Cui Zhengrong, Mumper Russell J
Division of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, Kentucky 40536-0082, USA.
Bioconjug Chem. 2002 Nov-Dec;13(6):1319-27. doi: 10.1021/bc0255586.
Nonviral gene therapy has been a rapidly growing field. However, delivery systems that can provide protection for pDNA and potential targeting are still desired. A novel pDNA-nanoparticle delivery system was developed by entrapping hydrophobized pDNA inside nanoparticles engineered from oil-in-water (O/W) microemulsion precursors. Plasmid DNA was hydrophobized by complexing with cationic surfactants DOTAP and DDAB. Warm O/W microemulsions were prepared at 50-55 degrees C with emulsifying wax, Brij 78, Tween 20, and Tween 80. Nanoparticles were engineered by simply cooling the O/W microemulsions containing the hydrophobized pDNA in the oil phase to room temperature while stirring. The nanoparticles were characterized by particle sizing, zeta-potential, and TEM. Nanoparticles were challenged with serum nucleases to assess pDNA stability. In addition, the nanoparticles were coincubated with simulated biological media to assess their stability. In vitro hepatocyte transfection studies were completed with uncoated nanoparticles or nanoparticles coated with pullulan, a hepatocyte targeting ligand. In vivo biodistribution of the nanoparticles containing I-125 labeled pDNA was monitored 30 min after tail-vein injection to Balb/C mice. Depending on the hydrophobizing lipid agent employed, uniform pDNA-entrapped nanoparticles (100-160 nm in diameter) were engineered within minutes from warm O/W microemulsion precursors. The nanoparticles were negatively charged (-6 to -15 mV) and spherical. An anionic exchange column was used to separate unentrapped pDNA from nanoparticles. Gel permeation chromatography of pDNA-entrapped and serum-digested nanoparticles showed that the incorporation efficiency was approximately 30%. Free 'naked' pDNA was completely digested by serum nucleases while the entrapped pDNA remained intact. Moreover, in vitro transfection studies in Hep G2 cells showed that pullulan-coated nanoparticles resulted in enhanced luciferase expression, compared to both pDNA alone and uncoated nanoparticles. Preincubation of the cells with free pullulan inhibited the transfection. Finally, 30 min after tail vein injection to mice, only 16% of the 'naked' pDNA remained in the circulating blood compared to over 40% of the entrapped pDNA. Due to the apparent stability of these pDNA-entrapped nanoparticles in the blood, they may have potential for systemic gene therapy applications requiring cell and/or tissue-specific delivery.
非病毒基因治疗一直是一个快速发展的领域。然而，仍需要能够为质粒DNA（pDNA）提供保护并具有潜在靶向性的递送系统。通过将疏水化的pDNA包裹在由水包油（O/W）微乳液前体制备的纳米颗粒内部，开发了一种新型的pDNA纳米颗粒递送系统。质粒DNA通过与阳离子表面活性剂DOTAP和DDAB复合而疏水化。在50 - 55摄氏度下，用乳化蜡、Brij 78、吐温20和吐温80制备温热的O/W微乳液。通过在搅拌的同时将油相中含有疏水化pDNA的O/W微乳液简单冷却至室温来制备纳米颗粒。通过粒度分析、zeta电位和透射电子显微镜（TEM）对纳米颗粒进行表征。用血清核酸酶处理纳米颗粒以评估pDNA的稳定性。此外，将纳米颗粒与模拟生物介质共同孵育以评估其稳定性。用未包被的纳米颗粒或用支链淀粉（一种肝细胞靶向配体）包被的纳米颗粒完成了体外肝细胞转染研究。在向Balb/C小鼠尾静脉注射含I - 125标记pDNA的纳米颗粒30分钟后，监测其体内生物分布。根据所使用的疏水化脂质试剂不同，在几分钟内就能从温热的O/W微乳液前体中制备出直径为100 - 160纳米、均匀包裹pDNA的纳米颗粒。这些纳米颗粒带负电荷（-6至-15 mV）且呈球形。使用阴离子交换柱将未包裹的pDNA与纳米颗粒分离。对包裹pDNA和经血清消化的纳米颗粒进行凝胶渗透色谱分析表明，掺入效率约为30%。游离的“裸”pDNA被血清核酸酶完全消化，而包裹的pDNA保持完整。此外，在Hep G2细胞中的体外转染研究表明，与单独的pDNA和未包被的纳米颗粒相比，支链淀粉包被的纳米颗粒导致荧光素酶表达增强。用游离支链淀粉对细胞进行预孵育会抑制转染。最后，在向小鼠尾静脉注射30分钟后，只有16%的“裸”pDNA仍留在循环血液中，而包裹的pDNA则超过40%。由于这些包裹pDNA的纳米颗粒在血液中具有明显的稳定性，它们在需要细胞和/或组织特异性递送的全身基因治疗应用中可能具有潜力。
