Ovalbumin and cholera toxin delivery to buccal mucus for immunization using microneedles and comparison of immunological response to transmucosal delivery.

The oral mucosa is an effective site for vaccination. However, for oral mucosal vaccines, delivery of the right dose of vaccine is not possible due to the water-rich environment. In this study, the buccal mucosa, which is easy to access using a microneedle array in the oral cavity, was selected as the administration site. The immune responses to the use of microneedles to conventional transmucosal delivery were compared. In addition, the adjuvant effect of the addition of cholera toxin (CT) to the drug formulation was observed. Two kinds of patches were prepared: (1) Ovalbumin (OVA) was dip coated only on the tips of microneedles (C-OVA-MN) and (2) OVA was coated on the surface of a flat disk patch substrate without microneedles (C-OVA-D). The drug delivery properties of C-OVA-MN and C-OVA-D were investigated using fluorescent-labeled OVA (OVA/FITC). Each patch was administered to mice twice, 2 weeks apart, and then antibody titers were measured. A microneedle patch can deliver vaccine into the epithelium of the buccal mucosa in a short period of time compared to transmucosal delivery. A microneedle system of C-OVA-MN showed a high serum IgG titer. In addition, CT triggered CD8+ and CD4+ T cell-mediated immune responses. Through this study, we present the possibility of a new method of vaccination to the buccal mucosa using microneedles and CT adjuvant. Illustration of delivery of vaccine to the oral mucosal epithelium using a microneedle patch: Ovalbumin (OVA)-coated microneedle (C-OVA-MN) consists of tip, step, and coating formulation. Microneedle patch coated with OVA formulation is targeting buccal mucosa, which is easy to access in the oral cavity. OVA is delivered to the buccal epithelium precisely using a microneedle patch, and OVA is delivered by transmucosal route using a disk patch.

Live Vaccinia Virus-Coated Microneedle Array Patches for Smallpox Vaccination and Stockpiling.

Although smallpox has been eradicated globally, the potential use of the smallpox virus in bioterrorism indicates the importance of stockpiling smallpox vaccines. Considering the advantages of microneedle-based vaccination over conventional needle injections, in this study, we examined the feasibility of microneedle-based smallpox vaccination as an alternative approach for stockpiling smallpox vaccines. We prepared polylactic acid (PLA) microneedle array patches by micromolding and loaded a second-generation smallpox vaccine on the microneedle tips via dip coating. We evaluated the effect of excipients and drying conditions on vaccine stability in vitro and examined immune responses in female BALB/c mice by measuring neutralizing antibodies and interferon (IFN)-γ-secreting cells. Approximately 40% of the virus titer was reduced during the vaccine-coating process, with or without excipients. At -20 °C, the smallpox vaccine coated on the microneedles was stable up to 6 months. Compared to natural evaporation, vacuum drying was more efficient in improving the smallpox vaccine stability. Microneedle-based vaccination of the mice elicited neutralizing antibodies beginning 3 weeks after immunization; the levels were maintained for 12 weeks. It significantly increased IFN-γ-secreting cells 12 weeks after priming, indicating the induction of cellular immune responses. The smallpox-vaccine-coated microneedles could serve as an alternative delivery system for vaccination and stockpiling.

Patchless administration of canine influenza vaccine on dog's ear using insertion-responsive microneedles (IRMN) without removal of hair and its in vivo efficacy evaluation.

Microneedles provide the advantages of convenience and compliance by avoiding the pain and fear of needles that animals often experience. Insertion-responsive microneedles (IRMN) were used for administration to a hairy dog without removing the dog's hair. Canine H3N2 vaccine was administered with IRMN attached to the dog's ears ex vivo and the conventional microneedle system (MN) was administered for 15 min to compare puncture performance and delivery efficiency. The vaccine was also administered to compare antibody formation using IRMN with the use of intramuscular injection. The veterinarian observed the behavior of the dog during the course of the administration and compared the response to IRMN with that of intramuscular administration. The tips of IRMN were separated from the base and delivered into the hairy skin successfully. Puncture performance of IRMN were the same as that of coated microneedles (95%), but delivery efficiency of IRMN were 95% compared to less than 1% for coated microneedles. The H3N2 vaccine inoculated into the dog's ears showed the same antibody formation as the intramuscular injection. The dog appeared to be more comfortable with IRMN administration compared to syringe administration. IRMN are the first microneedle system to deliver a canine vaccine successfully into a hairy dog without removal of the dog's hair. The use of IRMN can provide both convenience and compliance for both the dog and the owner.

Amino/Amido Conjugates Form to Nanoscale Cobalt Physiometacomposite (PMC) Materials Functionally Delivering Nucleic Acid Therapeutic to Nucleus Enhancing Anticancer Activity via Ras-Targeted Protein Interference.

Aberrant splicing and protein interaction of Ras binding domain (RBD) are associated with melanoma drug resistance. Here, cobalt or nickel doped zinc oxide (ZnO) physiometacomposite (PMC) materials bind to RNA and peptide shown by Ninhydrin staining, UV-vis, Fourier transform infrared, and circular dichroism spectroscopy. PMCs deliver splice switching oligomer (SSO) into melanoma cells or 3-D tumor spheroids shown by flow cytometry, fluorescence microscopy, and bioluminescence. Stability in serum, liver, or tumor homogenate up to 48 h and B16F10 melanoma inhibition ≥98-99% is shown. These data suggest preclinical potential of PMC for delivery of SSO, RBD, or other nucleic acid therapeutic and anticancer peptides.

Development of Botulinum Toxin A-Coated Microneedles for Treating Palmar Hyperhidrosis.

Hyperhidrosis is a disorder that is characterized by the production of excess amounts of sweat. The botulinum neurotoxin A (BoNT/A) has been used to treat hyperhidrosis through multiple intradermal injections at the site of the condition. However, because of BoNT/A toxicity, it is important to precisely deliver the proper dose of the toxin to the target site. In addition, the use of a conventional hypodermic needle for multiple injections in the palm makes the approach undesirable and painful. Here, we designed a BoNT/A-coated microneedle (BoNT-MN) array and tested its efficacy as a substitute pain-free method to treat hyperhidrosis. BoNT-MNs were prepared by coating polylactic acid microneedles with a BoNT/A formulation and were found to successfully penetrate into a thick skin in vitro. The coating formulations were then tested for their stability at 4, 25, and 37 °C for 24 h. BoNT-MNs were found to be much more stable than BoNT/A in a liquid state. Additionally, we carried out in vivo experiments by treating the right paws of mice with BoNT-MNs and found that the treatment induced a significant reduction in the sweating response in the mouse foot pad. Thus, BoNT/A treatment using microneedles is beneficial and may be used as a more efficient and less painful approach to treat hyperhidrosis.

Comparing the effects of physiologically-based metal oxide nanoparticles on ribonucleic acid and RAS/RBD-targeted triplex and aptamer interactions.

Physiological metals such as zinc, magnesium, and nickel facilitate nucleic acid and protein interactions and stability. In the nanoscale, the impact these have on nucleic acid structure-function is very poorly understood and was investigated here. Nanoparticles' (NP) RNA precipitation efficiency was in the order; NiO > MgO > ZnO > CaO > CaCO3>Cu. Gel mobility shift was observed for MgO and especially ZnO NP. Loss of staining intensity was shown for Cu suggesting this NP may denature RNA supported by the UV- and CD-spectroscopy patterns, change in area-under-the-curve (AUC) and abs260 nm measurements. Aptamer and triplex-forming oligomer (TFO) sequences were designed targeting RAS/Ras binding domain (RBD) and the impact of the NP on target interaction investigated. MgO NP promotes aptamer:RBD interaction and preserves triplex formation whereas NiO NP effects duplex migration and intensifies staining of the triplex suggesting a novel mechanism of interaction and conformation. These data strongly support the role of MgO, ZnO and NiO NP for nucleic acid nanobio interaction and suggest potential biomedical application for such novel interfaces.

Spray-Formed Layered Polymer Microneedles for Controlled Biphasic Drug Delivery.

In this study we present polymeric microneedles composed of multiple layers to control drug release kinetics. Layered microneedles were fabricated by spraying poly(lactic-co-glycolic acid) (PLGA) and polyvinylpyrrolidone (PVP) in sequence, and were characterized by mechanical testing and ex vivo skin insertion tests. The compression test demonstrated that no noticeable layer separation occurred, indicating good adhesion between PLGA and PVP layers. Histological examination confirmed that the microneedles were successfully inserted into the skin and indicated biphasic release of dyes incorporated within microneedle matrices. Structural changes of a model protein drug, bovine serum albumin (BSA), in PLGA and PVP matrices were examined by circular dichroism (CD) and fluorescence spectroscopy. The results showed that the tertiary structure of BSA was well maintained in both PLGA and PVP layers while the secondary structures were slightly changed during microneedle fabrication. In vitro release studies showed that over 60% of BSA in the PLGA layer was released within 1 h, followed by continuous slow release over the course of the experiments (7 days), while BSA in the PVP layer was completely released within 0.5 h. The initial burst of BSA from PLGA was further controlled by depositing a blank PLGA layer prior to forming the PLGA layer containing BSA. The blank PLGA layer acted as a diffusion barrier, resulting in a reduced initial burst. The formation of the PLGA diffusion barrier was visualized using confocal microscopy. Our results suggest that the spray-formed multilayer microneedles could be an attractive transdermal drug delivery system that is capable of modulating a drug release profile.

Interaction of Surface Energy Components between Solid and Liquid on Wettability, and Its Application to Textile Anti-Wetting Finish.

With various options of anti-wetting finish methods, this study intends to provide basic information that can be applied in selecting a relevant anti-wetting chemical to grant protection from spreading of liquids with different surface energy profiles. With such an aim, the anti-wetting effectiveness of fluorinated coating and silane coating was investigated for liquids having different surface energy components, water (WA), methylene iodide (MI) and formamide (FA). The wetting thermodynamics was experimentally investigated by analyzing dispersive and polar component surface energies of solids and liquids. The role of surface roughness in wettability was examined for fibrous nonwoven substrates that have varied surface roughness. The presence of roughness enhanced the anti-wetting performance of the anti-wetting treated surfaces. While the effectiveness of different anti-wetting treatments was varied depending on the liquid polarities, the distinction of different treatments was less apparent for the roughened fibrous surfaces than the film surfaces. This study provides experimental validation of wetting thermodynamics and the practical interpretation of anti-wetting finishing.

Comparative functional dynamics studies on the enzyme nano-bio interface.

INTRODUCTION: Biomedical applications of nanoparticles (NPs) as enzyme inhibitors have recently come to light. Oxides of metals native to the physiological environment (eg, Fe, Zn, Mg, etc.) are of particular interest-especially the functional consequences of their enzyme interaction. MATERIALS AND METHODS: Here, Fe2O3, zinc oxide (ZnO), magnesium oxide (MgO) and nickel oxide (NiO) NPs are compared to copper (Cu) and boron carbide (B4C) NPs. The functional impact of NP interaction to the model enzyme luciferase is determined by 2-dimensional fluorescence difference spectroscopy (2-D FDS) and 2-dimensional photoluminescence difference spectroscopy (2-D PLDS). By 2-D FDS analysis, the change in maximal intensity and in 2-D FDS area under the curve (AUC) is in the order Cu~B4C>ZnO>NiO>>Fe2O3>MgO. The induced changes in protein conformation are confirmed by tryptic digests and gel electrophoresis. RESULTS: Analysis of possible trypsin cleavage sites suggest that cleavage mostly occurs in the range of residues 112-155 and 372-439, giving a major 45 kDa band. By 2-D PLDS, it is found that B4C NPs completely ablate bioluminescence, while Cu and Fe2O3 NPs yield a unique bimodal negative decay rate, -7.67×103 and -3.50×101 relative light units respectively. Cu NPs, in particular, give a remarkable 271% change in enzyme activity. Molecular dynamics simulations in water predicted that the surfaces of metal oxide NPs become capped with metal hydroxide groups under physiological conditions, while the surface of B4C becomes populated with boronic acid or borinic acid groups. These predictions are supported by the experimentally determined zeta potential. Thin layer chromatography patterns further support this conception of the NP surfaces, where stabilizing interactions were in the order ionic>polar>non-polar for the series tested. CONCLUSION: Overall the results suggest that B4C and Cu NP functional dynamics on enzyme biochemistry are unique and should be examined further for potential ramifications on other model, physiological or disease-relevant enzymes.

Insertion-responsive microneedles for rapid intradermal delivery of canine influenza vaccine.

In this study, we present transcutaneous influenza vaccination using a novel tip-separable microneedle system called insertion-responsive microneedles (IRMNs). IRMNs are composed of dissolvable hyaluronic acid (HA) tips and biocompatible polycaprolactone (PCL) bases, the tip of which is instantly separated from the base during microneedle insertion and retraction. Vaccine antigens derived from canine influenza virus (A/canine/VC378/2012; H3N2) were successfully coated on HA tips by rapidly freezing the tips prior to coating. An ex vivo porcine skin insertion test showed that IRMNs were capable of penetrating the skin without tip breakage and releasing the coated materials within the skin. The thermal stability of the vaccine as determined by hemagglutination assay revealed that the coated vaccine partially maintained its activity when stored at 50 °C for 3 weeks, whereas the liquid form completely lost the activity. Immunization in guinea pigs showed that hemagglutination inhibition (HI) antibodies induced by IRMNs were two times higher than those induced by intramuscular (IM) injections. When challenged with influenza A/canine/Korea/01/2007 (H3N2) wild-type virus 2 weeks after the second vaccination, viral shedding was completely eliminated at 8 days post infection in both IRMNs and IM injection groups. Our results suggest that IRMNs have great potential for rapid and convenient vaccination, which will be particularly attractive for animal vaccinations.

Fabrication of Circular Obelisk-Type Multilayer Microneedles Using Micro-Milling and Spray Deposition.

In this study we present the fabrication of multilayer microneedles with circular obelisk and beveled-circular obelisk geometries, which have potential applications in implantable drug delivery devices. Micro-milling was adopted as an environmental-friendly and cost-effective way to fabricate primary metal microneedle masters. Polylactic acid (PLA) microneedles with sharp tips were then obtained by micromolding followed by oxygen plasma etching and used for preparing polydimethylsiloxane (PDMS) microneedle molds. A spray deposition process was employed for microneedle fabrication to facilitate the formation of multilayer microneedles while helping in maintenance of drug stability. Multilayer microneedles were successfully formed by sequential spraying of poly(lactic-co-glycolic acid) (PLGA) and polyvinylpyrrolidone (PVP) solutions into the mold. The fabricated PLGA-PVP multilayer microneedles penetrated the pig cadaver skin without breakage and released dyes in the skin at different rates, which reveals the potential for implantable microneedles enabling controlled release. Mechanical testing demonstrated that the obelisk-shaped microneedles were mechanically stronger than a pyramid-shaped microneedle and suggested that strong adhesion between PLGA and PVP layers was achieved as well. Structural stability and functionality of a model drug, horseradish peroxidase (HRP), upon spray deposition was examined using circular dichroism (CD) spectroscopy and enzyme activity assay. HRP retained its secondary structure and activity in PVP, whereas HRP in PLGA showed structural changes and reduced activity. Combination of micro-milling and spray deposition would be an attractive way of fabricating drug-containing polymer microneedles with various geometries while reducing prototyping time and process-induced drug instability.

Doxorubicin Release Controlled by Induced Phase Separation and Use of a Co-Solvent.

Electrospun-based drug delivery is emerging as a versatile means of localized therapy; however, controlling the release rates of active agents still remains as a key question. We propose a facile strategy to control the drug release behavior from electrospun fibers by a simple modification of polymer matrices. Polylactic acid (PLA) was used as a major component of the drug-carrier, and doxorubicin hydrochloride (Dox) was used as a model drug. The influences of a polar co-solvent, dimethyl sulfoxide (DMSO), and a hydrophilic polymer additive, polyvinylpyrrolidone (PVP), on the drug miscibility, loading efficiency and release behavior were investigated. The use of DMSO enabled the homogeneous internalization of the drug as well as higher drug loading efficiency within the electrospun fibers. The PVP additive induced phase separation in the PLA matrix and acted as a porogen. Preferable partitioning of Dox into the PVP domain resulted in increased drug loading efficiency in the PLA/PVP fiber. Fast dissolution of PVP domains created pores in the fibers, facilitating the release of internalized Dox. The novelty of this study lies in the detailed experimental investigation of the effect of additives in pre-spinning formulations, such as co-solvents and polymeric porogens, on the drug release behavior of nanofibers.

Early stage release control of an anticancer drug by drug-polymer miscibility in a hydrophobic fiber-based drug delivery system.

The drug release profiles of doxorubicin-loaded electrospun fiber mats were investigated with regard to drug-polymer miscibility, fiber wettability and degradability. Doxorubicin in hydrophilic form (Dox-HCl) and hydrophobic free base form (Dox-base) was employed as model drugs, and an aliphatic polyester, poly(lactic acid) (PLA), was used as a drug-carrier matrix. When hydrophilic Dox-HCl was directly mixed with PLA solution, drug molecules formed large aggregates on the fiber surface or in the fiber core, due to poor drug-polymer compatibility. Drug aggregates on the fiber surface contributed to the rapid initial release. The hydrophobic form of Dox-base was dispersed better with PLA matrix compared to Dox-HCl. When dimethyl sulfoxide (DMSO) was used as the solvent for Dox-HCl, the miscibility of drug in the polymer matrix was significantly improved, forming a quasi-monolithic solution scheme. The drug release from this monolithic matrix was slowest, and this slow release led to a lower toxicity to hepatocellular carcinoma. When an enzyme was used to promote PLA degradation, the release rates were closely correlated with degradation rates, demonstrating degradation was the dominant release mechanism. The possible drug release mechanisms were speculated based on the release kinetics. The results suggest that manipulation of drug-polymer miscibility and polymer degradability can be an effective means of designing drug release profiles.

Immediate separation of microneedle tips from base array during skin insertion for instantaneous drug delivery.

We have developed an insertion-responsive microneedle (IRMN) system that enables prompt drug delivery through the skin without attaching a skin patch. This system consists of square pyramidal hyaluronic acid (HA) microneedle tips and polycaprolactone (PCL) base arrays. During skin insertion, HA tips can be immediately separated from PCL base arrays due to the relatively weak adhesion strength between HA and PCL. Two base designs using truncated square pyramid stands, one without a wall (no-walled stand, NWS) and another with a wall on one side of the stand (single-walled stand, SWS), were prepared to study the effect of base geometry on the mechanical behavior of IRMNs. Ex vivo skin insertion tests showed successful separation of the tips from the base array upon insertion, regardless of the presence of a wall on the stand. However, only IRMNs-SWS were deeply embedded within the skin. Mechanical testing results demonstrated that the presence of a wall on the base enhanced the mechanical stability of the IRMNs. The wall also provided adequate adhesion between the tips and base, preventing the tips from breaking during insertion, while allowing the needle tip to separate upon removal. Histological examination confirmed that the tips were successfully separated from the base, embedded in the skin, and released fluorescent dyes within the skin. Our results suggest that the IRMN system is promising for the rapid and accurate delivery of various molecules through the skin, while improving user convenience by eliminating the need to attach microneedles to the skin.

Influence of shell compositions of solution blown PVP/PCL core-shell fibers on drug release and cell growth.

Developing a facile means of controlling drug release is of utmost interest in drug delivery systems. In this study, core-shell structured nanofibers containing a water-soluble porogen were fabricated via solution blow spinning, to be used as drug-loaded bioactive tissue scaffolds. Hydrophilic polyvinylpyrrolidone (PVP) and hydrophobic poly(ε-caprolactone) (PCL) were chosen to produce the core and the shell compartments of the fiber, respectively. In the core, a hydrophilic sulforhodamine B (SRB) dye was loaded as a model drug. In the PCL shell, two kinds of PVP with different molecular weights (40 kDa and 1300 kDa) were added, and the influence of PVP leaching on the SRB release and cell growth was investigated. The monolithic PCL-shelled fibers displayed a sustained SRB release with a weak burst effect. The addition of PVP in the shell induced a phase separation, forming microscale PVP domains. The PVP domain, acting as a porogen, was leached out in the medium and, as a result, the burst release of SRB was promoted. This burst effect was more prominent with the lower molecular weight PVP. The biocompatibility of the core-shell fibers was evaluated with human epidermal keratinocytes (HEK) by a cell viability assay and microscopic observation of cell proliferation. The HEK cells on fibers with a PVP/PCL composite shell formed self-assembled spherical clusters, displaying higher cell viability and proliferation than those on the monolithic PCL-shelled fibers that induced HEK cell growth in two-dimensional monolayers. The results demonstrate that the presence of hydrophilic porogens on tissue scaffolds can accelerate drug release and enhance cell proliferation by increasing surface wettability, roughness and porosity. The findings of this study provide a basic insight into the construction of bioactive three-dimensional tissue scaffolds.

Intracellular delivery of molecules using microfabricated nanoneedle arrays.

Many bioactive molecules have intracellular targets, but have difficulty crossing the cell membrane to reach those targets. To address this difficulty, we fabricated arrays of nanoneedles to gently and simultaneously puncture 10(5) cells and thereby provide transient pathways for transport of molecules into the cells. The nanoneedles were microfabricated by etching silicon to create arrays of nanoneedles measuring 12 µm in height, tapering to a sharp tip less than 30 nm wide to facilitate puncture into cells and spaced 10 µm apart in order to have at least one nanoneedle puncture each cell in a confluent monolayer. These nanoneedles were used for intracellular delivery in two ways: puncture loading, in which nanoneedle arrays were pressed into cell monolayers, and centrifuge loading, in which cells in suspension were spun down onto nanoneedle arrays. The effects on intracellular uptake and cell viability were determined as a function of nanoneedle length and sharpness, puncture force and duration, and molecular weight of the molecule delivered. Under optimal conditions, intracellular uptake was seen in approximately 50 % of cells while maintaining high cell viability. Overall, this study provides a comparative analysis of intracellular delivery using nanoneedle arrays by two different loading methods over a range of operating parameters.

Synthesis and Characterization of Biomimetic Hydroxyapatite Nanoconstruct Using Chemical Gradient across Lipid Bilayer.

In this study, we synthesized biomimetic hydroxyapatite nanoconstruct (nanosized hydroxyapatite, NHAp) using a double emulsion technique combined with a chemical gradient across a lipid bilayer for surface modification of a titanium (Ti) implant. The synthesized NHAp was characterized by dynamic light scattering, X-ray diffraction, transmission electron microscopy, and Fourier transform infrared (FTIR) spectroscopy, and it was further tested for its biocompatibility and in vitro proliferation efficacy using normal human osteoblasts (NHOst). The results showed that the synthesized NHAp had a hydrodynamic diameter of ∼200 nm with high aqueous stability. The chemistry of the NHAp was confirmed by FTIR spectroscopic analysis. Typical FTIR vibrational bands corresponding to the phosphate group (PO4(3-)) present in hydroxyapatite (HAp) were observed at 670, 960, and 1000 cm(-1). A broad band at 3500 cm(-1) confirmed the presence of a structural -OH group in the NHAp. Powder X-ray crystallographic diffraction further confirmed the formation of NHAp with characteristic reflections in (002), (211), (130), and (213) planes at respective 2θ degrees. These reflection planes are similar to those of typical HAp crystallized toward (002) and (211) crystallographic planes. The mechanism of the formation of NHAp was studied using the fluorescence resonance energy transfer (FRET) technique. The FRET study showed the fluorescent recovery of a donor fluorophore and the mechanism of the insertion of lipids into nanodroplets obtained from the first water-in-oil (w/o) emulsion during the formation of the second oil-in-water (o/w) emulsion. With these confirmations, we further studied NHOst cell proliferation on a Ti surface. When NHOst were cultured on the Ti surface coated with the NHAp, a distinct proliferation pattern and cell-cell communication via cytoplasmic extension on the substrate surface were observed. In contrast, a bare Ti surface showed diminished cell size with minimal adherence. This result indicates that our NHAp covered with a phospholipid bilayer provides a proper environment essential for cell adhesion, which is especially important for bone implants, and the inclusion of NHAp on the Ti substrate would be an effective support for long-term sustainability of implants.

Hollow microneedles for intradermal injection fabricated by sacrificial micromolding and selective electrodeposition.

Limitations with standard intradermal injections have created a clinical need for an alternative, low-cost injection device. In this study, we designed a hollow metal microneedle for reliable intradermal injection and developed a high-throughput micromolding process to produce metal microneedles with complex geometries. To fabricate the microneedles, we laser-ablated a 70 µm × 70 µm square cavity near the tip of poly(lactic acid) (PLA) microneedles. The master structure was a template for multiple micromolded poly(lactic acid-co-glycolic acid) (PLGA) replicas. Each replica was sputtered with a gold seed layer with minimal gold deposited in the cavity due to masking effects. In this way, nickel was electrodeposited selectively outside of the cavity, after which the polymer replica was dissolved to produce a hollow metal microneedle. Force-displacement tests showed the microneedles, with 12 µm thick electrodeposition, could penetrate skin with an insertion force 9 times less than their axial failure force. We injected fluid with the microneedles into pig skin in vitro and hairless guinea pig skin in vivo. The injections targeted 90 % of the material within the skin with minimal leakage onto the skin surface. We conclude that hollow microneedles made by this simple microfabrication method can achieve targeted intradermal injection.

Increased immunogenicity of avian influenza DNA vaccine delivered to the skin using a microneedle patch.

Effective public health responses to an influenza pandemic require an effective vaccine that can be manufactured and administered to large populations in the shortest possible time. In this study, we evaluated a method for vaccination against avian influenza virus that uses a DNA vaccine for rapid manufacturing and delivered by a microneedle skin patch for simplified administration and increased immunogenicity. We prepared patches containing 700-µm long microneedles coated with an avian H5 influenza hemagglutinin DNA vaccine from A/Viet Nam/1203/04 influenza virus. The coating DNA dose increased with DNA concentration in the coating solution and the number of dip-coating cycles. Coated DNA was released into the skin tissue by dissolution within minutes. Vaccination of mice using microneedles induced higher levels of antibody responses and hemagglutination inhibition titers, and improved protection against lethal infection with avian influenza as compared to conventional intramuscular delivery of the same dose of the DNA vaccine. Additional analysis showed that the microneedle coating solution containing carboxymethylcellulose and a surfactant may have negatively affected the immunogenicity of the DNA vaccine. Overall, this study shows that DNA vaccine delivery by microneedles can be a promising approach for improved vaccination to mitigate an influenza pandemic.