. Subsequent application of NPs in vivo after SCI demonstrated both a reduction in iNOS expression and cellular apoptosis.ConclusionProof of concept indicates that siRNA conjugated chitosan NPs can downregulate iNOS production and inhibit apoptosis following SCI. Our proposed gene silencing method putatively targets M1 macrophages as a means to attenuate secondary injury.
Highlights
Traumatic spinal cord injury (SCI) includes the primary insult as well as a sequela of biochemical and cellular cascades that amplifies the initial injury
Chitosan NPs The selection and volume of chitosan used during nanoparticle synthesis can lead to a large structural and functional versatility of the NPs
To optimize small interfering RNA (siRNA) conjugated chitosan NPs, the molecular weight (MW), degree of deacetylation (DD), pH, and N/P ratio as important criteria were considered in the design of NPs formation
Summary
Traumatic spinal cord injury (SCI) includes the primary insult as well as a sequela of biochemical and cellular cascades that amplifies the initial injury. This degenerative process, known as secondary injury, is often mediated by both reactive oxygen and nitrogen species released from damaged cells. Following spinal cord injury (SCI), a number of cellular and biochemical events are initiated that exacerbate the primary insult This secondary injury is often associated with dysregulated production of reactive oxygen and nitrogen species (ROS/RNS). These highly unstable compounds overwhelm the endogenous antioxidant system and perpetuate a vicious cycle of cellular bystander damage [1]. Evidence shows iNOS deficient mice demonstrate improved functional recovery following spinal cord damage [11] while the application of iNOS antisense oligonucleotides have increased the number surviving neurons after
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