Polypeptide point modifications with fatty acid and amphiphilic block copolymers for enhanced brain delivery

There is a tremendous need to enhance delivery of therapeutic polypeptides to the brain to treat disorders of the central nervous system (CNS). The brain delivery of many polypeptides is severely restricted by the blood-brain barrier (BBB). The present study demonstrates that point modifications of a BBB-impermeable polypeptide, horseradish peroxidase (HRP), with lipophilic (stearoyl) or amphiphilic (Pluronic block copolymer) moieties considerably enhance the transport of this polypeptide across the BBB and accumulation of the polypeptide in the brain in vitro and in vivo. The enzymatic activity of the HRP was preserved after the transport. The modifications of the HRP with amphiphilic block copolymer moieties through degradable disulfide links resulted in the most effective transport of the HRP across in vitro brain microvessel endothelial cell monolayers and efficient delivery of HRP to the brain. Stearoyl modification of HRP improved its penetration by about 60% but also increased the clearance from blood. Pluronic modification using increased penetration of the BBB and had no significant effect on clearance so that uptake by brain was almost doubled. These results show that point modification can improve delivery of even highly impermeable polypeptides to the brain.     

In vitro demonstration of a saturable transport system for leptin across the blood-brain barrier.

A saturable blood-to-brain transport system for leptin across the blood-brain barrier (BBB) has been observed in vivo. Since the main component of the non-fenestrated microvessels of the BBB is the endothelial cell, we established an in vitro culture system of these cerebrovascular cells to study leptin transport and to determine whether the self-inhibition of leptin transport characteristic of a saturable system occurs at this level. The results show that 125I-leptin crossed from the luminal to abluminal side of a monolayer of cerebral microvessel cells significantly faster than the albumin and lactalbumin controls. This transport of 125I-leptin across an in vitro BBB was significantly faster than in the opposite direction and was dose-relatedly inhibited by the addition of unlabeled leptin. Thus, the results establish that the saturable transport system for leptin across the BBB occurs at the level of the endothelial cells of the BBB.     

The blood–brain barrier transport and cerebral distribution of guanidinoacetate in rats: involvement of creatine and taurine transporters

Although the cerebral accumulation of guanidinoacetate (GAA) contributes to neurological complications in S -adenosylmethionine:guanidinoacetate N -methyltransferase (GAMT) deficiency, how GAA is abnormally distributed in the brain remains unknown. The purpose of this study was to investigate the transport of GAA across the blood–brain barrier (BBB) and in brain parenchymal cells in rats. [¹⁴C]GAA microinjected into the rat cerebrum was not eliminated from the brain, implying the negligible contribution of GAA efflux transport across the BBB. In contrast, in vivo analysis and an uptake study by TR-BBB cells, a rat in vitro BBB model, revealed that GAA was transported from the circulating blood across the BBB most likely via a creatine transporter (CRT). Although CRT at the BBB is almost saturated by endogenous creatine under physiological conditions, the creatine level in the blood significantly decreases in GAMT deficiency. This might lead to the increase of CRT-mediated blood-to-brain transport of GAA at the BBB. Furthermore, [¹⁴C]GAA was taken up by brain parenchymal cells in a concentrative manner most likely via taurine transporter and CRT. These characteristics of GAA transport across the BBB and in the brain parenchymal cells could be the key factors that facilitate GAA accumulation in the brains of patients with GAMT deficiency.     

Fatty acid transport protein expression in human brain and potential role in fatty acid transport across human brain microvessel endothelial cells

The blood-brain barrier (BBB), formed by the brain capillary endothelial cells, provides a protective barrier between the systemic blood and the extracellular environment of the CNS. Passage of fatty acids from the blood to the brain may occur either by diffusion or by proteins that facilitate their transport. Currently several protein families have been implicated in fatty acid transport. The focus of the present study was to identify the fatty acid transport proteins (FATPs) expressed in the brain microvessel endothelial cells and characterize their involvement in fatty acid transport across an in vitro BBB model. The major fatty acid transport proteins expressed in human brain microvessel endothelial cells (HBMEC), mouse capillaries and human grey matter were FATP-1, -4 and fatty acid binding protein 5 and fatty acid translocase/CD36. The passage of various radiolabeled fatty acids across confluent HBMEC monolayers was examined over a 30-min period in the presence of fatty acid free albumin in a 1 : 1 molar ratio. The apical to basolateral permeability of radiolabeled fatty acids was dependent upon both saturation and chain length of the fatty acid. Knockdown of various fatty acid transport proteins using siRNA significantly decreased radiolabeled fatty acid transport across the HBMEC monolayer. Our findings indicate that FATP-1 and FATP-4 are the predominant fatty acid transport proteins expressed in the BBB based on human and mouse expression studies. While transport studies in HBMEC monolayers support their involvement in fatty acid permeability, fatty acid translocase/CD36 also appears to play a prominent role in transport of fatty acids across HBMEC.     

Isolation of blood-brain barrier-crossing antibodies from a phage display library by competitive elution and their ability to penetrate the central nervous system

The blood-brain barrier (BBB) is a formidable obstacle for delivery of biologic therapeutics to central nervous system (CNS) targets. Whilst the BBB prevents passage of the vast majority of molecules, it also selectively transports a wide variety of molecules required to maintain brain homeostasis. Receptor-mediated transcytosis is one example of a macromolecule transport system that is employed by cells of the BBB to supply essential proteins to the brain and which can be utilized to deliver biologic payloads, such as antibodies, across the BBB. In this study, we performed phage display selections on the mouse brain endothelial cell line, bEND.3, to enrich for antibody single-chain variable fragments (scFvs) that could compete for binding with a known BBB-crossing antibody fragment, FC5. A number of these scFvs were converted to IgGs and characterized for their ability to bind to mouse, rat and human brain endothelial cells, and subsequent ability to transport across the BBB. We demonstrated that these newly identified BBB-targeting IgGs had increased brain exposure when delivered peripherally in mice and were also able to transport a biologically active molecule, interleukin-1 receptor antagonist (IL-1RA), into the CNS. The antagonism of the interleukin-1 system within the CNS can result in the relief of neuropathic pain. We demonstrated that the BBB-targeting IgGs were able to elicit an analgesic response in a mouse model of nerve ligation-induced hypersensitivity when fused to IL-1RA.     

The many lives of leptin

Leptin is a 16,000-Da protein which is secreted by fat but acts within the brain to regulate adiposity. Our Peptides Classic addressed the mystery of how such a large molecule could negotiate the blood-brain barrier (BBB), a structure which normally excludes proteins from the brain. We found that leptin was transported across the BBB by a saturable transport system. This finding was important to understanding how satiety-related peptides and proteins worked, but it was also important to the concept that the BBB is a regulatory interface important in brain-body communication. Obesity in humans and many animals is associated with a leptin resistant state rather than a leptin deficiency. Subsequent work has shown that a defect in the BBB transport of leptin is key in producing and reinforcing this state of resistance. Leptin is pluripotent and the concept of it being primarily an adipostat is being discarded for more encompassing views. Consideration of the BBB data would favor the view that ancestral levels of leptin were much lower than those currently considered normal and are consistent with leptin acting as a metabolic switch, informing the brain when fat reserves are adequate to direct energy expenditures towards activities other than seeking calories.     

Effects of triglycerides, obesity, and starvation on ghrelin transport across the blood-brain barrier

Human ghrelin is transported across the blood-brain barrier (BBB) of normal mice. Here, we studied the effects of triglycerides, obesity, and starvation in retired breeder mice maintained on a high fat diet, mice age-matched to the retired breeders but maintained on normal chow, and 8-week-old mice maintained on breeder chow. The rate of ghrelin transport across the BBB was studied by both the intravenous administration method of multiple-time regression analysis and by the brain perfusion method. We found that (1) obese, aged mice lost the ability to transport intravenously administered ghrelin across the BBB, resulting in an inverse relation between body weight and ghrelin BBB permeability; (2) serum triglycerides promoted transport of intravenously administered ghrelin across the BBB, whereas epinephrine had no effect; (3) fasting tended to promote ghrelin transport across the BBB as most readily shown in brain perfusion studies; (4) evidence suggested that a serum factor promoted ghrelin transport in 8-week-old mice. Overall, these results show that serum factors and physiological states influence the rate at which ghrelin is transported across the blood-brain barrier.     

聚乳酸纳米粒穿透血脑屏障的分析电镜研究

观察以聚乳酸 (D ,L-polylacticacid,PLA)为材料制备、经吐温-80(T-80)表面改性的纳米粒对血脑屏障的穿透效果并探讨其机制 ,分别将FITC-Dextran、叶绿素铜作为PLA纳米粒的示踪标记 ,应用荧光显微镜、透射电镜及分析电镜观察经静脉注射入小鼠体内的PLA纳米粒在脑组织中的分布、穿透血脑屏障的特性。荧光显微镜观察到小鼠脑组织中散在及沿毛细血管壁分布的荧光颗粒 ,透射电镜可观察到小鼠脑毛细血管内皮细胞及周围脑组织中圆形或类圆形的外源性纳米粒 ;进一步采用分析电镜对颗粒处组织进行能谱分析证实其为叶绿素铜标记的PLA纳米粒。证实了T-80修饰的PLA纳米粒具有穿透血脑屏障的特性 ,机制可能是毛细血管内皮细胞的胞吞转运作用 ,同时 ,为研究纳米粒在组织内的定位提供了新的标记方法.     

Blood-brain barrier is involved in the efflux transport of a neuroactive steroid, dehydroepiandrosterone sulfate, via organic anion transporting polypeptide 2

We have investigated the transport characteristics of dehydroepiandrosterone sulfate (DHEAS), a neuroactive steroid, at the blood-brain barrier (BBB) in a series of functional in vivo and in vitro studies. The apparent BBB efflux rate constant of [(3)H]DHEAS evaluated by the brain efflux index method was 2.68 x 10(-2) min(-1). DHEAS efflux transport was a saturable process with a Michaelis constant (K:(m)) of 32.6 microM: Significant amounts of [(3)H]DHEAS were determined in the jugular venous plasma by HPLC, providing direct evidence that most of the DHEAS is transported in intact form from brain to the circulating blood across the BBB. This efflux transport of [(3)H]DHEAS was significantly inhibited by common rat organic anion-transporting polypeptide (oatp) substrates such as taurocholate, cholate, sulfobromophthalein, and estrone-3-sulfate. Moreover, the apparent efflux clearance of [(3)H]DHEAS across the BBB (118 microl/min-g of brain) was 10.4-fold greater than its influx clearance estimated by the in situ brain perfusion technique (11.4 microl/min-g of brain), suggesting that DHEAS is predominantly transported from the brain to blood across the BBB. In cellular uptake studies using a conditionally immortalized mouse brain capillary endothelial cell line (TM-BBB4), [(3)H]DHEAS uptake by TM-BBB4 cells exhibited a concentration dependence with a K:(m) of 34.4 microM: and was significantly inhibited by the oatp2-specific substrate digoxin. Conversely, [(3)H]digoxin uptake by TM-BBB4 cells was significantly inhibited by DHEAS. Moreover, the net uptake of [(3)H]DHEAS at 30 min was significantly increased under ATP-depleted conditions, suggesting that an energy-dependent efflux process may also be involved in TM-BBB4. RT-PCR and sequence analysis suggest that an oatp2 is expressed in TM-BBB4 cells. In conclusion, DHEAS efflux transport takes place across the BBB, and studies involving in vitro DHEAS uptake and RT-PCR suggest that there is oatp2-mediated DHEAS transport at the BBB.     

Humanization of anti-human insulin receptor antibody for drug targeting across the human blood-brain barrier

A murine monoclonal antibody (MAb) to the human insulin receptor (HIR) has been engineered for use as a brain drug delivery system for transport across the human blood-brain barrier (BBB). The HIRMAb was humanized by complementarity determining region (CDR) grafting on the framework regions (FR) of the human B43 IgG heavy chain and the human REI kappa light chain. A problem encountered in the humanization process was the poor secretion of the CDR-grafted HIRMAb by myeloma cells. This problem was solved by the production of human/mouse hybrids of the engineered heavy chain variable region (VH), which led to the replacement of five amino acids in the FR3 of the VH with original murine amino acids. No replacement of FR amino acids in the light chain variable region (VL) was required. The affinity of the humanized HIRMAb for the HIR was decreased 27% relative to the murine HIRMAb. The humanized HIRMAb avidly bound to the HIR of isolated human brain capillaries, which are used as an in vitro model system of the human BBB. The HIRMAb cross reacts with the HIR of Old World primates such as the Rhesus monkey. The humanized HIRMAb was radiolabeled with 125-iodine, and injected intravenously into an adult, anesthetized Rhesus monkey. Brain scanning showed the humanized HIRMAb was rapidly transported into all parts of the primate brain after intravenous administration. The humanized HIRMAb may be used as a brain drug and gene delivery system for the targeting of large molecule therapeutics across the BBB in humans.     

Peptide-Based Delivery of Oligonucleotides Across Blood–Brain Barrier Model

Delivery of pharmaceutical agents across a blood–brain barrier (BBB) is a challenge for brain cancer therapy. In this study, an in vitro BBB model was utilized to study the delivery of oligonucleotides across brain endothelial cells targeting to glioma cells in a Transwell? setup. A series of novel peptides were synthesized by covalent conjugation of cell-penetrating peptides with targeting peptides for delivery of gene-based therapeutics. These peptides were screened for passage across the Transwell? and we found the most efficient peptide PepFect32 from originating PepFect 14 coupled with the targeting peptide angiopep-2. PepFect32/pDNA nanocomplexes exhibited high transcytosis across the BBB in vitro model and the highest transfection efficiency to glioma cells. In conclusion, PepFect32 revealed the most efficient peptide-based vector for pDNA delivery across in vitro BBB model.     

IgG‐single chain Fv fusion protein therapeutic for alzheimer's disease: Expression in CHO cells and pharmacokinetics and brain delivery in the rhesus monkey

Monoclonal antibodies (MAb) directed against the Abeta amyloid peptide of Alzheimer's disease (AD) are potential new therapies for AD, since these antibodies disaggregate brain amyloid plaque. However, the MAb is not transported across the blood–brain barrier (BBB). To enable BBB transport, a single chain Fv (ScFv) antibody against the Abeta peptide of AD was re‐engineered as a fusion protein with the MAb against the human insulin receptor (HIR). The HIRMAb acts as a molecular Trojan horse to ferry the ScFv therapeutic antibody across the BBB. Chinese hamster ovary (CHO) cells were stably transfected with a tandem vector encoding the heavy and light chains of the HIRMAb–ScFv fusion protein. A high secreting line was isolated following methotrexate amplification and dilutional cloning. The HIRMAb–ScFv fusion protein in conditioned serum‐free medium was purified by protein A affinity chromatography. The fusion protein was stable as a liquid formulation, and retained high‐affinity binding of both the HIR and the Abeta amyloid peptide. The HIRMAb–ScFv fusion protein was radiolabeled with the ¹²⁵I‐Bolton–Hunter reagent, followed by measurement of the pharmacokinetics of plasma clearance and brain uptake in the adult Rhesus monkey. The HIRMAb–ScFv fusion protein was rapidly cleared from plasma and was transported across the primate BBB in vivo. In conclusion, the HIRMAb–ScFv fusion protein is a new class of antibody‐based therapeutic for AD that has been specifically engineered to cross the human BBB. Biotechnol. Bioeng. 2010; 105: 627–635. © 2009 Wiley Periodicals, Inc.     

Afamin is synthesized by cerebrovascular endothelial cells and mediates α-tocopherol transport across an in vitro model of the blood–brain barrier

α-Tocopherol (αTocH), a member of the vitamin E family, is essential for normal neurological function. Despite the importance of αTocH transport into the CNS, transfer mechanisms across the blood–brain barrier (BBB) are not entirely clear. We here investigate whether afamin, a known αTocH-binding protein, contributes to αTocH transport across an in vitro model of the BBB consisting of primary porcine brain capillary endothelial cells (BCEC) and basolaterally cultured astrocytoma cells. Exogenously added afamin had no adverse effects on BCEC viability or barrier function and was transported across BCEC Transwell cultures. Furthermore, αTocH transport across polarized BCEC cultures to astrocytoma cells is facilitated by afamin, though to a lesser extent than by high-density lipoprotein-mediated transport, an essential and in vivo operating αTocH import pathway at the cerebrovasculature. We also demonstrate that porcine BCEC endogenously synthesize afamin. In line with these in vitro findings, afamin was detected by immunohistochemistry in porcine, human postmortem, and mouse brain, where prominent staining was observed almost exclusively in the cerebrovasculature. The demonstration of afamin mRNA expression in isolated brain capillaries suggests that afamin might be a new family member of binding/transport proteins contributing to αTocH homeostasis at the BBB in vivo .     

Efficient intracellular delivery of oligonucleotides formulated in folate receptor-targeted lipid vesicles

In this study, a novel lipid vector has been developed for targeted delivery of oligodeoxynucleotides (ODN) to tumors that overexpress folate receptor. This is based on a method developed by Semple et al. (1), which utilizes an ionizable aminolipid (1,2-dioleoyl-3-(dimethylammonio)propane, DODAP) and an ethanol-containing buffer system for encapsulating large quantities of polyanionic ODN in lipid vesicles. Folate is incorporated into the lipid vesicles via a distearoylphosphatidylethanolamine-poly(ethylene glycol) (DSPE-PEG) spacer. These vesicles are around 100-200 nm in diameter with an ODN entrapment efficiency of 60-80%. Folate mediated efficient delivery of ODN to KB cells that overexpress folate receptor. Uptake of folate-targeted lipidic ODN by KB cells is about 8-10-fold more efficient than that of lipidic ODN without a ligand or free ODN. This formulation is resistant to serum. Thus, targeted delivery of ODN via this novel lipid vector may have potential in treating tumors that overexpress folate receptors.     

Molecular modeling of blood-brain barrier nutrient transporters: in silico basis for evaluation of potential drug delivery to the central nervous system

For drugs that act in the brain, the blood-brain barrier (BBB) is a considerable physical barrier which influences the distribution of drugs to the brain. The BBB is essentially impermeable for hydrophilic and/or charged compounds. Nutrient membrane transporters have an important physiological role in the transport of essential substances across the BBB required for normal brain function. We and others have shown that these transporters may have utility as drug delivery vectors, thereby increasing brain distribution of these compounds via these systems. In this review, we evaluate molecular (in silico) models of BBB transport proteins. Few BBB membrane transporters have been crystallized, but their crystal structures have a possibility for use in homology modeling. Other techniques commonly used are 2D quantitative structure-activity relationships (QSAR), as well as 3D-QSAR techniques including comparative molecular field analysis (CoMFA) and comparative similarity index analysis (CoMSIA). Each of these models provides valuable information for ascertaining their potential basis for BBB transport and brain drug delivery.     

Human immunodeficiency virus-1 uses the mannose-6-phosphate receptor to cross the blood-brain barrier

HIV-1 circulates both as free virus and within immune cells, with the level of free virus being predictive of clinical course. Both forms of HIV-1 cross the blood-brain barrier (BBB) and much progress has been made in understanding the mechanisms by which infected immune cells cross the blood-brain barrier BBB. How HIV-1 as free virus crosses the BBB is less clear as brain endothelial cells are CD4 and galactosylceramide negative. Here, we found that HIV-1 can use the mannose-6 phosphate receptor (M6PR) to cross the BBB. Brain perfusion studies showed that HIV-1 crossed the BBB of all brain regions consistent with the uniform distribution of M6PR. Ultrastructural studies showed HIV-1 crossed by a transcytotic pathway consistent with transport by M6PR. An in vitro model of the BBB was used to show that transport of HIV-1 was inhibited by mannose, mannan, and mannose-6 phosphate and that enzymatic removal of high mannose oligosaccharide residues from HIV-1 reduced transport. Wheatgerm agglutinin and protamine sulfate, substances known to greatly increase transcytosis of HIV-1 across the BBB in vivo, were shown to be active in the in vitro model and to act through a mannose-dependent mechanism. Transport was also cAMP and calcium-dependent, the latter suggesting that the cation-dependent member of the M6PR family mediates HIV-1 transport across the BBB. We conclude that M6PR is an important receptor used by HIV-1 to cross the BBB.     

Ultrasound, microbubbles and the blood-brain barrier

The blood–brain barrier (BBB) is a specialized system of capillary endothelial cells that protects the brain from harmful substances in the blood stream, while supplying the brain with the required nutrients for proper function. The BBB controls transport through both tight junctions and metabolic barriers and is often a rate-limiting factor in determining permeation of therapeutic drugs into the brain. It is a significant obstacle for delivery of both small molecules and macromolecular agents. Although many drugs could be potentially used to treat brain disease, there has been no method that allows non-invasive-targeted delivery through the BBB. Recently, promising studies indicate that ultrasound can be used to locally deliver a drug or gene to a specific region of interest in the brain. If microbubbles are combined with ultrasound exposure, the effects of ultrasound can be focused upon the vasculature to reduce the acoustic intensity needed to produce BBB opening. Several avenues of transcapillary passage after ultrasound sonication have been identified including transcytosis, passage through endothelial cell cytoplasmic openings, opening of tight junctions and free passage through injured endothelium. This article reviews the topic of transient disruption of the BBB with ultrasound and microbubbles and addresses related safety issues. It also discusses possible roles of the BBB in brain disease and potential interactions with ultrasound and microbubbles in such disease states.     

Glycosylated Sertraline-Loaded Liposomes for Brain Targeting: QbD Study of Formulation Variabilities and Brain Transport

Effectiveness of CNS-acting drugs depends on the localization, targeting, and capacity to be transported through the blood–brain barrier (BBB) which can be achieved by designing brain-targeting delivery vectors. Hence, the objective of this study was to screen the formulation and process variables affecting the performance of sertraline (Ser-HCl)-loaded pegylated and glycosylated liposomes. The prepared vectors were characterized for Ser-HCl entrapment, size, surface charge, release behavior, and in vitro transport through the BBB. Furthermore, the compatibility among liposomal components was assessed using SEM, FTIR, and DSC analysis. Through a thorough screening study, enhancement of Ser-HCl entrapment, nanosized liposomes with low skewness, maximized stability, and controlled drug leakage were attained. The solid-state characterization revealed remarkable interaction between Ser-HCl and the charging agent to determine drug entrapment and leakage. Moreover, results of liposomal transport through mouse brain endothelialpolyoma cells demonstrated greater capacity of the proposed glycosylated liposomes to target the cerebellar due to its higher density of GLUT1 and higher glucose utilization. This transport capacity was confirmed by the inhibiting action of both cytochalasin B and phenobarbital. Using C6 glioma cells model, flow cytometry, time-lapse live cell imaging, and in vivo NIR fluorescence imaging demonstrated that optimized glycosylated liposomes can be transported through the BBB by classical endocytosis, as well as by specific transcytosis. In conclusion, the current study proposed a thorough screening of important formulation and process variabilities affecting brain-targeting liposomes for further scale-up processes.     

Development and brain delivery of chitosan-PEG nanoparticles functionalized with the monoclonal antibody OX26

The inhibition of the caspase-3 enzyme is reported to increase neuronal cell survival following cerebral ischemia. The peptide Z-DEVD-FMK is a specific caspase inhibitor, which significantly reduces vulnerability to the neuronal cell death. However, this molecule is unable to cross the blood-brain barrier (BBB) and to diffuse into the brain tissue. Thus, the development of an effective delivery system is needed to provide sufficient drug concentration into the brain to prevent cell death. Using the avidin (SA)-biotin (BIO) technology, we describe here the design of chitosan (CS) nanospheres conjugated with poly(ethylene glycol) (PEG) bearing the OX26 monoclonal antibody whose affinity for the transferrin receptor (TfR) may trigger receptor-mediated transport across the BBB. These functionalized CS-PEG-BIO-SA/OX26 nanoparticles (NPs) were characterized for their particle size, zeta potential, drug loading capacity, and release properties. Fluorescently labeled CS-PEG-BIO-SA/OX26 nanoparticles were administered systemically to mice in order to evaluate their efficacy for brain translocation. The results showed that an important amount of nanoparticles were located in the brain, outside of the intravascular compartment. These findings, which were also confirmed by electron microscopic examination of the brain tissue indicate that this novel targeted nanoparticulate drug delivery system was able to translocate into the brain tissue after iv administration. Consequently, these novel nanoparticles are promising carriers for the transport of the anticaspase peptide Z-DEVD-FMK into the brain.     

Involvement of the low-density lipoprotein receptor-related protein in the transcytosis of the brain delivery vector angiopep-2

The blood–brain barrier (BBB) restricts the entry of proteins as well as potential drugs to cerebral tissues. We previously reported that a family of Kunitz domain-derived peptides called Angiopeps can be used as a drug delivery system for the brain. Here, we further characterize the transcytosis ability of these peptides using an in vitro model of the BBB and in situ brain perfusion. These peptides, and in particular Angiopep-2, exhibited higher transcytosis capacity and parenchymal accumulation than do transferrin, lactoferrin, and avidin. Angiopep-2 transport and accumulation in brain endothelial cells were unaffected by the P-glycoprotein inhibitor, cyclosporin A, indicating that this peptide is not a substrate for the efflux pump P-glycoprotein. However, competition studies show that activated α₂-macroglobulin, a specific ligand for the low-density lipoprotein receptor-related protein-1 (LRP1) and Angiopep-2 can share the same receptor. In addition, LRP1 was detected in glioblastomas and brain metastases from lung and skin cancers. Fluorescent microscopy also revealed that Alexa488-Angiopep-2 co-localized with LRP1 in brain endothelial cell monolayers. Overall, these results suggest that Angiopep-2 transport across the BBB is, in part, mediated by LRP1.