Reversible Near-Infrared Fluorescence Photoswitching in Aqueous Media by Diarylethene: Toward High-Accuracy Live Optical Imaging.

Fluorescence imaging is an indispensable tool in modern biological research, allowing simple and inexpensive color-coded visualizations of real-time events in living cells and animals, as well as of fixed states of ex vivo specimens. The accuracy of fluorescence imaging in living systems is, however, impeded by autofluorescence, light scattering, and limited penetration depth of light. Nevertheless, the clinical use of fluorescence imaging is expected to grow along with advances in imaging equipment, and will increasingly demand high-accuracy probes to avoid false-positive results in disease detection. To this end, a water-soluble and relatively safe diarylethene (DAE)-based reversible near-infrared (NIR) fluorescence photoswitch for living systems is prepared here. Furthermore, to facilitate excellent switching performance, the photoirradiation results obtained is compared using three different visible light sources to turn on NIR fluorescence through cycloreversion of DAE. While photoswitching using 589 nm light leads to slightly higher cell viability, fluorescence quenching efficiency and fatigue resistance are higher when 532 nm light with low photobleaching is used in both aqueous solution and living systems. The authors anticipate that their reversible NIR fluorescence photoswitch mediated by DAE can be beneficial for fluorescence imaging in aqueous media requiring accurate detection, such as in the autofluorescence-rich living environment.

Modulating Lipid Nanoparticles with Histidinamide-Conjugated Cholesterol for Improved Intracellular Delivery of mRNA.

Recently, mRNA-based therapeutics, including vaccines, have gained significant attention in the field of gene therapy for treating various diseases. Among the various mRNA delivery vehicles, lipid nanoparticles (LNPs) have emerged as promising vehicles for packaging and delivering mRNA with low immunogenicity. However, while mRNA delivery has several advantages, the delivery efficiency and stability of LNPs remain challenging for mRNA therapy. In this study, an ionizable helper cholesterol analog, 3ß[L-histidinamide-carbamoyl] cholesterol (Hchol) lipid is developed and incorporated into LNPs instead of cholesterol to enhance the LNP potency. The pKa values of the Hchol-LNPs are ≈6.03 and 6.61 in MC3- and SM102-based lipid formulations. Notably, the Hchol-LNPs significantly improve the delivery efficiency by enhancing the endosomal escape of mRNA. Additionally, the Hchol-LNPs are more effective in a red blood cell hemolysis at pH 5.5, indicating a synergistic effect of the protonated imidazole groups of Hchol and cholesterol on endosomal membrane destabilization. Furthermore, mRNA delivery is substantially enhanced in mice treated with Hchol-LNPs. Importantly, LNP-encapsulated SARS-CoV-2 spike mRNA vaccinations induce potent antigen-specific antibodies against SARS-CoV-2. Overall, incorporating Hchol into LNP formulations enables efficient endosomal escape and stability, leading to an mRNA delivery vehicle with a higher delivery efficiency.

(-)-Nyasol, isolated from Anemarrhena asphodeloides suppresses neuroinflammatory response through the inhibition of I-κBα degradation in LPS-stimulated BV-2 microglial cells.

Microglial activation has been associated with neurodegenerative diseases by inducing the neuroinflammatory mediators such as nitric oxide (NO), TNF-α and IL-1ß. (-)-Nyasol, a norlignan isolated from a medicinal plant Anemarrhena asphodeloides, showed anti-inflammatory potential in lipopolysaccharide (LPS)-activated BV-2 microglial cells. (-)-Nyasol inhibited the production of NO and prostaglandin E2 (PGE2) and also the expression of inducible nitric oxide synthase and cyclooxygenase-2, which are responsible for the respective production of NO and PGE2. It also suppressed the mRNA levels of TNF-α and IL-1ß in activated microglial cells. These effects of (-)-nyasol were correlated with the inactivation of p38 MAPK and the suppression of LPS-induced I-κBα degradation. Taken together, these results suggest that (-)-nyasol can be a modulator in neuroinflammatory conditions induced by microglial activation.

A germacranolide sesquiterpene lactone suppressed inducible nitric oxide synthase by downregulating NF-κB activity.

A germacranolide sesquiterpene lactone, 2α,5-epoxy-5,10-dihydroxy-6α-angeloyloxy-9ß-(3-methylbutyloxy)-germacran-8α,12-olide (EDAG), isolated from Carpesium triste var. manshuricum, showed inhibitory activity in the production of nitric oxide (NO) and the expression of inducible nitric oxide synthase (iNOS) mRNA and protein in LPS-activated macrophage cells. Molecular analysis reveals that these suppressive effects are correlated with the inhibition of NF-κB activation by EDAG. Immunoblotting showed that EDAG suppressed the LPS-induced degradation of I-κBα and decreased nuclear translocation of p65. Furthermore, EDAG showed reduced phosphorylation of ERK1/2 and p38 MAPK, whereas activation of JNK was not changed. These data suggest, at least in part, that EDAG utilizes the signal cascades of ERK1/2, p38 MAPK, and NF-κB for the suppression of iNOS gene expression.

Carabrol suppresses LPS-induced nitric oxide synthase expression by inactivation of p38 and JNK via inhibition of I-kappaBalpha degradation in RAW 264.7 cells.

Carabrol, isolated from Carpesium macrocephalum, showed anti-inflammatory potential in LPS-induced RAW 264.7 murine macrophages. In present study, carabrol demonstrated the inhibitory activity on pro-inflammatory cytokines such as IL-1beta, IL-6 and TNF-alpha. In addition, mRNA and protein levels of iNOS and COX-2 were reduced by carabrol. Molecular analysis revealed that these suppressive effects were correlated with the inactivation of p38 and JNK via inhibition of NF-kappaB activation. Immunoblotting showed that carabrol suppressed LPS-induced degradation of I-kappaBalpha and decreased nuclear translocation of p65. Taken together, these results suggest that carabrol can be a modulator of pro-inflammatory signal transduction pathway in RAW 264.7 cells.

Tuning surface functionalities of sub-10 nm-sized nanocarriers to target outer retina in designing drug delivery agents for intravitreal administration.

Age-related macular degeneration (AMD) is one of the leading causes of irreversible blindness, generally affecting people over 50 years of age in industrialized countries. Despite the effectiveness of anti-vascular endothelial growth factor (VEGF) therapy in attenuating the growth of new blood vessels, substantial visual improvements are rare with this complex disease. Furthermore, the current regimen of repeated monthly intravitreal injections of drugs can result in serious side effects. Combination therapies-to complement anti-VEGF alone-with a prolonged therapeutic effect and efficient delivery to the intended site are urgently needed, which could be realized through the use of carefully designed nanocarriers. To understand the physicochemical effects (e.g., size, charge, geometry) of intravitreally administered nanocarriers on their bioavailability, distribution, and targeting efficiency across multiple layers of the retina, here we prepared seven different types of surface-functionalized water-soluble dendritic nanocarriers with hydrodynamic sizes mostly under 5 nm. A similar stoichiometric amount of fluorophore was covalently attached to each of these biocompatible nanocarriers for quantitative analyses by confocal microscopy of cryosectioned healthy mouse eyes. Interestingly, at 24 h post-injection, the nanocarrier with multiple copies of glucosamine on the surface (DNSG) accumulated predominantly in the photoreceptor layer and the retinal pigment epithelium (RPE), which are speculated to be associated with AMD pathogenesis (i.e., target sites). Furthermore, extended residence at these outer retinal layers was demonstrated by DNSG, which appeared to gradually turn into micron-scale particles potentially through aggregation. Our systematic findings may provide useful guidelines for the rational design of intravitreal nanocarriers to treat vision-threatening retinal diseases, including AMD.

Induction of neural stem cell-like cells (NSCLCs) from mouse astrocytes by Bmi1.

Recently, Bmi1 was shown to control the proliferation and self-renewal of neural stem cells (NSCs). In this study, we demonstrated the induction of NSC-like cells (NSCLCs) from mouse astrocytes by Bmi1 under NSC culture conditions. These NSCLCs exhibited the morphology and growth properties of NSCs, and expressed NSC marker genes, including nestin, CD133, and Sox2. In vitro differentiation of NSCLCs resulted in differentiated cell populations containing astrocytes, neurons, and oligodendrocytes. Following treatment with histone deacetylase inhibitors (trichostatin A and valproic acid), the potential of NSCLCs for proliferation, dedifferentiation, and self-renewal was significantly inhibited. Our data indicate that multipotent NSCLCs can be generated directly from astrocytes by the addition of Bmi1.

Toward redesigning the PEG surface of nanocarriers for tumor targeting: impact of inner functionalities on size, charge, multivalent binding, and biodistribution.

Achieving accurate and efficacious tumor targeting with minimal off-target effects is of paramount importance in designing diagnostic and therapeutic agents for cancer. In this respect, nanocarriers have gained enormous popularity because of their attainable multifunctional features, as well as tumor-targeting potential by extravasation. However, once administered into the bloodstream, nanocarriers face various in vivo obstacles that may significantly impair their performance needed for clinical translation. Herein, we demonstrate a strategy to enhance tumor-targeting efficiency by embedding functionalities in the interior region of partially PEGylated nanocarriers (ca. 10 nm in diameter), intended for active or passive targeting. The cooperative impact of these topologically inner functional groups (IFGs) was marked: enhancements of >100-fold in IC50in vitro (e.g., a high-avidity ligand with cationic IFGs) and >2-fold in tumor accumulation at 2 h post-injection in vivo (e.g., a high-avidity ligand with anionic IFGs), both against the fully PEGylated counterpart. Analogous to allosteric modulators, properly employed IFGs may substantially improve the process of effectively directing nanocarriers to tumors, which is otherwise solely dependent on avidity or extravasation.

High-performance dendritic contrast agents for X-ray computed tomography imaging using potent tetraiodobenzene derivatives.

The use of computed tomography (CT) for vascular imaging is critical in medical emergencies requiring urgent diagnostic decisions, such as cerebral ischemia and many cardiovascular diseases. Small-molecule iodinated contrast media are often injected intravenously as radiopaque agents during CT imaging to achieve high contrast enhancement of vascular systems. The rapid excretion rate of these agents is overcome by injecting a significantly high dose of iodine, which can have serious side effects. Here we report a simple method to prepare blood-pool contrast agents for CT based on dendrimers for the first time using tetraiodobenzene derivatives as potent radiopaque moieties. Excellent in vivo safety has been demonstrated for these small (13-22nm) unimolecular water-soluble dendritic contrast agents, which exhibit high contrast enhancement in the blood-pool and effectively extend their blood half-lives. Our method is applicable to virtually any scaffold with suitable surface groups and may fulfill the current need for safer, next-generation iodinated CT contrast agents.

One-pot synthesis of monodispersed silica nanoparticles for diarylethene-based reversible fluorescence photoswitching in living cells.

A small 29 nm monodispersed silica nanoparticle 1a was synthesized as a diarylethene-based reversible fluorescence photoswitch by copolymerizing silane precursors in one-pot including 3a and 4. Reversible photoswitching of nanoparticle 1a was successfully achieved in living cells to show its potential as a highly distinguishable and safe fluorescence probe for cell tracking.

Cell Death by Polyvinylpyrrolidine-Coated Silver Nanoparticles is Mediated by ROS-Dependent Signaling.

Silver nanoparticles (AgNPs) are widely used nanoparticles and they are mainly used in antibacterial and personal care products. In this study, we evaluated the effect of AgNPs on cell death induction in the murine dendritic cell line DC2.4. DC2.4 cells exposed to AgNPs showed a marked decrease in cell viability and an induction of lactate dehydrogenase (LDH) leakage in a time- and dose-dependent manner. In addition, AgNPs promoted reactive oxygen species (ROS)-dependent apoptosis and AgNP-induced ROS triggered a decrease in mitochondrial membrane potential. The activation of the intracellular signal transduction pathway was also observed in cells cultured with AgNPs. Taken together, our data demonstrate that AgNPs are able to induce a cytotoxic effect in DCs through ROS generation. This study provides important information about the safety of AgNPs that may help in guiding the development of nanotechnology applications.

NDRG2 Promotes GATA-1 Expression through Regulation of the JAK2/STAT Pathway in PMA-stimulated U937 Cells.

BACKGROUND: N-myc downstream-regulated gene 2 (NDRG2), a member of a newly described family of differentiation-related genes, has been characterized as a regulator of dendritic cells. However, the role of NDRG2 on the expression and activation of transcription factors in blood cells remains poorly understood. In this study, we investigated the effects of NDRG2 overexpression on GATA-1 expression in PMA-stimulated U937 cells. METHODS: We generated NDRG2-overexpressing U937 cell line (U937-NDRG2) and treated the cells with PMA to investigate the role of NDRG2 on GATA-1 expression. RESULTS: NDRG2 overexpression in U937 cells significantly induced GATA-1 expression in response to PMA stimulation. Interestingly, JAK2/STAT and BMP-4/Smad pathways associated with the induction of GATA-1 were activated in PMA-stimulated U937-NDRG2 cells. We found that the inhibition of JAK2 activation, but not of BMP-4/Smad signaling, can elicit a decrease of PMA-induced GATA-1 expression in U937-NDRG2 cells. CONCLUSION: The results reveal that NDRG2 promotes the expression of GATA-1 through activation of the JAK2/STAT pathway, but not through the regulation of the BMP-4/Smad pathway in U937 cells. Our findings further suggest that NDRG2 may play a role as a regulator of erythrocyte and megakaryocyte differentiation during hematopoiesis.

Photoreversible cellular imaging using photochrome-conjugated fullerene silica nanoparticles.

Photochromic compound-conjugated fluorescent fullerene-silica nanoparticles prepared by the reverse-microemulsion method was utilized for photoswitchable cellular imaging by repeatable irradiation of ultraviolet and visible light.

Isolation and characterization of multipotent human keloid-derived mesenchymal-like stem cells.

In this study, we report the isolation and characterization of a population of multipotent keloid-derived mesenchymal-like stem cells (KMLSCs) from keloid scalp tissues. These KMLSCs expressed the typical mesenchymal stem cell marker proteins CD13, CD29, CD44, CD90, fibronectin, and vimentin when they were cultured in serum-containing medium and when subsequent exposure to various differentiation media resulted in their differentiation into adipocytes, osteoblasts, chondrocytes, smooth muscle cells, and angiogenic endothelial cells. When KMLSCs were cultured in neural stem culture conditions (i.e., in the presence of epidermal growth factor and fibroblast growth factor 2 in substrate-free conditions), they produced large numbers of neurospheres containing nestin-, CD133-, and SOX2-positive cells that expressed neural-crest stem cell markers. Subsequent exposure of these cells to different differentiation conditions resulted in cells that expressed neuronal cell-, astrocyte-, oligodendrocyte-, or Schwann cell-specific markers. Our study suggests that KMLSCs may be an alternative adult stem cell resource for regenerative tissue repair and auto-transplantation.