Aldehyde dehydrogenase 2 deficiency reinforces formaldehyde-potentiated pro-inflammatory responses and glycolysis in macrophages.

Aldehyde dehydrogenase 2 (ALDH2) deficiency caused by   genetic variant is present in more than 560 million people of East Asian descent, which can be identified by apparent facial flushing from acetaldehyde accumulation after consuming alcohol. Recent findings indicated that ALDH2 also played a critical role in detoxification of formaldehyde (FA). Our previous studies showed that FA could enhance macrophagic inflammatory responses through the induction of HIF-1α-dependent glycolysis. In the present study, pro-inflammatory responses and glycolysis promoted by 0.5 mg/m3 FA were found in mice with Aldh2 gene knockout, which was confirmed in the primary macrophages isolated from Aldh2 gene knockout mice treated with 50 µM FA. FA at 50 and 100 µM also induced stronger dose-dependent increases of pro-inflammatory responses and glycolysis in RAW264.7 murine macrophages with knock-down of ALDH2, and the enhanced effects induced by 50 µM FA was alleviated by inhibition of HIF-1α in RAW264.7 macrophages with ALDH2 knock-down. Collectively, these results clearly demonstrated that ALDH2 deficiency reinforced pro-inflammatory responses and glycolysis in macrophages potentiated by environmentally relevant concentration of FA, which may increase the susceptibility to inflammation and immunotoxicity induced by environmental FA exposure.

Sequential Rocket-Mode Bioactivating Ticagrelor Prodrug Nanoplatform Combining Light-Switchable Diphtherin Transgene System for Breast Cancer Metastasis Inhibition.

The increased risk of breast cancer metastasis is closely linked to the effects of platelets. Our previously light-switchable diphtheria toxin A fragment (DTA) gene system, known as the LightOn system, has demonstrated significant therapeutic potential; it lacks antimetastatic capabilities. In this study, we devised an innovative system by combining cell membrane fusion liposomes (CML) loaded with the light-switchable transgene DTA (pDTA) and a ticagrelor (Tig) prodrug. This innovative system, named the sequential rocket-mode bioactivating drug delivery system (pDTA-Tig@CML), aims to achieve targeted pDTA delivery while concurrently inhibiting platelet activity through the sequential release of Tig triggered by reactive oxygen species with the tumor microenvironment. In vitro investigations have indicated that pDTA-Tig@CML, with its ability to sequentially release Tig and pDTA, effectively suppresses platelet activity, resulting in improved therapeutic outcomes and the mitigation of platelet driven metastasis in breast cancer. Furthermore, pDTA-Tig@CML exhibits enhanced tumor aggregation and successfully restrains tumor growth and metastasis. It also reduces the levels of ADP, ATP, TGF-ß, and P-selectin both in vitro and in vivo, underscoring the advantages of combining the bioactivating Tig prodrug nanoplatform with the LightOn system. Consequently, pDTA-Tig@CML emerges as a promising light-switchable DTA transgene system, offering a novel bioactivating prodrug platform for breast cancer treatment.

Accumulation of Intracellular Ferrous Iron in Inflammatory-Activated Macrophages.

Macrophages are important innate immune cells which can be polarized into heterogeneous populations. The inflammatory-activated M1 cells are known to be involved in all kinds of inflammatory diseases, which were also found to be associated with dysregulation of iron metabolism. While iron overload is known to induce M1 polarization, the valence states of iron and its intracellular dynamics during macrophage inflammatory activation have not been identified. In this study, THP-1-derived macrophages were polarized into M1, M2a, M2b, M2c, and M2d cells, and intracellular ferrous iron (Fe(II)) was measured by our previously developed ultrasensitive Fe(II) fluorescent probe. Significant accumulation of Fe(II) was only observed in M1 cells, which was different from the alterations of total iron. Time-dependent change of intracellular Fe(II) during the inflammatory activation was also consistent with the expression shifts of transferrin receptor CD71, ferrireductase Steap3, and Fe(II) exporter Slc40a1. In addition, accumulation of Fe(II) was also found in the colon macrophages of mice with ulcerative colitis, which was positively correlated to inflammatory phenotypes, including the productions of NO, IL-1ß, TNF-α, and IL-6. Collectively, these results demonstrated the specific accumulation of Fe(II) in inflammatory-activated macrophages, which not only enriched our understanding of iron homeostasis in macrophages, but also indicated that Fe(II) could be further developed as a potential biomarker for inflammatory-activated macrophages.

Arsenite inhibits M2a polarization of macrophages through downregulation of peroxisome proliferator-activated receptor gamma.

Arsenite (As+3) is a group one human carcinogen, which has been associated with many diseases. Previous studies indicated that As+3 could inhibit wound healing and repair. M2a cells are known as tissue remodeling macrophages, which play an important role in wound repair process. Peroxisome proliferator-activated receptor gamma (PPAR-γ), a key regulator of lipid and glucose metabolism, was found to mediate the IL-4-dependent M2a polarization of macrophages. In the present study, As+3 induced dose-dependent inhibition of M2a polarization starting from 0.1 µM in THP-1-derived macrophages stimulated with 20 ng/mL IL-4. Increased lipid accumulation and decreased PPAR-γ expression were also observed in As+3-treated M2a macrophages. Rosiglitazone (RSG), a potent PPAR-γ agonist, alleviated the suppressions of PPAR-γ and M2a polarization induced by 2 µM As+3. Collectively, these results not only demonstrated that As+3 was able to inhibit polarization of M2a cells through PPAR-γ suppression, but also indicated that PPAR-γ could be utilized as a target for the prevention and treatment of As+3-induced immunotoxicity.

An anthracenecarboximide-guanidine fluorescent probe for selective detection of glyoxals under weak acidic conditions.

An anthracenecarboximide-guanidine based turn-on fluorescent probe ANC-DCP-1 for selective detection of glyoxals (methylglyoxal and glyoxal, GOS) over formaldehyde under weak acidic conditions around pH 6.0 was reported. The probe showed great potential in studying relative GOS levels in weak acidic biological fluids such as in urine for diabetic diagnosis and prognosis, and also found application in the food industry such as for fast unique manuka factor (UMF) scale determination of Manuka honey.

Methylglyoxal produced by tumor cells through formaldehyde-enhanced Warburg effect potentiated polarization of tumor-associated macrophages.

Environmental exposure to formaldehyde is known to be associated with cancers and many other diseases. Although formaldehyde has been classified as a group I carcinogen, the molecular mechanisms of its carcinogenicity are still not fully understood. Formaldehyde is also involved in the folate-driven one­carbon metabolism, and excess amount of formaldehyde was found to interfere with other metabolic pathways including glycolysis, which can enhance Warburg effect and induce immunosuppression in tumor microenvironment. Therefore, different tumor cells and THP-1 derived macrophages were utilized to explore the metabolism-related effects induced by formaldehyde at environmentally relevant concentrations. Significant increases of glucose uptake, glycolysis levels, HIF-1α signaling and methylglyoxal production were observed in tumor cells treated with 20 and 50 µM formaldehyde for 24 h, and the overproduced methylglyoxal in the conditioned medium collected from the tumor cells treated with formaldehyde triggered macrophage polarization towards M2 cells. Myricetin, a flavonol scavenging methylglyoxal, reversed the polarization of macrophages induced by methylglyoxal at 50 µM. These results not only provided essential evidences to reveal the molecular mechanisms of Warburg effect and metabolism-related immunosuppression related to formaldehyde exposure, but also indicated that methylglyoxal could be utilized as a target for therapeutic treatment or prevention of formaldehyde-induced immunotoxicity.

A PET-based fluorescent probe for monitoring labile Fe(II) pools in macrophage activations and ferroptosis.

A fluorescent probe (COU-LIP-1) for monitoring labile Fe(II) pools (LIP) with high selectivity and sensitivity was developed utilizing coumarin 343 as the fluorophore and 3-nitrophenylazanyl ester as both the reactive group and the fluorescence quenching group. Fe(II)-induced reductive cleavage of the N-O bond results in a turn-on response via a photo-induced photon transfer (PET) mechanism. The probe was applied for monitoring labile iron(II) changes in M1 and M2a macrophage activations and also erastin-induced ferroptosis, providing a powerful tool for selectively sensing LIP under both physiological and stressed conditions.

Orlistat increases arsenite tolerance in THP-1 derived macrophages through the up-regulation of ABCA1.

Orlistat is an FDA-approved over-the-counter drug to treat obesity through the inhibition of lipase activity. Macrophages, which express high levels of lipoprotein lipase (LPL), are important phagocytes in the innate immune system. Our previous studies indicated that environmentally relevant concentrations of arsenite (As+3) could inhibit the major immune functions of macrophages. As the down-regulation of LPL is known to increase the expression of ABCA1, the cholesterol exporter demonstrated to be related to the resistance of arsenic toxicity. We examined if orlistat could reverse the inhibitive effects of As+3 on macrophage functions. The results showed that 50 µM orlistat reversed As+3-induced suppressions on phagocytosis, NO production and cytokine secretion in THP-1 derived macrophages. The expression of ABCA1 was significantly increased by orlistat in As+3 co-treated macrophages, which was associated with decreased intracellular As+3 levels. Collectively, these results indicated that orlistat could reverse the suppressive effects induced by As+3 in macrophages through the increased expression of ABCA1, which has the potential to be developed as a therapeutic agent for arsenic-induced immunosuppression.

Formaldehyde reinforces pro-inflammatory responses of macrophages through induction of glycolysis.

Formaldehyde (FA) is widely used in chemical industry, which is also known as a common indoor air pollutant. Exposure of FA has been associated with multiple detrimental health effects. Our previous study showed that FA could inhibit the development of T lymphocytes in mice, leading to impaired immune functions. Macrophages are important innate immune cells which trigger inflammatory responses in tissues. In the present study, FA exposure at 2.0 mg/m3 was found to enhance the pro-inflammatory responses of macrophages in male BALB/c mice, which was confirmed by elevated pro-inflammatory cytokine release and NO secretion in macrophages isolated from the FA-exposed mice and in vitro macrophage models upon lipopolysaccharide stimulation. Glycolysis is the key metabolic process for the classical activation of macrophages, which was found to be elevated in the in vitro macrophage models treated with FA at 50 and 100 µM concentrations for 18 h. HIF-1α and the associated proteins in its signaling cascade, which are known to mediate glycolytic metabolism and inflammatory responses, were found to be upregulated by 50 and 100 µM FA in THP-1 derived and RAW264.7 macrophage models, and the enhanced pro-inflammatory responses induced by 100 µM FA were reversed by inhibitory compounds interfering with glucose metabolism or suppressing HIF-1α activity. Collectively, the results in this study revealed that FA could enhance the pro-inflammatory responses of macrophages through the induction of glycolysis, which outlined the FA-triggered metabolic and functional alterations in immune cells.

An "AND"-logic-gate-based fluorescent probe with dual reactive sites for monitoring extracellular methylglyoxal level changes of activated macrophages.

An "AND"-logic-gate-based fluorescent probe NAP-DCP-4 with dual reactive sites is reported, which has improved selectivity for methylglyoxal over glyoxal, featuring formaldehyde-enhanced methylglyoxal detection and irreversible and reversible turn-on fluorescence responses at different excitation wavelengths. Its cell-impermeability enables facile monitoring of extracellular methylglyoxal level changes in the supernatant of activated macrophages.

Fluorophore-Promoted Facile Deprotonation and Exocyclic Five-Membered Ring Cyclization for Selective and Dynamic Tracking of Labile Glyoxals.

The lack of effective chemical tools capable of dynamic tracking of labile glyoxal species (GOS) [e.g., methylglyoxal (MGO) and glyoxal (GO)] levels with high selectivity over other relevant electrophilic species, particularly, formaldehyde (FA) and nitric oxide (NO), has significantly hampered the understanding of their roles in a complex metabolic network and disease progressions. Herein, we report the rational design of the bioinspired 4-(2-guanidino)-1,8-naphthalimide fluorescent probes NAP-DCP-1 and NAP-DCP-3 from arginine-specific protein modifications. These probes undergo facile reversible fluorophore-promoted deprotonation-cyclization of a guanidium ion with labile GOS to form exocyclic five-membered dihydroxyimidazolidines. The probe NAP-DCP-1 can differentiate GOS levels in the serum of diabetic mice and patients from nondiabetic ones, which correlate very well with glucose levels, providing the GOS level as a potential new biomarker for diabetes diagnosis. Notably, the endoplasmic reticulum (ER)-targeting probe NAP-DCP-3 enabled the study of GOS perturbation in ER under various stress conditions and led to the discovery that formaldehyde (FA), either exogenously added or endogenously generated, could induce GOS level increases in ER. This finding reveals the previous unknown connection of FA with upregulated GOS levels and suggests that GOS is a key metabolite in bridging one-carbon metabolism with glycolysis and the downstream cell redox status. Moreover, the probes also showed potentials in separate quantification of MGO and GO via ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) and unexpected selectivity modulation for GO over MGO via two-photon excitation. It is expected that probes reported herein provide powerful tools to study GOS level modulations in complex biological networks and would facilitate GOS-associated basic research and discovery.

Mebendazole is a potent inhibitor to chemoresistant T cell acute lymphoblastic leukemia cells.

Mebendazole (MBZ) is a tubulin-suppressive antihelmintic agent with low toxicity, which has been repurposed to treat different types of tumors. Chemoresistance is quite common in refractory or relapsed T cell acute lymphoblastic leukemia (T-ALL), which leads to dismal chances of recovery. In this study, MBZ was found to suppress the proliferation and reduce the viability of T-ALL cell line, CCRF-CEM, and its chemoresistant derivative, CEM/C1, at nanomolar concentrations. The inhibitive effects were found to be dose-dependent and not to be affected by the chemoresistance of CEM/C1 cells. Cell cycle arrest, caspase 3/7 activation and tubulin disruption were found in the MBZ-treated T-ALL cells. Notch1 signaling, which is often aberrantly activated in T-ALL cells, was showed to be suppressed by MBZ treatments. MBZ administration in murine T-ALL models also suppressed the growth of CEM/C1 cells, indicating that MBZ may be developed as a therapeutic agent for chemoresistant T-ALLs. The mRNA levels of the Notch1 and Hes1 were also confirmed to be suppressed by MBZ in vivo, which was consistent with the in vitro observations. This study demonstrated, for the first time, that MBZ could inhibit chemoresistant T-ALL cells both in vitro and in vivo, and the Notch1 signaling pathway was suppressed by MBZ treatment.

Safe and Efficacious Diphtheria Toxin-Based Treatment for Melanoma: Combination of a Light-On Gene-Expression System and Nanotechnology.

The controversy surrounding the use of diphtheria toxin (DT) as a therapeutic agent against tumor cells arises mainly from its unexpected harmfulness to healthy tissues. We encoded the cytotoxic fragment A of DT (DTA) as an objective gene in the Light-On gene-expression system to construct plasmids pGAVPO (pG) and pU5-DTA (pDTA). Meanwhile, a cRGD-modified ternary complex comprising plasmids, chitosan, and liposome (pG&pDTA@cRGD-CL) was prepared as a nanocarrier to ensure transfection efficiency. Benefiting from spatiotemporal control of this light-switchable transgene system and the superior tumor targeting of the carrier, toxins were designed to be expressed selectively in illuminated lesions. In vitro studies suggested that pG&pDTA@cRGD-CL exerted arrest of the S phase in B16F10 cells upon blue light irradiation and, ultimately, induced the apoptosis and necrosis of tumor cells. Such DTA-based treatment exerted enhanced antitumor activity in mice bearing B16F10 xenografts and displayed prolonged survival time with minimal side effects. Hence, we described novel DTA-based therapy combined with nanotechnology and the Light-On gene-expression system: such treatment could be a promising strategy against melanoma.

Spatiotemporally controllable diphtheria toxin expression using a light-switchable transgene system combining multifunctional nanoparticle delivery system for targeted melanoma therapy.

Gene therapy with external gene insertion (e. g. a suicide gene) and expression specifically in mutated tumor cells has shown to be a promising strategy in treatment of tumors. However, current tumor gene therapy often suffered from low efficiency in gene expression and off-target effects which may cause damage to normal tissues. To address these issues, in this study, a light-switchable transgene nanoparticle delivery system loaded with a diphtheria toxin A (DTA) segment encoded gene, a suicide gene for tumor cells, was developed. The nanoparticles contained vitamin E succinate-grafted polyethyleneimine core and arginylglycylaspartic acid (RGD)-modified pegylated hyaluronic acid shell for targeted delivery of the loaded gene to tumor cells via receptor-mediated (CD44 and αvß3) endocytosis. Notably, the expression of target proteins in tumor cells could be conveniently regulated by adjusting the blue light intensity in the Light-On system. In in-vitro studies in cultured B16-F10 cells, the pG-DTA-loaded nano-micelles showed greatly improved inhibitory rate compared with the pG-DTA group. Moreover, in the tumor-bearing C57BL/6 mice model, the pG-DTA-loaded nanoparticle exhibited greatly improved efficacy and reduced systemic toxicity with significantly increased survival rate after 21 days. Significantly suppressed tumor angiogenesis was also identified in the nanoparticle-treated group likely due to the targeting ability of the RGD-modified nanoparticle. All the above results indicated that the combination of a light-switchable transgene system with a nanoparticle-based targeted delivery system have great potentials in gene therapy of malignant tumors with improved precision and efficacy.

Vitamin E succinate-grafted-chitosan/chitosan oligosaccharide mixed micelles loaded with C-DMSA for Hg2+ detection and detoxification in rat liver.

AIM: To determine whether the use of a mixed polymeric micelle delivery system based on vitamin E succinate (VES)-grafted-chitosan oligosaccharide (CSO)/VES-grafted-chitosan (CS) mixed micelles (VES-g-CSO/VES-g-CS MM) enhances the delivery of C-DMSA, a theranostic fluorescent probe, for Hg2+ detection and detoxification in vitro and in vivo. METHODS: Mixed micelles self-assembled from two polymers, VES-g-CSO and VES-g-CS, were used to load C-DMSA and afforded C-DMSA@VES-g-CSO/VES-g-CS MM for cell and in vivo applications. Fluorescence microscopy was used to assess C-DMSA cellular uptake and Hg2+ detection in L929 cells. C-DMSA@VES-g-CSO/VES-g-CS MM was then administered intravenously. Hg2+ detection was assessed by fluorescence microscopy in terms of bio-distribution while detoxification efficacy in Hg2+-poisoned rat models was evaluated in terms of mercury contents in blood and in liver. RESULTS: The C-DMSA loaded mixed micelles, C-DMSA@VES-g-CSO/VES-g-CS MM, significantly enhanced cellular uptake and detoxification efficacy of C-DMSA in Hg2+ pretreated human L929 cells. Evidence from the reduction of liver coefficient, mercury contents in liver and blood, alanine transaminase and aspartate transaminase activities in Hg2+ poisoned SD rats treated with the mixed micelles strongly supported that the micelles were effective for Hg2+ detoxification in vivo. Furthermore, ex vivo fluorescence imaging experiments also supported enhanced Hg2+ detection in rat liver. CONCLUSION: The mixed polymeric micelle delivery system could significantly enhance cell uptake and efficacy of a theranostic probe for Hg2+ detection and detoxification treatment in vitro and in vivo. Moreover, this nanoparticle drug delivery system could achieve targeted detection and detoxification in liver.

A naphthalimide-aminal-based pH-sensitive fluorescent donor for lysosome-targeted formaldehyde release and fluorescence turn-on readout.

A naphthalimide-aminal-based fluorescent donor (NAP-FAD-1) is a pH-sensitive smart donor, which features accelerated FA release at pH 5.0 compared to that at pH 7.4 and fluorescence turn-on readout for facile tracking and quantification of FA release. Confocal imaging studies of HeLa cells co-incubated with Lyso-Tracker Red suggested that its FA release is lysosome-targeted.

Cyclodextrin/chitosan nanoparticles for oral ovalbumin delivery: Preparation, characterization and intestinal mucosal immunity in mice.

A novel oral protein delivery system with enhanced intestinal penetration and improved antigen stability based on chitosan (CS) nanoparticles and antigen-cyclodextrin (CD) inclusion complex was prepared by a precipitation/coacervation method. Ovalbumin (OVA) as a model antigen was firstly encapsulated by cyclodextrin, either ß-cyclodextrin (ß-CD) or carboxymethyl-hydroxypropyl-ß-cyclodextrin (CM-HP-ß-CD) and formed OVA-CD inclusion complexes, which were then loaded to chitosan nanoparticles to form OVA loaded ß-CD/CS or CM-HP-ß-CD/CS nanoparticles with uniform particle size (836.3 and 779.2 nm, respectively) and improved OVA loading efficiency (27.6% and 20.4%, respectively). In vitro drug release studies mimicking oral delivery condition of OVA loaded CD/CS nanoparticles showed low initial releases at pH 1.2 for 2 h less than 3.0% and a delayed release which was below to 30% at pH 6.8 for further 72 h. More importantly, after oral administration of OVA loaded ß-CD/CS nanoparticles to Balb/c mice, OVA-specific sIgA levels in jejunum of OVA loaded ß-CD/CS nanoparticles were 3.6-fold and 1.9-fold higher than that of OVA solution and OVA loaded chitosan nanoparticles, respectively. In vivo evaluation results showed that OVA loaded CD/CS nanoparticles could enhance its efficacy for inducing intestinal mucosal immune response. In conclusion, our data suggested that CD/CS nanoparticles could serve as a promising antigen-delivery system for oral vaccination.

Analyte Regeneration Fluorescent Probes for Formaldehyde Enabled by Regiospecific Formaldehyde-Induced Intramolecularity.

An important challenge for reaction-based fluorescent probes is that they generally require analyte consumption for fluorescence signal generation, thus creating potential perturbation of native analyte homeostasis or change of local concentrations. Herein, we reported two formaldehyde (FA) regeneration fluorescent probes, NAP-FAP-1 and NAP-FAP-2. An unprecedented regiospecific FA-induced intramolecularity strategy is implemented in the probe design, which adopts 3-(benzylamino)-succinimide as the FA-selective reaction group. The probes are able to capture the analyte molecule, induce regiospecific imide bond cleavage, and then release the captured FA molecule with simultaneous fluorescence turn-on response via a unique dual PeT/ICT quenching mechanism. The probes have shown potentials in detection, comparison, and imaging of FA levels intracellularly and inside lysosomes. These features make them useful for the study of FA homeostasis and functions in biological systems with minimal perturbation.

Recent developments in multimodality fluorescence imaging probes.

Multimodality optical imaging probes have emerged as powerful tools that improve detection sensitivity and accuracy, important in disease diagnosis and treatment. In this review, we focus on recent developments of optical fluorescence imaging (OFI) probe integration with other imaging modalities such as X-ray computed tomography (CT), magnetic resonance imaging (MRI), positron emission tomography (PET), single-photon emission computed tomography (SPECT), and photoacoustic imaging (PAI). The imaging technologies are briefly described in order to introduce the strengths and limitations of each techniques and the need for further multimodality optical imaging probe development. The emphasis of this account is placed on how design strategies are currently implemented to afford physicochemically and biologically compatible multimodality optical fluorescence imaging probes. We also present studies that overcame intrinsic disadvantages of each imaging technique by multimodality approach with improved detection sensitivity and accuracy.

A simple two-photon turn-on fluorescent probe for the selective detection of cysteine based on a dual PeT/ICT mechanism.

Herein, a simple two-photon turn-on fluorescent probe, N-(6-acyl-2-naphthayl)-maleimide (1), based on a dual PeT/ICT quenching mechanism is reported for the highly sensitive and selective detection of cysteine (Cys) over other biothiols. The probe was applied in the two-photon imaging of Cys in cultured HeLa cells, excited by a near-infrared laser at 690 nm.