Hybrid external-cavity lasers (ECL) using photonic wire bonds as coupling elements

Combining semiconductor optical amplifiers (SOA) on direct-bandgap III–V substrates with low-loss silicon or silicon-nitride photonic integrated circuits (PIC) has been key to chip-scale external-cavity lasers (ECL) that offer wideband tunability along with small optical linewidths. However, fabrication of such devices still relies on technologically demanding monolithic integration of heterogeneous material systems or requires costly high-precision package-level assembly, often based on active alignment, to achieve low-loss coupling between the SOA and the external feedback circuits. In this paper, we demonstrate a novel class of hybrid ECL that overcome these limitations by exploiting 3D-printed photonic wire bonds as intra-cavity coupling elements. Photonic wire bonds can be written in-situ in a fully automated process with shapes adapted to the mode-field sizes and the positions of the chips at both ends, thereby providing low-loss coupling even in presence of limited placement accuracy. In a proof-of-concept experiment, we use an InP-based reflective SOA (RSOA) along with a silicon photonic external feedback circuit and demonstrate a single-mode tuning range from 1515 to 1565 nm along with side mode suppression ratios above 40 dB and intrinsic linewidths down to 105 kHz. Our approach combines the scalability advantages of monolithic integration with the performance and flexibility of hybrid multi-chip assemblies and may thus open a path towards integrated ECL on a wide variety of integration platforms

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

Die Rolle von LRP1 in der Funktion des NMDA-Rezeptors

Ein funktionelles Zusammenspiel von LRP1, einem Mitglied der LDL-Rezeptorfamilie, mit dem NMDA-Rezeptor, einem Glutamat Rezeptor, wurde durch die Interaktion beider Proteine sowie eine tPa-vermittelte, LRP1-abhängige Signalübertragung durch den NMDA-Rezeptor belegt. Darüber hinaus zeigen Mäuse mit einem konditionellen neuronalen knock-out des Lrp1 Gens Verhaltensänderungen, die mit einer beeinträchtigten Signalübertragung durch NMDA-Rezeptoren assoziiert werden könnten. Die genaue Rolle von LRP1 in der NMDA-Rezeptor-Funktion bleibt allerdings noch unklar. In der vorliegenden Arbeit wurde die Rolle von LRP1 bei der Expression der NR2B-Untereinheit des NMDA-Rezeptors an der Zelloberfläche primärer kortikaler Neurone untersucht. Zu diesem Zweck wurde die knock-in Mauslinie LRP1ΔNPxY2, die sich durch eine Alanin Substitution im NPxY2 Motiv des LRP1 auszeichnet, eingesetzt. rnEs konnte gezeigt werden, dass diese knock-in Mutation in einer erhöhten Expression von LRP1 und der NMDA-Rezeptoruntereinheiten NR1 und NR2B an der Zelloberfläche primärer kortikaler Neurone resultiert. Der Effekt konnte durch eine reduzierte Endozytoserate von LRP1 und der NR1-und NR2B-Untereinheiten in primären LRP1ΔNPxY2 Neuronen erklärt werden. Darüber hinaus wurde ein verändertes Phosphorylierungsmuster der Internalisierungssignale der NR2B-Rezeptoruntereinheit Serin S1480 und Tyrosin Y1472 an der Zelloberfläche primärer LRP1ΔNPxY2 Neurone detektiert. Die verantwortlichen Kinasen Fyn und Kasein-Kinase II sind allerdings in LRP1ΔNPxY2 Neuronen im Vergleich zu den Wildtyp-Kontrollen nicht abweichend reguliert. In den Co-Immunopräzipitationsexperimenten wurde gezeigt, dass die Bindung von LRP1 mit NR2B durch die Phosphorylierung reguliert wird und dieser Regulationsmechanismus in LRP1ΔNPxY2 Neuronen beeinträchtigt ist. Dies resultiert in einer stärkeren Bindung von NR2B-Rezeptoruntereinheit an LRP1. Aufgrund reduzierter Internalisierungsraten von LRP1 in LRP1ΔNPxY2 Neuronen führt dieser Umstand zu einer Akkumulation beider Rezeptorproteine an der Zelloberfläche. Schließlich wurden die NMDA-Rezeptor-assoziierten Verhaltensänderungen wie die Hyperaktivität und die Defizite im direkten und umgekehrten räumlichen Lernvermögen in den LRP1ΔNPxY2 Tieren nachgewiesen. Zusammengefasst, demonstrieren diese Ergebnisse, dass LRP1 eine kritische Rolle in der Regulierung der NR2B-Expression an der Zelloberfläche spielt.A functional interplay of the Low Density Lipoprotein Receptor Related Protein 1 (LRP1) NMDA receptor has already been reported. Such abilities as interaction of LRP1 with NMDA receptor subunits or its important role in tPa-mediated NMDA receptor signaling were already demonstrated. Moreover, mice harboring a conditional neuronal knock-out mutation of the entire Lrp1 gene display NMDA-associated behavioral changes. However, the exact role of LRP1 on NMDA receptor function remains still elusive. rnTo provide a mechanistic explanation for such effects we investigated whether an inactivating knock-in mutation into the NPxY2 motif of LRP1 might influence the cell surface expression of LRP1 and NMDA receptors in primary cortical neurons. Here we demonstrate that a knock-in into the NPxY2 motif of LRP1 results in an increased surface expression of LRP1 and NR2B NMDA receptor subunit due to reduced endocytosis rates of LRP1 and the NR2B subunit in primary neurons derived from LRP1ΔNPxY2 animals. Furthermore, we demonstrate an altered phosphorylation pattern of S1480 and Y1472 in the NR2B subunit at the surface of LRP1ΔNPxY2 neurons, while the respective kinases Fyn and casein kinase II are not differently regulated compared with wild type controls. Performing co-immunoprecipitation experiments we demonstrate that binding of LRP1 to NR2B is phosphorylation dependent and this regulation mechanism is impaired in LRP1ΔNPxY2 neurons. Finally, we demonstrate hyperactivity and changes in spatial and reversal learning in LRP1ΔNPxY2 mice, confirming the mechanistic interaction in a physiological readout.rnIn summary, our data demonstrate that LRP1 plays a critical role in the regulation of NR2B expression at the cell surface and may provide a mechanistic explanation for the behavioral abnormalities detected in neuronal LRP1 knock-out animals reported earlier.r

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

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