The Construction Of Multi-enzyme Cascade Nanoreactor For Antibacterial Application

Due to unique nano-confined environment,nanoreactors can not only protect the enzymes in the inner space by separating them from the external environment,but also make different enzymes close to each other to achieve multi-enzyme cascade reaction for the enhanced synergistic effect and catalytic efficiency by shortening the substrate transportation distance.However,how to further improve the target recognition ability of the nanoreactors for selective enrichment and to adjust the permeability of its membrane based on the environment changes for mimicking the semi-permeability of biofilm that allows the substrates and products of the multi-enzyme cascade reaction freely exchanged inside/outside and simultaneously limits the escape of enzyme molecules,are still challenges in the design and preparation of nanoreactors.In addition,rational use of the multi-enzyme system to convert the substrates in the lesion site into toxic products in situ for killing the pathogenic microorganisms or the diseased cells is also crucial for promoting the biomedical applications of nanoreactors.In this thesis,a targeted,dual-responsive nanoreactor was constructed using supermolecular self-assembly strategy.This nanoreactor can produce highly toxic reactive oxygen species via a chain reaction of the encapsulated natural enzyme and nanoenzyme to realize a selective and efficient killing effect on Gram-positive/negative bacteria after the change of membrane permeability by environmental stimuli.The detailed content is as follows:1.Construction of a polyfunctional nanoreactor based on supra-amphiphilic assembly.A maleimide-modified viologen and azobenzene-linked helical quinoline oligomer were synthesized.CB[8]is able to form a 1:1:1 ternary complex with these two molecules to construct a supra-amphiphile for self-assembly into a novel vesicular nanoreactor.This nanoreactor shows dual responsiveness to p H/light stimuli and can control the switch of membrane permeability through structural rearrangement.When p H is acidic,the volume of the nanoreactor increases significantly and its outer membrane changes from non-permeable to semi-permeable for selective substrate transmembrane transport capacity;Under UV light irradiation condition,the nanoreactor undergoes the disassembly process and the formation of irregular aggregates leads to its outer membrane changed from semi-permeable to completely permeable,where all contents including enzymes can be released.Molecular encapsulation experiments indicate that GOx can be stabilized in the inner space of nanoreactor for several days and shows higher activity than that of GOx without protection,revealing the excellent protection ability of nanoreactor.2.Research on targeted antibacterial function of multi-enzyme cascade nanoreactor.Based on the click reaction between the thiol group of DAPT and the surface-coated maleimide group,the nanoreactor with the target recognition ability has been fabricated.After the regulation of the membrane permeability by UV light and p H,the cascade reaction catalyzed by the encapsulated GOx and Fe₃O₄ nanozyme was triggered to convert the glucose into highly toxic·OH,which shows a selective killing effect on Gram-positive/negative bacteria.The corresponding minimum inhibitory concentration for both bacteria were also determined,respectively.The antibacterial kinetic results confirm that the nanoreactor with a single enzyme has no significant antibacterial effect,while the nanoreactor with two types of enzymes（GOx and Fe₃O₄）exhibits highly efficient antibacterial ability.Antibacterial mechanism research indicates that the nanoreactor with multi-enzyme system can catalyze the production of a large amount of hydroxyl radicals（·OH）to attack the bacterial membrane.This will interfere with the potential of bacterial membrane and disrupt the permeability of bacterial membrane,leading to the reduction of the ATP synthesis ability and the outflow of cytoplasmic substances,and thus achieving a good antibacterial efficacy.