He Jiang, Liu Guozheng, Zhang Surong, Vanderheyden Jean-Luc, Liu Ning, Liu Changbin, Zhang Yumin, Gupta Suresh, Rusckowski Mary, Hnatowich Donald J
Department of Radiology, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, Massachusetts 01655, USA.
Bioconjug Chem. 2003 Sep-Oct;14(5):1018-23. doi: 10.1021/bc0341019.
The stability of hybridized duplexes is an important criterion for any radiopharmaceutical application of DNAs or their analogues such as phosphorodiamidate morpholinos (MORFs).
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.
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 (99m)Tc, 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-MAG(3). 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-(99m)Tc-MORF15 and PA-cMORF25-(99m)Tc-MORF25 homoduplexes were evaluated by size exclusion HPLC in saline, after incubation in 37 degrees C serum and in urine obtained 30 min post IV administration to normal mice. Biodistributions were obtained up to 18 h post administration.
By SPR, the affinity constants for the homoduplexes were both about 10(9) 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-(99m)Tc-MORF15 showed an immediate and pronounced low molecular weight peak that was identified by a shift assay to be (99m)Tc-MORF15. The comparable peak in both fluids was much less pronounced in the case of PA-cMORF25-(99m)Tc-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-(99m)Tc-MORF15 heteroduplex were more similar to that obtained with the 15-mer homoduplex than the 25-mer homoduplex.
Despite what is reported to be high hybridization affinities, both the homoduplex and heteroduplexes prepared with (99m)Tc-MORF15 were found to be unstable in serum and in vivo toward dissociation to free (99m)Tc-MORF15. By contrast, homoduplex prepared with (99m)Tc-MORF25 showed higher stability. These differences in hybridization stability may be important considerations in radiopharmaceutical design.
杂交双链体的稳定性是DNA或其类似物（如吗啉代磷酰胺（MORF））任何放射性药物应用的重要标准。
研究了两种不同链长的MORF与其互补链（cMORF）之间双链体在体外和小鼠体内的稳定性，将15聚体MORF与其相同但延长至25聚体的MORF进行比较。
使用表面等离子体共振（SPR）分析测量15聚体MORF与其互补15聚体以及25聚体与其互补25聚体MORF的杂交特性。为了用（99m）Tc进行放射性标记，将3'等效端通过10元连接子带有伯胺的15聚体和25聚体MORF与NHS-MAG（3）偶联。15聚体和25聚体cMORF通过其胺与经碳二亚咪唑处理的聚（甲基乙烯基醚-alt-马来酸）（PA）偶联，使得在两种情况下每个聚合物分子连接约50个cMORF（估计分子量分别约为300和450 kDa）。体外杂交后，在37℃血清中孵育以及静脉注射给正常小鼠30分钟后收集的尿液中，通过尺寸排阻HPLC评估PA-cMORF15-（99m）Tc-MORF15和PA-cMORF25-（99m）Tc-MORF25同型双链体。给药后长达18小时获得生物分布数据。
通过SPR，同型双链体的亲和常数均约为10（9）M（-）（1），25/25双链体仅比15/15双链体高约25％。然而，15/25和25/15异型双链体的亲和常数显示出惊人的13倍差异。通过HPLC分析，所有双链体在盐水中稳定；然而，对含有PA-cMORF15-（99m）Tc-MORF15的血清孵育物和尿液的分析显示出一个立即出现且明显的低分子量峰，通过位移分析鉴定为（99m）Tc-MORF15。在PA-cMORF25-（99m）Tc-MORF25的情况下，两种液体中的可比峰不太明显。接受15聚体偶联物的小鼠体内全身放射性水平下降也更快（18小时时消除65％对30％），生物分布结果显示15聚体偶联物的肾脏水平更高。PA-cMORF25-（99m）Tc-MORF15异型双链体的结果与15聚体同型双链体获得的结果比与25聚体同型双链体获得的结果更相似。
尽管据报道具有高杂交亲和力，但发现用（99m）Tc-MORF15制备的同型双链体和异型双链体在血清和体内对解离为游离（99m）Tc-MORF15均不稳定。相比之下，用（99m）Tc-MORF25制备的同型双链体显示出更高的稳定性。这些杂交稳定性的差异可能是放射性药物设计中的重要考虑因素。
