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Characterization of M1 and M2 polarization phenotypes in peritoneal macrophages after treatment with graphene oxide nanosheets
摘要： Macrophages play a key role in nanoparticle removal and are primarily responsible for their uptake and trafficking in vivo. Due to their functional plasticity, macrophages display a spectrum of phenotypes between two extremes identified as pro-inflammatory M1 and reparative M2 macrophages, characterized by the expression of specific cell surface markers and the secretion of different cytokines. The influence of graphene oxide (GO) nanosheets functionalized with poly(ethylene glycol-amine) and labelled with fluorescein isothiocyanate (FITC-PEG-GO) on polarization of murine peritoneal macrophages towards M1 and M2 phenotypes was evaluated in basal and stimulated conditions by flow cytometry and confocal microscopy through the expression of different cell markers: CD80 and iNOS as M1 markers, and CD206 and CD163 as M2 markers. Although FITC-PEG-GO did not induce M1 or M2 macrophage polarization after 24 and 48 h in basal conditions, this nanomaterial decreased the percentage of M2 reparative macrophages. We have also compared control macrophages with macrophages that have or have not taken up FITC-PEG-GO after treatment with these nanosheets (GO+ and GO- cells, respectively). The CD80 expression diminished in GO+ macrophages after 48 h of GO treatment but the CD206 expression in GO+ population showed higher values than in both GO- population and control macrophages. In the presence of pro-inflammatory stimuli (LPS and IFN-γ), a significant decrease of CD80+ cells was observed after treatment with GO. This nanomaterial also induced significant decreases of CD206+ and CD163+ cells in the presence of reparative stimulus (IL-4). The CD80, iNOS and CD206 expression was lower in both GO- and GO+ cells than in control macrophages. However, higher CD163 expression was obtained in both GO- and GO+ cells in comparison with control macrophages. All these facts suggest that FITC-PEG-GO uptake did not induce the macrophage polarization towards the M1 pro-inflammatory phenotype, promoting the control of the M1/M2 balance with a slight shift towards M2 reparative phenotype involved in tissue repair, ensuring an appropriate immune response to these nanosheets.
关键词： Graphene oxide nanosheets,Macrophage polarization,Peritoneal macrophages,Cytokine profiling
更新于2025-09-23 15:23:52
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A Fluorescence Based-Proliferation Assay for the Identification of Replicating Bacteria Within Host Cells
摘要： Understanding host pathogen interactions is paramount to the development of novel antimicrobials. An important facet of this pursuit is the accurate characterization of pathogen replication within infected host cells. Here we describe the use of a fluorescence-based proliferation assay to identify intracellular populations of replicating bacteria at the subcellular level. Using Staphylococcus aureus as a model Gram-positive bacterial pathogen and macrophages as a model host phagocyte, we demonstrate this assay can be used to reliably identify individual phagocytes that contain replicating bacteria. Furthermore, we demonstrate this assay is compatible with additional cellular probes that enable characterization of cellular compartments in which replicating bacteria reside. Finally, we demonstrate that this assay facilitates the investigation of both Gram-negative and Gram-positive bacteria within host cells.
关键词： microscopy,Staphylococcus,phagolysosome,fluorescence,phagocytosis,macrophage
更新于2025-09-23 15:22:29
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Preliminary Study of MR and Fluorescence Dual-mode Imaging: Combined Macrophage-Targeted and Superparamagnetic Polymeric Micelles
摘要： Purpose: To establish small-sized superparamagnetic polymeric micelles for magnetic resonance and fluorescent dual-modal imaging (MRI) and investigated the macrophage-targeted in vitro. Methods: A new class of superparamagnetic iron oxide nanoparticles (SPIONs) and Nile red-co-loaded mPEG-Lys3-CA4-NR/SPION polymeric micelles was synthesized to label Raw264.7 cells. The physical characteristics of the polymeric micelles were assessed, the T2 relaxation rate was calculated, and the effect of labeling on the cell viability and cytotoxicity was also determined in vitro. In addition, further evaluation of the application potential of the micelles was conducted via in vitro MRI. Results: The diameter of the mPEG-Lys3-CA4-NR/SPION polymeric micelles was 33.8 ± 5.8 nm on average. Compared with the hydrophilic SPIO, mPEG-Lys3-CA4-NR/SPION micelles increased transversely (r2), leading to a notably high r2 from 1.908 μg/mL-1S-1 up to 5.032 μg/mL-1S-1, making the mPEG-Lys3-CA4-NR/SPION micelles a highly sensitive MRI T2 contrast agent, as further demonstrated by in vitro MRI. The results of Confocal Laser Scanning Microscopy (CLSM) and Prussian blue staining of Raw264.7 after incubation with micelle-containing medium indicated that the cellular uptake efficiency is high. Conclusion: We successfully synthesized dual-modal MR and fluorescence imaging mPEG-Lys3-CA4-NR/SPION polymeric micelles with an ultra-small size and high MRI sensitivity, which were effectively and quickly uptaken into Raw 264.7 cells. mPEG-Lys3-CA4-NR/SPION polymeric micelles might become a new MR lymphography contrast agent, with high effectiveness and high MRI sensitivity.
关键词： macrophage-targeted,polymeric micelles,MRI,SPIONs,fluorescence imaging
更新于2025-09-23 15:21:01
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Novel ?2-1,3-D-glucan porous microcapsule enveloped folate-functionalized liposomes as a Trojan horse for facilitated oral tumor-targeted co-delivery of chemotherapeutic drugs and quantum dot
摘要： In this study, a new type of β-1,3-D-glucan porous microcapsule (GPM)-enveloped and folate conjugated chitosan-functional liposome (FCL), FCL@GPM, was developed for the potential oral co-delivery of chemotherapeutic drugs and quantum dots (QDs) with facilitated drug absorption and antitumor efficacy. In this dual-particulate system, multiple FCLs serve as the cores for effective loading, folate-mediated tumor-targeting, facilitated intracellular accumulation, and pH-responsive controlled release of chemotherapeutic agents, while a GPM acts as the shell for affording macrophage-mediated tumor selectivity. Gefitinib (GEF) was selected as a chemotherapeutic agent, while acid degradable ZnO QDs were selected due to its dual role both as an anticancer agent for synergistic chemotherapy and as a fluorescent probe for potential cancer cellular imaging. The GEF and ZnO QDs co-loaded FCL@GPMs (GEF/ZnO-FCL@GPMs) have a prolonged release manner with limited release before uptake by intestinal cells. Furthermore, the Peyer’s patches uptake, macrophages uptake, cytotoxicity, and biodistribution of FCL@GPMs were tested. In addition, GEF and ZnO QDs co-loaded FCLs (GEF/ZnO-FCLs) not only have a tumor acidity responsive release property, but also induce a superior cytotoxicity on cancer cells as compared to GEF. Moreover, a 1.75-fold increase in the bioavailability of GEF delivered from GEF/ZnO-FCL@GPMs as compared to its trademarked drug (Iressa?). As a result, GEF/ZnO-FCL@GPMs exerted a superior antitumor efficacy (1.47-fold) as compared to its trademarked drug in mice. Considered together, the developed FCL@GPMs, combining the unique physicochemical and biological benefits of FCLs and GPMs, possess a great potential as an efficient delivery system for the co-delivery of chemotherapeutic agents and quantum dots.
关键词： chemotherapeutic drugs,pH-responsive controlled release,tumor-targeting,oral co-delivery,macrophage-mediated tumor selectivity,β-1,3-D-glucan porous microcapsule,folate conjugated chitosan-functional liposome,quantum dots
更新于2025-09-23 15:19:57
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Essential contribution of macrophage Tie2 signal mediated autophagy in laser-induced choroidal neovascularization
摘要： Autophagy plays critical roles in various ocular diseases, including age-related macular degeneration (AMD). Tie2-expressing macrophages (TEMs) play crucial roles in angiogenesis. To investigate the role of TEMs and autophagy in the development of AMD, we employed macrophage-specific Tie2 knockout mice and used a laser-induced choroidal neovascularization (CNV). The results showed that TEMs can promote CNV formation by up-regulating the level of autophagy. These results were further verified by in vitro cell experiments that peritoneal macrophages from Tie2 knockout mice can inhibit the expression of autophagy-related factors and inhibit the expression of angiogenic factor of VEGF by activating AMPK signaling pathway. Our results suggest that TEMs and macrophage Tie2 signal mediated-autophagy play critical role in experimental CNV, and they may be novel preventive targets for AMD treatment.
关键词： Tie2-expressing macrophage (TEMs),Age-related macular degeneration,Choroidal neovascularization,Autophagy
更新于2025-09-23 15:19:57
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Aromatic secondary amine-functionalized fluorescent NO probes: improved detection sensitivity for NO and potential applications in cancer immunotherapy studies
摘要： Tumor-associated macrophages (TAMs), constituting up to 50% of the solid tumor mass and commonly having a pro-tumoral M2 phenotype, are closely associated with decreased survival in patients. Based on the highly dynamic properties of macrophages, in recent years the repolarization of TAMs from pro-tumoral M2 phenotype to anti-tumoral M1 phenotype by various strategies has emerged as a promising cancer immunotherapy approach for improving cancer therapy. Herein, we present an aromatic secondary amine-functionalized Bodipy dye 1 and its mitochondria-targetable derivative Mito1 as fluorescent NO probes for discriminating M1 macrophages from M2 macrophages in terms of their difference in inducible NO synthase (iNOS) levels. The two probes possess the unique ability to simultaneously respond to two secondary oxides of NO, i.e., N2O3 and ONOO-, thus being more sensitive and reliable for reflecting intracellular NO than most of the existing fluorescent NO probes that usually respond to N2O3 only. With 1 as a representative, the discrimination between M1 and M2 macrophages, evaluation of the repolarization of TAMs from pro-tumoral M2 phenotype to anti-tumoral M1 phenotype, and visualization of NO communication during the immune-mediated phagocytosis of cancer cells by M1 macrophages have been realized. These results indicate that our probes should hold great potential for imaging applications in cancer immunotherapy studies and relevant anti-cancer drug screening.
关键词： fluorescent NO probes,Bodipy dye,cancer immunotherapy,NO detection,macrophage polarization
更新于2025-09-19 17:15:36
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The Effect of Fluence on Macrophage Kinetics, Oxidative Stress, and Wound Closure Using Real-Time <i>In Vivo</i> Imaging
摘要： Objective: The aim of our study was to quantify the effect of doses delivered by a He:Ne laser on individual macrophage kinetics, tissue oxidative stress, and wound closure using real-time in vivo imaging. Background: Photobiomodulation has been reported to reduce tissue inflammation and accelerate wound closure; however, precise parameters of laser settings to optimize macrophage behavior have not been established. We hypothesized that quantitative and real-time in vivo imaging could identify optimal fluence for macrophage migration, reduction of reactive oxygen species, and acceleration of wound closure. Methods: Larval zebrafish Tg(mpeg-dendra2) were loaded with dihydroethidium for oxidative stress detection. Fish were caudal fin injured, treated with 635 nm continuous 5 mW He:Ne laser irradiation at 3, 9, or 18 J/cm2 and time-lapsed imaged within the first 120 min postinjury. Images taken 1 and 24-h postinjury were compared for percentage wound closure. Results: A fluence of 3 J/cm2 demonstrated significant increases in macrophage migration speed, fewer stops along the way, and greatest directed migration toward the wound. These findings were associated with a significant reduction in wound content reactive oxygen species when compared with control wounded fins. Both 3 and 9 J/cm2 significantly accelerated wound closure when compared with nonirradiated control fish. Conclusions: Wound macrophage activity could be manipulated by applied fluence, leading to reduced levels of wound reactive oxygen species and accelerated wound closure. The zebrafish model provides a means to quantitatively compare wound macrophage behavior in response to a variety of laser treatment parameters in real time.
关键词： zebrafish,macrophage,He:Ne laser,oxidative stress,time-lapse imaging
更新于2025-09-19 17:15:36
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Clinical Translation of [68Ga]Ga-NOTA-anti-MMR-sdAb for PET/CT Imaging of Protumorigenic Macrophages
摘要： Purpose: Macrophage mannose receptor (MMR, CD206) expressing tumor-associated macrophages (TAM) are protumorigenic and was reported to negatively impact therapy responsiveness and is associated with higher chances of tumor relapse following multiple treatment regimens in preclinical tumor models. Since the distribution of immune cells within the tumor is often heterogeneous, sampling errors using tissue biopsies will occur. In order to overcome this limitation, we propose positron emission tomography (PET)/X-ray computed tomography (CT) imaging using 68Ga-labeled anti-MMR single-domain antibody fragment (sdAb) to assess the presence of these protumorigenic TAM. Procedures: Cross-reactive anti-MMR-sdAb was produced according to good manufacturing practice (GMP) and conjugated to p-SCN-Bn-NOTA bifunctional chelator for 68Ga-labeling. Biodistribution and PET/CT studies were performed in wild-type and MMR-deficient 3LL-R tumor-bearing mice. Biodistribution data obtained in mice were extrapolated to calculate radiation dose estimates for the human adult using OLINDA software. A 7-day repeated dose toxicity study for NOTA-anti-MMR-sdAb was performed in healthy mice up to a dose of 1.68 mg/kg. Results: [68Ga]Ga-NOTA-anti-MMR-sdAb was obtained with 76 ± 2 % radiochemical yield, 99 ± 1 % radiochemical purity, and apparent molar activity of 57 ± 11 GBq/μmol. In vivo biodistribution analysis showed fast clearance via the kidneys and retention in MMR-expressing organs and tumor, with tumor-to-blood and tumor-to-muscle ratios of 6.80 ± 0.62 and 5.47 ± 1.82, respectively. The calculated effective dose was 0.027 mSv/MBq and 0.034 mSv/MBq for male and female, respectively, which means that a proposed dose of 185 MBq in humans would yield a radiation dose of 5.0 and 6.3 mSv to male and female patients, respectively. In the toxicity study, no adverse effects were observed. Conclusions: Preclinical validation of [68Ga]Ga-NOTA-anti-MMR-sdAb showed high specific uptake of this tracer in MMR-expressing TAM and organs, with no observed toxicity. [68Ga]Ga-NOTA-anti-MMR-sdAb is ready for a phase I clinical trial.
关键词： Single-domain antibody (sdAb),PET,Tumor-associated macrophages (TAM),Macrophage mannose receptor (MMR)
更新于2025-09-19 17:15:36
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Camouflaged Nanosilver with Excitation Wavelength Dependent High Quantum Yield for Targeted Theranostic
摘要： The present study shows the thorough investigations on optical properties and hydrodynamic diameters of glutathione (GSH) stabilized nanosilver clusters (AgNC) at different stages of synthesis and engineering for the optimized absolute quantum yield to generate fluorescent images of Dalton Lymphoma Ascites (DLA) tumour bearing mice. The initial increment of quantum yield was wavelength dependent and finally it became 0.509 which was due to the camouflaging or entrapment of AgNC in macrophages membranes. The potentiality of macrophages membrane camouflaged silver nanoclusters (AgM) was reflected in the cell viability assay and confocal based live dead cell assay where the AgM has better cell killing effect compared to AgNC with reduced dosage and in vivo mice imaging generated the clear visualization at the tumour sites. Therefore, from the present study, it can be considered that the camouflaged nanosilver can be used for targeted theranostic applications.
关键词： nanosilver clusters,macrophage membrane,theranostic,fluorescence imaging,quantum yield
更新于2025-09-10 09:29:36
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Lactobacillus paracasei KW3110 Prevents Blue Light-Induced Inflammation and Degeneration in the Retina
摘要： Age-related macular degeneration and retinitis pigmentosa are leading causes of blindness and share a pathological feature, which is photoreceptor degeneration. To date, the lack of a potential treatment to prevent such diseases has raised great concern. Photoreceptor degeneration can be accelerated by excessive light exposure via an inflammatory response; therefore, anti-inflammatory agents would be candidates to prevent the progress of photoreceptor degeneration. We previously reported that a lactic acid bacterium, Lactobacillus paracasei KW3110 (L. paracasei KW3110), activated macrophages suppressing inflammation in mice and humans. Recently, we also showed that intake of L. paracasei KW3110 could mitigate visual display terminal (VDT) load-induced ocular disorders in humans. However, the biological mechanism of L. paracasei KW3110 to retain visual function remains unclear. In this study, we found that L. paracasei KW3110 activated M2 macrophages inducing anti-inflammatory cytokine interleukin-10 (IL-10) production in vitro using bone marrow-derived M2 macrophages. We also show that IL-10 gene expression was significantly increased in the intestinal immune tissues 6 h after oral administration of L. paracasei KW3110 in vivo. Furthermore, we demonstrated that intake of L. paracasei KW3110 suppressed inflammation and photoreceptor degeneration in a murine model of light-induced retinopathy. These results suggest that L. paracasei KW3110 may have a preventive effect against degrative retinal diseases.
关键词： light,macrophage,Lactobacillus paracasei KW3110,retina
更新于2025-09-04 15:30:14