Materials and Methods
The 15-base, single stranded antisense oligonucleotides complementary to the translation beginning sites of c-myc oncogene messenger RNA and it's sense sequence were selected. The antisense oligonucleoptides (Sangon Biotech, CA) had a base sequence of 5'-amine-AAC,GTT,GAG,GGG,CAT-3'. The molecular weight was about 4.6 kDa. The melting temperature in physiological saline was calculated to be 46ºC. The DNAs were purchased unpurified and were used without further purification. They were generally handled under sterile conditions; all solutions were sterilized by terminal filtration through a 0.22µm filter, and sterile pipette tips were used. All other pipette tips and tubes were autoclaved prior to use.
Technetium-99m-pertechnetate was obtained from a 99Mo-99mTc nuclide generator (Atomic Energy Institute, China). The S-Acetyl-NHS-MAG3 was a gift from Dr. Donald J. Hnatowich (Massachusetts Medical Center, USA).
Oligonucleotides Conjugation and Labeling
Oligonucleotides conjugation and labeling procedure was performed according to the method of P. Winnard et al (2).
A solution of single stranded amine-derivatized DNA (165-990µg) was prepared and heated to 55ºC for 10 min and
immediately plunged into ice water to dissociate any DNA duplexes. The DMF solution of NHS-MAG3 was then added to the stirred DNA solution. The solution was incubated at room temperature for 15-20 min in the dark. After
purification, fractions off the P4 column (BioRad, Melville, NY) were collected and the absorbance at 260 nm of each measured (Gene Quant RNA/DNA Calculator, Pharmacia Biotech). The peak fractions were lyophilized and stored at -70ºC for periods from several days to more than four months before labeling.
To a sterile test tube containing the MAG3-DNA(about 10-100µg,10-100µl) was added sufficient 99mTc-pertechnetate solution(10-50µl) to provide about 50-100µCi/µg of DNA. To this was added the tartrate solution and stannous ion solution. After 15min at room temperature, the labeled DNA was purified on a 0.7×20cm gel filtration column of Sephadex G-25 using sterile 0.25 M ammonium acetate, pH 5.2, or saline, as eluant. Radioactivity and absorbency at 260nm were used to identify and quantitate peak fractions.
Control labeling was performed in which the native, unconjugated DNA was subjected to the identical labeling procedure to assess the extent of nonspecific labeling.
Radiochemical purity was estimated by Sep-Pak C-18 (Waters, Milford, MA) column chromatography. A column was conditioned with 10ml of absolute ethanol followed by 10ml of 1mM HCl. After the sample was loaded, the first elution, with 10ml of 1mM HCl, removed 99mTc-pertechnetate and 99mTc-tartrate. The second elution, with 10ml of ethanol/saline (1:1), removed the labeled DNA. Radiolabeled colloids remained on the column.
In addition to measuring radiochemical purity, Sep-Pak studies were also used to establish whether the conjugation and labeling procedure had diminished the ability of the labeled DNA to hybridize to its complement. The radiolabeled MAG3-DNA was analyzed as above by Sep-Pak column chromatography before and after the addition of the complementary DNA.
Serum Incubation Studies
Labeled MAG3-DNA was incubated at a concentration of 10µg/ml in fresh human serum at 37ºC. Samples were periodically removed over 24hr for Sep-Pak analysis.
Preparation of Animal Model
Ehrlich carcinoma mice (bearing mammary adenocarcinoma in the ascites) were obtained from the Department of Pharmacology, Tongji Medical College, Huazhong University of Science and Technology. The ascites was suctioned into a sterile syringe and diluted to a concentration of 5-10×106 tumor cells/ml. A colony of KM mice (15-20g) were inoculated with 1×106 tumor cells in the right thigh, and the tumors were allowed to grow for 6-7 days to a size of 1.0-1.5 cm in diameter.
A total of 40 mice(two groups:20 sense phosphodiester and 20 antisense phosphodiester) were injected intravenously in the tail vein with 50 µCi of 99mTc-labeled oncogene probes (sense and antisense). The animals were killed by cervical dislocation at different time points from 45min to 24hr. The tumor, blood, heart, liver, spleen, lung, kidney, intestinal bowel, muscle and bone were harvested and weighed. The tracer distribution in the viscera and tumor was determined with a gamma counter in the 130-150 keV window to include the 140 keV of the 99mTc radionuclide. The mean and s.d. values, relative radioactivity ratios of tumor-to-blood and tumor-to-muscle, percent of injected dose/gram were calculated for each tumor bearing mouse model.
Tumor Imaging Studies
A total of 8 mice (two groups: 4 sense phosphodiester and 4 antisense phosphodiester) were injected intravenously in the tail vein with 1-2 mCi of 99mTc-labeled oncogene probes, immobilized with ketamine hydrochloride and imaged at 30min, 1h, 2h, 4h, 6h and 24hr with a gamma camera fitted with a medium-energy pin-hole collimator (Sopha DSX SPECT, France). Images were acquired for a present time of 30 to 1000s using a 256×256 matrix with a 20% energy window set at 140 keV.
In a test of specificity, the unlabeled, unmodified antisense oligonucleotide was administered to block base-specific binding sites of mRNA 2 hr before the administration of 99mTc labeled antisense oligonucleotides. To eliminate the interference of free 99mTc, mice were also imaged after the same dose of 99mTc-pertechnetate was administered.