Nanoscale

PAPER

Cite this: Nanoscale, 2017, 9, 17318

Received 31st August 2017,Accepted 11th October 2017

DOI: 10.1039/c7nr06479a

rsc.li/nanoscale

Nanocarriers with multi-locked DNA valvestargeting intracellular tumor-related mRNAsfor controlled drug release†

Yanhua Li, Yuanyuan Chen, Wei Pan, Zhengze Yu, Limin Yang,Hongyu Wang, Na Li * and Bo Tang *

The fabrication of well-behaved drug delivery systems that can transport drugs to specifically treat cancer

cells rather than normal cells is still a tremendous challenge. A novel drug delivery system with two types

of tumor-related mRNAs as “keys” to open the multiple valves of the nanocarrier to control drug release

was developed. Hollow mesoporous silica nanoparticles were employed as the nanocarrier and dual

DNAs targeting two intracellular mRNAs were employed as “multi-locks” to lock up the nanocarrier. When

the nanocarrier enters the cancer cells, the overexpressed endogenous mRNA keys hybridize with the

DNA multi-locks to open the valves and release the drug. Each single mRNA could not trigger the

opening of the locks to release the cargo. Therefore, the nanocarrier can be applied for specific chemo-

therapy against cancer cells with minor side effects to normal cells. The current strategy could provide an

important avenue towards advancing the practical applications of drug delivery systems used for cancer

therapy.

1 Introduction

Chemotherapy, one of the most predominant strategies foreffective tumor therapy, is the use of chemotherapeutic agentsto destroy cancer cells. However, it is still severely limited byits unexpected biodistribution.1,2 Moreover, therapeutic agentsentering all cells in the body will indiscriminately cause rela-tively low drug potency and harm healthy cells, eventuallyresulting in severe adverse effects to patients.2,3 Therefore, it ishighly desirable to design drug delivery systems (DDSs) toincrease the local effective therapeutic concentration in cancercells but not in normal cells.4 In order to achieve this goal, twopromising strategies have been proposed: one is to establish aDDS for actively targeting cancer cells to convey the therapeuticagent using folic acid, etc.;5–9 the other is to develop a stimuli-responsive DDS responding to high ROS or low pH in thetumor microenvironment rather than normal tissues for con-trolled drug release.10–15 However, significant folate receptorexpression also occurs on the surface of normal cells and the

microenvironment difference between the tumor and normaltissues is negligible, which all lead to inevitable drug releasein normal cells.4,16,17 Thus, the fabrication of drug deliverysystems that can transport drugs to selectively treat the cancercells rather than the normal cells is the main goal andchallenge.

In recent years, tumor markers are usually used as the mosteffective biomolecules used to distinguish cancer cells fromnormal cells.18–21 However, certain tumor markers are alsooverexpressed in normal cells. Our group has designed variousnanoprobes to detect multiple tumor markers for identifyingcancer cells whilst preventing false positive results.22–28 Weproposed that either diagnosis or treatment of cancer willobtain imperfect results based on a single tumor marker.Therefore, developing DDSs with multiple tumor markers tosynergistically control drug release is a safer strategy.

Herein, we report a proof of concept for developing novelDDS with dual DNAs as “multi-locks” to lock up the nano-carrier and two types of tumor-related mRNAs as the “keys” tosynergistically open the valves to release the drug. This DDSuses amino modified hollow mesoporous silica nanoparticles(HMSNs) as the nanocarrier, mainly on account of their excel-lent biocompatibility, large surface area, high pore volume andtunable pore sizes.29–31 HMSNs are positively charged becauseof protonation of the amino groups, which is beneficial encap-sulation with negatively charged DNA.32,33 As depicted inScheme 1, once the HMSNs were loaded with drugs, the curly

†Electronic supplementary information (ESI) available. See DOI: 10.1039/c7nr06479a

College of Chemistry, Chemical Engineering and Materials Science, Collaborative

Innovation Center of Functionalized Probes for Chemical Imaging in Universities of

Shandong, Key Laboratory of Molecular and Nano Probes, Ministry of Education,

Institute of Molecular and Nano Science, Shandong Normal University, Jinan

250014, P. R. China. E-mail: lina@sdnu.edu.cn, tangb@sdnu.edu.cn

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DNA molecules on the surface of the nanoparticles can encap-sulate the mesopores and serve as the “locks” to lock up thechannels. The DNA multi-locks can specifically hybridize withthe intracellular tumor-related mRNA (TK1 mRNA and GalNAc-T mRNA). TK1 mRNA is a marker for tumor growth, which islinked to cell division; GalNAc-T mRNA (abbreviated as GTmRNA) is overexpressed in numerous cancer cells and is a vitalenzyme in the synthetic process of gangliosides GM2/GD2,while it is also overexpressed in certain normal cells.22,34,35

Only when TK1 and GT mRNA exist simultaneously in thesystem, will the DNA multi-locks hybridize with them, result-ing in the uncapping of the pores and release of the entrappedcargo from the nanocarrier. Each single mRNA key cannottrigger the opening of the valves to release the cargo.Therefore, this DDS can be applied for specific chemotherapyagainst cancer cells with little side effects to normal cells. Thedetails of this process are presented in Scheme 1.

2 Experimental2.1 Reagents and materials

Octoxinol (Tx-100), n-hexanol, cyclohexane, ammoniumhydroxide (NH4OH, 28%), tetraethyl orthosilicate (TEOS),ethanol and hydrofluoric acid (HF, 40%) were purchased fromChina National Pharmaceutical Group Corporation (Shanghai,China). TK1 DNA, GT DNA and doxorubicin hydrochloride (Dox)were purchased from Sangon Biotechnology Co., Ltd (Shanghai,China). 3-Aminopropyltriethoxysilane (APTS), Rhodamine B(RhB), 3-(4, 5-dimethyl-thiazol-2-yl)-2 and 5-diphenyltetrazoliumbromide (MTT) were purchased from Sigma Chemical Company(America). The human breast cancer cell line (MCF-7), humanmammary epithelial cell line (MCF-10A), human lung epithelialcell line (BEAS), human lung adenocarcinoma cell line (A549),human hepatocellular liver carcinoma cell line (HepG2) andhuman hepatocyte cell line (HL-7702) were purchased fromKeyGEN biotechnology Company (Nanjing, China). All thechemicals were of analytical grade and used without any further

purification. Sartorius ultrapure water (18.2 MΩ cm) was usedthroughout the experiments.

2.2 Apparatus

Transmission electron microscopy (TEM) was carried out on aJEM-100CX Π electron microscope. Fluorescence spectra wereobtained on a FLS-920 Edinburgh Fluorescence Spectrometerequipped with a Xenon lamp using 1.0 cm quartz cells. All pHmeasurements were performed with a pH-3c digital pH-meter(Shanghai LeiCi Device Works, Shanghai, China) using a com-bined glass-calomel electrode. The absorbance was measuredon a microplate reader (Synergy 2, Biotek, USA) in the MTTassay. Confocal fluorescence imaging studies were performedon a TCS SP5 confocal laser scanning microscope (Leica Co.,Ltd. Germany) equipped with an objective lens (×40). RT-PCRwas carried out with an ABI PRISM 7000 sequence detectionsystem (Applied Biosystems, Foster City, CA).

2.3 Preparation of silica coated SiO2-NH2 nanoparticles

Silica nanoparticles modified with amine groups (SiO2-NH2)were synthesized using a reversed-phase microemulsionmethod according to a reported protocol with some modifi-cation.36 A solution containing 5.3 mL of Tx-100, 22.5 mL ofcyclohexane, 5.4 mL of n-hexanol and 1.5 mL of sartorius ultra-pure water was prepared in a conical flask. Next, 350 μL ofNH4OH (28%) was added to the mixture with stirring. After30 min, 400 μL of TEOS and 40 μL of APTS were rapidly addedinto the above mixture and the resulting solution was stirredfor 24 h. The as-synthesized SiO2-NH2 were centrifuged andwashed with ethanol, and then redispersed in 60 mL ofethanol for the preparation of the silica coated SiO2-NH2 nano-particles (SiO2-NH2@SiO2). 20 mL of NH4OH was added intothe above solution with stirring. After that, 1 mL of TEOS (dis-persed in 9 mL of ethanol) was added dropwise to the sampleand the reaction was conducted for at least 12 h. The as-prepared SiO2-NH2@SiO2 was centrifuged and washed severaltimes with ethanol/deionized water and then redispersed indeionized water for further use.

2.4 Preparation of the HMSNs with various pore sizes

12 mL of the SiO2-NH2@SiO2 dispersion was added to a plastictube. Then 600 μL of hydrofluoric acid (8%, 6% or 4%, respect-ively) was rapidly added into the solution with vigorous stirringfor 25 min to obtain the hollow mesoporous silica nano-particles (HMSNs) with various pore sizes. The as-preparedHMSNs were immediately centrifuged and washed severaltimes with deionized water to remove the excess HF.

2.5 Preparation of HMSNs-NH2

HMSNs functionalized with amine groups (HMSNs-NH2) wereobtained as follows: the as-prepared HMSNs dispersion wasadded to a solution containing 16 mL of deionized water,40 mL of ethanol and 400 μL of NH4OH with stirring. Then,50 μL of APTS was added into the mixture and the reaction per-formed for 12 h. The product was centrifuged and washedseveral times with ethanol and deionized water.

Scheme 1 Schematic illustration of the preparation and application ofnanocarriers based on DNAs as “multi-locks” and tumor-related mRNAsas the “keys” for controlled drug release.

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To verify the existence of the amino groups in HMSNs-NH2,a ninhydrine assay was carried out. Ninhydrine (1%) wasadded to 1 mL of the HMSNs-NH2 solution and the mixturewas subject to boiling (the experiment group). The reaction ofninhydrine with the supernatant liquid of the centrifugedHMSNs-NH2 solution served as the control group. The experi-ment group gave rise to a purple color, however, the controlgroup remained pale yellow. This confirmed that the -NH2

groups were modified in HMSNs-NH2 instead of in the solu-tion (Fig. 2a).

2.6 Preparation of HMSNs(RhB) and HMSNs(Dox)

1 mg mL−1 HMSNs-NH2 was mixed with 0.5 mg mL−1 RhB andthen further stirred for 3 d. The precipitates were designatedas HMSNs(RhB). The preparation of HMSNs(Dox) was identicalto the method mentioned above. 100 μL of the above solutionwas centrifuged at 10 000 rpm for 10 min and the precipitateswere redispersed in 500 μL of deionized water for further use.

2.7 Preparation of HMSNs(RhB)-DNAs and HMSNs(Dox)-DNAs

1 OD of TK1 DNA and GT DNA were centrifuged, and thendissolved in 500 μL of deionized water, respectively. Afterthat, the two kinds of DNAs were mixed together and 500 μLof the mixture was added into the solutions of HMSNs(RhB)or HMSNs(Dox) in two portions under gentle stirring at30 min intervals. The mixture was stirred for at least 12 hin the dark to obtain HMSNs(RhB) coated with TK1 DNA andGT DNA (abbreviated as HMSNs(RhB)-DNAs) or HMSNs(Dox)coated with TK1 DNA and GT DNA (abbreviated as HMSNs(Dox)-DNAs). HMSNs(RhB) or HMSNs(Dox) coated with GTDNA (designated as HMSNs(RhB)-GT or HMSNs(Dox)-GT)were prepared by coating with GT DNA only, which servedas the control. The zeta potentials of the HMSNs-NH2

and HMSNs-NH2-DNAs were studied. As can be seenfrom Fig. 2b, the HMSNs-NH2 were highly positively charged,while the nanocarriers were negatively charged after beingcoated with DNAs (designated as HMSNs-NH2-DNAs),which indicated that the DNAs have been capped on theHMSNs. The DNA sequences employed in this work are shownin Table S1.†

2.8 The stability of the HMSNs(RhB)-DNAs

The fluorescence intensity of the as-prepared HMSNs(RhB)-DNAs or HMSNs(RhB) samples (0.2 mg mL−1) was measuredupon excitation at 532 nm and emission at 575 nm fordifferent time periods, respectively.

2.9 The quantitation of Dox loaded into the nanocarrier

1 mL of the as-prepared HMSNs(Dox) solution was heated at80 °C in a water bath for at least 30 min and then centrifugedat 10 000 rpm for 10 min. The supernatant was separated andthe precipitate was redispersed in 1 mL of deionized water. Tomake sure that the Dox was released from the HMSNs, theabove-mentioned process was repeated at least twice. The fluo-rescence of the supernatant was excited at 490 nm and

measured at 590 nm. The loading capacity of the HMSNs wascalculated to be 0.08 mg Dox per 1 mg HMSNs according tothe standard linear calibration curve of Dox (Fig. S1†).

2.10 Cell culture

MCF-7, MCF-10A, A549, BEAS, HepG2 and HL-7702 cellswere cultured in Dulbecco’s modified Eagles medium (DMEM)or RPMI-1640 with 10% fetal bovine serum and 100 U mL−1

1% antibiotics penicillin/streptomycin and maintained at37 °C in a 100% humidified atmosphere containing 5% CO2.In the confocal fluorescence imaging and MTT assay, the cellswere cultured on the chamber slides or in 96-well microtiterplates at 37 °C in 5% CO2 for 24 h. The cells (5000 cells perwell) were cultured in 96-well microtiter plates in the MTTassay.

2.11 Confocal fluorescence imaging

MCF-7, MCF-10A, A549, BEAS, HepG2 and HL-7702 cells wereused in this experiment. Then, the HMSNs, RhB, HMSNs(RhB)-DNAs (0.02 mg mL−1) and HMSNs(RhB)-GT (0.02mg mL−1) samples were respectively delivered into the cells inculture medium for 6 h. Then, the cells were washed threetimes with PBS buffer and examined using confocal laser scan-ning microscopy with excitation at 543 nm for RhB.

2.12 RT-PCR

The total RNA from the various cells was extracted usinga RNeasy Mini Kit (Qiagen, Valencia, CA). cDNA synthesiswas performed using an iScript kit (Bio-Rad). RT-PCR wascarried out using SYBR Green I (Qiagen) on an ABIPRISM 7000 sequence detection system. The relative levelsof mRNA were calculated from the quantity of mRNAPCR products and the quantity of GAPDH PCR products.The primers used in this experiment were as follows:TK1 forward, 5′-TATGCCAAAGACACTCGCTAC-3′; TK1 reverse,5′-GCAGAACTCCACGAT-GTCAG-3′; GalNAc-T forward, 5′-CCAAGACCTTCCTCCGTTAT-3′; GalNAc-T reverse, 5′-AACCGTTGGGTAGAAGCG-3′; GAPDH forward, 5′-GGGAAACTGTGGCGTGAT-3′;and GAPDH reverse, 5′-GAGTGGGTGTCGCTGTTGA-3′.

2.13 MTT assay

The therapeutic effect of the nanocarrier was determinedusing a 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) assay. MCF-7, MCF-10A, A549, BEAS, HepG2and HL-7702 cells were respectively incubated with the HMSNs(0.1 mg mL−1), Dox, HMSNs(Dox)-GT and HMSNs(Dox)-DNAs(0.1 mg mL−1) for 6 h. After being washed with PBS, the cellswere cultured at 37 °C for another 24 h. Then, the cells werewashed with PBS buffer and 150 µL of MTT solution (0.5mg mL−1 in PBS) was added. After the MTT solution wasremoved 4 h later, 150 µL of DMSO was added to dissolve theformazan crystals. Finally, the absorbance of the abovesolution was measured at 490 nm. Cells incubated without thenanocarrier served as the control.

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3 Results and discussion3.1 Synthesis and characterization of the HMSNs

Hollow mesoporous silica nanoparticles (HMSNs) were used asthe nanocarrier because of their unique large hollow cavitiesand intact porous shells, which are suitable to store cargo.37,38

The HMSNs were prepared via silica-etching chemistrywith some modification.39,40 In addition, HMSNs with threedifferent pore sizes were successfully synthesized by etchingwith different concentrations of hydrofluoric acid (HF). Fig. 1and Fig. S2† show that the as-prepared HMSNs are about120 nm and possess huge hollow cavities and uniform meso-porous silica shells (about 15 nm), which are beneficial fordrug loading. As shown in Fig. 1d, the HMSNs achieved byetching with HF exhibit typical Type IV hysteresis loops, illus-trating the existence of representative mesopores. Uing theBarrett–Joyner–Halenda (BJH) method, the average mesoporesizes of the HMSNs fabricated by etching with 4%, 6% and 8%HF were 3.9 nm, 7.6 nm and 9.2 nm, respectively (Fig. 1e). TheHMSNs with the larger pore size were employed in the follow-ing experiment because they could store more drugs and accel-

erate the intracellular drug release, which will give rise toincreased intracellular drug accumulation.41 Then, the HMSNswere treated with 3-aminopropyltriethoxysilane (APTS), whichcan react with the Si–OH bonds to modify the silica shell withamino groups. Ninhydrine can react with primary aminegroups to give a purple color after boiling, which can verify theexistence of amino groups. As can be seen from Fig. 2a, thepurple color in tube 1 indicates that the HMSNs were success-fully functionalized with amino groups.42 In addition, theHMSNs are positively charged because of the amino groupsbeing protonated, which is beneficial for adsorbing negativelycharged DNA via electrostatic interactions.

3.2 HMSNs-NH2 encapsulated with DNAs

The adsorption of DNAs to the surface of the nanoparticles isprimarily motivated by electrostatic interactions. In particular,DNA is highly negatively charged because of the negative phos-phate groups of the DNA backbone. The nanoparticles arehighly positively charged due to the amino groups on thesurface of the HMSNs. In a word, the DNA coats the surface ofthe nanoparticles via the interactions between the phosphategroups in the DNA backbone and the amino groups on thesurface of the HMSNs. Zeta potential experiments further veri-fied the successful adsorption of DNA onto the surface of theHMSNs, i.e., +15.1 ± 1.36 mV (HMSNs-NH2) and −21.2 ±0.25 mV (HMSNs-NH2-DNAs) (Fig. 2b).

3.3 Drug-loading and leaking from the HMSNs

Rhodamine B (RhB) was chosen as the model drug loaded intothe HMSNs (designated as HMSNs(RhB)) to conduct the fol-lowing experiments. Then, two single-stranded DNAs (target-ing to TK1 and GT mRNA) were simultaneously adsorbed onthe surface of the nanoparticles via electrostatic interactions toform the nanocarrier (HMSNs(RhB)-DNAs). The stability of thenanocarrier is of vital importance for transporting drugs to aspecific location. Therefore, the stability of the nanocarrierwas estimated by the variation in the fluorescence intensity atdifferent times. Fig. 2c shows that about 84% of RhB wasleaked from HMSNs(RhB) after 300 min, while only about 16%of RhB was leaked from the HMSNs(RhB)-DNAs within thesame timeframe, indicating that the DNA gatekeepers could

Fig. 1 TEM images of the HMSNs fabricated by etching with (a) 4%; (b)6% and (c) 8% HF. (d) The N2 adsorption–desorption isotherms. (e) Thepore size distribution of the HMSNs with various pore sizes. The scalebars represent 100 nm.

Fig. 2 (a) Verification of the amino groups: (1) HMSNs-NH2 and (2) the supernatant solution of HMSNs-NH2 after centrifugation. (b) The zeta poten-tials of HMSNs-NH2 and HMSNs-NH2-DNAs. (c) The leakage of RhB from HMSNs(RhB)-DNAs and HMSNs(RhB).

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lock up the channel and effectively prevents the cargo mole-cules from leaking from the nanocarriers.

3.4 Confocal fluorescence imaging in living cells

To utilize the nanocarrier for drug delivery in practical appli-cation, intracellular experiments were implemented with thehuman breast cancer cell line (MCF-7), human mammary epi-thelial cell line (MCF-10A), human lung epithelial cell line(BEAS), human lung adenocarcinoma cell line (A549), humanhepatocellular liver carcinoma cell line (HepG2) and humanhepatocyte cell line (HL-7702), respectively. Cells incubatedwith the HMSNs, RhB or HMSNs(RhB)-GT served as thevarious control groups, respectively. In addition, the cells incu-bated with the HMSNs(RhB)-DNAs act as the experimentalgroups. As shown in Fig. 3, all six kinds of cells incubated withRhB show higher intracellular fluorescence signals when com-pared to those incubated with the HMSNs. The MCF-7, A549and HepG2 cells incubated with HMSNs(RhB)-DNAs show sig-nificant intracellular fluorescence signals, while the MCF-10A,BEAS, HL-7702 cells incubated with HMSNs(RhB)-DNAs showweak intracellular fluorescence signals, suggesting that theDDS can be employed to selectively release drugs in cancercells rather than in normal cells. Interestingly, HL-7702 cells

incubated with HMSNs(RhB)-GT showed a significant intra-cellular fluorescence signal, while a negligible intracellularfluorescence signal with the HMSNs(RhB)-DNAs. This indi-cates that the nanocarrier with dual DNAs as valves generatelower release in HL-7702 cells than the nanocarrier with singleDNA as a valve. To further prove this conclusion, HepG2 andHL-7702 cells were incubated with the HMSNs(Dox)-DNAs. Thefaint fluorescence signal observed in the HL-7702 cells andbright fluorescent signal in the HepG2 cells, as shown inFig. S3,† indicate that the dual DNA gatekeepers were unlockedonly in the presence of both the tumor-related mRNAs andfurther confirmed that single mRNA could not open the valvesto release the cargo.

3.5 RT-PCR

The RT-PCR results reveal that TK1 and GT mRNA are overex-pressed in MCF-7 when compared with that in MCF-10A; TK1and GT mRNA are overexpressed in A549 when compared withthat in BEAS cells. Interestingly, the expression of TK1 mRNAwas low in HL-7702 when compared with that in the HepG2cells, while the expression of GT mRNA was as high as that inHepG2 (Fig. 4). The results are consistent with the fluo-rescence confocal imaging results and further indicated that

Fig. 3 Confocal fluorescence images: HMSNs, as the negative control; RhB, as the positive control; HMSNs(RhB)-GT and HMSNs(RhB)-DNAs inthree groups of cells: (a) MCF-7 and MCF-10A, (b) A549 and BEAS, and (c) HepG2 and HL-7702.

Fig. 4 The amount of mRNAs in the cells was quantified by QPCR: (a) MCF-7 and MCF-10A, (b) A549 and BEAS, and (c) HepG2 and HL-7702.

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the overexpression of mRNAs led to drug release. This furtherindicates that only when TK1 and GT mRNA were simul-taneously overexpressed in the MCF-7, A549 and HepG2 cells,will the DNA multi-locks be unlocked and the cargo releasedfrom the HMSNs(RhB)-DNAs. Moreover, GT mRNA is over-expressed in not only cancer cells but also in some normalcells, which can trigger the cargo to be released from theHMSNs(RhB)-GT, further verifying that a single DNA valve willcause the inevitable release of cargo in unexpected sites. Italso demonstrates that the simultaneous existence of bothtumor markers can open the valves and the dual DNA gate-keepers can avoid unexpected drug release in normal cells toreduce the occurrence of side effects.

3.6 Cytotoxicity assay

3-(4,5-Dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) tests43,44 were carried out in MCF-7, MCF-10A, A549,BEAS, HepG2 and HL-7702 cells to evaluate the cytotoxicity ofthe HMSNs and the therapeutic effect of the nanocarriers.Cells without treatment served as the control, with HMSNs asthe negative control and with Dox as the positive control. Thecell viability was all approximately 100% in both the controland the negative control, which indicates that the HMSNsexpress excellent biocompatibility for application in DDSs(Fig. 5). The therapeutic effects of HMSNs(Dox)-DNAs andHMSNs(Dox)-GT were further studied. As can be seen fromFig. 5, the viabilities of the cancer cells incubated with HMSNs(Dox)-GT and HMSNs(Dox)-DNAs were as low as the positivecontrol. While the viabilities of the MCF-10A, BEAS andHL-7702 cells incubated with the HMSNs(Dox)-DNAs wasmuch higher than the positive control. Interestingly, the viabi-

lity of HL-7702 incubated with HMSNs(Dox)-GT was lower thanthat found with the HMSNs(Dox)-DNAs. This further confirmsthat the dual DNA gatekeepers can effectively avoid unexpecteddrug release in normal cells. In addition, the MTT results werein accordance with those observed in the confocal fluorescencedataset.

4 Conclusions

In conclusion, we have demonstrated a novel DDS usingHMSNs as a nanocarrier and dual DNAs as multi-locks for con-trolled drug release. The two tumor-related mRNAs in cancercells serve as “keys” to specifically hybridize with the dual DNAmulti-locks to unlock the valves and release the drug, whichshow a low release in normal cells. The cargo leakage experi-ment suggested that the nanocarrier has good stability and issuitable for drug delivery. Confocal fluorescence imagesshowed that only TK1 mRNA and GT mRNA simultaneouslyexist in the cells will the DNA multi-locks be unlocked andproduce fluorescence signals. The cytotoxicity assay furtherindicates that this DDS presents an excellent therapeutic effectwith low damage to normal cells. We expect that this novelstrategy can provide a new avenue for effective drug deliveryand cancer therapy.

Conflicts of interest

There are no conflicts or competing financial interest.

Fig. 5 MTT assay: (1) Control: cells incubated without any nanocarrier; cells were incubated with (2) HMSNs, (3) Dox, (4) HMSNs(Dox)-GT and (5)HMSNs(Dox)-DNAs for 6 h and then cultured for 24 h at 37 °C (n = 3; mean ± SD).

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Acknowledgements

This work was supported by the National Natural ScienceFoundation of China (21535004, 21390411, 21422505, 21375081and 21505087) and the Natural Science Foundation forDistinguished Young Scholars of Shandong Province (JQ201503).

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