Recent advances in nanotechnology mediated mitochondria-targeted imaging

Abstract

Mitochondria play a critical role in cell growth and metabolism. And mitochondrial dysfunction is closely related to various diseases, such as cancers, and neurodegenerative and cardiovascular diseases. Therefore, it is of vital importance to monitor mitochondrial dynamics and function. One of the most widely used methods is to use nanotechnology-mediated mitochondria targeting and imaging. It has gained increasing attention in the past few years because of the flexibility, versatility and effectiveness of nanotechnology. In the past few years, researchers have implemented various types of design and construction of the mitochondrial structure dependent nanoprobes following assorted nanotechnology pathways. This review presents an overview on the recent development of mitochondrial structure dependent target imaging probes and classifies it into two main sections: mitochondrial membrane targeting and mitochondrial microenvironment targeting. Also, the significant impact of previous research as well as the application and perspectives will be demonstrated.

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Nannan Zheng

Dr Nannan Zheng obtained her PhD in Biomedical Engineering from Harbin Institute of Technology in 2021. After graduation, she started her postdoctoral career at Harbin Institute of Technology. Her research interests are mainly focused on the multifunctional nanoparticles for cancer diagnosis and therapy. Dr Zheng's research also focuses on nanoparticles for the treatment of Alzheimer's disease.

Liangcan He

Dr Liangcan He graduated from Tsinghua University and National Center for Nanoscience and Technology in 2014 under the guidance of Prof. Zhiyong Tang and Prof. Yadong Li. After that, he joined Prof. Jennifer N. Cha's group in the University of Colorado, Boulder, as a Research Associate, and later worked with Prof. Jennifer N. Cha and Prof. Christopher N. Bowman as a Senior Research Associate. In June 2018, he joined Dr Shawn Chen's lab in the National Institutes of Health as a Postdoc Research Scientist. In May 2021, he started his independent career at Harbin Institute of Technology. His research interests focus on design and synthesis of organic–inorganic nanomaterials for biomedical applications.

1. Introduction

Known to be the cell's main energy supply organelles, mitochondria are the primary source of intracellular reactive oxygen species and are responsible for modulation of apoptosis.^1,2 Also, mitochondria are closely associated with many diseases involving cellular energy disorders and oxidative damage, such as neurodegeneration, cardiovascular diseases and metabolic diseases.^3–5 To gain a deeper understanding of the diagnosis and treatment of these diseases, mitochondrial movement and dynamic changes have drawn intense research with recent advances in mitochondrial research, bioenergetics and metabolism, reactive oxygen species and cell signal detection. However, since the ultrastructure of mitochondria is typically close to the diffraction limit in optical microscopy, it would be challenging to directly observe submitochondrial architecture or protein distributions using standard light microscopes. As a result, there is a substantial lack of research and study on subcellular organelles. Thanks to the development of mitochondria-targeted imaging techniques and more advanced microscopy technology, changes in the internal microstructure of mitochondria are gradually becoming better understood. Currently, the techniques used in mitochondria-targeted imaging mainly include implementing small molecule fluorescent probes, polypeptide fluorescent probes, and nanoscale fluorescent probes. The fluorescent probes can be modified to exhibit multifunctional properties, such as anti-tumor therapy function, and selective targeting mitochondrial reactive oxygen species and reductive species attachment.^6–8 To be specific, small molecule fluorescent probes, such as commercial MitoTrack ®Green FM and Rhodamine 123, JC-1, are mainly modified by cations to enable them to locate and image mitochondria. Additionally, mitochondria targeted imaging can also be achieved by the introduction of mitochondrial targeting groups, such as tri-phenyl-phosphine (TPP)-Green,⁹ IR-780 iodide,¹⁰ CAI,¹¹etc. As regards polypeptide fluorescent probes, the mitochondrial localization and fluorescence imaging functions can readily be realized by linking the polypeptides with the fluorophore groups.¹² Although various organic fluorescent probes have been used for mitochondrial imaging, the development of fluorescent probes with low toxicity, high selectivity and excellent photostability still face challenges. In recent years, with the rapid development of nanotechnology, nanomaterial-based probes not only offer the advantages of commercial mitochondrial imaging, but also can be designed to respond to the changes in the mitochondrial microenvironment. Mitochondrial imaging based on nanotechnology mainly includes targeting mitochondrial structures (such as the mitochondrial outer membrane, mitochondrial inner membrane, cristae, and mtDNA) and targeting the mitochondrial microenvironment (ATP, pH, and ROS) and signaling molecules (H₂S). Mitochondria-targeted imaging is useful in a wide range of applications, including long-term imaging of living mitochondria, exploration of the changes in the mitochondrial structure, microenvironment investigation and treatment for diseases. Here, this paper will review the recent progress on the application of nanomaterials in mitochondrial imaging and elaborate the advantages of nanomaterial implemented mitochondrial imaging by exploring the structural and microenvironmental alterations in mitochondria. This review aims to understand and propose new directions of nanomaterial engineering for the study of mitochondria-related diseases. It is organized in three parts: the first one reviews and discusses the mitochondrial targeting imaging of mitochondrial membrane; the second part is dedicated to microenvironmental imaging. Last, our conclusions and proposals for new generation of technology will be presented in the final section.

2. Mitochondrial membrane targeting imaging

The structure of mitochondria can be divided into four functional regions: mitochondrial outer membrane (OMM), mitochondrial inner membrane (IMM), mitochondrial intermembrane space (IMS), and mitochondrial matrix (MM).^3,13,14 The design of various small-molecule mitochondrial targeting probes is realized by exploiting the negative potential of the mitochondrial membrane. Consequently, the fluorescent probes that target the mitochondrial outer membrane mainly include actin chromobody targeting,¹⁵ mitochondrial membrane potential^16–18 and monoamine oxidase.^19,20

Due to the oxidation pathway and proton pump operation, mitochondria have a very large membrane potential, reaching up to −180 mV, which is 5–6 times that of the plasma membrane and is quite different from those of other biological membranes.²¹ Therefore, cationic compounds are more easily labeled in mitochondria than in other organelles,²² which is the reason why cationic fluorescent compounds such as triphenylphosphorous (TPP⁺), tetramethylrhodamine methyl ester (TMRM), tetramethylrhodamine ethyl ester (TMRE) and 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolcarbocyanine iodide (JC-1),^23,24 (E)-4-(1H-indol-3-ylvinyl)-N-methylpyridiniumiodide (F16),^25,26 dequalinium (DQA),^27,28 Rhodamine^29,30 and guanidine/biguanidine³¹ have excellent mitochondrial targeting performance. Therefore, grafting mitochondrial targeting molecules such as triphenylphosphine onto the surface of nanoparticles is a simple strategy to obtain mitochondrial targeting nanoprobes.

Lanthanide doped upconversion nanoparticles (UCNPs) can emit high energy UV or visible light under excitation of near-infrared light, which has the potential for biomedical imaging applications.^32,33 However, undecorated UCNPs cannot localize to mitochondria to achieve the function of disease diagnosis and treatment. Recently, Zhao's group synthesized Janus nanostructures by growing porphyrin MOF on Nd³⁺ sensitized upconversion nanoparticles (UCMTs) and the mitochondrial targeting function was achieved by surface modification of triphenylphosphine³⁴ (Fig. 1). In this work, mitochondria-targeted PDT induces the initiation of endogenous apoptotic pathways, resulting in a superior therapeutic effect to that of untargeted therapies.

Fig. 1 (a) Schematic of mitochondria-targeted upconversion MOFs for amplified PDT. (b) Confocal fluorescence images of the 4T1 cells treated with UCMTs or UCMs. The green fluorescence is from the MOFs.³⁴ (c) Schematic illustration of the preparation and therapeutic functions of BP@PDA-Ce6&TPP NSs. (d) Confocal fluorescence images of HeLa cells incubated with BP@PDA-Ce6&TPP NSs and BP@PDA-Ce6 NSs (excited at 633 nm) and then Mito-Tracker Green (excited at 488 nm).³⁸ Copyright 2019, wiley and 2018, Royal Society of Chemistry.

Black phosphorus (BP) is a novel bioinorganic material with biocompatibility, biodegradability and biosafety. It has been used as a photothermal agent for cancer treatment due to its extensive near infrared absorption.^35–37 There is still plenty of potential to improve the therapeutic efficacy of nanomedicine based on BP. In 2018, Dong's group developed an image-guided mitochondrial targeted photothermal/photodynamic nanosystem³⁸ (Fig. 1). Based on dopamine-coated black phosphorus nanosheets, the system covalently linked with Ce6 and TPP to form BP@PDA-Ce6&TPP NSs. TPP modification significantly improved the mitochondrial targeting ability of BP@PDA-Ce6&TPP NSs, and its killing efficiency against tumor cells was significantly improved.

In addition, Zhuang et al. prepared a mitochondrial targeting fluorescent probe AuNC@CS-TPP by combining chitosan-modified Au nanocrystals (AuNC@CS) with TPP through covalent bonding.³⁹ The conjugated compound AuNCs@CS-TPP emits blue fluorescence at 440 nm with a high quantum yield of 8.5%. It is noteworthy that the fluorescence intensity of AuNCs@CS-TPP labeled HeLa cells showed insignificant quenching after irradiation for 8 min, demonstrating its high photostability. Moreover, the studied compound AuNC@CS-TPP can accumulate selectively in the mitochondria of HeLa and HepG2 cells, which suggests its potential for targeted imaging of mitochondria in live cells (Fig. 2). The laser confocal microscope is used in Fig. 2. Combining TPP with traditional nanomaterials, mitochondrial targeting function can be endowed with nanomaterials to achieve mitochondrial imaging and therapeutic effects.

Fig. 2 Fluorescent images of living HeLa cells cultured with AuNCs@CS-TPP (20 μg mL⁻¹) with various incubating times (0–8 min). Excitation wavelength: 405 nm. Reproduced with permission.³⁹ Copyright 2014, Elsevier.

Yu et al. constructed a multifunctional nanoplatform (called UR-HAPT) by combining ucNP-based nano-photosensitizer and mitochondrial targeting strategy with a reasonably designed DNA reporter (Fig. 3).⁴⁰ The platform enables simultaneous monitoring of the subcellular dynamics of human apurinic/apyrimidinic-free endonuclease 1 (APE1) during near-infrared (NIR) light-mediated PDT. APE1 accumulation in mitochondria can be visualized during PDT in vitro and in vivo. Thus, it can be used to screen and evaluate potential enzyme inhibitors to improve the efficacy of PDT.

Fig. 3 Schematic illustration of the design of UR-HAPT for precise imaging of subcellular enzymatic dynamics of APE1 during NIR light-mediated, mitochondria-targeted PDT.⁴⁰ Copyright 2022, Wiley-VCH GmbH.

There have been a wide variety of TPP-based compounds that have proved to be useful for mitochondria-targeted imaging, such as NIR Cy-5-TPP/FF,⁴¹ WSSe/MnO₂-INH-TPP@CM,⁴² C-dots-TPP⁴³ and Gold-TPP.⁴⁴

In addition to TPP, which can be used as a small molecular group targeting the mitochondrial outer membrane, PQC is also a group that can target mitochondria. Tang et al.⁴⁵ designed lipid small molecule hybrid nanoparticles (LPHNPs) for imaging and treatment of in situ glioma models. LPHNPs are prepared by designing a co-assembly of lipids and amphiphilic pheophorbide a-quinolinium conjugate (PQC), a small-molecule-specific mitochondria (Fig. 4). Unlike PQC-NFS, LPHNPs exhibit a spherical nanostructure and ideal particle size, which reduces potential systemic toxicity and enables the administration of nanoparticles by intravenous injection. In addition, compared to traditional liposomes, LPHNPs have a higher drug loading and better stability, and retain the excellent properties of PQC, such as mitochondrial targeting, fluorescence imaging ability and photosensitivity. By integrating the benefits of liposomes and PQC molecules, LPHNPs achieve minimal systemic toxicity, enhanced photodynamic therapy (PDT) efficacy, and the ability to visualize drug biologic distribution and tumor imaging. Mixed nanoparticles showed good efficacy and could significantly prolong the survival time of mice with glioma in situ. Fluorescence imaging showed that LPHNPs accumulated at the tumor site 2 hours after injection and remained there for at least 48 hours. In vitro imaging further confirmed the tumor-targeting ability of LPHNPs. In an in situ glioma model, single-dose LPHNPs combined with laser therapy have a good inhibitory effect on tumor progression. More importantly, they significantly extended the survival time of mice with glioma in situ. The unique co-assembly of lipids and small molecules offers a new potential for the construction of new liposome derived nanoagents and improved cancer therapy.

Fig. 4 Schematic illustration of hybrid nanoparticles (LPHNPs) based on amphiphilic lipids and mitochondria targeting PQC molecules.⁴⁵ Copyright 2022, Elsevier.

In addition, the construction of a positively charged surface environment for nanomaterials is another strategy for designing mitochondrial membrane targeted imaging nanoprobes. In 2017, Wu's group prepared fluorescent carbon quantum dots (APTMS CDs) with the mitochondrial targeted imaging function by solvent heat treatment of glycerol and organosilane molecules (3-aminopropyl)trimethoxysilane (APTMS).⁴⁶ The abundant amino groups on the surface of APTMS CDs lead to a zeta potential of +13.1 mV in water, endowing them with the ability for mitochondrial targeting. Compared with normal cells, cancerous cells have a more negative mitochondrial membrane potential of about −220 mV,^47,48 resulting in a stronger electrostatic interaction between APTMS CDs and mitochondrial membrane, and so a brighter fluorescence can be observed in cancerous cells (Fig. 5). Therefore, APTMS CDs can be used to distinguish normal cells and cancerous cells.

Fig. 5 One-step solvothermal synthesis of APTMS CDs using APTMS and glycerol and applications of APTMS CDs in mitochondrial tracking and normal/cancerous cell differentiation.⁴⁶ Copyright 2017, Royal Society of Chemistry.

3. Mitochondrial microenvironment imaging

3.1 Mitochondrial matrix targeted imaging

Mitochondria matrix is the main structure of mitochondria, and contains numerous proteins/enzymes, nucleic acids, hydrogen sulfide and other biological species for energy supply and signal transduction. These components serve as a medium for mitochondrial imaging.^49,50 In order to prolong the retention time of the fluorescence in mitochondria, Grzybowski and Kandere-Grzybowska et al. designed mixed-charged nanocarriers for selective targeting of mitochondria by otherwise nonselective dyes.⁵¹ In this work, the mixed-charged nanocarriers traverse an endolysosomal tract to selectively deliver non-covalently bound dye molecules into mitochondria and covalently link them to cysteines of mitochondrial proteins via disulfide bonds (Fig. 6). It is also demonstrated that the nanoparticles of interest can deliver low-toxicity anionic fluorophores into mitochondria to observe the dynamic remodeling process of mitochondrial network and even long-term tracking of mitochondria in dividing cells. Cell mitochondria are imaged using confocal laser scanning microscopy (CLSM).

Fig. 6 Mechanism of action of mixed-charge nanocarriers targeting mitochondria.⁵¹ Copyright 2021, American Chemical Society.

3.2 ATP targeting imaging

Adenosine triphosphate (ATP) consists of three main components: adenine, ribose, and three phosphate groups. When hydrolyzed, a large amount of energy is released, providing a direct source of energy for metabolism and activity.^52,53 Mitochondria are the major organelles that produce ATP;⁵⁴ therefore, mitochondrial imaging can be achieved by monitoring the fluctuation of ATP level in mitochondria or the fluctuation of cellular ATP levels. Chu and coworkers developed a fluorescent ATP probe out of titanium carbide (TC) nanosheets that had been modified with an ROX (X-rhodamine)-labeled ATP aptamer (TC/Apt).⁵⁵ TC in the TC/Apt shows superior quenching efficiency for ROX (e.g., ∼97%). In the presence of ATP, the ROX-tagged aptamer is released from the TC surface, resulting in the recovery of ROX fluorescence under 545 nm excitation (Fig. 7). Through this procedure, the researchers successfully obtained the fluorescence detection and imaging of ATP in living cells, body fluids (e.g., urine and serum), and a mouse tumor model.

Fig. 7 Scheme of TC/Apt fluorescent probes. (a) Schematic diagram of fabrication of TC/Apt-based fluorescent probes. (b) TC/Apt-based fluorescent probes for the accurate detection of ATP based on a signal off/on switch mechanism. (c) TC/Apt-based fluorescent probes for in vivo imaging of ATP in tumors of a mouse model. (d) TC/Apt-based fluorescent probes for imaging analysis of intracellular ATP in live cells. (e) TC/Apt-based fluorescent probes for quantitative detection of ATP in real samples, such as mouse and human serum. Reproduced with permission.⁵⁵ Copyright 2021, Biomedcentral.

Deng et al. reported the imaging of subcellular mitochondria and mitochondrial ATP in living cells⁵⁶ targeted by nano-ZIF-90 that self-assembled from Zn²⁺ and imidazole-2-carboxyaldehyde (2-ICA). After ATP triggering the breakdown of ZIF-90, the encapsulation of fluorescent Rhodamine B (RhB) into ZIF-90 inhibits the emission of RhB. The release of RhB for ATP induction is caused by a competitive coordination between ATP and the metal nodes of ZIF-90 (Fig. 8). The electrostatic interaction between the positively charged ZIF-90 and the negatively charged mitochondria inner membrane facilitates the accumulation of RhB/ZIF-90 within mitochondria. This method allows for imaging mitochondrial ATP in living cells and studying fluctuations in ATP levels during glycolysis and apoptosis.

Fig. 8 Schematic representation of modulating the host–guest chemistry of nanoscale RhB/ZIF-90 for fluorescent ATP sensing and mitochondrial ATP imaging. Reproduced with permission.⁵⁶ Copyright 2017, American Chemical Society.

In addition, graphene quantum dots (GQDs), as a new class of carbon-based quantum dots, have unique properties such as stable photoluminescence, low toxicity and good biocompatibility. It can effectively avoid the limitation of poor luminescence stability of organic probes and has a great potential for mitochondrial targeted imaging. Liu et al. synthesized single-layered graphene quantum dots (s-GQDs) with yellow fluorescence for ATP imaging in living cells.⁵⁷ Macrocyclic aromatic conjugated structure and positively charged sites in s-GQDS can achieve specific recognition of ATP/GTP and good mitochondrial targeting ability. The results showed that s-GQDS successfully monitored the ATP fluctuation in HeLa cells after Ca²⁺ and sodium azide treatment, respectively. This study offers an effective solution to monitor mitochondrial ATP fluctuations caused by ATP activation and inhibition in active cells. In other words, s-GQDs provide a new platform for studying cell metabolism diseases and mitochondrial dysfunction conditions.

3.3 H₂O₂ targeting imaging

As an important member of intracellular active species, hydrogen peroxide is one of the key by-products of energy metabolism. The physiological levels of hydrogen peroxide act as a molecular messenger and provide essential information for the regulation of intracellular processes, such as immune cell activation and vascular remodeling in mammals. There has been a link established between high levels of hydrogen peroxide and major diseases such as malignant tumors, neurodegenerative Parkinson's disease and Alzheimer's syndrome. Therefore, it is of great significance to detect the concentration levels of hydrogen peroxide in mitochondria for the prevention, diagnosis and pathological studies of certain diseases. The ratiometric fluorescence technique based on the fluorescence resonance energy transfer (FRET) mechanism is highly accurate and shows significantly low interference, thus enabling real-time detection and imaging of hydrogen peroxide in mitochondria. Wu and coworkers designed a multifunctional fluorescent nanoprobe for detecting mitochondrial H₂O₂.⁵⁸ A mitochondria-targeting ligand (TPP) and an H₂O₂ recognition element (PFl) are covalently linked onto carbon dots to construct the nanoprobe (CDs). Mito-CD-PF1 is non-cytotoxic and easily loaded into cells for ratiometric detection of mitochondrial H₂O₂ variations in the presence of exogenous or endogenous H₂O₂ (Fig. 9). This work offers some important insights into the design and construction of fluorescence detection and imaging system at the subcellular level. Another example of FRET based imaging was presented by Niu and Qiao.⁵⁹ In their work, the ratiometric detection of mitochondrial H₂O₂ in live cells was successfully realized by the introduction of a self-assembled polymeric micelle that carried tetraphenylethene (TPE), fluorescent boronate and triphenylphosphonium as the donor, a H₂O₂-responsive acceptor, and a mitochondria-targeted moiety, respectively. To further extend the study, one can incorporate multi-functional molecules into the nanoprobes to enable them to be accessible for targeting, sensing, and energy transfer.

Fig. 9 Confocal fluorescence images of Mito-CD-PF1-stained Raw 264.7 cells upon stimulation by PMA with varied concentrations. Columns from left to right: control (cells with no PMA), cells treated with 1 μg mL⁻¹ and 2 μg mL⁻¹ PMA. Rows from top to bottom: brightfield channel, blue channel, green channel, overlay of blue and green channel, and ratio image, respectively. Concentration of Mito-CD-PF1 in culture media: 0.15 mg mL⁻¹. Images were acquired under 405 nm excitation and fluorescent emission windows: blue = 425–475 nm, green = 500–550 nm. Scale bar: 15 μm. Reproduced with permission.⁵⁸ Copyright 2013, Wiley-VCH GmbH.

3.4 H₂S targeting imaging

Hydrogen sulfide (H₂S) is a multipurpose regulator in cells and is involved in a variety of diseases caused by mitochondrial dysfunction.^60,61 As the major metabolic locus of H₂S in organisms,⁶² the concentration change of H₂S indirectly reflects the function of mitochondria. Additionally, the active chemical properties of H₂S can also be used as a medium for mitochondrial imaging and reflect the physiological and pathological states of mitochondria. Therefore, various imaging probes have been developed in order to evaluate the function of mitochondria through monitoring the level of H₂S in mitochondria.^63–65 Currently, the organic molecule probes used for mitochondrial H₂S imaging are not suitable for long-term imaging in vivo for multiple reasons. First, the organic molecule probes cannot be effectively penetrated by excitation irradiation. Second, they exhibit short blood circulation time. Last, they easily bind with proteins before even reaching target cells. To address these problems, there have been a growing number of reports that apply upconversion nanoparticles (UCNPs) in the field of bioimaging, owing to their unique properties of negligible photobleaching, deep penetration depth, and high resistance to autofluorescence interference.⁶⁶ In 2018, Huang et al. developed a lysosome-assisted mitochondrial targeting probe based on triphenylamine-merocyanine (TPAMC) modified UCNPs (TPAMC-UCNPs@PHIS-PEG) with an acid-activated targeting strategy for ratiometric detection and imaging of H₂S in mitochondria.⁶⁷ In the imaging system, the absorption spectra of TPAMC matched well with the green upconversion luminescence (UCL), resulting in a strong luminescence resonance energy transfer (LRET) from UCNPs to TPAMC. Thus, only a weak UCL signal in the green channel was observed for TPAMC-UCNPs@PHIS-PEG nanoparticles under 980 nm excitation. In mitochondria, TPAMC on the surface of nanoparticles reacts with H₂S, and its adsorption decreases sharply, leading to the recovery of green UCL. In order to improve the stability of the nanoprobe in blood circulation, a poly(ethylene glycol) (PEG) shell composed of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy-(poly(ethylene glycol))-2000] (mDSPE-PEG) and poly(L-histidine)-b-PEG (PHIS-PEG) were further coated on the surface of TPAMC-UCNPs. PEG shells reduce the possibility that TPAMC-UCNPs will adhere to biomolecules and endow it with acid-activated properties (Fig. 10). In vitro and in vivo experiments indicated that TPAMC-UCNPs@PHIS-PEG nanoprobe was able to be used not only for ratiometric UCL imaging of mitochondrial H₂S generation in living cells, but also for monitoring mitochondrial H₂S levels in colon cancer mouse models.

Fig. 10 Design of TPAMC-UCNPs@PEG, and the illustration of lysosome-assisted mitochondrial targeting process and UCL monitoring of mitochondrial H₂S in cells. Reproduced with permission.⁶⁷ Copyright 2018, American Chemical Society.

3.5 Other mitochondrial microenvironment targeting imaging

In the above sections, we have reviewed the development of study in mitochondrial microenvironments by targeting matrix, ATP, H₂O₂, and H₂S. In this section, we will focus on nanoprobes that target other microenvironmental factors, such as pH and peroxynitrite. Xu and Zhou prepared a nanoprobe by covalently linking a mitochondria-targeted ligand (triphenylphosphonium, TPP) and a pH recognition fluorescence indicator (rhodamine, RhB) onto the surface of MoS₂ quantum dots (QDs). MoS₂ QDs serve as a fluorescent reference for the ratiometric signal as well as a nanocarrier for the targeted ligand and pH fluorescent indicator in this multifunctional fluorescent nanoprobe. The pH-sensitive fluorescence of the RhB-based group was compared to the pH-resistant fluorescence of MoS₂ QDs for ratiometric pH detection. After absorption in live cells, the nanoprobe was able to label mitochondria with great precision, allowing for imaging and pH monitoring.⁶⁸ Peroxynitrite (ONOO⁻) is a group of biological oxidants that play a role in both physiological and pathological processes. It has proved difficult to detect ONOO⁻ in biological systems due to its exceptionally short half-life and low steady-state concentration. Zhao and Yang prepared a ratiometric fluorescent nanoprobe for ONOO⁻ by covalently connecting graphene quantum dots (GQDs) with cyanine 5.5 (Cy5.5). This nanoprobe (GQD-Cy5.5) has two main fluorescence emission bands that peak at 520 and 694 nm, respectively. In the presence of ONOO⁻, the intensity of 520 nm centered emission increased whereas that of 694 nm centered emission decreased. The fluorescence intensity ratio of the two emission bands demonstrated a strong linear relationship with ONOO⁻ concentration.⁶⁹ These unique luminescent properties make the studied nanoprobe, a great tool to image endogenous ONOO⁻ in cell mitochondria using ratiometric fluorescence imaging.

3.6 Mitochondrial micro RNA targeting imaging

Mitochondrial DNA (mtDNA) has 16.5 kb base pairs and encodes 13 complexes, and it plays an important role in regulation of immune responses.^70–72 There is only one type of mtDNA in an organism, and mtDNA possesses a unique structural characteristic, an extremely small double-stranded circular structure. However, most of the mtDNA probes are small molecules, which were proved to have low photostability and broad emission bands. And there have been very few reported imaging probes and techniques constructed by nanotechnology. Therefore, it's highly necessary to develop more ideal imaging probes, especially with the aid of mitochondrial targeting microRNA (mitomiR) that can affect mitochondrial morphology, metabolism, redox homeostasis, autophagy and apoptosis by regulating the mitochondrial gene expression. The abnormal expression of mitomiR is closely related to metabolic diseases, cardiovascular diseases, neurodegenerative diseases and tumors. Studies have found that mitomiR is involved in chemotherapy tolerance, cancer metastasis, and recurrence. Therefore, the precise in situ imaging of mitomiR is of great significance for exploring the physiological and pathological functions of mitomiR and disease diagnosis. Li and coworkers presented a study that makes an original contribution to mitochondrial microRNA imaging.⁷³ Nanoreporters based on the combination of PD (photocleavable linker into a designed DNA sensor probe) and UCNPs were synthesized using the mitochondria targeting strategy. Upconverted NaGdF₄:Yb³⁺,Tm³⁺ core–shell nanocomposites efficiently convert NIR light to ultraviolet (UV) light. During the targeting procedure, the DNA nanoreporters could be localized to mitochondria in live cells with blocked sensing function. Then, the UV light-activatable PD sensing function could be triggered noninvasively by the UV emission emitted by UCNPs upon NIR excitation at a certain time and location. To be specific, the UV emission triggers strand displacement reactions (SDRs), thus ensuring accurate mitomiR imaging at a specific location in suborganelles.

4. Conclusion and perspectives

Mitochondria are the main energy supply organelles in cells, the main source of intracellular reactive oxygen species, and one of the regulatory hubs of cell apoptosis. It has now been demonstrated that the dynamic changes in the mitochondrial structure and function, as well as the microenvironment in mitochondria can cause cellular energy disturbances and oxidative damage, which may result in neurodegenerative diseases, metabolic problems, cardiovascular diseases and others. Mitochondrial imaging is a useful tool to track the changes in the mitochondrial internal structure and monitor the performance of mitochondria. Used in one of the three main techniques that are used for mitochondria targeted imaging, the small molecule fluorescent probes are easy to use, water-soluble, and can be passively transported across the cell membrane and directly aggregated on active mitochondria. However, one disadvantage regarding this technique is that the performance of small molecule fluorescent probes strongly depends on mitochondrial membrane potential. The probes will fall off from mitochondria if the mitochondrial membrane potential is lost. This finding significantly limits their use and application in tracking the environmental changes in mitochondria. In contrast, nanomaterials exhibit unique properties of robust photobleaching resistance and great photostability. More importantly, they enable us to detect the changes in mitochondrial microenvironment, making them a promising solution for mitochondrial imaging.

It is also noted that more advanced microscopy techniques make a crucial impact on the development of the mitochondria imaging. The cristae of mitochondria can only be observed by simultaneously applying novel photostable mitochondrial probes and state-of-the-art microscopic imaging techniques such as Hessian SIM, STED superresolution microscopy and algorithm-assisted super-resolution imaging. Despite the fact that there has been a rapid development of mitochondria targeted imaging by using fluorescent nanoprobes, there are several limitations that need further research and study. First, nanoprobes usually require complicated sample preparation processes. Second, the broad and visible only luminescence spectra result in strong background signal and weak signal-to-noise ratio. Therefore, it is necessary to develop new probes with a narrow luminescence interval, strong photostability and good biocompatibility, which would be of far-reaching significance to the understanding of mitochondria related diseases and the development of mitochondria targeting drugs.

Author contributions

All authors (N. Z., Q. W., S. Z., C. M., L. H., and S. L.) contributed to writing, reviewing and editing of this work and consented to the final version. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the National Science Fund for Distinguished Young Scholars (No. 51825202), the National Natural Science Foundation of China (U20A20339) and the Fundamental Research Funds for the Central Universities (AUGA5710052421).

Conflicts of interest

The authors declare no conflict of interest.

References

1. D. J. Pagliarini and J. Rutter, Genes Dev., 2013, 27, 2615–2627.
2. J. B. Spinelli and M. C. Haigis, Nat. Cell Biol., 2018, 20, 745–754.
3. J. Nunnari and A. Suomalainen, Cell, 2012, 148, 1145–1159.
4. S. B. Vafai and V. K. Mootha, Nature, 2012, 491, 374–383.
5. H. Wang, B. Fang, B. Peng, L. Wang, Y. Xue, H. Bai, S. Lu, N. H. Voelcker, L. Li, L. Fu and W. Huang, Front. Chem., 2021, 9, 683220.
6. Y. Chu, J. Park, E. Kim and S. Lee, Materials, 2021, 14, 4180.
7. J. Yang, Y. Guo, M. Pistolozzi and J. Yan, Dyes Pigm., 2021, 193, 109466.
8. C. Ma, F. Xia and S. O. Kelley, Bioconjugate Chem., 2020, 31, 2650–2667.
9. H. Yuan, H. Cho, H. H. Chen, M. Panagia, D. E. Sosnovik and L. Josephson, Chem. Commun., 2013, 49, 10361–10363.
10. C. Zhang, T. Liu, Y. Su, S. Luo, Y. Zhu, X. Tan, S. Fan, L. Zhang, Y. Zhou, T. Cheng and C. Shi, Biomaterials, 2010, 31, 6612–6617.
11. F. Miao, W. Zhang, Y. Sun, R. Zhang, Y. Liu, F. Guo, G. Song, M. Tian and X. Yu, Biosens. Bioelectron., 2014, 55, 423–429.
12. A. Martin, A. Byrne, C. S. Burke, R. J. Förster and T. E. Keyes, J. Am. Chem. Soc., 2014, 136, 15300–15309.
13. V. Jayashankar and S. M. Rafelski, Curr. Opin. Cell Biol., 2014, 26, 34–40.
14. T. G. Frey and C. A. Mannella, Trends Biochem. Sci., 2000, 25, 319–324.
15. C. R. Schiavon, T. Zhang, B. Zhao, A. S. Moore, P. Wales, L. R. Andrade, M. Wu, T. C. Sung, Y. Dayn, J. W. Feng, O. A. Quintero, G. S. Shadel, R. Grosse and U. Manor, Nat. Methods, 2020, 17, 917–921.
16. B. Lin, Y. Liu, X. Zhang, L. Fan, Y. Shu and J. Wang, ACS Sens., 2021, 6, 4009–4018.
17. L. Zhang, J. L. Wang, X. X. Ba, S. Y. Hua, P. Jiang, F. L. Jiang and Y. Liu, ACS Appl. Mater. Interfaces, 2021, 13, 7945–7954.
18. D. Ma, Q. Zong, Y. Du, F. Yu, X. Xiao, R. Sun, Y. Guo, X. Wei and Y. Yuan, Acta Biomater., 2021, 135, 628–637.
19. J. Wu, C. Han, X. Cao, Z. Lv, C. Wang, X. Huo, L. Feng, B. Zhang, X. Tian and X. Ma, Anal. Chim. Acta, 2022, 1199, 339573.
20. J. Huang, D. Hong, W. Lang, J. Liu, J. Dong, C. Yuan, J. Luo, J. Ge and Q. Zhu, Analyst, 2019, 144, 3703–3709.
21. A. T. Hoye, J. E. Davoren, P. Wipf, M. P. Fink and V. E. Kagan, Acc. Chem. Res., 2008, 41(1), 87–97.
22. G. Zhang, Y. Sun, X. He, W. Zhang, M. Tian, R. Feng, R. Zhang, X. Li, L. Guo, X. Yu and S. Zhang, Anal. Chem., 2015, 87(24), 12088–12095.
23. S. Jakobs, Biochim. Biophys. Acta, Mol. Cell Res., 2006, 1763(5), 561–575.
24. B. A. D. Neto, J. R. Corrêa and R. G. Silva, RSC Adv., 2013, 3(16), 5291–5301.
25. A. Rathinavelu, K. Alhazzani, S. Dhandayuthapani and T. Kanagasabai, Tumor Biol., 2017, 39, 1010428317726841.
26. M. V. Dubinin, A. A. Semenova, A. I. Ilzorkina, N. V. Penkov, D. A. Nedopekina, V. A. Sharapov, E. I. Khoroshavina, E. V. Davletshin, N. V. Belosludtseva, A. Y. Spivak and K. N. Belosludtsev, Free Radical Biol. Med., 2021, 168, 55–69.
27. P. Sancho, E. Galeano, E. Nieto, M. D. Delgado and A. I. Garcia-Perez, Leuk. Res., 2007, 31, 969–978.
28. Y. F. Song, D. Z. Liu, Y. Cheng, M. Liu, W. L. Ye, B. L. Zhang, X. Y. Liu and S. Y. Zhou, Sci. Rep., 2015, 5, 16125.
29. L. V. Johnson, M. L. Walsh and L. B. Chen, Proc. Natl. Acad. Sci. U. S. A., 1980, 77, 990–994.
30. R. L. Pan, W. Q. Hu, J. Pan, L. Huang, C. C. Luan and H. M. Shen, Neural Regener. Res., 2020, 15, 1086–1093.
31. S. A. Dyshlovoy, E. K. Kudryashova, M. Kaune, T. N. Makarieva, L. K. Shubina, T. Busenbender, V. A. Denisenko, R. S. Popov, J. Hauschild, S. N. Fedorov, C. Bokemeyer, M. Graefen, V. A. Stonik and G. von Amsberg, Sci. Rep., 2020, 10, 9764.
32. F. Auzel, Chem. Rev., 2004, 104(1), 139–174.
33. M. Haase and H. Schäfer, Angew. Chem., Int. Ed., 2011, 50(26), 5808–5829.
34. C. Liu, B. Liu, J. Zhao, Z. Di, D. Chen, Z. Gu, L. Li and Y. Zhao, Angew. Chem., Int. Ed., 2020, 59(7), 2634–2638.
35. X. Qian, Z. Gu and Y. Chen, Mater. Horiz., 2017, 4(5), 800–816.
36. Z. Sun, H. Xie, S. Tang, X.-F. Yu, Z. Guo, J. Shao, H. Zhang, H. Huang, H. Wang and P. K. Chu, Angew. Chem., Int. Ed., 2015, 54(39), 11526–11530.
37. J. Shao, H. Xie, H. Huang, Z. Li, Z. Sun, Y. Xu, Q. Xiao, X.-F. Yu, Y. Zhao, H. Zhang, H. Wang and P. K. Chu, Nat. Commun., 2016, 7(1), 12967.
38. X. Yang, D. Wang, J. Zhu, L. Xue, C. Ou, W. Wang, M. Lu, X. Song and X. Dong, Chem. Sci., 2019, 10(13), 3779–3785.
39. Q. Zhuang, H. Jia, L. Du, Y. Li, Z. Chen, S. Huang and Y. Liu, Biosens. Bioelectron., 2014, 55, 76–82.
40. F. Yu, Y. Shao and X. Chai, et al. , Angew. Chem., Int. Ed., 2022, 61, e202203238.
41. P. C. Saha, T. Bera, T. Chatterjee, J. Samanta, A. Sengupta, M. Bhattacharyya and S. Guha, Bioconjugate Chem., 2021, 32, 833–841.
42. Y. Cheng, F. Yang, K. Zhang, Y. Zhang, Y. Cao, C. Liu, H. Lu, H. Dong and X. Zhang, Adv. Funct. Mater., 2019, 29, 1903850.
43. X. Wu, S. Sun, Y. Wang, J. Zhu, K. Jiang, Y. Leng, Q. Shu and H. Lin, Biosens. Bioelectron., 2017, 90, 501–507.
44. O. Oladimeji, J. Akinyelu, A. Daniels and M. Singh, Int. J. Mol. Sci., 2021, 22, 5072.
45. B. Mta, L. Kai and B. Mr, et al., A mitochondria-targeting lipid-small molecule hybrid nanoparticle for imaging and therapy in an orthotopic glioma model, Acta Pharm. Sin. B, 2022, 12(6), 2672–2682.
46. G. Gao, Y.-W. Jiang, J. Yang and F.-G. Wu, Nanoscale, 2017, 9(46), 18368–18378.
47. C. Gui, E. Zhao, R. T. K. Kwok, A. C. S. Leung, J. W. Y. Lam, M. Jiang, H. Deng, Y. Cai, W. Zhang, H. Su and B. Z. Tang, Chem. Sci., 2017, 8(3), 1822–1830.
48. Q. Hu, M. Gao, G. Feng and B. Liu, Angew. Chem., Int. Ed., 2014, 53(51), 14225–14229.
49. M. H. Chen, F. X. Wang, J. J. Cao, C. P. Tan, L. N. Ji and Z. W. Mao, ACS Appl. Mater. Interfaces, 2017, 9, 13304–13314.
50. A. M. Aleardi, G. Benard, O. Augereau, M. Malgat, J. C. Talbot, J. P. Mazat, T. Letellier, J. Dachary-Prigent, G. C. Solaini and R. Rossignol, J. Bioenerg. Biomembr., 2005, 37, 207–225.
51. D. V. Kolygina, M. Siek, M. Borkowska, G. Ahumada, P. Barski, D. Witt, A. Y. Jee, H. Miao, J. C. Ahumada, S. Granick, K. Kandere-Grzybowska and B. A. Grzybowski, ACS Nano, 2021, 15, 11470–11490.
52. X. Li, X. Guo, L. Cao, Z. Xun, S. Wang, S. Li, Y. Li and G. Yang, Angew. Chem., Int. Ed., 2014, 53, 7809–7813.
53. J. L. Tang, C. Y. Li, Y. F. Li and C. X. Zou, Chem. Commun., 2014, 50, 15411–15414.
54. K. Y. Tan, C. Y. Li, Y. F. Li, J. Fei, B. Yang, Y. J. Fu and F. Li, Anal. Chem., 2017, 89, 1749–1756.
55. B. Chu, A. Wang, L. Cheng, R. Chen, H. Shi, B. Song, F. Dong, H. Wang and Y. He, J. Nanobiotechnol., 2021, 19, 187.
56. J. Deng, K. Wang, M. Wang, P. Yu and L. Mao, J. Am. Chem. Soc., 2017, 139, 5877–5882.
57. J. H. Liu, R. S. Li, B. Yuan, J. Wang, Y. F. Li and C. Z. Huang, Nanoscale, 2018, 10, 17402–17408.
58. F. Du, Y. Min, F. Zeng, C. Yu and S. Wu, Small, 2014, 10, 964–972.
59. J. Qiao, Z. Liu, Y. Tian, M. Wu and Z. Niu, Chem. Commun., 2015, 51, 3641–3644.
60. C. Szabo, Nat. Rev. Drug Discovery, 2007, 6, 917–935.
61. D. J. Lefer, Proc. Natl. Acad. Sci. U. S. A., 2007, 104, 17907–17908.
62. W. Guo, J. T. Kan, Z. Y. Cheng, J. F. Chen, Y. Q. Shen, J. Xu, D. Wu and Y. Z. Zhu, Oxid. Med. Cell. Longevity, 2012, 2012, 878052.
63. S. K. Bae, C. H. Heo, D. J. Choi, D. Sen, E.-H. Joe, B. R. Cho and H. M. Kim, J. Am. Chem. Soc., 2013, 135, 9915–9923.
64. X. Wang, J. Sun, W. Zhang, X. Ma, J. Lv and B. Tang, Chem. Sci., 2013, 4, 2551–2556.
65. Y. Chen, C. Zhu, Z. Yang, J. Chen, Y. He, Y. Jiao, W. He, L. Qiu, J. Cen and Z. Guo, Angew. Chem., Int. Ed., 2013, 52, 1688–1691.
66. S. Wen, J. Zhou, K. Zheng, A. Bednarkiewicz, X. Liu and D. Jin, Nat. Commun., 2018, 9, 2415.
67. X. Li, H. Zhao, Y. Ji, C. Yin, J. Li, Z. Yang, Y. Tang, Q. Zhang, Q. Fan and W. Huang, ACS Appl. Mater. Interfaces, 2018, 10, 39544–39556.
68. S. Xu, G. Zheng and K. Zhou, Anal. Biochem., 2022, 640, 114545.
69. K. Yang, L. Hou, Z. Li, T. Lin, J. Tian and S. Zhao, Talanta, 2021, 231, 122421.
70. K. Ishikawa, K. Takenaga, M. Akimoto, N. Koshikawa, A. Yamaguchi, H. Imanishi, K. Nakada, Y. Honma and J. Hayashi, Science, 2008, 320, 661–664.
71. K. McArthur, L. W. Whitehead, J. M. Heddleston, L. Li, B. S. Padman, V. Oorschot, N. D. Geoghegan, S. Chappaz, S. Davidson, H. San Chin, R. M. Lane, M. Dramicanin, T. L. Saunders, C. Sugiana, R. Lessene, L. D. Osellame, T. L. Chew, G. Dewson, M. Lazarou, G. Ramm, G. Lessene, M. T. Ryan, K. L. Rogers, M. F. van Delft and B. T. Kile, Science, 2018, 359, 883–895.
72. K. Gao, M. Cheng, X. Zuo, J. Lin, K. Hoogewijs, M. P. Murphy, X. D. Fu and X. Zhang, Cell Res., 2021, 31, 219–228.
73. J. Zhao, Z. Li, Y. Shao, W. Hu and L. Li, Angew. Chem., Int. Ed., 2021, 60, 17937–17941.

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