Intratumoral delivery of p2CMVmIL-12 using water-soluble lipopolymers.
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.
Mahato
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RI
Center for Controlled Chemical Delivery, Department of Pharmaceutics and Pharmaceutical Chemistry, University of Utah, Salt Lake City, Utah 84112, USA.
Lee
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M
Han
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S
Maheshwari
A
A
Kim
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SW
D000818:Animals; D003587:Cytomegalovirus / Q000235:genetics / Q000378:metabolism; D018450:Disease Progression; D005260:Female; D015316:Genetic Therapy; D005822:Genetic Vectors; D006801:Humans; D007267:Injections; D007371:Interferon-gamma / Q000096:biosynthesis; D018664:Interleukin-12 / Q000235:genetics* / Q000378:metabolism; D050356:Lipid Metabolism; D008055:Lipids / Q000737:chemistry*; D051379:Mice; D008807:Mice, Inbred BALB C; D015394:Molecular Structure; D009374:Neoplasms, Experimental / Q000628:therapy*; D009569:Nitric Oxide / Q000096:biosynthesis; D010316:Particle Size; D010957:Plasmids; D011108:Polymers / Q000737:chemistry* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D014162:Transfection; D019737:Transplants; D014407:Tumor Cells, Cultured; D014867:Water / Q000737:chemistry
D008055:Lipids; D011108:Polymers; D014867:Water; D018664:Interleukin-12; D009569:Nitric Oxide; D007371:Interferon-gamma