In vivo bioluminescence imaging of labile iron pools in a murine model of sepsis with a highly selective probe

Highlights

• A novel bioluminescent probe for visualizing Iron (II) has been developed with high sensitivity and selectivity.

• This probe was successfully appied to detect exogenous Fe²⁺ in living animal and monitor labile endogenous Fe²⁺ levels in animal disease model.

Abstract

Iron plays an essential role in biological system. An approach for in vivo imaging of this metal ion is needed to investigate its complex contributions to physiological and pathological processes. Herein, we present a bioluminescent probe FP-1 as a powerful tool for targeting Fe²⁺ detection in vitro and in vivo. The turn-on sensing scheme is based on the caged strategy of luciferin-luciferase system. FP-1 not only can detect accumulations of exogenous Fe²⁺ in living animal, but also has the capability of monitoring labile endogenous Fe²⁺ levels in animal model of sepsis. Implementation of this technique provides a valuable opportunity for understanding underlying mechanisms of Fe²⁺ in biological processes and disease states.

Introduction

Iron is not only a transition metal widely used in industry, but also an essential element for nearly all living organisms [1,2]. As a critical catalytic center in metalloproteins such as hemoglobin and myoglobin, iron performs versatile physiological functions involved in a wide range of biological processes including oxygen transport [2], electron transfer [3], cellular metabolism, immune response [4], and DNA synthesis [5]. When iron intake is insufficient to meet the physiological needs, iron deficiency may occur and lead to anemia [6], dysplasia, decreased immunity function [4], mental disorders [7], and impaired neural motor development [8]. In fact, iron deficiency is one of the most prevalent malnutrition in the world [9]. On the other hand, abnormalities in iron level and its homeostasis disruption are closely associated with a variety of diseases, including cardiovascular diseases [10], neurodegenerative disorders [8], infection [11], and cancers [12].

The indispensable role of iron in living organism prompts great interest in techniques for detecting this metal within living cells and other intact biological specimens and assessing its functions in diverse biological processes and disease states. Multiple methods for analyzing iron ion exist: atomic absorption-emission spectrometry [13], electrochemical methods [14], and inductively coupled plasma atomic emission spectroscopy [15]. However, these traditional techniques normally require sample pretreatment, sophisticated procedure and costly instrument. Compared to these methods, optical strategy stands out as a unique case, due to its high sensitivity and real-time response capability which make it suitable for biological analysis. Along this line, a number of fluorescent probes have been established [[16], [17], [18], [19], [20], [21], [22]]. Despite of ongoing efforts to the development of new fluorescent probes, inherent defects of fluorescence, including autofluorescence and photobleaching, greatly limit its further applications in bioimaging, especially in vivo imaging. The lack of follow-up approach for tracking iron in whole animal restricts the study of distribution, excretion, and other events of iron ion in biological system.

To fill this gap, we decided to seek for a novel strategy to construct an imaging platform to detect iron level in living animal. Bioluminescence is a phenomenon of luminescence widely found in nature. Some organisms can convert chemical energy into light energy through a series of oxidation reactions [23]. In this process, luminescence signal can be efficiently generated without external light excitation. The incomparable characteristics of bioluminescence, including excellent biocompatibility, high signal-to-noise ratio, and in vivo detection ability, provide an indispensable tool for probing cell functions, transgene expression, and molecular mechanisms in complex biological contexts. In the past decade, bioluminescence approach, as an alternative to fluorescence, was widely applied and greatly advanced the virtual concept of biological research. Bioluminescent probes were devised for the detection of analytes of interest [[24], [25], [26], [27], [28], [29], [30]]. Chang et al. developed a bioluminescence probe ICL-1 for in vivo imaging of iron accumulation in a murin model of Acinetobacter baumannii infection [30]. Herein, we reported our effort in developing new bioluminescent probe FP-1 (Scheme 1) with high selectivity for visualizing Fe (II) in cell culture, living animal and disease model. We also compared the differences of FP-1 and ICL-1 in Supporting Information.