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One step synthesis of quantum dot–magnetic nanoparticle heterodimers for dual modal imaging applications† Jiyeon Lee,*a,b Gyoyeon Hwang,a,b Yeon Sun Honga,c and Taebo Sim*a,b,c Dual modal nanoprobes are promising tools for accurately detecting target molecules as part of the diagnosis of diseases including cancers. We have explored a new dual modal bioimaging probe that is comprised of a quantum dot (QD)–magnetic nanoparticle (MNP) hybrid. The MNP–QD heterodimers explored are fabricated by using a platinum–guanine coordination bonding guided self-assembly process, employing the metal–DNA conjugation method. Investigations utilizing energy dispersive spectroscopy
Received 17th December 2014, Accepted 23rd February 2015 DOI: 10.1039/c4an02322f www.rsc.org/analyst
(EDS) equipped high resolution transmission electron microscopy (HRTEM) demonstrate that the heterodimer contains an iron (Fe) dominant MNP and a cadmium (Cd) dominant QD. Finally, the results of cell studies show that the MNP–QD conjugates display good HeLa cell uptake in the absence of non-specific binding to the cell membrane and, as such, they can be used to label cells in vitro and in vivo as part of a new cell imaging technique.
Introduction Quantum dots (QDs) have excellent fluorescence intensity and photostability and, as a result, they are widely used as bioimaging probes.1,2 In addition, QDs serve as substrates in reactions that lead to the introduction of surface functional groups that enable conjugation with various biomolecules.3 This capability enhances their use as bioimaging agents4,5 and biosensors.6,7 Because of the high potential applications of QDs in a wide range of therapeutic situations, improvement in the efficiency of their binding to a wide range of biomolecules remains a challenge. This issue has driven the development of a variety of new conjugation protocols.5,8,9 To widen their targeting ability, dual modal bioimaging probes, which employ fluorescence with magnetism as detecting methods, have been developed as part of attractive strategies to image biomolecules.10,11 Magnetic nanoparticle (MNP)–QD conjugates have attracted great interest owing to their unique fluorescence and magnetic properties.12 Probes of this type have been
a Chemical Kinomics Research Center, Future Convergence Research Division, Korea Institute of Science and Technology, Hwarangno 14-gil 5, Seongbuk-gu, Seoul 136-791, Korea. E-mail: [email protected], [email protected], [email protected] b Biological Chemistry, Korea University of Science and Technology, 217, Gajeong-ro, Yuseong-gu, Daejeon, Korea c KU-KIST Graduate School of Converging Science and Technology, 145, Anam-ro, Seongbuk-gu, Seoul, 136-713, Korea † Electronic supplementary information (ESI) available: Additional images of HRTEM, the results of gel mobility assay, TEM images of 10 nm MNP–QD605, and cellular uptake images of MNPs and 10 nm MNP–QD800. See DOI: 10.1039/ c4an02322f
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applied to the new fluorescence molecular tomography (FMT) technique that combines fluorescence imaging with positron emission tomography (PET), computed tomography (CT), and magnetic resonance imaging (MRI).13,14 Several approaches to prepare QD–MNP nanoconjugates for use in FMT have been described,10,15 but most display low fluorescence or magnetic detection sensitivities compared to those of the individual nanoparticles. Recently, we developed a new method for assembling QD–MNP nanoconjugates that relies on platinum–guanine bonding to link the component QDs and MNPs.12 In that effort, we observed that the fluorescence intensity of the QD component and the magnetism of the MNP component of the platinum containing QD–MNP are not significantly altered by dual modal nanoconjugate formation. Although the results of our earlier work demonstrated that QD–MNP nanoconjugates can be used as FMT probes, further studies are required to determine if the new nanoconjugates interact with and/or internalized by cells. In addition, in order to apply nanoconjugates of this type as in vivo probes, the QD components must be responsive to light in the near infrared region. These two goals have been addressed in the studies described below, in which near infrared MNP–QD nanoconjugates were prepared and evaluated for their cell uptake propensities.
Experimental Materials Quantum dots (QDs, QD605 and QD800), fetal bovine serum, and penicillin/streptomycin were purchased from Invitrogen
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(USA). Magnetic nanoparticles (MNPs) were purchased from Ocean Nano Tech (USA). Ethylenediamine, cis-PtII(DMSO)2Cl2, Prussian blue staining reagent, and N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) were obtained from Sigma-Aldrich (USA). N-Hydroxysulfosuccinimide sodium salt (Sulfo-NHS) was purchased from Pierce (USA). Amine-C12-GGGGGG oligonucleotide was synthesized by Bioneer (Korea). Dulbecco’s modified Eagle’s medium (DMEM) and Dulbecco’s phosphate buffered saline (DPBS) were obtained from Welgen Inc. (Korea). Platinum conjugation of QDs The starting QDs (QD605 and QD800), modified with amino polyethylene glycol (PEG), were modified to introduce ethylenediamine moieties using (10-(2,5-dioxopyrrolidin-1-yl)10-oxodecanoic acid) by using the procedure described earlier.12 cis-PtII(DMSO)2Cl2 was added to diamine modified QDs followed by incubation for 30 min at room temperature. The fluorescence intensities of the QD–Pt conjugates were analyzed by using a Flexstation 3 Microplate Reader (Molecular Devices, USA). Guanine conjugation of MNPs Carboxylic acid modified 10 nm iron oxide MNPs were reacted with amine-C12-GGGGGG using sulfo-NHS and the EDC coupling technique.12 The MNP to 6G conjugation ratio was adjusted to be 1 : 50. After 3 h incubation at room temperature, unreacted DNA and reagent were removed by washing the filtrate using a 100k centrifugal filter. Binding QD–Pt to 6G–MNP and characterization of the hybrid The nanoconjugates were formed using equimolar concentrations of the QD–Pt and 6G–MNP conjugates with 3 h incubation at room temperature. Nanoparticle concentrations were determined by using scanning UV spectroscopy with a Flexstation 3 Microplate Reader. DLS analysis was performed using Nano-ZS (Malvern, UK) at room temperature. QD–MNPs were applied to a carbon film TEM grid (EMS, Hatfield, PA, USA) and observed using cryo TEM and Technai TEM (KIST, Seoul, Korea). EDS equipped with Technai was used for analysis of the composition of the nanoparticles. Zeta potential measurements were analysed through Nano-ZS (Malvern, UK). Cell culture and imaging Human cervical adenocarcinoma (HeLa) cells were cultured with DMEM, supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. HeLa cells were applied to 8-well glass chamber slide dishes and the QD–MNP conjugates in cell complete medium were added. After 24 h, cells were fixed by using 4% formaldehyde. The cell nuclei were stained using DAPI (1 : 100 dilution of 1 mg mL−1 in DPBS) for fluorescence imaging and Prussian blue reagent for MNP imaging. The cells were then imaged using Nikon Ti and Olympus IX2, and the images were edited with Nikon element software and ImageJ program.
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In vitro MRI HeLa cells (2 × 105) were seeded in each well of a 12-well plate. After 24 hours incubation, 50 nM of MNP, MNP–QD605, and MNP–QD800 in only DMEM (without FBS) were added to each well and incubated for another 24 hours. Cells were harvested using trypsin-EDTA and fixed using 4% formaldehyde in PBS. After washing with PBS, cells were collected by centrifuging at 3000 rpm for 5 min. Three layers of gelatin, gelatin including cells, and gelatin were added to each tube. Phantoms were scanned in a 3 T magnetic resonance imaging scanner (MAGNETOM 3 T Trio, SIEMENS Medical Systems) in Korea Institute of Radiological and Medical Sciences (KIRAMS) using a T2-weighted turbo spin echo imaging sequence with a repetition time (TR) of 5000 ms and TE of 118 ms, with a field of view (FOV) of 130 × 56 mm2, imaging matrix of 226 × 320, and one slice with 3 mm slice thickness. The fluorescence intensity of cell pellets before and after applying a magnetic field was estimated through the OV100 imaging system (Olympus).
Results and discussion Conjugation of near infrared QD with platinum Previously, we reported that QD605 with a fluorescence maximum of 605 nm (red) can be conjugated with platinum with QD to Pt ratios of 1 : 5 and 1 : 10 without undergoing a significant decrease of emission efficiency.12 Here, we demonstrate that the near infrared (800 nm) emitting QD (QD800) can also be conjugated with platinum using the previously reported conjugation method without undergoing a significant change in its fluorescence efficiency. For this purpose, QD800– Pt conjugates in three different (1 : 5, 1 : 10 and 1 : 20) QD to Pt molar ratios were prepared by previously reported method.12 Fluorescence spectra of the QD800–diamine and QD800–Pt conjugates at the same molar concentration were recorded (Fig. 1(A)) in order to assess the effect of the Pt content on emission efficiencies. Interestingly, the QD800–diamine–Pt conjugate with the 1 : 5 QD to Pt ratio has the same fluorescence efficiency as QD800–diamine. However, because of the general phenomenon that the fluorescence intensity of QDs usually decreases when they are bound to other materials, increases in the ratio of platinum in the QD–diamine–Pt (1 : 10 and 1 : 20) causes the emission efficiencies to decrease. The number of Pt atoms on the surface of the QD800–Pt was analyzed by ICP-MS (Fig. 1(B)). Even though the fluorescence efficiency of the 1 : 10 QD800–Pt conjugate is slightly less than those of the starting QD800–diamine and the 1 : 5 QD800–Pt conjugate, the 1 : 10 QD800–Pt conjugate was employed because of the higher platinum ratio in the studies aimed at determining the binding of this conjugate to MNPs to form dual modal nanoprobes. Self-assembling QD800–Pt conjugates with 6G–MNP and their characterization In order to form dual modal nanoprobes with the QD800–Pt conjugate, the coordination bonding of Pt from QD800–Pt con-
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Fig. 1 QD800–Pt conjugates characterization. (A) Photoluminescence (PL) intensity of QD800–diamine and QD800–Pt conjugates with three different QD to Pt ratios (1 : 5, 1 : 10, 1 : 20). The fluorescence intensity is not decreased with the 1 : 5 QD800–Pt conjugate, but 1 : 10 and 1 : 20 QD800–Pt conjugates have slightly decreased fluorescence intensities compared to that of QD800–diamine. (B) ICP-MS measurement results indicating that the number of Pt atoms on each QD are 1.6, 2.7, and 5.6, respectively.
jugates to guanines from MNPs depicted in Fig. 2(A) was used. Simply, carboxylic acid modified MNPs were conjugated with amino-GGGGGG (6G) oligonucleotide using carbodiimide chemistry to yield the functionalized MNP–6G. A mixture containing a 1 : 1 ratio of the 1 : 10 QD800–Pt conjugate and MNP– 6G was incubated and unreacted reagents were removed by a centrifugal filter and exclusion column chromatography. To investigate the formation of the MNP–QD800 conjugate, TEM images were obtained from the conjugates (Fig. 2(B)) and the mixture of QD–diamine with carboxylic acid modified MNPs (Fig. 2(C)). We found more heterodimers in MNP–QD800 conjugates rather than in the mixture of QD–diamine and MNPs. Even if TEM images show that MNP–QD800 heterodimer is likely to be formed, it is hard to distinguish QD from MNP by using only the TEM imaging method because the average size and the shape of a QD800 and a MNP are similar. As a result, HRTEM and EDS analyses were carried out to ensure the formation of MNP–QD800 conjugates. In Fig. 3(A) an HRTEM image of the heterodimer that is generated by conjugated MNP–6G and QD800–Pt is shown. Furthermore, EDS spectral analysis (Fig. 3(B)) of the image displayed that the nanoparticle component of the conjugate numbered 1 in the figure is Fe rich while the nanoparticle numbered 2 is Cd rich. The presence of peaks associated with carbon (C) and copper (Cu) in the spectra is negligible because they were from TEM grids. Hence, the results confirm that a new 1 : 1 heterodimer, formed by platinum–guanine bonding, is generated by MNP–6G and QD800–Pt conjugates. MNP–QD800 conjugates were evaluated for the propensity for cellular uptake and used as a cell imaging probe. The size of a dual modal probe is an important factor determining whether or not it will undergo uptake by cells.16,17 Jiang et al.
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Fig. 2 Self-assembling MNP–QD800 conjugates. (A) Schematic representation of QD–MNP conjugation. Platinum of QD connects to guanine of MNP to form the coordination bonds. (B) TEM images of the conjugated MNP–QD800. Guanine modified MNPs (MNP–6G) bonded to platinum linked QD800 (QD800–Pt) to form heterodimers, MNP– QD800. (C) TEM images of the mixture of QD800–diamine and MNP. They did not form heterodimers.
reported that the most efficient uptake occurs with gold nanoparticles that are in the 25–50 nm size range.17 In addition, it is known that smaller nanoprobes more readily penetrate cell membranes than their larger counterparts.18 To investigate whether this new heterodimer has the optimal size as a cell imaging probe, the MNP–QD800 conjugates were characterized by using DLS. The DLS data show that the hydrodynamic size distribution of MNP–QD800 conjugates is shifted to the right with an average size of 50.05 nm (Fig. 3(C)). Because the average size of starting QD800–diamine and MNP–6G are each 30 nm in the DLS data, the fact that the average size of the conjugate is about 50 nm demonstrates that they were successfully conjugated for use in cell imaging with an efficient size for cellular uptake. Gel electrophoresis analysis showed that the band for MNP–QD800 migrates more slowly than the starting QD800– diamine (see Fig. S1 in ESI†). Interestingly, a mixture of nonfunctionalized QD800 and MNP displays a broad band that migrates more rapidly than the starting QD800, a likely result
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Fig. 4 Fluorescence microscopic images of HeLa cells following incubation with MNP–QD conjugates. (A) Cellular uptake by MNP–QD605 conjugate is similar to that of QD605–diamine. (B) QD800–diamine and MNP–QD800 conjugates are located next to the nuclei.
Fig. 3 MNP–QD800 conjugate characterization. (A) HRTEM image shows that the heterodimer is closely positioned. (B) EDS data show that the nanoparticle component 1 is Fe dominant, whereas the nanoparticle component 2 is dominant in Cd. (C) DLS measurements showing the hydrodynamic diameter of QD800–Pt, 6G–MNP and MNP–QD800 conjugates. The hydrodynamic size distribution of the MNP–QD800 conjugate is shifted to the right.
of the fact that the two nanoparticles have opposite charges which enable them to interact in a charge cancelling manner during electrophoretic migration. The Prussian blue and colloidal blue staining images following electrophoresis indicate that the mobility of the MNP–QD800 conjugate is slower than those of the other substances, a consequence of the fact that the net negative charge of the MNP is lost in the formation of the form MNP–QD800. Cellular imaging with the MNP–QD conjugates To use the MNP–QD800 conjugates in in vitro bioimaging probes, they must undergo cellular uptake. Firstly, we tested MNP–QD605 conjugate imaging in the HeLa cells through the fluorescence microscopy (TEM image of the MNP–QD605 conjugates in Fig. S2 (ESI†)). Red fluorescence arising from both QD605–diamine and MNP–QD605 was localized mainly in the nuclei of the cells (Fig. 4(A)). Moreover, because no binding ligands exist on the nanoprobes, cellular uptake of MNP– QD605 occurs through transport into the cell plasma membrane without specific binding to receptors and proteins. The results show that the MNP–QD605 hybrid can be used for in vitro cellular imaging without worrying about significant fluorescence quenching and abnormal cellular internalization. MNP–QD800 conjugates were also tested using HeLa cells. QD800 were visualized using a Cy5.5 filter set. Because the Cy5.5
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filter has a 635-675 nm excitation wavelength and 696–736 nm emission wavelength, QD800 fluorescence intensity using a Cy5.5 filter is weak to see through the fluorescence microscope. Nonetheless, we showed that QD800–diamine and MNP–QD800 were successfully internalized in the HeLa cells (Fig. 4(B)). Previously, we showed that the magnetism of MNPs was not changed even after QD conjugation.12 Here, MR imaging was performed to show that MNP–QD conjugates do not show significant decrease of MR properties in the cells (Fig. 5(A)). After MRI analysis, cellular fluorescence intensity was not significantly changed (Fig. 5(B)), which means that MNP–QD conjugates could be applied for the dual modal probe without a significant change in MR properties and fluorescence intensity. Also, Prussian blue staining was performed to see MNPs through the optical microscope images (Fig. S3 in the ESI†), which show that the starting MNPs are attached to the surfaces of the HeLa cells, whereas MNP–QD800 is localized within the cells next to the nuclei (white arrows in the Fig. S3(A) in ESI†). The different positioning of MNP and MNP–QD800 in the cells might be caused by the net charge difference, size variation, and functional moieties between these substances. As the results of the gel mobility assay (see Fig. S1 in ESI†) demonstrate, the net charge of MNPs seems to be less negative than QD800–diamine. The zeta potentials of QD800–diamine, MNP, and MNP–QD800 are −27.9 ± 3.68 mV, −6.17 ± 0.764 mV, and −6.00 ± 0.327 mV respectively. Although QD–diamine has a bigger negative value than MNP and MNP–QD800 from gel mobility assay and zeta potential measurements, the net charge does not solely affect the result of nanoconjugate uptake by the cells since cell membranes are negatively charged and zeta potential can affect nanoconjugates to penetrate in the cell membrane with cationic potential.19 Therefore, the aggregation or engulfment of nanoconjugates in the intracellular environment depends not only on the net charge, but
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ported by Korea Institute of Science and Technology (KIST), the Basic Science Research Program (NRF-2012R1A1A2008427) and the Creative/Challenging Research Program (20110028676) of the National Research Foundation of Korea (NRF), and a grant (D33400) of Korea Basic Science Institute.
Notes and references
Fig. 5 In vitro MRI results and cellular fluorescence intensity comparison after MR imaging. (A) MR images of HeLa cells, HeLa cells with MNP, HeLa cells with MNP–QD605, and HeLa cells with MNP–QD800 (MNP and MNP–QD conjugate concentrations were 50 nM). The contrast to noise ratio (CNR) values were obtained (CNR = (mean contrast of sample)/(background contrast)). MNP–QD605 and MNP–QD800 do not have significant decrease of MR properties in the cells. (B) Fluorescence intensity of the cells embedded in the gelatin which was used for MRI. There is no decrease of fluorescence intensity after applying a magnetic field.
also on other factors (size, surface moiety etc.). With this complicated mechanism of nanoconjugates engulfment in the cells, MNP–QD800 nanoconjugates have superior properties for cell imaging because of their excellent ability for entering inside the cells. Collectively, the heterodimer, MNP–QD800, would be an excellent dual modal imaging probe in vitro and possibly applicable to in vivo imaging.
Conclusions A method for dual modal MNP–QD heterodimer formation has been developed in the investigation described above. Near infrared QDs containing platinum on their surfaces were prepared by using a novel linking strategy. The QDs were observed to form heterodimers with guanine conjugated MNPs through strong coordinate bonds between Pt and guanine residues. By employing cell imaging using fluorescence microscopy, we have shown that the QD–MNP heterodimers are internalized into HeLa cells. MR images and cellular fluorescence intensity after MR analysis showed that there is no significant change in MR properties and fluorescence even after conjugation. Consequently, the results show that the MNP–QDs have the potential to be applied as dual modal probes in the FMT based cell imaging systems.
Acknowledgements We greatly thank Dr Tae Sup Lee in KIRAMS, Korea for support and technical assistance for MR imaging. This work was sup-
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One step synthesis of quantum dot–magnetic nanoparticle heterodimers for dual modal imaging applications† Jiyeon Lee,*a,b Gyoyeon Hwang,a,b Yeon Sun Honga,c and Taebo Sim*a,b,c Dual modal nanoprobes are promising tools for accurately detecting target molecules as part of the diagnosis of diseases including cancers. We have explored a new dual modal bioimaging probe that is comprised of a quantum dot (QD)–magnetic nanoparticle (MNP) hybrid. The MNP–QD heterodimers explored are fabricated by using a platinum–guanine coordination bonding guided self-assembly process, employing the metal–DNA conjugation method. Investigations utilizing energy dispersive spectroscopy
Received 17th December 2014, Accepted 23rd February 2015 DOI: 10.1039/c4an02322f www.rsc.org/analyst
(EDS) equipped high resolution transmission electron microscopy (HRTEM) demonstrate that the heterodimer contains an iron (Fe) dominant MNP and a cadmium (Cd) dominant QD. Finally, the results of cell studies show that the MNP–QD conjugates display good HeLa cell uptake in the absence of non-specific binding to the cell membrane and, as such, they can be used to label cells in vitro and in vivo as part of a new cell imaging technique.
Introduction Quantum dots (QDs) have excellent fluorescence intensity and photostability and, as a result, they are widely used as bioimaging probes.1,2 In addition, QDs serve as substrates in reactions that lead to the introduction of surface functional groups that enable conjugation with various biomolecules.3 This capability enhances their use as bioimaging agents4,5 and biosensors.6,7 Because of the high potential applications of QDs in a wide range of therapeutic situations, improvement in the efficiency of their binding to a wide range of biomolecules remains a challenge. This issue has driven the development of a variety of new conjugation protocols.5,8,9 To widen their targeting ability, dual modal bioimaging probes, which employ fluorescence with magnetism as detecting methods, have been developed as part of attractive strategies to image biomolecules.10,11 Magnetic nanoparticle (MNP)–QD conjugates have attracted great interest owing to their unique fluorescence and magnetic properties.12 Probes of this type have been
a Chemical Kinomics Research Center, Future Convergence Research Division, Korea Institute of Science and Technology, Hwarangno 14-gil 5, Seongbuk-gu, Seoul 136-791, Korea. E-mail: [email protected], [email protected], [email protected] b Biological Chemistry, Korea University of Science and Technology, 217, Gajeong-ro, Yuseong-gu, Daejeon, Korea c KU-KIST Graduate School of Converging Science and Technology, 145, Anam-ro, Seongbuk-gu, Seoul, 136-713, Korea † Electronic supplementary information (ESI) available: Additional images of HRTEM, the results of gel mobility assay, TEM images of 10 nm MNP–QD605, and cellular uptake images of MNPs and 10 nm MNP–QD800. See DOI: 10.1039/ c4an02322f
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applied to the new fluorescence molecular tomography (FMT) technique that combines fluorescence imaging with positron emission tomography (PET), computed tomography (CT), and magnetic resonance imaging (MRI).13,14 Several approaches to prepare QD–MNP nanoconjugates for use in FMT have been described,10,15 but most display low fluorescence or magnetic detection sensitivities compared to those of the individual nanoparticles. Recently, we developed a new method for assembling QD–MNP nanoconjugates that relies on platinum–guanine bonding to link the component QDs and MNPs.12 In that effort, we observed that the fluorescence intensity of the QD component and the magnetism of the MNP component of the platinum containing QD–MNP are not significantly altered by dual modal nanoconjugate formation. Although the results of our earlier work demonstrated that QD–MNP nanoconjugates can be used as FMT probes, further studies are required to determine if the new nanoconjugates interact with and/or internalized by cells. In addition, in order to apply nanoconjugates of this type as in vivo probes, the QD components must be responsive to light in the near infrared region. These two goals have been addressed in the studies described below, in which near infrared MNP–QD nanoconjugates were prepared and evaluated for their cell uptake propensities.
Experimental Materials Quantum dots (QDs, QD605 and QD800), fetal bovine serum, and penicillin/streptomycin were purchased from Invitrogen
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(USA). Magnetic nanoparticles (MNPs) were purchased from Ocean Nano Tech (USA). Ethylenediamine, cis-PtII(DMSO)2Cl2, Prussian blue staining reagent, and N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) were obtained from Sigma-Aldrich (USA). N-Hydroxysulfosuccinimide sodium salt (Sulfo-NHS) was purchased from Pierce (USA). Amine-C12-GGGGGG oligonucleotide was synthesized by Bioneer (Korea). Dulbecco’s modified Eagle’s medium (DMEM) and Dulbecco’s phosphate buffered saline (DPBS) were obtained from Welgen Inc. (Korea). Platinum conjugation of QDs The starting QDs (QD605 and QD800), modified with amino polyethylene glycol (PEG), were modified to introduce ethylenediamine moieties using (10-(2,5-dioxopyrrolidin-1-yl)10-oxodecanoic acid) by using the procedure described earlier.12 cis-PtII(DMSO)2Cl2 was added to diamine modified QDs followed by incubation for 30 min at room temperature. The fluorescence intensities of the QD–Pt conjugates were analyzed by using a Flexstation 3 Microplate Reader (Molecular Devices, USA). Guanine conjugation of MNPs Carboxylic acid modified 10 nm iron oxide MNPs were reacted with amine-C12-GGGGGG using sulfo-NHS and the EDC coupling technique.12 The MNP to 6G conjugation ratio was adjusted to be 1 : 50. After 3 h incubation at room temperature, unreacted DNA and reagent were removed by washing the filtrate using a 100k centrifugal filter. Binding QD–Pt to 6G–MNP and characterization of the hybrid The nanoconjugates were formed using equimolar concentrations of the QD–Pt and 6G–MNP conjugates with 3 h incubation at room temperature. Nanoparticle concentrations were determined by using scanning UV spectroscopy with a Flexstation 3 Microplate Reader. DLS analysis was performed using Nano-ZS (Malvern, UK) at room temperature. QD–MNPs were applied to a carbon film TEM grid (EMS, Hatfield, PA, USA) and observed using cryo TEM and Technai TEM (KIST, Seoul, Korea). EDS equipped with Technai was used for analysis of the composition of the nanoparticles. Zeta potential measurements were analysed through Nano-ZS (Malvern, UK). Cell culture and imaging Human cervical adenocarcinoma (HeLa) cells were cultured with DMEM, supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. HeLa cells were applied to 8-well glass chamber slide dishes and the QD–MNP conjugates in cell complete medium were added. After 24 h, cells were fixed by using 4% formaldehyde. The cell nuclei were stained using DAPI (1 : 100 dilution of 1 mg mL−1 in DPBS) for fluorescence imaging and Prussian blue reagent for MNP imaging. The cells were then imaged using Nikon Ti and Olympus IX2, and the images were edited with Nikon element software and ImageJ program.
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In vitro MRI HeLa cells (2 × 105) were seeded in each well of a 12-well plate. After 24 hours incubation, 50 nM of MNP, MNP–QD605, and MNP–QD800 in only DMEM (without FBS) were added to each well and incubated for another 24 hours. Cells were harvested using trypsin-EDTA and fixed using 4% formaldehyde in PBS. After washing with PBS, cells were collected by centrifuging at 3000 rpm for 5 min. Three layers of gelatin, gelatin including cells, and gelatin were added to each tube. Phantoms were scanned in a 3 T magnetic resonance imaging scanner (MAGNETOM 3 T Trio, SIEMENS Medical Systems) in Korea Institute of Radiological and Medical Sciences (KIRAMS) using a T2-weighted turbo spin echo imaging sequence with a repetition time (TR) of 5000 ms and TE of 118 ms, with a field of view (FOV) of 130 × 56 mm2, imaging matrix of 226 × 320, and one slice with 3 mm slice thickness. The fluorescence intensity of cell pellets before and after applying a magnetic field was estimated through the OV100 imaging system (Olympus).
Results and discussion Conjugation of near infrared QD with platinum Previously, we reported that QD605 with a fluorescence maximum of 605 nm (red) can be conjugated with platinum with QD to Pt ratios of 1 : 5 and 1 : 10 without undergoing a significant decrease of emission efficiency.12 Here, we demonstrate that the near infrared (800 nm) emitting QD (QD800) can also be conjugated with platinum using the previously reported conjugation method without undergoing a significant change in its fluorescence efficiency. For this purpose, QD800– Pt conjugates in three different (1 : 5, 1 : 10 and 1 : 20) QD to Pt molar ratios were prepared by previously reported method.12 Fluorescence spectra of the QD800–diamine and QD800–Pt conjugates at the same molar concentration were recorded (Fig. 1(A)) in order to assess the effect of the Pt content on emission efficiencies. Interestingly, the QD800–diamine–Pt conjugate with the 1 : 5 QD to Pt ratio has the same fluorescence efficiency as QD800–diamine. However, because of the general phenomenon that the fluorescence intensity of QDs usually decreases when they are bound to other materials, increases in the ratio of platinum in the QD–diamine–Pt (1 : 10 and 1 : 20) causes the emission efficiencies to decrease. The number of Pt atoms on the surface of the QD800–Pt was analyzed by ICP-MS (Fig. 1(B)). Even though the fluorescence efficiency of the 1 : 10 QD800–Pt conjugate is slightly less than those of the starting QD800–diamine and the 1 : 5 QD800–Pt conjugate, the 1 : 10 QD800–Pt conjugate was employed because of the higher platinum ratio in the studies aimed at determining the binding of this conjugate to MNPs to form dual modal nanoprobes. Self-assembling QD800–Pt conjugates with 6G–MNP and their characterization In order to form dual modal nanoprobes with the QD800–Pt conjugate, the coordination bonding of Pt from QD800–Pt con-
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Fig. 1 QD800–Pt conjugates characterization. (A) Photoluminescence (PL) intensity of QD800–diamine and QD800–Pt conjugates with three different QD to Pt ratios (1 : 5, 1 : 10, 1 : 20). The fluorescence intensity is not decreased with the 1 : 5 QD800–Pt conjugate, but 1 : 10 and 1 : 20 QD800–Pt conjugates have slightly decreased fluorescence intensities compared to that of QD800–diamine. (B) ICP-MS measurement results indicating that the number of Pt atoms on each QD are 1.6, 2.7, and 5.6, respectively.
jugates to guanines from MNPs depicted in Fig. 2(A) was used. Simply, carboxylic acid modified MNPs were conjugated with amino-GGGGGG (6G) oligonucleotide using carbodiimide chemistry to yield the functionalized MNP–6G. A mixture containing a 1 : 1 ratio of the 1 : 10 QD800–Pt conjugate and MNP– 6G was incubated and unreacted reagents were removed by a centrifugal filter and exclusion column chromatography. To investigate the formation of the MNP–QD800 conjugate, TEM images were obtained from the conjugates (Fig. 2(B)) and the mixture of QD–diamine with carboxylic acid modified MNPs (Fig. 2(C)). We found more heterodimers in MNP–QD800 conjugates rather than in the mixture of QD–diamine and MNPs. Even if TEM images show that MNP–QD800 heterodimer is likely to be formed, it is hard to distinguish QD from MNP by using only the TEM imaging method because the average size and the shape of a QD800 and a MNP are similar. As a result, HRTEM and EDS analyses were carried out to ensure the formation of MNP–QD800 conjugates. In Fig. 3(A) an HRTEM image of the heterodimer that is generated by conjugated MNP–6G and QD800–Pt is shown. Furthermore, EDS spectral analysis (Fig. 3(B)) of the image displayed that the nanoparticle component of the conjugate numbered 1 in the figure is Fe rich while the nanoparticle numbered 2 is Cd rich. The presence of peaks associated with carbon (C) and copper (Cu) in the spectra is negligible because they were from TEM grids. Hence, the results confirm that a new 1 : 1 heterodimer, formed by platinum–guanine bonding, is generated by MNP–6G and QD800–Pt conjugates. MNP–QD800 conjugates were evaluated for the propensity for cellular uptake and used as a cell imaging probe. The size of a dual modal probe is an important factor determining whether or not it will undergo uptake by cells.16,17 Jiang et al.
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Fig. 2 Self-assembling MNP–QD800 conjugates. (A) Schematic representation of QD–MNP conjugation. Platinum of QD connects to guanine of MNP to form the coordination bonds. (B) TEM images of the conjugated MNP–QD800. Guanine modified MNPs (MNP–6G) bonded to platinum linked QD800 (QD800–Pt) to form heterodimers, MNP– QD800. (C) TEM images of the mixture of QD800–diamine and MNP. They did not form heterodimers.
reported that the most efficient uptake occurs with gold nanoparticles that are in the 25–50 nm size range.17 In addition, it is known that smaller nanoprobes more readily penetrate cell membranes than their larger counterparts.18 To investigate whether this new heterodimer has the optimal size as a cell imaging probe, the MNP–QD800 conjugates were characterized by using DLS. The DLS data show that the hydrodynamic size distribution of MNP–QD800 conjugates is shifted to the right with an average size of 50.05 nm (Fig. 3(C)). Because the average size of starting QD800–diamine and MNP–6G are each 30 nm in the DLS data, the fact that the average size of the conjugate is about 50 nm demonstrates that they were successfully conjugated for use in cell imaging with an efficient size for cellular uptake. Gel electrophoresis analysis showed that the band for MNP–QD800 migrates more slowly than the starting QD800– diamine (see Fig. S1 in ESI†). Interestingly, a mixture of nonfunctionalized QD800 and MNP displays a broad band that migrates more rapidly than the starting QD800, a likely result
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Fig. 4 Fluorescence microscopic images of HeLa cells following incubation with MNP–QD conjugates. (A) Cellular uptake by MNP–QD605 conjugate is similar to that of QD605–diamine. (B) QD800–diamine and MNP–QD800 conjugates are located next to the nuclei.
Fig. 3 MNP–QD800 conjugate characterization. (A) HRTEM image shows that the heterodimer is closely positioned. (B) EDS data show that the nanoparticle component 1 is Fe dominant, whereas the nanoparticle component 2 is dominant in Cd. (C) DLS measurements showing the hydrodynamic diameter of QD800–Pt, 6G–MNP and MNP–QD800 conjugates. The hydrodynamic size distribution of the MNP–QD800 conjugate is shifted to the right.
of the fact that the two nanoparticles have opposite charges which enable them to interact in a charge cancelling manner during electrophoretic migration. The Prussian blue and colloidal blue staining images following electrophoresis indicate that the mobility of the MNP–QD800 conjugate is slower than those of the other substances, a consequence of the fact that the net negative charge of the MNP is lost in the formation of the form MNP–QD800. Cellular imaging with the MNP–QD conjugates To use the MNP–QD800 conjugates in in vitro bioimaging probes, they must undergo cellular uptake. Firstly, we tested MNP–QD605 conjugate imaging in the HeLa cells through the fluorescence microscopy (TEM image of the MNP–QD605 conjugates in Fig. S2 (ESI†)). Red fluorescence arising from both QD605–diamine and MNP–QD605 was localized mainly in the nuclei of the cells (Fig. 4(A)). Moreover, because no binding ligands exist on the nanoprobes, cellular uptake of MNP– QD605 occurs through transport into the cell plasma membrane without specific binding to receptors and proteins. The results show that the MNP–QD605 hybrid can be used for in vitro cellular imaging without worrying about significant fluorescence quenching and abnormal cellular internalization. MNP–QD800 conjugates were also tested using HeLa cells. QD800 were visualized using a Cy5.5 filter set. Because the Cy5.5
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filter has a 635-675 nm excitation wavelength and 696–736 nm emission wavelength, QD800 fluorescence intensity using a Cy5.5 filter is weak to see through the fluorescence microscope. Nonetheless, we showed that QD800–diamine and MNP–QD800 were successfully internalized in the HeLa cells (Fig. 4(B)). Previously, we showed that the magnetism of MNPs was not changed even after QD conjugation.12 Here, MR imaging was performed to show that MNP–QD conjugates do not show significant decrease of MR properties in the cells (Fig. 5(A)). After MRI analysis, cellular fluorescence intensity was not significantly changed (Fig. 5(B)), which means that MNP–QD conjugates could be applied for the dual modal probe without a significant change in MR properties and fluorescence intensity. Also, Prussian blue staining was performed to see MNPs through the optical microscope images (Fig. S3 in the ESI†), which show that the starting MNPs are attached to the surfaces of the HeLa cells, whereas MNP–QD800 is localized within the cells next to the nuclei (white arrows in the Fig. S3(A) in ESI†). The different positioning of MNP and MNP–QD800 in the cells might be caused by the net charge difference, size variation, and functional moieties between these substances. As the results of the gel mobility assay (see Fig. S1 in ESI†) demonstrate, the net charge of MNPs seems to be less negative than QD800–diamine. The zeta potentials of QD800–diamine, MNP, and MNP–QD800 are −27.9 ± 3.68 mV, −6.17 ± 0.764 mV, and −6.00 ± 0.327 mV respectively. Although QD–diamine has a bigger negative value than MNP and MNP–QD800 from gel mobility assay and zeta potential measurements, the net charge does not solely affect the result of nanoconjugate uptake by the cells since cell membranes are negatively charged and zeta potential can affect nanoconjugates to penetrate in the cell membrane with cationic potential.19 Therefore, the aggregation or engulfment of nanoconjugates in the intracellular environment depends not only on the net charge, but
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ported by Korea Institute of Science and Technology (KIST), the Basic Science Research Program (NRF-2012R1A1A2008427) and the Creative/Challenging Research Program (20110028676) of the National Research Foundation of Korea (NRF), and a grant (D33400) of Korea Basic Science Institute.
Notes and references
Fig. 5 In vitro MRI results and cellular fluorescence intensity comparison after MR imaging. (A) MR images of HeLa cells, HeLa cells with MNP, HeLa cells with MNP–QD605, and HeLa cells with MNP–QD800 (MNP and MNP–QD conjugate concentrations were 50 nM). The contrast to noise ratio (CNR) values were obtained (CNR = (mean contrast of sample)/(background contrast)). MNP–QD605 and MNP–QD800 do not have significant decrease of MR properties in the cells. (B) Fluorescence intensity of the cells embedded in the gelatin which was used for MRI. There is no decrease of fluorescence intensity after applying a magnetic field.
also on other factors (size, surface moiety etc.). With this complicated mechanism of nanoconjugates engulfment in the cells, MNP–QD800 nanoconjugates have superior properties for cell imaging because of their excellent ability for entering inside the cells. Collectively, the heterodimer, MNP–QD800, would be an excellent dual modal imaging probe in vitro and possibly applicable to in vivo imaging.
Conclusions A method for dual modal MNP–QD heterodimer formation has been developed in the investigation described above. Near infrared QDs containing platinum on their surfaces were prepared by using a novel linking strategy. The QDs were observed to form heterodimers with guanine conjugated MNPs through strong coordinate bonds between Pt and guanine residues. By employing cell imaging using fluorescence microscopy, we have shown that the QD–MNP heterodimers are internalized into HeLa cells. MR images and cellular fluorescence intensity after MR analysis showed that there is no significant change in MR properties and fluorescence even after conjugation. Consequently, the results show that the MNP–QDs have the potential to be applied as dual modal probes in the FMT based cell imaging systems.
Acknowledgements We greatly thank Dr Tae Sup Lee in KIRAMS, Korea for support and technical assistance for MR imaging. This work was sup-
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