Blood-brain Barrier Drug Research Articles

Transporter Expressions as Part of Required Scaling Factor to Support In vitro In vivo Extrapolation for Blood-Brain Barrier Drug Permeability.

Access of drugs to the central nervous system is limited by the blood-brain barrier, and this in turn affects drug efficacy/toxicity. To date, most drug discovery optimization paradigms have relied heavily on in vitro transporter assays and preclinical species pharmacokinetic evaluation to provide a qualitative assessment of human brain penetration. Because of the lack of human brain pharmacokinetic data, mechanistic models for preclinical species, combined with in vitro and in silico data, are useful for translation to human. These models require transporter expression data to be measured in both in vitro and in vivo systems. The purpose of this work was to quantify transporter expression and generate scaling factors (SFs) to enable in vitro in vivo extrapolation (IVIVE) of transporter-mediated processes and to support the development of a PBPK model of the brain in rats. SF represents the ratio of abundance of the relevant transporters in the tissue relative to transporter expressing cells. Using quantitative proteomics with QconCAT technology, the expression of human and rat P-gp (ABCB1/Abcb1) and BCRP/Bcrp (ABCG2/Abcg2) was measured in rat brain microvessels, mock and transfected cell lines including, (Madin-Darby canine kidney I (MDCK I), Madin-Darby canine kidney II (MDCK II) and pig kidney epithelial cells (LLC-PK1). P-gp expression ranged from 32 to 71 pmol/mg in rat brain microvessels, exceeding literature values of 14.1-25.2 pmol/mg microvessels proteins. Conversely, Bcrp expression ranged between 0.02-0.27 pmol/mg,proteinslower than the literature range (2-6.2 pmol/mg of proteins). P-gp expression in MDCK I and LLC-PK1 cells transfected with rat Mdr1a was similar (within 1.5-fold) as was human P-gp expression in MDR1 transfected LLC-PK1 and MDCK II cells. The generated SFs were 34.4 and 50.4 for brain P-gp (depending on the cell line used) and 0.53 for brain Bcrp. Endogenous P-gp transporter was detected in MDCK II cell lines when protein expression was measured using a surrogate peptide that was shared across species. The current work provides a framework for proteomics-informed translation of in vitro P-gp and BCRP-related kinetics of drugs and supports the development of PBPK models to predict drug disposition in the brain.

ROS-responsive quercetin-based polydopamine nanoparticles for targeting ischemic stroke by attenuating oxidative stress and neuroinflammation.

Ischemic stroke (IS), a prevalent cerebrovascular disorder, is characterized by high morbidity rates and significant disability. However, relevant drug therapy for IS still suffers from limitations such as limited blood-brain barrier (BBB) penetration efficiency, single therapeutic target, short half-life, and strong side effects. The development of multi-target neuroprotective agents using natural drug molecules with low toxicity and combining them with nanotechnology to improve BBB permeability and drug utilization is an important direction in the development of IS therapeutic strategies. Based on the anti-inflammatory and antioxidant properties of quercetin (Que), as well as the ROS-responsive degradation properties of polydopamine (PDA), an IS therapeutic strategy (Que@DAR NPs) was developed in this study. Que@DAR NPs were formed by dopamine wrapping Que by oxidative self-assembly and wrapping the rabies virus glycoprotein (RVG29) on the surface. The results showed that Que@DAR NPs greatly improved the dispersion stability of Que and exhibited ROS-responsive degradation properties. Cellular internalization assay in human neuroblastoma cells (SH-SY5Y) showed that RVG29 peptide substantially augmented the cellular uptake of Que@DAR NPs. Moreover, Que@DAR NPs can effectively reduce the oxidative damage of SH-SY5Y cells and induce the polarization of microglia to anti-inflammatory (M2) phenotype. In vivo studies further demonstrated that Que@DAR NPs inhibited neuroinflammation, reduced neuronal apoptosis, and significantly ameliorated neurological dysfunction in a rat model of middle cerebral artery occlusion (MCAO). In conclusion, Que@DAR NPs provide a safe and effective new strategy for the precision treatment of IS.

Fabrication of Biomimetic Hybrid Liposomes via Microfluidic Technology: Homotypic Targeting and Antitumor Efficacy Studies in Glioma Cells.

The treatment of glioblastoma is hindered by the blood-brain barrier (BBB) and rapid drug clearance by the immune system. To address these challenges, we propose a novel drug delivery system using liposomes modified with cell membrane fragments. These modified liposomes can evade the immune system, cross the BBB, and accumulate in tumor tissue through homotypic targeting, thereby delivering drugs like paclitaxel and carboplatin more effectively. In this work, the hybrid liposomes were synthesized using microfluidics and integrating 3D printing to produce the microfluidic devices. In vitro, we explored the homotypic targeting capability, BBB passing ability, and therapeutic efficacy of paclitaxel and carboplatin. The production of hybrid liposomes by microfluidics has been key to creating high-quality biomimetic nanoparticles, and the integration of 3D printing has simplified the production of microfluidic devices, making the process more efficient and economical. In vitro experiments have shown that these drug-loaded biomimetic hybrid liposomes are able to reach the homotypic target, cross the BBB, and maintain the efficacy of paclitaxel and carboplatin. The development of biomimetic hybrid liposomes represents a promising approach for the treatment of glioblastoma. By combining the advantages of liposomal drug delivery with the stealth properties and targeting capabilities of cell membrane fragments, these nanoparticles can potentially overcome the challenges associated with traditional therapies.

Latest Perspectives on Alzheimer's Disease Treatment: The Role of Blood-Brain Barrier and Antioxidant-Based Drug Delivery Systems.

There has been a growing interest recently in exploring the role of the blood-brain barrier (BBB) in the treatment of Alzheimer's disease (AD), a neurodegenerative disorder characterized by cognitive decline and memory loss that affects millions of people worldwide. Research has shown that the BBB plays a crucial role in regulating the entry of therapeutics into the brain. Also, the potential benefits of using antioxidant molecules for drug delivery were highlighted in Alzheimer's treatment to enhance the therapeutic efficacy and reduce oxidative stress in affected patients. Antioxidant-based nanomedicine shows promise for treating AD by effectively crossing the BBB and targeting neuroinflammation, potentially slowing disease progression and improving cognitive function. Therefore, new drug delivery systems are being developed to overcome the BBB and improve the delivery of therapeutics to the brain, ultimately improving treatment outcomes for AD patients. In this context, the present review provides an in-depth analysis of recent advancements in AD treatment strategies, such as silica nanoparticles loaded with curcumin, selenium nanoparticles loaded with resveratrol, and many others, focusing on the critical role of the BBB and the use of antioxidant-based drug delivery systems.

3,4-difluoro-2-(((4-phenoxyphenyl)imino)methyl)phenol with in silico predictions: Synthesis, spectral analyses, ADME studies, targets and biological activity, toxicity and adverse effects, site of metabolism, taste activity

In this study, a fluorinated Schiff base compound, named 3,4-difluoro-2-(((4-phenoxy phenyl)imino)methyl)phenol, was synthesized using the precursor compounds 4-phenoxyaniline and 2,3-difluoro-6-hydroxybenzaldehyde. This study aims to characterize the synthesized Schiff base and its structural properties and to investigate its pharmaceutical suitability. X-ray, 1H NMR, UV–vis, and FTIR spectroscopy were used to characterize the synthesized compound. The leading pharmaceutical properties investigated in this study include physicochemical properties, ADME analyses, target identification, prediction of biological activity, absorption in human gastrointestinal tract, blood-brain barrier permeability, drug classification, adverse and toxic effects, phase I and phase II metabolism, sites of metabolism and organoleptic properties. This paper also includes applications of many CADD methods that can be applied to small molecules. The results of the study show that the title compound has moderate physicochemical and ADME properties, moderate toxic and adverse effects, suitable intestinal absorption, and unsuitable BBB permeation. The most potential target of the title compound is the endoplasmic reticulum-associated amyloid-beta peptide-binding protein. The primary cleavage sites of the title compound are the oxygen atom in the phenol group, the nitrogen atom in the amine group, and the oxygen atom in the diphenyl ether.

Blood-Brain Barrier-Targeting Nanoparticles: Biomaterial Properties and Biomedical Applications in Translational Neuroscience.

Overcoming the blood-brain barrier (BBB) remains a significant hurdle in effective drug delivery to the brain. While the BBB serves as a crucial protective barrier, it poses challenges in delivering therapeutic agents to their intended targets within the brain parenchyma. To enhance drug delivery for the treatment of neurological diseases, several delivery technologies to circumvent the BBB have been developed in the last few years. Among them, nanoparticles (NPs) are one of the most versatile and promising tools. Here, we summarize the characteristics of NPs that facilitate BBB penetration, including their size, shape, chemical composition, surface charge, and importantly, their conjugation with various biological or synthetic molecules such as glucose, transferrin, insulin, polyethylene glycol, peptides, and aptamers. Additionally, we discuss the coating of NPs with surfactants. A comprehensive overview of the common in vitro and in vivo models of the BBB for NP penetration studies is also provided. The discussion extends to discussing BBB impairment under pathological conditions and leveraging BBB alterations under pathological conditions to enhance drug delivery. Emphasizing the need for future studies to uncover the inherent therapeutic properties of NPs, the review advocates for their role beyond delivery systems and calls for efforts translating NPs to the clinic as therapeutics. Overall, NPs stand out as a highly promising therapeutic strategy for precise BBB targeting and drug delivery in neurological disorders.

Machine Learning Exploration of the Relationship Between Drugs and the Blood-Brain Barrier: Guiding Molecular Modification.

This study aimed to improve the efficiency of pharmacotherapy for CNS diseases by optimizing the ability of drug molecules to penetrate the Blood-Brain Barrier (BBB). We established qualitative and quantitative databases of the ADME properties of drugs and derived characteristic features of compounds with efficient BBB penetration. Using these insights, we developed four machine learning models to predict a drug's BBB permeability by assessing ADME properties and molecular topology. We then validated the models using the B3DB database. For acyclovir and ceftriaxone, we modified the Hydrogen Bond Donors and Acceptors, and evaluated the BBB permeability using the predictive model. The machine learning models performed well in predicting BBB permeability on both internal and external validation sets. Reducing the number of Hydrogen Bond Donors and Acceptors generally improves BBB permeability. Modification only enhanced BBB penetration in the case of acyclovir and not ceftriaxone. The machine learning models developed can accurately predict BBB permeability, and many drug molecules are likely to have increased BBB penetration if the number of Hydrogen Bond Donors and Acceptors are reduced. These findings suggest that molecular modifications can enhance the efficacy of CNS drugs and provide practical strategies for drug design and development. This is particularly relevant for improving drug penetration of the BBB.

Abstract 5110: Exploring the effects of Indirubins in pediatric diffuse high grade glioma models

Abstract Diffuse midline glioma (DMG) is an invasive pediatric brain tumor which grows in the pons of the brainstem. The current course of treatment is focal radiation however, DMG continues to have a very poor prognosis with a median survival of less than 10 months. A major barrier for chemotherapeutic treatment of DMG is the blood brain barrier (BBB) which tightly controls access of molecules to the brain. This limits the efficacy of drugs to treat brain cancer and other diseases of the central nervous system. Previous studies from the Lawler lab and others have shown that the indirubin derivative, 6-bromoindirubin-3′-acetoxime(BIO-acetoxime/BIA), has anti-invasive properties and can enhance survival in glioblastoma xenograft models. Here, we investigated the effects of BIA on pediatric glioma models using a range of relevant assays. In an in vitro collagen migration assay, BIA was observed to significantly slow cell migration at a concentration of 1µM in DIPG 36 cells, over 72 hours. Furthermore, we have also shown that BIA modulates the tumor vasculature, which could limit tumor growth, and improves drug delivery to tumors by targeting tight junctions in tumor associated endothelium. The effect on BBB drug penetration was assessed in a multicellular 3D in vitro BBB model and effects of BIA on endothelial barrier functions was determined using trans-endothelial resistance assays (TEER). BIA treatment increased dextran uptake into 3D BBB models, and also lowered endothelial cell permeability in vitro via a reduction in the expression of tight junction proteins as shown by staining and a decrease in TEER values. In order to assess potential synergistic interactions, a drug screen was performed on a panel of distinct pediatric glioma cell lines (DIPG 36 and KNS42). This identified several FDA approved drugs that combine well with BIA. From this screen, BIA was shown to synergize with DNA damaging chemotherapy to enhance toxicity and in combination with cisplatin promote DNA damage in pediatric glioma cell lines. Other drugs identified in the screen are currently under investigation and details will be reported. Thus, our hypothesis is that BIA may provide an effective approach for further development as a DMG therapeutic through targeting invasion and enhancing drug potency and delivery via modulation of endothelial cell tight junctions. Interrogating the mechanisms involved and developing drug formulations informed by the drug screen for in vivo studies to determine the translational potential of this approach is our focus for future studies. Citation Format: Jasmine S. Clark, Jorge Jimenez-Macias, Philippa Vaughn-Beaucaire, Shengliang Zhang, Wafik El-Deiry, Rishi Lulla, Sean Lawler. Exploring the effects of Indirubins in pediatric diffuse high grade glioma models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5110.

Patient-Derived Blood-Brain Barrier Model for Screening Copper Bis(thiosemicarbazone) Complexes as Potential Therapeutics in Alzheimer's Disease.

Alzheimer's disease (AD) is the most prevalent cause of dementia characterized by a progressive cognitive decline. Addressing neuroinflammation represents a promising therapeutic avenue to treat AD; however, the development of effective antineuroinflammatory compounds is often hindered by their limited blood-brain barrier (BBB) permeability. Consequently, there is an urgent need for accurate, preclinical AD patient-specific BBB models to facilitate the early identification of immunomodulatory drugs capable of efficiently crossing the human AD BBB. This study presents a unique approach to BBB drug permeability screening as it utilizes the familial AD patient-derived induced brain endothelial-like cell (iBEC)-based model, which exhibits increased disease relevance and serves as an improved BBB drug permeability assessment tool when compared to traditionally employed in vitro models. To demonstrate its utility as a small molecule drug candidate screening platform, we investigated the effects of diacetylbis(N(4)-methylthiosemicarbazonato)copper(II) (CuII(atsm)) and a library of metal bis(thiosemicarbazone) complexes─a class of compounds exhibiting antineuroinflammatory therapeutic potential in neurodegenerative disorders. By evaluating the toxicity, cellular accumulation, and permeability of those compounds in the AD patient-derived iBEC, we have identified 3,4-hexanedione bis(N(4)-methylthiosemicarbazonato)copper(II) (CuII(dtsm)) as a candidate with good transport across the AD BBB. Furthermore, we have developed a multiplex approach where AD patient-derived iBEC were combined with immune modulators TNFα and IFNγ to establish an in vitro model representing the characteristic neuroinflammatory phenotype at the patient's BBB. Here, we observed that treatment with CuII(dtsm) not only reduced the expression of proinflammatory cytokine genes but also reversed the detrimental effects of TNFα and IFNγ on the integrity and function of the AD iBEC monolayer. This suggests a novel pathway through which copper bis(thiosemicarbazone) complexes may exert neurotherapeutic effects on AD by mitigating BBB neuroinflammation and related BBB integrity impairment. Together, the presented model provides an effective and easily scalable in vitro BBB platform for screening AD drug candidates. Its improved translational potential makes it a valuable tool for advancing the development of metal-based compounds aimed at modulating neuroinflammation in AD.

Microphysiological Blood-Brain Barrier Systems for Disease Modeling and Drug Development.

The blood-brain barrier (BBB) is a highly controlled microenvironment that regulates the interactions between cerebral blood and brain tissue. Due to its selectivity, many therapeutics targeting various neurological disorders are not able to penetrate into brain tissue. Pre-clinical studies using animals and other in vitro platforms have not shown the ability to fully replicate the human BBB leading to the failure of a majority of therapeutics in clinical trials. However, recent innovations in vitro and ex vivo modeling called organs-on-chips have shown the potential to create more accurate disease models for improved drug development. These microfluidic platforms induce physiological stressors on cultured cells and are able to generate more physiologically accurate BBBs compared to previous in vitro models. In this review, different approaches to create BBBs-on-chips are explored alongside their application in modeling various neurological disorders and potential therapeutic efficacy. Additionally, organs-on-chips use in BBB drug delivery studies is discussed, and advances in linking brain organs-on-chips onto multiorgan platforms to mimic organ crosstalk are reviewed.

A Giant Heterometallic Polyoxometalate Nanocluster for Enhanced Brain-Targeted Glioma Therapy.

Giant heterometallic polyoxometalate (POM) clusters with precise atom structures, flexibly adjustable and abundant active sites are promising for constructing functional nanodrugs. However, current POM drugs are almost vacant in orthotopic brain tumor therapy due to the inability to effectively penetrate the blood-brain barrier (BBB) and low drug activity. Here, we designed the largest (3.0 nm × 6.0 nm) transition-metal-lanthanide co-encapsulated POM cluster {[Ce10 Ag6 (DMEA)(H2 O)27 W22 O70 ][B-α-TeW9 O33 ]9 }2 88- featuring 238 metal centers via synergistic coordination between two geometry-unrestricted Ce3+ and Ag+ linkers with tungsten-oxo cluster fragments. This POM was combined with brain-targeted peptide to prepare a brain-targeted nanodrug that could efficiently traverse BBB and target glioma cells. The Ag+ active centers in the nanodrug specifically activate reactive oxygen species to regulate the apoptosis pathway of glioma cells with a low half-maximal inhibitory concentration (5.66 μM). As the first brain-targeted POM drug, it efficiently prolongs the survival of orthotopic glioma-bearing mice.

Nanotechnology as a new strategy for the diagnosis and treatment of gliomas.

Glioma is the most common malignant tumor of the central nervous system (CNS), and is characterized by high aggressiveness and a high recurrence rate. Currently, the main treatments for gliomas include surgical resection, temozolomide chemotherapy and radiotherapy. However, the prognosis of glioma patients after active standardized treatment is still poor, especially for glioblastoma (GBM); the median survival is still only 14.6 months, and the 5-year survival rate is only 4-5%. The current challenges in glioma treatment include difficulty in complete surgical resection, poor blood‒brain barrier (BBB) drug permeability, therapeutic resistance, and difficulty in tumor-specific targeting. In recent years, the rapid development of nanotechnology has provided new directions for diagnosing and treating gliomas. Nanoparticles (NPs) are characterized by excellent surface tunability, precise targeting, excellent biocompatibility, and high safety. In addition, NPs can be used for gene therapy, photodynamic therapy, and antiangiogenic therapy and can be combined with biomaterials for thermotherapy. In recent decades, breakthroughs in diagnosing and treating gliomas have been made with various functional NPs, and NPs are expected to become a new strategy for glioma diagnosis and treatment. In this paper, we review the main obstacles in the treatment of glioma and discuss the potential and challenges of the latest nanotechnology in the diagnosis and treatment of glioma.

Lab-on-a-chip models of the blood-brain barrier: evolution, problems, perspectives.

A great progress has been made in the development and use of lab-on-a-chip devices to model and study the blood-brain barrier (BBB) in the last decade. We present the main types of BBB-on-chip models and their use for the investigation of BBB physiology, drug and nanoparticle transport, toxicology and pathology. The selection of the appropriate cell types to be integrated into BBB-on-chip devices is discussed, as this greatly impacts the physiological relevance and translatability of findings. We identify knowledge gaps, neglected engineering and cell biological aspects and point out problems and contradictions in the literature of BBB-on-chip models, and suggest areas for further studies to progress this highly interdisciplinary field. BBB-on-chip models have an exceptional potential as predictive tools and alternatives of animal experiments in basic and preclinical research. To exploit the full potential of this technique expertise from materials science, bioengineering as well as stem cell and vascular/BBB biology is necessary. There is a need for better integration of these diverse disciplines that can only be achieved by setting clear parameters for characterizing both the chip and the BBB model parts technically and functionally.

Applications of Polymeric Nanoparticle in Nose to Brain Drug Delivery

The primary goal of developing novel formulations is to effectively deliver the drug the at the target site. A desirable, non-invasive method of enhancing medication penetration or delivering innovative drug or gene carriers into the brain is nose-to-brain administration. The main benefit of intranasal medication administration is that it avoids the blood-brain barrier and targets drug molecules directly to the brain. Due to their difficulty in crossing the blood-brain barrier, big molecular weight and hydrophilic compounds can also be transported to the brain by this drug delivery channel. By speeding the administration of treatments at the target site and preventing systemic adverse effects, intranasal delivery to the brain is helpful in treating many neurological disorders. Potential drug delivery systems, the drug-encapsulated polymeric nanoparticles can convey a sizable amount of medication from the nose to brain. The advantages of polymeric nanoparticles-mediated nose to brain targeting are discussed in this paper. Additionally, it provides an overview of the polymeric nanoparticles studied for the therapy of various brain disorders as well as the process of nanoparticle transport.

DDEL-14. EXPLORING THE ROLE OF WNT PATHWAY INHIBITION ON GLIOMA STEM CELLS PROGRESSION AND BRAIN ENDOTHELIAL INTEGRITY

Abstract The blood–brain barrier (BBB) is a specialized neurovascular unit evolved to maintain brain homeostasis and prevent effective agents from reaching malignant brain tumors such as glioblastoma. The Wnt/β-catenin signaling pathway helps to ensure BBB integrity and previous studies demonstrate Wnt pathway inhibition increases BBB permeability. High grade gliomas express increased Wnt expression compared with normal tissue. We hypothesize that Wnt pathway inhibition in glioma stem cells (GSCs) will hinder progression, increase BBB permeability, and enhance overall treatment response. In this study, we transduced primary pediatric-derived glioma stem cells with lentiviral vectors overexpressing Wnt inhibitors (DKK1 or WIF1) and then characterized migration, proliferation, and cell cycle progression. WNT inhibition on brain endothelial cell integrity was evaluated with GSC conditioned media (GSC-CM) +/- CHIR99021 (Wnt pathway activator) for function via electrical cell-cell impedance and junctional expression. Wnt inhibitor overexpression was verified using RNAseq, proteomic analysis, and immunoblotting. We found that GSC-DKK1 demonstrated significantly decreased cell migration (p< 0.005) and proliferation (p< 0.0001). We also found that CHIR99021 treated GSC-CM endothelial cells displayed decreased tight junctional proteins and increased fenestration protein expression (p< 0.0005), correlating with a more porous endothelium. Overall, our studies showed Wnt inhibitor overexpressing GSCs impaired glioma progression in vitro and indirectly disrupted endothelial cell integrity. Future studies will evaluate orthotopic Wnt inhibitor overexpressing transplant rodent models to assess changes in BBB integrity, drug permeability, model survival, and treatment response. Further understanding of the Wnt pathway in both GSCs and the BBB has the potential for improved central nervous system treatment penetration and prolonged disease response.

RTID-05. SURGICAL WINDOW OF OPPORTUNITY TRIAL REVEALS MECHANISMS OF RESPONSE AND RESISTANCE TO NAVTEMADLIN (KRT-232) IN PATIENTS WITH RECURRENT GLIOBLASTOMA

Abstract Over half of glioblastoma multiforme (GBM) patients retain wild-type p53, resulting in potential susceptibility to MDM2 inhibitors. These molecules disrupt the interaction between p53 and its negative regulator MDM2, resulting in downstream pathway signaling and cell fate decisions such as growth inhibition and death from cellular stress. We have analyzed clinical samples from 21 patients with recurrent GBM treated with the MDM2 inhibitor KRT-232, all of which relapsed despite the biological promise of this small molecule. By analyzing on-treatment biopsy samples, we detected upregulation of p53 transcriptional targets, suggesting blood-brain barrier penetrance and drug pharmacodynamics. However, in patient-derived cell lines, treatment with KRT-232 at the clinically achieved concentration was only sufficient to stall but not reduce tumor growth. Cell death via apoptosis was achieved in these cell line models when MDM2 inhibition was combined with the chemotherapeutic agent temozolomide. These drugs have non-overlapping mechanisms of action and exhibit synergy in our cell line models. Normal human bone marrow cells underwent a reversible growth inhibition phenotype when treated with KRT-232 and TMZ, supporting the idea that a therapeutic window exists with this combination. We additionally observed that tumor progression occurred in the absence of p53 inactivating mutations, suggesting that other mechanisms of resistance to KRT-232 may modulate drug response and resistance in GBM. Indeed, drug treatment induced an adaptive response in human tumors, characterized by the upregulation of genes involved in glial cell differentiation processes. Ongoing work is focused on understanding how tumor heterogeneity and cell state modulate response and resistance of GBMs to MDM2 inhibition and other therapeutic strategies that reactivate p53 signaling. Our study highlights the utility of tissue sampling during clinical trials and represents the first clinical effort to detect adaptive changes in human tumor tissue associated with p53 reactivation.

The Blood-brain Barrier, a Key Bridge to Treat Neurodegenerative Diseases.

As a highly selective and semi-permeable barrier that separates the circulating blood from the brain and central nervous system (CNS), the blood-brain barrier (BBB) plays a critical role in the onset and treatment of neurodegenerative diseases (NDs). To delay or reverse the NDs progression, the dysfunction of BBB should be improved to protect the brain from harmful substances. Simultaneously, a highly efficient drug delivery across the BBB is indispensable. Here, we summarized several methods to improve BBB dysfunction in NDs, including knocking out risk geneAPOE4, regulating circadian rhythms, restoring the gut microenvironment, and activating the Wnt/β-catenin signaling pathway. Then we discussed the advances in BBB penetration techniques, such as transient BBB opening, carrier-mediated drug delivery, and nasal administration, which facilitates drug delivery across the BBB. Furthermore, various in vivo and in vitro BBB models and research methods related to NDs are reviewed. Based on the current research progress, the treatment of NDs in the long term should prioritize the integrity of the BBB. However, a treatment approach that combines precise control of transient BBB permeability and non-invasive targeted BBB drug delivery holds profound significance in improving treatment effectiveness, safety, and clinical feasibility during drug therapy. This review involves the cross application of biology, materials science, imaging, engineering and other disciplines in the field of BBB, aiming to provide multi-dimensional research directions and clinical ideas for the treating NDs.

CNSC-02. INVESTIGATING THE ROLE OF THE WNT SIGNALING TRANSDUCTION PATHWAY IN GLIOMA BLOOD-BRAIN-BARRIER INTEGRITY

Abstract The WNT signaling transduction pathway has been linked to cancer stem cell self-renewal, differentiation, and blood brain barrier (BBB) development. Previous studies have shown that WNT pathway inhibition increased BBB permeability, specifically in WNT medulloblastoma. Glioblastoma, an aggressive malignant tumor with poor prognosis, has also been shown to have high WNT expression and variable BBB permeability. We hypothesize that activating WNT pathway inhibition in glioblastoma stem cells (GSCs) will inhibit glioma progression, increase BBB permeability within the tumor microenvironment, and enhance overall treatment responsiveness. We transduced primary pediatric derived glioma stem cells with overexpressing vectors for WNT inhibitors (DKK1 or WIF1). To characterize effects of DKK1 or WIF1 overexpression, we evaluated cell migration patterns via RTCA xCelligence, cell cycle progression via Annexin 5 assays, and cell viability via cell-titer glo. Brain endothelial cell integrity was evaluated with the treatment of CHIR99021, WNT pathway activator, or GSC condition media with immunoblotting for junctional protein expression (Claudin-5, Claudin-3, Occludin, ZO-1, VE-Cadherin). Verification of WNT inhibitory overexpressing GSCs for DKK1 and WIF1 was performed using RNAseq, proteomics, and immunoblotting. DKK1-GSCs demonstrated a statistically significant impairment in the migratory slope compared to empty vector GSCs. Cell cycle analysis also revealed that DKK1-GSCs were arrested in G0/1 phase while WIF1-GSCs were arrested in S/G2M phase. CHIR99021 endothelial treatment resulted in increased secretion of WNT inhibitors DKK1 and SFRP1 and resultantly, there was a decrease in junctional protein expression. Overall, WNT inhibitor overexpressing GSCs exhibited unique cellular patterns and indirectly disrupted endothelial cell integrity. Future studies will evaluate orthotopic WNT inhibitor overexpressing transplant rodent models to determine BBB integrity, drug permeability, model survival, and chemotherapeutic treatment response. By enhancing our understanding of the WNT pathway in GSCs, we anticipate understanding the use of future therapeutic WNT pathway targeting to increase chemotherapeutic responsiveness and improve glioblastoma prognosis.

Matrix stiffness regulates the tight junction phenotypes and local barrier properties in tricellular regions in an iPSC-derived BBB model

The blood-brain barrier (BBB) can respond to various mechanical cues such as shear stress and substrate stiffness. In the human brain, the compromised barrier function of the BBB is closely associated with a series of neurological disorders that are often also accompanied by the alteration of brain stiffness. In many types of peripheral vasculature, higher matrix stiffness decreases barrier function of endothelial cells through mechanotransduction pathways that alter cell-cell junction integrity. However, human brain endothelial cells are specialized endothelial cells that largely resist changes in cell morphology and key BBB markers. Therefore, it has remained an open question how matrix stiffness affects barrier integrity in the human BBB. To gain insight into the effects of matrix stiffness on BBB permeability, we differentiated brain microvascular endothelial-like cells from human induced pluripotent stem cells (iBMEC-like cells) and cultured the cells on extracellular matrix-coated hydrogels of varying stiffness. We first detected and quantified the junction presentation of key tight junction (TJ) proteins. Our results show matrix-dependent junction phenotypes in iBMEC-like cells, where cells on softer gels (1 kPa) have significantly lower continuous and total TJ coverages. We also determined that these softer gels also lead to decreased barrier function in a local permeability assay. Furthermore, we found that matrix stiffness regulates the local permeability of iBMEC-like cells through the balance of continuous ZO-1 TJs and no junction regions ZO-1 in tricellular regions. Together, these findings provide valuable insights into the effects of matrix stiffness on TJ phenotypes and local permeability of iBMEC-like cells. Statement of SignificanceBrain mechanical properties, including stiffness, are particularly sensitive indicators for pathophysiological changes in neural tissue. The compromised function of the blood-brain barrier is closely associated with a series of neurological disorders often accompanied by altered brain stiffness. In this study, we use polymeric biomaterials and provide new evidence that biomaterial stiffness regulates the local permeability in iPSC-derived brain endothelial cells in tricellular regions through the tight junction protein ZO-1. Our findings provide valuable insights into the changes in junction architecture and barrier permeability in response to different substrate stiffnesses. Since BBB dysfunction has been linked to many diseases, understanding the influence of substrate stiffness on junction presentations and barrier permeability could lead to the development of new treatments for diseases associated with BBB dysfunction or drug delivery across BBB systems.

Reduction in cardiolipin reduces expression of creatine transporter-1 and creatine transport in growing hCMEC/D3 human brain microvessel endothelial cells

The phospholipid cardiolipin (CL) regulates mitochondrial energy production. Endothelial cells of the blood-brain barrier (BBB) play a vital role in uptake of metabolites into the brain and are enriched in mitochondria. We examined how deficiency in BBB endothelial cell CL regulates the expression of selected drug and metabolite transporters and their function. Cardiolipin synthase-1 (hCLS1) was knocked down in a human brain microvessel endothelial cell line, hCMEC/D3, and CL levels and the mRNA expression of selected BBB drug and metabolite transporters examined. Mock transfected hCMEC/D3 cells served as controls. Incorporation of (14C)creatine and (14C)oleate into hCMEC/D3 cells was determined as a measure of solute metabolite transport. In addition, protein expression of the creatine transporter was determined. Knockdown of hCLS1 in hCMEC/D3 reduced CL and the mRNA expression of creatine transporter-1, p-glycoprotein and breast cancer resistance protein compared to controls. In contrast, mRNA expression of ATP binding cassette subfamily C members-1, -3, multidrug resistance-associated protein-4 variants 1, -2, and fatty acid transport protein-1 were unaltered. Although ATP production was unaltered by hCLS1 knockdown, incorporation of (14C)creatine into hCMEC/D3 cells was reduced compared to controls. The reduction in (14C)creatine incorporation was associated with a reduction in creatine transporter-1 protein expression. In contrast, incorporation of (14C)oleic acid into hCMEC/D3 cells and the mRNA expression of fatty acid transport protein-1 was unaltered by knockdown of hCLS1 compared to controls. Thus, knockdown of hCLS1 in hCMEC/D3, with a corresponding reduction in CL, results in alteration in expression of specific solute membrane transporters.