The Effect And Mechanism Of CuS-MnS₂ Nanoflowers As A Theranostic Agent On Ovarian Cancer A2780 Cel Line And Its Animal Model

Background:Ovarian cancer keeps a low early diagnosis rate and a high mortality,which seriously endangers women's health.Surgery and chemotherapy havn't ffectively improve the prognosis.As nanotechnology developing,the concept of theranostic was put forward in recent years,which means integration of diagnosis and therapy.The imaging function of nanomaterials can help diagnosis,guidance and monitoring the treatment.The photothermal conversion efficiency combined with NIR irradiation provides targeted,visible and manageable photothermal therapy.At present,there is little report of nanomaterials applied in obstetrics and gynecology,and many defects in the theranostic nanomaterials,and so it is urgent to develop new diagnostic agents that have better performance and multiple therapeutic effects.This study is aimed to evaluate the theranostic efficacy of newly produced nanomaterials for ovarian cancer cells and its animal model,and explore its mechanism,expecting to provide new chance for clinical treatment of ovarian cancer.Objectives:1.Synthesize CuS-MnS2 nanoflower and define its characterization;2.Evaluate the biocompatibility of CuS-MnS2 nanoflower;3.Evaluate in vitro and in vivo MRI imaging signal of CuS-MnS2;4.Investigate the PPT/PDT effect of CuS-MnS2 on ovarian cancer cells and animal model of ovarian cancer;5.Explore the molecular mechanism of CuS-MnS2+ NIR killing ovarian cancer cells.Methods:1.The CuS-MnS2 nanoflowers were synthesized by hydrothermal method.The crystal structure was observed by X-ray diffractometer.The morphology,size and element distribution were observed by transmission electron microscopy(TEM),scanning electron microscopy(SEM),and energy dispersive spectroscopy.The UV-Vis-NIR absorption spectrum of CuS-MnS2 is detected using a spectrophotometer.To evaluate its photothermal performance,CuS-MnS2 solution were irradiated by near infrared laser irradiation(NIR)at 808 nm,infrared thermal imaging was collected simultaneously,and temperature was recorded to mapping heating curves.2.Some kinds of human normal cells and cancer cells were co-cultured with CuS-MnS2 and the cell viability was tested by CCK-8 kit.Human red blood cell suspension were coincubated with different concentrations of CuS-MnS2,in order to evaluate and the erythrocyte toxicity of CuS-MnS2 by hemolysis.3.CuS-MnS2 nanoflowers were configured into agarose gel solutions to different concentration,then tested MRI imaging efficacy.Make ovarian cancer mice models by inoculating ovarian cancer cells into mice subcutaneously,subsequently inject CuS-MnS2 and PBS into the tumor respectively,then take and compare the MRI images of tumors.4.Pre-cultured human ovarian cancer cells were added CuS-MnS2,irradiated by near-infrared laser to observe the photothermal,photodynamic effect.A2780 cells were inoculated into mice to form tumor-bearing mice,then the tumors were injected CuS-MnS2 or PBS respectively,irradiated by NIR and test the tempreture change.After irradiation,tumor tissues were taken for H&E staining to assess tumor cell necrosis.5.A2780 cells were added CuS-MnS2 and irradiated with NIR,then detected the apoptosis-related protein,anti-apoptosis-related protein,and necrosis-related protein by Western Blot experiment.Results:1.The p repared material is a flower-like CuS-MnS2 composite,which possesses a photothermal conversion efficiency(?)of estimated 67.5%and high photothermal stability,also T1 enhanced MRI imaging function.2.The survival rate of A2780 and H460 cells decreases obviously as CuS-MnS2 concentration reach 0.5 mg/mL and above,while the survival rate of L02,MIHA and A549 cells keep higher than 93%when co-cultured with 1 mg/mL CuS-MnS2 concentration.In red blood cells co-incubated with different concentrations of CuS-MnS2,mild hemolysis was overseved at a lower concentration,while not obvious was found at the concentration of 0.5,1.0 mg/mL.3.CuS-MnS2 holds function of T1-weighted MRI imaging,and the signal intensity is proportional to concentration.After injection of CuS-MnS2 in tumors of ovarian cancer-bearing mice,the tumor showed high T1W1 signal.4.With concentration above 0.5 mg/mL,CuS-MnS2 combined laser irradiation can cause significant A2780 cell death,also induce intracellular reactive oxygen species.Temperature of the tumor in tumor-bearing mice treated with CuS-MnS2+NIR increased to above 55? rapidly during irradiation,while the mice treated with PBS+NIR onlu about 40?.With H&E staining,tumor tissue in CuS-MnS2+NIR group was found obvious cell damage.5.In WB,down-regulation of apoptosis-associated protein,up-regulation of anti-apoptosis-associated proteins,and up-regulation of necrosis-associated protein were detected in cells treated with CuS-MnS2+NIR.Conclusions:1.CuS-MnS2 nanoflowers possess a high photothermal conversion efficiency and photothermal stability,also photodynamic effect.2.CuS-MnS2 nanoflowers hold a low cytotoxicity to human normal cells and higher cytotoxicity to some kinds of human cancer cells.3.CuS-MnS2 nano flowers possess T1 enhanced MRI imaging efficacy,and the signal intensity is proportional to concentration.4.CuS-MnS2+NIR shouw both photothermal and photodynamic effect for ovarian cancer cells,perform good in the treatment of subcutaneous ovarian cancer-bearing mice.5.Activating the programmed necrotic pathway of cells is the main mechanism of CuS-MnS2+NIR inducing cell death.