Preparation And Evaluation Of Artemether Liposomes For Enhanced Anti-Tumor Therapy

Artemisinin is a compound of sesquiterpene lipids obtained from the leaves of artemisia annua.As the efficient anti-malarial drugs,artemisinin-based drugs also showed significant anti-tumor activity and were not readily drug-resistant.Artemether(ARM)is a methyl ether derivative of artemisinin.Because of its higher lipid solubility,fast absorption and high permeability,ARM has about 10 times stronger antimalarial activity than artemisinin.In addition,the high anti-tumor activity and low toxicity of ARM are particularly noticeable.ARM has obvious inhibition and killing effects on many kinds of tumor cells,with few toxic and side effects,good tolerability,and certain activity on some tumor cells that are easy to produce multidrug resistance.However,ARM has poor water solubility and short half-life,and it is difficult to maintain an effective concentration in vivo.Therefore,,it has a positive and far-reaching significance for the application of ARM in cancer by virtue of the nano drug delivery system to overcome its shortcomings of poor water solubility and further increase its bioavailability.In this study,we used ARM with highly effective and low toxicity as a model drug,soybean phospholipid and cholesterol as the carrier materiasl,and prepared ARM-Lips via ethanol injection method with simple process and high stability.The main contents of this research included preparation and physical and chemical properties of ARM-Lips;In vitro release,stability test and preliminary safety evaluation of ARM-Lips;Pharmacokinetics of ARM-Lips;Pharmacodynamics study of ARM-Lips on the H22 tumor models through intravenous injection.1.Preparation and physical and chemical properties of ARM-LipsFirst of all,the ultraviolet spectrophotometry(UV)method for the determination of ARM content in Lips was established and its methodology was investigated.Then,we chose the method of ultrafiltration centrifugation to separate the Lips and free drugs.We established the determination method of EE and DL of ARM-Lips and conducted a methodological study.With the particle size,EE and DL as the evaluation indexes,the prescriptions and process factors were investigated and optimized by single factor experiment.In the next part,we verified the rationality of formulation and preparation process through the reproducibility test;We chose the appropriate amount of lyoprotectant to prepare the injection of ARM-Lips freeze-dried preparation.Finally,the appearance of ARM-Lips,particle size,particle size distribution,Zeta potential,pH,EE and DL were evaluated.The results showed that the good linearity of the drug concentration was obtained within the range of 0.5-22.5 ?g/mL.The method of ultrafiltration and UV detection of EE and DL of the ARM-Lips had high precision,high recovery and good reproducibillity.The ethanol injection method for the preparation of ARM-Lips was a good preparation method for liposoluble medicine with some advantages such as simple operation and stable preparation process.The reproducibility test indicated that the composition of the ARM-Lips was reasonable and the preparation process was feasible.The ARM-Lips prepared by the method showed a spherical shape under TEM with an average particle size of(187.3±1.83)nm.The EE and DL were(94.49±1.18)%and(10.94±0.10)%,respectively;the average Zeta potential was(-11.6±0.26)mV and pH value of ARM-Lips was 6.70±0.62.2.In vitro release,stability test and preliminary safety study of ARM-LipsFirst of all,the method of determination of in vitro release of ARM-Lips was established and verified.Then,compared with the ARM-Sol,the in vitro drug release of ARM-Lips at different time points was determined via dynamic membrane dialysis with PBS(pH 7.4)containing 1%Tween as the release medium.The release curve was drawn and then fitted by mathematical models to study the in vitro release of ARM-Lips.Then,the stability of ARM-Lips in 10%plasma-physiological saline solution and pH 7.4 PBS solution was investigated via using the change in particle size of ARM-Lips as an indicator.Finally,the preliminary safety of ARM-Lips was investigated by in vitro hemolysis test and pathological experiment.The results showed that the determination method of ARM-Lips with the release medium as solvent had high precision,high recovery and good stability.The good linearity of the drug concentration was obtained within the range of 2.5-22.5 ?g/mL,which met the content determination requirements.The results of in vitro release showed that the release rate of ARM-Sol was faster and reached(81.30±2.26)%at 8 h.While the release of ARM-Lips was relatively slow.The release percentage was(50.76±2.01)%at 8 h and the cumulative release percentage reached(79.35±2.10)%at 48 h with good sustained release.In addition,the release of ARM-Lips in vitro was in accordance with the first-order kinetic equation.The stability test results showed that ARM-Lips had no significant change in particle size in PBS,and had good stability.In 10%of the plasma,its particle size increased significantly,and the maximum particle size was close to 1100 nm after 24 h.And the floe was visible to the naked eye.In hemolysis test using the naked eye observation and UV spectrophotometry,the results showed that ARM-Lips did not hemolysis,indicating that its safety,which could be applied to intravenous injection.The results of pathological experiment showed that there were no obvious pathological changes of the main organs in ARM-Lips group compared with NS group.Therefore,Lips as a carrier of ARM had lower toxicity and good biocompatibility.3.In vivo pharmacokinetic studies of ARM-Lips in miceFirst,with Kunming mice as an animal model,ARM-Sol and ARM-Lips were injected into tail vein,respectively.Eyedrops were removed from mice at different time points.The drug concentrations in the plasma of mice at different time points were determined and compared.We used pharmacokinetic parameters via DAS 2.0 to evaluate the sustained release of ARM-Lips.The advantages of ARM-Lips for intravenous administration could provide good prospects for ARM in the development of anti-tumor.The results showed that the drug concentration in the plasma in ARM-Lips group was significantly higher than the ARM-Sol group after intravenous injection.Compared with the ARM-Sol group,the t1/2? of the ARM-Lips group was prolonged from 0.352 h to 2.948 h and the MRT was prolonged from 0.457 h to 1.544 h,which were 8.375 and 3.38 times higher than that of the ARM-Sol group,respectively.The AUC was prolonged from 130.245(mg/L·h)to 404.43(mg/L·h),which was 3.11 times that of the ARM-Sol group.The CL was 0.41 times that of the ARM-Sol group and the bioavailability of the drug in the ARM-Lips group was significantly higher.These above pharmacokinetic parameters indicated that the ARM encapsulated in the lipid bilayer of the Lips could rely on the sustained release of the carrier to prolong the circulation time of the drug in the body and improve its bioavailability.4.The pharmacodynamics study of ARM-Lips in tumor-bearing miceFirstly,H22 hepatoma tumor cells in logarithmic phase were seeded in the right arm of Kunming mice to establish a tumor-bearing mouse model of H22 tumor.Then,NS,Blank-Lips,ARM-Sol and ARM-Lips were injected into tail vein with tumor-bearing mice as animal models and NS as control,respectively.We calculated the tumor volume by measuring the long and short diameters of tumor at different time points to evaluate the antitumor activity of each group.The toxicity of the informations was indirectly evaluated by measuring the body weight of the mice at different time points.The tumor tissues of each group were stained with HE and observed under a microscope for the pathological features.The anti-tumor efficacy of each group was evaluated according to the necrosis of tumor cells,and the tumor inhibitory effect of ARM-Lips was further evaluated.Pharmacodynamic results showed that the size of the tumors in the Blank-Lips group and the NS group were similar,which indicated that the Lips as a carrier of the drug could not inhibit tumor growth.Both ARM-Sol and ARM-Lips could inhibit the tumor to a certain extent.The TGI of ARM-Lips was 1.54 times that of ARM-Sol group,the SGR of tumor in ARM-Lips group was 0.51 times of that of ARM-Sol group,and the DT of tumor was 1.97 times of that of ARM-Sol group,indicating that the ARM-Lips group on the tumor inhibition was significantly stronger than the ARM-Sol group.During the course of treatment,the body weight of mice did not change much,indicating that ARM-Lips are less toxic to mice.The results of pathological experiment showed that the tumors of Blank-Lips group and NS group were intact and proliferated.Only a small part of the tumor cells in the ARM-Sol group showed necrosis,most of the tumor cells maintained the intact structure,whereas the ARM-Lips group had a severely damaged tumor cell structure with a large necrosis state.Therefore,the ARM made into Lips not only could improve water insoluble of ARM problems,but extend the half-life of ARM in the body and increase the body circulation time.In addition,the size of the Lips was used to form a passive target.The EPR effect of the tumor could increase the accumulation of drug in the tumor site,thereby further enhancing the tumor inhibitory effect.In summary,with the artemisinin-based plant-derived drugs-ARM as a model drug,nanocarrier-liposome technology could improve the water insoluble,short half-life and other problems of ARM.To explore new ideas for the treatment of liver cancer through the preparation and physical and chemical properties of ARM-Lips for intravenous injection,in vitro release,stability test and preliminary safety evaluation,pharmacokinetics and pharmacodynamics research,which had a high clinical value and broader clinical application prospects.