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Introduction

Tongue cancer is one of the most globally common malignant tumors in the oral and maxillofacial region (1,2). However, the clinical symptoms of early tongue cancer are not readily observed, thereby rendering a timely diagnosis difficult. Typically, an imaging examination is performed to ensure accurate preoperative staging and for surgical planning of the tongue mass excision. Magnetic resonance imaging (MRI) has been widely recognized as an ideal technique in the diagnosis of tongue cancer due to the markedly high resolution of soft tissue that it provides. Generally, abnormal MRI signals are flagged as lesions. In addition, neoplastic lesions in the tongue that are located towards the oral floor are readily detected on coronal images (3,4). However, it is difficult to detect potential neoplastic lesions and micrometastasis within lymph nodes using MRI, particularly neoplasms that measure <1 cm in diameter (5). Therefore, the sensitivity and histological specificity of traditional MR must be improved for the effective and early diagnosis of tongue cancer. Furthermore, imaging modalities that provide functional and molecular imaging would be more advantageous when compared with traditional morphological imaging (6,7). Accordingly, advances in nano-micelle science have led to the development of a non-invasive reagent that provides molecular imaging with the potential for sequential and longitudinal monitoring. In particular, super-paramagnetic iron oxide nanoparticles (termed SPIO) have been demonstrated as a highly effective contrast agent for MRI (8). Specifically, SPIO provide high magnetic signal strength and relatively low cytotoxicity (9,10). However, crude SPIO lack specificity towards a pathological site, which potentially limits their applications. Furthermore, SPIO have a highly hydrophobic surface, which may be efficiently coated with plasma components, thereby leading to the rapid endocytosis of these particles by the reticuloendothelial system (11). Therefore, surface modification of SPIO is essential for most bio-associated applications.

To date, various nano-sized particles, including liposomes, micelles and vesicles, have been utilized as carriers for the delivery of SPIO to sites of interest (12,13). Among these various modifications, micelles composed of polyethylene glycol (PEG) have been found to block copolymers with good biocompatibility. For example, a PEG outer shell for nanoparticles imparts a ‘stealth’ quality to hydrophobic SPIO, which helps to avoid potential exposure of the SPIO surface and adsorption of blood proteins (14). In addition, poly-caprolactone (PCL) has been widely investigated and has been applied to micelles due to its high colloidal stability compared with small molecular surfactants, and its enhanced solubilization (15,16). Furthermore, PEG-PCL micelles provide a stable interface between their inner core and the aqueous environment, and this enhances the biocompatibility and biodegradability of these micelles to improve their efficacy (17,18). In the present study, polymeric micelles were designed, which incorporate a diblock copolymer of PEG and PCL. The PEG-PCL block copolymers form bilayer micelles that are able to encapsulate SPIO into their hydrophobic cores (14,15).

Malignant cells, due to their high rate of cell division, have an increased requirement for folate, as it is as an essential component of single-carbon metabolism and nuclear glucoside synthesis. Accordingly, the folate receptor has been found to be overexpressed by a wide range of malignant tissues, including ovarian, breast, oropharyngeal and liver cancer tissues (15–17,19–21). Furthermore, the binding efficacy of folate to the folate receptors expressed by tumor cells is high, which renders folate an ideal candidate for the targeted delivery of vehicles to tumor cells. To date, folic acid (FA) has been applied as a targeting ligand in tumor imaging diagnosis and cancer chemotherapy studies (15,19,22).

To the best of our knowledge, only a small number of studies have described the targeted imaging of tongue cancer (19–23). Therefore, in the present study, folate was attached to the surface layer of SPIO-PEG-PCL copolymers to achieve functional micelles that target tongue cells. The aim was to produce SPIO-loaded micelles that would represent a T2-negative contrast agent, which could be detected by MRI to evaluate their tumor targeting efficiency noninvasively, thereby leading to the development of a method for the early detection of tongue cancer.

Materials and methods

Material preparation and characterization

Monomethoxy PEG and PCL were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Copolymers of PEG-PCL and FA-PEG-PCL were synthesized via multistep reactions and purified according to previously reported methods (23,24). The schematic illustration of folate-targeted SPIO-PEG-PCL micelle formation is presented in Fig. 1. SPIO were prepared and encapsulated inside the micelle cores as previously reported (25).

Figure 1. Schematic illustration of folate-targeted SPIO-PEG-PCL micelle formation. SPIO, superparamagnetic iron oxide nanoparticles; PEG, polyethylene glycol; PCL, poly-caprolactone; FA, folic acid.

Particle sizes of the obtained nanovesicles were determined using a Brookhaven BI-200SM dynamic light scattering (DLS) instrument (Brookhaven Instruments Corp., Holtsville, NY, USA). A 532-nm vertically polarized argon ion laser was used as the light source. Measurements were made at a 90° angle at 25°C and scattered light was collected using an autocorrelator. The particle size of each sample was measured five times. Transmission electron microscopy (TEM) images were obtained with a JEOL TEM-2010HR microscope (JEOL Ltd., Tokyo, Japan) operated at 160 kV.

The loading density of the SPIO polymeric micelles was determined using a polarized Zeeman Atomic Absorption Spectrophotometer (Z-2000; Hitachi High Technologies, Tokyo, Japan). Briefly, freeze-dried powders of 5-ml SPIO-loaded nanovesicle solutions were weighed and were combined with 1 M hydrochloric acid (HCl; 10 wt. % in water) to accelerate polymer degradation. Complete dissolution of the SPIO was achieved after 3 days. Fe concentrations were detected at 248.3 nm, the absorption wavelength specific for Fe, according to a previously established calibration curve (26). SPIO loading density was calculated as the ratio of the encapsulated SPIO over the total vesicle weight.

Cell culture

Tca-8113 (Institute of Biochemistry and Cell Biology; Chinese Academy of Sciences, Shanghai, China) is a human tongue cancer cell line that overexpresses cell surface receptors for folate. Tca-8113 cells were grown in Minimal Essential Media (MEM; Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% heat-inactivated fetal bovine serum (Invitrogen; Thermo Fisher Scientific, Inc.) and were maintained at 37°C in a humidified atmosphere with 5% CO2.

Biocompatibility assay

Methyl thiazolyl tetrazolium (MTT; Sigma-Aldrich; Merck KGaA) was used to assay the cytotoxicity of folate-targeted SPIO-PEG-PCL and folate-free SPIO-PEG-PCL micelles towards Tca-8113 cells. Briefly, Tca-8113 cells were seeded in 96-well plates (1×105 cells/well) and the cells were incubated with graded dilutions (1-, 2-, 4-, 8-, 16-, 32-, and 64-fold) of nanovesicle solutions in MEM medium. The plates were incubated at 37°C in a humidified atmosphere with 5% CO2 for 3 days. Subsequently, the folate-targeted SPIO-PEG-PCL micelles were washed twice with phosphate-buffered saline (PBS) and incubated in fresh medium containing 20 µl MTT solution in PBS (5 mg/ml) at 37°C. After 4 h, the supernatant in each well was discarded and 150 ml dimethyl sulfoxide (Sigma-Aldrich; Merck KGaA) was added to each well to dissolve the formazen. Following gentle agitation of the plates for 10 min, the optical density of each well at a wavelength of 570 nm was recorded using an Infinite F200 multimode plate reader (Tecan, Mȁnnedorf, Switzerland). Three duplicates were measured for each experimental sample.

In vitro MRI

Tca-8113 cells (1×106 cells/well) were incubated at 37°C with folate-targeted or folate-free SPIO-PEG-PCL micelles at an Fe concentration of 80 µg/ml in folate-free MEM. Following incubation periods of 0.5, 1, and 2 h in a humidified atmosphere with 5% CO2 at 37°C, the cells were washed with PBS three times, trypsinized, mixed with a 2% agarose solution in PBS and scanned with a 3.0 T MRI scanner (Magnetom Avanto; version Syngo MR B17, Siemens Healthcare Sector, Erlangen, Germany) at room temperature. A spin echo (SE) T1-weighted sequence [repetition time (TR), 300 msec; echo time (TE), 17 msec], a fast spin-echo (FSE) T2-weighted sequence (T2WI; TR, 3,000 msec; TE, 105 msec), and T2 mapping (TR, 2,000 msec, TE, 30, 60, 90, and 120 msec, flip angle, 180°, total duration, 4 min 13 sec) were acquired with the following parameters: Head coil; axial scanning; slice thickness, 2.0 mm; slice spacing, 0.2 mm; field-of-view, 180×180 mm2; and matrix, 256×256. MapIT software (version, Syngo MR B17; Siemens Healthcare Sector) was applied to measure the T2 relaxation time at the work station, with a circular region of interest (ROI; area, 44.9 mm2). The shape and area of the ROI were consistent. Relative T2 relaxation time were calculated as follows (26): T2=[(T'-T0)/T0]x100%, where T' indicates T2 relaxation time obtained at different points during incubation and T0 represents the T2 relaxation time prior to incubation.

Prussian blue staining

Tca-8113 cells (5×105 cells/ml) were incubated for 1 h at 37°C with folate-targeted and folate-free SPIO-PEG-PCL micelles in folate-free RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc.) containing 80 µg/ml Fe (Gibco; Thermo Fisher Scientific, Inc.; 61870-036). The cells were washed three times with folate-free RPMI-1640 medium and trypsinized (10-5 mol/l). The cell suspensions were subsequently washed with PBS three times before being fixed with 4% glutaraldehyde. After 10 min, the cells were incubated at 37°C with 2 ml Prussian blue solution that was composed of an equal volume of 2% HCl acid aqueous solution and 2% potassium ferrocyanide (II) trihydrate. After 30 min, the cells were briefly stained with 0.5% neutral red for 3 min. The resulting staining was observed under a light microscope. The intensity of staining was defined by eye with the following scoring system: i) 0, no staining; ii) +, weak staining; iii) ++, moderate staining; and iv) +++, strong staining (27).

Statistical analysis

Statistical analyses were conducted with SPSS software (version 11.0; SPSS, Inc., Chicago, IL, USA). One-way analysis of variance and a Bonferroni post hoc test were used to identify differences between groups, and P<0.05 was considered to indicate a statistically significant difference.

Results

Micelle size and biocompatibility

Sizes of the folate-free SPIO-PEG-PCL and folate-targeted SPIO-PEG-PCL micelles were estimated to be 45.5 and 50.2 nm, respectively, according to the DLS measurement (Fig. 2). In the MTT viability assays of the Tca-8113 cells, various SPIO concentrations were evaluated. No statistically significant differences were identified between the folate-free SPIO-PEG-PCL and folate-targeted SPIO-PEG-PCL groups (F=0.698; P=0.676), thereby demonstrating that increased concentrations of either type of SPIO-PEG-PCL micelle did not increase cytotoxicity (Fig. 3).

Figure 2. Size distribution of folate-free SPIO-PEG-PCL and folate-targeted SPIO-PEG-PCL micelles as determined by dynamic light scattering measurements. SPIO, superparamagnetic iron oxide nanoparticles; PEG, polyethylene glycol; PCL, poly-caprolactone.

Figure 3. In vitro cytotoxicity of graded dilutions (1-, 2-, 4-, 8-, 16-, 32- and 64-fold) of folate-targeted SPIO-PEG-PCL and folate-free SPIO-PEG-PCL micelles towards Tca-8113 cells, as detected by a methyl thiazolyl tetrazolium assay. SPIO, superparamagnetic iron oxide nanoparticles; PEG, polyethylene glycol; PCL, poly-caprolactone.

In vitro MRI

To validate the tumor targeting ability of the nanoparticles generated, MRI signal intensity was measured for each set of cells that was incubated with folate-targeted SPIO-PEG-PCL and folate-free SPIO-PEG-PCL micelles, respectively. The T2WI signal intensity of the two sets of cells decreased marginally after 0.5 h (Fig. 4), and a time-dependent decrease in signal intensity was observed for the two groups. However, compared with the folate-targeted SPIO-PEG-PCL micelles, the decrease in signal intensity for the folate-free SPIO-PEG-PCL micelles was less marked (Fig. 4). Relative T2 relaxation time was calculated and, under the same conditions, the MRI signals of the cells incubated with the folate-targeted SPIO-PEG-PCL micelles exhibited a significantly greater weakening in signal compared with the folate-free SPIO-PEG-PCL micelles (P=0.002; Fig. 5).

Figure 4. In vitro MRI of the Tca-8113 cells that were incubated with folate-free SPIO-PEG-PCL and folate-targeted SPIO-PEG-PCL micelles for various incubation durations. The MRI signal intensities of the folate-targeted cells were stronger than those of the cells that were incubated with the folate-free micelles. SPIO, superparamagnetic iron oxide nanoparticles; PEG, polyethylene glycol; PCL, poly-caprolactone; MRI, magnetic resonance imaging.

Figure 5. Relative T2 relaxation times of the Tca-8113 cells that were incubated with the folate-targeted SPIO-PEG-PCL and folate-free SPIO-PEG-PCL micelles. For each incubation period, the MRI signals associated with the folate-targeted SPIO-PEG-PCL micelle treatment were weaker than those of the folate-free SPIO-PEG-PCL micelle treatment. For the whole period, the MRI signals of the cells incubated with the folate-targeted SPIO-PEG-PCL micelles exhibited a significantly greater weakening in signal when compared with the folate-free SPIO-PEG-PCL micelles (P=0.002). *P<0.05, **P=0.01 and ***P<0.001 vs. folate-free. SPIO, superparamagnetic iron oxide nanoparticles; PEG, polyethylene glycol; PCL, poly-caprolactone; MRI, magnetic resonance imaging.

Prussian blue staining

Entrapment of the SPIO in the nanomicelles allowed direct visualization of their uptake by Tca-8113 cells. As shown in Fig. 6, Prussian blue staining experiments demonstrated that the cells incubated with the folate-targeted SPIO-PEG-PCL micelles exhibited strong staining (+++), which was markedly stronger staining than the cells that were incubated with the folate-free SPIO-PEG-PCL micelles (moderate staining, ++).

Figure 6. Prussian blue staining of Tca-8113 cells that were incubated with (A) folate-targeted SPIO-PEG-PCL micelles vs. (B) folate-free SPIO-PEG-PCL micelles for 2 h (magnification, ×200). SPIO, superparamagnetic iron oxide nanoparticles; PEG, polyethylene glycol; PCL, poly-caprolactone.

Discussion

Advances in imaging technology and their application to cellular biology have brought molecular imaging to the forefront (28). Simultaneously, the identification of molecular targets has been applied to the development and design of various molecular imaging probes for solid tumors (29). As a diagnostic technique, MRI is currently one of the most powerful, non-invasive imaging modalities that is used in clinical medicine today (30), and SPIO represent one of the most effective MRI contrast agents. In the present study, polymeric micelles were generated for the delivery of SPIO, and these SPIO were attached to folate to achieve a targeted-micelle system. The folate receptor is overexpressed by a wide range of malignant cells, including tongue cancer cells. Therefore, the aim of the present study was to combine folate with SPIO-PEG-PCL copolymers to provide tumor-specific imaging following folate receptor-mediated endocytosis of these micelles. In the MRI experiments conducted, this folate-targeted micelle system effectively weakened the MRI signal of Tca-8113 cells. Prussian blue staining further confirmed the endocytosis of the targeted micelles by Tca-8113 cells. The folate-targeted SPIO-PEG-PCL demonstrated potential as a platform for diagnostic imaging applications.

It has been shown that tumor vasculature exhibits poor architecture due to the absence of a pericyte lining and a poor lymphatic drainage system (31). Correspondingly, this leaky vasculature leads to a stronger differential interstitial pressure at the center of tumors when compared with the tumor periphery, and this is referred to as the enhanced permeability and retention (EPR) effect (31,32). For particles ranging in size from 10–100 nm, the EPR effect is consistent with the preferential accumulation of particles in a tumor and extended blood circulation times (33,34). In the present study, DLS measurement demonstrated that the mean diameter for the folate-targeted PEG-PCL-SPIO micelle was ~50.2 nm. This diameter is suitable for efficient targeting in MRI applications, it is highly desirable for achieving a longer blood circulation time, and it facilitates the uptake of SPIO by cancer cells (35).

In the present study, MTT assays were performed to evaluate the possible cytotoxicity of the generated micelles and no obvious decrease in cell viability was observed despite the increasing concentrations of the polymers in these assays. Furthermore, when the micelles were incubated in the 1-fold dilution, the level of cytotoxicity detected was considered to be acceptable. Thus, copolymers of the folate-targeted SPIO-PEG-PCL exhibited minimal cytotoxicity towards the Tca-8113 cells examined.

In the MRI conducted in the present study, incubation of the Tca-8113 cells with the folate-targeted SPIO-PEG-PCL micelles resulted in a significant decrease in signal intensity and relative T2 relaxation time. Furthermore, these same decreases were not observed for the Tca-8113 cells that were incubated with the folate-free SPIO-PEG-PCL micelles. Consistent with these results, stronger Prussian blue staining was observed for the folate-targeted SPIO-PEG-PCL micelles versus the folate-free SPIO-PEG-PCL micelles, thereby indicating a higher intracellular Fe concentration in the former. Thus, cellular uptake of the micelles appeared to be dependent on the presence of the folate ligand. These results are consistent with those of Yang et al (23) and Hong et al (29), who demonstrated that hydrophobic SPIO nanocrystals that were loaded into folate-targeted SPIO-PEG-PCL micelles provided targeted delivery of an anticancer drug to hepatocellular carcinoma.

Therefore, these folate-targeted SPIO-PEG-PCL micelles demonstrated high sensitivity for MRI. This indicates that this type of micelle has the potential capacity to be an ideal MRI contrast agent for tongue tumors. Furthermore, the results of the Prussian blue staining in the present study revealed that folate-targeted micelles revealed a superior targeting effect on tongue cancer cells than the folate-free micelles. Thus, folate-targeted micelles have a greater potential as a drug delivery system to treat tongue tumors than folate-free micelles.

However, there were two main limitations associated with the present study. The examination of folate targeting was only performed in vitro and was not investigated in vivo. In addition, the delivery efficiency of the micelles was not evaluated using an anti-cancer drug. Thus, future studies will include monitoring of the transfer efficiency of SPIO and anti-cancer drugs to tongue cancers in vivo in order to validate the present findings and to confirm the potential for this method to provide early detection of tongue cancer.

In conclusion, compared with folate-free SPIO-PEG-PCL micelles, folate-targeted SPIO-PEG-PCL micelles exhibited superior targeting for Tca-8113 cells and achieved an ideal imaging effect in vitro MRI. This novel targeted micelle may be used as an effective contrast agent for the early diagnosis of tongue cancer.

Acknowledgements

The authors received grants from the Natural Science Foundation of Guangdong Province (grant no. 2014A030310484) and the S&T Programs of Guangdong Province (grant no. 2012B031800086).

References