Optical characterization of colloidal CdSe quantum dots in endothelial progenitor cells
© Molnár et al; licensee BioMed Central Ltd. 2010
Received: 16 September 2009
Accepted: 4 February 2010
Published: 4 February 2010
We have quantitatively analyzed the confocal spectra of colloidal quantum dots (QDs) in rat endothelial progenitor cells (EPCs) by using Leica TCS SP5 Confocal Microscopy System. Comparison of the confocal spectra of QDs located inside and outside EPCs revealed that the interaction between the QDs and EPCs effectively reduces the radius of the exciton confinement inside the QDs so that the excitonic energy increases and the QD fluorescence peak blueshifts. Furthermore, the EPC environment surrounding the QDs shields the QDs so that the excitation of the QDs inside the cells is relatively weak, whereas the QDs outside the cells can be highly excited. At high excitations, the occupation of the ground excitonic state in the QD outside the cells becomes saturated and high-energy states excited, resulting in a large relaxation energy and a broad fluorescence peak. This permits, in concept, to use QD biomarkers to monitor EPCs by characterizing QD fluorescence spectra.
The use of colloidal quantum dots (QDs) is one of the most exciting developments in nanobiotechnology. Because of their high durability and unique optical properties QDs are widely used as fluorescent labelling agents for in vitro and in vivo bioimagings, such as cellular labeling, deep tissue imaging, and fluorescent resonance energy transfer donors . Surface modified and water-soluble QDs open a new era in cell imaging and bio targeting as transport vehicles for therapeutic drug delivery to different diseases such as cancer and atherosclerosis [2–5].
Endothelial progenitor cells (EPCs) are heterogeneous groups of endothelial cell precursors which are circulating in the blood vessel. These cells play an important role in atherogenesis and cardiovascular regeneration [6–9]. One of the important challenges in cardiovascular research is to develop a sensitive tool that allows non-invasive in vivo tracking of EPCs, which can provide important information about site specific EPCs incorporation throughout the vasculature and whether the stage of disease alters the way EPCs are targeted.
In this work we carefully characterized confocal microscopic spectra of QDs after uptaken by EPCs. The main aim of this work is to find quantitative indicators about the interaction between the QDs and the EPCs so that we can rely on these indicators to characterize chemical and physical interactions between QDs and EPCs for in vivo tracking of EPCs.
Materials and methods
Colloidal CdSe QDs with one monolayer CdS shell (the external CdS shell was introduced for COOH derivatization), with a nominal diameter 5.5 nm and an emission wavelength of 625 nm (denoted as QD625), were chemically synthesised following the common standard method (see detailed description in Ref.  and references therein), which were octadecylamine coated so that they were not water soluble. They were dissolved in chloroform and a same volume of a water solution containing 3-mercaptopropionic acid (3-MPA) (1 mol/L = M) was then added under vigorous stirring for 2 hours after which QDs become water soluble. After resting the mixture for a while, chloroform and water were separated and the aqueous layer, which contained mercapto-coated QDs, was extracted. After centrifugation and decantation with water twice, an aqueous Na2CO3 solution was added to form a clear solution which was washed to remove residual 3-MPA ligands. Successive re-dispersion of QDs into water at pH 10.8 yielded a clear solution containing water-soluble QDs coated with carboxyl groups. Similar QDs were purchased from Invitrogen. Same optical characterizations were obtained using our QDs and the ones from Invitrogen so that in the following presentation we do not make further distinctions between them. QD625 were diluted in the cell culture medium to a final concentration of 16 nano-M (nM) and were added to the EPCs. Here unlike conventional organic and inorganic chemicals, the concentration of colloidal QDs is difficult to determine by gravimetric methods. It is usually expressed as molar concentration determined via molar extinction coefficient measurement . The cells were incubated with QD625 for 30 hours. After QD incubation the cells were washed with phosphate-buffered saline (PBS, pH 7.2), fixed with 4% paraformaldehyde for 10 minutes and then stored in PBS at 5°C for confocal microscopy measurements (note that the confocal microscopy measurements were performed at room temperature).
Leica TCS SP5 Confocal Microscopy System was used to characterize the optical properties of these samples. Images were captured with a scanning speed of 400 Hz and image resolution of 512 × 512 pixels, and then analysed using Leica Application Suite 2.02.
Results and discussion
Note that the cells were washed after QD incubation so that QDs are not expected to remain outside cells in the sample. However, we occasionally observed QD clusters stuck to small particles. These particles remained loosely attached to the bottom of the dish after washing steps. The optical spectrum of one of such QD cluster outside EPCs is shown in Fig. 3 measured by using a wavelength scanning step of 12 nm. It was difficult to measure confocal spectra at smaller scanning steps since these loosely attached QDs were moving. In order to be able to do precise quantitative comparison, we prepared reference QD samples (QD cluster 3 and cluster 4) simply by drying one drop of 8 μ M carboxyl-coated QD625 solution on a glass-bottomed dish so that QDs are not mobile.
Note the difference in fluorescent emission peaks (616 nm and 613 nm) when 458 nm and 514 nm LASER wavelengths are used for cluster 1 and 2 in Figs. 4(a-b) and 6(a-b). The most probable reason is the merging of the laser signal with the QD fluorescent signal when the 514 nm laser is used, especially at high excitation powers. The fitted wavelength of the peak at about 613 nm in Figs. 6(a-b) actually blue shifts from 615 to 613 nm following the increase of the 514-nm laser power.
where E = ħω0 is the excitonic energy, r is the QD radius, E g is the energy bandgap of the QD material, δr and δE are modifications in radius and excitonic energy. For our CdSe QD625, the nominal diameter is 5.5 nm. Assuming one monolayer modification (about 0.3 nm ) in the radius, Eq. (2) gives us δE = 30 meV, which agrees very well with Figs. 8 and 9. Note that the fitted fluorescence peak position for 458 nm excitation is different from the 514 nm excitation, 616 nm vs 613 nm in Figs. 4 and 6, which we believe is due to the mixtures between the excitation signal and the QD fluorescence. For 514 nm excitation, the mixture is stronger so that the blue shift appeared to be larger.
Zhang et al. reported similar blue shift of fluorescence peak of thiol-capped CdTe QDs within less than 10 min of QD uptaking in living cells caused by surface photooxidation . The reported blue shift in CdTe QDs is much larger than our cases. Furthermore, the peak width of CdTe QDs is largely increased, while it remains basically unchanged for our CdSe QDs. The major differences between CdTe QDs and our CdSe QDs are probably due to the fact that the oxidation of Te atoms are relatively easy, therefore CdTe QDs are less chemically stable.
The other important finding is that the relaxation energy in the QDs inside cells is relatively small and independent of the excitation power, while it increases quickly in the QDs outside of cells then saturates as a function of the excitation power, see Figs. 8(b) and 9(b). The large relaxation energy is actually an indication of the saturation of the ground excitonic state occupation and the occupations of high-energy excitonic states due to the large optical pumping by the excitation radiation.
The same effects (blue shift and the relaxation energy behavior) were obtained for QD625 (emission wavelength 625 nm) under the excitations of 458 and 514 nm wavelengths. The insensitivity to the excitation wavelength can be theoretically expected when the excitation energy is not too high compared with the excitonic energy of QDs (i.e., in the range of one-photon and multiphoton excitations) . High energy radiation (larger than twice the excitonic energy) was shown to induce multicarrier excitation  so that it may induce different characterizations in the QD fluorescence spectrum.
Similar measurements were repeated two and four months late on randomly chosen QD clusters, and we found that both the samples and measurement results were very stable when the same measurement setups were used. We noticed that as long as measurement performances are careful, there are no significant changes in the confocal spectral characteristics (i.e., the fluorescence intensity, excitonic energy and relaxation energy).
We have shown that the uptaking of colloidal QDs by EPCs effectively reduces the radius of the exciton confinement inside the QDs so that the excitonic energy increases and the peak of the QD fluorescence blue shifts. Furthermore, the cell environment surrounding the QDs shields the QDs so that the excitation of the QDs inside the cells is usually weaker. QDs outside the cells are excited to higher degree, which leads to the saturation of the ground excitonic state. The excitation of high-energy states results in a broader fluorescence peak.
Our study shown that intracellular environment can affect optical characteristics of QDs and that such changes are quantifiable. Therefore, changes of QD fluorescence spectra should allow one to characterize the interaction between colloidal QDs and EPCs. This should facilitate the development of QD biomarkers for monitoring EPCs at sub-cellular level.
Swedish Vinnova support to project "Molecular study of early atherosclerosis with quantum dots" (Pro-jektnummer P35914-1) and computing resources from the Swedish National Infrastructure for Computing (SNIC 001-09-52) are acknowledged.
- Medintz IL, Uyeda HT, Goldman ER, Mattoussi H: Quantum dot bioconjugates for imaging, labelling and sensing. Nature Materials. 2005, 4: 435-446. 10.1038/nmat1390.View ArticleGoogle Scholar
- Vashist SK, Tewari R, Bajpai RP, Bharadwaj LM, Raiteri R: Review of Quantum Dot Technologies for Cancer Detection and Treatment. AZojono J Nanotechnology Online. 2006, 2: 1-14.Google Scholar
- Park JH, von Maltzahn G, Ruoslahti E, Bhatia SN, Sailor MJ: Micellar Hybrid Nanoparticles for Simultaneous Magnetofluorescent Imaging and Drug Delivery. Angew Chem Int Ed. 2008, 47: 7284-7288. 10.1002/anie.200801810.View ArticleGoogle Scholar
- Yezhelyev MV, Qi L, O'Regan RM, Nie S, Gao X: Proton-sponge coated quantum dots for siRNA delivery and intracellular imaging. J Am Chem Soc. 2008, 130: 9006-9012. 10.1021/ja800086u.View ArticleGoogle Scholar
- Derfus AM, Chen AA, Min DH, Ruoslahti E, Bhatia SN: Targeted Quantum Dot Conjugates for siRNA Delivery. Bioconjugate Chem. 2007, 18: 1391-1396. 10.1021/bc060367e.View ArticleGoogle Scholar
- Hirschi KK, Ingram DA, Yoder MC: Assessing identity, phenotype, and fate of endothelial progenitor cells. Arterioscler Thromb Vasc Biol. 2008, 28 (9): 1584-1595. 10.1161/ATVBAHA.107.155960.View ArticleGoogle Scholar
- Fadini GP, Baesso I, Albiero M, Sartore S, Agostini C, Avogaro A: Technical notes on endothelial progenitor cells: ways to escape from the knowledge plateau. Atherosclerosis. 2008, 197: 496-503. 10.1016/j.atherosclerosis.2007.12.039.View ArticleGoogle Scholar
- Kawamoto A, Losordo DW: Endothelial progenitor cells for cardiovascular regeneration. Trends Cardiovasc Med. 2008, 18: 33-37. 10.1016/j.tcm.2007.11.004.View ArticleGoogle Scholar
- Brunt KR, Hall SRR, Ward CA, Melo LG: Endothelial Progenitor Cell and Mesenchymal Stem Cell Isolation, Characterization, Viral Transduction. Methods in Molecular Medicine, Vascular Biology Protocols. Edited by: Sreejayan N, Ren J. 139: 197-210. full_text.Google Scholar
- Li JJ, Wang YA, Guo W, Keay JC, Mishima TD, Johnson MB, Peng X: Large-scale synthesis of nearly monodisperse CdSe/CdS core/shell nanocrystals using air-stable reagents via successive ion layer adsorption and reaction. J Am Chem Soc. 2003, 125: 12567-75. 10.1021/ja0363563.View ArticleGoogle Scholar
- Yu WW, Qu L, Guo W, Peng X: Experimental Determination of the Extinction Coefficient of CdTe, CdSe, and CdS Nanocrystals. Chem Mater. 2003, 15: 2854-2860. 10.1021/cm034081k.View ArticleGoogle Scholar
- Data in Science and Technology: Semiconductors other than Group IV Elements and III-V Compounds. Edited by: Madelung O. 1992, Springer, BostonGoogle Scholar
- Fu Y, Han TT, Luo Y, Ågren H: Dynamic analysis of multiple-photon optical processes in semiconductor quantum dots. J Phys Condens Matter. 2006, 18: 9071-82. 10.1088/0953-8984/18/39/033.View ArticleGoogle Scholar
- Zhang LW, Monteiro-Riviere NA: Mechanisms of quantum dot nanoparticle cellular uptake. Toxicological Sciences. 2009, 110: 138-55. 10.1093/toxsci/kfp087.View ArticleGoogle Scholar
- Fu Y, Han TT, Ågren H, Lin L, Chen P, Liu Y, Tang GO, Wu J, Yue Y, Dai N: Design of semiconductor CdSe core ZnS/CdS multishell quantum dots for multiphoton applications. Appl Phys Lett. 2007, 90 (3): 173102-10.1063/1.2731525.View ArticleGoogle Scholar
- Zhang Y, He J, Wang PN, Chen JY, Lu ZY, Lu DR, Guo J, Wang CC, Yang WL: Time-dependent photoluminescence blue shift of the quantum dots in living cells: effect of oxidation by singlet oxygen. J Am Chem Soc. 2006, 128: 13396-13401. 10.1021/ja061225y.View ArticleGoogle Scholar
- Fu Y, Han TT, Luo Y, Ågren H: Multiphoton excitation of quantum dots by ultrashort and ultraintense laser pulses. Appl Phys Lett. 2006, 88 (3): 221114-10.1063/1.2209209.View ArticleGoogle Scholar
- Schaller RD, Klimov VI: High efficiency carrier multiplication in PbSe nanocrystals: implications for solar energy conversion. Phys Rev Lett. 2004, 92 (4): 186601-10.1103/PhysRevLett.92.186601.View ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.