Extended delivery of vaccines to the skin improves immune responses.

Vaccines prevent 2-3 million childhood deaths annually; however, low vaccine efficacy and the resulting need for booster doses create gaps in immunization coverage. In this translational study, we explore the benefits of extended release of licensed vaccine antigens into skin to increase immune responses after a single dose in order to design improved vaccine delivery systems. By administering daily intradermal injections of inactivated polio vaccine according to six different delivery profiles, zeroth-order release over 28 days resulted in neutralizing antibody titers equivalent to two bolus vaccinations administered one month apart. Vaccinations following this profile also improved immune responses to tetanus toxoid and subunit influenza vaccine but not a live-attenuated viral vaccine, measles vaccine. Finally, using subunit influenza vaccine, we demonstrated that daily vaccination by microneedle patch induced a potent, balanced humoral immunity with an increased memory response compared to bolus vaccination. We conclude that extended presentation of antigen in skin via intradermal injection or microneedle patch can enhance immune responses and reduce the number of vaccine doses, thereby enabling increased vaccination efficacy.

Immunogenicity of pulsatile-release PLGA microspheres for single-injection vaccination.

The World Health Organization's Expanded Programme on Immunization has led to a dramatic rise in worldwide vaccination rates over the past 40years, yet 19.4 million infants remain underimmunized each year. Many of these infants have received at least one vaccine dose but may remain unprotected because they did not receive subsequent booster doses due to logistical challenges. This study aimed to develop injectable controlled release microparticles with kinetics that mimic common vaccine dosing regimens consisting of large antigen doses administered periodically over the course of months in order to eliminate the need for boosters. Sixteen poly(lactic-co-glycolic acid) (PLGA) microsphere formulations containing bovine serum albumin (BSA) as a model vaccine antigen were screened in vitro to determine their respective release kinetics. Three formulations that exhibited desirable pulsatile release profiles were then selected for studying immunogenicity in mice. Two low-dose microsphere formulations induced peak anti-BSA IgG antibody titers of 13.9±1.3 and 13.7±2.2 log2 compared to 15.5±1.5 log2 for a series of three bolus injections delivered at 0, 4, and 8weeks with an equivalent cumulative dose. Similarly, high-dose formulations induced peak antibody titers that were 16.1±2.1 log2 compared to 17.7±2.2 log2 for controls. All three microparticle formulations studied in vivo induced peak antibody titers that were statistically similar to bolus controls. These results suggest that pulsatile antigen release from polymeric microparticles is a promising approach for single-injection vaccination, which could potentially reduce the logistical burden associated with immunization in the developing world.

Fabrication of fillable microparticles and other complex 3D microstructures.

Three-dimensional (3D) microstructures created by microfabrication and additive manufacturing have demonstrated value across a number of fields, ranging from biomedicine to microelectronics. However, the techniques used to create these devices each have their own characteristic set of advantages and limitations with regards to resolution, material compatibility, and geometrical constraints that determine the types of microstructures that can be formed. We describe a microfabrication method, termed StampEd Assembly of polymer Layers (SEAL), and create injectable pulsatile drug-delivery microparticles, pH sensors, and 3D microfluidic devices that we could not produce using traditional 3D printing. SEAL allows us to generate microstructures with complex geometry at high resolution, produce fully enclosed internal cavities containing a solid or liquid, and use potentially any thermoplastic material without processing additives.

An economic model assessing the value of microneedle patch delivery of the seasonal influenza vaccine.

BACKGROUND: New vaccine technologies may improve the acceptability, delivery (potentially enabling self-administration), and product efficacy of influenza vaccines. One such technology is the microneedle patch (MNP), a skin delivery technology currently in development. Although MNPs hold promise in preclinical studies, their potential economic and epidemiologic impacts have not yet been evaluated. METHODS: We utilized a susceptible-exposed-infectious-recovered (SEIR) transmission model linked to an economic influenza outcomes model to assess the economic value of introducing the MNP into the current influenza vaccine market in the United States from the third-party payer and societal perspectives. We also explored the impact of different vaccination settings, self-administration, the MNP price, vaccine efficacy, compliance, and MNP market share. Outcomes included costs, quality-adjusted life years (QALYs), cases, and incremental cost-effectiveness ratios (ICERs; cost/QALY). RESULTS: With healthcare provider administration, MNP introduction would be cost-effective (ICERs ≤$23,347/QALY) at all MNP price points ($9.50-$30) and market shares (10-60%) assessed, except when compliance and efficacy were assumed to be the same as existing vaccines and the MNP occupied a 10% market share. If MNP self-administration were available (assuming the same efficacy as current technologies), MNP compliance or its efficacy would need to increase by ≥3% in order to be cost-effective (ICERs ≤$1401/QALY), assuming a 2% reduction in administration success with unsupervised self-administration. Under these conditions, MNP introduction would be cost-effective for all price points and market shares assessed. CONCLUSIONS: When healthcare providers administered the MNP, its introduction would be cost-effective or dominant (i.e., less costly and more effective) in the majority of scenarios assessed. If self-administration were available, MNP introduction would be cost-effective if it increased compliance enough to overcome any decrease in self-administration success or if the MNP presentation afforded an increase in efficacy over current delivery methods for influenza vaccines.

Microneedle patches: usability and acceptability for self-vaccination against influenza.

While therapeutic drugs are routinely self-administered by patients, there is little precedent for self-vaccination. Convenient self-vaccination may expand vaccination coverage and reduce administration costs. Microneedle patches are in development for many vaccines, but no reports exist on usability or acceptability. We hypothesized that naïve patients could apply patches and that self-administered patches would improve stated intent to receive an influenza vaccine. We conducted a randomized, repeated measures study with 91 venue-recruited adults. To simulate vaccination, subjects received placebo microneedle patches given three times by self-administration and once by the investigator, as well as an intramuscular injection of saline. Seventy participants inserted patches with thumb pressure alone and the remainder used snap-based devices that closed shut at a certain force. Usability was assessed by skin staining and acceptability was measured with an adaptive-choice analysis. The best usability was seen with the snap device, with users inserting a median value of 93-96% of microneedles over three repetitions. When a self-administered microneedle patch was offered, intent to vaccinate increased from 44% to 65% (CI: 55-74%). The majority of those intending vaccination would prefer to self-vaccinate: 64% (CI: 51-75%). There were no serious adverse events associated with use of microneedle patches. The findings from this initial study indicate that microneedle patches for self-vaccination against influenza are usable and may lead to improved vaccination coverage.

Reliability and accuracy of intradermal injection by Mantoux technique, hypodermic needle adapter, and hollow microneedle in pigs.

We compared the ability of three intradermal delivery devices to administer an intended dose to pig skin in vivo and target that dose to the dermal rather than subcutaneous layers. The three devices were a standard hypodermic needle and syringe for the Mantoux technique, an adapter designed to facilitate proper hypodermic needle and syringe use, and a hollow microneedle. Reliability was determined as the percentage of the administered dose that entered the skin, as opposed to remaining in the device or on the skin surface. The intradermal adapter (97.6 ± 1.5 % delivered, mean ± standard deviation), Mantoux technique (95.4 ± 4.9 %), and hollow microneedle (94.9 ± 0.3 %) exhibited similar reliability. Accuracy was determined as the percentage of the dose that entered the skin that localized in the dermis. All three devices achieved similar accuracy: hollow microneedle (99 ± 12 % delivered to the dermis, median ± standard deviation), Mantoux technique (97 ± 16 %), and intradermal adapter (92 ± 21 %). We conclude that intradermal injection by all three methods studied provided reliable delivery to the skin and provided accurate localization of delivery within the dermis. Next-generation designs of these devices have now received clearance from the FDA and are used as medical products and/or in clinical trials.

Faster pharmacokinetics and increased patient acceptance of intradermal insulin delivery using a single hollow microneedle in children and adolescents with type 1 diabetes.

OBJECTIVE: In an effort to improve compliance with insulin therapy and to accelerate insulin pharmacokinetics, we tested the hypothesis that intradermal insulin delivery using a hollow microneedle causes less pain and leads to faster onset and offset of insulin pharmacokinetics in children and adolescents with type 1 diabetes (T1DM) compared with a subcutaneous, insulin pump catheter. RESEARCH DESIGN AND METHODS: In this repeated measures study, 16 children and adolescents with T1DM received Lispro insulin by microneedle and subcutaneous administration on separate days. Subjects rated the pain of insertion and infusion using a visual analog scale. Blood specimens were collected over 4 h to determine insulin and glucose concentrations. RESULTS: Microneedle insertion pain was significantly lower compared with insertion of the subcutaneous catheter (p = 0.005). Insulin onset time was 22 min faster (p = 0.0004) and offset time was 34 min faster (p = 0.017) after hollow microneedle delivery compared with subcutaneous delivery. CONCLUSIONS: In this study, intradermal insulin delivery using a single, hollow microneedle device resulted in less insertion pain and faster insulin onset and offset in children and adolescents with T1DM. A reduction in pain might improve compliance with insulin delivery. The faster onset and offset times of insulin action may enable closed-loop insulin therapy.

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.

Improving patient acceptance of insulin therapy by improving needle design.

Improved needle designs could increase patient compliance with insulin therapy. In this issue of Journal of Diabetes Science and Technology, Hirsch and colleagues assessed patient pain and preference for a 5-bevel needle design among diabetes patients. A blinded comparison with traditional 3-bevel needles yielded no significant difference, but patients preferred the 5-bevel needle in unblinded home injection and clinical insertion studies. This suggests that important subjective/contextual factors contribute to preference in conjunction with the fundamental needle design change. While 5-bevel needles may increase patient acceptance, more dramatic changes of needle design, such as microneedles, could enable still greater patient acceptance through reduced pain as well as improved insulin pharmacokinetics.

Microsystems for biomechanical measurements.

The use of microtechnology to make biomechanical measurements allows for the study of cellular and subcellular scale mechanical forces. Forces generated by cells are in the few nanoNewton to several microNewton range and can change spatially over subcellular size scales. Transducing forces at such small size and force scales is a challenging task. Methods of microfabrication developed in the integrated circuit industry have allowed researchers to build platforms with cellular and subcellular scale parts with which individual cells can interact. These parts act as transducers of stresses and forces generated by the cell during migration or in the maintenance of physical equilibrium. Due to the size and sensitivity of such devices, quantitative studies of single cell and even single molecule biomechanics have become possible. In this review we focus on two classes of cellular force transducers: silicon-based devices and soft-polymer platforms. We concentrate on the biomechanical discoveries made with these devices and less so on the engineering behind their development because this is covered in great detail elsewhere.

Microstructures in 3D biological gels affect cell proliferation.

Controlling the microscale environment in three-dimensional (3D) matrices for tissue engineering applications is a challenging but necessary goal. In this work, the effect of discrete microscale structures (microrods) on cell proliferation was assessed in 3D gels. Microrods were fabricated out of SU-8 with dimensions of 100 x 15 x 15 microm (L x H x W) and incorporated into Matrigel seeded with fibroblasts. The 3D microrod-Matrigel composite system inhibited proliferation of both primary and cell-line fibroblasts compared to cells seeded in Matrigel alone. To rule out bulk mechanical effects, the bulk shear modulus (G') and loss modulus (G") were assessed between 0.1 and 5 Hz for both Matrigel and microrod-Matrigel composites. The incorporation of microrods did not change the bulk stiffness of the gel. Moreover, it was determined that the chemistry of the microrod material itself did not inhibit cell proliferation. Therefore, results indicate that the presence of suspended microscale structures in three dimensions can regulate cell proliferation in a dose-dependent manner. This system provides a biocompatible, long-term way to modulate cell growth in 3D cultures and is amenable to in vivo applications.

Surface modification of SU-8 for enhanced biofunctionality and nonfouling properties.

SU-8 is a chemically amplified, epoxy-based negative photoresist typically used for producing ultrathick resist layers during device manufacturing in the semiconductor industry. As a simple resist, SU-8 has garnered attention as a possible material for a variety of biomedical applications, including tissue engineering, drug delivery, as well as cell-based screening and sensing. However, as a hydrophobic material, the use of SU-8 is limited due to a high degree of nonspecific adsorption of biomolecules, as well as limited cell attachment. In this work, surface chemistry is utilized to modify the SU-8 surface by covalently attaching poly(ethylene glycol) (PEG) to increase biofunctionality and improve its nonfouling properties. Different molecular weights and concentrations of PEG were used to form films of various grafting densities on SU-8 surfaces. X-ray photoelectron spectroscopy (XPS) was used to verify the presence of PEG moieties on the SU-8 surface. High-resolution C1s spectra show that, with an increase in concentration and immobilization time, the grafting density of PEG also increases. Further, a standard overlayer model was used to calculate the thickness of the PEG films formed. The effect of PEG-modified SU-8 was examined in terms of protein adsorption on the surface and fibroblast-surface interactions.

Imaging cellular responses to mechanical stimuli within three-dimensional tissue constructs.

The cellular response to environmental cues is complex, involving both structural and functional changes within the cell. Our understanding of this response is facilitated by microscopy techniques, but has been limited by our ability to image cell structure and function deep in highly-scattering tissues or 3D constructs. A novel multimodal microscopy technique that combines coherent and incoherent imaging for simultaneous visualization of structural and functional properties of cells and engineered tissues is demonstrated. This microscopic technique allows for the simultaneous acquisition of optical coherence microscopy and multiphoton microscopy data with particular emphasis for applications in cell biology and tissue engineering. The capability of this technique is shown using representative 3D cell and tissue engineering cultures consisting of primary fibroblasts from transgenic green fluorescent protein (GFP) mice and GFP-vinculin transfected fibroblasts. Imaging is performed following static and dynamic mechanically-stimulating culture conditions. The microscopy technique presented here reveals unique complementary data on the structure and function of cells and their adhesions and interactions with the surrounding microenvironment.

Methods for fabrication of nanoscale topography for tissue engineering scaffolds.

Observations of how controlling the microenvironment of cell cultures can lead to changes in a variety of parameters has lead investigators to begin studying how the nano-environment of a culture can affects cells. Cells have many structures at the nanoscale such as filipodia and cytoskeletal and membrane proteins that interact with the environment surrounding them. By using techniques that can control the nano-environment presented to a cell, investigators are beginning to be able to mimic the nanoscale topographical features presented to cells by extracellular matrix proteins such as collagen, which has precise and repeating nano-topography. The belief is that these nanoscale surface features are important to creating more natural cell growth and function. A number of techniques are currently being used to create nanoscale topographies for cell scaffolding. These techniques fall into two main categories: techniques that create ordered topographies and those that create unordered topographies. Electron Beam lithography and photo-lithography are two standard techniques for creating ordered features. Polymer demixing, phase separation, colloidal lithography and chemical etching are most typically used for creating unordered surface patterns. This review will give an overview of these techniques and cite observations from experiments carried out using them.

Optical coherence tomography of cell dynamics in three-dimensional tissue models.

Three-dimensional cell-based tissue models have been increasingly useful in the fields of tissue engineering, drug discovery, and cell biology. While techniques for building these tissue models have been advanced, there have been increasing demands for imaging techniques that are capable of assessing complex dynamic three-dimensional cell behavior in real-time and at larger depths in highly-scattering scaffolds. Understanding these cell behaviors requires advanced imaging tools to progress from characterizing two-dimensional cell cultures to complex, highly-scattering, thick three-dimensional tissue constructs. Optical coherence tomography (OCT) is an emerging biomedical imaging technique that can perform cellular-resolution imaging in situ and in real-time. In this study, we demonstrate that it is possible to use OCT to evaluate dynamic cell behavior and function in a quantitative fashion in four dimensions (three-dimensional space plus time). We investigated and characterized in thick tissue models a variety of cell processes, such as chemotaxis migration, proliferation, de-adhesion, and cell-material interactions. This optical imaging technique was developed and utilized in order to gain new insights into how chemical and/or mechanical microenvironments influence cellular dynamics in multiple dimensions. With deep imaging penetration and increased spatial and temporal resolution in three-dimensional space, OCT will be a useful tool for improving our understanding of complex biological interactions at the cellular level.

Control of cellular organization in three dimensions using a microfabricated polydimethylsiloxane-collagen composite tissue scaffold.

Parallel channels of various dimensions have been shown to cause a monolayer of cells in culture to align in the direction of the channels. For the engineering of complex organ systems to become a reality, similar control over the cellular microenvironment in three dimensions must be achieved. Using microfabrication, a polydimethylsiloxane (PDMS) scaffold (40 microm wide, 70-microm-deep parallel channels separated by 25-microm-wide walls) was created. A fibroblast-seeded collagen matrix was then molded around this PDMS scaffold. The PDMS scaffold served as an internal skeleton to guide the cells to grow in the prescribed three-dimensional pattern. Organization, aspect ratio, and the z diameter of the cells were analyzed by confocal microscopy. Fibroblasts elongated and organized in the direction of the channels throughout the height of the scaffold. The mean angle of the cells off of the long axis of the channels was 4.3 +/- 0.7 degrees as opposed to 32.6 +/- 2.2 degrees in controls. The morphology of the cells was also affected by the PDMS scaffold. The nuclei were longer (1.25x) and thinner (0.75x) than in control gels; however, no changes in diameter of the cells in the z direction were seen.