Experimental Study Of Ce6 Encapsulated Nanoparticles For Inhibiting Growth And Metastasis Of Breast Cancer Based On Low Intensity Focused Ultrasound

PART Ⅰ Study of basic properties for preparation,characterization and drug releasing of Ce6 encapsulated nanoparticlesObjective To prepare the multifunctional nanoparticle encapsulated with Ce6,PFP and DTX,and to explore the basic properties including its characterization and drug releasing capacity.Methods The multifunctional nanoparticles(CPDP NPs)containing Ce6,PFP and DTX in the PLGA shell were established by the double emulsion method.The particle size and zeta potential were evaluated,and TEM was applied to determine the morphology of the nanoparticles,and the encapsulation efficiency was calculated and finally,the drug releasing efficiency was detected in different pH solutions.Results Ce6-PFP-DTX-PLGA nanoparticles(CPDP NPs)were successfully prepared with an excellent dispersion.The size and zeta potential were measured as 249.5±77.46 nm and-18.47±0.55 m V,respectively with a relatively good stability.TEM confirmed CPDP NPs owned spherical morphology and a stable core-shell structure.The encapsulation efficiency of Ce6 and DTX were 60.54±3.79%and 83.84±1.39%,respectively.The drug releasing rates of Ce6 in pH 7.4 and pH 5.5 solutions were 14.00±0.72%and 20.15±0.76%,and rates of DTX under the same conditions were 24.04±2.11%and 47.61±1.34%,respectively.Conclusion CPDP NPs were successfully prepared by the double emulsion method.The nanoparticles featured with typical core-shell structure,excellent size,zeta potential and relatively stable.With the superior encapsulation efficiency and drug releasing capability of CPDP NPs,it could lay a solid foundation for the subsequent experiments in this study.PART Ⅱ The inhibition of breast cancer growth and metastasis by Ce6 encapsulated multifunctional nanoparticles:the in vitro studyObjective To investigate the in vitro ultrasound imaging capability,biosafety,and the elimination of breast cancer cell progression and metastasis of CPDP NPs based on LIFU.Methods Using agarose gel model to verify the ultrasound imaging ability of nanoparticles under different LIFU irradiating intensity as well as time.To verify the biosafety as well as cell viability of CPDP NPs based on LIFU irradiation,CCK-8 assay was applied.DCFH-DA fluorescent probe was applied to verify the generation of ROS after co-culture of CPDP NPs and cells.To observe the effectiveness of synergistic therapy,the flow cytometry was conducted.The synergistic therapy of anti-metastasis capability of CPDP NPs combined with LIFU irradiation was testified through both wound healing and transwell assay.Results After 2 W/cm~2 LIFU irradiation of 120 s,both 2D and CEUS ultrasound imaging capability was remarkably elevated.As the CCK-8 assay showed,the highest level of CPDP NPs concentration(0.8 mg/m L)exhibited an over 80%of cell viability.Combined with LIFU irradiation,that number declined to merely 52.54±2.90%.The CLSM showed ROS in CPDP NPs+LIFU group was remarkably elevated in comparison with other groups.The necrotic and apoptotic rate exhibited by flow cytometry revealed CPDP NPs+LIFU group was 30.92±1.92%,which was obviously higher in comparison with other groups.The wound healing assay and transwell assay exhibited the cell migration and the inhibition rate of CPDP NPs+LIFU group were remarkably decreased.Conclusion CPDP NPs featured with excellent ultrasound imaging capability after LIFU irradiation,desirable ROS generation efficiency in vitro,and were highly effective and stable in synergistic therapy with biosafety.According to the in vitro results,it has been verified to the remark prohibition of 4T1 breast cancer cell promotion and metastasis,which could be the basis of the follow-up experiments.PART Ⅲ The inhibition of breast cancer growth and metastasis by Ce6 encapsulated multifunctional nanoparticles:the in vivo studyObjective To verify the in vivo ultrasound imaging ability and biosafety of CPDP NPs.To evaluate its ability of synergistic therapy of breast cancer and the distant metastasis combined with LIFU.Methods CPDP NPs was injected through tail vein to observe both the 2D and CEUS imaging capability combined with LIFU.During 18 days,weight and tumor size were documented every other day,and the growth and tumor volume curve were analyzed and calculated.After treatment,the mice were sacrificed and their tumor tissues were sent to H&E,PCNA and TUNEL,the number of lung metastatic nodules was observed and H&E staining was performed.Healthy Kunming mice were chosen were injected with CPDP NPs via tail vein to investigate the in vivo biosafety of CPDP NPs,and the body weight was summarized every two days.After one month,the mice were executed for blood examination,while the major organs were harvested for H&E staining.Results After LIFU irradiation,the 2D and CEUS imaging ability of mice was remarkably elevated,and the gray-scale intensity was 2.94 and 2.98 times superior than previous irradiation.The tumor volume of CPDP NPs+LIFU group was remarkably inferior in comparison with the rest groups,and no significant difference was found in mouse weight among all the groups.Histopathological results of the tumor observed a significant decrease in proliferation rate and increase in apoptosis rate in the CPDP NPs+LIFU group.The number of metastatic lung nodules and H&E staining were significantly reduced in the CPDP NPs+LIFU group.No significant differences were found in the blood indexes,body weight growth,and no significant damage was detected in all major organs.Conclusion CPDP NPs has a good biosafety at in vivo level and when combined with LIFU irradiation,the strategy shows an enhanced ultrasound imaging and effective synergistic treatment efficiency.The results indicate that the synergistic strategy not only remarkably curbs the promotion of primary tumors in tumor-bearing mice,but also strongly prevents the progression of lung metastases in tumor-bearing mice which could become a promising strategy in breast cancer treatment.