Lysosomal Localization Research Articles

Anticancer potential of hexamolybdenum clusters [{Mo6I8}(L)6]2− (L = CF3COO− and C6F5COO−) incorporated into different nanoparticulate forms

The work for the first time highlights the specific nanoparticulate form of hexamolybdenum cluster complexes [{Mo6I8}(L)6]2− with L = CF3COO− and C6F5COO− (Mo6) exhibiting high anticancer potential in the irradiation-free conditions. The diverse nanoparticulate forms developed from the Mo6-units include self-aggregated (Bu4N)2[{Mo6I8}(L)6 (Mo6-aggregates) and those deposited onto the specifically decorated silica nanoparticles (SNs). The chemical and colloid stabilities of the as prepared nanoparticles reveal both Mo6-aggregates and those deposited onto the preliminary hydrophilized by polyethyleneimine (PEI) SNs (Mo6-PEI-SNs) as optimal candidates for biomedical applications. High photodynamic activities of Mo6-aggregates and Mo6-PEI-SNs revealed by the spin trap facilitated ESR technique along with the efficient irradiation-induced cytotoxic effects of both nanoparticulate forms indicate their great potential in photodynamic therapy. However, “dark” cytotoxicity measurements for M−HeLa cancer and Chang Liver normal cell lines revealed significant specific anticancer effect of Mo6-aggregates, but the insignificant one of Mo6-PEI-SNs. Quantitative LysoTracker-facilitated colocalization analysis in lysosomal compartments highlighted the highest lysosomal localization of Mo6-aggregates for L = CF3COO−, to a lesser degree for L = C6F5COO−, for Mo6-PEI-SNs being the lowest. The great anticancer effect of the Mo6-aggregates correlates with their highest localization in lysosomal compartment followed by the enhanced aggregation, in turn, derived from the hydrophobic apical ligands.

Mitochondrial ROS Induced Lysosomal Dysfunction and Autophagy Impairment in an Animal Model of Congenital Hereditary Endothelial Dystrophy.

PurposeThe Slc4a11 knock out (KO) mouse model recapitulates the human disease phenotype associated with congenital hereditary endothelial dystrophy (CHED). Increased mitochondrial reactive oxygen species (ROS) in the Slc4a11 KO mouse model is a major cause of edema and endothelial cell loss. Here, we asked if autophagy was activated by ROS in the KO mice.MethodsImmortalized cell lines and mouse corneal endothelia were used to measure autophagy and lysosome associated protein expressions using Protein Simple Wes immunoassay. Autophagy and lysosome functions were examined in wild type (WT) and KO cells as well as animals treated with the mitochondrial ROS quencher MitoQ.ResultsEven though autophagy activation was evident, autophagy flux was aberrant in Slc4a11 KO cells and corneal endothelium. Expression of lysosomal proteins and lysosomal mass were decreased along with reduced nuclear translocation of lysosomal master regulator, transcription factor EB (TFEB). MitoQ reversed aberrant lysosomal functions and TFEB nuclear localization in KO cells. MitoQ injections in KO animals reduced corneal edema and decreased the rate of endothelial cell loss.ConclusionsMitochondrial ROS disrupts TFEB signaling causing lysosomal dysfunction with impairment of autophagy in Slc4a11 KO corneal endothelium. Our study is the first to identify the presence as well as cause of lysosomal dysfunction in an animal model of CHED, and to identify a potential therapeutic approach.

Engineering Mitochondriotropic Carbon Dots for Targeting Cancer Cells.

Aiming to understand and enhance the capacity of carbon dots (CDs) to transport through cell membranes and target subcellular organelles—in particular, mitochondria—a series of nitrogen-doped CDs were prepared by the one-step microwave-assisted pyrolysis of citric acid and ethylenediamine. Following optimization of the reaction conditions for maximum fluorescence, functionalization at various degrees with alkylated triphenylphosphonium functional groups of two different alkyl chain lengths afforded a series of functionalized CDs that exhibited either lysosome or mitochondria subcellular localization. Further functionalization with rhodamine B enabled enhanced fluorescence imaging capabilities in the visible spectrum and allowed the use of low quantities of CDs in relevant experiments. It was thus possible, by the appropriate selection of the alkyl chain length and degree of functionalization, to attain successful mitochondrial targeting, while preserving non-toxicity and biocompatibility. In vitro cell experiments performed on normal as well as cancer cell lines proved their non-cytotoxic character and imaging potential, even at very low concentrations, by fluorescence microscopy. Precise targeting of mitochondria is feasible with carefully designed CDs that, furthermore, are specifically internalized in cells and cell mitochondria of high transmembrane potential and thus exhibit selective uptake in malignant cells compared to normal cells.

Rodlike Particles of Polydopamine-CdTe Quantum Dots: An Actuator As a Photothermal Agent and Reactive Oxygen Species-Generating Nanoplatform for Cancer Therapy.

Herein, novel rodlike CdTe@MPA-PDA particles based on polydopamine (PDA) loaded with CdTe quantum dots (QDs) capped with mercaptopropionic acid (CdTe@MPA QDs) with atypical chemical features are evaluated as a potential actuator for photothermal therapy and oxidative stress induction. Under mild conditions established for the safe and efficient use of lasers, temperature increases of 10.2 and 7.8 °C, photothermal conversion efficiencies of 37.7 and 26.2%, and specific absorption rates of 99 and 69 W/g were obtained for CdTe@MPA-PDA and traditional PDA particles in water, respectively. The particles were set to interact with the human breast adenocarcinoma cell line MDA-MB-231. A significant cellular uptake with the majority of particles colocalized into the lysosomes was obtained at a concentration of 100 μg/mL after 24 h. Additionally, CdTe@MPA-PDA and CdTe@MPA QDs showed significantly different internalization levels and loading kinetics profiles. For the first time, the thermal lens technique was used to demonstrate the stability of particle-like CdTe@MPA-PDA after heating at pH 7 and their migration within the heating region due to the thermodiffusion effect. However, under acidic pH-type lysosomes, a performance decrease in heating was observed, and the chemical feature of the particles was damaged as well. Besides, the internalized rodlike CdTe@MPA-PDA notably enhanced the induction of oxidative stress compared with PDA alone and CdTe@MPA QDs in MDA-MB-231 cells initiating apoptosis. Combining these effects suggests that after meticulous optimizations of the conditions, the CdTe@MPA-PDA particles could be used as a photothermal agent under mild conditions and short incubation time, allowing cytoplasmatic subcellular localization. On the other hand, the same particles act as cell killers by triggering reactive oxygen species after a longer incubation time and lysosomal subcellular localization due to the pH effect on the chemical morphology features of the CdTe@MPA-PDA particles.

A novel lysosome‐localized fluorescent probe with aggregation‐induced emission without alkalinizing effect

AbstractHerein, a novel small molecule probe, tetraphenylethylene‐cystein (TPE‐Cys), was rationally designed and developed for the intracellular lysosome localization. The combination of tetraphenylethylene and cysteine serve as a novel strategy for lysosome‐targeting, with significant aggregation‐induced emission increases with the proportion of water by as high as 25‐folds. Biofluorescence imaging experiments show that the probe TPE‐Cys has a good colocalization effect with the commercially available lysotracker red, with a Pearson correlation coefficient of 0.91. In addition, we also demonstrate that TPE‐Cys has good light stability. TPE‐Cys, as a neutral compound, is free of alkalinizing effect when applied in lysosome localization. Moreover, molecular dynamics simulations and density function theories are employed to explore the detailed aggregation process and the mechanism of TPE‐Cys aggregation in water solution. The mechanism of lysosome localization of probe TPE‐Cys was explained by cellular endocytosis process and the formation of larger particle in the more acidic environment of lysosomes. This work not only demonstrates an efficient strategy for the construction of fluorescent probes for lysosome localization, but also indicates that they are promising as live cell imaging tools.

Luminescent Neutral Cyclometalated Iridium(III) Complexes Featuring a Cubic Polyhedral Oligomeric Silsesquioxane for Lipid Droplet Imaging and Photocytotoxic Applications

New neutral iridium(III) complexes featuring a cubic polyhedral oligomeric silsesquioxane (POSS) unit, [Ir(N∧C)2(L1-POSS)] [HN∧C = 2-phenylpyridine (Hppy; 1), 2-phenylbenzothioazole (Hbt; 2), and 2-(1-naphthyl)benzothiazole (Hbsn; 3); L1-POSS = (E)-4-[(2-hydroxybenzylidene)amino]benzyl 3-heptakis(isobutyl)POSS-propyl carbamate], were designed and synthesized. Their POSS-free counterparts, [Ir(N∧C)2(L1)] [L1 = (E)-N-(4-hydroxymethylphenyl)-1-(2-hydroxyphenyl)methanimine; HN∧C = Hppy (1a), Hbt (2a), and Hbsn (3a)], and the poly(ethylene glycol) (PEG) derivatives [Ir(N∧C)2(L1-PEG)] [L1-PEG = (E)-4-[(2-hydroxybenzylidene)amino]benzyl 3-[2-[ω-methoxypoly(1-oxapropyl)]ethyl]carbamate; HN∧C = Hppy (1b), Hbt (2b), and Hbsn (3b)] were also prepared. The photophysical, photochemical, and biological properties of the POSS complexes were compared with those of their POSS-free and PEG-modified counterparts. Upon irradiation, all of these complexes displayed orange-to-red emission and long emission lifetimes under ambient conditions. The bsn complexes 3, 3a, and 3b exhibited the highest singlet oxygen (1O2) generation quantum yields (ΦΔ = 0.85-0.86) in aerated CH3CN. Laser-scanning confocal microscopy images revealed that complexes 1-3 and 1a-3a showed exclusive lipid-droplet staining upon cellular uptake, while the PEG derivatives 1b-3b displayed lysosomal localization. Complex 3 was utilized to study various lipid-droplet-related biological events including lipid-droplet accumulation under oleic acid stimulation, the movement of lipid droplets, and preadipocyte differentiation. Notably, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays indicated that the ppy complexes 1 and 1b and the bt complexes 2 and 2b were noncytotoxic both in the dark and upon irradiation at 450 nm for 5 min (IC50 > 200 μM), while the bsn complexes 3, 3a, and 3b showed low dark cytotoxicity (IC50 = 52.9 to >200 μM) and high photocytotoxicity (IC50 = 1.1-5.3 μM). The cellular uptake, internalization mechanisms, and cell death pathways of these complexes were also investigated. This work not only offers promising luminescent probes for lipid droplets through the structural modification of iridium(III) complexes but also paves the way to the construction of new reagents for theranostics.

Impact of Endocytosis Mechanisms for the Receptors Targeted by the Currently Approved Antibody-Drug Conjugates (ADCs)-A Necessity for Future ADC Research and Development.

Biologically-based therapies increasingly rely on the endocytic cycle of internalization and exocytosis of target receptors for cancer therapies. However, receptor trafficking pathways (endosomal sorting (recycling, lysosome localization) and lateral membrane movement) are often dysfunctional in cancer. Antibody-drug conjugates (ADCs) have revitalized the concept of targeted chemotherapy by coupling inhibitory antibodies to cytotoxic payloads. Significant advances in ADC technology and format, and target biology have hastened the FDA approval of nine ADCs (four since 2019). Although the links between aberrant endocytic machinery and cancer are emerging, the impact of dysregulated internalization processes of ADC targets and response rates or resistance have not been well studied. This is despite the reliance on ADC uptake and trafficking to lysosomes for linker cleavage and payload release. In this review, we describe what is known about all the target antigens for the currently approved ADCs. Specifically, internalization efficiency and relevant intracellular sorting activities are described for each receptor under normal processes, and when complexed to an ADC. In addition, we discuss aberrant endocytic processes that have been directly linked to preclinical ADC resistance mechanisms. The implications of endocytosis in regard to therapeutic effectiveness in the clinic are also described. Unexpectedly, information on endocytosis is scarce (absent for two receptors). Moreover, much of what is known about endocytosis is not in the context of receptor-ADC/antibody complexes. This review provides a deeper understanding of the pertinent principles of receptor endocytosis for the currently approved ADCs.

Ratiometric fluorescent sensor based on MoS2 QDs and AuNCs for determination and bioimaging of alkaline phosphatase

Herein, a ratiometric fluorescent sensor based on MoS2 quantum dots (QDs) and glutathione-capped gold nanoclusters (AuNCs) was developed for determination and imaging of alkaline phosphatase (ALP). The sensor was developed by covalently linking QDs with AuNCs to form stable MoS2@AuNCs nanohybrids that exhibited the blue fluorescence of MoS2 QDs and the red fluorescence of AuNCs. In the presence of Ce3+, the fluorescence intensity of AuNCs was increased due to the aggregation-induced emission enhancement (AIEE), while that of MoS2 QDs remained unchanged, thus could be used as a reference signal. After adenosine 5′-monophosphate (AMP) and ALP were introduced into the system, AMP was hydrolyzed to adenosine and phosphate ions (PO43−). Owing to higher affinity between Ce3+ and PO43−, the AIEE effect was inhibited, in turn resulting in the decrease of AuNCs fluorescence. The developed ratiometric fluorescent sensor had a linear response to ALP concentration ranging from 0.5 to 50 U L−1 with a detection limit (LOD) of 0.08 U L−1. Moreover, the sensor had low cytotoxicity and was successfully employed in lysosome localization and bioimaging of intracellular ALP in living cells.

734-P: Cellular Internalization and Localization of Once-Weekly Basal Insulin Fc (BIF)

Basal Insulin Fc (BIF, LY3209590) is an insulin Fc-fusion protein in clinical testing as a once weekly treatment for diabetes mellitus. BIF is comprised of a human single-chain insulin fused to a human IgG2 Fc domain through a peptide linker. The cellular internalization characteristics of BIF compared to native insulin using U2OS cells expressing human insulin receptor (hIR) are reported in this work. Internalization and sub-cellular localization were visualized by immuno-fluorescent confocal microscopy in U2OS cells expressing human insulin receptor. Internalization of BIF was observed, and the insulin and Fc moieties remained co-localized. Co-localization studies using an antibody to the early endosomal marker Early Endosome Antigen 1 (EEA1) showed that human insulin and BIF (insulin and Fc moieties) were rapidly internalized and co-localized with EEA1. During ligand washout, the remaining insulin and Fc moieties of BIF remained largely co-localized with EEA1 and a concomitant loss of immunostaining was observed, similar to human insulin. Studies using an antibody to the lysosomal marker Lysosomal Associated Membrane Protein-1 (LAMP-1) revealed a low level of lysosomal localization for human insulin and BIF (insulin and Fc moieties), indicating a similar intracellular trafficking pattern of human insulin and BIF to the lysosomal degradative pathway. The co-localization of remaining ligand decreased over time. IR internalization and transport to early endosomes was measured using an enzyme complementation assay. The EC50 for BIF was 4.7 ± 2.1 nM (n = 3), and the EC50 for human insulin was 0.06 ± 0.01 nM (n = 3). In summary, BIF stimulated IR internalization to a similar maximum level; however, with decreased potency versus human insulin. The subcellular trafficking pattern of BIF is similar to human insulin. BIF undergoes rapid internalization and transport to early endosomes with limited transport to the lysosomes and undergoes loss of cellular immunostaining during ligand washout. Disclosure C. Volk: None. C. Zhang: Employee; Self; Eli Lilly and Company. J. S. Moyers: Employee; Self; Eli Lilly and Company, Stock/Shareholder; Self; Eli Lilly and Company. Funding Eli Lilly and Company

Multifunctional Heterometallic IrIII -AuI Probes as Promising Anticancer and Antiangiogenic Agents.

A new class of emissive cyclometallated IrIII−AuI complexes with a bis(diphenylphosphino) methanide bridging ligand was successfully synthesised from the diphosphino complex [Ir(N^C)2(dppm)]+ (1). The different gold ancillary ligand, a triphenylphosphine (2), a chloride (3) or a thiocytosine (4) did not reveal any significant effect on the photophysical properties, which are mainly due to metal‐to‐ligand charge‐transfer (3MLCT) transitions based on IrIII. However, the AuI fragment, along with the ancillary ligand, seemed crucial for the bioactivity in A549 lung carcinoma cells versus endothelial cells. Both cell types display variable sensitivities to the complexes (IC50=0.6–3.5 μM). The apoptotic pathway is activated in all cases, and paraptotic cell death seems to take place at initial stages in A549 cells. Species 2–4 showed at least dual lysosomal and mitochondrial biodistribution in A549 cells, with an initial lysosomal localisation and a possible trafficking process between both organelles with time. The bimetallic IrIII−AuI complexes disrupted the mitochondrial transmembrane potential in A549 cells and increased reactive oxygen species (ROS) generation and thioredoxin reductase (TrxR) inhibition in comparison with that displayed by the monometallic complex 1. Angiogenic activity assays performed in endothelial cells revealed the promising antimetastatic potential of 1, 2 and 4.

ArfGAP1 inhibits mTORC1 lysosomal localization and activation

The mammalian target of rapamycin complex 1 (mTORC1) integrates nutrients, growth factors, stress, and energy status to regulate cell growth and metabolism. Amino acids promote mTORC1 lysosomal localization and subsequent activation. However, the subcellular location or interacting proteins of mTORC1 under amino acid‐deficient conditions is not completely understood. Here, we identify ADP‐ribosylation factor GTPase‐activating protein 1 (ArfGAP1) as a crucial regulator of mTORC1. ArfGAP1 interacts with mTORC1 in the absence of amino acids and inhibits mTORC1 lysosomal localization and activation. Mechanistically, the membrane curvature‐sensing amphipathic lipid packing sensor (ALPS) motifs that bind to vesicle membranes are crucial for ArfGAP1 to interact with and regulate mTORC1 activity. Importantly, ArfGAP1 represses cell growth through mTORC1 and is an independent prognostic factor for the overall survival of pancreatic cancer patients. Our study identifies ArfGAP1 as a critical regulator of mTORC1 that functions by preventing the lysosomal transport and activation of mTORC1, with potential for cancer therapeutics.

DNA-Based pH Nanosensor with Adjustable FRET Responses to Track Lysosomes and pH Fluctuations.

Extensive attention has been recently focused on designing signal adjustable biosensors. However, there are limited approaches available in this field. In this work, to visually track lysosomes with high contrast, we used the i-motif structure as a pH-responsive unit and proposed a novel strategy to regulate the fluorescence resonance energy transfer (FRET) response of the pH sensor. By simply splitting the i-motif into two parts and modulating the split parameters, we can tune the pH transition midpoint (pHt) from 5.71 to 6.81 and the signal-to-noise ratio (S/N) from 1.94 to 18.11. To facilitate the lysosome tracking, we combined the i-motif split design with tetrahedral DNA (Td). The obtained pH nanosensor (pH-Td) displays appropriate pHt (6.12) to trace lysosomes with high S/N (10.3). Benefited from the improved stability, the superior cell uptake and lysosomal location of pH-Td, the visualization of the distribution of lysosomes, the lysosome-mitochondria interaction, and the pH changes of lysosomes in response to different stimuli were successfully achieved in NIH 3T3 cells. We believe that the design concept of controlling the split sequence distance will provide a novel insight into the design of i-motif-based nanosensors and even inspire the construction of smart DNA nanodevices for sensing, disease diagnosis, and controllable drug delivery.

CHIR99021 Augmented the Function of Late Endothelial Progenitor Cells by Preventing Replicative Senescence.

Endothelial progenitor cells (EPCs) are specialized cells in circulating blood, well known for their ability to form new vascular structures. Aging and various ailments such as diabetes, atherosclerosis and cardiovascular disease make EPCs vulnerable to decreasing in number, which affects their migration, proliferation and angiogenesis. Myocardial ischemia is also linked to a reduced number of EPCs and their endothelial functional role, which hinders proper blood circulation to the myocardium. The current study shows that an aminopyrimidine derivative compound (CHIR99021) induces the inhibition of GSK-3β in cultured late EPCs. GSK-3β inhibition subsequently inhibits mTOR by blocking the phosphorylation of TSC2 and lysosomal localization of mTOR. Furthermore, suppression of GSK-3β activity considerably increased lysosomal activation and autophagy. The activation of lysosomes and autophagy by GSK-3β inhibition not only prevented replicative senescence of the late EPCs but also directed their migration, proliferation and angiogenesis. To conclude, our results demonstrate that lysosome activation and autophagy play a crucial role in blocking the replicative senescence of EPCs and in increasing their endothelial function. Thus, the findings provide an insight towards the treatment of ischemia-associated cardiovascular diseases based on the role of late EPCs.

Red-Light-Responsive Ru Complex Photosensitizer for Lysosome Localization Photodynamic Therapy.

Photoresponsive ruthenium (Ru) complexes have been extensively studied in the photodynamic therapy (PDT) of cancer. The metal-to-ligand charge transfer (MLCT) absorption maximum of most Ru complexes is located in the short-wavelength visible region, which is well suited for superficial tumors but shows inefficient therapeutic effects for more deep-seated ones. Moreover, Ru complexes are primarily located in the mitochondria or nucleus, always resulting in high levels of dark toxicity and DNA mutation. Herein, we reported a new ruthenium complex (Ru-I) for red-light-triggered PDT. The activation wavelength of Ru-I is successfully extended to 660 nm. Importantly, the complex photosensitizer can be quickly taken up by cancer cells and selectively accumulated in the lysosome, an ideal localization for PDT purposes. Intratumoral injection of Ru-I into tumor-bearing mice achieved excellent therapeutic effects and thus holds great promise for applications in lysosome localization photodynamic therapy.

Mitochondrial C3a Receptor Activation in Oxidatively Stressed Epithelial Cells Reduces Mitochondrial Respiration and Metabolism.

Complement component 3 fragment C3a is an anaphylatoxin involved in promoting cellular responses important in immune response and host defense. Its receptor (C3a receptor, C3aR) is distributed on the plasma membrane; however, lysosomal localization in immune cells has been reported. Oxidative stress increases intracellular reactive oxygen species (ROS), and ROS activate complement signaling in immune cells and metabolic reprogramming. Here we tested oxidative stress and intracellular complement in mitochondrial dysfunction in RPE cells using high resolution live-cell imaging, and metabolism analysis in isolated mitochondria using Seahorse technology. While C3aR levels were unaffected by oxidative stress, its cell membrane levels decreased and mitochondrial (mt) localization increased. Trafficking was dependent on endocytosis, utilizing endosomal-to-mitochondrial cargo transfer. H2O2-treatment also increased C3a-mtC3aR co-localization dose-dependently. In isolated mitochondria from H2O2-treated cells C3a increased mitochondrial Ca2+ uptake, that could be inhibited by C3aR antagonism (SB290157), mitochondrial Ca2+ uniporter blocker (Ru360), and Gαi-protein inhibition (pertussis toxin, PTX); and inhibited mitochondrial repiration in an SB290157- and PTX-dependent manner. Specifically, mtC3aR activation inhibited state III ADP-driven respiration and maximal respiratory capacity. Mitochondria from control cells did not respond to C3a. Furthermore, transmitochondrial cybrid ARPE-19 cells harboring J haplogroup mitochondria that confer risk for age-related macular degeneration, showed high levels of mtC3aR and reduced ATP production upon C3a stimulation. Our findings suggest that oxidative stress increases mtC3aR, leading to altered mitochondrial calcium uptake and ATP production. These studies will have important implication in our understanding on the balance of extra- and intracellular complement signaling in controlling cellular health and dysfunction.

Lysosomal retargeting of Myoferlin mitigates membrane stress to enable pancreatic cancer growth.

Lysosomes must maintain the integrity of their limiting membrane to ensure efficient fusion with incoming organelles and degradation of substrates within their lumen. Pancreatic cancer cells upregulate lysosomal biogenesis to enhance nutrient recycling and stress resistance, but it is unknown whether dedicated programmes for maintaining the integrity of the lysosome membrane facilitate pancreatic cancer growth. Using proteomic-based organelle profiling, we identify the Ferlin family plasma membrane repair factor Myoferlin as selectively and highly enriched on the membrane of pancreatic cancer lysosomes. Mechanistically, lysosomal localization of Myoferlin is necessary and sufficient for the maintenance of lysosome health and provides an early acting protective system against membrane damage that is independent of the endosomal sorting complex required for transport (ESCRT)-mediated repair network. Myoferlin is upregulated in human pancreatic cancer, predicts poor survival and its ablation severely impairs lysosome function and tumour growth in vivo. Thus, retargeting of plasma membrane repair factors enhances the pro-oncogenic activities of the lysosome.

A near-infrared and lysosomal targeting thiophene-BODIPY photosensitizer: Synthesis and its imaging guided photodynamic therapy of cancer cells

In this study, a novel NIR and lysosomal targeting thiophene-BODIPY photosensitizer SBOP-Lyso was synthesized to explore its potential applications in photodynamic therapy of A549 cells. In the strategy of designing SBOP-Lyso, S atom in thiophene as well as heavy atom I were introduced to promote ISC efficiency to ensure high singlet oxygen yield. A common lysosome targeted group (M1: 1-(2-morpholinoethyl)-1H-indole-3-carbaldehyde) was linked to SBOP to extend its wavelength to the NIR region. Its absorption peak was at 660 nm (εmax = 5.2 × 104 cm−1 M−1) and its corresponding emission peak was located at 705 nm. Singlet oxygen could be quickly generated by SBOP-Lyso in the presence of 660 nm LED irradiation and the singlet oxygen yield was up to 44.1%. In addition, it also had good biocompatibility and could enter cells or zebrafish in a short time. SBOP-Lyso had negligible dark cytotoxicity (cell survival rate > 80%) and excellent phototoxicity (IC50 = 0.2 μM). DCFH-DA (ROS indicator) proved that SBOP-Lyso could generate singlet oxygen with 660 nm LED irradiation. Singlet oxygen produced by SBOP-Lyso could kill cancer cells in PDT process and it had the ability to effectively inhibit A549 cells migration. Besides that, lysosomal colocalization assay showed that it had good lysosomal localization ability (Pearson colocation coefficient, R = 0.93). Considering the above results, SBOP-Lyso as a unique lysosome-targeted photosensitizer with excellent properties would exhibit positive results in PDT process of cancer cells.

A septin GTPase scaffold of dynein-dynactin motors triggers retrograde lysosome transport.

The metabolic and signaling functions of lysosomes depend on their intracellular positioning and trafficking, but the underlying mechanisms are little understood. Here, we have discovered a novel septin GTPase-based mechanism for retrograde lysosome transport. We found that septin 9 (SEPT9) associates with lysosomes, promoting the perinuclear localization of lysosomes in a Rab7-independent manner. SEPT9 targeting to mitochondria and peroxisomes is sufficient to recruit dynein and cause perinuclear clustering. We show that SEPT9 interacts with both dynein and dynactin through its GTPase domain and N-terminal extension, respectively. Strikingly, SEPT9 associates preferentially with the dynein intermediate chain (DIC) in its GDP-bound state, which favors dimerization and assembly into septin multimers. In response to oxidative cell stress induced by arsenite, SEPT9 localization to lysosomes is enhanced, promoting the perinuclear clustering of lysosomes. We posit that septins function as GDP-activated scaffolds for the cooperative assembly of dynein-dynactin, providing an alternative mechanism of retrograde lysosome transport at steady state and during cellular adaptation to stress.

Chemosensitivity enhanced by autophagy inhibition based on a polycationic nano-drug carrier.

In recent years, with the increasing understanding of the role of autophagy in tumorigenesis and development, a steady stream of studies have demonstrated that both excessive induction and inhibition of autophagy could effectively improve the therapeutic efficacy against tumors during cytotoxic or molecularly targeted drug therapy. Among them, autophagy inhibition mediated by nanomaterials has become an appealing notion in nanomedicine therapeutics, since it can be exploited as an effective adjuvant in chemotherapy or as a potential anti-tumor agent. Herein, we constructed a pH-sensitive nanoplatform loaded with epirubicin (EPI) (mPEG-b-P(DPA-b-DMAEMA)/EPI), enabling effective autophagy inhibition in the process of tumor-targeting therapy and further sensitized the tumors to EPI. It was found that polycationic nanomicelles (PEDD-Ms) displayed specific localization in lysosomes after entering tumor cells and caused the impairment of lysosomal degradation capacity through lysosomal alkalization in a dose-dependent manner. HepG2 cells treated with PEDD-Ms displayed a large-scale accumulation of autophagosomes and LC3 (an autophagosome marker protein), and the degradation of the autophagy substrate p62 was also blocked, which indicated that these functional nanomicelles could significantly inhibit autophagy. Meanwhile, the typical morphological characteristics of autophagosomes were directly visualized by TEM. In vivo results also showed that the tumor-targeted and autophagy inhibition-associated nanoplatform therapy could effectively improve the therapeutic efficiency of EPI, which may be partially attributed to the fact that autophagy inhibition could enhance the sensitivity of tumor cells to EPI. Overall, we revealed the effect of polycationic nanomicelles on autophagic processes in tumor cells and explored their possible molecular mechanism, also considering the synergistic outcome between autophagy mediated by nanomaterials and chemotherapeutic drugs to improve the therapeutic effect on tumors.

Natural steroid-based cationic copolymers cholesterol/diosgenin-r-PDMAEMAs and their pDNA nanoplexes: impact of steroid structures and hydrophobic/hydrophilic ratios on pDNA delivery.

Using natural-based lipids to construct biocompatible, controllable and efficient nanocarriers and elucidating their structure–function relationships, was regarded as an important area for creating sustainable biomaterials. Herein, we utilized two natural steroids: cholesterol and diosgenin (bearing different hydrophobic tails) as the building blocks, to synthesize a series of natural steroid-based cationic random copolymers PMA6Chol-r-PDMAEMA and PMA6Dios-r-PDMAEMA via RAFT polymerization. The results demonstrated that the steroid-r-PDMAEMA copolymers could efficiently bind pDNA (N/P < 3.0) and then form near-spherical shape (142–449 nm) and positively-charged (+11.5 to +19.6 mV) nanoparticles. The in vitro cytotoxicity and gene transfection efficiency greatly depend on the steroid hydrophobic tail structures and steroid/PDMAEMA block ratios. Optimum transfection efficiency of the (Chol-P1/pDNA and Dios-P3/pDNA) nanoplexes could reach to 18.1–31.2% of the PEI-25K/pDNA complex. Moreover, all of the steroid-r-PDMAEMA/Cy3-pDNA nanoplexes have an obvious “lysosome localization” effect, indicating the steroid structures do not remarkably influence the intracellular localization behaviors of these nanoplexes.