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.

Keywords: dissolving MAPs; hydrogel‐forming MAPs; implantable MAPs; miniature pigs; repeated application; safety.

Figure 1

Microscopic images of different types of blank MAP systems. (A) hydrogel‐forming MAPs, (B) dissolving MAPs, and (C) implantable MAPs.

Figure 2

(A) Percentage reduction in needle height after compression with a 32 N force for 30 s (means + SD, n  =  20). (B) Insertion capabilities of MAPs into stacked Parafilm layers, measured by the number of holes created in each layer (means ± SD, n  =  3). Representative false color OCT images of MAP insertion into Parafilm and excised porcine skin for (C) hydrogel‐forming MAPs, (D) dissolving MAPs, and (E) implantable MAPs. Insertion depth (µm) and percentage insertion efficiency of (F) hydrogel‐forming MAPs, (G) dissolving MAPs, and implantable MAPs into Parafilm M and full‐thickness porcine skin, as measured by OCT (means + SD, n  =  20).

Figure 3

(A) MAPs supported by microfoam tape were applied to the miniature pig skin, while microfoam tape without MAPs was used for the control site. Both the MAP‐treated and control sites were covered with (B) an adhesive foam bandage, followed by (C) Omnifix elastic and a jacket‐type dressing made from an elastic tubular support bandage. (D) MAPs after removal from the skin. Representative images of skin from different sites under various conditions: covered with MAPs, covered without MAPs, and uncovered. These show no signs of erythema for the different MAP types: (E) hydrogel‐forming MAPs, (F) dissolving MAPs, and (G) implantable MAPs. Pictures were taken on day 7 of the study.

Figure 4

TEWL values were measured at predetermined time points during the study. (A) TEWL values at the MAP application site on miniature pig skin (means + SD, n = 12). (B) Comparison of TEWL values at different sites: MAP application site, covered skin without MAP, and uncovered skin (means + SD, n = 12).

Figure 5

Histological analysis of miniature pig skin tissue stained with H&E, collected at the conclusion of the in vivo study (Day 28). The images illustrate skin tissue from different groups, including hydrogel‐forming MAPs, dissolving MAPs, and implantable MAPs, highlighting the structural effects of repeated MAP application.

Figure 6

Weekly evaluation of urine parameters from miniature pigs treated with MAPs. Parameters assessed include (A) leukocytes, (B) urobilinogen, (C) protein, (D) bilirubin, (E) glucose, (F) specific gravity, (G) nitrites, (H) creatinine, (I) ketones, and (J) pH (means + SD, n = 3). The ‘#’ symbol indicates values below the lower limit of quantification (LLOQ) or undetected in the samples. Reference normal range of urinalysis using urine dipstick: leukocyte (negative), urobilinogen (<2.0 mg dL⁻¹), protein (negative), bilirubin (negative), glucose (negative), specific gravity (1.003–1.030), Nitrite (negative), creatinine (20–400 mg dL⁻¹), ketone (negative) and pH (5–9).

Figure 7

Weekly hematological evaluation of miniature pigs treated with MAPs. The parameters analyzed include (A) red blood cell (RBC) count, (B) hematocrit (HCT), (C) hemoglobin (HGB), (D) mean corpuscular volume (MCV), (E) mean corpuscular hemoglobin (MCH), (F) mean corpuscular hemoglobin concentration (MCHC), (G) red cell distribution width (RDWc), (H) white blood cell (WBC) count, (I,L) neutrophils (NEU), (J,M) lymphocytes (LYM), (K,N) monocytes (MON), and (O) platelets (PLT) (means + SD, n = 3). The red dashed line indicates the upper limit, while the green dashed line represents the lower limit of the reference range. Reference normal range of hematological evaluation: RBC (5.00–8.00 M µL⁻¹), HCT (32.0–50.0%), HGB (10.7–16.7 g dL⁻¹), MCV (50.0–68.0 fL), MCH (17.0–21.0 pg), MCHC (30.0–34.0 g dL⁻¹), WCB (11.00–22.00 K µL⁻¹), NEU (4.48–7.52 K µL⁻¹), LYM (6.60–18.70 K µL⁻¹), MON (0.30–1.25 K µL⁻¹), PLT (300–700 K µL⁻¹).

Figure 8

Weekly serum biochemical evaluation of miniature pigs treated with MAPs. The parameters analyzed include (A) glucose (GLU), (B) creatinine (CREA), (C) urea (BUN), (D) phosphorus (P), (E) sodium (Na), (F) potassium (K), (G) chloride (Cl), (H) total protein (TP), (I) albumin (ALB), (J) alkaline phosphatase (ALKP), (K) albumin‐to‐globulin ratio (A/G), (L) alanine aminotransferase (ALT), (M) gamma‐glutamyltransferase (GGT), (N) cholesterol (CHOL), and (O) triglycerides (TRIG) (means + SD, n = 3). The red dashed line indicates the upper limit, while the green dashed line represents the lower limit of the reference range. Reference normal range of biochemical evaluation: GLU (85–160 mg dL⁻¹), CREA (0.5–2.1 mg dL⁻¹), BUN (6–30 mg dL⁻¹), P (3.6–9.2 mg dL⁻¹), TP (6.0–8.0 g dL⁻¹), ALB (1.8–3.3 g dL⁻¹), ALKP (92–294 U L⁻¹), ALT (9–43 U L⁻¹), GGT (16–30 U L⁻¹), CHOL 18–79 mg dL⁻¹), TRIG (41–83 mg dL⁻).

Figure 9

Plasma levels of (A) TNF‐α, (B) IgE, (C) IgG, (D) CRP, and (E) IL‐1β measured at the beginning (Day 0) and the end (Day 28) of the study using ELISA (means ± SD, n  =  3).

Figure 10

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

Figure 11

Schematic diagram illustrating the preparation process of hydrogel‐forming MAPs.

Figure 12

Schematic diagram illustrating the preparation process of dissolving MAPs.

Figure 13

Schematic diagram illustrating the preparation process of implantable MAPs.

Figure 14

Representative image of the study areas.

Figure 15

Schematic representation of the health assessment protocol and sample collection in minipigs following repeated application of MAPs over a 28‐day period.