Preparation Of Functional Nanoparticles And Their Applications In Cellular Imaging

An in-depth understanding of biochemical processes occurring within biological systems is the key step for early diagnosis of disease and identification of appropriate treatments. Intracellular pH plays a significant role in many biological events. It can not only modulate the biological function of individual proteins but also affect the performance of subcellular organelles, such as lysosomes and mitochondria require proton gradients to function properly. In a normal mammalian cell, the intracellular pH value varies in a broad range, i.e., from 4.7 in lysosome to 8.0 in mitochondrial, which is heterogeneously distributed around the cell. An abnormal deviation of the pH units even by 0.1-0.2 in either direction can lead to dysfunction of the organelles that might finally result in cardiopulmonary and neurologic problems(e.g., cancer, stroke and Alzheimer’s disease). As a consequence, detecting and monitoring the pH values with subcellular spatial resolution in living cells is fundamentally important.Intracellular pH sensing using fluorescent materials represents a potentially useful tool for real-time, in vivo monitoring of important cellular analytes. The low immunogenicity, nontoxicity, biodegrability and preferential uptake in tumor make serum albumin an ideal candidate carrier for anticancer drug delivery. Through covalently crosslink a reference dye Alexa 594 to the surface of BNPs, a ratiometric pH sensor can then be readily obtained. In the thesis, we developed the synthesis and characterization of this pH responsive albumin nanoparticles for in vivo imaging of intracellular pH distribution in HeLa cells, and the development of the peptide modified AuNPs was added in the final chapter. The studies have been performed as follows:1. The research backgroundwere reviewed, which included the kinds of nanoparticles, the response principle of fluorescent probe, the approaches to realize pH responsive, the kinds of fluorescent probes for the measurement of pH(the direct fluorescence intensity-based measurement and ratiometric measurement), and the development of the BNPs and AuNPs for cell imaging.2. BSA nanoparticles were prepared by a desolvation technique as reported earlier. Two routes were commonly adopted for the fabrication of fluorescent ratiometric sensor: a) particles with dyes physically embedded in a polymer network and b) particles with dyes covalently conjugated on the nanoparticle surface.F-4600 fluorescence spectrophotometer,Nikon Ti-U inverted epi-fluorescence microscope,transmission electron microscopy(TEM),dynamic light scattering(DLS) and the FT-IR spectrum were introduced to study the characteristics of BSA nanoparticles for the sensitive and robust ratiometric pH sensing. The results indicated that the resultant BSA nanoparticles display bright fluorescence at 535 nm with excitation wavelength at 495 nm. Importantly, the fluorescent BNPs have a direct optical response to pH change in the range of 6.0-8.0, indicative of a good candidate for pH sensing.3. In order to explore the capability of this probe for biological sample measurement, we applied BNPs fluorescent probe for probing the pH in living HeLa cells. And then a series of cell imaging experiments were performed. The results showed that the probe can efficiently detect the intracellular pH in HeLa cells, which can afford deep insight for the better understanding of the cellular metabolism process as well as provide essential knowledge for the early diagnosis of fatal diseases.4. Single-particle tracking experiments were carried out with Tat peptide-labeled gold nanoparticles, which contained increasing concentrations ofTat peptide in living HeLa cells.