CDC42/RAC guanine nucleotide exchange factor, βPix, binds to β-catenin in colon cancer cells, augmenting β-catenin transcriptional activity. Using in silico, in vitro, and in vivo approaches, we explored whether these actions are regulated by M3R. At the invasive fronts of murine and human colon cancers, we detected co-localized nuclear expression of βPix and β-catenin in stem cells overexpressing M3R. Using immunohistochemistry, immunoprecipitation, proximity ligand, and fluorescent cell sorting assays in human tissues and established and primary human colon cancer cell cultures, we detected time-dependent M3R agonist-induced cytoplasmic and nuclear association of βPix with β-catenin. βPix knockdown attenuated M3R agonist-induced human colon cancer cell proliferation, migration, invasion, and expression of PTGS2, the gene encoding cyclooxygenase-2, a key player in colon neoplasia. Overexpressing βPix dose-dependently augmented β-catenin binding to the transcription factor TCF4. In a murine model of sporadic colon cancer, advanced neoplasia was attenuated in conditional knockout mice with intestinal epithelial cell deficiency of βPix. Expression levels of β-catenin target genes and proteins relevant to colon neoplasia, including c-Myc and Ptgs2, were reduced in colon tumors from βPix-deficient conditional knockout mice. Targeting the M3R/βPix/β-catenin axis may have therapeutic potential.M PTGS2, cyclooxygenase2 (COX2)]. In colon cancer cells, muscarinic receptor (MR) activation augments β-catenin signaling1. Of five MR subtypes, CHRM3, a conditional oncogene encoding M3R2, is overexpressed in 60–80% of colon cancers5. Post-M3R signaling selectively induces genes promoting cell proliferation, migration, and invasion13. Interconnecting mechanisms involving activation of protein kinase C-α (PKC-α) and transactivation of epidermal growth factor receptors (EGFR) mediate post-M3R signaling. M3R deficiency attenuates murine intestinal neoplasia14. Attenuated nuclear accumulation of β-catenin and intestinal tumor formation in M3R-deficient ApcMin/+ mice15, led us to explore functional crosstalk between M3R and β-catenin signaling.In quiescent intestinal epithelial cells, cytosolic β-catenin is sequestered in a multi-protein complex and targeted for degradation. In ~ 90% of colon cancers APC or β-catenin mutations destabilize β-catenin destruction complexes, freeing β-catenin to translocate to the cell nucleus. As a transcriptional coactivator of TCF4, nuclear β-catenin induces transcription of genes that promote neoplasia, e.g., prostaglandin-endoperoxide synthase 2   revealed frequent ARHGEF7 amplification in colorectal cancer  and 12 (71%) cancers, respectively; ARHGEF7 and CHRM3 were both over-expressed in seven of 17 cancers , and both βPix and M3R immunostaining were more intense in cancer compared to normal tissue /ERK pathway; there is extensive crosstalk between signaling pathways52. To explore post-receptor signaling underlying MR agonist-induced nuclear binding of βPix to β-catenin, we used previously validated chemical inhibitors52. Pre-treating HT-29 cells with Gö6976, a PKC inhibitor, or with AG-1478, an EGFR inhibitor, nearly abolished MR agonist-induced binding of βPix to β-catenin , using an antibody that recognizes activated β-catenin unphosphorylated at Ser-37 and Thr-41 residues55. Treating primary colon cancer cells with ACh stimulated striking cytoplasmic and nuclear colocalization of βPix and activated β-catenin . We used fluorescence cell sorting to explore if MR activation stimulated nuclear translocation of βPix or β-catenin. After treating HT29 cells with 200 µM ACh for 10 min, we measured shifts in nuclear localization of total β-catenin, activated β-catenin, and βPix. We failed to detect an increased nuclear signal for either total or activated β-catenin Fig. e, right.We were curious to see if β-catenin transcriptional activity modulated βPix expression. We verified that siRNA-treated HT-29 and SNU-C4 cells had reduced β-catenin expression; Fig. 3R agonists stimulate binding of pre-existing nuclear βPix to β-catenin and not cytoplasm-to-nucleus translocation of either βPix or β-catenin. Thus, our working model suggests M3R activation stimulates βPix binding to β-catenin independently within the cytoplasm and nucleus. We performed a pilot experiment to test whether nuclear βPix bound to β-catenin could participate as a TCF-4 transcriptional cofactor. We used anti-TCF-4 antibodies to determine whether exogenous Flag-tagged βPix augmented β-catenin binding to TCF-4. Immunoprecipitation assays using nuclear extracts derived from HCT116 colon cancer cells incubated with increasing concentrations of Flag-βPix revealed progressively augmented Flag-βPix and β-catenin binding to TCF-4 . We used the same approach to knock down βPix expression in HT-29 cells and confirmed ACh (100 μM for 24 h) did not alter βPix expression in naïve or transfected cells . Next, we examined the effects of stable knockdown of βPix expression on cell invasion. shRNA against ARHGEF7 robustly knocked down βPix expression in HT-29 cells; control shRNA had no effect  littermate controls with AOM/dextran sodium sulfate (DSS) and harvesting colons after 20 weeks  , a stronger effect of βPix deficiency than without MR agonist treatment .We explored the role of βPix in vivo by treating 30  eks Fig. a. Body weks Fig. b. Arhgefse) Fig. c − 55% fent Fig. a. ArhgefcMyc, Ccnd1, and Ptgs2 (Cox2)56, we compared expression of these genes in adenocarcinomas from CKO and control mice. mRNA expression levels for cMyc and Ccnd1, the genes encoding c-myc and cyclin D1 were reduced ~ 50% in tumors from Arhgef7 CKO mice . Removing an ‘outlier’ from the Ptgs2 dataset , uncovered 70% reduced Ptgs2 expression in βPix-deficient cancers .Having previously observed a relationship between MR activation and expression of Arhgef7 CKO and six control mice. Whereas β-actin loading controls were similar in gels from control and CKO mice, except for Cox2 signals in CKO mice 2 and 4, expression of colon cancer-related proteins was substantially reduced in βPix-deficient adenocarcinomas . We failed to detect a correlation between ARHGEF7 and PTGS2 mRNA expression , catalyzes the metabolism of arachidonic acid to prostaglandins and other bioactive molecules, must be carefully regulated. Elevated levels of COX2 are associated with the development and progression of colon neoplasia62 and reduced survival63; in both animal models and humans, inhibiting COX-2 activity with non-steroid anti-inflammatory drugs attenuates colon neoplasia. MR activation in human colon cancer cells up-regulates COX2 expression4. We found Ptgs2 expression was also greatly reduced in tumors from Arhgef7 CKO mice with intestinal epithelial cell-selective βPix deficiency. Finding a connection between the M3R/βPix/β-catenin and COX2 expression provides a mechanistic framework that may be clinically relevant. Indeed, the lack of a relationship between ARHGEF7 and PTGS2 expression in colon cancer suggests M3R activation is pivotal to augmenting COX2 expression by a βPix-dependent mechanism.In human colon cancer cells, we observed that βPix knockdown reduced PTGS2, important for cancer progression62. Reduced βPix expression attenuates β-catenin target gene expression, cell function, and murine colon tumor formation. Nonetheless, we acknowledge that our work has limitations. In vitro cell models may not recapitulate complex in vivo interactions and murine cancer models, while mimicking key features of human colon cancer progression, do not fully mirror the development and progression of human colon neoplasia. Lastly, a putative role for βPix as a cofactor in the β-catenin/TCF4 transcription factor complex must remain speculative until confirmed by complementary approaches. Pursuing this intriguing finding and confirming a functional interaction between endogenous βPix and the β-catenin/TCF-4 transcription factor complex that alters β-catenin target gene expression requires luciferase reporter, electrophoretic mobility shift, chromatin immunoprecipitation, and other approaches beyond the scope of the current project and will be pursued in future work.Based on the current findings, we propose a model wherein post-MR signal transduction, mediated primarily by PKC-α activation, stimulates βPix binding to β-catenin in the cytoplasm and nucleus. Our preliminary data suggest nuclear βPix binds β-catenin/TCF4 transcription complexes, augmenting key β-catenin target gene expression, e.g., 3R and β-catenin signaling that modulates colon cancer gene expression and cell function. Employing rigorous in vitro and in vivo experimental designs to test established human colon cancer cell lines and fresh human colon cancer cells using complementary innovative approaches, we uniformly observed M3R agonist-stimulated βPix/β-catenin binding in both the cytosolic and nuclear compartments and consistent effects on downstream expression of β-catenin target genes. Targeting the M3R/βPix/β-catenin axis has therapeutic potential. Our experience using Arhgef7 CKO mice with intestinal epithelial cell-selective βPix deficiency suggests targeting intestinal βPix would be safe; Arhgef7 CKO mice are healthy and fertile with modest phenotypic changes59. Lastly, since other upstream receptors act through PKC-α, our findings may be mechanistically relevant to other receptors and signaling pathways in colon neoplasia and other cancers in which muscarinic receptor signaling plays a prominent role.In conclusion, our findings identify a noncanonical role of βPix as an intermediary between MChemicals and reagents were obtained from the following sources: Matrigel from Corning #356,231), azoxymethane (AOM) from MRIGlobal (#0061); and dextran sodium sulfate (DSS) from MP Biomedical . If not otherwise specified, chemicals and reagents were from Sigma-Aldrich, St. Louis, MO, USA. Antibodies were obtained and used as follows: anti-βPix antibodies from Millipore , Santa Cruz , and Sigma-Aldrich ; anti-total β-catenin from Abcam (#ab19381); anti-activated-β-catenin from Millipore (05–665-AF555); Alexa Fluor 488 from Invitrogen (#A21206); Alexa Fluor 594 from Invitrogen (#A21203); anti-GAPDH from Cell Signaling (#2118); anti-FLAG antibody from Sigma-Aldrich (#F1365); and anti-Ki67 from Bethyl Labs (#IHC-00375). PCR primers are shown in Supplemental Table , azoxymeArhgef7flox/flox-/Tg(Villin-Cre) mice (Arhgef7 CKO mice) with intestinal epithelial βPix deficiency59. Mice were bred, housed, and treated under identical conditions in a pathogen-free room. To achieve microbiome equivalence, we co-housed animals throughout these studies until they were euthanized. Experiments were approved by the Institutional Animal Care and Use Committee of the University of Maryland, Baltimore, and VA Maryland Health Care System Research and Development Committee. All experiments were performed in accordance with institutional guidelines and regulations and the authors complied with the ARRIVE guidelines.We created 2 in a humidified incubator and passaged weekly at subconfluence after trypsinization.HT-29, H508, SNU-C4, and HCT116 human colon cancer cell lines were authenticated and purchased from American Type Culture Collection. HT-29 and HCT116 cells were grown in McCoy's 5A medium and H508 and SNU-C4 wells were grown in RPMI 1640 (Life Technologies-Thermo Fisher), both supplemented with 10% FBS. Cells were grown at 37ºC, with 5% CO64, after washing (HBSS), we cut tissues into 1–2 mm3 sections, and placed them in 0.1% trypsin at 4 °C overnight. We then placed tissues in complete growth media (RPMI 1640 supplemented with 10% FBS with an antibiotic–antimycotic) and incubated the material at 37 °C for 15 min. We then washed tissues (HBSS) before additional incubation in 0.1% collagenase for 45 min at 37 °C (5% CO2). After filtration (40-μm filters), we centrifuged cells at 150 × g for 5 min at room temperature, resuspended cells in culture media, and seeded cells in T25 flasks.We collected deidentified discarded fresh tissue samples from colon adenocarcinomas and adjacent normal colon from patients undergoing primary colon cancer resection at the University of Maryland Medical Center . As described previously65. Briefly, we grew fresh primary cancers on slides, fixed in cold MeOH for 10 min and permeabilized with 0.1% Triton-X-100 for 10 min on ice. We incubated cells with blocking solution for 60 min and incubated overnight (4 °C) with anti-βPix and -β-catenin antibodies. We then incubated cells with PLA probes for 1 h, with ligation solution for 30 min, and amplification solution for 100 min . To identify cell nuclei, we stained slides with 4',6-diamidino-2-phenylindole (DAPI). We acquired images using an LSM 510 inverted confocal microscope; dots in the cell nucleus represented βPix molecules within 40 nm of β-catenin molecules.We used the Duo-link In Situ-Fluorescence kit following both the manufacturer's instructions (Sigma-Aldrich) and published methods59, we synthesized first-strand cDNAs from 5 µg RNA  and performed qPCR using 50 ng cDNA, the SYBR Green PCR Master Mix (Applied Biosystems), and 0.5 μM in 20 μl forward and reverse primers. We designed primers . We performed qPCR using Step One (Applied Biosystems) with Power SYBR Green Master Mix (ABI). PCR conditions were as follows: 5 min at 95 °C followed by 37 cycles of 95 °C for 15 s, 60 °C for 20 s, 72 °C for 40 s, and final cycles at 95 °C for 15 s, 60 °C for 15 s, and 95 °C for 15 s. We normalized gene expression to β2-microglobulin (B2M) and analyzed qPCR data using the comparative CT (2C–ΔΔT) method.As described previously38, lysed cells in 20 mM Tris, pH 7.5, 100 mM NaCl, 5 mM MgCl2, 1 mM EDTA, 1% Triton X-100, 1 mM sodium fluoride, 1 mM sodium vanadate, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml pepstatin, and 1 µg/ml leupeptin. We separated equal amounts of protein by 7.5% sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transferred onto polyvinylidene difluoride membranes (Millipore), immunoblotted, and visualized bands by chemiluminescence (Amersham Biosciences). For immunoprecipitation, we added antibodies against β-catenin or βPix to cytoplasmic or nuclear lysates for 2 h, followed by protein A- or protein G-agarose beads for 1 h. We washed beads thrice in PBS, released immunoprecipitated proteins from beads by boiling in 1 × sample buffer (5 min), and immunoblotted. We employed β-actin and H2A loading controls to ascertain that equal amounts of cytoplasmic and nuclear proteins, respectively, were included in the extracted protein input. After immunoprecipitation, as a second loading control, we immunoblotted for expression of the bait protein ; this is marked by a vertical line to the left of the images obtained from the same gel.For immunoblotting, we treated cells with MR agonists and antagonists at the concentrations and times indicated and, as described previously38, we washed cells twice with ice-cold PBS, harvested cells with a rubber policeman, and lysed cells in 20 mM HEPES, pH 7.0, 10 mM KCl, 2 mM MgCl2, 0.5% Nonidet P-40, 1 mM Na3VO4, 10 mM NaF, 1 mM phenylmethanesulfonyl fluoride, and 2 µg/ml aprotinin. After a 10-min incubation on ice, we homogenized cells (20 strokes in a Dounce homogenizer) and sedimented nuclear homogenates by centrifuging at 1,500 × g for 5 min. Supernatants were centrifuged at 16,000 × g for 20 min; the resulting supernatant was the non-nuclear fraction. To remove contaminating cytoplasmic membranes, we washed nuclear pellets thrice with lysis buffer. To extract nuclear proteins, we resuspended isolated nuclei in 150 mM NaCl, 1 mM EDTA, 20 mM Tris–Cl, pH 8.0, 0.5% Nonidet P-40, 1 mM Na3VO4, 10 mM NaF, 1 mM phenylmethanesulfonyl fluoride, and 2 µg/ml aprotinin, and sonicated the material for nuclear lysis. We collected nuclear lysates after centrifugation  and electrophoresed the material on 7.5% SDS–polyacrylamide gels. We transferred proteins to polyvinylidene difluoride membranes, immunoblotted with antibodies, and detected bands by electrochemiluminescence.As described previously38.We performed FLAG-βPix and transient transfection of HCT116 colon cancer cells with FLAG-βPix using Trans-It transfection 2020 reagent  following manufacturer’s instructions66. We transfected HCT116 cells with siRNA targeting human βPix: siRNA #1, 5’-GAGCUCGAGAGACACAUGGTT-3’; siRNA #2, 5’-GGAUAUUAGUGUCGUGCAATT-3’ (Ambion)38. Silencer Negative Control siRNA #1 (Invitrogen) was used as control. For shRNA experiments using HT-29 cells, we targeted human GIPZ viral particles against the following sequence: AGGATGAAGTTCAAGAATT (Thermo Scientific)38. For shRNA, we used non-silencing-GIPZ lentiviral shRNAmir (Thermo Scientific) as control.We transfected HT-29 and SNU-C4 cells with 50 nM siRNA targeting human β-catenin67, we seeded HT-29 cells (4.5 × 106 cells) in 60-mm plates for 24 h. We serum starved cells for 24 h before adding 200 µM ACh for 10 min. After trypsinization, we fixed cells with 1% PFA on ice for 15 min and filtered them through 40-µm filters. We resuspended cells in 80% ethanol, incubated them for 4 min on ice, and washed with PBS. We blocked cells in PBS with 7% donkey serum and 0.1% TX-100 for 30 min at room temperature before adding primary anti-β-Pix and anti-activated β-catenin antibodies for 1 h at 4 °C. After washing with PBS, we incubated cells with secondary antibody . We washed cells with PBS, stained nuclei with DAPI at room temperature (15 min) and washed again with Ca2+/Mg2+-free DPBS. We performed flow cytometry and measured intracellular fluorescence . We collected bright field and fluorescent images  at a × 60 magnification. To determine nuclear localization of β-Pix (AF488 attached) and activated β-catenin (AF555 attached), we analyzed 5,000 gated cell singlets per sample and used IDEAS Analysis Software (Amnis) and the Similarity Morphology Feature.Applying methods described previously3 cells per well and transfected cells with 50 nM control or βPix siRNA using TransIT-siQuest reagent according to the manufacturer (Mirus). After 24 h, we stimulated cells with 100 µM ACh for 48 h and measured cell proliferation using the CellTiter-Glo assay kit (Promega). For cell migration assays, we placed HT-29 cells in the upper chamber of uncoated inserts (BD Biosciences). Cells on the underside of inserts were fixed, stained , and counted in five random high-power fields (HPF). We performed Invasion assays using Biocoat Matrigel Invasion Chambers (Corning) as described previously52.For cell proliferation assays, we seeded HCT116 cells at 7 × 10Arhgef7 CKO and littermate control male and female mice with once weekly intraperitoneal injection of 7.5 mg AOM/kg body weight for four weeks. Starting one week after the last AOM injection, 2.5% DSS was added to the drinking water for 5 days. In a separate experiment, after AOM/DSS treatment, we added bethanechol (400 µg/ml) to the drinking water for 15 weeks. Twenty weeks after the first AOM injection, investigators masked to genotype euthanized mice, harvested colons, and measured tumor number and size.We treated 10- to 17-week-old Oncomine™ (www.oncomine.org)44 to compare gene mRNA levels in colon cancer and normal adjacent tissue. GEPIA (gepia.cancer-pku.cn) analyzes RNA expression in tumor and normal samples from the National Cancer Institute Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) databases45. The Human Protein Atlas Program provided information on proteins in cells, tissues, and organs46 and used UALCAN  to analyze OMICS data47. We compared ARHGEF7, CHRM3, and PTSG2 mRNA expression levels as described57.We used t-test, Mann–Whitney U test, one-way ANOVA, with either Tukey’s HSD post hoc or Dunn’s tests using SigmaPlot 13.0  and two-tailed Fisher’s exact test using GraphPad Prism . We considered a p-value < 0.05 statistically significant.Unless indicated otherwise, we expressed data as mean ± SE from at least three experiments and analyzed results using Student’s two-tailed Supplementary Information.


TM S-97, and pectin) on the swelling property. The optimal discrete model was selected to predict the appropriate variables, due to the good fit of the experimental data and the model validity. The analysis of variance (ANOVA) of the model demonstrated p-value < 0.0001, R2 = 0.9923, adjusted R2 = 0.9894, and predicted R2 = 0.9831. Finally, the predicted film formulation containing 2.75% w/w hyaluronic acid, 1.321% w/w GantrezTM S-97, and 1.246% w/w pectin was used for further fabrication of MNs (525.4 ± 3.8 µm height and 157.4 ± 2.0 µm base width), which possessed 1508.2 ± 66.2% swelling, with 124.6 ± 7.4 µL of collection volume, and could withstand thumb pressure. Moreover, almost 50% of MNs achieved a skin insertion depth of approx. 400 µm, with 71.8 ± 3.2% to 78.3 ± 2.6% recoveries. The developed MNs show a promising prospect in microsample collection, which would be beneficial for POCT.Microneedles (MNs) have shown a great potential for the microsampling of dermal interstitial fluid (ISF) in a minimally invasive manner for point-of-care testing (POCT). The swelling properties of hydrogel-forming microneedles (MNs) allow for passive extraction of ISF. Surface response approaches, including Box-Behnken design (BBD), central composite design (CCD), and optimal discrete design, were employed for the optimization of hydrogel film by studying the effects of independent variables (i.e., the amount of hyaluronic acid, Gantrez The utilization of hypodermic needles for drug administration and sample extraction has a long-standing history. Despite their widespread usage, however, this traditional method has notable drawbacks, including pain, invasiveness, psychological distress, bio-hazardous waste, and the necessity for trained healthcare professionals. In recent times, the development of microneedle (MN) systems has been gaining increasing interest as a more effective approach for sample extraction ) and drug delivery, due to their various advantages over hypodermic needle-based systems. MN systems are characterized by micron-sized needles that offer a minimally invasive and more efficient method of drug delivery and sample extraction . FurtherMNs have been investigated for the delivery of drug molecules or the monitoring of biomarkers ,5, whichThe development of hydrogel-forming MNs for health monitoring assay purposes should approach a collection volume of ≥1 µL, with a collection time of ≤20 min; thus, the method should be applied in a simple and rapid manner . StatistTM S-97 have been utilized for hydrogel-forming MNs. Hyaluronic acid is a naturally occurring linear polysaccharide made up of repeating N-acetyl-d-glucosamine and d-glucuronic acid units, which has been widely used for hydrogel formulations in biomedical applications due to its excellent hydrophilicity, biocompatibility, biodegradability, nonimmunogenicity, and nontoxicity [TM S-97 is a synthetic copolymer of methyl vinyl ether and maleic anhydride (free acid), which is a non-toxic and biocompatible crosslinker with good mechanical strength. It has been widely used in denture adhesives, toothpastes, and mouthwash formulations because of its excellent film-forming ability [Hydrogels are commonly formulated from a variety of natural polymers, including chitosan, alginate, cellulose, hyaluronic acid, pectin, and starch, as well as synthetic polymers such as polyvinyl alcohol and polyethylene glycol. However, the irreparable impact of synthetic polymers on the environment has driven a recent increase in the use of natural polymers. This is due to their biodegradability, nontoxicity, widespread availability, and cost-effectiveness . Hyalurotoxicity ,19. Gant ability . Further ability .TM S-97 with the addition of pectin. A variety of RSM  were employed to study the effects of formulation variables on the significant characteristic of hydrogel film . Suitable models were evaluated by the analysis of variance (ANOVA). Next, the optimal hydrogel film was prepared according to the predicted values, and hydrogel-forming MNs were further fabricated by a micromolding method. Finally, the MNs were assessed by examining the morphology, mechanical strength, swelling and skin permeation abilities, and recoveries.This study aimed to develop hydrogel-forming MNs for the rapid collection of skin ISF in sufficient volumes by a formulation containing hydrogel with the main polymers of hyaluronic acid and Gantrezw/w hyaluronic acid and 0.5% w/w GantrezTM S-97 as the main polymer and crosslinker, respectively, which yielded a good swelling degree. However, the swelling degree dropped due to incomplete crosslinking of the film caused by an insufficient amount of GantrezTM S-97. Moreover, GantrezTM S-97 above 3% w/w resulted in a decrease in swelling. Moreover, the report suggested the use of 2% and 4% w/w pectin to enhance the film’s strength, but the results showed a decrease in swelling [w/w hyaluronic acid, 1–2% w/w GantrezTM S-97, and 0.5–1.5% w/w pectin) and the dependent variable . The experimental data obtained from the hydrogel film formulations listed in p-value, adjusted R2, predicted R2, and predicted residual sum of square (PRESS) value. Moreover, the ANOVA was applied to examine the significance of the model. All models were significant for the swelling degree with p-values < 0.05. The greater adjusted R2, predicted R2, and low PRESS value indicated the good fit of the model; therefore, the optimal (discrete) model was selected (1 + 76.75263X2 − 1090.60741X3 + 63.23516X1X2 + 100.12782X1X3 + 8.75302X2X3 − 25.37784(X1)2 − 330.53100(X2)2 + 153.67881(X3)2, where Y = % swelling, X1 = hyaluronic acid concentration (% w/w), X2 = GantrezTM S-97 concentration (% w/w), and X3 = pectin concentration (% w/w). In addition, the coefficient values in w/w hyaluronic acid, 1.321% w/w GantrezTM S-97, and 1.246% w/w pectin, offering the swelling degree of 958.76. The model was a good fit for the range of independent variables used to establish the model. However, the relationship between the independent and dependent variables can change outside of this range.The previous report demonstrated the preparation of hydrogel films using 5% swelling ,23. Therselected . The quaselected  is as foTM S-97, and pectin were utilized as the main polymer, crosslinking agent, and film-strength enhancer, respectively. The crosslinked film by esterification showed peaks at 1721 and 1150 cm−1, which are assigned to the C=O stretch and the C-O stretch, respectively. The peak at 3450 cm−1 is associated with free -OH groups of ingredients. The amide groups of hyaluronic acid show the peak of C = O stretch at 1555 and 1621 cm−1 and C-N stretch at 1403 cm−1. The peaks representing the C=O stretch and the C-O stretch were found to decrease when the film was not crosslinked. The swelling degree of the hydrogel films was 1033.5 ± 25.7% after placing them in the artificial ISF for 5 min  spectra of hydrogel ingredients and corresponding non-crosslinked and crosslinked films are shown in or 5 min , which wThe hydrogel-forming MNs featuring 100 pyramidal-shaped needles were fabricated by a simple micromolding method. The height and base width of MNs were 525.4 ± 3.8 µm and 157.4 ± 2.0 µm, respectively, which would penetrate through the epidermis (50–200 µm thickness) and reach the dermis (1500–3000 µm thickness) for the microsampling of ISF . TherefoThen, the MNs were placed in the artificial ISF for 5 min, which demonstrated the swelling degree of 1508.2 ± 66.2% and the collected volume of 124.6 ± 7.4 µL . After r® M (PF) membrane was used for MN testing as a stratum corneum simulant, which is the outermost layer of the epidermis. In order to investigate the influence of the manual application force in the MN insertion, the created holes on an eight-stacked PF layer were examined. The created holes of 100.0 ± 0.0%, 97.7 ± 3.2% and 45.3 ± 4.2% were observed for the first, second, and third layer, respectively  of 0.9966. The MNs were soaked with 0.3125, 1.25, and 5 µg/mL of the methylene blue. The percent recovery of the extracted ISF was in the range of 71.8 ± 3.2 to 78.3 ± 2.6%, indicating that the MNs were able to absorb the sufficient ISF volume.To study in vitro recovery of the extracted ISF extraction, the calibration curve of methylene blue in ISF was established in the range of 0.3125–5 µg/mL. The linear equation was y = 3459.5x + 3230 with correlation coefficient  (MW 1500 kDa), was provided by Ashland, Worcestershire, UK. Pectin from red apples was purchased from Chemipan, Bangkok, Thailand. MN molds  were purchased from Micropoint Technologies Pte Ltd., Singapore. Other chemicals used were analytical grade.Hyaluronic acid (MW 420 kDa) was purchased from Nanjing Gemsen International Co., Ltd., Nanjing, China. Gantrezp-value, adjusted R2, predicted R2, and PRESS values. The predicted optimal condition was tested experimentally in triplicate to validate the results.A randomized RSM, including CCD, BBD, and optimal (discrete) design, was used to study the effect of three variables on hydrogel films’ swelling properties. The dependent and independent variables, along with their levels, are listed in Ten grams of each aqueous formulation , Thermo Fisher Scientific, Waltham, MA, USA).The morphology, including length, width, and thickness of hydrogel films, was measured by the digital vernier caliper. Then, FTIR analysis of all ingredients and non-crosslinked and crosslinked films were carried out in the range of 4000–600 cm0 is the weight of the initial film, and mt is weight of the swollen film at 5 min.Next, the swelling test of the crosslinked hydrogel films was performed in artificial ISF (pH 7.4) . An emptv/v) by sonication for 20 min. The optimal formulation (0.04–0.05 g) was filled into the molds and centrifuged at 4500 rpm for 15 min at room temperature. Next, the molds were filled up to the weight of 0.150 ± 0.015 g. The MNs were dried at room temperature for 18 h, crosslinked at 80 °C for 24 h, and kept in the desiccator for further analysis.All the PDMS molds with 100 MNs obtained from the commercial molds were cleaned with the mixture of ethanol and water  that was capable of variable magnification of 20–220×. The accurate measurement of height and base width of the MNs was carried out by adjusting the magnification and focal height and setting the calibration.The swelling test procedure of the hydrogel-forming MNs was performed in the same manner as that of the hydrogel films. Then, the volume of ISF absorbed into each MN was also determined using the artificial ISF (density of 1.005 ± 0.001 g/mL). Also, the morphology of the MNs was evaluated before and after the swelling test.a) and after (Hb) the application of compressive force was measured, using the digital microscope. The percentage of height reduction of MNs was calculated by the following equation [The mechanical strength of hydrogel-forming MNs was investigated by the TA.XTplusC texture analyzer  in the compression mode. The MNs were attached to the cylindrical probe  by double-sided tape. The probe was lowered at a speed of 0.5 mm/s  until a pre-set force of 32 N, mimicking thumb pressure, was achieved. Subsequently, the force was analyzed to obtain a force-displacement curve. Furthermore, the height of MNs before  was placed on the base of the test machine, and the hydrogel-forming MNs were attached to the cylindrical probe. The procedure and all parameters were the same as mentioned in A calibration curve was established by plotting the fluorescence intensity against five different concentrations of methylene blue in the artificial ISF (0.3125–5 µg/mL). Then, the hydrogel-forming MNs were weighed and placed in the ISF containing 3 concentration levels  of methylene blue for 5 min. The MNs were taken out and weighed before transferring to a centrifuge tube containing 3 mL ISF. After centrifugation at 5000 rpm for 5 min, the supernatant was spectrofluorimetrically measured at the excitation wavelength of 650 nm and the emission wavelength of 685 nm. The percentage of recovery was calculated with the following equation:The RSM models employed Design-Expert software . All experiments were performed in triplicate. The data were expressed as mean ± standard deviation.