Research On X-PDT Of Breast Cancer Mediated By Nano-scintillator And Its Mechanism

Objective:1.The aim of this study was to prepare the nano-scintillators（NSs）using hydrothermal one-step method,detect of the morphological and optical characteristics of NSs,and explore the potential as scintillator for X-ray induced photodynamic therapy（X-PDT）.2.The aim of this study was to investigate the therapeutic effect of NSs for X-PDT on 4T1 breast cancer cells in vitro,explore the influence of X-PDT on proliferation,migration,and invasion ability of 4T1 cells.In this study,we investigated the inhibitory effect of X-PDT on cellular autophagy and the changes of the ubiquitin-dependent mitophagy pathway of PINK1/Parkin,mitochondrial membrane potential,PI3K/AKT/mTOR pathway,autophagy markers（LC3 and p62）,Golgi protein（GM130and p115）,and p53 during the radiosensitization process.3.It was aimed to explore the therapeutic effect of NSs on mouse model of 4T1tumor-bearing mice by X-PDT and the mechanisms on the genetic,protein,and histological level in this research.Methods:1.Preparation and characterization of NSs nano-scintillator.NSs were prepared using one-step hydrothermal method based on carboxymethyl cellulose（CMC）and p-bromobenzoic acid,with the photosensitizer of Rose bengal（RB）.Based on the structural characteristics of scintillators,transmission electron microscopy（TEM）,Fourier transform infrared spectroscopy（FT-IR）,and X-ray photoelectron spectroscopy（XPS）were used to analyze the particle morphology,infrared characteristics,and electron spectrum of scintillators.The optical characteristics of scintillators are tested using fluorescence emission,phosphorescence spectroscopy,and lifetime testing.Different concentrations of NSs were applied to treat 4T1 cells,and their effects on cell viability were measured using MTT assay.4T1 cells were divided into blank control group（Control）,NSs group,radiotherapy group（X-ray）,and X-PDT group（NSs+X-ray）according to different treatment methods.Using singlet oxygen sensor green（SOSG）probe to detect the ¹O₂ production of NSs induced X-PDT.2.The mechanism of NSs induced X-PDT in vitro.Applied MTT method to determine appropriate NSs concentration and X-ray irradiation dose,then covered the chicken breast to determine the tissue penetration depth of X-PDT mediated by NSs.Appling Calcein AM/PI live/dead cell dual staining,C11-BODIPY lipid peroxidation probe to detect the killing effect and lipid peroxidation of X-PDT.Utilized cloning formation,Ethynyl deoxyuridine（EdU）,wound healing and Transwell assay to investigate the proliferation,migration,and invasion ability of 4T1 cells.Real-time quantitative PCR（qPCR）was used to analyze the expression of PINK1,Parkin,p-PI3K,p-AKT,p-mTOR,LC3,p62,GM130,p115 and p53 mRNA.Western blot was used to detect the expression of PINK1/Parkin pathway,PI3K/AKT/mTOR pathway,autophagy markers（LC3 and p62）,Golgi protein（GM130 and p115）,and p53 in each group.TEM was used to detect the presence of autophagosomes,as well as the morphological changes of mitochondria and Golgi apparatus.3.The mechanism of NSs induced X-PDT in vivo.The 4T1-bearing BALB/c mice were divided into five groups according to different treatment methods,including blank control group,NSs group,radiotherapy group,intravenous injection and intratumoral injection group of X-PDT.Using small animal live imaging system to explore the enrichment of NSs in subcutaneous tumors of mice.Monitored tumor volumes and weights,and evaluated the effect of X-PDT in vivo by combining hematoxylin-eosin staining（HE）and immunohistochemistry（IHC）staining.Indicators such as weight,liver function,kidney function and routine blood test were detected in mice.Collected the heart,liver,spleen,lungs,and kidneys of each group for HE staining and observed the toxicity in vivo.qPCR technology was used to detect the expression of PINK1/Parkin pathway,PI3K/AKT/mTOR pathway,autophagy markers（LC3 and p62）,Golgi apparatus proteins（GM130 and p115）and p53 in genetic level.Western blot was used to further verify the changes of aforementioned proteins.Immunofluorescence staining was used to detect the distribution of LC3,Parkin,and GM130 in tumor tissue.Results:1.Successfully prepared nano-scintillator NSs and characterized them.（1）The TEM image showed that the average size of NSs was 3nm,and the lattice spacing was 0.21nm.The chemical structure and functional group composition of NSs were confirmed by FT-IR spectroscopy.XPS spectroscopy further confirmed the state and content of chemical elements in NSs.（2）Fluorescence spectroscopy revealed that NSs exhibited bimodal emission peaks of 450nm and 560nm.The phosphorescence analysis confirmed the phosphorescence characteristics of NSs,with phosphorescence lifetime of 134.24ms and 60.97ms at emission wavelengths of 475nm and 615nm,respectively.（3）Under fluorescence microscope,it was observed that NSs can be ingested by4T1 cells and exhibited red fluorescence with dose-dependent trend.When the concentrations of NSs were 1μg/ml,5μg/ml,10μg/ml,50μg/ml,100μg/ml,200μg/ml and 500μg/ml,the viabilities were（94.25±1.08）%,（93.42±2.13）%,（88.53±9.60）%,（80.30±6.04）%,（62.46±2.42）%,（57.79±6.21）%,and（53.60±0.83）%,respectively.The SOSG probe showed significant increase of green fluorescence intensity in the X-PDT group compared to those of other groups（P<0.05,n=3）.2.Results of NSs induced X-PDT in vitro.（1）The concentration of NSs in X-PDT was determined to be 10μg/ml by MTT method,with an effective irradiation dose of 4Gy,and have exhibited good tissue penetration.There was no significant difference in the relative viability of 4T1 cells covered with 3cm chicken breast compared to that of non-covered（P>0.05,n=3）.Significantly more dead cells,and a higher degree of lipid peroxidation in the X-PDT group than those in other groups could be observed（P<0.05,n=3）.（2）X-PDT mediated by NSs inhibited the biological behavior of proliferation,migration,and invasion of 4T1 cell.Colony formation,EdU,wound healing and Transwell assays showed that the proliferation,migration,and invasion abilities of 4T1cells in the X-PDT group were significantly reduced compared to those of other groups（P<0.05,n=3）.（3）X-PDT mediated by NSs downregulated PINK1/Parkin pathway for ubiquitin-dependent mitophagy,and inhibition of mitochondrial autophagy led to the accumulation of mitochondria with reduced membrane potential.The qPCR results showed the mRNA expression levels of PINK1 and Parkin decreased in X-PDT group than those in other groups（P<0.05,n=3）.Western blot results showed that the expression of PINK1 and Parkin decreased in the X-PDT group compared to other groups（P<0.05,n=3）.The decrease in fluorescence expression of JC-1 probe for mitochondrial membrane potential was more obvious in the X-PDT group than that in other groups（P<0.05,n=3）.TEM images showed that the accumulation of mitochondria with structural damage.（4）X-PDT mediated by NSs led to upregulation of PI3K/AKT/mTOR pathway and inhibition of cellular autophagy.The qPCR results showed increase in mRNA expression of p-PI3K,p-AKT,and p-mTOR in X-PDT group than those in other groups（P<0.05,n=3）.Western blot results showed that the expression of PI3K,AKT,and mTOR in the X-PDT group increased compared to other groups（P<0.05,n=3）.The qPCR results showed that the expression of LC3 mRNA was reduced and the expression of p62 mRNA was increased in the X-PDT group compared to other groups（P<0.05,n=3）.Western blot results showed that the autophagy markers of LC3-II/LC3-I increased in the X-PDT group compared to other groups,while the expression of p62protein decreased（P<0.05,n=3）.TEM images showed significant autophagosome formation in the blank control group and NSs group at the level of cell ultrastructure,while almost no autophagosome was observed in the radiotherapy and X-PDT group.（5）X-PDT mediated by NSs caused downregulation of Golgi protein GM130,degradation of vesicular transport protein p115,and structural damage to Golgi apparatus,further upregulated p53 protein,which resulted in autophagy inhibition.The qPCR results showed that the mRNA of Golgi protein GM130 and vesicle transport protein p115 were down regulated in the X-PDT group,while the expression of p53mRNA was increased（P<0.05,n=3）.Western blot results showed that the expressions of GM130 and p115 in the X-PDT group were significantly decreased compared to those of other groups,while the expression of p53 was upregulated（P<0.05,n=3）.TEM images showed significant swelling of the Golgi apparatus in the X-PDT group.3.Results of X-PDT induced by NSs in vivo.（1）The results of small animal imaging showed that NSs bounded to the fluorescent dye Cy5.5-COOH,and the effective accumulation could be detected in the tumor 0.5 hours after injection.The stable and high-level accumulation of NSs within the tumor could be observed from 1st hour to 24th hour,and it could also be observed that NSs had been rapidly metabolized through liver and kidney.（2）NSs exhibited good therapeutic effects in vivo X-PDT.The volumes of the tumors in the blank control,NSs,radiotherapy,intravenous injection and intratumoral injection X-PDT group were（1648.46±394.75）mm³,（1263.64±319.11）mm³,（821.81±179.40）mm³,（238.96±34.32）mm³and（256.15±29.54）mm³,respectively.The weights of the tumors in the above groups were（0.8604±0.2707）g,（0.5666±0.0920）g,（0.2011±0.0360）g,（0.0903±0.0522）g and（0.0701±0.0169）g,respectively.The spleen index of these groups were（2.87±0.42）%,（2.64±0.24）%,（1.13±0.26）%,（0.65±0.06）%,and（0.76±0.06）%,respectively.The tumor volumes,weights,and spleen indexes were significantly reduced in the intravenous and intratumoral injection of X-PDT groups than those of other groups（P<0.05）.HE staining and IHC staining of Ki67 both confirmed the satisfactory therapeutic effect of X-PDT group at the histological level.（3）NSs did not present obvious toxicity in vivo.There were no significant differences in the weight,liver function,kidney function,and blood routine of the mice in different groups（P>0.05）.No obvious abnormalities were observed in the HE staining slices of the hearts,livers,spleens,lungs,and kidneys of mice in different groups.（4）The ubiquitin-dependent pathway of PINK1/Parkin has been inhibited.The qPCR results showed the mRNA expression of PINK1 and Parkin decreased in X-PDT group compared to those of other groups（P<0.05）.Western blot showed that the expression of PINK1 and Parkin were reduced in X-PDT group than in others（P<0.05）.The immunofluorescence results showed decrease of Parkin protein expression in the X-PDT group compared to other groups（P<0.05）.（5）The PI3K/AKT/mTOR pathway has been activated,which inhibited cell autophagy.The qPCR results showed an increase in mRNA expression of p-PI3K,p-AKT and p-mTOR in X-PDT group compared to those of other groups（P<0.05）.Western blot showed that the expression of PI3K,AKT,and mTOR in the tumor tissue of X-PDT group were upregulated（P<0.05）.IHC staining showed upregulation of p-PI3K,p-AKT,and p-mTOR expression in the X-PDT group（P<0.05）.The qPCR results showed that the expression of LC3 mRNA in the X-PDT group was lower and the p62 mRNA in the X-PDT group was higher than those in other groups（P<0.05）.Western blot showed that LC3-II/LC3-I levels in the X-PDT group decreased compared to other groups（P<0.05）,while the expression of p62 increased（P<0.05）.Immunofluorescence staining confirmed that the expression of LC3 decreased in the X-PDT group compared to that of other groups（P<0.05）.（6）The damage of Golgi apparatus could be associated with p53 protein and autophagy inhibition in vivo experiments.The qPCR results showed that the mRNA expression of GM130 and p115 in the X-PDT group decreased,while the expression of p53 mRNA increased（P<0.05）.Western blot results revealed that the expressions of Golgi protein GM130 and vesicle transport protein p115 in X-PDT tumor tissue were downregulated,while the expression of p53 protein was upregulated（P<0.05）.Immunofluorescence showed decrease in GM130 expression in the X-PDT group compared to that of other groups（P<0.05）.Conclusions:1.The phosphorescent nano-scintillator NSs were successfully designed and prepared with excellent fluorescence and phosphorescence response,which could generate a large amount of ¹O₂ under X-ray excitation.2.X-PDT mediated by NSs was achieved by inhibiting cellular autophagy,particularly mitophagy.It was found that the damage of Golgi apparatus caused by X-PDT was also involved in the molecular mechanism of autophagy inhibition.3.NSs successfully mediated X-PDT in 4T1-bearing BALB/c mice,and autophagy inhibition played an important role in the therapeutic mechanism.