Early life cow's milk allergy prevention: PLGA Nanoparticles for the Oral Delivery of β-Lactoglobulin Derived Peptide and CpG Oligodeoxynucleotides

Abstract

The prevalence and lack of good solutions for cow's milk allergy (CMA) in young children are leading to public concern and increasing socioeconomic problems. This requires new preventive approaches. In this thesis, we investigated the potential of an oral formulation – based on poly(lactic acid-co-glycolic acid) (PLGA) nanoparticles (NP) – of selected peptides from whey proteins and CpG-Oligodeoxynucleotides for CMA prevention, using both in vitro and in vivo studies. In vitro, we showed, through Förster Resonance Energy Transfer (FRET) and imaging in living cells, that PLGA NP protected the encapsulated peptides for up to 6 hours in simulated gastric fluid and extended their residence time in isolated mouse dendritic cells to 96 hours. This demonstrates the hypothesis that PLGA nanoparticles improved the stability of the encapsulated peptide cargo in the gastrointestinal lumen and retention in intestinal DC upon internalization. In vivo, we showed that oral pretreatment with two PLGA-NP-encapsulated peptides, prior to allergic sensitization to whey, protected mice from acute allergic skin reactions upon intradermal exposure to whey. In addition, we have shown that this protection is dose-related and may be associated with whey-specific systemic silencing of ex vivo whey-stimulated TNF-α release by splenocytes. Intriguingly, in vivo, we showed the superior tolerogenic effect of orally administered PLGA nanoparticles co-loaded with T-cell epitopes containing peptide and a CpG adjuvant as compared to the separately encapsulated peptide and CpG counterpart. The combination of peptides and CpG prevented rise in specific IgE antibody levels in serum and whey-induced acute allergic skin reactions, and showed a beneficial immunomodulatory effect on immune cells. Finally, allergy-associated immunomodulatory effects of empty, peptide or CpG-containing PLGA nanoparticles were evaluated in vitro using dendritic cells derived from human monocytes. The immature moDCs primed with CpG-ODN loaded PLGA NP also effectively increased the ratios of Treg/Th2 and Th1/Th2. This finding supports the hypothesis that the co-encapsulated CpG-ODN facilitates the inhibition of Th2 immunity also in vivo. In conclusion, our findings substantiate the translational potential of PLGA nanoparticles in which T-cell epitopes containing peptides are encapsulated for future development of effective and safe strategies for early life CMA prevention (i.e., supplementing hydrolyzed formula milk for infants at risk). In addition, besides BLG-peptides also CpG-ODN motives, which may include bacterial DNA of selected bacterial strains, can be co-loaded in the PLGA NP to increase the efficacy of the formulations in oral tolerance induction. This strategy may be further developed as a modality to treat cow’s milk allergy for example as adjunct treatment for allergen specific oral immunotherapy.