Electrospun two-layer mucoadhesive patch for co-delivery of immunostimulatory Lactococcus lactis GEM particles and 33-mer gliadin peptide to the oral mucosa

• Two-layer patch effectively delivers immunostimulatory agents relevant for SLIT. • Loading 33-mer gliadin peptide and Gram-positive enhancer matrix particles in patch. • Two-layer patch showed strong adhesion to the oral mucosa and biocompatibility. • Two-layer patch was flexible and mechanically strong. • Loaded nanofibers in two-layer patch displayed “beads-on-a-string” morphology. Immunotherapy is conventionally managed via subcutaneous injections, yet recently sublingual immunotherapy (SLIT) has been established as an effective and safer route. However, short retention times at the mucosa, in addition to insufficient presentation to antigen-presenting cells can lead to inefficient performance of SLIT. To overcome these challenges, we aimed to co-deliver 33-mer gliadin peptide, the primary trigger of celiac disease, with immunostimulatory Lactococcus lactis Gram-positive enhancer matrix (GEM) particles in a mucoadhesive patch. Accordingly, 33-mer gliadin peptide was co-loaded with GEMs in a two-layer mucoadhesive patch prepared by electrospinning. The delivery system consisted of i) a mucoadhesive layer based on chitosan/polyethylene oxide (PEO) nanofibers to ensure adherence to the mucosa and ii) an active layer of PEO nanofibers for fast co-release of 33-mer gliadin peptide and GEMs. Both layers displayed smooth nanofibers with the GEMs appearing as ‘beads on a string’. Upon wetting, 33-mer gliadin peptide and GEMs were released within minutes in vitro , with no changes in structure or morphology. The two-layer patch showed optimal mechanical properties, maintaining flexibility and shape during handling. Prolonged residence time at the oral mucosa is crucial for SLIT efficacy by enhancing antigen presentation. Strong adhesion of the two-layer patch was demonstrated by measuring adhesion to ex vivo porcine oral mucosa. Furthermore, biocompatibility was demonstrated by exposure to human oral epithelial cell monolayers in vitro . In conclusion, the two-layer patch displayed appropriate properties to serve as a dosage form for SLIT, capable of co-presentation of immunostimulatory actives and enhancing retention at the mucosal surface.