A Nitric Oxide (NO) Nanoreporter for Noninvasive Real-Time Imaging of Macrophage Immunotherapy

Macrophage-centered therapeutic approaches that rely on immune modulation of tumor associated macrophages (TAMs) from a pro-tumorigenic phenotype (M2) to an anti-tumorigenic phenotype (M1) have facilitated a paradigm shift in macrophage immunotherapy. However, limited clinical success has been achieved due to the low response rates observed in different types of cancers. The ability to measure immune response in real time is critical in order to differentiate responders from non-responders; however, there are currently no platforms to monitor real-time macrophage immunotherapy response. Hence, there is an immediate need to develop imaging techniques that can longitudinally monitor macrophage immunotherapy response. Nitric oxide (NO) produced as a result of activation of macrophages to an anti-tumorigenic state is considered as a hallmark of M1 and can be a direct indication of response. In this study, a NO nanoreporter (NO-NR) is reported that enables real-time monitoring of macrophage immunotherapy drugs in vitro and in vivo. Furthermore, it is observed that sustained inhibition of colony stimulating factor 1 receptor (CSF1R) using a CSF1R inhibitor–NO-NR system leads to enhanced efficacy and better imaging signal. In conclusion, a first-of-its-kind NO nanoreporter tool is reported that can be used as an activatable imaging agent to monitor macrophage immunotherapy response in real time.     

Glycocalyx‐Mimicking Nanoparticles for Stimulation and Polarization of Macrophages via Specific Interactions

Malignant tumors develop multiple mechanisms to impair and escape from antitumor immune responses, of which tumor‐associated macrophages that often show immunosuppressive phenotype (M2), play a critical role in tumor‐induced immunosuppression. Therefore, strategies that can reverse M2 phenotype and even enhance immune‐stimulation function of macrophage would benefit tumor immunotherapy. In this paper, self‐assembled glyco‐nanoparticles (glyco‐NPs), as artificial glycocalyx, have been found to be able to successfully induce the polarization of mouse primary peritoneal macrophages from M2 to inflammatory type (M1). The polarization change was evidenced by the decreased expression of cell surface signaling molecules CD206 and CD23, and the increased expression of CD86. Meanwhile, secretion of cytokines supported this polarization change as well. More importantly, this phenomenon is observed not only in vitro, but also in vivo. As far as we known, this is the first report about macrophage polarization being induced by synthetic nanomaterials. Moreover, preparation, characterization of these glyco‐NPs and their interaction with the macrophages are also demonstrated.     

Comparative Effects of Graphene and Molybdenum Disulfide on Human Macrophage Toxicity

Graphene and other 2D materials, such as molybdenum disulfide, have been increasingly used in electronics, composites, and biomedicine. In particular, MoS₂ and graphene hybrids have attracted a great interest for applications in the biomedical research, therefore stimulating a pertinent investigation on their safety in immune cells like macrophages, which commonly engulf these materials. In this study, M1 and M2 macrophage viability and activation are mainly found to be unaffected by few‐layer graphene (FLG) and MoS₂ at doses up to 50 µg mL^?1. The uptake of both materials is confirmed by transmission electron microscopy, inductively coupled plasma mass spectrometry, and inductively coupled plasma atomic emission spectroscopy. Notably, both 2D materials increase the secretion of inflammatory cytokines in M1 macrophages. At the highest dose, FLG decreases CD206 expression while MoS₂ decreases CD80 expression. CathB and CathL gene expressions are dose‐dependently increased by both materials. Despite a minimal impact on the autophagic pathway, FLG is found to increase the expression of Atg5 and autophagic flux, as observed by Western blotting of LC3‐II, in M1 macrophages. Overall, FLG and MoS₂ are of little toxicity in human macrophages even though they are found to trigger cell stress and inflammatory responses.     

Cyclodextrin‐Derived Intrinsically Bioactive Nanoparticles for Treatment of Acute and Chronic Inflammatory Diseases

Inflammation is a common cause of many acute and chronic inflammatory diseases. A major limitation of existing anti‐inflammatory therapeutics is that they cannot simultaneously regulate pro‐inflammatory cytokine production, oxidative stress, and recruitment of neutrophils and macrophages. To overcome this limitation, nanoparticles (NPs) with multiple pharmacological activities are synthesized, using a chemically modified cyclic oligosaccharide. The manufacture of this type of bioactive, saccharide material‐based NPs (defined as LCD NP) is straightforward, cost‐effective, and scalable. Functionally, LCD NP effectively inhibits inflammatory response, oxidative stress, and cell migration for both neutrophils and macrophages, two major players of inflammation. Therapeutically, LCD NP shows desirable efficacies for the treatment of acute and chronic inflammatory diseases in mouse models of peritonitis, acute lung injury, and atherosclerosis. Mechanistically, the therapeutic benefits of LCD NP are achieved by inhibiting neutrophil‐mediated inflammatory macrophage recruitment and by preventing subsequent pro‐inflammatory events. In addition, LCD NP shows good safety profile in a mouse model. Thus, LCD NP can serve as an effective anti‐inflammatory nanotherapy for the treatment of inflammatory diseases mainly associated with neutrophil and macrophage infiltration.     

Secondary ion MS imaging to relatively quantify cholesterol in the membranes of individual cells from differentially treated populations

Time-of-flight secondary ion mass spectrometry (TOF-SIMS) is a well-established bioanalytical method for directly imaging the chemical distribution across single cells. Here we report a protocol for the use of SIMS imaging to comparatively quantify the relative difference in cholesterol level between the plasma membranes of two cells. It should be possible to apply this procedure to the study of other selected lipids. This development enables direct comparison of the chemical effects of different drug treatments and incubation conditions in the plasma membrane at the single-cell level. Relative, quantitative TOF-SIMS imaging has been used here to compare macrophage cells treated to contain elevated levels of cholesterol with respect to control cells. In situ fluorescence microscopy with two different membrane dyes was used to discriminate morphologically similar but differentially treated cells prior to SIMS analysis. SIMS images of fluorescently identified cells reveal that the two populations of cells have distinct outer leaflet membrane compositions with the membranes of the cholesterol-treated macrophages containing more than twice the amount of cholesterol of control macrophages. Relative quantification with SIMS to compare the chemical composition of single cells can provide valuable information about normal biological functions, causative agents of diseases, and possible therapies for diseases.     

Fluorescent liposomes as contrast agents for in vivo optical imaging of edemas in mice

This study assesses if specially designed fluorescent liposomes can be used as contrast agent for near-infrared fluorescence (NIRF) optical imaging of cultured macrophages in vitro and for NIRF imaging of inflammatory processes, like edema, in an in vivo mouse model. Fluorescent liposomes are prepared by the film hydration and extrusion method using cholesterol, L-phosphatidylcholine, and the NIR fluorescent dye DY-676-C(18) ester. Photon correlation spectroscopy and flow cytometry reveal that fluorescent liposomes are structurally stable for up to 133 days. Distinct uptake/labeling of cultured murine J774 macrophages is demonstrated by confocal laser scanning microscopy (CLSM), flow cytometry, and macroscopic NIRF imaging system at wavelengths >670 nm. Moreover, CLSM analysis reveals fluorescence signals within intracellular compartments. Ear edema is induced in mice (n = 16) by subcutaneous injection of zymosan A. Whole-body NIRF imaging is performed after intravenous injection (0-24 h) of fluorescent liposomes (55 nmol dye per kg body weight). Distinctly higher fluorescence intensities (1613.6 +/- 61.7 a.u.) are detected at inflamed areas of diseased mice as compared to controls (892.8 +/- 19.4 a.u.). Furthermore, cell isolated from ear lavage reveals the presence of labeled F4/80 positive tissue macrophages. Taken together, the results indicate both that mouse macrophages labeled with fluorescent liposomes can be detected in vitro with fluoro-optical methods and that in vivo optical imaging of inflammatory processes with fluorescent liposomes as contrast agent is feasible. Possibly, early stages of other inflammatory diseases could also be detected by the proposed diagnostic tool in the long term.     

CSF1R‐ and SHP2‐Inhibitor‐Loaded Nanoparticles Enhance Cytotoxic Activity and Phagocytosis in Tumor‐Associated Macrophages

Immune modulation of macrophages has emerged as an attractive approach for anti‐cancer therapy. However, there are two main challenges in successfully utilizing macrophages for immunotherapy. First, macrophage colony stimulating factor (MCSF) secreted by cancer cells binds to colony stimulating factor 1 receptor (CSF1‐R) on macrophages and in turn activates the downstream signaling pathway responsible for polarization of tumor‐associated macrophages (TAMs) to immunosuppressive M2 phenotype. Second, ligation of signal regulatory protein α (SIRPα) expressed on myeloid cells to CD47, a transmembrane protein overexpressed on cancer cells, activates the Src homology region 2 (SH2) domain ‐phosphatases SHP‐1 and SHP‐2 in macrophages. This results in activation of “eat‐me‐not” signaling pathway and inhibition of phagocytosis. Here, it is reported that self‐assembled dual‐inhibitor‐loaded nanoparticles (DNTs) target M2 macrophages and simultaneously inhibit CSF1R and SHP2 pathways. This results in efficient repolarization of M2 macrophages to an active M1 phenotype, and superior phagocytic capabilities as compared to individual drug treatments. Furthermore, suboptimal dose administration of DNTs in highly aggressive breast cancer and melanoma mouse models show enhanced anti‐tumor efficacy without any toxicity. These studies demonstrate that the concurrent inhibition of CSF1‐R and SHP2 signaling pathways for macrophage activation and phagocytosis enhancement could be an effective strategy for macrophage‐based immunotherapy.     

Dynamics of Salmonella infection of macrophages at the single cell level

Salmonella enterica causes a range of diseases. Salmonellae are intracellular parasites of macrophages, and the control of bacteria within these cells is critical to surviving an infection. The dynamics of the bacteria invading, surviving, proliferating in and killing macrophages are central to disease pathogenesis. Fundamentally important parameters, however, such as the cellular infection rate, have not previously been calculated. We used two independent approaches to calculate the macrophage infection rate: mathematical modelling of Salmonella infection experiments, and analysis of real-time video microscopy of infection events. Cells repeatedly encounter salmonellae, with the bacteria often remain associated with the macrophage for more than ten seconds. Once Salmonella encounters a macrophage, the probability of that bacterium infecting the cell is remarkably low: less than 5%. The macrophage population is heterogeneous in terms of its susceptibility to the first infection event. Once infected, a macrophage can undergo further infection events, but these reinfection events occur at a lower rate than that of the primary infection.     

Detection of inflammation in vivo by surface-enhanced Raman scattering provides higher sensitivity than conventional fluorescence imaging

The detection of inflammatory changes is a key aim for the early diagnosis and treatment of several autoimmune, infectious, and metastatic diseases. While surface-enhanced Raman scattering (SERS) has the capability to provide noninvasive, in vivo imaging at sufficient depth to achieve this goal, this approach has not been exploited in the study of inflammation. SERS-active nanoparticles were coded with a unique Raman signal that was protected under a wide range of conditions and stimuli. To detect early-stage inflammation, gold nanoparticle clusters containing Raman-active molecules were conjugated to intercellular adhesion molecule 1- (ICAM-1-) specific monoclonal antibodies. SERS allowed noninvasive measurement of ICAM-1 expression in vivo with twice the sensitivity of two-photon fluorescence. This is the first time SERS has been used for in vivo detection of inflammation and is a major advance in the ever-growing toolkit of approaches for use in noninvasive, next-generation in vivo imaging.     

Detection of magnetic nanoparticles in tissue using magneto-motive ultrasound

The purpose of this study was to demonstrate the magneto-motive ultrasonic detection of superparamagnetic iron oxide (SPIO) nanoparticles as a marker of macrophage recruitment in tissue. The capability of ultrasound to detect SPIO nanoparticles (core diameter ～20?nm) taken up by murine liver macrophages was investigated. Eight mice were sacrificed two days after the intravenous administration of four SPIO doses (1.5, 1.0, 0.5, and 0.1?mmol Fe/kg body weight). In the iron-laden livers, ultrasound Doppler measurements showed a frequency shift in response to an applied time-varying magnetic field. M-mode scan and colour power Doppler images of the iron-laden livers also demonstrated nanoparticle movement under focused magnetic field excitation. In the livers of two saline injected control mice, no movement was observed using any ultrasound imaging modes. The results of our experiments indicate that ultrasound imaging of magneto-motive excitation is a candidate imaging modality to identify tissue-based macrophages containing SPIO nanoparticles.     

A Light‐Activated Theranostic Nanoagent for Targeted Macrophage Ablation in Inflammatory Atherosclerosis

The synthesis and utility of a multimodal theranostic nanoagent based upon magnetofluorescent nanoparticles for the treatment of inflammatory atherosclerosis is described. These particles are modified with near‐infrared fluorophores and light‐activated therapeutic moieties, which allow for the optical determination of agent localization and phototoxic activation at spectrally distinct wavelengths. The resulting agent is readily taken up by murine macrophages in vitro and is highly phototoxic, with an LD₅₀ of 430 pM . Intravenous administration results in the localization of the nanoagent within macrophage‐rich atherosclerotic lesions that can be imaged by intravital fluorescence microscopy. Irradiation of the atheroma with 650 nm light activates the therapeutic component and results in eradication of inflammatory macrophages, which may induce lesion stabilization. Importantly, these agents display limited skin photosensitivity, are highly efficacious, and provide an integrated imaging and therapeutic nanoplatform for atherosclerosis.     

Human iPSC-Derived Proinflammatory Macrophages cause Insulin Resistance in an Isogenic White Adipose Tissue Microphysiological System

Chronic white adipose tissue (WAT) inflammation has been recognized as a critical early event in the pathogenesis of obesity-related disorders. This process is characterized by the increased residency of proinflammatory M1 macrophages in WAT. However, the lack of an isogenic human macrophage-adipocyte model has limited biological studies and drug discovery efforts, highlighting the need for human stem cell-based approaches. Here, human induced pluripotent stem cell (iPSC) derived macrophages (iMACs) and adipocytes (iADIPOs) are cocultured in a microphysiological system (MPS). iMACs migrate toward and infiltrate into the 3D iADIPOs cluster to form crown-like structures (CLSs)-like morphology around damaged iADIPOs, recreating classic histological features of WAT inflammation seen in obesity. Significantly more CLS-like morphologies formed in aged and palmitic acid-treated iMAC-iADIPO-MPS, showing the ability to mimic inflammatory severity. Importantly, M1 (proinflammatory) but not M2 (tissue repair) iMACs induced insulin resistance and dysregulated lipolysis in iADIPOs. Both RNAseq and cytokines analyses revealed a reciprocal proinflammatory loop in the interactions of M1 iMACs and iADIPOs. This iMAC-iADIPO-MPS thus successfully recreates pathological conditions of chronically inflamed human WAT, opening a door to study the dynamic inflammatory progression and identify clinically relevant therapies.     

Engineering Macrophages for Cancer Immunotherapy and Drug Delivery

Macrophages play an important role in cancer development and metastasis. Proinflammatory M1 macrophages can phagocytose tumor cells, while anti-inflammatory M2 macrophages such as tumor-associated macrophages (TAMs) promote tumor growth and invasion. Modulating the tumor immune microenvironment through engineering macrophages is efficacious in tumor therapy. M1 macrophages target cancerous cells and, therefore, can be used as drug carriers for tumor therapy. Herein, the strategies to engineer macrophages for cancer immunotherapy, such as inhibition of macrophage recruitment, depletion of TAMs, reprograming of TAMs, and blocking of the CD47-SIRPα pathway, are discussed. Further, the recent advances in drug delivery using M1 macrophages, macrophage-derived exosomes, and macrophage-membrane-coated nanoparticles are elaborated. Overall, there is still significant room for development in macrophage-mediated immune modulation and macrophage-mediated drug delivery, which will further enhance current tumor therapies against various malignant solid tumors, including drug-resistant tumors and metastatic tumors.     

Engineered Cytokine-Primed Extracellular Vesicles with High PD-L1 Expression Ameliorate Type 1 Diabetes

Type 1 diabetes (T1D), which is a chronic autoimmune disease, results from the destruction of insulin-producing β cells targeted by autoreactive T cells. The recent discovery that mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) function as therapeutic tools for autoimmune conditions has attracted substantial attention. However, the in vivo distribution and therapeutic effects of MSC-EVs potentiated by pro-inflammatory cytokines in the context of T1D have yet to be established. Here, it is reported that hexyl 5-aminolevulinate hydrochloride (HAL)-loaded engineered cytokine-primed MSC-EVs (H@TI-EVs) with high expression of immune checkpoint molecule programmed death-legend 1 (PD-L1) exert excellent inflammatory targeting and immunosuppressive effects for T1D imaging and therapy. The accumulated H@TI-EVs in injured pancreas not only enabled the fluorescence imaging and tracking of TI-EVs through the intermediate product protoporphyrin (PpIX) generated by HAL, but also promoted the proliferative and anti-apoptotic effects of islet β cells. Further analysis revealed that H@TI-EVs exhibited an impressive ability to reduce CD4⁺ T cell density and activation through the PD-L1/PD-1 axis, and induced M1-to-M2 macrophage transition to reshape the immune microenvironment, exhibiting high therapeutic efficiency in mice with T1D. This work identifies a novel strategy for the imaging and treatment of T1D with great potential for clinical application.     

Aurozyme: A Revolutionary Nanozyme in Colitis,Switching Peroxidase-Like to Catalase-Like Activity

A therapeutic strategy that could address colitis of multiple etiologies while restoring the dysbiosis of gut microbiota is attractive. Here, Aurozyme, a novel nanomedicine comprised of gold nanoparticles (AuNPs) and glycyrrhizin (GL) with a glycol chitosan coating layer, as a promising approach for colitis, is demonstrated. The unique feature of Aurozyme is the conversion of harmful peroxidase-like activity of AuNPs to beneficial catalase-like activity due to the amine-rich environment provided by the glycol chitosan. This conversion process enables Aurozyme to oxidize the hydroxyl radicals derived from AuNP, producing water and oxygen molecules. In fact, Aurozyme effectively scavenges reactive oxygen/reactive nitrogen species (ROS/RNS) and damage-associated molecular patterns (DAMPs), which can attenuate the M1 polarization of macrophage. It exhibits prolonged adhesion to the lesion site, promoting sustained anti-inflammatory effects and restoring intestinal function in colitis-challenged mice. Additionally, it increases the abundance and diversity of beneficial probiotics, which are essential for maintaining microbial homeostasis in the gut. The work highlights the transformative potential of nanozymes for the comprehensive treatment of inflammatory disease and represents an innovative switching technology of enzyme-like activity by Aurozyme.     

Biofilm Potentiates Cancer-Promoting Effects of Tumor-Associated Macrophages in a 3D Multi-Faceted Tumor Model (Small 19/2023)

Components of the tumor microenvironment (TME), such as tumor-associated macrophages (TAMs), influence tumor progression. The specific polarization and phenotypic transition of TAMs in the tumor microenvironment lead to two-pronged impacts that can promote or hinder cancer development and treatment. Here, a novel microfluidic multi-faceted bladder tumor model (TAM^PIEB) is developed incorporating TAMs and cancer cells to evaluate the impact of bacterial distribution on immunomodulation within the tumor microenvironment in vivo. It is demonstrated for the first time that biofilm-induced inflammatory conditions within tumors promote the transition of macrophages from a pro-inflammatory M1-like to an anti-inflammatory/pro-tumor M2-like state. Consequently, multiple roles and mechanisms by which biofilms promote cancer by inducing pro-tumor phenotypic switch of TAMs are identified, including cancer hallmarks such as reducing susceptibility to apoptosis, enhancing cell viability, and promoting epithelial-mesenchymal transition and metastasis. Furthermore, biofilms formed by extratumoral bacteria can shield tumors from immune attack by TAMs, which can be visualized through various imaging assays in situ. The study sheds light on the underlying mechanism of biofilm-mediated inflammation on tumor progression and provides new insights into combined anti-biofilm therapy and immunotherapy strategies in clinical trials.     

Screening of biomedical polymer biocompatibility in NMRI-mice peritoneal cavity: A comparison between ultra-high-molecular-weight polyethylene (UHMW-PE) and polyethyleneterephthalate (PET)

The peritoneal resident cell population is influenced by various inflammatory and immunogenic stimuli. The influence of intraperitoneal application of polyethyleneterephthalate (PET) (group A) and ultra-high-molecular-weight polyethylene (UHMW-PE) (group B) powders on peritoneal cell count and macrophage activity was investigated. Powders were tested to mimic wear particles from solid implant devices as these particles often cause chronic granulomatous inflammation. The results were compared with the inflammatory response following an abdominal midline incision (group C) and untreated animals (group D). On days 1, 7, 14 and 30 peritoneal cells were quantified and the number of active macrophages was assessed. Groups A and C mice showed a significant loss of macrophages in the peritoneal lavage at day 1 but this returned to normal values (group D) on day 7. In contrast, group B animals remained at low peritoneal cell counts but showed the highest number of active macrophages. Only in this latter group was adhesion formation and granulomatous clustering of polymer powder observed. Applying the parameters macrophage count and the number of active macrophages it can be concluded that PET elicits a weaker inflammatory reaction than UHMW-PE in mice peritoneal cavity. Thus this animal model may be used as a screening test for biomedical materials, especially their wear products.