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Inorganic manganese based particles are becoming attractive for molecular and cellular imaging, due to their ability to provide bright contrast on MRI, as opposed to the dark contrast generated from iron based particles. Using a single emulsion technique, we have successfully fabricated pH sensitive, poly(lactic-co-glycolic acid) (PLGA) encapsulated manganese oxide (MnO) nanocrystals. Two classes of particles were fabricated at ~ 140 nm and 1.7 μm, and incorporated 15 to 20 nm MnO nanocrystals with high encapsulation efficiencies. Intact particles at physiological pH cause little contrast in MRI, but following endocytosis into low pH compartments within the cells, the particles erode, and MnO dissolves to release Mn2+. This causes the cells to appear bright on MR images. The magnitude of the change in MRI properties is as high as 35-fold, making it the most dynamic ‘smart’ MRI contrast agent yet reported. Possible applications of these MnO particles include slow release Mn2+, tumor targeting, and confirmation of cell uptake.

The formation of neutrophil extracellular traps (NETs), known as NETosis, was first observed as a novel immune response to bacterial infection, but has since been found to occur abnormally in a variety of other inflammatory disease states including cancer. Breast cancer is the most commonly diagnosed malignancy in women. In breast cancer, NETosis has been linked to increased disease progression, metastasis, and complications such as venous thromboembolism. NET-targeted therapies have shown success in preclinical cancer models and may prove valuable clinical targets in slowing or halting tumor progression in breast cancer patients. We will briefly outline the mechanisms by which NETs may form in the tumor microenvironment and circulation, including the crosstalk between neutrophils, tumor cells, endothelial cells, and platelets as well as the role of cancer-associated extracellular vesicles in modulating neutrophil behavior and NET extrusion. The prognostic implications of cancer-associated NETosis will be explored in addition to development of novel therapeutics aimed at targeting NET interactions to improve outcomes in patients with breast cancer.

Epilepsy results from aberrant electrical activity that can affect either a focal area or the entire brain. In treating epilepsy with drugs, the aim is to decrease seizure frequency and severity while minimizing toxicity to the brain and other tissues. Antiepileptic drugs (AEDs) are usually administered by oral and intravenous (IV) routes, but these drug treatments are not always effective. Drug access to the brain is severely limited by a number of biological factors, particularly the blood-brain barrier (BBB), which impedes the ability of AEDs to enter and remain in the brain. To improve the efficacy of AEDs, new drug delivery strategies are being developed; these methods fall into the three main categories: drug modification, BBB modification, and direct drug delivery. Recently, all three methods have been improved through the use of drug-loaded nanoparticles.

Rationale: Intraerythrocytic polymerization of Hb S promotes hemolysis and vasoocclusive events in the microvasculature of patients with sickle cell disease (SCD). Although plateletneutrophil aggregate-dependent vasoocclusion is known to occur in the lung and contribute to acute chest syndrome, the etiological mechanisms that trigger acute chest syndrome are largely unknown.Objectives: To identify the innate immune mechanism that promotes platelet-neutrophil aggregate-dependent lung vasoocclusion and injury in SCD.Methods: In vivo imaging of the lung in transgenic humanized SCD mice and in vitro imaging of SCD patient blood flowing through a microfluidic system was performed. SCD mice were systemically challenged with nanogram quantities of LPS to trigger lung vasoocclusion.Measurements and Main Results: Platelet-inflammasome activation led to generation of IL-1b and caspase-1-carrying platelet extracellular vesicles (EVs) that bind to neutrophils and promote platelet-neutrophil aggregation in lung arterioles of SCD mice in vivo and SCD human blood in microfluidics in vitro. The inflammasome activation, platelet EV generation, and platelet-neutrophil aggregation were enhanced by the presence of LPS at a nanogram dose in SCD but not control human blood. Inhibition of the inflammasome effector caspase-1 or IL-1b pathway attenuated platelet EV generation, prevented platelet-neutrophil aggregation, and restored microvascular blood flow in lung arterioles of SCD mice in vivo and SCD human blood in microfluidics in vitro.Conclusions: These results are the first to identify that plateletinflammasome-dependent shedding of IL-1b and caspase-1carrying platelet EVs promote lung vasoocclusion in SCD. The current findings also highlight the therapeutic potential of targeting the platelet-inflammasome-dependent innate immune pathway to prevent acute chest syndrome.

Purpose To design, fabricate, characterize and in vivo assay clinically viable magnetic particles for MRI-based cell tracking. Methods PLGA encapsulated magnetic nano- and microparticles were fabricated. Multiple biologically relevant experiments were performed to assess cell viability, cellular performance and stem cell differentiation. In vivo MRI experiments were performed to separately test cell transplantation and cell migration paradigms, as well as in vivo biodegradation. Results Highly magnetic nano- (~100 nm) and microparticles (~1–2 μm) were fabricated. Magnetic cell labeling in culture occurred rapidly achieving 3–50 pg Fe/cell at 3 hrs for different particles types, and >100 pg Fe/cell after 10 hours, without the requirement of a transfection agent, and with no effect on cell viability. The capability of magnetically labeled mesenchymal or neural stem cells to differentiate down multiple lineages, or for magnetically labeled immune cells to release cytokines following stimulation, was uncompromised. An in vivo biodegradation study revealed that NPs degraded ~80% over the course of 12 weeks. MRI detected as few as 10 magnetically labeled cells, transplanted into the brains of rats. Also, these particles enabled the in vivo monitoring of endogenous neural progenitor cell migration in rat brains over 2 weeks. Conclusion The robust MRI properties and benign safety profile of these particles make them promising candidates for clinical translation for MRI-based cell tracking.

Sickle cell disease (SCD) is a genetic disorder that leads to red blood cell (RBC) sickling, hemolysis and the upregulation of adhesion molecules on sickle RBCs. Chronic hemolysis in SCD results in a hyper-inflammatory state characterized by activation of circulating leukocytes, platelets and endothelial cells even in the absence of a crisis. A crisis in SCD is often triggered by an inflammatory stimulus and can lead to the acute chest syndrome (ACS), which is a type of lung injury and a leading cause of mortality among SCD patients. Although it is believed that pulmonary vaso-occlusion could be the phenomenon contributing to the development of ACS, the role of vaso-occlusion in ACS remains elusive. Intravital imaging of the cremaster microcirculation in SCD mice has been instrumental in establishing the role of neutrophil-RBC-endothelium interactions in systemic vaso-occlusion; however, such studies, although warranted, have never been done in the pulmonary microcirculation of SCD mice. Here, we show that two-photon excitation fluorescence microscopy can be used to perform quantitative analysis of neutrophil and RBC trafficking in the pulmonary microcirculation of SCD mice. We provide the experimental approach that enables microscopic observations under physiological conditions and use it to show that RBC and neutrophil trafficking is comparable in SCD and control mice in the absence of an inflammatory stimulus. The intravital imaging scheme proposed in this study can be useful in elucidating the cellular and molecular mechanism of pulmonary vaso-occlusion in SCD mice following an inflammatory stimulus.

Manganese oxide (MnO) nanoparticles (NPs) can serve as robust pH-sensitive contrast agents for magnetic resonance imaging (MRI) due to Mn 2+ release at low pH, which generates a~30 fold change in T 1 relaxivity. Strategies to control NP size, composition, and Mn 2+ dissolution rates are essential to improve diagnostic performance of pH-responsive MnO NPs. We are the first to demonstrate that MnO NP size and composition can be tuned by the temperature ramping rate and aging time used during thermal decomposition of manganese (II) acetylacetonate. Two different temperature ramping rates (10˚C/min and 20˚C/min) were applied to reach 300˚C and NPs were aged at that temperature for 5, 15, or 30 min. A faster ramping rate and shorter aging time produced the smallest NPs of~23 nm. Shorter aging times created a mixture of MnO and Mn 3 O 4 NPs, whereas longer aging times formed MnO. Our results indicate that a 20˚C/min ramp rate with an aging time of 30 min was the ideal temperature condition to form the smallest pure MnO NPs of~32 nm. However, Mn 2+ dissolution rates at low pH were unaffected by synthesis conditions. Although Mn 2+ production was high at pH 5 mimicking endosomes inside cells, minimal Mn 2+ was released at pH 6.5 and 7.4, which mimic the tumor extracellular space and blood, respectively. To further elucidate the effects of NP composition and size on Mn 2+ release and MRI contrast, the ideal MnO NP formulation (~32 nm) was compared with smaller MnO and Mn 3 O 4 NPs. Small MnO NPs produced the highest amount of Mn 2+ at acidic pH with maximum T 1 MRI signal; Mn 3 O 4 NPs generated the lowest MRI signal. MnO NPs encapsulated within poly (lactide-co-glycolide) (PLGA) retained significantly higher Mn 2+ release and MRI signal compared to PLGA Mn 3 O 4 NPs. Therefore, MnO instead of Mn 3 O 4 should be targeted intracellularly to maximize MRI contrast.