Nonspecific Cellular Accumulation Of Liposome-coated ⁹⁹Tc^m-HYNIC-survivin ASODN Is Influenced By Guanine Content And Position Relative To The Promoter

Objective: survivin ASON(antisense oligonucleotide) sequence with two kinds of guanine content and two kingds of promotor's position was synthesized and liposome-coated (99)^Tc^m labeled, to explore whether nonspecific cellular accumulation of (99)^Tc^m -survivin ASODN (antisense oligodeoxynucleotide), in nude mice with human hepatocellular carcinoma, is influenced by guanine content and the position relative to the promoter.Methods:1 Modeling: 7×10⁶ hepatoma cells (SMMC-7721), in a final volume of 0.2 ml complete media, were subcutaneously inoculated into the right iliac fossa of nude mice under sterile conditions. Nude mice were reared in an specific pathogen free (SPF)environment for 6⁸ weeks when the tumor size reached 0.8¹.0 cm.2 Preparation of Liposome-encapsulated (99)^Tc^m -HYNIC-survivinASODN: A total of four groups of ASODNs were prepared (Table 1), including two 10-base, single stranded ASODNs (A'₁ and A'₂) that contained the same quantity of G and were located at different positions relative to the promoter. Additionally, we created two more ASODNs that were 16-base, single stranded ASODNs (A₁ and A₂) and contained different quantities of G and had the same position relative to the promoter. Phosphorothioate modifications were added to the three phosphates at the ends of A'₁ and A'₂ and to the five phosphates at the ends of A₁ and A₂. Amino modifications of all four groups of ASODNs were performed at each 5'-end, which were coupled to HYNIC and then labled by (99)^Tc^m. Then the purification of (99)^Tc^m -labeled ASODN was performed using Cellufine Mini-Columns, which contain a Chisso cellulose-based gel filler, CellufineGH-25, that can filter out molecules with a olecular weight > 3 kDa. The emission peaks of purified (99)^Tc^m -labeled ASODNs were measured with A ZD-6000 Technetium (99)^Tc^m analyzer. Then the ASODNs were encapsulated with liposomes.3 The quality assurance of liposome - encapsulated (99)^Tc^m - HYNIC -Sur- vivinASODN was measured by high performance liquid chromatography and paper chromatography. Liposome-encapsulated (99)^Tc^m - HYNIC - survivinAS- ODN respectively in saline and human fresh serum after 6 hours of incubation at 37℃was measured . The labeling rate,radiochemical purity and stability were assessed.4 Antisense tumor imaging and Bio-distribution in vivo: Mice were allocated to four experimental groups (A₁, A₂, A'₁ and A'₂) with five mice randomly in each group. Liposome - encapsulated (99)^Tc^m- HYNIC - Survivin - ASODN 37 MBq was injected by tail vein. At 2h and 4h after injection, static radionuclide imaging was performed with the SPECT device on mice in the supine position in a pinhole-type collimator device. A contralateral muscle to tumor (M/T,NT/T) ratio was calculated, using the regions of interest (ROI) technique, at each time point. At 4h after injection, static radionuclide imaging was performed and immediately the blood was obtained from retrobulbar venous plexus and then the animals were euthanized by cervical dislocation. After the acquisition the blood was obtained from retrobulbar venous plexus and then the animals were euthanized by cervical dislocation and samples from the heart, liver, spleen, lung, kidney, stomach, small intestine, skeletal muscle and tumor were obtained. Organs were quickly rinsed and activities of the tracers were assessed using a gamma-well counter. All tissue counts were corrected for background and decay during the time of counting. Tissue activities of the tracers were expressed as percent of the injected dose per gram of wet weight (%ID/g) and NT/T ratios for skeletalmuscle and tumor were calculated.5 Tumor samples of each group were fixed into 4% paraformaldehyde, embedded with paraffin wax and resected into 5μm section, Routine HE staining were performed to evaluate the hepatoma cells (SMMC-7721). Results:1 Formation and characterization of tumors:Twenty xenotransplantation models for human hepatocellular carcinoma in BALB/C nude mice were all successfully established. The tumor formation rate was 100%.Macroscopic observation: The mass of tumor were fleshpink, nodular, smooth-faced and section-offwhite. Haematoxylin Eosin staining revealed that the tumor cells were uneven in staining quality, seen such as epithelioid cells, irregular shape, with inverted N:C ratios, abundant heteromorphism and manifest caryocinesia, which matched the characteristic of malignant tumor.2 The results of HPLC and paper chromatography confirmed that the labeling rates of liposome-encapsulated (99)^Tc^m - HYNIC-SurvivinASODN were 70%±0.69%(n=5) and that the radiochemical purities were 95%±1.29%(n=5). The labeling rates were about 66% and 65% and the radiochemical purities were about 93% and 90% with liposome-encapsulated (99)^Tc^m - HYNIC - survivinASODN respectively in saline and human fresh serum after 6 hours of incubation at 37℃. The similar results in saline and human fresh serum authenticated the labels have high stability.3 Imaging and Bio-distribution results:Imaging: 2h and 4h after injection of liposome-encapsulated (99)^Tc^m -HYNIC-survivinASODN, planar and static imaging was performed using a SPECT device. At 2h, there was an apparent concentration of radionuclide in the abdomen of the tumor-bearing mice, none in the liver and kidneys, a slight concentration in the chest and a small amount of radionuclide in the limbs and head. The right iliac fossa tumor was clear imaging at 2h; the surrounding tissue decreased the background with time. At the first time point, the iliac fossa tumor had very little radionucleotide concentration compared to the background, which gradually increased and peaked at 4 h.Bio-distribution:The %ID/g values for the four (99)^Tc^m -HYNIC-survivinASODNs wre high in both the liver and the kidneys, indicating that theyweresecreted by the liver and kidneys, thus accounting for the high background in the abdomen.Comparison of imaging and bio-distribution results with the four ASODNs: The ratios of muscle-to-tumor were 0.243±0.005 and 0.405±0.007 2h after the intravenous injection of (99)^Tc^m -HYNIC-survivin ASODN (A1 and A₂); at 4h, the ratios were 0.204±0.004 and 0.267±0.007 in the antisense gene imaging study. The ratios were 0.256±0.004 and 0.479±0.015 at 2h after intravenous injection of (99)^Tc^m -HYNIC-survivin ASODN (A'1 and A'2); at 4h, the ratios were 0.227±0.004 and 0.360±0.013 in antisense gene imaging study. At 4h, tissue biodistribution was measured, and the contralateral muscle/tumor (M/T, NT/T) ratios were 0.173±0.035, 0.315±0.059, 0.293±0.049, 0.430±0.021 (for A1, A2, A'1, A'2, respectively). At each time point, all groups were significantly different (P <0.05).Conclusion:The closer the ASODN sequence was to the promoter position, the lower the rate of nonspecific cellular accumulation was and the better antisense gene imaging was. Lower guanine content of ASODN correlated with a lower rate of nonspecific cellular accumulation and better antisense gene imaging. These results may provide some guidance to improve clinical tumor antisense imaging research.