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Neutrophil extracellular traps (NETs) are highly associated with inflammatory response and vascular injury after ischemic stroke. As the primary degrader of NETs, DNase 1 is limited by easy deactivation and low efficiency of crossing the blood‐brain barrier (BBB). Here, CD206+ M2‐like macrophages‐derived exosomal (M2exo) system is developed for DNase 1 delivery to achieve enhanced ischemic stroke therapy. The nanoplatform can cross the BBB through transcytosis of exosomes, subsequently clearing NETs by DNase 1 to inhibit inflammatory factors release and prevent vascular injury. Moreover, M2exo induces the polarization of M1 microglia to M2 phenotype, alleviating neuroinflammation via producing anti‐inflammatory cytokines. This nanoplatform exhibits significant efficiency in reducing brain infarct area, improving long‐term neurologic outcomes, and promoting BBB remodeling. The mechanism for the synergistic effect from M2exo and DNase 1 is unveiled at the genetic level through transcriptome analysis. This work provides a paradigm for improving the effectiveness of ischemic stroke therapy. [ABSTRACT FROM AUTHOR]

Although numerous peroxidase (POD)‐like nanozymes have been designed for catalytic therapy of cancer, development of nanozymes with higher therapeutic efficacy and less adverse effects are challengeable. More importantly, the underlying antitumor mechanism remains largely unknown which hinders their application. Here, the nitrogen‐doped carbon nanozyme (N‐PCNS) as a model is utilized and demonstrated that its capacity to specifically activate the STING pathway in tumor cells through reactive oxgen species (ROS)‐mediated mitochondrial DNA (mtDNA) release, which provides the initial signals for STING‐dependent innate immune response. Further, a peptide‐nanozyme conjugate (PNEC, OPBP1‐N‐PCNS), comprising a PD‐L1 blocking/targeting dual‐functional peptide and a cationic‐coated N‐PCNS is constructed, which are conjugated through a matrix metalloproteinase responsive peptide linker. The cleaved derivate nanozyme with positive charge exhibits recyclable capacity and superior ability in capturing and enhancing the transcellular transport of tumor‐derived mtDNA toward DCs, thereby amplifying the STING signaling‐mediated anti‐tumor immune response. This study proposed a unique mechanism and design strategy for POD‐like nanozyme in cancer immunotherapy. [ABSTRACT FROM AUTHOR]

Aim: Blood–brain barrier (BBB) disorder is one of the early findings in cognitive impairments. We have recently found that Porphyromonas gingivalis bacteraemia can cause cognitive impairment and increased BBB permeability. This study aimed to find out the possible key virulence factors of P. gingivalis contributing to the pathological process. Materials and Methods: C57/BL6 mice were infected with P. gingivalis or gingipains or P. gingivalis lipopolysaccharide (P. gingivalis LPS group) by tail vein injection for 8 weeks. The cognitive behaviour changes in mice, the histopathological changes in the hippocampus and cerebral cortex, the alternations of BBB permeability, and the changes in Mfsd2a and Cav‐1 levels were measured. The mechanisms of Ddx3x‐induced regulation on Mfsd2a by arginine‐specific gingipain A (RgpA) in BMECs were explored. Results: P. gingivalis and gingipains significantly promoted mice cognitive impairment, pathological changes in the hippocampus and cerebral cortex, increased BBB permeability, inhibited Mfsd2a expression and up‐regulated Cav‐1 expression. After RgpA stimulation, the permeability of the BBB model in vitro increased, and the Ddx3x/Mfsd2a/Cav‐1 regulatory axis was activated. Conclusions: Gingipains may be one of the key virulence factors of P. gingivalis to impair cognition and enhance BBB permeability by the Ddx3x/Mfsd2a/Cav‐1 axis. [ABSTRACT FROM AUTHOR]

Aim: Although of potential biomedical relevance, dipeptide metabolism has hardly been studied. We found the dipeptidase carnosinase‐2 (CN2) to be abundant in human proximal tubules, which regulate water and solute homeostasis. We therefore hypothesized, that CN2 has a key metabolic role, impacting proximal tubular transport function. Methods: A knockout of the CN2 gene (CNDP2‐KO) was generated in human proximal tubule cells and characterized by metabolomics, RNA‐seq analysis, paracellular permeability analysis and ion transport. Results: CNDP2‐KO in human proximal tubule cells resulted in the accumulation of cellular dipeptides, reduction of amino acids and imbalance of related metabolic pathways, and of energy supply. RNA‐seq analyses indicated altered protein metabolism and ion transport. Detailed functional studies demonstrated lower CNDP2‐KO cell viability and proliferation, and altered ion and macromolecule transport via trans‐ and paracellular pathways. Regulatory and transport protein abundance was disturbed, either as a consequence of the metabolic imbalance or the resulting functional disequilibrium. Conclusion: CN2 function has a major impact on intracellular amino acid and dipeptide metabolism and is essential for key metabolic and regulatory functions of proximal tubular cells. These findings deserve in vivo analysis of the relevance of CN2 for nephron function and regulation of body homeostasis. [ABSTRACT FROM AUTHOR]

Transcytosis‐based tubular reabsorption of endogenous proteins is a well‐known energy‐saving pathway that prevents nutrient loss. However, utilization of this well‐known reabsorption pathway for the delivery of exogenous nanodrugs remains a challenge. In this study, using the surface mimic strategy of a specific PEPT1/2‐targeted Gly−Sar peptide as a ligand, renal‐clearable luminescent gold nanoparticles (P−AuNPs) were developed as protein mimics to investigate the transcytosis‐based tubular reabsorption of exogenous substances. By regulating the influential factors (H+ content in tubular lumens and PEPT1/2 transporter counts in tubular cells) of Gly−Sar‐mediated transcytosis, the specific and efficient interaction between P−AuNPs and renal tubular cells was demonstrated both in vitro and in vivo. Efficient transcellular transportation significantly guided the reabsorption of P−AuNPs back into the bloodstream, which enhanced the blood concentration and bioavailability of nanoparticles, contributing to high‐contrast tumor imaging. [ABSTRACT FROM AUTHOR]

Significant challenges persist in enhancing the delivery efficiency of tumor nanomedicines, predominantly due to the difficulty of successfully surpassing pathophysiological barriers. Enhancing tumor penetration of nanomedicines in such conditions represents a pivotal goal in advancing anticancer nanotherapeutics. Transcytosis emerges as a promising solution in this context, addressing the limitations of passive drug delivery. By harnessing diverse stimuli to induce transcytosis, nanocarriers can achieve precise drug delivery and deep tumor penetration, resulting in high therapeutic efficacy and reduced systemic exposure to the therapeutic compound. This review briefly examines various stimuli‐responsive nanosystems and offers an overview and outlook on the development of stimuli‐responsive nanocarriers for transcytosis‐based cancer drug delivery, aiming to provide informative insights for the design of nanomedicines capable of deep tissue penetration and enhanced therapeutic efficacy. [ABSTRACT FROM AUTHOR]

Hydrogels are prevailing drug delivery depots to improve antitumor efficacy and reduce systemic toxicity. However, the application of conventional free drug‐loaded hydrogel is hindered by poor drug penetration in solid tumors. Here, an injectable ferritin‐based nanocomposite hydrogel is constructed to facilitate tumor penetration and improve cancer chemoimmunotherapy. Specifically, doxorubicin‐loaded human ferritin (Dox@HFn) and oxidized dextran (Dex‐CHO) are used to construct the injectable hydrogel (Dox@HFn Gel) through the formation of pH‐sensitive Schiff‐base bonds. After peritumoral injection, the Dox@HFn Gel is retained locally for up to three weeks, and released intact Dox@HFn gradually, which can not only facilitate tumor penetration through active transcytosis but also induce immunogenic cell death (ICD) to tumor cells to generate an antitumor immune response. Combining with anti‐programmed death‐1 antibody (αPD‐1), Dox@HFn Gel induces remarkable regression of orthotopic 4T1 breast tumors, further elicits a strong systemic anti‐tumor immune response to effectively suppress tumor recurrence and lung metastasis of 4T1 tumors after surgical resection. Besides, the combination of Dox@HFn GelL with anti‐CD47 antibody (αCD47) inhibits postsurgical tumor recurrence of aggressive orthotopic glioblastoma tumor model and significantly extends mice survival. This work sheds light on the construction of local hydrogels to potentiate antitumor immune response for improved cancer therapy. [ABSTRACT FROM AUTHOR]

The therapeutic effects of pharmaceuticals depend on their drug concentrations in the cochlea. Efficient drug delivery from the systemic circulation into the inner ear is limited by the blood‐labyrinth‐barrier (BLB). This study investigated a novel noninvasive sound conditioning (SC) strategy (90 dB SPL, 8–16 kHz, 2 h sound exposure) to temporally enhance BLB permeability in a controllable way, contributing to maximizing the penetration of pharmaceuticals from blood circulation into the cochlea. Trafficking of Fluorescein Isothiocyanate conjugated dextran and bovine serum albumin (FITC‐dextran and FITC‐BSA) demonstrated that paracellular leakage of BLB sustained for 6 h after SC, providing a controllable time window for systemic administration. Cochlear concentrations of dexamethasone (DEX) and dexamethasone phosphate (DEX‐P), respectively transported by transcellular and paracellular pathways, showed a higher content of the latter one after SC, further confirming the key role of paracellular pathway in the SC‐induced hyperpermeability. Results of high‐throughput RNA‐sequencing identified a series of tight junction (TJ)‐associated genes after SC. The expressions of TJ (ZO‐1) were reduced and irregular rearrangements of the junction were observed by transmission electron microscopy after SC. We further determined the inhibiting role of Rab13 in the recruitment of ZO‐1 and later in the regulation of cellular permeability. Meanwhile, no significant change in the quantifications of endothelial caveolae vesicles after SC indicated that cellular transcytosis accounted little for the temporary hyperpermeability after SC. Based on these results, SC enhances the BLB permeability within 6 h and allows systemically applied drugs which tend to be transported by paracellular pathway to readily enter the inner ear, contributing to guiding the clinical medications on hearing loss. [ABSTRACT FROM AUTHOR]

Aim: Phosphorus is a critical constituent of bone as a component of hydroxyapatite. Bone mineral content accrues rapidly early in life necessitating a positive phosphorus balance, which could be established by a combination of increased renal reabsorption and intestinal absorption. Intestinal absorption can occur via a transcellular pathway mediated by the apical sodium‐phosphate cotransporter, Slc34a2/NaPiIIb or via the paracellular pathway. We sought to determine how young mammals increase dietary phosphorus absorption from the small intestine to establish a positive phosphorus balance, a prerequisite for rapid bone growth. Methods: The developmental expression profile of genes mediating phosphate absorption from the small intestine was determined in mice by qPCR and immunohistochemistry. Additionally, Ussing chamber studies were performed on small bowel of young (p7–p14) and older (8‐ to 17‐week‐old) mice to examine developmental changes in paracellular Pi permeability and transcellular Pi transport. Results: Blood and urinary Pi levels were higher in young mice. Intestinal paracellular phosphate permeability of young mice was significantly increased relative to older mice across all intestinal segments. NaPiIIb expression was markedly increased in juvenile mice, in comparison to adult animals. Consistent with this, young mice had increased transcellular phosphate flux across the jejunum and ileum relative to older animals. Moreover, transcellular phosphate transport was attenuated by the NaPiIIb inhibitor NTX1942 in the jejunum and ileum of young mice. Conclusion: Our results are consistent with young mice increasing phosphate absorption via increasing paracellular permeability and the NaPiIIb‐mediated transcellular pathway. [ABSTRACT FROM AUTHOR]

The silver‐based formulation has become one of the most widely used nanoagents for agrifood industry and clinical translation. However, the in vivo fate of silver nanoparticles (AgNPs) via oral exposure remains poorly understood. Here, a specific strategy is shown for tracking the gastrointestinal transformation, intestinal absorption, and organ distribution of particulate Ag after oral exposure by using the gold (Au)‐Ag core−shell NPs (Au@AgNPs). Taking advantage of both plasmonic and elemental characteristics of Au@AgNPs, the transformation of particulate Ag in the gastrointestinal tract (GIT), and later on, intestinal absorption are visualized at the single‐particle level. Furthermore, quantitative analysis reveals that the clearance rate of ingested particulate Ag from the blood and organs is much higher than that of ionic Ag, and at 6 h post‐oral administration, the former only contributed 0.08%, 1.10%, and 0.64% of the total Ag in the liver, spleen, and kidneys. Finally, the transcellular and paracellular pathways are identified as two possible mechanisms responsible for the transepithelial transport of particulate Ag. This study opens an avenue toward the examination of oral bioavailability of NPs and may have important implications in exploring the oral delivery technologies of nanoagents. [ABSTRACT FROM AUTHOR]

The hyperproliferative tumor stroma of pancreatic ductal adenocarcinoma (PDAC) severely limits drug permeation and constructs an immunosuppressive microenvironment, causing resistance to chemotherapy and immunotherapy. Traditional nanomedicine mainly focuses on manipulating nanoparticles' particle size or electrical characteristics to penetrate deep PDAC through the paracellular pathway, but the transcellular pathway is often ignored. Therefore, a versatile drug‐polymer conjugate PODEA‐Gem‐HMI is prepared and assembled into nanoparticles for the codelivery of chemotherapy drug gemcitabine and focal adhesion kinase (FAK) inhibitor defactinib. While sensing the mild acidity in the tumor microenvironment, the nanoparticle will disintegrate and release defactinib to modulate the tumor stroma. The PODEA block of the conjugate can bind with cell membranes reversibly and trigger adsorption‐mediated transcytosis (AMT) for promoted tumor penetration and cellular uptake. The internalized conjugates will release gemcitabine responding to the overexpressed glutathione (GSH) for enhanced chemotherapy, and PHMI can condensate the STING monomers for prolonged spontaneous immune stimulation. [ABSTRACT FROM AUTHOR]

The interest for developing antibody‐driven therapeutic interventions has exponentially grown over the last few decades. Even though there have been promising leaps in the development of efficacious antibody therapies, problems revolving around production and site‐directed delivery of these large macromolecules persist. This is especially pertinent when it comes to designing and producing antibodies to penetrate the blood–brain barrier (BBB) to tackle neurodegenerative diseases. One of the most effective approaches to alleviating this problem is to employ a "Trojan Horse" approach, using receptor‐mediated transcytosis, such as those governed by the transferrin receptor (TfR)‐mediated pathways, to deliver large protein payloads into the brain. Even though this method is effective, ideal limiting factors, related to how the antibody binds to the TfR, need to be elucidated to improve BBB penetrance. With this said, we have designed and produced a single‐chain Fc antibody, conjugated to an scFv8D3 TfR binding motif, creating a single‐chain monovalent BBB transporter (scFc‐scFv8D3). This recombinant protein is easy to produce and purify, demonstrates monovalent binding to the TfR and is structurally stable at physiologically relevant temperatures. Using an in vitro BBB model system, we show a positive correlation between the concentration of administered antibody and transcytosis efficacy, with scFc‐scFv8D3 demonstrating significantly higher transcytosis levels compared with scFv8D3‐conjugated bivalent antibodies at elevated administered concentrations. Furthermore, in vivo studies recapitulate the in vitro results, with the scFc‐scFv8D3 demonstrating an elevated brain uptake at higher therapeutic doses in wild‐type mice, comparable with that of the scFv8D3‐conjugated bivalent antibody control. In addition, the half‐life of the single‐chain monovalent BBB transporter is comparable with that of standard IgG antibodies, indicating that the scFc format does not exacerbate physiological degradation. Our results lead us to the conclusion that valency and affinity are important variables to consider when discerning optimal transport across the BBB using TfR‐mediated transcytosis pathways. In addition, we believe the single‐chain Fc antibody we have described, which can easily be manipulated to accommodate a bispecific target tactic, provides a simple and efficacious approach for delivering therapeutic payloads to the brain milieu. [ABSTRACT FROM AUTHOR]

The active transport of nanoparticles into the solid tumor through cell transcytosis has shown great promise in cancer nanomedicine, but it is challenging to develop efficient active transporting nanomedicines with the potential for clinical translation. Here, a type of tertiary amine oxide (TAO)‐containing zwitterionic liposomal nanocarriers is developed that can hitchhike red blood cells (RBCs) to tumor blood vessels and enter solid tumors through transcytosis. To boost the active‐transporting capability, a library of the TAO liposomes (TAOLs) with different chemical structures and particle sizes is constructed and screened by their stability and active transporting capability. Two types of TAOLs are identified that can induce efficient tumor cell transcytosis through rapid macropinocytosis and endoplasmic reticulum/Golgi‐involved exocytosis. It is found that these zwitterionic TAOLs can hitchhike RBCs to gain long blood circulation, get off the cell at the tumor site, effectively enter the tumor through transcytosis, and infiltrate the whole tumor. The chemotherapeutic drug‐loaded liposomes can stop the tumor progression of mice bearing human hepatocellular carcinoma HepG2 cells, exhibiting superior antitumor activity compared to the traditional liposomal drug. This study demonstrates a strategy to construct effective active transporting liposomal nanomedicines for efficient tumor entrance. [ABSTRACT FROM AUTHOR]

Tumor enzyme‐responsive charge‐reversal carriers can induce efficient transcytosis and lead to efficient tumor infiltration and potent anticancer efficacy. However, the correlations of molecular structure with charge‐reversal property, tumor penetration, and drug delivery efficiency are unknown. Herein, aminopeptidase N (APN)‐responsive conjugates were synthesized to investigate these correlations. We found that the monomeric unit structure and the polymer chain structure determined the enzymatic hydrolysis and charge‐reversal rates, and accordingly, the transcytosis and tumor accumulation and penetration of the APN‐responsive conjugates. The conjugate with moderate APN responsiveness balanced the in vitro transcytosis and in vivo overall drug delivery process and achieved the best tumor delivery efficiency, giving potent antitumor efficacy. This work provides new insight into the design of tumor enzyme‐responsive charge‐reversal nanomedicines for efficient cancer drug delivery. [ABSTRACT FROM AUTHOR]

The blood–retina barrier (BRB) is the term used to define the properties of the retinal capillaries and the retinal pigment epithelium (RPE), which separate the systemic circulation from the retina. More specifically, the inner blood–retina barrier (iBRB) is used to describe the properties of the endothelial cells that line the microvasculature of the inner retina, while the outer blood–retina barrier (oBRB) refers to the properties of the RPE cells that separate the fenestrated choriocapillaris from the retina. The BRB is not a fixed structure; rather, it is dynamic, with its components making unique contributions to its function and structural integrity, and therefore the retina. For example, while tight junction (TJ) proteins between retinal endothelial cells are the key molecular structures in the maintenance of the iBRB, other cell types surrounding endothelial cells are also important. In fact, this overall structure is termed the neurovascular unit (NVU). The integrity of the BRB is crucial in the maintenance of a 'dry', tightly regulated retinal microenvironment through the regulation of transcellular and paracellular transport. Specifically, breakdown of TJs can result in oedema formation, a hallmark feature of many retinal diseases. Here, we will describe the oBRB briefly, with a more in‐depth focus on the structure and function of the iBRB in health and diseased states. Finally, the contribution of the BRB to the pathophysiology of age‐related macular degeneration (AMD), diabetic retinopathy (DR) and other rarer retinal diseases will be discussed. [ABSTRACT FROM AUTHOR]

Understanding the intestinal transport of particles is critical in several fields ranging from optimizing drug delivery systems to capturing health risks from the increased presence of nano‐ and micro‐sized particles in human environment. While Caco‐2 cell monolayers grown on permeable supports are the traditional in vitro model used to probe intestinal absorption of dissolved molecules, they fail to recapitulate the transcytotic activity of polarized enterocytes. Here, an intestine‐on‐chip model is combined with in silico modeling to demonstrate that the rate of particle transcytosis is ≈350× higher across Caco‐2 cell monolayers exposed to fluid shear stress compared to Caco‐2 cells in standard "static" configuration. This relates to profound phenotypical alterations and highly polarized state of cells grown under mechanical stimulation and it is shown that transcytosis in the microphysiological model is energy‐dependent and involves both clathrin and macropinocytosis mediated endocytic pathways. Finally, it is demonstrated that the increased rate of transcytosis through cells exposed to flow is explained by a higher rate of internal particle transport (i.e., vesicular cellular trafficking and basolateral exocytosis), rather than a change in apical uptake (i.e., binding and endocytosis). Taken together, the findings have important implications for addressing research questions concerning intestinal transport of engineered and environmental particles. [ABSTRACT FROM AUTHOR]

Cancer photo‐immunotherapy (CPIT) as an ideal strategy can rapidly release hostile signals by appropriate dosage of focal laser irradiation to unmask primary tumor immunogenicity and can activate adaptive immunity to control distant metastases. However, many factors, including disordered immunometabolism, poor penetration of photothermal agents and immuno‐regulators, inadequate laser penetration into the deep tumor region, restrict the therapeutic outcomes of CPIT. Here, a second near‐infrared window (NIR‐II) photo‐immunometabolic cancer therapy (PICT) by a programmed raspberry‐structured nanoadjuvant (PRNMT) is presented that can potentiates efficient immunogenic cell death (ICD) in deep tumor tissue and alleviates immunometabolic disorder. The PRNMT is architected through self‐assembly of indoleamine 2,3‐dioxygenase 1 (IDO‐1) inhibitor modified small‐sized CuS nanoparticles (CuS5) and tumor microenvironment (TME) responsive cationized polymeric matrix. The TME can trigger the splitting and surface cationization of PRNMT into small cationized CuS5 that feature high transcytosis potential and TME immunometabolic regulation. Upon NIR‐II irradiation, CuS5 induce homogeneous ICD and release immunometabolic regulator in deep tumor tissues, which ameliorates IDO‐1 mediated immunometabolic disorder and further suppresses regulatory T cells infiltration. PRNMT mediated PICT effectively delays the primary murine mammary carcinoma 4T1 tumor growth and inhibits the lethal pulmonary metastasis in combination with programmed cell death protein 1 (PD1) blockade. [ABSTRACT FROM AUTHOR]

Angiotensin II (Ang II)‐dependent stimulation of the AT1 receptor in proximal tubules increases sodium reabsorption and blood pressure. Reabsorption is driven by the Na,K‐pump that is acutely stimulated by Ang II, which requires phosphorylation of serine‐938 (S938). This site is present in humans and only known to phosphorylated by PKA. Yet, activation of AT1 decreases cAMP required to activate PKA and inhibiting PKA does not block Ang II‐dependent phosphorylation of S938. We tested the hypothesis that Ang II‐dependent activation is mediated via increased phosphorylation at S938 through a PI3K/AKT‐dependent pathway. Experiments were conducted using opossum kidney cells, a proximal tubule cell line, stably co‐expressing the AT1 receptor and either the wild‐type (α‐1.wild‐type) or an alanine substituted (α‐1.S938A) form of rat kidney Na,K‐pump. A 5‐min exposure to 10 pM Ang II significantly activated Na,K‐pump activity (56%) measured as short‐circuit current across polarized α‐1.wild‐type cells. Wortmannin, at a concentration that selectively inhibits PI3K, blocked that Ang II‐dependent activation. Ang II did not stimulate Na,K‐pump activity in α‐1.S938A cells. Ang II at 10 and 100 pM increased phosphorylation at S938 in α‐1.wild‐type cells measured in whole cell lysates. The increase was inhibited by wortmannin plus H‐89, an inhibitor of PKA, not by either alone. Ang II activated AKT inhibited by wortmannin, not H‐89. These data support our hypothesis and show that Ang II‐dependent phosphorylation at S938 stimulates Na,K‐pump activity and transcellular sodium transport. [ABSTRACT FROM AUTHOR]

Summary: Chenopodium quinoa uses epidermal bladder cells (EBCs) to sequester excess salt. Each EBC complex consists of a leaf epidermal cell, a stalk cell, and the bladder.Under salt stress, sodium (Na+), chloride (Cl−), potassium (K+) and various metabolites are shuttled from the leaf lamina to the bladders. Stalk cells operate as both a selectivity filter and a flux controller.In line with the nature of a transfer cell, advanced transmission electron tomography, electrophysiology, and fluorescent tracer flux studies revealed the stalk cell's polar organization and bladder‐directed solute flow.RNA sequencing and cluster analysis revealed the gene expression profiles of the stalk cells. Among the stalk cell enriched genes, ion channels and carriers as well as sugar transporters were most pronounced. Based on their electrophysiological fingerprint and thermodynamic considerations, a model for stalk cell transcellular transport was derived. [ABSTRACT FROM AUTHOR]

Increasing awareness of the health benefits of specific constituents in fruits, vegetables, cereals, and other whole foods has sparked a broader interest in the potential health benefits of nutraceuticals. Many nutraceuticals are hydrophobic substances, which means they must be encapsulated in colloidal delivery systems. Oil‐in‐water emulsions are one of the most widely used delivery systems for improving the bioavailability and bioactivity of these nutraceuticals. The composition and structure of emulsions can be designed to improve the water dispersibility, physicochemical stability, and bioavailability of the encapsulated nutraceuticals. The nature of the emulsion used influences the interfacial area and properties of the nutraceutical‐loaded oil droplets in the gastrointestinal tract, which influences their digestion, as well as the bioaccessibility, metabolism, and absorption of the nutraceuticals. In this article, we review recent in vitro and in vivo studies on the utilization of emulsions to improve the bioavailability of nutraceuticals. The findings from this review should facilitate the design of more efficacious nutraceutical‐loaded emulsions with increased bioactivity. [ABSTRACT FROM AUTHOR]

Bile acid‐modified nanoparticles provide a convenient strategy to improve oral bioavailability of poorly permeable drugs by exploiting specific interactions with bile acid transporters. However, the underlying mechanisms are unknown, especially considering the different absorption sites of free bile acids (ileum) and digested fat molecules from bile acid‐emulsified fat droplets (duodenum). Here, glycocholic acid (GCA)‐conjugated polystyrene nanoparticles (GCPNs) are synthesized and their transport in Caco‐2 cell models is studied. GCA conjugation enhances the uptake by interactions with apical sodium‐dependent bile acid transporter (ASBT). A new pathway correlated with both ASBT and chylomicron pathways is identified. Meanwhile, the higher uptake of GCPNs does not lead to higher transcytosis to the same degree compared with unmodified nanoparticles (CPNs). The pharmacological and genomics study confirm that GCA conjugation changes the endocytosis mechanisms and downregulates the cellular response to the transport at gene levels, which works as a negative feedback loop and explains the higher cellular retention of GCPNs. These findings offer a solid foundation in the bile acid‐based nanomedicine design, with utilizing advantages of the ASBT‐mediated uptake, as well as inspiration to take comprehensive consideration of the cellular response with more developed technologies. [ABSTRACT FROM AUTHOR]

The blood–brain barrier (BBB) severely blocks the intracranial accumulation of most systemic drugs. Inspired by the contribution of the bacterial outer membrane to Escherichia coli K1 (EC‐K1) binding to and invasion of BBB endothelial cells in bacterial meningitis, utilization of the BBB invasion ability of the EC‐K1 outer membrane for brain‐targeted drug delivery and construction of a biomimetic self‐assembled nanoparticle with a surface featuring a lipopolysaccharide‐free EC‐K1 outer membrane are proposed. BBB penetration of biomimetic nanoparticles is demonstrated to occur through the transcellular vesicle transport pathway, which is at least partially dependent on internalization, endosomal escape, and transcytosis mediated by the interactions between outer membrane protein A and gp96 on BBB endothelial cells. This biomimetic nanoengineering strategy endows the loaded drugs with prolonged circulation, intracranial interstitial distribution, and extremely high biocompatibility. Based on the critical roles of gp96 in cancer biology, this strategy reveals enormous potential for delivering therapeutics to treat gp96‐overexpressing intracranial malignancies. [ABSTRACT FROM AUTHOR]

The cancer drug delivery process involves a series of biological barriers, which require the nanomedicine to exhibit different, even opposite properties for high therapeutic efficacy. The prevailing design philosophy, i.e., integrating these properties within one nanomedicine via on‐demand property transitions such as PEGylation/dePEGylation, complicates nanomedicines' composition and thus impedes clinical translation. Here, polyzwitterionic micelles of poly(tertiary amine‐oxide)‐block‐poly(ε‐caprolactone) (PTAO‐PCL) amphiphiles that enable all the required functions are presented. The zwitterionic nature and unique cell membrane affinity confer the PTAO micelles long blood circulation, efficient tumor accumulation and penetration, and fast cellular internalization. The mitochondrial targeting capability allows drug delivery into the mitochondria to induce mitochondrial dysfunction and overcome tumor multidrug resistance. As a result, the PTAO/drug micelles exhibit potent anticancer efficacy. This simple yet multipotent carrier system holds great promise as a generic platform for potential clinical translation. [ABSTRACT FROM AUTHOR]

To exert their therapeutic effects, nanoparticles (NPs) often need to travel into the tissues composed of multilayered cells. Accumulative evidence has revealed the crucial role of transcellular transport route (entry into one cell, exocytosis, and re‐entry into another) in this process. While NP endocytosis and subcellular transport are intensively characterized, the exocytosis and re‐entry steps are poorly understood, which becomes a barrier for NP delivery into complex tissues. Here, the authors term the exocytosis and re‐entry steps together as intercellular exchange. A collagen‐based three‐dimension assay is developed to specifically quantify the intercellular exchange of NPs, and distinguish the contributions of several potential mechanisms. The authors show that NPs can be exocytosed freely or enclosed inside extracellular vesicles (EVs) for re‐entry, while direct cell–cell contact is hardly involved. EVs account for a significant fraction of NP intercellular exchange, and its importance in NP transport is demonstrated in vitro and in vivo. While freely released NPs engage with the same receptors for re‐entry, EV‐enclosed ones bypass this dependence. These studies provide an easy and precise system to investigate the intercellular exchange stage of NP delivery, and shed the first light in the importance of EVs in NP transport between cells and into complex tissues. [ABSTRACT FROM AUTHOR]

Objective: Despite the absolute requirement of Delta/Notch signaling to activate lateral inhibition during early blood vessel development, many mechanisms remain unclear about how this system is regulated. Our objective was to determine the involvement of Epsin 15 Homology Domain Containing 2 (EHD2) in delta‐like ligand 4 (Dll4) endocytosis during Notch activation. Approach and Results: Using both in vivo and in vitro models, we demonstrate that EHD2 is a novel modulator of Notch activation in endothelial cells through controlling endocytosis of Dll4. In vitro, EHD2 localized to plasma membrane‐bound Dll4 and caveolae. Chemical disruption of caveolae complexes resulted in EHD2 failing to organize around Dll4 as well as loss of Dll4 internalization. Reduced Dll4 internalization blunted Notch activation in endothelial cells. In vivo, EHD2 is primarily expressed in the vasculature, colocalizing with junctional marker VE‐cadherin and Dll4. Knockout of EHD2 in zebrafish produced a significant increase in dysmorphic sprouts in zebrafish intersomitic vessels during development and a reduction in downstream Notch signaling. Conclusions: Overall, we demonstrate that EHD2 is necessary for Dll4 transcytosis and downstream Notch activation. [ABSTRACT FROM AUTHOR]

The breakdown of the blood‐brain barrier (BBB) is related to the occurrence and deterioration of neurological dysfunction in ischemic stroke, which leads to the extravasation of blood‐borne substances, resulting in vasogenic edema and increased mortality. However, a limited understanding of the molecular mechanisms that control the restrictive properties of the BBB hinders the manipulation of the BBB in disease and treatment. Here, we found that the glycocalyx (GCX) is a critical factor in the regulation of brain endothelial barrier integrity. First, endothelial GCX displayed a biphasic change pattern, of which the timescale matched well with the biphasic evolution of BBB permeability to tracers within the first week after t‐MCAO. Moreover, GCX destruction with hyaluronidase increased BBB permeability in healthy mice and aggravated BBB leakage in transient middle cerebral artery occlusion (t‐MCAO) mice. Surprisingly, ultrastructural observation showed that GCX destruction was accompanied by increased endothelial transcytosis at the ischemic BBB, while the tight junctions remained morphologically and functionally intact. Knockdown of caveolin1 (Cav1) suppressed endothelial transcytosis, leading to reduced BBB permeability, and brain edema. Lastly, a coimmunoprecipitation assay showed that GCX degradation enhanced the interaction between syndecan1 and Src by promoting the binding of phosphorylated syndecan1 to the Src SH2 domain, which led to rapid modulation of cytoskeletal proteins to promote caveolae‐mediated endocytosis. Overall, these findings demonstrate that the dynamic degradation and reconstruction of GCX may account for the biphasic changes in BBB permeability in ischemic stroke, and reveal an essential role of GCX in suppressing transcellular transport in brain endothelial cells to maintain BBB integrity. Targeting GCX may provide a novel strategy for managing BBB dysfunction and central nervous system drug delivery. [ABSTRACT FROM AUTHOR]

Blood brain barrier (BBB) protects homeostasis and sensitive environment of brain from several toxic substances coming from the systemic circulation. This barrier along with those substances also prevents therapeutic chemicals to reach brain tissues for several brain diseases. BBB consists of a number of cell types and junctions that help maintain its intricate structure and physiology. To open BBB for therapeutic purposes, researchers are keen to explore the use of nanomaterials as therapeutic agents. Nanomaterials have unique physio‐chemical properties such as, increased surface area to mass ratio, superior adsorption capacity, and a wide variety of functionalization possibilities in contrast to bulk materials, making them sought‐after for research pertaining to brain delivery of therapeutic substances. Both organic and inorganic nanomaterials have been researched in this regard with numerous interesting functionalizations, and their toxicity and distribution profiles have been well assessed. Different pathways taken up by nanomaterials to cross BBB like adsorptive‐mediated transcytosis, inhibition of active efflux pumps, receptor‐mediated transport, and cell‐mediated endocytosis have also been investigated. This review summarizes the structural and physiological properties and the modulation techniques of BBB for delivery of adsorbed/functionalized nano delivery platforms and imaging nanomaterials across. [ABSTRACT FROM AUTHOR]

Listeria monocytogenes is a food‐borne bacterium that causes gastroenteritis, meningitis, or abortion. L. monocytogenes induces its internalization (entry) into human cells and either spreads laterally in tissues or transcytoses to traverse anatomical barriers. In this review, we discuss mechanisms by which five structurally related proteins of the "internalin" family of L. monocytogenes (InlA, InlB, InlC, InlF, and InlP) interact with distinct host receptors to promote infection of human cells and/or crossing of the intestinal, blood–brain, or placental barriers. We focus on recent results demonstrating that the internalin proteins InlA, InlB, and InlC exploit exocytic pathways to stimulate transcytosis, entry, or cell‐to‐cell spread, respectively. We also discuss evidence that InlA‐mediated transcytosis contributes to traversal of the intestinal barrier, whereas InlF promotes entry into endothelial cells to breach the blood–brain barrier. InlB also facilitates the crossing of the blood–brain barrier, but does so by extending the longevity of infected monocytes that may subsequently act as a "Trojan horse" to transfer bacteria to the brain. InlA, InlB, and InlP each contribute to fetoplacental infection by targeting syncytiotrophoblast or cytotrophoblast layers of the placenta. This work highlights the diverse functions of internalins and the complex mechanisms by which these structurally related proteins contribute to disease. [ABSTRACT FROM AUTHOR]

Hypertension and its associated dysfunction of the blood–brain barrier (BBB) contribute to cerebral small vessel disease (cSVD). Angiotensin II (Ang II), a vasoactive peptide of the renin–angiotensin system (RAS), is not only a pivotal molecular signal in hypertension but also causes BBB leakage, cSVD, and cognitive impair. Harpagoside, the major bioactive constituent of Scrophulariae Radix, has been commonly used for the treatment of multiple diseases including hypertension in China. The effect of harpagoside on Ang II‐induced BBB damage is unclear. We employed an immortalized endothelial cell line (bEnd.3) to mimic a BBB monolayer model in vitro and investigated the effect of harpagoside on BBB and found that harpagoside alleviated Ang II‐induced BBB destruction, inhibited Ang II–associated cytotoxicity in a concentration‐dependent manner and attenuated Ang II‐induced reactive oxygen species (ROS) impair by downregulation of Nox2, Nox4, and COX‐2. Harpagoside prevented Ang II–induced apoptosis via keeping Bax/Bcl‐2 balance, decreasing cytochrome c release, and inactivation of caspase‐8, caspase‐9, and caspase‐3 (the mitochondria‐dependent and death receptor–mediated apoptosis pathways). Moreover, harpagoside can alleviate Ang II–induced BBB damage through upregulation of tight junction proteins and decrease of caveolae‐mediated endocytosis. Thus, harpagoside might be a potential drug to treat Ang II–induced cSVD. [ABSTRACT FROM AUTHOR]

Active tumor penetration has been recently recognized as a promising strategy to resolve the limitation of nanomedicine in terms of tumor penetration, but it is challenging to develop active transporting nanocarriers. Here, an ultrasonic cavitation‐assisted and acid‐activatable active transporting liposome for a broad range of tumors is reported. The active transporting liposome (size and charge dual‐conversional gemcitabine prodrug‐integrated liposomal nanodroplet (SCGLN)) overcomes the tight blood vessel walls with the aid of ultrasonic cavitation. The SCGLN subsequently transforms from micro‐size to nano‐size under prolonged ultrasound radiation. Once in the acidic tumor microenvironment, the nanosized SCGLN undergoes negative‐to‐positive charge‐reversal and triggers the cationization‐initiated transcytosis to penetrate deep into tumor parenchyma. The gemcitabine‐loaded SCGLN exhibits potent antitumor activity in multiple poorly permeable tumor models, which completely erases subcutaneous U251 glioma and stops the progression of orthotopic BxPC3 pancreatic ductal adenocarcinoma. This study presents a promising and universal strategy to develop active penetrating nanomedicines for efficient drug delivery in the low permeable tumor. [ABSTRACT FROM AUTHOR]

Aim: Fluid and macromolecule transport from the interstitium into and through lymphatic vessels is necessary for tissue homeostasis. While lymphatic capillary structure suggests that passive, paracellular transport would be the predominant route of macromolecule entry, active caveolae‐mediated transcellular transport has been identified in lymphatic endothelial cells (LECs) in vitro. Caveolae also mediate a wide array of endothelial cell processes, including nitric oxide regulation. Thus, how does the lack of caveolae impact "lymphatic function"? Methods: Various aspects of lymphatic transport were measured in mice constitutively lacking caveolin‐1 ("CavKO"), the protein required for caveolae formation in endothelial cells, and in mice with a LEC‐specific Cav1 gene deletion (Lyve1‐Cre x Cav1flox/flox; "LyCav") and ex vivo in their vessels and cells. Results: In each model, lymphatic architecture was largely unchanged. The lymphatic conductance, or initial tissue uptake, was significantly higher in both CavKO mice and LyCav mice by quantitative microlymphangiography and the permeability to 70 kDa dextran was significantly increased in monolayers of LECs isolated from CavKO mice. Conversely, transport within the lymphatic system to the sentinel node was significantly reduced in anaesthetized CavKO and LyCav mice. Isolated, cannulated collecting vessel studies identified significantly reduced phasic contractility when lymphatic endothelium lacks caveolae. Inhibition of nitric oxide synthase was able to partially restore ex vivo vessel contractility. Conclusion: Macromolecule transport across lymphatics is increased with loss of caveolae, yet phasic contractility reduced, resulting in reduced overall lymphatic transport function. These studies identify lymphatic caveolar biology as a key regulator of active lymphatic transport functions. [ABSTRACT FROM AUTHOR]

Lyme disease is the most common tick‐transmitted disease in the northern hemisphere and is caused by the spirochete Borreliaburgdorferi and related Borrelia species. The constellation of symptoms attributable to this malady results from vascular dissemination of B. burgdorferi throughout the body to invade various tissue types. However, little is known about the mechanism by which the spirochetes can breach the blood vessel wall to reach distant tissues. We have studied this process by direct observation of spirochetes in the microvasculature of living mice using multi‐laser spinning‐disk intravital microscopy. Our results show that in our experimental system, instead of phagocytizing B. burgdorferi, host neutrophils are involved in the production of specific cytokines that activate the endothelium and potentiate B. burgdorferi escape into the surrounding tissue. Spirochete escape is not induced by paracellular permeability and appears to occur via a transcellular pathway. Neutrophil repurposing to promote bacterial extravasation represents a new and innovative pathogenic strategy. [ABSTRACT FROM AUTHOR]

The blood‐brain barrier (BBB) is the most restrictive and complicated barrier that keeps most biomolecules and drugs from the brain. An efficient brain delivery strategy is urgently needed for the treatment of brain diseases. Based on the studies of brain‐targeting extracellular vesicles (EVs), the potential of using small apoptotic bodies (sABs) from brain metastatic cancer cells for brain‐targeting drug delivery is explored. It is found that anti‐TNF‐α antisense oligonucleotide (ASO) combined with cationic konjac glucomannan (cKGM) can be successfully loaded into sABs via a transfection/apoptosis induction process and that the sABs generated by B16F10 cells have an extraordinarily high brain delivery efficiency. Further studies suggest that ASO‐loaded sABs (sCABs) are transcytosed by b. End3 (brain microvascular endothelial cells, BMECs) to penetrate the BBB, which is mediated by CD44v6, and eventually taken up by microglial cells in the brain. In a Parkinson's disease (PD) mouse model, sCABs dramatically ameliorate PD symptoms via the anti‐inflammatory effect of ASO. This study suggests that sABs from brain metastatic cancer cells are excellent carriers for brain‐targeted delivery, as they have not only an extraordinary delivery efficiency but also a much higher scale‐up production potential than other EVs. [ABSTRACT FROM AUTHOR]

Transcytosis is a form of specialized transport through which an extracellular cargo is endocytosed, shuttled across the cytoplasm in membrane‐bound vesicles, and secreted at a different plasma membrane surface. This important process allows membrane‐impermeable macromolecules to pass through a cell and become accessible to adjacent cells and tissue compartments. Transcytosis also promotes redistribution of plasma membrane proteins and lipids to different regions of the cell surface. Here we review transcytosis and highlight in vivo studies showing how developing epithelial cells use it to change shape, to migrate, and to relocalize signaling molecules. [ABSTRACT FROM AUTHOR]

Mechanisms of virus transmission to the fetus. A) ascending transmission to upper reproductive tract. B) transmission through maternal macrophages to the placenta trophoblast. C) transcytosis transmission through attachment to the receptor in the apical surface and ejection to inner layers. D) transmission from maternal vascular endothelium to extravillous trophoblasts (EVTs). Further details can be seen in the article by Heydarifard et al. in this issue. [ABSTRACT FROM AUTHOR]

Nanoparticle transport across tumor blood vessels is a key step in nanoparticle delivery to solid tumors. However, the specific pathways and mechanisms of this nanoparticle delivery process are not fully understood. Here, the biological and physical characteristics of the tumor vasculature and the tumor microenvironment are explored and how these features affect nanoparticle transport across tumor blood vessels is discussed. The biological and physical methods to deliver nanoparticles into tumors are reviewed and paracellular and transcellular nanoparticle transport pathways are explored. Understanding the underlying pathways and mechanisms of nanoparticle tumor delivery will inform the engineering of safer and more effective nanomedicines for clinical translation. [ABSTRACT FROM AUTHOR]

Key points: Intestinal absorption of phosphate proceeds via an active/transcellular route mostly mediated by NaPi‐IIb/Slc34a2 and a poorly characterized passive/paracellular pathway.Intestinal phosphate absorption and expression of NaPi‐IIb are stimulated by 1,25(OH)2 vitamin D3 but whether NaPi‐IIb is the only target under hormonal control remains unknown.We report that administration of 1,25(OH)2 vitamin D3 to wild‐type mice resulted in the expected increase in active transport of phosphate in jejunum, without changing paracellular fluxes. Instead, the same treatment failed to alter phosphate transport in intestinal‐depleted Slc34a2‐deficient mice.In both genotypes, 1,25(OH)2 vitamin D3 induced similar hyperphosphaturic responses and changes in the plasma levels of FGF23 and PTH.While urinary phosphate loss induced by administration of 1,25(OH)2 vitamin D3 did not alter plasma phosphate, further studies should investigate whether chronic administration would lead to phosphate imbalance in mice with reduced active intestinal absorption. Intestinal absorption of phosphate is stimulated by 1,25(OH)2 vitamin D3. At least two distinct mechanisms underlie phosphate absorption in the gut, an active transcellular transport requiring the Na+/phosphate cotransporter NaPi‐IIb/Slc34a2, and a poorly characterized paracellular passive pathway. 1,25(OH)2 vitamin D3 stimulates NaPi‐IIb expression and function, and loss of NaPi‐IIb reduces intestinal phosphate absorption. However, it is remains unknown whether NaPi‐IIb is the only target for hormonal regulation by 1,25(OH)2 vitamin D3. Here we compared the effects of intraperitoneal administration of 1,25(OH)2 vitamin D3 (2 days, once per day) in wild‐type and intestinal‐specific Slc34a2‐deficient mice, and analysed trans‐ vs. paracellular routes of phosphate absorption. We found that treatment stimulated active transport of phosphate only in jejunum of wild‐type mice, though NaPi‐IIb protein expression was upregulated in jejunum and ileum. In contrast, 1,25(OH)2 vitamin D3 administration had no effect in Slc34a2‐deficient mice, suggesting that the hormone specifically regulates NaPi‐IIb expression. In both groups, 1,25(OH)2 vitamin D3 elicited the expected increase of plasma fibroblast growth factor 23 (FGF23) and reduction of parathyroid hormone (PTH). Treatment resulted in hyperphosphaturia (and hypercalciuria) in both genotypes, though mice remained normophosphataemic. While increased intestinal absorption and higher FGF23 can trigger the hyperphosphaturic response in wild types, only higher FGF23 can explain the renal response in Slc34a2‐deficient mice. Thus, 1,25(OH)2 vitamin D3 stimulates intestinal phosphate absorption by acting on the active transcellular pathway mostly mediated by NaPi‐IIb while the paracellular pathway appears not to be affected. Key points: Intestinal absorption of phosphate proceeds via an active/transcellular route mostly mediated by NaPi‐IIb/Slc34a2 and a poorly characterized passive/paracellular pathway.Intestinal phosphate absorption and expression of NaPi‐IIb are stimulated by 1,25(OH)2 vitamin D3 but whether NaPi‐IIb is the only target under hormonal control remains unknown.We report that administration of 1,25(OH)2 vitamin D3 to wild‐type mice resulted in the expected increase in active transport of phosphate in jejunum, without changing paracellular fluxes. Instead, the same treatment failed to alter phosphate transport in intestinal‐depleted Slc34a2‐deficient mice.In both genotypes, 1,25(OH)2 vitamin D3 induced similar hyperphosphaturic responses and changes in the plasma levels of FGF23 and PTH.While urinary phosphate loss induced by administration of 1,25(OH)2 vitamin D3 did not alter plasma phosphate, further studies should investigate whether chronic administration would lead to phosphate imbalance in mice with reduced active intestinal absorption. [ABSTRACT FROM AUTHOR]

Drug delivery to the brain is greatly hampered by the presence of the blood–brain barrier (BBB) which tightly regulates the passage of molecules from blood to brain and vice versa. Nanocarriers, in which drugs can be encapsulated, can move across the blood–brain barrier (BBB) via the process of transcytosis, thus showing promise to improve drug delivery to the brain. Here, we demonstrate the use of natural nanovesicles, that is, exosomes, derived from C17.2 neural stem cells (NSCs) to efficiently carry a protein cargo across an in vitro BBB model consisting of human brain microvascular endothelial cells. We show that the exosomes are primarily taken up in brain endothelial cells via endocytosis, while heparan sulfate proteoglycans (HSPGs) act as receptors. Taken together, our data support the view that NSC exosomes may act as biological nanocarriers for efficient passage across the BBB. Nanomedicines that target HSPGs may improve their binding to brain endothelial cells and, possibly, show subsequent transcytosis across the BBB. [ABSTRACT FROM AUTHOR]

The aim of the study was to understand the hydraulic response to salt stress of the root system of the comparatively salt‐tolerant crop barley (Hordeum vulgare L.). We focused on the transcellular path of water movement across the root cylinder that involves the crossing of membranes. This path allows for selective water uptake, while excluding salt ions. Hydroponically grown plants were exposed to 100 mM NaCl for 5–7 days and analysed when 15–17 days old. A range of complementary and novel approaches was used to determine hydraulic conductivity (Lp). This included analyses at cell, root and plant level and modelling of water flow. Apoplastic barrier formation and gene expression level of aquaporins (AQPs) was analysed. Salt stress reduced the Lp of root system through reducing water flow along the transcellular path. This involved changes in the activity and gene expression level of AQPs. Modelling of root‐Lp showed that the reduction in root‐Lp did not require added hydraulic resistances through apoplastic barriers at the endodermis. The bulk of data points to a near‐perfect semi‐permeability of roots of control plants (solute reflection coefficient σ ~1.0). Roots of salt‐stressed plants are almost as semi‐permeable (σ > 0.8). The hydraulic response of roots to salt stress is poorly understood. We show for barley that roots exhibit a very high degree of semi‐permeability. Salt stress reduces root water uptake along the transcellular path, and apoplastic barriers are not required to explain changes in plant water flow. [ABSTRACT FROM AUTHOR]

In eukaryotic cells, clathrin‐mediated endocytosis (CME) is a central pathway for the internalization of proteins from the cell surface, thereby contributing to the maintenance of the plasma membrane protein composition. A key component for the formation of endocytic clathrin‐coated vesicles (CCVs) is AP‐2, as it sequesters cargo membrane proteins, recruits a multitude of other endocytic factors and initiates clathrin polymerization. Here, we inhibited CME by depletion of AP‐2 and explored the consequences for the plasma membrane proteome. Quantitative analysis revealed accumulation of major constituents of the endosomal‐lysosomal system reflecting a block in retrieval by compensatory CME. The noticeable enrichment of integrins and blockage of their turnover resulted in severely impaired cell migration. Rare proteins such as the anti‐cancer drug target CA9 and tumor markers (CD73, CD164, CD302) were significantly enriched. The AP‐2 knockdown attenuated the global endocytic capacity, but clathrin‐independent entry pathways were still operating, as indicated by persistent internalization of specific membrane‐spanning and GPI‐anchored receptors (PVR, IGF1R, CD55, TNAP). We hypothesize that blocking AP‐2 function and thus inhibiting CME may be a novel approach to identify new druggable targets, or to increase their residence time at the plasma membrane, thereby increasing the probability for efficient therapeutic intervention. [ABSTRACT FROM AUTHOR]

Aims: Pre‐existing hyperglycemia (HG) aggravates the breakdown of blood–brain barrier (BBB) and increases the risk of hemorrhagic transformation (HT) after acute ischemic stroke in both animal models and patients. To date, HG‐induced ultrastructural changes of brain microvascular endothelial cells (BMECs) and the mechanisms underlying HG‐enhanced HT after ischemic stroke are poorly understood. Methods: We used a mouse model of mild brain ischemia/reperfusion to investigate HG‐induced ultrastructural changes of BMECs that contribute to the impairment of BBB integrity after stroke. Adult male mice received systemic glucose administration 15 min before middle cerebral artery occlusion (MCAO) for 20 min. Ultrastructural characteristics of BMECs were evaluated using two‐dimensional and three‐dimensional electron microscopy and quantitatively analyzed. Results: Mice with acute HG had exacerbated BBB disruption and larger brain infarcts compared to mice with normoglycemia (NG) after MCAO and 4 h of reperfusion, as assessed by brain extravasation of the Evans blue dye and microtubule‐associated protein 2 immunostaining. Electron microscopy further revealed that HG mice had more endothelial vesicles in the striatal neurovascular unit than NG mice, which may account for their deterioration of BBB impairment. In contrast with enhanced endothelial transcytosis, paracellular tight junction ultrastructure was not disrupted after this mild ischemia/reperfusion insult or altered upon HG. Consistent with the observed increase of endothelial vesicles, transcytosis‐related proteins caveolin‐1, clathrin, and hypoxia‐inducible factor (HIF)‐1α were upregulated by HG after MCAO and reperfusion. Conclusion: Our study provides solid structural evidence to understand the role of endothelial transcytosis in HG‐elicited BBB hyperpermeability. Enhanced transcytosis occurs prior to the physical breakdown of BMECs and is a promising therapeutic target to preserve BBB integrity. [ABSTRACT FROM AUTHOR]

Plasma membranes fulfil many physiological functions. In polarized cells, different membrane compartments take on specialized roles, each being allocated correct amounts of membrane. The Drosophila tracheal system, an established tubulogenesis model, contains branched terminal cells with subcellular tubes formed by apical plasma membrane invagination. We show that apical endocytosis and late endosome‐mediated trafficking are required for membrane allocation to the apical and basal membrane domains. Basal plasma membrane growth stops if endocytosis is blocked, whereas the apical membrane grows excessively. Plasma membrane is initially delivered apically and then continuously endocytosed, together with apical and basal cargo. We describe an organelle carrying markers of late endosomes and multivesicular bodies (MVBs) that is abolished by inhibiting endocytosis and which we suggest acts as transit station for membrane destined to be redistributed both apically and basally. This is based on the observation that disrupting MVB formation prevents growth of both compartments. Synopsis: Cells reorganize their plasma membranes to build complex structures. This study shows that the endocytic pathway is required for proper membrane allocation during epithelial tube morphogenesis in Drosophila, with late endosomes acting as hubs for membrane and protein redistribution. Subcellular tubulogenesis in embryonic trachea uses apical‐to‐basal transcytosis to distribute plasma membrane to the appropriate domains of the growing tube branch.Dynamin‐mediated apical endocytosis is required for apical membrane pruning, thereby providing material that allows the basal compartment to grow.Proteins targeted to the basal compartment transit from the apical to the basal compartment through late endosomes.Late endosomes are also required for the recycling of material back to the apical compartment. [ABSTRACT FROM AUTHOR]

The effective treatment of Alzheimer's disease (AD) is hindered due to the hard blood–brain barrier (BBB) penetration and non‐selective distribution of drugs in the brain. Moreover, the complicated pathological mechanism of AD involves various pathway dysfunctions that limit the effectiveness of a single therapeutic drug. Herein, a dendrigraft poly‐l‐lysines (DGL)‐based siRNA and D peptide (Dp) loaded nanoparticle is designed that could target and penetrate through the BBB, enter the brain parenchyma, and further accumulate at the AD lesion. In this system, T7 peptide, which specifically targets transferrin receptors on the BBB, is linked to DGL via acid‐cleavable long polyethylene glycol (PEG) to achieve high internalization, quick escape from endo/lysosome, and effective transcytosis. Then, the Tet1, which specifically targets diseased neurons, is modified onto DGL by short PEG. After being exposed, Tet1 could drive the nanoparticles to the AD lesion and release the drugs. As a result, the production of β amyloid plaques (Aβ) is inhibited. Neurotoxicity induced by Aβ plaques and tau proten phosphorylation (p‐tau) tangle is also alleviated, and the cognition of AD mice is significantly improved. Overall, this system programmatically targets BBB and neurons, thus, significantly enhances the intracephalic drug accumulation and AD treatment efficacy. [ABSTRACT FROM AUTHOR]

Hepatocytes, the main epithelial cells of the liver, organize their polarized membrane domains differently from ductal epithelia. They also differ in their biosynthetic delivery of single‐membrane‐spanning and glycophosphatidylinositol‐anchored proteins to the apical domain. While ductal epithelia target apical proteins to varying degrees from the trans‐Golgi network (TGN) to the apical surface directly, hepatocytes target them first to the basolateral domain, from where they undergo basolateral‐to‐apical transcytosis. How TGN‐to‐surface transport differs in both scenarios is unknown. Here, we report that the basolateral detour of a hepatocyte apical protein is due, in part, to low RhoA activity at the TGN, which prevents its segregation from basolateral transport carriers. Activating Rho in hepatocytic cells, which switches their polarity from hepatocytic to ductal, also led to apical‐basolateral cargo segregation at the TGN as is typical for ductal cells, affirming a central role for Rho‐signaling in different aspects of the hepatocytic polarity phenotype. Nevertheless, Rho‐induced cargo segregation was not sufficient to target the apical protein directly; thus, failure to recruit apical targeting machinery also contributes to its indirect itinerary. [ABSTRACT FROM AUTHOR]

Oral insulin administration still represents a paramount quest that almost a century of continuous research attempts did not suffice to fulfill. Before pre‐clinical development, oral insulin products have first to be optimized in terms of encapsulation efficiency, protection against proteolysis, and intestinal permeation ability. With the use of dendritic mesoporous silica nanoparticles (DMSNs) as an insulin host and together with a protein‐based excipient, succinylated β‐lactoglobulin (BL), pH‐responsive tablets permitted the shielding of insulin from early release/degradation in the stomach and mediated insulin permeation across the intestinal cellular membrane. Following an original in vitro cellular assay based on insulin starvation, direct cellular fluorescent visualization has evidenced how DMSNs could ensure the intestinal cellular transport of insulin. [ABSTRACT FROM AUTHOR]

The Junonia coenia densovirus rapidly traverses the gut epithelium of the host lepidopteran without replicating in the gut cells. The ability of this virus to transcytose across the gut epithelium is of interest for the potential use of virus structural proteins as delivery vehicles for insecticidal peptides that act within the insect hemocoel, rather than in the gut. In this study, we used fall armyworm, Spodoptera frugiperda to examine the binding of the virus to brush border membrane vesicle proteins by two‐dimensional ligand blot analysis. We also assessed the rate of flux of the primary viral structural protein, VP4 fused to eGFP with a proline‐rich linker (VP4‐P‐eGFP) through the gut epithelium ex vivo in an Ussing chamber. The mechanisms involved with transcytosis of VP4‐P‐eGFP were assessed by use of inhibitors. Bovine serum albumin (BSA) and eGFP were used as positive and negative control proteins, respectively. In contrast to BSA, which binds to multiple proteins on the brush border membrane, VP4‐P‐eGFP binding was specific to a protein of high molecular mass. Protein flux was significantly higher for VP4‐P‐eGFP after 2 h than for albumin or eGFP, with rapid transcytosis of VP4‐P‐eGFP within the first 30 min. In contrast to BSA which transcytosed following clathrin‐mediated endocytosis, the movement of VP4‐P‐eGFP was vesicle‐mediated but clathrin‐independent. The specificity of binding combined with the efficiency of transport across the gut epithelium suggest that VP4 will provide a useful carrier for insecticidal peptides active within the hemocoel of key lepidopteran pests including S. frugiperda. [ABSTRACT FROM AUTHOR]

Summary: Dairy protein‐derived bioactive peptides (DBPs) have potential benefits for human health. However, their transport mechanisms and bioavailability from intestinal lumen to bloodstream are not well understood. This review summarises current understanding of their transport mechanisms across the intestinal membrane (peptide transport 1, paracellular route, transcytosis and passive transcellular diffusion) and the bioavailability of DBPs in animal and human studies. Some DBPs can escape the degradation of peptidase and reach the bloodstream at concentrations of micromolar range, and keep intact for several minutes to hours. The presences of brush‐border peptidases at the site of absorption and other peptidases in the blood, along with the peptide properties, such as molecular size and weight, stability to proteases, hydrophobicity and charge, determine their bioavailability. Developing novel analytical tools for accurate measurement and study of the transport, metabolism, and bioavailability of DBPs in vivo are expected. [ABSTRACT FROM AUTHOR]

An endothelial cell monolayer separates interstitia from blood and lymph, and determines the bidirectional transfer of solutes and macromolecules across these biological spaces. We review advances in transport modalities across these endothelial barriers. Glucose is a major fuel for the brain and peripheral tissues, and insulin acts on both central and peripheral tissues to promote whole‐body metabolic signalling and anabolic activity. Blood‐brain barrier endothelial cells display stringent tight junctions and lack pinocytic activity. Delivery of blood glucose and insulin to the brain occurs through their respective carrier (Glucose transporter 1) and receptor (insulin receptor), enacting bona fide transcytosis. At supraphysiological concentrations, insulin is also likely transferred by fluid phase cellular uptake and paracellular transport, especially in peripheral microvascular endothelia. The lymphatic microvasculature also transports insulin but in this case from tissues to lymph and therefrom to blood. This serves to end the hormone's action and to absorb highly concentrated subcutaneously injected insulin in diabetic individuals. The former function may involve receptor‐mediated transcytosis into lymphatic endothelial cells, the latter fluid phase uptake and paracellular transport. Lymphatic capillaries also mediate carrier‐dependent transport of other nutrients and macromolecules. These findings challenge the notion that lymphatic capillaries only transport macromolecules through intercellular flaps. [ABSTRACT FROM AUTHOR]

The pulmonary epithelial and vascular endothelial cell layers provide two sequential physical and immunological barriers that together form a semi‐permeable interface and prevent alveolar and interstitial oedema formation. In this review, we focus specifically on the continuous endothelium of the pulmonary microvascular bed that warrants strict control of the exchange of gases, fluid, solutes and circulating cells between the plasma and the interstitial space. The present review provides an overview of emerging molecular mechanisms that permit constant transcellular exchange between the vascular and interstitial compartment, and cause, prevent or reverse lung endothelial barrier failure under experimental conditions, yet with a clinical perspective. Based on recent findings and at times seemingly conflicting results we discuss emerging paradigms of permeability regulation by altered ion transport as well as shifts in the homeostasis of sphingolipids, angiopoietins and prostaglandins. A summary of the molecular mechanisms that permit constant transcellular exchange between the vascular and interstitial compartment, and cause, prevent or reverse lung endothelial barrier integrity. The arrows indicate the positive (upward) or negative (downward) influence of altered ion transport, sphingolipids, angiopoietins and prostaglandins on endothelial barrier integrity. [ABSTRACT FROM AUTHOR]

Objective: Cortical spreading depolarizations (CSDs) are intense and ubiquitous depolarization waves relevant for the pathophysiology of migraine and brain injury. CSDs disrupt the blood-brain barrier (BBB), but the mechanisms are unknown.Methods: A total of six CSDs were evoked over 1 hour by topical application of 300 mM of KCl or optogenetically with 470 nm (blue) LED over the right hemisphere in anesthetized mice (C57BL/6 J wild type, Thy1-ChR2-YFP line 18, and cav-1-/- ). BBB disruption was assessed by Evans blue (2% EB, 3 ml/kg, intra-arterial) or dextran (200 mg/kg, fluorescein, 70,000 MW, intra-arterial) extravasation in parietotemporal cortex at 3 to 24 hours after CSD. Endothelial cell ultrastructure was examined using transmission electron microscopy 0 to 24 hours after the same CSD protocol in order to assess vesicular trafficking, endothelial tight junctions, and pericyte integrity. Mice were treated with vehicle, isoform nonselective rho-associated kinase (ROCK) inhibitor fasudil (10 mg/kg, intraperitoneally 30 minutes before CSD), or ROCK-2 selective inhibitor KD025 (200 mg/kg, per oral twice-daily for 5 doses before CSD).Results: We show that CSD-induced BBB opening to water and large molecules is mediated by increased endothelial transcytosis starting between 3 and 6 hours and lasting approximately 24 hours. Endothelial tight junctions, pericytes, and basement membrane remain preserved after CSDs. Moreover, we show that CSD-induced BBB disruption is exclusively caveolin-1-dependent and requires rho-kinase 2 activity. Importantly, hyperoxia failed to prevent CSD-induced BBB breakdown, suggesting that the latter is independent of tissue hypoxia.Interpretation: Our data elucidate the mechanisms by which CSDs lead to transient BBB disruption, with diagnostic and therapeutic implications for migraine and brain injury. [ABSTRACT FROM AUTHOR]