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Intact synaptic function and plasticity are fundamental prerequisites to a healthy brain. Therefore, synaptic proteins are one of the major targets for drugs used as neuro-chemical therapeutics. Unfortunately, the majority of drugs is not able to cross the blood–brain barrier (BBB) and is therefore distributed within the CNS parenchyma. Here, we report the development of novel biodegradable Nanoparticles (NPs), made of poly-lactide-co-glycolide (PLGA) conjugated with glycopeptides that are able to cross the BBB and deliver for example Zn2+ ions. We also provide a thorough characterization of loaded and unloaded NPs for their stability, cellular uptake, release properties, toxicity, and impact on cell trafficking. Our data reveal that these NPs are biocompatible, and can be used to elevate intracellular levels of Zn2+. Importantly, by engineering the surface of NPs with antibodies against NCAM1 and CD44, we were able to selectively target neurons or glial cells, respectively. Our results indicate that these biodegradable NPs provide a potential new venue for the delivery Zn2+ to the CNS and thus a means to explore the influence of altered zinc levels linked to neuropsychological disorders such as depression.
Citation: Grabrucker AM, Garner CC, Boeckers TM, Bondioli L, Ruozi B, Forni F, et al. (2011) Development of Novel Zn2+ Loaded Nanoparticles Designed for Cell-Type Targeted Drug Release in CNS Neurons: In Vitro Evidences. PLoS ONE 6(3): e17851. https://doi.org/10.1371/journal.pone.0017851
Editor: David Finkelstein, The Mental Health Research Institute of Victoria, Australia
Received: December 10, 2010; Accepted: February 10, 2011; Published: March 23, 2011
Copyright: © 2011 Grabrucker et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: AMG has been supported by a fellowship from the Deutsche Forschungsgemeinschaft (DFG) and the National Institutes of Health (PO1 NS053862; R21MH091471) to CCG. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
The majority of drugs used as neuro-chemical therapeutics target synaptic proteins. Unfortunately, a high number of drugs are not able to cross through the blood–brain barrier (BBB) [1]. The transmissivity of this epithelial structure is restricted by the presence of tight junctions (TJ) that connect the cerebral endothelial and epithelial cells of the choroids plexus. Additionally, glial cells are found surrounding the surface of the capillaries, which cohere the endothelial cells, producing an electrical resistance much higher than that of other systemic endothelia [2]. Recent studies have demonstrated a non-invasive method of drug delivery to the CNS, based on the use of biodegradable Nanoparticles (NPs). Injectable nanoparticulate drug carriers made of poly-lactide-co-glycolide (PLGA), and specifically modified with ligands were shown to be able to cross the blood–brain barrier (BBB), thus representing an important potential tool for treatment of neurological diseases [3], [4].
In particular, this new strategy for NPs-brain targeting is based on the surface engineering of NPs, using a glycopeptides (g7)–derived PLGA [3], [5], [6]. The attachment of ligands for CNS targeting and/or fluorescent markers on the surface of NPs allows evaluating and influencing their properties both in vitro and in vivo. These engineered NPs (BBB-NPs) bear the possibility to deliver different kinds of drugs to the brain with a high rate of efficiency (13–15% of the injected dose) [7]. Thus, by encapsulation of drugs into BBB-NPs a significant improvement of brain delivery can be expected.
In recent years, there has been mounting evidence that suggests a correlation between zinc deficiency and clinical depression [8], [9]. Clinical studies as well as work with animal models indicate that zinc levels and some neuropsychological disorders are functionally linked. Zinc deficiency, for example, has been shown to induce depression- and anxiety-like behaviors, while zinc supplementation has been used to treat depression. Intriguingly, zinc administration improves the efficacy of antidepressant drugs in depressed patients [10] and the level of zinc at synapses in the hippocampus [11]. Thus, while zinc deficiency may have a critical role in the development of mood disorders and may serve as a putative model of depression in mice, zinc supplementation may be important in the treatment of these disorders [12].
Given the therapeutic potential of NPs, in the present study, we have investigated whether un-modified PLGA NPs (P-NPs) or BBB-NPs specifically targeted to neurons or glia cells could be used to encapsulate and deliver Zn2+ into cells. Our data demonstrate that this strategy is feasible creating a novel platform not only for understanding the role of zinc in neuronal function, but also the delivery of drugs into the CNS and ultimately the treatment of neurological diseases.
NPs do not affect cell viability or neuronal morphogenesis
Although in previous studies, NPs have been shown to cross the BBB, whether they are actively taken up by neurons and/or adversely affect cell viability is poorly investigated. To explore these possibilities, we examined the effect of adding NPs to dissociated neuronal/glial cultures. In an initial set of experiments designed to assess their affect on cell viability, cultures were treated with one of four preparations [unloaded P-NPs, unloaded BBB-NPs, Zn2+ loaded P-NPs (Zn-P-NPs) or Zn2+ loaded BBB-NPs (Zn-BBB-NPs)] starting at DIV7 (625 µg NPs per ml) (Fig. 1A) and compared to untreated control cells at DIV14.
A) Mixed neuronal cultures containing glia cells and hippocampal neurons were plated and grown until DIV14. Cells were treated with the following NPs: "P-NPs" (empty NPs without ligand), "BBB-NPs" (un-loaded g7 ligand coated NPs), "Zn-P-NPs" (NPs encapsulating Zn2+) or "Zn-BBB-NPs" (g7 ligand coated NPs encapsulating Zn2+) (all: 625 µg NPs per ml), at DIV7. Upper panel: cells were stained with antibodies against the dendritic microtubule associated protein MAP2 and DAPI. The number of neurons (MAP2 positive cells) and the overall cell number (DAPI staining) per optic field was measured. Neither P-NPs nor BBB-NPs affected cell number as quantified in the lower panel. A small but significant reduction in cell number was observed in Zn-NPs (neurons) and Zn-BBB-NPs treated cells. B) Neuronal/glial cultures were plated at 20,000 cells per coverslip and treated for 7 days with different concentrations of P-NPs and BBB-NPs (e.g. multiples of 625 µg NPs per ml applied to control cells (Ctrl)). The cells were treated at 7DIV fixed at DIV14 and labeling for MAP2 and nuclei (DAPI) was performed. Lower panel: quantification of DAPI positive cells/optical field of view (40× mag). Only healthy nuclei were counted. At 10× 625 µg/ml (6.25 mg/ml), there is a significant reduction in cell number of cells treated with P-NPs. Cell-number reduction with BBB-NPs treated cells is seen at 8× 625 µg/ml (5 mg NP/ml). Arrows indicate neurons that underwent cell death.
Cells treated with unloaded P-NPs and BBB-NPs exhibited no difference in their morphology, e.g. dendrite branching measured by determining the "dendritic complexity index" DCI [13] or the number of synapses per unit length of dendrites (Fig. S1). The findings also allow to dismiss the possibility of a possible toxic effect of glycopeptides covering NPs surface (BBB-NPs) since there is no significant difference between viability-results with empty P-NPs and empty BBB-NPs. Furthermore, the number of neurons assessed by anti-MAP2 staining as well as overall cell number per field of view including glial cells assessed by DAPI staining was not changed compared to control cells. Cells treated with NPs containing Zn2+ (Zn-P-NPs and Zn-BBB-NPs) show a slight but significant reduction in glial and/or neuronal cell number following the addition of Zn-BBB-NPs or Zn-P-NPs, respectively. Since cells treated with empty P-NPs do not show this decrease in neurons, the effect is likely due to an increase in intracellular zinc levels and not by the treatment with NPs itself. The intracellular zinc concentration might have reached already toxic levels for neurons but not for glial cells (Zn-P-NPs) or both, neurons and glial cells (Zn-BBB-NPs) due to increased cellular uptake of Zn-BBB-NPs (Fig. 1A).
To further investigate the influence of NPs on cell viability, cells were plated with a density of 20,000 cells per coverslip and treated with different NP concentrations of "P-NPs" and "BBB-NPs", i.e. Ctrl (625 µg NPs per ml), 2×, 4×, etc. compared to the concentration used in the first cell viability experiment (Fig. 1B). The cells were fixed at DIV14 and the number of cells was assessed. Beginning with the 6× concentration, the standard-deviation becomes larger, meaning that there were already some optic fields per/coverslip, where cell viability was reduced while in other regions cells remained healthy. However, at 8× ("BBB-NPs") and 10× ("P-NPs" and "BBB-NPs") concentrations of NPs, there is a significant reduction in cell number in all fields of view.
Neurons and glial cells endocytose nanoparticles
To explore the fate of NPs added to our neuronal/glial cultures, we took advantage of the fluorescent labeling of both unloaded P-NPs and BBB-NPs (by using tetra-methyl rhodamine conjugated PLGA in the formulation of the NPs). The results reveal that unloaded P-NPs and BBB-NPs readily associated with cultured cells (Fig. 2A). Interestingly, BBB-NPs were found associated to cells in higher amount compared to P-NPs (Fig 2A), perhaps due to the presence of the glycopeptides coating on the surface of BBB-NPs. To investigate whether cells take up NPs, we used Zn2+ loaded NPs and Zinpyr-1, a zinc staining fluorophore, to visualize intracellular zinc-ions. Interestingly, in contrast to untreated as well as ZnCl2 supplemented cells (data not shown), bright fluorescent vesicular structures were visible in HEK293 cells and hippocampal neurons (Fig. 2B, arrows and Fig. S2A) after supplementation of the growth medium with Zn2+ loaded Zn-P-NPs or Zn-BBB-NPs (data not shown). Monitoring the distribution of the rhodamine-conjugated PLGA, we observed a strong colocalization with Zinpyr-1 fluorescent puncta. These data are consistent with the internalization of NPs into Zn2+ rich intracellular organelles/vesicles (Fig. 2C). This conclusion is furthermore consistent with the selective co-localization of the zinc signal with fluorescently tagged Zn-BBB-NPs treated cells (Fig. 2C, full arrows) but not cells treated with unloaded BBB-NPs (Fig. 2C, empty arrows).
A) Images of hippocampal cultures treated with NPs (left panel). Nanoparticles without ligand (P-NPs) or coated with BBB-ligand (BBB-NPs) associate with cells. The distribution pattern is consistent with NPs both on the surface and within cells. Quantification of Rhodamine fluorescent signals per cells shows a ratio of BBB-NPs to P-NPs of 3.15 (n = 10) (right panel). B) Changes in the intracellular zinc levels of HEK293 (upper panel) and neuronal (lower panel) cells after addition Zn-P-NPs were detected by loading cells with Zinypr-1. Bright fluorescent vesicular structures were visible (arrows) in HEK293 cells as well as hippocampal neurons after application of Zn-P-NPs compared to untreated (control) cells. The bottom row of images in each panel shows heat maps revealing regional differences in zinc levels. C) Fluorescent images of HEK293 (upper panels) or neurons (lower panel) assessing the relative distribution of NPs and zinc. Zinypr-1 was used to detect Zn2+, and Rhodamine fluorescence to detect the distribution of Rhodamine labeled NPs. The zinc-signal colocalizes with fluorescent NPs in Zn-BBB-NPs treated cells (HEK293: middle panel, full arrows and Neurons: lower panel) but not in unloaded BBB-NPs (empty arrows, upper panel). D) Fluorescent images of glial cells (upper panel) and hippocampal neurons (lower panel) treated with Rhodamine-conjugated BBB-NPs and FM1-34FX. BBB-NPs fluorescence is observed colocalizing with FM1-43 FX within endocytotic vesicles (arrows). E) Fluorescent P-NPs can - similar to BBB-NPs - be found inside the cell soma co-localizing with FM 1-43 FX within endocytotic vesicles. Quantitative analysis of the percent of vesicles colabeled with NPs and FM dye in comparison to the total pool of FM-labeled vesicles reveals that no significant differences between P-NPs and BBB-NPs can be measured. However, the total number of FM/NP vesicles is significantly increased in BBB-NP treated cells vs. P-NP treated cells.
In order to study the mechanism of entrance and the localization of BBB-NPs within cells, we used fluorescent BBB-NPs together with FM1-43FX dye (Fig. 2D). FM1-43FX is a fixable membrane probe widely used for monitoring recycling vesicles [14]. In these experiments, fluorescently labeled BBB-NPs (Fig. 2D) and P-NPs (Fig. 2E) were found to decorate the same vesicular structures labeled with FM1-43FX consistent with the model of NPs entering cells by endocytosis (Fig. 2A,D,E). Analysis of the amount of vesicles harboring NPs compared to the total pool of labeled vesicles shows that no differences between P-NPs and BBB-NPs can be seen. However, the total number of FM1-43FX+NP positive vesicles was significantly increased in BBB-NP treated cells (Fig. 2E). These findings suggest that the PLGA coating might enhance endocytosis of NPs. Additionally, we monitored the fluorescent pattern of BBB-NPs treated cells over a time-course of 7 d. This revealed that the initial punctate pattern becomes more diffuse over time, consistent with the slow degradation of BBB-NPs resulting in cells with higher background fluorescence (Fig. S2B).
Taken together, these data indicate that BBB-NPs have the capacity to release their content intracellularly.
Characterization of Zn2+ loaded NPs
As a biodegradable polymer, NPs are predicted to slowly release their content over time. We were thus keen to characterize the rate of zinc release both in vitro and in vivo. For all studies outlined below, batches of NPs were characterized for their size, surface charge and general shape by means of Photon Correlation Spectroscopy (PCS) methods and SEM analysis. All NPs (independently from the surface modifiers or the loading) show an average size of 190–210 nm, with overall surface charge (expressed as zeta-potential, z-p) close to neutrality (z-p ranging from −0.5 to −10 mV). This value was maintained also after re-suspension of all kinds of lyophilized NPs. Moreover, a study on the content and release in 7.4 pH buffer was performed on Zn2+-loaded P-NPs (Zn-P-NPs) and BBB-NPs (Zn-BBB-NPs) evidencing a mean of 2.6 mg of Zn2+ (detected both by colorimetric and atomic absorbance technologies) per 100 mg of NPs. Moreover, surface analysis (i.e. ESCA analysis) of the antibody-engineered NPs (Anti-NCAM1-NPs and Anti-CD44-NPs) demonstrated the efficacy of the surface modification process.
Characterization of the in vitro release of Zinc from NPs
ZnSO4. 7H2O was encapsulated in both P-NPs and BBB-NPs (mean size close to 200 nm, PDI of 0.204 and a z-p of −6.13 mV) with a final content of 2.6 mg of Zn2+-ions per 100 mg of NPs. Both NP samples showed similar features (with respect to loading and chemico-physical properties). To assess the timescale, in which Zn-P-NPs and Zn-BBB-NPs release Zn2+ in vitro, both types of Zn-loaded NPs were suspended in Neurobasal Medium +Glutamine +B27 (NB++). A 1.5 µM basic Zn2+ concentration of NB++ was measured using plasma mass-spectrometry.
A suspension with an amount of Zn-loaded NPs (Zn-P-NPs and Zn-BBB-NPs) approx. encapsulating 1 mM Zn2+ was prepared, added to the medium and incubated for 28 days at 37°C. Each day, the zinc concentration was measured using Zinpyr-1, (C46H36Cl2N6O5) a fluorescent sensor for Zn2+ with a high specificity and affinity for Zn2+ (Kd = 0.7±0.1 nM) (Fig. 3). Since Zinpyr-1 is not able to enter or to be adsorbed by NPs, the rise in fluorescence is due to NP-Zn2+ release, a value that correlates directly. After 28 days, TPEN (N,N,N′,N′-Tetrakis(2-pyridylmethyl)ethylenediamine) was added to the samples. TPEN is a water-soluble and cell membrane permeable zinc chelator with very high affinity for Zn2+ (Kd for Zn2+ binding to TPEN is 6.3 * 10−16 M at pH 7.6). A drop in fluorescence shows that the fluorescence of Zinpyr-1 indeed was due to the presence of Zn2+ in the medium. Since Zinpyr-1 only fluoresces upon binding Zn2+, the loss of Zn2+ due to chelation causes this drop in fluorescence.
Quantification of the rate of zinc released from NPs in vitro by monitoring free zinc levels with Zinypr-1 fluorescence. The blue line indicates the trend-line for the obtained data-points. The green line shows an overall drop of fluorescence after treatment with TPEN that competitively binds free zinc with higher affinity. The fluorescence intensity of Zinpyr-1 was measured every 24 hrs and correlated to the fluorescence of medium with known zinc concentration.
The results of the release of Zn2+ suggest that Zn-P-NPs and Zn-BBB-NPs degrade over time, producing a sustained release: in particular, during the first 16 days, a slightly faster release of Zn2+ is visible compared to day 16–22, while, after 22 days, nearly all Zn2+ is released (Fig. 3). The release profile could be due to a degradation/diffusion process happening on NPs architecture. Since the release of Zn2+ from both types of NPs is almost the same, we chose to show only the release of Zn-P-NPs, in order to avoid overlapping and redundancy of results.
Release of Zinc from NP in cultured cells
To assess whether an intracellular environment alters the time release properties of NPs, we monitored changes in intracellular Zn2+ in HEK293 cells and Hippocampal neurons treated with NPs loaded with Zn2+ (Zn-P-NPs and Zn-BBB-NPs). As above, the Zinpyr-1 fluorescent probe was loaded into cells by its addition to the culture medium. Zinpyr-1 is able to penetrate cell membranes.
In initial experiments, we examined the rate of intracellular NPs-Zn2+ release in HEK293 cells, a wildly used fibroblast cell line that shares many properties of immature neurons [11]. Here, HEK293 cells were incubated with a 30 µM ZnCl2 solution and a suspension of Zn-P-NPs and Zn-BBB-NPs (End concentration: 1 mM Zn2+ after NP degradation in DMEM) (Fig. 4A,B). A final zinc concentration was calculated using grey values of Zinpyr-1 fluorescence correlating to the local zinc concentration. The background fluorescence of untreated HEK293 cells was subtracted and cells treated only with 30 µM ZnCl2 used as reference (Fig. S4). After 3 days, a final zinc concentration of 180 µM released by Zn-BBB-NPs was reached (Fig. 4A,B). Compared to the expected Zn2+ release-curve (Fig. 3), this concentration should have been reached only after 6–7 days. Thus, compared to in vitro release of Zn2+ from NPs, the release appears to occur faster in cells. This might be due to a faster degradation of intracellular NPs. In line with this, Zn-BBB-NPs that show increased cellular uptake compared to Zn-P-NPs also lead to a significantly higher increase of the intracellular zinc concentration after 1 d. Not unexpectedly, after 3 days, HEK293 cells showed signs of distress and underwent cell death. This is consistent with published literature showing that an intracellular free zinc concentration of this magnitude is cyto-toxic [15], [16] (and Fig S3).
A) Changes in intracellular zinc levels of HEK293 cells incubated with Zn-P-NPs or Zn-BBB-NPs for 1–3 days (End concentration: 1 mM after complete degradation) monitored by Zinypr-1 fluorescence (upper row). Color-coding of Zinpyr-1 fluorescence (lower row) reveals changes in the intracellular zinc concentration. B) Quantification intracellular zinc with Zinypr-1 (for control and ZnCl2 reference see Fig S4.). Zinc levels in cells treated with Zn-BBB-NPs (41 µM) are significantly higher after one day compared to cells treated with Zn-P-NPs (22 µM). After 3 days, a final zinc concentration of 160 µM (Zn-P-NPs) and 180 µM (Zn-BBB-NPs) was reached. C) Hippocampal neurons were incubated with Zn-P-NPs and Zn-BBB-NPs at 6DIV and zinc levels determined by monitoring changes in Zinpyr-1 fluorescence (upper row and lower row heat-maps) at 7DIV and 14DIV (End concentration: 250 µM after complete degradation). D) After one day, Zn-P-NPs (7 µM) show a significantly lower level of intracellular zinc compared to Zn-BBB-NPs treated cells (10 µM). After 8 days, a final zinc concentration of 48 µM (Zn-P-NPs) and 55 µM (Zn-BBB-NPs) was reached.
Zinc release in rat hippocampal neurons
In a parallel set of cell-based experiments, we investigated the extent of Zn2+ release from NPs in Hippocampal neurons. As above, cells were incubated with a 30 µM ZnCl2 solution as zinc-loading control (Fig. S4) and a suspension of Zn2+ loaded NPs (Zn-P-NPs and Zn-BBB-NPs) (End concentration: 250 µM Zn2+ after NP degradation in Neurobasal +B27, +Glut) (Fig. 4C,D). To maintain cell viability and ensure the growth and maturation of primary hippocampal culture cells for 14DIV, the amount of NPs loaded was reduced compared to the solution taken for HEK293 cells. Although the local Zn2+ concentration at synapses after synaptic activity can reach 300 µM, prolonged exposure to high free Zn2+ concentration causes cell death [15]–[17]. This is nicely illustrated in supplemental data where we find that neurons are more sensitive to free Zn2+ compared to HEK293 cells, however concentrations higher than 160 µM lead to cell death in both cell cultures (Fig. S3).
In these experiments, the background and 30 µM ZnCl2 fluorescence was measured at 7DIV and 14DIV and since no difference was obtained (Fig. S4), the average is used for the quantification of intracellular Zn2+ elevation (Fig. 4C,D). After application of Zn2+ loaded NPs, neurons display an increase in intracellular zinc concentration. The zinc level can be reduced by replacing the growth medium with medium without NPs (data not shown). This leads to Zn2+ efflux due to lower extracellular Zn2+ concentrations in the culture medium (0.093 µg Zn2+/ml = 1.42 µM for Neurobasal medium with addition of B27, Pen/Strep and Glutamine measured by Plasma-Massspectrometry). As in HEK293 cells, Zn-BBB-NPs in neuron elicits a higher intracellular level of zinc compared to Zn-P-NPs treated cells (Fig. 4D).
Targeting Nanoparticles to specific neural cell populations
Based on these experiments, it can be assumed that the endocytosis of un-modified NPs (P-NPs and BBB-NPs) in the CNS will occur in neurons as well as glial cells. However, a more targeted drug release may be desired. Given that P-NPs can be modified with almost any combination of ligands, we explored the possibility of achieving cell type specific targeting. As a proof principle experiment, we coupled NCAM1 and CD44 antibodies to the surface of NPs that recognize antigens on the surface of neurons and glia, respectively. As a control for antibody specificity, neuronal cultures were immuno-stained with antibodies against NCAM1, CD44 and MAP2 a neuronal specific microtubule associated protein. As expected, NCAM1 antibodies immuno-labeled MAP2 positive cells, whereas CD44 antibodies labeled glia cells (Fig. 5A).
A) Immuncytochemistry of hippocampal neurons 14DIV stained with antibodies against NCAM1, CD44 and MAP2. The neuronal protein NCAM1 is found to label neurons that are also immuno-positive for MAP2 (upper row of images). CD44 positive glia are not labeled with MAP2 (lower panel). B) Fluorescent images of neuronal/glial cultures treated with TMR-labeled NPs (red) coated with antibodies against NCAM1 or CD44. Antibodies against MAP2 and the glial specific intermediate filament associated protein GFAP (green) were used to label neurons and glia in these cultures. Unmodified NPs (CNT-Ab-NPs) association with glial cells as well as neurons (upper panel). NCAM1-NPs preferentially label MAP2 positive neurons (arrow, middle panel) but not GFAP positive glia. In contrast, CD44-NPs preferentially label glial cells and a reduced labeling of neurons (arrow, lower panel) is seen. Note that the anti-CD44 ligand appears to promote NPs endocytosis. C) Quantification of percent colocalization (yellow) between NPs (red) and MAP2 (green) or GFAP (green) (n = 10). CD44-NPs show significantly higher colocalization with GFAP compared to NCAM1-NPs and CNT-AB-NPs. In contrast, NCAM1-NPs show significantly higher colocalization with MAP2 compared to GFAP and CNT-Ab-NPs. CD44-NPs and NCAM1-NPs show similar colocalization values like CNT-Ab-NPs for MAP2 (CD44-NPs) and GFAP (NCAM1-NPs).
We then applied fluorescent NPs conjugated with anti-NCAM1 (anti-NCAM1-NPs) or anti-CD44 antibodies (anti-CD44-NPs) to primary mixed hippocampal cultures to evaluate their targeting behavior in presence of both, glia and neuronal cells (Fig. 5B). As expected, control NPs without further modification (CNT-Ab-NPs) associated with glial cells and neurons to a similar degree (Fig. 5C). However, anti-NCAM1-NPs (NCAM-NPs) show reduced targeting to glial cells together with highly increased targeting to neurons stained for MAP2 (arrow Fig. 5B middle panel and C). In contrast, anti-CD44-NPs (CD44-NPs) show increased targeting to glial cells stained for Glial fibrillary acidic protein (GFAP) together with decreased targeting to neurons (arrow Fig. 5B lower panel and C). Interestingly, the anti-CD44 ligand not only increases targeting to glial cells, but appears to promote the endocytosis of NPs.
Non-invasive CNS drug delivery systems have been actively studied, especially with the development of colloidal carriers such as nanoparticles (NP) and liposomes. Indeed, reports in the literature show that these carriers if properly engineered, with a diameter around 100–200 nm, are able to cross the BBB without apparent damage [18], and can deliver drugs or genetic material into the brain [19], [20]. However, a selective biodistribution within the CNS is highly needed and this goal is far from being achieved. Nowadays, the use of a polymeric NP is one of the most promising approaches for CNS drug delivery [18], [21], [22], because polymeric NPs possess advantages with respect to free drug molecules or pro-drugs, such as a high drug-loading capacity [22]. In addition, NPs protect the embedded drugs against chemical or enzymatic degradation, thus increasing the chance for the active molecule to reach the target site. Only few polymers currently guarantee the safety of the polymer-based nanocarriers. Polylactide-co-glycolide (PLGA) or polylactide (PLA) polymers are biodegradable, biocompatible, FDA-approved and are therefore two of the most promising polymers for the preparation of NPs [18]. In this paper, we evaluated whether PLGA-based NPs could efficiently deliver Zn2+ to neuronal cells.
In brain chelatable Zn2+ has been detected in presynaptic vesicles of glutamatergic terminals and Zn2+ ions are released from the presynaptic site. At the postsynaptic site, Zn2+ ions are recruited into large macromolecular platforms within the PSD, assembled by scaffolding molecules such as ProSAP2/Shank3 and ProSAP1/Shank2, thus modulating the structure of the protein meshwork underneath the postsynaptic membrane [23]–[26]. In animal models, zinc exhibits antidepressant-like effects in the forced swim test, both in mice and rats and in tail suspension test [27]–[31], which are used for evaluation of antidepressant activity. Moreover, very low doses of zinc administered together with low, ineffective doses of imipramine or citalopram enhanced the antidepressant-like effect in this test [27], [32]. In humans, involvement of zinc in antidepressant therapy has some clinical correlates. It was shown that human depression is likely to be accompanied by lower serum zinc concentrations [33]–[35]. Subjects suffering from major depression showed significantly lower serum zinc levels than non-depressed controls, whereas patients with minor depression showed intermediate zinc levels [36]. Furthermore, a relationship between severity of depressive symptoms and decreased serum zinc concentration in postpartum depression was demonstrated [37] and zinc supplementation showed a significant reduction in anger-hostility and depression-dejection score in the Profile of Moods State (POMS) of women, suggesting that zinc supplementation may be effective in reducing anger and depression [38], [39].
Here, we show that PLGA-based NPs can be considered as a promising drug delivery system for the specific application of Zn2+ to CNS neurons. Several kinds of Zn2+ loaded or unloaded NPs (un-modified or modified for BBB crossing) were tested both in cultured fibroblast and primary neurons. The experiments show that NPs themselves are not toxic to cells, even at concentrations higher than those used for delivering Zn2+ (Fig. 1). In fact, cell viability is not affected below approx. 5000 µg/ml of NPs, a concentration much higher than that needed to efficiently deliver Zn2+ to cells. NPs decorated with the BBB-crossing ligand (BBB-NPs), a glycopeptide consisting of 7 aminoacids are slightly more toxic, however not at concentrations capable of dramatically increasing intracellular Zn2+ levels. Nevertheless, the data obtained should provide an important base line with respect to the evaluation of toxicity of unloaded NPs for future experiments.
Regarding NP mediated therapies, an important question is how, when and where NPs release their content, e.g. interstitially or perhaps following cellular uptake. In the event of endocytosis-mediated NP uptake, it is predicted that the forward trafficking of these endocytic vesicles to lysosomes could enhance release rates of zinc due to the low pH in lysosomes (Fig. 4). To test this hypothesis, we assessed the fate of Rhodamine labeled P-NPs and BBB-NPs after application to cells together with FM-43 labeling (Fig. 2D). Our results show that NPs associate with the surface of cells and then seem to appear in an endocytic compartment together with FM-1-43. This latter data indicates that they were endocytosed by these cells (Fig. 2D,E). Interestingly, it seems that the presence of BBB-ligand (glycopeptides) on the surface of BBB-NPs enhances the number of endocytotic events compared to un-modified NPs. The glycopeptides covering the surface of BBB-NPs exhibit a helix-like conformation [40] postulated to contribute to the entrance of BBB into cells by stimulating membrane curvature and thus endocytosis. To date, no data on the effect of this "membrane curvature effect" are available for neurons, but a membrane "hopping" type mechanism is likely [41]. Due to the fact that NPs are polymeric and not lipid based as liposomes, it is unlikely that they are able to fuse with cell lipid membranes. However, NPs stay in very close contact with the cell surface and released zinc could enter cells via specific or unspecific transporters/ion channel systems. While feasible, the rise in cell soma fluorescence associated with both Rhodamine-labeled BBB-NPs and P-NPs after 7 d, indicates that at least some of these NPs entered and released their content inside cells.
This conclusion is consistent with our results showing that BBB-NPs can be used to elevate the intracellular Zn2+ concentration in a cell-based assay (Fig. 4). Given that the release rate of encapsulated Zn2+ in NPs (Zn-NPs) measured in vitro (Fig. 3) showed that after approximately 3 weeks, nearly all NPs released their content, the elevation in zinc concentration might be due to an extracellular rise in Zn2+ or endocytosed NPs releasing Zn2+ inside the cell. However, in our cell-based experiments the release-rate of zinc is faster, and perhaps cell autonomous, due to their uptake. This is exemplified by the observed difference between BBB-NP and P-NP, where BBB-NPs are more readily taken up by cells, resulting in higher intracellular levels of zinc.
Since glial cells are known to clear the extracellular fluid of substances within the brain, having NPs that selectively target neurons or glial cells will be helpful in many ways. The extent to which glial cells participate in psychiatric disorders is currently only beginning to be explored, but a crucial role of glia in a wide range of neurological disorders has long been recognized. Although neurodegenerative disorders such as Parkinson's disease, Alzheimer's disease, ALS and Huntington's are caused by the death of neurons, glial cells have both a positive and negative influence on the progression of these diseases [42]–[45]. To differentially target neurons and glial cells, we designed antibody-engineered labeled NPs using antibodies directed against an extracellular epitope specific for neurons (NCAM1) and glial cells (CD44). These modified NPs demonstrate that a more selective targeting strategy can be used to enrich zinc or other drugs in a cell type specific manner. Intriguingly, in case of CD44, this ligand may influence endocytosis positively. Based on the results of this study, the targeting behavior, drug release properties and metabolism of the generated antibody-conjugated NPs can be investigated in an in vivo model as a next step for future application of NPs.
In this study we have characterized a novel nanoparticle technology, based on PLGA NPs modified with glycopeptides known to promote the ability of NPs to cross the BBB in vivo. We demonstrated that both un-modified and BBB-crossing modified NPs, harboring substances released over time, are able to influence neuronal cells in culture. We could show that these NPs can efficiently deliver Zinc to cells at non-toxic concentrations. We also provided evidence that NPs can be easily modified so that they preferentially target specific cell populations, e.g. neurons versus glial cells, after crossing the BBB. This strategy should improve the delivery of drugs to the CNS. Thus, this work provides important base line data for future in vivo application of Zn2+ delivery by using modified biodegradable NPs.
Materials and Methods
ZnCl2, the Zn2+ chelator TPEN (N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine) and Zinpyr-1 were purchased from Sigma-Aldrich. Primary antibodies were purchased from Chemicon (MAP2), Abcam (GFAP), Fisher Scientific (anti-CD44, clone OX-50), Lifespan Biosciences (NCAM1 extracellular domain) and Synaptic Systems (Homer1). Secondary antibodies Alexa488, 568 and 647 were from Invitrogen. Poly(D,L-lactide-co-glycolide) (PLGA RG502H) was used as received from the manufacturer (Boehringer-Ingelheim, Ingelheim am Rhein, Germany). BBB ligand (glycopeptides) was provided by EZ-Biolab, (Carmel, USA). PLGA conjugated with tetramethylrhodamine (TMR-PLGA) and conjugated with glycopeptides for BBB crossing (BBB-PLGA) was prepared as previously described [3], [6]. FM1-43FX dye was purchased from Invitrogen. A MilliQ water system (Millipore, Bedford, MA, USA), supplied with distilled water, provided high-purity water (18 MΩ). Unless otherwise indicated, all other chemicals were obtained from Sigma-Aldrich and were of analytical grade.
Preparation of Nanoparticles
NPs were prepared as described in literature [46] with some modifications in the preparation procedure. For a typical formulation, 225 mg of ZnSO4 were dissolved in 0.25 ml of distilled water and emulsified in 2.5 ml of CH2Cl2 (containing 50 mg of a mixture of PLGA 503 H and BBB-PLGA (80∶20 w/w) (with or without 5–10 mg TMR-PLGA) by sonication over an ice bath using a probe sonicator (Misonix, MicrosonTM Ultrasonic Cell Disruptor XL, Opto-lab, Concordia, Mo, Italy) at 60 W output for 45 sec. The resulting primary emulsion was added to 5 ml distilled water containing PVA 1% (15000 MW, Sigma-Aldrich) and was sonicated for 60 sec at 100 W amplitude over an ice bath to form the double emulsion. Organic solvent was removed by stirring at room temperature for at least 1 h and finally purified by Hi-Speed Refrigerated Centrifugation (Beckman J21) at 14000 rpm for 10 min. Alternatively, we also obtained unloaded NPs (P-NPs and BBB-NPs) by omission of drugs within the inner aqueous phase. With this procedure we were able to obtain Zn-P-NPs (when using un-modified PLGA) and Zn-BBB-NPs (when BBB-PLGA was used). In conclusion, labeled (TMR-conjugation) BBB-NPs as well as NPs were obtained and purified to be tested in cell cultures.
Antibody engineered NPs were prepared starting from PLGA NPs, obtained as described before for P-NPs, and applying well known methodologies for Ab-surface engineering of NPs [47]. Briefly, in the presence of EDC (1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide, EDC) (170 mg) and N–Hydroxy-succinimide (NHS, 30 mg) to conjugate the free primary amine groups on the NPs surface with the carboxylic groups on the antibody molecules, desired amount of TMR-labeled P-NPs (50 mg) was suspended and stirred at RT for 1.30 hr in MES (2-(N-morpholino)ethanesulfonic acid, Sigma Aldrich) buffer with designated volume of Anti-NCAM1 Ab (50 µl of a 1 mg/mL stock solution) or Anti-CD44 Ab (50 µl of a 0.5 mg/mL stock solution) stock solution or a corresponding amount of buffer solution, in order to obtain respectively Anti-NCAM1-NPs, Anti-CD44 NPs and control group (CNT-Ab-NPs). After the reaction, the suspension of the engineered NPs were collected by centrifugation and further washed twice by distilled water for purification. Some amount of engineered NPs was re-suspended in ultrapure water for further characterization, while other NPs were lyophilized to prepare stocks (trealose was added as cryoprotector).
NPs characterization
All the batches of NPs were characterized in their surface properties, size and shape. All the data collected (data not shown) demonstrated that the surface engineering (with Ab) of NPs or the loading with Zn did not affect the morphological features of the NPs, all having similar dimensions and shape. A scanning electron microscope (SEM) (XL-40 Philips, Eindhoven, The Netherlands) (×10,000) was used to evaluate the morphology of NPs. Before the SEM analysis, the samples were coated under argon atmosphere with a 10 nm layer of palladium gold (Emitech K550 Supper Coated, Emitech LTD, U.K.). NPs in distilled water were analyzed for particle size and zeta potential (z-p) by photon correlation spectroscopy and laser Doppler anemometry using a Zetasizer Nano ZS (Malvern, UK; Laser 4 mW He–Ne, 633 nm, Laser attenuator Automatic, transmission 100% to 0.0003%, Detector Avalanche photodiode, Q.E.>50% at 633 nm, T = 25°C). The results were normalized with respect to a polystyrene standard solution.
As scientifically established in literature, [47] the surface engineering of NPs (Anti-NCAM1 or Anti-CD44 NPs) was demonstrated by Electron Spectroscopy for Chemical Analysis (ESCA) study showing the presence of atoms (N) present on the surface of antibody-engineered NPs and thus proof of success of the surface engineering procedure. ESCA was performed on a 04-153 X-ray source analysis system (PHI, Uvalca-PHI, Tokyo, Japan) and an EA11 hemispherical electron analyzer (Leybold Optics, Germany), using MgKα1,2 radiations. The spectra were recorded in fixed retardation ratio (FAT) mode with 190 eV pass energy. The pressure in the sample analysis chamber was ca. 10−9 mbar. The data were acquired and processed using the RBD AugerScan 2. 1H-HRMAS NMR spectra were recorded on a Bruker Avance 400 instrument; D2O was added to the sample, which was then spun at 4000 Hz. All experiments were recorded at RT.
Determination of NPs content
10 mg of NPs (both batches of BBB and plain NPs) loaded with ZnSO4 were dissolved in 1 ml of DCM and 5 ml of milliQ water were added to the organic solution. The organic solvent was removed by stirring at room temperature for at least 3 h and finally the aqueous solution was filtered through a syringe filter to eliminate the polymer insoluble in water. The final volume of the aqueous solution was adjusted to 25 ml with distilled water and 1 ml of this solution was diluted to 25 ml. The final aqueous solution was analyzed through atomic absorption spectrophotometry. A 5 mM Zn2+ containing stock solution (12.5 mg NPs/ml) was prepared. The results shown represent the mean of at least 3 experiments.
Zn release from NP
10 mg of Zn-NPs (both BBB-NPs and P-NPs) loaded with ZnSO4 were suspended in 1 ml of milliQ water into a dialysis membrane tube (3500 MW cut-off). The dialysis membrane tube was then placed into a becker containing the release medium (25 ml of milliQ water), which was stirred continuously and maintained at 37°C. At determined intervals, 100 µl of release medium solution was collected and then replaced with 100 µl of fresh milliQ water. This sample was diluted to 25 ml with Milli-Q water and then the solution was analyzed with atomic absorption spectrophotometry. The results shown represent the mean of at least 3 experiments.
Hippocampal culture from rat brain
The preparation of hippocampal cultures was performed essentially as described by Goslin et al. [48] with some modifications as detailed in Dresbach et al. [49]. Cell culture experiments of hippocampal primary neurons from rat (embryonic day-18; E18) were performed as described previously [50]. After preparation the hippocampal neurons were seeded on poly-l-lysine (0.1 mg/ml; Sigma-Aldrich, Steinheim) glas coverlslips. Cells were grown in Neurobasal medium (Invitrogen), complemented with B27 supplement (Invitrogen), 0.5 mM L-Glutamine (Invitrogen) and 100 U/ml penicillin/streptomycin (Invitrogen) and maintained at 37°C in 5% CO2. All animal experiments were performed in compliance with the guidelines for the welfare of experimental animals issued by the Federal Government of Germany and the National Institutes of Health. All of the experiments were conducted in strict compliance with APLAC approved animal protocols from Stanford University (protocol # 14607) and by the local ethics committee (Ulm University) ID Number: O.103.
For immunofluorescence, HEK293 cells and primary cultures were fixed with 4% paraformaldehyde (PFA)/1.5% sucrose/PBS at 4°C for 20 min and processed for immunohistochemistry. After washing 3× 5 min with 1× PBS at RT, blocking was performed with 0.5% cold fish gelatine (Sigma) and 0.1% Ovalbumin (Sigma)/1× PBS for 30 min at RT and the cells were washed again 3× 5 min with 1× PBS at RT, followed by the primary antibody at 4°C overnight. After a 3× 5 min washing-step with 1× PBS, incubation with the second antibody coupled to Alexa488, Alexa568, Alexa647 or TexasRed (Molecular Probes) for 1 h followed. The cells were washed again in 1× PBS for 10 min and 5 min with aqua bidest and mounted in Vecta Shield mounting medium with or without DAPI (for staining the nucleus) for fluorescence microscopy. To test cell viability, the number of DAPI positive nuclei and neurons was counted from 10 optic fields for each condition.
Zinc staining
Zn-P-NPs and Zn-BBB-NPs were suspended in Neurobasal Medium (+Glutamine +B27) and applied to cells. As Control, different solutions with defined Zn2+ concentration were used. An aliquot of Zinpyr-1 DMSO stock solution (5 mM) was diluted to a final concentration of 1–5 µM in culture medium and the cells were incubated with Zinpyr-1 for 20 minutes at 37°C. After this, cells were washed in Ca/Mg Phosphate buffered saline and used for confocal microscopy. Images were taken with the same exposure time and grey values quantified using ImageJ v.1.44e. For quantification, the basic fluorescence of the medium (grey value) was measured and subtracted from the grey values of a defined ZnCl2 solution as well as the Zn-P-NPs and Zn-BBB-NPs-suspension. The ratio of the normalized ZnCl2 solution to the NPs suspension determined the final Zn2+ concentration. Measurements were performed three times for each time point using different exposure times.
Measurement of Zn2+ concentrations
The zinc-concentration of hippocampal cell culture medium was measured by Plasma-Massspectrometry (ICP-MS) at the "Spurenanalytisches Laboratorium Dr. Baumann" (Maxhütte-Haidhof, Germany).
Statistical analysis
Images were taken with a spinning disk confocal microscope (Zeiss) using MetaMorph (Universal Imaging, Downing-town, PA) software. Quantification of fluorescence data was performed using, Image J 1.44e software with JACoP plugin. Statistical analysis in this paper was performed using Microsoft Excel for Macintosh and data were tested for significance using two-tailed, Student's t-test followed by ANOVA and p-values <0.05 were stated as significant (<0.05*; <0.01**; <0.001***).
Supporting Information
Figure S1.
Cells treated with unloaded P-NPs and BBB-NPs exhibited no difference in their morphology and synapse density compared to untreated cells. A) Dendrite branching was measured using MAP2 stained neurons. The dendritic complexity index (DCI) [13] was calculated based on the equation: [DCI = (# of prim. Dendrites * 1+# of sec. Dendrites * 2+# of tert. Dendrites * 3)/(# of prim.+sec.+tert. Dendrites)]. B) The number of synapses per unit length of dendrites was evaluated using Homer1 as postsynaptic marker and Bassoon as presynaptic marker. The number of Homer1/Bassoon colocalizing signals per 10 µm dendrite length is shown.
Figure S2.
A) Overview of images of HEK293 (upper panel) and neurons (lower panel) shown in detail in Figure 2B. White squares indicate regions shown in Figure 2B. Bottom row of images in each panel are heat maps revealing regional differences in zinc levels. B) Fluorescent images of neurons 1, 2, 6 and 7 days following the addition of Rhodamine conjugated BBB-NPs. After 7 days the Rhodamine-BBB-NPs exhibit a diffuse pattern suggesting that endocytosed BBB-NPs are partially released into the cell soma.
Figure S3.
Prolonged exposure to high free Zn2+ concentration causes cell death in HEK293 cells and hippocampal neurons. To determine the range of viable intracellular Zn2+ concentrations and their limit for toxicity, we supplemented cell cultures with increasing Zn2+ concentrations for 8 h. The results show that neurons (upper row) are more sensitive to free Zn2+ compared to HEK293 cells (lower row). After supplementation of the medium with a concentration of 80 µM Zn2+ and higher, neurons undergo cell death visible through cell fractionation, which leads to a loss of intracellular fluid and thus Zn2+ (full arrows). Additionally, condensed and fractionated nuclei appear (open arrow). HEK293 cells undergo cell death at Zn2+ concentrations higher then 160 µM, visible through cells rounding up due to a loss of surface adhesion.
Figure S4.
Supplementation of HEK293 cells (left panel) and hippocampal neurons (right panel) with ZnCl2 was used as reference for the evaluation of zinc concentrations (Fig. 4). The brightness of the Zinpyr-1 signal of untreated cells and cells treated with a defined amount (30 µM) ZnCl2 was used to evaluate the increase in zinc concentration by Zn-P-NPs and Zn-BBB-NPs (Fig. 4). The final zinc concentration was calculated using grey values of Zinpyr-1 fluorescence. Background fluorescence of untreated neurons was measured and cells treated with 30 µM ZnCl2 used as reference. HEK293 cells were supplemented with ZnCl2 at DIV0 and the zinc concentration was evaluated after 1 and 3 days. Hippocampal neurons were supplemented with ZnCl2 at DIV6 and the zinc concentration was evaluated at DIV7 and DIV14.
Author Contributions
Conceived and designed the experiments: AMG CCG TMB GT FF. Performed the experiments: AMG LB BR MAV GT. Analyzed the data: AMG GT. Contributed reagents/materials/analysis tools: AMG GT. Wrote the paper: AMG CCG TMB FF GT.
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Sept. 6, 2010
Park Ridge, Ill. -
The conference office announced on Monday the second weekly field hockey award winners for the 2010 season. Northwestern sophomore Chelsea Armstrong earned honors on offense after posting a pair of two-goal games during the week, while Ayla Halus gave the Penn State Nittany Lions their second straight accolade as she earned Defensive Player of the Week. Michigan State's Katherine Jamieson earned her first Freshman of the Week honors.
Offensive Player of the Week
Chelsea Armstrong earns her fourth career Offensive Player of the Week award after posting a pair of two-goal games. Armstrong was a constant threat in Northwestern's 4-1 victory at Colgate on Monday, recording 15 total shots and eight shots on goal. Armstrong picked up one assist Friday vs. Virginia Commonwealth before closing the week by scoring twice in the first 12 minutes of Northwestern's match at William & Mary. She later assisted on the Wildcats' fourth goal of the game to raise her Big Ten-leading points total to 15. With six goals in five games this season, Armstrong now has 28 goals in 26 career games at Northwestern.
Last Northwestern Offensive Player of the Week: Chelsea Armstrong (Oct. 12, 2009)
Defensive Player of the Week
Ayla Halus earns her first career award after picking up her third straight double-digit save performance to start the season, all which have come against ranked foes. Halus recorded her second double-digit save half of the season, stopping 10 shots in the first half against No. 5 Connecticut to keep the Lions in the game. The sophomore tallied 12 saves overall in the contest. Halus has an 87.5 save percentage, stopping 35 of 40 shots thus far.
Last Penn State Defensive Player of the Week: Ali Meves (Oct. 5, 2009)
Freshman Player of the Week
Katherine Jamieson, Michigan State
M - Fr. - Toledo, Ohio - Ottawa Hills
Spartan freshman Katherine Jamieson earns the first weekly honor of her career after tallying one goal and a game-tying assist on the weekend. On Friday, she scored the game-winning goal with 1:48 remaining in regulation to help MSU to a 4-3 win over California. Jamieson also had an assist on a Joelle van Ireland score in the Spartans' 10-0 victory against Pacific on Saturday.
Last Michigan State Freshman of the Week: Molly Cassidy (Aug. 30, 2010)
Schools Illinois Indiana Iowa Maryland Michigan Michigan State Minnesota Nebraska Northwestern Ohio State Penn State Purdue Rutgers Wisconsin
Men's Sports Baseball Basketball Cross Country Football Golf Gymnastics Hockey Indoor Track & Field Lacrosse Soccer Swimming & Diving Tennis Outdoor Track & Field Wrestling
Women's Sports Basketball Cross Country Field Hockey Golf Gymnastics Indoor Track & Field Lacrosse Rowing Soccer Softball Swimming & Diving Tennis Outdoor Track & Field Volleyball

subset: commoncrawl


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Lamoille County's Legislative Breakfast Series continues on February 10th.
The Lamoille Region Chamber of Commerce and Stowe Area Association present the 2014 Lamoille Legislative Series. Light Breakfast is served at 7:45am and the meeting begins promptly at 8:10am and concludes at 9:30am for all breakfasts.
Second Session Sponsored by:
February 10 -
Session Sponsored by Union Bank
2014 Remaining Save the Dates:
- March 10 in Stowe, Town & Country Resort, $12 pp, buffet breakfast,
Session Sponsored by Lamoille Economic Development Corporation
- April 7 in The Family Table Restaurant, Jeffersonville $12 pp, buffet breakfast
Session Sponsored by Jack F Corse, Inc.
Our Legislative Breakfast Overall Presenting Sponsor is Edward Jones Investments, Rich Jacobs Financial Advisor


text:
Hey all,
First let me reference the site link....
The picture shows the spats basically having the same color as the the flight suit. Yet this is written for 501st credentials...
Now if you read what the belt pouches are supposed to be...
For 501st approval:
- The top of the boot is covered by a fabric spat of similar color to the belt pouches.
For level two certification (if applicable):
For level three certification (if applicable):
Hip Pouches
For 501st approval:
- A pouch is present at each hip.
- Pouches are beige and must be weathered.
- Pouches have angled flaps at the top.
- Pouches are mounted via fabric loops over the ammo belt. The loops are the full width of the pouches.
Basically you would think the spats are supposed to be beige in color. Which is correct because I am confused!


text:
An Apple patent (number 7971782) for a multi-point [of sale] transaction system has appeared at the US Patent & Trademark Office. It relates to temporarily pairing devices to complete transactions; and more specifically to temporarily pairing at least one handheld communication device selected from a plurality of handheld communication devices with at least one payment terminal selected from a plurality of payment terminals.
Computer implemented systems and methods for conducting transactions are disclosed in the patent. In one exemplary embodiment, a method includes receiving transaction information corresponding to a transaction initiated with one of a plurality of handheld computing devices. The handheld computing device is associated with one of a plurality of payment terminals. The transaction is completed and the association between the handheld computing device and the payment terminal is terminated. Khawaja Shams is the inventor.
Here's Apple's...| Read more »


text:
Chef McDang: Nobody wants to watch quality television in Thailand
As yet another soap opera gets thrown into the Thai morality ring for its less than family-friendly content –- in effect drawing more people to tune in -- one question comes to many people's minds.
Just how challenging is it to produce quality television in Thailand, given the obvious lack of it?
Well it depends on how you look at it. What are the parameters of quality, and what does the audience want to see?
Or, do we just say to hell with our principles. Let's make lots of money.
In Thailand, a nation addicted to "lakorns" and slapstick comedies, the general and unfortunate truth is that most TV viewers have not been taught to think for themselves. And the Thai education system is much to blame for this.
Therefore, television shows with any meat in them do not attract viewers. The Thai audience has been trained to be completely parochial. They want entertainment, and nothing more.
That's not to say there aren't people interested in learning about worldly things. But sadly, they're in the minority.
This is why television producers find it difficult to create quality programs. They have to weigh the heavy economics of producing the show against potential ratings and sponsorships.
Ratings do, quite often, come before a program's integrity. The most successful Thai television shows tend to offer pure entertainment as opposed to programs that educate, inform and broaden the horizons of the general audience in a meaningful way.
Here, the lion's share of society wants to know who sleeps with whom, what is the latest scandal, who's rich or poor. Consequently, these same people spend most of their time aspiring to be wealthy, bitchy and avaricious just like the characters portrayed on your average cheap television soap opera -- known locally as "lakorns."
But there is a second, smaller group of television producers. A group that's more concerned about education and social standards -- which is why that group isn't as successful.
Therein lies the challenge. When I produce my weekly television show, just like everybody else in the business I have to weigh the commercial aspects of production with the content and our role as educators.
If nobody's watching, I'm not going to have a TV show. So when filming, I find it difficult to be serious because, unfortunately, the general audience has a very short attention span when it comes to intellectual exercises.
It is very hard to win and maintain a time slot on television in Thailand, let alone produce a high quality TV show. But things get even more difficult when it comes to acquiring sponsors as they are understandably more concerned about your program's ratings than its contents.
Fortunately there are a few TV stations in Thailand that are attempting to educate the general public, such as Nation Channel and Thai PBS.
Their programs have gained measured success, but they are far from being as financially successful as the mainstream TV programs in Thailand.
But to change this, we have to change our whole system of education in this country.
Currently, educators favor rote memorization. Yet free thought and actual learning can only be encouraged by teaching the basics of any given field in a systematized fashion so as to lay a strong foundation for individuals to expand upon it in their own unique way.
Right now, this doesn't happen in Thai schools. And it will take a concerted effort by those in the government to change the way we teach and are taught.
Even if the government is truly serious and determined to change the education system, which I doubt it is, it will take at least one whole generation (that is 30-40 years) before we see any change in general perception.
For those of us who would like to see the public develop a bigger social conscience and demand Thai programs of value, we will have to wait until our next life. What a shame.
A household name in Thailand, Chef McDang is a chef, TV personality and writer. Born into the Thai royal family, McDang completed his early education in the United Kingdom and United States and at the Culinary Institute of America, which led to a career as an executive chef, restaurant owner and manager that saw him travel from Washington D.C. to Florida and California.Upon returning to Thailand in 1993, McDang began writing about food and appearing on TV cooking shows. Almost two decades on, McDang is Thailand's most famous food expert and a respected ambassador for Thai cuisine.


text:
- People
- Events
- Organizations
- Programs
- Eras
- Recollections
- Issues
- About
Eras in Social Welfare History
Social welfare policy and programs in America have roots all the way back to early Colonial days. Below is a list of distinct eras in American history into which posts have been sorted. Some entries may appear in more than one time period, as historical periods overlap and are not always simple to delineate.
Colonial and Post-Revolutionary America (approx. 1600-1800)
The Antebellum Period (approx. 1800-1860)
The Civil War, Reconstruction, and Progressivism (approx. 1870-1914)
World War I and the 1920s (1914-1929)
The Great Depression (approx. 1930-1940)
World War II and the 1950s (approx. 1941-1955)


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Team America (NASL)
|Full name
|Team America
|Team America
|RFK Stadium,
Washington, D.C.
|Robert Lifton
|Alketas Panagoulias
|North American Soccer League
|10–20, Last Place
Team America was a professional version of the United States men's national soccer team which played as a franchise in the defunct North American Soccer League (NASL) during the 1983 season. The team was based in Washington, D.C., played its home games at RFK Stadium, and was intended by the NASL and the United States Soccer Federation to build fan support for the league and create a cohesive and internationally competitive national team. It did not accomplish these objectives.
A "Team America" also played an unofficial exhibition match against England in 1976, to commemorate the bicentenary of American Independence. The players were drawn from North American Soccer League clubs and included Pele and Bobby Moore. The match was played in Philadelphia, and finished 3–1 to England.[1]
The origins of Team America came with the ascension of Howard Samuels to the positions of president and CEO of NASL on June 25, 1982. At the time, the league was on the verge of collapsing. The league had 21 teams at the end of the 1981 season, but began the 1982 season with only 14. Attendance was dropping and the league had lost its television contract.[2] To reverse these trends, Samuels proposed turning the league's focus away from international stars to domestic players in order to create a larger fan base.[3] Samuels' concept dovetailed with a desire on the part of the United States Soccer Federation (USSF) to build a more successful national team program. In order to fulfill the twin objectives of nurturing new U.S. talent to reach a greater fan base while feeding that talent into the national team, Samuels and the USSF decided to enter the national team into NASL as a franchise.[4] At the time, U.S. players found it difficult to compete for roster spots against the foreigners who stocked most of the league's teams. In theory, Team America would only feature U.S. players who would train with each other, fostering a sense of team cohesion which would pay dividends when the U.S. played international games.[3][5] The name Team America was a reference to both the U.S. national team as well as the 1976 Team America, a team which combined U.S. national team players with the addition of several NASL international stars, which played in the 1976 U.S.A. Bicentennial Cup Tournament.[2]
Creating the team[edit]
Samuels brought in Robert Lifton as the team owner, and in coordination with Lifton and USSF secured RFK Stadium in Washington, D.C. as the team's home field.[6] In January 1983, the NASL and USSF invited 39 players from the NASL, American Soccer League (ASL) and Major Indoor Soccer League (MISL) to tryouts for the 20 available roster spots.[7] However, several top U.S. players, including Rick Davis, Jimmy McAllister, Winston DuBose, David Brcic, and Juli Veee, refused to leave their teams to play for Team America.[8][9] This forced the NASL and USSF to turn to recently naturalized players and in the case of Alan Green a player who had not yet gained his citizenship.[10][11] Several top U.S. players joined the team, including Chico Borja and Jeff Durgan joined Team America as did Arnie Mausser and Perry Van der Beck.
Wearing a red, white and blue uniform with a horizontally striped jersey,[12] the team began well, going 8–5, but lost 15 of its last 17 games, finishing at the bottom of the standings with a 10–20 record. (Lack of scoring punch was the biggest problem: they scored just 33 goals on the season, barely one per game and easily the worst in the league.) None of the players were named to the league's All Star team. As the team stumbled through the regular season, Samuels and Lifton became desperate.[13] On July 27, 1983, they announced that they had approached the owners of the other NASL, as well as MISL, teams about loaning U.S. players to Team America on a game by game basis when the players' regular teams had an idle game day.[14] However, MISL refused the NASL/USSF request for player loans, citing the NASL policy forbidding teams in that league from loaning its players to MISL.[15] Lack of success on the field led to tensions among U.S. national team players. Some, such as Jeff Durgan, publicly criticized Rick Davis and Steve Moyers for choosing to remain with the Cosmos rather than signing with Team America.[16] Mark Peterson, the North American Soccer League's player of the year in 1982, began the season with the Seattle Sounders, but joined Team America during the 1983 season after receiving severe criticism from other U.S. players.[17]
The team averaged only 12,000 fans per home game, leading to severe financial losses. Additionally, USSF failed to finalize several potential marketing deals. At the end of the season, Robert Lifton pulled the plug on the franchise. Following the demise of the team, Lifton, Samuels and USSF engaged public rancour and wrangling regarding who was most responsible for this fiasco.[18]
After the failure of Team America, Washington, D.C. was without a professional soccer club until the birth of Major League Soccer and the creation of D.C. United in 1996.
Note: Flags indicate national team as defined under FIFA eligibility rules. Players may hold more than one non-FIFA nationality.
|Reg. Season
|4th, Southern Division
|Did not qualify
See also[edit]
- Pailan Arrows, a similar attempt by the All India Football Federation to involve the India U19 team in the I-League.
- ^ England Football Online (February 2008). "England's Unofficial Matches". Archived from the original on January 22, 2011. Retrieved February 26, 2012.
- ^ a b Litterer, Dave (August 15, 2012). "The Year in American Soccer – 1982". sover.net.
- ^ a b A Dream Comes True: Team America is Born Archived March 21, 2005, at the Wayback Machine.
- ^ Litterer, Dave (August 17, 2011). "The Year in American Soccer – 1983". sover.net.
- ^ Bell, Jack (September 27, 2006). "The Long, Hard Struggle to Mold an American Team". The New York Times.[permanent dead link]
- ^ Gammon, Clive (March 7, 1983). "Red, White, Blue And New". Sports Illustrated.[permanent dead link]
- ^ "Team America Has Field of 39". The New York Times. January 26, 1983.
- ^ "Team America's Plight". The New York Times. April 21, 1983.
- ^ Falla, Jack (August 22, 1983). "Red, White, Black And Blue". Sports Illustrated.[permanent dead link]
- ^ "Transactions". The New York Times. April 23, 1983.
- ^ "SPORTS PEOPLE; Reluctant Guests". The New York Times. January 28, 1983.
- ^ "Team America". nasljerseys.com.
- ^ "SPORTS PEOPLE; Soccer Complaint". The New York Times. August 3, 1983.
- ^ "Help for Team America". The New York Times. July 27, 1983.
- ^ "M.I.S.L. Ignores Team America". The New York Times. United Press International. May 12, 1983.
- ^ "SPORTS PEOPLE; Star to Team America". The New York Times. August 9, 1983.
- ^ Hutcherson, J. "NASL: The Last Commissioner". ussoccerplayers.com.
- ^ "NASL Team America Rosters". nasljserseys.com. Retrieved November 11, 2012.
External links[edit]
- North American Soccer League (1968–84)
- Defunct soccer clubs in the United States
- North American Soccer League (1968–84) teams
- Soccer clubs in Washington, D.C.
- History of the United States men's national soccer team
- 1983 establishments in Washington, D.C.
- 1983 disestablishments in Washington, D.C.
- 1976 in American soccer
- 1983 North American Soccer League season
- Association football clubs established in 1983
- Association football clubs disestablished in 1983


text: Matt Labash of The Weekly Standard has a very funny piece up called "The Adventures of Low Impact Man". Some of you have heard of Colin Beavan and his efforts to live what he calls a "low impact", non-wasteful life. Labash takes him up on his challenge and writes an often hilarious and quite lengthy [Read More…]


text:
Game Offers Elementary School Kids a Chance to Solve World Peace
Since 1978, educator John Hunter has been asking fourth graders to solve humanity's most complicated problems. So far, no big solution has emerged but the students learn a lot, he said in a Tuesday interview on WNYC.
"This game is designed to fail massively," Hunter said. "Often they go to war against my wishes. But they go through it and come out the other side with a deeper understanding than they could have had I stopped them."
He has a book out called World Peace and Other Fourth-Grade Achievements. You can hear the conversation on The Brian Lehrer Show:
"All 50 interlocking global problems has to be solved reasonably," Hunter said. "Everybody has to win if the game is to be won."
He said the keys to a successful game are to have open-minded participants who know and trust each other; it can be played with older students or adults too.
Over the past 35 years, Hunter said the tenor of the conversation has evolved, in a way that leaves him optimistic.
"The children started out back then, at the tail end of the Cold War, with much more militaristic intent. Over time they've become much more thoughtful, less inclined to war, which is surprising to me in general, much more thoughtful before engaging in those kinds of activities, particularly after 9/11."
Teachers and parents, any games you like to play with children that tackle real-life problems? Share your ideas and experiences here.


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MIAMI -- Sweaty and still soaking after a dominant pitching performance, Josh Johnson was met at his locker with a handshake and a smile that came all the way from the top.
"Wow," Florida Marlins owner Jeffrey Loria told him.
Johnson had a lot of people saying that.
The right-hander pitched seven innings of three-hit ball, Dan Uggla homered and the Marlins beat the New York Yankees 2-1 on Saturday.
Johnson (7-1) struck out five and walked two to outpitch former Marlins teammate A.J. Burnett (5-4). It was the sixth straight start Johnson has gone at least seven innings.
He's not only cemented himself as the staff ace, he's quickly becoming one of the most potent pitchers in the National League after years of injuries.
"That's huge for me," Johnson said about going deep in games. "That means I'm doing what I'm supposed to do."
The Yankees didn't do much to help Burnett.
Johnny Damon misplayed a ball hit to left field by Jorge Cantu in the sixth, allowing Hanley Ramirez to score from first to give the Marlins a 2-0 lead they would never lose.
The play was ruled an error, with Damon slow to read the ball, whiffing on the catch and letting the ball to trickle to the wall.
"I had a good read on the ball. I knew what the ball was doing," Damon said. "He kind of inside-outed the ball and it was going to center. I ended up missing the ball by a foot and that's totally unacceptable. It's amazing. There's been a lot of plays that I've messed up this year.
"I'm definitely a much better defender than that. It comes in bunches. For a while, I was pretty good out there, and then lately, I've been brutal."
Johnson quieted a Yankees team that kept a slumping Alex Rodriguez out of the starting lineup for the second straight game because of fatigue. But the three-time AL MVP did pinch-hit in the eighth, drawing a walk in his first plate appearance ever for a regular-season game in his hometown of Miami.
Matt Lindstrom pitched the ninth for his 13th save in 15 chances, getting Robinson Cano to ground into a game-ending double play. But it was Johnson that kept the Yankees off their game for most of the night.
"He didn't throw this hard when I was here," said Yankees manager Joe Girardi, who was at the helm in Florida in 2006.
The Yankees' fans turned out in scores for the second straight game with their team's rare visit to Miami. The Bronx buzz helped fill the stadium with 46,427 fans, the third-largest home crowd for a regular-season game in Marlins history.
The stadium's orange seats could barely be seen, replaced with fans in navy and pinstripes, who chanted and ranted with every swing. Florida fans fought back with cow bells that were handed out at the gate, creating a circus-like atmosphere rarely seen at baseball games in Miami.
They even to got cheer one of their own.
It was Rodriguez's first regular-season plate appearance in his hometown of Miami against the Marlins. The crowd stood throughout his at-bat and gave him nothing but cheers, the cameras flashed with every pitch.
He walked to put two runners on with no outs, but Derek Jeter grounded into a double play to put a major dent in the Yankees' rally.
It was one of many chances New York blew.
Nick Swisher hit a single to score Cano in the seventh to give the Yankees their only run. With runners on first and third, Johnson regrouped and struck out Melky Cabrera, pounding his glove and waving his hands in celebration.
"He's pretty good," Marlins manager Fredi Gonzalez said, sarcastically. "Twenty years from now, we'll say special."
Yankees OF Xavier Nady went 0 for 4 in five innings of an extended spring training game Saturday in Clearwater, Fla. It was the second consecutive day he played since hurting a ligament in his right elbow while making a throw April 14 at Tampa Bay. He hopes to start a minor league rehab assignment next week. ... Marlins RHP Anibal Sanchez played catch Saturday for the first time since aggravating his throwing shoulder during a mound session June 3. Sanchez, who threw a no-hitter in September 2006, is not expected to come off the DL when he is eligible on June 18.


text:
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AKC Yellow Lab Puppies - Labrador Retrievers in Nampa, Idaho - Michaels Labs
AKC Yellow Lab puppies. Father is a master hunter trained by Duck Buster Kennels. Mother is junior title ready and also trained by Duck Buster Kennels. We are asking $500.00 male or female. Please call with any questions. 208-863-4914
Ad location: Nampa, Idaho
Ad created: Jul 28, 2014
Contact: Michael
208-863-4914Contact breeder through email
Michaels Labs Breeder Information
My Classified Pictures


text:
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Adm. Robert Lee Dennison Dies; Was Atlantic Fleet's Commander; Naval Aide to Truman
Adm. Robert Lee Dennison, whose half-century as a much-decorated United States naval officer included posts such as Commander in Chief of the Atlantic Fleet and naval aide to President Truman, died Friday of a pulmonary embolism at the National Naval Medical ...
March 18, 1980
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text:
Airballin Body Painter
Erin Brittany ReidModel Female New Orleans, Louisiana, US
Mayhem # 2206268
About MeI am open to anything, very flexible and always available.
NOT interested in Pornography.
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text:
New 2017 Car Inventory Serving Elmurst, Downers Grove, & Chicago
Chicago, IL area drivers know that at Larry Roesch Chrysler Jeep Dodge RAM, they can find the newest Chrysler, Jeep, Dodge, and RAM car models in stock. Whether you want to check out the new re-designed minivan, the 2017 Chrysler Pacifica, drive the family on vacation in a 2017 Jeep Wrangler Unlimited or new Jeep Grand Cherokee SUV, or cruise around in a new Dodge Challenger, we are sure to have a vehicle for you. We even have some budget-friendly deals on new cars in our hand-picked featured new car lineup.
If you're curious how our trucks and cars compare with the competition, let us help you out with our guides for the new RAM 1500 truck and the RAM 2500. We can even help you decide which type of Jeep Cherokee or Jeep Grand Grand Cherokee is right for you.
When you're ready to get started on the new car buying process or want to trade in your current or old vehicle, get the value of your trade-in by using our Kelly Blue Book application. You can even estimate your monthly payments and see what you might be paying toward your new car on a loan or lease. Give us a call or visit our Elmhurst, IL Jeep dealership for a test drive today.
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*Images,prices, and options shown, including vehicle color, trim, options, pricing and other specifications are subject to availability, incentive offerings, current pricing and credit worthiness
*MSRP is manufacturer's Suggested Retail Price (MSRP) of the vehicle. It does not include any taxes, fees or other charges. Pricing and availability may vary based on a variety of factors, including options, dealer,specials,fees, and financing qualifications. Consult your dealer for actual price and complete details. Vehicles shown may have optional equipment at additional cost.
*The estimated selling price that appears after calculating dealer offers is for informational purposes, only. You may not qualify for the offers,incentives, discounts, or financing. Offers, incentives, discounts, or financing are subject are subject to expiration and other restrictions. See dealer qualifications and complete details.
* In transit means that vehicles have been built , but have not yet arrived at your dealer. Images shown may not necessarily represent identical vehicles in transit to your dealership. See your dealer for actual, price, payments and complete details.