- Nano Express
- Open Access
One-step fabrication of biocompatible chitosan-coated ZnS and ZnS:Mn2+ quantum dots via a γ-radiation route
Nanoscale Research Letters volume 6, Article number: 591 (2011)
Biocompatible chitosan-coated ZnS quantum dots [CS-ZnS QDs] and chitosan-coated ZnS:Mn2+ quantum dots [CS-ZnS:Mn2+ QDs] were successfully fabricated via a convenient one-step γ-radiation route. The as-obtained QDs were around 5 nm in diameter with excellent water-solubility. These QDs emitting strong visible blue or orange light under UV excitation were successfully used as labels for PANC-1 cells. The cell experiments revealed that CS-ZnS and CS-ZnS:Mn2+ QDs showed low cytotoxicity and good biocompatibility, which offered possibilities for further biomedical applications. Moreover, this convenient synthesis strategy could be extended to fabricate other nanoparticles coated with chitosan.
PACS: 81.07.Ta; 78.67.Hc; 82.35.Np; 87.85.Rs.
Recently, quantum dots [QDs] have shown great potential in biomedical applications [1–3]. How to minimize the cytotoxicity of QDs is still a great challenge for their clinical applications [4–6]. In the past few years, great efforts have been paid to reduce the toxicity of QDs. Various coatings, such as ZnS shell, thiol-containing molecules, PEG, and other polymers, have been developed to modify the QDs to reduce their cytotoxicity [7–10]. Moreover, a series of non-heavy-metal QDs, such as InGaP/ZnS, ZnS, and ZnS:Mn2+, have also been investigated to solve the toxicity problem [11–13]. Significant progresses have been made in producing high quality ZnS and ZnS:Mn2+ QDs by wet chemical methods [14–18]. However, most of these synthesis strategies require the use of an environmentally hazardous organic solvent or surfactant, which is not environmentally friendly and might not be suitable for biomedical applications.
Compared with traditional chemical methods, γ-radiation route, in which reactions can take place in an aqueous system at room temperature under ambient pressure, has been considered as a promising way to process traditional materials or synthesize new materials. Up to now, the γ-radiation route has been employed to prepare many types of novel nanomaterials, such as silver nanoparticles, CdS nanocrystal-modified fibers, CdSe quantum dots, and polyelectrolyte complex [19–23]. Herein, we present a novel strategy to fabricate chitosan-coated ZnS [CS-ZnS] and ZnS:Mn2+ [CS-ZnS:Mn2+] quantum dots [QDs] via γ-radiation for the first time.
In our synthesis experiments, chitosan oligomer, a kind of biocompatible polymer, was used as a stabilizer as well as coating molecules [24, 25]. By controlling the reaction conditions, the particle sizes and fluorescent properties of QDs could be well tuned. The obtained QDs were around 5 nm in diameter and exhibited excellent fluorescent properties and water solubility. These QDs had low cytotoxicity and high cell membrane penetrability, which offered potentials for biomedical applications.
γ-Radiation synthesis of CS-ZnS and CS-ZnS:Mn2+ QDs
CS-ZnS and CS-ZnS:Mn2+ QDs were directly fabricated in an aqueous system at room temperature under ambient pressure via a convenient γ-irradiation route. Chitosan oligomer, with the average polymerization degree of 30, was prepared through radiation degradation . All other reagents used in this study were of analytical grade. In a typical synthesis, 5 mL Zn(Ac)2 (10 mM), 5 mL Mn(Ac)2 (0.1 mM), and 10 mL Na2S2O3 (10 mM) were added into 70 mL of deionized water containing 0.1% chitosan (w/v, pH = 5.4) or 2% chitosan (w/v, pH = 5); then 10 mL (CH3)2CHOH was added into the above solution as a free radical scavenger. Afterwards, the solution was bubbled with pure nitrogen for 30 min to remove the dissolved oxygen; then the mixed solution was placed in the field of a 1.1 × 1015 Bq 60Co γ-ray source with a radiation dose rate of 3 kGy h-1. The radiation dose was set as 5, 10, 20, and 30 kGy, respectively. After irradiation, the synthesized QDs were separated from the solution with a centrifugation at 10,000 rpm for 5 min. The samples were washed three times with deionized water and stored in deionized water for further experiments. Thioglycolic acid-coated CdSe QDs [TGA-CdSe QDs] were also synthesized following the reported method .
Transmission electron microscope [TEM] and high-resolution TEM [HRTEM] images were taken using a JEOL JEM-200CX and JEM-2010 (JEOL Ltd., Akishima, Tokyo, Japan). Elementary analysis was carried out with an energy dispersion spectrum [EDS]. X-ray diffraction [XRD] patterns were recorded on a BRUKER D8-ADVANCE (BRUKER AXS GMBH, Karlsruhe, Germany) X-ray diffractometer. Ultraviolet-visible [UV-Vis] spectra were carried out using a PerkinElmer (Waltham, MA, USA) λ-17 spectrophotometer. The photoluminescence [PL] spectra were taken with a Hitachi 850 spectrofluorophotometer (Hitachi High-Tech, Minato-ku, Tokyo, Japan). The quantum yield [QY] values of QDs were calculated relative to rhodamine 6G in water using the following equation: QYs = (F s/ F r) × (A r/ A s) × QYr, where F and A were the measured fluorescence (area under the emission peak) and the absorbance at the excitation wavelength, respectively. Human pancreatic carcinoma cells (PANC-1, ATCC no. TIB-222) were cultured in a DMEM medium containing 10% (v/v) fetal calf serum at 37°C in humidified air containing 5% CO2. QDs dispersed in phosphate-buffered saline [PBS] were added to the culture medium to achieve a final concentration of 10, 50, 200 or 1,000 μg mL-1. An Olympus FV-1000 (Olympus Corporation, Shinjuku-ku, Tokyo, Japan) laser scanning confocal microscope [LSCM] was used to examine the PANC-1 cells labeled with QDs. For LSCM imaging, cells were seeded onto a glass cover slip placed in six well plates. After incubation in a medium containing QDs for 2 h, the PANC-1 cells were fixed with 4% paraformaldehyde for 15 min, and then washed three times with PBS buffer. To clearly observe the distribution and position of QDs in cells, nuclei were stained with 5 μg mL-1 of propidium iodide [PI] for 5 min. Before LSCM imaging, cells were washed with PBS again. MTT assays were performed to assess the metabolic activity of cells labeled with QDs. For MTT assays, cells were cultured in 96 well plates. Before MTT assays, cells were incubated in a medium containing QDs for 2 h, and then the medium was removed and replaced with a serum-free medium (200 μL/well). A total of 20 μL stock MTT (5 mg mL-1) was added to each well, and the cells were then incubated for 1 h at 37°C. The medium was removed, and the cells were lysed with DMSO. The absorbance was measured at 595 nm.
Results and discussion
Strategy and mechanism for the preparation of CS-ZnS and CS-ZnS:Mn2+ QDs
Figure 1 illustrated the basic strategy and mechanism for the preparation of CS-ZnS and CS-ZnS:Mn2+ QDs. Chitosan was used as a stabilizer as well as coating molecules in the synthesis processes. (CH3)2CHOH and excess S2O3 2- were necessary in order to obtain high quality products. First, Zn2+ and Mn2+ were arrested by chitosan through the interaction with -OH and -NH2 binding sites and other chemical structures . Subsequently, H2O was decomposed by the γ-ray . (CH3)2CHOH was used to eliminate the strongly oxidative free radical HO· and H2O2. Hydrated electron and hydrogen atoms with reduction potentials of -2.77 V and -2.13 V were used to reduce S2O3 2- and generate S2-. Then, S2- reacted with the bounded Zn2+ and Mn2+, and CS-ZnS:Mn2+ QDs were successfully produced. The main reaction processes were proposed as follows:
Morphology and structure of CS-ZnS and CS-ZnS:Mn2+ QDs
The morphology and structure of CS-ZnS and CS-ZnS:Mn2+ QDs fabricated in different conditions were characterized by TEM, EDS, and XRD. Without chitosan, the ZnS particles were seriously aggregated, and almost no monodispersed ZnS QDs were observed (Figure 2a). When the concentration of chitosan was 0.1% (w/v), monodispersed CS-ZnS QDs with the mean size of 4.5 nm were observed (Figure 2b). When the concentration of chitosan increased up to 2% (w/v), the dispersity of QDs was also very poor because of the redundant organic molecules (Figure 2c). The EDS spectrum demonstrated the existence of the Zn, S, C, N, and O elements in CS-ZnS QDs (Figure 2b). The as-synthesized CS-ZnS:Mn2+ QDs were about 5 nm in diameter and consisted of Zn, Mn, S, C, N, and O elements (Figure 2d). The obtained QDs were with narrow particle size distributions (insets in Figure 2b, d). There was no significant difference between the XRD patterns of CS-ZnS and CS-ZnS:Mn2+ QDs (Figure 2e). Three observed peaks could be indexed to (111), (220), and (311) reflections for cubic ZnS (JCPDS no. 77-2100). HRTEM images (insets in Figure 2b, d) indicated that as-synthesized CS-ZnS and CS-ZnS:Mn2+ QDs were highly crystalline, and the distances between the adjacent lattice fringes were measured as about 0.31 nm and 0.32 nm, respectively, which agree well with the literature value for the (111) d spacing (JCPDS no. 77-2100). These results indicated that the addition of Mn2+ did not affect the crystal structure of ZnS seriously.
Fluorescent properties of CS-ZnS and CS-ZnS:Mn2+ QDs
As shown in Figure 3A, the absorption peaks of Mn-doped and undoped CS-ZnS QDs were at 295 and 289 nm, respectively. The undoped CS-ZnS QDs showed a PL emission peak at about 447 nm, which is the typical luminescence of ZnS that resulted from the transition of electrons from shallow states near the conduction band to sulfur vacancies present near the valence band. A new strong emission peak at about 590 nm was observed in Mn-doped CS-ZnS QDs. On doping ZnS with Mn2+, the Mn2+ substitutes the Zn2+ in the ZnS crystal acting as trap sites, where the electrons and holes can be trapped. An electron can undergo a photoexcitation process in the host ZnS lattice of the nanoparticles and subsequently can decay via a transition from the 4T1 level to the 6A1 level, which results in the strong emission at around 590 nm. The photographs of samples (insets of Figure 3A) showed that the as-prepared QDs exhibited excellent water solubility and glowed a bright orange or blue fluorescence under UV illumination, respectively.
Preparation conditions have been proved to be very crucial to the properties of ZnS:Mn2+[16, 17]. Our results indicated that the fluorescence of as-synthesized QDs could be tuned by changing the dose of gamma radiation and the content of Mn in ZnS nanocrystals (Figure 3B, C). The PL emission peak of the sample was not very obvious when the radiation dose was 5 kGy. When the radiation dose was 10 kGy, the QY values of the as-obtained QDs were about 16.6%, and PL emission peaks appeared at around 442 and 584 nm. With the increase of radiation dose, the particle sizes were increasing, and the PL emission peaks appeared a slight red shift. When the radiation dose was 20 kGy, the QY values of the QDs reached 22.5%. However, when the radiation dose increased to 30 kGy, the QY values of the QDs decreased to 18.2% possibly because of the destruction of the crystal structure and chitosan. With the increase of Mn concentrations in ZnS nanocrystals, the PL emission peak around 590 nm was gradually enhanced with a slight red shift, and the QY values of the QDs were also increased. However, when the ratio of Mn to Zn reached 5:100, the QY values of the QDs decreased from 22.5% to 13% again because excessive amounts of Mn2+ in the reaction solution affected the generation and structure of ZnS nanocrystals.
Cellular labeling and cytotoxicity of CS-ZnS and CS-ZnS:Mn2+ QDs
LSCM imaging and MTT assays were carried out to examine the cellular labeling and cytotoxicity of as-synthesized QDs. As shown in Figure 4A, CS-ZnS and CS-ZnS:Mn2+ QDs were successfully taken by the PANC-1 cells. Most of the QDs were located in the cytoplasm around the nuclei; only very few QDs were present in the nuclei. Cells containing QDs emitted a bright blue light and orange light, respectively, under laser excitation. The metabolic activity of cells labeled with CS-ZnS and CS-ZnS:Mn2+ QDs were kept above 80% when their concentrations in the medium were below 200 μg mL-1 and were also kept above 70% when their concentrations reached 1,000 μg mL-1 (Figure 4B). The metabolic activity of cells labeled with TGA-CdSe QDs had decreased to 32% when their concentration was 10 μg mL-1, which showed a striking contrast. The cytotoxicity of as-prepared QDs was very low, which might be attributed to biocompatible chitosan coatings and nontoxic chemical composition. These results demonstrated the potential application of as-fabricated QDs in biomedical areas.
In summary, this study has presented an attempt to fabricate biocompatible chitosan-coated ZnS and ZnS:Mn2+ QDs by a γ-radiation technique. The synthesized QDs had good fluorescent properties which could be tuned by changing the dose of gamma radiation and the content of Mn in ZnS nanocrystals. The obtained QDs exhibited low cytotoxicity and high biocompatibility, which might be very useful in further biomedical applications. We claim that this convenient synthesis strategy could be also easily extended to fabricate various other functional nanoparticles coated with chitosan.
Michalet X, Pinaud FF, Bentolila LA, Tsay JM, Doose S, Li JJ, Sundaresan G, Wu AM, Gambhir SS, Weiss S: Quantum dots for live cells, in vivo imaging, and diagnostics. Science 2005, 307: 538–544. 10.1126/science.1104274
Mews A, Zhao J: Light-emitting diodes - a bright outlook for quantum dots. Nat Photonics 2007, 1: 682–683.
Bakalova R, Zhelev Z, Aoki I, Kanno I: Designing quantum-dot probes. Nat Photonics 2007, 1: 487–489. 10.1038/nphoton.2007.150
Gao XH, Cui YY, Levenson RM, Chung LWK, Nie SM: In vivo cancer targeting and imaging with semiconductor quantum dots. Nat Biotechnol 2004, 22: 969–976. 10.1038/nbt994
Oberdorster G, Oberdorster E, Oberdorster J: Nanotoxicology: An emerging discipline evolving from studies of ultrafine particles. Environ Health Persp 2005, 113: 823–839. 10.1289/ehp.7339
Wang XM, Zhang G, Shi LX, Selke M: CdTe quantum dots with daunorubicin induce apoptosis of multidrug-resistant human hepatoma HepG2/ADM cells: in vitro and in vivo evaluation. Nanoscale Res Lett 2011, 6: 418. 10.1186/1556-276X-6-418
Chen FQ, Gerion D: Fluorescent CdSe/ZnS nanocrystal-peptide conjugates for long-term, nontoxic imaging and nuclear targeting in living cells. Nano Lett 2004, 4: 1827–1832. 10.1021/nl049170q
Yang XT, Zhang Y: Encapsulation of quantum nanodots in polystyrene and silica micro-/nanoparticles. Langmuir 2004, 20: 6071–6073. 10.1021/la049610t
Nie QL, Tan WB, Zhang Y: Synthesis and characterization of monodisperse chitosan nanoparticles with embedded quantum dots. Nanotechnol 2006, 17: 140–144. 10.1088/0957-4484/17/1/022
Cui DX, Ruan J, Wang K, Song H, Xu X, Ji JJ: Biocompatibility of hydrophilic silica-coated CdTe quantum dots and magnetic nanoparticles. Nanoscale Res Lett 2011, 6: 299. 10.1186/1556-276X-6-299
Li H, Shih WY, Shih WH: Non-heavy-metal ZnS quantum dots with bright blue photoluminescence by a one-step aqueous synthesis. Nanotechnology 2007, 18: 205604. 10.1088/0957-4484/18/20/205604
Li H, Shih WY, Shih WH: Synthesis and characterization of aqueous carboxyl-capped CdS quantum dots for bioapplications. Ind Eng Chem Res 2007, 46: 2013–2019. 10.1021/ie060963s
Sandros MG, Behrendt M, Maysinger D, Tabrizian M: InGaP@ZnS-Enriched chitosan nanoparticles: a versatile fluorescent probe for deep-tissue imaging. Adv Funct Mater 2007, 17: 3724–3730. 10.1002/adfm.200700439
Mansi X, Karamjit S, Inderjeet SS, Harbhajan SB: Photo-catalytic activity of Zn 1-x Mn x S nanocrystals synthesized by wet chemical technique. Nanoscale Res Lett 2011, 6: 438. 10.1186/1556-276X-6-438
Quan ZW, Wang ZL, Yang PP, Lin J, Fang JY: Synthesis and characterization of high-quality ZnS, ZnS:Mn 2+ , and ZnS:Mn 2+ /ZnS (core/shell) luminescent nanocrystals. Inorg Chem 2007, 46: 1354–1360. 10.1021/ic061917n
Suyver JF, Wuister SF, Kelly JJ, Meijerink A: Synthesis and photoluminescence of nanocrystalline ZnS:Mn 2+ . Nano Lett 2001, 1: 429–433. 10.1021/nl015551h
Yang HS, Santra S, Holloway PH: Syntheses and applications of Mn-doped II-VI semiconductor nanocrystals. J Nanosci Nanotechnol 2005, 5: 1364–1375. 10.1166/jnn.2005.308
Yang HS, Holloway PH, Ratna BB: Photoluminescent and electroluminescent properties of Mn-doped ZnS nanocrystals. J Appl Phys 2003, 93: 586–592. 10.1063/1.1529316
Pan AL, Ma JG, Yan XZ, Zou BS: The formation of CdS nanocrystals in silica gels by gamma-irradiation and their optical properties. J Phys-Condens Mat 2004, 16: 3229–3238. 10.1088/0953-8984/16/18/023
Said AEA: Radiation synthesis of interpolymer polyelectrolyte complex and its application as a carrier for colon-specific drug delivery system. Biomaterials 2005, 26: 2733–2739. 10.1016/j.biomaterials.2004.07.049
Chang SQ, Kang B, Dai YD, Chen D: A novel route to synthesize CdS quantum dots on the surface of silk fibers via γ-radiation. Mater Lett 2008, 62: 3444–3446. 10.1016/j.matlet.2008.02.075
Chang SQ, Dai YD, Kang B, Chen D: Synthesis of antimicrobial silver nanoparticles on silk fibers via gamma-radiation. J Appl Polym Sci 2009, 112: 2511–2515. 10.1002/app.29716
Chang SQ, Dai YD, Kang B, Han W, Chen D: Gamma-radiation synthesis of silk fibroin coated CdSe quantum dots and their biocompatibility and photostability in living cells. J Nanosci Nanotechnol 2009, 9: 5693–5700. 10.1166/jnn.2009.1226
Wang JJ, Zeng ZW, Xiao RZ, Xie TA, Zhou GL, Zhan XR, Wang SL: Recent advances of chitosan nanoparticles as drug carriers. Int J Nanomed 2011, 6: 765–774.
Tan WB, Zhang Y: Surface modification of gold and quantum dot nanoparticles with chitosan for bioapplications. Journal of Biomedical Materials Research Part A 2005, 75A: 56–62. 10.1002/jbm.a.30410
Kang B, Dai YD, Zhang HQ, Chen D: Synergetic degradation of chitosan with gamma radiation and hydrogen peroxide. Polymer Degrad Stab 2007, 92: 359–362. 10.1016/j.polymdegradstab.2006.12.006
Su HL, Han J, Dong Q, Zhang D, Guo QX: In situ synthesis and photoluminescence of QD-CdS on silk fibroin fibers at room temperature. Nanotechnol 2007, 19: 025601.
This work was supported by the National Natural Science Foundation of China (11105073, 81101146, 81071256), the China Postdoctoral Science Foundation (20100471338), the Natural Science Foundation of Jiangsu Province (BK2008400, BK2011739, BK2011738) and NUAA Research Funding (NS2010152).
The authors declare that they have no competing interests.
SQC carried out the experimental design and analysis and drafted the manuscript. DC participated in the experimental design. BK and HXZ participated in the synthesis and characterization of quantum dots. YDD participated in the experimental analysis. All authors read and approved the final manuscript.
About this article
Cite this article
Chang, S., Kang, B., Dai, Y. et al. One-step fabrication of biocompatible chitosan-coated ZnS and ZnS:Mn2+ quantum dots via a γ-radiation route. Nanoscale Res Lett 6, 591 (2011) doi:10.1186/1556-276X-6-591
- quantum dots