Carboxymethylichitosan Coated Proliposomes Containing Coix Seed Oil: Preparation, Characterization, Stability And In Vitro Release Evaluation

Coix seed, an inexpensive, easy accessed Medicinal and Edible food, has important nutritional value and health effects, which can not be replaced by other general foods. Coix seed oil (CSO) as its main active ingredient has immunity, antitumor, hypolipidemic, and many other physiological functions. However, the oil have many drawbacks of biologically unstable, poorly soluble in water, and low absorption rate. All of which greatly limit the utilization of CSO in food industries.Liposomes as a new type of nano-capsule preparation, are used as carriers for the delivery of drug and functional ingredients. Employing liposome technology to embedded CSO can greatly improve its water solubility, targeting and bioavailability. However, conventional aqueous liposomes may be subject to a series of stability problems such as aggregation, fusion, phospholipids hydrolysis and the leakage of the encapsulated active agents. To overcome the shortcomings, modifying surface of liposomes with hydrophilic polymers followed by spray-drying is an effective method. And the additional convenience of the transportation, distribution, and storage would make'proliposomes'(PL) a promising industrial product.CSO loaded conventional liposomes were prepared by using five different methods:thin film evaportion, freezing-thawing, ether injection, ethanol injection, and reverse phase evaportion. Results showed that the preparetion methods had significant effects on structure, encapsulating efficientcy (EE), partile size and zeta potential. Liposomes prepared by thin film evaportion were multilamellar vesicles (MLV), liposomes prepared by freezing-thawing were irregular MLV, liposomes prepared by reverse phase evaporation were larger unilarmellar vesicles (LUV), and liposomes prepared by ether injection method and ethanol injection were smaller unilarmellar vesicles (SUV). The encapsulating efficientcy of liposomes was in the follows:thin film evaportion> ethanol injection> ether injection> reverse phase evaporation> freezing-thawing, when loading capacity was 20%. Liposomes prepared by thin film evaportion and ether injection displayed worse dispersivity, while liposomes prepared by ethanol injection showed best dispersivity and smallest particle size. In all cases, zeta potential of all samples were similarly in a rang of-1～3 mV. The stability experiment indicated that liposomes prepared by ethanol injection were most stable of oil is best had the lowest leakage rate and lowest changes in paticle size and pH values.By using ammonium thiocyanate method to determine coating efficiency for carboxymethyl chitosan (CMCT) modifying conventional liposomes prepared by ethanol injection. The results showed that the wavelength for determination of phospholipid was 470 nm.,The linear regression equation was A=10.963C+0.0156, R2=0.9991, and at the concentration range of 0.01-0.1 mg/mL showed good linear relationship. In order to fully open up the uncoated liposomes. The centrifugation method was applied for separation of uncoated liposomes from coated ones. The uncoated liposomes was then broken by alcohol-ultrasound, followed by calculating the phospholipid content according to linear regression equation and evaluating coating encapsulation rate. The recovery, precision, and reproducible experiments have proved this method was feasible.The effects of several key factors for preparing coating liposomes were investigated by setting coating efficiency as main index. The preparation techniques and formation were also optimized with coating efficiency as criteria by orthogonal array design. Results indicted that optimum coated liposomes were obtained with carboxymethyl chitosan to phospholipid mass ratio of 5:4, loading capacity of 15%, the medium pH was 6.5, twain to polyethylene glycol 400 mass ratio of 1:4 at the condition of stirring speed at 800 r/min, reaction temperature at 45℃, reaction time for 80 min, ultrasonic intensity at 200 W.The CMCT coated liposomes were dried by spray drying method, the obtained proliposomes powders showed good fluidity, easy to dissolve in water, with an encapsulation rate higher than 83%. The physi-chemical properties of spray-dried product were researched, the results were as followes:scanning electron microscopy (SEM) showed CMCT coated proliposomes (CM-PL) had smooth surface and heterogeneous size distribution ranging from 1 to 5μm. Transmission electron microscopy (TEM) observed that there were quite few changes between CM-L and rehydrated CM-PL, both of them with an carboxymethyl chitosan on the surface of conventional liposomes. X-ray diffraction (XRD) results showed that the crystalline character of conventional liposomes disappeared in the pattern of CM-PL indicated that the conventional liposomes was sufficiently entrapped by CMCT layer. The conventional liposomes could be coated sufficiently by CMCT via H-bonding interactions between CMCT and the interfacial region of the phosphatidylcholine bilayer. DSC showed that the endothermic peak of CM-PL was at 191.33℃greatly higher than that of the conventional PL, indicating their thermal stability was improved after coating.The effects of light, atmosphere, temperature, and coexisted food ingredients on the stability of CM-PL were also examined. The results indicated that all factors could promote the oxidation of CSO, and lead to leakage of the CSO to different degrees, but the products showed better stability.The in vitro release experiments for the samples of CSO dissolved liquid, conventional liposomes, rehydrated uncoated proliposomes and rehydrated CM-PL were investiged. The results showed that all liposomes showed controlled release of CSO, while the coated ones exhibted better sustained-release. And the release rate had tight releationship with release medium. CM-L and rehydrated CM-PL exhibited more sustained release in SIF than in SGF. This may be attributed to the presence of carboxyl groups and amino groups of amphiphilic CMCT which were sensitive to pH values, and could altering the swelling of CMCT layer furtur influence the release of CSO.