Preperation Of Folate-targeted Ursolic Acid Stealth Liposome And Its Antitumor Effects

Objectives:Ursolic acid (UA) has extensive biological effect and anti-cancer effect. However, its wide application was limited by poor hydrophilicity and low bioavailability. The purpose of this study is to take advantage of the characteristics of the multi-expression of folate receptors on some tumor cells, encapsulating UA in folate targeted liposome can increase the drug concentration in the tumor target tissue. Such approach will improve the solubility, bioavailability of UA, dramatically improve the efficacy and prolong the time of drug action, reduce drug toxicity and provide a new ideas and means for clinical cancer chemotherapy.Methods:Liposome of UA was prepared by thin-film dispersion method. And we investigated the physical and chemical properties of liposome such as particle size, drug loading capacity, encapsulation efficiency, various factors such as zeta potential and stability.Choosing appropriate targeted tumor cells, cellular uptake efficiency and other assays were performed to show that folate-receptor targeted liposomal UA (FTL-UA) has more cytotoxicity and higher apoptosis than non-targeted PEGylated liposomal UA (PL-UA) in vitro. we observed the antitumor effect on human oral epidermoid carcinoma tumors (KB) cell line was investigated by MTT method. The nuclear morphology of cells was evaluated using Hoechst33258(the cell-permeable DNA fluorescent dye). And the apoptosis and cell cycle distribution changes of KB cells induced by UA liposome was studied by Flow cytometry analysis.Then we investingated the pharmacyokinetic characterictics, distribution and targeting effect of UA liposme in vivo. We analyzed the drug concentration of plasma, heart, liver, spleen, stomach, kidney in mice, and inspected the difference between PEGylated stealth nano-liposomes and free drug in the body long circulation function and organization distribution. Therapeutic effect of folate targeted UA liposme on balb/c nude mice bearing KB tumor was also intensively studied.Results:1) In a typical preparation, liposomes were prepared by thin-film hydration dispersion method followed by high-pressure extrusion. The average particle diameter of folate-receptor targeted liposomal UA (FTL-UA) and non-targeted PEGylated liposomal ursolic acid (PL-UA) was (160.1±12.5) nm and (151.4±16.1) nm respectively. The entrapment efficiency was (88.9±7.9)%and (86.7±12.1)%respectively. The polydispersity index (PDI) was0.196±0.052and0.168±0.066respectively, with narrow particle size distribution and uniform size. And the zeta potential was-21.24±4.2,-23.15±6.7respectively. UA liposomes can maintain a better stability at4℃for3months.2) In vitro analysis of antitumor activity against human KB cell lines, showed a significant concentration and time dependent manner and the inhibition of the proliferation of KB cells following exposure to FTL-UA as compared to PL-UA (at72h, IC50:22.05μM vs.146.3μM). Effect of UA liposome induced apoptosis cycle in KB cells shows that FTL-UA mainly caused S cell cycle arrest. Briefly, UA leading to tumor cell apoptosis occurred in the DNA synthesis phase. Fluorescent staining analysis and flow cytometry also confirmed FTL-UA has a promoting role of inducing KB cell apoptosis.3) Six-weeks-old Kunming mice were treated tail intravenous injection of20mg/KG UA, the distribution half-life, elimination half-life, the drug area under the curve, mean residence time and elimination rate of UA liposome has significant differences than simply giving free UA, showed a better effect in vivo release and long-cycle. UA contents of liposomes in the liver and kidney are higher than that of other organizations. UA liposomes in other organizations were rarely distributed, only less residue.4) In vivo the antitumor activity of FTL-UA was investigated in balb/c nu/nu mice bearing KB xenografts. Mice were treated tail intravenous injection of free UA, PL-UA, or FTL-UA (4.5mg/Kg, respectively) for five times every other day. We observed a great higher inhibition of tumor growth in mice treated with FTL-UA versus other groups and there were necrosis voids in the surface of the KB tumor xenograft in each mice of FTL-UA group, but in other group, we haven’t seen this necrosis voids. The life cycle of FTL-UA group was significantly longer than any other groups.Conclusions:The experimental results suggest that FTL-UA may be used as a new type of chemotherapy drugs in cancer chemotherapy. It plays an active role in improving the solubility and bioavailability of UA, extending its duration of action, reducing their dosage and administration frequency. It also directed drug in targeted tumor and increase the drug concentration in the target tissue and significantly inhibited the growth of tumor tissue, exhibit an antitumor effect. Generally speaking, our study indicated that FTL-UA had a wide application of achieving great anticancer effects in clinical cancer treatment. Folate receptor (FR) targeting therapy could be a novel approach to the cancer treatment.