Porcine Brain Capillary Endothelial Cells Research Articles

Astaxanthin enhances autophagy, amyloid beta clearance and exerts anti-inflammatory effects in in vitro models of Alzheimer’s disease-related blood brain barrier dysfunction and inflammation

Defective degradation and clearance of amyloid-β as well as inflammation per se are crucial players in the pathology of Alzheimer’s disease (AD). A defective transport across the blood–brain barrier is causative for amyloid-β (Aβ) accumulation in the brain, provoking amyloid plaque formation. Using primary porcine brain capillary endothelial cells and murine organotypic hippocampal slice cultures as in vitro models of AD, we investigated the effects of the antioxidant astaxanthin (ASX) on Aβ clearance and neuroinflammation. We report that ASX enhanced the clearance of misfolded proteins in primary porcine brain capillary endothelial cells by inducing autophagy and altered the Aβ processing pathway. We observed a reduction in the expression levels of intracellular and secreted amyloid precursor protein/Aβ accompanied by an increase in ABC transporters ABCA1, ABCG1 as well as low density lipoprotein receptor-related protein 1 mRNA levels. Furthermore, ASX treatment increased autophagic flux as evidenced by increased lipidation of LC3B-II as well as reduced protein expression of phosphorylated S6 ribosomal protein and mTOR. In LPS-stimulated brain slices, ASX exerted anti-inflammatory effects by reducing the secretion of inflammatory cytokines while shifting microglia polarization from M1 to M2 phenotype. Our data suggest ASX as potential therapeutic compound ameliorating AD-related blood brain barrier impairment and inflammation.

Se supplementation to an in vitro blood-brain barrier does not affect Cu transfer into the brain

BackgroundDyshomeostasis of copper (Cu) accompanied by Cu accumulation in certain brain areas has been associated with neurodegenerative diseases. One proposed toxic mode of action following Cu overload is oxidative stress associated with neuronal damage, whereas Selenium (Se) is assumed to play here a protective role. This study investigates the relationship between adequate Se supplementation and the respective consequences for Cu transfer into the brain applying an in vitro model of the blood-brain barrier (BBB). MethodsPrimary porcine brain capillary endothelial cells (PBCECs) seeded on Transwell® inserts were supplemented with selenite starting at cultivation in both compartments. After apical application of 15 or 50 µM CuSO4, transfer of Cu to the basolateral compartment, the brain facing side, was assessed by ICP-MS/MS. ResultsIncubation with Cu did not negatively affect the barrier properties, whereas Se had a positive effect. Additionally, Se status improved after selenite supplementation. Transfer of Cu was not affected by selenite supplementation. Under Se-deficient conditions, Cu permeability coefficients decreased with increasing Cu concentrations. ConclusionThe results of this study do not indicate that under suboptimal Se supplementation more Cu transfers across the BBB to the brain.

Permeability of dopamine D2 receptor agonist hordenine across the intestinal and blood-brain barrier in vitro.

Hordenine, a bioactive food compound, has several pharmacological properties and has recently been identified as a dopamine D2 receptor (D2R) agonist. Since the pharmacokinetic profile of hordenine has been described to a limited extent, the present study focused on the transfer and transport of hordenine across the intestinal epithelium and the blood-brain barrier (BBB) in vitro. Hordenine was quickly transferred through the Caco-2 monolayer in only a few hours, indicating a rapid oral uptake. However, the high bioavailability may be reduced by the observed efflux transport of hordenine from the bloodstream back into the intestinal lumen and by first pass metabolism in intestinal epithelial cells. To determine the biotransformation rate of hordenine, the metabolite hordenine sulfate was synthesized as reference standard for analytical purposes. In addition, transfer studies using primary porcine brain capillary endothelial cells (PBCEC) showed that hordenine is able to rapidly penetrate the BBB and potentially accumulate in the brain. Thus, a D2R interaction of hordenine and activation of dopaminergic signaling is conceivable, assuming that the intestinal barrier can be circumvented by a route of administration alternative to oral uptake.

Bis-choline tetrathiomolybdate prevents copper-induced blood-brain barrier damage.

In Wilson disease, excessive copper accumulates in patients' livers and may, upon serum leakage, severely affect the brain according to current viewpoints. Present remedies aim at avoiding copper toxicity by chelation, for example, by D-penicillamine (DPA) or bis-choline tetrathiomolybdate (ALXN1840), the latter with a very high copper affinity. Hence, ALXN1840 may potentially avoid neurological deterioration that frequently occurs upon DPA treatment. As the etiology of such worsening is unclear, we reasoned that copper loosely bound to albumin, that is, mimicking a potential liver copper leakage into blood, may damage cells that constitute the blood-brain barrier, which was found to be the case in an in vitro model using primary porcine brain capillary endothelial cells. Such blood-brain barrier damage was avoided by ALXN1840, plausibly due to firm protein embedding of the chelator bound copper, but not by DPA. Mitochondrial protection was observed, a prerequisite for blood-brain barrier integrity. Thus, high-affinity copper chelators may minimize such deterioration in the treatment of neurologic Wilson disease.

Efflux at the Blood-Brain Barrier Reduces the Cerebral Exposure to Ochratoxin A, Ochratoxin α, Citrinin and Dihydrocitrinone.

Recent studies have implied that environmental toxins, such as mycotoxins, are risk factors for neurodegenerative diseases. To act directly as neurotoxins, mycotoxins need to penetrate or affect the integrity of the blood-brain barrier, which protects the mammalian brain from potentially harmful substances. As common food and feed contaminants of fungal origin, the interest in the potential neurotoxicity of ochratoxin A, citrinin and their metabolites has recently increased. Primary porcine brain capillary endothelial cells were used to investigate cytotoxic or barrier-weakening effects of ochratoxin A, ochratoxin α, citrinin and dihydrocitrinone. The transfer and transport properties of the mycotoxins across the barrier formed by porcine brain capillary endothelial cell monolayers were analysed using HPLC-MS/MS. High levels of Ochratoxin A caused cytotoxic and barrier-weakening effects, whereas ochratoxin α, citrinin and dihydrocitrinone showed no adverse effects up to 10 µM. Likely due to efflux transporter proteins, the transfer to the brain compartment was much slower than expected from their high lipophilicity. Due to their slow transfer across the blood-brain barrier, cerebral exposure of ochratoxin A, ochratoxin α, citrinin and dihydrocitrinone is low and neurotoxicity is likely to play a subordinate role in their toxicity at common physiological concentrations.

The blood-brain barrier studied in vitro across species.

The blood-brain barrier (BBB) is formed by brain capillary endothelial cells (BECs) supported by pericytes and astrocytes. The BBB maintains homeostasis and protects the brain against toxic substances circulating in the blood, meaning that only a few drugs can pass the BBB. Thus, for drug screening, understanding cell interactions, and pathology, in vitro BBB models have been developed using BECs from various animal sources. When comparing models of different species, differences exist especially in regards to the transendothelial electrical resistance (TEER). Thus, we compared primary mice, rat, and porcine BECs (mBECs, rBECs, and pBECs) cultured in mono- and co-culture with astrocytes, to identify species-dependent differences that could explain the variations in TEER and aid to the selection of models for future BBB studies. The BBB models based on primary mBECs, rBECs, and pBECs were evaluated and compared in regards to major BBB characteristics. The barrier integrity was evaluated by the expression of tight junction proteins and measurements of TEER and apparent permeability (Papp). Additionally, the cell size, the functionality of the P-glycoprotein (P-gp) efflux transporter, and the expression of the transferrin receptor were evaluated and compared. Expression and organization of tight junction proteins were in all three species influenced by co-culturing, supporting the findings, that TEER increases after co-culturing with astrocytes. All models had functional polarised P-gp efflux transporters and expressed the transferrin receptor. The most interesting discovery was that even though the pBECs had higher TEER than rBECs and mBECs, the Papp did not show the same variation between species, which could be explained by a significantly larger cell size of pBECs. In conclusion, our results imply that the choice of species for a given BBB study should be defined from its purpose, instead of aiming to reach the highest TEER, as the models studied here revealed similar BBB properties.

Capabilities of selenoneine to cross the in vitro blood-brain barrier model.

The naturally occurring selenoneine (SeN), the selenium analogue of the sulfur-containing antioxidant ergothioneine, can be found in high abundance in several marine fish species. However, data on biological properties of SeN and its relevance for human health are still scarce. This study aims to investigate the transfer and presystemic metabolism of SeN in a well-established in vitro model of the blood-brain barrier (BBB). Therefore, SeN and the reference Se species selenite and Se-methylselenocysteine (MeSeCys) were applied to primary porcine brain capillary endothelial cells (PBCECs). Se content of culture media and cell lysates was measured via ICP-MS/MS. Speciation analysis was conducted by HPLC-ICP-MS. Barrier integrity was shown to be unaffected during transfer experiments. SeN demonstrated the lowest transfer rates and permeability coefficient (6.7× 10-7cm s-1) in comparison to selenite and MeSeCys. No side-directed accumulation was observed after both-sided application of SeN. However, concentration-dependent transfer of SeN indicated possible presence of transporters on both sides of the barrier. Speciation analysis demonstrated no methylation of SeN by the PBCECs. Several derivatives of SeN detected in the media of the BBB model were also found in cell-free media containing SeN and hence not considered to be true metabolites of the PBCECs. In concluding, SeN is likely to have a slow transfer rate to the brain and not being metabolized by the brain endothelial cells. Since this study demonstrates that SeN may reach the brain tissue, further studies are needed to investigate possible health-promoting effects of SeN in humans.

Lipoprotein imitating nanoparticles: Lecithin coating binds ApoE and mediates non-lysosomal uptake leading to transcytosis over the blood-brain barrier.

Lipoproteins are naturally occurring nano sized transport vehicles in the human body. Therefore, lipoproteins could be applied as a drug carrier system. Additionally, several reports of apolipoprotein mediated blood-brain barrier (BBB) crossing suggest lipoprotein mimicking nanoparticles (NPs) as possible drug delivery vehicles to the brain. This could extend the therapy opportunities of various diseases of the central nervous system. A lipoprotein imitating NP system, consisting of a lecithin coated lipophilic cholesteryl oleate core with embedded fluorescent dye and adsorbed apolipoprotein E3 (ApoE) has been established using a two-step solvent injection method. Lecithin coating was proven to stabilize the NPs in isotonic saline solution and to bind ApoE in a highly efficient way. Fluorescent dye load (as model drug) and ApoE amount were varied, obtaining 100nm sized, monodisperse NPs. The NPs' interaction with the BBB formed by primary porcine brain capillary endothelial cells (PBCEC) was investigated by fluorescence microscopy observing that ApoE mediated a lysosome bypassing uptake mechanism. Using this in vitro BBB model, ApoE concentration dependent permeation over the cell layer could be proven in both directions. An ApoE mediated transcytosis could be achieved, as it had been observed earlier for low-density lipoproteins. These results show that the newly developed NP system successfully mimics endogenous lipoproteins. An ApoE dependent penetration of the BBB was confirmed and provided an indication of apolipoprotein mediated transcytosis, avoiding lysosomal degradation.

Lactoferrin-Loaded PEG/PLA Block Copolymer Targeted With Anti-Transferrin Receptor Antibodies for Alzheimer Disease.

Last few years, struggles have been reported to develop the nanovesicles for drug delivery via the brain–blood barrier (BBB). Novel drugs, for instance, iAβ5, are efficient to inhibit the aggregates connected to the treatment of Alzheimer disease and are being evaluated, but most of the reports reflect some drawbacks of the drugs to reach the brain in preferred concentrations owing to the less BBB penetrability of the surface dimensions. In this report, we designed and developed a new approach to enhance the transport of drug via BBB, constructed with lactoferrin (Lf)-coated polyethylene glycol-polylactide nanoparticles (Lf-PPN) with superficial monoclonal antibody-functionalized antitransferrin receptor and anti-Aβ to deliver the iAβ5 hooked on the brain. The porcine brain capillary endothelial cells were utilized as BBB typically to examine the framework efficacy and toxicity. The cellular uptake of the immuno-nanoparticles with measured conveyance of the iAβ5 peptide was significantly enhanced and associated with Lf-PPN without monoclonal antibody functionalizations.

Astaxanthin exerts protective effects similar to bexarotene in Alzheimer's disease by modulating amyloid-beta and cholesterol homeostasis in blood-brain barrier endothelial cells

The pathogenesis of Alzheimer's disease (AD) is characterized by overproduction, impaired clearance, and deposition of amyloid-β peptides (Aβ) and connected to cholesterol homeostasis. Since the blood-brain barrier (BBB) is involved in these processes, we investigated effects of the retinoid X receptor agonist, bexarotene (Bex), and the peroxisome proliferator-activated receptor α agonist and antioxidant, astaxanthin (Asx), on pathways of cellular cholesterol metabolism, amyloid precursor protein processing/Aβ production and transfer at the BBB in vitro using primary porcine brain capillary endothelial cells (pBCEC), and in 3xTg AD mice. Asx/Bex downregulated transcription/activity of amyloidogenic BACE1 and reduced Aβ oligomers and ~80 kDa intracellular 6E10-reactive APP/Aβ species, while upregulating non-amyloidogenic ADAM10 and soluble (s)APPα production in pBCEC. Asx/Bex enhanced Aβ clearance to the apical/plasma compartment of the in vitro BBB model. Asx/Bex increased expression levels of ABCA1, LRP1, and/or APOA-I. Asx/Bex promoted cholesterol efflux, partly via PPARα/RXR activation, while cholesterol biosynthesis/esterification was suppressed. Silencing of LRP-1 or inhibition of ABCA1 by probucol reversed Asx/Bex-mediated effects on levels of APP/Aβ species in pBCEC. Murine (m)BCEC isolated from 3xTg AD mice treated with Bex revealed elevated expression of APOE and ABCA1. Asx/Bex reduced BACE1 and increased LRP-1 expression in mBCEC from 3xTg AD mice when compared to vehicle-treated or non-Tg treated mice. In parallel, Asx/Bex reduced levels of Aβ oligomers in mBCEC and Aβ species in brain soluble and insoluble fractions of 3xTg AD mice. Our results suggest that both agonists exert beneficial effects at the BBB by balancing cholesterol homeostasis and enhancing clearance of Aβ from cerebrovascular endothelial cells.

Monocultures of primary porcine brain capillary endothelial cells: Still a functional in vitro model for the blood-brain-barrier

The main obstacle for the treatment of brain diseases is the restriction of the passage of pharmaceuticals across the blood-brain barrier. Endothelial cells line up the cerebral micro vessels and prevent the uncontrolled transfer of polar substances by intercellular tight junctions. In addition to this physical barrier, active transporters of the multi-drug-resistance prevent the passage of hydrophobic substances by refluxing them back to the blood stream. This paper reviews the development and selected applications of an in vitro porcine brain derived primary cell culture system established in the authors lab that closely resembles the BBB in vivo and could thus be used to study beyond other applications drug delivery to the brain. An essential technique to control the intactness or destruction of the barrier, the impedance spectroscopy, will be introduced. It will be shown that nanoparticles can cross the blood brain barrier by two mechanisms: opening the tight junctions and thus allowing parallel import of substances into the brain as well as receptor mediated endocytosis using brain specific target molecules. However cytotoxic effects have to be considered as well which beside standard cytotoxicity assays could be also determined by impedance technology. Moreover it will be shown that enzymes e.g. for enzyme replacement therapy could be transferred across the barrier by proper tuning or chemical modification of the enzyme. Since this review is based on a conference presentation it will mainly focus on applications of the monoculture system developed in the authors lab which under given culture conditions is useful due to its easy availability, robustness, good reproducibility and also due to its simplicity. Improvements of this model made by other groups will be acknowledged but not discussed here in detail.

Transport of enniatin B and enniatin B1 across the blood-brain barrier and hints for neurotoxic effects in cerebral cells.

Enniatins are common contaminants of food and feed and belong to the group of the “emerging” mycotoxins, which are produced by various Fusarium species. Although a wide range of toxic effects, like antibacterial, antifungal, insecticidal and cytotoxic properties, have been described in vitro, so far, no cases of mycotoxicosis connected to enniatins in vivo are reported. Among this group of mycotoxins, enniatin B and enniatin B1 are the most prevalent compounds and therefore are present in the human diet. Enniatins can reach systemic circulation, thus, the investigation of possible neurotoxic effects is of importance. Different cerebral cells were used to address effects on cell death having an impact on the blood-brain barrier. The influence of enniatin B and enniatin B1 on cellular viability was examined via Cell Counting kit-8 assay (CCK-8) in three different cell types of the blood-brain barrier: porcine brain capillary endothelial cells (PBCEC), human brain microvascular endothelial cells (HBMEC) and human astrocytoma cells (CCF-STTG1). CCF-STTG1 cells were more sensitive to enniatin B (IC50 = 8.9 μM) and enniatin B1 (IC50 = 4.4 μM) than both endothelial cell types. In CCF-STTG1 cells, caspase-3 activation and lactate dehydrogenase (LDH) release were evaluated. Both compounds did not induce any LDH release and only enniatin B increased caspase-3 activity as a marker for apoptosis. The transport kinetics of enniatin B and enniatin B1 across the blood-brain barrier in vitro were evaluated using PBCEC, cultivated on Transwell® filter inserts. Analysis of the apical and the basolateral compartment by high performance liquid chromatography-mass spectrometry revealed high influx rates for enniatin B and enniatin B1. Thus, both compounds can reach the brain parenchyma where neurotoxic effects cannot be ruled out.

Effects of arsenolipids on in vitro blood-brain barrier model.

Arsenic-containing hydrocarbons (AsHCs), a subgroup of arsenolipids (AsLs) occurring in fish and edible algae, possess a substantial neurotoxic potential in fully differentiated human brain cells. Previous in vivo studies indicating that AsHCs cross the blood-brain barrier of the fruit fly Drosophila melanogaster raised the question whether AsLs could also cross the vertebrate blood-brain barrier (BBB). In the present study, we investigated the impact of several representatives of AsLs (AsHC 332, AsHC 360, AsHC 444, and two arsenic-containing fatty acids, AsFA 362 and AsFA 388) as well as of their metabolites (thio/oxo-dimethylpropionic acid, dimethylarsinic acid) on porcine brain capillary endothelial cells (PBCECs, in vitro model for the blood-brain barrier). AsHCs exerted the strongest cytotoxic effects of all investigated arsenicals as they were up to fivefold more potent than the toxic reference species arsenite (iAsIII). In our in vitro BBB-model, we observed a slight transfer of AsHC 332 across the BBB after 6h at concentrations that do not affect the barrier integrity. Furthermore, incubation with AsHCs for 72h led to a disruption of the barrier at sub-cytotoxic concentrations. The subsequent immunocytochemical staining of three tight junction proteins revealed a significant impact on the cell membrane. Because AsHCs enhance the permeability of the in vitro blood-brain barrier, a similar behavior in an in vivo system cannot be excluded. Consequently, AsHCs might facilitate the transfer of accompanying foodborne toxicants into the brain.

Implications of cerebrovascular ATP-binding cassette transporter G1 (ABCG1) and apolipoprotein M in cholesterol transport at the blood-brain barrier

Impaired cholesterol/lipoprotein metabolism is linked to neurodegenerative diseases such as Alzheimer's disease (AD). Cerebral cholesterol homeostasis is maintained by the highly efficient blood-brain barrier (BBB) and flux of the oxysterols 24(S)-hydroxycholesterol and 27-hydroxycholesterol, potent liver-X-receptor (LXR) activators. HDL and their apolipoproteins are crucial for cerebral lipid transfer, and loss of ATP binding cassette transporters (ABC)G1 and G4 results in toxic accumulation of oxysterols in the brain. The HDL-associated apolipoprotein (apo)M is positively correlated with pre-β HDL formation in plasma; its presence and function in the brain was thus far unknown. Using an in vitro model of the BBB, we examined expression, regulation, and functions of ABCG1, ABCG4, and apoM in primary porcine brain capillary endothelial cells (pBCEC). RT Q-PCR analyses and immunoblotting revealed that in addition to ABCA1 and scavenger receptor, class B, type I (SR-BI), pBCEC express high levels of ABCG1, which was up-regulated by LXR activation. Immunofluorescent staining, site-specific biotinylation and immunoprecipitation revealed that ABCG1 is localized both to early and late endosomes and on apical and basolateral plasma membranes. Using siRNA interference to silence ABCG1 (by 50%) reduced HDL-mediated [3H]-cholesterol efflux (by 50%) but did not reduce [3H]-24(S)-hydroxycholesterol efflux. In addition to apoA-I, pBCEC express and secrete apoM mainly to the basolateral (brain) compartment. HDL enhanced expression and secretion of apoM by pBCEC, apoM-enriched HDL promoted cellular cholesterol efflux more efficiently than apoM-free HDL, while apoM-silencing diminished cellular cholesterol release. We suggest that ABCG1 and apoM are centrally involved in regulation of cholesterol metabolism/turnover at the BBB.

Cellular uptake of PLGA nanoparticles targeted with anti-amyloid and anti-transferrin receptor antibodies for Alzheimer’s disease treatment

During the last few decades, relevant efforts have been reported to design nanocarriers for drug transport through the blood brain barrier (BBB). New drugs, such as peptide iAβ5, capable to inhibit the aggregates associated with Alzheimeŕs disease (AD) are being tested but the most frequent drawback is to reach the brain in the desired concentrations due to the low BBB permeability-surface area. Our approach, as a proof of concept to improve drug transport through the BBB, is based on poly(lactic-co-glycolic acid) (PLGA) nanoparticles with surface functionalized with anti-transferrin receptor monoclonal antibody (OX26) and anti-Aβ (DE2B4) to deliver encapsulated iAβ5 into the brain. Porcine brain capillary endothelial cells (PBCECs) were used as a BBB model to evaluate the system efficacy and toxicity. The uptake of immune nanoparticles with a controlled delivery of the peptide iAβ5 was substantially increased compared to the nanoparticles (NPs) without monoclonal antibody functionalization.

Blood-brain barrier properties in vitro depend on composition and assembly of endogenous extracellular matrices.

Brain capillary endothelial cells, which constitute the blood-brain barrier (BBB), are enveloped by the extracellular matrix (ECM) produced by endothelial cells, pericytes and astrocytes. The contribution of matrix components secreted by the various cell types at the neurovascular unit, however, remains unclear with respect to their effect on endothelial barrier function. In this study, a new in vitro model was established by growing endothelial cells on an ECM produced by pericytes, astrocytes or a serial combination of both. The last-mentioned was found to be more in vivo-like. We investigated the role of the composition and morphology of ECM supra-structures in maintaining BBB function. The composition was analysed by protein analysis (enzyme-linked immunosorbent assay) and the ultrastructure of generated matrices was analysed by transmission electron microscopy including immunogold labelling. We could show by electric cell-substrate impedance sensing measurements that pericytes and combined matrices significantly improved the barrier tightness of porcine brain capillary endothelial cells (PBCEC). The increase of the resistance was verified by enhanced expression of tight junction proteins. Thus, for the first time, we have shown that barrier integrity is strictly controlled by the ECM, which is a product of all cells involved in the secretion of ECM components and their modification by corresponding cells. Moreover, we have demonstrated that complex matrices by the various cells of the BBB induce barrier marker enzymes in PBCEC, such as alkaline phosphatase.

Blood-Brain Barrier Effects of the Fusarium Mycotoxins Deoxynivalenol, 3 Acetyldeoxynivalenol, and Moniliformin and Their Transfer to the Brain.

BackgroundSecondary metabolites produced by Fusarium fungi frequently contaminate food and feed and have adverse effects on human and animal health. Fusarium mycotoxins exhibit a wide structural and biosynthetic diversity leading to different toxicokinetics and toxicodynamics. Several studies investigated the toxicity of mycotoxins, focusing on very specific targets, like the brain. However, it still remains unclear how fast mycotoxins reach the brain and if they impair the integrity of the blood-brain barrier. This study investigated and compared the effects of the Fusarium mycotoxins deoxynivalenol, 3-acetyldeoxynivalenol and moniliformin on the blood-brain barrier. Furthermore, the transfer properties to the brain were analyzed, which are required for risk assessment, including potential neurotoxic effects.MethodsPrimary porcine brain capillary endothelial cells were cultivated to study the effects of the examined mycotoxins on the blood-brain barrier in vitro. The barrier integrity was monitored by cellular impedance spectroscopy and 14C radiolabeled sucrose permeability measurements. The distribution of the applied toxins between blood and brain compartments of the cell monolayer was analyzed by high performance liquid chromatography-mass spectrometry to calculate transfer rates and permeability coefficients.ResultsDeoxynivalenol reduced the barrier integrity and caused cytotoxic effects at 10 μM concentrations. Slight alterations of the barrier integrity were also detected for 3-acetyldeoxynivalenol. The latter was transferred very quickly across the barrier and additionally cleaved to deoxynivalenol. The transfer of deoxynivalenol and moniliformin was slower, but clearly exceeded the permeability of the negative control. None of the compounds was enriched in one of the compartments, indicating that no efflux transport protein is involved in their transport.

Transport of treosulfan and temozolomide across an in-vitro blood-brain barrier model.

In vitro, treosulfan (TREO) has shown high effectiveness against malignant gliomas. However, a first clinical trial for newly diagnosed glioblastoma did not show any positive effect. Even though dosing and timing might have been the reasons for this failure, it might also be that TREO does not reach the brain in sufficient amount. Surprisingly, there are no published data on TREO uptake into the brain of patients, despite extensive research on this compound. An in-vitro blood-brain barrier (BBB) model consisting of primary porcine brain capillary endothelial cells was used to determine the transport of TREO across the cell monolayer. Temozolomide (TMZ), the most widely used cytotoxic drug for malignant gliomas, served as a reference. An HPLC-ESI-MS/MS procedure was developed to detect TREO and TMZ in cell culture medium. Parallel to the experimental approach, the permeability of TREO and the reference substance across the in-vitro BBB was estimated on the basis of their physicochemical properties. The detection limit was 30 nmol/l for TREO and 10 nmol/l for TMZ. Drug transport was measured in two directions: influx, apical-to-basolateral (A-to-B), and efflux, basolateral-to-apical (B-to-A). For TREO, the A-to-B permeability was lower (1.6%) than the B-to-A permeability (3.0%). This was in contrast to TMZ, which had higher A-to-B (13.1%) than B-to-A (7.2%) permeability values. The in-vitro BBB model applied simulated the human BBB properly for TMZ. It is, therefore, reasonable to assume that the values for TREO are also meaningful. Considering the lack of noninvasive, significant alternative methods to study transport across the BBB, the porcine brain capillary endothelial cell model was efficient to collect first data for TREO that explain the disappointing clinical results for this drug against cerebral tumors.

Dual ligand immunoliposomes for drug delivery to the brain

Drug delivery systems that can reach brain areas affected by amyloid deposits are still underdeveloped. We propose pegylated liposomes functionalized with two antibodies, the anti-transferrin receptor monoclonal antibody (OX26MAb) and the anti-amyloid beta peptide antibody (19B8MAb), as nanocarriers of drugs for Alzheimer’s disease therapy. Two distinct conjugation methods are investigated. In one formulation, the OX26MAb is conjugated to the tip of polyethylene glycol molecules through the maleimide group and the 19B8MAb is bound through the streptavidin–biotin complex. In the second system the conjugation reagents are swapped between the antibodies. Fluorescence spectroscopy experiments on porcine brain capillary endothelial cells show that the cellular uptake of the immunoliposomes is substantially more efficient if OX26MAb antibody is conjugated through the streptavidin–biotin complex instead of the maleimide group. The ability of the immunoliposomes to cross the blood brain barrier was established by in vivo studies in wild type rats. Our results demonstrate the importance of the conjugation method used to bind the antibody that targets the blood brain barrier to immunoliposomes for efficient drug delivery to the brain.

Liposomal conjugates for drug delivery to the central nervous system.

Treatments of central nervous system (CNS) diseases often fail due to the blood–brain barrier. Circumvention of this obstacle is crucial for any systemic treatment of such diseases to be effective. One approach to transfer drugs into the brain is the use of colloidal carrier systems—amongst others, liposomes. A prerequisite for successful drug delivery by colloidal carriers to the brain is the modification of their surface, making them invisible to the reticuloendothelial system (RES) and to target them to specific surface epitopes at the blood–brain barrier. This study characterizes liposomes conjugated with cationized bovine serum albumin (cBSA) as transport vectors in vitro in porcine brain capillary endothelial cells (PBCEC) and in vivo in rats using fluorescently labelled liposomes. Experiments with PBCEC showed that sterically stabilized (PEGylated) liposomes without protein as well as liposomes conjugated to native bovine serum albumin (BSA) were not taken up. In contrast, cBSA-liposomes were taken up and appeared to be concentrated in intracellular vesicles. Uptake occurred in a concentration and time dependent manner. Free BSA and free cBSA inhibited uptake. After intravenous application of cBSA-liposomes, confocal fluorescence microscopy of brain cryosections from male Wistar rats showed fluorescence associated with liposomes in brain capillary surrounding tissue after 3, 6 and 24 h, for liposomes with a diameter between 120 and 150 nm, suggesting successful brain delivery of cationized-albumin coupled liposomes.