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.

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.

Optimization of stereolithography 3D printing of microneedle micro-molds for ocular drug delivery.

Microneedles (MN) have emerged as an innovative technology for drug delivery, offering a minimally invasive approach to administer therapeutic agents. Recent applications have included ocular drug delivery, requiring the manufacture of sub-millimeter needle arrays in a reproducible and reliable manner. The development of 3D printing technologies has facilitated the fabrication of MN via mold production, although there is a paucity of information available regarding how the printing parameters may influence crucial issues such as sharpness and penetration efficacy. In this study, we have developed and optimized a 3D-printed MN micro-mold using stereolithography (SLA) 3D printing to prepare a dissolving ocular MN patch. The effects of a range of parameters including aspect ratio, layer thickness, length, mold shape and printing orientation have been examined with regard to both architecture and printing accuracy of the MN micro-mold, while the effects of printing angle on needle fidelity was also examined for a range of basic shapes (conical, pyramidal and triangular pyramidal). Mechanical strength and in vitro penetration of the polymeric (PVP/PVA) MN patch produced from reverse molds fabricated using MN with a range of shapes and height, and aspect ratios were assessed, followed by ex vivo studies of penetration into excised scleral and corneal tissues. The optimization process identified the parameters required to produce MN with the sharpest tips and highest dimensional fidelity, while the ex vivo studies indicated that these optimized systems would penetrate the ocular tissue with minimal applied pressure, thereby allowing ease of patient self-administration.

Integrating microneedles and sensing strategies for diagnostic and monitoring applications: The state of the art.

Microneedles (MNs) offer minimally-invasive access to interstitial fluid (ISF) - a potent alternative to blood in terms of monitoring physiological analytes. This property is particularly advantageous for the painless detection and monitoring of drugs and biomolecules. However, the complexity of the skin environment, coupled with the inherent nature of the analytes being detected and the inherent physical properties of MNs, pose challenges when conducting physiological monitoring using this fluid. In this review, we discuss different sensing mechanisms and highlight advancements in monitoring different targets, with a particular focus on drug monitoring. We further list the current challenges facing the field and conclude by discussing aspects of MN design which serve to enhance their performance when monitoring different classes of analytes.

Development and comparison of machine learning models for in-vitro drug permeation prediction from microneedle patch.

The field of machine learning (ML) is advancing to a larger extent and finding its applications across numerous fields. ML has the potential to optimize the development process of microneedle patch by predicting the drug release pattern prior to its fabrication and production. The early predictions could not only assist the in-vitro and in-vivo experimentation of drug release but also conserve materials, reduce cost, and save time. In this work, we have used a dataset gleaned from the literature to train and evaluate different ML models, such as stacking regressor, artificial neural network (ANN) model, and voting regressor model. In this study, models were developed to improve prediction accuracy of the in-vitro drug release amount from the hydrogel-type microneedle patch and the in-vitro drug permeation amount through the micropores created by solid microneedles on the skin. We compared the performance of these models using various metrics, including R-squared score (R2 score), root mean squared error (RMSE), and mean absolute error (MAE). Voting regressor model performed better with drug permeation percentage as an outcome feature having RMSE value of 3.24. In comparison, stacking regressor have a RMSE value of 16.54, and ANN model has shown a RMSE value of 14. The value of permeation amount calculated from the predicted percentage is found to be more accurate with RMSE of 654.94 than direct amount prediction, having a RMSE of 669.69. All our models have performed far better than the previously developed model before this research, which had a RMSE of 4447.23. We then optimized voting regressor model's hyperparameter and cross validated its performance. Furthermore, it was deployed in a webapp using Flask framework, showing a way to develop an application to allow other users to easily predict drug permeation amount from the microneedle patch at a particular time period. This project demonstrates the potential of ML to facilitate the development of microneedle patch and other drug delivery systems.

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.

Dissolvable microneedles in the skin: Determination the impact of barrier disruption and dry skin on dissolution.

Dissolvable microneedles (DMNs), fabricated from biocompatible materials that dissolve in both water and skin have gained popularity in dermatology. However, limited research exists on their application in compromised skin conditions. This study compares the hyaluronic acid-based DMNs penetration, formation of microchannels, dissolution, and diffusion kinetics in intact, barrier-disrupted (tape stripped), and dry (acetone-treated) porcine ear skin ex vivo. After DMNs application, comprehensive investigations including dermoscopy, stereomicroscope, skin hydration, transepidermal water loss (TEWL), optical coherence tomography (OCT), reflectance confocal laser scanning microscopy (RCLSM), confocal Raman micro-spectroscopy (CRM), two-photon tomography combined with fluorescence lifetime imaging (TPT-FLIM), histology, and scanning electron microscopy (SEM) were conducted. The 400 µm long DMNs successfully penetrated the skin to depths of ≈200 µm for dry skin and ≈200-290 µm for barrier-disrupted skin. Although DMNs fully inserted into all skin conditions, their dissolution rates were high in barrier-disrupted and low in dry skin, as observed through stereomicroscopy and TPT-FLIM. The dissolved polymer exhibited a more significant expansion in barrier-disrupted skin compared to intact skin, with the smallest increase observed in dry skin. Elevated TEWL and reduced skin hydration levels were evident in barrier-disrupted and dry skins compared to intact skin. OCT and RCLSM revealed noticeable skin indentation and pronounced microchannel areas, particularly in barrier-disrupted and dry skin. Additional confirmation of DMN effects on the skin and substance dissolution was obtained through histology, SEM, and CRM techniques. This study highlights the impact of skin condition on DMN effectiveness, emphasizing the importance of considering dissolvability and dissolution rates of needle materials, primarily composed of hyaluronic acid, for optimizing DMN-based drug delivery.

3D DLP-printed cannabinoid microneedles patch and its pharmacokinetic evaluation in rats.

OBJECTIVE: The objective of the present study was to enhance the bioavailability of cannabidiol (CBD) using 3D Digital Light Processing (DLP)-printed microneedle (MN) transdermal drug delivery system. METHODS: CBD MN patch was fabricated and optimized using 3D DLP printing using CBD (8% w/v), Lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) (0.49% w/v), distilled water (20% w/v), and poly (ethylene glycol) dimethacrylate 550 (PEGDAMA 550) (up to 100% w/v). CBD MNs were characterized for their morphology, mechanical strength, in vitro release study, ex vivo permeation study, and in vivo pharmacokinetic (PK) profile. KEY FINDINGS: Microscopic images showed that sharp CBD MNs with a height of ~800 µm, base diameter of ~250 µm, and tip with a radius of curvature (RoC) of ~15 µm were successfully printed using optimized printing parameters. Mechanical strength studies showed no significant deformation in the morphology of CBD MNs even after applying 0.5N/needle force. Ex vivo permeation study showed significant (P < .0001) permeation of CBD in the receiving media as compared to CBD patch (control). In vivo PK study showed significantly (P < .05) enhanced bioavailability in the case of CBD MN patch as compared to CBD subcutaneous inj. (control). CONCLUSION: Overall, systemic absorption of CBD was significantly enhanced using 3D-printed MN drug delivery system.

Polymeric Microneedles for Health Care Monitoring: An Emerging Trend.

Bioanalyte collection by blood draw is a painful process, prone to needle phobia and injuries. Microneedles can be engineered to penetrate the epidermal skin barrier and collect analytes from the interstitial fluid, arising as a safe, painless, and effective alternative to hypodermic needles. Although there are plenty of reviews on the various types of microneedles and their use as drug delivery systems, there is a lack of systematization on the application of polymeric microneedles for diagnosis. In this review, we focus on the current state of the art of this field, while providing information on safety, preclinical and clinical trials, and market distribution, to outline what we believe will be the future of health monitoring.

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.

Application of hydrogel microneedles in the oral cavity.

Microneedles are a transdermal drug delivery system in which the needle punctures the epithelium to deliver the drug directly to deep tissues, thus avoiding the influence of the first-pass effect of the gastrointestinal tract and minimizing the likelihood of pain induction. Hydrogel microneedles are microneedles prepared from hydrogels that have good biocompatibility, controllable mechanical properties, and controllable drug release and can be modified to achieve environmental control of drug release in vivo. The large epithelial tissue in the oral cavity is an ideal site for drug delivery via microneedles. Hydrogel microneedles can overcome mucosal hindrances to delivering drugs to deep tissues; this prevents humidity and a highly dynamic environment in the oral cavity from influencing the efficacy of the drugs and enables them to obtain better therapeutic effects. This article analyzes the materials and advantages of common hydrogel microneedles and reviews the application of hydrogel microneedles in the oral cavity.

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.

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.

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.

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.

Potential strategy of microneedle-based transdermal drug delivery system for effective management of skin-related immune disorders.

Skin-related immune disorders are a category of diseases that lead to the dysregulation of the body's immune response due to imbalanced immune regulation. These disorders exhibit diverse clinical manifestations and complicated pathogenesis. The long-term use of corticosteroids, anti-inflammatory drugs, and immunosuppressants as traditional treatment methods for skin-related immune disorders frequently leads to adverse reactions in patients. In addition, the effect of external preparations is not ideal in some cases due to the compacted barrier function of the stratum corneum (SC). Microneedles (MNs) are novel transdermal drug delivery systems that have theapparent advantages ofpenetrating the skin barrier, such as long-term and controlled drug delivery, less systemic exposure, and painless and minimally invasive targeted delivery. These advantages make it a good candidate formulation for the treatment of skin-related immune disorders and a hotspot for research in this field. This paper updates the classification, preparation, evaluation strategies, materials, and related applications of five types of MNs. Specific information, including the mechanical properties, dimensions, stability, and in vitro and in vivo evaluations of MNs in the treatment of skin-related immune disorders, is also discussed. This review provides an overview of the advances and applications of MNs in the effective treatment of skin-related immune disorders and their emerging trends.

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.

Design and analysis of a reservoir-based controllable microneedle for transdermal drug delivery applications.

Microneedle has made excellent contribution in the era of biomedical sector. This paper presents a reservoir-based out-of-plane silicon carbide (SiC) microneedle which has two lumens for delivering drug. The total height of the designed microneedle is 451 µm where the conical tip area is about 69.39 µm2. The additional part of this microneedle is a reservoir which is trapezium in shape having a height of 150 µm. This work use COMSOL Multiphysics software for the structural analysis and Ansys Workbench software to investigate the fluid analysis. The flow analyses are performed by releasing drugs from the reservoir where different viscosity based sample drugs are included. Although reservoir-based microneedles are existing, however, there is no system to control the fluid in those microneedles. Thus, this work proposes a controllable microneedle which able to control the drug flow by using a valve. For both the case of valveless and with a valve, the drug velocities are determined. As paracetamol has highest viscosity among other drugs, it provides lowest velocities. Conversely, the flow of aspirin shows high velocity of 6.51E-2 m/s without a valve and 4.26E-2 m/s with a valve. To analyze the skin insertion performance, a skin model including six layers is designed. The simulation results ensure that the proposed microneedle can penetrate the human skin successfully with less stress and deformation.

Chronically high stress hormone levels dysregulate sperm long noncoding RNAs and their embryonic microinjection alters development and affective behaviours.

Previous studies on paternal epigenetic inheritance have shown that sperm RNAs play a role in this type of inheritance. The microinjection of sperm small noncoding RNAs into fertilised mouse oocytes induces reprogramming of the early embryo, which is thought to be responsible for the differences observed in adult phenotype. While sperm long noncoding RNAs (lncRNAs) have also been investigated in a previous study, their microinjection into fertilised oocytes did not yield conclusive results regarding their role in modulating brain development and adult behavioural phenotypes. Therefore, in the current study we sought to investigate this further. We used our previously established paternal corticosterone (stress hormone) model to assess sperm lncRNA expression using CaptureSeq, a sequencing technique that is more sensitive than the ones used in other studies in the field. Paternal corticosterone exposure led to dysregulation of sperm long noncoding RNA expression, which encompassed lncRNAs, circular RNAs and transposable element transcripts. Although they have limited functional annotation, bioinformatic approaches indicated the potential of these lncRNAs in regulating brain development and function. We then separated and isolated the sperm lncRNAs and performed microinjections into fertilised oocytes, to generate embryos with modulated lncRNA populations. We observed that the resulting adult offspring had lower body weight and altered anxiety and affective behavioural responses, demonstrating roles for lncRNAs in modulating development and brain function. This study provides novel insights into the roles of lncRNAs in epigenetic inheritance, including impacts on brain development and behaviours of relevance to affective disorders.

Microneedles for advanced ocular drug delivery.

In the field of ocular drug delivery, topical delivery remains the most common treatment option for managing anterior segment diseases, whileintraocular injectionsare the current gold standard treatment option for treating posterior segment diseases. Nonetheless, topical eye drops are associated with low bioavailability (<5%), and theintravitreal administration procedure is highly invasive, yielding poor patient acceptability. In both cases, frequent administration is currently required. As a result, there is a clear unmet need for sustained drug delivery to the eye, particularly in a manner that can be localised. Microneedles, which are patches containing an array of micron-scale needles (<1 mm), have the potential to meet this need. These platforms can enable localised drug delivery to the eye while enhancing penetration of drug molecules through key ocular barriers, thereby improving overall therapeutic outcomes. Moreover, the minimally invasive manner in which microneedles are applied could provide significant advantages over traditional intravitreal injections regarding patient acceptability. Considering the benefitsofthis novel ocular delivery system, this review provides an in-depth overviewofthe microneedle systems for ocular drug delivery, including the types of microneedles used and therapeutics delivered. Notably, we outline and discuss the current challenges associated with the clinical translation of these platforms and offer opinions on factors which should be considered to improve such transition from lab to clinic.