Supporting Information Click-Functionalized Compact Quantum Dots Protected by Multidentate-Imidazole Ligands: Conjugation-Ready Nanotags for Living-virus Labeling and Imaging Pengfei Zhang, Shuhui Liu, Duyang Gao, Dehong Hu, Ping Gong, Zonghai Sheng, Jizhe Deng, Yifan Ma and Lintao Cai* CAS Key Laboratory of Health Informatics, Shenzhen Key Laboratory of Cancer Nanotechnology, Institute of Biomedicine and Biotechnology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, 518055, P. R. China. Experimental Section Materials: poly(maleic anhydride) (MW=5000 Da) was purchased from PolySciences, Inc, USA. N3-PEG8-CH2CH2NH2 (MW=438 Da) were obtained from Biomatrik Inc., China. DBCO-PEG4-NHS ester was purchased from Click Chemistry Tools (Scottsdale, FL, USA). Lab-Tek II coverglass-bottomed 8-well chamber slides were obtained from Nalgene Nunc International (Naperville, IL). Commercial CdSe/ZnS quantum dots (QDs) were acquired from three sources. Organic-soluble QD525 was purchased from Ocean Nanotech (Springdale, Arkansas, USA). Organic-soluble QD605, OPA-QD605 and SA-OPA-QD605 were commercially available from Jiayuan Quantum Dot Co. Ltd. (Wuhan, China), Organic-soluble QD705 was purchased from Invitrogen. SYBR gold nucleic acid gel stain and baculovirus transduction the Organelle Lights Tubulin-GFP were purchased from Invitrogen (Carlsbad, CA, USA). Unless specified, chemicals were purchased from Sigma-Aldrich (St. Louis, MO) and used without further purification. All reagents and solvents were obtained from commercial suppliers and were used as received. Milli-Q grade (R > 18MΩ cm) water was used throughout. Characterization: Ultraviolet–visible (UV-vis) absorbance spectra were taken using a PerkinElmer Lambda 25 UV-Vis absorption spectrophotometer. Photoluminescence (PL) spectra were recorded with a Edinburgh F900 fluorescent spectrometer. Transmission electron microscopy (TEM) images were taken on a JEOL JEM-1230 transmission microscope at 100 kV. Samples for TEM observation were prepared through dropped 10 µL of the solution on a carbon-coated grid and allowed the sample to dry. 1H NMR spectra were recorded on a Bruker SpectroSpin 400 MHz spectrometer and the chemical shifts are reported relative to tetramethylsilane (TMS, δ = 0.00 ppm). FTIR spectra were obtained on a Vertex 70 FTIR spectrometer (Bruke, Germany) scanning from 4000 cm-1 to 400 cm-1. Dynamic light scattering (DLS) analysis was performed using a Zetasizer Nano ZS (Malvern Instruments). All QDs samples were between 2-3 µM in concentration and filtered through a 0.2 µm filter before analysis. Gel Filtration Chromatography (GFC) was performed using an AKTAprimePlus chromatography system from Amersham Biosciences equipped with a Superose 6 10/300 GL column. PBS (pH 7.4) was used as the mobile phase with a flow rate of 0.5 mL/min. Typical injection volumes were 100µL. Detection was achieved by measuring the absorption at 280 nm. Calibration of hydrodynamic diameter was performed by injecting 100 µL of gel filtration protein standards containing blue dextran (2000 kDa, 29.5 nm), thyroglobulin (669 kDa, 17.0 nm), γ-globulin (158 kDa, 11.0 nm), ovalbumin (44 kDa, 5.5 nm), myoglobin (17 kDa, 3.8 nm), and
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vitamin B12 (1.4 kDa, 1.5 nm). Multidentate Polymer Ligand Synthesis: To synthesize the multidentate polymer, 1.0 g of poly(maleic anhydride) (MW=5000 Da) was dissolved in 5 mL of DMSO containing 20 mg of DMAP, and 1.2 g of histamine was added as solid. With the reaction going on at room temperature, the histamine solid gradually dissolved within 2 h. After another 4 h, the product, named as PMAH, was purified by precipitation in water followed by lyophilization. The obtained product has excellent solubility in strongly polar solvents, such as DMSO, DMF or water. To synthesize the azido-derivatized multidentate polymer, a mixture of histamine and N3-PEG-NH2 was used instead of pure histamine. Ligand Exchange with the Multidentate Polymer: In a typical procedure, organic-soluble QD nanoparticles solution in decane were first flocculated by using a methanol/isopropanol mixture (v/v 75/25), and resuspended in chloroform. The QD stock solution was mixed with PMAH/N3-PMAH solution of DMSO, and stirred for 10 min at RT, after which TMAOH was added and stirred for an additional 20 min to form a biphasic system. The solution was centrifuged at 5000 x g for 10 min to achieve phase separation. The organic layer was removed by pipette and any residual chloroform was removed from the aqueous phase by evaporation with stirring under reduced pressure. The PMAH/N3-PMAH coated QDs were precipitated by centrifugation twice with the addition of acetone and redispersed in PBS buffer at pH 7.4. Free unbound PMAH/N3-PMAH ligands were removed by an ultrafiltration device with a cutoff of 50 kDa. DHLA-QDs were prepared according procedures previously reported. OPA-QD and SA-QDs were commercially provided by Jiayuan Quantum Dot Co. Ltd. Evaluation of QD Stabilities: We conducted a series of parallel stability tests using DHLA-capped QDs as the control sample. For storage stability comparison of DHLA-capped QDs and PMAH-capped QDs, the two QDs samples were dissolved in PBS buffer (pH 7.4), respectively. Afterwards, the two samples were stored in 4 o
C refrigerator. The corresponding hydrodynamic diameter and PL intensity of the samples were
recorded by Malvern Zetasizer Nano ZS instrument and Edinburgh F900 fluorescent spectrometer at different interval times, respectively. For thermal stability comparison of DHLA-capped QDs and PMAH-capped QDs, the two samples were dissolved in PBS buffer and the DLS measurement was taken at a heating course from 25 oC to 75 oC, respectively. The PL intensity of the samples were recorded after treatment at 75 oC for different times. For the photostability experiment, the two samples were continuously exposed to the xenon lamp (365nm) over 60 min, PL intensity were recorded at 30s intervals, respectively. For chemical oxidation experiments, the two QDs samples were dissolved in freshly prepared PBS buffers with H2O2 concentration ranging from 17µM to 176µM. PL intensities were recorded after 10 minutes. For pH stability experiments, the PMAH-QDs samples were dissolved in buffer at the desired pH and stored at ambient temperature. The photograph of the dispersions was collected (when needed) upon illumination with a UV lamp. For salt solution stability experiments, the PMAH-QDs samples were dissolved in a series of NaCl solutions with concentration ranging from 0 to 2.0 M and stored at ambient temperature The photograph of the dispersions was collected (when needed) upon illumination with a UV lamp. For stability tests in polar organic solvents, the PMAH-QDs samples were dissolved in the common solvents including DMSO, DMF and methanol. The samples were stored at ambient temperature, and the photograph of the dispersions was collected (when needed) upon illumination with a UV lamp.
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Cell Cultures: A549 cells (human lung adenocarcinoma epithelial cell line), Hela cells (Human epithelial carcinoma cell line), MB-MDA-231 cells (human breast cancer cell line), 293T cells (Human embryonic kidney cell line), and RAW 264.7 cells (Mouse leukaemic monocyte macrophage cell line) were originally obtained from American Type Culture Collection (ATCC). Cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Sigma-Aldrich) and 100 UI/mL penicillin and 100 µg streptomycin (Lonza). Cell cultures were incubated in a 5% CO2 humidified incubator at 37°C. Cytotoxicity Assay: MTS
(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,
inner salt)-Based Cell Viability Assay was performed to assess the metabolic activity of cells. Cells were seeded in 96-well plates (Costar, IL, USA) at an intensity of 5 x 104 cells/mL. After 24 h incubation, the old medium was replaced by the QDs solution in medium at different QDs concentrations, and the cells were then incubated for 24h. After the designated time intervals, the wells were washed twice with PBS buffer and 20 µL of MTS Reagent was added into each well. After 3 h incubation at 37°C, The absorbance of MTS at 490 nm was monitored by the microplate reader. Cell viability was expressed by the ratio of absorbance of the cells incubated with QDs solution to that of the cells incubated with culture medium only. QD-Labeling of Virus: To realize viruses labeled with QDs, the dibenzocyclooctyne (DBCO, an strained cyclooctyne moiety) was first introduced to the viral surface. The viruses were incubated with DBCO-PEG4-NHS ester (Click Chemistry Tools) for 2 h at room temperature. Unbound DBCO-PEG4-NHS ester was removed by gel filtration on a NAP-5 column (GE Healthcare), followed by incubation with 10 nM of N3-PMAH-QDs for 60 min at room temperature. Virus-QDs conjugations were confirmed by gel electrophoresis and colocalization imaging analysis. The viral infectivity and gene expression were evaluated using confocal laser scanning microscope. Confocal Imaging: Fluorescent images were acquired on a Leica TCS SP5 laser scanning confocal microscope equipped with Argon, red HeNe, and green HeNe lasers. Images were collected using a Plan-apochromat 63X/1.4 oil immersion objective by sequential line scanning, with excitation at 488 nm along with a brightfield image. Emission was collected by photomultiplier tubes in the ranges 510-570 and 590-630 nm, respectively, obtained by tunable high-reflectance mirrors. To ensure the QDs were coupled to the virus, the virus particles labeled with QDs were further stained with a fluorescent dye, SYBR gold, to label the viral nucleic acids. QD-labeled viruses were overlaid upon coverslips for 60 min at 37 °C. The coverslips were then rinsed, fixed with 4% formaldehyde and imaged by confocal laser scanning microscope. For the study of viral infectivity, A549 cells were cultured in the confocal imaging chamber (LAB-TEK, Chambered Coverglass System) at 37 °C. After 80% confluence, the medium was removed and the adherent cells were washed twice with PBS buffer. The labeled virus in DMEM medium was then added to the chambers. After incubation for 2 h, the cells were washed three times with PBS buffer and imaged by confocal laser scanning microscope. The cells were further incubated overnight and the gene expression was imaged after an overnight period. Images were analyzed with the use of the Leica TCS SP5 software.
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Figure S1. Schematic illustration of a) the one-step synthesis of the imidazole multidentate polymer ligands (PMAH) and b) the procedure to prepare PMAH coated QDs.
Figure S2. 1HNMR spectra of the a) PMAH and b) N3-PMAH multidentate polymers measured in DMSO-d6, respectively. The peaks at ~6.95 ppm(b) and ~7.67ppm(a) correspond to the protons from the imidazole ring.
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Figure S3. FT-IR spectra of original oil-soluble QDs (bottom), PMAH (top) and as-received water-soluble PMAH-QDs (middle). Spectra show that the bands characteristic of hydrophobic ligand at 2922 cm−1 and 2852 cm−1 have disappeared following cap exchange, while new bands at 1689 cm−1 (ascribed to amide I, C=O stretching) and at 1574 cm−1 (ascribed to amide II, N−H bending) were measured. Moreover, the spectrum of the PMAH-QDs is identical to that collected from the pure PMAH.
Figure S4. a) - c) TEM images of three different-sized CdSe/ZnS QDs(QD525, QD605 and QD705) capped with PMAH, showing nonaggregated monodisperse samples. d) Dynamic light scattering measurement of QDs ligand exchanged with PMAH. e) - g) Normalized absorbance and photoluminescence spectra of the three different-sized QD before ligand exchange in chloroform (solid lines), and after ligand exchange with PMAH in PBS (dashed lines). The optical densities for each sample in CHCl3 and in water were matched at their excitation wavelengths in order to be able to directly compare the photoluminescence intensities of the two samples. Fluorescence spectrum measurement revealed that the emission peak of PMAH-capped QDs did not shift, which was the same as that of original QDs, and the photoluminescence intensities in water were compared to those in CHCl3. h) Relative optical intensities of the three QDs samples in water compared with CHCl3.
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Figure S5. Protein standards for gel filtration chromatography (GFC). From left to right: blue dextran ( 2000 kDa, 29.5 nm), thyroglobulin ( 669 kDa, 17.0 nm), gamma -globulin ( 158 kDa, 11.0 nm), ovalbumin ( 44kDa, 5.5 nm), myoglobin ( 17 kDa, 3.8 nm), and vitamin B12 ( 1.4 kDa, 1.7 nm).
Figure S6. Stability test of PMAH-QDs in different incubation conditions. a) Fluorescence photograph for a set of QDs capped with PMAH in various pH buffers. The visible formation of macroscopic aggregation was observed below pH 5 due to the protonation of the imidazole group. This was consistent with the binding of the polymer to the surface of QDs via metal-affinity interactions. b) Fluorescence photograph of PMAH-QDs under various concentrations of NaCl solutions. c) Fluorescence photograph of PMAH-QDs that instantly dissolved in polar organic solvents. Samples were excited using a hand-held UV lamp at 365 nm. The image was collected at room temperature.
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Table S1. The influence of pH value on the hydrodynamic diameters of PMAH-QDs.
pH
2
3
4
5
*
*
*
*
hydrodynamic diameters (nm)
6
7
8
9
10
11
10.43
9.95
10.05
10.28
10.20
10.37
± 0.16
± 0.16
± 0.31
± 0.23
± 0.36
± 0.15
* The visible formation of macroscopic aggregation was observed.
Figure S7. Cytotoxicity of PMAH-QDs. A549, Hela, MB-MDA-231, 293T, and RAW 254.7 cells were incubated with QDs at different concentrations ranging from 0 to 400 nM for 24 hours. The cell viabilities were measured with the MTS assay. All conditions were performed in triplicate. Data represent the average of three replicate wells with standard deviation.
References
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