Abstract
Purpose: :
To develop new treatments for disease of the lacrimal gland, new drug carriers are required that are biocompatible and easily modified with bioactive peptides. An emerging solution to this challenge utilizes genetically engineered polypeptides to drive the assembly of nanostructures. Elastin-like-polypeptide (ELP) engages in a unique phase transition behavior, which can mediate self-assembly of nanoparticles. In this study we characterized the properties and cellular uptake in lacrimal acinar cells of diblock ELP fusion peptides with and without peptides targeted to the polymeric immunoglobulin receptor (pIgR), which is responsible for selective uptake and transcytosis of dIgA in mucosal epithelial cells.
Methods: :
A targeting peptide known to bind to pIgR, mIgA, comprising 402-410 conserved amino acid residues of the Cα3 domain of dIgA, was genetically fused to diblock ELP at the N-terminus in the plasmid PET25b. The plasmid was expressed in E. coli, and polypeptides were purified using the phase behavior of the ELPs. The purified diblock ELP and mIgA-ELP were characterized with SDS-PAGE, transition temperature and dynamic light scattering. Cellular uptake was conducted with cultured rabbit lacrimal gland acinar cells (LGAC).
Results: :
Phase transition temperature experiments showed that diblock mIgA-ELP has two transition temperatures, one at 27°C and one at 45°C. Dynamic light scattering results indicated that above a critical temperature of 25°C, both diblock ELP and mIgA-ELP could self-assemble into nanoparticles of ~40 nm diameter. To evaluate cellular uptake, both conjugates were labeled with rhodamine at the N-terminus. Cellular uptake experiments indicate ELP was minimally internalized in LGAC which express abundant pIgR, while mIgA-ELP showed a higher uptake. Most internalized mIgA-ELP was co-localized with lysosomes.
Conclusions: :
Both diblock ELP and diblock mIgA-ELP are able to self assemble into nanoparticles. The diblock mIgA-ELP showed higher efficiency of uptake in LGAC which abundantly express pIgR, indicating the feasibility of this nanocarrier for gene therapy and drug delivery to LGAC and other mucosal epithelial cells.
Keywords: lacrimal gland • protein purification and characterization • receptors