Biological Membranes Research Articles

Direct detection of the chloride release and uptake reactions of Natronomonas pharaonis halorhodopsin

Membrane transport proteins undergo multistep conformational changes to fulfill the transport of substrates across biological membranes. Substrate release and uptake are the most important events of these multistep reactions that accompany significant conformational changes. Thus, their relevant structural intermediates should be identified to better understand the molecular mechanism. However, their identifications have not been achieved for most transporters due to the difficulty of detecting the intermediates. Herein, we report the success of these identifications for a light-driven chloride transporter halorhodopsin (HR). We compared the time course of two flash-induced signals during a single transport cycle. One is a potential change of Cl--selective membrane, which enabled us to detect tiny Cl--concentration changes due to the Cl- release and the subsequent Cl--uptake reactions by HR. The other is the absorbance change of HR reflecting the sequential formations and decays of structural intermediates. Their comparison revealed not only the intermediates associated with the key reactions but also the presence of two additional Cl--binding sites on the Cl--transport pathways. The subsequent mutation studies identified one of the sites locating the protein surface on the releasing side. Thus, this determination also clarified the Cl--transport pathway from the initial binding site until the release to the medium.

Localization and Aggregation of Honokiol in the Lipid Membrane.

Honokiol, a biphenyl lignan extracted from bark extracts belonging to Magnolia plant species, is a pleiotropic compound which exhibits a widespread range of antioxidant, antibacterial, antidiabetic, anti-inflammatory, antiaggregant, analgesic, antitumor, antiviral and neuroprotective activities. Honokiol, being highly hydrophobic, is soluble in common organic solvents but insoluble in water. Therefore, its biological effects could depend on its bioactive mechanism. Although honokiol has many impressive bioactive properties, its effects are unknown at the level of the biological membrane. Understanding honokiol's bioactive mechanism could unlock innovative perspectives for its therapeutic development or for therapeutic development of molecules similar to it. I have studied the behaviour of the honokiol molecule in the presence of a plasma-like membrane and established the detailed relation of honokiol with membrane components using all-atom molecular dynamics. The results obtained in this work sustain that honokiol has a tendency to insert inside the membrane; locates near and below the cholesterol oxygen atom, amid the hydrocarbon membrane palisade; increases slightly hydrocarbon fluidity; does not interact specifically with any membrane lipid; and, significantly, forms aggregates. Significantly, aggregation does not impede honokiol from going inside the membrane. Some of the biological characteristics of honokiol could be accredited to its aptitude to alter membrane biophysical properties, but the establishment of aggregate forms in solution might hamper its clinical use.

Tensing Flipper: Photosensitized Manipulation of Membrane Tension, Lipid Phase Separation, and Raft Protein Sorting in Biological Membranes.

The lateral organization of proteins and lipids in the plasma membrane is fundamental to regulating a wide range of cellular processes. Compartmentalized ordered membrane domains enriched with specific lipids, often termed lipid rafts, have been shown to modulate the physicochemical and mechanical properties of membranes and to drive protein sorting. Novel methods and tools enabling the visualization, characterization, and/or manipulation of membrane compartmentalization are crucial to link the properties of the membrane with cell functions. Flipper, a commercially available fluorescent membrane tension probe, has become a reference tool for quantitative membrane tension studies in living cells. Here, we report on a so far unidentified property of Flipper, namely, its ability to photosensitize singlet oxygen (1O2) under blue light when embedded into lipid membranes. This in turn results in the production of lipid hydroperoxides that increase membrane tension and trigger phase separation. In biological membranes, the photoinduced segregated domains retain the sorting ability of intact phase-separated membranes, directing raft and nonraft proteins into ordered and disordered regions, respectively, in contrast to radical-based photo-oxidation reactions that disrupt raft protein partitioning. The dual tension reporting and photosensitizing abilities of Flipper enable simultaneous visualization and manipulation of the mechanical properties and lateral organization of membranes, providing a powerful tool to optically control lipid raft formation and to explore the interplay between membrane biophysics and cell function.

Role of NOX1 and NOX5 in protein kinase C/reactive oxygen species‑mediated MMP‑9 activation and invasion in MCF‑7 breast cancer cells.

NADPH oxidases (NOXs) are a family of membrane proteins responsible for intracellular reactive oxygen species (ROS) generation by facilitating electron transfer across biological membranes. Despite the established activation of NOXs by protein kinase C (PKC), the precise mechanism through which PKC triggers NOX activation during breast cancer invasion remains unclear. The present study aimed to investigate the role of NOX1 and NOX5 in the invasion of MCF‑7 human breast cancer cells. The expression and activity of NOXs and matrix metalloprotease (MMP)‑9 were assessed by reverse transcription‑quantitative PCR and western blotting, and the activity of MMP‑9 was monitored using zymography. Cellular invasion was assessed using the Matrigel invasion assay, whereas ROS levels were quantified using a FACSCalibur flow cytometer. The findings suggested that NOX1 and NOX5 serve crucial roles in 12‑O‑tetradecanoylphorbol‑13‑acetate (TPA)‑induced MMP‑9 expression and invasion of MCF‑7 cells. Furthermore, a connection was established between PKC and the NOX1 and 5/ROS signaling pathways in mediating TPA‑induced MMP‑9 expression and cellular invasion. Notably, NOX inhibitors (diphenyleneiodonium chloride and apocynin) significantly attenuated TPA‑induced MMP‑9 expression and invasion in MCF‑7 cells. NOX1‑ and NOX5‑specific small interfering RNAs attenuated TPA‑induced MMP‑9 expression and cellular invasion. In addition, knockdown of NOX1 and NOX5 suppressed TPA‑induced ROS levels. Furthermore, a PKC inhibitor (GF109203X) suppressed TPA‑induced intracellular ROS levels, MMP‑9 expression and NOX activity in MCF‑7 cells. Therefore, NOX1 and NOX5 may serve crucial roles in TPA‑induced MMP‑9 expression and invasion of MCF‑7 breast cancer cells. Furthermore, the present study indicated that TPA‑induced MMP‑9 expression and cellular invasion were mediated through PKC, thus linking the NOX1 and 5/ROS signaling pathways. These findings offer novel insights into the potential mechanisms underlying their anti‑invasive effects in breast cancer.

Utilizing decellularized bio-membranes to optimize histopathological embedding of small tissues

In recent years, minimally invasive biopsy techniques have been widely used to generate small tissue samples that require processing in clinical pathology. However, small paraffin-embedded tissues are prone to loss due to their small size. To prevent the loss of small tissues, researchers have employed nonbiological embedding materials for preembedding, but this approach can lead to cumbersome experimental procedures and increase the chances of tissue loss. This study aimed to develop a convenient decellularized embedding material derived from biological membrane tissues to effectively protect small tissues from loss during paraffin embedding. This study decellularized three types of fresh animal-derived membrane tissues and selected the small intestine as the most suitable decellularized raw material through attempts at softening, comparing physical properties, and using tissue as the starting material. Subsequently, small tissues from various tissue sources were embedded, followed by H&E staining, Masson staining, immunofluorescence staining, and immunohistochemical staining. The decellularized material derived from biomembrane tissues (DMBT) developed in this study can reduce the loss of small tissues without the need for preembedding, thereby shortening the embedding process. This provides a new pathological embedding tool for future laboratory and clinical research and work.•The fat layer of the pig's small intestine is scraped off, and chemical reagents are used to defat and decellularize it.•Chemical reagents are used to soften and make the pig's small intestine transparent, and the decellularized pig's small intestine is dried.•DMBT is used for embedding and staining the biological tissue.

Aging and physiological barriers: mechanisms of barrier integrity changes and implications for age-related diseases.

The phenomenon of compartmentalization is one of the key traits of life. Biological membranes and histohematic barriers protect the internal environment of the cell and organism from endogenous and exogenous impacts. It is known that the integrity of these barriers decreases with age due to the loss of homeostasis, including age-related gene expression profile changes and the abnormal folding/assembly, crosslinking, and cleavage of barrier-forming macromolecules in addition to morphological changes in cells and tissues. The critical molecular and cellular mechanisms involved in physiological barrier integrity maintenance and aging-associated changes in their functioning are reviewed on different levels: molecular, organelle, cellular, tissue (histohematic, epithelial, and endothelial barriers), and organ one (skin). Biogerontology, which studies physiological barriers in the aspect of age, is still in its infancy; data are being accumulated, but there is no talk of the synthesis of complex theories yet. This paper mainly presents the mechanisms that will become targets of anti-aging therapy only in the future, possibly: pharmacological, cellular, and gene therapies, including potential geroprotectors, hormetins, senomorphic drugs, and senolytics.

Capturing RAS oligomerization on a membrane

RAS GTPases associate with the biological membrane where they function as molecular switches to regulate cell growth. Recent studies indicate that RAS proteins oligomerize on membranes, and disrupting these assemblies represents an alternative therapeutic strategy. However, conflicting reports on RAS assemblies, ranging in size from dimers to nanoclusters, have brought to the fore key questions regarding the stoichiometry and parameters that influence oligomerization. Here, we probe three isoforms of RAS [Kirsten Rat Sarcoma viral oncogene (KRAS), Harvey Rat Sarcoma viral oncogene (HRAS), and Neuroblastoma oncogene (NRAS)] directly from membranes using mass spectrometry. We show that KRAS on membranes in the inactive state (GDP-bound) is monomeric but forms dimers in the active state (GTP-bound). We demonstrate that the small molecule BI2852 can induce dimerization of KRAS, whereas the binding of effector proteins disrupts dimerization. We also show that RAS dimerization is dependent on lipid composition and reveal that oligomerization of NRAS is regulated by palmitoylation. By monitoring the intrinsic GTPase activity of RAS, we capture the emergence of a dimer containing either mixed nucleotides or GDP on membranes. We find that the interaction of RAS with the catalytic domain of Son of Sevenless (SOScat) is influenced by membrane composition. We also capture the activation and monomer to dimer conversion of KRAS by SOScat. These results not only reveal the stoichiometry of RAS assemblies on membranes but also uncover the impact of critical factors on oligomerization, encompassing regulation by nucleotides, lipids, and palmitoylation.

In vitro evolution driven by epistasis reveals alternative cholesterol-specific binding motifs of perfringolysin O

The crucial molecular factors that shape the interfaces of lipid-binding proteins with their target ligands and surfaces remain unknown due to the complex makeup of biological membranes. Cholesterol, the major modulator of bilayer structure in mammalian cell membranes, is recognized by various proteins, including the well-studied cholesterol-dependent cytolysins. Here, we use invitro evolution to investigate the molecular adaptations that preserve the cholesterol specificity of perfringolysin O, the prototypical cholesterol-dependent cytolysin from Clostridium perfringens. We identify variants with altered membrane-binding interfaces whose cholesterol-specific activity exceeds that of the wild-type perfringolysin O. These novel variants represent alternative evolutionary outcomes and have mutations at conserved positions that can only accumulate when epistatic constraints are alleviated. Our results improve the current understanding of the biochemical malleability of the surface of a lipid-binding protein.

Micro-scale quantitative analysis of sterol content in liposomes

The high complexity of biological membranes has driven the development and application of a wide range of model membrane systems. Among these models, liposomes are extensively used because of their versatility in mimicking cellular membranes with a wide range of lipid compositions. However, the accurate quantification of lipid components, such as sterols, within these models remains a critical requirement for validation, data interpretation, and comparison. Here, we present a reliable and sensitive colorimetric assay using the Zak color reaction, which we have specifically adapted for the quantification of sterols at the micro-scale level. The assay was evaluated using cholesterol, ergosterol, and sitosterol standards, reflecting the diversity of sterol species across organisms. The reaction mechanism involves the dehydration of sterols to form carbonium ions, which are oxidized to form various enylic carbonium ions with specific absorption peaks. Due to the different chemical structures of cholesterol, ergosterol, and sitosterol, the resulting spectra show that the colored reaction products are formed in different proportions. The stability and interconversion of these species over time were analyzed. Cholesterol and sitosterol showed a clear peak at 555 nm, while ergosterol had prominent peaks at shorter wavelengths. Sterol assays on liposomal preparations showed accurate sterol incorporation with minimal loss during processing steps. These results demonstrate that this assay provides a robust and accurate measurement of sterol content in large unilamellar vesicles, making it a valuable tool for liposomal studies.

Guidelines for naming and studying plasma membrane domains in plants.

Biological membranes play a crucial role in actively hosting, modulating and coordinating a wide range of molecular events essential for cellular function. Membranes are organized into diverse domains giving rise to dynamic molecular patchworks. However, the very definition of membrane domains has been the subject of continuous debate. For example, in the plant field, membrane domains are often referred to as nanodomains, nanoclusters, microdomains, lipid rafts, membrane rafts, signalling platforms, foci or liquid-ordered membranes without any clear rationale. In the context of plant-microbe interactions, microdomains have sometimes been used to refer to the large area at the plant-microbe interface. Some of these terms have partially overlapping meanings at best, but they are often used interchangeably in the literature. This situation generates much confusion and limits conceptual progress. There is thus an urgent need for us as a scientific community to resolve these semantic and conceptual controversies by defining an unambiguous nomenclature of membrane domains. In this Review, experts in the field get together to provide explicit definitions of plasma membrane domains in plant systems and experimental guidelines for their study. We propose that plasma membrane domains should not be considered on the basis of their size alone but rather according to the biological system being considered, such as the local membrane environment or the entire cell.

A synthetic method to assay polycystin channel biophysics.

Ion channels are biological transistors that control ionic flux across cell membranes to regulate electrical transmission and signal transduction. They are found in all biological membranes and their conductive state kinetics are frequently disrupted in human diseases. Organelle ion channels are among the most resistant to functional and pharmacological interrogation. Traditional channel protein reconstitution methods rely upon exogenous expression and/or purification from endogenous cellular sources which are frequently contaminated by resident ionophores. Here we describe a fully synthetic method to assay functional properties of polycystin channels that natively traffic to primary cilia and endoplasmic reticulum organelles. Using this method, we characterize their oligomeric assembly, membrane integration, orientation and conductance while comparing these results to their endogenous channel properties. Outcomes define a novel synthetic approach that can be applied broadly to investigate channels resistant to biophysical analysis and pharmacological characterization.

Electrochemical and Photoelectrochemical Reduction of Carbon Dioxide on Ruthenium-Cultured Bacterial Biofilms

Most of the bacterial species form biofilms, in which microorganisms are attached to a surface and they are held together by extracellular polymeric substances that they produce. They tend to grow almost everywhere both on living or non-living surfaces. Biofilms are able to propagate charge within their structures and to transfer effectively electrons at interfaces, as well as they could exhibit electrocatalytic properties (e.g. in Microbial Fuels Cells). The application of microbes provides better flexibility: experiments with fuel cells can be operated at normal conditions (temperatures and pressures). Wide variety of microbial metabolic pathways gives the possibility to use aggregates of bacteria in diverse processes. Proposed electrochemical studies using bacterial biofilms (in the form of thin coatings on the glassy carbon electrodes) can be considered as an attempt to find efficient methods of using the energy produced by microorganisms and converting it to electricity.The ultimate goal of the present research has been to determine whether it is possible under laboratory conditions to perform electrocatalytic processes using the hybrid (composite) layers composed of aggregates of bacteria in pristine or modified forms. A biofilm formed by a strain of Yersinia enterocolitica (Y. enterocolitica) is characterized by a high physicochemical stability over a wide pH range (4-10) and temperatures (0-40°C).The subject of interest is a fairly complex reaction, electroreduction of carbon dioxide. There has been growing interest in the search of electrocatalytic anf photoelectrochemical systems capable of efficient conversion of carbon dioxide into fuels and utility chemicals. Our previously performed studies have clearly shown that the Y. enterocolitica biofilm itself has no activity with respect to reduction of CO2, however it acts as a good matrix for the catalytic (e.g. noble metal or metaloorganic) centers, because it affects the reaction mechanism and appears to decrease overpotential of the electroreduction processes. The conducted research shows that the composite materials containing bacterial biofilms can be successfully used to construct systems that have an electrocatalytic reactivity in the reduction of carbon dioxide. We will also address the possibility of dispersing the organometallic ruthenium (II) complex in the biological layer (biofilm). Indeed, the ruthenium (II) complex has been immobilized in the biofilm matrix by successive modification of the liquid medium (Luria-Bertani medium) for culturing bacteria with a solution of the complex compound. In addition, the biological matrix was used (along with the ruthenium (II) complex molecules dispersed in its layer) as a protective coating, stabilizing the unstable p-type semiconductor - copper (I) oxide. The proposed hybrid co-catalytic system showed activity during the photoelectrochemical reduction of carbon dioxide and stability under semi-neutral experimental conditions. Finally, we are going to address the design of the above-mentioned catalytically active systems emphasizing the need to control the structure of the studied hybrid materials (in addition to their stability). Among important issues is the viability of bacteria in the biological membrane as well as elucidation of the role of the bacterial biofilm during the carbon dioxide reduction.

(Invited) Nanofluidic Ion Transport in Carbon Nanotube Porin Channels

Extreme spatial confinement in narrow fluidic channels strongly influences their transport properties and enables unconventional selectivity mechanisms that can often mimic some of the selectivity and transport characteristics of biological membrane channels. More than any modern nanomaterial, carbon nanotubes have enabled experimental platforms that can recreate such extreme confinement in a channel with defined geometry and controllable electronic properties. I will discuss ion transport in canon nanotube porin channels and show how confinement phenomena, coupled with the different electronic effects in these channels can shape their transport properties and ion selectivity characteristics. Overall, these observations contribute to our understanding of nanoscale transport and pave the way for developing a new generation of precision separation platforms for biomedical and industrial use.

Effect of Synthetic Vitreous Fiber Exposure on TMEM16A Channels in a Xenopus laevis Oocyte Model.

Many years ago, asbestos fibers were banned and replaced by synthetic vitreous fibers because of their carcinogenicity. However, the toxicity of the latter fibers is still under debate, especially when it concerns the early fiber interactions with biological cell membranes. Here, we aimed to investigate the effects of a synthetic vitreous fiber named FAV173 on the Xenopus laevis oocyte membrane, the cell model we have already used to characterize the effect of crocidolite asbestos fiber exposure. Using an electrophysiological approach, we found that, similarly to crocidolite asbestos, FAV173 was able to stimulate a chloride outward current evoked by step membrane depolarizations, that was blocked by the potent and specific TMEM16A channel antagonist Ani9. Exposure to FAV173 fibers also altered the oocyte cell membrane microvilli morphology similarly to crocidolite fibers, most likely as a consequence of the TMEM16A protein interaction with actin. However, FAV173 only partially mimicked the crocidolite fibers effects, even at higher fiber suspension concentrations. As expected, the crocidolite fibers' effect was more similar to that induced by the co-treatment with (Fe3+ + H2O2), since the iron content of asbestos fibers is known to trigger reactive oxygen species (ROS) production. Taken together, our findings suggest that FAV173 may be less harmful that crocidolite but not ineffective in altering cell membrane properties.

Biomembrane-Modified Biomimetic Nanodrug Delivery Systems: Frontier Platforms for Cardiovascular Disease Treatment.

Cardiovascular diseases (CVDs) are one of the leading causes of death worldwide. Despite significant advances in current drug therapies, issues such as poor drug targeting and severe side effects persist. In recent years, nanomedicine has been extensively applied in the research and treatment of CVDs. Among these, biomembrane-modified biomimetic nanodrug delivery systems (BNDSs) have emerged as a research focus due to their unique biocompatibility and efficient drug delivery capabilities. By modifying with biological membranes, BNDSs can effectively reduce recognition and clearance by the immune system, enhance biocompatibility and circulation time in vivo, and improve drug targeting. This review first provides an overview of the classification and pathological mechanisms of CVDs, then systematically summarizes the research progress of BNDSs in the treatment of CVDs, discussing their design principles, functional characteristics, and clinical application potential. Finally, it highlights the issues and challenges faced in the clinical translation of BNDSs.

Fine-tuned protein-lipid interactions in biological membranes: exploration and implications of the ORMDL-ceramide negative feedback loop in the endoplasmic reticulum.

Biological membranes consist of a lipid bilayer in which integral membrane proteins are embedded. Based on the compositional complexity of the lipid species found in membranes, and on their specific and selective interactions with membrane proteins, we recently suggested that membrane bilayers can be best described as "finely-tuned molecular machines." We now discuss one such set of lipid-protein interactions by describing a negative feedback mechanism operating in the de novo sphingolipid biosynthetic pathway, which occurs in the membrane of the endoplasmic reticulum, and describe the atomic interactions between the first enzyme in the pathway, namely serine palmitoyl transferase, and the product of the fourth enzyme in the pathway, ceramide. We explore how hydrogen-bonding and hydrophobic interactions formed between Asn13 and Phe63 in the serine palmitoyl transferase complex and ceramide can influence the ceramide content of the endoplasmic reticulum. This example of finely-tuned biochemical interactions raises intriguing mechanistic questions about how sphingolipids and their biosynthetic enzymes could have evolved, particularly in light of their metabolic co-dependence.

Bending-driven patterning of solid inclusions in lipid membranes: Colloidal assembly and transitions in elastic 2D fluids.

Biological or biomimetic membranes are examples within the larger material class of flexible ultrathin lamellae and contoured fluid sheets that require work or energy to impose bending deformations. Bending elasticity also dictates the interactions and assembly of integrated phases or molecular clusters within fluid lamellae, for instance enabling critical cell functions in biomembranes. More broadly, lamella and other thin fluids that integrate dispersed objects, inclusions, and phases behave as contoured 2D colloidal suspensions governed by elastic interactions. To elucidate the breadth of interactions and assembled patterns accessible through elastic interactions, we consider the bending elasticity-driven assembly of 1-10 μm solid plate-shaped Brownian domains (the 2D colloids), integrated into a fluid phospholipid membrane (the 2D fluid). Here, the fluid membranes of giant unilamellar vesicles, 20-50 μm in diameter, each contain 4-100 monodisperse plate-domains at an overall solid area fraction of 17 ± 3%. Three types of reversible plate arrangements are found: persistent vesicle-encompassing quasi-hexagonal lattices, persistent closely associated chains or concentrated lattices, and a dynamic disordered state. The interdomain distances evidence combined attractive and repulsive elastic interactions up to 10 μm, far exceeding the ranges of physio-chemical interactions. Bending contributions are controlled through membrane slack (excess area) producing, for a fixed composition, a sharp cooperative multibody transition in plate arrangement, while domain size and number contribute intricacy.

Evolutionary analysis of tonoplast intrinsic proteins (TIPs) unraveling the role of TIP3s in plant seed development

Tonoplast intrinsic proteins (TIPs) are crucial in facilitating the transportation of water and various small solutes across biological membranes. The evolutionary path and functional roles of TIPs is poorly understood in plants. In the present study, a total of 976 TIPs were identified in 104 diverse species and subsequently studied to trace their lineage-specific evolutionary path and tissue-specific function. Interestingly, TIPs were found to be absent in lower forms such as algae and fungi and they evolved later in primitive plants like bryophytes. Bryophytes possess a distant class of TIPs, denoted as TIP6, which is not found in higher plants. The aromatic/arginine (ar/R) selectivity filter found in TIP6 of certain liverworts share similarity with hybrid intrinsic protein (HIP), suggesting an evolutionary kinship. As plants evolved to more advanced forms, TIPs diversified into five different sub-groups (TIP1 to TIP5). Notably, TIP5 is a sub-group unique to angiosperms. The evolutionary history of the TIP subfamily reveals an interesting observation that the TIP3 subgroup has evolved within seed-bearing Spermatophyta. Further, TIPs exhibit tissue-specific expression that is conserved within various plant species. Specifically, the TIP3s were found to be exclusively expressed in seeds. Quantitative PCR analysis of TIP3s showed gradually increasing expression in soybean seed developmental stages. The expression of TIP3s in different plant species was also found to be gradually increasing during seed maturation. The results presented here address the knowledge gap concerning the evolutionary background of TIPs, specifically TIP3 in plants, and provide valuable insights for a deeper comprehension of the functions of TIPs in plants.

Development of ocular delivery systems for macitentan and ex vivo study of intraocular permeation

Macitentan is a drug that acts as a non-selective antagonist of endothelin receptors. It has been officially approved for the treatment of cases of pulmonary arterial hypertension. There are studies in animal models that indicate a therapeutic potential for this drug in the treatment of glaucoma, a degenerative eye disease marked by a progressive reduction in the visual field that can progress to irreversible blindness. In the present work, ophthalmic formulations of macitentan were developed with the aim of evaluating their potential to deliver the active ingredient to therapeutically relevant sites. An HPLC-DAD method was adapted from the literature and validated to quantify macitentan in each step. Physicochemical characterization studies of the formulations were performed, and a Franz vertical diffusion cell was used in an analysis of the in vitro/ex vivo permeation using cornea and sclera of porcine eyes to represent biological membranes. The developed formulations presented different permeation profiles according to the presence or absence of water in the base formulation and the type of viscosity promoting agent used. The formulations that showed the highest percentage of permeation were evaluated for mucus adhesiveness and the most promising were further analyzed for stability that included measuring drug content, pH, and viscosity. One formulation was developed for the ocular release of macitentan with a high percentage of drug permeation, adequate viscosity and stability that could represent an advancement in the treatment of glaucoma with a high potential for patient adherence.

Cell‐Anchored Lead‐Free Piezoelectric KNN NPs Resisting Washout for Low‐Intensity Ultrasound Driven Neuromodulation

AbstractPiezoelectric nanomaterials for wireless neuromodulation is a promising alternative to traditional electrical stimulation. However, the low‐avidity between piezoelectric nanomaterials and cellular membranes leads to low efficiency of electrical signal transmission, which requires high‐intensity thresholds of ultrasound stimulation (US). Here, lead‐free piezoelectric (K,Na)NbO3 (KNN) nanoparticles (NPs) with cholesterol coating (KNNC) are presented, in which Cholesterol can be accommodated in the membrane and make them append on the plasma membrane. Compared to non‐modified nanoparticles, cell‐anchored KNNC NPs highly resist convective washout owing to high affinity of cholesterol to biological membranes, which enables highly efficient wireless electrical stimulation to activate cell impulses under low‐intensity ultrasound. Meanwhile, after perfusion washing, the KNNC NPs distributed around the cells are washed away, while part of KNNC NPs remain on the surface of cell membrane still can induce significant Ca2+ influx under US, similar to the group without washing, indicating the KNNC NPs appended on the cell play a major role in wireless electrical stimulation. Furthermore, the highly efficient electrical transmission of KNNC enables neural differentiation of stem cells in regulating synaptic plasticity by modulating Ca2+ influx, demonstrating that KNNC NPs offer a perspective toward minimally invasive wireless neuromodulation therapies for neurological diseases.