| Antitumor effect of low-frequency ultrasound combined with autophagy blockade in the mouse model of 4T1 breast cancerObjective:To investigate the effect of low-frequency ultrasound combined with autophagy blockade on the growth and invasion of 4T1 breast cancer in mice.Methods:4T1 cells in logarithmic growth phase were divided into four groups:control group,ultrasound(US)group,hydroxychloroquine(HCQ)group and US combined with HCQ(US+HCQ)group.Western blot was performed to detect the protein expression levels of autophagy-related proteins LC3,p62.Transmission electron microscopy was used to observe the formation of autophagic vesicles.The cell proliferation and cell viability were determined by CCK-8.Transwell assay was performed to detect the effect of ultrasound combined with HCQ on the invasive ability of 4T1 cells.Flow cytometry was performed to detect the effect of ultrasound combined with HCQ on the apoptosis of 4T1 cells.The transplantation tumor model of 4T1 breast cancer mice was constructed.The mice were randomly divided into four groups(n=5),including:control group,ultrasound group,HCQ group,ultrasound combined with HCQ group.The tumor volume and mice body weight were evaluated and measured in each group every 2 days.Results:The expression of LC3-II and p62 protein levels increased in the ultrasound combined with HCQ group(all P<0.05),and intracellular autophagosome accumulation was evident by transmission electron microscopy.In cellular experiments,compared with the other groups,the ultrasound combined with HCQ group showed stronger growth inhibition,and significantly inhibited invasion(all P<0.05);In the in vivo experiments,compared with the control group,the tumor growth rate of all groups of mice decreased(all P<0.05),and the ultrasound combined with HCQ group had better therapeutic effects(all P<0.05).Conclusion:The combination of low-frequency ultrasound and autophagy blockade could synergistically inhibit tumor cell viability,promote apoptosis,and significantly inhibit tumor growth in 4T1 tumor-bearing mice.Preparation and study of pH/ultrasound dual-responsive nanodroplets loaded with HCQ Objective:To prepare HCQ-loaded nanodroplets(HCQ-NDs)and examine their characterization,pH-and ultrasound-responsive drug release,contrast-enhanced ultrasound imaging properties and tumor-targeting.Methods:HCQ-NDs were prepared by homogenization/emulsification method;Transmission electron microscopy was used to determine the morphology and structure of NDs.The particle size,polydispersity index and ζ potential of NDs under different pH conditions were examined by dynamic light scattering;The drug release under different pH and ultrasound conditions was examined by UV-vis spectroscope;The cellular uptake pathways of NDs were examined by flow cytometry and confocal microscopy;The effect of enhanced ultrasound development in vivo and in vitro was examined by ultrasound contrast imaging;The biodistribution and intratumor enrichment patterns were examined by fluorescence imaging.Results:NDs were small and homogeneous in a neutral environment with a negative ζ potential,while they expanded and aggregated in an acidic environment with a positiveζpotential;Acidic pH and ultrasound irradiation promoted the drug release of HCQ-NDs(all P<0.05);Confocal microscopy confirmed that NDs entered the cells through the lysosomal pathway and ultrasound irradiation promoted the uptake of NDs by cells(P<0.05).Both NDs and HCQ-NDs exhibited persistent high echo signals in ultrasound contrast mode.Fluorescence imaging results showed that NDs has a long intratumoral retention time and the highest tumor accumulation at 2 h after injection.Conclusion:Ultrasound nanodroplets loaded with HCQ were successfully prepared.HCQNDs show pH-responsive charge conversion,serum stability,contrast-enhanced ultrasound imaging properties and pH-and ultrasound-responsive drug release capability,which can effectively target and penetrate tumors.UTMD combined with HCQ-NDs for inhibition of tumor growth and metastasisObjective:To investigate the therapeutic effect and mechanism of UTMD combined with HCQ-NDs on tumor growth and metastasis.Methods:4T1 cells at logarithmic growth stage were divided into control group,NDs group,NDs+US group,free HCQ group,HCQ-NDs group and HCQ-NDs+US group.Western blot was performed to detect the protein expression levels of autophagy-related proteins LC3,p62.Transmission electron microscopy was used to observe the formation of autophagic vesicles.Autophagic flux was observed by laser confocal microscopy.CCK-8 was used to determine cell viability;Flow cytometry was used to detect the effect on apoptosis;Wound healing assay and transwell were used to determine cell migration and invasion;A subcutaneous transplantation tumor model of 4T1 breast cancer mice was constructed,and the tumor size and weight changes were monitored,and the tumor apoptosis,proliferation level and biosafety were detected by morphological analysis of histological sections and immunohistochemistry.The lung metastasis tumor model of 4T1 breast cancer mice was constructed,and the effect of combination therapy on tumor metastasis was verified by staining the lung surface nodules as well as H&E,and the expression of tumor MMP2 and MMP9 was detected by immunohistochemistry.Results:The autophagic process stalled at the autophagosome stage after combined treatment.In cellular experiments,the HCQ-NDs+US group had a stronger antitumor effect and significantly inhibited tumor migration and invasion(all P<0.05);In the in vivo experiments,the HCQ-NDs+US group had the slowest tumor growth rate and the lightest tumor weight(all P<0.05);Histological section staining showed that the HCQ-NDs+US group had the highest level of apoptosis and the lowest proliferation within the tumor(all P<0.05);All treatment groups had high safety and no significant effects on the major organs of mice;The HCQNDs+US group had the least lung metastasis(P<0.05),and the expression of tumor MMP2 and MMP9 was the lowest.Conclusion:The combination of HCQ-NDs and UTMD has synergistic antitumor effect with high biosafety.In addition,this strategy could significantly inhibit tumor metastasis,which is related to the decreased expression of MMP2 and MMP9. |
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