1007. Development of Nuclear Targeting System of Plasmid DNA; Impact of Sugar Display on the Surface of Lipid Envelope

Abstract

Top of pageAbstract [Objective] To develop a non-viral gene vector successfully, highly efficient nuclear delivery system of plasmid DNA (pDNA) is essential. In the present study, we developed a nuclear delivery system based on the novel concept of |[ldquo]|degradable particle on the nuclear membrane|[rdquo]|, which mimic the mechanism of nuclear entry of adenovirus by means of multifunctional envelope-type nano device (MEND). In this system, pDNA was condensed with polycation, followed by encapsulation with lipid membrane. Devices for the nuclear targeting were modified on the surface of lipid envelope. After the particle binds to the nuclear pore complex (NPC), lipid envelope was drawn into the nucleus with condensed pDNA particle. As a device for nuclear targeting, various sugars were modified. [Methods] To modify the sugars on the surface of the lipid envelope, sugars were conjugated with cholesterol, which enables it to insert in the lipid layer. To enhance a cellular uptake via macropinocytosis, which is advantageous to avoid a lysosomal degradation, stearylated octaarginine (STR-R8) was further modified on the envelope. We evaluated transfection activities of sugar chain modified MEND in HeLa cells which is synchronized in G1 phase by hydroxyurea. As a control, sugar-conjugated cholesterol was replaced with unconjugated cholesterol. [Results] Sugar modified MEND showed drastically higher transfection activity compared with sugar-unmodified MEND in dividing cells. Moreover, in synchronized cells, transfection activity of sugar- unmodified MEND was prominently decreased. Even this condition, high transfection activity was also observed with sugar-modified MEND. These results suggested that sugar chain improved the nuclear transfer of pDNA. Optimum density of sugar on the MEND was depended on the type of sugar. Furthermore it was demonstrated that trans-gene expression was decreased when lipid envelope was stabilized by increasing the cholesterol content. Therefore, destabilization of lipid envelope on the nuclear membrane was closely related to the transfection activity. [Conclusion] Collectively, we succeeded in developing the novel system for the nuclear delivery of pDNA with MEND by optimizing the density of sugar and the stability of lipid envelope.