| Theα-linolenic acid（ALA）,a polyunsaturated fatty acid（PUFA）essential for the body,plays a vital important role in human health and nutritional fortification.However,since ALA has three unsaturated double bonds,it is prone to be oxidized,greatly restricting its applications.Water-in-oil-in-water（W1/O/W2）microemulsion has a hydrophilic and hydrophobic multi-compartment structure.Due to this distinct structure,it has the unique advantage in embedding and delivering unstable active nutrients or drugs.In this study,the“two-step-heterotherm method”was used to prepare the W1/O/W2microemulsion.The water-in-oil（W/O）microemulsion was constructed at45°C using pseudoternary phase diagrams,and then cooled to room temperature.The W/O microemulsion was dispersed into the external water phase again to prepare W1/O/W2microemulsion.For ALA-loaded W1/O/W2（ALA-W1/O/W2）microemulsion,nuclear magnetic resonance（NMR）confirmed that during the encapsulation process of ALA,aggregation and diffusion effects could occur at the water-oil interface simultaneously.Compared with ALA-loaded O/W（ALA-O/W）microemulsion,ALA-W1/O/W2microemulsion increased its fluorescence intensity by7.75-20.15%.At 8 h,ALA-W1/O/W2microemulsion slowed down the release rate of ALA by 60.46-67.57%.The effects of multi-scale molecules on physical properties,antioxidant capacity and in vitro release kinetics of ALA-W1/O/W2microemulsion were revealed.The glucose（Glu）and carboxymethyl cellulose sodium（CMC）solutions were selected as the internal aqueous phase（W1）of the microemulsion,respectively,while the multi-scale molecules,such as sodium chloride（Na Cl）,glycine（Gly）,Glu,CMC,bovine serum albumin（BSA）and sodium caseinate（SC）,were added in the external aqueous phase（W2）to balance the osmotic pressure.Among these,the CMCW1（0.1%（w/w））-Na Cl W2（0.2 M）and Glu W1（0.2 M）-CMCW2（0.2%（w/w））microemulsion increased the antioxidant capacity of ALA by 5.57%and 4.20%,respectively.The release kinetic data were fitted and the Korsmeyer-Peppas equation was considered as the most suitable to explain the ALA release kinetics controlled by diffusion,erosion in the simulated gastric fluid（SGF,pH=2.0）and simulated intestinal fluid（SIF,p H=6.8）.The effects of ALA-O/W and ALA-W1/O/W2microemulsion on cell viability,lactic dehydrogenase（LDH）viability and reactive oxygen species（ROS）levels were examined using cell counting kit-8（CCK-8）,LDH assay kit and fluorescence microscope,respectively.The CCK-8 assay demonstrated that ALA inhibited MDA-MB-231 human breast cancer cells proliferation in a dose-dependent manner.Further,the results of LDH activity and ROS level revealed that ALA-induced cancer cell damage was closely related to oxidative stress.Under the irradiation of ultraviolet light,ALA-O/W and ALA-W1/O/W2microemulsion co-cultured with cancer cells exhibited long-term photostability,so that the release of ALA could be monitored in real time.Based on the self-assembled structure of the microemulsion,the fluorescence mechanism was explained by immobilizing surfactant molecules with aggregation-induced emission（AIE）properties at the water-oil interface. |
