scholarly journals Transport of ultrasmall gold nanoparticles (2 nm) across the blood–brain barrier in a six-cell brain spheroid model

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Viktoriya Sokolova ◽  
Gehad Mekky ◽  
Selina Beatrice van der Meer ◽  
Michael C. Seeds ◽  
Anthony J. Atala ◽  
...  
Keyword(s):  
Gold Nanoparticles ◽  
Blood Brain Barrier ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Brain Barrier ◽  
Confocal Laser ◽  
Hydrodynamic Diameter ◽  
Core Diameter ◽  
Blood Brain

Abstract The blood–brain barrier (BBB) is an efficient barrier for molecules and drugs. Multicellular 3D spheroids display reproducible BBB features and functions. The spheroids used here were composed of six brain cell types: Astrocytes, pericytes, endothelial cells, microglia cells, oligodendrocytes, and neurons. They form an in vitro BBB that regulates the transport of compounds into the spheroid. The penetration of fluorescent ultrasmall gold nanoparticles (core diameter 2 nm; hydrodynamic diameter 3–4 nm) across the BBB was studied as a function of time by confocal laser scanning microscopy, with the dissolved fluorescent dye (FAM-alkyne) as a control. The nanoparticles readily entered the interior of the spheroid, whereas the dissolved dye alone did not penetrate the BBB. We present a model that is based on a time-dependent opening of the BBB for nanoparticles, followed by a rapid diffusion into the center of the spheroid. After the spheroids underwent hypoxia (0.1% O2; 24 h), the BBB was more permeable, permitting the uptake of more nanoparticles and also of dissolved dye molecules. Together with our previous observations that such nanoparticles can easily enter cells and even the cell nucleus, these data provide evidence that ultrasmall nanoparticle can cross the blood brain barrier.

scholarly journals Functionalized Solid-Sphere PEG-b-PCL Nanoparticles to Target Brain Capillary Endothelial CellsIn Vitro

2016 ◽  
Vol 2016 ◽  
pp. 1-13 ◽  
Author(s):  
Philip Grossen ◽  
Gabriela Québatte ◽  
Dominik Witzigmann ◽  
Cristina Prescianotto-Baschong ◽  
Le-Ha Dieu ◽  
...  
Keyword(s):  
Blood Brain Barrier ◽  
Laser Scanning ◽  
Solid Sphere ◽  
Correlation Spectroscopy ◽  
Laser Scanning Microscopy ◽  
Brain Barrier ◽  
Critical Aggregation Concentration ◽  
Confocal Laser ◽  
Blood Brain ◽  
Human Liver Cell

Nanoparticles are increasingly used to implement drug targeting strategies. In the present study, solid-sphere nanoparticles (SNPs) made of poly(ethylene glycol)-b-poly(ε-caprolactone) (PEG-b-PCL) were covalently linked to a monoclonal antibody (83-14 mAb) targeted against the human insulin receptor that is highly expressed on human brain microvascular endothelial cells. Resulting targeted SNPs were characterized using transmission electron microscopy (TEM), cryo-TEM, dynamic light scattering, and fluorescence correlation spectroscopy. The critical aggregation concentration was determined using a fluorescence approach. Interaction with a well-characterized humanin vitromodel of the blood-brain barrier (hCMEC/D3) was analysed using an array of methods (flow cytometry, confocal laser scanning microscopy, and TEM). The toxicity on hCMEC/D3 cells and in addition on the human liver cell line HepG2 was assessed using the MTT assay. SNPs with a diameter of 80 nm and a homogeneous size distribution were obtained. Successful conjugation of 83-14 mAb using a heterobifunctional linker resulted in 5-6 molecules of fluorescently labeled 83-14 mAb per SNP. Functionalized SNPs were taken up by hCMEC/D3 cells efficiently without showing a significant toxic effect on cells of the blood-brain barrier and HepG2 cells. These results indicate that functionalized PEG-b-PCL SNPs are a promising candidate to deliver drugs to the CNS.

P10.02 Combined methods of a micropump system and a chronic cranial window allows tumor observation with multi photon laser scanning microscopy under continuous treatment

2021 ◽  
Vol 23 (Supplement_2) ◽  
pp. ii28-ii28
Author(s):  
S Weil ◽  
E Jung ◽  
D Domínguez Azorín ◽  
J Higgins ◽  
J Reckless ◽  
...  
Keyword(s):  
Drug Delivery ◽  
Tumor Cells ◽  
Blood Brain Barrier ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
New Drugs ◽  
Brain Barrier ◽  
Cranial Window ◽  
Blood Brain ◽  
The Brain

Abstract BACKGROUND Glioblastomas are notoriously therapy resistant tumors. As opposed to other tumor entities, no major advances in therapeutic success have been made in the past decades. This has been calling for a deeper biological understanding of the tumor, its growth and resistance patterns. We have been using a xenograft glioma model, where human glioblastoma cells are implanted under chronic cranial windows and studied longitudinally over many weeks and months using multi photon laser scanning microscopy (MPLSM). To test the effect of (new) drugs, a stable and direct delivery system avoiding the blood-brain-barrier has come into our interest. MATERIAL AND METHODS We implanted cranial windows and fluorescently labeled human glioblastoma stem-like cells into NMRI nude mice to follow up on the tumor development in our MPLSM model. After tumor establishment, an Alzet® micropump was implanted to directly deliver agents via a catheter system continuously over 28 days directly under the cranial window onto the brain surface. Using the MPLSM technique, the continuous delivery and infusion of drugs onto the brain and into the tumor was measured over many weeks in detail using MPLSM. RESULTS The establishment of the combined methods allowed reliable concurrent drug delivery over 28 days bypassing the blood-brain-barrier. Individual regions and tumor cells could be measured and followed up before, and after the beginning of the treatment, as well as after the end of the pump activity. Fluorescently labelled drugs were detectable in the MPLSM and its distribution into the brain parenchyma could be quantified. After the end of the micropump activity, further MPLSM measurements offer the possibility to observe long term effects of the applied drug on the tumor. CONCLUSION The combination of tumor observation in the MPSLM and concurrent continuous drug delivery is a feasible and reliable method for the investigation of (novel) anti-tumor agents, especially drugs that are not blood-brain-barrier penetrant. Morphological or even functional changes of individual tumor cells can be measured under and after treatment. These techniques can be used to test new drugs targeting the tumor, its tumor microtubes and tumor cells networks, and measure the effects longitudinally.

scholarly journals Studying the Neurovascular Unit: An Improved Blood–Brain Barrier Model

2009 ◽  
Vol 29 (12) ◽  
pp. 1879-1884 ◽  
Author(s):  
Christoph M Zehendner ◽  
Heiko J Luhmann ◽  
Christoph RW Kuhlmann
Keyword(s):  
Blood Brain Barrier ◽  
Laser Scanning ◽  
Neurovascular Unit ◽  
Cell Types ◽  
Laser Scanning Confocal Microscopy ◽  
Brain Barrier ◽  
Scanning Confocal Microscopy ◽  
Blood Brain

The blood–brain barrier (BBB) closely interacts with the neuronal parenchyma in vivo. To replicate this interdependence in vitro, we established a murine coculture model composed of brain endothelial cell (BEC) monolayers with cortical organotypic slice cultures. The morphology of cell types, expression of tight junctions, formation of reactive oxygen species, caspase-3 activity in BECs, and alterations of electrical resistance under physiologic and pathophysiological conditions were investigated. This new BBB model allows the application of techniques such as laser scanning confocal microscopy, immunohistochemistry, fluorescent live cell imaging, and electrical cell substrate impedance sensing in real time for studying the dynamics of BBB function under defined conditions.

scholarly journals Network Pharmacology-Based Approach to Revealing Biological Mechanisms of Qingkailing Injection against IschemicStroke: Focusing on Blood-Brain Barrier

2020 ◽  
Vol 2020 ◽  
pp. 1-16
Author(s):  
Shuang Zhang ◽  
Xueqian Wang ◽  
Fafeng Cheng ◽  
Chongyang Ma ◽  
Shuning Fan ◽  
...  
Keyword(s):  
Ischemic Stroke ◽  
Tight Junction ◽  
Blood Brain Barrier ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Brain Barrier ◽  
Neurological Deficits ◽  
Network Pharmacology ◽  
Ppi Network ◽  
Blood Brain

Ischemic stroke is the most common type of cerebrovascular accident worldwide. It causes long-term disability and death. Qingkailing (QKL) injection is a traditional Chinese patent medicine which has been clinically applied in the treatment of ischemic stroke for nearly thirty years. In the present study, network pharmacology combined with experimentation was used to elucidate the mechanisms of QKL. ADME screening and target prediction identified 62 active compounds and 275 targets for QKL. Topological screening of the protein-protein interaction (PPI) network was used to build a core PPI network consisting of 408 nodes and 17,830 edges. KEGG enrichment indicated that the main signaling pathway implicated in ischemic stroke involved hypoxia-inducible factor-1 (HIF-1). Experimentation showed that QKL alleviated neurological deficits, brain infraction, blood-brain barrier (BBB) leakage, and tight junction degeneration in a mouse ischemic stroke model. Two-photon laser scanning microscopy was used to evaluate BBB permeability and cerebral microvessel structure in living mice. HIF-1α, matrix metalloproteinase-9 (MMP-9), and tight junction proteins such as occludin, zonula occludins-1 (ZO-1), claudin-5, and VE-Cadherin were measured by western blotting. QKL upregulated ZO-1 and downregulated HIF-1α and MMP-9. QKL has a multiapproach, multitarget, and synergistic effect against ischemic stroke.

Abstract 388: Transcellular Transport of HDL Bearing Gold Nanoparticles Across the Blood Brain Barrier

2017 ◽  
Vol 37 (suppl_1) ◽  
Author(s):  
Skylar T Chuang ◽  
Siobanth Cruz ◽  
Julia Stab ◽  
Sylvia Wagner ◽  
Hagen Von Briesen ◽  
...  
Keyword(s):  
Gold Nanoparticles ◽  
Endothelial Cells ◽  
Blood Brain Barrier ◽  
Aqueous Solubility ◽  
Brain Barrier ◽  
Spherical Particles ◽  
Glioblastoma Cells ◽  
Transcellular Transport ◽  
Blood Brain

The overall objective of this study was to develop a HDL-based multifunctional platform for transport and delivery of highly hydrophobic gold nanoparticles (AuNP) bearing photothermic properties across the blood brain barrier (BBB). We exploited the ability of apolipoprotein E3 (apoE3) to act as a high affinity ligand for the low-density lipoprotein receptor to gain entry into endothelial and glioblastoma cells. The issue of poor aqueous solubility of AuNP of varying diameters (3, 10, or 10 nm) was overcome by integrating them with phospholipids and apoE3, yielding reconstituted rHDL bearing AuNP (rHDL-AuNP). Transmission electron microscopy (TEM) revealed the presence of AuNP embedded in spherical particles. Incubation of human brain microvasculature endothelial cells or glioblastoma cells with rHDL-AuNP bearing unlabeled or FITC-labeled apoE3 revealed robust uptake of particles that were localized in endocytic/lysosomal vesicles. The transport of rHDL-AuNP across an in vitro BBB model developed from primary porcine endothelial cells was examined. The addition of rHDL-AuNP to the luminal side of the cells did not affect the integrity of the BBB as assessed by the localization of key tight junction markers such as occludin, claudins and ZO-1 by immunofluorescence, and, by continual measurement of the transepithelial electrical resistance by impedance spectroscopy under physiological conditions. Lastly, the appearance of fluorescein fluorescence and AuNP in the abluminal side suggested transport of rHDL-AuNP across the neurovascular junction. These findings demonstrate that rHDL bearing apoE3 acts as a detergent in solubilizing and dramatically improving the aqueous solubility of AuNP, facilitates cellular uptake and transcellular transport of rHDL-AuNP across endothelial cells. They are significant since they present rHDL bearing apoE3 as an effective platform for delivering AuNP across the BBB.

Identifying Iron Oxide Based Materials that Can Either Pass or Not Pass through the in vitro Blood-Brain Barrier

2014 ◽  
Vol 1621 ◽  
pp. 33-38
Author(s):  
Di Shi ◽  
Linlin Sun ◽  
Gujie Mi ◽  
Soumya Bhattacharya ◽  
Suprabha Nayar ◽  
...  
Keyword(s):  
Glutamic Acid ◽  
Blood Brain Barrier ◽  
Magnetic Nanomaterials ◽  
Brain Barrier ◽  
Hydrodynamic Diameter ◽  
Brain Drug Delivery ◽  
Blood Brain ◽  
Blood Brain Barrier Model ◽  
Barrier Model

ABSTRACTIn this study, an in vitro blood-brain barrier model was developed using murine brain endothelioma cells (b.End3 cells). By comparing the permeability of FITC-Dextran at increasing exposure times in serum-free medium to such values in the literature, we confirm that the blood-brain barrier model was successfully established. After such confirmation, the permeability of five ferrofluid (FF) nanoparticle samples, GGB (ferrofluid synthesized using glycine, glutamic acid and BSA), GGC (glycine, glutamic acid and collagen), GGP (glycine, glutamic acid and PVA), BPC (BSA, PEG and collagen) and CPB (collagen, PVA and BSA), was determined using this model. In addition, all the five FF samples were characterized by zeta potential to determine their charge as well as TEM and dynamic light scattering for determining their hydrodynamic diameter. Results showed that FF coated with collagen had better permeability to the blood-brain barrier than FF coated with glycine and glutamic acid based on an increase of 4.5% in permeability. Through such experiments, magnetic nanomaterials, such as ferrofluids, that are less permeable to the blood brain barrier can be used to decrease neural tissue toxicity and magnetic nanomaterials with more permeable to the blood-brain barrier can be used for brain drug delivery.

A multiparametric study of gold nanoparticles cytotoxicity, internalization and permeability using an in vitro model of blood–brain barrier. Influence of size, shape and capping agent

2019 ◽  
Vol 13 (7) ◽  
pp. 990-1004 ◽  
Author(s):  
Maria Enea ◽  
Miguel Peixoto de Almeida ◽  
Peter Eaton ◽  
Diana Dias da Silva ◽  
Eulália Pereira ◽  
...  
Keyword(s):  
Gold Nanoparticles ◽  
Blood Brain Barrier ◽  
In Vitro Model ◽  
Brain Barrier ◽  
Capping Agent ◽  
Blood Brain ◽  
Vitro Model

scholarly journals Gold nanocarriers for transport of oligonucleotides across brain endothelial cells

2020 ◽  
Author(s):  
Nayab Fatima ◽  
Radka Gromnicova ◽  
Jane Loughlin ◽  
Basil Sharrack ◽  
David Male
Keyword(s):  
Gold Nanoparticles ◽  
Endothelial Cells ◽  
Blood Brain Barrier ◽  
Brain Barrier ◽  
Target Cells ◽  
Brain Endothelium ◽  
Mobility Shift ◽  
Dna Oligonucleotides ◽  
Blood Brain

AbstractTreatment of diseases that affect the CNS by gene therapy requires delivery of oligonucleotides to target cells within the brain. As the blood brain barrier prevents movement of large biomolecules, current approaches involve direct injection of the oligonucleotides, which is invasive and may have only a localised effect. The aim of this study was to investigate the potential of 2 nm galactose-coated gold nanoparticles (NP-Gal) as a delivery system of oligonucleotides across brain endothelium.DNA oligonucleotides of different types were attached to NP-Gal by the place exchange reaction and were characterised by EMSA (electrophoretic mobility shift assay). Several nanoparticle formulations were created, with single-or double-stranded (20nt or 40nt) DNA oligonucleotides, or with different amounts of DNA attached to the carriers. These nanocarriers were applied to transwell cultures of human brain endothelium in vitro (hCMEC/D3 cell-line) or to a 3D-hydrogel model of the blood-brain barrier including astrocytes. Transfer rates were measured by quantitative electron microscopy for the nanoparticles and qPCR for DNA.Despite the increase in nanoparticle size caused by attachment of oligonucleotides to the NP-Gal carrier, the rates of endocytosis and transcytosis of nanoparticles were both considerably increased when they carried an oligonucleotide cargo. Carriers with 40nt dsDNA were most efficient, accumulating in vesicles, in the cytosol and beneath the basal membrane of the endothelium. The oligonucleotide cargo remained attached to the nanocarriers during transcytosis and the transport rate across the endothelial cells was increased at least 50fold compared with free DNA. The nanoparticles entered the extracellular matrix and were taken up by the astrocytes in biologically functional amounts.Attachment of DNA confers a strong negative charge to the nanoparticles which may explain the enhanced binding to the endothelium and transcytosis by both vesicular transport and the transmembrane/cytosol pathway. These gold nanoparticles have the potential to transport therapeutic amounts of nucleic acids into the CNS.

Vacuoles Induced in Mammalian Cells by Helicobacter Cytotoxin are Acidic Organelles

1990 ◽  
Vol 48 (3) ◽  
pp. 168-169
Author(s):  
M. H. Chestnut ◽  
C. E. Catrenich
Keyword(s):  
Acridine Orange ◽  
Mammalian Cells ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Ulcer Disease ◽  
Gastroduodenal Disease ◽  
H Pylori

Helicobacter pylori is a non-invasive, Gram-negative spiral bacterium first identified in 1983, and subsequently implicated in the pathogenesis of gastroduodenal disease including gastritis and peptic ulcer disease. Cytotoxic activity, manifested by intracytoplasmic vacuolation of mammalian cells in vitro, was identified in 55% of H. pylori strains examined. The vacuoles increase in number and size during extended incubation, resulting in vacuolar and cellular degeneration after 24 h to 48 h. Vacuolation of gastric epithelial cells is also observed in vivo during infection by H. pylori. A high molecular weight, heat labile protein is believed to be responsible for vacuolation and to significantly contribute to the development of gastroduodenal disease in humans. The mechanism by which the cytotoxin exerts its effect is unknown, as is the intracellular origin of the vacuolar membrane and contents. Acridine orange is a membrane-permeant weak base that initially accumulates in low-pH compartments. We have used acridine orange accumulation in conjunction with confocal laser scanning microscopy of toxin-treated cells to begin probing the nature and origin of these vacuoles.

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