Critical role for the chemokine receptor CXCR6 in NK cell-mediated antigen-specific memory of haptens and viruses.

Hepatic natural killer (NK) cells mediate antigen-specific contact hypersensitivity (CHS) in mice deficient in T cells and B cells. We report here that hepatic NK cells, but not splenic or naive NK cells, also developed specific memory of vaccines containing antigens from influenza, vesicular stomatitis virus (VSV) or human immunodeficiency virus type 1 (HIV-1). Adoptive transfer of virus-sensitized NK cells into naive recipient mice enhanced the survival of the mice after lethal challenge with the sensitizing virus but not after lethal challenge with a different virus. NK cell memory of haptens and viruses depended on CXCR6, a chemokine receptor on hepatic NK cells that was required for the persistence of memory NK cells but not for antigen recognition. Thus, hepatic NK cells can develop adaptive immunity to structurally diverse antigens, an activity that requires NK cell-expressed CXCR6.

Drug coated microneedles for minimally-invasive treatment of oral carcinomas: development and in vitro evaluation.

Treatment of recurring oral cancers is challenging as common surgical approaches are not feasible for these patients. In addition, these patients do not respond well to systemic chemotherapy. Localized intratumoral injection of anti-cancer drugs is considered to be an attractive alternative treatment approach for these patients. However, conventional hypodermic injections result in poor distribution of the drug in the tumor and leakage of the drug from the injection site to systemic circulation, in addition to causing pain to the patient. The objective of this study was to develop coated microneedles as a novel device for direct and minimally invasive intratumoral delivery of anti-cancer drugs. Poly(lactic-co-glycolic) acid (PLGA) nanoparticles encapsulating doxorubicin (DOX) were prepared and coated on inplane (1D) microneedles. Microscopic evaluation of 3D tissue phantoms and porcine cadaver buccal tissues that were treated with 1D microneedle arrays coated with DOX-PLGA nanoparticles demonstrated that DOX could diffuse both laterally and vertically in to the tissues and produced cellular cytotoxicity. Out of plane (2D) microneedle arrays measuring 1 cm x 1 cm with 57 microneedles coated with free DOX could produce uniform distribution of DOX in a porcine cadaver buccal tissue up to a depth greater than 3 mm. Hypodermic injection of different volumes in to a porcine buccal tissue confirmed significant leakage of the injected volume (about 25% of the injected 80 µl). In summary, this study demonstrates that drug coated microneedles is an attractive microscale device that can uniformly and effectively deliver drugs to localized oral cancers. This microscale device has potential to impact the treatment of oral cancer patients.

Vaccine delivery to the oral cavity using coated microneedles induces systemic and mucosal immunity.

PURPOSE: The objective of this study is to evaluate the feasibility of using coated microneedles to deliver vaccines into the oral cavity to induce systemic and mucosal immune responses. METHOD: Microneedles were coated with sulforhodamine, ovalbumin and two HIV antigens. Coated microneedles were inserted into the inner lower lip and dorsal surface of the tongue of rabbits. Histology was used to confirm microneedle insertion, and systemic and mucosal immune responses were characterized by measuring antigen-specific immunoglobulin G (IgG) in serum and immunoglobulin A (IgA) in saliva, respectively. RESULTS: Histological evaluation of tissues shows that coated microneedles can penetrate the lip and tongue to deliver coatings. Using ovalbumin as a model antigen it was found that the lip and the tongue are equally immunogenic sites for vaccination. Importantly, both sites also induced a significant (p < 0.05) secretory IgA in saliva compared to pre-immune saliva. Microneedle-based oral cavity vaccination was also compared to the intramuscular route using two HIV antigens, a virus-like particle and a DNA vaccine. Microneedle-based delivery to the oral cavity and the intramuscular route exhibited similar (p > 0.05) yet significant (p < 0.05) levels of antigen-specific IgG in serum. However, only the microneedle-based oral cavity vaccination group stimulated a significantly higher (p < 0.05) antigen-specific IgA response in saliva, but not intramuscular injection. CONCLUSION: In conclusion, this study provides a novel method using microneedles to induce systemic IgG and secretory IgA in saliva, and could offer a versatile technique for oral mucosal vaccination.

Microneedles for oral mucosal delivery - Current trends and perspective on future directions.

INTRODUCTION: Oral cavity drug and vaccine delivery has the potential for local targeting, dose reduction, minimization of systemic side effects, and generation of mucosal immunity. To overcome current limitations of delivery into the oral cavity mucosa, microneedles (MNs) have emerged as a promising technology. AREAS COVERED: We reviewed the literature on MN application in the oral cavity, including in vitro studies, in vivo animal studies, and human clinical trials. EXPERT OPINION: MNs are sufficiently robust to cross the oral cavity epithelium and nearly painless when applied to different parts of the human oral mucosa including the lip, cheek, tongue, and palate. In recent years, MNs have been evaluated for different applications, including vaccination, topical anesthetic delivery, and treatment of local oral pathologies such as oral lesions or carcinomas. MNs are attractive because they have the potential to produce a better treatment outcome with reduced side effects. Over the coming years, we project a significant increase in research related to the development of MNs for use in dentistry and other medical conditions of the mouth.

Immunization by vaccine-coated microneedle arrays protects against lethal influenza virus challenge.

Influenza prophylaxis would benefit from a simple method to administer influenza vaccine into skin without the need for hypodermic needles. In this study, solid metal microneedle arrays (MNs) were investigated as a system for cutaneous vaccine delivery using influenza virus antigen. The MNs with 5 monument-shaped microneedles per array were produced and coated with inactivated influenza virus A/PR/8/34 (IIV). As much as 10 microg of viral proteins could be coated onto an array of 5 microneedles, and the coated IIV was delivered into skin at high efficiency within minutes. The coated MNs were used to immunize mice in comparison with conventional intramuscular injection at the same dose. Analysis of immune responses showed that a single immunization with IIV-coated MNs induced strong antibody responses against influenza virus, with significant levels of hemagglutination inhibition activities (>1:40), which were comparable to those induced by conventional intramuscular immunization. Moreover, mice immunized by a single dose of IIV coated on MNs were effectively protected against lethal challenge by a high dose of mouse-adapted influenza virus A/PR/8/34. These results show that MNs are highly effective as a simple method of vaccine delivery to elicit protective immune responses against virus infection.

Treating allergies via skin - Recent advances in cutaneous allergen immunotherapy.

Subcutaneous allergen immunotherapy has been practiced clinically for decades to treat airborne allergies. Recently, the cutaneous route, which exploits the immunocompetence of the skin has received attention, which is evident from attempts to use it to treat peanut allergy. Delivery of allergens into the skin is inherently impeded by the barrier imposed by stratum corneum, the top layer of the skin. While the stratum corneum barrier must be overcome for efficient allergen delivery, excessive disruption of this layer can predispose to development of allergic inflammation. Thus, the most desirable allergen delivery approach must provide a balance between the level of skin disruption and the amount of allergen delivered. Such an approach should aim to achieve high allergen delivery efficiency across various skin types independent of age and ethnicity, and optimize variables such as safety profile, allergen dosage, treatment frequency, application time and patient compliance. The ability to precisely quantify the amount of allergen being delivered into the skin is crucial since it can allow for allergen dose optimization and can promote consistency and reproducibility in treatment response. In this work we review prominent cutaneous delivery approaches, and offer a perspective on further improvisation in cutaneous allergen-specific immunotherapy.

Microneedles permit transdermal delivery of a skin-impermeant medication to humans.

Drugs with poor oral bioavailability usually are administered by hypodermic injection, which causes pain, poor patient compliance, the need for trained personnel, and risk of infectious disease transmission. Transdermal (TD) delivery provides an excellent alternative, but the barrier of skin's outer stratum corneum (SC) prevents delivery of most drugs. Micrometer-scale microneedles (MNs) have been used to pierce animal and human cadaver skin and thereby enable TD delivery of small molecules, proteins, DNA, and vaccines for systemic action. Here, we present a clinical study of MN-enhanced delivery of a medication to humans. Naltrexone (NTX) is a potent mu-opioid receptor antagonist used to treat opiate and alcohol dependence. This hydrophilic and skin-impermeant molecule was delivered from a TD patch to healthy human subjects with and without pretreatment of the skin with MNs. Whereas delivery from a standard NTX TD patch over a 72-h period yielded undetectable drug plasma levels, pretreatment of skin with MNs achieved steady-state plasma concentrations within 2 h of patch application and were maintained for at least 48 h. The MNs and NTX patch were well tolerated with mild systemic and application site side effects. The MN arrays were painless upon administration and not damaged during skin insertion, and no MNs were broken off into the skin. This human proof-of-concept study demonstrates systemic administration of a hydrophilic medication by MN-enhanced TD delivery. These findings set the stage for future human studies of skin-impermeant medications and biopharmaceuticals for clinical applications.

Trends of microneedle technology in the scientific literature, patents, clinical trials and internet activity.

The powerful and intriguing idea that drives the emerging technology of microneedles-shrinking the standard needle to a micron scale-has fostered an entire field of microneedle study and subsequent exponential growth in research and product development. Originally enabled by microfabrication tools derived from the microelectronic industry, microneedles are now produced through a number of methods in a variety of forms including solid, coated, dissolvable, and hollow microneedles. They are used to deliver a broad spectrum of molecules, including small molecules, biomolecules, and vaccines, as well as various forms of energy into the skin, eye, and other tissues. Microneedles are also being exploited for use in diagnostics, as well as additional medical, cosmetic, and other applications. This review elucidates the relative roles of different aspects of microneedle technology development, as shown through scientific papers, patents, clinical studies, and internet/social media activity. Considering >1000 papers, 750 patents, and almost 80 clinical trials, we analyze different attributes of microneedles such as usage of microneedles, types of microneedles, testing environment, types of patent claims, and phases of clinical trials, as well as which institutions and people in academia and industry from different locations and in different journals are publishing, patenting, and otherwise studying the potential of microneedles. We conclude that there is robust and growing activity in the field of microneedles; the technology is rapidly developing and being used for novel applications to benefit human health and well-being.

An electrically active microneedle array for electroporation.

We have designed and fabricated a microneedle array with electrical functionality with the final goal of electroporating skin's epidermal cells to increase their transfection by DNA vaccines. The microneedle array was made of polymethylmethacrylate (PMMA) by micromolding technology from a polydimethylsiloxane (PDMS) mold, followed by metal deposition, patterning using laser ablation, and electrodeposition. This microneedle array possessed sufficient mechanical strength to penetrate human skin in vivo and was also able to electroporate both red blood cells and human prostate cancer cells as an in vitro model to demonstrate cell membrane permeabilization. A computational model to predict the effective volume for electroporation with respect to applied voltages was constructed from finite element simulation. This study demonstrates the mechanical and electrical functionalities of the first MEMS-fabricated microneedle array for electroporation, designed for DNA vaccine delivery.

Pocketed Microneedles for Drug Delivery to the Skin.

Drug delivery to the skin is limited by the strong barrier properties of skin's outer layer of stratum corneum. Micron-scale needles have been developed to deliver drugs across this barrier layer and into the skin in a minimally invasive manner. One method of delivery involves coating these microneedles with a drug that rapidly dissolves off within the skin. As a variation on this approach, this study examines microneedles with holes cut through their shafts to form "pockets" that can be filled with drug formulations using a dip-coating method. Our results (i) demonstrated the filling of microneedle pockets having a variety of different sizes and shapes, (ii) quantified the amount of drug that can be filled into pockets and coated onto microneedle surfaces, (iii) developed composite microneedle structures that sequester one model drug within the microneedle pocket and coat another model drug on the microneedle surface and (iv) showed that pocketed microneedles can deliver a model drug to a targeted depth within the skin. We conclude that pocketed microneedles offer unique capabilities for controlled drug delivery to the skin.

Coated microneedles for drug delivery to the eye.

PURPOSE: To test the hypothesis that coated microneedles can deliver drugs into the eye via intrascleral and intracorneal routes in a minimally invasive manner. METHODS: Solid metal microneedles measuring 500 to 750 microm in length were coated with model drugs, protein, and DNA; inserted into nonpreserved human cadaveric sclera; and imaged. Microneedles coated with sodium fluorescein were then inserted into rabbit cornea in vivo. After needle removal, fluorescein concentration in the anterior segment of the rabbit eye was measured for 24 hours. Similar experiments were performed using pilocarpine-coated microneedles, and the rabbit pupil size was monitored afterward. RESULTS: In vitro insertion tests showed that microneedles were mechanically strong enough to penetrate into human cadaveric sclera and that the drug coating rapidly dissolved off the needles within the scleral tissue within 30 seconds after insertion. In vivo delivery from fluorescein-coated microneedles showed that fluorescein concentrations in the anterior chamber were 60 times greater than those achieved by topical application without microneedles. Similarly, microneedle delivery of pilocarpine caused rapid and extensive rabbit pupil constriction. There were no measurable inflammatory responses caused by microneedle insertion. CONCLUSIONS: This study demonstrated for the first time that coated microneedles can deliver drugs into the eye via intrascleral and intracorneal routes. This minimally invasive approach may avoid the complications associated with intraocular injection and systemic administration.

Coated microneedles for transdermal delivery.

Coated microneedles have been shown to deliver proteins and DNA into the skin in a minimally invasive manner. However, detailed studies examining coating methods and their breadth of applicability are lacking. This study's goal was to develop a simple, versatile and controlled microneedle coating process to make uniform coatings on microneedles and establish the breadth of molecules and particles that can be coated onto microneedles. First, microneedles were fabricated from stainless steel sheets as single microneedles or arrays of microneedles. Next, a novel micron-scale dip-coating process and a GRAS coating formulation were designed to reliably produce uniform coatings on both individual and arrays of microneedles. This process was used to coat compounds including calcein, vitamin B, bovine serum albumin and plasmid DNA. Modified vaccinia virus and microparticles of 1 to 20 micro m diameter were also coated. Coatings could be localized just to the needle shafts and formulated to dissolve within 20 s in porcine cadaver skin. Histological examination validated that microneedle coatings were delivered into the skin and did not wipe off during insertion. In conclusion, this study presents a simple, versatile, and controllable method to coat microneedles with proteins, DNA, viruses and microparticles for rapid delivery into the skin.

5-Aminolevulinic acid coated microneedles for photodynamic therapy of skin tumors.

This study evaluated the potential of coated microneedles for improved dermal delivery of 5-aminolevulinic acid (5-ALA), which naturally gets converted by cells of the tissue in to a photosensitizer called protoporphyrin IX (PPIX). Microneedle patches containing 57 microneedles were coated with 5-ALA using an in-house developed micro-precision dip coater. The coating process was optimized to achieve higher 5-ALA loading on microneedles and a high delivery efficiency into porcine cadaver skin. Using 5 dips with 25% w/v 5-ALA solution, a mass of about 350µg of 5-ALA was coated per patch, which gave a delivery efficiency of about 90% in porcine cadaver skin. Bright-field and scanning electron microscopy established that coatings of 5-ALA on microneedles of the patch were uniform. In vivo dermal pharmacokinetics showed that delivery of just 350µg of 5-ALA using coated microneedles led to about 3.2-fold higher PPIX formation after 4h, as compared to topical application of 20% w/w 5-ALA in a conventional cream formulation (25mg cream). Furthermore, with use of coated microneedles, PPIX was observed in deeper regions of the skin (~480µm) as compared to topical 5-ALA cream formulation (~150µm). The potential of PPIX for photodynamic therapy was tested in vivo. After light exposure (633nm; 118J/cm(2)), PPIX got photosensitized, and due to higher initial amount of PPIX in the coated microneedle group, about twice the amount of PPIX was photobleached compared to topical cream application. Finally, even with a lower dose of just 1.75mg 5-ALA, coated microneedles suppressed the growth of subcutaneous tumors by ~57%, while a topical cream containing 5mg of 5-ALA did not suppress the tumor volume and led to tumor growth comparable to the untreated control group. Overall, the strategy of delivering 5-ALA using coated microneedles could be a promising approach for photodynamic therapy of skin tumors.

M2e-immobilized gold nanoparticles as influenza A vaccine: Role of soluble M2e and longevity of protection.

Influenza virus causes seasonal epidemics and also poses a high risk for pandemics. To develop a broadly cross-protective influenza vaccine we have previously shown that a formulation consisting of the extracellular domain of M2 membrane protein (M2e) immobilized on gold nanoparticles (AuNPs) and soluble CpG as an adjuvant can elicit protective immunity against different influenza A subtypes. The vaccine formulation contains M2e that is immobilized on AuNPs, and an excess amount that is freely dissolved in solution, whose role in inducing protective immunity against virus infection is unclear. Using a mouse model, the current study shows that inclusion of excess soluble M2e antigen along with M2e immobilized on AuNPs is vital for inducing high levels of antibody response, and in providing complete protection against lethal influenza virus challenge. We also show that the vaccine induces long-lasting protection against mortality and morbidity upon lethal challenge with influenza A virus.

Coating solid dispersions on microneedles via a molten dip-coating method: development and in vitro evaluation for transdermal delivery of a water-insoluble drug.

This study demonstrates for the first time the ability to coat solid dispersions on microneedles as a means to deliver water-insoluble drugs through the skin. Polyethylene glycol (PEG) was selected as the hydrophilic matrix, and lidocaine base was selected as the model hydrophobic drug to create the solid dispersion. First, thermal characterization and viscosity measurements of the PEG-lidocaine mixture at different mass fractions were performed. The results show that lidocaine can remain stable at temperatures up to ∼130°C and that viscosity of the PEG-lidocaine molten solution increases as the mass fraction of lidocaine decreases. Differential scanning calorimetry demonstrated that at lidocaine mass fraction less than or equal to 50%, lidocaine is well dispersed in the PEG-lidocaine mixture. Uniform coatings were obtained on microneedle surfaces. In vitro dissolution studies in porcine skin showed that microneedles coated with PEG-lidocaine dispersions resulted in significantly higher delivery of lidocaine in just 3 min compared with 1 h topical application of 0.15 g EMLA®, a commercial lidocaine-prilocaine cream. In conclusion, the molten coating process we introduce here offers a practical approach to coat water-insoluble drugs on microneedles for transdermal delivery.

Gold nanoparticle-M2e conjugate coformulated with CpG induces protective immunity against influenza A virus.

AIM: This study aimed to develop a novel influenza A vaccine by conjugating the highly conserved extracellular region of the matrix 2 protein (M2e) of influenza A virus to gold nanoparticles (AuNPs) and to test the vaccine in a mouse influenza challenge model. MATERIALS & METHODS: Citrate-reduced AuNPs (diameter: 12 nm) were synthesized, and characterized by transmission electron microscopy and dynamic light scattering. M2e was conjugated to AuNPs through thiol-gold interactions to form M2e-AuNP conjugates. Particle stability was confirmed by UV-visible spectra, and M2e conjugation was further characterized by x-ray photoelectron spectroscopy. Mice were immunized with M2e-AuNPs with or without CpG (cytosine-guanine rich oligonucleotide) as an adjuvant with appropriate control groups. Sera was collected and M2e-specific immunoglobulin (IgG) was measured, and immunized mice were challenged with PR8-H1N1 influenza virus. RESULTS: M2e-capped AuNPs could be lyophilized and stably resuspended in water. Intranasal vaccination of mice with M2e-AuNP conjugates induced M2e-specific IgG serum antibodies, which significantly increased upon addition of soluble CpG as adjuvant. Upon challenge with lethal PR8, mice vaccinated with M2e-AuNP conjugates were only partially protected, while mice that received soluble CpG as adjuvant in addition to M2e-AuNP were fully protected. CONCLUSION: Overall, this study demonstrates the potential of using the M2e-AuNP conjugates with CpG as an adjuvant as a platform for developing an influenza A vaccine.

Synthesis and Immunogenicity Assessment of Elastin-Like Polypeptide-M2e Construct as an Influenza Antigen.

The 23 amino acid-long extracellular domain of the influenza virus transmembrane protein M2 (M2e) has remained highly conserved since the 1918 pandemic, and is thus considered a good candidate for development of a universal influenza A vaccine. However, M2e is poorly immunogenic. In this study we assessed the potential of increasing immunogenicity of M2e by constructing a nanoscale-designed protein polymer containing the M2e sequence and an elastin-like polypeptide (ELP) nanodomain consisting of alanine and tyrosine guest residues (ELP(A2YA2)24). The ELP nanodomain was included to increase antigen size, and to exploit the inherent thermal inverse phase transition behavior of ELPs to purify the protein polymer. The ELP(A2YA2)24 + M2e nanodomained molecule was recombinantly synthesized. Characterization of its inverse phase transition behavior demonstrated that attachment of M2e to ELP(A2YA2)24 increased its transition temperature compared to ELP(A2YA2)24. Using a dot blot test we determined that M2e conjugated to ELP is recognizable by M2e-specific antibodies, suggesting that the conjugation process does not adversely affect the immunogenic property of M2e. Further, upon vaccinating mice with ELP(A2YA2)24 + M2e it was found that indeed the nanodomained protein enhanced M2e-specific antibodies in mouse serum compared to free M2e peptide and ELP(A2YA2)24. The immune serum could also recognize M2 expressed on influenza virions. Overall, this data suggests the potential of using molecules containing M2e-ELP nano-domains to develop a universal influenza vaccine.

Cutaneous immunization: an evolving paradigm in influenza vaccines.

INTRODUCTION: Most vaccines are administered by intramuscular injection using a hypodermic needle and syringe. Some limitations of this procedure include reluctance to be immunized because of fear of needlesticks, and concerns associated with the safe disposal of needles after their use. Skin delivery is an alternate route of vaccination that has potential to be painless and could even lead to dose reduction of vaccines. Recently, microneedles have emerged as a novel painless approach for delivery of influenza vaccines via the skin. AREAS COVERED: In this review, we briefly summarize the approaches and devices used for skin vaccination, and then focus on studies of skin immunization with influenza vaccines using microneedles. We discuss both the functional immune response and the nature of this immune response following vaccination with microneedles. EXPERT OPINION: The cutaneous administration of influenza vaccines using microneedles offers several advantages: it is painless, elicits stronger immune responses in preclinical studies and could improve responses in high-risk populations. These dry formulations of vaccines provide enhanced stability, a property of high importance in enabling their rapid global distribution in response to possible outbreaks of pandemic influenza and newly emerging infectious diseases.

Microneedle delivery of an M2e-TLR5 ligand fusion protein to skin confers broadly cross-protective influenza immunity.

Influenza vaccines with broad cross-protection are urgently needed to prevent an emerging influenza pandemic. A fusion protein of the Toll-like receptor (TLR) 5-agonist domains from flagellin and multiple repeats of the conserved extracellular domain of the influenza matrix protein 2 (M2e) was constructed, purified and evaluated as such a vaccine. A painless vaccination method suitable for possible self-administration using coated microneedle arrays was investigated for skin-targeted delivery of the fusion protein in a mouse model. The results demonstrate that microneedle immunization induced strong humoral as well as mucosal antibody responses and conferred complete protection against homo- and heterosubtypic lethal virus challenges. Protective efficacy with microneedles was found to be significantly better than that seen with conventional intramuscular injection, and comparable to that observed with intranasal immunization. Because of its advantages for administration, safety and storage, microneedle delivery of M2e-flagellin fusion protein is a promising approach for an easy-to-administer universal influenza vaccine.

Enhanced influenza virus-like particle vaccines containing the extracellular domain of matrix protein 2 and a Toll-like receptor ligand.

The extracellular domain of matrix protein 2 (M2e) is conserved among influenza A viruses. The goal of this project is to develop enhanced influenza vaccines with broad protective efficacy using the M2e antigen. We designed a membrane-anchored fusion protein by replacing the hyperimmunogenic region of Salmonella enterica serovar Typhimurium flagellin (FliC) with four repeats of M2e (4.M2e-tFliC) and fusing it to a membrane anchor from influenza virus hemagglutinin (HA). The fusion protein was incorporated into influenza virus M1-based virus-like particles (VLPs). These VLPs retained Toll-like receptor 5 (TLR5) agonist activity comparable to that of soluble FliC. Mice immunized with the VLPs by either intramuscular or intranasal immunization showed high levels of systemic M2-specific antibody responses compared to the responses to soluble 4.M2e protein. High mucosal antibody titers were also induced in intranasally immunized mice. All intranasally immunized mice survived lethal challenges with live virus, while intramuscularly immunized mice showed only partial protection, revealing better protection by the intranasal route. These results indicate that a combination of M2e antigens and TLR ligand adjuvants in VLPs has potential for development of a broadly protective influenza A virus vaccine.