Biotinylated Glyco-Functionalized Quantum Dots: Synthesis

994

Bioconjugate Chem. 2009, 20, 994–1001

Biotinylated Glyco-Functionalized Quantum Dots: Synthesis, Characterization, and Cytotoxicity Studies Xiaoze Jiang, Marya Ahmed, Zhicheng Deng, and Ravin Narain* Department of Chemistry and Biochemistry, Biomolecular Sciences Program, Laurentian University, 935, Ramsey Lake Road, Sudbury, Ontario P3E 2C6, Canada. Received January 13, 2009; Revised Manuscript Received April 14, 2009

Quantum dots (QDs) containing surface carboxylic groups have been successfully modified using biotinylated glycopolymer and carbohydrate/biotin reagents via EDC coupling. The biotinylated glycopolymer was synthesized in controlled dimension via the reversible addition-fragmentation chain transfer (RAFT) polymerization of the three monomers containing biotin, sugar, and amine groups as pendent groups, respectively. The modified QDs were analyzed by dynamic light scattering and fluorescence spectrophotometry, and the data revealed no change in the physical properties of QDs after surface modification. Furthermore, the surface modified QDs showed excellent water solubility and colloidal stability. Subsequently, the availability of the biotin ligand on the surface of functionalized QDs was quantified using 4-hydroxyazobenzene 2-carboxylic acid (HABA)/avidin binding assay. Cell viability studies revealed that the cytotoxicity of QDs after surface functionalization is improved and that the biotinylated glycopolymer modified QDs showed an enhancement in biocompatibility as compared to that of the original QDs. The biotinylated glyco-functionalized quantum dots may act as new suitable fluorescent probes in biomedical applications.

INTRODUCTION Semiconductor nanocrystals (usually called quantum dots, QDs) have been receiving enormous attention in recent years (1-6). QDs display unique size dependent optical properties for potential applications from electronic to biomedical fields. In the field of biomedicine, the high luminescence, single excitation narrow emission, and low photobleaching properties of high quality water-soluble QDs with low toxicity are exploited for in ViVo labeling/imaging of cells (7-11), and their surfaces have been functionalized with DNA (12-14), peptides (15, 16), proteins (5, 6, 17, 18), and other small ligands (19-21) to realize their applications as a new class of size-tunable optical probes. Several strategies have previously been reported in the surface functionalization of water-soluble QDs with surface functional groups (22-27). The promised method generally used for the functionalization of QDs is encapsulating the QDs with watersoluble polymer chains (26, 27) or charged proteins (18), which improve their solubility, reduce toxicity, and essentially preserve their initial optical properties in physiological conditions. As the main application of functionalized QDs concern the labeling of cells, specific ligands have to be incorporated for site specific binding of the functionalized QDs, and the cytotoxicity of QDs have to be considered as well. Protein-carbohydrate interactions are involved in a wide variety of cellular recognition processes including cell growth regulation, differentiation, adhesion, cancer cell metastasis, cellular trafficking, inflammation by bacteria and viruses, and immune response (28, 29). However, individual carbohydrateprotein interactions are generally weak, and multivalent forms of carbohydrate ligands, such as glycopolymers (30-34), glycodendrimers (35-37), or glyconanoparticles (38, 39), have been used to enhance them through cooperative multiple interactions. Therefore, the functionalization of QDs with carbohydrates is of interest for the study of carbohydrate-cell * Corresponding author. Tel: 1 705-6751151 (2186). Fax: 1 7056754844. E-mail: [email protected].

interactions. The incorporation of carbohydrates on the surface of the QDs is also expected to improve the solubility and biocompatibility, and reduce the toxicity of fluorescent nanocrystals. Sun and co-workers (20) were the first to report glycofunctionalized QDs with biotin end-terminated glycopolymers synthesized via a cyanoxyl-mediated free radical polymerization. However, the glycopolymers with free hydroxyl groups were obtained via polymerization of the protected glycomonomer and deprotection after polymerization. Recently, Babu et al. (19) reported the functionalization of QDs with several carbohydrates bearing reactive thiol groups at the end chains in aqueous solution via the ligand exchange method, and the special binding of the fluorescent glyconanoparticles to glycoproteins was studied. However, in both of the reports, the biocompatibility of the carbohydrate functionalized QDs, which is important for potential biomedical applications, is still to be investigated. Biotinylated QDs have also been synthesized for the study of site-specific biomolecular interactions of biotin and avidin as a ligand-receptor pair, which was widely used in the fields of biology and medicine (40, 41). Two new ligands consisting of thiol and biotin terminal groups were synthesized to functionalize the surface of QDs reported by Reiss et al. (7) Another new series of ligands comprising a poly(ethylene glycol) chain, a dihydrolipoic acid, and a potential biological functional group such as biotin were designed and prepared by Mattoussi and co-workers (9). However, both of the biotinylated QDs were prepared via the ligand exchange method, which usually changes the optical properties of original QDs, and the ligands used generally need complicated synthesis. Therefore, designing welldefined biotinylated glycopolymers bearing biotin and carbohydrates moieties as pendent groups is interesting for the fabrication of high quality water-soluble and bioactive QDs with low toxicity. In order to maximize the biological functions of the QDs, we herein report two different strategies to prepare stable surface functionalized QDs with both biotin and carbohydrates moieties. Our motivation here is to incorporate carbohydrate and biotin moieties on the surface of QDs for improving biocompatibility

10.1021/bc800566f CCC: $40.75  2009 American Chemical Society Published on Web 04/29/2009

Biotinylated Glyco-Functionalized Quantum Dots

and cell viability of the original QDs. Two strategies are used to functionalize the surface of original carboxyl-capped QDs, and the bioavailability of the biotin ligands on the surface of functionalized QDs is quantified. The cytotoxicity of biotinylated glyco-functionalized QDs is clearly reduced as shown by MTT assay results.

EXPERIMENTAL SECTION Materials. 2-Aminoethyl methacrylamide hydrochloride (AEMA) (42, 43), gluconamidoethyl methacrylamide hydrochloride (GAEMA) (44), and S,S′-bis(R,R′-dimethyl-R′′-acetic acid) trithiocarbonate (CTA1) (45) were synthesized as previously described. 4,4′-Azobis(4-cyanovaleric acid) (ACVA, 97%) was purchased from Acros Organics and used as received. 1-Ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were purchased from Sigma-Aldrich and used as received. The synthesis and characterization of biotinyl-N-hydroxysuccinimide ester (biotinNHS), N-(2-aminoethyl) biotinamide (biotin-NH2), N-(2aminoethyl) gluconamide hydrochloride (sugar-NH2), and biotinyl2-aminoethyl methacrylamide hydrochloride (BAEMA) are given in Supporting Information. Quantum dots (yellow color, carboxy-functionalized CdScapped CdTe nanoparticle solution, sodium as carboxyl counterion; 20 mg/mL in water, and emission maxima wavelength and full-width at half-maximum (FWHM) are 563 and 90 nm at 350 nm of excitation, respectively) with surface carboxylic groups were obtained from Northern Nanotechnologies Inc., and their detailed information could be checked at https://store. vivenano.com/library/Qdots_specs.pdf. Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum, phosphate buffered solution at pH 7.4 (DPBS), trypsin, and a (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a tetrazole) assay kit (MTT assay kit) were purchased from SigmaAldrich Chemicals. Preparation of P(GAEMA-stat-AEMA-stat-BAEMA) Copolymer via RAFT Polymerization. The RAFT polymerization was achieved at 70 °C, employing ACVA as the initiator and CTA1 as the chain transfer agent. A typical protocol was as follows. In a 10 mL flask, GAEMA (0.5 g, 1.46 mmol) and BAEMA (0.21 g, 0.54 mmol) were dissolved in distilled water (3 mL) at 40 °C and then cooled to room temperature. AEMA (0.18 g, 1.09 mmol, dissolved in 1.0 mL water) and the mixture of ACVA (2.0 mg, 0.007 mmol) and CTA1 (10 mg, 0.035 mmol) dissolved into 2 mL of dioxane were added into the above solution. After degassing via three freeze-pump-thaw cycles, the flask was placed in an oil bath preheated at 70 °C to start the polymerization. After 18 h, the flask was placed into liquid nitrogen to quench the polymerization. The mixture was diluted with water and then precipitated into an excess of isopropanol. This purification cycle was repeated twice. The obtained slightly yellow powder (0.60 g, 67% yield) was dried in a vacuum oven overnight at room temperature. EDC Coupling. Quantum dots solution (1 mL) was added to PBS buffer solution (pH 7.4, 4 mL) of EDC (2.6 mg) and NHS (1.5 mg). The reaction mixture was stirred for 30 min at room temperature. Then the P(GAEMA-stat-AEMA-statBAEMA) copolymer (10.0 mg in 35 mL of PBS buffer solution) or the mixture of sugar-NH2 (2.6 mg, dissolved in 34 mL of PBS buffer solution) and biotin-NH2 (1.1 mg in 1 mL of DMF) was added to the above PBS buffer solution. After stirring the reaction mixture overnight at room temperature, the mixture was purified by dialysis against distilled water for 2 days using the dialysis membrane molecular weight cut-off (MWCO) of 12,000-14,000. The desired QDs were freeze-dried under vacuum for 2 days. Instrumentation. 1H NMR spectra of the monomers and polymers were recorded on a Varian 200 MHz instrument.

Bioconjugate Chem., Vol. 20, No. 5, 2009 995

Aqueous gel permeation chromatography analysis was performed on a Viscotek Instrument using a 0.5 M sodium acetate/ acetic acid buffer as eluent, two Waters WAT011545 columns, and a flow rate of 1.0 mL/min. Six near-monodisperse PEO standards (Mp ) 1,010-101,200 g mol-1) were used for calibration. The products were analyzed by an Agilent HPLC 1100 interfaced with an Electro-Spray Ionization Agilent Mass Spectrometer Model 6120 with a Chemstation data system LCMSD B.03.01. Dynamic light scattering (DLS) was performed at room temperature using a Viscotek DLS instrument with an He-Ne laser at a wavelength of 632 nm and a Pelletier temperature controller. The surface functionalized quantum dot aqueous solutions (20 nM) were filtered through Millipore membranes (0.45 µm pore size). The data was recorded with OmniSize Software. The 4-hydroxyazobenzene 2-carboxylic acid (HABA)/avidin binding assay was performed as described by Green group (46, 47). UV-visible absorption spectra were recorded on a Cary UV 100 spectrophotometer from the aqueous solution of surface functionalized quantum dots at room temperature. The absorbance values at 500 nm for the HABA/avidin reagent and for the solution after the addition of surface functionalized quantum dots were monitored. Before the samples were tested, the solutions were filtered through Millipore membranes (0.45 µm pore size) and then concentrated or diluted to the appropriate concentration. The change of absorbance at 500 nm of HABA/ avidin and after HABA/avidin added surface functionalized quantum dots should be at 0.1-0.4. Fluorescence measurements were performed on an OLISRSM1000 (Desa rapid-scanning monochromator spectrophotometry system). The spectra were recorded in the wavelength range of 500-650 nm upon excitation at 490 nm; a 1.00 cm path length rectangular quartz cell was used for this study. Very dilute solutions of surface functionalized quantum dots (20 nM) were used in the experiment. Cell viability/cytotoxicity of quantum dots was characterized using an MTT (3-(4,5-bimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a tetrazole) assay. HeLa cells were plated in a 96-well plate at a density of 15000 cells/well in 100 µL of growth medium consisting of Dulbecco’s modified Eagle’s medium (DMEM) with 5% fetal bovine serum and antibiotics (100U/mL penicillin and 100 µg/mL streptomycin). Cells were incubated for 24 h at 37 °C and 5% CO2, after which time the growth medium was replaced with DMEM medium containing different concentrations of quantum dots. The preparation of QD stock solutions was as follows. All aqueous quantum dot solutions were dissolved into Dulbecco’s phosphate buffer at a final concentration of 32 nM. The samples were autoclaved, and different volumes of prepared solution were added into different volumes of DMEM medium to obtain final concentrations of 2, 10, and 20 nM of QDs in the medium. The cells were further incubated for 24 h under the same conditions, after which 25 µL of MTT dye was added to each well, and the plate was incubated for 2 h. Next, 100 µL of MTT lysis solution was added to each well, and cells were incubated overnight. Absorbance was measured at 570 nm (Power wave X plate reader). Survival percentage was calculated by comparison to blank cells (100% survival). Confocal microsocopy was performed to determine the uptake of QDs by HeLa cells. HeLa cells were grown in Dubelcco’s modified Eagle’s medium, DMEM, containing 10% fetal bovine serum, and 1% penicillin/streptomycin, in a humidified atmosphere with 5% CO2 at 37 °C. Upon 80% confluency, cells were trypsinized and neutralized with an equal amount of medium. The cells were then reseeded in a 6 well plate, in which each

996 Bioconjugate Chem., Vol. 20, No. 5, 2009

Jiang et al.

Scheme 1. Schematic Illustration of the Synthesis of N-(2-Aminoethyl)gluconamide Hydrochloride (Sugar-NH2) and N-(2-Aminoethyl)biotinamide (Biotin-NH2)

well contained sterile glass coverslips. The plate was incubated in a humidified atmosphere at 37 °C, and upon 80% confluency, 75 nM QDs containing medium was introduced in chambered cover glass. The cells were further incubated for 3 h at 37 °C. The growth medium was then removed, and the cells were washed several times with phosphate buffered saline. The cells were fixed with 4% paraformaldehyde solution at room temperature for 5 min, followed by washing with PBS. The cover glass was then mounted on a microscopic glass slide and was studied under a microscope. The fluorescent images were obtained using a Zeiss LSM 510 Meta confocal microscope with a 63× objective. The wavelengths of excitation and emission used were 490 and 510 nm, respectively.

RESULTS AND DISCUSSION Quantum dots (QDs) have attracted tremendous interest because of their remarkable and unique properties such as photochemical stability, size dependent broad absorption, very high extinction coefficients, and high fluorescence quantum yield (1, 2, 48, 49). Such features make the QDs an indispensable tool for use in biomedical research, especially for biological assays and imaging. Recent advances in QD nanoparticles have led to the development of high-quality watersoluble biotin or sugar functionalized QDs to reduce the toxicity of QDs for biomedical applications (7, 20). To the best of our knowledge, there is only one report that describes the functionalization of QDs using both biotin and sugar groups (20). However, the reported method requires a multiple step synthetic process, and therefore, our goal was to find a simple method to fabricate biotin and sugar functionalized QDs without protecting group chemistry. The original quantum dots (yellow color) used in this study were donated by Northern Nanotechnologies, which were carboxy-functionalized in CdS-capped CdTe nanoparticle solution, with sodium as carboxy counterion, and stabilized in aqueous solution by the carboxyl terminated polymer chains. The polymers improve their water solubility in basic conditions and provide the surface for further functionalization. In order to obtain the surface biotin and sugar functionalized QDs from the carboxyl-capped QDs, two methods could be used. The first approach is to modify the COOH groups with small biotin and carbohydrate molecules bearing amine groups. Alternatively, the QD surface could be modified with biotinylated glycopolymers bearing biotin, amine, and carbohydrate as pendent moieties.

Synthesis of Biotin-NH2 and Sugar-NH2. Although, various biotin functionalized QDs have been reported in the literature, very few studies deal with the direct functionalization of the QD surface. The general approach deals with the fabrication of QDs with avidin groups, which are then conjugated with biotin to form the desired QDs. Recently, biotinylated CdSe/ZnSe nanocrystals were directly synthesized by Reiss et al. (7) with two new ligands consisting of a thiolated diethylene glycol derivative and an alkylthiol substituted biotin molecule by the ligand exchange method. They further demonstrated that functionalized QDs could be used for special fluorescent labeling. However, the fabrication process of two ligands needs multistep synthetic pathways in their study, and the ligand exchange method usually changes the optical properties of original QDs. Therefore, we have aimed at the synthesis of biotin and sugar reagents via a simple and straightforward method to obtain the biotinylated glyco-functionalized QDs. Herein, the biotin bearing terminal amino group was fabricated via a two-step synthesis to functionalize the carboxylcapped QDs from D-biotin. The D-biotin was first activated by the NHS to form biotin-NHS and then reacted with ethylenediamine to obtain biotin-NH2 as shown in Scheme 1a. In order to minimize side reactions in the fabrication process, the DMF solution of BNHS was added dropwise into the flask containing ethylenediamine to avoid any possible side reactions. The product was characterized by 1H NMR in d6-DMSO solvent and mass spectrometry to confirm the formation of the desired product (Figures S1 and S2, Supporting Information). Similarly, the sugar bearing terminal amino group (sugarNH2) was synthesized by the reaction of D-(+)-gluconic acid δ-lactone with ethylenediamine as shown in Scheme 1b. A sticky yellow product was obtained in high yield (∼90%), and the chemical structure was confirmed by mass spectrometry (as shown in Figure S3 in Supporting Information). Synthesis of Monomers and RAFT Polymerization. Quantum dots were usually encapsulated with water-soluble polymer chains to improve their water solubility and reduce their toxicity. The polymers bearing the sugar or biotin groups were found to be ideal to enhance biocompatibility and for their potential use in biosensing applications. These new types of nanoparticles linked with bioactive groups will maintain the optical properties of original QDs and offer new opportunities for multicolor imaging of molecular targets in living cells. We have therefore synthesized the monomers bearing sugar or biotin moieties and



Scheme 2. Schematic Illustration of the Synthesis of the 2-Gluconamidoethyl Methacrylamide (GAMEA) Monomer and the P(GAEMA-stat-AEMA-stat-BAEMA) Copolymers

the monomer containing amino groups for realizing the possibility to link the polymer chain onto the surface of original QDs. The detailed synthesis process of the amine-based monomer (AEMA) and carbohydrate-containing monomer (GAEMA) has been reported by our group (42-44). Similarly, a novel biotincontaining monomer, BAEMA, was prepared by the reaction of biotin-NHS with AEMA. The structure has been confirmed via 1H NMR spectroscopy and mass spectrometry (as shown in Figures S1d and S4, Supporting Information). Reversible addition-fragmentation chain transfer polymerization (RAFT) is a highly versatile method as it works well both in aqueous and organic media for the synthesis of polymers with controlled dimensions (50-54). The controlled RAFT polymerizations of AEMA and GAEMA monomers in aqueous media have been well documented in our group (42, 44). PBAEMA could also be obtained via RAFT polymerization for the characteristic RAFT processes. In preliminary experiments, RAFT polymerization of BAEMA was attempted in 1,4-dioxane and water (v/v ) 1:2) at 70 °C using CTA 1 as the chain transfer agent. The PBAEMA homopolymer could be obtained with narrow polydispersity (less than 1.3), but the conversion is very low (about 30% calculated from 1H NMR spectroscopy) even after 18 h. This may be related to the hindrance effect of the biotin group in the RAFT process. In our case, our aim is to obtain the copolymer-bearing biotin groups to functionalize desired QDs. As BAEMA was structurally similar to AEMA and GAEMA, the statistical RAFT copolymerization of AEMA, GAEMA, and BAEMA using CTA 1 as the RAFT agent was quite expected, leading to the formation of the P(GAEMAstat-AEMA-stat-BAEMA) copolymer (Scheme 2). Polymerization was carried out using CTA1 as the chain transfer agent and ACVA as the initiator. The ratio of CTA to initiator was set to 5. The AEMA monomer was used in the protonated form during polymerization to prevent any side

reactions of the primary amino group to the chain transfer groups. Furthermore, it has been demonstrated in previous reports that the amine groups of the AEMA monomer in the protonated form have no influence on the RAFT process (42, 43). The carbohydrate monomer used, GAEMA, is a kind of acrylamide monomer and is very stable in aqueous solution. After polymerization for 18 h, the final statistical glycopolymer was obtained and denoted P(GAEMA36-stat-AEMA24-statBAEMA4.4) from the 1H NMR and HABA/avidin binding assay results, and the GPC trace in Figure S5b (Supporting Information) shows a monomodal peak with a very low polydispersity index (1.19), which has a Mn about 12,900 g/mol using polyethylene oxide as standards. The details are given in Supporting Information. Bioconjugated Quantum Dots via Covalent Functionalization. Water-soluble QDs (yellow color) containing surface carboxylic groups were used in this study. The QDs are originally dispersed in basic aqueous solution, but they rapidly lost fluorescence below pH 6-6.5. Some of the carboxylic groups on the surface of the QDs are activated using the NHS/EDC coupling method once the pH of the QDs solution was fixed at 7.4 using PBS and subsequently reacted with the biotinylated glycopolymer or the mixture of biotin-NH2 and sugar-NH2 as shown in Figure 1. The primary amino group on the statistical copolymer or biotin and sugar reagents was targeted to react with the activated carboxylic groups on the QDs. Since we are only aiming at modifying the surface carboxyl groups of the QDs, we do not expect a significant change in the quantum yield of the original QDs. Fluorescence spectra were recorded on the original QDs, biotin and sugar functionalized QDs, and biotinylated glycopolymer functionalized QDs. Both surface functionalized QDs still show strong fluorescence indicating that the surface modification did not cause a significant change in the optical properties of the original QDs (Figure 2), which is as expected.


Jiang et al.

Figure 1. Schematic illustration of the fabrication of biotin and sugar surface functionalized or glycopolymer functionalized QDs.

Figure 2. Emission spectra of original QDs, biotin and sugar functionalized QDs, and biotinylated glycopolymer functionalized QDs, where the excitation wavelength is at 490 nm.

After surface functionalization, the QDs were further investigated by dynamic light scattering (DLS). Figure 3 shows the number size distribution of original QDs, biotin and sugar functionalized QDs, and biotinylated glycopolymer functionalized QDs. The size distributions of both surface functionalized QDs are relatively narrow (less than 0.15), and the size of biotin and sugar functionalized QDs is similar to that of original QDs, which further confirms that surface functionalization has no influence on the physical states of original QDs. The glycopolymer functionalized QDs revealed a larger size (10-11 nm), which confirms the successful functionalization of the glycopolymer to the QDs. It should be noted that the covalent attachment of the RAFT copolymer to a carboxylated polymer chain coating already present on the surface of the original QDs causes a significant increase in the size of QDs. The DLS data and fluorescence spectra confirm that surface functionalization of QDs does not alter their optical properties. The functionalized QDs are stable, and no precipitates were observed even after several weeks when dispersed in aqueous basic solution condition. This is not considered as a significant change in the water solubility of surface functionalized QDs, when compared to that of the original QDs. However, when the solution pH is changed to acidic conditions (pH 5-6) the surface functionalized QDs remain stable and maintain their fluorescence even after two weeks. It clearly shows that the QDs after surface modification have excellent water solubility and colloidal stability compared to those of the original QDs over a broad pH range. HABA/Avidin Binding Assay. After EDC coupling, the surface of functionalized QDs must contain the biotin groups, but the amount of surface available biotin groups was difficult to calculate directly especially for the surface modification

Figure 3. DLS number average distribution of original QDs, biotin and sugar functionalized QDs, and biotinylated glycopolymer functionalized QDs.

of QDs using the synthesized glycopolymer. To prevent possible flocculation of the QDs, the coupling reaction was carried out under high dilution to decrease the possibility of side reactions, and the amount and bioavailability of biotin presented from the functionalized QDs was evaluated and quantified using the HABA/avidin binding assay. HABA is a dye that binds to avidin, and when HABA is complexed with avidin in water, the aqueous solution has a maximum absorbance at 500 nm. Upon addition of biotin or biotinylated reagents to the HABA/avidin solution, HABA is displaced quantitatively by available biotin, as avidin’s affinity for biotin (Kd ) 10-15 M) is much higher than that of HABA (Kd ) 10-6 M). This displacement can be quantitatively monitored by the decrease in UV absorbance at a wavelength of 500 nm. Upon addition of the functionalized QDs, the absorbance value at 500 nm of HABA clearly decreases, suggesting that HABA is displaced from the HABA/avidin complex by available biotin presented on the QD surface. The results of these analyses are shown in the overlaid UV-vis spectra


Figure 4. UV-vis spectra of the HABA/avidin reagent before and after the addition of surface functionalized QDs, biotinylated glycopolymer QDs, and sugar and biotin functionalized QDs, where the QDs concentrations were 0.02 µM and 0.1 µM for biotinylated glycopolymer QDs and sugar and biotin functionalized QDs, respectively.

(Figure 4). The amount of biotin in each biotinylated QD solution could be calculated by the following formula: ∆A500 ) 0.9 AHABA / avidin + AQDs - AHABA / avidin+QDs µmol biotin/mL ) 10(∆A500 /34) where the dilution factor of HABA/avidin is 0.9, the mM extinction coefficient of biotin at 500 nm is 34, and the dilution factor of QDs is 10. The aqueous solution of QDs before or after surface functionalization is a colored solution and has an absorbance of about 0.238 at 500 nm after surface functionalization of QDs using polymer chains. So the total decreased absorbance of the HABA/ avidin complex after the addition of surface functionalized QDs should be considered the initial absorbance of QDs. Although the absorbance of HABA/avidin complex solution seems to increase after the addition of the glycopolymer functionalized QDs solution as shown in Figure 4, the total absorbance after mixing actually decreases, and this decrease can be calculated by the above formula to be 0.11. The amounts of available biotin per mL of QDs solution were thus calculated to be 1.29 µmol and 0.93 µmol for the glycopolymer functionalized QDs and biotin and sugar functionalized QDs, respectively. According to the concentration of original QDs, which is about 0.8 µM, the quantity of available biotin per QD could be calculated to be ∼1600 and ∼1100 for biotinylated glycopolymer functionalized QDs and biotin and sugar functionalized QDs, respectively. Cytotoxicity of the QD Solution. The actual application of QDs in biology needs to address the question about its toxicity.


Many methods including polymer or silica encapsulation have been reported to reduce the toxicity of QDs, and it has been shown that the toxicity of QDs can be reduced by modifying the surface area of QDs (55, 56). In a recent study, biotin or carbohydrate coated QDs have been synthesized and are shown to be biocompatible. The main idea is to find new strategies that can encapsulate the QDs using biocompatible polymer chains to reduce toxicity. We have addressed this issue by surface functionalization of carboxylic acid coated QDs with polymer chains bearing a variety of biocompatible materials. The in Vitro cytotoxicity of the QDs is obtained using the MTT assay on the HeLa cell line. The assay determines the number of viable cells based on the mitochondrial activity of the cells. The MTT dye produces formazan crystals based on the mitochondrial activity of the cells. The MTT lysis buffer dissolves the formazan crystals produced, and their absorbance can be read at 570 nm, giving the approximate number of viable cells in each well. The wells that contained only the media were used as the negative control, and the wells containing untreated cells were used as the positive control. Each experiment was performed in triplicate and, % cell viability was obtained by QD treated cells/absorbance of positive control. Confocal microsocopy was performed to determine the uptake of QDs by HeLa cells. The fluorescent images obtained were overlapped on bright field images to determine the site of localization of QDs in the cells shown in Figure 5. The images show the uptake of QDs by cells, after 3 h of incubation with cells. The images obtained confirm that MTT data provide the toxicity of QDs after their uptake by the cell line. The QDs are found to be mostly localized in the endosomes of the cells, thus indicating that the possible mechanism of uptake is receptor mediated endocytosis. The cytotoxicity of QDs has been studied as a function of concentration. The biological and biomedical applications of QDs are highly dependent upon their biocompatibility. We therefore, studied the toxicity of QDs before and after surface functionalization at different concentrations. As shown in Figure 6, QDs were coated with sugar and biotin reagents, and glycopolymer P(GAEMA36-stat-AEMA24-stat-BAEMA4.4) individually, and their cytotoxicity values were compared to those of the original QDs. At very low concentration (2 nM), the cell viability of all QDs are found to be above 75%. Increasing the concentration of QDs up to 20 nM causes a significant increase in the toxicity of the original QDs, and the cell viability is only 59%. However, the cell viability for the glycopolymer functionalized QDs is 89% at this concentration. It is also apparent from the graph that biotin and sugar coated QDs also show

Figure 5. Representative fluorescent and white cell images of HELA cells incubated with (A) sugar and biotin functionalized QDs and (B) biotinylated glycopolymer functionalized QDs.


Figure 6. Percent cell viability/cytotoxicity as a function of different QD concentrations before and after surface functionalization.

concentration dependent cytotoxicity, which is comparable to that of the original QDs, whereas the cytotoxicity is negligible for glycopolymer stabilized QDs, regardless of concentration. Although, all of the molecules used for the surface functionalization of QDs are biocompatible, the 12.9 kDa glycopolymer seems to mask the surface of QDs more efficiently, thus accounting for the low toxicity, when compared to small molecules such as sugar and biotin coated QDs. The biotinylated glycopolymer functionalized QDs may be suitable for use in some potential biomedical applications. It should also be noted that the original QDs have been functionalized by water-soluble polymer chains, which also prevent the release of metal and increase the cell viability of QDs.

CONCLUSIONS We have successfully synthesized biotin and carbohydrate functionalized reagents for the surface functionalization of QDs. The modified QDs have a high affinity biotin ligand as well a high density of surface carbohydrate moieties. The surface modified QDs showed excellent optical properties and colloid stability compared to those of the original QDs at whole pH conditions. The amount of available biotin was quantified via the HABA/avidin binding assay, and surface functionalization using the glycopolymer chains bearing biocompatible biotin and carbohydrate moieties clearly reduces the cytotoxicity of original QDs. Therefore, the designed biotinylated glyco-functionalized QDs have high potential for applications in biodiagnostic devices or in ViVo cellular imaging. In our current case, the amount of carbohydrate moieties and their interaction with protein have not been studied. We will study the bioavailability of the surface carbohydrate moieties on the QDs for various carbohydrateprotein interactions in our next work, which is of considerable interest in biomedicine.

ACKNOWLEDGMENT This work was supported jointly by Ontario Centres of Excellence, Emerging Materials and Knowledge (OCE-EMK), and Northern Nanotechnologies Inc. Supporting Information Available: Synthesis of biotinNHS, biotin-NH2, sugar-NH2, BAEMA, and GAEMA as well as compound characterization data. This material is available free of charge via the Internet at http://pubs.acs.org.

LITERATURE CITED (1) Murray, C. B., Norris, D. J., and Bawendi, M. G. (1993) Synthesis and characterization of nearly monodisperse CdE (E ) sulfur, selenium, tellurium) semiconductor nanocrystallites. J. Am. Chem. Soc. 115, 8706–8715.

Jiang et al. (2) Alivisatos, A. P. (1996) Semiconductor clusters, nanocrystals, and quantum dots. Science 271, 933–937. (3) Moriarty, P. (2001) Nanostructured materials. Rep. Prog. Phys. 64, 297–381. (4) Efros, A. L., and Rosen, M. (2000) The electronic structure of semiconductor nanocrystals. Annu. ReV. Mater. Sci. 30, 475– 521. (5) Bruchez, M., Moronne, M., Gin, P., Weiss, S., and Alivisatos, A. P. (1998) Semiconductor nanocrystals as fluorescent biological labels. Science 281, 2013–2016. (6) Chan, W. C. W., and Nie, S. M. (1998) Quantum dot bioconjugates for ultrasensitive nonisotopic detection. Science 281, 2016–2018. (7) Charvet, N., Reiss, P., Roget, A., Dupuis, A., Gru¨nwald, D., Carayon, S., Chandezon, S., and Livache, T. (2004) Biotinylated CdSe/ZnSe nanocrystals for specific fluorescent labeling. J. Mater. Chem. 14, 2638–2642. (8) Murray, C. B., Kagan, C. R., and Bawendi, M. G. (2000) Synthesis and characterization of monodisperse nanocrystals and close-packed nanocrystal assembles. Annu. ReV. Mater. Sci. 30, 545–610. (9) Susumu, K., Uyeda, H. T., Medintz, I. L., Pons, T., Delehanty, J. B., and Mattoussi, H. (2007) Enhancing the stability and biological functionalities of quantum dots via compact multifunctional ligands. J. Am. Chem. Soc. 129, 13987–13996. (10) Qi, L., and Gao, X. (2008) Emerging application of quantum dots for drug delivery and therapy. Expert Opin. Drug DeliVery 5, 263–267. (11) Gao, X., Chan, W. C. W., and Nie, S. (2002) Quantum-dot nanocrystals for ultrasensitive biological labeling and multicolor optical encoding. J. Biomed. Opt. 7, 532–537. (12) Gill, R., Willner, I., Shweky, I., and Banin, U. (2005) Fluorescence resonance energy transfer in CdSe/ZnS-DNA conjugates: probing hybridization and DNA cleavage. J. Phys. Chem. B 109, 23715–23719. (13) Peng, H., Zhang, L., Kja¨llman, T. H. M., Soeller, C., and Travas-Sejdic, J. (2007) DNA hybridization detection with blue luminescent quantum dots and dye-labeled single-stranded DNA. J. Am. Chem. Soc. 129, 3048–3049. (14) Charkrabarti, R., and Klibanov, A. M. (2003) Nanocrystals modified with peptide nucleic acids (PNAs) for selective selfassembly and DNA detection. J. Am. Chem. Soc. 125, 2531– 12540. (15) Åkerman, M. E., Chan, W. C. W., Laakkonen, P., Bhatia, S. N., and Ruoslahti, E. (2002) Nanocrystal targeting in vivo. Proc. Natl. Acad. Sci. U.S.A. 99, 12617–12621. (16) Pinaud, F., King, D., Moore, H.-P., and Weiss, S. (2004) Bioactivation and cell targeting of semiconductor CdSe/ZnS nanocrystals with phytochelatin-related peptides. J. Am. Chem. Soc. 126, 6115–6123. (17) Winter, J. O., Liu, T. Y., Korgel, B. A., and Schmidt, C. E. (2001) Recognition molecule directed interfacing between semiconductor quantum dots and nerve cells. AdV. Mater. 13, 1673– 1677. (18) Mattoussi, H., Mauro, J. M., Goldman, E. M., Anderson, G. P., Sundar, V. C., Mikulec, F. V., and Bawendi, M. G. (2000) Selfassembly of CdSe-ZnS quantum dot bioconjugates using an engineered recombinant protein. J. Am. Chem. Soc. 122, 12142– 12150. (19) Babu, P., Sinha, S., and Surolia, A. (2007) Sugar-quantum dot conjugates for a selective and sensitive detection of lectins. Bioconjugate Chem. 18, 146–151. (20) Sun, X.-L., Cui, W., Haller, C., and Chaikof, E. L. (2004) Site-specific multivalent carbohydrate labeling of quantum dots and magnetic beads. ChemBioChem. 5, 1593–1596. (21) Rosenthal, S. J., Tomlinson, I., Adkins, E. M., Schroeter, S., Adams, S., Swafford, L., McBbride, J., Wang, Y., DeFelice, L. J., and Blakely, R. D. (2002) Targeting cell surface receptors with ligand-conjugated nanocrystals. J. Am. Chem. Soc. 124, 4586– 4594.

Biotinylated Glyco-Functionalized Quantum Dots (22) Wuister, S. F., Swart, I., Driel, F. V., Hickey, S. G., and Donega, C. D. M. (2003) Highly luminescent water-soluble CdTe quantum dots. Nano Lett. 3, 503–507. (23) Aldana, J., Wang, Y. A., and Peng, X. (2001) Photochemical instability of CdSe nanocrystals coated by hydrophilic thiols. J. Am. Chem. Soc. 123, 8844–8850. (24) Gerion, D., Pinaud, F., Williams, S. C., Parak, W. J., Zanchet, D., Weiss, S., and Alivisatos, A. P. (2001) Synthesis and properties of biocompatible water-soluble silica-coated CdSe/ ZnS semiconductor quantum dots. J. Phys. Chem. B 105, 8861– 8871. (25) Tan, W., Wang, K., He, X., Zhao, X. J., Drake, T., Wang, L., and Bagwe, R. P. (2004) Bionanotechnology based on silica nanoparticles. Med. Res. ReV. 24, 621–638. (26) Fan, H., Leve, E. M., Scullin, C., Gabaldon, J., Tallant, D., Bunge, S., Boyle, T., Wilson, M. C., and Brinker, C. J. (2005) Surfactant-assisted synthesis of water-soluble and biocompatible semiconductor quantum dot micelles. Nano Lett. 5, 645–648. (27) Gao, X., Cui, Y., Levenson, R. M., Chung, L. W. K., and Nie, S. (2004) In vivo cancer targeting and imaging with semiconductor quantum dots. Nat. Biotechnol. 22, 969–976. (28) Goldstein, I. J., Iyer, R. N., Smith, E. E., and So, L. L. (1967) Protein-carbohydrate cnteraction. XX. the Interaction of concanavalin A with sophorose and some of its derivatives. Biochemistry. 6, 2373–2377. (29) Hong, C.-H. (2003) Carbohydrate-Based Drug DiscoVery, Wiley-VCH, Weinheim, Germany. (30) Choi, S.-K., Mammen, M., and Whitesides, G. M. (1997) Generation and in situ evaluation of libraries of poly(acrylic acid) presenting sialosides as side chains as polyvalent inhibitors of influenza-mediated hemagglutination. J. Am. Chem. Soc. 119, 4103–4111. (31) Bovin, N. V., and Gabius, H.-J. (1995) Polymer-immobilized carbohydrate ligands: versatile chemical tools for biochemistry and medical sciences. Chem. Soc. ReV. 413–421. (32) Kiessling, L. L., Gestwicki, J. E., and Strong, L. E. (2000) Synthetic multivalent ligands in the exploration of cell-surface interactions. Curr. Opin. Chem. Biol. 4, 696–703. (33) Bertozzi, C. R., and Kiessling, L. L. (2001) Chemical glycobiology. Science 291, 2357–2364. (34) Gestwicki, J. E., and Kiessling, L. L. (2002) Inter-receptor communication through arrays of bacterial chemoreceptors. Nature 415, 81–84. (35) Roy, R. (1996) Syntheses and some applications of chemically defined multivalent glycoconjugates. Curr. Opin. Struct. Biol. 6, 692–702. (36) Ro¨kendorf, N., and Lindhorst, T. K. (2001) Glycodendrimers. Top. Curr. Chem. 217, 201–238. (37) Turnbull, W. B., Kalovidouris, S. A., and Stoddart, J. F. (2002) Large oligosaccharide-based glycodendrimers. Chem. Eur. J. 8, 2988–3000. (38) Otsuka, H., Akiyama, Y., Nagasaki, Y., and Kataoka, K. (2001) Quantitative and reversible lectin-induced association of gold nanoparticles modified with R-lactosyl-ω-mercapto-poly(ethylene glycol). J. Am. Chem. Soc. 123, 8226–8230. (39) Lin, C.-C., Yeh, Y.-C., Yang, C. Y., Chen, C.-L., Chen, G.F., Chen, C.-C., and Wu, Y.-C. (2002) Selective binding of mannose-encapsulated gold nanoparticles to type 1 Pili in Escherichia coli. J. Am. Chem. Soc. 124, 3508–3509.

Bioconjugate Chem., Vol. 20, No. 5, 2009 1001 (40) Wilchek, M., and Bayer, E. A. (1988) The avidin-biotin complex in bioanalytical applications. Anal. Biochem. 171, 1–32. (41) Wilchek, M., and Bayer, E. A. (1990) Introduction to avidinbiotin technology. Methods Enzymol. 184, 5–13. (42) Deng, Z.-C., Bouche´kif, H., Babooram, K., Housni, A., Choytun, N., and Narain, R. (2008) Facile synthesis of controlledstructure primary amine-based methacrylamide polymers via the reversible addition-fragmentation chain transfer process. J. Polym. Sci., Part A: Polym. Chem. 46, 4984–4996. (43) Li, Y., Lokitz, B. S., and McCormick, C. L. (2006) Thermally responsive vesicles and their structural “locking” through polyelectrolyte complex formation. Angew. Chem., Int. Ed. 118, 5924–5927. (44) Deng, Z.-C., Ahmed, M., and Narain, R. (2009) Novel welldefined glycopolymers synthesized via the reversible addition fragmentation chain transfer process in aqueous media. J. Polym. Sci., Part A: Polym. Chem. 47, 614–627. (45) Lai, J. T., Filla, D., and Shea, R. (2002) Functional polymers from novel carboxyl-terminated trithiocarbonates as highly efficient RAFT agents. Macromolecules 35, 6754–6756. (46) Green, N. M. (1965) A spectrophotometric assay for avidin and biotin based on binding of dyes by avidin. Biochem. J. 94, 23c–24c. (47) Green, N. M. (1970) Spectrophotometric determination of avidin and biotin. Methods Enzymol. 18, 418–424. (48) Chen, Y., and Rosenzweig, Z. (2002) Luminescent CdSe quantum dot doped stabilized micelles. Nano Lett. 2, 1299–1302. (49) Farmer, S. C., and Patten, T. E. (2001) Photoluminescent polymer/quantum dot composite nanoparticles. Chem. Mater. 13, 3920–3926. (50) Chiefari, J., Chong, Y. K., Ercole, F., Krstina, J., Jeffery, J., Le, T. P. T., Mayadunne, R. T. A., Meijs, G. F., Moad, C. L., Moad, G., Rizzardo, E., and Thang, S. H. (1998) Living freeradical polymerization by reversible addition-fragmentation chain transfer: the RAFT process. Macromolecules 31, 5559. (51) Moad, G., Rizzardo, E., and Thang, S. H. (2005) Living radical polymerization by the RAFT process. Aust. J. Chem. 58, 379– 410. (52) Donovan, M. S., Lowe, A. B., Sumerlin, B. S., and McCormick, C. L. (2002) Raft polymerization of N,N-dimethylacrylamide utilizing novel chain transfer agents tailored for high reinitiation efficiency and structural control. Macromolecules 35, 4123–4132. (53) Narain, R., Housni, A., Gody, G., Boulanger, P., Charreyre, M. T., and Delair, T. (2007) Preparation of biotinylated glyconanoparticles via a photochemical process and study of their bioconjugation to streptavidin. Langmuir 23, 12835–12841. (54) Spain, S. G., Albertin, L., and Cameron, N. R. (2006) Facile in situ preparation of biologically active multivalent glyconanoparticles. Chem. Commun. 4198–4200. (55) Hoshino, A., Fujioka, K., Oku, T., Suga, M., Sasaki, Y. F., Ohta, T., Yasuhara, M., Suzuki, K., and Yamamoto, K. (2004) Physicochemical properties and cellular toxicity of nanocrystal quantum dots depend on their surface modification. Nano Lett. 4, 2163–2169. (56) Duan, H., and Nie, S. (2007) Cell-penetrating quantum dots based on multivalent and endosome-disrupting surface coatings. J. Am. Chem. Soc. 129, 3333–3338. BC800566F

Biotinylated Glyco-Functionalized Quantum Dots: Synthesis

Recommend Documents