when we consider the dendrites; respectively), in close accordance with probability-based design. (c). Showing that vaccination of mice yields equivalent total anti-influenza IgG responses as intramuscular injection, but with more than 100-fold less antigen delivered to the skin. Since a protective response has been elicited in this study, obviously the cells targeted have been above the threshold. Our work in this paper paves the way to finding this threshold in future work. We have now established a rational design and engineering approach for designing Nanopatches to target discrete and incremental ranges of APC populations (both epidermal and dermal). This will be applied in follow-up studies on mice and on humans. Furthermore, we have shown the actual number and location of targeted APCs – for both the epidermis and dermis – agrees with the probability-based prediction used to guide the device design. This is important, because it demonstrates that a mathematical/engineer- ing design approach ‘from the ground up’ does indeed achieve the desired APC targeting – thereby putting in place a rigorous template for further extending this uniquely precise delivery approach to targeting vaccines to selectively different quantities of APCs. However, these studies have only been performed with one NanopatchTM configuration and we believe there is much scope to apply this device, together with it’s underlying probability-based design approach, as a toolbox for further studies optimizing many vaccines – including those for influenza. We foresee, most importantly, that our study positions the NanopatchTM for extension beyond mice and into humans. In doing this, we will again apply the same idea of tailoring the NanopatchTM device to directly target vaccine to large populations of the epidermal and dermal cells within human skin – accounting for the physiological differences between the skin of the two species. We expect the spatial targeting considerations to be similar, because the density of the characterized APCs resident in the skin is comparable (e.g. Langerhans cell density is 542617 mm22 in mouse ear skin (for the strain tested in this study) [22]; and 730660 mm22 in human upper arm skin [32]). However, the human skin is thicker than mouse skin (e.g., mouse ear epidermis is 17.562.1 mm [21]; while a candidate vaccination site for humans is the forearm, with a dorsal epidermis is 61.3611.0 mm [33]). And correspondingly, the depth of Langerhans cell locations (for example), in the supra-basal region, is greater in humans. Reaching this human target, together with underlying dermal APCs, will require deeper penetration of the projections, which could be achieved by slightly longer projections, and exploiting the skin’s viscoelastic behavior (e.g. by altering the strain-rate of NanopatchTM application). It is worth noting that we have also engineered Nanopatches as practical delivery devices for potential use in humans. They are produced by a simple manufacturing process with the potential for high volume production. By dry-coating vaccine to the Nano- patchTM tips, we expect to minimize or eliminate the need to refrigerate vaccines, as is currently required for most vaccines. The basis for this expectation is that reformulating liquid vaccines into dry-form micro-particles makes them thermostable [34], and we are applying very similar coating principles with our NanopatchTM. Thermostable vaccines have the potential to remove the need for refrigeration – which particularly burden infrastructure in the developing world and/or a pandemic requiring very rapid transportation. The potential for self-application is an added advantage. Nanopatches are also anticipated to avoid needle- phobia, needle-stick injuries (causing more than 500,000 deaths per year), and cross-contamination (e.g. 21 million cases of hepatitis B transmission annually) associated with the hypodermic syringe [35], [36]. With these collective attributes, Nanopatches should be Skin Targeted Vaccinations suitable for general immunization programs for many vaccines, particularly where there is a need to avoid the use of needle and syringe as in vaccinations of infants and children. Nanopatches are safe to use in humans because it is made out of silicon and coated with gold and the microprojections do not break during insertion [37,38]. In a very unlikely case of breakage (expected to be %0.1% of projections, the effects on safety are expected to be minimal because both gold and silicon have been approved as safe by the FDA for skin use [39]. Furthermore gold particles have been used extensively in gene gun DNA immunisations approximately 1mg dose delivered into the skin without any deleterious effects (eg. Phase 1 safety and immune response studies of a DNA vaccine encoding hepatitis B surface antigen delivered by a gene delivery device [40]. Any microprojections remaining in the skin will eventually be sloughed off with the skin. The cost of a vaccination depends on many factors, including: the delivery device (e.g. needle and syringe, or alternative), the vaccine, the cost of transportation, cold chain and the use of a qualified medical practitioner to administer the vaccine and the disposal cost of the device (and packaging). For example, cold chain can contribute 80% of the cost of vaccination programs in developing countries [41]. Although the cost of the Nanopatch is comparable to the cost of the needle and syringe, we believe the ultimate overall cost of vaccine administration via Nanopatch will be considerably cheaper than the needle and syringe, because the Nanopatch is expected to outcompete the needle and syringe in each of the attributes listed. We speculate that in the future we could rollout a rapid response to pandemic influenza to a much greater population via highly-immunogenic NanopatchTM-delivered vaccine sent in the mail to individuals for self-administration. Materials and Methods Ethics Statement All animal experiments were conducted according to the University of Queensland animal ethics regulations. Vaccine used We used was the seasonal human influenza vaccine, Flu- vax2008H, manufactured by CSL Ltd, Parkville Australia which contains 15 mg hemagglutinin (HA) per 0.5ml of each of the three viral strains bearing the surface antigens of A/Brisbane/10/2007 (H3N2), A/Solomon Islands/3/2006 (H1N1) and B/Florida/4/ 2006. Coating of Nanopatches Each NanopatchTM (0.16 cm2) was coated with commercially- available trivalent influenza vaccine (Fluvax2008H CSL Ltd, Parkville Australia) using a nitrogen-jet drying coating method [37]. Briefly, 7 ml of coating solution, containing methylcellulose and the vaccine, was applied to each NanopatchTM. A nitrogen gas jet (6–8 m/s) evenly distributed the solution on the whole NanopatchTM, while simultaneously localizing the vaccine on the projections where it would be delivered to skin. Measurement of projection penetration in skin, using fluorescent dye Four DiD (1,19-dioctadecyl-3,3,39,39-tetramethylindocarbocya- nine per-chlorate) coated patches were applied to the inner earlobe of anaesthetized female 6–8 week old C57BL/6 mice (one patch per ear) with a custom spring based applicator device at 1.96 m/s. After application, each NanopatchTM was kept in place on the skin for 2 minutes. Penetration and release profiles were obtained by PLoS ONE | www.plosone.org 8 April 2010 | Volume 5 | Issue 4 | e10266