Albumin nanoparticles targeted with Apo E enter the CNS by transcytosis and are delivered to neurones
Abstract
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.
Graphical abstract
Electron-microscopy of mouse brain tissue shows that nanoparticles with covalently bound apolipoprotein E are endocytosed after intravenous injection by brain endothelial cells and transported into the surrounding tissue by transcytosis. 
Introduction
The blood–brain barrier (BBB) restricts the transport of many therapeutically important drugs from the blood into the brain, including anticancer drugs, antibiotics, and a wide variety of central nervous system (CNS)-active drugs, especially macromolecules such as neuropeptides and proteins. The BBB is formed by the endothelial cells of the cerebral capillaries which essentially comprise the major exchange interface between the blood and the brain. A barrier function also occurs at the arachnoid membrane and in the ependymal cells surrounding the circumventricular organs of the brain [1], [2]. The BBB is a vital element in the regulation of the constancy of the internal environment of the brain. The BBB also protects the CNS from harmful circulating substances and maintains an essential brain homeostasis but as a result represents a great impediment to the effective treatment of many brain diseases.
Over the last few years a number of different strategies have been devised to overcome this barrier such as osmotic opening of the tight junctions [3], and the direct surgical administration of drugs into the brain. The most notable progress, however, has been achieved by the use of nanotechnology. Liposomes as well as solid lipid nanoparticles or different polymeric nanoparticles [4], [5] have successfully been used for the transport of drugs across the BBB and into the brain. These drugs included neuroactive peptides [4], [6] as well as the cytostatic drug doxorubicin. Doxorubicin after binding to polysorbate 80-coated nanoparticles significantly reduced the growth of the very aggressive intracranially transplanted brain tumour glioblastoma 101/8 and led to the disappearance of the tumour in about 20–40% of the animals following intravenous injection [7], [8]. Aside from the enhanced transport of the drug into the brain, these nanocarriers also protect the active agent from enzymatic degradation, appear to by-pass ABC efflux transporters, and additionally and importantly, are able to reduce side effects [9], [10], [11]. The mechanism by which nanoparticle delivery systems achieve drug transport across the BBB, however, has so-far not been totally explained and has attracted considerable debate. It has been suggested that nanoparticle systems exert a generalised toxic affect on the BBB permeabilising tight junctions and allowing paracellular movement of solutes into the brain [12]. Later studies have shown no toxic effects, in vivo and in vitro, at the doses normally used. They also show that in order to be delivered to the CNS, a drug has to be bound to the nanoparticle before intravenous administration, which would not be a requirement if tight junction opening were involved [13]. These later studies clearly show that the nanoparticles are physically transporting a drug across the BBB, and an association/combination of drug with nanoparticle is a requirement for this transport. It has also been hypothesised that after injection into the blood stream apolipoproteins are adsorbed onto the surface of polysorbate 80-coated nanoparticles, and thus they resemble endogenously circulating lipoproteins [14], [15], [16]. A number of mechanisms exist, transporting lipoproteins across the BBB [17], [18]. The apolipoprotein-modified particles could then interact with these apolipoprotein receptors at the blood–brain barrier and result in their endocytotic uptake into endothelium and possibly transcytosis into the brain. This suggestion is supported by the observation that nanoparticles with adsorbed or covalently bound apolipoprotein E or AI (Apo E or AI) transport attached drugs equally well across the BBB [19], [20]. Nevertheless, a small modulation of tight junction integrity cannot be totally excluded since the so-called “inulin space”, corresponding to the normal vascular space of the brain, is approximately doubled after intravenous injection of polysorbate 80-coated polybutyl-cyanoacylate nanoparticles [21]. This small increase in the in vivo inulin space could be due to co-induction of non-specific endocytosis alongside specific uptake of nanoparticles. This phenomenon has been observed [22] where endocytosis of FITC-albumin has been demonstrated together with rhodamine 123-labelled polysorbate 80-coated PBCA nanoparticles. Albumin uptake at the BBB does not normally occur [23].
Therefore, in this present study electron dense human serum albumin (HSA) nanoparticles targeted with covalently bound apolipoprotein E were intravenously injected into SV 129 mice and the animals sacrificed after 15 and 30 min. The brains of the mice were examined by transmission electron microscopy.
In addition, in vitro cell culture experiments with b.End3 cells (derived from the SV 129 mouse) were performed, and these cells also were investigated by electron microscopy and by flow cytometry (FACS) to supplement and support the interpretation of the in vivo results.
Section snippets
Chemicals and reagents
Human serum albumin (HSA, fraction V, purity 96–99%, 65 000 Da) as well as glutaraldehyde 8% solution, paraformaldehyde 16% solution and collagen IV was purchased from Sigma-Aldrich (Schnelldorf, Germany). Traut's reagent (2-Iminothiolane HCL) and D-Salt™ Dextran Desalting columns were obtained from Pierce (Rockford USA). The different components for the Araldite® mixture as well as osmium tetroxide 4% solution and lead citrate were bought from SERVA GmbH (Heidelberg, Germany), the TAAB
Results and discussion
Dark spherical structures which are identified as human serum albumin nanoparticles were easily detectable in the brains of SV 129 mice by electron microscopy at 15 and 30 min after injection of HSA nanoparticles with bound Apo E into the jugular vein. The Apo E was covalently attached to the nanoparticles using a bifunctional Mal-PEG-NHS linker reacting on one side with amino groups on the particle's surface and on the other with thiol groups of the Apo E. Typical micrographs of the tissue
Conclusions
HSA nanoparticles with covalently bound Apo E are clearly taken up into the brain endothelial cells by endocytosis after injection into the blood stream. A certain proportion of these particles are also transported into the brain parenchyma. This movement into the interior compartments of the CNS can only be accomplished by transcytosis across the BBB. Although not observed, a re-uptake of the particles by neurones must occur. An opening or modulation of the tight junctions of the brain
Competing interests statement
The authors declare that they have no competing financial interests.
Acknowledgements
The authors would like to thank Marion Basoglu for the technical support during the electron microscopy studies as well as Sascha Wien for the technical support during cell culture experiments, Dr. Tikva Vogel for kindly providing the recombinant Apo E to us, and Dr. Alice Warley and Ken Brady of the Centre for Ultrastructural Imaging at King's College London for preparation of some of the specimens and electron micrographs. The authors also want to thank H. Wolburg (University of Tübingen) and
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