Preparation and Quality Test of Superparamagnetic Iron Oxide Labeled Antisense Oligodeoxynucleotide Probe: A Preliminary Study

Abstract

Molecular imaging of tumor antisense gene techniques have been applied to the study of magnetic resonance (MR) gene imaging associated with malignant tumors. In this study, we designed, synthesized, and tested a novel molecular probe, in which the antisense oligodeoxynucleotide (ASODN) was labeled with superparamagnetic iron oxide (SPIO), and its efficiency was examined by in vitro MR imaging after SK-Br-3 mammary carcinoma cell lines (oncocytes) transfection. The SPIO-labeled ASODN probe was prepared through SPIO conjugated to ASODN using a chemical cross linking method. Its morphology and size were detected by atomic force microscope, size distribution were detected by laser granulometer, the conjugating rate and biological activity were determined by high performance liquid chromatography, and the stability was determined by polyacrylamide gel electrophoresis. After that, the probes were transfected into the SK-Br-3 oncocytes, cellular iron uptake was analyzed qualitatively at light and electron microscopy and was quantified at atomic absorption spectrometry, and the signal change of the transfected cells was observed and measured using MR imaging. The morphology of the SPIO-labeled ASODN probe was mostly spherical with well-distributed scattering, and the diameters were between 25 and 40 nm (95%) by atomic force microscope and laser granulometer, the conjugating rate of the probe was 99%. Moreover, this probe kept its activity under physiological conditions and could conjugate with antisense oligodeoxynucleotide. In addition, light microscopy revealed an intracellular uptake of iron oxides in the cytosol and electron microscopic studies revealed a lysosomal deposition of iron oxides in the transfected SK-Br-3 oncocytes by antisense probes, some of them gathered stacks, and the iron content of the group of transfected SK-Br-3 oncocytes by antisense probe is significantly higher (18.37 +/- 0.42 pg) than other contrast groups, the MR imaging showed that transfected SK-Br-3 oncocytes by antisense probe had the lowest signal of all. The SPIO-labeled ASODN probe shows unique features including well-distributed spherical morphology, high conjugating rate and loading efficiency, and the signal intensity of SPIO-labeled ASODN-transfected SK-Br-3 oncocytes is reduced in MR imaging. These results indicate that the SPIO-labeled ASODN probe is potentially useful as a MR targeting contrast enhancing agent to specifically diagnose tumors which had over-expression of the c-erbB2 oncogene at an early stage.

Distribution of c-erbB2 Antisense Probe Labeled with Superparamagnetic Iron Oxide Nanoparticles in the Major Organs of Mice on MR Imaging

“…O ur preliminary results showed [7,8] that antisense probe spheres with a diameter of about 25 -40 nm disperse evenly, the rate of connection with the ASODN is 100% while still maintaining the original biological activity, and the connection is stable. In transfected SK-Br3 cells with high expression of the c-erbB2 oncogene, varying amounts of blue iron particles could be seen in the cytoplasm, and the intracellular iron content was significantly higher than in cells without the c-erbB2 oncogene.…”

“…We prepared the c-erbB2 antisense probe labeled with SPIO nanoparticles by chemical crosslinking following specific steps described previously [7,8] (Chinese patent No. ZL 200710092512.5).…”

“…However, SPIO enters and aggregates in the organism in a nontargeted manner, which leads to a low specificity. To solve this problem, we took the c-erbB2 oncogene as the target, and successfully prepared a c-erbB2 antisense probe labeled with SPIO nanoparticles based on the antisense gene theory [7,8]. Following preliminary experiments, we determined the related index and investigated the use of the probe as a contrast agent in MR imaging with the aim of revealing the rules governing its dynamic variation in animals to lay the foundation for its use in future MR studies in living organisms.…”

Bench-To-Bedside Translation of Magnetic Nanoparticles

“…Since its development, several research groups have optimized and implemented the concept of magnetic targeting and transfection of genetic materials. MNPs have been employed successfully for in vivo and in vitro transfection of plasmids, antisense oligodeoxynucleotides, siRNA and shRNA [48,80, 81]. …”

Antisense Oligonucleotides and Magnetic Nanoparticles for Targeted Diagnostic and Cancer Treatment

“…Recently, antisense ODN has been labeled with iron nanoparticles for tumor antisense gene detection with magnetic resonance gene imaging. This novel molecular probe could be potentially useful as magnetic resonance targeting contrast enhancing agent to diagnose tumors at an early stage [14] see in (Figure 1). …”