Matsumoto Takahiro, Numata Munenori, Anada Takahisa, Mizu Masami, Koumoto Kazuya, Sakurai Kazuo, Nagasaki Takeshi, Shinkai Seiji
Faculty of Engineering, Department of Chemistry and Biochemistry, Graduate School of Engineering, Kyushu University 6-10-1 Hakozaki, Higashi, Fukuoka, Fukuoka 812-8581, Japan.
Biochim Biophys Acta. 2004 Jan 22;1670(2):91-104. doi: 10.1016/j.bbagen.2003.10.019.
Schizophyllan is a natural beta-(1-->3)-D-glucan existing as a triple helix in water and as a single chain in dimethylsulfoxide (DMSO), respectively. As we already reported, when some homo-phosphodiester polynucleotide (for example, poly(dA) or poly(C)) is added to the schizophyllan/DMSO solution and subsequently DMSO is exchanged for water, the single chain of schizophyllan forms a complex with the polynucleotide. Furthermore, we have already demonstrated that one of the potential applications of this novel complex is an antisense-oligonucleotide (AS ODN) carrier. This work describes a versatile and universal modification technique which enables us to introduce various functional groups only to the side chain of schizophyllan. This technique consists of periodate oxidation of the glucose side chain (it does not react with the main chain because of the absence of the 1,2-diol group in beta-(1-->3)-glucan) and subsequent introduction of the functional groups into the formyl terminate. In the present work, the introduced functional groups were spermine, octa-arginine (R8), arginine-glycine-aspartic acid tripeptide (RGD) and some amino or alpha-amino acid compounds. Using these compounds, we made the complexes and carried out an in vitro antisense assay for them, administrating a phosphorothioate AS ODN to the melanoma A375 or leukemia HL-60 cell lines to depress their c-myb mRNA. When we used the R8 or RGD modified schizophyllan as the antisense carrier, the antisense effect was most enhanced among others. Their superiority can be ascribed to enhancement of endocytosis due to these functional peptides. Furthermore, the cytotoxicity for these two modified schizophyllans was negligibly as small as the natural (unmodified) schizophyllan. One of the peculiar features of our system is that the complex (i.e., carrier+AS ODN) is charged negatively in total, which is different from the conventional systems. The present work has thus clarified that schizophyllan can act as a new potential candidate for AS ODN carriers.
裂褶多糖是一种天然的β-(1→3)-D-葡聚糖，在水中以三螺旋形式存在，在二甲基亚砜（DMSO）中以单链形式存在。正如我们之前报道的，当向裂褶多糖/DMSO溶液中加入一些同型磷酸二酯多核苷酸（例如，聚(dA)或聚(C)），随后将DMSO换成水时，裂褶多糖的单链会与多核苷酸形成复合物。此外，我们已经证明这种新型复合物的潜在应用之一是作为反义寡核苷酸（AS ODN）载体。这项工作描述了一种通用且万能的修饰技术，使我们能够仅在裂褶多糖的侧链上引入各种官能团。该技术包括对葡萄糖侧链进行高碘酸盐氧化（由于β-(1→3)-葡聚糖中不存在1,2-二醇基团，所以它不会与主链反应），随后将官能团引入到醛基末端。在本工作中，引入的官能团有精胺、八聚精氨酸（R8）、精氨酸-甘氨酸-天冬氨酸三肽（RGD）以及一些氨基或α-氨基酸化合物。使用这些化合物，我们制备了复合物并对其进行体外反义测定，将硫代磷酸酯AS ODN施用于黑色素瘤A375或白血病HL-60细胞系以抑制它们的c-myb mRNA。当我们使用R8或RGD修饰的裂褶多糖作为反义载体时，反义效果在其他载体中最为显著。它们的优势可归因于这些功能性肽增强了内吞作用。此外，这两种修饰裂褶多糖的细胞毒性与天然（未修饰）裂褶多糖一样小，几乎可以忽略不计。我们系统的一个独特特征是复合物（即载体+AS ODN）总体带负电荷，这与传统系统不同。因此，本工作阐明了裂褶多糖可以作为AS ODN载体的一种新的潜在候选物。
