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Low-toxic Mn-doped ZnSe@ZnS Quantum Dots Conjugated with Nanohydroxyapatite for Cell Imaging Ronghui Zhou,1 Mei Li,2 Shanling Wang,2 Peng Wu,2 Lan Wu,2* Xiandeng Hou1, 2
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Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX DOI: 10.1039/b000000x Fluorescent bio-imaging has received great attention in a myriad of research disciplines, and QDs is playing an increasingly important role in these areas. Doped QDs, as an important alternative to conventional heavy metal-containing QDs, is appealing for biomedical applications. However, since QDs is exogenous substances to biological environment, the biocompatibility of QDs is expected to be problematic in some cases. Herein, nano fluorine-doped hydroxyapatite (FAp, a well-known biocompatible material) was introduced to embark biocompatibility to Cd-free Mn-doped ZnSe@ZnS QDs. A nano-FAp-QD conjugate was thus developed and the biocompatibility as well as potential cell imaging application was investigated. To construct the proposed conjugate, Cd-free highly luminescent Mn-doped ZnSe@ZnS QDs and monodispersed nano-FAp were first prepared in high-temperature organic media. For facilitating the conjugation, hydrophobic nano-FAp was made water-soluble via ophosphoethanolamine (PEA) coating, which further provides conjugating sites for QDs to anchor. Cytotoxicity studies indicated the developed conjugate indeed possesses good compatibility and low toxicity to cells. The nano-FAp-QDs conjugate was successfully employed for cancer cell staining for at least 24 h, demonstrating the potential usefulness of this material in future biomedical research.
1. Introduction As an effective method of optical bio-imaging technique, fluorescent imaging has drawn great attention due to its' high sensitivity, multicolor imaging capability and low cost.1-5 Owing to the complexity of the biological system, a high-quality fluorescence imaging agency for diagnosis and therapy must be characterized as low toxicity, good biocompatibility, high brightness, and good photostability. Compared with conventional organic dyes, fluorescent quantum dots (QDs) possess unique photophysical properties such as size-tunable emission, broad excitation, and high photostability, which make them a promising type of fluorescent imaging agency.6-8 However, heavy metals in typically used QDs (majorly CdSe and CdTe QDs) are reported to be released under biological stress, resulting in toxicity of these QDs in vivo.9 Doped QDs, especially the Cd-free Mn-doped ZnS or ZnSe QDs, is an important alternative to CdSe or CdTe QDs,10 thus appealing for bio-imaging applications.11-15 Moreover, doped QDs can be protected against photooxidation due to efficient and fast energy transfer from host to dopants, which quickly localizes the excitation and suppresses undesirable reactions on the surface of QDs.16 Therefore, the photostability of doped QDs is increased as compared to undoped counterparts. However, both the conventional Cd-containing QDs and the doped QDs are all exogenous substances to biological environment. In other words, the biocompatibility of these QDs is expected to be problematic in some cases. Hydroxyapatite (HAp) is the main inorganic component of bones and teeth and also an ideal biocompatible material. When This journal is © The Royal Society of Chemistry [year]
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decreasing the size of HAp to the nano-size regime, the perfect biocompatibility, low immunogenicity, and biodegradable property of this materials can be harvested for tissue engineering17 and drug and gene delivery.18-20 Also, use of nanoHAp as scaffolds for loading or compositing of inorganic functional materials has been shown to be an effective way for embarking biocompatibility to these materials, such as Fe3O4 magnetic nanoparticles,21, 22 carbon nanotube,23 zirconia,24 silica,25 and etc. For loading of QDs, Guo et al. conjugated CdSe@ZnS QDs with nano-HAp to improve the biocompatibility QDs for in vivo imaging;26 Chang et al. labeled nano-HAp with CdSe@ZnS QDs via compositing to investigate the interaction between nano-HAp and MC3T3-E1 osteoblast cells.27 Obviously, the biocompatibility of CdSe@ZnS QDs was largely increased after marriage with nano-HAp. But still, Cd-containing QDs was still used in the above two studies. Besides, the agglomeration of nano-HAp is expected and would possibly decrease the imaging sensitivity and resolution. Therefore, herein we conjugated Cd-free Mn-doped ZnSe (Mn:ZnSe) QDs with nano-HAp to develop a low-toxic and biocompatible fluorescent contrast material for bio-imaging. Highly fluorescent Mn:ZnSe QDs was synthesized in organic phase and shelled with a thin layer of ZnS after watersolubilization to further increase the photostability of the doped QDs. To ensure the monodispersity and crystallinity of HAp material, nano fluorine-doped HAp (nano-FAp) was first synthesized in high-temperature organic media with the liquidsolid-solution (LSS) strategy.28-30 Then, monodispersed nano[journal], [year], [vol], 00–00 | 1
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2.4 Investigation of the Cellular uptake of nano-FAp-QD nanoparticles
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2.1 Synthesis of Mn:ZnSe@ZnS D-Dots 15
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Mn-doped ZnSe QDs was prepared via the "nucleationdoping" approach developed by Peng et al. (Supporting Information).31-33 Briefly, MnSe nanoclusters was firstly formed with Mn2+ and Se precursors. Then, Zn2+ precursors was added to form ZnSe shell around the MnSe nanoclusters. The obtained QDs was purified using acetone/CHCl3 extraction with QDs in the CHCl3 layer. For overcoating of ZnS shell, Mn-doped ZnSe QDs was firstly subjected to a standard ligand exchange strategy with MPA,34 and the polar carboxylic groups renderd the QDs water-soluble. After ligand exchange, an additional ZnS shell was grew on the outer layer of the Mn:ZnSe QDs to form MPAcapped core/shell Mn:ZnSe@ZnS QDs.35 The final QDs product was stored in distilled water with a concentration of 5 mg/mL. 2.2 Synthesis of PEA modified monodispersed nFAp
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The monodispersed nano-FAp nanoparticles was synthesized via a hydrothermal synthetic route based on the LSS strategy (Supporting Information).28-30 Then, oleic acid-capped monodispersed nano-FAp was phase-transferred with PEA. Briefly, an amount of 50 mg hydrophobic nano-FAp nanoparticles was dispersed in 10 mL cyclohexane, a solution of phosphoethanolamine (0.5 g in 10 mL distilled water) was added under stirring. Thereafter, the nano-FAp was successfully transferred into the water layer due to the replacement of oleic acid with PEA. After decanting of the upper cyclohexane layer, the hydrophilic nano-FAp was collected via centrifugation and then washed with distilled water three times. The amino terminal groups on the surface of nano-FAp provide conjugation sites with MPA-capped Mn:ZnSe@ZnS QDs. 2.3 Synthesis of nano-FAp-QD nanoparticles
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MPA-capped Mn:ZnSe@ZnS QDs can be conjugated with amino-termined nano-FAp via covalent cross-linking. The weight ratios of the QDs to the nano-FAp were optimized through investigating their effects on the fluorescent intensities and photostabilities of the nano-FAp-QD nanoparticles (Fig. S2). Weight ratio of 1:2 (QDs: nano-FAp) was chosen for use based on the optimization results. Typically, PEA modified nano-FAp was suspended in Tris-HCl buffer (0.1 M, pH 7.4) at a concentration of 5 mg/mL. At the same time, carboxyl-terminated Mn:ZnSe@ZnS QDs (1 mL, 5 mg/mL) was activated with 1-(3dimethylamino-propyl)-3-ethylcarbodiimide hydrochloride (EDC, 8 mg) and N-hydroxysuccinimide (NHS, 4 mg) in Tris2 | Journal Name, [year], [vol], 00–00
HCl buffer (0.1 M, pH 7.4) for 20 min. Then 2 mL well-dispersed nano-FAp was added to the activated solution, and this mixture was stirred for 90 min. To ensure the conjugation, 0.1 M NaCl was added to shield the adsorption effect. The as-synthesized product was washed and re-dispersed with Tris-HCl buffer (0.1 M, pH 7.4) several times.
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The quantitative cellular uptake of nFAp-QDs nanoparticles with different incubation time was measured by flow cytometry. The Hela and HepG2 cells were seeded in 6-well plates at a density of 1 × 105 cells/well in 2 mL DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicilline streptomycin at 37 ºC for 24 h. Then, 50 µg/mL nFAp-QDs nanoparticles were added and incubated for 4 h, 8 h, 12 h and 24 h, respectively. The cells were washed with PBS for three times and harvested by trypsinization and centrifuged at 1000 rpm for 5 min, resuspended in 0.5 mL PBS medium and examined by flow cytometry (Cytomics FC 500, Beckman Coulter) with 360 nm excitation lasers. For uptake studies, the two cell lines were treated with the following inhibitors for 0.5 h: genistein (0.2 mM, the caveolaemediated endocytosis inhibitor), amiloride (1 mM, the macropinocytosis inhibitor), and chlorpromazine (10 µg/mL, the clathrin-mediated endocytosis inhibitor), respectively. Then, 50 µg/mL nFAp-QDs nanoparticles were added and further incubated for 12 h. Cells incubated only with the nFAp-QDs nanoparticles were used as the control. The fluorescent intensity of per cell was tested by flow cytometry at 360 nm excitation after the same process of cell wash, harvesting and re-suspending as the above section mentioned. 2.5 Cytotoxicity of nano-FAp-QD nanoparticles
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To examine the effects of nano-FAp-QD nanoparticles on the live cells, an inverted optical microscope was used to observe the cell morphology change after incubating with nano-FAp-QD in DMEM medium for different time. HeLa and HepG2 cells were dispersed in 6-well plates at a density of 1 × 105 cells/well in 2 mL DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicilline streptomycin at 37 ºC. After 24 h of cell adherence, the plates were washed with PBS and divided to two categories for treating with 200 µg/mL nano-FAp-QD in DMEM medium for 24 h and 48 h, respectively. Then all wells were washed with PBS to remove the excess nano-FAp-QD, and the morphologies of cells treated for 24 h and 48 h, respectively, were observed using the inverted optical microscope. Cell viability was investigated with MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) test. The HeLa and HepG2 cells were cultured in 96-well assay plates at a density of 1 × 104 cells/well (100 µL total volume per well). After cultured in DMEM medium containing 10% FBS and 1% penicilline streptomycin at 37 ºC and 5% CO2 for 24 h, different concentrations of nano-FAp-QD were added to the wells, and the cells were further cultured for 24 h. Thereafter, nanoparticles was removed and 10 µL MTT (5 mg/mL in PBS) following with 90 µL DMEM medium were added into each well. After another 4 h of incubation, 100 µL dimethyl sulfoxide (DMSO) was added to each well to dissolve the dark blue formazan crystal. The This journal is © The Royal Society of Chemistry [year]
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FAp was phase-transferred with o-phosphoethanolamine (PEA). PEA can be quickly absorbed on the surface of nano-FAp to endow it hydrophilic and provide functional amino groups. MPAcapped Mn:ZnSe@ZnS QDs can be thus easily conjugated with nano-FAp via covalent cross-linking. To investigate the biocompatibility of the developed nano-FAP-QD composite material, the interaction between nano-FAP-QD composite and HeLa and HepG2 cells was studied through cell viability and cellimaging. The results showed that the monodispersed nano-FAPQD nanoparticles possessed high luminescence, excellent biocompatibility and photostability, which can be explored for cell imaging applications.
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formazan concentration was finally quantified using a microplate reader by measuring the absorbance at 570 nm (Thermo Fisher Scientific, Varioskan Flash).
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The cell imaging experiments were performed by confocal laser scanning microscopy (CLSM, excited at 405 nm, Leica TCP SP5). HeLa and HepG2 cells were seeded in 35 mm glass culture dishes at a density of 1 × 104 cells/well and allowed to adhere for 24 h at 37 ºC. After removing the culture medium, the cells were incubated with nano-FAp-QD nanoparticles at a final concentration of 50 µg/mL for 24 h and 48 h. Afterward, the cells were washed with PBS and observed by using CLSM.
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3. Results and discussion 3.1 Characterization of Mn:ZnSe@ZnS QDs 55 15
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In this work, Mn:ZnSe@ZnS core/shell doped QDs was explored as the fluorescent material for cell imaging. In order to obtain highly bright and stable doped QDs, Mn-doped ZnSe QDs was firstly prepared via the "nucleation-doping" approach developed by Peng et al. (Supporting Information).31-33 As shown in Fig. 1A, the characteristic photoluminescent peak at around 590 nm indicates the successful doping of Mn2+ into ZnSe host, which can be ascribed to the Mn2+ 4T1 → 6A1 transition that gains energy from ZnSe host. Meanwhile, a small peak at about 450 nm was also observed possibly due to trap state emission of ZnSe QDs (Fig. 1A).31 Meanwhile, the Mn:ZnSe QDs can be successfully translated to be water-soluble through a routine ligand exchange process,34 and the solution of MPA-capped Mn:ZnSe QDs displayed orange fluorescence under UV irradiation (inset in Fig. 1A).
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Mn:ZnSe QDs (Supporting Information). Both the UV-vis absorption and the corresponding FL spectra of Mn:ZnSe@ZnS QDs exhibited a slight red shift compared to the Mn:ZnSe QDs due to the formation of ZnS shell outside of the Mn:ZnSe core (Fig. 1A). Besides, the Mn:ZnSe@ZnS QDs exhibited obviously higher fluorescence intensity as compared to the original Mn:ZnSe QDs, which can be ascribed to the elimination of surface defects by ZnS shelling. Meanwhile, the trap state emission at 450 nm was completely diminished. The fluorescence quantum yield (QY) of the Mn:ZnSe@ZnS QDs was determined as 25.6% (directly useing Rohdamine 6G as reference, Supporting Information) and 58.7% (used CdSe quantum dots as intermediate reference). After ZnS shelling, the fluorescence excitation spectrum of Mn:ZnSe@ZnS QDs also slightly redshifted (Fig. 1B), which can be well-excited with the 405-nm laser in CLSM for bio-imaging. The morphologies of both the Mn:ZnSe and the Mn:ZnSe@ZnS QDs were characterized with TEM and high HRTEM. As shown in Fig. 1C and 1D, both the Mn:ZnSe and the Mn:ZnSe@ZnS QDs showed high crystallinity and monodispersity, with particle sizes of about 2.6 nm and 3.5 nm, respectively. Upon growth of the ZnS shell, the increase of the particle size was clearly observed with HRTEM, and a strong S peak was also observed with the energy dispersive X-ray (EDX) characterization (Fig. S1), indicating successful growth of ZnS shell on the Mn:ZnSe QDs.
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Fig. 1 Characterization of the used doped QDs: (A) UV-vis absorption and FL spectra; (B) fluorescent excitation spectra; (C) TEM image of Mn:ZnSe QDs; (D) TEM image of Mn:ZnSe@ZnS QDs. The inset in (A) shows the fluorescent photo images of the Mn:ZnSe QDs before and after water-solubilization under a 365 nm UV lamp. HRTEM images of the QDs with clear lattice fringes of the corresponding QDs.
To suppress this trap state emission and enhance the dopant emission, a thin layer of ZnS was shelled on the surface of This journal is © The Royal Society of Chemistry [year]
Fig. 2 Characterization of the used nFAp nanoparticles: (A) TEM image of the hydrophobic nFAp nanoparticles, the inset shows nanoparticles dispersied in cyclohexane under room light; (B) TEM image of the hydrophilic nFAp nanoparticles, the inset shows nanoparticles dispersied in water under room light; (C) FT-IR spectroscopy of nFAp nanoparticles before and after PEA modification; and (D) XPS patterns of nFAp nanoparticles before and after PEA modification, and the inset is XPS pattern of N1s.
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To obtain high-quality and monodispersed nano-HAp precursors for conjugation with Mn:ZnSe@ZnS QDs, the high temperature LSS strategy developed by Li and Wang28-30 was employed here. Besides, previous reports showed that the incorporation of fluoride into HAp could enhance the stability
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term tracking study showed that the conjugates could be kept stable for at least 6 months without appreciable lost of the fluorescence. Furthermore, both the Mn:ZnSe@ZnS QDs and the nano-FAp-QD conjugates displayed good photostability (Fig. 4B), as compared with CdTe QDs (undoped) and Rhodamine B (one kind of traditional dyes), which may be ascribed to the good photostability of the doped QDs.10 The fluorescence intensity of Rhodamine B and CdTe QDs decreased after UV irradiation for 10 min and 5 min, respectively, while that of the Mn:ZnSe@ZnS QDs and the nano-FAp-QD conjugates was preserved by more than 95% of the original intensity even after irradiation for 60 minutes (Fig. 4B).
Fig. 3 (A) Schematic illustration of the modification procedure with PEA and the procedure for coupling the nano-FAp with MPA-capped QDs; (B) TEM imaging of nano-FAp-QD nanoparticles; (C) HRTEM imaging of QDs on the surface of the nano-FAp; and (D) EDX spectrum acquired from the nano-FAp-QD nanoparticles.
3.3 Characterization of nano-FAp-QDs Conjugation of the nano-FAp with the MPA-capped Mn:ZnSe@ZnS QDs was schematically illustrated in Fig. 3A, which is via the covalent reaction between amine of nano-FAp and carboxyl of QDs via the aid of EDC and NHS. TEM characterization showed that QDs were uniformly distributed on the surfaces of the nano-FAp (Fig. 3B), indicating successful conjugation. Unfortunately, the crystal structure of the nano-FAp is destroyed by high-energy electron beam during the obtaining of HRTEM image of the conjugates (Fig. S3), but the lattice structures of the QDs were clearly retained (Fig. 3C). EDX characterization further verified the conjugate formation, since elemental signals from both the QDs and the nano-FAp could be identified (Fig. 3D). After conjugation, the high fluorescence of Mn:ZnSe@ZnS QDs was largely retained, as indicated in Fig. 4A. Besides, the bright fluorescent photo image also reveal the uniform distribution of the conjugates in buffer solution (Tris-HCl, 0.10 M, pH 7.4), implying potential utility for bio-applications. Long4 | Journal Name, [year], [vol], 00–00
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Fig. 4 Characterization of the stability of the as-prepared nano-FAp-QD nanoparticles: (A) FL spectra of the nano-FAp-QD; (B) photostability comparison of Rhodamine B, CdTe QDs, Mn:ZnSe@ZnS QDs and nanoFAp-QD conjugate, with all samples continuously irradiated with the 365-nm excitation light (slit of 5 nm); (C) temporal evolution of fluorescence in DMEM medium of the nano-FAp-QD nanoparticles under different cultured time; and (D) temporal evolution of zinc ions concentration released in DMEM medium under different cultured time.
For an good fluorescent cell imaging agent, the stability of the material in cell culture media is a key issue for potential longterm cell tracking studies. Besides, previous studies also indicated that Cd2+ from Cd-containing QDs may be released in biological
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and uniformity of HAp.29 Therefore, here the monodispersed Fsubstituted HAp nanoparticles (nano-FAp) was synthesized as the biocompatible scaffold. As shown in Fig. 2A, uniform and monodispersed FAp nanorods was obtained, with size of 80~120 nm (length) and 13.6~16.4 nm (width), respectively. The hydrophobic nano-FAp can be well-dispersed in cyclohexane (inset of Fig. 2A), which might be attributed to the long alkyl chains of oleic acid absorbed on the surface of the nano-FAp. To render the nano-FAp water-soluble, PEA was used for phase-transfer of hydrophobic nano-FAp through the Ca-P coordination chemistry. PEA is a substrate for many phospholipids of the cell membranes,36 and it is expected to increase the affinity of nano-FAp to cells. Moreover, the functional amino groups of PEA can be easily conjugated with MPA-capped Mn:ZnSe@ZnS QDs via covalent cross-linking. After surface modification with PEA, the size and morphology of nano-FAp were well-retained (Fig. 2B). Moreover, the PEAfunctionalized nano-FAp can be readily dispersed in water layer, without appreciable residues in the cyclohexane layer (inset of Fig. 2B). These results suggested successful phase-transfer of nano-FAp from hydrophobic to hydrophilic. The interactions between the nano-FAp nanorods and PEA was further studied by FT-IR spectroscopy and XPS. From Fig. 2C, it can be seen that the peaks at 2923 and 2862 cm-1 ascribed to the C-H (possibly from oleic acid) stretching bands sharply decreased. Meanwhile, a new band at 1630 cm-1 attributed to N-H bending vibration is emerged after surface modification with PEA, implying replacement of C-H bond in oleic acid with N-H bond in PEA. Similarly, XPS data revealed that after PEA modification, the O 1s and N 1s peaks were increased in height, while that of C 1s was decreased. Moreover, the binding energy of N 1s changed from 398.8 to 399.7 eV after PEA modification (inset of Fig. 2D), suggesting that there should exist interactions between PEA and the nano-FAp. The decrease of Ca/P ratio from 1.54 to 1.48 after PEA adsorption (determined by XPS) further verified successful PEA loading. Besides, Ca2+ should be the main sites for interaction with PEA, possibly due to coordination and statistic attraction between Ca2+ of the nano-FAp and PO43of PEA.
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media.9 Here the stability of the conjugates and potential degradation of the QDs in cell culture media were studied, through detecting the corresponding fluorescent intensity and the concentration of Zn2+ in the supernatant after culturing the conjugates with DMEM medium for 2, 4, 8, 12, 24 and 48 hours. As shown in Fig. 4C, the fluorescence intensity of the supernatant increased less than 20% after 48 h, indicating good stability of the conjugates.37 Free Zn2+ concentrations in cell culture media were also increased only slightly (Fig. 4D), indicating the release of Zn2+ from QDs was minimal and the good stability of the QDs.
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imaging studies, only 50 µg/mL of the conjugate was used. These data indicated that the proposed nano-FAp-QD conjugates was suitable for biomedical applications, possibly because of the Cdfree nature of the Mn-doped ZnSe QDs as well as the embarked biocompatibility from the nano-FAp.
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As shown in Fig. 5A, for HepG2 cells and HeLa cells, the fluorescent intensities of per cell all increased with the incubation time, which not only confirmed the successful cellular uptake of the conjugates but also the good photostability of the conjugates in cell imaging. The cellular uptake and internalization of nanoparticles is a complicated process. Inhibitory test was usually used for investigating the endocytosis pathways and possible cell uptake mechanisms.38, 39 Fig. 5B provided the cellular uptake information of the nano-FAp-QD conjugates in the presence of specific endocytosis inhibitors. When HepG2 cells were preincubated with chlorpromazine, amiloride and genistein, the uptake decreased to 83.6%, 44.6% and 39.8% of the control, respectively. Therefore, macropinocytosis and caveolae-mediated endocytosis may be the main uptake pathways. However, for HeLa cells, the cellular uptake of nano-FAp-QD conjugates only decreased sharply to 28.9% after pre-incubated with genistein but not with chlorpromazine and amiloride. Accordingly, the main uptake pathway of nano-FAp-QD conjugate for HeLa cells could probably be ascribed to caveolae-mediated endocytosis.
Fig. 5 Quantitative analysis of the cellular uptake of nano-FAp-QD conjugates (50 µg/mL) by flow cytometry: (A) Cellular uptake kinetics of nano-FAp-QD conjugates by HeLa and HepG2 cells incubation for different time; (B) Cellular uptake of nano-FAp-QD conjugates (50 µg/mL, 12 h) in the presence of specific endocytosis inhibitors.
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The biocompatibility of the developed nano-FAp-QD conjugates was investigated through co-culturing with HeLa and HepG2 cells. Through microscopic observations, one can see all cells could still retain their normal morphologies after being incubated with 200 µg/mL nano-FAp-QD conjugates for 24 and 48 h (Fig. 6A), indicating good biocompatibility of the conjugates. Cell viability results from the MTT examinations also further confirmed the above claim. As shown in Fig. 6B, no obvious cytotoxicity was observed even when the concentration of the conjugate reached 100 µg/mL. Note that in the following cell This journal is © The Royal Society of Chemistry [year]
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Fig. 6 Characterization of the biocompatibility of the as-prepared nanoFAp-QD conjugates: (A) The morphologic investigations of HeLa and HepG2 cells incubated with nano-FAp-QD conjugates (200 µg/mL) for different time in DMEM media; and (B) MTT examinations of HeLa and HepG2 cells cultured for 24 h at 37 ºC.
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In vitro cell imaging of the nano-FAp-QD conjugates in HeLa and HepG2 cells using CLSM was further studied under bright field, 405 nm excitation, and overlap of both micrographs, respectively (Fig. 7). The bright fluorescence images confirmed the successful cellular uptake of the nano-FAp-QD conjugates and lighting up the cellular microenvironment with the conjugate. Even after incubation for 48 h, the fluorescence of the conjugates Journal Name, [year], [vol], 00–00 | 5
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could still be visualized inside the cells without noticeable fatigue effect, suggesting the good biocompatibility of the nano-FAp-QD conjugates. More importantly, it is worth noting that the concentration used for the cell imaging (50 µg/mL) is lower than the maximum concentration investigated in MTT test (100 µg/mL), reconfirming the excellent luminescent properties of the nano-FAp-QD conjugates for cell imaging studies.
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This work was financially supported by the National Natural Science Foundation of China (No. 21205082) and Chengdu Science and Technology Bureau Project (No. 12DXYB303JH002). We also grateful to Dr. Gang Wang and Prof. Zhongwei Gu of National Engineering Research Center for Biomaterials in Sichuan University for their help in cell studies.
Notes and references
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Fig. 7 CLSM images of HeLa and HepG2 cells incubated with 50 µg/mL of nano-FAp-QD nanoparticles for 24 and 48 h. (Left) bright field, (Middle) fluorescent images excited with a 405 nm laser, and (Right) merged images.
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In summary, a biocompatible conjugate of Cd-free Mndoped ZnSe@ZnS QDs and nano-FAp was developed for cell imaging application. To construct the proposed conjugates, highly luminescent Mn-doped ZnSe@ZnS QDs and monodispersed nano-FAp were first prepared. For facilitating the conjugation, hydrophobic nano-FAp was made water-soluble via PEA coating, which further provides conjugation sites for loading of the QDs. The proposed nano-FAp-QD conjugates preserved the high yet stable fluorescence and good biocompatibility as well as monodispersity of the doped QDs and the nano-FAp, respectively. Cytotoxicity studies confirmed the developed conjugates possessed good biocompatibility and exhibited low toxicity to cells. CLSM studies confirmed that the conjugates can be easily uptaken by cells and light up the microenvironment of cells, i.e., cell imaging. Given the bright future of both doped QDs and the nano-FAp in biomedical research, it is expected that this nano-FAp-QD will have promising prospect in clinical tumor diagnosis and treatment.
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College of Chemistry, 2Analytical & Testing Center, Sichuan University, Chengdu, 610064, China *Corresponding author, E-mail: [email protected] †Electronic Supplementary Information (ESI) available: Experimental details. See DOI: 10.1039/b000000x/ 1.
J. V. Frangioni, Current Opinion in Chemical Biology, 2003, 7, 626634. 2. A. R. Lippert, E. J. New and C. J. Chang, J. Am. Chem. Soc., 2011, 133, 10078-10080. 3. H. Kobayashi and P. L. Choyke, Accounts Chem. Res., 2011, 44, 8390. 4. Z. S. Yu, X. Y. Ma, B. Yu, Y. F. Pan and Z. G. Liu, J. Biomater. Appl., 2013, 28, 232-240. 5. Y. G. Zheng, S. J. Gao and J. Y. Ying, Adv. Mater., 2007, 19, 376-380. 6. B. H. Jun, D. W. Hwang, H. S. Jung, J. Jang, H. Kim, H. Kang, T. Kang, S. Kyeong, H. Lee, D. H. Jeong, K. W. Kang, H. Youn, D. S. Lee and Y. S. Lee, Adv. Funct. Mater., 2012, 22, 1843-1849. 7. M. E. Mathew, J. C. Mohan, K. Manzoor, S. V. Nair, H. Tamura and R. Jayakumar, Carbohydr. Polym., 2010, 80, 442-448. 8. Y. Zhang, J. M. Liu and X. P. Yan, Anal. Chem., 2013, 85, 228-234. 9. K. T. Yong, W. C. Law, R. Hu, L. Ye, L. W. Liu, M. T. Swihart and P. N. Prasad, Chem. Soc. Rev., 2013, 42, 1236-1250. 10. P. Wu and X.-P. Yan, Chem. Soc. Rev., 2013, 42, 5489-5521. 11. A. Jayasree, S. Sasidharan, M. Koyakutty, S. Nair and D. Menon, Carbohydr. Polym., 2011, 85, 37-43. 12. X. Gao, H. Zhang, Y. Li and X. G. Su, Anal. Bioanal. Chem., 2012, 402, 1871-1877. 13. K. Manzoor, S. Johny, D. Thomas, S. Setua, D. Menon and S. Nair, Nanotechnology, 2009, 20, 065102. 14. A. R. Maity, S. Palmal, S. K. Basiruddin, N. S. Karan, S. Sarkar, N. Pradhan and N. R. Jana, Nanoscale, 2013, 5, 5506-5513. 15. K. Ding, L. H. Jing, C. Y. Liu, Y. Hou and M. Y. Gao, Biomaterials, 2014, 35, 1608-1617. 16. D. J. Norris, A. L. Efros and S. C. Erwin, Science, 2008, 319, 17761779. 17. L. A. Hails, J. C. Babister, S. Inglis, S. A. Davis, R. O. C. Oreffo and S. Mann, Small, 2010, 6, 1986-1991. 18. P. P. Yang, Z. W. Quan, C. X. Li, X. J. Kang, H. Z. Lian and J. Lin, Biomaterials, 2008, 29, 4341-4347. 19. G. J. Wu, L. Z. Zhou, K. W. Wang, F. Chen, Y. Sun, Y. R. Duan, Y. J. Zhu and H. C. Gu, J. Colloid Interface Sci., 2010, 345, 427-432. 20. B. Palazzo, M. Iafisco, M. Laforgia, N. Margiotta, G. Natile, C. L. Bianchi, D. Walsh, S. Mann and N. Roveri, Adv. Funct. Mater., 2007, 17, 2180-2188. 21. H. C. Wu, T. W. Wang, M. C. Bohn, F. H. Lin and M. Spector, Adv. Funct. Mater., 2010, 20, 67-77. 22. A. Ashokan, D. Menon, S. Nair and M. Koyakutty, Biomaterials, 2010, 31, 2606-2616. 23. M. K. Singh, T. Shokuhfar, J. J. D. Gracio, A. C. M. de Sousa, J. M. D. Fereira, H. Garmestani and S. Ahzi, Adv. Funct. Mater., 2008, 18, 694-700. 24. Y. M. Sung, Y. K. Shin and J. J. Ryu, Nanotechnology, 2007, 18, 065602. 25. J. H. Chang, M. E. Park, Y. Shin, G. J. Exarhos, K. J. Kim, S. C. Lee and K. S. Oh, J. Mater. Chem., 2007, 17, 238-242. 26. Y. Guo, D. L. Shi, J. Lian, Z. Y. Dong, W. Wang, H. S. Cho, G. K. Liu, L. M. Wang and R. C. Ewing, Nanotechnology, 2008, 19, 175102. 27. M. F. Hsieh, J. K. J. Li, C. A. J. Lin, S. H. Huang, R. A. Sperling, W. J. Parak and W. H. Chang, J. Nanosci. Nanotechnol., 2009, 9, 2758-
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Low-toxic Mn-doped ZnSe@ZnS Quantum Dots Conjugated with Nanohydroxyapatite for Cell Imaging Ronghui Zhou,1 Mei Li,2 Shanling Wang,2 Peng Wu,2 Lan Wu,2* Xiandeng Hou1, 2
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Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX DOI: 10.1039/b000000x Fluorescent bio-imaging has received great attention in a myriad of research disciplines, and QDs is playing an increasingly important role in these areas. Doped QDs, as an important alternative to conventional heavy metal-containing QDs, is appealing for biomedical applications. However, since QDs is exogenous substances to biological environment, the biocompatibility of QDs is expected to be problematic in some cases. Herein, nano fluorine-doped hydroxyapatite (FAp, a well-known biocompatible material) was introduced to embark biocompatibility to Cd-free Mn-doped ZnSe@ZnS QDs. A nano-FAp-QD conjugate was thus developed and the biocompatibility as well as potential cell imaging application was investigated. To construct the proposed conjugate, Cd-free highly luminescent Mn-doped ZnSe@ZnS QDs and monodispersed nano-FAp were first prepared in high-temperature organic media. For facilitating the conjugation, hydrophobic nano-FAp was made water-soluble via ophosphoethanolamine (PEA) coating, which further provides conjugating sites for QDs to anchor. Cytotoxicity studies indicated the developed conjugate indeed possesses good compatibility and low toxicity to cells. The nano-FAp-QDs conjugate was successfully employed for cancer cell staining for at least 24 h, demonstrating the potential usefulness of this material in future biomedical research.
1. Introduction As an effective method of optical bio-imaging technique, fluorescent imaging has drawn great attention due to its' high sensitivity, multicolor imaging capability and low cost.1-5 Owing to the complexity of the biological system, a high-quality fluorescence imaging agency for diagnosis and therapy must be characterized as low toxicity, good biocompatibility, high brightness, and good photostability. Compared with conventional organic dyes, fluorescent quantum dots (QDs) possess unique photophysical properties such as size-tunable emission, broad excitation, and high photostability, which make them a promising type of fluorescent imaging agency.6-8 However, heavy metals in typically used QDs (majorly CdSe and CdTe QDs) are reported to be released under biological stress, resulting in toxicity of these QDs in vivo.9 Doped QDs, especially the Cd-free Mn-doped ZnS or ZnSe QDs, is an important alternative to CdSe or CdTe QDs,10 thus appealing for bio-imaging applications.11-15 Moreover, doped QDs can be protected against photooxidation due to efficient and fast energy transfer from host to dopants, which quickly localizes the excitation and suppresses undesirable reactions on the surface of QDs.16 Therefore, the photostability of doped QDs is increased as compared to undoped counterparts. However, both the conventional Cd-containing QDs and the doped QDs are all exogenous substances to biological environment. In other words, the biocompatibility of these QDs is expected to be problematic in some cases. Hydroxyapatite (HAp) is the main inorganic component of bones and teeth and also an ideal biocompatible material. When This journal is © The Royal Society of Chemistry [year]
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decreasing the size of HAp to the nano-size regime, the perfect biocompatibility, low immunogenicity, and biodegradable property of this materials can be harvested for tissue engineering17 and drug and gene delivery.18-20 Also, use of nanoHAp as scaffolds for loading or compositing of inorganic functional materials has been shown to be an effective way for embarking biocompatibility to these materials, such as Fe3O4 magnetic nanoparticles,21, 22 carbon nanotube,23 zirconia,24 silica,25 and etc. For loading of QDs, Guo et al. conjugated CdSe@ZnS QDs with nano-HAp to improve the biocompatibility QDs for in vivo imaging;26 Chang et al. labeled nano-HAp with CdSe@ZnS QDs via compositing to investigate the interaction between nano-HAp and MC3T3-E1 osteoblast cells.27 Obviously, the biocompatibility of CdSe@ZnS QDs was largely increased after marriage with nano-HAp. But still, Cd-containing QDs was still used in the above two studies. Besides, the agglomeration of nano-HAp is expected and would possibly decrease the imaging sensitivity and resolution. Therefore, herein we conjugated Cd-free Mn-doped ZnSe (Mn:ZnSe) QDs with nano-HAp to develop a low-toxic and biocompatible fluorescent contrast material for bio-imaging. Highly fluorescent Mn:ZnSe QDs was synthesized in organic phase and shelled with a thin layer of ZnS after watersolubilization to further increase the photostability of the doped QDs. To ensure the monodispersity and crystallinity of HAp material, nano fluorine-doped HAp (nano-FAp) was first synthesized in high-temperature organic media with the liquidsolid-solution (LSS) strategy.28-30 Then, monodispersed nano[journal], [year], [vol], 00–00 | 1
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2.4 Investigation of the Cellular uptake of nano-FAp-QD nanoparticles
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2.1 Synthesis of Mn:ZnSe@ZnS D-Dots 15
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Mn-doped ZnSe QDs was prepared via the "nucleationdoping" approach developed by Peng et al. (Supporting Information).31-33 Briefly, MnSe nanoclusters was firstly formed with Mn2+ and Se precursors. Then, Zn2+ precursors was added to form ZnSe shell around the MnSe nanoclusters. The obtained QDs was purified using acetone/CHCl3 extraction with QDs in the CHCl3 layer. For overcoating of ZnS shell, Mn-doped ZnSe QDs was firstly subjected to a standard ligand exchange strategy with MPA,34 and the polar carboxylic groups renderd the QDs water-soluble. After ligand exchange, an additional ZnS shell was grew on the outer layer of the Mn:ZnSe QDs to form MPAcapped core/shell Mn:ZnSe@ZnS QDs.35 The final QDs product was stored in distilled water with a concentration of 5 mg/mL. 2.2 Synthesis of PEA modified monodispersed nFAp
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The monodispersed nano-FAp nanoparticles was synthesized via a hydrothermal synthetic route based on the LSS strategy (Supporting Information).28-30 Then, oleic acid-capped monodispersed nano-FAp was phase-transferred with PEA. Briefly, an amount of 50 mg hydrophobic nano-FAp nanoparticles was dispersed in 10 mL cyclohexane, a solution of phosphoethanolamine (0.5 g in 10 mL distilled water) was added under stirring. Thereafter, the nano-FAp was successfully transferred into the water layer due to the replacement of oleic acid with PEA. After decanting of the upper cyclohexane layer, the hydrophilic nano-FAp was collected via centrifugation and then washed with distilled water three times. The amino terminal groups on the surface of nano-FAp provide conjugation sites with MPA-capped Mn:ZnSe@ZnS QDs. 2.3 Synthesis of nano-FAp-QD nanoparticles
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MPA-capped Mn:ZnSe@ZnS QDs can be conjugated with amino-termined nano-FAp via covalent cross-linking. The weight ratios of the QDs to the nano-FAp were optimized through investigating their effects on the fluorescent intensities and photostabilities of the nano-FAp-QD nanoparticles (Fig. S2). Weight ratio of 1:2 (QDs: nano-FAp) was chosen for use based on the optimization results. Typically, PEA modified nano-FAp was suspended in Tris-HCl buffer (0.1 M, pH 7.4) at a concentration of 5 mg/mL. At the same time, carboxyl-terminated Mn:ZnSe@ZnS QDs (1 mL, 5 mg/mL) was activated with 1-(3dimethylamino-propyl)-3-ethylcarbodiimide hydrochloride (EDC, 8 mg) and N-hydroxysuccinimide (NHS, 4 mg) in Tris2 | Journal Name, [year], [vol], 00–00
HCl buffer (0.1 M, pH 7.4) for 20 min. Then 2 mL well-dispersed nano-FAp was added to the activated solution, and this mixture was stirred for 90 min. To ensure the conjugation, 0.1 M NaCl was added to shield the adsorption effect. The as-synthesized product was washed and re-dispersed with Tris-HCl buffer (0.1 M, pH 7.4) several times.
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The quantitative cellular uptake of nFAp-QDs nanoparticles with different incubation time was measured by flow cytometry. The Hela and HepG2 cells were seeded in 6-well plates at a density of 1 × 105 cells/well in 2 mL DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicilline streptomycin at 37 ºC for 24 h. Then, 50 µg/mL nFAp-QDs nanoparticles were added and incubated for 4 h, 8 h, 12 h and 24 h, respectively. The cells were washed with PBS for three times and harvested by trypsinization and centrifuged at 1000 rpm for 5 min, resuspended in 0.5 mL PBS medium and examined by flow cytometry (Cytomics FC 500, Beckman Coulter) with 360 nm excitation lasers. For uptake studies, the two cell lines were treated with the following inhibitors for 0.5 h: genistein (0.2 mM, the caveolaemediated endocytosis inhibitor), amiloride (1 mM, the macropinocytosis inhibitor), and chlorpromazine (10 µg/mL, the clathrin-mediated endocytosis inhibitor), respectively. Then, 50 µg/mL nFAp-QDs nanoparticles were added and further incubated for 12 h. Cells incubated only with the nFAp-QDs nanoparticles were used as the control. The fluorescent intensity of per cell was tested by flow cytometry at 360 nm excitation after the same process of cell wash, harvesting and re-suspending as the above section mentioned. 2.5 Cytotoxicity of nano-FAp-QD nanoparticles
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To examine the effects of nano-FAp-QD nanoparticles on the live cells, an inverted optical microscope was used to observe the cell morphology change after incubating with nano-FAp-QD in DMEM medium for different time. HeLa and HepG2 cells were dispersed in 6-well plates at a density of 1 × 105 cells/well in 2 mL DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicilline streptomycin at 37 ºC. After 24 h of cell adherence, the plates were washed with PBS and divided to two categories for treating with 200 µg/mL nano-FAp-QD in DMEM medium for 24 h and 48 h, respectively. Then all wells were washed with PBS to remove the excess nano-FAp-QD, and the morphologies of cells treated for 24 h and 48 h, respectively, were observed using the inverted optical microscope. Cell viability was investigated with MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) test. The HeLa and HepG2 cells were cultured in 96-well assay plates at a density of 1 × 104 cells/well (100 µL total volume per well). After cultured in DMEM medium containing 10% FBS and 1% penicilline streptomycin at 37 ºC and 5% CO2 for 24 h, different concentrations of nano-FAp-QD were added to the wells, and the cells were further cultured for 24 h. Thereafter, nanoparticles was removed and 10 µL MTT (5 mg/mL in PBS) following with 90 µL DMEM medium were added into each well. After another 4 h of incubation, 100 µL dimethyl sulfoxide (DMSO) was added to each well to dissolve the dark blue formazan crystal. The This journal is © The Royal Society of Chemistry [year]
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FAp was phase-transferred with o-phosphoethanolamine (PEA). PEA can be quickly absorbed on the surface of nano-FAp to endow it hydrophilic and provide functional amino groups. MPAcapped Mn:ZnSe@ZnS QDs can be thus easily conjugated with nano-FAp via covalent cross-linking. To investigate the biocompatibility of the developed nano-FAP-QD composite material, the interaction between nano-FAP-QD composite and HeLa and HepG2 cells was studied through cell viability and cellimaging. The results showed that the monodispersed nano-FAPQD nanoparticles possessed high luminescence, excellent biocompatibility and photostability, which can be explored for cell imaging applications.
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formazan concentration was finally quantified using a microplate reader by measuring the absorbance at 570 nm (Thermo Fisher Scientific, Varioskan Flash).
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The cell imaging experiments were performed by confocal laser scanning microscopy (CLSM, excited at 405 nm, Leica TCP SP5). HeLa and HepG2 cells were seeded in 35 mm glass culture dishes at a density of 1 × 104 cells/well and allowed to adhere for 24 h at 37 ºC. After removing the culture medium, the cells were incubated with nano-FAp-QD nanoparticles at a final concentration of 50 µg/mL for 24 h and 48 h. Afterward, the cells were washed with PBS and observed by using CLSM.
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3. Results and discussion 3.1 Characterization of Mn:ZnSe@ZnS QDs 55 15
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In this work, Mn:ZnSe@ZnS core/shell doped QDs was explored as the fluorescent material for cell imaging. In order to obtain highly bright and stable doped QDs, Mn-doped ZnSe QDs was firstly prepared via the "nucleation-doping" approach developed by Peng et al. (Supporting Information).31-33 As shown in Fig. 1A, the characteristic photoluminescent peak at around 590 nm indicates the successful doping of Mn2+ into ZnSe host, which can be ascribed to the Mn2+ 4T1 → 6A1 transition that gains energy from ZnSe host. Meanwhile, a small peak at about 450 nm was also observed possibly due to trap state emission of ZnSe QDs (Fig. 1A).31 Meanwhile, the Mn:ZnSe QDs can be successfully translated to be water-soluble through a routine ligand exchange process,34 and the solution of MPA-capped Mn:ZnSe QDs displayed orange fluorescence under UV irradiation (inset in Fig. 1A).
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Mn:ZnSe QDs (Supporting Information). Both the UV-vis absorption and the corresponding FL spectra of Mn:ZnSe@ZnS QDs exhibited a slight red shift compared to the Mn:ZnSe QDs due to the formation of ZnS shell outside of the Mn:ZnSe core (Fig. 1A). Besides, the Mn:ZnSe@ZnS QDs exhibited obviously higher fluorescence intensity as compared to the original Mn:ZnSe QDs, which can be ascribed to the elimination of surface defects by ZnS shelling. Meanwhile, the trap state emission at 450 nm was completely diminished. The fluorescence quantum yield (QY) of the Mn:ZnSe@ZnS QDs was determined as 25.6% (directly useing Rohdamine 6G as reference, Supporting Information) and 58.7% (used CdSe quantum dots as intermediate reference). After ZnS shelling, the fluorescence excitation spectrum of Mn:ZnSe@ZnS QDs also slightly redshifted (Fig. 1B), which can be well-excited with the 405-nm laser in CLSM for bio-imaging. The morphologies of both the Mn:ZnSe and the Mn:ZnSe@ZnS QDs were characterized with TEM and high HRTEM. As shown in Fig. 1C and 1D, both the Mn:ZnSe and the Mn:ZnSe@ZnS QDs showed high crystallinity and monodispersity, with particle sizes of about 2.6 nm and 3.5 nm, respectively. Upon growth of the ZnS shell, the increase of the particle size was clearly observed with HRTEM, and a strong S peak was also observed with the energy dispersive X-ray (EDX) characterization (Fig. S1), indicating successful growth of ZnS shell on the Mn:ZnSe QDs.
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Fig. 1 Characterization of the used doped QDs: (A) UV-vis absorption and FL spectra; (B) fluorescent excitation spectra; (C) TEM image of Mn:ZnSe QDs; (D) TEM image of Mn:ZnSe@ZnS QDs. The inset in (A) shows the fluorescent photo images of the Mn:ZnSe QDs before and after water-solubilization under a 365 nm UV lamp. HRTEM images of the QDs with clear lattice fringes of the corresponding QDs.
To suppress this trap state emission and enhance the dopant emission, a thin layer of ZnS was shelled on the surface of This journal is © The Royal Society of Chemistry [year]
Fig. 2 Characterization of the used nFAp nanoparticles: (A) TEM image of the hydrophobic nFAp nanoparticles, the inset shows nanoparticles dispersied in cyclohexane under room light; (B) TEM image of the hydrophilic nFAp nanoparticles, the inset shows nanoparticles dispersied in water under room light; (C) FT-IR spectroscopy of nFAp nanoparticles before and after PEA modification; and (D) XPS patterns of nFAp nanoparticles before and after PEA modification, and the inset is XPS pattern of N1s.
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To obtain high-quality and monodispersed nano-HAp precursors for conjugation with Mn:ZnSe@ZnS QDs, the high temperature LSS strategy developed by Li and Wang28-30 was employed here. Besides, previous reports showed that the incorporation of fluoride into HAp could enhance the stability
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term tracking study showed that the conjugates could be kept stable for at least 6 months without appreciable lost of the fluorescence. Furthermore, both the Mn:ZnSe@ZnS QDs and the nano-FAp-QD conjugates displayed good photostability (Fig. 4B), as compared with CdTe QDs (undoped) and Rhodamine B (one kind of traditional dyes), which may be ascribed to the good photostability of the doped QDs.10 The fluorescence intensity of Rhodamine B and CdTe QDs decreased after UV irradiation for 10 min and 5 min, respectively, while that of the Mn:ZnSe@ZnS QDs and the nano-FAp-QD conjugates was preserved by more than 95% of the original intensity even after irradiation for 60 minutes (Fig. 4B).
Fig. 3 (A) Schematic illustration of the modification procedure with PEA and the procedure for coupling the nano-FAp with MPA-capped QDs; (B) TEM imaging of nano-FAp-QD nanoparticles; (C) HRTEM imaging of QDs on the surface of the nano-FAp; and (D) EDX spectrum acquired from the nano-FAp-QD nanoparticles.
3.3 Characterization of nano-FAp-QDs Conjugation of the nano-FAp with the MPA-capped Mn:ZnSe@ZnS QDs was schematically illustrated in Fig. 3A, which is via the covalent reaction between amine of nano-FAp and carboxyl of QDs via the aid of EDC and NHS. TEM characterization showed that QDs were uniformly distributed on the surfaces of the nano-FAp (Fig. 3B), indicating successful conjugation. Unfortunately, the crystal structure of the nano-FAp is destroyed by high-energy electron beam during the obtaining of HRTEM image of the conjugates (Fig. S3), but the lattice structures of the QDs were clearly retained (Fig. 3C). EDX characterization further verified the conjugate formation, since elemental signals from both the QDs and the nano-FAp could be identified (Fig. 3D). After conjugation, the high fluorescence of Mn:ZnSe@ZnS QDs was largely retained, as indicated in Fig. 4A. Besides, the bright fluorescent photo image also reveal the uniform distribution of the conjugates in buffer solution (Tris-HCl, 0.10 M, pH 7.4), implying potential utility for bio-applications. Long4 | Journal Name, [year], [vol], 00–00
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Fig. 4 Characterization of the stability of the as-prepared nano-FAp-QD nanoparticles: (A) FL spectra of the nano-FAp-QD; (B) photostability comparison of Rhodamine B, CdTe QDs, Mn:ZnSe@ZnS QDs and nanoFAp-QD conjugate, with all samples continuously irradiated with the 365-nm excitation light (slit of 5 nm); (C) temporal evolution of fluorescence in DMEM medium of the nano-FAp-QD nanoparticles under different cultured time; and (D) temporal evolution of zinc ions concentration released in DMEM medium under different cultured time.
For an good fluorescent cell imaging agent, the stability of the material in cell culture media is a key issue for potential longterm cell tracking studies. Besides, previous studies also indicated that Cd2+ from Cd-containing QDs may be released in biological
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and uniformity of HAp.29 Therefore, here the monodispersed Fsubstituted HAp nanoparticles (nano-FAp) was synthesized as the biocompatible scaffold. As shown in Fig. 2A, uniform and monodispersed FAp nanorods was obtained, with size of 80~120 nm (length) and 13.6~16.4 nm (width), respectively. The hydrophobic nano-FAp can be well-dispersed in cyclohexane (inset of Fig. 2A), which might be attributed to the long alkyl chains of oleic acid absorbed on the surface of the nano-FAp. To render the nano-FAp water-soluble, PEA was used for phase-transfer of hydrophobic nano-FAp through the Ca-P coordination chemistry. PEA is a substrate for many phospholipids of the cell membranes,36 and it is expected to increase the affinity of nano-FAp to cells. Moreover, the functional amino groups of PEA can be easily conjugated with MPA-capped Mn:ZnSe@ZnS QDs via covalent cross-linking. After surface modification with PEA, the size and morphology of nano-FAp were well-retained (Fig. 2B). Moreover, the PEAfunctionalized nano-FAp can be readily dispersed in water layer, without appreciable residues in the cyclohexane layer (inset of Fig. 2B). These results suggested successful phase-transfer of nano-FAp from hydrophobic to hydrophilic. The interactions between the nano-FAp nanorods and PEA was further studied by FT-IR spectroscopy and XPS. From Fig. 2C, it can be seen that the peaks at 2923 and 2862 cm-1 ascribed to the C-H (possibly from oleic acid) stretching bands sharply decreased. Meanwhile, a new band at 1630 cm-1 attributed to N-H bending vibration is emerged after surface modification with PEA, implying replacement of C-H bond in oleic acid with N-H bond in PEA. Similarly, XPS data revealed that after PEA modification, the O 1s and N 1s peaks were increased in height, while that of C 1s was decreased. Moreover, the binding energy of N 1s changed from 398.8 to 399.7 eV after PEA modification (inset of Fig. 2D), suggesting that there should exist interactions between PEA and the nano-FAp. The decrease of Ca/P ratio from 1.54 to 1.48 after PEA adsorption (determined by XPS) further verified successful PEA loading. Besides, Ca2+ should be the main sites for interaction with PEA, possibly due to coordination and statistic attraction between Ca2+ of the nano-FAp and PO43of PEA.
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media.9 Here the stability of the conjugates and potential degradation of the QDs in cell culture media were studied, through detecting the corresponding fluorescent intensity and the concentration of Zn2+ in the supernatant after culturing the conjugates with DMEM medium for 2, 4, 8, 12, 24 and 48 hours. As shown in Fig. 4C, the fluorescence intensity of the supernatant increased less than 20% after 48 h, indicating good stability of the conjugates.37 Free Zn2+ concentrations in cell culture media were also increased only slightly (Fig. 4D), indicating the release of Zn2+ from QDs was minimal and the good stability of the QDs.
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imaging studies, only 50 µg/mL of the conjugate was used. These data indicated that the proposed nano-FAp-QD conjugates was suitable for biomedical applications, possibly because of the Cdfree nature of the Mn-doped ZnSe QDs as well as the embarked biocompatibility from the nano-FAp.
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As shown in Fig. 5A, for HepG2 cells and HeLa cells, the fluorescent intensities of per cell all increased with the incubation time, which not only confirmed the successful cellular uptake of the conjugates but also the good photostability of the conjugates in cell imaging. The cellular uptake and internalization of nanoparticles is a complicated process. Inhibitory test was usually used for investigating the endocytosis pathways and possible cell uptake mechanisms.38, 39 Fig. 5B provided the cellular uptake information of the nano-FAp-QD conjugates in the presence of specific endocytosis inhibitors. When HepG2 cells were preincubated with chlorpromazine, amiloride and genistein, the uptake decreased to 83.6%, 44.6% and 39.8% of the control, respectively. Therefore, macropinocytosis and caveolae-mediated endocytosis may be the main uptake pathways. However, for HeLa cells, the cellular uptake of nano-FAp-QD conjugates only decreased sharply to 28.9% after pre-incubated with genistein but not with chlorpromazine and amiloride. Accordingly, the main uptake pathway of nano-FAp-QD conjugate for HeLa cells could probably be ascribed to caveolae-mediated endocytosis.
Fig. 5 Quantitative analysis of the cellular uptake of nano-FAp-QD conjugates (50 µg/mL) by flow cytometry: (A) Cellular uptake kinetics of nano-FAp-QD conjugates by HeLa and HepG2 cells incubation for different time; (B) Cellular uptake of nano-FAp-QD conjugates (50 µg/mL, 12 h) in the presence of specific endocytosis inhibitors.
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The biocompatibility of the developed nano-FAp-QD conjugates was investigated through co-culturing with HeLa and HepG2 cells. Through microscopic observations, one can see all cells could still retain their normal morphologies after being incubated with 200 µg/mL nano-FAp-QD conjugates for 24 and 48 h (Fig. 6A), indicating good biocompatibility of the conjugates. Cell viability results from the MTT examinations also further confirmed the above claim. As shown in Fig. 6B, no obvious cytotoxicity was observed even when the concentration of the conjugate reached 100 µg/mL. Note that in the following cell This journal is © The Royal Society of Chemistry [year]
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Fig. 6 Characterization of the biocompatibility of the as-prepared nanoFAp-QD conjugates: (A) The morphologic investigations of HeLa and HepG2 cells incubated with nano-FAp-QD conjugates (200 µg/mL) for different time in DMEM media; and (B) MTT examinations of HeLa and HepG2 cells cultured for 24 h at 37 ºC.
3.6 Cell imaging with the nano-FAp-QD conjugates
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In vitro cell imaging of the nano-FAp-QD conjugates in HeLa and HepG2 cells using CLSM was further studied under bright field, 405 nm excitation, and overlap of both micrographs, respectively (Fig. 7). The bright fluorescence images confirmed the successful cellular uptake of the nano-FAp-QD conjugates and lighting up the cellular microenvironment with the conjugate. Even after incubation for 48 h, the fluorescence of the conjugates Journal Name, [year], [vol], 00–00 | 5
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could still be visualized inside the cells without noticeable fatigue effect, suggesting the good biocompatibility of the nano-FAp-QD conjugates. More importantly, it is worth noting that the concentration used for the cell imaging (50 µg/mL) is lower than the maximum concentration investigated in MTT test (100 µg/mL), reconfirming the excellent luminescent properties of the nano-FAp-QD conjugates for cell imaging studies.
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This work was financially supported by the National Natural Science Foundation of China (No. 21205082) and Chengdu Science and Technology Bureau Project (No. 12DXYB303JH002). We also grateful to Dr. Gang Wang and Prof. Zhongwei Gu of National Engineering Research Center for Biomaterials in Sichuan University for their help in cell studies.
Notes and references
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Fig. 7 CLSM images of HeLa and HepG2 cells incubated with 50 µg/mL of nano-FAp-QD nanoparticles for 24 and 48 h. (Left) bright field, (Middle) fluorescent images excited with a 405 nm laser, and (Right) merged images.
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In summary, a biocompatible conjugate of Cd-free Mndoped ZnSe@ZnS QDs and nano-FAp was developed for cell imaging application. To construct the proposed conjugates, highly luminescent Mn-doped ZnSe@ZnS QDs and monodispersed nano-FAp were first prepared. For facilitating the conjugation, hydrophobic nano-FAp was made water-soluble via PEA coating, which further provides conjugation sites for loading of the QDs. The proposed nano-FAp-QD conjugates preserved the high yet stable fluorescence and good biocompatibility as well as monodispersity of the doped QDs and the nano-FAp, respectively. Cytotoxicity studies confirmed the developed conjugates possessed good biocompatibility and exhibited low toxicity to cells. CLSM studies confirmed that the conjugates can be easily uptaken by cells and light up the microenvironment of cells, i.e., cell imaging. Given the bright future of both doped QDs and the nano-FAp in biomedical research, it is expected that this nano-FAp-QD will have promising prospect in clinical tumor diagnosis and treatment.
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College of Chemistry, 2Analytical & Testing Center, Sichuan University, Chengdu, 610064, China *Corresponding author, E-mail: [email protected] †Electronic Supplementary Information (ESI) available: Experimental details. See DOI: 10.1039/b000000x/ 1.
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