Cyclosporin A-loaded dissolving microneedles for dermatitis therapy: Development, characterisation and efficacy in a delayed-type hypersensitivity in vivo model

Abstract

Several drugs can be used for treating inflammatory skin pathologies like dermatitis and psoriasis. However, for the management of chronic and long-term cases, topical administration is preferred over oral delivery since it prevents certain issues due to systemic side effects from occurring. Cyclosporin A (CsA) has been used for this purpose; however, its high molecular weight (1202 Da) restricts the diffusion through the skin structure. Here, we developed a nano-in-micro device combining lipid vesicles (LVs) and dissolving microneedle array patches (DMAPs) for targeted skin delivery. CsA-LVs allowed the effective incorporation of CsA in the hydrophilic DMAP matrix despite the hydrophobicity of the drug. Polymeric matrix composed of poly (vinyl alcohol) (5% w/v), poly (vinyl pyrrolidine) (15% w/v) and CsA-LV dispersion (10% v/v) led to the formation of CsA-LVs@DMAPs with adequate mechanical properties to penetrate the stratum corneum barrier. The safety and biocompatibility were ensured in an in vitro viability test using HaCaT keratinocytes and L929 fibroblast cell lines. Ex vivo permeability studies in a Franz-diffusion cell setup showed effective drug retention in the skin structure. Finally, CsA-LVs@DMAPs were challenged in an in vivo murine model of delayed-type hypersensitivity to corroborate their potential to ameliorate skin inflammatory conditions. Different findings like photon emission reduction in bioluminescence study, normalisation of histological damage and decrease of inflammatory cytokines point out the effectivity of CsA-LVs@DMAPs to treat these conditions. Overall, our study demonstrates that CsA-LVs@DMAPs can downregulate the skin inflammatory environment which paves the way for their clinical translation and their use as an alternative to corticosteroid-based therapies.

Keywords: Cyclosporin A; Dermatitis; Dissolving microneedles; Lipid vesicles; Skin delivery; Skin inflammatory conditions.

Fig. 1

Schematic illustration of the delayed-type hypersensitivity (DTH) murine in vivo model

Fig. 2

a Representative TEM image of CsA-LVs. b Size and PDI of the blank LVs and CsA-LVs. Results are expressed as mean ± SD (n = 3). c ζ-potential of the blank LVs and CsA-LVs. Results are expressed as mean ± SD (n = 3). No statistical differences were found between CsA-LVs and blank LVs. * denotes that the measurement has been performed after dissolving CsA-LVs@DMAPs for releasing the CsA-LVs in aqueous media

Fig. 3

a Macroscopic image of CsA-LVs@DMAPs; b, c SEM images of the CsA-LVs@DMAPs. Scale bars: 400 µm (b), 100 µm (c); d compression percentage of the different DMAP prototypes. Results are expressed as mean ± SD (n = 3); e insertion percentage from different DMAP prototypes in Parafilm-M^® artificial skin model. Results are expressed as mean ± SD (n = 3); f release profile from different DMAP prototypes. Results are expressed as mean ± SD (n = 3); g TGA of the F3 formulation: (i) after fabrication, (ii) 1 week after fabrication and (iii) 6 weeks after fabrication. * denotes statistically significant differences in comparison with the other prototypes

Fig. 4

a L929 fibroblast cell viability after 24 h of treatment with CsA-LVs@DMAP components. Results expressed as mean (percentage) ± SD; b HaCaT keratinocyte cell viability after 24 h of treatment with CsA-LVs@DMAP components. Results expressed as mean (percentage) ± SD. Red line indicates 80% cell viability

Fig. 5

a Cumulative ex vivo amounts of CsA found in the receptor FDC chamber after 1.5, 3, 12, 24 and 48 h. Results expressed as mean (percentage of initial dose) ± SD (n = 5); b Cumulative amount of CsA found in the skin structure after drug extraction at 2, 12, 24 and 48 h (n = 3). Results expressed as mean (percentage of initial dose) ± SD. ** and **** denote significant differences between the comparison pair (p < 0.01 and p < 0.0001, respectively); c representative pictures pre-insertion and post post-insertion of CsA-LVs@DMAPs and skin staining (internal side) after CsA-LVs@DMAP dissolution

Fig. 6

a Representative histology image CsA-LVs@DMAP insertion in the DTH murine in vivo model. Arrows indicate the place where DMAPs were inserted exactly; b representative images of BLI luminescence 24 h after OXA challenge of the different groups involved in the experiment: Healthy control (unchallenged), dexamethasone-treated group (DEXA), CsA-LVs@DMAP-treated group, non-treated group (OXA) and blank DMAP–treated group. Scale was set up from 200 up to 4000 photons; c ROI counts of BLI induced after the different treatments. ROI area was in the exact application place of the DMAPs and set equal for each subject under study. Results are expressed as mean ± SD (n = 6); d MPO quantification in skin tissue after the different treatments. MPO activity was expressed as absorbance/mg of protein. Results are expressed as mean ± SD (n = 3). * denotes statistically significant differences in comparison to healthy control (p < 0.05). # denotes statistically significant differences in comparison to the DEXA group (p < 0.05)

Fig. 7

a Representative images from the histological sections stained with H&E stain (× 20 magnifications); b representative images from the histological sections stained with MT stain (× 20 magnifications); black arrows indicate the epidermal thickness, and red arrows indicate spongiotic patterns and neutrophile infiltration. c Collagen thickness measured from histological observation 24 h after the administration of the different treatments on the MT stains at × 10 magnifications; d epidermal thickness measured from histological observation 24 h after the administration of the different treatment H&E stains at × 20 magnifications. Results are expressed as mean ± SD (n = 3); results are expressed as mean ± SD (n = 3). * denotes statistically significant differences in comparison to healthy control (p < 0.05). # denotes statistically significant differences in comparison to the DEXA group (p < 0.05)

Fig. 8

a TNF-α quantification detected by ELISA immunoassay in skin samples; b IL-6 quantification detected by ELISA immunoassay in skin samples; c IL-4 quantification detected by ELISA immunoassay in skin samples; d IL-1β quantification detected by ELISA immunoassay in skin samples. Results are expressed as mean (pg of cytokine/mg of protein) ± SD (n = 3). * denotes statistically significant differences in comparison to healthy control (p < 0.05). # denotes statistically significant differences in comparison to the DEXA group (p < 0.05)