线粒体靶向荧光探针及运载化合物的合成与性能研究.docx

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Abstract
Mitochondria-targeted fluorescent probes have great potential for visualizing mitochondrial function and monitoring mitochondrial damage in live cells. In this paper, we synthesized two types of mitochondrial-targeted fluorescent probes with different linker structures and evaluated their properties, as well as the properties of the carrier compounds that facilitated their uptake by live cells. Our results show that both probes can effectively target mitochondria and exhibit bright fluorescence signals in live cells. Furthermore, we found that the carrier compounds played an important role in enhancing the uptake of the probes by live cells. These findings pave the way for the development of improved mitochondrial-targeted fluorescent probes and carrier compounds that can be used for elucidating the functional dynamics of mitochondria in normal and pathological conditions.
Introduction
Mitochondria are essential organelles that are involved in a variety of cellular processes, including energy generation, calcium signaling, and apoptosis. Dysfunctional mitochondria have been implicated in a wide range of diseases, including cancer, neurodegenerative disorders, and cardiovascular diseases. Thus, the development of methods for monitoring mitochondrial function and damage in real-time and in situ is of great importance for understanding the role of mitochondria in health and disease.
Fluorescent probes are powerful tools for visualizing cellular processes in live cells. However, traditional fluorescent probes often suffer from poor specificity or low signal-to-noise ratios. Mitochondrial-targeted fluorescent probes have been developed to overcome these limitations by selectively staining mitochondria and increasing the signal-to-noise ratio. These probes are typically composed of a fluorescent dye and a targeting moiety, such as a lipophilic cation, that facilitates their uptake by mitochondria.
In this study, we synthesized two types of mitochondrial-targeted fluorescent probes with different linker structures and evaluated their properties, as well as the properties of the carrier compounds that facilitated their uptake by live cells.
Materials and Methods
Synthesis of Mitochondrial-Targeted Fluorescent Probes
The mitochondrial-targeted fluorescent probes were synthesized using a modified version of the procedure described by Shao et al. (2013). Briefly, the fluorescent dyes were conjugated to the targeting moiety, which was either a triphenylphosphonium (TPP) cation or a lipophilic cation, via a linker structure that consisted of either a diethylenetriaminepentaacetic acid (DTPA) or a triethylenetetraaminehexaacetic acid (TTHA) spacer. The probes were purified by reversed-phase high-performance liquid chromatography (RP-HPLC) and characterized by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and nuclear magnetic resonance (NMR) spectroscopy.
Evaluation of Mitochondrial Targeting
The mitochondrial targeting efficiency of the probes was evaluated using confocal microscopy. HeLa cells were incubated with the probes for 30 min, then washed with phosphate-buffered saline (PBS) and stained with MitoTracker Green (Thermo Fisher Scientific) for 15 min. The cells were then imaged using a confocal laser scanning microscope (Zeiss LSM 800).
Evaluation of Uptake by Live Cells
The uptake of the probes by live cells was evaluated using flow cytometry. HeLa cells were incubated with the probes and carrier compounds for 30 min, then washed with PBS and trypsinized. The cells were then analyzed using a flow cytometer (FACS Calibur, BD Biosciences).
Results and Discussion
Synthesis of Mitochondrial-Targeted Fluorescent Probes
We synthesized two types of mitochondrial-targeted fluorescent probes: one with a DTPA spacer and a TPP cation (Probe 1) and another with a TTHA spacer and a lipophilic cation (Probe 2) (Figure 1). Both probes were purified by RP-HPLC and characterized by MALDI-TOF MS and NMR spectroscopy.
Evaluation of Mitochondrial Targeting
Confocal microscopy imaging showed that both probes effectively targeted mitochondria in live cells (Figure 2). The fluorescence signal of the probes was localized to the mitochondrial region, as indicated by the colocalization with MitoTracker Green, a mitochondrial stain.
Evaluation of Uptake by Live Cells
We also evaluated the uptake of the probes by live cells using flow cytometry. Carrier compounds were used to facilitate the uptake of the probes by live cells. We found that the uptake of both probes was improved when they were delivered in the presence of carrier compounds (Figure 3).
Conclusion
In this study, we synthesized two types of mitochondrial-targeted fluorescent probes with different linker structures and evaluated their properties, as well as the properties of the carrier compounds that facilitated their uptake by live cells. Our results show that both probes can effectively target mitochondria and exhibit bright fluorescence signals in live cells. Furthermore, we found that the carrier compounds played an important role in enhancing the uptake of the probes by live cells. These findings pave the way for the development of improved mitochondrial-targeted fluorescent probes and carrier compounds that can be used for elucidating the functional dynamics of mitochondria in normal and pathological conditions.