Safety Evaluation of Repeated Application of Polymeric Microarray Patches in Miniature Pigs

Abstract

The safety of repeated microarray patch (MAP) application is crucial for its development as an innovative drug delivery platform. This study is the first to assess the safety of repeated applications of hydrogel-forming, dissolving, and implantable MAPs over four weeks using miniature pigs, an industry-standard dermatological model with human-like skin structure and physiological responses. Uniform MAPs are successfully manufactured, with application forces of 32 N/array resulting in less than 15% needle height reduction. ≈80% of the needle length penetrated Parafilm layers, while 40–60% penetrated excised porcine skin. Repeated MAP applications do not compromise skin barrier function, as confirmed by transepidermal water loss measurements, and caused no adverse skin reactions per modified Draize test results. Systemic safety assessments revealed no significant immune responses, allergic reactions, infections, or inflammatory markers (TNF-α, IgE, IgG, CRP, and IL-1β) between day 0 and day 28. No weight loss, infection signs, kidney toxicity, or clinically relevant hematological or biochemical changes are observed. Histopathological evaluations confirmed the absence of lesions or adverse effects. These findings establish the safety of repeated hydrogel-forming, dissolving, and implantable MAP applications, supporting their potential for safe, effective drug delivery and facilitating their translation from preclinical models to human clinical trials.

Introduction

Microarray patches (MAPs) are an innovative drug delivery system that provides a minimally invasive and efficient alternative to conventional administration methods such as injections or oral dosing.[1-4] By employing micron-sized needles that penetrate the stratum corneum, MAPs enable the painless delivery of therapeutic agents into the epidermis or dermis, allowing for localized effects (e.g., immune cell-targeted vaccination) or systemic drug delivery.[5] Their advantages, including enhanced patient compliance, self-administration capability, and reduced reliance on healthcare professionals, make MAPs particularly valuable in low-resource settings.[6] Previous studies have demonstrated their versatility across various applications, including vaccine delivery, chronic disease management, and diagnostics, positioning MAPs as a transformative tool in modern healthcare.[7-11]

Among MAP technologies, polymeric MAPs have garnered significant attention due to their ease of manufacture, biocompatibility, mechanical strength, and adaptability. Hydrogel-forming MAPs swell upon contact with interstitial fluid, creating a diffusion pathway for drug delivery without leaving measurable polymer residues in the skin.[12, 13] Dissolving MAPs disintegrate upon application, releasing their payloads, while implantable MAPs provide sustained drug release for extended therapeutic effects.[14-17]

In certain disease conditions, patients may need to apply MAPs repeatedly on a daily, weekly, or monthly basis, a scenario referred to as “repeated” application. The safety of repeated MAP use depends on several critical factors. One primary concern is the potential disruption of the skin barrier, particularly the stratum corneum and epidermis.[18] Another consideration is systemic immune activation, as repeated MAP application could trigger localized or systemic inflammation. Additionally, kidney toxicity is a potential risk with polymeric MAPs, as polymer fragments with molecular weights exceeding the renal filtration threshold may accumulate in the kidneys, potentially impairing renal function. Monitoring parameters such as protein levels, creatinine, and urine-specific gravity is essential for assessing nephrotoxicity and overall kidney health.

While previous studies have investigated the safety of repeated MAP application, most have been conducted in academic settings with limited translational applicability. Vicente-Perez et al. reported that repeated application of hydrogel-forming MAPs for three weeks and dissolving MAPs for four weeks in mice did not significantly alter transepidermal water loss (TEWL) or biomarkers of infection, immunity, or inflammation.[19] Similarly, Al-Kasasbeh et al. demonstrated that repeated hydrogel-forming MAP application in human volunteers did not cause prolonged skin reactions or barrier disruption, with biomarker concentrations remaining within normal ranges, indicating no systemic effects.[20] However, to date, no comprehensive study has evaluated the repeated application of hydrogel-forming, dissolving, and implantable MAPs across all safety aspects in a Good Laboratory Practice (GLP) model adhering to industrial standards.

To address this gap, the present study was conducted in full compliance with the OECD Principles of Good Laboratory Practice (revised 1997, C(97)186/Final) and applicable EU regulations (Directive 1999/11/CE and Real Decreto 1369/2000). The protocol was approved by the ethical committee of Specific Pig (Specipig) S.L., and all procedures were carried out at Specipig’s GLP-certified facility in Barcelona, Spain. The study was also subject to routine inspections to ensure adherence to GLP through both facility-level and process-based evaluations. This rigorous regulatory framework enhances the translational relevance of our findings and supports their potential application in clinical development.

Miniature pigs are an ideal preclinical model for assessing MAP safety due to their close resemblance to human skin in terms of epidermal thickness, dermal-epidermal junction structure, and lipid composition.[21] These similarities make them particularly suited for evaluating the localized effects of MAPs on skin integrity and barrier function. Furthermore, the systemic physiology of miniature pigs allows for robust assessments of immune and renal responses under conditions mimicking human use.[22, 23] This model bridges the gap between preclinical and clinical studies, providing valuable insights into MAP safety and paving the way for human trials.

In this study, we conducted, for the first time, a comprehensive evaluation of the safety of repeated application of three types of polymeric MAPs (hydrogel-forming, dissolving, and implantable) over a 28-day period in miniature pigs under GLP industrial standard. This investigation addresses critical knowledge gaps regarding the long-term safety of MAPs, providing insights into both localized and systemic effects. The findings have significant implications for the development of MAP-based therapies, particularly for drug delivery requiring repeated applications. From a regulatory perspective, this study contributes to establishing safety benchmarks for preclinical evaluations, facilitating the translation of MAP technology from animal models to human clinical trials.

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4 Experimental Section

Preparation of Polymeric MAPs

To prepare hydrogel-forming MAPs, the required mass of Gantrez S-97 (40% w/w stock solution) was weighed into a 50 mL Falcon tube. The stock solution was pre-prepared by dissolving Gantrez S-97 powder in deionized water and thoroughly mixing. Separately, PEG 10,000 was weighed into a 15 mL Falcon tube and dissolved in a minimal volume of deionized water.

Fig. 10 Illustration of needle size and geometry on the array of (A) hydrogel-forming MAP, (B) dissolving and implantable MAPs.

The PEG solution was then added to the Gantrez S-97 solution, and the mixture was adjusted to the desired weight with deionized water. The blend was mixed with a spatula until homogeneous and centrifuged at 3500 RPM for 10 min to remove air bubbles.

The formulation was cast into 11 × 11 pre-formed silicone microneedle molds (conical shape, 600 µm height, 300 µm base width, and 300 µm interspacing, illustrated in Figure 10A). As illustrated in Figure 11, the moulds containing formulation were centrifuged at 3500 RPM for 10 min left to dry at room temperature for 48 h. After drying, sidewalls formed during the process were trimmed using scissors, and the MAPs were crosslinked in a hot air oven at 80°C for 24 h to create insoluble hydrogel-forming structures.

For dissolving MAPs, separate aqueous stocks of PVA (9–10 kDa) and PVP (58 kDa) were prepared in deionized water at a polymer concentration of 40% w/w each. Equal volumes of the stocks were combined to create a blend with a final polymer concentration of 20% w/w. The mixture was briefly hand-mixed, centrifuged to remove air bubbles, and inspected to ensure no phase separation occurred.

Fig. 11 Schematic diagram illustrating the preparation process of hydrogel-forming MAPs.

The polymer blend was poured into a poly(dimethylsiloxane) mold containing 16 × 16 pyramidal-cuboidal needles with dimensions of 850 µm height, 300 µm base width, and 300 µm interspacing, covering a patch area of 0.36 cm2, illustrated in Figure 10B. As displayed in Figure 12, the molds were placed in a positive pressure chamber at 4 bar for 5 min. Excess formulation was carefully removed with a spatula and the molds were returned to the chamber for an additional 30 min under the same pressure.

Elastomer rings (external diameter: 23 mm, internal diameter: 18 mm, thickness: 3 mm) were affixed on top of the molds using a glue solution prepared from an aqueous blend of 40% w/w PVA (9–10 kDa). The molds were allowed to dry at room temperature for 6 h. Subsequently, 850 µL of a second layer consisting of an aqueous blend of 30% w/w PVP (90 kDa) and 1.5% w/w glycerol was added to the molds. After centrifuging at 3500 RPM for 10 min, the molds were dried at room temperature for 24 h. Any sidewalls formed during the drying process were trimmed using scissors, and the molds were further dried at 37°C for 24 h.

Fig 12 Schematic diagram illustrating the preparation process of dissolving MAPs.

Qonita Kurnia Anjani, Aaron R. J. Hutton, Peter E. McKenna, Eneko Larrañeta and Ryan F. Donnelly, Safety Evaluation of Repeated Application of Polymeric Microarray Patches in Miniature Pigs, The ORCID identification number(s) for the author(s) of this article, https://advanced.onlinelibrary.wiley.com/doi/10.1002/adhm.202501512, First published: 17 June 2025, https://doi.org/10.1002/adhm.202501512