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Intratumoral delivery of p2CMVmIL-12 using water-soluble lipopolymers
R I Mahato1, M Lee, S Han
1Center for Controlled Chemical Delivery, Department of Pharmaceutics and Pharmaceutical Chemistry, University of Utah, Salt Lake City, Utah 84112, USA.
Abstract:
Our objective was to design a water-soluble lipopolymer (WSLP) and an interleukin-12 (IL-12) expression plasmid for enhanced delivery of the IL-12 gene. We synthesized WSLP using branched polyethylenimine (PEI) of 1800 Da and cholesteryl chloroformate, and constructed p2CMVmIL-12, encoding the IL-12 subunits p35 and p40, each under the transcriptional control of a separate cytomegalovirus (CMV) promoter. The percentage of cholesterol conjugated to PEI was about 47% and the average molecular weight of WSLP was approximately 2000 Da. The mean particle size of WSLP/p2CMVmIL-12 complexes formulated in 5% glucose was 26 to 62 nm and xi potential was 8 to 60 mV. The WSLP/p2CMVmIL-12 complexes were nontoxic to CT-26 colon carcinoma cells at the N/P ratio (nitrogen atoms of WSLP/phosphate of plasmid DNA) of 20 and below; PEI25000/pDNA complexes were highly toxic. WSLP/p2CMVmIL-12 complexes demonstrated higher transfection in CT-26 cells compared with the DNA formulations prepared using PEI of molecular weights 1800, 10,000 and 25,000 Da. Transfection efficiency increased with an increase in N/P ratios from 5 to 15, then there was no significant increase in transfection up to the N/P ratio of 30/1. There was an increase in the level of IL-12 when free or complexed p2CMVmIL-12 was compared with free or complexed pIRESmIL-12 in which the p35 and p40 subunits were linked to the internal ribosome entry sites (IRES). At 48 hours post-injection of WSLP/p2CMVmIL-12 complexes into BALB/c mice bearing CT-26 subcutaneous tumors, the levels of IL-12, IFN-gamma, and nitric oxide (NO) in the supernatant of the cultured tumors were higher for the WSLP/p2CMVmIL-12 complexes than for the naked p2CMVmIL-12, WSLP, and 5% glucose injected groups. There was a significant improvement in the survival rate and the inhibition of tumor growth after a single injection of WSLP/p2CMVmIL-12 complexes. We have designed an effective, nontoxic WSLP and an IL-12 expression plasmid with two CMV promoters.