-
Scientific Reports Jan 2017Insertion of foreign molecules such as functionalized fluorescent probes, antibodies, or plasmid DNA to living cells requires overcoming the plasma membrane barrier...
Insertion of foreign molecules such as functionalized fluorescent probes, antibodies, or plasmid DNA to living cells requires overcoming the plasma membrane barrier without harming the cell during the staining process. Many techniques such as electroporation, lipofection or microinjection have been developed to overcome the cellular plasma membrane, but they all result in reduced cell viability. A novel approach is the injection of cells with a nanopipette and using electrophoretic forces for the delivery of molecules. The tip size of these pipettes is approximately ten times smaller than typical microinjection pipettes and rather than pressure pulses as delivery method, moderate DC electric fields are used to drive charged molecules out of the tip. Here, we show that this approach leads to a significantly higher survival rate of nanoinjected cells and that injection with nanopipettes has a significantly lower impact on the proliferation behavior of injected cells. Thus, we propose that injection with nanopipettes using electrophoretic delivery is an excellent alternative when working with valuable and rare living cells, such as primary cells or stem cells.
Topics: Carbocyanines; Cell Line, Tumor; Cell Nucleus; Cell Survival; Electricity; Electrophoresis; Eukaryotic Cells; Fluorescence; Humans; Microinjections; Nanotechnology; Time Factors; Time-Lapse Imaging
PubMed: 28120926
DOI: 10.1038/srep41277 -
Respiratory Physiology & Neurobiology Dec 2017The modulation of cough by microinjections of codeine in 3 medullary regions, the solitary tract nucleus rostral to the obex (rNTS), caudal to the obex (cNTS) and the...
The modulation of cough by microinjections of codeine in 3 medullary regions, the solitary tract nucleus rostral to the obex (rNTS), caudal to the obex (cNTS) and the lateral tegmental field (FTL) was studied. Experiments were performed on 27 anesthetized spontaneously breathing cats. Electromyograms (EMG) were recorded from the sternal diaphragm and expiratory muscles (transversus abdominis and/or obliquus externus; ABD). Repetitive coughing was elicited by mechanical stimulation of the intrathoracic airways. Bilateral microinjections of codeine (3.3 or 33mM, 54±16nl per injection) in the cNTS had no effect on cough, while those in the rNTS and in the FTL reduced coughing. Bilateral microinjections into the rNTS (3.3mM codeine, 34±1 nl per injection) reduced the number of cough responses by 24% (P<0.05), amplitudes of diaphragm EMG by 19% (P<0.01), of ABD EMG by 49% (P<0.001) and of expiratory esophageal pressure by 56% (P<0.001). Bilateral microinjections into the FTL (33mM codeine, 33±3 nl per injection) induced reductions in cough expiratory as well as inspiratory EMG amplitudes (ABD by 60% and diaphragm by 34%; P<0.01) and esophageal pressure amplitudes (expiratory by 55% and inspiratory by 26%; P<0.001 and 0.01, respectively). Microinjections of vehicle did not significantly alter coughing. Breathing was not affected by microinjections of codeine. These results suggest that: 1) codeine acts within the rNTS and the FTL to reduce cough in the cat, 2) the neuronal circuits in these target areas have unequal sensitivity to codeine and/or they have differential effects on spatiotemporal control of cough, 3) the cNTS has a limited role in the cough suppression induced by codeine in cats.
Topics: Abdominal Muscles; Animals; Antitussive Agents; Blood Pressure; Cats; Codeine; Cough; Diaphragm; Disease Models, Animal; Dose-Response Relationship, Drug; Electromyography; Female; Male; Medulla Oblongata; Microinjections; Respiratory Muscles
PubMed: 28778649
DOI: 10.1016/j.resp.2017.07.011 -
Physiological Research Mar 2020As stated by Korpáš and Tomori (1979), cough is the most important airway protective reflex which provides airway defensive responses to nociceptive stimuli. They... (Review)
Review
As stated by Korpáš and Tomori (1979), cough is the most important airway protective reflex which provides airway defensive responses to nociceptive stimuli. They recognized that active expiratory efforts, due to the activation of caudal ventral respiratory group (cVRG) expiratory premotoneurons, are the prominent component of coughs. Here, we discuss data suggesting that neurons located in the cVRG have an essential role in the generation of both the inspiratory and expiratory components of the cough reflex. Some lines of evidence indicate that cVRG expiratory neurons, when strongly activated, may subserve the alternation of inspiratory and expiratory cough bursts, possibly owing to the presence of axon collaterals. Of note, experimental findings such as blockade or impairment of glutamatergic transmission to the cVRG neurons lead to the view that neurons located in the cVRG are crucial for the production of the complete cough motor pattern. The involvement of bulbospinal expiratory neurons seems unlikely since their activation affects differentially expiratory and inspiratory muscles, while their blockade does not affect baseline inspiratory activity. Thus, other types of cVRG neurons with their medullary projections should have a role and possibly contribute to the fine tuning of the intensity of inspiratory and expiratory efforts.
Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Cough; Excitatory Amino Acid Antagonists; Exhalation; Humans; Inhalation; Medulla Oblongata; Microinjections; Neurons; Phrenic Nerve; Reflex; Respiratory Mechanics
PubMed: 32228008
DOI: 10.33549/physiolres.934396 -
Advanced Science (Weinheim,... Sep 2021Microneedles are regarded as an emerging and promising transdermal drug delivery strategy. Great efforts are devoted to getting rid of their material restrictions and...
Microneedles are regarded as an emerging and promising transdermal drug delivery strategy. Great efforts are devoted to getting rid of their material restrictions and imparting them with abilities to carry various drugs. Here, inspired by ice formation in nature and based on characteristics of different frozen materials, the authors present novel ice microneedles made from versatile soft materials using a simple freezing template-based fabrication stratagem for effective transdermal delivery of diverse actives. Their strategy can convert microneedles with almost all water-containing components from softness into hardness for guaranteeing satisfactory penetration, thus removing their material component limitations. As all fabrication procedures are mild and actives can maintain activity during these processes, the ice microneedles can carry and deliver various actives from small molecules and macromolecules to even living organisms. They have demonstrated that these ice microneedles can easily penetrate mouse and swine skins using a microneedle injector, with their active-carried tips left inside after their ice base melts. Thus, by loading heparin, erythropoietin, or biosafe Bacillus subtilis (B. subtilis) inside the ice microneedles to treat mouse models, the practical values of these microneedles are well displayed, indicating their bright prospects in universal drug delivery systems.
Topics: Administration, Cutaneous; Animals; Cold Temperature; Drug Administration Routes; Ice; Mice; Microinjections; Models, Animal; Needles; Swine
PubMed: 34218532
DOI: 10.1002/advs.202101210 -
Pharmaceutical Research Jun 2020The success of protein, peptide and antibody based therapies is evident - the biopharmaceuticals market is predicted to reach $388 billion by 2024 [1], and more than... (Review)
Review
The success of protein, peptide and antibody based therapies is evident - the biopharmaceuticals market is predicted to reach $388 billion by 2024 [1], and more than half of the current top 20 blockbuster drugs are biopharmaceuticals. However, the intrinsic properties of biopharmaceuticals has restricted the routes available for successful drug delivery. While providing 100% bioavailability, the intravenous route is often associated with pain and needle phobia from a patient perspective, which may translate as a reluctance to receive necessary treatment. Several non-invasive strategies have since emerged to overcome these limitations. One such strategy involves the use of microneedles (MNs), which are able to painlessly penetrate the stratum corneum barrier to dramatically increase transdermal drug delivery of numerous drugs. This review reports the wealth of studies that aim to enhance transdermal delivery of biopharmaceutics using MNs. The true potential of MNs as a drug delivery device for biopharmaceuticals will not only rely on acceptance from prescribers, patients and the regulatory authorities, but the ability to upscale MN manufacture in a cost-effective manner and the long term safety of MN application. Thus, the current barriers to clinical translation of MNs, and how these barriers may be overcome are also discussed.
Topics: Administration, Cutaneous; Animals; Antibodies; Drug Delivery Systems; Epidermis; Humans; Microinjections; Needles; Peptides; Pharmaceutical Preparations; Proteins; Skin
PubMed: 32488611
DOI: 10.1007/s11095-020-02844-6 -
Acta Biomaterialia Mar 2018The osteogenic and angiogenic responses of organisms to the ionic products of degradation of bioactive glasses (BGs) are being intensively investigated. The promotion of...
UNLABELLED
The osteogenic and angiogenic responses of organisms to the ionic products of degradation of bioactive glasses (BGs) are being intensively investigated. The promotion of angiogenesis by copper (Cu) has been known for more than three decades. This element can be incorporated to delivery carriers, such as BGs, and the materials used in biological assays. In this work, Cu-containing mesoporous bioactive glass (MBG) in the SiO-CaO-PO compositional system was prepared incorporating 5% mol Cu (MBG-5Cu) by replacement of the corresponding amount of Ca. The biological effects of the ionic products of MBG biodegradation were evaluated on a well-known endothelial cell line, the bovine aorta endothelial cells (BAEC), as well as in an in vivo zebrafish (Danio rerio) embryo assay. The results suggest that ionic products of both MBG (Cu free) and MBG-5Cu materials promote angiogenesis. In vitro cell cultures show that the ionic dissolution products of these materials are not toxic and promote BAEC viability and migration. In addition, the in vivo assay indicates that both exposition and microinjection of zebrafish embryos with Cu free MBG material increase vessel number and thickness of the subintestinal venous plexus (SIVP), whereas assays using MBG-5Cu enhance this effect.
STATEMENT OF SIGNIFICANCE
Mesoporous bioactive glasses (MBGs) with high specific surface area, well-ordered pores, large pore volumes and controllable amount of ions are interesting to develop controlled drug delivery systems for bone tissue regeneration. Copper (Cu) incorporation to the basic SiO-CaO-PO composition has attracted high interest due to its multifunctional biological properties. Promotion of angiogenesis is one of these properties, which can be integrated to the biomaterial with lower cost and higher stability when compared with growth factors. This work reports the synthesis and characterization of Cu-containing MBG evaluating its angiogenic properties in the subintestinal vessel zebrafish assay. This transgenic in vivo assay is merging as an alternative model providing short-time consuming protocols and facilities during pro-angiogenic drug screenings. The report shows that the ionic products of this MBG material delivered to the zebrafish incubation media significantly enhance angiogenesis in comparison with control groups. Besides, results indicate Cu ions may exhibit a synergic effect with Si, Ca, and P ions in angiogenesis stimulation both in vitro and in vivo. To our knowledge, this is the first time that zebrafish in vivo assays are used to evaluate angiogenic activity of ionic dissolution products from MBG materials.
Topics: Animals; Aorta; Cattle; Cell Movement; Cell Proliferation; Ceramics; Copper; Embryo, Nonmammalian; Endothelial Cells; Intestines; Ions; Microinjections; Models, Animal; Neovascularization, Physiologic; Porosity; Zebrafish
PubMed: 29288822
DOI: 10.1016/j.actbio.2017.12.032 -
STAR Protocols Sep 2021LINE1 is the most active and abundant family of retrotransposons; it is implicated in a number of pathologies, as well as in early embryo development. We present a...
LINE1 is the most active and abundant family of retrotransposons; it is implicated in a number of pathologies, as well as in early embryo development. We present a protocol to specifically knockdown LINE1 in mouse embryonic stem cells and embryos, including details for the nucleofection and zygote microinjection of LINE antisense oligos, followed by RNA FISH validation. This protocol can be used in development, as well as other cell types where LINE1 is believed to be expressed. For complete information on the use and execution of this protocol, please refer to Percharde et al. (2018).
Topics: Animals; Embryo, Mammalian; Embryonic Development; Gene Expression Regulation, Developmental; Gene Knockdown Techniques; Long Interspersed Nucleotide Elements; Mice; Microinjections; Mouse Embryonic Stem Cells; Oligonucleotides, Antisense; RNA; RNA, Nuclear; Zygote
PubMed: 34409306
DOI: 10.1016/j.xpro.2021.100726 -
Methods in Enzymology 2019Caged morpholino oligonucleotides (cMOs) are useful research tools in developmental biology because they allow spatiotemporal control of gene expression in whole...
Caged morpholino oligonucleotides (cMOs) are useful research tools in developmental biology because they allow spatiotemporal control of gene expression in whole organisms. While cMOs are usually triggered by light of a single wavelength, the introduction of spectrally distinct chromophores can enable combinatorial regulation of multiple genes. This chapter describes the general principles and methods of wavelength-selective cMO design and synthesis from commercially available reagents. Synthetic protocols for the linkers and the two-step cMO assembly are described in detail, as well as the microinjection and photoactivation techniques. Following these protocols, spectrally separated cyclic cMOs for multiple genes of interest can be prepared, enabling their inhibition in zebrafish embryos and other animal models.
Topics: Animals; Chemistry Techniques, Synthetic; Gene Silencing; Light; Microinjections; Morpholinos; Oligonucleotides, Antisense; Photochemical Processes; Zebrafish
PubMed: 31370936
DOI: 10.1016/bs.mie.2019.04.007 -
Journal of Visualized Experiments : JoVE Oct 2016Simple model organisms are instrumental for in vivo studies of developmental and cellular differentiation processes. Currently, the evolutionary distance to man of...
Simple model organisms are instrumental for in vivo studies of developmental and cellular differentiation processes. Currently, the evolutionary distance to man of conventional invertebrate model systems and the complexity of genomes in vertebrates are critical challenges to modeling human normal and pathological conditions. The chordate Ciona intestinalis is an invertebrate chordate that emerged from a common ancestor with the vertebrates and may represent features at the interface between invertebrates and vertebrates. A common body plan with much simpler cellular and genomic composition should unveil gene regulatory network (GRN) links and functional genomics readouts explaining phenomena in the vertebrate condition. The compact genome of Ciona, a fixed embryonic lineage with few divisions and large cells, combined with versatile community tools foster efficient gene functional analyses in this organism. Here, we present several crucial methods for this promising model organism, which belongs to the closest sister group to vertebrates. We present protocols for transient transgenesis by electroporation, along with microinjection-mediated gene knockdown, which together provide the means to study gene function and genomic regulatory elements. We extend our protocols to provide information on how community databases are utilized for in silico design of gene regulatory or gene functional experiments. An example study demonstrates how novel information can be gained on the interplay, and its quantification, of selected neural factors conserved between Ciona and man. Furthermore, we show examples of differential subcellular localization in embryonic cells, following DNA electroporation in Ciona zygotes. Finally, we discuss the potential of these protocols to be adapted for tissue specific gene interference with emerging gene editing methods. The in vivo approaches in Ciona overcome major shortcomings of classical model organisms in the quest of unraveling conserved mechanisms in the chordate developmental program, relevant to stem cell research, drug discovery, and subsequent clinical application.
Topics: Animals; Ciona intestinalis; Electroporation; Gene Transfer Techniques; Humans; Microinjections
PubMed: 27805579
DOI: 10.3791/54313 -
Sheng Wu Gong Cheng Xue Bao = Chinese... Feb 2024In recent years, microneedles have emerged as a drug delivery technology that holds great research value and application potential due to their minimally invasive,... (Review)
Review
In recent years, microneedles have emerged as a drug delivery technology that holds great research value and application potential due to their minimally invasive, painless, user-friendly, and efficient characteristics. The technology of microneedles has rapidly evolved over the past 20 years, allowing customization of shape, composition, mechanical properties, and unique functions to meet diverse needs. With the ability to minimally invasively traverse various biological barriers, researchers have explored the applications of microneedles in various tissues and organs beyond the skin. This article summarizes the research progress on the use of microneedles for drug delivery in tissues such as eyes, blood vessel, and heart. By presenting these cutting-edge research to readers, we hope to promote the development and application of microneedle technology.
Topics: Administration, Cutaneous; Microinjections; Needles; Skin; Drug Delivery Systems
PubMed: 38369832
DOI: 10.13345/j.cjb.230617