Microneedle patch casting using a micromachined carbon master for enhanced drug delivery.

For successful treatment of diseases, sufficient therapeutics must be provided to the body. Microneedle applications in therapeutic delivery and analytics sampling are restricted because of various issues, including smaller area for drug loading and analytics sampling. To achieve sufficient drug loading and analytics sampling and improve drug penetration while maintaining painless administration, patch-type microneedle arrays were designed and fabricated using polymer casting from a conical cavity mold. Microcavities were formed on a carbon plate via micromechanical machining. A porous polymer layer was coated on a microneedle patch (MNP). The pores of the porous polymer layer provided space and channels for drug delivery. A pH-sensitive polymer layer was employed to cap the porous polymer layer, which prevented drug leakage during storage and provided a stimulus drug release in response to body pH conditions. The drug can be delivered through holes connected to both sides of the patch. The drug release of the MNP was investigated in vitro and in vivo and showed conceptual proof that these MNs have the potential to enhance treatment protocols for various diseases with the flexibility of coating and therapeutic materials and offer significant scope for further variations and advancement.

State-of-the-art strategies to enhance the mechanical properties of microneedles.

Microneedles (MNs) have gained increasing attention in the biomedical field, owing to their notable advantages over injectable and transdermal preparations. The mechanical properties of MNs are the key to determine whether MNs can puncture the skin for efficient drug delivery and therapeutic purposes. However, there is still lacking of a systemic summary on how to improve the mechanical properties of MNs. Herein, this review mainly analyzes the key factors affecting the mechanical properties of MNs from the theoretical point of view and puts forward improvement approaches. First, we analyzed the major stresses exerted on the MNs during skin puncture and described general methods to evaluate the mechanical properties of MNs. We then provided detail examples to elucidate how the physicochemical properties of single polymer, formulation compositions, and geometric parameters affected the mechanical properties of MNs. Overall, the mechanical strength of MNs can be enhanced by tuning the crosslinking density, crystallinity degree, and molecular weight of single polymer, introducing polysaccharides and nano-microparticles as reinforcers to form complex with polymer, and optimizing the geometric parameters of MNs. Therefore, this review will provide critical guidance on how to fabricate MNs with robust mechanical strength for successful transdermal drug delivery.

Finite Element Analysis of Skin Deformation and Puncture for Microneedle Array Design.

The mechanics of microneedle insertion have thus far been studied in a limited manner. Previous work has focused on buckling and failure of microneedle devices, while providing little insight into skin deformation, puncture, and the final positioning of needle tips under full microneedle arrays. The current study aims to develop a numerical approach capable of evaluating deformation and puncture conditions for full microneedle array designs. The analysis included a series of finite element submodels used to calibrate the microneedle-epidermal interface for failure properties using traction-separation laws. The single needle model is validated using experimental data and imaging, including results from a customized nanoindentation procedure to measure loads and displacements during microneedle insertion. Upon validation, full microneedle arrays are implemented in a 3 D finite element model and a design framework is developed, allowing evaluation of different design variables (i.e. needle shape, material, spacing) with respect to outputs relevant to successful microneedle performance. Results from the model include skin deformation, force to puncture, penetration depth, and the punctured state at each microneedle tip. In addition to microneedle parameters, patient parameters such as subcutaneous tissue thickness are included to evaluate the sensitivity of different microneedle designs to expected patient and anatomical region variability.

Microneedle system for tissue engineering and regenerative medicines: a smart and efficient therapeutic approach.

The global demand for an enhanced quality of life and extended lifespan has driven significant advancements in tissue engineering and regenerative medicine. These fields utilize a range of interdisciplinary theories and techniques to repair structurally impaired or damaged tissues and organs, as well as restore their normal functions. Nevertheless, the clinical efficacy of medications, materials, and potent cells used at the laboratory level is always constrained by technological limitations. A novel platform known as adaptable microneedles has been developed to address the abovementioned issues. These microneedles offer a solution for the localized distribution of various cargos while minimizing invasiveness. Microneedles provide favorable patient compliance in clinical settings due to their effective administration and ability to provide a painless and convenient process. In this review article, we summarized the most recent development of microneedles, and we started by classifying various microneedle systems, advantages, and fundamental properties. Subsequently, it provides a comprehensive overview of different types of microneedles, the material used to fabricate microneedles, the fundamental properties of ideal microneedles, and their applications in tissue engineering and regenerative medicine, primarily focusing on preserving and restoring impaired tissues and organs. The limitations and perspectives have been discussed by concluding their future therapeutic applications in tissue engineering and regenerative medicines.

Recent progress of microneedles in transdermal immunotherapy: A review.

Since human skin is an immune organ, a large number of immune cells are distributed in the epidermis and the dermis of the skin. Transdermal immunotherapy shows great therapeutic advantages in innate immunotherapy and adaptive immunotherapy. To solve the problem that macromolecules are difficult to penetrate into the skin, the microneedle technology can directly break through the skin barrier using micron-sized needles in a non-invasive and painless way for transdermal drug delivery. Therefore, it is considered to be an effective technology to increase drug transdermal absorption. In this review, the types of preparation, the combinations with different techniques and the mechanisms of microneedles in transdermal immunotherapy were summarized. Compared with traditional immunotherapy like intramuscular injection and subcutaneous injection, the microneedle has many advantages in transdermal immunotherapy, such as reducing patient pain, enhancing vaccine stability, and inducing stronger immune responses. Although there are still some limitations to be solved, the application of microneedle technology in transdermal immunotherapy is undoubtedly a promising means of drug delivery.

Role of L- and T-type voltage-dependent calcium channels in the hierarchical organization of defensive responses to electrical stimulation of the rat dorsolateral periaqueductal gray.

Stimulation of the dorsal half of the rat periaqueductal gray (DPAG) with 60-Hz pulses of increasing intensity, 30-µA pulses of increasing frequency, or increasing doses of an excitatory amino acid elicits sequential defensive responses of exophthalmia, immobility, trotting, galloping, and jumping. These responses may be controlled by voltage-gated calcium channel-specific firing patterns. Indeed, a previous study showed that microinjection of the DPAG with 15 nmol of verapamil, a putative blocker of L-type calcium channels, attenuated all defensive responses to electrical stimulation at the same site as the injection. Accordingly, here we investigated the effects of microinjection of lower doses (0.7 and 7 nmol) of both verapamil and mibefradil, a preferential blocker of T-type calcium channels, on DPAG-evoked defensive behaviors of the male rat. Behaviors were recorded either 24 h before or 10 min, 24 h, and 48 h after microinjection. Effects were analyzed by both threshold logistic analysis and repeated measures analysis of variance for treatment by session interactions. Data showed that the electrodes were all located within the dorsolateral PAG. Compared to the effects of saline, verapamil significantly attenuated exophthalmia, immobility, and trotting. Mibefradil significantly attenuated exophthalmia and marginally attenuated immobility while facilitating trotting. While galloping was not attenuated by either antagonist, jumping was unexpectedly attenuated by 0.7 nmol verapamil only. These results suggest that T-type calcium channels are involved in the low-threshold freezing responses of exophthalmia and immobility, whereas L-type calcium channels are involved in the trotting response that precedes the full-fledged escape responses of galloping and jumping.

Nanoformulation-assisted microneedle transdermal drug delivery system: An innovative platform enhancing rheumatoid arthritis treatment.

A transdermal delivery system offers high bioavailability and favorable patient adherence, constituting an optimal approach for localized administration in rheumatoid arthritis (RA) treatment. However, the stratum corneum (SC) impedes the delivery efficiency of conventional transdermal drug delivery systems. Microneedles (MNs) can temporarily create micropores within the SC, enabling drug distribution via bypassing this barrier and enhancing transdermal delivery effectiveness. Notably, MNs provide a painless method of drug delivery through the skin and may directly modulate inflammation in immune cells by delivering drugs via the lymphatic system during transdermal administration. However, the MN delivery system is not suitable for drugs with low water solubility and stability. Additionally, major concerns exist regarding the safety of using MN delivery for highly cytotoxic drugs, given that it could result in high local drug concentration at the delivery site. While MNs exhibit some degree of targeted delivery to the immune and inflammatory environment, their targeting efficiency remains suboptimal. Nanoformulations have the potential to significantly address the limitations of MNs in RA treatment by improving drug targeting, solubility, stability, and biocompatibility. Therefore, this review provides a concise overview of the advantages, disadvantages, and mechanisms of different types of MNs for RA treatment. It specifically focuses on the application and advantages of combining nanoformulation with MNs for RA treatment and summarizes the current trends in the development of nanoformulations combined with MNs in the field of RA treatment, offering theoretical support for future advancements and clinical applications.

Additive manufacturing of microneedles for sensing and drug delivery.

INTRODUCTION: Microneedles (MNs) are miniaturized, painless, and minimally invasive platforms that have attracted significant attention over recent decades across multiple fields, such as drug delivery, disease monitoring, disease diagnosis, and cosmetics. Several manufacturing methods have been employed to create MNs; however, these approaches come with drawbacks related to complicated, costly, and time-consuming fabrication processes. In this context, employing additive manufacturing (AM) technology for MN fabrication allows for the quick production of intricate MN prototypes with exceptional precision, providing the flexibility to customize MNs according to the desired shape and dimensions. Furthermore, AM demonstrates significant promise in the fabrication of sophisticated transdermal drug delivery systems and medical devices through the integration of MNs with various technologies. AREAS COVERED: This review offers an extensive overview of various AM technologies with great potential for the fabrication of MNs. Different types of MNs and the materials utilized in their fabrication are also discussed. Recent applications of 3D-printed MNs in the fields of transdermal drug delivery and biosensing are highlighted. EXPERT OPINION: This review also mentions the critical obstacles, including drug loading, biocompatibility, and regulatory requirements, which must be resolved to enable the mass-scale adoption of AM methods for MN production, and future trends.

Zygote cryobanking applied to CRISPR/Cas9 microinjection in mice.

Microinjection of CRISPR/Cas9 requires the availability of zygotes that implies animal breeding, superovulation schemes, and embryo collection. Vitrification of zygotes may allow having ready-to-use embryos and to temporally dissociate the workload of embryo production from microinjection. In this study, fresh (F group) or vitrified (V group) zygotes were microinjected with CRISPR/Cas9 system to test the hypothesis that vitrified zygotes could be a suitable source of embryos for microinjection. In Experiment 1 (in vitro evaluation), B6D2F1/J zygotes were microinjected and cultured until blastocyst stage. Embryo survival and cleavage rates after microinjection were similar between groups (~50% and ~80% respectively; P = NS). Development rate was significantly higher for F than V group (55.0% vs. 32.6%, respectively; P<0.05). Mutation rate did not show statistical differences among groups (P = NS). In Experiment 2 (in vivo evaluation), C57BL/6J zygotes were microinjected and transferred to recipient females. Embryo survival was significantly lower in fresh than in vitrified zygotes (49.2% vs. 62.7%, respectively; P<0.05). Cleavage rate did not show statistical differences (~70%; P = NS). Pregnancy rate (70.0% vs. 58.3%) and birth rate (11.9% vs. 11.2%) were not different between groups (F vs. V group; P = NS). Offspring mutation rate was higher for F than V group, in both heterodimer analysis (73.7% vs. 33.3%, respectively; P = 0.015) and Sanger sequencing (89.5% vs. 41.7%, respectively; P = 0.006). In conclusion, vitrified-warmed zygotes present a viable alternative source for CRISPR/Cas9 microinjection when the production of fresh embryos is impeded by limited technical support. The possibility of zygote cryobanking to perform microinjection sessions on demand seems to be a suitable alternative to avoid the breeding and maintenance of animals all over the year, enhancing the implementation of CRISPR technology.

Spatiotemporal fate of nanocarriers-embedded dissolving microneedles: the impact of needle dissolving rate.

OBJECTIVE: Dissolving microneedles (DMNs) have shown great potential for transdermal drug delivery due to their excellent skin-penetrating ability and combination with nanocarriers (NCs) can realize targeted drug delivery. The objective of this study was to investigate the impact of microneedle dissolving rate on the in vivo fate of NC-loaded DMNs, which would facilitate the clinical translation of such systems. METHODS: Solid lipid nanoparticles (SLNs) were selected as the model NC for loading in DMNs, which were labeled by P4 probes with aggregation-quenching properties. Sodium hyaluronate acid (HA) and chitosan (CS), with different aqueous dissolving rates, were chosen as model tip materials. The effects of needle dissolving rate on the in vivo fate of NC-loaded DMNs was investigated by tracking the distribution of fluorescence signals after transdermal exposure. RESULTS: P4 SLNs achieved a deeper diffusion depth of 180 µm in DMN-HA with a faster dissolution rate, while the diffusion depth in DMN-CS with a slower dissolution rate was lower (140 µm). The in vivo experiments demonstrated that P4 SLNs had a T1/2 value of 12.14 h in DMN-HA, whilst a longer retention time was found in DMN-CS, with a T1/2 of 13.12 h. CONCLUSIONS: This study confirmed that the in vivo diffusion rate of NC-loaded DMNs was determined by the dissolving rate of DMNs materials and provided valuable guidance for the design and development of NC-loaded DMNs in the future.

Fabrication and Characterization of Microneedle Patches for Loading and Delivery of Exosomes.

Exosomes, as emerging "next-generation" biotherapeutics and drug delivery vectors, hold immense potential in diverse biomedical fields, ranging from drug delivery and regenerative medicine to disease diagnosis and tumor immunotherapy. However, the rapid clearance by traditional bolus injection and poor stability of exosomes restrict their clinical application. Microneedles serve as a solution that prolongs the residence time of exosomes at the administration site, thereby maintaining the drug concentration and facilitating sustained therapeutic effects. In addition, microneedles also possess the ability to maintain the stability of bioactive substances. Therefore, we introduce a microneedle patch for loading and delivering exosomes and share the methods, including isolation of exosomes, fabrication, and characterization of exosome-loaded microneedle patches. The microneedle patches were fabricated using trehalose and hyaluronic acid as the tip materials and polyvinylpyrrolidone as the backing material through a two-step casting method. The microneedles demonstrated robust mechanical strength, with tips able to withstand 2 N. Pig skin was used to simulate human skin, and the tips of microneedles completely melted within 60 s after skin puncture. The exosomes released from the microneedles exhibited morphology, particle size, marker proteins, and biological functions comparable to those of fresh exosomes, enabling dendritic cells uptake and promoting their maturation.

Microneedle-mediated drug delivery for neurological diseases.

Neurological disorders, including brain injury, brain tumors, and neurodegenerative diseases, rank as the second leading cause of death worldwide. Exploring effective new treatments for neurological disorders has long been a hot research issue in clinical practice. Recently, microneedles (MNs) have attracted much attention due to their designation as a "painless and non-invasive" novel transdermal delivery method, characterized by their biocompatibility and sustainability. The advantages of MNs open an avenue for potential therapeutic interventions targeting neurological disorders. This review presents a concise overview of progress in the field of MNs, with highlights on the application in the treatment of neurological disorders. Notably, trends in the development of MNs and future challenges are also discussed.

Converting Short-Acting Insulin into Thermo-Stable Longer-Acting Insulin Using Multi-Layer Detachable Microneedles.

The detachable dissolving microneedles (DDMNs) feature an array of needles capable of being separated from the base sheet during administration. Here they were fabricated to address delivery efficiency and storage stability of insulin. The constructed insulin-DDMN is multi-layered, with 1) a hard tip cover layer; 2) a layer of regular short-acting insulin (RI) mixed with hyaluronic acid (HA) and sorbitol (Sor) which occupies the taper tip region of the needles; 3) a barrier layer situated above the RI layer; and 4) a fast-dissolving layer connecting the barrier layer to the base sheet. RI entrapped in DDMNs exhibited enhanced thermal stability; it could be stored at 40 °C for 35 days without losing significant biological activity. Differential scanning calorimetric analysis revealed that the HA-Sor matrix could improve the denaturation temperature of the RI from lower than room temperature to 186 °C. Tests in ex vivo porcine skin demonstrated RI delivery efficiency of 91±1.59 %. Experiments with diabetic rats revealed sustained release of RI, i.e., when compared to subcutaneous injection with the same RI dose, RI-DDMNs produced slower absorption of insulin into blood circulation, delayed onset of hypoglycemic effect, longer serum insulin half-life, and longer hypoglycemic duration.

The dose-dependent effect of the D2R agonist quinpirole microinjected into the ventral pallidum on information flow in the limbic system.

The ventral pallidum (VP) receives its primary inputs from the nucleus accumbens (NAC) and the basolateral amygdala (BLA). We demonstrated recently that in the VP, the D2 DA receptor (D2R) agonist quinpirole dose-dependently facilitates memory consolidation in inhibitory avoidance and spatial learning. In the VP, D2R can be found both on NAC and BLA terminals. According to our hypothesis, quinpirole microinjected into the VP can facilitate memory consolidation via modulation of synaptic plasticity on NAC and/or BLA terminals. The effect of intra-VP quinpirole on BLA-VP and NAC shell-VP synapses was investigated via a high frequency stimulation (HFS) protocol. Quinpirole was administered in three doses into the VP of male Sprague-Dawley rats after HFS; controls received vehicle. To examine whether an interaction between the NAC shell and the BLA at the level of the VP was involved, tetrodotoxin (TTX) was microinjected into one of the nuclei while stimulating the other nucleus. Our results showed that quinpirole dose-dependently modulates BLA-VP and NAC shell-VP synapses, similar to those observed in inhibitory avoidance and spatial learning, respectively. The lower dose inhibits BLA inputs, while the larger doses facilitates NAC shell inputs. The experiments with TTX demonstrates that the two nuclei do not influence each others' evoked responses in the VP. Power spectral density analysis demonstrated that independent from the synaptic facilitation, intra-VP quinpirole increases the amplitude of gamma frequency band after NAC HFS, and BLA tonically suppresses the NAC's HFS-induced gamma facilitation. In contrast, HFS of the BLA results in a delayed, transient increase in the amplitude of the gamma frequency band correlating with the LTP of the P1 component of the VP response to BLA stimulation. Furthermore, our results demonstrate that the BLA plays a prominent role in the generation of the delta oscillations: HFS of the BLA leads to a gradually increasing delta frequency band facilitation over time, while BLA inhibition blocks the NAC's HFS induced strong delta facilitation. These findings demonstrate that there is a complex interaction between the NAC shell region and the VP, as well as the BLA and the VP, and support the important role of VP D2Rs in the regulation of limbic information flow.

Embryo Microinjection for Transgenesis in Drosophila.

Transgenesis in Drosophila is an essential approach to studying gene function at the organism level. Embryo microinjection is a crucial step for the construction of transgenic flies. Microinjection requires some types of equipment, including a microinjector, a micromanipulator, an inverted microscope, and a stereo microscope. Plasmids isolated with a plasmid miniprep kit are qualified for microinjection. Embryos at the pre-blastoderm or syncytial blastoderm stage, where nuclei share a common cytoplasm, are subjected to microinjection. A cell strainer eases the process of dechorionating embryos. The optimal time for dechorionation and desiccation of embryos needs to be determined experimentally. To increase the efficiency of embryo microinjection, needles prepared by a puller need to be beveled by a needle grinder. In the process of grinding needles, we utilize a foot air pump with a pressure gauge to avoid the capillary effect of the needle tip. We routinely inject 120-140 embryos for each plasmid and obtain at least one transgenic line for around 85% of plasmids. This article takes the phiC31 integrase-mediated transgenesis in Drosophila as an example and presents a detailed protocol for embryo microinjection for transgenesis in Drosophila.

Iontophoresis-coupled rapidly dissolving polymeric microneedle patch of naloxone for its enhanced transdermal delivery.

The transdermal delivery of naloxone for opioid overdose emergency purposes is a challenge due to its poor rate of diffusion through the layers of skin. This results in delayed delivery of an insufficient amount of the drug within minimal time as is desired to save lives. The ability of dissolving polymeric microneedles to shorten the lag time significantly has been explored and shown to have prospects in terms of the transdermal delivery of naloxone. This is an option that offers critical advantages to the ongoing opioid crisis, including ease of distribution and easy administration, with little to no need for intervention by clinicians. Nonetheless, this approach by itself needs augmentation to meet pharmacokinetic delivery attributes desired for a viable clinical alternative to existing market dosage forms. In this study, we report the success of an optimized iontophoresis-coupled naloxone loaded dissolving microneedle patch which had facilitated a 12- fold increase in average cumulative permeation and a 6-fold increase in drug flux over a conventional dissolving microneedle patch within 60 min of application (p < 0.05). This translates to a 30 % decrease in dose requirement in a mechanistically predicted microneedle patch established to be able to achieve the desired early plasma concentration time profile needed in an opioid overdose emergency. Applying a predictive mathematical model, we describe an iontophoresis-coupled microneedle patch design capable of meeting the desired pharmacokinetic profile for a viable naloxone delivery form through skin.

Development of ropivacaine hydrochloride-loaded dissolving microneedles as a local anesthetic agent: A proof-of-concept.

Ropivacaine hydrochloride (RPL) is a local anesthetic agent that has been widely used for the treatment of pain during or after surgery. However, this drug is only available in parenteral dosage form and may contribute to the infiltration of RPL into the plasma, causing some undesirable side effects. Intradermal delivery of RPL using dissolving microneedles may become a promising strategy to deliver such drugs into the skin. This research aimed to develop RPL-loaded dissolving microneedles (DMN-RPLs) as a proof of the concept of intradermal delivery of a local anesthetic. The DMN-RPLs were fabricated using either centrifugation or air-pressurized chamber methods. Several polymers, such as poly(vinyl pyrrolidone) (PVP), poly(vinyl alcohol) (PVA), and sodium hyaluronate (SH), were utilized for manufacturing the DMN-RPLs. The prepared DMN-RPLs were assessed for their thermal properties, chemical bonds, mechanical strength, insertion ability, skin-dissolution study, and drug content. Furthermore, in-skin deposition and dermatokinetic studies were also performed. The results showed that F9 (30 % w/w PVP-4 % w/w SH) and F10 (30 % w/w PVP-5 % w/w PVA) containing 5 % w/w of RPL were the most promising formulations, as shown by their needle height reduction (<10 %) and insertion depth (∼400 µm). Both formulations were also able to deliver more than 60 % of the RPL contained in the DMNs into the epidermis, dermis, and receiver compartment. This study, for the first time, has provided a proof concept to deliver RPL as a local anesthetic using DMNs and the intradermal route, aiming to minimize pain and discomfort during administration and improve the patient's experience.

Advances in microneedles for transdermal diagnostics and sensing applications.

Microneedles, the miniaturized needles, which can pierce the skin with minimal invasiveness open up new possibilities for constructing personalized Point-of-Care (POC) diagnostic platforms. Recent advances in microneedle-based POC diagnostic systems, especially their successful implementation with wearable technologies, enable biochemical detection and physiological recordings in a user-friendly manner. This review presents an overview of the current advances in microneedle-based sensor devices, with emphasis on the biological basis of transdermal sensing, fabrication, and application of different types of microneedles, and a summary of microneedle devices based on various sensing strategies. It concludes with the challenges and future prospects of this swiftly growing field. The aim is to present a critical and thorough analysis of the state-of-the-art development of transdermal diagnostics and sensing devices based on microneedles, and to bridge the gap between microneedle technology and pragmatic applications.

Micro-Infusion of 5-HT1a Receptor Antagonists into the Ventral Subiculum Ameliorate MK-801 Induced Schizophrenia-Like Behavior in Rats.

Recent studies have shown that the 5-HT1a receptor (5-HT1aR) in the central 5-HT (Serotonergic) system is involved in the pathophysiology of schizophrenia through its various receptors, and the dysfunction of the ventral hippocampus may be a key causative factor in schizophrenia. To date, whether the 5-HT1a receptor is involved in ventral hippocampal dysfunction and its internal mechanism remain unclear. In this study, schizophrenia-like animal model was induced by intraperitoneal injection of aspartate receptor antagonist MK-801 in male Sprague Dawley rats, and the role of 5-HT1aR in this animal model was investigated by bilaterally micro-infusing the 5-HT1aR antagonist WAY100635 into the ventral subiculum (vSub) of the hippocampus of rats. Behavioral experiments such as open field test (OFT) and prepulse inhibition (PPI) were performed. The results showed that MK-801 induced hyperactivity and impaired prepulse inhibition in rats, whereas, micro-infusion of 5-HT1aR antagonist WAY100635 into the vSub ameliorated these phenomena. Immunofluorescence analysis revealed that WAY100635 significantly increased the c-Fos expression in vSub. Western blot and immunohistochemical analysis showed that MK-801 induced up-regulation of 5-HT1aR and phospho-extracellular regulated protein kinase (p-ERK) pathway, while micro-infusion of the WAY100635 down-regulated 5-HT1aR and p-ERK in the vSub. Therefore, the results of the present study suggested that in vSub, the 5-HT1aR antagonist WAY100635 may attenuate MK-801-induced schizophrenia-like activity by modulating excitatory neurons and downregulating p-ERK.

Microinjection of the CRISPR/Cas9 editing system through the germ pore of a wheat microspore induces mutations in the target Ms2 gene.

BACKGROUND: Microinjection is a direct procedure for delivering various compounds via micropipette into individual cells. Combined with the CRISPR/Cas9 editing technology, it has been used to produce genetically engineered animal cells. However, genetic micromanipulation of intact plant cells has been a relatively unexplored area of research, partly due to the cytological characteristics of these cells. This study aimed to gain insight into the genetic micromanipulation of wheat microspores using microinjection procedures combined with the CRISPR/Cas9 editing system targeting the Ms2 gene. METHODS AND RESULTS: Microspores were first reprogrammed by starvation and heat shock treatment to make them structurally suitable for microinjection. The large central vacuole was fragmented and the nucleus with cytoplasm was positioned in the center of the cell. This step and an additional maltose gradient provided an adequate source of intact single cells in the three wheat genotypes. The microcapillary was inserted into the cell through the germ pore to deliver a working solution with a fluorescent marker. This procedure was much more efficient and less harmful to the microspore than inserting the microcapillary through the cell wall. The CRISPR/Cas9 binary vectors injected into reprogrammed microspores induced mutations in the target Ms2 gene with deletions ranging from 1 to 16 bp. CONCLUSIONS: This is the first report of successful genome editing in an intact microspore/wheat cell using the microinjection technique and the CRISPR/Cas9 editing system. The study presented offers a range of molecular and cellular biology tools that can aid in genetic micromanipulation and single-cell analysis.