Facile construction of dual-response super-resolution probes for tracking organelles dynamics.

Super-resolution imaging techniques, such as structured illumination microscopy (SIM), have enabled researchers to obtain nanoscale organelle-level outputs in living systems, but they impose additional stringent requirements on fluorescence probes. However, high-performance, custom-designed SIM probes that can explain underlying biological processes remain unavailable. Herein, a customizable engineering toolkit is developed for the facile assembly of SIM probes suitable for subcellular component detection. This toolkit is used to customize a fluorescent molecule, CPC (coumarin-phenylhydrazine-carboxyl), capable of simultaneously monitoring peroxynitrite (ONOO-) and polarity distribution in mitochondria and lipid droplets (LDs), respectively, through functional ON-OFF mechanisms. The customized CPC molecule demonstrated excellent imaging capabilities under SIM, enabled the successful localization of multiple organelles, and reliably tracked the distribution of different components, thus facilitating the study of the interplay between organelles. Using CPC, the physical transition of intracellular LDs is demonstrated from heterogeneity to homogeneity. This was specifically observed during ferroptosis where the polarity of the LDs increased and their morphology became more contracted. Furthermore, the loss of LDs functionality could not counteract the accumulation of ONOO- within the mitochondria, leading to the decoupling of mitochondrial LDs during ferroptosis. These results confirmed the potential mechanism of LDs dysfunction and decoupling triggered via cumulative mitochondrial oxidative stress during ferroptosis. To summarize, this toolkit will be a powerful tool for examining subtle variations among components during the interplay between different organelles, thus offering novel avenues for understanding and treating related diseases.

Super-resolution imaging for in situ monitoring sub-cellular micro-dynamics of small molecule drug.

Small molecule drugs play a pivotal role in the arsenal of anticancer pharmacological agents. Nonetheless, their small size poses a challenge when directly visualizing their localization, distribution, mechanism of action (MOA), and target engagement at the subcellular level in real time. We propose a strategy for developing triple-functioning drug beacons that seamlessly integrate therapeutically relevant bioactivity, precise subcellular localization, and direct visualization capabilities within a single molecular entity. As a proof of concept, we have meticulously designed and constructed a boronic acid fluorescence drug beacon using coumarin-hemicyanine (CHB). Our CHB design includes three pivotal features: a boronic acid moiety that binds both adenosine triphosphate (ATP) and adenosine diphosphate (ADP), thus depleting their levels and disrupting the energy supply within mitochondria; a positively charged component that targets the drug beacon to mitochondria; and a sizeable conjugated luminophore that emits fluorescence, facilitating the application of structured illumination microscopy (SIM). Our study indicates the exceptional responsiveness of our proof-of-concept drug beacon to ADP and ATP, its efficacy in inhibiting tumor growth, and its ability to facilitate the tracking of ADP and ATP distribution around the mitochondrial cristae. Furthermore, our investigation reveals that the micro-dynamics of CHB induce mitochondrial dysfunction by causing damage to the mitochondrial cristae and mitochondrial DNA. Altogether, our findings highlight the potential of SIM in conjunction with visual drug design as a potent tool for monitoring the in situ MOA of small molecule anticancer compounds. This approach represents a crucial advancement in addressing a current challenge within the field of small molecule drug discovery and validation.

Lysosome passivation triggered by silver nanoparticles enhances subcellular-targeted drug therapy.

Frequently, subcellular-targeted drugs tend to accumulate in lysosomes after cellular absorption, a process termed the lysosomal trap. This accumulation often interferes with the drug's ability to bind to its target, resulting in decreased efficiency. Existing methods for addressing lysosome-induced drug resistance mainly involve improving the structures of small molecules or enveloping drugs in nanomaterials. Nonetheless, these approaches can lead to changes in the drug structure or potentially trigger unexpected reactions within organisms. To address these issues, we introduced a strategy that involves inactivating the lysosome with the use of Ag nanoparticles (Cy3.5@Ag NPs). In this method, the Cy3.5@Ag NPs gradually accumulate inside lysosomes, leading to permeation of the lysosomal membrane and subsequent lysosomal inactivation. In addition, Cy3.5@Ag NPs also significantly affected the motility of lysosomes and induced the occurrence of lysosome passivation. Importantly, coincubating Cy3.5@Ag NPs with various subcellular-targeted drugs was found to significantly increase the efficiency of these treatments. Our strategy illustrates the potential of using lysosomal inactivation to enhance drug efficacy, providing a promising therapeutic strategy for cancer.

Single Image Capture of Bioactive Ion Crosstalk within Inter-Organelle Membrane Contacts at Nanometer Resolution.

Rapid bioactive ion exchange is a form of communication that regulates a wide range of biological processes. Despite advances in super-resolution optical microscopy, visualizing ion exchange remains challenging due to the extremely fast nature of these events. Here, a "converting a dynamic event into a static image construction" (CDtSC) strategy is developed that uses the color transformation of a single dichromatic molecular probe to visualize bioactive ion inter-organelle exchange in live cells. As a proof of concept, a reactive sulfur species (RSS) is analyzed at the mitochondria-lysosome contact sites (MLCs). A non-toxic and sensitive probe based on coumarin-hemicyanine structure is designed that responds to RSS localized in both mitochondria and lysosomes while fluorescing different colors. Using this probe, RSS give-and-take at MLCs is visualized, thus providing the first evidence that RSS is involved in inter-organelle contacts and communication. Taken together, the CDtSC provides a strategy to visualize and analyze rapid inter-organelle ion exchange events in live cells at nanometer resolution.

A Dual-Labeling Probe for Super-Resolution Imaging to Detect Mitochondrial Reactive Sulfur Species in Live Cells.

Background: Mitochondria are the main sites of reactive sulfur species (RSS) production in living cells. RSS in mitochondria play an important role in physiological and pathological processes of life. In this study, a dual-labeling probe that could simultaneously label the mitochondrial membrane and matrix was designed to quantitatively detect RSS of mitochondria in living cells using nano-level super-resolution imaging. Methods: A fluorescent probe CPE was designed and synthesized. The cytotoxicity of CPE was determined and co-localization of CPE with a commercial mitochondrial probe was analyzed in HeLa cells. Then, the uptake patterns of CPE in HeLa cells at different temperatures and endocytosis levels were investigated. The staining characteristics of CPE under different conditions were imaged and quantitated under structured illumination microscopy. Results: A fluorescence probe CPE reacting to RSS was developed, which could simultaneously label the mitochondrial membrane with green fluorescence and the mitochondrial matrix with red fluorescence. CPE was able to demonstrate the mitochondrial morphology and detect the changes of RSS in mitochondria. With the increase of mitochondrial RSS concentration, the light of the red matrix will be quenched. Conclusion: CPE provides a strategy for the design of probes and an attractive tool for accurate examination to changes of mitochondrial morphology and RSS in mitochondria in living cells at the nanoscale.

Lysosome-Targeted Biosensor for the Super-Resolution Imaging of Lysosome-Mitochondrion Interaction.

Background: The interaction between lysosomes and mitochondria includes not only mitophagy but also mitochondrion-lysosome contact (MLC) that enables the two organelles to exchange materials and information. In our study, we synthesised a biosensor with fluorescence characteristics that can image lysosomes for structured illumination microscopy and, in turn, examined morphological changes in mitochondria and the phenomenon of MLC under pathological conditions. Methods: After designing and synthesising the biosensor, dubbed CNN, we performed an assay with a Cell Counting Kit-8 to detect CNN's toxicity in relation to H9C2 cardiomyocytes. We next analysed the co-localisation of CNN and the commercial lysosomal probe LTG in cells, qualitatively analysed the imaging characteristics of CNN in different cells (i.e. H9C2, HeLa and HepG2 cells) via structured illumination microscopy and observed how CNN entered cells at different temperatures and levels of endocytosis. Last, we treated the H9C2 cells with mannitol or glucose to observe the morphological changes of mitochondria and their positions relative to lysosomes. Results: After we endocytosed CNN, a lysosome-targeted biosensor with a wide, stable pH response range, into cells in an energy-dependent manner. SIM also revealed that conditions in high glucose induced stress in lysosomes and changed the morphology of mitochondria from elongated strips to round spheres. Conclusion: CNN is a new tool for tracking lysosomes in living cells, both physiologically and pathologically, and showcases new options for the design of similar biosensors.

A near-infrared, colorimetric and ratiometric fluorescent sensor with high sensitivity to hydrogen peroxide and viscosity for solutions detection and imaging living cells.

Considering to the importance of hydrogen peroxide (H2O2) in a number of pathological processes and fluorescence sensor as a powerful tool for imaging and therapy of H2O2-related diseases, herein, a near-infrared fluorescent sensor for colorimetric and ratiometric detecting H2O2 and viscosity, named NCR, was constructed and reported. Its long emission wavelength (670 nm) avoids the interference of biological autofluorescence. When NCR interacted with 10 equiv. of H2O2, colorimetric characteristic provides a clear naked eye recognition (colourless turned to green under UV light and light purple turned to colorless under visible light). In addition, NCR shows high sensitivity to viscosity with or without the addition of H2O2. It was also found alkaline environment contribute to enhance fluorescence response to H2O2. A suitable water-octanol partition coefficient (logP, 0.521 ± 0.003) and low cytotoxicity displayed that NCR is very favorable to image living cells. Furthermore, exogenous and endogenous H2O2 was detected successfully by NCR in living Hela cells. These studies indicate that NCR has great potential to develop as a practical tool for monitoring H2O2 level in biological process.

Novel detection method for gallic acid: A water soluble boronic acid-based fluorescent sensor with double recognition sites.

As one of the widespread phenols in nature, gallic acid (GA) has attracted a subject of attention due to its extensive biological properties. It is very important and significant to develop a sensitive and selective gallic acid sensor. In recent years, owing to their reversible covalent binding with Lewis bases and polyols, boronic acid compounds have been widely reported as fluorescence sensors for the identification of carbohydrates, ions and hydrogen peroxide, etc. However, boronic acid sensors for specific recognition of gallic acid have not been reported. Herein, a novel water-soluble boronic acid sensor with double recognition sites is reported. When the concentration of gallic acid added was 1.1 × 10-4 M, the fluorescence intensity of sensor 9b decreased by 80%, followed by pyrogallic acid and dopamine. However, the fluorescence of the sensor 9b combined with other analytes such as ATP, sialic acid, and uridine was basically unchanged, indicating that the sensor 9b had no ability to recognize these analytes. Also, sensor 9b has a fast response time to gallic acid at room temperature, and has a high binding constant (12355.9 ± 156.89 M-1) and low LOD (7.30 × 10-7 M). Moreover, gallic acid content of real samples was also determined, and the results showed that this method has a higher recovery rate. Therefore, sensor 9b can be used as a potential tool for detecting biologically significant gallic acid in actual samples such as food, medicine, and environmental analysis samples.

A highly selective and sensitive boronic acid-based sensor for detecting Pd2+ ion under mild conditions.

Herein, a boronic acid-based sensor was reported selectively to recognize Pd2+ ion. The fluorescence intensity increased 36-fold after sensor binding with 2.47 × 10-5 M of Pd2+ ion. It was carried out in the 99% aqueous solution for binding tests, indicating sensor having good water solubility. In addition, it is discernible that Pd2+ ion turned on the blue fluorescence of sensor under a UV-lamp (365 nm), while other ions (Ag+, Al3+, Ba2+, Ca2+, Cr2+, Cd2+, Co2+, Cs2+, Cu2+, Fe2+, Fe3+, K+, Li+, Mg2+, Mn2+, Na+, Ni2+ and Zn2+) did not show the similar change. Furthermore, sensor has a low limit of detection (38 nM) and high selectivity, which exhibits the potential for the development of Pd2+ recognition in practical environments.

Boronic acid sensors with double recognition sites: a review.

Boronic acids reversibly and covalently bind to Lewis bases and polyols, which facilitated the development of a large number of chemical sensors to recognize carbohydrates, catecholamines, ions, hydrogen peroxide, and so on. However, as the binding mechanism of boronic acids and analytes is not very clear, it is still a challenge to discover sensors with high affinity and selectivity. In this review, boronic acid sensors with two recognition sites, including diboronic acid sensors, and monoboronic acid sensors having another group or binding moiety, are summarized. Owing to double recognition sites working synergistically, the binding affinity and selectivity of sensors can be improved significantly. This review may help researchers to sort out the binding rules and develop ideal boronic acid-based sensors.

A water-soluble boronic acid sensor for caffeic acid based on double sites recognition.

Due to reversibly and covalently binding with Lewis bases and polyols, boronic acid compounds as fluorescent sensors have been widely reported to recognize carbohydrates, ions, hydrogen peroxide, and so on. However, boronic acid sensors for highly selective recognition of caffeic acid rather than catechol or catechol derivatives have not been reported yet. Herein a novel water-soluble sensor 5c with double recognition sites based on a boronic acid was reported. When 2.3 × 10-4 M of caffeic acid was added, the fluorescence intensity of sensor 5c decreased by 99.6% via inner filter effect (IFE) because its excitation spectrum well overlaps with the absorption spectrum of caffeic acid under neutral condition, while the fluorescence increased or did not change obviously after binding with other analytes including carbohydrates and other catechol derivatives. In addition, the response time to caffeic acid is fast at room temperature, and a high binding constant (9245.7 ± 348.3 M-1) and low LOD (1.81 × 10-6 M) was calculated. Moreover, determination of caffeic acid content in caffeic acid tablets was studied, and the recovery rate is sufficient. Therefore, sensor 5c can be used as a potential tool for detecting biologically significant caffeic acid in real samples.

A novel boronic acid-based fluorescent sensor for selectively recognizing Fe3+ ion in real time.

Boronic acid provides faster fluorescence response to Fe3+ compared to other reported sensors, which is critical for continuous dynamic detection. Herein, we reported a novel boronic acid-based sensor 4 that could recognize Fe3+ ion in real time. After 10 equiv. of Fe3+ ion (1 mM) was added, the fluorescence of sensor 4 was immediately quenched by 96%. While other ions, including Ba2+, Ca2+, Cr2+, Cd2+, Co2+, Cs2+, Cu2+, Fe2+, K+, Li+, Mg2+, Mn2+, Na+, Ni2+ or Zn2+, respectively, did not change the fluorescence significantly. Further tests indicated that the high selectively sensing Fe3+ ion benefits from the two boronic acid functionalities in the structure. Moreover, interference experiments showed this sensor has an excellent anti-interference ability. In addition, we performed binding activity test in rabbit plasma and other real samples for practical applications, obtaining similar results. And the thin layer loading sensor 4 was also successfully applied to recognize Fe3+ ion among various ions. Therefore, 4 may serve as a potential sensor for continuous monitoring and detecting Fe3+ ion in real time.

Recent development of boronic acid-based fluorescent sensors.

As Lewis acids, boronic acids can bind with 1,2- or 1,3-diols in aqueous solution reversibly and covalently to form five or six cyclic esters, thus resulting in significant fluorescence changes. Based on this phenomenon, boronic acid compounds have been well developed as sensors to recognize carbohydrates or other substances. Several reviews in this area have been reported before, however, novel boronic acid-based fluorescent sensors have emerged in large numbers in recent years. This paper reviews new boron-based sensors from the last five years that can detect carbohydrates such as glucose, ribose and sialyl Lewis A/X, and other substances including catecholamines, reactive oxygen species, and ionic compounds. And emerging electrochemically related fluorescent sensors and functionalized boronic acid as new materials including nanoparticles, smart polymer gels, and quantum dots were also involved. By summarizing and discussing these newly developed sensors, we expect new inspiration in the design of boronic acid-based fluorescent sensors.