Elaeagnus umbellata is a plant commonly used in traditional Asian medicine for its many health benefits and strong antioxidative activity. Its therapeutic potential is believed to be connected to its effect on fibroblasts. This study aimed to investigate E. umbellata methanol-acetone extract’s (EUE) defense against hydrogen peroxide (H2O2)-induced fibroblast damage.E. umbellata are water-insoluble, we evaluated the effects of methanol-acetone fruit extracts using liquid chromatography (for ascorbic acid and beta-carotene) and spectrophotometry . The extract’s antioxidative activity was measured using DPPH radical inhibition, and EUE’s effect on human fibroblasts was also evaluated. We assessed the metabolic activity and apoptosis of HFFF-2 fibroblasts exposed to EUE and/or H2O2using the XTT test and flow cytometry, respectively. Superoxide dismutase activity and reactive oxygen species (ROS) production were evaluated using colorimetric and fluorometric assays, respectively. We measured pro-inflammatory cytokine  expression in HFFF-2 cells using immunocytochemistry.Because the main biologically active compounds of 2O2-induced stress. H2O2-treated fibroblasts had greater ROS production than cells treated with both H2O2 and EUE. Additionally, the group treated with H2O2 alone showed higher pro-inflammatory cytokine  expression.The extract increased HFFF-2 cell proliferation and reduced cell death caused by H2O2-induced oxidative stress and reduced the fibroblast-mediated inflammatory response triggered by ROS.EUE protected human fibroblasts from H Fibroblasts are the most common type of cells in mammalian connective tissue. In addition to their role as a structural ingredient, fibroblasts contribute to various immune and inflammatory responses. Human chronic inflammatory diseases can develop into a number of debilitating fibrotic disorders, proving that permanent activation of the immune system can lead to strong disturbance in fibroblast function . FibroblElaeagnus genus includes 70 to 80 species, the most studied of which being Elaeagnus angustifolia L. (Russian olive). Another oft-mentioned variety is Elaeagnus umbellata, frequently referred to as the autumn olive, silverberry, or spreading oleaster. It can be found in Asia, North America, or its place of origin, Southeast Asia. The Elaeagnus sp. berry is an excellent source of antioxidative compounds, including carotenoids, phenolic, flavonoids, and vitamins, specifically vitamins A, C, E, and D (α-tocopherol work with reactive oxygen species (ROS), especially peroxyl radicals, and singlet oxygen. Beta-carotene and lycopene act as antioxidants during lipid phases by neutralizing singlet oxygen or ROS  is mainly generated from superoxide anions produced by mitochondria and NADPH oxidases. Physiological levels are involved in normal cell metabolism, whereas increased levels of superoxide or hydroxyl radicals can invoke oxidative stress. Accordingly, H2O2 has been shown to be involved in the formation of numerous pathologies. More specifically, H2O2 can exert its toxic effects through the ferrous iron-dependent formation of highly reactive hydroxyl radicals, which in turn can alter the structure of lipids, proteins, and DNA (2O2 on epidermis cells was found to vary depending on the dose amount. 2O2 over 100 µM significantly increased the expression of tumour necrosis factor α (TNF-α) mRNA in human middle ear epithelial cells, suggesting that this dose was appropriate for studying anti-inflammatory effects.Hydrogen peroxide . The fruits were then lyophilized.Fruits of A field permit was not required as the fruit was bought at an ornamental plant farm.First, samples were incubated with methanol and acetone (1:50:10) in an ultrasound bath for 30 min. Then, extracts were filtered and the process of extraction was repeated. Extracts were combined and methanol was evaporated under vacuum in a rotary evaporator (40 °C). Next, they were kept in −20  °C until analysis. All extraction procedures were performed in darkness.E. umbellata berry extracts was performed according to the AOAC procedure (1990) at 25  °C. Analysis of carotenoids, beta-carotene, D (group), E (α-tocopherol), K (phylloquinone) in crude fat was conducted using an Agilent 1,100 high performance liquid chromatography (HPLC) system . Separations were performed at 25  °C using a 4.6 × 150 mm and 5 µm Zorbax Eclipse XDB C8 column (Agilent). Solvent flow rate was maintained at 1.2 mL/min in a linear gradient of 90:10 (vol./vol.) methanol to water, with an injection volume of 10 µL. The temperature of the column oven was set at 30  °C. Detection with a UV–vis detector was carried out at 230 nm for α-tocopherol, and 265 nm for K, carotenoids, β-carotene, and vitamin D. The standard solution (1 mg/mL) was prepared separately by dissolving 10 mg of each individual vitamin in 10 mL of methanol and storing in darkness at 4  °C. N = 3Fat extraction and saponification of N = 3).L-ascorbic acid levels was determined by HPLC using the method described by E. umbellata extract dry matter .The total phenolic (TC) content was determined using the standard Folin–Ciocalteu method. The extract was was combined with 2% sodium carbonate in deionized water and vortexed the mixture well. Next, 50% Folin–Ciocalteau reagent was added to the mixture and vortexed. It was left to settle for 45 min and the absorbance was measured at 750 nm. TC contents were calculated with the use of calibration curve of quercetin. The results were presented as quercetin equivalent milligram per gram (mg/g) of N = 3).The spectrophotometric method was used to determine the amount of lycopene . Lycopenα-tocopherol were diluted in methanol and L-ascorbic acid in deionized water before the analysis.The anti-oxidant in vitro properties of extracts were measured using DPPH free radical scavenging test, as previously shown by α-tocopherol). After 30 min in darkness (25 °C), samples were centrifuged  and then the absorbance was measured at 517 nm (N = 6).Regarding the scavenging activity of the DPPH? test, 0.2 mM DPPH? in methyl alcohol was mix with each extracts or standard antioxidant . Cells that reached about 70% confluence were used for the experiment.The HFFF-2 human foetal foreskin fibroblast cell line was obtained from the European Collection of Authenticated Cell Cultures  and maintained in Dulbecco’s modified medium  supplemented with 10% foetal bovine serum , 1% penicillin, and streptomycin  at 37  °C in a humidified atmosphere of 5% COTo measure cell viability, the XTT Kit , a colorimetric assay for the nonradioactive quantification of cellular proliferation, viability, and cytotoxicity, was used. The assay is based on the extracellular reduction of XTT by NADH produced in the mitochondria. XTT is only reduced to formazan by metabolically active cells. Cells were plated into 96-well plates at 17 000 cells/well and 100 µL DMEM was added to each well. After 24 h, 90 µL of fresh culture medium and 10 µL of extract at concentrations ranging from 100 to 500 µL/mL were added to each well.The following groups were created:2O2I. Control without extract or H2O2II. 100 µg/mL of extract without H2O2III. 150 µg/mL of extract without H2O2IV. 200 µg/mL of extract without H2O2V. 250 µg/mL of extract without H2O2VI. 500 µg/mL of extract without H2O2VII. 100 µg/mL of extract with H2O2VIII. 150 µg/mL of extract with H2O 2IX. 200 µg/mL of extract with H2O2X. 250 µg/mL of extract with H2O2XI. 500 µg/mL of extract with H2O2 alone.XII. H2O2 was added to induce oxidative stress.The extract was mixed with PBS and stirred until completely dissolution. Equal volumes of PBS were added to control cells. After 72 h, 100 µM HThen, after another 24 h, the XTT test was performed according to the manufacturer’s protocol.N = 6).Optical density (OD) was measured at 450 nm using an enzyme-linked immunosorbent assay reader . Cell viability was expressed as the percentage of (ODtest −ODblank)/(ODcontrol −ODblank), where “ODtest” is the OD of cells incubated with the extract, “ODcontrol” is the OD of the control sample, and “ODblank” is the OD of the wells without cells  and 100 µg/mL of 10% extract in PBS  or PBS alone . After 72 h, 100 µM of H2O2 was added to two groups (one with extract and another without extract). After 6 h of incubation, the cells were collected and centrifugated. Each cell pellet was then mixed with 100 µL of Annexin binding buffer. 5 µL of fluorescein isothiocyanate-labelled Annexin V and 1 µL of propidium iodide were pipetted into each vial. The samples were left for 15 min at 25  °C. Finally 400 µL of binding buffer and the samples on ice. A total of 10,000 events were recorded per sample and the apoptosis plots were generated using Flowing Software 2.5.1 .Annexin V/PI Apoptosis Kit  was used to estimate cell death pathways according to 2O2 incubation times of 12 and 24 h, respectively.This protocol was repeated twice and in triplicate with HTo measure cell-generated ROS, the fluorometric intracellular ROS assay kit was used. The following groups were created:2O2I. Control without extract or H2O2II. 100 µg/mL of extract with H2O2III. 500 µg/mL of extract with H2O2 alone.IV. HE. umbellata extract at concentrations of 100 mg/L and 500 mg/L in PBS or appropriate volume of PBS. After 72 h, oxidative stress was induced by addition of 100 µM H2O2 to the groups receiving the extract and the group treated only with H2O2. H2O2 dose was chosen based on literature data  or an equivalent volume of PBS was added to cells. After 72 h incubation, oxidative stress was induced by addition of 100 µM H2O2. Cell pellets from individual groups were pooled to obtain three samples: 100 µg/mL extract + H2O2, H2O2 alone, and control. Total protein was isolated and measured using the BCA method. The same amount of protein was used from each group. The isolated protein was analyzed using the SOD determination KIT (Sigma-Aldrich) according to the manufacturer’s instructions. This KIT allowed us to evaluate SOD activity by utilizing WST-1 , a highly water-soluble tetrazolium salt that produces a water-soluble formazan dye when reduced with a superoxide anion. The O2 reduction rate was linearly related to xanthine oxidase (XO) activity and was inhibited by SOD. The results were expressed as SOD activity (%), according to protocol.Cells were seeded at 75 cmThe expression of selected cytokines was measured using cytokine arrays, which are antibody-pair-based assays that are analogous to Western blots, but using a membrane as a substrate.The following groups were created:2O2I. 100 µg/mL of extract with H2O2 aloneII. HIII. Control –PBS only.3 flask in complete medium as described in section ‘Cell cultures and treatments’, except for the FBS concentration, which was reduced to 1%. After 24 h, extract (100 µg/mL in PBS) or an equivalent volume of PBS was added to cells. Following a 72 h incubation period, oxidative stress was induced by adding 100 µM H2O2. Cell pellets from individual groups were pooled to obtain 3 samples: 100 µg/mL extract + H2O2, H2O2 alone, and the control group. Total protein was isolated and measured using the BCA method. The isolated protein was analyzed using the human cytokine antibody array (Abcam), according to the manufacturer’s instructions. he same amount of protein was applied to each membrane. Chemiluminescence detection was performed using the ChemiDoc1 Imaging System and Quantity One Basic Software . The captured images of each membrane were analysed using Fiji software . The results from each group were optimized according to the Abcam protocol (N = 4).Cells at 75 cmStatistical analyses were performed using the Statistica 13 program.In order to determine antioxidant potential and viability statistical significance of differences between control and individual concentrations of the extract was examined using the ANOVA test. Equality of variances was confirmed using the Brown-Forsythe test. The differences between individual concentrations were checked using the Tukey’s post hoc test.E. umbellata fruit is known to contain large amounts of phenolic compounds, carotenes, ascorbic acid, and fat-soluble vitamins. ethanol-acetone extracts from E. umbellata fruit were evaluated regarding total phenols [1749 (± 5.2) mg], lycopene [50 (± 0.2) mg], beta-carotene [73 (± 0.2) mg], and vitamins D [1.38 (± 0.1) mg], E (α-tocopherol) [1124 (± 3.2) mg], K [0.44 (± 0.08) mg], and C (L-ascorbic acid) [64 (± 1.8) mg] levels per 100 g of d.m.E. umbellata fruit extract and the standard antioxidants. The results were expressed as the % of DPPH inhibition  of hibition .α-tocopherol had similar antioxidant activity. The extract showed lower potential to reduce DPPH when compared to the standard antioxidants; the extract showed 54.78% potential in the 100 µg/ml dose versus 78.30% for vitamin C and 76.31% for vitamin E. However, this difference was less significant in a higher dose , reaching 75.25% potential for the extract and 86.24% and 88.37% for L-ascorbic acid and α-tocopherol, respectively.L-ascorbic acid and E. umbellata fruit extract showed any cytotoxic effects on HFFF-2 fibroblast cells. After 72 h of treatment, all of the extract concentrations increased cell viability when compared to the non-treated control. The observed effect was statistically significant (p < 0.05) across all tested doses  of the ed doses . In conted doses . The dif2O2 had the highest rate of necrosis in the total mortality. Across different time intervals, the control or extract-only cells had the lowest necrosis rates, and the highest proportion of necrosis was found in the H2O2-only cells.E. umbellata extract concentrations to cells 72 h before oxidative stress induction resulted in a statistically significant (p < 0.05) decrease in ROS production (Adding both oduction .p < 0.05).Notably, the 500 mg/L extract concentration had a greater effect in inhibiting ROS production than the 100 mg/L concentration , fractalkine, monocyte chemoattractant protein 4 (MCP-4), B lymphocyte chemoattractant (BLC), granulocyte chemotactic protein 2 (GCP-2), neutrophil-activating peptide 2 (NAP-2), Eotaxin-2, and Eotaxin-3 than in cells treated only with H2O2. The non-treated control samples showed the lowest expression of chemokines, of which fractillin, BLC, GCP-2, and Eotaxin-3 were negligible. Cells treated with the extract and H2O2 had more than twice the expression of fibroblast growth factor 7 (FGF-7) and brain-derived neurotrophic factor (BDNF) after H2O2 administration than cells treated only with H2O2. Likewise, TIMP metallopeptidase inhibitor 1 (TIMP-1) and HGF expression was higher by over 40% and 5%, respectively , acetone, ethyl acetate, or boiling ethanol are the recommended solvents. However, this study’s temperature of extraction was 40 °C, and we used methanol and acetone as solvents in a 80:20 (vol/vol) ratio. Concurrently, beta-carotene has been found to more effectively dissolve in ethanol at lower temperatures (maximum extraction at 25 °C) . TherefoE. umbellata fruit extract contained strong anti-oxidative properties. E. umbellata methanol, ethanol, and water extracts had more than 99.5% DPPH scavenging activity, whereas the extract described in this study had 65.55% DPPH scavenging potential. This difference could have been influenced by the use of acetone as a co-solvent for the extract, but also from subtle changes during the experiments .The results of our study confirmed that α-tocopherol, which are among the strongest known antioxidants. Although the extract contained both of these vitamins, it should be noted that our extraction protocol did not remove all of the fruit’s non-antioxidant compounds, as the procurement of pure bioactive compounds was outside the scope of the study.The results showed that the extract was a weaker antioxidant than L-ascorbic acid and Nutraceuticals and functional foods are becoming increasingly popular and contribute to the public’s growing fear of potential side effects of synthetic compounds. Consumers also have low confidence in supplements containing pure, isolated substances. Therefore, this study focused on evaluating the health benefits of an extract containing a mixture of bioactive compounds and other residues.E. umbellata extract significantly reduced the H2O2-induced secretion of ROS in HFFF-2 fibroblast cells. As expected, administering H2O2 significantly increased ROS secretion in the cells. However, pre-treating the cells with the extract caused up to 4 times lower ROS secretion when compared to cells only treated with H2O2.In this study, we found that E. umbellata extract’s ability to protect cells against oxidative stress might be a result of the presence of phenols, particularly polyphenols, which have the ability to reduce the oxidation rates of organic matter by transferring an H atom (from their OH groups) to chain-carrying ROO* radicals.The α-tocopherol. When delivered to a cell, α-tocopherol has been shown to protect membranes against lipid peroxidation  reactions resulting from H2O2 reacting with iron (II) ions (the Fenton reaction) or with iron and copper ions (the Haber-Weiss reaction). The strong OH oxidizing effect was directed against mitochondrial DNA. The reaction of these metal ions with AA is also important for this process, as L-ascorbic acid acts as a co-substrate of hydroxylases and oxygenases, which are enzymes involved in collagen biosynthesis. Overall, AA keeps the metal ions in a reduced state in the enzyme active site, which in turn enables optimal enzyme activity. L-ascorbic acid reduction of free transition metal ions generates oxygen radicals. Reduced metal ions, such as iron, react with H2O2to generate highly reactive OH or O2− ions. However, due to the limited availability of iron and copper ions in living organism tissue, it has not been confirmed that this reaction takes place in vivo. These metal ions are bound by proteins that are present in cells, such as ferritin, transferrin, and ceruloplasmin . In some instances, physiological inducers of apoptosis  can promote inflammatory cytokine expression in conjunction with apoptosis, resulting in an apoptotic process that appears pro-inflammatory. Conversely, necrosis is considered pro-inflammatory due to DAMP .2O2), while cells exposed to both the extract and H2O2 showed significantly lower necrosis rates just 6 h after H2O2-induced stress. However, only 24 h after H2O2, administration, the group that received the extract and H2O2 showed similar necrotic activation as the group that received the extract without the stress factor. These results indicated that H2O2 causes rapid inflammation-related cellular changes, which HFFF-2 cells can partially neutralize. On the other hand, the extract reduced overall cell death and the incidence of necrosis at rates that were similar to the untreated control group, where no oxidative stress was induced.We evaluated the cell death pathways that were activated by the extract. The percentage of necrosis decreased gradually across all groups except for the non-treated control group. Necrosis was most pronounced in cells treated with only the stress factor  activity in liver tissue and decreased malondialdehyde (MDA), nitric oxide (NO), interleukin (IL) 1 β, 6 (IL-6), TNF-α, and cyclooxygenase-2 (COX-2) levels in paw oedema tissue after λ-carrageenan-induced inflammatory reactions. E. angustifolia fruit powder in women with knee osteoarthritis. They observed a significant decrease in serum pro-inflammatory cytokine  levels and a rise in serum anti-inflammatory cytokine (IL-10) levels after 8 weeks of supplementation. No significant changes were observed in the IL-1 β and MMP-13 serum levels , IL-1 α, IL-1 β, and the majority of chemokines, such as C-C motif chemokine ligand 2 (CCL2), CCL22 (MCP-1) and C-X-C motif chemokine ligand 10  in LPS-stimulated murine macrophage cells.The mechanisms that affect pro-inflammatory cytokine expression E. umbellata extract. Chemokines play an important role in connective tissue diseases by mediating the recruitment of eosinophils to the inflammation site and ROS release, leading to tissue damage and inflammatory response propagation . One theE. umbellata extract decreased the expression of four cytokines that induce MMP expression: NAP-2, CPG-2, MIF/CXCR2, and Fractalkine/CX3CL1. As previously mentioned, MMPs are associated with collagen degradation during oxidative stress. Scutellaria baicalensis root) decreased MMP-1 expression and improved the integrity of type I collagen in foreskin fibroblasts. In recent years, there has been a greater use of natural substances which, instead of reducing MMP expression, increase the expression of their inhibitors . The imbalance between MMPs and TIMPs has been characteristic for inflammatory diseases, cancer, and neurodegenerative diseases  because of its inflammatory-regulating properties. Moreover, the extract was demonstrated to increase the expression of TIMP-1, an MPP-9 inhibitor, indicating that collagen may be protected from degradation during oxidative stress. These properties, combined with the increase in BDNF expression, suggest that E. umbellata extracts may be effective in the treatment of neuro-inflammatory disorders, such as Alzheimer’s or Parkinson’s diseases. However, further studies are necessary to delineate the precise mechanisms underlying these activities.This study was the first to explore the antioxidant effects of 10.7717/peerj.10760/supp-1Supplemental Information 1Click here for additional data file.


PIEZO1 was showed to be abundantly expressed, and Piezo1 agonist Yoda1 and mechanical stretch caused a Piezo1-dependent Ca2+ influx and release of arachidonic acid and PGE2. Treatment with Yoda1 or PGE2 significantly inhibited hTM cell contraction. These results suggest that mechanical stretch stimuli in TM activates Piezo1 and subsequently regulates TM cell contraction by triggering Ca2+ influx and release of arachidonic acid and PGE2. Thus, Piezo1 could acts as a regulator of intraocular pressure (IOP) within the conventional outflow pathway and could be a novel therapeutic strategy to modulate IOP in glaucoma patients.The trabecular meshwork (TM) constitutes the main pathway for aqueous humor drainage and is exposed to complex intraocular pressure fluctuations. The mechanism of homeostasis in which TM senses changes in intraocular pressure and leads to normal levels of outflow resistance is not yet well understood. Previous reports have shown that Piezo1, a mechanically-activated cation channel, is expressed in TM and isolated TM cells. Therefore, we tested hypothesis that Piezo1 may function in response to membrane tension and stretch in TM. In human trabecular meshwork (hTM) cells,  Conventional outflow tissues are constantly subjected to fluctuations in IOP, and cells in these tissues sense alterations in IOP as mechanical stretch or strain3. Changes in pressure of 8–30 mmHg can cause length extension by 50% in cells in the outflow pathway5. In response to mechanical stretch or strain, TM cells activate intracellular signaling pathways leading to changes in gene expression, extracellular matrix (ECM) turnover, contractile properties6.Intraocular pressure (IOP) is regulated by the production and outflow of aqueous humor and has been associated with increased risk of glaucoma development. The main route of aqueous humor drainage is the conventional outflow pathway, which involves the flow of aqueous humor to the Schlemm's canal through the juxtacanalicular trabecular meshwork (TM)10. Following membrane stretch, TM-resident TRPV4 channels initiate calcium (Ca2+) signals and cytoskeletal reorganization, and TRPV4 antagonists increased the outflow facility and lowered IOP in glaucomatous mouse eyes10. Recent studies showed that TREK-1 collaborates with TRPV4, regulating TM tensile homeostasis8. Although numerous studies highlighted the importance of mechanosensation in modulating TM outflow, the molecular links between mechanotransduction, Ca2+ homeostasis, and reorganization of the conventional outflow pathway remain unclear. Hence, the mechanosensory mechanisms mediating force-coupling and IOP regulation should be further explored.The non-selective cation channel TRPV4 and the two-pore domain potassium channel TREK1 are expressed in TM cells, sense mechanical stretch generated by fluctuations in IOP, and play an essential role in IOP adjustment11. Piezo1 has been implicated in mechanical stretch response and extracellular Ca2+ influx regulation in the bladder urothelium12, red blood cell13, and neural stem cells14. Nevertheless, its role in TM remains poorly understood. We hypothesized that in TM, Piezo1 might respond to mechanical stress and regulate IOP by locally releasing lipid mediators. Lipid mediators are well known to have an important role in various physiological events through rapid production from the cell membrane and working transiently and locally in the restricted tissue. Notably, several arachidonic acid-derived metabolites have been implicated in mechanical signaling16. Among lipid mediators, prostaglandins such as PGF2α and PGE2, lysophospholipids, or cannabinoids are associated to the IOP regulation20. Thus, we investigated the involvement of Piezo1 on the mechanical stress and the release of lipid mediators in primary cultured human TM cells.Piezo1 and Piezo2 were first described in 2010 as components of a mechanically activated cation channel expressed in neurons and neuronal cell linesFirst, we examined the expression of human Piezo and TRPV family members in primary hTM cells by qPCR. Piezo1 mRNA levels were profoundly higher than Piezo2 and TRPV family mRNA levels in hTM cells  compared with control cells  group was 32.96 ± 4.24 pg/ml, 29.13 ± 1.60 pg/ml, and 28.69 ± 2.89 pg/ml, and in the Stretch group was 196.70 ± 15.15 pg/ml, 176.80 ± 21.27 pg/ml, and 122.40 ± 3.85 pg/ml. PGD2 release was not significantly altered by stretch  group was 46.82 ± 2.27 pg/ml, 46.39 ± 3.43 pg/ml, and 35.28 ± 4.09 pg/ml, and in the Yoda1 group was 102.27 ± 1.12 pg/ml, 106.83 ± 4.52 pg/ml, and 100.25 ± 2.75 pg/ml. No other lipid mediators were detected. These data suggest that Piezo1 activation triggers the secretion of arachidonic acid and PGE2.To assess the direct effect of Piezo1 activation, we measured lipid mediators in hTM cell supernatants after Yoda1 treatment. This analysis revealed that Yoda1 treatment significantly increased the levels of arachidonic acid and PGEGD2 Fig. . The amo2 on hTM cells contraction, we performed an in vitro collagen gel contraction assay. This assay correlates that cells have contraction ability, which is supported by the fact that collagen alone does not cause contraction22. Compared to controls, Yoda1 significantly suppressed collagen gel contraction after 48 h and later, and PGE2 at 10 and 100 nM PGE2 after 24 h and later  not only activates Piezo1 but may also have toxic effects. As shown in Fig. 2 significantly inhibited hTM collagen gel contraction in a dose-dependent manner, suggesting PGE2 production after Piezo1 activation contributes to IOP regulation. On the other hand, activation of EP2  receptors have been demonstrated to increase TM cell contractility34. However, this result is a response within 1 h of agonist exposure. PGE2 lowers IOP, and also temporarily increases IOP in the early stages after instillation18. The increase in TM cell contractility due to activation of EP2 receptor may reflect this transient increase in IOP and change at later time. EP2 receptor was also expressed in Schlemm's canal (SC) and its activation was shown to reduce SC cell stiffness34. Accordingly, PGE2 released from hTM cells may target SC in addition to TM.To assess the effect of direct activation of Piezo1 in hTM cells, we used the Piezo1-selective activator Yoda1. Yoda1 was identified among approximately 3.25 million compounds as a synthetic small molecule that acts as an agonist of both human and mouse Piezo1exposure 2 h not o12. In chondrocyte, Piezo1 was largely responsible for the stretch-activated current, whilst TRPV4 had no involvement in the specific mechanoelectrical transduction pathway35. However, there are no reports comparing the importance of Piezo1 and TRPV4 in TM. Thus, future studies are required to compare the sensitivity of Piezo1 and TRPV4 to stretch stimuli, as well as the relationship between Piezo1 and TRPV4 mRNA levels and function. We focused on lipid mediator and its involvement in the TM contraction, and didn’t further explore whether Piezo1 are involved in the stretch-induced cytoskeletal remodeling in hTM cells, which should be determined in the future study. In addition to lipid mediators, nitric oxide (NO) is a candidate that link intracellular Ca2+ elevation and regulation of TM outflow. NO is formed by the enzyme called nitric oxide synthase (NOS) and Three different isoforms have been identified including neuronal NOS or brain NOS , endothelial NOS (eNOS or NOS3), and inducible NOS (iNOS or NOS2). nNOS1 and eNOS are constitutively expressed in cells and are activated by an increase in intracellular Ca2+36. iNOS is induced by a variety of stimuli, such as endotoxins and inflammatory cytokines, regardless of Ca2+ level. Three isoforms of NOS has been reported to be expressed in TM37. Several studies in vitro and in vivo have demonstrated IOP-lowering effects of NO, and treatment with the ROCK inhibitor Y-26732 increased the expression of eNOS and NO production more significantly39. Increased visual field damage results in decreased eNOS expression and Ca2+-dependent NOS activity in TM of POAG patients40. Thus, elevated intracellular Ca2+ in TM may increase eNOS expression and NO production, leading to decreased IOP. In this study, we applied a uniaxial stretch stimuli to Piezo1 knockdown cells, but by applying a multiaxial stretch stimuli to knockout cells, the exact contribution of Piezo1 in the cellular response to stretch stimuli could be obtained under more physiological conditions. Moreover, since our conclusions are based solely on evidence from in vitro experiments, we couldn’t determine the pathophysiological relevance of our in vitro observations in regulation of IOP or outflow facility. Further study using animal models or perfusion experiment will be needed to determine this issue.While our findings highlighted the critical role of Piezo1 in IOP regulations upon TM stretch, there were several limitations in the present study. Piezo1 and TRPV4 were compared only for mRNA expression in this study. There are several reports comparing the role of Piezo1 and TRPV4 in response to stretch stimulation. In bladder urthelium, the sensitivity of Piezo1 to stretch stimuli was higher than that of TRPV42+ influx in response to mechanical stretching and that PGE2 is essential for Piezo1-mediated hTM cell contraction inhibition. Our study revealed a novel role for Piezo1 in hTM cells and proposed a new therapeutic strategy for the conventional outflow pathway.In conclusion, this study clarified the role of new aqueous humor outflow regulation mediated by Piezo1 in TM. The present data has shown that Piezo1 regulates Ca41. All experiments were conducted according to the principles of the Declaration of Helsinki and approved by the ethics committee of the University of Tokyo. Written informed consent was obtained from donors. Briefly, corneal scleral rims maintained in Optisol were cut and divided into 6 to 8 sections. The trabecular meshwork strip was picked and isolated with forceps from sections. TM strips were cultured in a dish coated with collagen gel . After the TM cells migrated from the tissue, the gel was digested with collagenase, and the collected TM cells were grown on dishes coated with 1 μg/mL fibronectin. hTM cells from passages 3 to 6 were used in subsequent experiments and maintained in Trabecular Meshwork Cell Medium  ) containing 2% fetal bovine serum (ScienCell), 1% Trabecular Meshwork cell growth supplement (ScienCell) and 1% penicillin/streptomycin solution (ScienCell). The identity of the isolated cells was confirmed by the dexamethasone-induced upregulation of MYOC. After 7 days of exposure to vehicle (DMSO), 100 or 500 nM dexamethasone, dexamethasone enhanced MYOC expression  or MISSION siRNA Universal Negative Control #1 siRNAs (Merck). siRNA transfections were performed using MISSION siRNA Transfection Reagent (Merck) according to the manufacturer’s instructions.Primary human trabecular meshwork (hTM) cells were isolated from human corneal scleral rims obtained from the Rocky Mountain Lions Eye Bank from 8 donors , as described previouslyTRPV1-6 and PIEZO1-2 were designed by us or purchased from Takara Bio Inc., as detailed in Table Total RNA was extracted from hTM cells at 24 h after siRNA transfection using ISOGEN . The concentration of Total RNA was measured by the absorbance at a wavelength of 260 nm using a spectrophotometer NanoDrop 2000 . Complementary DNA (cDNA) was prepared using ReverTra Ace qPCR RT Master Mix with gDNA remover . Quantitative real-time PCR was run on a Thermal Cycler Dice Realtime System  using SYBR Premix Ex Taq™ II (Tli RNaseH Plus) (Takara Bio Inc.). Standard curves were used to determine mRNA transcript copy number in individual reactions. Primers specific for 2+ indicator Fluo-8 AM  for 20 min. Subsequently, 10 μM Yoda1 32 was applied directly to the cover glass chambers. STB-CH-24 was attached to a stretch device STB-150  on the stage of a fluorescence microscope and subjected to a single uniaxial stretch with a nominal 30% extension  at room temperature. As evaluated from images of the chamber at 30% extension, the actual extension of the cell culture surface was about 20%. Fluorescent images were acquired under a fluorescence microscope . The fluorescence intensity of Fluo-8 was quantified using ImageJ software (https://imagej.nih.gov/ij/download.html). First, the captured images were stacked and the misalignment was corrected with the StackReg plugin. Next, the image with the highest fluorescence intensity from the cells was binarized by setting a threshold value by the triangle method, and the cells were selected with Analyze particles. Finally, the fluorescence intensity of the selected cells was measured on all images. The change ratio  was calculated using peak and basal values. FT, Fmax, and F0 represent the fluorescence intensity at that time, the maximum fluorescence intensity, and the fluorescence intensity before stimulation, respectively.hTM cells or those 24 h after siRNA transfection were seeded on cover glass chambers  or stretch chamber STB-CH-24  coated with 1 μg/mL fibronectin. After washing with PBS, hTM were loaded with 0.1% BSA (Sigma) and 5 μM fluorescent Ca42. Cell supernatant was collected 10, 30, and 60 min after 10 μM Yoda1 treatment or stretch stimulation, and stored at − 80 ℃. Methanol was added to each sample at a ratio of 1:1, and 10 μL of internal standard was added. The internal standard contained 6-keto-PGF1α-d4, AEA-d4, arachidonic acid-d8, docosahexaenoic acid-d5, eicosapentaenoic acid-d5, OEA-d4, tetranor-PGEM-d6, TxB2-d4, PGF2α-d9, PGE2-d4, PGD2-d4, LTB4-d4, LTC4-d5, LTD4-d5, LTE4-d5, 5(S)-HETE-d8, 12-HETE-d8, 15(S)-HETE-d8, and PAF-C16-d4 . After vortexing, samples were centrifuged at 15,000×g and 4 °C for 15 min to obtain phase separation. Supernatants were transferred into 15 mL glass tubes. The supernatant and 0.03% formic acid were mixed at a ratio of 1:4 with vortex. Samples were loaded onto a solid-phase extraction column  pre-conditioned with methanol and 0.03% formic acid. The column was centrifuged at 800 rpm and 4 °C for 2 min, and washed with petroleum ether. Methanol containing 0.2% formic acid was added to the column, followed by centrifugation at 800 rpm and 4 °C for 2 min to elute lipids. After evaporation, eluents were reconstituted in methanol.Lipid analysis was performed as previously described42.The samples were analyzed on a Nexera ultra-high performance liquid chromatograph connected to a triple quadrupole mass spectrometer LCMS-8060 . Chromatographic separation was performed using a Kinetex C8 column . The mass spectrometer was operated in selected reaction monitoring mode allowing for simultaneous detection of target lipid mediators. Quantification was performed by internal standard calibration method using chromatographic peak areas, as previously described6 cells/mL. The resultant mixture and cell suspension were mixed at a 9:1 ratio and 500 μL of the mixture was added to each well of 24-well plates and incubated at 37 °C to allow for collagen gel formation. After 60 min, gels were placed in 6-cm dishes containing 5 mL Trabecular Meshwork Cell Medium with DMSO, 10 μM Yoda1, or PGE2 . After 0, 12, 24, 48, and 72 h, gels were imaged with a gel imaging device, and the area was quantified with ImageJ software (https://imagej.nih.gov/ij/download.html).Gel contraction assay was performed using the Collagen Gel Culturing Kit  as per the manufacturer’s instructions. Collagen type I, 10 × MEM, and reconstitution buffer (pH 7.3) were mixed at a ratio of 7:1:1 at 4 °C. The final hTM cell concentration was adjusted to 1 × 10t-test or Dunnett’s test. A difference was considered statistically significant when p < 0.05.Data were represented as mean ± standard deviation (SD) in Fig. Supplementary Figure Legends.Supplementary Figure S1.Supplementary Figure S2.


The ALD window, i.e., the region where the growth per cycle (GPC) is constant, shifted from high temperatures (150–200 °C) to lower temperatures (80–150 °C) in PEALD. HfO2 deposited at 80 °C by PEALD showed higher density (8.1 g/cm3) than those deposited by thermal ALD (5.3 g/cm3) and a smooth surface (RMS Roughness: 0.2 nm). HfO2 deposited at a low temperature by PEALD showed decreased contaminants compared to thermal ALD deposited HfO2. Values of refractive indices and optical band gap of HfO2 deposited at 80 °C by PEALD  were higher than those obtained by using thermal ALD . Transparency of HfO2 deposited at 80 °C by PEALD on polyethylene terephthalate (PET) was high (> 84%). PET deposited above 80 °C was unable to withstand heat and showed deformation. HfO2 deposited at 80 °C by PEALD showed decreased leakage current from 1.4 × 10−2 to 2.5 × 10−5 A/cm2 and increased capacitance of approximately 21% compared to HfO2 using thermal ALD. Consequently, HfO2 deposited at a low temperature by PEALD showed improved properties compared to HfO2 deposited by thermal ALD.HfO However, scaled-down devices can show many problems, such as direct tunneling, high gate leakage current and poor reliability ,2. Theres high-k ,4. HfO2  control ,6,7,8.2 thin films has been required [2 thin films normally must be deposited at around 200 °C because the metal–organic precursors used as sources during the ALD process fully decompose at high temperatures [2 in ALD [2 thin films deposited at low temperatures had problems, such as a high level of carbon impurities (5.15%–8.9% carbon impurity in HfO2 thin films deposited at 150 °C) or low density ; these problems cause high leakage current and poor reliability in electronic devices [Recently, as the importance of wearable devices has increased, low-temperature deposition of HfOrequired ,10. Howeeratures . Many me2 in ALD ,14,15,16 devices ,13,14.2 gas as a reactant; this is in contrast to the conventional thermal ALD process, which uses O3 as a reactant. In the previous studies, it was found that the electrochemical oxidation potential, a measure of the sensitivity of the oxidation reaction, of atomic oxygen radicals (2.42 V) is higher than that of O3 (2.08 V) [Using plasma to produce oxygen radicals with high reactivity can solve these problems, and plasma has been used in other low-temperature deposition processes . Consequ(2.08 V) ,19,20. I(2.08 V) ,22,23,24(2.08 V) .2 thin films were deposited by PEALD at 80 °C, and their variable properties, such as film structures, surface morphology and surface components, were compared with thin films deposited by using thermal ALD and PEALD at various temperatures . Moreover, values of densities, refractive index, optical bandgap determined by Tauc plot and transmittance of HfO2 deposited at 80 °C by thermal ALD and PEALD were compared. In our study, the HfO2 deposited at a low temperature (80 °C) by PEALD showed a low carbon ratio (3.5%) and high film density (8.1 g/cm3). Finally, electrical characteristics, such as capacitance–voltage (C–V) curve, current–voltage (I–V) and fixed-charge density (Qf) of HfO2 deposited at 80, 150 and 250 °C were analyzed, using an MOS capacitor. The HfO2 thin films deposited at a low temperature (80 °C), using PEALD, showed improved structural, chemical, optical and electrical properties, without any degradation.In this study, HfO2 thin films were fabricated on doped (ρ ~ 15 Ω·cm) p-type Si (100) wafers. Substrates were cleaned for 10 min with acetone, 10 min with ethanol and 10 min with IPA in an ultrasonic generator; they were immediately dried by blowing argon over the sample. The substrates were loaded at different temperatures, in a range of 80–250 °C. The main pump was an MVP-90 , and the base pressure was 10 mtorr. An ISP-90  was used as a by-pass pump for constant flow. In this experiment, direct plasma was used; the plasma power was fixed at 150 W, using a 13.56 MHz RF power supply . Tetrakis(ethylmethylamino) hafnium  was used as a precursor. High-purity O3 and O2 were used as oxidants. O3 was produced from O2 by an ozone generator . Ar gas, used as a carrier gas and purge gas, also had a purity of 99.999%. TEMAH precursor canister was maintained at 75 °C. The precursor flow line and the chamber were also maintained at 80 °C, to prevent condensation and clogging.Using an automated ALD system , HfO2 thin films was measured by using a Reflectometer  and Spectroscopic Ellipsometry . In addition, the film structures and density in HfO2 were examined by Grazing Incidence X-ray diffraction and X-ray reflectivity, respectively . The root mean square (RMS) roughness values of the HfO2 films (50 nm) were obtained by Atomic Force Microscope  images and scanned at 2 µm × 2 µm size. The chemical bonding states and components were examined by using X-ray photoelectron spectroscopy  To remove carbon- and nitrogen-contaminant layers from air, approximately 7 to 10 nm of the HfO2 films was removed via Ar etching, at 1 keV, for 30 s [2 (50 nm) were extracted from the Ellipsometry  data. The transmittance of HfO2 (50 nm) at 550 nm on the PET substrate  was measured in a range from 190 to 1100 nm, which was measured in the normal incidence of light by UV-vis spectroscopy . To measure the electrical properties (I–V and C–V), MOS capacitors were fabricated. Cu/Ti top electrodes were deposited on HfO2/p-Si, using an E-beam evaporator . Cu/Ti circular electrodes were patterned, using a shadow mask. Electrical properties, as indicated by the I–V and C–V curves, were measured by using a Manual Probe Station . The C–V curve was obtained at 1 MHz in the range of −7 to +7 V, and the I–V curve was obtained from −2 to 2 V.The thickness of the HfOfor 30 s ,26. Refr2 thin films deposited at 80 °C, To show the experimental conditions of HfO2 deposition consisted of 2 s source feeding, 15 s Ar purging, 1.5 s O3 reactant feeding and 15 s Ar purging. Additionally, the PEALD cycle for HfO2 deposition consisted of 3 s source feeding, 25 s Ar purging, 1.5 s O2 reactant feeding, a 1.5 s O2 plasma-on state and 25 s Ar purging. Since direct plasma was used in the experiment, O2 plasma was used for a relatively short time compared with remote plasma. 2 thickness increased linearly as the cycle increased, without a growth delay problem; GPC values were similar to those obtained from using the reflectometer.GPC curves, which changed according to the feeding and purge times, were clearly saturated at the same time with sufficient feeding and purge times. The experimental periods were determined according to these saturation times, as indicated by the arrows in 2 thin film according to the deposition temperatures of the thermal ALD and PEALD processes. The temperature section in which GPC shows constant temperature is called the ALD window and is a problem-free deposition region. The region between 150 and 200 °C is the ALD window in thermal ALD. In PEALD, the ALD window shifted to lower temperatures (80–150 °C) from high temperatures (150–200 °C) because of the high reactivity of O2 plasma; this allowed more stable low-temperature deposition. When thin films were deposited at 80 °C, using thermal ALD, GPC increased and exhibited condensation because of the insufficient thermal energy. Above 250 °C in thermal ALD, because of source decomposition due to high thermal energy, the GPC increased as the temperature increased. Conversely, in PEALD, GPC decreased, and desorption occurred in a manner different from that in thermal ALD [2 thin film and caused desorption [rmal ALD . The reasorption . 2 when deposited by thermal ALD and PEALD [nd PEALD . 2 deposited at 80–250 °C by thermal ALD showed a broad peak at 2θ = 32°, indicating a dominantly amorphous structure HfO2 thin film [2 deposited at 80–150 °C by PEALD also had an amorphous structure, but HfO2 deposited at 250 °C by PEALD contained a polycrystalline structure. This means that the crystallization of HfO2 thin film deposited by PEALD started at a lower temperature than that of HfO2 deposited by thermal ALD [2 deposited at 80 °C, obtained from a period and critical angle of reflectivity oscillation pattern, as measured by XRR. Thickness was measured and found to be approximately 50 nm for both thermal ALD and PEALD samples, and density increased in PEALD from 5.3 to 8.1 g/cm3. This means that the HfO2 thin film deposited by PEALD at a low temperature was denser than that deposited to the same thickness by thermal ALD. 2 deposited according to temperature (80–250 °C) in thermal ALD and PEALD. In the PEALD samples, there was no difference of roughness compared to the thermal ALD samples, and the HfO2 thin film was still flat at 80 °C (0.2 nm). Additionally, no large particles were seen when HfO2 was deposited at a low temperature. As the temperature rose, the roughness of the thin film rapidly increased due to the formation of crystallite [XRD patterns of HfOhin film . Moreovermal ALD . Figure 2 [2−x. The atomic concentration of hafnium in HfO2 thin film deposited at a low temperature by thermal ALD was the lowest at 23.7%, because many defects, such as carbon, nitrogen and hydroxyl groups (−OH), were located in the HfO2 thin films. Conversely, the atomic concentration of hafnium in HfO2 deposited at 80 °C by PEALD was high, at 30.0%, due to the low level of contaminants, similar to the sample deposited at 250 °C by thermal ALD. 2 at 530.03 eV and an additional peak associated with carbon and oxygen at 531.68 eV [2 was similar, except for the thin film deposited at a low temperature by thermal ALD. As mentioned previously for elemental Hf, the presence of many contaminants can lower the atomic concentration of oxygen in thin films.The deconvoluted Hf 4f spectra show the doublet of peaks at binding energy of 18.31 and 19.99 eV, which is associated with HfO2 . Moreove31.68 eV . The C–O31.68 eV ,35. Acco2 thin films. Carbon and nitrogen inside the film act as defects, causing a decrease of density or degradation of properties. At a low temperature, HfO2 deposited through thermal ALD had high ratios of carbon (13.8%) and nitrogen (7.0%) because of incomplete source decomposition. Conversely, in the case of thin films deposited through PEALD, both carbon (3.5%) and nitrogen (2.8%) ratios were low, even at low temperatures. This suggests that, in PEALD, because the precursor was decomposed more by O2 plasma than by O3, the number of inner defects was lower than in thermal ALD at a low temperature. 2 films (50 nm) deposited at 80 °C. 1 + iε2), respectively [2 are associated with the density of HfO2 thin films [2 film deposited by PEALD had less carbon content and a lower O/Hf ratio than that obtained from using thermal ALD, it is expected that the HfO2 film deposited by PEALD has a higher density than that deposited by using thermal ALD. Therefore, the n value of the HfO2 deposited with PEALD was higher than that of HfO2 deposited with thermal ALD in all photon energy ranges. 2 thin films (50 nm) deposited at 80 °C. The band gap of HfO2 thin film in the previous studies were typically between 5.6 and 5.7 eV [2 deposited by thermal ALD was lower at 5.1 eV. When HfO2 was deposited by PEALD, the optical band gap increased to 5.6 eV. If the optical band gap is small, HfO2 thin films cannot function properly as insulators. Optical band gap was plotted by using the Tauc method, as described in Equation (1) [α(= 4πκ/λ) is the absorption coefficient, h is Planck’s constant, ν is photon frequency, A is a proportionality constant and gE is the optical band gap.Using ellipsometry, values of n and k were calculated from the real and imaginary parts of the complex dielectric function  :(1)(αhν)2 (50 nm) deposited at 80 °C on PET substrate. The transmittance of HfO2 deposited by PEALD was high (>84%) in the visible region . The transmittance decreased slightly for PEALD compared to thermal ALD because the HfO2 film deposited by PEALD was denser [2 was deposited at more than 80 °C on PET substrate, PET could not endure the heat, and deformation occurred. s denser . When Hf2 deposited by thermal ALD and PEALD, respectively.2 deposited at 80 °C by PEALD increased from 444.9 to 540.1 nF/cm2, an approximately 21% increase. The dielectric constant  of HfO2 deposited at 80 °C in PEALD (12.6) was higher than those of samples deposited by thermal ALD (8.7). Since the native oxide was not etched on the Si substrate, the κ-value was calculated by considering the native oxide thickness (~3 nm) [2 thin film was calculated from the value of CHfO2, using the following formula, Equation (2):HfO2 and CSiO2 are the capacitance of HfO2 and SiO2, respectively. Cox is the overall capacitance of the MOS capacitor.Capacitance of HfO (~3 nm) ,44. More2 deposited by thermal ALD and PEALD. Leakage currents at negative voltage in PEALD were reduced overall compared to those for thermal ALD. Significantly, at 80 °C, the leakage current decreased from 1.4 × 10−2 A/cm2 to 2.5 × 10−5 A/cm2 at −2 V, which was lower than that of HfO2 deposited at 250 °C by thermal ALD. Because HfO2 films deposited by PEALD at a low temperature were denser, contaminants in the thin films were reduced [There was no significant improvement at temperatures other than 80 °C.  reduced . Becausefb) and fixed charge density (Qf), extracted from the C–V curves in fb of HfO2 deposited by PEALD at 80 °C was lower than those of sample formed by thermal ALD. Qf of HfO2 deposited at 80 °C by PEALD decreased about 90%, from 9.5 × 1012 to 1.0 × 1012, the lowest value in all temperature ranges (80–250 °C). Vfb and Qf showed almost identical values at all temperatures, except 80 °C. Qf has a (+) charge and is distributed at the interface between Si and the HfO2 thin film, which puts the device into a (+) state and makes it to work at higher voltage. This suggests that, at low temperatures of around 80 °C, capacitors using HfO2 deposited by PEALD will have better electrical properties than those using HfO2 deposited by thermal ALD. However, the Qf value of HfO2 deposited by PEALD tended to increase as the temperature increased. This means that, as the deposition temperature rose, the substrate was damaged by increased plasma ion energy during deposition [position ,47.2 thin films deposited at a low temperature (80 °C), using PEALD with O2 plasma, showed improved properties compared to films deposited by using thermal ALD. The ALD window shifted from high temperatures (150–200 °C) to low temperatures (80–150 °C) when using PEALD, allowing stable deposition at a low temperature. HfO2 deposited by low-temperature PEALD showed a flat surface and higher density than films deposited by thermal ALD. Moreover, HfO2 deposited at 80 °C by PEALD showed a decreased presence of contaminants, such as carbon and nitrogen, compared to films deposited by thermal ALD. HfO2 thin films deposited by PEALD showed an increased refractive index, improved optical band gap (5.6 eV) and high transparency of ~84%. Denser and lower-contaminant HfO2 thin films deposited by PEALD contributed to capacitance improvement of about 21%, low leakage current of 2.5 × 10−5 A/cm2 and the lowest fixed charge density (1.0 × 1012). As a result, due to the higher decomposition power of O2 plasma, HfO2 thin films deposited at a low temperature by PEALD showed improved properties compared to those of films deposited by thermal ALD.In this study, HfO


These results provide useful information for the design of improved dimeric aptamers based on further optimization of the linker joining the two consecutive V7t1 sequences.In the optimization process of nucleic acid aptamers, increased affinity and/or activity are generally searched by exploring structural analogues of the lead compound. In many cases, promising results have been obtained by dimerization of the starting aptamer. Here we studied a focused set of covalent dimers of the G-quadruplex (G4) forming anti-Vascular Endothelial Growth Factor (VEGF) V7t1 aptamer with the aim of identifying derivatives with improved properties. In the design of these covalent dimers, connecting linkers of different chemical nature, maintaining the same polarity along the strand or inverting it, have been introduced. These dimeric aptamers have been investigated using several biophysical techniques to disclose the conformational behavior, molecularity and thermal stability of the structures formed in different buffers. This in-depth biophysical characterization revealed the formation of stable G4 structures, however in some cases accompanied by alternative tridimensional arrangements. When tested for their VEGF VEGF121 ,8,9, whi VEGF121 . The recisoforms ,8,9.Using Systematic Evolution of Ligands by EXponential enrichment (SELEX) strategies, various nucleic acid-based aptamers  have bee5′TGT GGG GGT GGA CGG GCC GGG TAGA3′ [165 compared to Vap7 [Aiming at the development of VEGF inhibitors as anticancer agents, Nonaka et al.  identifiG TAGA3′ . This G- to Vap7 , and hig to Vap7 .+-rich solutions [+-rich medium  [The intriguing biological properties showed by V7t1 have stimulated structural studies to better understand the effective conformations of this aptamer and thus design improved analogues. However, due to its marked polymorphism, no definitive structural data could be obtained on this VEGF-targeting aptamer, in any case investigated mainly in Kolutions . In thisH = 7.4) , which b+-containing buffer, showed a behavior dramatically influenced by the sample preparation procedure [165, while the monomeric V7t1 species did not bind the protein under the same conditions [These recent investigations provided new insights on this aptamer, which, in the tested Narocedure . Indeed,rocedure . Notablynditions .165 is a homodimeric protein [Our results definitively proved that the protein showed a marked preference for dimeric G4 species also when the monomolecular species were concomitantly present in solution, reasonably due to multivalency effects, further favored by the fact that VEGF protein ,4.165 for bivalent aptamers was indeed already demonstrated. In fact, Nonaka et al. also identified a mutant of V7t1, called 3R02 (sequence: 5′TGT GGG GGT GGA CTG GGT GGG TAC C3′), by three rounds of an in silico maturation [The great affinity of VEGFturation . Startinturation .5′ATA CCA GTC TAT TCA ATT GGG CCC GTC CGT ATG GTG GGT GTG CTG GCC AGA TAG TAT GTG CAA TCA3′). In this case the best results in terms of Kd values were obtained when no linker was introduced, while the presence of a linker containing from 10 to 20 T residues proved to be detrimental to the protein binding [Furthermore, the same research group prepared a targeted construct for the heparin binding site of the homodimeric VEGF protein made of two identical anti-VEGF DNA-based aptamers , able to recognize the heparin-binding domain [Kd values in the nanomolar range [More recently, Manochehry and coworkers studied the affinity for VEGFt length . Among tg domain  with Kd ar range . The oveThese are not the unique literature examples of aptamer optimization obtained by dimerization of known aptamer sequences. Indeed, in the last decade a number of works described improved aptamers realized by engineering bivalent or multivalent analogues of the selected oligonucleotide, not only for VEGF, but also for thrombin ,25,26,27In addition to therapeutic approaches, an increasing interest has been also devoted to the use of dimeric aptamers in sensing applications. In thrombin targeting, the dimerization approach has been exploited also to achieve a sensitive and highly efficient acoustic-based protein detection, constructing bivalent aptamers by immobilization of a thrombin binding sequence on a gold surface. Then, a partially complementary aptamer sequence was hybridized with the immobilized one .165, we here investigated a focused set of covalent V7t1 dimers in order to identify novel anti-VEGF G-quadruplex-based aptamers with improved performance. In the absence of high-resolution structural data on aptamer/VEGF165 complexes, no rational design can be performed to select optimal linkers connecting the two aptamer sequences, and previous literature examples show that essentially a trial-and-error procedure was followed thus far. Therefore, we designed new dimeric V7t1 constructs introducing either all-thymidine or oligoethylene glycol-based linkers, following typical strategies used in aptamer dimerization and/or oligonucleotide bioconjugation [In this scenario, and particularly starting from our recent findings on the enhanced affinity of dimeric vs. monomeric V7t1 towards VEGFjugation .165 was also investigated by EMSA experiments in order to evaluate possible differences among the studied analogues in terms of affinity towards the protein. Finally, in preliminary in vitro investigations, their cytotoxic activity on human breast adenocarcinoma MCF-7 cells was evaluated.By using several biophysical techniques, i.e., electrophoretic, chromatographic as well as spectroscopic methods, the conformational behavior of these V7t1 dimers was studied in comparison with V7t1, particularly analyzing the influence of the different linkers on aptamer structuring. The ability of these V7t1 dimers to bind VEGF165 than its monomeric species, we here studied covalently linked V7t1 dimers. As main objectives, we aimed at possibly enhancing the V7t1 bioactive form and making the aptamer conformational behavior not strictly dependent on the specific pre-treatment used, e.g., the annealing procedure.Based on our recent findings that V7t1, when not subjected to annealing, tends to form dimeric G-quadruplex structures having higher affinity for VEGFbisV7t1T7, bisV7t1HEG2 and bisV7t1TEG2D, were ad hoc designed introducing proper connecting linkers with similar overall lengths but different chemical features  phosphoramidites  or K+  ions, respectively mimicking the extra- and intracellular environment. It is indeed well known that buffer composition, and particularly ion nature and concentration, can produce dramatic changes in the aptamers secondary structure, finally affecting their stability and activity [In order to evaluate the effects of different saline conditions on the structuring ability of these aptamers, all the biophysical investigations were carried out in two different buffer solutions, containing a high content of Naactivity ,40,41,42+-rich buffer mimes the blood salt composition and is indeed more appropriate for studying the structure-activity relationships of these aptamers in the extracellular media, although the stability of the G4 module is generally decreased in these saline conditions. In addition, the VEGF165 protein was provided folded in this solution in which we had previously performed the key experiment demonstrating the preference of the protein for dimeric V7t1 [The selected Naric V7t1 .Taking into account the different conformational behavior and VEGF binding properties of V7t1 if subjected to annealing or not, all the covalent V7t1 dimers were analyzed in both forms to verify in detail their behavior. Thus, the not-annealed samples (here abbreviated as N.A.) were obtained by simple dissolution of the required oligonucleotide amount in the selected buffer from a concentrated stock solution in water. The annealed samples (here indicated as A.) were in turn prepared by dissolving the oligonucleotides at the desired concentration in the selected saline solution, keeping them at 100 °C for 5 min and then leaving them to slowly cool at room temperature overnight.bisV7t1T7 and to a 50-mer for bisV7t1HEG2 and bisV7t1TEG2D.To further check the purity of the here studied V7t1 dimers, also assessed by HPLC and Matrix-Assisted Laser Desorption/Ionization (MALDI) data provided from the commercial suppliers, a 20% denaturing PAGE analysis was carried out, in comparison with the 25-mer V7t1 . In denaThen, agarose  and acry+ buffer, all the investigated V7t1 dimers showed a detectable difference if in N.A. or A. forms, as in the case of V7t1 [bisV7t1T7 and bisV7t1HEG2 in the N.A. samples migrated as two distinct bands with a marked difference in their electrophoretic mobility , as K+-rich buffer -HPLC recently emerged as an attractive and useful technique to study the different species formed by polymorphic G4s ,47, ofte+ buffer . In addition, a very weak peak (tR ca. 10 min) was present (tR = 11.3 min) suggesting that it may be correlated to other V7t1 dimeric conformations. A slightly different behavior was found for bisV7t1TEG2D  were obtained recording spectra at low and high temperatures  for both N.A. and A. form at 2 μM concentration. Differential spectra—representing the spectral difference between the unfolded and the folded oligonucleotide—have peculiar patterns allowing identifying different nucleic acid secondary structures, and in particular G-quadruplexes ,50,51.+ buffer . The only exception to this trend in the UV thermal profiles at 295 nm was bisV7t1TEG2D, its A. sample showed sigmoidal denaturation pathways with almost superimposable melting/cooling profiles and only limited hysteresis, indicating that under the experimental conditions used (scan rate: 1 °C/min), the related processes were essentially reversible  analysis by exploiting the software recently developed by del Villar-Guerra et al. . By usin+ buffer N.A. V7t1 and N.A. bisV7t1TEG2D almost exclusively formed parallel G4 structures, while additional conformations were evidenced for all the other investigated systems , accompanied by a small percentage of antiparallel G4 topology  and also a little amount of hybrid G4 conformations (ca. 7%) for bisV7t1T7.In terms of tertiary structure parameters , the deconvolution analysis performed on the CD spectra indicated that in the selected HEPES/Na systems . In detabisV7t1T7 and bisV7t1HEG2 reflected the presence of a mixture of parallel and antiparallel G4 conformations, with prevalence of the latter ones (ca. 63–67%), and no contribution of hybrid G4 structures. Conversely, A. bisV7t1TEG2D preferentially adopted a parallel G4 (ca. 75%), accompanied by a ca. 25% hybrid G4.In contrast, SVD analysis performed on the CD spectra of A. V7t1, +-rich medium, the deconvolution analysis showed that the CD spectra of N.A. V7t1 as well as bisV7t1HEG2 and bisV7t1TEG2D in both N.A. and A. form were indicative of the exclusive presence in solution of parallel G4 structures. In turn, beside the prevalent parallel G4 conformation, additional G4 topologies were also predicted for the other samples , along with a low percentage of antiparallel conformations (ca. 16%). Notably, these predictions are in agreement with the different spectral features observed for A. bisV7t1T7, particularly considering the CD signal maximum at 268 nm  and then one specific for proteins (Coomassie) [VEGF-binding properties of covalent V7t1 dimers were studied by EMSA experiments under non-denaturing conditions, which is a sensitive and widely used method to detect protein-nucleic acid interactions . In factomassie) .bisV7t1T7 and bisV7t1HEG2 did not completely disappear . Thus, in parallel experiments, both N.A. and A. samples—dissolved in the selected HEPES/Na+ buffer—were incubated with BSA following the same protocol exploited for VEGF165 binding and then the aptamer/protein mixtures (in 1:1.3 ratio) were analyzed by EMSA assays, including the free oligonucleotides as well as pure BSA as negative controls  assay after 48 h of treatment. The MTT assay results showed a slight cytotoxicity of all the covalent V7t1 dimers starting from 5 µM concentration, producing a ca. 20% cell viability reduction. At the highest concentration tested (20 µM), enhanced antiproliferative effects for all these compounds were found, in line with the bioactivity increase of V7t1, thus demonstrating a dose-dependent effect .5′TCACACACACACACACACACACTT3′) non-containing guanines and representing a useful DNA model system [We also tested a single-stranded oligonucleotide, selected as a negative control, i.e., the 24-mer  is the concentration of a drug required to obtain 50% inhibition of cell proliferation in vitro and measures the effectiveness of a substance in inhibiting a specific biological function. All the analyzed compounds displayed IC50 values slightly higher than 20 µM, except bisV7t1T7, for which an IC50 value of 19 µM was obtained from the experimental results  with the aim to identify better performing analogues with enhanced affinity or activity. A dimeric structure represents the simplest multivalent system and nucleic acid aptamers are especially suitable for the design of these constructs as they can be easily and ad hoc chemically modified.165 compared to the monomeric V7t1 species, in this study we selected a focused set of covalent V7t1 dimers in order to identify novel anti-VEGF aptamers with improved affinity and/or bioactivity. In our expectations, the covalent connection of two V7t1 sequences via suitable linkers should somehow pre-organize its G4 structures, in principle reducing the structural polymorphism of V7t1 and enhancing its effective bioactive form.Starting from our recent and intriguing findings on the ability of V7t1 to form a dimeric G4 structure with preferential binding capability toward VEGFbisV7t1T7, bisV7t1HEG2 and bisV7t1TEG2D, were ad hoc designed with linkers connecting the V7t1 sequences of similar overall lengths, but different chemical nature. In particular, bisV7t1T7 and bisV7t1HEG2 presented two tandem V7t1 sequences, both in the 5′→3′ direction, connected respectively by a T7 and a HEG-based linker. In turn, in bisV7t1TEG2D, a TEG-based spacer joined the 3′ extremities of two V7t1 25-mers providing the final dimer with a 3′-3′ inversion of polarity.In detail, three covalent V7t1 dimers, here named +-rich and a K+-rich buffer, indicated respectively as HEPES/Na+ and TRIS/K+), with and without prior annealing.Different biophysical techniques were used to elucidate the conformational behavior, thermal stability and molecularity of the G4 species formed in two different solutions ;V7t1 (▪5′V7t13′TTTTTTT5′V7t13′);bisV7t1T7 (▪5′V7t13′C24H51O20P35′V7t13′);bisV7t1HEG2 (▪5′V7t13′C25H53N2O24P53′V7t15′).bisV7t1TEG2D , Gel Loading Buffer 4X, 6X Orange DNA Loading Dye and Tris-Borate-EDTA (TBE) 10X were purchased from VWR. Ammonium persulfate (APS) and tetramethylethylenediamine (TEMED) were purchased from Sigma Aldrich. All the oligonucleotides here studied  were purEvidence of the oligonucleotide identity and purity was obtained by MALDI-TOF mass spectrometry and HPLC data, provided by the commercial suppliers. The purity of these oligonucleotides was further confirmed by denaturing 20% PAGE analysis.5′TCACACACACACACACACACACTT3′), used as negative oligonucleotide control in the MTT assays, was obtained as reported in previous works [The 24-mer sequence  was purchased from TwinHelix srl  and prepared according to the manufacturer’s instructions.Recombinant human VEGFBovine serum albumin (BSA) was purchased from Thermo Scientific™ .bisV7t1T7, bisV7t1HEG2 and bisV7t1TEG2D, 260 = 273.522 M−1·cm−1 for V7t1, 619.962 M−1·cm−1 for bisV7t1T7 and 547.044 M−1·cm−1 for both bisV7t1HEG2 and bisV7t1TEG2D, as calculated for the unstacked oligonucleotides, assuming that the inserted non-nucleotide linkers did not provide any significant contribution to the UV absorbance at 260 nm. The UV spectra were recorded in the range 200–320 nm with a medium response, a scanning speed of 100 nm/min and a 2.0 nm bandwidth with the appropriate baseline subtracted. Taking a suitable aliquot from the initial stock solutions in H2O, all the investigated oligonucleotides were then diluted in the selected Na+-  or K+-rich  buffer. In particular, the not-annealed (N.A.) samples were prepared by simple dilution in the selected buffer at r.t. from a concentrated stock solution in pure water. In contrast, the annealed (A.) samples were obtained by heating the appropriate aptamer solution at 100 °C for 5 min and then allowing it to slowly cool to r.t. overnight, to allow their structuring into the thermodynamically most stable conformations [Lyophilized V7t1 and covalent V7t1 dimers , heated at 95 °C for 5 min, then left in contact with ice until subsequent loading. Thereafter, all the samples—supplemented with 6X Orange DNA Loading Dye immediately before loading—were analyzed by electrophoresis on 20% denaturing polyacrylamide gels using 8 M urea in TBE (Tris-Borate-EDTA) 1X as running buffer. The gels were run at r.t., at constant 200 V for 3.5 h, then stained with GelGreen Nucleic Acid Stain for 30 min and finally visualized with a UV transilluminator . The experiment was performed in triplicate.Native PAGE. Not-annealed and slowly annealed samples of V7t1 and its covalent dimers, dissolved at 4 μM concentration in both the selected HEPES/Na+ or TRIS/K+ buffer solutions, were loaded on 10% polyacrylamide gels in TBE 1X as running buffer. All the samples were supplemented with BPB Gel Loading Buffer 4X immediately before loading and then run, under native conditions, at constant 70 V at r.t. for 1.75 or 2 h, as specified. Gels were stained with a GelGreen solution (supplemented with NaCl 0.1 M) for 30 min and finally visualized with a UV transilluminator. Each experiment was performed at least in triplicate.Native agarose gel electrophoresis. Agarose solution in TBE 1X (2% w/v) was mixed with 0.01 % (v/v) of GelGreen stock reagent before agarose solidification (pre-cast protocol). Not-annealed and slowly annealed samples of V7t1 and covalent V7t1 dimers were dissolved at 4 μM concentration in both the selected HEPES/Na+ or TRIS/K+ buffer solutions as well as in the amine-free buffers, i.e., 150 mM NaCl (pH = 7.4), as Na+-rich buffer and 100 mM KCl (pH = 7.3), as K+-rich buffer.w/v in the final solution) immediately before loading them on the gel and then run, under native conditions, at constant 60 V at r.t. for 2 h in TBE 1X as running buffer. The gel was then visualized with a UV transilluminator. Each experiment was performed at least in triplicate.All the samples were supplemented with glycerol  or TRIS/K+  buffer solutions. Both N.A. and A. V7t1 and the covalent V7t1 dimers were investigated at 2 µM concentration in the selected buffer solutions, using V7t1 as control. Each experiment was performed in triplicate. The error associated with the retention time (tR) determination is within ± 5%.SE-HPLC analyses were performed using an Agilent HPLC system, equipped with a UV/vis detector, on a Yarra 3 µm analytical column . The elution was monitored at λ = 254 nm with 0.35 mL·minm (15 °C), at which the aptamer is fully structured, from that obtained at a temperature above the Tm (90 °C), when the oligonucleotide is fully destructured [The UV spectra measurements were performed on a JASCO V-630 UV-vis spectrophotometer equipped with a Peltier Thermostat JASCO ETCS-761, using 1 cm path length cuvette . All the covalent V7t1 dimers were dissolved in the selected buffer solution, so to obtain 2 μM solutions, and analyzed as such or in the annealed form. The thermal difference spectra (TDS) were obtained by subtracting the UV spectrum recorded at a temperature below the Tructured ,50,51. Iructured ,50.m values, where possible, were calculated as the maxima of the first derivative plots of the melting curves (associated error: ±1 °C). Each experiment was performed in duplicate.The absorbance vs. temperature profiles of N.A. and A. covalent V7t1 dimers were recorded following the absorbance changes  in the temperature range 15–90 °C ,68. The + and 220–320 nm for TRIS/K+, data pitch 1 nm, band width 2 nm, response 4 s, scanning speed 100 nm/min, 3 accumulations, with the appropriate subtraction of the background scan with proper blank [CD spectra and CD-monitored melting/annealing curves were recorded on a Jasco J-715 spectropolarimeter equipped with a PTC-348WI Peltier-type temperature control system , Italy), using a quartz cuvette with a path length of 1 cm . CD parameters for spectra recording were the following: spectral window of 235-320 for HEPES/Naer blank .+ and TRIS/K+ buffer solutions. Each experiment was performed in duplicate. The molar ellipticity (θ) (deg·cm2·dmol−1) was calculated from the equation (θ) = θobs/10 × l × C, where θobs is the observed ellipticity (mdeg), C is the oligonucleotide molar concentration and l is the optical path length of the cell (cm). The Tm values were estimated as the maxima of the first derivative plots of the melting/annealing curves and the error associated with the Tm determination was ± 1 °C.All the oligonucleotides were analyzed at 2 μM concentration in the selected buffer solutions. Thermal denaturation/renaturation curves were recorded following the CD signal—at the maximum ellipticity observed for each oligonucleotide system—vs. the temperature (scan rate of 1.0 °C/min) and recording spectra in 5 °C steps, in the 15–90 and 15–95 °C temperature range, respectively for HEPES/Na−1·cm−1) = θ/(32980 × c × l) based on G4 strand concentration, where θ is the CD ellipticity in millidegrees, c is DNA concentration in mol·L−1 and l is the path length in cm. Then, the obtained spectra were analyzed by the software developed by del Villar-Guerra et al. [For the singular value decomposition (SVD) analysis, CD spectra were also normalized to molar circular dichroism, Δε . All the oligonucleotide + protein samples were incubated in HEPES/Na+ buffer for 30 min at 4 °C. Glycerol was added to all the samples to a final concentration of 5% immediately before loading on the gel. Electrophoresis was performed on 7% polyacrylamide gels in TAE (Tris Acetate EDTA) 1X, pH = 7.8, at constant 45 V for 2.3 h [For EMSA experiments, 30 pmol of each oligonucleotide in both N.A. and A. form were incubated with 40 pmol of VEGFor 2.3 h ,61. Gelsor 2.3 h .Eukaryotic cell cultures and cell viability MTT assays. Human breast adenocarcinoma MCF-7 cells were cultured in high-glucose Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% penicillin-streptomycin at 37 °C in the presence of 5% carbon dioxide (CO2). MTT cell viability assays were performed upon seeding MCF-7 cells on 96-well plates at a density of 3 × 103 cells/well in 0.1 mL of complete DMEM. After 24 h, the cells were incubated in the presence of increasing concentrations (0.6–20 μM) of samples and incubated for 48 h. Cell viability was evaluated by adding the MTT reagent -2,5-diphenyltetrazolium bromide) diluted at 0.5 mg/mL in DMEM without red phenol (0.1 mL/well). After 4 h of incubation at 37 °C, the resulting insoluble formazan salts were solubilized in 0.01 N HCl in anhydrous isopropanol and quantified by measuring the absorbance at 570 nm, using an automatic plate reader spectrophotometer . Cell survival was expressed as percentage of viable cells in the presence of the aptamers, with respect to control cells grown in their absence. In all the experiments, controls were performed by supplementing cell cultures with identical volumes of the sole buffer for the same incubation time. Data were obtained from at least three independent experiments.Statistical analysis. Results are presented as the mean ± standard deviation (SD) of at least three independent experiments. Statistical significance was assessed by using either Student’s t-Test or ANOVA test. All samples were compared to the control (in the absence of treatment) and to V7t1-tretaed cells; significant differences were indicated as * P < 0.05, ** P < 0.01 or *** P < 0.001 in the case of Student’s t-Test and as * P < 0.05 in the case of ANOVA test.


This berberine mixed micelle formulation had a mean size of 12 nm and increased the cellular accumulation of digoxin via P-gp inhibition. It also inhibited berberine metabolism in rat intestinal microsomes, without significant cytotoxicity, up to a berberine concentration of 100 μM. Next, we compared the pharmacokinetics of berberine and its major metabolites in rat plasma following the oral administration of the berberine formulation (50 mg/kg) in rats with the oral administration of berberine alone (50 mg/kg). The plasma exposure of berberine was significantly greater in rats administered the berberine formulation compared to rats administered only berberine, which could be attributed to the increased berberine absorption by inhibiting the P-gp-mediated berberine efflux and intestinal berberine metabolism by berberine formulation. In conclusion, we successfully prepared berberine mixed micelle formulation using P85 and tween 80 that has inhibitory potential for P-gp and CYPs  and increased the berberine plasma exposure. Therefore, a mixed micelle formulation strategy with P85 and tween 80 for drugs with high intestinal first-pass effects could be applied to increase the oral absorption and plasma concentrations of the drugs.We aimed to develop a berberine formulation to enhance the intestinal absorption and plasma concentrations of berberine through the inhibition of P-glycoprotein (P-gp)-mediated efflux and the intestinal metabolism of berberine in rats. We used pluronic P85 (P85) and tween 80, which have the potential to inhibit P-gp and cytochrome P450s . A berberine-loaded mixed micelle formulation with ratios of berberine: P85: tween 80 of 1:5:0.5 ( Berberine , an isoqmax and AUC of berberine by 2.9- and 1.9-fold, respectively. A self-microemulsifying drug delivery system using labrasol increased the Cmax and AUC of berberine in rats by 1.63- and 1.54-fold, respectively, compared with those of commercial tablets Digoxin (1.103 TBq/mmol) was purchased from Perkin Elmer Inc. .Berberine chloride, pluronic P85 (P85), tween 80, Hanks’ Balanced Salts Solution (HBSS), cyclosporine A, β-Glucuronidase from Helix pomatia (containing 300KU β-Glucuronidase and 10KU sulfatase/g), sodium acetate, acetaminophen, quinidine, ketoconazole, propranolol, diclofenac, hydroxydiclofenac, and KHCaco-2 cells (passage number 38–42) were purchased from American Type Culture Collection . LLC-PK1-P-gp cells (passage number 22–23), ultrapooled human liver microsomes, reduced nicotinamide adenine dinucleotide phosphate (NADPH) generating solution, and fetal bovine serum were purchased from BD-Corning . Rat intestinal microsomes from SD rats were purchased from XenoTech . All other reagents were of reagent grade.2PO4 (pH 7.4) buffer was added to dissolve the substrate. Aliquots (20 μL) of 0.1 M KH2PO4 (pH 7.4) buffer containing 0.25 mg/mL of liver microsomal protein and aliquots (5 μL) of 0.1 M KH2PO4 (pH 7.4) buffer containing P85 (2 and 20%), tween 80 (2 and 20%), or representative inhibitors of 5 CYPs (20-fold working solution dissolved in 2% DMSO) were added to the substrate solution and preincubated for 5 min. The reaction mixtures were further incubated for 15 min at 37 °C in a thermoshaker, after a NADPH generating system (10 μL) had been added. The final concentration of the 5 CYPs inhibitors were as follows: 0.1 μM of α-Naphthoflavone (CYP1A2), 5 μM of sulfaphenazole (CYP2C9), 5 μM of S-benzylnirvanol (CYP2C19), 5 μM of quinidine (CYP2D6), and 5 μM of ketoconazole (CYP3A), and the final DMSO content was 0.1%. To examine the effects of P85 and tween 80 on the 5 CYP activities, the metabolites of CYP substrates were measured in the presence of P85 (0.1 and 1%) and tween 80 (0.1 and 1%). Briefly, the final concentrations for the 5 CYPs substrates were as follows: 50 μM of phenacetin (CYP1A2), 10 μM of diclofenac (CYP2C9), 100 μM of mephenytoin (CYP2C19), 5 μM of dextromethorphan (CYP2D6), and 5 μM of midazolam (CYP3A). This was prepared by adding 8.5 μL of 10-fold-concentrated methanol solution into reaction tubes, the methanol was dried using a vacuum concentrator, and 65 μL of 0.1 M KHg for 5 min at 4 °C. The supernatants were analyzed using the Shimadzu 8040 Triple Quadrupole liquid chromatography-mass spectrometry (LC-MS/MS) system . The separation was performed on a Kinetex XB-C18 column  with a gradient elution of a mobile phase that consisted of acetonitrile and water with 0.1% formic acid  at a flow rate of 0.2 mL/min. The retention time was 3.18 min for acetaminophen, 5.08 min for hydroxydiclofenac, 4.56 min for hydroxymephenytoin, 4.02 min for dextrorphan, 4.67 min for 1′-hydroxymidazolam, and 5.35 min for propranolol (IS). The mass spectra were recorded by electrospray ionization with a positive mode. The quantification of metabolite formation was carried out using multiple reaction monitoring (MRM) at m/z 152 → 110 for acetaminophen, m/z 312 → 231 for hydroxydiclofenac, m/z 235 → 150 for hydroxymephenytoin, m/z 258 → 201 for dextrorphan, m/z 342 → 203 for 1′-hydroxymidazolam, and m/z 260.0 → 116.0 for propranolol.All the reactions were terminated by the addition of 50 μL of cold acetonitrile containing propranolol ), and the mixtures were vortex-mixed for 5 min, then centrifuged at 16,000× 5 cells/well and grown for 24 h. After washing the cells twice with pre-warmed HBSS, the cellular accumulation of digoxin was initiated by the addition of 0.5 mL of HBSS containing 0.1 μM of [3H]digoxin and P85 (0.1% and 1%), tween 80 (0.1% and 1%), or cyclosporine A . Cyclosporine A stock solution in DMSO (10 mM) was 400-fold diluted in 0.5 mL of pre-warmed HBSS to make a final concentration of 25 μM of cyclosporine A. The reaction mixture was incubated for 30 min at 37 °C. After 30 min of incubation, the cells were washed three times with 1 mL of ice-cold HBSS and aspirated. The cells were then lyzed with 0.1 mL of 10% SDS solution, mixed with 0.5 mL Optiphase cocktail solution , and stabilized for overnight incubation. The radioactivity of [3H]digoxin in the cell lysate was measured using a Microbeta 2 liquid scintillation counter .LLC-PK1-P-gp cells were seeded onto 24-well plates  at a density of 1 × 10g for 20 min and filtered through a nylon membrane filter (0.45 μm) [An excess amount of berberine chloride (10 mg) with various combinations of the berberine: P85: tween 80 mixtures (from 1:1:0.1 to 1:10:2) were incubated in 2 mL of distilled water for 4 h at 37 °C in a thermoshaker (300 rpm). Undissolved berberine powder was removed by centrifugation at 16,000× 0.45 μm) . The berg for 20 min. The size distribution of the mixed micelle formulations of berberine: P85: tween 80 (from 1:1:0.1 to 1:10:1) was determined using Delsa Max Pro Light Scattering Analyzer .Berberine-loaded P85 and tween 80 mixed micelles were prepared using the thin-film hydration method . Briefly5 cells/well, and cultured for 24 h at 37 °C in a 5% CO2 incubator. The cells were exposed to 100 μL of culture medium containing various mixed micelle formulations of berberine: P85: tween 80 (from 1:5:0.1 to 1:10:0.5). As a control group, berberine was treated at concentrations of 0, 1, 5, 10, 50, 100, 500, and 1000 μM. After 24 h of treatment, 20 μL of CellTiter 96 aqueous one solution from a cell proliferation assay kit  was added to the culture medium and incubated for 1 h. The absorbance rate of the reaction mixture was measured at 492 nm using an Infiniti M200Pro microplate reader .Caco-2 cells were seeded onto 96-well plates  at a density of 1 × 10g for 20 min.Berberine-loaded P85 and tween 80 mixed micelles with the ratio of 1:5: 0.5 were prepared using the thin-film hydration method . Briefly5 cells/well, and cultured for 24 h at 37 °C in a 5% CO2 incubator. The cells were grown for 21 days and the culture medium was changed every 2 days. The integrity of the cell monolayers was evaluated prior to the transport experiments by measuring the transepithelial electrical resistance (TEER). Values in the range of 300–650 Ω·cm2 were used in the transport experiment [Caco-2 cells were seeded onto filter inserts from 12-well transwell plates  at a density of 5 × 10periment .To characterize the absorption transport of berberine in Caco-2 cells, the apical to basal (A to B) and basal to apical (B to A) transport of berberine was measured. To measure the A to B transport of berberine, 0.5 mL of HBSS (pH 7.4) medium containing berberine (10 μM) in the presence or absence of cyclosporine A (25 μM) was added to the apical side, and 1.5 mL of HBSS medium without berberine was added to the basal side of the insert. The insert was then transferred to a well containing fresh HBSS medium every 15 min for 1 h. To measure the basal to apical (B to A) transport, 1.5 mL of HBSS medium containing berberine (10 μM) in the presence or absence of cyclosporine A (25 μM) was added to the basal side, and 0.5 mL of HBSS medium without berberine was added to the apical side. The transport medium on the apical side was replaced with 0.4 mL of fresh incubation medium every 15 min for 1 h. A 100 μL aliquot of each sample was stored at −80 °C for the analysis of berberine.The concentration-dependent transport of berberine in Caco-2 cells was measured in a concentration range of 1–100 μM using a same procedure described above. A 100 μL aliquot of each sample was stored at −80 °C for the analysis of berberine.To measure the A to B and B to A permeability of berberine and berberine formulation, 0.5 mL of HBSS supplemented with 10 mM of hydroxyethyl piperazine ethane sulfonicacid (pH 7.4) medium containing berberine  or berberine formulation  was added to the apical side, and 1.5 mL of HBSS medium without berberine was added to the basal side of the insert. The insert was then transferred to a well containing fresh HBSS medium every 15 min for 1 h. To measure the B to A transport, 1.5 mL of HBSS medium containing berberine  or berberine formulation  was added to the basal side, and 0.5 mL of HBSS medium without berberine was added to the apical side. The transport medium on the apical side was replaced with 0.4 mL of fresh incubation medium every 15 min for 1 h. A 100 μL aliquot of each sample was stored at −80 °C for the analysis of berberine. The TEER values were monitored before and after the transport experiments with berberine formulation using an epithelial TEER meter .g for 10 min, the supernatant was injected directly into the LC-MS/MS system.A 100 μL aliquot of each sample was added to a 100 μL aliquot of ice-cold methanol containing 10 ng/mL of propranolol (IS). After vortexing and centrifuging the samples at 16,000× 2PO4 (pH 7.4) buffer that contained rat intestinal microsomes (0.25 mg/mL) and a NADPH generating system at 37 °C in a thermoshaker. All the reactions were terminated by the addition of 50 μL of ice-cold methanol containing 10 ng/mL of propranolol (IS), and were centrifuged at 16,000× g for 5 min at 4 °C. The supernatants were then analyzed via LC-MS/MS analysis.To examine the formation of berberine metabolites, berberine (10 μM) was incubated for up to 1 h in 0.1 M of KH2PO4 buffer  contained 0.25 mg/mL of protein from rat intestinal microsomes. The berberine or berberine formulations were preincubated for 5 min and the reaction mixtures were then incubated for another 15 min at 37 °C in a thermoshaker after a NADPH generating system (10 μL) had been added. All the reactions were terminated by the addition of 50 μL of ice-cold methanol containing 10 ng/mL of propranolol (IS), and were centrifuged at 16,000× g for 5 min at 4 °C. The supernatants were then analyzed via LC-MS/MS analysis.To examine the effects of berberine formulation on the metabolism of berberine in intestinal microsomes, the formations of thalifendine and berberrubine, two major metabolites of berberine , were meAll the animal procedures were approved by the animal care and use committee of Kyungpook National University . Male Sprague–Dawley rats weighing 250–270 g (8 weeks old) were purchased from Samtako Bio Korea . When the rats arrived, they were housed with a 12 h light/dark cycle. Food and water were supplied ad libitum for 1 week prior to the animal experiment.g for 1 min at 4 °C. Bile and urine samples were collected every 4 h for 12 h. After 12 h, the bile and urine samples were pooled and weighed. Aliquots (50 µL each) of plasma, urine, and bile samples were stored at −80 °C for the analysis of berberine and its metabolites.The rats were fasted for 12 h before the oral administration of the berberine or berberine formulation. The femoral vein, femoral arteries, and bile ducts of the rats were cannulated with polyethylene tubes  under anesthesia with isoflurane (30 mmol/kg) on the warming surgery table , and the rats were remained on the warming table to maintain body temperature during the pharmacokinetic study. We suspended berberine chloride in a 0.5% carboxymethylcelluose suspension (50 mg/8 mL/kg of berberine), and the berberine formulation was hydrated in distilled water (50 mg/8 mL/kg of berberine). The prepared berberine suspension or berberine formulation was administered to rats via oral gavage. Blood samples (approximately 150 µL) were taken via the femoral artery at 0, 0.25, 0.5, 1, 1.5, 2, 4, 8, and 12 h after the oral administration, and pre-warmed saline (approximately 150 µL) was injected into the femoral vein to compensate the blood loss. The blood samples were centrifuged at 10,000× g for 5 min at 4 °C. An aliquot (10 µL) of the supernatant was injected into the LC–MS/MS system.Plasma, urine, and bile samples (50 µL) were added to 150 µL of methanol containing propranolol (10 ng/mL), vortex-mixed for 15 min, and centrifuged at 10,000× Agilent 6430 Triple Quad LC/MS system  coupled with an Agilent 1260 series HPLC system was used for the analysis of berberine and its metabolites using the modified method of Kwon et al. [g for 5 min at 4 °C. An aliquot (10 µL) of the supernatant was injected into the LC–MS/MS system.To identify the berberine metabolites in in vivo samples, 50 µL of bile samples were incubated with 100 µL of sodium acetate buffer  with or without 900 U of β-Glucuronidase and 30 U of sulfatase for 16 h at 300 rpm and 37 °C. The reaction was terminated by adding 150 µL of ice-cold methanol containing propranolol (10 ng/mL), vortex-mixed for 5 min, and centrifuged at 10,000× m/z 498.1 → 322.1 for thalifendine-glucuronide and berberrubine-glucuronide, m/z 402.0 → 322.1 for thalifendine-sulfate, m/z 336.1 → 320.0 for berberine, m/z 322.1 → 307.1 for thalifendine and berberrubine, and m/z 260.0 → 116.0 for propranolol. Calibration was applied on a standard curve in the range of 0.1–200 ng/mL of berberine, thalifendine, and berberrubine, respectively. The thalifendine-glucuronide, berberrubine-glucuronide, and thalifendine-sulfate were relatively quantitated using berberine’s standard curve, since the authentic standard compounds were not available. The separation was performed on a Synergy polar RP column  using a gradient elution of the mobile phase that consisted of methanol and water with 0.1% formic acid at a flow rate of 0.2 mL/min. The composition of the mobile phase was as follows: 55% methanol for 0–2.5 min, 70% methanol for 3–5 min, 55% methanol for 5.3–12 min. The retention time was 3.01 min and 3.53 min for thalifendine-glucuronide and berberrubine-glucuronide, 6.30 min for thalifendine-sulfate, 7.79 min for berberine, 5.47 min for thalifendine, 6.38 min for berberrubine, and 2.53 min for propranolol. Mass spectra were recorded by electrospray ionization with a positive mode. Quantification was carried out using MRM at The pharmacokinetic parameters were calculated by non-compartmental analysis using the WinNonlin Version 5.1 software . Statistical analysis was performed using SPSS for Windows .We investigated the inhibitory effect of P85 and tween 80 on five major CYP enzyme activities in human liver microsomes and on P-gp efflux function using LLC-PK1-P-gp cells. For the system validation, representative inhibitors for the five CYPs and P-gp were used as a positive control (PC). The CYP1A2-mediated phenacetin O-deethylase, CYP2C9-catalyzed diclofenac 4-hydroxylase, CYP2C19-mediated mephenytoin 4-hydroxylase, CYP2D6-mediated dextromethorphan demethylase, and CYP3A-mediated midazolam 1-hydroxylase activities in ultra-pooled human liver microsomes were inhibited by the use of the PC. The use of P85 (0.1% and 1%) and tween 80 (0.1% and 1%) partially inhibited the activities of CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 A–E. Amonw/w). The addition of tween 80 with a ratio of 0.1 and 0.5 (w/w/w) in the mixture of berberine: P85 from 1:1 to 1:10 greatly increased the solubility of berberine. However, the addition of tween 80 with a ratio over 1 gradually increased the solubility of berberine  of 933 μM. Berberine was also known for its anticancer effect in various cancer cells, which was mediated by the induction of apoptosis by the upregulation of p53 and the downregulation of Bcl-2, etc. [50 value in Caco-2 cells were comparable with its IC50 value in human colon cancer HCT-8 cells (470 μM) [The cytotoxicity of P85 and tween 80 was investigated using the cell viability test in Caco-2 cells. Berberine itself did not induce cell toxicity at a dose of up to 100 μM, but the cell viability decreased with the increasing concentrations of berberine with the half maximal inhibitory concentration  . 50 values of berberine formulations with that of berberine itself  and investigated the effect of this berberine formulation on the P-gp-mediated berberine efflux and intestinal metabolism of berberine using Caco-2 cell monolayers and rat intestinal microsomes, respectively. Based on the solubility, size distribution, and cell viability results, we selected a final berberine formulation containing a berberine:P85:tween 80 ratio of 1:5:0.5  [Berberine has long been used against diarrhea and microbial activities in the gut, and has had a good clinical efficacy . Berbering∙h/mL) .w/w/w) using a thin-layer film hydration method. This berberine formulation increased the berberine solubility by 800% and had a mean particle size of 12 nm, without significant cytotoxicity for up to 100 μM  decreased from 7.54 to 1.05 C, suggescompared , the sumcompared . We founCollectively, our berberine-loaded mixed micelle formulation could increase berberine plasma concentrations by increasing berberine intestinal absorption through the use of pharmaceutical excipients that inhibit P-gp function and berberine metabolism. This could contribute to the beneficial effect of berberine therapy in regard to antidiabetic effects and antihyperlipidemic actions by increasing the plasma exposure of berberine.