Preparation of liposome-coated oligonucleotide labeled with ^99mTc and its uptake in vascular smooth muscle cells

Preparation of liposome-coated oligonucleotide labeled with 99m Tc and its uptake in vascular smooth muscle cells 1 first-author 1 corresp Corresponding author. E-mail: zhyx1229@163.com Corresponding author. E-mail: zhyx1229@163.com zhyx1229@163.com 1 1 Department of Nuclear Medicine, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Key Laboratory of Molecular Imaging of Hubei Province, Wuhan 430022, China Department of Nuclear Medicine, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Key Laboratory of Molecular Imaging of Hubei Province, Wuhan 430022, China To explore the preparation method of liposome-coated 99m Tc-labeled antisense oligonucleotide (ASON), targeteing the proliferating cell nuclear antigen (PCNA), and to explore the biological characteristics and the uptake kinetics of a radiolabeled probe in vascular smooth muscle cells, an 18-base single-stranded antisense oligonucleotide targeting PCNA mRNA and the complementary strand (sense oligonucleotide, SON) were synthesized. The ASON (SON) was labeled with 99m Tc, by conjugating the bifunctional chelator (hydrazino nicotinamide, HYNIC), and purified through a gel filtration column of Sephadex G-25. The product was then encapsulated in cationic liposome (oligofectamineTM). The radiolabeling efficiency, radiochemical purity, stability of the liposome-coated 99m Tc-HYNIC- ASON in a phosphate buffered solution (PBS), and fresh human serum and its uptake rate were studied. There was no significant difference between the 99m Tc radiolabeling efficiencies of HYNIC-ASON and HYNIC-SON, which were 60.04% ± 1.92% and 59.60% ± 2.53%, respectively ( P > 0.05, n = 5). The radiochemical purity of the liposome-coated 99m Tc-HYNIC-ASON was 94.70% ± 1.90% ( n = 5). And after incubation with PBS and fresh human serum at a concentration of 1.2 µg·mL -1 for 120 min, the radiochemical purities were 92.27% and 91.55% respectively. At 90 min after transfection, the uptake rate of the liposome-coated 99m Tc-HYNIC-ASON reached its peak of 83.8% ± 5.92% in vascular smooth muscle cells (VSMCs) and was much higher than that of the nonliposome-coated 99m Tc- HYNIC-ASON, which was 11.16% ± 0.54% ( P < 0.01, n = 4). The labeling method of PCNA ASON (SON) conjugated by HYNIC has been proved successful. The liposome was able to enhance the ASON (SON) uptake in VSMCs, and could be widely used as a safe, convenient, effective gene transfer carrier. Proliferating cell nuclear antigen Oligonucleotides Antisense Isotope labeling Liposome Vascular smooth muscle cells References Scovassi A I , Prosperi E . Methods Mol Biol , 2006 , 314 : 457 - 475 . Methods Mol Biol Pawlikowski M , Gruszka A , Kurnatowska I , et al . Folia Histochem Cytobiol , 2006 , 44 ( 1 ): 37 - 41 . Folia Histochem Cytobiol Sampaio-Goes F C , Oliveira D T , Dorta R G , et al . Head Neck , 2005 , 27 ( 11 ): 982 - 989 . Head Neck Miniati D N , Hoyt E G , Feeley B T , et al . Circulation , 2000 , 102 ( 3 ): 237 - 242 . Circulation Abrams M J , Juweid M , Tenkate C I , et al . J Nucl Med , 1990 , 31 ( 12 ): 2022 - 2028 . J Nucl Med Lavezzi A M , Milei J , Grana D R , et al . Int J Cardiol , 2003 , 92 ( 1 ): 59 - 63 . Int J Cardiol Tamm I , Dorken B , Hartmann G . Lancet , 2001 , 358 ( 9280 ): 489 - 497 . Lancet Qin G , Zhang Y , Cao W , et al . Eur J Nucl Med Mol Imaging , 2005 , 32 ( 1 ): 6 - 14 . Eur J Nucl Med Mol Imaging Zhang Y R , Zhang Y X , Cao W , et al . J Nucl Med , 2005 , 46 ( 6 ): 1052 - 1058 . J Nucl Med Zhang Y M , Liu N , Zhu Z H , et al . Eur J Nucl Med , 2000 , 27 ( 11 ): 1700 - 1707 . Eur J Nucl Med Cornelis S , Vandenbranden M , Ruysschaert J M , et al . DNA Cell Biol , 2002 , 21 ( 2 ): 91 - 97 . DNA Cell Biol Igarashi S , Hattori Y , Maitani Y . J Control Release , 2006 , 112 ( 3 ): 362 - 368 . J Control Release 10.1016/S1001-8042(07)60027-X R730.44,R817.4 Supported by Natural Science Foundation of China (No.30271439) 2006-09-15 2007-04-20 2021-01-19 2007 Nuclear Science and Techniques 02 18