Inflammatory stimuli impact on cellular uptake and biodistribution of perfluorocarbon nanoemulsions.

Intravenously administered perfluorocarbon nanoemulsion (PFC) are taken up by phagocytic immune cells which enables the non-invasive visualization of inflammatory hot spots by combined 1H/19F magnetic resonance imaging (MRI). However, little is known about the influence of inflammatory stimuli on cellular uptake and biodistribution of PFCs. Here, we systematically investigated the impact of inflammation induced by subcutaneous implantation of Matrigel/lipopolysaccharide (Matrigel/LPS) or myocardial infarction (MI; 50 minutes ischemia reperfusion) on PFC-uptake and biodistribution in C57BL/6J mice. We detected strong 19F signals in Matrigel/LPS plugs and infarcted hearts, which were completely absent in controls. Cellular uptake of PFCs was increased in neutrophils isolated from the blood and Matrigel/LPS plugs, whereas uptake by monocytes was only slightly elevated. In contrast, MI caused only a moderate early increase of PFC-uptake in monocytes and neutrophils. Interestingly, the inflammatory model did also affect the biodistribution of the PFCs. The blood half-life of PFCs was slightly increased after Matrigel/LPS implantation, whereas it was reduced after MI. Compared to controls, the 19F signal of the liver was significantly stronger in Matrigel/LPS, but not in MI animals. Interestingly, stimulation of primary immune cells and RAW264.7 macrophages with LPS had no effect on PFC-uptake, whereas CRP-incubation elevated internalization of PFCs at least in RAW264.7 cells. In conclusion, we show that the cellular PFC-uptake can differ between individual inflammatory conditions. This is an important aspect that has to be considered for the proper interpretation of 1H/19F MRI data obtained from inflammatory hot spots.

CD73 deficiency does not aggravate angiotensin II-induced aortic inflammation in mice.

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.

Noninvasive assessment of metabolic turnover during inflammation by in vivo deuterium magnetic resonance spectroscopy.

Background: Inflammation 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. Methods: For 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. Results: After 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. Conclusion: 1H/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.

Biodegradable polyphosphoester micelles act as both background-free 31P magnetic resonance imaging agents and drug nanocarriers.

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 31P centers in the polymer backbone. We overcome challenges in 31P MRI, including background interference and low sensitivity, by modifying the molecular environment of 31P, 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.

Cardiac injection of USSC boosts remuscularization of the infarcted heart by shaping the T-cell response.

Regenerating the injured heart remains one of the most vexing challenges in cardiovascular medicine. Cell therapy has shown potential for treatment of myocardial infarction, but low cell retention so far has limited its success. Here we show that intramyocardial injection of highly apoptosis-resistant unrestricted somatic stem cells (USSC) into infarcted rat hearts resulted in an unprecedented thickening of the left ventricular wall with cTnT+/BrdU+ cardiomyocytes that was paralleled by progressively restored ejection fraction. USSC induced significant T-cell enrichment in ischemic tissue with enhanced expression of T-cell related cytokines. Inhibition of T-cell activation by anti-CD28 monoclonal antibody, fully abolished the regenerative response which was restored by adoptive T-cell transfer. Secretome analysis of USSC and lineage tracing studies suggest that USSC secrete paracrine factors over an extended period of time which boosts a T-cell driven endogenous regenerative response mainly from adult cardiomyocytes.

Non-invasive mapping of systemic neutrophil dynamics upon cardiovascular injury.

Neutrophils play a complex role during onset of tissue injury and subsequent resolution and healing. To assess neutrophil dynamics upon cardiovascular injury, here we develop a non-invasive, background-free approach for specific mapping of neutrophil dynamics by whole-body magnetic resonance imaging using targeted multimodal fluorine-loaded nanotracers engineered with binding peptides specifically directed against murine or human neutrophils. Intravenous tracer application before injury allowed non-invasive three-dimensional visualization of neutrophils within their different hematopoietic niches over the entire body and subsequent monitoring of their egress into affected tissues. Stimulated murine and human neutrophils exhibited enhanced labeling due to upregulation of their target receptors, which could be exploited as an in vivo readout for their activation state in both sterile and nonsterile cardiovascular inflammation. This non-invasive approach will allow us to identify hidden origins of bacterial or sterile inflammation in patients and also to unravel cardiovascular disease states on the verge of severe aggravation due to enhanced neutrophil infiltration or activation.

Beyond Vessel Diameters: Non-invasive Monitoring of Flow Patterns and Immune Cell Recruitment in Murine Abdominal Aortic Disorders by Multiparametric MRI.

The pathophysiology of the initiation and progression of abdominal aortic aneurysms (AAAs) and aortic dissections (AADs) is still unclear. However, there is strong evidence that monocytes and macrophages are of crucial importance in these processes. Here, we utilized a molecular imaging approach based on background-free 19F MRI and employed perfluorocarbon nanoemulsions (PFCs) for in situ 19F labeling of monocytes/macrophages to monitor vascular inflammation and AAA/AAD formation in angiotensin II (angII)-treated apolipoproteinE-deficient (apoE-/-) mice. In parallel, we used conventional 1H MRI for the characterization of aortic flow patterns and morphology. AngII (1 µg/kg/min) was infused into apoE-/- mice via osmotic minipumps for 10 days and mice were monitored by multiparametric 1H/19F MRI. PFCs were intravenously injected directly after pump implantation followed by additional applications on day 2 and 4 to allow an efficient 19F loading of circulating monocytes. The combination of angiographic, hemodynamic, and anatomical measurements allowed an unequivocal classification of mice in groups with developing AAAs, AADs or without any obvious aortic vessel alterations despite the exposure to angII. Maximal luminal and external diameters of the aorta were enlarged in AAAs, whereas AADs showed either a slight decrease of the luminal diameter or no alteration. 1H/19F MRI after intravenous PFC application demonstrated significantly higher 19F signals in aortae of mice that developed AAAs or AADs as compared to mice in which no aortic disorders were detected. High resolution 1H/19F MRI of excised aortae revealed a patchy pattern of the 19F signals predominantly in the adventitia of the aorta. Histological analysis confirmed the presence of macrophages in this area and flow cytometry revealed higher numbers of immune cells in aortae of mice that have developed AAA/AAD. Importantly, there was a linear correlation of the 19F signal with the total number of infiltrated macrophages. In conclusion, our approach enables a precise differentiation between AAA and AAD as well as visualization and quantitative assessment of inflammatory active vascular lesions, and therefore may help to unravel the complex interplay between macrophage accumulation, vascular inflammation, and the development and progression of AAAs and AADs.

MRI-based molecular imaging of epicardium-derived stromal cells (EpiSC) by peptide-mediated active targeting.

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 19F cargo to EpiSCs and enabled their visualization by 19F 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.

In vivo clearance of 19F MRI imaging nanocarriers is strongly influenced by nanoparticle ultrastructure.

Perfluorocarbons hold great promise both as imaging agents, particularly for 19F MRI, and in therapy, such as oxygen delivery. 19F MRI is unique in its ability to unambiguously track and quantify a tracer while maintaining anatomic context, and without the use of ionizing radiation. This is particularly well-suited for inflammation imaging and quantitative cell tracking. However, perfluorocarbons, which are best suited for imaging - like perfluoro-15-crown-5 ether (PFCE) - tend to have extremely long biological retention. Here, we showed that the use of a multi-core PLGA nanoparticle entrapping PFCE allows for a 15-fold reduction of half-life in vivo compared to what is reported in literature. This unexpected rapid decrease in 19F signal was observed in liver, spleen and within the infarcted region after myocardial infarction and was confirmed by whole body NMR spectroscopy. We demonstrate that the fast clearance is due to disassembly of the ~200 nm nanoparticle into ~30 nm domains that remain soluble and are cleared quickly. We show here that the nanoparticle ultrastructure has a direct impact on in vivo clearance of its cargo i.e. allowing fast release of PFCE, and therefore also bringing the possibility of multifunctional nanoparticle-based imaging to translational imaging, therapy and diagnostics.

Hot spot 19 F magnetic resonance imaging of inflammation.

Among the preclinical molecular imaging approaches, lately fluorine (19 F) magnetic resonance imaging (MRI) has garnered significant scientific interest in the biomedical research community, due to the unique properties of fluorinated materials and the 19 F nucleus. Fluorine is an intrinsically sensitive nucleus for MRI-there is negligible endogenous 19 F in the body and, thus, no background signal which allows the detection of fluorinated materials as "hot spots" by combined 1 H/19 F MRI and renders fluorine-containing molecules as ideal tracers with high specificity. In addition, perfluorocarbons are a family of compounds that exhibit a very high fluorine payload and are biochemically as well as physiologically inert. Perfluorocarbon nanoemulsions (PFCs) are well known to be readily taken up by immunocompetent cells, which can be exploited for the unequivocal identification of inflammatory foci by tracking the recruitment of PFC-loaded immune cells to affected tissues using 1 H/19 F MRI. The required 19 F labeling of immune cells can be accomplished either ex vivo by PFC incubation of isolated endogenous immune cells followed by their re-injection or by intravenous application of PFCs for in situ uptake by circulating immune cells. With both approaches, inflamed tissues can unambiguously be detected via background-free 19 F signals due to trafficking of PFC-loaded immune cells to affected organs. To extend 19 F MRI tracking beyond cells with phagocytic properties, the PFC surface can further be equipped with distinct ligands to generate specificity against epitopes and/or types of immune cells independent of phagocytosis. Recent developments also allow for concurrent detection of different PFCs with distinct spectral signatures allowing the simultaneous visualization of several targets, such as various immune cell subtypes labeled with these PFCs. Since ligands and targets can easily be adapted to a variety of problems, this approach provides a general and versatile platform for inflammation imaging which will strongly extend the frontiers of molecular MRI. This article is categorized under: Diagnostic Tools > in vivo Nanodiagnostics and Imaging Therapeutic Approaches and Drug Discovery > Emerging Technologies Therapeutic Approaches and Drug Discovery > Nanomedicine for Cardiovascular Disease.

Dissociation of 19F and fluorescence signal upon cellular uptake of dual-contrast perfluorocarbon nanoemulsions.

OBJECTIVE: Perfluorocarbon nanoemulsions (PFCs) tagged with fluorescence dyes have been intensively used to confirm the in vivo 19F magnetic resonance imaging (MRI) localization of PFCs by post mortem histology or flow cytometry. However, only limited data are available on tagged PFCs and the potential dissociation of fluorescence and 19F label after cellular uptake over time. MATERIALS AND METHODS: PFCs were coupled to rhodamine (Rho) or carboxyfluorescein (Cfl) and their fate was analyzed after in vitro uptake by J774, RAW and CHO cells by flow cytometry and 19F MRI. In separate in vivo experiments, the dual-labelled emulsions were intravenously applied into mice and their distribution was monitored in spleen and liver over 24 h. In a final step, time course of fluorescence and 19F signals from injected emulsions were tracked in a local inflammation model making use of a subcutaneous matrigel depot doped with LPS (lipopolysaccharide). RESULTS: Internalization of fluorescence-labelled PFCs was associated with a substantial whitening over 24 h in all macrophage cell lines while the 19F signal remained stable over time. In all experiments, CflPFCs were more susceptible to bleaching than RhoPFCs. After intravenous injection of RhoPFCs, the fluorescence signal in spleen and liver peaked after 30 min and 2 h, respectively, followed by a successive decrease over 24 h, whereas the 19F signal continuously increased during this observation period. Similar results were found in the matrigel/LPS model, where we observed increasing 19F signals in the inflammatory hot spot over time while the fluorescence signal of immune cells isolated from the matrigel depot 24 h after its implantation was only marginally elevated over background levels. This resulted in a massive underestimation of the true PFC deposition in the reticuloendothelial system and at inflammatory hot spots. CONCLUSION: Cellular uptake of fluorescently tagged PFCs leads to a dissociation of the fluorescence and the 19F label signal over time, which critically impacts on interpretation of long-term experiments validated by histology or flow cytometry.

IL-23R Signaling Plays No Role in Myocardial Infarction.

Ischemic heart diseases are the most frequent diseases in the western world. Apart from Interleukin (IL-)1, inflammatory therapeutic targets in the clinic are still missing. Interestingly, opposing roles of the pro-inflammatory cytokine IL-23 have been described in cardiac ischemia in mice. IL-23 is a composite cytokine consisting of p19 and p40 which binds to IL-23R and IL-12Rß1 to initiate signal transduction characterized by activation of the Jak/STAT, PI3K and Ras/Raf/MAPK pathways. Here, we generate IL-23R-Y416FΔICD signaling deficient mice and challenged these mice in close- and open-chest left anterior descending coronary arteria ischemia/reperfusion experiments. Our experiments showed only minimal changes in all assayed parameters in IL-23R signaling deficient mice compared to wild-type mice in ischemia and for up to four weeks of reperfusion, including ejection fraction, endsystolic volume, enddiastolic volume, infarct size, gene regulation and α smooth muscle actin (αSMA) and Hyaluronic acid (HA) protein expression. Moreover, injection of IL-23 in wild-type mice after LAD ischemia/reperfusion had also no influence on the outcome of the healing phase. Our data showed that IL-23R deficiency has no effects in myocardial I/R.

Synthetic Cargo Internalization Receptor System for Nanoparticle Tracking of Individual Cell Populations by Fluorine Magnetic Resonance Imaging.

Specific detection of target structures or cells lacking particular surface epitopes still poses a serious problem for all imaging modalities. Here, we demonstrate the capability of synthetic "cargo internalization receptors" (CIRs) for tracking of individual cell populations by 1H/19F magnetic resonance imaging (MRI). To this end, a nanobody for green fluorescent protein (GFP) was used to engineer cell-surface-expressed CIRs which undergo rapid internalization after GFP binding. For 19F MR visibility, the GFP carrier was equipped with "contrast cargo", in that GFP was coupled to perfluorocarbon nanoemulsions (PFCs). To explore the suitability of different uptake mechanisms for this approach, CIRs were constructed by combination of the GFP nanobody and three different cytoplasmic tails that contained individual internalization motifs for endocytosis of the contrast cargo (CIR1-3). Exposure of CIR+ cells to GFP-PFCs resulted in highly specific binding and internalization as confirmed by fluorescence microscopy as well as flow cytometry and enabled visualization by 1H/19F MRI. In particular, expression of CIR2/3 resulted in substantial incorporation of 19F cargo and readily enabled in vivo visualization of GFP-PFC recruitment to transplanted CIR+ cells by 1H/19F MRI in mice. Competition experiments with blood immune cells revealed that CIR+ cells are predominantly loaded with GFP-PFCs even in the presence of cells with strong phagocytotic capacity. Importantly, binding and internalization of GFP-PFCs did not result in the activation of signaling cascades and therefore does not alter cell physiology. Overall, this approach represents a versatile in vivo imaging platform for tracking of individual cell populations by making use of cell-type-specific CIR+ mice.

Ion transporter NKCC1, modulator of neurogenesis in murine olfactory neurons.

Olfaction is one of the most crucial senses for vertebrates regarding foraging and social behavior. Therefore, it is of particular interest to investigate the sense of smell, its function on a molecular level, the signaling proteins involved in the process and the mechanism of required ion transport. In recent years, the precise role of the ion transporter NKCC1 in olfactory sensory neuron (OSN) chloride accumulation has been a controversial subject. NKCC1 is expressed in OSNs and is involved in chloride accumulation of dissociated neurons, but it had not been shown to play a role in mouse odorant sensation. Here, we present electro-olfactogram recordings (EOG) demonstrating that NKCC1-deficient mice exhibit significant defects in perception of a complex odorant mixture (Henkel100) in both air-phase and submerged approaches. Using next generation sequencing (NGS) and RT-PCR experiments of NKCC1-deficient and wild type mouse transcriptomes, we confirmed the absence of a highly expressed ion transporter that could compensate for NKCC1. Additional histological investigations demonstrated a reduced number of cells in the olfactory epithelium (OE), resulting in a thinner neuronal layer. Therefore, we conclude that NKCC1 is an important transporter involved in chloride ion accumulation in the olfactory epithelium, but it is also involved in OSN neurogenesis.