Gao Wen, Li Jianming
Department of Basic Medical Sciences, Center for Paralysis Research, College of Veterinary Medicine, Purdue University, 408 S. University St., West Lafayette, IN, 47907, USA.
J Nanobiotechnology. 2017 May 8;15(1):38. doi: 10.1186/s12951-017-0272-7.
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. 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.
siRNA 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.
Ab-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.
Proof 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.
创伤性脊髓损伤（SCI）包括原发性损伤以及一系列生化和细胞级联反应的后遗症，这些反应会加剧初始损伤。这种退行性过程，即继发性损伤，通常由受损细胞释放的活性氧和氮物质介导。先前的数据表明，通过诱导型一氧化氮合酶（iNOS）产生的一氧化氮失调对脊髓恢复有害。M1巨噬细胞被认为在脊髓损伤后会过度表达iNOS。在这项研究中，我们提议通过与壳聚糖纳米颗粒（NPs）复合的小干扰RNA（siRNA）来抑制iNOS的表达，该纳米颗粒主要靶向M1巨噬细胞。
制备带有和不带有抗体（Ab）靶向部分的siRNA共轭壳聚糖复合物，并在体外筛选其降低iNOS表达的效率。随后将最佳制剂应用于啮齿动物急性脊髓损伤后的体内实验。在损伤后24小时，使用iNOS表达以及Bax和Bcl-2生物标志物来评估损伤部位的细胞凋亡。
Ab-siRNA共轭壳聚糖纳米颗粒在体外显著降低了M1极化巨噬细胞中的iNOS表达。结果显示转染效率高且细胞毒性低。脊髓损伤后在体内随后应用纳米颗粒表明iNOS表达和细胞凋亡均减少。
概念验证表明，siRNA共轭壳聚糖纳米颗粒可以下调脊髓损伤后iNOS的产生并抑制细胞凋亡。我们提出的基因沉默方法可能以M1巨噬细胞为靶点，作为减轻继发性损伤的一种手段。
