Harvinder S. Gill

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.

Transdermal Delivery of Insulin Using Microneedles in Vivo

AbstractPurpose. The purpose of this study was to design and fabricate arrays of solid microneedles and insert them into the skin of diabetic hairless rats for transdermal delivery of insulin to lower blood glucose level. Methods. Arrays containing 105 microneedles were laser-cut from stainless steel metal sheets and inserted into the skin of anesthetized hairless rats with streptozotocin-induced diabetes. During and after microneedle treatment, an insulin solution (100 or 500 U/ml) was placed in contact with the skin for 4 h. Microneedles were removed 10 s, 10 min, or 4 h after initiating transdermal insulin delivery. Blood glucose levels were measured electrochemically every 30 min. Plasma insulin concentration was determined by radioimmunoassay at the end of most experiments. Results. Arrays of microneedles were fabricated and demonstrated to insert fully into hairless rat skin in vivo. Microneedles increased skin permeability to insulin, which rapidly and steadily reduced blood glucose levels to an extent similar to 0.05-0.5 U insulin injected subcutaneously. Plasma insulin concentrations were directly measured to be 0.5-7.4 ng/ml. Higher donor solution insulin concentration, shorter insertion time, and fewer repeated insertions resulted in larger drops in blood glucose level and larger plasma insulin concentrations. Conclusions. Solid metal microneedles are capable of increasing transdermal insulin delivery and lowering blood glucose levels by as much as 80% in diabetic hairless rats in vivo.

Effect of microneedle design on pain in human volunteers.

ObjectivesTo design microneedles that minimize pain, this study tested the hypothesis that microneedles cause significantly less pain than a 26-gauge hypodermic needle, and that decreasing microneedle length and the number of microneedles reduces pain in normal human volunteers. MethodsSingle microneedles with lengths ranging from 480 to 1450 μm, widths from 160 to 465 μm, thicknesses from 30 to 100 μm, and tip angles from 20 to 90 degrees; and arrays containing 5 or 50 microneedles were inserted into the volar forearms of 10 healthy, human volunteers in a double-blinded, randomized study. Visual analog scale pain scores were recorded and compared with each other and to the pain from a 26-gauge hypodermic needle. ResultsAll microneedles investigated were significantly less painful than the hypodermic needle with microneedle pain scores varying from 5% to 40% of the hypodermic needle. Microneedle length had the strongest effect on pain, where a 3-fold increase in length increased the pain score by 7-fold. The number of microneedles also affected the pain score, where a 10-fold increase in the number of microneedles increased pain just over 2-fold. Microneedle tip angle, thickness, and width did not significantly influence pain. DiscussionMicroneedles are significantly less painful than a 26-gauge hypodermic needle over the range of dimensions investigated. Decreasing microneedle length and number of microneedles reduces pain.

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 skins 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.

Coating Formulations for Microneedles

PurposeTo develop a rational basis for designing coating solution formulations for uniform and thick coatings on microneedles and to identify coating strategies to form composite coatings, deliver liquid formulations, and control the mass deposited on microneedles.Materials and MethodsMicroneedles were fabricated using laser-cutting and then dip-coated using different aqueous, organic solvent-based or molten liquid formulations. The mass of riboflavin (vitamin B2) coated onto microneedles was determined as a function of coating and microneedle parameters. Coated microneedles were also inserted into porcine cadaver skin to assess delivery efficacy.ResultsSharp-tipped microneedles, including pocketed microneedles, were fabricated. Excipients that reduced coating solution surface tension improved coating uniformity, while excipients that increased solution viscosity improved coating thickness. Evaluation of more than 20 different coating formulations using FDA approved excipients showed that hydrophilic and hydrophobic molecules could be uniformly coated onto microneedles. Model proteins were also uniformly coated on microneedles using the formulations identified in the study. Pocketed microneedles were selectively filled with solid or liquid formulations to deliver difficult-to-coat substances, and composite drug layers were formed for different release profiles. The mass of riboflavin coated onto microneedles increased with its concentration in the coating solution and the number of coating dips and microneedles in the array. Coatings rapidly dissolved in the skin without wiping off on the skin surface.ConclusionsMicroneedles and coating formulations can be designed to have a range of different properties to address different drug delivery scenarios.

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 μg 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.

Kinetics of Skin Resealing After Insertion of Microneedles in Human Subjects

Over the past decade, microneedles have been shown to dramatically increase skin permeability to a broad range of compounds by creating reversible microchannels in the skin. However, in order to achieve sustained transdermal drug delivery, the extent and duration of skins increased permeability needs to be determined. In this study, we used electrical impedance spectroscopy to perform the first experiments in human subjects to analyze the resealing of skins barrier properties after insertion of microneedles. Microneedles having a range of geometries were studied in conjunction with the effect of occlusion to test the hypothesis that increasing microneedle length, number, and cross-sectional area together with occlusion leads to an increase in skin resealing time that can exceed one day. Results indicated that in the absence of occlusion, all microneedle treated sites recovered barrier properties within 2 h, while occluded sites resealed more slowly, with resealing windows ranging from 3 to 40 h depending on microneedle geometry. Upon subsequent removal of occlusion, the skin barrier resealed rapidly. Longer microneedles, increased number of needles, and larger cross-sectional area demonstrated slower resealing kinetics indicating that microneedle geometry played a significant role in the barrier resealing process. Overall, this study showed that pre-treatment of skin with microneedles before applying an occlusive transdermal patch can increase skin permeability for more than one day, but nonetheless allow skin to reseal rapidly after patch removal.

Cutaneous vaccination using microneedles coated with hepatitis C DNA vaccine

The skin is potentially an excellent organ for vaccine delivery because of accessibility and the presence of immune cells. However, no simple and inexpensive cutaneous vaccination method is available. Micron-scale needles coated with DNA were tested as a simple, inexpensive device for skin delivery. Vaccination with a plasmid encoding hepatitis C virus nonstructural 3/4A protein using microneedles effectively primed specific cytotoxic T lymphocytes (CTLs). Importantly, the minimally invasive microneedles were as efficient in priming CTLs as more complicated or invasive delivery techniques, such as gene gun and hypodermic needles. Thus, microneedles may offer a promising technology for DNA vaccination.

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.

Does Needle Size Matter

Hypodermic needles are in widespread use, but patients are unhappy with the pain, anxiety, and difficulty of using them. To increase patient acceptance, smaller needle diameters and lower insertion forces have been shown to reduce the frequency of painful injections. Guided by these observations, fine needles and microneedles have been developed to minimize pain and have found the greatest utility for delivery of vaccines and biopharmaceuticals such as insulin. However, pain reduction must be balanced against limitations of injection depth, volume, and formulations introduced by reduced needle dimensions. In some cases, needle-free delivery methods provide useful alternatives.