The Construction Of Curcumin-loaded Lactoferrin Peptides Self-assembled Nano-micelles And Their Intervention On The Oxidative Stress-mediated Injury Of BEnd.3 Cells

Curcumin（Curcumin,Cur）is a natural polyphenol with antioxidant,anti-inflammatory,neuroprotective,and cardioprotective effects,and has been widely used as an immune enhancer and antioxidant.However,curcumin has poor water solubility and low stability,which reduces its biological activity and limits its application in functional foods and health care products.Food-derived proteins and their hydrolyzates have become a research hotspot in the design of delivery systems in the field of food and medicine due to their good biocompatibility,biodegradability and low toxicity.Lactoferrin peptides have important physiological functions,and studies have shown that more than 60%of bovine lactoferrin peptides can enter the small intestine in intact form through the stomach without being digested.The research on the formation mechanism of peptide self-assembled nano-micelles and their interaction with hydrophobic bioactive compounds remains to be further explored.Based on this,enzymatically partially hydrolyzed lactoferrin polypeptide nano-micelles were constructed by self-assembly in this study,explored the structure,physicochemical properties,and application for encapsulating curcumin,and clarified this nano-delivery system can improve the bioavailability of curcumin.The main research contents and results are as follows:（1）Construction and characterization of enzymatic partially hydrolyzed lactoferrin hydrolysate nano-micelles（blank nano-micelles,M）to explore its formation mechanism and properties of delivering curcumin.Partially enzymatically hydrolyzed lactoferrin peptides could form self-assembled nano-micelles（CMC was 0.03 mg/m L）with the assistance of ultrasound.The optimized process conditions were as follows:the protein concentration during hydrolysis was 1.0 mg/m L,p H6.0,and after the hydrolysis was completed,the micelles were formed by sonicating at 50 W for 60 s in an ice bath and then vertically stirring for 10min.The optimal mixing volume ratio of Cur to M was 1:10 and stirred overnight.The formed nano-micelles could encapsulate Cur with high loading efficiency,and the encapsulation rate was about 93.44%.The Cur-loaded nano-micelles（M（Cur））were relatively small（approximately 125 nm）and positively charged and it had a uniform spherical shape.Circular dichroism and fluorescence quenching experiments proved that the bonding of Cur and micelles did not alter the conformation of nano-micelles and that Cur was incorporated into the hydrophobic core of nano-micelles.（2）In order to explore the stability,in vitro antioxidant activity and bioaccessibility of M（Cur）,the results of DPPH·and ABTS+·scavenging assays demonstrated that the antioxidant capacity of M（Cur）was superior to that of free Cur or LF,and M（Cur）has good light stability,thermal stability,dilution stability,storage stability and reconstitution stability of lyophilized powder.Meanwhile,in vitro simulated digestion results demonstrated that M（Cur）improved the bioaccessibility and transformation rate of curcumin.Furthermore,cytotoxicity tests confirmed that free Cur and M（Cur）were not cytotoxic to human colon cancer cells（Caco-2）when the curcumin concentration was lower than 150μg/m L,and had good biocompatibility.（3）The effect and mechanism of M（Cur）on H₂O₂-induced cellular oxidative stress injury were studied using mouse brain capillary endothelial cells（bEnd.3）as the research subject.Free Cur and M（Cur）intervention containing equal amounts of Cur could restore the cell morphology of bEnd.3 cells under oxidative stress,increase cell viability,restore mitochondrial membrane potential,and effectively inhibit the generation of reactive oxygen species to significantly alleviate oxidative stress damage.The results showed that free Cur and M（Cur）could effectively promote the expression of antioxidant defense enzymes and maintain intracellular redox balance by regulating the m RNA expression levels of HO-1,COX-2,PSD-95,and BDNF.And compared with free Cur,M（Cur）had more obvious intervention effect.In conclusion,as compared to free Cur,M（Cur）has improved water solubility,stability,antioxidant activity,and bioavailability,and the nano-micelles may considerably reduce H₂O₂-induced oxidation of bEnd.3 cells stress damage.The research indicated that the self-assembled nano-micelles of lactoferrin hydrolysate could be used as novel delivery vehicles for hydrophobic bioactive compounds,which provided a scientific basis for improving the utilization of hydrophobic bioactive substances.