Mitochondria‐Targeting BODIPY Probes for Imaging of Reactive Oxygen Species

Reactive oxygen species (ROSs) are an important class of signaling molecules that play a critical role in regulating physiological processes in the human body. Mitochondria are the primary site of ROSs production, and abnormal concentrations of ROSs can lead to the malignant proliferation of cells, resulting in diseases. Therefore, it is crucial to detect ROSs in mitochondria. Fluorometric methods have gained significant attention from scientists because of their ease of observation, simplicity of operation, and noninvasiveness. Among the fluorescent dyes, boron‐dipyrromethene (BODIPY) stands out for its high quantum yield, large molar extinction coefficient, and excellent chemical stability, making it widely used in fluorescent imaging. The common design principle of fluorescent probe for the detection of ROSs includes photo‐induced electron transfer, intramolecular charge transfer, and fluorescence resonance energy transfer. This paper provides an overview of BODIPY‐based fluorescent probes designed for imaging ROSs in mitochondria, covering the sensing mechanisms, molecular engineering strategies, and recent advancements. Additionally, the review provides insight into the potential clinical applications of mitochondria‐targeting BODIPY probes in disease diagnosis.


Introduction
Reactive oxygen species (ROSs) refer to a group of oxygencontaining radicals and oxides found in living organisms that ROSs in mitochondria for several reasons. First, BODIPY exhibits low toxicity, making it compatible with mitochondrion, which is a fragile organelle in the cell. Second, while other dyes may be hindered by the negative potential of mitochondria, BODIPY remains unaffected. [39] Third, BODIPY's exceptional brightness enables the detection of trace concentrations of ROSs in mitochondria with high precision.
The use of BODIPY and its derivatives for detecting ROSs has proven valuable in disease diagnosis and prognosis. [40] However, there is a lack of specific articles that discuss mitochondrial ROSs detection. Given the pivotal role of mitochondria in ROSs production, a review article summarizing recent progress in the use of BODIPY as probes for mitochondrial ROSs detection could provide timely information for readers across various areas. Therefore, this review aims to summarize the BODIPY probes capable of targeting mitochondria for ROSs detection, with a focus on their application in bioimaging. The goal is to encourage the development of BODIPY probes for detecting ROSs in mitochondria, which may aid in the future monitoring of mitochondria-related diseases due to their fast response and high selectivity.

A Brief Introduction to BODIPY Molecules
Since its discovery in 1968 by Treibs and Kreuzer, BODIPY has become widely used in various fields due to its high fluorescence quantum yield and large molar extinction coefficient. [41] The BODIPY molecular core can be numbered using either numbers or letters, in accordance with IUPAC nomenclature, as illustrated in Figure 1. Both numbering systems are acceptable, but letter-based nomenclature is more convenient and widely used, and thus is used for naming BODIPY in this paper. BODIPY offers multiple modification sites. Modifications at the meso position are less likely to affect the absorption and emission wavelengths of the molecule. Hydrophilic groups, such as sulfonates or carboxylates, are usually introduced at the position to enhance the hydrophilicity of BODIPY molecules. Modifications at the position are usually used to increase the emission wavelength, while the modification at the position can be challenging due to the large spatial hindrance. [42][43] BODIPY is highly resistant to changes in both polarity and pH, remaining extremely stable under physiological conditions. For this reason, BODIPY has wide-ranging applications in various fields, including fluorescent labeling, chemosensing, and photodynamic therapy. [44][45][46][47] This review paper specifically focuses on the utilization of BOD-IPY for constructing fluorescent probes that can detect ROSs in mitochondria.

The Methodology for Modifying BODIPY to Enable Mitochondrial Targeting
Several ATPases in the inner mitochondrial membrane's respiratory chain act as proton pumps, continuously exporting protons out of the mitochondria, thereby establishing a highly negative potential of about 160-180 mV inside the organelle. [41] To target BODIPY to mitochondria, conjugating cations to the molecule is a highly effective and widely employed strategy. Cationic triphenylphosphine (TPP + ) and pyridinium are commonly used groups, and aptamers are also utilized as targeting options. [48][49][50][51] Of the various mitochondrial-targeting groups mentioned above, TPP + is an off-domain lipophilic cation that consists of benzene rings and stands out due to its straightforward synthesis and exceptional in vivo stability. TPP + contains a phosphorus (P) atom that enhances its lipid solubility. Furthermore, the positive charge on the P atom is distributed to the neighboring benzene ring, generating a positive charge on the molecule. This characteristic facilitates TPP + to cross the mitochondrial bilayer and enables it to penetrate the inner mitochondria. [52][53] As an example, Wang et al. synthesized a novel BODIPY conjugate by attaching TPP + and varying lengths of ethylene glycol. [54] This modification not only enhanced the photothermal and photoacoustic effects but also enabled more precise and efficient targeting of mitochondria. Meanwhile, Gao et al. synthesized BODIPY derivatives by introducing a TPP + unit, which exhibited exceptional mitochondrial targeting efficacy while retaining high stability and low cytotoxicity. [55] Another commonly used cationic group for targeting mitochondria is pyridinium, which has good water solubility and can compensate for the poor solubility of BODIPY in water. The pyridine cation is another type of delocalized lipophilic cation. The positive charge on the nitrogen (N) atom delocalizes to the attached benzene ring, aiding its accumulation inside the mitochondria. In a study by Cao et al., two pyridiniums were coupled to BODIPY. [49] This modification not only improved the derivative's mitochondrial targeting but also reduced its cytotoxicity. Colocalization experiments showed that the pyridiniummodified BODIPY effectively targeted the mitochondria of HeLa cells.
Aptamers are oligonucleotide sequences obtained in vitro, enabling rapid batch production. Aptamers fold into a specific shape that matches the target configuration upon binding to their target, resulting in a greatly improved binding affinity for the target. [56] Aptamers are stable and nontoxic, making them an attractive option for targeting mitochondria. G-quadruplex aptamers were reported being capable of targeting mitochondria, albeit the underlying mechanism remains unclear. Zhang et al. synthesized BPTPA, a BODIPY derivative, by coupling triphenylamine to BODIPY, [51] and found that BPTPA automatically binds to G 3 T 3 to form the complex BPTPA-G 3 T 3 , which can be untied for targeting and imaging mitochondria.

Design Principle of ROSs Probe
The molecular structure is critical in determining the sensing performance of a probe, and understanding the detection mechanisms involved can help in designing better probes. Various mechanisms have been reported for designing fluorescence probes for ROSs, including photoinduced electron transfer (PET), intramolecular charge transfer (ICT), and fluorescence resonance energy transfer (FRET), which can be explained using the highest occupied molecular orbital (HOMO)-lowest unoccupied molecular orbital (LUMO) orbital theory. In this section, we provide a brief introduction to these mechanisms and highlight representative examples for each. [57][58][59]

ROSs Probes Based on PET Mechanism
In the case of PET, there are two types of processes, a-PET and d-PET. For the detection of ROSs using BODIPY, fluorescence can be activated by blocking the PET between the fluorophores and the acceptors, i.e., the quenching group. When the fluorophore is excited by a specific wavelength of light, electrons on the HOMO will transfer to the LUMO, resulting in quenched fluorescence due to the HOMO of the acceptor lying between the HOMO and LUMO of the fluorophore. However, upon reacting with ROSs, the acceptor gets oxidized, lowering its HOMO level below that of the fluorophore, which leads to the acceptance of electrons from the LUMO of the fluorophore, blocking the PET process and turning on fluorescence (Figure 2a). In the case of d-PET, the LUMO of the acceptor lies between the HOMO and LUMO of BODIPY, and electron transfer occurs via the PET process. Upon reacting with ROSs, the LUMO of the acceptor moves up to the LUMO of BODIPY, blocking the PET process and emitting fluorescence ( Figure 2b). [60] Based on the principles of PET, a wide variety of BODIPY probes have been developed for detecting ROSs. For instance, Zhu et al. reported the BODIPY probe (BClO) for the rapid detection of HClO by coupling 2, 4-dimethylpyrrole with BODIPY. [61] The pyrrole ring, rich in electrons, enhances the PET process, resulting in a probe with low background fluorescence. Upon detection of HClO, the pyrrole reacts specifically with HClO to form pyrrolidone, inhibiting the PET process and restoring fluorescence. And, the detection limit of BClO was as low as 0.56 nm, which was the lowest achievable detection limit.
In another study, Sun et al. reported a probe called HKOCl-1, which modified BODIPY with p-methylphenol at the meso position for the specific detection of HClO. [62] Prior to detecting HClO, the HOMO energy level of p-methylphenol was −8.71 eV, higher than the HOMO energy level of BODIPY (−9.14 eV), resulting in PET of p-methylphenol for the BODIPY fluorophore. Upon detection of HClO, p-methylphenol was oxidized to benzoquinone, which has a HOMO energy level of −10.9 eV, lower than the HOMO energy level of the fluorophore. As a result, the PET effect was inhibited, and intense fluorescence was observed upon excitation.

ROSs Probes Based on ICT Mechanism
Fluorescent probes based on the ICT mechanism have attracted considerable attention from scientists in recent years due to their ease of synthesis and utilization. In ICT processes, once excited by light, electrons on the donor with strong electron-donating ability will be transferred to the acceptor with strong electronwithdrawing ability, attributed to the push-pull electron effect between the donor and the acceptor. Meanwhile, the electrons in the molecule will be rearranged and the dipole moment may change, thus causing the emission spectrum to change.
In the detection of ROSs using BODIPY based probes, the BODIPY part serves as an electron donor. A strong electronwithdrawing acceptor is then attached to form a D--A system through -conjugation. After reacting with ROSs, the molecular structure is altered, leading to changes in the electronic structure of the molecules. As a consequence, the ICT process will be weakened or strengthened, accompanied by a shift in the emission wavelength. If the electron-withdrawing capacity of the receptor becomes stronger after the reaction, the ICT process will be enhanced, resulting in a redshifted wavelength. On the contrary, if the electron-withdrawing capacity becomes weaker, the ICT process will be weakened, leading to a blueshifted wavelength (Figure 3a). [63][64] Based on the ICT mechanism, Xu et al. synthesized a probe named BCPA-BODIPY-TCF for the detection of ClO − . [65] The acceptor group was a tricyanofuran unit, and the acceptor group was linked to the donor group via a vinyl bond. Compared with BCPA-BODIPY without the coupled tricyanofuran, BCPA-BODIPY-TCF exhibited an ICT phenomenon attributed to the introduction of strong electron-withdrawing tricyanofuran. As a result, the spectrum was redshifted from 495 to 580 nm. Upon reaction with ClO − , the C═C bond close to the acceptor side was oxidized into the aldehyde group, releasing the acceptor group. Then, the ICT mechanism was suppressed. Correspondingly, the absorption peak at 580 nm was reduced, and the absorption band at 500 nm was broadened. Meanwhile, the color of the probe solution changed from purple to pink within a short time. Similarly, Leng et al. developed the probe (BODIPY-DAMN) by coupling the acceptor diaminomaleonitrile (DAMN) to BODIPY using a C═N bond as a reactive site for ClO − . [66] The difference was that the authors modified the molecule with a hydrophilic carboxyl group to increase the water solubility of the probe, making the probe better applicable in vivo. In the absence of ClO − , the fluorescence remained quenched due to the strong electron-absorbing ability of DAMN. Reaction with ClO − caused C═N bond oxidation to release the DAMN group, resulting in inhibition of the ICT process and enhancing fluorescence.

ROSs Probes Based on FRET Mechanism
The FRET mechanism is a widely used sensing mechanism in spectroscopy. Unlike PET and ICT, which receive light directly, the energy transfer of FRET is nonradiative. When the donor receives excitation light, its electrons leap to emit fluorescence, which acts as a light source for the acceptor and transfers energy to the acceptor in the form of resonance through the dipole and dipole interaction between them. The acceptor is excited and emits longer-wavelength light. Since the acceptor needs to be excited by the resonance energy of the donor, the mutual distance between the donor and the acceptor must be less than 10 nm (Figure 3b). [67] Deng et al. constructed a fluorescent probe (Rho-Bob) by coupling a BODIPY derivative with rhodamine. [68] In the molecule, rhodamine acted as a donor, while BODIPY acted as an acceptor. After irradiation with light at the excitation wavelength of rhodamine, Rho-Bob emitted intense red fluorescence upon detection of ·OH.

ROSs Probes Based on Multiple Mechanisms
In rare cases, some probes are reported to utilize multiple sensing mechanisms for the detection of ROSs, in addition to probes that utilize a single sensing mechanism. Compared to probes with a single sensing mechanism, probes that utilize multiple sensing mechanisms can simultaneously detect changes in Reproduced with permission. [73] Copyright 2013, American Chemical Society.
signal intensity and wavelength. This allows for the detection of multiple analytes by a single probe and can improve the sensitivity and selectivity of the probes. [69][70] However, BODIPY probes utilizing multiple sensing mechanisms for the detection of mitochondrial ROSs have not yet been reported, suggesting a promising direction for future development.
While there are other luminescence processes of fluorescent probes, such as metal-ligand charge transfer (MLCT), chelationenhanced fluorescence (CHEF), and excited state intramolecular proton transfer (ESIPT), these mechanisms are not commonly used in the detection of ROSs and, therefore, will not be discussed in this review article. This section outlines three common mechanisms for detecting ROSs using BODIPY. However, there are currently limited probes available for specifically detecting ROSs in mitochondria using the ICT and FRET mechanisms.

Detection of ROSs in Mitochondria
Mitochondria are the primary site of aerobic respiration, which results in the production of various ROSs, such as ·OH, O 2 − , ClO − , H 2 O 2 , 1 O 2 . However, while the detection of 1 O 2 using BODIPY has progressed to the cellular level, it has not yet penetrated deeper into the mitochondria. Therefore, this paper focuses on the detection of the other four types of ROSs.

Superoxide Radical
During aerobic respiration in mitochondria, most of the oxygen reacts with protons to produce water in complex IV, but only about − in mitochondria is of great importance. [71][72] The detection of O 2 − and the removal of excess O 2 − would provide a double benefit. Krumova et al. developed a novel probe, Mito-BODIPY-TOH, by coupling -tocopherol ( -TOH) to the position of BODIPY using a double bond as the reaction site (Figure 4a). [73] The double bond can be oxidized to produce a separate TOH portion and a BODIPY portion. The ability of Mito-BODIPY-TOH to scavenge peroxyl radicals was comparable to that of natural TOH, and its absorption peak was located at 520 nm, while its emission peak was 570 nm (Figure 4b). Furthermore, after coupling with BODIPY, the fluorescence was quenched due to the PET process from the tryptophan portion to BODIPY. When detecting O 2 − , the double bond was cleaved, inhibiting the PET process and restoring fluorescence. In subsequent fibroblast imaging experiments, an emission enhancement of about eight folds was observed after incubating NIH373 cells with the probe in the presence of methyl viologen (paraquat). In contrast, no enhancement was observed in the blank group that served as a control (Figure 4c).
Similarly, Tracy A. Prime et al. also used a double bond as a reaction site and developed a new probe, MitoPrOx (Figure 4d), consisting of a BODIPY fluorophore, a phenyl group, and a TPP + unit. [74] The BODIPY and phenyl group are bridged with a diene bond, and the maximum fluorescence emission of the probe was located at 590 nm. Upon detecting O 2 − , the diene bond was oxidized, and the maximum fluorescence emission of the spectrum can blueshift to 520 nm. MitoPrOx was useful for assessing lipid peroxidation specifically on mitochondrial membranes due to its ability to easily target mitochondria.  [68] Copyright 2020, Royal Society of Chemistry.

Hydroxyl Radical
In the mitochondrial matrix, O 2 − reacts with manganesecontaining SOD to produce H 2 O 2 , which in turn reacts with ferrous ions to form ·OH via the Fenton reaction. ·OH is highly oxidizing and poses a great threat to intracellular macromolecules such as DNA, proteins, and lipids.
Deng et al. constructed an ·OH probe (Rho-Bob) based on the FRET mechanism using rhodamine B as the donor and BODIPY as the acceptor (Figure 5a). [68] In the absence of ·OH, photoirradiation caused rhodamine B to transfer energy to BODIPY in the form of resonance, resulting in the emission of red light. Upon detection of ·OH, ·OH attacked and released BODIPY, and the red fluorescence was diminished to emit yellow-green light. Fluorescence intensity increased with the increase of ·OH concentration (Figure 5b). Rho-Bob can effectively target mitochondria due to the positive ions on this probe (Figure 5c). In vivo imaging of zebrafish showed that the probe emitted rather weak fluorescence when N-acetylcysteamine (NAC) was used to neutralize in vivo ·OH, but emitted a bright red fluorescence when ·OH was present in the absence of NAC (Figure 5d).

Hypochlorite
In mitochondria, the generated H 2 O 2 reacts with chloride ions to form the active ClO − catalyzed by the enzyme heme myeloperoxidase (MPO). [75,76] As a potent oxidizing agent, ClO − is known to inflict tissue damage and leads to the onset of various diseases such as atherosclerosis, arthritis, and cancers.
In contrast to TPP + , pyridinium-substituted BODIPY derivatives can also increase solubility while targeting mitochondria. Ji et al. developed BODIPY-DMPC, a probe that used pyridinium as the targeting group and dimethylthiocarbamate (DMPC) to protect the phenolic hydroxyl group on BODIPY. [77] The fluorescence remained in the quenching state due to the PET mechanism in the absence of ClO − . After reacting with ClO − , DMPC deprotection occurred, and subsequent PET inhibition resulted in a 158fold fluorescence enhancement (Figure 6a). The detection limit was found to be as low as 95 nm. Moreover, the fluorescence intensity of this probe increased with the increase in ClO − concentration (Figure 6b). The presence of pyridinium enabled the probe to target mitochondria and achieved ClO − detection with high organelle-level precision, as revealed by a colocalization experiment using MTR as a commercial mitochondrial-targeting dye (Figure 6c). In addition, ClO − produced endogenously in mitochondria was successfully detected using this probe in zebrafish embryo experiments.
Li et al. developed a novel probe that used pyridinium as the targeting group and phenylboronic acid as the reactive group. [78] This probe also utilized the PET mechanism to achieve fluorescence off-on, realizing a detection limit of 0.6 μm, a response time of less than 5 min, and high selectivity for ClO − . The presence of the pyridinium unit allowed successful application of the probe to detect ClO − in the mitochondria of HepG2 cells. Cheng et al. reported the probe (MitoClO, Figure 6d) using the C═N bond as a reaction site of ClO − . [79] The C═N bond of the oxime group was modified at the position of BODIPY. Isomerization of the C═N bond decayed the fluorescence of the excited state complex. Upon detection of ClO − , the C═N bond was disrupted, and fluorescence was restored. High selectivity for ClO − with a detection limit as low as 0.52 μm was achieved. The incorporation of the targeting group TPP + enabled ClO − detection in the mitochondria of MCF-7 cells. In a recent study by Li et al., a probe (PI-Py, Figure 6d) with a thienyl group was designed and synthesized to detect mitochondrial ClO − . [80] Like BODIPY-DMPC, this probe achieved fluorescence off-on by the PET mechanism and demonstrated a detection limit of 1.7 nm. Furthermore, PI-Py was able to visualize ClO − in exogenous HeLa cells and target mitochondria.
When it comes to the reactive groups for ClO − detection, in addition to the molecular groups mentioned above, reductants such as organic sulfides and selenium-containing compounds have also been used for probes design. Xu et al. reported on the use of the meso position-substituted BODIPY selenide (BSe-Bz, Figure 6d) as a fluorescent probe for detecting ClO − . [81] In the absence of ClO − , the selenide quenched the emission by posing a PET effect on the BODIPY fluorophore. When selenide underwent ClO − initiated oxidation, the PET process was inhibited, and an intense fluorescence was triggered. The probe showed a high selectivity toward ClO − , with a detection limit of 0.8 nm. Finally, the probe was successfully applied to the detection of ClO − in the mitochondria of RAW 264.7 cells.
In contrast to visible light, NIR light offers a higher degree of specificity when imaging target biomolecules and generates lower background fluorescence, resulting in minimal interference. Therefore, it is of paramount interest in the field of biological imaging. Shen et al. reported a two-photon near-infrared fluorescent probe (NCS-BOD-OCH 3 , Figure 6d) for the detection of ClO − . [82] The probe introduced anisaldehyde at positions of BODIPY to extend the emission to the NIR region. Due to the two-photon excited mechanism, the probe can be excited with a wavelength of 800 nm to obtain an emission peak at ≈658 nm. A thiosemicarbazone group that underwent desulfurization upon reaction with ClO − was introduced at the meso position as the sensing group. The desulfurization resulted in the formation of 1, 3, 4-oxadiazole, which has a strong electron-withdrawing ability, causing a significant decrease in fluorescence intensity. In addition, clear yellow-green or red fluorescence can be observed under the electron microscope when the probe detected ClO − , resulting in clear mitochondrial imaging in A357 cells. The probe was demonstrated to be an effective NIR visualizer in imaging experiments of live cells.

Hydrogen Peroxide (H 2 O 2 )
H 2 O 2 is generated by the reaction between O 2 − and SOD. Copper-containing SOD generates H 2 O 2 in the cytoplasm, while manganese-containing SOD generates H 2 O 2 in the mitochondria. As an important signaling molecule, H 2 O 2 plays a critical role in various cellular processes that regulate physiological responses such as cell proliferation, differentiation, and migration. However, excessively high levels of H 2 O 2 in mitochondria can lead to malignant diseases such as Alzheimer's disease, inflammations, and cancers. [83][84][85] A commonly used moiety for detecting H 2 O 2 is the phenylboronic acid pinacol ester, where the boron unit is converted to the corresponding phenolic hydroxyl group upon H 2 O 2 detection. Song et al. reported a NIR probe, Mito-Bor, which coupled a phenylboronic acid pinacol ester as a reaction site with H 2 O 2 and TPP + as a mitochondria-targeting group (Figure 7a). [40] Mito-Bor was highly selective for H 2 O 2 and had a detection limit as low as 0.1 μm, and its fluorescence intensity increased with increasing H 2 O 2 concentration (Figure 7b). In addition, Mito-Bor could detect mitochondrial H 2 O 2 in lung fibroblasts, and was found to be a reliable monitor of elevated H 2 O 2 in mitochondria during pulmonary fibrosis (Figure 7c), which could provide a reliable basis for future pulmonary fibrosis treatment. The blue part is the active part that can react with ROSs, and the red part is the mitochondria-targeting unit. Reproduced with permission. [40] Copyright 2021, American Chemical Society. . c) Mitochondrial colocalization imaging of P-HP-FR-tpp and MTG. The blue part is the active part that can react with ROSs, and the red part is the mitochondria-targeting unit. Reproduced with permission. [86] Copyright 2017, American Chemical Society.
Similarly, Chen et al. reported the probe (P-HP-FR-tpp) for mitochondrial H 2 O 2 detection, which also utilized a phenylboronic acid pinacol ester as the reaction site (Figure 8a). [86] The main part of the probe was composed of BODIPY, TPP + , and phenylboronic acid pinacol ester. To improve water solubility, P-HP-FR-tpp was modified with a carboxyl group at the meso position of BODIPY and two side chains at the position. In addition, the probe introduced a self-immolating linker. Upon detection of H 2 O 2 , the electron-withdrawing ability of the easter group at the meso position was significantly reduced and converted into an unstable intermediate product, which further converted to form BODIPY-COO − , a carboxyl substituent at the meso position of BODIPY emitting intense fluorescence. The fluorescence emission wavelength was 660 nm and increased with time (Figure 8b). In the cell imaging experiments of HeLa cells, P-HP-FR-tpp emitted clearly red fluorescence upon detection of endogenous H 2 O 2 www.advancedsciencenews.com www.advsensorres.com and revealed good colocalization with commercial mitochondrial dye (MTG, Figure 8c). This result demonstrated that this probe can detect endogenous H 2 O 2 in the mitochondria of live cells.

Conclusion
Most ROSs in humans are produced in the mitochondria, and excessive concentrations of these species can trigger diseases related to mitochondrial dysfunction. Therefore, it is crucial to detect ROSs in mitochondria. Among the methods for detecting ROSs, fluorescence imaging technology is a prevalent method due to its noninvasiveness and high sensitivity. BODIPY, a smallmolecule fluorescent dye, is highly favored for its excellent photostability and chemical stability, high molar extinction coefficient, and high photoluminescence quantum yield. This paper provides a comprehensive review of the common motifs for targeting mitochondria, along with the detection mechanisms employed, and summarizes recent advances in the detection of ROSs in mitochondria. Additionally, this paper reviews three common mechanisms for detecting ROSs, namely PET, ICT, and FRET. Most current probes for detecting ROSs in mitochondria employ the PET mechanism, whereas ICT and FRET probes are relatively few. Therefore, introducing ICT and FRET probes for detecting ROSs based on BODIPY may provide inspiration for future mitochondrial targeting. Although significant progress has been made in the detection of ROSs in mitochondria in the past few years, challenges remain in terms of targeting and disease detection.
First, currently, BODIPY based probes are only capable of detecting 1 O 2 at the cellular level and not yet in mitochondria. Nevertheless, intracellular BODIPY probes can still provide valuable reference significance for BODIPY probes targeting mitochondria. Several BODIPY probes have been developed to detect 1 O 2 , including heavy atom-free BODIPY-anthracene dyads developed by Filatov et al., a 1, 3-diphenylisobenzofuran-BODIPY conjugate developed by Kaya et al., and a luminescent platinum complex developed by Geist et al. [87][88][89] Although some organic fluorophores, such as naphthalimide, have been reported to detect mitochondrial 1 O 2 , significant challenges persist in achieving reliable detection. The concentration of 1 O 2 in healthy cells is exceptionally low. Even in diseased cells, although it does increase, it still remains at a very low level, necessitating probes with remarkably high sensitivity. Additionally, since 1 O 2 is the lowest excited state of molecular oxygen, there are no chemical reactions or energy transfers involved in its formation. Consequently, 1 O 2 has an extremely short lifetime, typically lasting only a few tens of nanoseconds in a biological environment. This short half-life restricts its diffusion to a maximum radius of about 20 nm, making it less prone to be captured by probes. [90][91][92][93][94] To address this issue, one potential solution is to couple a mitochondrial targeting motif to an excellent probe that detects 1 O 2 . In the future, we may explore the use of mitochondrial penetrating peptides or nanoparticles to wrap the probe and release the BODIPY probes after targeting the mitochondria. This approach could enable in situ release of the probe molecules, thus reducing the interference of cytoplasm/other organelles and shortening the detection time for ROSs with short lifespans.
Second, since ROSs are also present in some amounts in the cytoplasm, there is a possibility that the probe may react directly with ROSs before reaching the mitochondria, resulting in the failure to exactly detect ROSs concentration in the mitochondria. To address this issue, a pH-activating dual-response BODIPY probe can be considered to improve efficiency by exploiting the difference in mitochondrial and cytoplasmic acidity. When the probe enters the cytoplasm, the pH does not reach the requirement for probe release, so the probe will not react with ROSs and presents an "off" state. By the time the probe enters the mitochondria, the special microenvironment stimulates the probe to switch from "off" to "on," thus allowing the probe to function.
Third, the diagnosis of ROS-related diseases, including cancer, pulmonary fibrosis, atherosclerosis, and diabetes mellitus, is among the notable applications of fluorescent probes. However, the complex physiological structures of the human body pose a significant challenge. NIR probes offer advantages such as high penetration, making them suitable for disease diagnosis. One potential method to develop such probes is by extending the absorption and emission of BODIPY based probes to the NIR region. It is believed that BODIPY probes with NIR properties could significantly contribute to disease detection by enabling the detection of ROSs in the future. Moreover, once introduced into the body, these probes must overcome multiple barriers. Ensuring that they remain intact and accurately targeted during this process is a crucial prerequisite for advancing them toward clinical applications.
BODIPY-based probes have proven to be highly versatile in biomedicine, beyond their use in disease diagnosis. One promising application of BODIPY is in photodynamic therapy, a nontoxic tumor treatment method that has gained attention due to its high efficiency in 1 O 2 production. By combining both 1 O 2 detection and induction, BODIPY probes have demonstrated great potential for cancer theranostics. However, these types of BOD-IPY probes often have complex structures and require timeconsuming synthesis protocols. Therefore, developing BODIPY probes with simpler structures is an important area to explore for future research.
In conclusion, significant advancements have been made in the research of ROSs detection, and this technology is expected to become an invaluable tool for disease monitoring in the future.