Evaluation of Barrier Integrity Using a Two-Layered Microfluidic Device Mimicking the Blood-Brain Barrier.

The blood-brain barrier (BBB) plays an essential role in maintaining the homeostasis of the brain microenvironment by controlling the influx and efflux of biological substances that are necessary to sustain the neuronal metabolic activity and functions. This barrier is established at the blood-brain interface of the brain microcapillaries by different cells. These include microvascular endothelial cells, astrocytes, and pericytes besides other components such as microglia, basal membrane, and neuronal cells forming together what is commonly referred to as the neurovascular unit; different in vivo and in vitro platforms are available to study the BBB where each system provides specific benefits and drawbacks. Recently, organ-on-a-chip platforms combine the elegance of microengineering technology with the complexity of biological systems to create near-ideal experimental models for various diseases and organs. These microfluidic devices with micron-sized channels allow the cells to be grown in a more biologically relevant environment, enabling cell to cell communications with continuous bathing in biological fluids in a tissue-like fashion. They also closely represent tissue and organ functionality by recapitulating mechanical forces as well as vascular perfusion. Here, we describe the use of humanized BBB model created with microfluidic organ-on-a-chip technology where human brain microvascular endothelial cells (BMECs) are cocultured with primary human pericytes and astrocytes. We thoroughly described the method to assess BBB integrity using a microfluidic chip and various sizes of labeled dextran as permeability markers. In addition, we provide a detailed protocol on how to microscopically investigate the tight junction proteins expression between hBMECs.

Advanced Microfluidic Vascularized Tissues as Platform for the Study of Human Diseases and Drug Development.

The vascular system plays a critical role in human physiology and diseases. It is a complex subject to study using in vitro models due to its dynamic and three-dimensional microenvironment. Microfluidic technology has recently become a popular technology in various biological fields for its advantages in mimicking complex microenvironments to an extent not achievable by more conventional platforms. Microfluidic technologies can reproduce different vascular system-related structures and functions that can be utilized for drug development and human diseases studies. Herein, we first review the relevant structural and functional vascular biology systems of various organ systems and then the fabrication methods to reproduce these vascular districts. We provide a thorough review of the latest achievement in vascular organ-on-chip modeling specific to lung, heart, and the brain microvasculature for drug screening and the study of human disorders.

Comparative assessment of in vitro BBB tight junction integrity following exposure to cigarette smoke and e-cigarette vapor: a quantitative evaluation of the protective effects of metformin using small-molecular-weight paracellular markers.

BACKGROUND: The blood-brain barrier (BBB) plays a critical role in protecting the central nervous system (CNS) from blood-borne agents and potentially harmful xenobiotics. Our group's previous data has shown that tobacco smoke (TS) and electronic cigarettes (EC) affect the BBB integrity, increase stroke incidence, and are considered a risk factor for multiple CNS disorders. Metformin was also found to abrogate the adverse effects of TS and EC. METHODS: We used sucrose and mannitol as paracellular markers to quantitatively assess TS and EC's impact on the BBB in-vitro. Specifically, we used a quantitative platform to determine the harmful effects of smoking on the BBB and study the protective effect of metformin. Using a transwell system and iPSCs-derived BMECs, we assessed TS and EC's effect on sucrose and mannitol permeability with and without metformin pre-treatment at different time points. Concurrently, using immunofluorescence (IF) and Western blot (WB) techniques, we evaluated the expression and distribution of tight junction proteins, including ZO-1, occludin, and claudin-5. RESULTS: Our data showed that TS and EC negatively affect sucrose and mannitol permeability starting after 6 h and up to 24 h. The loss of barrier integrity was associated with a reduction of TEER values. While the overall expression level of ZO-1 and occludin was not significantly downregulated, the distribution of ZO-1 was altered, and discontinuation patterns were evident through IF imaging. In contrast to occludin, claudin-5 expression was significantly decreased by TS and EC, as demonstrated by WB and IF data. CONCLUSION: In agreement with previous studies, our data showed the metformin could counteract the negative impact of TS and EC on BBB integrity, thus suggesting the possibility of repurposing this drug to afford cerebrovascular protection.

Method for Investigating Fibroblast Durotaxis.

Durotaxis is the phenomena of directed cell migration driven by gradients of extracellular matrix stiffness. Durotaxis has been recently involved in the development of fibrosis by promoting the recruitment of pathological fibroblasts to areas of established fibrosis, thus amplifying the fibrotic response. Here, we describe the fabrication of mechanically patterned hydrogels that can be used to investigate molecular mechanisms controlling durotaxis of fibroblasts and other cells with mechanosensing properties. This method effectively creates a stiffness gradient of 275 Pa/µm, mimicking the natural spatial stiffness variations we observed in fibrotic tissues from mouse models of fibrosis and human fibrotic diseases.

A blood-brain barrier overview on structure, function, impairment, and biomarkers of integrity.

The blood-brain barrier is playing a critical role in controlling the influx and efflux of biological substances essential for the brain's metabolic activity as well as neuronal function. Thus, the functional and structural integrity of the BBB is pivotal to maintain the homeostasis of the brain microenvironment. The different cells and structures contributing to developing this barrier are summarized along with the different functions that BBB plays at the brain-blood interface. We also explained the role of shear stress in maintaining BBB integrity. Furthermore, we elaborated on the clinical aspects that correlate between BBB disruption and different neurological and pathological conditions. Finally, we discussed several biomarkers that can help to assess the BBB permeability and integrity in-vitro or in-vivo and briefly explain their advantages and disadvantages.

Pulmonary-arterial-hypertension (PAH)-on-a-chip: fabrication, validation and application.

Currently used animal and cellular models for pulmonary arterial hypertension (PAH) only partially recapitulate its pathophysiology in humans and are thus inadequate in reproducing the hallmarks of the disease, inconsistent in portraying the sex-disparity, and unyielding to combinatorial study designs. Here we sought to deploy the ingenuity of microengineering in developing and validating a tissue chip model for human PAH. We designed and fabricated a microfluidic device to emulate the luminal, intimal, medial, adventitial, and perivascular layers of a pulmonary artery. By growing three types of pulmonary arterial cells (PACs)-endothelial, smooth muscle, and adventitial cells, we recreated the PAH pathophysiology on the device. Diseased (PAH) PACs, when grown on the chips, moved of out their designated layers and created phenomena similar to the major pathologies of human PAH: intimal thickening, muscularization, and arterial remodeling and show an endothelial to mesenchymal transition. Flow-induced stress caused control cells, grown on the chips, to undergo morphological changes and elicit arterial remodeling. Our data also suggest that the newly developed chips can be used to elucidate the sex disparity in PAH and to study the therapeutic efficacy of existing and investigational anti-PAH drugs. We believe this miniaturized device can be deployed for testing various prevailing and new hypotheses regarding the pathobiology and drug therapy in human PAH.

Statistical optimization and validation of a novel ultra-performance liquid chromatography method for estimation of nintedanib in rat and human plasma.

Aim: A high throughput ultra-performance liquid chromatography (UPLC)-ultraviolet method for quantification of nintedanib in rat and human plasma was developed and optimized using chemometrical approach. Method: Design of experiment and multivariate statistical approach was used for definition of optimized method. Final separation was performed using protein precipitation method on ACQUITY HSS T3 C18 column in isocratic mode using potassium phosphate buffer (pH 7.5): acetonitrile. Results: Method was validated as per US-FDA guidelines linearly from 15-750 ng/ml. All quality control samples showed <15% relative standard deviation for precision and 85-115% accuracy along with >98% extraction recovery. Conclusion: The developed method is easily applicable in determining pharmacokinetic parameters in preclinical subjects along with successful implementation for quantification in human plasma samples.

Newer approaches and novel drugs for inhalational therapy for pulmonary arterial hypertension.

Introduction: Pulmonary arterial hypertension (PAH) is a progressive disease characterized by remodeling of small pulmonary arteries leading to increased pulmonary arterial pressure. Existing treatments acts to normalize vascular tone via three signaling pathways: the prostacyclin, the endothelin-1, and the nitric oxide. Although over the past 20 years, there has been considerable progress in terms of treatments for PAH, the disease still remains incurable with a disappointing prognosis.Areas covered: This review summarizes the pathophysiology of PAH, the advantages and disadvantages of the inhalation route, and assess the relative advantages various inhaled therapies for PAH. The recent studies concerning the development of controlled-release drug delivery systems loaded with available anti-PAH drugs have also been summarized.Expert opinion: The main obstacles of current pharmacotherapies of PAH are their short half-life, stability, and formulations, resulting in reducing the efficacy and increasing systemic side effects and unknown pathogenesis of PAH. The pulmonary route has been proposed for delivering anti-PAH drugs to overcome the shortcomings. However, the application of approved inhaled anti-PAH drugs is limited. Inhalational delivery of controlled-release nanoformulations can overcome these restrictions. Extensive studies are required to develop safe and effective drug delivery systems for PAH patients.

Digital light processing (DLP) 3D-printing technology and photoreactive polymers in fabrication of modified-release tablets.

In this study, we assessed the feasibility of using digital light processing (DLP) 3D printers (3DP) in fabrication of solid oral dosage forms. The DLP technology uses a digital micromirror device (DMD) that reflects and focuses ultraviolet (UV) light on the surfaces of photoreactive materials that polymerize in a layer-by-layer fashion. Using poly(ethylene glycol) diacrylate (PEGDA) and poly(ethylene glycol) dimethacrylate (PEGDMA) as photoreactive polymers and theophylline as a model drug, we deployed a DLP printer to fabricate tablets. After optimizing various printing parameters including UV intensity and exposure time, layer thickness, and polymer concentration, we printed various types of tablets with and without perforation. We then assessed the tablets for drug content, mechanical strengths, swellability, weight variation, microscopic features, drug-polymer interactions and drug release profiles. The loading of theophylline was 1%, which was independent of tablet weights. The drug content and weight variation were within the acceptable range, as recommended by the United States Pharmacopeia (USP). Scanning electronic microscopic (SEM) pictures showed tablets with distinct layers and smooth outer surfaces. The spectral scans, obtained using Fourier Transform Infrared Spectroscopy (FTIR), showed no chemical interactions between the drug and polymers. Similarly, drug content determined using a UV spectrophotometer was the same as that determined by a high performance liquid chromatography (UPLC). The extent of drug release increased with the increase in the number of perforations in the tablets. Overall, this study demonstrates that DLP 3DP can be used as a platform for fabricating oral tablets with well-defined shapes and different release profiles.

Multi-purposable filaments of HPMC for 3D printing of medications with tailored drug release and timed-absorption.

Three-dimensional printing (3DP), though developed for nonmedical applications and once regarded as futuristic only, has recently been deployed for the fabrication of pharmaceutical products. However, the existing feeding materials (inks and filaments) that are used for printing drug products have various shortcomings, including the lack of biocompatibility, inadequate extrudability and printability, poor drug loading, and instability. Here, we have sought to develop a filament using a single pharmaceutical polymer, with no additives, which can be multi-purposed and manipulated by computational design for the preparation of tablets with desired release and absorption patterns. As such, we have used hydroxypropyl-methylcellulose (HPMC) and diltiazem, a model drug, to prepare both drug-free and drug-impregnated filaments, and investigated their thermal and crystalline properties, studied the cytotoxicity of the filaments, designed and printed tablets with various infill densities and patterns. By alternating the drug-free and drug-impregnated filaments, we fabricated various types of tablets, studied the drug release profiles, and assessed oral absorption in rats. Both diltiazem and HPMC were stable at extrusion and printing temperatures, and the drug loading was 10% (w/w). The infill density, as well as infill patterns, influenced the drug release profile, and thus, when the infill density was increased to 100%, the percentage of drug released dramatically declined. Tablets with alternating drug-free and drug-loaded layers showed delayed and intermittent drug release, depending on when the drug-loaded layers encountered the dissolution media. Importantly, the oral absorption patterns accurately reproduced the drug release profiles and showed immediate, extended, delayed and episodic absorption of the drug from the rat gastrointestinal tract (GIT). Overall, we have demonstrated here that filaments for 3D printers can be prepared from a pharmaceutical polymer with no additives, and the novel computational design allows for fabricating tablets with the capability of producing distinct absorption patterns after oral administration.