Study On Zoledronic Acid Cationic Liposomes As Targeting Anti-tumor Agents

Objective To prepare Zoledronic acid Cationic Liposomes (ZOL-CL), investigate its physicochemical properties and release behavior in vitro, and research anti-tumor effect in vitro and the pharmacodynamic in tumor-bearing mice.Methods The HPLC method was selected to evaluate the content of ZOL. ZOL-CL was prepared by film dispersion method with DPPC and DC-Chol as the carriers. The formulation was optimized based on single factor analysis including amount of the mass ratio of DPPC and DC-Chol, Chloroform volume, Rotary evaporation time, PBS volume, ultrasonography time. Morphology of ZOL-CL was observed by transmission electron microscope. Mean particle size, PDI and Zeta potential was estimated by Laser particle size analyzer. Entrapment efficiency and drug loading were investigated by ultracentrifugation and HPLC. Release behavior in vitro was studied by dialysis. SRB method was used to investigate the proliferation of A549tumor cells, and evaluate the anti-tumor effect of ZOL-CL in vitro. Evaluated the tumor-suppressing effect of tumor-bearing mice through ZOL-CL. Researched the immunohistochemical study of tumor tissue by determined the expression of vascular endothelial growth factor(VEGF) protein and cyclooxygenase-2(COX-2) protein.Results The HPLC method was established to evaluate the content of ZOL-CL and the calibration curve showed a good linear relationship in the concentration range of15.0～200μg·mL-1(r=0.9999). The morphology of ZOL-CL was spherical. The mean particle size, polydispersity index, Zeta potential, entrapment efficiency and drug loading were (106.76±1.94) nm,(0.262±0.027),(42.37±2.60) mV,(38.54±0.99)%and (3.42±0.27)%, respectively. The release behavior in vitro showed a significant sustained release characteristics in NaOH solution at pH10.0. The profiles of release in vitro was expressed well by Weibull equationin which was lnln (1/1-Q)=0.4472lnt-1.3514, r=0.9863. In the study of anti-tumor experiment in vitro, the IC50of ZOL group, ZOL-Lipo group, ZOL-CL group were (184.6±12.31)μg·mL-1. (66.92±3.75)μg·mL-1,(6.38±1.66)μg·mL-1, respectively. The growth curve of A549cells and the observation of A549cells by interted microscope also showed that ZOL-CL was better than ZOL-Lipo and ZOL to inhibit the proliferation of A549cells. In the study of pharmacodynamics, The average tumor weight of saline group, ZOL group, ZOL-Lipo group, ZOL-CL group were (1.73±0.57) g,(1.69±0.69)g,(1.09±0.37) g,(0.90±0.38)g, respectively. Compared with the control group, the tumor inhibition rate of ZOL group, ZOL-Lipo group, ZOL-CL group were2.10%,36.66%,48.18%, respectively. Immunohistochemical study showed that the expression of VEGF protein and COX-2protein were high in control group, medium in ZOL group, low in ZOL-Lipo group, and lower in ZOL-CL group.Conclusion The results of physicochemical properties and release behavior in vitro showed that ZOL-CL was successfully achieved. ZOL, ZOL-Lipo, ZOL-CL could inhibit the proliferation of A549tumor cells when compared with the PBS control group, and ZOL-CL had the strongest inhibition of cell proliferation. Antitumor test in vivo suggested that tumor-bearing mice via intravenous administration with ZOL-CL, can significently inhibit tumor angiogenesis, through down-regulating the expression of VEGF protein and COX-2protein in the tumor tissue, thus suppressing the proliferation of tumor. The experimental results showed that the ZOL-CL has targeted, anti-tumor effect.