Studies On Mitoxantrone Folate-targeted Liposomes

The liposomal drug can increase the stability and decrease the toxicity.Meanwhile,the accumulation of encapsulated drug into tumor area is increased by enhanced permeation and retention(EPR)effect of the liposome.Thus the targeting effect of the drug towards tumor is enhanced.The active targeting liposome constructed by PEGylated phospholipids with folate(FA)modification can change the delivery pathway of the liposome by the specific binding of the folate and the over-expressed folate receptor on the tumor cell surface,and uptake the liposomes into tumor cells by endocytosis.The encapsulated drug is release from the liposome after accumulating into the tumor cells so as to exhibit its antitumor effects.Objective:This study intended to increase the accumulation of the drug in the tumor cells and to achieve effective release by preparing folate targeting liposome.Thus the therapeutic index is improved and the toxicity is reduced.Moreover,reversing of multidrug resistance of the tumor cell by folate targeting liposome was studied,which conducted to provide a method for the therapy of multidrug resistance of tumor.Method:Western Blot was employed in detecting the protein expression of folate receptor,and the real time quantitative PCR was adopted to determine the mRNA expression of the folate receptor.Therefore,the tumor cell hyper-expressed folate receptor was selected and used in the subsequent studies.The preparation of folate-targeted liposomes:The empty liposome with ammonium sulphate as the inner phase was firstly prepared.And then the outer phase was replaced by passing the Sephadex G-75 column.Thus an ammonium sulphate gradient was formed.Finally the drug solution was incubated with the empty liposomes after dialysis.Using cell uptake rate as an evaluation criterion and doxorubicin as an indicator drug for it is easy to dectect,orthogonal experiment of folate-targeted liposomes was designed in order to investigate how differences in content of DSPE-mPEG2000,type and content of folate excipient,particle size of the liposome,would affect the targeting of liposome to the tumor cell.Finally,the optimal formulation would be selected.The mitoxantrone folate-targeted liposome was prepared by the optimal formulation selected.The quality study,including the characteristics,and the determination of pH value,particle size,osmolality,encapsulation efficiency,content and the release rate,was conducted.Meanwhile,a two month stability study was carried out too.The cell uptake and inhibition study studies of free mitoxantrone,liposomal mitoxantrone(MIT-L)and folate-targeted liposomal mitoxantrone(MIT-FL)were carried out on human breast cancer cell MCF-7 and its multidrug resistant cell MCF-7/ADR to investigate the reverse of multidrug resistant by the folate-targeted liposome.In order to evaluate the special targeting effect of folate targeting liposome to folate receptor hyper-expressed tumor cells,the cell uptake and inhibition study was conducted on tumor cells hyper-and hypo-expressed folate a receptor and expressed ? receptor,respectively.Human KB Oral epidermoid carcinoma xenograft model built on NOD-SCID female mice was used in the pharmacological study of the mitoxantrone folate-targeted liposome,and the survival of the experimental animal,change of weight and change of tumor volume were observed.The therapeutic effects were evaluated by the inhibition rate of the tumor volume of the experimental animals.The toxicity of the drug was evaluated by the decrease of body weight.Results:The tumor cells hyper-and hypo-expressed folate a receptor and expressed ? receptor in protein and mRNA level were selected.The amount of expression is as follows:KB>BEL-7402>SPC-A-1>SMMC-7721>MCF-7,and tumor cells with folate-P expression,human Myelogenous leukemia MV4-11 and mice Lymphoma leukemia L1210.The results at both levels were substantially the same.The encapsulate efficiency of the folate-targeted liposomes prepared by ammonium sulphate gradient pathway was more than 99%,and of good reproducibility.The optimal formulations are:When calculated by the molar ratio in HSPC,1.DSPE-mPEG2000 is 2.6%?DSPE-PEG3350-FA is 0.4%,and the particle size is 90nm;2.DSPE-mPEG2000 is 2.6%?DSPE-PEG3350-FA is 0.4%,and the particle size is 70nm.The results of quality studies were:the pH,osmolality,particle size and encapsulation efficiency for 6 batches of 2 different formulations liposomes mentioned above were all consistent with the requirement.The in vitro release rate of 8 hr was 0.69%and 9.30%.The stability study result showed that:there were not any significant changes in all the indicators above after two months'storage at 2-8?In comparision of the content of free mitoxantrone and MIT-L entered into the MCF-7 cell and MCF-7/ADR cell,it was exhibited that the MCF-7 cell uptake of free mitoxantrone was much higher than that of the MCF-7/ADR,demonstrating that MCF-7/ADR was free mitoxantrone resistant;as to MIT-FL,the MCF-7/ADR cells uptake was higher than the MCF-7 cells.The reason might be that the liposome with folate linkage could bind to the folate receptor and be internalized in to the cell by endocyrosis,whose content are associated with the expression of folate receptor on the surface of the cell.The result of cell inhibition study showed that both MIT-FL and MIT-L can inhibit the growth of MCF-7 and MCF-7/ADR cell when enter the cell in liposomal encapsulated form.There is no significant difference for inhibition rate of MIT-FL and MIT-L at same concentration level to MCF-7 cell.As for MCF-7/ADR resistant cell,the inhibition rate for MIT-FL was significantly higher than that of the MIT-L at the same dose level.The result demonstrated that the liposome with a mechanism of folate-targeted can reverse the multidrug resistance.The result of cell uptake study demonstrated that the folate-targeted liposome is of higher uptake rate and targeting effect to tumor cells hyper-expressed folate receptor.As to the tumor cells hypo-expressed folate receptor,there's no significant difference in the targeting effect.Moreover,the uptake rates of the targeting liposome into the tumor cells hypo-expressed folate receptor are much larger than that hypo-expressed,suggesting that when the surface of liposome is modified by folate,the targeting ability to the tumor cells with hyper-expressed folate receptor.The result of MTT experiment showed that the 2 LUV formulations have no influence on the cells growth,while the mitoxantrone folate-targeted SUV liposome(MIT-FSL)can significantly inhibit proliferation of the cells,with an IC50 value of 3644.3ng/mL.Meanwhile the mitoxantrone SUV liposome(MIT-SL)free of folate showed some inhibiting effects two,whose IC50 value is 32583.8 ng/mL.So the inhibition effect of MIT-FSL is stronger compared to MIT-SL.Transmembrane delivery of the drug is increased and intracellular drug release is effective by using the small unilamellar vesicle(SUV)modified by FA,thus the antitumor effect of drug was enhanced by encapsulating in folate modified active targeting liposome.The toxicity result of KB Oral epidermoid carcinoma xenograft model in mice demonstrated that the weight loss of animal in group of free drug was significant compared to the liposome group.That mean the drug loaded in liposome is less toxic than free drug,suggesting toxicity can be decreased by encapsulating mitoxantrone in liposome.As to result of pharmacological study,inhibiting effects can be observed for all the groups.However,the inhibiting effect of liposome was slightly lower than that of the free drug.Since the accumulation of liposomes in tumor area depends mainly on the ability of diffusion from the vascular system into the tumor,the possible reason might be that the extravasation is the rate-limiting step of liposome accumulation in tumors compared to free drug,moreover,the relatively slow release maybe another reason.Conclusion:The folate-targeted liposome which surface modified with folate is prepared with high encapsulated efficiency and good reproduction.The folate-targeted liposomes have two main advantages:Firstly,it can effectively increase the drug accumulation into the multidrug resistant tumor cells to reverse the multidrug resistance.Secondly,it can increase drug accumulation into tumor cells hyper-expressed folate receptor and effectively release drug in cells,thereby kill the tumor cells.