Lysosome-targeted Aza-BODIPY photosensitizers for anti-cancer photodynamic therapy.

Developing effective near-infrared (NIR) photosensitizers (PSs) has been an attractive goal of photodynamic therapy (PDT) for cancer treatment. In this study, we synthesized N, N-diethylaminomethylphenyl-containing Aza-BODIPY photosensitizers and comprehensively investigated their photophysical/photochemical properties, as well as cell-based and animal-based anti-tumor studies. Among them, BDP 1 has strong NIR absorption at 680 nm and higher singlet oxygen yield in PBS which showed favorable pH-activatable and lysosome-targeting ability. BDP 1 could be easily taken up by tumor cells and showed negligible dark activity (IC50 > 50 µM), however strong phototoxicity upon exposure to light irradiation. The acceptable fluorescence emission from BDP 1 allowed convenient in vivo fluorescence imaging for organ distribution studies in mice. After PDT treatment with upon single time PDT treatment at the beginning using relatively low light dose (54 J/ cm2), BDP 1 (2 mg/kg, 0.1 mL) was found to have strong efficacy to inhibit tumor growth and even to ablate off tumor without causing body weight loss. Therefore, pH-activatable and lysosome-targeted PS may become an effective way to develop potent PDT agent.

Synthesis and biological evaluation of zwitterionic half-sandwich Rhodium(III) and Ruthenium(II) organometallic complexes.

Herein we present the synthesis and characterization of a panel of structurally related zwitterionic piano-stool rhodium(III) and ruthenium(II) complexes. The identities of these novel complexes have been determined by NMR spectroscopy, mass spectrometry, elemental analysis and single-crystal X-ray crystallography. The stability and fluorescence property of these zwitterionic complexes were also confirmed. Zwitterionic rhodium(III) complexes Rh1-Rh4 displayed potent cytotoxic activity against A549 and HeLa human cancer cells. On the contrary, zwitterionic ruthenium(II) complexes Ru1-Ru4 presented no obvious cytotoxic activity to the test cell lines. Moreover, the trend that the introduction of fluorinated substituent and phenyl ring in the η5-CpR ring and N,N-chelating ligand, respectively, could enhance the cytotoxicity of these zwitterionic rhodium(III) complexes, were observed. The exploration of mechanism using flow cytometry displayed that the cytotoxicity of these rhodium(III) complexes was associated with the perturbation of the cell cycle and the induction of cell apoptosis. Furthermore, microscopic analysis using confocal microscopy indicated that the representative rhodium(III) complex Rh4 entered A549 cells via energy-dependent pathway and predominantly accumulated in lysosomes, thus leading to the disruption of lysosomal integrity.

Development of Lysosome-Targeted Fluorescent Probes for Cys by Regulating the Boron-dipyrromethene (BODIPY) Molecular Structure.

Our previous discovery suggested that substituents on the 1,7 positions delicately modulate the sensing ability of the meso-arylmercapto boron-dipyrromethene (BODIPY) to biothiols. In this work, the impact of delicate modulations on the sensing ability is investigated. Therefore, 1,7-dimethyl, 3,5-diaryl substituted BODIPY is designed and developed and its conformationally restricted species with a meso-arylmercapto moiety (DM-BDP-SAr and DM-BDP-R-SAr) as selective fluorescent probes for Cys. Moreover, the lysosome-target probes (Lyso-S and Lyso-D) based on DM-BDP-SAr carrying one or two morpholinoethoxy moieties were developed. They were able to detect Cys selectively in vitro with low detection limits. Both Lyso-S and Lyso-D localized nicely in lysosomes in living HeLa cells and exhibited red fluorescence for Cys. Moreover, a novel fluorescence quenching mechanism was proposed from the calculations by density functional theory (DFT). The probes may go through intersystem crossing (from singlet excited state to triplet excited state) to result in fluorescence quenching.

Lysosome-Targeted Single Fluorescence Probe for Two-Channel Imaging Intracellular SO2 and Biothiols.

As the members of reactive sulfur species, SO2 and biothiols play a significant role in physiological and pathological processes and directly influence numerous diseases. Furthermore, SO2 and biothiols can provide a reductive environment for lysosomes to carry out their optimal functionality. To this end, the development of single fluorescent probes for imaging SO2 and biothiols from different emission channels is highly desirable for understanding their physiological nature. Here, a lysosome-targeted fluorescent probe (BPO-DNSP) with a dual reaction site for SO2 and biothiols was presented. BPO-DNSP can sensitively and selectively respond to SO2 in the green channel with a large Stokes shift over 105 nm, and to biothiols in the near-infrared emission channel with a large Stokes shift over 109 nm. The emission shift for the two channels was as high as 170 nm. Colocalization experiments verified that BPO-DNSP can selectively enrich lysosomes. Notably, BPO-DNSP can not only be used to image intracellular SO2 and biothiols from two different channels, but also to monitor the conversion of biothiols to SO2 without adding exogenous enzymes in living HeLa cells.

Supramolecular Assemblies with Near-Infrared Emission Mediated in Two Stages by Cucurbituril and Amphiphilic Calixarene for Lysosome-Targeted Cell Imaging.

A two-stage mediated near-infrared (NIR) emissive supramolecular assembly for lysosome-targeted cell imaging is presented. 4,4'-Anthracene-9,10-diylbis(ethene-2,1-diyl))bis(1-ethylpyridin-1-ium) bromide (ENDT) was synthesized as an organic dye with weak fluorescence emission at 625 nm. When ENDT complexes with cucurbit[8]uril (CB[8]), this binary supramolecular complex assembles into nanorods with a near-infrared fluorescence emission (655 nm) and fluorescence enhancement as the first stage. Such supramolecular complexes interact with lower-rim dodecyl-modified sulfonatocalix[4]arene (SC4AD) to form nanoparticles for further fluorescence enhancement as the second stage. Furthermore, based on a co-staining experiment with LysoTracker Blue, such nanoparticles can be applied in NIR lysosome-targeted cell imaging.

An Acid-Activatable Fluorescent Probe for Sulfur Dioxide in Traditional Chinese Medicines and Living Cells.

Excessive sulfur dioxide (SO2) disturbs physiology of lysosomes causing diseases and threatening human health. A fluorescent probe has been regarded as one of the most attractive approaches, which is compatible with living cells and possesses high sensitivity. However, most of fluorescent probes' reaction sites are activated before they reach the destination. In this work, an acid-activatable fluorescent probe PT1 was synthesized, characterized, and used for SO2 detection. The introduction of oxazolines in PT1 enables the intelligent response of probe to release the activation stie for SO2 derivatives through Michael addition upon exposure to acid. In vitro studies showed a remarkable selectivity of PT1 to SO2 derivatives than other biothiols with a limit of detection as low as 62 nM. By using this acidic pH-controlled fluorescence responsiveness to SO2, precise spatiotemporal identification of lysosomal SO2 fluctuations has been successfully performed. Furthermore, probe PT1 can be applied for monitoring SO2 derivatives in traditional Chinese medicines.

Palbociclib-derived multifunctional molecules for lysosomal targeting and diagnostic-therapeutic integration.

Aim: Lysosomal pH changes are associated with drug resistance, cell growth and invasion of tumors, but effective and specific real-time monitoring of lysosomal pH compounds for cancer therapy is lacking. Materials & methods: Here, based on the covalent linkage of the anticancer drug palbociclib and fluorescent dye fluorescein isothiocyanate (FITC), we designed and developed a novel palbociclib-derived multifunctional molecule (Pal-FITC) for lysosomal targeting and diagnostic therapeutic integration. Results & discussion: Pal-FITC fluoresces is 20-fold stronger than that of FITC and shows a linear response in the pH range of 4.0-8.2 (R2 = 0.9901). Pal-FITC blocks cells in G1 phase via Cyclin D-CDK4/6-Rb. Conclusion: Our study provides new strategies for tumor-targeted imaging and personalized therapy.

Based on the covalent linkage of the anticancer drug and the fluorescent dye, we designed and developed a novel palbociclib-derived multifunctional molecule (Pal-FITC) for lysosomal targeting and diagnostic therapeutic integration. Pal-FITC responded linearly in the pH range of 4.0­8.2. In addition, Pal-FITC was able to effectively treat lung cancer without toxic side effects on normal cells. It has a significant cell cycle blocking phenomenon and blocks G1 phase cells via Cyclin D-CDK4/6-Rb. Our study provides a new strategy for tumor-targeted imaging and personalized therapy.

A lysosome-targeted fluorescent probe for fluorescence imaging of hypochlorous acid in living cells and in vivo.

Lysosomes, as crucial acidic organelles in cells, play a significant role in cellular functions. The levels and distribution of hypochlorous acid (HOCl) within lysosomes can profoundly impact their biological functionality. Hence, real-time monitoring of the concentration of HOCl in lysosomes holds paramount importance for further understanding various physiological and pathological processes associated with lysosomes. In this study, we developed a bodipy-based fluorescent probe derived from pyridine and phenyl selenide for the specific detection of HOCl in aqueous solutions. Leveraging the probe's sensitive photoinduced electron transfer effect from phenyl selenide to the fluorophore, the probe exhibited satisfactory high sensitivity (with a limit of detection of 5.2 nM and a response time of 15 s) to hypochlorous acid. Further biological experiments confirmed that the introduction of the pyridine moiety enabled the probe molecule to selectively target lysosomes. Moreover, the probe successfully facilitated real-time monitoring of HOCl in cell models stimulated by N-acetylcysteine (NAC) and lipopolysaccharide (LPS), as well as in a normal zebrafish model. This provides a universal method for dynamically sensing HOCl in lysosomes.

Dual-site lysosome-targeted fluorescent sensor for fast distinguishing visualization of HClO and ONOO- in living cells and zebrafish.

As two of important highly reactive species / nitrogen species, hypochloric acid (HClO) and peroxynitrite (ONOO-) are involved in various pathological and physiological processes, which are important factors that affect and reflect the functional state of lysosome. Nevertheless, many of their roles are still indefinite because of lack of suitable analytical methods for HClO and ONOO- detection in lysosome. Herein, we designed a lysosome-targeted probe to monitor HClO and ONOO-, which was a hydrid of the benzothiazole derivative, methyl thioether (HClO recognition site) and morpholino hydrazone (ONOO- recognition and lysosome target site). The probe exhibited high sensitivity, good selectivity and fast response toward HClO and ONOO- without spectral crosstalk, and can be employed for quantitative monitoring HClO and ONOO- with LOD of 63 and 83 nM, respectively. In addition, the dual-site probe was lysosome targetable and could be used for detection of HClO and ONOO- in living cells. Furthermore, the excellent behavior made it was suitable for imaging of HClO and ONOO- in zebrafish. Thus, the present probe provides a potent tool for distinguishing monitoring HClO and ONOO- and exploring the role of HClO and ONOO- in biological systems.

Lysosome-targeted silicon quantum dots theranostics for simultaneous fluorescent imaging and photodynamic therapy.

As an emerging treatment method, photodynamic therapy (PDT) has attracted considerable interest due to the characteristics of non-invasiveness, repeatable treatment, high spatiotemporal resolution and few side effects. However, the life span (<40 ns) and diffusion distance (<20 nm) of reactive oxygen species such as singlet oxygen (1O2) in tumor cells are extremely short, which has seriously limited therapeutic efficacy of PDT. The enrichment site of photosensitizers in cancer cells is usually the first site of PDT action, which will not only affect the biological signaling pathway of cancer cell death, but also is closely related to the final therapeutic effect. Therefore, the design and preparation of photosensitizers targeting specific subcellular organelles can directly break the biological function of the organelle and trigger the corresponding cell death signaling pathway, which can significantly improve the efficacy of PDT. Herein, a lysosome-targeted silicon quantum dots (L-Si QDs) was first made by diethylene glycol-mediated synthetic route as a multicolor fluorescent imaging reagents and a new photosensitizer. The as-prepared L-Si QDs exhibit bright fluorescence with excellent pH stability and time stability, excitation-dependent emission, and good biocompatibility. Furthermore, the results of cell experiments showed that L-Si QDs was accumulated in lysosomes after being taken up by cancer cells, and can efficiently produce1O2upon 635 nm laser irradiation, which can damage lysosomes, up-regulate cleavage caspase-3, increase Bax release, down-regulate Bcl-2 and induce cell apoptosis finally. This study significantly broadens the biomedical applications of silicon quantum dots and provides excellent nanomaterials candidates for tumor phototherapy.

Configurationally regulated half-sandwich iridium(III)-ferrocene heteronuclear metal complexes: Potential anticancer agents.

Half-sandwich iridium(III) (IrIII) complexes and ferrocenyl (Fc) derivatives are becoming the research hotspot in the field of anticancer because of their good bioactivity and unique anticancer mechanism different from platinum-based drugs. Then, a series of half-sandwich IrIII-Fc pyridine complexes have been prepared through the structural regulation in this study. The incorporation of half-sandwich IrIII complex with Fc unit successfully improves their anticancer activity, and the optimal performance (IrFc5) is almost 3-fold higher than that of cisplatin against A549 cells, meanwhile, which also shows better anti-proliferative activity against A549/DDP cells. Complexes can aggregate in the intracellular lysosome of A549 cells and induce lysosomal damage, disrupt the cell cycle, increase the level of intracellular reactive oxygen species, and eventually lead to cell apoptosis. Half-sandwich IrIII-Fc heteronuclear metal complexes possess a different anticancer mechanism from cisplatin, which can serve as a potential alternative to platinum-based drugs and show a good application prospect.

Two simple but effective turn-on benzothiazole-based fluorescent probes for detecting hydrogen sulfide in real water samples and HeLa cells.

Two simple turn-on fluorescent probes, containing a benzothiazole and the 2,4-dinitrobenzenesulfonyl group, were designed for detecting H2S. Two probes exhibited good selectivity and high sensitivity, which were applied to detect the H2S in real water samples. Probe P2 with a positive charge had better solubility than probe P1 in water; therefore, probe P2 was successfully applied to detect both the endogenous and exogenous H2S in lysosomes of living HeLa cells.

Lysosome-targeted cyclometalated iridium(III) complexes: JMJD inhibition, dual induction of apoptosis, and autophagy.

A series of cyclometalated iridium(III) complexes with the formula [Ir(C^N)2 L](PF6) (C^N = 2-phenylpyridine (ppy, in Ir-1), 2-(2-thienyl)pyridine (thpy, in Ir-2), 2-(2,4-difluorophenyl)pyridine (dfppy, in Ir-3), L = 2-(1H-imidazo[4,5-f][1,10]phenanthrolin-2-yl)quinolin-8-ol) were designed and synthesized, which utilize 8-hydroxyquinoline derivative as N^N ligands to chelate the cofactor Fe2+ of the Jumonji domain-containing protein (JMJD) histone demethylase. As expected, the results of UV/Vis titration analysis confirm the chelating capabilities of Ir-1-3 for Fe2+, and molecular docking studies also show that Ir-1-3 can interact with the active pocket of JMJD protein, and treatment of cells with Ir-1-3 results in significant upregulation of trimethylated histone 3 lysine 9 (H3K9Me3), indicating the inhibition of JMJD activity. Meanwhile, Ir-1-3 exhibit much higher cytotoxicity against the tested tumor cell lines compared with the clinical chemotherapeutic agent cisplatin. And Ir-1-3 can block the cell cycle at the G2/M phase and inhibit cell migration and colony formation. Further studies show that Ir-1-3 can specifically accumulate in lysosomes, damage the integrity of lysosomes, and induce apoptosis and autophagy. Reduction of mitochondrial membrane potential and elevation of reactive oxygen species also contribute to the antitumor effects of Ir-1-3. Finally, Ir-1 can inhibit tumor growth effectively in vivo and increase the expression of H3K9Me3 in tumor tissues. Our study demonstrates that these iridium(III) complexes are promising anticancer agents with multiple functions, including the inhibition of JMJD and induction of apoptosis and autophagy.

Lysosome­targeted drug combination induces multiple organelle dysfunctions and non­canonical death in pancreatic cancer cells.

Pancreatic cancer is one of the leading causes of cancer­related mortality and has the lowest 5­year survival rate. Therefore, novel strategies are urgently required to treat pancreatic cancer. Pancreatic ductal adenocarcinoma (PDAC) cells rely on enhanced lysosomal function for survival and proliferation to facilitate the degradation of contents accumulated via autophagy and macropinocytosis. Previously, we have reported that the combination of epidermal growth factor receptor/HER2 inhibitor lapatinib and sphingosine analog fingolimod (FTY720) confers a significant cytostatic effect in lung cancer cells. In the present study, the combined effects of these drugs on PDAC cell lines, BxPC­3, KP­4, PANC­1 and MIA PaCa­2, were examined. It was observed that FTY720 enhanced the lapatinib­induced cytotoxic effect and caused non­canonical and lysosome­dependent death in PDAC cells. Lapatinib and FTY720 induced lysosomal swelling and inhibited lysosomal acidification. Combination treatment with lapatinib and FTY720 increased lysosomal membrane permeability, induced mitochondrial depolarization, induced endoplasmic reticulum stress and disturbed intracellular calcium homeostasis. Additionally, the cytotoxic effect of lapatinib was enhanced by hydroxychloroquine or the CDK4/6 inhibitor abemaciclib, both of which induce lysosomal dysfunction. Collectively, these results indicated that the lysosome­targeted drug combination induces multiple organelle dysfunction and exerts a marked cytotoxic effect in PDAC cells.

A lysosome-targeted fluorescent probe for the specific detection and imaging of formaldehyde in living cells.

We presented herien the rational design, synthesis, and photophysical property studies of the lysosome-targeted fluorescence FA probe NP-Lyso, an isopropyl group modified ortho-diaminonaphthalimide derivative. After the reaction of FA and ortho-phenylenediamine modified with the isopropyl group in NP-Lyso, the probe exhibited favorable features such as a large fluorescence enhancement, specific selectivity and high sensitivity for the detection of FA. More importantly, NP-Lyso could be used to detect and image endogenous FA in lysosomes. In light of these prominent properties, we envision that NP-Lyso will be an efficient optical imaging approach for investigating the biofunctions of FA in living systems.

Intracellular protein delivery using QRPL - A vacuolar targeting signal on carboxypeptidase Y.

The signal peptide sequence is known to increase transport efficiency to organelles in eukaryotic cells. In this study, we focus on the signal peptide of the vacuolar protein for vacuolar targeting. The signal peptide sequence QRPL of carboxypeptidase Y (CPY) was inserted inside the interest protein that does not locate in the vacuole for vacuolar targeting. We constructed recombinant strains MBTL-Q-DJ1 and MBTL-Q-DJ2 containing QRPL and green florescent protein (GFP) or aldehyde dehydrogenase 6 (ALD6), respectively. The protein location was then confirmed by confocal microscopy. Fascinatingly, the green fluorescent protein that contains QRPL inside the sequence could be expressed faster than its natural form (within 1 h after induction). Also, the aldehyde removal activity of ALD6 protein in the recombinant yeast was then analyzed by measuring the luminescent intensity in Vibrio fischeri. We confirmed that MBTL-Q-DJ2 containing ALD6 protein has the aldehydes-reducing ability, and in particular, the highest efficiency showed at 500 µg/µL of vacuolar enzyme. In summary, the signal peptide QRPL could be used not only to transport proteins accurately to vacuole but also to improve the protein activity and shorten the induction time.

Near-Infrared Light-Triggered Lysosome-Targetable Carbon Dots for Photothermal Therapy of Cancer.

Photothermal therapy (PTT) has inherent advantages in the treatment of hypoxic tumors due to its optically controlled selectivity on tumor ablation and oxygen-independent nature. The subcellular organelle-targeting capability and photothermal conversion efficiency (PCE) at near-infrared (NIR) wavelength are the key parameters in the assessment of the photothermal agent (PTA). Here, we report that carbon dots (CDs) prepared by the hydrothermal treatment of coronene derivatives show a high PCE of 54.7% at 808 nm, which can be attributed to the narrow band gap and the presence of amounts of continuous energy bands on CDs. Moreover, the vibrations in the layered graphite structures of the CDs also increase the rate of nonradiative transition and thus enhance the PCE. Furthermore, the CDs also possess excellent photostability, biocompatibility, and cell penetration capability and could mainly accumulate in the lysosomes. These experiment results have proved that the CDs are suitable as an efficient NIR light-triggered PTA for efficient PTT against cancer.

A two-photon multi-emissive fluorescent probe for discrimination of Cys and Hcy/GSH via an aromatic substitution-rearrangement.

Cys (Cysteine), Hcy (homocysteine), and GSH (glutathione) are three important kinds of biothiols, playing crucial roles in the variety of pathological and physiological processes. It is greater challenges to simultaneously identify different biothiols due to their similar molecular structures and chemical characteristics. In this work, we employed a multi-emissive fluorescent probe by sulfonyl benzoxadiazole (SBD) with halogen chloride unit as the interaction site based on aromatic substitution-rearrangement strategy to discriminate Cys and Hcy/GSH. The response of probe 1 to Cys would generate FRET and cause a red-shift of fluorescence emission, while Hcy/GSH only lead to different degrees of fluorescence enhancement owing to PET. The probe showed good selectivity, high sensitivity, and low detection limits to three biothiols (Cys: 0.86 µM, Hcy: 0.48 µM and GSH: 0.54 µM). Such capability of the probe could be demonstrated to successfully quantitatively detect the concentrations of Cys/Hcy/GSH in human plasmas. In addition, the probe was also successfully applied for imaging biothiols in lysosomes and real-time monitoring GSH changes in living cells through two-photon fluorescence microscopy.

A lysosome-targeted ratiometric fluorescent probe with a large blue shift for monitoring hypochlorous acid in living cells and zebrafish.

A new phenothiazine derivative as lysosome-targeted fluorescent probe with a large blue-shift (128 nm) for ClO- detection in a fine ratiometric manner has been designed and synthesized. Probe Lyso-PTB has remarkable fluorescence ratiometric variations (98-fold), low cytotoxicity, rapid response time (50 s) and a low detection limit (23 nM). In particular, the application of Lyso-PTB for ClO- detection was successfully demonstrated in lysosome and in zebrafish.

Labeled-protein corona-coated Bi2S3 nanorods targeted to lysosomes for bioimaging and efficient photothermal cancer therapy.

One of the main diseases contributing to human death are malignant tumors. Phototherapy is a promising approach for cancer therapy, and functional nanoparticles with targeting ligands are commonly used to improve the therapeutic efficiency. However, recent studies have shown that nanoparticles in contact with a biological fluid can rapidly form a "protein corona" on their surface, which will remarkably decrease the targeting ability. Here, we describe the preparation of hybrid nanomaterials with Bi2S3 nanorods as the core, and fluorescein-isothiocyanate and folic acid-modified human serum albumin (HSA-FITC-FA) as the shell. By using fluorescent binding label (FITC) and imaging techniques, we discovered the image of the cell lysosomes, indicating that the photothermal therapy agent was predominantly targeted to and accumulated in lysosomes. Combined with photothermal therapy agent (Bi2S3 nanorods) and targeting ligand (FA), the obtained product shows enhanced photothermal therapy under near-infrared region laser irradiation. Additionally, SDS-PAGE shows that the modified HSA shell could remarkably reduce the reabsorption of protein corona from blood serum, minimized the adverse effect of protein corona on targetability. Taken together, the results indicate that our strategy has the potential for preparing efficient photothermal nanomaterials with image-guided subcellular organelle-targeting cancer cell ablation ability.