Skin Targeted Vaccinations Figure 1. The concept of targeting antigen directly to skin antigen presenting cells (APCs) (in the epidermis and dermis) using NanopatchesTM – compared to existing needle-based delivery methods. Drawn to scale is the structure of human skin (which is thicker than mouse skin; including the location of Langerhans cells and dermal APCs). We also show, to scale, the geometry of different needle-based delivery devices (a–c) and the NanopatchTM (d). (a) Intramuscular (IM) route directly inject a vaccine into muscle, which contains a low density of antigen presenting cells. (b) Intradermal (ID) injection delivers vaccine to the dermis of the skin, where there is an extensive network of resident professional APCs. These APCs can capture antigen, migrate to the draining lymph node, and orchestrate potent systemic immune responses. Therefore, ID delivery can achieve comparable immune responses to IM, but at about one-tenth of the vaccine dose. However, it is technically challenging to do an ID injection. (c) Microneedle techniques use sparsely packed needles with dimensions of hundreds of micrometers to deliver a liquid or dry form of the vaccine to the skin. This method is technically easier and is as efficient as ID injection. (d) The NanopatchTM technique uses a very small and densely packed array (over 20,000 projections/cm2) to directly deposit vaccine material into the immediate vicinity of a large population of APCs in a small area. Therefore, numerous APCs can be directly involved in raising an immune response. doi:10.1371/journal.pone.0010266.g001 use of a needle or microneedle(s) to deliver the vaccine to different tissue sites; and we illustrate these schematically in Fig. 1 b–c. It is compared to the current convention of most vaccines being delivered with a needle into muscle (Fig 1a). The closest alternative to this – and the most tested [7]– is delivering liquid vaccine with a fine needle to the dermis (Fig 1b); with, for example, influenza vaccines requiring one tenth of the antigen for a comparable immune response as standard intramuscular injection [3]. With a thinner target area, intradermal injection is technically difficult to administer. Recently, this difficulty has been mitigated by a more controlled fixed-penetration injection mechanism [8] (.1mm in length) – with a finer needle (,100 mm in diameter) – while still yielding similar immunological results that are functional in the protection against disease (some work was also presented with even greater dose-sparing than 1:10, but this is limited to total antibody readouts – which do not yield insights into protective efficacy against a particular disease). However, needle-based intradermal vaccine approaches pierce through the tightly-defined epidermal immune cell populations [2] (abundant in a class of APCs called Langerhans cells), delivering liquid vaccine as a single bolus into the dermis (Fig 1b) [2]. This may not be the most effective way of targeting antigens to the skin immune system: the epidermal immune cells are largely missed; and further below, in the dermis, the ‘pooled’ vaccine reaches dermal APCs, with much of it in the surrounding region away from these cells – and not directly accessible to them. By increasing the number of similarly shaped needles up to 5 – to deliver either liquid or solid-coated vaccine (e.g. Fig 1c) – vaccine interaction with the skin target cell populations is not fundamentally different; and therefore the resultant immune responses are also similar to individual needle injection to the dermis [4], [9], [10]. And by further increasing the number of micro-needles in an array to 190 on a 1 cm2 patch (then extended to 1314 microneedles on a 2 cm2 patch) [11], [12], better immune responses have been achieved following chicken egg albumin delivery (a non pathogenic protein; functional protection against a disease has therefore not been tested) in comparison with intramuscular injection. However, the induced immune responses are no higher than achieved by needle intradermal delivery. And dose-sparing effects were not explored [11]. Recently, other methods of vaccine delivery moving completely away from the needle (e.g., diffusion/permeation delivery [13], liquid jet injection [14], and ballistic microparticle injection [15]) have been pursued to meet these delivery challenges. However, their ability to consistently and directly deliver vaccines directly to a high population of skin immune cells with minimal cell damage is limited (resulting in comparable dose sparing gains as needle or microneedle-based systems). Also, these devices are hindered by practical considerations, such as size, expense of manufacture or complexity of operation Here, we present a new method of controlled and targeted delivery of vaccine, to directly contact thousands of APCs residing within the skin, without widespread cell death – and exploiting the network of epidermal and dermal APCs [16] to significantly improve vaccine potency over existing delivery approaches. To achieve this goal, we designed a NanopatchTM device (shown conceptually in Fig 1d; and Fig 2 a–c), displaying an array of densely-packed (21025 projections/cm2) gold-coated silicon projec- tions (,110 mm in total length, including a cone of 40 mm height; tapering to tip designs at ,1000 nm) coated with vaccine antigen in dry form (Fig 1 d and 2 c). When applied to the skin, the concept is for the NanopatchTM projections to penetrate the epidermis and upper dermis, depositing antigen directly to high populations of APCs residing within these skin layers (Fig 1 d). However, we also design the slender projections at the micro-nanoscale (with much of the length below the diameter of target cells), so that when they are slowly applied to the skin (i.e. around 1 m/s, which is much lower than the very high gene gun speeds), they induce low stresses to the target cells, and thus are likely to induce a low incidence of cell death near the tips [17], [18]. In doing so, we are combining the benefits of a more accurately directed delivery of vaccines to a high population of APCs, without widespread cell death. As shown to scale in Fig 1, PLoS ONE | www.plosone.org 2 April 2010 | Volume 5 | Issue 4 | e10266