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1～50ng.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0.5～10μg.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 establish a method for the determination of trace DNA and chitosan, and to determine the entrapment efficiency and drug loading of pDNA in pDNA loaded chitosan nanoparticles. Methods: The specific fluorescence intensity of PicoGreen fluorescent dye was detected by double-stranded DNA. The fluorescence intensity was measured and the standard curve was established. The content of free pDNA in the nanoparticle suspension was measured. Using ninhydrin and chitosan The reaction of chromogenic reaction occurred, and the absorbance was determined by UV method. The standard curve was established and the free chitosan content in the nanoparticle suspension was determined. The entrapment efficiency of pDNA in the nanoparticle was calculated according to the content of free pDNA and chitosan Dosage. Results: The linear range of PicoGreen fluorescence method was 1 ~ 50ng.mL-1, the recoveries were 103.4%, 97.6% and 98.7% respectively with corresponding RSDs of 1.0%, 0.1%, 0.2% (n = 3). The linear range of ninhydrin colorimetry was 0.5 ~ 10μg.mL-1, the recoveries of low, medium and high concentrations were 104.0%, 98.6% and 97.9% respectively, and the corresponding RSDs were 3.3%, 0.6%, 1.7 % (n = 3). The RSDs for intra-day and inter-day precision of the two methods were all less than 5% (n = 5). The entrapment efficiency of pDNA in the nanoparticles was (99.67 ± 0.12)% and the RSD was 0.12% (n = 3). The drug loading was (50.90 ± 1.71)% and the RSD was 3.4% (n = 3). Conclusion: The established PicoGreen fluorescence method and ninhydrin colorimetric method have high sensitivity, accuracy and reliability and can be used to determine the entrapment efficiency and drug loading of pDNA in pDNA chitosan nanoparticles.