Co-encapsulation of two plasmids in chitosan microspheres as a non-viral gene delivery vehicle.
Co-encapsulation of two plasmids in chitosan microspheres as a non-viral gene delivery vehicle.
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发表时间：
2003
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通讯作者：
S. Ozbaş-Turan;C. Aral;L. Kabasakal;M. Keyer‐Uysal;J. Akbuğa
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作者：
S. Ozbaş-Turan;C. Aral;L. Kabasakal;M. Keyer‐Uysal;J. Akbuğa
目的将两种不同的质粒DNA（pGL 2和pMK 3）包裹在同一微球结构中，研究壳聚糖微球的体内转染特性。研究了壳聚糖浓度、质粒DNA用量等因素对微球体外性能的影响。方法采用复凝聚法制备双质粒壳聚糖微球。释放研究在37 ℃的磷酸盐缓冲盐水中进行，释放的质粒DNA用荧光光度法测定。通过琼脂糖凝胶电泳检查质粒DNA的完整性。对于体内转染研究，将微球注射到小鼠的肌肉中，并测量蛋白质（β-半乳糖苷酶和荧光素酶）的表达。结果壳聚糖微球的包封率可达90%。颗粒大小约为1.15 - 1.28 μ m。在大小和制剂变量（壳聚糖浓度和质粒量）之间没有观察到依赖性。包封后，两种质粒的完整性没有改变。质粒DNA从壳聚糖微球中持续释放。壳聚糖浓度和质粒量影响体外释放特性。小鼠肌肉注射双质粒微球后，获得共表达。高β-半乳糖苷酶和荧光素酶的生产与这些微球后，转染时间长（12周）。结论壳聚糖微球可将两种质粒包封于壳聚糖微球中，且不影响其结构和功能的完整性。因此，用这些微球获得了持续和高的蛋白质产量。
PURPOSE The aims of this study are to encapsulate two different plasmid DNAs (pGL2 and pMK3) in the same microsphere structure and to investigate in vivo transfection characteristics of chitosan microspheres. Furthermore, the effect of formulation factors, such as chitosan concentration and plasmid DNA amount on in vitro properties of microspheres were studied. METHODS Double plasmid-loaded chitosan microspheres were prepared by complex coacervation. Release studies were done in phosphate buffered saline at 37 degrees C and released plasmid DNA was determined spectrophotometrically. Integrity of plasmid DNAs was checked by agarose gel electrophoresis. For in vivo transfection studies, microspheres were injected into the muscle of the mice and expression of proteins (beta-galactosidase and luciferase) was measured. RESULTS High encapsulation efficiency was obtained with chitosan microspheres (90%). The size of particles was about 1.15 - 1.28 m. No dependence was observed between the size and formulation variables (chitosan concentration and the amount of plasmid). After encapsulation process, integrity of two plasmids did not change. Plasmid DNAs were continuously released from chitosan microspheres. Chitosan concentrations and plasmid amounts affected in vitro release properties. After intramuscular injection of double plasmids loaded microspheres into muscle of the mice, co-expression was obtained. High beta-galactosidase and luciferase productions were determined with these microspheres after a long post-transfection period (12 weeks). CONCLUSIONS Our results showed that two plasmids could be encapsulated in chitosan microspheres without affecting their structural and functional integrity. Thus, sustained and high protein production was obtained with these microspheres.