Functional Fluorescent Nanoprobes For Tumor Cell Imaging, Detection And Therapy

Cancer is a major disease that threats human life and social development seriously,is also a leading cause of deaths worldwide. The incidence of cancer is increasing yearby year. Therefore, it is a great challenge to prevent and treat cancer for current publichealth. On the one hand, early detection and diagnosis are critical for prevention andtreatment of cancer. Recent studies have shown that the cure rate and survival ratecould be increased greatly if the cancer can be detected and diagnosed early and thecorresponding treatments are taken. However, the specificity and sensitivity of theconventional diagnostic methods are very low, it is difficult to detect early intumorigenesis. When the cancer was diagnosed, the invasion and metastasis have beenoccurred, thus the best time for treatment was missed. To detect and diagnose thecancer as soon as possible, the researchers devoted to detect and identify cancer cellsusing the expression of tumor markers at the cellular level. On the other hand, toimprove the therapeutic effect for cancer, much attention has been paid on thedevelopment of new drugs and treatments. Nevertheless, there are still manydrawbacks for chemotherapy such as the severe side effects of chemotherapy drugs,poor targeted therapy, drug resistantance for tumor cells, and so on. Hence, there is anurgent need for us to develop nanocarriers for controlled release, to target drugdelivery for tumor cells, to reduce the dose and side effects, to enhance the therapeuticeffect, and to improve the therapeutic effect of chemotherapy.In recent years, with the development of nanotechnology, fluorescent nanoprobeshave been widely applied in the fields of chemistry, biology and medicine, especiallyfor diagnosis, imaging and treatment of cancer cells. Among these nanomaterials, goldnanoparticles possess unique optical properties and biocompatibility, so it hasattracted widespread attention in biological detection, cellular and in vivo imaging,drug delivery. In particular, it is hot and difficult to detect and image multiple tumormarkers with high sensitivity and high selectivity. The huge challenge in current studyis to apply multi-component strategy for detecting biological models, and determineintracellular multiple tumor markers dynamically and in real time. A variety of studieshave shown that there is no nanoprobe to simultaneously detect four or more tumor markers for biological imaging in living cells. Most of the multicolor nanoprobesdetect tumor markers in the same type. In order to avoid "false positive" resultseffectively, it is very necessary to develop the nanoprobes that can simultaneouslydetect multiple and different types of intracellular tumor markers for early cancerdetection. Moreover, the current drug carriers mainly focus on the single targeted drugrelease can not recognize the tumor cells specifically and release drugs in a controlledmanner. Therefore, the development of multi-targeted drug carriers has importanttheoretical and practical value for specific recognition of tumor cells, controlledrelease in tumor cells, the dosage reduction, the improvement of therapeutic effect.In this dissertation, a variety of nanoprobes were designed and synthesized fortumor cell detection, imaging, targeted delivery and therapy, based on intracellularmRNA, matrix metalloproteinase (MMP) and cell surface receptor. It mainly includesthe following sections:1. A four-color nanoprobe was devoloped based on the gold nanoparticles andmolecular beacons, that can simultaneously detect and image four kinds of mRNAs inliving cells. The nanoprobe composed of gold nanoparticles functionalized with adense shell of molecular beacons, which can identify multiple intracellular mRNAtranscripts. It shows rapid response, high specificity, nuclease stability and goodbiocompatibility. Intracellular experiments indicated that the nanoprobe couldeffectively distinguish tumor cells from their normal cells, even some mRNAs areoverexpressed in normal cells. Moreover, it can identify the changes of the expressionlevels of mRNA in living cells. The current strategy could provide more accurateinformation for early cancer detection and effectively avoid false positive results.2. We prepare a four-color nanoprobe using step by step assembly. The molecularbeacons and peptides were assembled on the surface of gold nanoparticles. Thefluorescence of the dye-functionalized molecular beacons and peptides can bequenched by the gold nanoparticles. In the presence of target mRNA，the stem-loopstructure of molecular beacon was opened, recovering the fluorescence. In thepresence of MMP, the peptides were cut and the fluorescence was recovered.Simultaneous detection and imaging for different types of tumor markers ofintracellular mRNA and MMP can be achieved. This is the first time that a nanoprobewas successfully applied for detection and imaging of multiple different types oftumor markers in living cells. The nanoprobe could effectively discriminate normal cells and tumor cells and evaluate the change of expression of different tumor markers.It can prevent "false positive" results and improve the accuracy of early cancerdetection.3. Based on the mechanism of fluorescence resonance energy transfer (FRET), weintroduce a novel strategy for simultaneous detection of two tumor markers using asingle wavelength excitation. The nanoprobe was prepared based on two molecularbeacons. The molecular beacons were functionalized with two fluorophores (AlexaFluor488and Cy3), in which the FRET can be generated for the two fluorophores. Inthe presence of an mRNA, no fluorescent signal was produced. When the two mRNAwere both overexpressed, fluorescent signal can be detected. The nanoprobe candetect two intracellular mRNAs simultaneously, which can reduce the cell injury andbackground noise of multiple excitation and avoid the "false positive" results causedby detection of a single mRNA.4. A novel dual-targeted nanocarrier was developed based on cell surface receptorand intracellular mRNA. The gold nanoparticles were modified with a dense shell ofsynthetic oligonucleotides. The folic acid functionalized single-stranded DNA wasdesigned to target the folate receptor on the cancer cell surface and the molecularbeacon was employed as drug carrier for activated release associated with intracellulartumor mRNA. The nanocarrier possesses good stability, nuclease resistance and goodbiocompatibility. Intracellular experiments indicated that the dual-targeted nanocarriercould be preferentially internalized into cancer cells due to the folate receptortargeting and release Dox selectively in cancer cells because of the activated releasewith intracellular mRNA. The nanocarrier could reduce the dosage and greatlyimprove the therapeutic effect of drugs in cancer cells.