柔性多价装订肽构建的自组装寡核苷酸递送系统用于治疗老年黄斑变性

Age-related macular degeneration (AMD) is a kind of ocular fundus disease that has high incidence and severe impairment in visual function, and is clinically treated by oligonucleotide via intravitreal injection. Our previous study has validated that penetratin, a cell-penetrating peptide (CPP) derived from natural protein, had an outstanding capability of enhancing ocular absorption, which could rapidly distribute in the retina after topical administration. On this basis, the present project intends to improve the cellular membrane permeability of penetratin using a stapled peptide technology by increasing the hydrophobicity of peptide chain and the rigidity of spatial structure. Then, the stapled penetratin will be covalently conjugated at the ends of multi-arms polyethylene glycol to construct a flexible multivalent stapled penetratin (FMSP). FMSP has an octopus-like structure, and its flexible arms stretch out before being loaded with drugs. When mixed with oligonucleotides, several stapled penetratins at the ends of arms from one FMSP will tightly encapsulate these oligonucleotides via electrostatic interaction, and form a self-assembled noncovalent nanocomplex. The other unbound stapled penetratins at the ends of arms from the same FMSP will independently function to penetrate cellular membrane, therefore, the nanocomplex will be wholly endocytosed by cells. The FMSP is able to simultaneously improve the capabilities of CPP to carry and deliver oligonucleotides, and furthermore facilitate the oligonucleotides to escape from endosome after cellular uptake. When instilled in the conjunctival sac, the FMSP has a potential to deliver the oligonucleotides to the retina, which will provide a novel and practical, as well as non-invasive method for the treatment of ocular fundus diseases, including AMD. In addition, this project will attempt to clarify the way for the FMSP to transport from the ocular surface to fundus, which will be helpful in building a theoretical foundation for the research on drug delivery to the posterior segment of the eye.

老年黄斑变性（AMD）是一种发病率高且严重威胁视功能的眼底病变，临床上采取玻璃体内注射寡核苷酸治疗。本项目拟采用装订肽技术对天然来源的穿膜肽penetratin进行结构改造，通过增强肽链的疏水性与空间结构的刚性进一步提升其穿膜能力，并将其修饰于多臂聚乙二醇末端，构建柔性多价装订penetratin（FMSP）。FMSP具有章鱼样结构，其柔性手臂载药前处于伸展状态；当与寡核苷酸混合后，FMSP的部分手臂借助静电相互作用将寡核苷酸紧密包裹，自组装形成纳米复合物，其余游离手臂末端的装订penetratin独立地发挥穿膜功能，促进复合物整体被细胞摄取。FMSP可同时改善穿膜肽携载和递送寡核苷酸的能力，并且借助质子海绵效应帮助其从内涵体逃逸。眼表滴入FMSP将寡核苷酸送达视网膜，可为AMD提供一种新颖实用且非损伤性的药物治疗方法。本项目将探讨FMSP的体内吸收途径，为眼后段递药研究奠定理论基础。

眼底疾病是导致视力严重受损甚至失明的主要原因，极大地影响患者生活质量。基因治疗眼底疾病前景广阔，但基因药物分子量大、亲水性强，在眼部几乎不吸收。为实现眼底疾病的无创基因治疗，本课题主要开展了两方面工作：基于前期发现的促进眼部吸收的野生型穿膜肽，对其进行结构优化，以获得更高效的多肽类眼部吸收促进剂；设计一种柔性多价穿膜肽类基因载体，实现基因药物安全、高效的眼内递送，探讨相关吸收途径，并考察其对眼内疾病模型的治疗效果。.保持野生型penetratin序列中碱性氨基酸不变，对其部分氨基酸进行突变得到一系列衍生肽，采用圆二色谱及计算机模拟技术，证明疏水性突变利于增加多肽空间结构中α-螺旋比例；通过细胞摄取、离体组织透过、眼内分布与消除等实验证明疏水性突变能够有效增强多肽的眼部渗透能力；细胞及组织水平的评价结果表明，衍生肽眼部应用安全性良好。.以多臂聚乙二醇为核心，末端修饰penetratin得到章鱼状柔性8价penetratin（8VP），以市售基因转染载体阳离子脂质体为对照，分别以反义寡核苷酸（ASO）、小干扰RNA（siRNA）为模型药物构建基因复合物。体外表征结果表明，8VP对基因压缩能力更强，体外稳定性更好；细胞实验结果表明，8VP介导ASO或siRNA细胞摄取或转染效果不低于市售载体，同时安全性更佳，对血-视网膜屏障模型透过能力优于市售载体；建立了荷眼内肿瘤小鼠模型，经滴眼给药，8VP基因复合物可以有效抑制眼内肿瘤蛋白表达，长期应用未见眼组织损伤，显著优于市售载体。所构建的基因复合物经滴眼给药后主要是通过非角膜途径吸收至眼底。.除此之外，本项目还采用野生型penetratin及优化后的衍生肽分别构建了携载质粒DNA、ASO或siRNA的基因复合物，并利用报告基因表达、小梁网切除术后瘢痕形成、眼内原位瘤等动物模型验证了上述基因递送系统通过滴眼给药治疗眼后段和眼底疾病的有效性。相关工作发表SCI论文7篇（1篇IF>10，5篇IF>5）；授权专利1项，申请专利6项，向企业授权使用1项；2项成果被国内外媒体广泛报道，超额完成预期任务。

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