Epicutaneous Delivery of Allergens Using Dual Stimuli-Responsive Liposomes-Loaded Dissolving Microneedles

There has been a growing interest in allergen-specific epicutaneous immunotherapy (EPIT) as an alternative to oral immunotherapy due to safety concerns with oral approaches. A key challenge in EPIT is the effective delivery of macromolecular allergens across the stratum corneum (SC) without inducing skin inflammation or irritation. To enhance the efficacy of macromolecular allergen delivery through the SC, allergen-loaded liposomes incorporated into dissolving microneedles (dMN) were developed. The compatibility of food allergens with stigmasterol (STG)-based liposomes, their release kinetics in response to various stimuli, and the suitability of liposomes in microneedle formulations were studied. Peanut allergen-loaded thermo- and phospholipase (sPLA)-responsive liposomes were synthesised using the thin-film hydration method, and dMNs encapsulating these liposomes were fabricated using micromoulding method. Liposomes’ structure post-fabrication was characterised by transmission electron microscopy (TEM), and stability was assessed using dynamic light scattering (DLS). Fluorescein-conjugated allergens were used to simulate temperature- and enzyme-dependent release across skin layers, and allergen release profiles were evaluated immunochemically using anti-allergens IgG antibodies, human peanut sIgE, and rat basophil leukemia (RBL) assays. Microchannels created by dMNs were also characterised. The synthesised peanut allergen-loaded liposomes, using thermo-responsive (DPPC/DOPE/STG) and dual heat- and sPLA-responsive (DPPG/DPPC/STG), had average diameters of 154.6 ± 1.1 nm and 164.8 ± 2.0 nm, respectively. Encapsulation efficiency in DPPC/DOPE/STG was lower at 20% with mixed allergens (e.g., crude peanut protein), and higher at 30 - 35% with single allergens (e.g., Ara h 1 and Ara h 2). DPPG/DPPC/STG liposomes showed a significantly improved encapsulation efficiency of 35.9% for crude peanut protein. The thermo-responsive liposomes released 50% of their encapsulated allergens over 3 days at 37°C, while the sPLA-responsive liposomes released 50% within 3h at the same temperature. The profiles of allergens released from these liposomes via stimuli (i.e., heat or/and sPLA) were assessed by sodium dodecyl-sulphate polyacrylamide gel electrophoresis (SDS-PAGE), showing the presence of all clinically relevant peanut allergens (Ara h 1, Ara h2 and Ara h 3). This was further confirmed immunochemically using allergen-specific antibodies, demonstrating reduced immunoreactivity due to structural modification through interaction between allergen-lipid components. The liposomes showed no significant cytotoxicity at 100 µg/mL. They were also stable for 4 weeks at 4 °C with minimal size increase (-0.8% and -0.5% growth). The allergen-loaded liposomes incorporated dMNs (the dimensions of height = 598.4 ± 7.6 µm, diameter = 284.7 ± 4.1 µm, interspacing = 302.7 ± 12.7 µm) were successfully fabricated. The TEM analysis of dMNs showed no significant changes in liposome size or structure following microneedle encapsulation, showing the feasibility and compatibility of encapsulating liposomes in these dMNs. In vitro diffusion studies using a cellulose acetate membrane revealed a 6.4% increase in allergen release after 8 h for liposomes alone, while liposome incorporation in dMNs resulted in an 81.4% increase. This implied that dMNs greatly increased the penetrability of allergens. Released allergen profiles exhibited reduced binding to polyclonal anti-allergens IgG antibodies and human sIgE, suggesting some structural modifications in the released allergens resulting in decreased allergenic potential as tested by rat basophil leukemia (RBL) assay. These findings highlight the potential of peanut allergen-loaded liposomes encapsulated in dMNs as a novel, safe and efficient vehicle for EPIT. By demonstrating a hypoallergenic effect and enhanced skin delivery, this approach paves the way for a transformative advancement in allergen immunotherapy.

Grantor dc:publisher

UNSW, Sydney

Year dc:date

2025

Author dc:creator

• Lee, Annabelle

Statement dc:rights

• embargoed access
• CC BY 4.0

Licence

• http://purl.org/coar/access_right/c_f1cf
• https://creativecommons.org/licenses/by/4.0/

Language dc:language

en

Identifier

https://doi.org/10.26190/unsworks/31650

OAI identifier oai:identifier

oai:unsworks.library.unsw.edu.au:1959.4/106020