Targeted Molecular Imaging And Gene Delivery In Tumor Cells Based On Quantum Dots Nanoparticle

As a new class of fluorescent materials, quantum dots (QDs) possess unique physico-chemical properties, such as broad absorption spectra with continuous distribution, narrow emission spectra with symmetrical distribution, size-and composition-tunable emission from ultraviolet to infrared wavelengths, high fluorescence quantum yields, anti-photobleaching and good biocompatibility after surface modification. These excellent properties make QDs as superior probes for long-term observation in cell trafficking, tumor targeting, and visualization of gene expression and have wide application in the field of life science. This thesis mainly focuses on core-shell CdSe/ZnS QDs as a fluorescence probe in targeted tumor cells imaging and as a gene delivery vehicle in tracking the gene expression. The main contents and results are summarized as follows:(1) CdSe/ZnS QDs probes for targeted delivery to mouse and human cells using aptamer GS24and peptide T7specific to mouse/human transferrm receptors were developed. Fluorescence imaging revealed QD-GS24and QD-T7probes were able to specifically recognize B16cells and HeLa cells, respectively. Quantitative flow cytometry analysis indicated the transportation of QD-GS24or QD-T7into cells could be promoted by corresponding free transferrin. Transmission electron microscopy (TEM) confirmed the uptake of probes in cells and the effective intracellular delivery. MTT assay suggested the cytotoxicity of probes was related to the surface ligand, and aptamer GS24(or peptide T7) could reduce the cytotoxicity of probes to a certain degree. The study has great significance for preparing QDs fluorescent probes using non-antibody target molecules.(2) Based on the multivalent binding capability of streptavidin (SA) to biotin, a multifunctional quantum dot probe QD-(AS-ODN+p160) coupled with antisense oligonucleotide (AS-ODN) and peptide p160is designed for real-time tracking of targeted delivery of AS-ODN and regulation of folate receptor-a (hFR-a) in MCF-7breast cancer cells. Confocal imaging and quantitative flow cytometry show that QD-(AS-ODN+pl60) is able to specifically target human breast cancer MCF-7cells. The effects of inhibition agents and co-localization imaging further confirm the endocytic pathway is mainly receptor mediated. Transmission electron microscopy (TEM) shows the intracellular delivery and endosomal escape of QD probe along with incubation time extended. Real-time PCR, Weatern blot and quantitive ELISA investigate the gene silence efficiency from mRNA to protein level. These results demonstrate that the QD-(AS-ODN+pl60) probe not only achieves gene silence in a cell-specific manner but also achieves real-time tracking during AS-ODN intracellular delivery.(3) Incorporating ligands with nanoparticle-based carriers for specific delivery of therapeutic nucleic acids (such as antisense oligonucleotide and siRNA) to tumor sites is a promising approach in anti-cancer strategies. However, nanoparticle-based carriers remain insufficient in terms of the selectivity and transfection efficiency when compare to viral carriers. Here, we designed a dual receptor targeted QD gene carrier QD-(AS-ODN+GE11+c(RGDfK)) which could increase the cellular uptake efficiency of QDs and further enhance the transfection efficiency. The receptor targeting ligands we chose were peptides GE11and c(RGDfK) which could recognize epidermal growth receptors (EGFR) and integrin αvβ3receptors with high binding ability, respectively. Results of quantitative flow cytometry and ICP/MS showed that the synergistic effect between EGFR and integrin αvβ3increased the cellular uptake of QDs carriers. Western blot confirmed that this dual targeted probe QD-(AS-ODN+GE11+c(RGDfK)) could further enhance gene silencing efficiency compared to its two corresponding single targeted probes QD-(AS-ODN+GE11) and QD-(AS-ODN+c(RGDfK. In this gene delivery system, QDs could not only be as a gene vehicle but also as fluorescence probe, allowing for localization and tracking during the delivery progess. This transport model has very well reference to enhance the targeting ability and transfection efficiency of non-viral gene carriers.