Introduction

These days, melanoma, a malignant conversion of melanin-producing cells, is presented as one of the most aggressive and metastatic skin cancers [ 1 , 2 ]. While ordinary disease treatment experiences low accuracy, high risks, and troublesome reactions in healthy cells, improvement of sheltered, viable and helpful modalities with moderated antagonistic impacts is significant in melanoma cancer treatment. Today, we look for rational treatments with the least amount of invasion and best results.

Photothermal (PTT) and photodynamic (PDT) therapies are groundbreaking therapeutic modalities that leverage nanotechnology and light-based interventions to address the limitations of conventional melanoma treatments [ 3 - 5 ]. Nano systems enhance treatment outcomes by protecting sensitive therapeutic agents, optimizing pharmacokinetics, increasing bioavailability, and serving as carriers for natural compounds, drugs, and genes or acting directly as anti-melanoma agents [ 5 ]. The success of nanomedicine is evident in the approval and ongoing clinical trials of various formulations [ 4 - 6 ]. Activation by external stimuli, in PTT and PDT, underscores the versatility of nano systems in melanoma therapy. PTT and PDT utilize laser irradiation and photosensitizing agents to trigger a cascade of chemical, biological, and physiological reactions, leading to the demise of neoplastic cells. PTT is based on the conversion of visible or near-infrared (NIR) light energy into thermal energy, increase in temperature at the tumor location, and induction of apoptosis and necrosis. However, the killing mode of PDT on tumor cells is through light absorption of photosensitizer and formation of its excited singlet and triplet states that are followed by electron transfer to other molecules (such as molecular oxygen), and final reactive oxygen species (ROS) generation [ 5 , 6 ]. The mechanisms include endoplasmic reticulum stress, ROS-mediated autophagy, release of damage-associated molecular patterns, and immunogenic tumor cell death [ 7 ]. Indeed, PTT and PDT, coupled with nanotechnology advancements, present a multifaceted and promising approach for melanoma treatment, addressing challenges and offering new horizons in therapeutic efficacy and safety. Although some pre-clinical investigations are being followed in the field of PTT/PDT in a wide range of cancers, including melanoma, there are still outstanding issues which should be clarified in future studies regarding the approval of these modalities in melanoma cancer treatment. Ongoing research in Nano systems, immunomodulation, and combination therapies continue to drive innovation in the field [ 5 ].

Among a variety of NIR photothermal transducers, gold nanoparticles (AuNPs) have critical functions including surface plasmon resonance (SPR) activity to convert the NIR light into local heat, size ward shading, biocompatibility, low toxicity, and simple surface functionalization [ 8 ]. Therefore, it is expected to achieve advanced anti-cancer property by combining PTT and chemotherapeutic drugs based on AuNPs as a photosensitizer. Up to now, researchers have orchestrated different sorts of AuNPs with various properties of continuous imaging, and photothermal/photodynamic and nanozyme oxidative treatments [ 9 ]. Synergistic PTT/chemotherapy of 4T1 cells using AuNPs coupled with metformin [ 10 ], mesoporous silica-coated gold nanorods for gene/immunotherapy of MC38 cells [ 11 ], green synthesized AuNPs for PTT of basal cell carcinoma [ 12 ], PTT of Colorectal Cancer in a Mouse Model using gold nanoshells-combined liposomal doxorubicin (DOX) [ 13 ], a series of thiol-terminated PEG-paclitaxel derivatives-conjugated AuNPs [ 14 ] and multifunctional gold-DOX nanoparticles system for pH-triggered intracellular anticancer drug release, and provision of a promising platform for intracellular delivery of a variety of anticancer drugs [ 15 ] have been the routes for PTT/chemotherapy of cancer.

Recently, conducting polymers is being considered for a range of biomedical applications, including the development of artificial muscle [ 16 ], controlled drug release [ 17 ], stimulation of nerve regeneration [ 18 ] and biosensing [ 19 - 21 ]. Low cytotoxicity and high biocompatibility of these materials are evident from the growth of cells in conducting polymers and for indicating small degree of inflammation in test animals for several weeks [ 22 ]. Conducting polymers are redox-active with the potential to act as reducing agents and scavengers of free radicals. However, their antioxidant ability in biological media needs to be examined to assess their likely activities in biomedical applications [ 23 ]. A review of literature indicated that certain polymeric carriers by inhibiting the P-glycoprotein (P-gp) drug efflux system will sensitize drug-resistant cells to a group of cytotoxic drugs [ 24 - 26 ]. This would increase intracellular concentration of intracellular drug to the concentrations required for induction of cytotoxicity [ 27 , 28 ].

Based on a literature review, some studies have been carried out for applications of poly (2-amino phenol) (P2AO) in drug sensing [ 20 ], biosensing [ 21 ], antibacterial composites [ 29 ], biomimetic coatings [ 30 ] and protein-repellence membranes [ 31 ]. However, no attempt was made for synthesis of nanoparticles of (P2AO)/gold (P2AO/AuNPs) as a photosensitizer as well as DOX intensifier in a synergistic manner for melanoma cancer cells killing.

Material and Methods

Materials

In this experimental study, all chemicals were obtained from Sigma Chemicals Co. (USA), Scharlau Chemie Co. (Spain), or Merck Co. (Germany), and used without further purification. Before use, all glassware was washed with fresh aqua regia and then deionized (DI) water. DI water was used throughout the experiments.

A C540 (B16-F10) cell line derived from mouse malignant melanoma was prepared from Pasteur Institute of Iran. The cells were cultured in Roswell Park Memorial Institute-1640 (RPMI) medium, which contained 1% antibiotics (penicillin-streptomycin, 10,000 µg mL^-1) and 10% fetal bovine serum (FBS) in a humidified atmosphere comprising 5% CO₂ at 37 °C. Cell incubator conditions of 5% CO₂ at 37 °C were followed throughout the study.

Synthesis and characterization of P2AO/AuNPs and P2AO/AuNPs-DOX mixture (P2AO/AuNPs-DOX)

Firstly, AuNPs were prepared by the citrate method, as described previously [ 32 , 33 ]. Chloroauric acid (250 mL, 1.0 mmol L^-1) was mixed with sodium citrate (25 mL, 39 mmol L^-1), and the resultant mixture was boiled in a glass round bottom flask in a reflux equipment for 15 min. The resultant AuNPs solution was then cooled to room temperature. Then, into 4.5 mL AuNPs solution of 13.3 nmol L^-1, 300 μL sodium dodecyl sulfate solution of 40 mmol L^-1 and 1.5 mL 2-aminophenol solution of 2.0 mmol L^-1 were added. The resultant solution was mixed by a vortex mixture for one minute followed by addition of 1.5 mL acidic ammonium persulfate solution of 2 mmol L^-1 in 10 mmol L^-1 hydrochloric acid. After mixing again for one minute, the obtained solution was mixed by a stirrer for 4 h. The obtained P2AO/AuNPs were washed three times by DI water.

Zeta potential, size, and morphology of P2AO/AuNPs were evaluated by a SZ-100 HORIBA zeta potentiometer (Japan) and a Zeiss, TESCAN Mira 3-XMU field emission scanning electron microscope (FESEM, Czech Republic).

Laser instrument

A diode laser system from Thorlabs (Germany) working at 808 nm with an optical output power of 1000 mW with a bench top temperature controller was used. Output power density of the diode laser was fixed at 1.0 W cm^-2 by changing the spot size of the output mounting lens and the distance between the lens and the target. Irradiation was performed over the 96-well culture plates for 10 min.

Temperature changes of P2AO/AuNPs upon laser light irradiation

A digital thermoprobe of Lutron (Taiwan) with precision of 0.01 °C was placed in the center of the glass cuvette contained P2AO/AuNPs suspension (50 µg mL^-1) to measure temperature changes upon laser irradiation. P2AO/AuNPs suspension were irradiated by an 808-nm diode laser 1.0 W cm^-2 and temperature was recorded after 10 min.

In vitro cytotoxicity evaluation of P2AO/AuNPs, DOX and P2AO/AuNPs-DOX

In vitro toxicity effect of P2AO/AuNPs, DOX, and P2AO/AuNPs-DOX on the C540 cells was measured using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay. 24-h cultured C540 cells in the culture plates (5.0×10⁴ cells well^-1) were treated with different concentrations of P2AO/AuNPs: 0 (Ctrl), 10 (N10), 50 (N50), 100 (N100) and 250 (N250) µg mL^-1, DOX: 0 (Ctrl), 0.15 (D0.15), 0.3 (D0.3), 1.0 (D1) and 3.0 (D3) µg mL^-1, or P2AO/AuNPs-DOX with different contents of the components of P2AO/AuNPs: 50 µg mL^-1+DOX: 0 µg mL^-1 (N50), P2AO/AuNPs: 50 µg mL^-1+DOX: 0.15 µg mL^-1 (N50+D0.15), P2AO/AuNPs: 50 µg mL^-1+DOX: 0.3 µg mL^-1 (N50+D0.3), P2AO/AuNPs: 50 µg mL^-1+DOX: 1.0 µg mL^-1 (N50+D1), P2AO/AuNPs: 50 µg mL^-1+DOX: 3.0 µg mL^-1 (N50+D3), P2AO/AuNPs: 50 µg mL^-1+DOX: 0 µg mL^-1 (N100), P2AO/AuNPs: 100 µg mL^-1+DOX: 0.15 µg mL^-1 (N100+D0.15), P2AO/AuNPs: 100 µg mL^-1+DOX: 0.3 µg mL^-1 (N100+D0.3), P2AO/AuNPs: 100 µg mL^-1+DOX: 1.0 µg mL^-1 (N100+D1), and P2AO/AuNPs: 100 µg mL^-1+DOX: 3.0 µg mL^-1 (N100+D3), for 4 h. Then, the cell media were rinsed and refreshed with fresh medium culture, followed by incubation overnight in a humidified atmosphere comprising 5% CO₂ at 37 °C. The cytotoxicity was then measured by the by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. For this assay, the wells medium was washed with 100 mmol L^-1 phosphate buffer saline (PBS) three times, substituted with 100 µL of a MTT solution of 0.5 mg mL^-1 dissolved in PBS, and finally incubated for 4 h at 37 °C. Afterward, the plates were centrifuged and the wells medium was substituted with 100 μL of dimethyl sulfoxide to dissolve the MTT formazan crystals. The optical intensity of each well was recorded at 570 nm using a microplate reader of Stat Fax (USA). Cytotoxicity values were reported as the ratio of absorbance of each well and control. All measurements were performed in triplicate.

In vitro cytotoxicity evaluation of P2AO/AuNPs, DOX, and P2AO/AuNPs-DOX upon laser light irradiation

In vitro photosensitizing effect of P2AO/AuNPs, DOX, and P2AO/AuNPs-DOX on C540 cells was investigated by cytotoxicity evaluation using the MTT assay upon laser light irradiation. 24-h cultured C540 cells in the culture plates (5.0×10⁴ cells well^-1) in RPMI medium, which contains 1% antibiotics (penicillin-streptomycin, 10,000 µg mL^-1); also, 10% FBS were treated with different concentrations of P2AO/AuNPs: 0 (Ctrl), 10 (N10), 50 (N50), 100 (N100) and 250 (N250) µg mL^-1, DOX: 1.0 µmol L^-1 (D1) and P2AO/AuNPs-DOX of P2AO/AuNPs: 50 µg mL^-1+DOX: 1.0 µmol L^-1 (N50+D1) for 4 h. Then, the cell media were rinsed and refreshed with fresh medium culture, followed by laser light irradiation. After irradiation, the cells were incubated overnight in a humidified atmosphere comprising 5% CO₂ at 37 °C. Cells without any treatment were considered as controls.

Domination of any synergism effect between laser light irradiation and P2AO/AuNPs, DOX, or P2AO/AuNPs-DOX to cell killing was inspected by calculation of combination indices (CIs). CI is represented as [ 34 ]:

$\mathrm{CI}=V_A\times V_B/V_{\mathrm{AB}}$ (1)

Where V_A, V_B, and V_AB are the cell viabilities under effects of A, B (alone) and their combination, respectively. For, CI>1, CI=1, or CI<1, domination of synergism, additive or antagonism effects of A and B agents, respectively, is witnessed.

Detection of intracellular generation of ROS

As a signal of intracellular ROS production, intensity of fluorescence emission of 2′,7′-dichlorodihydrofluorescein diacetate (H2DCF-DA) was measured. The cells were seeded in 96-well plates at a density of (1×10⁵ cells well^-1) in RPMI medium, which contains 1% antibiotics (penicillin-streptomycin, 10,000 µg mL^-1) and 10% FBS, and prepared according to the procedure mentioned in sections 2.5 with the difference that after 3.5 h of incubation, the cells were treated by 100 μL of a fresh H2DCF-DA solution (50 μmol L^-1) followed by incubation continuation for 30 min. Then, laser light was the irradiation used in the related cells, and after 30 min, the extracellular H2DCF-DA was rinsed thrice with PBS; ultimately, 100 mL of a lysis buffer (containing 0.1% triton X-100, 150 mmol L^-1 NaCl, 50 mmol L^-1 Tris-HCl buffer, pH=8.0) was added into each well. The intensity of fluorescence emission at 520 nm was measured upon excitation at 485 nm in black plates using a microplate reader of Biotek (USA).

Apoptosis assay

Apoptosis was evaluated by fluorescence-activated cell sorting (FACS analysis), using Annexin V-FITC/PI staining kit from BD Pharmingen (USA). The C540 cells were seeded in 12-well plates at a density of 1×10⁵ cells well^-1 and prepared according to the procedure mentioned in sections 2.5. After 24 h of incubation, the cells were harvested, washed with PBS, and stained with Annexin V-FITC (5 µL) and propidium iodide (5 µL), followed by flowcytometry analysis by BD FACSCalibur (USA). The apoptosis rate was evaluated based on the fluorescence of 10,000 events.

In vitro evaluation of P2AO/AuNPs uptake using inductively coupled plasma optical emission spectrometry (ICP-OES)

For determination of the uptake quantity of P2AO/AuNPs by C540 cells cultured in RPMI medium, which contains 1% antibiotics (penicillin-streptomycin, 10,000 µg mL^-1) and 10% FBS, 24-h post-seeding cells were treated with P2AO/AuNPs. Then, they were washed with PBS thrice and lysed and digested with aqua regia. Gold content of the cells was determined using a Varian 730-ES ICP-OES (USA) in peak hopping gas mode.

Statistical analysis

For each determination, a minimum of three parallel measurements were done, and final values were expressed as mean±standard deviation. Non-parametric Kruskal-Wallis- and T-tests were employed for the analysis of statistical significance of the values using the Prism 6 software. P-values less than 0.05 were considered statistically significant.

Results

Although new drugs and expectations have arisen for patients with melanoma in recent years, it is generally still incurable over the last 30 years. The new discovered PTT has the potential to treat this type of cancer in some stages and prolong the patient’s life and can shrink the tumor and relieve symptoms. PTT is not as aggressive as conventional chemotherapy because plasmonic AuNPs used which can absorb light at specific wavelengths result in hyperthermic cancer treatments. It would be expected that P2AP with biocompatibility and low cytotoxicity can extend the blood half-life of P2AO/AuNPs, enhance cellular uptake, and reduce non-specific protein adsorption to some extent. We made an attempt to introduce P2AO/AuNPs-DOX as a dual therapy including photothermal therapy and chemotherapy in a synergistic manner working at reduced concentrations of both the photosensitizer and drug.

Morphology and size of P2AO/AuNPs were inspected by FESEM. A typical FESEM image of P2AO/AuNPs is presented in Figure 1. The FESEM image showed that P2AO/AuNPs consisted of spherical gold nanoparticles covered with an amorphous layer of P2AP which were connected together. The average size of P2AO/AuNPs was 9.5±5 nm. Particle size analysis by dynamic light scattering for Au@POAP NPs (Figure 2) showed hydrodynamic diameter of 11±4 nm, with a polydispersity index of 0.5. Zeta potential of P2AO/AuNPs was also measured to be -0.5 mV.

Figure 1. A Field emission scanning electron microscopy (FESEM) image of P2AO/AuNPs. The image consisted of spherical gold nanoparticles covered with an amorphous layer of P2AP

Figure 2. Particle size analysis of Au@POAP NPs by dynamic light scattering. Au@POAP nanoparticles showed hydrodynamic diameter of 11±4 nm, with a polydispersity index of 0.5

To evaluate the photothermal effect of P2AO/AuNPs, their temperature increment at concentration of 50 µg mL^-1 was measured under 808-nm irradiation at a power density of 1 W cm^-2 for 10 min and a temperature increment of 5.32 °C was recorded.

Figure 3 shows cytotoxicity effects of P2AO/AuNPs, DOX, and P2AO/AuNPs-DOX toward C540 cells evaluated by the MTT assay. The results of cytotoxicity of different concentrations indicated that P2AO/AuNPs had a relative toxicity (cell viability >56% in the presence of 10, 50 and 100 µg mL^-1, and ~29% in the presence of 250 µg mL^-1) with an estimated half-maximal inhibitory concentration (IC50) of 140 µg mL^-1. Cytotoxicity of different concentrations of DOX also resulted in an IC50 of ~4.6 µmol mL^-1. Combination of a fixed amount of P2AO/AuNPs of 50 µg mL^-1 with various concentrations of DOX (0.15, 0.3, 1.0 and 3.0 µmol mL^-1) led to a decreased IC50 of ~0.17 µmol mL^-1 for DOX. Moreover, combination of P2AO/AuNPs of 50 µg mL^-1) with various concentrations of DOX (0.15, 0.3, 1.0 and 3.0 µmol mL^-1) led to a decreased IC50 of ~0.10 µmol mL^-1 for DOX.

Figure 3. Cytotoxicity effects of P2AO/AuNPs, DOX and Au@POAP NPs +DOX toward C540 cells. Different concentrations of P2AO/AuNPs (0, 10 (N10), 50 (N50), 100 (N100) and 250 μg mL^-1 (N250)), DOX (0.15 (D0.15), 0.3 (D0.3), 1.0 (D1) and 3.0 µmol L^-1 (D3)), and P2AO/AuNPs-DOX (Au@POAP NPs of 50 μg mL^-1+DOX of 0.15, 0.3, 1.0 and 3.0 µmol L^-1 (N50+D0.15, N50+D0.3, N50+D1 and N50+D3), and Au@POAP NPs of 100 μg mL^-1+DOX (0.15, 0.3, 1.0 and 3.0 µmol L^-1 (N100+D0.15, N100+D0.3, N100+D1 and N100+D3)) was evaluated by the MTT assay

Figure 4 shows cytotoxicity effects of P2AO/AuNPs and P2AO/AuNPs-DOX upon laser light irradiation toward C540 cells evaluated by the MTT assay. The results showed a cell viability of 91% upon laser light irradiation, indicating that the irradiation had a non-toxic effect. However, laser light irradiation of P2AO/AuNPs induced cell toxicities more that treatment with irradiation or P2AO/AuNPs alone, and the viability values went through CIs>1. CIs for all the combination treatments are presented in Table 1. The results showed that laser light irradiation of DOX represented an additive effect arising from the effects of irradiation and the drug separately. Also, while P2AO/AuNPs had lower toxicity than DOX, laser light irradiation induced higher cell killing ability of P2AO/AuNPs than DOX. This further confirmed the photosensitizing ability of P2AO/AuNPs. In addition, cell treatment with P2AO/AuNPs-DOX, either with or without laser light irradiation, represented additive effects, when compared to treatment with P2AO/AuNPs or DOX alone. However, when laser light irradiation and treatment with P2AO/AuNPs-DOX of the cells were considered as two independent parameters, the results indicated domination of a synergistic effect for PTT, using P2AO/AuNPs-DOX. Regarding the C540 cellular uptake of P2AO/AuNPs during 4 h of incubation time, a value of 4.47 ng cell^-1 was obtained.

Figure 4. Cytotoxicity effects of P2AO/AuNPs, DOX and Au@POAP NPs+DOX without () and with laser light irradiation () toward C540 cells. Different concentrations of P2AO/AuNPs (0, 10 (N10), 50 (N50), 100 (N100) and 250 μg mL^-1 (N250)), and DOX (1.0 µmol L^-1 (D1)), P2AO/AuNPs-DOX (Au@POAP NPs of 50 μg mL^-1+DOX of 1.0 µmol L^-1(N50+D1)), without () and with laser light irradiation () was evaluated by the MTT assay

Intracellular ROS generation during PTT treatment of C540 cells was evaluated using the intensity of fluorescence emission of H2DCF-DA. H2DCF-DA passively penetrates the cells, and it is deacetylated by esterases to non-fluorescent H2DCF. Intracellular ROS interacts with H2DCF and produces fluorescent DCF. Figure 5 shows the intensities of fluorescence emission at 520 nm arising from intracellular ROS generation upon C540 cell treatment with P2AO/AuNPs, DOX, and P2AO/AuNPs-DOX, either with or without laser light irradiation, while the intensity of fluorescence emission of the control cells was considered as a base signal. Without laser light irradiation, the results and a comparison with those presented for cytotoxicity effects indicated that the generated ROS levels for cell treatment with P2AO/AuNPs and DOX, as presented in Figure 5, were in disagreement with the corresponding results presented in Figures 3 and 4; while ROS level for treatment with P2AO/AuNPs was greater than DOX, cell killing ability of DOX was better. The quantities of generated ROS also indicated that P2AO/AuNPs and DOX could play a synergistic role to generate ROS, either with or without laser light irradiation. Laser light irradiation of P2AO/AuNPs-DOX produced the highest ROS level.

Figure 5. The impact of NIR on ROS production in C540 cells treated with P2AO/AuNPs. Intensities of fluorescence emission at 520 nm arising from intracellular ROS generation upon C540 cell treatment with P2AO/AuNPs (50 μg mL^-1 (N50)), DOX (1.0 µmol L^-1 (D1)) and P2AO/AuNPs-DOX (Au@POAP NPs of 50 μg mL^-1+DOX of 1.0 µmol L^-1 (N50+D1)), without () and with laser light irradiation ()

Figure 6A shows the occurrence of apoptosis in the C540 cells upon PTT was evaluated by FACS analysis; scatter plots containing Q1 to Q4 areas indicate necrotic, late apoptotic, early apoptotic, and live cells, respectively, so that the sum of Q1, Q2 and Q3 was considered as necroptotic. Figure 6B and C show population of different cells upon treatments. Without laser light irradiation, the controls showed an insignificant number of cells in necroptotic stages (7%) with 93% of live cells. Cell treatment with P2AO/AuNPs resulted in an insignificant increase in the necroptotic cells (16%) with slight decrease in the percentage of live cells (84%). Cell treatment with DOX resulted in necroptotic population of 27%. Cell treatment with P2AO/AuNPs-DOX resulted in moderate increase in necroptotic population percentage of 48%. Laser light irradiation alone also resulted in a slight increase in the necroptotic cells (15%) and a slight decrease in live cells (85%). With laser light irradiation, cell treatment with P2AO/AuNPs and DOX resulted in moderate increase in necroptotic cells with 46 and 31%, respectively. Cell treatment with P2AO/AuNPs-DOX, however, resulted in a considerable increase in necroptotic cells to 61% with a significant decrease in the live cells (39%).

Figure 6.A: Evaluation of the apoptotic effect induced by P2AO/AuNPs in C540 cells. FACS analysis results as scatter plots for treatment of C540 cells with P2AO/AuNPs (50 μg mL^-1 (N50)), DOX (1.0 µmol L^-1 (D1)) and P2AO/AuNPs-DOX (Au@POAP NPs of 50 μg mL^-1+DOX of 1.0 µmol L^-1 (N50+D1)), without () and with laser light irradiation (). B, C: Quantitative assessment of apoptotic responses in C540 cells induced by P2AO/AuNPs. Percentages of viability of live, early apoptotic, late apoptotic and necrotic C540 cells

Discussion

The size, relatively uniform size distribution, and zeta potential of P2AO/AuNPs show that it can directly and effectively penetrate the cancer cells and tumor tissues; among other things, due to its small size, it can be removed from the body faster through renal filtration [ 35 , 36 ].

Cytotoxicity assessment and IC50 determination indicated that combination of P2AO/AuNPs with DOX resulted in better efficiency of DOX as chemotherapy agent on melanoma cancer and very low concentration of DOX in this combination needs for killing melanoma cancer cells. Therefore, combination of P2AO/AuNPs with DOX resulted in decreased consumption of the drug to attain similar cell toxicity obtained with higher doses. Laser light irradiation at 808 nm at 1.0 W cm^-2 power density of P2AO/AuNPs killed the cells in a synergistic manner at all the P2AO/AuNPs concentrations. However, the results confirmed that laser light irradiation had further effect on DOX cytotoxicity. Therefore, P2AO/AuNPs had lower toxicity than DOX; laser light irradiation induced higher cell killing ability of P2AO/AuNPs than DOX. This further confirmed the photosensitizing ability of P2AO/AuNPs, and synergism behavior of P2AO/AuNPs-DOX upon laser light irradiation indicated that P2AO/AuNPs uptake in cells absorbed 808-nm light to generate heat on one hand and accelerated DOX affection upon light irradiation. The light of 700- to 1000-nm is a preferable source of energy for cancer cell treatment because it is not absorbed by proteins and DNA and has a deep penetration in tissues [ 37 ]. The order of more intracellular ROS generation occurred in the presence of P2AO/AuNPs-DOX, P2AO/AuNPs, and then DOX upon laser light irradiation. While cell killing ability of DOX was better, the results of intracellular ROS indicated that the routes of cell killing of P2AO/AuNPs and DOX were different, and that for P2AO/AuNPs it was mainly based on ROS generation. Also, synergistic effects arising from combinations of P2AO/AuNPs and DOX, P2AO/AuNPs and light irradiation, and DOX and light irradiation induced ROS generation. While chemotherapy of melanoma cells with DOX induced less killing via necroptotic, its combination in P2AO/AuNPs-DOX upon laser light irradiation resulted in significant cell loss via necroptotic.

C540 cells PTT using P2AO/AuNPs-DOX works with three different paths: i) generates a hyperthermal microenvironment leading to induction of necrosis and/or apoptosis, ii) retards the repair of DNA damage induced with AuNPs [ 38 , 39 ], and iii) intercalation of DOX into DNA and disruption of topoisomerase-II-mediated DNA repair. This study supports the idea that P2AO/AuNPs-DOX provides a good laser-responsive activity with milder chemotherapeutic conditions.

However, as with many studies, the design of the current study would be followed by in vivo investigations and clinical trials, both of which being essential for converting research results from laboratories into useful medicinal uses. Future research can also explore the effectiveness of P2AO/AuNPs in various melanoma subtypes or other types of cancers.

Conclusion

In this study, a photothermal therapy using P2AO/AuNPs was developed based on 808-nm light absorbing of both P2AO and AuNPs and energy conversion preliminary into heat. The photosensitizer P2AO/AuNPs and laser light killed C540 cancer cells through heat and ROS generation. We showed that P2AO coating of AuNPs provided a suitable platform to carry DOX, and light irradiation of P2AO/AuNPs-DOX led to intensifying DOX therapeutic effect. The employed components provided a new photothermal/chemotherapy by domination of synergistic effects attained by combination of laser light, P2AO, AuNPs, and DOX. Generation of hyperthermal microenvironment upon light irradiation of both P2AO and AuNPs, retardation of DNA damage repairing by AuNPs and DOX, and disrupting the structure of DNA led to intensifying DOX efficacy.

Although PTT holds promise in its selectivity and reduced invasiveness compared to conventional therapies, currently, using the results of this research in vivo is facing challenges. Continuous NIR energy could lead to overheating, thermal injury, inflammation, and uncomfortable and pain for patients. Moreover, challenges in intracellular delivery of P2AO/AuNPs pose additional concerns. Large tumors often feature hypoxic regions with limited blood flow and high interstitial pressure, retarding effective NP delivery. This unequal distribution and restricted laser depth induce heterogeneous heat dispersal in tumor during PTT, reducing treatment effectiveness and complicating simultaneous cancer cell eradication. Therefore, it is necessary to consider the limitations of the present research and seek a solution and opportunities for future research.

Acknowledgment

We would like to thank the Research Council of Shiraz University of Medical Sciences for supporting this research.

Authors’ Contribution

Regarding the authors’ contributions, N. Sattarahmady and Z. Kayani contributed to the study conception and design. N. Sattarahmady conceived the original idea and H. Heli supervised the project. Z. Kayani played a pivotal role in designing the procedure and fabrication of the nanoparticles. Material preparation, data collection, and analysis were performed by Z. Kayani, P. Faghani-Eskandarkolaei, and H. Haghighi. All authors actively contributed in writing and reviewing the manuscript, ensuring a comprehensive and well-rounded study. All authors read, modified, and approved the final version of the manuscript.

Ethical Approval

The national ethics committee confirmed the study with the ethical code of IR.SUMS.REC.1398.1179. We did not perform any intervention in therapeutic procedures. Therefore, gathering the consent forms was waived due to the nature of this study.

Funding

This work was supported by Shiraz University of Medical Sciences (Shiraz, Iran) with the grant number of “18253”.

Conflict of Interest

None

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