Plasmid DNA-Entrapped Nanoparticles Engineered from Microemulsion Precursors: In Vitro and in Vivo Evaluation
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- 作者：Zhengrong Cui and Russell J. Mumper
- 刊名：Bioconjugate Chemistry
- 出版年：2002
- 出版时间：November 2002
- 年：2002
- 卷：13
- 期：6
- 页码：1319 - 1327
- 全文大小：170K
- 年卷期：v.13,no.6(November 2002)
- ISSN：1520-4812
文摘
Nonviral gene therapy has been a rapidly growing field. However, delivery systems that can provideprotection for pDNA and potential targeting are still desired. A novel pDNA-nanoparticle deliverysystem was developed by entrapping hydrophobized pDNA inside nanoparticles engineered from oil-in-water (O/W) microemulsion precursors. Plasmid DNA was hydrophobized by complexing withcationic surfactants DOTAP and DDAB. Warm O/W microemulsions were prepared at 50-55 
C withemulsifying wax, Brij 78, Tween 20, and Tween 80. Nanoparticles were engineered by simply coolingthe O/W microemulsions containing the hydrophobized pDNA in the oil phase to room temperaturewhile stirring. The nanoparticles were characterized by particle sizing, zeta-potential, and TEM.Nanoparticles were challenged with serum nucleases to assess pDNA stability. In addition, thenanoparticles were coincubated with simulated biological media to assess their stability. In vitrohepatocyte transfection studies were completed with uncoated nanoparticles or nanoparticles coatedwith pullulan, a hepatocyte targeting ligand. In vivo biodistribution of the nanoparticles containingI-125 labeled pDNA was monitored 30 min after tail-vein injection to Balb/C mice. Depending on thehydrophobizing lipid agent employed, uniform pDNA-entrapped nanoparticles (100-160 nm indiameter) 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 usedto separate unentrapped pDNA from nanoparticles. Gel permeation chromatography of pDNA-entrapped and serum-digested nanoparticles showed that the incorporation efficiency was ~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 nanoparticlesresulted 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 veininjection to mice, only 16% of the 'naked' pDNA remained in the circulating blood compared to over40% of the entrapped pDNA. Due to the apparent stability of these pDNA-entrapped nanoparticles inthe blood, they may have potential for systemic gene therapy applications requiring cell and/or tissue-specific delivery.
C withemulsifying wax, Brij 78, Tween 20, and Tween 80. Nanoparticles were engineered by simply coolingthe O/W microemulsions containing the hydrophobized pDNA in the oil phase to room temperaturewhile stirring. The nanoparticles were characterized by particle sizing, zeta-potential, and TEM.Nanoparticles were challenged with serum nucleases to assess pDNA stability. In addition, thenanoparticles were coincubated with simulated biological media to assess their stability. In vitrohepatocyte transfection studies were completed with uncoated nanoparticles or nanoparticles coatedwith pullulan, a hepatocyte targeting ligand. In vivo biodistribution of the nanoparticles containingI-125 labeled pDNA was monitored 30 min after tail-vein injection to Balb/C mice. Depending on thehydrophobizing lipid agent employed, uniform pDNA-entrapped nanoparticles (100-160 nm indiameter) 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 usedto separate unentrapped pDNA from nanoparticles. Gel permeation chromatography of pDNA-entrapped and serum-digested nanoparticles showed that the incorporation efficiency was ~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 nanoparticlesresulted 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 veininjection to mice, only 16% of the 'naked' pDNA remained in the circulating blood compared to over40% of the entrapped pDNA. Due to the apparent stability of these pDNA-entrapped nanoparticles inthe blood, they may have potential for systemic gene therapy applications requiring cell and/or tissue-specific delivery.