The Function Of Metal Nanoparticle And Annexin A2 Receptor In Angiogenesis

Part 1 Gold nanorods inhibit angiogenesisPurpose: The present study investigated the cellular uptake mechanism, the specific inhibition of GNRs on HREC and the possible mechanism.Methods: We synthesize GNRs with seed mediated method, decorated GNRs with PEG and examine the characteristics of PEGYlated GNRs. Then we investigate the cellular uptake mechanism using chemical inhibitors and si RNAs. Furthermore, we investigate the effect of PEGYlated GNRs on HREC with cell proliferation assay, cell migration assay and tube formation assay. Then, we use confocal microscope to study the cytoskeletal changes of HREC treated with PEGYlated GNRs. Furthermore, we use flow cytometry to detect cell cycle changes of HREC treated with PYGYlated GNRs. Finally, we investigate the possible mechanism using Western blot.Results The GNRs we synthesized are of high purity and well decorated with PEG, which could adsorb serum proteins forming protein corona that changes the characteristics of PEGYlated GNRs time dependently. We further proved that cellular uptake mechanism of PEGYlated GNRs is time dependent. Then we found that PEGYlated GNRs could suppress HREC proliferation, migration, tube formation and induce cytoskeletal change. The possible mechanism might be that PEGYlated GNRs could induce cell cycle arrest at G1 phase and affect the expression of cell cycle related genes.Conclusion GNRs could specifically inhibit vascular related cells proliferation via inducing cell cycle arrest while had little effect on other cells, which makes it possible for further clinical application.Part 2 Cuprous oxide nanoparticles suppress angiogenesis via inhibiting VEGFR2 expressionPurpose: To examined the effect of Cuprous oxide nanoparticles(CO-NPs) on angiogenesis and the possible mechanism.Methods: In the present study, the anti-angiogenic function of CO-NPs in primary HUVEC had been investigated in detail using cell proliferation assay, cell migration assay and tube formation assay. Furthermore, we examined the effect of CO-NPs on the incidence of apoptosis and cell cycle phase. In order to better understand the molecular mechanism, we studied the expression of VEGF and VEGFR2 in both protein and m RNA level and the status of VEGFR2/ERK phosphorylation at the same time.Results: CO-NPs can induce HUVEC morphology changes and inhibit cell proliferation, migration and tube formation dose dependently. Furthermore, CO-NPs are able to induce HUVEC cell cycle arrest at S phase and cell apoptosis at both the early and late apoptotic stage. Finally, we reveal that the plausible anti-angiogenic mechanism of CO-NPs is through down regulating the expression of VEGFR2 but not VEGF or VEGFR1.Conclusion: The results obtained from this study provide novel understanding of CO-NPs in biomedical application which may lead to the emergence of CO-NPs as a promising therapeutic molecule to suppress angiogenesis.Part 3 Disrupt Annexin A2 receptor expression suppress angiogenesisPurpose: The present study aimed to examine the effect of disrupting AXIIR expression on angiogenesis and the possible mechanism.Methods: Firstly, we designed and synthesized small interfering RNA targeted to AXIIR and examined the interfering efficiency using western blot and RT-PCR. Then we studied the effect of si AXIIR on angiogenesis using cell proliferation assay,cell migration assay, cell adhesion assay and angiogenesis assay in vitro and in vivo. Furthermore, we use flow cytometry to detect cell cycle and apoptosis changes of cells treated with si AXIIR.Results: We found AXIIR was expressed in HUVECs and knockdown of AXIIR expression using RNA interference technique could inhibit HUVECs proliferation, adhesion, migration and tube formation significantly. Furthermore, we found AXIIR si RNA could induce cell cycle arrest in the S/G2 phase while had no effect on cell apoptosis and could inhibit MMP2 and MMP9 expression significantly.Conclusion: The results obtained from this study demonstrate an important role of AXIIR in endothelial cell proliferation, migration, adhesion, which may be a novel target for angiogenesis therapy.Part 4 Cuprous oxide nanoparticles inhibit uveal melanoma migration and invasionPurpose: The present study aim to investigate the effect of CO-NPs on uveal melanoma(UM) and possible mechanism.Methods: We examined the changes of CO-NPs using western blot and protein mass spectrometry, and examined the effect of CO-NPs on multiple cells proliferation. Then we studied melanoma cell migration ability and invasion ability using transwell assay. Furthermore, we investigated the internalization pathway of CO-NPs using chemical inhibitors. The skeletal changes and autophagy were detected using confocal microscopy. The apoptosis was determined using TUNEL assay. Finally, the possible mechanism was investigated using western blot.Results: The formation and composition of protein corona had much effect on cellular uptake pathway and particle intracellular localization. Furthermore, CO-NPs could specifically inhibit cancer cell growth and suppressed uveal melanoma cell migration and invasion. The possible mechanism was that CO-NPs localized in and damage mitochondria and lysosomes leading to programmed cell death.Conclusion: The comprehensive data revealed high specificity of CO-NPs and particular mechanism of CO-NPs inhibited cancer cell growth which may provide new insights on uveal melanoma therapy.