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

Abstract

To understand how chloroquine (CQ) enhances transgene expression in polycation-based, nonviral gene delivery systems, a number of CQ 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 CQ 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 CQ analogues. Quinacrine (QC), a CQ analogue with a fused acridinyl structure that can strongly intercalate DNA, enhances transfection similarly to CQ at a concentration 10 times lower, while N(4)-(4-pyridinyl)-N(1),N(1)-diethyl-1,4-pentanediamine (CP), a CQ 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 CQ analogues to enhance transgene expression. Transfection in the presence of N(4)-(7-trifluoromethyl-4-quinolinyl)-N(1),N(1)-diethyl-1,4-pentanediamin e (CQ7a) shows expression efficiency 10 times higher than in the presence of CQ at same concentration, while transfection in the presence of N(4)-(4-quinolinyl)-N(1),N(1)-diethyl-1,4-pentanediamine (CQ7b) does not reveal any enhancing effects on expression. Through a number of comparative studies with CQ and its analogues, we conclude that there are at least three mechanistic features of CQ 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.

Efficient polyethylenimine-mediated gene delivery proceeds via a caveolar pathway in HeLa cells

“…Because of its aromatic ring structure, chloroquine is able to intercalate DNA, thereby causing a conformational change that is capable of inhibiting enzymatic DNA degradation. 35,36 The addition of 5 mM chloroquine prevents total DNA digestion, as evidenced by the faint pGL3…”

Generation of Carrier Peptides for the Delivery of Nucleic Acid Drugs in Primary Cells

“…These data indicate a distinct increase in uptake efficacy of both of the novel branched hCT derivatives relative to the unbranched and nearly uncharged CF-hCTA C H T U N G T R E N N U N G (9-32) as well as to CF-hCTA C H T U N G T R E N N U N G (9-32)-br. Thereby, the efficacy of peptide uptake was similar to that of CF-TatA C H T U N G T R E N N U N G (48)(49)(50)(51)(52)(53)(54)(55)(56)(57)(58)(59)(60). However, in contrast to CF-2br, which showed no significant cellular uptake, the peptide CF-k7 was effectively internalised.…”

“…[30] It is thought to protect the endocytotically internalised nucleic acids from intracellular degradation through neutralisation of the acidic compartments, decreasing endosomal delivery to lysosomes, and destabilising the complex between carrier peptide and cargo. [60] Erbacher et al detected this complex dissociation in the presence of > 20 mm CQ, a concentration described to be easily reached inside endocytotic vesicles of HepG2 cells. Indeed, both transfection rates and expression levels increased proportionally with CQ concentration (up to 125 mm).…”

“…Images were taken using either fluorescence (A-C, E, G) or confocal (D, F, H) microscopy; scale bars are indicated. [48][49][50][51][52][53][54][55][56][57][58][59][60]. Samples were separated in a 1 % agarose gel and stained with EtBr.…”

Chloroquine-enhanced gene delivery mediated by carbon nanotubes

“…There was a clear enhancement of transfection efficiency by using chloroquine-loaded DWCNTs, of about two orders of magnitude when compared with controls. Note that this result was obtained at a chloroquine concentration in the entire transfection mixture lower than 1 µM (estimated from the DWCNT concentration in the transfection mixture and loading yield of the nanotubes with chloroquine) that was much lower than the concentration normally added to culture media as free drug for optimum transfection (of 25 -100 µM) [36,37,38]; and so was highly unlikely to be due to leeching out of the chloroquine prior to entry of CNTs inside cells. The other forms of DWCNTs assayed for optimizing chloroquine loading were also tested to investigate the effect of chloroquine loading on DWCNTs on the transfection efficiency.…”