Study On Carotenoid-loaded Liposomes

Carotenoids are a large group of naturally-occurring pigments that are applied in food,pharmaceutical and cosmetic industries. They are important provitamin A sources and playimportant physiological roles in antioxidant activities, scavenging free radical and adjustingimmune function. However, their physiological benefits have been restricted which isattributed to the low aqueous solubility and oral bioavailability. Additionally, the inherentchemical reactivity leads them to be sensitive to O2, light and heat, which make them havegreat limitation in processing and application. This study selected the mixed lipid includingnative phospholipid (egg yolk phosphatidylcholine, EYPC) and nonionic surfactant Tween80to prepare a new type of liposomes as efficient carriers for four typical carotenoids (lycopene,carotene, lutein and canthaxanthin). The influencing factors to stabilization and physiologicalfunction of carotenoid-loaded liposomes were determined from physicochemical properties,antioxidant activity and biological effects. On the basis of these findings, the contrastingmodulation of biopolymer chitosan decoration on the stabilization of lipid vesicles wasinvestigated. The fabrication and stabilization mechanisms of chitosan/liposome core–shellnanocomplexes were elucidated aimed to stabilize and control the delivery of encapsulatedbioactive substancesFirstly, the influence of carotenoid incorporation on the particle size, morphology anddispersion was investigated by dynamic (DLS) and atomic force microscopy (AFM). It wasfound that the liposomal particles loading lycopene, β-carotene or lutein appeared sphericalshape and the particle size was homogeneous ranging from80to100nm, whereascanthaxanthin incorporation resulted in particle aggregation and heterogeneous suspension.Subsequently, the loading ability of various carotenoids into liposomal membrane wasevaluated by extraction and spectrophotometer method. It was demonstrated that carotenoidsexhibited various incorporating abilities into liposomes ranging from the strongest to theweakest: lutein> β-carotene> lycopene> canthaxanthin. Storage measurements revealed thatliposomes loading β-carotene and lutein underwent slight particle increase and the retentionrates exceeded60%and75%, respectively. By contrast, liposomes loading lycopene andcanthaxanthin seriously aggregated with low retention rate less than50%. Additionally,incorporation of β-carotene or lutein could suppress the aggregation and fusion of particlesunder heat treatment, whereas the effect was opposite for lycopene and canthaxanthin.The dynamic and structural changes due to the presence of carotenoid in the bilayer wereexamined by fluorescence probe, Raman spectroscopy, and electron paramagnetic resonancein order to illustrate the stability mechanism of carotenoid liposomes. The incorporation oflycopene and canthaxanthin within the concentration range of0.25~1.0%and0.25~0.5%,respectively, did not significantly influence the membrane fluidity of liposomal membrane.Beyond the corresponding range, they would disturb the organization of lipid chains, decreasethe membrane microviscosity. To the contrary, β-carotene or lutein can rigidify the liposomesand significantly decrease the membrane fluidity within the studied concentration range.These modulations were closely correlated with the stabilization of liposomes, including mediating particle aggregation and fusion.To understand how carotenoids exerted antioxidant activity in different medium, threeassays were selected to achieve a wide range of technical principles, including2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging, ferric reducing antioxidant powder (FRAP),and lipid peroxidation inhibition capacity (LPIC) during liposome preparation, auto-oxidation,or when induced by ferric iron/ascorbate. Antioxidant activity of carotenoids was measuredeither after they were mixed with preformed liposomes or after their incorporation into theliposomal system. Whatever the antioxidant model was, carotenoids displayed differentantioxidant activities in suspension and in liposomes. The encapsulation could enhance theDPPH scavenging and FRAP activities of carotenoids. The strongest antioxidant activity wasobserved with lutein, followed by β-carotene, lycopene and canthaxanthin. The antioxidantactivity of carotenoids after liposome encapsulation was not only related to their chemicalreactivity, but also to their orientation in liposomal membrane. Furthermore, lipidperoxidation assay revealed a mutual protective relationship: the incorporation of either luteinor β-carotene not only exert strong LPIC but also protect themselves against pro-oxidationelements; however, the LPIC of lycopene and canthaxanthin on liposomes was weak or evenpro-oxidation effect appeared, concomitantly, leading to the considerable depletion of theseencapsulated carotenoids.The morphology of liposomes, lipid digestibility, and carotenoids release were investigatedduring incubation in a simulated gastrointestinal tract. Analysis on the liposome size andmorphology by DLS and AFM observation showed that after digestion, the majority ofparticles maintained spherical shape with only an increase of size in liposomes loadingβ-carotene or lutein. However, a large proportion of heterogeneous particles were visible inthe micelle phase of liposomes loading lycopene or canthaxanthin. It was also found that therelease of lutein and β-carotene from liposomes was inhibited in a simulated gastric fluid,while was slow and sustained in a simulated intestinal fluid. The release rates were around45%and55%after digestion, respectively. By contrast, lycopene and canthaxanthin exhibited fastand considerable release in the gastrointestinal media. Both carotenoid bioaccessibility andmicellization content decreased with the increase of incorporated concentration. Anyway, thebioaccessibility of carotenoids after encapsulated in liposomes was in the following order:lutein> β-carotene> lycopene> canthaxanthin. Bivariate correlation analysis revealed thatcarotenoid bioaccessibility depended strongly on the incorporating ability of carotenoids intoa lipid bilayer, loading content, and nature of the system.The concentration dependence of chitosan conformation in aqueous solution was illustratedby surface tension, fluorescence spectra and viscosity measurements. As the concentrationincreased, the extended chains of chitosan would partially crimp and then self-aggregatedforming irregular coils. Unfolded chains adsorbed on membrane surface via electrostaticattraction, forming the thin coated layer of10nm by means of turbidimetric, DLS andmicroscopic observations. However, the interactions of crimpled chains and coils withliposomes were controlled by both electrostatic and hydrophobic interactions, leading toheterogeneous suspension dispersion. Fluorescence spectra, Raman spectra and electronparamagnetic resonance were subsequently employed to investigate the dynamic and structural change of liposomal membrane resulting from chitosan decoration. Results showed,in addition to providing the physical barrier for membrane surface, the decoration of chitosanextended chains decreased the membrane fluidity and enhanced the order of the lipidmolecules. Such stabilizing effects were more pronounced for crimpled chains. However,chitosan coils tended to act as a “polymeric surfactant”. The presence of chitosan coilsinduced the increase in membrane fluidity, the intrachain disorder in lipid molecules, and theincrease in membrane permeability.