Kidney Cell Monolayers Research Articles

Qualification of In Vitro Dissolution Absorption System 2 (IDAS2) with Caco-2 and MDCK Cell Monolayers: Dose Sensitivity Study Using BCS Class I and III Drugs.

This study aimed to validate the In vitro Dissolution Absorption System 2 (IDAS2) containing a biological barrier of Caco-2 or Madin-Darby canine kidney (MDCK) cell monolayer through dose sensitivity studies. Metoprolol and propranolol were selected as Biopharmaceutics Classification System (BCS) Class I model drugs, and atenolol as a Class III model drug. The IDAS2 is comprised of a dissolution vessel (500 mL) and two permeation chambers (2 × 8.0 mL) mounted with Caco-2 or MDCK cell monolayer. One or two immediate-release tablet(s) of the model drug were added to the dissolution vessel, and the time profiles of dissolution and permeation were observed. Greater than 85% of metoprolol and propranolol (tested at two dosing concentrations) were dissolved by 15 min, and all drugs were fully dissolved by 30 min. All three drugs were more permeable across Caco-2 cells than MDCK cells with a linear increase in permeation across both cells at both dose concentrations. Thus, the dose sensitivity of the IDAS2 was demonstrated using both cell barriers. These results indicate a successful qualification of IDAS2 for the development/optimization of oral formulations and that MDCK cells can be utilized as a surrogate for Caco-2 cells.

Activation of farnesoid X receptor retards expansion of renal collecting duct cell-derived cysts via inhibition of CFTR-mediated Cl- secretion.

The transcription factor farnesoid X receptor (FXR) regulates energy metabolism. Specifically, FXR functions to regulate cystic fibrosis transmembrane conductance regulator (CFTR)-mediated Cl- secretion in intestinal epithelial cells. Therefore, this study aimed to investigate the role of FXR in CFTR-mediated Cl- secretion in renal tubular cells and to further elucidate its effects on renal cyst formation and growth. CFTR-mediated Cl- transport was evaluated via short-circuit current (ISC) measurements in Madin-Darby canine kidney (MDCK) cell monolayers and primary rat inner medullary collecting duct cells. The role of FXR in renal cyst formation and growth was determined by the MDCK cell-derived cyst model. Incubation with synthesized (GW4064) and endogenous (CDCA) FXR ligands reduced CFTR-mediated Cl- secretion in a concentration- and time-dependent manner. The inhibitory effect of FXR ligands was not due to the result of reduced cell viability and was attenuated by cotreatment with an FXR antagonist. FXR activation significantly decreased CFTR protein but not its mRNA. In addition, FXR activation inhibited CFTR-mediated Cl- secretion in primary renal collecting duct cells. FXR activation decreased ouabain-sensitive ISC without altering Na+-K+-ATPase mRNA and protein levels. Furthermore, FXR activation significantly reduced the number of cysts and renal cyst expansion. These inhibitory effects were correlated with a decrease in the expression of protein synthesis regulators mammalian target of rapamycin/S6 kinase. This study shows that FXR activation inhibits Cl- secretion in renal cells via inhibition of CFTR expression and retards renal cyst formation and growth. The discoveries point to a physiological role of FXR in the regulation of CFTR and a potential therapeutic application in polycystic kidney disease treatment.NEW & NOTEWORTHY The present study reveals that farnesoid X receptor (FXR) activation reduces microcyst formation and enlargement. This inhibitory effect of FXR activation is involved with decreased cell proliferation and cystic fibrosis transmembrane conductance regulator-mediated Cl- secretion in renal collecting duct cells. FXR might represent a novel target for the treatment of autosomal dominant polycystic kidney disease.

Hexanematic crossover in epithelial monolayers depends on cell adhesion and cell density

Changes in tissue geometry during developmental processes are associated with collective migration of cells. Recent experimental and numerical results suggest that these changes could leverage on the coexistence of nematic and hexatic orientational order at different length scales. How this multiscale organization is affected by the material properties of the cells and their substrate is presently unknown. In this study, we address these questions in monolayers of Madin-Darby canine kidney cells having various cell densities and molecular repertoires. At small length scales, confluent monolayers are characterized by a prominent hexatic order, independent of the presence of E-cadherin, monolayer density, and underlying substrate stiffness. However, all three properties affect the meso-scale tissue organization. The length scale at which hexatic order transits to nematic order, the “hexanematic” crossover scale, strongly depends on cell-cell adhesions and correlates with monolayer density. Our study demonstrates how epithelial organization is affected by mechanical properties, and provides a robust description of tissue organization during developmental processes.

Topological properties and shape of proliferative and nonproliferative cell monolayers.

During embryonic development, structures with complex geometry can emerge from planar epithelial monolayers; studying these shape transitions is of key importance for revealing the biophysical laws involved in the morphogenesis of biological systems. Here, using the example of normal proliferative monkey kidney (COS) cell monolayers, we investigate global and local topological characteristics of this model system in dependence on its shape. The obtained distributions of cells by their valence demonstrate a difference between the spherical and planar monolayers. In addition, in both spherical and planar monolayers, the probability of observing a pair of neighboring cells with certain valences depends on the topological charge of the pair. The zero topological charge of the cell pair can increase the probability for the cells to be the nearest neighbors. We then test and confirm that analogous relationships take place in a more ordered spherical system with a larger fraction of 6-valent cells, namely, in the nonproliferative epithelium (follicular system) of ascidian species oocytes. However, unlike spherical COS cell monolayers, ascidian monolayers are prone to nonrandom agglomeration of 6-valent cells and have linear topological defects called scars and pleats. The reasons for this difference in morphology are discussed. The morphological peculiarities found are compared with predictions of the widely used vertex model of epithelium.

Actin-rich lamellipodia-like protrusions contribute to the integrity of epithelial cell–cell junctions

Metastasis-suppressor 1 (MTSS1) is a membrane-interacting scaffolding protein that regulates the integrity of epithelial cell-cell junctions and functions as a tumor suppressor in a wide range of carcinomas. MTSS1 binds phosphoinositide-rich membranes through its I-BAR domain and is capable of sensing and generating negative membrane curvature invitro. However, the mechanisms by which MTSS1 localizes to intercellular junctions in epithelial cells and contributes to their integrity and maintenance have remained elusive. By carrying out EM and live-cell imaging on cultured Madin-Darby canine kidney cell monolayers, we provide evidence that adherens junctions of epithelial cells harbor lamellipodia-like, dynamic actin-driven membrane folds, which exhibit high negative membrane curvature at their distal edges. BioID proteomics and imaging experiments demonstrated that MTSS1 associates with an Arp2/3 complex activator, the WAVE-2 complex, in dynamic actin-rich protrusions at cell-cell junctions. Inhibition of Arp2/3 or WAVE-2 suppressed actin filament assembly at adherens junctions, decreased the dynamics of junctional membrane protrusions, and led to defects in epithelial integrity. Together, these results support a model in which membrane-associated MTSS1, together with the WAVE-2 and Arp2/3 complexes, promotes the formation of dynamic lamellipodia-like actin protrusions that contribute to the integrity of cell-cell junctions in epithelial monolayers.

Effects of Citrullus colocynthis Seed Aqueous Extracts upon Sodium Transport across A6 Kidney Cell Monolayers

Various extracts from Citrullus colocynthis seeds were recently examined for their possible favorable effects on glucose homeostasis, other metabolic variables, and pancreatic islet size in streptozotocin-induced diabetic rats. Considering the effectiveness of insulin upon target cells as another process susceptible to being influenced by plant extracts, the present study is used to compare the effects of either crude or defatted aqueous extracts from C. colocynthis seeds upon Na + transport in A6 kidney cells. The intensity of electrical currents, taken as the potential difference divided by the epithelial resistance was measured in monolayers of A6 kidney cells exposed at the basolateral side to either crude untreated aqueous (EI) or defatted aqueous (EII) extracts, as well as their fractions EI1and EII1 containing components with high molecular weight (↑5 kDa) and EI2 and EII2 containing components with low molecular weight (↓5 kDa). The effect of insulin was to assess the functional integrity of the A6 monolayer. All extracts caused a rapid and sustained increase in current intensity. The defatted aqueous extract (EII) was more efficient than the non-defatted aqueous extract (EI) in terms of increasing the intensity of current in the A6 monolayer. The components of the defatted aqueous extract responsible for such a greater efficiency were located in the fraction containing components with low molecular weight and acted in a time-related progressive fashion upon Na + transport. These data suggest that colocynth seed extracts mainly extract containing components with low molecular weight increased the current intensity in A6 cells, thus suggesting that colocynth may affect Na + transport in kidney cells.

UV light-induced spatial loss of sialic acid capping using a photoactivatable sialyltransferase inhibitor.

Sialic acids cap glycans displayed on mammalian glycoproteins and glycolipids and mediate many glycan-receptor interactions. Sialoglycans play a role in diseases such as cancer and infections where they facilitate immune evasion and metastasis or serve as cellular receptors for viruses, respectively. Strategies that specifically interfere with cellular sialoglycan biosynthesis, such as sialic acid mimetics that act as metabolic sialyltransferase inhibitors, enable research into the diverse biological functions of sialoglycans. Sialylation inhibitors are also emerging as potential therapeutics for cancer, infection, and other diseases. However, sialoglycans serve many important biological functions and systemic inhibition of sialoglycan biosynthesis can have adverse effects. To enable local and inducible inhibition of sialylation, we have synthesized and characterized a caged sialyltransferase inhibitor that can be selectively activated with UV-light. A photolabile protecting group was conjugated to a known sialyltransferase inhibitor (P-SiaFNEtoc). This yielded a photoactivatable inhibitor, UV-SiaFNEtoc, that remained inactive in human cell cultures and was readily activated through radiation with 365 nm UV light. Direct and short radiation of a human embryonic kidney (HEK293) cell monolayer was well-tolerated and resulted in photoactivation of the inhibitor and subsequent spatial restricted synthesis of asialoglycans. The developed photocaged sialic acid mimetic holds the potential to locally hinder the synthesis of sialoglycans through focused treatment with UV light and may be applied to bypass the adverse effects related to systemic loss of sialylation.

Comparisons of in Vitro Models to Evaluate the Membrane Permeability of Amorphous Drug Nanoparticles.

The spontaneous formation of amorphous drug nanoparticles following the release of a drug from a supersaturating formulation is gaining increasing attention due to their potential contribution to increased oral bioavailability. The formation of nanosized drug particles also has considerable implications for the interpretation of in vitro and in vivo data. However, the membrane transport properties of these drug particles remain less well understood. Herein, the membrane permeation of nanosized amorphous drug particles of a model drug atazanavir was evaluated using different artificial membrane-based, cell-based, and animal tissue-based models. Results showed that flux enhancement by particles was different for the various systems used. Generally, good agreement was obtained among experiments performed using the same apparatus with different model membranes, with the exception of the Madin-Darby canine kidney cell monolayer and the Long-Evans rat intestine tissue, which showed lower flux enhancements. Franz cell-based models showed slightly higher flux enhancements by particles compared to Transwell and intestinal tissue sac models. Mass transport analysis suggested that the extent of flux enhancement by particles is dependent on the geometry of the apparatus as well as the properties of the membrane and buffer used, whereas the flux plateau concentration is dependent on the unstirred water later (UWL) asymmetry. These results highlight the complexity in characterizing the permeability advantage of these nonmembrane permeable drug particles and suggest that caution should be used in selecting the appropriate in vitro model to evaluate the overall permeability of colloidal drug particles.

Transepithelial delivery of insulin conjugated with phospholipid-mimicking polymers via biomembrane fusion-mediated transcellular pathways

Epithelial barriers that seal cell gaps by forming tight junctions to prevent the free permeation of nutrients, electrolytes, and drugs, are essential for maintaining homeostasis in multicellular organisms. The development of nanocarriers that can permeate epithelial tissues without compromising barrier function is key for establishing a safe and efficient drug delivery system (DDS). Previously, we have demonstrated that a water-soluble phospholipid-mimicking random copolymer, poly(2-methacryloyloxyethyl phosphorylcholine30-random-n‑butyl methacrylate70) (PMB30W), enters the cytoplasm of live cells by passive diffusion manners, without damaging the cell membranes. The internalization mechanism was confirmed to be amphiphilicity-induced membrane fusion. In the present study, we demonstrated energy-independent permeation of PMB30W through the model epithelial barriers of Madin-Darby canine kidney (MDCK) cell monolayers in vitro. The polymer penetrated epithelial MDCK monolayers via transcellular pathways without breaching the barrier functions. This was confirmed by our unique assay that can monitor the leakage of the proton as the smallest indicator across the epithelial barriers. Moreover, energy-independent transepithelial permeation was achieved when insulin was chemically conjugated with the phospholipid-mimicking nanocarrier. The bioactivity of insulin as a growth factor was found to be maintained even after translocation. These fundamental findings may aid the establishment of transepithelial DDS with advanced drug efficiency and safety. Statement of significanceA nanocarrier that can freely permeate epithelial tissues without compromising barrier function is key for successful DDS. Existing strategies mainly rely on paracellular transport associated with tight junction breakdown or transcellular transport via transporter recognition-mediated active uptake. These approaches raise concerns about efficiency and safety. In this study, we performed non-endocytic permeation of phospholipid-mimicking polymers through the model epithelial barriers in vitro. The polymer penetrated via transcytotic pathways without breaching the barriers of biomembrane and tight junction. Moreover, transepithelial permeation occurred when insulin was covalently attached to the nanocarrier. The bioactivity of insulin was maintained even after translocation. The biomimetic design of nanocarrier may realize safe and efficient transepithelial DDS.

781. Antiviral Effectiveness of Different Disinfectants on Surfaces Contaminated with Surrogate Non-enveloped Viruses

Abstract Background Contamination of surfaces with virions remains an important medical issues because they can serve as fomites for viral transmission. Long acting disinfectants are important because viruses can remain viable for extended durations. Here we assessed the anti-viral efficacy of various environmental spray disinfectants against F specific coliphage MS2, and Feline Calicivirus (FCV) as surrogate non-enveloped viruses. Methods MS2 was grown and titrated according to Environmental Protection Agency method 1601 and counted as plague forming unit per milliliter (PFU/mL.). Strain F9 of FCV was propagated and titrated in monolayers of Crandell-Reese Feline Kidney cells. Commercially available and an experimental disinfectant based on a biguanide combination were tested. The disinfectants, both wet and as dried residues, were compared on silicone surfaces for reduction of viral loads following 1-minute exposure to high viral load challenges (106-107 PFU/ml). Sterilized deionized water was used as a control. Results Results (as log10 reduction relative to sterilized deionized water control) are tabulated below for the disinfectants in wet and dry states following 1-minute viral challenges. Log10 reductions of MS2 and FCV viral loads following 1-minute exposures to disinfectants in wet and dry residue states. Conclusion The disinfectants were generally much less effective as dried residues than wet. The biguanide-combination and hydrogen peroxide-combination disinfectants were the most effective disinfectants tested in both wet and dry residue states. Both warrant further study for extended eradication of highly infectious viruses on surfaces. Disclosures All Authors: No reported disclosures

In Vitro Infection Dynamics of Japanese Encephalitis Virus in Established Porcine Cell Lines.

Japanese encephalitis virus (JEV) is a zoonotic mosquito-borne pathogen that regularly causes severe neurological disease in humans in Southeast Asia and the Western Pacific region. Pigs are one of the main amplifying hosts of JEV and play a central role in the virus transmission cycle. The objective of this study was to identify in vitro cell systems to investigate early effects of JEV infection including viral replication and host cell death. Here, we demonstrate the susceptibility of several porcine cell lines to the attenuated genotype III JEV strain SA14-14-2. Monolayers of porcine nasal turbinate (PT-K75), kidney (SK-RST), testis (ST), and monocyte-derived macrophage (C2+) cells were infected with SA14-14-2 for up to five days at a multiplicity of infection (MOI) of 0.1. The hamster kidney cell line BHK-21, previously shown to be susceptible to SA14-14-2, was used as a positive control. Culture supernatants and cells were collected between 0 and 120 h post infection (hpi), and monolayers were observed for cytopathic effect (CPE) using brightfield microscopy. The number of infectious virus particles was quantified by plaque assay and cell viability was determined using trypan blue staining. An indirect immunofluorescence assay was used to detect the presence of JEV NS1 antigens in cells infected at 1 MOI. All four porcine cell lines demonstrated susceptibility to SA14-14-2 and produced infectious virus by 12 hpi. Virus titers peaked at 48 hpi in C2+, BHK-21, and SK-RST cells, at 72 hpi in PT-K75, and at 120 hpi in ST cells. CPE was visible in infected C2+ and BHK-21 cells, but not the other three cell lines. The proportion of viable cells, as measured by trypan blue exclusion, declined after 24 hpi in BHK-21 and 48 hpi in C2+ cells, but did not substantially decline in SK-RST, PT-K75 or ST cells. At 48 hpi, JEV NS1 was detected in all infected cell lines by fluorescence microscopy. These findings demonstrate several porcine cell lines which have the potential to serve as useful research tools for investigating JEV infection dynamics and host cell mechanisms in a natural amplifying host species, such as pigs, in vitro.

Effect of serum starvation on rheology of cell monolayers

The rheological properties of cells and tissues are central to embryonic development and homeostasis in adult tissues and organs and are closely related to their physiological activities. This work presents our study of rheological experiments on cell monolayer under serum starvation compared to healthy cell monolayer with full serum. Serum starvation is one of the most widely used procedures in cell biology. However, the effect of deprivation of serum concentration on the material properties of cells is still unknown. Therefore, we performed macro-rheology experiments to investigate the effect of serum starvation on a fully confluent Madin–Darby Canine Kidney cell monolayer. The material properties, such as linear and non-linear viscoelastic moduli, of the monolayer, were measured using oscillatory shear experiments under serum-free [0% fetal bovine serum (FBS)] and full serum (10% FBS) conditions. Our results indicate that a serum-starved cell monolayer shows a different rheological behavior than a healthy cell monolayer. The loss and storage moduli decrease for the step-change in oscillatory strain amplitude experiments for a serum-starved cell monolayer and do not recover fully even after small deformation. In comparison, a healthy cell monolayer under full serum condition remains flexible and can fully recover even from a large deformation at higher strain. The effect of adhesion due to fibronectin was also studied in this work, and we found a significant difference in slip behavior for cell monolayer with and without serum.

Stretchable electrical cell-substrate impedance sensor platform for monitoring cell monolayers under strain

Stretchable microelectrodes paired with an ultra-elastic substrate can be used for electrical sensing of mechanically stretched cells and cell monolayers. Here, we present the development of a cell-stretching platform with thin-film interdigitated microelectrodes. Up to 35 % cyclic stretch are feasible with a novel interlaced meander design connected to the microelectrodes and using Poly(dimethylsiloxane) (PDMS) with a Young’s modulus of 50 kPa as an ultra-elastic substrate. Reliable electrical contacting of the microelectrodes under stretch was achieved by perforation of the contact pads. The novel platform enables label-free, real-time electrical cell-substrate impedance (ECIS) monitoring of cell monolayers. Proof-of-concept experiments indicated that electrical impedance of Madin-Darby canine kidney (MDCK) cell monolayers increased sharply by uniaxial mechanical strain above 20 %. For comparison, human alveolar basal epithelial adenocarcinoma (A549) cell monolayers, which are known to lack mature cell junctions, showed a continuous decrease of electrical impedance over the whole applied strain range of 35 %. The data reveal that impedance changes upon stretching depend on epithelial cell types and existence of tight cellular junctions. The system provides the basis for reliable continuous long-term monitoring of electrical properties of cell monolayers under strain by electrical impedance spectroscopy, e.g., to monitor epithelial permeability changes in real time and under label-free conditions to screen the influence of pharmacological substances.

Transepithelial Delivery of Insulin Conjugated with Phospholipid-Mimicking Polymers via Biomembrane Fusion-Mediated Transcellular Pathways

Epithelial barriers that seal cell gaps by forming tight junctions to prevent the free permeation of nutrients, electrolytes, and drugs, are essential for maintaining homeostasis in multicellular organisms. The development of nanocarriers that can permeate epithelial tissues without compromising barrier function is key for establishing a safe and efficient drug delivery system (DDS). Previously, we have demonstrated that a water-soluble phospholipid-mimicking random copolymer, poly(2-methacryloyloxyethyl phosphorylcholine30-random-n-butyl methacrylate70) (PMB30W), enters the cytoplasm of live cells by passive diffusion mechanisms, without damaging the cell membranes. The internalization mechanism was confirmed to be amphiphilicity-induced membrane fusion. In the present study, we demonstrated nonendocytic permeation of PMB30W through the model epithelial barriers of Madin-Darby canine kidney (MDCK) cell monolayers in vitro. The polymer penetrated epithelial MDCK monolayers via transcellular pathways without breaching the barrier functions. This was confirmed by our unique assay that can monitor the leakage of the proton as the smallest indicator across the epithelial barriers. Moreover, nonendocytic transepithelial permeation was achieved when insulin was chemically conjugated with the phospholipid-mimicking nanocarrier. The bioactivity of insulin as a growth factor was found to be maintained even after translocation. These fundamental findings may aid the establishment of transepithelial DDS with advanced drug efficiency and safety.

Effect of double bond in unsaturated long-chain monoglyceride in self-emulsified nanoemulsion on tight junction opening

Unsaturated long chain monoglycerides (LCMs) used to formulate self-emulsified nanoemulsion (SEN) are varied in their degree of unsaturation which may influence their activities on membrane permeability. Therefore, this study highlights the impact of double bond in LCMs on the tight junctions of Madin-Darby Canine Kidney (MDCK) cell monolayer. Two LCMs, Glyceryl monooleate (one double bond) and Glyceryl monolinoleate (two double bonds) were formulated into SENs (SEN2 and SEN3, respectively) with Glyceryl tricaprylate and PEG-40 hydrogenated castor oil (1:1:2), and compared to the SEN (SEN1) consisted of only Glyceryl tricaprylate and PEG-40 hydrogenated castor oil (1:4). Lucifer Yellow (LY, MW 457) or Fluorescein isothiocyanate–Dextran4K (FD4, MW 3000-5000) was added to the aqueous phase of the SENs and tested for their transport across the cell monolayer. The cumulative transport of LY and FD4 achieved by SEN2 and SEN3 were significantly (p < 0.05) higher than those in phosphate buffer, 0.5% PEG-40 hydrogenated castor oil, and SEN1, indicating that the tight junctions were opened by the SENs containing LCMs. The cumulative transport of FD4 achieved by SEN3 was also 2-3 folds higher than SEN2 (p < 0.05). Therefore, LCM with two double bonds have greater potency of opening tight junction than LCM with a single double bond in SEN formulation.

Understanding the Effect of Hydroxypropyl-β-Cyclodextrin on Fenebrutinib Absorption in an Itraconazole-Fenebrutinib Drug-Drug Interaction Study.

Cyclodextrins are widely used pharmaceutical excipients, particularly for insoluble compounds dosed orally, such as the oral solution of itraconazole, which is frequently used in clinical drug–drug interaction studies to inhibit cytochrome P450 3A. Since cyclodextrins act by forming inclusion complexes with their coformulated drug, they could have an unintended consequence of affecting absorption if they form a strong complex with the potential victim drug in an itraconazole drug–drug interaction study. This observation was made in a drug–drug interaction study with the Bruton’s tyrosine kinase (BTK) inhibitor fenebrutinib and itraconazole, in which, relative to the control group, the expected increase in fenebrutinib maximum plasma concentration (Cmax) was not observed in the itraconazole group, and a delay in time to reach maximum plasma concentration (Tmax) was observed in the itraconazole group. The in vitro binding constant between fenebrutinib and hydroxypropyl‐β‐cyclodextrin was determined to be 2 × 105 M−1, and the apparent permeability of fenebrutinib across a Madin‐Darby canine kidney cell monolayer decreased in a cyclodextrin concentration‐dependent manner. This observation was confirmed in vivo, in a pentagastrin‐pretreated dog model, in which fenebrutinib was administered with or without cyclodextrin; a reduction in Cmax, a prolonged Tmax, and increased fenebrutinib recovery in feces replicated the previous observation in healthy volunteers and supported the hypothesis that complexation with cyclodextrin decreased rate and extent of fenebrutinib absorption. Physiologically‐based pharmacokinetic modeling was used to translate the in vitro effect of cyclodextrin on fenebrutinib apparent permeability to the in vivo effect on absorption, which was then confirmed using the in vivo dog pharmacokinetic data.

Effect of fatty acid ester structure on cytotoxicity of self-emulsified nanoemulsion and transport of nanoemulsion droplets

Although the cytotoxic and permeation enhancing activities of some fatty acid esters (FAEs) have been studied individually, there lacks systemic studies on how their molecular structures can affect these activities and how the activities would change when FAEs form micelles or nanoemulsion droplets. Therefore, this study aims to address these issues by investigating the cytotoxic and permeation enhancing effects of twenty-six FAEs in lipid droplets on Madin-Darby Canine Kidney (MDCK) cell monolayer. The effect of FAEs on the cytotoxicity and the transport of nanoemulsion droplets depends on how easy the FAE monomers can diffuse out of the droplets and perturbate into the cellular membrane, which determined by the critical structures of FAEs including hydrophilic head size, chain length, number of chains, and number of double bond(s) in the chain. The less the intermolecular attraction, the easier the monomer to diffuse out of the lipid droplets and the more interaction with the cell monolayer. Fatty acid monoester (FAME) with less intermolecular attraction than di- and tri-esters had more cytotoxic and higher permeation enhancing effects. Among the FAMEs, the cytotoxicity and permeation enhancing effects were in the order: medium-chain with small hydrophilic head (propylene glycol and glycol) > long-chain with two double bonds > medium-chain with large head (sorbitan, polyethylene glycol, sucrose, and PEGylated sorbitan) ≈ long-chain with single double bond > saturated long-chain, which are consistent with the intermolecular attraction force (low-high). However, the more kinks in the monomer’s tail caused by the double bond, the less the monomer can perturbate into the cell membrane, resulting in less cytotoxicity and permeation enhancement. In conclusion, the FAEs with less intermolecular attraction and straight chain have more cytotoxic and permeation enhancing effects when formulated in nanoemulsion.

Mechanical adaptions of collective cells nearby free tissue boundaries

Mechanical adaptions of cells, including stiffness variation, cytoskeleton remodeling, motion coordination, and shape changing, are essential for tissue morphogenesis, wound healing, and malignant progression. In this paper, we take confluent monolayers of Madin-Darby canine kidney (MDCK) and mouse myoblast (C2C12) cells as model systems to probe how cells collectively adapt their mechanical features in response to a free tissue boundary. We show that the free boundary not only can trigger unjamming transition but also induces cell fluidization nearby the boundary. The Young’s moduli of cells near the boundary are found to be much lower than those of interior cells. We demonstrate that the stiffness of cells in monolayers with a free tissue boundary exhibits negative dependence on the projected cell area, in contrast to previous studies where cells were found to stiffen as cellular area increases in a confluent MDCK monolayer without boundary. In addition, the free tissue boundary may activate cell remodeling, rendering volume enlargement and cell-specified cytoskeleton organization. Our study emphasizes the important role of geometrical boundary in regulating biomechanical properties of cell aggregates.

Improving Eflornithine Oral Bioavailability and Brain Uptake by Modulating Intercellular Junctions With an E-cadherin Peptide

Eflornithine has been used to treat second-stage human African trypanosomiasis. However, it has inadequate oral bioavailability and low blood-brain barrier permeation, thus requiring a lengthy and complicated intravenous infusion schedule. Here, we investigated the feasibility of using an intercellular junction-modulating E-cadherin peptide HAV6 to enhance the oral bioavailability and blood-brain barrier permeation of eflornithine. Eflornithine was not metabolized in liver microsomes, nor was it a substrate for the human efflux transporter P-glycoprotein. Furthermore, HAV6 and HAV6scr (sequence scrambled HAV6) were stable in simulated gastric fluid with pepsin and rat intestinal mucosal scrapings. Both peptides were stable in human plasma, albeit less stable in rat and mouse plasma. HAV6 increased eflornithine permeability across Madin-Darby canine kidney and Caco-2 cell monolayers (5- and up to 8.5-fold), whereas HAV6scr had little effect. Using an in situ rat brain perfusion model, HAV6, but not HAV6scr, significantly increased eflornithine concentrations in different brain regions up to 4.9-fold. In rats, coadministration of HAV6 increased eflornithine oral bioavailability from 38% to 54%, brain concentrations by up to 83%, and cerebrospinal fluid concentrations by 40%. In conclusion, coadministration of HAV6, either during intravenous infusion or as an oral formulation, has the potential to improve eflornithine-based treatment for second-stage human African trypanosomiasis.

Label-free self-referenced sensing of living cells by terahertz metamaterial-based reflection spectroscopy.

Terahertz (THz) metamaterial-based reflection spectroscopy is proposed for label-free sensing of living cells by a self-referenced method. When sensing the living Madin-Darby canine kidney cell monolayer and phosphate buffered saline solution, self-referenced signals showed significant differences in peak intensity because of inherent discrepancy in the imaginary part of their complex refractive indices, as confirmed by 3D-FDTD simulations. The resonance peak intensity was unaffected by cell monolayer thickness variation, demonstrating feasibility for sensing various cells. Simulations and experiments showed that saponin-induced changes in cell permeability could be monitored in real-time. The self-referenced signal was linearly dependent on the adherent cell density, illustrating a label-free in situ THz metamaterial-based cell sensor.