GPP-TSAIII nanocomposite hydrogel-based photothermal ablation facilitates melanoma therapy

ABSTRACT Background Photothermal therapy (PTT) is a promising cancer treatment, but its application is limited by low photoconversion efficiency. In this study, we aimed to develop a novel graphene oxide (GO)-based nanocomposite hydrogel to improve the bioavailability of timosaponin AIII (TSAIII) while maximizing PTT efficacy and enhancing the antitumor effect. Methods GO was modified via physical cross-linking with polyvinyl alcohol. The pore structure of the gel was adjusted by repeated freeze-thawing and the addition of polyethylene glycol 2000 to obtain a nanocomposite hydrogel (GPP). The GPP loaded with TSAIII constituted a GPP-TSAIII drug delivery system, and its efficacy was evaluated by in vitro cytotoxicity, apoptosis, migration, and uptake analyses, and in vivo antitumor studies. Results The encapsulation rate of GPP-TSAIII was 66.36 ± 3.97%, with slower in vitro release and higher tumor cell uptake (6.4-fold) compared to TSAIII. GPP-TSAIII in combination with PTT showed better bioavailability and antitumor effects in vivo than did TSAIII, with a 1.9-fold higher tumor suppression rate than the TSAIII group. Conclusions GPP is a potential vehicle for delivery of TSAIII-like poor water-soluble anticancer drugs. The innovative PTT co-delivery system may serve as a safe and effective melanoma treatment platform for further anticancer translational purposes. GRAPHICAL ABSTRACT


Introduction
Melanoma is a malignant tumor derived from transformed melanocytes, and its incidence continues to increase worldwide [1].Although its incidence is not common, melanoma is very lethal, accounting for approximately 73% of skin cancer-related deaths [2].The main cause of death is widespread metastasis to the lymphatic system and other important organs [3].The current standard therapy for melanoma includes surgical treatment, immunotherapy, targeted therapy, and combination therapy.Because melanoma has a very poor prognosis and spreads quickly and easily at an early stage, chemotherapy is frequently used in combination with other adjuvant therapies, including biological therapy, skin-directed therapy, and radiotherapy.The effectiveness of these treatments, however, is constrained due to poor sensitivity, debilitating side effects, and strong drug resistance.It is important to investigate novel and efficient treatments for progressive melanoma.Pathogenic skin infections acquired due to a relatively weak immune system of the skin tissue can exacerbate the complexity of melanoma and increase the mortality of patients [4].Composite hydrogels are ideal for skin wound dressings due to their multifunctional properties [5] and are also often employed to encapsulate drugs and as a delivery system for sustained drug release [6].Composite hydrogels can absorb body fluids in moist environments, accelerate healing, reduce inflammation, and impede bacterial growth through their biocompatibility [6].However, hydrogels also have the obvious disadvantage that the loaded therapeutic drugs will leak and collect.To overcome this drawback, researchers have attempted to modify hydrogels for better mechanical properties that maintain their structural integrity and achieve the kinetic properties required for sustained drug release [7].Several scientific studies have found that hydrogels containing graphene oxide (GO) have more mechanical properties and can maintain their structural integrity while having good antibacterial, antiviral, antitumor and anticancer activities [8][9][10].
Graphene is an atomically thin sheet of a two-dimensional honeycomb monolayer of sp 2 hybridized carbon atoms.More specifically, it has a poly-aromatic surface structure with a large specific surface area, which provides free π-electrons and reaction sites for a large number of surface reactions [11].Therefore, graphene-based photosensitive nanoparticles (NPs) have been highlighted as an efficient delivery system in photothermal therapy (PTT) [12].GO, a water-soluble derivative of graphene, is capable of drug delivery and non-covalent surface modification via π-π stacking and hydrophobic interactions, and is widely used for drug delivery, near-infrared (NIR) PPT, and bioimaging studies [13].
PTT utilizes NPs to convert light into heat energy to generate local high temperatures and thus eliminate malignant tumors, and this method is also suitable for the treatment of superficial cancer [14].Therefore, as a superficial cancer, melanoma is a tumor suitable for PTT-induced antitumor therapy, because the penetration depth of light is not an issue.NPs can also transfer the absorbed photon energy to the surrounding oxygen molecules to generate reactive oxygen species (ROS), including singlet oxygen or free radicals, which can kill cancer cells and destroy tumor tissue, achieving an antitumor effect [15,16].
In the form of NPs, GO absorbs light in the near-infrared region and can convert light energy into heat energy, which can be used for photothermal therapy [17,18].The exposed GO cannot, however, undergo active or prolonged accumulation at the tumor site due to a low photothermal conversion efficiency, lack of an active targeting capability, and rapid innate immune system elimination.The hydrophilic polymer polyvinyl alcohol (PVA) was added to the nanocomposite hydrogel in the current study to impart the hydrogel with porous and hydrophilic structures.The nanocomposite hydrogel hydrophilicity and porosity can be changed by varying the mass percentages of PVA and GO.These features gave the nanocomposite hydrogel a higher water -wetting capacity and improved light-toheat conversion efficiency.We also used hydrophilic polyethylene glycol (PEG) to modify the surface of the nanocomposite hydrogel, and many reports have reported good biocompatibility [19][20][21].
Melanoma is a cancer with a strong mutational burden that can resist and evade currently available therapies [22].Therefore, despite the significant progress made in the clinical treatment of cancer, the treatment of metastatic melanoma is still ineffective and tumors often recur [23,24].Timosaponin AIII (TSAIII) is a steroidal saponin whose sugar chain is mainly located at the C3 position.The chemical structure is shown in the Figure 1.TSAIII was preliminarily isolated from the dried rhizomes of the Liliaceae plant Anemarrhena asphodeloides Bge.In China, Anemarrhena asphodeloides Bge has been used for the treatment of various diseases, including joint pain, blood in the stool, cough, and hemoptysis [25].TSAIII was shown to affect the migration potential of melanoma cells [26].In a previous study, we found that TSAIII acted on the mitochondria of B16F10 cells and induced their apoptosis.Most mitochondrial ion channels are differentially expressed and/or regulated in cancer cells compared to healthy cells, so pharmacological targeting of mitochondrial ion channels is emerging as a promising approach to eliminating cancer cells [27].The mitochondrial structure is the main source of ROS and plays a variety of roles in cancer [28,29].Oxidative phosphorylation may underlie cancer and cancer stem cell survival.Perturbations in the mitochondrial membrane and inner membrane ion flux are associated with changes in membrane potential, redox state, and bioenergetic efficiency, which were found to lead to indirect regulation of oxidative phosphorylation, affecting cancer cell survival [30].Mitochondria also play an important role in endogenous apoptosis, and targeting mitochondria may be very beneficial to induce apoptosis in cancer cells.However, the application of TSAIII is limited by poor water solubility.Therefore, we propose to construct a drug delivery system loaded with TSAIII to improve the bioavailability of TSAIII while making it more available for uptake by tumor cells, acting on tumor cell mitochondria and inducing their apoptosis.
In this study, we prepared a novel nanocomposite hydrogel GPP-TSAIII drug delivery system (this system included PVA, GO, and TSAIII) as a novel drug delivery system for the treatment of melanoma in order to improve the encapsulation rate, water solubility, and bioavailability of TSAIII, and to improve the PTT therapeutic efficiency of GO.To our knowledge, this drug delivery system strategy has never been reported in the published literature.We prepared GPP by adding PVA to GO with repeated freeze-thawing, and the synthesized GPP was characterized using Fourier transform infrared (FTIR), transmission electron microscopy (TEM), scanning electron microscopy (SEM), and water contact angle analyses.The encapsulation rate and release properties of GPP in TSAIII were tested, and the cellular uptake and in vitro cytotoxicity, cell migration, and apoptosis of TSAIII and GPP-TSAIII were evaluated.Finally, antitumor efficacy and safety studies of GPP-TSAIII were performed in nude mice using a B16F10 ×enograft model.The prepared nanocomposite hydrogels were shown to be potential biomaterials for melanoma treatment, providing promising ideas for the clinical treatment of melanoma and corresponding infections.

Cell culture
B16F10 mouse melanoma cells were obtained from Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences.B16F10 cells were maintained in a DMEM medium supplemented with 10% FBS, 100 U/mL penicillin, and 100 U/ mL streptomycin.The conditions of the incubator were 90% relative humidity and 5% CO 2 at 37°C.

Viability assay
B16F10 cells (1.0 × 10 4 ) were seeded in 96-well plates.Samples, including TSAIII, GO, GPP, and GPP-TSAIII, were diluted with cellular medium to different concentrations (n = 6).Following incubation for 24 h, 10 µL of Cell Counting kit-8 (CCK-8) reagent was added to each well for 1 h.A microplate reader (i3×, Thermo Fisher Scientific) was used to determine the absorbance values (OD) at 450 nm.The calculation formula for cell viability was as follows:

Wound healing assay
B16F10 cells (1.0 × 10 5 ) were seeded in six-well plates.When the cell density reached 90%, a 200 µL pipette tip was used to quickly draw two vertical lines at the bottom of the sixwell plates.The cells were washed thrice three times with PBS to remove the loosened cells.Incubation was continued after treatment.Images of the cells were taken under an inverted fluorescence microscope (Leica, DMi1, Germany) at 0, 24, and 48 h.

Mitochondrial membrane potential assay
B16F10 cells (1.0 × 10 4 ) were seeded in a laser confocal culture dish.After the cells were fully adhered, the culture was continued for 4 h after treatment.Then, the cells were stained using a JC-1 kit and observed under a confocal laser scanning microscope (Leica TCS SP8, Leica, Germany).

ROS assay
B16F10 cells (1.0 × 10 4 ) were seeded in a laser confocal culture dish.After the cells fully adhered, the culture was continued for 4 h after the intervention treatment.Then, the cells were stained using a ROS assay kit and observed under a confocal laser scanning microscope.

Apoptosis assay
B16F10 cells (1.0 × 10 4 ) were seeded in a laser confocal culture dish.After the cells fully adhered, the culture was continued for 24 h after treatment.Then, the cells were stained with Hoechst 33,258 and observed under a confocal laser scanning microscope.

Metabolic assay
The oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured using a Seahorse XF96 extracellular flux analyzer.Briefly, 1.0 × 10 5 cells per well were seeded overnight in XF 96-well plates inaserum-free medium.On the same day that cells were plated, 1 mL of hydration solution was added to the lower layer of the XF24 extracellular flux assay plate.The plate contents were hydrated overnight in a 37°C CO 2 -free incubator.The required drugs and hippocampal XF basic medium were prepared, and the pH was adjusted to 7.4.While using the hippocampal XF basic medium, it was placed in a 37°C water bath for 1 h.On the second day, the cells were washed twice with hippocampal XF basic medium in a water bath.Then, 450 µL of hippocampal XF basic medium was added to each port, and the plate was placed in a 37°C CO 2 -free incubator for 1 h.The drug was diluted to the required concentration, and then added to the upper layer of the XF24 extracellular flux assay plate assembly (75 µL, per well), and the plate was placed in the machine.After 30 min, the lower layer of XF24 extracellular flux assay plate assembly was replaced with a cell plate that had been cultured in a CO 2 -free incubator at 37°C for 1 h, and then analyzed on the machine.

Preparation and characterization of GPP-TSAIII
The drug delivery system described in this article is based on functionalized GO loaded with TSAIII.Specifically, 1 mL of PVA with a concentration of 80 mg/mL was placed in a flatbottomed glass vial, and 10 wt% PEG solid particles were added to promote the full dissolution of the PEG solid to be fully dissolved in the PVA aqueous solution.Eighty milligrams of GO were accurately weighed, 20 mL of distilled water was added, and the solution was fully dissolved using ultrasonication for 30 min to prepare an aqueous dispersion of GO with a concentration of 4 mg/mL Approximately 5 mL of the GO aqueous dispersion was added to a mixed aqueous solution of PEG and PVA, and then stirred.The solution was mixed uniformly by ultrasonication and treated at 50°C.After the liquid reached room temperature, it was placed in a refrigerator at −20°C and frozen for 8 h.After removing it from the refrigerator, the sample was melted at room temperature for 4 h, and then the freeze-thaw cycle was repeated eight times.After all the samples had formed a gel, the remaining PEG was washed with deionized water to obtain a freeze-thaw cycle GO-PVA-PEG hydrogel, which was placed in a − 80°C refrigerator for 12 h, and then placed in a freeze dryer for 48 h.Then, freeze-thaw cycle GO-PVA-PEG (GPP) nanocomposite hydrogel was obtained.

Drug loading and in vitro drug release
A GPP aqueous solution and TSAIII methanol solution were prepared by dissolving each at room temperature (23-25°C), and the final concentrations were fixed at 2 and 0.5 mg/mL, respectively.Then, 10 mL of GPP aqueous solution was transferred into a beaker (50 mL) and stirred at 400 rpm at room temperature.Afterward, 20 mL of TSAIII methanol solution was added dropwise.After the methanol was completely volatilized, 20 mL of a blank methanol solution was used as a control and magnetically stirred under the same conditions, and the time required for its complete volatilization was measured.Stirring was then stopped, and the resulting reaction solution was centrifuged at a rate of 800 rpm at room temperature for 15 min.The excess unreacted TSAIII precipitate was removed.The supernatant was collected and transferred into a new centrifuge tube, placed in a refrigerator at −80°C and frozen for 12 h, and then placed in a freeze dryer for 48 h.The GO-carrying TSAIII composite NPs (GPP-TSAIII) were obtained.
A total of 5.0 mg of TSAIII and an certain amount of the GPP-TSAIII delivery system that contained 5.0 mg TSAIII were accurately weighed with 30% ethanol (v:v) in phosphate buffer (pH = 7.4) as the release medium.Parallel samples (n = 3) were incubated in a dissolution apparatus at 37°C.The drug release medium (2.0 mL) was collected, and fresh medium was then added to the vials at preset time intervals.The amount of TSAIII released was confirmed using highperformance liquid chromatography-evaporative light scattering detection (HPLC-ELSD).

Cellular uptake
To observe the uptake of TSAIII by B16F10 cells, we used the fluorescent dye indocyanine green to label TSAIII.After incubation for 2, 4, or 6 h at 37°C, confocal microscopy and CytoFLEX flow cytometry (Beckman Coulter, CytoFLEX, U.S. A) were used to study the uptake behavior of TSAIII by cancer cells.

Flow cytometry-based apoptosis assay
B16F10 cells were used to evaluate the in vitro apoptosis of GPP, TSAIII, and GPP-TSAIII.Briefly, the cells (1.0 × 10 5 cells) were seeded in six-well plates with 1.0 mL of culture medium.After 24 h, GPP, TSAIII, and GPP-TSAIII culture medium (1.0 mL) was added to the six-well plate and incubated at 37°C for 6 h.At the same time, the near-infrared light (NIR) irradiation group (808 nm, 2 W cm −2 , 8 min) was treated.After the incubation was completed, the Annexin V-FITC cell apoptosis detection kit was used for staining according to the instructions, and the detection of apoptotic cells was performed on a flow cytometer.

Western blot
Approximately 25 µg of cell-extracted protein from both B16F10 cells was loaded onto a premade sodium dodecylsulfate-polyacrylamide gel, subjected to electrophoresis, and electrotransferred onto a nitrocellulose membrane.The membrane was then probed with a primary antibody overnight at 4°C.The following day, the membrane was washed, and an appropriate secondary antibody was added.The signal was visualized using SuperSignal West Pico enhance chemiluminescent detection reagent (Biyuntian Biotechnology, Shanghai, China).

Tumor models
Nude mice (female, 4-6 weeks old) were obtained from Shanghai Sipuer-Bikai Experimental Animal Co., Ltd.All animal operations were performed in accordance with the 'Guidelines for the Care and Use of Laboratory Animals' of Shanghai University of Traditional Chinese Medicine and were approved by the Animal Ethics Committee of Shanghai University of Traditional Chinese Medicine.B16F10 cells (4.0 × 10 5 ) in 100 μL PBS were inoculated into the armpits of nude mice to prepare xenograft tumor models.When the average tumor volume exceeded approximately 100 mm 3 , additional experiments were performed.

Flow cytometry
The different immune cells in the tumors were investigated by cutting the tumors collected from mice after different treatments into small pieces and homogenizing them to form single cells in a cold staining buffer.The sample was stained with antibodies and evaluated using a flow cytometer.

In vivo anticancer efficacy
The tumor-bearing mice were randomly divided into six groups.Different pharmaceutical preparations with a final volume of 100 μL were administered by injection at multiple sites within the tumor.Then, the tumor volume and body weight of the treated mice were monitored every two days.Formula (2) was used to calculate the tumor volume, while formula (3) was used to calculate the tumor inhibition rate.
where a and b represent the length and width of the tumor, and c and d represent the average tumor weight of the experimental group and the average tumor weight of the model group, respectively.

Histology and assay
After topical treatment with different drugs, the treated mice were sacrificed on the 15th day.Then, the tumor tissues and various organ tissues were used for further studies.The tissue sections were cut into 5-μm-thick slices and stained with hematoxylin and eosin (H&E) for histological analysis.

Evaluation of the in vitro efficacy of TSAIII on B16F10 cells
TSAIII plays an important role in inhibiting the growth of various tumor cells (e.g.colorectal [31], hepatocellular [32], and breast cancer cells [33]).TSAIII remarkably inhibited the growth of cancer cells at low concentrations and selectively reduced the viability of cancer cells, but it did not reduce the viability of normal cells [34].We aimed to verify whether TSAIII can effectively inhibited the proliferation, migration, and apoptosis of B16F10 cells.After treating B16F10 cells with TSAIII for 24 h, the IC 50 value of TSAIII, which inhibited the proliferation of B16F10 cells, was 3.398 μg/mL (Figure 2a).The IC 50 concentration was lower than that of the current first-line chemotherapeutic drug DITC for melanoma (IC 50 = 30.1 μmol/L, 72 h) [35].As shown in Figure 2b, after incubating B16F10 cells with TSAIII for 24 h, the wound healing rates of the 4, 6, and 8 μg/mL TSAIII administration groups were 19.45% ± 0.82%, 1.36% ± 0.67%, and−0.25%± 2.50%, respectively (p < 0.01).Moreover, after 48 h of incubation, the wound healing rates of the 4, 6, and 8 μg/mL TSAIII administration groups were 31.62% ± 4.91%, 5.51% ± 1.10%, and−0.30%± 0.20%, respectively, p < 0.01.These results confirmed that TSAIII can inhibited the migration of B16F10 cells and suppresses the expression of migrationassociated proteins(Supplementary Figure S1).
After Hoechst 33,258 staining, the nuclei of apoptotic cells were densely stained with some fragmentation.As shown in Figure 2c, when 4, 6, or 8 μg/mL TSAIII was applied to B16F10 cells, the nuclei of the B16F10 cells showed different degrees of chromatin contraction, and the color of the nuclei became whitish and densely stained (red arrow) or appeared dense in fragments (green arrow).The nuclei of the blank control group showed a normal, uneven blue color.The experimental results indicated that TSAIII may induce the apoptosis of B16F10 cells, and its inhibitory effect on the proliferation of B16F10 cells may be achieved by inducing B16F10 cell apoptosis.
Changes in cell morphology are important features for testing the mode of cell death.Apoptosis refers to the autonomous and orderly death of cells controlled by genes to maintain the stability of the internal environment.Morphological changes in apoptotic cells are mainly characterized by pyknosis of the nucleus, condensation of chromatin, and the appearance of nuclear fragments or apoptotic bodies [36,37].After TSAIII acted on B16F10 cells, swelling occurred (red arrow), a large number of vacuoles appeared in the cytoplasm (black arrow), and bubbles appeared on the cell membrane (green arrow) (Figure 3a).The results showed that the vacuoles may have originated from the mitochondria, and that further studies on cellular respiration, mitochondrial function, and mitochondrial respiratory status were needed.
As shown in Figure 3b, based on the comparison of the fluorescence intensity of the treated and blank group under a confocal microscope, the fluorescence intensity in B16F10 cells after treatment with the TSAIII drug solution was significantly higher than that in the blank group, and the level of intracellular ROS was high.Moreover, after incubating B16F10 cells with the TSAIII drug solution for 4 h, the red fluorescence on the surface of the B16F10 cells decreased, and the green fluorescence increased (Figure 3d).The results showed that TSAIII drugs reduced the mitochondrial membrane potential of B16F10 cells.
These data indicate that TSAIII disrupted the mitochondrial function of B16F10 cells and promoted their apoptosis.We next measured ECAR, which indicates glycolysis, and OCR, which indicates mitochondrial oxidative phosphorylation by using a Seahorse XF96 extracellular flux analyzer.The levels of non-mitochondrial respiration, basal respiration, respiratory capacity, proton leakage and indirectly measured ATP production were all changed after TSAIII was applied to B16F10 cells.The basal respiration, respiratory capacity, proton leakage, and indirectly measured ATP production levels were significantly decreased at a concentration of 5 µg/mL of TSAIII, which was statistically significant compared with the control group (Figure 4a).The assays of the main indicators of cellular metabolism, including the glycolytic flux, maximum glycolytic capacity, and glycolytic reserve capacity, all revealed changes following TSAIII action on B16F10 cells.When the concentration of TSAIII was 5 µg/mL, the non-glycolytic acidification and glycolytic levels decreased significantly, which was statistically significant compared with the control group.These results indicated that the metabolic level of B16F10 cells decreased significantly after TSAIII shift from aerobic respiration to glycolysis.However, TSAIII only induced B16F10 cells to shift from aerobic respiration to glycolysis for energy supply, and did not improve the glycolytic capacity of B16F10 cells.Further testing clarified that TSAIII significantly decreased mitochondrial aerobic respiration and reduced the ATP production capacity of B16F10 cells (Figure 4b).

Characterization of GPP-TSAIII
To verify the successful formation of GPP, we used FTIR to characterize each component material, and the chemical functional groups that may exist inside GPP were analyzed.As shown in Figure 5a, PEG had a unique and strong stretching vibration peak of CO at 1,101.86 cm −1 , and GO shows its phase with its oxygen-containing functional group.The corresponding characteristic FTIR peaks included the C=O tensile vibration peak at 1,716.47 cm −1 , the CO (epoxy) tensile vibration peak at 1,218.72 cm −1 , and the CO (alkoxy) peak at 1,048.88 cm −1.The vibration peak of the OH group was observed at 3,027.67 cm −1 , and the deformation peak was observed at 1,652.52 cm −1 , As shown in Figure 5a, PVA exhibited distinct hydroxyl absorption peaks at 3,389.99 cm −1 with obvious stretching vibration peaks of saturated carbon chain CH near 1,391 and 2,940.09cm −1 and CO absorption peaks at 1,097.60 cm −1 .GPP was prepared by adding PEG (10 wt% PVA) to the mixture of GO and PVA (w/w, 4:1) and analyzed.The results are shown in Figure 5a.In the infrared spectrum of pure PEG, PEG had a unique and strong C−O stretching vibration peak at 1,101.86 cm −1 .In GPP, obvious C=O stretching vibration peaks were observed at 1,733.76 cm −1 and CO stretching vibration peaks and obvious PVA molecular chains were observed near 1,094 and 2,940.09cm −1 , respectively.The stretching vibration peak of the saturated carbon chain CH was also observed.In summary, the FTIR characterization results showed that in the GPP system, PEG only played a role in modifying pores and was removed after washing with sufficient water.The final product GPP system was based on a gel composed of PVA.
As shown in Figure 5b, the GPP system had a distinct diffraction peak at 20°, and the XRD curve of pure PVA also had an obvious diffraction peak near 20°.The results showed that PVA was a matrix material in the GPP system.The XRD curve of pure PEG had clear diffraction peaks near 20° and 25°, and these diffraction peaks did not affect the number or the position of XRD diffraction peaks of the GPP system.In summary, PEG did not participate in any reaction during the preparation of the GPP system and was removed during the preparation.
Raman spectroscopy can be used to study the characteristic peaks of carbon atom crystals.The D peak representing defects of the carbon atom lattice was located near 1,300 cm −1 , while the G peak representing the in-plane stretching vibration of sp2 hybridization of carbon atoms was located near 1,600 cm −1 .The ratio of these two peaks is ID/IG, which is commonly used to characterize the changes in graphene structure.Many factors affect the change in the ID/IG value, and the most common ones are the change of oxygencontaining functional groups caused by the reduction or oxidation of graphene or the structure of graphene caused by repairing defects.As shown in Figure 5c, the ID/IG values of GO and GPP were 0.76 and 0.60, respectively.In comparison with GO, the ID/IG value of GPP decreased because defect repair.This parameter further decreased, indicating that the defects of GO were further repaired to form GPP after modification with PVA and PEG.The ID/IG values of GPP and GPP-TSAIII composite NPs were 0.60 and 0.80, respectively.In comparison with GPP, the ID/IG value of GPP-TSAIII increased because of the large number of hydroxyl groups on TSAIII, as shown in Figure 5c.When TSAIII was loaded on GPP, the hydroxyl groups on GPP increased, and the ID/IG value increased accordingly.
Figure 5d shows the SEM image of GO, revealing its massive crystal structure with distinct edge effects formed by stacking of many layers.PVA molecular chains were dispersed around the GO sheet, which is equivalent to intercalation on the GO sheet.During repeated freezethaw cycles and freezing and crystallization, crystals smaller than ice crystals can be formed.GO formed graphene walls around these crystals, and a small spindle-shaped hole was formed.The addition of PEG adjusted the internal pore structure of the GPP system to produce pores and generate the GPP system.The GPP composite NPs had a porous structure with a large number of pore structures inside, while TSAIII had a massive crystal structure, and many layers were stacked together to form massive crystals.As shown in Figure 5d, the GPP-TSAIII composite NPs no longer had a hole-like structure similar to the GPP system but were covered with TSAIII lamellar crystals.TEM images further showed that GO had a lamellar structure with a flaky shape with obvious edge effects, indicating that the structure was smooth and had no adsorption phenomena of the other shapes.The GPP system had an internal pore structure, the edge effect was not obvious, and the material coating was visible on the surface, while the surface of the GPP-TSAIII composite NPs had TSAIII adsorption on the surface, and no longer had the pore-like structure of the GPP system.
These experiments confirmed that in the entire preparation process, PVA acted as a 'foaming agent,' and the GPP system was based on a gel composed of PVA.PVA is a water-soluble hydrophilic polymer with a large number of hydroxyl groups in its molecular chain.The PVA coating makes the GPP system more hydrophilic, and with the addition of PEG, the internal pore structure of the GPP system is modified.GPP has a porous structure and an increased specific surface area, making it more conducive to water absorption.Therefore, the prepared GPP has good water solubility and can quickly absorb water.A contact angle test was performed on the GO material and the prepared GPP system to verify the difference in hydrophilic properties between them.The test results are shown in Figure 5e.The GO material, which was not modified by PVA and PEG, showed poor hydrophilic properties, with a contact angle of 109.942°.With the addition of PVA and PEG, the hydrophilic properties of GO improved, and the contact angle of the GPP system was immediately reduced to 17.505°.Water droplets applied to the surface of the sample were immediately absorbed within 13 ms, showing excellent hydrophilic properties.TSAIII is difficult to dissolve in water, and its contact angle was 121.129°.Water droplets on the surface of the TSAIII were not completely absorbed, and the TSAIII hydrophilic properties were extremely poor.When TSAIII was loaded on the GPP system with good hydrophilicity and prepared into GPP-TSAIII composite NPs, the contact angle was 22.532°, which was slightly higher than that of the GPP system.Water droplets were also applied to the surface of GPP-TSAIII, and were quickly absorbed within 13 ms.GPP-TSAIII exhibited excellent hydrophilic properties.In summary, the experimental results showed that the preparation of GPP-TSAIII composite NPs greatly improved the water solubility of TSAIII, thus providing the possibility to improve the results of in vivo administration.

Evaluation of cytotoxicity in vitro
The cytotoxicities of GO and GPP with or without laser treatment were investigated in B16F10 cells.As shown in Figure 6a, the results showed that the blank GPP showed good cell compatibility in vitro.In comparison with GO and GPP, GPP showed lower cytotoxicity to B16F10 cells.Treatment with GPP+Laser showed higher cytotoxicity than GO+Laser.After subjecting the B16F10 cells to GO+Laser and GPP+Laser treatment for 24 h, their IC 50 values were 35.173 and 7.263 µg/mL, respectively.This finding indicated that GO and GPP had low cytotoxicity and good photothermal conversion effects.As shown in Figure 6b, the IC 50 values of B16F10 cells treated with TSAIII and GPP-TSAIII +Laser for 24 h were 3.398 and 3.090 µg/mL, respectively.The above results confirmed that GPP-TSAIII composite NPs were not only effective as a chemotherapy alone, but also combined with light and heat to further enhance cytotoxicity and achieve the purpose of combined therapy.Notably, compared with the free TSAIII treatment group, GPP-TSAIII induced less cell death possibly because of the prolonged drug release time of the GPP carrier material.

Evaluation of uptake in vitro
As shown in Figure 6c, the observation results under a laser confocal microscope show that the uptake of TSAIII and GPP-TSAIII by B16F10 cells was different at different depending on incubation times.When the cells were incubated for 2 h, only weak fluorescence was observed in the TSAIII-free drug administration group, while the GPP-TSAIII composite NP administration group had relatively strong fluorescence, indicating that the GPP-TSAIII composite NP drug was likely internalized.Similarly, when the cells were incubated for 4 and 6 h, the fluorescence of the GPP-TSAIII composite NP administration group was stronger than that of the TSAIII free drug administration group, suggesting that the GPP-TSAIII composite NPs enhanced intracellular drug accumulation.The cell uptake results of the quantitative analysis by using flow cytometry are shown in Figure 6d and 6e, and the statistical results are shown in Figure 6f.When the cells were incubated for 2 h, the uptake rate by B16F10 cells incubated with the TSAIII free drug was 3.95 ± 0.24%, while the uptake rate of B16F10 cells incubated with GPP-TSAIII composite NPs was 99.67 ± 0.06%.A significant difference (p < 0.01) was observed, indicating that the GPP-TSAIII composite NP drug was more likely to be endocytosed by B16F10 cells.Similarly, when the cells were incubated for 4 h, the uptake rate by B16F10 cells incubated with TSAIII-free drug was 8.39% ± 0.32%, while that of B16F10 cells incubated with GPP-TSAIII composite NPs was 99.8% ± 0.10%.A significant difference was observed between the two samples (p < 0.01).After cells and drug were co-incubated for 4 h, the TSAIII free drug uptake rate by incubated B16F10 cells was 15.53% ± 0.47%, while that by B16F10 cells incubated with GPP-TSAIII composite NPs was 99.86% ± 0.58%.A significant difference was observed between the two samples (p < 0.01).The experimental results are consistent with those observed under a laser confocal microscope and further suggest that GPP-TSAIII composite NPs enhanced the accumulation of drugs in cells.

Evaluation of cell migration in vitro
A wound healing experiment was performed to observe cell migration that gradually decreased the size of a scratch area and to investigate the effect of the drug on cell migration [38].As shown in Figure 7a, after 24 h of incubation, scattered cells were observed in the scratches of the control group, and the scratch area remarkably decreased.The healing rate of the control group (52.76 ± 6.82%) was significantly higher than that of the other treatment groups (TSAIII: 3.08 ± 2.01%; GPP-TSAIII+Laser: 13.08% ± 1.49%; GPP-TSAIII: 22.69% ± 1.66%, p < 0.05), but no significant difference was observed with the GPP+Laser (43.61 ± 2.59%) group.The healing rate of the TSAIII group was lower than that of the GPP-TSAIII+Laser drug delivery system group and GPP-TSAIII group, and the difference was statistically significant (p < 0.05), which may be caused by the sustained release effect of GPP-TSAIII.Although a difference was observed in the scratch healing rate between the GPP-TSAIII+Laser group and the GPP-TSAIII group, the difference was not statistically significant (p > 0.05, Figure 7b).In summary, the experimental results show that TSAIII (6 µg/mL) can significantly inhibited the migration of B16F10 cells, and the inhibitory effect was not necessarily related to whether a laser was applied.The GPP carrier material itself did not inhibit the migration of B16F10 cells, nor did it significantly inhibit the migration of B16F10 cells with the laser treatment.However, the healing rate decreased slightly with the laser treatment, which may have been caused by the death or deterioration of B16F10 cells caused by the photothermal conversion of GPP materials.After 48 h of treatment and incubation, the scratches of the blank group were covered with cells, and the scratches were no longer obvious.The healing rate of the blank group (89.03% ± 2.42%) was significantly higher than that of the other treatment groups (TSAIII: 31.27%± 3.65%; GPP-TSAIII+Laser: 38.76% ± 0.66%; GPP-TSAIII: 44.24% ± 2.65%, p < 0.01, Figure 7b), but no significant difference was observed with the GPP+Laser (87.59% ± 2.16%) group.The healing rate of the TSAIII drug group was lower than that of the GPP-TSAIII+Laser drug delivery system group and the GPP-TSAIII composite NP group, but the difference was not statistically significant (p > 0.05), confirming that the difference in the healing rate after 24 h of drug incubation was caused by the slow-release effect of GPP-TSAIII composite NPs.These results show that TSAIII (6 µg/mL) incubation for 48 h significantly inhibited the migration of B16F10 cells, and the inhibition was independent of laser treatment.The GPP carrier material itself did not inhibit the migration of B16F10 cells and did not significantly inhibit the migration of B16F10 cells with laser treatment.

Evaluation of cell apoptosis detection in vitro
As shown in Figure 7c, the apoptosis rate of B16F10 cells was detected using flow cytometry.The TSAIII, GPP-TSAIII, and GPP-TSAIII+Laser treatment groups all induced the apoptosis of B16F10 cells after the same incubation time, but to different degrees.After 6 h of TSAIII incubation, the prophase apoptotic rate of B16F10 cells was 4.80% ± 0.40%, the metaphase apoptotic rate was 13.30% ± 1.10%, the total cell apoptosis rate was 18.14% ± 0.70%, and the cell death rate was 22.10% ± 0.30%.In the GPP-TSAIII group, the prophase apoptosis rate was 15.66% ± 0.16%, the metaphase apoptosis rate was 16.41% ± 0.45%, the total cell apoptosis rate was 32.07%± 0.60%, and the cell death rate was 8.66% ± 0.71%.In the GPP-TSAIII+Laser group, the prophase apoptosis rate was 2.01% ± 0.90%, the metaphase apoptosis rate was 9.11% ± 2.32%, the total cell apoptosis rate was 11.12% ± 3.18%, and the cell death rate was 26.19% ± 1.64% (Figure 7d).The above results revealed that TSAIII, GPP-TSAIII, and GPP-TSAIII+Laser induced the apoptosis of B16F10 cells.The prophase and metaphase, and total apoptosis rates of the TSAIII group were lower than those of the GPP-TSAIII group, while the cell death rate was higher than that of the GPP-TSAIII group (p < 0.01), which revealed the slow-release effect of GPP-TSAIII.In the GPP-TSAIII+Laser group, although prophase, metaphase, and total apoptotic rates were lower than those of the TSAIII-free drug group, the mortality rate was significantly higher than that of the TSAIII-free drug group.The experimental results revealed that the GPP-TSAIII+Laser drug delivery system induced photothermal therapy to kill tumor cells.

Effect on the tumor microenvironment
To evaluate the antitumor activity, we performed PTT treatment every other day for five treatments.The experiment was terminated when the tumor in the blank control model group was larger than 500 mm 3 (Figure 8a).In comparison with the other treatment groups, GPP-TSAIII+Laser treatment effectively delayed tumor growth (Figure 8a).Moreover, the body weight of mice treated with GPP-TSAIII+Laser did not change significantly (<10%, Figure 8b), indicating that this new PTT therapeutic drug delivery system had good biocompatibility.In comparison with the other groups, the tumor weight of the B16F10 tumor-bearing mouse model treated with GPP-TSAIII+Laser showed that tumor growth was significantly reduced (Figure 8c).
The positive rate of immunohistochemical sections reflects the number of positive cells [39].Tissue was stained for CD11B, CD11C, and CD45 and the results were quantified.CD11B, CD11C, and CD45 levels increased after treatment with GPP-TSAIII (Figure 8d and Supplementary Figure S2) and at a higher level than the other groups.In addition, after GPP+Laser treatment, CD11B, CD11C, and CD45 had no significant changes.The results showed that in the GPP-TSAIII+Laser group, TSAIII stimulated an immune response and further inhibited tumor growth.
Evaluating the immune response after treatment in the B16F10 tumor model was extremely challenging [40,41].After treatment, all B16F10 tumors were collected and analyzed using flow cytometry.Due to improved bioavailability of TSAIII, the GPP-TSAIII-treated group had more CD11C+ cells infiltrated into the tumors of the mice and downregulated T-Treg cell expression in the tumor tissue compared to the control group (Figure 8e and Supplementary Figure S3).These results were consistent with other three other treatment groups.In general, the observation results showed that when using GPP-TSAIIII+Laser combination therapy, TSAIII triggered an enhanced T cell-mediated anticancer immune response and inhibited Treg cell-mediated tumor progression, generating a more significant antitumor effect.
When tumors occur, the supply of nutrients to the body, especially the supply of protein, is very important.Alterations of amino acid metabolism pathways in tumor cells are often driven by multiple signaling pathways and transcription factors.Currently, methods for targeting amino acid metabolism include the inhibition of amino acid transporters, biosynthesis, and consumption.Cancer cells meet the increased demand for amino acids by upregulating the expression of amino acid transporters [42].We collected the serum of tumor-bearing mice after the experiment and extracted water-soluble metabolites.Mass spectrometry was performed to detect the amino acid levels.According to the mass spectrometry results, we detected a total of 21 amino acids, among which 12 had obvious changes.As shown in Figure 9, compared with the control group, in the serum of mice in the TSAIII model group, the levels of L-leucine (Leu), L-histidine (His), L-phenylalanine (Phe), L-Glutamine (Gln), L-isoleucine (Lie), L-aspartic acid (Asp), L-tryptophan (Trp), L-valine (Val), L-serine (Ser), and L-arginine (Arg) tend to increased significantly, whereas that of L-methionine (Met), and L-ornithine hydrochloride (Orn) had a downward trend, indicating that TSAIII can regulated the abnormal levels of amino acid metabolites in the tumor microenvironment.This difference affects the proliferation and migration of tumors, which has positive significance for finding new treatment strategies for melanoma.This topic needs to be further explored.

Combination antitumor efficacy in vivo
Controlling the growth of large tumors is one of the urgent challenges of traditional anticancer therapies [43][44][45][46].Functionalized graphene materials are ideal candidates for local combined cancer therapy.Therefore, in the present work, tumorbearing mice with an initial tumor size of approximately 110 mm 3 were used to evaluate the combined antitumor efficacy.
The timeline of the modeling and drug administration in the nude mice is shown in Figure 10a.The results of the laser treatment and heating of the tumor site are shown in Figure 10b.After an intratumoral injection of GPP-TSAIII composite NPs and NIR irradiation (808 nm, 2 W cm −2 ) for 3 min, the temperature of the tumor site in the mice increased significantly to approximately 42.5°CWhen the ambient temperature is higher than 42°C, tumor cells begin to die. Figure 10c shows that during the administration period, the body weights of the mice in the saline group, cisplatin group, GPP+Laser group, and GPP-TSAIII+Laser group did not change significantly (<10%).However, during the treatment period, the mice in the cisplatin group became thinner as the tumor volume became larger, and their mental and activity status were not as good as those of the other groups.Although the body weights of TSAIII and GPP-TSAIII-treated mice increased significantly (>10%) at the end of the administration, the mice's body changes were not obvious during the period, and the mental and activity status of the mice were good.The weight change was mainly caused by the change in tumor volume.Figure 10d shows that compared with the saline group, the tumor volume of the other treatment groups grew slowly.The tumor volume growth rate in the GPP-TSAIII+Laser administration group was the slowest, and the tumor volume did not change significantly compared with the initial tumor volume.Figure 10e shows that after administration, the mice in the saline group had the largest tumor volume and the greatest mass.In comparison with the other administration groups, significant differences were observed (p < 0.05).The tumor volume in the GPP-TSAIII+Laser administration group was the smallest and the lightest in terms of mass.In comparison with the other groups, significant differences were observed (p < 0.05).The tumor inhibition rate data show that the tumor inhibition effect of the GPP-TSAIII+Laser group is the best, and a significant difference was observed compared with other groups.Based on the above experimental results, the GPP-TSAIIII+Laser drug delivery system combined the advantages of GPP+Laser photothermal therapy and TSAIII antitumor therapy and achieved the drug effect of combined drug delivery.The tumor suppression rate was determined using TSAIII treatment without laser irradiation.The rate was 1.9 times that of the GPP+Laser treatment group without drugs and 1.1 times that of the GPP+Laser treatment group, which effectively inhibited the growth of tumors.
From the H&E sections of the mouse pathological tissues (Figure 10f), tumor necrosis (red arrow) and a small amount of local hemorrhage (blue arrow) were seen in the saline group.The tumor cells had a high nucleoplasmic ratio, and the nuclear fission phase was occasionally seen (black arrow).The positive CIS group had about 5% local tumor necrosis, high nucleoplasmic ratio of tumor cells, loose and lightly stained cytoplasm, and in some cases even vacuolated cytoplasm (green arrow).No obvious necrosis was seen in the tumor cells of GPP-TSAIII+Laser group.However, mice in the CIS administration group showed obvious lung inflammation and kidney injury, so GPP-TSAIII+Lase is a promising low toxicity anti-melanoma delivery system.

Discussion
Taking advantage of its dense near-infrared light absorption, PTT with graphene as a substrate material is widely used in antitumor applications [47,48].The large surface area derived from the two-dimensional structure of graphene produces enough active sites and space for multifunctional groups and therapeutic modalities for combined cancer therapy.However, when in contact with biological media, PTT with graphene allows more protein uptake to form 'protein crowns,' leading to rapid elimination and lack of tumor targeting [49,50].To overcome this shortcoming, researchers have made various attempts to artificially modify graphene to modulate protein crown formation and enhance tumor distribution [51].Although research on graphene has progressed significantly over the years, it has only been evaluated in animal models and no clinical applications have been reported.
We functionalized GO sheets with PEG and PVA in a stable and controlled manner, thereby increasing their hydrophilicity and surface area, and significantly improving drug encapsulation rates and photothermal conversion to amplify thermochemotherapy.Mitochondria, as the 'energy supply station' for all living cells in the body, are considered to be one of the key targets for cancer therapy.Mitochondria are widely used as targets because they are not only the cellular powerhouse for the production of adenosine triphosphate, but also the catalyst for the activation of programmed cell death.In living cells mitochondria are the main organelle for the production of ROS, and up to 90% of ROS originate from mitochondria [52,53].Imbalance of ROS in mitochondria can lead to mitochondrial dysfunction, which is a determinant of apoptosis [54].In a previous study, we found that TSAIII could induce mitochondrial dysfunction in melanocytes, leading to apoptosis and autophagy and thus inhibited the proliferation and migration of tumor cells [55].However, its clinical application is limited by poor water solubility, and there is an urgent need to develop new drug delivery systems to improve its bioavailability.Based on this, we constructed a GPP-TSAIII drug delivery system.We demonstrated that GPP-TSAIII not only improved the aqueous solubility and bioavailability of TSAIII, but also improved the uptake of the drug by tumor cells, resulting in effective inhibition of tumor growth and metastasis under NIR irradiation.We hope that this combined drug delivery strategy can provide inspiration for multimodal applications of graphene oxide to enhance anticancer activity and reduce side effects, providing a basis for early clinical application.
Currently, researchers are still very much committed to the development of inorganic 2D materials for PTT [56][57][58][59] to meet the urgent need for therapeutic agents with good photostability, and excellent biocompatibility and photothermal conversion efficiency [60].However, as with graphene-based materials, a comprehensive safety assessment of these materials including toxicity, biodistribution and biodegradability, as well as reliable synthesis methods on an industrial scale, remain the primary issues to be addressed before clinical translation [61,62].Although many nanocarriers have been developed to improve this phenomenon to some extent, the fundamental problem is still unsolved.In their research work, Zhao et al. [63]proposed the concept of 'drug-free therapy,' i.e. without the use of traditional toxic drugs.The maximizes therapeutic benefits without consuming therapeutic agents, providing inexhaustible therapeutic capacity during the treatment process.This therapeutic strategy provides a new way to achieve efficient and low-toxic cancer treatment, which is worthy of further research and exploration.

Conclusions
We developed a novel strategy for a GO-and PVA-based TSAIII delivery system to enhance the local treatment of melanoma.Compared with TSAIII, GPP-TSAIII exhibited good biocompatibility, demonstrating its flexibility in biomedical applications.GPP-TSAIII achieved better antitumor activity by promoting tumor cell uptake, enhancing intratumor drug accumulation, and enhancing antitumor immunity.Furthermore, both in vivo and in vitro antitumor results demonstrated that GPP-TSAIII+Laser, a combined photothermal therapy and drug treatment strategy, showed the highest antitumor efficacy in both a B16F10 cell model and animal model.It also showed significantly lower tissue toxicity and achieved better biosafety in vivo compared to the positive drug cisplatin.This suggests that functionalized GO is a potential carrier for TSAIII, providing superior biosafety with enhanced antitumor activity while ensuring high encapsulation rates.This delivery strategy based on photothermal therapy and drug treatment provides an encouraging avenue for the treatment of melanoma.

Figure 4 .
Figure 4. TSAIII affects mitochondrial respiration and glycolysis in B16F10 cells.(a and b) ECARs and OCRs in the indicated B16F10 cells were detected using a Seahorse XF96 extracellular flux analyzer under basal conditions or in response to the indicated inhibitors.Basal glycolytic and respiration rates are summarized in the right panel (n=6).(*p<0.05,**p<0.01,***p<0.001).

Figure 6 .
Figure 6.(a and b) Results of the cell proliferation inhibition experiment after incubating the cells with the drugs for 24 h.B16F10 cell uptake measurement results of the different drugs: (c) observation under a laser confocal microscope of uptake of TSAIII and GPP-TSAIII by B16F10 cells after incubation for 2, 4, or 6 h.(d) flow cytometry measurement of the uptake of TSAIII by B16F10 cells after incubation for 2, 4, or 6 h.(e) flow cytometry measurement of the uptake of GPP-TSAIII by B16F10 cells after incubation for 2, 4, or 6 h (*p < 0.05, **p < 0.01, ***p < 0.001).

Figure 7 .
Figure 7. Results of the cell scratch test and the apoptotic rate.(a) Migration of B16F10 cells after incubating the cells with the drug solution for 24 or 48 h.(b) Three wells were used for each concentration, five photo fields per well were obtained, the void area was measured, and quantitative statistics were conducted to determine the percentage of cell migration.(c, d) Flow cytometry was used to detect the apoptotic rate of B16F10 cells treated with TSAIII, GPP-TSAIII, or GPP-TSAIII +Laser for 24 h (n = 3, *p < 0.05, **p < 0.01, ***p < 0.001).

Figure 10 .
Figure 10.Evaluation of antitumor efficacy in vivo.(a) Timeline of administration and treatment of mice.(b) laser irradiation induced the temperature rise of tumor sites in mice as a result of changes over time.(c) changes in body weight of mice in each group during the administration period.(d) changes in tumor volume of mice in each group during the administration period, (E) comparison of tumor size of mice in each group (*p < 0.05, **p < 0.01, ***p < 0.001).(F) Histological characteristics of tumor, heart, liver, spleen, lung and kidney excised from B16F10 tumor-bearing mice.The images were obtained using a digital microscope at 400 ×, magnification scale bar = 50 μm.