|其他摘要||The emerging nanoscience and nanotechnology hold great promise in advancing biomedicine. As the increasing mortality, the early diagnosis and therapy of malignant tumor has drawn great attention in current cancer treatment. In order to improve the sensitivity and accuracy in cancer diagnosis, and efficiently inhibit the tumor proliferation and migration, it is in urgent need to explore tumor-targeting ligands with high specificity, imaging contrast agents with high sensitivity and resolution, and efficient therapy method with minimum side effects. As an attractive alternative to antibodies, specific peptides show higher stability and selectivity, smaller molecular weight and easier to scale up synthesis. The random foreign peptides can be fused to phage coat proteins by genetic engineering and the constructed phage display library can be used to select specific peptides in a high-throughput way.
Gold nanostructures with near-infrared (NIR) absorption (650-900 nm) have been widely used in tumor photothermal ablation owing to their unique SPR property that can convert the absorbed light energy into hyperthermia. Besides, gold nanostructures have been proved to be biocompatible, which promotes their clinical applications. In this project, using the colorectal carcinoma cell SW620 as a model, the specific phage coat proteins-conjugated gold nanocomposites were assembled for cancer cell-specific multimodal imaging and efficiently photothermal therapy.
Firstly, the SW620 cells were biopanned with f8/8 landscape phage library, from which several landscape phage monoclones were isolated. According to the results of phage capture test, the SW620 cell-specific phage displaying octapeptide DDAGNRQP was successfully identified. The specific phage was amplified and its pVIII fusion coat proteins (fusion pVIII) were isolated in large quantities. The C terminus of fusion pVIII protein is positively charged because of the existing four lysines, and the N terminus is negatively charged because of the displayed two aspartic acids. Comparing with the traditional antibodies, the specific fusion pVIII proteins not only exhibit the exact charge distribution, but also have advantages of low molecular weight, high stability, high specificity and affinity, and low cost, so they are able to be used as SW620 cell-targeting ligands.
Secondly, as the hemoglobin and water in vivo have lowest absorption in NIR region, the Au@Ag heterogenous NRs with longitudinal SPR absorption peak at 803 nm were synthesized by controllable one-pot method, using the cationic polyelectrolyte as the surface stabilizer. The fluorescent molecules and SW620 cell-specific pVIII proteins were attached on the Au@Ag heterogeneous NRs through a layer-by-layer (LbL) assembly. In the self-assembly process, the absorption peak of the nanostructure gradually shifted to longer wavelength, and the ultimately assembled bio-mimetic nanostructures showed maximum absorption peak at 810 nm, which made them ideal agents for in vivo tumor therapy. Due to the unique dipole properties of fusion pVIII proteins, their N terminuses with negative charge that were fused with DDAGNRQP were exposed outwardly on the bio-mimetic nanostructures, which was characterized by the change of zeta potential. The fluorescent result further confirmed that the assembled fusion pVIII proteins can specifically direct the constructed nanostructures into SW620 cells, and successfully used for controllably optical imaging of cancer cells. The cell viability test showed that the bio-mimetic nanostructures exhibited higher photon-thermal conversion efficiency than gold nanorods and silver nanotriangle, and were able to specifically ablate SW620 cells after 10 min illumination with a NIR laser in the light intensity of 4 W/cm2.
Thirdly, MRI has become the most important imaging mode of tumor because of the advantages of high spatial resolution and nonionizing radiation. Therefore, it is in great need to explore multifunctional nanocomposites for multimodal imaging and photothermal therapy of tumor. The superparamagnetic Fe3O4 nanospheres were prepared through solvothermal method and the morphology was intensely related to the surfactant and precursor. TEM and XRD datas indicated that Fe3O4 nanospheres were aggregated from many Fe3O4 nanocrystals of small size that can be decreased by adding surfactant. The crystalline water in the precursor inhibited the aggregation of single Fe3O4 nanocrystals. By modification with tetraethoxysilane and RhB, Fe3O4@RhB/SiO2 nanoparticles were formed and their fluorescent intensity was tested. Then, gold nanospheres were synthesized using surcose as reductant and stably assembled onto the surface of Fe3O4@RhB/SiO2 nanoparticles via the Au-S chemical bond to improve the stability and reproducibility of Au shells. As the absorbed gold nanospheres grow in the reduction process, Au nanoshells were formed and their absorption peak shifted to the longer wavelength gradually. By controllable synthesis, Fe3O4@RhB/SiO2@Au nanocomposites with the maximum absorption at 747 nm were successfully prepared. The C terminuses of fusion pVIII proteins were activated and assembled onto the surface of Fe3O4@RhB/SiO2@Au nanocomposites, leaving their N terminuses fused with DDAGNRQP outwardly. The obtained Fe3O4@RhB/SiO2@Au@fusion-pVIII nanocomposites showed SPR peak at 774 nm, which made them suitable for in vivo applications. The T2-weighted imaging showed that the r2 value of the nanocomposite was as hihg as 176.2 mM-1s-1. Moreover, the fluorescent images confirmed that the nanocomposites were specifically endocytosed by SW620 cells. Therefore, Fe3O4@RhB/SiO2@Au@fusion-pVIII nanocomposites can be simultaneously used as constrast agents for both MRI and fluorescent imaging. The cell viability showed that the prepared nanocomposites were able to specifically ablate SW620 cells after 10 min illumination with the light intensity of 3 W/cm2. Therefore, the as-prepared Fe3O4@RhB/SiO2@Au@fusion-pVIII nanocomposites were successfully applied to specifically bimodal imaging and efficiently photothermal therapy of cancer cells.|