PicoGreen荧光法结合茚三酮比色法测定载pDNA壳聚糖纳米粒中pDNA的包封率和载药量___

目的：建立微量DNA和壳聚糖的含量测定方法，用于测定载pDNA壳聚糖纳米粒中pDNA的包封率和载药量。方法：利用PicoGreen荧光染料与双链DNA特异结合后激发产生荧光，检测荧光强度，建立标准曲线，测定纳米粒混悬液中游离的pDNA含量 利用茚三酮和壳聚糖分子上的伯氨基发生显色反应，紫外法测定吸收度，建立标准曲线，测定纳米粒混悬液中游离的壳聚糖含量 按照游离的pDNA和壳聚糖含量分别计算纳米粒中pDNA的包封率和载药量。结果：PicoGreen荧光法的线性范围1~50 ng•mL-1，低、中、高3种浓度的回收率分别为103.4%，97.6%，98.7%，相应RSD分别为 1.0%，0.1%，0.2% (n=3)。茚三酮比色法的线性范围0.5~10 μg•mL-1，低、中、高3种浓度的回收率分别为104.0%，98.6%，97.9%，相应RSD分别为 3.3%，0.6%，1.7% (n=3)。2种分析方法的日内和日间精密度RSD均小于5% (n=5)。测定纳米粒中pDNA的包封率为(99.67 ± 0.12)%，RSD 为0.12% (n=3) 载药量为(50.90 ± 1.71)%，RSD 为3.4% (n=3)。结论：本文建立的PicoGreen荧光法和茚三酮比色法灵敏度高，准确可靠，可以用于载pDNA壳聚糖纳米粒中pDNA的包封率和载药量的测定。

Objective:To develop the analytical methods for determining pDNA encapsulating and loading efficiency of pDNA loaded chitosan nanoparticles.Methods:The fluorescence intensity of the mixture of PicoGreen and pDNA was detected by fluoromerter.A standard curve was established between the fluorescence intensities and the pDNA concentrations,and the concentration of free pDNA in the nanoparticle suspension was determined.The absorbance of the mixture of chitosan and ninhydrin was detected by an UV/Vis spectrophotometer.A standard curve was established between the absorbance and the chitosan concentrations,and the concentration of free chitosan in the nanoparticle suspension was determined.The encapsulating and loading efficiency were calculated according to the free concentrations of pDNA and chitosan,respectively.Results:The PicoGreen-fluorometry method had a good linear correlation in the range 1-50 ng•mL-1 of DNA,and the recovery at the low,middle,high concentrations were 103.4%,97.6%,98.7% with corresponding RSD value 1.0%,0.1% and 0.2%,respectively (n=3).The ninhydrin-colorimetry method had a good linear correlation in the range 0.5-10 μg•mL-1 of chitosan,and the recovery at the low,middle,high concentrations were 104.0%,98.6%and 97.9% with corresponding RSD value 3.3%,0.6%and 1.7%,respectively (n=3).For these two analytical methods,the RSD values of intra-day and inter-day precision were all less than 5% (n=5).The average encapsulating and loading efficiency were (99.67 ± 0.12)% and (50.90 ± 1.71)% with corresponding RSD value 0.12%and 3.4%,respectively (n=3).Conclusion:The developed PicoGreen-fluorometry and ninhydrin-colorimetry methods were sensitive and accurate,and suitable for determining pDNA encapsulating and loading efficiency of pDNA loaded chitosan nanoparticles.