Biocompatible Phosphorescent O2 Sensors Based on Ir(III) Complexes for In Vivo Hypoxia Imaging

Abstract

In this work, we obtained three new phosphorescent iridium complexes (Ir1–Ir3) of general stoichiometry [Ir(N^C)2(N^N)]Cl decorated with oligo(ethylene glycol) fragments to make them water-soluble and biocompatible, as well as to protect them from aggregation with biomolecules such as albumin. The major photophysical characteristics of these phosphorescent complexes are determined by the nature of two cyclometallating ligands (N^C) based on 2-pyridine-benzothiophene, since quantum chemical calculations revealed that the electronic transitions responsible for the excitation and emission are localized mainly at these fragments. However, the use of various diimine ligands (N^N) proved to affect the quantum yield of phosphorescence and allowed for changing the complexes’ sensitivity to oxygen, due to the variations in the steric accessibility of the chromophore center for O2 molecules. It was also found that the N^N ligands made it possible to tune the biocompatibility of the resulting compounds. The wavelengths of the Ir1–Ir3 emission maxima fell in the range of 630–650 nm, the quantum yields reached 17% (Ir1) in a deaerated solution, and sensitivity to molecular oxygen, estimated as the ratio of emission lifetime in deaerated and aerated water solutions, displayed the highest value, 8.2, for Ir1. The obtained complexes featured low toxicity, good water solubility and the absence of a significant effect of biological environment components on the parameters of their emission. Of the studied compounds, Ir1 and Ir2 were chosen for in vitro and in vivo biological experiments to estimate oxygen concentration in cell lines and tumors. These sensors have demonstrated their effectiveness for mapping the distribution of oxygen and for monitoring hypoxia in the biological objects studied.

Electrogenerated Chemiluminescence Biosensor for Quantization of Matrix Metalloproteinase-3 in Serum via Target-Induced Cleavage of Oligopeptide

“…[(3-pba) 2 Ir(bpy-COOH)](PF 6 ) was synthesized through two steps [23,24], in which 3-pba was used as the main ligand and bpy-COOH was used as the auxiliary ligand (Supplementary Materials, the synthetic route shown in Scheme S1). First, the dichlorobridged iridium dimer [(3-pba) 2 Ir(µ-Cl)] 2 was synthesized by adding IrCl 3 •3H 2 O and 3-pba (CˆN ligand) to a mixture of 2-ethoxyethanol and H 2 O.…”

“…The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/bios14040181/s1, Methods: Synthesis of AuNPs [25] and fabrication of AuNPs/Nafion/GCE [16]; Synthesis of Ir [23,24]; Scheme S1: The synthetic route for Ir; Figure S1: ESI-MS spectra of Ir; Figure S2: 1 H NMR spectrum of Ir; Figure S3: Characterization of modified electrodes using CVs and Nyquist plots of electrochemical impedance spectra; Figure S4: TEM image of AuNPs; Figure S5: ECL intensity vs. time curve of one ECL biosensor obtained from continuous potential scanning or six independent ECL biosensors for one scan and effect of storage time on the ECL intensity of the ECL biosensors; Figure S6: ECL intensity vs. potential profiles of one gold electrode-based ECL biosensor for continuous potential scanning over ten cycles; Figure S7: ECL intensity vs. potential profiles of gold electrode-based ECL biosensor before and after incubation with MMP-3; Figure S8: The effects of the self-assembly time of Ir-peptide and the cleavage time of MMP-3 on the ECL intensity of the ECL biosensor; Figure S9: ECL intensity vs. potential profiles of the ECL biosensor in the absence and presence of original serum samples; Table S1: Parameter values obtained from the fit of the impedance spectra represented with the equivalent circuit; Table S2: MMP-3 levels in the serum of patients [29]; Table S3: Analytical results of MMP-3 in 20-fold diluted serum of healthy people, measured using ELISA and the developed ECL biosensor; Table S4: Comparison of different reported methods for the determination of MMP-3 [8,11,12,31].…”

Biocompatible Phosphorescent Ir(III) Complexes for Hypoxia Sensing in PLIM Mode

“…This is why time resolved luminescence microscopy, in partic-ular, phosphorescence lifetime measurement mode (PLIM) [12,26,[28][29][30] is getting popular for application in quantitative analysis of biological systems with phosphorescent sensors. [31][32][33][34][35][36][37][38][39] The PLIM mode proved to be particularly effective instrument in oxygen concentration monitoring in plain cell cultures, [40,41] 3D spheroids [42,43] and various in vivo systems, [36,[44][45][46][47] where the sensors based on platinum and palladium porphyrins dominate over the other types of chromophores due to extremely strong sensor response onto the target analyte. However, these excellent sensors, e. g. Oxyphors family, [32,33,[48][49][50] require challenging synthesis, are impenetrable into cells and show a very long excited state lifetime (10-270 μs under physiological conditions).…”

“…Therefore, the phosphorescent iridium orthometalated complexes are the object of choice for intracellular studies [34,[51][52][53][54] due to ability to internalize into cells, substantially shorter lifetimes and much easier synthetic procedures. It is worth noting that red and near-infrared (NIR) iridium emitters can be effectively used for in vivo imaging [17,47,55] due to deep penetration of NIR emission signal. Moreover, using a more complicated design of the iridium based NIR chromophores it is also possible to impart them theranostics characteristics, i. e. ability to simultaneously visualize the target and also serve as a photodynamic sensitizer.…”

The Dual Luminescence Lifetime pH/Oxygen Sensor: Evaluation of Applicability for Intravital Analysis of 2D- and 3D-Cultivated Human Endometrial Mesenchymal Stromal Cells

“…In this context, the sensors allowing simultaneous determination of several parameters, for example, pH level and oxygen pressure, in the same intracellular structures are of particular interest. Phosphorescent cyclometallated Ir(III) complexes are a convenient basis for creating highly sensitive biolabels and sensors that visualize the O 2 level in tissues [16][17][18]. It is also worth noting that cyclometallated Ir(III) complexes usually remain stable under exposure to photoexcitation that makes them suitable candidates for prolonged biological experiments.…”