| Autor: |
He, J., Liu, G., Zhang, S., Vanderheyden, J.-L., Liu, N., Liu, C., Zhang, Y., Gupta, S., Rusckowski, M., Hnatowich, D. J. |
| Zdroj: |
Bioconjugate Chemistry; September 2003, Vol. 14 Issue: 5 p1018-1023, 6p |
| Abstrakt: |
The stability of hybridized duplexes is an important criterion for any radiopharmaceutical application of DNAs or their analogues such as phosphorodiamidate morpholinos (MORFs). Objective: The stabilities in vitro and in mice of the duplex between MORF and its complement (cMORF) were investigated for two different chain lengths, a 15-mer MORF compared to the identical MORF but elongated to a 25-mer. Methods: The hybridization characteristics of the 15-mer MORF with its complementary 15-mer and that of the 25-mer with its complementary 25-mer MORF were measured using surface plasmon resonance (SPR) analysis. For radiolabeling with 99mTc, the 15- and 25-mer MORF, both with a primary amine via a 10-member linker on the 3 equivalent end, were conjugated with NHS-MAG3. The 15- and 25-mer cMORFs were conjugated via their amines to carbodiimidazole treated poly(methyl vinyl ether-alt-maleic acid) (PA) such that about 50 cMORFs were attached to each polymer molecule in both cases (estimated MWs about 300 and 450 kDa, respectively). After hybridization in vitro, both the PA-cMORF15-99mTc-MORF15 and PA-cMORF25-99mTc-MORF25 homoduplexes were evaluated by size exclusion HPLC in saline, after incubation in 37 °C serum and in urine obtained 30 min post IV administration to normal mice. Biodistributions were obtained up to 18 h post administration. Results: By SPR, the affinity constants for the homoduplexes were both about 109 M-1 with the 25/25 only about 25% higher than the 15/15. However, the affinity constants for the 15/25 and 25/15 heteroduplexes showed a surprisingly 13-fold difference. By HPLC analysis, all duplexes were stable in saline; however, analysis of serum incubates and urine containing PA-cMORF15-99mTc-MORF15 showed an immediate and pronounced low molecular weight peak that was identified by a shift assay to be 99mTc-MORF15. The comparable peak in both fluids was much less pronounced in the case of PA-cMORF25-99mTc-MORF25. Whole body radioactivity levels also fell much more rapidly in mice receiving the 15-mer conjugate (65 vs 30% eliminated at 18 h) and biodistribution results showed higher kidney levels for the 15-mer conjugate. Results with the PA-cMORF25-99mTc-MORF15 heteroduplex were more similar to that obtained with the 15-mer homoduplex than the 25-mer homoduplex. Conclusion: Despite what is reported to be high hybridization affinities, both the homoduplex and heteroduplexes prepared with 99mTc-MORF15 were found to be unstable in serum and in vivo toward dissociation to free 99mTc-MORF15. By contrast, homoduplex prepared with 99mTc-MORF25 showed higher stability. These differences in hybridization stability may be important considerations in radiopharmaceutical design. |
| Databáze: |
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