| Multivesicular liposomes (MVLs) were prepared by double emulsion method.The first step was making a W/O emulsion by emulsifing lipid solution with firstaqueous solution which contained drugs or other biologically active substances usinga high-speed homogenate machine. Then the W/O emulsion was emulsified with thesecond aqueous solution by a vortex suspension instrument to form a W/O/Wemulsion. MVLs were prepared after the organic solvent was volatilized out by arotary evaporator. Mitoxantrone hydrochlotide (DHAD) and horseradish peroxidase(HRP) were chosen as small molecule and macromolecule drugs respectively toprepare DHAD-loading, HRP-loading and DHAD/HRP-loading MVLs. Then themorphology, Zeta potential, the phase transition temperature and drug leakage rate ofdrug-loading MVLs were studied and the in vitro release performances of them werealso investigated. Furthermore, the in vitro tumor suppressing activity of drug-loadingMVLs were also studied.Firstly, dichloromethane was used as the organic solvent to dissolve soybeanlecithin and cholesterol and form the oil phase to prepare MVLs. The encapsulationefficiency of DHAD was taken as a optimization goal, and the preparation processtogether with the prescription of MVLs were optimized by single factor experimentsand the uniform design experimen, the results were as follows: concentration ofsoybean lecithin, cholesterol, triolein, L-Lysine, sucrose and glucose were16mg/mL,12mg/mL,12mg/mL,40mmol/L,5%(w/v) and7.5%(w/v) respectively; volumeratio of oil phase and first water phase was1:1; volume ratio of W/O emulsion andsecond water phase was2:3; speed of homogenate machine was20000rpm; the firstemulsification time was10min and the second emulsification time was10s. Afteroptimization, the encapsulation efficiency of DHAD achieved90%.Secondly, the qualities of DHAD loaded multivesicular liposomes(DHAD-MVLs) were estimated. DHAD-MVLs were round or oval with granularinternal structrues. The average particle size of DHAD-MVLs was58.75μm with aspan of1.10. Zeta potential of DHAD-MVLs was-47.1mV while the phase transition temperature was38.8oC. The leakage rate of DHAD in168h were55.9%and23.68%respectively when stored at37oC and4oC. Within the scope of the study, cumulativerelease rate of DHAD could be reduced effectively with appropriate usage ofcholesterol while with improving the amount of triolein, the release rate of DHADincreased. Throughout the release process, the release rate of DHAD was below30%in first0.5h and higher than80%in120h, which indicated that there was no initialburst release and the release was complete.Thirdly, HRP-loaded multivesicular liposomes (HRP-MVLs) were prepared andthe qualities were estimated. The effect of speed of homogenate machine onecapsulation efficiency and activity of HRP was studied first. When the speedchanged from10000rpm to20000rpm, the ecapsulation efficiency of HRP increasedto larger than90%with the specific activity of HRP decreased to70%. The averageparticle size of HRP-MVLs was19.36μm with a span of2.41. Zeta potencial ofHRP-MVLs was-52.7mV while the phase transition temperature was38.32oC. Theleakage of HRP in168h were32.8%and45.4%respectively when HRP-MVLs werestored at4oC and37oC. The effect of cholesterol and triolein on cumulative releaserate of HRP was similar to that of DHAD. During the release process, there was noinitial burst release either and the release was complete.Fourthly, DHAD and HRP co-loaded multivesicular liposomes(DHAD/HRP-MVLs) were prepared and the qualities were studied. The effect ofratio of DHAD to HRP on the ecapsulation efficiency of DHAD/HRP and the activityof HRP was studied. The results showed that with the accretion of HRP, encapsulationefficiency of DHAD and HRP were both reduced, but the activity of HRP was almostunaffected. Quality assessment of DHAD/HRP-MVLs with different ratio of DHADto HRP indicated that the change of drug ratio had little influence on the properties ofDHAD/HRP-MVLs. In vrito release study showed that DHAD and HRP could gain asustained release from MVLs, and the release rate of DHAD was faster than that ofHRP, DHAD/HRP-MVLs exhibited an orderly release behavior.Finally, the in vitro tumor suppressing activity study showed that HRP had someinhibitory effect on Bcap-37cells, but it could not improve the inhibitory effect ofDHAD. Blank MVLs of high concentration showed an apparent inhibitory effect on Bcap-37which had no significant difference with that of HRP-MVLs. DHAD-MVLsand DHAD/HRP-MVLs both had good inhibitory effect on Bcap-37, and the resultsindicated that DHAD and HRP could be released slowly from MVLs. |
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