Synthesis Of Phosphorescent Oxygen Nanosensors For Assessment Of Tumor Mitochondrial Dysfunction

Measuring dissolved oxygen is of major importance in various physiological and pathological processes. In terms of in vivo oxygen sensing, fluorescent oxygen nanosensors are very attractive. Mitochondria are crucial for numerous vital cellular processes, and mitochondrial dysfunctions might lead to the development of cancers. Mitochondrial respiration rate has long been employed to evaluate mitochondrial dysfunction by measuring dissolved oxygen levels. However, most of the existing sensing strategies merely report extracellular or intracellular O2rather than intramitochondrial O2. Herein techniques are needed that can exclusively detect intramitochondrial levels of oxygen in living cells to differentiate the subtle dysfunction of tumor mitochondria.The main research of this thesis is as follows:1. Ratiometric fluorescent nanoparticles (NPs) are prepared by a one-step reprecipitation-encapsulation method for sensing dissolved oxygen. The surface of the NPs is modified with poly-L-lysine (PLL), which renders good biocompatibility and enables easy internalization into living cells. Firstly, PMMA and BTD are used as matrix and silica-based encapsulation agent respectively to incapsulate the oxygen probe platinum (II) octaethylporphine (PtOEP) and the reference dye coumarin6(C6) into the ratiometric NPs. Then positively charged PLL molecules are electrostatically absorbed onto the surface of negatively charged NPs (with silanol groups), and PLL modified PMMA:PtOEP/C6nanosensor is complished. This ratiometric fluorescence of nanosensor has a quenching response of77%, and can be well fitted by a nonlinear Stern-Volmer equation. Because of the PLL modification, the sensor NPs could be easily introduced inside living cells. Afterwards, PS is used in place of PMMA. In addition, the oxygen probe PtOEP, the reference dye C6and a third fluorophore DNM (transfer energy) are entrapped in a hydrophobic hybrid core (PS-BTD) to prepare highly sensitive PLL modified PS:PtOEP/DNM/C6oxygen nanosensor. The ratiometric nanosensor is very sensitive to oxygen with a quenching response of94%, and follows a linear Stern-Volmer behavior, which is fundamental for practical sensing. The resultant sensing NPs exhibit low cytotoxic effects as well as effortless cellular uptake, indicating targeted intracellular oxygen sensing is very promising using the oxygen nanosensors.2. The TPP-conjugated NPs are developed to selectively target mitochondria in living cells. Firstly, the mechanism of the PLL-assisted self-assembly of NPs is studied, and MH-PLL (Mw=30-70kDa) with a dosage of10mg/mL is found to yield the best PLL-NPs in terms of morphology, size and zeta potential. According to the zeta potentials of MH-PLL-NPs that are crosslinked by GA, three types of amino groups are present in the MH-PLL shell:deep interior, interior and surface amino groups (SAGs). SAGs are thought to be responsible for TPP-conjugation, whose concentration is determined to be12nmol/mL in MH-PLL-NPs using a fluorescamine assay. Then TPP groups are successfully conjugated via a carbodiimide reaction process, which is conformed by NMR and quantitatively characterized by absorption spectra. The TPP-conjugated NPs are found to be efficiently taken up by living cells and to selectively target mitochondria by the merged z-stacked images in co-localization experiments with MitoTracker-stained mitochondria.3. Three targetable O2nanosensors are prepared in order to respond to extracellular (ec-), intracellular (ic-) and intramitochondrial (im-) O2. Then, the cellular respiration of HepG2and LO2cells is monitored using the time-resolved fluorescence microplate reader. These nanosensors possess the same sensing core doped with an oxygen-sensitive optical probe, but differ in their surfaces. The silica groups on the surface of the Si-NPs carry a negative charge, and such anionic nanoparticles remain in the extracellular space. The PLL-NPs modified by poly-L-lysine warrant an easy cellular uptake. The lipophilic TPP-NPs bear large-area-dispersed cationic charges, and accumulate within the mitochondrial matrix. The results of confocal microscopic and TEM indicate that TPP-NPs mainly dwell in mitochondria rather than towards peri-mitochondria. In vivo calibration plots were established for the determination of ec-, ic-and im-O2in both HepG2cells and LO2cells. The cellular O2gradients can be detected in real-time by the three targetable nanosensors. By inspection of the data of respective oxygen consumption rates and (intra)cellular O2gradients, the mitochondria in tumor cells are found to be distinctly less active than those in healthy cells. The reduced activity of tumor mitochondria is attributed not only to restrained glucose utilization but also to physical defects in the respiratory chain.