Blood-brain Barrier Receptors Research Articles

Receptor-mediated drug delivery of bispecific therapeutic antibodies through the blood-brain barrier.

Therapeutic antibody drug development is a rapidly growing sector of the pharmaceutical industry. However, antibody drug development for the brain is a technical challenge, and therapeutic antibodies for the central nervous system account for ~3% of all such agents. The principal obstacle to antibody drug development for brain or spinal cord is the lack of transport of large molecule biologics across the blood-brain barrier (BBB). Therapeutic antibodies can be made transportable through the blood-brain barrier by the re-engineering of the therapeutic antibody as a BBB-penetrating bispecific antibody (BSA). One arm of the BSA is the therapeutic antibody and the other arm of the BSA is a transporting antibody. The transporting antibody targets an exofacial epitope on a BBB receptor, and this enables receptor-mediated transcytosis (RMT) of the BSA across the BBB. Following BBB transport, the therapeutic antibody then engages the target receptor in brain. RMT systems at the BBB that are potential conduits to the brain include the insulin receptor (IR), the transferrin receptor (TfR), the insulin-like growth factor receptor (IGFR) and the leptin receptor. Therapeutic antibodies have been re-engineered as BSAs that target the insulin receptor, TfR, or IGFR RMT systems at the BBB for the treatment of Alzheimer's disease and Parkinson's disease.

DH5α Outer Membrane-Coated Biomimetic Nanocapsules Deliver Drugs to Brain Metastases but not Normal Brain Cells via Targeting GRP94.

Receptor-mediated vesicular transport has been extensively developed to penetrate the blood-brain barrier (BBB) and has emerged as a class of powerful brain-targeting delivery technologies. However, commonly used BBB receptors such as transferrin receptor and low-density lipoprotein receptor-related protein 1, are also expressed in normal brain parenchymal cells and can cause drug distribution in normal brain tissues and subsequent neuroinflammation and cognitive impairment. Here, the endoplasmic reticulum residing protein GRP94 is found upregulated and relocated to the cell membrane of both BBB endothelial cells and brain metastatic breast cancer cells (BMBCCs) by preclinical and clinical investigations. Inspired by that Escherichia coli penetrates the BBB via the binding of its outer membrane proteins with GRP94, avirulent DH5α outer membrane protein-coated nanocapsules (Omp@NCs) are developed to cross the BBB, avert normal brain cells, and target BMBCCs via recognizing GRP94. Embelin (EMB)-loaded Omp@EMB specifically reduce neuroserpin in BMBCCs, which inhibits vascular cooption growth and induces apoptosis of BMBCCs by restoring plasmin. Omp@EMB plus anti-angiogenic therapy prolongs the survival of mice with brain metastases. This platform holds the translational potential to maximize therapeutic effects on GRP94-positive brain diseases.

Molecular Heterogeneity of the Brain Endothelium

The blood–brain barrier (BBB) is part of a neurovascular structure located in the brain’s micro vessels, that is essential to maintain brain homeostasis, but prevents the brain uptake of most drugs. Because of its importance in neuro-pharmacotherapy, the BBB has been the subject of extensive research since its discovery over 100 years ago. Major advances in understanding the structure and function of the barrier have been made. Drugs are re-designed to cross the BBB. However, despite these efforts, overcoming the BBB efficiently to treat brain diseases safely remains challenging. The majority of BBB research studies focus on the BBB as a homogenous structure throughout the different brain regions. However, this simplification may lead to an inadequate understanding of the BBB function with significant therapeutic consequences. From this perspective, we analyzed the gene and protein expression profiles of the BBB in the micro vessels from the brains of mice that were isolated from two different brain regions, namely the cortex and the hippocampus. The expression profile of the inter-endothelial junctional protein (claudin-5), three ABC transporters (P-glycoprotein, Bcrp and Mrp-1), and three BBB receptors (lrp-1, TRF and GLUT-1) were analyzed. Our gene and protein analysis showed that the brain endothelium in the hippocampus exhibits different expression profiles compared to the brain cortex. Specifically, brain endothelial cells (BECs) of the hippocampus express higher gene levels of abcb1, abcg2, lrp1, and slc2a1 compared to the BECs of the cortex regions with a trend of increase for claudin-5, while BECs of the cortex express higher gene levels of abcc1 and trf compared to the hippocampus. At the protein levels, the P-gp expression was found to be significantly higher in the hippocampus compared to the cortex, while TRF was found to be up-regulated in the cortex. These data suggest that the structure and function of the BBB are not homogeneous, and imply that drugs are not delivered similarly among the different brain regions. Appreciation of the BBB heterogeneity by future research programs is thus critical for efficient drug delivery and the treatment of brain diseases.

Exhaustive in silico design and screening of novel antipsychotic compounds with improved pharmacodynamics and blood-brain barrier permeation properties

Antipsychotic drugs or neuroleptics are widely used in the treatment of psychosis as a manifestation of schizophrenia and bipolar disorder. However, their effectiveness largely depends on the blood-brain barrier (BBB) permeation (pharmacokinetics) and drug-receptor pharmacodynamics. Therefore, in this study, we developed and implemented the in silico pipeline to design novel compounds (n = 260) as leads using the standard drug scaffolds with improved PK/PD properties from the standard scaffolds. As a result, the best candidates (n = 3) were evaluated in molecular docking to interact with serotonin and dopamine receptors. Finally, haloperidol (HAL) derivative (1‐(4‐fluorophenyl)‐4‐(4‐hydroxy‐4‐{4‐[(2‐phenyl‐1,3‐thiazol‐4‐yl)methyl]phenyl}piperidin‐1‐yl)butan‐1‐one) was identified as a “magic shotgun” lead compound with better affinity to the 5-HT2A, 5-HT1D, D2, D3, and 5-HT1B receptors than the control molecule. Additionally, this hit substance was predicted to possess similar BBB permeation properties and much lower toxicological profiles in comparison to HAL. Overall, the proposed rational drug design platform for novel antipsychotic drugs based on the BBB permeation and receptor binding might be an invaluable asset for a medicinal chemist or translational pharmacologist. Communicated by Ramaswamy H. Sarma

O‐GlcNAcylation modulates GLUT1 levels at the blood‐brain barrier

AbstractBackgroundGlucose transporter 1 (GLUT1) at the blood‐brain barrier (BBB) is essential for glucose uptake into the brain. Previous studies have shown that GLUT1 levels are reduced at the BBB in Alzheimer’s disease (AD) post‐mortem brain, and perhaps explains the decreased glucose utilization observed in AD (Sweeney et al., Nat Rev Neurol, 2018). Individuals with a genetic reduction of GLUT1, known as GLUT1‐deficiency syndrome, were treated with increased blood glucose with no long‐lasting increase in brain glucose. Diabetes is a risk factor for AD and AD is sometimes referred to as Type‐3 diabetes due to elevated blood glucose levels. Taken together, these ideas provoked us to investigate connections between glucose and GLUT1 levels. Here, we tested if the glucose‐dependent post‐translational modification, O‐linked‐N‐acetylglucosaminylation (O‐GlcNAcylation), is the missing link. We hypothesized that increasing glucose concentrations will elevate O‐GlcNAcylation and decrease GLUT1 levels at the BBB. Furthermore, previous studies have shown that decreased GLUT1 levels result in reduced levels of the low‐density lipoprotein receptor‐related protein‐1 (LRP‐1) (Winkler, Nat Neurosci, 2015). Thus, we also are determining if altering glucose levels not only changes GLUT1 levels, but also other key BBB receptors implicated in AD.MethodsMouse brain endothelial (bEnd.3) cells were treated with vehicle (glucose 5.6 mmol), DMSO, the O‐GlcNAcase inhibitor Thiamet‐G (TMG, 1 µM), or the O‐GlcNAc transferase inhibitor OSMI‐1 (20 µM), for 24 and 48 hours. Next, the protein expression of O‐GlcNAc and GLUT1 were measured by western blot.ResultsAs previously reported, TMG increased and OSMI‐1 decreased O‐GlcNAcylation at both 24 and 48 h, compared to vehicle. Notably, TMG treatment significantly reduced and OSMI‐1 increased GLUT1 expression at both time points.ConclusionGLUT1 levels are modulated through O‐GlcNAcylation at BBB. When blood glucose levels are high, O‐GlcNAcylation will be high, thereby reducing GLUT1 at the BBB and brain glucose uptake. This is likely a homeostatic mechanism to prevent spikes in brain glucose levels when blood sugar levels are elevated.

2-(2-Cholesteroxyethoxyl)ethyl-3′-S-glutathionylpropionate (COXP) for brain-targeting liposomes

The blood-brain barrier (BBB) prevents a drug from reaching its therapeutic concentration in the brain resulting in a failure for the treatment of most brain diseases. Various approaches have been explored to develop brain-targeting agents that can help a drug or a drug delivery system to cross the BBB to reach the brain. A ligand or ligand analog of a BBB transporter or BBB receptor has been commonly employed to develop a brain targeting agent.We reported early 2-(2-cholesteroxyethoxyl)ethyl-3′-S-glutathionylpropionate (COXP) as a novel glutathione (GSH) transporter-mediated brain targeting agent based on the over-expression of GSH transporters at the BBB. COXP was obtained by connecting a molecule of GSH with a molecule of cholesterol via a two-ethylene glycol unit. COXP can self-assemble resulting in the formation of brain targeting micelles.Since cholesterol is one of the major components of liposomes, this work was designed to investigate if COXP could be used as a component of liposomes to prepare brain-targeting liposomes (COXP liposomes). Our results demonstrated that COXP liposomes effectively delivered DiR, a fluorescence tracking agent, to the brain of a mouse. The whole-body imaging of mice showed that the fluorescence intensity of DiR in the brain could reach 11 times higher for DiR delivered by COXP liposomes than that delivered by a cationic liposome which served as a control. The results show COXP's potential for developing novel brain-targeting liposomes.

Functional analysis of human brain endothelium using a microfluidic device integrating a cell culture insert.

The blood-brain barrier (BBB) is a specialized brain endothelial barrier structure that regulates the highly selective transport of molecules under continuous blood flow. Recently, various types of BBB-on-chip models have been developed to mimic the microenvironmental cues that regulate the human BBB drug transport. However, technical difficulties in complex microfluidic systems limit their accessibility. Here, we propose a simple and easy-to-handle microfluidic device integrated with a cell culture insert to investigate the functional regulation of the human BBB endothelium in response to fluid shear stress (FSS). Using currently established immortalized human brain microvascular endothelial cells (HBMEC/ci18), we formed a BBB endothelial barrier without the substantial loss of barrier tightness under the relatively low range of FSS (0.1–1 dyn/cm2). Expression levels of key BBB transporters and receptors in the HBMEC/ci18 cells were dynamically changed in response to the FSS, and the effect of FSS reached a plateau around 1 dyn/cm2. Similar responses were observed in the primary HBMECs. Taking advantage of the detachable cell culture insert from the device, the drug efflux activity of P-glycoprotein (P-gp) was analyzed by the bidirectional permeability assay after the perfusion culture of cells. The data revealed that the FSS-stimulated BBB endothelium exhibited the 1.9-fold higher P-gp activity than that of the static culture control. Our microfluidic system coupling with the transwell model provides a functional human BBB endothelium with secured transporter activity, which is useful to investigate the bidirectional transport of drugs and its regulation by FSS.

Proteome of the Luminal Surface of the Blood-Brain Barrier.

Interrogation of the molecular makeup of the blood–brain barrier (BBB) using proteomic techniques has contributed to the cataloguing and functional understanding of the proteins uniquely organized at this specialized interface. The majority of proteomic studies have focused on cellular components of the BBB, including cultured brain endothelial cells (BEC). Detailed proteome mapping of polarized BEC membranes and their intracellular endosomal compartments has led to an improved understanding of the processes leading to internalization and transport of various classes of molecules across the BBB. Quantitative proteomic methods have further enabled absolute and comparative quantification of key BBB transporters and receptors in isolated BEC and microvessels from various species. However, translational studies further require in vivo/in situ analyses of the proteins exposed on the luminal surface of BEC in vessels under various disease and treatment conditions. In vivo proteomics approaches, both profiling and quantitative, usually rely on ‘capturing’ luminally-exposed proteins after perfusion with chemical labeling reagents, followed by analysis with various mass spectrometry-based approaches. This manuscript reviews recent advances in proteomic analyses of luminal membranes of BEC in vitro and in vivo and their applications in translational studies focused on developing novel delivery methods across the BBB.

Thiolated Chitosan-Centella asiatica Nanocomposite: A Potential Brain Targeting Strategy Through Nasal Route.

The major challenge associated with the treatment of neurological disorders is the inefficiency of drugs to enter the Central Nervous System (CNS). Polymer-drug conjugates are now being tailored to overcome this hindrance associated with conventional drugs. The study aimed at developing polymer hybrid nasal nanocomposite for enhanced delivery of Centella to the CNS. Thiolated chitosan was complexed with Centella to form a composite using EDAC hydrochloride. The composite was characterized by FTIR, XRD, NMR, and MS. Further, this composite was converted into a nanoformulation by the ionic-gelation method, characterized, and subjected to ex vivo permeation studies. Additionally, MTT assay was performed using Human Uumbilical cord Vein Endothelial Cells (HUVECs) mimicking Blood-Brain Barrier (BBB) to establish the safety of nanocomposite. The targeting efficacy was predicted by molecular docking studies against receptors associated with BBB. The FTIR, XRD, NMR, and MS studies confirmed the chemical conjugation of thiolated chitosan with Centella. Nanocomposite characterization through SEM, AFM, and DLS confirmed the size and stability of the developed nanocomposite having a zeta potential of - 14.5mV and PDI of 0.260. The nanocomposite showed no signs of nasal ciliotoxicity and good permeation of 89.44 ± 1.75% (mean ± SD, n = 3) at 8h across the nasal mucosa. MTT assay showed that the nanocomposite had lesser toxicity compared to the free drug (IC50 of Centella-269.1μg/mL and IC50 of CTC nanocomposite-485.375μg/mL). The affinity of polymer to the BBB receptors as proved by docking studies suggests the ability of polymer-based nanocomposite to concentrate in the brain post nasal administration.

Highly Specific Blood-Brain Barrier Transmigrating Single-Domain Antibodies Selected by an In Vivo Phage Display Screening.

A major bottleneck in the successful development of central nervous system (CNS) drugs is the discovery and design of molecules that can cross the blood-brain barrier (BBB). Nano-delivery strategies are a promising approach that take advantage of natural portals of entry into the brain such as monoclonal antibodies (mAbs) targeting endogenous BBB receptors. However, the main selected mAbs rely on targeting broadly expressed receptors, such as the transferrin and insulin receptors, and in selection processes that do not fully mimic the native receptor conformation, leading to mistargeting and a low fraction of the administered dose effectively reaching the brain. Thus, there is an urgent need to identify new BBB receptors and explore novel antibody selection approaches that can allow a more selective delivery into the brain. Considering that in vitro models fail to completely mimic brain structure complexity, we explored an in vivo cell immunization approach to construct a rabbit derived single-domain antibody (sdAb) library towards BBB endothelial cell receptors. The sdAb antibody library was used in an in vivo phage display screening as a functional selection of novel BBB targeting antibodies. Following three rounds of selections, next generation sequencing analysis, in vitro brain endothelial barrier (BEB) model screenings and in vivo biodistribution studies, five potential sdAbs were identified, three of which reaching >0.6% ID/g in the brain. To validate the brain drug delivery proof-of-concept, the most promising sdAb, namely RG3, was conjugated at the surface of liposomes encapsulated with a model drug, the pan-histone deacetylase inhibitor panobinostat (PAN). The translocation efficiency and activity of the conjugate liposome was determined in a dual functional in vitro BEB-glioblastoma model. The RG3 conjugated PAN liposomes enabled an efficient BEB translocation and presented a potent antitumoral activity against LN229 glioblastoma cells without influencing BEB integrity. In conclusion, our in vivo screening approach allowed the selection of highly specific nano-antibody scaffolds with promising properties for brain targeting and drug delivery.

ApoE-Targeting Increases the Transfer of Solid Lipid Nanoparticles with Donepezil Cargo across a Culture Model of the Blood-Brain Barrier.

Pharmacological treatment of central nervous system (CNS) disorders is difficult, because the blood–brain barrier (BBB) restricts the penetration of many drugs into the brain. To solve this unmet therapeutic need, nanosized drug carriers are the focus of research efforts to develop drug delivery systems for the CNS. For the successful delivery of nanoparticles (NPs) to the brain, targeting ligands on their surface is necessary. Our research aim was to design a nanoscale drug delivery system for a more efficient transfer of donepezil, an anticholinergic drug in the therapy of Alzheimer’s disease across the BBB. Rhodamine B-labeled solid lipid nanoparticles with donepezil cargo were prepared and targeted with apolipoprotein E (ApoE), a ligand of BBB receptors. Nanoparticles were characterized by measurement of size, polydispersity index, zeta potential, thermal analysis, Fourier-transform infrared spectroscopy, in vitro release, and stability. Cytotoxicity of nanoparticles were investigated by metabolic assay and impedance-based cell analysis. ApoE-targeting increased the uptake of lipid nanoparticles in cultured brain endothelial cells and neurons. Furthermore, the permeability of ApoE-targeted nanoparticles across a co-culture model of the BBB was also elevated. Our data indicate that ApoE, which binds BBB receptors, can potentially be exploited for successful CNS targeting of solid lipid nanoparticles.

Single domain antibody-based vectors in the delivery of biologics across the blood–brain barrier: a review

Biologics are a promising and effective method for the treatment of central nervous system (CNS) diseases. The blood-brain barrier (BBB) is a natural barrier for the delivery of biologics into the brain, which decreases the effective concentration of drugs in the CNS. A range of strategies has been explored to transport biologics across the BBB endothelium, typically via receptor-mediated transcytosis (RMT), which involving molecules for endogenous BBB receptors to be fused with biologics. This review emphasized a category of novel alternative RMT-targeting vectors: single domain antibodies (sdAb). SdAbs are a unique category of antibodies derived from naturally occurring heavy-chain-only antibodies. Herein, we describe their properties, mechanisms, modifications, and translational perspectives for their ability to transmigrate across the BBB in vitro and in vivo in detail.

Human induced pluripotent stem cells (BIONi010-C) generate tight cell monolayers with blood-brain barrier traits and functional expression of large neutral amino acid transporter 1 (SLC7A5)

The barrier properties of the brain capillary endothelium, the blood-brain barrier (BBB) restricts uptake of most small and all large molecule drug compounds to the CNS. There is a need for predictive human in vitro models of the BBB to enable studies of brain drug delivery. Here, we investigated whether human induced pluripotent stem cell (hiPSC) line (BIONi010-C) could be differentiated to brain capillary endothelial- like cells (BCEC) and evaluated their potential use in drug delivery studies.BIONi010-C hIPSCs were differentiated according to established protocols. BCEC monolayers displayed transendothelial electrical resistance (TEER) values of 5,829±354 Ω∙cm2, a Papp,mannitol of 1.09±0.15 ∙ 10−6 cm∙s−1 and a Papp,diazepam of 85.7 ± 5.9 ∙ 10−6 cm ∙s−1. The Pdiazepam/Pmannitol ratio of ~80, indicated a large dynamic passive permeability range. Monolayers maintained their integrity after medium exchange. Claudin-5, Occludin, Zonulae Occludens 1 and VE-Cadherin were expressed at the cell-cell contact zones. Efflux transporters were present at the mRNA level, but functional efflux of substrates was not detected. Transferrin-receptor (TFR), Low density lipoprotein receptor-related protein 1 (LRP1) and Basigin receptors were expressed at the mRNA-level. The presence and localization of TFR and LRP1 were verified at the protein level. A wide range of BBB-expressed solute carriers (SLC's) were detected at the mRNA level. The presence and localization of SLC transporters GLUT1 and LAT1 was verified at the protein level. Functional studies revealed transport of the LAT1 substrate [3H]-L-Leucine and the LRP1 substrate angiopep-2.In conclusion, we have demonstrated that BIONi010-C-derived BCEC monolayers exhibited, BBB properties including barrier tightness and integrity, a high dynamic range, expression of some of the BBB receptor and transporter expression, as well as functional transport of LAT1 and LRP1 substrates. This suggests that BIONi010-C-derived BCEC monolayers may be useful for studying the roles of LAT-1 and LRP1 in brain drug delivery.

Peptide Mediated Brain Delivery of Nano- and Submicroparticles: A Synergistic Approach.

The brain is a complex, regulated organ with a highly controlled access mechanism: The Blood-Brain Barrier (BBB). The selectivity of this barrier is a double-edged sword, being both its greatest strength and weakness. This weakness is evident when trying to target therapeutics against diseases within the brain. Diseases such as metastatic brain cancer have extremely poor prognosis due to the poor permeability of many therapeutics across the BBB. Peptides can be designed to target BBB receptors and gain access to the brain by transcytosis. These peptides (known as BBB-shuttles) can carry compounds, usually excluded from the brain, across the BBB. BBB-shuttles are limited by poor loading of therapeutics and degradation of the peptide and cargo. Likewise, nano- submicro- and microparticles can be fine-tuned to limit their degradation and with high loading of therapeutics. However, most nano- and microparticles’ core materials completely lack efficient targeting, with a few selected materials able to cross the BBB passively. Combining the selectivity of peptides with the high loading potential of nano-, microparticles offers an exciting strategy to develop novel, targeted therapeutics towards many brain disorders and diseases. Nevertheless, at present the field is diverse, in both scope and nomenclature, often with competing or contradictory names. In this review, we will try to address some of these issues and evaluate the current state of peptide mediated nano,-microparticle transport to the brain, analyzing delivery vehicle type and peptide design, the two key components that must act synergistically for optimal therapeutic impact.

Delivery of Biologics Across the Blood–Brain Barrier with Molecular Trojan Horse Technology

Biologics are potential new therapeutics for many diseases of the central nervous system. Biologics include recombinant lysosomal enzymes, neurotrophins, decoy receptors, and therapeutic antibodies. These are large molecule drugs that do not cross the blood-brain barrier (BBB). All classes of biologics have been tested, without success, in clinical trials of brain disease over the last 25years. In none of these past clinical trials was the biologic re-engineered to enable transport across the BBB. If the biologic does not cross the BBB, the drug cannot reach the target site in brain, and success in a clinical trial is not expected. Biologics can be re-engineered for BBB transport with the use of molecular Trojan horse technology. A BBB molecular Trojan horse is a monoclonal antibody (MAb) against an endogenous BBB receptor transporter, such as the insulin receptor or transferrin receptor. The receptor-specific MAb penetrates the brain via transport on the endogenous BBB receptor. The MAb acts as a molecular Trojan horse to deliver across the BBB the biologic pharmaceutical that is genetically fused to the MAb. The lead Trojan horse is a MAb against the human insulin receptor (HIR), and HIRMAb-derived fusion proteins have entered clinical trials for the treatment of brain disease.

Targeting Malignant Brain Tumors with Antibodies.

Antibodies have been shown to be a potent therapeutic tool. However, their use for targeting brain diseases, including neurodegenerative diseases and brain cancers, has been limited, particularly because the blood–brain barrier (BBB) makes brain tissue hard to access by conventional antibody-targeting strategies. In this review, we summarize new antibody therapeutic approaches to target brain tumors, especially malignant gliomas, as well as their potential drawbacks. Many different brain delivery platforms for antibodies have been studied such as liposomes, nanoparticle-based systems, cell-penetrating peptides (CPPs), and cell-based approaches. We have already shown the successful delivery of single-chain fragment variable (scFv) with CPP as a linker between two variable domains in the brain. Antibodies normally face poor penetration through the BBB, with some variants sufficiently passing the barrier on their own. A “Trojan horse” method allows passage of biomolecules, such as antibodies, through the BBB by receptor-mediated transcytosis (RMT). Such examples of therapeutic antibodies are the bispecific antibodies where one binding specificity recognizes and binds a BBB receptor, enabling RMT and where a second binding specificity recognizes an antigen as a therapeutic target. On the other hand, cell-based systems such as stem cells (SCs) are a promising delivery system because of their tumor tropism and ability to cross the BBB. Genetically engineered SCs can be used in gene therapy, where they express anti-tumor drugs, including antibodies. Different types and sources of SCs have been studied for the delivery of therapeutics to the brain; both mesenchymal stem cells (MSCs) and neural stem cells (NSCs) show great potential. Following the success in treatment of leukemias and lymphomas, the adoptive T-cell therapies, especially the chimeric antigen receptor-T cells (CAR-Ts), are making their way into glioma treatment as another type of cell-based therapy using the antibody to bind to the specific target(s). Finally, the current clinical trials are reviewed, showing the most recent progress of attractive approaches to deliver therapeutic antibodies across the BBB aiming at the specific antigen.

Novel Peptides Derived from Dengue Virus Capsid Protein Translocate Reversibly the Blood-Brain Barrier through a Receptor-Free Mechanism.

The delivery of therapeutic molecules to the central nervous system is hampered by poor delivery across the blood-brain barrier (BBB). Several strategies have been proposed to enhance transport into the brain, including invasive techniques and receptor-mediated transport (RMT). Both approaches have several drawbacks, such as BBB disruption, receptor saturation, and off-target effects, raising safety issues. Herein, we show that specific domains of Dengue virus type 2 capsid protein (DEN2C) can be used as trans-BBB peptide vectors. Their mechanism of translocation is receptor-independent and consistent with adsorptive-mediated transport (AMT). One peptide in particular, named PepH3, reaches equilibrium distribution concentrations across the BBB in less than 24 h in a cellular in vitro assay. Importantly, in vivo biodistribution data with radiolabeled peptide derivatives show high brain penetration. In addition, there is fast clearance from the brain and high levels of excretion, showing that PepH3 is a very good candidate to be used as a peptide shuttle taking cargo in and out of the brain.

Immunoliposomes doubly targeted to transferrin receptor and to α-synuclein.

Aim:The present study was designed to test the cellular uptake of PEGylated liposomes targeted to transferrin receptor and to α-synuclein by a cell model of the blood–brain barrier (BBB).Materials & methods:PEGylated immunoliposomes were prepared with anti-transferrin receptor OX26 and anti-α-synuclein LB509 antibodies to overcome the BBB in Parkinson's disease.Results:The doubly targeted immunoliposomes bind to transferrin receptor and to α-synuclein protein, as assessed by ELISA assays. We establish that 40% of an encapsulated tested drug (epigallocatechin-3-gallate) is released in a time frame of 44 h, which is reasonable for sustained release. The cellular uptake of doubly targeted immunoliposomes in cultured brain endothelial cells hCMEC/D3 was two-times more efficient than that of PEGylated liposomes.Conclusion:Immunoliposomes targeted to BBB receptors and to α-synuclein could potentially enable the transport of drugs across the BBB and reach one of the drug targets in Parkinson's disease.

Setting-up an <em>In Vitro</em> Model of Rat Blood-brain Barrier (BBB): A Focus on BBB Impermeability and Receptor-mediated Transport

The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and characterize a highly reproducible rat syngeneic in vitro model of the BBB using co-cultures of primary rat brain endothelial cells (RBEC) and astrocytes to study receptors involved in transcytosis across the endothelial cell monolayer. Astrocytes were isolated by mechanical dissection following trypsin digestion and were frozen for later co-culture. RBEC were isolated from 5-week-old rat cortices. The brains were cleaned of meninges and white matter, and mechanically dissociated following enzymatic digestion. Thereafter, the tissue homogenate was centrifuged in bovine serum albumin to separate vessel fragments from nervous tissue. The vessel fragments underwent a second enzymatic digestion to free endothelial cells from their extracellular matrix. The remaining contaminating cells such as pericytes were further eliminated by plating the microvessel fragments in puromycin-containing medium. They were then passaged onto filters for co-culture with astrocytes grown on the bottom of the wells. RBEC expressed high levels of tight junction (TJ) proteins such as occludin, claudin-5 and ZO-1 with a typical localization at the cell borders. The transendothelial electrical resistance (TEER) of brain endothelial monolayers, indicating the tightness of TJs reached 300 ohm·cm2 on average. The endothelial permeability coefficients (Pe) for lucifer yellow (LY) was highly reproducible with an average of 0.26 ± 0.11 x 10-3 cm/min. Brain endothelial cells organized in monolayers expressed the efflux transporter P-glycoprotein (P-gp), showed a polarized transport of rhodamine 123, a ligand for P-gp, and showed specific transport of transferrin-Cy3 and DiILDL across the endothelial cell monolayer. In conclusion, we provide a protocol for setting up an in vitro BBB model that is highly reproducible due to the quality assurance methods, and that is suitable for research on BBB transporters and receptors.

Setting-up an in vitro model of rat blood-brain barrier (BBB): a focus on BBB impermeability and receptor-mediated transport.

The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and characterize a highly reproducible rat syngeneic in vitro model of the BBB using co-cultures of primary rat brain endothelial cells (RBEC) and astrocytes to study receptors involved in transcytosis across the endothelial cell monolayer. Astrocytes were isolated by mechanical dissection following trypsin digestion and were frozen for later co-culture. RBEC were isolated from 5-week-old rat cortices. The brains were cleaned of meninges and white matter, and mechanically dissociated following enzymatic digestion. Thereafter, the tissue homogenate was centrifuged in bovine serum albumin to separate vessel fragments from nervous tissue. The vessel fragments underwent a second enzymatic digestion to free endothelial cells from their extracellular matrix. The remaining contaminating cells such as pericytes were further eliminated by plating the microvessel fragments in puromycin-containing medium. They were then passaged onto filters for co-culture with astrocytes grown on the bottom of the wells. RBEC expressed high levels of tight junction (TJ) proteins such as occludin, claudin-5 and ZO-1 with a typical localization at the cell borders. The transendothelial electrical resistance (TEER) of brain endothelial monolayers, indicating the tightness of TJs reached 300 ohm·cm2 on average. The endothelial permeability coefficients (Pe) for lucifer yellow (LY) was highly reproducible with an average of 0.26 ± 0.11 x 10-3 cm/min. Brain endothelial cells organized in monolayers expressed the efflux transporter P-glycoprotein (P-gp), showed a polarized transport of rhodamine 123, a ligand for P-gp, and showed specific transport of transferrin-Cy3 and DiILDL across the endothelial cell monolayer. In conclusion, we provide a protocol for setting up an in vitro BBB model that is highly reproducible due to the quality assurance methods, and that is suitable for research on BBB transporters and receptors.