Facile synthesis of folate-conjugated magnetic/fluorescent bifunctional microspheres
© Shen et al.; licensee Springer. 2014
Received: 26 May 2014
Accepted: 1 October 2014
Published: 8 October 2014
In this paper, we investigated the functional imaging properties of magnetic microspheres composed of magnetic core and CdTe quantum dots in the silica shell functionalized with folic acid (FA). The preparation procedure included the preparation of chitosan-coated Fe3O4 nanoparticles (CS-coated Fe3O4 NPs) prepared by a one-pot solvothermal method, the reaction between carboxylic and amino groups under activation of NHS and EDC in order to obtain the CdTe-CS-coated Fe3O4 NPs, and finally the growth of SiO2 shell vent the photoluminescence (PL) quenching via a Stöber method (Fe3O4-CdTe@SiO2). Moreover, in order to have a specific targeting capacity, the magnetic and fluorescent bifunctional microspheres were synthesized by bonding of SiO2 shell with FA molecules via amide reaction (Fe3O4-CdTe@SiO2-FA). The morphology, size, chemical components, and magnetic property of as-prepared composite nanoparticles were characterized by ultraviolet-visible spectroscopy, fluorescent spectroscopy, Fourier transform infrared spectroscopy (FTIR), X-ray powder diffraction (XRD), scanning transmission electron microscopy (SEM), transmission electron microscopy (TEM), thermogravimetric analysis (TGA), and vibrating sample magnetometer (VSM), respectively. The results show that the magnetic and fluorescent bifunctional microspheres have strong luminescent which will be employed for immuno-labeling and fluorescent imaging of HeLa cells.
KeywordsMagnetic nanoparticles Chitosan Solvothermal Fluorescent
In the past few years, a variety of ferroferric oxide magnetic nanoparticles (Fe3O4 NPs) have been widely used in biomedical applications, such as targeted drug delivery, rapid biological separation, biosensors, and magnetic hyperthermia therapy[1–4]. At the same time, quantum dots (QDs) have been actively studied for bioimaging applications due to their excellent optical properties such as narrow emission bands, continuous broad absorption band, and high resistance to photobleaching in comparison with organic dyes. Highly luminescent QDs could serve as luminescent markers, while Fe3O4 NPs could be easily manipulated under the external magnetic field. Therefore, combining QDs and Fe3O4 NPs to get fluorescent/magnetic bifunctional composite nanoparticles has attracted intense attention due to its appealing applications[5–7]. However, the preparation of fluorescent/magnetic bifunctional composite nanoparticles is challenging. There are also problems associated with their low chemical stability and biocompatibility. A specific difficulty in the preparation of fluorescent/magnetic bifunctional composite nanoparticles is the risk of quenching of the fluorophore on the particle surface by the magnetic core and the complicated synthesis procedures. These problems can be solved by coating the magnetic core with a stable isolating shell prior to the introduction of luminescent QDs (Fe3O4@X@QDs, X = SiO2, carbon, polymers, et al.). However, most of these conversion processes are very complicated and could cause dramatic changes on the size, shapes, and phases of the NPs, which are not conducive to biological applications. Thus, it is desirable to find a simple and flexible synthesis method to prepare the stable isolating shell.
In addition, folic acid is a small-molecule vitamin that is essential for the human body, especially the single carbon metabolism of eukaryotic cells and nucleoside synthesis. Folate receptor (FR) is a well-known tumor marker; it has limited expression in normal tissues, and is greatly overexpressed in a variety of carcinomas. Folic acid (FA) conjugated of imaging agents have high binding affinity for cell surface FRs, which allows for selective targeting of tumor cells.
Ferric chloride hexahydrate (FeCl3 · 6H2O, >99%), anhydrous sodium acetate (NaOAc), ethylene glycol (EG), cadmium chloride (CdCl2 · 2.5H2O), tellurium dioxide (TeO2, 99.99%), 3-mercaptopropionic acid (MPA, 99%), glucose, polyvinylpyrrolidone (PVP), FA, N-hydroxysuccinimide (NHS), and N-ethyl-N-(3-(dimethylamino) propyl) carbodiimide (EDC) were purchased from Aldrich (Wyoming, IL, USA). HeLa cells were supplied by Zhejiang University. Sodium hydroxide (NaOH) and aqueous ammonia solution (25 wt %) were analytical grade. The pure water was obtained from a Milli-Q synthesis system (Millipore, Billerica, MA, USA).
Fluorescence spectra were obtained at room temperature using a CARY ECLIPSE (Agilent Technologies, Santa Clara, CA, USA) fluorescence spectrometer. Transmission electron microscopy (TEM) images were obtained on a JEM-2100 TEM (Jeol Ltd., Tokyo, Japan). X-ray powder diffraction (XRD) analysis was performed using a Dmax-2500 (CuKα =1.5406 Å; Rigaku Corporation, Tokyo, Japan). Magnetic characteristics were studied using a vibrating sample magnetometer (VSM) (Lake Shore Company, Westerville, OH, USA) at room temperature. Scanning transmission electron microscopy (SEM) was carried out on a Philips XL30 microscope (Philips, Amsterdam, The Netherlands). The zeta potential of these particles was measured by dynamic light scattering (DLS) with a Delsa™ NanoC Particle Size Analyzer (Beckman Coulter, Pasadena, CA, USA). Thermogravimetric analysis (TGA) of nanocomposite and chitosan was performed on the TGA Q500 from TA Instruments (New Castle, DE, USA). Analyzed samples were heated from 100 to 800°C at a heating rate of 10°C/min under a nitrogen flow of 50 ml/min. The Fourier transform infrared spectroscopy (FTIR) of as-prepared composite nanoparticles was performed by Nicolet 5700 (Thermo Nicolet, Waltham, MA, USA).
Synthesis of CS-coated Fe3O4 NPs
Functionalized magnetite nanoparticles were synthesized via a versatile solvothermal reaction as previously reported with a slight modification. Typically, FeCl3 · 6H2O (1.50 g), chitosan (0.5 g), NaOAc (3.6 g), and PVP (1.0 g) were added to 70 ml of ethylene glycol to give a transparent solution via vigorous stirring. This mixture was then transferred to a Teflon-lined autoclave (80 ml) for treatment at 200°C for 8 h. The products were obtained with the help of a magnet and were exhaustively washed, in order for all the chitosan in the final products to be chemically bound to the magnetic nanoparticles. Finally, the products were collected with a magnet and dried in a vacuum oven at 60°C for further use.
Synthesis of MPA-stabilized CdTe QDs
MPA-stabilized CdTe QDs were synthesized by the reaction of tellurium dioxide as a tellurium source and 3-mercaptopropionic acid as a reductant following our reported procedures. Briefly, 2 mmol CdCl2 · 2.5H2O was dissolved in 100 ml of deionized water in a beaker, and 5.4 mmol MPA was added under stirring. The pH of the solution was then adjusted to 10.0 by dropwise addition of 0.1 mol/l NaOH solution. Under stirring, 0.5 mmol TeO2 was added to the original solution. The typical molar ratio of Cd2+/Te2-/MPA was 1:0.25:2.7. The monomer was heated in a XO-SM100 microwave-assisted heating system (XO-SM100 Microwave and Ultrasonic combination response system, MW-50%; Xianou Company, Nanjing, China). The solution was refluxed for 3.5 h and the reaction was terminated to obtain CdTe QDs with red-emitting colors.
Synthesis of CdTe-CS-coated Fe3O4 NPs
The CdTe-CS-coated Fe3O4 microspheres were synthesized by the reaction between carboxylic and amino groups under activation of NHS and EDC. Briefly, 0.1 g chitosan-coated (CS-coated) Fe3O4 microspheres were dispersed in 100 ml deionized water, mixed with 0.1 g NHS, and 0.158 g EDC by ultrasonication for 30 min, then different amounts of previously obtained CdTe NCs solutions were added to the suspension under continuous mechanical stirring for 24 h. Finally, the products were collected with a magnet and dried in a vacuum oven at 50°C for further use.
Synthesis of Fe3O4-CdTe@SiO2-NH2 core/shell microspheres
The Stöber method was employed to coat the as-produced spherical particles with a SiO2 layer. In this process, 0.1 g of CdTe-CS-coated Fe3O4 powder was dispersed in a mixture of ethanol (80 ml) and deionized water (20 ml) by ultrasonication for 10 min. Then, 1 ml of ammonia solution (25%) and 0.1 ml of TEOS were added into the solution quickly under continuous mechanical stirring. After 6 h of stirring at room temperature, 0.1 ml of 3-aminopropyltriethoxysilane (APTES) was added into the mixture solution and refluxed for 12 h. At last, the Fe3O4-CdTe@SiO2-NH2 microspheres were separated by a magnet and repeatedly washed with ethanol for several times and dried in a vacuum oven at 50°C for further use.
Conjugation of Fe3O4-CdTe@SiO2-NH2 magnetic/fluorescent microspheres with FA
In order to test the applications of Fe3O4-CdTe@SiO2-NH2 microspheres in immuno-labeling and fluorescent imaging of cancer cells, the microspheres were conjugated with FA. The conjugation of FA with Fe3O4-CdTe@SiO2-NH2 microspheres was completed through the reaction between carboxylic and amino groups under activation of NHS and EDC. Typically, 50 mg Fe3O4-CdTe@SiO2-NH2 microspheres was dissolved in 100 ml dimethylsulphoxide (DMSO), mixed with 0.1 g FA, 0.05 g NHS, and 0.06 g EDC by ultrasonication for 30 min, then stirred overnight. The mixed solution was separated with an external magnetic field, alternately rinsed with ultra-pure water and ethanol four times, then dispersed in 50 ml deionized water for further use.
Results and discussion
Structure characterization of the nanoparticles
Magnetic property and fluorescent spectra of the as-synthesized NPs
Figure 8a (inset) illustrates the magnetic separation process of the Fe3O4-CdTe@SiO2 NPs under normal light (the upper left corner) and 365-nm excitation (the lower right corner). In the absence of an external magnetic field, the solution of Fe3O4-CdTe@SiO2-NH2 NPs is red and the NPs are well dispersed in the aqueous solution under both normal light and UV irradiation. When a magnetic field is placed near the solution, the as-prepared nanoparticles are attracted and accumulated toward the magnet, and the bulk solution becomes a clear phase, indicating that magnetic separation occurs. These results suggest that the as-prepared nanocomposites can find potential applications in magnetic guiding and separation. Figure 8b showed the fluorescent spectra of the CdTe-CS-coated Fe3O4 NPs with different content of CdTe QDs (Vol =10, 20, 30, 50, 60 ml) constructed by amide reaction. It is clearly seen that the photoluminescence (PL) intensity gradually increased first and then decreased with the content of CdTe QDs changing from 10 to 60 ml, which suggests the formation of more homogeneous QDs.
In summary, a facile synthesis method was developed to prepare Fe3O4-CdTe@SiO2-FA NPs with tunable magnetism, sizes, suspension stability, and surface charge. The magnetic and fluorescent bifunctional microspheres have strong luminescent. Because these brightly luminescent beads exhibited high stability and super-paramagnetic behavior, we will next focus on their bio-applications, such as magnetic resonance imaging, drug delivery, cell labeling, and magnetic cell separation.
The authors gratefully acknowledge the support for this research from the Zhejiang Provincial Natural Science Foundation of China under Grant No. LQ13B070002, the Science and Technology Plan Project of Zhejiang Province under Grant No. 2012C37028, 2013C37052, the State Key Laboratory of Chemical Resources Engineering under Grant No. CRE-2012-C-303, and the National Natural Science Foundation of China under Grant No. 81201530.
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