Integrating FRET And AIE For In Situ Probing Intracellular Drug Release From Redox-responsive Micelles

Stimuli-responsive nanocarriers are appealing in triggered drug release and sensitive analyte assay.Among these,redox-sensitive micelles are robust nanoplatforms for on-demand drug delivery and biosensing due to the intracellular reducing environment.However,the in situ evaluation of drug release is challenging.Fluorescence resonance energy transfer(FRET)offers a unique mechanism shows potential for addressing this,as the molecular assembly and disassembly in the nanoscale range can induce the variation of fluorescence signals.One big problem of FRET is that the clump of traditional chromophores in micelles could initiate fluorescence quenching,i.e.,the aggregation-caused quenching(ACQ)effect.AIE probes are intrinsically superior to traditional ACQ dyes due to their high resolution,resistance to photo-bleaching and large Stokes shift.Therefore,incorporating AIE molecules in FRET pairs offers a sensitive fluorescence “turn-on” technique that shows a broad of chemical and biological applications.The aim of the current work is to combine aggregation-induced emission(AIE)probe with FRET to realize drug release assessment from micelles.Tetraphenylethylene(TPE)is selected as AIE dye and curcumin(Cur)was chosen as the model drug as well as the FRET receptor.The drug was covalently linked to a block copolymer via the disulfide bond linker and TPE is also chemically linked to the polymer via an amide bond;the obtained amphiphilic polymer conjugate self-assembles into micelles with a hydrodynamic size of ca.125 nm and the critical micelle concentration(CMC)was determined to be at 8.7 ± 0.3 ?g/mL,the low CMC indicates good micelle stability,which is beneficial to maintain the micelle integrity under in vivo situations.Upon the supplement of glutathione or tris(2-carboxyethyl)phosphine trigger(10 mM),the drug release induces the fluorescence increase of both TPE and Cur.The intensity of absorption of micelles also increased with time,which was a result of drug release.Accompanied with Cur liberation,the size of micelles also increased over time.This was because the release of Cur altered the polymer amphiphilicity and reduced the driving force of micelle assembly.The same phenomenon is observed in MCF-7 cells.As GSH is an endogenous molecule in the cytosol,no supplementation of external GSH is needed.The confocal laser scanning microscope(CLSM)analysis clearly demonstrated that the fluorescence intensity of both TPE and Cur increased over time,which was believed as a result of GSH-mediated Cur release and the concomitant decline of FRET effect between TPE and Cur.In addition,the cells treated by GSH or buthionine sulfoximine(BSO)were used as the positive and negative controls,respectively.Increasing intracellular GSH concentration could speed up drug release with corresponding rapid upsurge of Cur fluorescence intensity.By contrast,the pretreatment by BSO would decrease the intracellular GSH level,and thus the rate of fluorescence change was slow.Interestingly,regarding the drug release within cells,the fluorescence intensity of TPE increased linearly against time,which was a clear contrast and advantage against that of Cur.This phenomenon was contributed to the high resolution and photo-bleaching resistance of TPE.The presence of TPE moiety could not only aid the monitoring in situ release of fluorescent drugs,but also provide information of non-fluorescent agents' release.The FRET-AIE coupling approach can be a useful addition to the spectrum of available methods for monitoring drug release from stimuli-responsive nanomedicine.When the excitation wavelength of both the drug and AIE probe locates within the near infrared range,such strategy could be utilized for in situ assessing drug release in vivo.