Radiolabeling PDGFR-β MRNA Antisense Oligodeoxynucleotide With ～(99m)Tc/～(188)Re And Its Biodistribution In Normal Mice

Given the prevalence of coronary artery disease and the widespread dissemination of technology and expertise, the number of percutaneous coronary interventions will continue to increase aggressively. Although many patients can benefit from percutaneous intervention, many can also suffer from its limitations, with restenosis causing recurrent angina, ischemic events, and repeat interventions. However, restenosis after angioplasty remains the major limitation to the long-term effectiveness of percutaneous transluminal coronary angioplasty, with a rate of between 30% and 50%. Stent implantation can obviously reduce the incidence of restenosis after PTCA. In recent years Stent implantation has become the standard angioplasty procedure, but in-stent restenosis remains the major limitation to coronary stenting.It is estimated that in-stent restenosis rate after implantation is up to 20%-30% after six months and become a predominating problem which limited its use in clinical application. Despite an exhaustive search for an effective therapy to treat or prevent restenosis, however, the effective therapy is still in exploring.Therefore, expanding our understanding of restenosis and treatment options becomes more and more pressing.Restenosis reflects a cascade of molecular and cellular events within the vascular wall which induced by a complex interaction of multiple growth factors that are activated by vascular injury after percutaneous intervention surgery. Within this complicate cascade, two major processes can be discerned: arterial remodeling and vascular smooth muscle cell (VSMC) hyperplasia. Platelet Derived Growth Factor (PDGF) and Platelet Derived Growth Factor Receptor-β (PDGFR-β) play a very important role in this process.With the great development of Molecular biology technique in recent years, Gene therapy technique is emerging as a potential strategy for the treatment of restenosis. The possibility of antisense oligonucleotide-based therapy for restenosis was expected, and its effect had been proven in animal models by many researchers.Some antisense oligonucleotide-based gene therapy trials for restenosis had shown that an AODN complimentary to the mRNA of PDGFR-β can inhibit VSMC hyperplasia, which is one of the major mechanisms of restenosis. They offered the exciting possibility of blocking the expression of PDGFR-β gene without any change in functions of other genes because of AODN's specificity. Therefore, AODN are useful tools in the study of restenosis and may be potential therapeutic agents for restenosis. In some other trials, intracoronary radiation had been shown to be effective in proventing restenosis through inhibiting VSMC hyperplasia. In 2002, the Food and Drug Administration of American approved imtracoronary radiation (brachytherapy) as a viable therapeutic option for clinical restenosis patients.Accordingly, we developed a new strategy combining radiotherapy andgene therapy. Theoretically, this new strategy is superior to either of the two methods. Radiolabeled AODN is not only a platform for the delivery of radiation, but also can inhibit targeted gene expression. It is a kind of dual effects. This new strategy may be a new potential therapeutic method for restenosis. All Our studies are based on this new strategy. First of all, we must find a good method of radiolabeling AODN with radionuclides and lay a good groundwork for future research work. AIM:To explore a better method of radiolabeling AODN complimentary to the mRNA of PDGFR-p with ^99mTc/¹88Re, and to investigate the stability of radiolabeled AODN and biodistribution of radiolabeled AODN in normal mice. METHODS:1.The synthesis of NHS-MAG₃ from purified MAG₃ was as followed: MAG3 was dissolved in DMF and mixed at room temperature with dry DMF containing NHS. The reaction mixture was placed in an ice bath, DMF containing DCC added and the mixture stirred for 20 hours. The residue was washed with DMF. Ethylether was added into the solvent. The residue was NHS-MAG3 which we wanted. We used mass spectrogram method for assessment;2.A 18mer single-chain AODN, complimentary to PDGFR-β mRNA, was conjugated with chelator NHS-MAG3 and labeled with ^99mTc/¹88Re. We used orthogonal design method to find the relative best conditions from the multiple conditions for radiolabeling;3.Labeling efficiency and radiochemical purity were measured by paper chromatography. The stability of ^99mTc/¹88Re-MAG₃-AODN in vitro wasinvestigated and biodistribution was carried out in normal mice. RESULTS:1. We synthesized NHS-MAG3 successfully. The Yield was 48.6%. The mass spectrogram showed that the NHS-MAG3 was purified. It was good enough for our experiments;2. As proved by paper chromatography and P4 column analysis, AODN was successfully radiolabeled by ^99mTc.With orthogonal design analysis, we found that the importance rank of the four major conditions of radiolabeling were as follows: PH value, stannous chloride's dose, reaction temperature and reaction time. The relative best radiolabeling conditions were as follows: PH value 6, stannous chloride dose 5μg, reaction temperature 90℃, reaction time 30min.The results showed that the average labeling efficiency was approaching to 70% under the relative best conditions.After purified on a P₄ column, the radiochemical purity was above 95%, the specific activity was about 7.4-11.1 MBq(200-300μCi)/ μg.As proved by paper chromatography and Sep-Pak C₁8 columns analysis, AODN was successfully radiolabeled by ¹88Re. With orthogonal design analysis, we found that the importance rank of the four major conditions of radiolabeling were as follows: stannous chloride's dose, PH value, reaction temperature and reaction time. The relative best radiolabeling conditions were as follows: stannous chloride dose 500μg, PH value 4, reaction temperature 90℃, reaction time 30min.The results showed that the average labeling efficiency was approaching to 70% under the relative best conditions.After purified on Sep-Pak C₁8 columns, the radiochemical purity was above 95%, the specific activity was about 3.7-7.4MBq(100-200μCi)/μg;3. In saline at room temperature and in 37℃ serum, radiochemical purity...