Identification of novel superior polycationic vectors for gene delivery by high-throughput synthesis and screening of a combinatorial library

• Published in: Pharmaceutical research 2007-08, Vol.24 (8), p.1564-1571
• Main Authors: Thomas, Mini, Lu, James J, Zhang, Chengcheng, Chen, Jianzhu, Klibanov, Alexander M
• Format: Article
• Language: English
• Subjects: Acrylates - chemistry; Animals; beta-Galactosidase - genetics; beta-Galactosidase - metabolism; Cell Line, Tumor; Cell Survival; Cercopithecus aethiops; Combinatorial Chemistry Techniques; COS Cells; Cross-Linking Reagents - chemistry; Gene expression; Gene therapy; Genetic Vectors - chemistry; Genetic Vectors - genetics; Humans; Imines - chemistry; Luciferases - genetics; Luciferases - metabolism; Male; Mice; Molecular Structure; Pharmaceuticals; Polyamines - chemistry; Polyethylenes - chemistry; Toxicity; Transfection - methods
• ISSN: 0724-8741; 1573-904X
• DOI: 10.1007/s11095-007-9279-3

Low efficiency and toxicity are two major drawbacks of current non-viral gene delivery vectors. Since DNA delivery to mammalian cells is a multi-step process, generating and searching combinatorial libraries of vectors employing high-throughput synthesis and screening methods is an attractive strategy for the development of new improved vectors because it increases the chance of identifying the most overall optimized vectors. Based on the rationale that increasing the effective molecular weight of small PEIs, which are poor vectors compared to the higher molecular weight homologues but less toxic, raises their transfection efficiency due to better DNA binding, we synthesized a library of 144 biodegradable derivatives from two small PEIs and 24 bi- and oligo-acrylate esters. A 423-Da linear PEI and its 1:1 (w/w) mixture with a 1.8-kDa branched PEI were cross-linked with the acrylates at three molar ratios in DMSO. The resulting polymers were screened for their efficiency in delivering a beta-galactosidase expressing plasmid to COS-7 monkey kidney cells. Selected most potent polymers from the initial screen were tested for toxicity in A549 human lung cancer cells, and in vivo in a systemic gene delivery model in mice employing a firefly luciferase expressing plasmid. Several polycations that exhibited high potency and low toxicity in vitro were identified from the library. The most potent derivative of the linear 423-Da PEI was that cross-linked with tricycle-[5.2.1.0]-decane-dimethanol diacrylate (diacrylate 14), which exhibited an over 3,600-fold enhancement in efficiency over the parent. The most potent mixed PEI was that cross-linked with ethylene glycol diacrylate (diacrylate 4) which was over 850-fold more efficient than the physically mixed parent PEIs. The relative efficiencies of these polymers were even up to over twice as high as that of the linear 22-kDa PEI, considered the "gold standard" for in vitro and systemic gene delivery. The potent cross-linked polycations identified were also less toxic than the 22-kDa PEI. The optimal vector in vivo was the mixed PEI cross-linked with propylene glycol glycerolate diacrylate (diacrylate 7); it mediated the highest gene expression in the lungs, followed by the spleen, with the expression in the former being 53-fold higher compared to the latter. In contrast, the parent PEIs mediated no gene expression at all under similar conditions, and injection of the polyplexes of the 22-kDa PEI at its optimal N/P of