Abstract: Multifunctional poly(glycidyl methacrylate) (PGMA) microspheres containing magnetic, fluorescent and cancer-cell specific moieties were prepared in four steps: (1) Preparation of parent PGMA microspheres by dispersion polymerization and their reaction with ethylene diamine to obtain amino groups; (2) Precipitation of iron ions (Fe2+ and Fe3+) to form Fe3O4 nanoparticles within the microspheres; (3) Consecutive reactions of folic acid with the amino groups on PGMA; (4) Incorporation of fluorescein isothiocyanate (FITC) into the microspheres. The microspheres were superparamagnetic, highly monodispersive, strongly fluorescent, and capable of recognizing and binding cancer-cells that overexpress folic acid receptors. It was demonstrated that with these microspheres Hela cells could be captured from their suspension and moved in the direction of the externally applied magnetic field in ease.
Keywords: Microspheres; Fe3O4 nanoparticles; Fluorescence; Folic acid; Cell detection and separation
In recent years, multifunctional particles possessing mainly magnetism and fluorescence have attracted much attention [1-3]. Magnetic moieties allow for manipulation of the particles by external magnetic fields, and luminescent moieties provide a possibility to detect the presence and movements of the particles. Therefore, many multifunctional particles have been prepared and studied extensively [4, 5]. Among them, FePt nanoparticles coated with CdS shell , Au-Fe3O4 dumbbell nanoparticle , Co/CdSe core-shell nanocomposites  are typical examples. Usually, their magnetism comes from the magnetic metallic elements or compounds. Their fluorescence originates from "quantum dots (QD)3" or gold nanoparticles. But they are easy to aggregate to cause fluorescence quenching. Meanwhile, because the metallic elements and oxides are reactive under the biomedical environments, they are easily etched in practical applications. Therefore, protective coatings, inorganic or organic, are developed for these particles, including silica [9, 10], polyelectrolytes , lipid layers , micelles , etc. Among them polymer particles were widely used because of its monodispersivity, desirable shapes and various functional groups. Usually, they are applied to the magnetic or fluorescent nanoparticles when the latter were already fabricated. This procedure may be called as "coating" method. Special coating materials or coating techniques are often needed, e.g., emulsion polymerization of vinyl monomers in the presence of the magnetic and/or fluorescent nanoparticles to form a thin polymer shell around the nanoparticles [14-16]. An alternative procedure is insertion , i.e., to prepare carrier microparticles first, and then to incorporate magnetic and fluorescent moieties into them . This process provides the possibility for preparing monodisperse and compact particles of desired size. Success in the latter method depends on the choice of proper carrier materials and on the effectiveness of incorporating the functional components into the carrier spheres. Reactive and swellable polymers are suitable carrier materials because magnetic and/or fluorescent components can diffuse into their particles and undergo correspondent reactions to form final functional moieties.
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In the present study, poly(glycidyl methacrylate) (PGMA) was chosen as a particle-forming material. PGMA is a well-known polymer in both industrial and biomedical applications because it is reactive, inexpensive, hydrophilic, biocompatible, and nontoxic. Procedures of producing its uniform microspheres have been documented . Ethylene diamine was used to react with the epoxy groups of PGMA to introduce more reactive amino groups for the subsequent syntheses, including formation of superparamagnetic Fe3O4 nanoparticles and incorporation of FITC fluorophores. Furthermore, the amino groups introduced were used to conjugate folic acid (FA) to endow the microspheres with ability of specific adhesion to cancer cells that overexpress folic acid receptors (FRs). This was the third function of the microparticles, i.e., detection and separation of specific cells. It was demonstrated with HeLa cells and rabbit chondrocytes. By culture with the multifunctional spheres for 2 h, the spheres adhered to the HeLa cells and the cells were visually observed by fluorescence microscope. When a magnet is placed aside, the movement of the HeLa cells towards the magnet was observed even with naked eyes because of the yellow color of the microspheres.
Materials and methods
Glycidyl methacrylate (GMA, 99%) was purchased from Sigma-Aldrich and distilled under reduced pressure before use. Folic acid (FA) was also obtained from Sigma-Aldrich. FITC was supplied by Beijing Solarbio Science & Technology Co. Ltd.. Dicyclohexylcarbodiimide (DCC) and N-hydroxysuccinimide (NHS) were obtained from GL Biochem (Shanghai) Ltd.. 2,2'-Azobis(isobutyronitrile) (AIBN) was purchased from Beijing Chemical Works and purified by recrystallizaton. Analytical grade polyvinylpyrrolidone (PVP K-30, Mw 40,000), ferric chloride hexahydrate (FeCl3âˆ™6H2O), ferrous chloride tetrahydrate (FeCl2âˆ™4H2O), ammonium hydroxide (25% w/w) and ethylene diamine were used as purchased from Beijing Chemical Works. All other reagents were of analytical grade and were used without further purification.
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Preparation of monodisperse PGMA microspheres with amino groups (NH2-PGMA)
PGMA microspheres with amino groups were prepared according to our previous paper . Briefly, A 250 ml four-neck round-bottom flask which was equipped with a mechanical stirrer, condenser, nitrogen inlet and thermometer was placed in a water bath at 50 Â°C. PVP (3 g) dissolved in a mixture of ethanol (120.5 g) and water (13.5 g) were introduced into the flask under stirring. Nitrogen gas was bubbled through the solution to remove oxygen from the system. The monomer GMA (12 g) including AIBN (0.24 g) was added into the flask and was polymerized at 70 Â°C for 16 h under stirring and nitrogen atmosphere. The reaction was terminated by cooling and the formed microspheres were collected by centrifugation, washed with ethanol and water, and dried by lyophilization.
To introduce amino groups, PGMA microspheres were reacted with ethylene diamine. Typically, 5 g of dried PGMA microspheres were treated with a mixture of 50 ml water and 50 ml ethylene diamine at 80 Â°C for 12 h. After the reaction, the particles were separated by centrifugation, washed several times with water to remove excess of ethylene diamine, and dried by lyophilization.
Synthesis of magnetic PGMA microspheres (M-PGMA)
To form Fe3O4 nanoparticles within the polymer microspheres, iron ions (Fe2+ and Fe3+) were impregnated into NH2-PGMA spheres and subsequently precipitated with ammonium hydroxide. 5 g of dried NH2-PGMA were dispersed into 50 ml water at room temperature to form the NH2-PGMA latex and the latex was cooled down to 10 Â°C. 811 mg of FeCl3âˆ™6H2O and 338 mg of FeCl2âˆ™4H2O were dissolved in 20 ml of water and cooled down to 10 Â°C, respectively. The two iron chloride solutions were mixed together and then added into the latex under continuous stirring. After mixing, the container was rapidly evacuated down to 10 mm Hg. After 20 minutes, 10 ml cold (10 Â°C) ammonia solution (25%) were added by suction. The vacuum was then eliminated, and the temperature was raised to 80 Â°C. The reaction was continued for 30 minutes at 80 Â°C. The mixture was cooled to room temperature and the particles were washed several times with water to remove excess of ammonia. The particles were then dried by freeze-drying.
Synthesis of FA-conjugated magnetic PGMA microspheres (FA-M-PGMA)
FA was conjugated onto the microspheres as reported elsewhere . In brief, NHS ester of folic acid (NHS-FA) was first prepared by esterification of folic acid (5 g) with NHS (2.6 g) in 100 ml of dry dimethylsulfoxide (DMSO) in the presence of DCC (4.7 g) and triethylamine (2.5 ml) as catalyst overnight at room temperature. The by-product, dicyclohexylurea, was removed by filtration. The DMSO solution was then concentrated under reduced pressure and heating, and NHS-FA was precipitated in diethyl ether. The product, NHS-FA, was washed several times with anhydrous ether, dried under vacuum, and stored as yellow powders. Then, NHS-FA (23 mg) was dissolved in DMSO (0.4 mL) and the solution was added to the microsphere suspension of M-PGMA (4 mL, 5 mg/mL, in carbonate/bicarbonate buffer, pH 9.0). The reaction was carried out for 30 min at room temperature. FA-M-PGMAs were separated by a commercial magnet and washed several times with carbonate/bicarbonate buffer. FA-M-PGMA powders were obtained by lyophilization.
Preparation of fluorescent-magnetic FA-conjugated microspheres (FA-F-M-PGMA)
FA-M-PGMA powders (20 mg) were swelled using a methylene chloride (20%)/ethanol (80%) (v/v) solvent mixture for 1 hour. FITC (1 mg) was added to the suspension. After 24 hours of constant shaking in dark and subsequent evaporation of the organic solvent, the particles were washed several times with water to remove excessive FITC, collected with a magnet and finally freeze-dried.
HeLa (human cervical cancer) cells and rabbit chondrocytes were used to examine cancer-cell specific adhering ability of the spheres. They were first cultured in DMEM (Dulbecco's modified Eagle's medium, GIBCO) supplemented with 10% (v/v) heat-inactivated fetal bovine serum, 100 units/mL penicillin, and 100 mg/mL streptomycin. Then they were seeded into 6-well plates (0.5Â´105 cells in 2.50 mL of DMEM per well) and incubated for 24 h (HeLa cells) and 48 h (rabbit chondrocytes), respectively. The culture medium was replaced with the fresh one every day. After FA-F-M-PGMA suspension (200 mL, 2 mg/mL) was added, incubation was continued for another 2 h. Then the medium was removed. The cells were washed directly five times with PBS (100 mM, pH 7.4) and imaged immediately via inverted fluorescence microscopy. Then, HeLa cells were trypsinized with 0.25% trypsin. A commercial magnet was used to collect the cells that carried the FA-F-M-PGMA particles.
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A JEOL JXA-840 scanning electron microscope was used to observe the morphology of the microspheres under an accelerating voltage of 20 kV. FT-IR spectra were measured by a vacuum FT-IR spectrophotometer (Bruker, IFS 66V/S). Fluorescence images of microspheres were collected using an inverted TE-2000-U digital fluorescence microscopy (Nikon) attached with a digital camera (DXM1200F). Confocal laser scanning microscopy (CLSM) images were collected with a Leica TCS SP2 CLSM (Leica Microsystems Heidelberg GmbH, Germany) equipped with a 20Â´ dry objective (NA 0.7) using digital zooms of 1Â´ to 32Â´ attached to a Leica DMIRE2 inverted microscope. Confocal optical sections were collected in the image-scan x-y-z mode. The samples were excited with a 492 nm He/Ne laser. Steady-state fluorescence spectra were obtained by Perkin-Elmer LS50B luminescence spectrometer.
Results and Discussion
Preparation of FA-F-M-PGMA microspheres.
Preparation procedure of the FA-F-M-PGMA microspheres is shown in Scheme 1. The blank PGMA microspheres were prepared using dispersion polymerization. Their size was ~ 2 mm with a narrow distribution (see the Supporting Information, Figure S1). Then, ethylene diamine was attached to the polymer chain to obtain surface amino groups. An excessive amount of ethylene diamine was used to avoid crosslinking between microspheres. The amount of surface amino groups was determined by titration (3 mmol/g). Iron ions (Fe2+ and Fe3+) were impregnated into the microspheres and subsequently precipitated with ammonium hydroxide. XRD spectrum of M-PGMA (see the Supporting Information, Figure S2) confirmed the formation of magnetic Fe3O4 nanoparticles in the polymer microspheres. The microspheres are dispersed uniformly in aqueous solution in the absence of a magnetic field (Figure 1, left). When a magnet is placed beside the glass vial, the particles get accumulated on the vial wall near the magnet in a few minutes (Figure 1, right). After removal of the external magnetic field, the aggregates were rapidly re-dispersed by gentle stirring. The magnetization hysteresis loops of M-PGMA (see the Supporting Information, Figure S3) shows that the microspheres are superparamagnetic.
For selective adhesion to cancer cells, FA molecules were conjugated onto the microspheres. FA, a high-affinity ligand to folate receptors (FRs), has been widely used for many biomedical applications [20,21], because of its high stability, low-cost, nonimmunogenic character and compatibility with both organic and aqueous solvent. The FA molecules were conjugated onto the magnetic microspheres in two steps: (1) activation of FA with NHS in DMSO; (2) reaction with the amino groups of the microspheres in a mixed solvent (DMSO//carbonate/bicarbonate buffer = 1/10 v/v). It is well known that NHS-FA is capable of reacting with primary amine in high yield [20, 22]. Most of previous researches employed DMSO, a good solvent for NHS-FA, as a reaction medium. In this work, a solvent mixture was used instead of pure DMSO because the polymer matrix can dissolve in DMSO. By using such mixture solvent, the microsphere morphology was maintained and leakage of Fe3O4 nanoparticles from the microspheres was effectively avoided. As shown in Figure 2, the microspheres after FA-conjugation remain spherical, highly monodispersive and smooth. Successful FA-conjugation of the magnetic PGMA microspheres was confirmed by FT-IR spectroscopy. As shown in Figure 3, after ethylene diamine attachment, the characteristic epoxy peak of PGMA at 910 cm-1 in Figure 3(a) almost disappears in Figure 3(b). The spectrum of NH2-PGMA shows clear peaks at 3400 cm-1 and 1564 cm-1, indicating the existence of -NH2 and -NH groups. The FT-IR spectrum of FA-M-PGMA (Figure 3(c)) exhibits two benzene ring absorptions at 1604 cm-1 and 1510 cm-1 characteristic of the folate moieties.
For rapid and effective detection of the target cancer cells, the microspheres were labeled with organic fluorescent dye FITC (green color, lmax(em) = 518 nm). The microspheres were first swelled in a mixed solvent consisting of 20% in volume of methylene chloride and 80% in volume of ethanol. The swollen microspheres could absorb quite amount of FITC from the solution, presumably due to the reaction between the isothiocyanate group of FITC and the amino groups on the PGMA chains. As shown in Figure 4(a), the aqueous dispersion of the microspheres shows intense green fluorescence. Optical sectioning by CLSM (inset in Figure 4(a)) reveals homogeneous distribution of FITC inside the microspheres. Moreover, the fluorescence remained quite strong even after the microspheres were washed six times with ethanol, confirming chemical combination of FITC with NH2-PGMA. On the other hand, a considerable red shift (about 10 nm) in the fluorescence peak is observed from pure FITC to FITC-containing spheres (Figure 4(b)), due to the environmental change and concentration increase of FITC from aqueous solution to organic PGMA matrix . A similar red-shift has been observed for rhodamine 6G in clay minerals [23,24].
Selective Cell Separation and Imaging.
In order to demonstrate how cancer cells are selectively detected and separated by using FA-F-M-PGMA spheres, cancer cells (HeLa cells) and normal cells (rabbit chondrocytes) were incubated with FA-F-M-PGMA for 2 h, respectively. It was found that after thorough washing many FA-F-M-PGMA spheres adhere to HeLa cell surface (Figures 5a and 5b) whereas there are very few FA-F-M-PGMA spheres on the surface of rabbit chondrocytes (Figures 5c and 5d). It indicated that the microspheres can specifically recognize the cancer cells that overexpress FRs. Therefore, HeLa cells can be identified and separated from other cells that do not overexpress FRs. The final separation and collection of Hela cells were demonstrated by applying an external magnetic field. As shown in Scheme 2, when a magnet was put aside the glass vial, HeLa cells went towards the magnet and got accumulated on the side wall of the vial. When the vial was turned around over 180 Â°, the HeLa cells moved across the vial towards the magnet quickly. This process was recorded in a short movie which is available as Supporting Information. The separation efficiency of this system and the viability of the cells are in further research.
Multi-functional FA-F-M-PGMA microspheres were fabricated starting from blank monodispersive PGMA microspheres. Firstly, ethylene diamine was reacted with the epoxy groups on PGMA to incorporate amino groups onto the spheres. These amino groups were subsequently used to couple with folic acid and FITC. Secondly Fe3O4 nanoparticles were formed inside the spheres to gain superparamagnetism. Thirdly, folic acid and FITC were introduced to PGMA to achieve the ability of selectively recognizing and detecting cancer cells and to realize fluorescent detection of the spheres themselves respectively. With these FA-F-M-PGMA spheres, selective separation of HeLa cells from their mixtures with other cells is possible. If folic acid is replaced with other ligands, other cells that overexpress corresponding receptors can be detected and separated surely in a similar manner. Therefore, this study provided an example of preparing multifunctional particles with an "insertion" method, i.e., preparing carrier microspheres first and then incorporating individual functional moieties into the microspheres afterwards.