[Effect of gene transfer using nanoparticles as gene vector in different animal models]

To evaluate the effect of antisense monocyte chemotactic protein-1 (A-MCP-1) nanoparticles (NPs) as gene carrier on gene transfer in two kinds of animal models.

Poly (lactic acid-co-glycolic acid) (PLGA) was used to make the NPs loaded with A-MCP-1 through a double-emulsion/solvent evaporation technique. NPs size was assessed by dynamic laser defractometer. The particle morphology was observed by scanning electron microscopy. DNA content in the NPs was measured by dissolving known amounts of NPs in chloroform and extracting DNA with water. In vitro release was performed in tris-EDTA buffer at 37 degrees C using double-chamber diffusion cells. The receiver buffer was replaced daily. The A-MCP-1 NPs was transfected into the cultured smooth muscle cells. PCR was used to evaluate the transfection of A-MCP-1. Cationic lipid (Lipofectamine) was used to transfect A-MCP-1 as control. After 48 hours incubation, cells were digested and examined by polymerase chain reaction. Twenty New Zealand white rabbits under jugular vein to artery bypass grafting procedure were divided into four groups: the first group received grafts treated with A-MCP-1 NPs, the second group received grafts treated with cationic liposome (dioleoyl trimethyl ammonium propane)-A-MCP-1, the third group received grafts treated with plasmid DNA, and the fourth group received grafts without transfection as control. Fourteen days after surgery the grafts were harvested. The expression of A-MCP-1 and its effect on MCP-1 in vein grafts were detected by dot blotting. The morphology of the grafts was investigated. To establish abdominal aortic aneurysms rats model, rats were randomly divided into three groups: A-MCP-1 NPs injection group, shame NPs injection group and control groups (without injection). Two weeks after surgery, diameter of abdominal aorta was measured and aortic tissue was obtained for PCR analysis to evaluate the A-MCP-1 expression. Western blot were applied to detect the inhibitory effect to the expression of MCP-1 mRNA and CD68 protein by A-MCP-1 NPs.

NPs size ranged 198nm to 205nm with average around 201.4 nm. DNA content in the NPs was 4.14%. NPs showed steady release rate in vitro in Tris-EDTA solution. It released faster in the first week then maintained a slowly sustained release up to 16 days. In cell culture A-MCP-1 gene successfully transfected into smooth muscle cells by NPs vector. In vein grafting animal model, A-MCP-1 expression was detected in the vascular walls of NPs and cationic lipid treated groups. The degree of vascular hyperplasia in the gene NPs treated group was significantly lower than that in control group. There was no significant difference in the inhibition of intimal hyperplasia between NPs and cationic lipid treated groups. Two weeks after transfection in abdominal aortic aneurysm rats models, the abdominal aortic diameter of A-MCP-1 NPs injection group was (1.79 +/- 0.12) mm, significantly smaller than that of control groups [shame NPs group was (2.58 +/- 0.21) mm, and saline group was (2.63 +/- 0.29) mm] (P < 0.01). The expressions of MCP-1 mRNA and CD68 protein in A-MCP-1 NPs injection group were 12.5 +/- 1.5 and 17.6 +/- 2.1, which were much lower than those in control group [in shame NPs group, which were 35.7 +/- 4.5, 42.3 +/- 5.7 (P < 0.01), and saline group which is 32.4 +/- 3.9, 39.8 +/- 4.8 (P < 0.01)]. Specific band of A-MCP-1 was detected only in the A-MCP-1 NPs injection group by PCR.

A-MCP-1 gene NPs can be successfully used in rabbit vein grafting model and abdominal aortic aneurysm rats models, and may be potentially applied in clinical practice.