171. PEG-b-PPA/DNA Micelles for Liver Targeted-Gene Delivery
Authors: Xuan Jiang, Hui Dai, Kam W. Leong, Hai-Quan Mao
Journal: Molecular Therapy (2006)

Liver is a critically important target for gene medicine applications because of the access of the transgene product to systemic circulation, and because it is the site of many metabolic genetic disorders, viral infection and malignancies. At present, the full potential of liver- targeted gene transfer is hindered by a lack of safe and efficient gene carriers. Recently, we have developed a new class of polyphosphoramidates (PPAs) as non-viral gene carriers. These PPA carriers exhibit favorable biocompatibility, DNA compaction and protection capacity, high transfection efficiency in several cell types in culture [1]. However, PPA/DNA complexes, like many polycationic carriers, aggregate severely upon incubation in serum containing medium. This aggregation will lead to reduced cellular uptake by parenchymal cells and lead to sequestration by reticuloendothelial system following intravenous injection, rendering the complexes inefficient for liver-targeted gene delivery. To improve the in vivo stability of PPA/DNA complexes, we synthesized PEG-b-PPA copolymers by initiating the cyclic monomer, 4-methyl-2-oxo-2-hydro-1,3,2-dioxaphospholane, with poly(ethylene glycol)- ate (PEGate) anion macroinitiators with Mws of 2,000 and 12,000, followed by side chain conjugation before deprotection of the sidechain amine groups [1]. The average diameters of PEG2000-b-PPA/DNA micelles and PEG12000-b-PPA/DNA micelles at N/P=10 were 80 nm and 150 nm, respectively. Transmission electron microscopic examination of PEG2000-b-PPA/DNA micelles confirmed the size measurement and revealed the spherical or rod-like morphology of the micelles. In HEK293 cells, the transfection efficiency of PEG2000-b-PPA/DNA micelles was similar to that of PPA/DNA complexes, and comparable to PEI; whereas the transfection efficiency of PEG12000-b-PPA/DNA micelles was about 4-fold lower than that of PEG2000-b-PPA/DNA micelles. For the in vivo study, PEG2000-b-PPA/DNA micelle was chosen and injected through tail vein of Balb/c mice at a DNA dose of 25 μg in 250 μl saline in 30 seconds. Following tail vein injection, PEG2000-b-PPA/DNA micelles mediated 5-, 8- and 150-fold higher luciferase expression than PPA/DNA complexes in the liver, spleen and lung. Although the highest transgene expression was found in the lung, this improved transfection efficiency, over PPA/DNA complexes, is likely the result of increased stability of PEG2000-b- PPA/DNA micelles in serum. Intrabiliary infusion in Wistar rats was performed at a DNA dose of 20 μg in 4 ml of PEG2000-b-PPA/ DNA micelle solution over 20 minutes using an infusion pump. After intrabiliary infusion, PEG2000-b-PPA/DNA micelles also mediated 4-fold higher average luciferase expression in the liver than PPA/DNA complexes on day 3. No expression was detected in other major organs (lung, kidney, heart, spleen). Moreover, luciferase expression was only found in one of the liver lobes for PPA/DNA complexes, in contrast to a relatively widespread expression among different lobes of the liver in the micelle group. References: 1. Mao HQ and Leong KW. Adv. Genet. 2005; 53: 275–306.