Diphtheria toxoid dissolving microneedle vaccination: Adjuvant screening and effect of repeated-fractional dose administration.

In this study the effect of repeated-fractional intradermal administration of diphtheria toxoid (DT) compared to a single administration in the presence or absence of adjuvants formulated in dissolving microneedles (dMNs) was investigated. Based on an adjuvant screening with a hollow microneedle (hMN) system, poly(I:C) and gibbsite, a nanoparticulate aluminum salt, were selected for further studies: they were co-encapsulated with DT in dMNs with either a full or fractional DT-adjuvant dose. Sharp dMNs were prepared regardless the composition and were capable to penetrate the skin, dissolve within 20 min and deposit the intended antigen-adjuvant dose, which remained in the skin for at least 5 h. Dermal immunization with hMN in repeated-fractional dosing (RFrD) resulted in a higher immune response than a single-full dose (SFD) administration. Vaccination by dMNs led overall to higher responses than hMN but did not show an enhanced response after RFrD compared to a SFD administration. Co-encapsulation of the adjuvant in dMNs did not increase the immune response further. Immunization by dMNs without adjuvant gave a comparable response to subcutaneously injected DT-AlPO4 in a 15 times higher dose of DT, as well as subcutaneous injected DT-poly(I:C) in a similar DT dose. Summarizing, adjuvant-free dMNs showed to be a promising delivery tool for vaccination performed in SFD administration.

Anesthetic protocol for microinjection-related handling of Siberian sturgeon (Acipenser baerii; Acipenseriformes) prolarvae.

An anesthetic protocol was optimized for microinjection-related handling of Siberian sturgeon (Acipenser baerii; Acipenseriformes) prolarvae, an extant primitive fish species commonly grown in aquaculture. Comparative examinations of three selected anesthetics (clove oil, lidocaine, and MS-222) with a dosage regime of 50, 100, 200, and 400 mg/L indicated that MS-222 was the most efficient agent for Siberian sturgeon prolarvae, as evidenced by the fast induction of anesthesia with quick and uniform recovery. Meanwhile, clove oil should be avoided, due to prolonged recovery times varying widely between individuals. None of the tested anesthetics significantly affected prolarval viability at any of the dosage regimes tested in this study. Based on an analysis of the duration of an unconscious state in air, we recommend a dose of 200 mg/L MS-222 for microinjection. Recovery time after use of this dose was influenced by the prolarval age and the development of gills, in which prolarvae older than 3 days after hatching required longer recovery times than did younger prolarvae. Post-recovery behavioral assessment showed no apparent difference between MS-222-anesthetized and non-anesthetized prolarvae in their swimming behavior and phototactic responses. Applicability of currently developed anesthetic protocol using MS-222 in larval microinjection was demonstrated with the injection of a visible dye to the anesthetized prolarvae, followed by the analysis of post-recovery viability. Taken together, the present anesthetic protocol based on 200 mg/L of MS-222 could provide researchers with practical usefulness with good safety margins for the micromanipulation and other related handlings of Siberian sturgeon prolarvae.

Depressed scar after filler injection successfully treated with pneumatic needleless injector and radiofrequency device.

Fillers are known to be associated with a number of side effects, one of the most severe being skin necrosis. The most vulnerable areas are those that are supplied by a single arterial branch; for example, the glabellar and nasolabial folds are susceptible. In this study, we report good cosmetic outcomes were produced by utilizing the pneumatic needleless injector and radiofrequency device to treat depressed scars that occurred after necrosis following filler injection. Initially, applying light-emitting diode treatment and following through with the two devices appears to have synergistic effects for scar remodeling when dealing with treatment of depressed scars with irregular borders.

Ex vivo evaluation of a microneedle array device for transdermal application.

A new approach of transdermal drug delivery is the use of microneedles. This promising technique offers the potential to be broadly used for drug administration as it enables the dramatic increase in permeation of medicaments across the stratum corneum. The potential of microneedles has evolved to spawn a plethora of potential transdermal applications. In order to advance the microneedle capabilities and possibly revolutionize advanced drug delivery, this study introduces a novel transdermal electro-modulated hydrogel-microneedle array (EMH-MNA) device composed of a nano-porous, embeddable ceramic microneedle array as well as an optimized EMH for the electro-responsive delivery of indomethacin through the skin. The ex vivo permeation as well as drug release experiments were performed on porcine skin tissue to ascertain the electro-responsive capabilities of the device. In addition, the microbial permeation ability of the microneedles across the viable epidermis in both microneedle-punctured skin as well as hypodermic needle-punctured skin was determined. Ex vivo evaluation of the EMH-MNA device across porcine skin demonstrated that without electro-stimulation, significantly less drug release was obtained (±0.4540mg) as compared to electro-stimulation (±2.93mg).

Novel use of pectin as a microneedle base.

Hydrocolloid pectin formulation was utilized as a novel base for fabricating biodegradable micro-needle (MN) arrays. The pectin MNs were, on average, found to be 897.71 ± 3.48 µm in height and 234.31 ± 2.27 µm in base width, with an inter base spacing of 498.66 ± 1.60 µm, and corresponding to an aspect ratio of 3.83 ± 0.04. Bovine serum albumin (BSA) and pectin gel interaction was found to be dependent on the loaded protein amount. By contrast, regardless of the amount of BSA incorporated, pectin MNs competed with BSA to form a complex with Cu(2+) ions in a bicinchoninic acid (BCA) kit. The glass transition of the pectin MN base was found to be 145.15 ± 12.09, with a delta Cp of 2.60 ± 0.02 J g(-1) K(-1). Because pectin MNs are skin friendly and naturally occurring, with biodegradable and hydrocolloidal features, they are promising vehicle for the controlled release of macromolecules.

Design and physicochemical characterisation of novel dissolving polymeric microneedle arrays for transdermal delivery of high dose, low molecular weight drugs.

We describe formulation and evaluation of novel dissolving polymeric microneedle (MN) arrays for the facilitated delivery of low molecular weight, high dose drugs. Ibuprofen sodium was used as the model here and was successfully formulated at approximately 50% w/w in the dry state using the copolymer poly(methylvinylether/maleic acid). These MNs were robust and effectively penetrated skin in vitro, dissolving rapidly to deliver the incorporated drug. The delivery of 1.5mg ibuprofen sodium, the theoretical mass of ibuprofen sodium contained within the dry MN alone, was vastly exceeded, indicating extensive delivery of the drug loaded into the baseplates. Indeed in in vitro transdermal delivery studies, approximately 33mg (90%) of the drug initially loaded into the arrays was delivered over 24h. Iontophoresis produced no meaningful increase in delivery. Biocompatibility studies and in vivo rat skin tolerance experiments raised no concerns. The blood plasma ibuprofen sodium concentrations achieved in rats (263µgml(-1) at the 24h time point) were approximately 20 times greater than the human therapeutic plasma level. By simplistic extrapolation of average weights from rats to humans, a MN patch design of no greater than 10cm(2) could cautiously be estimated to deliver therapeutically-relevant concentrations of ibuprofen sodium in humans. This work, therefore, represents a significant progression in exploitation of MN for successful transdermal delivery of a much wider range of drugs.

Influence of an apical negative pressure irrigation system on bacterial elimination during endodontic therapy: a prospective randomized clinical study.

INTRODUCTION: Recent in vitro studies that use an apical negative pressure irrigation system, EndoVac, have demonstrated promising results in the production of debris-free root canals, while also preventing potential extrusion of irrigants into the periapical region. We conducted a randomized, controlled, prospective clinical study to determine whether the use of EndoVac irrigation (EndoVac group) was more efficient compared with standard needle irrigation (control group) in obtaining canals from which microbes could not be cultivated. METHODS: Routine endodontic therapy was performed in 48 patients with necrotic, single-rooted, single-canal teeth. The patients were randomly assigned to either the EndoVac group (n = 25) or control group (n = 23). Irrigation with either method was carried out with 0.5% sodium hypochlorite. After surface disinfection, before instrumentation and on completion of chemomechanical preparation, intracanal microbial samples were obtained and cultured under anaerobic conditions. The frequency of microbial cultivability by using either irrigation system was analyzed. RESULTS: The frequency of obtaining culture-negative root canals was 90.9% and 82.6% for the control group and EndoVac group, respectively. There was no significant difference in the antimicrobial efficacy of either control group or EndoVac group (Fisher exact test, P = .665). Furthermore, no significant association between study variables and the irrigation systems' antimicrobial efficacy was found (P > .05). CONCLUSIONS: The results of this prospective in vivo study demonstrate that the antimicrobial efficacy of EndoVac irrigation is comparable to that of standard irrigation.

Subretinal delivery and electroporation in pigmented and nonpigmented adult mouse eyes.

Subretinal injection offers one of the best ways to deliver many classes of drugs, reagents, cells and treatments to the photoreceptor, Müller, and retinal pigment epithelium (RPE) cells of the retina. Agents delivered to this space are placed within microns of the intended target cell, accumulating to high concentrations because there is no dilution due to transport processes or diffusion. Dilution in the interphotoreceptor space (IPS) is minimal because the IPS volume is only 10-20 µl in the human eye and less than 1 µl in the mouse eye. For gene delivery purposes, we wished to transfect the cells adjacent to the IPS in adult mouse eyes. Others transfect these cells in neonatal rats to study the development of the retina. In both neonates and adults, electroporation is found to be effective. Here we describe the optimization of electroporation conditions for RPE cells in the adult mouse eye with naked plasmids. However, both techniques, subretinal injection and electroporation, present some technical challenges that require skill on the part of the surgeon to prevent untoward damage to the eye. Here we describe methods that we have used for the past 10 years (Johnson et al. Mol Vis 14: 2211-2226, 2008).

Femtosecond laser-assisted microinjection into living neurons.

A Ti:sapphire femtosecond laser micro-manipulation system was used to perforate vital cells. Propidium iodide was successfully transferred into both astrocytes and PC12 cells via the channel of a femtosecond laser, which induced transiently recoverable opening of the cell membrane. The transfection efficiency reached almost 100%. This method also had the advantage of contact-free, non-disruptive and stable transfection.

An automated system for intracellular and intranuclear injection.

The Xenopus oocyte expression system has played an important role in the study of cellular proteins, particularly in the field of membrane physiology; expression of transporters and ion channels has significantly advanced our knowledge of these membrane proteins and the rapid and easy expression of mutants has been crucial in many structure-function studies. Xenopus oocytes are an expression system in many ligand-binding assays and in functional screening for ion channel modulators. Several commercially available automated technologies use this system, generating a demand for large numbers of oocytes injected with ion channel genes. Injection of oocytes with genetic material is generally carried out manually. Here we describe an automated system capable of injecting up to 600 oocytes per hour. Oocytes are contained in microplates with conical wells, a simple calibration procedure by the operator is required and pipette filling and oocyte injection are carried out automatically. Following intracellular injection of mRNA coding for ligand-gated ion channels close to 100% of oocytes tested positive for expression, and intranuclear injection of cDNA gave a rate of expression >50%. Moreover, we demonstrate that this method can also be successfully applied to inject zebrafish embryos and could be extended to other cell types.

Validation of a method for localised microinjection of drugs into thalamic subregions in rats for epilepsy pharmacological studies.

OBJECTIVES: To validate a method for the chronic implantation of micro-cannulae to examine the effect of drug administration to two small brain regions critical to the control of generalised seizures, the reticular nucleus of the thalamus (Rt) and the ventrobasal thalamus (VB), in a genetically epileptic rat model. METHOD: Micro-cannulae guides (length 9 mm, 26G, i.d. 0.24 mm, o.d. 0.46 mm) were implanted bilaterally into either the Rt or the VB of 11- to 13-week-old Genetic Absence Epilepsy Rats from Strasbourg (GAERS) using a stereotaxic head frame. After a seven-day recovery period the animals were injected with 0.2 microl of methylene blue. The animals were allowed to move freely in their cages for a further 90 min while a post-drug EEG recording was acquired and then brains were perfused with 4% paraformaldehyde and extracted. Twenty-micrometer slices were cut on a cryostat and the site and extent of the methylene blue staining in the brain determined. The implantation co-ordinates were adjusted accordingly, and then a validation study was performed on a further cohort of rats (n=8 Rt, n=7 VB). RESULTS: The co-ordinates that were found to most accurately localise the Rt were: AP -3mm, ML 3.6mm, DV -5.8mm (relative to Bregma). Those that accurately localised the VB were: AP -3mm, ML 2.6mm, DV -5.5mm. In the validation study, the dye staining was measured to diffuse an average radius of 520+/-120 microm from the centre of the injection site for the 0.2 microl injection in both brain hemispheres. For the VB injections the dye remained confined within the structure, however, for the smaller Rt there was spread to surrounding structures in the axial plane. The radial diffusion for the 0.5 microl injection was similar, but more of the dye was found to spread back up the cannula tract away from the target zone. CONCLUSION: These studies have validated a method for accurate and localised injection of drugs into the VB and Rt for neuropharmacological studies in a rat model of generalised epilepsy. This method allows the measurement of localised drug effects on EEG and generalised seizure activity at these sites.

A technique for long-term implantation of a microcatheter into the third ventricle of post-pubertal Chinese Meishan pigs based on ventriculography.

The objectives of this study were to implant a microcatheter into the third ventricle of post-pubertal Chinese Meishan pigs, and to maintain the microcatheter for a long time without causing stress. Fourteen pigs (45-60 kg BW) were used. Each pig was anesthetized and the head was orientated on the stereotaxic apparatus. A radiopaque dye was placed into the ventricle via a guide cannula inserted 3.5 mm forward of the bregma. A microcatheter was inserted into the third ventricle using ventriculography, and fixed with dental cement to a metal-mesh protector and screw anchors embedded into the skull. The opposite end of the microcatheter was externalized from the dorsal neck so that corticotropin-releasing hormone (CRH) could be injected easily. Simultaneously, a catheter was fitted in the jugular vein, and the free end of the catheter was externalized from the dorsal neck. Microcatheter-implanted pigs showed a normal progesterone concentration profile, and a constant cortisol level during at least two estrous cycles. Furthermore, intracerebroventricular injections of CRH (25 microg/500 microl) resulted in an increased plasma cortisol concentration (P < 0.05). Thus, the technique developed in this study enables us to approach the third ventricle in post-pubertal freely-moving pigs effectively over a long time, without causing stress.

A simple method for focally delivering multiple drugs or ligands to cells.

A simple venturi-based "spritzer" device is described that is capable of repeatedly and rapidly delivering small volumes of different solutions to cells or tissues. This is particularly advantageous in situations when the appropriate ligand or drug is not known a priori and has to be determined "on the fly" during the limited lifetime of a preparation. The utility of the device is demonstrated for applying odorants to olfactory receptor neurons (ORNs).

Modification of an automated liquid-handling system for reagent-jet, nanoliter-level dispensing.

Reducing the scale of biochemical reactions is becoming commonplace. Examples include the screening of large libraries of chemical compounds or gene sequences. These applications demand the ability to transfer sub-microliter volumes of fluid. To this end, we have modified a Hamilton MICROLAB 2200 with high-speed solenoids and a liquid pressurization system to modulate volume delivery down to the nanoliter level. Additional modifications include the use of sapphire-tipped dispensing nozzles and a high-resolution substage to assist in the construction of DNA microarrays. Techniques for characterizing the dispensed volume are presented.

Xenopus adenine nucleotide translocase mRNA exhibits specific and dynamic patterns of expression during development.

We report the isolation and characterization of the Xenopus homolog to human T1 ANT (adenine nucleotide translocase). The 1290-nucleotide sequence contains initiation and termination signals, and encodes a conceptual protein of 298 amino acids. The sequence shares high amino acid identity with the mammalian adenine translocases. The transcript is present in unfertilized eggs, and it is expressed at higher levels during formation of the antero-posterior dorsal axis in embryos. Although low levels are expressed constitutively except in endodermal cells, adenine nucleotide translocase (ANT) expression is dynamically regulated during neurulation. At this stage, expression in ectoderm rapidly diminishes as the neural folds form, and then ANT expression increases slightly in mesoderm. At the culmination of neurulation, the neural tube briefly expresses ANT, and thereafter its expression predominates in the somitic mesoderm and also the chordoneural hinge. In addition, ANT expression is particularly high in the prosencephalon, the mesencephalon, the branchial arches, eye, and the otic vesicle. Treatment of embryos with retinoic acid has the effect of diminishing constitutive expression of ANT, but microinjection studies demonstrate that immediate and local repression cannot be induced in dorsal structures.

A galinstan expansion femtosyringe for microinjection of eukaryotic organelles and prokaryotes.

A galinstan expansion femtosyringe enables femtoliter to attoliter samples to be introduced into prokaryotes and subcellular compartments of eukaryotes. The method uses heat-induced expansion of galinstan (a liquid metal alloy of gallium, indium, and tin) within a glass syringe to expel samples through a tip diameter of about 0.1 microm. The narrow tip inflicts less damage than conventional capillaries, and the heat-induced expansion of the galinstan allows fine control over the rate of injection. We demonstrate injection of Lucifer Yellow and Lucifer Yellow-dextran conjugates into cyanobacteria, and into nuclei and chloroplasts of higher organisms. Injection of a plasmid containing the bla gene into the cyanobacterium Phormidium laminosum resulted in transformed ampicillin-resistant cultures. Green fluorescent protein was expressed in attached leaves of tobacco and Vicia faba following injection of DNA containing its gene into individual chloroplasts.

Microcatheter adhesion of cyanoacrylates: comparison of normal butyl cyanoacrylate to 2-hexyl cyanoacrylate.

PURPOSE: To compare the catheter adhesion properties of 2-hexyl cyanoacrylate (Neuracryl M), a new agent, to those of normal butyl cyanoacrylate (Histoacryl), the most widely used liquid acrylic agent for microcatheter embolization. MATERIALS AND METHODS: 2-hexyl cyanoacrylate (Neuracryl M1) was tested in pure form and mixed with either a proprietary polymerization retardant/contrast agent (Neuracryl M2) or ethiodized oil (Ethiodol). Histoacryl was tested in pure form and mixed with Ethiodol. The cyanoacrylate mixtures were injected through microcatheters into wells partially filled with heparinized whole blood. The cyanoacrylates were allowed to polymerize around the microcatheter tips for 1-3 minutes. The microcatheters were then pulled at a constant rate until they were extracted from the polymerized cyanoacrylates. The peak forces required for extraction were recorded. RESULTS: The peak forces required to extract the microcatheters from either pure Histoacryl or Histoacryl mixed with 33% Ethiodol were significantly higher (P < .01; P < .05) than those for pure Neuracryl M1. When Neuracryl M1 and M2 were mixed together (as intended for clinical use), the force required for microcatheter extraction was significantly lower than that for either pure Histoacryl, Histoacryl mixed with 33% Ethiodol, or Neuracryl M1 alone (P < .01; P < .01; P < .01, respectively). The force required to extract microcatheters from the Neuracryl M1 and M2 mixture was not, however, significantly different from that of Histoacryl mixed with 50% Ethiodol. The force of extraction for the Neuracryl M1 and 50% Ethiodol mixture was below our ability to obtain precise measurements. CONCLUSION: When Neuracryl M1 was mixed with its proprietary polymerization retardant/contrast agent (Neuracryl M2), catheter adhesion was not significantly different from that of Histoacryl mixed with 50% Ethiodol, a mixture common in clinical use. When Neuracryl M1 was tested alone or mixed with Ethiodol (not intended by the manufacturer), catheter adhesion was significantly decreased relative to pure Histoacryl or equivalent mixtures of Histoacryl and Ethiodol.

A simple pressure microinjecting system for delivery of small substance volumes to the brain: application to the developmental study of thalamo-cortical projections in foetal and neonatal rats.

We describe a reliable and inexpensive method for placing injections of anatomical tracers into the brain of lower mammals. The pressure microinjecting system we developed is specifically designed to deliver very small amount of substances. The injecting portion of the system is relatively easy to assemble and can be repeatedly used for multiple experimental sessions. The system has been validated with experiments of multiple fluorescent retrograde tracing. In these experiments the populations of thalamo-cortical neurons were consistently labeled by the tracers injected bilaterally and symmetrically in the cortex of foetal and neonatal rats.