Heterosubstituted Derivatives of PtPFPP for O₂ Sensing and Cell Analysis: Structure–Activity Relationships

Abstract

Biological applications of phosphorescent probes for sensing molecular oxygen (O2) and bioimaging have gained popularity, but their choice is rather limited. We describe a family of new heterosubstituted phosphorescent bioprobes based on the Pt(II)-tetrakis(pentafluorophenyl)porphyrin (PtPFPP) dye. The probes are produced by simple click modification of its para-fluorine atoms with thiols, such as 1/2-thio-glucose, thio-poly(ethylene glycol) (PEG), or cysteamine. The probes were designed to have one cell-targeting moiety and three polar moieties forming a hydrophilic shell. Their chemical synthesis and purification were optimized to produce high reaction yields and easy scale-up. The ability to perform as cell-permeable or -impermeable probes was tuned by the polarity and molecular charge of the bioconjugate. The new PtPFPP derivatives were characterized for their spectral properties and cell-penetrating ability in the experiments with mammalian cell cultures, using a time-resolved fluorescence reader and PLIM imaging detection. Structure–activity relationships were established. Thus, the tri- and tetra-PEGylated structures showed low cell internalization allowing their use as extracellular probes, while cysteamine derivatives performed as efficient intracellular probes. No significant cytotoxicity was observed for all of the probes under the experimental conditions used.

Hetero-substituted derivative of PtPFPP for intracellular O2 sensing of mammalian cells

“…Cells were then incubated with a range of probe concentrations (5, 10, and 20 μM) for 3 or 18 h, then washed twice and measured in respiration medium containing 10 mM glucose (DMEM, serum free). Phosphorescent intensity signals were recorded in time-resolved fluorescence (TR-F) mode at two different delay times, using the settings described in (20). Subsequently, measured TR-F intensity signals were converted into lifetime values according to the formula LT = where t1 (25μs) and t2 (50μs) are the delay times, and I1 and I2 the corresponding intensity signals (23).…”

“…EGTA was then added at 2.5mM and the plate was measured for further 60 min. Subsequently, antimycin A at 5μM was added and intensity recorded for further 30 min (20,24).…”

“…The medium was replaced with complete DMEM and cells were imaged using a confocal TCSPC-PLIM microscope (Becker & Hickl) using an immersion lens adapter at 63× magnification and recorded using SPCImage software (Becker & Hickl). A 488 nm laser was used for the excitation of the Calcein green probe, and a 405 nm laser in PLIM mode was used for the PtPFPP-based O2 probe (12,20).…”

“…Following up with our previous study (20), we decided to further study the effect of di-glycosylation (using 1-Thioglucose) and di-PEGylation (using the neutral methoxy-PEG) in trans-position of PtPFPP. In fact, previous studies in the photodynamic therapy field, demonstrated the superiority of the trans-conformation for cellular uptake and subsequent cytotoxicity when exposed to light (16,21).…”

“…PEGs are often used to increase hydrophilicity and reduce non-specific binding interactions (18,19). It was also shown that tri-and tetra-PEGylation reduces the cell penetration ability of such derivatives, thus allowing their use as extracellular O2 sensing probes (20). In this study, we aimed to investigate the other hetero-substitution geometries, particularly the trans-di-PEGylated-di-glucosylated tetrasubstituted derivative of PtPFPP.…”