Photo-immuno nano-bomb for co-delivery of Ce6 and R837 potentiates immunogenic cell death and amplifies anti-tumor efficacy in cutaneous squamous cell carcinoma

Abstract

Cutaneous squamous cell carcinoma (cSCC) remains a formidable clinical challenge, constrained by the drawbacks of current treatments such as functional loss from surgery, radioresistance, and low adherence to protracted topical regimens. To address these issues, we designed a “photo-immuno nano-bomb” composed of polydopamine nanoparticles (PDA NPs) for co-delivering the photosensitizer chlorin e6 (Ce6) and toll-like receptor 7 agonist imiquimod (R837), thereby integrating photothermal (PTT), photodynamic (PDT), and immunotherapeutic modalities. The system utilizes π–π stacking to achieve high drug loading and stability while exhibiting a dual stimuli-responsive release profile – governed by pH-dependent surface charge alteration and photothermally triggered payload liberation – enabling more precise spatiotemporal control over combination therapy. Remarkably, the nanoformulation potently suppressed tumor cell proliferation, migration, and invasion in vitro by activating apoptotic pathways. Mechanistic studies revealed that under dual-wavelength laser irradiation (660 + 808 nm), PDA-mediated PTT enhanced cellular internalization of the nanoplatform and further augmented singlet oxygen generation, ultimately inducing mitochondrial dysfunction and cytoskeletal disintegration. This synergistic action provoked severe cellular oxidative stress and organelle damage, culminating in robust immunogenic cell death (ICD). Additionally, the platform demonstrated excellent biocompatibility, achieving complete tumor regression in vivo, outperforming all mono- and combination therapy controls. Thus, this “photo-immuno nano-bomb” multimodal strategy represents a promising therapeutic alternative for advanced cSCC, delivering superior efficacy through coordinated molecular mechanisms and minimized systemic toxicity.

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1. Introduction

Cutaneous squamous cell carcinoma (cSCC) represents one of the most prevalent non-melanoma skin cancers globally, with steadily increasing incidence due to cumulative ultraviolet exposure and aging populations.^1–3 While surgical resection remains the standard treatment, a significant number of patients – particularly those with large, poorly demarcated, or recurrent tumors in cosmetically sensitive areas – are left with functional impairments and suboptimal aesthetic outcomes.^4,5 Non-surgical alternatives such as radiotherapy, cryotherapy, and topical agents often suffer from inconsistent efficacy, high recurrence rates, and damage to adjacent healthy tissues.^6,7 Thus, the development of minimally invasive yet highly effective treatment strategies that achieve complete tumor eradication while preserving normal tissue architecture remains an urgent clinical priority.

Photodynamic therapy (PDT) has emerged as a promising modality for superficial skin cancers due to its selective cytotoxicity, minimal scarring, and repeatable application.^8–12 It relies on photosensitizers to generate cytotoxic reactive oxygen species (ROS) – particularly singlet oxygen (¹O₂) – upon light activation, inducing apoptosis and necrosis in malignant cells. Despite its advantages, the efficacy of PDT in thicker (>2 mm) and invasive cSCC lesions is limited by two major factors, including inadequate light penetration depth and tumor hypoxia. Conventional visible light (e.g., 630–660 nm) used in PDT penetrates only 1–2 mm into tissue, restricting treatment to superficial lesions. Furthermore, the oxygen-dependent nature of PDT renders it less effective in hypoxic tumor microenvironments, a common feature of rapidly growing skin tumors.^13–16 Hence, although PDT has been endorsed by European guidelines for the management of in situ cSCC (Bowen disease),^17,18 its application is not advised for the treatment of invasive cSCC, owing to the inadequate accumulation of photosensitizers within the tumor tissue and the suboptimal penetration of external light sources.

To overcome these limitations, combination strategies incorporating photothermal therapy (PTT) have gained considerable attention.^19–25 PTT employs photothermal agents that convert near-infrared (NIR) light into heat, inducing hyperthermic damage in tumors. Notably, NIR light offers superior tissue penetration compared to visible wavelengths, enabling treatment of deeper lesions. More importantly, PTT can synergize with PDT through multiple mechanisms. Firstly, mild hyperthermia increases vascular perfusion and oxygen supply, alleviating tumor hypoxia and enhancing ROS generation. Secondly, heat-induced membrane permeability improves cellular uptake of photosensitizers. Last but not least, thermal effects can disrupt lysosomal and mitochondrial integrity, amplifying PDT-induced apoptosis. Beyond enhancing direct tumor ablation, PTT can also potentiate anti-tumor immunity. Local hyperthermia promotes the release of damage-associated molecular patterns (DAMPs), such as heat shock protein 70 (HSP70) and adenosine triphosphate (ATP), which facilitate dendritic cell maturation and antigen presentation – a process central to immunogenic cell death (ICD).^26–28 When combined with immunomodulators such as imiquimod (R837), a TLR7 agonist, this effect can be substantially amplified. R837 not only triggers innate immune activation but also reverses immunosuppressive microenvironments by promoting M1 macrophage polarization and inhibiting angiogenesis.^29–32 As reported, PTT-induced ICD enhances tumor immunogenicity, while R837 sustains T-cell mediated anti-tumor memory, creating a powerful feedback loop for durable tumor restriction.³³ Despite these advances, a unified nanoplatform that concurrently delivers PTT, PDT, and immunotherapy in a spatiotemporally controlled manner has not been fully realized for cSCC.

In this study, we engineered a multifunctional “photo-immuno nano-bomb” based on polydopamine nanoparticles (PDA NPs) for co-delivery of the photosensitizer chlorin e6 (Ce6) and the toll-like receptor 7 agonist imiquimod (R837), thereby combining PTT, PDT, and immunotherapy within a single platform. Specifically, PDA NPs have been well reported as photothermal agents in tumor therapy due to their good biocompatibility and tunable light absorption, in particular, at near-infrared region.^34,35 The nanosystem exploits π–π stacking to achieve high drug loading and stability, and demonstrates a dual stimuli-responsive release behavior – mediated by pH-dependent surface charge changes and NIR-triggered payload release – allowing spatiotemporally controlled combination therapy. The nanoformulation significantly inhibited tumor cell proliferation, migration, and invasion in vitro through activation of apoptotic pathways. Mechanistic investigations indicated that under dual-wavelength laser irradiation (660 + 808 nm), PDA-facilitated PTT enhanced cellular uptake and amplified Ce6-derived singlet oxygen generation, leading to mitochondrial damage and cytoskeletal breakdown. This synergistic effect induced severe cellular oxidative stress and organelle disruption, resulting in prominent ICD. Moreover, the platform showed excellent biocompatibility and achieved complete tumor regression in vivo, surpassing all mono- and combination therapy groups. Collectively, this multimodal approach thus offers a highly promising strategy for advanced cSCC treatment, delivering enhanced antitumor efficacy through well-orchestrated mechanisms and reduced systemic toxicity.

2. Experimental

2.1 Materials

Dopamine hydrochloride, R837 and Tris buffer were obtained from Sigma-Aldrich, Missouri, USA. Ce6 was purchased from J&K Scientific. The singlet oxygen sensor green (SOSG) reagent was obtained from Invitrogen. Annexin V-FITC/PI Apoptosis Kit, CCK8 assay kit, and 2′,7′-dichlorodihydrofluorescein diacetate were purchased from Sigma-Aldrich. Matrigel and Transwell were obtained from Corning, Inc.

2.2 Preparation of polydopamine nanoparticles (PDA NPs)

PDA NPs were synthesized through dopamine monomer polymerization in an alkaline environment as reported.^36,37 In detail, 7 mg of dopamine hydrochloride was dissolved in 10 mL of 0.05 mM Tris buffer, and the pH was adjusted to 11 with 5 M NaOH. The solution was stirred at 500 rpm at 25 °C for 4 h, then filtered with a 0.22 µm filter and centrifuged at 18 000 rpm for 15 min. The precipitate was washed with deionized water, and the supernatant was discarded. The black–brown precipitate was collected, transferred to a glass dish, and vacuum freeze-dried for 48 h to produce black–brown PDA NP powder.

2.3 Fabrication of PDA-Ce6-R837 NPs

To synthesize PDA-Ce6-R837 NPs, equal volumes of Ce6 solution (5 mg mL⁻¹), R837 solution (1 mg mL⁻¹), and PDA NP solution (1 mg mL⁻¹) were combined and subjected to continuous stirring for 48 h. Subsequently, the resultant PDA-Ce6-R837 NPs underwent three cycles of centrifugal washing. Following this, the black–brown precipitates were collected, and subjected to vacuum freeze-drying for 48 h. Similarly, PDA-Ce6 NPs or PDA-R837 NPs were obtained by incubating PDA NPs with Ce6 or R837 at various concentrations, respectively, under continuous stirring for 48 h.

2.4 Characterization of PDA NPs and PDA-Ce6-R837 NPs

The hydrodynamic diameter, zeta potential, and polydispersity index (PDI) of both PDA NPs and PDA-Ce6-R837 NPs were assessed via dynamic light scattering (DLS) using a Zetasizer Nano ZS instrument. The morphological characteristics of the nanoparticles were examined using scanning electron microscopy (SEM).

2.5 Ce6 and R837 loading efficiency and release profile assays

The quantification of Ce6 and R837 loading efficiency was achieved by calculating the difference between the initial drug amount introduced to the synthesized NPs and the residual drug quantity in the supernatant, as determined through fluorescence measurements. In detail, various concentrations of Ce6 solution (1, 2, 3, 4, and 5 mg mL⁻¹) and R837 solution (0.2, 0.4, 0.6, 0.8, and 1.0 mg mL⁻¹) were prepared. A 1 mL aliquot of each solution was combined with 1 mL of PDA solution and mixed at room temperature using a magnetic stirrer for 48 h. Subsequently, the supernatant was collected and further centrifuged at a high speed of 18 000 rpm for 10 min to precipitate the Ce6 or R837-loaded NPs. The supernatant from each centrifugation step was collected, and the absorbance of the unbound drugs was measured at wavelengths of 660 nm and 320 nm using an ultraviolet spectrophotometer, respectively.

The drug release from PDA-Ce6-R837 NPs was investigated at pH levels of 5.8 and 7.2 over various time intervals. The released drug quantity was assessed using UV-Vis spectroscopy. Additionally, drug release was simulated using near-infrared (NIR) irradiation with an 808 nm laser. In particular, PDA-Ce6-R837 NPs were dissolved in 15 mL of PBS with varying pH values. At specified time points, the solution was subjected to 808 nm laser irradiation at a power density of 2 W cm⁻² for 5 min. Subsequently, 1 mL of the solution was collected and centrifuged to isolate the released drug, which was then quantified using UV-Vis spectroscopy. The experiment was conducted in triplicate.

2.6 Photothermal efficacy evaluation

The PDA-Ce6-R837 NP solution, at varying concentrations of 0.5, 1, 1.5, and 2 mg mL⁻¹, was subjected to irradiation using an 808 nm laser at a power density of 2 W cm⁻² for a duration of 10 min. A thermal imaging device was employed to monitor and record the temperature of the solution at 10 s intervals throughout the irradiation process. Additionally, the 808 nm laser was applied at different power densities (0.5, 1, 1.5, and 2 W cm⁻²) to irradiate a 1 mg mL⁻¹ PDA-Ce6-R837 solution for 10 min, with temperature measurements taken every 10 s. To assess the reproducibility and stability of nanoparticle-mediated photothermal ability, 1 mg mL⁻¹ PDA-Ce6-R837 aqueous solution was irradiated using an 808 nm near-infrared laser at a power density of 2.0 W cm⁻² for 600 s, followed by a 600 s cessation of laser application. This cycle was repeated five times, with thermal imaging being used to record the solution temperature at 10 s intervals during each irradiation phase.

2.7 Detection of reactive oxygen species production

The production of ¹O₂ was assessed using the SOSG reagent as a fluorescent probe, measuring the generation of ¹O₂ from PDA-Ce6-R837 NPs based on time, power density, and nanoparticle concentration. In detail, various concentrations of PDA-Ce6-R837 NPs (0.5, 0.75, 1.0, and 1.5 mg mL⁻¹) were prepared in solution, and 20 µL of SOSG (50 µM) was added to each sample within a quartz cuvette. Each solution was subjected to irradiation by a 660 nm laser at a power density of 1.5 W cm⁻² for a duration of 5 min, after which the fluorescence spectra of SOSG were recorded using a fluorescence spectrometer with an excitation wavelength of 504 nm. Additionally, solutions containing 1 mg mL⁻¹ of PDA-Ce6-R837 NPs and 20 µL of SOSG (50 µM) were irradiated for varying durations (1, 2, and 5 min) under the same laser conditions, and the corresponding fluorescence spectra were obtained. Furthermore, solutions with 1 mg mL⁻¹ of PDA-Ce6-R837 NPs and 20 µL of SOSG (50 µM) were exposed to a 660 nm laser at varying power densities (50, 100, 250, and 500 mW cm⁻²) for 2 min, followed by fluorescence spectra analyses.

2.8 In vitro cytotoxicity assay

The A431 cell line (human skin squamous cell carcinoma, ATCC) was purchased from the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences. The cell line was cultured and subcultured in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% FBS and 1% penicillin/streptomycin at 37 °C and 5% CO₂ in a humidified atmosphere.

The in vitro cytotoxicity of NPs was examined. In detail, A431 cells were cultured in 24-well plates at 5 × 10⁵ cells per well for 24 h. Then the experimental groups were respectively added with solutions of different concentrations (0, 25, 50, 100, 150, 200 µg mL⁻¹) of PDA-Ce6-R837 NPs or equal amounts of free Ce6, PDA NPs and R837 solutions. Samples were incubated in the dark for 4 h. The PDA + L group was exposed to an 808 nm laser (2 W cm⁻²) for 5 min, the Ce6 + L group to a 660 nm laser (0.25 W cm⁻²) for 5 min, and the PDA-Ce6-R837 + L group to both lasers sequentially, each for 5 min with a 5-min gap. After 24 h, cell viability was assessed using the CCK8 assay. Meanwhile, intracellular ROS was detected using the DCFH-DA probe and fluorescence intensity was measured using a fluorescence microplate reader (GloMax® Discover Microplate Reader, Promega).

2.9 Intracellular uptake of PDA-Ce6-R837 NPs

Qualitative analysis of cellular uptake was conducted through fluorescence imaging and transmission electron microscopy (TEM). In detail, A431 cells were seeded at a density of 5000 cells per confocal dish and incubated for 24 h. Then, cells were treated with 100 µg mL⁻¹ of PDA-Ce6-R837 NPs in culture media or an equivalent amount of free Ce6 and incubated for 4 and 8 h, respectively. Post-incubation, the cells were fixed with 4% formaldehyde for 15 min, washed three times with PBS, and stained with 4′,6-diamidino-2-phenylindole (DAPI) for 5 minutes prior to imaging via confocal laser scanning microscopy (LSM 780, Carl Zeiss, Germany). The fluorescence intensities of Ce6 in the images, indicative of Ce6 uptake by the cells, were quantified using Image J software by converting the images to grayscale and calculating their mean gray intensity levels.

To investigate the photothermal effect on the cellular uptake of nanoparticles, a similar experiment was performed with minor modification. After the introduction of NPs, one group of cells was co-cultured with the NPs for 4 h. And another group was exposed to the NPs for 2 h, followed by irradiation with an 808 nm laser at an intensity of 1.5 W cm⁻² for 5 min, and then incubated for an additional 2 h.

2.10 Cell scratching assays

A431 cells were seeded at 2 × 10⁵ cells per well in a 6-well plate and cultured for 24 h before scratching. After washing with PBS three times, 100 µg mL⁻¹ of PDA-Ce6-R837 NPs, and equal amounts of free Ce6, R837, and PDA solutions were added and incubated for 24 h. Then, cells were incubated in the dark for 4 h. The PDA + L group was exposed to an 808 nm laser (2 W cm⁻²) for 5 min, the Ce6 + L group to a 660 nm laser (0.25 W cm⁻²) for 5 min, and the PDA-Ce6-R837 + L group to both lasers for 5 min each, with a 5-min interval. Incubation was continued until 48 h.

2.11 Cell invasion assay

A431 cells were seeded at 1 × 10⁵ cells per well in 12-well plates and incubated for 24 h. After removing the supernatant, experimental groups received 2 mL of medium with either 100 µg mL⁻¹ PDA-Ce6-R837 NPs or equivalent concentrations of free Ce6, R837, and PDA NPs. Then, the PDA + L group was exposed to an 808 nm laser (0.5 W cm⁻²) for 5 min, the Ce6 + L group to a 660 nm laser (0.1 W cm⁻²) for 5 min, and the PDA-Ce6-R837 + L group to both lasers sequentially for 5 min each, with a 5-min interval. After 24 h, cells were collected, adjusted to 2.5 × 10⁶ cells per mL, and 200 µL was placed in the upper chamber, with 600 µL of DMEM with 20% FBS in the lower chamber. Samples were incubated at 37 °C with 5% CO₂ for 48 h. Then, stained cells were counted from five independent fields using an inverted fluorescence microscope.

2.12 Cell apoptosis assay

An Annexin V-FITC/PI apoptosis kit was employed to assess cell apoptosis following the manufacturer's instructions. In brief, cells were grouped and treated as previously described. After that, cells were collected, washed with PBS, and stained with 5 µL FITC-Annexin V and 10 µL PI for 20 min at room temperature. Apoptotic cells, including cells at early and late stages, were detected via flow cytometry.

2.13 Xenograft tumour model

The animal study received approval from the West China Hospital's ethics committee at Sichuan University (no. 20220329003). Female BALB/c nude mice from Beijing Virton Li Hua were kept in an SPF facility and acclimated for a week before the experiment. Thirty-five mice (18–22 g, ∼4 weeks old) were divided into 7 groups (n = 5): blank control (PBS), three groups without laser (i.e., PDA-Ce6 NPs, R837, and PDA-Ce6-R837 NPs), and three groups with laser (Ce6 + L, PDA NPs + L, and PDA-Ce6-R837 NPs+ L). A subcutaneous A431 cell xenograft tumor model was created by subcutaneously injecting ∼2 × 10⁶ A431 cells in a 50 µL suspension with 20% Matrigel on the right hip of mice.

Upon reaching a tumor volume of 150 mm³, treatment was initiated and designated as day 1. On days 1, 7, 14, and 21, an intratumoral injection of 100 µL of various solutions was administered to the respective groups, with concentrations of PDA-Ce6-R837 NPs at 10 mg mL⁻¹, Ce6 at 2.5 mg mL⁻¹, and R837 at 1.2 mg mL⁻¹. The laser irradiation group was treated on day 8, where Ce6 + L with a 660 nm laser (0.5 W cm⁻²), PDA + L with an 808 nm laser (2 W cm⁻²), and PDA-Ce6-R837 + L with both lasers, each for 5 min and a 10-min interval. The control group received intratumoral injections of 100 µL of PBS, while the R837 group was administered 100 µL of a 0.3 mg mL⁻¹ R837 solution intratumorally on the 1st, 3rd, 5th and 7th days of each week for a total of four weeks. Multiple injection sites were utilized during the intratumoral administration, and post-injection, the site was compressed with an iodophor cotton swab for 30 seconds to mitigate drug leakage.

The tumor dimensions, specifically the width and length, in each mouse were measured daily using a Vernier caliper, and the tumor volume was calculated using the formula tumor volume (mm³) = (length × width²)/2. Following a 4-week observation period, the animals were euthanized, and the tumors were excised and weighed.

2.14 Biosafety assessment

After 4 weeks, all mice were euthanized, and tissue samples from the heart, liver, spleen, lungs, and kidneys were collected for H&E staining. Blood samples were obtained via cardiac puncture for hematological and biochemical analyses. Hematological parameters, including white blood cell count (WBC), erythrocytes (RBC), hemoglobin (HGB), lymphocytes (LYM), neutrophils (NEUT), and platelet count (PLT), were measured using a hematological autoanalyzer (ADVIA 2120i, Siemens, Germany). The serum was isolated by centrifuging whole blood at 1800 G for 10 min. Biochemical analyses of the serum samples were conducted using an automated biochemistry analyzer (cobas c 311, Roche, Switzerland). Measurements were taken for serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatinine (CREA), blood urea nitrogen (BUN), blood calcium (CA), and blood phosphorus (PHOS).

2.15 Statistical analysis

Statistical analyses and graphical representations were executed using GraphPad Prism version 6.01 software (GraphPad Software Inc., USA). Quantitative data were expressed as mean ± standard deviation (SD). Group comparisons were performed using Student's t-test and one-way ANOVA, followed by Dunnett's t-test. P < 0.05 was considered to indicate statistical significance.

3. Results and discussion

3.1 Synthesis and characterization of PDA-Ce6-R837 NPs

Cutaneous squamous cell carcinoma (cSCC) presents significant clinical challenges due to the limitations of existing treatments, including functional loss from surgery, radioresistance, and poor adherence to prolonged topical therapies. Monotherapy is often inadequate for effective cSCC management. Photothermal therapy (PTT) offers a promising alternative by generating localized heat to destroy tumor tissue and enhance drug delivery. Specifically, PTT complements photodynamic therapy (PDT) by mitigating tumor hypoxia – mild hyperthermia improves oxygen supply, thereby boosting ROS production. Additionally, PTT induces immunogenic cell death (ICD) through the release of DAMPs like HSP70 and ATP, promoting dendritic cell maturation and antigen presentation. Hence, integrating immunotherapy with phototherapy not only enhances antitumor immunity but also establishes long-term immune memory, reducing recurrence. Thus, a combined “photo-immuno nano-bomb” strategy incorporating PTT, PDT, and immunotherapy is proposed to achieve synergistic antitumor efficacy with a “1 + 1 + 1 > 3” effect.

As illustrated in Fig. 1(A), a multifunctional “photo-immuno nano-bomb” based on polydopamine nanoparticles (PDA NPs) for co-delivery of the photosensitizer chlorin e6 (Ce6) and the toll-like receptor 7 agonist imiquimod (R837), thereby combining PTT, PDT, and immunotherapy within a single platform, was developed, which is expected to enhance cell ICD and further enhance antitumor effect. First of all, PDA NPs were synthesized via oxidative polymerization of dopamine under alkaline Tris buffer (pH 11), using atmospheric oxygen as a green oxidant. These NPs serve as an efficient and versatile drug delivery platform owing to their strong binding properties, enabling the loading of therapeutic agents through both covalent and non-covalent interactions. Specifically, Ce6 and R837, both featuring aromatic rings, could be effectively loaded onto PDA NPs via π–π stacking and electrostatic interaction.³⁸ SEM and DLS analyses (Fig. 1(B)–(D)) reveal that the resulting PDA NPs and PDA-Ce6-R837 NPs are spherical, monodisperse (PDI < 0.2), and exhibit average sizes of 123 nm and 240 nm, respectively. The increase in diameter and slightly rougher surface after drug loading are attributed to the formation of a stable drug layer. Zeta potentials remain moderately negative around −26 mV (Fig. 1(D)), indicating good colloidal stability. FTIR and UV-Vis spectroscopic analyses further confirmed the successful fabrication of PDA NPs and PDA-Ce6-R837 NPs (Fig. S1 and Fig. 1(E)). As shown in Fig. 1(F) and (G), the drug loading of Ce6 and R837 in PDA NPs increases as the drug input increases, achieving maximum loading capacities of approximately 35% for Ce6 and 13% for R837, respectively. This observed plateau in the drug loading ratio is attributed to the limited surface area of the PDA NPs. Meanwhile, the drug loading efficiency of PDA-Ce6-R837 NPs was determined to be 24.6% for Ce6 and 11.4% for R837 (Fig. 1(H)). A comparative decrease relative to single-drug loading suggests competitive adsorption between the two molecules during incorporation. Hence, this physical adsorption enables high and multiple drug loading under mild conditions, allowing robust combination therapy with potential for synergistic photodynamic and immune modulation applications.

Fig. 1 Preparation and characterization of PDA-Ce6-R837 NPs. (A) Schematic images of PDA-Ce6-R837 NPs preparation and its application in photo-immunotumor therapy; (B) SEM images, (C) size and (D) zeta potential of PDA NPs and PDA-Ce6-R837 NPs; (E) UV spectra of Ce6, R837, PDA NPs and PDA-Ce6-R837 NPs; (F) Ce6 and (G) R837 loading ratio curves; (H) Ce6 or/and R837 loading ratio in PDA NPs and PDA-Ce6-R837 NPs; (I) Ce6 and (J) R837 accumulative release profiles under different conditions.

Following this, the drug release profiles were further examined under different conditions. As shown in Fig. 1(I) and (J), the release profiles of both Ce6 and R837 exhibited distinct pH- and NIR-responsive characteristics. Notably, a relatively higher release ratio was observed for both drugs at neutral pH than under acidic conditions, regardless of laser exposure. Moreover, upon exposure to an 808 nm laser, the release of both agents was markedly accelerated. At pH 5.8, approximately 48% of Ce6 and 73% of R837 were released; at pH 7.2, the release ratios reached about 58% for Ce6 and 70% for R837. This enhancement is likely due to NIR-induced photothermal heating, which disrupts the π–π stacking and causes structural loosening of the nanoparticles.³⁹ Collectively, these results underscore the dual stimuli-responsive drug release mechanism-encompassing both pH-dependent surface charge modulation and photothermal-triggered payload liberation-enabling spatiotemporally controlled combination therapy with promising applications in photo-immunotherapy.

3.2 Photothermal and photodynamic properties of PDA-Ce6-R837 NPs

As well documented, PDA NPs have excellent photothermal characteristics, stemming from the inherent optical and structural properties of polydopamine – a synthetic analogue of eumelanin – which exhibits broad and strong NIR absorption due to its extensive π-electron system and catechol-derived chromophores. Upon irradiation, the energy is efficiently converted into heat through non-radiative relaxation processes. In this study, the photothermal properties of PDA-Ce6-R837 NPs were systematically evaluated under 808 nm laser irradiation (Fig. 2(A)–(C) and Fig. S2). In detail, PDA-Ce6-R837 NPs solutions at varying concentrations (0.5–2 mg mL⁻¹) were exposed to laser light (1 W cm⁻²) for 10 min, revealing a concentration-dependent temperature increase. The highest concentration (2 mg mL⁻¹) reached around 50 °C, while deionized water showed no significant change. Similarly, higher laser power densities resulted in more rapid and pronounced heating, confirming the strong and tunable photothermal conversion capability of the NPs. Furthermore, the PDA-Ce6-R837 NPs demonstrated remarkable photothermal stability over four on/off laser cycles with no significant loss in heating efficiency (Fig. 2(C)), indicating high durability for repeated applications. Importantly, the photothermal performance remained robust after drug loading, underscoring the stability of PDA NPs as a photothermal matrix and the compatibility of the loading process.

Fig. 2 Photothermal and photodynamic properties of PDA-Ce6-R837 NPs. Temperature increase curves at varying (A) concentrations, (B) laser power density of PDA-Ce6-R837 NPs; (C) photothermal stability after four photothermal cycles of PDA-Ce6-R837 NPs; (D) singlet oxygen sensor green (SOSG) intensity curves of different samples under 808 or 660 nm laser irradiation; SOSG intensity curves with the change of (E) concentration and (F) laser-irradiation time PDA-Ce6-R837 NPs.

Additionally, the singlet oxygen (¹O₂) generation capability of PDA-Ce6-R837 NPs was examined under laser irradiation, which is essential for effective PDT. In general, Ce6 acts as a potent photosensitizer that, upon photoexcitation, transfers energy to ambient oxygen molecules via a Type II photochemical pathway, generating cytotoxic singlet oxygen with high yield.⁴⁰ As shown in Fig. 2(D), similar ¹O₂ production profiles could be detected in Ce6 and PDA-Ce6-R837 NPs under 660 nm laser irradiation, suggestive of the good activity maintenance of Ce6 during NP fabrication. Moreover, the ¹O₂ production exhibited strong dependence on NP concentration, laser power density, and irradiation time, highlighting the tunability and efficiency of the system (Fig. 2(E) and (F) and Fig. S3).

Overall, the nanoplatform combines multiple therapeutic modalities, including sustained drug release with pH- and NIR-triggered kinetics, efficient photothermal conversion, and robust photodynamic activity, representing an integrated and controllable system for synergistic photothermal–photodynamic–immunotherapy, offering a promising strategy for targeted cancer treatment with minimal invasiveness and high efficacy.

3.3 In vitro effects of PDA-Ce6-R837 NPs on cancer cell bioactivities

Inspired by the excellent physiochemical properties of PDA-Ce6-R837 NPs, the in vitro biocompatibility and therapeutic efficacy were further systematically evaluated using human squamous cell carcinoma (A431) and immortalized keratinocyte (HaCaT) cell lines. In detail, PDA NPs exhibited excellent biocompatibility, with cell viability remaining above 85% even at a high concentration of 400 µg mL⁻¹ (Fig. 3(A) and Fig. S4), confirming their suitability as a drug carrier for biological applications. Under laser irradiation, PDA NPs and Ce6 displayed concentration-dependent photothermal and photodynamic antitumor effects, respectively. The PDA-Ce6-R837 NPs inhibited cell viability to some extent at higher concentration primarily due to R837. When subjected to dual-wavelength laser irradiation (808 nm + 660 nm), the highest cytotoxicity was observed, underscoring synergistic PTT and PDT effects.

Fig. 3 In vitro effects of PDA-Ce6-R837 NPs on cancer cell bioactivities, including viability, migration and invasion. (A) Cytotoxicity study of PDA-Ce6-R837 NPs with or without laser irradiation on A431 Cells; (B) wound scratch test of healing inhibition effects induced by PDA-Ce6-R837 NPs and their components on A431 cells with or without laser irradiation after 48 h, ×40 magnification. (C) Quantification of migration distance rate of A431 cells in wound scratch test (**p < 0.01, ***p < 0.001, ****p < 0.0001 versus control, n = 3); (D) Transwell migration assay of A431 cells (violet) migrating through the chamber after being treated with PDA-Ce6-R837 NPs and their components with/without laser irradiation, ×200 magnification. (E) Quantification of migrating cell number of A431 cells in Transwell migration assay (***p < 0.001, ****p < 0.0001 versus control, n = 3).

Furthermore, the PDA-Ce6-R837 NPs + L significantly impeded cancer cell migration and invasion (Fig. 3(B)–(E)). While PDA or Ce6 alone showed no inhibitory effect in the dark, laser irradiation markedly reduced cell motility through photothermal and photodynamic mechanisms. R837 and PDA-Ce6-R837 NPs also attenuated migration and invasion to some extent, attributable to R837's biological activity. The most profound suppression occurred after dual-laser treatment, with the PDA-Ce6-R837 NPs + L group exhibiting near-complete inhibition of invasive capacity in Transwell assays. In summary, PDA-Ce6-R837 NPs combine high biocompatibility with multimodal therapeutic functions, including PTT, PDT, and immunotherapy, resulting in potent antitumor and anti-metastatic efficacy.

3.4 Synergistic induction of cellular apoptosis by PDA-Ce6-R837 NPs

Furthermore, Annexin V/PI staining was performed to examine the influence of PDA-Ce6-R837 NPs on A431 cells activities, including viability, early apoptosis, late apoptosis and necrosis (Fig. 4). In the absence of laser irradiation, neither individual components (PDA, Ce6, or R837) nor the integrated NPs induced significant apoptosis compared to the control group (p > 0.05), indicating low basal cytotoxicity and suggesting that single drug at low concentrations does not initiate apoptotic pathways independently. However, upon laser irradiation, profound apoptosis was triggered through PTT and PDT mechanisms. PDA NPs under 808 nm laser exposure significantly increased early apoptosis from around 4% to 26% and late apoptosis from around 2% to 11%. Similarly, Ce6 irradiated with 660 nm laser elevated early apoptosis from around 4% to 27% and late apoptosis from around 4% to 11%, confirming the efficacy of both PTT and PDT in inducing early apoptotic cell death. Notably, the PDA-Ce6-R837 NPs under dual-wavelength irradiation demonstrated a remarkable synergistic effect. While the early apoptosis rate around 16% was comparable to the PDA + L group though slightly lower than Ce6 + L, the late apoptosis rate surged to 60%, significantly exceeding all monotherapy groups. This was accompanied by a substantial increase in necrotic cell death, resulting in only 13% of cells remaining viable – the lowest across all conditions. In conclusion, PDA-Ce6-R837 NPs under laser irradiation elicit potent tumor cell apoptosis and necrosis via synergistic PTT/PDT mechanisms, highlighting their potential as an effective combinatory platform for cancer therapy.

Fig. 4 Synergistic induction of cellular apoptosis by PDA-Ce6-R837 NPs. (A) Representative flow cytometry plots of A431 cells treated with PDA-Ce6-R837 NPs and their components with/without laser irradiation; statistical data of (B) early apoptosis rate, (C) late apoptosis rate and (D) cell survival rate after different treatments (*p < 0.05, **p < 0.01, ****p < 0.0001 versus control, n = 3).

3.5 Molecular mechanisms of synergistic therapy potentiated by PDA-Ce6-R837 NPs

In general, PTT and PDT always are related to cell ICD, which is important for tumor ablation. Specially, PDT is a powerful physical stressor that efficiently kills tumor cells through oxidative damage. This specific type of damage is a potent trigger for the cellular pathways that leads to the emission of danger signals (DAMPs). Moreover, PTT can also potentiate anti-tumor immunity. Local hyperthermia promotes the release of damage-associated molecular patterns DAMPs, such as HSP70 and ATP, which facilitate dendritic cell maturation and antigen presentation – a process central to immunogenic cell death (ICD). Hence, in order to further examine the underlying mechanism of PDA-Ce6-R837 NP-induced tumor cell apoptosis, we evaluated the cellular uptake and intracellular behavior of PDA-Ce6-R837 NPs systematically (Fig. 5). CLSM imaging revealed time- and energy-dependent internalization of the NPs into A431 cells, with Ce6-derived red fluorescence localized predominantly in the cytoplasm, indicating endo/lysosomal encapsulation without nuclear entry. The cellular uptake of PDA-Ce6-R837 NPs was significantly enhanced compared to free Ce6, with fluorescence intensity increasing over time and being markedly higher at both 4 h and 8 h (Fig. 5(A) and (B)). This underscores the role of PDA NPs as an efficient nanocarrier that improves drug delivery through enhanced endocytosis and accumulation.

Fig. 5 Synergistic therapeutic mechanisms of PDA-Ce6-R837 NPs for cancer cell killing, where PTT potentiates intracellular NP uptake, triggers massive ROS generation, and induces irreversible cellular structure collapse. (A) Fluorescence imaging of the cellular uptake and localization of free Ce6 and PDA-Ce6-R837 NPs loaded with the same Ce6 over a range of incubation time; (B) corresponding quantification using ImageJ (*p < 0.05 versus control, **p < 0.01 versus control, n = 3); (C) fluorescence imaging of the cellular uptake of PDA-Ce6-R837 NPs with or without 808 nm laser irradiation; (D) corresponding quantification (*p < 0.05 versus control, n = 3); (E) production of ROS within A431 cells after different treatments (**p < 0.01, ****p < 0.0001 versus control, n = 3); (F) the effect of PDA-Ce6-R837 NPs on A431 cell morphology observed by TEM under conditions without laser irradiation (i and ii) and with laser irradiation (iii and iv). N, cell nucleus; M, mitochondria; RER, rough endoplasmic reticulum.

Notably, photothermal treatment via 808 nm laser irradiation further increased cellular uptake (Fig. 5(C) and (D)), attributed to laser-induced membrane permeability enhancement and temperature-mediated endocytic activation, facilitating accelerated NP internalization. This synergistic effect was corroborated by ROS generation assays, where PDA-Ce6-R837 NPs under irradiation produced significantly higher intracellular ROS than free Ce6 (Fig. 5(E)), confirming improved photodynamic efficacy through enhanced photosensitizer delivery and more efficient energy transfer owing to the confined micro-environment within NPs.

TEM analysis provided ultrastructural insights into the cellular response. Without irradiation, cells treated with PDA-Ce6-R837 NPs exhibited mild cytotoxicity features, including mitochondrial contraction, cristae disorganization, and chromatin marginalization (Fig. 5(F) and Fig. S5), suggesting initial stress response without acute lethality. In contrast, dual laser irradiation (808 + 660 nm) induced severe cellular damage, including loss of membrane integrity, cytoplasmic vacuolization, mitochondrial swelling with cristae dissolution, dilation of the rough endoplasmic reticulum, and nucleolar disintegration. These morphological alterations are consistent with irreversible necrotic and apoptotic cell death, reinforced by extensive ROS-mediated oxidative stress, thermal injury, and organelle-specific damage.

Overall, this multifunctional platform represents a promising strategy for synergistic cancer therapy with heightened efficacy and subcellular precision; the photothermal effect induces local hyperthermia, causing protein denaturation and membrane disruption, while PDT generates cytotoxic ROS, promoting oxidative damage and apoptosis. R837 further augments treatment by stimulating immune responses and directly suppressing metastatic behaviors. The nanoplatform also leverages pH- and NIR-triggered drug release, enhancing site-specificity and reducing off-target effects. Its multimodal action-combining PTT, PDT, and immunotherapy-enables comprehensive tumor suppression and inhibits metastasis more effectively than any monotherapy.

3.6 Synergistic triple-modality antitumor therapy and biosafety of PDA-Ce6-R837 NPs

In vivo antitumor efficacy of PDA-Ce6-R837 NPs was systematically evaluated in A431 tumor-bearing nude mice. As illustrated in Fig. 6, the PBS and PDA-Ce6 NPs groups exhibited progressive tumor growth, while significant suppression was observed in those treated with laser irradiation and/or R837-containing formulations. The PDA-Ce6-R837 NP group without laser irradiation still inhibited tumor growth to some extent, which is attributed to the cytotoxicity induced by R837. Most notably, the PDA-Ce6-R837 + L group (with dual 660 + 808 nm laser irradiation) demonstrated the most potent tumor inhibition capacity, showing a markedly reduced tumor volume by day 28 (Fig. 6(A)–(C)). This superior therapeutic efficacy was further confirmed by the measured tumor weights (Fig. 6(D)). Mechanistically, the nanocomplex enables spatiotemporally controlled triple therapy, where PDA mediates hyperthermia under 808 nm light, disrupting cellular structures and promoting apoptosis/necrosis. Meanwhile, Ce6 generates cytotoxic ROS under 660 nm irradiation, inducing oxidative damage. Furthermore, R837 independently induces tumor cell death via non-immunogenic pathways. The combination strategy not only synergizes these effects but also allows sustained drug release and reduced dosing frequency. The once-weekly intratumoral injection of PDA-Ce6-R837 NPs, combined with monthly PTT/PDT, demonstrated superior efficacy compared to the high-frequency R837 regimens used clinically, significantly improving treatment practicality and patient compliance.

Fig. 6 PDA-Ce6-R837 NPs mediate robust anti-tumor efficacy in vivo. (A) Schematic image of the experimental process; (B) observation of the resected tumor from A431 tumor-bearing mice on the 28th day; (C) tumor growth curves after different treatment until day 28; (D) mass quantification of the resected tumor from A431 tumor-bearing mice on the 28th day (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 versus control, n = 3).

Moreover, the biosafety of PDA-Ce6-R837 NPs + L was assessments, and the results revealed no significant histological abnormalities in major organs (Fig. 7) nor deviations in hematological/biochemical parameters (WBC, RBC, HGB, ALT, AST, UREA, etc.) (Fig. 8), confirming the high biocompatibility and clinical potential of the nanoplatform. In summary, PDA-Ce6-R837 NPs represent a robust and safe combinatory system that integrates PTT, PDT, and immuno-agent therapy into a single platform, enabling potent tumor suppression with reduced dosing and enhanced treatment efficiency.

Fig. 7 H&E staining of lungs, liver, spleen, kidneys, and heart of A431 tumor-bearing mice after different treatments.

Fig. 8 Statistical data of the main blood parameters, including (A) WBC, (B) RBC, (C) HGB, (D) LYM, (E) NEUT, (F) PLT, (G) ALT, (H) AST, (I) UREA, (J) CREA, (K) PHOS, (M) CA, of tumor-bearing mice after different treatments (n = 3).

4. Conclusions

In this study, we developed a multifunctional “photo-immuno nano-bomb” based on polydopamine nanoparticles (PDA NPs) for the co-delivery of Ce6 and R837, which synergistically integrates photothermal (PTT), photodynamic (PDT), and immunotherapy into a unified platform for the treatment of cutaneous squamous cell carcinoma (cSCC). The nanoplatform demonstrated high drug loading and dual stimuli-responsive release properties, governed by pH-dependent charge switching and near-infrared-triggered payload liberation, enabling spatiotemporally controlled therapeutic delivery. Our results indicate that the nanocomposite significantly inhibits tumor cell proliferation, migration, and invasion in vitro, primarily through the initiation of cellular apoptosis. Under dual-wavelength NIR irradiation, the photothermal effect enhanced cellular internalization and singlet oxygen generation, leading to severe oxidative stress and organelle damage, and ultimately robust immunogenic cell death (ICD). In vivo experiments confirmed the high biocompatibility and superior therapeutic efficacy of the system, achieving complete tumor regression, outperforming all mono- and combination therapy controls. This multi-mechanistic strategy effectively addresses key limitations of existing cSCC treatments, such as functional damage, recurrence, and poor compliance, by offering a minimally invasive yet potent alternative. Hence, this work provides a compelling translational paradigm for next-generation cSCC therapy, highlighting the potential of nanotechnology to unify diverse treatment modalities into a single, controllable, and highly effective platform.

Author contributions

Xiaoyan Zhang: writing – original draft, visualization, validation, project administration, methodology, investigation. Jiahui Zhu, Tinghua Li: visualization, Huan Cao: writing – original draft, visualization, writing – review & editing, Lin Wang: writing – review & editing, supervision, funding acquisition, conceptualization.

Conflicts of interest

There are no conflicts to declare.

Data availability

The data supporting this article have been included as part of the supplementary information (SI). The supplementary information includes the FTIR spectrum of PDA NPs, the temperature increase observed at various concentrations following PDA-Ce6-R837 NPs combined with laser treatment, the variation in SOSG intensity as a function of laser power density post-treatment, a cytotoxicity analysis of PDA NPs on A431 and HaCaT cell lines, and TEM images illustrating the intracellular localization of PDA-Ce6-R837 NPs. See DOI: https://doi.org/10.1039/d5tb02242h.

Acknowledgements

This work was supported by the Sichuan Science and Technology Program (2024NSFSC1021) and the Cross-disciplinary Innovation Project of the 1·3·5 Project for the Outstanding Development of Disciplines at West China Hospital, Sichuan University (ZYJC21051). We would like to thank the Analytical & Testing Center of Sichuan University for material characterization work.

Notes and references

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