| Purpose:Prepare gold nanoparticles (GNPs) for the experiment of radiation enhancement in A549cells in vitro.Materials and methods:1. Chemical reduction of gold salt in aqueous is the classical method to prepare GNPs, which was introduced by Turkevich. Gold salt in aqueous can be reduced by several reducing agents, like NaBH4, PEG, and sodium citrate. Here, in the experiment, GNPs were synthesized by the reduction of aqueous chloroaurate ions by sodium citrate. After GNPs were prepared, thio-glucose was added to form thio-glucose capped GNPs (Glu-GNPs);2. The diameter and morphology of the particles were viewed by transmission electron microscopy (TEM). The size distribution was measured by dynamic light scattering (DLS). The concentration of GNPs and Glu-GNPs was tested by inductively coupled plasma atomic emission spectroscopy (ICP-AES). X-ray photoelectron spectroscopy (XPS) was used to verify the binding of GNPs with thio-glucose.Results:According to the picture in TEM, GNPs were13±2.2nm in uniform size with analysis by Origin Software, and spherical with good disperse. The sized distribution was also measured by DLS, which is15±1.8nm, about2nm larger than that observed by TEM. The difference is attributed to that TEM identifies the core particle, while DLS measures the hydrodynamic radius. The concentration of the synthesized GNPs was36.9mg/l measured by ICP-AES. The average number of coating molecules (approximately2.3×104thio-glucose) on nanoparticle surface (calculated by measuring the gold-to-sulfur atom ratio) was measured by XPS.Conclusion:GNPs capped with thio-glucose were successfully prepared by the method above according to the measurement of instruments. Purpose:Study the uptake of naked GNPs and Glu-GNPs by A549cells; their distribution in cells, and the cytotoxicity of Glu-GNPs to A549cells; observe the effect of radiation enhancement of Glu-GNPs to A549cells; explore the mechanisms of radiosensitivity including cell cycle distributi and cell apoptosis, the further molecular mechanisms involved in apoptosis.Materials and methods:1. After A549cells were cultured with Glu-GNPs for24hours, they were centrifuged and collected to make paraffin section. The section was observed by TEM to confirm distribution of Glu-GNPs in cells;2. A549cells were exposed to naked GNPs and Glu-GNPs with a concentration of5nM. Collect cells at different time intervals, lyse them and centrifuge. Then measure the gold mass using ICP-AES. Thus quantities and numbers of GNPs and Glu-GNPs in a cell were obtained;3. Expose cells to Glu-GNPs with different concentrations (0nM,5nM,10nM and20nM) for different time (24h,48h and72h), and then cell viability was tested by MTT assay;4. Determine viability of cells exposed to Glu-GNPs with different concentrations (0nM,5nM,10nM and20nM) combined with radiation (dose:10Gy) by MTT assay;5. A549cells with different numbers (50,200,400,1000,2000and5000) were incubated with20nM Glu-GNPs for24h. Then replace culture medium and expose cells to different radiation doses (0Gy,2Gy,4Gy,6Gy,8Gy, and10Gy) according to different cell numbers.2weeks later, count the clonies and protract clonogenic survival assay curves;6. Divide A549cells to four groups:control, Glu-GNPs, radiation (IR), and Glu-GNPs+IR, respectively. Collect cells after treatment and analyze changes of cell cycle by PI staining using flow cytometry;7. Divide A549cells to four groups:control, Glu-GNPs, radiation (IR), and Glu-GNPs+IR, respectively. Apoptosis of each sample was analyzed by Annexin V and PI staining by flow cytometry;8. Divide A549cells to four groups:control, Glu-GNPs, radiation (IR), and Glu-GNPs+IR respectively. Extract protein and test the expression of several critical proteins related to apoptosis, including Bcl-2, Bax, and cleaved caspase-3. Results:1. Glu-GNPs were localized in cytoplasm of A549cells, mainly on the membrane of membrane-coated vesicles including endosomes and lysosomes in aggregated states by TEM;2. The uptake of both naked GNPs and Glu-GNPs by A549cells reached a peak after incubation for24h, with much more Glu-GNPs than GNPs at any time interval (except6h)(P<0.05);3. Glu-GNPs did not induce obvious growth inhibition on A549cells, with the concentration below20nM (P>0.05);4. Cell viability decreased with increasing concentrations of Glu-GNPs at any radiation dose (P<0.05);5. Clonogenic survival assay showed Dq, D0and SF2of Glu-GNPs+IR group were all lesser than IR group, and the SER of the combined group was1.49. Glu-GNPs can significantly increase the radiation effect of A549cells (P<0.05);6. Glu-GNPs increased radiation effect by arresting A549cells at G2/M phase which is the most sensitive phase to radiotherapy (P<0.05);7. Flow cytometry indicated that Glu-GNPs helped radiation to inhibit A549cell growth by increasing cell apoptosis (P<0.05);8. Western blot showed that Glu-GNPs play a important role in apoptosis by upregulating the expression of Bax and cleaved caspase-3and downregulating the expression of Bcl-2.Conclusion:Glu-GNPs are mainly distributed in the membrane-coated vesicles of A549cells like endosomes and lysosomes. Thio-glucose can help GNPs easily enter to A549cells. Glu-GNPs with concentrations below20nM induce no influence on cell growth; however the combination of Glu-GNPs with radiation results in a significant growth inhibition, compared with radiation alone. Glu-GNPs enhanced radiation effect by increasing the ratio of A549cells in the G2/M phase, which is the most sensitive phase to radiation, and inducing more apoptosis. Furthermore, when combined with radiation, Glu-GNPs resulted in downregulation of Bcl-2and upregulation of Bax and cleaved caspase-3. Our results suggest that Glu-GNPs, as a new radiosensitizer, combined with radiation, can increase cytotoxicity on A549cells not only by arresting of the G2/M phase, but also by increasing apoptosis—probably via promoting caspase-3activity and mitochondrial apoptotic pathway. |
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