基于葡聚糖修饰的铁蛋白-白藜芦醇纳米颗粒的构建及其缓释特性研究

As a kind of plant polyphenol, resveratrol has a wide range of physiological effects. However, its bioavailability is not high due to its poor water solubility and stability. At present, the biological utilization rate of resveratrol can be improved by constructing nano carriers, but most of these methods have some disadvantages, such as poor stability, the weak ability of nanoparticles core material to be protected, the high cost of preparation process and the organic solvent residue. As a kind of naturally existing cage like protein with nano cavity, ferritin has a wide application prospect in the field of nano encapsulation. Our group has been successfully prepared ferritin nanoparticles embedding calcium, anthocyanins, curcumin and so on. However, when natural ferritin is used as a carrier alone, it is vulnerable to environmental factors. The combination of polysaccharide and protein can modify the functional characteristics of protein. However, reports on the grafting on ferritin using polysaccharides by Maillard reaction have not been reported yet. So based on the above results, the study will construct the ferritin-resveratrol nanoparticle at first. Then through grafting dextran to modify ferritin, and after that, the structure, stability and the interaction mechanism of different nanoparticals will be discussed. Finally, the slow release characteristics of the nanoparticals will be further evaluated by simulated gastrointestinal fluid, Caco-2 cell absorption and intestinal perfusion model in vitro. The research results will construct a new approach through grafting dextran on ferritin to embedding hydrophobic substances, and lay the foundation for the practical application of ferritin in the field of nanotechnology.

白藜芦醇作为非黄酮类多酚化合物，具有广泛的生理学效应，但因其水溶性与稳定性差，其生物利用率不高。目前国内外对于白藜芦醇的纳米包埋技术主要着眼于白藜芦醇微粉、纳米脂质载体等，但现有方法大多存在纳米粒子稳定性差、芯材保护弱、制备工艺成本高、有机溶剂残留等缺点。铁蛋白作为一种天然存在的具有纳米空腔的笼状蛋白，在纳米包埋领域具有广泛的应用前景。申请者博士期间所在课题组已成功制备出铁蛋白含钙、花青素、姜黄素等纳米颗粒，但是天然铁蛋白单独用作载体时，易受环境因素等影响。多糖与蛋白质复配可对蛋白质进行改性，然而，目前铁蛋白与多糖通过美拉德反应进行接枝而改变其特性的报道还不多见。因此本研究拟通过制备铁蛋白-白藜芦醇纳米颗粒，再以葡聚糖对其进行共价修饰，并进一步通过体外实验评价纳米粒子的缓释及吸收特性。研究结果将构建一种新的以铁蛋白为基础的包埋疏水性物质的方法，为铁蛋白用于纳米技术领域提供一定的理论依据。

白藜芦醇具有广泛的生理学效应，因其水溶性与稳定性差，其生物利用率不高。铁蛋白作为天然存在的具有纳米空腔的笼状蛋白，在纳米包埋领域具有广泛的应用前景。天然铁蛋白单独用作载体时，易受环境因素等影响，而多糖与蛋白质复配可对蛋白质改性。基于此，本研究首先制备得到铁蛋白-白藜芦醇纳米颗粒，分析可知每个铁蛋白分子可以包埋21.8个白藜芦醇分子，透射电镜显示白藜芦醇存在于铁蛋白纳米空腔中，且其不影响铁蛋白的流体动力学半径（6.75 nm）；铁蛋白-白藜芦醇纳米粒子具有较好的光、热等稳定性；紫外-可见光谱、傅里叶转换红外光谱均显示铁蛋白-白藜芦醇纳米粒子特征峰形发生了改变，圆二色谱显示其二级结构亦发生变化，提示二者发生了相互作用。之后以葡聚糖对其进行共价修饰，结果表明铁蛋白通过干热法接枝葡聚糖的最佳条件为：葡聚糖40 kD，铁蛋白、葡聚糖摩尔比 1:2，pH 7.8，相对湿度40%，温度50℃，时间30 h；SDS-PAGE及扫描电镜均显示葡聚糖的接枝使得铁蛋白结构发生了改变；相较于单独铁蛋白，葡聚糖-铁蛋白接枝物随pH的变化较为稳定；紫外-可见光谱及傅里叶转换红外光谱显示，葡聚糖接枝后铁蛋白特征峰形发生了变化；圆二色谱显示，铁蛋白经葡聚糖接枝后，α-螺旋及无规则卷曲比例降低，而β-折叠和β-转角比例增加。最后，制备得到了葡聚糖-铁蛋白-白藜芦醇纳米粒子，透射电镜显示白藜芦醇存在于铁蛋白纳米空腔中，扫描电镜显示葡聚糖接枝后铁蛋白表面结构发生了改变；葡聚糖-铁蛋白-白藜芦醇纳米粒子同样具有较好的光、热等稳定性；紫外-可见光谱及傅里叶红外光谱显示，葡聚糖接枝后铁蛋白-白藜芦醇纳米粒子峰形发生了变化；圆二色谱显示其二级结构同样发生改变。对白藜芦醇及其他两种纳米粒子的释放、吸收特性进行研究，结果显示葡聚糖修饰后的铁蛋白-白藜芦醇纳米粒子具有较好的缓释特性。本项目构建了一种新的以铁蛋白为基础的包埋疏水性物质的方法，结果可为铁蛋白用于纳米技术领域提供一定的理论依据。