MAGE-Targeted Gold Nanoparticles for Ultrasound Imaging-Guided Phototherapy in Melanoma

Gold nanorods exhibit a wide variety of applications such as tumor molecular imaging and photothermal therapy (PTT) due to their tunable optical properties. Several studies have demonstrated that the combination of other therapeutic strategies may improve PTT efficiency. A method called optical droplet vaporization (ODV) was considered as another noninvasive imaging and therapy strategy. Via the ODV method, superheated perfluorocarbon droplets can be vaporized to a gas phase for enhancing ultrasound imaging; meanwhile, this violent process can cause damage to cells and tissue. In addition, active targeting through the functionalization with targeting ligands can effectively increase nanoprobe accumulation in the tumor area, improving the sensitivity and specificity of imaging and therapy. Our study prepared a nanoparticle loaded with gold nanorods and perfluorinated hexane and conjugated to a monoclonal antibody (MAGE-1 antibody) to melanoma-associated antigens (MAGE) targeting melanoma, investigated the synergistic effect of PTT/ODV therapy, and monitored the therapeutic effect using ultrasound. The prepared MAGE-Au-PFH-NPs achieved complete eradication of tumors. Meanwhile, the MAGE-Au-PFH-NPs also possess significant ultrasound imaging signal enhancement, which shows the potential for imaging-guided tumor therapy in the future.


Introduction
Melanoma is a malignant neoplasm sourced from melanocytes skin cells-with poor prognosis at advanced stages. Standard cancer treatments can be highly toxic to healthy tissues without differentiating malignant from normal cells, causing significant adverse effects in patients. Nanoparticlebased photothermal ablation therapy assisted by nearinfrared (NIR) laser holds a promise to eliminate tumors noninvasively, reduce tumor resistance, and prevent recurrence [1][2][3]. In addition, active targeting ability through functionalization with specific ligands can effectively enable nanoprobes to accumulate in the tumor area [2]. Melanomaassociated antigens (MAGE) are a specific and highly expressed family of antigens in malignant melanoma [4][5][6].
Therefore, MAGE proteins could also be used to functionalize nanoprobes for molecular imaging and accurate treatment of melanoma.
Among the available nanoparticle systems, gold nanorods (GNRs/Au-NRs) have attracted particular attention in cancer imaging and photothermal therapy [7][8][9] due to several advantages: biocompatibility, high photothermal conversion efficiency, well-established methods for synthesis in a wide range of sizes, and ease of biomodification [10]. However, complete tumor eradication is so far difficult to achieve with the introduction of these photothermal nanomaterials. In particular, their low tissue bioabsorption and utilization result in limited curative effect in deeply located tumors [11]. Recently, it has been reported that PTT efficiency may be improved with the combination of other therapeutic strategies [12][13][14]. Phase-changeable liquid fluorocarbon emulsions can be vaporized and transformed from droplets to microbubbles under optical irradiation (ODV) [15][16][17][18] which has become an attractive noninvasive theranostic protocol based on ultrasound imaging and physical therapy [19]. The emulsion significantly increased acoustic impedance between the tumor and surrounding tissues [20] and causes physical damage to tumor cells [21].
Poly(D,L-lactide-co-glycolide) (PLGA), as a type of biodegradable nanomaterial with an excellent safety profile in humans [22] and good film-forming ability, has been approved by FDA for vaccine and drug delivery as well as tissue engineering [23][24][25]. Thus, we prepared a MAGEtargeted PLGA nanomolecular probe encapsulating liquid perfluorohexane (PFH) and Au-NRs (MAGE-Au-PFH-NPs) in this work, specifically targeting melanoma cells, which have been confirmed to possess the potential to enhance photoacoustic (PA) and ultrasonic imaging in melanoma in our previous study. The results from our previous study showed that MAGE-Au-PFH-NPs could accumulate and retain in the tumor area, allowing for therapeutic guidance and monitoring [26]. This study went further in exploring the role of the MAGE-Au-PFH-NPs in the treatment of melanoma based on our previous study. Benefiting from the high photothermal-conversion efficiency and ODV effect in MAGE-Au-PFH-NPs, the efficient tumor ablation was achieved in melanoma-bearing mice, which provides a promising alternative strategy for imaging-guided phototherapy of cancer.

Cell
Culture and Animal Experiment. B16 mouse melanoma cells were cultured in T75 flasks containing Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% foetal bovine serum and 1% penicillin and streptomycin (antibiotics) and incubated at 37°C under 5% CO 2 , with medium changes every 2-3 days. For all the experiments, the cells were harvested using 0.25% trypsin solution and were then resuspended in fresh medium before plating.
All the animals (male BALB/c nude mice:~20 g, 4-6 weeks) were purchased from the Experimental Animal Center of Chongqing Medical University and bred at constant temperature and humidity, with food and water provided ad libitum. The animals were maintained in accordance with the National Guidelines for Experimental Animal Welfare (MOST, China, 2006) at the Centre for Animal Experiments, and all the experiments and procedures were approved by the Institutional Animal Care and Use Committee of Chongqing Medical University. B16 cells were suspended in PBS (1 × 10 6 B16 cells in 100 μL of PBS per mouse) and then injected subcutaneously into the buttock of the BALB/c nude mice to establish tumor-bearing mice.
2.3. Characterization of the NPs. By reference to our previous study [26], MAGE-Au-PFH-NPs were prepared by the double emulsion method. The carbodiimide method was employed to modify the Au-PFH-NPs with MAGE antibody to prepare the targeted nanoparticles (MAGE-Au-PFH-NPs).
The size and zeta potential of MAGE-Au-PFH-NPs were measured by a Malvern laser particle size analyzer (Malvern, England). Furthermore, to assess the stability of MAGE-Au-PFH-NPs, the NP size and zeta potential changes were tested for 72 hours while incubating in plasma at 37°C. Meanwhile, the NPs underwent PAI scanning at different wavelengths ranging from 680 nm to 970 nm (interval = 5 nm) to determine the maximum absorbance for optimized PAI by a Vevo LAZR Photoacoustic Imaging System (Vevo LAZR, Toronto, Canada).

2.4.
In Vitro Photothermal Properties of NPs. In our previous study, MAGE-Au-PFH-NP temperature was increased to 70°C through photothermal conversion after absorbing near infrared light [26]. In this study, we evaluated photothermal capability of NPs in different concentrations at different power densities by laser irradiation using an 808 nm laser for 5 minutes in vitro. The infrared radiation (IR) thermal images and temperature changes were recorded by an infrared thermal-imaging camera.
2.5. In Vitro Photothermal Ablation against B16 Cells. To test the photothermal ablation effects of MAGE-Au-PFH-NPs in vitro, B16 cancer cells were divided into 4 groups: control (normal saline (NS) only), NPs only, laser only, and NPs +laser, and were seeded onto four confocal-specific cellculture dishes (1 × 10 5 cells per dish) overnight. After cell adhesion, NP suspension was added into two dishes (two groups: NPs only and NPs+laser), and equal volume serum-free RPMI 1640 medium was added into the other two dishes and incubated for another 12 h; cells of two groups (laser only and NPs+laser) were exposed to laser (1.00 W/cm 2 ) for 10 min. Then, the medium was removed, and the cells of each dish were washed three times with PBS. The cells of four groups were scanned by confocal microscopy after costaining with Calcein-AM and propidium iodide.  3 BioMed Research International photothermal efficiency of MAGE-Au-PFH-NPs, twenty B16 tumor-bearing mice were randomly divided into four groups (5 mice per group) when the tumor volumes reached about 100 mm 3 , including MAGE-Au-PFH-NP, Au-PFH-NP, Au-NP, and NS groups. The mice were intravenously injected with 100 μL MAGE-Au-PFH-NPs, Au-PFH-NPs, and Au-NPs at a concentration of 25 mg/mL, respectively. The same volume of normal saline was injected into the mice in the control NS group. The tumors were exposed to the 808 nm laser (1.00 W/cm 2 ) for 10 min after injection of NPs and NS 2 minutes later. The temperature changes in tumors and infrared radiation (IR) thermal images were recorded by an infrared thermal-imaging camera. To further analyze the effect of photothermal therapy on local hyperthermia, we detected the HSP70 expression within tumors by immunofluorescent staining. All the dose and laser irradiation conditions were adjusted to the same level as mentioned above. At the second day after treatment, mice were sacrificed to collect tumors for HSP70 immunofluorescent staining. Anti-HSP70 rabbit pAb as primary antibodies was added in the section and incubated overnight at -4°C; Cy3 conjugated goat antirabbit IgG as secondary antibodies was added in the section away from light for 50 min at room temperature, followed by DAPI hyperchromatic nucleus and sealing piece. Eventually, the sections were observed and imaged under a fluorescence microscope.

Targeting Ability In Vitro and In Vivo.
To assess the targeting ability in vitro, immunofluorescence imaging observed under confocal microscopy has been performed. The melanoma-associated antigens were combined to the targeted nanoparticles to obtain the blocking group (MAGE-R-Au-PFH-NPs). MAGE-Au-PFH-NPs and the blocking group (MAGE-R-Au-PFH-NPs) were all treated with DiI fluorescent dye in the first step of synthesis before the sonica-tion. B16 cells were seeded in confocal laser dishes at 1 × 10 5 and coincubated with dyed NPs (MAGE-Au-PFH-NPs, MAGE-R-Au-PFH-NPs) at 37°C for 30 min and then observed under a laser scanning confocal microscope after being fixed with 4% paraformaldehyde and stained with 20 μL of 4′,6-diamidino-2-phenylindole (DAPI).
To detect whether MAGE-Au-PFH-NPs have a long circulation time compared to that of the Au-PFH-NPs, 6 tumor-bearing mice were randomly divided into two groups (MAGE-Au-PFH-NPs and Au-PFH-NPs); the two groups of mice were intravenously injected with 100 μL of MAGE-Au-PFH-NPs and Au-PFH-NPs (25 mg/mL). The dynamic distribution of nanoparticles within the whole body was measured using fluorescence spectrum.

Photothermal/ODV Efficiency and Detection In Vivo.
To evaluate the in vivo photothermal and ODV efficiency of MAGE-Au-PFH-NPs, twenty B16 tumor-bearing mice were used when the tumor volumes reached about 100 mm 3 . The mice were divided into four groups (5 mice per group) randomly including the laser only, Au-NP+laser, Au-PFH-NP +laser, and MAGE-Au-PFH-NP+laser groups, which were intravenously injected with normal saline solution (100 μL), Au-NP (25 mg/mL, 100 μL), Au-PFH-NP (25 mg/mL, 100 μL), and MAGE-Au-PFH-NP suspension (25 mg/mL, 100 μL), respectively. Then, each mouse was exposed to the 808 nm laser for 10 min at a power density of 1.00 W/cm 2 .
The treatment was performed every other day, and one of the mice in each group was sacrificed on the third day for pathological section and staining. All the tumor tissues were collected and fixed in a 4% paraformaldehyde solution, stained with H&E, TdT-mediated dUTP Nick-End Labeling (TUNEL), and Proliferating Cell Nuclear Antigen (PCNA) for histopathology analysis.

BioMed Research International
The remaining four mice were maintained for 15 days, and the mouse weight and tumor volume were measured every other day after PTT. The tumor-volume changes were normalized using the relative tumor volumes V/V 0 (V 0 : the initial tumor volume before the treatment).
In our previous study, CEUS imaging was significantly enhanced at the tumor site in the MAGE-Au-PFH-NP group after laser irradiation [26]. Thus, contrast-enhanced ultrasonography (Esaote MyLab 90, Genoa, Italy) was performed in tumor-bearing mice after the treatment to evaluate the therapeutic effect of MAGE-Au-PFH-NPs in vivo in this study.
CEUS-mode images of the tumors were recorded after exposure to the 808 nm laser (1.00 W/cm 2 , 10 mins). Echo intensity was acquired and analyzed using a DFY-ultrasonic image quantitative analyzer (Institute of Ultrasound Imaging of Chongqing Medical University, Chongqing, China).

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BioMed Research International BUN), and H&E staining of the major organs (heart, liver, and kidney) after intravenous injection and laser irradiation have been performed to evaluate the biocompatibility in vivo.

Statistical Analysis
Data were presented as the mean ± standard deviation. Statistical analyses were done using the SPSS Ver. 19.0. P < 0:05 was considered statistically significant according to one-way ANOVA and Student's t-test.

Characterization of NPs and Photothermal Conversion
Efficiency of NPs In Vitro. The size and zeta potential of MAGE-Au-PFH-NPs were 324:54 ± 21:76 nm and −4:76 ± 3:7 mV, respectively, measured by a Malvern laser particle size analyzer. Furthermore, results showed that the size and zeta potential changes measured at 48 h and 72 h had no statistic difference compared with 12 h that could confirm the stability of MAGE-Au-PFH-NPs (Figures 1(a)-1(d)). The result of the PAI scanning showed that the maximum absorbance of three groups (Au-NPs, Au-PFH-NPs, and MAGE-Au-PFH-NPs) had been detected at 780 nm which conformed to the spectral properties of gold nanorods (GNRs/Au-NRs) (Figure 1(e)).
The NPs showed a good dispersity under a confocal laser scanning microscope and were mainly distributed in the cytoplasm after uptake by cells under a transmission electron microscope (Figures 2(a) and 2(b)).
The temperature change of NPs after laser irradiation was recorded by an infrared thermal-imaging camera. The

Photothermal Conversion Efficiency and HSP Evaluation
In Vivo. As shown in Figures 3(a) and 3(b), the surface temperature of tumors in the MAGE-Au-PFH-NP+laser group increased from 31:6 ± 1:09°C to 56:1 ± 2:6°C under irradiation for 10 min. The Au-PFH-NP+laser group increased from 30:1 ± 1:3°C to 52:0 ± 2:1°C under irradiation for 10 min. The temperature in the Au-NP+laser group increased from 31:2 ± 0:9°C to 51:4 ± 1:7°C under irradiation for 10 min. Comparatively, only a slight temperature increase was found in the tumor region in the laser only group. The HSP70 expression level was analyzed and is shown in Figures 3(c) and 3(d). The mice that received MAGE-Au-PFH-NPs plus laser irradiation showed a remarkably higher HSP70 expression compared to those treated with Au-PFH-NPs and Au-NPs followed by laser irradiation. In the control group (NS with laser irradiation), no evident expression of HSP70 was found.       (Figure 4(c)). These results confirmed that the MAGE-Au-PFH-NPs could specifically target B16 cells.

Photothermal/ODV Efficiency and Detection In Vivo.
Based on the in vivo photoacoustic imaging experiment, MAGE-Au-PFH-NPs actively accumulated in the tumor region, and the signal reached a peak at 2 h post injection [26]. Therefore, the mice were treated for 10 min after 2 h post injection until the 5th day. Tumor tissue necrosis was found in mice in the MAGE-Au-PFH-NP+laser group the second day after treatment, leaving black scars in the initial tumor regions (Figure 5(a), red arrow). Then, they disappeared 15 days later, leading to complete tumor eradication ( Figure 5(a), black arrow), while the tumors in the remaining three groups grew rapidly. The ultrasound imaging was significantly enhanced at the tumor site in the MAGE-Au-PFH-NP group under the laser irradiation. Then, the enhanced ultrasound signals gradually decreased with the increase of treatment times and disappeared by day 15 (Figure 5(b)). The weight and tumor volume of each mouse were recorded every other day ( Figure 5(c)). Then, the tumor-volume change was normalized as V/V 0 ( Figure 5(d)). H&E and TUNEL staining results further confirmed tumor necrosis in mice in the MAGE-Au-PFH-NP+laser group, which was more serious compared to those in the remaining three groups ( Figure 5(e)). From the result of the PCNA assay, the MAGE-Au-PFH-NP+laser group exhibited a significant suppression effect on tumor cell proliferation. In contrast, no evident proliferative inhibition was observed in the remaining three groups. 4.6. Toxicity Test In Vitro and Biocompatibility In Vivo. The cytotoxicity of MAGE-Au-PFH-NPs was investigated by the CCK-8 assay. After 24 h incubation, inconspicuous cytotoxicity of the NPs on endothelial cells and hepatic cells was observed since cell viability remained above 80% at NP concentration of 25 mg/mL. And no significant difference was found among the groups (NS, Au-NPs, Au-PFH-NPS, and MAGE-Au-PFH-NPs) (Figure 6(c)). The test of liver functional markers (AST and ALT) and kidney functional markers (CR and BUN) and H&E staining of the major organs (heart, liver, and kidney) after intravenous injection and laser irradiation showed no significant physiological toxicity (Figures 6(a), 6(b), and 6(d)).

Discussion
Photothermal therapy (PTT) is a minimally invasive technique for cancer treatment which uses laser-activated photoabsorbers to convert photon energy into heat sufficient to induce cell destruction via apoptosis, necroptosis, and/or necrosis. From the current studies, photothermal therapy cannot ablate the tumor thoroughly using photothermal materials due to nonuniform tumor internal heat distribution. The combination of PTT and other methods may overcome this disadvantage. Gold NPs (Au-NPs) designed to act as photothermal sensitizing agents are widely used in cancer therapy due to their high optical absorption coefficients. In our previous study, the Au-NRs and PFH were encapsulated in a PLGA shell through the double emulsion method with a high encapsulation efficiency and conjugated with the 9 BioMed Research International MAGE-1 antibody for targeting melanoma cells [26]. With the application of MAGE-Au-PFH-NPs, the temperature can be raised up to 71°C under laser irradiation, showing their excellent photothermal effect [27]. These targeted nanoparticles actively accumulated in the tumor area and enhanced the PTT effect. In addition, the PFH can be vaporized by laser irradiation [28] with the application of MAGE-Au-PFH-NPs [16], which had also been confirmed in our in vitro study [26]. Meanwhile, from the in vivo results, the temperature in the tumor region increased up to 56:1 ± 2:6°C (Figure 3(b)), which was sufficient to ablate the tumor tissue [29]; meanwhile, it could convert MAGE-Au-PFH-NPs into bubbles. Then, the physical and mechanical damage induced by the phase change process can directly kill tumor cells [21].
Heat shock protein is produced under the induction of stress agents such as high temperature to induce thermoresistance for cells [30]. In our study, the results showed that HSP70 expression in the MAGE-Au-PFH-NP group was significantly higher compared to that in the groups of Au-PFH-NPs and Au-NPs with laser irradiation. Only little expression of HSP70 was found in the NS plus laser group. The results of HSP70 immunofluorescent staining corresponded well with those of photothermal conversion effect.
In our study, complete tumor eradication was observed in the MAGE-Au-PFH-NP+laser group. In contrast, the tumors of the other three groups grew rapidly. The results showed that the combination of targeted photothermal therapy and ODV phase transition physical therapy could achieve better tumor ablation effect. In vivo fluorescence imaging confirmed that targeted nanoparticles (MAGE-Au-PFH-NPs) had a longer circulation time compared to the nontargeted Au-PFH-NPs. Tumor recurrence was found in the Au-PFH-NP and Au-NP plus laser therapy groups indicating limited PTT effect leading to residues in tumor tissue. By combining the PTT effect and ODV physical damage from MAGE-Au-PFH-NPs, the tumor ablation was greatly improved. Meanwhile, damage to normal tissues caused by long time laser irradiation can be avoided.
Besides, when the phase-changeable nanoparticles were transformed from droplets to microbubbles, the acoustic impedance of the tumor and surrounding tissues was increased, which thereby enhanced ultrasound imaging. In this study, the CEUS imaging was significantly enhanced at the tumor site in the MAGE-Au-PFH-NP group under laser irradiation and decreased with the increase of treatment times, providing a method for monitoring tumor ablation effect by CEUS.

Conclusion
We successfully constructed MAGE-targeted phasechangeable gold nanoparticles which could accumulate at tumor sites. With the combination of PTT and ODV effects, complete tumor eradiation was achieved and could be monitored by contrast-enhanced ultrasonography. Several advantages such as noninvasiveness, short recovery time, low complication rate, and monitorable treatment process were included with this protocol. These novel targeted nanoparticles could be used as a multifunctional theranostic agent for imaging-guided tumor ablation.

Data Availability
The data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
There are no conflicts to declare.