基于FRET效应对纳米递药系统胞内释药检测方法的建立

Establishment of a detection method for intracellular drug release of nano drug delivery systems based on FRET effect

目的:以2种荧光标记物之间的荧光共振能量转移效应(FRET)为基础,建立一种纳米递药系统细胞内释药的测定方法,并对该方法进行实验验证。方法:以香豆素-6(Cou-6)为荧光探针标记的药物模型,采用二硫键还原法以罗丹明B异硫氰酸酯(RBITC)标记的白蛋白制备载Cou-6的白蛋白纳米粒。根据FRET效应建立纳米粒释药的荧光检测方法,分别采用该法和超滤离心法对载药纳米粒在缓冲液中的释药行为进行测定,通过比较2种方法的检测结果对FRET法进行验证,并且以FRET法对载Cou-6的白蛋白纳米粒在MCF-7细胞内的Cou-6释放进行考察。结果:载Cou-6的白蛋白纳米粒具有显著的FRET效应,药载比为0.000 5时,FRET指数为0.33。缓冲液中的释药结果显示FRET法与超滤离心法的测定结果无显著差异。MCF-7细胞中的Cou-6释放实验表明Cou-6在12 h可持续从递药系统中释放,释药半衰期为27.3 h。结论:本文建立的FRET法,可实现对该递药系统胞内释药的定量测定。

Objective： To establish and validate a detection method for intracellular drug release of nano drug delivery systems based on fluorescence resonance energy transfer effects（ FRET） of dyes. Methods： Coumarin-6（ Cou-6） was selected as the model drug,and Cou-6-loaded albumin nanoparticles were formulated from rhodamine B isothiocyanate（ RBITC）-labeled albumin using a disulfide bond reducing method. A FRET-based fluorescence method was established to determine the intracellular drug release of this delivery system. This method was validated by comparing the release of Cou-6 in buffers determined with the FRET method and the centrifugal ultrafiltration method,and the established method was used to detect the release of Cou-6 in MCF-7 cells. Results： The Cou-6-loaded albumin nanoparticles possessed significant FRET effects,and the FRET index of the nanoparticles was 0. 33 when the ratio of Cou-6 to albumin was set at 0. 000 5. The release of Cou-6 in buffers showed no significant difference between the determination results of the FRET method and the centrifugal ultrafiltration method. The Cou-6 release test in MCF-7 cells showed that Cou-6 was released from the carriers gradually within 12 h,and the half-life of drug release was calculated to be 27. 3 h. Conclusion： The established FRET-based method can be usedto determine the intracellular drug release of the system prepared in this study quantitatively.