David J. Begley

Structure and function of the blood-brain barrier.

Neural signalling within the central nervous system (CNS) requires a highly controlled microenvironment. Cells at three key interfaces form barriers between the blood and the CNS: the blood-brain barrier (BBB), blood-CSF barrier and the arachnoid barrier. The BBB at the level of brain microvessel endothelium is the major site of blood-CNS exchange. The structure and function of the BBB is summarised, the physical barrier formed by the endothelial tight junctions, and the transport barrier resulting from membrane transporters and vesicular mechanisms. The roles of associated cells are outlined, especially the endfeet of astrocytic glial cells, and pericytes and microglia. The embryonic development of the BBB, and changes in pathology are described. The BBB is subject to short and long-term regulation, which may be disturbed in pathology. Any programme for drug discovery or delivery, to target or avoid the CNS, needs to consider the special features of the BBB.

Direct Evidence That Polysorbate-80-Coated Poly(Butylcyanoacrylate) Nanoparticles Deliver Drugs to the CNS via Specific Mechanisms Requiring Prior Binding of Drug to the Nanoparticles

AbstractPurpose. It has recently been suggested that the poly(butylcyanoacrylate) (PBCA) nanoparticle drug delivery system has a generalized toxic effect on the blood-brain barrier (BBB) (8) and that this effect forms the basis of an apparent enhanced drug delivery to the brain. The purpose of this study is to explore more fully the mechanism by which PBCA nanoparticles can deliver drugs to the brain. Methods. Both in vivo and in vitro methods have been applied to examine the possible toxic effects of PBCA nanoparticles and polysorbate-80 on cerebral endothelial cells. Human, bovine, and rat models have been used in this study. Results. In bovine primary cerebral endothelial cells, nontoxic levels of PBCA particles and polysorbate-80 did not increase paracellular transport of sucrose and inulin in the monolayers. Electron microscopic studies confirm cell viability. In vivo studies using the antinociceptive opioid peptide dalargin showed that both empty PBCA nanoparticles and polysorbate-80 did not allow dalargin to enter the brain in quantities sufficient to cause antinociception. Only dalargin preadsorbed to PBCA nanoparticles was able to induce an antinociceptive effect in the animals. Conclusion. At concentrations of PBCA nanoparticles and polysorbate-80 that achieve significant drug delivery to the brain, there is little in vivo or in vitro evidence to suggest that a generalized toxic effect on the BBB is the primary mechanism for drug delivery to the brain. The fact that dalargin has to be preadsorbed onto nanoparticles before it is effective in inducing antinociception suggests specific mechanisms of delivery to the CNS rather than a simple disruption of the BBB allowing a diffusional drug entry.

Albumin nanoparticles targeted with Apo E enter the CNS by transcytosis and are delivered to neurones

The blood-brain barrier (BBB) represents a considerable obstacle to brain entry of the majority of drugs and thus severely restricts the therapy of many serious CNS diseases including brain tumours, brain HIV, Alzheimer and other neurodegenerative diseases. The use of nanoparticles coated with polysorbate 80 or with attached apolipoprotein E has enabled the delivery of drugs across the BBB. However, the mechanism of this enhanced transport is still not fully understood. In this present study, human serum albumin nanoparticles, with covalently bound apolipoprotein E (Apo E) as a targetor as well as without apolipoprotein E, were manufactured and injected intravenously into SV 129 mice. The animals were sacrificed after 15 and 30 min, and their brains were examined by transmission electron microscopy. Only the nanoparticles with covalently bound apolipoprotein E were detected in brain capillary endothelial cells and neurones, whereas no uptake into the brain was detectable with nanoparticles without apolipoprotein E. We have also demonstrated uptake of the albumin/ApoE nanoparticles into mouse endothelial (b.End3) cells in vitro and their intracellular localisation. These findings indicate that nanoparticles with covalently bound apolipoprotein E are taken up into the cerebral endothelium by an endocytic mechanism followed by transcytosis into brain parenchyma.

The blood-brain barrier : Principles for targeting peptides and drugs to the central nervous system

The presence of the blood‐brain barrier (BBB), reduces the brain uptake of many drugs, peptides and other solutes from blood. Strategies for increasing the uptake of drugs and peptide‐based drugs include; structural modifications to increase plasma half‐life; improving passive penetration of the BBB by increasing the lipophilicity of the molecule; designing drugs which react with transporters present in the BBB; and reducing turnover and efflux from the central nervous system (CNS).

Central Nervous System Drug Disposition: The Relationship between in Situ Brain Permeability and Brain Free Fraction

The dispositions of 50 marketed central nervous system (CNS) drugs into the brain have been examined in terms of their rat in situ (P) and in vitro apparent membrane permeability (Papp) alongside lipophilicity and free fraction in rat brain tissue. The inter-relationship between these parameters highlights that both permeability and brain tissue binding influence the uptake of drugs into the CNS. Hydrophilic compounds characterized by low brain tissue binding display a strong correlation (R2 = 0.82) between P and Papp, whereas the uptake of more lipophilic compounds seems to be influenced by both Papp and brain free fraction. A nonlinear relationship is observed between logPoct and P over the 6 orders of magnitude range in lipophilicity studied. These findings corroborate recent reports in the literature that brain penetration is a function of both rate and extent of drug uptake into the CNS.

Significant entry of tubocurarine into the brain of rats by adsorption to polysorbate 80–coated polybutylcyanoacrylate nanoparticles: An in situ brain perfusion study

The possibility of using polysorbate 80-coated polybutylcyanoacrylate nanoparticles to deliver low molecular polar hydrophilic drugs to the CNS has been studied. Tubocurarine (a quaternary ammonium salt) does not penetrate the normal intact blood-brain barrier. However, the injection of this drug directly into the cerebral ventricles of the brain provokes the development of epileptiform seizures as assessed by electroencephalogram (EEG). An in situ perfused rat brain technique was used as an experimental technique together with a simultaneous recording of the EEG. Nanoparticles were prepared by butylcyanoacrylate polymerization in an acidic medium. Fifteen minutes after the introduction of tubocurarine-loaded polysorbate 80-coated nanoparticles into the perfusate, epileptiform spikes in the EEG appeared. Intraventricular injection of tubocurarine caused the appearance of the EEG seizures 5 min after administration. Neither tubocurarine solution nor tubocurarine-loaded nanoparticles without polysorbate 80 or a mixture of polysorbate 80 and tubocurarine were able to influence the EEG. Thus only the loading of tubocurarine onto the polysorbate 80-coated nanoparticles appears to enable the transport of this quaternary ammonium compound through the blood-brain barrier.

Measurement of Solute Transport Across the Blood–Brain Barrier in the Perfused Guinea Pig Brain: Method and Application to N‐Methyl‐α‐Aminoisobutyric Acid

Abstract: A technique for the vascular perfusion of the guinea pig head in vivo, suitable for measurements of blood‐to‐brain transport under controlled conditions of arterial inflow, has been developed. With a perfusion pressure ranging between 13 and 18 kPa and Pco2 in the arterial inflow of 5 and 5.5 kPa, cerebral blood flow, measured with [14C]butanol, was about 1 ml min−1 g−1 in the cerebral cortex, hippocampus, and caudate‐putamen of the ipsilateral hemisphere; in the cerebellum and pontine white matter it was considerably less, and much higher perfusion pressures were required to establish equal blood flow throughout the whole brain. Regional water content, Na+/K+ ratio, ATP, energy charge potential, and lactate content of the ipsilateral side of perfused and nonperfused brain were not significantly different after 10 min perfusion. The D‐[3H]mannitol space did not exceed 1% after 30 min of perfusion, indicating the integrity of the barrier. Over this period, EEG, ECG, and respiratory waveform remained normal. When [14C]N‐methyl‐α‐aminoisobutyric acid (MeAIB), and D‐[3H]mannitol were perfused together over periods extending to 30 min progressive uptakes of both solutes by the parietal cortex could be measured, and the unidirectional transfer constants estimated from multiple time‐uptake data. The Kin for MeAIB (0.75 × 10−3 ml min−1 g−1) was some three times that for mannitol. It is concluded that the technique provides a stable, well‐controlled environment in the cerebral microvasculature of the ipsilateral perfused brain hemisphere suitable for examining the transport of slowly penetrating solutes into the brain.

Interaction of Poly(butylcyanoacrylate) Nanoparticles with the Blood-Brain Barrier in vivo and in vitro

Poly(butylcyanoacrylate) nanoparticles were produced by emulsion polymerisation and used either uncoated or overcoated with polysorbate 80 (Tween® 80). [3H]-dalargin bound to nanoparticles overcoated with polysorbate 80 or in the form of saline solution was injected into mice and the brain concentrations of radioactivity determined. Statistically significant, three-fold higher brain concentrations with the nanoparticle preparations were obtained after 45 minutes, the time of greatest pharmacological response assessed as analgesia in previous experiments. In addition the brain inulin spaces in rats and the uptake of fluoresceine isothiocyanate labelled nanoparticles in immortalised rat cerebral endothelial cells, (RBE4) were measured. The inulin spaces after i.v. injection of polysorbate 80-coated nanoparticles were significantly increased by 1% compared to controls. This is interpreted as indicating that there is no large scale opening of the tight junctions of the brain endothelium by the polysorbate 80-coated nanoparticles. In in vitro experiments endocytic uptake of fluorescent nanoparticles by RBE4 cells was only observed after polysorbate 80-overcoating, not with uncoated particles. These results further support the hypothesis that the mechanism of blood-brain barrier transport of drugs by polysorbate 80-coated nanoparticles is one of endocytosis followed by possible transcytosis. The experiments were conducted in several laboratories as part of an EEC/INTAS collaborative program. For various procedural and regulatory reasons this necessitated the use of both rats and mice as experimental animals. The brain endothelial cell line used for the in vitro studies is the rat RBE4.

Polysorbate-80 coating enhances uptake of polybutylcyanoacrylate (PBCA)-nanoparticles by human and bovine primary brain capillary endothelial cells

Certain drugs such as dalargin, loperamide or tubocurarine are not transported across the blood–brain barrier (BBB) and therefore exhibit no effects on the central nervous system. However, effects on the central nervous system can be observed when these drugs are loaded onto polybutylcyanoacrylate (PBCA)‐nanoparticles and coated with polysorbate 80. The mechanism by which these complexed nanoparticles cross the BBB and exhibit their effects has not been elucidated. Cultured microvessel brain endothelial cells of human and bovine origin were used as an in vitro model for the BBB to gain further insight into the mechanism of uptake of nanoparticles. With cells from these species we were able to show that polysorbate 80‐coated nanoparticles were taken up by brain endothelial cells much more rapidly and in significantly higher amounts (20‐fold) than uncoated nanoparticles. The process of uptake was followed by fluorescence and confocal laser scanning microscopy. The results demonstrate that the nanoparticles are taken up by cells and that this uptake occurs via an endocytotic mechanism.

Immunologic privilege in the central nervous system and the blood-brain barrier.

The brain is in many ways an immunologically and pharmacologically privileged site. The blood–brain barrier (BBB) of the cerebrovascular endothelium and its participation in the complex structure of the neurovascular unit (NVU) restrict access of immune cells and immune mediators to the central nervous system (CNS). In pathologic conditions, very well-organized immunologic responses can develop within the CNS, raising important questions about the real nature and the intrinsic and extrinsic regulation of this immune privilege. We assess the interactions of immune cells and immune mediators with the BBB and NVU in neurologic disease, cerebrovascular disease, and intracerebral tumors. The goals of this review are to outline key scientific advances and the status of the science central to both the neuroinflammation and CNS barriers fields, and highlight the opportunities and priorities in advancing brain barriers research in the context of the larger immunology and neuroscience disciplines. This review article was developed from reports presented at the 2011 Annual Blood-Brain Barrier Consortium Meeting.