Research article Received: 23 October 2013,
Revised: 27 February 2014,
Accepted: 21 March 2014
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/bio.2679
Characterization and cancer cell targeted imaging properties of human antivascular endothelial growth factor monoclonal antibody conjugated CdTe/ZnS quantum dots Lili Pang,a Jian Xu,b Chang Shu,a Jin Guo,a Xiaona Ma,a Yu Liub* and Wenying Zhonga* ABSTRACT: High luminescence quantum yield water-soluble CdTe/ZnS core/shell quantum dots (QDs) stabilized with thioglycolic acid were synthesized. QDs were chemically coupled to fully humanized antivascular endothelial growth factor165 monoclonal antibodies to produce fluorescent probes. These probes can be used to assay the biological affinity of the antibody. The properties of QDs conjugated to an antibody were characterized by ultraviolet and visible spectrophotometry, fluorescent spectrophotometry, sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transmission electron microscopy and fluorescence microscopy. Cell-targeted imaging was performed in human breast cancer cell lines. The cytotoxicity of bare QDs and fluorescent probes was evaluated in the MCF-7 cells with an MTT viability assay. The results proved that CdTe/ZnS QD–monoclonal antibody nanoprobes had been successfully prepared with excellent spectral properties in target detections. Surface modification by ZnS shell could mitigate the cytotoxicity of cadmium-based QDs. The therapeutic effects of antivascular endothelial growth factor antibodies towards cultured human cancer cells were confirmed by MTT assay. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: quantum dots; human anti-VEGF165 antibody; cell imaging; cytotoxicity
Introduction Over the past few decades, the applications of quantum dots (QDs) have evolved rapidly, particularly in areas of bioimaging and bioanalysis. QDs, as the next generation fluorophores, have the potential to lead to major advancement in biological applications. With the aim of realizing the application of QDs in nanobiotechnology and nanomedicine as tools for diagnostic and therapeutic applications, the biofunctionalization progress of QDs is indispensable (1). QDs can be functionalized by conjugation to a number of biological molecules, including oligonucleotides, DNA, aptamers, peptides, proteins and antibodies (2). QDs were first linked with bio-recognition molecules by Nie’s group (3). After labeling with transferrin, CdSe/ZnS QDs can enter the cultured HeLa cells by endocytosis or by specific binding based on the antibody–antigen reaction. The resultant bioconjugates can be imaged. Since then, attractive fluorescent probes composed of QDs and bio-recognitive molecules (4,5) were widely used in biological and medical fields, such as immunolabeling (6), cell tracking (7) and biosensing (8,9). Angiogenesis plays a critical role for solid tumor growth and metastasis (10–12). Vascular endothelial growth factor (VEGF) is an endothelial cell-specific mitogen and an angiogenic stimulant released by a variety of tumor cells. Its binding to VEGF receptor (VEGFR) is important for endothelial cell proliferation. Kim et al. (13) demonstrated that monoclonal antibodies (mAbs) specific for VEGF could inhibit the action of VEGF and suppress tumor
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growth. However, the immunogenicity of rodent antibodies in humans prevents their application in clinical procedures. To overcome this problem, we produced humanized anti-VEGF165 mAbs by technologies using phage display and transgenic mice expressing human immunoglobulin genes (14). To detect the biological affinity of humanized anti-VEGF mAbs to VEGF at cellular level, we aimed to use QD-conjugated antibodies in consideration of QD high photobleaching thresholds compared with conventional dyes (15). At present, the most widely used semiconductor QDs in biological applications is the CdTe QDs. However, the dissociation of Cd2+ from CdTe QDs was found not only to be detrimental to cells, but also to weaken the luminescence intensity from the QDs. As a result, CdTe QDs are not suitable for in vitro and/
* Correspondence to: W. Zhong, Department of Analytical Chemistry, China Pharmaceutical University, Nanjing 210009, People’s Republic of China. E-mail: [email protected] * Y. Liu, Department of Biochemistry, School of Life Science and Technology, China Pharmaceutical University, Nanjing, 210009, People’s Republic of China. E-mail: [email protected] a
Department of Analytical Chemistry, China Pharmaceutical University, Nanjing, 210009, People’s Republic of China
b
Department of Biochemistry School of Life Science and Technology, China Pharmaceutical University, Nanjing, 210009, People’s Republic of China
Copyright © 2014 John Wiley & Sons, Ltd.
L. Pang et al. or in vivo imaging. It is, therefore, important to introduce a shell that protects the core (e.g. core/shell CdTe/ZnS, core/shell/shell CdSe/CdS/ZnS) from degradation (16,17). In comparison to CdTe QDs, core/shell CdTe/ZnS QDs evidently suppress the toxicity of the QDs itself in biological environments with outstanding stability. Liu and Yu found that CdTe/ZnS QDs were of less toxicity and better biocompatibility in hemolysis assays (18). In the present work, we covalently linked CdTe/ZnS core/shell QDs to anti-VEGF mAbs for tumor-specific imaging. The probe we synthesized consists of QDs as fluorescent tracers and an antibody targeting to VEGF with tumor-killing effect (19). The conjugates make it facile to detect the bioactivity, biospecificity and tumor inhibition effect of the antibody. The targeting ability of QD–mAb fluorescent probe was confirmed in vitro; the antitumor effect of anti-VEGF mAbs was confirmed by 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays.
Experimental Materials Anti-VEGF165 mAbs were produced in Yu Liu’s laboratory as described before (14). The water used in the experiment was purified by distillation of deionized water. Tellurium powder (300 mesh, 99.95%), sodium borohydride (NaBH4 98%), thioglycolic acid (TGA, 98%), N-ethyl-N′-[3 (dimethylamino) propyl] carbodiimide hydrochloride (98.5%) and N-hydroxysuccinimide (98%) were purchased from Aladdin Reagent Corporation (Shanghai, China). Human breast carcinoma cell line (MCF-7) and human embryonic kidney cell line (HEK293) was obtained from American Type Culture Collection (ATCC, VA, USA).
The synthesis of fluorescent CdTe quantum dots The CdTe QDs were synthesized by a modified method based on a procedure described previously (20). 0.171 g CdCl2 · 2.5H2O and 125 μL of TGA were dissolved in 150 mL of water in a three-necked flask, and then the pH was adjusted to 8.0 with 1 mol/L NaOH solution. The NaHTe solution was prepared by dissolving tellurium powder (47.85 mg) and NaBH4 (50.00 mg) in 3 mL of anhydrous ethyl alcohol. The air in the system was then pumped off and replaced with N2. Fifty minutes later, freshly prepared NaHTe solution was added into the above-mentioned three-necked flask in the presence of TGA as a stabilizing agent. The mixture was re-fluxed for 2 h at 100°C to support for the growth of nanocrystals.
Shell growth of ZnS over CdTe core The crude CdTe were heated with gentle stirring for 10 min. ZnSO4 solution (2.5 mmol/L) and Na2S solution (2.5 mmol/L) were added to the above solution drop by drop. The reaction mixture was heated and stirred for 30 min. The mixture was then re-fluxed for 5 min at 100°C for the annealing process of the CdTe/ZnS QDs. Preparation of quantum dot–monoclonal antibody conjugates One hundred microliters of QDs solution were activated by adding100 μL of a mixture of N-ethyl-N′-[3 (dimethylamino) propyl] carbodiimide hydrochloride (0.6 mg/mL in H2O) and N-hydroxysuccinimide (1.4 mg/mL in H2O) for 15 min at room temperature. Four microliters of anti-VEGF mAbs (25 mg/mL) in phosphate-buffered saline (PBS, 0.01 M, pH 7.4) was added to the above QD solution for reaction at 37°C for 2 h in a reciprocating oscillator. The excess unreacted coupling agents were separated using a molecular weight cutoff filter (Amicon Ultra 4 mL, 100 kDa; Millipore, MA, USA). The reaction solution was stored in a refrigerator at 4°C before use. The schematic representation of anti-VEGF antibodies functionalized QD nanoparticles was shown in Fig. 1. Characterization of quantum dots and quantum dot–monoclonal antibody conjugates Emission spectra were collected using a RF-5301 spectrophotometer (Shimadzu Corp., Tokyo, Japan). Absorption spectra were measured using a UV-1800 spectrophotometer (Shimadzu Corp., Tokyo, Japan). Transmission electron microscopy (TEM) images were collected by a JEM-2100 (JEOL Ltd., Tokyo, Japan). Dynamic light scattering (DLS) analysis was done by ZetaPlus Zeta Potential Analyzer (Brookhaven, NY, USA). Samples mixed with × 5 concentrated sample buffer containing 10% sodium dodecyl sulfate (w/v) and 5% β-mercaptoethanol (v/v) were loaded on a 12% acrylamide gel. The voltage was set at 120 V, after then the gel plate was stained with Coomassie Blue. Electrophoretic gel was imaged using a ChemiDoc XRS system (Bio-Rad, CA, USA). Cell culture and fluorescence microscopic imaging The cultured MCF-7 cancer cells were seeded into 24-well plates. The cells were then kept in a CO2 incubator at 37°C for 24 h. QDs and QD–mAb bioconjugates were incubated with the MCF-7 cells or HEK293 cells for 2 h at 37°C before samples were washed
Figure 1. The schematic illustration of antivascular endothelial growth factor antibodies functionalized quantum dots. EDC, N-ethyl-N′-[3 (dimethylamino) propyl] carbodiimide hydrochloride; NHS, N-hydroxysuccinimide; TGA, thioglycolic acid.
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Copyright © 2014 John Wiley & Sons, Ltd.
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Characterization of antibody conjugated QDs for cancer cell imaging three times with PBS and photographed using an inverted fluorescence microscope equipped with CCD camera (Olympus IX71, Tokyo, Japan). MTT viability assay The MCF-7 human breast cancer cells were cultured on 96-well microtiter plates and incubated with different concentrations of CdTe, CdTe/ZnS and CdTe/ZnS-mAb for 48 h, with three duplicates for each concentration. Then the cells were incubated with MTT reagents (5 mg/mL in PBS) for 4 h at 37°C. After incubation, the cells were lysed with dimethyl sulfoxide on the plate. The absorbance at 490 nm was measured on a microplate reader (Bio-Rad).
Results and discussion Spectral characterization of CdTe and CdTe/ZnS quantum dots Fluorescence and absorption spectra were used to characterise the optical properties of the QDs. Figure 2(a,b) shows the excitonic absorption peak of nanoparticles shifted to the longer wavelengths from 499 nm to 540 nm, indicating the growth of a ZnS shell on top of the CdTe core. Meanwhile, the emission peak (Fig. 2c,d) shifted from 545 nm to 583 nm as the particles’ size increased (21). The fluorescence spectra also displayed that the peak width at half-maximum were maintained during the shell growth, about 39 nm, which indicated the aforementioned core/shell QDs had a good monodispersity. Moreover, the luminescent quantum yields of core/shell CdTe/ ZnS at room temperature was measured and calculated as up to 59% , while it was only 36% for naked CdTe QDs, using rhodamine 6G as a reference (22). Bioconjugation of quantum dots with antivascular endothelial growth factor monoclonal antibodies Emission spectra of CdTe/ZnS QDs and QD–mAbs were shown in Fig. 3. The maximum emission peak of QD–mAb was blueshifted from 583 nm to 577 nm after conjugation. We also observed that the fluorescent intensity was enhanced because of the rearranged gross electrostatic environment on the surface
Figure 3. Emission spectra of CdTe/ZnS QDs and QD–mAb conjugates. mAb, monoclonal antibody; QD, quantum dot.
of QDs. The two changes in the maximum emission peak were attributed to the conjugation between the carboxylic groups in QDs and the amino groups of the antibody by covalent bonds, which could weaken the interaction between dipole–dipole and shorten the Stokes shifts (23,24).
Size, morphology and stability Figure 4(a,b) shows the TEM and high-resolution TEM images of QD–mAb conjugates prepared in this study. The size distribution of conjugates based on TEM (Fig. 4a) was mono-dispersed. As shown in the high-resolution TEM image (Fig. 4b), the nanoparticles also had a good crystal structure (25). Compared to the TEM images of CdTe/ZnS QDs that were reported previously by our group (20), the size and morphology of QDs were not aggregated after the coupling of antibodies. The DLS technique (Fig. 4c) confirmed that the mean hydrodynamic diameter of QD–mAb conjugates in aqueous phase was about 10.9 nm. The results of the DLS experiments demonstrated that the hydrodynamic diameter included the size of the solvated capping agent (TGA) layer and anti-VEGF mAbs, which made the DLS size larger than the measured TEM size (26). Figure 4d shows the luminescence intensity of QD–mAb conjugates during different time. When the conjugates were stored at 4°C, over 94% of the initial response remained after a storage period of 5 days. These results indicated that the conjugates had acceptable reliability and stability.
Sodium dodecyl sulfate–polyacrylamide gel electrophoresis gel electrophoresis
Figure 2. Absorption spectra of CdTe and CdTe/ZnS QDs (a,b); emission spectra of CdTe and CdTe/ZnS QDs (c,d). FL, fluorescence.
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The electrophoresis technique is employed here to identify further the bioconjugation of QDs and proteins. Figure 5 shows the image of the stained gel plate. In sodium dodecyl sulfate– polyacrylamide gel electrophoresis, the covalent bonds between the antibody and the QD surface were stable (27), while the disulfide bonds used to link the heavy chain and the light chain in IgG were deoxidized by mercaptoethanol. As a result, two proteins (25 kDa, 50 kDa) were detected on the gel. Because the molecular weight of CdTe/ZnS–mAb was larger than the pure antibody, both of the two chains’ electrophoretic velocities of
Copyright © 2014 John Wiley & Sons, Ltd.
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L. Pang et al.
Figure 4. (a,b) Transmission electron microscopy image of QD–mAb conjugates at different magnification(c) hydrodynamic diameter distribution graph of QD–mAb conjugates(d) luminescence intensity of QD–mAb conjugates during different storage time. mAb, monoclonal antibody; PL, photoluminescence; QD, quantum dot.
QD–mAb conjugates (lane a) were slightly slower than that of the anti-VEGF antibodies (lane b).
Imaging in live cells using quantum dots–monoclonal antibody conjugates Figure 6(a) shows the image of MCF-7 cancer cells in bright fields. When MCF-7 cells were incubated with QDs alone (Fig. 6b), almost no signal was detected. However, when QD–mAb conjugates were incubated with MCF-7 cells, a high-intensity fluorescent signal was detected based on the antibody–antigen reaction, as shown in Fig. 6(d) (28–30). To evaluate further the specificity of QD–mAb probes, human embryonic kidney cells (HEK293) were incubated with QD–mAb conjugates. As shown in Fig. 6(c), following such treatment no signal was observed, indicating that the QD–mAb conjugates can be used as a detectable probe for VEGF antigen detection in cancer cells (31,32).
Cytotoxicity evaluation As shown in Fig. 7, a dose-dependent decrease in cell viability was observed in bare CdTe sample. As the CdTe QDs’ concentration increased to 75 μM, the cell viability decreased to 27.2% sharply. In contrast, the viability of cells incubated with CdTe/ZnS QDs maintained greater than 66% even at nanoparticles loadings as high as 150 μM. We also observed that the growth trend of the MCF-7 cells was not significantly altered in all the CdTe/ZnS concentrations. It could be explained by the fact that CdTe QDs were encapsulated by the ZnS shell, which could reduce the possible loss of cadmium ions from the nanoparticles (33). It is worth noting that the cytotoxicity of CdTe/ZnS-mAb was also concentration dependent. With the increase of
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Figure 5. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis photography. Lane a: quantum dot–monoclonal antibody conjugates; lane M: protein MW marker; lane b: antivascular endothelial growth factor antibodies.
CdTe/ZnS-mAb in concentration, the cell viability decreases, which might be due to the therapeutic value of anti-VEGF mAbs. This therapeutic value can be attributed to the blocking effect of mAbs on VEGF165 binding to VEGFR.
Copyright © 2014 John Wiley & Sons, Ltd.
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Characterization of antibody conjugated QDs for cancer cell imaging
Figure 6. (a) Image of MCF-7 cancer cells in bright field; (b) fluorescence image of MCF-7 cancer cells incubated with CdTe/ZnS QDs (30 μM) for 2 h; fluorescence images of HEK293 cells (c) and MCF-7 cancer cells (d) incubated with quantum dot–monoclonal antibody conjugates (30 μM) for 2 h, respectively.
with a bio-recognitive component can be applied in immunological cell imaging (34). It also could be used to detect the therapeutic effect of mAbs. Promising emerging fluorescent labels based on QDs and functional biological macromolecules were both specific to recognize cellular targets and bright for effective detection in real applications, such as cellular tracking (35), cancer diagnosis (36) and even cancer therapy applications (37). Acknowledgements The authors gratefully acknowledge the financial support received from the National Natural Science Foundation of China (grant no. 81173023), National Science and Technology Major Project (grant no. 2013ZX09301303-004) and National High Technology Research and Development Program 863 (grant no. 2012AA02A303).
Figure 7. Cell viabilities of MCF-7 cells by MTT viability assay relative to untreated controls (mean ± SD; n = 3). mAb, monoclonal antibody.
Conclusions Highly fluorescent CdTe/ZnS QDs were prepared by an aqueous approach and used as fluorescent labels for anti-VEGF mAbs for the detection of human VEGF. The results demonstrated that CdTe capped with ZnS experimentally was not cytotoxic to live cells. The CdTe/ZnS QD-conjugated anti-VEGF mAbs are capable of detecting the presence of human VEGF. The QD–mAb probes
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Copyright © 2014 John Wiley & Sons, Ltd.
Luminescence 2014
Revised: 27 February 2014,
Accepted: 21 March 2014
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/bio.2679
Characterization and cancer cell targeted imaging properties of human antivascular endothelial growth factor monoclonal antibody conjugated CdTe/ZnS quantum dots Lili Pang,a Jian Xu,b Chang Shu,a Jin Guo,a Xiaona Ma,a Yu Liub* and Wenying Zhonga* ABSTRACT: High luminescence quantum yield water-soluble CdTe/ZnS core/shell quantum dots (QDs) stabilized with thioglycolic acid were synthesized. QDs were chemically coupled to fully humanized antivascular endothelial growth factor165 monoclonal antibodies to produce fluorescent probes. These probes can be used to assay the biological affinity of the antibody. The properties of QDs conjugated to an antibody were characterized by ultraviolet and visible spectrophotometry, fluorescent spectrophotometry, sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transmission electron microscopy and fluorescence microscopy. Cell-targeted imaging was performed in human breast cancer cell lines. The cytotoxicity of bare QDs and fluorescent probes was evaluated in the MCF-7 cells with an MTT viability assay. The results proved that CdTe/ZnS QD–monoclonal antibody nanoprobes had been successfully prepared with excellent spectral properties in target detections. Surface modification by ZnS shell could mitigate the cytotoxicity of cadmium-based QDs. The therapeutic effects of antivascular endothelial growth factor antibodies towards cultured human cancer cells were confirmed by MTT assay. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: quantum dots; human anti-VEGF165 antibody; cell imaging; cytotoxicity
Introduction Over the past few decades, the applications of quantum dots (QDs) have evolved rapidly, particularly in areas of bioimaging and bioanalysis. QDs, as the next generation fluorophores, have the potential to lead to major advancement in biological applications. With the aim of realizing the application of QDs in nanobiotechnology and nanomedicine as tools for diagnostic and therapeutic applications, the biofunctionalization progress of QDs is indispensable (1). QDs can be functionalized by conjugation to a number of biological molecules, including oligonucleotides, DNA, aptamers, peptides, proteins and antibodies (2). QDs were first linked with bio-recognition molecules by Nie’s group (3). After labeling with transferrin, CdSe/ZnS QDs can enter the cultured HeLa cells by endocytosis or by specific binding based on the antibody–antigen reaction. The resultant bioconjugates can be imaged. Since then, attractive fluorescent probes composed of QDs and bio-recognitive molecules (4,5) were widely used in biological and medical fields, such as immunolabeling (6), cell tracking (7) and biosensing (8,9). Angiogenesis plays a critical role for solid tumor growth and metastasis (10–12). Vascular endothelial growth factor (VEGF) is an endothelial cell-specific mitogen and an angiogenic stimulant released by a variety of tumor cells. Its binding to VEGF receptor (VEGFR) is important for endothelial cell proliferation. Kim et al. (13) demonstrated that monoclonal antibodies (mAbs) specific for VEGF could inhibit the action of VEGF and suppress tumor
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growth. However, the immunogenicity of rodent antibodies in humans prevents their application in clinical procedures. To overcome this problem, we produced humanized anti-VEGF165 mAbs by technologies using phage display and transgenic mice expressing human immunoglobulin genes (14). To detect the biological affinity of humanized anti-VEGF mAbs to VEGF at cellular level, we aimed to use QD-conjugated antibodies in consideration of QD high photobleaching thresholds compared with conventional dyes (15). At present, the most widely used semiconductor QDs in biological applications is the CdTe QDs. However, the dissociation of Cd2+ from CdTe QDs was found not only to be detrimental to cells, but also to weaken the luminescence intensity from the QDs. As a result, CdTe QDs are not suitable for in vitro and/
* Correspondence to: W. Zhong, Department of Analytical Chemistry, China Pharmaceutical University, Nanjing 210009, People’s Republic of China. E-mail: [email protected] * Y. Liu, Department of Biochemistry, School of Life Science and Technology, China Pharmaceutical University, Nanjing, 210009, People’s Republic of China. E-mail: [email protected] a
Department of Analytical Chemistry, China Pharmaceutical University, Nanjing, 210009, People’s Republic of China
b
Department of Biochemistry School of Life Science and Technology, China Pharmaceutical University, Nanjing, 210009, People’s Republic of China
Copyright © 2014 John Wiley & Sons, Ltd.
L. Pang et al. or in vivo imaging. It is, therefore, important to introduce a shell that protects the core (e.g. core/shell CdTe/ZnS, core/shell/shell CdSe/CdS/ZnS) from degradation (16,17). In comparison to CdTe QDs, core/shell CdTe/ZnS QDs evidently suppress the toxicity of the QDs itself in biological environments with outstanding stability. Liu and Yu found that CdTe/ZnS QDs were of less toxicity and better biocompatibility in hemolysis assays (18). In the present work, we covalently linked CdTe/ZnS core/shell QDs to anti-VEGF mAbs for tumor-specific imaging. The probe we synthesized consists of QDs as fluorescent tracers and an antibody targeting to VEGF with tumor-killing effect (19). The conjugates make it facile to detect the bioactivity, biospecificity and tumor inhibition effect of the antibody. The targeting ability of QD–mAb fluorescent probe was confirmed in vitro; the antitumor effect of anti-VEGF mAbs was confirmed by 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays.
Experimental Materials Anti-VEGF165 mAbs were produced in Yu Liu’s laboratory as described before (14). The water used in the experiment was purified by distillation of deionized water. Tellurium powder (300 mesh, 99.95%), sodium borohydride (NaBH4 98%), thioglycolic acid (TGA, 98%), N-ethyl-N′-[3 (dimethylamino) propyl] carbodiimide hydrochloride (98.5%) and N-hydroxysuccinimide (98%) were purchased from Aladdin Reagent Corporation (Shanghai, China). Human breast carcinoma cell line (MCF-7) and human embryonic kidney cell line (HEK293) was obtained from American Type Culture Collection (ATCC, VA, USA).
The synthesis of fluorescent CdTe quantum dots The CdTe QDs were synthesized by a modified method based on a procedure described previously (20). 0.171 g CdCl2 · 2.5H2O and 125 μL of TGA were dissolved in 150 mL of water in a three-necked flask, and then the pH was adjusted to 8.0 with 1 mol/L NaOH solution. The NaHTe solution was prepared by dissolving tellurium powder (47.85 mg) and NaBH4 (50.00 mg) in 3 mL of anhydrous ethyl alcohol. The air in the system was then pumped off and replaced with N2. Fifty minutes later, freshly prepared NaHTe solution was added into the above-mentioned three-necked flask in the presence of TGA as a stabilizing agent. The mixture was re-fluxed for 2 h at 100°C to support for the growth of nanocrystals.
Shell growth of ZnS over CdTe core The crude CdTe were heated with gentle stirring for 10 min. ZnSO4 solution (2.5 mmol/L) and Na2S solution (2.5 mmol/L) were added to the above solution drop by drop. The reaction mixture was heated and stirred for 30 min. The mixture was then re-fluxed for 5 min at 100°C for the annealing process of the CdTe/ZnS QDs. Preparation of quantum dot–monoclonal antibody conjugates One hundred microliters of QDs solution were activated by adding100 μL of a mixture of N-ethyl-N′-[3 (dimethylamino) propyl] carbodiimide hydrochloride (0.6 mg/mL in H2O) and N-hydroxysuccinimide (1.4 mg/mL in H2O) for 15 min at room temperature. Four microliters of anti-VEGF mAbs (25 mg/mL) in phosphate-buffered saline (PBS, 0.01 M, pH 7.4) was added to the above QD solution for reaction at 37°C for 2 h in a reciprocating oscillator. The excess unreacted coupling agents were separated using a molecular weight cutoff filter (Amicon Ultra 4 mL, 100 kDa; Millipore, MA, USA). The reaction solution was stored in a refrigerator at 4°C before use. The schematic representation of anti-VEGF antibodies functionalized QD nanoparticles was shown in Fig. 1. Characterization of quantum dots and quantum dot–monoclonal antibody conjugates Emission spectra were collected using a RF-5301 spectrophotometer (Shimadzu Corp., Tokyo, Japan). Absorption spectra were measured using a UV-1800 spectrophotometer (Shimadzu Corp., Tokyo, Japan). Transmission electron microscopy (TEM) images were collected by a JEM-2100 (JEOL Ltd., Tokyo, Japan). Dynamic light scattering (DLS) analysis was done by ZetaPlus Zeta Potential Analyzer (Brookhaven, NY, USA). Samples mixed with × 5 concentrated sample buffer containing 10% sodium dodecyl sulfate (w/v) and 5% β-mercaptoethanol (v/v) were loaded on a 12% acrylamide gel. The voltage was set at 120 V, after then the gel plate was stained with Coomassie Blue. Electrophoretic gel was imaged using a ChemiDoc XRS system (Bio-Rad, CA, USA). Cell culture and fluorescence microscopic imaging The cultured MCF-7 cancer cells were seeded into 24-well plates. The cells were then kept in a CO2 incubator at 37°C for 24 h. QDs and QD–mAb bioconjugates were incubated with the MCF-7 cells or HEK293 cells for 2 h at 37°C before samples were washed
Figure 1. The schematic illustration of antivascular endothelial growth factor antibodies functionalized quantum dots. EDC, N-ethyl-N′-[3 (dimethylamino) propyl] carbodiimide hydrochloride; NHS, N-hydroxysuccinimide; TGA, thioglycolic acid.
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Characterization of antibody conjugated QDs for cancer cell imaging three times with PBS and photographed using an inverted fluorescence microscope equipped with CCD camera (Olympus IX71, Tokyo, Japan). MTT viability assay The MCF-7 human breast cancer cells were cultured on 96-well microtiter plates and incubated with different concentrations of CdTe, CdTe/ZnS and CdTe/ZnS-mAb for 48 h, with three duplicates for each concentration. Then the cells were incubated with MTT reagents (5 mg/mL in PBS) for 4 h at 37°C. After incubation, the cells were lysed with dimethyl sulfoxide on the plate. The absorbance at 490 nm was measured on a microplate reader (Bio-Rad).
Results and discussion Spectral characterization of CdTe and CdTe/ZnS quantum dots Fluorescence and absorption spectra were used to characterise the optical properties of the QDs. Figure 2(a,b) shows the excitonic absorption peak of nanoparticles shifted to the longer wavelengths from 499 nm to 540 nm, indicating the growth of a ZnS shell on top of the CdTe core. Meanwhile, the emission peak (Fig. 2c,d) shifted from 545 nm to 583 nm as the particles’ size increased (21). The fluorescence spectra also displayed that the peak width at half-maximum were maintained during the shell growth, about 39 nm, which indicated the aforementioned core/shell QDs had a good monodispersity. Moreover, the luminescent quantum yields of core/shell CdTe/ ZnS at room temperature was measured and calculated as up to 59% , while it was only 36% for naked CdTe QDs, using rhodamine 6G as a reference (22). Bioconjugation of quantum dots with antivascular endothelial growth factor monoclonal antibodies Emission spectra of CdTe/ZnS QDs and QD–mAbs were shown in Fig. 3. The maximum emission peak of QD–mAb was blueshifted from 583 nm to 577 nm after conjugation. We also observed that the fluorescent intensity was enhanced because of the rearranged gross electrostatic environment on the surface
Figure 3. Emission spectra of CdTe/ZnS QDs and QD–mAb conjugates. mAb, monoclonal antibody; QD, quantum dot.
of QDs. The two changes in the maximum emission peak were attributed to the conjugation between the carboxylic groups in QDs and the amino groups of the antibody by covalent bonds, which could weaken the interaction between dipole–dipole and shorten the Stokes shifts (23,24).
Size, morphology and stability Figure 4(a,b) shows the TEM and high-resolution TEM images of QD–mAb conjugates prepared in this study. The size distribution of conjugates based on TEM (Fig. 4a) was mono-dispersed. As shown in the high-resolution TEM image (Fig. 4b), the nanoparticles also had a good crystal structure (25). Compared to the TEM images of CdTe/ZnS QDs that were reported previously by our group (20), the size and morphology of QDs were not aggregated after the coupling of antibodies. The DLS technique (Fig. 4c) confirmed that the mean hydrodynamic diameter of QD–mAb conjugates in aqueous phase was about 10.9 nm. The results of the DLS experiments demonstrated that the hydrodynamic diameter included the size of the solvated capping agent (TGA) layer and anti-VEGF mAbs, which made the DLS size larger than the measured TEM size (26). Figure 4d shows the luminescence intensity of QD–mAb conjugates during different time. When the conjugates were stored at 4°C, over 94% of the initial response remained after a storage period of 5 days. These results indicated that the conjugates had acceptable reliability and stability.
Sodium dodecyl sulfate–polyacrylamide gel electrophoresis gel electrophoresis
Figure 2. Absorption spectra of CdTe and CdTe/ZnS QDs (a,b); emission spectra of CdTe and CdTe/ZnS QDs (c,d). FL, fluorescence.
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The electrophoresis technique is employed here to identify further the bioconjugation of QDs and proteins. Figure 5 shows the image of the stained gel plate. In sodium dodecyl sulfate– polyacrylamide gel electrophoresis, the covalent bonds between the antibody and the QD surface were stable (27), while the disulfide bonds used to link the heavy chain and the light chain in IgG were deoxidized by mercaptoethanol. As a result, two proteins (25 kDa, 50 kDa) were detected on the gel. Because the molecular weight of CdTe/ZnS–mAb was larger than the pure antibody, both of the two chains’ electrophoretic velocities of
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L. Pang et al.
Figure 4. (a,b) Transmission electron microscopy image of QD–mAb conjugates at different magnification(c) hydrodynamic diameter distribution graph of QD–mAb conjugates(d) luminescence intensity of QD–mAb conjugates during different storage time. mAb, monoclonal antibody; PL, photoluminescence; QD, quantum dot.
QD–mAb conjugates (lane a) were slightly slower than that of the anti-VEGF antibodies (lane b).
Imaging in live cells using quantum dots–monoclonal antibody conjugates Figure 6(a) shows the image of MCF-7 cancer cells in bright fields. When MCF-7 cells were incubated with QDs alone (Fig. 6b), almost no signal was detected. However, when QD–mAb conjugates were incubated with MCF-7 cells, a high-intensity fluorescent signal was detected based on the antibody–antigen reaction, as shown in Fig. 6(d) (28–30). To evaluate further the specificity of QD–mAb probes, human embryonic kidney cells (HEK293) were incubated with QD–mAb conjugates. As shown in Fig. 6(c), following such treatment no signal was observed, indicating that the QD–mAb conjugates can be used as a detectable probe for VEGF antigen detection in cancer cells (31,32).
Cytotoxicity evaluation As shown in Fig. 7, a dose-dependent decrease in cell viability was observed in bare CdTe sample. As the CdTe QDs’ concentration increased to 75 μM, the cell viability decreased to 27.2% sharply. In contrast, the viability of cells incubated with CdTe/ZnS QDs maintained greater than 66% even at nanoparticles loadings as high as 150 μM. We also observed that the growth trend of the MCF-7 cells was not significantly altered in all the CdTe/ZnS concentrations. It could be explained by the fact that CdTe QDs were encapsulated by the ZnS shell, which could reduce the possible loss of cadmium ions from the nanoparticles (33). It is worth noting that the cytotoxicity of CdTe/ZnS-mAb was also concentration dependent. With the increase of
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Figure 5. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis photography. Lane a: quantum dot–monoclonal antibody conjugates; lane M: protein MW marker; lane b: antivascular endothelial growth factor antibodies.
CdTe/ZnS-mAb in concentration, the cell viability decreases, which might be due to the therapeutic value of anti-VEGF mAbs. This therapeutic value can be attributed to the blocking effect of mAbs on VEGF165 binding to VEGFR.
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Characterization of antibody conjugated QDs for cancer cell imaging
Figure 6. (a) Image of MCF-7 cancer cells in bright field; (b) fluorescence image of MCF-7 cancer cells incubated with CdTe/ZnS QDs (30 μM) for 2 h; fluorescence images of HEK293 cells (c) and MCF-7 cancer cells (d) incubated with quantum dot–monoclonal antibody conjugates (30 μM) for 2 h, respectively.
with a bio-recognitive component can be applied in immunological cell imaging (34). It also could be used to detect the therapeutic effect of mAbs. Promising emerging fluorescent labels based on QDs and functional biological macromolecules were both specific to recognize cellular targets and bright for effective detection in real applications, such as cellular tracking (35), cancer diagnosis (36) and even cancer therapy applications (37). Acknowledgements The authors gratefully acknowledge the financial support received from the National Natural Science Foundation of China (grant no. 81173023), National Science and Technology Major Project (grant no. 2013ZX09301303-004) and National High Technology Research and Development Program 863 (grant no. 2012AA02A303).
Figure 7. Cell viabilities of MCF-7 cells by MTT viability assay relative to untreated controls (mean ± SD; n = 3). mAb, monoclonal antibody.
Conclusions Highly fluorescent CdTe/ZnS QDs were prepared by an aqueous approach and used as fluorescent labels for anti-VEGF mAbs for the detection of human VEGF. The results demonstrated that CdTe capped with ZnS experimentally was not cytotoxic to live cells. The CdTe/ZnS QD-conjugated anti-VEGF mAbs are capable of detecting the presence of human VEGF. The QD–mAb probes
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