Fluorescence Nanoprobes For Detection And Imaging Of Subcellular PH In Living Cells Based On Silica Nanoparticles

Intracellular pH balance plays many vital roles in various biological processes,including cell proliferation, apoptosis, multidrug resistance, phagocytosis, and endocytosis. Anomalous pH values in cells and tissues are associated with the cellular dysfunctions, which are correlated with many severe diseases such as cancer and Alzheimer's disease. Moreover, intracellular pH distribution is heterogeneous in different subcellular compartments and the unique functions of organelles determine the pH values of each separate compartment. For instance, lysosomes and endosomes are acidic ranging from 4.7 to 6.5, the cytoplasm and nucleus have a neutral pH of 7.2-7.4,and mitochondria display a slightly basic pH about 8.0. To better understand the roles of pH in physiology and pathology, it is of great significance to exploit effective approaches for monitoring subcellular pH values and their fluctuations in living cells.The silica nanoparticles have many advantages, including good chemical stability,uniform and tunable size, which make them ideal candidates as nanocarriers. Recently,various chemical or biological compounds such as fluorescence dyes, DNA, enzymes and anti-bodies can be modified on the surface of silica nanoparticles through physical absorption or chemical conjugation due to their easy functionalization property. Besides,the silica nanoparticles exhibit good biocompatibility, low toxicity and no pharmacological activities, therefore have been widely used in biological systems.In this thesis, based on silica nanoparticles, we design and synthesize a series of fluorescence nanoprobes for accurate and quantitative detection and imaging of pH in living cells. We mainly carried out two aspects of the investigation:1. Based on the strategy of ratiometric detection, we assemble two dyes into one nanoparticle to construct a variety of ratiometric fluorescent nanoprobes. By conjugating the targeting moieties, quantitative detection and imaging of the subcellularpH fluctuations in different organelles using a single-wavelength excitation could be achieved in living cells. Mesoporous silica nanoparticles(MSNs) were selected as the carrier due to their good biocompatibility, and easy functionalization. The carboxyl-coated mesoporous silica nanoparticles were conjugated with aminofluorescein(AF) for pH measurements and ethidium bromide(EB) for reference.Fluorescein has been widely used to determine intracellular pH values owing to the good biocompatibility and high sensitivity under physiological conditions. The ethidium bromide was served as the reference to form a ratiometric fluorescent sensor for pH detection with good stability and high selectivity. Further conjugation of the triphenylphosphonium(TPP) or 3-(4-morpholinyl) propanoic acid hydrochloride(MPP)moiety on the surface of amino-functionalized silica by amide bonds endows the nanoprobes with specific targeting ability for mitochondria or lysosome. The designed nanoprobes are capable of accurately monitoring the changes of pH in cytoplasm,lysosomes, and mitochondria of living cells by ratio imaging.2. We design and synthesize a variety of ratiometric fluorescent nanoprobes with different sizes(25 nm, 50 nm, 85 nm and 135 nm), which can detect the effect of different sizes of silica on cell physiological activities. The nanoprobes consist of mesoporous silica nanoparticles, pH-sensitive dye AF and pH-insensitive dye EB. In order to prevent the dyes from leaking, the MSNs-EB-AF was coated by amino-functionalized mesoporous silica(MSNs-EB-AF@SiO2-NH2). The designed nanoprobes are able to accurately determine the changes of pH in living cells by ratio imaging. In this research, we found that the fluorescence response of various nanoprobes for different pH values is consistent.