PEG-b-PPA/DNA micelles improve transgene expression in rat liver through intrabiliary infusion

Abstract

We have developed a new block copolymer gene carrier that comprises of a polyethylene glycol segment and a degradable cationic polyphosphoramidate (PPA) segment. This PEG-b-PPA copolymer carrier formed micelles upon condensation with plasmid DNA in aqueous solution. PEG-b-PPA/DNA micelles exhibited uniform and reduced particle size ranging from 80 to 100 nm and lowered surface charge, compared with complexes of DNA with the corresponding cationic PPA carrier. PEG-b-PPA/DNA micelles maintained similar transfection efficiency as PPA/DNA complexes, which was comparable to that of PEI/DNA complexes in HepG2 cells, but yielded about 16-fold lower transgene expression in primary rat hepatocytes than PPA/DNA complexes. Following bile duct infusion in Wistar rats, PEG-b-PPA/DNA micelles mediated 4-fold higher and more uniform gene expression in the liver than PPA/DNA complexes. Liver function tests and histopathological examination indicated that PEG-b-PPA/DNA micelles showed low toxicity and good biocompatibility in the liver. This study demonstrated the potential of PEG-b-PPA/DNA micelles as an efficient carrier for liver-targeted gene delivery.

Figure 1

Synthetic scheme of PEG-b-PPA copolymer.

Figure 2

Gel electrophoresis mobility of DNA in PPA/DNA complexes and PEG-b-PPA/DNA micelles. Plasmid DNA (500 ng) and various amounts of PPAs or PEG-b-PPA were mixed at the indicated N/P ratio (primary amino groups in polymers to phosphate groups in DNA), followed by electrophoresis on an agarose gel (0.8% w/v) and stained with ethidium bromide.

Figure 3

Average particle size (a) and average zeta potential (b) of PEG-b-PPA/DNA micelles and PPA-DPA/DNA complexes.

Figure 4

TEM images of (a) PPA/DNA complexes (N/P = 10) and (b) PEG-b-PPA/DNA micelles (N/P = 10).

Figure 5

Transfection efficiency of PEI/DNA complexes (N/P = 10), PPA/DNA complexes and PEG-b-PPA/DNA micelles at various N/P ratios in (a) HepG2 cells and (b) primary rat hepatocytes 48 h after transfection. Each bar represents average ± standard division (n = 4). * denotes statistically significant comparison (p < 0.05) by the Student's t test.

Figure 6

Luciferase expression in rats on day 3 following intrabiliary infusion of PPA/DNA complexes (N/P = 10) and PEG-b-PPA/DNA micelles (N/P=10) at a DNA dose of 20 μg per rat. Each bar represents average ± standard division (n = 3).

Figure 7

Serum alanine transaminase (ALT) and aspartate transaminase (AST) levels in rats following intrabiliary infusion of PPA/DNA complexes (N/P = 10) and PEG-b-PPA/DNA micelles (N/P = 10) at a DNA dose of 20 μg per rat. Each bar represents average ± standard division (n = 3).

Figure 8

H&E stained tissue sections of the portal triad region of liver tissue harvested on day 3 from rats received (a) PPA/DNA complexes (N/P = 10) or (b) PEG-b-PPA/DNA micelles (N/P = 10).