Prepration Of Nutrient Proliposomes Based On Solution Enhanced Dispersion By Supercritical Fluids Technology

The concept of proliposomes is introduced to solve problems caused by the liquid form of liposomes, such as aggregation, fusion, hydrolysis of lipid, and so on. Proliposomes are defined as a kind of dry and free-flowing particles with loading ingredients. The liposome suspension can be easily obtained from proliposomes when they are dispersed in water. However, there are some shortcomings in the traditional methods: such as proper cryoprotectants needed, many steps involved, and high processing temperatures, which limit their wide application. To solve the problems above, solution enhanced dispersion by supercritical CO₂ widely used in food industry should be developed to prepare proliposomes, because of its lower residual solvents, simpler steps and mild operation temperatures. In the aspect of the preparation of proliposomes using supercritical CO₂ technique, limited papers are published. A few papers just involved in the preparation of phospholipid powders by supercriticalCO₂. Only one paper involved in the preparation of proliposomes containing miconazole by the aerosol solvent extraction system (ASES) process.This research was to prepare proliposomes using solution enhanced dispersion by supercritical CO₂ (SEDS) and to make liposomes via the hydration of the proliposomes. Effects of the process parameters were investigated, and the animal experiments were introduced to determine the antioxidant activity of the liposomes. The purpose of our study was to provide the technical parameters and the theory basis for the industrialization of the liposomes. The conclusions were as follows:1. Experimental data for the phase behavior of the four systems CO₂ + DCM + EtOH (ethanol), CO₂ + EtOH + C₆H₁₄ (n-hexane) and HPC + EtOH + CO₂ (dichloromethane) with different compositions at temperatures from 308.5K to 328.5K were investigated. The bubble point pressure increases with increasing temperature at constant CO₂ mass fraction. In addition, the modeling results indicate that PR-EOS (Peng-Robinson equation of state) with one interaction parameter can correlate the experimental data for the bubble points of the system CO₂ + EtOH + C₆H₁₄ (n-hexane).2. The coenzyme Q10 was chosen as the model drug. The mixture of cholesterol and soybean phosphatidylcholine (PC) was chosen as wall materials. The effects of operation conditions (temperature, pressure and components) on the recovery of CoQ10 and the CoQ10 loading in CoQ10 proliposomes were studied. At the optimum conditions of pressure of 8.0MPa, temperature of 35℃, the weight ratio of 1/10 between CoQ10 and PC, and the weight ratio of 1/3 between cholesterol and PC, the CoQ10 loading reached 8.92%. CoQ10 liposomes were obtained by hydrating CoQ10 proliposomes and the entrapment efficiency of CoQ10 reached 82.28%. The morphology of CoQ10 proliposomes were characterized by SEM, and their solid state was characterized by XRD. The structure of CoQ10 liposomes were characterized by TEM. The particle size distribution of CoQ10 liposomes was determined by DLS. The results indicate that CoQ10 liposomes with particle sizes about 50nm can be easily got from hydrating CoQ10 proliposomes prepared by SEDS.3. Vitamin D3 (VD₃) proliposomes, consisted of hydrogenated phosphatidycholine (HPC) and VD₃, were prepared using SEDS. The effects of operation conditions (temperature, pressure and components) on the VD₃ loading in VDP were studied. At the optimum conditions of pressure of 8.0MPa, temperature of 45℃, and the weight ratio of 15.0% between VD₃ and HPC, the VD₃ loading reached 12.89%. VD₃ liposomes were obtained by hydrating VD₃ proliposomes and the entrapment efficiency of VD₃ in VD₃ liposomes reached 98.5%. The morphology and structure of proliposomes and liposomes were characterized by SEM, TEM and XRD. The structure of VD₃ nanoparticles in HPC matrix was formed. The size of liposome was determined by Dynamic Light Scattering instrument (DLS). The average diameter of liposomes was about 1μm. The results indicate that VDP can be made by SEDS and liposomes with high entrapment efficiency can be formed easily via the hydration of proliposomes.4. Proliposomes composed of lutein and hydrogenated phosphatidylcholine were prepared using SEDS. The effects of the process parameters on the lutein loading and the particle sizes of the proliposomes were investigated. HPLC was applied to determine the content of lutein in the samples. At the optimum conditions—temperature of 35℃, pressure of 8MPa and the solution flow rate of 1 ml/min—the lutein loading of the proliposomes reached 55mg/g. The images characterized by SEM were evaluated for the different proliposomes samples in order to study the influences of operational conditions on the particle sizes and morphology. When proliposome was hydrated, the lutein liposome suspensions were formed automatically. The crystallinity of proliposomes was analyzed using DSC to analyze the distribution of lutein in proliposomes. The structure of proliposomes and the lutein liposome was detected by TEM. The results indicate that proliposomes with the high lutein loading was made successfully and the lutein liposome was obtained with the encapsulation efficiency of more than 90% after hydrating proliposomes. The animal experiment results show that there is dose dependence on the lutein liposomes. These results demonstrate that SEDS technique is a simple and effective process for the preparation of proliposomes from which liposome can be easily formed.