BRCAA1 antibody- and Her2 antibody-conjugated amphiphilic polymer engineered CdSe/ZnS quantum dots for targeted imaging of gastric cancer
- Chao Li†1,
- Yang Ji†2,
- Can Wang3,
- Shujing Liang1,
- Fei Pan1,
- Chunlei Zhang1,
- Feng Chen1,
- Hualin Fu1,
- Kan Wang1 and
- Daxiang Cui1Email author
© Li et al.; licensee Springer. 2014
Received: 19 March 2014
Accepted: 3 May 2014
Published: 19 May 2014
Successful development of safe and highly effective nanoprobes for targeted imaging of in vivo early gastric cancer is a great challenge. Herein, we choose the CdSe/ZnS (core-shell) quantum dots (QDs) as prototypical materials, synthesized one kind of a new amphiphilic polymer including dentate-like alkyl chains and multiple carboxyl groups, and then used the prepared amphiphilic polymer to modify QDs. The resultant amphiphilic polymer engineered QDs (PQDs) were conjugated with BRCAA1 and Her2 monoclonal antibody, and prepared BRCAA1 antibody- and Her2 antibody-conjugated QDs were used for in vitro MGC803 cell labeling and in vivo targeted imaging of gastric cancer cells. Results showed that the PQDs exhibited good water solubility, strong photoluminescence (PL) intensity, and good biocompatibility. BRCAA1 antibody- and Her2 antibody-conjugated QD nanoprobes successfully realized targeted imaging of in vivo gastric cancer MGC803 cells. In conclusion, BRCAA1 antibody- and Her2 antibody-conjugated PQDs have great potential in applications such as single cell labeling and in vivo tracking, and targeted imaging and therapeutic effects' evaluation of in vivo early gastric cancer cells in the near future.
Gastric cancer is the second most common cancer and the third leading cause of cancer-related death in China [1, 2]. It remains very difficult to cure effectively, primarily because most patients present with advanced diseases . Therefore, how to recognize and track or kill early gastric cancer cells is a great challenge for early diagnosis and therapy of patients with gastric cancer.
We have tried to establish an early gastric cancer prewarning and diagnosis system since 2005 [4, 5]. We hoped to find early gastric cancer cells in vivo by multimode targeting imaging and serum biomarker detection techniques [6–9]. Our previous studies showed that subcutaneous and in situ gastric cancer tissues with 5 mm in diameter could be recognized and treated by using multifunctional nanoprobes such as BRCAA1-conjugated fluorescent magnetic nanoparticles, Her2 antibody-conjugated RNase A-associated CdTe quantum dots, folic acid-conjugated upper conversion nanoparticles, RGD-conjugated gold nanorods, Ce6-conjugated carbon dots, and Ce6-conjugated Au nanoclusters (Au NCs) [10, 11]. However, clinical translation of these prepared nanoprobes is always confounded by their in vivo biosafety. Development of safe and highly effective nanoprobes for targeted imaging and tracking of in vivo early gastric cancer cells has become our concern.
In the recent 10 years, quantum dots have been subjected to intensive investigations because of their unique photoluminescence properties and potential applications. So far, quantum dots have been used successfully in cellular imaging [12, 13], immunoassays , DNA hybridization [15, 16], and optical barcoding . Quantum dots also have been used to study the interaction between protein molecules or to detect the dynamic course of signal transduction in live cells by fluorescence resonance energy transfer (FRET) [18, 19]. These synthesized quantum dots have significant advantages over traditional fluorescent dyes, including better stability, stronger fluorescence intensity, and different colors, which are adjusted by controlling the size of the dots . Therefore, quantum dots provide a new functional platform for bioanalytical sciences and molecular imaging. However, some studies also showed that some kinds of quantum dots exhibited toxic effects such as cytotoxicity, tissue toxicity, and in vivo residues [21, 22]. How to develop safe quantum dots has become the concern of many scientists.
In our previous work, we also synthesized safe quantum dots such as Ag2S and AgSe [23, 24] and used them for in vitro cell labeling and targeted imaging of in vivo gastric cancer cells. However, their fluorescence signals are too weak to be used for long-time imaging and single cell tracking . How to prepare safe quantum dots with strong fluorescence signals has become a great challenge.
Cadmium oxide (CdO, AR), stearic acid (98%), selenium powder, octylamine (OA, 99%), 1-hexadecylamine (HAD, 90%), and diethylzinc (ZnEt2) were obtained from Aladdin Co., Ltd. (Xi'an, China). Trioctylphosphine oxide (TOPO, 98%), trioctylphosphine (TOP, 95%), poly(acrylic acid) (PAA, molecular weight (MW) 1,800), 1-ethyl-3-[3-dimethylaminoporpyl] carbodiimide hydrochloride (EDC, 98.5%), and N-hydroxysuccinimide (NHS, 98%) were obtained from Sigma-Aldrich Co., Ltd. (St. Louis, MO, USA). Bovine serum albumin (BSA, 99.9%) was purchased from MP Biomedicals Company (Santa Ana, CA, USA). Bis(trimethylsilyl) sulfide ((TMS)2S) was purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Liquid paraffin, chloroform, ethanol, hydrochloric acid (HCl), 2-(4-morpholino)ethanesulfonic acid (MES), N,N-dimethylformamide (DMF), paraformaldehyde, and Tween-20 were purchased from Sinopharm Chemical Regent Co., Ltd. (Shanghai, China).
Synthesis of CdSe and CdSe/ZnS core-shell QDs
Highly luminescent core-shell CdSe/ZnS QDs were prepared in high temperature via the pyrolysis of organometallic reagents in a coordinating solvent [26–28]. We select 200°C with and without HAD for synthesis of green- and red-emitting CdSe QDs. The molar ratio of CdO/Se/stearic acid in liquid paraffin was 1:1:4, and the crude QD products were purified by chloroform and ethanol. For the ZnS shell, equal molar ratios of (TMS)2S and ZnEt2 as precursors of Zn and S, and TOP/TOPO were used, and 90°C was used for shell growth. The final core-shell product was repurified and redispersed into aliquot chloroform for later use. About 10 ml of deionized water was added to the solution to prevent evaporation of chloroform for long-period storage (see Additional file 1 for synthesis details of QDs).
Synthesis and characterization of amphiphilic polymer
The amphiphilic polymer is synthesized as follows: in ambient temperature, 0.2 g of PAA (MW 1,800) was added to a flask containing 10 ml DMF. Under slight stirring for 1 h, 137 μl of OA was added, and the solution was continuously stirred for another 30 min. In an individual vial, 0.47 g EDC was dissolved in 0.5 ml DMF and injected to the reaction solution dropwisely. The reaction solution was mixed vigorously overnight to produce amphiphilic polymers (with 50% of the carboxylic acid functional groups modified with an aliphatic chain). Next, 0.25 M HCl was added drop by drop to the polymer solution under vigorous stirring, resulting in a milky and opaque colloid solution. The colloid was centrifuged at 8,000 rpm/min for 10 min. The supernatant was discarded, and the jelly-like precipitant was washed with 0.25 M HCl twice to remove any by-products and impurities. The final precipitate was collected and freeze dried to remove trace amounts of water, giving a dry, white powder. Fourier transform infrared (FTIR) spectroscopy (Equinox 55, Bruker, Karlsruhe, Germany) was used to verify the formation of amide bond and carboxylic groups.
Preparation and characterization of amphiphilic polymers conjugated with QDs
An aliquot of amphiphilic polymer powder was resuspended in MES buffer (0.1 mol/l, pH 6.0) for later use. As-prepared QDs (200 μl, 0.15 mmol) dissolved in chloroform and amphiphilic polymer solution (2.0 ml, 0.45 mmol) were added to 8 ml of deionized water in an open container. The solution was stirred and sonicated for 30 min until the chloroform evaporated completely in the final products. Afterward, the hydrated colloid (polymer-coated QDs, PQDs) was further purified by size exclusion chromatography (Superdex 75, Pharmacia Biotech, AB, Uppsala, Sweden), yielding a transparent, homogeneous, and strong fluorescent solution.
After purification, the purified solution was then concentrated under reduced pressure using a rotary evaporator at approximately 15°C. For assessment of the size distribution and monodispersity of the PQDs, the primal QDs of CdSe, CdSe/ZnS, and purified PQDs were pipetted onto a carbon transmission electron microscopy (TEM) grid; the solvents were wicked away slowly after 15 min. For the PQDs, the grids were counterstained with a 1% phosphotungstic acid solution (pH adjusted to 6) for 30 s. The staining solution was wicked away similarly. All of the prepared grids were imaged (TEM, JEM-2100 F system, JEOL Ltd., Tokyo, Japan) and compared to determine size distribution of the QDs and the degree of polymer coating. For further size analysis, the as-prepared QDs and PQDs were measured using Zetasizer Nano ZSP (Malvern Instruments, Ltd., Worcestershire, UK). In addition, the optical properties of the prepared CdSe, CdSe/ZnS, and PQDs were measured using UV-visible and fluorescence spectrophotometer (Cary 50 Conc, Varian, Palo Alto, CA, USA; F-4600, Hitachi, Tokyo, Japan). The QD concentration was determined using Beer's law after measuring the absorbance value using spectrophotometry [29, 30].
In order to estimate the surface charge and functional group character, we further characterized the polymer and PQDs by using 1% agarose gel electrophoresis. The agarose gel was prepared using standard techniques, and the prepared polymer and PQDs were added into the loading well. The gel was run in 0.5× TBE buffer (pH 8.0) for 30 min at 100 V and imaged with Tanon 2500 gel imaging system (Tanon, Shanghai, China) under 365-nm exciting light. Afterward, the gel was stained with lead acetate (1%) and potassium chromate (1%) for 5 min, respectively, and imaged in the Tanon 2500 system with white light to visualize the carboxyl group contained in the amphiphilic polymer. The migration rates of polymer and PQDs were compared to validate the success of QDs' surface coating.
Effects of pH and ionic strength on the stability of PQDs
In order to evaluate the effects of a wide pH range and high salt concentration on the colloidal stability of the PQDs, the PQD colloids were dispersed in varied pH buffers, PQDs/buffer = 1:1 (v/v), and pH ranged from 2 to 13 (Additional file 1: details of preparation of a series of buffer solutions). The resulting PL spectra were background-corrected, integrated, and normalized to the intensity of PQDs in pH = 7, set as 100%. The stability effect of ionic strength was carried out as follows: dispersions of PQDs were placed in fluorescence cuvettes (1-cm optical path) containing an equal concentration of PQDs but various concentrations of sodium chloride. The lack of volumes was replenished with deionized water (pH = 7). The PL emission from PQDs without NaCl added was set to 100%. The resulting PL spectra were normalized to the emission form slat-free solution.
Preparation of BRCAA1 antibody- and Her2 antibody-conjugated QD nanoprobes
The BRCAA1 monoclonal antibody was conjugated with red PQDs, whereas humanized Her2 monoclonal antibody was conjugated with green PQDs. The optimum mole ratio of PQDs to antibody is 5:3 . The cross-linking reaction was done by using standard EDC-NHS procedure in ambient temperature and dark place for 2 h with continuous mixing. The mixture was then purified by chromatography (Superdex 75, Pharmacia Biotech, AB, Uppsala, Sweden) to remove the free antibody residues. The resultant BRCAA1 antibody- and Her2 antibody-conjugated PQDs were stored at 4°C for later use.
Afterward, the prepared PQDs and specific monoclonal antibody conjunction were analyzed in 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, Beyotime, Shanghai, China). The gel was run in a standard SDS buffer for 90 min at 120 V. Firstly, the gel was imaged with UV light to determine PQD position, and then, the gel was stained with Coomassie Brilliant Blue fast staining solution and imaged with white light to determine protein position.
BRCAA1 antibody- and Her2 antibody-conjugated QDs for targeted imaging of MGC803 cells in vitro
The overnight incubated MGC803 and GES-1 cells were fixed with 4% paraformaldehyde for 10 min and permeated with 0.5% (v/v) Tween-20 for 20 min. Then, these cells were blocked for 20 min in PBS containing 1% (w/v) BSA. After being washed with PBS twice, these cells were incubated at 4°C with BRCAA1 antibody- and Her2 antibody-conjugated QDs overnight. Unbound probes were removed by washing three times with PBS. Afterward, these cells were imaged under a fluorescence microscope (TS100, ×400, Nikon Co., Tokyo, Japan) and laser scanning confocal microscope in oil immersion objective (Nikon A1si+, ×1,000).
After attaining the fluorescence images, the gastric cancer cells were dissociated from the glass culture dish and sectioned as routine for TEM imaging.
BRCAA1 antibody- and Her2 antibody-conjugated QDs for targeted imaging of gastric cancer cells in vivo
To quantitatively analyze the fluorescence intensity from PQD-labeled MGC803 cells, macro fluorescence images were acquired using PQD-labeled MGC803 cells which were diluted with PBS to a final concentration from 2 × 102 to 2,048 × 102 cells/200 μl. Afterward, 200 μl of the prepared cell solutions were added to polystyrene TC-treated 96-well microplates (Corning® Life Sciences, Corning, NY, USA, #3603). Fluorescence intensity was measured in a Bruker In-Vivo F PRO system (Bruker Corporation, UK), and the resulting background-corrected data was curve fitted to single exponentials. Signal curve fitting was done using the software Origin (OriginLab, Northampton, MA, USA; http://www.originlab.com/).
All of the following animal studies complied with current ethical considerations: Approval (SYXK-2007-0025) of the Institutional Animal Care and Use Committee of Shanghai JiaoTong University (Shanghai, China) was obtained. Nude mice (male, 18 to 22 g, 4 to 5 weeks old) were obtained from the Shanghai LAC Laboratory Animal Co. Ltd., Chinese Academy of Sciences (Shanghai, China, SCXK2007-0005), and housed in a SPF-grade animal center.
Pathogen-free athymic nude mice were housed in a vivarium accredited by our University. Male athymic nude mice (4 to 6 weeks old) were used to establish subcutaneous gastric cancer models; 1.5 × 106 MGC803 cells suspended in 100 μl DMEM were subcutaneously injected into the left anterior flank area of each mouse. Four weeks later, tumors were allowed to grow to approximately 5 mm in diameter, and the prepared Her2 antibody-conjugated QDs (red, emission peak 657 nm) were injected into the mice via the tail vein for 6 h. Whole-animal imaging and ex vivo organ imaging were performed using the Bruker In-Vivo F PRO system. The excitation and emission filters were set to 410 and 700 nm (band pass, ±15 nm), respectively, and exposure time was set to 3 s. Collected images were analyzed using the imageJ software (NIH ImageJ; http://rsb.info.nih.gov/ij/), which uses spectral unmixing algorithms to separate autofluorescence from quantum dot signals.
Results and discussion
Characterization of synthesized CdSe, CdSe/ZnS QDs, and PQDs
The emission peaks of synthesized CdSe, CdSe/ZnS, and PQDs (nm)
The FTIR spectrum of the primary CdSe, CdSe/ZnS, and PQDs shows that (Additional file 1: Figure S1, details of FTIR) the peak of CdSe at 2,760 ~ 2,930 cm-1 is the characteristic symmetric and asymmetric methylene stretching (v C-H) that comes from the cosolvent material used in synthesis  (Additional file 1: Figure S1a). In the FTIR spectrum of CdSe/ZnS QDs (Additional file 1: Figure S1b), the peak at 1,183 cm-1 is the characteristic symmetric and asymmetric stretching vibrations from TOPO (vP=O) [37, 41]. After transferring from the hydrophobic phase to the hydrophilic phase, for PQDs (Additional file 1: Figure S1c), many peaks emerged. The peak at 1,728 cm-1 is the vibration from C = O of the synthesized polymer (v C = O), and the peaks emerging at 1,609 and 1,310 cm-1 are the characteristic asymmetric and symmetric stretching vibrations from COO- groups (v COO-) . The difference in the FTIR spectrum of these QDs is an excellent evidence to prove that the PQDs had been successfully modified by the amphiphilic polymer.Figure 3d shows a comparison of the mobility shift of the amphiphilic polymer and 657-nm-emitting PQDs capped with the amphiphilic polymer. After 30 min of electrophoresis, the amphiphilic polymer cannot been seen in this UV condition (Figure 3d, left panel, lane 1). Meanwhile, the PQDs show a bright and narrow predominant band under the 365-nm UV light (Figure 3d, left panel, lane 2). This suggests that the synthesized PQDs are homogeneous. Afterward, the gel was stained with lead acetate and potassium chromate, and the carboxyl group was stained with lead chromate and had a dark yellow color. Under room light, the amphiphilic polymer and PQD (containing carboxyl groups) migrations can be seen clearly (Figure 3d, right panel).
Stability of synthesized PQDs
In addition to the pH stability, we investigated the behavior of the PQDs in aqueous solutions with different ionic strengths. In the experiment, the PL properties of PQDs dispersed in PB buffer solutions at neutral pH were monitored, with NaCl concentration increased from 0 to 300 mM. Over the concentration range of NaCl, we observed little decrease in PL intensity and no change of the emission spectra for PQDs (Figure 4b, the PL intensity without NaCl added was set to 100%). This result is very similar with the previous reports [44, 45]. These results of pH and ionic strength stability further highlight that the PQDs may be completely tolerant to intracellular and in vivo environments, where the ionic concentration is known to be less than 150 mM .
The efficiency of PQDs conjugated with antibody
The prepared PQDs and PQD-antibody probe characteristics were assessed by 8% polyacrylamide gel electrophoresis (SDS-PAGE) . In this experiment, the synthesized PQDs, monoclonal antibody, and PQD-antibody conjugation were added to specimen insertion ports, named lanes 1, 2, and 3, respectively. To avoid the acidic quenching effect on PQDs (the destaining solution contains acetic acid, based on the anterior results), after running with SDS buffer for 90 min, the gel was imaged on the Tanon 2500 gel imaging system with UV light (365 nm) in advance. To validate the coupling reaction, the gel was stained with Coomassie Brilliant Blue fast staining solution and washed with destaining solution. The stained gel was imaged again in white light. A comparison of the UV image with the image obtained by staining with Coomassie Blue is shown in Figure 3e. Apparently, in lane 1, the PQDs showed a clear bond which cannot be seen in bright fields (Figure 3e, left and right panels, lane 1). For monoclonal antibody, no signal can be detected in UV light but it is fairly visible in bright fields (Figure 3e, left and right panel, lane 2). However, in the conjugation of PQD-antibody, the band clearly can be seen both in UV light and bright fields; both of the migration ratios in different imaging conditions are identical (Figure 3e, left and right panels, lane 3). This result suggested that the conjugation between monoclonal antibody and PQDs is successful.
Coupling rate measurements of PQD-antibody
Total concentration (ng/ml)
The residue concentration (ng/ml)
Coupling rate (%)
Total concentration (ng/ml)
The residue concentration (ng/ml)
Coupling rate (%)
Effects of PQDs on cellular viability
BRCAA1 monoclonal antibody-conjugated QDs for in vitro targeted imaging
BRCAA1 monoclonal antibody-conjugated QDs for in vivo targeted imaging
As shown in Figure 9c, after subcutaneous injection of different amounts of PQD-labeled MGC803 cells in the athymic nude mouse, the fluorescence signals were observed in the injection sites and there was a steady increase of the fluorescence intensity in the injection sites with the increment of PQD-labeled MGC803 cells. As can be seen in injection site 1, merely 32 × 102 PQD-labeled cells could provide a significant fluorescence signal. The fluorescence signal of in vivo imaging shows that MGC803 cells were successfully labeled with PQDs.
In conclusion, BRCAA1 monoclonal antibody- and Her2 antibody-conjugated amphiphilic polymer-modified core-shell CdSe/ZnS quantum dots were successfully prepared, exhibited good biocompatibility and strong stable fluorescence signals, and were successfully used for in vitro and in vivo targeted imaging of gastric cancer MGC803 cells. High-performance BRCAA1 antibody- and Her2 antibody-conjugated amphiphilic polymer-modified core-shell CdSe/ZnS quantum dot nanoprobes exhibit great potential in applications such as molecular imaging and therapeutic effect evaluation of early gastric cancer in the near future.
This work is supported by the National Key Basic Research Program (973 Project) (No. 2011CB933100), National Natural Scientific Fund (Nos. 81225010, 81327002, and 31100717), 863 project of China (2012AA022703), Shanghai Science and Technology Fund (No. 13NM1401500), and Shanghai Jiao Tong University Innovation Fund for Postgraduates (No. AE340011).
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