Preparation And Release Of Anti-tuberculosis PaMZ Liposomes In Vitro

Objective The goal of this study is to construct liposomes of PaMZ,a new triple anti-tuberculosis drug combination[Pa（Pretomanid,Pa-824）,M（Moxifloxacin）,Z（Pyrazinamide）],by the ammonium sulfate gradient method and to observe the in vitro drug release of PaMZ liposomes.Methods High performance liquid chromatography（HPLC）was used to establish the in vitro determination and analysis method for the three drugs in PaMZ liposomes prepared by the ammonium sulfate gradient method.The evaluation indicator was the encapsulation efficiency of pyrazinamide.The optimal conditions for the preparation of PaMZ liposomes were screened by a four-factor,three-level L₉（3⁴）orthogonal experimental design,in which the four influencing factors of cholesterol to phosphate ratio（A,1:1、1:2、1:4）,drug to phospholipid ratio（B,1:1、1:2、1:4）,ammonium sulfate concentration（mol/L）（C,0.1、0.2、0.3）and incubation temperature（℃）（D,50、55、60）were investigated.The PaMZ liposomes were prepared based on the optimal manufacturing conditions described above and their physicochemical properties were determined.In vitro drug release experiments were performed by membrane dialysis,and the cumulative drug release efficiencies of PaMZ liposomes were measured at the specified times of 1h,4h,8h,12h,24h,36h,48h and 72h,fitted with Zero-Order Kinetics,First-Order Kinetics and Higuchi equation,and the cumulative drug release time curves were plotted to observe their in vitro drug release performance.Results（1）The experimental results showed that the drug concentrations of the three drugs Pa-824,moxifloxacin and pyrazinamide had a good linear relationship with the detection peak areas when the three drugs were detected in the range of 1～200μg/m L.The average spiked recoveries were 100.38%for Pa-824,100.48%for moxifloxacin and 100.30%for pyrazinamide,with small relative standard deviations for the spiked recoveries and intra-day and inter-day precision for all three drugs.（2）Nine different PaMZ liposome production protocols were developed according to the orthogonal L₉（3⁴）experiment,and the measured liposome inclusion rates were 39.8%,69.2%,73.2%,36.8%,71.2%,70.6%,33.2%,56.5%and 60.4%,in that order.The comparison of the magnitude of the extreme difference R values of the four factors shows that the superiority of the effect on the encapsulation efficiency is factor B（drug to phospholipid ratio）>factor A（cholesterol to phospholipid ratio）>factor C（concentration of ammonium sulfate）>factor D（incubation temperature）.The results of multi-factor ANOVA showed that the effects of factor A（cholesterol to phospholipid ratio）,factor B（drug to phospholipid ratio）and factor C（ammonium sulfate concentration）on the liposome encapsulation efficiency were statistically significant,while the effect of factor D（incubation temperature）on the liposome encapsulation efficiency was not statistically significant.（3）The release profile of PaMZ liposomes exhibited a biphasic drug release profile,with the first phase being a rapid release phase for the first 12 h,a slower release process follows.PaMZ liposomes exerted a slow-release effect on the release of Pa-824,moxifloxacin and pyrazinamide.The release data of Pa-824,moxifloxacin and pyrazinamide from PaMZ liposomes were fitted to the Zero-Order Kinetics,the First-Order Kinetics and Higuchi equation release curves,respectively.The results showed that the in vitro drug release process of PaMZ liposomes complied with the first-order pharmacokinetic equation based on the R² of each model.Conclusion（1）PaMZ liposomes were successfully prepared with high encapsulation efficiency.（2）PaMZ liposomes showed good slow-release performance and showed biphasic release within 72 hours,which laid the foundation for subsequent anti-tuberculosis studies and provided a new idea for clinical transformation of spinal tuberculosis treatment in the future.