Lectin-Tagged Fluorescent Polymeric Nanoparticles for Targeting of

Apr 24, 2014 - Jaebum Cho, Keiichiro Kushiro, Yuji Teramura, and Madoka Takai*. Department of Bioengineering, Grad10-ate School of Engineering, The ...
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Lectin-Tagged Fluorescent Polymeric Nanoparticles for Targeting of Sialic Acid on Living Cells Jaebum Cho, Keiichiro Kushiro, Yuji Teramura, and Madoka Takai* Department of Bioengineering, Grad10-ate School of Engineering, The University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-8656, Japan S Supporting Information *

ABSTRACT: In this study, we fabricated lectin-tagged fluorescent polymeric nanoparticles approximately 35 nm in diameter using biocompatible polymers conjugated with lectins for the purpose of detecting sialic acid on a living cell surface, which is one of the most important biomarkers for cancer diagnosis. Through cellular experiments, we successfully detected sialic acid overexpression on cancerous cells with high specificity. These fluorescent polymeric nanoparticles can be useful as a potential bioimaging probe for detecting diseased cells.

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developed based on bioimaging probes.13,14 Additionally, a surface engineering approach has been examined for the imaging.15−17 These studies involved the observation of sialic acids on living cells using fluorescence materials conjugated with phenylboronic acid, which is a derivative of boronic acid. Nonetheless, many fluorescence materials like CdSe/ZnS quantum dots and some organic materials have cell toxicity, so these materials contribute to the decrease of cell viability. Furthermore, boronic acid, which is a well-known sialic acid detection probe, also has an affinity for other monossacharides such as glucose,18,19 which leads to a decrease in signal-to-noise ratio (S/N). Also, due to its nonspecificity, it cannot be used to analyze different forms of bound sialic acids. In this study, we aimed to overcome these issue concerning cell toxicity and target specificity in the previous studies by using bioimaging probes based on the biocompatible polymeric nanoparticles and lectins. Here we designed biocompatible bioimaging probes, which consist of lectins and fluorescenceconjugated polymeric nanoparticles, to specifically monitor the expression level of sialic acids on a living cell (Scheme 1a). Major constituents of the nanoparticle are (1) lectins and (2) 2methacryloyloxyethyl phosphorylcholine (MPC) polymer with rhodamine derivative. Controlling the monomer compositions and molecular weights, we synthesized water-soluble, MPC-based polymers, poly(MPC-co-n-butyl methacrylate (BMA)-co-o-nitrophenyloxycarbonyl polyethleneglycol methacrylate (MEONP)-co-pmethacyloxyethyl thiocarbonyl rhodamine B (MTR))

INTRODUCTION Protein glycosylation, a kind of enzymatic process in the cell membrane, is one of the most abundant and structurally diverse post-translational modifications in organisms.1 Glycoproteins, products from glycosylation processes, play key roles in a wide variety of biological processes like cell adhesion, cell signaling, and immune response.2−6 Furthermore, various kinds of glycoproteins on the cell membrane and these derivatives have been used as disease biomarkers. For example, epidermal growth factor receptor (EGFR) and dysadherin are some of the most common tumor indicators.7,8 In recent years, sialic acids, one of the glycans attached to glycoproteins on the cell, have been studied as disease-associated carbohydrate derivatives, because the expression of sialic acids provides many opportunities for the appraisal of the cell processes.9 Thus, the comparative research of expression level of sialic acids on the cell membrane for normal and diseased cells can help to comprehend their behavior for application of cancer diagnosis and treatment. In addition, the sialic acid combines with other monossacharides and proteins on the cell membrane. These types are different depending on the cell species and cell organ types. Therefore, it is not only important to analyze the expression levels of total sialic acid on the cell membrane, but also distinguish the specific forms of bound sialic acid. So far, some papers have reported various methods of glycan analysis like mass spectrometry, chromatography, and polymerase chain reaction (PCR).10−12 However, these methods are unsuitable for detection and analysis on living cells, because they require extracted DNA or proteins through cell lysis, in addition to having complex and time-consuming pretreatment steps prior to analysis. Recently, various studies for the analysis of sialic acid expression level on living cells have been © XXXX American Chemical Society

Received: January 31, 2014 Revised: April 23, 2014

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Scheme 1. (a) The Fabrication Step of the Bioimaging Probe for Sialic Acid on a Living Cell and (b) the Structure of the Synthesized MPC Polymer (PMBNR)

Fabrication of PMBNR Nanoparticles and Lectin-Immobilization. Combining the PMBNR and polylactic acid (PLA), we fabricated fluorescent polymeric nanoparticles based on the solvent evaporation technique22 and later immobilized lectins on the surfaces of nanoparticles. The polymeric nanoparticles were prepared as follows. 0.1 wt % of PLA in dichloromethane solution was added in 0.1 wt % of PMBNR aqueous solution. Under stirring conditions at 400 rpm and 0 °C, the mixture was sonicated using a probe-type sonicator (VP-5S, TAITEC, Japan) for 5 min. The formed nanoparticles were collected by an ultracentrifuge (XL-A, Beckman Coulter, U.S.A.) used at 50 000 rpm at 4 °C for 2 h. The collected nanoparticles were dispersed in deionized water (DW) and stored at 4 °C. For immobilization of lectins on the nanoparticle surfaces, we utilized the avidin−biotin reaction. Ten milligrams per milliliter of the nanoparticles were reacted with 10 μg/mL of streptavidin for 3 h. Then we immobilized 10 μg/mL of biotinylated lectins, which can recognize α-2,6-sialic acids, on the streptavidin-immobilized nanoparticles for 3 h. In each step, the products were collected by centrifugation at 50 000 rpm at 4 °C. Characterization of the Properties of PMBNR, PMBNR Nanoparticles, and Lectin-Tagged Nanoparticles. The molecular weight of PMBNR and monomer compositions in PMBNR were determined by gel permeation chromatography (GPC; Jasco, Tokyo, Japan) in a 3:7 (v/v ratio) mixture of water and methanol, respectively, with a poly(ethylene oxide) (PEO) standard using a PU-2000 plus pump, a SB-804 HQ column and a refractive index detector RI-2031 plus, and hydrogen-1 nuclear magnetic resonance (1H NMR; JEOL, Tokyo, Japan), respectively. For the molecular characterization of the nanoparticles, we measured the average size and zeta-potential of fluorescent nanoparticles using dynamic light scattering (DLS, Zetasizer NanoZS, Malvern, England). And the fluorescence intensity of the nanoparticles was measured by spectrofluorometer (FP-6500, JASCO, Japan). Also, we used UV/vis spectrophotometer (V-560, JASCO, Japan) to confirm the reaction between the active ester group and streptavidin. For the observation of nanoparticle morphology, we used transmission electron microscope (TEM, JEM-1400, JEOL, Japan) at 120 kV. To confirm the quenching effect of fluorescence intensity of PMBNR nanoparticles, PMBNR1 and the polymeric nanoparticles based on PMBNR1 and PLA, we dried 0.5 μL of polymer and polymeric nanoparticles solution in DW on the slide glass, and measured the change of the fluorescence intensity using the fluorescence microscopy (Zeiss, Germany) for 90 min. Cell Culturing and Cell Experiments with Nanoparticles. For culturing human breast epithelial MCF-10A cells, 5% horse serum, 20 ng/mL of epidermal growth factor, 0.5 μg/mL of hydrocortizone, 0.1 μg/mL of cholera toxin, and 10 μg/mL of insulin were added in Dulbecco’s modified Eagle’s medium (DMEM) with 1% of penicillin/ streptomycin. For culturing human breast cancer epithelial MCF-7

(PMBNR) (Scheme 1b). The roles of the components of PMBNR are the following. Rhodamine derivatives have been readily used for the detection of mitochondria in the living cell as a bioimaging probe.20 Thus, MTR, also being a rhodamine derivative, can play a role as the fluorescence molecule in the polymer. The active ester group of MEONP has the ability to conjugate other biomolecules. Lectins are often used for profiling glycoproteins and have been studied as imaging probes for the detection of sialic acids, recently.21,22 MPC polymers have found many uses in various biomedical fields due to their advantageous properties of protein adsorption inhibition and tissue biocompatibility,23,24 and also there are several previous reports on MPC polymers being used as biocompatible imaging materials.25−27 With the combination of these monomers and lectins, the fabricated polymeric nanoparticles can provide a highly sensitive and selective platform as bioimaging probes for the analysis of sialic acid expression level on living cells.

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EXPERIMENTAL SECTION

Materials. MPC was provided by NOF Co. Ltd. (Tokyo, Japan), and was synthesized by a previously reported method.28 n-Butyl methacrylate (BMA), α-2,2′-azobis(isobutyronitrile) (AIBN), diethyl ether, chloroform, and dichloromethane were purchased from Kanto Chemical Co., Inc. (Tokyo, Japan). Streptavidin was purchased from Sigma-Aldrich Corp. (St. Louis, MO, U.S.A.), and biotinylated Sambucus nigra bark lectin was purchased from Vector Laboratory (Burlingame, U.S.A.). Streptavidin conjugated Alexa488 was purchased from Invitrogen (Carlsbad, CA, U.S.A.). n-Methacyloxyethyl thiocarbonyl rhodamine B (MTR) was purchased from Polysciences, Inc. (Philadelphia, PA, U.S.A.). PLA was purchased from Wako Pure Chemical Industries Co., Ltd. (Osaka, Japan). The MEONP monomer was synthesized by a previously reported method.29 Biotinylated Sambucus nigra bark Lectin (biotin-lectin) was purchased from Vector Lab (Burlingame, CA, U.S.A.). Synthesis of PMBNR. We synthesized the water-soluble biocompatible PMBNR via free radical polymerization with AIBN as an initiator, intended for use in biological conditions. For each PMBNR synthesis, MPC, BMA, MEONP, and MTR were dissolved in 30 mL of ethanol at 0.5 M, and the solution was purged with argon gas to remove dissolved oxygen for 20 min after adding 10 mM of AIBN. The solution was reacted in the 65 °C oil bath for 15 h to initiate polymerization. After the free radical polymerization, the polymer was precipitated in the 8:2 (v/v ratio) mixture of diethyl ether and chloroform, respectively. The collected PMBNR was stored in solid state at 4 °C. B

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Table 1. Molecular Characterization of the Synthesized PMBNR mole fraction in polymera

mole fraction in feed MPC PMBNR1 PMBNR2 PMBNR3

0.40 0.60 0.70

BMA 0.49 0.29 0.19

MEONP 0.10 0.10 0.10

MTR 0.01 0.01 0.01

MPC 0.45 0.62 0.76

BMA 0.50 0.32 0.20

MEONP

MTR

0.05 0.06 0.04

c

-c -c

yield (%)

Mwb

Mnb

88 82 82

5.8 × 10 5.6 × 104 6.9 × 104 4

Mw/Mnb

1.3 × 10 1.5 × 104 1.7 × 104 4

4.5 3.7 4.1

a

Mole fraction in polymer was determined by 1H NMR spectrum. bMolecular weight (Mw and Mn) was determined by GPC in water: methanol (v/ v ratio =3:7); PEO standard. cMTR in polymer was not identified from 1H NMR because other peaks were overlapped.

cells, human cervical cancer HeLa cells and mouse L929 fibroblasts, 10% of fetal bovine serum (FBS) was added in the DMEM with 1% of penicillin/streptomycin. For observation with confocal microscope, cultured cells were seeded in 35 mm glass-bottom culture dish (MatTek, Ashland, U.S.A.) and cultured overnight at 37 °C in 5% CO2 and 95% air. For the viability assay, we used the trypan blue exclusion method. We reacted 200 μL of the fluorescent polymeric nanoparticles (10 mg/ mL) in each dish of the cultured cells for 1 h. Then the cultured cells with the nanoparticles were rinsed with Dulbecco’s phosphate-buffered saline (DPBS, pH 7.4), and were collected by the centrifuge spun at 1000 rpm for 3 min. After collection, we dyed the cells using 0.4% of trypan blue stain solution (Gibco, Oklahoma, U.S.A.) for 1 min, and counted the number of dyed cells. For the cellular experiments to detect the sialic acid expression levels on the cell surface, 10 mg/mL of lectin-tagged fluorescent polymeric nanoparticles were reacted with precultured cells for 30 min. After washing three times with DPBS for the removal of residual nanoparticles, the fluorescence images of the cells were observed by confocal laser scanning microscope (LSM510, Zeiss, Germany). For the specificity experiment of lectin-tagged fluorescent polymeric nanoparticles for silaic acids, we used cells pretreated with sialidase at a concentration of 0.016 mg/mL for 16 h before the addition of the lectin-tagged fluorescent polymeric nanoparticles. For quantification of expression level of sialic acids on each cell membrane, the fluorescence intensities of the cultured cells with the lectin-tagged fluorescent polymeric nanoparticles were measured by a BD LSR II flow cytometer (BD Bioscience, New Jersey, U.S.A.). As a control experiment, lectin-conjugated Alexa488 was used as a probe. The lectin-conjugated Alexa488 was prepared by mixing 10 μg/ mL of streptavidin conjugated Alexa488 and 10 μg/mL of biotin-lectin. The solution of lectin-conjugated Alexa488 was added to MCF-7 and HeLa for 30 min. After washing with DPBS, the fluorescence images of reacted cells were observed by confocal microscopy. Protein Adsorption onto Nanoparticles. After 10 mg/mL of lectin-tagged fluorescent polymeric nanoparticles were incubated in culture medium containing 10% FBS at 37 °C for 1 h, those nanoparticles were collected by ultracentrifuge by 50 000 rpm at 4 °C for 2 h three times. Then protein concentration was determined by a Micro BCA Protein Assay Kit (Thermo scientific, Rockford, U.S.A.). The absorbance of optical density was measured at 560 nm by a micro plate reader (Wallec Arvo SX 1420 Multilabel Counter, PerkinElmer, Waltham, MA, U.S.A.).

nanoparticles have good dispersion in anqueous solution is an important factor for use in biological conditions. Because PLA, which aggregates with PMBNR during nanoparticle fabrication, was insoluble in water, PMBNR nanoparticles were prepared using a solvent evaporation technique with dichloromethane that has a low boiling temperature. As a result, fabricated nanoparticles formed PLA cores with PMBNR coatings, in which the hydrophobic part was linked with PLA and the hydrophilic part shaped the surface layer. The average size of each type of PMBNR nanoparticle is shown in Figure 1. The size of PMBNR1

Figure 1. Average size of different PMBNR nanoparticles with different monomer compositions (Error bars represent standard deviation with n = 5, *p < 0.05, and **p < 0.01).

nanoparticles was 13 nm (SD = ± 2 nm) as measured by DLS, while those fabricated with PMBNR2 and PMBNR3 were 14 nm (SD = ± 3 nm) and 18 nm (SD = ± 5 nm), respectively. The average size and the size distribution of PMBNR1 nanoparticles were smaller than the PMBNR2 or PMBNR3 nanoparticles due to the increased hydrophobic interaction between PLA and BMA with the change in the hydrophobic monomer ratio. Because of the small size and size distribution, we proceeded to use PMBNR1 nanoparticles for the cellular experiments. In addition, the fluorescence of MTR monomer in PMBNR was also confirmed by the presence of a near 570 nm peak of rhodamine, as measured by the fluorescence spectrometer. To further confirm the DLS results, we observed PMBNR1 nanoparticles by TEM on a carbon support membrane in dried state, and as shown in Figure 2a, we could examine the size of PMBNR1 nanoparticles to be similar to that measured by DLS. As for the water-solubility of PMBNR, the fluorescent polymeric nanoparticles dispersed well in water and DPBS (pH 7.4), and still maintained good fluorescence intensity. The fluorescence spectrum of PMBNR1 and PMBNR1 nanoparticles in DW was almost identical to that of MTR in ethanol (Figure 2b). Because MTR was insoluble in water, the fluorescence spectrum of MTR was measured with ethanol as

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RESULTS AND DISCUSSIONS Fabrication and Analysis of PMBNR Nanoparticles. The free radical polymerization of MPC, BMA, MEONP and MTR progressed homogeneously, and the molecular characterization (monomer compositions, molecular weights and polydispersity) of the synthesized PMBNR as measured by GPC and 1H NMR are summarized in Table 1. Three types of PMBNR (PMBNR1, PMBNR2, and PMBNR3) were synthesized with different BMA composition ratios in order to vary the size of the nanoparticles, and each PMBNR had a similar final composition to the initial feed composition based on 1H NMR. In addition, each PMBNR was soluble in water at high concentration (above 1 wt %), and the fact that the polymeric C

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substrate, and the change in fluorescence intensity with the light exposure to rhodamine’s excitation wavelength was measured by fluorescence microscope. As a result, the fluorescence of rhodamine B of the nanoparticles was more stable than that of naked rhodamine B (MTR) after exposure to the light for 90 min, consistent with a previous study.30 By shielding the rhodamine core with the polymer, the photostability was increased compared to the unshaped organic molecule. The fluorescence intensity of PMBNR1 nanoparticles showed a decline of 65% compared to the unexposed PMBNR1 nanoparticles from UV light, while the bare PMBNR1, not as nanoparticles, decreased about 85% (Figure 3). Because the quantum yield of fluorescent materials such as rhodamine is affected from hydrophobic surroundings, the fabricated polymeric nanoparticles proved to be more ideal for quantifying the fluorescence intensity for detecting the expression of sialic acids on the cell membrane compared to the unshaped polymer. In addition, because rhodamine has an innate specificity for mitochondria in living cells,17 this specificity may be nullified by trapping rhodamine in the nanoparticle coated by the hydrophilic MPC polymer on the surface. Furthermore, surface zeta potential confirmed by DLS to be approximately zero charge also supports that the nanoparticles were coated by the hydrophilic MPC segments. Lectin-Conjugated PMBNR1 Nanoparticles for Cell Studies. For tagging lectins to the nanoparticles, we utilized the avidin−biotin reaction. After immobilization of streptavidin with the active ester group of MEONP on the PMBNR1 nanoparticles, the biotinylated lectin was reacted on the streptavidin-tagged nanoparticles. In this step, the surface reaction mechanisms to produce the lectin-tagged fluorescent polymeric nanoparticles were also confirmed. In the reaction of active ester group of MEONP with an amine group of a biomolecule, the p-nitrophenoxy group of MEONP is separated from PMBNR1. This p-nitrophenoxy ion has a specific peak in the UV spectrum at 400 nm. The solution of PMBNR1 nanoparticles showed the specific peak at 400 nm after reaction with streptavidin for 3 h, while the spectrum of the unreacted nanoparticle solution did not show any peak around 400 nm (Data is not shown). After confirmation of PMBNR1 nanoparticles reacted with streptavidin, we reacted streptavidin-tagged nanoparticles with biotinylated lectins and measured the size of lectin-tagged nanoparticles using DLS. The size of lectin-tagged nanoparticles was measured to be 35 nm, indicating that the size increased from the original size (13 nm) as lectin molecules were immobilized around the nanoparticle surface. Also, the zeta potential of lectin-tagged nanoparticles was −15.8 mV, whereas that of nanoparticles was −5.7 mV. This also indicates that lectin molecules were conjugated to the surface of nanoparticles. For the purpose of cellular diagnosis, we fabricated polymeric nanoparticles containing the MPC polymer. Therefore, it is important that we confirm the biocompatibility of the lectintagged nanoparticles as probes for biological conditions. To check cell viability, we reacted 200 μL of various concentrations of PMBNR1 nanoparticles for 1 h with mouse fibroblasts (L929), human epithelial normal cells (MCF-10A), and human epithelial cancerous cells (MCF-7), and examined the cell viability based on the trypan blue exclusion method. For all concentrations of fluorescent polymeric nanoparticles, more than 90% of the probe-bounded cells survived. The survival rate of cells with the nanoparticles was similar to the cases without the nanoparticles. The result indicated that the polymeric

Figure 2. (a) Bright field TEM image (scale bar =50 nm) of the PMBNR1 nanoparticles. The red circle indicates a single nanoparticle. (b) Fluorescence intensity comparison of PMBNR1 in DW (blue line), PMBNR1 nanoparticles in DW (red dotted line), and MTR in ethanol (green dashed line).

solvent. In Figure 2b, the peak shift is due to the different hydrophobic environment of rhodamine B. To assess the potential as a bioimaging probe, we also examined the quenching effect by photobleaching of PMBNR1 and PMBNR1 nanoparticles for short exposure time (Figure 3). PMBNR1 and PMBNR1 nanoparticles were dried on glass

Figure 3. Change of fluorescence intensity versus light exposure time for 90 min (Error bars represent standard deviation with n = 3). D

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acids began responding to lectin-tagged nanoparticles from 5 min after incubation, which is faster than the response time of MCF-10A and L929 (Data is not shown). It is also due to the absolute amount of overexpressed sialic acids on the cell membrane as a target. For the actual quantification of sialic acid expression level on each cell line, we measured the fluorescence intensity of each cell type reacted with lectin-tagged fluorescent polymeric nanoparticles using flow cytometer. The high and accurate sensitivity of the fabricated nanoparticles allowed them to be further used in determining the sialic acid expression level on the living cell membrane. For the measurement, each cell line reacted with lectin-tagged nanoparticles was collected, resuspended in 1 mL of DPBS, and quantified by flow cytometer. The result also showed that the intensity of fluorescent polymeric nanoparticles reacted with MCF-7 is higher than that of other cell lines (Figure 6). In the quantitative value, the intensity of MCF-7 is roughly twice than that of MCF-10A and L929. It means that the amount of sialic acid on the cancerous cell surface is more than that on noncancerous cells. The overexpression of sialic acids can be a promising marker on cancerous cells.31−33 As a control experiment, lectin-conjugated Alexa488 was used as a probe for MCF-7 and HeLa cells. After reaction for 30 min, strong fluorescence was observed for both cell lines when lectin-tagged nanoparticles were used. On the other hand, there was a weak fluorescence signal from cells in case lectinconjugated Alexa488 was used (Supporting Information, Figure S1 and S2). Additionally, the fluorescence from lectin-tagged nanoparticles could be observed after 2 days; however, lectinconjugated Alexa488 disappeared at that time. These results indicate that lectin-tagged nanoparticles have higher affinity as a lot of lectin molecules were immobilized on a nanoparticles compared to lectin-conjugated Alexa488. Moreover, nanoparticles were slowly excluded from cells. Therefore, there is an advantage in higher affinity and stability for lectin-tagged nanoparticles compared to lectin-conjugated Alexa488. Finally, the specificity of the fabricated nanoparticles for sialic acid was confirmed using sialidase. Treatment with sialidase removes sialic acids on the cell membrane, so that there remains no target for lectin-tagged nanoparticles. Sialidase can even cleave sialic acids immobilized with other biomolecules on the cell membrane. By treatment of sialidase to the living cells, we could examine the specificity of lectin-tagged fluorescent polymeric nanoparticles for sialic acids. As expected, no fluorescence was observed in all types of sialidase-treated cells (Figure 7). All in all, we find that lectins of the nanoparticles specifically recognize sialic acids on the cell membrane and differentially bind cells with different sialic acid expression levels. When lectin-tagged nanoparticles were incubated in culture medium containing 10% FBS at 37 °C for 1 h, there was small increase of serum protein adsorption. However, lectin-tagged nanoparticles specifically recognized sialic acid expressed on the cell surface, indicating that nonspecifically adsorbed proteins did not impair the function of lectin. Since the nanoparticles were made of MPC polymer, the surface was well protected from nonspecific protein adsorption.

nanoparticles are biocompatible enough for the analysis of living cells (Figure 4).

Figure 4. Cell viability of each cell line for lectin-tagged fluorescent polymeric nanoparticles (Error bars represent standard deviation with n = 3).

Analysis of the Sialic Acid Expression Levels of the Different Cell Types Using the PMBNR1 Bioimaging Probe. We investigated the expression level of sialic acids on the living cell membrane using the fabricated lectin-tagged fluorescent polymeric nanoparticles. After rinsing the living cells reacted with lectin-tagged fluorescent polymeric nanoparticles for 30 min using DPBS, we observed the fluorescence images. The result showed that the fluorescence intensity of the nanoparticles on MCF-7 cells, the cancerous human epithelial cell type, was stronger than those on any other noncancerous cell types (Figure 5). It shows that indeed the sialic acids are overexpressed on the cancerous cells, as mentioned in previous studies.31−33 In addition, MCF-7 with the overexpressed sialic

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CONCLUSION We fabricated lectin-tagged fluorescent polymeric nanoparticles to specifically detect sialic acid on the cell membrane. With our biocompatible nanoparticles, it is possible to perform fast and

Figure 5. Fluorescence images (a,c,e) and bright-field images (b,d,f) of each cell line ((a,b) L929; (c,d) MCF-10A; (e, f) MCF-7) reacted with lectin-tagged fluorescent polymeric nanoparticles (scale bar = 20 μm). E

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Figure 6. Quantification of fluorescence intensity of nanoparticle-reacted (a) L929, (b) MCF-10A, and (c) MCF-7 using flow cytometry. The mean fluorescence intensities (a.u.) of gray sections (P1) were (a) 176, (b) 188, and (c) 334.

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Figure 7. Fluorescence images (a,c,e) and bright-field images (b,d,f) of each cell type ((a,b) L929; (c,d) MCF-10A; (e,f) MCF-7) pretreated with sialidase and reacted with lectin-tagged fluorescent polymeric nanoparticles (scale bar = 20 μm).

simple detection for cancerous cells. Our approach will be promising for bioimaging with high sensitivity and selectivity of cancerous cells.

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ASSOCIATED CONTENT

S Supporting Information *

Fluorescence images of HeLa and MCF-7 cells reacted with lectin-conjugated Alexa488 and lectin-tagged fluorescent polymeric nanoparticles. This material is available free of charge via the Internet at http://pubs.acs.org.

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*Phone: +81-3-5841-7125; Fax: +81-3-5841-0621; E-mail: [email protected] and [email protected]. Notes

The authors declare no competing financial interest. F

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