Peroxiredoxin II regulates exosome secretion from dermal mesenchymal stem cells through the ISGylation signaling pathway.

BACKGROUND: Exosomes are small extracellular vesicles that play important roles in intercellular communication and have potential therapeutic applications in regenerative medicine. Dermal mesenchymal stem cells (DMSCs) are a promising source of exosomes due to their regenerative and immunomodulatory properties. However, the molecular mechanisms regulating exosome secretion from DMSCs are not fully understood. RESULTS: In this study, the role of peroxiredoxin II (Prx II) in regulating exosome secretion from DMSCs and the underlying molecular mechanisms were investigated. It was discovered that depletion of Prx II led to a significant reduction in exosome secretion from DMSCs and an increase in the number of intracellular multivesicular bodies (MVBs), which serve as precursors of exosomes. Mechanistically, Prx II regulates the ISGylation switch that controls MVB degradation and impairs exosome secretion. Specifically, Prx II depletion decreased JNK activity, reduced the expression of the transcription inhibitor Foxo1, and promoted miR-221 expression. Increased miR-221 expression inhibited the STAT signaling pathway, thus downregulating the expression of ISGylation-related genes involved in MVB degradation. Together, these results identify Prx II as a critical regulator of exosome secretion from DMSCs through the ISGylation signaling pathway. CONCLUSIONS: Our findings provide important insights into the molecular mechanisms regulating exosome secretion from DMSCs and highlight the critical role of Prx II in controlling the ISGylation switch that regulates DMSC-exosome secretion. This study has significant implications for developing new therapeutic strategies in regenerative medicine. Video Abstract.

Hypoxia-Directed and Self-Immolative Theranostic Agent: Imaging and Treatment of Cancer and Bacterial Infections.

The impact of bacteria on cancer progression and treatment is becoming increasingly recognized. Cancer-associated bacteria are linked to metastases, reduced efficacy, and survival challenges. In this study, we present a sensitive hypoxia-activated prodrug, NR-NO2, which comprises an antibiotic combined with a chemotherapeutic. This prodrug demonstrates rapid and robust fluorescence enhancement and exhibits potent antibacterial activity against both Gram-positive and Gram-negative bacteria as well as tumor cells. Upon activation, NR-NO2 produces a distinct "fluorescence-on" signal, enabling real-time drug release monitoring. By leveraging elevated nitroreductase in cancer cells, NR-NO2 gives rise to heightened bacterial cytotoxicity while sparing normal cells. In A549 solid tumor-bearing mice, NR-NO2 selectively accumulated at tumor sites, displaying fluorescence signals under hypoxia superior to those of a corresponding prodrug-like control. These findings highlight the potential of NR-NO2 as a promising cancer therapy prodrug that benefits from targeted release, antibacterial impact, and imaging-based guidance.

Universal Emission Characteristics of Upconverting Nanoparticles Revealed by Single-Particle Spectroscopy.

Upconverting nanoparticles (UCNPs) have been extensively investigated for nanophotonics and biomedical applications. However, establishing a unified view of their emission characteristics to elucidate the underlying photophysics and expand the application fields of these materials is a great challenge due to their sophisticated internal energy transfer and lack of standardized single-particle spectroscopy (SPS) platform. Here, we present an SPS technique called multiband single-particle irradiance-dependent imaging (multiband SPIDI). We demonstrate that the emission characteristics of Yb3+,Tm3+-doped UCNPs are universal for three emission bands over a wide range of irradiance and dependent on the Tm3+ doping concentration, indicating that the number of emitted photons of each band is proportional to the number of activator ions and is dependent on the number of absorbed photons and the activator interionic distance. We also suggest a cooperative energy transfer upconversion (CETU) mechanism for transition to a higher-energy state through photon accumulation. For a single UCNP, the emission at 800 nm is detectable at an ultralow irradiance of 4.9 W cm-2; moreover, that at 450 nm is measurable at 98 W cm-2, based on the optimal concentration. These findings based on the multiband SPIDI platform can provide insights into the interionic energy transfer by studying irradiance-dependent steady-state dynamics to achieve brighter UCNPs and their broader applications.

Correction: Visualization of intercellular cargo transfer using upconverting nanoparticles.

Correction for 'Visualization of intercellular cargo transfer using upconverting nanoparticles' by Yeongchang Goh et al., Nanoscale, 2022, https://doi.org/10.1039/d2nr01999j.

Visualization of intercellular cargo transfer using upconverting nanoparticles.

Cell-cell communication is important for cellular differentiation, organ function, and immune responses. In intercellular communication, the extracellular vesicles (EVs) play a significant role in delivering the cargo molecules such as genes, proteins, and enzymes, to regulate and control the ability of the recipient cells. In this study, the observation of intercellular cargo transfer via dual-colour imaging using upconverting nanoparticles (UCNPs) has been demonstrated. Using this technique, the intercellular transport via contact-dependent and contact-independent signaling in live HeLa cells was clearly visualized with real-time, long-term single-vesicle tracking. Furthermore, it was demonstrated that the endocytosed UCNPs can be transmitted with the encapsulation of EVs labelled with fluorescent proteins.

Heterotypic cell-in-cell structures between cancer and NK cells are associated with enhanced anticancer drug resistance.

The heterotypic CIC structures formed of cancer and immune cells have been observed in tumor tissues. We aimed to assess the feasibility of using heterotypic CICs as a functional biomarker to predict NK susceptibility and drug resistance. The heterotypic CIC-forming cancer cells showed a lower response to NK cytotoxicity and higher proliferative ability than non-CIC cancer cells. After treatment with anticancer drugs, cancer cells that formed heterotypic CICs showed a higher resistance to anticancer drugs than non-CIC cancer cells. We also observed the formation of more CIC structures in cancer cells treated with anticancer drugs than in the non-treated group. Our results confirm the association between heterotypic CIC structures and anticancer drug resistance in CICs formed from NK and cancer cells. These results suggest a mechanism underlying immune evasion in heterotypic CIC cancer cells and provide insights into the anticancer drug resistance of cancer cells.

Hypoxia-Responsive Luminescent CEST MRI Agent for In Vitro and In Vivo Tumor Detection and Imaging.

Hypoxia is a feature of most solid tumors and a key determinant of cancer growth and propagation. Sensing hypoxia effectively could lead to more favorable clinical outcomes. Here, we report a molecular antenna-based bimodal probe designed to exploit the complementary advantages of magnetic resonance (MR)- and optical-based imaging. Specifically, we describe the synthesis and evaluation of a dual-action probe (NO2-Eu) that permits hypoxia-activated chemical exchange saturation transfer (CEST) MR and optical imaging. In CT26 cells, this NO2-Eu probe not only provides an enhanced CEST MRI signal but also turns "on" the optical signal under hypoxic conditions. Time-dependent in vivo CEST imaging in a hypoxic CT26 tumor xenograft mouse model revealed probe-dependent tumor detection by CEST MRI contrast in the tumor area. We thus suggest that dual-action hypoxia probes, like that reported here, could have a role to play in solid tumor diagnosis and monitoring.

Monitoring in vivo behavior of size-dependent fluorescent particles as a model fine dust.

BACKGROUND: There has been growing concern regarding the impact of air pollution, especially fine dust, on human health. However, it is difficult to estimate the toxicity of fine dust on the human body because of its diverse effects depending on the composition and environmental factors. RESULTS: In this study, we focused on the difference in the biodistribution of fine dust according to the size distribution of particulate matter after inhalation into the body to predict its impact on human health. We synthesized Cy7-doped silica particulate matters (CSPMs) having different particle sizes and employed them as model fine dust, and studied their whole-body in vivo biodistribution in BALB/c nude mice. Image-tracking and quantitative and qualitative analyses were performed on the ex vivo organs and tissues. Additionally, flow cytometric analysis of single cells isolated from the lungs was performed. Smaller particles with a diameter of less than 100 nm (CSPM0.1) were observed to be removed relatively rapidly from the lungs upon initial inhalation. However, they were confirmed to accumulate continuously over 4 weeks of observation. In particular, smaller particles were found to spread rapidly to other organs during the early stages of inhalation. CONCLUSIONS: The results show in vivo behavioral differences that arisen from particle size through mouse experimental model. Although these are far from the human inhalation studies, it provides information that can help predict the effect of fine dust on human health. This study might provide with insights on association between CSPM0.1 accumulation in several organs including the lungs and adverse effect to underlying diseases in the organs.

A modifiable universal cotinine-chimeric antigen system of NK cells with multiple targets.

Natural killer (NK) cells are immune effector cells with outstanding features for adoptive immunotherapy. Immune effector cells with chimeric antigen receptors (CARs) are promising targeted therapeutic agents for various diseases. Because tumor cells exhibit heterogeneous antigen expression and lose cell surface antigen expression during malignant progression, many CARs fixed against only one antigen have limited efficacy and are associated with tumor relapse. To expand the utility of CAR-NK cells, we designed a split and universal cotinine-CAR (Cot-CAR) system, comprising a Cot-conjugator and NK92 cells (α-Cot-NK92 cells) engineered with a CAR containing an anti-Cot-specific single-chain variable fragment and intracellular signaling domain. The efficacy of the Cot-CAR system was assessed in vitro using a cytolysis assay against various tumor cells, and its single- or multiple- utility potential was demonstrated using an in vivo lung metastasis model by injecting A549-Red-Fluc cells. The α-Cot-NK92 cells could switch targets, logically respond to multiple antigens, and tune cytolytic activation through the alteration of conjugators without re-engineering. Therefore the universal Cot-CAR system is useful for enhancing specificity and diversity of antigens, combating relapse, and controlling cytolytic activity. In conclusion, this universal Cot-CAR system reveals that multiple availability and controllability can be generated with a single, integrated system.

In vivo MRI detection of intraplaque macrophages with biocompatible silica-coated iron oxide nanoparticles in murine atherosclerosis.

Identification of a vulnerable atherosclerotic plaque before rupture is an unmet clinical need. Integrating nanomedicine with multimodal imaging has the potential to precisely detect biological processes in atherosclerosis. We synthesized silica-coated iron oxide nanoparticles (SIONs) coated with rhodamine B isothiocyanate and polyethylene glycol and investigated their feasibility in the detection of macrophages in inflamed atherosclerotic plaques of apolipoprotein E-deficient (ApoE-/-) mice via magnetic resonance (MR) and fluorescence reflectance (FR) imaging. In vitro cellular uptake of SIONs was assessed in macrophages using confocal laser scanning microscopy (CLSM). In vivo MR imaging was performed 24 h after SION injection via the tail vein in 26-week-old ApoE-/- mice fed a high-cholesterol diet (HCD). We also performed FR imaging of the extracted aortas from four different mice: two normal-diet-fed C57BL/6 mice injected with saline or 10 mg/kg SIONs and two HCD-fed ApoE-/- mice injected with 5 or 10 mg/kg SIONs. The harvested aortas were cryosectioned and stained with immunohistochemical staining. The CLSM images at 24 h after incubation showed efficient uptake of SIONs by macrophages, with no evidence of cytotoxicity. The in vivo and ex vivo MR and FR images demonstrated SION deposition in the atheroma. Upon immunohistochemical staining of the aorta, CLSM images revealed colocalization of macrophages and SIONs in the atherosclerotic plaque. These results demonstrate that polyethylene glycosylated SIONs could be a highly effective method to identify macrophage activity in atherosclerotic plaques as a multimodal imaging agent.

Therapeutic Effect of BDNF-Overexpressing Human Neural Stem Cells (F3.BDNF) in a Contusion Model of Spinal Cord Injury in Rats.

The most common type of spinal cord injury is the contusion of the spinal cord, which causes progressive secondary tissue degeneration. In this study, we applied genetically modified human neural stem cells overexpressing BDNF (brain-derived neurotrophic factor) (F3.BDNF) to determine whether they can promote functional recovery in the spinal cord injury (SCI) model in rats. We transplanted F3.BDNF cells via intrathecal catheter delivery after a contusion of the thoracic spinal cord and found that they were migrated toward the injured spinal cord area by MR imaging. Transplanted F3.BDNF cells expressed neural lineage markers, such as NeuN, MBP, and GFAP and were functionally connected to the host neurons. The F3.BDNF-transplanted rats exhibited significantly improved locomotor functions compared with the sham group. This functional recovery was accompanied by an increased volume of spared myelination and decreased area of cystic cavity in the F3.BDNF group. We also observed that the F3.BDNF-transplanted rats showed reduced numbers of Iba1- and iNOS-positive inflammatory cells as well as GFAP-positive astrocytes. These results strongly suggest the transplantation of F3.BDNF cells can modulate inflammatory cells and glia activation and also improve the hyperalgesia following SCI.

In Situ Biosynthesis of a Metal Nanoparticle Encapsulated in Alginate Gel for Imageable Drug-Delivery System.

Development of drug-delivery systems that allow simultaneous in vivo imaging has gained much interest. We report a novel strategy to encapsulate metal nanoparticles (NPs) within alginate gel for in vivo imaging. The cell lysate of recombinant Escherichia coli strain, expressing Arabidopsis thaliana phytochelatin synthase and Pseudomonas putida metallothionein genes, was encapsulated within the alginate gel. Incubation of alginate gel with metal ion precursors followed by UV irradiation resulted in the synthesis of high concentrations of metal NPs, such as Au, Ag, CdSe, and EuSe NPs, within the gel. The alginate gel with metal NPs was used as a drug-delivery system by further co-encapsulating doxorubicin and rifampicin, the release of which was made to be pH-dependent. This system can be conveniently and safely used for in vitro and in vivo bioimaging, enabled by the metal NPs formed within the gel matrix without using toxic reducing reagents or surfactants.

An Activatable T1-Weighted MR Contrast Agent: A Noninvasive Tool for Tracking the Vicinal Thiol Motif of Thioredoxin in Live Cells.

We have synthesized new magnetic resonance imaging (MRI) T1 contrast agents (CA1 and CA2) that permit the activatable recognition of the cellular vicinal thiol motifs of the protein thioredoxin. The contrast agents showed MR relaxivities typical of gadolinium complexes with a single water molecule coordinated to a Gd3+ center (i.e., ~4.54 mM-1s-1) for both CA1 and CA2 at 60 MHz. The contrast agent CA1 showed a ~140% relaxivity enhancement in the presence of thioredoxin, a finding attributed to a reduction in the flexibility of the molecule after binding to thioredoxin. Support for this rationale, as opposed to one based on preferential binding, came from 1H-15N-HSQC NMR spectral studies; these revealed that the binding affinities toward thioredoxin were almost the same for both CA1 and CA2. In the case of CA1, T1-weighted phantom images of cancer cells (MCF-7, A549) could be generated based on the expression of thioredoxin. We further confirmed thioredoxin expression-dependent changes in the T1-weighted contrast via knockdown of the expression of the thioredoxin using siRNA-transfected MCF-7 cells. The nontoxic nature of CA1, coupled with its relaxivity features, leads us to suggest that it constitutes a first-in-class MRI T1 contrast agent that allows for the facile and noninvasive monitoring of vicinal thiol protein motif expression in live cells.

Near-Infrared Fluorescent Probe Activated by Nitroreductase for In Vitro and In Vivo Hypoxic Tumor Detection.

Tumor hypoxia is correlated with increased resistance to chemotherapy and poor overall prognoses across a number of cancer types. We present here a cancer cell-selective and hypoxia-responsive probe (fol-BODIPY) designed on the basis of density functional theory (DFT)-optimized quantum chemical calculations. The fol-BODIPY probe was found to provide a rapid fluorescence "off-on" response to hypoxia relative to controls, which lack the folate or nitro-benzyl moieties. In vitro confocal microscopy and flow cytometry analyses, as well as in vivo near-infrared optical imaging of CT26 solid tumor-bearing mice, provided support for the contention that fol-BODIPY is more readily accepted by folate receptor-positive CT26 cancer cells and provides a superior fluorescence "off-on" signal under hypoxic conditions than the controls. Based on the findings of this study, we propose that fol-BODIPY may serve as a tumor-targeting, hypoxia-activatable probe that allows for direct cancer monitoring both in vitro and in vivo.

Effectual Labeling of Natural Killer Cells with Upconverting Nanoparticles by Electroporation for In Vivo Tracking and Biodistribution Assessment.

Natural killer (NK) cells, which are cytotoxic lymphocytes of the innate immune system and recognize cancer cells via various immune receptors, are promising agents in cell immunotherapy. To utilize NK cells as a therapeutic agent, their biodistribution and pharmacokinetics need to be evaluated following systemic administration. Therefore, in vivo imaging and tracking with efficient labeling and quantitative analysis of NK cells are required. However, the lack of the phagocytic capacity of NK cells makes it difficult to establish breakthroughs in cell labeling and subsequent in vivo studies. Herein, an effective labeling of upconverting nanoparticles (UCNPs) in NK cells is proposed using electroporation with high sensitivity and stability. The labeling performance of UCNPs functionalized with carboxy-polyethylene glycol (PEG) is better than with methoxy-PEG or with amine-PEG. The labeling efficiency becomes higher, but cell damage is greater as electric field increases; thus, there is an optimum electroporation condition for internalization of UCNPs into NK cells. The tracking and biodistribution imaging analyses of intravenously injected NK cells show that the labeled NK cells are initially distributed primarily in lungs and then spread to the liver and spleen. These advances will accelerate the application of NK cells as key components of immunotherapy against cancer.

Intracerebral Transplantation of BDNF-overexpressing Human Neural Stem Cells (HB1.F3.BDNF) Promotes Migration, Differentiation and Functional Recovery in a Rodent Model of Huntington's Disease.

Huntington's disease (HD) is a dominantly inherited neurodegenerative disorder caused by abnormally expanded CAG repeats in the huntingtin gene. The huntingtin gene mutation leads to the progressive degeneration of striatal GABAergic medium spiny neurons (MSN) and reduces the level of brain-derived neurotrophic factor (BDNF) in HD patient's brain. BDNF is an essential neurotrophic factor for the cortico-striatal synaptic activity and the survival of GABAergic neurons. In this study, we transplanted BDNF-overexpressing human neural stem cells (HB1.F3.BDNF) into the contra-lateral side of unilateral quinolinic acid (QA)-lesioned striatum of HD rat model. The results of in vivo transplantation were monitored using various behavioral tests, 4.7 T animal magnetic resonance imaging (MRI) and immunohistochemical staining. We observed that the QA-lesioned rats receiving HB1.F3.BDNF cells exhibited significant behavioral improvements in the stepping, rotarod and apomorphine-induced rotation tests. Interestingly, contralaterally transplanted cells were migrated to the QA-lesioned striatum and the size of lateral ventricle was reduced. Histological analyses further revealed that the transplanted cells, which had migrated to the QA lesion site, were differentiated into the cells of GABAergic, MSN-type neurons expressing DARPP-32, and neural networks were established between the transplanted cells and the host brain, as revealed by retrograde tracing. Finally, there was a significant reduction of inflammatory response in HB1.F3.BDNF-transplanted HD animal model, compared with vehicle-transplanted group. Taken together, these results suggest that HB1.F3.BDNF can be an effective therapeutic strategy to treat HD patients in the future.

A three-dimensional hyaluronic acid-based niche enhances the therapeutic efficacy of human natural killer cell-based cancer immunotherapy.

Adoptive transfer of natural killer (NK) cells is becoming one of the most important parts of cancer immunotherapy. However, recent accomplishments have focused on the improvement of the targeting effects based on the engineering of chimeric antigen receptors (CARs) on cell surfaces. Despite the large quantity of therapeutic cells required for clinical applications, the technology for ex vivo expansion is not well developed. Herein, a three-dimensional (3D) engineered hyaluronic acid-based niche for cell expansion (3D-ENHANCE) is introduced. Compared with the conventional two-dimensional (2D) method, NK-92 cell lines and human EGFR-specific (CAR)-NK cells cultured in 3D-ENHANCE yield favorable mRNA expressions, elevated cytokine release, upregulated proliferative and tumor-lytic abilities, and result in enhanced antitumor efficacy. Furthermore, controllable degradation rates can be realized by tuning the formulation of 3D-ENHANCE so that it can be applied as an implantable cell reservoir at surgical sites. In vivo results with the incompletely resected MDA-MB-231 model confirm that the peri-operative implantation of 3D-ENHANCE prevents the relapse and metastases after surgery. Overall, 3D-ENHANCE presents an effective cytokine-free niche for ex vivo expansion and postsurgical treatment that enhances the low-therapeutic efficacy of human NK cells.

Oxaliplatin Pt(IV) prodrugs conjugated to gadolinium-texaphyrin as potential antitumor agents.

Described here is the development of gadolinium(III) texaphyrin-platinum(IV) conjugates capable of overcoming platinum resistance by 1) localizing to solid tumors, 2) promoting enhanced cancer cell uptake, and 3) reactivating p53 in platinum-resistant models. Side by side comparative studies of these Pt(IV) conjugates to clinically approved platinum(II) agents and previously reported platinum(II)-texaphyrin conjugates demonstrate that the present Pt(IV) conjugates are more stable against hydrolysis and nucleophilic attack. Moreover, they display high potent antiproliferative activity in vitro against human and mouse cell cancer lines. Relative to the current platinum clinical standard of care (SOC), a lead Gd(III) texaphyrin-Pt(IV) prodrug conjugate emerging from this development effort was found to be more efficacious in subcutaneous (s.c.) mouse models involving both cell-derived xenografts and platinum-resistant patient-derived xenografts. Comparative pathology studies in mice treated with equimolar doses of the lead Gd texaphyrin-Pt(IV) conjugate or the US Food and Drug Administration (FDA)-approved agent oxaliplatin revealed that the conjugate was better tolerated. Specifically, the lead could be dosed at more than three times (i.e., 70 mg/kg per dose) the tolerable dose of oxaliplatin (i.e., 4 to 6 mg/kg per dose depending on the animal model) with little to no observable adverse effects. A combination of tumor localization, redox cycling, and reversible protein binding is invoked to explain the relatively increased tolerability and enhanced anticancer activity seen in vivo. On the basis of the present studies, we conclude that metallotexaphyrin-Pt conjugates may have substantial clinical potential as antitumor agents.

Elevated aldolase 1A, retrogene 1 expression induces cardiac apoptosis in rat experimental autoimmune myocarditis model.

Acute myocarditis is an unpredictable heart disease that is caused by inflammation-associated cell death. Although viral infection and drug exposure are known to induce acute myocarditis, the molecular basis for its development remains undefined. Using proteomics and molecular analyses in myosin-induced rat experimental autoimmune myocarditis (EAM), we identified that elevated expression of aldolase 1A, retrogene 1 (Aldoart1) is critical to induce mitochondrial dysfunction and acute myocarditis development. Here, we demonstrate that cardiac cell death is associated with increased expressions of proapoptotic genes in addition to high levels of glucose, lactate, and triglyceride in metabolite profiling. The functional protein association network analysis also suggests that Aldoart1 upregulation correlates with high levels of dihydroxyacetone kinase and triglyceride. In H9c2 cardiac cells, lipopolysaccharides (LPS) or high glucose exposure significantly increases the cytochrome c release and the conversion of pro-caspase 3 into the cleaved form of caspase 3. We also found that LPS- or glucose-induced toxicities are almost completely reversed by siRNA-mediated knockdown of Aldoartl, which consequently increases cell viability. Together, our study strongly suggests that Aldoart1 may be involved in inducing mitochondrial apoptotic processes and can be a novel therapeutic target to prevent the onset of acute myocarditis or cardiac apoptosis.