Microprojection array (MPA) patches are an attractive approach to selectively capture circulating proteins from the skin with minimal invasiveness for diagnostics at the point-of-care or in the home. ...A key challenge to develop this technology is to extract sufficient quantities of specific proteins from within the skin to enable high diagnostic sensitivity within a convenient amount of time. To achieve this, we investigated the effect of MPA geometry (i.e. projection density, length and array size) on protein capture. We hypothesised that the penetrated surface area of MPAs is a major determinant of protein capture however it was not known if simultaneously increasing projection density, length and array size is possible without adversely affecting penetration and/or tolerability. We show that increasing the projection density (5000–30,000 proj. cm−2) and array size (4–36 mm2) significantly increases biomarker capture whilst maintaining of a similar level tolerability, which supports previous literature for projection length (40–190 μm). Ultimately, we designed a high surface area MPA (30,000 proj. cm−2, 36 mm2, 140 μm) with a 4.5-fold increase in penetrated surface area compared to our standard MPA design (20,408 proj. cm−2, 16 mm2, 100 μm). The high surface area MPA captured antigen-specific IgG from mice in 30 s with 100% diagnostic sensitivity compared with 10–30 min for previous MPA immunoassay patches, which is over an order of magnitude reduction in wear time. This demonstrates for the first time that MPAs may be used for ultra-rapid (<1 min) protein capture from skin in a time competitive with standard clinical procedures like the needle and lancet, which has broad implications for minimally invasive and point-of-care diagnostics.
Abstract Microprojection array (MPA) skin patches selectively capture circulating biomarkers from the dermal layers of the skin, avoiding the need to extract, handle or process blood. Here we ...investigate the effect of improving biomarker capture in vivo on MPA detection of a model biomarker (antigen-specific-IgG raised in response to Fluvax vaccine) in a murine model. First, we investigate targeting MPA penetration to biomarker rich regions of the skin by varying MPA penetration depth. We observed a 4-fold increase in biomarker capture from predominantly epidermal to deep dermal penetration (27 ± 9 μm–153 ± 30 μm penetration range). We then study the kinetics of biomarker capture by varying the contact time with skin from rapid application (less than 20 min) to long term application (up to 24 h) with a wearable MPA patch. We observed MPAs reproducibly captured detectable amounts of our model biomarker after 10 min application and a greater than 6-fold increase in capture was observed up to 6 h application. Combining the effect of penetration depth and application time we obtained comparable early detection (after vaccination) of our model biomarker as a standard enzyme-linked immunosorbent assay (ELISA). We expect that integration of these devices with existing detection technologies has potential advantages in rapid diagnostic tests, particularly in cases where laboratory-based sample collection and processing is not available.
Vaccines delivered to the skin by microneedles—with and without adjuvants—have increased immunogenicity with lower doses than standard vaccine delivery techniques such as intramuscular or intradermal ...injection. However, the mechanisms underlying this skin-mediated “adjuvant” effect are not clear. Here, we show that the dynamic application of a microprojection array (the Nanopatch) to skin generates localized transient stresses invoking cell death around each projection. Nanopatch application caused significantly higher levels (∼65-fold) of cell death in murine ear skin than i.d. injection using a hypodermic needle. Measured skin cell death is associated with modeled stresses ∼1–10MPa. Nanopatch-immunized groups also yielded consistently higher anti-immunoglobulin G endpoint titers (up to 50-fold higher) than i.d. groups after delivery of a split virion influenza vaccine. Importantly, colocalization of cell death with nearby live skin cells and delivered antigen was necessary for immunogenicity enhancement. These results suggest a correlation between cell death caused by the Nanopatch with increased immunogenicity. We propose that the localized cell death serves as a “physical immune enhancer” for the adjacent viable skin cells, which also receive antigen from the projections. This natural immune enhancer effect has the potential to mitigate or replace chemical-based adjuvants in vaccines.
Abstract Surface modified microprojection arrays are a needle-free alternative to capture circulating biomarkers from the skin in vivo for diagnosis. The concentration and turnover of biomarkers in ...the interstitial fluid, however, may limit the amount of biomarker that can be accessed by microprojection arrays and ultimately their capture efficiency. Here we report that microprojection array insertion induces protein extravasation from blood vessels and increases the concentration of biomarkers in skin, which can synergistically improve biomarker capture. Regions of blood vessels in skin were identified in the upper dermis and subcutaneous tissue by multi-photon microscopy. Insertion of microprojection array designs with varying projection length (40–190 μm), density (5000–20,408 proj.cm−2 ) and array size (4–36 mm2 ) did not affect the degree of extravasation. Furthermore, the location of extravasated protein did not correlate with projection penetration to these highly vascularised regions, suggesting extravasation was not caused by direct puncture of blood vessels. Biomarker extravasation was also induced by dynamic application of flat control surfaces, and varied with the impact velocity, further supporting this conclusion. The extravasated protein distribution correlated well with regions of high mechanical stress generated during insertion, quantified by finite element models. Using this approach to induce extravasation prior to microprojection array-based biomarker capture, anti-influenza IgG was captured within a 2 min application time, demonstrating that extravasation can lead to rapid biomarker sampling and significantly improved microprojection array capture efficiency. These results have broad implications for the development of transdermal devices that deliver to and sample from the skin.
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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.
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.
Many types of microneedle (MN) arrays have been tested for delivery of vaccines to the skin. However, the effect of MN geometry/array design on antibody production is still unclear. Reports suggest ...that systemic immune responses may be affected by how MN arrays “mechanically” deliver vaccines, which can induce cell damage and act as an adjuvant. This includes parameters such as MN length/insertion depth, delivery energy/velocity, MN shape, and density. However, these effects have not been systematically investigated. Herein, the effect of MN density on antibody responses to influenza vaccination is assessed, keeping all other variables constant. MN arrays are manufactured within the “high‐density range” from 5 k microneedles cm−2 (n cm−2) to the “ultrahigh” 30 k n cm−2. Prior to this study, the highest MN density used for vaccination is 20 k n cm−2. Thus, MN array vaccination is evaluated over an unprecedented range. Cell viability is assessed in the skin after application and antibody responses at days 21/63. It is demonstrated that increasing MN density from 5 to 30 k n cm−2 increases both epidermal cell death and anti‐influenza IgG1, without an increase in anti‐influenza IgG2a. This suggests that MN density has a direct adjuvant effect on immune responses through Th2‐mediated signalling—a response critical for human vaccination.
“Physical adjuvantation”—a process caused by the mechanical disruption of tissue, inducing cell death and the release of immune stimulating danger signals during vaccination (similar to that of traditional skin scarification techniques)—is proposed herein to have been achieved by increasing microneedle array density during transdermal vaccination and was shown to increase IgG1 but not IgG2a antibody responses in mice.
Herein we demonstrate the use of a wearable device that can selectively capture two distinct circulating protein biomarkers (recombinant P. falciparum rPfHRP2 and total IgG) from the intradermal ...fluid of live mice in situ, for subsequent detection in vitro. The device comprises a microprojection array that, when applied to the skin, penetrates the outer skin layers to interface directly with intradermal fluid. Because of the complexity of the biological fluid being sampled, we investigated the effects of solution conditions on the attachment of capture antibodies, to optimize the assay detection limit both in vitro and on live mice. For detection of the target antigen diluted in 20% serum, immobilization conditions favoring high antibody surface density (low pH, low ionic strength) resulted in 100-fold greater sensitivity in comparison to standard conditions, yielding a detection limit equivalent to the plate enzyme-linked immunosorbent assay (ELISA). We also show that blocking the device surface to reduce nonspecific adsorption of target analyte and host proteins does not significantly change sensitivity. After injecting mice with rPfHRP2 via the tail vein, we compared analyte levels in both plasma and skin biopsies (cross-sectional area same as the microprojection array), observing that skin samples contained the equivalent of ∼8 μL of analyte-containing plasma. We then applied the arrays to mice, showing that surfaces coated with a high density of antibodies captured a significant amount of the rPfHRP2 target while the standard surface showed no capture in comparison to the negative control. Next, we applied a triplex device to both control and rPfHRP2-treated mice, simultaneously capturing rPfHRP2 and total IgG (as a positive control for skin penetration) in comparison to a negative control device. We conclude that such devices can be used to capture clinically relevant, circulating protein biomarkers of infectious disease via the skin, with potential applications as a minimally invasive and lab-free biomarker detection platform.
Immunoassays are ubiquitous across research and clinical laboratories, yet little attention is paid to the effect of the substrate material on the assay performance characteristics. Given the ...emerging interest in wearable immunoassay formats, investigations into substrate materials that provide an optimal mix of mechanical and bioanalytical properties are paramount. In the course of our research in developing wearable immunoassays which can penetrate skin to selectively capture disease antigens from the underlying blood vessels, we recently identified significant differences in immunoassay performance between gold and polycarbonate surfaces, even with a consistent surface modification procedure. We observed significant differences in PEG density, antibody immobilization, and nonspecific adsorption between the two substrates. Despite a higher PEG density formed on gold-coated surfaces than on amine-functionalized polycarbonate, the latter revealed a higher immobilized capture antibody density and lower nonspecific adsorption, leading to improved signal-to-noise ratios and assay sensitivities. The major conclusion from this study is that in designing wearable bioassays or biosensors, the design and its effect on the antifouling polymer layer can significantly affect the assay performance in terms of analytical specificity and sensitivity.
Selective capture of disease-related proteins in complex biological fluids and tissues is an important aim in developing sensitive protein biosensors for in vivo applications. Microprojection arrays ...are biomedical devices whose mechanical and chemical properties can be tuned to allow efficient penetration of skin, coupled with highly selective biomarker capture from the complex biological environment of skin tissue. Herein, the authors describe an improved surface modification strategy to produce amine-modified polycarbonate arrays, followed by the attachment of an antifouling poly(sulfobetaine-methacrylate) (pSBMA) polymer or a linear polyethylene glycol (PEG) polymer of comparative molecular weight and hydrodynamic radius. Using a "grafting to" approach, pSBMA and linear PEG coatings yielded comparative antifouling behavior in single protein solutions, diluted plasma, or when applied to mouse flank skin penetrating into the vascularized dermal tissue. Interestingly, the density of immobilized immunoglobulin G (IgG) or bovine serum albumin protein on pSBMA surfaces was significantly higher than that on the PEG surfaces, while the nonspecific adsorption was comparable for each protein. When incubated in buffer or plasma solutions containing dengue non-structural protein 1 (NS1), anti-NS1-IgG-coated pSBMA surfaces captured significantly more NS1 in comparison to PEG-coated devices. Similarly, when wearable microprojection arrays were applied to the skin of dengue-infected mice using the same coatings, the pSBMA-coated devices showed significantly higher capture efficiency (>2-fold increase in signal) than the PEG-coated substrates, which showed comparative signal when applied to naïve mice. In conclusion, zwitterionic pSBMA polymers (of equivalent hydrodynamic radii to PEG) allowed detection of dengue NS1 disease biomarker in a preclinical model of dengue infection, showing significantly higher signal-to-noise ratio in comparison to the PEG controls. The results of this study will be useful in the future development of a range of protein biosensors designed for use in vivo.
Vaccines delivered to the skin by microneedles – with and without adjuvants – have increased immunogenicity with lower doses than standard vaccine delivery techniques such as intramuscular (i.m.) or ...intradermal (i.d.) injection. However, the mechanisms behind this skin-mediated ‘adjuvant’ effect are not clear. Here, we show that the dynamic application of a microprojection array (the Nanopatch) to skin generates localized transient stresses invoking cell death around each projection. Nanopatch application caused significantly higher levels (~65-fold) of cell death in murine ear skin than i.d. injection using a hypodermic needle. Measured skin cell death is associated with modeled stresses ~1–10 MPa. Nanopatch-immunized groups also yielded consistently higher anti-IgG endpoint titers (up to 50-fold higher) than i.d. groups after delivery of a split virion influenza vaccine. Importantly, co-localization of cell death with nearby live skin cells and delivered antigen was necessary for immunogenicity enhancement. These results suggest a correlation between cell death caused by the Nanopatch with increased immunogenicity. We propose that the localized cell death serves as a ‘physical immune enhancer’ for the adjacent viable skin cells, which also receive antigen from the projections. This natural immune enhancer effect has the potential to mitigate or replace chemical-based adjuvants in vaccines.
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