Experimental Study Of Multifunctional Porphyrin Liposome-mediated Sonochemotherapy In Triple-negative Breast Cancer

Triple negative breast cancer(TNBC)is a special type of breast cancer,which is characterized by strong aggression,high metastasis rate,easy recurrence and poor prognosis.Due to the lack of effective molecular targets,chemotherapy is still the main stratege for TNBC treatment.However,the severe side effects of chemotherapeutic drugs greatly limit its clinical application.Therefore,the exploration of new targeted,low toxicity and high efficiency strategies is a major challenge in the field of TNBC treatment.Sonodynamic therapy(SDT)with the synergism of ultrasound and sensitizers is one of the emerging therapeutic methods in tumor research in recent years.Ultrasound,as a non-invasive and deep-penetration external stimulus,can focus on the tumor precisely to achieve targeted activation of sonosensitizers and the controlled release of drugs.With the development of research,the design of sonosensitizers is also toward "intelligence"and multi-functionality.Bying combining with imaging methods for achieving tumor visualization has become important issues of SDT research.In this study,we proposed a imaging-guided sonodynamic and chemotherapy collaborative treatment scheme for TNBC,based on the cross-application of ultrasound medicine,tumor biology,nanomedicine and other disciplines.We have constructed amulti-functional porphysome,which can perform NIR imaging for image recognition of TNBC and guide ultrasound to focus on the lesion area to excite porphyrin.The sonodynamic effect promoted the release of anti-tumor drug DOX and CHK1 inhibitor SRA737 from liposomes in a specific time and space,which can facilited the complementary synergy in anti-tumor therapy,so as to realize the non-invasive,controllable and precise sonochemotherapy application of TNBC.Research contents and results(1)Synthesis,characterization and drug release potential of DOX/SRA737 porphysomesPorphysomes containing DOX and SRA737 with different drug quality ratios were prepared by thin film hydration and ammonium sulfate gradient method.By measuring its particle size,PDI and encapsulation efficiency,DOX loading by adding a drug:lipid with a weight ratio of 1:20,while SRA737:lipid at 1:50 were obtained.Altogether,we prepared four liposomal nano-sonosensitizers:PL(liposomes loaded with Pp18-lipid),PSL(Pp-liposomes loaded with SRA737),PDL(Pp 18-liposomes loaded with DOX),and PSDL(Pp 18-liposomes co-loaded with SRA737 and DOX).The prepared PL,PSL,PDL and PSDL were uniform in size between 114 nm and 132 nm with good dispersibility.The potential of liposomes ranged from-38 to-45V,showing good biocompatibility and circulation stability.The encapsulation efficiencies of DOX and SRA737 in PSDL were 60.52%and 31.34%of their initial amounts respectively.The cumulative drug release rates of SRA737 and DOX in PSDL under ultrasound were?40%and 70%.Compared with ordinary liposomes(without porphyrin phospholipids),porphyrin liposomes showed better ultrasound-response features,and showed the potential of controlled drug-release depending on ultrasonic intensity and duration time.(2)In vitro anti-tumor effect of ultrasound combined with PSDLThe results showed that the uptake of PL in MDA-MB-231 cells was time-dependent by using flow cytometry and fluorescence microscopy.Using laser confocal microscopy,it was observed that US exposure not only enhanced the MDA-MB-231 cellular uptake efficiency of PDL,but also promoted the nuclear translocation of DOX.CCK8 and calcein AM/PI double staining results showed that PSDL combined with US could kill tumor cells more significantly at the same drug dose and ultrasound intensity.The results of flow cytometry showed that PSDL combined with US could significantly enhance the apoptosis of cells.DCFH-DA combined with fluorescence microscopy was used to detect the changes of intracellular reactive oxygen species.The results showed that US combined with PSDL induced more reactive oxygen species than ultrasound combined with PL and chemotherapy.The results of DNA damage experiments showed that US combined with PSDL could promote the transfer of DOX to the nucleus,and the synergistic effect with SRA737 could accurately regulate DNA damage in situ and induce serious cell apoptosis.The cell cycle experiment results showed that the G2/M phase arrest of cells was aggravated by the combination of US and PDL,while the addition of SRA737 alleviated the cycle arrest and suppressed,DNA damage repair,thus ehnhancing cell damage,and improving the anti-tumor effect.(3)In vivo synergistic anti-tumor effect of US combined with PSDLWe evaluated the anti-tumor efficacy of US combined with PSDL in an MDA-MB-231 xenograft model of nude mice in situ.IVIS spectrum imaging system was used to detect the distribution and enrichment of PL in tumor bearing nude mice after tail vein injection of PL.The results showed that PL had a strong fluorescence signal in the organs and tumor tissues of nude mice,and the fluorescence intensity reached the maximum at 12 h.At 24 h,the fluorescence intensity in all organs decreased,but there was still a strong fluorescence signal in tumor tissues.Continuous frozen sections and fluorescence microscope were used to analyze the penetration of PDL into the tumor after ultrasonic treatment.It was found that ultrasound could promote the enrichment of DOX in tumor cells and enhance the distribution of DOX in the nucleus,so as to achieve the effect of DNA damage in situ.In vivo tumor tissue inhibition experiments showed that ultrasound combined with PSDL mediated sonochemotherapy could significantly inhibit tumor tissue growth in nude mice compared with chemotherapy alone and sonodynamic therapy under the same drug dose and treatment conditions.The toxicity of the treatment mode of ultrasound plus PSDL on the tissues of nude mice was preliminarily evaluated by H?E staining and analysis of serum biochemical indexes.The results showed that the treatment strategy had less toxic effects on other organs of nude mice and had relative biological safety.In conclusionIn this study,Pp 18-liposomes co-loaded with SRA737 and DOX were successfully constructed and focused ultrasound was used as an external stimulus to achieve targeted delivery and controlled release of drugs.Both in vivo and in vitro studies confirmed that ultrasound combined with PSDL had obvious anti-tumor effects,and DOX and SRA737 could play a synergism and cooperate well in this system.The designed and constructed PSDL can realize the multi-modal theranostics based on image guidance of porphyrin liposome,acoustic-controlled drug-release and sonochemotherapy,which can provide data support and theoretical basis precision for the treatment of TNBC.