An Experimental Study Of Liposome-mediated Sonochemotherapy In Breast Cancer

Breast cancer is the second most common cancer in the world after lung cancer,with the highest incidence of malignant tumors in women.At present,the treatment of breast cancer is mostly surgery,radiotherapy and chemotherapy,etc.,but it is very prone to recurrence and metastasis and the toxic and side effects of the systemic system cause its poor prognosis.Therefore,exploring new safe and effective treatment strategies to suppress their recurrence and metastasis is still a major challenge for basic and clinical research.Sonodynamic therapy(SDT),as a new tumor treatment method,mainly uses ultrasound to activate the sensitizer to produce reactive oxygen species(ROS)and other killing tumor cells.Acoustic sensitizers are the key factors that affect the antitumor effect of SDT,and porphyrins and porphyrin derivatives have been used as acoustic sensitizers to carry out many basic anticancer researches.In the early stage of the laboratory,porphyrin liposomes with purpurin 18-membrane chimera have been prepared,and it is initially shown that they can be used as acousto-optic stimuli-responsive controllable drug release carriers.Angiogenesis is an important reason for the recurrence and metastasis of solid tumors.It is expected to become a promising new strategy for breast cancer treatment by inhibiting tumor angiogenesis.Anlotinib hydrochloride(AL)is a small-molecule tyrosine kinase inhibitor that can block tumor neovascularization by down-regulating vascular endothelial growth factor(VEGF),thereby inhibiting tumor growth.Based on the above research background,iRGD-Plipo-AL was designed and synthesized.After ultrasonic treatment,on the one hand,it promotes the local fixed release of the loaded AL to achieve tumor cell damage and inhibit tumor angiogenesis;On the other hand,the sensitizing agent purpurin 18 on the liposome membrane responded to ultrasound stimulation and produced an acoustic dynamic effect to kill tumor cells.The targeted acoustic chemotherapy effect of SDT combined with AL effectively inhibited the growth and metastasis of breast cancer.The specific experimental research is as follows:Part ? iRGD-Plipo-AL preparation,characterizationand its drug-release performance by ultrasound stimulationThe iRGD-Plipo-AL was prepared by thin-film hydration,and purpurin 18 was selected as the loading platform for the anti-tumor angiogenesis inhibitor AL.and AL was loaded in the liposome hydrophilic core.The particle size and potential of iRGD-Plipo-AL were determined by dynamic light scattering method,the particle morphology of iRGD-Plipo-AL was observed by transmission electron microscope,and the encapsulation efficiency and drug loading of iRGD-Plipo-AL were determined by high performance liquid chromatography.At the same time,in order to investigate the stability of iRGD-Plipo-AL.its particle size and polydispersity coefficient(PDI)were measured in vitro for 7 consecutive days.The production of free radicals by stimulating purpurin 18 was detected by terephthalic acid method.The results show that the preparation steps of iRGD-Plipo-AL are simple and stable.Transmission electron microscopy observed that liposomes were spherical vesicles and monodispersed.The diameter of iRGD-Plipo-AL measured by dynamic light scattering method was about 130 nm.The drug loading of AL in iRGD-Plipo-AL is 0.86%.the encapsulation rate is 43%,and it remains relatively stable for a certain period of time;In addition,the potential of iRGD-Plipo-AL is not significantly different from that of blank liposomes.IRGD-Plipo-AL can produce ROS under ultrasound stimulation,indicating that the iRGD-Plipo-AL prepared by the study has good acoustic sensitivity.To further explore the ultrasound-responsive drug release of iRGD-Plipo-AL,the experiment used iRGD-Plipo-AL with different intensities to determine the release of AL by dialysis and high performance liquid chromatography.The results showed that ultrasound treatment promoted the release of AL from iRGD-Plipo-AL,and as the ultrasound intensity increased,the drug release rate increased significantly.Part ? Anti-tumor effect of iRGD-Plipo-AL combined with ultrasound dielectricThe study selected mouse breast cancer 4T1,human breast cancer MBA-MB-231 and MCF-7 as the cell model,using flow cytometry and fluorescence microscope to observe the difference in the uptake of iRGD modified liposomes between 4T1 cells and normal cells NIH3T3;The MTT and Calcein-AM/PI double staining experiments were used to detect the change of survival rate of 4T1 cells after different treatments;Use AnnexinV/PI to detect the apoptosis of cells after different treatments;The generation of intracellular reactive oxygen species after treatment was studied by flow cytometry combined with DCFH-DA probe.The results showed that breast cancer 4T1 cells were more likely to take up iRGD-targeted Plipo than normal cells NIH3T3,and iRGD-Plipo uptake in 4T1 cells was higher than that of Plipo.When the AL concentration was 1?g/mL,there was no obvious cytotoxicity with ultrasound and AL alone.After iRGD-Plipo-AL treatment alone,the cell survival rate decreased by approximately 10.86%,and after iRGD-Plipo-AL combined with 2 W/cm2 ultrasound treatment,the cell survival rate decreased to 43.64%(p<0.01).The results of AnnexinV/PI also showed that iRGD-Plipo-AL combined with ultrasound can mediate more cell apoptosis compared with the single treatment groups.Flow cytometry measurement of DCF fluorescence intensity shows that iRGD-Plipo-AL combined with ultrasound can generate ROS.Part III Effect of iRGD-Plipo-AL combined with ultrasound on tumor angiogenesisThe effect of iRGD-Plipo-AL+US on the expression of VEGF in 4T1 cells was observed by immunofluorescence in vitro.In addition,the expression of vascular endothelial growth factor receptor VEGFR and platelet endothelial adhesion molecule CD31 in tumor tissue sections after different treatments were detected by immunohistochemistry.The results showed that the VEGF immunofluorescence of tumor cells in the control group was strong,and almost no VEGF fluorescence was observed after iRGD-Plipo-AL+US treatment;indicating that the expression of VEGF became weak;Histochemical results showed that the strong positive staining of microvessels marked with CD31/VEGFR was significantly weakened.After iRGD-Plipo-AL+US treatment,the positive staining almost disappeared,indicating that the expression of CD31/VEGFR decreased and angiogenesis was effectively suppressed.Part ? Study on anti-tumor effect of iRGD-Plipo-AL combined with ultrasound dielectricFemale Balb/c mice were selected as animal models,and 4T1 cells were subcutaneously inoculated.The tumor volume,mouse weight,serum biochemical indicators,immunohistochemical analysis,main organ observation,and morphological observation were comprehensively explored in iRGD-Plipo-AL+US antitumor effect.The results show that,compared with Plipo,iRGD-Plipo can reach the maximum enrichment in vivo at 12h;The iRGD-Plipo-AL+US treatment group has a significant inhibitory effect on tumors,and also inhibits lung metastasis to a certain extent;Observation of mouse body weight,serum biochemical indicators and main organ sections suggest that this experimental treatment is safe.To sum up,this thesis designed and synthesized a targeted Purpurin 18 liposomecontaining anlotinib hydrochloride(iRGD-Plipo-AL)and applied it in acoustic chemotherapy and anti-tumor research.Studies have found that iRGD-Plipo-AL can achieve controlled release of ultrasound and kill tumor cells from both aspects of inhibiting tumor angiogenesis and anti-tumor.It has also proved preliminary safety in in vivo experiments.Phased experimental results show that the treatment method developed in this paper can provide a certain theoretical basis and reference for the subsequent research on ultrasound and vascular suppression.