Chemically modified polysaccharide schizophyllan for antisense oligonucleotides delivery to enhance the cellular uptake efficiency.
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.
Matsumoto
Takahiro
T
Faculty of Engineering, Department of Chemistry and Biochemistry, Graduate School of Engineering, Kyushu University 6-10-1 Hakozaki, Higashi, Fukuoka, Fukuoka 812-8581, Japan.
Numata
Munenori
M
Anada
Takahisa
T
Mizu
Masami
M
Koumoto
Kazuya
K
Sakurai
Kazuo
K
Nagasaki
Takeshi
T
Shinkai
Seiji
S
D000818:Animals; D002240:Carbohydrate Sequence; D045744:Cell Line, Tumor; D016503:Drug Delivery Systems; D015316:Genetic Therapy; D018922:HL-60 Cells; D006801:Humans; D008081:Liposomes; D008546:Melanoma, Experimental; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D016376:Oligonucleotides, Antisense / Q000008:administration & dosage / Q000737:chemistry* / Q000494:pharmacology; D010504:Periodic Acid; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012566:Sizofiran / Q000008:administration & dosage / Q000737:chemistry* / Q000633:toxicity; D013873:Thionucleotides / Q000737:chemistry
D008081:Liposomes; D016376:Oligonucleotides, Antisense; D013873:Thionucleotides; D010504:Periodic Acid; D012566:Sizofiran; C009288:metaperiodate