Gliadin, the alcohol-soluble protein fraction of wheat, contains the factor toxic for celiac disease (CD), and its toxicity is not reduced by digestion with gastro-pancreatic enzymes. Importantly, it is proved that an innate immunity to gliadin plays a key role in the development of CD. The immune response induces epithelial stress and reprograms intraepithelial lymphocytes into natural killer (NK)-like cells, leading to enterocyte apoptosis and an increase in epithelium permeability. In this contribution, we have reported that in Caco-2 cells the administration of enzymatically digested gliadin (PT-gliadin) reduced significantly the expression of the autophagy-related marker LC3-II. Furthermore, electron and fluorescent microscope analysis suggested a compromised functionality of the autophagosome apparatus. The rescue of the dysregulated autophagy process, along with a reduction of PT-gliadin toxicity, was obtained with a starvation induction protocol and by 3-methyladenine administration, while rapamycin, a well-known autophagy inducer, did not produce a significant improvement in the clearance of extra- and intra-cellular fluorescent PT-gliadin amount. Altogether, our results highlighted the possible contribution of the autophagy process in the degradation and in the reduction of extra-cellular release of gliadin peptides and suggest novel molecular targets to counteract gliadin-induced toxicity in CD.

Extracellular vesicles (EVs) are considered as promising nanoparticle theranostic tools in many pathological contexts. The increasing clinical employment of therapeutic nanoparticles is contributing to the development of a new research area related to the design of artificial EVs. To this aim, different approaches have been described to develop mimetic biologically functional nanovescicles. In this paper, we suggest a simplified procedure to generate plasma membrane-derived nanovesicles with the possibility to efficiently encapsulate different drugs during their spontaneously assembly. After physical and molecular characterization by Tunable Resistive Pulse Sensing (TRPS) technology, transmission electron microscopy, and flow cytometry, as a proof of principle, we have loaded into mimetic EVs the isoquinoline alkaloid Berberine chloride and the chemotherapy compounds Temozolomide or Givinostat. We demonstrated the fully functionality of these nanoparticles in drug encapsulation and cell delivery, showing, in particular, a similar cytotoxic effect of direct cell culture administration of the anticancer drugs. In conclusion, we have documented the possibility to easily generate scalable nanovesicles with specific therapeutic cargo modifications useful in different drug delivery contexts.

Celiac disease (CD) is a chronic systemic autoimmune disorder that is triggered by the ingestion of gliadin peptides, the alcohol-soluble fraction of wheat gluten. These peptides, which play a key role in the immune response that underlies CD, spontaneously form aggregates and exert a direct toxic action on cells due to the increase in the reactive oxygen species (ROS) levels. Furthermore, peptic-tryptic digested gliadin peptides (PT-gliadin) lead to an impairment in the autophagy pathway in an in vitro model based on Caco-2 cells. Considering these premises, in this study we have analyzed different mTOR-independent inducers, reporting that the disaccharide trehalose, a mTOR-independent autophagy activator, rescued the autophagy flux in Caco-2 cells treated with digested gliadin, as well as improved cell viability. Moreover, trehalose administration to Caco-2 cells in presence of digested gliadin reduced the intracellular levels of these toxic peptides. Altogether, these results showed the beneficial effects of trehalose in a CD in vitro model as well as underlining autophagy as a molecular pathway whose modulation might be promising in counteracting PT-gliadin cytotoxicity.