Antitumor Effects Of Chlorin E6 Magnetic Sonicate Nanoliposomes(Ce6/SML)on A549 Lung Cancer Xenografts

This subject is part of the project of Department of Science and Technology of Guangdong Provincial(Project Name:Chlorine6 Magnetic Sonicate Nanoliposomes-SDT-Delivery System and Tumor Pharmacokinetics;No.2014A020210022).In the previous study of our research group,the prescription and preparation technology of Chlorin e6 magnetic sonicate nanoliposomes were determined.Based on this,Chlorine6 Magnetic Sonicate Nanoliposomes(Ce6/SML)-SDT-Targeted Delivery System were established for the first time,and its antitumor activity in vitro and in vivo was studied.After administration of Ce6/SML,Ce6 can be slowly released from the liposomes,and Ce6/SML can be guided to the tumor site under an external magnetic field.After the liposomes are accumulated at the tumor site,the targeted release of the Ce6 from liposomes was achieved under ultrasonic treatment,which was also used to activate Ce6.1 ObjectTo improve the tumor targeting of Ce6 for achieving its tumor killing effect in this study,we used magnetic sound-sensitive liposomes as a drug carrier,coupled with ultrasound,which was used as a magnetic sensitizer liposomes release conditions and chlorin e6 excitation conditions.Then the effective Chlorin e6 Magnetic Sonicate Nanoliposomes(Ce6/SML)-SDTTargeted Delivery System were established:1.Magnetic targeting under the magnetic field;2.Ce6 sustained-release effect of liposomes;3.Ultrasound to promote the release of Ce6 from liposomes;4.Ultrasound on the Ce6 activation.Pharmacodynamic studies included in vivo and in vitro anti-tumor activity,as well as the effect of ultrasound and Ce6/SML on the activity of CAT,SOD and GSH-PX,the expression of VEGF and ANG in tumor tissue and the expression of TNF-α in blood.2 Methods2.1 Establishing the detection method of Ce6Method of HPLC was applied to detect the content of Ce6.Including the determination of chromatographic condition,standard curves,linear range,precision test,stability test,quantification limit and detection limit.2.2 Preparation and quality evaluation of the chlorin e6 magnetic sonicate nanoliposomes(Ce6/SML)Chlorin e6 magnetic sonicate nanoliposomes(Ce6/SML)were prepared using egg-yolk lecithin and cholesterol as the carrier material,according to the membrane dispersion-vacuum freeze-drying technique.The encapsulation efficiency,drug loading,particle size and morphology of the prepared chlorin e6 magnetic sonicate nanoliposomes are evaluated.2.3 Study on cytotoxicity and cytolytic activity of chlorin e6 magnetic sonicated nanoliposomes(Ce6/SML)The A549 lung cancer cells were used as the research object,and the experiments were divided into control group,blank liposome group,single ultrasound group,chlorin e6 group(Ce6),chlorin e6-sonodynamic group(Ce6-SDT),chlorin e6 magnetic sonicate nanoliposomes group(Ce6/SML)and chlorin e6 magnetic sonicate nanoliposomes-sonodynamic group(Ce6/SML-SDT).MTT colorimetric method was used to investigate the cytotoxicity of chlorin e6 and chlorin e6 magnetic sonicate nanoliposomes(Ce6/SML)on A549 lung cancer cells.The activities of SOD,GSH-PX activity are determined to confirm the activation of chlorin e6 by ultrasound.2.4 Establishment of A549 xenograft modelA549 lung cancer cells were injected subcutaneously in the right axilla of nude mice,and the infection in inoculation sites were observed daily.Whether there was any natural subsidence after tumor growth was observed;tumor growth was recorded daily.2.5 Anti-tumor activity of chlorin e6 magnetic sonicate nanoliposomes(Ce6/SML)on A549 xenograftsThe small animal live imaging system was used to determine the specific ultrasonic treatment time after the administration of each experimental group,and the experiments were divided into control group(Control),chlorin e6 group(Ce6),chlorin e6-sonodynamic group(Ce6-SDT),chlorin e6 magnetic sonicate nanoliposomes group(Ce6/SML)、chlorin e6 magnetic sonicate nanoliposomes-sonodynamic group(Ce6/SML-SDT)、 chlorin e6 magnetic sonicate nanoliposomes-magnetic targeted sonodynamic group(Ce6/SML-MSDT).Each experimental group was given every other day for 16 days at a concentration of 10 mg/kg Ce6,control group injected with an equal volume of saline.The tumor diameter and tumor weight of nude mice are measured every day.At the end of administration,blood was taken from anesthetized eyes and HE stained sections were prepared from the tumor tissue.2.6 Mechanism of anti-tumor activity of chlorin e6 magnetic sonicate nanoliposomes(Ce6/SML)on A549 xenograftsTNF-α was detected by serum of each control group and experiment group.The activity of CAT,SOD,GSH-PX and the expression of VEGF and ANG protein in tumor tissue were detected.The effects of ultrasonic combined with chlorin e6 magnetic sensitized liposomes on the activity of related enzymes and protein expression were examined.2.7 The microdialysis probe recovery study of Ce6In order to prove the effect of ultrasound on the release of chlorin e6 in liposomes,the microdialysis method of chlorin e6 in vivo and in vitro are established to determine the concentration of Ce6 in the tumor tissues of nude mice.3 Results3.1 Establishing the detection method of Ce6The method of detecting Ce6 by HPLC-UV was as follow: Agilent 1260 Chromatograph;Aglient HC-C18 ODS(4.6 mm×150 mm,5 μm)column;Mobile phase:Acetonitrilepotassium dihydrogen phosphate(60:40;p H 2.1);Flowing rate: 1m L/min;Detecting wavelength:403nm;Column temperature : Room temperature;Column pressure:80bar.Under the chromatographic condition,Ce6 had symmetrical peaks with retention time at 7.53 min.The linear range,limit of quantification and the limit of detection could meet the analytical requirements.3.2 Preparation and quality evaluation of the chlorin e6 magnetic sonicate nanoliposomes(Ce6/SML)Prepared chlorin e6 magnetic sonicate nanoliposomes(Ce6/SML)freeze-dried powder is light green uniform dry powder.The average encapsulation efficiency,drug loading and particle size were respectively 93.80 %,2.33 % and 166.40 nm,showing good in vitro acoustic sensitivities.3.3 Study on cytotoxicity and cytolytic activity of chlorin e6 magnetic sonicated nanoliposomes(Ce6/SML)Alone-ultrasound group and blank liposome group had no inhibitory effect on the growth of A549 cells.The drug concentration in the range of 200～1000 μg/m L,tumor cell killing:Ce6-SDT>Ce6;Ce6/SML-SDT>Ce6/SML.It shows that ultrasound with certain frequency and intensity(1 MHz,2.5 W/cm2)can activate chlorin e6 accumulated in tumor cells and inhibit tumor cell growth.Compared with Control group,the enzyme activity of Ce6,Ce6-SDT,Ce6/SML,Ce6/SML-SDT group showed significant difference(p <0.01);Compared with Ce6 group and Ce6/SML group,the enzyme activity of Ce6-SDT group and Ce6/SMLSDT group was significantly decreased(p<0.01).This result shows that ultrasound can activate chlorin e6,which is consistent with the cytotoxicity test results.3.4 Establishment of A549 xenograft modelThe experimental tumor formation rate was 100 %;the tumor grew evenly.3.5 Anti-tumor activity of chlorin e6 magnetic sonicate nanoliposomes(Ce6/SML)on A549 xenograftsThe small animal live imaging system to determine the ultrasound treatment time: Ultrasound1 hour after administration.In vivo antitumor activity:Ce6-SDT> Ce6;Ce6/SMLMSDT>Ce6/SML-SDT>Ce6/SML>Ce6,which preliminary confirmed that the feasibility of the Chlorin e6 Magnetic Sonicated Nanoliposomes(Ce6/SML)-SDT-Targeted Delivery System.Although in vitro experiments demonstrated the acoustic sensitivities of Ce6/SML,this experiment was unable to determine the effect of ultrasound to promote the release of Ce6 from liposomes in vivo.3.6 Mechanism of anti-tumor activity of chlorin e6 magnetic sonicate nanoliposomes(Ce6 / SML)on A549 xenograftsEnzyme activity(SOD,CAT,GSH-PX)experiments:Control>Ce6>Ce6-SDT;Control>Ce6>Ce6/SML>Ce6/SML-SDT>Ce6/SML-MSDT.VEGF and ANG protein expression:Control>Ce6>Ce6-SDT;Control> Ce6>Ce6/SML>Ce6/SML-SDT>Ce6/SMLMSDT.It further proves that the Chlorin e6 Magnetic Sensitized Nanoliposomes(Ce6/SML)-SDT-Targeted Delivery System is feasible.However,the effect of ultrasound to promote the release of chlorin e6 from liposomes in vivo has not been proved yet.In this study,the in vivo pharmacokinetic study of chlorin e6 microdialysis sampling was proposed to solve this problem.3.7 The microdialysis probe recovery study of Ce6The in vivo and in vitro microdialysis sampling method of chlorin e6 have been successfully established in our previous experiments,and high performance liquid chromatography were used for sample analysis.Because of the lipophilic properties of Ce6,we used different concentrations of ethanol to modify Ce6 in this study,but the existing conditions do not allow Ce6 to effectively penetrate the microdialysis membrane.4 ConclusionThe experiments confirmed the triple effect of the Chlorin e6 Magnetic Sonicate Nanoliposomes(Ce6/SML)-SDT-Delivery System:Magnetic targeting under the magnetic field;Chlorin e6 Sustained-release effect of liposomes;Ultrasound on the chlorin e6 activation.However,the effect of ultrasound to promote the release of Ce6 from liposomes in vivo has not been proved yet.