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Fluorine-containing nanoparticles (FNPs) are widely used for inflammation imaging by fluorine-19 magnetic resonance imaging ( 19 F MRI) due to their biocompatibility and suitability to track immune cells via phagocytic uptake. For targeting approaches beyond passive incorporation, surface PEGylation of FNPs is required to reduce cellular uptake, but is known to prolong blood half-life of the particles. This study investigates the efficacy of FNP PEGylation for inflammation imaging in vivo. FNPs and PEGylated FNPs ( PEG FNPs) of different size were synthesized and characterized for particle properties and fluorine content. Cellular uptake was explored in CHO, RAW, and J774 cells as well as in whole blood using flow cytometry. For in vivo imaging, a murine lipopolysaccharide (LPS)-induced inflammation model was employed, followed by intravenous injection of FNPs or PEG FNPs and 19 F MRI to monitor inflammation. PEGylation significantly reduced the uptake of FNPs by macrophages and blood immune cells, as observed through reduced fluorescence and 19 F signals. Despite reduced cellular uptake in vitro, in vivo 19 F MRI showed similar signal intensities in inflamed tissues for both FNPs and PEG FNPs, suggesting contributions from both immune cell-associated and non-cell-associated signals for small particles. However, for bigger particles significantly more 19 F signal was observed in inflamed tissue for FNP compared to PEG FNP. In conclusion, increase in particle size can abolish the non-specific accumulation of FNPs in inflammatory lesions and additionally increase the phagocytosis of FNPs by murine immune cells. This results in a specific immune-cell dependent 19 F signal with rather no background due to non-specific diffusion.

In vivo monitoring of polymers is crucial for drug delivery and tissue regeneration. Magnetic resonance imaging (MRI) is a whole-body imaging technique, and heteronuclear MRI allows quantitative imaging. However, MRI agents can result in environmental pollution and organ accumulation. To address this, we introduce biocompatible and biodegradable polyphosphoesters, as MRI-traceable polymers using the 31 P centers in the polymer backbone. We overcome challenges in 31 P MRI, including background interference and low sensitivity, by modifying the molecular environment of 31 P, assembling polymers into colloids, and tailoring the polymers’ microstructure to adjust MRI-relaxation times. Specifically, gradient-type polyphosphonate-copolymers demonstrate improved MRI-relaxation times compared to homo- and block copolymers, making them suitable for imaging. We validate background-free imaging and biodegradation in vivo using Manduca sexta . Furthermore, encapsulating the potent drug PROTAC allows using these amphiphilic copolymers to simultaneously deliver drugs, enabling theranostics. This first report paves the way for polyphosphoesters as background-free MRI-traceable polymers for theranostic applications. MRI agents can result in environmental pollution and organ accumulation. Here, the authors show that modifying the molecular structure of biodegradable polyphosphoesters and tailoring the polymers’ microstructure to adjust MRI relaxation times can overcome challenges in 31 P MR imaging.

BackgroundInflammation and metabolism exhibit a complex interplay, where inflammation influences metabolic pathways, and in turn, metabolism shapes the quality of immune responses. Here, glucose turnover is of special interest, as proinflammatory immune cells mainly utilize glycolysis to meet their energy needs. Noninvasive approaches to monitor both processes would help elucidate this interwoven relationship to identify new therapeutic targets and diagnostic opportunities.MethodsFor induction of defined inflammatory hotspots, LPS-doped Matrigel plugs were implanted into the neck of C57BL/6J mice. Subsequently, 1H/19F magnetic resonance imaging (MRI) was used to track the recruitment of 19F-loaded immune cells to the inflammatory focus and deuterium (2H) magnetic resonance spectroscopy (MRS) was used to monitor the metabolic fate of [6,6-2H2]glucose within the affected tissue. Histology and flow cytometry were used to validate the in vivo data.ResultsAfter plug implantation and intravenous administration of the 19F-containing contrast agent, 1H/19F MRI confirmed the infiltration of 19F-labeled immune cells into LPS-doped plugs while no 19F signal was observed in PBS-containing control plugs. Identification of the inflammatory focus was followed by i.p. bolus injection of deuterated glucose and continuous 2H MRS. Inflammation-induced alterations in metabolic fluxes could be tracked with an excellent temporal resolution of 2 min up to approximately 60 min after injection and demonstrated a more anaerobic glucose utilization in the initial phase of immune cell recruitment.Conclusion1H/2H/19F MRI/MRS was successfully employed for noninvasive monitoring of metabolic alterations in an inflammatory environment, paving the way for simultaneous in vivo registration of immunometabolic data in basic research and patients.

Histological analysis with 2,3,5-triphenyltetrazolium chloride (TTC) staining is the most frequently used tool to detect myocardial ischemia/reperfusion injury. However, its practicality is often challenged by poor image quality in gross histology, leading to an equivocal infarct-boundary delineation and potentially compromised measurement accuracy. Here, we introduce several crucial refinements in staining protocol and sample processing, which enable TTC images to be analyzed with light microscopy. The refined protocol involves a two-step TTC staining process (perfusion and immersion) and subsequent Zamboni fixation to differentiate myocardial viability and necrosis, and use of Coomassie brilliant blue to label area-at-risk. After the duo-staining steps were completed, the heart sample was embedded and sliced transversally by a cryostat into a series of thin sections (50 µm) for microscopic analysis. The refined TTC (redTTC) assay yielded remarkably high-quality images with striking color intensity and sharply defined boundaries, permitting unambiguous and reliable delineation of the infarct and area-at-risk. In the same animals, the redTTC assay showed good agreement with the in-vivo gold standard measurements (LGE and MEMRI). Meanwhile, redTTC imaging allows tracking of viable cardiomyocytes at cellular resolution, and with this enhanced capability, we convincingly demonstrated the pro-survival action of stem cells based-therapy. Therefore, the redTTC assay represents a significant technical advance that permits precise detection of the true extent of cardiac injury and cardiomyocyte viability. This approach is cost-effective and may be adapted for use in diverse applications, making it highly appealing to many laboratories performing ischemia/reperfusion injury experiments.

After myocardial infarction (MI), epicardial cells reactivate their embryonic program, proliferate and migrate into the damaged tissue to differentiate into fibroblasts, endothelial cells and, if adequately stimulated, to cardiomyocytes. Targeting epicardium-derived stromal cells (EpiSC) by specific ligands might enable the direct imaging of EpiSCs after MI to better understand their biology, but also may permit the cell-specific delivery of small molecules to improve the post-MI healing process. Therefore, the aim of this study was to identify specific peptides by phage display screening to enable EpiSC specific cargo delivery by active targeting. To this end, we utilized a sequential panning of a phage library on cultured rat EpiSCs and then subtracted phage that nonspecifically bound blood immune cells. EpiSC specific phage were analyzed by deep sequencing and bioinformatics analysis to identify a total of 78 300 ± 31 900 different, EpiSC-specific, peptide insertion sequences. Flow cytometry of the five most highly abundant peptides (EP1, -2, –3, -7 or EP9) showed strong binding to EpiSCs but not to blood immune cells. The best binding properties were found for EP9 which was further studied by surface plasmon resonance (SPR). SPR revealed rapid and stable association of EpiSCs with EP9. As a negative control, THP-1 monocytes did not associate with EP9. Coupling of EP9 to perfluorocarbon nanoemulsions (PFCs) resulted in the efficient delivery of 19 F cargo to EpiSCs and enabled their visualization by 19 F MRI. Moreover, active targeting of EpiSCs by EP9-labelled PFCs was able to outcompete the strong phagocytic uptake of PFCs by circulating monocytes. In summary, we have identified a 7-mer peptide, (EP9) that binds to EpiSCs with high affinity and specificity. This peptide can be used to deliver small molecule cargos such as contrast agents to permit future in vivo tracking of EpiSCs by molecular imaging and to transfer small pharmaceutical molecules to modulate the biological activity of EpiSCs.

Vascular inflammation plays a key role in the development of aortic diseases. A potential novel target for treatment might be CD73, an ecto-5′-nucleotidase that generates anti-inflammatory adenosine in the extracellular space. Here, we investigated whether a lack of CD73 results in enhanced aortic inflammation. To this end, angiotensin II was infused into wildtype and CD73 −/− mice over 10 days. Before and after infusion, mice were analyzed using magnetic resonance imaging, ultrasound, flow cytometry, and histology. The impact of age and gender was investigated using female and male mice of three and six months of age, respectively. Angiotensin II infusion led to increased immune cell infiltration in both genotypes’ aortae, but depletion of CD73 had no impact on immune cell recruitment. These findings were not modified by age or sex. No substantial difference in morphological or functional characteristics could be detected between wildtype and CD73 −/− mice. Interestingly, the expression of CD73 on neutrophils decreased significantly in wildtype mice during treatment. In summary, we have found no evidence that CD73 deficiency affects the onset of aortic inflammation. However, as CD73 expression decreased during disease induction, an increase in CD73 by pharmaceutical intervention might result in lower vascular inflammation and less vascular disease.

Neutrophils are crucial for fighting invading pathogens and for the clearance of sterile wounds. However, in cases of overwhelming pathogens or excessive inflammatory responses, fine‐tuning of neutrophil functions can help to either enhance bacterial killing or prevent unwanted tissue damage. Thus, the present study is aimed at developing a nanoparticle‐based toolbox to direct neutrophils toward either a proinflammatory or reparative phenotype tailored to the specific inflammatory environment. For this, neutrophil‐specific fluorine nanoparticles additionally equipped with either activating or inhibiting peptides for a background‐free 19F MRI‐based theranostic approach are engineered. It is demonstrated that with this i) distinct effector functions can be specifically modulated in both murine and human neutrophils, ii) the disease‐specific degree of neutrophil infiltration can be non‐invasively monitored in vivo, and in turn iii) neutrophil effector functions can either be attenuated during a pharmacological challenge or amplified to ameliorate tissue damage during bacteria‐driven acute colitis. Thus, this nanotheranostic approach is suitable to visualize and concomitantly direct neutrophil functionality depending on the specific requirements to improve disease course and outcome, thus paving the way for a personalized neutrophil immunotherapy. Here, neutrophil‐specific fluorine nanoparticles additionally equipped with either activating or inhibiting peptides for a background‐free 19F MRI‐based nanotheranostic approach are engineered. It is demonstrated that this approach is suitable to visualize and concomitantly direct neutrophil functionality depending on the specific requirements to improve disease course and outcome, thus paving the way for a personalized neutrophil immunotherapy.

Abdominal aortic aneurysms and dissections (AAA/AD) are vascular disorders with high mortality due to aortic ruptures. Critical pathomechanisms involve immune cell infiltration and degradation of the vascular extracellular matrix (ECM). Hyaluronan (HA), a major constituent of the ECM synthesized by three HA synthase isoenzymes (HAS1-3), plays a role in both processes. Specifically, HAS3 is crucially involved in inflammatory conditions. Here, we aimed to elucidate the role of HAS3-derived HA in AAA/AD. Mice double-deficient for and ( -DKO) and littermate controls ( -KO) were studied in a model of angiotensin II (AngII)-induced AAA/AD. deficiency improved survival in -DKO mice reducing aortic ruptures. This was associated with decreased monocyte infiltration into the vessel wall. Aortic RNA-Seq analysis indicated disturbed immune cell adhesion and diapedesis. Transfer of deficient bone marrow into -DKO mice largely normalized the -DKO phenotype. While gene expression in endothelial cells (ECs) was not affected, AngII-induced upregulation of proinflammatory cytokines, adhesion receptors and the HA receptor CD44 was attenuated in DKO monocytes. This reduced CD44 cell surface expression in double-deficient monocytes, ultimately inhibiting their transmigration. Our results show that HAS3 plays a key role in AAA/AD formation and suggest the HAS3/CD44 axis as promising therapeutic target to reduce monocyte recruitment and aortic rupture.

Fluorine-containing nanoparticles (FNPs) are widely used for inflammation imaging by fluorine-19 magnetic resonance imaging ( F MRI) due to their biocompatibility and suitability to track immune cells via phagocytic uptake. For targeting approaches beyond passive incorporation, surface PEGylation of FNPs is required to reduce cellular uptake, but is known to prolong blood half-life of the particles. This study investigates the efficacy of FNP PEGylation for inflammation imaging in vivo. FNPs and PEGylated FNPs ( FNPs) of different size were synthesized and characterized for particle properties and fluorine content. Cellular uptake was explored in CHO, RAW, and J774 cells as well as in whole blood using flow cytometry. For in vivo imaging, a murine lipopolysaccharide (LPS)-induced inflammation model was employed, followed by intravenous injection of FNPs or FNPs and F MRI to monitor inflammation. PEGylation significantly reduced the uptake of FNPs by macrophages and blood immune cells, as observed through reduced fluorescence and F signals. Despite reduced cellular uptake in vitro, in vivo F MRI showed similar signal intensities in inflamed tissues for both FNPs and FNPs, suggesting contributions from both immune cell-associated and non-cell-associated signals for small particles. However, for bigger particles significantly more F signal was observed in inflamed tissue for FNP compared to FNP. In conclusion, increase in particle size can abolish the non-specific accumulation of FNPs in inflammatory lesions and additionally increase the phagocytosis of FNPs by murine immune cells. This results in a specific immune-cell dependent F signal with rather no background due to non-specific diffusion.

The close interplay between thrombotic and immunologic processes plays an important physiological role in the immune defence after tissue injury and has the aim to reduce damage and to prevent the spread of invading pathogens. However, the uncontrolled or exaggerated activation of these processes can lead to pathological thromboinflammation. Thromboinflammation has been shown to worsen the outcome of cardiovascular, autoinflammatory, or even infectious diseases. Imaging of thromboinflammation is difficult because many clinically relevant imaging techniques can only visualize either inflammatory or thrombotic processes. One interesting option for the noninvasive imaging of thromboinflammation is multispectral 19F magnetic resonance imaging (MRI). Due to the large chemical shift range of the 19F atoms, it is possible to simultaneously visualize immune cells as well as thrombus components with specific 19F tracer that have individual spectral 19F signatures. Of note, the 19F signal can be easily quantified and a merging of the 19F datasets with the anatomical 1H MRI images enables precise anatomical localization. In this review, we briefly summarize the background of 19F MRI for inflammation imaging, active targeting approaches to visualize thrombi and specific immune cells, introduce studies about multispectral 19F MRI, and summarize one study that imaged thromboinflammation by multispectral 19F MRI.