ARTICLE pubs.acs.org/JPCC
Biolabeling Hematopoietic System Cells Using Near-Infrared Fluorescent Gold Nanoclusters Xin Huang,†,|| Yi Luo,‡,|| Zhen Li,§ Buyi Li,† Hui Zhang,† Luo Li,† Irfan Majeed,† Ping Zou,‡ and Bien Tan†,* †
School of Chemistry and Chemical Engineering, Huazhong University of Science and Technology, Wuhan 430074, People's Republic of China ‡ Institute of Hematology, Union Hospital, Tongji Medical College, Huazhong Universty of Science and Technology, Wuhan 430022, People's Republic of China § ARC Centre of Excellence for Functional Nanomaterials, Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, QLD 4072, Australia
bS Supporting Information ABSTRACT: Highly bright fluorescent gold nanoclusters (Au NCs) have been prepared by one-step reduction of aqueous precursor solution in the presence of multidentate thioether-terminated poly(methacrylic acid) (PTMP-PMAA). The fluorescence quantum yield of the resultant Au NCs is 4.8% higher than that of the similarly sized Au NCs prepared by the etching method (1.84.0%). These Au NCs show excellent photostability and have been successfully applied to label the hematopoietic cells first. The results show that Au NCs were endocytosed by the cancer cells significantly more than the normal cells, in comparison with control experiments labeled with fluorescent quantum dots (CdTe). The cytotoxicity experiments demonstrate the excellent biocompatibility of Au NCs, proven by a relatively lower cytotoxicity than CdTe. These robust near-infrared Au NCs show great potential in biolabeling, tracking, and imaging of other cells and diseases, especially in the diagnosis and treatment of chronic myeloid leukemia.
’ INTRODUCTION Fluorescence bioimaging has become an indispensable tool in cancer research, clinical trial, and medical practice,1 and the used fluorophores can be organic dyes,2 fluorescent proteins,3 and inorganic nanoparticles.4 Special attention has been paid to fluorescent inorganic nanocrystals, e.g., IIVI and IIIVI quantum dots (QDs) during the past decades because their emissions can be well controlled over a wide range by tuning particle size and compositions.5 In addition, QDs show a larger Stokes shift and a higher photostability than other fluorophores. But the toxicity of QDs makes people doubtful about their future clinical applications, so the development of safe and nontoxic fluorescent biomarkers has been a subject of intensive research. In terms of toxicity6 and biocompatibility,7gold nanoparticles (Au NPs) are less-toxic metals and have strong optical absorption and scattering in the visible range as caused by collective oscillation of free electrons within the NPs (called localized surface plasmon resonance, LSPR) and become the alternative to quantum dots.8 Therefore, Au NPs have been extensively used for many applications including cell labeling,9tracking and imaging,10 and target drug delivery.11 According to Mie’s theory,12 Au NPs show a specific sizedependent plasma absorption when their size is smaller than average free path length of conduction electrons (i.e., < 20 nm). However, this exceptional property is dampened or disappears r 2011 American Chemical Society
when their size is further decreased to the regime of Au nanoclusters (Au NCs) with a size smaller than 2.0 nm,13due to the “spill-out” of conduction electrons and the influence of d-electrons and the effect of surface ligands.14 These effects lead to their novel optical properties in comparison with conventional Au NPs, i.e., tunable bright fluorescence which makes them very attractive in near-infrared (NIR) bioimaging,15photodynamic therapy,16 and fabricating biological sensing devices.17For example, Au NCs have been successfully used to label various cells such as human embryo kidney cells,14 human aortic endothelial cells,18 and human hepatoma (HepG2) cells.19 Small-sized fluorescent Au NCs can either be prepared by direct-reduction approaches or etching routes. The first example of colloidal fluorescent Au NCs was reported by Wilcoxon et al.,20 in which they observed a blue emission at 440 nm from small Au NPs. The breakthrough of preparing fluorescent Au NCs has been done by Zheng and his co-workers.21 They prepared a series of Au5, Au8, Au13, Au23, and Au31NCs in the presence of Polymer(amindoamine) (PAMAM) dendrimers through the direct reduction method. By adjusting the molar Received: March 19, 2011 Revised: July 23, 2011 Published: July 27, 2011 16753
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Scheme 1. (a) Reaction Scheme for the Synthesis of Thioether Polymer Ligand PTMP-PMAA; (b) Preparation of Au NCs Stabilized by Polymer Ligandsa; and (c) Process of Biological Labeling in Hematopoietic Normal and Cancer Cells
Insert is photographs of Au NCs aqueous solution under the irradiation of 365 nm ultraviolet light (left, red fluorescence) and day light (right, yellow color).
a
ratio between Au3+and PAMAM from 1:1 to 1:15, the emissions of these Au NCs were tuned from UV to the NIR range with a quantum yield (QY) of 10 to 70%, however, the latter experiments proved that PAMAM dendrimers made contributions to the solution fluorescence. Recently, Ying et al.22 prepared Au25NCs with 6.0% QY using bovine serum albumin (BSA) as a reducing agent. The reduction process was induced by adjusting the solution pH value. Besides the direct reduction method, an alternative approach of preparing fluorescent nanoclusters is etching “larger Au NPs”. For example, Duan et al.23etched 8 nm Au NPs with polyethylenimine (PEI) to produce Au8 clusters with an emission of 445 nm and a quantum yield of 1020%. Similarly, Parak's group18 etched 5.6 nm Au particles by adding Au-precursor solution to result in 3.2 nm particles. After replacing didodecyldimethylammonium bromide (DDAB) with dihydrolipoic acid, these Au NPs were further decreased to 1.6 nm and showed a red emission around 700 nm. The fluorescence QY of their nanoclusters was 3.4% in methanol and 1.8% in water (pH = 9). In contrast to previous reports, we have used multidentate thioether-terminated poly(methacrylic acid) (PTMP-PMAA) as ligands to prepare water-soluble fluorescent Au NCs (QY = 3.0%) with a diameter of 1.11.7 nm.24The results confirmed
that the multidentate polymer does not display any fluorescence and the solution fluorescence is only from Au NCs themselves. It is worth mentioning that, in addition to fluorescent Au NCs, this versatile polymer was used to synthesize monodispersed watersoluble Co NPs25 and Fe3O4 NPs.26 In this work, we first systematically optimize reaction conditions (e.g., polymer concentration and molecular weight) to produce highly bright Au NCs which show a quantum yield of 4.8%, higher photostability, and narrower size distribution (0.9 ( 0.2 nm). Moreover, multifunctional thioether polymer (PTMP-PMAA) as the stabilizer to prepare Au NCs has many excellent properties including weaker bonding force, biocompatibility, easy modification, and ligand exchange.27 We used these Au NCs to label cord blood mononuclear cells (CBMC), a type of relatively normal cells in hematopoietic system, and hematopoietic cancer cells K562 for the first time, by comparing them with CdTe QDs under the same conditions. These cells were chosen as targets because of their significance in leukemia research. Compared with other cell systems, the hematopoietic system is composed by a series of differentiating pluripotent, multipotent, and unipotent cellular intermediates which evolve into complicated mature blood cells with distinct functions.28 Additionally, there is no effective 16754
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The Journal of Physical Chemistry C treatment for leukemia except bone marrow transplant which is limited by donor resources and transplant rejection. Our investigation demonstrates the potential of these NIR fluorescent Au NCs in labeling, tracking, and imaging of cancer cells and other diseases, especially for chronic myeloid leukemia, due to the environmental safety, low cytotoxicity, biological specificity, and biocompatibility.
’ EXPERIMENTAL SECTION Materials. All chemicals were of analytical grade and used asreceived without any further purification, unless otherwise described. Methacrylic acid (MAA, 99%), 2,2-azobisisobutyronitrile (AIBN, 98%), hydrogen tetrachloroaurate (HAuCl4 3 4H2O, 47.8%), sodium borohydride (NaBH4, 99%), Rhodamine 6 G (Rh 6G), anhydrous ether, toluene, anhydrous ethanol, methanol, and tetrahydrofuran were purchased from National Medicines Corporation Ltd. of P. R. China. Pentaerythritol tetrakis 3-mercaptopropionate (PTMP, 97%) was obtained from Aldrich, CdTe@MPA were obtained from the Institute of Hematology, Union Hospital. Preparation of Gold Nanoclusters (Au NCs). Fluorescent Au NCs were prepared according to our previous report. Seventeen mL HAuCl4 solution was added into a polymer aqueous solution (3 mL) under vigorous stirring to give a final concentration of HAuCl4 of 0.5 mM. After the gold/polymer mixture solution was stirred for 0.5 h, freshly prepared NaBH4 solution (2 mL, 50 mM) was added into the mixture. It should be noted that the reducing agent was added two times (1 mL each time). The reaction was then allowed to continue overnight under vigorous stirring. The resultant Au NCs were dialyzed overnight using a 96-well microplate dialyzer (the molecular weight cutoff is 10 000 g/mol) in order to remove impurities and free polymer ligands. The weight contents of Au NCs in the final product was 12.1% determined by thermo gravimetric analysis (TGA, See Supporting Information, Figure S2), which was approximately equal with the theoretical value. Thus, the concentration of Au precursor can be used as the concentration of Au NCs. In order to prepare highly fluorescent Au NCs, we optimized the reaction conditions by changing polymer concentration (from 0.6 to 30 mM), polymer molecular weight, and the molar ratio between Au-precursor to polymer. Cell Culture and Treatment with Au NCs. Heparinized cord blood was loaded on Ficoll-Hypaque (TBD Sciences) in 15 mL tubes, and then centrifuged with a speed of 350 g for 30 min at room temperature, and mononuclear cells at the interface were collected. The CBMC (cord blood monouclear cells) and K562 cells were suspended in RPMI 1640 medium(GIBCO) containing 10% (v/v) fetal calf serum (FCS, GIBCO), 2 mM/L glutamine, 100 units of penicillin per mL, and 100 μg of streptomycin per mL. Cells were incubated in 25 cm2 tissue culture flasks (cell density is 1 106 cells per mL) at 37 °C under a condition of humidified atmosphere with 5% CO2. Half of the culture medium was weekly replaced with freshly prepared medium. The cultured cells were then split into the 24-well tissue culture plates with a density of 2 105 per well, and Au NCs were added into the medium directly with a concentration of 30 and 50 nM, respectively. After incubation for 24 h, cells were collected for various experiments. The process of cell labeling with QDs (CdTe@MPA) was the same to the case of Au NCs. In addition to CBMC and K562 cells, Jurkat cells and HeLa cells were labeled with fluorescent Au NCs. Jurkat cells were first suspended in the RPMI 1640 without FCS (HeLa cells
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were cultured in DMEM without FCS), and then Au NCs (1 μM) were added into the medium and incubated with cells for 5 h at 37 °C. Detection the Uptake of Au NCs and QDs. After 5 and 24 h of incubation, the uptake of Au NCs (or QDs) by cells was tested. Cells were fixed with 4% paraformaldehyde, washed with phosphate buffered saline (PBS), followed by staining nuclei with bisbenzimide (18.7 μmol/L) for 15 min at room temperature, then washed with PBS again, and finally immersed in 50% glycerol (v/v). Fluorescence images of the cells were then recorded using a confocal microscope (OLYMPUS) with 488/ 560 nm (excitation/emission) for AuNCs (or QDs) and bisbenzimide, respectively (Scheme 1c). MTT Assay. CBMC and K562 cells were placed in 96-well plates at a density of 50006000 cells per well, then Au NCs and QDs were added into cell suspensions with different concentrations (30 nM, 50 nM), respectively. One-well cells were treated without Au NCs (or QDs) for control experiment. After these cells were cultured at 37 °C for 24 h, 3-(4,5-dimethylthiazol2-yl)2,5-diphenyltetrazolium bromide (MTT) was added to each well and incubated for 4 h. After incubation, removed the media and added DMSO to extract the MTT in the cells. Then absorbance was measured at 570 nm for each well. For each concentration, the experiments were repeated in triplet wells five times. Cell Apoptosis Assay. Apoptosis rate of CBMC and K562 cells was analyzed using an Apoptosis AssayKit (Gaigene) according to the manufacturer’s protocol. Briefly, after being treated with 50nM Au NCs, the cells were washed and collected. Then the cells (1 106) were resuspended in 500 μL of Annexin-binding buffer and fluorescein isothiocyanate Annexin-V/propidiumiodide. After 15 min of incubation at room temperature, samples were analyzed by a flow cytometry measuring the fluorescence emission at 530 nm. At least, 10 000 events were counted and experiments were repeated for five times. Transmission Electron Microscope (TEM). Transmission electron microscopy images were recorded on a JEOL-2100 electron microscope operating at an acceleration voltage of 200 kV. TEM samples were prepared by dropping a diluted water solution of Au NCs onto carbon-coated copper grids (400 mesh). Dynamic Light Scattering (DLS). The particle size was measured by a high-performance particle sizer (Zetasizer Nano S) with DLS (Dynamic Light Scattering) and NIBS (Noninvasive Back Scatter) technology from Malvern Instruments (Malvern, UK) with an effective detection capability from 0.6 to 6000 nm. The samples were put into the plastic cells and measured under the condition of 25 °C. MALODI-TOF. Samples were analyzed on a Bruker Autoflex MALDI-TOF/TOF instrument. Samples (0.5 μL) were spotted to a MALDI target plate with 0.5 μL CHCA matrix (10 mg/mL CHCA in 50% ACN/0.1%TFA/25 mM diammonium citrate) and externally calibrated with a mixture of angiotensin I (5 pmol/μL)/ ACTH (5 pmol/μL)/insulin (100 pmol/μL)/cytochrome C (100 pmol/μL). Data was processed using Flex Analysis (Bruker). XPS. The XPS of Au NCs was measured by AXIS-ULTRA DLD high-performance imaging X-ray photoelectron spectroscopy (Shimadzu, Japan). The energy resolution was set to 1.7 eV to minimize data acquisition time, and the photoelectron takeoff angle was 37°. Fluorescence Spectroscopy. The fluorescence of Au NCs was determined by a FP-6500 fluorescence spectrometer (Jasco, JPN) at room temperature. The emission spectra were obtained with an excited wavelength of 480 nm and the excitation 16755
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Figure 1. (a) Color photographs of gold nanoclusters prepared from different molecular weights and concentrations of PTMP-PMAA under daylight (left) and UVvisible light at 365 nm (right). From groups AE, the used polymer ligands are PTMP-PMAA0.5%, PTMP-PMAA1.0%, PTMPPMAA2.0%, PTMP-PMAA3.0%, and PTMP-PMAA4.0%, respectively; (b) The 3D emission spectra (excitation at 480 nm) of Au
[email protected]% obtained from various polymer concentrations. (c) The emission spectra of Au
[email protected]% (15 mM) powder (red line) and aqueous solution (green line) and corresponding photographs under visible light (left) and UV light (right).
spectra were measured at the corresponding maximum emission wavelength. Zeta (ζ) Potential. The ζ potential values were determined by a Malvern nanoZS90 (Malvern Instruments, UK) at 25 °C using the folded capillary cells. Data were obtained using a monomodal acquisition and was fit according to the Smoluchowski theory. After the process of filtration with aqueous membrane (Φ = 13 mm, 0.22 μm), the samples were measured with ultrapure water as solvent (pH = 7). These measurements were run at least three times with independent particle batches. Quantum Yield (QY) Measurement. The fluorescent QY of a compound is defined as the fraction of molecules that emits a
photon after direct excitation by the source.29 This quantity is not the same as the total number of emitted photons which escape a bulk sample divided by the total number of absorbed photons, although in many instances, the two quantities are nearly equal. The measurement of QY was employed the compared method which is described below (eq 1): Φunk ¼
Astd Funk nunk 2 Φstd Aunk Fstd nstd 2
ð1Þ
where Φ = quantum yield; unk = unknow sample; std = standard; n = refractive index of solvent; A = absorption at the selected 16756
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Figure 2. Transmissionelectron microscopy (TEM) image of Au NCs prepared from polymer ligand PTMP-PMAA2% with a concentration of 15 mM. Insert is the high resolution transmission electron microscopy (HR-TEM) and the scale bar is 1 nm.
excitation wavelength; F = integrated fluorescence signal in the emission region. To calculate the QY of Au NCs protected by polymer ligands at different concentrations, a series of the samples and the standard Rhodamine 6G (Rh 6G, Φ = 0.95) were measured. All of the samples were diluted to ensure the optical densities less than 0.02 (in order to reduce the error) measured by Lambda 35 UVvis spectrophotometer (Perkin-Elmer, U.S.) to get the value of A (absorption at the selected excitation wavelength). The emission spectra of samples and Rh 6G were recorded on FP-6500 fluorescence spectrometer (Jasco, JPN) under the same excitation of 480 nm light. Then the emission curve were integrated using the software to obtain the F (integrated fluorescence signal in the emission region) mentioned in the eq 1. Eventually, eq 1 was used to measure the QY of the Au NCs. Each sample was measured three times to obtain the final result.
’ RESULTS AND DISCUSSION Synthesis and Characterization of Au NCs. Au NCs were prepared by employing multidentate thioether-terminated poly(methacrylic acid) (PTMP-PMAA) as stabilizers (Scheme 1). The samples were divided into five groups (AE) based on the different polymer molecular weights. Figure 1(a) shows the obtained solutions of Au NCs under daylight and under excitation of 365 nm UV light. At low polymer concentration (0.6 mM), Au NCs do not exhibit fluorescence in each group, but the fluorescence is apparent when the polymer concentration is increased to 3 mM. The intensities increase to the maximum value and then decrease, with the increase of polymer concentration from 3 to 30 mM. The variation of particle size and distribution is a crucial factor influencing the fluorescence of Au NCs. Tailoring the polymer molecular weights (Mn) and concentrations, we can produce a
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series of Au NCs with different sizes and size distributions. Figures S4,5 of the Supporting Information, SI, show the effects of polymer Mn and polymer concentration on the average hydrodynamic size of Au NCs, determined by dynamic light scattering (DLS) measurement. Most of the samples have a small hydrodynamic size (
