Array tomography of in vivo labeled synaptic receptors.

Array tomography (AT) allows one to localize sub-cellular components within the structural context of cells in 3D through the imaging of serial sections. Using this technique, the z-resolution can be improved physically by cutting ultra-thin sections. Nevertheless, conventional immunofluorescence staining of those sections is time consuming and requires relatively large amounts of costly antibody solutions. Moreover, epitopes are only readily accessible at the section's surface, leaving the volume of the serial sections unlabeled. Localization of receptors at neuronal synapses in 3D in their native cellular ultrastructural context is important for understanding signaling processes. Here, we present in vivo labeling of receptors via fluorophore-coupled tags in combination with super-resolution AT. We present two workflows where we label receptors at the plasma membrane: first, in vivo labeling via microinjection with a setup consisting of readily available components and self-manufactured microscope table equipment and second, live receptor labeling by using a cell-permeable tag. To take advantage of a near-to-native preservation of tissues for subsequent scanning electron microscopy (SEM), we also apply high-pressure freezing and freeze substitution. The advantages and disadvantages of our workflows are discussed.

Nanosuspension-Loaded Dissolving Microneedle Patches for Enhanced Transdermal Delivery of a Highly Lipophilic Cannabidiol.

Purpose: Transdermal Drug Delivery System (TDDS) offers a promising alternative for delivering poorly soluble drugs, challenged by the stratum corneum's barrier effect, which restricts the pool of drug candidates suitable for TDDS. This study aims to establish a delivery platform specifically for highly lipophilic drugs requiring high doses (log P > 5, dose > 10 mg/kg/d), to improve their intradermal delivery and enhance solubility. Methods: Cannabidiol (CBD, log P = 5.91) served as the model drug. A CBD nanosuspension (CBD-NS) was prepared using a bottom-up method. The particle size, polydispersity index (PDI), zeta potential, and concentration of the CBD-NS were characterized. Subsequently, CBD-NS was incorporated into dissolving microneedles (DMNs) through a one-step manufacturing process. The intradermal dissolution abilities, physicochemical properties, mechanical strength, insertion depth, and release behavior of the DMNs were evaluated. Sprague-Dawley (SD) rats were utilized to assess the efficacy of the DMN patch in treating knee synovitis and to analyze its skin permeation kinetics and pharmacokinetic performance. Results: The CBD-NS, stabilized with Tween 80, exhibited a particle size of 166.83 ± 3.33 nm, a PDI of 0.21 ± 0.07, and a concentration of 46.11 ± 0.52 mg/mL. The DMN loaded with CBD-NS demonstrated favorable intradermal dissolution and mechanical properties. It effectively increased the delivery of CBD into the skin, extended the action's duration in vivo, and enhanced bioavailability. CBD-NS DMN exhibited superior therapeutic efficacy and safety in a rat model of knee synovitis, significantly inhibiting TNF-α and IL-1ß compared with the methotrexate subcutaneous injection method. Conclusion: NS technology effectively enhances the solubility of the poorly soluble drug CBD, while DMN facilitates penetration, extends the duration of action in vivo, and improves bioavailability. Furthermore, CBD has shown promising therapeutic outcomes in treating knee synovitis. This innovative drug delivery system is expected to offer a more efficient solution for the administration of highly lipophilic drugs akin to CBD, thereby facilitating high-dose administration.

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.

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.

[Research advances of biomedical polymeric microneedles for non-transdermal local drug delivery].

Microneedles have emerged as the new class of local drug delivery system that has broad potential for development. Considering that the microneedles can penetrate tissue barriers quickly, and provide localized and targeted drug delivery, their applications have gradually expanded to non-transdermal drug delivery recently, which are capable of providing rapid and effective treatment for injuries and diseases of organs or tissues. However, a literature search revealed that there is a lack of summaries of the latest developments in non-transdermal drug delivery research by using biomedical polymeric microneedles. The review first described the materials and fabrication methods for the polymeric microneedles, and then reviewed a representative application of microneedles for non-transdermal drug delivery, with the primary focus being on treating and repairing the tissues or organs such as oral cavity, ocular tissues, blood vessels and heart. At the end of the article, the opportunities and challenges associated with microneedles for non-transdermal drug delivery were discussed, along with its future development, in order to provide reference for researchers in the relevant field.

Parotid hypersalivation after inferior salivatory nucleus glutamate/NMDA receptor excitation in the rat.

Although salivation is essential during eating behavior, little is known about the brainstem centers that directly control the salivary glands. With regard to the inferior salivatory nucleus (ISN), the site of origin of the parasympathetic preganglionic cell bodies that innervate the parotid glands, previous anatomical studies have located it within the rostrodorsal medullary reticular formation. However, to date there is no functional data that shows the secretory nature of the somas grouped in this region. To activate only the somas and rule out the activation of the efferent fibers from and the afferent fibers to the ISN, in exp. 1, NMDA neurotoxin was administered to the rostrodorsal medullary region and the secretion of saliva was recorded during the following hour. Results showed an increased secretion of parotid saliva but a total absence of submandibular-sublingual secretion. In exp. 2, results showed that the hypersecretion of parotid saliva after NMDA microinjection was completely blocked by the administration of atropine (a cholinergic blocker) but not after administration of dihydroergotamine plus propranolol (α and ß-adrenergic blockers, respectively). These findings suggest that the somata of the rostrodorsal medulla are secretory in nature, controlling parotid secretion via a cholinergic pathway. The data thus functionally supports the idea that these cells constitute the ISN.

Nanocarrier-Integrated Microneedles: Divulging the Potential of Novel Frontiers for Fostering the Management of Skin Ailments.

The various kinds of nanocarriers (NCs) have been explored for the delivery of therapeutics designed for the management of skin manifestations. The NCs are considered as one of the promising approaches for the skin delivery of therapeutics attributable to sustained release and enhanced skin penetration. Despite the extensive applications of the NCs, the challenges in their delivery via skin barrier (majorly stratum corneum) have persisted. To overcome all the challenges associated with the delivery of NCs, the microneedle (MN) technology has emerged as a beacon of hope. Programmable drug release, being painless, and its minimally invasive nature make it an intriguing strategy to circumvent the multiple challenges associated with the various drug delivery systems. The integration of positive traits of NCs and MNs boosts therapeutic effectiveness by evading stratum corneum, facilitating the delivery of NCs through the skin and enhancing their targeted delivery. This review discusses the barrier function of skin, the importance of MNs, the types of MNs, and the superiority of NC-loaded MNs. We highlighted the applications of NC-integrated MNs for the management of various skin ailments, combinational drug delivery, active targeting, in vivo imaging, and as theranostics. The clinical trials, patent portfolio, and marketed products of drug/NC-integrated MNs are covered. Finally, regulatory hurdles toward benchtop-to-bedside translation, along with promising prospects needed to scale up NC-integrated MN technology, have been deliberated. The current review is anticipated to deliver thoughtful visions to researchers, clinicians, and formulation scientists for the successful development of the MN-technology-based product by carefully optimizing all the formulation variables.

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.

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.

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.

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.

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.

Comparing robotic and manual injection methods in zebrafish embryos for high-throughput RNA silencing using CRISPR-RfxCas13d.

In this study, the authors compared the efficiency of automated robotic and manual injection methods for the CRISPR-RfxCas13d (CasRx) system for mRNA knockdown and Cas9-mediated DNA targeting in zebrafish embryos. They targeted the no tail (TBXTA) gene as a proof-of-principle, evaluating the induced embryonic phenotypes. Both Cas9 and CasRx systems caused loss of function phenotypes for TBXTA. Cas9 protein exhibited a higher percentage of severe phenotypes compared with mRNA, while CasRx protein and mRNA showed similar efficiency. Both robotic and manual injections demonstrated comparable phenotype percentages and mortality rates. The findings highlight the potential of RNA-targeting CRISPR effectors for precise gene knockdown and endorse automated microinjection at a speed of 1.0 s per embryo as a high-throughput alternative to manual methods.

[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.

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.

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.

Advancements in microneedle fabrication techniques: artificial intelligence assisted 3D-printing technology.

Microneedles (MNs) are micron-scale needles that are a painless alternative to injections for delivering drugs through the skin. MNs find applications as biosensing devices and could serve as real-time diagnosis tools. There have been numerous fabrication techniques employed for producing quality MN-based systems, prominent among them is the three-dimensional (3D) printing. 3D printing enables the production of quality MNs of tuneable characteristics using a variety of materials. Further, the possible integration of artificial intelligence (AI) tools such as machine learning (ML) and deep learning (DL) with 3D printing makes it an indispensable tool for fabricating microneedles. Provided that these AI tools can be trained and act with minimal human intervention to control the quality of products produced, there is also a possibility of mass production of MNs using these tools in the future. This work reviews the specific role of AI in the 3D printing of MN-based devices discussing the use of AI in predicting drug release patterns, its role as a quality control tool, and in predicting the biomarker levels. Additionally, the autonomous 3D printing of microneedles using an integrated system of the internet of things (IoT) and machine learning (ML) is discussed in brief. Different categories of machine learning including supervised learning, semi-supervised learning, unsupervised learning, and reinforced learning have been discussed in brief. Lastly, a brief section is dedicated to the biosensing applications of MN-based devices.

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.

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.