Ponente
Descripción
Non-viral gene delivery systems should carry functional components that can facilitate overcoming the endosomal barrier [1, 2]. Sticholysin I (StI) shows permeabilizing activity on lipid membranes containing sphingomyelin by forming oligomeric pores [3]. The aim of this work was to study the ability of the bio-responsive conjugate based on the StI mutant, StIW111C, to increase the endosomal release of a plasmid DNA (pDNA) and, consequently, to improve its expression. The reducible conjugate obtained by linking StIW111C to a polylysine peptide was purified and the DNA-polylysine complexes generated with or without the conjugate was characterized by EMSA, DLS and Cryo-TEM. The uptake and the expression of the reporter gene was evaluated by flow cytometry and confocal microscopy. Positive nanometric complexes were obtained mixing pDNA with the conjugate and the polylysine peptide. However, the heterogeneity was higher compared with samples obtained from the binary mix of DNA and peptide. In in vitro assays, the StIW111C conjugate was able to permeabilize endosomes although the expression of the reporter gene was not observed. This could be consequence of a reduced capacity of StIW111C to be internalized since it might remain trapped into the large aggregates observed by cryo-TEM. These results suggest that only small quantities of the reducible conjugate are able to reach the endosomal compartment. A limited number of pores formed in the endosomal membranes could not be enough to destabilize the endosomal compartment to release the particles into the cytosol. Hence it is necessary to change the design of complexes to reduce the aggregates.
