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Wheat Germ Agglutinin-Modified Trifunctional Nanospheres for Cell Recognition Hai-Yan Xie,†,‡ Min Xie,‡ Zhi-Ling Zhang,‡ Yan-Min Long,‡ Xin Liu,‡ Ming-Liang Tang,§ Dai-Wen Pang,*,‡ Zheng Tan,§ Calum Dickinson,| and Wuzong Zhou*,| School of Life Science and Technology, Beijing Institute of Technology, Beijing 100081, P. R. China; College of Chemistry and Molecular Sciences, State Key Laboratory of Virology and College of Life Sciences, Wuhan University, Wuhan 430072, P. R. China; and EaStChem, School of Chemistry, University of St. Andrews, St. Andrews, Fife KY16 9ST, United Kingdom. Received December 14, 2006; Revised Manuscript Received August 5, 2007
A simple and convenient strategy has been put forward to fabricate smart fluorescent magnetic wheat germ agglutinin-modified trifunctional nanospheres (WGA-TFNS) for recognition of human prostate carcinoma DU145 cells which are surface-expressed with sialic acid and N-acetylglucosamine. These TFNS can be easily manipulated, tracked, and conveniently used to capture and separate target cells. The presence of wheat germ agglutinin on the surface of WGA-TFNS was confirmed by FTIR, biorecognition of carboxymethyl chitin-modified quantum dots (CM-CT-QDs), and bacterium Staphylococcus aureus. The success in recognizing DU-145 cells by the WGA-TFNS indicates that WGA-TFNS could be applicable.
INTRODUCTION Lectins, which can be found in most organisms, ranging from viruses and bacteria to plants and animals (1), form a family of natural nonenzymatic proteins/glycoproteins that bind monosaccharides and oligosaccharides with high specificity, although they are not products of an immune response. Each lectin molecule contains typically two or more carbohydrate binding sites. When they interact with cells, they will not only specifically recognize and bind to the carbohydrate residues on the cell surface, but also cause crosslinking of the cells and their subsequent agglutination (1). Lectins also have cytoadhesive and cytoinvasive properties, and can initiate vesicular transport processes based on their recognizing and binding to carbohydrate residues on a cell surface (2). Therefore, lectins have been widely used in drug delivery systems (3). Wheat germ agglutinin (WGA), derived from cereals and one of the most widely characterized and least immunogenic lectins, is a dimer consisting of two identical 18 kDa subunits and four carbohydrate binding sites. Since WGA receptors, such as N-acetylglucosamine (GlcNAc) and sialic acid, are commonly expressed on surface plasma membranes of most types of neurons in the brain, among a number of lectins with different carbohydrate-binding specificities (4), WGA can be used as an effective tracer in a variety of neural systems. As WGA can also recognize some types of cancer cells, it has already been used in some drug delivery applications (5). Nanomaterials, especially nanomaterial-based functional nanodevices, are increasing in their popularity with researchers and manufacturers. Among them, magnetic nanoparticles are extensively studied and widely used for drug delivery, magnetic * Corresponding authors. Phone 0086-27-68756759; Faxes 008627-68754067, +44 1334 463808; E-mail addresses:
[email protected] or
[email protected]. † Beijing Institute of Technology. ‡ College of Chemistry and Molecular Sciences and State Key Laboratory of Virology, Wuhan University. § College of Life Sciences, Wuhan University. | University of St. Andrews.
treatment, and magnetic separation due to their small size, single magnetic domain structures, and superparamagnetism (6–14). Fluorescent semiconductor quantum dots (QDs) are another important type of nanoparticles, which have broad excitation and size-dependent photoluminescence with narrow emission bandwidth covering a wide spectral range. By using a single wavelength, many particles with different sizes and different compositions can be simultaneously excited. Furthermore, the QDs exhibit unusual photochemical stability and relatively high photoluminescent quantum yields (15–19). By embedding QDs with different emission colors into polymer spheres, a large number of encoded and distinguishable microbeads can be obtained. QD-embedded polymer microspheres are expected to be used in gene expression, high-throughput screening, and medical diagnostics (20). On the other hand, magnetic nanoparticle filled beads have been used in the biomedical field, such as immunomagnetic separation and nucleic acid hybridization (11, 21). However, in many biomedical and clinical diagnostic fields, there has always been some need for materials with the functions of both magnetic separation and fluorescence tracking. The common strategy is to anchor magnetic nanoparticles and organic fluorophores, respectively, to two kinds of molecules that can interact with each other. The method is complicated, time-consuming, and inefficient (11, 12, 14). Several new methods have been developed recently based on the use of QDs and magnetic nanoparticles (22–30). Among them, the method developed by us is to construct fluorescent–magnetic bifunctional nanospheres by co-embedding the fluorescent quantum dots and γ-Fe2O3 magnetic nanoparticles into single swellingpoly(styrene/acrylamide) copolymer nanospheres (22, 26–28). The nanospheres are fluorescence-trackable and magnetomanipulatable, and demonstrate their capability of specific cell targeting after surface-modification with some specific reagents such as folic acid (22, 26, 27). Subsequently, they were successfully used in apoptotic Hela cell recognition (28). Here, we present WGA-modified trifunctional nanospheres for recognition of human prostate carcinoma DU-145 cells based on a new strategy of convenient modification of fluorescent–mag-
10.1021/bc060387g CCC: $37.00 2007 American Chemical Society Published on Web 09/26/2007
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netic bifunctional nanospheres, which will be universally applicable for GlcNAc and sialic acid recognition.
EXPERIMENTAL PROCEDURES Fabrication of Fluorescent–Magnetic Bifunctional Nanospheres. In order to fabricate fluorescent–magnetic bifunctional nanospheres (BFNS) (22, 26, 27), some related building materials should be first synthesized. CdSe/ZnS QDs, with the diameter between 3 and 6 nm, were synthesized by the method described in a previous report (31). The synthesis of nano-γFe2O3 (ca. 8 nm in diameter) was as reported (27, 32). Poly(styrene/acrylamide) copolymer nanospheres (St-AAm, 100–250 nm in diameter) were produced from styrene (St) and acrylamide (AAm) by means of emulsifier-free polymerization by the previously reported method (33). St-AAm was subsequently modified with hydrazine through the surface amide functional groups, producing a reactive nanosphere surface for future modification and attachment (27). The hydrazinized StAAm was denoted as H2N-St-AAm. On the basis of the above building materials, the bifunctional nanospheres were fabricated as described below (Scheme 1) (22, 26, 27). Both the QDs and the nano-γ-Fe2O3 dispersed in hexane were washed several times to remove any impurities, and the nanoparticles and copolymer nanospheres were dried before use. Hydrazinized bifunctional nanospheres (H2N-BFNS) were produced by swelling the H2N-St-AAm in a solvent mixture containing 5% (vol) chloroform and 95% (vol) butanol, and adding a controlled amount of CdSe/ZnS QDs and nanoγ-Fe2O3 to the mixture. For example, a typical ratio used in the present work was 3:1 QDs/Fe2O3. The mixture was then ultrasonicated until it became a well-dispersed suspension. The mixture was ultrasonicated for a further 30 min, centrifuged, and washed with butanol three times. The resultant H2N-BFNS with both fluorescence and magnetism were dispersed in ultrapure water for use. Construction of Wheat Germ Agglutinin-Modified Trifunctional Nanospheres. 0.1 g of succinic anhydride was added to 5 mL of 10 mg/mL H2N-BFNS aqueous solution. The pH of the reaction system was adjusted to 4 with 1 M NaOH. The reaction lasted for 2–3 h. Then, the product was centrifuged at 12 000 rpm for 10 min several times to remove the excess succinic anhydride. The final product HOOC-BFNS was kept in 0.1 M phosphate buffer solution (PBS) (pH 6.5). The tube with the solution containing the HOOC-BFNS was put onto a magnetic scaffold for ca. 5 min, and then the HOOCBFNS were attracted by magnetic force, allowing the solvent to be removed easily. The attachment method was similar to that previously reported (2, 34). The HOOC-BFNS were thoroughly washed with 0.1 M PBS (pH 6.5). After that, ca. 5 mg of HOOC-BFNS were activated with 100 mM 1-ethyl-3(3-dimethylaminopropyl)carbodiimide hydrochloride (EDAC) and 100 mM N-hydroxysuccinimide (NHS) in l mL of 0.1 M PBS (pH 6.5) for 30 min at room temperature with continuous shaking. The activated HOOC-BFNS were then washed with l mL of 0.1 M PBS (pH 7.4). To covalently immobilize WGA onto the BFNS surface, 90 µL of activated HOOC-BFNS were reacted with 10 µL of WGA (3.6 mg/mL, dissolved in 0.1 M PBS at pH 7.4) for 4 h at room temperature with continuous shaking to form fluorescent magnetic wheat germ agglutinin modified trifunctional nanospheres (WGA-TFNS). The tube containing WGA-TFNS was put onto the magnetic scaffold to remove any unreacted WGA and then washed with 0.1 M Tris–HCl (pH 7.5, containing 1 mM CaCl2 and 1 mM MgCl2). The resultant WGA-TFNS were dispersed in 400 µL of the same Tris–HCl solution. Because nano-γ-Fe2O3 and QDs in WGA-TFNS would influence the characterization of WGA on the surface of WGA-
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TFNS, WGA-St-AAm was constructed to facilitate the characterization. WGA-St-AAm was prepared according to the procedure similar to that of WGA-TFNS. The washed HOOCSt-AAm was dispersed in 1 mL of 0.1 M PBS (pH 6.5), then activated with 100 mM EDAC and 100 mM NHS in l mL of 0.1 M PBS (pH 6.5) for 30 min at room temperature with continuous shaking, followed by centrifuging at 12 000 rpm for 40 min to remove excess activation reagents and further washing with l mL of 0.1 M PBS (pH 7.4) by centrifuging at 12 000 rpm for 40 min. Subsequently, 950 µL of activated HOOC-StAAm was reacted with 50 µL of WGA (3.6 mg/mL, dissolved in 0.1 M PBS at pH 7.4) for 4 h at room temperature with continuous shaking to form WGA-St-AAm. WGA-St-AAm was purified by centrifugation at 12 000 rpm for 40 min to remove excess WGA, and finally dispersed in l mL of 0.1 M PBS (pH 7.4). Characterization. An Acc. V Spot Maqn scanning electron microscope (SEM) operated at 20 kV was used to characterize the surface morphology of the H2N-St-AAm nanospheres. The size and dispersity of the nano-γ-Fe2O3 and H2N-St-AAm were characterized by transmission electron microscopy (TEM) on a JEOL-JEM 2010 microscope operated at 200 kV using a Gatan Dual Vision 300 W CCD camera. High-resolution TEM (HRTEM) images of BFNS were obtained on a JEOL-JEM 2011 microscope operated at 200 kV. Chemical composition of the products was examined by energy-dispersive X-ray microanalysis (EDX) using an Oxford Link ISIS system attached to the JEM-2011 microscope. A small electron beam (5–10 nm) was normally chosen for the EDX studies. The X-ray diffraction pattern of the nano-γ-Fe2O3 was determined on a D/max-RC diffractometer with Cu (40 kV, 50 mA) as the target. Fluorescence images were recorded with a Zeiss HRC Axiocam (3900 × 3900 pixels) camera attached to a Zeiss inverted optical microscope (Axiovert 200M) or an Olympus IX70 inverted fluorescence microscope with a 100 W mercury arc lamp, U-MWU (330–385/400/420nm) and U-MWB filters (450–480/500/515 nm) and an oil-immersion objective (100×). WGA on the surface of WGA-TFNS was characterized by FTIR spectroscopy. H2N-St-AAm, HOOC-St-AAm, and WGASt-AAm were lyophilized for FTIR analysis. The infrared spectra of the samples were obtained on a Thermo Nicolet 360 FT-IR spectrophotometer. Pressed pellets were prepared by grinding the powder specimens with KBr in an agate mortar. Cell Culture and Recognition of Cells by WGA-TFNS. Staphylococcus aureus (S. aureus) was precultured overnight in 5 mL of Luria-Bertain (LB) culture medium at 37 °C. Then, 100 µL of precultured S. aureus suspension was added to 5 mL of LB culture medium, incubated at 37 °C for 6 h with shaking, and kept at 4 °C for further experiments. S. aureus agglutination was used to confirm the presence of WGA on the WGA-TFNS surface. 0.5 mL of S. aureus suspension was harvested by centrifugation at 8000 rpm for 5 min, washed twice with 0.1 M Tris–HCl (pH 7.5, containing 1 mM CaCl2 and 1 mM MgCl2), and resuspended in 400 µL of the same Tris–HCl solution. The mixture of 150 µL of WGATFNS and harvested S. aureus suspension was incubated at 37 °C for 25 min, magnetically separated on a magnetic scaffold, washed with the 0.1 M Tris–HCl solution, and resuspended in 200 µL of the 0.1 M Tris–HCl solution for imaging. DU-145 cells (a human prostate carcinoma cell line) were used for the recognition by WGA-TFNS. Cells were routinely cultured at 37 °C in a flask containing RPMI 1640, supplemented with 10% fetal bovine serum (FBS), 100 µg/mL penicillin G, 100 µg/mL streptomycin sulfate, 1.8 mg/mL NaHCO3 and 25 mM HEPES in a humidified atmosphere with 5% CO2 (in air).
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Scheme 1. Construction of a Fluorescent Magnetic Wheat Germ Agglutinin Trifunctional Nanosphere and Its Capturing a Human Prostate Carcinoma Du-145 Cell
Experiments of DU-145 cell capture and separation by WGATFNS were carried out as described below. About 1 × 106 DU145 cells were first cultured in a flask and then detached using trypsin–EDTA solution, and collected by centrifugation at approximately 1000 rpm for 5 min at room temperature. Subsequently, the cells were washed with cold 1 × PBS, resuspended in 900 µL of 1 × PBS and incubated with 100 µL of 5 mg/mL WGA-TFNS for 30 min at room temperature. Besides WGA, Dolichos biflorus agglutinin (DBA), another kind of lectin that can bind with N-acetyl-D-galactosamine (GalNAc), was used as a control for WGA. DBA-TFNS were fabricated according to the same procedure as for WGA-TFNS mentioned above. Examination of the Capacity of WGA-TFNS to Capture DU-145 Cells. DU-145 cells were first cultured in a flask and then detached using trypsin–EDTA solution, and collected by centrifugation at approximately 1000 rpm for 5 min at room temperature. Subsequently, the cells were washed with cold 1 × PBS, resuspended in 1 × PBS, and the cell concentration was determined by hemocytometry. Then, WGATFNS were added to DU-145 cell suspension and incubated for 30 min at room temperature and isolated with a magnetic scaffold, and then the uncaptured DU-145 cells in solution were counted. Cytotoxicity of WGA-TFNS Examined by Trypan Blue Dye Exclusion Assay. DU-145 cells were plated in a 6-well tissue culture plate. After the cells became confluent, the cells were washed twice with 1 × PBS and added with WGA-TFNS, then incubated for 30 min at room temperature. After incubation, DU-145 cells were washed twice with 1 × PBS after redundant WGA-TFNS were removed, then trypsinized and resuspended in RPMI 1640 culture media. DU-145 cells were centrifuged at 1000 rpm for 5 min and dispersed in 1 × PBS, then DU-145 cell solution was mixed with Trypan blue dye (0.8% w/v) in equal volume, staining for 2 min. More than 200 stained and unstained cells (viable cells could not be stained by Trypan blue) were counted by hemocytometry. The cell viability was calculated below. Cell viability [%] ) (number of viable cells/total cell number) × 100 DU-145 cells cultured without WGA-TFNS under the same experimental conditions were used as the control.
by embedding both the CdSe/ZnS QDs and the nano-γ-Fe2O3 into the swelling H2N-St-AAm nanospheres simultaneously. The hydrophilic amide (-CONH2) groups of the copolymer tend to be located towards the outer surface of the nanospheres, while the hydrophobic moieties are found in the interior, leading to the formation of hydrophobic hollow cavities, since the copolymer nanospheres are synthesized in an aqueous solution. Both hydrophobic QDs and γ-Fe2O3 can be embedded in weakly polar organic solvent (22). Therefore, it is believed that the embedment is completed via hydrophobic interactions. As shown in Figure 1, the nanoparticles are widely distributed in the nanospheres. The strong ultrasonication is usually used to prevent the nanoparticles from adsorbing on the surface, and consequently, the surface of the nanospheres is relatively clean as observed from the TEM images. It is reasonable to believe that the particles are actually embedded in the copolymer nanospheres rather than deposited on the surface. When the nanospheres, embedded with QDs and γ-Fe2O3, are dispersed in a polar aqueous solution, the embedded hydrophobic nanoparticles are unable to leak from the hydrophobic cavities. No detectable leakage was observed in the resultant H2N-BFNS, even after 1 week of consecutive ultrasonication. Therefore, the H2N-BFNS are stable if no chemical that can decompose and denature the nanospheres is present in solution, and any operation, which can denature the nanospheres, is avoided. H2N-BFNS were found to have good dispersivity as can be seen from Figure 2. If the numbers of embedded QDs and γ-Fe2O3 nanoparticles increase, the fluorescence of the nanospheres will become stronger, and the magneto-manipulation time will be shorter. Strong fluorescence of QDs enables us to observe the light spot from a single QD with the naked eye. Therefore, there are no rigorous requirements for the number of embedded nanoparticles and the reproducibility of the exact embedding amounts.
RESULTS AND DISCUSSION Fluorescent Magnetic Bifunctional Nanospheres. Trifunctional nanospheres or smart nanobiodevices simultaneously with fluorescence, magnetism, and biotargeting properties will be a powerful tool for biotracking, bioimaging, bioseparation, clinical diagnosis, and biomedical fundamental research. In view of the structural features (Scheme 1), the formation of the fluorescent magnetic bifunctional nanospheres is an important basis for construction of a variety of smart nanobiodevices. By our simple and convenient approach (22, 26, 27), perfect bifunctional nanospheres (Figure 1) can be readily fabricated
Figure 1. TEM image of a fluorescent magnetic bifunctional nanosphere embedded with both QDs and nano-γ-Fe2O3.
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Figure 2. Microscopic images of H2N-BFNS: (a) bright field and (b) fluorescent. 40× objective.
Strategy for the Modification and Biofunctionalization of the BFNS. As mentioned above, the reactive group of StAAm for the coupling purpose is the amide on its surface. Since the scheme for the synthesis of the copolymer nanospheres is emulsifier-free, the surface is relatively clean and convenient for conjugating with other molecules. In most cases, it is difficult to anchor any other molecules, especially biomacromolecules, onto the surface of St-AAm, either due to the steric hindrance or due to insufficient reactivity of the amide group on the surface. Therefore, keeping the reactive group away from the surface of St-AAm or converting the amide into another more reactive group is crucial for further biofunctionalization. As shown in Scheme 1, modification of St-AAm with hydrazine through the surface amide functional groups will produce a relatively reactive nanosphere surface with hydrazide groups. Subsequent co-embedment of CdSe/ZnS QDs and nanoγ-Fe2O3 into the H2N-St-AAm will create H2N-BFNS to facilitate both future application and construction of trifunctional nanospheres. End carboxyl groups can be readily generated on the surface via another amide linkage to form HOOC-BFNS by reaction of the surface hydrazide of H2N-BFNS with succinic anhydride. It is due to the magnetism of HOOC-BFNS that the resulting product can be easily separated. The end carboxyl functionalization enables the nanospheres to decrease in steric hindrance and supply a flexible choice of functional groups for attachment to many molecules. The coupling via carboxyl groups can retain the bioactivity of proteins, and the resultant products containing BFNS can be easily separated and purified. WGA with four carbohydrate binding sites can be conveniently attached to the nanosphere surface through an amide bond between its amino group and the carboxyl on the surface of HOOC-BFNS to produce WGA-TFNS targeting GlcNAcor sialic acid-containing species. Characterization of WGA-TFNS. The characterization of nanomaterials, especially nanosized bioconjugates or bioprobes, is extremely important for nanobiotechnology but is also very difficult. Consequently, any attempt to carry this out is highly significant. In the present work, we tried to employ FTIR spectroscopy and also biorecognition assay to characterize the WGA-TFNS. FTIR Confirmation. FTIR is a powerful characterization tool with the capability of discriminating among various types of covalent bonds. In the present work, FTIR was used to confirm the conjugation of WGA onto the St-AAm surface. As mentioned in the Experimental Procedures, due to the influence of nano-γ-Fe2O3 and QDs in WGA-TFNS on the characterization of WGA on the surface of WGA-TFNS, WGA-St-AAm was used as the substitute for WGA-TFNS in the characterization experiments. Figure 3 gives the FTIR spectra of H2N-St-AAm, HOOCSt-AAm, and WGA-St-AAm. For H2N-St-AAm, FTIR spectra showed a broad band near 3442 cm-1, which corresponds to the amine stretching vibration. After pretreating with succinic anhydride (spectrum b), there were two enhanced peaks at 1159
Figure 3. FTIR spectra of H2N-St-AAm (a), HOOC-St-AAm (b), and WGA-St-AAm (c).
Figure 4. Microscopic images of CM-CT-QDs in the presence of WGASt-AAm (a,b) or HOOC-St-AAm (c,d), CM-CT-QDs only (e,f) (as the control), and MAA-QDs in the presence of WGA-St-AAm (g,h): bright field (a,c,e,g); fluorescence (b,d,f,h).
and 1072 cm-1 associated with the stretching vibration of the C–N bond of the amide group, showing that succinic anhydride had reacted with H2N-St-AAm to simultaneously produce HOOC-St-AAm with more amide linkages and carboxyl groups on the surface of the nanospheres for further binding to WGA. For all the three kinds of nanosphere samples, two peaks of phenyl out-of-plane bending vibration at 754 and 698 cm-1 could be seen. However, for WGA-St-AAm (spectrum c), the peak at 860 cm-1 was obviously enhanced, which may be ascribed to the phenylalanine and tyrosine in the WGA (35).
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Figure 5. Microscopic images of WGA-TFNS having been incubated with Staphylococcus aureus (a,b) at 37 °C for 25 min. Magnetically separated on a magnetic scaffold, washed with 0.1 M Tris–HCl (pH 7.5, containing 1 mM CaCl2 and 1 mM MgCl2), and resuspended in 0.1 M Tris–HCl. Control experiment: WGA-TFNS and E. coli were incubated at 37 °C for 30 min. Bright field (a,c); fluorescence (b,d).
Biorecognition Assays. Since WGA can recognize chitin (36), which has four carbohydrate binding sites to chitin (1), carboxymethyl chitin-modified QDs (37) (CM-CT-QDs) can be an ideal probe for the presence of WGA on the surface of WGA-St-AAm. In order to further validate the conjugation of WGA onto the StAAm surface, CM-CT-QDs were added to WGA-St-AAm and HOOC-St-AAm, respectively (all of them were dispersed in 0.1 M PBS at pH 7.4), and then allowed to react for 30 min. The mixture was centrifuged to remove excess CM-CT-QDs and washed with 0.1 M PBS (pH 7.4). The final product was dissolved in 0.1 M PBS and imaged with a microscope. Because the isoelectric point of WGA is about pH 9, it will be positively charged at pH 7.4, while CM-CT-QDs are negatively charged. In order to confirm that the binding of carboxymethyl chitin to WGA results from specific recognition rather than simply electrostatic interaction, negatively charged mercaptoacetic acid modified QDs (MAA-QDs) was used to examine the electrostatic interaction with WGA-St-AAm. As shown in Figure 4, when WGA-St-AAm reacted with CMCT-QDs, the apparent aggregation of WGA-St-AAm was observed in the same field of vision whether in bright or fluorescent field (Figure 4a,b). The pattern (Figure 4b) of fluorescence from CM-CT-QDs evidently indicates that the chitin of CM-CT-QDs had bound to WGA on the WGA-StAAm surface, further resulting in aggregation due to synchronous exertion of multiple binding sites of WGA. But under the same conditions, no aggregation of HOOC-St-AAm was found (Figure 4c), and also no fluorescence from CM-CT-QDs was observed (Figure 4d), suggesting that CM-CT-QDs could not interact with HOOC-St-AAm without WGA. On the other hand, MAA-QDs could only make WGA-St-AAm slightly aggregate, not so remarkably as with CM-CT-QDs, and no fluorescence from MAA-QDs was observed (Figure 4g,h). This is because MAA-QDs could not strongly bind to WGA-St-AAm and had been washed off. The particle size of CM-CT-QDs was much smaller than WGA-St-AAm. Therefore, CM-CT-QDs could not be observed in bright field. However, the fluorescence of CMCT-QDs could be seen clearly (Figure 4e,f), implying that CMCT-QDs were of good dispersivity without aggregation. These experiments confirmed that WGA was indeed present on the surface of WGA-St-AAm, namely, on the surface of WGA-TFNS. In order to reconfirm the conjugation of WGA onto the surface of WGA-TFNS, S. aureus was used. As can be seen from Figure 5a,b, the WGA-TFNS had agglutinated after S. aureus suspension was incubated with WGA-TFNS at 37 °C for 25 min, indicative of the presence of WGA on WGA-TFNS surface. It is the multiple binding sites of WGA that one WGA molecule can at most bind to four S. aureus cells, resulting in the agglutination. However, this agglutination phenomenon could not be observed with Escherichia coli (Figure 5c,d) because no N-acetylglucosamine is present on the Escherichia coli surface. Recognition of Human Prostate Carcinoma DU-145 Cells by WGA-TFNS. The four carbohydrate-binding sites of WGA can recognize sialic acid and N-acetylglucosamine. Therefore, WGA can accordingly recognize human prostate
Figure 6. Microscopic images for the recognition of human prostate carcinoma DU-145 cells by WGA-TFNS under an incubation of 30 min with subsequent magnetic scaffold separation (a,b). Control experiment (c–f): DU-145 cells incubated with DBA-TFNS (c,d) and HOOC-BFNS (e,f) for 30 min with magnetic separation. Fluorescence (a,c,e); bright field (b,d,f).
carcinoma DU-145 cells because they can express sialic acid and N-acetylglucosamine on their surface (38). Thus, the recognition of DU-145 cells by WGA-TFNS was expected. Figure 6 shows that, after 30 min incubation with WGA-TFNS and subsequent magnetic isolation, the cells had been collected and separated (Figure 6a,b). Accordingly, the cells captured by WGA-TFNS could fluoresce orange (with a maximum emission wavelength of ca. 580 nm), which came from the WGA-TFNS binding to the cancer cells. Dolichos biflorus agglutinin (DBA) was used for the control, which is a glycoprotein with a molecular weight of about 120 000 and consists of 4 subunits of approximately equal size. This lectin has an isoelectric point of about pH 5.5 and 2 binding sites which are specific for glycoconjugates containing N-acetyl-D-galactosamine (GalNAc) in the terminal position (39). As reported in the literature (38), DBA did not have specificity for DU-145 cells; thus, DBATFNS could not capture DU-145 cells as shown in Figure 6c and d. At the same time, COOH-BFNS without WGA cannot recognize DU-145 cells as well (Figure 6e,f). Clearly, WGATFNS with WGA on the nanosphere surface can indeed recognize specifically DU-145 cells within 30 min. In particular, the cells captured by WGA-TFNS can be isolated readily with
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investigate the mechanism of killing cancer cells by lectin and drug release.
CONCLUSION
Figure 7. Histogram for capturing capacity of WGA-TFNS.
A simple and convenient strategy has been put forward to fabricate smart WGA-TFNS with excellent fluorescence, magnetism, and recognition of cancer cells surface-expressed with sialic acid and N-acetylglucosamine, which can be easily manipulated, tracked, and conveniently used to capture target cancer cells. WGA-TFNS have the specificity to recognize DU145 cells, but this is not the case for DBA-TFNS. They have good capture capacity for DU-145 and no cytotoxicity on DU145 in the present case. The success in capturing DU-145 cells by the WGA-TFNS has opened up a new field: visualizeable and recognizable manipulation and sorting of cells. Alterable functional groups on the BFNS surface such as -NH2 or -COOH will facilitate their bioconjugation with a variety of biomolecules on demand for the purposes of bioanalysis, biomedical imaging, diagnosis, and combinatorial screening of drugs. Among all the possible applications, simultaneous sorting of different categories of target cells will be the most fascinating, based on multicolor labeling by embedding different sizes and/or types of QDs in different batches of the trifunctional nanospheres.
ACKNOWLEDGMENT
Figure 8. Cytotoxicity of WGA-TFNS examined by Trypan blue dye exclusion assay.
a magnetic scaffold. It is worth pointing out that the magnetic separation is very fast. It only takes tens of seconds to collect the WGA-TFNS-captured cells. The capacity of WGA-TFNS to capture DU-145 cells is shown in Figure 7. About 1.06 × 105 DU-145 cells were captured by only 0.33 mg of WGATFNS. By increasing the WGA-TFNS concentration, the number of captured DU-145 cells increased. The cytotoxicity of WGA-TFNS was examined by Trypan blue dye exclusion assay (40). As shown in Figure 8, the cell viability was almost the same both in the presence of WGA-TFNS and in their absence for the control, indicating that WGA-TFNS have scarcely any cytotoxicity on DU-145 cells at WGA concentrations below 2.0 mg/mL and with 30 min incubation. In general, the relative abundances and branch structures of glycans on the membrane surface of cancer cells are often altered compared with normal cells. These alterations in glycosylation may be indicative of the different stages of the disease, thus being useful for diagnosis. Glycosylation changes in human carcinomas are thought to contribute to the malignant phenotype observed downstream of certain oncogenic events (41). Glycans are also the key structures involved in biological processes such as cell attachment, migration, and invasion. Information coded on cell surface glycans is frequently deciphered by proteins, such as lectins, that recognize specific carbohydrate topology (42). So, lectins always play an important role in the identification of glycoprotein and the phases of disease, which are very useful for disease diagnosis. Mo and Lim have successfully modified poly(D,L-lactide-co-glycolide) particles with WGA to interact with A549 cells (2) and made use of lectin receptormediated endocytosis for drug release (5) based on its capability of binding to membrane-bound carbohydrates. Heinrich et al. (43) found that lectin can kill cancer cells, so lectin could be used as a drug candidate for cancer therapy. Hopefully, WGATFNS could be used to capture and separate cancer cells, and
This work was supported by the National Science Fund for Distinguished Young Scholars (no. 20025311), the National Key Scientific Program - Nanoscience and Nanotechnology (no. 2006CB933100), the Science Fund for Creative Research Groups of NSFC (no. 20621502), the 863 Program (no. 2006AA03Z320), the National Natural Science Foundation of China (grant nos. 20505001 and 30570490), the Ministry of Education (nos. 306011 and IRT0543) and Beijing Institute of Technology Fund for Excellent Youth (000Y06-24). The authors thank Professor Dao-Cheng Wu for his helpful discussion and Dr. Wei-Hua Huang for FL imaging. W.Z. thanks EPSRC for a studentship to CD. Supporting Information Available: A comparison of the difference in recognition of DU-145 cells among WGA-TFNS, DBA-TFNS, and HOOC-BFNS; the cytotoxicity of WGA. This material is available free of charge via the Internet at http:// pubs.acs.org/BC.
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