847. Hyaluronic Acid as a Self-Assembled Coating of Plasmid / Polycation Complexes for Cell-Specific Gene Delivery

Abstract

Top of pageAbstract As non-viral vectors for gene therapy, uncontrolled interaction of the cationic DNA/polycation complexes with the extracellular matrix or the serum proteins has been obstacle to the in vivo gene therapy. We have reported that the poly(ethylene glycol) derivatives having carboxylic acid-side chains (PEG-C) coated the DNA/polycation complexes, and protect them from these uncontrollable interactions1. Introducing the ligand into the side chains of the polymer allowed the receptor-mediated cell-specific and highly effective gene transfection2. PEG-C could be deposited onto the surface of the plasmid/ polycation complexes, and the ternary complex was formed. But common polyanions like polyacrylic acid, or heparin would decompose the complex and substitute with the plasmid releasing the free DNA molecules. Hyaluronic acid (HA) is a natural acidic mucopolysaccharide, and was found to coat the DNA/polycation complexes without decomposition. CD44, a receptor for HA, is known to be overexpressed on the various tumor cell surfaces, and HA is thus expected to form ternary complexes with plasmid and polycation, expressing the binding site to the receptors on the malignant cell surfaces. In this study, we investigated the effect of the HA-coating of the plasmid/polycation complex on the suppression of the undesired interaction with serum protein, and the gene-transfection efficiency on the CD44-presenting CHO cells. (1) Microscopic observation of the plasmid/polycation/HA ternary complex: The fluorescence microscopic images of the DAPI-DNA, fluorescein-PEI and rhodamine-HA mixture were all observed at the same position as brightening dots, indicating the formation of the ternary complexes. Albumin induced the aggregation of the plasmid/polycation binary complexes, but not the HA-coated ternary complexes, which remained to fluctuate for a long time after the albumin addition, showing the protecting effect of HA against the serum protein. The efficient binding of the fluorescence-labeled plasmid/PEI/HA ternary complex to the CHO cells was observed under the fluorescence microscopic observation. (2) Gene expression study: HA obviously enhanced the gene-expression on CHO cells mediated by HelΔ1 (a cationic amphiphilic peptide) up to 30-fold higher than those without HA. Transfection efficiency mediated by chitosan, or linear-PEI was also 3-4 times improved by HA. Such enhancing effect was, however, not observed on the CD44-negative COS-7 cells. Far excess addition of HA or the pretreatment by the polysaccharide diminished the gene-expression, indicating the receptor-mediated mechanism of the HA-coated ternary complex.