| Mitoxantrone(MIT)is an anthracenedione antineoplastic agent,which is a cell-cycle non-specific broad-spectrum anticancer drug.MIT may cause several adverse reactions with a varying severity,e.g.cardiotoxicy.As carrier of anticancer drugs,Liposome has many excellent characteristics such as tumor targeting,toxicity reducing and efficacy enhancing.However,the fact that release of MIT from liposomes is difficult to control limits the application of liposome technology in MIT drug development.Thermosensitive liposomes use the phospholipids whose phase transition temperature is slightly higher than the body temperature to encapsulate drugs.After administration in vivo,the encapsulated drugs can be rapidly released to certain area via a local heating method which triggers targeted release to heating tissue.Thermosensitive liposomes can be modified by long circulation materials which formed hydrophilic layer and sterically hindered on the surface of liposomes to avoid being phagocytized by RES.This effect reduces blood clearance rate,prolongs circulating time and enhances targeting efficacy.Objective:This study selected MIT as the model drug to prepare the long circulating-thermosensitive liposomes.MIT liposomes combined with hyperthermia which increase concentration of liposomal drugs in tumor tissues and trigger rapid release can improve targeting and therapeutic efficacy.Methods:The preparation method of MIT long circulating thermosensitive liposome was preliminarily determined based on the preformulation study.First,the blank liposomes which contain citrate solution were prepared.Secondly,the extraliposomal buffer was exchanged by column dialysis method to form transmembrane pH gradient.Thirdly,drug solution and liposomes were mixed and incubated for a period to realize the encapsulation of MIT.Single factor experiment was adopted to optimize the preparing process of MIT long circulating-thermosensitive liposome on the basis of the encapsulation efficiency(EE)%and content of the liposomes.The factors,such as different dialysis solution,the incubation temperature,the incubation time and the ratio of drug to lipid,which affect EE%were investigated to determine the best prescription of MIT long circulating-thermosensitive liposome.Plasma pharmacokinetic analysis was performed in BDF1 mice.A HPLC-MS method was developed for detecting the MIT in mice plasma.The toxicity of thermosensitive liposome was evaluated in BDF1 mice.The antitumor efficacy between free MIT and MIT encapsulated in thermosensitive liposome were compared in RM-1 bearing mice.To investigate whether accelerated blood clearance(ABC)phenomenon could be induced after repeated injection of MIT long circulating-thermosensitive liposome,Plasma pharmacokinetic analysis was performed in normal Wistar rat.A HPLC-MS method was developed for detecting the MIT in rat plasma.Furthermore,anti-PEG IgM in plasma was detected by ELISA to reveal the reason why the pharmacokinetic parameters were changed.RM-1 bearing mice were used to study the distribution of MIT in liver and tumor after administration of the thermosensitive liposome coordinating with different ways of hyperthermia.Results:The best prescription determined by the experiments was:DPPC:MSPC:mPEG2000-DSPE was 90:10:4(molar ratio),the ratio of drug to lipid was 1:10(w/w),the inner phase solution was 0.3 mol/L citrate solution(pH=4.0),dialysis solution was 20mmol/L histidine solution,the incubation temperature was 35?,the incubation time was 45 min.The EE%of thermosensitive liposome was more than 98%.The in vitro release and cellular experiments have shown the thermal properties of thermosensitive liposomes.After incubating 1 h,durg released from mitoxantrone long circulating thermosensitive liposomes less than 10%at 37?,while almost all of the drug released at 41?.In cell experiments,after being treated by mitoxantrone long circulating thermosensitive liposomes at 37? for 45 min,no significant suppression on cell growth was observed,indicating very little durg release from the liposomes;however,after being treated at 41? for 10 min or 45 min,a significant suppression on cell growth was observed,which proved that the drug released quickly at this temperature.The results of quality study of three batches thermosensitive liposomes demonstrate were as follows:pH value is 6.53,osmotic pressure is 309 mOsmol/Kg,paticle size is 100.1 nm,EE%is 98.41%.The stability of thermosensitive liposomes has indicated as follows:the liposomes showed preferable stability for 2 month under 4-8 ? storage;there was no significant change in EE%,pH,paticle size and content;but the thermal properties disappeared during the long storage time.These resluts indicated that the disappearance of thermal properties was due to the existence of slow sedimentation between MIT and inner citrate solution.The pharmacokinetic studies showed that:there is a significant difference of plasma pharmacokinetic between free MIT and MIT liposomes:the AUC,elimination half-life,and clearance rate of them were 5.332 ?g·h·ml-1,1.294 h,0.719 ml·h-1·g-1 vs.0.583 ?g·h·ml-1,0.412 h,6.691 ml·h-1·g-1,respectively.These results demonstrated that the MIT encapsulated in long circulating thermosensitive liposomes could increase the circulation time in vivo.Toxicity and pharmacodynamic experiment demonstrated that MIT liposome did not reduce the toxicity compared with free MIT,and the MTD of both were less than 10.2 mg/kg.In the pharmacodynamic experiment,the inhibition rate on tumor of free MIT and MIT long circulating-thermosensitive liposomes were 27.9%and 86.1%,respectively.Thus,MIT long circulating-thermosensitive liposomes could increase the therapeutic index by improving the antitumor effect.The studies of ABC phenomenon show that:after the first dose,AUC was 1.905±0.121?g·h·mL-1 and CL was 1251.291±229.132 mL·h-1·kg-1;while after the second dose,AUC was 0.581±0.082 ?g·h·mL-1 and CL was 3567.959±374.738 mL·h-1·kg-1;The detection of antibody in vivo indicated the existence of high level of anti-PEG IgM in blood at 7th day after first intravenous injection of MIT long circulating-thermosensitive liposome,and the anti-PEG IgM could bind to the liposome drug administered at the second injection.Combining with pharmacokinetic studies and the change of antibody level,it is proved that repeated administration of MIT long circulating-thermosensitive liposomes in rat caused the ABC phenomenon.Tissue distribution experiment showed that the time of hyperthermia could influence the drug distribution after administration.Drug concentration in tumor tissue increased more significantly when heating immediately after injection.Conclusion:MIT was used as a model drug to prepare long circulating-thermosensitive liposomes,which had high EE%and significant thermal properties.Combining with hyperthermia,this liposome could significantly increase the antitumor effect and improve the therapeutic index.We also use simulation experiment to explain the disappearance of the thermal properties characteristics after long-term storage.It was found that the osmotic pressure of dialysate buffer could affect the durg loading of long circulating-thermosensitive liposomes.Combining with pharmacokinetic studies and the change of antibody level,it is proved that repeated administration of MIT long circulating-thermosensitive liposomes in rat caused the ABC phenomenon. |
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