Mitochondria-targeting Au Nanoclusters:Cell Imaging And Enhanced Radiosensitivity Of Cancer Cells

With the rapid development of nanotechnology recently,nanomaterials have attracted much attention in the applications of molecular detection,biological imaging,diagnosis and therapecutics for diseases.Au nanoclusters are a new type of fluorescent nanomaterials,exhibiting attractive features such as ultra small size,large Stokes shift,long-term stable fluorescence and good biocompatibility.It has found a broad range of applications as biological sensors,nano probes,cell markers,imaging contrast reagents,or theranostic nanomaterials.In the literature reports,Au nanomaterials have been proposed as promising radiotherapy sensitization agent.Radiotherapy has been widely utilized in clinics,and brought the hope of survival to the patients.But the traditional radiotherapy suffers from low selectivity in differentiating tumor sites from normal tissues,or high dosage with significant side effects.Interestingly,Au nanomaterials can produce photoelectrons,compton electrons and secondary charger particles under X-ray irradiation,which can dramatically produce cellular reactive oxygen species(ROS),and induce cellular damages such as double-stranded DNA breaks inside cells.Therefore,gold nanomaterials can reduce the radiation dose effectively as radiotherapy sensitization agents.However,it remains a challenge whether the radiosensitization effect can further elevated by new designs.Mitochondrion is an important cell organelle for energy production and maintainence of cellular ROS equilibrium.My thesis mainly focuses on the development of Au nanoclusters with targeting mitochondrial function in radiotherapy sensitization.A new peptide with the sequence of CCYKFR was designed,in which the peptide segment of KFR can assist mitochondria targeting.The unique composition of CCY peptide segment allows the reduction of Au ions into nanocdusters via the phenolic group of tyrosine(Y)under the alkaline condition,and simultaneously stabilizes Au nanoclusters with the sulfydryl groups of cysteine(C).By adjusted the pH value of the reaction,I successfully prepared peptide-templated Au nanoclusters(CCYKFR-AuNCs)with the ability of targeting mitochondria through one-step synthesis.In addition,the peptide sequence CCY without targeting mitochondrial function CCY was used to synthesize Au nanoclusters(CCY-AuNCs)as the control.Fluorescent images were acquired by laser confocal scanning microscopy,and compared the intracellular distribution of these two different types of Au nanoclusters.The distribution of Au nanoclusters among the cell organelles was also analyzed by TEM imaging of the cell slices.These experiments suggested that CCYKFR-AuNCs were internalized by the cells effectively,and accumulated onto mitochondria under the mediationg of mitochondrial-targeting peptide.It provided evidence that the as-prepared Au nanoclusters could serve as a mitochondria fluorescent probe.In the experiments to investigate the internalization pathways of CCYKFR-AuNCs,we tested different cellular pathway inhibitors,demonstrating that the CCYKFR-AuNCs internalization was dominated by the caveolae-mediated endocytic pathway.The radiosensitive effect of AuNCs was investigate in the scenario of mitochondria accumulation.A series of experiments were designed to exam the performance of peptide-AuNCs as radiosensitizers with or without mitochondria targeting.Indeed,AuNCs can improve X-ray killing effect on cells,as validated by cytotoxicity assays and cloning assays preliminary.CCYKFR-AuNCs performed better than CCY-AuNCs,attributed to the mitochondria-targeting effect.The intracellular ROS level was tested in the cell experiments by using dedicated ROS probe,which further revealed that the mitochondria-targeted AuNCs can promote ROS levels in the cells significantly under X-ray irradiation.Immunofluorescence staining method was employed to detect cell DNA damages induced by the combination of AuNCs and X-ray.The results indicated that the mitochondria-targeting Au NCs can generate much more DNA damages than the control.Therefore,CCYKFR-AuNCs is a good radiotherapy sensitization agent by enhancing the radiosensitivity of cancer cells under the exposure to lower dosage of X-ray irradiation.My work provides a new strategy for the development of theranostic nanomaterials,by integrating mitochondrial targeting imaging and enhanced radiosensitization into the biocompatible Au nanoclusters.It promises important applications in the development of targeted anticancer therapies.