Abstract
The encapsulation of biopharmaceuticals into micro‐ or nanoparticles is a strategy frequently used to prevent degradation or to achieve the slow release of therapeutics and vaccines. Protein bodies (PBs), which occur naturally as storage organelles in seeds, can be used as such carrier vehicles. The fusion of the N‐terminal sequence of the maize storage protein, γ‐zein, to other proteins is sufficient to induce the formation of PBs, which can be used to bioencapsulate recombinant proteins directly in the plant production host. In addition, the immunostimulatory effects of zein have been reported, which are advantageous for vaccine delivery. However, little is known about the interaction between zein PBs and mammalian cells. To better understand this interaction, fluorescent PBs, resulting from the fusion of the N‐terminal portion of zein to a green fluorescent protein, was produced in Nicotiana benthamiana leaves, recovered by a filtration‐based downstream procedure, and used to investigate their internalization efficiency into mammalian cells. We show that fluorescent PBs were efficiently internalized into intestinal epithelial cells and antigen‐presenting cells (APCs) at a higher rate than polystyrene beads of comparable size. Furthermore, we observed that PBs stimulated cytokine secretion by epithelial cells, a characteristic that may confer vaccine adjuvant activities through the recruitment of APCs. Taken together, these results support the use of zein fusion proteins in developing novel approaches for drug delivery based on controlled protein packaging into plant PBs.
Highlights
Oral administration of pharmaceuticals is often the desired drug delivery route for reasons such as safety, patient compliance, and socioeconomic advantages (De Smet, Allais, & Cuvelier, 2014; Jennifer Schwestka and Marc Tschofen contributed to the study.Sastry, Nyshadham, & Fix, 2000)
We show that fluorescent Protein bodies (PBs) were efficiently internalized into intestinal epithelial cells and antigen‐presenting cells (APCs) at a higher rate than polystyrene beads of comparable size
We explore a downstream procedure based on two consecutive tangential flow filtrations (TFFs) as a means to enrich the zein PBs from larger amounts of leaf tissue, and we investigate the internalization efficiency of zein PBs into cells of the mucosal lining by comparing the uptake of fluorescent gz93 PBs and polystyrene beads of comparable size
Summary
Oral administration of pharmaceuticals is often the desired drug delivery route for reasons such as safety, patient compliance, and socioeconomic advantages Zein‐containing protein storage organelles, so‐ called zein protein bodies (PBs), found in maize endosperm cells (Lending & Larkins, 1989), may offer natural bioencapsulation strategies for recombinant oral pharmaceuticals. This assumption has been substantiated by experiments with rice seeds showing that the sequestration of recombinant proteins in endogenous storage organelles containing rice prolamins confers protection from digestive proteolysis after oral administration in an animal model (Nochi et al, 2007). A faster and more versatile method for encapsulating proteins into the protective environment of zein micro/nanocarriers is to create a fusion protein in which the protein of interest is fused to a partial sequence of zein Expression of such fusion protein results in in vivo bioencapsulation in various production hosts, within newly induced storage organelles. We analyze whether the epithelial cells secrete cytokines, which are known to recruit APCs
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