Graphene Quantum Dots

Aptamer/Graphene Quantum Dots Nanocomposite Capped Fluorescent Mesoporous Silica Nanoparticles for Intracellular Drug Delivery and Real-Time Monitoring of Drug Release

Fen-Fen Zheng,Peng-Hui Zhang,Yu Xi,Jing-Jia Chen,Ling-Ling Li,*and Jun-Jie Zhu*

State Key Laboratory of Analytical Chemistry and Collaborative Innovation Center of Chemistry for Life Sciences,School of Chemistry&Chemical Engineering,Nanjing University,Nanjing210093,People’s Republic of China

*Supporting Information

system features high speci?city of dual-target

FRET-based monitoring strategy.Thus,the

applications for versatile drug-release monitoring,

suggested with the use of thermosensitive liposomes for the local release of drug through hyperthermia.1Since then,a lot of research has been carried out on stimuli-responsive systems for drug delivery.2?8An ideal stimuli-responsive drug delivery system should have the following characteristics:(i) recognize tumor microenvironment and target tumor cells in high selective manner,(ii)allow for tailored release pro?les with spatial,temporal,and dosage control in response to exogenous or endogenous stimulus,(iii)monitor drug release in real time to ascertain actual drug concentrations at targeted area for avoiding insu?cient or excess drug dosing.

To date,noninvasive and biocompatible mesoporous silica nanoparticles(MSNs)as e?cient drug delivery carriers have attracted tremendous attention by virtue of their tunable pore size,unique porous structure,high speci?c surface area,good biocompatibility,and ease of surface functionalization.9,10Great progress in structural control and functional design has been achieved for bioapplications.11,12The ordered pore network of these MSNs can entrap drug within the pores.Importantly,the pores could be gated with various valves such as nano-particles,13?17polymer multilayers,18DNA,19,20or proteins,21 which were designed to trigger the release of the entrapped drug in the presence of external or internal stimuli including light,22?24temperature,25?27pH,28?30and biomolecules.31?33

numerous stimuli-responsive MSNs for drug delivery have been reported,few has the capability of real-time monitoring,which remains a critical challenge.

Among all of the current real-time monitoring strategies,?uorescence imaging is one of the most sensitive techniques.34 To follow the optical signal of the drug molecules themselves is the most ideal mode of monitoring drug release.35,36However, the majority of the drug candidates are neither?uorescent nor light-absorbent.Another possible way is to conjugate the drugs with caged dyes,37,38which may lead to drug structural change, thus a?ecting their therapeutic e?cacy.Lee’s group developed a novel FRET(?uorescence resonance energy transfer)based real-time monitoring system in drug delivery,39which consisted of coumarin-labeled-cysteine tethered mesoporous silica nano-particles(MSNs)as the drug carrier,?uorescein isothiocyanate-β-cyclodextrin(FITC-β-CD)as the redox-responsive molecular valve to block the pores,and a FRET donor?acceptor pair of coumarin and FITC integrated within the pore-unlocking event.This provides a new strategy for real-time monitoring of drug release by using a nondrug based FRET donor?acceptor pair.

Received:August4,2015

Accepted:November2,2015

Owing to its unique ability for DNA adsorption 40,41as well as its super quenching capability,graphene is known to be a robust arti ?cial nanomaterial in DNA analysis,42,43protein assays,44drug delivery,45etc.Graphene quantum dots (GQDs)maintain the intrinsic layered structural motif of graphene,but with smaller lateral size and abundant periphery carboxylic groups,and are more compatible with biological systems,46,47thus emerging as promising nanomaterials for DNA adsorbing and ?uorescence quenching.Recently,Wang ’s group reported that GQDs had an attractive ability in drug delivery without any premodi ?cation due to their unique structural properties.48The GQDs could e ?ciently adsorb doxorubicin (Dox)molecules and quench their ?uorescence.Therefore,the smaller size,excellent adsorption,and quenching capability of GQDs make them to be ideal capping motifs and FRET acceptors.

In this article,a multifunctional FRET-nanocarrier was fabricated by assembling the ATP aptamer (functionalization aptamer,FA)on the surface of ?uorescent mesoporous silica nanoparticles (FMSNs)to form FA-FMSNs,which could strongly adsorb GQDs through π?πstacking interaction and e ?ciently cap the pores of FMSNs as well as quench the ?uorescence of FMSNs via FRET (Scheme 1).Moreover,ATP is present in low concentrations (<0.4mM)in the extracellular environment but is relatively concentrated within the intra-cellular cytosol (1?10mM).49?52This striking di ?erence is bene ?cial in the design of ATP-mediated drug release systems.53?55Thus,once the FRET-nanocarriers were exclusively internalized into the target tumor cells by AS1411aptamer (target aptamer,TA)recognition,the intracellular ATP served as a speci ?c key to bind with ATP aptamer,inducing gradual desorption of GQDs from the mesopores and thereby triggering controlled drug release.The increase of ?uorescence intensity due to the reduction of FRET from FMSNs to GQDs could be simultaneously used to monitor the drug release in real time.

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EXPERIMENTAL SECTION

Preparation of FAG-FMSNs-TA-Dox-PEG (FRET-Nano-carriers).The synthesis of GQDs and amino-functionalized ?uorescent mesoporous silica nanoparticles (FMSNs-NH 2)

were described in the Supporting Information .The EDC/NHS method was used to conduct the conjugation of the aptamer molecules (ATP aptamer (FA)and AS1411aptamer (TA),molar ratio1:5)and FMSNs-NH 2to form FA-FMSNs-TA.The loading amounts of the aptamer strands on the FMSNs have been quanti ?ed by monitoring the ?uorescence spectra of the aptamer (modi ?ed with FAM)in the supernatant (n 1)and in the stock solutions (n 2),the surface densities of the conjugated aptamer (d 3)on the FMSNs (m 3=1mg)were calculated as follows:d 3=(n 2?n 1)/m 3,where the units of n ,m ,and d are nmol,mg,and nmol mg ?1,respectively.According to the amounts of the aptamer (10nmol)added,the surface densities of the conjugated aptamer were 9.4nmol mg ?1.First,Dox molecules were loaded into the FA-FMSNs-TA by incubating the nanoparticles (1.0mg mL ?1)with di ?erent concentrations of Dox stock solutions in PBS (10mM,5mM MgCl 2,100mM NaCl,pH 7.4)for 24h.By centrifugation,the FA-FMSNs-TA-Dox and the supernatant were separated and collected.The Dox loading capacities were calculated by measuring the UV ?vis spectra of the supernatant and stock solutions.Second,the sediment of FA-FMSNs-TA-Dox was dissolved in PBS (10mM,5mM MgCl 2,100mM NaCl,pH 7.4),then di ?erent concentrations of GQDs were added and shaken at 37°C.After 6h,the FAG-FMSNs-TA-Dox were obtained by centrifugation and washing with PBS for three times.Through the interaction between FA and GQDs,the GQDs were capped onto the surfaces of the FA-FMSNs-TA-Dox to form FAG-FMSNs-TA-Dox.On the one hand,the GQDs could prevent the leakage of DOX from the mesopores.On the other hand,the GQDs also could quench the ?uorescence of FMSNs by FRET from FMSNs to GQDs.Finally,FAG-FMSNs-TA-Dox-PEG was obtained by shaking the mixture of FAG-FMSNs-TA-Dox solution and mPEG-SPA stock solution (2mg mL ?1in pH 7.4PBS)at a speed of 200rpm for 6h.The mPEG-SPA,readily hydrolyzed in water to yield N -hydroxysuccinimide,was rapidly conjugated onto the nanoparticles by reacting with the amine residues originating from the APTES functionalization.The unreacted mPEG-SPA molecules were removed by two centrifugation/washing cycles.

Dox Release Experiments.The Dox loaded FRET-nanocarriers were incubated in 10mM pH 7.4,6.5,and 5.0PBS for di ?erent amounts of time,respectively.After the addition of di ?erent concentrations of ATP stock solution,the Dox released from the FRET-nanocarriers was collected by centrifugation at 9300rpm.The amount of released Dox in the supernatant solutions was measured by UV ?vis spectropho-tometry.

Cell Culture and Drug Release Monitoring.Human cervical carcinoma cells (HeLa cells)were obtained from Nanjing KeyGen Biotech Co.Ltd.and cultured in Dulbecco ’s Modi ?ed Eagle Medium (DMEM)at 37°C under 5%CO 2atmosphere,supplemented with L -glutamine (2mM),penicillin (100units mL ?1),streptomycin (100μg mL ?1),and 10%fetal bovine serum (FBS).At the logarithmic growth phase,the cells were incubated with di ?erent nanoparticles in cultured medium for di ?erent times,then washed with PBS three times and cultured with fresh medium.The amounts of the FRET-nanocarriers internalized into the cells were evaluated by ?ow cytometry,while the drug release inside HeLa cells was monitored by CLSM.Cell viability was measured by MTT assay.Brie ?y,HeLa cells were cultured in a 96-well plate at a density of 10000cells in each well.After incubation for 24h,the medium was replaced with 100μL of fresh medium

Scheme 1.Preparation and Application of ATP-Responsive FRET

Nanocarriers

containing di ?erent concentrations of the nanoparticles.At the indicated time points (24or 48h),the medium was removed,and fresh medium (100μL)containing MTT (10μL,5mg mL ?1)was added into each well.After incubation for 4h,the absorbance of the solution was measured to assess the relative viability of the cells using a ThermoFisher Scienti ?c Varioskan Flash multifunctional microplate reader.The optical density (OD)was read at a wavelength of 492nm.Relative cell viability was expressed as ([OD]test /[OD]control )×100%.Each experiment was repeated at least three times.

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RESULTS AND DISCUSSION

Interaction between FA and GQDs.GQDs was synthesized according to our previously reported method.56The high-resolution transmission electron microscopy (HRTEM)shows that the GQDs have the average diameter of 4.5nm (Figure S1a ).The topographic heights of GQDs are mostly located between 0.5and 1.5nm with an average height of 1nm,suggesting that most of GQDs are single layered or bilayered (Figure S1b ).The cytotoxicity of the GQDs was measured by in vitro MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo-lium bromide)assay (Figure S1c ).The cell viability of HeLa cells remained above 80%when they were treated by GQDs with a concentration up to 800μg mL ?1for 48h,indicating that GQDs have excellent biocompatibility.To investigate the feasibility of the FA/GQD nanocomplexes as a stimuli-responsive gate,ATP-responsive experiment was carried out in vitro ?rst.Brie ?y,ATP aptamer labeled with FAM (carboxy ?uorescein)was incubated with GQDs to form FAM-FA/GQD nanocomplexes.The quenching ability of GQDs was estimated by measuring the ?uorescence intensity of FAM-FA solution (100nM)with di ?erent amounts of GQDs,ranging from 1to 100μg mL ?1(Figure 1b).Owing to the FRET

process between FAM and GQDs,a gradual decrease of ?uorescence intensity was observed with increasing amounts of GQDs assembled on the complex.Here,100μg mL ?1of GQDs was chosen as the optimized concentration with a quenching e ?ciency of 70%.In the presence of ATP,ATP aptamer could target ATP to form a hairpin con ?guration,thus dissociating ATP aptamer from the surface of GQDs and invalidating the FRET process.Consequently,obvious ?uorescence recovery

was observed by injecting ATP solution into the reaction system (Figure 1c).

Fabrication and Characterization of FRET-Nanocarr-riers.The FMSNs were synthesized by doping ?uorescein isothiocyanate (FITC)into the mesoporous silica nanoparticles following the reported protocol with some modi ?cation.57The obtained FMSNs had a uniform size of 40nm and displayed strong ?uorescence and remarkable photostability,thus facilitating the tracking of the intracellular drug delivery and the real-time monitoring of drug release (Figure S2).These FMSNs were then functionalized with APTES and tethered with FA and TA via an amide bond.The average diameter of the GQDs was 4.5nm,which could be preferable to cap the 2.7nm-wide-pore.The Dox was selected as a model cargo,which was loaded into the pores of FMSNs by mixing FA-FMSNs-TA and Dox overnight (FA-FMSNs-TA-Dox).The Dox molecules with positive charge could di ?use into the pores of FMSNs and were trapped by the silanol residuals through hydrogen bonds and electrostatic interactions.58,59Then,the pores were capped with GQDs through π?πstacking interaction between FA and GQDs (FAG-FMSNs-TA-Dox),and the mPEG-SPA (SPA =succinyl propionate)was further conjugated to the FMSNs via the reaction between amine residues and SPA.Finally,the FRET-nanocarrriers (FAG-FMSNs-TA-Dox-PEG)was isolated by centrifugation after repeated washing.The ?uorescence of the entrapped Dox could also be quenched by GQDs which could be used to verify the real-time monitoring e ?ciency of the FRET-nanocarriers.The amount of Dox loaded into FMSNs was determined to be 168mg g ?1(Figure S8).And the drug amount remains nearly the same after it is treated with GQDs and PEG (Figure S9).The FRET-nanocarriers were well-dispersed in aqueous solutions due to the conjugation of hydrophilic PEG moieties on their surface.

Next,the assembly process of the FRET-nanocarrriers was characterized by TEM.Figure 2a shows that FMSNs and FA-FMSNs-TA has similar size with a mean diameter of 40nm,while the size of FAG-FMSNs-TA-Dox-PEG has increased to 50nm.Besides,the dynamic light scattering (DLS)result reveals that the hydrodynamic diameters of FMSNs,FA-FMSNs-TA,and FAG-FMSNs-TA are 58nm,72.5nm,and 79nm,respectively (Figure S3).The gradual increasing of the hydrodynamic diameter indicates the successful conjugation of FA and GQDs.The wormlike mesoporous structures with pore sizes of 2?3nm and relatively high speci ?c surface areas (373.427m 2g ?1)could be observed from the absence of corresponding small-angle X-ray di ?raction peak(s)(Figure S4)and nitrogen adsorption ?desorption isotherm measurements (Figure S5).Furthermore,the successful surface functionaliza-tion was con ?rmed by Fourier transform-infrared (FT-IR)spectroscopy and zeta potential measurement (Figures S6and S7).As expected,the ?uorescence of FMSNs-FA could be quenched by GQDs based on FRET similar to FAM-FA (Figure 2c).The loading amounts of the GQDs on the FA-FMSNs have been optimized to be 149μg mg ?1by monitoring the ?uorescence spectra of the GQDs in the supernatant and in the stock solutions (Figure S10).In addition,the remarkable ?uorescence recovery of FMSNs further demonstrated that these FRET-nanocarriers could be used to monitor ATP-triggered drug release in real time (Figure 2d).

ATP-Responsive Controlled Drug Release and Corre-lating Drug Release to the FRET Signal in Vitro .Furthermore,to investigate the ATP-triggered uncapping e ?ciency of the FRET-nanocarrriers,drug release

experiments

Figure 1.(a)Scheme of the interaction between ATP aptamer and GQDs.(b)Fluorescence emission spectra of 100nM FAM-FA quenched by addition of GQDs with concentrations ranging from 10to 100μg mL ?1.(c)Fluorescence recovery of 10nM FAM-FA/GQD nanocomplexes by addition of ATP (0,0.05,0.2,0.8,2mM).

were carried out at di ?erent levels of ATP.Real-time drug release pro ?les of the samples were recorded using a UV ?vis spectrophotometer under the wavelength of 480nm (Figure 3a,1?4).In the absence of ATP,about 20%release of Dox was observed over a period of 48h,indicating that the FRET-nanocarriers almost remained intact.While in the presence of di ?erent ATP amounts,an increase in the released Dox could

be observed as time progressed.The percentage of Dox released from the FRET-nanocarriers was dependent on ATP concentrations,wherein concentrations of 5mM or higher led to signi ?cantly faster and greater release of Dox.The release of Dox could reach a plateau within 4h due to the rapid interaction between high concentrations of ATP and ATP aptamer.As a comparison test,G-FMSNs-Dox-PEG was prepared by the electrostatic interactions between GQDs and NH 2?FMSNs.Figure 3a,5shows that Dox entrapped in G-FMSNs-Dox-PEG cannot be released e ?ectively even in high concentrations of triggers,which indicates that the dissociation of GQDs from the FRET-nanocarrers is due to the speci ?c recognition and binding between ATP and FA.Considering the acidic extracellular microenvironment of tumor tissue,the stability of the drug delivery system was evaluated by determining the drug release in di ?erent bu ?ers.As shown in Figure S11,the FA/GQD (FAG)valve could e ?ectively prevent the release of Dox from the FRET-nanocarriers even in acidic environments (pH =5).With the addition of ATP,rapid drug release was observed at the given pH values.It is worth noting that upon the addition of ATP,the drug release rate was independent of the pH changes.Additionally,selectivity studies toward ATP have been presented by probing the e ?ects of di ?erent thymidine triphosphate analogues,TTP,CTP,GTP,on the unlocking process.The changes in ?uorescence spectra of the Dox released from the nanocarriers treated by ATP,TTP,CTP,and GTP are described in Figure https://www.sodocs.net/doc/b217364231.html,pared to the analogues,only ATP stimulates the e ?ective release of Dox with a sharp increase of ?uorescence intensity,indicating that the highly selective release of the nanocarriers was attributed to the speci ?c recognition between ATP and FA.

In addition to behaving as three-dimensional gatekeepers to completely encapsulate the drugs in the nanocarriers,the aptamer/GQD nanocomplexes were proven to be electron acceptors for e ?ciently quenching the ?uorescence of the FMSNs based on FRET (see above).This FRET property of the FRET-nanocarriers could be used to monitor the drug release from the pores.Because the modulation of FRET is integrated within the uncapping event,the corresponding change in the FRET signal can be used for monitoring the drug release at a temporal level.Meanwhile,since the release of Dox is rapid enough and only occurs when the pores are uncapped as a consequence of GQDs dissociation from the FRET-nanocarriers,there was probably a positive correlation between the drug release and the ?uorescence recovery of FMSNs.As shown in Figure 3b,the addition of ATP to Dox-loaded FRET-nanocarriers induced the Dox release that was indicated by the ?uorescence recovery of Dox.Simultaneously,a decrease in the FRET signal and thus the ?uorescence turn-on of FMSNs was synchronized to the release of Dox.On the basis of this result,a positive correlation between the released Dox and FRET signal was obtained,which strongly indicated that the drug release could be monitored in real-time by the FRET signal change of the FRET-nanocarriers.

Speci ?c Targeting and Traceability.Targeted delivery to cancer cells is essential in chemotherapy.Herein,the AS1411aptamer,currently an anticancer agent under phase II clinical trials and also an aptamer to speci ?cally recognize nucleolin overexpressed on the surfaces of some cancer cells,60was tethered to the FRET-nanocarriers as the target aptamer.The target e ?ciency and speci ?city of the FRET-nanocarriers were assessed by confocal laser scanning microscopy (CLSM)

and

Figure 2.(a)Schemes (top)and TEM images (bottom)of FMSNs,FA-FMSNs-TA,and FAG-FMSNs-TA-Dox-PEG.(b)Scheme of the interaction between FA-FMSNs-TA and GQDs.(c)Fluorescence emission spectra of 1mg mL ?1FA-FMSNs quenched by addition of GQDs with increased concentration (0,10,20,50,100,200μg mL ?1,respectively).(d)Fluorescence recovery of 1mg mL ?1FAG-FMSNs by addition of ATP (0,1,2,5,10mM,

respectively).

Figure 3.(a)(1?4)Release pro ?les of the FRET-nanocarrriers with addition of di ?erent concentrations of ATP in phosphate-bu ?ered saline (PBS,10mM,pH =5.0),the addition amounts of ATP were 0,1,5,10mM,respectively.(5)Release pro ?les of the G-FMSNs-Dox-PEG with addition of 10mM of ATP in phosphate-bu ?ered saline.(b)Fluorescence emission spectra of 0.1mg mL ?1FA-FMSNs-TA-Dox (1)quenched with 200μg mL ?1GQDs (2)and ?uorescence recovery by adding of 5mM ATP (incubated for 2h (3)or for 4h (4)).

?ow cytometry analysis by determining the?uorescence of the loaded Dox(Figure S13).The?uorescence emission of released Dox for HeLa cells was higher than that for NIH 3T3cells,which re?ected the enhanced nanocarriers uptake due to the speci?c interaction between AS1411aptamer and nucleolin.To further alleviate the side e?ects during cancer therapy,the FRET-nanocarriers was PEGylated,which could signi?cantly minimize its nonspeci?c adsorption onto the NIH 3T3cells.

CLSM was used to investigate the traceability of the FRET-nanocarriers during intracellular delivery and drug release. HeLa cells were cultured and incubated in cultured medium containing20μg mL?1of the FRET-nanocarriers at37°C for a period of time.The cellular uptake and intracellular tra?cking were investigated by monitoring the recovered?uorescence of FMSNs and Dox.After2h of incubation,FMSNs(green)and Dox(red)?uorescence signals were observed in HeLa cells. The FMSNs were mostly distributed in the cytoplasm,and the Dox was con?rmed mostly in the cell nucleus by costaining with DAPI molecules(Figure S14).Lysosome staining experiment showed that most of the FRET-nanocarriers were trapped inside the endosome/lysosome,while some of them had successfully escaped into the cytoplasm(Figure S15).The high cytosolic ATP level led to a signi?cant release of Dox, which speci?cally accumulated into the nuclei,intercalated into the twin-screw structure of DNA,inhibited macromolecular biosynthesis,and eventually induced cells death. Intracellular ATP-Triggered Drug Release and Mon-itoring in Real-Time.We further investigated whether the FRET-nanocarriers could be used to monitor intracellular ATP-triggered drug release by detecting the?uorescence recovery of FMSNs and Dox.The HeLa cells were incubated with the FRET-nanocarriers for a di?erent length of time.As expected,a time-course enhancement of Dox?uorescence in the cancer cells clearly indicated the controlled release of drugs from the FRET-nanocarriers.In the meantime,the gradual?uorescence recovery of FMSNs induced by DOX release was clearly detectable(Figure S16).This proves that the FRET-nano-carriers can respond to the presence of exogenous ATP by releasing the entrapped drug with concurrent change in the FRET signal.To demonstrate the intracellular ATP concen-

tration-dependent Dox release from FRET-nanocarriers,we inhibited the ATP production in the cells by physically lowering the temperature(4°C)and adding a chemical inhibitor, iodoacetic acid(IAA),prior to the addition of the FRET-nanocarriers.Cell incubation at4°C and adding IAA at37°C both led to a signi?cant decrease in ATP generation within the cells(Figure4a).The?uctuation in intracellular ATP concentration can result in a change in the extent of GQDs dissociation from the FRET-nanocarriers,which shall deter-mine the FRET signal as well as the amount of Dox released. Correspondingly,?ow cytometric results demonstrated the decrease of FMSNs?uorescence signal,which could be attributed to the down-regulation of ATP contents and reduced Dox release(Figure4b).CLSM revealed that Dox release from FRET-nanocarriers was remarkable reduced in the cells that were incubated at4°C or treated with IAA at37°C,further con?rming the monitoring capability of the FRET-nanocarriers for intracellular ATP-mediated drug release(Figure4c). Selective Toxicity.MTT tests were conducted to assess the tumoricidal potential of the established FRET-nanocarriers. HeLa cells and NIH-3T3cells were incubated with di?erent FRET-nanocarriers,respectively,in culture medium at37°C for24or48h.As shown in Figure S17a,blank FRET-nanocarriers without Dox did not show cytotoxicity within the tested concentration range.Meanwhile,the e?cient intra-cellular release of Dox from FAG-FMSNs-TA-Dox-PEG triggered by ATP provided higher cytotoxicity as compared to G-FMSNs-Dox-PEG which could not be uncapped by ATP. Speci?c lethality of the FRET-nanocarriers was investigated by incubating target cells(HeLa)with Dox,Dox-loaded FA-FMSNs and Dox-loaded FA-FMSNs-TA(Figure S17b).The lethality of free Dox at the microgram level was very small due to the drug e?ux pump e?ect by ATP-binding cassette transporters from cytoplasm.61On the other hand,thanks to the AS1411aptamer,the cellular uptake of FA-FMSNs-TA was higher than FA-FMSNs,which led to better anticancer activity. The MTT results shown in Figure5demonstrated that the toxicity of the FRET-nanocarriers without AS1411aptamer and PEG modi?cation was approximately equal toward NIH-3T3 cells and HeLa cells.After AS1411aptamer and PEG modi?cation,the FRET-nanocarriers exhibited much higher toxicity to HeLa cells than to NIH-3T3cells due to the speci?c recognition of AS1411aptamer and the antinonspeci?c adsorption by PEG.CLSM results have displayed

the Figure4.(a)ATP content in HeLa cells after di?erent treatments;(b)?uorescence recovery of FMSNs obtained by?ow cytometry(lines from left to right,control;incubation at4°C;incubation with IAA at 37°C;incubation at37°C);(c)Dox released in HeLa cells of FAG-FMSNs-TA-Dox-PEG obtained using the CLSM.The cells were incubated with FAG-FMSNs-TA-Dox-PEG at37°C for1h and then incubated with the fresh culture medium at37°C for additional2h (i),incubated with IAA at37°C for additional2h(ii),or incubated at 4°C for additional2h(iii)after removal of the excess FRET-nanocarriers.

therapeutic e ?cacy of the FRET-nanocarriers more visually (Figure 5e).A burst release of Dox was observed after the ?rst 6h owing to the uncapping by ATP.A mass of apoptotic bodies appeared after 24h,which indicated that most of the cells were induced to apoptosis,suggesting a high therapeutic e ?cacy of the FRET-nanocarriers.

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CONCLUSIONS

In summary,we have fabricated an ATP-responsive FRET-nanocarrier for intracellular drug delivery and real-time monitoring of drug release,wherein ?uorescent mesoporous silica nanoparticles acted as sca ?olds and aptamer ?GQD nanocomplexes as capping and quenching motifs.It has been demonstrated that any exogenous or endogenous ?uctuation in the ATP concentration can result in a change in drug release as well as concurrent variation in the FRET signal.The FRET-nanocarriers possess various attractive features.First,with the dual-target of AS1411and ATP aptamer,the FRET-nano-carriers could release the drug more selectively in the cytoplasm of cancer cells.Second,the aptamer ?GQD nanocomplexes behave as three-dimensional gatekeepers to completely encapsulate the drugs in the nanocarriers as well as electron acceptors to e ?ciently quench the ?uorescence of the FMSNs.Third,the drug release was directly re ?ected by the recovery of the FMSNs along with the dissociation of the GQDs,which can be extended to any cargo without relying on the optical properties or the structural modi ?cation of the drugs.It is

hoped this strategy could monitor intracellular drug concen-trations and bene ?t successful chemotherapy for cancers.

■ASSOCIATED CONTENT

*

Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI:10.1021/acs.anal-chem.5b03131.

Additional experimental details and data (PDF )

■AUTHOR INFORMATION

Corresponding Authors

*E-mail:lll-100@https://www.sodocs.net/doc/b217364231.html, .*E-mail:jjzhu@https://www.sodocs.net/doc/b217364231.html, .

Notes

The authors declare no competing ?nancial interest.

■ACKNOWLEDGMENTS

We gratefully appreciate the support from the National Basic Research Program (Grant 2011CB933502)of China and the National Natural Science Foundation (Grant Nos.21335004,21427807,and 21405078).

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