Gold Nanomaterials For Cancer Therapy And Cell Autophagy Regulation

Among the metallic nanomaterials, gold nanomaterials have become one of the most outstanding candidates for fundamental research and practical applications due to their unique optical properties, facile synthesis, easy surface functionalization, and excellent biocompatibility.Recently, gold nanomaterials with the high-Z gold atoms (Z = 79) could enhance the radiation effect over a large tumor area, making them the ideal radiosensitizers for cancer radiotherapy.Furthermore, some gold nanomaterials that can absorb and convert near-infrared (NIR) light into heat are widely used in synergistic tumor therapy. In addition, radiation therapy has been shown =to induce the up-regulation of cell autophagy. However, the relationship between autophagy and tumor proliferation provides two different outcomes for tumor growth inhibition: (1) Inhibition of autophagy may be employed to increase the efficiency of cancer therapy; (2) Induction of autophagy can induce autophagic programmed cell death. Therefore, autophagy inhibitors or inducers cannot be applied blindly in clinical applications, otherwise the opposite results will be observed.In this thesis,we successfully synthesized gold nanospikes (GNSs) with hollow interior and ultrahigh density of nanotips followed by performing a series of investigations on their applications in cancer radiotherapy and photothermal therapy. The main contents are as follows:1. Shape effect of Au nanostructures on radiation enhancement effect in cancer radiotherapyTo elucidate the shape effect of gold nanostructures on their radiosensitization performance in cancer radiation therapy, spherical gold nanoparticles (GNPs), gold nanospikes (GNSs), and gold nanorods (GNRs) with the same average diameter (-50 nm) and surface coating(polyethylene glycol, PEG) were prepared. The cellular uptake of these materials increased in the order of GNPs > GNSs > GNRs. The sensitization enhancement ratio (SER) calculated by a novel multi-target single-hit model was 1.62, 1.37, and 1.21 corresponding to the treatment of GNPs, GNSs, and GNRs, respectively, suggesting the excellent radiation enhancement effect of these gold nanostructures. A positive correlation between SER and cellular uptake efficiency was observed. Besides, nearly the same values were obtained if we divided the SER of the three types of gold nanomaterials by their corresponding cellular uptake amount. This indicates that the Au-based nanomaterials with the specific geometric structure should be applied to increase the cellular internalization of gold atoms and the radiosensitization performance in cancer radiotherapy. Taken together, the maximum cellular internalization can be achieved through designing a kind of gold nanostructures with specific shape. These findings provide useful guidance for the applications of gold nanostructures in cancer radiotherapy.2. Surface-modified gold nanospikes induce enhanced cancer radiotherapy and autophagy via efficient intracellular uptakeTo increase the cellular internalization of gold nanospikes (GNSs) for a better radiation enhancement effect, a series of different surface-modified gold nanospikes (e.g., GNSs,NH2-GNSs, FA-GNSs, and TAT-GNSs) were prepared through covalent conjugation with PEG molecules and specific ligands (e.g., amine, folic acid, cell-penetrating peptides). Among these,GNSs refer to the PEGylated gold nanospikes without functional ligands. The differences of these modified GNSs on both cellular uptake efficiency and X-ray radiosensitization were investigated. The cellular uptake in cells increased in the order of TAT-GNSs > FA-GNSs >NH2-GNSs > GNSs. The calculated SER for TAT-GNSs, FA-GNSs, NH2-GNSs, and GNSs was 2.30, 1.84, 1.57, and 1.34, respectively, which were in good consistence with their cellular uptake amounts. The results confirmed that the different surface-modified GNSs (TAT-GNSs,FA-GNSs, and NH2-GNSs) drastically enhanced the ionizing radiation effect as compared with GNSs. Furthermore, the underlying mechanisms of the radiation enhancement induced by surface-modified GNSs were explored, including endocytosis pathways, reactive oxygen species generation, mitochondrial membrane potential, cell cycle distribution, and apoptosis. The results demonstrated that surface-modified GNSs combined with X-ray radiation induced increased reactive oxygen species generation, mitochondrial depolarization, and cell cycle redistribution.Among these, reactive oxygen species as the executor played a crucial role in radiation enhancement.Meanwhile, the relationship between cell autophagy and modified GNSs-mediated radiosensitization was also investigated. Autophagy as a lysosome-based degradative pathway played an essential role in GNSs-induced radiotherapy. Western blotting assays confirmed that the surface-modified GNSs could induce the up-regulation of autophagy-related protein (LC3-?)and apoptosis-related protein (caspase-3) in treated samples. Through investigating the degradation of the autophagy substrate p62 protein,it was found that the surface-modified GNSs could impair the autolysosome degradation capacity and autophagosome accumulation.Particularly, our data demonstrated that either GNSs or GNSs + X-ray irradiation with the presence of autophagy inhibitor (3-MA) could increase the cell apoptotic ratio. That is to say,autophagy process played a protective role in caner radiotherapy,and the inhibition of protective autophagy with inhibitor would increase cell apoptosis. This work provides a better understanding of the effect of surface modification on the cellular uptake of gold nanostructures and has demonstrated that autophagy inhibitor can enhance the X-ray radiation effect of gold nanospikes.3. Enhanced radiosensitization of gold nanospikes via hyperthermia in combined cancer radiation and photothermal therapyBased on the unique near-infrared (NIR) absorption performance of PEGylated GNSs, the synergistic NIR photothermal and X-ray radiation therapeutic approach was developed. Herein,GNSs-mediated hyperthermia as an adjuvant therapy was adopted to enhance the efficiency of radiotherapy in vitro and in vivo. The SER of GNSs alone was calculated to be about 1.38, which increased to 1.63 when combined with NIR laser irradiation, which clearly confirmed that GNSs-mediated hyperthermia efficiently enhanced the radiosensitizing effect of hypoxic cancer cells. For the in vitro study, colony formation assay demonstrated that GNSs-mediated photothermal therapy and X-ray radiotherapy reduced the cell survival fraction to 89% and 51%,respectively. In contrast, the cell survival fraction of the combined X-ray radiation and photothermal treatment decreased to 33%. In vivo tumor growth study indicated that the tumor growth inhibition (TGI) in the synergistically treated group reached 92.2%, which was much higher than that of the group treated with the GNSs-enhanced X-ray radiation (TGI = 29.8%) or the group treated with the GNSs-mediated photothermal therapy (TGI = 70.5%). This research demonstrated that the superior anticancer effect could be achieved through combining both NIR absorption performance and radiation enhancement effect of GNSs, thus providing a new guideline for developing multifunctional nanotheranostics.