The Effect Of AuNPs-PEG-AS1411 Nanoparticles On Radiosensitization Of He La Cancer Cells

Objective:To study the effect of Au NPs@MPA-PEG 、Au NPs-PEG-AS1411 and Au NPs-PEG-c Apt on radiosensitization of human uterine cervix cancer cell line(He La).Gold nanoparticles(Au NPs) were functioned with MPA-PEG 、 PEG-AS1411 、PEG-c Apt,respectively. He La cells were exposed to different concentrations of Au NPs@MPA-PEG 、 Au NPs-PEG-AS1411 and Au NPs-PEG-c Apt combined with radiation, then the cell survival rate of He La cells was detected;the intracellular uptake of Au NPs@MPA-PEG 、 Au NPs-PEG-AS1411 and Au NPs-PEG-c Apt was measured and the DNA damage of He La cells was observed.Methods: Gold nanoparticles(Au NPs) were synthesized by reducing gold chloride in the presence of sodium citrate.And the grain sizes distribution was homogeneous.Then they were functioned with MPA-PEG 、PEG-AS1411 、PEG-c Apt,respectively.The UV-Vis absorption spectra of Au NPs@MPA-PEG 、Au NPs-PEG-AS1411 and Au NPs-PEG-c Apt were collected by UV-VIS-NIR spectrophotometer(UV-3600).The size and size distribution were measured by using dynamic light scattering.The drug concentrations were measured by inductively coupled plasma mass spectrometry(ICP-MS).He La cells were cultured in different concentrations of Au NPs@MPA-PEG、 Au NPs-PEG-AS1411 and Au NPs-PEG-c Apt in vitro.CCK-8 assay was used to measure the acute toxicity effect of He La cells and colon forming assay was used to measure the chronic toxicity effect.At the same time,colon forming assay was applied to study the cell survival rate of He La cells after exposuring to Au NPs@MPA-PEG、 Au NPs-PEG-AS1411 and Au NPs-PEG-c Apt combined with radiation.The intracellular uptake of Au NPs@MPA-PEG、 Au NPs-PEG-AS1411 and Au NPs-PEG-c Apt were detected by ICP-MS.γ-H2 AX fluorescence spot test was used to observe the number of DNA double-stranded breaks of He La cells,which was applied to study the effect of Au NPs@MPA-PEG、 Au NPs-PEG-AS1411 and Au NPs-PEG-c Apt on DNA double-stranded breaks of He La cells caused by X-ray.Results:⑴ Gold nanoparticles(Au NPs) were synthesized by reducing gold chloride in the presence of sodium citrate. The grain sizes distribution was homogeneous and the partcle size was 44 nm.Then they were functioned with MPA-PEG 、PEG-AS1411 、PEG-c Apt,respectively. And the nanoparticle sizes were 48nm、50nm and 50 nm.⑵ The CCK-8 experimental results showed that the acute toxicity effect of Au NPs@MPA-PEG 、 Au NPs-PEG-AS1411 、 Au NPs-PEG-c Apt was small; all the survival rates of He La cells were more than 95% when the Au NPs@MPA-PEG 、Au NPs-PEG-AS1411 、 Au NPs-PEG-c Apt with different concentrations were used to treat the cells for 24h、48h and 72 h, respectively. Colon forming assay was used to measure the chronic toxicity effect. With the increasing of the concentrations of Au NPs@MPA-PEG、Au NPs-PEG-AS1411、Au NPs-PEG-c Apt,the cell survival rate of He La cells was reducing significantly. When the concentrations of Au NPs@MPA-PEG、Au NPs-PEG-AS1411、Au NPs-PEG-c Apt comed to 10 mg/L,the cell survival rate of He La cells was 84.7%、78.2%、80.6%, respectively.The experimental result indicated that Au NPs@MPA-PEG、Au NPs-PEG-AS1411 and Au NPs-PEG-c Apt had the chronic toxicity effect.⑶ The results of the intracellular uptake experiment showed that when the concentrations was at 10 mg/L,the number of Au NPs-PEG-AS1411 in cell and nuclei was the largest. Au NPs-PEG-c Apt and Au NPs@MPA-PEG made no difference.⑷ The results of the survival rate of cells after radiation which was detected by colon forming assay showed that: there was a significant difference in survival rate curve of He La cells pretreating by different concentrations of Au NPs@MPA-PEG 、Au NPs-PEG-AS1411 、 Au NPs-PEG-c Apt and exposuring to X ray by different irradiation doses(P<0.05) when the concentrations were ranging from 0 to 10mg/L. The low-dose zone curve became smaller and the linear slope increased significantly. The survival rate of all dose points was lower than the control group, indicating Au NPs@MPA-PEG 、 Au NPs-PEG-AS1411 and Au NPs-PEG-c Apt had a significant radiosensitivity effect on the He La cells. When the concentrations of Au NPs@MPA-PEG、Au NPs-PEG-AS1411、Au NPs-PEG-c Apt coming to 10 mg/L,the SERDo of Au NPs@MPA-PEG、Au NPs-PEG-AS1411、Au NPs-PEG-c Apt was 1.12、1.19、1.12, respectively. The SERDo of Au NPs-PEG-AS1411 was the largest.⑸ DNA damage experimental results showed that after the pretreatment of 10mg/L of Au nanoparticles,the number of fluorescence spot in the nuclei was much more than the irradiation alone group. And the group of Au NPs-PEG-AS1411 was the largest.When the irradiation dose was 8Gy, the number of fluorescence spot of Au NPs-PEG-AS1411 was 34.69±3.04,the irradiation alone group was 20.78±2.73,while Au NPs@MPA-PEG、Au NPs-PEG-c Apt was 26.05±1.86、27.05±2.16,respectively.The number of DSBs of Au NPs-PEG-AS1411 was the largest,Au NPs@MPA-PEG and Au NPs-PEG-c Apt made no difference.Conclusions:⑴ At room temperature,Au NPs、Au NPs@MPA-PEG、Au NPs-PEG-AS1411、Au NPs-PEG-c Apt were synthesized and the particle size were 44nm、48nm、50nm and50 nm,respectively.⑵ Au NPs@MPA-PEG、Au NPs-PEG-AS1411、Au NPs-PEG-c Apt did not show the acute toxicity effect on He La cells.In the colon forming assay, when the concentrations of Au NPs@MPA-PEG、Au NPs-PEG-AS1411、Au NPs-PEG-c Apt comed to 10 mg/L,the cell survival rate of He La cells were 84.7%、78.2%、80.6%, respectively; indicating that Au NPs@MPA-PEG 、 Au NPs-PEG-AS1411 and Au NPs-PEG-c Apt had the chronic toxicity effect.⑶ The uptake of Au NPs-PEG-AS1411 in He La cell and nuclei was the largest.Au NPs were functioned with AS1411 which could promote He La cells to absorb themselves.⑷ When the concentrations were ranging from 0 to 10mg/L, the colon forming assay showed that Au NPs@MPA-PEG 、Au NPs-PEG-AS1411、Au NPs-PEG-c Apt had a significant radiosensitivity effect on He La cells. The SER of Au NPs-PEG-AS1411 was largest, the SERDo of Au NPs-PEG-AS1411 was 1.19 at the concentrations of 10mg/L.⑸ After the pretreatment of Au nanoparticles,the number of DSBs was much more than the irradiation alone group. The group of Au NPs-PEG-AS1411 was the largest,Au NPs@MPA-PEG and Au NPs-PEG-c Apt made no difference.