Design And Synthesis Of A FRET-based Small Molecular Drug Delivery System And Its Application Research

Due to its relatively high reactivity with the disulfide bond,GSH has been extensively utilized as a trigger to cleave the disulfide-containing prodrug and release the active drug.To date,there are some reports on the use of GSH to trigger the release of active drug from stimuli-responsive prodrug.While the majority of those GSH-triggered prodrug systems are based on the fluorescence “turn-on” mechanism;it is preferable to design novel drug delivery systems(DDS)with ratiometric fluorescence response capability,where the disease diagnosis and/or drug release can be reported via more than one fluorescence signals.Herein,we designed a prodrug strategy based on the fluorescence resonance energy transfer(FRET)mechanism.The prodrug was constructed with an anticancer drug camptothecin(CPT),a cleavable linker based on disulfide bonds(SS)and a fluorophore naphthalimide derivative(NAP).For this drug delivery and reporting system(NAP-SS-CPT),FRET occurs between CPT(energy donor)and NAP(energy receptor);and upon cellular uptake by GSH-overexpressing cancer cells,disulfide bonds can be successfully cleaved by the high concentration of GSH,and FRET process is interrupted to achieve dual fluorescence response and specifically release of CPT.In this paper,the main research contents and conclusions are as follow:(1)The small molecular drug release system NAP-SS-CPT was synthesized in four steps under mild conditions.Firstly,the bromine in the 4-bromo-1,8-naphthalic anhydride is replaced with dimethylamine,and then it reacts with β-alanine to introduce carboxyl group.One end of bis(2-hydroxyethyl)disulfide is attached to the last step product by esterification reaction,and the other end is attached to drug CPT through carbonic ester under the action of triphosgene to obtain final product.The structure of the intermediate and final product was characterized by 1H-NMR and ESI-MS,and the results showed that we successfully obtained prodrug NAP-SS-CPT.(2)In the spectroscopy properties research,when excited at 400 nm,an emission band at544 nm which belongs to the NAP moiety,while the fluorescence of CPT moiety is almostcompletely quenched;after treatment with GSH,the fluorescence intensity at 448 nm increases significantly and the fluorescence intensity at 544 nm decreases obviously.The result indicate GSH is capable of cleaving the disulfide bond in the prodrug and subsequently releasing CPT,and it also confirms that the FRET process is disrupted upon addition of GSH.In addition,We also tried to investigate the effect of pH on the drug delivery systems in the presence or absence of GSH and its selectivity.All results demonstrate that the prodrug we design is stable under physiological conditions and CPT can be released efficiently,and the prodrug NAP-SS-CPT possesses high selectivity towards GSH.(3)In the biological applications studies of this prodrug,intracellular fluorescence imaging studies indicate that the prodrug can be efficiently internalized in HeLa cells and used for real-time monitoring of drug release.Moreover,MTT and flow cytometry assay indicate that NAP-SS-CPT exhibits high pro-apoptotic effect for cancer cells.This strategy may provide a new approach for development of small molecular drug delivery system.