Human Blood-brain Barrier Research Articles

Development and Blood–Brain Barrier Penetration of Nanovesicles Loaded with Cannabidiol

Background: Cannabidiol (CBD) is a highly lipophilic compound with potential therapeutic applications in neurological disorders. However, its poor aqueous solubility and bioavailability, coupled with instability in physiological conditions, significantly limit its clinical use. Objectives: This study aimed to develop and characterize nanovesicles incorporating Tween 20 to enhance CBD encapsulation, stability, and the performance across the blood–brain barrier (BBB). Methods: Nanovesicles were prepared via thin-film hydration followed by sonication and optimized for size, polydispersity index, and zeta potential. Stability studies were conducted under physiological conditions and during storage at 4 °C. In vitro release studies employed the dialysis bag method, while permeability across the BBB was assessed using PAMPA-BBB and the hCMEC/D3-BBB cell line, characterized for brain endothelial phenotype and largely employed as a model of human blood–brain barrier (BBB) function. Cytotoxicity was evaluated via MTT and LDH assays. Results: The quantification of CBD was carried out by HPLC-DAD and HPLC-MS/MS. Nanovesicles with Tween 20 (VS-CBD) exhibited smaller size (65.27 ± 1.27 nm vs. 90.7 ± 0.2), lower polydispersity (0.230 ± 0.005 vs. 0.295 ± 0.003), and higher stability compared to conventional liposomes (L-CBD). VS-CBD achieved high encapsulation efficiency (96.80 ± 0.96%) and recovery (99.89 ± 0.52%). Release studies showed sustained CBD release with Higuchi model fitting (R2 = 0.9901). Both PAMPA-BBB and hCMEC/D3-BBB cell lines demonstrated an improved controlled permeability of the formulation compared to free CBD. Cytotoxicity tests confirmed the good biocompatibility of VS-CBD formulations. The addition of Tween 20 to nanovesicles enhanced CBD encapsulation, stability, and controlled release. Conclusions: These nanovesicles represent a promising strategy to improve CBD delivery to the brain, offering sustained therapeutic effects and reduced dosing frequency, potentially benefiting the treatment of neurological disorders.

Nanoparticle Interactions with the Blood Brain Barrier: Insights from Drosophila and Implications for Human Astrocyte Targeted Therapies.

This review explores the intricate connections between Drosophila models and the human blood-brain barrier (BBB) with nanoparticle-based approaches for neurological treatment. Drosophila serves as a powerful model organism due to its evolutionary conservation of key biological processes, particularly in the context of the BBB, which is formed by glial cells that share structural and functional similarities with mammalian endothelial cells. Recent advancements in nanoparticle technology have highlighted their potential for effective drug delivery across the BBB, utilizing mechanisms such as passive diffusion, receptor-mediated transcytosis, and carrier-mediated transport. The ability to engineer nanoparticles with specific physicochemical properties-such as size, surface charge, and functionalization-enhances their targeting capabilities, particularly towards astrocytes, which play a crucial role in maintaining BBB integrity and responding to neuroinflammation. Insights gained from Drosophila studies have informed the design of personalized nanomedicine strategies aimed at treating neurodegenerative diseases, including Alzheimer's, Parkinson's disease etc. As research progresses, the integration of findings from Drosophila models with emerging humanized BBB systems will pave the way for innovative therapeutic approaches that improve drug delivery and patient outcomes in neurological disorders.

Essential Oils from Citrus Peels Promote Calcium Overload-Induced Calcicoptosis in U251 Cells.

Citrus peel essential oils (CPEOs) have demonstrated substantial medicinal potential for glioblastoma treatment because of their extensive antitumor effects, low potential for drug resistance, and ability to cross the human blood-brain barrier. In this study, the chemical compositions of five CPEOs were analyzed via gas chromatography-mass spectrometry (GC-MS). CCK8 assays were used to evaluate the ability of five CPEOs to inhibit U251 human glioblastoma cells, and XLB and RA were selected for further investigation. Through wound healing assays and cell cycle and apoptosis analyses via flow cytometry, it was revealed that these CPEOs inhibited cell migration, arrested the cell cycle at G1/G0, and induced apoptosis with similar levels of inhibition. After CPEOs treatment, the intracellular Ca2+ content and reactive oxygen species levels in U251 cells increased significantly, whereas the mitochondrial membrane potential decreased. Additionally, the antioxidant enzyme system (SOD, POD, CAT, and GR) and the nonenzymatic defense system (GSH) were inhibited, leading to an increase in lipid peroxidation. qRT-PCR indicated the significant upregulation of intracellular calcium ion signaling pathways and the upregulation of mitochondrial apoptosis-related genes. Additionally, the activation of calcicoptosis-related indicators induced by the CPEOs could be reversed by inhibitor treatment, confirming that both of the selected CPEOs inhibit tumors by activating calcicoptosis-related pathways. These findings highlight the immense potential of CPEOs in healthcare and pharmaceutical applications by not only providing a scientific basis for the potential application of CPEOs in the treatment of glioblastoma but also offering new insights for the development of novel antitumor drugs.

The Aβ42:Aβ40 ratio modulates aggregation in beta-amyloid oligomers and drives metabolic changes and cellular dysfunction

The pathophysiological role of Aβ42 oligomers in the onset of Alzheimer’s disease (AD) is heavily disputed, pivoting research toward investigating mixed oligomers composed of Aβ42 and Aβ40, which is more abundant but less aggregation-prone. This study investigates Aβ42:Aβ40 oligomers in different ratios, examining their adverse effects on endothelial cells, neurons, astroglia, and microglia, as well as in a human blood–brain barrier (BBB) model. Combining label-free Raman microscopy with complementary imaging techniques and biochemical assays, we show the prominent impact of Aβ40 on Aβ42 fibrillation, suggesting an inhibitory effect on aggregation. Mixed oligomers, especially with low proportions of Aβ42, were equally detrimental as pure Aβ42 oligomers regarding cell viability, functionality, and metabolism. They also differentially affected lipid droplet metabolism in BBB-associated microglia, indicating distinct pathophysiological responses. Our findings demonstrate the overarching significance of the Aβ42:Aβ40 ratio in Aβ oligomers, challenging the traditional focus on Aβ42 in AD research.

VCPP2319 interacts with metastatic breast cancer extracellular vesicles (EVs) and transposes a human blood-brain barrier model

Brain metastases (BM) are frequently found in cancer patients and, though their precise incidence is difficult to estimate, there is evidence for a correlation between BM and specific primary cancers, such as lung, breast, and skin (melanoma). Among all these, breast cancer is the most frequently diagnosed among women and, in this case, BM cause a critical reduction of the overall survival (OS), especially in triple negative breast cancer (TNBC) patients. The main challenge of BM treatment is the impermeable nature of the blood-brain barrier (BBB), which shields the central nervous systems (CNS) from chemotherapeutic drugs. Extracellular vesicles (EVs) have been proposed as ideal natural drug carriers and these may exhibit some advantages over synthetic nanoparticles (NPs). In this work, we isolate breast cancer-derived EVs and study their ability to carry vCPP2319, a peptide with dual cell-penetration and anticancer activities. The selective cytotoxicity of anticancer peptide-loaded EVs towards breast cancer cells and their ability to translocate an in vitro BBB model are also addressed. Overall, it was possible to conclude that vCPP2319 naturally interacts with breast cancer-derived EVs, being retained at the surface of these vesicles. Moreover, the results revealed a cytotoxic activity for peptide-loaded EVs similar to that obtained with the peptide alone and the ability of peptide-loaded EVs to translocate an in vitro BBB model, which contrasts with the results obtained with the peptide alone. In conclusion, this work supports the use of EVs in the development of biological drug-delivery systems (DDS) capable of translocating the BBB.

Potential MAO-B Inhibitors from Cissampelos Capensis L.f.: ADMET, Molecular Docking, Dynamics, and DFT Insights.

This study explores the therapeutic potential of three proaporphine alkaloids-cissamaline, cissamanine, and cissamdine, which were recently isolated from Cissampelos capensis L.f., against Parkinson's disease (PD). Using computational techniques, we investigated their efficacy as inhibitors of a key protein in PD. ADMET analysis demonstrated that these alkaloids conform to the Lipinski, Pfizer, Golden Triangle, and GSK rules, indicating favorable safety, oral bioavailability, and a high probability of passing the human intestinal and blood-brain barriers. They were neither substrates nor inhibitors of any CYP enzymes tested, indicating minimal metabolic interference and an enhanced safety profile. Molecular docking studies revealed binding energies of -9.05 kcal/mol (cissamaline), -9.95 kcal/mol (cissamanine), and -10.65 kcal/mol (cissamdine) against MAO-B, a critical PD target, surpassing the control (zonisamide, -6.96 kcal/mol). The molecular interaction analyses were also promising, with interactions comparable to the control. Molecular dynamics (MD) simulations confirmed stable protein-ligand interactions, with root-mean-square deviation (RMSD) values ranging from 1.03 Å to 3.92 Å, root-mean-square fluctuation (RMSF) values remaining below 1.14 Å, and radius of gyration (RGyr) values between 20.20 Å and 20.50 Å, indicating compact structures. Hydrogen bonding analysis revealed maximum hydrogen bond counts of 6 (cissamanine), 5 (cissamaline), and 4 (cissamdine), demonstrating robust interactions with MAO-B. Density Functional Theory (DFT) calculations revealed the highest electrophilicity (ω =0.151), highest electron affinity (EA =0.075), and smallest HOMO-LUMO gap (ΔE =0.130) for cissamanine, indicating enhanced reactivity. These results advocate for further in vitro and in vivo studies to evaluate the compounds' potential as PD therapeutics.

In vitro characterization of taurine transport using the human brain microvascular endothelial cell line as a human blood-brain barrier model

Taurine, a sulfur-containing β-amino acid, has various roles in the brain including cellular osmoregulation and neuroprotection. For adequate supply to the brain, taurine has to pass through the blood-brain barrier (BBB); however, the associated mechanism behind crossing the human BBB is not fully understood. Therefore, we characterized taurine transport in vitro using the human brain microvascular endothelial (hCMEC/D3) cell line, a model of human BBB function. [3H]Taurine uptake by hCMEC/D3 cells exhibited time-, as well as extracellular Na+- and Cl−-dependence. The uptake was saturable with a Km of 19 μM and was inhibited by GABA at an IC50 of 328 μM, which were similar to Km values of taurine transporter (TauT)-mediated transport of taurine and GABA, respectively, suggesting that TauT is a major contributor to taurine uptake. For distribution to the brain, taurine must undergo cellular efflux after uptake. Taurine efflux from hCMEC/D3 cells increased for at least 60 min, and monocarboxylate transporter 7 (MCT7)-targeted siRNA significantly reduced MCT7 mRNA levels and [3H]taurine efflux by 93% and 12%, respectively, suggesting that MCT7 partly contributes to taurine efflux from hCMEC/D3 cells. Taken together, these results suggest that TauT and MCT7 function cooperatively in the human BBB.

Blood-brain barrier permeability increases with the differentiation of glioblastoma cells in vitro

BackgroundGlioblastoma multiforme (GBM) is an aggressive tumor, difficult to treat pharmacologically because of the blood-brain barrier (BBB), which is rich in ATP-binding cassette (ABC) transporters and tight junction (TJ) proteins. The BBB is disrupted within GBM bulk, but it is competent in brain-adjacent-to-tumor areas, where eventual GBM foci can trigger tumor relapse. How GBM cells influence the permeability of BBB is poorly investigated.MethodsTo clarify this point, we co-cultured human BBB models with 3 patient-derived GBM cells, after separating from each tumor the stem cell/neurosphere (SC/NS) and the differentiated/adherent cell (AC) components. Also, we set up cultures of BBB cells with the conditioned medium of NS or AC, enriched or depleted of IL-6. Extracellular cytokines were measured by protein arrays and ELISA. The intracellular signaling in BBB cells was measured by immunoblotting, in the presence of STAT3 pharmacological inhibitor or specific PROTAC. The competence of BBB was evaluated by permeability assays and TEER measurement.ResultsThe presence of GBM cells or their conditioned medium increased the permeability to doxorubicin, mitoxantrone and dextran-70, decreased TEER, down-regulated ABC transporters and TJ proteins at the transcriptional level. These effects were higher with AC or their medium than with NS. The secretome analysis identified IL-6 as significantly more produced by AC than by NS. Notably, AC-conditioned medium treated with an IL-6 neutralizing antibody reduced the BBB permeability to NS levels, while NS-conditioned medium enriched with IL-6 increased BBB permeability to AC levels. Mechanistically, IL-6 released by AC GBM cells activated STAT3 in BBB cells. In turn, STAT3 down-regulated ABC transporter and TJ expression, increased permeability and decreased TEER. The same effects were obtained in BBB cells treated with STA-21, a pharmacological inhibitor of STAT3, or with a PROTAC targeting STAT3.ConclusionsOur work demonstrates for the first time that the degree of GBM differentiation influences BBB permeability. The crosstalk between GBM cells that release IL-6 and BBB cells that respond by activating STAT3, controls the expression of ABC transporters and TJ proteins on BBB. These results may pave the way for novel therapeutic tools to tune BBB permeability and improve drug delivery to GBM.

Involvement of Proton-Coupled Organic Cation Antiporter in Human Blood-Brain Barrier Transport of Mesoridazine and Metoclopramide.

Mesoridazine and metoclopramide are cationic drugs that are distributed in the human brain despite being substrates of multidrug resistance protein 1 (MDR1), an efflux transporter expressed at the blood-brain barrier (BBB). We investigated their transport mechanisms at the BBB using hCMEC/D3, a human cerebral microvascular endothelial cell line often used as an in vitro BBB model. The cells exhibited time- and concentration-dependent uptake of mesoridazine and metoclopramide, with Km values of 34 and 277 µM, respectively. The uptake of both drugs significantly decreased in the presence of typical inhibitors and/or substrates of the H+-coupled organic cation (H+/OC) antiporter but not in the presence of inhibitors or substrates of organic cation transporters (OCTs), OCTN2, OATPs, SLC35F2, or the plasma membrane monoamine transporter (PMAT). Furthermore, metoclopramide uptake by hCMEC/D3 cells was pH- and energy-dependent, whereas mesoridazine uptake was unaffected by intracellular acidification and treatment with metabolic inhibitors. These results suggest that the H+/OC antiporter is involved in the influx of mesoridazine and metoclopramide into the brain across the BBB.

High-Density Lipoprotein Mimetic Peptide 4F Reduces Toxic Amyloid-Beta Exposure to the Blood-Brain Barrier Endothelium in Alzheimer's Disease Transgenic Mice.

Aβ accumulation in the blood-brain barrier (BBB) endothelium, which lines the cerebrovascular lumen, is a significant contributor to cerebrovascular dysfunction in Alzheimer's disease (AD). Reduced high-density lipoprotein (HDL) levels are associated with increased AD risk, and the HDL mimetic peptide 4F has been developed as a promising therapeutic agent to improve cerebrovascular health in AD. In this study, we evaluated the impact of 4F on 125I-Aβ42 blood-to-brain distribution using dynamic SPECT/CT imaging in both wild-type and APP/PS1 transgenic mice. Graphical analysis of the imaging data demonstrated that 4F significantly reduced the blood-to-brain influx rate in wild-type mice and the distribution of 125I-Aβ42 in the BBB endothelium in APP/PS1 mice. To elucidate the molecular mechanisms underlying the effect of 4F, we evaluated its impact on the p38 pathway and its role in mediating Aβ42 trafficking in human BBB endothelial cell monolayers. Treatment with 4F significantly decreased Aβ42 induced p38 activation in BBB endothelial cells. Furthermore, inhibition of p38 kinase significantly reduced endothelial accumulation of fluorescence-labeled Aβ42 and luminal-to-abluminal permeability across the cell monolayer. While our previous publication has hinted at the potential of 4F to reduce Aβ accumulation in the brain parenchyma, the current findings demonstrated the protective effect of 4F in reducing Aβ42 accumulation in the BBB endothelium of AD transgenic mice. These findings revealed the impact of a clinically tested agent, the HDL mimetic peptide 4F, on Aβ exposure to the BBB endothelium and offer novel mechanistic insights into potential therapeutic strategies to treat cerebrovascular dysfunction in AD.

Hypoxia-Induced Inflammation in In Vitro Model of Human Blood-Brain Barrier: Modulatory Effects of the Olfactory Ensheathing Cell-Conditioned Medium.

Hypoxia compromises the integrity of the blood-brain barrier (BBB) and increases its permeability, thereby inducing inflammation. Olfactory ensheathing cells (OECs) garnered considerable interest due to their neuroregenerative and anti-inflammatory properties. Here, we aimed to investigate the potential modulatory effects of OEC-conditioned medium (OEC-CM) on the response of human brain microvascular endothelial cells (HBMECs), constituting the BBB, when exposed to hypoxia. HBMECs were utilized to establish the in vitro BBB model. OECs were isolated from mouse olfactory bulbs, and OEC-CM was collected after 48h of culture. The effect of OEC-CM treatment on the HBMEC viability was evaluated under both normoxic and hypoxic conditions at 6h, 24h, and 30h. Western blot and immunostaining techniques were employed to assess NF-κB/phospho-NF-κB expression. HIF-1α, VEGF-A, and cPLA2 mRNA expression levels were quantified using digital PCR. ELISA assays were performed to measure PGE2, VEGF-A, IL-8 secretion, and cPLA2 specific activity. The in vitro formation of HBMEC capillary-like structures was examined using a three-dimensional matrix system. OEC-CM attenuated pro-inflammatory responses and mitigated the HIF-1α/VEGFA signaling pathway activation in HBMECs under hypoxic condition. Hypoxia-induced damage of the BBB can be mitigated by novel therapeutic strategies harnessing OEC potential.

Endothelial Neurogranin Regulates Blood-Brain Barrier Permeability via Modulation of the AKT Pathway.

Neurogranin (Ng) expression is a biomarker for Alzheimer's disease. A loss of brain Ng and an increase in CSF Ng positively correlate with cognitive decline. Ng is known to regulate neuronal calcium-calmodulin binding and synaptic plasticity, which are critical for learning/memory. Interestingly, we discovered that Ng is also expressed in mouse and human blood-brain barrier (BBB). However, the role of Ng expression in brain vasculature remains largely undefined. In this study, we investigated the role of Ng expression on neurovascular structure and function using Ng null mice and human cerebral microvascular endothelial (hCMEC/D3) cells. We performed brain clearing and immunolabeling of blood vessels from whole brains and brain slices. Deletion of Ng significantly decreases neurovascular density in mice. Using in vivo permeability assays, we found increased neurovascular permeability in Ng null mice. We also observed significant changes in the expression of tight junction proteins using western blot and immunofluorescent staining. To identify the molecular pathways involved, we carried out label-free proteomics on brain lysates from endothelial-specific Ng knockout mice. Ingenuity Pathway Analysis indicated that the AKT pathway is attenuated in the vasculature of endothelial-specific Ng knockout mice. To validate these in vivo findings, we pharmacologically manipulated AKT signaling in hCMEC/D3 cells and observed that inhibition of AKT activation causes increased permeability. Our results indicate that the loss of Ng expression alters neurovascular structure and permeability, potentially contributing to neurological dysfunction. Therefore, modulating Ng expression in the BBB may offer a novel therapeutic approach for Alzheimer's disease.

Rift Valley fever virus is able to cross the human blood-brain barrier in vitro by direct infection with no deleterious effects.

Rift Valley fever (RVF) is a zoonotic arboviral disease that causes recurrent epidemics in Africa that may trigger fatal neurological disorders. However, the mechanisms of neuroinvasion by which the RVF virus (RVFV) reaches the human central nervous system (CNS) remain poorly characterized. In particular, it is not clear how RVFV is able to cross the human blood-brain barrier (hBBB), which is a neurovascular endothelium that protects the brain by regulating brain and blood exchanges. To explore these mechanisms, we used an in vitro hBBB model to mimic in vivo hBBB selectiveness and apicobasal polarity. Our results highlight the ability of RVFV to cross the hBBB by direct infection in a non-structural protein S (NSs)-independent but strain-dependent manner, leading to astrocyte and pericyte infections. Interestingly, RVFV infection did not induce hBBB disruption and was associated with progressive elimination of infected cells with no impairment of the tight junction protein scaffold and barrier function. Our work also shows that NSs, a well described RVFV virulence factor, limited the establishment of the hBBB-induced innate immune response and subsequent lymphocyte recruitment. These results provide in vitro confirmation of the ability of RVFV to reach human CNS by direct infection of the hBBB without altering its barrier function, and provide new directions to explore human RVFV neurovirulence and neuroinvasion mechanisms.IMPORTANCEThe RVF virus (RVFV) is capable of infecting humans and inducing severe and fatal neurological disorders. Neuropathogenesis and human central nervous system (CNS) invasion mechanisms of RVFV are still unknown, with only historical studies of autopsy data from fatal human cases in the 1980s and exploration studies in rodent models. One of the gaps in understanding RVFV human pathogenesis is how RVFV is able to cross the blood-brain barrier (BBB) in order to reach the human CNS. For the first time, we show that RVFV is able to directly infect cells of the human BBB in vitro to release viral particles into the human CNS, a well-characterized neuroinvasion mechanism of pathogens. Furthermore, we demonstrate strain-dependent variability of this neuroinvasion mechanism, identifying possible viral properties that could be explored to prevent neurological disorders during RVFV outbreaks.

Erratum for Gaume et al., "Enterovirus A71 crosses a human blood-brain barrier model through infected immune cells".

Erratum for Gaume et al., "Enterovirus A71 crosses a human blood-brain barrier model through infected immune cells".

NX210c drug candidate peptide strengthens mouse and human blood-brain barriers

BackgroundAlterations of blood-brain barrier (BBB) and blood-spinal cord barrier have been documented in various animal models of neurodegenerative diseases and in patients. Correlations of these alterations with functional deficits suggest that repairing barriers integrity may represent a disease-modifying approach to prevent neuroinflammation and neurodegeneration induced by the extravasation of blood components into the parenchyma. Here, we screened the effect of a subcommissural organ-spondin-derived peptide (NX210c), known to promote functional recovery in several models of neurological disorders, on BBB integrity in vitro and in vivo.MethodsIn vitro, bEnd.3 endothelial cell (EC) monolayers and two different primary human BBB models containing EC, astrocytes and pericytes, in static and microfluidic conditions, were treated with NX210c (1-100 µM), or its vehicle, for 4 h and up to 5 days. Tight junction (TJ) protein levels, permeability to dextrans and transendothelial electrical resistance (TEER) were evaluated. In vivo, young and old mice (3- and 21-month-old, respectively) were treated daily intraperitoneally with NX210c at 10 mg/kg or its vehicle for 5 days and their brains collected at day 6 to measure TJ protein levels by immunohistochemistry.ResultsNX210c induced an increase in claudin-5 protein expression after 24-h and 72-h treatments in mouse EC. Occludin level was also increased after a 24-h treatment. Accordingly, NX210c decreased by half the permeability of EC to a 40-kDa FITC-dextran and increased TEER. In the human static BBB model, NX210c increased by ∼ 25% the TEER from 3 to 5 days. NX210c also increased TEER in the human 3D dynamic BBB model after 4 h, which was associated with a reduced permeability to a 4-kDa FITC-dextran. In line with in vitro results, after only 5 days of daily treatments in mice, NX210c restored aging-induced reduction of claudin-5 and occludin levels in the hippocampus, and also in the cortex for occludin.ConclusionsIn summary, we have gathered preclinical data showing the capacity of NX210c to strengthen BBB integrity. Through this property, NX210c holds great promises of being a disease-modifying treatment for several neurological disorders with high unmet medical needs.

Meta-analysis of the make-up and properties of in vitro models of the healthy and diseased blood-brain barrier.

In vitro models of the human blood-brain barrier (BBB) are increasingly used to develop therapeutics that can cross the BBB for treating diseases of the central nervous system. Here we report a meta-analysis of the make-up and properties of transwell and microfluidic models of the healthy BBB and of BBBs in glioblastoma, Alzheimer's disease, Parkinson's disease and inflammatory diseases. We found that the type of model, the culture method (static or dynamic), the cell types and cell ratios, and the biomaterials employed as extracellular matrix are all crucial to recapitulate the low permeability and high expression of tight-junction proteins of the BBB, and to obtain high trans-endothelial electrical resistance. Specifically, for models of the healthy BBB, the inclusion of endothelial cells and pericytes as well as physiological shear stresses (~10-20 dyne cm-2) are necessary, and when astrocytes are added, astrocytes or pericytes should outnumber endothelial cells. We expect this meta-analysis to facilitate the design of increasingly physiological models of the BBB.

Molecular dynamics simulation of the brain-isolated single-domain antibody/nanobody from camels through in vivo phage display screening.

During the last decade, there has been a significant rise in the use of therapeutic antibodies or passive immunotherapy for treating various conditions like inflammation and cancer. However, these proteins face challenges reaching the brain and often require specialized delivery methods such as single-domain antibodies (sdAbs). Traditional antibodies struggle to efficiently cross the blood-brain barrier (BBB), hindering their effectiveness. Receptor-mediated transcytosis (RMT) offers a promising pathway for transporting large molecules essential for brain function and treatment across the BBB. SdAbs and peptide ligands with an affinity for RMT receptors are commonly employed to enhance the transport of biotherapeutics compounds across the BBB. This research used a sdAbs phage-displayed library from 13 camelus dromedarius samples to identify sdABs that specifically bind to and are internalized by human BBB endothelial cells (ECs) through in vivo panning. One sdAb, defined as FB24, was isolated, sequenced, translated into an open reading frame (ORF), and subjected to three-dimensional (3D) modeling. Molecular docking and molecular dynamics simulations were carried out by the HADDOCK web server and GROMACS, respectively, to evaluate the interaction between FB24 and EC receptors in silico. The docking results revealed that FB24 exhibited binding activity against potential EC receptors with -1.7 to -2.7 ranged z score and maintained a stable structure. The docked complex of FB24-RAGE (receptor for advanced glycation end products, also known as advanced glycation end product receptor [AGER]) showed 18 hydrogen bonds and 213 non-bonded contacts. It was chosen for further analysis by molecular dynamics simulations by GROMACS. This complex showed a stable condition, and its root mean square deviation (RMSD) was 0.218 nm. The results suggest that FB24 could serve as a suitable carrier vector for transporting therapeutic and diagnostic agents across the BBB to the brain through a non-invasive route.

Developing a Triple Cell Culture Model of the Human Blood-Brain Barrier

Developing a Triple Cell Culture Model of the Human Blood-Brain Barrier

Developing a Triple Cell Culture Model of the Human Blood-Brain Barrier

Developing a Triple Cell Culture Model of the Human Blood-Brain Barrier

Single-cell dissection of the human blood-brain barrier and glioma blood-tumor barrier

The blood-brain barrier (BBB) serves as a crucial vascular specialization, shielding and nourishing brain neurons and glia while impeding drug delivery. Here, we conducted single-cell mRNA sequencing of human cerebrovascular cells from 13 surgically resected glioma samples and adjacent normal brain tissue. The transcriptomes of 103,230 cells were mapped, including 57,324 endothelial cells (ECs) and 27,703 mural cells (MCs). Both EC and MC transcriptomes originating from lower-grade glioma were indistinguishable from those of normal brain tissue, whereas transcriptomes from glioblastoma (GBM) displayed a range of abnormalities. Among these, we identified LOXL2-dependent collagen modification as a common GBM-dependent trait and demonstrated that inhibiting LOXL2 enhanced chemotherapy efficacy in both murine and human patient-derived xenograft (PDX) GBM models. Our comprehensive single-cell RNA sequencing-based molecular atlas of the human BBB, coupled with insights into its perturbations in GBM, holds promise for guiding future investigations into brain health, pathology, and therapeutic strategies.