We hypothesized that small molecule transcriptional perturbation could be harnessed to target a cellular dependency involving protein arginine methyltransferase 5 (PRMT5) in the context of methylthioadenosine phosphorylase (MTAP) deletion, seen frequently in malignant pleural mesothelioma (MPM). Here we show, that MTAP deletion is negatively prognostic in MPM. In vitro, the off-patent antibiotic Quinacrine efficiently suppressed PRMT5 transcription, causing chromatin remodelling with reduced global histone H4 symmetrical demethylation. Quinacrine phenocopied PRMT5 RNA interference and small molecule PRMT5 inhibition, reducing clonogenicity in an MTAP-dependent manner. This activity required a functional PRMT5 methyltransferase as MTAP negative cells were rescued by exogenous wild type PRMT5, but not a PRMT5E444Q methyltransferase-dead mutant. We identified c-jun as an essential PRMT5 transcription factor and a probable target for Quinacrine. Our results therefore suggest that small molecule-based transcriptional perturbation of PRMT5 can leverage a mutation-selective vulnerability, that is therapeutically tractable, and has relevance to 9p21 deleted cancers including MPM. MTAP is a critical enzyme in the methionine salvage pathway that metabolizes the substrate methylthioadenosine (MTA), leading to regeneration of methionine and adenosine3. Deletion of MTAP has been reported to confer a vulnerability to inhibition of the epigenetic regulator protein arginine methyltransferase 5 (PRMT5), which symmetrically methylates arginine on histone H4, leading to chromatin remodelling5.Deletion of chromosome 9p21 encompasses the tumour suppressors CDKN2A and methylthioadenosine phosphorylase (MTAP)7. Here we report a negative prognostic impact of MTAP on survival of patients with MPM, and demonstrate potential to selectively target somatic deletion of MTAP using a small-molecule based strategy, involving PRMT5 transcriptional suppression.Malignant pleural mesothelioma (MPM) is an incurable cancer caused by asbestos that lacks effective targeted therapies, and harbours high frequency deletion of 9p21p = 0.042, HR 0.609 (95%CI 0.376–0.987),   (Supplementary Table 7), Fig. A. In an p = 0.02); iMig stage (p = 0.009) and MTAP status (p = 0.008). These variables were taken forward into a Cox multivariate analysis showing that iMig stage (HR 1.36 (95%CI 1.11–1.68), p = 0.003) and MTAP status (HR 1.41 (95%CI 1.03–1.93), p = 0.032) retained statistical significance whilst age (HR 1.32 (95%CI 0.97–1.79), p = 0.08) did not.To explore MTAP as a covariate, we performed a univariate analysis showing a statistically significant effect between overall survival with: age  implicated by PROMOine Fig. A. c-JUN ine Fig. B, but noine Fig. B. Quinacine Fig. C. The efine Fig. D, withouine Fig. E. Intereine Fig. F.Figure 10 and has been reported to be a dependency in MTAP negative cells11, which we have verified in MPM. We confirmed global epigenetic modification associated with reduced H4R3me2S and re-expression of tumour suppressors, such as EIF3F, FOXP4, ZBTB4, GANAB, TMEM141, in association with loss of clonogenicity.Copy number loss of MTAP is one of the most frequent events in MPM. Our data confirmed a marked negative prognostic effect, which warrants novel targeted therapy. It should be noted that given the co-deletion of MTAP with CDKN2A, which has been also shown to be negatively prognostic, it was not possible to deconvolute the impact of MTAP in isolation from our data involving chromosome 9p21.3 deleted cel lines. PRMT5 plays a key role in the regulation of several pathways including DNA damage response, apoptosis, inhibition of tumour suppressors, and activation of survival pathways10. To address this limitation, we used the connectivity map approach8 to screen for small molecules with PRMT5 downregulating activity and identified the off-patent small molecule Quinacrine, which was primarily used as an antimalarial drug as well as an intrapleural sclerosing treatment for malignant pleural effusions with an excellent safety profile12. Quinacrine-mediated growth arrest was PRMT5 dependent as confirmed by a methyltransferase dead PRMT5 mutant.SAM competitive PRMT5 inhibitors, such as EPZ015666 and GSK3326595, have shown limitations in recapitulating the vulnerability of MTAP negative cells in response to PRMT5 inhibition14. Whether this axis is exploited by Quinacrine requires further exploration.Apoptosis was not observed as a mechanism of reduced clonogenicity; the phenotype was cytostatic, but not restricted to any phase of the cell cycle. Recent studies suggest that small molecule PRMT5 inhibition, either directly or indirectly through inhibition of cyclin dependent kinases 4 and 6, alters RNA splicing, leading to an MDM4 dependent activation of p53/p2115. We showed that c-jun is essential in driving PRMT5 expression and mediating Quinacrine-induced apoptosis. This study therefore implicates c-jun or SAPK pathway regulation as a mechanism underlying PRMT5 transcriptional repression by Quinacrine.Quinacrine has been reported to regulate c-jun phosphorylation at positions 349 to 340 and 266 to 257Based on early pharmacokinetic studies conducted with Quinacrine in the 1940′s, plasma concentration was measured as 225 nM at a dose of 100 mg, 3 times a day. However, hepatic and leucocyte distributions are high, with high plasma binding. This agent can however be safely instilled into the pleural cavity at a much higher dose of 5 mM, at which dose it acts as a sclerosant. This implies that lower micromole doses are achievable and could be locally administered, achieving localised exposure to mesothelioma at concentrations capable of suppressing PRMT5 expression.Our results provide a proof of concept to support the use of small molecule transcriptional perturbation to leverage a somatic-mutation based vulnerability, suggesting a repurposing potential that warrants further study.16 was used for validation and clinico-pathological characteristics are described in Supplementary Table Seventy-nine patient MPM samples were obtained at the time of extended pleurectomy decortication (EPD) under ethical approval. This study was approved by the London-Fulham Research Ethics Committee (reference 14/LO/1527), The Northampton Research Ethics Committee (reference 14/EM/1159) and University Hospitals Leicester NHS Trust Research and Innovation Department . Patient clinico-pathological characteristics are outlined in Supplementary Table 17. The BioDiscovery Nexus Express 10.0 for OncoScan software was then used to define copy number alterations and loss of heterozygosity as previously described18.DNA was extracted with the GeneRead DNA FFPE kit . 80 ng of gDNA were analysed using the OncoScan FFPE Assay Kit , which utilizes molecular inversion probe (MIPs) technologyQuinacrine was purchased from Sigma . The antibodies against MEP50, PRMT5 and c-JUN were obtained from Cell Signaling , MTAP antibody was purchased from Santacruz , H4R3me2S and β-tubulin were obtained from Abcam . Goat anti-rabbit HRP and donkey anti-mouse HRP secondary antibodies were from Cell Signaling .MPM cell lines: NCI-H2052, were purchased from ATCC . MPP89 and NCI-H2591 were kindly provided by Dr. P.W. Szlosarek, Institute of Cancer at Barts, London, UK. Cell lines were grown in RPMI Medium 1640, 1% L-Glutamine and 10% FBS .19. Each treatment condition was measured in triplicate.5000 cells per well were seeded in 12 well plates and left untreated or treated with Quinacrine . Cells were fixed between days five and seven (once enough colonies had formed in the control) on ice in methanol for 10 min. Cells were then stained with crystal violet  for 20 min. Colonies were dissolved in 30% acetic acid to allow quantificationThe non-silencing control (NT), siPRMT5, siWDR77, sic-JUN, siCEBPB, siNF-YA were obtained from Qiagen . siRNA transfections (20 nM) were performed using the RNAiMAX transfection reagent  according to manufacturer’s instructions. Validation of siPRMT5 and siWDR77 was carried out with a second sequence from Dharmacon .The xCELLigence RTCA DP instrument  was used as described in the manufacturer´s instruction manual. Cells  were seeded in E16-Plates. The cell indices were measured every 15 min for 120 h. Each treatment condition was measured in triplicate.18. The genes’ ranks were then combined across these datasets to obtain an overall rank for each gene to determine its inclusion to the PRMT5 gene signature for subsequent connectivity mapping analysis. A gene signature progression approach19 determined that an 8-gene signature was the optimal length including PRMT5 and its 7 strongest co-expression correlates PSMB5, HNRNPC, APEX1, HNRNPC, IPO4, TOX4, and TUBB. This 8-gene signature was used as an input to query a collection of 83,939 reference drug gene expression profiles20 covering 1353 FDA approved drugs (http://www.lincscloud.org). This connectivity mapping analysis was conducted in the framework of sscMap22. In our analysis all the individual reference profiles with the same drug formed a reference set. A set score was then calculated between the gene signature and each reference set, and the associated p-value was estimated by generating a large number of random gene signatures of the same length. Any signature-drug connections with a p-value no greater than a pre-set threshold (1/1353 = 7.4e−4) were declared as statistically significant. Additionally, gene signature perturbation analysis23 was performed to obtain the robustness (perturbation stability) of the significant signature-drug connections. Only the significant drugs that had 100% perturbation stability were selected for further consideration. Finally, significant drugs were ranked by the absolute value of their connection z-score to the PRMT5 gene signature.A PRMT5-centred gene signature was created from co-expression analyses of 9 independent Gene Expression Omnibus (GEO) datasets GSE37745, GSE50081, GSE28571, GSE77803, GSE43580, GSE19804, GSE18842, GSE10245, and GSE19188. Within each dataset, PRMT5 was used as the seed gene, with which the gene expression correlation coefficients for other genes (probes) were calculated. All genes were then ranked based on the magnitude and statistical significance of their correlations with the seed, following the ranking method described inPRMT5 chemoluminescent assay was purchased from AMS Biotechnology (Europe) Ltd . Quinacrine or EPZ015666 was added to the plate pre-coated with histone H4 peptide substrate. PRMT5 enzymatic activity was measured after reaction with the antibody against methylated arginine3 residue of Histone H4, the secondary HRP-labeled antibody, S-adenosylmethionine, methyltransferase assay buffer, and purified PRMT5 enzyme, according to the manufacturer instructions.Seventy-two hours after treatment cells were lysed in RIPA buffer containing protease inhibitors . Lysates were clarified by centrifugation at 4 °C at 13,000 rpm for 10 min. 40 µg of total cell lysates were loaded on SDS-PAGE gels. Signal detection was performed with ECL-plus chemiluminescent system .Total RNA was extracted using Trizol  according to manufacturer’s instructions. Reverse transcription was performed with High Capacity RNA-to-cDNA Kit . Real-Time PCR was carried out using Power SYBR Green PCR Master Mix  after 72 h of silencing or treatment with Quinacrine. QuantiTect primer assays  were used for PRMT5, c-JUN and Actin.. The luciferase activity was then measured by a Dual-Luciferase reporter assay system . Luciferase activity was normalized to Renilla activity19.Cells were transfected with pGL2 basic or pGL2-PRMT5 and Renilla by using the Xtreme gene transfection reagent  according to the manufacturer’s instructions. After 24 h cells were treated with Quinacrine and 72 h after transfection cells were lysed and stored at -80 °C for at least 24 hNCI-H2591 cells were transfected with 2 µg of PRMT5 (WT or mutant) or empty vector, using the Xtreme gene transfection reagent  according to the manufacturer’s instructions. Selection of stable clones was performed with G418 500 μg/ml .The Quick change II site directed mutagenesis kit  was used according to manufacturer instructions to introduce a mutation in PRMT5 (E444Q).t-test (two tails), or One-way ANOVA. All data are representative of the mean and standard deviation for at least three independent experiments. Kaplan–Meier curves for overall survival were used to assess survival estimates of the cohorts. Univariate analyses comparing the clinical variables with overall survival were performed using the log rank test and all statistically significant clinical variables were taken forward into a multivariate Cox regression model. The data was analysed using SPSS Version 25 . All p-values less than or equal to 0.05 were considered significant.Dose–response curves were fitted using non-linear regression . The significance of the data has been assessed with Supplementary Information


For this purpose, emulsion polymerization technique was exploited to fabricate poly core-poly(methyl methacrylate-glycidyl methacrylate) shell impact modifier particles with an average particle size of 407 nm. It was revealed that using a combination of the prepared CSIMPs and MWCNTs could significantly enhance the toughness and tensile properties of the epoxy resin. Also, it was observed that the dominant factors for improving the fracture toughness of the ternary composites are crack deflection/arresting as well as enlarged plastic deformation around the growing crack tip induced by the combination of rigid and soft particles. The Response Surface Methodology (RSM) with central composite design (CCD) was utilized to study the effects of the amounts of CSIMPs and MWCNTs on the physicomechanical properties of the epoxy resin. The proposed quadratic models were in accordance with the experimental results with correlation coefficient more than 98%. The optimum condition for maximum toughness, elastic modulus, and tensile strength was 3 wt% MWCNT and 1.03 wt% CSIMPs. The sample fabricated in the optimal condition indicated toughness, elastic modulus, and tensile strength equal to 2.2 MPa m However, due to their high density of three-dimensional molecular structure, hardly can they resist toward growth of crack. This serious drawback limits their applications in many critical fields where a notable fracture strength or low-temperature toughness is required, such as aerospace and automotive industries3. To deplete the severity of this issue, a plethora of different ingredients such as liquid rubbers, core–shell impact modifier particles (CSIMPs), and rigid organic and inorganic ingredients were used as impact modifiers particles (IMPs) of epoxy resin7.Epoxy resins, due to their great physicomechanical attributes such as good chemical resistance, outstanding adhesion, and exceptional dimensional stability, are of paramount importance in various structural applications8. Nonetheless, incorporation of such IMPs in epoxy formulation could result in deterioration of other desired properties of the host polymer 9. On the other hand, rigid IMPs not only could enhance the toughness of epoxy resin by crack pinning, crack deflection, and deflection/bifurcation effect of growing crack, but also could boost stiffness and modulus of the host polymer10. However, to reach a satisfying level of toughness, it is inevitable to use a relatively high dosage of such IMPs in the epoxy formulation. This obligation causes a dramatic increase in viscosity and subsequently inroads serious difficulties in the processability of the obtained composite11.Generally speaking, soft IMPs (i.e. liquid rubbers and CSIMPs) could endanger significant improvement in the toughness of epoxy resin through various mechanisms such as deboning, cavitation, and crazing16. The obtained ternary nanocomposite could overcome the mentioned drawbacks and cause synergistic influences on the toughness and other physicomechanical properties of the obtained composite products.Regarding the mentioned limitations of IMPs and their inherent differences from the viewpoint of toughening mechanisms, in the last decade, hybridization of different rigid and soft IMPs embedded into epoxy polymer matrix provided outstanding opportunities to the researchers for developing high-performance nanocomposites with enhanced toughness and physicomechanical properties17. Thus, among different rigid particles, it has been demonstrated that using carbon nanotubes as rigid fillers along with other soft IMPs could culminate a notable enhancement in fracture toughness of the obtained composites. Río et al. showed that the addition of a mixture of rigid carbon nanotube and soft polystyrene-polybutadiene-poly (methyl methacrylate) triblock copolymers led to the dramatic increase in both fracture toughness and fracture energy of the epoxy resin18. In another attempt, it was revealed that using block copolymer could ameliorate the dispersion state of carbon nanotubes and causes a synergistic effect on the toughness of epoxy resin19. Jojibabu et al. showed that ternary composite of triblock copolymer, modified carbon nanotubes, and epoxy resin possess a remarkable improved toughness in comparison with binary composites20.It is no doubt that outstanding attributes of carbon nanotubes such as their high tensile strength and stiffness, could remarkably enhance fracture toughness and physicomechanical properties of polymers21. Among different methods, the response surface method, which is a collection of mathematical and statistical techniques, has been used extensively to study and optimize the effect of diverse determining factors on the various parameters22. To the best of current authors' knowledge, there is no single published numerical and systematic study about the synergistic effects of MWCNTs and CSIMPs on the toughness and tensile properties of epoxy resin. Thus, a systematic study of the influence of multiwalled carbon nanotubes (MWCNTs) and CSIMPa on the mentioned attributes of epoxy resin is the main objective of this work. For this purpose, response surface methodology was used to find an applicable approximation function for predicting and optimization of the toughness and tensile properties of composites with various amounts of CSIMPs and MWCNTs.The technique of experimental design, due to its valuable assistance for comprehensive understanding of the process and interactive relationships between different variables, is known as a distinguished method in the engineering fields4. 7H2O), and ammonium persulphate (APS) from Aldrich, were used without any further purification. MWCNTs with the purity of > 95%, length of 5–15 µm, and outer diameter of 10–20 nm was purchased from Nutrino . The epoxy resin was standard diglycidylether of bisphenol A (DGEBA) (Epon828 from HEXION) with an epoxy equivalent molecular weight between 185 g/eq and 192 g/eq. The curing agent was cycloaliphatic polyamine hardener (Epicure F205 from HEXION) with hydroxyl equivalent weight of 102–106 g/eq. Deionized water (DIW) was used in all recipes.Monomers, glycidyl methacrylate (GMA), methyl methacrylate (MMA), allyl methacrylate (ALMA) from Merck Chemical Co., and butyl acrylate (BA) from Fluka were purchased and used as received. Emulsogen APS 100 from Clariant, ter-butyl perbenzoate (TBPB) from Merck Chemical Co., sodium formaldehyde sulfoxylate (SFS), ethylene diamine tertaacetic acid (EDTA), iron (II) sulfate heptahydrate (FeSOg) of the core and shell portions of the prepared CSIMPs were determined according to ASTM D 3418-08, by differential scanning calorimeter, DSC-Netzsch (England). The prepared dried sample was scanned in the temperature range between − 100 and 140 °C at a heating rate of 10 °C/min under nitrogen atmosphere. The Tg was defined as the midpoint of deflection of the baseline from the DSC thermogram. Size and external morphology of the prepared latex particles were investigated by scanning electron microscopy (SEM) with MIRA3 instrument from Tescan (The Czech Republic). Also, SEM images of the fracture surfaces of the epoxy specimens were used to assess the toughening mechanisms. The internal morphology of the prepared latex particles was studied by transmission electron microscopy (TEM) with CEM 902A ZEISS with an accelerating voltage of 80 keV . Tensile properties of the dog-bone-shaped specimens were obtained by a universal testing machine according to ASTM D-638. For each sample, the data reported are the average of five measurements. Fracture energy (GIC) and fracture toughness (KIC) were determined using a single-edge notch bend (SENB) specimen with dimensions of 6 × 12 × 52.8 mm. The tests were performed at a rate of 10 mm/min.FT-IR spectrum was recorded on FT-IR BRUKER-IFS 48 spectrophotometer (Germany) using KBr pellet. Glass transition temperature  were prepared through batchwise emulsion polymerization. For this purpose, 160 g DIW, 1 g Emulsogen APS 100, 30 g BA, and 0.16 g ALMA were premixed and charged into a 250 mL, four-necked round bottom glass reactor equipped with reflux condenser, thermocouple, crescent Teflon-steel mechanical stirrer, and feeding inlets. The content of the reactor was deoxygenated by purging with nitrogen for 15 min. The temperature was raised to 80 °C and the polymerization reaction was started by the addition of solution of the initiator (0.04 g APS dissolved at 10 mL water). The conversion of the polymerization reaction was followed by gravimetric analysis of samples withdrawn from the reactor at regular time intervals. After 120 min, the final conversion of the polymerization reaction was above 99%.4·7H2O, and 0.1 g SFS were added into the reactor. The redox emulsion copolymerization was proceeded for 3 h at 30 °C to reach conversion of > 98%. Then, the prepared latex was filtered through a 53 mm sieve and the filtrate was freezing for 12 h and freeze-drying for another 12 h.In the second stage, copolymerization of MMA and GMA was occurred on the surface of the prepared elastomeric cores. For that, 200 g of the prepared latex, 9 g MMA, 1 g GMA, and 0.1 g TBPB charged into the reactor. The content of the reactor was mixed for 30 min at room temperature. Then, 10 mL of the aqueous solution of 0.02 g EDTA, 0.01 g FeSOThe CCD of RSM was used to assess the effects of the contents of MWCNTs and CSIMPs as independent factors on the fracture toughness and tensile properties of epoxy composites. Table o is the constant, bi is the linear effect of the factor xi , bii is the quadratic effect of the factor xi and bij is the linear interaction effect between the input factors, xi and xj.Table To evaluate the effect of CSIMPs and MWCNTs on the physicomechanical properties of epoxy resin, specimens with different contents of these reinforcing fillers were fabricated according to the Table 23. Here, thanks to these features, impact modifier particles with elastomeric core and epoxy-functionalized plastic shell were prepared successfully. A schematic illustration of the preparation steps of the CSIMPs is shown in Fig. The capability of emulsion polymerization in preparing colloidal particles with disparate morphologies, physical, and chemical attributes makes it a unique and enticing polymerization method24. Linked to these rationales, in this work, ALMA was chosen as a graflinking monomer in the formulation of the prepared elastomeric cores of CSIMPs. To fabricate the plastic shell, redox emulsion copolymerization of MMA and GMA was occurred at the interfacial region of elastomeric cores and aqueous medium. It was suggested that using a swelling time for dissolving monomers in poly (BA-ALMA) colloidal particles, as well as proceeding the radical formation reactions at the interfacial region, could effectively minimize the probability of micellar and homogeneous nucleation25. Also, it is surmised that such swelling time could significantly enhance the grafting reaction between the elastomeric core and the glassy shell. This reaction could enhance the stability of the formed shell toward dissolution and separation from the elastomeric core during mixing of CSIMPs with epoxy polymer.It is well known that using monomers such as ALMA, which has unsaturated bonds with different reactivity, could significantly enhance grafting efficiency, gel content, and structural stability of CSIMPs29. For example, it has been demonstrated that by decreasing the particle size of various elastomeric impact modifiers such as carboxyl-terminated butadiene acrylonitrile (CTBN) or CSIMPs their resistance towards cavitation increased26. Kim et al. showed that due to the difficulty in cavitation of rubbery core, toughening effectiveness of poly (butyl acrylate) core- poly (methyl methacrylate) shell particles decreased for particle sizes smaller than 0.2 µm27. However, fracture toughness was constant in the bigger particle sizes ranges. Here, the measured average particle size of the prepared poly (ALMA-BA) core is in good accord with the size of the elastomeric core of the other prepared CSIMPs which could remarkably enhance the fracture toughness of epoxy polymer29. Thus, it is surmised that the fabricated elastomeric particles could efficiently ameliorate the toughness of epoxy resin through toughening mechanisms such as induced plastic deformation and cavitation.The electron microscopy results of the prepared CSIMPs Fig.  manifest29. Thus, the lower thickness of the shell could remarkably facilitate the transferring stress from polymer matrix toward the rubbery core and enhance the performance of the CSIMPs. On the other hand, the adequate thickness of the formed glassy shell could guarantee the compatibility between CSIMPs and epoxy matrix and enhance the dispersion state of the CSIMPs by preventing their coalescence during the drying and compounding process. The low measured average thickness of the shell and the free-flowing state of the synthesized CSIMPs at their powdery form substantiated that the thickness of the shell is in a suitable range for meeting the cited challenges30.The most prominent role of the glassy shell of CSIMPs is making a compatible layer between the elastomeric core and epoxy matrix. This layer provides a stress bridge for transferring of the energy of the imposed load from the matrix to the rubbery core of CSIMPs−1 and 1160 cm−1 are attributed to the C=O and C–O stretching vibrations of ester groups, respectively. The absorption peaks at 1230–1280 cm−1, 815–950 cm−1, and 750–770 cm−1, correspond to the symmetric stretching vibration and bending mode of the epoxy groups and confirm that the exploited GMA monomer could successfully functionalize the CSIMPs by epoxy rings20.Figure g of the elastomeric core. The observed difference between the measured Tg of the elastomeric core and reported values for poly (butyl acrylate) could be due to its crosslinked structure and induced restriction by the grafting glassy shell of CSIMPs25. The second glass transition temperature (≈ 109 °C) corresponds to the grafted poly (MMA-GMA) shell. The observed distinct differences between measured Tgs of the core and shell portions of CSIMPs confirmed that exploited monomers in each step of the preparation procedure could form discrete phases31.DSC thermogram of the prepared CSIMPs revealed two main transition temperatures Fig. b. The fiIC) of the toughened specimens (responses) as a function of the contents of MWCNTs  and CSIMPs . The following equations express the overall predictive model in terms of the coded variables:The results in Table 2, and adjusted R2. The ANOVA results , which are listed in Table The importance and statistical significance of the quadratic models were evaluated by the analysis of variance (ANOVA), which has been presented in Table 32. Also, it can be seen that the addition of CSIMPs could not effectively alter Young’s modulus of the host polymer up to 2.2 wt%. The plausible explanation for this observation could be due to the relatively low dosage of the used CSIMPs and also their ability to establish covalent linkages with epoxy matrix33. The latter phenomena could effectively form spatial hindrance for polymer chains and increase the crosslink density of the final composite34. Nevertheless, this effect could partially be compensated by the elastomeric core of the CSIMPs and result in the observed plateau region of modulus as a function of the content of CSIMPs. However, at higher levels of CSIMPs (> 2.2 wt%), an increase in the modulus of the prepared composites was observed which could be attributed to the increasing in the content of the glassy functional shell of the CSIMPs in the composites structures35. As it can be seen clearly from the obtained contour plot, the measured modulus of the fabricated ternary composites could be explained by the cited rationales for individual effects of MWCNTs and CSIMPs.The obtained response surface and contour plots provide a procedure to predict the mechanical properties of the prepared epoxy specimens reinforced with various amounts of MWCNTs and CSIMPs. As it can be seen obviously in Fig. 36. This influence could be seen as a remarkable improvement in the tensile strength by increasing the content of MWCNTs in Fig. 28. The obtained S values of ternary composites revealed that the effect of CSIMPs on the obtained results is more prominent than MWCNTs. In other words, at a constant concentration of MWCNTs, the addition of CSIMPs render a significant decrease in the tensile strength of the obtained nanocomposite, which could be attributed to the elastomeric portion of CSIMPs.It is well known that introducing MWCNTs in the epoxy matrix could exert significant influence over the tensile strength of the obtained compositeIC of epoxy resin. However, as it is expected, the influence of CSIMPs is more prominent than MWCNTs. Moreover, it is observed that for the binary composites of MWCNTs/epoxy the measured fracture toughness is not directly proportional to the amounts of MWCNTs. This observation is probably due to the dominant aggregation of MWCNTs at higher concentration of this material37. To put it in another way, it is well-known that due to the enormous specific surface area of MWCNTs, they have strong tendency for creating clusters within the epoxy matrix. The formed clusters of MWCNTs could decrease the interfacial surface area between the epoxy resin and rigid fillers, which consequently can reduce their interactions with growing cracks and decrease the toughening performance of MWCNTs. An enticing additional insight is the synergistic toughening effects of MWCNTs and CSIMPs. It can be seen conspicuously that the ternary composite with 3 wt% MWCNTs and 3 wt% CSIMPs possesses the highest value of fracture toughness. This observation shows that the employed rigid and soft fillers could exert significant positive influence over the fracture toughness of epoxy resin. It is substantiated that the dispersion state of MWCNTs could be improved significantly by the addition of polymeric impact modifiers18. Here, it is suggested that the added CSIMPs could decrease the aggregated structures of MWCNTs and facilitate their dispersion in the epoxy matrix.The influence of the contents of MWCNTs and CSIMPs on the fracture toughness of epoxy resin is shown in Fig. IC. The optimum values of selected factors were obtained by solving the regression equations  for the studied properties. The optimum condition for maximum Young’s modulus, tensile strength, and toughness of the epoxy composite obtained from RSM was 3 wt% MWCNTs and 1.03 wt% CSIMPs in the epoxy matrix. The sample prepared in optimal conditions indicated module = 3014 MPa, tensile strength = 40.57 MPa, and KIC = 2.2 MPa m1/2. The obtained results indicated that the optimized ternary composites possess a good balance between Young’s modulus, tensile strength, and toughness, which could not be achieved by other binary ones. The SEM images of the fractographes substantiated that combination of MWCNTs and CSIMPs could merge their toughening mechanisms and cause a synergistic toughening effect on the obtained ternary composite.In this study, for the first time CCD/RSM design was adopted to determine the optimum condition for tensile properties and fracture toughness of epoxy/MWCNT/CSIMPs ternary composite. The CCD/RSM prepared sufficient statistical data to fit a quadratic model in terms of the wt% MWCNTs and wt% CSIMPs in epoxy resin, on the tensile and toughness properties. The significance of the proposed models was indicated by the p-value of Regression < 0.0001 and P-value of LOF > 0.05. Analysis of variance showed good coefficient of determination values, R


We assessed an effect of an embedded electro-conductive multiwalled carbon nanotube nanopaper in an epoxy matrix on the release of the frozen actuation force and the actuation torque in the carbon nanotube nanopaper/epoxy composite after heating above its glass transition temperature. The presence of the nanopaper augmented the recovery of the actuation stress by the factor of two in comparison with the pure epoxy strips. We proposed a procedure that allowed us to assess this composite strengthening mechanism. The strengthening of the composite was attributed to the interlocking of the carbon nanotubes with the epoxy. When reheated, the composite samples, which contained stretched mutually intertwined nanotubes and epoxy segments, released a greater actuation stress then the epoxy samples, which comprised of less elastic networks of crosslinked segments of pure epoxy. A carbon nanotube (CNT) nanopaper, also called buckypaper, is an aggregate of entangled carbon nanotubes (CNTs) produced by a filtering of a CNT suspension onto a membrane support. After drying, the nanopaper is removed from the support, leaving a free-standing electrically and thermally conductive structure. Properties of the nanopaper can be tailored by a selective attachment of organic or inorganic moieties to CNT surfaces . Such moRecently, we have combined a multiwalled carbon nanotube nanopaper and an epoxy resin into a reinforced thermosetting polymer matrix composite and demonstrated its versatile use for the self-regulation of the epoxy curing temperatures as well as for the fast and efficient debonding of objects glued by this MWCNT nanopaper/epoxy composite . FurtherTg and subjected to an external load, bonding between its components rearrange to adapt to the external load. Such an altered configuration is locked when the composite cools below its Tg, yet, once the composite is re-heated above its Tg, the original shape is fully or partially recovered ). The prepolymer was prepared with a sufficient excess of epichlorohydrin, so the termination was by free oxirane rings, which were capable of reacting chemically with amine groups. The component B (Trimethylolpropane tris  ether [CAS: 39423-51-3]) was a hardener or a curing agent containing three polymeric chains of poly(propylene glycol) terminated by amine groups. Structural formulae of components are shown in To prepare the electroconductive MWCNT nanopaper/epoxy composite strip (40 mm × 20 mm), at first were two Cu strip electrodes glued at the opposite sides of the about 300 μm-thick nanopaper. Next, the porous nanopaper was laid on a polytetrafluorethylene (PTFE) foil and its pores were filled up with ca. 0.2 mL of the above described epoxy resin and curing agent at the A:B ratio 100:75. After filling, the nanopaper permeated by the epoxy resin and curing agent mixture was covered with another PTFE foil and loaded with a pressure of 300 kPa to avoid wrinkling in the course of curing by Joule heating at temperature around 60 °C for 15 min. The measured electrical conductivity of the resulting MWCNT nanopaper/epoxy composite was 3.16 S/cm. The glass transition temperature of the epoxy matrix with the embedded nanopaper, measured as the peak temperature by the DSC, was 53.9 °C.The structure of the MWCNT nanopaper and the cross-section of the nanopaper/epoxy composite were analyzed by the scanning electron microscope (SEM) Nova NanoSEM 450 . The surface temperature of the specimens was measured by means of the thermal camera Flir E5-XT , which was able to create a detailed temperature pattern.The sensor resistance was measured lengthwise by the two-point technique using the fast-scanning data logger Multiplex datalogger 34980A , which stored the readouts once per second. The DC power supply AX 502  was used to power Joule heating of the composites. Tensile tests were carried out using the Testometric M350-5CT system .Tg, and then let to cool down at ambient temperature while subjected to a given tensile strain. Afterwards, the stretched glassy strips were reheated to the rubbery state while measuring an increase of the actuation force in the strips. The composite strips were heated by Joule heating and the epoxy strips by warm air. In the former, two electrodes were attached lengthwise to the opposite sides of the strip. These electrodes were made of Cuprexit (a thin copper layer supported by a 0.22 mm thick fiberglass plate) to which conductive wires were soldered. In the latter, a hot air blower Triac PID  was used for heating. The distance of the blower from the epoxy strip was about 30 cm and the temperature of the outgoing air was 200 °C. In both cases, the temperature was measured in the center of the strip by a thermal camera as well as times necessary for reaching of given temperature values.Two different tests of a strengthening mechanism of electrothermal actuation were done to assess the role of an embedded MWCNT nanopaper. In the first test, the stretched MWCNT nanopaper/epoxy composite strips and the epoxy strips were heated above their respective Tg, i.e., to 65 °C and 47 °C, respectively. At these temperatures, the samples went from being rigid and glassy to being rubbery, flexible, and highly formable. When a bending force was applied, the strips were easily bent by hand at right angles around a cylinder with a 5 mm diameter. The altered shape held after the strips had cooled down to room temperature. The bent strips were attached to a pad by a double-sided tape, heated again to 65 °C and 47 °C, respectively, and fully or partially straightened. The straightening angle was measured visually by a comparison with a scale.The second test assessed a straightening of bent strips, which was induced by Joule heating in the MWCNT nanopaper/epoxy composite samples or by warm air in the pure epoxy. The composite and epoxy strip were warmed about 12 °C above their gT. According to the supplier, the ratio of the epoxy components (A/B-hard/elastic) 100:50 yields a hard epoxy of hardness 79 Shore D, while the component ratio of 100:100 yields a flexible one (44 Shore A). The glass transition temperatures Tg of the epoxy with component ratio 100:100, 100:75 and 100:50 were determined by the differential scanning calorimetry (DSC) analysis  to be 18.4, 35.1 and 58.1 °C, respectively. The chosen mixing epoxy component ratio of 100:75 enabled that once the experimental epoxy samples were heated above their Tg of 35.1 °C, they were able to be formed manually. The curing time of such experiments samples was 48 h at room temperature. The exothermic heat of the curing was determined by the DSC analysis as −275 J/g and the peak temperature 140 °C at the heating rate 10 °C/min. The cured epoxy was elastic and flexible (without any damage at bending over radius 70 mm), its ultimate tensile strain was 3.45% and hardness 66 Shore D.The poly(propylene glycol) chains themselves are very movable, capable to rotate around a single chemical bond. The chains contain oxygen, which has a relatively low rotational barrier and therefore the conformation of the chains can change easily under, e.g., an applied mechanical stress. The reaction of the two components A and B leads to a formation of a three-dimensional polymer network of a final epoxy matrix. At the A:B ratio 100:100, an excess of the component B causes that some poly(propylene glycol) branches remain unbounded because of a lack of oxirane rings with a high molecular mobility potential, and the epoxy is elastic. At the ratio 100:50, the proportion of functional groups for crosslinking between components A and B is more equimolecular. The epoxy contains less free unbounded poly(propylene glycol) branches, which results in a resistance against conformation changes, stiffness and a higher Tg temperatures. The composite elongated with strain up to the fracture stress of 2.4 MPa at the strain 5.5% and the cured epoxy up to the fracture stress of 0.95 MPa at the strain 7.5%. The strain dependency of stresses for the rubbery MWCNT nanopaper/epoxy composite and the epoxy was linear and the Young’s moduli, which are defined as E = σ/ε  were 46 MPa and 24 MPa, respectively.A tensile test was carried out to determine the fracture tensile stress and the strain of the MWCNT nanopaper/epoxy composite and the cured epoxy at the temperature 75 °C, which was above their respective Tg temperatures, are shown also in Similar data taken for illustration from our paper  for the Tg transition between the glassy and the rubbery strip state as the raising temperature gradually released frozen arrangements of the epoxy crosslinked structure and concurrently longitudinally stretched and transversely compressed the nanotube structure of the MWCNT nanopaper. The recovered stress in the rubbery composite strips between the minimum and the maximum stress values plotted in Tg temperature of 35.1 °C was exceeded in about 3 s after the start of heating, the stress in the epoxy strips regained rapidly. On the other hand, a stress recovery in the composite strips proceeded slightly slower, since the Tg temperature of 53.9 °C was not exceeded until 8 s after the start of heating. Obviously, the sooner the frozen actuation stress was released, the shorter was the stress recovery time.Next, we assessed a strengthening mechanism of the electrothermal actuation in the MWCNT nanopaper/epoxy composite. The extended MWCNT nanopaper/epoxy composite and the epoxy strips were cooled down from 75 °C to the ambient temperature of 25 °C and then reheated to the rubbery state at 75 °C at the rate 2.7 °C/s and 3.3 °C/s for the composite and the pure epoxy strips, respectively. When the strips were heated again, the stress response in the pre-deformed strips initially began to decrease due to the thermal expansion of the strips. Then, the actuation stress started to increase at the The final actuation stress in the composite strips was two-times higher than the actuation stress in the epoxy strips at the corresponding strain pre-deformation, which corresponded to the ratio of their Young’s moduli as shown in After an extension of the composite over the nanopaper ultimate strain 0.8% , the embtransverse/εaxial. The measured Poisson’s ratio ranged from 0.5 at a small compression to 0.29 at the final compression by the stress of 4 MPa, which induced a compressive strain of around 50%.The structural integrity of the nanopaper was tested only in compression. In the course of the compression, the nanopaper extended in the direction transverse to the direction of the compression force. The deformation of a material transverse to the direction of loading is called the Poisson effect. Its size is characterized by the Poisson’s ratio, which is the ratio of the positive transverse strain to the negative axial strain ν= −εA SEM analysis of the nanopaper surface after the compressive deformation indicated that the integrity of the nanotube network was disrupted by randomly located cracks. These local cracks ran transverse to the extension direction and they were partially bridged by the remaining interconnected nanotubes . The vieTg, the straight strips were bent at a right angle, and once the samples cooled to the room temperature, this shape was fixed.In the second shape recovery test, the return of the bent MWCNT nanopaper/epoxy composite strip and the epoxy strip to their original straight shapes was assessed. After heating above Sr (%) = (αi − αf)/ αi × 100, where αi denotes the initial bending angle (90°) and αf the final bending angle. At the final transformation temperature of 76.2 °C the straightening of the bent strip to the straightened one (Sr = 100%) was achieved in about 43 s. At 47.1 °C, i.e., at the temperature below Tg (53.6 °C), the final unbent angle 64° and the maximum Sr only 29% was achieved in about 600 s. The corresponding straightening speed, which was defined as Srmax/t, where Srmax denoted the maximum straightening ratio, was Srmax/t = 100/43 = 2.3 at the temperature of 76.2 °C and Srmax/t = 29/621 = 0.05 at the temperature of 47.1 °C.When the bent strip was reheated by Joule heating with an electric current of different actuating voltages, the frozen actuation stress in the embedded MWCNT nanopaper and the epoxy matrix was released, which drove the bent strip to restore to some degree its original straight shape. Depending on the final transformation temperature, the rearrangement of the deformed networks of the embedded nanotubes and the crosslinking of epoxy molecules was fully or partially restored to their arrangement before bending . Such a Tg exerted a torque, which unbent the composite strip. Specifically, the strength of the unbending torque in the MWCNT nanopaper/epoxy composite straightened the strips from the right-angled shape bent down , and then the strips were released to unbend. The strip straightening ratio started with the full  straightening at the zero load and ended with the incomplete straightening of 49% at the maximum load of 4.5 g  than in the composite strips (Tg = 53.9 °C), while in the second tests, the underlying mechanism was mainly affected by the entanglement of the epoxy segments and the carbon nanotubes of the porous nanopaper as discussed in the following Section. The differences found in the times and straightening ratio values of the strips with comparable loads suggested higher actuation torques for composite strips than for epoxy strips. On the other hand, the straightening of the composite strips proceeded more slowly than that of the epoxy strips.The straightening of the heated bent composite strips was compared to the straightening of the epoxy strips under comparable conditions, see Tg of the composite in comparison to the pure epoxy . The DSC analysis after the physical aging of the epoxy and the MWCNT nanopaper/epoxy composite specimens suggested only one enthalpy relaxation peak, whose area represented the amount of energy involved in the specimen transition from the glassy state to the rubbery state. The physical aging was initiated by a change in the temperature of the pure epoxy and the composite from equilibrium at approximately Tg + 20 °C for 20 min to the annealing temperature Tg − 5 °C, which was reached by a cooling at the rate 10 °C/min. After the required period of aging ta the samples were reheated at the rate of 10 °C/min to Tg + 20 °C. The obtained DSC curves and the peak temperature plot shown in Tp as the aging time increased [The embedded nanopaper substantially increased the glass transition temperature ncreased . A shiftTg. At corresponding deformations, the recovered actuation stress in the composite strips was almost two-times higher than the recovered actuation stress in the pure epoxy strips, which corresponded to the ratio of their Young’s moduli as shown in Tg, the shape recovery is initiated by detaching of the secondary crosslinks and releasing of the stored strain energy. After the elongation and the subsequent locking of the shape of the MWCNT nanopaper/epoxy composite strip by a cooling below Tg the embedded nanopaper was held in the stretched state by the frozen epoxy. Apparently, the individual nanotubes of the extended nanopaper network, which were intertwined with each other and with epoxy segments, were straightened longitudinally and their mechanical bonds shifted. When the nanopaper elongated longitudinally, it simultaneously compressed in the transverse direction. Successive cycles of loading and unloading causing a compressive deformation up to about 55% of a self-standing MWCNT nanopaper proved that the nanotube rearrangement is steady and reversible [Tg and the nanotube network remained conductive, since it was still possible to use Joule heating to heat the composite as shown in Tg to the transformation temperature of the shape memory, the epoxy matrix softened and the shape recovery stress in the embedded MWCNT nanopaper was released by detaching of the secondary crosslinks and by releasing of the stored epoxy strain energy of stretched segments within chemical crosslinks. At the same time, when the frozen actuation force in the epoxy was released, the deformed nanopaper nanotubes began to relax to their initial positions before deformation and thus the actuation force of the composite increased when compared to the pure epoxy.We embedded an electro-conductive MWCNT nanopaper in an epoxy matrix, and assessed its effect on the release of the frozen actuation force and the actuation torque after heating of the composite above its versible . Similarversible . When thThe dependence of the release of the reversible force in the composite on the release of frozen polymer epoxy segments was confirmed by the measurement of different degrees of release of the frozen force at different temperatures . An inteWe proposed a measurement that allowed us to assess and analyze the strengthening mechanism of the electrothermal actuation by Joule heating in a composite consisting of a multiwalled carbon nanotube nanopaper embedded in an epoxy matrix. The measurement was performed by reheating of the straight glassy composite and pure epoxy strips, which held in the pre-stretched state by the frozen actuation stress, and by monitoring the release of the actuation stress in the strip samples during the glass/rubber transition. We found that the regained actuation stress in the composite strips was two-times higher than the similar stress in the pure epoxy strips at corresponding pre-stretched levels. This was because the more elastic structure of the stretched mutually intertwined epoxy segments and nanotubes of the nanopaper released when reheated a greater actuation stress than the less elastic stretched network of crosslinked segments of the pure epoxy.


Three variants of the aging time in the above-mentioned operating environments were adopted: one month, two months, and three months. After the specified maturing time, samples of epoxy adhesive compositions were subjected to the strength tests on the Zwick/Roell 150 testing machine, which is in accordance with ISO 604 standard, determining the compressive strength. (3) Results: On the basis of the obtained strength test results and their analysis, it was noticed, inter alia, that the strength of the epoxy compounds decreases with the aging time in all used aqueous environments. Moreover, in the case of both types of the epoxy compounds, the highest strength was achieved after aging in demineralized water. A significant advantage of epoxy compounds as adhesive is the possibility of joining together most of the available materials (in any combinations and arrangements), such as glass, metal, wood, ceramics, polymer materials, etc. due to their excellent adhesion. There is no second method that allows for so many possibilities of joining the different materials. Another advantage of epoxy compounds is good mechanical properties and resistance to many chemical factors, and the adhesive layer is characterized by excellent dielectric properties and allows the exclusion of moisture from entering the joint ,2,3,4.− and Na+ ions can accelerate hydrolization of the epoxy adhesive and enhance water ingress ability in the epoxy adhesive. This statement is also emphasized by Wang et al. [The operating conditions of epoxy compounds may have a large impact on their strength, as well as the strength of adhesive joints made with the use of various epoxy compounds ,5,6,7,8.g et al. .ca. 100% RH) at various elevated temperatures. Based on the results, it can be noticed that long-term exposure of the epoxy adhesive to water leads to both physical and chemical degradation of the material. Fernades et al. [Under conditions of high humidity, epoxy compounds may change their strength reversibly or irreversibly, which was presented and confirmed in many works ,10,12,13s et al.  presenteIn order to prevent the epoxy compounds from weakening the strength of the epoxy compounds, this problem should be anticipated at the beginning of the formulation.The issues analyzed in the study concern selected operating factors and their impact on the strength of epoxy compound. The subject of this study is to compare the effect of operating factors on the strength of epoxy compounds made of epoxy resin based on bisphenol A and two different curing agents: tritethylenetetramine and polyaminoamide C. A comparative analysis of the operating factors of the epoxy compounds will be carried out in terms of the compressive strength. This is to determine which operating environment is more favorable for the strength results obtained in the epoxy compounds.Two types of the epoxy compounds were used in the experimental tests, containing the epoxy resin made of bisphenol A  as the basic component and two types of curing agent: triethylentetraamine , and polyaminoamide C . The epoxy resin used is a synthetic resin made of epichlorohydrin (ECH) and dian . The epoxy number of the used epoxy resin amounts to 0.40 mol/100 g. This resin belongs to the group of low molecular weight resins. The prepared epoxy compounds are presented in Epidian 57 epoxy resin is a mixture of Epidian 5 epoxy resin  with modified polyester resin . It is aThe triethylenetetramine (TECZA) and polyaminoamide C curing agents were used to cure the epoxy compounds. The selected properties of the curing agents were presented in 6H18N4, and the structural formula is shown in The triethylenetetramine  is used for curing reactive resins . The cheIt is most often used with low molecular weight (liquid) epoxy resins or in various compounds based on them. The spatial cross-linking of epoxy resins occurs as a result of chemical reactions of functional groups contained in the resin (epoxy and hydroxide) with a properly selected curing agent. The curing process takes place when the curing agent is added to the resin and the gelling time begins from that moment. After the gelling period, the resin is quickly cured. It is important to remember that the use of the triethylenetetramine curing agent requires precise dosing and weighing, as any excess curing agent will shorten its gelation time. Incorrect weight proportions also result in deterioration of the properties of the cured material. The gel time of the triethylenetetramine curing at room temperature is about 35 min. Initial curing is obtained after about 3–4 h, and after 48 h, the degree of curing of the composition is about 80–90%. It takes 7 to 14 days to fully cure. When there is a need to shorten the curing time of a given compound, the triethylenetetramine curing agent can also be used for the curing at an increased temperature . The triPolyaminoamide C curing agent (PAC trade name) is used with the low molecular weight (liquid) epoxy resins and with various compounds based on them. The weight ratio of the polyaminoamide C curing agent to the epoxy resin can be varied within a fairly wide range by controlling the reaction rate and properties of the cured material . The polOne of these epoxy compounds shows the characteristics of a more rigid material (Epidian 57/Z-1/10:1), while the other composition (Epidian 57/PAC/1:1) shows the characteristics of a more flexible material than the first epoxy compounds.d = 20 mm and length L = 60 mm  was used as a silicone anti-adhesive agent, preventing the prepared the epoxy compounds from sticking to the molds. After spraying, the preparation creates a thin layer of silicone oil on the surface of the mold, which has an anti-adhesive effect. Silicone oil does not in any way affect the properties of the prepared epoxy compounds and does not cause some defects in the finished products. Before use, the agent was shaken and then sprayed onto the mold surface from a distance of about 30 cm. Three minutes after the release of the release agent, the mold was filled with the liquid epoxy compound. This process was carried out at a temperature of 22 ± 2 °C, with an air humidity of 21–23%.Proper preparation of the cylinder-shaped molds with dimensions Before applying the epoxy compounds to the molds, the selected components should be carefully combined. The combined components of the compound were mixed in the appropriate proportions resulting from the stoichiometric ratio  for E57/Z-1/10:1 epoxy compound and above the stoichiometric ratio for E57/PAC/1:1 epoxy compound. The mixture of Epidian 57 epoxy resin and polyaminoamide C curing agent was mixed in the ratio of 1: 1, while with the polyaminoamide C curing agent was mixed in the ratio of 10: 1. A TP-2/1 scale  was used to measure the ingredients, with a maximum weight of 2000 g and an accuracy of 0.1 g. The components of epoxy compounds were mixed for 90 s with a disk agitator at 128 m/min in a polymer container on the mixing station. The mixing continued for 2 min; then, the epoxy compounds were kept under vacuum for a further 2 min, in order to remove gases and avoid the formation of bubbles. An important element in creating the epoxy compound is to avoid the formation of air bubbles. Air bubbles can significantly reduce the strength of the epoxy compound. The epoxy compound preparation process was done at 22 ± 2 °C and 21–23% RH.In order for the epoxy compounds to be carefully made, special attention should be paid to the correct application of the compounds to the cylindrical molds, after prior careful preparation of the molds. Then, the epoxy compounds were subjected to a one-stage curing process for 7 days at a room temperature of 22 ± 2 °C and air humidity of 21–23%.2O. The water does not exist in nature as a pure hydrogen–oxygen compound. As one of the few substances, it can exist in three states of aggregation: gas, as water vapor, liquid, as rain, and solid, as ice. In each state of aggregation, water has the same chemical composition and similar structure, but its behavior may be different. Water is always a very dilute solution of acids, bases, salts, and gases. The chemical composition of any water depends on its origin. The rainwater contains a small amount of various admixtures. Surface waters are characterized by a high content of substances of industrial and natural origin. The water for the production of the drinking water undergoes various treatment processes. The treatment process depends on the amount and type of contaminants removed from it. There are several compounds in the chemical language that are called water, but they have a completely different composition than real water.The water (hydrogen oxide) is a compound composed of two hydrogen atoms and one oxygen atom: HThe experimental studies used the three types of water and a sweetened drink. The aqueous environments’ characteristics are presented in The rainwater is formed by the condensation of water vapor that is formed in the atmosphere and falls back to the Earth’s surface in the form of precipitation such as rain, snow, or hail. The composition of the rainwater depends on the purity of the air. The rainwater is characterized by a large amount of gases . It may also contain the microorganisms, the industrial dust, the soot, and small amounts of the mineral salts. The pH content in rainwater is about 6, which is acidic due to the content of dissolved carbon dioxide. The demineralized water is the water devoid of mineral salts and most of the other pollutants by repeated distillation. It contains dissolved gases .Tests of the cured epoxy compounds were carried out in accordance with ISO 604 on a test stand consisting of a Zwick/Roell Z150 testing machine  and a computer set connected to a testing machine. The samples were successively subjected to the strength tests at predetermined dates.The samples of the epoxy compounds were placed in a special holder, which in turn was placed in the flat jaws of the testing machine. Mounting the samples in an additional holder allowed maintaining the coaxiality of the sample in relation to the axis of the traverse and the action of the axial force as well as perpendicularity to the surface of the holders of the testing machine. The holder with mounted cylindrical samples of the epoxy compounds placed in the flat jaws of the testing machine is presented in The strength tests concerned the determination of the compressive strength, taking into account four different variants of the aqueous environments: rainwater, demineralized water, tap water, and sweetened drink. During the strength test, the initial force was 10 N, the traverse speed was 10 mm/min, and the maximum deformation was set at 15%.In all variants of aging, the highest compressive strength was achieved after aging in the demineralized water, and the lowest was achieved after aging in the tap water;Increasing the aging time reduces the value of the compressive strength of the samples of Epidian 57/Z-1/10 1 epoxy compound;The percentage decrease after 2 months of sample aging in the rainwater is 4.4%; for the demineralized water, it is 3%; for the tap water, it is 5.9%; and it is 4.9% for the sweetened drink;The percentage decrease after 3 months of the sample aging in the rainwater is 4.2%, while it is 4.7% for the demineralized water, 4% for the tap water, and 12.7% for the sweetened drink.Based on a comparison of the compressive strength test results , it was The highest maximum strength was achieved after aging in demineralized water; it amounts to 412 MPa (V1-DW);The lowest compressive strength of the epoxy compound in the cured state was achieved after aging in the tap water, amounting to 257 MPa, which is 62% of the highest value of the strength achieved after aging in the demineralized water;The compressive strength after aging in the rainwater is 62% of the highest compressive strength (258 MPa);After aging in the sweetened drink (in V1-SD), the average value of the compressive strength was achieved at the level of 408 MPa, which is 98% of the strength of the epoxy compounds aged in demineralized water.Comparing the results of the research in relation to the aging variant I (1 month), the following can be noted:The highest strength achieved after aging in the demineralized water (V2-DW) is 404 MPa;The lowest compressive strength of the epoxy compound in the cured state was achieved after aging in tap water (V2-TW) (242 MPa), which is 60% of the highest strength value (404 MPa);The compressive strength after aging in rainwater (V2-RW) is 39% lower than the highest compressive strength;After aging in the sweetened drink (V2-SD), the compressive strength is lower by 4% compared with the compounds aged in the demineralized water.When analyzing the test results for the 2nd variant of aging (2 months), it was noticed that:The highest maximum strength was achieved after aging in the demineralized water (V3-DW), and it amounts to 385 MPa, and the lowest compressive strength of the epoxy compounds in the hardened state was achieved after aging in the tap water (V3-TW), amounting to 232 MPa, which is 60.2% of the highest strength value;After aging in the rainwater (V3-RW), the compressive strength (237 MPa) is nearly 40% lower than the highest strength;After aging in the sweetened drink (V3-SD), the average value of the compressive strength was achieved at the level of 339 MPa, which is 88.0% of the strength of the epoxy compounds aged in the demineralized water.When considering the obtained test results in the case of the 3rd variant of aging (V3-3 months), it was noticed that:The strength of the considered epoxy compounds decreases with the increase of the aging time in various environments;The epoxy compounds aged in the demineralized water achieved the highest strength at any time of aging;The lowest strength is characteristic of the epoxy compounds immersed in the tap water, at any time of aging;The percentage decrease after 2 months of the epoxy compound samples aging in the rainwater is 7.7%; for the demineralized water, it is 0.11%; for the tap water, it is 5.7%; and for the sweetened drink, it is 15.1%;The percentage decrease after 3 months of sample aging in the rainwater is 13.5%, for the demineralized water, it is 2.4%, for the tap water, it is 28.7%, and for the sweetened drink, it is 13%.Considering the obtained results of the compressive strength of Epidian 57/PAC/1:1 epoxy compounds, presented in The highest maximum strength was achieved after aging in the demineralized water, and it amounts to 95.0 MPa. On the other hand, the lowest compressive strength of the epoxy compound samples in the hardened state was achieved after seasoning in the tap water, amounting to 8.7 MPa, which is 9.2% of the highest value of strength achieved in the demineralized water;The compressive strength of aging in the rainwater is 10.9% of the highest compressive strength;After aging in the sweetened drink, the average value of the compressive strength was achieved at the level of 34.5 MPa, which is 36.3% of the strength of the epoxy compounds aged in the demineralized water.Comparing the results of the research in relation to the aging variant I (V1), the following can be noted:The highest maximum strength was achieved after aging in the demineralized water and it amounts to 94.9 MPa, and the lowest was achieved after aging in the tap water, which was 8.2 MPa, and it constitutes 8.6% of the highest strength value;After aging in the rainwater for two months (V2-RW), the compressive strength is 90% lower than the compressive strength after aging in the demineralized water (V2-DW);The second variant of the aging in the sweetened drink (V2-SD) allowed achieving the compressive strength of 29.3 MPa, which is 30.9% of the strength of the epoxy compounds aged in the demineralized water (V2-DW).When analyzing the obtained values of the compressive strength for the 2nd variant of aging during 2 months (V2), the following can be noted:The compressive strength after aging for three months reached the highest value after aging in the demineralized water (V3-DW)—67.7 MPa, and the lowest was achieved in tap water, amounting to 8.0 MPa (V3-TW), which is 11.8% of the highest strength value;After aging in rainwater (V3-RW), the compressive strength is 12.3% of the highest compressive strength;After aging in the sweetened drink (V3-SD), the average value of the compressive strength was achieved at the level of 25.5 MPa, which constitutes 37.7% of the strength of the epoxy compounds seasoned in the demineralized water (V3-DW).The compressive strength for the 3rd aging variant (V3) was as follows:The images of Epidian 57/Z-1/10:1 epoxy compounds samples after the strength tests  are presented in The visual analysis of the samples of the epoxy compounds subjected to various aging variants  was madeIt was noticed that in the case of water, the color changed very little with regard to the different types of water used. On the other hand, the use of aging in the sweetened drink caused a significant change in the color of the samples. Initially, the samples had a milky white color, while after aging in the sweetened drink, the samples turned brown. The intensity of this color increased with the aging time in this solution. The epoxy compound samples aged in the sweetened drink also show a similar nature of damage for variant I and variant II. On the other hand, aging for 3 months in the sweetened drink (V3-SD) seems to have less influence on the cohesive failure of the samples than in the case of other water environments . In thisThe Epidian 57/PAC/1:1 epoxy compounds samples after the strength tests are presented in When analyzing the shape and appearance of the Epidian 57PAC/1:1 epoxy compound samples after the strength tests, minor scratches with no distinct signs of samples deformation were observed . There aThere is a significant difference in the color of the samples when comparing samples aged in the three types of the water and in a sweetened drink. The epoxy compound samples aged in a sweetened drink take on a brown color, which is likely due to the type of aging time in the sweetened drink. Color intensity increases with increasing aging time. The darkest brown color occurred in the samples aged for 3 months (V3-SD). From the photos of aging in the sweetened drink in On the other hand, no significant differences in the type of deformation of the samples of epoxy compounds subjected to the aging in the sweetened drink were observed, when comparing individual variants of aging. There is also a larger mesh of cracks during the 3 months of seasoning in a sweetened drink as well as when aging in different types of water for 3 months. However, cracks and delamination at the ends of the epoxy compound samples can be noticed.By observing the results, it is possible to notice a different nature of the damage of the Epidian 57ZPAC/1:1 epoxy compounds than the samples made of the Epidian 57Z-1/10:1 epoxy compounds. The samples of the Epidian 57Z-1/10:1 epoxy compound are more prone to deformation, which is revealed by the test shapes buckling and spalling during compression.Differences were also noticed in the susceptibility to color change under the influence of dyes in the sweet drink. A significant color change was noticed in the case of the Epidian 57/PAC/1:1 epoxy compounds, with the intensity of the color (up to dark brown) increasing along with the aging time.From this, it can be assumed that the type of curing agent in the epoxy compounds contributes to the compound being more or less given a color change. This can be aesthetically important when making certain articles of this epoxy compounds .Okba et al.  underlin3O +) and hydroxyl (OH-) ions on the material. Polymer degradation processes are most often associated with structural changes in the polymer chain (and thus with a reduction in molecular weight). This is related to changes in the physicochemical properties of polymers . It was found that the samples subjected to aging in dematerialized water, i.e., water free of foreign ions, showed higher strength in the case of both type of epoxy compounds. It can be seen that in the analyzed case, differences in the strength values of epoxy compounds are noticeable. Tap water and rainwater have different ions, and in addition, rainwater may have additional impurities that may weaken the structure of the epoxy cross-linked material;Degradation is a process of structural changes that may be the result of physical or chemical changes occurring in polymer materials under the influence of long-term external factors. Most often, these interactions are synergistic and result in interactions between individual stimuli. The aqueous environment (mainly water) was the degradation factor in the work. Most of the materials subjected to long-term exposure to the aquatic environment show a change in some properties. The reduction of mechanical parameters, surface changes observed macroscopically and microscopically, as well as chemical changes are the result of the interaction of hydronium  the type of epoxy composition—the type of curing agent included in the epoxy compounds composition, containing the same epoxy resin, (ii) the type of the environment, and (iii) the aging time.It can be noticed that in many studies, various issues related to the influence of aging in various environments on the strength of epoxy compounds are considered and investigated. Both unmodified compositions and those modified with various fillers are tested. The research methodology is also different; therefore, not all research results are consistent in all aspects with the results presented in the literature. However, the trends or the differences in outcomes are worth noting, which may contribute to a more complete interpretation of the research or design of the experiment.The experimental tests concerned the compressive strength of the epoxy compounds in the cured state of the appropriate shape of the samples, where the aging time in various aqueous environments was an important factor.The compressive strength of the epoxy compounds decreases with the aging time in all used aqueous environments;The more flexible epoxy compound (Epidian 57/PAC/1:1) is characterized by lower compressive strength than the more rigid epoxy compound (Epidian 57/Z1/10:1);In the case of both types of prepared epoxy compounds, the highest strength was achieved after aging in the demineralized water, and the lowest was achieved after aging in the tap water, and this is independent of the type of aqueous environment. Therefore, it can be assumed that the content of various types of substances in the tap water adversely affects the mechanical properties of epoxy materials ;It can also be assumed that the rainwater (similarly to tap water) containing various substances also adversely affects the reduction of the compressive strength of the epoxy compounds compared to the strength of the epoxy compounds aged in the demineralized water, free of many substances and impurities;The results of the strength tests obtained after aging in the sweetened drink seem interesting, because this exploitation factor allows for obtaining better strength results than tap water or rainwater. Perhaps the chemical composition of this substance  contributes to such results, although the color should be mentioned after aging the samples of epoxy compound in the sweetened drink, which took a very unsightly appearance, obtaining a brown color (the base color is milk white—Epidian 57/Z-1/10:1 or milky-yellowish of Epidian 57/PAC/1:1).Based on the results of the strength tests and their analysis, the following conclusions can be drawn:In conclusion, it can be assumed that the strength of epoxy compounds is significantly influenced by both the type of epoxy compounds, the aging time, and the type of environment in which the compounds will be used. The knowledge of the factors influencing the strength of epoxy compounds allows creating the epoxy compound in such a way that they are characterized by the highest possible strength in selected operating environments. Based on the results obtained, further and extended testing of the epoxy compounds is planned.


Instead, in wild-type cells, moderate chemical pre-activation of the Wnt/β-catenin pathway promotes EpiLC in vitro derivation. Finally, we suggest that moderate β-catenin levels in β-catenin null mouse ESCs favor early stem cell commitment toward mesoderm if the exogenous protein is induced only in the “ground state” of pluripotency condition, or endoderm if the induction is maintained during the differentiation. Overall, our results confirm previous findings about the role of β-catenin in pluripotency and differentiation, while indicating a role for its doses in promoting specific differentiation programs.The Wnt/β-catenin pathway is involved in development, cancer, and embryonic stem cell (ESC) maintenance; its dual role in stem cell self-renewal and differentiation is still controversial. Here, by applying an   •in vitroModerate β-catenin levels promote EpiLCs derivation •Chemical pre-activation of the Wnt pathway enhances ESC-EpiLC transition•β-catenin overexpression tips the balance between mesoderm and endoderm•Cell fate is influenced by the extent of β-catenin induction Cell biology; Stem cells research The first step for stem cell differentiation is the exit from the pluripotent state, tightly controlled by signaling pathway and gene regulatory networks which can drive specific lineage commitment. During murine development epiblast , convertin vitro as an intermediate of ESC differentiation , but presented some differentiation defects when LIF deprived  and moderate chemical pre-activation of the Wnt/β-catenin pathway in wild-type ESCs promote efficient EpiLC in vitro derivation. Finally, the transcriptome of β-catenin−/− ESCs expressing different doses of exogenous β-catenin before and/or during differentiation confirmed what we and others reported about the dispensable requirement of β-catenin transcriptional activity for pluripotency establishment  ESC line we previously generated  resulting from endogenous protein induction or chemical pathway activation.To study the role of the Wnt/β-catenin pathway in EpiLC enerated . Briefly−/− ESCs  were modCs (S33Y A (Sadot Cs (S33Y . The indCs (S33Y A (PedoneCs (S33Y . The useWe confirmed in C1 cells the correct induction ), intrac−/− cell line we used had already been characterized for having a transcriptional profile similar to wild-type cells in pluripotent (serum/LIF) cultures, with Wnt signaling activation repressing ESC spontaneous differentiation in dependence of β-catenin  or in 2i/L. As prolonged culture in 2i/L results in epigenetic changes impairing normal differentiation in vitro and development in vivo , showed mixed behaviors upon Wnt/β-catenin pathway perturbations A; howeverbations E, suggesNext, we differentiated cells after pre-activation of the Wnt/β-catenin pathway in FBS/L condition A or 1 weAltogether, these results indicate that intermediate chemical pre-activation of the Wnt pathway in FBS/L-cultured wild-type cells favors the ESC-EpiLC transition using the standard differentiation protocol (AFD).Next, we studied C1 ESCs exit from the “ground state” of pluripotency, i.e., upon 2i/L withdrawal, by RNA sequencing; such monolayer differentiation protocol does not induce a specific cell fate and is well suited to observe possible β-catenin-dependent differentiation bias .C1 ESCs cultured in 2i/L for 3 passages  were treated for 48 h with saturating concentrations of dox and TMP  to induce the expression of the exogenous fusion protein A. TakingTo investigate the extent of the batch effect, we applied the ComBat-Seq method  considerPrincipal components analysis PCA, B showed Next, to explore the biological processes associated with β-catenin perturbations, we used weighted gene correlation network analysis (WGCNA) , a gene Next, to gain more information about the effects of β-catenin at the exit from pluripotency, we also correlated each module with β-catenin doses  analysis between specific pairs of samples and performed GOEA to identify the involved biological processes  and/or dynamic β-catenin levels on cell decision making. Moreover, we did not consider comparing transcriptionally competent lucia.marucci@bristol.ac.uk).Further information and requests can be addressed to Lucia Marucci (This study did not generate new unique reagents.−/− ESCs (S33Y(Puromycin). Cells were selected with Neomycin after the first round and with Puromycin after the last infection.C1 cell lines were previously derived in −/− ESCs  with theESCs were cultured on gelatin-coated dishes in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 15% fetal bovine serum (FBS), 1x nonessential amino acids, 1x GlutaMax, 1 x 2-mercaptoethanol, 1x Penicillin-Streptomycin and 1000 U/mL LIF. To note, for the 2i/L culture, cells were kept for 3 passages (around 1 week) in serum-free NDiff227-based media supplemented with 1000 U/mL LIF, 3μM of the GSK-3α/β inhibitor Chiron-99021 and 1μM of the MEK inhibitor PD0325901.in vitro we adapted the protocol for EpiSCs culture reported in 4 cells/cm2, on 10 μg/mL Fibronectin-coated 12-well plates in NDiff227. According to the experiment in For EpiLC derivation 4 cells/cm2 on gelatin-coated 12-well plates in plain NDiff227 and stimulated with DMSO or TMP10μM±dox10–100ng/mL for 4 days with the media and drugs refreshed after the first 2 culture days  and High (C1H) expressing ESCs were plated at 1.5×10ure days A.Some experimental conditions required pre-treatment of cells. For β-catenin induction in 2i/L pre-cultured ESCs were washed with sterile phosphate-buffered saline , trypsinised for 2–3′ at room temperature and centrifuged at 1000 × g for 5′. Pelleted cells were resuspended in 500μL of plain NDiff227 media supplemented with DAPI. The mCherry positive fraction was sorted from DAPI negative using the BD Influx high-speed 16-parameter fluorescence activated cell sorter.For quantitative PCR, the total RNA, extracted from cells using the PureLink RNA Mini Kit (Invitrogen), was retrotranscribed  and the cDNA used as template for each qPCR reaction in a 15μL reaction volume. iTaq Universal SYBR Green Supermix was used with the Qiagen Rotor-Gene System. To eliminate the contamination from genomic DNA, the RNeasy Plus Mini Kit (Qiagen) was used to purify the total RNA used for the RNA Sequencing. Oligos are reported in Preparation of libraries was performed with a total of 100ng of RNA from each sample using QuantSeq 3'mRNA-Seq Library prep kit  according to manufacturer's instructions. Total RNA was quantified using the Qubit 2.0 fluorimetric Assay (Thermo Fisher Scientific). Libraries were prepared from 100ng of total RNA using the QuantSeq 3' mRNA-Seq Library Prep Kit FWD for Illumina (Lexogen GmbH). Quality of libraries was assessed by using screen tape High sensitivity DNA D1000 (Agilent Technologies). Libraries were sequenced on a NovaSeq 6000 sequencing system using an S1, 100 cycles flow cell (Illumina Inc.). Amplified fragmented cDNA of 300 bp in size were sequenced in single-end mode with a read length of 100 bp.Illumina novaSeq base call (BCL) files are converted in fastq file through bcl2fastq.GSE148879). The lists of differentially expressed genes (DEGs), for each comparison, with a threshold of logFC >2 for the induced and logFC <−2 for the inhibited transcripts  to remove adapter sequences, poly-A tails and low-quality end bases . Alignment was performed with STAR 2.6.0a3  on mm10 nscripts  were use2) ≥ 0.85 was selected to build the weighted adjacency matrix. The weighted adjacency matrix was further transformed into a topological overlap matrix (TOM)  from theix (TOM)  and the ix (TOM)  statistiix (TOM) . The solDifferentially expressed genes (either logFC >2 or logFC <−2) and module “hubs” having high module membership  within the module were analysed for the enrichment in GO Biological Processes  and KEGGDifferences between samples were analysed by two-tailed unpaired t-test and one-way ANOVA with Bonferroni’s multiple comparison test using GraphPad. A p-value lower than 0.05 was considered statistically significant.Clustergram over heatmaps were generated using the clustergram function in Matlab that applies the Euclidean distance metric and average linkage. The data have been standardized across all samples for each gene and have 0 as mean and 1 as standard deviation.


Liquid rubber toughened epoxy resins are widely used in electrical equipment and electronic packaging. Previous studies have only investigated the relaxation process of epoxy resins through dielectric spectroscopy. The trap characteristics of the relaxation process by thermally stimulated depolarization current (TSDC) analysis are less studied. In this work, TSDC and broadband dielectric spectroscopy techniques were used to complementarily characterize the dielectric relaxation process of hydroxyl-terminated liquid nitrile-butadiene rubber (HTBN) toughened epoxy resin polymers. The experimental results show that HTBN introduces two new relaxation processes in the epoxy matrix, which are attributed to the α polarization of the rubber molecule and the interfacial polarization based on the correlation between the TSDC and the dielectric spectroscopy data, respectively. The trap parameters of each TSDC current peak were obtained using the multi-peak fitting method. The addition of rubber increases the trap density in epoxy composites significantly, especially for traps with energy levels in the range of 0.5–0.9 eV. The trap energy level of the DC conductivity process increases with increasing rubber concentration. The above results provide analytical ideas for rubber-toughened epoxy resins’ polarization and trap characteristics and theoretical guidance for formulation improvement. Epoxy resins are widely used in power equipment and electronic devices because of their excellent adhesion, corrosion resistance, electrical insulation and ease of processing and forming . HoweverStudies have shown that liquid rubber doped epoxy resin can effectively improve the mechanical and dielectric properties. The commonly used liquid rubbers are carboxyl-terminated liquid nitrile-butadiene rubber (CTBN), HTBN, hydroxyl-terminated liquid polybutadiene rubber (HTPB) and carboxyl-terminated liquid polybutadiene rubber (CTPB) . Dong anSince epoxy resin materials are primarily applied in complex physical fields such as electricity, heat and force, which are prone to aging causing more physical and chemical defects. The dielectric spectroscopy technique cannot reflect the trap properties of the material, and scholars have investigated the connection between polarization and trap distribution using the TSDC method . J. KataIn this paper, TSDC and broadband dielectric spectroscopy techniques were used to complementarily characterize the dielectric relaxation process of HTBN toughened epoxy resin polymers. The relaxation process corresponding to each current peak was analyzed by splitting the TSDC current peak and verified using the dielectric spectrum data. The trap parameters of each current peak were calculated, and the effect of the introduction of HTBN on the trap characteristics of the relaxation process of epoxy resin was studied. The study results can provide analytical ideas for the polarization and trap characteristics of rubber-toughened epoxy resins and theoretical guidance for formulation improvement.The epoxy resin used in this study is diglycidyl ether of bisphenol A (DGEBA) with the epoxy value of 3.9 mmol/g, which was produced by Xingchen Synthetic Materials Co., Ltd., Nantong, China. The curing agent was polyoxide propylene diamine, a room temperature amine curing agent, purchased from purchased from Zhongsi Industrial Co. Ltd., Shanghai, China. And the average molecular weight (Mn) adopted in our research is 400. The liquid rubber was selected as terminal hydroxyl liquid nitrile butadiene rubber with a hydroxyl content of 0.60 mmol/g. All reagents were not specially treated before use.Samples of different rubber contents were prepared according to the following steps. First, HTBN was added to the epoxy resin and the mixture was homogeneously mixed, followed by low-speed stirring in an oil bath at 150°C to make it homogeneously mixed. After 1 h, the blends were removed and cooled naturally, and then the curing agent was added, stirred homogeneously, and then vacuum degassed. The prepolymer was injected into the mould under vacuum environment and then left for 2 h at room temperature for initial curing, followed by placing the mould in an oven and curing under the conditions of 40°C/2 h, 60°C/2 h, 80°C/3 h and 110°C/4 h. After curing, the temperature was cooled down for the polymer gradient and then removed from the oven.6 Hz, and the temperature range was -60–200°C.The dielectric spectrum measurements were performed on the specimens using a Concept80 dielectric spectrum tester from Novocontrol, Germany. The size of the sample is 40 mm in diameter and 1 mm in thickness. By the ion sputtering, one side of the sample is coated with gold all, and the other side is coated with a circle of 30 mm. The measurement frequency range was 0.1 Hz–10Tp (160°C), then maintain the temperature constant. Ep (0.3 kV/mm) DC polarization field strength is applied to both ends of the specimen for a duration of tp (40 min). Then the specimen temperature is rapidly reduced to −120°C, when the applied voltage is withdrawn and the specimen is short-circuited at both ends for tp (5 min). Finally, the temperature was increased linearly at 3°C/min, while the current in the external circuit was measured using a Keithley 6517B electrostatic meter, which is the depolarization current.TSDC measurement has been performed to determine the trap depth and density in the epoxy/rubber composites. Both sides of vacuum dried samples were sputtered with golden electrodes with a diameter of 30 mm. The tests have been carried out with a Novocontrol Quatro Cryosystem. TSDC experimental scheme is shown in n is the carrier concentration in the trap, s is the frequency factor, n0 means the initial concentration of carriers in the trap, E is the activation energy, k is the Boltzmann constant, T is the absolute temperature, and b is the kinetic level.The calculation of the thermal stimulation current is complex, and the trap parameters are closely related to the thermal motion of the molecules. The following kinetic equation can describe the thermal stimulation process.The solution of β is the linear rate of temperature increase, and Tm is the temperature corresponding to the peak current. Taking the logarithm of If a linear temperature rise is used during the experiment, the following equation can be derived.The integrals for According to the derivation in As shown in the TSDC curves in Moreover, the temperature below −100°C, all samples have the same change trend. There is a broader current peak, which is judged to be caused by β relaxation of epoxy resin according to the peak’s size and position, consistent with the literature . The aboSince the glass transition peak of HTBN is close to the epoxy secondary transition peak in the dielectric spectrum, meanwhile the epoxy secondary transition peak is gradually masked as the rubber content increases. To further analyze the trap characteristics of dielectric relaxation at low temperatures, the trap energy levels of β relaxation and α relaxation of rubber molecules were calculated for different samples according to the relevant results in As the curing cross-linking reaction proceeds, the molecular weight of the epoxy resin increases, making the compatibility between the two poor. The unstable phase separation mechanism separates the rubber phase and the epoxy resin phase and gradually precipitate, forming an intercalated phase dispersed in the cured epoxy resin . At the According to the dielectric spectrum analysis in At present, the influence of rubber molecules on the dielectric properties of epoxy resins is mainly studied in academia by introducing interfacial properties . It has Rubber molecules entering the epoxy resin matrix become entangled and chained into spherical (or sphere-like) particles on the nanometer (or micron) scale, and the number of particles increases with increasing rubber content . The parAs can be seen from the table, the peak temperature of interfacial polarization peaks with different rubber contents varied less. With the increase of rubber content, the peak current and trap charge of interfacial polarization of EP1 and EP2 increased. The trap energy level and trap charge introduced by the interfacial polarization of EP2 formulation was higher than those of EP1 formulation at the same rubber concentration. This is since HTBN is hardly involved in the curing reaction of the amine formulation system, resulting in a larger amount of rubber precipitation in the EP2 formulation than in the EP1 formulation. Therefore, the rubber filler introduced more traps for the same liquid rubber addition. In addition, due to the difference in epoxy values, the bonding of the epoxy matrix to the rubber particles is also different between the two formulations, resulting in different trap energy levels introduced at the interface. The above differences were also verified in the dielectric spectra .Tg) of the formulation used in this paper is about 70°C according to the report in literature  toughened epoxy resin polymers and the trap characteristics of each polarization process were analyzed, leading to the following conclusions:HTBN introduces two new relaxation processes in the composite. Rubber molecular α polarization and interfacial polarization, both trap energy levels, are proportional to the rubber concentration. The interfacial polarization introduces trap energy levels from 0.5 to 0.9 eV. The introduction of HTBN has a negligible effect on the trap characteristics of epoxy resin’s high-temperature electrode polarization process due to the conductivity effect. The trapped charge of electrode polarization is maximum at 5% HTBN content. DC conductivity is the main factor leading to the variation of the dielectric loss factor in the high-temperature region. The variation with temperature follows the Arrhenius law, whose activation energy increases first and then decreases with increasing rubber concentration and whose trap energy level decreases with increasing rubber concentration.