基于仿生纳米凝胶囊的酶纳米反应器的构建及评价

Enzyme nanoreactors are a class of nanosized enzyme encapsulation systems with cell-like structures and enzyme-catalyzed reaction functions. The systems can be constructed using liposomes and polyersomes,but the former have poor stability,easily leading to enzyme leakage, while there is a thicker hydrophobic layer formed by hydrophobic long chains in the bilayer membranes of the latter, which is unfavorable for the permeation of hydrophilic small-molecules (e.g. enzyme substrates ). It requires to insert channel proteins into their membranes (or shells). In this project, a novel enzyme nanoreator will be constructed based on shell cross-linked nanogel capsules because of their high stability and the semi-permeablity of hydrophilic cross-linking networks, and its enzyme-catalyzed reaction function will be evaluated on biology using urease as a model enzyme. First，novel nano-sized pro-vesicles with bilayer membranes are constructed by self-assembly in aqueous solution using a photo-cross-linkable amphiphilic biomacromolecule derivative,namely carboxymethyl chitosan with 4-azidobenzylidene groups linked with the amino groups of its main chain, instead of amphiphilic block copolymer.When the pro-vesicle solution is exposed to UV light to induce in situ cross-linking of the hydrophobic 4-azidobenzylidene groups in their shells, biomimetic nanocapsules can be obtained, which have stabler structure and good hydrophilicity or semipermeability of shell. The design is particularly suitable for the in situ encapsulation of enzymes, that is, by adding an enzyme into aqueous solution before self-assembly, enzyme-encapsulated biomimetic nanogel capsules (enzyme nanoreactor) can be obtained. This method is simple, its preparation conditions are mild, without adding any cross-linking agent or other substances. Therefore, this study may provide technical support and theoretical basis for the construction of novel nanocapsules and the development of enzyme nanoreactors.

酶纳米反应器是一类具有仿生细胞结构及酶催化功能的纳米级酶封装系统。基于脂质体和聚质体构建的酶纳米反应器，前者缺乏稳定性，易发生酶的泄漏；后者因其双层膜中疏水链段形成的疏水层较厚，不利于亲水性小分子（如酶的底物）的渗透，需要在膜（或壳）中插入通道蛋白。本项目基于壳交联纳米凝胶囊的高稳定性和亲水交联网络的良好半透性构建一种新型酶纳米反应器，并以脲酶为模型对其催化功能展开生物学评价。首先采用可光交联的两亲性生物大分子衍生物，即主链氨基上带有叠氮苯亚甲基的羧甲基壳聚糖替代双亲性嵌段共聚物，在水溶液中组装成具有双层膜的纳米前囊；然后，经紫外光引发壳中叠氮基交联锁定构造，获得结构更稳定、壳半透性良好的仿生纳米凝胶囊；若在自组装前的水溶液中加入活性酶，就可得到封装酶的仿生纳米凝胶囊即酶纳米反应器。该方法简易，制备条件温和，无需加入交联剂或其他物质，能为新型纳米囊和酶纳米反应器的发展提供理论及技术支撑。

酶纳米反应器(enzmye nanoreactors)是一类模仿细胞内酶代谢功能的纳米级酶包囊系统。它不仅可用于体内某种酶缺失病人的替补治疗，而且还可用于体内某些靶物质的催化代谢，延长病人的生存期。基于脂质体和聚质体构建的酶纳米反应器，前者稳定性差、易发生酶的泄漏，后者不利于酶底物的穿透、需要在膜（或壳）中插入通道蛋白。本项目以此为出发点设计构建了高稳定性和良好半透性的新型纳米凝胶囊：首先采用羧甲基壳聚糖制备出了可光交联的两亲性生物大分子，并随后在水溶液中自组装形成了纳米囊，紧接着采用紫外光辐射快速壳交联锁定构造、获得了高稳定的纳米凝胶囊。最后，我们采用投射电镜及动态光散射等手段或方法表征了纳米凝胶囊的形态、大小及稳定性。. 精氨酸剥夺疗法是通过精氨酸代谢酶降解或剥夺体循环中的精氨酸，切断恶性肿瘤细胞生长和增殖所必需的氨基酸或营养，实现靶向治疗某些肿瘤的一种新方法。精氨酸脱亚胺酶（ADI）对精氨酸的降解或剥夺具有高活性，但该酶的体内免疫原性高、循环半衰期短，限制了其应用。本项目以光交联纳米凝胶囊的制备方法为基础，通过在囊组装前的水溶液中加入ADI制备出了ADI酶纳米反应器，并对其进行了稳定性、催化活性及体外生物学效应等评价。研究结果表明，该纳米反应器不仅在体外血浆中显示出高稳定性和高活性，而且能显著抑制小鼠肿瘤肝癌细胞（MH134）的生长。该研究为发展新型高效酶纳米反应器及其应用提供了新思路和尝试。