Study On The Liposomal Nutriment And Microbubble Ultrasound Contrast Agents

Liposome is a new technique that microcapsule and nanotechnology combine together. It can encapsulates nutriment, such as CoQ₁₀, to prepare CoQ₁₀ liposomes; Also it can encapsulates gas, such as SF6, air, etc., to prepare microbubble ultrasound contrast agent. Furthermore, with microbubbles as the carrier of CoQ₁₀, it can attain the effect of heart-targeted release, which can improve the bioavailability of CoQ₁₀.Firstly, filming-rehydration method was selected to prepare CoQ₁₀ liposomes. The optimization of the preparation parameters of CoQ₁₀ liposomes were as follows: ultrasound power (50%), pulse ultrasound (1 s/1 s) time (5 min), hydration pH (7.4), hydration temperature (40℃) and hydration time (1 h). Achieved by single factor experiments, the best composition of wall materials was as follows: lecithin (2%), cholesterol (0.4%), PEG4000 (0.2%), palmitic acid (0.2%); and the largest loading capacity of CoQ₁₀ was ₁₀%. CoQ₁₀ liposomes employing these parameters had homogeneous shape with average particle size 116.8 nm, and Zeta potential -35.0 mV. The encapsulation efficiency of CoQ₁₀ liposomes was greater than 90%, and retention rate was higher than 90% after storage at 4℃for 60 days.Also using filming-rehydration method to prepare microbubble ultrasound contrast agents. The average diameter, concentration, and half life of microbubbles were taken as criteria, the optimization of the preparation parameters of microbubbles were as follows: hydration pH (7.4), hydration temperature (45℃) and hydration time (1-2 h), ultrasound power (50 %) and ultrasound time (5 min); The best formulation of wall materials consisted of lecithin/cholesterol/PEG/palmitic acid (20:5:2:2, w/w). Microbubbles employing such parameters had the average diameter 2.54μm, which 99% microbubbles remained 1-5μm, and the concentration was graeter than 4.55×₁₀9/mL with half life above 1 h. The air content of microbubbles was 7.01μL/mL, which was propitious to microvasculature imaging.Furthermore, by CoQ₁₀ incorporated in microbubbles, we were able to prepare nutriment-combined microbubbles, whose average diameter was 2.02μm. The largest loading capacity of CoQ₁₀ was ₁₀%. After preparing suitable microbubbles, we carried on a stability test in vitro. As the result, microbubbles were very stable to pressure and temperature, while microbubbles made of DSPC were more stable than those made of EPC. After PEG was added, the circulating time of microbubbles in the body was increased. Imaging test in vitro indicated: Microbubbles can scatter ultrasound waves very well, thus they were the ideal ultrasound contrast agents. The result of animal test indicated: PEG4000 was the best protectant of lipid membrane. Adding 0.2% palmitic acid can help to improve the stability of microbubbles and the effect of imaging. When EPC was used as the primary wall material, it got the best imaging effect at EPC content of 2%, but it still had no advantages over microbubbles made of DSPC. Even the content of DSPC reduced to 0.5%, microbubbles can got a very good imaging effect, and imaging lasted for 2 to 3 minutes. The gas encapsulated also influenced the imaging effect. SF6 was the ideal gas, which had large molecular weight, low diffusibility and solubility. Based on imaging, Nutriment-combined microbubbles can delievered nutriment to the target position, and then release the nutriment. In order to store microbubble ultrasound contrast agents for long time, proliposomes were prepared byfreeze-drying. The optimization of the preparation parameters were as follows: Using galactose (0.5%) as the cryoprotectant; Without foaming process; The thickness of the material was 3 mm; Prefreeze for 12 h at -80℃. The temperature control programme was as follows: Keep the temperature at -30℃for 28 h, then heat up to -₁₀℃at the rate of 1.5℃/min, at last keep the temperature at -₁₀℃for 25 h. After rehydration, the average diameter of microbubbles was 2.93μm, and the concentration was graeter than 2.25×10⁸/mL. It can be used as ultrasound contrast agent, and imaging lasted for 2 to 3 minutes.