Aptamer Functionalized Novel Nanoprobe For Diagnosis And Treatment Of Cancer

With the highly development of modern science and medical technology, great progress has been made in our understanding of molecular biology, chemotherapy, radiotherapy and conventional surgical procedures. However, cancer still remains one of the leading causes of death in the world. Early detection and treatment of cancer is the key to improve the survival rate of cancer patients and has become one of the top priorities for biotechnological research and pharmaceutical innovation worldwide. With the traditional diagnostic and treatment technique, false-positive diagnostic results and toxic side effects are always happened due to the presence of nonspecific interaction. Since the discovery of aptamer in 1990s, they have been developed as a new recognition molecule during the past thirty years and become a class of novel targeting ligand for accurate diagnosis and effective treatment of cancer. On the other hand, nanomaterials are widely used in the fields of clinical diagnostics, drug analysis, energy and the environment, due to their small sizes, large specifc surface areas, good biocompatibility, unique physical and chemical properties and easy modifications etc..Based on the biotechnologies and nanotechnologies above, this disserttation presentes an exploration of the use of aptamer functionalized nanomaterial in early diagnosis and treatment of cancer. The main results were summarized as follows:1. Nanoarchitectured Electrochemical Cytosensors for Selective Detection of Leukemia Cells and Quantitative Evaluation of Death Receptor Expression on Cell SurfacesThe variable susceptibility to the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) treatment observed in various types of leukemia cells is related to the difference in the expression levels of death receptors, DR4 and DR5, on the cell surfaces. Quantifying the DR4/DR5 expression status on leukemia cell surfaces is of vital importance to the development of diagnostic tools to guide death receptor-based leukemia treatment. Taking the full advantages of novel nanobiotechnology, we have developed a robust electrochemical cytosensing approach toward ultrasensitive detection of leukemia cells with detection limit as low as ～40 cells and quantitative evaluation of DR4/DR5 expression on leukemia cell surfaces. The optimization of electron transfer and cell capture processes at specifically tailored nanobiointerfaces and the incorporation of multiple functions into rationally designed nanoprobes provide unique opportunities of integrating high specificity and signal amplification on one electrochemical cytosensor. The high sensitivity and selectivity of this electrochemical cytosensing approach also allows us to evaluate the dynamic alteration of DR4/DR5 expression on the surfaces of living cells in response to drug treatments. Using the TRAIL-resistant HL-60 cells and TRAIL-sensitive Jurkat cells as model cells, we have further verified that the TRAIL susceptibility of various types of leukemia cells is directly correlated to the surface expression levels of DR4/DR5. This versatile electrochemical cytosensing platform is believed to be of great clinical value for the early diagnosis of human leukemia and the evaluation of therapeutic effects on leukemia patients after radiation therapy or drug treatment.2. Robust Nonenzymatic Hybrid Nanoelectrocatalysts for Signal Amplification toward Ultrasensitive Electrochemical CytosensingWe have discovered that magnetic Fe3O4 nanoparticles exhibit an intrinsic catalytic activity toward the electrochemical reduction of small dye molecules. Metallic nanocages, which act as efficient signal amplifiers, can be attached to the surface of Fe3O4 beads to further enhance the catalytic electrochemical signals. The Fe3O4@ nanocage core-satellite hybrid nanoparticles show significantly more robust electrocatalytic activities than the enzymatic peroxidase/H2O2 system. Using breast cancer cell lines as the model CTCs, we have further demonstrated that these nonenzymatic nanoelectrocatalysts can be used as signal-amplifying nanoprobes for ultrasensitive electrochemical cytosensing.3. Multiplex acute leukemia cytosensing using multifunctional hybrid electrochemical nanoprobes at a hierarchically nanoarchitectured electrode interfaceWe have developed a robust, nanobiotechnology-based electrochemical cytosensing approach with high sensitivity, selectivity, and reproducibility toward the simultaneous multiplex detection and classification of both acute myeloid leukemia and acute lymphocytic leukemia cells. The construction of the electrochemical cytosensor involves the hierarchical assembly of dual aptamer-functionalized, multilayered graphene-Au nanoparticle electrode interface and the utilization of hybrid electrochemical nanoprobes co-functionalized with redox tags, horseradish peroxidase, and cell-targeting nucleic acid aptamers. The hybrid nanoprobes are multifunctional, capable of specifically targeting the cells of interest, amplifying the electrochemical signals, and generating distinguishable signals for multiplex cytosensing. The as-assembled electrode interface not only greatly facilitates the interfacial electron transfer process due to its high conductivity and surface area but also exhibits excellent biocompatibility and specificity for cell recognition and adhesion. A superstructured sandwich-type sensor geometry is adopted for electrochemical cytosensing, with the cells of interest sandwiched between the nanoprobes and the electrode interface. Such an electrochemical sensing strategy allows for ultrasensitive, multiplex acute leukemia cytosensing with a detection limit as low as ～350 cells per mL and a wide linear response range from 5×102 to 1×107 cells per mL for HL-60 and CEM cells, with minimal cross-reactivity and interference from non-targeting cells. This electrochemical cytosensing approach holds great promise as a new point-of-care diagnostic tool for early detection and classification of human acute leukemia and may be readily expanded to multiplex cytosensing of other cancer cells.4. Ultrasensitive dual-channel detection of matrix metalloproteinase-2 in human serum using gold-quantum dot core-satellite nanoprobesThis work constructed a robust enzymatic peptide cleavage-based assay for the ultrasensitive dual-channel detection of matrix metalloproteinase-2 (MMP-2) in human serum using gold-quantum dot (Au-QD) core-satellite nanoprobes. The core-satellite signaling probe composed of QDs, AuNPs, and streptavidin was first conjugated using a single-stranded DNA. Then, a smart, MMP-2-responsive, and biotinylated PLGVR peptide was designed and immobilized on the gold-labeled polydimethylsiloxane (PDMS) suface, which could be specifically recognized and cleaved by MMP-2. Streptavidin-functionalized Au-QD nanoprobes were then conjugated to the uncleaved peptide molecules through the streptavidin-biotin interaction. By dissolving the conjugated QDs with HNO3, the number of uncleaved peptide molecules, which was related to the MMP-2 concentration, could be quantified using ASV and fluorenscence analysis. Using this dual-channel sensing strategy, we have been able to detect MMP-2 in the concentration range of 1～500 pg mL-1 with a detection limit down to sub-pg mL-1 (～10 fM), which is the lowest detection limit for MMP-2 quantification ever reported so far. By selecting the substrate peptide sequences, this approach can be readily developed into a general method with high sensitivity, selectivity, and accuracy for the detection of MMPs and other proteases in clinical samples.5. Aptamer Functionalized Large-sized Gold Nanostucture for pH, Temperature and NIR-Activatable Targeted Cancer Stem Cell (CSC) TheranosticsA multi-stimuli responsive system based on dual-controllable copolymer-liposome capped large-sized porous Au nanostructures was developed for CSC-target theranostics. The newly fabricated Au nanostructures possess capabilities of high drug loading; drug release is controlled by the pH and temperature sensitive copolymer-liposome, as well as by the NIR irradiation of Au nanostructures. As compared to either of the two therapies independently, a complete inhibition of tumor growth treated with the combination of chemotherapy and photothermal therapy was observed in vivo. Besides, a novel electrochemical anti-cancer drug sensitivity test was first developed to evaluate the drug delievery system above. Taken together, our present study provides new insights into developing multi-stimuli responsive intracellular drug release systems for synergistic cancer therapy.