MEG3) is known to be involved in the pathogenesis of preeclampsia by inhibiting the migration and invasion of trophoblasts and promoting their apoptosis. Nevertheless, the specific underlying downstream molecular mechanism of MEG3 is less well characterized. In this study, we detected lower expression levels of MEG3 and β-Catenin and higher expression of nod-like receptor pyrin domain-containing 3 (NLRP3) in placental tissues of pregnant women with severe preeclampsia (sPE) than in normal pregnancies. Elevated serum levels of IL-1β and TNF-α were also observed in the sPE group. Then, we established a hypoxia/reoxygenation (H/R) model to mimic preeclampsia. Similar results with sPE group were found in the H/R group compared with the control group. In addition, suppressive trophoblast proliferation, migration and invasion and increases in the apoptotic rate and inflammation were also detected in the H/R group. Notably, overexpressing MEG3 markedly improved trophoblast dysfunction and inflammation caused by H/R. However, the effects of MEG3 on trophoblasts, whether upregulated or downregulated, can be reversed by DKK-1 (Wnt/β-Catenin inhibitor) and MCC950 (NLRP3 inhibitor). The current study revealed that MEG3 regulates trophoblast function and inflammation through the Wnt/β-Catenin/NLRP3 axis and provided new insights into the pathogenesis of preeclampsia.Inadequate trophoblastic infiltration and resulting placental hypoxia and inflammation comprise the core pathological basis of preeclampsia (PE). Maternally expressed gene 3 ( Preeclampsia (PE) is a common and serious obstetrical disease that occurs in approximately 2%–8% of all pregnancies . It not MEG3), located on human chromosome 14q32, is a pregnancy-related gene  are commonly defined as RNA transcripts with lengths of more than 200 nucleotides that cannot encode corresponding proteins . In receted gene . It has poptosis . These rβ-Catenin is a highly conserved signaling pathway centered on β-Catenin, which is broadly implicated in numerous biological processes and human diseases -challenged human trophoblasts as an in vitro model to show that upregulation of MEG3 contributed to enhanced trophoblast proliferation, migration and invasion and attenuated the apoptotic rate and inflammatory status through the Wnt/β-Catenin/NLRP3 axis.In this study, we first measured the expression pattern of the lncRNA Twenty patients with severe preeclampsia (sPE) and 20 normotensive pregnant women who delivered by cesarean section at Shandong Provincial Hospital between April 2019 and September 2021 were enrolled in the current study. PE was defined as the presence of blood pressure ≥140/90 mmHg after 20 weeks of gestation and urinary protein level ≥300 mg/24 h. According to the criteria of NHBPEP (2000), diagnosis of sPE was considered when the following criteria were present: blood pressure ≥160/110 mmHg, proteinuria ≥2.0 g/24 h or + 2 dipstick, serum creatinine >1.2 mg/dl, acute pulmonary edema, HELLP syndrome, or eclampsia. Exclusion criteria included maternal history of hypertension, premature rupture of membranes, chemical dependency, diabetes mellitus, renal diseases, autoimmune and other complications that may lead to hypoxic changes.Placental tissues were taken from the center area around the umbilical cord immediately after cesarean section, avoiding regions of infarct and calcification. The specimens were then washed in chilled phosphate-buffered saline (PBS) and stored at −80°C until further use. Maternal venous blood was collected prior to delivery, followed by serum separation and storage at −80°C.The clinical data of all pregnant women were retrospectively collected from medical records. This study was conducted in accordance with the Declaration of Helsinki and was approved by the Ethics Committee of the Shandong Provincial Hospital (2020-805). All participants signed written informed consent forms. The clinical features of all patients in the sPE and normal groups are shown in 2 and 5% CO2. To construct an in vitro hypoxia/reoxygenation model, cells were maintained in 2% O2, 93% N2, and 5% CO2 for 8 h (hypoxia) before being returned to standard culture conditions  for 16 h  were cultured in RPMI 1640 (Gibco) supplemented with 10% fetal bovine serum  at 37°C in an atmosphere of 20% Ofor 16 h . RecombiMEG3, sh-MEG3 and their corresponding negative controls, which were referred to as pcDNA-NC and sh-NC, were designed and synthesized by Shanghai GeneChem Medical Technology Co., Ltd. . When the cell confluence reached approximately 50%, the abovementioned sequences were transiently transfected into HTR8/SVneo cells using Lipofectamine 3000  in Opti-MEM culture medium (Gibco) according to the manufacturer’s instructions. After 6 h, the transfection was replaced with 2 ml RPMI 1640 medium containing 10% FBS, and the cells were further cultured for 48 h and for subsequent experimentation.pcDNA-® Green Pro Taq HS Premix qPCR kit  on a Light Cycler 480 . Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was considered to be an endogenous control, and the original data were calculated by the 2-ΔΔCt method. The primer sequences used in this study are listed in Total RNA was extracted from the placental tissues or cells using TRIzol reagent , and a Nanodrop 2000 spectrophotometer  was used to measure the purity and concentration of the extracted RNA. One microgram of RNA was reverse transcribed into complementary DNA (cDNA) using a reverse transcription kit with gDNA removal reagent . Subsequently, we conducted quantitative real-time PCR (qRT‒PCR) to detect the relative expression level of the target gene using a SYBRβ-Catenin , NLRP3 , c-Myc , Vimentin , Bax , Bcl-2 , and GAPDH  overnight at 4°C on a shaker. Then, HRP-conjugated AffiniPure goat anti-rabbit IgG  diluted in 5% skim milk powder was added for 1 h of incubation at room temperature. Finally, the transferred membrane was covered with Immobilon Western Chemiluminescent HRP Substrate , and images were developed using an Amersham Imager 680 . ImageJ software (NIH) was employed for quantitative analysis of protein bands. GAPDH served as the internal reference.Total protein in placental tissues and cells was extracted with RIPA buffer containing protease inhibitors  and quantified by a BCA protein assay kit . Equal amounts of protein were separated by 10% SDS‒PAGE  and transferred onto polyvinylidene fluoride (PVDF) membranes. After blocking with 5% skim milk powder at room temperature for 1 h, the membranes were incubated with the following primary antibodies : rabbit monoclonal antibodies against β-Catenin antibody  at 4°C overnight. As a secondary antibody, fluorescein isothiocyanate-conjugated goat anti-rabbit antibody  was added for incubation at room temperature for 50 min. Nuclei were stained with DAPI  under the conditions of protection from light for 10 min, and then the sections were sealed with antifading mounting medium . Immunofluorescent images were visualized with confocal fluorescence microscopy , and the pixel intensity of the immunoreactive signal was measured using ImageJ (NIH).For placental tissues, paraffin-embedded sections were subjected to a standard deparaffinization and rehydration process and incubated in citric acid for antigen retrieval. After that, the sections were blocked with 1% bovine serum albumin (BSA) at 37°C for 30 min and then incubated with primary NLRP3 antibody  or Serum samples and culture media were centrifuged for supernatant collection and stored in a −80°C freezer for subsequent use. Human IL-1β and TNF-α enzyme-linked immunosorbent assay (ELISA) kits  were employed to detect the levels of the cytokines IL-1β and TNF-α following the manufacturer’s instructions. The optical density (OD) value of each well was measured, and the concentration of cytokines was calculated by the standard curve generated from the standard solutions.The migratory ability of HTR8/SVneo cells was determined using the scratch-wound healing method. In short, cells were seeded in six-well culture plates and cultured for 24 h to ensure that the cells adhered to the wall. A wound-like gap was made on the cell layers using a 200-μl sterile pipette tip and rinsed three times with PBS. Then, the cells were cultured in serum-free RPMI-1640 medium to reduce the effect of cell proliferation on migration. Randomly selected fields were photographed under an inverted microscope  at 0 and 48 h. ImageJ software was used to measure the scratch rate.5 cells in 200 µl serum-free RPMI-1640 medium was plated onto the upper chamber, and 600 µl complete medium containing 10% FBS was added to the lower chamber. After 48 h of incubation, the noninvading cells on the upper surface of the membrane were removed, while the invading cells were fixed in methanol, stained with 0.1% crystal violet, and counted microscopically.A transwell chamber  with a membrane pore diameter of 8 µm was used to conduct the transwell assay. The membrane was covered with 50 µl Matrigel  diluted with serum-free RPMI 1640 medium at a ratio of 1:8. A cell suspension of 1 × 103 cells per well and were cultured for 0, 24, 48, and 96 h. Then, 10 µl of CCK-8 reagent  was added to each well, and the incubation was continued in the incubator at 37°C for 2 h. The absorbance value at 450 nm of all samples was determined using a microplate reader  and then used to plot the growth curve of cells.HTR8/SVneo cells grown to the log phase were seeded in 96-well plates at a density of approximately 1 × 10Cell apoptosis analysis was performed using the Annexin V-fluorescein isothiocyanate (FITC) Apoptosis Detection Kit . Cells were detached from the culture plates with trypsin without EDTA (Gibco) and washed twice in PBS. Next, 100 µl binding buffer was added to resuspend the cells, followed by 5 µl FITC Annexin V and 5 µl propidium iodide (PI) solution for a 20 min incubation in the dark. Finally, the cell suspension was mixed with 400 µl binding buffer and examined using a BD FACSCalibur . The data were analyzed by FlowJo software .t test. Comparisons among multiple groups were performed employing one-way analysis of variance (ANOVA), followed by Tukey’s multiple comparison post hoc analysis. A p value of less than 0.05 was considered an indicator of statistical significance.All data are presented as the mean ± SEM and were analyzed by using GraphPad Prism 7 software . The data conforming to normal distribution and homogeneity of variance between two groups were analyzed by using an unpaired MEG3, β-Catenin, NLRP3 and the pathogenesis of preeclampsia, we first measured the expression levels of MEG3, β-Catenin and NLRP3 in placental tissues by qRT‒PCR. The results indicated that the mRNA expression levels of MEG3 (β-Catenin (β-Catenin and NLRP3 were the same as the changes in the mRNA levels. ELISA results revealed that the serum levels of IL-1β (To establish the relationships between  of MEG3  and β-Ca-Catenin  were sig-Catenin  expressi-Catenin  and immu-Catenin  further of IL-1β  and TNF-of IL-1β  were sigin vitro, trophoblasts were cultured with H/R treatment. Compared to the control group, MEG3 and β-Catenin levels were downregulated and NLRP3 expression was upregulated in HTR-8/SVneo cells cultured in H/R, as shown by qRT-PCR (To simulate the microenvironment of PE  qRT-PCR  and West qRT-PCR . Subsequ qRT-PCR , transwe qRT-PCR , wound h qRT-PCR , and flo qRT-PCR  assays w qRT-PCR . ELISA a qRT-PCR  and TNF- qRT-PCR . Taken tMEG3 on trophoblast cell function, MEG3 expression levels were upregulated by introducing pcDNA-MEG3. Compared to the H/R and H/R + NC groups, HTR8/SVneo cells transfected with pcDNA-MEG3 after H/R manifested enhanced mRNA levels of MEG3 (β-Catenin (β-Catenin and NLRP3 were consistent with the changes in their mRNA levels (MEG3 overexpression group versus the other two groups. Related proteins varied similarly to these results (To uncover the specific effect of  of MEG3  and β-Ca-Catenin  and sign-Catenin . At the A levels . FurtherA levels , invasioA levels  and migrA levels  of trophA levels  and inflA levels  were sig results .β-Catenin pathway to trophoblast function and the MEG3 regulatory pathway, we treated HTR8/SVneo cells with DKK-1, a Wnt/β-Catenin pathway inhibitor. We found that the MEG3-induced upregulation of β-Catenin and downregulation of NLRP3 were reversed by DKK-1 at the mRNA (MEG3 contributes to improving trophoblast cell function through the Wnt/β-Catenin pathway.To verify the contribution of the Wnt/the mRNA  and protthe mRNA . Similarthe mRNA , invasiothe mRNA , migratithe mRNA , apoptosthe mRNA  and relathe mRNA , as wellthe mRNA and TNF-αthe mRNA . These rMEG3/Wnt/β-Catenin axis on trophoblast function. Compared to the H/R group, targeting MEG3 with shRNA substantially increased the expression of NLRP3 and further aggravated cell dysfunction induced by H/R. However, the upregulation of NLRP3 by sh-MEG3 was reversed by the NLRP3 inhibitor MCC950 (Next, we confirmed whether NLRP3 is involved in the effect of the r MCC950 . Moreover MCC950 , invasior MCC950  and migrr MCC950 , evidentr MCC950  and inflr MCC950 , in addir MCC950 .PE remains an important cause of maternal morbidity and mortality . AlthougMEG3 was underexpressed in preeclamptic placentas, which was in line with our results. Its downregulation has been demonstrated to decrease migration and invasion while promoting the apoptosis of trophoblasts (MEG3 plays a vital role in the initiation and progression of PE through its effects on trophoblast cells. However, little is known about the specific downstream molecular mechanism of MEG3 regulation in trophoblast cells. In the present investigation, we focused on the function and mechanism of lncRNA MEG3 and indicated that MEG3 reduces NLRP3 levels by targeting Wnt/β-Catenin and ultimately improves trophoblast cell function by promoting cell proliferation, migration, invasion and suppressing cell apoptosis and the inflammatory response.In recent years, lncRNAs have been extensively studied for their relationship with trophoblast function and placental development. The study conducted earlier revealed that hoblasts . Combineβ-Catenin and NLRP3 in placental tissues were detected using PCR, Western blotting, and immunofluorescence. All of the experiments showed significantly lower expression of β-Catenin and higher expression of NLRP3 in preeclampsia placental tissues compared to normal pregnancies, implying that they might have functions related to preeclampsia. At the same time, the upregulation of the proinflammatory cytokines IL-1β and TNF-α in the serum of preeclampsia patients suggested the activation of systemic inflammation in preeclampsia patients. To investigate the detailed mechanisms, we performed in vitro experiments using HTR8/SVneo cell line.First, the expression profiles of MEG3 and β-Catenin expression decreased and NLRP3 expression increased in HTR-8/SVneo cells stimulated by H/R, similar to the levels observed in the placental tissue of preeclampsia. Subsequently, we verified that H/R induced trophoblast functional impairment and inflammation activation, which are not conducive to uterine spiral artery remodeling. Interestingly, pcDNA-MEG3-mediated overexpression of MEG3 notably reversed changes in trophoblast cell function and molecular proteins triggered by H/R. These changes mainly manifest as elevated β-Catenin expression and decreased NLRP3 expression, intensifying the capacities of proliferation, migration and invasion as well as attenuating cell apoptosis and inflammation. MEG3 overexpression also improves trophoblast cell function by regulating proteins related to proliferation, migration, invasion and apoptosis, such as c-Myc, Vimentin, Bcl-2, and Bax. Thus, we concluded that MEG3 alleviates preeclampsia progression not only by improving trophoblast dysfunction but also by inhibiting inflammatory activation.H/R can give rise to oxidative stress, apoptosis and inflammation and is often used to mimic preeclampsia . In our β-Catenin is a pivotal regulator of placental formation by facilitating the migration and invasion of trophoblast cells, which is corroborated by previous studies (β-Catenin is a target of MEG3 and is involved in a variety of human diseases downstream of MEG3, including Wilms’ tumor, glioma, and oral squamous cell carcinoma (β-Catenin was prominently strengthened after MEG3 overexpression. To further verify whether MEG3 affects the biological functions of trophoblast cells through the Wnt/β-Catenin signaling pathway, we used DKK1 to block Wnt signaling. Our data illustrated that blocking β-Catenin partially eliminated the recovery effect of MEG3, suggesting that β-Catenin mediates the regulation of MEG3 on trophoblast cell function.Wnt/ studies . Recentlarcinoma . Similarβ-Catenin pathway, is involved in the regulation of melatonin on osteoblast differentiation MEG3/Wnt/β-Catenin may be mediated by NLRP3. Thus, we used an NLRP3-specific inhibitor, MCC950, to downregulate NLRP3 levels and observed that when NLRP3 expression was decreased, superimposed injury of trophoblast cells by sh-MEG3 and H/R was greatly weakened.Consistent with previous reports, we found that NLRP3 was substantially increased in PE and was relevant to impaired trophoblast function and aggravated inflammation . IdentifMEG3 expression should be investigated to validate our results in the HTR8/SVneo cell line. Additionally, the data generated by our experiments only illustrated the critical role of MEG3 in the placenta of PE. Maternal vascular wall dysfunction caused by placental factors is also considered to be the key pathogenesis of PE (MEG3 involved in maternal and placental vascular dysfunction in PE deserve to be further explored in future work.The present study has several limitations. First, the gestational age of preeclampsia was earlier than that of the normal pregnancy group due to the difference in optimal termination time. Second, further confirmation of this discovery is needed in animal models of preeclampsia and other trophoblast cell lines. Third, the correlation between placental tissue apoptosis and is of PE . Thus, tMEG3 and β-Catenin and high levels of NLRP3 in the placental tissue of PE and demonstrated that MEG3 can participate in the proliferation, migration, invasion, apoptosis and inflammatory response of trophoblast cells through the Wnt/β-Catenin/NLRP3 pathway. Our research revealed a promising target for the clinical treatment of preeclampsia and a new direction for the study of its pathogenesis.Collectively, our research implied low levels of


Soft lithography has permitted rapid prototyping of precise microfluidic features by patterning a deformable elastomer such as polydimethylsiloxane (PDMS) with a photolithographically patterned mold. In microfluidics applications where the flexibility of PDMS is a drawback, a variety of more rigid materials have been proposed. Compared to alternatives, devices fabricated from epoxy and glass have superior mechanical performance, feature resolution, and solvent compatibility. Here we provide a detailed step-by-step method for fabricating rigid microfluidic devices from soft lithography patterned epoxy and glass. The bonding protocol was optimized yielding devices that withstand pressures exceeding 500 psi. Using this method, we demonstrate the use of rigid high aspect ratio spiral microchannels for high throughput cell focusing. The relatively soft and tough PDMS part is lifted off from the rigid silicon mold. The major advantages of soft lithography, as compared with alternative rapid prototyping methods such as 3D printing, stem from the excellent feature resolution afforded by thin film photolithography on silicon wafers, as well as the ability to quickly produce multiple elastomeric devices from a single wafer mold.Rapid prototyping techniques accelerate early-stage development of microfluidic technologies by reducing iteration time and upfront costs. Perhaps the most widely used rapid prototyping technique for microfluidics research is soft lithography, which typically involves patterning a polydimethylsiloxane (PDMS) elastomer part from a mold of micropatterned photoresist film on a silicon wafer2. Therefore, the use of PDMS can jeopardize both research results, where deformability can be a substantial source of experimental variability, as well as translational development, since scaled-up manufacturing processes overwhelmingly use thermoplastics which are much more rigid than PDMS. In these cases, it would be prudent to first validate microfluidic designs in a rigid material before bearing the substantial tooling costs for injection molding or embossing.However, the deformability of PDMS is unfavorable for microfluidics applications that involve moderate pressures and where the channel geometry is important. For example, this is the case for virtually all studies of inertial microfluidics phenomena, which generally involve relatively high-pressure flows (P > 30 psi) in relatively long microchannels (> 1 cm). PDMS devices start to deform at as little as 15 psi, and can rupture at pressures of about 40–60 psi3. These efforts demonstrated the fabrication of rigid devices from a photolithographically defined pattern by transfer molding using an intermediate PDMS replica. Among the materials evaluated, the thermoset recipe first described in 2007 exhibited the highest stiffness and best bonding performance (at least 150 psi)4. Later, another transparent thermoset, the epoxy resin EpoxAcast 690, was utilized to measure particle focusing at very high flow rates at operating pressures approaching 10,000 psi6. This same material was also shown to have excellent chemical inertness and gas impermeability7. It was also shown that an epoxy chip had the capability to capture circulating tumor cells from whole blood based on their size with an efficiency of about 80%8. Based on these studies, epoxy-glass devices may be considered to have equal or superior characteristics to all other studied rapid prototyping materials with regards to feature fidelity, rigidity, and bond strength. Altogether, compared to alternative rapid prototyping methods such as 3D printing or patterned laminate films, photolithographically patterned devices have the highest feature resolution and wall smoothness9. Likewise, commercially available epoxies are more accessible to researchers than custom materials10.These considerations motivated the development of new techniques for rigid device prototyping by several groups, systematically reviewed in 2011Despite the many advantages of rigid devices, rigid materials are rarely utilized in the literature even when warranted. A detailed step-by-step protocol may greatly improve the accessibility of rigid device prototyping to microfluidics laboratories that are already using soft lithography and PDMS. To address this need, we describe the most common pitfalls in mold preparation and device bonding, how they may be addressed. Furthermore, we optimize several key process parameters to maximize bond strength while maintaining the ease and limited resources of the protocol.1. Before you begin, you will need a master mold with the desired patterned features, such as a patterned SU-8 thin film on a silicon wafer. As described below, the channels in the final device should be raised features on the master mold, just like for conventional PDMS devices.The procedure consists of three main steps: 1) PDMS mold preparation, 2) epoxy pouring, and (3) mold release, post-processing, and bonding  will not react with the epoxy.Regarding the protocol as a whole, we had several important observations. First, the TCPFOS-treated replicas which are used to make the PDMS mold can be reused, but only 2–3 times. Beyond that, the surface of the replica can become warped. On the other hand, the PDMS device molds can be reused many (10+) times. Over time, the mold can acquire an opaque white color. It does not affect the device or the PDMS mold. Furthermore, cutting the Polytetrafluoroethylene (PFTE) rods at a slight angle can make it easier to insert them into the molds. Finally, the tubing material should be selected to be compatible with epoxy bonding. We use polyethylene tubing, which forms a strong bond with the epoxy. Common tubing materials such as TygonWe optimized several process variables with the goal of being able to reliably make devices that could withstand high pressures. Cure time was optimized before mold removal, the plasma treatment time, and the duration of resting time on the hot plate after bonding. To test the bond strength of a device, we fabricated the device with just a single port (i.e. no outlets) and connected it to the high-pressure syringe pump model 100Dx with an integrated pressure transducer. The device was gradually pressurized with DI water over the course of several minutes until failure, and the peak pressure before failure was recorded.First, we hypothesized that using a shorter cure time than 24 h may increase the bond strength if the epoxy has more available reactive groups. For cure times less than 20 h, the epoxy was too soft and tacky to remove from the mold. On the other hand, for cure times over 30 h, we observed that the epoxy had almost fully hardened and would not bond to the glass slide. Within this window, we observed a trend towards higher bonding pressures at lower cure times, with a maximum of 670 psi at the lowest tested cure time of 20 h , we also tested whether devices would withstand similarly high pressures for a prolonged period. We fabricated a device using the protocol described above at a 24-h cure, and pressurized at increments of 100 psi, increasing by 100 psi every 10 min. We observed that the device was able to withstand a pressure of 100 psi for an hour. It was also able to last for at least 10 min at 200 and 300 psi. The device failed at 400 psi after 6 min, which was similar to the device strength expected based on the pressure sweep experiments . This resulted in a device that could withstand an average of 714.5 psi based on two chips, about 12% higher than the prior highest condition during optimization.We used the optimized fabrication protocol to make spiral microchannels with a very high aspect ratio and test their performance for high throughput inertio-elastic cell focusing. For biomedical applications of inertial focusing involving large sample volumes, the maximum shear stress on the cells must remain within limits to avoid damage. For reasons discussed later, we hypothesized that a very high aspect ratio microchannel (i.e. 10:1 or more) would have several advantages. However, high aspect ratio channels are also more prone to inflating under pressure if they are fabricated from deformable materials.We fabricated a single microchannel 100 µm tall, 1 mm wide, and about 140 mm long, coiled into a spiral about 17 mm wide altogether  may be roughly estimated as a zero-shear viscosity of about 5 mPa s, and a relaxation time of about 10 ms. With a characteristic channel width of 100 µm, the highest tested flow rate of 2.4 ml/min would correspond to a Reynolds number of about 8 and to a Weissenberg number of about 40. The observed focusing characteristics are consistent with results by others investigating flows with a similar Elasticity Number, here El = Wi/Re = 4.813.Using a rigid epoxy device, we studied the focusing performance in high aspect ratio microchannels. Compared with moderate aspect ratio microchannels , high aspect ratio curved microchannels have the advantage of suppressing the onset of Dean flow secondary vortices at higher Reynolds numbers, which permits efficient cell focusing at higher flow ratesThis protocol has several potential limitations. One potential limitation of this procedure is that it is not ideal for high aspect ratio features such as pillars or thin walls because the mold is released when the epoxy part is still pliable. Releasing and bonding devices with very high aspect ratio features may take additional optimization and practice. Nevertheless, epoxy provides excellent mechanical properties and surface finish for high throughput applications. This detailed protocol will allow more laboratories to easily integrate rigid devices into their existing PDMS-based prototyping workflows.Channels in the final device should be raised features on the master mold, just like when using it to mold a conventional PDMS device.The pattern master mold, e.g. a micropatterned SU-8 thin film silicon wafer. https://shop.smooth-on.com/epoxacast-690).*EpoxAcastTM 690 transparent epoxy kit .*Trichlorosilane (TCPFOS) .*Sylgard 184 silicone elastomer kit .Polytetrafluoroethylene (PFTE) Rod 0.07″ (https://www.mcmaster.com/5648K67-5648K226/).Polyethylene (PE) tubing 1/16″ ID (Plastic Petri dishes 100 mm.Petri Dishes 175 mm with lid.Weigh boat.Hot plate.https://www.fishersci.com/shop/products/ultraclean-microarray-slides-enhanced-surface/22037213?searchHijack=true&searchTerm=+C22-5128-M20&searchType=RAPID&matchedCatNo=+C22-5128-M20).Ultraclean glass slides .Clean metal spatula with rounded end, e.g. (Pipettes.https://www.fishersci.com/shop/products/bel-art-scienceware-space-saver-vacuum-desiccators-3/0859416A?gclid=CjwKCAiAvK2bBhB8EiwAZUbP1OvPjQ-ZJAhbEmrVHglNDxRCoS1kEZrG47Yro7f3h4sNqjjlgiL0dRoCh7YQAvD_BwE&ef_id=CjwKCAiAvK2bBhB8EiwAZUbP1OvPjQ-ZJAhbEmrVHglNDxRCoS1kEZrG47Yro7f3h4sNqjjlgiL0dRoCh7YQAvD_BwE:G:s&ppc_id=PLA_goog_2086145680_81843405274_0859416A__386247001354_9967139677844655410&ev_chn=shop&s_kwcid=AL!4428!3!386247001354!!!g!827721591040!0859416A).Separate Vacuum chamber for TCPFOS vapor deposition (https://www.wpiinc.com/14140-g-graefe-forceps-7cm-straight-07mm-1x2-teeth-german ).Toothed Forceps, also called ‘tissue’ or ‘rat tooth’ forceps, e.g. (https://www.enerconind.com/plasma-treating/products/vintage-treaters/dyne-a-mite-hp.aspx).*Plasma Generator .Electric Ultra-High-Vacuum Pump  times.In this step, we will first prepare a PDMS 2) it is possible to cast a single PDMS mold from multiple PDMS replicas.This protocol is for making devices sized similar to or smaller than a standard glass slide. For small devices  from the PFTE rod diameter, to allow a tight press to fit and prevent epoxy from seeping through. On the other hand, if the punched holes are too undersized, it may still be possible to fit the rod inside, however this will stretch the PDMS surface and cause it to pull down, by virtue of the Poisson effect. This pulldown will then be carried over to the epoxy part and may cause issues with bonding.A second potential pitfall with mold preparation, also associated with hole punching, is hole shape. A conventional round biopsy punch can compress a PDMS part substantially before biting through, causing a necking-down of the hole with depth in the relaxed PDMS part. This hole shape can leave a small ‘countersunk’ perimeter around a press-fit PFTE rod, and this feature can be carried over to the epoxy part and cause issues with bonding.To avoid these two issues, we use a 2 mm thick sacrificial PDMS spacer on top of the PDMS mold during punching. In our experience, a 2.0 mm biopsy punch can be used to make straight-walled holes 1.8 mm in diameter, which are suitable for 0.07″ PFTE rod stock and 1/16″ inner diameter tubing.Time: 15 minMake a PDMS mixture total of 50 g . Mix well.Pour the PDMS over the SU-8 wafer.Place the dish with the SU-8 wafer and PDMS into vacuum chamber. Leave the dish for 1–3 h, then transfer to a 60 °C oven to cure the PDMS overnight.Time: 15 min4.Gently remove the PDMS casting from the silicon mold.5.Cut out the replica to size from the PDMS.Time: 35 min6.Perform oxygen plasma treatment of the PDMS replica.7.Place replica in a clean 100 mm petri dish.NOTE: If the devices are small, there may be space to fit multiple replicas in a single 100 mm petri dish mold. Make sure the replicas do not touch each other in the dish, ideally at least 5 mm of space between replicas as well as between any replicas and the petri dish wall.8.Pipette and lightly spread 2 μl of Tricholorperfluorooctylsilane (TCPFOS) onto the petri dish and to make a border around the replicas.NOTE: TCPFOS is toxic. Use appropriate PPE.9.Place the petri dish into a dedicated vacuum chamber for 30 min.NOTE: Small amounts of TCPFOS may be deposited on both the inner and outer surfaces of the petri dish and should be handled accordingly following appropriate safety precautions. Do not handle the dish without gloves.Time: 15 min10.Make a PDMS mixture total of 50 g . Mix well.11.Pour the mixture into the 100 mm dish with the devices. Be sure they are fully covered.NOTE: Make note of how many times this replica has been used. Do not got past 2 uses. Future devices will become warped.12.Place the dish into vacuum chamber. Let the dish sit for 1–3 h, then transfer to s 60 °C oven to cure PDMS overnight.OVERNIGHT.Time: 15 min13.Using a metal spatula, gently remove the PDMS mold completely out of the petri dish.NOTE: It may help to squeeze opposite sidewalls of the plastic petri dish very gently towards one another, to slightly flex the entire dish and allow the PDMS to separate from the dish. The replica may start to fall out of the mold during this step. Take care not to damage or tear the mold or the replica at this step.14.Using a clean metal spatula or tweezers, gently remove the PDMS replica from the mold.NOTE: It may help to gently flex the entire PDMS mold until the replica begins to separate from the mold. Be sure not to damage or tear the mold or the replica at this step.15.Cut off the extra lip from the edge of the PDMS mold. Test the level of it on the table and lightly tap; it should be flush with the table.16.Using a 2.0 mm hole punch, punch out the holes necessary. Lift the PDMS and punch out the PDMS plug from the other side.NOTE: We recommend using a spare piece of PDMS on top of the hole being punched. This prevents the necking-down of the hole described in “® and FEP do not react with epoxy. If other materials outside this protocol are used, make sure they react with epoxy.As noted in the key points, certain materials like TygonThe purpose of inserting the PFTE rod into the PE tubing is to hold the PE tubing in place during the epoxy pouring and curing. It also ensures an open connection between the tubing and the microfeatures in case any epoxy leaks into the hole. In “Be sure to clean the surface being used and the tubing with 70% ethanol before starting.Time: 5 min17.Cut the PFTE rod to 6 mm and the PE tubing to 2 cm.NOTE: Cutting the PFTE tubing at an angle can help guide the tubing during insertion.18.Place the PFTE rod into the PE tubing. The rod does not need to go all the way into the larger tube; 1 mm deep is adequate.19.Place the tubing, rod first, into the holes of the mold in one smooth motion. The tubing should be at least 1–2 mm below the top of the PDMS mold. Do not push the tubing all the way down through the mold. Halfway down or less into the mold is sufficient; the tubing should have about 1–2 mm of space above the mold. See Fig. NOTE: You may leave the petri dish with mold and tubing in a vacuum chamber for about 15 minutes while preparing the epoxy mixture below. In our experience, degassing the PDMS mold before pouring the epoxy may help prevent small bubbles in the final epoxy part.All epoxy work should take place in a well-ventilated area. Read over all safety data sheets before using epoxy. The epoxy used in this protocol has a potting life of 5 h. Epoxy spills can be cleaned with 70% ethanol. If a micropipette is used to pour the epoxy for smaller molds, be sure to use tips with filters to avoid epoxy entering into the micropipette.Time: 10 min20.Measure out the required amount of mixture into a weigh boat. Shake Epoxy mixture bottles well before use. The ratio of Part A to Part B to use is 10 g:3 g.NOTE: The densities of both Parts are different from water. The mass of added parts must be measured directly. Part A is much more viscous than Part B. One strategy for accurate preparation is to pour an approximate amount of Part A directly from the bottle, then calculate the precise amount of Part B to match the 10:3 ratio and add Part B gradually.21.Bring the mixture into a fume hood. Mix very well for 3 min.22.Place the epoxy mixture into the electric ultra-high-vacuum pump to degas the epoxy.23.Watch for air bubbles forming. Bubbles will rise to the surface, combine, and then pop. Turn off machine when the first bubble begins to boil.TROUBLESHOOT: If there are still many bubbles leftover after turning the pump off, let the bubbles sit and pop by themselves for a few minutes before releasing the air. If there are still bubbles in the epoxy after degassing, run the vacuum again.24.Aliquot the epoxy mixture very slowly to the lowest point of the mold. Let the epoxy spread into the mold on its own. Do not let the epoxy overflow or bubble over. It should be leveled with the top of the PDMS mold.® can also be used to absorb excess epoxy by touching the top of the epoxy mold until it becomes level.NOTE: If the mold bubbles over, you can use a pipette to remove excess epoxy. A KimwipeTROUBLESHOOT: Small air bubbles trapped on the PDMS features may be visible. It may help to use a plastic 10 μl pipette tip and gently brush the mold to release bubbles from the mold surface and let them rise to the top of the epoxy. Any bubbles floating on top of the epoxy are acceptable and usually burst during the cure.25.Place the petri dish in the fume hood. Leave any excess epoxy in the fume hood for 24 h before discarding it.26.Leave the epoxy to cure for overnight, typically about 22–24 h depending on ambient temperature and epoxy thickness. As we describe above, devices cured for longer times are generally easier to release from the mold but have lower bond strength.OVERNIGHTVariability in bonding ability is dependent on this section of the procedure. These effects from cure times, plasma treatment time, and resting time on the hot plate are all observed and described in the results. Pay attention to small details such as “feathering”, extra epoxy still attached to the device that may have emerged from plucking out the rod, occurring after the removal of the PFTE rod. Full bonding could be affected by tampering with the bottom of the device after removal from the PDMS mold. Bonding devices with thin walls and other delicate features will require practice.When using the plasma generator, take appropriate safety precautions.Time: 10 min27.Perform quick little bends around the edges of the devices. Partially-cured epoxy should feel firm, rubbery, and tacky. It is visible from under the device if the edges are releasing from the PDMS.TROUBLESHOOT: If the devices are not clearly releasing from the mold at this stage, they are too soft and require more time to cure. Check back in 1 to 2 h and try again.28.Holding onto the tubes, quickly pull the devices straight out of the PDMS. The epoxy part may deform during mold removal, but it will begin to relax back to its molded shape within a few seconds after release.NOTE: Do not turn the rod. With your other hand, you can also try bending the mold back away from the epoxy device. This helps to partially release the device and pull the PDMS walls away as you pull the device out.29.Holding onto the corresponding tube, pluck out the PFTE rods very quickly from underneath using tweezers. Do not touch the bottom of the chip. Adding a small bend to the tube helps, though do not twist the tube. Leave the device in dish on its side once the pieces are out.NOTE: Going slowly can leave a bend in the chip. It is recommended to use the tweezers shown in the “TROUBLESHOOT: For any leftover “feathering”, use the tube recently removed to gently push the feathering back into the hole.30.Bring the hot place to about 70 °C.31.Perform the oxygen plasma treatment of a clean glass slide.NOTE: Plasma treated glass gradually loses its reactivity in atmosphere and should be used as quickly as possible, ideally within minutes.32.Move the glass slide to the hot plate.NOTE: Avoid touching the surface to be bonded.33.Gently drop the device onto the glass slide. Leave on the hot plate for 20–30 s. Tap the top of the epoxy lightly to ensure the device is on the glass and there are no air bubbles. If pressed too hard, channels can collapse. Bonded parts of the device are visible from above.34.Using a light source behind you, check in the reflection of the glass for air bubbles. Parts that are bonded to the glass are darker than the unbonded areas. Most can be taken out by pressing the chip firmly into the glass slide directly on top of the bubble. Beware that thin walls and channels can also easily collapse here if the device is pressed too hard.NOTE: This step is more challenging for high aspect ratio channels and may require practice.35.Let the new epoxy-made microfluidic device sit for 24 h to fully cure before using it for any experiments. Devices can be stored at an ambient temperature while it finishes curing and after completing the full procedure.


Protein arginine methyltransferase 5 (PRMT5) is upregulated in multiple tumors and plays a pivotal role in cancer cell proliferation. However, the role of PRMT5 in colorectal cancer remains poorly understood.We detected the expression level of PRMT5 and glycolytic enzymes using online databases and colorectal cancer cell lines by immunohistochemical staining, quantitative real-time polymerase chain reaction (qRT-PCR), and western blotting. And MTT and colony formation assays were conducted to investigate cell proliferation. Then, we evaluated ECAR and OCR levels using a biological energy analyzer to investigate the energy status of colorectal cancer, and the transcriptional regulation was detected by dual luciferase reporter assay and ChIP assay. Finally, the efficacy of combined treatment of tadalafil and 5-FU was verified.PRMT5 was highly expressed in colorectal cancer tissues compared with their normal counterparts and correlated with poor prognosis in CRC patients. Then, we demonstrated that PRMT5 knockdown or loss of function attenuated the viability of CRC cells, while overexpression of PRMT5 promoted cell proliferation. Mechanistically, PRMT5 enhanced glycolysis through transcriptionally activating LDHA expression. In addition, the PRMT5 inhibitor, tadalafil, rendered CRC cells sensitive to antitumor agent 5-FU in vitro and in vivo.Our data indicates that PRMT5 promoted colorectal cancer proliferation partially through activating glycolysis and may be a potential target for colorectal cancer therapy.The online version contains supplementary material available at 10.1186/s40170-022-00299-4. Colorectal cancer (CRC) is one of the most common tumor types all over the world; despite the decline in CRC incidence, it still ranks the third in cancer-related death , 2. A seRecent studies showed that protein arginine methyltransferases (PRMTs) family played an important role in cancer progression . As a tyIn several decades, the most important theory in cancer metabolism area is the “Warburg effect” , 18, whiIn the present study, we demonstrated that the expression level of PRMT5 was elevated in CRCs, and PRMT5 promoted glycolysis-induced cell proliferation through transcriptionally activating LDHA. Finally, we found that the novel PRMT5 inhibitor tadalafil could enhanced the efficiency of 5-FU in CRC cells. Collectively, the results of our present study uncovered a novel PRMT5 role in colorectal cancer, and targeting PRMT5 might be a strategy for the improvement treatment of colorectal cancer.2. All of the medium was added 10% fetal bovine serum. JNJ-64619178 , tadalafil , 2-deoxy-D-glucose (2-DG) , and oligomycin  were purchased from MedChemExpress.The CRC cell lines used in this study include HCT116, HT29, SW480, and SW620. HCT116 and HT29 cells were cultured in McCoy’s 5A medium, and SW480 and SW620 were cultured in L15 medium supplemented with 10% fetal bovine serum . Cells were incubated at 37 °C, 5% COSurgical resected cancerous colon tissues and their normal counterparts were collected by Xijing Hospital of The Fourth Military Medical University. They were stored in liquid nitrogen. In addition, all these patients did not receive any radio- or chemotherapy before surgery. Then, the total 103 paired tissue samples were positioned in a tissue microarray. Detailed information about this can be seen in a previous work . All theTissue microarrays were stained with primary antibodies against PRMT5 . The Aperio ScanScope XT automated slide scanner was used to scan the tissue microarrays; after that, the expression levels of PRMT5 were scored semiquantitatively based on staining intensity and distribution using the immunoreactive score. Briefly, IHC score = SI (staining intensity) × PP (percentage of positive cells). SI was assigned as 0 = negative; 1 = weak; 2 = moderate; and 3 = strong. PP was defined as 0 = 0–15%; 1 = 15–50%; 2 = 50–75%; and 3 = 75–100%.−ΔΔCt method, and β-actin was used as an internal reference.To detect mRNA expression, total RNA was extracted with TRIzol kit , and the complementary DNA was synthesized with the M-MLV First-Strand Kit (Life Technologies), followed by performing qPCR with the SYBR Green (Solarbio). Primer sequences used in the study were listed in Table S1. Relative expression of mRNA was evaluated by 2Protein extracts were resolved through 10–15% SDS-PAGE, transferred to NC membranes, and probed with primary antibodies. Peroxidase-conjugated anti-mouse or rabbit antibody was used as secondary antibody, and the antigen–antibody reaction was visualized by enhanced chemiluminescence assay. The following commercial antibodies were used: β-actin , PRMT5 , LDHA , H3R8me2s , and H4R3me2s .3 cells/mL) were seeded into 96-well plates and cultured for 24 h, 48 h, or 72 h. The MTT solution  was added to each well, after incubating for 4 h. Then, discarding the culture medium, the purple crystals were dissolved with dimethyl sulfoxide . Next, the absorbance at 562 nm was evaluated by the microplate reader .Cells  were seeded into Seahorse XF24 cell culture microplates. When pre-treated with the PRMT5 inhibitor 0.5 μM JNJ-64619178, 10 μM of tadalafil was added to the corresponding group 12 h before the assay started (or DMSO for control). For the ECAR analysis, glucose, oligomycin, and 2-DG were sequentially injected into each well at the specified time point. At last, the data were analyzed with the Seahorse XF-24 Wave software, and ECAR was presented in mpH/min. Experiments were performed in triplicate for three independent experiments.The ECAR of cells were analyzed using the Seahorse XF Glycolysis Stress Test Kit  on an XF24 Extracellular Flux analyzer (Seahorse Bioscience), and raw ECAR values were normalized to cell numbers. In short, the cells  was used to determine glucose uptake according to the manufacturer’s protocols. In total, 5 × 10To explore PRMT5 effect on the LDHA promoter transcriptional activity, the fragments of LDHA promoter were synthesized and inserted into the pGL3 vectors . The luciferase activity in the lysate of HCT116 cells co-transfected with a luciferase plasmid and pCDH-PRMT5 or pCDH-NC was evaluated by using a FLUOstar device , with the Dual-Luciferase Reporter Assay System (Promega). Transfection efficiency was normalized by dividing the luciferase activity of the construct by the corresponding Renilla luciferase activity.A ChIP assay kit  was used according to the manufacturer’s instructions. Cells cultured in a 10 cm dish were fixed with 4% formaldehyde to cross-link proteins to DNA. Chromatin was extracted from the cells using anti-H3R8me2s , and cross-linked DNA was sheared into 250–500 bp fragments. IgG was used as negative control.3) was estimated by the formula: tumor volume (mm3) = short diameter2 × long diameter/2. Three weeks after inoculation, tumor-bearing mice were sacrificed by cervical dislocation under anesthesia , and the subcutaneous xenograft tumors were excised. Finally, all tumors were kept in formalin for LDHA  staining.To illustrate the effect of PRMT5 on tumor growth in vivo, 6-week-old male nude mice were used in our study. HCT116 cells were suspended in 100 μL PBS and injected into the right flank of mice subcutaneously to establish the colorectal cancer xenograft model. Nude mice were divided into four groups: control group, 5-FU group, tadalafil group, and combination group. 5-FU administration was at a concentration of 20 mg/kg body weight and intraperitoneal injection every other day, while tadalafil was given at 2 mg/kg body weight concentration by daily oral infusion. We monitored the tumor size every 3 days, and tumor volume . We obtained our data from at least three independent tests, and each test was conducted in triplicate. One representative experimental result was selected to display in present study from three or more independent experiments. Data were presented as the mean ± SEM. Unpaired Student’s We firstly analyzed the role of PRMT5 in CRC using TCGA database and two GEO datasets (GSE8671 and GSE32323). We found that PRMT5 expression was higher in colorectal tumors than the adjacent normal counterparts , although we did not detect obvious alteration of cell proliferation by LDHA silencing per se , the cell proliferation showed a significant drop in PRMT5 overexpressing group  treatment . Our datThis study demonstrated the function of PRMT5 in CRC, but there are some limitations. First, we need more clinical samples to verify the expression and prognostic function of PRMT5 in CRC. Second, tadalafil enhanced the effectiveness of 5-FU in CRC cells, but we did not explore how is the function mode. Thus, further studies relating to this are needed.Our work indicates that PRMT5 promotes cell proliferation of CRC by regulating glycolysis. What’s more, blockade of PRMT5 with tadalafil inhibits the growth of CRC cells and enhances 5-FU’s antitumor effect. Therefore, PRMT5 may be a potential target for CRC treatment.Additional file 1.Additional file 2.Additional file 3.


Barrier resistance was performed by immersing the coated steel specimens in salt solution and in a citric acid medium. Adding alumina (Al2O3) particles in micron and nano size to epoxy coatings improved the barrier resistance, tensile, and hardness under dry and wet conditions as compared to pure epoxy coating. Further increases in Al2O3 micro/nanoparticles cause deterioration in tensile strength and barrier resistance. The steel lined with epoxy filled with 1 wt% Al2O3 nanoparticles has a maximum tensile strength of 299.5 MPa and 280.9 MPa under dry and wet conditions, respectively. However, the steel lined with epoxy filled with 1 wt% Al2O3 microparticles has a tensile strength of 296.5 MPa and 275.4 MPa under dry and wet conditions, respectively. Good properties were observed with stepwise graded micro/nanocomposite coatings. The steel lined with epoxy filled with 3 wt% Al2O3 nanoparticles has maximum hardness of 46 HV and 40 HV under dry and wet conditions, respectively.This work studied comparison of the mechanical and barrier resistance properties between different structures of three multilayers polymeric coating on each side of the steel coupons. Epoxy filled with 1 wt%, 2 wt%, and 3 wt% micron or nano-sized alumina (Al Steel has high mechanical strength with low-cost fabrication. Consequently, it is utilized in drilling equipment, shipbuilding, and pipelines. In marines, corrosion results in 30% of the total failure and thus needed to be repaired or replaced parts. In a marine environment, the corrosion of steel is influenced by salinity and alkalinity2. Subsequently, the coating was performed on steel faces to avoid the corrosion of new or existing steel construction. The corrosion of steel attracted many research interests as it is costly, particularly in the oilfield and marine environments3. Recently, polymer composite liners to steel were used to decrease the diffusion of oxygen and moisture. The protective organic coating as an epoxy coating to metal is characterized by its excellent weather ability4. Protected epoxy coating has attracted great attention in wet environments due to its very good toughness, durability, and adhesion to metal substrates1. However, the highly cross-linking density and the barrier behavior of the epoxy coating can be undesirably affected when exposed to corrosion. The polymer coating weakening results in the creation of holes, and defects in the epoxy coating surface. During exposure to corrosive media, holes and defects become larger in width and depth. Holes are considered as conductive paths as the electrolyte diffuses in the polymeric coating5. Moreover, the protective coating fails by the cause of delamination which is the separation at the polymeric coating/metal interface6. The deterioration of polymeric coating decreases the barrier properties thus the mechanical properties of the polymeric coating5. Therefore, it is essential to enhance the properties of epoxy resin by replacing epoxy with epoxy composite coatings to achieve the requirements of real applications4.The corrosion of metal is considered one of the vital issues for steel structures when these structures are subjected to corrosion2. Nanoparticles are considered a good water barrier and thus effectively obstruct water absorption improving the service life of metals2. Different nanomaterials are involved at various levels in the food industry having both positive and negative effects on human health. Alumina can also be present due to contamination or migration from other food contact materials such as processing machinery, utensils, and devices7. The coatings containing Al2O3 particles showed enhancement in scratch and abrasive resistance compared with that of the polymer coating. This enhancement in scratch and abrasive resistance is attributed to the dispersion hardening of Al2O3 nanoparticles in polymer coatings8. Enhancement in environmental impact can be attained by utilizing nanosized particulates in polymeric coating and eliminating the requirement for toxic solvents9. Nanoparticles embedded in polymeric coatings are well known for their outstanding physical, mechanical, and thermal properties11.Embedded inorganic fillers to epoxy coating are one of the methods to enhance the anti-corrosion characterization of organic polymeric coatings. Adding smaller filler particles in micron or nano size may improve the barrier properties of the introduced polymeric coating. Size, morphology, shape, and the weight percentage of the fillers greatly affect the intrinsic characteristics of composite5 investigated the corrosion resistance of the epoxy coating containing micron and nano-sized ZnO fillers. The specimens were submerged in 3.5 wt% NaCl solution. The corrosion resistance of the coupons was significantly decreased after immersion for 15 days. The corrosion resistance of the epoxy coating was enhanced as reinforced with nano-sized ZnO fillers. The results showed that the lowest reduction in cross-linking density and reducing the hardness of the polymeric coating submerged in 3.5 wt% NaCl solution was attained as the epoxy coating was reinforced with the 3.5 wt% nano ZnO particles. Moreover, the adhesion was also increased at 3.5 wt%. Furthermore, Anaki and Xavier1 studied the dispersibility of reinforcing epoxy coating on mild steel with 2 wt% of nano Al2O3. The resultant specimens have been submerged in a 3.5% NaCl solution. The improved anticorrosion performance was conducted by the modified nanocomposite coating as compared to epoxy coating. The reinforced epoxy coating resulted in good adhesive strength, increasing in hardness, tensile strength, and better corrosion resistance than epoxy coating. In addition, Golru et al.12 prepared epoxy/polyamide reinforced with 1, 2.5 and 3.5 wt% nano-alumina filler coted AA1050 substrate. The results showed that the nanofillers dispersed uniformly in the polymeric coating even when loading at high percentages. The polymeric coating corrosion resistance was more improved by increasing the weight percentage nanofillers.Ramezanzadeh and Attar4. Al2O3 filler in micron size is commercially available and has a lower cost than Al2O3 in nano-size. So, the objective of the study is to develop multilayers of epoxy liners to steel filled with micro and nano Al2O3 particles with different percentages and differentiate between them. Three percentages of alumina micro and nanoparticles  were introduced to epoxy with different configurations. The specimens were immersed in salt solution and in citric acid media. Barrier resistance and mechanical properties were investigated under dry and wet conditions.In recent times, multilayered nanocomposites have gained great attention due to their required characteristics as microwave absorbing, mechanical properties, permittivity constructed on the interfaces between adjacent layers, and the synergistic impacts of fillers. Nevertheless, the application of multilayered micro/nanocomposite coatings has not been reported yet2O3 fillers in micron and nano size with a purity of about 99%. The size of microparticles and nanoparticles are 90 µm and 70 nm, respectively.Mild steel was used as a metal substrate supplied from Al Ezz-Dekheila Steel Company Alexandria. The steel sheets were cut to the required dimensions of the specimens by a laser machine. The specimens were polished in order to roughen the surface of the steel substrate. After polishing, the top and bottom side of the coupon's surface was cleaned with acetone before coating. Chemicals including sodium hydroxide, citric acid, and acetone were supplied by El Nasr Pharmaceutical Chemicals, Egypt. The coating is Epoxy resin (Kemapoxy RGL150) which is supplied by CNB Company, Egypt. The reinforcements are Al14. For epoxy resin protection from degradation, the mixture was cooled by placing it on an ice water bath during sonication15. Afterward, the blend and the hardener were mixed with the recommended ratio at a temperature of 25 °C. The protective layer was prepared on steel by a metallic roller to remove excess resin and reduce void content and any entrapped air bubbles. Painting on one side of the steel specimen is left for 24 h to cure. Subsequently, the second layer on the same side was constructed and left for a day to cure. and the same with the third layer. The same technique was done for the other three layers on the bottom face of the coupons. The final stepwise graded and non-graded micro/nanocomposite coatings on steel substrate were constructed as illustrated in Fig. Epoxy protective films were made by adding hardener carefully to the epoxy and blended thoroughly with a ratio of 1:2 by mass of epoxy resin. The micro/nanocomposite protective films were performed as micro, or nanoparticles were added to the epoxy resin by sonication process. The sonication was performed with Hielscher ultrasonic processor UP 200 S. The sonication was conducted at 0.5 cycles per second on/off with an amplitude of 40% for 2 h as recommended byThe tensile behavior of the steel coated with micro/nanocomposites coupons were tested according to ASTM D3039. The tensile test was achieved with a computerized universal testing machine (Jinan Test Machine WDW 100 kN). The cross-head speed was set at 2 mm/min and the stress–strain curve was recorded by a computer data acquisition system. All tests were performed at ambient temperature.The hardness was determined via the PCE-1000N Hardness instrument measured at ten different places of the micro/nanocomposite coated steel and the average value was taken.−4 g to monitor the weight change during the absorption process. The solution content M(t) absorbed by micro/nanocomposite protective coating was then calculated as the mass gain percentage referring to its initial weight (w0) as follows16:wt is the coupon mass after time t. Coated coupons were immersed up to 21 days.Some test coupons were immersed in salt solution and in citric acid solution to estimate the corrosion media of the steel lined with micro/nanocomposite. The salt solution was performed as 3.5% NaCl dissolved in water. Citric acid solution with a concentration of 2 N was prepared with double distilled water. Uptake tests were performed according to ASTM D5229/D5229M-14. The coated coupons were periodically withdrawn from the solutions, wiped dry, and weighed using an analytical balance of accuracy up to 102O3 microparticles and Al2O3 nanoparticles under dry and wet condition, respectively. In wet conditions, the coated steel specimens were immersed in a salt solution for 35 days. The improvement in hardness in dry and wet conditions was attained in both sizes of Al2O3 particles as compared to pure epoxy coating. Moreover, as the weight percentage of the micron and nanoparticles increased, the hardness increased. This increase in hardness is due to increasing the Al2O3 particles content up to 3 wt% on the surface of coated steel specimens may be attributed to the high value of the hardness of ceramic particles as Al2O3 particles as compared to the hardness of the polymer. Furthermore, during measuring with the hardness indenter, the force results in the load applied increasing which in turn presses the epoxy causing the particles to touch each other giving more resistance to the force applied. As the weight percent of Al2O3 particles content increases, micro/nanoparticles fill in the gaps presented in the polymeric matrix as cracks and voids thus increasing hardness18. In addition, the hardness of the stepwise graded micro/nanocomposite coating gives high hardness as compared to composite liners filled with 1 wt% and 2 wt% micro/nano Al2O3 particles. This may be attributed to the higher percentage of micro/nano Al2O3 particles (3 wt%) on the outer surface of coated steel specimens, followed by 2 wt% Al2O3 particles then 1 wt% Al2O3 particles.Figure 2O3 particles and epoxy got weakened. In addition, the epoxy got swelled due to the water absorption hence generating pressure to Al2O3 particles that results in pulling out of the particles from the epoxy forming micro-cracks inside the coated specimen. This reduces the hardness of the specimens in wet conditions as compared to the hardness of equivalent specimens in dry conditions19.It can be depicted from Fig. 2O3 particles either in dry or wet conditions. Followed by stepwise graded nanocomposite coating. This indicates the high effect of Al2O3 nanometer-sized particles in strengthening the epoxy matrix. It is attributed to the great surface area of Al2O3 nanoparticles compared to Al2O3 microparticles20. The specimen N3 exhibited the highest hardness value with an improvement of 48.4% and 90.48% in dry and wet conditions, respectively.Figure 2O3 microparticles and Al2O3 nanoparticles under dry and wet conditions is shown in Fig. 2O3 micro/nanoparticles and the epoxy matrix. Water that was introduced inside the layers of the coatings caused the interface to be damaged and thus polymer matrix cracked resulting in degrading the mechanical properties of the polymeric composite coating21. The debonding between coating layers and at the particle/matrix interface affected the stress transfer and thus the reinforcing effect of micro/nano Al2O3 particles on the epoxy matrix22. As the weight percentage of the micron and nano Al2O3 particles increased, the tensile strength decreased. Adding 1 wt% micro/nano Al2O3 particles gives the maximum enhancement of tensile strength in dry and wet conditions as compared to pure epoxy coating. Incorporation of small weight percentages of fillers leads to substantial improvement in mechanical properties of polymeric composite23. From Fig. 29.The tensile strength of multilayer epoxy coating to steel substrate filled with Al24. The specimen N1 exhibited the maximum improvement in tensile strength under dry and wet conditions, respectively. The least improvement was obtained with specimen M3. Both the stepwise graded steel lined with micro/nanocomposite are close to specimens coated with epoxy filled with 1 wt% Al2O3 micro/nanoparticles.Figure 30.The mechanical properties of polymeric coating filled with particles depend on the filler size, filler/polymeric matrix interface adhesion, and filler content. For a given filler content, the polymeric composite strengths are enhanced by decreasing the filler size. Nanocomposite coatings such as N1, N2, N3, and N123 have a high total surface area than microcomposite coatings such as M1, M2, M3, and M123. Hence, the hardness and tensile strength are enhanced by increasing the total surface area of the reinforced particles with a further effective stress transfer mechanism. The filler/matrix interface's adhesion strengths control the load transfer between the constituents. Effective stress transfer is considered the most essential factor that contributes to the strength of the two constituents of the polymeric composite materials. For weak bonded fillers, the stress transfer at the filler/polymer interface is ineffective. Discontinuities occurred in the form of debonding because of poor adherence of filler to the polymeric matrix. Consequently, the filler cannot carry any load thus the polymeric composite strength reduces with increasing filler content. Nevertheless, for polymeric composite reinforced with well-bonded fillers, the addition of fillers to a polymeric matrix resulted in an increase in mechanical properties mainly for nanofillers with high surface areas2O3 microparticles and nanoparticles. When adding the Al2O3 micro/nanoparticles to the epoxy, the tensile strain improved as compared to pure epoxy under dry and wet conditions. As the weight percentage of Al2O3 fillers increases, the tensile strain increases. Immersing the specimens in a salt solution leads to increasing the tensile strain of both sizes of Al2O3 particles as compared to pure epoxy coating. The ductility for both unfilled epoxy and Al2O3 filled micro/nanocomposite was enhanced as a result of water absorption. This can be attributed to the plasticization effect of water as the immersion time increased which can improve the ductility of the epoxy resin33. Figure Figure 2O3 nanoparticles is the best attractive issue accordingly developing a large interface in a polymeric composite coating37. With adding 3 wt% Al2O3 nanoparticles to epoxy coating, the amount of Al2O3 nanoparticles is very high resulting in particle-to-particle interaction rather than the particle-to-epoxy interaction. Consequently, the Al2O3 particles begin to aggregate and form clusters that influence the Van der Waals interaction between the polymeric matrix chains reducing the cross-linking and increasing the void content. So, the resulting mechanical properties are therefore degraded35.The high surface area of Al2O3 micro/nanoparticles to the epoxy matrix led to the development of a good filler/polymeric matrix interface hence reducing the delamination between coating layers and consequently increasing the mechanical properties20.The fracture occurred in the steel coated with multilayers of filled epoxy is accompanied by a delamination between coating layers, matrix cracking and delamination between steel and coated layers. Delamination occurred at the interface between adjacent layers. Delamination occupies the most failure in polymeric composites as subjected to different kinds of testing. As the specimens loaded, further growth of interlayer delamination leads to final failure. Delamination is formed from interlaminar stresses being developed at the interfaces between adjacent layers. The fracture of filler/polymeric matrix interface stresses formed layer cracks that subsequently act as initiation places for delamination. Good interfacial bonding occurred between polymeric composite coating layers hindering the formation of delamination. Adding Al2O3 micro/nanoparticles immersed in salt solution and citric acid for 35 days. It is clear from the figures that the uptake% is significantly higher than that of seawater for both Al2O3 micro and nanocomposite coatings.Figure 2O3 micro/nanoparticles. The uptake for both solutions was decreased with decreasing the size of the Al2O3 particles. The rate of water absorption increases with the increase in Al2O3 micro/nanoparticles content. This may be attributed to the increase in voids that formed with the presence of high content of Al2O3 micro/nanoparticles content and also due to the poor Al2O3/epoxy adhesion resulted in microcrack formation due to Al2O3 micro/nanoparticles agglomeration formed in the polymeric matrix. Furthermore, under the effect of seawater, the Al2O3 micro/nanoparticles tend to leave their places forming voids that are filled with seawater due to the capillary effect37. The decrease in water uptake rate is higher for epoxy coating filled with Al2O3 nanoparticles. This may be attributed to the good barrier properties of Al2O3 nanoparticles that form tortuous paths that hindered the seawater movement so reducing the water absorption rate38. Improved corrosion and mechanical properties were observed by filling the cracks presented in epoxy coats. The nanoparticles act as a strong barrier that can avoid penetration of aggressive ions to the steel surface2. Consequently, nanoparticles with very fine grain size and high boundary volume offer enhanced barrier properties as compared to conventional fillers9.Figure 2O3 nanoparticles. This can be attributed to the good dispersion of low weight percentage (1 wt%) of Al2O3 nanoparticles as shown in Fig. 39. The inclusion of small weight percentages of nanofillers indicated substantial improvement in properties40.The least uptake % was attained with steel coated with 1 wt% Al2O3 micro/nanoparticles to the epoxy, the uptake percentage increased. This may be attributed to the presence of agglomeration caused by the addition of more Al2O3 particles to the epoxy that helps in more absorption of water as shown in Fig. 2O3 particles and epoxy resin, the more water permeable for the micro/nanocomposites are and the worse barrier properties can be attained. Furthermore, extra free volume presented at the interface also assisted the water permeability inside micro/nanocomposites. Seawater is more likely to diffuse along the epoxy/Al2O3 interface and destroy the interfacial bonding rather than diffuse through the epoxy matrix. So, as the free volume increased, water permeability increased41. Aggregation possesses rise to lower surface interactions of Al2O3-epoxy and higher stress concentration. This lead to lower the mechanical and barrier properties of the composites filled with nanofillers. However, a smaller aggregate size resulted in highly improved mechanical properties42. The uniform dispersion of Al2O3 nanoparticles verified more surface area of nanoparticles in the epoxy matrix. This increases the exposed surface area of Al2O3 nanoparticles to epoxy molecules resulting in cross-linking between Al2O3 nanoparticles and epoxy coating. This cross-linking enabled transferring the stress from epoxy to Al2O3 nanoparticles. The high strength of Al2O3 nanoparticles made them effective to carry extra loads when introduced to the polymeric matrix43.It is observed that with a further increase in Al2O3 nanoparticles in the composites, surface analysis of the coated specimen N123 was done by FESEM and the composition scanning (EDX) images shown in Fig. The stepwise graded nanocomposite coating (N123) possess good barrier and mechanical properties. In order to determine the distribution of Al46. Epoxy is considered to be the most conventional and superior coating because of its good adhesion properties, excellent scratch hardness, etc.27. Nevertheless, epoxy coatings may fail under severe environmental conditions for prolonged exposure28. Poor adhesions of the coating can cause not only delamination of the coating layers but also the corrosion of the steel beneath the polymeric coating48. The alumina thin films coating have high mechanical properties and corrosion resistance, so, they have been applied in many industrial fields such as gas diffusion barriers, surface passivation, antireflection layers, etc.49. Producing protective coating with the inclusion of Al2O3 micro/nanoparticles into epoxy coating could have a great potential for commercial applications using metallic surfaces50. Nanocoatings to metallic surfaces can be used in equipment design thus lowering the maintenance and the working cost51.Economic losses due to metallic corrosion reached billions of dollars per year worldwide2O3 particles in micron and nano size were investigated. The results showed that barrier resistance against salt and citric acid media were significantly enhanced by adding either nano or micron-sized Al2O3 particles to epoxy coatings. Nanocomposite coating has higher mechanical and barrier properties than microcomposite coatings. A maximum improvement of 48.4% and 90.48% was attained with epoxy liner filled with 3 wt% Al2O3 under dry and wet conditions, respectively. However, a maximum enhancement in tensile strength of 6.92% and 4.33% was obtained with epoxy liner filled with 1 wt% Al2O3 nanoparticles under dry and seawater conditions, respectively. With further increase in Al2O3 micro/nanoparticles to the epoxy liner, the uptake percentage of salt and citric acid solution increased.In this study, the tensile, hardne+ss, and barrier properties of steel lined with multilayered epoxy filled with Al


Drug delivery through the skin has immense advantages compared to other routes of administration and offers an optimal way to treat inflammatory skin diseases, where corticosteroids are the cornerstone of topical therapy. Still, their therapeutic efficiency is limited due to inadequate skin permeability, potential side effects, and reduced patient compliance. To overcome these drawbacks, we propose a drug delivery system consisting of dexamethasone (DEX)-loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) incorporated in sodium alginate (SA) microneedles (MNs) as a minimally invasive dosage form for controlled drug release. Drug-loaded PLGA NPs were prepared by a nanoprecipitation method with a high encapsulation yield. They exhibited a controlled release pattern over 120 h. A modified vacuum-deposition micromolding method was used to load the obtained DEX-NPs into the tips of dissolving MNs. The NP-MNs showed improved insertion capabilities into the skin-simulant parafilm model and enhanced mechanical strength when tested against different static forces compared to their counterparts (SA-MNs). The results of an MN dissolution study following application to ex vivo chicken skin and agarose gel indicate that the NP-loaded segments of MNs dissolve within 15 s, in which the NPs are released into the skin. Taken together, the incorporation of DEX-NPs into SA-MNs could be a promising approach to bypass the limitations of conventional topical treatment of skin diseases, allowing for self-administration, increased patient compliance, and controlled drug release. The use of microneedles (MNs) is an innovative and minimally invasive approach that has been developed in the last two decades to deliver active pharmaceutical substances through the skin by overcoming the main skin barrier, the stratum corneum (SC). MNs are micron-sized structures in the height range of 50 to 1500 μm allowing for painless penetration into the skin via the SC, viable epidermis, and upper dermis without contacting nerve fibers or blood vessels. Drug-loaded MNs create micron-sized pores in the skin to enhance drug delivery to the different layers of the skin for local or systemic treatment. Thus, numerous factors should be taken into account when designing MN delivery, such as geometry, needle height, thickness and tip radius, base diameter, needle density, and MN material. Compared to traditional transdermal delivery systems, MNs possess several advantages, including enhanced bioavailability and drug permeation, self-administration, and improved patient compliance .Different types of MNs were developed, such as solid, hollow, coated, dissolving, and hydrogel-forming MNs, which differ in their delivery strategy, fabrication methods, geometry, and the materials used in their manufacturing. Several constituent materials, such as metals, ceramics, sugars, silicon, and biodegradable polymers, are widely used to fabricate MNs due to their advantages, including low cost, simple manufacturing, biocompatibility, stability, folding ability, and favorable drug loading capacity ,4,5.Dissolving MNs can encapsulate therapeutic agents and release their content once inserted into the skin. Sodium alginate (SA) is widely used in the pharmaceutical and biomedical fields for the fabrication of wound dressings, drug encapsulation, and coating due to its unique properties, such as biocompatibility, biodegradability, and low cost . The chePLGA is one of the most frequently used polymers for drug delivery due to its FDA approval, biodegradability, and biocompatibility ,9. NanopSince MNs can penetrate the skin easily and provide a site-specific delivery in a minimally invasive manner, they can serve as a vehicle for the treatment of several skin diseases, such as psoriasis, dermatitis, eczema, acne, and skin cancer . TopicalTherefore, a nano-delivery system may be advantageous by facilitating direct drug delivery at the site of interest and the release of the drug in a sustained manner. Hence, the purpose of this study is to achieve controlled drug release by the incorporation of a model drug, dexamethasone, into PLGA nanoparticles and embedding it in rapidly dissolving microneedles made of sodium alginate (NP-MNs) for improved treatment of skin diseases, in terms of both therapeutic effect and patient compliance. Blank sodium alginate microneedles (SA-MNs) were prepared and used as controls.Dexamethasone (Alfa Aesar) and sodium alginate  were purchased from Holland-Moran Inc. Israel. PLGA-Purasorb PDLG 5010 (50:50) was donated by Corbion Purac . Solutol HS 15 was supplied by BASF . Silicone MPatch microneedle templates were purchased from Micropoint Technologies Pte Ltd.  and were pyramidal in shape with a dimension of 10 × 10 needle array, 200 µm base, 500 µm height, and 500 µm pitch. Phosphate buffer saline (PBS) was purchased from Hyclone Laboratories. Organic solvents were obtained from Sigma-Aldrich .w/v) solutol HS 15, which was continuously stirred at room temperature  for 24 h to evaporate the organic solvent, followed by adjustment of the formulation volume to 2 mL by distilled water. The vials were wrapped with aluminum foil to avoid drug degradation due to light exposure. Blank PLGA NPs were prepared using the same method but without adding DEX at any stage of the preparation.DEX-loaded NPs were prepared using the nanoprecipitation method with modification ,18. Briev/v) with water and added to folded capillary zeta cells (DTS1070) to analyze at a 90° angle at room temperature (25 °C). For each sample, the mean value ± s.d. of three determinations was established.Particle size (hydrodynamic diameter), polydispersity index (PDI), and zeta potential were determined by the dynamic light scattering (DLS) method using a Malvern ZetaSizer nano-ZS laser particle size distribution analyzer . Samples were diluted at 1:100 (® Ultra-15 (molecular weight cut-off 100 kDa) centrifugal filter unit . The formulation samples were added to the upper chamber of the Amicon® tube and then washed with an equal volume of distilled water 3 times by centrifugation at 3000 rpm for 1 min each. Finally, the amount of free DEX was determined from the filtrate using a UV-Vis spectrophotometer  at a wavelength of 242 nm [Encapsulation efficiency (EE) and drug loading content (DLC) were determined by first separation of the unloaded drug (free DEX) by filtration/centrifugation using an Amiconf 242 nm . The con® tube were centrifuged at 3000 rpm for 1 min to remove the polymeric debris, and polymeric debris was analyzed to ensure that no drug residues were present.Afterward, the nanoparticles from the upper chamber of the Amicon−1, and the scan range was between 4000 and 650 cm−1.ATR-FTIR spectra were measured by a PerkinElmer Spectrum 100S spectrometer equipped with a universal ATR sampling accessory. For each spectrum, 4 scans were collected with a resolution of 2 cmThe thermal behavior of DEX-loaded nanoparticles, PLGA, dexamethasone, and the physical mixture was analyzed by a DSC 1 Star System equipped with Star Software  and a DSC131 Evo . Weighed samples of 6–11 mg were placed into 100 μL aluminum crucibles and the samples were scanned from 25 °C to 300 °C at a constant heating rate of 20 °C/min.X-ray powder diffraction measurements were performed on the D8 Advance diffractometer with a LYNXEYE-XE-T detector  operating in 1D mode. Low-background quartz sample holders were carefully filled with the powder samples. XRD patterns within the range 2° to 75° 2θ were recorded at room temperature using CuKα radiation (λ = 1.5418 Å) with the following measurement conditions: tube voltage of 40 kV, tube current of 40 mA, step-scan mode with a step size of 0.02° 2θ, and counting time of 0.5 s/step.w/v SA solution was injected under vacuum through the septum into the micromold surface, held for 10 min, and allowed to dry at 37 °C for 1 h. Afterward, 100 µL of 4% w/v SA solution was added to form the base of MNs and dried at 37 °C for 1.5 h before being demolded. SA-MNs were fabricated by the same method as NP-MNs without adding NPs at any stage of the preparation. The MNs were characterized as depicted in NP-MNs were prepared using a vacuum-deposition micromolding method  with a mMorphological evaluation of NP-MNs and SA-MNs was performed by a scanning electron microscope .SA-MNs and NP-MNs were manually pushed into 8 stacked parafilm layers as a skin stimulant for 30 s and observed under a stereomicroscope  to study the insertion efficiency and MN insertion depth . The insThe mechanical strength of MNs against static forces was measured by placing different weights against the MNs. Briefly, SA-MNs and NP-MNs were placed upward, onto which weights of 50 g, 500 g, and 1000 g were placed gently on the top of each patch, respectively. After 5 min, the weights were removed, and morphological changes were evaluated by a HAYEAR 4K UHD microscope camera. The rate of change of the height (µm) of the MNs was calculated and plotted as the percentage of reduction in MN height ,24.w/v) served as a skin simulant. A weight of 500 g was placed above the arrays, and MNs were removed after predetermined times of 15, 45, and 120 s. The MNs were imaged before and after dissolution using a microscope camera [The MN arrays were applied on ex vivo chicken skin obtained from a local slaughterhouse, and agarose gel 3% (e camera .DEX NPs samples (200 µL) were placed into dialysis bags with a molecular cut-off of 8000 Da, soaked in 10 mL of PBS pH 7.4, and maintained at 37 °C (50 rpm) in a rotary incubator. At predetermined time intervals, 0.5 mL of the release medium was sampled and was replenished immediately with the same volume of fresh prewarmed PBS (37 °C), maintaining sink condition throughout the experiment. Then, DEX concentration was determined using a UV–Vis spectrophotometer at a wavelength of 242 nm . The conIn this study, DEX-loaded PLGA nanoparticles were prepared by nanoprecipitation with a mean particle size of 93.7 nm, narrow size distribution (PDI < 0.3), and negative zeta potential values. DEX was successfully incorporated into the PLGA NPs, exhibiting an encapsulation efficiency of 80% and adequate drug loading content of 9.1% . Previou−1, in agreement with previous reports [−1 due to the ester group [−1 that corresponds to the axial deformation of the C-F group in DEX. The spectrum of the DEX-NPs evidenced the contribution of the organic groups of PLGA and solutol HS 15 and did not fully demonstrate the bands equivalent to DEX. This was attributed to the superposition of the bands from all the NP components and could be further explained by the loading content of DEX as well as the relatively low peak intensity. The spectra of blank-NPs and DEX-NPs showed a peak at 1735 cm−1, which was ascribed to the used surfactant, solutol HS 15  was chosen from preliminary tests for the fabrication of dissolving MNs. In the case of NP-MNs, the obtained nanoparticles were encapsulated into the tips of SA MNs, being concentrated at the tips rather than being dispersed in the base layer to provide more efficient delivery by decreasing drug wastage [Sodium alginate (SA) is one of the most widely used materials in the biomedical field ,39. SA a wastage ,41. The When characterized by SEM, the MN formulations had a quadrangular pyramidal shape and were uniformly distributed on the substrate . SA-MNs To evaluate the insertion properties of MNs and to analyze MN insertion depth, commercial parafilm was used as a skin simulant for preliminary assessment instead of biological tissues . In thisTo further shed light on the mechanical strength of the MNs, the morphological changes of MN tips against static force were microscopically observed by placing different weights on the top of the MN arrays for 5 min, and the height reduction percentages were calculated. As shown in The dissolving capability of the polymeric tips affects the release rate of NPs from MNs. Herein, ex vivo chicken skin, frequently used for the characterization of microneedle arrays ,44,45, aDue to the high-water solubility of SA, the MNs could dissolve very quickly and thoroughly. The presence of a surfactant plays a vital role in formulating dissolving MNs . It is pDespite the significant value of these skin-simulant models for the characterization of MNs, there are some limitations to be considered, such as the lack of pharmacopeial standards for MN-based products , the absBased on the dissolution study findings, in vitro release studies of DEX from PLGA NPs were performed using a dialysis bag in phosphate buffer saline (pH 7.4) at 37 °C for 120 h. The release data presented in The main challenges of the current topical treatment modalities for inflammatory skin diseases, such as psoriasis and atopic dermatitis, are the lack of selectivity in delivering the drug to the inflammation site, side effects associated with steroids, inadequate skin penetration, and patient adherence ,57. NanoRecent studies have highlighted the beneficial use of polymeric nanoparticles for anti-inflammatory topical treatment due to their ability to form a drug reservoir, retaining the drug locally at the site of action ,61. It wThis suggests that the delivery system proposed in this study, based on the combination of minimally invasive dissolving MNs with PLGA NPs, can be foreseen as an interesting strategy to treat various inflammatory skin diseases by means of increased patient compliance, controlled drug release, improved safety, and efficacy of topical steroid therapy.The work presented demonstrates a useful step-by-step strategy, based on a modified vacuum-deposition micromolding method to fabricate rapidly dissolving MNs loaded with biodegradable NPs for the treatment of skin diseases. DEX-loaded PLGA NPs were successfully prepared, with acceptable drug loading content exhibiting a controlled release profile. The incorporation of DEX-NPs into the tips of dissolving MNs, made of SA, significantly improves their mechanical strength and insertion capabilities, as demonstrated by the skin-simulant parafilm model and static force tests. The MN dissolution study following application to ex vivo chicken skin and skin mimicking agarose gel reveals that the NP-loaded segments of MNs have been dissolved within 15 s. Taken together, the combination of dissolving MNs and biodegradable NPs offers an interesting minimally invasive approach to bypass the limitations of conventional topical treatment of skin diseases, allowing for self-administration, increased patient compliance, and controlled drug release. Thus, the potential of this delivery system warrants further ex vivo and in vivo investigations.