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. 2011 Dec 4;11(1):82-90.
doi: 10.1038/nmat3187.

Biodegradable Poly(amine-Co-Ester) Terpolymers for Targeted Gene Delivery

Free PMC article

Biodegradable Poly(amine-Co-Ester) Terpolymers for Targeted Gene Delivery

Jiangbing Zhou et al. Nat Mater. .
Free PMC article


Many synthetic polycationic vectors for non-viral gene delivery show high efficiency in vitro, but their usually excessive charge density makes them toxic for in vivo applications. Here we describe the synthesis of a series of high molecular weight terpolymers with low charge density, and show that they exhibit efficient gene delivery, some surpassing the efficiency of the commercial transfection reagents Polyethylenimine and Lipofectamine 2000. The terpolymers were synthesized via enzyme-catalyzed copolymerization of lactone with dialkyl diester and amino diol, and their hydrophobicity adjusted by varying the lactone content and by selecting a lactone comonomer of specific ring size. Targeted delivery of the pro-apoptotic TRAIL gene to tumour xenografts by one of the terpolymers results in significant inhibition of tumour growth, with minimal toxicity both in vitro and in vivo. Our findings suggest that the gene delivery ability of the terpolymers stems from their high molecular weight and increased hydrophobicity, which compensates for their low charge density.


Figure 1
Figure 1. Synthesis and characteristics of polymers and terpolymer/DNA complexes
a, Two-stage process for terpolymerization of lactone with DES and MDEA. b, Visualization of |||-20% PDL/DNA polyplexes at 100:1 weight ratio using TEM. Scale bar represents 1 µm.
Figure 2
Figure 2. Gene delivery efficiency and toxicity of terpolymer
a, Gene delivery efficiency of terpolymers on HEK293 cells (open square) and A549 cells (open diamond). Polyplexes of DNA and terpolymer were prepared at a weight ratio of 1:100. Transfections by Lipofectamine 2000 and PEI were performed according to the manufacturer’s standard protocols. The same amount of DNA was used for all transfection experiments. b, The effect of III-20% PDL to DNA ratio on transfection efficiency on HEK293 cells. The same amount of DNA but various amount of polymer, as indicated, was used for forming polyplexes. c, d, Toxicity of PEI (open square) and III-20% PDL (open diamond) on HEK293 cells (c) and A549 cells (d). Toxicity is given as the percentage of viable cells remained after treatment for three days, compared against the control vehicle treated cells. Cell number was determined by the standard MTT assay. All experiments were carried out in triplicate and the standard deviation is shown by the error bars. Luciferase signal was detected 48 h after transfection. Luciferase signal is normalized by the amount of protein for comparison.
Figure 3
Figure 3. Coating III-20% PDL/DNA polyplexes with peptide polyE–mRGD for improved stability in vitro and gene delivery in vivo
a, Comparison between coated and uncoated polyplexes for in vivo luciferase transfection. Polyplexes were administrated through tail vein injection. Luciferase expression was determined 48 h after the last treatment of three consecutive daily treatments. b, Coating with polyE–mRGD prevented change of surface charge in serum. Polyplexes was prepared by mixing polymer and DNA and incubated at room temperature for 10 min. Then, polyE–mRGD was added at the indicated concentrations and coating allowed for 5 min. The zeta potential of the coated polyplexes was determined 5 min after their incubation in NaAc buffer containing 10% FBS. c, Change of coated and non-coated polyplex size in NaAc buffer containing 10% FBS. Coated polyplexes were prepared as described in b. Sizes of polyplexes were determined by dynamic light scattering at various time intervals. d, The effect of coating on transfection efficiency in vitro. When polyE–mRGD was added at 2.5:1 and 5:1 peptide/DNA weight ratios, a slight decrease in transfection efficiency of III-20% PDL/pLucDNA polyplex was observed. In contrast, when the peptide to DNA ratio increased to 10:1, the transfection efficiency of the polyplex decreased by over 3,000 times. All experiments were carried out in triplicate and the standard error are shown by the error bars.
Figure 4
Figure 4. Evaluation of coated III-20%PDL/TRAIL DNA polyplexes in vivo
a, Flow cytometry analysis of A549 cells transfected with pEGFP, using III-20% PDL, Lipofectamine 2000 and PEI. A549C: control untransfected A549 cells. Cell transfection was performed in a 6-well plate. DNA complexes with polymer or lipofectamine were prepared as described in the main text. 4 µg of plasmid was used for all vectors. GFP analysis was performed two days after transfection. b, Change of mouse weight during systemic administration of III-20% PDL/TRAIL polyplexes. Polyplexes were administrated through tail vein injection three days a week, at the dose of 1.7 mg per mouse, for 6 weeks. The dose was chosen based on the maximum amount of the polymer that can be used in 200 µl buffer solution for injection. Luc: luciferase. c, Antitumour effects of III-20% PDL/TRAIL polyplexes. Treatment started when the tumour reached a size of ∼50 mm3. Data are given as mean (n = 5). d, TUNEL staining demonstrated a marked increase in the number of apoptotic cells following treatment with TRAIL, as indicated by the significantly increased number of TUNEL-positive cells in the coated III-20% PDL/TRAIL group (right), as compared with the control group (left); ×20 magnification.

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