In this account, we varied PEGylation density on the surface of hydrogel PRINT nanoparticles and systematically observed the effects on protein adsorption, macrophage uptake, and circulation time. Interestingly, the density of PEGylation necessary to promote a long-circulating particle was dramatically less than what has been previously reported. Overall, our methodology provides a rapid screening technique to predict particle behavior in vivo and our results deliver further insight to what PEG density is necessary to facilitate long-circulation.

It has long been hypothesized that elastic modulus governs the biodistribution and circulation times of particles and cells in blood; however, this notion has never been rigorously tested. We synthesized hydrogel microparticles with tunable elasticity in the physiological range, which resemble red blood cells in size and shape, and tested their behavior in vivo. Decreasing the modulus of these particles altered their biodistribution properties, allowing them to bypass several organs, such as the lung, that entrapped their more rigid counterparts, resulting in increasingly longer circulation times well past those of conventional microparticles. An 8-fold decrease in hydrogel modulus correlated to a greater than 30-fold increase in the elimination phase half-life for these particles. These results demonstrate a critical design parameter for hydrogel microparticles.biomimetic | deformability | drug carriers | long circulating | red blood cell mimic

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Highlights d 3D imaging defines ILC2 niches in perivascular regions of multiple tissues d ILC2s localize with fibroblast-like adventitial stromal cells (ASCs) d Lung ASCs produce IL-33 and TSLP to support ILC2 and Th2s d ILC2s promote ASC expansion and IL-33 production after helminth infection

We report here several unusual features of inactivation of the rat Kv2.1 delayed rectifier potassium channel, expressed in Xenopus oocytes. The voltage dependence of inactivation was U-shaped, with maximum inactivation near 0 mV. During a maintained depolarization, development of inactivation was slow and only weakly voltage dependent (tau = 4 s at 0 mV; tau = 7 s at +80 mV). However, recovery from inactivation was strongly voltage dependent (e-fold for 20 mV) and could be rapid (tau = 0.27 s at -140 mV). Kv2.1 showed cumulative inactivation, where inactivation built up during a train of brief depolarizations. A single maintained depolarization produced more steady-state inactivation than a train of pulses, but there could actually be more inactivation with the repeated pulses during the first few seconds. We term this phenomenon "excessive cumulative inactivation." These results can be explained by an allosteric model, in which inactivation is favored by activation of voltage sensors, but the open state of the channel is resistant to inactivation.

Coating nanoparticles with polyethylene glycol (PEG), which reduces particle uptake and clearance by immune cells, is routinely used to extend the circulation times of nanoparticle therapeutics. Nevertheless, due to technical hurdles in quantifying the extent of PEG grafting, as well as in generating very dense PEG coatings, few studies have rigorously explored the precise PEG grafting density necessary to achieve desirable "stealth" properties. Here, using polymeric nanoparticles with precisely tunable PEG grafting, we found that, for a wide range of PEG lengths (0.6-20 kDa), PEG coatings at densities substantially exceeding those required for PEG to adopt a "brush" conformation are exceptionally resistant to uptake by cultured human macrophages, as well as primary peripheral blood leukocytes. Less than 20% of these nanoparticles were cleared from the blood after 2 h (t1/2 ∼ 14 h) in BALB/c mice, whereas slightly less densely PEGylated and uncoated control particles were both virtually eliminated within 2 h. Our results suggest that the stealth properties of PEG-coated nanoparticles are critically dependent on achieving PEG grafting at densities exceeding those required for brush conformation.

We have examined the kinetics of whole-cell T-current in HEK 293 cells stably expressing the α1G channel, with symmetrical Na+ i and Na+ o and 2 mM Ca2+ o. After brief strong depolarization to activate the channels (2 ms at +60 mV; holding potential −100 mV), currents relaxed exponentially at all voltages. The time constant of the relaxation was exponentially voltage dependent from −120 to −70 mV \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}({\mathrm{e-fold\;for}}\;31\;{\mathrm{mV}};\;{\mathrm{{\tau}}}\;=\;2.5\;{\mathrm{ms\;at}}\;-100\;{\mathrm{mV}})\end{equation*}\end{document}, but \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathrm{{\tau}}}\;=\;12{\raisebox{1mm}{\line(1,0){6}}}17\;{\mathrm{ms\;from}}-40\;{\mathrm{to}}\;+60\;{\mathrm{mV}}\end{equation*}\end{document}. This suggests a mixture of voltage-dependent deactivation (dominating at very negative voltages) and nearly voltage-independent inactivation. Inactivation measured by test pulses following that protocol was consistent with open-state inactivation. During depolarizations lasting 100–300 ms, inactivation was strong but incomplete (∼98%). Inactivation was also produced by long, weak depolarizations \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}({\mathrm{{\tau}}}\;=\;220\;{\mathrm{ms\;at}}\;-80\;{\mathrm{mV}};\;{\mathrm{V}}_{1/2}\;=\;-82\;{\mathrm{mV}})\end{equation*}\end{document}, which could not be explained by voltage-independent inactivation exclusively from the open state. Recovery from inactivation was exponential and fast \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}({\mathrm{{\tau}}}\;=\;85\;{\mathrm{ms\;at}}\;-100\;{\mathrm{mV}})\end{equation*}\end{document}, but weakly voltage dependent. Recovery was similar after 60-ms steps to −20 mV or 600-ms steps to −70 mV, suggesting rapid equilibration of open- and closed-state inactivation. There was little current at −100 mV during recovery from inactivation, consistent with ≤8% of the channels recovering through the open state. The results are well described by a kinetic model where inactivation is allosterically coupled to the movement of the first three voltage sensors...

Extended circulation of nanoparticles in blood is essential for most clinical applications. Nanoparticles are rapidly cleared by cells of the mononuclear phagocyte system (MPS). Approaches such as grafting polyethylene glycol onto particles (PEGylation) extend circulation times; however, these particles are still cleared, and the processes involved in this clearance remain poorly understood. Here, we present an intravital microscopybased assay for the quantification of nanoparticle clearance, allowing us to determine the effect of mouse strain and immune system function on particle clearance. We demonstrate that mouse strains that are prone to Th1 immune responses clear nanoparticles at a slower rate than Th2-prone mice. Using depletion strategies, we show that both granulocytes and macrophages participate in the enhanced clearance observed in Th2-prone mice. Macrophages isolated from Th1 strains took up fewer particles in vitro than macrophages from Th2 strains. Treating macrophages from Th1 strains with cytokines to differentiate them into M2 macrophages increased the amount of particle uptake. Conversely, treating macrophages from Th2 strains with cytokines to differentiate them into M1 macrophages decreased their particle uptake. Moreover, these results were confirmed in human monocyte-derived macrophages, suggesting that global immune regulation has a significant impact on nanoparticle clearance in humans. IntroductionThe potential clinical applications of nanoparticles and nanoformulations have been investigated for more than 30 years. Nanoparticle approaches have the potential to revolutionize drug delivery by allowing for the encapsulation of drugs with poor solubility or stability in a stable carrier particle. In addition, targeting nanoparticles to specific pathological sites may allow an increased effective dose of drug at the needed site, while decreasing systemic drug exposure, and therefore side effects. However, to date, only 2 nanoformulations for cancer treatment have been approved for clinical use (liposomal doxorubicin [Doxil] and protein-bound paclitaxel [Abraxane]) (1-3). One major obstacle for the use of nanoparticles in vivo is rapid clearance by the cells of the reticuloendothelial system (RES)/mononuclear phagocyte system (MPS) (4-7). In addition to rapid clearance, variable activity of the MPS among patients leads to widely variable pharmacokinetics of nanoformulations in the clinic, reducing the efficacy of both approved and future experimental nanoformulations (8).