Skin Targeted Vaccinations Figure 2. NanopatchTM appearance, application, and targeted delivery of antigen to antigen presenting cells (APCs). Nanopatches (a) were fabricated (cone length 40 mm) with Deep Reactive Ion Etching and then visualized by scanning electron microscopy (SEM) uncoated (b) or dry coated with FluvaxH2008 and methylcellulose (c). The skin following NanopatchTM application was visualized using Cryo-SEM (d), the arrows show entry points through the epidermis (SC: stratum corneum; E: viable epidermis; D: dermis). The localization of vaccine (red, panel e side view, panel i top view; Cy3 labeled FluvaxH) and MHC class II +ve cells (green, f side view, panel j top view) was determined by confocal microscopy to the depth of 46 mm. A hair can be seen in j and l as a large diagonal bar. Nuclear staining (blue, panel g and k; stained with Hoechst 33342) in the epidermis was used to determine the epidermal-dermal boundary. Co-localization is shown in panel h and l (arrow heads). The distal tip penetration values are shown in the panel m. Quantification of MHC class II +ve cells, antigen and co-localized antigen and MHC class II +ve cells was carried out using data from 9 areas within each of three treated ears (n,o,p ). An example of a confocal microscopy image in three dimensions is presented in Video S1. doi:10.1371/journal.pone.0010266.g002 Nanopatches are distinct from existing microneedle devices, by having very high packing density of projections (.20000 projec- tions/cm2) tailored by a probability analysis to deposit antigen directly to thousands of epidermal and dermal APCs mapped within the skin, with the smaller diameter far less likely to damage cells near the projections. In this study, we tested the ability of Nanopatches to deliver seasonal influenza vaccine with delivered dose reduction. We chose commercially-available seasonal human trivalent influenza vaccine (FluvaxH 2008) as a test case, because an antigen-efficient vaccine delivery system for influenza might overcome seasonal vaccine shortages and allow a greater number of people to be vaccinated quickly following the arrival of a pandemic strain [19], [20]. In the event of a pandemic, the current influenza vaccine production capacity of about 900 million doses [20] falls far short of the global population; most of whom might need the vaccine. Conceptually, however, we believe the Nanopatch is broadly applicable to many other vaccines. PLoS ONE | www.plosone.org 3 April 2010 | Volume 5 | Issue 4 | e10266