Loading Proteins into Extracellular Vesicles to Camouflage Protein Allergens

Extracellular vesicles (EVs) are cell-derived membranous nanoparticles with a high potential as drug delivery systems due to their intrinsic capability to vehicle biological materials and information. Beyond the transport of small drugs and therapeutics, a biotechnological challenge consists of loading nanoparticles and macromolecules with large size, including antibodies and other proteins. Here, we use microalgal-derived EVs, named nanoalgosomes, that are biocompatible, sustainable, and green EVs derived from microalgae and thoroughly characterized in our previous work, and a recombinant calciumbinding protein (CBP), Par j 4, a minor allergen from Parietaria judaica (Pj). We set up an efficient method to load Par j 4 into nanoalgosomes, using extrusion as the loading technique, affinity chromatography as the purification method, and fluorescence spectroscopy to quantify loaded cargo. Confocal microscopy is used to show protein and EVs colocalization; specific dot blot demonstrates that loaded Par j 4 is detectable only after EVs lysis, being masked by intact nanoalgosomes. The achieved camouflage of an allergen opens the perspective of addressing an unmet need in the treatment of allergies, that is, the possibility to present allergens in a controlled manner and without side effects. Further, we showed that nanoalgosomes may be efficiently exploited to carry large size macromolecules.