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Nanomedicine: Nanotechnology, Biology, and Medicine 3 (2007) 266–272www.nanomedjournal.com

Diagnostic Nanomedicine

Rhodamine B isothiocyanate doped silica-coated fluorescent nanoparticles(RBITC-DSFNPs)–based bioprobes conjugated to Annexin V

for apoptosis detection and imagingHui Shi, PhD, Xiaoxiao He, PhD, Kemin Wang, PhD,⁎ Yin Yuan, MS, Ke Deng, MS,

Jiyun Chen, MS, Weihong Tan, PhDState Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Engineering Center for Biomedicine,

Institute of Life Science and Biological Technology, Hunan University,Key Laboratory for Bio-Nanotechnology and Molecule Engineering of Hunan Province, Changsha, China

Received 19 April 2007; accepted 27 August 2007

Abstract We report here a novel bioprobe based on Rhodamine B isothiocyanate doped silica-coated

No conflict of inteThis work was pa

Program (2002CB513Ministry (107084), KeProgram of China (20Talents in University (China (90606003, 204Province (06JJ10004)

⁎ Corresponding aing, Hunan University

E-mail address: k

1549-9634/$ – see frodoi:10.1016/j.nano.20

fluorescent nanoparticles (RBITC-DSFNPs) for early-stage apoptosis detection and imaging.RBITC-DSFNPs were constructed by synchronous hydrolysis of APTES-RBITC and tetraethox-ysilane in water-in-oil microemulsion, and the bioprobes were prepared through modifying AnnexinV with RBITC-DSFNPs. The characterization of RBITC-DSFNPs and the bioprobes' application forapoptosis detection and imaging were investigated. It was demonstrated that RBITC-DSFNPs areuniform and possess excellent photostability. The bioprobes could specifically recognize early-stageapoptotic cells through the binding between Annexin V and phosphatidylserine (the externalizationof which from the inner to the outer membrane is an early and major event in the apoptotic process)on the outer membrane of apoptotic cells. Moreover, the RBITC-DSFNPs labeling method was alsoused to monitor the increase of the number of early-stage apoptotic cells along with the extendedinduction time. Compared with the conventional fluorochrome such as Cy3-labeled Annexin V forstaining apoptotic cells, the RBITC-DSFNPs labeling method possesses much better photostability,which offers a promising approach for tumor-related research.© 2007 Elsevier Inc. All rights reserved.

Key words: Apoptosis; Silica-coated fluorescent nanoparticles; Photostable; Biological fluorescent label; Image detection

Apoptosis, or programmed cell death, is a distinct form ofcell death that is characterized by specific morphologicalfeatures and regulated by complex molecular mechanisms. It

rest was reported by the authors of this article.rtially supported by the National Key Basic Research100-10), Key Project Foundation of China Educationy Project of International Technologies Collaboration03DF000039), Program for New Century ExcellentNCET-06-0697), National Science Foundation of P.R.05005) and Outstanding Youth Foundation of Hunan.uthor. College of Chemistry and Chemical Engineer-, Yuelu-Qu, Changsa, Hunan 410082, China.mwang@hnu.cn (K. Wang).

nt matter © 2007 Elsevier Inc. All rights reserved.07.08.004

is a universal and important process in development ofmulticellular organisms, regulation of the immune system,and clearance of abnormal cells [1,2]. Dysfunction inapoptosis is linked to disease states such as tumors, andmoreover, anticancer therapies suppress tumor growthmainly by induction of cell apoptosis [3-7]. On account ofits wide-range implications and possible therapeutic inter-ventions, the importance of apoptosis research has beenrealized by numerous biological scientists, and many effortshave been made to develop specific methods that can beroutinely used to detect apoptosis [8-18].

As a complicated biological phenomenon, apoptosisinvolves many subprocesses and changes that can be dividedinto three phases: initiation, decision, and execution. A

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wealth of changes in the cells that occur in the executionphase can be available for apoptosis detection. There aremorphological criteria that can be used to define cellapoptosis, including cell shrinkage, chromatin condensationand margination, membrane blebbing, and finally theproduction of apoptotic bodies containing the remains fromthe cell. Also, apoptosis can be detected biochemically byenzymatic internucleosomal DNA destruction. But unfortu-nately, early-stage apoptotic cells may not show theseclassical morphological features, and biochemical evaluationof apoptosis thus not only lacks sensitivity, but also could notbe used to detect early-stage apoptosis. Therefore, it isimportant to develop highly sensitive and exact detectionsystems for early-stage apoptosis, which can also provide agood basis for anticancer drug screening.

The externalization of phosphatidylserine from theinner to the outer membrane is an early and major event inthe apoptotic process. Because phosphatidylserine externa-lization precedes any change in the nucleus and acts as aspecific feature of apoptosis that does not occur in the case ofnecrosis, it has been used to detect and stain early-stageapoptosis by binding phosphatidylserine sitting on the outermembrane of apoptotic cells [19-24]. By conjugating afluorochrome such as fluorescein isothiocyanate (FITC),Cy5, or Cy3 to Annexin V (a small protein that binds tophosphatidylserine in the presence of Ca2+), it is possible todetect and image early-stage apoptotic cells. But conven-tional fluorochrome labels present limitations in photostabil-ity, sensitivity, and other aspects. First, conventional organicdyes are easily photobleached, which will reduce theirstaining sensitivity; second, their toxicity to living cellscannot be ignored either. Finally, only a limited numberof dyes can be used simultaneously in the conventionalfluorochrome label method.

Recently, fluorescent nanomaterials have been developedinto bioprobes for bioanalysis and bioimaging, and shownmany advantages over conventional fluorochrome labels[25-37]. In this article we report a technique for early-stageapoptosis detection and imaging based on Rhodamine Bisothiocyanate doped silica-coated fluorescent nanoparticles(RBITC-DSFNPs) as the bioprobes. RBITC-DSFNPs werecreated through synchronous hydrolysis of tetraethoxysilaneand APTES-RBITC that was prepared by covalentlybinding of RBITC molecules with the amino groups of3-aminopropyltriethoxysilane (APTES), in water-in-oilmicroemulsion. Annexin V was then conjugated toRBITC-DSFNPs for detecting and imaging the paclitaxel-induced early-stage apoptotic MCF-7 cells. Our results showthat the RBITC-DSFNP–based bioprobes could specificallyrecognize apoptotic MCF-7 cells through the binding ofAnnexin V to phosphatidylserine located on the outermembrane of the apoptotic cells. Moreover, compared withCy3-labeled Annexin V, RBITC-DSFNP–based bioprobespossess higher photostability in apoptosis detection andimaging, thus providing a promising approach for tumor-related research.

Materials and methods

Materials

RBITC, Hoechst33258, Annexin V, and Cy3-labeledAnnexin V were purchased from Sigma (St. Louis, MO).APTES was obtained from the Institute of Organic SiliconMaterials (Wuhan, China). Tetraethoxysilane was purchasedfrom Chemistry and Reagents Corporation (Shanghai, China).MCF-7 (breast cancer) cells were provided by the cell center inour laboratory. All the other chemicals were purchased fromReagent & Glass Apparatus Corporation (Changsha, China)and were used without further purification.

Preparation of RBITC-DSFNPs

RBITC dye was prepared in aqueous solution and thenmixed with an equimolar quantity of APTES, reactingovernight at room temperature. The monomer precursorproducts (RBITC-APTES) were directly used to preparesilica-coated fluorescent nanoparticles using water-in-oilmicroemulsion technique as reported elsewhere [35-37].Briefly, a water-in-oil microemulsion was prepared by mixing1.77 mL of Triton X-100 (a nonionic surfactant), 7.5 mL ofcyclohexane, 1.8 mL of n-hexanol, 400 μL of water and100 μL of the RBITC-APTES solutions mentioned above. Inthis method, RBITC was successfully doped as a result of thecovalent binding of the RBITC dye molecules with the aminogroups of APTES, as in our earlier study on FITC doped silica-coated nanoparticles [37]. After stirring for 1 hour, 200 μL oftetraethoxysilane were then added as a precursor for silicaformation, followed by the addition of 100 μL NH4OH toinitiate the polymerization process. The reaction was allowedto continue for 24 hours at room temperature. When it wascompleted the nanoparticles were isolated.

Characterization of RBITC-DSFNPs

The sample for transmission electron microscopy wasprepared by placing a few drops of microemulsion containingRBITC-DSFNPs on carbon-coated copper grids. After eva-poration of the solvent, the particles were observed by aHitachi-800 transmission electron microscope. To investigatethe photostability ofRBITC-DSFNPswhen they are exposed toan aqueous environment for biological applications, a photo-bleaching experiment for RBITC-DSFNPs–containing suspen-sion was performed with mercury lamp HG-100W (Olympus,Tokyo, Japan) excitation.

Biological modification of RBITC-DSFNPs

The biomolecules were coupled to RBITC-DSFNPs byusing the following method as reported elsewhere [29].Prepared dried RBITC-DSFNPswere suspended in 9.0mL of2 M sodium carbonate solution (activation buffer) byultrasonication. A solution of CNBr in acetonitrile was thenadded dropwise to the RBITC-DSFNPs suspension understirring at room temperature. When the mixture becameclotted, activated RBITC-DSFNPs were washed three times

Fig 1. Transmission electron micrograph of Rhodamine B isothiocyanatedoped silica-coated fluorescent nanoparticles (RBITC-DSFNPs) at100,000× magnification.

Fig 2. Photobleaching of (A) RBITC-DSFNPs (λem = 550 nm) and (B) pureRBITC (λem = 550 nm) in solution phase with a 100-W mercury lampexcitation.

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with ice-cold water and three times with ice-cold phosphate-buffered saline (PBS) buffer (pH 7.3), followed byresuspension in 500 μL PBS buffer (pH 7.3). Subsequently,10 μL of Annexin V was added into the surface-modifiedRBITC-DSFNPs suspension, and stirring was continued for24 hours at 4°C. Annexin V–conjugated nanoparticles werethen treated with 10 mL of 0.03 M glycine solution overnightat 4°C. Finally, Annexin V–functionalized RBITC-DSFNPswere washed, resuspended in PBS buffer (pH 7.3), and storedat 4°C for future usage.

Preparation of early apoptotic MCF-7 cells

This study was carried out with MCF-7 cell lines. TheMCF-7 cells were plated at a density of 1 × 105 cells/wellin six-well plates, and then cultured at 37°C in the RPMI1640 (GIBCO, Invitrogen, Carlsbad, CA) medium with10% fetal calf serum. After 24 hours had passed, paclitaxelwas added into the medium with the final concentration of4 μM. Cells were continuously cultured in the medium withpaclitaxel for 6, 12, 18, 24, 30, and 48 hours, respectively,to select the best time for induction of early apoptoticMCF-7 cells.

Detection of early apoptotic MCF-7 cells

The MCF-7 cells were plated at a density of 1 × 105 cells/well in six-well plates at 37°C in the RPMI 1640 (GIBCO)medium with 10% fetal calf serum. After 24 hours had passed,paclitaxel was added into the medium and cells werecontinuously cultured for some hours. Subsequently, theinduced MCF-7 cells were washed twice with D-hanksbalanced salt solution to remove the dead cells, and thenincubated with the Annexin V–functionalized RBITC-DSFNPs suspension at 37°C for 30 minutes. After washingwith PBS buffer (pH 7.3), the cells were fixed by methanol-

acetic acid (3:1) and then stained with 0.03 mmol/LHoechst33258 for labeling the nucleus.

Results and discussion

Characterization of RBITC-DSFNPs

RBITC-DSFNPs were uniform in size—50 ± 5 nm indiameter (Figure 1), as characterized by transmission electronmicroscopy. It was observed from the solution-phasephotobleaching experiment that there was only minimalphotobleaching for RBITC-DSFNPs over continuous inten-sive excitation with a mercury lamp HG-100W. The resultswere also compared with a RBITC aqueous solution. Asshown in Figure 2, a small amount of photobleaching wasobserved for RBITC-DSFNPs in solution for a period of1500 seconds, the emission intensity of which decreased to70% of that upon initial excitation, and the red signals werestill clearly visible to the naked eye. However, a significantdecrease was observed for RBITC aqueous solution, theemission intensity of which dropped to 18% of that uponinitial excitation. It is clearly demonstrated that thephotostability is sharply improved as a result of the silicashell, which separated the potential quenching substancesfrom the dye molecules in the nanoparticles.

Application of bioprobes based on RBITC-DSFNPsconjugated to Annexin V for apoptosis detection and imaging

Preparation of early-stage apoptotic MCF-7 cellsPhosphatidylserine externalization precedes any change

in the nucleus, so it is possible to specifically recognizethe early-stage apoptotic cells by targeting phosphatidyl-serine located on the outer membrane. At the same time,once the translocation of phosphatidylserine from theinner to the outer membrane occurs, Annexin V can beused to monitor the progression of apoptosis from early-stage apoptosis to late-stage apoptosis, and finally to theproduction of apoptotic bodies. To avoid the interference,

Fig 3. Fluorescence images of MCF-7 cells that were cultured in the medium with paclitaxel for different time periods: (A) 0 hours, (B) 6 hours, (C) 12 hours,(D) 18 hours, (E) 24 hours, (F) 30 hours, and (G) 48 hours. The white arrow points to the nucleolus that has obviously changed morphology.

Fig 4. Confocal microscopy images of (A) Early-stage apoptotic MCF-7 cells induced with paclitaxel and (B) MCF-7 cells untreated with paclitaxel, incubatedwith RBITC-DSFNPs conjugated to Annexin Vand then stained with Hoechst33258. a is an optical image; b and c are fluorescence images of Hoechst33258 andRBITC-DSFNPs, respectively; d is the merged images of b and c. Photomultiplier tube (PMT) 503, gain power 1.8%, offset 10%.

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MCF-7 cells were continuously cultured for 6, 12, 18, 24,30, and 48 hours, respectively, in the medium withpaclitaxel to optimize the induction time for early-stageapoptotic cells. As shown in Figure 3, the nucleolar shapeof MCF-7 cells that were cultured in the medium withpaclitaxel for 6 hours was almost the same as that ofcontrol cells, with well-proportioned dispersed bluefluorescence. A few nucleoli became crimped withasymmetrical pigmentation when MCF-7 cells werecultured in the medium with paclitaxel for 12 hours, andthe number of cells with crimped nucleoli increased alongwith the extension of culture time in the medium withpaclitaxel as shown in Figure 3, D. As the culture time inthe medium with paclitaxel was further extended to 24 and30 hours, cell apoptosis became much more evident—namely condensation and margination of chromatin and theloss of membrane integrity. Moreover, apoptotic bodies

from the cells were observed after MCF-7 cells werecultured in the medium with paclitaxel for 48 hours. Inview of these results, we selected MCF-7 cells that werecultured in the medium with paclitaxel from 7 hours to 11hours for the application of bioprobes based on RBITC-DSFNPs conjugated to Annexin V in early-stage apoptosisdetection and imaging.

Detection and imaging of early-stage apoptosisbased on the RBITC-DSFNPs labeling method

Annexin V–functionalized RBITC-DSFNPs were firstused to distinguish between early-stage apoptotic MCF-7cells and the control MCF-7 cells untreated with paclitaxel.As shown in Figure 4, A, early-stage apoptotic MCF-7cells were stained with Annexin V–functionalized RBITC-DSFNPs as a result of the specific phosphatidylserinebinding, and the staining pattern was a red fluorescent

Fig 5. Confocal microscopy images of early-stage apoptotic MCF-7 cells incubated with (A) RBITC-DSFNPs conjugated to Annexin V and (B) RBITC-DSFNPs. (a and b are optical and fluorescence images, respectively. PMT 503, gain power 1.8%, offset 10%.

Fig 6. Confocal microscopy fluorescence images of early-stage apoptotic MCF-7 cells that were treated with the same concentration of paclitaxel for differenttime periods: (a) 0 minutes, (b) 100 minutes, (c) 210 minutes, and (d) 400 minutes, incubated with RBITC-DSFNPs conjugated to Annexin V. Images of groupsA and B represent the photographs taken at the lower and higher magnifications, respectively. White arrows point to the cells detected by RBITC-DSFNPsconjugated to Annexin V. PMT 565, gain power 2.0%, offset 10%.

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Fig 7. Photostability comparison of the stained early-stage apoptotic MCF-7 cells by using (A) pure Cy3-labeled Annexin Vand (B) RBITC-DSFNPs conjugatedto Annexin V. Images a, b, c, d, and e were taken after 3 minutes, 6 minutes, 9 minutes, 15 minutes, and 20 minutes of continually intense excitation, respectively.

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circle on the outer membrane of the apoptotic cell. On thecontrary, the control MCF-7 cells untreated with paclitaxeldid not show any obvious labeling signals other than thesymmetrical blue fluorescence from nucleoli and someinconspicuous red signals that result from the unspecificadsorption of Annexin V–functionalized RBITC-DSFNPs,as shown in Figure 4, B. It is clearly indicated thatRBITC-DSFNPs conjugated to Annexin V are able toperform as a specific biomarker for early-stage apoptoticcells through receptor-ligand recognition. To further avoidthe possibility of RBITC-DSFNPs staining the apoptoticcells themselves, Annexin V–functionalized RBITC-DSFNPs and RBITC-DSFNPs were incubated withapoptotic MCF-7 cells, respectively, and the results aredisplayed in Figure 5. It was observed that there were noobvious red fluorescence signals around the cell membranewhen the apoptotic MCF-7 cells were incubated with pureRBITC-DSFNPs, in contrast to the results with theapoptotic MCF-7 cells incubated with Annexin V–functionalized RBITC-DSFNPs. It is thus illustrated thatonly Annexin V–functionalized RBITC-DSFNPs couldspecifically recognize the apoptotic cells.

The results from these experiments lead us to believe thatbioprobes based on RBITC-DSFNPs conjugated to AnnexinV are absolutely competent for apoptosis detection andimaging. Hence, early-stage apoptotic MCF-7 cells that wereinduced with the same concentration of paclitaxel fordifferent time periods were further detected and imaged byusing the RBITC-DSFNPs labeling method. As shown inFigure 6, A, not one apoptotic cell was detected amongnumerous cells viewed at lower magnification when MCF-7cells were untreated with paclitaxel. When MCF-7 cells weretreated with paclitaxel for 100 minutes, several apoptoticcells were stained. With the extension of culture time from210 minutes to 400 minutes, the number of the apoptoticcells stained by Annexin V–functionalized RBITC-DSFNPsincreased ever more. Furthermore, another phenomenon wasobserved at higher magnification (Figure 6, B)—that the redfluorescence on the outer membrane of the recognized

apoptotic cells changed as the treatment time with paclitaxelwas prolonged, from weak to strong, from partially tocompletely surrounding the cell membrane.

Photostability of the RBITC-DSFNPs labeling method

To test the photostability of the RBITC-DSFNPslabeling method, early-stage apoptotic MCF-7 cells werestained with RBITC-DSFNPs conjugated to Annexin Vand pure Cy3-labeled Annexin V, respectively. Thesamples were excited for 20 minutes by successive intenseirradiation, and fluorescent images were acquired everyfew minutes. As displayed in Figure 7, the fluorescence onthe outer membrane of early apoptotic cells stained bypure Cy3-labeled Annexin V was bleached very quicklyduring 9 minutes, and the red signals of Cy3 were almostinvisible. In contrast, the fluorescence intensity of theapoptotic cells stained by Annexin V–functionalizedRBITC-DSFNPs decreased very slowly, and the redsignals were still clearly distinguishable to the naked eyeafter 20 minutes' continuous intensive irradiation. It isdemonstrated that the bioprobes based on RBITC-DSFNPspossess much better photostability in comparison with thepure organic dye labels.

Conclusions

In conclusion, a novel bioprobe based on RBITC-DSFNPs has been developed for the detection andimaging of early-stage apoptotic cells by using AnnexinV–functionalized RBITC-DSFNPs specifically binding tophosphatidylserine on the outer membrane of theapoptotic cell. The RBITC-DSFNPs labeling methodpossesses the unique advantage of better photostabilityover the conventional fluorochrome-labeled Annexin Vfor apoptosis detection and imaging. It is indicated thatRBITC-DSFNPs are potential biomarkers that would playimportant roles in bioanalysis and bioimaging, especiallyin monitoring the changes of biochemical informationover a long time.

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