Fluorescence-On Imaging of Reticulophagy Enabled by an Acidity-Reporting Solvatochromic Probe.

Aberrant autophagy of the endoplasmic reticulum (reticulophagy) is engaged in diverse pathological disorders. Herein, we reported sensitive imaging of reticulophagy with ER-Green-proRed, a diad combining a solvatochromic entity of trifluoromethylated naphthalimide for long-term ER tracking by green fluorescence and an entity of rhodamine-lactam fluorogenic to lysosomal acidity. Stringently accumulated in the ER to give green fluorescence, ER-Green-proRed exhibits robust red fluorescence upon codelivery with the ER subdomain into lysosomes. The relevance of turn-on red fluorescence to reticulophagy was validated by reticulophagy modulated by starvation, reticulophagic receptors, and autophagy inhibition. This imaging method was successfully employed to discern reticulophagy induced by various pharmacological agents. These results show the potential of ER-targeted pH probes, as exemplified by ER-Green-proRed, to image reticulophagy and to identify reticulophagy inducers.

A dual biomarker-targeting probe enables signal-on surface labeling of Staphylococcus aureus.

Imaging or killing of a specific pathogen is of significance for precise therapy. Staphylococcus aureus (S. aureus) is an infectious gram-positive bacteria relying on Sortase A (SrtA) to anchor cell surface protein on peptidoglycan. We herein report signal-on labeling of S. aureus with self-quenched optical probes featuring vancomycin-conjugated SrtA substrate that is flanked by a dabcyl moiety paired with either fluorescein or eosine photosensizer (PS). SrtA-mediated cleavage of the substrate motif releases the dabcyl quencher, leading to covalent labeling of peptidoglycan with fluorescein or PS of restored photophysical property. The dual biomarked-enabled peptidoglycan labeling enables signal-on imaging and effective photodynamic destruction of S. aureus, suggesting a protheranostic approch activatable to SrtA-positive bacteria engaged in myriad diseases.

Biocapture-Directed Chemical Labeling for Discerning Stressed States of Organelles.

Lysosomal rupture engaged in diverse diseases remains poorly discerned from lysosomal membrane permeabilization (LMP). We herein reported biocapture-directed chemical labeling (BCCL) for the discern of lysosomal rupture by tracking the release of optically labeled cathepsins from damaged lysosomes into the cytosol. BCCL entails covalent anchoring of an azide-tagged suicide substrate (Epo-LeuTyrAz) to the enzyme active site and bioorthogonal ligation of the introduced azide with DBCORC, a ratiometric sensor featuring an acidity-reporting red emissive X-rhodamine-lactam (ROX), blue emissive coumarin (CM) inert to pH, and DBCO reactive to azide. Aided with fluorescein isocyanate-labeled sialic acid (FITC-Sia), a probe remained in pH-elevated lysosomes but dissipated from LMP+ lysosomes, BCCL enables optical discern of four states of lysosomes: ruptured lysosomes (blue in cytosol), LMP+ lysosomes (blue in lysosomes), pH-elevated lysosomes (blue and green in lysosomes), and physiological lysosomes (blue, green and red in lysosomes). This approach could find applicability to study lysosome rupture over LMP in diseases and to evaluate lysosome rupture-inducing drugs.

Metabolic installation of macrophage-recruiting glycan ligand on tumor cell surface for in vivo tumor suppression.

Synthetic probes that could direct immune cells against tumors are potential immunotherapeutics. We herein report in vivo tumor suppression via an intravenously injected abiotic sialic acid (TCCSia) that could be metabolically incorporated into tumor cell surface to yield of a high affinity ligand (TCCSiaα2,3-Gal) of Siglec-1 specifically expressed on macrophages. We observed marked suppression of pulmonary metastasis and subcutaneous tumor growth of B16F10 melanoma cells in mice with TCCSia, suggesting the utility of abiotic sialic acid to modulate tumor immunity via recruiting Siglec+ immune cells.

Galectin Trafficking Pathway-Enabled Color-Switchable Detection of Lysosomal Membrane Permeabilization.

Lysosomal membrane permeabilization (LMP) engaged in multiple human diseases is accompanied by relocation of cytosolic galectin into LMP+ lysosomes. We herein reported a galectin trafficking-targeted method to image LMP using two kinds of glyco-dendrimers, a sialic acid-terminated dendrimer labeled with pH-inert rhodamine and a lactose-terminated dendrimer labeled with fluorescein that becomes green-emissive in pH-elevated lysosomes. Albeit both accumulated in physiological lysosomes, the former is released from LMP+ lysosomes while the latter binds to galectin accumulated in LMP+ lysosomes and thus trapped in LMP+ lysosomes. Accordingly, LMP+ lysosomes exhibit loss of red fluorescence and turn-on green fluorescence due to loss of lysosomal acidity. This red-to-green color switch enables discernment of LMP+ lysosomes from physiological lysosomes and pH-elevated lysosomes and can be further utilized to detect LMP in distinct cell death pathways. These results suggest the utility of galectin trafficking pathway-integrated synthetic probes for detection of LMP, a key factor for diseased cells.

Imaging of Mitophagy Enabled by an Acidity-Reporting Probe Anchored on the Mitochondrial Inner Membrane.

Classical chemical probes are prone to dissipation from stressed organelles, as evidenced by the incapability of mitochondrial dyes to image mitophagy linked to multiple diseases. We herein reported mitophagy imaging via covalent anchoring of a lysosomal probe to the mitochondrial inner membrane (CALM). Utilizing DBCORC-TPP, an azide-conjugatable probe with acidity-triggered fluorescence, CALM is operated via ΔΨm-promoted probe accumulation in mitochondria and thereby bioorthogonal ligation of the trapped probe with azido-choline (Azcholine) metabolically installed on the mitochondrial membrane. Overcoming the limitation of synthetic probes to dissipate from stressed organelles, CALM enables signal-on fluorescence imaging of mitophagy induced by starvation and is further employed to reveal mitophagy in ferroptosis. These results suggest the potential of CALM as a new tool to study mitophagy.

Installation of high-affinity Siglec-1 ligand on tumor surface for macrophage-engaged tumor suppression.

Siglecs that binds cell surface sialoglycans are a family of immunomodulatory receptors, of which, Siglec-7 expressed on natural killer (NK) cells promotes tumor immunoevation while the role of Siglec-1 expressed on macrophages on tumor development remains largely unexplored. Herein, we selectively introduced high affinity sialoside ligands of Siglec-1 and Siglec-7 to tumor cell surface via in vivo Strain-promoted Azide-Alkyne cyclization of TCCSiaα2,3-Lactose or FITCSiaα2,6-Lactose with 9-azido sialic acid (AzSia) metabolically installed on tumor cell surface. We found that TCCSiaα2,3-Lactose conjugated on tumor surface moderately inhibited tumor growth while FITCSiaα2,6-Lactose promote tumor growth. These results suggest high-affinity ligand of Siglec-1 dispalyed on tumors surface provide a new perspective for tumor immunotherapy.

Organelle-Directed Metabolic Glycan Labeling and Optical Tracking of Dysfunctional Lysosomes Thereof.

Metabolic glycan labeling (MGL) has been employed for diverse purposes, such as cell surface glycan imaging and tumor surface engineering. We herein reported organelle-specific MGL (OMGL) for selective tagging of the inner limiting membrane of lysosomes over the cell surface. This is operated via acidity-promoted accumulation of optical probes in lysosomes and bioorthogonal ligation of the trapped probes with 9-azidosialic acid (AzSia) metabolically installed on lysosomal membrane proteins. Overcoming the limitation of classical organelle probes to dissipate from stressed organelles, OMGL enables optical tracking of pH-elevated lysosomes in exocytosis and membrane-permeabilized lysosomes in different cell death pathways. Thus, OMGL offers a new tool to study lysosome biology.

Imaging stressed organelles via sugar-conjugated color-switchable pH sensors.

Stressed organelles are often challenging to image with canonical organelle probes owing to their propensity to dissipate from stressed organelles. We herein report the imaging of stressed lysosomes with color-switchable glyco-probes that contain an entity of mannose-6-carboxylate or sialic acid for targeting to and long-term retention in stressed lysosomes, and a diad of fluorescein/rhodamine-X-lactam exhibiting dramatic red-to-green fluorescence shift upon pH elevation. Relative to acidotropic dyes prone to dissipate from pH-elevated lysosomes, both glyco-probes are stably trapped in lysosomes without resorting to lysosomal acidity. Importantly, these probes are red emissive in acidic lysosomes (pH 4.5-5.8), but switched to green fluorescence in lysosomes of pH 6.0-7.4, allowing dual color discrimination of lysosomal pH alterations in cell starvation. These results support the use of sugar-armed sensors to investigate stressed lysosomes in biology and disease.

Organelle-Redirected Chameleon Sensor-Enabled Live Cell Imaging of Mitochondrial DNA.

Mitochondrial DNA (mtDNA) plays important roles in diverse physiological processes and myriad diseases. We herein report mtDNA imaging with a chameleon sensor containing a cationic rhodamine B (RB) entity for mitochondria targeting and a fluorogenic SYBR Green-I (SG) entity for DNA sensing. SG-RB selectively binds to mtDNA and gives green SG fluorescence in mitochondria of living cells but gives red RB fluorescence upon delivery of mitochondria into lysosomes in mitophagy. With the dual-color imaging, mtDNA aggregation and elevated mitophagy were identified in HeLa cells stressed with anticancer doxorubicin. These results suggest the utility of organelle-redirected DNA sensors for live cell imaging of mtDNA involved in myriad pathological disorders.

An in cellulo-activated multicolor cell labeling approach used to image dying cell clearance.

Dying cell clearance is critical for myriad biological processes such as tissue homeostasis. We herein report an enzyme-activated fluorescence cell labeling approach and its use for multicolor imaging of dying cell clearance. Diacetylated 4-hydroxymandelic acid (DHA)-conjugated dyes give rise to reactive quinone methides upon deacetylation in live cells, which in turn covalently labels cellular proteins. With partner cells tagged with distinct fluorescence, apoptotic cell clearance by Raw 264.7 macrophages and epithelial HeLa cells was captured by confocal microscopy, showing the potential of DHA-based cell labeling for investigating cell-cell interactions.

Bifunctional Super-resolution Imaging Probe with Acidity-Independent Lysosome-Retention Mechanism.

Spatiotemporal imaging is of enormous use to explore organelle biology, necessitating organelle-tracing techniques reliable in varied cell stress. We herein reported lysosomal imaging using rhodamine-X-integrated sialic acid (ROXSA), which is stably maintained in lysosomes irrespective of lysosomal pH changes. Exhibiting bright fluorescence and superior photostability, ROXSA enables 120 h continual tracking of fusion/fission of lysosomes and mitochondrion-lysosome interaction in mitophagy. Relative to conventional acidotropic probes prone to dissipation from stressed lysosomes, ROXSA offers a new route for long-term tracking of stressed lysosomes relevant to diverse pathological conditions.

Organelle-Directed Staudinger Reaction Enabling Fluorescence-on Resolution of Mitochondrial Electropotentials via a Self-Immolative Charge Reversal Probe.

Organelles often feature parameters pertinent to functions and yet responsive to biochemical stress. The electropotential across the mitochondrial membrane (ΔΨm) is a crucial mediator of cell fates. Herein we report a bioorthogonal reaction enabled fluorescence-on probing of ΔΨm alterations featuring anionic fluorescein-triphenylphosphonium diad (F-TPP), which is released via intramitochondria Staudinger reaction triggered self-immolation of o-azidomethylbenzoylated F-TPP. Compared to classical cationic mitochondria-specific dyes, F-TPP is hydrophilic and negatively charged. Effectively discerning ΔΨm changes upon diverse stress inducers, the organelle-directed bioorthogonal imaging strategy offers unprecedented choices to probe mitochondrial biology with functional molecules that are otherwise inaccessible via physiological organelle-probe affinity.

Liposome-aided metabolic engineering of tumor surface immunogenicity.

Approaches to increase tumor immunogenicity are of therapeutic potentials. We herein reported the use of liposomes for covalent incorporation of neoantigen on tumor surfaces with DNP-conjugated sialic acid (DNPSia). Relative to free DNPSia, sugar-encapsulated biotinylated liposomes (DNPSia@LP@biotin) enables effective cell surface expression of DNPSia on biotin receptor (BR)-expressing cells over BR-free cells in vitro, and on tumor cell surfaces with high tumor-to-normal tissue contrast in a mice model. These findings suggest the potentials of targetable liposomes for modulating tumor surface immunity via metabolic oligosaccharide engineering.

Bioorthogonal Conjugation Directed by a Sugar-Sorting Pathway for Continual Tracking of Stressed Organelles.

Selective and continuous tracking of dynamic organelles is crucial for modern biology. We herein report a ship-in-a-bottle strategy for tagging lysosomes by a strain-promoted azide-alkyne cycloaddition to couple a pH sensor (RC) with mannose-6-carboxylate (M6C) actively transported into lysosomes through cell sorting. In contrast to classical acidotropic sensors, which are prone to dissipate from lysosomes, M6C-RC formed in situ is stably trapped in lysosomes without resort to lysosomal acidity and exhibits "always-on" blue fluorescence to pinpoint lysosomes and red-to-blue fluorescence ratios indicative of the lysosomal pH value. These advantages enable tracking of stressed lysosomes, and necrosis to be differentiated from apoptosis on the basis of lysosomal pH changes. The cell-sorting-mediated bioorthogonal tagging strategy offers a new route to track stressed organelles with disrupted physiological organelle-probe affinity.

Defining Cancer Cell Bioenergetic Profiles Using a Dual Organelle-Oriented Chemosensor Responsive to pH Values and Electropotential Changes.

Cell fate is largely shaped by combined activity of different types of organelles, which often feature functionally critical parameters that succumb to pathological inducers. We herein report the analysis of cell bioenergetic profiles with a dual organelle-oriented chemosensor (RC-AMI), partitioning in mitochondria to give blue fluorescence and in lysosomes to give red fluorescence. Responsive to lysosomal pH and mitochondrial transmembrane potential (ΔΨm), two parameters crucial to cell bioenergetics, RC-AMI enables dual colored reporting of lysosomal acidity and ΔΨm, revealing upregulated ΔΨm and imbalance dramatically shifted favoring ΔΨm over lysosomal acidity in cancer cells whereas the tendency is reversed in starved cells. Complementing classical homo-organelle-specific sensors, this dual organelle-oriented and fluorescently responsive probe offers a new tool to detect imbalance between lysosomal acidity and mitochondrial ΔΨm, an index critical for cancer bioenergetics.

Optical tracking of phagocytosis with an activatable profluorophore metabolically incorporated into bacterial peptidoglycan.

Phagocytosis is critical for immunity against pathogens. Prior imaging using dye-labeled synthetic beads or green fluorescent protein-expressing bacteria is limited by "always-on" signals which compromise discerning phagocytosed particles from adherent particles. Targeting cellular internalization of pathogens into acidic phagolysosomes, we herein report "turn-on" fluorescence imaging of phagocytosis with viable bacteria featuring peptidoglycans covalently modified with rhodamine-lactam responsive to acidic pH. Culturing of Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) with d-lysine conjugated rhodamine-lactam and fluorescein isocyanate (FITC) leads to efficient metabolic incorporation of FITC and rhodamine-lactam into bacterial peptidoglycan. E. coli and S. aureus become red-emissive upon phagocytosis into Raw 264.7 macrophages. With FITC as the reference signal, the mono- and dual-color emission allow efficient in situ distinction of ingested bacteria from extracellular bacteria. Given the ease of optical peptidoglycan labeling, the prevalence of microbial peptidoglycan and preservation of microbial surface landscape, this approach would be of use for investigation on microbial pathogenesis and high-throughput screening of immunomodulators of phagocytosis.

Lysosomal pH Decrease in Inflammatory Cells Used To Enable Activatable Imaging of Inflammation with a Sialic Acid Conjugated Profluorophore.

Inflammation causes significant morbidity and mortality, necessitating effective in vivo imaging of inflammation. Prior approaches often rely on combination of optical agents with entities specific for proteinaceous biomarkers overexpressed in inflammatory tissues. We herein report a fundamentally new approach to image inflammation by targeting lysosomes undergoing acidification in inflammatory cells with a sialic acid (Sia) conjugated near-infrared profluorophore (pNIR). Sia-pNIR contains a sialic acid domain for in vivo targeting of inflamed tissues and a pNIR domain which isomerizes into fluorescent and optoacoustic species in acidic lysosomes. Sia-pNIR displays high inflammation-to-healthy tissue signal contrasts in mice treated with Escherichia coli, Staphylococcus aureus, or lipopolysaccharide. In addition, inflammation-associated fluorescence is switched off upon antibiotics treatment in mice. This report shows the potentials of Sia-pNIR for activatable dual-modality inflammation imaging, and particularly the use of lysosomes of inflamed cells as a previously unappreciated biomarker for inflammation imaging.

Centrifugation aided highly sensitive detection of nitrite with a dye-silica conjugate featuring cleavable linkages.

Rhodamine-silica nanocomposite bridged by a cleavable linker was used for highly sensitive nitrite detection via analyte triggered release of rhodamine from silica particles. Centrifugal removal of pristine nanoconjugate from the assay medium effectively decreased background signals in the supernatant whereas rhodamine unleashed from silica platform is retained in the supernatant, enabling facile detection of nitrite with an assay limit of 50 nM which is 400 folds lower than legislated maximum contaminant levels of nitrite in drinking water. Assays based on small molecular chemosensors are often compromised by their intrinsic fluorescence signals and low aqueous compatibility. The performance of water compatible rhodamine-silica nanocomposite suggests broad analytical potentials of centrifugal nanoparticulate systems with dyes conjugated via appropriate cleavable linkers.

A near-infrared fluorescence dye for sensitive detection of hydrogen sulfide in serum.

Cy-Cl, a cationic near-infrared cyanine dye, readily reacts with hydrogen sulfide (H2S) via nucleophilic thiolation to give dose-dependent 'turn-off' fluorescence and colorimetric read-out, allowing selective detection of low levels of H2S in serum and imaging of mitochondrial H2S in living cells.