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.

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.

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

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

Microneedle-Mediated Cell Therapy.

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

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.

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.

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.

Advances in microneedle-based therapy for bone disorders.

Bone-related disorders treatment is a serious public health concern, imposing a significant social and economic burden on patients and healthcare systems. Although conventional drug delivery systems have made advances in bone diseases prevention and management, the limited delivery efficiency and convoluted focal environment lead to inadequate drug absorption and lack of specificity to achieve the intended therapeutic impact. Microneedle-based therapy represents an extraordinarily safe and well-tolerable therapeutic approach for treating bone disorders, providing improved efficacy by breaking down the barriers and delivery of therapeutic components to the target sites with programable release profiles in a less invasive manner. Over the past decades, numerous significant achievements in the development of various types of drug-carried microneedles have been made to address the obstacles encountered in the bone-treating procedure, enabling the microneedle-based therapy to take an important step in practical applications. In this light, this review summarizes these remarkable researches in terms of microneedles types and drug delivery strategies, with the goal of demonstrating the benefits of exploiting microneedle-based therapy as a novel strategy for treating bone-related disorders. The remaining challenges and future perspectives are also discussed in the hope of inspiring more efficient and intelligent bone treatment strategies.

Mathematical modelling of microneedle-mediated transdermal delivery of drug nanocarriers into skin tissue and circulatory system.

Microneedle-mediated transdermal delivery using nanocarriers can successfully overcome the barrier of the stratum corneum and protect drugs from elimination in skin tissues. However, the effectiveness of drug delivery to different layers of skin tissues and the circulatory system varies considerably, subject to the properties of the drug delivery system and delivery regime. How to maximise delivery outcomes remains unclear. In this study, mathematical modelling is employed to investigate this transdermal delivery under various conditions, using the skin model that is reconstructed based on the realistic skin anatomical structure. Treatment efficacy is evaluated in terms of drug exposure over time. The modelling results demonstrate the complex dependence of drug accumulation and distribution on the nanocarrier properties, microneedle properties and environment in different skin layers and blood. Specifically, delivery outcomes in the entire skin and blood can be improved by increasing the loading dose and reducing microneedle spacing. However, several parameters need to be optimised with respect to the specific location of the target site in the tissue for better treatment; these include the drug release rate, nanocarrier diffusivity in microneedle and skin tissue, nanocarrier transvascular permeability, nanocarrier partition coefficient between tissue and microneedle, microneedle length, wind speed and relative humidity. The delivery is less sensitive to the diffusivity and physical degradation rate of free drugs in microneedle, and their partition coefficient between tissue and microneedle. Results obtained from this study can be used to improve the design of the microneedle-nanocarrier combined drug delivery system and delivery regime.

A simple method to dramatically increase C. elegans germline microinjection efficiency.

Genome manipulation methods in C. elegans require microinjecting DNA or ribonucleoprotein complexes into the microscopic core of the gonadal syncytium. These microinjections are technically demanding and represent a key bottleneck for all genome engineering and transgenic approaches in C. elegans. While there have been steady improvements in the ease and efficiency of genetic methods for C. elegans genome manipulation, there have not been comparable advances in the physical process of microinjection. Here, we report a simple and inexpensive method for handling worms using a paintbrush during the injection process that nearly tripled average microinjection rates compared to traditional worm handling methods. We found that the paintbrush increased injection throughput by substantially increasing both injection speeds and post-injection survival rates. In addition to dramatically and universally increasing injection efficiency for experienced personnel, the paintbrush method also significantly improved the abilities of novice investigators to perform key steps in the microinjection process. We expect that this method will benefit the C. elegans community by increasing the speed at which new strains can be generated and will also make microinjection-based approaches less challenging and more accessible to personnel and labs without extensive experience.

Effects of Intradermal Administration Volume Using a Hollow Microneedle on the Pharmacokinetics of Fluorescein Isothiocyanate Dextran (M.W. 4,000).

PURPOSE: Hollow microneedles (hMNs) have been gaining attention as a tool to enable the intradermal (i.d.) administration of pharmaceutical products. However, few reports have examined the effect of administration volume on distribution in the skin and pharmacokinetics parameters after i.d. injection. In the present study, a model middle molecular weight compound, fluorescein isothiocyanate dextran (M.W. 4,000, FD-4), was selected, and blood concentration-time profiles after i.d. and subcutaneous (s.c.) injections with different administration volumes were compared. METHODS: FD-4 solution was injected i.d. using a hMN or injected s.c. with a 27 G needle. Pharmacokinetics and dermatokinetics of FD-4 were analyzed using a compartment model. The skin distribution of iodine, as an X ray tracer, was used to evaluate drug disposition. RESULTS: With the administered drug assumed to be absorbed from the broad injection site into blood vessels in the upper and lower dermis by rapid (krapid) and slow (kslow) first-order absorption rate constants, respectively, better agreement of observed and theoretical values was obtained. Furthermore, the fraction, F, of the administered dose absorbed with krapid decreased with the increase in injection volume after i.d. injection, although the pharmacokinetics parameters were almost the same regardless of administration volume after s.c. injection. CONCLUSION: The drug distribution in the skin may be related to the obtained pharmacokinetics parameters suggested that the number of needles in the MN system and the total administration volume should be considered in designing hMN systems. The present results provide useful information that may support effective drug delivery with hMNs.

A Rate-Dependent Cell Microinjection Model Based on Membrane Theory.

As an effective method to deliver external materials into biological cells, microinjection has been widely applied in the biomedical field. However, the knowledge of cell mechanical property is still inadequate, which greatly limits the efficiency and success rate of injection. Thus, a new rate-dependent mechanical model based on membrane theory is proposed for the first time. In this model, an analytical equilibrium equation between the injection force and cell deformation is established by considering the speed effect of microinjection. Different from the traditional membrane-theory-based model, the elastic coefficient of the constitutive material in the proposed model is modified as a function of the injection velocity and acceleration, effectively simulating the influence of speeds on the mechanical responses and providing a more generalized and practical model. Using this model, other mechanical responses at different speeds can be also accurately predicted, including the distribution of membrane tension and stress and the deformed shape. To verify the validity of the model, numerical simulations and experiments were carried out. The results show that the proposed model can match the real mechanical responses well at different injection speeds up to 2 mm/s. The model presented in this paper will be promising in the application of automatic batch cell microinjection with high efficiency.

Microneedles: a novel strategy for wound management.

Wound management is a serious concern worldwide, inflicting a huge social and economic burden on patients and healthcare systems, and research into efficient wound-management measures is crucial. Although advances have been made in traditional wound dressings for wound management to date, the complicated environment near the wound leads to inadequate drug absorption for achieving the intended therapeutic impact. Microneedles, a novel transdermal drug delivery method, can improve wound-healing efficacy by breaking down the barriers at the wound site and enhancing drug delivery efficiency. In recent years, there have been many advanced types of research on the application of microneedles to wound management to address the difficulties encountered in the wound-healing process. This article summarizes and analyzes these research efforts, classifying them according to their distinct efficacy, and addresses them in five areas: hemostasis, antibacterial effects, proliferation, anti-scar, and wound monitoring. The article concludes with a review of the current state and limitations of microneedle patches and an outlook on the future direction of microneedles in wound management as a way to inspire more efficient and smarter wound-management strategies.

Cleanroom-Free Fabrication of Microneedles for Multimodal Drug Delivery.

Microneedles have recently emerged as a powerful tool for minimally invasive drug delivery and body fluid sampling. To date, high-resolution fabrication of microneedle arrays (MNAs) is mostly achieved by the utilization of sophisticated facilities and expertise. Particularly, hollow microneedles have usually been manufactured in cleanrooms out of silicon, resin, or metallic materials. Such strategies do not support the fabrication of microneedles from biocompatible/biodegradable materials and limit the capability of multimodal drug delivery for the controlled release of different therapeutics through a combination of injection and sustained diffusion. This study implements low-cost 3D printers to fabricate relatively large needle arrays, followed by repeatable shrink-molding of hydrogels to form high-resolution molds for solid and hollow MNAs with controllable sizes. The developed strategy further enables modulating surface topography of MNAs to tailor their surface area and instantaneous wettability for controllable drug delivery and body fluid sampling. Hybrid gelatin methacryloyl (GelMA)/polyethylene glycol diacrylate (PEGDA) MNAs are fabricated using the developed strategy that can easily penetrate the skin and enable multimodal drug delivery. The proposed method holds promise for affordable, controllable, and scalable fabrication of MNAs by researchers and clinicians for controlled spatiotemporal administration of therapeutics and sample collection.

Advances in dermatological application of GelMA hydrogel microneedles.

BACKGROUND: Compared with systemic administration methods like injection and oral administration, traditional transdermal drug delivery has the advantages of rapid onset of activity and low side effects. However, hydrophilic drugs and bioactive substances are often unsuitable for traditional transdermal drug delivery. METHODS: The application of microneedles made from gelatin methylacryloyl (GelMA) has greatly expanded thepossibilities for skin transdermal drug delivery. We have reviewed the latest literatures about the dermatological application of GelMA hydrogel microneedles in recent years using Google Scholar, PubMed and Springer. RESULTS: GelMA hydrogel microneedles exhibit huge potency in the diagnosis and treatment of skin diseases, and this technology also brings broad application prospects for subcutaneous micro-invasive transdermal targeted drug delivery, which mainly used in skin tissue fluid collection, local substance delivery and wound healing. CONCLUSION: With in-depth research on GelMA hydrogel, this technology will bring more breakthroughs and developments in the clinical diagnosis and treatment of skin diseases.

Hollow silicon microneedles, fabricated using combined wet and dry etching techniques, for transdermal delivery and diagnostics.

Microneedle-based technologies are the subject of intense research and commercial interest for applications in transdermal delivery and diagnostics, primarily because of their minimally invasive and painless nature, which in turn could lead to increased patient compliance and self-administration. In this paper, a process for the fabrication of arrays of hollow silicon microneedles is described. This method uses just two bulk silicon etches - a front-side wet etch to define the 500 µm tall octagonal needle structure itself, and a rear-side dry etch to create a 50 µm diameter bore through the needle. This reduces the number of etches and process complexity over the approaches described elsewhere. Ex-vivo human skin and a customised applicator were used to demonstrate biomechanical reliability and the feasibility of using these microneedles for both transdermal delivery and diagnostics. Microneedle arrays show no damage even when applied to skin up to 40 times, are capable of delivering several mL of fluid at flowrates of 30 µL/min, and of withdrawing 1 µL of interstitial fluid using capillary action.

Research progress of physical transdermal enhancement techniques in tumor therapy.

The advancement and popularity of transdermal drug delivery (TDD) based on the physical transdermal enhancement technique (PTET) has opened a new paradigm for local tumor treatment. The drug can be directly delivered to the tumor site through the skin, thus avoiding the toxic side effects caused by the first-pass effect and achieving high patient compliance. Further development of PTETs has provided many options for antitumor drugs and laid the foundation for future applications of wearable closed-loop targeting drug delivery systems. In this highlight, the different types of PTETs and related mechanisms, and applications of PTET-related tumor detection and therapy are highlighted. According to their type and characteristics, PTETs are categorized as follows: (1) iontophoresis, (2) electroporation, (3) ultrasound, (4) thermal ablation, and (5) microneedles. PTET-related applications in the local treatment of tumors are categorized as follows: (1) melanoma, (2) breast tumor, (3) squamous cell carcinoma, (4) cervical tumor, and (5) others. The challenges and future prospects of existing PTETs are also discussed. This highlight will provide guidance for the design of PTET-based wearable closed-loop targeting drug delivery systems and personalized therapy for tumors.

Efficient CRISPR/Cas9-Assisted Knockin of Large DNA Donors by Pronuclear Microinjection During S-Phase in Mouse Zygotes.

In the CRISPR/Cas9-mediated gene cassette knockin (KI) strategy, a gene cassette is integrated into a target locus through a proper DNA repair pathway after the Cas9-induced double-strand DNA breaks; the activation of the DNA repair pathway is known to be correlated with the cell cycle. Recently, we have reported a new KI approach named SPRINT (S-phase pronuclear injection for targeting)-CRISPR, focusing on the correlation between the cell cycle and the KI efficiency in the mouse zygote microinjection. Our results suggest that the CRISPR-mediated KI with a homologous recombination-based donor vector during S-phase enhances the KI efficiency. For SPRINT-CRISPR, the uniformity of the zygotes in the cell cycle is achieved by in vitro fertilization, and the zygotes are cryopreserved until use. These reproductive techniques are necessary for efficient KI. Furthermore, Piezo-assisted microinjection has been successful in improving the survival rate of the injected embryos. In this chapter, we describe the protocols that focus on the zygote preparation and Piezo-assisted microinjection of the SPRINT-CRISPR method.

Dissolving microneedles for effective and painless intradermal drug delivery in various skin conditions: A systematic review.

Intra- and transdermal administration of substances via percutaneous injection is effective but considered painful, and inconvenient in addition to bringing forth biohazardous waste material. In contrast to injection, topical drug application, which includes ointments, creams and lotions, increases the local drug load. Moreover, it has reduced side effects compared to systemic administration. However, the epidermis poses a barrier to high molecular weight substances, limiting the delivery efficiency. Dissolving microneedles (DMN) are hydrophilic, mostly polymer-based constructs that are capable of skin penetration and were developed to provide painless and direct dermal drug delivery. This systematic review provides a comprehensive overview of the available clinical evidence for the use of DMN to treat various skin conditions. According to the PRISMA statement, a systematic search for articles on the use of DMN for dermatological indications was conducted on three different databases (Pubmed, Embase, and the Cochrane library). Only human clinical trials were considered. Qualitative assessment was done by two separate reviewers using the Cochrane risk of bias (RoB 2) and Chambers' criteria assessment tools. The search yielded 1090 articles. After deduplication and removal of ineligible records, 889 records were screened on title and abstract. Full text screening was done for 18 articles and ultimately 17 articles were included of which 15 were randomized controlled trials and two were case series. The quality assessment showed that the majority of included studies had low to no risk of bias. Clinical data supports that DMN are an excellent, effective, and pain free drug delivery method for multiple dermatological disorders including skin aging, hyperpigmentation, psoriasis, warts, and keloids by supplying a painless and effective vehicle for intradermal/intralesional drug administration. Microneedle technology provides a promising non- to minimally-invasive alternative to percutaneous injection.

Producing Genetically Engineered Macrophages With Enhanced Immunity via Microinjection.

Many virus-mediated and chemical-based methods for delivering foreign genes into target cells, such as recombinant lentivirus transfection and cationic lipid transfection, are remarkably challenging to use on immune cells because of low efficiency and high toxicity. Microinjection is a promising method to deliver foreign gene expression plasmids into single macrophages directly. This paper reports a new method that can be used to produce a genetically engineered macrophage cell line with enhanced immunity through a home-made high-throughput microinjection system. Microinjection of the expression plasmid carrying a mouse-derived toll-like receptor 4 (Tlr4) gene into a mouse macrophage cell line (Raw264.7) can construct a new stable cell line overexpressing the target gene. The expression efficiency of the target gene in the injected Raw264.7 cells reached 90%, which was measured by injecting a particular plasmid carrying a fused enhanced green fluorescent protein (eGFP) gene fragment with the Tlr4 gene and counting the proportion of cells that emitted green fluorescence. Further assessment of the messenger RNA (mRNA) and protein produced by the Tlr4 gene indicated that its expression was up-regulated remarkably in successfully injected cells. The expression of downstream genes of Tlr4 in injected cells was higher than in untouched cells. Microinjection can avoid polarization effects, which are common when traditional transfection methods are used. A case study was conducted to verify that the injected macrophages overexpressing Tlr4 could activate downstream signaling pathways and showed enhanced inhibition effect on tumor cell migration and invasion. The success of this research will verify that microinjection can be an efficient and safe method in cell transfection applications.