Noncovalent Modification of Cycloparaphenylene by Catenane Formation Using an Active Metal Template Strategy.

The active metal template (AMT) strategy is a powerful tool for the formation of mechanically interlocked molecules (MIMs) such as rotaxanes and catenanes, allowing the synthesis of a variety of MIMs, including π-conjugated and multicomponent macrocycles. Cycloparaphenylene (CPP) is an emerging molecule characterized by its cyclic π-conjugated structure and unique properties. Therefore, diverse modifications of CPPs are necessary for its wide application. However, most CPP modifications require early stage functionalization and the direct modification of CPPs is very limited. Herein, we report the synthesis of a catenane consisting of [9]CPP and a 2,2'-bipyridine macrocycle as a new CPP analogue that contains a reliable synthetic scaffold enabling diverse and concise post-modification. Following the AMT strategy, the [9]CPP-bipyridine catenane was successfully synthesized through Ni-mediated aryl-aryl coupling. Catalytic C-H borylation/cross-coupling and metal complexation of the bipyridine macrocycle moiety, an effective post-functionalization method, were also demonstrated with the [9]CPP-bipyridine catenane. Single-crystal X-ray structural analysis revealed that the [9]CPP-bipyridine catenane forms a tridentated complex with an Ag ion inside the CPP ring. This interaction significantly enhances the phosphorescence lifetime through improved intermolecular interactions.

In Vivo Imaging of Lipid Droplets and Oxygen Status in Hepatic Tissues of Nonalcoholic Fatty Liver Model Mice Using a Lipophilic Ir(III) Complex.

Nonalcoholic fatty liver disease (NAFLD) is becoming common worldwide. In pathophysiological studies of NAFLD, an in vivo optical probe that enables visualization of lipid droplets (LDs) and imaging of oxygen status in hepatic tissues simultaneously would be very useful. Here, we present the phosphorescent Ir(III) complex BTP ((btp)2Ir(acac) (btp = benzothienylpyridine, acac = acetylacetone)) as the first probe that meets this requirement. BTP was efficiently taken up into cultured 3T3-L1 adipocytes and selectively accumulated into LDs. Quantifying oxygen levels in LDs based on the phosphorescence lifetime of BTP allowed us to track changes in cellular oxygen tension after treatment with metabolic stimulants. Phosphorescence lifetime imaging microscopy combined with intravenously administered BTP in mice enabled specific visualization of LDs in hepatic lobules and simultaneous imaging of the oxygen gradient that decreased from the portal vein (PV) to the central vein (CV). NAFL model mice were created by feeding a high-fat diet (HFD) to mice for 3 or 7 days. The mice fed an HFD showed a marked increase in the amount and size of LDs in hepatocytes compared with those fed a normal diet, leading to abnormal microvascular structures. In addition, HFD-fed mice also exhibited reduced oxygen tension in areas other than the CV. Multicolor imaging with the LD-accumulated oxygen probe BTP and vasculature-staining FITC-lectin suggested that structural distortions of the sinusoidal microvasculature caused by enlarged LDs were associated with partial hypoxia in NAFL.

Near-Infrared Emitting Ir(III) Complexes Bearing a Dipyrromethene Ligand for Oxygen Imaging of Deeper Tissues In Vivo.

Phosphorescence lifetime imaging microscopy (PLIM) using a phosphorescent oxygen probe is an innovative technique for elucidating the behavior of oxygen in living tissues. In this study, we designed and synthesized an Ir(III) complex, PPYDM-BBMD, that exhibits long-lived phosphorescence in the near-infrared region and enables in vivo oxygen imaging in deeper tissues. PPYDM-BBMD has a π-extended ligand based on a meso-mesityl dipyrromethene structure and phenylpyridine ligands with cationic dimethylamino groups to promote intracellular uptake. This complex gave a phosphorescence spectrum with a maximum at 773 nm in the wavelength range of the so-called biological window and exhibited an exceptionally long lifetime (18.5 µs in degassed acetonitrile), allowing for excellent oxygen sensitivity even in the near-infrared window. PPYDM-BBMD showed a high intracellular uptake in cultured cells and mainly accumulated in the endoplasmic reticulum. We evaluated the oxygen sensitivity of PPYDM-BBMD phosphorescence in alpha mouse liver 12 (AML12) cells based on the Stern-Volmer analysis, which gave an O2-induced quenching rate constant of 1.42 × 103 mmHg-1 s-1. PPYDM-BBMD was administered in the tail veins of anesthetized mice, and confocal one-photon PLIM images of hepatic tissues were measured at different depths from the liver surfaces. The PLIM images visualized the oxygen gradients in hepatic lobules up to a depth of about 100 µm from the liver surfaces with a cellular-level resolution, allowing for the quantification of oxygen partial pressure based on calibration results using AML12 cells.

A distinctive distribution of hypoxia-inducible factor-1α in cultured renal tubular cells with hypoperfusion simulated by coverslip placement.

Chronic hypoxia in the renal tubulointerstitium plays a key role in the progression of chronic kidney disease (CKD). It is therefore important to investigate tubular hypoxia and the activity of hypoxia-inducible factor (HIF)-1α in response to hypoxia. Rarefaction of the peritubular capillary causes hypoperfusion in CKD; however, the effect of hypoperfusion on HIFs has rarely been investigated. We induced hypoperfusion caused by coverslip placement in human kidney-2 cells, and observed an oxygen gradient under the coverslip. Immunocytochemistry of HIF-1α showed a doughnut-shaped formation on the edge of a pimonidazole-positive area, which we named the "HIF-ring". The oxygen tension of the HIF-ring was estimated to be between approximately 4 mmHg and 20 mmHg. This result was not compatible with those of past research showing HIF-1α accumulation in the anoxic range with homogeneous oxygen tension. We further observed the presence of a pH gradient under a coverslip, as well as a shift of the HIF ring due to changes in the pH of the culture medium, suggesting that the HIF ring was formed by suppression of HIF-1α related to low pH. This research demonstrated that HIF-1α activation mimics the physiological state in cultured cells with hypoperfusion.

In vivo O2 imaging in hepatic tissues by phosphorescence lifetime imaging microscopy using Ir(III) complexes as intracellular probes.

Phosphorescence lifetime imaging microscopy (PLIM) combined with an oxygen (O2)-sensitive luminescent probe allows for high-resolution O2 imaging of living tissues. Herein, we present phosphorescent Ir(III) complexes, (btp)2Ir(acac-DM) (Ir-1) and (btp-OH)3Ir (Ir-2), as useful O2 probes for PLIM measurement. These small-molecule probes were efficiently taken up into cultured cells and accumulated in specific organelles. Their excellent cell-permeable properties allowed for efficient staining of three-dimensional cell spheroids, and thereby phosphorescence lifetime measurements enabled the evaluation of the O2 level and distribution in spheroids, including the detection of alterations in O2 levels by metabolic stimulation with an effector. We took PLIM images of hepatic tissues of living mice by intravenously administrating these probes. The PLIM images clearly visualized the O2 gradient in hepatic lobules with cellular-level resolution, and the O2 levels were derived based on calibration using cultured cells; the phosphorescence lifetime of Ir-1 gave reasonable O2 levels, whereas Ir-2 exhibited much lower O2 levels. Intravenous administration of NH4Cl to mice caused the hepatic tissues to experience hypoxia, presumably due to O2 consumption to produce ATP required for ammonia detoxification, suggesting that the metabolism of the probe molecule might affect liver O2 levels.

Intravital phosphorescence lifetime imaging of the renal cortex accurately measures renal hypoxia.

Renal tubulointerstitial hypoxia is recognized as a final common pathway of chronic kidney disease and is considered a promising drug target. However, hypoxia in the tubules is not well examined because of limited detection methods. Here, we devised a method to visualize renal tubular oxygen tension with spatial resolution at a cellular level using the cell-penetrating phosphorescent probe, BTPDM1 (an iridium-based cationic lipophilic dye), and confocal phosphorescence lifetime imaging microscopy to precisely assess renal hypoxia. Imaging with BTPDM1 revealed an oxygen gradient between S1 and S2 segments in mouse kidney. We also demonstrated that our microscopy system can detect subtle changes of hypoxemia and reoxygenation, and the acquired phosphorescence lifetime can be converted to partial pressure of oxygen. This new method allows, for the first time, visualization of intravital oxygen gradients at the renal surface with high spatial resolution. Thus, the confocal phosphorescence lifetime imaging microscopy platform, combined with BTPDM1, will promote an accurate understanding of tissue hypoxia, including renal hypoxia.