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Introduction

A total of 90% of all cases of oral cancer are classified as squamous cell carcinoma (SCC), with SCC of the tongue comprising 50-60% cases worldwide, making it the most common type of oral cancer (1,2). Tongue cancer can metastasize from the primary site to cervical regional lymph nodes in the early stage (3), resulting in a 5-year survival rate of <50% (4,5). Therefore, the early diagnosis and treatment of cervical lymph node metastasis in patients with SCC of the tongue is crucial in order to improve patient survival rates.

The treatment of oral cancer typically includes surgery, post-operative adjuvant radiotherapy and chemotherapy (6). Pre-operative ultrasound is a non-invasive method used to examine lymph nodes; however, ultrasound cannot distinguish lymphadenitis from metastatic lymph nodes (7). Moreover, the current imaging strategies often result in a missed diagnosis or misdiagnosis of cancer, especially in patients with early metastatic lymphoma (8,9). Establishing reliable diagnostic procedures and standards for the primary foci of tongue cancer and the detection of metastatic SCC in lymph nodes has important potential diagnostic and prognostic value for patients with oral cancer.

Multimodal imaging has become a frequently used technique in molecular imaging as it can be used to obtain more accurate images and improve the detection rate of diseases, including thyroid cancer (10) and liver fibrosis (11). There has been an increased interest worldwide in nano-composite materials with multimodal imaging functions (12-14). Poly(lactic-co-glycolic acid)(PLGA) is commonly used in the preparation of ultrasound contrast agents. PLGA is a biodegradable organic polymer compound with hydrophobic properties, high spheronization, film formation rates and good biocompatibility (15,16). As a Food and Drug Administration-approved material, PLGA has been widely used in protein research and clinical applications for the delivery of small molecule drugs and other large molecules (17).

Liquid fluorocarbon perfluorohexane (PFH) is an aliphatic fluorine-containing compound with a low boiling point (56˚C) (6). PFH displays good biocompatibility and is eliminated directly via respiration without degradation in vivo (18). Furthermore, the diameter of a nanoparticle contrast agent coated with liquid fluorocarbon can be increased following liquid-gas phase change, which results in increased acoustic impedance followed by increased enhanced ultrasonic signal development (19).

Superparamagnetic iron oxide (SPIO) nanoparticles can be coated with a variety of biological macromolecules due to their small particle size and strong surface modifiability (20). Additionally, SPIO nanoparticles display good biological safety and a long circulation half-life (21). SPIOs integrate magnetic resonance (MR), ultrasonic and photoacoustic (PA) multimodal imaging, providing novel ideas and strategies for disease diagnosis and treatment (22-26). Norton et al (27) demonstrated that magnetic SPIO particles vibrate under the action of acoustic waves, which alters the acoustic impedance of tissues and enhances backscatter signals and ultrasound imaging. A previous study reported that PA imaging technology based on the PA effect has received increasing interest (28). PA imaging technology is a safe and nondestructive imaging technology that uses a pulsed laser as the excitation source to irradiate the medium with a light absorber to generate a PA effect. Moreover, the imaging method reconstructs and calculates the collected acoustic signals via a signal processing system to obtain information regarding the internal structure of tissues (29).

PA imaging technology combines the advantages of pure optical and acoustic imaging of tissues, thus obtaining tissue images with high contrast and spatial resolution (30). As a light absorber, SPIO can absorb laser light at specific wavelengths (31). Laser irradiation of SPIO-coated particles causes the temperature of the surrounding medium to rise, causing the medium to expand, generating PA signals (32).

The specific receptor for stromal cell-derived factor (SDF)1 is CXC chemokine receptor 4 (CXCR4), which is expressed by fibroblasts, endothelial cells, some inflammatory cells (natural killer cells, neutrophils, plasma cells and B and T lymphocytes) and various cancer cells (33). Delilbasi et al (34) demonstrated that all tongue SCC cells expressed CXCR4 whereas normal squamous epithelium had no or little expression of CXCR4 and that metastatic SCC cells from the lymph nodes displayed stronger expression compared with lymphocytes. At present, CXCR4 is the only known receptor of SDF-1(6).

The CXCR4-C-X-C motif chemokine (CXCL)12 axis serves an important role in the invasion and metastasis of SCC and has been indicated to contribute to tumor development. Due to this (35), it is often used as a potential target in SCC treatment (36).

The aim of the present study was to prepare SDF-1-modified nanoparticle contrast agents coated with iron (II,III) oxide (Fe3O4), the main component of SPIO (21) and PFH for targeted multi-modal imaging and to examine their properties in vitro. The present study provided a potential foundation for the development of procedures and materials for the diagnosis of primary tongue cancer and lymph node metastasis.

Materials and methods

Materials

Fe3O4 (cat. no. SOR-10-50) modified by oleic acid was obtained from Ocean NanoTech, LLC. Maleimide-polyethylene glycol-poly(lactide co-glycolide)(MAL-PEG-PLGA; 75:25; 12,000 Da MW) was purchased from Jinan Daigang Biomaterial Co., Ltd. Polyvinyl alcohol (PVA; 25,000 Da) and PFH were obtained from Sigma-Aldrich, Merck KGaA. Methylene chloride (CH2Cl2) and isopropyl alcohol were purchased from Chongqing East Sichuan Chemical Co., Ltd. SDF-1 modified with FITC was obtained from Shanghai Qiangyao Biotechnology Co., Ltd. The human tongue SCC cell line (SCC-15) was obtained from the Chongqing Key Laboratory of Oral Diseases and Biomedical Sciences.

Preparation of SDF-1/Fe3O4/PLGA/PFH nanoparticles

A double emulsification method was used to prepare PLGA shells loaded with Fe3O4 and PFH. A total of 100 mg PLGA and 5 mg Fe3O4 were added into 2 ml CH2Cl2 and the mixture was placed in an ice bath with an ultrasound probe at 125 W for 60 sec until it was fully dissolved. Subsequently, 100 µl PFH and 5 ml of 50 g/l PVA were added to the emulsion, followed by ultrasound treatment for 5 min (on: 5 sec; off: 5 sec) in an ice bath to obtain the first emulsion. A total of 10 ml isopropyl alcohol (20 ml/l) was then poured into the first emulsion to solidify the surface of the nanoparticles. A total of 2 magnetic beads (Beyotime Institute of Biotechnology,) were placed into the mixture and the liquid was stirred on a magnetic stirrer for 3 h in an ice bath to extract CH2Cl2. The sample was then centrifuged with pure water in a low-temperature (4˚C) high-speed centrifuge (5 min; 9,391 x g) and the supernatant was discarded. The precipitate was washed with pure water and the process was repeated in triplicate. A total of 200 µl SDF-1 (terminal sulfhydrylation modification and FITC labeling) was added into the final emulsion and stirred for 4 h at 4˚C. SDF-1 was conjugated to nanoparticles via thioether bonds. Particles were then centrifuged at 9,391 x g for 5 min at 4˚C and washed with pure water. The process was repeated twice to obtain SDF-1/Fe3O4/PLGA/PFH nanoparticles, which were stored in the dark at 4˚C until further analysis.

The blank PLGA nanoparticles, Fe3O4/PLGA/PFH nanoparticles and SDF-1/PLGA/PFH nanoparticles were all prepared according to the aforementioned method.

Determination of the basic properties of the SDF-1/Fe3O4/PLGA/PFH nanoparticles

Samples were gently stirred with a pipette until they were fully dispersed and assessed for surface morphology, distribution and size using a CKX41 light microscope (magnification, x100); Olympus Corporation) and a JEOL JSM-7800F scanning electron microscope (magnification, x2,000; Hitachi, Ltd.). A drop of the nanoparticles (50 ug/ml) was placed on a silicon panel (5x5 mm) until it dried naturally in the dark overnight at room temperature. The silicon panel then analyzed via scanning electron microscopy. A TCS-SP5 laser confocal microscopy (magnification, x400; Leica Microsystems GmbH) was used to observe SDF-1 conjugation. A laser particle size meter (Zetasizer 3000HS; Malvern Instruments, Inc.) was used to detect particle size and Zeta potential. Determination of the iron content in the samples (3) was performed via graphite furnace atomic absorption spectrometry. The wavelength used for detection was 248.3 nm. The absorption spectrum of the nanoparticles was detected using an ultraviolet-visible (UV-Vis) spectrophotometer (CARY 50; Varian Medical Systems) and blank PLGA nanoparticles were used as the control group.

In vitro imaging experiments

The absorption peaks of nanoparticles were measured by UV-Vis spectrophotometry, which was performed in previous experiments (21). The absorption peaks provided the basis for selecting the excitation laser wavelength for PA imaging experiments. The SDF-1/Fe3O4/PLGA/PFH, Fe3O4/PLGA/PFH, SDF-1/PLGA/PFH and blank PLGA nanoparticles were divided into 4 groups according to PLGA concentration (20, 15, 10 and 5 mg/ml). A total of 200 µl of mixture from each group was placed in 3% agarose gel phantoms and the PA images and signals values of the groups were collected at the optimal wavelength (680 nm) using a VEVO LAZR PA imaging system (Vevo® LAZR; FUJIFILM VisualSonics, Inc.).

Ultrasonic imaging experiment

Ultrasound images of SDF-1/Fe3O4/PLGA/PFH nanoparticles were recorded according to 4 different PLGA concentrations (100, 50, 25 and 12.5 mg/ml) at a mechanical index (MI) of 0.6 and under different MIs (0.2-0.6) at the PLGA concentration of 12.5 mg/ml using an ultrasound apparatus (Philips iU22; Philips Healthcare). A total of 1 ml SDF-1/Fe3O4/PLGA/PFH nanoparticles were injected into a transparent rubber tube and placed into a water bath with an initial temperature of 37˚C for 6 min to obtain the images. During the process, the temperature was slowly raised (to 65˚C) and monitored in real time. Using the B-mode and contrast mode of the ultrasound apparatus, images of SDF-1/Fe3O4/PLGA/PFH nanoparticles were obtained at the temperature of 56˚C when the two-dimensional grayscale ultrasound imaging and contrast-enhanced ultrasound imaging were strongest. The blank PLGA nanoparticles were treated using the same method. A DFY ultrasonic image quantitative analysis diagnostic instrument (Sonomath; Chongqing Ambition Science & Technologies Co., Ltd.) measured the echo intensity (EI) value of the samples. Additionally, alterations to SDF-1/Fe3O4/PLGA/PFH nanoparticle volume in response to increasing temperature were observed and recorded via an IX71 light microscope (magnification, x40; Olympus Corporation).

Cell culture

SCC-15 cells were cultured in DMEM/Nutrient Mixture F-12 (DMEM-12; Gibco; Thermo Fisher Scientific, Inc.) containing 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin or streptomycin at 37˚C with 5% CO2. The culture medium was changed every 2 days. At 80% confluence, cells were sub-cultured as follows: The culture medium was removed, cells were washed with PBS 3 times, 1 ml 0.25% trypsin was added and cells were returned to the incubator for 5 min. A total 3 ml DMEM-12 culture medium was added to the cells for neutralization. The mixture was pipetted up and down to form a well-mixed suspension and centrifuged for 3 min at 200 x g and 4˚C. The supernatant was removed and cells were resuspended in DMEM-12 medium.

Detection of CXCR4 localization and expression of SCC-15 cells

SCC-15 cells in the logarithmic phase were digested with 1 ml 0.25% trypsin for 5 min and then centrifuged at 200 x g for 3 min at 4˚C. Subsequently, cells were inoculated (5x104 cells/well) into 6-well plates with coverslips. Cells were cultured in DMEM-12 medium and incubated overnight with 5% CO2 at 37˚C. Cells were washed twice with PBS, fixed with 4% paraformaldehyde for 15 min at room temperature, washed with PBS 3 times and blocked with goat serum blocking solution (1% BSA; Beyotime Institute of Biotechnology) at 37˚C for 30 min to block non-specific antibody binding. Subsequently, rabbit anti-human CXCR4 primary antibody (1:200) was added to the cover glass, which was incubated at 4˚C overnight. After washing 3 times with PBS, FITC-labelled goat anti-rabbit IgG secondary antibody (1:100; A0562; Beyotime Institute of Biotechnology) was added to the cells and incubated at 37˚C for 50 min in the dark. After rinsing 3 times with PBS, cells were stained with DAPI for 5 min at room temperature, washed 3 times with PBS and stored at -20˚C until observation. Cells were observed under a TCS-SP5 laser confocal microscopy (magnification, x200; Leica Microsystems GmbH).

Targeted experiment

SCC-15 cells in the logarithmic phase were inoculated (1x105 cells/dish) into a 15 mm laser confocal dish. Cells were divided into 2 groups: i) SDF-1/Fe3O4/PLGA/PFH nanoparticle group; and ii) the blank control group. A total of 2 ml DMEM-12 medium was added to each dish. Following incubation for 12 h, the medium in the dish was changed. A total of 2 ml nanoparticles prepared in DMEM-12 (200 µg/ml) was added to the experimental group and 2 ml DMEM/F-12 medium was added to the blank control group. Following incubation for 2 h at 37˚C, cells were washed once with PBS and nanoparticle targeting was observed using a fully automatic fluorescence microscope (magnification, x200; Axio Imager.Z2; Carl Zeiss AG).

Flow cytometry test

SCC-15 cells in the logarithmic phase were inoculated (1x106 cells/well) into 6-well plates. Cells were divided into 2 groups: i) SDF-1/Fe3O4/PLGA/PFH nanoparticle group; and ii) the blank control group. A total of 2 ml DMEM-12 medium was added to each dish. Following incubation for 12 h at 37˚C, the medium in the dish was changed. A total of 2 ml nanoparticles prepared in DMEM/F-12 medium (200 µg/ml) was added to the experimental group and 2 ml DMEM/F-12 medium was added to the blank control group. Following incubation for 2 h at 37˚C, cells were washed 3 times with PBS, digested with 0.25% pancreatin for 5 min, centrifuged 3 times at 200 x g for 3 min at 4˚C and the obtained precipitate was resuspended in PBS for flow cytometry (BD INFLUX; Becton, Dickinson and Company). Nanoparticles with SDF-1 were selected as the analyte detector and FITC was used as the analyte reporter. FlowJo software (version 7.6.5; Becton, Dickinson and Company) was used for the analysis of flow cytometer data.

Statistical analysis

Statistical analyses were performed using SPSS software (version 22.0; IBM Corp.). Data are presented as the mean ± standard deviation. Experiments were performed in triplicate. One-way ANOVA followed by Tukey's post hoc test was used to determine significant differences between multiple groups. The Student's t-test was used to analyze differences between two groups The difference between PA signal values of SDF-1/Fe3O4/PLGA/PFH and Fe3O4/PLGA/PFH nanoparticles was analyzed with unpaired Student's t-test. Difference in EI values of SDF-1/Fe3O4/PLGA/PFH nanoparticles before and after the water bath was analyzed using paired Student's t-test. P<0.05 was considered to indicate a statistically significant difference.

Results

Basic properties of SDF-1/Fe3O4/PLGA/PFH nanoparticles

The nanoparticle solution was dark brown in appearance (data not shown). Under a light or scanning electron microscope, the prepared SDF-1/Fe3O4/PLGA/PFH nanoparticles were well dispersed when diluted with pure water, spherical in shape and uniform in size (Fig. 1A and B). Scanning electron microscopy demonstrated that the surfaces of the nanoparticles were incomplete and smooth with numerous pores (Fig. 1B). Laser confocal microscopy indicated that SDF-1 was attached to the PLGA shell of nanoparticles alongside bright green fluorescence on the surface (Fig. 1C). The average size of the nanoparticles, as measured using a Malvern particle size meter, was 586.5±124.7 nm (Fig. 1D) and the average zeta potential was-21.5±2.17 mV. Atomic absorption spectrometry measurements indicated that there was 35.74 µg/ml of iron in the sample (data not shown). UV-Vis spectrophotometry detected 2 slight absorption peaks near 556 and 623 nm for the SDF-1/Fe3O4/PLGA/PFH nanoparticles (Fig. 1F), whereas the control group displayed no obvious absorption peaks in detection wavelength bands (Fig. 1E).

Figure 1 Basic properties of SDF-1/Fe3O4/PLGA/PFH nanoparticles. SDF-1/Fe3O4/PLGA/PFH nanoparticles were visualized by (A) light, (B) scanning electron and (C) laser confocal microscopy. (D) SDF-1/Fe3O4/PLGA/PFH nanoparticle size distribution. (E) Absorption spectra of blank PLGA nanoparticles in the blank control group. (F) Absorption spectra of SDF-1/Fe3O4/PLGA/PFH nanoparticles. SDF-1, stromal cell-derived factor 1; Fe3O4, iron (II,III) oxide; PLGA, poly(lactic-co-glycolic acid); PFH, fluorocarbon perfluorohexane.

PA imaging experiment

UV-V is spectrophotometry was used to detect absorption peaks for the SDF-1/Fe3O4/PLGA/PFH nanoparticles at 556 and 623 nm in the wavelength range of 300-840 nm. However, the excitation laser wavelength range of the PA instrument used was 680-960 nm and the absorption peak of the nanoparticles was not within this range. According to its absorption spectrum, 680 nm was selected as the optimal excitation wavelength for the PA imaging experiment. The PA images of 4 groups and the mean PA value of SDF-1/Fe3O4/PLGA/PFH and Fe3O4/PLGA/PFH groups are presented in Figs. 2 and 3 respectively. The SDF-1/Fe3O4/PLGA/PFH and Fe3O4/PLGA/PFH nanoparticles displayed obvious PA signals, whereas there were no obvious PA signals from SDF-1/PLGA/PFH or blank PLGA nanoparticles. Furthermore, the PA images of SDF-1/Fe3O4/PLGA/PFH and Fe3O4/PLGA/PFH nanoparticles were enhanced compared with SDF-1/PLGA/PFH and blank PLGA nanoparticles (Fig. 2) and the average PA signal values of each group significantly increased (Fig. 3) with increasing PLGA concentration. Therefore, the results indicated that Fe3O4 displayed excellent PA imaging capability and 20 mg/ml nanoparticles emitted the strongest PA signals in the set of nanoparticles.

Figure 2 Photoacoustic images of the different groups of nanoparticles at different PLGA concentrations. (A) SDF-1/Fe3O4/PLGA/PFH, (B) Fe3O4/PLGA/PFH, (C) SDF-1/PLGA/PFH and (D) blank PLGA nanoparticles. PLGA, poly(lactic-co-glycolic acid); SDF-1, stromal cell-derived factor 1; Fe3O4, iron (II,III) oxide; PFH, fluorocarbon perfluorohexane.

Figure 3 Mean PA signal values of SDF-1/Fe3O4/PLGA/PFH and Fe3O4/PLGA/PFH nanoparticles. *P<0.05, as indicated. PA, photoacoustic; SDF-1, stromal cell-derived factor 1; Fe3O4, iron (II,III) oxide; PLGA, poly(lactic-co-glycolic acid); PFH, fluorocarbon perfluorohexane.

Ultrasonic imaging experiment

The SDF-1/Fe3O4/PLGA/PFH nanoparticles were divided into 4 groups according to PLGA concentration (100, 50, 25 and 12.5 mg/ml) and PBS solution was used as a control group. Samples of the different groups were embedded into an agarose gel model. The ultrasonic mode of the ultrasonic diagnostic instrument and an MI value of 0.6 were used to investigate the imaging conditions of PBS and the different concentrations of nanoparticles (Fig. 4A-E). Furthermore, ultrasound images for 12.5 mg/ml PLGA in nanoparticles with a decreased MI value (0.6 to 0.2) were obtained (Fig. 4E-I). and the corresponding EI values were measured by DFY quantifier. The results demonstrated that when the MI value was constant, the ultrasonic signal displayed a decreasing trend in response to a decrease in nanoparticle concentration (Table I; P<0.05). The PBS solution emitted no obvious ultrasonic signal. Furthermore, when the concentration of nanoparticles was constant, the ultrasonic signal value decreased in response to a decrease in MI (EI values were measured in triplicate for each MI value; Table II; P<0.05). In summary, the ultrasound imaging performance for SDF-1/Fe3O4/PLGA/PFH nanoparticles was satisfactory and the EI value was highest when the MI value was 0.6 and the concentration of PLGA was 100 mg/ml.

Figure 4 Ultrasonic imaging of SDF-1/Fe3O4/PLGA/PFH nanoparticles at different PLGA concentrations and MI values. Images of (A) PBS and SDF-1/Fe3O4/PLGA/PFH nanoparticles at PLGA concentrations of (B) 100, (C) 50, (D) 25 and (E) 12.5 mg/ml with a MI of 0.6. Images of SDF-1/Fe3O4/PLGA/PFH nanoparticles at MI value of (E) 0.6, (F) 0.5, (G) 0.4, (H) 0.3 and (I) 0.2 at a PLGA concentration of 12.5 mg/ml. Arrows indicate that the ultrasound images are shown within the circles. SDF-1, stromal cell-derived factor 1; Fe3O4, iron (II,III) oxide; PLGA, poly(lactic-co-glycolic acid); PFH, fluorocarbon perfluorohexane; MI, mechanical index.

Table I EI values of different PLGA concentrations at a mechanical index of 0.6.

Table I

EI values of different PLGA concentrations at a mechanical index of 0.6.

PLGA concentration (mg/ml)	EI
100a-c	163.8±3.42
50a,b	137.77±1.25
25a	116.2±2.98
12.5	99.17±2.76

[i] A decrease in PLGA concentration resulted in significantly decreased EI values in each group.

[ii] ^aP<0.05 vs. 12.5 mg/ml;

[iii] ^bP<0.05 vs. 25 mg/ml;

[iv] ^cP<0.05 vs. 50 mg/ml. EI, echo density; PLGA, poly(lactic-co-glycolic acid).

Table II EI values of different MIs at a concentration of 12.5 mg/ml PLGA.

Table II

EI values of different MIs at a concentration of 12.5 mg/ml PLGA.

MI	EI
0.6a-d	99.17±2.76
0.5a-c	78.96±3.15
0.4a,b	66.25±2.36
0.3a	54.59±2.42
0.2	43.73±2.59

[i] A decrease in MI resulted in significantly decreased EI values in each group.

[ii] ^aP<0.05 vs. 0.2 MI;

[iii] ^bP<0.05 vs. 0.3 MI;

[iv] ^cP<0.05 vs. 0.4 MI;

[v] ^dP<0.05 vs. 0.5 MI. EI, echo density; MI, mechanical index; PLGA, poly(lactic-co-glycolic acid).

In vitro phase change experiment

The temperature of the water bath used during the ultrasonic imaging experiment was slowly increased from 37˚C. Images of SDF-1/Fe3O4/PLGA/PFH nanoparticles and blank controls were taken before the water bath, when the contrast-enhanced ultrasound imaging was strongest and collected using the contrast mode of the ultrasound diagnostic instrument (Fig. 5). The controls did not display enhanced images; however, the experimental group displayed clear two-dimensional grayscale ultrasound images and contrast-enhanced ultrasound image signals during the heating process. The degree of two-dimensional grayscale ultrasound imaging and contrast-enhanced ultrasound imaging in the experimental group before and after heating, as detected by a DFY quantitative instrument, was significantly increased (grayscale values were measured in triplicate for each image; Table III; P<0.05).

Figure 5 Two-dimensional grayscale ultrasound imaging and contrast-enhanced ultrasound imaging of SDF-1/Fe3O4/PLGA/PFH nanoparticles and controls. (A) Blank PLGA and (B) SDF-1/Fe3O4/PLGA/PFH nanoparticles. Two-dimensional grayscale ultrasound images are displayed within the white dotted frame (as indicated by the white arrow). Contrast-enhanced ultrasound images are presented within the red dotted frame (as indicated by the red arrow). SDF-1, stromal cell-derived factor 1; Fe3O4, iron (II,III) oxide; PLGA, poly(lactic-co-glycolic acid); PFH, fluorocarbon perfluorohexane.

Table III Grayscale values before and after the water bath in the experimental group.

Table III

Grayscale values before and after the water bath in the experimental group.

Temperature	Two-dimensional grayscale ultrasound imaging	Contrast-enhanced ultrasound imaging
Before (37˚C)	68±1.73	32±1.67
After (56˚C)	152±2.75a	101±3.09a

[i] ^aP<0.05 vs. before.

Using an optical microscope, it was observed that nanoparticles were spherical and well dispersed prior to heating (Fig. 6A). Following 1 min in the hot water bath, certain nanoparticles underwent phase transformation and generated microbubbles (Fig. 6B). When the temperature was increased to 56˚C, large amounts of microbubbles with increased volume were observed and the bubbles gradually converged (Fig. 6C). As the temperature continued to increase to 65˚C, the microbubbles gradually burst and disappeared (data not shown).

Figure 6 Images of SDF-1/Fe3O4/PLGA/PFH nanoparticles before and after water bath treatment. (A) Nanoparticles before the water bath. (B) Following 1 min in the water bath, certain nanoparticles underwent phase transformation and generated microbubbles. (C) When the temperature of the water bath reached ~56˚C, a large amount of microbubbles converged. SDF-1, stromal cell-derived factor 1; Fe3O4, iron (II,III) oxide; PLGA, poly(lactic-co-glycolic acid); PFH, fluorocarbon perfluorohexane.

Detection of CXCR4 localization and expression in SCC-15 cells

In Fig. 7, blue indicated the nuclei of SCC-15 cells re-stained with DAPI and green indicated CXCR4 labeled with FITC (as indicated by arrows). The target protein was expressed in the cell membrane and cytoplasm, which indicated that CXCR4 was an ideal membrane target protein.

Figure 7 Immunofluorescence assay of SCC-15 cells. (A) The nuclei of SCC-15 cells were stained with DAPI, as indicated by the white arrow. (B) CXC chemokine receptor 4 was stained with FITC, as indicated by the white arrow. (C) Merged image.

Targeted experiment with SCC-15 cells

SCC-15 cells were observed under a light microscope and were found to exhibit a tadpole-shaped morphology and were evenly distributed (Fig. 8A). Following SDF-1/Fe3O4/PLGA/PFH nanoparticle addition and incubation for 2 h, SCC-15 cells were observed under a light microscope. A large number of nanoparticles adhered around SCC-15 cell membranes and to the cytoplasm (Fig. 8B). A fully-automatic fluorescence pattern of Fig. 8B from the same view was generated and bright green fluorescence was observed in and around the SCC-15 cells (as indicated by the arrows; Fig. 8C). The results demonstrated that SDF-1/Fe3O4/PLGA/PFH nanoparticles specifically bound to CXCR4 in SCC-15 cells.

Figure 8 Targeted experiments with SCC-15 cells and SDF-1/Fe3O4/PLGA/PFH. (A) SCC-15 cells without nanoparticles. (B) Light microscope image of SCC-15 cells with nanoparticles. (C) Fluorescence microscope image of SCC-15 cells with nanoparticles. SDF-1, stromal cell-derived factor 1; Fe3O4, iron (II,III) oxide; PLGA, poly(lactic-co-glycolic acid); PFH, fluorocarbon perfluorohexane. The SDF-1/Fe3O4/PLGA/PFH nanoparticles bind specifically to SCC-15 cells, as indicated by the white arrows.

Flow cytometry test

The rate of binding of the targeted nanoparticle group to SCC-15 cells was 84.44%, while that of the controls was only 1.1%. The results indicated that the nanoparticles possessed strong targeted binding ability to SCC-15 cells (Fig. 9).

Figure 9 Flow cytometric analysis of SDF-1/Fe3O4/PLGA/PFH nanoparticles. Blank control nanoparticles and SDF-1/Fe3O4/PLGA/PFH nanoparticles. SDF-1, stromal cell-derived factor 1; Fe3O4, iron (II,III) oxide; PLGA, poly(lactic-co-glycolic acid); PFH, fluorocarbon perfluorohexane.

Discussion

In the present study, a double emulsification method was used to prepare multifunctional, targeted polymeric nanoparticles using PLGA as the carrier. PLGA contains rich functional groups that can promote functionalization of PLGA-based nanoparticles and biomolecules, and can improve their stability and functionality (17). PFH and Fe3O4 were used concurrently as a coating and the surface of the PGLA shell was linked to FITC-labeled SDF-1 via thioether bonds, thus forming a nanoparticle with multiple functions.

The physical characteristics of the particles were tested using various methods. The solution of the nanoparticles was observed to be dark brown to the naked eye and the nanoparticles appeared round and spherical with numerous surface pores when observed via scanning electron microscopy. The particle size was measured to be 586.48±124.70 nm. It has been reported that the microvascular endothelial gap in SCC is 300-780 nm (30); therefore, the nanoparticles could potentially pass through the microvascular endothelial gap and be deposited into the target tissue via the CXCR4-CXCL12 axis. The therapeutic application of the nanoparticles may aid in locating tumors and metastatic lymphoma more accurately and blocking further metastasis via these channels. Contrary to the structure of tumor tissue, the vascular endothelium of normal tissue is tightly connected and extravasation of nanoparticles is not possible; however, tumors display enhanced vascular permeability and poor lymph drainage, which facilitates the retention of nanoparticles that extravasate into tumor tissue for a prolonged time (37).

Multi-modal imaging has served an increasingly important role in medical diagnosis and increased focused research on numerous multi-modality contrast agents with multiple imaging functions has been conducted (38). With the rapid development of medical imaging technology, ultrasound, computed tomography (CT), X-rays and MR imaging have been widely used. Imaging technology can be used for molecular and functional imaging of target organs and tissues to obtain pathological information, thus aiding in the early diagnosis of diseases (39). MR is frequently used to diagnose breast cancer (40) and CT is often used for localized brain tumors, head and neck cancer, and multiple myeloma (41). However, the imaging techniques currently used in clinical practice have limitations, including missed diagnosis, misdiagnosis and they are not specific to or target lesions (8).

PA imaging is a novel biomedical imaging mode that combines the advantages of optics and acoustics to improve the spatial resolution of traditional ultrasound (30). Combining data from two or more imaging techniques can improve diagnosis accuracy (42).

Fe3O4, a photosensitive material, absorbs laser light of a specific wavelength and causes the ambient temperature to rise (31). The thermal expansion of the surrounding objects produces a PA effect and the signal device generates PA signals after receiving sound waves (32). Simultaneously, Fe3O4 magnetic particles can vibrate in response to acoustic waves, altering the acoustic impedance of surrounding tissues and enhancing ultrasonic development (43). Additionally, numerous experimental studies have demonstrated that Fe3O4 improves the transverse relaxation rate of MR and improves MR imaging capability (44-46). Fe3O4 can integrate enhanced PA ultrasound and MR imaging, is an ideal molecular imaging material and is biodegradable and safe (21). After entering the body, Fe3O4 is metabolized by red blood cells and enters the normal plasma iron pool to participate in the synthesis of hemoglobin, myoglobin and cytochrome oxidase, or participate in energy metabolism and hematopoietic functions (47). In the in vitro imaging experiments performed in the present study, the Fe3O4-coated nanoparticles displayed PA and ultrasound imaging capabilities, which indicated that they may serve as a multi-modal contrast agent with potential clinical use for lesion visualization.

PFH is liquid at normal temperature, but when the temperature rises to its boiling point (56˚C) or above, or the external pressure decreases to its gasification pressure threshold, a liquid-gas phase change occurs (6). When PFH enters the gas phase, the increase of microsphere volume results in the increase of acoustic impedance, then ultrasonic development is enhanced (48). Commonly used methods to promote liquid-gas phase transition of liquid fluorocarbon include temperature-induced phase change (49), acoustic droplet vaporization (50,51) and optical droplet vaporization (32). In the present study, a water bath heating method was used to induce PFH liquid-gas phase change. When the temperature rose to ~56˚C, numerous phase change microbubbles were observed using a microscope. Additionally, the EI values of two-dimensional grayscale and contrast-enhanced ultrasound images were significantly increased compared with before heating in the water bat (37˚C). When the temperature rose to 65˚C, the microbubbles burst and disappeared, weakening the image. As the phase change temperature of PFH is higher than the body temperature of the human body, an external water bath heating was used to promote the phase change; however, this would be difficult to implement in the human body. Fe3O4, a photosensitive material, can undergo photo-thermal conversion under laser radiation to increase the temperature of the surrounding medium, which may serve as a potential mechanism to promote PFH phase change in the body (52). Moreover, a laser has stability and directionality, and can treat tumors without damaging the surrounding normal tissues via the principle of thermal ablation, thus providing a direction for future in vivo experiments (6).

SCC of the tongue accounts for the majority of oral SCC cases and its high lymph node metastasis rate results in the low 5-year survival rate (<30%) of patients (53). Several studies have reported that CXCR4 is widely and highly expressed in oral SCC cells, including SCC cells of the tongue (30,54,55). Additionally, it has been demonstrated that the CXCR4-CXCL12 axis serves an important role in the proliferation, invasion, immune evasion and metastasis of several types of cancer, including oral SCC (56,57). The interaction between CXCR12 and its receptor stimulates downstream signaling pathways to affect tumor angiogenesis, tumor cell proliferation and chemical resistance, which suggest that CXCR12 may serve as a potential target for cancer therapy (58). More importantly, when SDF-1 on targeted nanoparticles combines with CXCR4, it can antagonize the tumor-promoting effect caused by CXCR4-SDF-1(36). The active targeting effect of SDF-1 allows nanoparticles to remain in tumor tissues for a prolonged time and allows the rapid release of drugs using laser local fixed-point radiation, which would increase local drug concentrations and achieve a chemotherapy effect while reducing systemic adverse reactions; therefore, the aforementioned strategy may be used for targeted treatment with long-term chemotherapy drugs (6). Cellular immunofluorescence experiments demonstrated that CXCR4 is expressed in the cell membrane and cytoplasm of SCC-15 cells and is an ideal membrane protein target. In the present study, SDF-1 was attached to a PLGA shell by a thioether bond and SDF-1/Fe3O4/PLGA/PFH nanoparticles were successfully generated. SCC-15 targeting experiments indicated that SDF-1/Fe3O4/PLGA/PFH nanoparticles specifically combined with CXCR4 in SCC-15 cells and bright green fluorescence was microscopically observed in and around the cells. Flow cytometry assays indicated that the generated nanoparticles displayed a high targeting ability. However, whether the expected target effect can be achieved in vivo requires further investigation.

In the present study, a targeted nanoparticle contrast agent with a PA/ultrasonic bimodal imaging function was successfully generated. Basic physical characteristics of the nanoparticles were studied in vitro and it displayed PA and ultrasonic imaging capabilities. The nanoparticles underwent in vitro phase change under certain conditions to enhance ultrasonic imaging and specific adherence to tongue SCCs in vitro. The present work provided support for establishing procedures and standards to be used for the diagnosis of primary foci of tongue carcinoma and metastatic SCC of the lymph nodes and established a foundation for subsequent in vivo experiments.

Acknowledgements

Not applicable.

Funding

The present study was funded by the Chongqing Social Livelihood Science and Technology Innovation Project (grant no. cstc2016shmszx00010), the Science and Technology Research Project of Chongqing Education Commission (grant no. KJ1600231) and the Program for Innovation Team Building at Institutions of Higher Education in Chongqing (grant no. CXTDG201602006).

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Authors' contributions

FW performed the majority of the experiments and wrote the manuscript. ZW, LP and SW analyzed and interpreted data. LQ designed and supervised the study. All authors read and approved the final manuscript.

Ethics approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References