Effective cellular internalization of silica-coated CdSe quantum dots for high contrast cancer imaging and labelling applications
© Vibin et al.; licensee Springer 2014
Received: 10 June 2013
Accepted: 29 April 2014
Published: 27 June 2014
The possibility of developing novel contrast imaging agents for cancer cellular labelling and fluorescence imaging applications were explored using silica-coated cadmium selenide (CdSe) quantum dots (QDs). The time dependent cellular internalization efficiency study was carried out using Inductively Coupled Plasma-Optical Emission Spectroscopy (ICP-OES) and Confocal Laser Scanning Microscopy (cLSM) after exposing QDs to stem cells and cancer cells. The strong fluorescence from the cytoplasm confirmed that the QDs were efficiently internalized by the cells. The internalization maxima were observed at the fourth hour of incubation in both stem and cancer cells. Further, the in vitro fluorescence imaging as well as localization study of QDs were performed in various cells. Moreover, high contrast in vivo tumor imaging efficiency of silica-coated CdSe QDs was performed in ultrathin sections of tumor mice, and the results confirmed its effective role in cellular imaging and labelling in cancer and other diseases.
The development of highly sensitive and specific biological probes which lack the intrinsic limitations of organic dyes and fluorescent proteins is focus of many areas of research like molecular and cellular biology [,] and medical diagnostics [–]. The conventional organic fluorescent tags mainly suffer from low photo stability, poor quantum yield under biological conditions and interference from auto fluorescence. The exceptional photophysical properties of quantum dots (QDs), particularly photostability and emission as a function of size, make them superior to organic dyes for biological applications. These properties have opened new possibilities for advanced molecular and cellular imaging as well as for ultrasensitive bioassays and diagnostics [–]. Besides, QDs are better labelling agents in long-term imaging such as fluorescence marking of transport processes in cells and in tracking the path of single membrane-bound molecules [,].
Although imaging of fixed cells is useful and sufficient for many applications, live cell microscopy is ideal for visualizing cellular processes, which is considerably more difficult. It has been shown that many cell types naturally engulf QDs through a non-specific uptake mechanism. This mechanism was used to track the migration of breast tumor cells on a substrate coated with red emitting QDs; the fluorescence within the cells were increased due to the uptake of QDs, leaving behind a dark path [–].
Many reports highlight the use of QDs as a fluorescent probe to visualize the biological processes both in vitro and in vivo [–]. With proper surface functionalization using peptides, proteins or antibodies, QDs shows specificity in targeting and imaging tumors in living subjects. The fluorescence imaging can easily be achieved by monitoring the stable and strong fluorescence from the QDs []; Law et al. []. These studies confirm that QDs have opened up a new avenue for investigating biomolecular processes inside the cells.
The inherent hydrophobicity of QDs can be overcome by various approaches; among these overcoating with silica is most preferred method [–]. Besides, the cytocompatibility of CdSe QDs on overcoating with silica has been well established by many groups [–]. This is based on the observation that the core constituents viz., cadmium and selenium ions are well encapsulated with in the silica shell preventing surface oxidation, even in the biological media. Thus a silica shell plays dual role i.e., it makes QDs dispersible in aqueous media and it eliminates the toxicity. A detailed investigation of the cytocompatibility of silanised QDs was reported by our group []. By time and concentration dependent studies we observed that the internalized silica-coated CdSe QDs were non-toxic proving the cytocompatibility even at higher concentrations and longer incubation periods. We have also reported the non-specific cellular uptake and subcellular localization of silica-coated CdSe quantum dots [].
Several research groups have described the use of QDs for sensitive bioassays and cellular imaging in vitro and in vivo [–]. But many aspects of this approach need to be further optimized particularly for in vivo applications. Our preliminary in vitro investigations proved the cytocompatibility and stability of silica-coated CdSe QDs under biological conditions []. The objective of this study was to demonstrate that the silica-coated CdSe QDs can be used as labelling agents for long-life cellular imaging, cancer cellular imaging using cLSM and fluorescence microscopy. We use Inductively Coupled Plasma-Optical Emission (ICP-OES) Spectroscopy to quantify the in vitro cellular internalization efficiency of silica-coated CdSe QDs in a couple of cell models, New Zealand rabbit adipose derived mesenchymal stem cells (RADMSCs) and Human cervical cancer cells (HeLa). Imaging using Confocal microscopy is also performed to obtain a visual image of the QDs internalised by cells after incubation for a time interval, assessed from the results of ICP-OES studies. Simple fluorescence imaging and localization of the silica-coated CdSe QDs studies using fluorescent microscopy after staining with 4,6-diamidino-2-phenylindole (DAPI) have also been carried out in RADMSCs and HeLa cells. The high cellular internalisation efficiency as well as the high contrast confocal images of tumor sections obtained indicates the role of silica-coated CdSe QDs in imaging and labelling of cancer cells and other diseases.
Chemicals for QD synthesis and silica overcoating such as trioctylphosphine oxide (TOPO), trioctylphosphine, cadmium oxide (CdO), selenium powder, dodecylamine, igepal and aminopropyl silane (APS) were purchased from Sigma–Aldrich and tetradecylphosphonic acid (TDPA) from Alfa Aesar and used as such without further purification.
Synthesis and characterization of silica-coated CdSe QDs
QDs were synthesized by following a reported procedure [,]. Cadmium oxide (0.067 g, 0.52 mmol), dodecylamine (3.8 g, 20.72 mmol), trioctylphosphine oxide (2.7 g, 6.9 mmol) and tetradecylphosphonic acid (0.40 g, 1.44 mmol) were heated to 300°C under vacuum, until CdO dissolved completely to produce an optically clear solution. At this temperature, an injection mixture containing trioctylphosphine (5.2 mL) and Se (0.083 mmol) was introduced. After desired crystal growth, the reaction was arrested by reducing the reaction temperature down to ambient conditions. The QDs were purified by reprecipitation with methanol and redispersed in dry chloroform for silica overcoating.
For overcoating CdSe QDs with silica, we have modified the reported procedure [18, 24] as follows: A mixture of TOPO capped CdSe QDs in chloroform (400 μL) and APS (0.075 g, 0.36 mM) was vortexed for 30 min, in an inert atmosphere. This mixture was added to Igepal CO-520 (1.3 mL) in cyclohexane (10 mL) and stirred for 30 min under dry conditions. Ammonia solution (150 μL, 33 wt.%) was added drop wise and the stirring was continued for 1 day. The silica-coated QDs were purified by washing repeatedly with dry chloroform and redispersed in PBS buffer (pH 7.3). Dots were stored in the dark at room temperature and the emission stability was investigated as a function of time, pH and ionic strength of the medium.
The electronic absorption spectra were recorded on a Shimadzu UV-3101 scanning spectrophotometer; emission spectra were collected using SPEX-Fluorolog F112X spectrofluorimeter. For HRTEM studies, a drop of nanoparticle solution was placed on a carbon coated Cu grid and the solvent was allowed to evaporate. Specimens were imaged on a FEI Tecnai G2 S-TWIN 300 kV high resolution transmission electron microscope.
Materials and animals
For cellular imaging experiments, all chemicals and reagents used were purchased from Sigma–Aldrich, USA. The New Zealand rabbit adipose tissue-derived mesenchymal stem cells (RADMSCs) and Human cervical cancer cells (HeLa) were maintained at 37°C and 5% CO2 in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and antibiotics.
The animal models used for the tumor imaging study (Normal male, Swiss albino mice of 6 weeks old, weighing 20–25 g) were from the animal house facility, Department of Biochemistry, University of Kerala. For maintaining the experimental animals, the institutional ethical guidelines were absolutely followed as per Committee for the Purpose of CPCSEA rules [Sanction No: IAEC-KU-9/06-07/BC-AA (8) (ii)], Government of India. All animal experiments were performed in triplicate.
Solid tumor development
The solid tumor model was established by subcutaneous injection of DLA cells (1 × 106 cells/animal) into the back right hind limb of mice []. The mice were subjected to imaging studies when the tumor volume reached 200–450 mm3 (3–4 weeks after inoculation).
Percentage of cellular internalization of silica-coated CdSe QDs – ICP-OES
For silica-coated CdSe QDs internalization studies, HeLa and RADMSCs were seeded 1 × 106 cells/well in 6-well plates and incubated overnight to allow for cell attachment. After the overnight incubation, cell medium was removed and replaced with 1 mL of DMEM medium. Cells were serum starved for 30 min and fresh medium containing the silica-coated CdSe QDs was added. Cells were incubated for different time interval (0, 1, 2, 3, 4 and 5 h) in the presence of silica-coated CdSe QDs. At each time point, cell supernatant was removed and cells were washed three times with PBS. Samples were digested in 500 μL of nitric acid for 30 min at 70°C and diluted in 3 mL of double distilled water. Quantum dots internalization was quantified using ICP-OES (Optima 5300 DV instrument, Perkin Elmer). All experiments were performed in triplicate.
Cellular internalization studies of silica-coated CdSe QDs – cLSM
To study the cellular internalization efficiency of the silica-coated CdSe QDs in vitro, RADMSCs and HeLa cells were cultured on coverslips and the medium was changed on every 2 days, till 85% cell confluence was achieved. Then the cells were incubated at 37°C with 5% CO2 for 2 and 4 h after adding with silica-coated CdSe QD solution (100 nM) in PBS. After incubation, the coverslips were taken out, rinsed thrice at 37°C using pre-heated PBS, fixed with 3.7% paraformaldehyde and fluorescence images were observed in a confocal laser scanning microscope (cLSM). An argon laser (488 nm) was used for QD excitation under confocal fluorescence microscope (Carl Zeiss LSM 510 META). All experiments were performed in triplicate.
Cellular imaging and localization study using silica-coated CdSe QDs – Fluorescence microscopy
For cellular imaging experiment, stem cells (RADMSC) and cancer cells (HeLa) were used. RADMSCs and HeLa were cultured on coverslips, and the medium was changed every 2 days, until 85% cell confluence was achieved. Then the cells were incubated at 37°C with 5% CO2 for 4 h after adding with silica-coated CdSe QD solution (100 nM in PBS). Then, silica-coated CdSe QD solution (100 nM) was added to the cells. After incubation, the coverslips were taken out, rinsed thrice at 37°C using pre-heated PBS, fixed with 3.7% paraformaldehyde, after staining with 4,6-diamidino-2-phenylindole (DAPI) and fluorescence cellular images were observed using a DM 6000 fluorescence microscope (Leica, Germany, 20x objective, equipped with DFC 300 FX digital camera).
In the case of fluorescence imaging of cancer cells - Daltons Lymphoma Ascites (DLA) cells, were procured from Amala Cancer Research Centre, Thrissur, India and maintained in the peritoneal cavity of mice. Approximately 2 weeks were taken for the development of tumor in the peritoneal cavity and matured cells were aspirated from the peritoneum, washed with PBS and seeded on coverslips. Then the cells were incubated at 37°C with 5% CO2 for 2 and 4 h after adding with silica-coated CdSe QD solution (100 nM) in PBS. After incubation, the coverslips were taken out, rinsed thrice at 37°C using pre-heated PBS, fixed with 3.7% paraformaldehyde and fluorescence images were observed in a DM 6000 fluorescence microscope (Leica, Germany, 20x objective, equipped with DFC 300 FX digital camera).
Fluorescence imaging of ultrathin sections of tumor– cLSM
Tumor mice were anesthetized by intra-abdominal injection of 3% pentobarbital sodium (45 mg/kg). QD and QD-Ab probe were (10 nM) injected at the dosage of 20 ml/kg into the tail vein. After the injection, the mice were put in a dark chamber for 4 h, and then tumor region were stripped from the mice. Then tumor tissues was fixed and sectioned by ultramicrotome (LKB; Bromma-2088-Ultratome®V, Sweden), to obtain 100 nm ultrathin sections of tumor. For fluorescence imaging, these ultrathin tissue sections were directly examined with a cLSM. The ultrathin sections were stained with toludine blue and observed under a light microscope (Leica-DMIL, Germany).
Results and discussion
The in vitro cellular internalization efficiency of silica-coated CdSe QDs is systematically followed in this report. Cytocompatibility assessment experiments based on various assays showed that the silanised QDs were non-toxic, aqueous soluble and showed stable fluorescence under biological conditions. The ICP-OES measurements in stem cells (RADMSC) and in cancer cells (HeLa) confirmed that silica-coated CdSe QDs have excellent effective internalization efficiency and the peak concentration was observed after 4 hours. In addition, the high contrast images obtained in confocal laser scanning microscopy (cLSM) from the in vitro cellular imaging study on stem cells and cancer cells and in vivo tumor imaging studies further confirmed that these QDs are highly useful for cellular imaging and labelling applications using their relatively stable fluorescence emission under biological conditions. Overall, this study implies that silica-coated CdSe QDs could be used as labelling and imaging agents for cancer cellular imaging and cell tracking applications for the study of cancer and other diseases.
This work was supported by the research grant from the Department of Biotechnology, Ministry of Science and Technology, Govt. of India, New Delhi (Order No. BT/PR9904/NNT/28/63/2007) to the corresponding author Prof. Annie Abraham. We thank Prof. K. George Thomas, Dean, Indian Institutes of Science Education and Research (IISER), Thiruvananthapuram, India for providing nanomaterials for the study. Thanks are also due to Dr. T.V. Anilkumar, SCTIMST, Thiruvananthapuram for confocal microscopy, and Dr. H. K. Varma for the ICP-OES analysis.
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