Development of liposomal formulation containing phytosterols and tocopherols with the aim of reducing low density lipoprotein cholesterol (LDL-C)

Background and Rationale: Phytosterols can reduce low-density lipoprotein cholesterol (LDL-C) in serum from the range of 8-14% and have been approved by FDA and Health Canada for their cholesterol-lowering abilities. They can be obtained from vegetable oilseeds and their deodorizer distillates. Canola oil deodorizer distillate (CODD) contains 15-25% of phytosterols, making it an excellent underutilized source of these bioactives. Phytosterols obtained from the CODD can be developed into nutraceutical products in order to attain their commercial potential. However, there are three major challenges in utilizing phytosterols: 1) their lipophilic nature; 2) their thermo-sensitivity leading to the generation of phytosterol oxidation products (POPs); and 3) their formulation-dependent therapeutic efficiency. Nanotechnology-based liposomal formulation can possibly address all these challenges. In addition, the incorporation of antioxidants, namely tocopherols in phytosterol formulation, can enhance phytosterols’ oxidative stability. This study aims to develop liposomes containing phytosterols along with antioxidants into a nutraceutical formulation. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods have been developed to assess liposomal formulations. Furthermore, the LDL-C lowering efficiency of the developed formulation was assessed using the hamster animal model. For the first time, the efficacy of brassicasterol, a phytosterols unique to canola oil, in combination with campesterol and β-sitosterol will be assessed.\nMethods: Three different approaches were utilized for the preparation of liposomes containing phytosterols and tocopherols, namely, thin layer hydration homogenization, thin layer hydration ultrasonication, and the Mozafari method. Two LC-MS/MS methods were developed to determine the entrapment efficiency of phytosterols and tocopherols inside the liposomes as well as POPs present in liposomal formulations. Reversed-phase chromatography with isocratic elusion and atmospheric pressure chemical ionization (APCI) was used in LC-MS/MS method. The methods were validated as per the ICH and FDA guidelines to ensure the accuracy of analytical measurements. An accelerated stability study (microwave heating) was conducted for three different products: crude phytosterols, liposomal phytosterols, and liposomal phytosterols containing tocopherols to monitor the presence of POP during long term storage. The optimized formulations were orally fed to diet-induced hyperlipidemic hamsters for four weeks to assess the extent of LDL-C reduction by the liposomal phytosterols compared to control and marketed phytosterols-containing product. \nResults: Liposomal vesicles prepared via homogenization and ultrasonication methods were significantly lower in size (<200 nm) compared to the ones produced by the Mozafari method (>200 nm). All three methods showed comparable zeta potential values (-9 to -14 mV), which was adequate for the physical stability of the vesicles. LC-MS/MS method developed for the determination of phytosterols, and tocopherols had a run time of only seven minutes with excellent linearity (R2 = 0.998). An entrapment efficiency of >89% was obtained for target analytes. LC-MS/MS method for the determination of POPs had a run time of five minutes which is the shortest run time among reported methods for the determination of POPs. Only one POP (7-ketobrassicasterol) was in the quantifiable range in the liposomal formulations indicating that the preparation method results in negligible levels of POPs. In fact, the quantified value for 7-ketobrassicasterol was too low to cause cytotoxicity. Microwave heating (i.e., accelerated stability study) showed the presence of various POPs with the following ascending order for their generation: liposomal formulation of phytosterol and tocopherols < liposomal formulation of phytosterols < crude phytosterols. This confirms that the generation of POPs is prevented by the protective action of liposomes as well as antioxidants. Finally, animal testing showed a significant reduction of LDL-C by the liposomal phytosterols compared to the control.\nConclusion: In conclusion, liposomes are an excellent carrier to deliver phytosterols as they imparted high entrapment efficiency, prevented phytosterols’ oxidation in the presence of tocopherols, and showed a significant effect in reducing LDL-C in animals.

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