Early life high fructose exposure disrupts microglia function and impedes neurodevelopment.

Despite the success of fructose as a low-cost food additive, recent epidemiological evidence suggests that high fructose consumption by pregnant mothers or during adolescence is associated with disrupted neurodevelopment 1-7 . An essential step in appropriate mammalian neurodevelopment is the synaptic pruning and elimination of newly-formed neurons by microglia, the central nervous system's (CNS) resident professional phagocyte 8-10 . Whether early life high fructose consumption affects microglia function and if this directly impacts neurodevelopment remains unknown. Here, we show that both offspring born to dams fed a high fructose diet and neonates exposed to high fructose exhibit decreased microglial density, increased uncleared apoptotic cells, and decreased synaptic pruning in vivo . Importantly, deletion of the high affinity fructose transporter SLC2A5 (GLUT5) in neonates completely reversed microglia dysfunction, suggesting that high fructose directly affects neonatal development. Mechanistically, we found that high fructose treatment of both mouse and human microglia suppresses synaptic pruning and phagocytosis capacity which is fully reversed in GLUT5-deficient microglia. Using a combination of in vivo and in vitro nuclear magnetic resonance- and mass spectrometry-based fructose tracing, we found that high fructose drives significant GLUT5-dependent fructose uptake and catabolism, rewiring microglia metabolism towards a hypo-phagocytic state. Importantly, mice exposed to high fructose as neonates exhibited cognitive defects and developed anxiety-like behavior which were rescued in GLUT5-deficient animals. Our findings provide a mechanistic explanation for the epidemiological observation that early life high fructose exposure is associated with increased prevalence of adolescent anxiety disorders.

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.

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.

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.