The biomechanics of wounds at physiologically relevant levels: Understanding skin's stress-shielding effect for the quantitative assessment of healing.

Wounds are responsible for the decrease in quality of life of billions of people around the world. Their assessment relies on subjective parameters which often delays optimal treatments and results in increased healthcare costs. In this work, we sought to understand and quantify how wounds at different healing stages (days 1, 3, 7 and 14 post wounding) change the mechanical properties of the tissues that contain them, and how these could be measured at clinically relevant strain levels, as a step towards quantitative wound tracking technologies. To achieve this, we used digital image correlation and mechanical testing on a mouse model of wound healing to map the global and local tissue strains. We found no significant differences in the elastic and viscoelastic properties of wounded vs unwounded skin when samples were measured in bulk, presumably as these were masked by the protective mechanisms of skin, which redistributes the applied loads to mitigate high stresses and reduce tissue damage. By measuring local strain values and observing the distinct patterns they formed, it was possible to establish a connection between the healing phase of the tissue (determined by the time post-injury and the observed histological features) and the overall mechanical behaviour. Importantly, these parameters were measured from the surface of the tissue, using physiologically relevant strains without increasing the tissue's damage. Adaptations of these approaches for clinical use have the potential to aid in the identification of skin healing problems, such as excessive inflammation or lack of mechanical progression over time. An increase, decrease, or lack of change in the elasticity and viscoelasticity parameters, can be indicative of wound state, thus ultimately leading to improved diagnostic outcomes.

Rapid mechanosensitive migration and dispersal of newly divided mesenchymal cells aid their recruitment into dermal condensates.

Embryonic mesenchymal cells are dispersed within an extracellular matrix but can coalesce to form condensates with key developmental roles. Cells within condensates undergo fate and morphological changes and induce cell fate changes in nearby epithelia to produce structures including hair follicles, feathers, or intestinal villi. Here, by imaging mouse and chicken embryonic skin, we find that mesenchymal cells undergo much of their dispersal in early interphase, in a stereotyped process of displacement driven by 3 hours of rapid and persistent migration followed by a long period of low motility. The cell division plane and the elevated migration speed and persistence of newly born mesenchymal cells are mechanosensitive, aligning with tissue tension, and are reliant on active WNT secretion. This behaviour disperses mesenchymal cells and allows daughters of recent divisions to travel long distances to enter dermal condensates, demonstrating an unanticipated effect of cell cycle subphase on core mesenchymal behaviour.

The developmental basis of fingerprint pattern formation and variation.

Fingerprints are complex and individually unique patterns in the skin. Established prenatally, the molecular and cellular mechanisms that guide fingerprint ridge formation and their intricate arrangements are unknown. Here we show that fingerprint ridges are epithelial structures that undergo a truncated hair follicle developmental program and fail to recruit a mesenchymal condensate. Their spatial pattern is established by a Turing reaction-diffusion system, based on signaling between EDAR, WNT, and antagonistic BMP pathways. These signals resolve epithelial growth into bands of focalized proliferation under a precociously differentiated suprabasal layer. Ridge formation occurs as a set of waves spreading from variable initiation sites defined by the local signaling environments and anatomical intricacies of the digit, with the propagation and meeting of these waves determining the type of pattern that forms. Relying on a dynamic patterning system triggered at spatially distinct sites generates the characteristic types and unending variation of human fingerprint patterns.

In vivo, in situ and ex vivo comparison of porcine skin for microprojection array penetration depth, delivery efficiency and elastic modulus assessment.

With the development of wearable technologies, the interfacial properties of skin and devices have become much more important. For research and development purposes, porcine skin is often used to evaluate device performance, but the differences between in vivo, in situ and ex vivo porcine skin mechanical properties can potentially misdirect investigators during the development of their technology. In this study, we investigated the significant changes to mechanical properties with and without perfusion (in vivo versus in vitro tissue). The device focus for this study was a skin-targeting Nanopatch vaccine microneedle device, employed to assess the variance to key skin engagement parameters - penetration depth and delivery efficiency - due to different tissue conditions. The patches were coated with fluorescent or 14C radiolabelled formulations for penetration depth and delivery efficiency quantification in vivo, and at time points up to 4 h post mortem. An immediate cessation of blood circulation saw mean microneedle penetration depth fell from ∼100 µm to ∼55 µm (∼45%). Stiffening of underlying tissues as a result of rigor mortis then augmented the penetration depths at the 4 h timepoint back to ∼100 µm, insignificantly different (p = 0.0595) when compared with in vivo. The highest delivery efficiency of formulation into the skin (dose measured in the skin excluding leftover dose on skin and patch surfaces) was also observed at this time point of ∼25%, up from ∼2% in vivo. Data obtained herein progresses medical device development, highlighting the need to consider the state and muscle tissues when evaluating prototypes on cadavers.

Cellular metabolism and pore lifetime of human skin following microprojection array mediation.

Skin-targeting microscale medical devices are becoming popular for therapeutic delivery and diagnosis. We used cryo-SEM, fluorescence lifetime imaging microscopy (FLIM), autofluorescence imaging microscopy and inflammatory response to study the puncturing and recovery of human skin ex vivo and in vivo after discretised puncturing by a microneedle array (Nanopatch®). Pores induced by the microprojections were found to close by ~25% in diameter within the first 30 min, and almost completely close by ~6 h. FLIM images of ex vivo viable epidermis showed a stable fluorescence lifetime for unpatched areas of ~1000 ps up to 24 h. Only the cells in the immediate puncture zones (in direct contact with projections) showed a reduction in the observed fluorescence lifetimes to between ~518-583 ps. The ratio of free-bound NAD(P)H (α1/α2) in unaffected areas of the viable epidermis was ~2.5-3.0, whereas the ratio at puncture holes was almost double at ~4.2-4.6. An exploratory pilot in vivo study also suggested similar closure rate with histamine administration to the forearms of human volunteers after Nanopatch® treatment, although a prolonged inflammation was observed with Tissue Viability Imaging. Overall, this work shows that the pores created by the microneedle-type medical device, Nanopatch®, are transient, with the skin recovering rapidly within 1-2 days in the epidermis after application.

A low inflammatory, Langerhans cell-targeted microprojection patch to deliver ovalbumin to the epidermis of mouse skin.

In a low inflammatory skin environment, Langerhans cells (LCs) - but not dermal dendritic cells (dDCs) - contribute to the pivotal process of tolerance induction. Thus LCs are a target for specific-tolerance therapies. LCs reside just below the stratum corneum, within the skin's viable epidermis. One way to precisely deliver immunotherapies to LCs while remaining minimally invasive is with a skin delivery device such as a microprojection arrays (MPA). Today's MPAs currently achieve rapid delivery (e.g. within minutes of application), but are focussed primarily at delivery of therapeutics to the dermis, deeper within the skin. Indeed, no MPA currently delivers specifically to the epidermal LCs of mouse skin. Without any convenient, pre-clinical device available, advancement of LC-targeted therapies has been limited. In this study, we designed and tested a novel MPA that delivers ovalbumin to the mouse epidermis (eMPA) while maintaining a low, local inflammatory response (as defined by low erythema after 24 h). In comparison to available dermal-targeted MPAs (dMPA), only eMPAs with larger projection tip surface areas achieved shallow epidermal penetration at a low application energy. The eMPA characterised here induced significantly less erythema after 24 h (p = 0.0004), less epidermal swelling after 72 h (p < 0.0001) and 52% less epidermal cell death than the dMPA. Despite these differences in skin inflammation, the eMPA and dMPA promoted similar levels of LC migration out of the skin. However, only the eMPA promoted LCs to migrate with a low MHC II expression and in the absence of dDC migration. Implementing this more mouse-appropriate and low-inflammatory eMPA device to deliver potential immunotherapeutics could improve the practicality and cell-specific targeting of such therapeutics in the pre-clinical stage. Leading to more opportunities for LC-targeted therapeutics such as for allergy immunotherapy and asthma.

Space- and time-resolved investigation on diffusion kinetics of human skin following macromolecule delivery by microneedle arrays.

Microscale medical devices are being developed for targeted skin delivery of vaccines and the extraction of biomarkers, with the potential to revolutionise healthcare in both developing and developed countries. The effective clinical development of these devices is dependent on understanding the macro-molecular diffusion properties of skin. We hypothesised that diffusion varied according to specific skin layers. Using three different molecular weights of rhodamine dextran (RD) (MW of 70, 500 and 2000 kDa) relevant to the vaccine and therapeutic scales, we deposited molecules to a range of depths (0-300 µm) in ex vivo human skin using the Nanopatch device. We observed significant dissipation of RD as diffusion with 70 and 500 kDa within the 30 min timeframe, which varied with MW and skin layer. Using multiphoton microscopy, image analysis and a Fick's law analysis with 2D cartesian and axisymmetric cylindrical coordinates, we reported experimental trends of epidermal and dermal diffusivity values ranging from 1-8 µm2 s-1 to 1-20 µm2 s-1 respectively, with a significant decrease in the dermal-epidermal junction of 0.7-3 µm2 s-1. In breaching the stratum corneum (SC) and dermal-epidermal junction barriers, we have demonstrated practical application, delivery and targeting of macromolecules to both epidermal and dermal antigen presenting cells, providing a sound knowledge base for future development of skin-targeting clinical technologies in humans.

Allometric scaling of skin thickness, elasticity, viscoelasticity to mass for micro-medical device translation: from mice, rats, rabbits, pigs to humans.

Emerging micro-scale medical devices are showing promise, whether in delivering drugs or extracting diagnostic biomarkers from skin. In progressing these devices through animal models towards clinical products, understanding the mechanical properties and skin tissue structure with which they interact will be important. Here, through measurement and analytical modelling, we advanced knowledge of these properties for commonly used laboratory animals and humans (~30 g to ~150 kg). We hypothesised that skin's stiffness is a function of the thickness of its layers through allometric scaling, which could be estimated from knowing a species' body mass. Results suggest that skin layer thicknesses are proportional to body mass with similar composition ratios, inter- and intra-species. Experimental trends showed elastic moduli increased with body mass, except for human skin. To interpret the relationship between species, we developed a simple analytical model for the bulk elastic moduli of skin, which correlated well with experimental data. Our model suggest that layer thicknesses may be a key driver of structural stiffness, as the skin layer constituents are physically and therefore mechanically similar between species. Our findings help advance the knowledge of mammalian skin mechanical properties, providing a route towards streamlined micro-device research and development onto clinical use.

The hyperelastic and failure behaviors of skin in relation to the dynamic application of microscopic penetrators in a murine model.

In-depth understanding of skin elastic and rupture behavior is fundamental to enable next-generation biomedical devices to directly access areas rich in cells and biomolecules. However, the paucity of skin mechanical characterization and lack of established fracture models limits their rational design. We present an experimental and numerical study of skin mechanics during dynamic interaction with individual and arrays of micro-penetrators. Initially, micro-indentation of individual skin strata revealed hyperelastic moduli were dramatically rate-dependent, enabling extrapolation of stiffness properties at high velocity regimes (>1ms-1). A layered finite-element model satisfactorily predicted the penetration of micro-penetrators using characteristic fracture energies (∼10pJµm-2) significantly lower than previously reported (≫100pJµm-2). Interestingly, with our standard application conditions (∼2ms-1, 35gpistonmass), ∼95% of the application kinetic energy was transferred to the backing support rather than the skin ∼5% (murine ear model). At higher velocities (∼10ms-1) strain energy accumulated in the top skin layers, initiating fracture before stress waves transmitted deformation to the backing material, increasing energy transfer efficiency to 55%. Thus, the tools developed provide guidelines to rationally engineer skin penetrators to increase depth targeting consistency and payload delivery across patients whilst minimizing penetration energy to control skin inflammation, tolerability and acceptability. STATEMENT OF SIGNIFICANCE: The mechanics of skin penetration by dynamically-applied microscopic tips is investigated using a combined experimental-computational approach. A FE model of skin is parameterized using indentation tests and a ductile-failure implementation validated against penetration assays. The simulations shed light on skin elastic and fracture properties, and elucidate the interaction with microprojection arrays for vaccine delivery allowing rational design of next-generation devices.

Characterising the material properties at the interface between skin and a skin vaccination microprojection device.

UNLABELLED: The rapid emergence of micro-devices for biomedical applications over the past two decades has introduced new challenges for the materials used in the devices. Devices like microneedles and the Nanopatch, require sufficient strength to puncture skin often with sharp-slender micro-scale profiles, while maintaining mechanical integrity. For these technologies we sought to address two important questions: 1) On the scale at which the device operates, what forces are required to puncture the skin? And 2) What loads can the projections/microneedles withstand prior to failure. First, we used custom fabricated nanoindentation micro-probes to puncture skin at the micrometre scale, and show that puncture forces are ∼0.25-1.75mN for fresh mouse skin, in agreement with finite element simulations for our device. Then, we used two methods to perform strength tests of Nanopatch projections with varied aspect ratios. The first method used a nanoindenter to apply a force directly on the top or on the side of individual silicon projections (110µm in length, 10µm base radius), to measure the force of fracture. Our second method used an Instron to fracture full rows of projections and characterise a range of projection designs (with the method verified against previous nanoindentation experiments). Finally, we used Cryo-Scanning Electron Microscopy to visualise projections in situ in the skin to confirm the behaviour we quantified, qualitatively. STATEMENT OF SIGNIFICANCE: Micro-device development has proliferated in the past decade, including devices that interact with tissues for biomedical outcomes. The field of microneedles for vaccine delivery to skin has opened new material challenges both in understanding tissue material properties and device material. In this work we characterise both the biomaterial properties of skin and the material properties of our microprojection vaccine delivery device. This study directly measures the micro-scale puncture properties of skin, whilst demonstrating clearly how these relate to device design. This will be of strong interest to those in the field of biomedical microdevices. This includes work in the field of wearable and semi-implantable devices, which will require clear understanding of tissue behaviour and material characterisation.

Inactivated poliovirus type 2 vaccine delivered to rat skin via high density microprojection array elicits potent neutralising antibody responses.

Polio eradication is progressing rapidly, and the live attenuated Sabin strains in the oral poliovirus vaccine (OPV) are being removed sequentially, starting with type 2 in April 2016. For risk mitigation, countries are introducing inactivated poliovirus vaccine (IPV) into routine vaccination programs. After April 2016, monovalent type 2 OPV will be available for type 2 outbreak control. Because the current IPV is not suitable for house-to-house vaccination campaigns (the intramuscular injections require health professionals), we developed a high-density microprojection array, the Nanopatch, delivered monovalent type 2 IPV (IPV2) vaccine to the skin. To assess the immunogenicity of the Nanopatch, we performed a dose-matched study in rats, comparing the immunogenicity of IPV2 delivered by intramuscular injection or Nanopatch immunisation. A single dose of 0.2 D-antigen units of IPV2 elicited protective levels of poliovirus antibodies in 100% of animals. However, animals receiving IPV2 by IM required at least 3 immunisations to reach the same neutralising antibody titres. This level of dose reduction (1/40th of a full dose) is unprecedented for poliovirus vaccine delivery. The ease of administration coupled with the dose reduction observed in this study points to the Nanopatch as a potential tool for facilitating inexpensive IPV for mass vaccination campaigns.

Formulations for microprojection/microneedle vaccine delivery: Structure, strength and release profiles.

To develop novel methods for vaccine delivery, the skin is viewed as a high potential target, due to the abundance of immune cells that reside therein. One method, the use of dissolving microneedle technologies, has the potential to achieve this, with a range of formulations now being employed. Within this paper we assemble a range of methods (including FT-FIR using synchrotron radiation, nanoindentation and skin delivery assays) to systematically examine the effect of key bulking agents/excipients - sugars/polyols - on the material form, structure, strength, failure properties, diffusion and dissolution for dissolving microdevices. We investigated concentrations of mannitol, sucrose, trehalose and sorbitol from 1:1 to 30:1 with carboxymethylcellulose (CMC), although mannitol did not form our micro-structures so was discounted early in the study. The other formulations showed a variety of crystalline (sorbitol) and amorphous (sucrose, trehalose) structures, when investigated using Fourier transform far infra-red (FT-FIR) with synchrotron radiation. The crystalline structures had a higher elastic modulus than the amorphous formulations (8-12GPa compared to 0.05-11GPa), with sorbitol formulations showing a bimodal distribution of results including both amorphous and crystalline behaviour. In skin, diffusion properties were similar among all formulations with dissolution occurring within 5s for our small projection array structures (~100µm in length). Overall, slight variations in formulation can significantly change the ability of our projections to perform their required function, making the choice of bulking/vaccine stabilising agents of great importance for these devices.

Microprojection arrays to immunise at mucosal surfaces.

The buccal mucosa (inner cheek) is an attractive site for delivery of immunotherapeutics, due to its ease of access and rich antigen presenting cell (APC) distribution. However, to date, most delivery methods to the buccal mucosa have only been topical-with the challenges of: 1) an environment where significant biomolecule degradation may occur; 2) inability to reach the APCs that are located deep in the epithelium and lamina propria; and 3) salivary flow and mucous secretion that may result in removal of the therapeutic agent before absorption has taken place. To overcome these challenges and achieve consistent, repeatable targeted delivery of immunotherapeutics to within the buccal mucosa (not merely on to the surface), we utilised microprojection arrays (Nanopatches-110 µm length projections, 3364 projections, 16 mm2 surface area) with a purpose built clip applicator. The mechanical application of Nanopatches bearing a dry-coated vaccine (commercial influenza vaccine, as a test case immunotherapeutic) released the vaccine to a depth of 47.8±14.8 µm (mean±SD, n=4), in the mouse buccal mucosa (measured using fluorescent delivered dyes and CryoSEM). This location is in the direct vicinity of APCs, facilitating antigenic uptake. Resultant systemic immune responses were similar to systemic immunization methods, and superior to comparative orally immunised mice. This confirms the Nanopatch administered vaccine was delivered into the buccal mucosa and not ingested. This study demonstrates a minimally-invasive delivery device with rapid (2 min of application time), accurate and consistent release of immunotherapeutics in to the buccal mucosa-that conceptually can be extended in to human use for broad and practical utility.

Dry-coated live viral vector vaccines delivered by nanopatch microprojections retain long-term thermostability and induce transgene-specific T cell responses in mice.

The disadvantages of needle-based immunisation motivate the development of simple, low cost, needle-free alternatives. Vaccine delivery to cutaneous environments rich in specialised antigen-presenting cells using microprojection patches has practical and immunological advantages over conventional needle delivery. Additionally, stable coating of vaccine onto microprojections removes logistical obstacles presented by the strict requirement for cold-chain storage and distribution of liquid vaccine, or lyophilised vaccine plus diluent. These attributes make these technologies particularly suitable for delivery of vaccines against diseases such as malaria, which exerts its worst effects in countries with poorly-resourced healthcare systems. Live viral vectors including adenoviruses and poxviruses encoding exogenous antigens have shown significant clinical promise as vaccines, due to their ability to generate high numbers of antigen-specific T cells. Here, the simian adenovirus serotype 63 and the poxvirus modified vaccinia Ankara--two vectors under evaluation for the delivery of malaria antigens to humans--were formulated for coating onto Nanopatch microprojections and applied to murine skin. Co-formulation with the stabilising disaccharides trehalose and sucrose protected virions during the dry-coating process. Transgene-specific CD8(+) T cell responses following Nanopatch delivery of both vectors were similar to intradermal injection controls after a single immunisation (despite a much lower delivered dose), though MVA boosting of pre-primed responses with Nanopatch was found to be less effective than the ID route. Importantly, disaccharide-stabilised ChAd63 could be stored for 10 weeks at 37°C with less than 1 log10 loss of viability, and retained single-dose immunogenicity after storage. These data support the further development of microprojection patches for the deployment of live vaccines in hot climates.

Elastic modulus and viscoelastic properties of full thickness skin characterised at micro scales.

The recent emergence of micro-devices for vaccine delivery into upper layers of the skin holds potential for increased immune responses using physical means to target abundant immune cell populations. A challenge in doing this has been a limited understanding of the skin elastic properties at the micro scale (i.e. on the order of a cell diameter; ~10 µm). Here, we quantify skin's elastic properties at a micro-scale by fabricating customised probes of scales from sub- to super-cellular (0.5 µm-20 µm radius). We then probe full thickness skin; first with force-relaxation experiments and subsequently by elastic indentations. We find that skin's viscoelastic response is scale-independent: consistently a ~40% decrease in normalised force over the first second, followed by further 10% reduction over 10 s. Using Prony series and Hertzian contact analyses, we determined the strain-rate independent elastic moduli of the skin. A high scale dependency was found: the smallest probe encountered the highest elastic modulus (~30 MPa), whereas the 20 µm radius probe was lowest (below 1 MPa). We propose that this may be a result of the load distribution in skin facilitated by the hard corneocytes in the outermost skin layers, and softer living cell layers below.

The viscoelastic, hyperelastic and scale dependent behaviour of freshly excised individual skin layers.

Micro-devices using mechanical means to target skin for improved drug and vaccine delivery have great promise for improved clinical healthcare. Fully realizing this promise requires a greater understanding of key micro-biomechanical properties for each of the different skin layers - that are both the mechanical barriers and biological targets of these devices. Here, we performed atomic force microscopy indentation on a micro-nano scale to quantify separately, in fresh mouse skin, the viscous and elastic behaviour of the stratum corneum, viable epidermis and dermis. By accessing each layer directly, we examined the response to nanoindentation at sub-cellular and bulk-cellular scale. We found that the dermis showed greatest mechanical stiffness (elastic moduli of 7.33-13.48 MPa for 6.62 µm and 1.90 µm diameter spherical probes respectively). In comparison, the stratum corneum and viable epidermis were weaker at 0.75-1.62 MPa and 0.49-1.51 MPa respectively (again with the lower values resulting from indentations with the large probe 6.62 µm). The living cell layer of the epidermis (viable epidermis) showed greatest viscoelasticity - almost fully relaxing from shallow indentation - whilst the other layers reached a plateau after relaxing by around 40%. With small scale (sub-micron) AFM indentation, we directly determined the effects of different layer constituents - in particular, the dermis showed that some indents contacted collagen fibrils and others contacted ground substance/cellular areas. This work has far reaching implications for the design of micro-devices using mechanical means to deliver drugs or vaccines into the skin; providing key characterized mechanical property values for each constituent of the target delivery material.

Improving the reach of vaccines to low-resource regions, with a needle-free vaccine delivery device and long-term thermostabilization.

Dry-coated microprojections can deliver vaccine to abundant antigen-presenting cells in the skin and induce efficient immune responses and the dry-coated vaccines are expected to be thermostable at elevated temperatures. In this paper, we show that we have dramatically improved our previously reported gas-jet drying coating method and greatly increased the delivery efficiency of coating from patch to skin to from 6.5% to 32.5%, by both varying the coating parameters and removing the patch edge. Combined with our previous dose sparing report of influenza vaccine delivery in a mouse model, the results show that we now achieve equivalent protective immune responses as intramuscular injection (with the needle and syringe), but with only 1/30th of the actual dose. We also show that influenza vaccine coated microprojection patches are stable for at least 6 months at 23°C, inducing comparable immunogenicity with freshly coated patches. The dry-coated microprojection patches thus have key and unique attributes in ultimately meeting the medical need in certain low-resource regions with low vaccine affordability and difficulty in maintaining "cold-chain" for vaccine storage and transport.

Improved DNA vaccination by skin-targeted delivery using dry-coated densely-packed microprojection arrays.

HSV-2-gD2 DNA vaccine was precisely delivered to immunologically sensitive regions of the skin epithelia using dry-coated microprojection arrays. These arrays delivered a vaccine payload to the epidermis and the upper dermis of mouse skin. Immunomicroscopy results showed that, in 43 ± 5% of microprojection delivery sites, the DNA vaccine was delivered to contact with professional antigen presenting cells in the epidermal layer. Associated with this efficient delivery of the vaccine into the vicinity of the professional antigen presenting cells, we achieved superior antibody responses and statistically equal protection rate against an HSV-2 virus challenge, when compared with the mice immunized with intramuscular injection using needle and syringe, but with less than 1/10th of the delivered antigen.

Surface-modified microprojection arrays for intradermal biomarker capture, with low non-specific protein binding.

Minimally invasive biosensors are of great interest for rapid detection of disease biomarkers for diagnostic screening at the point-of-care. Here we introduce a device which extracts disease-specific biomarkers directly from the upper dermis, without the needle and syringe or resource-intensive blood processing. Using antigen-specific antibodies raised in mice as a model system, we confirm the analytical specificity and sensitivity of the antibody capture and extraction in comparison to the conventional methods based on needle/syringe blood draw followed by processing and antigen-specific ELISAs.

Targeted, needle-free vaccinations in skin using multilayered, densely-packed dissolving microprojection arrays.

Targeting of vaccines to abundant immune cell populations within our outer thin skin layers using miniaturized devices-much thinner than a needle and syringe, could improve the efficacy of vaccines (and other immunotherapies). To meet this goal, a densely packed dissolving microprojection array (dissolving Nanopatch) is designed, achieving functional miniaturization by 1) formulating small microneedles (two orders of magnitude smaller than a standard needle and syringe) and 2) multiple layering of the payload within microprojections with tight tolerances (of the order of a micrometer). The formulation method is suitable to many vaccines because it is without harsh or complex chemical processes, and it is performed at low temperatures and at a neutral pH. When the formulated dNPs are applied to skin, consistent and robust penetration is achieved, rapidly targeting the skin strata of interest (<5 min; significantly faster than larger dissolving microneedles that have been previously reported). Resultant diffusion is significantly enhanced within the dermis compared with the epidermis. Using two different antigens (ovalbumin and a commercial trivalent influenza vaccine [Fluvax2008]), the administration of these dissolving patches generate robust systemic immune responses in a mouse model. To the authors' knowledge, this is the first report of successful vaccination with any form of dissolving microneedles. The patches made by this method therefore have the potential for pain-free, needle-free, and effective vaccination in humans.