Structure−Function Correlation of Chloroquine and Analogues as Transgene Expression Enhancers in Nonviral Gene Delivery

To understand how chloroquine (<b>CQ</b>) enhances transgene expression in polycation-based, nonviral gene delivery systems, a number of <b>CQ</b> analogues with variations in the aliphatic amino side chain or in the aromatic ring are synthesized and investigated. Our studies indicate that the aliphatic amino moiety of <b>CQ</b> is essential to provide increased gene expression. Further, the enhancements are more dramatically affected by changes to the aromatic ring and are positively correlated to the strength of intercalation between DNA and the <b>CQ</b> analogues. Quinacrine (<b>QC</b>), a <b>CQ</b> analogue with a fused acridinyl structure that can strongly intercalate DNA, enhances transfection similarly to <b>CQ</b> at a concentration 10 times lower, while <i>N</i><sup>4</sup>-(4-pyridinyl)-<i>N</i>,<i>N</i><sup>1</sup>-diethyl-1,4-pentanediamine (<b>CP</b>), a <b>CQ</b> analogue that has a weakly intercalating pyridinyl ring, shows no effect on gene expression. Subtle change on the 7-substituent of the chloroquine aromatic structure can also greatly affect the ability of the <b>CQ</b> analogues to enhance transgene expression. Transfection in the presence of <i>N</i><sup>4</sup>-(7-trifluoromethyl-4-quinolinyl)-<i>N</i><sup>1</sup>,<i>N</i><sup>1</sup>-diethyl-1,4-pentanediamin e (<b>CQ7a</b>) shows expression efficiency 10 times higher than in the presence of <b>CQ</b> at same concentration, while transfection in the presence of <i>N</i><sup>4</sup>-(4-quinolinyl)-<i>N</i><sup>1</sup>,<i>N</i><sup>1</sup>-diethyl-1,4-pentanediamine (<b>CQ7b</b>) does not reveal any enhancing effects on expression. Through a number of comparative studies with <b>CQ</b> and its analogues, we conclude that there are at least three mechanistic features of <b>CQ </b>that lead to the enhancement in gene expression:  (i) pH buffering in endocytic vesicles, (ii) displacement of polycations from the nucleic acids in polyplexes, and (iii) alteration of the biophysical properties of the released nucleic acid.