Construction Of Peptide-loaded Liposome Nanocarriers And Research On Improving The Bioavailability Of Active Peptide

Bioactive peptides are functional compounds of proteins which are closely related to the physiological functions of humans,such as digestion and absorption,nutrient metabolism and immune regulation.However,they are easily degraded by proteases in the digestive tract after oral administration and encounter various physiological barriers during the absorption process in vivo,which highly reduces the bioavailability of active peptides.The choice of liposomes for encapsulation and delivery of active peptides has shown promise in improving the bioavailability of active peptides.Liposomes are spherical enclosed vesicles composed of phospholipid bilayers,which effectively encapsulate hydrophilic,lipophilic and amphiphilic substances,protect the active substance from external damage and act as a nutrient fortification.Liposomes have the advantages of low toxicity,good biocompatibility and slow release.Therefore,they are widely used in food,pharmaceutical,chemical and agricultural applications.In order to investigate the effect of liposomes on the encapsulation and protection of active peptides,Ala-HisLeu-Leu(AHLL),an angiotensin converting enzyme(ACE)inhibitor peptide,was chosen as the model peptide in this study.L-ascorbyl palmitate and chitosan were modified on their surfaces to investigate the uptake transportation and mechanism of different peptide-loaded liposomal nanoparticles.The results of the study were as follows.(1)The effects of oviduct ratio,drug-lipid ratio,sonication time and AHLL concentration on the particle size and encapsulation rate of liposomes were investigated by single-factor experiments.The results showed that the better formulation was selected based on the comprehensive results of the experiment,which was 5:1(w/w)liposomal oocyte ratio,1:8(w/w)drug-lipid ratio,5 min sonication time,and 1 mg/m L total drug concentration of AHLL.The liposomes prepared on this basis have the characteristics of high encapsulation efficiency,small average particle size and good stability.The average particle size of the liposome was(115.88 ± 6.35)nm,the encapsulation efficiency was(81.16 ± 1.31)%,and the potential was-24.1 m V.L-ascorbyl palmitate and chitosan were modified on the surface of the optimized liposomes,and the storage stability of the three liposomes was investigated.The results of transmission electron microscopy showed that the three liposomes were spherical in structure,with uniform particle size and clear appearance,and the particle size was about 100 ～ 200 nm.In addition,L-ascorbyl palmitate modified liposomes(AP-Lipo)and L-ascorbyl palmitate and chitosan modified liposomes(CS-AP-Lipo)can improve liposomes to a certain extent.In addition,the storage stability of CS-AP-Lipo was significantly greater than the previous two during storage,which could effectively protect the liposomes from being degraded.The changes in ACE inhibitory activity of liposomes and the cumulative release of AHLL were measured in simulated gastrointestinal fluid.The results showed that the ACE inhibition rate of free AHLL decreased to less than 50%after digestion,and the ACE inhibition rate of AP-Lipo and Lipo was about 60%.On the other hand,CS-AP-Lipo retained 85% ACE inhibition rate.In addition,in the in vitro release experiment,the total release of free AHLL after 24 h digestion was about 95%,the total release of AP-Lipo and Lipo was about 78%,and the total release of CS-AP-Lipo was 70%.The following shows that the entrapment of liposomes can have a good sustained release effect.(2)The toxicity,uptake,cellular uptake mechanism,intracellular transport pathway and transmembrane transport ability of three liposomes in Caco-2 cells were investigated.The results showed that AHLL,Lipo,AP-Lipo and CS-AP-Lipo were not cytotoxic in the concentration range of 100 μg/m L.The cellular uptake of L-ascorbyl palmitate-modified liposome AP-Lipo was significantly higher than that of unmodified liposome Lipo,and slightly higher than that of CS-AP-Lipo added with chitosan coating.The experimental results of the uptake mechanism showed that the uptake of the three liposomes involved energy-dependent pathways.Lipo mainly relies on clathrin and macropinocytosis-mediated endocytosis to enter cells.AP-Lipo mainly relies on caveolin and macropinocytosis-mediated endocytosis to enter cells.CS-AP-Lipo mainly relies on clathrin and macropinocytosis-mediated endocytosis to enter cells.Among the three liposomes,only AP-Lipo interacted with SVCT1 expressed on the top of Caco-2 and was efficiently internalized into cells.The fluorescence co-localization results showed that the co-localization fluorescence intensity of AP-Lipo and lysosome mediated by SVCT1 into cells was weaker than that of the other two groups,indicating that it can directly bypass the lysosome in the process of intracellular transport and reach the lysosome.Endoplasmic reticulum and Golgi apparatus.The results of transmembrane transport experiments showed that the order of AHLL concentration on the BL side is: CS-AP-Lipo> AP-Lipo>Lipo> free AHLL,which indicates that liposomes can promote the transport of AHLL,and chitosan can promote permeability and reversibly open tight junctions,greatly increased the amount of liposome transmembrane transport.(3)The in vivo absorption of three liposomes loaded with the active peptide of AHLL was studied at the animal level,and the improvement in the in vivo bioavailability of AHLL was determined by examining the distribution and the degree of transit absorption of the three liposomes in animals.The results showed that AP-Lipo and CS-AP-Lipo retained fluorescence longer in the gastrointestinal tract and the fluorescent material was retained longer in the jejunum compared to the unmodified liposomes.It was also shown that CS-AP-Lipo modified with chitosan showed significant fluorescence accumulation in the lungs of rats,which may suggest the existence of a specific lung targeting function of CS-AP-Lipo.In addition,in vivo unidirectional intestinal perfusion experiments showed that the effective membrane permeability of AP-Lipo nanoparticles in the duodenum,jejunum and ileum was increased by approximately 2.28-fold,2.63-fold and 2.63-fold,respectively,compared to Lipo The effective membrane permeability of CSAP-Lipo nanoparticles in the duodenum,jejunum and ileum increased by 3.05,3.31 and 3.41 times,respectively,and it was inferred that the main absorption site of APLipo and CS-AP-Lipo was the ileum.In summary,this study concluded that liposomes can effectively encapsulate the active peptide AHLL with good storage stability and good slow release in vivo.The surface modification of L-ascorbyl palmitate can effectively enhance the intracellularization of the nanocarrier,avoiding the lysosomal pathway and the degradation of the active peptide in the cell.In addition,the chitosan coating can also enhance the stability and pro-permeability of the liposomes,thus improving the bioavailability of the active peptide in vivo.