Nanoparticulate Transport of Oximes over an In Vitro Blood-Brain Barrier Model

Background: Due to the use of organophosphates (OP) as pesticides and the availability of OP-type nerve agents, an effective medical treatment for OP poisonings is still a challenging problem. The acute toxicity of an OP poisoning is mainly due to the inhibition of acetylcholinesterase (AChE) in the peripheral and central nervous systems (CNS). This results in an increase in the synaptic concentration of the neurotransmitter acetylcholine, overstimulation of cholinergic receptors and disorder of numerous body functions up to death. The standard treatment of OP poisoning includes a combination of a muscarinic antagonist and an AChE reactivator (oxime). However, these oximes can not cross the blood-brain barrier (BBB) sufficiently. Therefore, new strategies are needed to transport oximes over the BBB. Methodology/Principal Findings: In this study, we combined different oximes (obidoxime dichloride and two different HI 6 salts, HI 6 dichloride monohydrate and HI 6 dimethanesulfonate) with human serum albumin nanoparticles and could show an oxime transport over an in vitro BBB model. In general, the nanoparticulate transported oximes achieved a better reactivation of OP-inhibited AChE than free oximes. Conclusions/Significance: With these nanoparticles, for the first time, a tool exists that could enable a transport of oximes over the BBB. This is very important for survival after severe OP intoxication. Therefore, these nanoparticulate formulation

Uptake Mechanism of ApoE-Modified Nanoparticles on Brain Capillary Endothelial Cells as a Blood-Brain Barrier Model

Background: The blood-brain barrier (BBB) represents an insurmountable obstacle for most drugs thus obstructing an effective treatment of many brain diseases. One solution for overcoming this barrier is a transport by binding of these drugs to surface-modified nanoparticles. Especially apolipoprotein E (ApoE) appears to play a major role in the nanoparticle-mediated drug transport across the BBB. However, at present the underlying mechanism is incompletely understood. Methodology/Principal Findings: In this study, the uptake of the ApoE-modified nanoparticles into the brain capillary endothelial cells was investigated to differentiate between active and passive uptake mechanism by flow cytometry and confocal laser scanning microscopy. Furthermore, different in vitro co-incubation experiments were performed with competing ligands of the respective receptor. Conclusions/Significance: This study confirms an active endocytotic uptake mechanism and shows the involvement of low density lipoprotein receptor family members, notably the low density lipoprotein receptor related protein, on the uptake of the ApoE-modified nanoparticles into the brain capillary endothelial cells. This knowledge of the uptake mechanism of ApoE-modified nanoparticles enables future developments to rationally create very specific and effective carriers to overcome the blood-brain barrier

Interaction of plasminogen-related protein B with endothelial and smooth muscle cells in vitro

Plasminogen-related protein B (PRP-B) closely resembles the N-terminal plasminogen activation peptide, which is released from plasminogen during conversion to plasmin. We have previously demonstrated that the steady-state level of mRNA encoding PRP-B is increased within tumor tissues, and that recombinant PRP-B antagonizes neoplastic growth when administered systemically to mice harboring tumors, but no insights into the cell targets of PRP-B have been presented. Employing serum-free medium optimized for culturing human endothelial or smooth muscle cells, we show that recombinant PRP-B inhibits basic fibroblast growth factor-dependent cell migration for both cell types, as well as tube formation of endothelial cells. Comparison with the angiogenesis inhibitors angiostatin and endostatin revealed similar results. Recombinant PRP-B is effective in promoting cell attachment of endothelial and smooth muscle cells, and antibody interference experiments reveal that the interaction of recombinant PRP-B with endothelial cells is mediated at least in part by α v-containing integrins. Inhibition of angiogenesis in vivo by PRP-B was demonstrated in the chicken chorioallantoic membrane assay. PRP-B and other antiangiogenic molecules may elicit metabolic perturbations in endothelial cells as well as perivascular mesenchymal cells such as smooth muscle cells and pericytes

Effects of particle size on cell uptake of model triglyceride-rich particles with and without apoprotein E

The effect of apoprotein E on cellular uptake of "VLDL-size" and "IDL-size" triacylglycerol-phospholipid emulsion particles was studied in J-774 macrophages and fibroblasts. In the absence of apoprotein E (apo E), uptake of the smaller IDL-size particles was up to 2-fold higher by mass and 100-fold higher as calculated by particle number. Apo E enhanced the uptake of both VLDL-size and IDL-size emulsion particles, but the effect was greater on the uptake of larger particles (4-5-fold) as compared to up to a 2-fold increase in the uptake of IDL-size particles. In fibroblasts, particle uptake was less than in macrophages (30-50%), but preferential uptake of smaller particles was similarly observed. Particle internalization was demonstrated by 125I-apo E degradation and resistance to particle release by heparin-suramin. In the absence of apo E, cholesteryl ester of emulsion particles (prepared with trace amounts of [3H]cholesteryl ester) was hydrolyzed to free cholesterol, proving internalization and intracellular metabolism. Double-label experiments using Dil-labeled emulsion particles, in the absence and presence of apo E, showed that emulsion particles are rapidly targeted to perinuclear lysosomes. Thus, at physiological concentrations of triglyceride-rich particles, non-receptor-mediated uptake is a mechanism for the uptake of VLDL-size and IDL-size particles into cells. © 1994 American Chemical Society.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Omega-3 triglycerides modify blood clearance and tissue targeting pathways of lipid emulsions

Omega-3-rich (n-3) triglycerides (TG) are increasingly recognized as having modulating roles in many physiological and pathological conditions. We questioned whether the catabolism of lipid emulsions would be changed after enrichment with fish oil (n-3) TG as compared to enrichment with omega-6-rich soy oil (n-6) TG. Phospholipid-stabilized emulsions of n-3 TG and n-6 TG were labeled with [ 3H]cholesteryl oleoyl ether and administered by bolus injection to wild-type (WT) mice, mice lacking the low-density lipoprotein receptor (LDL-R) (LDL-R -/-), and apolipoprotein E (apoE) knockout mice (apoE -/-). The effects of exogenous apoE, heparin, Triton WR 1339, and lactoferrin on catabolism of emulsions were also assayed. n-3 TG emulsions were cleared faster from blood and had different extrahepatic tissue targeting compared to n-6 TG emulsions. In apoE -/- and LDL-R -/- mice, blood clearance of n-6 TG emulsions slowed with decreased liver uptake, but no changes were observed in n-3 TG emulsion clearance and tissue uptake compared to WT mice. In WT mice, addition of exogenous apoE to the emulsion increased liver uptake of n-6 TG emulsions but had no impact on n-3 TG emulsions. Pre-injection of heparin increased and Triton WR 1339 and lactoferrin decreased blood clearance of n-6 TG emulsions with little or no effect on n-3 TG emulsions. Liver uptake of n-6 TG emulsions increased after heparin injection and decreased after Triton WR 1339 injection, but uptake of n-3 TG emulsions was not changed. These data show that the catabolism of n-3 TG emulsions and the catabolism of n-6 TG emulsions occur via very different mechanisms. Removal of chylomicron-sized n-6 TG emulsions is modulated by lipoprotein lipase (LPL), apoE, LDL-R, and lactoferrin-sensitive pathways. In contrast, clearance of chylomicron-sized n-3 TG emulsions relies on LPL to a very minor extent and is independent of apoE, LDL-R, and lactoferrin-sensitive pathways.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Effects of apoprotein E on intracellular metabolism of model triglyceride-rich particles are distinct from effects on cell particle uptake

Apoprotein E (apoE) enhances uptake of triglyceride-rich lipoprotein particles (TGRP). We questioned whether apoE would also modulate intracellular metabolism of TGRP in addition to its effects on particle uptake. We prepared model TGRP with triolein and cholesteryl oleate (1:1, w/w) as the core lipids, emulsified by egg yolk phosphatidylcholine, and containing a non-degradable marker, [3H]cholesteryl hexadecyl ether. Particles were intermediate density lipoprotein-sized as determined by core lipid/phospholipid ratios (2.0-3.0/1) and gel filtration chromatography on Sepharose CL-2B. Emulsions were incubated with J774 macrophages for 5 min to 6 h at core lipid concentrations of 300-1200 μg/ml and 0-0.2 μg recombinant apoE/mg core lipid. Particle uptake was determined by [3H]cholesteryl ether uptake and fluorescence microscopy in the absence and presence of apoE. Similar uptake of particles with and without apoE was achieved by utilizing a 4 times higher particle concentration in the absence of apoE. At equivalent levels of uptake, particles with apoE lead to one-half of the triglyceride mass accumulation and twice the triglyceride utilization as compared to particles without apoE. Further, apoE doubles cell cholesteryl ester hydrolysis and to a lesser extent (∼30%) increases cholesteryl ester resynthesis by acyl-CoA cholesterol acyltransferase. Particles, both with and without apoE, reach the lysosomal compartment as determined by colocalization with fluorescein-labeled α2-macroglobulin. These results suggest that, in addition to its role in enhancing TGRP uptake, apoE has additional effects on modulating the cellular metabolism of both triglyceride and cholesteryl ester, after particle internalization.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Antiangiogenesis treatment combined with chemotherapy produces chondrosarcoma necrosis

A combination therapy protocol using a marine chemotherapeutic and an antiangiogenic molecule was tested in a mouse tumor xenograft model for the ability to curtail the growth of a human chondrosarcoma (CHSA). Ecteinascidin-743 (ET-743), a marine-derived chemotherapeutic, was effective at slowing the growth of a primary CHSA. Plasminogen-related protein B, which antagonizes various endothelial cell activities, also elicited a significant inhibition of neoplastic growth, albeit with reduced effectiveness. The combination of the two agents resulted in only a modest further repression of tumor growth over that associated with ET-743 treatment alone, as measured by tumor volume (82% versus 76% inhibition, respectively). However, analysis of the extent of tumor necrosis and vascularization of the tumor revealed that the coadministration of the two compounds was clearly more effective, eliciting a 2.5-fold increase in tumor necrosis relative to single-agent treatment. The combination therapy also was most effective at antagonizing tumor-associated microvessel formation, as assessed by CD31 immunostaining, suggesting that combination therapy may hold promise for treating CHSA. Tumor necrosis produced by combination therapy of ET-743 and recombinant plasminogen-related protein B was also significantly greater than that produced by conventional doxorubicin treatment, further corroborating the efficacy of combination therapy