Dimeric Lectin Chimeras as Novel Candidates for Gb3-Mediated Transcytotic Drug Delivery through Cellular Barriers

by Selleckchem | Nov 19 2023

Receptor-mediated transcytosis is an elegant and promising strategy for drug delivery across biological barriers. Here, we describe a novel ligand-receptor pair based on a dimeric, engineered derivative of the Pseudomonas aeruginosa lectin LecA, here termed Di-LecA, and the host cell glycosphingolipid Gb3. We characterized the trafficking kinetics and transcytosis efficiencies in polarized Gb3-positive and -negative MDCK cells using mainly immunofluorescence in combination with confocal microscopy. To evaluate the delivery capacity of dimeric LecA chimeras, EGFP was chosen as a fluorescent model protein representing macromolecules, such as antibody fragments, and fused to either the N- or C-terminus of monomeric LecA using recombinant DNA technology. Both LecA/EGFP fusion proteins crossed cellular monolayers in vitro. Of note, the conjugate with EGFP at the N-terminus of LecA (EGFP-LecA) showed a higher release rate than the conjugate with EGFP at the C-terminus (LecA-EGFP). Based on molecular dynamics simulations and cross-linking studies of giant unilamellar vesicles, we speculate that EGFP-LecA tends to be a dimer while LecA-EGFP forms a tetramer. Overall, we confidently propose the dimeric LecA chimeras as transcytotic drug delivery tools through Gb3-positive cellular barriers for future in vivo tests.

Comments:

Your description outlines a sophisticated approach to drug delivery utilizing receptor-mediated transcytosis, a process that involves the transportation of substances across biological barriers within cells. In this particular study, a novel ligand-receptor pair was identified, consisting of a dimeric engineered derivative of the Pseudomonas aeruginosa lectin LecA (referred to as Di-LecA) and the host cell glycosphingolipid Gb3. This pairing demonstrated promising potential for drug delivery across Gb3-positive cellular barriers.

The study involved the characterization of the trafficking kinetics and transcytosis efficiencies in polarized Gb3-positive and -negative MDCK cells. The evaluation of dimeric LecA chimeras' delivery capacity was conducted using EGFP (Enhanced Green Fluorescent Protein) as a fluorescent model protein, representing macromolecules like antibody fragments. EGFP was fused to either the N- or C-terminus of monomeric LecA using recombinant DNA technology, resulting in LecA/EGFP fusion proteins that successfully crossed cellular monolayers in vitro.

An interesting observation was made regarding the orientation of EGFP fusion in the LecA structure. EGFP-LecA, where EGFP was attached to the N-terminus of LecA, exhibited a higher release rate compared to LecA-EGFP, where EGFP was attached to the C-terminus. Molecular dynamics simulations and cross-linking studies of giant unilamellar vesicles suggested that EGFP-LecA tends to form dimers, while LecA-EGFP forms tetramers.

Based on these findings, the study proposes dimeric LecA chimeras as effective tools for transcytotic drug delivery across Gb3-positive cellular barriers. The potential applications of this research include the development of targeted drug delivery systems for future in vivo testing, showcasing the promising advancements in the field of drug delivery strategies.