Preparation And Evaluation Of Pemetrexed Disodium Loaded Cationic Liposomes In Vitro And In Vivo

Non-small cell lung cancer(NSCLC)is a most common type of lung cancer and a major cause of cancer death.Although the diagnosis and treatment of NSCLC has made great progress,the current status of clinical treatment is still not optimistic.Pemetrexed disodium(PMX)is a novel multi-target anti-folate drug that blocks the synthesis of purines and pyrimidines and inhibits the growth of cancer cells.PMX can significantly prolong the survival of patients with NSCLC and is well tolerated.However,PMX has a short half-life and a limited time to maintain an effective drug concentration after intravenous injection,and the drug itself does not have lung targeting properties,which may cause toxic side effects such as hematology,liver and kidney function damage.As an anti-tumor drug delivery vehicle,liposome has become a hot spot in this field.It has the following advantages:(1)It can be degraded in vivo and has good biocompatibility;(2)It can prolong the action time of drug and has a sustained release effect;(3)It has EPR effect and can improve the accumulation of drugs in the tumor site;(4)It can encapsulate lipid-soluble and water-soluble drugs and improve bioavailability;(5)The surface can be modified by targeting factors to improve targeting efficiency.In addition to the above characteristics,cationic liposomes can also increase adhesion,prevent ciliary movement and macrophage phagocytosis,and can actively target to the lungs by positive and negative charge attraction.Therefore,cationic liposomes have lung targeting property.In this study,in order to reduce the adverse reactions,increase the accumulation in the lungs and prolong the duration of PMX,PMX was prepared as PMX cationic liposome.The PMX cationic liposome was prepared by thin-film hydration using egg yolk lecithin and cholesterol as lipid membranes and stearylamine(SA)as the positive component of charge-regulating charge,and studied in vitro and in vivo.The main contents of this study include:preparation and characterization of pemetrexed disodium liposome(PMX-Lips),preparation and characterization of cationic pemetrexed disodium liposome(SA-PMX-Lips),in vitro release and preliminary safety evaluation,pharmacokinetics and tissue distribution1.Preparation and characterization of pemetrexed disodium liposomeThe content of PMX was determined by ultraviolet spectrophotometry(UV).The methodological verification showed that the method met the measurement requirements.PMX-Lips were prepared by thin film dispersion method.The optimal formulation and technology were obtained by single factor investigation,taking particle size and entrapment efficiency as indexes.Then the feasibility of the optimal prescription and process was verified by repeatability test.The results showed that the optimal prescription and process had good stability and reproducibility.The average particle size of liposome PMX-Lips was(180.4 ±2.4)nm,zeta potential was(-5.17±0.29)mV,entrapment efficiency was(58.29±1.24)%,drug loading was(7.37±0.36)%and pH value was(6.69 ±0.17).Transmission electron microscopy(TEM)showed that PMX-Lips were spherical or spheroidal with uniform distribution and no adhesion.2.Preparation and characterization of pemetrexed disodium cationic liposomeSA-PMX-Lips were prepared by modifying PMX-Lips with SA and based on the preparation and process of PMX-Lips.The particle size,zeta potential and encapsulation efficiency as the index,the optimal amount of SA was determined by single factor investigation.The reproducibility experiment showed that the formulation process was stable and reliable.The prepared SA-PMX-Lips were spherical or spheroidal and has no adhesion with good homogeneity.The particle size was(219.7±4.97)nm,the zeta potential was(22.2±0.52)mV,the particle size and potential distribution were uniform,the encapsulation efficiency was(92.39±1.94)%,and the drug loading was(9.15±0.07)%,the pH value was(8.65±0.07).It can meet the requirements of intravenous injection.3.In vitro release and preliminary safety assessmentThe content of PMX in the release medium was determined by UV.The methodological study showed that the method is suitable for measurement.In vitro release experiments were performed by dynamic membrane dialysis.The cumulative release percentages of PMX solution(PMX-Sol),PMX-Lips and SA-PMX-Lips were determined at different time points.The release curve was drawn,and then the in vitro release of the three groups of preparations was fitted by a mathematical model.In vitro release results showed that PMX-Lips and SA-PMX-Lips released slowly compared to PMX-Sol.The PMX-Sol group was completely released in 6 hours,and the cumulative release percentage reached(97.59±1.00)%.The PMX-Lips group and the SA-PMX-Lips group released only 78%in 48 hours.The release processes of PMX-Sol,PMX-Lips and SA-PMX-Lips are in accordance with the Weibull equation.Then PMX-Lips and SA-PMX-Lips were evaluated for preliminary safety.The pathological sections of the PMX-Lips group and the SA-PMX-Lips group showed no significant changes compared with the normal saline group.The results showed that PMX-Lips and SA-PMX-Lips had no damage to various tissues and organs and had good safety4.Pharmacokinetics and tissue distribution studiesThe content of PMX in vivo was determined by high performance liquid chromatography(HPLC).The methodological study showed that the method met the requirements of biological samples.Kunming mice were used as animal models,PMX-Sol was used as control,and PMX-Lips and SA-PMX-Lips were injected into the tail vein.Three mice were sacrificed in each group at a given time point after administration,and plasma,heart,liver,spleen,lung,and kidney were measured for the drug content.The pharmacokinetic results showed that the plasma concentrations of the PMX-Lips group and the SA-PMX-Lips group were always higher than those of the PMX-Sol group.After encapsulation of PMX in liposomes,the mean residence time(MRT)of PMX-Lips group and SA-PMX-Lips group increased 2.29 times and 5.14 times than that of PMX-Sol group,respectively.The results showed that the reaction time was prolonged after PMX was prepared into liposomes.In addition,the area under the curve(AUC)of the PMX-Lips group and the SA-PMX-Lips group was 1.71 times and 2.91 times that of the PMX-Sol group,respectively.The results showed that the preparation of PMX into liposomes could improve the bioavailability and therapeutic effect of PMX,and the effect of the cationic liposome is superior to that of the ordinary liposome(the bioavailability of cationic liposomes was 1.70 times higher than that of common liposomes).The results of tissue distribution experiments showed that the encapsulation of liposomes changed the tissue distribution of the drug,increased the distribution in the spleen,and reduced the distribution in the heart and kidney.In addition,the concentration of SA-PMX-Lips in the lungs was always higher than that of the PMX-Sol group and the PMX-Lips group.The AUC of the SA-PMX-Lips group in the lung was 2.47 times that of PMX-Sol,which was 1.94 of PMX-Lips.These results indicate that the liposome modified by SA can greatly increase the targeting efficiency of the lung,which is of great significance for improving the effect of PMX in the treatment of non-small cell lung cancer.In summary,this study successfully prepared pemetrexed disodium cationic liposome,and systematically evaluated it in vitro and in vivo,the prepared preparations met the experimental expectations,can significantly prolong the action time,reduce toxic side effects and have certain lung targeting effect.It is expected to provide new ideas for the effective delivery of PMX and the treatment of NSCLC.