Intracellular O₂ Sensing Probe Based on Cell-Penetrating Phosphorescent Nanoparticles

Abstract

A new intracellular O(2) (icO(2)) sensing probe is presented, which comprises a nanoparticle (NP) formulation of a cationic polymer Eudragit RL-100 and a hydrophobic phosphorescent dye Pt(II)-tetrakis(pentafluorophenyl)porphyrin (PtPFPP). Using the time-resolved fluorescence (TR-F) plate reader set-up, cell loading was investigated in detail, particularly the effects of probe concentration, loading time, serum content in the medium, cell type, density, etc. The use of a fluorescent analogue of the probe in conjunction with confocal microscopy and flow cytometry analysis, revealed that cellular uptake of the NPs is driven by nonspecific energy-dependent endocytosis and that the probe localizes inside the cell close to the nucleus. Probe calibration in biological environment was performed, which allowed conversion of measured phosphorescence lifetime signals into icO(2) concentration (μM). Its analytical performance in icO(2) sensing experiments was demonstrated by monitoring metabolic responses of mouse embryonic fibroblast cells under ambient and hypoxic macroenvironment. The NP probe was seen to generate stable and reproducible signals in different types of mammalian cells and robust responses to their metabolic stimulation, thus allowing accurate quantitative analysis. High brightness and photostability allow its use in screening experiments with cell populations on a commercial TR-F reader, and for single cell analysis on a fluorescent microscope.

Microneedle-array patches loaded with hypoxia-sensitive vesicles provide fast glucose-responsive insulin delivery

“…To examine the glucose-responsive disassembly of GRVs, vesicles were incubated with PBS buffer [137 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 , 2 mM KH 2 PO 4 (pH 7.4)] containing different concentrations of glucose, including a typical hyperglycemic level (400 mg/dL), a normoglycemic level (100 mg/dL), and a control level (0 mg/dL). The oxygen consumption, caused by the oxidation of glucose catalyzed by GO x , was measured using an oxygensensitive phosphorescent molecular probe (39,40). As presented in Fig.…”

Exceptional Oxygen Sensing Properties of New Blue Light‐Excitable Highly Luminescent Europium(III) and Gadolinium(III) Complexes

“…The Eu(HPhN) 3 dpp complex was successfully incorporated in two types of oxygen-permeable nanoparticles, namely neutral poly(styrene-block-vinylpyrrolidone) (PS-PVP) [98] and Rl-100, which is a poly(methyl methacrylate) derivative bearing positively charged quaternary ammonium groups. [99] These materials were demonstrated to be suitable for extracellular [98] and intracellular measurements, [99] respectively. In good agreement with the previous work involving other oxygen indicators the size of the PS-PVP beads is significantly larger than that of the Rl-100 beads (Z av = 184 nm, PDI = 0.062 and Z av = 33 nm, PDI = 0.14 for PS-PVP and Rl-100 particles, respectively).…”

The effect of nanoparticle uptake on cellular behavior: disrupting or enabling functions?

“…16,17 More recently, they also have been applied in real-time live-cell monitoring of a number of cell parameters such as intracellular oxygen concentration. 18 NPs can be made of different inorganic and organic materials: they can be lipid-based (such as liposomes, micelles, solid lipid, or lipoprotein-based NPs), [18][19][20][21] polymeric (such as polylactic acid, poly[lactic-co-glycolic acid], and poly[alkylcyanoacrylate]), 22,23 chitosan-based, 24 quantum dots, 25 silicon-based 26 gold NPs, 27 and magnetic NPs. 17 Since the intrinsic characteristics and thus the relevant applications of NPs are closely related to their size, shape, and surface properties, great efforts have been devoted to control the synthesis of NPs.…”