影响生物大分子稳定的纳米载体中营养素吸收途径及效率的生化机制研究

In order to solve the problem based on the poor reliability of the data and the challenges in analyzing the involving potential mechanisms induced by random selection of parameters of in vitro digestion models in evaluating the biological routes and efficiencies of the nutraceutical encapsulated in nano-delivery systems by biopolymers, and the poor differences of the current micellization transport and directly cellular uptake of nanocarriers evaluation methods in distinguishing bile salt micelles and nanocarriers, in this project a dynamic in vitro nutrient absorption model to achieve real-time control and effective absorption system which evaluate orientation inhibition of micellization or differential transport of bile salts micelles and nanocarriers were established. On the basis of above scheme, the relationship between the structure and digestion properties of biopolymers and structural stability of carriers, the relationship between structure and surface properties of carriers and the level of cellular uptake, and the relationship among oils type, oils digestibility, and micellization efficiency of bioactive components would be analyzed. And, the mechanisms that the compatibility between interfacial biopolymers, oils phase, and bile salts micelles and nutrients influences on the efficiency of nutrients through micellization transport would also be evaluated. Comprehensive understanding of physicochemical and biological characteristics of nano-delivery systems which would affect the competition and coexistence relationship between cellular uptake and micellization uptake would be formed. Information obtained from all the work could provide guidance to the valid building of nanodelivery systems with high bioavailability of active components encapsulated, and lay the foundation of the research on the effect of biological utilization pathways on bioavailability of nutraceuticals.

针对目前生物大分子稳定的纳米载体营养素生物利用途径与效率研究中普遍存在的体外模拟消化模型参数选择随意，胶束化转运与细胞直接摄取效率评价方法对胆盐胶束和纳米载体选择性差，以及由此带来的数据可靠性存疑和相关机制分析困难问题，本项目拟在建立动态实时调控的连续消化模型以及胶束化定向抑制与胶束、载体分速渗透相结合的营养素吸收模式评价方案的基础上，分析和确立生物大分子结构及消化特征与纳米载体结构稳定性的相关性；载体结构及表面特性与细胞直接摄取水平的相关性；油相组成与水解速率营养素胶束化效率相关性；以及载体界面大分子、油相及胆盐胶束与营养素的相容性对胶束化转运效率的影响机制。全面了解影响细胞直接摄取和胶束化转运两种生物利用途径之间竞争与共存关系的纳米载体物理化学和生物学特性，用于指导具有高生物利用率的营养素纳米载体的有效构建，并为进一步分析营养素生物利用途径对生物有效性的影响奠定基础。

针对目前生物大分子稳定的纳米载体营养素生物利用途径与效率研究中普遍存在的体外模拟消化模型参数选择随意，胶束化转运与细胞直接摄取效率评价方法对胆盐胶束和纳米载体选择性差，以及由此带来的数据可靠性存疑和相关机制分析困难问题。本项目以乳清蛋白、大豆蛋白和酪蛋白酸钠作为生物大分子乳化剂，以β-胡萝卜素作为脂溶性营养素的代表，完成了生物大分子乳化剂纳米乳液、纳米颗粒载体的构建，并对载体中β-胡萝卜素的保留率特性、释放特性及存在状态进行了研究，获得了储藏稳定性良好的纳米载体。同时，搭建了纳米载体的连续化消化模型，并与细胞穿透和摄取实验结合，确立了营养素纳米载体消化-吸收完整评价方案，分析了消化过程中各因素对纳米载体消化特性的影响。在此基础上，对可消化变形和不可消化变形纳米载体的消化吸收特性进行分析，明确了影响脂溶性营养素纳米载体细胞直接摄取和胶束化转运两种生物利用途径之间竞争与共存关系的重要因素。结果显示，对于可消化变形载体的营养素细胞摄取率仅与胶束中营养素的浓度相关，而与胶束的类型及结构无关。而胶束化效率又与和液滴的初始粒径、乳化蛋白的消化特性、油相类型及浓度密切相关。而未经消化或不可消化变形的纳米载体，可显著提高营养素的吸收效率，且颗粒粒径与细胞吸收效果呈负相关。且颗粒的主要内吞形式为网格蛋白与小窖蛋白（脂阀）介导的大胞饮，转运模式为受体介导的主动运输。最后，对比两种途径下营养素的细胞转运效率，结果显示以颗粒直接摄取的模式具备比胶束形式吸收更强的营养素转运能力，此结论可用于指导具有高生物利用率的营养素纳米载体的有效构建，并为进一步分析营养素生物利用途径对生物有效性的影响奠定基础。