Carboxymethyl chitosan-folic acid-conjugated Fe3O4@SiO2 as a safe and targeting antitumor nanovehicle in vitro
© Li et al.; licensee Springer. 2014
Received: 22 January 2014
Accepted: 11 March 2014
Published: 25 March 2014
A synthetic method to prepare a core-shell-structured Fe3O4@SiO2 as a safe nanovehicle for tumor cell targeting has been developed. Superparamagnetic iron oxide is encapsulated inside nonporous silica as the core to provide magnetic targeting. Carboxymethyl chitosan-folic acid (OCMCS-FA) synthesized through coupling folic acid (FA) with OCMCS is then covalently linked to the silica shell and renders new and improved functions because of the original biocompatible properties of OCMCS and the targeting efficacy of FA. Cellular uptake of the nanovehicle was assayed by confocal laser scanning microscope using rhodamine B (RB) as a fluorescent marker in HeLa cells. The results show that the surface modification of the core-shell silica nanovehicle with OCMCS-FA enhances the internalization of nanovehicle to HeLa cells which over-express the folate receptor. The cell viability assay demonstrated that Fe3O4@SiO2-OCMCS-FA nanovehicle has low toxicity and can be used as an eligible candidate for drug delivery system. These unique advantages make the prepared core-shell nanovehicle promising for cancer-specific targeting and therapy.
Recently, considerable effort has been devoted to magnetic nanoparticles (NPs) as novel nanovehicles  and targeting agents  for biological and biomedical applications [3, 4]. Iron oxide (Fe3O4) has emerged as one of the appealing candidates for drug delivery system  and magnetic fluorescence imaging [6, 7]. However, the aggregations of naked Fe3O4 NPs decrease their interfacial areas, thus resulting in the loss of magnetism  and dispersibility . Therefore, extensive work has been done to stabilize the NPs [10, 11]. Huang synthesized uniform Fe3O4@SiO2 NPs with well-controlled shell thickness . Kaskel developed a homogeneous Fe3O4@SiO2 with hollow mesoporous structure for drug delivery . Unfortunately, the common challenge among these applications is to ensure sufficient uptake of NPs by specific cells [14, 15]. The outer shell of silica not only protects the inner magnetite core from aggregation [16, 17] but also provides sites for flexible surface modification such as poly(ethylene glycol) to render NP biocompatibility by preventing the nonspecific adsorption of proteins  and various targeting biomolecules [19, 20] to improve the targeting efficiency. Kim reported Fe3O4@SiO2 NPs using CTAB as a template and PEG to prolong the short blood half-life of NPs . However, the safety of drug carriers is one of the most critical factors to ensure its efficacy. Carboxymethyl chitosan (OCMCS) is a water-soluble chitosan which receives a great deal of interest because of favorable biocompatibility, safety, nonimmunogenicity, as well as reasonable cost . Shi reported the OCMCS-Fe3O4 easily internalized into cells via endocytosis . Fan developed the Fe3O4 NPs with OCMCS which significantly reduced the cytotoxicity and the capture of NPs. Moreover, folic acid (FA)-modified OCMCS-Fe3O4 NPs combined receptor-mediated targeting and magnetic targeting together . It is noted that folic acid, as an effective target ligand [25, 26], shows high binding affinity with folate receptor, which over-expressed on the membranes of many human malignant cells, but limited on the normal cells. To the best of our knowledge, the general synthetic protocols to combine silica with diverse functional modification used as a safe drug delivery system are seldom reported. With regard to the above effects, we develop a novel carboxymethyl chitosan-based, silica-coated iron oxide nanovehicle (Fe3O4@SiO2-OCMCS-FA) with dual-targeting function (magnetic/folate) in this study. Fe3O4 core serves as a carrier for magnetic targeting, while silica coating on the iron oxide NPs offers sites for further modifications. OCMCS-FA was conjugated firstly to perform a folate receptor (FR)-mediated cellular endocytose and acted as the biocompatible segment and then subsequently coupled through acylation to the surface of animated Fe3O4@SiO2 which was modified with (3-aminopropyl) triethoxysilane (APTES) to obtain the multifunctional nanovehicle (Fe3O4@SiO2-OCMCS-FA). Its uptake by human cervical carcinoma cell lines (HeLa cells) is traced, and the cytotoxicity on the human tumor cells and normal cells are both evaluated. The results show that it is nontoxic to them, which reveal that it could be used as a promising candidate for drug target delivery system.
All chemicals are analytical reagent grade and were used as received. Folic acid is a biological reagent purchased from Sinopharm Chemical Reagent Co., Ltd., Shanghai, China.
Synthesis of magnetic Fe3O4@SiO2 NPs
Polyoxyethylene(5) nonylphenyl ether (5 mL, Igepal CO-520, Sigma-Aldrich, St. Louis, MO, USA) was firstly dispersed in cyclohexane (40 mL). Then, 2 mL Fe3O4 solution (50 mg mL-1 in cyclohexane) was added. After 10 min, ammonium hydroxide (292 μL) was added to form a transparent brown solution of reverse microemulsion. Next, tetraethylorthosilicate (TEOS) was added and the reaction was continued at room temperature for 24 h. When isopropanol was added into the reaction solution, Fe3O4@SiO2 NPs were precipitated. They were collected by centrifugation and washed with ethanol. Fe3O4@SiO2 NPs were then dried in vacuum at 60°C.
Synthesis of OCMCS-FA conjugate
Synthesis of Fe3O4@SiO2-OCMCS-FA NPs
APTES was anchored to the surface of Fe3O4@SiO2 through refluxing at 110°C in toluene to develop amide in the surface of silica in order to introduce carboxyl groups of OCMCS-FA conjugate. Fifty milligrams of APTES-modified Fe3O4 was added to 10 mL of a 2-(N-morpholino) ethanesulfonic acid buffer (0.1 M, pH 6.5) containing 50 mg of OCMCS-FA, EDC (20 mM), and NHS (50 mM). The mixture suspension was then sonicated for 10 min in ultrasonic disrupter and shaken for 24 h at room temperature. The OCMCS-FA bound Fe3O4@SiO2 were collected under centrifugation, washed with ethanol, and dried in vacuum at 60°C.
Cell culture and uptake
HeLa cell lines were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum, 100 units mL-1 penicillin, and 100 mg mL-1 streptomycin in 37°C, 5% CO2. For investigation on targeting of nanovehicles, nanovehicles were labeled with RB to form RBFe3O4@SiO2 and RBFe3O4@SiO2-OCMCS-FA nanoparticles . In a typical procedure, 2.5 × 104 cells were seeded in a 35-mm dish with a glass bottom for 24 h to allow the cells to attach. After the cells were washed twice with PBS, the samples were added to the dishes in a concentration of 100 μg mL-1. After 2 h of incubation, the cells were washed several times with PBS to remove the remaining samples and dead cells. Finally, the cells were observed under a confocal laser scanning microscope (CLSM; Carl Zeiss LSM 710, Oberkochen, Germany). Cells with the addition of Fe3O4@SiO2 were imaged as control.
Bio-TEM observations for HeLa cells
The HeLa cells were incubated with 2.5 μg mL-1 nanovehicle inDMEM in 5% CO2 at 37°C for 24 h. Afterwards, cells were washed three times with PBS and subsequently fixed with 2.5% glutaraldehyde in 0.03 M potassium phosphate buffer for at least 24 h. Cells were then washed in PBS, postfixed with 1% osmium tetroxide in sodium carboxylate buffer, washed with 0.05 mol L-1 maleate, and stained with 0.5% uranylacetate (Sigma-Aldrich) in maleate buffer. After washing the cells in 0.05 mol L-1 maleate, the cells were dehydrated in a grading series of ethanol followed by acetone, embedded in Epon (Momentive Specialty Chemicals, Inc., Columbus, OH, USA), and dried in an oven at 60°C for 4 days. Ultrathin sections of approximately 50 nm thick were cut with a diamond knife on a Leica Ultracut R ultramicrotome (Milton Keynes, UK) and transferred to the copper grid. The images were viewed on JEOL-2100 electron microscope (Akishima, Tokyo, Japan).
The in vitro cytotoxicity was measured by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay in HeLa cells. Cells were initially seeded into a 96-well cell culture plate at 1 × 104 per well and then incubated for 24 h at 37°C under 5% CO2. DEME solutions of nanovehicle at concentrations of 100 mg mL-1 were added to the wells. The cells were further incubated for 72 h at 37°C under 5% CO2. The cells were washed three times with 0.2 mL PBS to remove the unbound nanoparticles. Subsequently, 0.2 mL DEME and 25 mL MTT (5 mg mL-1) were added to each well and incubated for an additional 4 h at 37°C under 5% CO2. Then, the medium solution was replaced by 0.15 mL DMSO solution. After 10 min, the optical density at 490 nm (absorption value) of each well was measured on a Tecan Infinite M 200 monochromator-based multifunction microplate reader (Männedorf, Switzerland). The corresponding nanovehicle with cells but not treated by MTT were used as controls. The cell vitality after labeling was compared with that of unlabeled cells and expressed as the relative ratio.
1H NMR spectra was recorded at 300 MHz on a Bruker ARX 300 spectrometer (Ettlingen, Germany). Infrared spectra (4,000 to 400 cm-1) were recorded on Bruker Fourier transform infrared (FTIR) spectrometer in KBr pellets. The X-ray powder diffraction patterns were recorded on an X'Pert diffractometer (PANalytical B.V., Almelo, The Netherlands) with CuKα radiation (λ = 1.54060 Å) at 45 kV and 40 mA. X-ray photoelectron spectroscopy (XPS) analysis was performed with a ESCALB MK-II (Physical Electronics Instruments, Chanhassen, MN, USA). The source was the monochromatic MgKα radiation. The surface charge of the nanovehicles was investigated on Malvern Zetasizer Nano ZS 90 zeta potential analyzer (Westborough, MA, USA). Transmission electron microscopy (TEM) was performed on a JEOL-2100 with an accelerating voltage of 200 kV. TEM samples were prepared by drop-casting dispersion onto copper grids covered by carbon film. Ultrathin sections for bio-TEM were cut with a diamond knife on a Leica Ultracut R ultramicrotome. Scanning electron microscopy (SEM) was performed on a JEOL-S-3400 N II. Magnetic property measurements were performed using a Quantum Design MPMS XL-7 superconducting quantum interference device (SQUID; Olomouc, Czech Republic). Confocal images were acquired using a Zeiss confocal laser scanning unit mounted on an LSM 710 fixed-stage upright microscope.
Results and discussion
In vitro targeting of nanovehicle
Biocompatibility of nanovehicles (hemolysis assay and cytotoxicity)
In summary, we presented a rational method of preparing folic acid-conjugated carboxymethyl chitosan by homogeneous synthesis characterized by 1H NMR and FTIR. Moreover, a novel, safe, and tumor-targeting nanovehicle with iron oxide as core and silica as shell has been fabricated showing good dispersion. It was firstly reported that OCMCS-FA conjugated on the surface of Fe3O4@SiO2 via amide reaction to form the layer of compatibility and receptor-mediated targeting. Fe3O4@SiO2-OCMCS-FA nanovehicle exhibits high uptake of HeLa cells which imply low cytotoxicity and good biocompatibility because of the dual targeting and OCMCS serving as a long circulation. Furthermore, the silica moiety of Fe3O4@SiO2-OCMCS-FA nanovehicle could be extended to fabricate mesoporous nanovehicle which may increase surface area and pore volume. Thus, we believe that this strategy may provide a safe and efficient platform for antitumor drug delivery.
We gratefully acknowledge the assistance of Professor Zheng Xu from the State Key Laboratory of Coordination Chemistry in Nanjing University. The work was financially supported by the Fundamental Research Funds for the Central Universities (JKZD2013003).
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