In this study, we found an acetyltransferase KAT8 in grass carp  that acetylated IRF3/IRF7 and then resulted in inhibition of IFN 1 response. CiKAT8 expression was up-regulated in the cells under poly I:C, B-DNA or Z-DNA stimulation as well as GCRV(strain 873) or SVCV infection. The acetyltransferase domain (MYST domain) of KAT8 promoted the acetylation of IRF3 and IRF7 through the direct interaction with them. So, the domain is essential for KAT8 function. Expectedly, KAT8 without MYST domain (KAT8-△264-487) was granularly aggregated in the nucleus and failed to down-regulate IFN 1 expression. Subcellular localization analysis showed that KAT8 protein was evenly distributed in the nucleus. In addition, we found that KAT8 inhibited the recruitment of IRF3 and IRF7 to ISRE response element. Taken together, our findings revealed that grass carp KAT8 blocked the activities of IRF3 and IRF7 by acetylating them, resulting in a low affinity interaction of ISRE response element with IRF3 and IRF7, and then inhibiting nucleic acids-induced innate immune response.Post-translational modifications (PTMs), such as phosphorylation and ubiquitination,  KAT8, serves as an inhibitor for IFN 1 response.KAT8 blocks IRF3/IRF7-induced IFN 1 response.KAT8 directly interacts with IRF3/IRF7 and promotes their acetylation through MYST domain.KAT8 inhibits the interaction between IRF3/IRF7 and ISRE within target gene.Innate immunity is the early line of defense against microbial infection in cell. It recognizes and eliminates pathogenic molecules through its pattern recognition receptors , 2. HoweThe post-translational modifications (PTMs) of innate immune signaling molecules affect their activities in signal transduction, which are critical in regulating the expression of type I interferon –5. CurreAcetylation is involved in many key cellular processes, including gene transcription, signal transduction, DNA damage repair, cell apoptosis, metabolism, and so on . Acetylaϵ-acetylation of histones and other non-histone proteins, and generate dynamic Nϵ-acetylation based on cell signals . They all have highly conserved lysine acetyltransferase domain , 23. The signals , 25.etc. (Lysine (K) acetyltransferase 8 (KAT8), also termed as MOF, MYST1, MOZ, YBF2, SAS2 or TIP60 1, can acetylate histone H4 lysine 16 (H4K16) in mammalian cells . KAT8 coetc. –34.There are still a few studies on the involvement of KAT8 in innate immune response. KAT8 acetylates IRF3 to negatively regulate antiviral immunity in mice . HoweverIn this study, the experimental evidences provided a new insight into how grass carp acetyltransferase KAT8 functions in the IFN 1 pathway. We found grass carp KAT8 interacted with IRF3 and IRF7 and then acetylated them, resulting in a weak interaction between IRF3/IRF7 and ISRE within target gene, finally suppressing IFN 1 expression.C. idella) ovary cells (CO), Grass carp kidney cells (CIK) and epithelioma papulosum cyprini (EPC) cells were maintained in medium 199 supplemented with 10% FCS and 1% penicillin/streptomycin solution. CIK cells are usually used in subcellular localization assay and gene knockout experiment because of their stronger adhesion and better cell morphology. CO cells are usually used in overexpression experiments due to their higher transfection efficiency.Grass carp (50) assay on CIK or EPC cells. For viral infection, CIK cells were plated for 12 h in advance and then infected with GCRV-873 or SVCV at 10-8 TCID50.GCRV (strain 873) and SVCV were gifts from Professor Wuhan Xiao . GCRV-873, a strain of GCRV virus (dsRNA virus), was propagated in CIK cells at 28°C SVCV, a ssRNA virus, was propagated in EPC cells at 28°C. M199 medium was collected and stored at 80°C until use. Viral titers were determined by a 50% tissue culture-infective dose (TCIDPoly(I:C) was purchased from Sigma-Aldrich (USA). B-DNA and Z-DNA were synthesized by Sangon Biotech . The sequences are presented in CiIFN 1 (1:1000) and anti-CiGAPDH (1:10000) antibodies were produced by our laboratory previously (® Rabbit mAb (1:1000) were purchased from Beyotime (China) and CST (USA), respectively. Due to the high conservation between grass carp and human KAT8 (86%), anti-human KAT8 rabbit monoclonal antibody  was used to detect grass carp KAT8. Anti-IRF7 Ab (1:1000) was from HUABIO Biological and used as previously described (The antibodies used (including the place of production and the dilution ratio) are as follows: Rabbit polyclonal anti-eviously . Phosphoescribed . GFP-TagCiKAT8, CiIRF3 and CiIRF7 were separately inserted into pEGFP-C1  vector for co-IP, subcellular localization and DNA pull-down assays. The amino acid truncations KAT8-(△1-151), KAT8-(△151-264) and KAT8-(△264-487) were generated by PCR-based amplification and constructed into the pCMV-FLAG, pEGFP-C1 vector respectively. The ORF of CiKAT8, CiIRF3 and CiIRF7 were separately inserted into pcDNA3.1 (+) vector  for over-expression experiments. IFN 1 and ISG15 promoter luciferase reporter plasmids were prepared in our lab  eukaryotic expression vector. The ORF frame of  our lab ; pISRE-T™ transfection reagent  according to the manufacturer’s protocol.For transient transfection of poly(I:C), B-DNA or Z-DNA into CIK cells, Lipofectamine 2000  was used according to the manufacturer’s instructions. Empty plasmid or various expression plasmids were transfected in CIK cells and CO cells using Lipo8000−8 TCID50 viruses (GCRV or SVCV) were separately cultured in 6-well plates for 6, 12, 24, 48, and 72 h, as described in our previous studies (CIK cells transfected with 2 µg oligomeric nucleic acids [poly(I:C) or B-DNA or Z-DNA] or 50 µl of 10 studies –39.β-actin, the results relative mRNA abundance was calculated by 2-∆∆CT method. Gene-specific primer sequences for qRT-PCR were shown in Total RNA was isolated from cells using RNAsimple Total RNA Kit  following the instruction. cDNA was then synthesized from 1 µg RNA using PrimeScript RT Reagent Kit . The qRT-PCR reactions were as follows: 7.2 µl of ddH2O, 10 µl of TB Green premix Ex Taq , and 0.4 µl upstream and downstream primers for each. The cycling systems were as follows: 1 cycle of 5 min at 95°C, followed by 40 cycles of 30 s at 94°C, 30 s at 55°C, and 30 s at 72°C. The mRNA levels of KAT8, IFN 1 and ISG15 were detected by CFX Connect™ Real-Time System . The expression value of each gene was normalized to CiKAT8 and negative control RNA (N.C) oligonucleotides were synthesized by Suzhou GenePharma Co., Ltd  and transfected with the indicated plasmids. At 24 h post transfection, cells protein was extracted and luciferase activity was measured by the dual Luciferase reporter gene assay kit . The plasmid of PRL-TK was served as the reference in the assay.http://nls-mapper.iab.keio.ac.jp/cgi-bin/NLS_Mapper_form.cgi). CIK cells, plated on microscopic cover glass , were transfected with the following plasmids , pEGFP-KAT8-(△151-264), pEGFP-KAT8-(△264-487), respectively. After 24 h of transfection, the cells were fixed in 4% (wt/vol) paraformaldehyde for 20 min and then stained with 0.1 µg/ml DAPI (Sigma-Aldrich) at 37˚C for 15 min. Cells were observed with a confocal laser microscope .Nuclear localization of KAT8 protein in grass carp was predicted by cNLS Mapper software  or IgG tagged agarose . The immunoblotting method was used to detect the interaction according to the manufacturer’s protocol, and the detailed experimental steps were performed as described previously   for 30min. Then they were centrifuged at 12000rpm for 10min, the supernatant was collected and incubated with GFP Ab tagged agarose  overnight at 4°C. The beads were washed with PBS three times and eluted with 100 µl 2 × SDS loading buffer by boiling for 10 min at 95°C. The precipitates were detected by immunoblotting with the acetylated-lysine antibody.DNA pull-down assays were carried out as described previously , 42. In CiKAT8, CIK cells were seeded in 6-well plates and transfected with 2 μg of pcDNA3.1-KAT8 for 24 h. Then cells were infected with 50 µl 10-8 TCID50 GCRV-873 for 5 day. Among them, MOCK group was the no treatment group. Then the cells were fixed with 4% paraformaldehyde for 30 min, and stained with 1% (w/v) crystal violet for 30 min for visualizing cytopathic effect (CPE). The three viral genes of GCRV-873, vp5 (Genbank ID: AF403391.1), vp6 (Genbank ID: HQ018818.1) and vp7 (Genbank ID: HQ018819.1), were detected by qRT-PCR. Gene-specific primer sequences for qRT-PCR were shown in To investigate the antiviral activity of p value of less than 0.05 considered statistically significant. Each figure of western blot, cytopathic effect assay and confocal microscopy was produced using Image J, which was represented by three independent experiments in this study.Each data of qRT-PCR and dual-luciferase assays were presented as mean and ± SD (n = 3). Statistical significance was determined with the two-tailed Student’s t-test, with a CiKAT8 was significantly up-regulated in cells transfected with 2 µg poly I:C, B-DNA and Z-DNA respectively  promoter activity in CO cells   assays. Overexpression of KAT8 in CIK cells caused CPE to enhance upon challenged with GCRV-873  and co-transfection group . In a single transfection group, overexpression of grass carp IRF3 or IRF7 led to a significant activation of IFN 1 response, which is opposite to the cells with KAT8-pcDNA3.1. However, the IFN 1 response was weakened when cells were co-transfected with pcDNA3.1-KAT8 and pcDNA3.1-IRF3 or pcDNA3.1-IRF7 , C2HC zinc fingers (△151-264) and the enzymatic MYST domain (△264-487) (Co-IP assay showed that FLAG-tagged KAT8 separately interacted with GFP-tagged IRF3 and GFP-tagged IRF7 in CO cells (The above conclusions have proved that KAT8 interacted with IRF3 and IRF7 through MYST domain (acetyltransferase domain), so whether KAT8 regulates IFN 1 expression by affecting the acetylation level of IRF3 and IRF7. We then analyzed the acetylation of IRF3/IRF7 in CO cells co-transfected Flag-tagged KAT8 (WT) with GFP-tagged IRF3 or GFP-tagged IRF7. We used acetylated-Lysine antibody to detect the acetylation of IRF3 and IRF7 and found that KAT8 enhanced the acetylation level of IRF3 and IRF7  and the three different Flag-tagged KAT8 truncations. Each of KAT8-WT, KAT8-(△1-151) and KAT8-(△151-264) can enhance IRF3/IRF7 acetylation. This effect disappeared when the MYST domain of KAT8 (KAT8-(△264-487) was deleted  did not affect IFN 1 expression (both at mRNA and protein levels)  can be found in the article/CH and XX supervised the research. DL revised the manuscript. ML conceived the study, designed and performed the experiments. ML, XX, HM and JH analyzed the experiments and data. ZJ, ZS, and TY provided reagents, technical assistance and contributed to completion of the study. ML wrote the manuscript. All authors reviewed the results and approved the final version of the manuscript.This work supported by research grants from the National Natural Science Foundation of China , Jiangxi Agriculture Research System (JXARS-06), China Postdoctoral Science Foundation (2019M662279), Jiangxi Postdoctoral Science Foundation .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Cancer stem cell (CSC)-related chemoresistance leads to poor outcome of the patients with colorectal cancer (CRC). In this study, we identified the chemoresistance-relevant molecules and decipher the involved mechanisms to provide potential therapeutic target for CRC. We focused on Sec62, a novel target with significantly increased expression in chemoresistant CRC tissues, and further investigated its role in the progression of CRC.Through analyzing the differentially-expressed genes between chemoresistant and chemosensitive CRCs, we selected Sec62 as a novel chemoresistance-related target in CRC. The expression and clinical significance of Sec62 were determined by immunoblotting and immunohistochemistry in tissues and cell lines of CRC. The roles of Sec62 in drug resistance, stemness and tumorigenesis were evaluated in vitro and in vivo using functional experiments. GST pull-down, western blot, coimmunoprecipitation and Me-RIP assays were performed to further explore the downstream molecular mechanisms.6A-mediated stabilization of Sec62 mRNA.Sec62 upregulation was associated with the chemoresistance of CRC and poor outcome of CRC patients. Depletion of Sec62 sensitized CRC cells to chemotherapeutic drugs. Sec62 promoted the stemness of CRC cells through activating Wnt/β-catenin signaling. Mechanistically, Sec62 bound to β-catenin and inhibited the degradation of β-catenin. Sec62 competitively disrupted the interaction between β-catenin and APC to inhibit the β-catenin destruction complex assembly. Moreover, Sec62 expression was upregulated by the m6A modification promotes the stemness and chemoresistance of CRC by binding to β-catenin and enhancing Wnt signalling. Thus, m6A modification-Sec62-β-catenin molecular axis might act as therapeutic targets in improving treatment of CRC.Sec62 upregulated by the METTL3-mediated mThe online version contains supplementary material available at 10.1186/s13046-021-01934-6. Colorectal cancer (CRC) is the fourth leading cause of cancer-related mortality . SurgeryAPC gene is mutated and produces the N-terminal truncated APC lacking of APC-AXIN1 interaction. Thus, the regulation of the APC-β-catenin interaction is critical in the activation of β-catenin in the CRC cells [Wnt/β-catenin signaling plays crucial roles in the maintenance of CSCs . The turRC cells , 14. HowHere, we screened the chemosensitivity-related genes and identified Sec62 as a chemoresistant target. Sec62 was originally found to locate in the membrane of endoplasmic reticulum (ER) . Recent 6A modification of Sec62 mRNA in CRC.In the present study, we firstly evaluated the effect of Sec62 on the chemosensitivity and stemness of CRC. We further demonstrated that Sec62 activates β-catenin signaling to potentiate the stemness and attenuate the chemosensitivity in CRC. Additionally, Sec62 is upregulated by the METTL3-mediated m2 at 37 °C in incubators with 100% humidity. Cell line authentications were performed by the provider. The antibodies and reagents for this study are listed in supplementary Table SCRC cell lines were obtained from the Cell Resource Center  and cultured in DMEM or RPMI 1640 medium. All medium were supplemented with 10% fetal bovine serum. Cells were maintained in 5% COTissue microarrays consist of 102 formalin-fixed, paraffin-embedded CRC tissues and non-tumorous colorectal tissues obtained from the CRC patients who underwent curative surgical resection without prior neoadjuvant therapy from January 2004 to December 2008 in Peking University Cancer Hospital. The clinical pathologic characteristics of patients including age, gender, tumor location, carcinoembryonic antigen (CEA) level, tumor size, clinical stage, and distant metastasis are summarized in Supplemental Table SCells were transfected with plasmid DNA or siRNA RNA duplexes by Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. In transient transfection experiments, plasmid DNA was kept constant with empty vector. shRNAs were delivered by lentiviral infection with lentiviruses produced by transfection of HEK293T cells with the vector pLKO.1. Cells infected with lentiviruses delivering scrambled shRNA (shCtrl) were used as negative control cells. Short interfering RNA (siRNA) sequences were directly synthesized . The sequences of shRNAs and siRNAs are listed in the Table SFor sphere formation assay, a total of 800 cells were suspended in a serum-free medium and were plated into an ultralow attachment plate. Then, the cells were cultured in DMEM/F12 medium (Invitrogen) supplemented with insulin (Sigma), B27 (GIBCO), EGF (Sigma) and basic FGF (Sigma). For the serial passaging, the primary spheres were collected and resuspended in DMEM/F12 medium with the above supplements after trypsin dissociation. Finally, the number of spheres was counted under microscope and the size of spheres was estimated using Image J software.For MTT assay, cells were seeded in a 96-well plate and cultured with indicated drugs for 72 h. Then, MTT assay was employed to assess cell viability according to the manufacturer’s protocol (Promega). For colony formation, cells were treated with DMSO or chemotherapeutic agents for 24 h, and subsequently seeded into 6-well plate (500 cell per well). After cultured for 12 days, the colonies were fixed with 4% paraformaldehyde and stained with 0.5% crystal violet. The visible colonies were counted and summarized.+/CD44+ cells, CD133-PE (#130–090-853) and CD44-APC (#130–098-110) antibodies (Miltenyi Biotec.) were utilized to label cells. Then, labelled cells were subjected to flow cytometric analyses.Apoptosis assay was performed using Annexin V-FITC and propidium iodide staining kit  according to the manufacturer’s protocol. To detect CD1333, the nude mice were randomly divided into 6 groups. Group 1, 3 and 5 received an intratumoral injection of lentivirial-ctrl shRNA once per week for 5 weeks. Group 2, 4 and 6 received an intratumoral injection of lentivirial-Sec62 shRNA once per week for 5 weeks. At the same time, group 3 and 4 received an additional intraperitoneal injection of 5-Fu (0.15 mg/kg) twice per week for 4 weeks. Group 5 and 6 received an additional intraperitoneal injection of oxaliplatin (30 mg/kg) twice per week for 5 weeks. The tumor size was measured every 5 days and tumor volume was calculated using the formula V = 0.5 × W2 × L .DLD-1 cells were subcutaneously implanted into 4–6 weeks old female nude mice. When tumors reached a size of about 50 mmCells were harvested and cell lysates were prepared in Buffer A  and used for immunoprecipitation. The indicated antibodies were coupled with a 50% suspension of protein A-Sepharose beads  in Buffer IPP500 . Coupled beads were incubated with cell lysates for 2 h at 4 °C. After washing, the precipitates were examined by Western blot using the indicated antibodies.RIP assay was carried out as previously described , 20. Brihttp://bioinf.wehi.edu.au/software/elda.To investigate the effect of Sec62 on tumor self-renewal, an in vivo limiting dilution assay was performed. Female BABL/c nude mice (4 to 5 weeks old) were randomly divided into 4 groups (5 mice per group). Sec62-knockdown or control DLD1 cells were injected subcutaneously in the flank with a serial dilution of cells. At the end of 80 days, the tumor incidence of each group was observed and the stem cell frequency was estimated using an online tool at 6A-specific antibody  in IP buffer  supplemented with BSA. The mixture was then incubated with protein-A beads and eluted with elution buffer (1 × IP buffer and 6.7 mM m6A). Eluted RNA was precipitated by 75% ethanol. RNA was finally extracted by phenol-chloroform RNA extraction methods. The relative expression of RNA was assessed by qPCR and normalized to the input.Me-RIP assay was performed following published protocols , 21. BriTissue sections were deparaffinized in xylene and rehydrated in ethanol. Then, the sections were treated with peroxidase solution and citrate buffer. After treatment with blocking buffer, sections were incubated with a primary antibody at 4 °C overnight. Next, the sections were visualized using an UltraVision Quanto Detection System HRP DAB Kit  according to the manufacturer’s protocols. The staining intensity were evaluated independently by two observers blinded to the clinical outcome. The percentages of positive tumor cells were scored as follows: 1%, 0 points; 1–25%, 1 point; 26–50%, 2 points; 51–75%, 3 points; and 75%, 4 points. The staining intensity was scored as follows: no staining, 0 points; weak staining, 1 point; moderate staining, 2 points; and strong staining, 3 points. Then, the two scores were multiplied to acquire a combined score ranging from 0 to 12.Cells were fixed with 4% paraformaldehyde for 15 min and permeabilized using 0.2% Triton X-100 for 10 min at room temperature. After blocking with 10% goat serum, the cells were incubated with primary antibodies overnight at 4 °C. After washing with PBS, a FITC-conjugated anti-rabbit antibody and a TRITC-conjugated anti-mouse antibody were added, and the samples were incubated for 1 h at room temperature. Finally, the cells were stained with DAPI to visualize the nuclei.P < 0.05 was considered to indicate statistical significance. *P < 0.05 and **P < 0.01 for all the analyses.The significance of the differences was determined via one-way ANOVA or Student’s t-test. Spearman’s correlation coefficient was used to calculate the correlations between the two groups. Kaplan-Meier analysis was employed for survival analysis and the differences in the survival probabilities were estimated using the log-rank test. The statistical analyses were performed using GraphPad Prism or SPSS version 17.0 . All data was presented as mean ± SEM. A two-sided SEC62 is of particular interest since its upregulation has been found to be associated with tumorigenesis [To explore novel targets in chemosensitivity, we analyzed the DEGs  in two CRC cohorts consisting of chemoresistant and chemosensitive CRCs in dataset GSE28702. Eighteen genes were upregulated and 10 genes were downregulated in chemoresistant CRCs  in CRC cell lines and evaluated response of these cells to 5-Fu (fluorouracil) or oxaliplatin. Depletion of Sec62 sensitized the CRC cells to 5-Fu or oxaliplatin treatment  Fig. b. The coKnockdown of Sec62 significantly decreased the β-catenin protein level without affecting its mRNA level in DLD1, HT29 and LOVO cells Fig. a and b. The ubiquitin-proteasome system is critical for determination of protein stability, including β-catenin stability . Thus, wThe activity of β-catenin destruction complex plays essential role in controlling β-catenin stability , 28. To Since Sec62 binds to the 8–9 ARM repeat domain of β-catenin which also interacts with APC, Sec62 might compete with APC for binding to β-catenin. Flag-Sec62, but neither the Flag-Sec62 DA nor Flag-Sec62 NM mutant, inhibited the interaction between APC and β-catenin Fig. B, C, indGiven that β-catenin bridges the interaction between the truncated APC and AXIN1 in the assembly of the β-catenin destruction complex , we wondBased on the observed effect of Sec62 on β-catenin levels, we further investigated whether Sec62 regulated the nuclear translocation of β-catenin and activation of β-catenin signaling. Overexpression of Sec62 increased the nuclear accumulation of β-catenin  or Sec62 shRNA (shSec62)-lentivirus. Meanwhile, 5-Fu or oxaliplatin was injected. The results showed that Sec62 knockdown effectively attenuated tumor growth as 5-Fu or oxaliplatin did Fig. g and h. 6A sites were found in Sec62 mRNA by RMBase prediction, suggesting that Sec62 mRNA might be controlled by m6A modification. We show that m6A was significantly more enriched in Sec62 mRNA in DLD1 or SW480 cells than that in normal colorectal cell NCM460 , a“reader” of m6A, plays a specific role in reading m6A and controlling the stability of m6A-modified mRNA [6A-modified Sec62 mRNA to maintain the Sec62 mRNA stability. Additionally, METTL3 or IGF2BP1 expression was positively correlated with Sec62 expression in CRC tissues  C sequence and enhances the stability of targeted mRNAs in an m6A-dependent way [6A-dependent manner, which is consistent with previous findings.Aberrant overexpression of Sec62 has been found in various tumors, but the underlying mechanism was unknown. Recently, accumulated studies show that mficiency . Dysregut manner , 47. Herdent way . In the 6A modification of Sec62 mRNA upregulated Sec62 expression in CRC. Subsequently, Sec62 potentiates Wnt signaling through repressing β-catenin binding to APC complex, thus facilitates CRC cell stemness and chemoresistance  to determine the sphere-initiating cell frequency. Frequency and probability estimates were computed using the ELDA software. ***, P < 0.001. c, Sec62-overexpressed clones and the vector transcfected control cells were generated from DLD1 or HT29 cells. The protein levels of Sec62 in the stable clones were examined by Western blot. d, Western blot was performed as indicated using mice xenografts in Fig. Figure S2. Sec62 interacts with β-catenin. a, The amino acid sequences of Sec62 BCBL motif from different species were aligned. The critical conserved site of Sec62 is indicated by the red box. b, DA mutant lacking BCBL motif. Figure S3. Sec62 inhibits the APC-β-catenin interaction. a, DLD1 or HT29 cells were transfected as indicated. Then, cells were harvested and total proteins were subjected to Western blot using the indicated antibodies. b, RKO cells were transfected with the indicated plasmids. Co-immunoprecipitation assay was performed with anti-Flag antibody and the indicated proteins were evaluated by Western blot. c, DLD1 cells were transfected with the indicated plasmids. Co-immunoprecipitation assay was performed with anti-β-catenin antibody and the indicated proteins were evaluated by Western blot. Figure S4. Sec62 maintains the stemness of CRC by activating β-catenin signaling. a, Western blot analysis of Sec62, β-catenin, CD44 and c-Myc expression in 16 individual paired CRC tissues. b, Western blot was performed as indicated using mice xenografts in Fig. Supplemental Table S1. Reagents and antibodies. Supplemental Table S2. Correlation of Sec62 expression with clinicopathologic status in 102 cases of patients with colorectal cancer. Supplemental Table S3. shRNA sequence. Supplemental Table S4. Primer used for RT-qPCR.


Non-alcoholic fatty liver disease (NAFLD) is the leading chronic liver disease worldwide. Agonists of the glucagon-like peptide-1 receptor (GLP-1R), currently approved to treat type 2 diabetes, hold promise to improve steatosis and even steatohepatitis. However, due to their pleiotropic effects, the mechanisms underlying their protective effect on NAFLD remain elusive. We aimed to investigate these mechanisms using an in vitro model of steatosis treated with the GLP-1R agonist Exendin-4 (Ex-4). We established steatotic HepG2 cells by incubating the cells with 400 µM oleic acid (OA) overnight. Further treatment with 200 nM Ex-4 for 3 h significantly reduced the OA-induced lipid accumulation (p < 0.05). Concomitantly, Ex-4 substantially reduced the expression levels of Fatty Acid-Binding Protein 1 (FABP1) and its primary activator, Forkhead box protein A1 (FOXA1). Interestingly, the silencing of β-catenin with siRNA abolished the effect of Ex-4 on these genes, suggesting dependency on the Wnt/β-catenin pathway. Additionally, after β-catenin silencing, OA treatment significantly increased the expression of nuclear transcription factors SREBP-1 and TCF4, whereas Ex-4 significantly decreased this upregulation. Our findings suggest that direct activation of GLP-1R by Ex-4 reduces OA-induced steatosis in HepG2 cells by reducing fatty acid uptake and transport via FABP1 downregulation. NAFLD refers to a group of liver diseases that includes simple steatosis (benign fatty infiltration), non-alcoholic steatohepatitis (NASH) (fatty infiltration plus inflammation), fibrosis, and cirrhosis, which occasionally progresses to hepatocellular carcinoma4. NAFLD is associated with several comorbidities, including type 2 diabetes (T2D), cardiovascular diseases (CVD), and chronic kidney disease (CKD)5. The mechanisms underlying the above associations remain elusive. However, given the liver's crucial role in many aspects of the metabolism of lipids, carbohydrates, and proteins, it is appreciated that any injury to the liver will potentially impact several organs6. NAFLD's etiology is not fully elucidated. However, it is accepted that visceral adiposity, insulin resistance, T2D, hypertension, and dyslipidemia are significant contributors to NAFLD development7.Non-alcoholic fatty liver disease (NAFLD), defined as the excessive accumulation of lipids in the liver, is the most common cause of chronic liver disease in industrialized nations8. Losing 5% of one's bodyweight improves abnormal liver tests and reduces liver fat9, whereas losing 7 to 10% of one's body weight appears to reduce inflammation and injury to liver cells and may even reverse some fibrosis damage10. Unfortunately, most people find it difficult to lose the weight they need to improve NAFLD and much more challenging to keep it off. Hence, there is an urgent need for novel therapeutic approaches to improve NAFLD independently of weight loss.There is currently no approved pharmacotherapy for NAFLD. Hitherto, weight loss is the only intervention proven to be significantly beneficial for NAFLD patients11. GLP-1 is a multifaceted hormone secreted by the L cells of the intestine12. Among other things, GLP1 regulates blood glucose levels by stimulating glucose-dependent insulin release and decreasing glucagon secretion, promotes proliferation of pancreatic b-cells, slows gastric emptying, and inhibits satiety and food intake through effects on central nervous system centers14. This pleiotropic effect is due to the expression of the GLP-1 receptor by various organs such as the pancreas, brain, kidney, gut, lung, heart, muscle, and liver15. Some GLP-1R agonists, like Liraglutide (taken once daily) or Dulaglutide (taken once weekly), are already licensed for T2D and obesity management in humans due to their ability to mimic the effects of GLP-118.Agonists of the glucagon-like peptide-1 receptor (GLP-1R) have recently been investigated to treat NAFLD due to their bodyweight-lowering effects35. As a result, these drugs are suggested as potential options for treating and slowing the progression of NAFLD. Nonetheless, it is unclear whether the protective effect of GLP-1R agonists on fat content stems from weight loss, which, among other things, increases insulin sensitivity and improves glycemia and lipid profile, or from direct activation of the hepatic GLP-1R. Gupta and colleagues36 were the first to report GLP-1 receptor expression in human hepatocytes and proposed that they play a direct role in reducing hepatic steatosis in vitro through the modulation of effectors of the insulin signaling pathway. Recently, Seo and coworkers37 suggested that the GLP-1R agonist Exendin-4 (Ex-4) reduces fat content in an in vitro cell model of steatosis by inhibiting hepatic lipogenesis through activation of β-catenin signaling and modulation of the expression of several lipogenesis genes. β-catenin was also suggested to mediate the effect of GLP-1 receptor agonist Exenatide on ameliorating hepatic steatosis induced by a high fructose diet in rats38. The β-catenin is an intracellular signal transducer in the Wnt signaling pathway, which maintains hepatic homeostasis and contributes to specific hepatic characteristics, including liver metabolism39 and metabolic zonation regeneration40.Given their weight loss-inducing effect, by reducing satiety and food intake, the impact of the GLP-1R agonists on liver fat content has been investigated in numerous in vivo studies and yielded promising results42. We used HepG2 cells treated with Oleic Acid (OA) as a model of hepatic steatosis in this study to see if direct activation of the GLP-1R with Ex-4 affects any of the four processes listed above and thus improves steatosis. The use of the in vitro model allows us to demonstrate the direct effect of Ex-4 on GLP-1R and to overcome the pleiotropic effect of GLP-1R agonism in vivo.Hepatic lipid content and homeostasis are determined by: (a) circulating free fatty acid uptake, (b) hepatic de novo lipogenesis, (c) hepatic β-oxidation, and (d) hepatic lipid export via very-low-density lipoprotein (VLDL)We obtained the human hepatoma HepG2 cell line  from ATCC  and maintained it in Dulbecco's modified Eagle's medium (DMEM)  at 37 °C and 5% CO2. DMEM was supplemented with 10% FBS  and 1% penicillin/streptomycin . We performed all the experiments with cells passaged fewer than 25 times.43. Briefly, we dissolved the powder OA  at a final concentration of 12 mM in phosphate-buffered saline  that contained 11% fatty acid-free bovine serum albumin . The solution was then sonicated and shaken at 37 °C overnight using an OM10 Orbital Shaking Incubator . The OA solution was filtered using a 0.22 µm filter, aliquoted, and stored at 4 °C. We used a fresh aliquot for each experiment.We prepared the oleic acid solution as in5 cells/well until 70% confluence. We then starved the cells for 6 h in DMEM containing 1% fatty-acid-free bovine serum albumin. Following the starvation, a 16-h incubation in DMEM containing increasing concentrations of OA (0–500 µM) at 37 °C was performed, and steatosis was quantified . To this aim, we cultured HepG2 cells in 6-well plates at a density of 4 × 10After steatosis induction, the cells were washed and incubated in fresh DMEM containing 400 μM OA in the absence or presence of Ex-4 . To determine the optimal concentration of Ex-4, we treated the steatotic cells with increasing concentrations of Ex-4 from 0 to 1 mM and with different incubation periods . We then quantified the TG content as above. We used a fresh aliquot of EX-4 for each experiment.Quantification of triglyceridesWe measured total TGs levels using a commercial fluorometric assay kit  and a microplate reader . The kit converts triglycerides to free fatty acids and glycerol. Glycerol is then oxidized to generate a product that reacts with a probe to generate fluorescence when excited at 535 nm. The emitted fluorescence is collected at 587 nm. We calculated the TGs content from a standard curve prepared for each assay using known TGs concentrations. We normalized the data to total cellular protein content.Staining of neutral lipids with BODIPY 493/50344. Briefly, we grew HepG2 on 12 mm coverslips until 70% confluence, starved them, and then treated them with OA and Ex-4 as needed. After a quick wash, we fixed the cells with 4% paraformaldehyde for 7 min, washed them with PBS, and then incubated them for 10 min with 0.2 μM BODIPY 493/503. We further labeled the nuclei by incubating the cells with 1 μM DAPI for 1 min. After a final wash with PBS, we mounted the coverslips on microscope slides used for imaging on a Zeiss LSM 870 confocal microscope, as we reported recently45. To analyze the images, we used ImageJ software . The intracellular lipid accumulation was calculated by dividing the BODIPY fluorescence intensity by that of DAPI. Two independent researchers analyzed 200 individual cells for each condition  from three different experiments.To visualize the accumulation of lipids in response to OA treatment, we used boron-dipyrromethene (BODIPY) 493/503 , which labels specifically intracellular neutral lipidsRelative expression of perilipin genes46. We used qRT-PCR to quantify the relative expression of PLIN1, 2, and 3 and estimate the lipid accumulation in response to OA and EX-4 treatments. The primers we utilized for the genes are listed in Table Perilipin family proteins, with five recognized members PLIN1-5), are found on the surfaces of intracellular lipid droplets-5, are fWe used three methods to quantify steatosis in HepG2 cells:-ΔΔCT method to calculate the relative expression. We have quantified the expression level of the following genes: Fatty Acid Synthase (FAS), Acyl-CoA Dehydrogenase Long Chain (ACADL), Carnitine Palmitoyltransferase 1A (CPT1A), Stearoyl-CoA Desaturase 1 (SCD-1), Acetyl-CoA Carboxylase Alpha (ACC), Diacylglycerol O-acyltransferase 1 (DGAT1), Diacylglycerol O-acyltransferase 2 (DGAT2), Sterol Regulatory Element Binding Transcription Factor 1 (SREBP-1), Peroxisome Proliferator-Activated Receptor Gamma (PPARγ), Fatty Acid Binding Protein 1 (FABP1), Forkhead box A1 (FOXA1) and Apolipoprotein B (APOB). Table https://www.ncbi.nlm.nih.gov/tools/primer-blast/) to design specific primers that met the following criteria: (1) Primer pairs are unique. They will not bind to other locations in the genome except the intended gene or DNA fragment. (2) Primer pairs do not bind to each other (forming primer dimer): self-or hetero-dimer. (3) The possibility of forming the secondary structure of the primers, which may cause difficulties for PCR amplification, is very low. (4). Tm (temperature of mismatch) of two primers is designed to be close to each other. (5) TA  is much lower than Tm. Moreover, our primers were analyzed by 'OligoAnalyzer 3.1' program from IDT company .To quantify gene expression, we used the Pure Link RNA Mini kit , Hilden, Germany) to extract total RNA from untreated and treated HepG2 cells and used High-Capacity cDNA Reverse Transcription kit  and 2 μg total RNA to prepare cDNA. We quantified gene expression by qRT-PCR on QuantStudio 6 Flex system , using PowerUp™ SYBR™ Green Master Mix . We normalized the data to β-actin as an internal control and used the comparative 2® Kits  and the Lipofectamine RNAiMAX transfection kit  to transfect HepG2 cells with 20 nM of GLP-1R specific siRNA or negative scrambled siRNA, according to the manufacturer's instructions. The DsiRNAs-TriFECTa® kit contains three Dicer-substrate 27-mer RNA duplexes specific for a single target gene. A pool of the three duplexes was used to silence GLP-1R. After the silencing of GLP-1R, a qRT-PCR was performed for the following genes: PPARγ, FAS, DGAT1, DGAT2, and ACC. We normalized the data to β-actin as an internal control and used the comparative 2-ΔΔCT method to calculate the relative expression.For siRNA-mediated β-catenin gene silencing, we transfected HepG2 cells with 5 nM of β-catenin-specific siRNA or Stealth siRNA negative control, obtained from Dharmacon , using Lipofectamine RNAiMAX transfection kit  according to the manufacturer's instructions. After transfection, cells were cultured under normal growth conditions  for 24 h without antibiotics. The silencing efficiency was checked by quantifying the expression of β-catenin with qRT-PCR. For GLP-1R gene silencing, we used the Dicer-Substrate Short Interfering RNAs (DsiRNAs) and TriFECTaAfter treating HepG2 cells with OA and Ex-4, we extracted nuclear and cytoplasmic proteins using the PARIS™ Kit . We resolved 20 mg of proteins on 10% Tris–Glycine Mini Gels  and then transferred them to a 0.2 mM polyvinylidene difluoride (PVDF) membrane using the Trans-Blot Turbo . After the transfer, we incubated the membranes for 1 h at room temperature with the following primary antibodies: anti-β-catenin , anti-TCF4 , anti-SREBP-1 , anti-β-actin , anti-Lamin B1 , anti-FOXA1 , anti-FABP1  . After 3 × 10 min washes with PBS, we incubated the membranes with the appropriate horseradish peroxidase-conjugated secondary antibody. We developed the membranes with the super signal west Femto Maximum Sensitivity Substrate , and Immunoreactive bands were detected by chemiluminescence on Biorad ChemiDOC XRS  machine. We normalized the results to β-actin as an internal control for total proteins and Lamin-B1 for nuclear proteins. We used the dilutions recommended by the manufacturer for all antibodies unless otherwise stated., we used a two-way analysis of variance (ANOVA) to evaluate the significance of differences between the mean values of different experimental groups. Unless otherwise specified, a p-value of < 0.05 was considered significant.We performed the statistical analysis and the graphing with GraphPad Prism 9.0 software . Data are presented as the mean ± SEM. We used unpaired one-way ANOVA analysis (ANOVA) to assess the significance of differences in mean values between experimental groups, and Tukey's posthoc test was used to adjust multiple comparisons between experimental groups. When we silenced β-cateninBy treating HepG2 cells with increasing OA concentrations for 16 h and measuring TG accumulation, we determined the optimal concentration of OA required to induce steatosis  mice treated with taspoglutide, a long-lasting GLP-1R agonist56. However, the authors suggested the effect of taspoglutide was indirect as they could detect neither the protein nor the mRNA of GLP-1R in liver cells. However, it is worth noting that several studies have reported GLP-1R expression in both human and rodent hepatocytes57. We have also detected GLP-1R expression in HepG2 cells by western blotting and quantitative PCR (data not shown). Additionally, a recent study investigating the effect of the GLP-1R agonist liraglutide on obesity-induced chronic kidney injury in obese rats showed that the agonist significantly reduced the lipid content and, concomitantly, the expression level of FABP1 protein in the obese kidney, relative to untreated rats58.The Ex-4-induced FABP1 downregulation correlates with the significant reduction in TGs content observed under the same treatment Fig. C. Intere42. Therefore, one explanation for the Ex-4-induced improvement in steatosis observed in our model could be a decreased fatty acid uptake by FABP1. This explanation is consistent with the fact that FABP1 silencing in mice reduces liver weight and hepatic TG content60, whereas FABP1 overexpression increases hepatic fatty acid uptake61. Moreover, the expression of FABP1 is significantly higher in the liver in obese patients with simple steatosis than in the obese healthy group62.In principle, four separate mechanisms may lead to hepatic lipid accumulation: (a) enhanced uptake of circulating free fatty acids, (b) increased hepatic de novo lipogenesis, (c) diminished hepatic β-oxidation, and (d) decreased hepatic lipid export via VLDL64. Previous research on DGAT1-/- mice demonstrated that DGAT1 was required for hepatic steatosis caused by a high-fat diet or fasting, both of which promote hepatic uptake of exogenous FAs, but not for hepatic steatosis caused by upregulation of endogenous de novo FA synthesis65. As a result, the low DGAT1 expression observed in the presence of Ex-4 is most likely a response to reduced FAs uptake rather than reduced de novo lipogenesis, ruling out a role for reduced de novo lipogenesis in the Ex-4-induced steatosis improvement.We have also observed that the presence of Ex-4 decreases the expression of ACC and DGAT1 Fig. A, which 66. Thus, an increased β-oxidation might explain the improved steatosis we observe in the presence of Ex-4. Nevertheless, this possibility is ruled out by the fact that Ex-4 decreases the expression of CPT1, the rate-limiting enzyme for mitochondrial β-oxidation67.A decrease in ACC expression stimulates lipid β-oxidation by reducing the production of the β-oxidation inhibitor malonyl-CoA68. This increase in ApoB expression likely reflects a compensatory mechanism to enhance the secretion of VLDL and hence reduce the content of TGs. Nonetheless, Ex-4 significantly reduces the OA-induced upregulation of ApoB (Fig. 69. Thereby, this observation excludes the significant contribution of enhanced lipid export to the Ex-4-induced steatosis reduction. Interestingly, the Ex-4-induced reduction of ApoB expression was blunted by the silencing of β-catenin (Fig. OA treatment significantly increases the expression of ApoB, an essential protein for the assembly and secretion of TG-rich ApoB-containing lipoproteins, such as VLDLpoB Fig. C. This fnin Fig. G,H, indi70. We show that the presence of Ex-4 significantly reduces the FOXA1 expression relative to OA alone (Fig. 71, probably as a feedback mechanism to reduce FAs uptake by FABP1. Furthermore, FOXA1 promotes fatty acid breakdown by inducing peroxisomal fatty acid b-oxidation71. Nonetheless, given the reduced FOXA1 expression induced by Ex-4 in our study, it is unlikely that the observed Ex-4-induced TG content reduction is due to the stimulation of peroxisomal fatty acid -oxidation. Ex-4 induces a significant downregulation of FOXA1 (Fig. The transcription factor FOXA1 is among the most effective activators of human FABP1one Fig. C, which XA1 Fig. C compareXA1 Fig. G,H, sugg37 who showed that the β-catenin inhibitor IWR-1 abrogates the protective effect of Ex-4 against palmitate-induced steatosis. Our results also indicate the potential involvement of the β-catenin signaling pathway by showing the impact of Ex-4 on the expression of nuclear transcription factors SREBP-1, a key regulator of lipid metabolism in the liver72, and TCF4, a central transcription factor in the β-catenin pathway, when β-catenin is silenced. Hence, after β-catenin knockdown, OA treatment significantly upregulates both SREBP-1 and TCF4 (Fig. 73. Therefore, GLP-1R stimulation may activate the β-catenin pathway, which may result in a concerted action by TCF4 and FOXA1 to regulate the expression of FABP1 and hence prevent the lipid accumulation induced by OA (Fig. 74. However, further investigations are warranted to decipher the complete mechanism underlying the protective effect of GLP1R agonists against hepatic steatosis.The involvement of the β-catenin signaling in the Ex-4-induced improvement in hepatic steatosis was suggested previously by Seo and coworkers OA Fig. . It is wIn conclusion, the present study proposes that the direct activation of GLP-1R by Ex-4 reduces OA-induced steatosis in HepG2 cells by stimulating the Wnt/β-catenin signaling pathway, which reduces FOXA1 expression. FOXA1 downregulation, in turn, reduces FABP1 expression, which ultimately leads to a decrease in FFAs uptake. Targeting FABP1 expression in the liver could be beneficial as a medical treatment for fatty liver disease.Supplementary Information 1.Supplementary Information 2.Supplementary Information 3.Supplementary Information 4.Supplementary Information 5.Supplementary Information 6.Supplementary Information 7.Supplementary Information 8.Supplementary Information 9.Supplementary Information 10.Supplementary Information 11.Supplementary Information 12.


Arabidopsis thaliana. As a change is detected in impedance due to lighting and hydration, this raises the possibility for their widespread use in precision farming.Precision farming has the potential to increase global food production capacity whilst minimizing traditional inputs. However, the adoption and impact of precision farming are contingent on the availability of sensors that can discern the state of crops, while not interfering with their growth. Electrical impedance spectroscopy offers an avenue for nondestructive monitoring of crops. To that end, it is reported on the deployment of impedimetric sensors utilizing microneedles (MNs) that can be used to pierce the waxy exterior of plants to obtain sensitive impedance spectra in open‐air settings with an average relative noise value of 3.83%. The sensors are fabricated using a novel micromolding and release method that is compatible with UV photocurable and thermosetting polymers. Assessments of the quality of the MNs under scanning electron microscopy show that the replication process is high in fidelity to the original design of the master mold and that it can be used for upward of 20 replication cycles. The sensor's performance is validated against conventional planar sensors for obtaining the impedance values of  Arabidopsis thaliana.A microneedle electrode is developed, based on a scalable, versatile, and high‐resolution fabrication process for minimally invasive impedimetric plant biosensors. The metal‐coated polymeric microneedles can pierce the waxy coating layer of the plant tissue to establish direct contact with the extracellular fluid and assess its physiological state via impedance. A demonstration of the sensor's performance is conducted on  It is estimated that about half of the habitable land and 70% of annual freshwater withdrawals are dedicated to agriculture alone. Against the backdrop of an impending food shortage, the development of precision farming (PF) techniques, which seek to link the real‐time needs of crops to the administered supply, has accelerated. By utilizing an array of technologies that span drones equipped with spectral cameras to soil‐implanted sensors, PF may enable farmers to address heterogeneities in their fields, thereby increasing their productivity with little to no increase in the resource footprint. The impact and adoption of PF can be greatly accelerated by the procurement of sensitive and accurate information regarding the physiological status of the crop. With that said, current commercially available options do not achieve this goal as it requires direct nondestructive monitoring of a plant's extracellular fluid (EF). Due to the high spatial and temporal heterogeneity of farm fields and their scale, soil‐implanted sensors are not suitable to accurately monitor crops either, as they tend to aggregate averaged information about the content of the soil, which may be correlated to but is not directly reflective of the EF status of the crop. To circumvent this challenge, utilizing electrical impedance spectroscopy (EIS) on plant tissue has been a long‐standing subject of interest. Structural changes that occur in the plant, which reflect its physiological state, manifest in the tissue's ionic content, membrane permeability, or viscosity. As such, in effect, the use of EIS can allow for quick and nondestructive assessment of such structural changes and, by extension, the physiological state of the plant tissue. However, the utility of such an approach is limited by the lipid‐rich barrier layer, known as the cuticle that encapsulates the above‐ground exterior of plants. Such layers act as large electrical insulators that effectively mask the underlying physiologically relevant impedance. This is especially troublesome at the lower frequency range where electrode polarization (EP) is at its highest. Polarization occurs due to the buildup of an electric double layer of ions at the interface between the electrode and the electrolytes in the tissue. It results in a potential drop of the electrode relative to its value in an open‐circuit configuration by behaving as an additive impedance. As the EP has been noted to decrease with frequency via power‐law relation by capacitive shorting, at low frequencies below 1 kHz, it can contribute significantly to the measured impedance. Borrowing a page from medicine, minimally invasive microneedles (MNs) were developed to directly probe the relevant plant tissue, thereby bypassing the cuticle. As this approach gained traction, MNs were used for DNA extraction from leaves, EIS measurement of plant tissue to probe the content of the EF and to assess sap flow through the plant's stem.To keep up with the constantly growing global demand for food, farmers are forced into adopting technologies to uniformly manage large fields. While this approach yields many economy‐of‐scale benefits, as the quantity of arable land is exhausted, due to resource constraints, production growth may plateau, potentially leading to a surging nutritional deficit. To keep pace with the rising demands of a growing population, the food production capacity must double by 2050. Yet, studies have shown that the current yield rates are projected to fall short of this goal. or serial approaches such as backside exposure. Even the first to market MNs‐based products were fabricated using serial processes such as laser ablation of metal. Conventionally, anisotropic wet etching and isotropic dry etching methods have been combined to yield more control over the MN geometry, but these processes are often labor‐intensive and are limited in their applicability to a limited set of materials. Alternative approaches had to be devised as the aforementioned techniques do not lend themselves to scalable production or often limit the achievable MN design. In pursuit of cost effective and batch manufacturing techniques for MNs, silicone micromolding using an MN array template has been extensively explored in literature. Silicone varnish can be used to fabricate elastomeric molds via hot‐embossing, which can then be subsequently used for scalable replication whilst not sacrificing the high fidelity of the master template. The elastomeric properties of these intermediary molds are crucial for the final release of the MNs. During demolding, a lateral bending force is applied to the mold to release the MNs. Using elastomeric molds ensures that the mold can be easily bent at a low bending force to avoid plastic deformation of the MNs. The versatility of the strategy allows it to be compatible with several thermosetting polymers, such as cycloolefin polymers and polyimide, organically modified ceramics, and UV‐curable polymers, such as SU‐8.Due to the breadth of their applications, several MN fabrication techniques were devised. Earlier iterations relied on existing micromachining strategies of Silicon the MNs fabricated using such a technique have a tip radius of 20–40 µm, which increases the force needed for insertion. With the advent of two‐photon polymerization (TPP) based laser lithography, the gap between scalable production and control over MNs design and geometry can be bridged. With a resolution limit of 900 nm, MN master templates can be fabricated for micromolding. However, regardless of the strategy employed for the fabrication of the master template, the lateral bending force applied for demolding considerably limits the achievable geometry. This is especially evident in the thickness of the support bed of the MNs used previously. For example, micromolded SU‐8 MNs utilize a 1 mm thick bed to ensure minimal deformation during demolding. The thickness of the support bed influences both the conformability and the weight of the array on soft tissue. The former is needed to ensure adequate contact and adhesion to the surface and the latter should be minimized to facilitate the application on delicate leaf structures.As the replication fidelity of micromolding is high, the achievable geometry is limited only by the fabrication process used to generate the master template and the release technique for demolding. To fabricate the master template, both traditional micromachining strategies and additive manufacturing techniques, such as 3D printing, have been explored. Whilst the latter offers a high degree of control over the geometry of the MNs with high‐aspect ratios, it is subject to limits set by the resolution of the printer resolution. Although a two‐step “Print and Fill” method was devised to overcome this limit on low‐cost 3D printers,Arabidopsis thaliana for validation as a plant impedimetric sensor  on a master mold fabricated using TPP‐based laser lithography. A photosensitive resist (SU‐8) or a thermosetting polymer (polyimide) is then cast over the intermediary mold to fill its conical cavities and subsequently baked or UV‐crosslinked. To gently release the polymeric MNs, the elastomeric molds are submerged in a chlorinated hydrocarbon solvent. This causes swelling in the PDMS matrix, which translates to volume expansion of the matrix. Upon expansion, the mold gently and gradually releases the MNs from the conical cavities, pushing them out. Combined with a short burst of gentle sonication, the MNs can be released with minimal bending force. We demonstrate how this strategy can be used to fabricate MNs. These MNs‐equipped electrodes are employed for EIS measurements of 22.1 Furthermore, the thermal stability of the PDMS matrix crosslinking allows for its use in molding thermosetting polymers up to a temperature of 250 °C, which can be elevated by the introduction of copolymers. Lastly, the stability of its mechanical properties for several months allows for the repeatable use of the elastomeric molds. Submersion of PDMS in chlorinated hydrocarbons, such as chloroform, swells the polymer matrix by up to 40%. The original shape of the mold can be restored postswelling by submerging it in a solution of diluted ethanol in deionized water (50% by volume). Upon submerging the PDMS mold in the solution, chloroform exits the polymer matrix. The miscibility of chloroform in ethanol allows it to remain in a unique phase that does not mix with water in solution. The PDMS molds can then be reused after being cleaned in isopropanol. This approach allows for scalable and high‐fidelity production of MNs that can be more easily integrated with a host of devices, which include but are not limited to electrochemical sensors, drug delivery platforms, and scaffolds for cell culture. Uniquely, it also allows for the fabrication of high aspect ratio free‐standing structures as thin as 65 µm, such as interdigitated arrays of MNs relevant for electrochemical sensing applications or shadow masks  to further anchor it to the Silicon substrate and to smooth the stitching lines between the slices for easier detachment of the PDMS after curing.The master molds are fabricated using TPP‐based laser lithography using the photonic professional GT instrument . IP‐S is chosen as the photoresist of choice due to its low shrinkage, mechanical properties, and adherence to Silicon. The master mold consists of an array of 100 MNs arranged with a spacing of 300 µm from tip to tip in a 10 by 10 configuration. The spacing is chosen to mitigate the bed‐of‐needles effect and to ensure that no MN will lie at the boundary of two or more rectangular slicing windows, which are set at a maximum size of 300 by 300 µm More importantly, the low viscosity of the chosen formulations for SU‐8 and polyimide coupled with a relatively high solid content enables more flow into the conical cavities of the PDMS mold with little shrinkage after crosslinking or polymerization. As both polymers are dielectrics, they can be subsequently patterned with the use of shadow masks and direct metal sputtering.SU‐8 2005 (MicroChem) and polyimide PI‐2611 (DuPont) are chosen as photosensitive and thermosetting polymers for micromolding MNs. The crosslinking of SU‐8 can be initiated by exposure to 365 nm light whilst polyimide polymerizes at 200 °C, both of which are compatible with PDMS's optical transmission and thermal stability limit, respectively. The robust mechanical properties and biocompatibility of these polymers also allow for their use to penetrate the cuticle of living plants without triggering any cytotoxic effects.−2 for 11 s. Finally, the SU‐8 MNs are baked again at 95 °C for 30 min to ensure complete crosslinking. For the polyimide MNs, after outgassing, the molds are baked on a hot plate at 90 °C for 1 min and 30 s and then baked at 150 °C for another 1 min and 30 s. After being allowed to cool to room temperature, the polyimide MNs are polymerized by baking them on a hot plate from room temperature to 200 °C where they are held for 45 min. Upon demolding, the SU‐8 and polyimide MNs can be annealed at 150 and 350 °C, respectively, for optimal mechanical performance. To assess the reliability of the master template during hot embossing of elastomeric molds, the template is used for several replication cycles in PDMS and was imaged by scanning electron microscopy (SEM). After the 32th replication cycle, the template demonstrably deforms, with the supporting layer curving upward from the Silicon die  before and after compression. Normalizing the failure force to the MNs number in the array yields a force of 1.5 ± 0.13 and 1.8 ± 0.06 N per needle for SU‐8 and polyimide, respectively. The peak load was sustained for 5 and 11 µm of compression for polyimide and SU‐8 MNs, respectively. As the polyimide MNs proved to be more mechanically resilient, subsequent tests were not performed on the SU‐8 MNs. The crash test was repeated on the polyimide MNs with 10, 20, and 30 µm s−1 compression rates  as a reference, which is 0.8 N, the polyimide MNs should be able to withstand the piercing event.To ensure that the MNs can pierce the cuticle, the MN arrays of SU‐8 and polyimide were tested in a crash‐test configuration shown in s Figure . The fai2 laser is used to cut the film with the desired shape of the electrode bed on top of the Silicon die. The film is then subsequently peeled off the die to utilize its flexibility. To confer electrical conductivity, the MNs are coated with a layer of 10 nm of titanium followed by 150 nm of gold using sputter deposition. A laser‐cut poly(methyl methacrylate) shadow mask is used to ensure that each MN‐equipped electrode in the sensor was spaced by 2 mm to the closest neighboring electrode  covered by spin coating a 50 µm layer of uncured polyimide varnish. The die is heated to 350 °C to adhere the MNs to the film via cross polymerization. A COe Figure. An equie Figure. As the e Figure, the sen2.2Arabidopsis thaliana and then promptly removed to inspect the site for signs of the piercing. As can be seen from FigureTo experimentally verify that the MNs can pierce the cuticle, the sensor is pressed against a freshly cut leaf of The sensor is attached to an intact leaf in a plant growth chamber under controlled environmental conditions using 3M micropore tape . The hypoallergic nature of the tape ensures that the attachment process does not result in the leaching of materials into the leaf after removal. Furthermore, the porous nature of the adhesive allows the leaf to be aerated. Two rounds of testing are conducted to characterize the sensors and assess their potential for long‐term deployment. For long‐term testing, the sensor terminals are connected to the working (WE), counter (CE), and reference (RE) electrode alligator clips of a portable potentiostat.−2 s−1 with spectral peaks at 630, 520, and 450 nm) of 12 h at a temperature of 23 °C and 12 h of darkness at a temperature of 20 °C each day. The transition from light to darkness is abrupt as the hydroponic lights are turned off or on at full intensity during the cycling period. The soil is unchanged during the experimental test and the water level in the tray is maintained unless otherwise stated. The impedance of the tissue is acquired in situ from 10 Hz up to 100 kHz to probe the α and β dispersion regions with a 10 mV peak‐to‐peak sinusoidal stimulus. 360 frequencies are probed within the desired range and are spaced apart on a logarithmic scale. Prior to running each test, the open circuit potential is estimated with a stability tolerance of 0.01 mV s−1. The voltammetric test is thereby done with respect to the open circuit potential to aid in the reduction of the noise due to the plant's action potentials. During the test, the minimum sampling time, which is the period over which the stimulating potential was applied at each frequency, is set to 0.5 s. The impedance and phase shifts measured in the 0.5 s window are averaged and recorded.The plant specimens are exposed to a photoperiod  has shown that the impedance at 10 Hz cycles in a diurnal fashion entrained by light. Although the physiological phenomena explaining this cycling pattern are unclear, it may serve as a useful tool to assess light exposure of crops in a field, which is crucial for photosynthetic activities. As such, a specimen of Arabidopsis thaliana is monitored using the MNs‐equipped sensor for 12 d to discern if there is a cycling pattern that is also entrained by light similar to that of Barley. This experiment also serves to illustrate the long‐term effects of the sensor attachment to the leaf as it has been reported previously that plants tend to seal epidermal wounds to maintain turgor pressure in the leaf, which could impact the impedance measured overtime. The plants were regularly watered to maintain a constant level of water in the growth tray.Previously, we demonstrated that analyzing the impedance spectrum of Barley leaves  file using Describe software . The software splits the structure in the horizontal and vertical axes to configure the exposure path of the laser. The vertical and horizontal distances between the slices are referred to as the slicing and hatching distances, respectively. Both the slicing distance and hatching distance were set to 0.5 µm. A shell and scaffold configuration was chosen to accelerate the TPP‐direct laser lithography process. The outermost layer of the structure is referred to as the shell and the lines demarcating its slices are fully exposed by the laser. The thickness of the shell was chosen to be 10 µm. The interior of the structure was filled with a triangular scaffold, which was also hatched and sliced with distances of 0.5 µm. The writing field and piezoelectric motor used to articulate the laser in the instrument limits the voxel size to 300 by 300 by 300 µm3. A secondary galvo motor is engaged to move the laser between each voxel, which necessitates stitching of the voxels. To ensure adhesion of the individual voxel, a block overlap distance of 2 µm is used. At the interface of the voxels, a stitching line can be seen due to the minute misalignments during movement of the stage and exposure. A 25 by 25 mm2 single‐sided polished Silicon die was sequentially cleaned in acetone, isopropanol, and DI water. IP‐S resin was decanted into the center of the die, which was subsequently loaded into the Photonic Professional GT system . The exposure process of the master template was completed in 2 h and 35 min.The master mold computer‐aided design (CAD) file was designed using AutoCAD software suite (AutoDesk) to incorporate a 10 by 10 array of conical MNs with a radius of 70 µm and height of 350 µm and a spacing of 300 µm on a flat rectangular 2 by 2 mmUpon completion of the polymerization process, the die was submerged in mer‐Dev 600 (MicroChem) to remove the uncured resin and then washed in isopropanol.2 frame was cut using a CO2 laser. The frame was placed around the master mold and attached to the Silicon die using drops of chloroform ejected from a hypodermic needle nozzle. PDMS (Sylgard 184) was mixed in a 10:1 ratio (monomer to curing agent) vigorously and then outgassed for 45 min. The PDMS was then cast on the master mold and the excess was removed with a surgical blade. The mold was then outgassed for an additional 20 min and then moved to a convection oven set at 90 °C where the PDMS was cured for 30 min. To remove the replica from the master mold, a surgical blade was used to trim the edges and release the PDMS.An acrylic rectangular 20 by 20 mm−1 under a stationary blade fixed perpendicular to the surface of the mold. As the mold passed under the blade, the varnish was spread on the surface of the mold. A surgical blade was used to remove the excess around the mold cavity. The resulting thickness was characterized using a profilometer after the MNs were released. The mold was then soft‐baked at 65 °C for 3 min on a hot plate. The temperature was then ramped at a speed of 3° C min−1 to 95 °C and was held at that point for 5 min. Depending on the size of the design of the master mold, evaporation of the SU‐8 may cause significant shrinkage and may necessitate a supplementary addition of another layer of SU‐8 on top, which is then soft‐baked. This may be repeated until the dry SU‐8 fills the mold cavities. The mold was then exposed to UV light (365 nm) with the exposure dose depending on the thickness of the design. It was found that for designs that were 500, 300, and 100 µm thick, exposure doses of 600, 350, and 150 mJ cm−2 were adequate, respectively. If the design exceeds 500 µm in thickness then a dual exposure is necessary, where the first exposure round exposes the upper surface of the mold and the second round exposing through the PDMS surface at the bottom of the mold. Finally, the mold was hard‐baked at 95 °C for 30 min to crosslink SU‐8 and was subsequently cooled to room temperature at a ramp speed of 10 °C min−1.150 µL of SU‐8 2005  was cast onto the PDMS mold under yellow light using a transfer pipette. The mold was outgassed for 20–30 min to ensure adequate flow of the varnish into the cavities. The mold was fixed in a glass holder at ambient temperature. The holder was moved at a speed of 2.5 mm s−1. The mold was held at 250 °C for 30 min to complete the imidization process and was subsequently cooled gradually to return to room temperature at a ramp speed of 10 °C min−1.Polyimide varnish (PI‐2611)  was cast onto the PDMS mold and was outgassed for 20–30 min to allow the varnish to seep in completely into the cavities of the mold. The same blade coating technique used for SU‐8 was also used to spread the polyimide varnish on the mold. The excess varnish around the mold cavity was removed using a surgical blade. The mold was then baked at 90 °C on a hot plate for 1 min and 30 s and then on 150 °C for 1 min and 30 s. The mold was then allowed to return to room temperature and was then hard baked from 23C to 250 °C at a ramp speed of 4 °C minThe mold was submerged into chloroform in a glass beaker (80 mL). The beaker was sealed to prevent evaporation of the solution and minimize toxic fume release. Inside a chemical fume hood, the beaker was placed in a water bath, which was ultrasonicated for 10–15 min at a frequency of 76 Hz. The MNs array was gradually deflected out of the PDMS matrix. Depending on the size of the design, a surgical blade may be needed to tip the sides of the array to allow chloroform to seep into the cavities. Finally, the mold was submerged in a solution of 50% ethanol in DI water to restore its previous shape and the molded polymer could be cleaned in isopropanol to remove any residual chloroform.The authors declare no conflict of interest.Supporting InformationClick here for additional data file.


The ongoing search for biodegradable and biocompatible microneedles (MNs) that are strong enough to penetrate skin barriers, easy to prepare, and can be translated for clinical use continues. As such, this review paper is focused upon discussing the key points  for the translation of MNs from laboratory to clinical practice. The review reveals that polymers are most appropriately used for dissolvable and swellable MNs due to their wide range of tunable properties and that natural polymers are an ideal material choice as they structurally mimic native cellular environments. It has also been concluded that natural and synthetic polymer combinations are useful as polymers usually lack mechanical strength, stability, or other desired properties for the fabrication and insertion of MNs. This review evaluates fabrication methods and materials choice, disease and health conditions, clinical challenges, and the future of MNs in public healthcare services, focusing on literature from the last decade. Following the approval of the first transdermal patch for scopolamine administration by the FDA in 1979, as an alternative to oral administration of medications and hypodermic injections, the distribution of transdermal pharmaceuticals has gained considerable interest . Currentstratum corneum (SC) to these drugs [The transdermal drug delivery (TDD) systems such as the above have a range of constraints in their applications . In partse drugs ,8. The sse drugs . Therefose drugs . Alternase drugs , sonophose drugs , and elese drugs  can physse drugs . Withoutse drugs . Thus, Mse drugs .MNs can be produced from a range of materials such as metals, silicon, carbohydrates, and polymers ,18,19,20In the last two decades, MNs have become an increasingly popular study topic. This was evidenced in our analysis, which demonstrated that the number of publications obtained using the database Scopus with the search term ‘‘microneedle’’ has increased steadily. However, several challenges must be addressed for these MNs to find widespread medical applications. Challenges include skin irritation, microbial contamination, lack of mechanical strength of biomaterials, the quantity of drug loading, and the delivery of macromolecules with high hydrophilicity . There iWith the ongoing MNs’ progress toward commercialization, there is a greater need to address the issues surrounding their translation from the laboratory to the end-user. In addressing this point, this review aims to discuss the latest trends, progresses, and challenges of polymeric MNs specifically, e.g., key marketed products, drug delivery mechanisms, polymer kinetics, fabrication techniques, materials used and classifications, MNs evaluation techniques, preclinical and clinical trials, and others. Various drugs administered using MNs for different diseases and ailments along with MN compositions are also discussed briefly to motivate the review topics further. At the end of the review, we conclude that for a successful translation of MNs, one needs to consider all these aspects successfully.Polymeric MNs have been shown to be a strong technology to provide chemical molecules with clinical effectiveness and large complex biotherapeutic systems . The polThe choice of material for MN preparation and drug release kinetics has key roles in translating polymeric MNs to commercial applications for the treatment of human diseases. Becton Dickinson (BD), United States of America (USA), Georgia Institute of Technology, USA, and Alza Company, USA, led studies on the development of MNs for medication administration in the 1990s. Metal or silicone was initially used as the raw material to produce MNs . NeverthDue to its cost, consistency, biodegradability, hygienic application, swelling, and dissolving capability, polymers are favored . PolymerEach polymer used within an MN is characterized by its potential for strength, penetration, and continuous release of drugs . MN peneMoreover, the active ingredient loaded into the polymeric MNs may improve mechanical strength . The mecKinetics can be defined as the study of the rate of change of concentration. Dissolution is described as the process of a substance moving from a solid state to forming an aqueous solution. Hence, dissolution kinetics is the study of the rate of change of concentration due to a substance dissolving . Therefo2) nanostructures incorporated. The purpose of the LaB6@SiO2 was to absorb near-infrared ray (NIR) wavelengths. Therefore, when exposed to NIR, the structure heated, causing the MN to melt at 50 °C. This approach allows for the release of active pharmaceutical ingredients upon demand using externally applied NIR [Photothermal Responsive. Photothermal responsive polymers are polymers that melt in the presence of photons of a specific range of wavelengths due to the absorption converting light energy into thermal energy . Chen etlied NIR .d,l-lactic-co-glycolic acid) (PLGA) external membrane, which protons could diffuse through. Thus, when the hollow MN array delivered the microspheres into the skin, which is naturally acidic, the protons reacted with the sodium bicarbonate within the microspheres. This formed CO2, which generated pressure within the microsphere. The pressure increased until the microsphere membrane ruptured, releasing the encapsulated drugs [pH-Responsive. pH-responsive polymers are polymers that allow for the transfer of a substance as a direct result of acidic or alkaline conditions. Ke et al.  developeed drugs . Ullah eed drugs  implemened drugs  created ed drugs .Moisture Responsive. Moisture responsive polymers are polymers that undergo dissolution due to hydrolysis. Moisture responsive dissolving MNs can either be designed for an instant release or a controlled release system . CommonlMNs are ultra-small and minimally invasive drug-delivery devices requiring regulated, precise, and repeatable injections into human skin . During There are different parameters which shape the design and effectiveness of MN devices, as shown in Numerous factors, including age, gender, ethnicity, anatomical area, and hormonal balance affect the thickness of the skin . MN patcstratum corneum (SC), increasing drug permeability through the pathways created by the MNs [Longer MNs can penetrate deeper and create longer micro-holes in the skin. As a result, more drugs can effectively move across the  the MNs ,48,114.  the MNs ,48. High the MNs ,116.To visualize and evaluate intradermal penetration depth, there are techniques such as histological sectioning, confocal microscopy, fluorescence microscopy, optical coherence tomography (OCT), X-ray computed tomography (micro-CT), and high-speed X-ray imaging ,120,121.The mathematical models, in general, have been used mostly to verify the performance of the designed MNs . The maiWhen focusing on the skin, the major barrier-to-mass transfer within the skin is the SC. However, when modeling the MN-skin system, the full epidermis should be considered . AndrewsWhen modeling polymeric MNs, there are several factors associated solely with the MN that needs to be considered. The main consideration for the MN is the hydrolysis reaction occurring between the steric bonds of the polymer. The reaction results in a reduction of the molecular weight and describe the degradation of the MN array . The geo® and COMSOL Multiphysics® [The other key component of modeling is the selection of physics that allows for the evaluation of drug-release profiles. The governing equation for the mass transfer of the drug is Fick’s law . Once thphysics® ,134. Thephysics® . Gomaa ephysics®  demonstrZhang et al.  developeKim et al.  developeRecent research has focused on the advancement of dissolving MN technology to increase the therapeutic effects of drug molecules, which requires a further understanding of the transport mechanisms within the skin. Ronnander et al.  developeChavoshi et al.  developeThe drug is normally embedded inside the needle through polymeric MNs, but swellable MNs may also distribute the medication from an external reservoir . The druMicro molding is the preferred method for the preparation of polymeric MNs due to high reproducibility, convenience for scalable production, and cost-effectiveness . This isDroplet-born air blowing (DAB) is a novel technique of fabrication where the droplet of a polymer is shaped into the MN through air blowing , as showDrawing lithography involves creating microstructures that are three-dimensional from two-dimensional (2D) materials . The draMost importantly, the mentioned conventional MN fabrication techniques can only fabricate MNs on a flat substrate surface; consequently, it is difficult to fabricate uneven or curved skin surfaces due to its decreased penetration efficiency and drug delivery amount . The reaA drug can be loaded in suspension or dispersion form or encapsulated into MNs . The medMorphological Evaluation. Scanning electron microscopic (SEM) studies of MN patches are the most common techniques to understand MN morphology ,182. PreDimensional Evaluation. Different techniques have been used for the measurements of MN geometry and for calculating the MN tip radius, length, and height . OpticalMechanical Testing. An MN should be sharp and thin enough to reach the skin quickly and firmly enough not to rupture within the skin . The forIn vitro and In vivo Studies. For the in vitro study, diffusion cells have been used to determine drug permeation across the skin . The skiContent Uniformity Studies. According to FDA guidelines, the drug contents of the MNs need to be evaluated accurately . For exaStability Evaluation. The stability of MNs depends on the stability of the polymers and drugs individually ,208. To −1 using DSC that was calibrated previously with standard materials [−1, and the weight loss was recorded during this time. Fourier transform infra-red (FTIR) spectroscopic analysis was used to record the vibrational changes of different functional groups present in the samples in following interactions with light over a specific wavelength range (400–4000 cm−1) [Chemical Stability. Differential scanning calorimetry (DSC) has been used to observe the enthalpy-related changes in the constituent materials of MNs by increasing the temperature. The constituent materials were heated to 300 °C at a rate of 10 °C minaterials ,217. The00 cm−1) .Polymeric MN technology has been successfully used to improve drug penetration through the skin layer . This haAnimal Models. Most pharmacological studies are conducted through in vivo animal models ,222,223.Porcine Skin Cargo Delivery Tests. The amount of drug delivered from the MNs to the skin was assessed using Franz diffusion cells with abdominal porcine skin ,200,223.Skin Recovery Process and Irritation. When an MN device is implanted and removed from the skin following treatment, it leaves behind micron-size pants . The skiPolymeric MNs have been studied for the treatment of various diseases such as cancer, HIV, diabetes, etc. They have also been used for vaccination and immunization. A successful translation of MN technology requires careful consideration for the targeted disease. To motivate this point, we discuss some key diseases targeted for polymeric MNs application in this section. Furthermore, Diabetes. Diabetes mellitus is a common metabolic disorder that involves hyperglycemia or an excess of glucose in the bloodstream due to insulin deficiency or abnormality in the use of insulin . Early w® MN . p < 0.05 two-tailed test) improve the in vitro skin permeability and in vivo anti-tumor activity of topical 5-FU [Basal Cell Carcinoma. Skin cancer is a worldwide threat . Basal ccal 5-FU .HIV and Vaccination . In 2018, it was estimated that over 37.9 million people across the world had human immunodeficiency virus (HIV) . HIV attContraception—Transdermal Patches. While the introduction of the contraceptive pill in 1974 to the National Health Service (NHS), United Kingdom (UK) was pivotal to women’s health, the current state of reproductive and sexual health in the UK highlights the inconsistencies in contraception and effectiveness . Althoug® and topical bleomycin to treat plantar warts. Plantar warts are viral proliferations caused by human papillomavirus (HPV) infection [p < 0.05) to improve wrinkles and skin hydration. This is attributed to its high-water binding and uptake capacity, biocompatibility, and biodegradable properties, making it suitable for anti-wrinkle treatment [Dermatological Conditions and Cosmetics. The advances in the transdermal application of MNs to systemically deliver drugs through the skin have provoked the use of MNs for the treatment of dermatological conditions and nonmedical cosmetic therapeutics . The appnfection . Currentnfection . This clnfection . The cosnfection . MNs havnfection . It is mnfection . It is anfection . This isnfection . Hyaluroreatment . The delreatment . Adenosireatment .By the year 2025, the market for TDD is estimated to be worth approximately $95.57 billion . Consequ® (no-pass) and Soluvia® (BD), but they are not genuine MN arrays and are instead small hollow needles that can be used to insert a traditional syringe barrel with effective ID. These devices work in the same manner as traditional syringes. MicronJet®, a hollow MN device for intradermal injection, was developed by Nano pass Technology. The device consists of four hollow silicon needles smaller than 500 μm, which are attached to a plastic device that can be connected to any regular syringe. This method was used to vaccinate influenza with the immunogenicity of at least 20% of the traditional vaccine dosage. In 2010, the FDA approval was granted to Micronjet® [®, which consists of coated MN arrays, was developed by 3M, which allows quicker delivery of medicines and vaccines. This machine demonstrated quick distribution, with a manageable administration for up to 90 min [® and uses the BD technologies Soluvia® injector. This device includes a hypodermic needle 1.5 mm long attached to a syringe injector [® and MicronJet® are currently the only therapeutic-based MN-basic devices on the market. This injector is currently sold globally under the names IDflu® and Fluzone Intradermal® in addition to Intanza® [® has been designed with coated titanium micro-projections as an optimized delivery of biopharmaceuticals. In this system, the skin penetration depth can be reproductively regulated due to the integration of an applicator device system. Moreover, the ovalbumin delivery system has been successfully tested [MNs have important advantages relative to traditional transdermal pads, particularly in the delivery of biopharmaceuticals . Thus, icronjet® ,303. Theo 90 min ,305,306.o 90 min ,309,310.o 90 min ,222,235.o 90 min ,312. Saninjector ,305. SolIntanza® ,314,315.Intanza® ,317,318.Intanza® ,320,321.Intanza® ,323. Alzy tested . Rodan+Fy tested . Furthery tested .Although polymeric MNs are biocompatible, when the accumulation of polymers occurs in our bodies through repeated application of the MN arrays, hepatic impairment, immunological reaction, and build-up of polymers in the dermal tissue are probable health implications with the use of this device . One of To develop polymeric MNs, environmental factors need to be taken into consideration as well. The polymers used in MN fabrication can be hygroscopic . This hyClinicalTrials.gov database. Data from 116 clinical trials are available on the database, using the keyword “microneedle”. The selected number of clinical trial studies in this review was 76 based on the “complete” status . Depending on the availability of data, an overview of the percent of MNs in the clinical stage state and selected clinical trials are listed in http://patft.uspto.gov/netahtml/PTO/search-bool.html) using the term “microneedle” and field “Title”. From there, specific to each year, 2000–2020 were searched and is presented in There are several clinical trials related to MN technology available on the In the last two decades, a significant effort has persisted for the alternative use of MNs in clinical practice to be more concrete . As reseStudies on diabetes, psoriatic plaques, topical anesthetics, and influenza vaccines have involved hundreds of MN devices for clinical trials. Without laboratory trials with volunteer human subjects, clinical translation of the emerging medical innovations is unlikely to be successful. Dissolvable but also permanent, MNs prove to be easy to use and well-tolerated in clinical trials. Various study groups have recently published the results of MNs in dermatological disorders such as scars and keloids. In both cases, MNs have improvedimproved the efficiency of treatments by self-administration and painlessness. In several experiments, hollow MN systems that are commercially available were used, although clinical trials on polymer MNs are limited. The penetration potential and protection of the HA-base dissolving MNs have been investigated in two Phase I experiments. The findings indicate that the MNs can be reproducibly absorbed through the skin with the aid of an applicator and no apparent side effects. The protection and immunogenicity of dissolvable MNs dependent upon HA were analyzed in another phase trial. In the clinical trial for the delivery of strains of seasonal H1, H3N2, and B influenza virus vaccines, results revealed that the MN patch is tolerable, and a strong immune response is possible. These experiments have shown that polymer MNs can be translated clinically. However, we know that to date, no clinical trials on the continuous release of MNs are published ,365,366.The MN dosage forms microbiological requirements. In certain instances, the MN dose formulations pierce the SC to the dermis. Microbiological requirements can also represent a significant regulatory concern. If there is also a proven antimicrobial activity, it may be appropriate for a substance to have low bioburden ,373,374;Content standardization: this is a standard prerequisite for pharmacopeia to be used in MN schemes. This condition can, however, be extended to either the entire system or to individual drug-loaded MN in an array, depending on the system configuration ,376;Certain MN devices cannot be dissolved or biodegraded and can be re-inserted easily. This can be dangerous and requires a proper disposal process ,378;Certain MN chemicals accumulate on the skin. Polymeric MN dissolvable deposits can deposit materials, causing adverse effects on the skin, such as the formation of granulomas or local erythema. By alternating the application platform, this may be mitigated. As stated earlier, this problem must be solved, for the long-term use of MN goods ,380,381.Patient ease of use and reliability: without risks, patients should be able to use the goods as possible ,84;Correct delivery insertion: since MN injection does not induce discomfort or apparent emotion, the patient has no feedback. A proper program framework may be appropriate ,382,383;Repetitive use of immunological effects: repeated insertion of MN into the skin may cause immune reactions. The regulators should be assured of immunological protection ,384,385;Profile for long-term security: to date, human volunteers have conducted short-term security experiments. The long-term protection of the MN program should therefore be taken into consideration from the point of view of sporadic and repetitive demands ,386,387.Consequently, Because of its creativity and technologies, there are no regulatory criteria specified now for MN array-based products . The difThe combined efforts of engineering, pharmaceuticals, and immunology researchers can promote the future success of this technology and clinical translation . A microSustained drug release MNs demonstrated higher performance relative to fast dissolving MNs . NeverthThe effectiveness of polymer MNs relies heavily upon the polymer form or combination of polymers, their biocompatibility with drugs, their nature, and their mechanical ability . FurtherThe production of MN-based multifunctional smart bioprocessing is still desperately needed for potential biomedical applications, considering the impressive achievements around MNs . It is bResearchers aim to make use of MNs to provide organ repair , as illuDespite major developments in the development, detection, and control of MNs for the delivery of transdermal drugs, biomedical applications are still early in existence and have not advanced toward large-scale manufacturing and practicality . ShortlyPatients should not take anti-inflammatory drugs for a full week after treatment ;Patients should not use ice on the face or use bromelain/arnica. This will impair a normal mechanism of inflammation that is essential to the rejuvenation of the skin ,329;Prevention of sun tanning and extended direct sunshine use for at least 2 weeks. Use sunblock (30 SPF or higher) always after 24 h and wear a hat when outdoors ;Patients with anxiety should take a painkiller or Tylenol .This review paper demonstrates the value of polymeric MN as a promising technology, and it has a great future ahead. However, there are major obstacles in the immediate challenges of manufacturability, cost factors, and regulatory clearance. Science in this area is potentially advancing to safer supplies of transdermal drugs using the polymeric MNs method ,406. The