160. Additive Nanocomplexes of Cationic Lipopolymers for Improved Non-Viral Gene Delivery to Mesenchymal Stem Cells

Abstract

It has been a challenging task to transform MSC into therapeutically useful cells. Most studies were conducted with viral vectors, despite serious concerns on immunogenicity of the viral approach [1]. Cationic polymers display greater chemical diversity than lipids and accommodate versatile chemical schemes to incorporate functional moieties for better transfection[2]. Herein, we have developed a ternary nano-formulation for gene delivery to umbilical cord blood MSC and bone marrow MSC by using lipid-modified small (1.2 kDa) molecular weight polyethylenimine (PEI1.2) (Figure 1). Linoleic acid (LA) was end-capped with carboxyl functionality by coupling with mercaptopropionic acid through thio-ester linkage, and then grafted onto PEI1.2 via N-acylation. Structural functionalities of modified LA (tLA) and thio-ester LA grafted PEI1.2 (PEI-tLA) were analyzed through 1H-NMR spectroscopy and TNBS assay. To create polymer/pDNA complexes that more conducive for dissociation, polyanionic additive (hyaluronic acid, HA) was incorporated along with pDNA to PEI-tLA complexes. The binding capacity of the polymers and unpacking of resultant complexes was elucidated by an agarose gel retardation assay.PEI-tLA displayed a significantly lower (up to 6-fold) pDNA binding capability and a higher dissociation propensity. The dissociation ability of PEI-tLA/pDNA complexes was further enhanced by HA incorporation. The effect of HA was negligible in hydrodynamic size of the complexes but it significantly decreased surface charge which is essential to minimize cellular toxicity of cationic polymers [3]. PEI-tLAs displayed better compatibility to MSC compared to commercial targeting agents and conventional PEI-LA polymers. In vitro transfection efficiency of PEI-tLAs in MSC was substantially higher than positive control. The higher gene expression was the consequence of better cellular uptake, observed through confocal microscopy and flowcytometric study. Transfection efficiency of PEI-tLA was further increased using ternary complexes with HA, which was comparable to or higher than Lipofectamine™ 2000 and PEI25. The synergism between polyanionic additive (e.g. HA) and electronegative functionality (e.g. thio-ester, -S-CO-) of aliphatic lipids can generates a supersensitive nano-formulation which could be a potential carrier for the modification of primary cells. View Large Image | Download PowerPoint Slide