遺伝子デリバリーを目的としたナノ粒子キャリアの設計

抄録

Purpose: For gene therapy, development of non-viral vectors delivered more efficiently and safely into cell has long been awaited. We have successfully prepared poly (D,L-lactide-glycolide) nanospheres (PLGA NSs) for peptide delivery to improve drug absorption through the mucous membrane by modifying the surface of NSs with chitosan. The aim of this study is to establish the preparation method of pDNA loading PLGA NSs to improve loading efficiency and to enhance their cellular uptake and gene expression.

Methods: pDNA (pCMV-Luciferase) loaded PLGA NSs were prepared by emulsion solvent diffusion method. For the preparation of chitosan coated PLGA NSs (CS-PLGA NSs), chitosan-PVA mixed solution was used as the dispersing phase during evaporation process. Particle size and zeta potential of NSs were measured by a laser scattering method and zeta master (Zetasizer 3000HS, Malvern Inc.). pDNA loading efficiency into NS and release behavior of pDNA from NSs were investigated. In vitro transfection tests were performed with human lung adenocarcinoma cells (A549 cells) by luciferase assay system. In vitro cellular uptake tests of NS were performed with A549 cells by using 6-coumarin labeled NSs.

Results: The particle size of NSs was c.a. 250nm. Plasmid DNA could be dispersed in polymeric organic solution. Loading efficiency into nanosphere was increased by forming ion complex with cationic material (DOTAP or chitosan). CS-PLGA NSs had a positive charge, while PLGA NSs being negatively charged, suggesting that the NSs were coated successfully with chitosan. pDNA loading efficiency was increased by coating with chitosan. CS-PLGA NSs showed higher cellular uptake of NSs with A549 cells than uncoated NSs. The transfection efficiency of CS-PLGA NSs was higher than PLGA NSs. This result might be caused by higher cellular uptake ability of CS-PLGA NSs due to electrostatic interaction.