Modulation Of Tight Junctions Research Articles

Poly(amido amine) dendrimers as absorption enhancers for oral delivery of camptothecin

Oral delivery of camptothecin has a treatment advantage but is limited by low bioavailability and gastrointestinal toxicity. Poly(amido amine) or PAMAM dendrimers have shown promise as intestinal penetration enhancers, drug solubilizers and drug carriers for oral delivery in vitro and in situ. There have been very limited studies in vivo to evaluate PAMAM dendrimers for oral drug delivery. In this study, camptothecin (5 mg/kg) was formulated and co-delivered with cationic, amine-terminated PAMAM dendrimer generation 4.0 (G4.0) (100 and 300 mg/kg) and anionic, carboxylate-terminated PAMAM generation 3.5 (G3.5) (300 and 1000 mg/kg) in CD-1 mice. Camptothecin associated to a higher extent with G4.0 than G3.5 in the formulation, attributed to an electrostatic interaction on the surface of G4.0. Both PAMAM G4.0 and G3.5 increased camptothecin solubilization in simulated gastric fluid and caused a 2-3 fold increase in oral absorption of camptothecin when delivered at 2 h. PAMAM G4.0 and G3.5 did not increase mannitol transport suggesting that the oral absorption of camptothecin was not due to tight junction modulation. Histologic observations of the epithelial layer of small intestinal segments of the gastrointestinal tract (GIT) at 4 h post dosing supported no evidence of toxicity at the evaluated doses of PAMAM dendrimers. This study demonstrates that both cationic (G.4) and anionic (G3.5) PAMAM dendrimers were effective in enhancing the oral absorption of camptothecin. Results suggest that drug inclusion in PAMAM interior controlled solubilization in simulated gastric and intestinal fluids, and increased oral bioavailability.

Human chorionic gonadotropin controls luteal vascular permeability via vascular endothelial growth factor by down-regulation of a cascade of adhesion proteins

To study the functional interactions of junctional proteins acting as regulators of vascular permeability in the human corpus luteum. We investigated the role of vascular endothelial (VE)-cadherin, nectin 2, and claudin 5 as controllers of vascular endothelial cell permeability. Performing immunohistochemical dual staining, we colocalized the above-mentioned proteins in the human corpus luteum. Not applicable. Not applicable. Not applicable. Using a granulosa-endothelial coculture system, we revealed that hCG-treatment down-regulates VE-cadherin, nectin 2, and claudin 5 in endothelial cells via vascular endothelial growth factor (VEGFA). Furthermore, the interaction of VE-cadherin, nectin 2, and claudin 5 was investigated by silencing these proteins that perform siRNA knockdown. Interestingly, knockdown of VE-cadherin and claudin 5 induced a decrease of the respective other protein. This down-regulation was associated with changed rates of vascular permeability: hCG induced a VEGFA-dependent down-regulation of VE-cadherin, nectin 2, and claudin 5, which increased the endothelial permeability in the coculture system. Furthermore, knockdown of VE-cadherin, nectin-2, and claudin 5 also resulted in a consecutive increase of endothelial permeability for each different protein. These results demonstrate for the first time that VE-cadherin, nectin 2, and claudin 5 are involved in the regulation of vascular permeability in a mutually interacting manner, which indicates their prominent role for the functionality of the human corpus luteum.

Morphine Induces Bacterial Translocation in Mice by Compromising Intestinal Barrier Function in a TLR-Dependent Manner

Opiates are among the most prescribed drugs for pain management. However, morphine use or abuse results in significant gut bacterial translocation and predisposes patients to serious infections with gut origin. The mechanism underlying this defect is still unknown. In this report, we investigated the mechanisms underlying compromised gut immune function and bacterial translocation following morphine treatment. We demonstrate significant bacterial translocation to mesenteric lymph node (MLN) and liver following morphine treatment in wild-type (WT) animals that was dramatically and significantly attenuated in Toll-like receptor (TLR2 and 4) knockout mice. We further observed significant disruption of tight junction protein organization only in the ileum but not in the colon of morphine treated WT animals. Inhibition of myosin light chain kinase (MLCK) blocked the effects of both morphine and TLR ligands, suggesting the role of MLCK in tight junction modulation by TLR. This study conclusively demonstrates that morphine induced gut epithelial barrier dysfunction and subsequent bacteria translocation are mediated by TLR signaling and thus TLRs can be exploited as potential therapeutic targets for alleviating infections and even sepsis in morphine-using or abusing populations.

Percolation Analysis in Electrical Conductivity of Madin-Darby Canine Kidney and Caco-2 Cells by Permeation-Enhancing Agents

The control of permeability through the paracellular route has been paid great attention to for enhanced bioavailability of macromolecular and hydrophilic drugs. The paracellular permeability is controlled by tight junctions (TJ), and claudins are the major constituents of TJ. Despite numerous studies on TJ modulation, the dynamics is not well understood, although it could be crucial for clinical applications. Here, we studied the time (t) course of electrical conductivity (Σ) in a monolayer of Madin-Darby canine kidney (MDCK) and Caco-2 cells upon treatment with modulators, the C-terminus fragments of Clostridium perfringens enterotoxin (C-CPE) and sodium caprate (C10). For C-CPE treatment, Σ remains approximately constant, then starts increasing at t=tc (percolation threshold). For C10, on the other hand, Σ increases to 1.6-2.0 fold of the initial value, stays constant, and then starts increasing again for both MDCK and Caco-2 cells at t=tc. We find that this behavior can be explained within a framework of percolation, where Σ shows a logarithmic dependence on t-tc with the power of μ; μ denotes the critical exponent. We obtain μ=1.1-1.2 regardless of cell type or modulator. Notably, μ depends only on the dimensionality (d) of the system, and these values correspond to those for d=2. Percolation is thus the operative mechanism for the increase in Σ through TJ modulation. The findings provide fundamental knowledge, not only on controlled drug delivery, but also on bio-nanotechnologies including the fabrication of biological devices.

Bufalin prevents the migration and invasion of T24 bladder carcinoma cells through the inactivation of matrix metalloproteinases and modulation of tight junctions

Bufalin, a cardiotonic steroid extracted from toad venom, has generally been known to possess a range of biological activities; however, only a few studies have reported the anti-metastatic activity of bufalin. In the present study, we investigated the inhibitory effects of bufalin on cell migration and invasion, two critical cellular processes that are often deregulated during metastasis, using the human bladder cancer cell line, T24. Within the concentration range that was not cytotoxic, bufalin markedly inhibited the cell motility and invasiveness of T24 cells. The inhibitory effects of bufalin on cell invasiveness were associated with the tightening of tight junctions (TJs), which was demonstrated by an increase in transepithelial electrical resistance (TER). Bufalin treatment also repressed the levels of claudin proteins (claudin-2, -3 and -4) and the major components of TJs that play key roles in the control and selectivity of paracellular transport. Furthermore, the activities of matrix metalloproteinase (MMP)‑2 and -9 in T24 cells were dose‑dependently inhibited by treatment with bufalin and this also correlated with a decrease in their mRNA and protein expression levels; however, the mRNA and protein levels of the tissue inhibitor of metalloproteinase (TIMP)‑1 and -2 were increased. In addition, these effects were related to the increased phosphorylation of the extracellular signal-regulated protein kinase (ERK) pathway. The inhibition of ERK (PD98059) significantly prevented the bufalin‑induced suppression of T24 cell migration. These findings suggest that bufalin inhibits the migration and invasion of T24 cells by modulating the activity of TJs and MMPs, possibly in association with the activation of ERK.

Transport properties of puerarin and effect of Radix Angelicae Dahuricae extract on the transport of puerarin in Caco-2 cell model

Ethnopharmacological relevanceAngelicae Dahurica (Hoffm.)Benth.& Hook.f.ex Franch.&Sav combined with Pueraria labota (Willd.)Ohwi has been widely used as herb-pairs in traditional Chinese medicine (TCM) for utilization of antipyretic analgesic and anti-inflammatory drugs, and modern pharmacological studies have shown that application compatibility of the two drugs has the effects of cardiovascular disease treatment. The previous study has proved that Radix Angelicae Dahuricae extract could enhance the intestinal absorption of puerarin in Pueraria. But the underlying compatibility mechanism of the two herbs remains unknown. In this study we tried to further evaluate the improvement of Radix Angelicae Dahuricae extract on the puerarin using the Caco-2 cell model and explore the transport properties of puerarin through the above research to discuss the possible effect mechanism of Radix Angelicae Dahuricae extract on the transport of puerarin and the underlying compatibility mechanism of the two herbs. Aim of studyThe aim of this work was to study the transport properties of puerarin in Radix Pueraria across Caco-2 cell membrane and to explore how the Radix Angelicae Dahuricae extract affected the transport of puerarin using the well-characterized, human-based intestinal Caco-2 cell model as a platform. Materials and methodsThe bidirectional transport, and the effects of time, drug concentration, pH, P-gp inhibitors (Verapamil, Cyclosporin A), MRP inhibitor (MK-571) and EDTA-Na2 (tight junction modulator) on the absorption of puerarin were observed. Then the influence of extract of Radix Angelicae Dahuricae on the transport of puerarin was studied. Drug concentration was measured by HPLC and the apparent permeability coefficients (Papp) and apparent permeability ratio (PDR) were calculated. Results and conclusionsThe results showed that the transport (Papp) of puerarin in Caco-2 cell monolayer model had time and concentration dependence, and the transport showed saturation characteristics with the time and concentration of puerarin to a certain degree. The Papp of puerarin transported on Caco-2 cell monolayer model was significantly changed when the specified inhibitors of P-gp were added to the model and the PDR decreased from 1.74 to 0.43. The absorption of puerarin was improved when combined with Radix Angelicae Dahuricae. The intestinal absorption of puerarin is by passive diffusion as the dominating process and active transportation was mediated by P-gp and MRP transporter in Caco-2 cell monolayer model, and Radix Angelicae Dahuricae could enhance the intestinal absorption of puerarin.

Intestinal Permeability and Its Regulation by Zonulin: Diagnostic and Therapeutic Implications

One of the most important and overlooked functions of the gastrointestinal tract is to provide a dynamic barrier to tightly controlled antigen trafficking through both the transcellular and paracellular pathways. Intercellular tight junctions (TJ) are the key structures regulating paracellular trafficking of macromolecules. Although steady progress has been made in understanding TJ ultrastructure, relatively little is known about their pathophysiological regulation. Our discovery of zonulin, the only known physiological modulator of intercellular TJ described so far, increased understanding of the intricate mechanisms that regulate gut permeability and led us to appreciate that its up-regulation in genetically susceptible individuals may lead to immune-mediated diseases. This information has translational implications, because the zonulin pathway is currently exploited to develop both diagnostic and therapeutic applications pertinent to a variety of immune-mediated diseases.

Transepithelial Transport of Theasinensins through Caco-2 Cell Monolayers and Their Absorption in Sprague–Dawley Rats after Oral Administration

The aim of this study is to illustrate the in vivo and in vitro absorption of theasinensins B and A that are (-)-epigallocatechin-3-O-gallate (EGCG)-(-)-epigallocatechin (EGC) dimer and EGCG dimer, respectively, and their transport pathway across the intestinal membrane. Our animal study by a single oral administration to rats demonstrated the intact absorption of theasinensins into the blood system, which was estimated to be a >10-fold lower absorption amount than EGCG. The in vitro absorption study indicated that theasinensins can be transported across Caco-2 cell monolayers, while their permeability coefficients were also >10-fold lower than those of EGCG and EGC. Transport experiments using cytochalasin D or quercetin as a tight junction (TJ) modulator and a non-saturable permeation revealed that theasinensins were transported across Caco-2 cells in a TJ paracellular diffusion route. In conclusion, the dimers of condensed catechins, theasinensins B and A, can be absorbed intact into rat blood and transported across Caco-2 cell monolayers probably through a TJ paracellular pathway.

Zonulin, regulation of tight junctions, and autoimmune diseases.

Recent studies indicate that besides digestion and absorption of nutrients and water and electrolytes homeostasis, another key function of the intestine is to regulate the trafficking of environmental antigens across the host mucosal barrier. Intestinal tight junctions (TJs) create gradients for the optimal absorption and transport of nutrients and control the balance between tolerance and immunity to nonself antigens. To meet diverse physiological challenges, intestinal epithelial TJs must be modified rapidly and in a coordinated fashion by regulatory systems that orchestrate the state of assembly of the TJ multiprotein network. While considerable knowledge exists about TJ ultrastructure, relatively little is known about their physiological and pathophysiological regulation. Our discovery of zonulin, the only known physiologic modulator of intercellular TJs described so far, has increased our understanding of the intricate mechanisms that regulate the intestinal epithelial paracellular pathway and has led us to appreciate that its upregulation in genetically susceptible individuals leads to autoimmune diseases.

Tight junctions on the move: molecular mechanisms for epithelial barrier regulation

Increasing evidence suggests that the tight junction is a dynamically regulated structure. Cytoskeletal reorganization, particularly myosin light chain phosphorylation--induced actomyosin contraction, has increasingly been recognized as a mediator of physiological and pathophysiological tight junction regulation. However, our understanding of molecular mechanisms of tight junction modulation remains limited. Recent studies using live cell and live animal imaging techniques allowed us to peek into the molecular details of tight junction regulation. At resting conditions, the tight junction is maintained by dynamic protein-protein interactions, which may provide a platform for rapid tight junction regulation. Following stimulation, distinct forms of tight junction protein reorganization were observed. Tumor necrosis factor (TNF-α) causes a myosin light chain kinase (MLCK)--mediated barrier regulation by inducing occludin removal from the tight junction through caveolar endocytosis. In contrast, MLCK- and CK2-inhibition--caused tight junction regulation is mediated by altered zonula occludens (ZO)-1 protein dynamics and requires ZO-1--mediated protein-protein interaction, potentially through regulating claudin function. Although some of the molecular details are missing, studies summarized above point to modulating protein localization and dynamics that are common mechanisms for tight junction regulation.

Tight Junction in Blood‐Brain Barrier: An Overview of Structure, Regulation, and Regulator Substances

Blood-brain barrier (BBB) is a dynamic interference that regulates the nutrition and toxic substance in and out of the central nervous system (CNS), and plays a crucial role in maintaining a stable circumstance of the CNS. Tight junctions among adjacent cells form the basic structure of BBB to limiting paracellular permeability. In the present review, the constituents of tight junction proteins are depicted in detail, together with the regulation of tight junction under stimulation and in pathological conditions. Tight junction modulators are also discussed.

Tight junction proteins expression and modulation in immune cells and multiple sclerosis

The tight junction proteins (TJPs) are major determinants of endothelial cells comprising physiological vascular barriers such as the blood–brain barrier, but little is known about their expression and role in immune cells. In this study we assessed TJP expression in human leukocyte subsets, their induction by immune activation and modulation associated with autoimmune disease states and therapies. A consistent expression of TJP complexes was detected in peripheral blood leukocytes (PBLs), predominantly in B and T lymphocytes and monocytes, whereas the in vitro application of various immune cell activators led to an increase of claudin 1 levels, yet not of claudin 5. Claudins 1 and 5 levels were elevated in PBLs of multiple sclerosis (MS) patients in relapse, relative to patients in remission, healthy controls and patients with other neurological disorders. Interestingly, claudin 1 protein levels were elevated also in PBLs of patients with type 1 diabetes (T1D). Following glucocorticoid treatment of MS patients in relapse, RNA levels of JAM3 and CLDN5 and claudin 5 protein levels in PBLs decreased. Furthermore, a correlation between CLDN5 pre-treatment levels and clinical response phenotype to interferon-β therapy was detected. Our findings indicate that higher levels of leukocyte claudins are associated with immune activation and specifically, increased levels of claudin 5 are associated with MS disease activity. This study highlights a potential role of leukocyte TJPs in physiological states, and autoimmunity and suggests they should be further evaluated as biomarkers for aberrant immune activity and response to therapy in immune-mediated diseases such as MS.

Lipid Polarity Is Maintained in Absence of Tight Junctions

The role of tight junctions (TJs) in the establishment and maintenance of lipid polarity in epithelial cells has long been a subject of controversy. We have addressed this issue using lysenin, a toxin derived from earthworms, and an influenza virus labeled with a fluorescent lipid, octadecylrhodamine B (R18). When epithelial cells are stained with lysenin, lysenin selectively binds to their apical membranes. Using an artificial liposome, we demonstrated that lysenin recognizes the membrane domains where sphingomyelins are clustered. Interestingly, lysenin selectively stained the apical membranes of epithelial cells depleted of zonula occludens proteins (ZO-deficient cells), which completely lack TJs. Furthermore, the fluorescent lipid inserted into the apical membrane by fusion with the influenza virus did not diffuse to the lateral membrane in ZO-deficient epithelial cells. This study revealed that sphingomyelin-cluster formation occurs only in the apical membrane and that lipid polarity is maintained even in the absence of TJs.

Modulation of epithelial tight junctions by TGF‐beta 3 in cultured oral epithelial cells

Previous studies have indicated that transforming growth factor beta 3 (TGF-β3) was strongly expressed both in the gingival epithelium and the poorly structured pocket epithelium. A comprehensive analysis of the profile of tight junction proteins was carried out by quantitative real-time RT-PCR, Western blot and paracellular permeability assays. Active TGF-β3 protein added to monolayers of cultured oral epithelial cells initially reduced the permeability to dextran (10 kDa), followed by an increase in permeability. Three hours after the addition of TGF-β3, expression of genes encoding tight junction components was selectively up- or down-regulated. In addition, up- or down-regulation of expression of several tight junction associated proteins was observed, although the protein changes did not parallel changes in gene expression. To confirm that TGF-β3 plays a role in epithelial barrier function, a selective Src family kinase inhibitor saracatinib (AZD0530) was added to cells treated with active TGF-β3. Tight junction proteins claudins-2, -20 and ZO-2 were significantly decreased, but claudin-4 and -18 were significantly increased. These results suggest that TGF-β3 is involved in the modulation of epithelial barrier function by regulating assembly of tight junctions.

Doxycycline-Mediated Protective Effect Against Focal Cerebral Ischemia–Reperfusion Injury Through the Modulation of Tight Junctions and PKCδ Signaling in Rats

The strategy for the development of effective and safe neuroprotective agents has great potential to reduce cerebral ischemia-reperfusion injury and improve the functional outcome in stroke patients. Recently, doxycycline, a tetracycline antibiotic, has been shown to have neuroprotective efficiency in reduction of a variety of ischemia-reperfusion injuries as well as ischemic brain damage. We used the rat models of middle cerebral artery occlusion (MCAO) and reperfusion to investigate the effects of treatments with doxycycline against the blood-brain barrier (BBB) leakage at 3, 12, 72, and 120h of reperfusion. Male Sprague-Dawley rats were subjected to MCAO for 2h followed by reperfusion for 3, 12, 72, and 120h and received either doxycycline (45mg/kg) or saline. The results showed that the treatment of doxycycline significantly reduced the BBB leakage and cerebral infarct volume, which were proved by Evans blue assay and TTC staining. Real-time PCR, immunohistochemistry, and western blot assay verified that the administration of doxycycline significantly up-regulated the expression of tight junction claudin-5, occludin, and ZO-1 from 3 to 120h after reperfusion. The results of real-time PCR, western blot, and gelatin zymography analyses revealed that the gene and protein expression and activities of matrix metalloproteinases (MMPs) MMP-2 and MMP-9 were significantly elevated in a different time-dependent manner after ischemia-reperfusion but significantly inhibited by doxycycline treatment. Moreover, doxycycline could also significantly down-regulate the expression of PKCδ mRNA and protein after ischemia-reperfusion. These results suggested that the protective effects of doxycycline against BBB damage induced by reperfusion might be related to the up-regulation of tight junction proteins and inhibition of MMP-2, MMP-9, and PKCδ.

Leaky Gut and Autoimmune Diseases

Autoimmune diseases are characterized by tissue damage and loss of function due to an immune response that is directed against specific organs. This review is focused on the role of impaired intestinal barrier function on autoimmune pathogenesis. Together with the gut-associated lymphoid tissue and the neuroendocrine network, the intestinal epithelial barrier, with its intercellular tight junctions, controls the equilibrium between tolerance and immunity to non-self antigens. Zonulin is the only physiologic modulator of intercellular tight junctions described so far that is involved in trafficking of macromolecules and, therefore, in tolerance/immune response balance. When the zonulin pathway is deregulated in genetically susceptible individuals, autoimmune disorders can occur. This new paradigm subverts traditional theories underlying the development of these diseases and suggests that these processes can be arrested if the interplay between genes and environmental triggers is prevented by re-establishing the zonulin-dependent intestinal barrier function. Both animal models and recent clinical evidence support this new paradigm and provide the rationale for innovative approaches to prevent and treat autoimmune diseases.

Chloride channel ClC-2 modulates tight junction barrier function via intracellular trafficking of occludin

Previously, we have demonstrated that the chloride channel ClC-2 modulates intestinal mucosal barrier function. In the present study, we investigated the role of ClC-2 in epithelial barrier development and maintenance in Caco-2 cells. During early monolayer formation, silencing of ClC-2 with small interfering (si)RNA led to a significant delay in the development of transepithelial resistance (TER) and disruption of occludin localization. Proteomic analysis employing liquid chromatography-mass spectrometry /mass spectrometry revealed association of ClC-2 with key proteins involved in intracellular trafficking, including caveolin-1 and Rab5. In ClC-2 siRNA-treated cells, occludin colocalization with caveolin-1 was diffuse and in the subapical region. Subapically distributed occludin in ClC-2 siRNA-treated cells showed marked colocalization with Rab5. To study the link between ClC-2 and trafficking of occludin in confluent epithelial monolayers, a Caco-2 cell clone expressing ClC-2 short hairpin (sh)RNA was established. Disruption of caveolae with methyl-β-cyclodextrin (MβCD) caused a marked drop in TER and profound redistribution of caveolin-1-occludin coimmunofluorescence in ClC-2 shRNA cells. In ClC-2 shRNA cells, focal aggregations of Rab5-occludin coimmunofluorescence were present within the cytoplasm. Wortmannin caused an acute fall in TER in ClC-2 shRNA cells and subapical, diffuse redistribution of Rab5-occludin coimmunofluorescence in ClC-2 shRNA cells. An endocytosis and recycling assay for occludin revealed higher basal rate of endocytosis of occludin in ClC-2 shRNA cells. Wortmannin significantly reduced the rate of recycling of occludin in ClC-2 shRNA cells. These data clearly indicate that ClC-2 plays an important role in the modulation of tight junctions by influencing caveolar trafficking of the tight junction protein occludin.

Peptides as Tight Junction Modulators

The first step in drug absorption is the passage of drug molecules across epithelial cell sheets. Epithelial cell sheets are pivotal for the maintenance of homeostasis in the body by acting as a biological barrier that separates the inside of the body from the outside environment. Intercellular space between the adjacent epithelial cells is tightly sealed by tight junctions (TJs), which prevent solutes from freely moving across the epithelial cell sheets. Modulation of the TJ barrier has been a potent strategy for drug absorption. Absorption enhancers have been investigated since the 1980s, and sodium caprate is clinically used as an absorption enhancer. However, the biochemical constituents and structures of TJs were not elucidated until 1993. Occludin, a tetra-transmembrane protein, was identified to be a structural component of TJs in 1993. Claudin, another tetra-transmembrane protein, was identified as a structural and functional component of TJs in 1998. Modulation of occludin- or claudin-barrier is novel methods to enhance drug absorption. Recently, synthetic TJ-binding peptides, a kinase of claudin and peptide fragments of toxins have been developed. In the present review, we summarize the recent progress in TJ-modulating peptides and discuss their potencies.

Could antioxidants be the “magic pill” for cirrhosis-related complications? A pathophysiological appraisal

Patients with liver cirrhosis are prone to serious complications by almost all systems, leading to high morbidity rates and even death. Although the functional and structural derangement of diverse vital organs developed in the course of advanced liver disease is the result of one entity (cirrhosis) there are various treatment modalities for each system's complications, which are often ineffective. Identification of the link which connects the complications of cirrhosis from diverse systems might lead to a global, simple and more effective treatment approach for patients with cirrhosis. Accumulating evidence from experimental and clinical studies suggests that intestinal barrier dysfunction and subsequent gut-derived endotoxemia represent an important common pathogenetic mechanism in the development of diverse complications of cirrhosis. Intestinal oxidative stress seems to be a pivotal factor of gut barrier dysfunction in cirrhosis through promotion of enterocyte apoptosis, modulation of intestinal tight junctions and impairment of intestinal brush border function. In parallel, oxidative stress plays a fundamental role in the aggravation of liver injury and in the structural and/or functional derangements of diverse organs complicating the course of cirrhosis. Our hypothesis is that antioxidant treatments could prevent in a global way virtually all cirrhosis-related complications acting in two crucial levels in the pathophysiological cascade of events: (a) in a primary level, which is the gut-liver axis by ameliorating gut-derived endotoxemia, through prevention of intestinal oxidative stress and its associated gut barrier dysfunction, concurrently conferring direct antioxidant protection in the liver tissue and (b) in a secondary level, which refers to the diverse organs whose function is affected by liver cirrhosis, by preventing their oxidant-related structural and functional derangements.

Development of an Efficient Transdermal Delivery System of Small Interfering RNA Using Functional Peptides, Tat and AT-1002

Topical use of small interfering RNA (siRNA) as a therapeutic nucleic acid is increasingly studied for the treatment of skin diseases and for the improvement of skin properties. However, naked siRNA transdermal delivery is limited by its low stability in the body and low permeability into target cells. This is due to various skin barriers such as the stratum corneum that has multiple lipid bilayers and epidermal layers that have tight junctions. In this study, we investigate non-invasive transdermal siRNA delivery using two functional peptides: AT1002, which is a tight junction modulator and 6-mer synthetic peptide belonging to a novel class of compounds that reversibly increases paracellular transport of molecules across the epithelial barrier; and Tat, which is a cell-penetrating peptide applicable as a transdermal siRNA delivery enhancer. We examined whether expression of the tight junction protein zonula occludens protein 1 (ZO-1) was detected in mouse skin applied with AT1002. Additionally, siRNA stabilities for RNaseA using Tat and AT1002 were assessed. We also determined the intradermal delivery efficiency of siRNA using functional peptides by confocal laser microscopy of fluorescently labeled siRNA in mouse skin. We found that the Tat analog and AT1002 strongly increased siRNA stability against RNaseA. In addition, ZO-1 disappeared from the skin after treatment with AT1002, yet recovered with time after washing. Finally, we also found that Tat and AT1002 peptides accelerate transdermal siRNA delivery both widely and effectively. Thus, combination of Tat and AT1002 is expected to be a transdermal delivery enhancer of siRNA.