| 1 ObjectSound sensitive liposomes is a novel drug carrier,can carry drugs to specific tissues and organs,and release the drugs under the local ultrasound stimulation,so as to achieve the purpose of targeting drug release quickly,further improve the targeting and local drug concentration,at the same time,nan-liposome particles can also be as HIFU sensitizer.It can effectively reduce the intensity threshold of HIFU ablation and greatly enhance the effect of tumor treatment[1].High intensity focused ultrasound(HIFU)is a kind of tumor hyperthermia technology,it was used to produce a multiple killing effect with instantaneous high temperature,which could kill tumor cells[2].However,because of the high degree of localization of HIFU,the tumor cells around the solid tumor could not be effectively killed.Magnetic nano liposomes as a novel drug delivery system can be used to applying external magnetic field to change the distribution of drugs in vivo,which to improve the treatment of drug concentration,and reduce the drug concentration at the site of the other treatment,reaching the effect of targeted therapy[3].Both joint used in the treatment of tumor,can achieve synergistic effect of killing tumor cells.This study based on the previous work[4],using chlorin e6 as sonosensitizer,egg yolk lecithin(EPC)and cholesterol as a sensitive liposome membrane materials,and prescription preparation technology by orthogonal test,at the same time,adding oleic acid modified Fe304 nanoparticles to made Ce6 sound sensitive nano liposomes by Ce6 magnetic nano liposomes.The related quality items and the related indexes such as targeting,acoustic sensitivity and cytotoxicity were evaluated.In order to provide the support of dosage forms to further micro dialysis technology in local tumor pharmacokinetics and PK-PD combined model research.2 Methods2.1 The content of Ce6 was detected by UV spectrophotometer,and the method was investigated.2.2 Selection of appropriate preparation methods and materials for the preparation of magnetic sensitive liposomes,and optimize the prescription preparation process by orthogonal test,evaluation of Ce6 magnetic sound sensitive liposomes prepared by encapsulation efficiency,particle size,morphology of magnetic targeting and sound properties.2.3 Because Ce6 has a better therapeutic effect on human lung adenocarcinoma and breast cancer so that the human lung adenocarcinoma cell line SPC-A-1 and human breast cancer cell line MDA-MB-231 were used as the object of study.The experiment was divided into pure hydrogen cells,pure ultrasound group,Chlorin e6 group,blank liposome group,simple liposome group 1(normal liposome:Ce6 ordinary magnetic nano-liposomes),simple liposome group 2(sound sensitive liposome:Ce6 magnetic sound sensitive nano-liposomes),sonodynamic therapy group(Ce6 solution + ultrasound treatment),sonodynamic therapy group 1(normal liposomes + ultrasound treatment),Sonodynamic therapy group 2(sound sensitive liposomes + ultrasound treatment).MTT colorimetric method was used to investigate the inhibitory effect of Ce6 magnetic sensitive liposome on tumor cells under different ultrasonic parameters,and to calculate the inhibition rate,and comparing with ultrasound.Investigate the antitumor activity of different concentrations of the two groups.3 Result3.1 According to the properties of the drug,the UV spectrophotometry was established and the method was investigated.The results showed that the linear range and precision,stability and recovery rate are accordance with the requirements of the experiments.3.2 Through the screening of the preparation method and the preparation materials,EPC and cholesterol were chosen as the carrier materials in this experiment,and were prepared by the film hydration-freeze drying technique,screening and optimization by orthogonal test.3.3 Optimum preparation technology was as following:chloroform as the organic phase,water for hydration medium,hydration temperature of 50 ℃,60 min.3.4 Optimum formula was as following:ratio of egg yolk lecithin-cholesterol 5:1,ratio of egg yolk lecithin-Chlorin e6 1:50,ratio of Chlorin e6-magnetic particlel:2,the dosage of Twain 80 was 0.5%.Morphology of prepared liposome showed sphere structure and rounded appearance with uniform diameter.The average encapsulation efficiency was 90.28%,rate of leakage was 3.28%and the particle size was 176.9 nm.Compared with the free Ce6 solution,the release rate of Ce6 magnetic sound sensitive nano-liposomes showed a slow release effect in vitro.When the ultrasonic treatment instrument was fixed with 1.0MHz ultrasonic frequency,the cumulative release rate of Ce6 magnetic sound sensitive nano-liposomes was also increased,showing good sensitivity in vitro with ultrasonic time and ultrasonic intensity(0～150s)(0～2.0 W/cm2)increased.3.5 The experiment found that sonodynamic treatment group compared with the other group of drugs showed better inhibition effect in the two kinds of tumor cells.The drug concentration was 2.5～7.5 μg/mL and 2.5～10 μg/mL,respectively,the SPC-A-1 and MDA-MB-231 tumor cells were inhibited without the acoustic power.For SPC-A-1 cells,fixed 1.0MHz ultrasonic frequency,when the ultrasonic time 60s,ultrasonic intensity was 0.5 W/cm2,sonodynamic treatment group 2 on the inhibition rate increased with the increase of drug concentration(2.5～7.5 μg/mL)increased;When the ultrasonic intensity was 0.5W/cm2,the inhibition rate of sonodynamic treatment group 2(1.25～7.5～g/mL)was increased with the increase of ultrasonic time(30～120s)(P<0.05).When the ultrasonic time was 60s,the inhibition rate of sonodynamic treatment group 2(1.25～7.5 μg/mL)was significantly increased with the increase of ultrasonic intensity(0.5～2.OW/cm2)(P<0.05).In addition,for MDA-MB-231 tumor cells,fixed 1.0MHz ultrasonic frequency,when the ultrasonic time 60s,ultrasonic intensity was 1.0W/cm2,sonodynamic treatment group 2 on the inhibition rate increased with the increase of drug concentration(2.5～10～g/mL)increased;When the ultrasonic intensity was1.0W/cm2,the inhibition rate of sonodynamic treatment group 2(2.5～10 μg/mL)was increased with the increase of ultrasonic time(30～120s)(P<0.05).When the ultrasonic time was 60s,the inhibition rate of sonodynamic treatment group 2(2.5～10 μg/mL)was significantly increased with the increase of ultrasonic intensity(0.5～2.0W/cm2)(P<0.05).4 Conclusion4.1 The technology of the film hydration-freeze drying technique was applied to the preparation of Ce6 magnetic sound sensitive nano-liposome.Egg yolklecithin was used as the material for the preparation of the liposome,and the encapsulation efficiency was taken as the index.Through the orthogonal experiment to select the influencing factors,the liposome with good sensitivity was made.4.2 Modification of magnetic Fe3O4 surface with oleic acid,was conducive to the improvement of magnetic lipophilicity,combined with the magnetic powder and liposomes showed easier,stable magnetic liposomes formed in the suspension system,under the guidance of an external magnetic field,can be gathered in the tumor site.4.3 Study on cytotoxicity of Ce6 magnetic sound sensitive nano-liposome,in the range of the concentration of ultrasound,the ultrasonic angel therapeutic apparatus was used under different ultrasonic time(0～150s)and ultrasonic intensity(0～2.0 W/cm2),the sonodynamic treatment group 2 compared to the other experimental group on the inhibition rate of human lung adenocarcinoma cell line SPC-A-1 cells and human breast cancer cell line MDA-MB-231 cells were improved,and the inhibition of the SPC-A-1 tumor cells was greater than the latter under the same ultrasonic parameters(P<0.05). |
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