New phosphorescence based probes and techniques for the analysis of cellular oxygen and respiration

Real time monitoring of oxygenation and respiration is on the cutting edge of bioanalysis, including studies of cell metabolism, bioenergetics, mitochondrial function and drug toxicity. This thesis presents the development and evaluation of new luminescent probes and techniques for intracellular O2 sensing and imaging. A new oxygen consumption rate (OCR) platform based on the commercial microfluidic perfusion channel μ-slides compatible with extra- and intracellular O2 sensitive probes, different cell lines and measurement conditions was developed. The design of semi-closed channels allowed cell treatments, multiplexing with other assays and two-fold higher sensitivity to compare with microtiter plate. We compared three common OCR platforms: hermetically sealed quartz cuvettes for absolute OCRs, partially sealed with mineral oil 96-WPs for relative OCRs, and open 96-WPs for local cell oxygenation. Both 96-WP platforms were calibrated against absolute OCR platform with MEF cell line, phosphorescent O2 probe MitoXpress-Intra and time-resolved fluorescence reader. Found correlations allow tracing of cell respiration over time in a high throughput format with the possibility of cell stimulation and of changing measurement conditions. A new multimodal intracellular O2 probe, based on the phosphorescent reporter dye PtTFPP, fluorescent FRET donor and two-photon antennae PFO and cationic nanoparticles RL-100 was described. This probe, called MM2, possesses high brightness, photo- and chemical stability, low toxicity, efficient cell staining and high-resolution intracellular O2 imaging with 2D and 3D cell cultures in intensity, ratiometric and lifetime-based modalities with luminescence readers and FLIM microscopes. Extended range of O2 sensitive probes was designed and studied in order to optimize their spectral characteristics and intracellular targeting, using different NPs materials, delivery vectors, ratiometric pairs and IR dyes. The presented improvements provide useful tool for high sensitive monitoring and imaging of intracellular O2 in different measurement formats with wide range of physiological applications.

Organometallic derivatives of cyclotriphosphazene as precursors of nanostructured metallic materials: a new solid state method

The cyclic phosphazene trimers [N3P3(OC6H5)5OC5H4N·Ti(Cp)2Cl][PF6] (3), [N3P3(OC6H4CH2CN·Ti(Cp)2Cl)6][PF6]6 (4), [N3P3(OC6H4-But)5(OC6H4CH2CN·Ti(Cp)2Cl)][PF6] (5), [N3P3(OC6H5)5C6H4CH2CN·Ru(Cp)(PPh3)2][PF6] (6), [N3P3(OC6H5)5C6H4CH2CN·Fe(Cp)(dppe)][PF6] (7) and N3P3(OC6H5)5OC5H4N·W(CO)5 (8) were prepared and characterized. As a model, the simple compounds [HOC5H5N·Ti(Cp)2Cl]PF6 (1) and [HOC6H4CH2CN·Ti(Cp)2Cl]PF6 (2) were also prepared and characterized. Pyrolysis of the organometallic cyclic trimers in air yields metallic nanostructured materials, which according to transmission and scanning electron microscopy (TEM/SEM), energy-dispersive X-ray microanalysis (EDX), and IR data, can be formulated as either a metal oxide, metal pyrophosphate or a mixture in some cases, depending on the nature and quantity of the metal, characteristics of the organic spacer and the auxiliary substituent attached to the phosphorus cycle. Atomic force microscopy (AFM) data indicate the formation of small island and striate nanostructures. A plausible formation mechanism which involves the formation of a cyclomatrix is proposed, and the pyrolysis of the organometallic cyclic phosphazene polymer as a new and general method for obtaining metallic nanostructured materials is discussed.