Permeability of Epithelial/Endothelial Barriers in Transwells and Microfluidic Bilayer Devices

Lung-on-a-chip (LoC) models hold the potential to rapidly change the landscape for pulmonary drug screening and therapy, giving patients more advanced and less invasive treatment options. Understanding the drug absorption in these microphysiological systems, modeling the lung-blood barrier is essential for increasing the role of the organ-on-a-chip technology in drug development. In this work, epithelial/endothelial barrier tissue interfaces were established in microfluidic bilayer devices and transwells, with porous membranes, for permeability characterization. The effect of shear stress on the molecular transport was assessed using known paracellular and transcellular biomarkers. The permeability of porous membranes without cells, in both models, is inversely proportional to the molecular size due to its diffusivity. Paracellular transport, between epithelial/endothelial cell junctions, of large molecules such as transferrin, as well as transcellular transport, through cell lacking required active transporters, of molecules such as dextrans, is negligible. When subjected to shear stress, paracellular transport of intermediate-size molecules such as dextran was enhanced in microfluidic devices when compared to transwells. Similarly, shear stress enhances paracellular transport of small molecules such as Lucifer yellow, but its effect on transcellular transport is not clear. The results highlight the important role that LoC can play in drug absorption studies to accelerate pulmonary drug development.

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A physiologically based biopharmaceutical analysis of zolpidem

10.21248/gups.61326 ◽

2021 ◽

Author(s):

◽

Rafael Leal Monteiro Paraiso

Keyword(s):

Drug Development ◽

Drug Absorption ◽

Oral Absorption ◽

Food Effect ◽

Simulation Software ◽

Oral Drug ◽

Oral Drug Absorption ◽

Physiologically Based

Computational oral absorption models, in particular PBBM models, provide a powerful tool for researchers and pharmaceutical scientists in drug discovery and formulation development, as they mimic and can describe the physiologically processes relevant to the oral absorption. PBBM models provide in vivo context to in vitro data experiments and allow for a dynamic understanding of in vivo drug disposition that is not typically provided by data from standard in vitro assays. Investigations using these models permit informed decision-making, especially regarding to formulation strategies in drug development. PBBM models, but can also be used to investigate and provide insight into mechanisms responsible for complex phenomena such as food effect in drug absorption. Although there are obviously still some gaps regarding the in silico construction of the gastrointestinal environment, ongoing research in the area of oral drug absorption (e.g. the UNGAP, AGE-POP and InPharma projects) will increase knowledge and enable improvement of these models. PBBM can nowadays provide an alternative approach to the development of in vitro–in vivo correlations. The case studies presented in this thesis demonstrate how PBBM can address a mechanistic understanding of the negative food effect and be used to set clinically relevant dissolution specification for zolpidem immediate release tablets. In both cases, we demonstrated the importance of integrating drug properties with physiological variables to mechanistically understand and observe the impact of these parameters on oral drug absorption. Various complex physiological processes are initiated upon food consumption, which can enhance or reduce a drug’s dissolution, solubility, and permeability and thus lead to changes in drug absorption. With improvements in modeling and simulation software and design of in vitro studies, PBBM modeling of food effects may eventually serve as a surrogate for clinical food effect studies for new doses and formulations or drugs. Furthermore, the application of these models may be even more critical in case of compounds where execution of clinical studies in healthy volunteers would be difficult (e.g., oncology drugs). In the fourth chapter we have demonstrated the establishment of the link between biopredictive in vitro dissolution testing (QC or biorelevant method) PBBM coupled with PD modeling opens the opportunity to set truly clinically relevant specifications for drug release. This approach can be extended to other drugs regardless of its classification according to the BCS. With the increased adoption of PBBM, we expect that best practices in development and verification of these models will be established that can eventually inform a regulatory guidance. Therefore, the application of Physiologically Based Biopharmaceutical Modelling is an area with great potential to streamline late-stage drug development and impact on regulatory approval procedures. Freie Schlagwörter / Tags

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Microfluidic models of physiological or pathological flow shear stress for cell biology, disease modeling and drug development

TrAC Trends in Analytical Chemistry ◽

10.1016/j.trac.2019.06.023 ◽

2019 ◽

Vol 117 ◽

pp. 186-199 ◽

Cited By ~ 7

Author(s):

Huaying Chen ◽

Zhihang Yu ◽

Siwei Bai ◽

Huaxiu Lu ◽

Dong Xu ◽

...

Keyword(s):

Shear Stress ◽

Drug Development ◽

Cell Biology ◽

Disease Modeling ◽

Flow Shear ◽

Flow Shear Stress

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Quantitative Visualization of Molecular Transport through Porous Membranes: Enhanced Resolution and Contrast Using Intermittent Contact-Scanning Electrochemical Microscopy

Analytical Chemistry ◽

10.1021/ac201489c ◽

2011 ◽

Vol 83 (17) ◽

pp. 6447-6454 ◽

Cited By ~ 24

Author(s):

Kim McKelvey ◽

Michael E. Snowden ◽

Massimo Peruffo ◽

Patrick R. Unwin

Keyword(s):

Molecular Transport ◽

Scanning Electrochemical Microscopy ◽

Porous Membranes ◽

Enhanced Resolution ◽

Quantitative Visualization ◽

Intermittent Contact ◽

Electrochemical Microscopy

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Latrophilins are essential for endothelial junctional fluid shear stress mechanotransduction

10.1101/2020.02.03.932822 ◽

2020 ◽

Cited By ~ 1

Author(s):

Keiichiro Tanaka ◽

Andrew Prendergast ◽

Jared Hintzen ◽

Abhishek Kumar ◽

Minhwan Chung ◽

...

Keyword(s):

Shear Stress ◽

G Proteins ◽

Fluid Shear Stress ◽

Affinity Purification ◽

Cell Junctions ◽

Heterotrimeric G Proteins ◽

Fluid Shear ◽

Vascular Morphogenesis ◽

Flow Activation

AbstractEndothelial cell (EC) responses to fluid shear stress (FSS) are crucial for vascular development, adult physiology and disease. PECAM1 is an important transducer but earlier events remain poorly understood. We therefore investigated heterotrimeric G proteins in FSS sensing. Knockdown (KD) in ECs of single Gα proteins had little effect but combined depletion of Gαi and Gαq/11 blocked all known PECAM1-dependent responses. Re-expression of Gαi2 and Gαq but not Gαi1 and Gαi3 rescued these effects. Sequence alignment and mutational studies identified that K307 in Gαi2 and Gq/11 (Q306 in Gαi1/3), determines participation in flow signaling. We developed pull-down assays for measuring Gα activation and found that this residue, localized to the GPCR interface, determines activation by FSS. We developed a protocol for affinity purification of GPCRs on activated Gα’s, which identified latrophilins (ADGRLs) as specific upstream interactors for Gαi2 and Gq/11. Depletion of latrophilin-2 blocked EC activation of Gαi2 and Gαq, downstream events in vitro, and flow-dependent vascular morphogenesis in zebrafish embryos. Surprisingly, latrophilin-2 depletion also blocked flow activation of two additional pathways activated at cell-cell junctions, Smad1/5 and Notch1, independently of Gα proteins. Latrophilins are thus central mediators of junctional shear stress mechanotransduction via Gα protein-dependent and -independent mechanisms.

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NANO-BIOTECHNOLOGY AND ITS INNOVATIVE PERSPECTIVE IN DIABETES MANAGEMENT

Mini-Reviews in Medicinal Chemistry ◽

10.2174/1389557521666210623164052 ◽

2021 ◽

Vol 21 ◽

Author(s):

Jigar Raval ◽

Riddhi Trivedi ◽

Sonali Suman ◽

Arvind Kukrety ◽

Prajesh Prajapati

Keyword(s):

Cellular Uptake ◽

Glucose Homeostasis ◽

Diabetes Management ◽

The Body ◽

Paracellular Transport ◽

Specific Gene ◽

Future Prospects ◽

Transcellular Transport ◽

Routes Of Administration ◽

Two Stages

: Diabetes occurs due to the imbalance of glucose in the body known as glucose homeostasis, thus leading to metabolic changes in the body. The two stages hypoglycemia or hyperglycemia classify diabetes into various categories. Various bio-nanotechnological approaches are coupled up with nano particulates, polymers, liposome, various gold plated and solid lipid particulates, regulating transcellular transport, non specific cellular uptake, and paracellular transport, leading to oral, trans-dermal , pulmonary, buccal , nasal , specific gene oriented administration to avoid the patient’s non compliance with the parental routes of administration. Phytochemicals are emerging strategies for the future prospects of diabetes management.

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Shear stress regulates occludin content and phosphorylation

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.2001.281.1.h105 ◽

2001 ◽

Vol 281 (1) ◽

pp. H105-H113 ◽

Cited By ~ 82

Author(s):

Lucas DeMaio ◽

Yong S. Chang ◽

Thomas W. Gardner ◽

John M. Tarbell ◽

David A. Antonetti

Keyword(s):

Shear Stress ◽

Kinase Activity ◽

Total Content ◽

Time Dependent ◽

Paracellular Transport ◽

Bovine Aortic Endothelial Cell ◽

Dependent Manner ◽

Tyrosine Kinase Activity ◽

Phosphorylation State ◽

Junction Protein

Previous studies determined that shear stress imposed on bovine aortic endothelial cell (BAEC) monolayers increased the hydraulic conductivity ( L P); however, the mechanism by which shear stress increases L Premains unknown. This study tested the hypothesis that shear stress regulates paracellular transport by altering the expression and phosphorylation state of the tight junction protein occludin. The effect of shear stress on occludin content was examined by Western blot analysis. Ten dyn/cm2 significantly reduced occludin content in a time-dependent manner such that after a 3 h exposure to shear, occludin content decreased to 44% of control. Twenty dyn/cm2 decreased occludin content to 50% of control and increased L P by 4.7-fold after 3 h. Occludin expression and L P depend on tyrosine kinase activity because erbstatin A (10 μM) attenuated both the shear-induced decrease in occludin content and increase in L P. Shear stress increased occludin phosphorylation after 5 min, 15 min, and 3 h exposures. The shear-induced increase in occludin phosphorylation was attenuated with dibutyryl (DB) cAMP (1 mM), a reagent previously shown to reverse the shear-induced increase in L P. We conclude that shear stress rapidly (≤5 min) increases occludin phosphorylation and significantly decreases the expression of occludin over 1–4 h. Alterations in the occludin phosphorylation state and occludin total content are potential mechanisms by which shear stress increases L P.

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Sphingolipids affect fibrinogen-induced caveolar transcytosis and cerebrovascular permeability

AJP Cell Physiology ◽

10.1152/ajpcell.00305.2013 ◽

2014 ◽

Vol 307 (2) ◽

pp. C169-C179 ◽

Cited By ~ 12

Author(s):

Nino Muradashvili ◽

Syed Jalal Khundmiri ◽

Reeta Tyagi ◽

Allison Gartung ◽

William L. Dean ◽

...

Keyword(s):

Energy Transfer ◽

Endothelial Cell ◽

Total Internal Reflection ◽

De Novo ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Vascular Wall ◽

Paracellular Transport ◽

Transcellular Transport ◽

Cerebrovascular Permeability

Inflammation-induced vascular endothelial dysfunction can allow plasma proteins to cross the vascular wall, causing edema. Proteins may traverse the vascular wall through two main pathways, the paracellular and transcellular transport pathways. Paracellular transport involves changes in endothelial cell junction proteins, while transcellular transport involves caveolar transcytosis. Since both processes are associated with filamentous actin formation, the two pathways are interconnected. Therefore, it is difficult to differentiate the prevailing role of one or the other pathway during various pathologies causing an increase in vascular permeability. Using a newly developed dual-tracer probing method, we differentiated transcellular from paracellular transport during hyperfibrinogenemia (HFg), an increase in fibrinogen (Fg) content. Roles of cholesterol and sphingolipids in formation of functional caveolae were assessed using a cholesterol chelator, methyl-β-cyclodextrin, and the de novo sphingolipid synthesis inhibitor myriocin. Fg-induced formation of functional caveolae was defined by association and colocalization of Na+-K+-ATPase and plasmalemmal vesicle-associated protein-1 with use of Förster resonance energy transfer and total internal reflection fluorescence microscopy, respectively. HFg increased permeability of the endothelial cell layer mainly through the transcellular pathway. While MβCD blocked Fg-increased transcellular and paracellular transport, myriocin affected only transcellular transport. Less pial venular leakage of albumin was observed in myriocin-treated HFg mice. HFg induced greater formation of functional caveolae, as indicated by colocalization of Na+-K+-ATPase with plasmalemmal vesicle-associated protein-1 by Förster resonance energy transfer and total internal reflection fluorescence microscopy. Our results suggest that elevated blood levels of Fg alter cerebrovascular permeability mainly by affecting caveolae-mediated transcytosis through modulation of de novo sphingolipid synthesis.

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