BCL9L, and the role of BCL9L and Wnt/β-catenin signalling in BC cells. UTR mutations were analysed by a luciferase reporter. BCL9L protein was assessed by immunohistochemistry in BC tissues. Cell proliferation was examined by crystal violet staining and by the spheroid model. Moreover, migration and invasion were analysed in real-time using the xCelligence RTCA system. The A > T mutation at 3′ UTR of BCL9L reduces the luciferase reporter mRNA expression and activity. BCL9L is predominantly increased in dysplastic urothelial cells and muscle-invasive BC. Knockdown of BCL9L and inhibition of Wnt/β-catenin signalling significantly repress the proliferation, migration and invasion of Cal29 and T24. In addition, BCL9L knockdown reduces mRNA level of Wnt/β-catenin target genes in Cal29 but not in T24 cells. BCL9L and Wnt/β-catenin signalling play an oncogenic role in bladder cancer cells and seems to be associated with BC progression. Nevertheless, the involvement of BCL9L in Wnt/β-catenin signalling is cell-line specific.Bladder cancer (BC) is characterised by a high recurrence and progression rate. However, the molecular mechanisms of BC progression remain poorly understood. BCL9L, a coactivator of β-catenin was mutated in the 5′ and 3′ untranslated regions (UTRs). We assessed the influence of UTRs mutations on  Bladder cancer (BC) was the tenth most commonly cancer worldwide in 2020, however, the ranking was raised to the seventh place in the male population. The incidence in men is higher than in women ,2. The mThe Wnt signalling pathway is involved in several cellular processes such as proliferation, differentiation, migration, polarisation and self-renewal during development. Wnt pathway is distinguished in the canonical Wnt pathway (referred as Wnt/β-catenin pathway) and non-canonical Wnt pathway, which is independent of β-catenin ,12. Wnt/BCL9L/BCL9L is overexpressed in several tumour entities and it is associated with the development of tumourigenicity, tumour stage, and cancer progression [BCL9L expression is increased in hepatocellular carcinoma (HCC). Moreover, knockdown of BCL9L negatively affects the malignant behaviour of HCC cells through reduction of Wnt/β-catenin signalling [BCL9L protein contains three conserved regions known as homology domains: HD1, HD2, and HD3 . The congression ,31,32. Fgnalling ,34. The gnalling ,38,39,40BCL9L gene has not yet been analysed in bladder cancer. BCL9L was identified to be mutated at the 5′ and 3′ untranslated regions (UTR) of BC patients in our previous study [BCL9L as well as the functional role of BCL9L in BC cell lines. Given the known interaction between BCL9L and β-catenin proteins, we also aimed to decipher the role of BCL9L on the Wnt/β-catenin signalling in BC cells. The current study now suggests that BCL9L and ß-catenin also play an oncogenic role in bladder cancer.us study . TherefoBCL9L, a coactivator of β-catenin, carries frequent mutations in the regulatory 5′ and 3′ UTR regions of mRNA transcript in three patients out of eleven. Furthermore, the Cancer Genome Atlas Study of Bladder Cancer 2017, whole-exome sequencing of 412 MIBC detected that BCL9L is mutated in 11 patients out of 412, indicating that BCL9L might have a role in bladder cancer [From a previous study, the whole-exome sequencing analysis of progressive NMIBC and their corresponding MIBC of eight patients identified frequent mutations in the coding sequence as well as in the 5′ and 3′ UTRs of several genes to be part of distinct pathways, including Wnt/β-catenin signalling pathway . The tumr cancer .BCL9L gene in the regulation of gene expression was addressed by the luciferase reporter system. The whole wildtype or mutated 5′ UTR sequences (965 bp) was cloned upstream, while a fragment of wildtype or mutated 3′ UTR (783 bp) was cloned downstream of the luciferase reporter gene in the pmirNanoGLO vector (BCL9L due to the big sequence size of 4540 bp. The double mutated 5′ UTR (G > T and A > T) does not influence the luciferase activity and luciferase mRNA in T24 cells (p < 0.05), it did not affect the luciferase mRNA level. Interestingly, the single mutated 3′ UTR (A > T) of BCL9L significantly reduced the luciferase activity (16-fold reduction for T24 and 27-fold reduction for TCCsup) and luciferase mRNA expression  compared to the wildtype sequence  compared to adjacent non-tumour areas. Moreover, a high level of BCL9L is correlated with poor overall survival of HCC patients, suggesting the role of BCL9L in the progression and poor prognosis of HCC [BCL9L has not yet been linked with bladder cancer, the human protein atlas platform was utilised to investigate the association of BCL9L in bladder cancer. A high mRNA level of BCL9L is associated with lower survival of bladder cancer patients  tissues from 11 MIBC and 7 matched NMIBC samples . OverallBCL9L expression with BC progression, we investigated the functional role of BCL9L knockdown in BC cells. It remains unknown to our knowledge so far whether BCL9L has an oncogenic effect on BC cells. This was addressed by functional analysis of BCL9L in bladder cancer cells after knockdown experiments using siRNA technology. Cal29 and T24 cells were selected for analysis as a model for invasive bladder cancer cells. The mRNA level of BCL9L was significantly reduced in Cal29  and T24  after knockdown compared to siControl  and T24 (1.7-fold reduction) cells compared to siControl  and T24  compared to siControl  and T24  cells compared to siControl  and siBCL9L in Cal29 and T24 cells. Thereby the activator SKL2001 binds to AXIN2 protein and it interferes with the interaction of AXIN2 and GSK3-β proteins, resulting in the release of β-catenin from the destructive complex and consequently in the activation of Wnt/β-catenin signalling [AXIN2 and LEF1 target genes was significantly increased with the increase of the SKL2001 concentration, which was significantly reversed by the knockdown of BCL9L. SP5 mRNA level was significantly increased after SKL2001 but siBCL9L was not able to reduce SP5 expression. No differences were observed for the BIRC5 and MMP14 targets after SKL2001 treatment, however, co-treatment with siBCL9L led to a significant reduction of mRNA expression of BIRC5. MMP9 mRNA was significantly increased after SKL2001 treatment and surprisingly was further induced after transfection with siBCL9L in Cal29 cells. Without ß-Catenin activation by SKL2001, the knockdown by siBCL9L only affected the expression of AXIN2 and MMP9 on a significant level  and T24  compared to control  and T24  cells  cells C,D. ThesBCL9L, as a coactivator of β-catenin is involved in Wnt/β-catenin signalling, was identified to be exclusively mutated at the 5′ and 3’ UTR of BC patients. Here, we showed that one mutation (A > T) at 3′ UTR of BCL9L could influence the luciferase reporter activity in BC cells, suggesting that this mutation might be responsible for an aberrant expression of BCL9L. Other studies have shown that mutations at the 5′ and 3′ UTR of several genes might have an impact on gene expression and are strongly associated with various tumour entities [BCL9L.Several studies from whole-exome DNA and RNA sequencing analysis have shown that bladder cancer has a high mutation load and have identified distinct pathways and genes to be mutated. However, it is essential to identify progression-associated genes and proteins as possible biomarkers or targets for the therapy against progressive BC ,54,55. Ientities ,45,46,47BCL9L is overexpressed in several cancer types and is associated with tumour progression. BCL9L protein is overexpressed and correlated with poor prognosis and poor overall survival in various tumour entities, suggesting the role of BCL9L in the cancer’s progression [BCL9L has not yet been analysed and it is not known whether it plays a role in BC progression. Survival analysis of BC patients indicates that high mRNA level of BCL9L is significantly correlated with worse overall survival in bladder cancer patients, suggesting that a BCL9L high level might not be beneficial in BC patients. Furthermore, we showed that BCL9L mRNA expression was significantly increased in MIBC compared to normal ureters. Interestingly, BCL9L protein was increased on average as well as in some subpopulations of cells in MIBC and in dysplastic urothelial cells compared to NMIBC and non-dysplastic urothelium, suggesting an association with BC tumour stage and malignancy of carcinoma cells. In addition, the localisation of BCL9L was observed to be both in the nucleus and cytoplasm of tumour cells but with stronger staining in the nucleus, in agreement with its role as transcriptional cofactor of Wnt/β-catenin. Furthermore, we observed that BCL9L protein is mainly localised in the nucleus of Cal29 cells compared to T24 and TCCsup, where it is stronger expressed in the cytoplasm. This agrees with the observation that BCL9L is expressed in the nuclei of invasive lesions in breast cancer tissues, suggesting that strong staining of BCL9L in the nucleus might be related to cancer progression [It has been well demonstrated by some studies that gression ,38,56,57gression . We hypoBCL9L has any functional role in BC cells in vitro. Knockdown of BCL9L significantly repressed the proliferation, migration, and invasion in T24 and Cal29 cells, without induction of apoptosis, maybe through a cell-cycle arrest mechanism. Here, we suggest that BCL9L has an oncogenic role in BC cells. Epithelial-mesenchymal transition (EMT) plays a crucial role in the migration and invasion of tumour cells. Therefore, the influence of BCL9L on EMT has to be analysed in further studies using 3D cell culture or organ models. In general, the oncogenic effect of BCL9L in cancer is also confirmed by other studies for several tumour entities. In breast cancer, depletion of BCL9L by knockdown experiments inhibits the cell proliferation of MCF7 cells, while overexpression of BCL9L promotes tumourigenicity in transgenic mice, confirming the oncogenic effect of BCL9L in cell culture and animal models [BCL9L through knockdown and overexpression experiments in other cancer entities [BCL9L could promote the malignant behaviour of BC.Next, we focused on addressing whether l models . Other ientities ,50,56,59BCL9L could promote malignancy in BC. It is known that BCL9L protein interacts with β-catenin and activates the Wnt/β-catenin signalling, which is involved in tumourigenesis [BCL9L knockdown repressed only a subset of the analysed Wnt/β-catenin target genes, such as AXIN2, LEF1, and BIRC5 in Cal29, especially after the activation of the signalling by the specific activator SKL2001. In T24 cells, BCL9L knockdown did not influence the analysed Wnt/β-catenin target genes. Given the observation that BCL9L has an oncogenic effect in both Cal29 and T24 cells, we hypothesised that the action of BCL9L might be dependent of Wnt/β-catenin signalling in Cal29 and independent of Wnt/β-catenin signalling in the case of T24 cells. However, it remains unclear how BCL9L promotes proliferation, migration and invasion of T24 cells.Moreover, we focused on investigating the potential molecular mechanism of how igenesis ,35. Afteigenesis ,60,61. Digenesis ,38,39,40BCL9L mostly affects a subset of Wnt/β-catenin target genes [BCL9L could mediate its function independent of Wnt/β-catenin signalling. For example, the mechanism of action of BCL9L protein appears to be dependent on molecular subtypes of breast cancer and cell lines. In oestrogen receptor alpha (ER) positive breast cancer, BCL9L mechanism is independent of Wnt/β-catenin, however, it is dependent on ER signalling [Various BCL9L signalling pathways are suggested by other studies. et genes ,31,38,50gnalling .BCL9L mediates the malignancy of TNBC through Wnt/β-catenin and TGF-β signalling pathways [In triple-negative breast cancer (TNBC), pathways . The menof BCL9L knockdown [Furthermore, we examined the functional role of Wnt/β-catenin signalling in BC, independent of BCL9L. It has been shown that β-catenin is more strongly expressed in high grade and invasive BC, and Wnt/β-catenin signalling is involved in the maintenance and regulation of urothelial cancer stem cells suggesting that β-catenin is associated with aggressiveness and poor prognosis ,16,17. βnockdown . iCRT3 tBCL9L influence on Wnt/β-catenin signalling is cell line specific in BC cells. This is addressed by the hypothesis that a certain Wnt/β-catenin threshold is required in order BCL9L to affect Wnt/β-catenin signalling. In hepatocellular carcinoma, it has already been shown that a certain Wnt/β-catenin signalling threshold is required to be functional [A reasonable question is why nctional . Curtainnctional . Cancer nctional ,69. In lnctional ,73,74,75However, our results have to be confirmed by further experiments to analyse the function of BCL9L/β-catenin signalling in the progression of BC. For example, Yang et al. showed that in 3D spheroid model of T24 cells, β-catenin was significantly increased compared to 2D monolayer model . Our nexTumour tissue samples were obtained from patients subjected to transurethral resection of the bladder or radical cystectomy, and ureter samples were obtained from patients subjected to nephrectomy at the Department of Urology, Jena University Hospital, Germany. Samples for immunohistochemistry were formalin-fixed, embedded in paraffin for pathological examination, and stored at the Institute of Pathology, Jena University Hospital, Jena, Germany, until use. Frozen tissue from the same patients used for mRNA expression analysis originated from the biobank of the above-mentioned Department of Urology. The baseline characteristics  of the bladder cancer subjects are summarised in Protein expression of tumour samples was analysed by immunohistochemical staining with Dako REAL EnVision Detection System K5007  according to the manufacturer’s protocol against BCL9L . Briefly, 4 µm sections from formalin-fixed paraffin-embedded (FFPE) tumour samples underwent deparaffinisation and the heat-induced epitope retrieval was done in Tris-EDTA Buffer  at 98 °C for 20 min. A consecutive section was Hematoxylin–Eosin stained. Immunohistochemical staining was assessed by intensity: (no staining (−), weak (+), moderate (++) and strong (+++)) of the tumour cells. Moreover, the immunohistochemical staining was assessed by the H-Score. The H-Score is calculated by the formula: 3× percentage of strong staining + 2× percentage of moderate staining + percentage of weak staining [human bladder cancer cell lines T24 and TCCsup were cultured in RPMI 1640 medium with Glutamax  supplemented with 10% fetal bove serum (FBS), penicillin (100 U/mL) and streptomycin (100 µg/mL). The human bladder cancer cell line Cal29 was cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bove serum (FBS) , penicillin (100 U/mL), and streptomycin (100 µg/mL). All cells were incubated in a humidified atmosphere at 37 °C with 5% CO2.The Short tandem repeat (STR) analysis was done by the Institute of Forensic Medicine, Jena University Hospital, Germany, using PowerPlex 16 System  for amplification and capillary gel electrophoresis for analysing allele profiles. Profiles were checked by the German Collection of Microorganisms and Cell Cultures online STR analysis.BCL9L were synthesised by Eurofins Genomics Germany GmbH  and cloned between EcoRV and BstEII restriction sites upstream of the NLucP luciferase gene in the pmirNanoGLO vector. The wildtype 3′ UTR fragment of 783 bp  was amplified using cDNA from HEK293 cells. Briefly, 2 μg of total RNA from HEK293 cells was reverse transcribed into cDNA using the GoScript Reverse Transcription System  according to the manufacturer’s protocol, using oligo dT primers. The desired fragment of 783 bp was amplified by using specific cloning primers which contain the corresponding restriction sites . The fragment was cloned between NheI and SalI restriction sites downstream of the NLucP generating the 3´ UTR wildtype plasmid. The fragment was also used as a template to generate the single mutated 3′ UTR of BCL9L  by using the Q5 site-directed mutagenesis´s protocol and specific mutagenesis primers . The mutated UTR was cloned in the pmirNanoGLO vector and the sequence was verified by Sanger sequencing.For mutation analysis of untranslated regions (UTR), the luciferase reporter plasmid pmirNanoGLO  was used. The whole wildtype and double mutated 5′ UTR sequences  of For transfection, T24 or TCCsup cells were seeded in a 24-well plate and cultured until the cells reached about 80% confluency. Transfection with the wildtype and mutated UTR luciferase reporter plasmids using jetPRIME reagent was done according to the manufacturer’s protocol. Twenty-four hours post-transfection, the cells were lysated in 200 μL Passive Lysis Buffer  and 80 μL of the lysate was used for the measurement of the luciferase activity using the Dual-Glo Luciferase assay system  on the Tecan Infinite M200 Pro reader . Moreover, RNA was extracted in order to analyse the luciferase mRNA level. The NLucP luciferase activity and mRNA level was normalised to Luc2 luciferase to eliminate variations in the transfection efficiency (NLucP/Luc2). Fold change was calculated to emphasize the effects of mutation on luciferase activity and mRNA level. All experiments were repeated at least three times.RNA extraction from tissues and transfected cells with siBCL9L or treated with iCRT3 or SKL2001 was performed as described elsewhere . BrieflyThe total RNA concentration was quantified using a Qubit 3.0 Fluorometer  according to the manufacturer’s protocols. The RNA quality was analysed by an Agilent 2200 TapeStation  instrument according to the manufacturer’s instruction.RPS13 and RPS23 was calculated using the ΔΔCp method, including primer efficiency in the calculation [2 μg of total RNA was transcribed into cDNA using the GoScript Reverse Transcription System kit  according to the manufacturer’s protocol. The cDNA was diluted 1:8 with nuclease-free water and stored at −20 °C. The mRNA expression analysis was performed using a LightCycler 480 SYBR Green I master mix and the LightCycler 480 instrument  utilising specific primers for each target . The Cp culation ,79.mouse IgG-HRP  and anti-rabbit IgG-HRP  were used as secondary antibodies. The membrane was incubated with chemiluminescence (ECL) reagent  and the produced light emission was detected by a GBox Chemi XX6 .Western blotting was performed as described elsewhere . Briefly2. After reaching 60–70% confluence, the cells were washed one time with PBS and incubated in pre-chilled (−20 °C) 100% methanol for 5 min at room temperature, followed by a blocking step in 1% BSA/10% normal goat serum/0.3 M glycine in 0.1% PBS-Tween for 1 h. Then, the cells were incubated with 1 μg/mL anti-BCL9L antibody  diluted in 1% BSA/0.1% PBS-T at 4 °C overnight. The AlexaFluor®®488 goat anti-rabbit antibody  was used as a secondary antibody at 2 μg/mL diluted in 1% BSA/0.1% PBS-T for 1 h at room temperature in the dark, followed by DAPI (2.5 µM) incubation for 30 min in the dark. The slides were stored at 4 °C and were analysed by microscopy .For immunofluorescence, Cal29, T24 or TCCsup cells were seeded in 4-well Falcon culture slides  and incubated in a humidified atmosphere at 37 °C with 5% COBCL9L on Wnt/β-catenin signalling, Cal29 and T24 cells were first transfected with 40 nΜ pooled siBCL9L  or siControl . 24 h after transfection, the cells were treated with 0, 20, and 40 μM SKL2001 (specific Wnt/β-catenin activator) in 0.1% DMSO, and after 2 days, the mRNA expression of Wnt/β-catenin target genes AXIN2, LEF1, SP5, BIRC5, MMP9 and MMP14 was analysed by qRT-PCR. For the inhibition of Wnt/β-catenin signalling, the Cal29 and T24 cells were treated with 0 µM, 20 μM and 40 μM iCRT3 (specific Wnt/β-catenin inhibitor) in 0.1% DMSO. After 2 days, the mRNA expression of Wnt/β-catenin target genes was analysed.For the analysis of the effect of For proliferation assay, Cal29 or T24 cells were seeded on 6-well cell culture plates and after 24 h transfected with 40 nM pooled siBCL9L  or negative control siRNA  using INTERFERin reagent according to the manufacturer’s protocol . For inhibition of Wnt/β-catenin signalling, the cells were treated with 0, 20, or 40 μM iCRT3 in 0.1% DMSO. After 6 or 8 days, the cells were washed once in PBS buffer, fixed with 2% glutaraldehyde solution in PBS for 10 min and stained with 0.1% crystal violet solution for 30 min. Cells were gently washed with distilled water, dried overnight, and solubilised with lysing solution  for 30 min. The absorbance was measured on an Infinite M200 Pro reader at 590 nm .g for 10 min and incubated in a humidified atmosphere at 37 °C with 5% CO2. After 24 h, the spheroids were treated with 0 µM, 20 μM or 40 μM iCRT3 in 0.1% DMSO. Images were taken using a Cell observer . The software MATLAB R20019a and AnaSP were used to extract the volume from spheroid formation experiments. All experiments for proliferation and spheroid formation were repeated at least three times.For spheroid formation, T24 cells were gently trypsinated and collected at 100 g for 10 min. The cell pellet was resuspended with fresh growth medium, and 1000 cells were seeded in a cell repellent surface 96-well microplate . The plate was centrifuged at 100× Cal29 and T24 cells were transfected in 6-well plates, transfected with siRNA, or treated with iCRT3 as described above (proliferation and spheroid formation assay). Apoptosis was detected by an FITC Annexin V apoptosis detection kit  according to the manufacturer’s protocol. Data acquisition and analysis were performed by the BD Accuri C6 Plus software, using a specific template for the annexin V-FITC  and PI . At least 10,000 events were used for the analysis, excluding cell debris by setting appropriate light scatter gates. All experiments were repeated at least three times.t-test was performed for the whole time frame.Migration and invasion of cells were analysed using the xCELLigence RTCA System  according to the manufacturer’s protocol (Cell Migration and Invasion protocol). The cells were transfected with 40 nM pooled siBCL9L or siControl on a 6-well plate for three days or treated with 40 µM iCRT3 in 0.1% DMSO for 2 days as previously described. The transfected or treated cells were trypsinated and resuspended in serum-free medium. For migration, 20,000 cells in 100 μL were added to the upper chamber of CIM -plates. In the case of invasion, 40,000 cells in 100 μL were added to the upper chamber wells, pre-coated with 1:12 or 1:40 diluted Matrigel . In the lower chamber, medium with 10% serum was added. All experiments were repeated at least three times. For statistical analysis, the mean of three independent biologicals was combined and a two-tailed Student’s t-test and the non-parametric Mann–Whitney U-test were performed to compare the two groups. The type of test that was used is mentioned specifically in the result part. A 95% confidence interval  was considered as statistically significant (*).Statistical analysis was performed using the software IBM SPSS Statistics Version 25 by combined independent biological replicates. Two-tailed unpaired Student’s BCL9L, providing an additional mechanism for the aberrant expression in cancer. This study showed for the first time that BCL9L is overexpressed in dysplastic urothelial cells and MIBC, suggesting an association with tumour stage and invasiveness. Moreover, in vitro analysis demonstrated that both Wnt/β-catenin signalling and its cofactor BCL9L promote malignant behaviour in BC cells, indicating an oncogenic role and involvement in BC progression. The oncogenic effect of BCL9L in BC cells could be dependent or independent of Wnt/β-catenin signalling. The development of specific inhibitors which interfere with the normal function BCL9L and Wnt/β-catenin might provide a potential therapeutic strategy against progressive BC.BCL9L, a coactivator of β-catenin, was identified to be mutated at UTR in our patients with progressive BC. One mutation at 3′ UTR could influence the gene expression of


As a minimally invasive drug delivery platform, microneedles (MNs) overcome many drawbacks of the conventional transdermal drug delivery systems, therefore are favorable in biomedical applications. Microneedles with a combined burst and sustained release profile and maintained therapeutic molecular bioactivity could further broaden its applications as therapeutics. Here, we developed a double-network microneedles (DN MNs) based on gelatin methacrylate and acellular neural matrix (GelMA-ACNM). ACNM could function as an early drug release matrix, whereas the addition of GelMA facilitates sustained drug release. In particular, the double-network microneedles comprising GelMA-ACNM hydrogel has distinctive biological features in maintaining drug activity to meet the needs of application in treating different diseases. In this study, we prepared the double-network microneedles and evaluated its morphology, mechanical properties, drug release properties and biocompatibility, which shows great potential for delivery of therapeutic molecules that needs different release profiles in transdermal treatment. Due to a variety of challenges faced by oral drug delivery, such as hepatic first pass effect, the gastric irritation, and poor patient compliance, transdermal drug delivery (TDD) has been used as an alternative strategy for delivering therapeutics . One of Selection of MNs matrix material could significantly affect the drug loading and release profiles of the encapsulated therapeutics. From a drug release perspective, MNs could be largely classified as burst release and sustained release ones, which mainly depend on the dissolution and degradation properties of the MNs materials within the skin. Natural hydrogels, such as gelatin, cellulose, alginate, chitosan and hyaluronic acid, have been utilized to fabricate MNs for rapid drug release due to their innate water solubility . In contCompared to the above materials, acellular matrix (ACM) is distinguished by its ability to store, protect and release therapeutic molecules . The hybin vivo studies. As a result, our DN MNs presents a potentially valuable method for delivery of therapeutic molecules that needs different release profiles in transdermal treatment.Specifically, we designed a double-network MNs (DN MNs) that combined ACNM, as an early drug release matrix, and GelMA for sustained release . After t2) and Pepsin (1:3000) was purchased from Acmec Biochemical Co., Ltd. . Methacrylic anhydride (94%) were purchased from Sigma-Aldrich . Dialysis bag (12–14 kDa) was purchased from Yuanye Bio-Technology Co., Ltd. . Phosphate buffer (PBS) solution was self-prepared, and the other materials were purchased from Wuhan Servicebio Technology Co., Ltd. .Gelatin (porcine skin) and Rhodamine B was purchased from Aladdin . Photoinitiator (2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone), Agarose  were first agitated in the following decellularization baths sequentially: deionized water , 0.02% trypsin/0.05% EDTA (1 h), 3.0% Triton X100 (12 h), deionized water (rinse 4 times), 4% SDS solution , deionized water (rinse for 1 h), 4% SDS solution , deionized water (rinse for 1 h). Subsequently, the decellularized ACNM were dissolved with pepsin (1.0 mg/ml) in HCl (0.01 M) at a concentration of 10 mg/ml after lyophilized for 48 h. Finally, the solution was stirred at room temperature for 48 h and neutralized to pH 7.4 with 0.1 M NaOH to form a ACNM hydrogel.10% (w/v) gelatin solution was made by dissolving 20 g gelatin in 200 ml preheated PBS at 50°C for 1 h. After gelatin fully dissolved, 16 ml of methacrylic anhydride was slowly added to the gelatin solution and stirred at 50°C for 2 h. Subsequently, the reaction was stopped by an additional 200 ml of PBS was added and the solution was dialyzed for 7 days at 40°C to remove impurities by using dialysis bag with a molecular weight cut-off of 12–14 kDa. The dialyzed product was filtered and frozen to −20°C in 50 ml conical tubes for lyophilization. After 3 days of lyophilization, GelMA was obtained and stored with airtight and light-free until further use.2) for 30 s. The stability of CGRP II in GelMA-ACNM hydrogel were further evaluated. “Original”: 40 μg CGRP II was dissolved in 200 μl GelMA-ACNM hydrogel. “1, 4, 7 day”: above hydrogels were stored at 26°C for 1, 4, 7 day. As a control, 40 μg CGRP II was dissolved in 200 μl PBS and stored at 26°C for 1, 4, 7 day. Each sample consisted of three replicate measurements and the results was expressed as an average value.2.5 g of GelMA was dissolved in 10 ml of ACNM  to prepare the GelMA-ACNM hydrogel. The morphology of the GelMA-ACNM hydrogel was investigated through a scanning electron microscope . The GelMA-ACNM hydrogel was freeze-dried and sputtered with gold before observing by SEM. The effective amounts of intact CGRP II before and after UV irradiation by using EIA. “Before UV irradiation”: 40.0 μg CGRP II was dissolved in 200 μl GelMA-ACNM hydrogel. “After UV irradiation”: above hydrogels were irradiated by UV (270 mW/cm2) for 30 s, the solution of backing layer material was added to cover the tips and dried at room temperature overnight. Finally, the patch named as CN MNs  and DN MNs (double-network) was peeled off from the mold. For PN MNs , 2.5 g of GelMA was dissolved in 10 ml of ACNM , the rest of procedures were similar to the preparation of the DN MNs but without UV irradiation.The MNs were fabricated according to a two-step template replication method. Specifically, 20% (w/v) gelatin was dissolved in deionized water as the backing layer solution; 2.5 g of GelMA, 50 mg of ultraviolet (UV) photoinitiator were dissolved in 10 ml of PBS solution to prepare the GelMA hydrogel; 2.5 g of GelMA, 50 mg of UV photoinitiator were dissolved in 10 ml of ACNM  to prepare the GelMA-ACNM hydrogel. To load Rhodamine B or CGRP II, 5 mg Rhodamine B or 2 mg CGRP II was dissolved in above hydrogel solution as the tip material. Then, 200 μl of the tip solution was poured over a female polydimethylsiloxane  mold and remove the bubbles up the MN tips by vacuumize, and then, centrifugation at 3,500 rpm for 10 min to draw the tip solutions filled the cavities of the template. After removing the redundant solution and solidifying the tips by UV irradiation . The MNs were sputtered with gold before observing by SEM.The mechanical strength of the dry MNs was tested by a universal tensile compression tester . MNs were placed on a fixed station with their tips facing upward. Under a constant 2 mm/min speed of force sensor, and the maximum loading force was set to 5 N, the mechanical properties of MNs were profiled. For wet MNs mechanical strength test, the MN tips were first immersed in PBS solution for 10 s. The rest of the procedure is the same as the dry MNs procedure.Agarose hydrogel was used to simulate the skin to investigate the Rhodamine B release properties of the CN MNs, PN MNs and DN MNs. The agarose hydrogel  was first prepared by dissolving agarose powder in boiled deionized water and cooling. The MNs were then pressed to the surface of the agarose hydrogel for 7 days. Finally, cut the agarose hydrogel to capture the depth wise color change images by cameras every day and analyzed with Photoshop software. Furthermore, The CGRP II release test was determined with CGRP II - EIA Kit . The MN patches was first immersed in 2 ml PBS at 37°C, and then, all of release medium was collected at pre-set intervals and replaced with the same amount of fresh PBS. The CGRP II concentration of each release medium was measured with the EIA according to the standard protocol of the product. In addition, the morphology of the MNs with different time points  was observed with an optical microscope by incubating the MNs in PBS at 37°C.All the animal work was approved by the Animal Care Committee of Guangzhou Medical University and conducted in alignment with relevant guidelines. Female Sprague-Dawley rats (150–200 g body weight) were purchased from the Guangdong Medical Laboratory Animal Center and acclimatized for 1 week in an approved animal facility.DN MNs were inserted into the dorsal skin of the rat for 5 min and 24 h and the animals were euthanized by cervical vertebra dislocation. Skin tissue subject to MNs application was removed and immersed in 4% paraformaldehyde. The fixed tissues were then dehydrated, embedded in paraffin, and made into serially sections for further histology and immunofluorescent staining. Briefly, the fluorescent images were imaged using a Zeiss inverted microscope after the paraffin-embedded skin tissues were cut into 8-μm-thick sections and stained with H&E staining. For fluorescence images, a series of skin tissues sections were deparaffinized and underwent heatinduced antigen retrieval using citrated buffer according to the standard protocol. After immersed in antigen retrieval buffer, permeabilized in 0.3% Triton PBST, and blocked with goat serum for 30 min, sections were incubated with primary antibody against CD68  overnight at 4°C. Then, the sections rinsed with PBST and incubated with Alexa 555-conjugated secondary antibody at a dilution of 1:1000 for 60 min at room temperature. After incubation, the sections were counterstained with DAPI for 5 min and the fluorescent images were captured under a Zeiss inverted microscope.t-test. p < 0.05 were considered statistically significant .The statistical analysis was performed by SPSS software version 25.0. Experiments were run in triplicate unless stated otherwise. All the data presented in this study were expressed as mean ± standard deviation (SD). Statistical analysis was performed using Student’s via EIA. As shown in The microstructure and biological functions of ACNM-GelMA hydrogel were analyzed. After UV irradiation, the gel can be rapidly formed by cross-linking . The SEMvia a two-step template replication method . During the test, the MNs were first placed on a horizontally positioned glass slide with tips facing a force sensor that slowly approached the MNs. Then the force measurements started when the sensor touched the MN tips and lasted until it applied force 5 N . MechaniTo evaluate the drug release profile, a preliminary test was performed on the agarose hydrogel that acted as an artificial skin model  Chang e. MNs werIn addition, the degradation of the DN MNs was measured by incubating them in PBS solution. As shown in To investigate the biocompatibility of the DN MNs, we assessed the topical application of the DN MNs to the Sprague-Dawley rat skin. The DN MNs were applied to the dorsal skin of the rat and the rat were then sacrificed at 5 min and 24 h for histological and immunofluorescence analysis. As shown in In this study, we demonstrated that double-network GelMA-ACNM hydrogels can be used for the fabrication of a DN MNs to achieve a tunable drug release profile over time. We also showed that the DN MNs exhibited sufficient mechanical strength to penetrate the skin, and the DN MNs released their loaded therapeutic agents through both swelling and degradation of the MNs tips. Compared with sustained release for a long period by using some synthetic materials, the DN MNs exhibited a two-phase release: rapid release phase and sustained release phase, which could meet the treatment requirement of certain diseases at the early stages. As a minimally invasive platform, the DN MNs has the potential to be further optimized for different applications. We envisioned that developing a fabrication strategy with modularity in selecting different ACM and the versatility in loading various biomolecules could meet the needs of its application in treating different diseases. In comparison with other drug delivery systems fabricated through complexed processes with dedicated devices, physicochemical co-crosslinked DN MNs is an effective and convenient way to achieve tunable drug release.


Compared to conventional DPP molecules, Ex-DPPs feature strong absorption with a fivefold extinction coefficient, good molecular rigidity to effectively restrain non-radiative deactivation, and an expanded π-skeleton which endow them with well-suited intermolecular packing geometries for achieving efficient SF process. These results not only provide a new type of high-efficiency SF chromophore but also address some basic guidelines for the design of potential SF materials targeting practical light harvesting applications.Singlet fission (SF) has drawn tremendous attention as a multiexciton generation process that could mitigate the thermal loss and boost the efficiency of solar energy conversion. Although a SF-based solar cell with an EQE above 100% has already been fabricated successfully, the practical efficiency of the corresponding devices is plagued by the limited scope of SF materials. Therefore, it is of great importance to design and develop new SF-capable compounds aiming at practical device application. In the current contribution,  Via π-expanded design strategy, a new robust SF system, Ex-DPP, has been successfully developed with high extinction coefficient, good molecular rigidity and excellent SF properties. E(S1) ≈ 2E(T1), a SF molecule should possess a large molar extinction coefficient, suitable triplet energy and good stability.4,5,9,10 So far, only a few of the previously reported molecules meet these criteria, such as para-azaquinodimethane10 and diimide derivatives.11,12 Therefore, designing and developing new SF-capable compounds aiming at practical application remains an urgent but challenging task.3–19As a multiexciton generation pathway, singlet fission (SF) has drawn more and more attention on account of its great potential that could mitigate the thermal loss and boost the efficiency of solar cells.i.e. organic solar cells and organic field effect transistors.20–29 In contrast to acene series, DPP and its derivatives have many properties, such as good structural tunability, synthetic flexibility, strong visible absorption and substantial chemical stability, which make them well-suited for SF-based light harvesting applications.20–23,30,31 Importantly, a DPP molecule possesses a triplet excited state energy E(T1) of ∼1.1 eV, roughly half that of its singlet state, E(S1) ∼2.2 eV, that fulfils the relationship E(S1) ≈ 2E(T1) suggesting that DPP should be an ideal SF chromophore.32–35 Recently, Hartnett et al. and Mauck et al. sequentially and systematically investigated the SF dynamics in a series of 3,6-diaryl-substituted DPP derivatives and presented a close correlation between the intermolecular packing geometries and SF performances in thin films.32,33 The phenyl substituted DPP (PhDPP) does not exhibit SF but excimer decay,32 while thiophen-2-yl substituted DPP (TDPP) derivatives turn out to be good SF chromophores with SF rates of up to (20 ps)−1 and high triplet yields  represent a series of promising organic semiconductors in optoelectronic applications, t yields .33 Howevications .36 All reagents and solvents of the best grade available were purchased from commercial suppliers and used without further purification. Thin films were prepared by the vapor deposition method on a sapphire substrate at a rate of 0.2–0.3 Å s−1 under a vacuum of 1 × 105 mbar. X-ray diffraction (XRD) was carried out in the reflection mode at room temperature using a 2 kW Rigaku XRD system. Grazing incidence wide-angle X-ray scattering (GIWAXS) measurements were performed on a Xeuss 3.0 SAXS/WAXS system using a Cu X-ray source (1.542 Å) with incidence angles of 0.18° at the Vacuum Interconnected Nanotech Workstation (Nano-X).The studied Ex-DPP molecules, Ex-DPP1 and Ex-DPP2, were synthesized according to the previously reported routes.37 The ground state geometries were optimized at the B3LYP/6-311G* level. The vertical excitation energies of the lowest triplet and singlet states were evaluated using density functional theory (DFT) and time-dependent DFT (TDDFT) methods at the B3LYP/6-311G* level based on the optimized ground state geometries, respectively.All the calculations were carried out with the Gaussian 16 program package.38–40 All the spectra were measured at room temperature if no further notification is given. Species-associated spectra and kinetic analyses of TA data were performed using a global fitting method with lab-written MATLAB programs. A three-state sequential kinetic model was applied to interpret TA data from thin films.38,41 Besides, the other analyses of the kinetic traces from TA and TRPL data were performed individually and globally using nonlinear least-square fitting to a general sum-of-exponentials function after de-convolution of the instrument response function (IRF).UV-visible absorption and fluorescence spectra were measured on a Shimadzu UV-3600 spectrometer and Hitachi F-4500 spectrophotometer, respectively. Time-resolved photoluminescence (TRPL) spectra were detected using an FLS980 spectrometer equipped with an EPL-375 picosecond pulsed diode laser (Edinburgh Instruments Ltd.). Femtosecond transient absorption (fs-TA) and nanosecond laser flash photolysis (ns-TA) measurements were all performed using the previously described instruments and experimental conditions.2-catalyzed one-step annulation of the unsubstituted DPP skeleton using the diaryliodonium salt reagent.36 We selected two Ex-DPP molecules as the study subjects, i.e.Ex-DPP1 and Ex-DPP2 with bulky tert-butyl and straight n-octyl substituents, respectively ectively . The sin1) and triplet (T1) energies are 2.33 and 2.42, 2.62 and 1.15, and 1.00 and 1.19 eV for Ex-DPP, TDPP and PhDPP, respectively. Compared to the obviously exothermic condition of conventional DPPs, Ex-DPP possesses near-isoergic SF energetics of ΔESF = E(S1) − 2E(T1) = 0.03 eV and is even slightly endothermic taking into account the aggregation effect in thin films. The slightly endothermic condition could effectively mitigate the energy loss to a certain extent, and the triplet energy of ∼1.1 eV was deemed to be optimized for maximizing the advantages of a SF material.4,10 That is, the current π-expanded strategy not only does not compromise the SF energetics of these Ex-DPPs, especially triplet energy, but decreases the energy loss resulting from the excessively exothermic SF energetics of conventional DPP derivatives.Subsequently, the computed results from DFT calculations suggest that the lowest singlet excited state features the electronic excitation of the HOMO → LUMO, where both frontier molecular orbitals are delocalized to the entire Ex-DPP skeleton . More im2Cl2, the spectrum of each Ex-DPP is nearly indistinguishable from each other, and presents the absorption peak maximum at 498, 537 and 585 nm , the extinction coefficient could be obtained by calculating the slope of the absorbance vs. concentration plot  measurements for these Ex-DPPs in both solution and thin films . In dilud 585 nm . Compareion data . Similar39,40,43 In detail, Ex-DPP1 molecules exhibit slightly red shifted and vibrational mode-increased absorption bands, and Ex-DPP2 molecules present an obvious red shifted and enhanced absorption peak at the long wavelength. The spectral difference might be rooted in the different molecular packing geometries in these thin films as discussed in the above structural analysis from single crystals and GIWAXS data  and τ2 = 8.4 ns (64%)  pathway.32,33Using transient absorption (TA) and PL measurements, we first investigated the excited state dynamics of these Ex-DPPs in dilute solution . As showns (64%) , which wurements  provide 32,33 We ruled out the long-lived charge separation species given the absence of the characteristic absorption signatures of the Ex-DPP charge species in the NIR region  and τ2 = 4.56 ± 0.28 μs (24%), and τ1 = 0.28 ± 0.01 μs (88%) and τ2 = 4.37 ± 0.26 μs (12%) in Ex-DPP1 and Ex-DPP2 thin films, respectively. Namely, the majority of the SF-formed triplets relax over hundreds of nanoseconds and the rest could persist for several microseconds. Then the triplet yields of 167% and 175% were estimated for the studied Ex-DPP1 and Ex-DPP2 thin films using the singlet depletion method previously applied in determining triplet yields in DPP systems −1. We attributed this to the similar intermolecular arrangements, especially the close π–π distance of ∼3.3 Å, observed in these Ex-DPPs, which lead to the well-suited intermolecular coupling interaction for achieving an ultrafast and efficient SF process in thin films. These analyses are also in agreement with the previous SF reports by Hartnett et al. and Mauck et al.32,33 They presented a close correlation between the intermolecular packing geometries and SF properties in thin films of TDPP derivatives.33 The methyl-substituted TDPP with a π–π distance of ∼3.3 Å exhibits the most rapid and efficient SF process featuring a SF rate of (23 ps)−1, while the other substituted derivatives with a π–π distance more than 3.5 Å display SF dynamics that are one or two orders of magnitude slower. Therefore, the SF process in DPP skeletons turns out to be highly dependent on the intermolecular packing distances. Herein the expanded π-skeleton endows these Ex-DPPs with well-suited intermolecular packing geometries, especially a π–π distance of ∼3.3 Å, for achieving rapid and efficient SF process.10,32,33,39,41Then global analyses are performed for the TA data based on a sequential three-state kinetic model, 44–51 Herein we address that the π-expanded strategy can be utilized to develop new practical SF materials from the following points: (I) Increasing the molecular light absorbing capacity, i.e. achieving a high extinction coefficient, to reduce device thickness and mitigate problems with triplet diffusion, (II) Improving the molecular rigidity to effectively restrain non-radiative loss and fully utilize high-energy photons and (III) Adjusting the molecular packing and optimizing intermolecular interaction to trigger and implement efficient SF process. Future work points to fabricating these Ex-DPPs into practical photovoltaic devices and studying the applicability of the π-expanded strategy to design new practical SF materials.Previous studies suggested that the π-expanded strategy could be successfully applied in adjusting the optical and electronic properties as well as the intermolecular interactions to improve the fluorescence emission, two-photon absorption capacity, and charge carrier mobility of organic semiconductor materials.In this work, we demonstrate that the π-expanded strategy can be utilized to develop potential SF materials targeting practical light harvesting applications. A new series of robust SF chromophores, Ex-DPPs, have been successfully developed. Compared to conventional DPP molecules, the current π-expanded Ex-DPPs feature strong absorption with a fivefold extinction coefficient, good molecular rigidity to effectively restrain the non-radiative deactivation, and well-suited intermolecular packing geometries for achieving efficient SF process. These results not only provide a new type of high-efficiency SF chromophore but also address some basic guidelines for the design of potential SF materials targeting practical photovoltaic applications.All of the related experimental data are provided in the ESI.†L. W. conceived the project, which was supervised by L. W., D. Z., H. F. and J. Y. W. J. synthesized the compounds, which was supervised by Z. L. and D. Z. L. W. prepared samples, and conducted theoretical simulations and photophysical characterization studies including TA experiments with the assistance of S. G., S. W., M. Z. and Z. L. G. W., Y. M. and L. Y provided assistance in the GIWAXS measurements and data analysis. J. Y. S. and Y. W. Z. performed spectroelectrochemistry experiments. L. W. wrote the manuscript, and all authors participated in the data analysis and discussions.There are no conflicts to declare.SC-013-D2SC05580E-s001


In this study, the curing kinetics of epoxy nanocomposites containing ultra-fine full-vulcanized acrylonitrile butadiene rubber nanoparticles (UFNBRP) at different concentrations of 0, 0.5, 1 and 1.5 wt.% was investigated. In addition, the effect of curing temperatures was studied based on the rheological method under isothermal conditions. The epoxy resin/UFNBRP nanocomposites were characterized via Fourier transform infrared spectroscopy (FTIR). FTIR analysis exhibited the successful preparation of epoxy resin/UFNBRP, due to the existence of the UFNBRP characteristic peaks in the final product spectrum. The morphological structure of the epoxy resin/UFNBRP nanocomposites was investigated by both field emission scanning electron microscopy (FESEM) and transmission electron microscopy (TEM) studies. The FESEM and TEM studies showed UFNBRP had a spherical structure and was well dispersed in epoxy resin. The chemorheological analysis showed that due to the interactions between UFNBRP and epoxy resin, by increasing UFNBRP concentration at a constant temperature , the curing rate decreases at the gel point. Furthermore, both the curing kinetics modeling and chemorheological analysis demonstrated that the incorporation of 0.5% UFNBRP in epoxy resin matrix reduces the activation energy. The curing kinetic of epoxy resin/UFNBRP nanocomposite was best fitted with the Sestak–Berggren autocatalytic model. Among thermosetting polymers, epoxy resins are one the most notable thermosets due to their distinguished electrical, chemical and mechanical properties ,2. EpoxyOne of the most practical methods in the polymer industry, especially in epoxy resins, is enhancing the toughening of polymers through the addition of nanoparticles ,8,9. ElaThe incorporation of UFNBRP in epoxy resin matrix can affect the curing kinetics of the system. Understanding the curing behavior of epoxy resins is essential to achieve any enhancement in their curing and in the properties of the final product . The ratThe rheological behavior plays an important role in controlling the curing process, which leads to improving the quality of the final product ,26,27. O3) nanoparticles in the curing kinetics of polyester/epoxy resin was investigated. The results exhibited that adding 5 wt.% of CaCO3 decreases the activation energy (Ea) and the curing rate [a) during the curing process [In a previous work by our team, the rheological curing kinetics of nanoclay/vinyl ester resin nanocomposites were studied. The results showed that, by increasing the curing temperature, the addition of clay nanoparticles decreased the curing rate, as well as the curing time . The curing rate . The cur process .According to our previous studies, the physical and mechanical properties of epoxy resins are affected by elastomeric nanoparticles . Howevera, different models were investigated and, finally, the Sestak–Berggren autocatalytic model showed the best fit on the experimental data of the nanocomposites. This work has novelty for academic and technological purposes, because the curing kinetics of epoxy/UFNBRP nanocomposites is still an open window for new achievements and research. Practically, most epoxy resin nanocomposites are processed by thermal curing, so the reported curing kinetics data of this work can be useful for industrial applications to set the optimum time and temperature of the curing process.Herein, the epoxy/UFNBRP nanocomposites were prepared successfully by sonication process. The morphology of the nanocomposites was studied using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The isothermal rheological experiments at different temperatures  and different amounts of UFNBRP  were used to investigate the curing kinetics of epoxy/UFNBRP nanocomposites. To choose the right model for the reaction rate and EHuntsman Araldite LY564 epoxy resin with Huntsman Aradur 2954 amine hardener were supplied by Huntsman Co. . The NBR nanoparticles were obtained from SINOPEC Beijing  research institute. NBR nanoparticles size is smaller than 100 nm with narrow distribution and fully of vulcanization.A certain amount  of UFNBRP was pre-mixed with epoxy resin at a mixing rate of 2000 rpm to obtain the optimum mixing condition. After approximately 20 min, the solution was completely sonicated and mixed again with a homogenizer for 20 min at 10,000 rpm. After adding the hardener, the mixing was continued for 2 min at 2000 rpm. The mixture was vacuum-dried at environment temperature for 1 h before mixing and 2 h after mixing to obtain bubble-free samples. Afterwards, the samples were immediately used for the rheological test. The illustration of the structure of epoxy resin intercalated with UFNBRP is shown in Scanning electron microscopy (SEM) images were captured from the fractured surfaces in nitrogen using a TESCAN , and transmission electron microscopy (sliced specimens from the core) was used for morphological study using PHILIPS, TECNAI20, The Netherlands. Rheological studies carried out on the MCR300, Anton Paar rheometer , tests were performed at 65, 70 and 75 °C under isothermal conditions at 1 Hz frequency. The viscoelastic range was considered linear to record viscoelastic functions and to calculate the curing kinetics of the crosslinking at isothermal conditions.a is the activation energy in Mathematical models can be used for the analysis of the degradation or curing kinetics of thermoplastic or thermoset polymers . The cur−1 related to OH stretching vibration. The absorption bands at 2862–3060 cm−1 are related to the stretching vibration of aromatic CH and CH (CH2 and CH3) in aliphatic groups in the epoxy resin structure. The multiple stretching for C=C vibrations in aromatic rings appeared at 1509–1734 cm−1. The absorption bands related to the bending vibration of aliphatic groups can be found at 1244–1373 cm−1, and the absorption bands at 1036–1180 cm−1 are related to the C–O and C–O–C groups. The spectrum of epoxy resin is compatible with the literature [−1 is related to the CN group in the rubber structure. Because of the appropriate interaction between the epoxy resin and UFNBRP, the intensity of the OH group was decreased, which revealed an interaction between CN and OH groups. As can be seen, there is no other obvious change in absorption bands in the FTIR spectrum of epoxy/UFNBRP compared to the neat epoxy resin, which could possibly be due to the physical interaction between the UFNBRP and the epoxy. In fact, UFNBRP and epoxy are available as a blend.To investigate the interaction between the epoxy resin and UFNBRP, the FTIR technique images have been used, and the images are given in terature . In the The SEM images of neat epoxy and the epoxy resin/1% UFNBRP nanocomposite at 50 kx magnifications and the TEM image of epoxy resin/1% UFNBRP are given in The SEM analysis shows that the UFNBRP is uniformly dispersed in epoxy resins, due to the great interface and intense interactions, including hydrogen bonding and chemical reaction on the interface between UFNBRP and epoxy resin . The epoChemorheology is the study of the change in viscoelastic properties in reacting systems, including an analysis of the alteration in viscosity as a function of the chemical reaction’s conversion ,41. The As shown in The gel point is usually determined as the time or the degree of cure at which the solution no longer remains liquid. Originally, qualitative rheological investigation was used to define the gel point. The intercept of loss modulus and storage modulus can be considered the gel point . The crig), they are equal (G″ = G′) and the system shows elastic behavior (G′ > G″) [a of curing epoxy/UFNBRP are presented in At the gel point, the viscosity of epoxy resin increases sharply and the rate of reaction is controlled by the penetration phenomenon, while the resin processability is reduced. Glassing usually occurs after the gel point, when the degree of molecular weight increases and further crosslinking reduces the degree of crosslink . Many reG′ > G″) . The gela changed upon UFNBRP addition, as well as with temperature. Ea of neat epoxy is 29.00 As could be observed in eratures .Onset of cure and The degree of curing (α), is defined according to Equation (5) where G′(t) is known as the storage modulus at any time, G′(0) is the modulus at the initial time, and G′(∞) is named as the storage modulus at infinite time:As shown in a initially decreased (until 1%), and then increased (from 1 to 1.5%). This is probably due to the accumulation of nanoparticles, which can also be seen in the SEM and TEM images. In general, Ea was lower in the epoxy/UFNBRP nanocomposites than in the neat resin [Kinetics parameters of epoxy resins and epoxy/UFNBRP nanocomposites at temperatures of 65, 70 and 75 °C are shown in at resin ,59,60.The curing rate of epoxy resin and epoxy nanocomposites are depicted in The final equations, which are predictive equations for the curing reaction, were obtained for neat epoxy and nanocomposites. The experimental results that were obtained from the reaction rate and the amounts achieved from the following relationships are given in The effect of elastomeric nanoparticles on the cure behavior of epoxy nanocomposites was studied using morphological and chemorheological methods and curing kinetics. The viscoelastic functions,


Herein, PS‐loaded microneedle (MN) patches with O2 propellant for a deeper and faster transdermal delivery of PS and improved PDT by embedding sodium percarbonate (SPC) into dissolving poly(vinyl pyrrolidone) MNs are presented. It is shown that SPC in the MNs can react with surrounding fluid to generate gaseous oxygen bubbles, forming vigorous fluid flows and thus greatly enhancing PS of chlorin e6 (Ce6) penetration in both hydrogel models and skin tissues. Reactive oxygen species (ROS) in hypoxic breast cancer cells (4T1 cells) are greatly increased by rapid penetration of PS and relief of hypoxia in vitro, and Ce6‐loaded SPC MNs show an excellent cell‐killing effect. Moreover, lower tumor growth rate and tumor mass after a 20‐d treatment in tumor‐bearing mice model verify the improved PDT in gaseous oxygen‐droved delivery of PS. This study demonstrates a facile yet effective route of MN delivery of PSs for improved PDT in hypoxic tumor treatment.Photodynamic therapy (PDT) is an emerging technique for treating tumors. Especially, topical administration of photosensitizers (PSs) is more favorable for superficial tumor treatments with low systematic phototoxicity. Yet, ineffective migration of PSs to targeted tumor tissues and rapid consumption of O 2 propellant that can actively deliver photosensitizer to the tumor by generating gaseous oxygen bubbles are developed, greatly enhancing the penetration of photosensitizer (PS) and relieving the hypoxia for improved photodynamic therapy.Photosensitizer‐loaded microneedles with O PDT has been widely applied in the clinical local treatment of various cancers, especially superficial tumors considering the limited penetration depth of the light irradiation. In general, the successful implementation of the PDT for the tumor treatment replies on the delivery and the accumulation of PS in the targeted tumor tissues. Especially, the transdermal delivery of the PS to the targeted sites holds great promise in tumor treatments due to the low systematic toxicity.Photodynamic therapy (PDT) has become a promising and efficient strategy for the treatment of various tumors and non‐neoplastic diseases due to its noninvasive nature and spatiotemporal selectivity.1 In a nanoemulsions, iontophoresis, and microneedle (MN)‐based patches delivery. Among these, MN patches have received widespread attention in transdermal drug delivery due to their advantages of painlessness, minimal invasion, uniform drug delivery, safety, and high efficiency. Although dissolving MNs show good PDT effects for tumors, the traditionally tapered MNs usually show imperfect skin insertion due to the viscoelasticity of the skins, which would significantly reduce its delivery effectiveness. Besides, the passive diffusion of payloads in the skin also limits the penetration depth of the loaded PSs. Therefore, a significantly prolonged treating duration and increased amount of PSs are usually required to achieve the therapeutical effect. On the other hand, the abnormal metabolism and rapid growth of the tumor cells may consume a lot of oxygen, and disordered tumor blood vessels cannot supply sufficient oxygen which is essential for the PDT process. As a result, the partial pressure of oxygen at the tumor site is usually lower than that at normal tissues, and the hypoxic environment will severely inhibit the effect of PDT. Therefore, an all‐in‐one MN delivery platform that combines the characteristics for both active delivery and hypoxia relief is highly desirable but has been never explored.Recently, many techniques have been explored to further increase the transdermal delivery of PSs, including the help of penetration enhancers,6 nano2 propellant (PS‐loaded active DMNs) that could actively deliver PS to the tumor site by generating gaseous oxygen bubbles, greatly enhancing the penetration depth and the rate of PS and relieving the hypoxia of the tumor during the laser irradiation for improved PDT. The PS‐loaded active DMNs are made of a dissolving polymer of polyvinyl pyrrolidone (PVP) loaded with PS of chlorin e6 (Ce6) and sodium percarbonate (SPC) particles, which could effectively deliver PS to the tumor with high targeting efficiency. Upon skin insertion, the MNs dissolved immediately, and the encapsulated SPC particles can instantaneously react with the dermal interstitial fluid (ISF) in the surrounding environment and generate oxygen bubbles, producing a violent vortex in the ISF and powering up the penetration of the PSs. The improved release and migration of PS were verified by both theoretical simulation and experimental observation in vitro by recording the movement of fluorescence signal in water, Pluronic F‐127 gel, and pigskin model, and a greatly enhanced penetration depth was demonstrated via active delivery of SPC MNs. Different delivery behaviors were verified in hypoxic breast cancer cells (4T1 cells) and tumor‐bearing mice. Due to the enhanced penetration of PS driven by oxygen bubbles and relief of tumor hypoxia, the cell‐killing effect can be significantly increased from 29.2% to 99.0%. In vivo experiments in tumor‐bearing mice models demonstrated that tumor growth was significantly inhibited, and the survival rate increased 50% by the active delivery. The current study provides an efficient and rapid delivery of PSs, and the generated gaseous oxygen could relieve the tumor hypoxia, providing a new strategy for a high‐efficient PDT effect.Herein, we present PS‐loaded dissolving MN patches with O2Figure The generated oxygen bubbles can form convection at the localized site for enhancing penetration of PS to the deep tumor and meanwhile relieve tumor hypoxia transitorily. As shown in Figure  thus confirming its potential ability to pierce the skin and its scalability for in vivo applications. According to the ratio of the destructive force to the minimum force for MN insertion, the safety factor of our current MNs is calculated to be 2.44, higher than the minimum safety factor of 2, ensuring the integrality of MNs during the insertion process. Besides, the MN tip was observed through an optical microscope, and it could be seen that the tip structure of the MN was damaged after applying loads > 0.1 N per needle   to optimize the gas generation of the MN patches. It was found that once the MN patch contacted with phosphate buffer (PBS) solution, the MN shaft quickly dissolved and exposed the SPC particles to the surrounding fluid, producing gaseous oxygen bubbles rapidly  can be formed with the O2 pressure, which accelerated the migration of Ce6  and the results (0 and 25 min) confirmed that the significantly accelerated diffusion of Ce6 was observed, showing a larger penetration depth  due to the gaseous oxygen bubbles generated by SPC particles  replacing the SPC. We found that once being in contact with the PBS solution, the polymeric matrix quickly dissolved with clearly observable gas bubbles within 10 s  breast cancer cells (4T1) as models, we first evaluated the intracellular ROS level by using a 2,7‐dichlorofluorescin diacetate (DCFH‐DA) probe. The DCFH‐DA was non‐fluorescent, it could be hydrolyzed by esterase and followed by being oxidized by ROS in cell to form a compound with strong green fluorescence signal. Compared to the SC group, we found that the addition of SPC could increase the average fluorescence intensity of the probe that reflects the intracellular ROS level, by 2.37 times, suggesting that a more efficient cell‐killing effect was achieved . Without Ce6, the green fluorescence signal of probe in the SPC (+) group had a slight increase compared with that in the laser group, which could be attributed to the relief of hypoxia by gaseous oxygen released from active DMNs. Once the Ce6 was added, the ROS in hypoxic 4T1 cells was significantly increased under sufficient PS, especially with increased gaseous oxygen from Ce6‐loaded active DMNs, which could be attributed to the enhanced penetration and the relief of hypoxia , different treatment methods were adopted every other day: no treatment (control), laser (+), Ce6‐loaded SPC MNs (SPC MNs (−)), Ce6‐loaded MNs with laser irradiation (MNs (+)), Ce6‐loaded SC MNs with laser irradiation (SC MNs (+)), Ce6‐loaded SPC MNs with laser irradiation (SPC MNs (+)). The MN patches were inserted into the tumor on the right side of the mouse and kept for 5 min, followed by irradiation of 655 nm laser at 0.56 W cm−2 for 3 min after 30 min  was used to evaluate the in vivo PDT effect of the Ce6‐loaded DMNs. Specifically, 4T1 cells were cultured in the dermis of Balb/c mice, when the subcutaneous tumors grew to 5–6 mm in diameter  and sodium percarbonate (SPC) were bought from Aladdin Biochemical Technology Co., Ltd . Sodium carbonate (SC), dimethyl sulfoxide , and agarose (BR) were provided by Sinopharm Chemical Reagent Co., Ltd . Chlorins e6 (Ce6) was purchased from Frontier. F‐127 was purchased from Sigma (USA). 2,7‐Dichlorofluorescin diacetate (DCFH‐DA) was purchased from Beyotime Biotechnology Co., Ltd (China). Calcein‐AM/PI live/dead cell staining kit was provided by Yeasen Biotechnology Co., Ltd .Polydimethylsiloxane  was purchased from Dow Corning Corp. (USA). PVP (−1), PVP (400 mg mL−1), and Ce6 (1 mg mL−1) in DMSO was filled into the PDMS female mold under vacuum at a pressure of −0.08 MPa for 10 min, and the excess solution was removed. The mixture with the mold was then dried at 50 °C for 8 h. Subsequently, PVP solution in DMSO solution (500 mg mL−1) was added to the above PDMS mold under vacuum at a pressure of −0.05 MPa for 5 min to form a base, and then the sample was dried in an oven at 50 °C overnight, followed by being peeled off from the mold and stored in a desiccator at room temperature. The preparation of SC MNs was similar to that of SPC MNs with SPC being replaced by SC microparticles.MNs were fabricated by a two‐step process by a micro‐molding method. Briefly, a mixture of SPC . Then, the detector was put into the PBS. Taking the dissolving oxygen of PBS as a contrast, the generated oxygen of the active DMNs is obtained by the increment of the dissolving oxygen.Morphologies of MNs were observed by a digital microscope  and a scanning electron microscope . The energy‐dispersive X‐ray spectroscopy (EDX) mapping analysis was carried out using a voltage of 18 kV. Bright‐field and fluorescent microscopy images of MNs were obtained by Olympus fluorescence microscope with a fluorescence filter for a blue‐light excitation. The gaseous oxygen generated during the dissolving process of active DMNs was tested by an oxygen dissolution tester . First, eight active DMN patches were put into a 50 mL centrifuge tube containing 15 mL PBS solution  and stored at 4 °C until the solution became transparent. The solution was transferred to a plastic cuvette and cured at room temperature to obtain the F‐127 gel. Then, the Ce6‐loaded SPC MNs were inserted into the F‐127 gel, and the evolution of the red fluorescence signal was recorded by a microscope.An aqueous solution of F‐127 (30 wt%) was weighed in a 50 mL centrifuge tube in phosphate buffer solution  with a constant loading speed of 4 µm s−10 m2 s−1. Next, the boundary conditions were set: where the concentration of the inflowing payload at each capsule was 5.77×10−5 mol m−3, and the flow rate of gas was 2×10−4 kg (m2 s)−1. To simplify the geometry, the diffusion process was simulated of the three capsules, thus the boundaries were set periodically, and the top of the calculation region was set as the gas outlet condition. Finally, the transient flow process with a total duration of 120 s and time step of 0.1 s was calculated.A transient laminar flow model was used in COMSOL to simulate the flow process of payload in water. First, a 2D geometry was constructed and unstructured triangular meshes were created. The size of the meshes was controlled by physical field and there were 6318 meshes in total. Since the flow was multiphase, the Bubbly Flow (bf) interface was used to simulate the influence of bubbles on the velocity field, and employed the Transport of Diluted Species in Fractures (dsf) interface to simulate the convection and diffusion of payload. The coefficient of diffusion of the payload in water was set as 4.56×102) at 37 °C for another 30 min. After removing the gel, the wells were irradiated with 655 nm laser with an intensity of 0.1 W cm−2 for 3 min and observed under the fluorescent microscope. The average fluorescence intensity (FL intensity) was calculated using Image J by the equation: average FL intensity = (Integrated Density)/area.The 4T1 cells were cultured in 12‐well plates for 8 h and transferred to an anaerobic bag at 37 °C for 4 h, and were divided into 6 groups: (1) control, (2) laser, (3) Ce6‐loaded MNs + laser, (4) Ce6‐loaded SC MNs + laser, (5) Ce6‐loaded SPC MNs + laser, (6) SPC MNs + laser. A piece of 2 mm agarose gel was embedded into each well and added the DCFH‐DA probe for 30 min incubation, then treated it with MNs and sealed it in the anaerobic bag (1% Oty)/area.212) for another 30 min, and the wells were illuminated with 655 nm laser at 0.1 W cm−2 for 3 min after the removal of agarose gel. Lastly, the cell viability was tested by CCK8 according to the user's guide.To evaluate the cell‐killing ability of Ce6‐loaded SPC active MNs under 655 nm laser, the groups were divided as: (1) control, (2) laser, (3) Ce6‐loaded MNs + laser, (4) Ce6‐loaded SC MNs + laser, (5) Ce6‐loaded SPC MNs + laser, and (6) SPC MNs + laser. The 4T1 cell monolayers were cultured in 24‐well plates for 8 h and transferred into an anaerobic bag for 4 h to create a hypoxic atmosphere. The 2 mm agarose gel was tiled above the monolayers before punctuating the MNs. Then, each well was sealed and the plate was kept in the anaerobic bag (1% O2) for 4 h before PDT treatment . After the treatment, the cells were placed at 37 °C for a 4 h‐incubation. The Calcein‐AM/PI staining was performed according to the manufacture's instructions. Cells stained green  and red (dead) were imaged under the fluorescence microscope.The 4T1 cells were plated in 12‐well plates and the wells were put into an anaerobic bag for 4 h, followed by being classified as: (1) control, (2) laser, (3) Ce6‐loaded MNs + laser, (4) Ce6‐loaded SC MNs + laser, (5) Ce6‐loaded SPC MNs + laser, (6) SPC MNs + laser, and were put into an anaerobic bag  were purchased  and were housed in a temperature‐controlled room (22 ± 1 °C) on a 12 h light‐dark cycle with free access to food and water, and raised in the specific pathogen‐free condition. All animal experiments were carried out in accordance with the Guide for the Care and Use of Laboratory Animals of Huazhong University of Science and Technology, approved by the Institutional Animal Care and Use Committee, Tongji Medical College, Huazhong University of Science and Technology (IACUC Number: 2680).6 cells per mouse on the right buttocks. The PDT treatment was started when the tumor reached 100 mm3. The mice were randomly divided into 6 groups (8–10 mice/group): (1) control, (2) laser, (3) Ce6‐loaded MNs + laser, (4) Ce6‐loaded SC MNs + laser, (5) Ce6‐loaded SPC MNs, and (6) Ce6‐loaded SPC MNs + laser. The tumors were treated by MNs for 5 min to allow the MNs to dissolve completely, and the tumor was irradiated with 655 nm laser (0.56 W cm−2) for 3 min after 0.5 h. All the treatments were given every other day. The other group without treatment was employed as a control. The volume of the tumor was monitored by a caliper every other day and calculated by the following equation: V = Ltumor length × (Wtumor width)2/2. Simultaneously, the weight of the tumor was recorded. After treatment was completed, the mice were sacrificed, and then the tumor and major organs were dissected for further H&E staining and Ki67 immunohistochemical staining.The 4T1 tumor‐bearing mice were established by intradermal injection of 1×10T‐test. The probability value of *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 was considered significant. GraphPad Prism 7 was used for statistical analysis.All data are obtained from at least three independent experiments with three or more parallel samples per condition in each experiment and are expressed as mean ± standard deviations (SD). Significance was assessed with two‐way ANOVA and Student's The authors declare no conflict of interest.Supporting InformationClick here for additional data file.Supplemental Video 1Click here for additional data file.Supplemental Video 2Click here for additional data file.


Good dispersion of carbon nanotubes (CNTs) together with effective curing were obtained in epoxy/CNT nanocomposites (NCs) using three different ionic liquids (ILs). Compared to conventional amine-cured epoxy systems, lower electrical percolation thresholds were obtained in some of the IL-based epoxy systems. For example, the percolation threshold of the trihexyltetradecylphosphonium dicyanamide (IL-P-DCA)-based system was 0.001 wt.%. The addition of CNTs was not found to have any significant effect on the thermal or low-strain mechanical properties of the nanocomposites, but it did improve their adhesive properties considerably compared to the unfilled systems. This study demonstrates that ILs can be used to successfully replace traditional amine-based curing agents for the production of electrically conductive epoxy/CNT NCs and adhesives, as a similar or better balance of properties was achieved. This represents a step towards greater sustainability given that the vapor pressure of ILs is low, and the amount needed to effectively cure epoxy resins is significantly lower than any of their counterparts. Epoxy resins are widely used due to their excellent mechanical and adhesive properties. One way to further improve these properties and acquire others—such as electrical conductivity—is by adding carbon-based nanofillers such as carbon nanotubes (CNTs). CNTs are an allotropic form of carbon made up of one (SWCNT) or multiple (MWCNT) one-atom-thick graphene sheets that are rolled into hollow cylinders with a nanometric diameter. They present outstanding mechanical and electrical properties. However, the nanofiller must be properly dispersed for the nanocomposites (NCs) to obtain a good balance of properties.Epoxy resin/CNT NCs cured with traditional curing agents, such as amines, anhydrides, polyamides and imidazoles, have been widely studied ,2,3,4,5.In fact, the strong propensity of CNTs to form bundles and aggregates  is one of their main drawbacks, as it limits their potential for improving mechanical and adhesive properties. In recent years, the use of ionic liquids (ILs) has been reported to enhance the dispersion of CNTs, thereby improving the properties of NCs based on different thermoplastic ,13,14 anILs are generally defined as salts that melt at temperatures below 100 °C. They have a low vapor pressure, are ionically conductive, and are thermally and chemically resistant. Due to these properties, they are used in a wide variety of applications including as solvents in synthesis , electroRegarding the role of ILs as curing agents in epoxy-based systems , imic) was reached at 0.016 vol% when 10 phr of IL was used and at 0.047 vol% when 30 phr of IL was used. Using a different phosphonium-based IL (tri(hexyl)tetradecyl phosphonium bis phosphinate) as the dispersing and curing agent in epoxy/MWCNT NCs, Soares et al. [c-s of between 0.25 and 0.5 phr MWCNTs [Santos et al.  investigs et al.  and Makas et al.  achievedr MWCNTs  and belor MWCNTs , respectAlves et al.  and LopeWaters et al. ,18 and TKleinschmidt et al.  analyzed−8 S/cm to 10−5 S/cm). They attributed this to the ability of the IL to arrange the electrically conductive CNTs in the matrix and form a more complete conductive network. The enhanced dispersion of the CNTs also led to a 12% improvement in the tensile strength (from 64 MPa to 72 MPa) of the epoxy/CNT NCs.Gholami et al.  also anaHowever, while the dispersive effect of ILs in epoxy/CNT systems has been discussed quite widely in the literature, the role of ILs as effective curing agents has received less attention. Moreover, to the best of our knowledge, no studies have been conducted on the effect of different ILs and CNT concentrations on the final mechanical, electrical, and adhesive properties of epoxy NCs. Therefore, in this study, three different ionic liquids were selected (based on a previous study ) and use3; viscosity (at 25 °C): 11,500–13,500 mPa·s). A traditional amine-based curing agent, 2,2′-dimethyl-4,4′-methylenebis(cyclohexylamine) (Aradur) (Huntsman), was used as a reference. The carbon nanotubes used in this work were of the Nanocyl NC7000 series  supplied by Nanocyl.The ionic liquids used in this study were as follows: (a) trihexyltetradecylphosphonium bisphosphinate (IL-P-TMPP), (b) 1-ethyl-3-methylimidazolium dicyanamide (IL-I-DCA) from Sigma Aldrich, and (c) trihexyltetradecylphosphonium dicyanamide (IL-P-DCA) from IoLiTec-Ionic Liquid Technologies GmbH. The epoxy resin was previously degassed in a vacuum oven at 80 °C for one hour. For the reference unfilled epoxy/IL and epoxy/Aradur systems, 10 phr of IL (selected as the optimum content based on our previous work ) and theFor the epoxy/CNT/IL systems, after degassing the DGEBA, the CNTs were added at concentrations ranging from 0 to 0.25% and mechanically mixed at 2000 rpm for 20 min using a EUROSTAR power control-visc digital stirrer. They were then ultrasonicated in a Hielscher UP400s at an amplitude of 100% for 20 min. Next, the IL (10 phr) was added and mechanically mixed at 50 °C for 5 min using a Heidolph RZR2000 digital rod stirrer. Finally, the mixture was poured into molds or placed between lap shear test substrates and the appropriate curing protocol was applied . Figure 3 were tested using a TA Q800 viscoelastometer in single-cantilever bending mode, with a frequency of 1 Hz and an amplitude of 15 μm. The heating rate was set at 4 °C/min and the temperature interval ranged from −100 °C to 250 °C. One specimen was tested per composition. The elasticity theory (Equation (1)) was used to calculate the crosslinking density (eν):rE is the storage modulus in the rubbery state at a reference temperature (rT = 245 °C) and R is the ideal gas constant (8.314 J/mol·K).Dynamic mechanical analysis (DMA) was used to determine the glass transition temperature and to calculate the crosslinking density of the systems. Rectangular, nominally sized specimens measuring 17.5 × 6.0 × 2.0 mmThe nanostructure and the dispersion level of the CNTs were analyzed by transmission electron microscopy (TEM). A Tecnai G2 20 Twin microscope was used at an accelerating voltage of 200 kV. The samples were cut at a 45° angle using a Leica EM UCG ultramicrotome with a diamond blade.σ) was calculated using Equation (2):ρ is the resistivity, V is the voltage applied, I is the intensity of the current, and 22.9 is the geometrical constant (specific area between electrodes). Three measurements were made for each reported value.The electrical conductivity through the sample was measured using a digital Keithley 6487 picoammeter on circular samples (Ø70 mm × 2 mm thick), to which 1 V was applied for 1 min. The electrical conductivity (σ(p) is the conductivity, B is a constant, t is the critical exponent, p is the nanofiller concentration, and cp is the percolation concentration. The experimental data were fitted by plotting log(σ) vs. log(p − cp) and increasing cp until the best linear fit was obtained.The percolation threshold was calculated using Equation (3):Three-point bending tests were carried out in an Instron 5569 universal testing machine. The span was set at 64 mm with a crosshead speed of 2 mm/min. At least 5 specimens were tested for each composition.fE), the flexural strength (Fσ), and the deformation at break (Fε) were calculated according to the ISO 178 standard, using Equations (4), (5) and (6), respectively:F is the load, maxF is the maximum load, L is the span, s is the degree of deflection, and b and h are the width and thickness of the specimen, respectively.The flexural modulus . The maximums of the peaks for the IL-based systems, along with the calculated crosslinking density values, are summarized in g values have been reported when CNTs were added to epoxy resins, so there is no general consensus in the literature regarding the impact of CNTs on the Tg of conventionally cured epoxy/CNT systems. Decreases in Tg are generally attributed to the steric hindrance of the CNTs towards the curing reaction [g are usually linked to decreases in the mobility of the macromolecular chains caused by the presence of CNTs. This effect is more pronounced the greater the degree of dispersion or adhesion, i.e., the two parameters reported to most heavily impact the Tg [g have been reported. The effect of the presence of ILs is not completely clear in these cases because they are also known to act as effective plasticizers. Both decreases [g have also been reported for epoxy/CNT NCs where the ILs acted as both curing and dispersing agents. The behavior of the epoxy/Aradur system in this study was similar to that of the epoxy/IL systems; the addition of CNTs did not significantly affect the Tg or the crosslinking density of the unfilled system .As reaction ,55, whict the Tg . Regardit the Tg  and decrt the Tg ,48,56 inecreases  and increcreases ,37 in TgThe nanostructures of the epoxy/IL systems were analyzed by transmission electron microscopy (TEM). 2 orbitals perpendicular to the layers, which lead to the reaggregation of the CNTs. ILs have been reported to be effective in terms of dispersing CNTs, although how they actually execute this is a controversial topic judging from the bibliography contained herein. In the case of imidazolium-based ILs, some authors indicate that the π–cation interaction [The achievement of good dispersion of CNTs is known to be complicated due to interactions between the speraction ,20 is lieraction  cause theraction . Moreoveeraction ,36,37.−7, 5.67 × 10−8, 1.96 × 10−9, and 9.99 × 10−9 S/cm for the IL-P-TMPP, IL-P-DCA, IL-I-DCA, and Aradur-based systems, respectively. The percolation thresholds are shown in B and t (Equation (3)) along with the conductivity values corresponding to the pc are presented in cp at 0.001 wt.%. This is consistent with the best dispersion levels on the TEM micrographs. At high CNT shares, the conductivity values of the systems cured with the phosphonium-based ILs  were two orders of magnitude higher than those cured with the imidazolium-based IL , suggesting that the long chains in the structures of the former two are more effective at preventing the formation of CNT aggregates.cp values do not exceed 1–2 wt.% [−7 to 10−2 S/cm) can be found in our bibliography depending on the experimental technique or conditions used [In the literature, a wide range of percolation threshold concentrations have been reported for epoxy/CNT NCs. This variation is probably due to the myriad factors that affect the formation of percolated networks, namely, the aspect ratio of the CNTs. Usually, the reported 1–2 wt.% , with mo1–2 wt.% ,60,61. H1–2 wt.% ,63 or 0.1–2 wt.%  have als1–2 wt.%  studied ons used ,36,37.As No significant trends in flexural strength were observed in any of the systems b. As witWith respect to high-strain mechanical properties, the deformation at break c and theEpoxy resins that have been mechanically improved by adding CNTs have been extensively documented in the literature ,2. In amFew papers on the roles of ILs as both curing and dispersing agents in epoxy systems have examined their mechanical behavior. For those that have, organoclays , silica Our findings are consistent with the results reported in the literature for epoxy/CNT systems ,5,11. InConsidering the results in The dual role of ionic liquids (ILs) as effective curing and dispersing agents produced volatile-amine-free epoxy/CNT nanocomposites with a better balance of mechanical, electrical, and adhesive properties. Three different ILs were tested, and all three led to good dispersion of the nanofiller, featuring individually dispersed CNTs as well as some small aggregates. Overall, with a percolation threshold of 0.001 wt.%, the IL-P-DCA system was the most effective.The addition of CNTs had no effect on the thermal or low-strain mechanical properties of the epoxy/IL systems. However, it did improve the systems’ adhesive properties. The epoxy/IL-P-DCA system containing 0.025 wt.% CNTs improved by 30% and was the best of the three. This work proves that, using very small amounts of CNTs, it is possible to obtain electrically conductive, amine-free epoxy adhesives with similar mechanical properties but greater lap shear strength than the reference amine-cured epoxy system. ILs have a lower vapor pressure, and a significantly lower amount is needed to effectively cure epoxy resins. Therefore, replacing conventional epoxy resin curing agents  with ILs is a major step forward in the development of more sustainable materials.


The restoration of the Wnt/β-catenin pathway to alleviate alcoholic fatty liver disease (AFLD) progression is under study as a new strategy for alcoholic liver disease (ALD) treatment. Recent studies have indicated that interferon-stimulated gene 15 (ISG15) can covalently bind to β-catenin by HECT E3 ubiquitin ligase 5 (HERC5), leading to ISG degradation and downregulation of β-catenin levels. However, the relationship between β-catenin and the ISG15 system in AFLD remains unclear.Here, we explored the roles of the ISG15 system in β-catenin activation and in the pathogenesis of alcohol-induced liver injury and steatosis.In this study, HERC5 silencing upregulated β-catenin protein expression and inhibited lipid metabolism disorders and cell apoptosis. Reduced β-catenin protein expression, increased lipid metabolism disorders, and cell apoptosis were detected in cells induced with HERC5 overexpression, which was reversible with the reactive oxygen species (ROS) inhibitor. All the above results were statistically analyzed. Thus, these observations demonstrate that β-catenin ISGylation is a prominent regulator of ALD pathology, which works by regulating ROS to induce lipid metabolism disorders and cell apoptosis.Our findings provided the mechanism involved in the β-catenin ISGylation, allowing for future studies on the prevention or amelioration of liver injury in ALD. Excessive alcohol consumption is fatal to the liver, causing alcoholic liver disease (ALD) which is characterized by lipid metabolism disorders and liver injury ,2. GenerRecent studies have highlighted that Wnt/β-catenin signaling is required for liver protection against oxidative stress-induced apoptosis. Furthermore, pharmacological restoration of Wnt/β-catenin signaling has been proven to be effective in attenuating ALD progression in a rat model, indicating that it plays a protective role in ALD ,8. The cAn important novel insight was obtained in recent research articles, showing that HECT E3 ubiquitin ligase 5 (HERC5)-mediated ISG15 conjugation is involved in the degradation of β-catenin ,13. InteTherefore, an in-depth study on the regulatory mechanism of the ISGylation of β-catenin is crucial to understanding the pathogenesis of AFLD with abnormal Wnt signaling pathways. The current study was designed to test the effect of ISGylation of β-catenin on liver lipid metabolism and hepatocyte apoptosis as well as its mechanisms in the progression of AFLD.The adult male C57BL/6J mice (18–22 g weight) were purchased from the Experimental Animal Center of Anhui Medical University. All animal procedures were reviewed and approved by the Institutional Animal Experimental Ethics Committee. Mice were randomly divided into control diet (CD)-fed and EtOH-fed groups. The CD-fed mice were fed with isocaloric maltose-dextrin (Sigma-Aldrich), and the EtOH-fed mice were fed ethanol (5% v/v) liquid diets (LD) for 10 days with a single binge ethanol administration  by gavage. The mice were anesthetized after the last gavage alcohol administration, and their liver tissues were harvested for further analysis. The liver tissues were either immediately frozen in liquid nitrogen, fixed in 10% neutral-buffered formalin for hematoxylin and eosin (H&E) staining and Oil red O staining, or embedded in frozen specimen medium . All experiments were performed at Anhui Medical University according to the institutional ethical guidelines for laboratory animal care. Procedures involving animals and their care were conducted in conformance with NIH guidelines and approved by the Animal Care and Use Committee (number: LLSC20150348).2 incubator. The medium was supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 0.1 mg/mL streptomycin.The human liver L02 and mouse hepatocyte AML-12 cell lines were obtained from the Institute for Cell Bank of the Chinese Academy of Sciences . These were cultured in DMEM medium  at 37°C in a 5% COTM 2000  according to the manufacturer’s instructions. Transfection efficiency was determined by qPCR analysis and western blotting. The oligonucleotides against the HERC5 gene or scrambled sequences were designed and synthesized by Gene Pharma Company  and had the following sequences: HERC5-siRNA (human): 5-GGAAGGAAAUUUCCCUCAATT-3, 5-UUGAGGGAAAUUUCCUUCCTT-3; and negative control: 5-UUCUCCGAACGUGUCACGUTT-3, 5-ACGUGACACGUUCGGAGAATT-3’.The L02 cells were cultured in 6-well plates and transfected with LipofectamineHERC5 was amplified from human genomic DNA using the following primers: Forward: 5-TACAAGTCCGGACTCAGATCTATGGAGCGGAGGTCGCGGAGGAA-3; Reverse: 5-GTACCGTCGACTGCAGAATTCGTTGGACAAGCAAGCTGGTC -3. The amplified products were inserted into the pEGFP-C2 empty vector and verified with direct DNA sequencing. Cell transfection was performed with LipofectamineTM 2000 according to the manufacturer’s protocol. The efficiency of overexpression was determined by western blotting and qPCR analysis.The coding region of GAPDH gene was used as an internal control for normalization. The primers used are presented in supplementary table 1.The total RNA was extracted from the cells or liver tissues using TRIzol (Invitrogen). These were then reverse transcribed to cDNA using the Transcriptor First Strand cDNA Synthesis Kit . The relative expressions of mRNA were determined by real-time PCR with SYBR-Green Master Mix (TaKaRa) according to the manufacturer’s protocol. The The total protein was extracted from cells or liver tissues using RIPA lysis buffer . Each sample was isolated via electrophoresis through 8–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto a polyvinylidene fluoride (PVDF) membrane . The membranes were incubated with the following primary antibodies at 1:300 dilutions: HERC5, ISG15, β-catenin, SREBP-1, PPAR-α, FOXO3a, BIM, Bax and Bcl-2 . Caspase3  and β-actin  was diluted 1:1000. The protein bands were detected using an ECL-chemiluminescent kit .The liver tissues were fixed with 4% paraformaldehyde for 24 h and embedded in paraffin blocks or embedded in frozen specimen medium. H&E and Oil Red staining were performed according to the standard procedures. The pathological changes were assessed, and the samples were photographed under a microscope.Cell homogenates and cell supernatants were collected to determine the levels of triglycerides (TGs) using the corresponding biochemical assay kits .reactive oxygen species (ROS). The L02 cell lines were cultured in 6-well plates in DMEM containing 10% FBS. For apoptosis analysis, the density of cells used was 106 cells/ml after addition of 400 ul Annexin V binding fluid. Cells were re-stained with 10ul Propidium iodide for 5 min and immediately measured by Flow cytometry. For ROS analysis, the cells were collected after transfection and ethanol stimulation and then processed with a reactive oxygen species (ROS) assay kit . The ROS in cells were detected by flow cytometry  using the excitation wavelength of 488 nm.Flow cytometric analysis was performed to evaluate the percentage of apoptotic cells and the level of We downloaded the three-dimensional analytical structures of ISG15 and β-catenin from the Protein Data Bank (PDB) to study the initial interaction between ISG15 and β-catenin. AUTODOCK 4.0 was used on the Discovery Studio 2017R2 operating platform for docking research. In the experiment, molecular docking of receptor ISG15 and ligand β-catenin protein was carried out. After the docking was completed, only one construct was retained to be selected as the optimal protein docking construct.p < 0.05 was considered statistically significant.Data are presented as mean ± SD of all replicates and analyzed using SPSS16.0 software. Statistical analysis was performed using two-tailed unpaired t-test or one-way ANOVA. In all cases, All male C57BL/6J mice with chronic alcohol feeding were characterized by inflammation, injury, and steatosis in the liver. H&E staining showed that the liver tissues in the EtOH-fed mice exhibited fat vacuoles, liver cell cord derangement, intercellular space dilatation, and inflammatory cell infiltration, whereas the liver tissues of the CD-fed mice showed normal lobular architecture with central veins and radiating hepatic cords. Oil red staining showed that the hepatic lipid deposition in the EtOH-fed group was significantly higher than in the CD-fed mice A. AdditiThen, we stimulated AML-12 cells with 100 mM EtOH and L02 cells with 150 mM EtOH for 24 h, wherein the concentrations were confirmed by the preliminary test. The results revealed that ISG15 and HERC5 mRNA and protein levels were elevated A-E and βTo determine the effect of EtOH ingestion on lipid homeostasis, the levels of SREBP-1 and PPAR-α were determined by qRT-PCR and western blotting. The results suggest that with the lower expression of PPAR-α, there was a significant increase in SREBP-1 expression in the AML-12 cells G-H. FurtAs other studies have reported, ISG15 binds to β-catenin through HERC5 (ISG15 E3 ligase) causing ISGylation, which leads to β-catenin degradation through the 20S proteasome pathway. To further study the connection between ISG15 and β-catenin, we used computer simulation docking technology to verify the interaction of ISG15 and β-catenin. The results showed that the protein molecules of the receptor ISG15 (PDB:6YVA) and the ligand β-catenin (PDB:4R0Z) form a stable docking structure  by salt HERC5 was used to knockdown HERC5 expression  of WHO , and conStudies have shown that blocking the Wnt/β-catenin pathway leads to the development of oxidative defects of fatty acids, increased oxidative stress, decreased expression of ethanol metabolic enzymes, the development of liver gluconeogenesis defects, etc . High exHERC5 was silenced using siRNA, causing an upregulation of β-catenin levels. Moreover, the overexpression of HERC5 with pEGFP-HERC5 caused a downregulation of β-catenin levels. These findings further confirm that the protein levels of β-catenin are downregulated by HERC5. SREBP-1, an important transcription factor that accelerates fatty acid synthesis, has been implicated in the pathogenesis of AFLD [In this study, we found that HERC5 and ISG15 levels were significantly higher and β-catenin levels were significantly lower in the liver tissue from the EtOH-fed mice than in the liver tissue from the CD-fed mice. To further illustrate the potential function of HERC5, ISG15, and β-catenin in the progression of ALD, we induced liver injury in the AML-12 and L02 cells with EtOH in vitro. The results showed that HERC5 and ISG15 levels were significantly upregulated and β-catenin levels were significantly downregulated after the EtOH stimulation. Moreover, we studied a computer simulation using docking technology to confirm that β-catenin forms a stable complex structure with ISG15. This indirectly proves that the protein stability of β-catenin affects its expression. It has been reported that β-catenin can be modified by ISG15. Furthermore, HERC5, an E3 ligase, was found to be necessary for the ISGylation of β-catenin. This was observed in this study when  of AFLD ,24. Simi of AFLD . The abo of AFLD . Our stuHERC5-siRNA or pEGFP-HERC5. The results showed that HERC5-siRNA reduced the levels of ROS while pEGFP-HERC5 increased the levels of ROS. Moreover, inhibition of ROS production with NAC can reverse excessive lipid metabolism disorders and apoptosis. Thus, HERC5-mediated ISGylation decreased β-catenin expression and enhanced ROS production.Alcohol exposure increases ROS production and reduces antioxidant levels in cells, leading to hepatocyte apoptosis and hepatic inflammation . AccumulTaken together, we found that the expression of β-catenin is closely related to the lipid metabolism, apoptosis, and levels of ROS in AFLD. Our study contributed to the understanding of the regulatory mechanism of the Wnt/β-catenin signaling pathway in AFLD, preliminarily revealed the effect and possible mechanism of β-catenin ISGylation on AFLD, and provided a new therapeutic strategy for the clinical prevention and treatment of ALD.