抗氧化纳米粒子靶向抑制果实采后线粒体结构和功能丧失的规律和机制研究

Excessive accumulation of ROS results in structural and functional losses of fruits mitochondria, which account for the major causes of fruits senescence and quality deterioration during postharvest stages. However, exogenous anti-oxidants treatments cannot efficiency scavenging fruit’s endogenous ROS, probably because these exogenous anti-oxidants are prevented from transporting into cell by its sealed membrane structure. Moreover, the anti-oxidants might be degraded by the metabolic enzymes while transporting from extracellular matrix to intracellular target (mitochondria). Thus, we hypothesize that improving antioxidants transmembrane transportation efficiency and enhancing its protection from enzyme degradation while transporting will lead to an enhancement of its intracellular ROS scavenging activity, and that will be have a better anti-senescence and preventing quality deterioration effect of fruits. In this study, several anti-oxidants will be chemical conjugated with triphenylphosphine, given them as positive charged antioxidants to confer a mitochondria targeting property. The anti-oxidative nanoparticle will be prepared using charge driven self-assembly strategy by mixing positive charged anti-oxidants with negative charged polymers in aqueous buffer. These antioxidants encapsulated within nanoparticles are cellular uptake via endocytosis, which dramatically enhanced of theirs transmembrane transportation efficiency, and confer a mitochondria targeting antioxidants delivery. The interaction behaviors between the plant cell and these nanoparticles will be furtherly insight studied. Within this project, we adopt mitochondria protection strategy installed of traditional “whole fruit” protection by using anti-oxidative nanoparticle treatment rather than free antioxidant treatment, which have important theory and practice contributions in nano-biotechnology for fruit preservation application. And will benefit for food industry by increasing economic effectiveness and enhancing food safety.

活性氧（ROS）失衡诱导线粒体结构和功能丧失是果实采后衰老和品质劣变的关键因素。外源抗氧化物无法有效清除果实内源性ROS，因此难以有效延缓果实采后衰老和品质劣变的发生。我们认为细胞封闭的膜结构和胞内多样化的代谢酶是限制外源抗氧化物胞内生物功能的主要生物学屏障。如能将外源抗氧化物高效靶向递送至线粒体，增加外源抗氧化物胞内抗氧化效果，可从源头上抑制ROS失衡以及线粒体结构、功能丧失，延缓果实衰老及品质劣变。基于上述假设，我们拟对抗氧化物进行三苯基膦化修饰，使其带上正电，并与带负电的高分子材料自组装，形成抗氧化纳米粒子；而纳米粒子通过內吞作用进入细胞，增加抗氧化物跨膜转运效率，并加强其转运过程中的保护，促进其在线粒体内聚集，达到清除ROS目标。本项目用果实“亚细胞结构保护”替代传统的“整果保护”策略，为纳米生物技术用于果实保鲜提供理论和实践参考。

果实采后衰老和品质劣变导致极大的果实损耗及资源浪费。活性氧自由基失衡诱发的线粒体结构和功能损伤在果实采后衰老和品质劣变中扮演着关键角色。自由基爆发引发的微生物感染及采后果实失水萎蔫也会缩短采后果实货架期。因此，通过施加“抗氧化剂、抗菌剂、保湿剂”处理，有望成为延长采后果实货架期的重要策略。本研究利用三苯基膦（TPP）修饰合成新的线粒体靶向谷胱甘肽（Mito-GSH），；并利用具有抗微生物作用材料壳寡糖（COS）为基础材料，制备负载Mito-GSH纳米粒，并通过与果皮进行自组织，形成抗氧化和抗菌涂层；进一步通过与透明质酸（HA）自组装，形成保湿涂层。该涂层高效清除细胞内源性ROS，使其表皮达到较好的抗氧化、抗菌、保湿效果来抑制果实采后衰老和品质劣变。本课题制备的第一层保鲜涂层材料为PEG-PGA/COS纳米溶液，第二层为HA溶液。DLS测定发现随着投料比的增加，PEG-PGA/COS纳米粒Zeta电位逐渐降低，Size逐渐增加。扫描电镜结果显示在仅组装纳米材料时，香蕉果皮表面可见一层致密的附着物，且随着纳米粒投料比的增加，果皮表面附着物也明显增多。然而，继续组装HA溶液后，果皮表面变光滑。在接触角测试中发现香蕉果皮本身接触角在70°左右，涂抹纳米溶液后接触角变成20°左右，亲水性明显增强。为更清楚的了解涂层材料分布情况，本研究又进行了荧光分析。其中，激光共聚焦结果显示纳米粒进入果皮细胞，HA黏附在果皮表面。细胞实验表明，Mito-GSH NPs可显著降低BY-2细胞线粒体内ROS水平，更有利于维持线粒体膜电位的能力，保持线粒体正常生物学功能。香蕉外观光学图片显示Mito-GSH NPs和HA均可达到较好的保鲜效果，与MDA结果保持一致，且二者联合使用效果更佳。以上研究结果显示Mito-GSH NPs能够较好地清除细胞线粒体内ROS，并可保护线粒体内膜，从而使果实细胞免受氧化损伤；HA在果皮表面形成致密保护层，防止果实水分丢失，达到保湿效果。两种涂层材料通过电荷驱动自组装，最终达到较好的保鲜效果，为未来水果保鲜发展提供了一种新思路。