Mitochondrial Targeting Research Articles

Yellow Emissive Carbon Dots - A Robust Nanoprobe for Highly Sensitive Quantification of Jaundice Biomarker and Mitochondria Targeting in Cancer Cells.

The abnormally high level of bilirubin (BR) in biofluids (human serum and urine) indicates a high probability of jaundice and liver dysfunction. However, quantification of BR as the Jaundice biomarker is difficult due to the interference of various biomolecules in serum and urine. To address this issue, we developed a fluorescence-based detection strategy, for which yellow emissive carbon dots (YCDs) were produced from a one-step solvothermal process using phloroglucinol and thionin acetate as chemical precursors. The as-fabricated YCDs exhibited a strong fluorescence peak at the wavelength of 542 nm upon excitation at 390 nm. We used YCDs for detecting BR through the fluorescence turn-off mechanism, unveiling the excellent sensitivity in the linear range of 0.5-12.5 μM with a limit of detection (LOD) of 9.62 nM, which was far below the clinically relevant range. The analytical nanoprobe also offered excellent detection specificity for quantifying BR in real samples. Moreover, the biocompatible fluorescent nanoprobe was successfully employed to target mitochondria in live cancer cells. A colocalization study confirmed that YCDs possessed the ability to target mitochondria and overlapped completely with MitoTracker Red. The developed nanoprobe of YCDs turned out to be straightforward in their synthesis, noninvasive, and can be utilized for biomedical sensors to diagnose the onset of jaundice as well as for mitochondria targeting.

Investigating Therapies Targeting Druggable Targets of Mitochondria: Exploring the Efficacy for the Treatment of Cancer

AbstractCancer is a growing problem that has not yet been fully harnessed. Frequent mutations make this a highly variable and challenging pathology. Interestingly, mitochondria have currently emerged as a new target for cancer therapy. A group of agents with anticancer activity, termed mitocans that encourage apoptosis through mitochondrial disruption is currently important in research. From conventional glycolytic inhibitors to recent radiolabeled compounds, mitochondrial‐targeted therapies have marked their presence in cancer. Several TCA cycle and OXPHOS inhibitors are being established for their possible anticancer properties. There are so many distinctive differences in mitochondrial structure and function between normal and cancer cells that offer the potential for the clinical use of mitochondria as targets for novel and site‐specific anticancer agents. Mitochondrial targeting can be made possible if the bioactive molecule is specifically and selectively delivered to the mitochondria of the correct cell type using cell‐specific ligands and mitochondriotropic molecules.

Uncovering crosstalk between AMPK and the ATF4-Integrated Stress Response

The reprograming of cellular metabolism in cancer cells is directly inter-related to their capacity for uncontrolled growth. As such, targeting of mitochondrial function has been explored as a therapeutic strategy in cancer. However, inhibition of mitochondria inevitably leads to activation of cellular stress pathways including the integrated stress response (ISR). The ISR includes as a key feature, increased activity of activating transcription factor 4 (ATF4), which goes on to up-regulate a range of gene targets to restore cellular homeostasis. Inhibition of mitochondria also can impair bioenergetic and biosynthetic capacity, which might activate AMP-activated protein kinase (AMPK), a cellular energy sensor that also orchestrates metabolic homeostasis. While the ISR and AMPK both critically regulate metabolism in cancer cells, the link between these mechanisms remains unclear. To address this gap, we tested effects of various energetic stress conditions in MEF cell systems deficient in AMPK activity. To comprehensively impair metabolic networks, we used nutrient starvation treatments in combination with silencing of the protein OPA1 which maintains organisational integrity of the mitochondrial cristae. Our results suggest that AMPK may be involved in suppressing the ISR upon mitochondrial disruption. To further explore, we took the alternative approach to test a range of mitochondrial targeting drugs in wildtype or AMPK-deficient MEF. To gain insight into roles of AMPK in the ISR and related adaptive gene responses, we analysed resulting transcriptomes. These results further revealed a regulatory link between AMPK and the ATF4-ISR, showing that AMPK loss hypersensitizes the cell to energetic stress. Our work uncovering the crosstalk between AMPK and the ATF4-ISR has potential to reveal novel interactions that could suggest metabolic liabilities that may be relevant for targeting in cancer cells.

Small molecules targeting mitochondria as an innovative approach to cancer therapy

Cellular death evasion is a defining characteristic of human malignancies and a significant contributor to therapeutic inefficacy. As a result of oncogenic inhibition of cell death mechanisms, established therapeutic regimens seems to be ineffective. Mitochondria serve as the cellular powerhouses, but they also function as repositories of self-destructive weaponry. Changes in the structure and activities of mitochondria have been consistently documented in cancer cells. In recent years, there has been an increasing focus on using mitochondria as a targeted approach for treating cancer. Considerable attention has been devoted to the development of delivery systems that selectively aim to deliver small molecules called “mitocans” to mitochondria, with the ultimate goal of modulating the physiology of cancer cells. This review summarizes the rationale and mechanism of mitochondrial targeting with small molecules in the treatment of cancer, and their impact on the mitochondria. This paper provides a concise overview of the reasoning and mechanism behind directing treatment towards mitochondria in cancer therapy, with a particular focus on targeting using small molecules. This review also examines diverse small molecule types within each category as potential therapeutic agents for cancer.

Mitochondria-targeted fluorescent probes based on coumarin-hemicyanine for viscosity changes and their applications in cells and mice

As an important parameter of the cellular microenvironment, the changes in mitochondrial viscosity are closely related to various life activities. Therefore, the development of fluorescent probes for test the changes of mitochondrial viscosity has great significance. In this study, we developed two fluorescent probes for the detection of the mitochondrial viscosity changes. The probes exhibited different fluorescence intensities at different viscosity based on the twisted intramolecular charge transfer process. The characteristics of high anti-interference performance, wide pH applicability, low cytotoxicity and excellent mitochondrial targeting performance made the probes successfully used to distinguish normal cells from cancer cells, achieving visualization of viscosity changes. Furthermore, probes P1 and P2 can also be used as early diagnosis of tumors in mice and reveal the pathology of tumor development. The probes could be serve as a promising viscosity detection tool for discriminating normal cells and cancer cells in biology-related research.

Near-Infrared Fluorescent Probe Reveals Elevated Mitochondrial Viscosity during Acute Alcoholic Liver Injury.

Acute alcoholic liver injury (AALI) has become an important cause of liver disease worldwide, and there is an urgent need to develop noninvasive and sensitive methods to detect and evaluate AALI. We report herein three novel but readily available mitochondrial targeting fluorescence probes (ICR, ICJ, and ICQ) for AALI detection. These probes contain different electron-donating groups, among which ICQ exhibits NIR fluorescence (740 nm), a large Stokes shift (110 nm), and a sensitive response to viscosity (73-fold enhancement in fluorescence from water to glycerol), making it suitable for in vivo imaging. ICQ also exhibits an excellent ability to image mitochondrial viscosity changes in cells. More importantly, ICQ can target the liver selectively and image the viscosity changes in the liver noninvasively. Through establishing an AALI mouse model, ICQ was successfully applied to the in situ imaging changes in liver viscosity during the AALI process. The results showed a significant increase in liver viscosity in AALI mice, indicating that viscosity can serve as a marker for AALI, and ICQ is a promising noninvasive and sensitive tool for detecting and evaluating AALI.

MCAUSE: Prioritizing mitochondrial targets that alleviate pancreatic cancer cell phenotypes

Substantial changes in energy metabolism are a hallmark of pancreatic cancer. To adapt to hypoxic and nutrient-deprived microenvironments, pancreatic cancer cells remodel their bioenergetics from oxidative phosphorylation to glycolysis. This bioenergetic shift makes mitochondria an Achilles' heel. Since mitochondrial function remains essential for pancreatic cancer cells, further depleting mitochondrial energy production is an appealing treatment target. However, identifying effective mitochondrial targets for treatment is challenging. Here, we developed an approach, mitochondria-targeted cancer analysis using survival and expression (mCAUSE), to prioritize target proteins from the entire mitochondrial proteome. Selected proteins were further tested for their impact on pancreatic cancer cell phenotypes. We discovered that targeting a dynamin-related GTPase, OPA1, which controls mitochondrial fusion and cristae, effectively suppresses pancreatic cancer activities. Remarkably, when combined with a mutation-specific KRAS inhibitor, OPA1 inhibition showed a synergistic effect. Our findings offer a therapeutic strategy against pancreatic cancer by simultaneously targeting mitochondria dynamics and KRAS signaling.

Biocompatible bright orange emissive carbon dots: Multifunctional nanoprobes for highly specific sensing toxic Cr(VI) ions and mitochondrial targeting cancer cell imaging

Although several luminescent-based nanostructured materials are used as cellular imaging probes, creating a biocompatible subcellular imaging probe can be challenging. Instantaneously, it is crucial and urgently needed for certain fluorescent nanoprobes to identify possibly harmful heavy metal ions. We present a straightforward one-pot preparation of bright orange emissive (quantum yield approximately 16 %) Nitrogen/Sulfur co-doped carbon dots (N/S CDs) from citric acid and methylene blue as raw materials that will serve as a specific Cr(VI) ions sensor and an effective mitochondrial labeling in cancer cells. They had several benefits including low ecological impact, facile synthesis, good water solubility, photostability, and high stability. We found that N/S CDs photoluminescence (PL) could be reduced when Cr(VI) ions were present near them, and the PL reduction occurred highly sensitively to the presence of Cr(VI) compared to other metal ions including Cr(III) ions. This specific reduction of PL was due to the non-fluorescent complex formation through the inner filter effect (IFE). The established fluorescence-based sensing technique could serve for Cr(VI) ion quantification with exceptional sensing efficiency in the wide linear range of 7 to 70 μM (R2 = 0.9873), with the limit of detection of 53.5 nM. It was also revealed that the current fluorescent probe could be encouragingly utilized to quantify the concentration of Cr(VI) ions in various water specimens such as tap water. In addition, they were shown to function as a mitochondria-targeting nanoprobe in human cancer cells (ME 180 cells and Hela cells) for cell imaging. Concludingly, it was envisioned that these fluorescent nanoprobes could find a use in real-time monitoring of Cr(VI) ions in water-based ecosystems with ultrahigh sensitivity and cell image tracking via mitochondria labeling as biocompatible nanoprobes.

Overcoming cisplatin resistance in ovarian cancer: A novel approach via mitochondrial targeting peptide Pal-pHK-pKV

Cisplatin (DDP) resistance in advanced stages of ovarian cancer significantly reduces survival rates. Mitochondria may serve as a potential therapeutic target for ovarian cancer. Pal-pHK-pKV is a mitochondrial targeting peptide synthesized by supramolecular assembly. Our study aims to investigate whether Pal-pHK-pKV serves as a useful strategy to reverse DDP resistance in ovarian cancer. Subcutaneous tumor implantation of the DDP-resistant ovarian cancer cell line A2780CP was conducted in nude mice, and drugs were administered intraperitoneally to compare the inhibitory effects of Pal-pHK-pKV and DDP on A2780CP cells in vivo. Combination index values were calculated for various concentrations of DDP and Pal-pHK-pKV to determine the optimal combination concentration. Mitochondrial membrane potential, cytochrome C distribution and immunofluorescence were also measured. Our studies demonstrated that Pal-pHK-pKV treatment reduced the proliferation, invasion and metastasis of ovarian cancer cells and impaired mitochondrial function. Furthermore, the combination of Pal-pHK-pKV and DDP exhibited a synergistic effect. Mechanistically, Pal-pHK-pKV can impair mitochondrial function, reduce mitochondrial membrane potential and release ROS. On the other hand, Pal-pHK-pKV can affect ERK pathway activation and inhibit tumor development. In conclusion, the mitochondria-specific amphiphilic peptide Pal-pHK-pKV provides a novel approach for treating ovarian cancer and may potentially overcome DDP drug resistance.

Development of selectable markers for mitochondrial transformation in yeast

Mitochondria, present in most eukaryotic organisms, are crucial for energy production and essential for cellular functions. Sequencing of the complete mitochondrial genome of Saccharomyces cerevisiae in 1998 has paved the way for mtDNA gene editing, enabling the study of mitochondrial function and potential gene therapies for mitochondrial diseases. Effective selectable markers are crucial for addressing heteroplasmic mtDNA issues after mitochondrial transformation. Antibiotic resistance (AbR) marker genes aadA1, cat, and hph confer resistance to streptomycin, chloramphenicol, and hygromycin B, respectively. This study aimed to explore the feasibility of employing these AbR markers for selecting transformed yeast cells. Additionally, the usefulness of these AbR genes as selectable markers for yeast mitochondrial transformation was assessed by fusing a mitochondrial targeting signal (MTS) to the N-terminus of these genes using overlapping PCR. The minimal inhibitory concentration (MIC) of yeast transformants expressing various AbR genes, with or without MTS fusion, was determined using the agar dilution method. Yeast transformants expressing aadA1, cat, and hph, with or without MTS fusion, displayed resistance to streptomycin (>10 mg/mL), chloramphenicol (up to 6 mg/mL), and hygromycin B (up to 4 mg/mL), respectively. MICs were similar between AbR and MTS-tagged AbR yeast transformants. To assess mitochondrial targeting, GFP was fused to the C-terminus of cat and MTS-cat gene constructs. Fluorescence microscopy confirmed MTS-tagged CAT-GFP localization to yeast mitochondria, while CAT-GFP showed cytoplasmic localization. The fluorescence microscopy results were confirmed by Western blotting. This study demonstrated that yeast transformants expressing aadA1 exhibit a significant level of streptomycin resistance (>10 mg/mL), suggesting that aadA1-mediated streptomycin resistance has the potential to serve as a selectable marker for mitochondrial transformation in yeast.

A Mitochondria-Targeting SIRT3 Inhibitor with Activity against Diffuse Large B Cell Lymphoma.

Diffuse large B-cell lymphomas (DLBCLs) are heterogeneous cancers that still require better and less toxic treatments. SIRT3, a member of the sirtuin family of NAD+-dependent protein deacylase, is critical for DLBCL growth and survival. A mitochondria-targeted SIRT3 small-molecule inhibitor, YC8-02, exhibits promising activity against DLBCL. However, YC8-02 has several limitations including poor solubility. Here, we report our medicinal chemistry efforts that led to an improved mitochondria-targeted SIRT3 inhibitor, SJ-106C, achieved by using a triethylammonium group, which helps to increase both solubility and SIRT3 inhibition potency. SJ-106C, while still inhibiting SIRT1 and SIRT2, is enriched in the mitochondria to help with SIRT3 inhibition. It is more active against DLBCL than other solid tumor cells and effectively inhibits DLBCL xenograft tumor growth. The findings provide useful insights for the development of SIRT3 inhibitors and mitochondrial targeting agents and further support the notion that SIRT3 is a promising druggable target for DLBCL.

A multichannel nucleic acid-based Ca2+ nanomodulator induces multilevel destruction of mitochondria for cancer therapy

Mitochondria play a crucial function in tumor proliferation and apoptosis, and inducing mitochondrial dysfunction in cells has emerged as a promising therapeutic approach for tumors. Here, curcumin (CUR) is enrolled in amorphous calcium phosphate (ACP) through the coprecipitation method, followed by Cy5.5-labeled DNA was adsorbed on its surface to propose an acidity-responsive nucleic acid-based nanomodulator (ACP@C-D) to enhance Ca2+ overload and mitochondrial biomineralization for cancer therapy by amplifying intra-mitochondrial Ca2+ concentration. After tumor cell administration, the ACP@C-D will disintegrate in the acidic environment to enhance Ca2+ overload by the combined interaction of a dramatic increase in Ca2+ concentration and Ca2+ efflux inhibition by Cur. Moreover, the mitochondrial targeting ability of Cy5.5 allows DNA enrichment at mitochondrial, and the phosphate on DNA provides reaction sites for Ca2+ to achieve mitochondrial biomineralization thus mitochondrial dysfunction, which is reported for the first time. The facile and functional strategy of the nanomodulator will provide new insights into inmitochondria-based cancer therapy.

Extremely Sensitive Genetically Encoded Temperature Indicator Enabling Measurement at the Organelle Level.

Intracellular temperature is a fundamental parameter in biochemical reactions. Genetically encoded fluorescent temperature indicators (GETIs) have been developed to visualize intracellular thermogenesis; however, the temperature sensitivity or localization capability in specific organelles should have been further improved to clearly capture when and where intracellular temperature changes at the subcellular level occur. Here, we developed a new GETI, gMELT, composed of donor and acceptor subunits, in which cyan and yellow fluorescent proteins, respectively, as a Förster resonance energy transfer (FRET) pair were fused with temperature-sensitive domains. The donor and acceptor subunits associated and dissociated in response to temperature changes, altering the FRET efficiency. Consequently, gMELT functioned as a fluorescence ratiometric indicator. Untagged gMELT was expressed in the cytoplasm, whereas versions fused with specific localization signals were targeted to the endoplasmic reticulum (ER) or mitochondria. All gMELT variations enabled more sensitive temperature measurements in cellular compartments than those in previous GETIs. The gMELTs, tagged with ER or mitochondrial targeting sequences, were used to detect thermogenesis in organelles stimulated chemically, a method previously known to induce thermogenesis. The observed temperature changes were comparable to previous reports, assuming that the fluorescence readout changes were exclusively due to temperature variations. Furthermore, we demonstrated how macromolecular crowding influences gMELT fluorescence given that this factor can subtly affect the fluorescence readout. Investigating thermogenesis with gMELT, accounting for factors such as macromolecular crowding, will enhance our understanding of intracellular thermogenesis phenomena.

The mitochondrial protein Bcs1A regulates antifungal drug tolerance by affecting efflux pump expression in the filamentous pathogenic fungus Aspergillus fumigatus.

Aspergillus fumigatus is the predominant pathogen responsible for aspergillosis infections, with emerging drug-resistant strains complicating treatment strategies. The role of mitochondrial functionality in fungal resistance to antifungal agents is well-documented yet not fully understood. In this study, the mitochondrial protein Bcs1A, a homolog of yeast Bcs1, was found to regulate colony growth, ion homeostasis, and the response to antifungal drugs in A. fumigatus. Microscopic observations revealed substantial colocalization of Bcs1A-GFP fusion protein fluorescence with mitochondria. Bcs1A deletion compromised colony growth and the utilization of non-fermentable carbon sources, alongside causing abnormal mitochondrial membrane potential and reduced reactive oxygen species production. These findings underscore Bcs1A's vital role in maintaining mitochondrial integrity. Phenotypic analysis and determinations of minimum inhibitory concentrations indicated that the Δbcs1A mutant was more resistant to various antifungal agents, such as azoles, terbinafine, and simvastatin, compared to wild-type strain. RNA sequencing and RT-qPCR analysis highlighted an upregulation of multiple efflux pumps in the Δbcs1A mutant. Furthermore, loss of the principal drug efflux pump, mdr1, decreased azole tolerance in the Δbcs1A mutant, suggesting that Bcs1A's modulated of azoles response via efflux pump expression. Collectively, these results establish Bcs1A as essential for growth and antifungal drug responsiveness in A. fumigatus mediated through mitochondrial regulation.IMPORTANCEDrug resistance presents a formidable obstacle in the clinical management of aspergillosis. Mitochondria are integral to various biochemical pathways, including those involved in fungi drug response, making mitochondrial proteins promising therapeutic targets for drug therapy. This study confirms that Bcs1A, a mitochondrial respiratory chain protein, is indispensable for mitochondrial functionality and multidrug tolerance in Aspergillus fumigatus. Mutation of Bcs1A not only leads to a series of drug efflux pumps upregulated but also shows that loss of the primary efflux pump, mdr1, partial reduction in drug tolerance in the Bcs1A mutant, highlighting that Bcs1A's significant influence on mitochondria-mediated drug resistance.

Cardiac overexpression of a mitochondrial SUR2A splice variant impairs cardiac function and worsens myocardial ischemia reperfusion injury in female mice

The small splice variant of the sulfonylurea receptor protein isoform 2 A (SUR2A-55) targets mitochondria and enhances mitoKATP activity. In male mice the overexpression of this protein promotes cardioprotection, reducing myocardial injury after an ischemic insult. However, it is unclear what impact SUR2A-55 overexpression has on the female myocardium. To investigate the impact of SU2R2A-55 on the female heart, mice with cardiac specific transgenic overexpression of SUR2A-55 (TGSUR2A-55) were examined by resting echocardiography and histopathology. In addition, hearts were subjected to ischemia reperfusion (IR) injury. Female TGSUR2A-55 mice had resting LV dysfunction and worse hemodynamic recovery with increased infarct size after IR injury. RNA-seq analysis found 227 differential expressed genes between WT and TGSUR2A-55 female mouse hearts that were enriched in pathways of cellular metabolism. This was in direct contrast to male mice that had only four differentially expressed genes. Female TGSUR2A-55 mice compared to female WT mice had reduced cardiomyocyte mitochondrial membrane potential without a change in electron transport chain protein expression. In addition, isolated mitochondria from female TGSUR2A-55 hearts displayed reduced sensitivity to ATP and diazoxide suggestive of increased mitoKATP activity. In conclusion, our data suggests that female TGSUR2A-55 mice are unable to tolerate a more active mitoKATP channel leading to LV dysfunction and worse response to IR injury. This is in direct contrast to our prior report showing cardioprotection in male mice overexpressing SUR2A-55 in heart. Future research directed at examining the expression and activity of mitoKATP subunits according to sex may elucidate different treatments for male and female patients.

Upstream alternative polyadenylation in SCN5A produces a short transcript isoform encoding a mitochondria-localized NaV1.5 N-terminal fragment that influences cardiomyocyte respiration.

SCN5A encodes the cardiac voltage-gated Na+ channel, NaV1.5, that initiates action potentials. SCN5A gene variants cause arrhythmias and increased heart failure risk. Mechanisms controlling NaV1.5 expression and activity are not fully understood. We recently found a well-conserved alternative polyadenylation (APA) signal downstream of the first SCN5A coding exon. This yields a SCN5A-short transcript isoform expressed in several species (e.g. human, pig, and cat), though rodents lack this upstream APA. Reanalysis of transcriptome-wide cardiac APA-seq and mRNA-seq data shows reductions in both upstream APA usage and short/full-length SCN5A mRNA ratios in failing hearts. Knock-in of the human SCN5A APA sequence into mice is sufficient to enable expression of SCN5A -short transcript, while significantly decreasing expression of full-length SCN5A mRNA. Notably, SCN5A -short transcript encodes a novel protein (NaV1.5-NT), composed of an N-terminus identical to NaV1.5 and a unique C-terminus derived from intronic sequence. AAV9 constructs were able to achieve stable NaV1.5-NT expression in mouse hearts, and western blot of human heart tissues showed bands co-migrating with NaV1.5-NT transgene-derived bands. NaV1.5-NT is predicted to contain a mitochondrial targeting sequence and localizes to mitochondria in cultured cardiomyocytes and in mouse hearts. NaV1.5-NT expression in cardiomyocytes led to elevations in basal oxygen consumption rate, ATP production, and mitochondrial ROS, while depleting NADH supply. Native PAGE analyses of mitochondria lysates revealed that NaV1.5-NT expression resulted in increased levels of disassembled complex V subunits and accumulation of complex I-containing supercomplexes. Overall, we discovered that APA-mediated regulation of SCN5A produces a short transcript encoding NaV1.5-NT. Our data support that NaV1.5-NT plays a multifaceted role in influencing mitochondrial physiology: 1) by increasing basal respiration likely through promoting complex V conformations that enhance proton leak, and 2) by increasing overall respiratory efficiency and NADH consumption by enhancing formation and/or stability of complex I-containing respiratory supercomplexes, though the specific molecular mechanisms underlying each of these remain unresolved.

Gaining Insight into Mitochondrial Targeting: AUTAC-Biguanide as an Anticancer Agent.

AUTAC-Biguanide is a hybrid compound designed to target mitochondria, inducing their degradation by mitophagy. This study unveils the potential of biguanides as cancer cell-targeting agents, emphasizing AUTAC-Biguanide's superior antiproliferative properties compared to metformin and its selectivity for cancer cells. The mechanism behind this heightened effect includes the ability of AUTAC-Biguanide to trigger mitophagy. By providing a comprehensive analysis of these findings, this study adds valuable insights to the field of mitochondrial-targeting anticancer agents.

A mitochondria-targeted dual-response fluorescent probe with large Stokes shift for visualizing viscosity and cysteine in cancer cells and zebrafish

Mitochondrial cysteine (Cys) and viscosity are closely associated with numerous cellular processes and diseases. Developing a method capable of real-time tracking of viscosity and Cys in mitochondria is crucial for understanding cell status and mitochondria-related diseases. In this study, we developed a mitochondria-targeted fluorescent probe (Mito-QP) with a large Stokes shift of 171 nm for detecting Cys and viscosity. The acryloyl group enabled Mito-QP specific response to Cys with high sensitivity. The probe exhibited significant red fluorescence enhancement with increasing viscosity due to restricted intramolecular rotation. Additionally, Mito-QP showed low cytotoxicity and excellent mitochondrial-targeting ability. The probe was successfully employed to monitor fluctuations in mitochondrial viscosity and Cys in cancer cells and zebrafish. This work provides a convenient and effective sensor for studying mitochondrial dysfunction.

Real time precisely tracing the fluctuations of mitochondrial SO2 in cells during ferroptosis and tissues using a mitochondrial-immobilized ratiometric fluorescent probe

Mitochondrial sulfur dioxide (SO2) plays important roles in physiological and pathological activities. Unfortunately, it is lack of a reliable tool to precisely visualize the mitochondrial SO2 and elaborate its complicated functions in various cytoactivities. Here we report a mitochondrial-immobilized fluorescent probe PM-Cl consisting of coumarin and benzyl chloride modified benzothiazole, which enables selective visualization of mitochondrial SO2via chemical immobilization. The spectral results demonstrated that probe PM-Cl could respond to SO2 with high selectivity and sensitivity. Co-localization and the fluorescence of cytolysis extraction verified the excellent mitochondrial targeting and anchoring abilities. Due to the chemical immobilization, probe PM-Cl could firmly retain into mitochondria after stimulation of carbonyl cyanide m-chlorophenyl hydrazone (CCCP) and H2O2. Significantly, a series of fluorescence images are indicative of capability for detecting the fluctuations of SO2 in mitochondria during ferroptosis. Furthermore, PM-Cl also could visualize SO2 in myocardium and muscle tissues after the stimulation of CCCP. Taken together, probe PM-Cl is a very potential molecular tool for precisely detecting mitochondrial SO2 to explore its complex functions in physiological and pathological activities.

Development of cantharidin/baicalin co-delivery system based on mitochondrial targeting strategy for enhanced hepatocellular carcinoma therapy

Cantharidin (CTD) as a treatment for primary liver cancer are well known, and although structural alteration and nano-delivery are efficient in diminishing toxicity, its urinary and digestive toxicity has hindered its clinical application. Based on the synergistic effect of CTD and baicalin (BA) on proliferation inhibition of liver tumor cells, the highly expressed folic acid (FA) receptor of liver cancer cell membrane and the high negative membrane potential of tumor cell mitochondria, we synthesized CHEMS-FRFK and prepared a dual-ligand modified co-delivery liposomes (FA/FRFK-CTD/BA-Lips). Liposomes were spherical particles with uniform particle size, high encapsulation rate, good stability and no hemolysis. The results confirmed that FA/FRFK-CTD/BA-Lips had good targeting to mitochondria of tumor cells, and its target effect on mitochondria of HepG2 was better than that of Huh-7 cells. Compared with CTD, the cytotoxicity of liposomes on HepG2 is 7.0 times higher, while the cytotoxic effect on normal liver cells Thle-2 is 2.1 times lower. It may be related to increasing clathrin-mediated endocytosis, inhibiting tumor cell migration, arresting cell cycle in G1/G0 phase, and promoting mitochondria-mediated endogenous Bcl-2/Caspase 3 apoptosis pathway. Meanwhile, FA/FRFK-CTD/BA-Lips could alter the pharmacokinetic behavior of CTD and BA by increasing concentrations in vivo, slowing release and excretion, good anti-tumor with no obvious toxicity, and successfully delivered CTD/BA to liver tissue, tumor cells and mitochondria. Therefore, FA/FRFK-CTD/BA-Lips, as a delivery system targeting the mitochondria of liver cancer cells, has promising development potential, and could provide a new strategy for solving the problem of CTD toxicity and the treatment of hepatocellular carcinoma.