自组装菜籽肽纳米载体的靶向机制及生物学效价研究

Self-assembly peptide nanoparticles are nano-sized, well permeable, and biocompatible active substance delivery systems spontaneously assembled by polypeptide molecules using hydrogen bonds and other noncovalent bonds, and are capable of releasing controlled delivery of active substances to increase its bioavailability. However, it is unclear whether the peptide nanoparticles can be targeted and the biological effects and mechanism of small molecule peptide hydrolyzed by the targeted enzyme at the targeted point which is a bottleneck problem faced in the current research field of polypeptide self-assembly nanoparticles. The proposed project intends to focus on the enzyme-dependent targeting and biological activity of rapeseed peptides. Nanoparticles loaded curcumin will be constructed using self-assembly to study and characterize the topological structure of the nanoparticles. Cellular uptake of the nanoparticles will be studied to clarify the possibility of enzyme-dependence targeting. Mechanism of enzyme-hydrolyzed nanoparticles will be studied to determine the enzyme-dependent biological activity. Targeted enzyme hydrolysis of the nanoparticles produced small peptides and mechanism of their activity will be studied to explain the biological potency of rapeseed peptide nanoparticles. The research aims to provide a theoretical basis for the application of rapeseed peptide self-assembly nanoparticles.

多肽自组装纳米载体是指多肽分子利用氢键等非共价键力自发组装形成的纳米级、具有良好渗透性和生物相容性的活性物质输送系统，能够很好地实现输送物质的可控释放，提高其生物利用度。然而，肽类载体能否具有靶向性，以及到达靶标后被靶点酶水解成小分子肽而发挥的生物学效价及作用机理不清楚，是目前多肽自组装纳米载体基础研究领域面临的瓶颈问题。拟申请项目着眼于菜籽肽的酶依赖靶向性和生物活性，通过自组装构建可以负载姜黄素的纳米载体，研究载体的拓扑结构及其表征；研究载体的细胞吸收方式，明确载体的酶依赖性靶向性的可能性；研究靶点酶分解载体的机制，确定菜籽肽纳米载体的酶依赖性生物活性；研究靶点酶分解载体形成小分子肽及其活性机理，阐明菜籽肽纳米载体生物学效价。研究可为菜籽肽自组装纳米载体的应用提供理论依据。

蛋白肽自组装纳米载体具有良好渗透性和生物相容性，作为载体能够很好地实现物质输送、可控释放、提高生物利用度等目的，本项目研究了自组装菜籽蛋白肽纳米载体的靶向机制及生物学效价。研究获得了高品质菜籽蛋白质，粗蛋白含量高达88.26%；采用碱性蛋白酶等水解菜籽蛋白，通过分离纯化、鉴定，获得了具有抗炎、抗肿瘤生物活性的菜籽肽LY、RALP、GHS、DHHAPQL、SVIRPPL、GVIRPPL、NDGNQPL、THPGVAQ和VTDGEAH。研究了负载姜黄素的菜籽蛋白纳米载体的制备及吸收机制，当芯壁比为5%时，纳米载体对姜黄素包埋率和载量分别是72%和5.4%，纳米载体的渗透系数约为姜黄素的2.5倍，提高了姜黄素的利用率。研究了自组装菜籽肽纳米载体的构建、表征及吸收机制，SVIRPPL/NP具有最好的活性，其对HepG2、MKN-28、A549和MCF-7细胞IC50分别为0.57、0.81、0.97和1.10mM，制备并证明了SVIRPPL-HCPT通过网格蛋白依赖性内吞途径提高了细胞对HCPT的吸收。研究了依赖CathB的自组装菜籽肽纳米载体靶向机制，在强酸性溶酶体（pH 5.0）和弱酸性肿瘤（pH 6.5）微环境中，SVIRPPL-HCPT/NP可通过pH和CathB的双重响应实现包埋HCPT的控制释放，有利于提高HCPT的生物利用度；研究了自组装菜籽肽纳米载体的协同生物学作用机制，SVIRPPL-HCPT/NP可以通过线粒体途径并与HCPT产生的协同效应影响肿瘤细胞增殖过程，促进细胞凋亡，增强纳米载体的抗肿瘤活性。项目研究为菜籽蛋白肽纳米载体在食品领域的开发应用提供理论依据。