Study On Preparation, Quality Evaluation And Pharmacokinetics Of Liposomal Bufalin

Objective:Bufalin, an active single-entity in traditional Chinese material Chan Su, is a promising drug for several kinds of malignancies. However, the low water solubility, cardiotoxicity, neurotoxicity and rapid metabolism always limit its research and application. In present study, we adopted liposomes as drug carrier, prospectively not only improve the low solubility properties, but also have controlled release in sight. We designed PEGylated liposomal bufalin by surface modifying DSPE-PEG2000 on liposomal bufalin, and thus provided a thought of extending its blood circulation, prolonging half-life and expanding therapeutic window for glioma.The subject is to study on preparation,quality evaluation and pharmacokinetics of bufalin liposomes. Our purpose is to set up the high performance liquid chromatography(HPLC) method to determine bufalin in liposomes, compare and illuminate the pharmacokinetic rule of bufalin, common liposomal bufalin and PEGylated liposomal bufalin, and consequently provide a basis for pharmaceutic development of bufalin.Methods:A HPLC method was developed to detect bufalin in liposomes. The optimized preparation formulation and condition of liposomal bufalin were assessed by single-factor study and orthogonal test. The PEGylated liposomal bufalin was prepared by transforming the distearoyl phosphatidylethanolamine(DSPE) to the ramification of polyethylene glycol(PEG) and DSPE, DSPE-PEG2000 in the prescription, so as to stop plasma proteins adsorbing to surface of liposomes, avoid the mononuclear phagocyte system swallowing liposomes rapidly, and extend the blood circulation time, which will be in favor of relative accumulation of drug-carrying liposomes at lesion location. And the characterization, stability, pharmacokinetics and anticancer effects in vitro were estimated.Results:With a good linearity in concentration region of 0.80- 40 ?g/m L, the linear regression equation of bufalin is A=17991x-2183.6,R2=0.9999. The results of specificity, precision, stability, repeatability, recovery and sample detection accorded with the measure requirements. The liposomes, which were prepared by homogenization-film rehydration method, had uniform particle size and high entrapment efficiency. The selected and optimized preparation formulation and condition were: ratio of drug to lipid is 1:10; ratio of phosphatidylcholine(PC) to cholesterol(CH) was 4:1, and the volume of deinozed water was 30.0 m L.The above compositions in formulation were totally dissolved in 3.0 m L chloroform, and immediately evaporated under decompress condition, following with hydration using vortex by deionized water(H2O) at 50 degree centigrade for 15 min and high pressure homogenization at 500 bar for recycling 10 times. After that, H2 O was supplied in liposomes suspension to 30.0 m L. Then liposomes suspension was extruded through 200 nm Polycarbonate membrane filtration with an extrusion rapidly.Liposomal bufalin: percentage encapsulation efficiency(%EE) was 76.31 ± 3.40 %; mean particle size was 172.1 ± 3.4 nm(PI = 0.237); surface charge was 2.24 ± 0.33 m V. Towards PEGylated liposomal bufalin, %EE was 78.40 ± 1.62 %; mean particle size was 178.9 ± 4.21 nm(PI = 0.285); surface charge was-18.05 ± 2.57 m V. The results showed that both liposomes suspension had approximate spherical shape and good dispersion.The liposomes suspension had good stability and could be stored steadily at 4 ?. The release of liposomes in vitro was slowed down compared with free bufalin entity. In the meantime, the inhibition effect of PEGylated liposomal bufalin on U251 glioma cancer cells was enhanced.The plasma area under the curve(AUC) of liposomes was greater and elimination rate of liposomes were lower than that of free bufalin. The pharmacokinetic parameters calculated of bufalin, liposomal bufalin and PEGylated liposomal bufalin were: Cmax=1326.628±24.519, 1254.022±16.604, 1071.332±36.311 ng/m L; AUC(0-t)=25012.029 ±2246.286, 57137.521±1453.14, 138046.006±2771.79 ng/m L·min; T1/2z=40.516±10.607, 54.396±3.329, 87.842±7.79 min.Conclusion:Compared with common liposomal bufalin, PEGylated liposomal bufalin have high stability as well. Moreover, PEGylated liposomal bufalin have better biological activity, controlled release properties and antitumor effect on U251 glioma cells. PEGylated liposomes can effectively reduce metabolic rate, prolong the elimination half-life, and increase bioavailability of bufalin.It is concluded that PEGylated liposomal bufalin have promising potential applying to the cure of glioma cancer.