Our objective was to design a water-soluble lipopolymer (WSLP) and an inter
leukin-12 (IL-12) expression plasmid for enhanced delivery of the IL-12 gen
e. 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 t
o PEI was about 47% and the average molecular weight of WSLP was approximat
ely 2000 Da. The mean particle size of WSLP/p2CMVmIL-12 complexes formulate
d in 5% glucose was 26 to 62 nm and zeta 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 dem
onstrated higher transfection in CT-26 cells compared with the DNA formulat
ions prepared using PEI of molecular weights 1800, 10,000 and 25,000 Da. Tr
ansfection 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 rati
o of 30/1. There was an increase in the level of IL-12 when free or complex
ed 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 (IRE
S). At 48 hours post-injection of WSLP/p2CMVmIL-12 complexes into BALB/c mi
ce bearing CT-26 subcutaneous tumors, the levels of IL-12, IFN-gamma, and n
itric 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 surviv
al 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.