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

PDMS electrodes for recording and stimulation

The usability of flexible electrodes in moving environment is limited due to different mechanical characteristics of their metallic and polymeric components. To achieve structure compatible electrodes, all used materials need to have similar Young’s moduli as the surrounding tissue. This paper describes the characterization of macroscopic as well as miniaturized electrodes entirely made out of modified silicone (PDMS). Electrochemical, mechanical, biological, optical, and applicative methods were used. It could be shown, that PDMS electrodes are capable to be used for recording electrocardiograms with similar form and amplitude as with standard electrodes

Exogenous application of glucose induces aging in rat cerebral oligodendrocytes as revealed by alteration in telomere length

To investigate aspects of aging on rat oligodendrocytes, cells of an oligodendrocyte cell line, so-called OLN-93, were cultured either in the presence or absence of glucose. Our data demonstrated that glucose-induced aging in vitro caused an elongation and thickening of cell processes and significantly increased the expression of netrin reflecting a more mature state of oligodendrocyte development. A possible age-inducing effect of glucose is also supported by the decrease of ras protein expression and shortening of telomeres in glucose-treated oligodendrocytes. The present study clearly shows that OLN-93 cells are an exciting and suitable model system for the investigation of age-inducing molecules and the analysis of signaling pathways involved in cerebral aging and degenerations. © 2004 Elsevier Ireland Ltd. All rights reserved

Co-incubation of bEnd3 cells with the different nanoparticulate formulations and LDL+RAP.

<p>*: cells without NP incubation, with LDL+RAP;</p><p>One representative experiment out n>3 is shown.</p

Determination of the receptor state of the bEnd3 cells.

<p>C: control only with the secondary antibody.</p

Co-incubation of bEnd3 cells with the different nanoparticulate formulations and LDL.

<p>*: cells without NP incubation, with LDL.</p><p>One representative experiment out n>3 is shown.</p

Specific cellular binding of the ApoE-modified nanoparticles studied by flow cytometry.

<p>bEnd3 cells were incubated with ApoE-modified nanoparticles (NP-ApoE) or control nanoparticles without ApoE modification (NP-PEG) for 4 h at 37°C and 4°C, respectively. Flow cytometry analysis was performed to quantify their cellular binding. The data are shown as histograms of the FL1-H-channel (autofluorescence of the nanoparticles) as well as in the table with the analysis of the Y mean fluorescence and the percentage of positive cells. Green: NP-ApoE, red: NP-PEG, blue: untreated control.</p

Cellular uptake and intracellular distribution of the nanoparticles studied by CLSM: split of the fluorescence channels. bEnd3 cells were incubated for 4 h with 0.1 mg/ml of the different nanoparticulate formulations at 37°C.

<p>The green autofluorescence of the nanoparticles was used for detection. The cytosol was stained in red with CellTracker™ Red CMTPX, and the nucleus was stained in blue with DAPI. Pictures were taken within inner sections of the cells. Untreated control cells: a) overlay of all fluorescence channels, b) display of the blue nucleus channel, c) display of the green nanoparticle channel, d) display of the red cytosol channel. Cells with the unspecific control NP-PEG: e) overlay of all fluorescence channels, f) display of the blue nucleus channel, g) display of the green nanoparticle channel, h) display of the red cytosol channel. Cells with the specific NP-ApoE: i) overlay of all fluorescence channels, j) display of the blue nucleus channel, k) display of the green nanoparticle channel, l) display of the red cytosol channel.</p

Co-incubation of bEnd3 cells with the different nanoparticulate formulations and LRP1 Dom II and LRP1 Dom IV, respectively.

<p>One representative experiment out n = 3 is shown.</p