Preparation Of Sodium Porphyrin-loaded Liposome-microbubble Complex And Its Experimental Study On Acoustic-activated Breast Cancer

Background and Objective:With the rapid development of economy,serious environmental problem,and the increase of population growth and aging,cancer is the leading cause of death in China and is a major public health problem.According to the report of "Cancer Statistics in China,2015”,breast cancer has become the common cancer among Chinese women.Currently,clinical treatment of breast cancer includes surgery,radiotherapy,chemotherapy,endocrine therapy,and so on.However,these methods are still difficult to obtain satisfactory results.So,prevention and treatment of breast cancer has always been a major issue of biomedicine research.Sonodynamic therapy(SDT),which utilizes synergistic effect of ultrasound(US)and sonosensitizers,has emerged as a promising option for the minimally invasive treatment of cancerous tumours.US has an appropriate tissue attenuation coefficient for penetrating the ability to focus energy into small volumes and activate sonosensitizer,which is tumor-specific accumulation.This makes it possible to damage the pathological site but not peripheral healthy tissues,suggesting that SDT has a valuable application for targeted cancer therapy.In particular,for some patients who are difficult to undergo surgery,deep tissue treatment of cancer,palliative treatment of advanced cancer,SDT has strong targeting,safety,minimally invasive and actual practice.SDT involves in many fields,including biology,physics,medicine,and chemistry.To date,SDT has been widely investigated about the development of sonosensitizer,ultrasonic equipment and mechanisms of killing effects.Exploring how to facilitate SDT to achieve a more significant anti-tumor effect is also one of popular field.Recently,ultrasound microbubble destruction(UTMD)technology is a hotspot of drug delivery systems.Studies have shown that UTMD can promote the local release of the sonosensitizer in the lesion and enhance its anti-tumor activity of SDT.Therefore,the present study selected a new porphyrin dimers sodium salt(sinoporphyrin sodium,referred to DVDMS)as sonosensitizer,to synthesis DVDMS-loaded Liposomes-Microbubbles complexes(DLMBs).The controlled-release dynamic characteristics of DLMBs assisted by UTMD were determined.Human breast cancer MDA-MB-231 cells and mouse breast cancer 4T1 tumor-bearing mice were used as experimental models to study the combined anti-tumor effect of UTMD and SDT mediated by DVDMS.DLMBs assisted by UTMD will finally allow us to lay a theoretical and experimental foundation for such a promising therapy in clinical cancer treatment.Methods and Results:1.The DVDMS liposome was prepared through filming-rehydration method using phospholipid DPPC,DSPE-PEG 2000-Biotin and cholesterol.Liposomal size and encapsulation efficiency as indicators,the synthesis process of DVDMS liposomes was optimized through screening the factors such as the ratio of each component,the amount of DVDMS,and so on.The average size of DVDMS liposomes was(150.8 ± 23.3)nm,the encapsulation efficiency of DVDMS was(68.73± 6.99)%,and it remained stable within 96h.The microbubbles(MBs)were prepared through mechanical oscillation method using phospholipid DPPC,DSPE-PEG 2000-Biotin and cholesterol.MBs size as indicator,the synthesis process of MBs was optimized.The average size of MBs was 733.5 ± 49.2 nm,and it remained stable within 24h.2.Biotin-avidin system(BAS)is a combination of biotin and avidin,which has high sensitivity,high specificity and high stability.It is widely used in the synthesis of nano-carrier of drugs.In this study,biotinylated DVDMS liposomes were linkaged with biotinylated MBs using BAS to obtain DLMBs.After optimization,the average size of of DLMBs was(1009.2 ± 62.81)nm and the encapsulation efficiency of DVDMS was(40.67± 8.43)%.The DVDMS liposome was attached onto the shell of the MBs using a laser scanning confocal microscope.The rate of MBs linked with liposomes was approximate 67.02%monitored by flow cytometry.3.Under US exposure,DVDMS could be effectively released from DLMBs,showing the characteristic of "ultrasonic burst",and the relative release increased with the intensity of ultrasound.The bright-field microscopy images revealed bubble-like structure almost disappeared after sonication,suggesting that integrity of the DLMBs complex was completely destructed by US in the current setting of the experimental conditions.The generation of hydroxyl radicals was detected by Terephthalic acid method.The changes of lipid peroxide caused by ultrasound were tested through FTC assay and MDA detection.Results showed that the level of lipid oxidized product increased significantly after US treatment,which may lead to instantaneous instability of this drug-carried system and subsequent boosted release of DVDMS.4.Human breast cancer MDA-MB-231 cells was used as experimental models to study the combined anti-tumor effect of UTMD and SDT mediated by DVDMS.Compared with free DVDMS and DVDMS liposomes,the sono-cytotoxic effect of DLMBs was dose-and US intensity-dependent.On this basis,the experimental parameters of half inhibition of tumor cell survival were selected for subsequent experimental studies.5.Compared with free DVDMS and DVDMS liposomes,the inhibition effect of DLMBs combined with US on MDA-MB-231 cell proliferation was significantly continued to decline as the incubation after treatment.The SEM results showed that combined treatment could cause serious membrane damage.Flow cytometry showed that the apoptosis rate of cells was significantly improved,and the mitochondrial membrane potential was significantly decreased.At the same time,the intracellular level of reactive oxygen species(ROS)in cells increased;Inhibition experiments indicated that ROS played an important role in the killing process of breast cancer cells by DLMBs combined with ultrasound.6.Mouse breast cancer 4T1 tumor-bearing mice were used as experimental models,and the content of DVDMS in tumor tissue after DLMBs combined with ultrasound was analyzed by autologous control experiment.First,DLMBs were administered to tumor-bearing mice through tail vein injection,and one tumor was sonicated of each mouse.The results of Xenogen IVIS spectrum system imaging observation and fluorescence spectrophotometry showed that in the US-treated tumor,DVDMS content was much more than that of untreated one,suggesting that DLMBs combined with US could improve the level of DVDMS in tumor tissue.Second,DLMBs were successfully injected into the tumor tissue in situ in the same depth with the help of stereotaxic instrument and then sonication.The tumor tissues were collected and continuously frozen sectioned,then observed the DVDMS fluorescence by Stereo Fluorescence Microscope.Results showed that the red fluorescence of DVDMS diffused throughout the tumor section after US treatment,while the fluorescence was just gathered brightly in untreated group,suggesting that combination of DLMBs with US could obviously promote intratumor diffusion of DVDMS.7.To investigate the sonodynamic anti-tumor efficacy of composite DLMBs with US,a 4T1 mouse mammary tumor model was utilized.Results showed that compared with free DVDMS and DVDMS liposomes,the inhibition ration of tumor growth was much significant after DLMBs plus US.H&E staining revealed obvious tissue damage caused by combined treatment.Results of TUNEL staining and immunochemistry analysis of PCNA indicated that the inhibition of tumor growth appeared to result from increased apoptosis and reduced proliferation activity in mice 4T1 tumor.Simultaneously,body weight of tumor-bearing mice was measured during experiment and no major changes were observed following the various treatments.Histological examination of the major organs(i.e.,heart,liver,spleen and kidney)presented a good biosafety,as negligible organ damage was caused.