Fluoropolymer Functionalization of Organ-on-Chip Platform Increases Detection Sensitivity for Cannabinoids.

Microfluidic technology is applied across various research areas including organ-on-chip (OOC) systems. The main material used for microfluidics is polydimethylsiloxane (PDMS), a silicone elastomer material that is biocompatible, transparent, and easy to use for OOC systems with well-defined microstructures. However, PDMS-based OOC systems can absorb hydrophobic and small molecules, making it difficult and erroneous to make quantitative analytical assessments for such compounds. In this paper, we explore the use of a synthetic fluoropolymer, poly(4,5-difluoro-2,2-bis(trifluoromethyl)-1,3-dioxole-co-tetrafluoroethylene) (Teflon™ AF 2400), with excellent "non-stick" properties to functionalize OOC systems. Cannabinoids, including cannabidiol (CBD), are classes of hydrophobic compounds with a great potential for the treatment of anxiety, depression, pain, and cancer. By using CBD as a testing compound, we examined and systematically quantified CBD absorption into PDMS by means of an LC-MS/MS analysis. In comparison to the unmodified PDMS microchannels, an increase of approximately 30× in the CBD signal was detected with the fluoropolymer surface modification after 3 h of static incubation. Under perfusion conditions, we observed an increase of nearly 15× in the CBD signals from the surface-modified microchannels than from the unmodified microchannels. Furthermore, we also demonstrated that fluoropolymer-modified microchannels are compatible for culturing hCMEC/D3 endothelial cells and for CBD perfusion experiments.

Recent Advances in Formulations for Long-Acting Delivery of Therapeutic Peptides.

Recent preclinical and clinical studies have focused on the active area of therapeutic peptides due to their high potency, selectivity, and specificity in treating a broad range of diseases. However, therapeutic peptides suffer from multiple disadvantages, such as limited oral bioavailability, short half-life, rapid clearance from the body, and susceptibility to physiological conditions (e.g., acidic pH and enzymolysis). Therefore, high peptide dosages and dose frequencies are required for effective patient treatment. Recent innovations in pharmaceutical formulations have substantially improved therapeutic peptide administration by providing the following advantages: long-acting delivery, precise dose administration, retention of biological activity, and improvement of patient compliance. This review discusses therapeutic peptides and challenges in their delivery and explores recent peptide delivery formulations, including micro/nanoparticles (based on lipids, polymers, porous silicon, silica, and stimuli-responsive materials), (stimuli-responsive) hydrogels, particle/hydrogel composites, and (natural or synthetic) scaffolds. This review further covers the applications of these formulations for prolonged delivery and sustained release of therapeutic peptides and their impact on peptide bioactivity, loading efficiency, and (in vitro/in vivo) release parameters.

Blood-brain barrier (BBB)-on-a-chip: a promising breakthrough in brain disease research.

The blood-brain barrier (BBB) represents a key challenge in developing brain-penetrating therapeutic molecules. BBB dysfunction is also associated with the onset and progression of various brain diseases. The BBB-on-a-chip (µBBB), an organ-on-chip technology, has emerged as a powerful in vitro platform that closely mimics the human BBB microenvironments. While the µBBB technology has seen wide application in the study of brain cancer, its utility in other brain disease models ("µBBB+") is less appreciated. Based on the advances of the µBBB technology and the evolution of in vitro models for brain diseases over the last decade, we propose the concept of a "µBBB+" system and summarize its major promising applications in pathological studies, personalized medical research, drug development, and multi-organ-on-chip approaches. We believe that such a sophisticated "µBBB+" system is a highly tunable and promising in vitro platform for further advancement of the understanding of brain diseases.

Bio-Mimicking Brain Vasculature to Investigate the Role of Heterogeneous Shear Stress in Regulating Barrier Integrity.

A continuous, sealed endothelial membrane is essential for the blood-brain barrier (BBB) to protect neurons from toxins present in systemic circulation. Endothelial cells are critical sensors of the capillary environment, where factors like fluid shear stress (FSS) and systemic signaling molecules activate intracellular pathways that either promote or disrupt the BBB. The brain vasculature exhibits complex heterogeneity across the bed, which is challenging to recapitulate in BBB microfluidic models with fixed dimensions and rectangular cross-section microchannels. Here, a Cayley-tree pattern, fabricated using lithography-less, fluid shaping technique in a modified Hele-Shaw cell is used to emulate the brain vasculature in a microfluidic chip. This geometry generates an inherent distribution of heterogeneous FSS, due to smooth variations in branch height and width. hCMEC/D3 endothelial cells cultured in the Cayley-tree designed chip generate a 3D monolayer of brain endothelium with branching hierarchy, enabling the study of the effect of heterogeneous FSS on the brain endothelium. The model is employed to study neuroinflammatory conditions by stimulating the brain endothelium with tumor necrosis factor-α under heterogeneous FSS conditions. The model has immense potential for studies involving drug transport across the BBB, which can be misrepresented in fixed dimension models.

Development of Polymeric Nanoparticles for Blood-Brain Barrier Transfer-Strategies and Challenges.

Neurological disorders such as Alzheimer's disease, stroke, and brain cancers are difficult to treat with current drugs as their delivery efficacy to the brain is severely hampered by the presence of the blood-brain barrier (BBB). Drug delivery systems have been extensively explored in recent decades aiming to circumvent this barrier. In particular, polymeric nanoparticles have shown enormous potentials owing to their unique properties, such as high tunability, ease of synthesis, and control over drug release profile. However, careful analysis of their performance in effective drug transport across the BBB should be performed using clinically relevant testing models. In this review, polymeric nanoparticle systems for drug delivery to the central nervous system are discussed with an emphasis on the effects of particle size, shape, and surface modifications on BBB penetration. Moreover, the authors critically analyze the current in vitro and in vivo models used to evaluate BBB penetration efficacy, including the latest developments in the BBB-on-a-chip models. Finally, the challenges and future perspectives for the development of polymeric nanoparticles to combat neurological disorders are discussed.

In Situ Surface Modification of Microfluidic Blood-Brain-Barriers for Improved Screening of Small Molecules and Nanoparticles.

Here, we have developed and evaluated a microfluidic-based human blood-brain-barrier (µBBB) platform that models and predicts brain tissue uptake of small molecule drugs and nanoparticles (NPs) targeting the central nervous system. By using a photocrosslinkable copolymer that was prepared from monomers containing benzophenone and N-hydroxysuccinimide ester functional groups, we were able to evenly coat and functionalize µBBB chip channels in situ, providing a covalently attached homogenous layer of extracellular matrix proteins. This novel approach allowed the coculture of human endothelial cells, pericytes, and astrocytes and resulted in the formation of a mimic of cerebral endothelium expressing tight junction markers and efflux proteins, resembling the native BBB. The permeability coefficients of a number of compounds, including caffeine, nitrofurantoin, dextran, sucrose, glucose, and alanine, were measured on our µBBB platform and were found to agree with reported values. In addition, we successfully visualized the receptor-mediated uptake and transcytosis of transferrin-functionalized NPs. The BBB-penetrating NPs were able to target glioma cells cultured in 3D in the brain compartment of our µBBB. In conclusion, our µBBB was able to accurately predict the BBB permeability of both small molecule pharmaceuticals and nanovectors and allowed time-resolved visualization of transcytosis. Our versatile chip design accommodates different brain disease models and is expected to be exploited in further BBB studies, aiming at replacing animal experiments.

Multiparameter toxicity screening on a chip: Effects of UV radiation and titanium dioxide nanoparticles on HaCaT cells.

Microfluidic screening is gaining attention as an efficient method for evaluating nanomaterial toxicity. Here, we consider a multiparameter treatment where nanomaterials interact with cells in the presence of a secondary exposure (UV radiation). The microfluidic device contains channels that permit immobilization of HaCaT cells (human skin cell line), delivery of titanium dioxide nanoparticles (TNPs), and exposure to a known dose of UV radiation. The effect of single-parameter exposures (UV or TNP) was first studied as a benchmark, and then multiparameter toxicity (UV and TNP) at different concentrations was explored. The results demonstrate a concentration-dependent protective effect of TNP when exposed to UV irradiation.

Water permeation drives tumor cell migration in confined microenvironments.

Cell migration is a critical process for diverse (patho)physiological phenomena. Intriguingly, cell migration through physically confined spaces can persist even when typical hallmarks of 2D planar migration, such as actin polymerization and myosin II-mediated contractility, are inhibited. Here, we present an integrated experimental and theoretical approach ("Osmotic Engine Model") and demonstrate that directed water permeation is a major mechanism of cell migration in confined microenvironments. Using microfluidic and imaging techniques along with mathematical modeling, we show that tumor cells confined in a narrow channel establish a polarized distribution of Na+/H+ pumps and aquaporins in the cell membrane, which creates a net inflow of water and ions at the cell leading edge and a net outflow of water and ions at the trailing edge, leading to net cell displacement. Collectively, this study presents an alternate mechanism of cell migration in confinement that depends on cell-volume regulation via water permeation.

Physical confinement alters tumor cell adhesion and migration phenotypes.

Cell migration on planar surfaces is driven by cycles of actin protrusion, integrin-mediated adhesion, and myosin-mediated contraction; however, this mechanism may not accurately describe movement in 3-dimensional (3D) space. By subjecting cells to restrictive 3D environments, we demonstrate that physical confinement constitutes a biophysical stimulus that alters cell morphology and suppresses mesenchymal motility in human breast carcinoma (MDA-MB-231). Dorsoventral polarity, stress fibers, and focal adhesions are markedly attenuated by confinement. Inhibitors of myosin, Rho/ROCK, or ß1-integrins do not impair migration through 3-µm-wide channels (confinement), even though these treatments repress motility in 50-µm-wide channels (unconfined migration) by ≥50%. Strikingly, confined migration persists even when F-actin is disrupted, but depends largely on microtubule (MT) dynamics. Interfering with MT polymerization/depolymerization causes confined cells to undergo frequent directional changes, thereby reducing the average net displacement by ≥80% relative to vehicle controls. Live-cell EB1-GFP imaging reveals that confinement redirects MT polymerization toward the leading edge, where MTs continuously impact during advancement of the cell front. These results demonstrate that physical confinement can induce cytoskeletal alterations that reduce the dependence of migrating cells on adhesion-contraction force coupling. This mechanism may explain why integrins can exhibit reduced or altered function during migration in 3D environments.

Selectin-mediated adhesion in shear flow using micropatterned substrates: multiple-bond interactions govern the critical length for cell binding.

Receptor-ligand adhesive interactions play a pivotal role in diverse biological processes including inflammation and cancer metastasis. Cell adhesion is mediated by the molecular recognition of membrane-bound receptors by their cognate ligands on apposing cells. Cell-cell binding is regulated by distinct parameters such as the receptor-ligand binding kinetics, the tensile strength of individual bonds, the involvement of multiple bonds and their modulation by hydrodynamic shear. This work aims to investigate the interplay of these parameters on selectin-mediated cell adhesion in shear flow. We designed a microfluidic device that delivers cells in a single file over a receptor-functionalized substrate, thereby permitting accurate determination of the cell flux. The selectin(s) was presented on striped patches of fixed width and varying length. We identified the critical patch lengths of P- and L-selectin for the initiation of HL-60 cell binding in shear flow. This characteristic length is governed by the time required to form multiple-bond interactions, as revealed by a multiple-bond mathematical model. The number of bonds required to support cell binding increases with the applied shear stress (0.5-2 dyn cm(-2)) for L- but not P-selectin. This finding is explained by differences in the tensile strength of P- and L-selectin for PSGL-1. Our integrated experimental and mathematical approach advances our understanding of receptor-mediated cell adhesion in the vasculature. Detailed knowledge of how molecular interactions modulate macroscopic cell binding behavior pertinent to inflammation and metastasis would facilitate the development of promising diagnostic tools to combat these diseases.

Chemotaxis of cell populations through confined spaces at single-cell resolution.

Cell migration is crucial for both physiological and pathological processes. Current in vitro cell motility assays suffer from various drawbacks, including insufficient temporal and/or optical resolution, or the failure to include a controlled chemotactic stimulus. Here, we address these limitations with a migration chamber that utilizes a self-sustaining chemotactic gradient to induce locomotion through confined environments that emulate physiological settings. Dynamic real-time analysis of both population-scale and single-cell movement are achieved at high resolution. Interior surfaces can be functionalized through adsorption of extracellular matrix components, and pharmacological agents can be administered to cells directly, or indirectly through the chemotactic reservoir. Direct comparison of multiple cell types can be achieved in a single enclosed system to compare inherent migratory potentials. Our novel microfluidic design is therefore a powerful tool for the study of cellular chemotaxis, and is suitable for a wide range of biological and biomedical applications.

Response of chondrocytes to shear stress: antagonistic effects of the binding partners Toll-like receptor 4 and caveolin-1.

Osteoarthritis (OA) is often a consequence of excessive mechanical loading of cartilage, which produces hydrostatic stress, tensile strain, and fluid flow. Application of high fluid shear to chondrocytes recapitulates the earmarks of OA, as evidenced by the induction of cyclooxygenase-2, prostaglandins (PGs), and interleukin-6 (IL-6). Here, we delineated the signaling pathway by which high fluid shear mediates the temporal regulation of IL-6 synthesis in human chondrocytes. We determined that Toll-like receptor 4 (TLR4) and caveolin-1 are binding partners in chondrocytes. Their expression is temporally regulated by fluid shear via the sequential up-regulation of microsomal PGE synthase-1 (mPGES-1) and L-PGDS. High shear stress rapidly induces an 8-fold up-regulation of TLR4 expression via an mPGES-1-dependent pathway, whereas prolonged shear exposure concurrently down-regulates TLR4 by >4-fold and up-regulates caveolin-1 expression by > 2.5-fold in an L-PGDS-dependent manner. TLR4 and caveolin-1 exert opposing effects on the activation of ERK1/2, PI3-K and PKA signaling pathways, which, in turn, regulate the NF-κB-dependent IL-6 synthesis in a time-dependent fashion. Reconstructing the signaling network regulating shear-induced IL-6 expression in chondrocytes may provide insights for developing therapeutic strategies to combat osteoarthritis.

Sialyl Lewisx-dependent binding of human monocyte-derived dendritic cells to selectins.

The limited efficacy of monocyte-derived dendritic cell (mo-DC)-based vaccines is primarily attributed to the reduced mo-DC migratory capacity. One undefined aspect is the initial binding of mo-DCs to endothelial cells and vascular selectins. In this study, we investigated the role and modulation of the selectin binding determinant sialyl Lewis(x) (sLe(x)) in selectin-dependent mo-DC binding. Our data reveal that sLe(x) is required for maximal binding of mo-DCs to tumor necrosis factor (TNF)-α-activated endothelial cells under static conditions, as evidenced by the use of sialidase. Sialidase treatment also abrogated mo-DC cell tethering to immobilized, purified P-, L-, or E-selectin under flow. The requirement of sLe(x)-dependent binding of mo-DC to selectins was further substantiated by using sLe(x) free sugar and anti-sLe(x) antibody, which significantly suppressed mo-DC-selectin binding. P-selectin glycoprotein ligand-1 is required for mo-DC binding to both P- and L-selectin, but it is dispensable for E-selectin recognition. Interestingly, the extent of mo-DC tethering was maximal on P-selectin, followed by E- and L- selectin. Accordingly, L-selectin mediated faster mo-DC rolling than E- or P-selectin. Interferon (IFN)-γ induces a significant increase in mo-DC surface sLe(x) expression, which is probably due to the enhanced synthesis of C2GnT-I. These findings may contribute to improving mo-DC-based vaccination protocols.

Identification, characterization and utilization of tumor cell selectin ligands in the design of colon cancer diagnostics.

The efficiency of secondary tumor establishment is controlled by the ability of tumor cells to withstand a barrage of mechanical and immunological stresses during their passage through the circulatory system. Accumulating evidence suggests that the selectin-dependent interactions of circulating tumor cells with host cells promote their survival and extravasation from the vasculature, therefore representing a critical checkpoint for colonization of distant organs. These observations have motivated the identification and biochemical characterization of functional selectin ligands such as CD44 variant isoforms, carcinoembryonic antigen and podocalyxin-like protein, present on the surface of metastatic colon carcinoma cells. Understanding the molecular underpinnings of selectin-ligand interactions involved in heterotypic tumor cell-host cell adhesion events may provide guidelines for developing novel cancer diagnostics. Recent advancements in diagnostic device fabrication and design integrated with novel biomarkers exploiting the tissue specific biochemistry of malignant versus normal tissue-expressed selectin ligands may hold promise in providing effective alternatives to current cancer diagnostic technologies.