A family of multi-functional phosphorescent bioprobes based on the PtPFPP scaffolding moiety

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Zanetti, Chiara
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University College Cork
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Accurate monitoring of O2 levels is crucial for understanding the growth, differentiation, and function of individual cells and tissues. While phosphorescence-based oxygen sensing offers great potential for such investigations, current probes still show limitations in terms of their bio-distribution and analytical performance. New improved O2 probes are highly needed. In this project we synthesized a family of new hetero-substituted phosphorescent derivatives of Pt(II)-tetrakis(pentafluorophenyl)porphyrin (PtPFPP) dye, through click-modification of its para-fluorine atoms with 1-β-D-thio-glucose, neutral (mPEG) and carboxy (cPEG) thiol-PEGs, named as PtmPEG31Glc and PtcPEG31Glc respectively, or cysteamine (CA), named PtCA31Glc. Derivatisation with 2-thioelthyl-β-D-glucopyranoside was also performed to evaluate the effects of alternative SH-Glucose structures on cellular uptake, obtaining the corresponding 2Glc derivatives. The new probes comprise one cell-targeting moiety and three polar moieties, forming a hydrophilic shell. We also synthesized the di-glucosilated-di-PEGylated derivative, named as PtmPEG21Glc2 (trans), in which 1-thio-β-D-glucose (1Glc) and thio-methyl-polyethylene-glycol (mPEG) moieties were covalently attached to PtPFPP in trans- position. Tetra-substituted derivatives Pt1Glc4, Pt2Glc4, PtmPEG4 and PtcPEG4 were also synthesised and characterized for benchmarking. The chemical synthesis and purification procedures were optimized for all the new derivatives to achieve high reaction yields and scalability, and new chemical structures were confirmed by HR-MS and NMR. Then, the most promising derivatives were assessed for their spectral properties and cell penetrating ability in 2D mammalian cell cultures using time-resolved fluorescence (TRF) on a standard plate reader, and confocal microscopy in PLIM mode (phosphorescence lifetime imaging). Probes' ability to function as either cell permeable or impermeable was found to depend on polarity, molecular charge, size, and substitution position of the bioconjugate. Notably, the hetero-substituted tri-PEGylated (PtcPEG31Glc, PtmPEG31Glc) and tetra-PEGylated (PtcPEG4, PtmPEG4) derivatives showed reduced cell internalization compared to the 3:1 cysteamine (PtCA31Glc) derivative and 4:1 glycosylated probe (Pt1Glc4). This finding opens possibilities for their extracellular applications. In contrast, PtmPEG21Glc2 (trans), demonstrated efficient cell staining and suitability for intracellular oxygen sensing (IcO2) in murine embryonic fibroblast (MEFs) cells. Moreover, biocompatibility studies indicated no significant cytotoxicity for all probes at any tested concentrations, except for PtCA31Glc which caused reduced cell viability at concentrations above 10 µM. The chemical modifications did not alter absorption and emission spectra of the PtPFPP moiety but reduced its brightness and lifetimes. Some of the PEGylated derivatives showed tendency to aggregate in biological media. PtmPEG4 and Pt1Glc4 were also evaluated in microbial respirometric assays with Escherichia coli (E. coli) cells, however due to the lack of shielding, their performance in complex media was not as good as the macromolecular MitoXpress probe and the nanoparticle based NanO2 probe. Overall, our new phosphorescent bioprobes, particularly the hetero-substituted structures, extend the library of extracellular and intracellular probes for monitoring and imaging O2 in mammalian cell cultures. They are compatible with standard TRF plate readers and PLIM microscopes, making them promising candidates for physiological studies with cells. The established structure-activity relationships (SARs) will also help to develop new probe structures for future studies.
Phosphorescent porphyrins , Oxygen sensitive probes , PtPFPP derivatives and bioconjugates , Thiol click modification , Intracellular probes , Bioimaging
Zanetti, C. 2023. A family of multi-functional phosphorescent bioprobes based on the PtPFPP scaffolding moiety. PhD Thesis, University College Cork.
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