| Lactoferrin (Lf) is a protein with multiple biological functions, and it is notonly involved in iron transport, but also in immune response, antioxidant activities,antimicrobial activities, and anticarcinogenic activities. However, Lf is susceptible tobeing digested and the protein-based drugs are easy to be hydrolyzed and neutralizedby serum proteins. Nanoliposomes have been extensively studied in an attempt toenhance the therapeutic efficacy of various materials in the field of drug and fooddelivery system. The nanoliposomes enhance the stability of the encapsulatedmaterial by protecting them from the environment and make them into idealcandidates for delivery. To improve the effectiveness of lactoferrin, nanoliposomeswere used as a carrier in this study. The aim of the present study was to choose thebest method and the optimization of the preparation process to develop Lfnanoliposomes. We investigated the stability and residues of Lf nanoliposomes. Also,the effect on Caco-2activities was evaluated. The main findings are as follows:(1) Three different methods were compared for preparing Lf nanoliposomes basedon encapsulation efficiency and size distribution, reverse-phase evaporationmethod was fit for Lf nanoliposomes.(2) The effect of cholesterol to phosphatidylcholine ratio, lactoferrin concentration,tween80and aqueous phase to organic phase ratio on preparing lactoferrinnanolipsomes were studied. Response surface design experiments were used tooptimize the preparation conditions based on encapsulation efficiency and sizedistribution. Numerical optimization determined the optimum preparationconditions, which were phosphatidylcholine to cholesterol ratio of5.77,lactoferrin concentration of11.71mg/mL, tween80of1.18mg/mL and organicphase to aqueous phase ratio of4.59. At this optimum point, particle size andencapsulation efficiency were found to be214nm and60.21%, respectively.(3) Temperature as well as the external environments were too acid, should beeasy to destroy the lipid bilayer structure of nanoliposomes, therefore,nanoliposmes should be avoided stored at above the conditions. The effects ofcommon food additives, such as glucose, sucrose and the citric acid on the stability of lactoferrin nanoliposome which showed no significantly.(4) The effect of the sonication on the stability of Lf nanoliposomes was evaluatedbased on leakage ratio and size distribution. Sonication was fit to preparingthe nanoliposomes, which can be appropriate to reduce the particle size, buttoo long sonication would result in leakage of the entrapped substances. Aboveall, the20-min sonication time of Lf nanoliposomes may be fit for itspreparation. pH, leakage ratio and MDA values of nanoliposomes were testedfor the period of30days. The lactoferrin nanoliposomes showed an acceptablestability and no distinct differences in the physical and chemical properties.The study about the effect of simulated gastrointestinal environment onlactoferrin nanoliposome showed that liposome technology could protectlactoferrin from the destruction of the external factors such as pepsin.(5) To determin ether and chloroform in the lactoferrin nanoliposomes byheadspace gas chromatography, residues of the sample after testing were lessthan0.006%.(6) The activities of Caco-2cells were compared with lactoferrin (Lf) and Lfnanoliposomes. For anticancer activity evaluations, mitochondrial function(MTT assay), count kit-8(CCK-8), detection of intracellular reactive oxygenspecies (ROS) and apoptosis induction (AO/EB staining) assays were assessedunder control and exposed conditions. MTT results demonstrated that Lfnanoliposomes and Lf activated in the cells in a manner of dose and time effectrelationship. ROS results showed that when lactoferrin nanoliposomes at theconcentration of1mg/mL, ROS showed more obvious significantly thanlactoferrin. The above results were also supported by CCK-8evaluation andAO/EB double staining. |
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