Optimization of the preparation of sonogenic phospholipids-based microbubbles by using central composite experimental design and response surface methodology.

Sonogenic microbubble agent is a newly developed drug targeting delivery system, which uses ultrasonic beam to enhance the delivery of drug and gene to targeted cells and tissues. In this paper, the preparation of sonogenic phospholipids-based microbubbles was optimized by using central composite experimental design (CCD) and response surface methodology (RSM). Hydrogenated egg phosphatidylcholine (EPC), Tween 80 and polyethylene glycol 1500 (PEG 1500) were important components affecting the concentration of 2 - 8 microm microbubbles in the preparation. The combined effects of these three factors were analyzed by CCD and optimized by RSM. Evaluation variable was the concentration of 2 - 8 microm microbubbles. Overall desirability was fitted to a second-order polynomial equation, through which three dimensional response surface graphs were produced. Optimal experimental conditions were selected from the stationary point of the response surfaces. The stability of the sonogenic phospholipids-based microbubbles by the optimal formulation was investigated by accelerated experiment. The contrast effect in vivo of the optimal formulation was investigated. Foreign market product SonoVue was used as the control. From the results, all the three factors had positive effects on the concentration of 2 - 8 microm microbubbles. The optimal condition in the preparation of phospholipids-based microbubbles was obtained as following: EPC 8.35 mg, Tween 80 21.68 mg and PEG 1500 201 mg. The mean value of the concentration of 2 - 8 microm microbubbles in rechecking experiment reached 8.60 x 10(9) x mL(-1). From the accelerated experiment, phospholipids-based microbubbles showed good physical stability. The intensity (relative unit) and duration of the contrast effect by the optimal formulation were 4.47 +/- 0.15 and (302 +/- 7) s respectively, which showed little difference with foreign market product SonoVue [4.28 +/- 0.13, (309 +/- 8) s]. The optimal formulation selected by CCD and RSM showed high microbubble concentration, good physical stability and effective sonogenic contrast effect.