| Liposome was a delivery system consisting of a phospholipid bilayer with a closed vesicle structure.With the advantages of targeting,slow release and high biocompatibility,liposomes have been extensively studied as a promising delivery vehicle in the fields of medicine,biology,food,and agriculture.However,the membrane structure(size and composition)of liposomes significantly affects their performance.Based on the structural differences between infant formula milk fat globule membranes(small size,single composition)and breast milk fat globule membranes(large size,complex phospholipids),four biphasic liposomes with different sizes and membrane compositions were prepared,using the essential nutrients docosahexaenoic acid(DHA,hydrophobic)and lactoferrin(LF,hydrophilic)as core materials,namely small-sized single phospholipid liposome(S),small-sized mimicking breast milk fat globule membrane phospholipid liposome(S-C),large-sized single phospholipid liposome(L)and large-sized mimicking breast milk fat globule membrane phospholipid liposome(L-C).The physicochemical properties of the biphasic liposomes with different membrane structures were investigated.Besides,the effects of biphasic liposomes on infants’ in vitro semi-dynamic digestive behaviour and2 D intestinal organoid growth and nutrient absorption and utilization were evaluated.The main results are as follows.(1)Preparation and characterization of liposomes with different membrane structures.The single phospholipid fraction [100% phosphatidylcholine(PC)] and the mimic breast milk fat globule membrane phospholipid fraction [45% PC,30%phosphatidylethanolamine(PE),25% sphingomyelin(SM)] were used as wall materials,and small-sized liposomes closed to the particle size of infant formula fat globules were prepared by reverse evaporation combined sonication,and large-sized liposomes closed to the particle size of breast milk fat globules were prepared by a modified film dispersion combined sonication method.Four types of biphasic liposomes,S,S-C,L and L-C,were obtained.The particle sizes of S and S-C were 180.27±1.79 nm and171.17±0.98 nm,respectively,while those of L and L-C were 5.33±0.83 μm and5.24±1.58 μm,respectively.The MDA results showed that the small-sized liposomes were more stable than the large liposomes,and the backscattered light intensity over 24 hours was significantly less than that of the single liposomes(S and L)due to the higher zeta potential and increased interparticle repulsion.The oxidation rate was greater for the smaller liposomes than the larger liposomes,and the liposomes with single phospholipids were greater than the liposomes with mimicking mothers’ milk fat globule membrane phospholipids.The protective effect on DHA showed a significant size effect,with S and S-C liposomes showing more than 30%(4 °C storage)and 50%(room temperature storage)retention of DHA after 30 days of storage compared to the larger sized liposomes.The S-C liposomes had the highest environmental stability.(2)In vitro infants’ semi-dynamic digestion of liposomes with different membrane structure.The changes in particle size and zeta potential of the four liposomes were not significant during simulated gastric digestion,while remarkable changes occurred during small intestine digestion.The TEM results also revealed that the liposomal structure remained stable and the bilayer structure was clearly visible during gastric digestion,whereas after small intestinal digestion,the liposomal structure was significantly disrupted,with only a few fragments remaining.The membrane fluidity of undigested L and L-C was greater than that of S and S-C.The physical factors such as p H and ionic strength during the gastric digestion influence the membrane fluidity,while the degree apparently increased owing to the pancreatic hydrolysis of phospholipid and bile salts,forming lysophospholipids with higher polarity.The S-C and L-C groups were more rapidly hydrolysed at the beginning of digestion due to the incorporation of PE and SM,whereas,the fatty acid release rate was higher in the smallsized liposomes than in the large-sized liposomes at the end of digestion.Similar results were obtained for fatty acid fraction analysis,with higher total fatty acid(64.3±2.8%and 83.4±6.6%)and DHA release rates(83.7±0.7% and 91.7±1.3%)for S and S-C than for L and L-C(52.4±2% and 79.2±2.9% for total fatty acid(80.2±2.3% and 87±3.6%for DHA).Total fatty acid and DHA release rates were higher in S-C and L-C than in S and L,and DHA release rates were higher in all four liposomes than in the control group(64.6±3.4%).The results of structural degradation of lactoferrin and the liposome digestion process showed a high correlation,with approximately 10% of amino acids released during the gastric digestion,and a large amount of lactoferrin released and hydrolysis by pancreatic enzymes during the small intestine phase.(3)Effect of different membrane structured liposomes on the absorption and transport of intestinal organoids in suckling pigs.The crypt from the jejunal segment of the suckling pig was successfully isolated and cultured as a continuous transmissible organoid,and the organoid with 2D monolayer structure was further cultivated using Transwell plates.Toxicity results showed that the viability of the organoids remained above 80% for the 100 μg/m L of liposomal digestion product treatment and was not affected by the 50 μg/m L liposomal digestion product treatment.Four liposomes increased the efficiency of amino acid transport by more than 0.5-fold and fatty acid transport by more than 1.4-fold compared to the control group(0.95±0.02 μg/mg protein and 1.99±0.04 μg/mg protein).The liposomal transport efficiency and Papp were significantly higher in the phospholipid mimicking group than in the single phospholipid group,and slightly higher in the small-sized group than in the large-sized group,while the differences were not significant,and the effect of component was greater than that of size.m RNA expression results showed that the four liposomes increased the uptake and transport of fatty acids and amino acids mainly by regulating the expression of amino acid and fatty acid transport proteins,and S-C and L-C showed more significant effect.In summary,liposomes with different membrane structures had significant difference in environmental stability,simulated infant digestive behaviour and intestinal organoid absorption and transport.Smaller liposomes oxidized faster but retained more DHA than larger liposomes.In vitro digestion indicated S-C group had the lowest release rates of amino acids and DHA.The composition of liposomes influenced more significant on the 2D organoid absorption than the effect of size.Therefore,S-C liposomes have the best performance in terms of environmental stability and digestion and absorption.Liposomes with the small and phospholipid fraction with mimicking breast milk fat globule membranes can be used as an efficient nutrient delivery system to improve the deficiencies of unstable and low absorbable nutrients for infant foods and help newborns lacking breastfeeding to overcome some of the health problems caused by traditional infant formula feeding. |
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