(491a) Biodegradable, Multifunctional Poly (amine-co-ester) with Ortho Ester in the Main Chain for the Delivery of Plasmid DNA and siRNA

Zhang, J., Yale University
Deng, Y., Yale University
Saltzman, W. M., Yale University

Safe and effective delivery of genetic materials remains a most challenging problem. Viral vectors are commonly used and highly efficient, but there are many safety issues. Polymers are promising non-viral vectors because of their safety and high chemical versatility, but they usually suffer from low efficiency. Here, we report a family of biodegradable, multifunctional poly(amine-co-ester)s that include ortho ester groups in the main chain (PACEO). These polymers have low toxicity and high efficiency in the delivery of both plasmid DNA and siRNA. The best members of the PACEO family can deliver both plasmid DNA and siRNA with efficiency higher than or comparable to Lipofectamine, which is a standard for in vitro DNA/siRNA transfection.   

The PACEOs were designed with several features: 1) biodegradability; 2) positive charge provided by tertiary amine in the main chain, which is used to bind DNA/RNA; 3) high hydrophobicity to assist in the complex formation; 4) acid-sensitivity provided by the ortho ester groups; 5) high tunability of the different functions, which enables the easy optimization of the polymer to accommodate different cargos. We have previously shown that high hydrophobicity allows stable gene binding with a greatly lowered positive charge density on the polymer, making the polymer less toxic. One major barrier for the delivery of genetic materials is endosomal escape. Polyplexes usually enter cells via endocytosis. Thus, the escape from intracellular vesicles is crucial since the high acidity (pH=4~6) in these vesicles can degrade the genes. It has been reported that acid sensitive polymeric vectors can facilitate the escape of the loaded genes from endosomes because they promptly break apart into low molecular weight products, increasing intra-endosomal osmotic pressure, and enhancing breakdown of the endosomes.

In this study, a novel lipase-catalyzed polymerization enables the synthesis of PACEOs with all these features. A series of novel, lipase-compatible ortho ester monomers were synthesized and copolymerized with several different monomers: diester to provide biodegradability, amino-diol to provide tertiary amine group, and lactone to provide high hydrophobicity. The lipase-catalyzed polymerization enables the fine tune of these functions simultaneously by adjusting the feed ratio of different monomers. Also, to our knowledge, this is the first use of ortho ester bearing monomers in lipase-catalyzed polymerization.

We have shown that PACEOs can complex with plasmid DNA or siRNA and form stable nanocomplexes with size ranging from 120nm to 200nm. The polymers also exhibit pH-dependent degradation: in a weight loss study, the representative PACEO showed less than 10% weight lost at pH7.4 during a 6-day incubation, but had a near complete weight loss at pH 4 within 4 hours. Also, these polymers have minimal toxicity: when tested on HEK293 cell line, PEI killed almost all cells after incubation with 50 ug/mL polymer for 48 hr, whereas cells exposed to PACEO were 100% viable, even up to 500ug/mL polymer.

Several PACEOs were highly efficient for in vitro transfection of plasmid DNA and siRNA. For plasmid DNA, the best PACEO showed an 8-times higher efficiency than Lipofectamine 2000 when delivering plasmid DNA encoding the luciferase gene to HEK293 cell line.  When loaded with plasmid DNA encoding GFP, a single treatment with the best PACEO transfected over 60%  of the treated U87 glioblastoma cells, while Lipofectamine2000 only transfected 30% of the cells. In the delivery of siRNA, the same PACEO showed a comparable efficiency to Lipofectamine RNAiMAX in knocking down luciferase expression in a HEK293 cell line stably transfected with Luciferase gene.

In summary, the PACEOs we report here are novel in chemistry, biodegradable, multifunctional, non-toxic, and highly efficient in the delivery of both plasmid DNA and siRNA. These polymers provide a potent and universal tool for gene therapy. 


Zhou, J., J. Liu, C.J. Cheng, T. Patel, C. Weller, J.M. Piepmeier, Z. Jiang, and W.M. Saltzman, Biodegradable poly(amine-co-ester) terpolymers for targeted gene delivery. Nature Materials, 2012. 11(1): p. 82-90.