Surface Functionalization of Hydrophobic Nanocrystals with One

Prasanta Panja , Pradip Das , Kuheli Mandal , and Nikhil R. Jana. ACS Sustainable Chemistry & Engineering 2017 5 (6), 4879-4889. Abstract | Full Text ...
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Surface Functionalization of Hydrophobic Nanocrystals with One Particle per Micelle for Bioapplications Mingliang Deng,† Nina Tu,† Feng Bai,‡ and Leyu Wang†,* †

State Key Laboratory of Chemical Resource Engineering, School of Science, Beijing University of Chemical Technology, Beijing 100029, Peoples Republic China; ‡ Key Laboratory for Special Functional Materials of the Ministry of Education, Henan University, Kaifeng 475004, People′s Republic of China S Supporting Information *

ABSTRACT: A facile and general strategy was successfully developed for the surface modification of kinds of hydrophobic inorganic nanomaterials with various chemical compositions, shapes, and sizes. Via this ultrasonication assistant encapsulation technology, these hydrophobic inorganic nanocrystals were successfully encapsulated into the carboxylated phospholipids and polymers micelles with one particle per micelle. The surface modified nanocrystals were characterized by transmission electron microscopy (TEM), Fourier-transform infrared (FTIR), and thermogravimetric analysis (TGA). After encapsulation, the particle size, shape, and optical and magnetic properties were effectively retained. These functionalized nanocrystals are highly water-stable and biocompatible. After being bioconjugated with the antibodies, the functionalized quantum dots (QDs) have been successfully used as biolabels for targeted cell fluorescence imaging. KEYWORDS: hydrophobic nanocrystals, surface functionalization, cell imaging

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Gao and co-workers successfully functionalized the hydrophobic quantum dots with amphiphilic polymer and applied them for bioimaging.12,29−31 With the assistant of surfactants, Fan successfully encapsulated the hydrophobic gold nanoparticles and quantum dots into hydrophilic micelles.32,33 Based on the oil phase evaporation-induced self-assembly technology, hydrophobic nanocrystals can be successfully transferred into hydrophilic spheric clusters.34−38 However, the large size and highly cytotoxic hexadecyltrimethy ammonium bromide (CTAB) of these hydrophilic nanospheres partly limits their in vivo applications.39,40 Therefore, it would be of particular interest to develop a facile and general strategy to encapsulate the hydrophobic inorganic nanocrystals with various shape, size, and chemical compositions into hydrophilic micelles with one particle per micelle. Herein, we developed a method for the surface functionalization of hydrophobic NPs with an ultrasonication assistant encapsulation technology, by employing 1, 2-dioleoyl-snglycero-3-phosphoethanolamine-N-(succinyl) (carboxylated phospholipid) to graft onto the surface of hydrophobic NPs.

urface functionalization of hydrophobic inorganic nanoparticles (INPs) is prerequisite for biomedical applications, not only to render them reasonably water-stable and biocompatible but also to provide active sites for subsequent functional conjugation with biological or chemical moieties.1−9 Ligand exchange2,10,11 is supposed to be the most primary strategy for surface functionalization and phase transfer. In spite of its simplicity, however, the ligand exchange often compromises the luminescence efficiency and photochemical stability because the small molecule ligands tend to detach from the surface of the luminescent INPs. Silanization is another general approach for surface modification by coating an inorganic silica shell.12−14 Via the strong coordination interactions between metal cations and thiol groups, Yin and co-workers developed a facile and general strategy to assemble the hydrophobic nanoparticles onto the surface of functional silica spheres.13 In addition, Caruso and co-workers have developed a versatile layer-by-layer (LbL) strategy for the surface modification of nanoparticles and the fabrication of drug delivery capsule.15−23 They also successfully developed some facile strategies for the encapsulation of drugs or nanoparticles.23,24 Apart from the mentioned strategies, polymer or peptide coating has also become one of the most popular strategies for the surface modification of INPs.14,25−28 © 2012 American Chemical Society

Received: April 26, 2012 Revised: June 12, 2012 Published: June 12, 2012 2592

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The carboxylated phospholipid was selected as the coating material because of its outstanding amphiphilicity, biocompatibility, and bioconjugatability. To increase the water stability of the NPs and simultaneously decrease the nonspecific adsorption of biomolecules, the polyethylene-block-poly-(ethylene glycol) (PE-b-PEG) was also used as an assistant coating composition. Biocompatible N-lauroyl sarcosine sodium (NLSS) was used in place of CTAB, whose cytotoxicity has been identified,39,40 as a surfactant during the surface functionalization process. As shown in Scheme 1, with the Scheme 1. Surface Modification and Functionalization of NPs and Immunolabeling of HepG2 Liver Cancer Cells

Figure 1. TEM images and luminescence photographs of the nanocrystals before and after surface modification.

prone to aggregate on the interface between water and oil during the functionalization process. As shown in Figure 2, all three kinds of small particles were encapsulated into the phospholipid/polymer hybrid micelle and no aggregation was observed. In addition, the optical and magnetic properties were retained well after the surface modification. For example, after encapsulation, the shape and emission center of the fluorescence spectrum of ZnS:Mn2+ NPs have no change (Figure S1, Supporting Information). Only a slight decrease in the fluorescence intensity was observed under the identical excitation and at the same particle concentration. The results indicate that the optical property is well reserved after the surface modification. The versatility of this strategy was further identified by the test results on the large nanocrystals such as LaVO4:Eu3+ (∼26 nm) nanobricks,43 YPO4 (∼36 nm) nanoplates,44 and hydroxyapatite (∼150 nm)45 nanorods (Figure 2). In spite of large differences in particle size, shape, and chemical compositions, these nanocrystals were successfully immobilized in a single hydrophilic micelle, and demonstrated good water stability. As shown in the TEM images (Figure 2), the resulting core/shell nanocomposites are monodispersed and maintain discrete form without aggregation, which is highly desirable for bioapplications. FTIR Analysis of the Hydrophilic Nanocrystals. The coating shell on the inorganic nanoparticles was first investigated via the Fourier transform infrared (FTIR) spectroscopy. The N-lauroyl sarcosine sodium (NLSS) (spectrum A), 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(succinyl) (carboxylated phospholipid, PL) (spectrum B), oleic acid coated ZnS:Mn2+ quantum dots (spectrum C), and the phospholipid functionalized ZnS:Mn2+ quantum dots (spectrum D) were characterized, and the FTIR spectra are shown in Figure 3. As can be seen, the distinct difference between spectrum A and D is that the sharp and strong vibration peaks located at 3425, 1603, and 1392 cm−1 of NLSS (spectrum A) are not found in the spectrum of hydrophilic quantum dots (spectrum D). This indicates that the surfactant (NLSS) has already been removed after a thorough washing and cannot be detected on the surface of the hydrophilic nanoparticles. The vibration peaks in spectrum D (hydrophilic QDs) are in accordance with those in spectrum B (carboxylated

assistance of ultrasonication, the mixed chloroform solution of carboxylated phospholipids (PL), PE-b-PEG and hydrophobic inorganic NPs were transferred into the oil-in-water (O/W) micelle. After removing the chloroform via rotary evaporation, the PL and PE-b-PEG self-assembled into a thin layer on the nanoparticle surface, rendering the NPs not only water-soluble but also biocompatible and bioconjugatable.

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RESULTS AND DISCUSSION Transmission Electron Microscopy (TEM) Images. It is notable that this surface functionalization process is very rapid and simple, and the whole process can be finished in 20 min. As shown in Figure 1, the NaYF4:Yb3+-Er3+ upconverting nanocrystals with rodlike (∼24 nm) or spheric (∼15 nm) shape were encapsulated into the PL/PE-b-PEG hybrid micelle. No aggregation was observed, and the upconverting luminescence property was not influenced after the surface modification. Thereafter, we also successfully extended this method to the modification of LaF3:Ce3+-Tb3+ (∼10 nm) NPs, and the luminescence was not influenced, too. It is worth noting that only one fluoride NP is encapsulated in each lipid/polymer hybrid micelle. The shape, size, and chemical composition of the NPs has no influence on the surface functionalization. To investigate the universality of this facile modification strategy, we chose the small nanoparticles (∼7 nm) including gold,41 quantum dots (QDs),37 and magnetite42 NPs as the model because the small hydrophobic particles were highly 2593

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Figure 2. TEM images and optical photographs of the inorganic nanocrystals before and after surface modification.

Figure 4. TGA results for ZnS:Mn2+ QDs before (hydrophobic) and after (hydrophilic) surface modification.

Figure 3. FTIR spectra of (A) NLSS, (B) carboxylated phospholipids, (C) oleic acid coated ZnS:Mn2+, and (D) phospholipid functionalized ZnS:Mn2+ quantum dots.

modification) and hydrophilic (part a, after surface modification) QDs, respectively. The slight weight loss for hydrophobic QDs can be attributed to the decomposition of the coated oleic acids. Meanwhile, the sharp mass loss for hydrophilic QDs suggests the decomposition of the coating oleic acid, phospholipids, and block polymers on the particle surface. In addition, due to the hydrophilic coating shell, the hydrodynamic diameter of the QDs reaches c.a. 69.8 nm (Figure S3, Supporting Information). These results further suggest that the surface coating on the hydrophobic nanocrystals is successful. Due to the enriched carboxyl (in phospholipid) and hydroxyl (in PE-b-PEG) groups in the coating layer, these nanocomposites are found to be very stable in various aqueous solution including bovine serum albumin (BSA) solution, phosphate buffer solution (PBS), DMEM, and 1640 cell culture media (Figure S4, Supporting Information), which facilitates their chemical and biomedical applications. Cytotoxicity and Cell Imaging. To demonstrate their potential bioapplications, the surface functionalized QDs were further evaluated as cellular imaging probes by first examining their cytotoxic effects in a MTT cell proliferation assay, which showed that more than 94% of the HepG2 liver cancer cells survived at the particle concentration up to 100 μg/mL after incubation for 24 h (Figure 5). The QDs were bioconjugated with anti-α-fetoprotein (AFP) antibodies (Ab), which could specifically recognize the AFP (antigen) on the HepG2

phospholipids), primarily indicating that the phospholipids have been coated onto the nanoparticles. Compared spectrum C with D, the strong O−H and N−H vibration (around 3380 cm−1) and CO vibration (1730 cm−1) in the spectrum of hydrophilic QDs (spectrum D) were not found in the spectrum of oleic acid coated QDs (spectrum C), suggesting that the phospholipid coating is successful. It should be mentioned that the main vibration of the polyethylene-block-poly-(ethylene glycol) (PE-b-PEG) are O−H and C−H vibration, but it is not easy to be distinguished from the corresponding vibration of carboxylated phospholipids. Therefore, we can not identify the existence of PE-b-PEG on the surface of QDs after surface modification. However, if no PE-b-PEG was used in the surface functionalization, the water-stability of the QDs was poor, which confirmed in reverse the existence of PE-b-PEG on the particle surface. Therefore, the results forcefully indicate that the surface modification of hydrophobic nanocrystals is successful. Thermogravimetric Analysis (TGA). The hydrophilic shell on the nanoparticles was further identified with the test results of the energy dispersive spectrum (EDS) (Figure S2, Supporting Information) and thermogravimetric analysis (TGA) analysis (Figure 4). As shown in Figure 4, the weight loss is 24.6% and 57.7% for hydrophobic (part b, before surface 2594

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CONCLUSIONS

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MATERIALS AND METHODS

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In summary, we developed a facile and general strategy for the surface functionalization by grafting carboxylated phospholipids and amphiphilic polymers onto the surface of hydrophobic inorganic nanocrystals with various chemical compositions, shapes, and sizes. The nanoparticles that capped with carboxylated phospholipids and polymers hold their original properties with no shape and size change. In addition, because of the carboxylic group and PEG enriched hydrophilic shell, the functionalized nanocrystals are water-stable, biocompatible, and bioconjugatable with targeting biomolecules for cellular imaging. This simple and highly reproducible modification process can serve as a general protocol for the surface functionalization of hydrophobic inorganic nanocrystals, and it will draw wide attention in the field of nanobiotechnology and nanomedicine.

Figure 5. Cell viability tests of surface functionalized ZnS:Mn2+ quantum dots on HepG2 cell line at different concentration after incubation for 24 h (black) and 48 h (red). The results indicated that the as-obtained hydrophilic QDs were nontoxic (94% cells survived) even the concentration reached 100 μg/mL after incubation for 24 h. However, if the incubation time was prolonged to 48 h, 80% cells survived. When the concentration decreased to 60 μg/mL, about 90% cells were still alive after incubation for 48 h.

Chemicals and Reagents. The polyethylene-block-poly-(ethylene glycol) (PE-b-PEG), oleic acid, sodium stearate, 1-octadecene, hydrofluoric acid, lauryl mercaptan, dimethyl sulfoxide (DMSO), and oleylamine were purchased from Aldrich. All rare-earth nitrates utilized in this work were purchased from Beijing Ouhe Chemical Reagent Co. 1-ethyl-3-(3-dimethly aminopropyl) carbodiimide (EDC, Sigma) and N-hydroxysuccinimide (NHS, Acros) were used for bioconjugation. Ethanol, Na2HPO4, Na3PO4, MnCl2, Na2S, ZnCl2, HAuCl4·2H2O, NaF, Ca(NO3)2, paraformaldehyde, NaOH, and cyclohexane were obtained from Beijing Chemical Reagent Co. Fe(NH4)2·(SO4)·6H2O and NH4VO3 were from Tianjin Fuchen Chemicals Co. (Tianjin, China). 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(succinyl) (sodium salt) (carboxylated phospholipid, PL) was supplied by Avanti, and the N-lauroyl sarcosine sodium (NLSS) was supplied by Amresco. All chemicals were analytical grade and used without further purification. Deionized (DI) water was used throughout. Characterization. Fourier-transform infrared (FTIR) spectra were performed on a JASCO FT/IR-460 PLUS spectrometer (Tokyo). The size and morphology of the nanocrystals were characterized by a JEM1200 EX transmission electron microscope (JEOL). Dynamic light scattering (DLS) particle size analysis was carried out on a Malvern Master 2000 ζ and size analyzer. The emission spectra of the QDs were measured via a Hitachi F-4600 fluorescence spectrophotometer. The cell imaging was performed on a TCS SP5 two-photon Confocal Microscopes (Leica). The TGA was carried out on a Henven HCT-1 thermogravimetric analyzer. Synthesis of NaYF4:Yb3+-Er3+ Short Nanorods and Nanoparticles.46 To synthesize the β-phase NaYF4:Yb3+-Er3+ nanorods, a mixture of 6 mL of oleic acid, 9 mL of octadecene, and 0.35 g of sodium stearate were added into a three-necked flask and stirred thoroughly. Then, 4 mL of the as-prepared rare-earth oleate solution and 1.6 mL of the HF−oleylamine solution were quickly injected into the flask and kept at 80 °C for 20 min. Then, the solution was heated to 180 °C for 10 min before it was heated to 310 °C for 1 h. The mixture solution was kept under a protective nitrogen flow throughout. The reaction solution was cooled to room temperature, and the product was collected by centrifugation. The as-prepared nanorods were redispersed into cyclohexane and precipitated by addition of ethanol for further purification. The final product was redispersed in 5 mL of chloroform and stored for later use. For the synthesis of nanoparticles, 8 mL of oleic acid, 7 mL of octadecene, 0.35 g of sodium stearate, 3 mL of the as-prepared rare-earth oleate solution, and 1.05 mL of the HF−oleylamine solution were used. Synthesis of LaF3:Ce3+-Tb3+ Nanoparticles.47 0.6 g of NaOH was dissolved into 5 mL of deionized water in a 50-mL autoclave under stirring, and then 10 mL of ethanol and 10 mL of oleic acid were added. Thereafter, 1.0 mL of mixture Ln(NO3)3 solution (0.5M, La3+:Ce3+:Tb3+ = 90:5:5 mol %) was added and the mixture was

(hepatocellular carcinoma, human) cancer cells. As the nonspecific binding was effectively blocked by bovine serum albumin (BSA) and PEG, the confocal fluorescence image shown in Figure 6 clearly illustrated that the anti-AFP

Figure 6. Confocal fluorescence images of cells stained with QDs: (a− c) fixed HepG2 cells stained by QDs-Ab (anti-AFP); (d−f) fixed HepG2 cells incubated with QDs-BSA; (g−i) fixed HeLa cells incubated with QDs-Ab (anti-AFP).

conjugated QDs (QDs-Ab) specifically labeled the HepG2 cells (Figure 6a−c). As a control, if the QDs were conjugated with BSA only, the QDs-BSA could not label the HepG2 cells, and only very weak red fluorescence was observed (Figure 6d− f). In another control experiment (Figure 6g−i), the QDs-Ab also could not stain the HeLa cells. The cellular imaging results suggest that the nanocrystals functionalized via this facile and general surface modification strategy are capable of biomedical applications. 2595

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Surface Modification of Nanocrystals. Taking the surface modification of ZnS:Mn2+ nanoparticles as an example, into 8.0 mL of NLSS (0.1 g) aqueous solution, the mixture chloroform solution of PE-b-PEG (0.2 mg), carboxylated phospholipids (0.15 mg), and QDs (3.0 mg) were added under ultrasonication (400 W, 6 min) and stirring. Thereafter, the chloroform containing the hydrophobic nanocrystals, phospholipids, and PE-b-PEG was successfully transferred into the oil-in-water (O/L) microemulsion. It should be mentioned that, owing to the diversity of the particle size and shape, the dosage of the nanocrystals has slight difference and is tuned in the range 3−5 mg. After the removal of the chloroform by rotary evaporation at 40−60 °C for 10 min, the carboxylated phospholipids and PE-b-PEG block polymer self-assembled into a thin layer on the surface of the inorganic nanocrystals, and impart the nanoparticles water dispersible, biocompatible, and bioconjugatible. The hydrophilic nanocrystals were collected and purified by dialysis to remove the surfactants and excess chemicals. For FTIR and TGA tests, the nanoparticles were collected via centrifugation after dialysis. Cytotoxicity Assay of Surface Functionalized QDs. The cytotoxicity of the surface functionalized ZnS:Mn2+ quantum dots (QDs) (PL-ZnS:Mn2+) was evaluated by using the methyl thiazolyl tetrazolium (MTT) assay on the HepG2 (Hepatocellular carcinoma, human) cell line. About 5 × 104/well cells were seeded into a 96-well cell culture plate under recommended conditions at 37 °C in a 5% CO2-humidified incubator. The plate was stayed overnight to make sure the cells have been adherent, and various amounts (0−100 μg/ mL) of QDs were added into each well. Then, the plate was incubated at 37 °C for 24 or 48 h. After removing the culture media, 20 μL of MTT (5 mg/mL in PBS, pH = 7.4) was added into each well and incubated for another 4 h at 37 °C in a 5% CO2-humidified incubator. After removing the media carefully, 150 μL of dimethyl sulfoxide (DMSO) was added into each well to dissolve the newly formed formazan. The absorbance of the dissolved formazan in each well was measured at 490 nm by an ELISA plate reader (F50, TECAN). Due to the quantity of formazan product as measured by the amount of 490 nm absorbance is directly proportional to the number of living cells in culture, the cell viability can be calculated through the detected absorbance. Bioconjugation of PL-ZnS:Mn2+ with Anti-α-fetoprotein (anti-AFP) Antibody. The as-coated hydrophilic shell, which allows for the QDs water dispersible and biocompatible, combined with the carboxylic groups in the phospholipid structure, enabled easy surface modification and efficient labeling with targeting antibodies for their cellular imaging applications. Therefore, the carboxylic groups on the hydrophilic shell were activated with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide (EDC/NHS), and then conjugated with the anti-α-fetoprotein (AFP) antibodies (Ab), which could specifically recognize the AFP (antigen) on the HepG2 (hepatocellular carcinoma, human) cancer cells. To investigate the potential application for biotagging and fluorescence imaging, the carboxylated phospholipid coated quantum dots (PL-ZnS:Mn2+) were bioconjugated with the anti-AFP antibodies (Ab). In brief, 200 μL of ZnS:Mn2+ QDs (0.5 mg/mL, PBS, pH 7.4), 10 μL of ethyl dimethylaminopropyl carbodiimide solution (EDC, 20 mg/mL, PBS, pH 7.4), and 10 μL of N-hydroxysuccinimide (NHS, 50 mg/mL, PBS, pH 7.4) were mixed and then incubated at 25 °C for 30 min. Thereafter, 5 μL of anti-AFP antibody (1 mg/mL) was added, and the mixture was kept incubating at 25 °C for another 2 h. Excess carboxyl groups on the modified nanoparticles were blocked by adding 20 μL of bovine serum albumin (BSA, 50 mg/mL, in PBS, pH 7.4). Finally, the QDs-Ab bioconjugates were collected by centrifugation (10000 rpm, 10 min) and washed with PBS, and this purification process was repeated three times. The obtained QDs-Ab was redispersed in PBS buffer (pH = 7.4, 100 μL) and stocked at 4 °C for later use. The final concentration of QDs-Ab is 1.0 mg/mL. As a control, the hydrophilic QDs were bioconjugated with BSA only without anti-AFP antibodies via the same protocol for the antibody conjugation. Cell Imaging. HepG2 liver cancer cells were seeded on a sterilized glass cover slide and cultured in a 12-well cell culture plate overnight under recommended conditions at 37 °C in 5% CO2-humidified

stirred for 10 min. 1.8 mL of NaF solution (1.0 M) was then added dropwise under vigorous stirring. Finally, 10 mL of ethanol was added, and the autoclave was solvothermally treated at 190 °C for 6 h. The final product was redispersed in 2 mL of chloroform. Preparation of ZnS:Mn2+ Nanoparticles.37 Into a 50-mL Teflon-lined autoclave, 0.6 g of sodium hydroxide, 5 mL of DI water, 8 mL of ethanol, and 10 mL of oleic acid were added in sequence under vigorous stirring. Thereafter, the mixture aqueous solution of MnCl2 (0.1 mL, 1 M) and ZnCl2 (1.9 mL, 1 M) were added before the addition of 2.0 mL of Na2S solution (1 M). Finally, the autoclave was sealed and heated at 160 °C for 8 h. The luminescent quantum dots were collected by washing and centrifugation, and then dispersed into 5 mL of chloroform. Synthesis of Gold (Au) Nanocrystals.41 To synthesize the Au particle with the size of ∼7 nm, 0.1 g of sodium hydroxide and 5 mL of DI water were added into a 50-mL autoclave under stirring. Thereafter, 15 mL of ethanol, 2 mL of oleic acid, 1 mL of oleylamine, and 5 mL of cyclohexane were added into the autoclave in sequence. Finally, 5 mL of chloroauric acid aqueous solution (2 mg/mL) and 50 μL of lauryl mercaptan were added under vigorous stirring. The autoclave was stirred for another 10 min and then heated at 100 °C for 10 h. The final product was washed with cyclohexane and ethanol and redispersed into 2 mL of chloroform. Preparation of Fe3O4 Nanoparticles. The Fe3O4 magnetic nanoparticles (MNPs) were synthesized according to the articles.42 In a 50-mL Teflon-lined autoclave, 1.0 g of NaOH was dissolved into 5 mL of deionized water under strong stirring. Then, 15 mL of ethanol and 10 mL of oleic acid were added, and the mixture was stirred for 10 min. Thereafter, 10 mL of aqueous solution containing 0.784 g of Fe(NH4)2(SO4)2 ·6H2O was dripped, and the mixture was stirred for another 10 min. The autoclave was treated at 180 °C for 10 h before it was allowed to cool to room temperature. The black products were collected and washed with cyclohexane and ethanol. This purification cycle was repeated for twice. The final products were dispersed in chloroform (5 mL) and stored for use. Synthesis of Luminescent LaVO4:Eu3+ Nanocrystals. LaVO4:Eu3+ luminescent nanobricks were prepared according to the reported method with slight alteration.43 In brief, 0.06 g of NH4VO3 and 0.6 g of NaOH were first dissolved in 5 mL of DI water, followed by adding 20 mL of ethanol and 10 mL of oleic acid under vigorous stirring. Then, 1.0 mL of Ln(NO3)3 solution (1 M, La3+/Eu3+ = 98:2 mol %) were added, and the mixture was kept stirring for 10 min before it was transferred into a Teflon-lined autoclave. The autoclave was sealed and solvothermally treated at 140 °C for 5 h before it was allowed to cool to room temperature. The product deposited onto the bottom of the autoclave was collected and washed with cyclohexane and ethanol. Then, the white product was dispersed in 5 mL of chloroform. Synthesis of YPO4 Nanocrystals.44,48 In a 50-mL autoclave, 0.6 g of NaOH was dissolved into 5 mL of deionized water under stirring. Then, 10 mL of ethanol and 10 mL of oleic acid were added under vigorous stirring. Thereafter, 5 mL of NaH2PO4 aqueous solution (0.2 M), and 5 mL of Y(NO3)3 aqueous solutions (0.2 M) were added in sequence. Finally, 10 mL of ethanol was added into the autoclave after stirring for another 10 min, and the autoclave was sealed and heated at 140 °C for 8 h. The solution was cooled to room temperature and the nanoparticles were collected by centrifugation. The final product was redispersed in 5 mL of chloroform. Synthesis of HAp Nanorods. The synthesis of F-substituted hydroxyapatite (HAp) nanorods was according to the reported method.45 In a Teflon-lined autoclave, 16 mL of ethanol was added into the mixture solution of 0.5 g of octadecylamine and 4 mL of oleic acid. Then, 7 mL of Ca(NO3)2 (0.28 M) and 0.35 mL of Eu(NO3)3 (0.28 M) aqueous solution were added under agitation. Finally, 0.35 mL of NaF (0.28 M,) and 7 mL of Na3PO4 (0.16 M) were dripped into the solution. The mixture was agitated for about 5 min before being transferred into the autoclave. The solution was then heated at 150 °C for 12 h. The obtained nanorods were collected by centrifugation, washed with cyclohexane and ethanol for several times, and finally redispersed into 2 mL of chloroform. 2596

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incubator. Then, the QDs-Ab stock solution was added into the cell culture well with a final concentration of 100 μg/mL. The HepG2 cells were incubated with QDs-Ab for another 2 h. As a control, the QDsBSA, in place of QDs-Ab, was incubated with the HepG2 cells under the same conditions. Also, to further investigate the specific recognition ability of QDs-Ab, in another control experiment, the QDs-Ab was incubated with HeLa cells instead of HepG2 cells. Thereafter, the cells on the glass slide were washed with PBS and fixed in 4% paraformaldehyde solution for 15 min. The fluorescence imaging was conducted on a TCS SP5 two-photon confocal microscopes (Leica).

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ASSOCIATED CONTENT

S Supporting Information *

Fluorescence spectra, EDS analysis, dynamic light scattering (DLS), water-stability test of ZnS:Mn2+. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Email: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported in part by the National Natural Science Foundation of China (20175009), the State Key Project of Fundamental Research of China (2011CB932403 and 2011CBA00503), and the Program for New Century Excellent Talents in University of China (NCET-10-0213).

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dx.doi.org/10.1021/cm301285g | Chem. Mater. 2012, 24, 2592−2597