AIE-enabled transfection-free identification and isolation of viable cell subpopulations differing in the level of autophagy.

ABBREVIATIONS: 3-MA, 3-methyladenine; AIE, aggregation-induced emission; AIEgens, aggregation-induced emission luminogens; ATG5, autophagy related 5; BMDM, bone marrow-derived macrophage; CQ, chloroquine; DiD, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine perchlorate; DiO, 3,3'-dioctadecyloxacarbocyanine perchlorate; DMSO, dimethyl sulfoxide; d-THP-1, differentiated THP-1; FACS, fluorescence activated cell sorting; FBS, fetal bovine serum; FCCP, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone; GABARAP, GABA type A receptor-associated protein; GFP, green fluorescent protein; HBSS, Hanks' balanced salt solution; HPLC, high-performance liquid chromatography; HRP, horseradish peroxidase; IL1B, interleukin 1 beta; KT, an AIE probe composed of a cell-penetrating peptide and an AIEgen tetraphenyl ethylene; LC3-II, lipidated LC3; LDH, lactate dehydrogenase; LIR, LC3-interacting region; LKR, engineered molecular probe composed of an LC3-interacting peptide, a cell-penetrating peptide and a non-AIE fluorescent molecule rhodamine; LKT, engineered molecular probe composed of an LC3-interacting peptide, a cell-penetrating peptide and an AIEgen tetraphenyl ethylene; LPS, lipopolysaccharide; MAP1LC3/LC3, microtubule associated protein 1 light chain 3; MEF, mouse embryonic fibroblast; mRFP, monomeric red fluorescent protein; NHS, N-hydroxysuccinimide; NLRP3, NLR family pyrin domain containing 3; PBS, phosphate-buffered saline; PCC, pearson's correlation coefficient; PL, photoluminescence; PMA, phorbol 12-myristate 13-acetate; RAP, rapamycin; RIM, restriction of intramolecular motions; s.e.m., standard error of the mean; SPR, surface plasmon resonance; SQSTM1/p62, sequestosome 1; TAX1BP1, Tax1 binding protein 1; TPE, tetraphenylethylene; TPE-yne, 1-(4-ethynylphenyl)-1,2,2-triphenylethene; Tre, trehalose; u-THP-1: undifferentiated THP-1; UV-Vis, ultraviolet visible.

Nickle-cobalt alloy nanocrystals inhibit activation of inflammasomes.

Activation of inflammasomes-immune system receptor sensor complexes that selectively activate inflammatory responses-has been associated with diverse human diseases, and many nanomedicine studies have reported that structurally and chemically diverse inorganic nanomaterials cause excessive inflammasome activation. Here, in stark contrast to reports of other inorganic nanomaterials, we find that nickel-cobalt alloy magnetic nanocrystals (NiCo NCs) actually inhibit activation of NLRP3, NLRC4 and AIM2 inflammasomes. We show that NiCo NCs disrupt the canonical inflammasome ASC speck formation process by downregulating the lncRNA Neat1, and experimentally confirm that the entry of NiCo NCs into cells is required for the observed inhibition of inflammasome activation. Furthermore, we find that NiCo NCs inhibit neutrophil recruitment in an acute peritonitis mouse model and relieve symptoms in a colitis mouse model, again by inhibiting inflammasome activation. Beyond demonstrating a highly surprising and apparently therapeutic impact for an inorganic nanomaterial on inflammatory responses, our work suggests that nickel- and cobalt-containing nanomaterials may offer an opportunity to design anti-inflammatory nanomedicines for the therapeutics of macrophage-mediated diseases.

Melatonin attenuates prostatic inflammation and pelvic pain via Sirt1-dependent inhibition of the NLRP3 inflammasome in an EAP mouse model.

BACKGROUND: Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) is a common male genitourinary system disease. As a neuroendocrine hormone, melatonin possesses a variety of biological functions, among which its anti-inflammatory effects have recently drawn substantial attention. The purpose of the current research was to study the effect of melatonin on CP/CPPS and the underlying mechanisms using a mouse model of experimental autoimmune prostatitis (EAP). METHODS: The EAP mouse model was successfully established by subcutaneously injecting a mixture of prostate antigen and complete Freund's adjuvant. On Day 42, hematoxylin-eosin staining was used to evaluate the histological appearance of prostate tissues. Chronic pelvic pain development was assessed by suprapubic allodynia. The levels of inflammation-related cytokines, such as interferon-γ, interleukin (IL)-17, and IL-1ß, were detected by enzyme-linked immunosorbent assay. Then, we explored the anti-inflammatory effects of melatonin on CP/CPPS by Western blotting and immunohistochemical staining, by measuring the expression of silent information regulator 1 (Sirt1) and NLRP3 inflammasome-related proteins in EAP mice. RESULTS: The EAP model mice exhibited severe diffuse leukocyte infiltration and significantly increased pelvic pain compared to the control mice. In the melatonin treatment group, the histological appearance of the prostate tissues, pelvic pain development, and the levels of proinflammatory cytokines were significantly alleviated compared to the EAP + dimethyl sulfoxide group. Furthermore, we found that the protective effects of melatonin were achieved through activation of the Sirt1 pathway and downregulation of the NLRP3 inflammasome. CONCLUSIONS: The results indicated that melatonin could attenuate prostate inflammation and pelvic pain by inhibiting the NLRP3 inflammasomes signaling pathway through the activation of Sirt1 in mice with EAP, and these efforts should provide a promising therapeutic strategy for CP/CPPS.

Glutathionylation-dependent proteasomal degradation of wide-spectrum mutant p53 proteins by engineered zeolitic imidazolate framework-8.

Point mutations within the DNA-binding domain of the TP53 gene occur in a significant percentage of human cancer, leading to cellular accumulation of highly stabilized mutant p53 proteins (mutp53) with tumor-promoting properties. Depletion of mutp53, through inducing either autophagic or proteasomal degradation, is an attractive strategy for the therapy of p53-mutated cancer, but the currently-known degradation inducers, almost exclusively small molecules, are inadequate. Here we show that pH-responsive zeolitic imidazolate framework-8 (ZIF-8) offers a novel solution to mutp53 degradation. ZIF-8 facilitated ubiquitination-mediated and glutathionylation-dependent proteasomal degradation of all of the nine mutp53 we tested, including six hot-spot mutp53, but not the wild-type p53 protein. Sustained elevation of intracellular Zn++ level, resulted from decomposition of the internalized ZIF-8 in the acidic endosomes, decreased the intracellular reduced glutathione (GSH): oxidized glutathione (GSSG) ratio and was essential for mutp53 glutathionylation and degradation. ZIF-8 modified with an Z1-RGD peptide, exhibiting enhanced cellular internalization and improved decomposition behavior, preferentially killed mutp53-expressing cancer cells and demonstrated remarkable therapeutic efficacy in a p53 S241F ES-2 ovarian cancer model as well as in a p53 Y220C patient-derived xenograft (PDX) breast cancer model. The ability to induce wide-spectrum mutp53 degradation gives ZIF-8 a clear advantage over other degradation-inducers, and engineered nanomaterials may be promising alternatives to small molecules for the development of mutp53-targeting drugs.

Rationally designed rapamycin-encapsulated ZIF-8 nanosystem for overcoming chemotherapy resistance.

Zeolitic imidazolate framework-8 (ZIF-8) nanoparticles are widely reported as a pH-sensitive drug delivery carrier with high loading capacity for tumor therapy. However, the mechanism of intracellular corrosion of ZIF-8 and the corresponding biological effects especially for autophagy response have been rarely reported. Herein, the as-synthesized ZIF-8 was demonstrated to induce mTOR independent and pro-death autophagy. Interestingly, the autophagic process participated in the corrosion of ZIF-8. Subsequently, zinc ion release and the generation of reactive oxygen species due to its corrosion in the acidic compartments were directly responsible for tumor cell killing. In addition, ZIF-8 could sensitize tumor cells to chemotherapy by switching cytoprotective to death promoting autophagy induced by doxorubicin. The mTOR signaling pathway activation was demonstrated to restrict tumor chemotherapy efficiency. Hence, a combined platform rapamycin encapsulated zeolitic imidazolate frameworks (Rapa@ZIF-8) was constructed and demonstrated a more significant chemo-sensitized effect relative to ZIF-8 nanoparticles or rapamycin treatment alone. Lastly, the combined administration of Rapa@ZIF-8 and doxorubicin exhibited an outstanding synergistic antitumor effect without any obvious toxicity to the major organs of mice. Collectively, the optimized nanoplatform, Rapa@ZIF-8, provides a proof of concept for intentionally interfering mTOR pathway and utilizing the switch of survival-to death-promoting autophagy for adjunct chemotherapy.

Enhancing tumor chemotherapy and overcoming drug resistance through autophagy-mediated intracellular dissolution of zinc oxide nanoparticles.

Autophagy may represent a common cellular response to nanomaterials. In the present study, it was demonstrated that zinc oxide nanoparticle (ZON)-elicited autophagy contributes to tumor cell killing by accelerating the intracellular dissolution of ZONs and reactive oxygen species (ROS) generation. In particular, ZONs could promote Atg5-regulated autophagy flux without the impairment of autophagosome-lysosome fusion, which is responsible for ZON-elicited cell death in cancer cells. On the other hand, a further study revealed that a significant free zinc ion release in lysosomal acid compartments and sequential ROS generation in cells treated with ZONs were also associated with tumor cytotoxicity. Intriguingly, the colocalization between FITC-labeled ZONs and autophagic vacuoles indicates that the intracellular fate of ZONs is associated with autophagy. Moreover, the chemical or genetic inhibition of autophagy significantly reduced the level of intracellular zinc ion release and ROS generation separately, demonstrating that ZON-induced autophagy contributed toward cancer cell death by accelerating zinc ion release and sequentially increasing intracellular ROS generation. The modulation of autophagy holds great promise for improving the efficacy of tumor chemotherapy. Herein, ZONs were verified to enhance chemotherapy in both normal and drug-resistant cancer cells via synergistic autophagy elicitation. Further, this elicitation resulted in tremendous zinc ion release and ROS generation, which accounted for enhancing the tumor chemotherapy and overcoming drug resistance. No obvious changes in the expression level of P-gp proteins or the amount of doxorubicin uptake induced by ZONs in MCF-7/ADR cells also indicated that the increased zinc ion release and ROS generation via synergistic autophagy induction were responsible for overcoming the drug resistance. Finally, in vivo experiments involving animal models of 4T1 tumor cells revealed that the antitumor therapeutic effect of a combinatory administration obviously outperformed those of ZONs or free doxorubicin treatment alone at the same dose, which could be attenuated by the autophagy inhibitor wortmannin or ion-chelating agent EDTA. Taken together, our results reveal the mechanism wherein the autophagy induction by ZONs potentiates cancer cell death and a novel biological application for ZONs in adjunct chemotherapy in which autophagy reinforces zinc ion release and ROS generation.

MnFe2O4 nanoparticles accelerate the clearance of mutant huntingtin selectively through ubiquitin-proteasome system.

Neurodegenerative disorders such as Huntington's disease (HD) are fundamentally caused by accumulation of misfolded aggregate-prone proteins. Previous investigations have shown that these toxic protein aggregates could be degraded through autophagy induced by small molecules as well as by nanomaterials. However, whether engineered nanomaterials have the capacity to degrade these protein aggregates via the ubiquitin-proteasome system (UPS), the other major pathway for intracellular protein turnover, was unknown. Herein, we have synthesized biocompatible MnFe2O4 nanoparticles (NPs) and demonstrated their unique effect in accelerating the clearance of mutant huntingtin (Htt) protein exhibiting 74 glutamine repeats [Htt(Q74)]. UPS, rather than autophagy, was responsible for the efficient Htt(Q74) degradation facilitated by MnFe2O4 NPs. Meanwhile, we demonstrated that MnFe2O4 NPs enhanced K48-linked ubiquitination of GFP-Htt(Q74). Moreover, ubiqinlin-1, but not p62/SQSTM1, served as the ubiquitin receptor that mediated the enhanced degradation of Htt(Q74) by MnFe2O4 NPs. Our findings may have implications for developing novel nanomedicine for the therapy of HD and other polyglutamine expansion diseases.

Blood Circulation-Prolonging Peptides for Engineered Nanoparticles Identified via Phage Display.

Sustaining blood retention for theranostic nanoparticles is a big challenge. Various approaches have been attempted and have demonstrated some success but limitations remain. We hypothesized that peptides capable of increasing blood residence time for M13 bacteriophage, a rod-shaped nanoparticle self-assembled from proteins and nucleic acids, should also prolong blood circulation for engineered nanoparticles. Here we demonstrate the feasibility of this approach by identifying a series of blood circulation-prolonging (BCP) peptides through in vivo screening of an M13 peptide phage display library. Intriguingly, the majority of the identified BCP peptides contained an arginine-glycine-aspartic acid (RGD) motif, which was necessary but insufficient for the circulation-prolonging activity. We further demonstrated that the RGD-mediated specific binding to platelets was primarily responsible for the enhanced blood retention of BCP1. The utility of the BCP1 peptide was demonstrated by fusion of the peptide to human heavy-chain ferritin (HFn), leading to significantly improved pharmacokinetic profile, enhanced tumor cell uptake and optimum anticancer efficacy for doxorubicin encapsulated in the HFn nanocage. Our results provided a proof-of-concept for an innovative yet simple strategy, which utilizes phage display to discover novel peptides with the capability of substantially prolonging blood circulation for engineered theranostic nanoparticles.

Plasmonic MoO3-x nanoparticles incorporated in Prussian blue frameworks exhibit highly efficient dual photothermal/photodynamic therapy.

Development of near infrared (NIR) light-responsive nanomaterials for high performance multimodal phototherapy within a single nanoplatform is still challenging in technology and biomedicine. Herein, a new phototherapeutic nanoagent based on FDA-approved Prussian blue (PB) functionalized oxygen-deficient molybdenum oxide nanoparticles (MoO3-x NPs) is strategically designed and synthesized by a facile one-pot size/morphology-controlled process. The as-prepared PB-MoO3-x nanocomposites (NCs) with a uniform particle size of ∼90 nm and high water dispersibility exhibited strong optical absorption in the first biological window, which is induced by plasmon resonance in an oxygen-deficient MoO3-x semiconductor. More importantly, PB-MoO3-x NCs not only exhibited a high photothermal conversion efficiency of ∼63.7% and photostability but also offered a further approach for the generation of reactive oxygen species (ROS) upon singular NIR light irradiation which significantly improved the therapeutic efficiency of the PB agent. Furthermore, PB-MoO3-x NCs showed a negligible cytotoxic effect in the dark, but an excellent therapeutic effect toward two triple-negative breast cancer (TNBC) cell lines at a low concentration (20 µg mL-1) of NCs and a moderate NIR laser power density. Additionally, efficient tumor ablation and metastasis inhibition in a 4T1 TNBC mouse tumor model can also be realized by synergistic photothermal/photodynamic therapy (PTT/PDT) under a single continuous NIR wave laser. Taken together, this study paved the way for the use of a single nanosystem for multifunctional therapy.

Impact of Morphology on Iron Oxide Nanoparticles-Induced Inflammasome Activation in Macrophages.

Inflammasomes, a critical component of the innate immune system, mediate much of the inflammatory response manifested by engineered nanomaterials. Iron oxide nanoparticles (IONPs), a type of nanoparticles that have gained widespread acceptance in preclinical and clinical settings, are known to induce inflammasome activation, but how morphology affects the inflammasome-activating property of IONPs has not been addressed. In this report, we have synthesized four morphologically distinct IONPs having the same aspect ratio and similar surface charge, thus offering an ideal system to assess the impact of morphology on nanoparticle-elicited biological effect. We show that morphology was a critical determinant for IONP-induced IL-1ß release and pyroptosis, with the octapod and plate IONPs exhibiting significantly higher activity than the cube and sphere IONPs. The inflammasome-activating capacity of different IONPs correlated with their respective ability to elicit intracellular reactive oxygen species generation, lysosomal damage, and potassium efflux, three well-known mechanisms for nanoparticle-facilitated inflammasome activation. Furthermore, we demonstrate that the release of IL-1ß induced by IONPs was only partly mediated by NLRP3, suggesting that inflammasomes other than NLRP3 are also involved in IONP-induced inflammasome activation. Our results may have implications for designing safer nanoparticles for in vivo applications.

Harnessing copper-palladium alloy tetrapod nanoparticle-induced pro-survival autophagy for optimized photothermal therapy of drug-resistant cancer.

Chemo-PTT, which combines chemotherapy with photothermal therapy, offers a viable approach for the complete tumor eradication but would likely fail in drug-resistant situations if conventional chemotherapeutic agents are used. Here we show that a type of copper (Cu)-palladium (Pd) alloy tetrapod nanoparticles (TNP-1) presents an ideal solution to the chemo-PTT challenges. TNP-1 exhibit superior near-infrared photothermal conversion efficiency, thanks to their special sharp-tip structure, and induce pro-survival autophagy in a shape- and composition-dependent manner. Inhibition of autophagy with 3-methyl adenine or chloroquine has a remarkable synergistic effect on TNP-1-mediated PTT in triple-negative (4T1), drug-resistant (MCF7/MDR) and patient-derived breast cancer models, achieving a level of efficacy unattainable with TNP-2, the identically-shaped CuPd nanoparticles that have a higher photothermal conversion efficiency but no autophagy-inducing activity. Our results provide a proof-of-concept for a chemo-PTT strategy, which utilizes autophagy inhibitors instead of traditional chemotherapeutic agents and is particularly useful for eradicating drug-resistant cancer.

The Ethyl Acetate Extract of Gynura formosana Kitam. Leaves Inhibited Cervical Cancer Cell Proliferation via Induction of Autophagy.

Gynura formosana Kitam. belongs to the Compositae family and has been traditionally used for the prevention of cancer, diabetes, and inflammation in China. Previous studies had indicated that the ethyl acetate extract of Gynura formosana Kitam. leaves (EAEG) exhibited antioxidant and anti-inflammatory activity. In this report, we demonstrated that EAEG possessed potent anticancer activity through autophagy-mediated inhibition of cell proliferation. EAEG induced a strong cytostatic effect towards HeLa cells and, to a lesser extent, HepG2 and MCF-7 cells. This cytostatic effect of EAEG was not a consequence of increased apoptosis, as neither DNA fragmentation nor change in protein expression level for a number of apoptosis-related genes including Bid, Bax, Bcl-2, and caspase-3 was observed after EAEG treatment, and the apoptosis inhibitor Z-VAD-FMK did not inhibit the EAEG-elicited cytostatic effect. On the other hand, EAEG induced autophagy in a dose-dependent fashion, as shown by increased GFP puncta formation, enhanced conversion of the microtubule-associated protein light chain LC3-I to LC3-II, and downregulation of the p62 protein. Treating the HeLa cells with EAEG together with Chloroquine (CQ) further accelerated LC3 conversion and p62 clearance, indicating that EAEG induced complete autophagy flux. Importantly, the autophagy inhibitor 3-methyladenine (3MA) significantly abrogated the cytostatic effect of EAEG, strongly suggesting that EAEG inhibited HeLa cell proliferation through the induction of autophagy rather than apoptosis. Our results provided a novel and interesting mechanistic insight into the anticancer action of EAEG, supporting the traditional use of this plant for the treatment of the cancer.

Caspase mediated beclin-1 dependent autophagy tuning activity and apoptosis promotion by surface modified hausmannite nanoparticle.

Hidden effects of nano-materials to induced autophagy, a lysosomal degradative pathway, remain an exciting topic, in the level of material-protein interaction and subsequent cellular signaling features. Here, our studies show that surface modified hausmannite nanoparticles (Mn3 O4 NPs) can uniformly cleave/splice Beclin-1 protein and alter cellular mechanism on the emphasis of tuning autophagy and subsequently promote enhancement of apoptosis. Details investigation of Beclin-1 dependency and its uniform cleavage/splice pattern by surface modified Mn3 O4 NPs, shows tuning of cellular mechanism on emphasis of caspase mediated autophagy tuning. Our findings will also clarify the conflict between apoptosis-autophagy on the basis of its unique property derived from surface chemistry modulation, in context of Beclin-1 eminent cleavage/splice which remarks novel effect of Beclin-1 dependent tuning of autophagosomes formation and switch to enhance apoptotic index, mediates by PI3KC3 cleavage and caspase activation. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1299-1310, 2017.

Persistency of Enlarged Autolysosomes Underscores Nanoparticle-Induced Autophagy in Hepatocytes.

The diverse biological effects of nanomaterials form the basis for their applications in biomedicine but also cause safety issues. Induction of autophagy is a cellular response after nanoparticles exposure. It may be beneficial in some circumstances, yet autophagy-mediated toxicity raises an alarming concern. Previously, it has been reported that upconversion nanoparticles (UCNs) elicit liver damage, with autophagy contributing most of this toxicity. However, the detailed mechanism is unclear. This study reveals persistent presence of enlarged autolysosomes in hepatocytes after exposure to UCNs and SiO2 nanoparticles both in vitro and in vivo. This phenomenon is due to anomaly in the autophagy termination process named autophagic lysosome reformation (ALR). Phosphatidylinositol 4-phosphate (PI(4)P) relocates onto autolysosome membrane, which is a key event of ALR. PI(4)P is then converted into phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2 ) by phosphatidylinositol-4-phosphate 5-kinase. Clathrin is subsequently recruited by PI(4,5)P2 and leads to tubule budding of ALR. Yet it is observed that PI(4)P cannot be converted in nanoparticle-treated hepatocytes cells. Exogenous supplement of PI(4,5)P2 suppresses the enlarged autolysosomes in vitro. Abolishment of these enlarged autolysosomes by autophagy inhibitor relieves the hepatotoxicity of UCNs in vivo. The results provide evidence for disrupted ALR in nanoparticle-treated hepatocytes, suggesting that the termination of nanoparticle-induced autophagy is of equal importance as the initiation.

Inhibition of lanthanide nanocrystal-induced inflammasome activation in macrophages by a surface coating peptide through abrogation of ROS production and TRPM2-mediated Ca(2+) influx.

Lanthanide-based nanoparticles (LNs) hold great promise in medicine. A variety of nanocrystals, including LNs, elicits potent inflammatory response through activation of NLRP3 inflammasome. We have previously identified an LNs-specific surface coating peptide RE-1, with the sequence of 'ACTARSPWICG', which reduced nanocrystal-cell interaction and abrogated LNs-induced autophagy and toxicity in both HeLa cells and liver hepatocytes. Here we show that RE-1 coating effectively inhibited LNs-induced inflammasome activation, mostly mediated by NLRP3, in mouse bone marrow derived macrophage (BMDM) cells, human THP-1 cells and mouse peritoneal macrophages and also reduced LNs-elicited inflammatory response in vivo. RE-1 coating had no effect on cellular internalization of LNs in BMDM cells, in contrast to the situation in HeLa cells where cell uptake of LNs was significantly inhibited by RE-1. To elucidate the molecular mechanism underlying the inflammasome-inhibiting effect of RE-1, we assessed several parameters known to influence nanocrystal-induced NLRP3 inflammasome activation. RE-1 coating did not reduce potassium efflux, which occurred after LNs treatment in BMDM cells and was necessary but insufficient for LNs-induced inflammasome activation. RE-1 did decrease lysosomal damage induced by LNs, but the inhibitor of cathepsin B did not affect LNs-elicited caspase 1 activation and IL-1ß release, suggesting that lysosomal damage was not critically important for LNs-induced inflammasome activation. On the other hand, LNs-induced elevation of intracellular reactive oxygen species (ROS), critically important for inflammasome activation, was largely abolished by RE-1 coating, with the reduction on NADPH oxidase-generated ROS playing a more prominent role for RE-1's inflammasome-inhibiting effect than the reduction on mitochondria-generated ROS. ROS generation further triggered Ca(2+) influx, an event that was mediated by Transient Receptor Potential M2 (TRPM2) and was necessary for inflammasome activation, and this event was completely inhibited by RE-1 coating. We conclude from these studies that inhibition of ROS production, and the subsequent abrogation of TRPM2-mediated Ca(2+) influx, is the primary mechanism underlying RE-1's inhibitory effect on LNs-induced inflammasome activation. The ability of regulating the inflammatory response of nanocrystals through peptide surface coating may be of great value for in vivo applications of LNs and other engineered nanomaterials.

Giant Cellular Vacuoles Induced by Rare Earth Oxide Nanoparticles are Abnormally Enlarged Endo/Lysosomes and Promote mTOR-Dependent TFEB Nucleus Translocation.

Many nanomaterials are reported to disrupt lysosomal function and homeostasis, but how cells sense and then respond to nanomaterial-elicited lysosome stress is poorly understood. Nucleus translocation of transcription factor EB (TFEB) plays critical roles in lysosome biogenesis following lysosome stress induced by starvation. The authors previously reported massive cellular vacuolization, along with autophagy induction, in cells treated with rare earth oxide (REO) nanoparticles. Here, the authors identify these giant cellular vacuoles as abnormally enlarged and alkalinized endo/lysosomes whose formation is dependent on macropinocytosis. This vacuolization causes deactivation of mammalian target of rapamycin (mTOR), a TFEB-interacting kinase that resides on the lysosome membrane. Subsequently, TFEB is dephosphorylated at serine 142 and translocated into cell nucleus. This nucleus translocation of TFEB is observed only in vacuolated cells and it is critical for maintaining lysosome homeostasis after REO nanoparticle treatment, as knock-down of TFEB gene significantly compromises lysosome function and enhances cell death in nanoparticle-treated cells. Our results reveal that cellular vacuolization, which is commonly observed in cells treated with REOs and other nanomaterials, represents a condition of profound lysosome stress, and cells sense and respond to this stress by facilitating mTOR-dependent TFEB nucleus translocation in an effort to restore lysosome homeostasis.

Nanoparticle-facilitated autophagy inhibition promotes the efficacy of chemotherapeutics against breast cancer stem cells.

Cancer stem cells (CSCs) have garnered increasing attention over the past decade, as they are believed to play a crucial role in tumor initiation, progression and metastasis, relapse and drug resistance. Therapeutic strategies which simultaneously exterminate both bulk tumor cells and the rare CSC subpopulation may produce striking response and result in long-term tumor remission. Accumulating evidence provides insight into the function of autophagy in maintenance, plasticity and survival of CSCs. The role of autophagy in the susceptibility of breast CSCs to chemotherapeutics was investigated in the present work, reduced 'stemness' and increased susceptibility to chemotherapy drugs (doxorubicin, DOX and docetaxel, DTXL) were observed after chloroquine (CQ)-mediated autophagy inhibition in sorted ALDH(hi) cells of breast cancer cell line MDA-MB-231. We further proved that nanoparticle-mediated autophagy inhibition promoted the efficacy of chemotherapeutics against ALDH(hi) MDA-MB-231 cells in vitro. Administration of drug delivery systems significantly prolonged the circulation half-life and augmented enrichment of two different drugs in tumor tissues and ALDH(hi) cells. More importantly, compared with single treatment, the combined delivery systems NPCQ/NPDOX and NPCQ/DOX (NPCQ/NPDTXL and NPCQ/DTXL) showed most effective and persistent tumor growth inhibitory effect by eliminating bulk tumor cells as well as CSCs (p < 0.01) in an MDA-MB-231 orthotopic tumor murine model. Therefore, our research provides new insights into the nanoparticle-facilitated combination of autophagy inhibition and chemotherapy for effective therapy of breast cancer.

Lanthanide co-doped paramagnetic spindle-like mesocrystals for imaging and autophagy induction.

We synthesized two novel lanthanide doped spindle-like mesocrystals, YF3:Ce,Eu,Gd and YF3:Ce,Tb,Gd (abbreviated as YEG and YTG mesospindles, respectively). Both of them possess paramagnetic and fluorescent properties, and their excellent cyto-compatibility and low haemolysis are further confirmed. Therefore, they could act as dual mode contrast agents for magnetic resonance imaging (MRI) and fluorescence imaging. Furthermore, YEG and YTG mesospindles induce dose and time dependent autophagy by activating the PI3K signaling pathway. The autophagy induced by YEG and YTG mesocrystals is confirmed by enhanced autophagosome formation, normal cargo degradation, and no disruption of lysosomal function. This work is important to illustrate how rare-earth mesocrystals affect the autophagic pathway, indicating the potential of the YEG and YTG mesospindles in diagnosis and therapy.

Differential ERK activation during autophagy induced by europium hydroxide nanorods and trehalose: Maximum clearance of huntingtin aggregates through combined treatment.

Accelerating the clearance of intracellular protein aggregates through elevation of autophagy represents a viable approach for the treatment of neurodegenerative diseases. In our earlier report, we have demonstrated the enhanced degradation of mutant huntingtin protein aggregates through autophagy process induced by europium hydroxide nanorods [EHNs: Eu(III)(OH)3], but the underlying molecular mechanism of EHNs mediated autophagy was unclear. The present report reveals that EHNs induced autophagy does not follow the classical AKT-mTOR and AMPK signaling pathways. The inhibition of ERK1/2 phosphorylation using the specific MEK inhibitor U0126 partially abrogates the autophagy as well as the clearance of mutant huntingtin protein aggregates mediated by EHNs suggesting that nanorods stimulate the activation of MEK/ERK1/2 signaling pathway during autophagy process. In contrast, another mTOR-independent autophagy inducer trehalose has been found to induce autophagy without activating ERK1/2 signaling pathway. Interestingly, the combined treatment of EHNs and trehalose leads to more degradation of mutant huntingtin protein aggregates than that obtained with single treatment of either nanorods or trehalose. Our results demonstrate the rational that further enhanced clearance of intracellular protein aggregates, needed for diverse neurodegenerative diseases, may be achieved through the combined treatment of two or more autophagy inducers, which stimulate autophagy through different signaling pathways.

Oxidative stress, apoptosis, and cell cycle arrest are induced in primary fetal alveolar type II epithelial cells exposed to fine particulate matter from cooking oil fumes.

Epidemiological studies demonstrate a linkage between morbidity and mortality and particulate matter (PM), particularly fine particulate matter (PM2.5) that can readily penetrate into the lungs and are therefore more likely to increase the incidence of respiratory and cardiovascular diseases. The present study investigated the compositions of cooking oil fume (COF)-derived PM2.5, which is the major source of indoor pollution in China. Furthermore, oxidative stress, cytotoxicity, apoptosis, and cell cycle arrest induced by COF-derived PM2.5 in primary fetal alveolar type II epithelial cells (AEC II cells) were also detected. N-acetyl-L-cysteine (NAC), a radical scavenger, was used to identify the role of oxidative stress in the abovementioned processes. Our results suggested that compositions of COF-derived PM2.5 are obviously different to PM2.5 derived from other sources, and COF-derived PM2.5 led to cell death, oxidative stress, apoptosis, and G0/G1 cell arrest in primary fetal AEC II cells. Furthermore, the results also showed that COF-derived PM2.5 induced apoptosis through the endoplasmic reticulum (ER) stress pathway, which is indicated by the increased expression of ER stress-related apoptotic markers, namely GRP78 and caspase-12. Besides, the induction of oxidative stress, cytotoxicity, apoptosis, and cell cycle arrest was reversed by pretreatment with NAC. These findings strongly suggested that COF-derived PM2.5-induced toxicity in primary fetal AEC II cells is mediated by increased oxidative stress, accompanied by ER stress which results in apoptosis.