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Research Techniques Made Simple: Skin-Targeted Drug and Vaccine Delivery Using Dissolvable Microneedle Arrays

  • Author Footnotes
    6 These authors contributed equally to this work.
    Stephen C. Balmert
    Footnotes
    6 These authors contributed equally to this work.
    Affiliations
    Department of Dermatology, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, USA
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  • Author Footnotes
    6 These authors contributed equally to this work.
    Zohreh Gholizadeh Ghozloujeh
    Footnotes
    6 These authors contributed equally to this work.
    Affiliations
    Department of Dermatology, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, USA
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  • Cara Donahue Carey
    Affiliations
    Department of Dermatology, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, USA
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  • Oleg E. Akilov
    Affiliations
    Department of Dermatology, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, USA
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  • Author Footnotes
    7 These authors contributed equally to this work.
    Emrullah Korkmaz
    Footnotes
    7 These authors contributed equally to this work.
    Affiliations
    Department of Dermatology, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, USA

    Department of Bioengineering, Swanson School of Engineering, University of Pittsburgh, Pittsburgh, Pennsylvania, USA
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  • Author Footnotes
    7 These authors contributed equally to this work.
    Louis D. Falo Jr.
    Correspondence
    Correspondence: Louis D. Falo Jr, Department of Dermatology, School of Medicine, University of Pittsburgh, Thomas E. Starzl Biomedical Science Tower, 200 Lothrop Street, Suite W1152, Pittsburgh, Pennsylvania 15261, USA.
    Footnotes
    7 These authors contributed equally to this work.
    Affiliations
    Department of Dermatology, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, USA

    Department of Bioengineering, Swanson School of Engineering, University of Pittsburgh, Pittsburgh, Pennsylvania, USA

    Clinical and Translational Science Institute, University of Pittsburgh, Pittsburgh, Pennsylvania, USA

    The UPMC Hillman Cancer Center, Pittsburgh, Pennsylvania, USA

    The McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, USA
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  • Author Footnotes
    6 These authors contributed equally to this work.
    7 These authors contributed equally to this work.
      Skin-targeted drug delivery is broadly employed for both local and systemic therapeutics and is an important tool for discovery efforts in cutaneous biology. Recently, emerging technologies support efforts toward skin-targeted biocargo delivery for local and systemic therapeutic benefit. Effective targeting of bioactive molecules, including large (molecular weight > 500 Da) or complex (hydrophilic and charged) molecules, to defined cutaneous microenvironments is intrinsically challenging owing to the protective barrier function of the skin. Dissolvable microneedle arrays (MNAs) have proven to be a promising technology to address the unmet need for controlled, minimally invasive, and reliable delivery of a wide range of biocargos to the skin. In this paper, we describe the unique properties of the skin that make it an attractive target for vaccine delivery, for immune-modulating therapies, and for systemic drug delivery and the structural characteristics of the skin that present obstacles to efficient intracutaneous and transdermal delivery of bioactive molecules. We provide an overview of MNA fabrication and the characteristics and mechanisms of dissolvable MNA cargo delivery to the cutaneous microenvironment. We present a representative example of a clinical application of MNAs and discuss future directions for MNA development and applications.

      Abbreviations:

      3D (three-dimensional), APC (antigen-presenting cell), CMC (carboxymethylcellulose), CTCL (cutaneous T-cell lymphoma), DOX (doxorubicin), MNA (microneedle array), OCT (optical coherence tomography), TEWL (transepidermal water loss)

      Summary Points

      • Skin offers a readily accessible target for painless biocargo delivery for either local or systemic applications.
      • Skin-targeted delivery of bioactive macromolecules has long been challenging owing to the protective characteristics of superficial cutaneous layers and the lack of safe, convenient, inexpensive, and effective skin-targeted biocargo delivery systems.
      • Dissolvable microneedle arrays (MNAs) are an emerging, clinically viable technology that enables efficient and reproducible skin-targeted delivery of a broad range of vaccine components and therapeutics by mechanically piercing the superficial cutaneous layers in a minimally invasive fashion.
      • Transepidermal water loss and skin impedance measurements as well as optical coherence tomography are noninvasive tools that are commonly used to evaluate breaching of the skin barrier by microneedles and its subsequent recovery kinetics.
      • Dissolving MNAs, manufactured from biomaterials suggested by the Food and Drug Administration, are safe for human use, with potential advantages in terms of delivery with spatial and temporal control, improved thermostability and logistics, reduced medical waste, enhanced efficacy, and patient acceptability for some applications compared with other drug delivery methods.

      Limitations

      • Relatively low-dose capacity for systemic administration.
      • Relatively long administration time compared with pills and conventional parenteral injections.
      • Lack of feedback for consistent delivery confirmation.

      Introduction

      Intracutaneous and transdermal routes of administration aim to deliver biomolecules to targeted skin microenvironments or the systemic circulation, respectively. Regardless of the ultimate target (i.e., local or systemic administration), the outermost cutaneous layer, or stratum corneum, poses substantial challenges for simple, precise, reproducible, and efficient skin-targeted delivery of biocargos, especially those that are complex, charged, or large (
      • Prausnitz M.R.
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      Transdermal drug delivery.
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      Intradermal and transdermal drug delivery using microneedles - fabrication, performance evaluation and application to lymphatic delivery.
      ;
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      Drug delivery through the skin: molecular simulations of barrier lipids to design more effective noninvasive dermal and transdermal delivery systems for small molecules, biologics, and cosmetics.
      ). Although biocargos with certain physical and chemical characteristics (e.g., molecular weight < 500 Da and sufficient lipophilicity) may penetrate intact skin passively when applied topically or by conventional transdermal patches, inconsistencies due to variations in skin properties within and between individuals can introduce variability in delivery. A large number of therapeutics and vaccine components require chemical or physical skin permeability enhancers to facilitate reproducible and efficient delivery of these biomolecules to viable cutaneous microenvironments to achieve sufficient bioavailability (
      • Prausnitz M.R.
      • Langer R.
      Transdermal drug delivery.
      ;
      • Sabri A.H.
      • Kim Y.
      • Marlow M.
      • Scurr D.J.
      • Segal J.
      • Banga A.K.
      • et al.
      Intradermal and transdermal drug delivery using microneedles - fabrication, performance evaluation and application to lymphatic delivery.
      ;
      • Torin Huzil J.
      • Sivaloganathan S.
      • Kohandel M.
      • Foldvari M.
      Drug delivery through the skin: molecular simulations of barrier lipids to design more effective noninvasive dermal and transdermal delivery systems for small molecules, biologics, and cosmetics.
      ).
      Dissolvable microneedle arrays (MNAs) are a clinically feasible, patient-friendly platform technology for safe, precise, and consistent cutaneous and transdermal biocargo delivery (
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      Trends of microneedle technology in the scientific literature, patents, clinical trials and internet activity.
      ;
      • Korkmaz E.
      • Balmert S.C.
      • Sumpter T.L.
      • Carey C.D.
      • Erdos G.
      • Falo Jr., L.D.
      Microarray patches enable the development of skin-targeted vaccines against COVID-19.
      ;
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      • Paine M.
      • Mosley R.
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      • Kalluri H.
      • et al.
      The safety, immunogenicity, and acceptability of inactivated influenza vaccine delivered by microneedle patch (TIV-MNP 2015): a randomised, partly blinded, placebo-controlled, phase 1 trial.
      ). Dissolvable MNAs are typically fabricated from water-soluble biomaterials to incorporate drug or vaccine components and contain many sharp, micron-scale projections integrated with a backing layer or substrate, forming ready-to-use, potentially self-administered dry-dosage units (
      • Balmert S.C.
      • Carey C.D.
      • Falo G.D.
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      • Erdos G.
      • Korkmaz E.
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      Dissolving undercut microneedle arrays for multicomponent cutaneous vaccination.
      ;
      • Korkmaz E.
      • Balmert S.C.
      • Carey C.D.
      • Erdos G.
      • Falo Jr., L.D.
      Emerging skin-targeted drug delivery strategies to engineer immunity: a focus on infectious diseases.
      ,
      • Korkmaz E.
      • Balmert S.C.
      • Sumpter T.L.
      • Carey C.D.
      • Erdos G.
      • Falo Jr., L.D.
      Microarray patches enable the development of skin-targeted vaccines against COVID-19.
      ;
      • Lee J.W.
      • Park J.H.
      • Prausnitz M.R.
      Dissolving microneedles for transdermal drug delivery.
      ). Geometric and material properties of MNAs are designed to achieve optimal and painless skin-targeted biocargo delivery as well as to retain the bioactivity of integrated molecules (
      • Korkmaz E.
      • Balmert S.C.
      • Carey C.D.
      • Erdos G.
      • Falo Jr., L.D.
      Emerging skin-targeted drug delivery strategies to engineer immunity: a focus on infectious diseases.
      ;
      • Makvandi P.
      • Kirkby M.
      • Hutton A.R.J.
      • Shabani M.
      • Yiu C.K.Y.
      • Baghbantaraghdari Z.
      • et al.
      Engineering microneedle patches for improved penetration: analysis, skin models and factors affecting needle insertion.
      ;
      • Mistilis M.J.
      • Joyce J.C.
      • Esser E.S.
      • Skountzou I.
      • Compans R.W.
      • Bommarius A.S.
      • et al.
      Long-term stability of influenza vaccine in a dissolving microneedle patch.
      ). Dissolving MNAs can be applied to the skin simply by providing sufficient pressure to their backing layer either manually or using an external applicator device. Microneedles reproducibly breach the stratum corneum in a minimally invasive fashion, rapidly dissolve in the aqueous skin microenvironment, and actively deliver their contents to defined skin layers (
      • Balmert S.C.
      • Carey C.D.
      • Falo G.D.
      • Sethi S.K.
      • Erdos G.
      • Korkmaz E.
      • et al.
      Dissolving undercut microneedle arrays for multicomponent cutaneous vaccination.
      ;
      • Lee J.W.
      • Park J.H.
      • Prausnitz M.R.
      Dissolving microneedles for transdermal drug delivery.
      ;
      • Sullivan S.P.
      • Koutsonanos D.G.
      • del Pilar Martin M.
      • Lee J.W.
      • Zarnitsyn V.
      • Choi S.O.
      • et al.
      Dissolving polymer microneedle patches for influenza vaccination.
      ). Besides dissolvable MNAs, other types of MNAs, such as solid, hydrogel-forming, coated, porous, and hollow MNAs, each employing different delivery mechanisms, have been utilized to enhance the permeability of the skin to a broad range of biomolecules (
      • Amani H.
      • Shahbazi M.A.
      • D'Amico C.
      • Fontana F.
      • Abbaszadeh S.
      • Santos H.A.
      Microneedles for painless transdermal immunotherapeutic applications.
      ;
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      3D-printed microneedles in biomedical applications.
      ;
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      • Balmert S.C.
      • Sumpter T.L.
      • Carey C.D.
      • Erdos G.
      • Falo Jr., L.D.
      Microarray patches enable the development of skin-targeted vaccines against COVID-19.
      ;
      • Paredes A.J.
      • McKenna P.E.
      • Ramöller I.K.
      • Naser Y.A.
      • Volpe-Zanutto F.
      • Li M.
      • et al.
      Microarray patches: poking a hole in the challenges faced when delivering poorly soluble drugs.
      ). Owing to recent progress in this field, microneedles have been identified as one of the top 10 emerging technologies of 2020 by the World Economic Forum, thereby supporting their potential for clinical deployment for several applications in the coming years.
      In this paper, we briefly discuss the key characteristics of the skin that render intracutaneous and transdermal administration of several biocargos challenging while making the skin microenvironment an ideal target for vaccination and immune engineering approaches. We describe important features of dissolvable MNAs and associated design principles and fabrication methods. We also present fundamental aspects of skin-targeted biocargo delivery using dissolvable MNAs and the biophysical tools commonly used to investigate dissolvable MNA‒based biocargo delivery to the skin. In addition, we provide an overview of applications of MNAs and show a clinical application of dissolvable MNAs for targeted delivery of a chemotherapy drug, doxorubicin (DOX), for treatment of cutaneous T-cell lymphoma (CTCL) lesions (ClinicalTrials.gov Identifier: NCT02192021). Finally, we conclude with our perspective on future directions for MNA technology.

      Structural and functional characteristics of the skin

      The skin is a sophisticated organ that integrates multifunctional structural and immune cells, a complex lymphatic system, and a pervasive vascular network. Besides its crucial protective, sensory, and thermoregulatory roles, the skin is an active immunological organ, communicating with local lymph nodes to drive the induction of adaptive immune responses, and provides a means for systemic delivery of biocargos owing to its easy accessibility and microvasculature system. These important characteristics of the skin make the viable cutaneous layers an attractive target for localized or systemic biocargo delivery. Despite these opportunities, skin-targeted delivery is challenging owing to the inherently protective outer cutaneous layers that hinder realization of the full potential of the skin for systemic and local delivery of bioactive molecules.
      The cutaneous layers that play key barrier and physiological roles that affect skin-targeted biocargo delivery strategies are depicted in Figure 1. The stratum corneum constitutes a physical barrier to environmental threats. Intrinsically, this mechanical layer also precludes the reliable delivery of complex and large molecules to intact skin. The deeper layers of the epidermis form a viable microenvironment that houses multifaceted structural and immune cells. The dermal layer supports the epidermis and harbors the cutaneous microvasculature, immune-accessory cells, and antigen-presenting cells (APCs). Hence, overcoming the stratum corneum barrier provides reliable access to viable skin layers for the treatment of local skin disorders, cutaneous vaccination, and systemic delivery of biocargos. However, traditional topical applications with creams, ointments, or conventional transdermal patches fail to provide access to viable cutaneous layers for a high percentage of therapeutics and vaccines owing to their reliance on passive diffusion through intact skin. Therefore, the true potential of transdermal and intracutaneous routes has yet to be realized because of the limitations of prevailing skin-targeted delivery strategies, necessitating improved methods for the administration of biocargos to targeted skin microenvironments. Accordingly, recent years have witnessed major advances in skin-targeted vaccine and drug delivery systems to more efficiently exploit the cutaneous and transdermal routes.
      Figure thumbnail gr1
      Figure 1Transdermal and intracutaneous delivery of bioactive molecules. The skin is an obvious target for local treatment of cutaneous disorders, with minimal systemic exposure. Moreover, the skin microenvironment, which is an immunologically rich site and harbors a pervasive vascular network, offers an attractive target for vaccination and immunotherapy to harness systemic immunity as well as for delivery of biocargos to the systemic circulation. The superficial cutaneous layers, especially the stratum corneum, pose significant challenges for transdermal and intracutaneous administration of bioactive molecules, necessitating patient-friendly strategies to consistently breach these skin layers for reproducible and effective skin-targeted biocargo delivery. Image was created with biorender.com.

      Dissolvable MNAs

      Dissolvable MNAs are physical, single-unit, skin-targeted biocargo delivery platforms that can both preserve the embedded biocargo(s) and mechanically penetrate the superficial cutaneous layers in a minimally invasive fashion to actively deposit their contents in the viable skin microenvironment. Biocargo-loaded dissolvable MNAs, which combine drugs or biologics into a device as a single entity, are typically defined as combination products by regulatory agencies. Figure 2 shows a three-dimensional (3D) drawing of a representative dissolvable MNA platform that consists of an array of tip-loaded microscale needles integrated with a backing substrate or layer (Figure 2a) and illustrates the diverse microneedle shapes used for various biocargo delivery applications (Figure 2b). Several design parameters (Figure 2c) are chosen to achieve optimal skin penetration of the maximum number of microneedles without mechanical failure and subsequent efficient dissolution of microneedles in the cutaneous microenvironment as well as functional preservation of the integrated biocargos, especially for an extended period of time without refrigeration. These design parameters, which control the biocargo delivery performance of dissolvable MNAs, include microneedle dimensions (e.g., tip radius, width/diameter, height, taper or apex angle, and fillet radius) and material as well as the MNA patch size and the density of needles across an MNA patch. For instance, sharper tips decrease the skin penetration forces, and fillets at the bases of needles reduce stress concentrations, improving the mechanical performance of microneedles and, in turn, the penetration and delivery efficiencies of MNAs (
      • Balmert S.C.
      • Carey C.D.
      • Falo G.D.
      • Sethi S.K.
      • Erdos G.
      • Korkmaz E.
      • et al.
      Dissolving undercut microneedle arrays for multicomponent cutaneous vaccination.
      ;
      • Faraji Rad Z.
      • Nordon R.E.
      • Anthony C.J.
      • Bilston L.
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      High-fidelity replication of thermoplastic microneedles with open microfluidic channels.
      ). Similarly, too closely spaced, short, and/or blunt microneedles, when applied manually, often fail to overcome the viscoelasticity of the skin owing to the bed of nails effect, resulting in the lack of skin penetration, or necessitating an external applicator device for successful penetration (
      • Makvandi P.
      • Kirkby M.
      • Hutton A.R.J.
      • Shabani M.
      • Yiu C.K.Y.
      • Baghbantaraghdari Z.
      • et al.
      Engineering microneedle patches for improved penetration: analysis, skin models and factors affecting needle insertion.
      ). Furthermore, poor material choices may result in MNAs that require extended application times for sufficient dissolution of microneedles or compromise biocargo viability during storage. In addition, the temperature stability of dissolvable MNA-integrated biocargos depends on the preservative capabilities of the structural material that can be enriched with different cargo stabilizers, such as trehalose or sucrose. Ultimately, reliability, reproducibility, and efficiency of skin insertion and biocargo administration by dissolvable MNAs are controlled by these microneedle and array design parameters. As such, geometric and biomaterial parameters of MNAs should be engineered to ensure effective and reproducible skin delivery and stability of specific cargos while considering the challenges of large-scale manufacturability of the selected MNA designs.
      Figure thumbnail gr2
      Figure 2Design parameters that are engineered for the development of dissolvable MNA‒based biocargo delivery systems. Blue color indicates the structural material of MNAs, and other colors indicate biocargos that are typically mixed with different biomaterials to ensure the mechanical integrity of microneedles and thermostability of MNA-embedded cargos. (a) Three-dimensional CAD drawing of an MNA device with a predetermined patch size (e.g., 1 cm × 1 cm) and needle density (tip-to-tip distance and the total number of needles [e.g., 10 × 10 = 100]). (b) Various microneedle shapes and biocargo-loading strategies that are used for engineering dissolvable MNA‒based skin-targeted delivery systems. Single or multiple biocargo(s) can be integrated throughout the whole needles or at the tip region of microneedles. When multiple biocargos are loaded, they can be either mixed or separated in a layered fashion. (c) Geometric parameters of individual microneedles, including needle height and width/diameter, taper angle, and fillet radii at the needle bases. In addition to these parameters, the material choice and the interaction between biocargo and structural material affect the mechanical integrity and dissolution behavior of needles. Tip sharpness of needles is dictated by several factors, including the resolution of the manufacturing process used to fabricate master MNAs, the nature of the micromolding methods utilized, and the initial concentration and drying characteristics of the water-soluble structural material, with or without biocargo(s). CAD, computer-aided design; MNA, microneedle array.
      Several manufacturing strategies have been devised to create dissolvable MNAs with diverse design parameters (
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      ). Although these strategies exploit various techniques, they typically involve common processing steps that include (i) production of MNA master molds that comprise microneedle-shaped protrusions, with predefined needle and array geometries, using a suitable micromanufacturing method; (ii) generation of MNA production molds that consist of microneedle-shaped wells through soft lithography or other molding strategies; and (iii) casting of water-soluble structural material and biocargo(s) into MNA production molds to manufacture mechanically strong dissolvable MNAs integrating single or multiple bioactive cargos. During these processes, the biomaterials interacting with biocargos and the process conditions used to fill MNA production molds and dry cargos and structural materials are chosen carefully to fabricate high-quality biocargo-loaded dissolvable MNAs with mechanical integrity as well as to retain the viability of MNA-embedded biocargos. Importantly, establishing standard operating procedures is essential for the fabrication and quality control of biocargo-loaded dissolvable MNAs for clinical applications.
      Figure 3 shows the representative optical stereomicroscopy images of master MNA molds produced using 3D microadditive fabrication or 3D printing in microscale (Figure 3a and b); elastomer MNA production molds fabricated through soft lithography or micromolding (Figure 3c and d); single or multiple cargo-loaded dissolvable MNAs created using different solvent-based spin-casting strategies through centrifugation (Figure 3e and f); and a 20 × 20 (400 microneedles) MNA manufactured from carboxymethylcellulose (CMC), a water-soluble biomaterial that is considered safe by the United States Food and Drug Administration (Figure 3g).
      Figure thumbnail gr3
      Figure 3Representative products from the distinct processing steps of a traditional three-stage MNA manufacturing strategy toward engineering cargo-loaded, dissolvable MNAs. (a, b) Optical stereomicroscopy images of additively manufactured master MNAs with (a) square obelisk or (b) undercut microneedles with filleted bases. (c, d) MNA production molds with (c) square obelisk and (d) undercut needle-shaped wells. (e, f) Dissolving MNAs with (e) square obelisk and (f) undercut microneedles incorporating multiple or single cargos, respectively. Bars = 250 μm. (g) A representative 20 × 20 dissolvable MNA created from CMC, a biomaterial designated as Generally Recognized as Safe by the FDA. CMC, carboxymethylcellulose; FDA, Food and Drug Administration; MNA, microneedle array.

      Dissolving MNA‒directed drug delivery

      Dissolvable MNAs not only increase the permeability of the skin by physically breaching the superficial cutaneous layers but also precisely deliver their contents to defined skin microenvironments. The basic mechanism of skin-targeted cargo delivery with dissolving microneedles relies on mechanical and painless penetration of the outermost skin layers to generate transient microchannels and subsequent dissolution of microneedles with embedded biocargos in the targeted cutaneous microenvironment. Because dissolvable microneedles breach the outermost skin layers, they can effectively deliver a broad range of bioactive compounds, including proteins, hydrophobic and hydrophilic small-molecule agents, and even recombinant viral vectors (
      • Amani H.
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      Microneedles for painless transdermal immunotherapeutic applications.
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      ). Application-driven optimization of the geometry and materials of dissolving MNAs affords the capacity to achieve spatially and temporally controlled skin-targeted delivery of vaccine components and therapeutics in a reproducible manner. As such, MNAs can be used for effective and reproducible delivery of a myriad of bioactive molecules for different applications, such as in the treatment of cutaneous diseases (e.g., acne vulgaris, alopecia, and psoriasis), intracutaneous vaccination, management of diabetes, and immunotherapy for skin cancer (
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      Psoriasis-associated impairment of CCL27/CCR10-derived regulation leads to IL-17A/IL-22–producing skin T-cell overactivation.
      ;
      • Wan T.
      • Pan Q.
      • Ping Y.
      Microneedle-assisted genome editing: a transdermal strategy of targeting NLRP3 by CRISPR-Cas9 for synergistic therapy of inflammatory skin disorders.
      ;
      • Wang C.
      • Ye Y.
      • Hochu G.M.
      • Sadeghifar H.
      • Gu Z.
      Enhanced cancer immunotherapy by microneedle patch-assisted delivery of anti-PD1 antibody.
      ;
      • Yang G.
      • Chen Q.
      • Wen D.
      • Chen Z.
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      • et al.
      A therapeutic microneedle patch made from hair-derived keratin for promoting hair regrowth.
      ).
      Figure 4 shows the fundamental mechanism of skin-targeted delivery with dissolvable MNAs. All experiments with C57BL/6 mice were conducted in accordance with institutional animal care and use committee guidelines at the University of Pittsburgh (Pittsburgh, PA). Protective cutaneous barriers can be seen in histology images from intact murine and human skin before MNA application (Figure 4a and b). After MNA application, representative histology images show needle traces in murine skin (Figure 4c; single-needle trace) and in human skin (Figure 4d; two-needle traces), demonstrating that dissolvable MNAs physically disrupt the superficial skin barriers and create microscale pathways to enable reproducible and effective delivery of a myriad of compounds. These MNAs typically dissolve in the aqueous skin microenvironment within minutes, and their dissolution behavior can be modified by altering their structural material. In addition, cutaneous delivery of dry cargos by MNAs typically improves skin residence compared with intradermal injection of liquid biocargos (
      • Zhao X.
      • Birchall J.C.
      • Coulman S.A.
      • Tatovic D.
      • Singh R.K.
      • Wen L.
      • et al.
      Microneedle delivery of autoantigen for immunotherapy in type 1 diabetes.
      ). Figure 4e and f show a representative optical stereomicroscopy image of multiple cargo-loaded dissolving MNAs before and 5 minutes after application to freshly excised human skin, respectively, revealing nearly complete dissolution of microneedles. Figure 4g shows microneedle traces on the top surface of human skin after application of a multiple cargo-loaded dissolvable MNA, and Figure 4h shows the merged bright-field and fluorescent images of the cryosectioned human skin explant after MNA application, with the delivery of multiple cargos in two distinct microneedle tracks.
      Figure thumbnail gr4
      Figure 4Enhancing skin-targeted cargo delivery using dissolvable MNAs. Bright-field microscopy images of H&E-stained sections of intact (a) mouse ear and (b) human skin. Bright-field microscopy images H&E-stained tissue sections of (c) mouse ear and (d) human skin after dissolving MNA application. Bars = 100 μm. (e, f) Optical stereomicroscopy images of dissolvable MNAs loaded with multiple fluorescently labeled cargos (AF555-labeled virus and AF647-labeled protein) and created from a mixture of two biocompatible materials, carboxymethylcellulose and trehalose, (e) before and (f) after application to freshly excised human skin. Bars = 250 μm. (g) Optical stereomicroscopy image of the top surface of human skin after application of a multiple cargo-loaded dissolvable MNA. Bar = 1 mm. (h) Merged bright-field and fluorescent images of the cryosectioned human skin explant after MNA application, showing delivery of AF555-labeled virus and AF647-labeled protein in two distinct parallel microneedle tracks. Bar = 250 μm. (i) TEWL measurements (mean ± SEM) after application of blank (no cargo) dissolvable MNAs to the abdominal skin of live C57BL/6 mice (n = 5). h, hour; MNA, microneedle array; TEWL, transepidermal water loss.
      Several biophysical methods, such as transepidermal water loss (TEWL), skin impedance measurement, and optical coherence tomography (OCT), have been used to noninvasively evaluate MNA-based cutaneous penetration (
      • Enfield J.
      • O'Connell M.L.
      • Lawlor K.
      • Jonathan E.
      • O'Mahony C.
      • Leahy M.J.
      In-vivo dynamic characterization of microneedle skin penetration using optical coherence tomography.
      ;
      • Ogunjimi A.T.
      • Carr J.
      • Lawson C.
      • Ferguson N.
      • Brogden N.K.
      Micropore closure time is longer following microneedle application to skin of color.
      ). TEWL quantifies the amount of water that evaporates through the skin as an indicator of skin barrier integrity (
      • Alexander H.
      • Brown S.
      • Danby S.
      • Flohr C.
      Research techniques made simple: transepidermal water loss measurement as a research tool.
      ) and is commonly used to confirm both the generation of micropores by microneedles and the recovery of the skin barrier after MNA application. Representative TEWL measurements from an in vivo experiment with mice that received blank (no biocargo) dissolving MNAs on their abdomens are shown in Figure 4i. The results indicate the expected rapid increase in TEWL after the application of dissolving MNAs, compared with the baseline values before MNA application and followed by a return to baseline values a few hours after MNA application. Thus, this representative TEWL analysis confirms the transient generation of microscale channels by applied dissolving MNAs across the superficial skin layers and the rapid recovery of skin barrier properties. Skin impedance measurements and OCT analysis are alternative methods used to monitor cutaneous barrier characteristics. Disruption of the skin barrier results in a decrease in electrical impedance, compared with that of the intact skin, and thus, skin impedance measurements can be used as a noninvasive approach to evaluate transient breaching of the protective cutaneous layers and their subsequent recovery after MNA applications (
      • Ogunjimi A.T.
      • Carr J.
      • Lawson C.
      • Ferguson N.
      • Brogden N.K.
      Micropore closure time is longer following microneedle application to skin of color.
      ). OCT is a noninvasive imaging technology that can be used to visualize cutaneous layers, not only to monitor breaching of the top skin layers but also to measure the penetration depth of microneedles after skin application (
      • Enfield J.
      • O'Connell M.L.
      • Lawlor K.
      • Jonathan E.
      • O'Mahony C.
      • Leahy M.J.
      In-vivo dynamic characterization of microneedle skin penetration using optical coherence tomography.
      ). Ultimately, TEWL and skin impedance measurements as well as OCT analysis are noninvasive, complementary tools that have been successfully used to monitor skin barrier breaches by MNAs and subsequent barrier recovery kinetics in human subjects in research studies (
      • Enfield J.
      • O'Connell M.L.
      • Lawlor K.
      • Jonathan E.
      • O'Mahony C.
      • Leahy M.J.
      In-vivo dynamic characterization of microneedle skin penetration using optical coherence tomography.
      ;
      • Gupta J.
      • Gill H.S.
      • Andrews S.N.
      • Prausnitz M.R.
      Kinetics of skin resealing after insertion of microneedles in human subjects.
      ;
      • Ogunjimi A.T.
      • Carr J.
      • Lawson C.
      • Ferguson N.
      • Brogden N.K.
      Micropore closure time is longer following microneedle application to skin of color.
      ); however, their widespread adoption for dissolving MNA‒based clinical immunization and treatment strategies is problematic. Thus, simple and standard methods that can rapidly confirm skin penetration and effective delivery of MNA biocargos from dissolvable MNAs will be needed, especially for self-administration of dissolving MNAs at home.

      Translational application of dissolvable MNAs for the treatment of skin cancers

      The simplicity of dissolving MNA fabrication, ease of incorporating various types of drugs into dissolving MNAs, and thermostability of MNA‒embedded biocargos make this technology an exciting candidate for the development of rapidly translatable therapies and immunization strategies. In addition to vaccine applications, we are currently using dissolving MNAs to deliver a chemotherapy drug, DOX, to malignant cells in the skin microenvironment in an investigator-initiated phase 1 clinical trial for patients with treatment-refractory CTCL (ClinicalTrials.gov Identifier: NCT02192021). DOX is an immunogenic cell death‒inducing chemotherapeutic that has been shown to result in innate immune activation, including the attraction and activation of APCs, and a cell death process that facilitates the internalization and processing of dying tumor cell derivatives (
      • Galluzzi L.
      • Buqué A.
      • Kepp O.
      • Zitvogel L.
      • Kroemer G.
      Immunogenic cell death in cancer and infectious disease.
      ). Thus, DOX can both kill tumor cells and induce a proinflammatory microenvironment conducive to the induction of tumor-specific immune responses. The current trial utilizes dissolvable MNAs fabricated from CMC for targeted delivery of DOX and is designed as a single-arm, placebo-controlled (within-patient), open-label, dose-escalation study to determine the maximum tolerated dose of dissolving MNA‒delivered DOX. Regulatory requirements relevant to dissolving MNA delivery in general include reproducibility and consistency of (i) MNA structure, including both needle and patch geometries; (ii) cargo loading and integrity; and (iii) needle dissolution and drug delivery. Dosing from 25 μg to 200 μg per MNA is being evaluated over 4-weekly administrations. Figure 5 shows a representative clinical application of DOX-loaded MNAs, illustrating that MNA placement is accomplished simply by applying pressure with the fingertip, without the use of an external applicator device (Figure 5a and b). MNA dissolution and drug delivery are rapid, occurring within minutes and immediately after MNA placement (Figure 5c); drug delivery is evident by dermatoscopic evaluation of the MNA application site, which reveals the distribution of red-colored DOX in a characteristic geometric pattern corresponding to microneedle distribution over the MNA surface (Figure 5d). In this phase 1 study, systemic and local toxicity are being evaluated as primary outcome measures. It is anticipated that the low doses of DOX administered by MNAs will not result in systemic toxicity; however, local effects, including acute inflammatory reactions, are expected, and extensive monitoring is included to identify potential skin toxicities, should they develop.
      Figure thumbnail gr5
      Figure 5Dissolvable MNA‒based delivery of the chemotherapeutic drug DOX to the tumor microenvironment in patients with treatment-refractory cutaneous T-cell lymphoma. (a) DOX-loaded MNA that is being placed by a healthcare provider for the application. (b) Application of DOX-loaded MNAs by providing pressure with the fingertip. (c) Skin image after DOX-loaded MNA application. (d) Dermatoscopic evaluation of the DOX MNA application site showing the distribution of DOX (inherently red-colored drug) in a characteristic geometric pattern corresponding to that of the array of microneedles. DOX, doxorubicin; MNA, microneedle array.
      Besides their broad utilization for cutaneous vaccination and skin cancer immunotherapy, MNAs are being evaluated for several other applications in preclinical and clinical studies as comprehensively reviewed recently (
      • Amani H.
      • Shahbazi M.A.
      • D'Amico C.
      • Fontana F.
      • Abbaszadeh S.
      • Santos H.A.
      Microneedles for painless transdermal immunotherapeutic applications.
      ;
      • Guillot A.J.
      • Cordeiro A.S.
      • Donnelly R.F.
      • Montesinos M.C.
      • Garrigues T.M.
      • Melero A.
      Microneedle-based delivery: an overview of current applications and trends.
      ;
      • Halder J.
      • Gupta S.
      • Kumari R.
      • Gupta G.D.
      • Rai V.K.
      Microneedle array: applications, recent advances, and clinical pertinence in transdermal drug delivery.
      ;
      • Ingrole R.S.J.
      • Azizoglu E.
      • Dul M.
      • Birchall J.C.
      • Gill H.S.
      • Prausnitz M.R.
      Trends of microneedle technology in the scientific literature, patents, clinical trials and internet activity.
      ;
      • Jeong S.Y.
      • Park J.H.
      • Lee Y.S.
      • Kim Y.S.
      • Park J.Y.
      • Kim S.Y.
      The current status of clinical research involving microneedles: a systematic review.
      ;
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      • Sun Y.
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      • et al.
      Microneedle-mediated transdermal drug delivery for treating diverse skin diseases.
      ).

      Conclusions and future directions

      Dissolvable MNAs represent a minimally invasive, active physical skin penetration technology that can considerably improve intracutaneous and transdermal delivery of biocargos. Optimally designed dissolvable MNAs with favorable geometric and material properties could be self-administered without the need for medical expertise, could be stored and distributed without refrigeration, and would leave no biohazardous sharps waste, thereby offering practical and logistic advantages over traditional drug or vaccine delivery approaches. Targeting biocargos to the skin with dissolvable MNAs in a painless manner could bring compliance and efficacy benefits compared with conventional biocargo delivery technologies. MNA delivery technology is now available for investigational use and could be particularly valuable as a tool to advance studies in skin biology. In particular, defined and controlled delivery of drugs to a specific skin microenvironment could enable purposeful interventions to provide novel insight into biologic mechanisms.
      To fully exploit the translational advantages of dissolvable MNAs for a wide range of therapies and vaccine applications, clinical evaluation of MNAs for skin-targeted drug and vaccine delivery is emerging. Large clinical trials with the most promising dissolvable MNA candidates are expected for skin-targeted delivery of the most suitable vaccines and therapeutics. Identifying noninvasive biophysical tools and/or simpler alternatives to confirm the penetration of microneedles and delivery of cargos will be critical for reproducible clinical applications and/or for self-administration. As certain MNA-based vaccines and drugs reach later-phase clinical trials, standards for regulatory approval will need to be comprehensively defined, and the infrastructure necessary for manufacturing will need to be established using scalable fabrication technologies. In addition, other factors contributing to revolutionizing drug and vaccine delivery with dissolvable MNAs, such as supply chain challenges, evolving economic models, and the acceptability of MNA-based biocargo delivery by patients and healthcare providers, will need to be broadly considered.

      Conflict of Interest

      EK and LDF are inventors of related intellectual property. LDF is a cofounder and scientific advisor of SkinJect, a company that is developing dissolvable microneedle arrays for nonmelanoma skin cancer treatment.

      Multiple Choice Questions

      • 1.
        Which of the following statements about dissolvable microneedle arrays (MNAs) is TRUE?
        • A.
          They are used to deliver only small-molecule biocargos through passive diffusion.
        • B.
          They are chemical skin penetration enhancers.
        • C.
          They are physical penetration enhancers that can actively deliver small-molecule cargos and macromolecules into the skin.
        • D.
          They inject liquid biocargo solutions into the skin.
      • 2.
        Potential advantages of dissolvable MNAs include
        • A.
          Improved skin-targeted delivery efficiencies compared with traditional topical creams and transdermal patches.
        • B.
          Enhanced spatial and temporal controllability compared with conventional needle injections.
        • C.
          Extended thermostability compared with liquid biocargos.
        • D.
          All of the above.
      • 3.
        Which of the following MNA design parameters is the most critical for temperature stability of dissolvable MNA‒embedded biocargos?
        • A.
          Tip radii of microneedles
        • B.
          Structural material choice
        • C.
          Microneedle width
        • D.
          Microneedle length
      • 4.
        Which of the following molecules can be delivered with rationally formulated dissolving MNAs?
        • A.
          Hydrophobic small-molecule drugs
        • B.
          Proteins
        • C.
          Recombinant viral vectors
        • D.
          All of the above
      • 5.
        Which of the following methods can be used to measure the penetration depth of microneedles during MNA application?
        • A.
          Transepidermal water loss
        • B.
          Optical coherence tomography
        • C.
          Electrical impedance
        • D.
          All of the above

      Acknowledgments

      EK is supported by a grant from the Institute for Infection, Inflammation, and Immunity in Children (i4Kids). EK and LDF are supported by National Institutes of Health grants ( UM1-AI106701 and R01-AR079233 ). LDF is also supported by National Institutes of Health grants ( R01-AR074285 and R01-AR071277 ) and by a grant from UPMC Enterprises (IPA 25565). The authors acknowledge the Petersen Institute of Nanoscience and Engineering at the University of Pittsburgh Swanson School of Engineering. This project used the UPMC Hillman Cancer Center and Tissue and Research Pathology/Pitt Biospecimen Core shared resource, which is supported in part by award P30-CA047904.

      Author Contributions

      Conceptualization: SCB, ZGG, EK, LDF; Investigation: SCB, ZGG, CDC, OEA; Visualization: SCB, ZGG, CDC, OEA; Writing - Original Draft Preparation: SCB, ZGG; Writing - Review and Editing: SCB, ZGG, CDC, OEA, EK, LDF

      Supplementary Material

      Detailed Answers

      • 1.
        Which of the following statements about dissolvable microneedle arrays (MNAs) is TRUE?
      • CORRECT ANSWER: C. They are physical penetration enhancers that can actively deliver small-molecule cargos and macromolecules into the skin.
      • Dissolvable MNAs constitute single-unit physical dosage systems that can successfully breach the superficial skin layers and actively deliver a broad range of small- and large-molecule biocargos.
      • 2.
        Potential advantages of dissolvable MNAs include
      • CORRECT ANSWER: D. All of the above.
      • Dissolvable MNAs promise (i) improved skin delivery efficiencies compared with topical applications and transdermal patches, (ii) more spatiotemporally controlled release of biocargos than that delivered by conventional needle injections, and (iii) extended temperature stability compared with liquid biocargos.
      • 3.
        Which of the following MNA design parameters is the most critical for temperature stability of dissolvable MNA‒embedded biocargos?
      • CORRECT ANSWER: B. Structural material choice.
      • Dissolvable MNAs are fabricated from water-soluble biomaterials as structural materials, and the thermostability of dissolving MNA‒embedded cargos depends highly on the preservative capabilities of the structural material of MNAs.
      • 4.
        Which of the following molecules can be delivered with rationally formulated dissolving MNAs?
      • CORRECT ANSWER: D. All of the above.
      • Dissolvable MNAs mechanically breach the outermost skin layers, and thus, they can effectively deliver a broad range of bioactive molecules, including proteins, recombinant viral vectors, and hydrophobic and hydrophilic small-molecule compounds.
      • 5.
        Which of the following methods can be used to measure the penetration depth of microneedles during MNA application?
      • CORRECT ANSWER: B. Optical coherence tomography.
      • Optical coherence tomography is a noninvasive imaging technology that can be used to visualize cutaneous layers during MNA application and can not only monitor breaching of the top skin layers but also measure the penetration depth of microneedles.

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