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Simultaneous Visualization of Multiple mRNAs and MatrixMetalloproteinases in Living Cells Using a FluorescenceNanoprobe

Wei Pan, Huijun Yang, Na Li, Limin Yang, and Bo Tang*[a]

Abstract: Simultaneous monitoring of multiple tumourmarkers is of great significance for improving the accuracyof early cancer detection. In this study, a fluorescencenanoprobe has been prepared that can simultaneouslymonitor and visualize multiple mRNAs and matrix metallo-proteinases (MMPs) in living cells. Confocal fluorescenceimaging results indicate that the nanoprobe could effec-tively distinguish between cancer cells and normal cellseven if one tumour maker of normal cells was overex-pressed. Furthermore, it can detect changes in the expres-sion levels of mRNAs and MMPs in living cells. The currentapproach could provide new tools for early cancer detec-tion and monitoring the changes in expression levels ofbiomarkers during tumour progression.

Cancer is undoubtedly a pervasive and destructive disease inwhich the body cells begin to grow and divide in an uncon-trollable way.[1] The survival rate of cancer patients can begreatly increased if the cancer is detected at the cell level in anearly stage. For this purpose, a major target is to identify theexpression abnormalities of tumour markers in living cells.[2–4]

In recent years, many efforts have been made for the develop-ment of fluorescent probes for detection and imaging oftumour markers in living cells.[5–9] Although these nanoprobeshave performed well in detecting a single type of tumourmarker, it is difficult to detect multiple, different types oftumour markers in a single assay. To comprehend the complex-ity of cancer, researchers try to monitor the alterations of mul-tiple tumour markers in living cells. However, it remains a chal-lenge, as cancer commonly involves multiple types of tumourmarkers and the expression levels of these markers change dy-namically in tumour progression. Moreover, assembling manydifferent types of recognition units in a single probe is hard toachieve. Therefore, simultaneous detection of various different

targets is of crucial importance to improve the accuracy ofearly cancer detection and in obtaining a better understandingin the changes of tumour marker expression levels in livingcells.

Herein, we have developed a fluorescence nanoprobe for si-multaneous detection and imaging of multiple mRNAs andmatrix metalloproteinases (MMPs) in living cells. As a tumour-related marker, mRNA has been widely utilized in the diagnosisand treatment of cancer.[10–12] The MMPs have been associatedwith the invasive properties of cancer cells and are expressedat statistically higher levels in tumours.[13–15] The nanoprobewas prepared using gold nanoparticles (AuNPs) functionalizedwith a dense shell of synthetic DNA molecular beacons (MBs)and peptides, step-by-step via gold¢thiol bond formation,which allows good dispersibility in aqueous systems, and goodbiocompatibility. Firstly, the AuNPs were modified with DNAMBs before the next assembly. The MB was designed to forma unique stem–loop configuration with recognition sequencesand reporter fluorophores.[16–19] The recognition sequences canidentify two specific mRNA transcripts: TK1 mRNA and Gal-NAc-T mRNA, which are important markers in cancer cells.[20, 21]

The ends of the MBs are labelled with reporter fluorophores:Alexa Fluor 405 and Cy5, respectively. Next, the synthetic pep-tides are linked to the surface of AuNPs. The peptides are tar-geted to MMP-2 and MMP-7,[22, 23] which are tagged with FITCand rhodamine B (RhB). In the absence of the targets, the fluo-rophores are close to the AuNPs, resulting in the quenching ofthe fluorescence of the dyes by fluorescence resonance energytransfer. The hairpin of the MBs was forced to open and thepeptide can be specifically cleaved by the MMPs when thenanoprobe meets the corresponding targets, thereby separat-ing the fluorophores and AuNPs and restoring the fluores-cence. The details of the assembly are depicted in Scheme 1.

Large-sized AuNPs with diameter of 20 nm were employedin this study, because they cause stronger surface plasmon res-onance (SPR) absorption and assemble more recognition unitsthan small-sized AuNPs. As shown in Figure S1 in the Support-ing Information, the AuNPs were typical spherical with clearborders, while the borders of the nanoprobe was ambiguousdue to the assembly of recognition units on the surface of theAuNPs. The UV/Vis absorption spectra (Figure S2 in the Sup-porting Information) further confirmed that the nanoprobewas successfully assembled owing to the red shift of the maxi-mum absorption from 519 nm (for AuNPs) to 525 nm (for thenanoprobe). According to the previous method,[24] each AuNPwas calculated to carry 17�1 Alexa Fluor 405 labelled MB1 tar-

[a] Dr. W. Pan, H. Yang, Dr. N. Li, L. Yang, Prof. B. TangCollege of Chemistry, Chemical Engineering and Materials ScienceCollaborative Innovation Center of Functionalized Probes forChemical Imaging in Universities of ShandongKey Laboratory of Molecular and Nano Probes, Ministry of EducationShandong Provincial Key Laboratory of Clean Production of Fine ChemicalsShandong Normal University, Jinan 250014 (P.R. China)E-mail : tangb@sdnu.edu.cn

Supporting information for this article is available on the WWW underhttp ://dx.doi.org/10.1002/chem.201500365.

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geting TK1 mRNA, 33�1 FITC labelled peptide 1 targetingMMP-2, 38�1 RhB labelled peptide 2 targeting MMP-7 and18�1 Cy5 labelled MB2 targeting GalNAc-T mRNA.

The ability of the nanoprobe for simultaneous detection oftwo DNA targets and two MMP targets was evaluated by incu-bating the nanoprobe with the four corresponding targets.Figure 1 shows that the fluorescence intensity of each dye in-creased with the increase of its corresponding target concen-tration, thus demonstrating that the response of nanoprobetoward the targets led to fluorescence recovery.

Moreover, the fluorescence intensity is correlated positivelywith the concentrations of the targets in a concentration-de-pendent manner. Selectivity is a key property for application inliving cells, because the activity of each target is influenced bythe other three targets co-existing in living cells. Figure S4 in

the Supporting Information shows that every MB specificallybound to its own DNA target and generated a high fluores-cence signal. By comparison, the signals did not obviouslychange in the presence of the other three targets or its single-base mismatched DNA target, which were of comparable mag-nitude to background fluorescence. Similar results were ob-tained when the nanoprobe was incubated with the two MMPtargets. These results indicated that the nanoprobe was capa-ble of simultaneously monitoring four different targets withoutmutual interference, suggesting that each recognition unit ofthe nanoprobe retained high sequence specificity. Next, the ki-netic studies of the nanoprobe toward four different targetswere investigated. Figure 2 shows that the nanoprobe re-sponded rapidly to the perfectly matched DNA targets within5–10 min and it responded to the MMP targets within 20–30 min. The matched response time for the two types of tar-gets makes the nanoprobe highly attractive for simultaneousmonitoring of two kinds of tumour makers in living cells.

The cytotoxicity of the nanoprobe was tested with the MTTassay in the human breast cancer cell line, MCF-7.[8] Figure S5in the Supporting Information indicates that the cell viabilitieswere all more than 95 % when incubated with free AuNPs aswell as the nanoprobe for 3, 6, 12 and 24 h. The results revealthat the nanoprobe shows almost no cytotoxicity or side ef-fects in living cells. This confirms that the nanoprobe can beused for intracellular multimarker diagnosis.

The internalization pathways of the nanoprobe in living cellswere studied using different endocytosis inhibitors.[25] Asshown in Figure 3, the fluorescence intensities of all four signalchannels did not show obvious change for EIPA-treated cellsand chlorpromazine-treated cells, indicating that the endocyto-sis of the nanoprobe is not mediated by macropinocytosis orclathrin. Remarkably, after the cells were treated with filipin

Scheme 1. Schematic illustration of the designed nanoprobe for detectionand visualization of intracellular mRNAs and MMPs.

Figure 1. The fluorescence intensity of the nanoprobe (1 nm) in the presenceof various concentrations of : a) TK1 mRNA target, b) GalNAc-T mRNA target,c) MMP-2, and d) MMP-7 measured with different excitation wavelengths.

Figure 2. Kinetics of the nanoprobe. The nanoprobe (1 nm) was incubatedwith: a) TK1 mRNA target, b) GalNAc-T mRNA target, c) MMP-2, and d) MMP-7. The mRNA target concentrations are 200 nm and the MMP activities are0.4 mg mL¢1.

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and dynasore, all of the four fluorescence signals decreasedsharply, suggesting that the caveolae-mediated pathwaymainly contributes to cellular uptake of the nanoprobe.

To evaluate the ability of the nanoprobe to simultaneouslydetect multiple mRNAs and MMPs, two different cell lines werefirstly chosen: normal immortalized human mammary epithe-lial cell line (MCF-10A) and MCF-7 cells. After MCF-7 cells wereincubated with the nanoprobe (1 nm), strong blue fluorescencesignal for TK1 mRNA, green fluorescence signal for MMP-2,yellow fluorescence signal for MMP-7 and red fluorescencesignal for GalNAc-T mRNA were observed with confocal laserscanning microscopy (CLSM; Figure 4). When MCF-10A cellswere incubated with the nanoprobe under the same condi-tions, all of the four fluorescence signals were very low. Inter-estingly, strong green and yellow signals for MMPs were ob-served when the MCF-10A cells cultured with the MCF-7medium were incubated with the nanoprobe, indicating thatthe content of MMPs increased when MCF-10A cells were influ-enced by cancer cell culture medium. Next, human hepatocel-lular liver carcinoma cell line (HepG2) and human hepatocytecell line (HL-7702) were also chosen to evaluate the nanop-robe. Figure 4 shows that when HepG2 cells were incubatedwith the nanoprobe, strong blue, green, yellow and red fluo-rescence signals for the four tumour markers were observed,which was similar with the MCF-7 cells. Notably, after HL-7702cells were incubated with the nanoprobe under the same con-ditions, the blue, green and yellow signals were faint and thered signal was strong, indicating GalNAc-T mRNA was alsooverexpressed in HL-7702 cells, which is consistent with theprevious study.[8] When HepG2 medium cultured HL-7702 cellswere incubated with the nanoprobe, only the blue fluores-

cence signal was faint, while the green, yellow and red fluores-cent signals were strong. The results reveal that multiplexeddetection of intracellular mRNAs or MMPs could avoid falsepositive results produced by identifying one mRNA or MMP.Furthermore, these results suggest that the nanoprobe was ca-pable of distinguishing tumour cells and normal cells even ifone tumour maker of normal cells was overexpressed or thenormal cells were influenced by the environment.

The ability of the nanoprobe to detect relative mRNA andMMP levels in living cells is very significant for estimating thetumour stage and making treatment decisions, because the ex-pression levels of mRNA and MMP in cancer cells change dy-namically in different stages of tumour progression. Next, weinvestigated if the nanoprobe could detect the changes ofmRNA and MMP expression levels in living cells. MCF-7 cellswere chosen as an example. It has been reported that tamoxi-fen induces down-regulation of TK1 mRNA expression,[26] b-es-tradiol induces up-regulation of TK1 mRNA expression,[27] andilomastat can inhibit the expression of MMP-2 and MMP-7.[28]

MCF-7 cells were parallel divided into four groups. Group 1was treated with tamoxifen to decrease the TK1 mRNA expres-sion, group 2 was treated with b-estradiol to increase the TK1mRNA expression and group 3 was treated with ilomastat toinhibit the expression of MMP-2 and MMP-7. The untreatedgroup was used as control. Then the four groups of cells wereincubated with the nanoprobe (1 nm). As shown in Figure 5,the blue fluorescence signal for TK1 decreased in the tamoxi-fen-treated MCF-7 cells and increased in the b-estradiol-treated

Figure 3. Intracellular imaging of TK1 mRNA, GalNAc-T mRNA, MMP-2, andMMP-7 of MCF-7 cells treated with EIPA, chlorpromazine, filipin and dyna-sore. The four channels were recorded with 405 nm excitation, 488 nm exci-tation, 543 nm excitation, 633 nm excitation, from left to right. Scale bars :50 mm.

Figure 4. Intracellular imaging of TK1 mRNA, GalNAc-T mRNA, MMP-2, andMMP-7 with CLSM. The cells were incubated with the nanoprobe (1 nm) for4 h at 37 8C. Scale bars: 100 mm.

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MCF-7 cells compared with the untreated cells. The other threefluorescence signals did not show obvious change. The greenfluorescence signal for MMP-2 and yellow signal for MMP-7 de-creased in the ilomastat-treated MCF-7 cells and the other twofluorescence signals for mRNAs were unchanged. This revealsthat the nanoprobe was able to detect changes in the tumourmarker expression levels in cancer cells.

In summary, we report a fluorescence nanoprobe, which cansimultaneously monitor and image multiple mRNAs and MMPsin living cells. The nanoprobe consists of gold nanoparticles as-sembled with a dense shell of molecular beacons and pep-tides, step-by-step, which target intracellular TK1 mRNA,GalNAc-T mRNA, MMP-2 and MMP-7, respectively. The nanop-robe could signal the presence of target mRNAs and MMPswith the open form of the stem–loop structure of MBs and thecleavage of peptides, leading to the recovery of fluorescence.The nanoprobe exhibits high specificity, rapid response, andgood biocompatibility. The cellular internalization results re-vealed that the cellular uptake of the nanoprobe was primarilythrough the caveolae-mediated pathway. Confocal imaging ex-periments displayed that the nanoprobe could discriminate be-tween cancer cells and normal cells. Additionally, it can deter-mine changes in the expression levels of mRNAs and MMPs inliving cells, which is beneficial for evaluating the stage oftumour progression and making treatment decisions. We antic-ipate that this strategy could provide new avenues for moni-toring multiple biomarkers in living cells.

Acknowledgements

This work was supported by 973 Program (2013CB933800) andNational Natural Science Foundation of China (21227005,21390411, 91313302, 21422505, 21375081).

Keywords: analytical methods · imaging agents · matrixmetalloproteinases · mRNA · nanoprobes

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Received: January 28, 2015Published online on March 9, 2015

Figure 5. Intracellular imaging of different levels of mRNAs and MMPs withCLSM. The treated and untreated MCF-7 cells were incubated with thenanoprobe for 4 h at 37 8C. The MCF-7 cells were treated with tamoxifen todecrease the TK1 mRNA expression, treated with b-estradiol to increase theTK1 mRNA expression and treated with ilomastat to inhibit the expressionof MMP-2 and MMP-7. Scale bars : 100 mm.

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