Recent progress of polymeric microneedle-assisted long-acting transdermal drug delivery.

Microneedle (MN)-assisted drug delivery technology has gained increasing attention over the past two decades. Its advantages of self-management and being minimally invasive could allow this technology to be an alternative to hypodermic needles. MNs can penetrate the stratum corneum and deliver active ingredients to the body through the dermal tissue in a controlled and sustained release. Long-acting polymeric MNs can reduce administration frequency to improve patient compliance and therapeutic outcomes, especially in the management of chronic diseases. In addition, long-acting MNs could avoid gastrointestinal reactions and reduce side effects, which has potential value for clinical application. In this paper, advances in design strategies and applications of long-acting polymeric MNs are reviewed. We also discuss the challenges in scale manufacture and regulations of polymeric MN systems. These two aspects will accelerate the effective clinical translation of MN products.

Liquid-Driven Microinjection System for Precise Fundus Injection.

Microinjection is usually applied to the treatment of some retinal disorders, such as retinal vein cannulation and displaced submacular hemorrhage. Currently, the microinjection procedure is usually performed by using the viscous fluid control of a standard vitrectomy system, which applies a fixed air pressure through foot pedal activation. The injection process with the fixed pressure is uncontrollable and lacks feedback, the high flow rate of the injected drug may cause damage to the fundus tissue. In this paper, a liquid-driven microinjection system with a flow sensor is designed and developed specifically for fundus injection. In addition, a PID sliding mode control (SMC) method is proposed to achieve precise injection in the injection system. The experimental results of fundus simulation injection demonstrate that the microinjection system meets the requirements of fundus injection and reduces the impact of the injection process on the fundus tissue.

Fertility testing of preserved epididymal sperm by microinjection: A model for the rescue and utilization of genetically superior animals.

Background: Epididymal sperm preservation is a simple conservation approach that can help prevent the loss of high genetic quality of farm animals. The chance of loss increases, especially during disease outbreaks or other interruptions to normal reproduction function. Aim: This study looked into the ability of preserved ram epididymal sperm to fertilize oocytes. Due to motility becoming an issue following sperm storage for fertilization, the sperm microinjection known as intracytoplasmic sperm injection approach was employed. Methods: The study was divided into two parts. First, involved the preservation of epididymal sperm at 5°C for 12 days. During preservation, sperm quality parameters namely motility, viability, intact membrane, acrosome, and Deoxyribonucleic acid (DNA) are evaluated every three days. For the fertility test in the second experiment, matured oocytes were injected with immotile sperm discovered in the last days of preservation. The presence of pronucleus development following in vitro culture is used as an indicator of sperm's ability to activate and fertilize oocytes. Results: All sperm quality parameters significantly (p < 0.05) declined during preservation time. On day 12, motility was discovered to be 0%, but viable sperm, sperm with intact membrane, acrosome, and DNA remained at 41.86% ± 9.30%, 31.18% ± 5.15%, 21.88% ± 1.93%, and 33.35% ± 8.74%, respectively. On the fertility test, we inject immotile sperm from day 12 of preservation, which has the lowest motility found, into matured oocytes. Those sperms are able to activate (52.05% ± 7.15%) and fertilize (31.37% ± 1.75%) the injected oocytes, but their fertilizing ability is significantly lower (p < 0.05) when compared to the sperm derived from the ejaculate. Conclusion: In this study, simple preservation of epididymal sperm reduces all sperm quality criteria, particularly motility. Using the microinjection approach preserved sperm which had no motility, still demonstrated its ability to activate and fertilize the oocytes. According to that, this study provides potential approaches and tools for using genetically superior animals that have lost their ability to execute regular fertilization, and also prolong reproduction function.

A Hollow Microneedle Equipped with a Micropillar for Improved Needle Insertion and Injection of Drug Solution.

PURPOSE: Hollow-type microneedles (hMNs) are a promising device for the effective administration of drugs into intradermal sites. Complete insertion of the needle into the skin and administration of the drug solution without leakage must be achieved to obtain bioavailability or a constant effect. In the present study, several types of hMN with or without a rounded blunt tip micropillar, which suppresses skin deformation, around a hollow needle, and the effect on successful needle insertion and administration of a drug solution was investigated. Six different types of hMNs with needle lengths of 1000, 1300, and 1500 µm with or without a micropillar were used. METHODS: Needle insertion and the disposition of a drug in rat skin were investigated. In addition, the displacement-force profile during application of hMNs was also investigated using a texture analyzer with an artificial membrane to examine needle factors affecting successful insertion and administration of a drug solution by comparing with in vivo results. RESULTS: According to the results with the drug distribution of iodine, hMN1300 with a micropillar was able to successfully inject drug solution into an intradermal site with a high success rate. In addition, the results of displacement-force profiles with an artificial membrane showed that a micropillar can be effective for depth control of the injected solution as well as the prevention of contact between the hMN pedestal and the deformed membrane. CONCLUSION: In the present study, hMN1300S showed effective solution delivery into an intradermal site. In particular, a micropillar can be effective for depth control of the injected solution as well as preventing contact between the hMN pedestal and the deformed membrane. The obtained results will help in the design and development of hMNs that ensure successful injection of an administered drug.

Applications and prospects of microneedles in tumor drug delivery.

As drug delivery devices, microneedles are used widely in the local administration of various drugs. Such drug-loaded microneedles are minimally invasive, almost painless, and have high drug delivery efficiency. In recent decades, with advancements in microneedle technology, an increasing number of adaptive, engineered, and intelligent microneedles have been designed to meet increasing clinical needs. This article summarizes the types, preparation materials, and preparation methods of microneedles, as well as the latest research progress in the application of microneedles in tumor drug delivery. This article also discusses the current challenges and improvement strategies in the use of microneedles for tumor drug delivery.

Microinjection With Nanoparticles to Deliver Drugs in Prenatal Lung Explants - A Pilot Study for Prenatal Therapy in Congenital Diaphragmatic Hernia.

BACKGROUND: Fetoscopic endoluminal tracheal occlusion (FETO) improves the survival rate in fetuses with severe congenital diaphragmatic hernia (CDH). We hypothesize that prenatal therapies into the trachea during FETO can further improve outcomes. Here, we present an ex vivo microinjection technique with rat lung explants to study prenatal therapy with nanoparticles. METHODS: We used microsurgery to isolate lungs from rats on embryonic day 18. We injected chitosan nanoparticles loaded with fluorescein (FITC) into the trachea of the lung explants. We compared the difference in biodistribution of two types of nanoparticles, functionalized IgG-conjugated nanoparticles (IgG-nanoparticles) and bare nanoparticles after 24 h culture with immunofluorescence (IF). We used IF to mark lung epithelial cells with E-cadherin and to investigate an apoptosis (Active-caspase 3) and inflammatory marker (Interleukin, IL-6) and compared its abundance between the two experimental groups and control lung explants. RESULTS: We detected the presence of nanoparticles in the lung explants, and the relative number of nanoparticles to cells was 2.49 fold higher in IgG-nanoparticles than bare nanoparticles (p < 0.001). Active caspase-3 protein abundance was similar in the control, bare nanoparticles (1.20 fold higher), and IgG-nanoparticles (1.34 fold higher) groups (p = 0.34). Similarly, IL-6 protein abundance was not different in the control, bare nanoparticles (1.13 fold higher), and IgG-nanoparticles (1.12 fold higher) groups (p = 0.33). CONCLUSIONS: Functionalized nanoparticles had a higher presence in lung cells and this did not result in more apoptosis or inflammation. Our proof-of-principle study will guide future research with therapies to improve lung development prenatally. LEVELS OF EVIDENCE: N/A TYPE OF STUDY: Animal and laboratory study.

High-throughput genetic manipulation of multicellular organisms using a machine-vision guided embryonic microinjection robot.

Microinjection is a technique used for transgenesis, mutagenesis, cell labeling, cryopreservation, and in vitro fertilization in multiple single and multicellular organisms. Microinjection requires specialized skills and involves rate-limiting and labor-intensive preparatory steps. Here, we constructed a machine-vision guided generalized robot that fully automates the process of microinjection in fruit fly (Drosophila melanogaster) and zebrafish (Danio rerio) embryos. The robot uses machine learning models trained to detect embryos in images of agar plates and identify specific anatomical locations within each embryo in 3D space using dual view microscopes. The robot then serially performs a microinjection in each detected embryo. We constructed and used three such robots to automatically microinject tens of thousands of Drosophila and zebrafish embryos. We systematically optimized robotic microinjection for each species and performed routine transgenesis with proficiency comparable to highly skilled human practitioners while achieving up to 4× increases in microinjection throughput in Drosophila. The robot was utilized to microinject pools of over 20,000 uniquely barcoded plasmids into 1,713 embryos in 2 days to rapidly generate more than 400 unique transgenic Drosophila lines. This experiment enabled a novel measurement of the number of independent germline integration events per successfully injected embryo. Finally, we showed that robotic microinjection of cryoprotective agents in zebrafish embryos significantly improves vitrification rates and survival of cryopreserved embryos post-thaw as compared to manual microinjection. We anticipate that the robot can be used to carry out microinjection for genome-wide manipulation and cryopreservation at scale in a wide range of organisms.

Progress in the Application of Microneedles in Eye Disorders and the Proposal of the Upgraded Microneedle with Spinule.

PURPOSE: In the local administration methods for treating eye diseases, the application of microneedles has great potential due to the shortcomings of low efficacy and significant side effects of local administration preparations. This article provides ideas for the research on the application of ophthalmic microneedle in the treatment of eye diseases. RESULTS: This article analyzes the physiological structures of the eyes, ocular diseases and its existing ocular preparations in sequence. Finally, this article reviews the development and trends of ocular microneedles in recent years, and summarizes and discusses the drugs of ocular microneedles as well as the future directions of development. At the same time, according to the inspiration of previous work, the concept of "microneedle with spinule" is proposed for the first time, and its advantages and limitations are discussed in the article. CONCLUSIONS: At present, the application of ocular microneedles still faces multiple challenges. The aspects of auxiliary devices, appearance, the properties of the matrix materials, and preparation technology of ophthalmic microneedle are crucial for their application in the treatment of eye diseases.

Recent advances in hydrogel microneedle-based biofluid extraction and detection in food and agriculture.

Microneedle (MN) technology has been extensively studied for its advantages of minimal invasiveness and user-friendliness. Notably, hydrogel microneedles (HMNs) have garnered considerable attention for biofluid extraction due to its high swelling properties and biocompatibility. This review provides a comprehensive overview of definition, materials, and fabrication methods associated with HMNs. The extraction mechanisms and optimization strategies for enhancing extraction efficiency are summarized. Moreover, particular emphasis is placed on HMN-based biofluid extraction and detection in the domains of food and agriculture, encompassing the detection of small molecules, nucleic acids, and other relevant analytes. Finally, current challenges and possible solutions associated with HMN-based biofluid extraction are discussed.

[Applications of microneedles in non-transdermal drug delivery: a review].

In recent years, microneedles have emerged as a drug delivery technology that holds great research value and application potential due to their minimally invasive, painless, user-friendly, and efficient characteristics. The technology of microneedles has rapidly evolved over the past 20 years, allowing customization of shape, composition, mechanical properties, and unique functions to meet diverse needs. With the ability to minimally invasively traverse various biological barriers, researchers have explored the applications of microneedles in various tissues and organs beyond the skin. This article summarizes the research progress on the use of microneedles for drug delivery in tissues such as eyes, blood vessel, and heart. By presenting these cutting-edge research to readers, we hope to promote the development and application of microneedle technology.

Generation of glial cell-derived neurotrophic factor (gdnf) morphants in zebrafish larvae by cerebroventricular microinjection of vivo morpholino.

Dopaminergic neurons in the brain are an important source of dopamine, which is a crucial neurotransmitter for wellbeing, memory, reward, and motor control. Deficiency of dopamine due to advanced age and accumulative dopaminergic neuron defects can lead to movement disorders such as Parkinson's disease. Glial cell-derived neurotrophic factor (GDNF) is one of many factors involved in dopaminergic neuron development and/or survival. However, other endogenous GDNF functions in the brain await further investigation. Zebrafish is a well-established genetic model for neurodevelopment and neurodegeneration studies. Importantly, zebrafish shares approximately 70% functional orthologs with human genes including GDNF. To gain a better understanding on the precise functional role of gdnf in dopaminergic neurons, our laboratory devised a targeted knockdown of gdnf in the zebrafish larval brain using vivo morpholino. Here, detailed protocols on the generation of gdnf morphants using vivo morpholino are outlined. This method can be applied for targeting of genes in the brain to determine specific spatiotemporal gene function in situ.

Comparison of Embryological Results of Microinjection in Two Groups of Men with and without Requesting Sperm DNA Fragmentation Index Measurement.

Introduction: The sperm DNA fragmentation index (DFI) is considered a valuable measure to assess male fertility, but the predictive value of DFI for the outcomes in assisted reproductive technology (ART) is still controversial. Therefore, this study is aimed at investigating the effect of requesting a DFI test or performing ART without DFI on the results observed in the embryology laboratory (number of embryos, fertilization rate, and embryo quality) after intracytoplasmic sperm injection (ICSI). Methods: This retrospective study was conducted on infertile men who underwent ICSI and were referred to the Avicenna Infertility and Recurrent Abortion Treatment Center in Tehran from 2019 to 2022. The samples were categorized into two groups: a case group with DFI measurement and a control group without DFI measurement. We conducted a comparative analysis of the embryology results between the two groups, focusing on parameters such as fertilization rate, number of embryos, and embryo quality. t-tests and Mann-Whitney U tests were used to conduct single variable analysis. Potential confounding effects were adjusted to use the multivariate linear and logistic regression. Results: Data analysis showed no significant statistical difference between the case group and the control group in terms of the number of embryos (95% confidence interval for the regression coefficient (ß) = -0.257-0.123), and embryo quality (95% confidence interval for ß = -0.199-0.114). There was no significant statistical difference between the two groups due to the fertilization rate (95% confidence interval for ß = -3.42-3.42), except for the variables of woman's age and sperm count after ICSI, as determined by adjusted linear regression. Conclusions: Although DFI measurement is used to assess male infertility, its importance as a predictor for the embryology outcomes after ICSI requires further evaluation and the determination of a cut-off point for predicting results. This study was based on retrospectively collected DFI data, and prospective studies confirming the superiority of ICSI outcomes are necessary.

Establishing a Zebrafish Model for Borrelia burgdorferi Infection Using Immersion and Microinjection Methods.

Borrelia burgdorferi is the spirochetal bacterium that causes Lyme disease. Even though antimicrobial sensitivity of B. burgdorferi has been widely studied, there is still a need to develop an affordable, practical, high-throughput in vivo model which can be used to find effective antibiotic therapies, especially for the recently discovered persister and biofilm forms. Here, we describe the immersion and microinjection methods to introduce B. burgdorferi spirochetes into zebrafish larvae. The B. burgdorferi-zebrafish model can be produced by immersing 5-day post-fertilization (dpf) zebrafish in a B. burgdorferi culture, or by injecting B. burgdorferi into the hindbrain of zebrafish at 28 h post-fertilization (hpf). To demonstrate that B. burgdorferi indeed infect the fish, nested polymerase chain reaction (PCR), reverse transcription PCR (RT-PCR), live fluorescence imaging, histological staining, and wholemount immunohistochemical (IHC) methods can be used on B. burgdorferi-infected zebrafish.

A micro-fabricated device (microICSI) improves porcine blastocyst development and procedural efficiency for both porcine intracytoplasmic sperm injection and human microinjection.

PURPOSE: Intracytoplasmic sperm injection (ICSI) imparts physical stress on the oolemma of the oocyte and remains among the most technically demanding skills to master, with success rates related to experience and expertise. ICSI is also time-consuming and requires workflow management in the laboratory. This study presents a device designed to reduce the pressure on the oocyte during injection and investigates if this improves embryo development in a porcine model. The impact of this device on laboratory workflow was also assessed. METHODS: Porcine oocytes were matured in vitro and injected with porcine sperm by conventional ICSI (C-ICSI) or with microICSI, an ICSI dish that supports up to 20 oocytes housed individually in microwells created through microfabrication. Data collected included set-up time, time to align the polar body, time to perform the injection, the number of hand adjustments between controllers, and degree of invagination at injection. Developmental parameters measured included cleavage and day 6 blastocyst rates. Blastocysts were differentially stained to assess cell numbers of the inner cell mass and trophectoderm. A pilot study with human donated MII oocytes injected with beads was also performed. RESULTS: A significant increase in porcine blastocyst rate for microICSI compared to C-ICSI was observed, while cleavage rates and blastocyst cell numbers were comparable between treatments. Procedural efficiency of microinjection was significantly improved with microICSI compared to C-ICSI in both species. CONCLUSION: The microICSI device demonstrated significant developmental and procedural benefits for porcine ICSI. A pilot study suggests human ICSI should benefit equally.

Leveraging novel innovative thermoresponsive polymers in microneedles for targeted intradermal deposition.

Microneedles have garnered considerable attention over the years as a versatile pharmaceutical platform that could be leveraged to deliver drugs into and across the skin. In the current work, poly (N-isopropylacrylamide) (PNIPAm) is synthesized and characterized as a novel material for the development of a physiologically responsive microneedle-based drug delivery system. Typically, this polymer transitions reversibly between a swell state at lower temperatures and a more hydrophobic state at higher temperatures, enabling precise drug release. This study demonstrates that dissolving microneedles patches made from PNIPAm, incorporating BIS-PNIPAm, a crosslinked polymer variant, exhibit enhanced mechanical properties, evident from a smaller height reduction in microneedle (∼10 %). Although microneedles using PNIPAm alone were achievable, it displayed poor mechanical strength, requiring the inclusion of additional polymeric excipients like PVA to enhance mechanical properties. In addition, the incorporation of a thermoresponsive polymer did not have a significant (p > 0.05) impact on the insertion properties of the needles as all formulations inserted to a similar depth of 500 µm into ex vivo skin. Furthering this, the needles were loaded with a model payload, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine perchlorate (DID) and the deposition of the cargo was monitored via multiphoton microscopy that showed that a deposit is formed at a depth of ≈200 µm. Also, it was revealed that crosslinked-PNIPAm (Bis-PNIPAm) formulations exhibited notable skin accumulationof the dye only after 4 h, independent of the excipient matrix used. This phenomenon was absent in non-crosslinked PNIPAm formulations, indicating a deposit formation in Bis-PNIPAm microneedle formulation. Collectively, this proof-of-concept study has advanced our understanding on the possibility to use PNIPAm for dissolving microneedle fabrication which could be harnessed for the deposition of nanoparticles into the dermis, for extended drug release within the skin.

Alpha1- and Beta-norepinephrinergic receptors of dorsomedial and ventromedial hypothalamic nuclei modulate panic attack-like defensive behaviour elicited by diencephalic GABAergic neurotransmission disinhibition.

Gamma-aminobutyric acid (GABA) disinhibition in medial hypothalamus (MH) nuclei of rats elicits some defensive reactions that are considered panic attack-like behaviours. Recent evidence showed that the norepinephrine-mediated system modulates fear-related defensive behaviours organised by MH neurons at least in part via noradrenergic receptors recruitment on midbrain tegmentum. However, it is unknown whether noradrenergic receptors of the MH also modulate the panic attack-like reactions. The aim of this work was to investigate the distribution of noradrenergic receptors in MH, and the effects of either α1-, α2- or ß-noradrenergic receptors blockade in the MH on defensive behaviours elaborated by hypothalamic nuclei. Defensive behaviours were evaluated after the microinjection of the selective GABAA receptor antagonist bicuculline into the MH that was preceded by microinjection of either WB4101, RX821002, propranolol (α1-, α2- and ß-noradrenergic receptor selective antagonists, respectively), or physiological saline into the MH of male Wistar rats. The α1-, α2- and ß-noradrenergic receptors were found in neuronal perikarya of all MH nuclei, and the α2-noradrenergic receptor were also found on glial cells mainly situated in the ventrolateral division of the ventromedial hypothalamic nucleus. The α1- and ß-noradrenergic receptors blockade in the MH decreased defensive attention and escape reactions elicited by the intra-MH microinjections of bicuculline. These findings suggest that, despite the profuse distributions of α1-, α2- and ß-noradrenergic receptors in the MH, both α1- and ß-noradrenergic receptor- rather than α2-noradrenergic receptor-signalling in MH are critical for the neuromodulation of panic-like behaviour.

4D-printed microneedles from dual-sensitive chitosan for non-transdermal drug delivery.

Microneedles are a promising micro-scale drug delivery platform that has been under development for over two decades. While 3D printing technology has been applied to fabricate these systems, the challenge of achieving needle sharpness remains. In this study, we present an innovative approach for microneedle fabrication using digital light processing (DLP) 3D printing and smart chitosan biomaterial. For the first time, we used hydroxybutyl methacrylated chitosan (HBCMA), which possesses dual temperature- and photo-sensitive properties, to create microneedles. The DLP approach enabled a quick generation of HBCMA-based microneedles with a high resolution. The microneedles exhibited 4D properties with a change in needle dimensions upon exposure to temperature, which enhances resolution, sharpens needles, and improves mechanical strength. We demonstrated the ability of these microneedles to load, deliver, sustained release small molecular drugs and penetrate soft tissue. Overall, the HBCMA-based microneedles show promising potential in non-dermal drug delivery applications.

Microneedle-Mediated Cell Therapy.

Microneedles have emerged as a promising platform for transdermal drug delivery with prominent advantages, such as enhanced permeability, mitigated pain, and improved patient adherence. While microneedles have primarily been employed for delivering small molecules, nucleic acids, peptides, and proteins, recent researches have demonstrated their prospect in combination with cell therapy. Cell therapy involving administration or transplantation of living cells (e.g. T cells, stem cells, and pancreatic cells) has gained significant attention in preclinical and clinical applications for various disease treatments. However, the effectiveness of systemic cell delivery may be restricted in localized conditions like solid tumors and skin disorders due to limited penetration and accumulation into the lesions. In this perspective, an overview of recent advances in microneedle-assisted cell delivery for immunotherapy, tissue regeneration, and hormone modulation, with respect to their mechanical property, cell loading capacity, as well as viability and bioactivity of the loaded cells is provided. Potential challenges and future perspectives with microneedle-mediated cell therapy are also discussed.

An Ultraswelling Microneedle Device for Facile and Efficient Drug Loading and Transdermal Delivery.

The advancement and extensive demand for transdermal therapies can benefit from a safe, and efficient and user-friendly transdermal technology with broad applicability in delivering various hydrophilic drugs. Here the design and proof of concept applications of an ultraswelling microneedle device that enables the facile and efficient loading and transdermal delivery of hydrophilic drugs with different molecular weights is reported. The device consists of a super-hydrophilic hydrogel microneedle array and a resin base substrate. Using a special micromolding technique that involves hydrated crosslinking and cryogenic-demolding, the microneedle part displays a rapid swelling ratio of ≈3800%, enabling the loading of drugs up to 500 kDa in molecular weight. The drug loading process using the device just involves incubating the microneedle part in a drug solution for 1 min, followed by 15 min of drying. The microneedles can easily penetrate the skin under press and detach from the base substrate under shear, thereby releasing the payload. Administration of desired therapeutic agents using the device outperformed conventional administration methods in mitigating psoriasis and eliciting immunity. This biocompatible device, capable of withstanding ethylene oxide sterilization, can enhance the efficacy and accessibility of transdermal therapies in research institutes, hospitals, and even home settings.

Advances in biomedical systems based on microneedles: design, fabrication, and application.

Wearable devices have become prevalent in biomedical studies due to their convenient portability and potential utility in biomarker monitoring for healthcare. Accessing interstitial fluid (ISF) across the skin barrier, microneedle (MN) is a promising minimally invasive wearable technology for transdermal sensing and drug delivery. MN has the potential to overcome the limitations of conventional transdermal drug administration, making it another prospective mode of drug delivery after oral and injectable. Subsequently, combining MN with multiple sensing approaches has led to its extensive application to detect biomarkers in ISF. In this context, employing MN platforms and control schemes to merge diagnostic and therapeutic capabilities into theranostic systems will facilitate on-demand therapy and point-of-care diagnostics, paving the way for future MN technologies. A comprehensive analysis of the growing advances of microneedles in biomedical systems is presented in this review to summarize the latest studies for academics in the field and to offer for reference the issues that need to be addressed in MN application for healthcare. Covering an array of novel studies, we discuss the following main topics: classification of microneedles in the biomedical field, considerations of MN design, current applications of microneedles in diagnosis and therapy, and the regulatory landscape and prospects of microneedles for biomedical applications. This review sheds light on the significance of microneedle-based innovations, presenting an analysis of their potential implications and contributions to the community of wearable healthcare technologies. The review provides a comprehensive understanding of the field's current state and potential, making it a valuable resource for academics and clinicians seeking to harness the full potential of MN applications.