Preparation And Functional Properties Of ?-Carotene-Coix Seed Oil Composite Liposomes

As a new type of carrier,liposomes attracted so much attention,and the stability of active ingredients which encapsulated in liposomes can be improved because liposomes could protect them by reducing the damage from light and oxygen.As the ideal carriers in the food and pharmaceutical industries,liposomes have characteristics of sustained release,targeting,non-toxicity,cell affinity,and tissue compatibility.Currently,a large number of studies focus on the preparation and characterization of different liposomes containing single active ingredient,but few studies try to encapsulate two ingredients in liposomes.In actual research work,it has been found that many active ingredients can work synergistically to improve physiological functions.Therefore,incorporating two or more ingredients in liposomes to form novel composite liposomes could enhance the physiological function of the ingredients,and in general,this is very meaningful.?-carotene(?C)and Coix Seed Oil(CSO)are good nutrients in the human diet and play an important role in promoting human health.?-carotene has physiological functions of anti-aging,anti-cancer,and the CSO also has anti-inflammatory and anticancer effects.Studies have shown that the preparation of complexes of carotenoids and vegetable oils can effectively enhance the physiological functions of both.Considering that both ?-carotene and CSO contain unsaturated bonds and are easily degraded under light,heat,and oxygen conditions,?-carotene-CSO composite liposomes(L-?C-CSO),single ?-carotene liposomes(L-?C),single CSO liposomes(L-CSO)and blank liposomes(un-L)were prepared using ethanol injection method,and their physicochemical properties are selectively compared and analyzed.Based on the encapsulation efficiency of ?-carotene and particle size distribution of the L-?C-CSO,the single factor experiment was used to determine the most important factors to prepare L-?C-CSO.On this basis,the Box-Behnken response surface optimization method was used to determine the optimal process conditions to prepare L-?C-CSO.The single factor results showed that the preparation temperature,the mass ratio of lecithin to cholesterol,and the volume of PBS buffer used had a great influence on the L-?C-CSO.Box-Behnken response surface optimization results showed the optimal preparation conditions as follows: temperature was at 43?,massratio of lecithin to cholesterol was 7:1,volume of PBS buffer was 25 mL/10 m L ethanol,and under this condition,the entrapment efficiency of ?-carotene was 84.39%,The average particle size was 187.9±7.5 nm,the polydispersity coefficient(PDI)was0.104±0.09,and the average Zeta potential was-29.33±4.77 mv.In this study,L-?C-CSO(1:1,1:5,1:10)with mass ratios of 1:1,1:5,and 1:10 of?-carotene and CSO was prepared using abovementioned optimized process conditions,and L-?C,L-CSO and un-L were also prepared.The particle size distribution,entrapment efficiency of ?-carotene,apparent morphology,antioxidation,storage stability,in vitro release characteristics and inhibition effects of human colon cancer cell growth were investigated and compared.The results showed that the addition of CSO can occupy a certain hydrophobic space of liposomes,leading to a certain difference in the particle size distribution and ?-carotene entrapment efficiency between L-?C-CSO and L-?C.Transmission electron microscopy(TEM)results showed that both L-?C-CSO(1:1)and L-?C showed a monolayer vesicle structure and had a liposomes-specific fingerprint structure,but the dispersion of L-?C-CSO was more uniform.All liposomes had scavenging effect on DPPH free radicals.The DPPH-scavenging activity of L-CSO,L-?C,L-?C-CSO(1:1,1:5,1:10)were49.2±3.32%,41.5±7.1%,51.93±6.63%,66.74±2.23% and 73.48±3.66% respectively,indicating that the freshly prepared L-?C-CSO are more resistant to oxidation than single liposomes.In addition,the oxidation of lipids in liposomes can be effectively slowed down at 4°C,but the high content of CSO may accelerate the degradation of?-carotene in L-?C-CSO at 25°C.In vitro simulated release experiments showed that in artificial simulated gastric fluid(SGF),the release rate of ?-carotene in L-?C-CSO(1:10)was the lowest,but the value of L-?C was the highest,indicating that the addition of CSO can reduce the damaging effect of gastric fluid on ?-carotene.In artificial simulated intestinal fluid(SIF),the release rate of ?-carotene in L-?CCSO is higher than that of L-?C,and this trend is positively correlated with the content of CSO.In SGF,the release behavior of all samples fit to the first-order model,while in SIF,fit to the Korsmeyr-peppas model.MTT assay showed that all samples containing ?-carotene or CSO showed a concentration and time dependent inhibition rates to the Caco-2 cancer cells.The inhibition rates of L-?C-CSO alwayshigher than L-CSO and L-?C in spite of the liposomes concentration and culture time,indicating that the proper mass ratio of ?-carotene to CSO in L-?C-CSO may lead to better inhibition to cancer cell growth than single liposomes.In summary,it's can be found that L-?C-CSO has obvious advantages compared with single L-?C,which has important significance for the application of ?-carotene and CSO in food and medicine industry.To learn more about the stability and sustained-release effect of L-?C-CSO,L-?C-CSO(1:1)was selected as the initial liposomes(L),and the chitosan monolayer modified liposomes(C-L)and pectin-chitosan double-layered modified liposomes(PC-L)were prepared.The physicochemical properties of L,C-L and P-C-L were investigated and compared.The results of dynamic light scattering(DLS)showed that the particle sizes of L,C-L,and P-C-L were increased in order,the protective layer formed on the surface of the L after modificated may lead to the increase of particle size,but the transmission electron microscopy(TEM)showed that all samples exhibited regular spherical structure.Fourier Transform infrared spectroscopy(FTIR)showed that the amino groups on chitosan could form hydrogen bonds with the polar groups on the liposome phospholipid bilayer and successfully prepared C-L,and in the same,the carboxyl group on the pectin and the amino group on the chitosan can also interact to form a polyelectrolyte and successfully prepare P-C-L.Differential scanning calorimetry(DSC)measurements showed that the phase transition temperatures of L,C-L,and P-C-L increase sequentially,indicating that P-C-L has the best thermal stability and this may be due to the protection of the double-layered shell on the surface of L formed by pectin and chitosan.Thermogravimetric analysis(TGA)also showed that the thermal stability of L,C-L,and P-C-L increased in order.The results of storage stability test showed that the degradation rate of ?-carotene stability of L,C-L and P-C-L was lower at 4°C,therefore,all samples are best stored at 4?.However,the degradation rate of ?-carotene in P-C-L was the lowest whether the storage temperature is 4°C or 25°C,indicating that double modification can effectively enhance the storage stability of liposomes.In vitro simulated release results showed that both in SGF and SIF,the release rate of ?-carotene in L was the highest,while that of P-C-L was the lowest,indicating that the protective layerformed after the modification of pectin and chitosan had a certain sustained release effect.In SGF,the release behavior of L,C-L,and P-C-L all fit the first-order model,in SIF,the release behavior of L is consistent with the Korsmeyr-peppas model,whereas the release behavior of C-L and P-C-L fit the first-order model.