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OBJECTIVE: To testify that the asialoorosomucoid (ASOR) prepared by us has liver-targeting specificity and to investigate its pharmacokinetic characteristics. METHODS: The distribution of 125I-ASOR in vivo was determined by single photon emission computed tomography (SPECT) and immunohistochemical technique after 125I-ASOR was injected into Sprague-Dawley (S–D) rats through their caudal veins. In vitro, different doses of pEGFP-N1 plasmid were transfected into both HepG2 cells and HT1080 cells with the use of ASOR-poly-L-lysine. At 24 and 48 h after transfection, the expression of green fluorescent protein (GFP) was determined under fluorescent microscope. Pharmacokinetic parameters were calculated according to two-compartment open system model with first-order kinetics. RESULTS: SPECT images showed that 125I-ASOR was located only in liver/stomach and root of caudal vein / bladder at 10 min after injection. The 125I-ASOR radioactivities of organs taken out from S–D rats were different at different times, and about 63% of 125I-ASOR was located in the liver at 10 min after injection. At 30 min after injection a peak of radioactivity was seen in stomach. The times of these two radioactivity peaks were different. Immunohistochemical study of liver frozen sections showed that ASOR was combined mainly with hepatocyte membrane, especially in areas with rich blood flow. In vitro study showed that ASOR targeted specifically cells with asialoglycoprotein receptor (ASGr). GFP expression was detected in HepG2 cells but not in HT1080 cells. Furthermore, the more quantity of pEGFP-N1 transfected and the longer expression time, the higher GFP expression level was in HepG2 cells. The 125I-ASOR pharmacokinetics equation for liver was Ct = 662216e−3.362t  + 8896e−2343t. 125I-ASOR was excreted from liver slowly after an initial rapid decrease. The pharmacokinetic equation for stomach was Ct = –114815e−1.7t  + 1148153e−15t and the half-life of 125I-ASOR in stomach was 4.62 h. CONCLUSIONS: ASOR prepared by us could be an efficient gene transfer vector, ASOR was distributed mainly in the liver and stomach and had high targeting specificity to hepatocytes or hepatic originating cells. [ABSTRACT FROM AUTHOR]

To construct different conformations of a plasmid DNA/vector complex (pcDNA3.1/IFN-γ-ASOR-PLL) and transfect cells of the hepatoma cell line BEL7402 to investigate the optimal conformation of the complex for improved expression efficiency in the target cell.Double-distilled water and adjuvant were added to the naked pcDNA3.1/IFN-γ, target vector ASOR-PLL and the ASOR-PLL–pcDNA3.1/IFN-γ complex to create different conformations; molecules that were transfected into BEL7402 cells and the expression efficiency was determined by measuring theIFN-gconcentration in the culture supernatant by ELISA.Naked pcDNA3.1/IFN-γ DNA distributed linearly in double-distilled water and condensed into a mica configuration in adjuvant; ASOR-PLL had a net-like distribution without adjuvant and a spider-like form in the adjuvant-treated group; the ASOR-PLL–pcDNA3.1/IFN-gcomplex had a divaricate form without adjuvant, but a bead-like or granular conformation in 0.1 and 0.2 mol/L of adjuvant, a homogeneous bacilliform or chromatoid-shaped conformation in 0.3 mol/L adjuvant, and varied shapes in 0.4 and 0.5 mol/L adjuvant. The supernatantIFN-γ expression in the bacilliform/chromatoid conformation complex group was the highest among the different conformation groups and controls. When chloroquine was added the supernatantIFN-γ concentration increased in the liposome group and decreased in the bacilliform/chromatoid conformation group .The two structural molecules and their complex, ASOR-PLL–pcDNA3.1/IFN-γ, were adjustable in the liquid mode. The specific bacilliform/chromatoid conformation of complex was lysosome enzyme-resistant and could play an active role in improving the efficiency of gene expression. The hypothesis that a chromosome-like conformation of the target gene molecule is involved in enhancing exogenous gene expression is proposed. [ABSTRACT FROM AUTHOR]