Michael L. Crichton

Dry-coated microprojection array patches for targeted delivery of immunotherapeutics to the skin

Dry-coated microprojections (MPs) deliver vaccine to abundant immunogenic cells within the skin to induce immune responses. Success in this targeted vaccine delivery relies on overcoming the challenges of dry-coating the vaccine onto the very small (<or=90 microm length) and densely packed (approximately 20,000 cm(-2)) MPs. In this paper, we show that a gas-jet drying coating method achieves the desired uniform coating. The coating approach is robustly demonstrated on compounds representative of a range of immunotherapeutics (e.g. DNA, proteins), with each uniformly coated on thousands of MPs. Furthermore, the dry-coating remains intact during skin insertion, and then releases within the wet skin cellular environment within 3 min. Finally, we applied ovalbumin protein coated MP patches to immunise mice, achieving comparable antibody levels (p=0.08) with needle and syringe intramuscular injection. In summary, this paper presents a simple, practical and versatile method to achieve uniform coating on very small and densely packed MPs for a needle-free and targeted vaccine delivery technology.

Potent Immunity to Low Doses of Influenza Vaccine by Probabilistic Guided Micro-Targeted Skin Delivery in a Mouse Model

Background Over 14 million people die each year from infectious diseases despite extensive vaccine use [1]. The needle and syringe—first invented in 1853—is still the primary delivery device, injecting liquid vaccine into muscle. Vaccines could be far more effective if they were precisely delivered into the narrow layer just beneath the skin surface that contains a much higher density of potent antigen-presenting cells (APCs) essential to generate a protective immune response. We hypothesized that successful vaccination could be achieved this way with far lower antigen doses than required by the needle and syringe. Methodology/Principal Findings To meet this objective, using a probability-based theoretical analysis for targeting skin APCs, we designed the Nanopatch™, which contains an array of densely packed projections (21025/cm2) invisible to the human eye (110 µm in length, tapering to tips with a sharpness of <1000 nm), that are dry-coated with vaccine and applied to the skin for two minutes. Here we show that the Nanopatches deliver a seasonal influenza vaccine (Fluvax® 2008) to directly contact thousands of APCs, in excellent agreement with theoretical prediction. By physically targeting vaccine directly to these cells we induced protective levels of functional antibody responses in mice and also protection against an influenza virus challenge that are comparable to the vaccine delivered intramuscularly with the needle and syringe—but with less than 1/100th of the delivered antigen. Conclusions/Significance Our results represent a marked improvement—an order of magnitude greater than reported by others—for injected doses administered by other delivery methods, without reliance on an added adjuvant, and with only a single vaccination. This study provides a proven mathematical/engineering delivery device template for extension into human studies—and we speculate that successful translation of these findings into humans could uniquely assist with problems of vaccine shortages and distribution—together with alleviating fear of the needle and the need for trained practitioners to administer vaccine, e.g., during an influenza pandemic.

The effect of strain rate on the precision of penetration of short densely-packed microprojection array patches coated with vaccine

If skins non-linear viscoelastic properties are mechanically exploited for precise antigen placement, there is tremendous promise for improved vaccines. To achieve this, we designed a Nanopatch-densely packed micro-nanoprojections (>20,000/cm(2)) to directly deposit antigen to large numbers of epidermal Langerhans cells and dermal dendritic cells. Here, we controllably applied our Nanopatches with discrete conditions between peak strain rates of approximately 100 s(-1)-7000 s(-1) and quantified resulting penetration depths, delivery payloads and skin mechanics. Increasing the strain rate of application, we overcame key skin variability, achieving increases in both projection penetration depth (by over 50% length) and area coverage of a full array (from 50% to 100%). This delivery depth precision opens the way for more fully utilizing the skins immune function. Furthermore, we yielded new insights on mechanical behaviour of skin, including: 1) internal skin property changes that could affect/facilitate penetration; 2) projection design to dictate penetration depth; 3) puncture mechanics of skin in this strain rate range. Indeed, we show delivery of a model vaccine using our tested range of strain rates achieved functionally relevant tunable systemic antibody generation in mice. These findings could be of great utility in extending skin strata vaccine targeting approaches to human use.

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.

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.

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 skins 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 skins 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.

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.

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.

Novel coating of micro-nanoprojection patches for targeted vaccine delivery to skin

This study presents a simple, versatile and controllable method to achieve uniform coating on very small and densely packed micro-nanoprojections for epidermal immunotherapeutic delivery.