Development Of Functional Nucleic Acid Fluorescent Nanoprobes Suitable For Complex Biological Systems

Monitoring the change and distribution of intracellular biological molecules are critical for understanding their physiological and pathological functions, validating disease biomarkers and diagnosing disease in its early stages. Fluorescent imaging through staining with a molecular probe might be the most attractive molecular imaging technique for the detection of intracellular species by virtue of its high sensitivity, real-time spatial imaging, and detection of targets in living cells or tissues with minimal damaging effects. However, the conventional fluorescent probe delivery inefficiency restricts its application in intracellular. Meanwhile, most of these probes are single-photon excited at short wavelengths, which resulted in photobleaching, interference from autofluorescence in cells and tissues, and shallow penetration depth. Two-photon microscopy（TPM）, which utilizes two photons of lower energy for the excitation, is a new technique that can realize deep-tissue imaging with prolonged observation time, and provide minimized fluorescence background and less light scattering with better three-dimensional spatial localization and less tissue injury. These advantages make the two-photon fluorescence probe more favorable for complex biological systems.Functional nucleic acids mainly include two types of nucleic acid molecules with specific molecular recognition function, one type of them have been shown to perform catalytic reactions（called DNAzymes, or deoxyribozymes） like protein enzymes, and the other can bind a broad of analytes including metal ions, small molecules, drugs, proteins, and even whole cel ls（called aptamers） like antibodies. FNAs are naturally high affinity, high specificity, wide target range, good biocompatible as well as easy synthesis and modification. The discovery of functional nucleic acids has provided a new tool for molecular reco gnition in constructing fluorescent probe of application in complex biological system. Nanomaterials, such as gold nanoparticles（NP）, semiconductor nanocrystals（quantum dots, QDs）, upconversion nanoparticles（UCNPs）, DNA nanostructures, mesoporous silica nanoparticles（MSNs） and MnO₂ nanosheets normally possess unique light properties, high delivery efficiency, excellent biocompatibility, and large loading ability. Meanwhile, these nanomaterials have undergone many advances in synthesis and characterizati on in the past few years. As a consequence, the fluorescent nanoprobe combined FNAs and nanotechnology will have a broad spectrum of applications in complex biological syste m.In order to solve the fluorescent probes poor biological stability, limited membrane-permeability, as well as poor cell imaging, taking the advantages of fluorescent imaging technology and FNAs, using nucleic acid molecular engineering and nanotechnology, we have developed a series of functional nucleic acid fluorescent nanoprobes for complex biological systems. The major contents are as follows:（1） In chapter 2, based on Zr4+ could selectively bind with two phosphate-functionalized molecules, we report an efficient fluorescence turn-on probe for zirconium via a target-triggered DNA molecular beacon strategy. Because molecular beacon has a unique hairpin structure and γ-cyclodextrin was introduced to afford an amplified fluorescence signal, therefore, provided an improved sensitivity for the target Zr4+. The proposed fluorescent nanoprobe has also been used for detection of Zr4+ in river water samples with satisfactory result.（2） In chapter 3, based on DNA dendrimer scaffold as an efficient nanocarrier, we report a fluorescent nanoprobe to deliver FNAs and to conduct in situ monitorin g of biological molecules in living cells. The fluorescent nanoprobe maintained the catalytic activity of the DNAzyme or the aptamer recognition function toward ATP in the cellular environment. Meanwhile, these DNA dendrimeric nanocarriers show excellent biocompatibility, high intracellular delivery efficiency, and sufficient stability in a cellular environment. Therefor, this fluorescent nanoprobe may find a broad spectrum of applications in biomedical diagnosis and therapy.（3） Currently, most of probes are single-photon excited at short wavelengths, which resulted in photobleaching, interference from autofluorescence in cells and tissues, and shallow penetration depth. In chapter 4, based on two-photon microscopy can realize deep-tissue imaging with prolonged observation time, and provide minimized fluorescence background, less light scattering and less tissue injury, we report a “turn-on” TP fluorescence nanoprobe for efficient detection of glutathione（GSH） in aqueous solutions. The nanoprobe was successfully applied in monitoring the change of the intracellular GSH in living cells and tissues via TP fluorescence imaging, demonstrating its value of practical application in biological systems.（4） Based on the unique advantages of two-photon imaging techniques, manganese dioxide excellent physical and chemical properties, and aptamer’s specifically binding ability to the target molecules. In chapter 5, we report a multiple functional nanoprobe for contrast-enhanced bimodal cellular imaging and targeted therapy. In this nanoprobe, MSNs was prepared as both a signal reporter and nanocarrier, MnO₂ nanosheets were adopted as gatekeeper, quencher for TP excited fluorescence, and MRI contrast agent. Guided by aptamers, the nanoprobe is rapidly internalized into the target cells. Next, intracellular glutathione reduces Mn O 2 to Mn2+, resulting in contrast-enhanced TP fluorescence and magnetic resonance signal for cellular imaging. Meanwhile, preloaded doxorubicin and Chlorin e6 are released for chemotherapy and photodynamic therapy, with a synergistic effect and significantly enhanced therapeutic efficacy.