基于纳米生物材料的miRNA两阶段释放系统抑制椎间盘纤维化

Degeneration of the intervertebral disc (IVD) is clinically related to chronic low back pain, while TGF-β signaling pathway associated fibrosis plays a central role in degeneration of the IVD. MicroRNAs (miRNAs) are being developed to enhance tissue regeneration. However, miRNA and their mimics, which are negatively charged, cannot easily cross the cell membrane, which is also negatively charged. In addition, ‘naked’ miRNAs are highly susceptible to degradation in serum. Given the side effects caused by burst release or the off-target effect, safe and efficient delivery system is highly desired. In this work, a hyperbranched polymer (HP) with high miRNA 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 miRNA into nucleus pulposus (NP) cells. These polyplexes are then encapsulated in biodegradable nanoparticles to enable two-stage delivery: 1) temporally-controlled release of miRNA-carrying polyplexes and 2) highly efficient delivery of miRNA into cells by the released polyplexes. These biodegradable nanoparticles are attached to nanofibrous spongy microspheres (NF-SMS) to spatially control the release of miRNA. This technology is used to reverse fibrotic degeneration and support IVD regeneration by targeting TGF-β signaling pathway, thus addressing a critical challenge in regenerative medicine of achieving cell-free biomaterials-based miRNA therapy for tissue engineering.

TGF-β信号通路调控的病理性纤维化是导致椎间盘退变进而引起下腰痛等临床症状的重要原因之一。微小RNA(miRNA) 作为基因治疗新的靶点，将其应用到再生领域调控疾病信号通路正成为新的研究热点。miRNA难以通过细胞膜，在体内稳定性差，容易被各种生理屏障清除降解；短时间内的暴释或者非靶向释放带来的脱靶效应可能造成全身/局部毒副作用。组装具有高效低毒性的生物材料控释miRNA并实现细胞内递送，是应用miRNA再生椎间盘亟待解决的问题。我们构筑新型双壳层超支化聚酯/纳米颗粒/多孔微球体系，能形成独特双壳层三维环状结构保护miRNA，通过两阶段释放控制miRNA的作用时间，同时通过具有纳米纤维结构的多孔微球控制miRNA空间分布。基于前期的工作基础，本项目将进一步探索生物材料控释miRNA，调节椎间盘内TGF-β信号通路活性，抑制纤维化进程，为椎间盘退变治疗策略提供思路。

MiRNA参与椎间盘退变的众多病理生理过程，已成为基因治疗新靶点。但单纯miRNA难以穿过细胞膜、易降解以及爆释等特点使其难以达到治疗效果。因此，本项目合成和表征了作为miRNA载体的超支化聚酯PEG-H40-PEI，模拟病毒载体高效基因传输的机理,实现对miRNA 的高度有序压缩，形成独特双壳层三维环状结构保护miRNA，再分布载入PLGA纳米颗粒及纳米纤维多孔微球形成两阶段释放体系递送miRNA，维持miRNA的稳定性及达到缓释miRNA延长治疗时间的目的。在体外、体内及动物模型上验证该体系递送miRNA的有效性和准确性。通过Western Blot，实时定量 PCR 以及免疫共沉淀实验解析TGF-β信号通路，证明通过miRNA抑制椎间盘纤维化的分子机理。最后，通过兔腰椎间盘退变模型，验证超支化聚酯/多孔微球递送miRNA治疗椎间盘退变的可行性，取得了较好的前期治疗效果。本项目为miRNA提供有效的控释载体，并阐述相关具体分子机制，为其应用于椎间盘退变治疗奠定基础，为椎间盘退变的生物治疗策略提供新的思路。