Intratumoral Delivery of p2CMVmIL-12 Using Water-Soluble Lipopolymers

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.

Effective transfer of interleukin-12 gene to solid tumors using a novel gene delivery system, poly [D,L-2,4-diaminobutyric acid]

“…This is smaller than that of cationic liposome complexes (11357235 nm), 24 but larger than that of PAGA (100 nm) 18 or WSLP (26-62 nm). 19 In our preliminary experiments, the size can be decreased to 100 nm, when the complex was prepared in MQ water (not in PBS). However, the luciferase activity of the complex in MQ water was extremely (1/180) lower than that of the complex prepar in PBS (data not shown).…”

“…Furthermore, nonviral vectors may be good clinical options because they may be more efficient and less labor intensive than viral vectors, although the present gene transfection efficiencies still need to be improved. Recently, nonviral gene delivery systems with polyvinylpyrrolidone (PVP), 16 poly[a-(4-aminobutyl)-L-glycolic acid] (PAGA), 17,18 and water-soluble lipopolymers (WSLP) 19 have been developed and reported. However, the best method for intratumoral delivery of the IL-12 gene remains to be determined.…”

Different mechanisms of saturated versus polyunsaturated FFA-induced apoptosis in human endothelial cells

“…Confluent monolayers of primary HUVECs were transiently transfected (4 h) with 2 mg/ml In373-Myc (generously provided by V. Sexl, Vienna) or with the empty vector (pSRaMSV) using polyethyleneimine 25,000 (pH 7.0, Aldrich; Milwaukee, WI) (22) in serum-free M199. After overnight equilibration in full growth medium, HUVECs were trypsinized and seeded into 35 mm culture plates (2 3 10 7 cells/plate).…”

Water-soluble lipopolymer as a gene carrier to corpus cavernosum

“…[24][25][26] WSLP has high transfection efficiency to A7R5 smooth muscle cells, mouse insulinoma MIN6 cells, CT-26 colon cancer cells in vitro. [24][25][26][27] Corpus cavernosum has high smooth muscle cell content, and therefore, the high transfection efficiency of WSLP to smooth muscle cells suggests its usefulness for gene delivery to corpus cavernosum. In this research, we evaluated WSLP as a gene carrier to corpus cavernosum and the gene delivery condition was optimized.…”

“…24,25 However, the conjugation of cholesterol to PEI1800 increased the transfection efficiency, which was comparable to that of PEI25000. [24][25][26][27] Furthermore, WSLP has low cytotoxicity. [24][25][26] WSLP has high transfection efficiency to A7R5 smooth muscle cells, mouse insulinoma MIN6 cells, CT-26 colon cancer cells in vitro.…”