pH-activatable Fluorescent Probe Research Articles

Development of a Versatile Protein Labeling Tool for Live-Cell Imaging Using Fluorescent β-Lactamase Inhibitors.

To understand the function of protein in live cells, real-time monitoring of protein dynamics and sensing of their surrounding environment are important methods. Fluorescent labeling tools are thus needed that possess fast labeling kinetics, high efficiency, and long-term stability. We developed a versatile chemical protein-labeling tool based on fluorophore-conjugated diazabicyclooctane β-lactamase inhibitors (BLIs) and wild-type TEM-1 β-lactamase protein tag. The fluorescent probes efficiently formed a stable carbamoylated complex with β-lactamase, and the labeled proteins were visualized over a long period of time in live cells. Moreover, use of an α-fluorinated carboxylate ester-based BLI prodrug enabled the probe to permeate cell membranes and stably label intracellular proteins after unexpected spontaneous ester hydrolysis. Lastly, combining the labeling tool with a pH-activatable fluorescent probe allowed visual monitoring of lysosomal protein translocation during autophagy.

Development of near-infrared lysosomal pH-activatable fluorescent probe for real-time visualization of autophagy progression

As the central role in maintaining physiological homeostasis, tracking the autophagy in living cells is highly desired in biological research and pharmacologic development. Herein, a lysosome-targeted pH-activatable probe (Lyso-QL) with emission at near-infrared region (NIR) was developed for monitoring the progression of autophagy. During autophagy progress, the double-membraned autophagosomes are engulfed into lysosomes to provide autolysosomes, exhibiting significantly enhanced acidification. Based on the characteristic, the real-time monitoring of autophagic flux were realized by evaluating the pH changes in these acidic compartments with Lyso-QL under nutrient-deprivation conditions as well as pharmacological induction. Importantly, through the NIR changes, the modulation of tamoxifen on autophagic levels in estrogen receptor-negative cancer cells was disclosed. Further, the elevated hepatic autophagy in mice during acetaminophen-induced liver injury was revealed, and the N-acetylcysteine pretreatment was demonstrated with a certain protective effect to restore the autophagic levels. The study demonstrates probe Lyso-QL would provide an edge for mechanistic insights into autophagy and evaluating the activities of autophagy modulators.

β-Carboline-Based pH Fluorescent Probe and Its Application for Monitoring Enzymatic Ester Hydrolysis.

A novel pH-activatable fluorescent probe, 1-(propan-2-yl)-9H-pyrido[3,4-b]indole-3-carboxylic acid (L-1), based on β-carboline derivatives, has been developed, which displays significant fluorescent response toward pH variation with high selectivity, good photo-stability and favorable pKa value. Moreover, L-1 can dynamically monitor the release of protons during ester hydrolysis reaction in consistent with enzymatic kinetics manner.

In Vivo Multicolor Imaging with Fluorescent Probes Revealed the Dynamics and Function of Osteoclast Proton Pumps.

In vivo two-photon fluorescence imaging is a powerful modality to monitor cell dynamics in biomedical studies. To detect protein functions in living animals in real-time, fluorescent probes must show a quick response to the target function in specific tissues. Here, we developed a rhodamine-based small-molecule fluorescent probe called Red-pHocas (red pH-activatable fluorescent probe for osteoclast activity sensing) to reversibly detect the acidic environments for the spatiotemporal analysis of the function of osteoclast proton pumps. The introduction of electron-withdrawing N-alkyl substituents in the rhodamine spirolactam fluorophore remarkably increased the kinetics of the fluorescence response to acidic pHs, which allowed the rapid and reversible monitoring of acidic compartments and the analysis of the dynamics of osteoclast proton pumps during osteoclastic bone resorption. In vivo multicolor two-photon imaging using Red-pHocas in fluorescent reporter mice revealed that bone acidification occurred synchronously with the accumulation of proton pumps onto the bone surfaces. To our knowledge, this is the first study to demonstrate the direct involvement of osteoclast proton pumps in bone acidification under intravital conditions by means of an imaging probe.

Aggregation-induced emission spectral shift as a measure of local concentration of a pH-activatable rhodamine-based smart probe

Generating activatable probes that report about molecular vicinity through contact-based mechanisms such as aggregation can be very convenient. Specifically, such probes change a particular spectral property only at the intended biologically relevant target. Xanthene derivatives, for example rhodamines, are able to form aggregates. It is typical to examine aggregation by absorption spectroscopy but for microscopy applications utilizing fluorescent probes it is very important to perform characterization by measuring fluorescence spectra. First we show that excitation spectra of aqueous solutions of rhodamine 6G can be very informative about the aggregation features. Next we establish the dependence of the fluorescence emission spectral maximum shift on the dimer concentration. The obtained information helped us confirm the possibility of aggregation of a recently designed and synthesized rhodamine 6G-based pH-activatable fluorescent probe and to study its pH and concentration dependence. The size of the aggregation-induced emission spectral shift at specific position on the sample can be measured by fluorescence microspectroscopy, which at particular pH allows estimation of the local concentration of the observed probe at microscopic level. Therefore, we show that besides aggregation-caused quenching and aggregation-induced emission also aggregation-induced emission spectral shift can be a useful photophysical phenomenon.

Two-photon imaging of formaldehyde in live cells and animals utilizing a lysosome-targetable and acidic pH-activatable fluorescent probe.

Lyso-TPFP presents lysosomal targetability and an acidic pH-activatable response toward formaldehyde. Thus, it exclusively visualizes lysosomal formaldehyde and is immune against it in neutral cytosol and other organelles. In addition, two-photon fluorescence imaging endows Lyso-TPFP with the capability of in situ tracking formaldehyde in live cells and animals.

Engineering a nanolab for the determination of lysosomal nitric oxide by the rational design of a pH-activatable fluorescent probe.

Nitric oxide (NO) is often involved in many different physiological processes including the regulation of lysosomal functions. However, it remains a great challenge to explore the variations of NO levels in lysosomes, limiting the understanding behind its biological functions in cellular signaling pathways and various diseases. Herein, a pH-activatable fluorescent probe, Rhod-H-NO, was designed and synthesized for the determination of lysosomal NO, in which the activation response model is beneficial towards getting accurate biological information. To ensure that Rhod-H-NO can accumulate effectively and exist stably in lysosomes without interference and degradation from other active species, Rhod-H-NO was engineered into the nanopores of mesoporous silica nanoparticles (MSNs) with β-cyclodextrin (β-CD) as the gatekeeper to obtain a nanolab. The nanolab was successfully applied to detect lysosomal NO in living cells and in vivo with high time and spatial resolution. This nanolab could serve as an excellent molecular tool to exploit and elucidate the function of NO at sub-cellular levels.

A Multifunctional Nanomicelle for Real‐Time Targeted Imaging and Precise Near‐Infrared Cancer Therapy

AbstractSimultaneous targeted cancer imaging, therapy and real‐time therapeutic monitoring can prevent over‐ or undertreatment. This work describes the design of a multifunctional nanomicelle for recognition and precise near‐infrared (NIR) cancer therapy. The nanomicelle encapsulates a new pH‐activatable fluorescent probe and a robust NIR photosensitizer, R16FP, and is functionalized with a newly screened cancer‐specific aptamer for targeting viable cancer cells. The fluorescent probe can light up the lysosomes for real‐time imaging. Upon NIR irradiation, R16FP‐mediated generation of reactive oxygen species causes lysosomal destruction and subsequently trigger lysosomal cell death. Meanwhile the fluorescent probe can reflect the cellular status and in situ visualize the treatment process. This protocol can provide molecular information for precise therapy and therapeutic monitoring.

A multifunctional nanomicelle for real-time targeted imaging and precise near-infrared cancer therapy.

Simultaneous targeted cancer imaging, therapy and real-time therapeutic monitoring can prevent over- or undertreatment. This work describes the design of a multifunctional nanomicelle for recognition and precise near-infrared (NIR) cancer therapy. The nanomicelle encapsulates a new pH-activatable fluorescent probe and a robust NIR photosensitizer, R16FP, and is functionalized with a newly screened cancer-specific aptamer for targeting viable cancer cells. The fluorescent probe can light up the lysosomes for real-time imaging. Upon NIR irradiation, R16FP-mediated generation of reactive oxygen species causes lysosomal destruction and subsequently trigger lysosomal cell death. Meanwhile the fluorescent probe can reflect the cellular status and in situ visualize the treatment process. This protocol can provide molecular information for precise therapy and therapeutic monitoring.

Development of a pH-activatable fluorescent probe and its application for visualizing cellular pH change

Novel pH-activatable fluorescent probes with various pK(a) values have been developed utilizing 4-nitro-benzo[1,2,5]oxadiazole derivatives (NBD) as fluorophores and piperazine moieties as proton receptors. Under acidic conditions, probe FoPz displayed significant fluorescent enhancement of about 30 fold with a pK(a) value of 5.70 and it responds rapidly and sensitively to intracellular pH distributions and cellular pH fluctuations.