PHARAMACEUTICAL STUEDIES ON GENE MATERIALS-CARRIER SYSTEM

2001 Fiscal Year Final Research Report Summary

• Project/Area Number: 10672023
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Physical pharmacy
• Research Institution: Nagoya City University
• Principal Investigator: YOTSUYANAGI Toshihisa NAGOYA CITY UNIVERSITY, PHARMACEUTICAL SCIENCE, PROFESSOR, 薬学部, 教授 (06672150)
• Co-Investigator(Kenkyū-buntansha): HAZEMOTO Norio NAGOYA CITY UNIVERSITY, PHARMACEUTICAL SCIENCE, ASSOCIATE PROFESSOR, 薬学部, 助教授 (40192273)
• Project Period (FY): 1998 – 2000
• Keywords: GENE DELIVERY / DENDRITIC PEPTIDE / LIPOSOME / POLYAMINO ACID / FREEZE-DRYING / TRANSFECTION EFFICIENCY / PLASMID DNA / SUGAR

Research Abstract

The development of liposome and ionic polymer proceeds as gene carriers for the non-virus genetic treatment. In this research, we studied on a reagent consisting of liposome and peptides and on the freezing dry of the gene material. Liposome containing cholesterol derivative was used to improve the gene transfer efficiency of the basic polypeptide. Furthermore, dendritic peptide which had the branched structure was synthesized, and the genetic transfer efficiency under co-existence of liposome was examined. As an evaluation of gene expression, CAT activity (pSV2CAT) and luciferase activity (pGL3) in the HeLaS3 cell were assayed. The dendritic-peptide together with liposomes increased 2-3 times genetic expression compared with liposome only. The length of the side chain of dendritic peptide influenced gene expression. In four branched peptide, dendritic peptide of nine lysine chains accelated gene expression than three lysine chains. A remarkable difference was not observed in genetic e xpression between poly-L-lysine and dendritic peptide, though a optimal concentration of dendritic peptide in gene expression was shifted to the lower concentration compared with the case of poly-L-lysine. Complexes of plasmid DNA and polyamino acid showed high luciferase activity of the eight order of 10. Amounts of genetic expression decreased to about one-hundredths when complexes were freeze-dried and it dispersed again. Gene expression was recovered by addition of sugar in freeze-drying. The sugar effect in gene transfer was large in sucrose and trehalose. Effect on addition of the sugar in freezing dryness may be to aid the dispersion DNA complexes in freezing media, and to prevent the injury of the DNA molecule by a crystallization of water and to suppress a irreversible adhesion between DNA complexes at the time of dryness, and to suppress a irreversible adhesion between DNA complexes at the time of dryness. In this study, we characterized the DNA medicine manufactured by freezing dryness on biological aspect of the genetic expression as well as physicochemical aspect of complex dispersion.