The properties of epoxy can be adapted depending on the selection of bio-based diluents and crosslinkers to balance the appropriate viscosity for processing and the resulting mechanical properties for coating applications. This work presents a comprehensive study on the structure–property relationships for epoxy coatings with various diluents of mono-, di-, and bio-based trifunctional glycidyl ethers or bio-based epoxidized soybean oil added in appropriate concentration ranges, in combination with a traditional fossil-based amine or bio-based phenalkamine crosslinker. The viscosity of epoxy resins was already reduced for diluents with simple linear molecular configurations at low concentrations, while higher concentrations of more complex multifunctional diluents were needed for a similar viscosity reduction. The curing kinetics were evaluated through the fitting of data from differential scanning calorimetry to an Arrhenius equation, yielding the lowest activation energies for difunctional diluents in parallel with a balance between viscosity and reactivity. While the variations in curing kinetics with a change in diluent were minor, the phenalkamine crosslinkers resulted in a stronger decrease in activation energy. For cured epoxy resins, the glass transition temperature was determined as an intrinsic parameter that was further related to the mechanical coating performance. Considerable effects of the diluents on coating properties were investigated, mostly showing a reduction in abrasive wear for trifunctional diluents in parallel with the variations in hardness and ductility. The high hydrophobicity for coatings with diluents remained after wear and provided good protection. In conclusion, the coating performance could be related to the intrinsic mechanical properties independently of the fossil- or bio-based origin of diluents and crosslinkers, while additional lubricating properties are presented for vegetable oil diluents. Epoxy resins are frequently used in various sectors of industrial construction and the manufacturing of adhesives, coatings and paints, composites, primers and sealants, flooring, and tooling . Due to p-tert-butylphenyl glycidyl ether were identified [The high reactivity of the epoxide ring in combination with crosslinking agents results in the formation of a three-dimensional polymer network with properties that depend on the molecular structure of the reactants and/or co-reactants. The structure provides thermoset properties, where the mechanical properties depend on the crosslinking density and the flexibility of the polymer segments between the anchoring points. The visco-elastic properties can be controlled by a combination of curing conditions   and the entified . Therefoentified , vegetabentified , cardanoentified , or glycentified . The latentified , resultiThe reactive diluents participate in the crosslinking process of epoxy resins and are covalently bonded into the polymer network; hence, they do not migrate, in contrast with traditional solvents or non-reactive diluents. The reactive diluents contain reactive epoxy groups organized in aliphatic or aromatic glycidyl ethers of alcohols and alkylphenols, while the non-reactive diluents typically include aromatic hydrocarbons, such as toluene, xylene, phthalates, styrene, or phenolic compounds. Although the non-reactive diluents are expected to not decrease the reactivity of the epoxy and can be added in relatively large concentrations, it was observed that the mechanical properties of cured epoxy systems were reduced after adding toluene . AlternaIn this work, epoxy coatings were formulated with a range of diluents, including those with various origins, functionalities, and suitable concentrations, to evaluate their effects on the processing and mechanical performance of coatings, while their compatibility with a fossil amine (FA) and a bio-based phenalkamine (PK) crosslinker was evaluated. Complementary to existing knowledge summarized before, the effects of diluents and crosslinker combinations on the properties of epoxy coatings are not uniquely known and depend on a balance between the reduction in viscosity, softening, and lubrication. Therefore, a comprehensive study is presented where the coating performance was related to the intrinsic mechanical properties for both fossil- or bio-based origin of diluents and crosslinkers. The present results indicate good opportunities for a transition of fossil-based into bio-based epoxy coating formulations with controllable performance.The bisphenol A diglycidyl ether (DGEBA) epoxy resin was purchased from Resion Resin Technology  with the commercial name EP101. The reactive diluents with different functionalities were purchased from Merck , including a grade of fossil-based monofunctional glydicyl ether (MGE), fossil-based di-functional glycidyl ether (DGE), and a bio-based tri-functional glycidyl ether (TGE) that was synthesized from glycerol. As an alternative reactive diluent, the epoxidized soybean oil (ESBO) was obtained from Merck , with a molecular weight of 950 g/mol and epoxide equivalent of 230 g/mol. The latter indicates an average of 4.2 oxirane groups per molecule, therefore representing a multifunctional diluent with higher functionality than the previous diluents. The chemical structures and epoxy equivalent weight (EEW) for the epoxy resin and diluents are detailed in m-phenylene bis(methylamine) (10 to 30 wt.-%), with the trade name EP113 . The bio-based phenalkamine (PK) was obtained after a reaction between cardanol and 1,2-ethylenediamine and is commercially available under the trade name H811 . The chemical structures and amine hydrogen equivalent weight (AHEW) for crosslinkers are detailed in Two types of crosslinkers with optimized composition for epoxy coatings were used with either fossil-based or bio-based origin. The fossil amine (FA) was a commercially available fast crosslinking cycloaliphatic amine containing a mixture of 3-aminomethyl-3,5,5-trimethylcyclohexylamine (30 to 50 wt.-%) and mixEEW of the epoxy resin mixtures  was calculated according to Formula (1) and the values are summarized in DGEBAw) and diluent (diluentw) were determined on an analytical balance with 0.001 g accuracy. The weight of the added crosslinker (PK/FAw) per 10 g epoxy mixture was calculated from a 1:1 stoichiometric ratio between functional epoxy groups and amine crosslinker resulting from the respective mixEEW and AHEW values, according to Formula (2).The coating formulations were prepared by mixing the epoxy resin with different concentrations of MGE, DGE, TGE , or ESBO diluent , as well as a stoichiometric ratio of FA or PK crosslinker. The coating compositions were made by first stirring the epoxy resin with the diluent and subsequently adding the crosslinker after 10 min stirring time, followed by a 5 min stirring time in the presence of the crosslinker. A combination of diluents was applied for the resin mixtures of DGEBA + 30 wt.-% ESBO with TGE diluent . The The coatings were deposited via blade coating onto softwood beech substrates (10 cm × 10 cm × 5 cm), which were primarily planed and dried in a hot air oven overnight at 60 °C. The use of a constant blade speed at 5 mm/s resulted in a wet thickness of 70 µm and was verified with a coating thickness gauge indicating a 68 ± 2 µm dry thickness. The coatings were fully cured under controlled laboratory conditions of 25 °C and 60% relative humidity for one month before further testing. No thermal curing was applied in agreement with practical on-site application in the wood-coating industry. Similarly, films of the same thickness and compositions  were obtained after deposition and peeling off the coating from a non-sticky aluminum support.gT. For the liquid coatings, a sample size of 4 mg was heated in hermetically sealed aluminum pans between 10 and 210 °C at 5 °C/min under a nitrogen atmosphere. For the solid samples, a sample size of 7 mg was heated during two heating cycles between 20 and 110 °C at 10 °C/min under a nitrogen flow. The thermal characteristics were determined from the second heating cycle.The viscosity measurements were performed according to ADTM D2196 using a DV-III Ultra viscosimeter with spindle SC4-27RD  under a constant rotational shear rate of 100 rpm over a time of 1 min at a controlled temperature of 25 °C. The DSC measurements were performed on a DSC 3+  on a liquid sample to follow the curing reaction as a function of temperature, or on a solid cured sample to determine the glass transition temperature The abrasive wear of coatings was evaluated via Taber testing according to ASTM D4060-10 using a circular rotary platform  with calibrated CS-10 abrasive wheels loaded under a 250 g or 500 g load and 72 rpm rotational speed. The tests ran over 1000 cycles and the weight loss was determined on an analytical balance with an accuracy of 0.001 g . The microhardness was measured according to ASTM D2240  with a h2).The tensile testing of films was done on a universal testing machine  and impact testing was done on an Izod impact tester measuring the absorbed energy according to ASTM D256. The mechanical tests were repeated on 10 samples and reported as an average value for stress at break σ (MPa), strain at break ε (%), and impact strength  before adding crosslinker are presented in During the measurement, the viscosity stabilized after about 10 s and remained constant during the full recording time of 60 s, as no reaction between the epoxy resin and diluents happened in the absence of the crosslinker, while good pre-mixing of the resin and diluent had been achieved. The native epoxy resin inherently had the highest viscosity of 15,000 mPa·s, in agreement with the supplier’s data sheets, which posed problems in further application. The ability to reduce the viscosity in the presence of reactive diluents is demonstrated depending on the functionality and concentration of the diluent, where values can be reduced below 1000 mPa·s for practical coating application. The presence of reactive diluents favorably reduces and stabilizes the viscosity of epoxy resin such that it becomes systematically lower at higher concentrations. The viscosity of epoxy resin with an MGE diluent was the lowest, in agreement with the high flexibility of the linear polymer chain. It is known that monofunctional diluents are most efficient at reducing the viscosity of paints and coatings, as demonstrated for alkyds , but theThe crosslinking of epoxy resin with different reactive diluents was followed by the exothermal peak during DSC analysis, after adding stoichiometric amounts of FA or PK crosslinker. Although reaction kinetics are traditionally studied using isothermal DSC analysis, the crosslinking of epoxy coatings was explicitly monitored in this study using non-isothermal data, as the optimum temperature ranges for FA and PK crosslinker agents may be different and can be more efficiently detected. A detail of the exothermal peak during heating of liquid coating samples between 20 and 200 °C is illustrated for coating compositions containing epoxy with 7.5 wt.-% MGE, TGE, and DGE a, or varR), was lower in parallel with a slower crosslinking reaction that allowed for better control of heat dissipation.Depending on the reactive diluent, the crosslinking reaction was postponed for the epoxy coatings with MGE: although possessing the lowest viscosity and high molecular mobility, the low functionality limited the availability of the epoxy rings for ring-opening reactions. In parallel, the crosslinking of epoxy coatings with TGE diluent shifted to lower temperatures due to the higher reactivity of the trifunctional epoxy molecules, but the intensity of the curing reaction was lower in parallel with the higher viscosity and more difficult diffusion processes of reactive moieties. The DSC characteristics for diluents with FA crosslinker were evaluated at 10 °C/min and 20 °C/min curing rates, where the exothermal peak evidently became stronger and shifted toward higher temperatures at the higher heating rates, while the curing properties for different epoxy coating compositions were repeatable. For the ESBO diluent, the crosslinking shifted toward lower temperatures for concentrations up to 30 wt.-%, while the highest concentrations of 40 wt.-% did not yield favorable crosslinking. The latter may be explained through the steric hindrance caused by the long fatty acid molecules. The differences between FA and PK crosslinkers were clear for all epoxy compositions with a shift toward lower temperatures for PK: this is in line with the higher reactivity of PK at low temperatures, where the crosslinking may partially start at 30 °C. The higher reactivity of PK crosslinkers was noticed before , which cT) relative to the total exothermal heat during the entire curing reaction (ΔHR), i.e., α = ΔHT/ΔHR, where both values of heat enthalpy were calculated via the integration of the heat flow curve over the appropriate temperature range. The reaction rate da/dt can be expressed as a generalized Equation (3) [th-order curing kinetics with a specific reaction rate constant k(T) at a given temperature T. These parameters can be calculated from experimental data according to known procedures in the literature based on the kinetic modeling of DSC data after fitting to a model that assumes an Arrhenius-type expression of k(T) following Equation (4), with gas constant R = 8.314 J/(mol K) [R), peak temperature (pT), activation energy (aE), kinetic factor A, and reaction order n.The quantitative data were obtained after calculating the conversion degree α as a function of curing temperature b,d and ftion (3) , assumin/(mol K) . The paraE represents an input value required as a barrier to initiate the crosslinking. A higher aE would generally postpone the crosslinking reaction; however, the reaction rate after initiation also strongly depends on the molecular structure of the reactants. For regular epoxy/amine systems, a value for activation energy aE = 50 to 70 kJ/mol is reported [aE enables an easy crosslinking toward a high degree of conversion. Depending on selected diluents, the aE decreased and A increased compared with a pure epoxy coating due to the lower viscosity and reactivity of the diluent. It can be reasoned that crosslinking was easier for the more flexible polymer chains and accessible functional groups with, consequently, a lower aE, but this had to be balanced against the mobility of reactive groups and the viscosity of the medium. For the DGE diluent, the linear structure and accessibility of terminal epoxy groups favorably increased the reactivity of the system, resulting in a low aE. For the MGE diluent, the high aE may relate to the monofunctional epoxy groups and more frequent termination of the crosslinking reaction. It was indeed confirmed for other types of difunctional diluents that the activation energy for the curing process reduced most favorably [aE may have resulted from the formation of a dense three-dimensional molecular network from the beginning of the curing reaction, which may have hindered the reaction kinetics. Other studies showed the fastest curing and coating drying times for epoxy resins with trifunctional diluents [aE and A for PK crosslinkers were mainly due to the high reactivity and intrinsic molecular properties of the PK crosslinker versus FA crosslinker. In particular, the aE for PK-epoxy with diluents was within the lower range or incidentally slightly below the ones reported for traditional epoxy/amine curing. The crosslinking kinetics in the presence of ESBO diluent are more complex and were also documented before [aE and low kinetic factors were reported in parallel with the relatively high viscosity and more complex entanglements of the long fatty acid chains hindering the diffusion of reactive moieties. Irrespective of the used FA or PK crosslinkers, the kinetics evidently followed first-order kinetics with n = 0.9 to 1.0, as expected for amine crosslinking.The activation energy reported , which ireported . Dependiavorably . For thediluents . Alternadiluents . Howeverd before , where tgT values for fully cured coatings were determined from the second DSC heating scan, where some exemplary graphs  and high load (500 g) and is represented for coatings of FA-epoxy a and PK-The morphology of the wear tracks (after wear at the highest load) was evaluated through laser interferometric microscopy and three-dimensional surface topography , illustrgT are demonstrated  and strain at break (ε) were determined from standard tensile testing for FA-epoxy a and PK-In summary, the relationships between the intrinsic mechanical properties and tribological performance of the epoxy coatings are presented in gT values. The exposure of free oil molecules at the surface after wear evidently provided the highest hydrophobicity. For the other diluents, the hydrophobicity increased with higher concentration and higher functionality of the diluent with consistent trends for both crosslinker types and constantly some lower contact angles on PK-epoxy relative to FA-epoxy. Some higher contact angles on worn coatings of PK-epoxy compared with FA-epoxy were observed due to both chemical and topographical changes to the coating surface after wear. In particular, the polarity of C-O bonds or long aliphatic tails in the polymer chain of diluents was expressed in the high coating hydrophobicity.The hydrophobicity of epoxy coatings was characterized through static water contact angles , which wThe surface gloss for epoxy coatings with various diluents was compared after crosslinking with FA and PK . RelativThe use of reactive diluents with different functionalities and bio-based or fossil-based origins in combination with a fossil-based and bio-based crosslinker provides a toolbox to optimize the processing conditions and performances of epoxy coatings. The viscosity reduction was related to a combination of flexibility and functionality of the reactive diluent, where lower concentrations of the glycidyl ether diluents were more efficient compared with the higher concentrations of the vegetable oil diluents. The reduction in viscosity for mono- and di-functional diluent was within comparable ranges for fluent coating processing, where a more branched structure of the bio-based trifunctional diluent evidently increased the viscosity due to the enhanced ability for molecular entanglements. The curing kinetics for epoxy with difunctional diluent showed the lowest activation energy and highest reaction rates due to a favorable combination of accessibility and reactivity of the epoxy groups, while the kinetics were slowed down for a bio-based trifunctional diluent in parallel with the higher viscosity of the reaction mixture. All types of diluents induced first-order reaction kinetics, irrespective of their source. Although the variations in reaction kinetics for different diluents were minor, the use of a bio-based phenalkamine crosslinker had more of an effect on accelerating the crosslinking, mainly at lower temperatures.The mechanical coating properties were strongly related to the glass transition temperature of the respective epoxy formulations, which was lowered in the presence of the diluents and phenalkamine crosslinkers. The latter indeed introduced more ductile properties reflected in their mechanical properties. The reduction in abrasive wear for trifunctional diluents and phenalkamine crosslinkers was the highest in parallel with the high microhardness and scratch resistance. Any further reduction in abrasive wear in the presence of vegetable oil diluents was due to additional lubrication of the diluents rather than related to the intrinsic mechanical parameters. In particular, the long-term protection of the coating was retained by high hydrophobicity, which was evidently the highest in the presence of vegetable oil diluents, but also remained high for the trifunctional bio-based diluent.Overall, good relationships could be drawn between the intrinsic mechanical properties and coating properties for glycidyl ether diluents of different functionalities and two types of amine crosslinkers, independent of their bio-based or fossil-based origin. As such, the differences in the intrinsic chemical structure of the latter allow for tuning the coating performance and select new formulations with higher bio-based content and appropriate properties.


Prime objective of the current research was to develop a stable nimesulide emulgel with the help of arabinoxylan, a natural gelling agent extracted from Plantago ovata. The response surface methodology was used by a Design Expert 10 software to formulate and optimize the emulgel. The experimental design approach evaluated the impact of independent and dependent variables. Independent variables were different concentrations of arabinoxylan, span 80 and tween 20, whereas, dependent variables were viscosity, pH, and content uniformity. FTIR demonstrated the compatibility of nimesulide with the excipients. Stability study indicated no phase separation and no change in pH for formulation F1, F3 and F4. The negative values of zeta potential revealed the excellent stability of emulgel. Viscosity, spreadability and extrudability values were in desired range. Ex-vivo permeation study illustrated 86%, 55% and 66% release of the drug over a period of 24 h from the formulations F1, F3 and F4, respectively. Analgesic effect of the optimized emulgel was significantly higher in test group as compared to control and did not produce any sort of irritation. Therefore, it can be concluded that the newly developed emulgel based on arabinoxylan, as gelling agent, appear to be an effective drug delivery system. HoweverEmulgel is an innovative and promising topical drug delivery system that offers various advantages over other conventional topical formulations. It is characterized by its greaseless nature, easy spreadability, and soothing properties, making it safe, biocompatible, and visually appealing. Compared to other topical drug delivery systems, emulgel provide a superior and effective means of delivering drugs. The formulation of emulgel involves the combination of an oil phase, gelling agent, aqueous phase, and emulsifying agent, which significantly influences the drug release profile from the dosage form . The uniIn recent times, emulgel have gained considerable attention for the delivery of a wide range of drugs with diverse pharmacological effects. They have been successfully utilized for delivering hydrophilic and hydrophobic drugs, including non-steroidal anti-inflammatory drugs, anti-emetics, anti-fungal agents, antiviral drugs, antibacterial, and local anesthetics . The verNatural polymers and their derivatives have gained significant attention in drug delivery research due to their biodegradability, cost-effectiveness, and potential for chemical modification –14. ThesOur research work focused on utilizing the gelling properties of arabinoxylan (AX) to develop an efficient emulgel for topical drug delivery. We incorporated nimesulide, a model drug effective for pain associated with osteoarthritis and backache, into the emulgel formulation. Through comprehensive evaluations, including pH, viscosity, spreadability, swelling index, content uniformity, ex-vivo permeation study, as well as in vivo skin irritation and analgesic activity, we investigated the performance and potential therapeutic applications of the nimesulide emulgel. The developed emulgel formulation combines the advantages of both an emulsion and a gel, offering improved drug solubility, enhanced drug permeation, and extended residence time on the skin. This emulgel formulation holds promise for topical application, delivering targeted relief from pain and inflammation to the affected area. Hence, present study provides unique insights into the formulation development, optimization, and performance evaluation of the emulgel containing nimesulide.The inclusion of two emulsifying agents, Span 80 and Tween 20, in our emulgel formulation is based on the rationale of harnessing their synergistic effects to achieve stable emulsions and desirable rheological properties. Span 80, a lipophilic emulsifier, stabilizes oil-in-water emulsions and ensures uniform distribution of the oil phase. Tween 20, a hydrophilic emulsifier, promotes oil-in-water emulsion formation and provides a uniform dispersion of the water phase. By combining these emulsifiers, we optimize the emulsification process, enhance emulsion stability and improve product appearance. The use of both emulsifying agents contributes to the overall effectiveness and stability of our emulgel formulation.Plantago ovata) that was purchased from the local market of Sargodha . Acetic acid, span80, tween 20, and sodium dihydrogen phosphate and sodium hydroxide were purchased from Sigma Aldrich, Germany. Propylene glycol was purchased from Anala R. England. Ethanol was purchased from Riedel-de Haen, Germany. Liquid paraffin was purchased from Merck Millipore and methylparaben was purchased from Sigma Aldrich, USA. Distilled water was taken from the Industrial plant of the University of Sargodha . All other chemicals used in this study were of analytical grade.Nimesulide was received as a gift sample from Unixo Labs pharmaceuticals. AX was extracted from , medium (0), and higher (+1). For AX and span 80, concentration -1 was assigned to designate minor level concentration of 1%, for medium-level 0 was used for 1.5% and maximum concentration +1 was used for 2.0%. Similarly, for the concentration of tween 20, -1 indicates a lower concentration 0.5%, 0 indicates a medium concentration 0.75% and +1 indicates a higher concentration of 1%. Intercept is indicated as follows;From the formulation perspective, polymeric gel base dispersion was initially prepared by dispersing AX in distilled water with constant stirring at 510–550 RPM until the gel was formed and the pH was maintained at 6.5 ± 0.2 using 2.5% aqueous NaOH. For the emulsion, oil phase was prepared by dissolving span 80 in liquid paraffin and aqueous phase by dissolving tween 20 in distilled water. Separately, nimesulide was dissolved in ethanol and methylparaben in propylene glycol owed to the solubility of both ingredients in respective solvent. Both solutions of nimesulide and methylparaben were added to the aqueous phase. In the following, the oily and aqueous phases were separately heated in a water bath at 70°C. Upon obtaining uniformity, drug loaded aqueous phase was added to the oil phase with continuous stirring until the temperature reached to room temperature. The drug-loaded emulsion was incorporated into the polymeric gel base dispersion of AX steadily with continuous stirring to obtain homogenized emulgel. ,20. Afte2 values and statistically significant coefficients. The optimal formulations were chosen based on their attractiveness as determined by Design-Expert software acceptance criteria. The Design-Expert software featured a variety of 3D reaction surface graphs. To validate the experimental model and polynomial equations, three optimal checkpoint formulations were chosen over the experimental region. The response qualities of the improved checkpoint formulation variables were tested. To compute the percentage prediction error, the experimental values of the answers were quantitatively compared to the projected values.Design Expert 10, software , completed statistical validation of the polynomial equations, which was determined using the software’s ANOVA requirement. The models were assessed using RVisual inspection of formulations was done for their homogeneity, appearance, color, phase separation, and consistency .A viscosity test of all formulations was performed on a Brookfield viscometer  at 50 rpm and 100 rpm over a period of 10 min. The pH of all formulations was measured by using a digital pH meter .The amount of drug entrapped into the emulgel formulations was determined photometrically. Briefly, 1 g of each formulation was mixed with phosphate buffer (pH 7.4) (1:100 w/v) in a volumetric flask and continued stirring for 30 min. The mixture was further diluted with phosphate buffer (1:10 v/v) before measuring the absorbance using a UV spectrophotometer  against 392nm. A standard curve of the pure drug within the range of 1–10 μg/mL in phosphate buffer was used for quantification.The stability studies were performed for all the formulations stored in well-closed glass containers for a period of 90 days at room temperature. Samples were collected at predetermined intervals 0, 30, 60 and 90 days, and their physicochemical parameters were evaluated .A spreadability test based on ‘Slip’ and ‘drag’ characteristics of emulgel was performed for the optimized formulations. For this purpose, a glass slide was fixed on the wooden box and 1 g of each formulation was placed on this slide, separately. A second glass slide was used to cover in such a way that the emulgel was sandwiched between two glass slides. Afterwards, a standard weight of 1 Kg was placed on the sandwiched slides in order to remove any entrapped air and to get a uniform layer emulgel. After an incubation of 5 min, the excess emulgel that drained out of the slides was scrapped off and the weight was uplifted. In the following, the time required for the separation of two slides was noted in seconds .The following formula was used to calculate spreadability,2) of each formulation was done in triplicates [As a measure of thickness of the emulgel formulations, extrusion from lacquered aluminum collapsible tube on application of weight (in g) required to extrude at least 0.5 cm ribbon of emulgel in 10 s was measured. The measurement of extrudability  was used to determine the average globule size, polydispersity index, and zeta potentials of optimized emulgel formulations as per the concepts of dynamic light scattering and electrophoretic mobility theories, respectively.Under a light microscope, the morphology of the emulsion was examined. A formulation that displayed the best release was examined for shape, globule size, and agglomeration. The formulation was mounted on a glass slide and viewed under a microscope.−1 under similar operating conditions [FTIR of pure Nimesulide, AX, and physical mixture of AX and nimesulide was done by KBr disc method by employing Bruker FTIR  instrument, using attenuated total reflectance (ATR) technology accompanying software OPUS data collection. All the spectrum was obtained at 400-4000cmnditions .The ex-vivo permeation study for the emulgel formulation was performed using a Franz diffusion cell , following established protocols described in the literature (28–30).Rabbit skin required for present study was obtained from the local animal house. The hair at the dorsal part of the animal was shaved carefully using an electric clipper followed by scarifying the rabbits and separating the skin. The adipose tissue was removed from the skin then it was preserved at 4°C in phosphate buffer maintained at pH 7.4. For experiment, rabbit skin was thoroughly rinsed with normal saline and then clamped between the donor and receptor chambers of the Franz cell.A predetermined amount of the emulgel formulation (1 gram) was applied evenly over the rabbit skin. Phosphate buffer solution (pH 7.4) was used as the diffusion medium and maintained at a temperature of 37°C±0.5°C throughout the experiment. The diffusion medium was continuously stirred using a magnetic stirrer to ensure uniform distribution. Samples (0.1 mL) were collected at specific time intervals , and an equal volume of pre-warmed buffer was immediately replaced after each sampling to maintain sink conditions. The collected samples were appropriately diluted and their absorbance was measured using a UV spectrophotometer. The cumulative amount of drug permeation through the skin was calculated as a function of time, allowing for the evaluation of the emulgel formulation’s permeation characteristics .All the protocols for the in-vivo study were pre-approved by the Ethical Committee of the Faculty of Pharmacy, University of Sargodha (PREC4532). The in vivo experiment for analgesic activity was conducted on healthy adult Wistar rats weighing 150–200 g, whereas skin irritation was conducted on healthy albino rabbits weighing about 1.5 ± 0.5 kg. Animals were allowed to acclimatize for one week before the experiment. Animals were kept at ambient conditions  and had free access to standard pellet diet and fresh water . Guideli2.5.11.1. Analgesic activity. Two groups of rats were made, three rats per group. Group 1 was considered as standard group and group 2 was considered as test group. Group 1 or the standard group was treated with the marketed formulation of nimesulide. Group 2 or the test group was treated with the topical formulation of emulgel. In-vivo analgesic activity of nimesulide was examined by the Hot plate method. Each rat was kept on a hot plate (55°C) for 10 sec and the latency period  or PRT (pain reaction time) was measured. 0.5 g of the respective formulation was applied on the dorsal surface of the right hind paw of rat with gentle rubbing. The time (in seconds), spent licking the paw was considered an indicator of pain response.2.5.11.2 Skin irritation test. The test was conducted to explore the safety and skin compatibility of the optimized formulation. Two groups of rabbits were made, three rabbits each group. Group I was considered as control group, received no treatment while group II was considered as test group, and received topical treatment of emulgel.Before the study, rabbits were shaved and circumscribed dorsal skin area. A sample (0.5 g) of the formulation was applied once daily for 7 days, and the rabbit’s skin was inspected for any signs of irritation, redness, and inflammation. A scoring system was considered and average scores were recorded. The scoring system was designed as follows; 0: no signs of irritation, 1: slight irritation, 2: moderate irritation, and 3: severe irritation .In-vivo study, the statistical analysis of all the result was accomplished utilizing GraphPad Prism 7.0 Graph Pad Software . The data were analyzed using student t test. P value of <0.05 was considered as significant. All the results are depicted as value Mean± SEM.For The experimental design was used for the optimization of the formulation variables, as the response surface methodology required 18 experiments. As illustrated in Y1) and pH (Y2), and percent drug diffusion (Y3) were found to be significantly higher  in F8; (Y2 6.8) and (Y3 115%) in F3 respectively. The response surface models were developed with Design-Expert software by applying coded values of factor levels to estimate the quantitative impacts of various combinations of variables on Viscosity, pH, and percent drug diffusion. The following is a representation of the model described:Optimal conditions for three dependent variables (responses) were predicted by the desirability function and appropriate concentration ranges were chosen. Five options that are used by the program to shape the desirability indices include none, minimum, maximum, within range, and target. Our focus was to make formulations with good viscosity, acceptable pH range, and content uniformity. According to established criteria, the optimum situations for our formulation’s desired response were; the concentration of AX at 1.5%, the concentration of span 80 was 2% and the concentration of tween 20 was 0.75%. Design-Expert software was used to obtain optimized formulations and create the mathematical equations shown in (3), (4), and (5). The responses, viscosity (Y3) and pH (Y2) and Viscosity (Y1) among the formulations and demonstrated good quality with the best spreadability. Using Design Expert 10, software , the responses observed for 18 different formulations were fit to a design model at the same time as in Formulation F2 showed a significant percent drug diffusion (2) of viscosity and pH was determined to be 0.9535 and 0.9273 respectively, suggesting that the selected variables for each are well matched. Moreover, the "Pred R-Squared" values fairly agree with the "Adj R-Squared" for both of them. The percentage of drug diffusion values tested for various formulations exhibited a broad range of values (i.e. from a minimum of 60.8% to a maximum of 115.5%), and the R2 was determined to be 0.7951. The "Pred R-Squared" is 0.6459, which is reasonably close to the "Adj R-Squared" of 0.7010. In general, the findings demonstrate that the factors chosen for the investigation have a significant impact on the percentage drug diffusion and when two variables are adjusted at the same time, the interaction terms reveal how the percentage drug diffusion varies. Positive coefficients for interactions between two variables indicate an adverse influence on percent drug diffusion, whereas negative coefficients for all three independent variables suggest a beneficial effect on percent drug diffusion.Multiple regression was used to generate a polynomial equation using the percentage drug diffusion and viscosity acquired at various values of the three independent variables , the results are displayed in et al. The molar mass of AX was determined using Gel Permeation Chromatography (GPC), which is a technique commonly employed to analyze the size and distribution of macromolecules. The results of the GPC analysis revealed a weight average molar mass (Mw) of 364,470 g/mol. Weight average molar mass considers the contribution of each molecule’s weight to the overall average, providing a measure of the overall size of the molecules in the sample. Additionally, a number average molar mass (Mn) of 12,800 g/mol was determined. Number average molar mass considers the total number of molecules in the sample, providing an average size of the individual molecules. These results suggest that the arabinoxylan molecules extracted through alkali extraction have a wide range of molecular weights, with the weight average molar mass being significantly higher than the number average molar mass [AX was obtained from Plantago ovata using the alkali extraction method, resulting in a yield of 39%. Mild alkali treatment resulted in a strong gel formation, while harsh alkali treatment led to decreased viscosity and elasticity of the gel. This decrease in gel properties was attributed to the reduced ferulic acid (FA) content in AX caused by harsh alkali treatment. The FA content in AX plays a crucial role in determining the gel structure, with higher FA content leading to smaller mesh sizes, lower molecular weight, and increased cross-linking capacity, resulting in more compact gel structures. –32 The mlar mass .A viscosity test is recommended for semi-solid dosage forms in order to investigate the flow properties. The viscosity of the different emulgel formulations was in the range of 10000-30000cps, whereas, the ideal range for the viscosity is 12500–21100 cps. The outcomes of the viscosity studies, as illustrated in The pH values of the emulgel formulations, as indicated in The emulgel was examined visually for phase separation, pH and content uniformity. Among all formulation, F1, F3 and F4 were found to be incubated under given conditions, whereas all other formulations undergone phase separation. Formulations appear to be creamy with a smooth homogeneous texture and glossy appearance. Spreadability is inversely proportional to the viscosity, the less viscous formulation exhibits more spreadability and vice versa ,37. The When formulating a stable emulgel system, zeta potential, droplet size distribution, and PDI are the most important factors to consider . The emuWhen formulating a stable emulgel system, several factors are crucial to consider, including zeta potential, droplet size distribution, and Polydispersity Index (PDI) . Zeta poThe optimal range for zeta potential in ensuring emulgel stability is typically between +50mV and -50mV. It is crucial to note that the specific zeta potential value within this range is significant for ensuring the stability of emulgel formulations In The particle size of droplets is usually greater than 400 nm, which leads to the opacity of emulgel. The measured size of the particles of F1, F3 and F4 was 872 ± 7.4525 nm, 1168 ± 9.4525 nm and 1828 ± 17.4525 nm. The term PDI represents the system’s polydispersity and its values are 0.0 to 1.0. The PDI for nimesulide emulgel formulations ranged from 0.945,0.975 and 1.00 for F1, F3 and F4, respectively and these values depict the heterogenous dispersion of droplets in the formulation .The release of drug from emulgels depends on multiple factors including gelling agent concentration, spreadability, emulsifying agent, and viscosity. Optimized formulation F1 having maximum ex-vivo cumulative drug permeation was therefore selected for FTIR, in-vivo analgesic activity and skin irritation test. Ex-vivo cumulative drug permeation studies provide valuable insights into the release profile of the drug from the formulation. Formulation F1 demonstrated the highest ex-vivo cumulative drug permeation compared to other formulations under investigation. This indicates that F1 has the potential to deliver the drug effectively and efficiently to the target site, which is crucial for achieving the desired therapeutic effect. Furthermore, selecting formulation F1 for additional tests, such as FTIR analysis, in-vivo analgesic activity, and skin irritation testing, provides further insights into its performance and suitability. Additionally, conducting skin irritation tests is crucial to evaluate the formulation’s compatibility with the skin. By assessing the potential for skin irritation, we can ensure that formulation F1 is safe for topical application and minimizes the risk of adverse reactions or irritations.-1 indicate OH-stretching, the main band at 2866cm-1 is linked with the aliphatic CH-stretching and the bands from 1750–2000 cm-1 illustrate the deformation by water absorption. A further look indicates the presence a peak at 1483 cm-1 (conforming to the CH2 group), 1352 cm-1 (CH), 1134 cm-1 (arabinosyl side chain) and the peak at 1010 cm-1 showing C-C, C-O, and C-O-H group stretching [-1. The individual strong peak was observed at 3423 cm-1 that revealed the N-H stretching (amide group). The peak of the drug at 1490 cm-1 confirms the presence of NO2 group, while the peak at 1315. 2 cm-1 reveals the presence of CH3 group and 1157. 20 cm-1 is for S = O group that confirm the drug purity [-1is clear in the spectrum, revealing the presence of amide group of drugs in emulgel.FTIR of AX, pure nimesulide, and optimized emulgel formulation F1 is described in retching . FTIR spg purity . FTIR ofp < 0.05) for the treated group as compared to control group in the student’s t-test.Analgesic effect in treated groups of rats and control was evaluated by observing the response  to pain stimulus and outcomes of the study are described in For skin irritation test, procedure for the skin irritation grading was conducted every day extended over a period of one week. As depicted in the The outcomes of the skin irritation via the scoring system are demonstrated in This study was designed to evaluate natural biomaterial AX as gelling agent in emulgel formulation.The findings demonstrated that AX extracted from Plantago ovatago exhibited excellent gelling properties in emulgel formulations. The formulated emulgel, incorporating AX, exhibited desirable characteristics such as homogeneity, spreadability, stability, and gradual drug permeation. The application of response surface methodology aided in optimizing the formulation, and the most promising formulation, in terms of drug permeation, was selected for further investigations. The treated group using the optimized formulation (F1) showed a significant three-fold delay in the onset of analgesic effect compared to the control group. This result clearly indicated the enhanced analgesic activity of the developed formulation. Therefore, it can be concluded that the developed emulgel containing nimesulide demonstrated improved analgesic activity by effectively penetrating the stratum corneum. This formulation holds potential as a carrier for delivering other drugs in future applications. Additionally, the cost-effectiveness of the formulation is achieved through the utilization of readily available and cost-efficient ingredients, making it a viable option for commercial production. These cutting-edge features of our research contribute to advancing the field of topical drug delivery, offering improved efficacy and economic benefits, which can have a significant impact on patient care and treatment outcomes.S1 File(RAR)Click here for additional data file.


Diabetes mellitus (DM) increases the destruction of periodontal tissue and impairs osteogenesis differentiation. Exendin-4 (Ex-4), a glucagon-like peptide-1 (GLP-1) analogue, can be used for treating DM and promotes bone regeneration. The aim of this study was to explore the effect and mechanism of Ex-4 on improving the osteogenesis of periodontal ligament stem cells (PDLSCs) in a high glucose environment. Alkaline phosphatase staining and alizarin red staining were used to detect the osteogenic differentiation of PDLSCs. The results showed that 10 nM Ex-4 could reduce the osteogenesis inhibition of PDLSCs induced by high glucose. RT-PCR and western blot results showed that Ex-4 increased the osteogenesis-related gene expression of ALP, Runx2, and Osx, and upregulated the phosphorylation of P38, JNK, and ERK1/2; the peak effect was observed in the range 0.5–1.0 h. Mitogen-activated protein kinase (MAPK) inhibitors PD98059, SB203580, and SP600125 blocked the effects of Ex-4 on MAPK activation and decreased the expression of ALP, Runx2, and Osx in PDLSCs. Moreover, after Ex-4 treatment, the total β-catenin, p-GSK3β, LEF, and Runx2 protein levels increased under normal or high glucose environments. In conclusion, our results indicated that Ex-4 regulates the MAPK and WNT signaling pathways to alleviate the osteogenic inhibition of PDLSCs in a high glucose environment. Curre 16,647) ,9. Therein vivo, easily degraded by DPP-4, and its serum half-life in vivo is only 2 min; therefore, its action time is very short [In vivo, Ex-4 has a similar biological function to that of GLP-1 but its half-life is longer and its degradation tendency is lower, so it is currently widely used in the clinical treatment of type 2 diabetes [GLP-1 (glucagon-like peptide 1) is a kind of incretin synthesized and secreted by L cells of the intestine stimulated by food. It has a wide range of physiological functions, such as promoting the synthesis and release of insulin and reducing blood sugar concentration. However, GLP-1 is extremely unstable ry short . Exendindiabetes . Recent diabetes ,13,14. Adiabetes . Our prediabetes , althougdiabetes . Ex-4 cadiabetes . The Wntdiabetes . TherefoThe purpose of this study was to explore the osteogenesis regulatory mechanisms of Ex-4 in PDLSCs under high glucose conditions and to provide a new method for the clinical treatment of diabetic periodontitis.22.12. After culturing for 4 h, alpha modification of eagle’s medium (α-MEM)  supplemented with 10% fetal bovine serum (FBS) and 1% streptomycin solution  was added to the culture medium and changed every 3 days. PDLSCs from P3 to P5 were used for subsequent experiments.The premolars of healthy people were collected from orthodontic patients (18–26 years of age) who visited the Department of Orthodontics of the First Affiliated Hospital of Chongqing Medical University. Consent was obtained from all patients. After the removal of excess epithelial and granulation tissue, the teeth were washed repeatedly with phosphate-buffered saline . The periodontal membrane was scraped softly from a third of the root and then digested with collagenase type I  in a water bath at 37°C for 10 min. Finally, the tissues were transferred to 25 T cell culture flasks containing fetal bovine serum  at 37°C in a humidified atmosphere containing 5% COEthics approval and consent to participate: This study was carried out in accordance with the Declaration of Helsinki principles and the guidelines of the Ethics Committee of the First Affiliated Hospital of Chongqing Medical University, and the approval reference number is 201810401. Furthermore, all subjects have given their written informed consent.2.22 cell culture dishes. They were then cultured in a growth medium for 14 days, and about 2 mL of crystal violet dye solution  was added to each dish and the clone formation rate was calculated. When the number of cells in an aggregation exceeded 50, it was scored as a colony. For osteogenic experiments, PDLSCs were cultured in an osteogenic induction medium comprising α-MEM, 10% FBS, 1% streptomycin solution, 1 µM dexamethasone, 50 µg/mL ascorbic acid, and 3 µM β-glycerophosphate . After 21 days, alizarin red staining was used to assess the degree of osteogenic differentiation. For adipogenesis, PDLSCs were cultured in an adipogenic induction medium consisting of α-MEM, 10% FBS, 1% streptomycin solution, 1 µM dexamethasone, 10 µM insulin, 200 µM indomethacin, and 0.5 mM 3-isobutyl-1-methylxanthine . After 14 days, Oil Red O staining was used to detect the lipid droplets.To determine the characteristics of PDLSCs, we detected the colony-forming ability and multi-directional differentiation ability of PDLSCs. PDLSCs were inoculated into 6 cm2.3d-glucose  (NG), NG + 10 nM Ex-4 (NG + Ex-4), 30 mM d-glucose (HG), or HG + 10 nM Ex-4 (HG + Ex-4) for 7 days. ALP staining solution  was prepared according to the protocol of the alkaline phosphatase staining kit, and 500 μL of the ALP staining solution was added to the well to be tested, and incubated for 15 min at room temperature in the dark. Subsequently, all staining solution was aspirated, and the well was gently rinsed with ultrapure water, photographed, and observed.PDLSCs were induced in an osteogenic induction medium containing 5.5 mM 2.4PDLSCs were cultured in the osteogenic induction medium for 21 days. Then, the cells were fixed with 4% paraformaldehyde at room temperature in the dark for 30 min and stained with alizarin red  for 30 min; the dye solution was then discarded. After careful washing with PBS three times, the cells were observed and photographed under an inverted microscope.2.5−ΔΔCt method.PDLSCs were osteogenically cultured with NG or 30 mM mannitol for 7 days. PDLSCs were also cultured with NG + 10 nM Ex-4, HG, or HG + 10 nM Ex-4 for 7 or 14 days, and osteogenesis was induced. The MAPK-specific inhibitors, 25 µM PD98059, 25 µM SB203580, and 10 µM SP600125 , were also used to treat the cells. The expression of ALP, Osx, and Runx2 was detected with RT-PCR. Total RNA was extracted from the PDLSCs with TRIzol reagent . RNA  was then transcribed into cDNA using a PrimeScript™ RT reagent kit with gDNA Eraser . Finally, an SYBR Premix Ex Taq II kit  was used for RT-PCR. A total of 10 µL PCR system contained 2 µL of cDNA, 0.5 µL of each diluted primer, 5 µL of SYBR green, and 2 µL of distilled water. The primer sequences used in this research are listed in 2.6The levels of key proteins in MAPK and WNT pathways were tested by western blotting. PDLSCs were treated with 10 nM Ex-4 for 0, 0.5, 1, 1.5, 2, or 3 h. For inhibition experiments, cells were pretreated with the specific inhibitors PD98059 (25 µM), SB203580 (25 µM), or SP600125 (10 µM)  for 4 h, then incubated with Ex-4 (10 nM) for 0.5 or 1 h, and cellular proteins were extracted. A BCA protein assay kit (Beyotime Biotechnology) was used to detect and quantify the protein concentrations of PDLSCs. Then, 20 μg of total protein was separated with 12% SDS-PAGE and transferred to polyvinylidene fluoride membranes . The membrane was blocked in 5% skim milk and incubated on a shaker at room temperature for 2 h. Membranes were incubated overnight at 4°C with primary antibodies to p38, p-p38, ERK1/2, p-ERK1/2, JNK, p-JNK, GSK3β, p-GSK3β (Ser9), LEF, β-catenin, GAPDH, tubulin  and Runx2  diluted 1:1,000, and was then incubated with secondary antibodies  for 1 h at room temperature. Proteins were visualized with Hypersensitive ECL Chemiluminescence kits  and analyzed using Image J software.2.7t-test, and multiple groups of data were evaluated with one-way ANOVA. Differences were considered significant at p < 0.05.All statistical calculations were analyzed using Prism software (version 8.0). The differences between the two groups of data were compared with Student’s 33.1Primary periodontal ligament cells were successfully extracted from periodontal ligament tissue . Under m3.2p > 0.05)  for 7 days. RT-PCR indicated no significant differences in the expression of osteogenic differentiation-associated genes  between the 30 mM mannitol group and control group ( > 0.05) . Our finp < 0.01). Under HG conditions, the mRNA levels of ALP, Osx, and Runx2 were lower than that of the control group on the 7th day, and the mRNA levels of ALP and Runx2 were markedly lower than that of the control group on the 14th day (p < 0.05). The RT-PCR results also indicated that the effects of inhibition of high glucose on osteogenic genes in PDLSCs were clearly decreased by co-treatment with Ex-4 for 14 days (p < 0.01)  after treatment with 10 nM Ex-4 was markedly higher than that of the control group on the 14th day ( < 0.01) . These rWe further detected the changes in osteogenesis by ALP staining and alizarin red staining. After 7 and 21 days of osteogenic culture, the results indicated that the ALP activity and number of calcium nodules formed in the high glucose group strongly decreased. After treatment with Ex-4, the ALP activity and the number of calcium nodules in HG + Ex-4 were greater than those in the HG group . These r3.3p < 0.01)  and their phosphorylation were detected at different times. Western blotting indicated that Ex-4 promoted the phosphorylation of MAPKs in PDLSCs between 0.5 and 1.0 h. The phosphorylation of p38 peaked around 1.0 h, those of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) both peaked around 0.5 h ( < 0.01) .To further verify the effects of Ex-4 via the MAPK pathway, we pretreated cells with MAPK inhibitors  for 4 h, and then treated the cells with 10 nM Ex-4 or 30 mM HG for approximately 30 min. Western blotting detection showed that Ex-4 promoted greater MAPK phosphorylation of ERK1/2, p38, and JNK than that in the control group, and the phosphorylation of p38 and ERK1/2 in the HG group decreased. Compared with that in the HG group, the phosphorylation of MAPKs in the HG + Ex-4 group was also elevated. Furthermore, MAPK inhibitor treatment significantly decreased the phosphorylation of MAPKs by Ex-4. Thus, Ex-4 activates the MAPK pathway in a high glucose environment .3.4p < 0.01). Similarly, for SP600125, compared with the NG + Ex-4 group, the mRNA levels of ALP, Runx2, and OSX were reduced to approximately 98, 53, and 28%, respectively, in the NG + Ex-4 + SP600125 group, and compared with HG + Ex-4, they were reduced to about 95, 38, and 20% (p < 0.05) in the HG + Ex-4 + SP600125 group. PD98059 likewise reduced all the mRNA expressions; compared with the NG + Ex-4 group, it decreased the expressions of ALP, Runx2, and OSX to about 89, 70, and 73% in the NG + Ex-4 + PD98059 group, and compared with the HG + Ex-4 group the expressions decreased to 59, 65, and 38% in the HG + Ex-4 + PD98059 group, respectively (p < 0.05)  . Alizari < 0.05) .3.5p < 0.05)  .4In the present study, we found the GLP1 receptor agonist Ex-4 exerted a direct action on PDLSCs in a high glucose environment. Ex-4 alleviated the osteogenic inhibition of PDLSCs induced by high glucose, and promoted the osteogenic differentiation of PDLSCs in a normal glucose environment. Ex-4 also upregulated the activity of MAPK and WNT signaling pathways, which are known to play a key role in osteoblast differentiation and bone development. Taken together, these results imply that Ex-4 may play an active role in the osteogenesis of PDLSCs by regulating MAPK and WNT pathways in a high glucose environment.Studies have shown a bidirectional relationship between diabetes and periodontitis. For patients with periodontitis and diabetes, hyperglycemia can accelerate the destruction of periodontal tissue and promote alveolar bone absorption . ALP is in vitro, our data showed that 10 nM Ex-4 rapidly increased the expression of the osteogenic genes ALP, Runx2, and Osx. Ex-4 restored the expression of damaged osteogenic genes under high glucose conditions. Nonetheless, we conclude that Ex-4 has a positive effect on the PDLSC differentiation under a high glucose environment. This conclusion is in line with many previous research findings.Ex-4, an analogue of GLP-1, is used as a drug to treat type 2 diabetes. Recent studies have demonstrated that GLP-1 and its analogues play active roles in bone metabolism and have attracted increasing attention. In animal experiments, Nuche-Berenguer et al. have found that GLP-1 and Ex-4 had osteogenic roles in diabetes and can reverse the osteopenia associated with hyperlipidemia . MoreoveTo further investigate the osteogenic mechanism of Ex-4 in PDLSCs, we studied the effects of Ex-4 on the activation of the MAPK and WNT signaling pathways. MAPK signaling pathways regulate a series of biological activities, such as growth, reproduction, and death. It is particularly important for the differentiation of osteoblasts and the regulation and control of bone development . Four MAThe classical WNT signaling pathway is closely associated with the regulation of osteoblast differentiation. The pathway is mainly composed of ligands (WNT family molecules), transmembrane receptors (Frizzled family molecules and LRP-5/6), cytoplasmic regulatory proteins , and nuclear transcription factor families (TCF/LEF) . GSK-3β,5Our experiments demonstrated that the osteogenesis of PDLSCs was inhibited in a high glucose environment, but Ex-4 (10 nM) alleviated the inhibition of osteogenesis in PDLSCs by regulating the MAPK and WNT signaling pathways . Our fin


Downregulation of INa, as occurs in Brugada syndrome (BrS), causes ventricular arrhythmias. The present study investigated whether the Wnt/β‐catenin signaling regulates Nav1.5 in human‐induced pluripotent stem cell‐derived cardiomyocytes (iPSC‐CMs). In healthy male and female iPSC‐CMs, activation of Wnt/β‐catenin signaling by CHIR‐99021 reduced (p < 0.01) both Nav1.5 protein and SCN5A mRNA. In iPSC‐CMs from a BrS patient, both Nav1.5 protein and peak INa were reduced compared to those in healthy iPSC‐CMs. Treatment of BrS iPSC‐CMs with Wnt‐C59, a small‐molecule Wnt inhibitor, led to a 2.1‐fold increase in Nav1.5 protein (p = 0.0005) but surprisingly did not affect SCN5A mRNA (p = 0.146). Similarly, inhibition of Wnt signaling using shRNA‐mediated β‐catenin knockdown in BrS iPSC‐CMs led to a 4.0‐fold increase in Nav1.5, which was associated with a 4.9‐fold increase in peak INa but only a 2.1‐fold increase in SCN5A mRNA. The upregulation of Nav1.5 by β‐catenin knockdown was verified in iPSC‐CMs from a second BrS patient. This study demonstrated that Wnt/β‐catenin signaling inhibits Nav1.5 expression in both male and female human iPSC‐CMs, and inhibition of Wnt/β‐catenin signaling upregulates Nav1.5 in BrS iPSC‐CMs through both transcriptional and posttranscriptional mechanisms.The voltage‐gated Na Joseph C. Wu's lab at Stanford Cardiovascular Institute with Institutional Ethics Committee approved protocols (IRB 29904 and SCRO 485) and informed written consents were given prior to the inclusion of subjects in the study. Investigations involving human cells conformed to the principles outlined in the Declaration of Helsinki and were approved by the institutional review committee at the University of Ottawa Heart Institute.2.1SCN5A alleles  containing 1x B‐27 supplement . Cells at Day 30–45 of differentiation were used in this study. An additional line of healthy iPSC‐CMs  was purchased from Axolbio . iPSCs were derived from cord blood CD34+ cells collected from a healthy male newborn by reprogramming with episomal vectors expressing the reprogramming factors. Cardiac differentiation was performed in iPSC monolayer culture in a chemically defined medium.iPSC lines were maintained and differentiated into cardiomyocytes in a chemically defined medium according to standard operating procedures (SOPs) established in Dr. J.C. Wu's lab  or control adenovirus expressing a non‐silencing shRNA  at a MOI of 10. At Day 5–8 after virus transduction, cells were used for analyses . In another group of studies, Wnt signaling was inhibited in BrS iPSC‐CMs by including Wnt‐C59  in culture medium for 3 days. Control cells were cultured in medium containing equal volume of DMSO .To activate Wnt signaling, iPSC‐CMs were treated with 5 μM CHIR‐99021  for 48 h before RNA extraction, qRT‐PCR, and patch‐clamp studies as described below. Control cells were cultured in medium containing equal volume of DMSO . To suppress Wnt signaling, BrS iPSC‐CMs were transduced with adenovirus expressing an shRNA targeting human 2.3HPRT1 or the mean values of HPRT1, RPL32, and PPIA) in the same sample. Some qPCR experiments were performed with TaqMan Gene Expression Assays . Results were analyzed with the 2−ΔΔ C(t) method.Total RNA was isolated from iPSC‐CMs with a RNeasy mini kit  and cDNA was synthesized with a High‐Capacity cDNA Reverse Transcription Kit . Real‐time quantitative PCR was performed on a CFX Connect Real‐Time PCR Detection System (Bio‐Rad) using iTaq Universal SYBR Green Supermix . Information on the qPCR primers was included in Table 2.4v1.5 antibody targeting the C‐terminus , monoclonal mouse anti‐α‐sarcomeric actinin antibody , rabbit anti‐β‐catenin antibody , and mouse anti‐N‐Cadherin antibody . Secondary antibodies used were goat anti‐mouse IgG Alexa Fluor‐568  and anti‐rabbit IgG Alexa Fluor‐488 . Fluorescent cellular images were taken with a ZEISS confocal microscope equipped with the Airyscan technique for high‐resolution imaging (Zeiss Elyra S.1 LSM 880).iPSC‐CMs cultured on 8‐chamber culture slides  were fixed with 100% methanol at −20°C for 10 min. Cells were blocked and permeabilized in Dako protein block  containing 0.1% (w/v) saponin  at room temperature for 90 min. Cells were then incubated with primary antibodies (see below) diluted in the same blocking solution at 4°C for overnight. Then cells were washed with phosphate‐buffered saline (PBS) and incubated with secondary antibodies (see below) at room temperature for 1 h. Cells were washed with PBS and mounted with ProLong gold antifade reagent containing DAPI . Primary antibodies used were monoclonal rabbit anti‐Na2.5v1.5 antibody that binds to the intracellular loop between domains I and II  or monoclonal rabbit anti‐Nav1.5 antibody targeting the C‐terminus , rabbit anti‐Cav1.2 antibody , rabbit anti‐β‐catenin antibody , mouse anti‐α‐sarcomeric actinin antibody , rabbit anti‐Kir2.1 antibody , and rabbit anti‐Tbx3 antibody . Immunoreactivity was detected using SuperSignal West Pico PLUS Chemiluminescent Substrate . Equal protein loading of the gels was assessed by re‐probing the membranes with rabbit anti‐calnexin antibody . Band densities in western blot experiments were quantified using the “Measurements” function within ImageJ  or the “Gel Analyzer Protocol” function of ImageJ  and presented in the manuscript after normalization to calnexin band densities in the same membranes.iPSC‐CMs were homogenized in RIPA buffer  supplemented with Halt protease inhibitor cocktail . Protein concentration was determined by BCA assay  NaCl 20, TEA‐Cl 50, CsCl 67, MgCl2 1, CaCl2 1, glucose 10, and HEPES 10 (pH = 7.4 with CsOH) (ENa = +35.3 mV). In some studies, NaCl in the bath solution was increased to 100 mM (ENa = +75.7 mV) with reductions in TEA‐Cl and CsCl to keep the osmolarity constant. Either 0.1 mM CdCl2 or 10 μM nifedipine was included in the bath solution to inhibit L‐type Ca2+ current during the whole‐cell recording. Series resistance was compensated by 70%–80%. Cells were held at −120 mV and INa was elicited by a family of 50‐ms (or 200‐ms) voltage steps to potentials ranging from −90 to +30 mV (when 20 mM Na+ bath solution was used) or from −90 to +70 mV with 5‐mV increments and a cycle length of 600 ms. In some recordings, cells were held at −90 mV and stepped to −120 mV for 200 ms before the voltages steps to elicit INa  and the number of cell differentiations (N), is included in the figure legends. Differences between two means were evaluated by two‐tailed Student's t‐test. Differences among multiple means were assessed by one‐way analysis of variance (ANOVA). When significance was detected by ANOVA, differences among individual means were evaluated post hoc by Bonferroni's test.Data are expressed as mean ± standard deviation (SD) with 33.1AXIN2 mRNA  in stock solution. Therefore, CHIR stock solutions that were prepared within 3 months were used in subsequent studies to reduce variations among the experiments. Regardless, the changes in SCN5A and AXIN2 mRNA were highly correlated  by treatment with CHIR‐99021 (CHIR) for 48 h. CHIR is a cell‐permeant small‐molecule inhibitor of GSK‐3β and is a commonly used Wnt/β‐catenin pathway activator Figure . The incA Figure , a knownA Figure , suggestd Figure . Resultsv1.5 in iPSC‐CMs with one band near 260 kDa and another band with a higher molecular weight  in higher band and a 63% reduction (p = 0.0035) in lower band , and an 80% reduction in total Nav1.5 (p = 0.0002). To investigate if reduced Nav1.5 protein is associated with changes in voltage‐gated Na+ current (INa), whole‐cell patch‐clamp recording was performed in two different ionic conditions in male healthy iPSC‐CMs. In the first experiment, INa was recorded with a bath solution  and 0.1 mM Cd2+ (to inhibit L‐type Ca2+ current in whole‐cell recording). CHIR treatment led to an 82% reduction (p < 0.05) in peak INa density  without Cd2+ . CHIR treatment led to an 84% reduction in peak INa density , suggesting that the recorded current was primarily carried by Na+.Western blot revealed two distinct bands for Nad Figure , as wellt Figure . In botht Figure , the appINa density in CHIR‐treated iPSC‐CMs is consistent with our previous study  was increased by 13.9 mV  were also increased . In addition, the steady‐state inactivation curve of INa showed a small (4.8 mV) shift to the left  by CHIR  by CHIR treatment in female iPSC‐CMs. Regardless, total Nav1.5 was consistently reduced by CHIR in both male and female iPSC‐CMs. The higher and lower bands for Nav1.5 were further investigated by probing the same western blot samples of female iPSC‐CMs with two different anti‐Nav1.5 antibodies: a monoclonal antibody that binds to the C‐terminus of the Nav1.5 protein, and a polyclonal antibody that binds to the intracellular loop between domains I and II  to Scn5a promoter via two sites that are highly conserved among species including human and rats  who had unstable ventricular tachycardia and two missense variants in the SCN5A gene  were identified in this patient  is associated with the maturation degree of iPSC‐CMs  was affected by Wnt‐C59 treatment . Successful knockdown was confirmed by an 84% reduction in β‐catenin mRNA  showed small increases only in one batch of the cells (Batch‐2) after β‐catenin knockdown . INa was initially recorded in the presence of 0.1 mM Cd2+ (to block L‐type Ca2+ current) and INa was increased (p < 0.01) by 13‐fold in the cells after β‐catenin knockdown , and INa was increased (p < 0.01) by 4.9‐fold in the cells after β‐catenin knockdown  of INa in control‐shRNA group (+70 mV) was close to ENa (+76 mV) when Cd2+ was omitted  to 78% of that in healthy iPSC‐CMs. This is consistent with observations in immunocytostaining that Nav1.5 is increased in the plasma membrane of cardiomyocytes in BrS iPSC‐CMs after β‐catenin knockdown.Compared with s Figure . β‐caten3.6v1.5 by β‐catenin knockdown is reproduced in cardiomyocytes of other BrS patients, we tested an additional BrS iPSC‐CM line (BrS Line 2) that contains a mutation in one of the two SCN5A alleles  mechanism underlying Nav1.5 reductions. This aligns with the well‐established role of the Wnt/β‐catenin signaling as a regulator of gene transcriptions  and a lower band (near or slightly lower than 260 kDa) when a polyclonal anti‐Nav1.5 in BrS iPSC‐CMs suggesting that this is a potential therapeutic strategy. The increased INa density, as measured in single cells, is consistent with immunocytostaining results showing upregulation of Nav1.5 proteins at the plasma membrane in individual cardiomyocytes after β‐catenin knockdown. This also suggests that the increased total Nav1.5 protein in BrS iPSC‐CMs after β‐catenin knockdown, as demonstrated by western blot, reflects increased Nav1.5 expression in the individual cardiomyocytes, instead of increased number of Nav1.5‐expressing cells due to altered cell proliferation or survival. Despite the clear and consistent upregulation of Nav1.5 protein, the SCN5A mRNA was, surprisingly, either not increased or increased to a smaller degree after Wnt signaling inhibition. This suggests that, in addition to the known effect of Wnt signaling on Scn5a transcription  also showed downregulation of Nav1.5 in healthy adult human heart slices after treatment with a GSK‐3β inhibitor (SB216763)  , McDonald Scholarship and New Investigator Award from Heart and Stroke Foundation of Canada (to W.L.), University of Ottawa Faculty of Medicine Translational Research Grant (to D.R.D. and W.L.), Endowed Fellowship (to A.L.) and Endowed Scholarship (to Y.X. and J.W.) from the University of Ottawa Heart Institute.The authors have no competing interest.


Facile formation of carbon-heteroatom bonds is a long-standing objective in synthetic organic chemistry. However, direct cross-coupling with readily accessible alkenyl acetates via inert C‒O bond-cleavage for the carbon-heteroatom bond construction remains challenging. Here we report a practical preparation of stereoselective tri- and tetrasubstituted alkenyl silanes and stannanes by performing cobalt-catalyzed C‒O silylation and stannylation of alkenyl acetates using silylzinc pivalate and stannylzinc chloride as the nucleophiles. This protocol features a complete control of chemoselectivity, stereoselectivity, as well as excellent functional group compatibility. The resulting alkenyl silanes and stannanes show high reactivities in arylation and alkenylation by Hiyama and Stille reactions. The synthetic utility is further illustrated by the facile late-stage modifications of natural products and drug-like molecules. Mechanistic studies suggest that the reaction might involve a chelation-assisted oxidative insertion of cobalt species to C‒O bond. We anticipate that our findings should prove instrumental for potential applications of this technology to organic syntheses and drug discoveries in medicinal chemistry. Carbon-heteroatom bond formation reactions are of high interest in synthetic organic chemistry. Here, the authors report the cobalt-catalyzed synthesis of tri- and tetrasubstituted alkenyl silanes and stannanes via C–O functionalization of vinyl acetates using silylzinc pivalate and stannylzinc chloride as the nucleophiles. For practical applications and easy availability in synthesis, oxygen-based electrophiles have been recognized as halide-free alternatives for coupling with organometallic reagents10. Among them, alkenyl esters represent an attractive candidate for non-aromatic functionality due to their versatile occurrence in biomass-derived chemicals and convenient preparation. However, most of the advances in cross-couplings were accomplished with activated esters, such as triflate, tosylate, sulfamate, carbamate, phosphate, and carbonate. Hence, numerous contributions have been made to further expand the library of electrophiles16. Alkenyl acetates19, as a class of (pseudo)halide electrophile bearing a small and nonhazardous leaving group, have received considerable recent attention20. While unactivated enol acetates in coupling reactions remain a significant challenge due to the selective cleavage issue between vinyl Csp2–O and acetyl–O bonds (445 vs 335 kJ/mol)21. As such, using mild nucleophilic reagents as the coupling partners or/and devising highly active catalytic systems, which operate under kinetic control rather than a thermodynamic pathway, are the key for achieving cross-couplings via selective vinyl Csp2‒O bond cleavage  bonds, which play a vital role in the synthesis of pharmaceuticals, agrochemicals, and feedstock commodity chemicalsage Fig. .Fig. 1Ba22 and Garg23 independently pioneered the feasibility of nickel-catalyzed Negishi- and Suzuki-coupling reactions by using alkenyl pivalates as the electrophiles, alkenyl acetates have been extensively exploited for transition metal-catalyzed C–C bond formation cross-coupling reactions with B, Zn, Mg-based organometallics using Ni24, Fe21, Co25, Cr26, Rh27, and Pd catalysis30. Moreover, Ackermann31 disclosed the chelation-assisted cobalt-catalyzed Csp2–H alkenylations of indoles with alkenyl acetates, which underwent a remarkably mild C–O bond cleavage as compared to the ruthenium catalysis32. Gosmini35 and Shu36 also reported the applications of vinyl acetates in Co- and Ni-catalyzed reductive arylation and alkylation. Despite these major advances, major challenges in the area of C–O functionalization with alkenyl acetates still need to be overcome: (i) in contrast to the common alkenyl C–C bond formation, rather rare examples of alkenyl C–heteroatom bond formation were developed;37 (ii) most of the progresses have been made with cyclic, non- or mono-substituted alkenyl acetates as the coupling partners, rather poor Z/E-selectivity is observed when employing acyclic tri- or tetrasubstituted alkenyl acetates as the coupling partners. Hence, the methods aimed at C–heteroatom bond formation and stereo-controllable C–O cross-coupling are highly desirable  and trans-, or syn-elimination. Therefore, avoiding the latter pathway, selective oxidative insertion to form a single diastereomer intermediate (I) is the key for achieving stereoselective C–O functionalization (OPiv) and (nBu)3Sn‒ZnCl•LiCl show superior reactivity over the other commonly available Si- and Sn-based organometallic reagents. Moreover, notable features of our strategy are excellent functional group tolerance, and complete control of regio- and stereoselectivity, thus providing expedient access to the stereodefined alkenyl silanes and stannanes, which can be further utilized for the synthesis of stereochemically defined acyclic tri- and all-carbon tetrasubstituted alkenes.Towards the mechanism of C–O bond activation, two plausible pathways have been given: initial transmetalation between organometallics and transition metal catalyst forms the C–[TM] species, which undergoes (i) oxidative insertion of C–O bond to the [TM]-center, along with following reductive elimination; or (ii) 1,2-carbometalation of enol carboxylates, in tandem with isomerization  and Sn‒Met (2) reagents  through transmetalation of corresponding Si‒Li and Sn‒Li reagents with MgBr2, ZnX2, and Zn(OPiv)2, or AlCl3. Thereafter, we tested their reactivity for the cobalt-catalyzed silylation and stannylation of alkenyl acetates via C‒O bond activation (OPiv) (1a) as the nucleophile [PhMe2Si‒ZnOPiv•2Li(OPiv)(Cl) was abbreviated as Me2PhSi‒ZnOPiv for the sake of clarity], which displayed unique reactivity in carbonsilylation of alkenes by nickel catalysis45, the desired product 4 was obtained in 72% yield. Interestingly, the typical halides-supported Si‒Zn reagents 1b–1d, as well as Si‒Mg reagent 1e failed to undergo the C‒O silylation process under the identical cobalt catalysis. These findings are consistent with the previous nickel-catalyzed carbosilylation of alkenes, which demonstrated the crucial importance of OPiv-coordination for improving the reactivity of Si‒Zn reagents. Furthermore. cross-coupling product was not observed in the treatment of Sn‒Li (2d), Sn‒Zn (2b), or Sn‒Al (2 f) with indenyl acetate (3b) in the presence of CoBr2/2,2′-bipyridine (10 mol%) in MeCN at 0 °C. Whereas a two-step sequence including cobalt-catalyzed stannylation and nucleophilic addition/elimination occurred when employing Sn‒Mg (2e) as the nucleophile, affording the bis-stannylated product 5 in 24% yield. The envisioned alkenyl stannane 7 was obtained using Sn‒Zn 2a or 2c as the nucleophiles, and thus (nBu3)Sn‒ZnCl•LiCl yielding the desired product 7 in 20% yield, along with 24% of the destannylative product 6.We initiated our studies by preparing different Si‒Met (d Sn‒Met  reagents2PhSi‒ZnOPiv•Li2(Cl)(OPiv) (1a) and (nBu)3Sn‒ZnCl•LiCl (2a) as the nucleophilic partners (Table L1) or 5,5′-dimethylbipyridine (L4) as the ligands of choice for the cobalt-catalyzed C–O silylation, leading to the desired alkenyl silane 4 in 81% yield (Table L1‒L7), tridentate 2,6-bis(N-pyrazolyl)pyridine (L8) phosphines (L9‒L11), and bipyridine (L1) gave the optimal conversion of 7 . A significant breakthrough was made when performed the reaction at −20 °C, affording 7 in 53% yield, while no reaction was observed at −30 °C (entry 14). Notably, shortening the reaction time to 15 min led to a further yield increase to 66% yield (entries 15‒16), which highlighted the robustness of the cobalt catalyst and unique reactivity of (nBu)3Sn‒ZnCl•LiCl reagent (2a).These results encouraged us to further optimize the reaction conditions for cobalt-catalyzed C–Si and C–Sn bonds formation using Mel)(OPiv) a and (nBl)(OPiv)  and   exhibited good reactivity under remarkably mild reaction conditions at 23 °C, thereby delivering the corresponding tetrasubstituted alkenyl silanes 9‒30 in 50‒86% yields. Importantly, the cobalt catalysis displayed excellent functional group compatibility, such as fluoro, chloro, bromo, ester, nitrile, acetate, piperidine, and alkenyl substituents, were well tolerated under the mild reaction conditions. Notably, the catalytic system showed excellent chemoselectivity to realize C‒O bond activation, rather than C‒Hal (Cl and Br) cross-couplings. Moreover, the C‒O bond cleavage of all-carbon tetrasubstituted acyclic alkenyl acetates 5 smoothly underwent a silylation process in a highly stereoretentive fashion, the corresponding (Z)-alkenyl silanes 9‒31 were obtained with excellent stereoselectivity. A similar result was also observed when employing (Z)-alkenyl acetate bearing a lactone fragment as the electrophile, furnishing the (Z)-stereoisomer 32 as the sole product in 81% yield, which could be further reduced to the ring-opening diol 33 in a stereoretentive fashion. In sharp contrast, the lactam substrate failed to deliver the silylated product. The trisubstituted acyclic acetates failed to generate the desired product because a significant amount of the corresponding hydrodesilylated product 34 was formed, which is likely owing to the poor stability of the alkenyl silane under the reaction conditions.With the optimized cobalt catalyst in hand, the generality of our protocol was subsequently explored in a range of C‒O bond-silylation reactions with various polyfunctionalized alkenyl acetates 1a) Fig. . We werenBu3)Sn‒ZnCl•LiCl (2a). Indeed, a variety of cyclic enol acetates, including indenyl acetate, naphthalenyl acetate, chromenyl acetate, as well as cyclopentenyl and cycloheptenyl acetates, were smoothly stannylated with 2a, delivering the alkenyl stannanes 7, 35‒39 in 47‒85% yields. Moreover, the desired C‒O bond stannylation can be successfully scaled up to the gram scale with a slightly reduced yield (38)  Fig. .Fig. 4Su40‒51 in 49‒78% yields through excellent chemo- and stereoselective C‒O bond activation. Moreover, a variety of valuable functional groups such as trifluoromethyl, ester, nitrile, and terminal alkene were well tolerated under otherwise identical reaction conditions. A similar result was also observed when employing (Z)-acetoxyheptenyl carboxylate as the electrophilic partner, giving the desired product 52 in 76% yield -alkenyl acetates underwent cobalt-catalyzed stannylation in a stereoretentive fashion, generating the (Z)-stereoisomer as the sole product. As expected, various trisubstituted acrylates bearing electron-rich as well as electron-deficient substituents on the aryl side were identified as viable substrates for direct stannylations under remarkably mild reaction conditions, furnishing the (Z)-diastereomers eld Fig. .53‒68 in 47‒84% yields with complete control of stereoselectivity. It is worth noting that tertiary amide, lactam, as well as lactone were found to be effective chelation groups for the envisioned stereoselective C‒O bond-stannylation reaction, thereby providing (Z)-selective 69‒72 as the sole products , delivering the corresponding alkenyl silanes 73‒76 in moderate yields with high (Z)-selectivity. Moreover, the enol acetates derivatives of citronellol, ibuprofen, probenecid, celecoxib, and indomethacin were well stannylated with (nBu)3Sn‒ZnCl•LiCl, leading to the (Z)-diastereomers 77‒81 in 47‒71% yields, thus displaying the potential utility of this technology to drug discovery in medicinal chemistry  and (nBu)3Sn‒ZnCl•LiCl (2a) for cobalt-catalyzed stereoselective C‒O bond silyl- and stannylation of alkenyl acetates, we sought to unravel the reaction mechanism. To this end, a series of control experiments with radical scavengers 82 were performed, and both silylated and stannylated products were isolated with slightly reduced yields -83 as the electrophile. Moreover, a (E)-alkenyl acetate (85) bearing a conjugated amide only delivered the desired product 86 in a negligible conversion -83 to be inherently more reactive -88 led to a significantly decreased yield of the corresponding product 89 and 90 (0 and 31%) -91 only gave the desired stannane 93 in 43% yield with complete control of geometry. Moreover, an enol acetate 94 bearing a conjugated nitrile was also examined under standard conditions. However, only a trace amount of silylated product 95 and a low yield of stannylated product 96 were formed. As expected, no desired C‒O bond functionalization was observed when using the substrate without a conjugated ester group as the electrophilic coupling partner  Fig. . In addi29, a plausible mechanism for the cobalt-catalyzed stannylation of alkenyl acetates is proposed, as shown in Fig. nCoI-species by the [Si/Sn]‒ZnX (1a or 2a). Alkenyl acetate 3 then coordinates to the in situ formed LnCoI-species (A) with the assistance of ester chelation. The chelation effect can facilitate the oxidative addition of CoILn into C–O bond, thus forming Co(OAc) complex B as a single diastereomer. Subsequent coordination and transmetalation with [Si/Sn]‒ZnX (1a or 2a) affords the intermediate D. Final reductive elimination of D releases the desired alkenyl silanes and stannanes in a stereoretentive fashion and regenerates the catalytically active cobalt species A to close the catalytic cycle.Based on our mechanistic studies and previous insights97–100 in moderate to excellent yields with complete control of stereoselectivity, the conformation of 100 was confirmed by X-ray crystal analysis. 1,3-Dienes are key important structural motifs of natural products with activities of relevance to biology chemistry62. Such building blocks could be expediently constructed by palladium-catalyzed stereoselective alkenyl‒alkenyl coupling reactions; the desired 1,3-dienes 101‒105 were afforded in a stereoselective fashion. Beyond that, the potential applications of our protocol to the late-stage modifications of drug-like molecules, such as probenecid, duloxetine, and fluoxetine derivatives, were further demonstrated by a facile Stille-alkenylation under room temperature to afford the desired products 106‒108 in 56‒72% yields, thus proving instrumental technology for the synthesis of highly complex molecules in drug discovery. Moreover, these alkenyl silanes can be further converted to the corresponding silalactones 109‒110 through selective Si–C bond electrophilic cleavage. Interestingly, the selectivity was largely dominated by the reaction temperature.Finally, the synthetic utility of the resulting alkenyl silanes and stannanes was further described in the palladium-catalyzed Hiyama and Stille coupling reactions Fig. . RemarkaIn conclusion, we have demonstrated the utility of Si‒Zn and Sn‒Zn reagents for the efficient cobalt-catalyzed cross-coupling of alkenyl acetates through chelation-assisted C‒O bond cleavage under mild reaction conditions, thus providing a practical and efficient strategy for the preparation of various tri- and tetrasubstituted alkenyl silanes and stannanes in a stereoretentive fashion. Indeed, the solid silylzinc pivalate and stannylzinc chloride showed excellent reactivity and chemoselectivity toward diverse alkenyl acetates, as well as an ample substrate scope and high functional group compatibility. Preliminary mechanism studies suggested that the stereoselectivity presumably is caused by the chelation-assisted oxidative addition of cobalt to the C–O bond of alkenyl acetates. Finally, we have also illustrated the synthetical versatility of the polyfunctionalized alkenyl silanes and stannanes by the facile transformations of Hiyama and Stille reactions with complete control of stereoselectivity, such as arylation and alkenylation. Further development of the related applications with Si‒Zn and Sn‒Zn reagents are currently ongoing in our laboratory and will be reported in due course.2 for three times. A suspension of CoBr2 (10 mol%), 2,2′-bipyridyl (11 mol%) in THF or MeCN (0.5 mL) was charged and stirred at 0 °C for 5 min, then the alkenyl acetate , and PhMe2Si‒ZnOPiv•Li2(Cl)(OPiv)  or (nBu3)Sn‒ZnCl•LiCl  were sequentially added to the catalyst suspension. The reaction mixture was stirred at the identified temperature  under an atmosphere of N2. At ambient temperature, the solvent was evaporated under reduced pressure and the remaining residue was purified by column chromatography on silica gel to yield the desired products.To an oven-dried 25 mL screw-cap tube equipped with a stir bar was vacuumed and backfilled with NSupplementary InformationPeer Review File