Chemically modified polysaccharide schizophyllan for antisense oligonucleotides delivery to enhance the cellular uptake efficiency
Chemically modified polysaccharide schizophyllan for antisense oligonucleotides delivery to enhance the cellular uptake efficiency
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DOI：
10.1016/j.bbagen.2003.10.019
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发表时间：
2004-01-22
影响因子：
3
通讯作者：
Shinkai, S
中科院分区：
文献类型：
--
作者：
Matsumoto, T;Numata, M;Shinkai, S
裂殖酵母多糖是一种天然的β-(1--)-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。正如我们已经报道的，当一些同型磷二酯多核苷酸(例如，聚(DA)或聚(C))被添加到裂叶聚糖/DMSO溶液中并随后将DMSO交换为水时，裂叶聚糖的单链与多核苷酸形成络合物。此外，我们已经证明了这种新型复合体的一个潜在应用是作为反义寡核苷酸(AS ODN)载体。这项工作描述了一种通用的修饰技术，它使我们能够只在裂叶多糖的侧链上引入各种官能团。该技术包括葡萄糖侧链的高碘酸盐氧化(由于β-(1-gt；3)-葡聚糖中不存在1，2-二醇基团，所以它不与主链反应)和随后将官能团引入甲酰末端。在本工作中，引入的官能团是精胺、八-精氨酸(R8)、精氨酸-甘氨酸-天冬氨酸三肽(RGD)和一些氨基酸或α-氨基酸化合物。利用这些化合物，我们制备了这些化合物，并对它们进行了体外反义实验，给黑色素瘤A375或白血病HL-60细胞株以硫代磷酸作为ODN来抑制它们的c-myb mRNA。以R8或RGD修饰的裂叶多糖为反义载体时，反义效果最强。它们的优越性可以归因于这些功能肽对内吞作用的增强。此外，这两种修饰的裂叶多糖的细胞毒性与天然的(未修饰的)裂叶多糖一样小，可以忽略不计。与传统体系不同的是，该体系的特点之一是络合物(即载体+反义寡核苷酸)全部带负电荷。因此，本工作阐明了裂叶聚糖可作为ODN载体的一种新的潜在候选者。(C)2004爱思唯尔B.V.保留所有权利。
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. (C) 2004 Elsevier B.V. All rights reserved.