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SUPPORTING INFORMATION
Single Quantum Dot-Micelles Coated with Silica Shell as Potentially Non-Cytotoxic Fluorescent Cell Tracers Zhivko Zhelev, Hideki Ohba, Rumiana Bakalova* On-Site Sensing and Diagnosis Research Laboratory, AIST-Kyushu, 807-1 Shuku-machi, Tosu 8410052, Japan
Abbreviations. TOPO – Trioctylphosphine oxide (99%), TOP – Trioctylphosphine (90%), ODA – Octadecylamine (>99%), HDA – Hexadecylamine (>99%), DDA – Dodecylamine (98%), OTS – Octyltriethoxysilane (>97%), TEVS – Triethoxyvinylsilane (97%), AEAP-TMS – [3-(2-Aminoethylamino)propyl]trimethoxysilane (>98%), (TMS)2S – Hexamethyldisilathiane. Synthesis of CdSe/ZnS and CdSe/ZnSe/ZnS QDs. The QDs were routinely synthesized in our Lab. Briefly, the synthesis of six size-defined colors of TOPO/HDA or TOPO/ODA-capped CdSe QDs was carried out using selenium pellets (0.7896 g in 7.4 g TOP) and cadmium oxide (0.45 g in 8 g stearic or oleic acid) as precursors. The synthesis was provided in argon atmosphere and in the presence of the following coordinating ligands (TOPO – 8 g, and HDA – 12 g, or ODA – 12 g). Different colors CdSe core QDs was obtained in specified time intervals. They were characterized with HRTEM, spectrophotometry and spectrofluorimetry. Synthesis of ZnS shell was carried out using Zn-stearate (0.2 g dissolved in 2 g TOP and 2 g HDA) and elemental sulfur (0.1 g melted in 2 g DDA) as precursors and 30-50 mg CdSe . The synthesis of ZnS shell was carried out during 70 min at 150 o C, under argon atmosphere. The obtained CdSe/ZnS core/shell QDs were characterized by X-ray, absorbance and fluorescence spectroscopy. Synthesis of ZnSe/ZnS shell was carried out using Zn-stearate (0.2 g dissolved in 2 g TOP and 2 g HDA), selenium precursor (selenium pellets 0.7896 g in 7.4 g TOP), hexamethyldisilathiane [(TMS)2S 0.568 g in 2 g TOP] and 30-50 mg CdSe . The synthesis of ZnSe shell was carried out during 20 min at 200 oC, under argon atmosphere. After the formation of ZnSe shell, (TMS)2S was added and the synthesis of ZnS shell was carried out during 70 min at 150 oC. The obtained CdSe/ZnSe/ZnS core/shell QDs were characterized by X-ray, absorbance and fluorescence spectroscopy. Synthesis of silica-shelled single QD micelles. In principle, the synthesis was carried out in 4 steps, using consecutively 3 different silica precursors (Figure 1S).
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Figure 1S. Scheme of synthesis of silica-shelled single QD micelles. First step - a transfer of TOPO/HDA or TOPO/ODA-capped QDs (CdSe, CdSe/ZnS, or CdSe/ZnSe/ZnS) from the chloroform phase into the water phase with formation of QD/detergent micelles consisting of single nanocrystal capped with bilayer of coordinating ligands and negatively charged detergent molecules. Second step - a formation of single silica-stabilized QD/detergent micelles, using the first (“hydrophobic”) silica precursor n-octyltriethoxysilane (OTS). OTS has a hydrophobic chain that incorporates into the micelle and interacts with the coordinating ligands. The molecules of OTS can also polymerize on the surface of the micelle with formation of oxygen bridges and thus to stabilize it. Third step - an addition of the second (“amphiphilic”) silica precursor – triethoxyvinylsilane (TEVS), a growing of silica shell, and an edge-out of the detergent molecules. TEVS polymerizes over the first silica precursor, which is already integrated into the micelle structure. On this step, it is possible to control the thickness of silica shell, varying the amount of the second silica precursor. Detergent molecules gradually edge-out of the micelle and in the end of the polymerization process they are not a part of the micelle structure any more.
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The fourth step - an amino-functionalization of the silica-shelled single QD micelles, using third “hydrophilic” silica precursor - [3-(2-Aminoethylamino)propyl]-trimethoxysilane. (3-Aminopropyl)triethoxysilane was also used successfully. This silica precursor polymerizes over the silica shell and adds hydrophilic NH2-groups on the surface. The transfer of ligand-caped QDs from the chloroform phase into the water phase with formation of QD/detergent micelles was carried out by the procedure described in Fan et al. (ref. 4 in the main text). Briefly, ~ 5 mg QDs was dissolved in 800 µL chloroform and were transferred to 10 mL detergent solution (15 mM SDS or 50 mM Docusate sodium salt in distilled water; other co-surfactants or negatively charged lipids were also used). The sample was mixed intensively on a magnetic stirrer, and subsequently heated till 50 oC for complete evaporation of the chloroform. An additional ultrasound treatment for 3 min was applied. During this process, the surfactant formed micelles over the QDs. The sample became slightly turbid in the process of micelles formation. The use of negatively charged detergents minimized the possibility for formation of dimers, trimers, and larger and non-uniform silica structures. The use of positively charged detergents facilitated the aggregation and resulted in formation of duplets, triplets, and multiplets (two, three, and more QDs into one micelle structure), as well as of dimers, trimers, etc. (two, three or more mixed, aggregated micelles) after addition of the silica precursors. For the formation of silica-stabilized QD/detergent micelles, 16 µL of the first silica precursor (OTS) were added in 10 mL of the reaction mixture, containing QD/detergent micelles. The mixture was stirred for 2 hours at 22 oC. Hundred and ten µL of the second silica precursor (TEVS) were added in 10 mL of the reaction mixture. The mixture was stirred for 24 hours at 22 o C. Silica shell is formed and growths during this time. Addition of TEVS resulted in comparatively turbid solution, coming from partial insolubility of the precursor in water-phase, but expansion of the polymerization process enhanced the transparency of the sample. The sample was subjected to dialysis for 48 hours at 22 oC to remove the free ingredients. After the third step, the sample can be lyophilized and kept in the dark at room temperature for a long time. The sample can be also kept in distilled water in the dark at room temperature. The amino-functionalization of silica spheres was carried out by the third silica precursor - [3-(2Aminoethylamino)propyl]-trimethoxysilane (AEAP-TMS). If the nanoparticles are in aqueous solution, 18 µL of AEAP-TMS were added in 10 mL of the reaction mixture. The mixture was stirred for 12 hours. Finally, the product was purified by several washings using dialysis (48 hours at 22 oC) by Slide-A-Lyzer® Dialysis Cassette – 10 000 MWCO (Pierce). If the nanoparticles are lyophilized, 10 mg of the nanoparticles (dry weight) were dissolved in 10 mL of distilled water. In the case of preparation of silica-shelled single QD micelles for in vivo application, the surface of the structure was additionally modified with PEG-containing NHS-esters (e.g., NHS-m-dPEG precursors and S-acetyl-dPEG NHS esters Quanta Biodesign, Ltd.). Hundred mg PEG-containing NHS-ester was directly added to the reaction mixture and stirred for 16 hours. The product was purified by several washings using dialysis by Slide-A-Lyzer® Dialysis Cassette – 10 000 MWCO (Pierce). Analysis of the cytotoxic effect and intracellular delivery of silica-shelled single QD micelles Treatment of cells with silica-shelled single quantum dot micelles. HeLa, Jurkat and K-562 cells were cultured in RPMI1640 Medium, supplemented with 10 % heat-inactivated fetal bovine serum (FBS) and antibiotics (100 µg/mL streptomycin and 100 U/mL penicillin in the case of Jurkat and K-562 cells, and 0.3 mg/mL kanamycin in the case of HeLa cells) in a humidified atmosphere at 37 oC with 5 % CO2. The cells (in PRMI-1640 Medium) were dispensed in 96-well plates (90 µL in each well containing 5x104 cells per well). Ten µL of silica-shelled single quantum dot micelles (dissolved in distilled water and containing different concentrations of the product – two-serial dilutions starting from 5 µM to 312 nM – the concentration was calculated from the absorbance maximum of QD by the method of Yu et al. [ref. 6 in the main text]) were added to each well. Incubation was carried out during 30 min, 3 hours, 24 hours and 48 hours, at 37 oC in humidified atmosphere, in the dark. Flow cytometric analysis of cell viability. The cells were washed twice and re-suspended in new medium. The viability of cells was analyzed using flow cytometry (flow cytometer Beckman Coulter - Epics XL). The flow cytometer was operated in accordance with the manufacturer’s recommendations after fine adjustments for optimization. The forward- and side-scatter parameters were adjusted to accommodate the inclusion of both leukemia cells and normal lymphocytes within the acquisition
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data. No cells were excluded from the analysis, and 10,000 cells were counted. Data were collected and analyzed by “XL System II” software. The results were presented as a dot plot of quantum dot-fluorescence (green light) and PE-fluorescence (red light) with quadrant markers drawn to distinguish quantum dot- and PE-labeled cells. Percent of dead cells was calculated before and after treatment with silica-shelled single QDs. The results are expressed as cell viability as percent from the control (nontreated) cells – the viability of control cells was considered 100%. Intracellular delivery of silica-shelled single QD micelles - fluorescent confocal microscopy. Twenty µL of silica-shelled single QD micelles (QD concentration 0.5 µM) were incubated with 200 µL HeLa cells (1x105 cells per 200 µL) in Medium for 8 hours at RT. Cell suspensions were washed twice by PBS and analyzed by fluorescence confocal microscopy for intracellular delivery of QDs. Olympus IX70 microscope was used in the analysis.
Additional data Figure 2S. Image of silica-shelled single QD micelles under visible light and UV-irradiation. The image was obtained from sample kept in the dark at room temperature during three months. The sample contains very low concentration of silica-shelled single QD micelles – under visible light, the sample was almost uncolored and completely transparent (for comparison, a sample containing distilled water is shown). Under UV-irradiation, a bright fluorescence was detected from the same sample containing silica-shelled single QD micelles.
Table 1S. Amplitudes of normalized PL-spectra of different types silica-shelled single QD micelles in distilled water.
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Table 2S. Cytotoxicity of different types silica-shelled single QD micelles on leukemia cells (Jurkat, K-562) and normal lymphocytes. The cells (5x105 cells per well) were treated with 5 µM of the respective product during 30 min, 3 h, 24 h, and 48 h in humidified atmosphere at 37 oC. Cell viability was calculated as a percentage from the viability of control (non-treated) cells. The viability of control cells was considered 100%. The results are mean +/- SD from 4 independent experiments. Incubation time
Viability of Jurkat cells,
Viability of K-562 cells,
(% from control)
(% from control)
Viability of normal lymphocytes, (% from control)
CdSe/ZnS inside the silica sphere 30 min
99 +/- 10
100 +/- 9
103 +/- 12
3 hours
101 +/- 12
100 +/ 10
105 +/ 9
24 hours
102 +/- 10
98 +/- 12
101 +/- 9
48 hours
98 +/- 11
97 +/- 8
100 +/- 11
30 min
105 +/- 9
104 +/- 8
102 +/- 10
3 hours
105 +/- 11
102 +/ 12
105 +/ 10
24 hours
99 +/- 12
106 +/- 11
99 +/- 13
48 hours
98 +/- 10
100 +/- 9
98 +/- 10
104 +/- 10
101 +/- 11
105 +/- 8
CdSe/ZnSe/ZnS inside the silica sphere
CdSe inside the silica sphere 30 min
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3 hours
100 +/- 13
103 +/- 8
98 +/- 10
24 hours
97 +/- 11
98 +/- 11
96 +/- 11
48 hours
101 +/- 8
97 +/- 10
95 +/- 12
24 hours
99 +/- 11
106 +/- 10
105 +/- 10
48 hours
101 +/- 7
103 +/- 12
100 +/- 11
24 hours
104 +/- 12
101 +/- 14
105 +/- 7
48 hours
104 +/- 9
103 +/- 8
103 +/- 9
24 hours
99 +/- 10
102 +/- 10
100 +/- 14
48 hours
97 +/- 11
98 +/- 11
102 +/- 9
CdSe/ZnS + cholesterol
CdSe/ZnS + hexane
CdSe/ZnS + toluene
