Cell Culture Model Of Blood-brain Barrier Research Articles

Delivery of RNA to the Blood-Brain Barrier Endothelium Using Cationic Bicelles.

Blood-brain barrier (BBB) dysfunction is prevalent in Alzheimer's disease and other neurological disorders. Restoring normal BBB function through RNA therapy is a potential avenue for addressing cerebrovascular changes in these disorders that may lead to cognitive decline. Although lipid nanoparticles have been traditionally used as drug carriers for RNA, bicelles have been emerging as a better alternative because of their higher cellular uptake and superior transfection capabilities. Cationic bicelles composed of DPPC/DC7PC/DOTAP at molar ratios of 63.8/25.0/11.2 were evaluated for the delivery of RNA in polarized hCMEC/D3 monolayers, a widely used BBB cell culture model. RNA-bicelle complexes were formed at five N/P ratios (1:1 to 5:1) by a thin-film hydration method. The RNA-bicelle complexes at N/P ratios of 3:1 and 4:1 exhibited optimal particle characteristics for cellular delivery. The cellular uptake of cationic bicelles laced with 1 mol% DiI-C18 was confirmed by flow cytometry and confocal microscopy. The ability of cationic bicelles (N/P ratio 4:1) to transfect polarized hCMEC/D3 with FITC-labeled control siRNA was tested vis-a-vis commercially available Lipofectamine RNAiMAX. These studies demonstrated the higher transfection efficiency and greater potential of cationic bicelles for RNA delivery to the BBB endothelium.

Spermidine/Spermine N1-Acetyltransferase 1 (SAT1)-A Potential Gene Target for Selective Sensitization of Glioblastoma Cells Using an Ionizable Lipid Nanoparticle to Deliver siRNA.

Spermidine/spermine N1-acetyltransferase 1 (SAT1) responsible for cell polyamine catabolism is overexpressed in glioblastoma multiforme (GB). Its role in tumor survival and promoting resistance towards radiation therapy has made it an interesting target for therapy. In this study, we prepared a lipid nanoparticle-based siRNA delivery system (LNP-siSAT1) to selectively knockdown (KD) SAT1 enzyme in a human glioblastoma cell line. The LNP-siSAT1 containing ionizable DODAP lipid was prepared following a microfluidics mixing method and the resulting nanoparticles had a hydrodynamic size of around 80 nm and a neutral surface charge. The LNP-siSAT1 effectively knocked down the SAT1 expression in U251, LN229, and 42MGBA GB cells, and other brain-relevant endothelial (hCMEC/D3), astrocyte (HA) and macrophage (ANA-1) cells at the mRNA and protein levels. SAT1 KD in U251 cells resulted in a 40% loss in cell viability. Furthermore, SAT1 KD in U251, LN229 and 42MGBA cells sensitized them towards radiation and chemotherapy treatments. In contrast, despite similar SAT1 KD in other brain-relevant cells no significant effect on cytotoxic response, either alone or in combination, was observed. A major roadblock for brain therapeutics is their ability to cross the highly restrictive blood-brain barrier (BBB) presented by the brain microcapillary endothelial cells. Here, we used the BBB circumventing approach to enhance the delivery of LNP-siSAT1 across a BBB cell culture model. A cadherin binding peptide (ADTC5) was used to transiently open the BBB tight junctions to promote paracellular diffusion of LNP-siSAT1. These results suggest LNP-siSAT1 may provide a safe and effective method for reducing SAT1 and sensitizing GB cells to radiation and chemotherapeutic agents.

Distinct Uptake Kinetics of Alzheimer Disease Amyloid-β 40 and 42 at the Blood-Brain Barrier Endothelium.

Blood-brain barrier (BBB) endothelial cells lining the cerebral microvasculature maintain dynamic equilibrium between soluble amyloid-β (Aβ) levels in the brain and plasma. The BBB dysfunction prevalent in Alzheimer disease contributes to the dysregulation of plasma and brain Aβ and leads to the perturbation of the ratio between Aβ42 and Aβ40, the two most prevalent Aβ isoforms in patients with Alzheimer disease. We hypothesize that BBB endothelium distinguishes between Aβ40 and Aβ42, distinctly modulates their trafficking kinetics between plasma and brain, and thereby contributes to the maintenance of healthy Aβ42/Aβ40 ratios. To test this hypothesis, we investigated Aβ40 and Aβ42 trafficking kinetics in hCMEC/D3 monolayers (human BBB cell culture model) in vitro as well as in mice in vivo. Although the rates of uptake of fluorescein-labeled Aβ40 and Aβ42 (F-Aβ40 and F-Aβ42) were not significantly different on the abluminal side, the luminal uptake rate of F-Aβ42 was substantially higher than F-Aβ40. Since higher plasma Aβ levels were shown to aggravate BBB dysfunction and trigger cerebrovascular disease, we systematically investigated the dynamic interactions of luminal [125I]Aβ peptides and their trafficking kinetics at BBB using single-photon emission computed tomography/computed tomography imaging in mice. Quantitative modeling of the dynamic imaging data thus obtained showed that the rate of uptake of toxic [125I]Aβ42 and its subsequent BBB transcytosis is significantly higher than [125I]Aβ40. It is likely that the molecular mechanisms underlying these kinetic differences are differentially affected in Alzheimer and cerebrovascular diseases, impact plasma and brain levels of Aβ40 and Aβ42, engender shifts in the Aβ42/Aβ40 ratio, and unleash downstream toxic effects. SIGNIFICANCE STATEMENT: Dissecting the binding and uptake kinetics of Aβ40 and Aβ42 at the BBB endothelium will facilitate the estimation of Aβ40 versus Aβ42 exposure to the BBB endothelium and allow assessment of the risk of BBB dysfunction by monitoring Aβ42 and Aβ40 levels in plasma. This knowledge, in turn, will aid in elucidating the role of these predominant Aβ isoforms in aggravating BBB dysfunction and cerebrovascular disease.

Abstract 1721: Hypoxia penetrating ultra-small nanoparticle for anti-cancer effect in Glioblastoma

Abstract Glioblastoma multiforme (GBM) is a lethal brain tumor currently without successful treatment options. Chemotherapeutic agents are the frontline in the management of GBM. Nonetheless, accessibility of the brain tumor is greatly impeded by the poor blood-brain barrier (BBB) penetration of drugs, which needs immediate attention. Ultra-small multifunctional nanoparticle offers a great promise in overcoming the delivery barriers to GBM. The primary goal of our research is to develop BBB targeted nanoparticle that can selectively deliver drugs to GBM and trigger cell death. We developed Anti-GBM drug, such as temozolomide (TMZ) encapsulated biocompatible, ultra-small nanoparticle using Lactoferrin (LAC) protein. Further, we chemically conjugated LAC with tumor hypoxia biomarker homing molecule, Acetazolamide (ATZ) to obtain LAC-ATZ nanoparticle. To validate our hypothesis of hypoxia targeting, we evaluated the carbonic anhydrase-IX (CAIX) receptor expression in human brain tumor section. The IHC data demonstrated significant overexpression of CAIX in tumor tissue compared to healthy human brain tissue section. Literatures have reported overexpression of LRP-1 in endothelial blood vessel of GBM tumor. Thus, use of LAC-ATZ will enhance its penetration across the BBB and delivery of TMZ in hypoxic core of GBM. To achieve the effective BBB penetration, we engineered LAC-ATZ@TMZ nanoparticle that demonstrated homogenous ultra-small particle size of average diameter of 20-30 nm using TEM analysis. The SDS-PAGE analysis demonstrated the conjugation of LAC with ATZ. Our preliminary data in GBM cells, U87MG suggested the dose dependent cell uptake of DiD-fluorescence labeled LAC-ATZ nanoparticle. The nanoparticles are selective in targeting the U87MG cell when compared to healthy microglial BV2 cells. Thus, it is supporting the potential of LAC-ATZ nanoparticle for targeting the GBM tumor with limited effect in healthy brain tissue. We developed conditioned BBB cell culture model to demonstrate the penetration of LAC-ATZ across the BBB. Based on the exciting preliminary outcome in this study builds a strong rational of using LAC-ATZ nanoparticle for overcoming the bottle neck barrier of BBB with improved anti-GBM response. Keywords: Glioblastoma multiforme (GBM), blood-brain barrier (BBB), Lactoferrin (LAC), Acetazolamide (ATZ) Citation Format: Mohd A. Rauf, Katyayani Tatiparti, Samaresh Sau, Arun K. Iyer. Hypoxia penetrating ultra-small nanoparticle for anti-cancer effect in Glioblastoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1721.

PH responsive granulocyte colony-stimulating factor variants with implications for treating Alzheimer's disease and other central nervous system disorders.

Systemic injection of granulocyte colony-stimulating factor (G-CSF) has yielded encouraging results in treating Alzheimer's Disease (AD) and other central nervous system (CNS) disorders. Making G-CSF a viable AD therapeutic will, however, require increasing G-CSF's ability to stimulate neurons within the brain. This objective could be realized by increasing transcytosis of G-CSF across the blood brain barrier (BBB). An established correlation between G-CSF receptor (G-CSFR) binding pH responsiveness and increased recycling of G-CSF to the cell exterior after endocytosis motivated development of G-CSF variants with highly pH responsive G-CSFR binding affinities. These variants will be used in future validation of our hypothesis that increased BBB transcytosis can enhance G-CSF therapeutic efficacy. Flow cytometric screening of a yeast-displayed library in which G-CSF/G-CSFR interface residues were mutated to histidine yielded a G-CSF triple His mutant (L109H/D110H/Q120H) with highly pH responsive binding affinity. This variant's KD, measured by surface plasmon resonance (SPR), increases ∼20-fold as pH decreases from 7.4 to below histidine's pKa of ∼6.0; an increase 2-fold greater than for previously reported G-CSF His mutants. Cell-free protein synthesis (CFPS) enabled expression and purification of soluble, bioactive G-CSF triple His variant protein, an outcome inaccessible via Escherichia coli inclusion body refolding. This purification and bioactivity validation will enable future identification of correlations between pH responsiveness and transcytosis in BBB cell culture model and animal experiments. Furthermore, the library screening and CFPS methods employed here could be applied to developing other pH responsive hematopoietic or neurotrophic factors for treating CNS disorders.

Comparison of Five Peptide Vectors for Improved Brain Delivery of the Lysosomal Enzyme Arylsulfatase A

Enzyme replacement therapy (ERT) is a treatment option for lysosomal storage disorders (LSDs) caused by deficiencies of soluble lysosomal enzymes. ERT depends on receptor-mediated transport of intravenously injected recombinant enzyme to lysosomes of patient cells. The blood-brain barrier (BBB) prevents efficient transfer of therapeutic polypeptides from the blood to the brain parenchyma and thus hinders effective treatment of LSDs with CNS involvement. We compared the potential of five brain-targeting peptides to promote brain delivery of the lysosomal enzyme arylsulfatase A (ASA). Fusion proteins between ASA and the protein transduction domain of the human immunodeficiency virus TAT protein (Tat), an Angiopep peptide (Ang-2), and the receptor-binding domains of human apolipoprotein B (ApoB) and ApoE (two versions, ApoE-I and ApoE-II) were generated. All ASA fusion proteins were enzymatically active and targeted to lysosomes when added to cultured cells. In contrast to wild-type ASA, which is taken up by mannose-6-phosphate receptors, all chimeric proteins were additionally endocytosed via mannose-6-phosphate-independent routes. For ASA-Ang-2, ASA-ApoE-I, and ASA-ApoE-II, uptake was partially due to the low-density lipoprotein receptor-related protein 1. Transendothelial transfer in a BBB cell culture model was elevated for ASA-ApoB, ASA-ApoE-I, and ASA-ApoE-II. Brain delivery was, however, increased only for ASA-ApoE-II. ApoE-II was also superior to wild-type ASA in reducing lysosomal storage in the CNS of ASA-knock-out mice treated by ERT. Therefore, the ApoE-derived peptide appears useful to treat metachromatic leukodystrophy and possibly other neurological disorders more efficiently.

Metalloproteinase mediated occludin cleavage in the cerebral microcapillary endothelium under pathological conditions

Reactive oxygen species (ROS) as well as matrix metalloproteinases (MMPs) induce modifications in the tight junction (TJ) protein occludin which is crucial for the blood–brain barrier (BBB) function. We investigated the role of ROS and MMPs in endothelial autoregulatory response on oxidative stress with respect to occludin and the BBB integrity. The ROS hydrogen peroxide (H 2O 2) was applied to our well-established BBB cell culture model based on primary porcine brain capillary endothelial cells (PBCEC). At low concentrations (2.5 mM), H 2O 2-induced barrier impairment correlated with an altered occludin phosphorylation. At high, cell toxic H 2O 2 concentrations (≥ 10 mM) occludin cleavage occurred and elevated levels of active MMP-2 were detected. Under those conditions intercellular gaps were formed within the monolayer visualizing the barrier breakdown also determined by impedance analysis. A primary structure sequence analysis revealed potential type IV collagenase sensitive motives in the first extracellular loop, thus providing evidence that occludin might be an MMP-2 substrate. MMP inhibition by the metalloproteinase inhibitor GM6001 prevented occludin degradation and reduced the intercellular gap formation. However, the barrier function quantified by impedance measurement could not be maintained despite MMP inhibition. When we applied an enzymatic activity level which caused occludin cleavage in injured PBCEC to intact PBCEC, neither occludin cleavage nor barrier impairment was observed. Thus, in our model occludin cleavage is not an autoregulatory mechanism of microcapillary endothelium in barrier modulation under oxidative stress, but only occurs upon endothelial damage.

Role of the purinergic system in controlling blood-brain barrier functions upon ischemic conditions: focus on ATP-metabolizing enzymes

Event Abstract Back to Event Role of the purinergic system in controlling blood-brain barrier functions upon ischemic conditions: focus on ATP-metabolizing enzymes Stefania Ceruti1*, Laura Colombo1, Giuila Magni1, Francesca Vigano1, Zsófia Gazdag2, Marta Boccazzi1, Beáta Sperlágh2, Mária Deli3, Maria P. Abbracchio1 and Ágnes Kittel2 1 Università degli Studi di Milano, Department of Pharmacological Sciences, Italy 2 Hungarian Academy of Science, Institute of Experimental Medicine, Hungary 3 Hungarian Academy of Science, Institute of Biophysics, Biological Research Center, Hungary The blood-brain barrier (BBB) is composed of endothelial cells, pericytes and astrocytes, and serves as interface between the blood flow and the central nervous system (CNS). Since BBB deregulation plays important role in the pathogenesis of several CNS diseases spanning from brain tumors to stroke, understanding the molecular mechanisms controlling BBB functions might help unveiling new therapeutic targets to brain pathologies. Extracellular nucleotides are important signaling molecules both in physiological and pathological conditions. Their actions are mediated by 7 ionotropic P2X and 8 metabotropic P2Y purinergic receptors, and terminated by metabolizing enzymes, namely ectonucleotidases (NTPDases) and 5’-nucleotidase. To date the role of purinergic transmission in controlling BBB functions is not fully understood. Therefore, we used a new in vitro cell culture model of BBB to investigate the expression and distribution of NTPDases and P2Y receptors either in control conditions or following exposure to ischemia. RT-PCR analysis showed that astrocytes and pericytes expressed all the cloned P2Y receptors, that endothelial cells showed only the P2Y1,2,4 subtypes, and that NTPDase1 and 2 were expressed by all the three types of cell. Application of oxygen-glucose deprivation (OGD), which mimics cytotoxicity induced by ischemia in vivo, showed that endothelial cells were extremely susceptible to cell death, whereas astrocytes and pericytes were more resistant. A semi-quantitative assay highlighted increased ecto-ATPase activity following exposure to OGD in the three types of cell, both when grown separately and in triple co-culture. Our data show the usefulness of this new in vitro model to demonstrate a role for extracellular nucleotides in modulating BBB responses to ischemic events, and to determine if the purinergic system could represent a new target for the development of effective therapies to brain pathologies. Conference: IBRO International Workshop 2010, Pécs, Hungary, 21 Jan - 23 Jan, 2010. Presentation Type: Poster Presentation Topic: Disorders of the nervous system Citation: Ceruti S, Colombo L, Magni G, Vigano F, Gazdag Z, Boccazzi M, Sperlágh B, Deli M, Abbracchio MP and Kittel Á (2010). Role of the purinergic system in controlling blood-brain barrier functions upon ischemic conditions: focus on ATP-metabolizing enzymes. Front. Neurosci. Conference Abstract: IBRO International Workshop 2010. doi: 10.3389/conf.fnins.2010.10.00030 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 19 Apr 2010; Published Online: 19 Apr 2010. * Correspondence: Stefania Ceruti, Università degli Studi di Milano, Department of Pharmacological Sciences, Milan, Italy, stefania.ceruti@unimi.it Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Stefania Ceruti Laura Colombo Giuila Magni Francesca Vigano Zsófia Gazdag Marta Boccazzi Beáta Sperlágh Mária Deli Maria P Abbracchio Ágnes Kittel Google Stefania Ceruti Laura Colombo Giuila Magni Francesca Vigano Zsófia Gazdag Marta Boccazzi Beáta Sperlágh Mária Deli Maria P Abbracchio Ágnes Kittel Google Scholar Stefania Ceruti Laura Colombo Giuila Magni Francesca Vigano Zsófia Gazdag Marta Boccazzi Beáta Sperlágh Mária Deli Maria P Abbracchio Ágnes Kittel PubMed Stefania Ceruti Laura Colombo Giuila Magni Francesca Vigano Zsófia Gazdag Marta Boccazzi Beáta Sperlágh Mária Deli Maria P Abbracchio Ágnes Kittel Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.