Carboxymethyl chitosan-folic acid-conjugated Fe3O4@SiO2 as a safe and targeting antitumor nanovehicle in vitro

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.


Background
Recently, considerable effort has been devoted to magnetic nanoparticles (NPs) as novel nanovehicles [1] and targeting agents [2] for biological and biomedical applications [3,4]. Iron oxide (Fe 3 O 4 ) has emerged as one of the appealing candidates for drug delivery system [5] and magnetic fluorescence imaging [6,7]. However, the aggregations of naked Fe 3 O 4 NPs decrease their interfacial areas, thus resulting in the loss of magnetism [8] and dispersibility [9]. Therefore, extensive work has been done to stabilize the NPs [10,11]. Huang synthesized uniform Fe 3 O 4 @SiO 2 NPs with well-controlled shell thickness [12]. Kaskel developed a homogeneous Fe 3 O 4 @SiO 2 with hollow mesoporous structure for drug delivery [13]. 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 [18] and various targeting biomolecules [19,20] to improve the targeting efficiency. Kim reported Fe 3 O 4 @SiO 2 NPs using CTAB as a template and PEG to prolong the short blood half-life of NPs [21]. 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 [22]. Shi reported the OCMCS-Fe 3 O 4 easily internalized into cells via endocytosis [23]. Fan developed the Fe 3 O 4 NPs with OCMCS which significantly reduced the cytotoxicity and the capture of NPs. Moreover, folic acid (FA)-modified OCMCS-Fe 3 O 4 NPs combined receptor-mediated targeting and magnetic targeting together [24]. 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 (Fe 3 O 4 @SiO 2 -OCMCS-FA) with dualtargeting function (magnetic/folate) in this study. Fe 3 O 4 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 Fe 3 O 4 @SiO 2 which was modified with (3-aminopropyl) triethoxysilane (APTES) to obtain the multifunctional nanovehicle (Fe 3 O 4 @SiO 2 -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.

Reagent materials
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.
Next, tetraethylorthosilicate (TEOS) was added and the reaction was continued at room temperature for 24 h. When isopropanol was added into the reaction solution, Fe 3 O 4 @SiO 2 NPs were precipitated. They were collected by centrifugation and washed with ethanol. Fe 3 O 4 @SiO 2 NPs were then dried in vacuum at 60°C.

Synthesis of OCMCS-FA conjugate
The synthesis of OCMCS-FA conjugate was adopted by homogeneous synthesis through acylation ( Figure 2). Folic acid (0.884 g) was dissolved in 20 mL of anhydrous dimethylsulfoxide (DMSO) to which dicyclohexylcarbodiimide (DCC; 0.784 g) and N-hydroxysuccinimide (NHS; 0.256 g) were added. The reaction mixture was stirred for 24 h at 45°C in the dark [29]. The by-product dicyclohexylurea was filtered off, and 20 mL of 30% acetone in diethyl ether was added with stirring. A yellow precipitate (NHS-FA) formed and was collected after washing with diethyl ether several times. Then, 100 mg OCMCS was dissolved in acetate buffer (pH 4.7). A mixture solution of NHS-FA and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) was prepared by dissolving NHS-FA and EDC simultaneously in DMSO. Finally, the mixture solution was dropped into the OCMCS solution. After 24 h, the solution was adjusted to pH 9 with NaOH and purified by centrifugation followed by 2 days of dialysis against phosphate-buffered solution (PBS) and extensive dialysis against water using a 3,500-Da cutoff dialysis membrane. OCMCS-FA was then dried in vacuum at 60°C.

Hemolysis assay
Two milliliters of rat blood was injected from the eye socket vein. The red blood cells (RBCs) were obtained by removing the serum from the blood by centrifugation and suction. After being washed with PBS solution five times, the RBCs were diluted to 1/10 of their volume with PBS solution. Diluted RBC suspension (0.3 mL) was then mixed with the following: (a) PBS (1.2 mL) as a negative control, (b) deionized water (1.2 mL) as a positive control, and (c) nanovehicle suspensions (1.2 mL) at concentrations ranging from 40 to 500 μg mL −1 . The mixtures were then vortexed and kept for 2 h at room temperature. Finally, the mixtures were centrifuged for 2 min at 4,000 rpm and the absorbance of the upper supernatants at 541 nm was measured by UV-visible (UV-vis) characterization. The percentage of hemolysis was calculated using the following equation (A is the absorbance of UV-vis spectra) [30]:

Cytotoxicity
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 × 10 4 per well and then incubated for 24 h at 37°C under 5% CO 2 . DEME solutions of nanovehicle at concentrations of 100 mg mL −1 were added to the wells.

Results and discussion
The 1 H NMR spectra of OCMCS-FA conjugate was shown in Figure 3. The signals at δ 1.65, 2.88, and 3.08 to 3.64 ppm was assigned to the resonance of the monosaccharide residue protons, -COCH 3 , -CH-NH-, and -CH 2 -O-, respectively. The signals appearing at δ 6.3 to 8.5 ppm were attributed to the resonance of the folate aromatic protons. So, it revealed that the couple of the FA residue to the OCMCS could be achieved via EDC mediation [32]. FTIR spectroscopy shown in Figure 4 confirmed that OCMCS-FA was successfully immobilized on the Fe 3 O 4 @SiO 2 NPs. In the spectrum of OCMCS-FA (Figure 4b), the 1,635 cm −1 peak of COO-stretching vibration shifted to 1,590 cm −1 compared to OCMCS (Figure 4a). Moreover, a shoulder peak around 1,710 cm −1 is observed in OCMCS-FA which verified that FA conjugated to the OCMCS successfully [33]. The bare Fe 3 O 4 NPs showed characteristic bands related to the Fe-O vibrations near 569 cm −1 (Figure 4b,c). The peak at 1,100 cm −1 indicated Si-O bonding on the NP surface ( Figure 4c). Unsurprisingly, the FTIR spectra for Fe 3 O 4 @-SiO 2 -OCMCS-FA nanovehicle presented similar peaks at 1,710, 1,590, 1,100, and 569 cm −1 (Figure 4d). What is more, the FTIR spectrum of Fe 3 O 4 @SiO 2 -OCMCS-FA nanovehicle displayed an intense peak at 1,650 cm −1 which might result from the -CONH-due to the reaction between the carboxyl group of the OCMCS and amide on the surface of silica.
The XRD measurements were performed with the dried powder samples of bare, silica-coated and OCMCS-FA-conjugated iron oxide to identify the crystal phases. The pattern of OCMCS-FA-conjugated NPs ( Figure 5) showed all the major peaks corresponding to Fe 3 O 4 which could be assigned to the (311), (511), and (440) planes, respectively [34]. Additionally, the peak around 2θ = 25°due to the silica [35] was observed in the case of the silica-coated NPs, but disappeared in the Fe 3 O 4 @SiO 2 -OCMCS-FA nanovehicle which may attribute to the OCMCS-FA conjugated. These results confirmed the surface modification of the Fe 3 O 4 NPs with OCMCS-FA.
The surface composition was also ascertained by XPS as it is recognized as a quantitative surface elemental analysis and chemical state information. Wide-scan spectra were acquired for NPs with high-resolution C 1s, O 1s, and N 1s. Spectral calibration was carried out by setting the main C 1s peak at 285 eV. The high-resolution scans for C 1s (Figure 6a) (Figure 7b). The SEM image shows that the nanovehicles are very uniform in both size and shape (Figure 7b, inset).
The magnified hysteresis loop of Fe 3 O 4 @SiO 2 -OCMCS-FA nanovehicle which clearly showed that no remanence and hysteresis were detected demonstrated the superparamagnetism of the nanovehicle (Figure 8). After coating with silica, the magnetization of Fe 3 O 4 @SiO 2 was undoubtedly decreased compared with the Fe 3 O 4 nanoparticles for the shell and relatively low Fe 3 O 4 amount. However, after the final modification of OCMCS-FA, the magnetization of the nanovesicles was not apparently decreased due to the thin outer layer. Factually, superparamagnetism is usually highly desired because it can prevent the magnetic composite particles from irreversible aggregation and ensure an excellent dispersibility once the applied magnetic field is removed [37].

In vitro targeting of nanovehicle
The ability of nanoparticles to target specific locations is one of the most important factors for their prospective application in drug delivery and biomedicine. To investigate the uptake possibility of Fe 3 O 4 @SiO 2 -OCMCS-FA, CLSM was applied to trace the process of this nanovehicle. Therefore, RB is labeled on the surface of the nanovehicle to distinguish it. To explore the practical application of this nanovehicle in the targeting of tumor cells, the particles were incubated in physiological conditions with HeLa cells bearing the over-expressed folate receptor. Figure 9 shows DAPI, RB, and merged images of HeLa cells incubated with RBFe 3 O 4 @SiO 2 (20 μg mL −1 , control) and RBFe 3 O 4 @SiO 2 -OCMCS-FA (20 μg mL −1 ) for 2 h. Interestingly, even at the very low     Figure 9b also testifies that the nanovehicle was mainly distributed in the cytoplasm after cellular uptake. The confocal laser scanning microscope observation confirms that the nanovehicle could be effectively taken up by the HeLa cells as the folate modified.
To further reveal that the nanovehicle was internalized in HeLa cells rather than being bound to the cell surface, bio-TEM was used to analyze the nanovehicle-treated cells. Unlike the untreated cells (Figure 10a), some aggregates of nanovehicles were observed as black patches inside the cell cytoplasm which maintained their core-shell structure ( Figure 10b and the inset), while no nanovehicle was found in the nucleus which coincided with the results of CLSM. Based on the cell morphology, it is plausible that the nanovehicle accumulates on the membrane (Figure 10c) by the high specific interaction between folic acid on the nanovehicle and FR on HeLa cells which may increase the uptake through folate receptor-mediated endocytosis. Afterwards, majority of the internalized nanovehicle will be processed in lysosomes and are eventually released into the cytoplasm (Figure 10d). Therefore, in vitro CLSM and bio-TEM images present evidence about the target effects of nanovehicle with the OCMCS-FA modification.

Biocompatibility of nanovehicles (hemolysis assay and cytotoxicity)
It is important to investigate the biocompatibility of Fe 3 O 4 @SiO 2 -OCMCS-FA nanovehicles when materials are administrated by vein injection. Hemolysis assay is a primary approach to assess the biocompatibility for in vivo applications. The hemolysis percentage of the nanovehicles was quantified based on the absorbance of the supernatant  at 541 nm with isotonic PBS and distilled water as control. From Figure 11, Fe 3 O 4 @SiO 2 -OCMCS-FA nanovehicle exhibits good biocompatibility, and the hemolysis percentage of Fe 3 O 4 @SiO 2 -OCMCS-FA even at a high concentration of 500 μg mL −1 was 6.3% lower than the value of traditional nanoparticles (70% of 500 μg mL −1 ) [38]. Thus, the obtained results showed that no visible hemolysis effect was observed visually for nanovehicle to evidence the good blood compatibility for the introduction of OCMCS.
In order to verify the toxicity of nanovehicle, in vitro cytotoxicity of the nanovehicle on HeLa and human liver cells (L-O2) was evaluated using a traditional MTT assay. The results (Figure 12) showed that there was a relatively high cell viability (more than 80% at a concentration of 100 μg mL −1 ) in HeLa which displays low cytotoxicity and favorable cell compatibility which is consistent with hemolysis assay. In addition, the viability of the L-O2 cells was similar to that of the HeLa after incubating with nanovehicle which demonstrates that Fe 3 O 4 @SiO 2 -OCMCS-FA possesses safety for normal cells as a drug carrier. The mesoporous silica layer of this nanovehicle is currently studied by our group, which may offer the platform for insoluble drugs in biomedical application.

Conclusions
In summary, we presented a rational method of preparing folic acid-conjugated carboxymethyl chitosan by homogeneous synthesis characterized by 1 H 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 Fe 3 O 4 @SiO 2 via amide reaction to form the layer of compatibility and receptor-mediated targeting. Fe 3 O 4 @SiO 2 -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 Fe 3 O 4 @SiO 2 -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.