Microneedle (MNs) technology is a recent advancement in biomedical science across the globe. The current limitations of drug delivery, like poor absorption, low bioavailability, inadequate skin permeation, and poor biodistribution, can be overcome by MN-based drug delivery. Nanotechnology made significant changes in fabrication techniques for microneedles (MNs) and design shifted from conventional to novel, using various types of natural and synthetic materials and their combinations. Nowadays, MNs technology has gained popularity worldwide in biomedical research and drug delivery technology due to its multifaceted and broad-spectrum applications. This review broadly discusses MN’s types, fabrication methods, composition, characterization, applications, recent advancements, and global intellectual scenarios. Oral delivery is the most accepted route of administration for the treatment of disease, diagnosis, treatment, and the most widely studied topic by formulation scientists during the first and second generations of drug delivery. Oral drug delivery has the disadvantages of systemic metabolism, poor absorption, and a lack of tissue selectivity, leading to decreased therapeutic effects . This neMNs are micro projections ranging 25–2500 μm in height available in different shapes with the attachment of a base for support. MNs are used to sample fluid from the body and deliver therapeutic agents to cells . MNs areIn this present article, we review various types of MNs, different materials used for fabrication, properties of the MNs, characterization of MNs for biological and mechanical properties, toxicity assessment of MNs with various in vivo and in vitro methods, application of MNs in several diseases, as well as the regulatory aspect, marketed product, and patent of the MNs.Several types of MNs are majorly categorized based on the fabrication method and applications of MNs ,11. EachThe drug delivery from solid MNs is based on the “poke-and-patch” approach. The drug delivery mechanism is the disruption of the stratum corneum and the creation of microchannels by the solid MNs. After the formation of microchannels, the drug patch is applied to the skin, through which the drug is efficiently diffused through the microchannels into the skin .The solid MNs were used to transfer the drug through micronized channels formed inside the skin layer and improved drug diffusion ,16. In s“Poke-and-release” is the drug delivery approach seen with dissolving MNs. In this approach, the drug is encapsulated within MNs. After insertion into the skin, these MNs are retained in the skin and not removed. The encapsulated drug is released when these MNs are degraded within the skin . The disFurthermore, these MNs do not leave any biologically harmful waste behind after dissolution, so the drug delivery is safe. The first research work on dissolving MNs and their utility were reported by Miyano et al. in 2015 as a pioneering study within this field ,31. The The literature has reported various examples regarding dissolving MNs for its synergistic drug delivery system and other techniques ,33. The The drug delivery through the coated MNs is by a “coat-and-poke approach”. In this approach, the drug coating is applied to the MNs, and then these MNs are inserted into the skin. The drug coating present on inserted MNs gets dissolved into the skin, and after the dissolution of the drug, the MNs are removed. The advantage of this approach is that it only requires one step and has simple delivery, while a disadvantage is that a much smaller amount of the drug is delivered by this technique . Coated Hydrogel forming MNs fabricated with cross-linking polymers. The drug release approach of hydrogel-forming MNs is “poke-and-release”. The factors affecting MNs fabrication for solution parameters include a swelling index, molecular weight, and concentration of the foaming agent. This strategy was first established by Donnelly et al. for highly swellable polymers . IontophThis type of MNs can overcome the pitfalls of the conventional microarray technique by reducing drug loading capacity and modifying release . These hThe drug delivery approach used in Hollow MN is the “poke-and-flow”. The drug delivery from Hollow MNs is similar to the hypodermic injection. The micropump is generally used to execute them under pressure drug delivery into the skin. The advantage of hollow MN is the fast drug delivery as compared to other approaches as the drug delivery is pressure-driven. Another advantage of this technology is the painless and precisely controlled drug delivery into the skin . The HolVarious materials have fabricated MNs like silicon, zeolite, glass, metals, polymers, and sugars. After more continuous research, the fabrication complexity gets reduced and improves their mechanical strength, geometrical shapes, and sharpness of needles. MNs can be divided into two categories based on their fabrication material: biodegradable and non-biodegradable MNs. Silicon was the first fabrication material for MNs; author Henry et al. reported the first silicon MN in 1998. The specific reason behind silicon MNs fabrication is that silicon MNs were precise, 3D structures and widely used for target drug delivery. Still, there is a high risk of breaking the needle during insertion into the skin due to its brittleness .https://www.ncbi.nlm.nih.gov/pubmed accessed on 30 March 2022. Some of the keywords utilized are MNs of metal glass, ceramic, silicon, and polymer [The percentage of MNs was published in various materials. Based on hits for various keywords, percentages were determined. Best Match sorted search results at  polymer .The initial metal utilized in the fabrication of MN arrays was stainless steel. Metal MNs are designed by physically pushing the smallest size of stainless steel hypodermic needles through a predetermined thickness supporting material or laser cutting metal sheets into MN forms and twisting them out of the plane . This tyPolymers are the most promising material for MNs fabrication compared to metals and inorganic materials. The polymeric material has been used to make solid, coated and hollow MNs . The polThe variety of polymers used for the fabrication of MNs are polylactide-co-glycolide acid (PLGA) , poly-L-The polymeric biodegradable MNs are made up of natural and synthetic polymers to improve drug delivery at the targeted site with a prolonged or sustainable release. The synthetic materials are mainly Polyglycolic acid (PGA), polylactic acid (PLA), polycarbonate and their copolymers (poly(lactic-co-glycolic acid) (PLGA), polycaprolactone (PCL), polystyrene (PS)), and silk, chitin, chitosan are natural biodegradable polymers used for the fabrication of MNs, which dissolves or degrades into the body by a metabolic process without producing any poisonous side effects . Some otThe fabrication of MNs using biodegradable polymers by a molding method is a novel approach that provides inexpensive and robust mass production. The polyglycolic acid (PGA), Poly-L-lactic acid (PLA) and their co-polymers polylactide-co-glycolide acid (PLGA), polycaprolactone (PCL) are used for the manufacture of biodegradable MNs as this polymer has biocompatibility, is mechanically robust, cost-effective, and resorbable. Eventually, biodegradable MNs break when poked into the skin, which has additional safety concerns as it degrades and is transported into the skin. Biodegradable polymers have minimal severe side effects. The drug can be encapsulated into MNs and then inserted into the dermal layer; as the polymer degrades or dissolves simultaneously, the drug gets released into the skin. This method does not produce any biological hazard like sharp tips waste. This polymer material can process at a low melting temperature, so the micro-molding technique is mostly used as, with these certain advantages, the polymer becomes a promising material for the fabrication of MNs. The downside with polymers is that most polymers are soft and induce catastrophic buckling during injection or blood sampling. The author Chu and Prausnitz et al. prepared the biodegradable polymer MN of an arrowhead sharp tip with the base of a metal shaft. This intellectual design fabrication requires complex processing and extra cost. Some carbohydrates are an excellent natural material resource for the fabrication of MNs as they are very effective, cheap and safe. Carbohydrates have significant biocompatibility and less toxicity and produce products with great strength. Carbohydrates are a good source of a biodegradable polymer as they show biocompatibility, are less costly and have good mechanical strength for insertion into the skin. This includes various sugars, maltose, sucrose, trehalose, mannitol, and galactose used for MN fabrication ,78.The non-biodegradable polymers are usually synthesized using living organisms in one way or another. They have been found in the xenobiotic class, which are originally synthetic, i.e., these compounds are chemicals and do not fall into a natural polymer. Not all xenobiotics are non-biodegradable; there are many examples of biodegradable xenobiotics. We cannot say that all-natural polymers are biodegradable; lignin is the amplest natural polymer material, but it degrades by selective micro-organisms at a very slow rate. The polythioester is a biopolymer obtained by recombinant E. coli bacterial strain fermentation and can produce large quantities . There aPolymers have lower tensile strength than metal and silicon materials, but have a tough nature. Many naturally occurring polymers are used to prepare MNs, including polysaccharides, proteins, and synthetic and semisynthetic polymers. These polymers are mainly used to fabricate solid, dissolvable MNs and coat the other material .Carbohydrates are an excellent natural material resource for MNs fabrication as they are very effective, cheap, and safe. Carbohydrates have significant biocompatibility and less toxicity and produce products with great strength . By usin(1)Hyaluronic acid MNs;(2)Chondroitin sulphate MNs;(3)Cellulose-based MNs;(4)Chitin and chitosan MNs;(5)Starch-based MNs.Polysaccharides are primarily used in transdermal drug delivery due to their biocompatibility, biodegradability, cost, easy availability, ease of fabrication and sustainable delivery. They are obtained from natural sources such as plants, animals, microorganisms, etc. These include hyaluronic acid, dextran, Chitosan, and other biopolymers ,88,89: linkage of glucose monomers. Cellulose was reported for biomedical application. Cellulose nitrate is a film former in cosmetics . The appIn addition, some scientists at the University of Pittsburgh and Carnegie Mellon University studied carboxymethyl cellulose (CMC) MNs for incorporating various chemotherapeutic agents and immune-stimulating agents for skin cancer . These MChitosan is a polysaccharide prepared by deacetylation from chitin. Its linear structure of β- linkage contains D-glucosamine and N-acetyl-D-glucosamine units. It is water-insoluble and has a molecular weight of this polymer in the range of 300 and 1000 kDa. The lower molecular weight corresponds to poor mechanical strength, and this bottleneck can be overcome by blending it with PLGA . HoweverStarch is a versatile biomaterial and has been explored in various applications in the biomedical field. It provides brittleness and is applicable for various topical purposes. There are numerous reports on starch-based MNs, such as that by Yujie Zhang et al., who fabricated dissolving glucose-responsive insulin-releasing MN patches for diabetes. Starch improved the mechanical strength of MNs . The staInvestigators have used various methods over the past few years to manufacture a wide variety of MNs. The applications of the MN are considered first while designing an MN, which includes the type of drug, its dose, desirable pharmacokinetics/pharmacodynamics, and the targets and properties of the material used for MN . AchieviMNs are fabricated in various ways for different applications . The twoMicrofabrication technology  systems (MEMS) is the most encouraging method used for MNs fabrication and precise application . MEMS teThin-film deposition leads to the addition of the material in the thin-film layer over the substrates. These thin layers could act as spacers with a few nm thicknesses to 700 µm . Thin-fiMost of the developments in microelectronics and micromachining fabrication start with lithography. The different types of lithography include ion beam lithography, photolithography, X-ray lithography and electron beam lithography. The maximum widely utilized type of lithography is photolithography . PhotoliEtching is an important process to fabricate the final and optimized functional type of MEMS structure onto the substrate after lithography. This technique creates a design on a substrate surface by incising the thin films (unprotected parts) previously deposited/placed on a substrate and/or removing layers of the substrate itself by using strong acid or a physical process. It is mainly carried out after the photolithography process, through which a uniform layer of photoresist is deposited over the substrate and patterned for fabricating the etching pattern . Any phoWet etching is a patterning method. The material  or metal is removed/etched by submerging in a liquid bath containing a chemical solution or etchant . In thisThe isotropic etchants engrave the material, such as oxide, aluminum, nitride, polysilicon gold and silicon, at the same rate in all directions. Hence, they take off material horizontally under the etch mask at the same rate as they etch through it. For instance, a thin oxide film on a silicon wafer is etched with isotropic etchants (hydrofluoric acid) that etch the oxide quicker than the underlying silicon, as shown in Rounded side wall microchannels are produced by isotropic wet chemical etching. The application of titanium as a receding mask through the wet etching process leads to adjusting the shape and angle of the sidewall of the microchannels . The etcDuring the wet etching isotropic process, if the etching time is prolonged or when the etchant is the strong base solution, the photoresist could not offer enough protection and easily get damaged. For example, the photoresist layers are effortlessly etched in a wet etching of silicon where potassium hydroxide (KOH) or tetramethylammonium hydroxide (TMAH) is utilized. Hence, they have not functioned as the masking layer. In such a case, a multistep etching process is employed where the silicon dioxide is designed through a photoresist as the masking layer and HF as the etchant. As a result, silicon dioxide is the vastly resistant mask to KOH or TMAH, and the silicon layer is then etched .HF is employed as the main etchant for all types of silicate glass. The chemical reaction for etching is shown below:It is usually used to etch the oxides, and in strong concentrations, it speedily strips oxides . BufferiAnisotropic etchants attack the material at diverse rates in different directions, so they are faster in a favored direction at producing more controlled shapes ,155. TheDepending upon the exposure of the crystal faces of a few crystalline materials, they remain etched at diverse rates. Silicon is a single-crystal material having different crystal planes that allows very high anisotropic etching at different rates. KOH or TMAH is regularly used as an anisotropic etchant. Using KOH, the anisotropic etching of silicon wafers results in the most common crystal orientation (100). The etch rate of a 30% KOH solution in ˂100> directions is 0.8 μm/min, which is >150 times higher than the ˂111> directions , while tThe amount of boron in silicon affects its rate of etching. An increased amount of boron in silicon reduces the etching rate in KOH by a greater extent and further stops the etching of the boron-rich silicon, called as concentration-dependent etching method . The difA typical anisotropic etching process with a (100) silicon wafer is shown in Wet etching is used to produce silicon MN, metallic MN and hollow MN arrays of a sharp tip . HoweverDry etching, or plasma etching, is the etching process in which an accelerating motion of an ion species to the substrate combined with a masking process is used to physically or chemically etch the target materials. This form of etching is carried out at little pressure using inert or reactive gases. The most commonly used dry etchants are hydrogen fluoride , fluorocIn RIE, a plasma generates high-energy ions in a chamber that reacts chemically with the substrate to be etched. RIE is a form of isotropic etching, primarily embodied as barrel etching; conversely, it is often utilized as an anisotropic etch. In this form, the reactive ions are accelerated toward the substrate to be etched . The eleThe etching rate in RIE is low; hence, to achieve a high width-to-height ratio, a deep RIE (DRIE) process was introduced. A deep RIE (DRIE), often called the Bosch process, was introduced to generate high aspect ratios, height-to-width ratios, and structures in amalgamation with chemical vapor deposition (film forming process). Fabrication parameters can be optimized using the BOSCH process to achieve a high aspect ratio; high etch rate, straight sidewalls and small sidewall scalloping. The possibility of getting non-vertical, tapered sidewalls is limited .4H8) to preserve the etching of the side walls when the plasma etching phase is repeated for the next cycle of fabrication. The Bosch process typically creates undulating sidewalls due to the above two steps. The Bosch method is appropriate for the manufacture of off-plane MNs. This method is also used to yield hollow MNs with a lumen of several hundred µm widths to height ratio of 30:1 [In order to generate high aspect ratio troughs or beams with vertical walls in a substrate, two different phases that work repetitively at a reasonable frequency are employed : (1) Etc of 30:1 . Althoug of 30:1 .6 and O2 gas as an etchant at a pressure of 150 mTorr and power of 150 W for a run time of about 250 min. These parameters resulted in deep vertical and lateral etching forming MNs from the regions protected by the chromium masks [Research by Henry et al., in 1998, fabricated solid silicon out-of-plane MNs arrays employing the DRIE process. The chromium dots formed the masks on the silicon wafers. The patterned silicon wafers were etched using a reactive ion etcher using SFum masks . Howellsum masks . Figure 2, N2, Xe [The Ion-Beam Milling method, in which inert ions are accelerated from a source to physically eliminate the material etched from the wafer. They are of two types; showered-ion-beam milling (SIBM), and concentrated ion-beam milling (FIBM) . In SIBM, N2, Xe . The proThe characteristic feature of MN advancement is its diversified application. The basic characteristic includes dimensions, i.e., size, shape and geometry. The material  used in designing MNs also has a significant role in the characterization of MNs .These characteristics include: (1) Deep penetration of intact MN into the skin tissue. (2) Optimized dimensions, as the short needle would not be completely pierced into the skin tissue, and with a long needle size, there might be a risk of breaking (due to lack of strength and rigidness) before insertion into the skin ,200.Morphological characteristics like shape, length, base diameter, and a tip diameter of MN are observed.The study of geometry and dimensions was conducted on two types of MN, i.e., maltose MN and DermaRollerTM , on a Scanning electron microscope (Hitachi S-4100) for MN imaging. These MNs samples were directly imaged without coating with DermaRollerTM, and detachment of the head embedded with MN from a needle holder was executed, and samples were placed in SEM with 15 eV accelerated voltage. The measurement of the dimensions was recorded by software—Vantage 1.3 . The observed results were as follows; maltose MN consisted of pyramidal shape, 497.41 ± 31.10 μm length, 197.60 ± 17.53 μm base diameter and ~8 μm tip diameter, whereas DermaRollerTM MN had conical shape, 699.38 ± 70.72 μm length, 127.88 ± 15.96 μm base diameter, ~15 μm tip diameter .Amer RI et al. characterized polymeric MNs of Sodium alginate by conducting an SEM analysis. The geometry of the MN was confirmed from the master mold where the visualization of MN length is 600–650 μm, the base width of 300–350 μm with 100–125 μm interspacing. Deformation was observed slightly, i.e., needles were more wide and flat; this was associated with sampling preparation with a low level of electron beam and magnification, which resulted in the interaction of both the sample and electron beam, causing sample degradation by melting.Uniform sharp tips inter-spacing dimensions of MN were observed and confirmed by a photograph of a light microscope (MN cross-section sample) .A TGA was performed for sorbitol, polyvinyl alcohol, and MN patch to ensure the thermal stability of components of MNs where a 3% loss in mass, caused by the evaporation of adsorbed water, was observed for a powdered sample of polyvinyl alcohol (PVA) with temperatures ranging from 25 °C to 200 °C. More than 60% mass loss in PVA was recorded at 300–370 °C, indicating thermal degradation of PVA, and an additional 30% loss in mass at 370–470 °C occurred due to the decomposition mechanism. Sorbitol under thermal exposure reported mass loss <1% at 230 °C and represents thermal stability. A further 50% reduction in mass was observed with a temperature rise of 230–350 °C, representing thermal degradation. The MN patch showed the approximate 85% mass loss is similar to PVA as it consists of PVA in large amounts. About 6% mass reduction at 65–240 °C occurred due to water removal and melting. At 300–370 °C, organic combustion caused mas sample reduction of ~48%. At 380–480 °C the sample showed second phase degradation. This study concluded the efficient thermal stability of sorbitol, polyvinyl alcohol, and MN patch .sW), and the determination of the percentage of swelling was calculated using the following formula:The swelling property signifies the mechanical integrity of the MN. The swelling study is carried out as follows; where a blank and dry patch was taken from a known mass (Wd), and was immersed in a petri dish containing phosphate buffer saline solution with pH 6.8. The immersion continued until the swelling caused disruption to the structure and thus the structural integrity collapsed due to water uptake (approx. 30 min). The patch was taken out of the petri dish, and using filter paper, surface water removal was conducted . The swow/w Gantrez S-97, 7.5% w/w PEG 10,000 and 3% w/w Na2CO3 were subjected to swelling studies PBS pH 7.4 buffer. First, the weight of the MN was measured in a dry state at a zero time point and then immersed in PBS pH 7.4. The MN films were removed at specific time intervals, surface fluid was wiped, and the MN film’s swollen mass was measured. The results showed the highest swelling and fast achievement of the equilibrium state by the super swelling MN compared to the control formulation . At equilibrium, the super swelling percentage of the super swelling MN compared to the control MN was 1708% and 1071%, respectively. Thus, the above study proves the mechanical integrity of the hydrogel MN array when administered transdermally [The super swelling MN arrays fabricated using an aqueous mixture containing 20% dermally . SwellinMN design aims to ensure drug delivery into the epidermis and dermis of the skin by piercing the stratum corneum. The skin staining technique uses methylene blue stain to confirm these MN holes by piercing the MN. Preparation of methylene blue solution of 10 mg/mL by adding methylene blue powder and distilled water. For biological stains, methylene blue dye is used. It acts as it binds with the protein in the tissue; due to its hydrophilic nature, it is not absorbed by the stratum corneum hydrophobic nature ,206.When coated, PLA MN and dissolvable MN were removed from a skin sample, the deposition of methylene blue on the skin sample was conducted for 10 min on the skin surface, and leftover methylene blue solution was wiped out with ethanol , leaving dye stains in the punctured region of the stratum corneum and highlighting them .MN drug delivery aided the rapid dissolution of the drug into the body. Drug distribution enhances the efficiency of drug release. The interaction of polymer and drug molecules was carried out by multiscale simulation influencing MN drug distribution. The study was carried out with sulforhodamine B (SRB) as a model drug, along with Hyaluronic acid (HA) and polyvinyl alcohol (PVA) for fabricating MNs. Dissolvable MNs were produced to study controlled drug distribution in those MN patches. These observations were studied using an optical microscope under an objective lens by viewing the side and top angle of the MN array. The analysis of SRB in both MN patches was studied and observations made were as follows; limited drug distribution in PVA, as drug molecules, are seen concentrated at the tip, and mild concentration was seen in the middle portion, with no drug molecules at the base of the needle. Conversely, Hyaluronic acid showed poor drug diffusion control ability. Here, SRB distributes largely into the needle body and bottom plate; it has a poor concentration at the needle tip; owing to the high water solubility of HA with a high compatibility drug molecule, Hyaluronic acid faced a significant challenge in achieving controlled control drug distribution .Mechanical strength is an important aspect for MNs to perform their function. Mechanical property is often determined by compression strength . The comThe calculation of compressive strength was made when the MNs patch was penetrated to a length of 50% into porcine skin. There was a slight MNs patch stress change by a 43% compression rate. An optical microscope represented MNs patch deformation. Flat PVP- hydrogel has a compression strength of 268 kPa, while PVP-MNP has 284 kPa of compression strength; PVP-MNP’s higher compression strength is due to the larger force applied per unit of arrangement. The compressive strength of Ag coated PVP MNs 278 kPa, and Au coated PVP MNs 276 kPa are slightly less than PVP-MNP due to polymeric chain degradation with polymeric chain heat created during the process of degradation. These Ag and Au coated PVP MNs have higher strength than flat PVP hydrogel. The MNPs have perfectly penetrated the porcine skin, seldom broke or deformed. Furthermore, complete skin penetration occurred at the surface without breaking at contact. The slight reduction in length < 1% when penetrated to 700 μm was observed before (~701–703 μm) and after (~692–695 μm) MNs length skin penetration. The shape was retained well (slight deformation). Results represented effectiveness in drug delivery as PVP-based MNPs are prepared by radiation and have MNs of sufficient strength and flexibility required for insertion through the stratum corneum and to reach the dermis .In vitro studies for an assessment of the MNs include penetration and permeability studies. The skin distribution technique evaluates the uniformity and depth of penetration by MNs. The patch is placed on the hairless skin of a Rat or Pig and immediately removed. The skin site is dyed with a cotton swab soaked with India ink. This skin is then stored at −20 °C on dry ice for three days up to processing. The skin is cut in a section of uniform size . The number of stained pathways in each section is counted. The percentage of MNs to have penetrated the skin can be plotted as a function of depth, and the depth at which 50% of the MNs penetrated can be extrapolated . Permeabp > 0.05) than the time controlled group, approving in-efficiency of passive insulin absorption through the transdermal route. The subcutaneous administration of insulin rapidly decreased the blood glucose level at 1 h, to around 18%. For the 500 micron group, the glucose level was 18% at 3 h, significantly different from the negative control rats (p < 0.05). In comparison to the positive control after 3 h, the changes in blood glucose levels persuaded through the microneedle roller are higher (p < 0.05), but there was no significant difference 3 h . This specifies that microneedle rollers might increase the skin permeability in long-term delivery, which is suitable for the delivery of repeated amounts of pharmacologically active insulin.The visualization of MNs patches and drug uptake is carried out by an in vivo imaging system (IVIS). It observes the subject animal at a predetermined time interval . InsulinThe MNs were first introduced for drug delivery applications, and the major objective was the enhanced permeation in the skin using solid and hollow MNs compared to conventional hypodermic needles. The MNs were filled with drug solutions or formulations, or they were coated for improved intradermal drug delivery. Nowadays, MNs are the leading novel technology for several fields of drug delivery, such as intradermal, ocular and intracellular drug delivery. However, the transdermal route is still the leading application area for MNs, especially vaccine-based delivery.Drug delivery to the skin is challenging as it may be a local application or systemic delivery as a result of the stratum corneum’s highly tough and barrier properties. However, the human stratum corneum thickness is (10–15 µm), and it still prohibits the drugs at therapeutic levels . The FooThe small molecule or low molecular weight drugs have higher skin diffusion coefficients than the larger molecule or biomolecule, which could easily penetrate the skin. This is so that the small molecule is quickly delivered into the skin using MNs.Rojekar et al. have formulated dissolving MNs containing the etravirine and etravirine nanosuspension for long-acting drug delivery and improved HIV infection therapy. They have demonstrated the robust nature of MNs, with significant drug deposition of 12.84 ± 1.33% ex vivo, in neonatal porcine skin for 6 h. The in vivo pharmacokinetic studies demonstrated improved parameters; Cmax exhibited by DMNs containing ETR powder and ETR NS was 158 ± 10 ng/mL and 177 ± 30 ng/mL, respectively. It was also revealed that the improved t1/2, Tmax, and mean residence time (MRT) compared to intravenous ETR solutions indicated the long-acting nature of etravirine delivery using DMNs .Lin Zhu et al. have developed estriol-loaded EMNs to effectively treat radiation-induced injury. For the development of EMNs, biocompatible polymer polyvinyl pyrrolidone K90 was used. The drug is dissolved in methanol and mixed with polymer gel to cast into a mold to obtain the conical-shaped EMNs. The developed EMNs are robust and easily penetrate 200 μm into mouse skin. Most interestingly, these EMNs dissolve very quickly in 5 min, which could help immediately permeate the drug into the skin. The mouse model of the ionizing radiation-induced injury was developed by the source of 6.5 Gy radiation of 60 Co γ ray. Furthermore, EMNs enhanced the peripheral blood leukocytes count in irradiated mice, which protected the bone marrow hematopoietic system and 80% increased the survival rate of the irradiated mice .Alyaa et al. have investigated and developed dissolving MNs using biocompatible and biodegradable polymers, poly(vinylpyrrolidone) (PVP) and hyaluronic acid (HA) to intradermally deliver the Amphotericin-B (AMP-B) to treat the fungal infection. It was found that both polymers used in development reduced the AMP-B cytotoxicity, compared to the drug-free solution. Moreover, it was found that AMP-B-loaded dissolving MNs showed significant antifungal activity compared to plain drugs. Furthermore, MNs maintain the activity of drugs .2). The MAP was robust with the skin penetration potential. The tips were dissolved in 30 min, giving immediate deposition of the drug in the skin. Moreover, the in vivo dermatokinetic study in Sprague Dawley rats of both forms of drug-loaded MAP deposited into the skin, forming the depot. Both drug forms are released slowly, maintaining the therapeutic concentrations in the blood for one month for a single application [Ismaiel A. Tekko et al., have for the first time, developed the MNs array patches (MAP) with the higher Cabotegravir or micronized sodium salt loading of  loaded dissolving and implantable MAPs to systematically deliver or release the drug. The developed MNs are mechanically strong enough and could pierce the excised neonatal full-thickness porcine skin and deposit the drug as a depot form. The release study performed using dialysis methods demonstrated the relatively fast drug release in both the formulations. The in vivo studies in rats showed rapid metabolization of the TAF into tenofovir, along with quick elimination of the metabolite from the blood plasma .Maelíosa Crudden et al. have developed Rilpivirine nanosuspension-loaded, dissolving MN array patches (MAPs) for the long-acting delivery in HIV for improved therapy and patient compliance. MAPs were mechanically strong enough to pierce the skin and load the drug as depot formulation for a prolonged effect. In vivo pharmacokinetic studies demonstrated that the mean plasma concentration in rats is 431 ng/mL at seven days, which is about ten-fold larger than the trough concentration found after a single dose administered in the previous clinical studies .Mingshan Li et al. developed a novel strategy for co-formulating the dexamethasone and pro-drug dexamethasone sodium phosphate in the DMNs. That could have led to the immediate effect to achieve long-term drug delivery. The 3D printing technique was used the first time to fabricate the baseplate of the MN. The 3D printed base plate is robust, providing excellent support to the drug encapsulated or loaded tips. These novel trilayer-based MNs have shown the effective drug delivery of dexamethasone, which could be the novel promising drug option for oral and injectable drug delivery .Protein and peptides are very unstable and degraded after oral administration. Transdermal drug delivery could avoid this issue; however, delivering all kinds of molecules is difficult due to challenging skin barriers . Using MDesmopressin is a synthetic form of vasopressin, the potent peptide hormone used to replace the low vasopressin levels in the therapy. This is used to treat diabetes insipidus, which causes bedwetting in children and hemophilia A. The MNs formulation is a novel approach to deliver the desmopressin, which showed an effective and safe delivery compared to the other conventional routes .w/w of Cyclosporin A in the porcine skin for 60 min. Approximately 65% of MNs were dissolved with a 34 ± 6.5 μg drug delivery [Liu et al. have formulated GAP-26, a gap-junction blocker containing polyethylene glycol diacrylate MNs to deliver peptides by swelling effect. The developed MNs formulation has improved the permeation of peptides, which leads to improved inhibition of the keloid fibroblasts and the collagen I expression ,229. Cycdelivery ,230.Insulin is the hormone for modulating blood glucose levels with a 51-amino-acid peptide. However, the exceptionally high pain triggered by frequent subcutaneous injections could adversely affect the patient’s compliance ,231. DesZhou et al. demonstrated the applicability of the stainless steel MNs with different needle lengths, which were used to evaluate the delivery efficacy of insulin in diabetic rats. The results demonstrated that the skin permeability increased significantly with a rapidly decreasing glucose level within 1 h of application. It is also seen that solid MNs associated with the iontophoresis could effectively improve the intradermal delivery of the insulin . McAllis1/2 and three times shorter Tmax than the conventional injectable therapy [Ye and co-workers have studied MNs association with pancreatic β-cell capsules, which could sense the glucose level in the blood and secrete insulin as per the requirements. It was found that the patch was not effective enough. MNs matrix-loaded synthetic glucose signal amplifiers were developed. These MNs include α- amylase, glucoamylase, and glucose oxidase; this indicates insulin secretion from the β-cells capsules ,234. The therapy . These s therapy ,236. FigThe DNA and RNA are short oligonucleotides, basically smaller units than proteins. Delivery of the oligonucleotide is difficult due to their properties, so numerous techniques were employed to deliver these agents. The delivery of the 20-merphosphorothioated oligodeoxynucleotide was conducted using the MNs formulations approach. The solid MNs, made from stainless steel, were used to deliver these oligonucleotides via the poke with the patch approach. It was found that more drugs were delivered using this approach compared to intact skin ,239.A vaccine is a complex biological preparation or formulation. It successfully offers active acquired immunity to a specific disease. Vaccines consist of the killed or weakened form of disease triggering microorganisms, toxins or one of its surface proteins . VaccineThe painless withdrawal of the biological fluids from the body is the major advantage of MNs over conventional blood collection techniques. Various biomarkers present in interstitial fluid beneath the skin can be useful for diagnosing various diseases like diabetes, cancer, arthritis, etc., and helpful, timely medical intervention. Multiple research studies demonstrated the usefulness of MNs for disease diagnosis . Chang eAlong with vaccination, the MNs can be used to diagnose various skin diseases in pediatrics like psoriasis and other inflammatory conditions . One of MNs provide significant efficiency in the biosensing of various biomarkers and metabolites. Collection of biofluid is more convenient with MNs than with conventional hypodermic needles. Recent advancements in MNs are resulting in improved biosensing . StrambiMNs technology creates a new horizon in cancer therapy through efficient drug delivery of anticancer vaccines and drugs. Various chemotherapeutic agents, genes and proteins can be efficiently delivered through MN-based devices . Hao et Bypassing the ocular barrier with minimum invasion is the advantage of MNs over intravitreal injection. Several studies demonstrated the application of MNs in ocular drug delivery. Patel et al. demonstrated the successful delivery of micro and nanoparticle suspension in the suprachoroidal space of pig, rabbit and human eye (ex vivo) using hollow MNs. Optimizing dimension and process parameters concluded that efficient drug delivery could be achieved with a needle length of 800–1000 µm and pressure of 250–300 kPa . ThroughThe toxicity of MNs is checked by examining the materials used in their fabrication, such as silicon and stainless steel, and polymers, i.e., poly-methyl methacrylate, poly-lactic acid, etc., cause the toxicity of MNs. Sometimes the presence of materials such as photoinitiators can result in the toxic effects of MNs .4 per well. The incubation media used was 90% of DMEM and 10% of fetal bovine serum. The sterilized MN patches were put into the wells, and this was kept for incubation for 24 h. Untreated cells were used as control. For analysis, 100 µL of culture media from each well was transferred to a 96-well plate, and the absorbance was checked with a microplate reader at 450 nm [4 for 12 h. The MNs patch of 0.5 cm2 was soaked in 1 mL DMEM media for 24 h and this was used as a soaking solution. Next, 200 µL of soaking solution was poured to each well and incubated for 24 h. Supernatant was replaced by 200 MTT µL agent. Then, 200 µL of DMSO was added to each plate and gently shaken for 15 min. The absorbance was checked at 570 and 630 nm and relative cell viability was calculated [In vitro methods for toxicity assessment of MNs are carried out using cell line assays. Commonly used cell lines are human adult low calcium high temperature (HaCaT) keratinocytes and human embryonic kidney (HEK293) . Piu et t 450 nm . In anotlculated .Biocompatibility of MNs by in vivo methods is usually assessed by acute dermal toxicity study. An MNs patch is placed on to the shaved skin of rats and observed visually to see whether the inflammation has occurred or not . The matFor genuine MNs goods, there are currently no agreed regulatory requirements. Precise quality requirements in the framework of Good Manufacturing Practice need to be developed for MNs to be made on an industrial scale. Currently, standardized testing and equipment utilized to validate MNs mechanical characteristics and insertion capacity are missing, making it hard to compare MNs and necessitating the adoption of standardized tests and equipment to evaluate product quality and adequate least requirements. There is the need to perfect the implementation of a pharmaceutical quality system with good manufacturing practice and quality risk management for well-designed MNs to commercialization. A stringent rule and regulation should be applied to meet all drug product requirements before being released for human use. The complex regulatory specifications and time requirements will impact the cost of the end finished product ,270. TheMNs are a new approach to administered medications via the stratum corneum . MNs areThis review article extensively describes the type of microneedle, fabrication material, detailed casting methods and techniques, and its applications. Microneedles have been fabricated using different materials, like silicon, metals, polymers, and ceramics, by several fabrication methods, i.e., lithography, wet and dry etching, laser cutting and micro molding. The practical use of microneedles has been acknowledged and gained widespread attention. Optimization of sharpness, length, insertion force and velocity, and other parameters have allowed reliable microneedle insertion into the skin. Nowadays, MNs are the leading novel technology for several fields of drug delivery, such as intradermal, ocular and intracellular drug delivery. However, the transdermal route is still the leading application area for MNs, especially vaccine-based delivery. MNs technology creates a new horizon in cancer therapy through efficient drug delivery of anticancer vaccines and drugs. Various chemotherapeutic agents, genes and proteins can be efficiently delivered through MN-based devices. Patients and clinical workers are highly inclined to prefer microneedle-based delivery over hypodermic injections according to surveys. Human subjects report little or no pain associated with most microneedle designs. After microneedle treatment, the skin often shows mild, transient erythema, but there is currently no evidence of increased infection risk at the treatment site. Big pharmaceutical giants are currently working on the developments and commercialization of microneedle-based drug delivery systems; this technology is rated in the top 10 recent technologies. Patients, healthcare providers and companies have established interest in the technology. Microneedles are poised to make an expanded impact on clinical medicine over the coming years.Microneedles are being studied worldwide as a drug delivery system for various ailments, including treatment of all diseases and vaccination. The development of sophisticated delivery systems like MNs could boost the efficiency of drug administration and lower the total dose concentration to minimize side effects. Some characteristics, such as the needle dissolution rate, can affect bioavailability. As a result, changing the needle geometry and size could assist us in achieving a more profound transdermal drug release. Novel MNs production technologies have resulted from much research in this field. MNs devices improve patient compliance by avoiding site-specific infections caused by conventional needles. However, the discovery of innovative micro-fabrication processes and stability medicines for MNs devices remains a challenge. Some of the primary reasons driving the market expansion include the increased prevalence of chronic hyperpigmentation and skin infections worldwide. Another factor driving the market over the forecast period is the low cost of microneedle devices compared to plastic surgery. MNs are also anticipated to be a revolutionary tool in the Cosmetic Industry. Furthermore, changing lifestyles are predicted to increase skin infections, and the availability of at-home micro-needling devices is expected to drive market expansion. Lack of experience, lack of public consciousness, and treatment side effects are some of the limitations on market growth.


This paper presents a modified unified critical state model to predict the mechanical responses of both clays and sands under over-consolidation and cyclic loading conditions on the basis of clay and sand model (CASM), which is named as CASM-kII. Through the application of subloading surface concept, CASM-kII is able to describe the plastic deformation inside the yield surface and the reverse plastic flow, and is thus expected to capture the over-consolidation and cyclic loading behaviours of soils. CASM-kII is numerical implemented by the using of the forward Euler scheme with automatic substepping and error control. Then, a sensitivity study is carried out to check the influences of the three new parameters of CASM-kII on the mechanical response of soils in over-consolidation and cyclic loading conditions. Through the comparisons of experimental data and simulated results, it is found that CASM-kII is able to satisfactorily describe the mechanical responses of both clays and sands in over-consolidation and cyclic loading conditions. In order to unify the constitutive modelling of both clays and sands, Yu7 developed a unified critical state model for clay and sand  on the basis of Cam-clay plasticity and state parameter concept. Owing to its concise mathematical expression and clear physical meaning of material constants, the CASM has been further developed to capture the more complex mechanical behaviours of soils12.An accurate and concise description of the mechanical properties of soils is required for the design of civil engineering. As one of the most significant achievements in the field of geomechanics, Cam-clay plasticity15, classic elastoplastic constitutive models with a single yield surface enclosing the elastic domain possess many limitations in describing the mechanical response of soils. First, the original CASM predicts pure elastic behaviour inside the yield surface while the experimental data show that recoverable deformation only occurs within a very small range19. CASM describes discontinuous variation of the tangent stiffness modulus from elastic to plastic of over-consolidated soils and predicts an unsmooth stress–strain relation, and thus is unable to describe softening behaviour accurately20. Second, the ability to characterize the effect of stress history during cyclic loading process is significant to the constitutive modeling of soils25, since it has been discovered in cyclic loading tests that elastic and plastic deformations both develop during unloading before the stress path is completely reversed27. When the effect of reverse plastic flow is critical, the traditional isotropic hardening CASM model with a single yield surface is unable to provide appropriate solutions for boundary value problems because the reversed plastic flow fails to be considered just by expanding the single yield locus. Therefore, the above significant issues must be considered before further developing CASM to describe a variety of more complex properties of soils. Khong9 has attempted to introduce the concept of kinematic hardening into CASM within the context of bounding surface theory(i.e. CASM-k) but failed because of the difficulty of numerical implementation, thus Khong9 highly recommended that further development of CASM should be conducted in future research.However, as stated by Hashiguchi15 proposed the subloading surface concept with rigorous physical backgrounds. During last decades, subloading surface concept has been widely adopted in the constitutive modeling of soils. Hashiguchi et al.17 and Yao et al.18 adopted the subloading surface to describe the over-consolidated behaviour of soils. Asaoka et al.28 and Zhang et al.29 described the mechanical properties of structured soils through the combination of superloading and subloading surfaces. Since the concept of rotational hardening mechanism has been widely adopted in the constitutive modeling to capture the induced anisotropic properties of soils32, Hashiguchi and Chen33, Yamakawa et al.34 and Hashiguchi et al.35 combined the subloading surface concept with rotational hardening theory to characterize the cyclic mobility mechanism of anisotropic soils.To extend the prediction ability of conventional elastoplastic constitutive model under complex loading conditions , HashiguchiIn this study, a new modified unified critical state model called CASM-kII is developed by introducing the subloading surface theory into the original CASM. This new model is established to extend the prediction ability of CASM for both clays and sands under over-consolidation and cyclic loading conditions. The plastic modulus of this new model is derived through the consistency condition of subloading function and varies smoothly from elastic to plastic response during loading procedure, which makes it possible for CASM-kII to characterize the plastic deformation inside the yield surface and the reverse plastic flow and thus to describe the over-consolidated behaviour and cyclic loading response of soils in a flexible fashion. Then, the forward Euler scheme with automatic substepping and error control is adopted in this study to numerical implement this newly proposed model. Compared with original CASM, CASM-kII adds three material parameters to control the evolution law of subloading surface, and the influences of these new parameters on the mechanical properties of soils under drained and undrained conditions is checked through a sensitivity study. At last, CASM-kII is validated through the comparisons of experimental data and simulation results in monotonic and cyclic loading tests under both drained and undrained conditions.35 or soil fabric and its evolution rule39 in the model. The extension work for the issue of anisotropy can be referred to the work of Gao et al.40 Second, the bold-faced characters are used to represent vectors and tensors and the italics are used to represent scalars.First, soil behaviour is assumed to be isotropic in this paper for the sake of simplicity. If the inherent and induced anisotropy behaviours of soils need to be considered, CASM-kII may be extended to account for anisotropy by incorporating anisotropic mechanism such as rotational hardening behaviourp and deviatoric stress q can be expressed as:In this section, the subloading surface theory is introduced in CASM. The effective mean stress 15. The subloading surface passes always through the current stress state p and q axes, respectively; As illustrated in Fig. 20, the stress-state relation for soils can be given as:According to the work of YuBy substituting Eqs. –6) into into6) iR = 1. In this case, the subloading function becomes identical to the normal-yield function, and the subloading surface model exhibits a response similar to the conventional plasticity model.It should be noted that subloading surface coincides with the normal-yield surface when E denotes the elastic stiffness matrix; I is a fourth-order symmetric identity tensor; K and G denote the bulk and shear modulus, respectively. It should be noted that the elastic behaviour of granular materials is non-linear and depend on the stress level. There are two types of formulations are widely adopted to describe the non-linear elastic behaviour of granular materials, i.e., hypoelasticity42 and hyperelasticity48 formulations. Under the framework of hypoelasticity, K and G related to the mean stress p can be defined as:v − ln p space; In general stress space, the elastic stress–strain relation can be given as:49. Therefore, the dilatancy rule of CASM-kII follows the relation suggested by Rowe50:D is dilatancy rate; It is well known that the associated flow rule adopted by Cam-clay would lead to an over-prediction of shear strain for soils subjected to normal compressionThe plastic potential function of subloading surface can be obtained by integrating the Rowe’s relationship:Therefore, the plastic strain increment can be given as: to Eqs.  and 18)18\documeR and R, similar to the Cam-clay plasticity, the variable The hardening rule of the subloading surface are controlled by entclass1pt{minimaIntegrating Eqs.  and 19)19) over U is a monotonically decreasing function of the normal-yield ratio R, and it must satisfy the following rules:Considering that the stress approaches the normal-yield surface progressively, i.e., the subloading surface approaches the yield surface gradually during the plastic loading procedure, the rate of U that can satisfy Eq. , which makes it impractical to obtain the analytical solution of R. Hence, the Newton–Raphson method might be appropriate for calculation of R. The complete calculation procedure of R during elastic unloading process is listed in Table It should be noted that during the elastic unloading process (the loading criterion will be given in the next section), near Eq.  formed bause Eq.  only holmentclass2pt{minimBesides, it should be noted that the similarity center should be located inside the normal-yield surface during the whole loading process. Following the concept of subloading surface, the similarity center surface can be defined as follows:Then, it can be assumed that the rate of similarity center can be written as:By Substituting Eq.  into Eq.13. According to Eq. , , , 22) an an22) an15 is given as:By substituting Eq.  into Eq.54 and forward Euler57 schemes. The backward Euler scheme with the return-mapping technique is accurate because the resulting stress automatically satisfy the yield function to a specified tolerance. However, this backward Euler method requires the second derivatives of the yield and the plastic potential functions, which makes it difficult to the implement for complex constitutive relations58. Compared with back Euler scheme, forward Euler scheme have the advantage of being more straightforward to implement. Therefore, the forward Euler scheme with automatic substepping and error control is adopted in this study because its simplicity and efficiency. The forward Euler scheme with automatic substepping and error control can divide the loading step into several substeps according to the local computational accuracy required. It is assumed that the strain increment input by the analysis system is Existing numerical implementation schemes of the constitutive models are generally classified as backward Eulerentclass1pt{minimabasic constants”. 20, Rios et al.12 and Navarro et al.59. Hardening constants” in this study. The variable O for initial isotropy15. Once The newly proposed model CASM-kII requires ten material parameters  with same void ratio e0 = 0.71. The initial values of internal variables can be referred to the work of Zhang et al.61. As shown in Fig. confining-stress dependency of sand”64.Then, the experimental data of Toyoura sand observed by Verdugo and Ishihara21 is used here. The material constants and initial conditions of these samples can be found in Table q = 116 kPa. Figure To verify the applicability of the new model under cyclic loading conditions, the experiment data observed by Li and MeissnerIn this paper, a modified unified critical state model  to predict the mechanical properties of both clays and soils under over-consolidation and cyclic loading conditions is developed on the basis of original CASM model and subloading surface concept. Through the introduction of subloading surface, this newly proposed model is able to describe the plastic deformation inside the yield surface and the reverse plastic flow, and is thus expected to accurately capture the over-consolidated and cyclic behaviours. CASM-kII is numerical implemented by the using of the forward Euler scheme with automatic substepping and error control. Then, a sensitivity study is carried out to check the influences of these three new parameters (


Microneedles (MNs) have attracted great interest as a drug delivery alternative to subcutaneous injections for treating diabetes mellitus. We report MNs prepared from polylysine-modified cationized silk fibroin (SF) for responsive transdermal insulin delivery. Scanning electron microscopy analysis of MNs’ appearance and morphology revealed that the MNs were well arranged and formed an array with 0.5 mm pitch, and the length of single MNs is approximately 430 μm. The average breaking force of an MN is above 1.25 N, which guarantees that it can pierce the skin quickly and reach the dermis. Cationized SF MNs are pH-responsive. MNs dissolution rate increases as pH decreases and the rate of insulin release are accelerated. The swelling rate reached 223% at pH = 4, while only 172% at pH = 9. After adding glucose oxidase, cationized SF MNs are glucose-responsive. As the glucose concentration increases, the pH inside the MNs decreases, the MNs’ pore size increases, and the insulin release rate accelerates. In vivo experiments demonstrated that in normal Sprague Dawley (SD) rats, the amount of insulin released within the SF MNs was significantly smaller than that in diabetic rats. Before feeding, the blood glucose (BG) of diabetic rats in the injection group decreased rapidly to 6.9 mmol/L, and the diabetic rats in the patch group gradually reduced to 11.7 mmol/L. After feeding, the BG of diabetic rats in the injection group increased rapidly to 33.1 mmol/L and decreased slowly, while the diabetic rats in the patch group increased first to 21.7 mmol/L and then decreased to 15.3 mmol/L at 6 h. This demonstrated that the insulin inside the microneedle was released as the blood glucose concentration increased. Cationized SF MNs are expected to replace subcutaneous injections of insulin as a new modality for diabetes treatment. Diabetes is considered one of the world’s most complex health problems in the 21st century and a severe chronic disease ,4. This The transdermal drug delivery system , one of The original insulin delivery MNs came from drug coatings . As a deHydrogel-forming microneedles is an exciting and recent development in microneedling technology. Donnelly et al.  first idSF is a typical biological material with various applications, including drug delivery and scaffold synthesis in multiple forms, particularly in hydrogel ,25,26. IDue to the slow degradation and swelling of the polymer, MNs need to release the encapsulated drug over a more extended period . AccordiIn order to meet the daily insulin dose requirement of diabetic patients and avoid the risk of hypoglycemia caused by insulin overdose, an insulin-loaded modified SF MNs with both high dose and intelligent drug delivery effect was developed in this paper. In the previous stage, our research group used polylysine to modify SF and prepared SF hydrogel sensitive to acidic pH values, the swelling degree of which is different under different pH values . Based oSilkworm cocoons were purchased from Suzhou Xiancan silk Biotechnology Co., Ltd. . Horseradish peroxidase (HRP), Gox, and human recombinant insulin (100 IU/mg) were obtained from Shanghai Alading Biotechnology Co., Ltd. . An insulin detection kit (chemiluminescence immunoassay (CLIA)) was acquired from Henan Meikai Biotechnology Co., Ltd. . Male SD rats were provided by the Experimental Animal Center of Suzhou University, . The Ethics Committee of Soochow University approved all animal experiments, which followed the Guidelines for Ethical Conduct in the Care and Use of Research Animals established by Soochow University. All other chemicals used in the experiments were analytically pure. Ultra-pure deionized water was used for all experiments.3), 0.3 wt% sodium carbonate (Na2CO3) solution, heated to boiling, removed after 30 min, and cleared of silk glue using deionized water. The above experimental steps were repeated three times. After drying SF in an oven at 60 °C, SF was dissolved in 9.3 mol/L lithium bromide solution for about 1 h, cooled, and put into dialysis bags, dialyzed with deionized water for 3–4 d, then removed and centrifuged to obtain the filamentous solution.As shown in As described in 2O2) with a concentration of 1 mM, and Gox with a concentration of 0, 2, 4, and 6 mg/mL, respectively. FITC-INs were added to the mixed solution to ensure the insulin content was 10 IU/tablet.In the cationized SF solution (polylysine-modified solution accounting for 10% of the SF quantity), add HRP with a concentration of 10 IU/mL, Hydrogen peroxide (HThe hybrid solution was poured into a polydimethylsiloxane (PDMS) mold and pumped into a vacuum-drying oven . The mixPipette 1 mL each of cationized and pure SF solution (concentration 10 mg/mL) to the measurement cell of Malvern ZetasiZer Nano ZS90 particle size potential meter . Each sample was measured three times at a controlled temperature of 25 °C to take the average value as the final result.−1 was scanned on a Nicolet 5700 FT-IR Fourier transform infrared spectrometer  as required to obtain the infrared absorption spectra.The cationized SF solution was frozen in liquid nitrogen and dried in a freeze-dryer for 48 h. After that, the sample was processed and cut into powder form with scissors, and then the required sample was prepared by the KBr compression method. The absorbance in the range of 400–4000 cmAn MZFL III fluorescence microscope  was used in observation for the preparation of microinjection. The MNs were put into liquid nitrogen for quick freezing and vacuum drying. Part of the MNs was cut and taken out after spraying gold on the surface for 90 s. The morphology was observed on an S-4800 scanning electron microscope .Glucose-responsive SF MNs and pure SF MNs were assayed according to method Glucose-responsive SF MNs and pure SF MNs were freeze-dried and ground, processed into powder, pressed into a sample holder, and scanned for diffraction intensity profiles between 2θ = 5° and 45° using an X’PERT PRO MPD fully automated X-ray diffractometer  with CuKα radiation, a stable and fluctuation-free tube current of 35 mA, a stable and fluctuation-free tube voltage of 40 kV, and a scanning speed of 8°/min as the environment for their determination.The mechanical testing and insertion analysis techniques used to characterize microneedles differed between groups . The gluAfter blending with serine polylysine, the hydrogel was used as the control group. The prepared hydrogel was placed in a constant temperature and humidity environment, dried into a film for 1 d, weighed, and recorded as M0. The prepared citric acid/disodium hydrogen phosphate buffer solutions of different pH values were added according to the bath ratio of 1:100 after being placed in a constant temperature water bath at 37 °C for approximately 1 day to reach the dissolution equilibrium, removed, and blotted with filter paper. The surface water of the dry gel film was blotted with filter paper, and the wet weight M1 was recorded. The swelling rate was calculated using the swelling rate formula, as follows.2O2 were added to make a total system of 4 mL, and Gox (4 mg/mL) was added to make a hydrogel. The gel was wrapped on the surface of the pH meter probe and placed in a constant temperature with humidity environment for 1 d to dry, and then a dry gel film was made on the pH meter probe. The dry gel on the head of the SF MNs was simulated to test the dynamic changes of pH inside the dry gel. The dry gels were incubated in different concentrations of glucose solutions . The pH inside the dry gels was monitored at specified times using a BP 3000 pH meter . In contrast, the pH of the external solution was observed at the same time point for comparison. The specified times were: 0, 30, 60, 90, 120, 150, 180, 210, and 240 min.Since the internal pH of SF MNs is challenging to monitor, the pH of the SF MNs substrate, i.e., the hydrogel, was tested. An appropriate amount of cationized SF solution was taken, HRP and HA standard curve was made to measure the insulin content. FITC-INs were dissolved in PBS buffer solution at gradient concentrations of 0.05~1 μg/mL. The fluorescence intensity of the samples was measured at an excitation wavelength of 485 nm. Then, a linear regression curve of the fluorescence intensity measured by fluorescence spectroscopy versus the mass concentration of FITC-Ins was established, and finally, the following equation was obtained.Glucose-responsive SF MNs were pricked into fresh rabbit skin, fixed, and placed in a 12 mL transdermal release cell. The release solution was set as a citric acid/disodium hydrogen phosphate buffer solution with three pH values of 4, 7, and 9. Pure SF MNs without insulin loading was used as a blank control group. Hence, 1 mL of samples was taken at 1, 2, 4, 8, 12, 24 and 36 h for testing, and fresh 1 mL of buffer solution was added after each sample to ensure the total volume of explanation in the release cell was constant at 12 mL.i is the ith release pool FITC-INS concentration, i is the number of sampling times, V is the release pool capacity, and Vi is the volume required at each sampling. n is the total number of sampling times and C0 is 0 IU.Fluorescence values of the collected samples  were measured using an FM4P-TCSPC fluorescence spectrometer , and the data were recorded to calculate the corresponding FITC-Ins concentrations. According to the conversion of 1 IU = 45.5 μg, the unit of drug release concentration was converted from μg to IU and recorded as Ci. The cumulative release rate was calculated and the cumulative release rate curve of MNs with time was depicted according to the following equation. The calculation formula is as follows.The glucose-responsive SF MNs were pricked into fresh rabbit skin, fixed, and placed in a 12 mL transdermal drug release cell, and the designed release solutions were four concentrations of 0, 100, 200 and 400 mg/dL of glucose solution. Hence, 1 mL of samples was taken at 1, 2, 4, 8, 12, 24, and 36 h for testing, and a fresh 1 mL of release solution was added after each sample to ensure that the total volume in the release cell was constant at 12 mL. The formula for calculating the cumulative release rate is given in Equation (3).(1) Diabetic rat modelingDiabetic rats were induced with Streptozotocin (STZ), and the BG concentration was tested by a BG meter. The modeling was successful if the BG was stable and higher than 300 mg/dL .(2) Experimental groupingGroup I: experimental diabetic rats group with MNs: diabetic rats 1, 2 and 3 were anesthetized with an appropriate amount of ether, shaved, attached with glucose-responsive SF MNs (human insulin content 10 IU/tablet), and bandaged.Group II: control diabetic rats group injected: diabetic rats 4, 5 and 6 were directly injected with human insulin (10 IU/each).Group Ⅲ: experimental regular rats group with MNs: rats 7, 8, 9 with glucose-responsive SF MNs (10 IU/tablet).Group Ⅳ: control regular rats group with blank MNs: rats 10, 11, 12 affixed with pure SF MNs (0 IU/tablet).(3) Sampling and testing (BG and insulin content)Blood was collected intravenously from 12 rats separately, and the BG concentration was tested by a BG meter. 20 μL of blood was taken, diluted 10 times by PBS solution, centrifuged at 3000 rpm for 10 min, and the serum was separated. The diabetic rats 1–3 and normal rats 7–9 were attached with glucose-responsive SF MNs, and normal rats 10–12 were attached with pure SF MNs to start fasting; diabetic rats 4–6 were directly injected with insulin (10 IU/each) and fasting was started. Blood samples were taken at 1, 2, 3, 4, and 6 h to measure BG concentration and insulin concentration. After 6 h, feeding was started, rats were monitored, and blood samples were taken at 1, 2, 4, and 6 h after feeding. Blood samples were taken at 1, 2, 4, and 6 h after feeding to measure BG and insulin concentrations.Insulin levels in rats were determined using a chemiluminescence assay kit. The samples to be measured were equilibrated at room temperature for 30 min. The luminescent substrates A and B were mixed equally and prepared for use. The pieces were added to the 96-well assay plate. Then, 50 μL of standards of different concentrations were added to the first five wells, 50 μL of samples were added to the remaining wells, and 50 μL of enzyme conjugate was added to each well. The wells were shaken in a shaker to mix the liquid in the wells and placed at 37 °C for 60 min. The wells were washed 5 times with washing solution using a plate washer. 100 μL of the mixed luminescent substrate was added and reacted in a luminometer  for 5 min. The luminescence intensity was detected, the standard curve of the luminescence method was plotted, and the insulin content in rats was calculated.MN indicates the area under the blood concentration-time curve after insulin-laden MNs administration, AUCIP indicates the area under the blood concentration-time curve after insulin injection, and DoseIP and DoseMN both suggest the dose administered.The relative bioavailability of insulin was calculated by the insulin curve integration method with the following formula.The Zeta potential of the polylysine-modified SF in −1, the characteristic peak belonging to the amino group (3500–3300 cm−1), indicating the successful introduction of more amino groups. Furthermore, the comparison revealed that the wave number of the characteristic peak of the amide bond of the SF changed. A small peak appeared at the typical peak position at 1635 cm−1. This indicates the formation of some β-fold structure in the wave number range of amide I. Comparing the IR spectra of the SF before and after cationization, it can be shown that the polylysine grafting modified SF did increase the amino group, and the polylysine was successfully grafted to the SF.As shown in As shown in In order to compare the differences in mechanical properties between glucose-responsive MNs and pure SF MNs, the effect of Gox incorporation on the mechanical properties of MNs was investigated. −1 (amide I), 1540 cm−1 (amide II), and 1235 cm−1 (amide III), while pure SF MNs have distinct and sharp absorption peaks. These are the characteristic peaks of the β-fold structure. As can be seen from The microstructural changes of glucose-responsive SF MNs and pure SF MNs were observed. Meanwhile, glucose-responsive SF MNs showed acidic pH sensitivity. In contrast to pure SF MNs, the swelling properties are enhanced in an acidic environment. The reverse situation is exhibited in alkaline environments. The reason for this is that grafted SF is positively charged. The pH is smaller, the more positive charge there is. The repulsion of the positive charges increases the solubility of the MNs when it is acidic. As seen in We prepared a fluorescent value-concentration standard curve of fluorescently labeled human insulin to calculate the released drug concentration, shown in As shown in The MNs attachment method is provided in The earlier insulin MNs that were studied had soluble tips. After the patient applies the MNs, the immediate release of the drug from the front can satisfy the need for blood sugar reduction. However, SF also dissolves into the body. SF has good biocompatibility, but the effects caused by long-term accumulation in the body are unpredictable. Dissolving MNs can avoid this risk but they do not control the release of the drug, while excessive discharge may have the risk of causing hypoglycemia. Innovative, responsive MNs bridge this gap. In this study, cationized SF was obtained by graft copolymerizing SF with polylysine as MNs substrate, which gave the MNs acidic pH sensitivity.The blood level of insulin will significantly affect the BG concentration. In order to prevent hypoglycemic shock caused by an overdose of insulin, the MNs system is required to control the rate of insulin release autonomously and be able to respond to BG concentration. Glucose-responsive systems using Gox are always associated with pH-sensitive materials. It is possible to introduce Gox, which converts glucose to gluconic acid, creating an enzyme-mediated change in the pH environment. As illustrated in Equation (5) below, the regulation of the change in glucose concentration leads to the production of gluconic acid, which provides an acidic environment.3 and Gox, where NaHCO3 acts as a pH-sensitive component, to enable controlled insulin release. Furthermore, previous studies have demonstrated that silk protein can be used as an enzyme immobilization substrate. Lu et al. [This opens up the possibility of developing glucose-responsive SF MNs. It can be changed from pH-sensitive to glucose-sensitive, which enables controlled release of the drug at different BG concentrations. Ye et al.  reportedu et al.  revealed2O2, Gox, and insulin to the polylysine-modified SF solution. The MNs swell but do not dissolve when inserted into the skin. MNs have high mechanical strength, which can pierce the human epidermis under dry conditions and reach the dermis, creating a minimally invasive array of microchannels on the skin surface and enabling easy transdermal drug delivery. MNs piercing the skin rehydrate by absorbing interstitial fluid to form a semi-solid hydrogel with a porous cross-linked mesh structure and release internal insulin into the skin tissue in a controlled manner through the porous 3D mesh structure.Glucose-responsive SF MNs were obtained by adding HRP, H+ at low pH. Hydrogen ions enter the hydrogel to bind to the polylysine amino group, which positively charges the modified serine protein. Same-charged phase repulsion occurs, and it dissolves the MNs. Since MNs contain a large number of -OH, -COOH, -NH2, and other groups, the weakly ionized group -COOH in the SF fraction undergoes proton transfer at different pH. In acidic media, -OH and -NH2 groups are easily protonated to -H2O+ and -NH3+, which causes ionic and intermolecular interactions with water molecules and enhance the hydrophilic properties of the macromolecular chains inside the MNs. The lower the pH, the more H+, and with the greater the repulsion of the same charge, swelling degree increased. The number of protonated -OH and -NH2 groups decreased gradually with the increase in pH. The hydrophobicity of MNs is strengthened, the concentration of H+ in solution gradually decreases, and the repulsion of the same charge becomes smaller with less and less dissolution. As the glucose concentration increases, the transdermal drug diffusion capacity increases. The reason for this is the oxidation of glucose to gluconic acid under the action of Gox, which changes the local pH environment of the MNs and gradually increases the H+ concentration in the solution, promoting MNs lysis. Hence, transdermal insulin release is improved.The principle of transdermal drug release from glucose-responsive SF MNs under different pH and glucose concentration conditions was investigated by in vitro experiments. As An SD diabetic rat model was further established while using normal rats as a control group, testing the glucose responsiveness of MNs in vivo. A gradual decrease in BG level in diabetic rats after MNs application, dropped to 11.7 mmol/L after 6 h, indicates the MNs can effectively release insulin and lower blood sugar. The BG level in normal rats after MNs only slightly decreased and maintained stable. The comparison demonstrates that MNs have fast insulin release rate and large release volume at high BG concentrations with insulin-controlled release capability. Unlike direct insulin injection, modified SF MNs cause no hypoglycemia problems with only a slight insulin release at low BG concentrations. This indirectly proves the advantages of MNs for the sustained release of drugs. After rats were fed, it was noted that all three groups had elevated BG at 1 h after the meal, with the most significant increase in the experimental group of rats with insulin injection.For this reason, the digestion of food intake after eating can cause a sudden rise in BG. The insulin in the injection group has been degraded and challenging, making it difficult to lower blood glucose. Two hours after the meal, the BG of diabetic rats started to decrease gradually after the MNs, resulting from insulin release within the MNs. The comparison revealed that two normal rats’ blood glucose remained stable after the MNs, which reinforced the idea that these developed glucose-responsive SF MNs are indeed glucose-responsive with different insulin release rates at different BG concentrations.2O2 formed a pH-responsive cationic gel. The use of super-swelling polymers as a foundation material to SF MNs ensures a high swelling rate [In summary, we prepared SF MNs with pH and insulin responsiveness for biomedical applications. Cationized SF was obtained by graft copolymerization modification with polyllysine. Crosslinking with HRP/Hing rate . The inting rate ,46. Gox ing rate ,48 and ping rate  are wideing rate . We prepIn this paper, cationized SF was obtained by graft copolymerization modification of polylysine, and glucose-responsive SF MNs were prepared by adding insulin and Gox to this base material. The breaking strength of a single MN is more than 1.25 N, which can penetrate the human epidermis smoothly under dry conditions and achieve transdermal drug delivery. The MNs pierce the skin and absorb interstitial fluid to form a semi-solid hydrogel, which controls the release of internal insulin into the skin tissue through a porous 3D mesh structure. In vitro studies indicated that MNs exhibit excellent pH-responsive solubility and glucose-responsive insulin recovery. In vivo studies have shown that MNs control BG levels before and after feeding in diabetic SD rats as compared to conventional injections. This study demonstrates the potential application of glucose-responsive SF MNs in treating diabetes mellitus through transdermal insulin uptake. The research results of this paper lay a certain foundation for the intelligent transdermal release of insulin.


RING finger protein 43 (RNF43) encodes the transmembrane E3 ubiquitin ligase, which targets the Wnt receptor Frizzled (FZD). RNF43 mutations have been discovered in various human cancers including colon, pancreatic, stomach, ovarian, and liver cancers. Functional studies on RNF43 missense mutations have shown that they negatively regulate Wnt signaling; however, there are few functional studies on RNF43 frameshift mutations. In this study, we showed that R117fs and P441fs mutants enhanced Wnt/β-catenin signaling, whereas Q409fs and G659fs mutants retained the ability to suppress Wnt/β-catenin signaling. Specifically, R117fs was unable to ubiquitinate FZD5 due to lack of the RING domain, although it was able to interact with FZD5. Immunofluorescence showed that R117fs failed to internalize FZD5 expressed on the cell surface. We also showed that LGK974, a potent Wnt inhibitor, decreased the Wnt/β-catenin activity by R117fs and P441fs mutations. Together, these results demonstrate that RNF43 frameshift mutations retain normal functionality; thus, targeted anti-cancer therapy can be developed according to the mutation type of RNF43. Mutations of Wnt pathway components, such as APC, β-catenin, and Axin 1, result in aberrant activation of Wnt/β-catenin signaling. These mutations have been identified in various tumors and diseases3; therefore, precise regulation of Wnt signaling is important.Wnt signaling plays critical roles in the regulation of diverse processes including cell proliferation, survival, migration, and polarity. The Wnt ligand binds to its two receptors, Frizzled (FZD) and low-density lipoprotein-related receptors 5 and 6, to initiate intracellular signaling via β-catenin. In the absence of binding between Wnt ligand and its receptors, cytoplasmic β-catenin is targeted by a degradation complex composed of adenomatous polyposis coli (APC), Axin, and glycogen synthase kinase 3 α/β4. RING finger protein 43 (RNF43) is an E3 ligase that acts as a negative regulator of Wnt signaling by inducing degradation of the Wnt receptor FZD6. RNF43 mutations have been identified in various cancers, such as colorectal adenocarcinoma, pancreatic cancer, and gastric cancer (GC)7. The vast majority of RNF43 mutations are deletions or insertions8. We identified five frameshift mutations in patients with GC. Previous studies have used missense mutations to elucidate the functional role of RNF43; however, there have been no reports on the function of frameshift mutations.Ubiquitin-mediated regulation plays an essential role in Wnt signalingIn this study, we cloned the mutant types identified in GC patients and investigated whether truncated proteins due to frameshift mutations affect Wnt signaling. The results showed that R117fs is a positive regulator; R337*, Q409fs, and G659fs are negative regulators; and P441fs is a partial positive regulator of Wnt signaling. The positive effects induced by R117fs and P441fs were inhibited by the Wnt inhibitor LGK974, indicating that R117fs and P441fs are potential targets for Wnt inhibitors.9. To determine the function of these RNF43 truncating mutations, we cloned WT RNF43 and four truncating mutations  into expression plasmids, followed by transfection into HEK293T cells and subsequent western blot analysis with RNF43 antibody to detect the corresponding protein expression . As shown in Fig. Next, we determined whether LGK974, a Porcupine inhibitor that specifically inhibits Wnt secretion12. Therefore, we evaluated the effects of WT RNF43 and RNF43 truncated mutants on FZD5 expression by western blot analysis. We co-transfected C-terminal FLAG-tagged WT RNF43 or RNF43 truncated mutants with mFZD5-HA in HEK293T cells. Consistent with previous studies, overexpression of WT RNF43 selectively reduced the expression of mature mFZD5, the complex glycosylated form of FZD5, whereas the immature form remained unaffected5. However, treatment with bafilomycin A1, an inhibitor of autophagosome-lysosome fusion, prevented the degradation of mFZD5 by WT RNF43   domain of RNF43 interacts with FZD5, resulting in the suppression of Wnt/β-catenin signalingNext, we investigated whether the RNF43 truncated mutants can mediate the ubiquitination of FZD5. As shown in Fig. 14, leading to constitutive activation of β-catenin and abnormal activation of Wnt signaling. Therefore, the development of Wnt/β-catenin pathway inhibitors is very important for cancer therapies. The recent cancer genome atlas project and whole-genome sequencing analysis showed that genes associated with Wnt signaling are frequently mutated in gastric tumors15. Regulation of FZD receptor expression also influences Wnt signaling and plays an important role in cancer13. Several FZD receptors are upregulated in human tumors and cell lines18; therefore, turnover of Wnt receptors and cytoplasmic components of Wnt/β-catenin signaling is a critical regulatory mechanism in the Wnt signaling pathway. Ubiquitin-mediated degradation of Wnt receptors19, dishevelled20, Axin21, APC22, and β-catenin23 plays an important role in the regulation of Wnt signaling6. Ubiquitin-mediated protein degradation occurs in a three-step process via the sequential action of ubiquitin-activating (E1), ubiquitin-conjugating (E2), and ubiquitin-ligating (E3) enzymes24. RNF43, a cell surface transmembrane E3 ubiquitin ligase, shuts off the Wnt signaling pathway by promoting the ubiquitination and lysosomal degradation of FZD5. Thus, loss of function of RNF43 results in abnormal Wnt signaling. In this study, we demonstrated that the R117fs mutation led to loss of negative regulation of Wnt/β-catenin signaling, whereas other truncating mutations, including Q409fs and G659fs, maintained the function of RNF43.The components of the Wnt signaling pathway, such as APC and β-catenin, are frequently mutated in tumors25. In this study, we confirmed that FZD5 is localized at the cell surface in the absence of RNF43. However, FZD5 expression on the cell surface was decreased and localized in the cytoplasm in the presence of WT RNF43. This was not observed when the R117fs mutant lacking the RING domain was co-expressed with FZD5, while FZD5 expression at the cell surface was decreased upon co-expression with other mutants  and M51 Super 8 × FOPflash (TOPflash mutant) (Addgene plasmid #12457) were a gift from Randall Moon36. pcDNA-Wnt3A was a gift from Marian Waterman (Addgene plasmid #35908)37.Expression plasmids encoding full-length untagged and wild-type (WT) FLAG-tagged RNF43 were generated by PCR subcloning. Expression plasmids with point mutations were constructed by the introduction of these mutations by site-directed mutagenesis  according to the manufacturer’s instructions. Hemagglutinin (HA)-tagged ubiquitin and Renilla constructs were kindly provided by Dr. Kim . The pLEX307-mFzd5 plasmid was a gift from Henry Ho 2. HEK293T cells were seeded on laminin-coated glass coverslips. At 24 h after seeding, cells were co-transfected with WT FLAG-tagged RNF43 and various FLAG-tagged mutants or mFZD5-Myc using polyethyleneimine 38. At 48 h after seeding, cells were fixed in 4% paraformaldehyde, permeabilized with 0.2% Triton X-100 in phosphate-buffered saline (PBS) for 15 min at room temperature, and incubated with 1% bovine serum albumin to block nonspecific binding sites. Cells were labeled overnight at 4 °C with the following primary antibodies: anti-Myc , anti-FLAG , and anti-RNF43 (C252) . The cells were then incubated for 1 h at room temperature with Alexa Fluor-conjugated secondary antibodies, followed by staining with 4′,6-diamidino-2-phenylindole for 30 min. Finally, immunofluorescence was observed by confocal microscopy .HEK293T cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin at 37 °C in 5% COLuciferase assays were performed using the Dual-Luciferase Reporter Assay System  according to the manufacturer’s protocol. Briefly, HEK293T cells were transiently co-transfected with 100 ng WT RNF43 or RNF43 truncating mutations with 1 ng Renilla, 100 ng Wnt3A, and 100 ng of either TOP or FOPflash luciferase reporter. After 48 h, cells were lysed with passive lysis buffer and pelleted in a microcentrifuge. The luciferase activity of each lysate was measured using the Lumat LB 9507 luminometer  and normalized to the Renilla signal. All samples were measured in triplicate in each of three independent experiments. None of the constructs displayed significant FOPflash activity. LGK-974 was purchased from AdooQ Bioscience .HEK293T cells were transfected with the expression plasmids and treated overnight with bafilomycin A1  or MG132 . Then, transfected HEK293T cells were harvested and lysed in lysis buffer . Whole-cell lysates were centrifuged at 4 °C, and supernatants were incubated with anti-FLAG , anti-Myc , or anti-HA antibody (Santa Cruz) for 4 h, and with Protein G Agarose  for 12 h at 4 °C. After centrifugation, the Protein G Agarose beads were washed several times with PBS and the immunoprecipitated proteins were eluted from the beads with sample buffer. Protein samples were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and electrotransferred to nitrocellulose membranes. Membranes were probed with anti-FLAG M2-HRP , anti-HA-HRP , anti-ubiquitin (Santa Cruz), or anti-Myc (Thermo Fisher Scientific). Proteins were detected using Signal Light ECL Substrate  and quantified with ImageJ software . Images were captured using the Amersham Imager 600 system . The protein levels were quantified using Image J .Supplementary Information 1.Supplementary Information 2.


Td) which were both much higher than those of a typical bisphenol A dicyanate ester  after thermal polymerization. Here, the inorganic DDSQ nanomaterial improved the thermal behavior through a nano-reinforcement effect. Blending the inorganic DDSQ-OCN cage into the epoxy resin improved its thermal and mechanical stabilities after the ring-opening polymerization of the epoxy units during thermal polymerization. The enhancement in the physical properties arose from the copolymerization of the epoxy and OCN units to form the organic/inorganic covalently bonded network structure, as well as the hydrogen bonding of the OH groups of the epoxy with the SiOSi moieties of the DDSQ units. For example, the epoxy/DDSQ-OCN = 1/1 hybrid, prepared without Cu(II)-acac as a catalyst, exhibited a glass transition temperature, thermal decomposition temperature (Td), and char yield  that were significantly higher than those obtained when applying typical organic curing agents in the epoxy resin. The addition of Cu(II)-acac into the epoxy/BADCy and epoxy/DDSQ-OCN hybrids decreased the thermal stability  because the crosslinking density and post-hardening also decreased during thermal polymerization; nevertheless, it accelerated the thermal polymerization to a lower curing peak temperature, which is potentially useful for real applications as epoxy molding compounds.In this study, we prepared a difunctionalized cyanate ester double-decker silsesquioxane (DDSQ-OCN) cage with a char yield and thermal decomposition temperature ( The thermal polymerization peaks for the epoxy/BADCy and epoxy/BADCy/Cu(II)-acac hybrids appeared at 244 and 212 °C, respectively. Upon increasing the thermal polymerization temperature of the epoxy resin, the thermal polymerization peaks vanished. When we performed the thermal polymerization at 210 °C, the ring-opening polymerization of the remaining epoxy groups was characterized by peaks at 312 and 310 °C, respectively. Increasing the thermal polymerization temperature to 240 and 270 °C, however, caused the thermal polymerization peaks to disappear, which was indicative of complete thermal curing. These DSC analyses revealed that the addition of Cu(II)-acac as a catalyst decreased the thermal curing temperature; −1. The spectra of the pure DGEBA-type of epoxy resin exhibited absorption peaks at 914 cm−1 for the epoxy units in both −1, representing the stretching of secondary OH groups, suggested that the ring-opening thermal polymerization of the epoxy led to isocyanurate -acac, we recorded the FTIR spectra of these hybrids before and after thermal polymerization at 210, 240, and 270 °C  as a result of the O-C≡N units forming triazine structures, as displayed in Td10 and the char yield increased from 378 to 386 °C and from 17.5 to 21.5 wt%, respectively, for the epoxy/BADCy hybrid, and from 327 to 362 °C and from 11.2 and 18.5 wt%, respectively, for the epoxy/BADCy/Cu(II)-acac hybrid after thermal polymerization. The values of Td10 and the char yield of the epoxy/BADCy/Cu(acac)2 hybrid system were lower than those of the epoxy/BADCy hybrid; therefore, the catalyst decreased the thermal polymerization temperature but also decreased the crosslinking density and degree of post-hardening during the thermal polymerization procedure.3 units at 1261 cm−1, and a strong signal at 1097 cm−1 for the Si–O–Si units; the signals for the C=O groups of DDSQ-4OH were located at 1709 and 1772 cm−1, with a broad signal centered at 3420 cm−1 representing the phenolic OH units. When the substitution reaction was complete, the spectrum of DDSQ-OCN featured signals at 2201, 2239, and 2277 cm−1 for the O-C≡N units, which were similar to those of the BADCy monomer in −1. Upon increasing the amount of epoxy resin in the DDSQ-OCN monomer, the thermal polymerization temperature decreased, again indicating that the epoxy units themselves could catalyze the cyclotrimerization of the O-C≡N units, this time for the DDSQ-OCN monomer. For example, the thermal polymerization peak appeared at 192 °C for the epoxy/DDSQ-OCN = 1/1 hybrid; this peak shifted significantly to 158 °C for the epoxy/DDSQ-OCN = 7/1 hybrid. Furthermore, the values of Td10 and the char yield also decreased significantly, from 408 °C and 44.8 wt%, respectively, for the epoxy/DDSQ-OCN = 1/1 hybrid, and to 299 °C and 15.5 wt%, respectively, for the epoxy/DDSQ-OCN = 7/1 hybrid, with the inorganic DDSQ nanomaterial improving these thermal properties through its nano-reinforcement effect.−1, while that of DDSQ-OCN displayed signals for the O-C≡N units at 2201, 2239, and 2277 cm−1; both sets of signals disappeared after thermal polymerization at each temperature. Furthermore, a strong, broad signal for OH stretching appeared at 3100–3450 cm−1, indicating that the ring opening of the epoxy had occurred, with signals for isocyanurateamine, oxazolidinone, and triazine units appearing at 1753, 1703, 1614, 1505, 1362, and 1251 cm−1, arising from the O-C≡N units. In addition, the signal for the Si–O–Si units of the epoxy/DDSS-OCN hybrid at 1092 cm−1 shifted to 1103 cm−1 after thermal polymerization, suggesting that the OH units of the epoxy resin formed hydrogen bonds with the Si–O–Si units of the DDSQ cage [DSQ cage . We obseDSQ cage ,48; hereTd10 and the char yield both increased after applying the various thermal polymerization temperatures, which is consistent with a crosslinking structure of DDSQ-OCN having formed in the epoxy matrix, thereby enhancing the thermal properties, similarly to the behavior of the epoxy/BADCy systems. Furthermore, the values of Td10 and the char yield of the epoxy/DDSQ-OCN/Cu(acac)2 hybrids were lower than those of the epoxy/DDSQ-OCN hybrid; again, this behavior is similar to that of the epoxy/BADCy systems, with decreases in the crosslinking density and the degree of post-hardening occurring during thermal polymerization. All of the values of Td10 and the char yield of the various epoxy/DDSQ-OCN hybrids, in the presence and absence of Cu(II)-acac, are summarized in 2 hybrids caused the thermal stability to increase. Furthermore, the thermal stability of the epoxy/DDQQ-OCN hybrids was greater than that of the epoxy/BADCy hybrids because the inorganic DDSQ cages exerted a nano-reinforcement effect; in addition, the epoxy resin presumably formed hydrogen bonds with the DDSQ cages. For example, the values of Td10 and the char yield of epoxy/DDSQ-OCN = 1/1 were 427 °C and 51.0 wt%, respectively; these values are much higher than those  of epoxy/BADCy = 1/1 after thermal polymerization at 270 °C in the absence of Cu(II)-acac. We found that increasing the concentration of DDSQ-OCN in both the epoxy/DDSQ-OCN and epoxy/DDSQ-OCN/Cu(acac)Tg value and storage modulus (E′) due to the mechanical property. The initial storage modulus of epoxy/BADCy = 5/1 at 25 °C was 11,608 MPa, with a value of Tg of 162 °C. The epoxy/DDSQ-OCN = 5/1 hybrid exhibited lower initial values of E′ and Tg , presumably because the molecular weight (Mw) of DDSQ-OCN (1713 g/mol) was approximately six times that of BADCy (278 g/mol), and thus the molar ratio of DDSQ-OCN relative to epoxy was lower than that of BADCy at the same weight ratio. As a result, the poorer mechanical properties and lower value of Tg arose because of a lower crosslinking density in the epoxy/DDSQ-OCN hybrid at the same weight ratio, compared with that in the epoxy/BADCy hybrid. Further increasing the concentration of DDSQ-OCN in the epoxy resin caused the values of E′ and Tg to increase to 11,110 MPa and 105 °C, respectively, for epoxy/DDSQ-OCN = 3/1, and to 12,410 MPa and 166 °C, respectively, for epoxy/DDSQ-OCN = 1/1, due to the rigid inorganic DDSQ nanoparticles enhancing the nano-reinforcement effect, while also increasing the crosslinking of the structure and decreasing the mobility of the epoxy resin. Comparing the epoxy/BADCy= 5/1 and epoxy/DDSQ-OCN = 1/1 hybrids with similar molar ratios of their cyanate ester functional units, the presence of DDSQ-OCN appeared to provide superior mechanical properties and values of Tg when compared with the effects of BADCy; thus, incorporating the cubic DDSQ cages could indeed improve the thermal and mechanical properties.Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) revealed the dispersion of inorganic DDSQ-OCN cages in the epoxy resin. The bisphenol A, tetrahydrofuran (THF), and Cu(II) 2,4-pentanedionate (Cu(II)-acac) were purchased from Alfa–Aesar. The triethylamine, cyanogen bromide (BrCN), methanol (MeOH), and cyclohexane were purchased from Sigma–Aldrich. The DDSQ-OCN was synthesized as described previously . The epo2. THF (100 mL) was added slowly while stirring rapidly. The solution was cooled to −25 °C, and then triethylamine (3.41 mL) was added slowly over 30 min. The temperature was stabilized at −30 °C by immersion in a Dewar flask containing a MeOH/liquid N2 mixture. The reaction was complete after 4 h. The white salt was filtered off. Ice water (500 mL) was added to the filtrate to form a white precipitate. This crude product was recrystallized from cyclohexane to obtain a white powder .Bisphenol A  and BrCN  were placed in a flask under a blanket of NVarious epoxy/BADCy and epoxy/DDSQ-OCN hybrids, in the presence and absence Cu(II)-acac (0.01 wt%), were stirred for 48 h at 60 °C under a vacuum. Each casting sample was placed into an aluminum tray and subjected to thermal polymerization at 210, 240, or 270 °C for 2 h. The epoxy hybrids were obtained with a dark brown color.Td of the DDSQ-OCN monomer were higher than those of the typical BADCy monomer (without DDSQ cages) after thermal polymerization, because the inorganic DDSQ cages enhanced the thermal behavior through a nano-reinforcement effect. SEM and TEM images revealed that the inorganic DDSQ-OCN cages were dispersed homogeneously in the resulting epoxy resins. Therefore, the values of Tg and Td and the storage modulus of these epoxy/DDSQ hybrids all increased significantly as a result of restricted chain mobility, which arose from hydrogen bonding between the OH units of the epoxy resin  and the SiOSi units of the DDSQ cages (based on FTIR spectroscopy), as well as covalent bonding after the copolymerization of the epoxy and cyanate ester units. The values of Tg and Td for the epoxy/DDSQ-OCN = 1/1 hybrid, prepared without Cu(II)-acac as a catalyst  were significantly higher than those obtained when adding typical organic curing agents to DGEBA-type epoxy resin, because of the effect of the rigid inorganic DDSQ cage nanomaterials.We prepared BADCy and DDSQ-OCN cyanate monomers through the substitution of the phenolic functional groups of BPA and DDSQ-4OH with BrCN. The char yield and value of