Study On The Bufalin Liposome

Chan'su is a traditional Chinese medicine. Recent studies found an active antitumor component bufalin (BF) in chan'su. The bufalin selectively induces differentiation and apoptosis of human leukemia cells, and enhances retinoic acid induced differentiation of acute promyecytic Leukemia cells in primary culture. In combination with other anticancer drugs the bufalin may be very useful in the differentiation of human leukemia. Due to its high toxicity and low therapeutic index, we designed BF liposomal formulations to explore the possibility for anti-cancer therapy using bufalin. Bufalin was isolated from the chan'su by silica gel column chromatography and confirmed by ?C-NMR and C-NMR. The encapsulation efficiency, size distribution and Leakage of bufalin liposomes were studied. The results showed that the encapsulation efficiency of bufalin in liposomes were more than 98%, the mean liposome sizes were 840.6 nm and the Leakage amount of BF from the liposomes were about 45% at 37 0C in 36h. A combination of high-resolution solid-state ?C-NMR, 31P-NMR and differential scanning calorimetry (DSC) was used to study the thermotropic and dynamic properties of bufalin in egg phosphatidylcholine model membranes. DSC of bufalin liposomes showed that the bufaLin decreases the main phase transition temperature and increases the span of temperature of the phase transition. The ?C-CP/MAS revealed that the specific peaks disappeared and the relative intensities of the specific peaks changed in NMR spectra of bufalin liposomes. These results suggest the bufalin was perturbated into the lipid bilayer. The 31P-NMR revealed that the bufalin didn affect the lipid biLayer structure of the liposome, when it was untreated by ultrasonic waves. The liposome ultrasonded, the lipid bilayer of liposome was transformed into hexagonal (H), and the transformation extent was increased with the amounts of bufaLin in the liposome. The growth inhibitory effect of BE, PC-BF-ChoL-L, PC-BF-OA-L and PC-BF- DSPG-L on human leukemia cells was studied with a MTT method. The results showed that after three days of continuous exposure to PC-BF-Chol-L, PC-BF-QA-L, BE, and PC-BF-DSPG-L, the IC50 for HL-60 cells were found to be 2.5x108M, 16xlO~8M 2.0xl08M, and 2.0xl08M, respectively. Chromatin condensation and nuclear fragmentation were observed in HL-60 and U937 cells stained with Giemsa under microscopy. Agarose gel eLectrophoresis revealed typical DNA fragmentation (ladder patter) of HL-60 following treatment with the BF and BE liposoms. Cell cycle distribution exhibited that the BF and BF liposoms arrested cells in G2/M phase in HL-60 cells. The cellular differentiation, as assessed by morphological criteria, was induced by culture in the presence of PC-BF-DSPG-L. The ability of PC-BF-DSPG-L treated HL-60 cells to reduce nitrobluetetrazolium demonstrated that they were functionally different- iated cells. The acute toxicity of PC-BF-DSPG-L in mice showed that the LD50 was 2.68 mg/kg, it exceeded that of the bufalin, which had a LD50 of 1.64 mg/kg by ip administration. The antitumor activity of PC-BF-DSPG-L was evaluated against Lewis lung tumor. The equal dose of BF and PC-BF-DSPG-L showed similar antitumor effect, the equal toxicity dose of bufalin exhibited the PC-BF-DSPG-L was more potent than that of the BF, the rates of inhibiting against tumor were 46.74% and 39.13%, respectively.