Results A probabilistically guided approach for directly depositing antigen to more than 50% of both epidermal and dermal APCs, beneath the patch area As our starting point we chose Langerhans cells, a key antigen- presenting cell in the skin [16], as just one example target because their depth and location are very well quantified [21]. First, we determined the probability of a single projection contacting a Langerhans cell: Pcontact~Vtarget Vlayer 1 ð Þ Where: Vlayer is the volume of the tissue layer containing the Langerhans cells, Vtarget the volume, including the target to which the vaccine can be delivered. In solving (1), we applied the knowledge of Langerhans density and location: e.g. the mouse ear (C57BL/6 Strain) has 542617 Langerhans cells per square mm of skin [22], tightly distributed in the vertical plane at a mean depth of 14.9 mm below the skin surface for the mouse ear (in humans the density of these cells is similar, but they are deeper), less than 3 mm above the dermo- epidermal boundary and with a normal histogram distribution [21]. We calculated that the chance of a tapering projection contacting a Langerhans cell body was Pcontact = 0.275. This probability calculation is simplified and therefore conservatively low: we assume that the contact event will be an antigen touching the Langerhans cell body, and have not included the other contact events with the network of dendrites extending from the Langerhans cell. With each Langerhans cell possessing between five and nine dendrites – some measuring twice as long as the cell body – this difference is significant (e.g. overall, Langerhans cells can cover a quarter of the total projected skin surface area, while their bodies alone cover just ,5%) [23]. Therefore, if all the projections penetrate the skin to the Langerhans cell layer, 6728 projections (i.e. using two Nanopatches; 3364 projections per NanopatchTM covering 0.16 cm2 of skin) will accordingly target the bodies of 2000 Langerhans cells – or, when we also take into account the dendrites, about 10000 Langerhans cells (of an available 17350 resident under the NanopatchTM). Penetrating deeper into the dermis will also target vaccine directly to other classes of APCs [24], that have a less tightly- defined location [25]. Using the knowledge of there being a third of dermal APCs – compared to Langerhans cells in the epidermis – their location, and shorter dendrites [25] we again applied our probability analysis to estimate that our NanopatchTM designs would target the bodies of approximately 500 dermal APCs, or about 3700 APCs (of an available 7360 residing in the dermis) when we also take into account contact with the cell dendrite arms. In summary, our NanopatchTM design for this experiment was theoretically configured to deliver antigen directly to more than 50% of both the epidermal and dermal APCs available under the patch area within the skin – without relying on antigen diffusing away from the projections. Translating theoretical skin APC targeting into a practical NanopatchTM design Guided by our theoretical probability-based analysis, we designed devices with arrays of densely-packed silicon projections (each ,110 mm in length; with a cone height of 40 mm, at a density of 21025/cm2; distributed in 464 mm arrays): also Skin Targeted Vaccinations designed to be slender enough to puncture individual cells without inducing significant cell death, but at the same time mechanically strong enough to be pushed through the stratum corneum without breaking. To achieve this balance, the projection tips for this experiment were designed to be sharp, tapering down to ,1000 nm at the tips, which, when applied to skin at low speeds (,1 m/s), were predicted to induce minimal cell death [17], [18]. We designed the spacing between the projections to be sufficiently close for the Nanopatches to be practically sized for skin application; while providing enough space for the skin to deflect and compress around the projections during penetration. We fabricated Nanopatches from silicon wafers with Deep Reactive Ion Etching – a method typically used in industry and potentially suitable to high volume manufacture at low unit cost [26]. NanopatchTM projections consistently penetrate the skin surface, through to the upper dermis We then applied the Nanopatches to the skin, first with the goal of establishing the desired consistent and repeatable projection penetration through the epidermis and into the upper dermis. First, using scanning electron microscopy images of the skin surface following application, we observed 9362.9% (mean 6 SD; n = 350 projections) of the projections penetrated the skin. These data show consistently, that most of projections breach the skin barrier. We then progressed to measure how far the projections penetrate into the skin. Using the delivery of lipophilic dye by the NanopatchTM (fluorescent DiD; imaged by confocal micros- copy), Fig. 2m shows we measured the resultant distal tip penetration depth of 42610 mm (mean 6 SD; n = 350 projections, 5 ears) – extending through the epidermis (17.1 mm in thickness [21]) and into the upper dermis by 25 mm. And in separate experiments, we observed the in situ projection track pathways (i.e. the ‘holes’ left behind in the skin by projections; not reliant on dyes; obtained with cryo-scanning electron microscopy (SEM) in which, C57BL/6 mouse ear skin was frozen when the patch was in place). Fig. 2d shows a representative projection track pathway piercing fully through the stratum corneum and epidermis – and into the upper dermis. These qualitative observations are consistent with the quantitative lipophilic dye measurements. Influenza antigen is directly deposited to thousands of epidermal and dermal APCs, in close accordance with probability-based design Having established the NanopatchTM projections achieve the desired penetration into skin, we then measured the resultant delivery of influenza vaccine directly to skin APCs – in practice. Each NanopatchTM was coated with commercially-available trivalent influenza vaccine (FluvaxH2008; CSL Ltd, Parkville, Australia) and applied to the inner earlobe of anaesthetized female 6–8 week old C57BL/6 mice. Using immunofluorescence microscopy (Fig 2e–h side view and 2i–l top view); and, in three dimensions, (Video S1), we found that 40619% of all the projections directly deposited antigen in contact with epidermal and upper dermal MHC class II positive cells. We also examined the skin area subjected to NanopatchTM application and found the Nanopatches directly targeted antigen to 9.265.2% of the cell bodies of resident MHC class II cells imaged within the ‘targeting zone’ (i.e. the skin area the NanopatchTM is applied to) of epidermis and upper dermis. We measured the overall density of MHC class II cells at 2 7726212 cells/mm , which is nearly identical with the sum of PLoS ONE | www.plosone.org 4 April 2010 | Volume 5 | Issue 4 | e10266