| Objective:Breast cancer has surpassed lung cancer,becoming the most common cancer in the world,and patients tend to be younger.Therefore there is an urgent need to develop more effective treatment methods to improve the survival rate of patients.According to the molecular classification,breast cancer can be divided into the following four types including Luminal A type,Luminal B type,HER-2 overexpression type and triple negative breast cancer.At present,the main treatment methods of breast cancer are operation,radiotherapy,chemotherapy,endocrine therapy(expression of estrogen receptor(ER)and progesterone receptor(PR))and targeted therapy(anti-Her-2 therapy).Due to the low expression of ER,PR and HER-2there is still a lack of effective targeted drugs to the triple negative breast cancer,hence,to find out potential therapeutic targets is of research significance and clinical application value.Heat shock protein 60(HSP60)belongs to the heat shock protein(HSP)family and is an important molecular chaperone.It exists in all prokaryotes and the mitochondria,Golgi apparatus,cytoplasm and cell membrane of eukaryotes.HSP60 can inhibit mitochondrial apoptosis in tumorigenesis,and promote angiogenesis in tumor tissues and cancer metastasis.An increasing number of studies have shown that HSP60 content in breast cancer tissues or cells is significantly higher than that in normal tissues or cells.HSP60 content increases gradually from normal breast epithelial cells though ductal carcinoma in situ to invasive breast cancer.This suggests that HSP60 may become a diagnostic biomarker or a potential therapeutic target for breast cancer.Our laboratory has developed a peptide(named P17)that can specifically bind to HSP60.The aim of this study is to combine this peptide with gold nanoparticles(AuNPs)to construct targeted gold nanoparticles for photothermal therapy,and explore its photothermal killing effect on mouse triple negative breast cancer cell line 4T1 cells.AuNP as a kind of photothermal conversion agent have the advantages of high photothermal conversion efficiency,good biocompatibility and easy surface modification.Photothermotherapy is a new method of tumor treatment with the advantages of high selectivity and low side effects.Methods:Firstly,AuNP were prepared by reducing HAuCl4 with sodium citrate;the morphology of AuNPs was characterized by transmission electron microscope(TEM).Then,AuNP-P17 was prepared by modifying AuNP with synthetic peptide P17.The characteristic spectrum of AuNP-P17 was obtained by UV-vis-NIR spectroscopy;the particle size and zeta potential of AuNP-P17 were characterized by dynamic light scattering(DLS).Flow cytometry was used to detect the HSP60 expression on the surface and inside of 4T1 cells and the binding of P17 to 4T1 cells.The uptake of AuNP and AuNP-P17 by 4T1 cells were observed by inverted microscope.The temperature of 4T1 cells incubated with AuNP or AuNP-P17 for 24 h was measured by a portable thermocouple thermometer after 2 or 4 min laser irradiation.CCK-8 kit was used to determine the effects of different concentrations of P17,AuNP and AuNP-P17 on the cell viability of 4T1 cells.Results:AuNP was granular,uniform in size and well dispersed;AuNP had a classical SPR single peak at about 528 nm.After incubation with P17,the intensity of SPR absorption peak of AuNP decreased and the absorption value at long wave increased with the increase of P17 concentration,indicating that P17 was connected to AuNP.DLS results showed that the average particle size increased with the increase of P17 ratio,from(51.8±0.1)nm to(52.1±2.2)、(60.8±1.9)and(64.5±2.5)nm。The zeta potential changed from(-29.3±0.3)mV to(-29.6± 0.6)、(-26.4±0.4)and(-26.9±2.5)mV.The flow cytometry results showed that HSP60 was expressed on the surface and inside of 4T1 cells,and P17 could bind to the surface and intracellular HSP60 of 4T1 cells.The results of cell viability experiment showed that P17 had little effect on the cell viability of 4T1 cells in the range of 0-20 μM;at the same time,AuNP or AuNP-P17 had no significant effect on the cell viability of 4T1 cells when the concentration of gold atom was less than 100μg/mL.These results showed that P17,AuNP and AuNP-P17 had good biocompatibility.The results of inverted microscope showed that when the two nanoparticles were co-incubated with 4T1 cells for 4,6or 8 h,there was no significant change in cell color in AuNP treated group compared with the control group,indicating that the binding of AuNP to 4T1 cells was not obvious;however,the red color of AuNP could be seen in the 4T1 cells treated with AuNP-P17,which indicated that the combination of AuNP-P17 and 4T1 cells was very obvious.P17 promoted the uptake of AuNP by 4T1 cells,indicating that AuNP-P17 had strong ability of targeting.When 4T1 cells were co-incubated with AuNP and AuNP-P17 and irradiated by laser,the temperature of culture medium changed.The temperture of AuNP-17 treated group increased from(22.9±0.4)℃ to(52.0±3.9)℃ after 4 minutes of laser irradiation;for the AuNP treated group,the temperature of cell culture medium only increased to(47.2±0.6)℃,while the control group only warmed up to(38.2±2.4)℃.These results of cell viability experiment showed that there was no significant change in cell viability of AuNP and AuNP-P17 treated groups compared with the control group after 2 minutes of laser irradiation.After 4 minutes of laser irradiation,the cell viability of cells in AuNP-P 17 treatment group decreased to(1.3± 2.8)%,and the cell viability of AuNP treated group decreased to(64.1±6.8)%,while the cell viability of the control group was similar to the group without laser irradiation(100.0±3.2)%.There was significant difference between any two groups of the three(***:P<0.001).The results showed that compared with AuNP,the photothermal killing effect of AuNP-P17 on 4T1 cells was significantly stronger.Conclusion:Based on the high expression of HSP60 in breast cancer cells,we modified AuNP with HSP60 binding peptide P17 to construct a photothermal killing system AuNP-P17,targeting the breast cancer cell 4T1.The nanoparticles had low cytotoxicity and good biocompatibility.4T1 cells showed stronger uptake of AuNP-P17 than AuNP.The temperature of 4T1 cells co-incubated with AuNP-P17 increased significantly under laser irradiation,which indicated that AuNP-P17 had targeting and photothermal effects,and achieved efficient photothermal killing of 4T1 cells.Therefore,targeting HSP60 can enhance the photothermal killing effect of AuNP on 4T1 cells,which has the potential for further research and transformation in the treatment of triple negative breast cancer. |
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