| Toad venom, a traditional Chinese medicine, has been exploited for thousands of years in China. The principal antitumor-active components of toad venom are bufadienolides. In this paper, the major bufadienolides extracted from toad venom including bufalin (B), cinobufagin (C) and resibufogenin (R) were loaded into liposomes and nanostructured lipid carriers in order to reduce the vascular irritation, improve the short half-life and widely biodistribution in vivo which was caused by toad venom after intravenous administration. The preparations will also increase the drug concentration in the tumor and avoid the unwanted uptake by heart.The TLC and HPLC methods were developed for the qualitation and quantitation assay of raw material, extract and the final separated products. The raw material were separated by 80% ethanol reflux extraction, ethyl acetate extraction and loaded on silica gel column using the content and the transfer rate of bufadienolides as index. The final separated products contained mainly B, C and R (purity>95%) with a transfer rate above 70%. Preformulation study showed the solubilities of bufadienolides in water were very low, the solubility and oil/water partition coefficient of bufadienolides were not affected by pH. Their log P values were all around 3. The stability study showed the solid bufadienolides were stable at different stress conditions. But bufadienolides solution at pH below 4 or above 8 were not stable, the suitable pH would be between pH 6 and 7. In the enzymolysis study, B and R were very stable, whereas C was readily degradable in rat plasma. The enzyme kinetics of C followed pseudo-first-order kinetics.Regarding the appearance, particle size and entrapment efficieny (EE) of formulation as index, bufadienolides-loaded liposome (BU-lipo) was investigated in detail. In the optimal formulation, Lipoid E80(?) was at 1.25%, the mass ratio of cholesterol and a-tocopherol to lipid were at 1:20 and 1%.10% trehalose was used as cryoprotectants in the lyophilization process. The lyophilization cycle consisted of primary freezing at -75℃for 12 h, drying at -35℃for 1 h, then, the shelf temperature was increased to -25℃for 12 h and secondary drying at 20℃for 4 h. BU-lipo were mainly unilamellar vesicle. The media diameters of BU-lipo was about 96.5±50.6 nm, the range of zeta potential was -8 to -4 mV, the pH value was around 6.5, and the EEs of B, C and R were 86.5%,90.0% and 92.1%, respectively. The in vitro release of BU-lipo followed the Weibull equation and the stability of cinobufagin (C) in rat plasma was enhanced by being encapsulated in particles of BU-lipo. Additionally, the precise and reproducible microdialysis method was applied to measure the EE of microcarrier systems.Bufadienolides-loaded nanostructured lipid carriers (BU-NLC) was prepared by a modified melt-emulsification ultrasonic technique, with glyceryl monostearate, medium-chain triglyceride and oleic acid as lipid carrier, Lipoid E80(?), Pluronic F68 and sodium deoxycholate as emulsifiers. The mean diameters and zeta potential of BU-NLC were 104.1±51.2 nm and -15 to -20 mV, respectively. The EEs of the bufadienolides were all above 85% with pH at 6.8 to 7.2. The particles of BU-NLC were spheric under transmission electron microscopy and refrigeration etcher transmission electron microscope. DSC and XRD showed that BU-NLC was in an amorphous state after lyophilization. The in vitro release of BU-NLC followed the Weibull equation and entered the platform stage after 72 h. BU-NLC has a good performance of controlled release of bufadienolides. The half-life of C (9.34 h) in BU-NLC was 1.72- and 17-fold than that in BU-lipo and bufadienolides solution (BU-S) at the plasma concentration of 80%. The lipid concentration and cryoprotectants were very important during the freeze-drying process. The final concentrations of lipid materials and sucrose were 0.3% and 10%, then, the aggregation of BU-NLC could be prevented completely.A high sensitivity, rapid and specific ultra performance liquid chromatography tandem mass spectrometry method has been developed for simultaneous determination of three bufadienolides in biological sample. The pharmacokinetics study showed BU-NLC exhibited a linear pharmacokinetic behavior at doses ranging from 0.25 to 1.0 mg/kg. The overall plasma concentrations of B, C and R following administration of BU-NLC were slightly higher than those of BU-S. The AUC(0-∞) of B, C and R was 1.87-,1.96- and 2.84-fold higher than that of BU-S, and the Cmax was increased (P<0.05). The Vss and CL of BU-NLC were also decreased. The biodistribution study showed BU-NLC resulted in higher drug concentrations in brain, spleen and liver, but lower concentrations in heart compared with BU-S at dose of 1.5 mg/kg for normal Kunming mice. As for the S180-tumor-bearing mice after multiple or single-dose of BU-NLC and BU-S at dose of 1.0 mg/kg, the relative distribution of drug in different tissues was similar to the normal mice. The three bufadienolides have been distributed in tumor in sequence of R>C>B. BU-NLC also resulted in higher drug concentrations in tumor, brain, spleen and liver, but lower concentrations in heart. However the result was based on insufficient data without statistical significant difference. It was noteworthy that for both BU-NLC and BU-S bufadienolides were distributed in the sequence of lung>brain>spleen>kidney>heart>liver. Bufadienolides were different from other drugs, the lowest concentrations were observed in liver and a relative higher accumulation in brain with the longest retention.The in vitro cytotoxicity of BU-NLC was examined by microculture tetrazolium (MTT) colorimetric assay using two tumor cell lines, human astrocytoma cell line (U87-MG) and human gastric carcinoma cell line (HGC-27). BU-NLC had a similar cytotoxicity to the free drug against the two cell lines, which was time- and concentration- dependent. The in vivo antitumor effect of BU-NLC was assessed using S180-tumor-bearing mice as a model. BU-S at the dose of 1.0 mg/kg/d showed a lower inhibition rate (36.79%) which was equal to that of BU-NLC at the dose of 0.25 mg/kg/d. The grown of S180-tumor could be effectively inhibited at doses of 0.25,0.5 and 1.0 mg/kg/d BU-NLC. The tumor inhibition effect (up to 67%) rose with increase of dose. Meanwhile the bufadienolides showed no decrease in the thymus and spleen index in S180-tumor-bearing mice which was occurred in the cyclophosphamide group. The acute toxicity experiment showed the LD5o of BU-NLC was 2.95 mg/kg, which was 1.28-fold higher than that of BU-S. In the safety study, the BU-NLC aqueous suspensions were non-haemolytic up to the tested concentration of 1.2 mg/mL.In was showed that the BU-NLC was able to improve the in vivo behavior and antitumor activity of bufadienolides. BU-NLC was with good physical stability and safe for the intravenous administration. We hope to give reliable scientific basis including formulation, pharmacology and toxicology are provided for clinical application of toad venom. |
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