Short-term exposure of female BALB/cJ mice to e-cigarette aerosol promotes neutrophil recruitment and enhances neutrophil-platelet aggregation in pulmonary microvasculature.

Despite the perception that e-cigarettes are safer than conventional cigarettes, numerous findings demonstrated that e-cigarette aerosol (EC) exposure induced compromised immune functionality, vascular changes even after acute exposure, and lung injury. Notably, altered neutrophil functionality and platelet hemodynamics have been observed post-EC exposure. It was hypothesized that EC exposure initiates an inflammatory response resulting in altered neutrophil behavior and increased neutrophil-platelet interaction in the pulmonary microvasculature. Neutrophil and platelet responses were examined up to 48 hrs following whole-body, short-term EC exposure without flavorants or nicotine in a murine model, which most closely modeled secondhand exposure. This study is the first to investigate the impact of EC exposure through lung intravital imaging. Compared to room air-exposed mice, EC-exposed mice displayed significantly increased 1.7‒1.9-fold number of neutrophils in the pulmonary microvasculature associated with no marked change in neutrophils within whole blood or bronchoalveolar lavage fluid (BALF). Neutrophil-platelet interactions were also significantly elevated 1.9‒2.5-fold in exposed mice. Plasma concentration of myeloperoxidase was markedly reduced 1.5-fold 48 hr following exposure cessation, suggesting suppressed neutrophil antimicrobial activity. Cytokine expression exhibited changes indicating vascular damage. Effects persisted for 48 hr post-EC exposure. Data demonstrated that EC exposure repeated for 3 consecutive days in 2.5 hr intervals in the absence of flavorants or nicotine resulted in modified pulmonary vasculature hemodynamics, altered immune functionality, and a pro-inflammatory state in female BALB/cJ mice.

Platelet Extracellular Vesicles Drive Inflammasome-IL-1ß-Dependent Lung Injury in Sickle Cell Disease.

Rationale: Intraerythrocytic polymerization of Hb S promotes hemolysis and vasoocclusive events in the microvasculature of patients with sickle cell disease (SCD). Although platelet-neutrophil 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-1ß 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-1ß 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 platelet-inflammasome-dependent shedding of IL-1ß and caspase-1-carrying 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.

E-Cigarette Use: Device Market, Study Design, and Emerging Evidence of Biological Consequences.

Electronic cigarettes are frequently viewed as a safer alternative to conventional cigarettes; however, evidence to support this perspective has not materialized. Indeed, the current literature reports that electronic cigarette use is associated with both acute lung injury and subclinical dysfunction to the lung and vasculature that may result in pathology following chronic use. E-cigarettes can alter vascular dynamics, polarize innate immune populations towards a proinflammatory state, compromise barrier function in the pulmonary endothelium and epithelium, and promote pre-oncogenic phenomena. This review will summarize the variety of e-cigarette products available to users, discuss current challenges in e-cigarette study design, outline the range of pathologies occurring in cases of e-cigarette associated acute lung injury, highlight disease supporting tissue- and cellular-level changes resulting from e-cigarette exposure, and briefly examine how these changes may promote tumorigenesis. Continued research of the mechanisms by which e-cigarettes induce pathology benefit users and clinicians by resulting in increased regulation of vaping devices, informing treatments for emerging diseases e-cigarettes produce, and increasing public awareness to reduce e-cigarette use and the onset of preventable disease.

Neutrophil extracellular traps in breast cancer and beyond: current perspectives on NET stimuli, thrombosis and metastasis, and clinical utility for diagnosis and treatment.

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.

Intelligent and automatic in vivo detection and quantification of transplanted cells in MRI.

PURPOSE: Magnetic resonance imaging (MRI)-based cell tracking has emerged as a useful tool for identifying the location of transplanted cells, and even their migration. Magnetically labeled cells appear as dark contrast in T2*-weighted MRI, with sensitivities of individual cells. One key hurdle to the widespread use of MRI-based cell tracking is the inability to determine the number of transplanted cells based on this contrast feature. In the case of single cell detection, manual enumeration of spots in three-dimensional (3D) MRI in principle is possible; however, it is a tedious and time-consuming task that is prone to subjectivity and inaccuracy on a large scale. This research presents the first comprehensive study on how a computer-based intelligent, automatic, and accurate cell quantification approach can be designed for spot detection in MRI scans. METHODS: Magnetically labeled mesenchymal stem cells (MSCs) were transplanted into rats using an intracardiac injection, accomplishing single cell seeding in the brain. T2*-weighted MRI of these rat brains were performed where labeled MSCs appeared as spots. Using machine learning and computer vision paradigms, approaches were designed to systematically explore the possibility of automatic detection of these spots in MRI. Experiments were validated against known in vitro scenarios. RESULTS: Using the proposed deep convolutional neural network (CNN) architecture, an in vivo accuracy up to 97.3% and in vitro accuracy of up to 99.8% was achieved for automated spot detection in MRI data. CONCLUSION: The proposed approach for automatic quantification of MRI-based cell tracking will facilitate the use of MRI in large-scale cell therapy studies. Magn Reson Med 78:1991-2002, 2017. © 2016 International Society for Magnetic Resonance in Medicine.

Clinically viable magnetic poly(lactide-co-glycolide) particles for MRI-based cell tracking.

PURPOSE: To design, fabricate, characterize, and in vivo assay clinically viable magnetic particles for MRI-based cell tracking. METHODS: Poly(lactide-co-glycolide) (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 h for different particles types, and >100 pg Fe/cell after 10 h, 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.

Poly(lactic-co-glycolic acid) encapsulated gadolinium oxide nanoparticles for MRI-based cell tracking.

Superparamagnetic iron oxide particles have proven useful for cell tracking applications by monitoring cell transplantation and migration in living organisms. However, one perceived drawback is that these particles cause dark contrast in MRI, sometimes yielding confusion with other biological phenomena, which also yield dark contrast. To that end, researchers have investigated the use of gadolinium oxide (Gd2O3) based contrast agents for MRI-based cell tracking, as Gd2O3 has favorable r1 molar relaxivity. We synthesized Gd2O3 nanocrystals and encapsulated them within PLGA matrices to form approximatley to 150 nm nanoparticles. r1 was 1.9 mM(-1) sec(-1) and r2 was 8.4 mM(-1) sec(-1). Cell labeling with particles was well tolerated by cells except at very high doses. MRI of labeled cells showed that labeled cells could achieve both R1 and R2 enhancements due to the internalized particles. R2 enhancements were approximately to twice that of R1 enhancements suggesting the use of very short echo times when using Gd2O3 based contrast agents for MRI-based cell tracking.

Caveat Emptor: Commercialized Manganese Oxide Nanoparticles Exhibit Unintended Properties.

Nano-encapsulated manganese oxide (NEMO) particles are noteworthy contrast agents for magnetic resonance imaging (MRI) due to their bright, pH-switchable signal ("OFF" to "ON" at low pH), high metal loading, and targeting capability for increased specificity. For the first time, we performed a head-to-head comparison of NEMO particles from In-house and commercialized sources (US Nano vs Nanoshel) to assess their potential as bright T1 MRI contrast agents. Manganese oxide nanocrystals (MnO, Mn2O3, and Mn3O4) were systematically evaluated for size, chemistry, release of manganese ions, and MRI signal pre- and post-encapsulation within poly(lactic-co-glycolic acid) (PLGA). Suprisingly, a majority of the commercialized formulations were not as advertised by displaying unintended sizes, morphologies, chemistry, dissolution profiles, and/or MRI signal that precludes in vivo use. US Nano's Mn3O4 and Mn2O3 nanocrystals contained impurities that impacted Mn ion release as well as micron-sized rodlike structures. Nanoshel's MnO and Mn2O3 nanoparticles had very large hydrodynamic sizes (>600 nm). In-house MnO and Nanoshel's Mn3O4 nanoparticles demonstrated the best characteristics with brighter T1 MRI signals, small hydrodynamic sizes, and high encapsulation efficiencies. Our findings highlight that researchers must confirm the properties of purchased nanomaterials before utilizing them in desired applications, as their experimental success may be impacted.

PEGylation of Metal Oxide Nanoparticles Modulates Neutrophil Extracellular Trap Formation.

Novel metal oxide nanoparticle (NP) contrast agents may offer safety and functionality advantages over conventional gadolinium-based contrast agents (GBCAs) for cancer diagnosis by magnetic resonance imaging. However, little is known about the behavior of metal oxide NPs, or of their effect, upon coming into contact with the innate immune system. As neutrophils are the body's first line of defense, we sought to understand how manganese oxide and iron oxide NPs impact leukocyte functionality. Specifically, we evaluated whether contrast agents caused neutrophils to release web-like fibers of DNA known as neutrophil extracellular traps (NETs), which are known to enhance metastasis and thrombosis in cancer patients. Murine neutrophils were treated with GBCA, bare manganese oxide or iron oxide NPs, or poly(lactic-co-glycolic acid) (PLGA)-coated metal oxide NPs with different incorporated levels of poly(ethylene glycol) (PEG). Manganese oxide NPs elicited the highest NETosis rates and had enhanced neutrophil uptake properties compared to iron oxide NPs. Interestingly, NPs with low levels of PEGylation produced more NETs than those with higher PEGylation. Despite generating a low rate of NETosis, GBCA altered neutrophil cytokine expression more than NP treatments. This study is the first to investigate whether manganese oxide NPs and GBCAs modulate NETosis and reveals that contrast agents may have unintended off-target effects which warrant further investigation.

Manganese Oxide Nanoparticle Synthesis by Thermal Decomposition of Manganese(II) Acetylacetonate.

For biomedical applications, metal oxide nanoparticles such as iron oxide and manganese oxide (MnO), have been used as biosensors and contrast agents in magnetic resonance imaging (MRI). While iron oxide nanoparticles provide constant negative contrast on MRI over typical experimental timeframes, MnO generates switchable positive contrast on MRI through dissolution of MnO to Mn2+ at low pH within cell endosomes to 'turn ON' MRI contrast. This protocol describes a one-pot synthesis of MnO nanoparticles formed by thermal decomposition of manganese(II) acetylacetonate in oleylamine and dibenzyl ether. Although running the synthesis of MnO nanoparticles is simple, the initial experimental setup can be difficult to reproduce if detailed instructions are not provided. Thus, the glassware and tubing assembly is first thoroughly described to allow other investigators to easily reproduce the setup. The synthesis method incorporates a temperature controller to achieve automated and precise manipulation of the desired temperature profile, which will impact resulting nanoparticle size and chemistry. The thermal decomposition protocol can be readily adapted to generate other metal oxide nanoparticles (e.g., iron oxide) and to include alternative organic solvents and stabilizers (e.g., oleic acid). In addition, the ratio of organic solvent to stabilizer can be changed to further impact nanoparticle properties, which is shown herein. Synthesized MnO nanoparticles are characterized for morphology, size, bulk composition, and surface composition through transmission electron microscopy, X-ray diffraction, and Fourier-transform infrared spectroscopy, respectively. The MnO nanoparticles synthesized by this method will be hydrophobic and must be further manipulated through ligand exchange, polymeric encapsulation, or lipid capping to incorporate hydrophilic groups for interaction with biological fluids and tissues.

Live Imaging of the Lung.

Live imaging is critical to determining the dynamics and spatial interactions of cells within the tissue environment. In the lung, this has proven to be difficult due to the motion brought about by ventilation and cardiac contractions. A previous version of this Current Protocols in Cytometry article reported protocols for imaging ex vivo live lung slices and the intact mouse lung. Here, we update those protocols by adding new methodologies, new approaches for quantitative image analysis, and new areas of potential application. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Live imaging of lung slices Support Protocol 1: Staining lung sections with fluorescent antibodies Basic Protocol 2: Live imaging in the mouse lung Support Protocol 2: Intratracheal instillations Support Protocol 3: Intravascular instillations Support Protocol 4: Monitoring vital signs of the mouse during live lung imaging Support Protocol 5: Antibodies Support Protocol 6: Fluorescent reporter mice Basic Protocol 3: Quantification of neutrophil-platelet aggregation in pulmonary vasculature Basic Protocol 4: Quantification of platelet-dependent pulmonary thrombosis Basic Protocol 5: Quantification of pulmonary vascular permeability.

Tuning the size and composition of manganese oxide nanoparticles through varying temperature ramp and aging time.

Manganese oxide (MnO) nanoparticles (NPs) can serve as robust pH-sensitive contrast agents for magnetic resonance imaging (MRI) due to Mn2+ release at low pH, which generates a ~30 fold change in T1 relaxivity. Strategies to control NP size, composition, and Mn2+ 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 Mn3O4 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, Mn2+ dissolution rates at low pH were unaffected by synthesis conditions. Although Mn2+ production was high at pH 5 mimicking endosomes inside cells, minimal Mn2+ 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 Mn2+ release and MRI contrast, the ideal MnO NP formulation (~32 nm) was compared with smaller MnO and Mn3O4 NPs. Small MnO NPs produced the highest amount of Mn2+ at acidic pH with maximum T1 MRI signal; Mn3O4 NPs generated the lowest MRI signal. MnO NPs encapsulated within poly(lactide-co-glycolide) (PLGA) retained significantly higher Mn2+ release and MRI signal compared to PLGA Mn3O4 NPs. Therefore, MnO instead of Mn3O4 should be targeted intracellularly to maximize MRI contrast.

Intravascular hemolysis triggers ADP-mediated generation of platelet-rich thrombi in precapillary pulmonary arterioles.

Patients with hereditary or acquired hemolytic anemias have a high risk of developing in situ thrombosis of the pulmonary vasculature. While pulmonary thrombosis is a major morbidity associated with hemolytic disorders, the etiological mechanism underlying hemolysis-induced pulmonary thrombosis remains largely unknown. Here, we use intravital lung microscopy in mice to assess the pathogenesis of pulmonary thrombosis following deionized water-induced acute intravascular hemolysis. Acute hemolysis triggered the development of αIIbß3-dependent platelet-rich thrombi in precapillary pulmonary arterioles, which led to the transient impairment of pulmonary blood flow. The hemolysis-induced pulmonary thrombosis was phenocopied with intravascular ADP- but not thrombin-triggered pulmonary thrombosis. Consistent with a mechanism involving ADP release from hemolyzing erythrocytes, the inhibition of platelet P2Y12 purinergic receptor signaling attenuated pulmonary thrombosis and rescued blood flow in the pulmonary arterioles of mice following intravascular hemolysis. These findings are the first in vivo studies to our knowledge to suggest that acute intravascular hemolysis promotes ADP-dependent platelet activation, leading to thrombosis in the precapillary pulmonary arterioles, and that thrombin generation most likely does not play a significant role in the pathogenesis of acute hemolysis-triggered pulmonary thrombosis.

Lung vaso-occlusion in sickle cell disease mediated by arteriolar neutrophil-platelet microemboli.

In patients with sickle cell disease (SCD), the polymerization of intraerythrocytic hemoglobin S promotes downstream vaso-occlusive events in the microvasculature. While vaso-occlusion is known to occur in the lung, often in the context of systemic vaso-occlusive crisis and the acute chest syndrome, the pathophysiological mechanisms that incite lung injury are unknown. We used intravital microscopy of the lung in transgenic humanized SCD mice to monitor acute vaso-occlusive events following an acute dose of systemic lipopolysaccharide sufficient to trigger events in SCD but not control mice. We observed cellular microembolism of precapillary pulmonary arteriolar bottlenecks by neutrophil-platelet aggregates. Blood from SCD patients was next studied under flow in an in vitro microfluidic system. Similar to the pulmonary circulation, circulating platelets nucleated around arrested neutrophils, translating to a greater number and duration of neutrophil-platelet interactions compared with normal human blood. Inhibition of platelet P-selectin with function-blocking antibody attenuated the neutrophil-platelet interactions in SCD patient blood in vitro and resolved pulmonary arteriole microembolism in SCD mice in vivo. These results establish the relevance of neutrophil-platelet aggregate formation in lung arterioles in promoting lung vaso-occlusion in SCD and highlight the therapeutic potential of targeting platelet adhesion molecules to prevent acute chest syndrome.

Quantitative intravital two-photon excitation microscopy reveals absence of pulmonary vaso-occlusion in unchallenged Sickle Cell Disease mice.

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.

Specific chemotaxis of magnetically labeled mesenchymal stem cells: implications for MRI of glioma.

PURPOSE: Glioblastoma multiforme (GBM) is a lethal disease marked by infiltration of cancerous cells into the surrounding normal brain. The dire outcome of GBM patients stems in part from the limitations of current neuroimaging methods. Notably, early cancer detection methodologies are lacking, without the ability to identify aggressive, metastatic tumor cells. We propose a novel approach for tumor detection using magnetic resonance imaging (MRI) based on imaging specific tumor tropism of mesenchymal stem cells (MSCs) labeled with micron-sized iron oxide particles (MPIOs). PROCEDURES: MPIO labeled and unlabeled MSCs were compared for viability, multi-lineage differentiation, and migration, where both chemotactic and chemokinetic movement were assessed in the presence of serum-free medium, serum-containing medium, and glioma-conditioned medium. MRI was performed on agarose samples, consisting of MPIO-labeled single MSCs, to confirm the capability to detect single cells. RESULTS: We determined that MPIO-labeled MSCs exhibit specific and significant chemotactic migration towards glioma-conditioned medium in vitro. Confocal fluorescence microscopy confirmed that MPIOs are internalized and do not impact important cell processes of MSCs. Lastly, MPIO-labeled MSCs appear as single distinct, dark spots on T(2)*-weighted MRI, supporting the robustness of this contrast agent for cell tracking. CONCLUSIONS: This is the first study to show that MPIO-labeled MSCs exhibit specific tropism toward tumor-secreted factors in vitro. The potential for detecting single MPIO-labeled MSCs provides rationale for in vivo extension of this methodology to visualize GBM in animal models.

Biocompatible and pH-sensitive PLGA encapsulated MnO nanocrystals for molecular and cellular MRI.

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 Mn(2+). 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 Mn(2+), tumor targeting, and confirmation of cell uptake.

Nanotechnology for delivery of drugs to the brain for epilepsy.

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 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, 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, blood-brain barrier modification, and direct drug delivery. Recently, all three methods have been improved through the use of drug-loaded nanoparticles.