NIR-activatable aza-BODIPY nanoparticles for photoacoustic imaging and synergistic NO–photothermal cancer therapy

Abstract

Near-infrared (NIR) photothermal therapy (PTT) has emerged as a promising modality for cancer treatment due to its minimal invasiveness, precise spatiotemporal control, and potent therapeutic outcomes. However, the clinical application of photothermal agents (PTAs) remains limited by issues such as poor biodegradability, long-term toxicity, and insufficient photothermal conversion efficiency. Herein, we report the development of a novel amphiphilic aza-boron-dipyrromethene (aza-BODIPY)-based photothermal agent, C₈-NBDP-OEG₄, which self-assembles into monodisperse nanoparticles in aqueous solution. These nanoparticles exhibit excellent chemical and photostability, along with a high photothermal conversion efficiency of 39.8% under 808 nm laser irradiation. To endow the system with multifunctionality, the nitric oxide (NO) donor S-nitroso-N-acetylpenicillamine (SNAP) was co-encapsulated within the nanoparticles, enabling NIR-triggered NO release. This design achieves a dual-mode therapeutic strategy, combining localized hyperthermia and NO-mediated modulation of the tumor microenvironment, thereby significantly enhancing anticancer efficacy. Importantly, the released NO was found to amplify the photoacoustic (PA) signal intensity, facilitating photoacoustic imaging-guided therapy. Both in vitro and in vivo studies demonstrated pronounced tumor growth inhibition with minimal systemic toxicity. Collectively, our study introduces C₈-NBDP-OEG₄@NO nanoparticles as a multifunctional theranostic nanoplatform, offering NIR-activated, PA imaging-guided synergistic NO–photothermal therapy and showcasing strong potential for precise and effective cancer treatment.

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1. Introduction

Near-infrared (NIR) photothermal materials have garnered considerable attention in clinical applications, including biomedical imaging, light-triggered drug delivery, and photothermal therapy (PTT).^1–3 As a promising strategy in the realm of precision medicine, PTT offers minimal invasiveness, excellent spatiotemporal control, and potent therapeutic efficacy. Upon NIR irradiation, photothermal agents (PTAs) convert absorbed light energy into localized heat through nonradiative relaxation processes, thereby inducing hyperthermia at tumor sites to ablate cancer cells.^4,5 To date, a wide range of photoactive nanomaterials, such as noble metals,^6,7 carbon materials,⁸ organic dyes,^9–14 and semiconducting polymers,^15–17 have been investigated as PTAs for PTT.

However, conventional inorganic PTAs often suffer from poor biodegradability and potential long-term toxicity due to their persistent retention in vivo, which poses significant challenges for clinical translation. In contrast, organic PTAs, such as cyanine dyes, conjugated polymers, and dye-loaded micelles or liposomes, exhibit better biocompatibility and are thus considered more suitable for medical applications.^18–21

Despite these advantages, organic PTAs still face several limitations, including poor photostability, susceptibility to photobleaching, and suboptimal photothermal conversion efficiency. For example, cyanine dyes are prone to rapid photodegradation under prolonged NIR exposure, which compromises therapeutic efficacy.²² Similarly, while conjugated polymers offer high photothermal performance, their limited aqueous solubility hinders biological applicability.²³ Dye-loaded micelles and liposomes, although effective carriers, may suffer from premature drug leakage or instability in complex physiological environments.²⁴ Various strategies such as chemical modification, J-aggregate formation, and hybridization with inorganic components have been employed to improve photostability and performance.²⁵ Nevertheless, the rational design of amphiphilic organic PTAs with robust solubility, high stability, and efficient photothermal performance remains a formidable challenge.

Meanwhile, nitric oxide (NO), a versatile gaseous signaling molecule, plays a pivotal role in modulating the tumor microenvironment by increasing vascular permeability, promoting cancer cell apoptosis, and enhancing the therapeutic efficacy of chemo- and radiotherapies. Besides, at the cellular level, NO has been reported to induce DNA damage and mitochondrial dysfunction.^26,27 Controlled NO release at tumor sites is therefore highly desirable for synergistic cancer treatment. In particular, NIR-triggered NO release offers an attractive strategy by enabling spatiotemporal control, sensitizing tumor cells to hyperthermia, and improving drug delivery through vasodilation.^28,29 When combined with PTT-induced localized heating, this dual-modal approach can significantly amplify therapeutic outcomes through synergistic action.

Among organic photothermal materials, BODIPY and aza-BODIPY derivatives have attracted increasing interest due to their strong NIR absorption, high molar extinction coefficients, excellent photostability, and favorable physicochemical properties.^30,31 These features make them suitable for applications in NIR fluorescence imaging, photodynamic therapy (PDT), and PTT. However, their inherent hydrophobicity often compromises aqueous dispersibility and biocompatibility, limiting their practical biomedical use. A viable strategy to overcome this limitation involves the introduction of hydrophilic chains to create amphiphilic structures, thereby promoting self-assembly into stable nanoparticles in aqueous environments.

In this study, we report the design and synthesis of a novel amphiphilic aza-BODIPY-based photothermal agent, C₈-NBDP-OEG₄, functionalized with two hydrophilic oligo(ethylene glycol) (OEG₄) chains and four hydrophobic octyloxy groups, allowing for spontaneous self-assembly into uniform nanoparticles (Scheme 1). To introduce gas therapeutic functionality, the nitric oxide donor S-nitroso-N-acetylpenicillamine (SNAP) was co-encapsulated into the nanoparticle core. The resulting nanoparticles exhibit excellent photothermal performance with a photothermal conversion efficiency of 39.8% under 808 nm laser irradiation, along with NIR-triggered NO release. Notably, the generated NO not only contributes to tumor microenvironment modulation and therapeutic enhancement but also improves photoacoustic imaging (PAI) signals, enabling a theranostic platform for simultaneous diagnosis and treatment. Both in vitro and in vivo studies confirmed significant tumor inhibition with minimal systemic toxicity.

Scheme 1 Schematic illustration of the fabrication of C₈-NBDP-OEG₄@NO nanomaterials and the synergistic cancer photothermal and gas therapy.

2. Materials and methods

2.1. Materials

4-Aminobenzaldehyde, 1-bromooctane, 4-hydroxyacetophenone, and tetraethylene glycol monomethyl ether were purchased from TCI. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), bovine serum albumin (BSA), N,N-diisopropylethylamine (DIPEA), and boron trifluoride etherate (BF₃.Et₂O) were purchased from Sigma-Aldrich and used as received. Fetal bovine serum (FBS), penicillin, streptomycin, and Dulbecco's modified Eagle's medium (DMEM) were purchased from GIBCO and used as received. Potassium hydroxide, ammonium acetate, 1,4-dioxane, ethyl acetate (EtOAc), acetonitrile (MeCN), dichloromethane (DCM), methanol, acetone, ethanol, diethyl ether, N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), n-hexane, and all other reagents were purchased from Adamas-beta Co. Ltd and used as received. Water was deionized with a Milli-Q SP reagent water system (Millipore) to a specific resistivity of 18.4 MΩ cm. Nitromethane, NaOH, and other reagents were purchased from Energy Chemical Co., Ltd and used as received. The 4T1 mouse breast cancer cell line was purchased from Shanghai Institute of Biochemistry and Cell Biology (SIBCB) (Shanghai, China). 5-week-old female BALB/c mice were purchased from Vital River Laboratory Animal Co. Ltd (Guangzhou, China). Ethical approval of animal experiments was obtained from the Institutional Animal Care and Use Committee of the Affiliated Hospital of Beijing Institute of Technology.

2.2. Synthesis and characterization of C₈-NBDP-OEG₄

Synthetic routes for the relevant precursors are provided in the SI. Ammonium acetate (323 mg, 4.2 mmol, 10.0 equiv.) was added to a stirred solution of compound D (300 mg, 0.42 mmol, 1.0 equiv.) in n-BuOH (20 mL) and the mixture was heated at 110 °C for 3 h under a N₂ atmosphere. After cooling to room temperature, the solvent was removed under reduced pressure, the residue was diluted with CH₂Cl₂ (100 mL) and washed with saturated NaCl (50 mL) twice, the organic phase was dried with anhydrous Na₂SO₄ and concentrated under reduced pressure. The resulting solid was subsequently dissolved in dry CH₂Cl₂ (20 mL), cooled to −20 °C under a N₂ atmosphere, and treated with DIPEA (163 mg, 1.26 mmol, 3.0 equiv.), followed by slow addition of BF₃.Et₂O (298 mg, 2.1 mmol, 5.0 equiv.) and then stirring at room temperature for 3 h. The reaction mixture was quenched with ice and extracted with CH₂Cl₂ (50 mL). The organic layer was dried with anhydrous Na₂SO₄ and the solvent was removed under reduced pressure. The residues were purified by silica gel column chromatography (DCM : CH₃OH = 4 : 1), affording C₈-NBDP-OEG₄ as a blue solid (73 mg, yield: 25%). ¹H NMR (500 MHz, chloroform-d) δ 8.04 (dd, J = 22.4, 8.6 Hz, 6H), 7.45 (m, 2H), 6.97 (dd, J = 17.7, 8.3 Hz, 4H), 6.78 (s, 2H), 6.69 (d, J = 8.6 Hz, 4H), 4.19 (q, J = 5.0 Hz, 4H), 3.89 (t, J = 4.8 Hz, 6H), 3.81–3.45 (m, 30H), 3.38 (s, 6H), 1.39–1.24 (m, 48H), 0.89 (t, J = 6.7 Hz, 12H). ¹³C NMR (126 MHz, chloroform-d) δ 160.68, 156.87, 149.13, 145.51, 143.23, 131.46, 131.36, 125.61, 120.82, 114.87, 114.71, 114.61, 111.76, 72.30, 71.22, 71.0, 70.89, 70.00, 70.00, 67.79, 59.42, 51.53, 32.23, 30.53, 30.08, 29.91, 29.87, 29.76, 27.82, 27.63, 27.57, 23.05, 14.50. MALDI-TOF-MS (m/z): calcd for [M + H]⁺, 1390.96, found, 1390.03.

2.3. Nanoparticle fabrication

Typical procedures are described as follows. C₈-NBDP-OEG₄ (2 mg) was dissolved in 1 mL of DMSO and quickly added into deionized water (9 mL) under vigorous stirring at 25 °C. The organic solvent was removed by dialysis (molecular weight cutoff, MWCO, ∼3.5 kDa) against deionized water and fresh water was replaced approximately every 4 h; the final concentration is 0.14 mM. Nanoparticle suspensions can be concentrated to elevated levels via optimized centrifugation protocols to achieve high-concentration dispersions requisite for advanced nanomedicine applications. Note: the initial concentration of the C₈-NBDP-OEG₄ DMSO solution that is higher than 2 mg mL⁻¹ is not suitable for the preparation of uniform nanoparticles with diameters below 100 nm. Except for DMSO, other organic solvents such as DMF, acetonitrile, THF, etc., which are miscible with water, can be used to prepare nanoparticles.

As for the preparation of C₈-NBDP-OEG₄ @NO nanoparticles, S-nitroso-N-acetyl-DL-penicillamine (SNAP), a heat-sensitive NO donor with limited water solubility, was used in the work. Typical procedures are as follows: SNAP (2 mg) and C₈-NBDP-OEG₄ (2 mg) were dissolved in 1 mL of DMSO and then quickly added into deionized water (9 mL) under vigorous stirring. The unloaded free SNAP was removed by dialysis against cooled deionized water (MWCO ∼3.5 kDa) for 24 h. Fresh, cooled deionized water was replaced approximately every 4 h. To determine the SNAP loading content, an aliquot of the dispersion was placed in an ultraviolet cuvette, and the Griess reagent was added and sealed, followed by incubation overnight at 60 °C, which was then subjected to further UV-Vis measurements against the standard NO calibration curve at 540 nm. The encapsulation efficiency (EE%) was calculated as

EE% = [W_total − W_unloaded]/W_total × 100%

The loading content (LC%) was calculated as

LC% = [W_loaded]/[W_C8-NBDP-OEG4 + W_loaded] × 100%

where W_total, W_unloaded, W_loaded, and W_C8-NBDP-OEG4 refer to the weights of SNAP used, unloaded SNAP, SNAP encapsulated into the nanoparticles, and C₈-NBDP-OEG₄, respectively. Based on the standard calibration curve, the loading content of SNAP was determined to be 5.2 wt%. The encapsulation efficiency of SNAP was calculated to be 5.5%. The relatively low loading efficiency was attributed to the partial water solubility and rapid diffusion into the aqueous phase during the nanoprecipitation process.

2.4. Photothermal conversion efficiency

The photothermal conversion efficiency of C₈-NBDP-OEG₄ nanoparticles was calculated based on the reported methods. 0.05 g L⁻¹ C₈-NBDP-OEG₄ nanoparticle dispersions were irradiated with 808 nm laser irradiation at a power density of 1.8 W cm⁻² for 1800 s. After that, the laser was turned off and the solution was allowed to cool to ambient temperature. The temperature was recorded during the whole process. The photothermal conversion efficiency (η) was calculated using the following equation (eqn (1)):

η = [hA(T_max − T_surr) − Q_dis]/[I(1 − 10^{−A₈₀₈})] (1)

where h and A represent the heat transfer coefficient and the surface area of the container, respectively. T_max and T_surr represent the maximum temperature and the surrounding temperature. Q_dis represents the heat dissipation of the solvent (deionized water). I represents the laser power (1.0 W) and A₈₀₈ is the absorbance of C₈-NBDP-OEG₄ at 808 nm. The value of hA was calculated using eqn (2):

τ_s = M_DC_D/hA (2)

where τ_s represents the time constant for heat transfer in the system; M_D and C_D are the solvent weight (deionized water, 1.0 g) and solvent heat capacity (deionized water, 4.2 J g⁻¹), respectively.

The value of τ_s was calculated using eqn (3)

t = −τ_s(ln θ) (3)

where θ is defined as the ratio of ΔT and ΔT_max, and θ could be obtained using eqn (4):

θ = (T − T_surr)/(T_max − T_surr) (4)

Q _dis was calculated using eqn (5):

Q_dis = [C_DM_D(T_{max (water)} − S_urr)]/τ_{s (water)} (5)

Note: 808 nm laser power density exceeding 1.8 W cm⁻² is generally not recommended, as exposure at this level for 10 minutes can induce a significant temperature increase even in pure water. Of course, this photothermal effect can be effectively achieved at lower laser power levels by increasing the sample concentration.

2.5. In vitro NO release

The NO released was detected using the urine nitrite qualitative test reagent (GRIESS reagent) in vitro. The GRIESS reagent was dissolved completely in H₂O and added to C₈-NBDP-OEG₄@NO aqueous solution. Then, the mixed solution was immediately transferred into a UV quartz cuvette with a lid. After 808 nm laser irradiation for a period of time, the UV absorption of the solution at 540 nm was measured using a UV spectrophotometer.

2.6. In vitro cytotoxicity assay

4T1 cells were used for determining in vitro dark toxicity via the standard MTT assay. 4T1 cells were first cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), penicillin (100 units per mL), and streptomycin (100 μg mL⁻¹) at 37 °C in a CO₂/air (5 : 95) incubator for 2 days. For a typical cytotoxicity assay, the cells were first seeded in two 96-well plates at a density of 5 × 10³ cells per well in 150 μL of complete DMEM. After incubating at 37 °C for 24 h, the medium was replaced with fresh culture medium and the cells were classified into the following groups: (1) the control group without any treatment. (2) the DSPE-PEG_2k@NO group; (3) the C₈-NBDP-OEG₄@NO group; (4) the C₈-NBDP-OEG₄ + laser group; (5) the DSPE-PEG_2k@NO + laser group; and (6) the C₈-NBDP-OEG₄@NO + laser group. The treated cells were incubated in a humidified environment with 5% CO₂ at 37 °C for 24 h and then the medium was removed and fresh medium was added. As for the laser irradiation groups, 808 nm (1.8 W cm⁻²) laser irradiation was performed for 5 min, followed by further incubation for 4 h. The culture medium in each well was carefully removed and then 200 μL of DMSO was added to each well. The plate was gently agitated for 15 min before recording the absorbance at 570 nm in each well using a microplate reader (Thermo Fisher Scientific Co. Ltd). Cell viability was calculated as A_{570, treated}/A_{570, control} × 100%. Each experimental condition was performed in quadruplicate and the data are shown as the mean value plus or minus standard deviation (±SD). The MTT assay of C₈-NBDP-OEG₄@NO with various concentrations was conducted with similar procedures.

2.7. Intracellular NO detection

4T1 cells were inoculated in a cell culture dish and incubated with complete DMEM (containing 10% FBS) for 24 h under standard conditions (37 °C, 5% CO₂) in the dark. Then, DSPE-PEG_2k@NO (0.2 mg mL⁻¹) or C₈-NBDP-OEG₄@NO was added to fresh DMEM. After co-incubation with the mixed medium for 4 h in the dark, the cells were irradiated with an 808 nm laser (1.8 W cm⁻²) for 5 min. Then, the medium was replaced with fresh DMEM, and further incubation was performed for 4 h, followed by incubation with DAF-FM DA working solution at 37 °C for 20 min. The cells were washed twice with PBS buffer, and then 1 mL of fresh DMEM was added to the cells. Finally, the cells were imaged by CLSM, following excitation with a 488 nm laser, and their emission was recorded from 500 nm to 550 nm.

2.8. Apoptosis assay

4T1 cells were inoculated in a cell culture dish and incubated with complete DMEM (containing 10% FBS) for 24 h under a 5% CO₂ atmosphere at 37 °C. Then, DSPE-PEG_2k@NO (0.2 mg mL⁻¹) and C₈-NBDP-OEG₄@NO (0.2 mg mL⁻¹) were added to fresh DMEM. After coincubation with the mixed medium for 4 h in the dark, the cells were irradiated with an 808 nm laser (1.8 W cm⁻²) for 5 min. Then, the medium was removed, and the cells were stained with fresh DMEM (1 mL) and further incubated for 4 h. Then, the cells were incubated with calcein-AM/PI working solution at 37 °C for 20 min. The cells were washed with PBS buffer twice, and then fresh DMEM (1.0 mL) was added to the cells. Finally, the cells were imaged by confocal laser scanning microscopy (CLSM). The green channel was excited with a 488 nm laser and its emission was recorded from 500 nm to 550 nm. The red channel was excited with a 543 nm laser and its emission was recorded from 600 nm to 630 nm.

2.9. In vivo tumor imaging

All animal procedures were performed in accordance with the Guidelines for Care and Use of Laboratory Animals of Beijing Institute of Technology and approved by the Animal Ethics Committee of Beijing Institute of Technology. The mice were intravenously injected with C₈-NBDP-OEG₄@NO in a PBS dispersion (0.2 mg mL⁻¹, 100 μL). Then, photothermal imaging (808 nm laser irradiation, 1.8 W cm⁻²) and photoacoustic imaging were conducted on the mice over time.

2.10. In vivo antitumor activity

The 4T1 tumor-bearing mice with a tumor volume (80–100 mm³) were randomly divided into six groups (n = 5): (1) PBS; (2) C₈-NBDP-OEG₄@NO; (3) only laser irradiation; (4) DSPE-PEG_2k@NO + laser; (5) C₈-NBDP-OEG₄ + laser; and (6) C₈-NBDP-OEG₄@NO + laser. Each mouse was intravenously injected with a PBS dispersion (0.2 g L⁻¹, 100 μL); 12 h after administration, the tumor sites were irradiated with an 808 nm laser at a power intensity of 1.8 W cm⁻² for 5 min. Tumor volumes and body weights of all mice were monitored every 3 days. Tumor volumes of all mice were measured using a vernier caliper; the maximum diameter (length) and the largest transverse diameter (width) were used to calculate the tumor volume, V = width × width × length/2. The relative tumor volume was defined as V/V₀, where V₀ is the tumor volume when the treatment was initiated. After 21 days, the mice were euthanized, and major organs including the heart, liver, spleen, lungs, and kidneys were harvested for histological analysis.

3. Results and discussion

3.1. Synthesis and characterization

C₈-NBDP-OEG₄ is synthesized by the typical O'Shea's method (Scheme 2).³² Firstly, aldol condensation was used to obtain E-chalcone, Compound C, from benzaldehyde and acetophenone. The obtained compound C was then treated with nitromethane, and the Michael addition product D was condensed with ammonium acetate to form aza-dipyrromethene, which was then treated with boron trifluoride etherate in a basic medium to yield the target compound, C₈-NBDP-OEG₄. The overall yield was about 13.0%. C₈-NBDP-OEG₄ and the corresponding precursors were fully characterized by ¹H NMR, ¹³C NMR and HRMS (Fig. S1–S15).

Scheme 2 The synthetic routes employed for the preparation of C₈-NBDP-OEG₄.

The synthesized C₈-NBDP-OEG₄ demonstrated broad absorption from 600 nm to 900 nm in various organic solvents (Fig. S16). Notably, comparative spectral analysis revealed no detectable bathochromic or hypsochromic shifts upon transition of C₈-NBDP-OEG₄ from DMSO to aqueous media (Fig. 1a). Furthermore, C₈-NBDP-OEG₄ could self-assemble into uniform nanoparticles, as confirmed by the TEM observation, with an average diameter of ∼91 nm and a polydispersity index (μ₂/Γ²) of 0.11 based on the DLS measurement (Fig. 1b). UV-Vis spectroscopy, DLS, and TEM images collectively indicated that the SNAP loading had negligible effects on the dye's absorption profile or the assembled particles’ size (Fig. S17). The fluorescence intensity of C₈-NBDP-OEG₄ nanoparticles in aqueous solution decreased ∼40-fold relative to that in DMSO (Fig. 1c), indicating the strong fluorescence quenching effect among the aggregated dyes, which is beneficial for enhancing photothermal efficiency by promoting non-radiative transitions during excited-state relaxation.³³ The self-assembled C₈-NBDP-OEG₄ nanoparticles exhibited exceptional photostability, retaining ∼90% absorption after 90 min of 808 nm laser irradiation, whereas indocyanine green (ICG) maintained <20% absorption within 25 min under identical conditions (Fig. 1d). Moreover, the nanoparticles demonstrated superior chemical stability against various reactive oxygen species (ROSs, including H₂O₂, O₂⁻, ClO⁻, and ONOO⁻), while ICG underwent significant degradation (Fig. 1e). Additionally, C₈-NBDP-OEG₄@NO nanoparticles exhibited long-term colloidal stability in deionized water, phosphate-buffered saline (PBS) and 10% (v/v) fetal bovine serum (FBS) for 48 h without obvious size fluctuation (Fig. 1f), demonstrating it as a stable formulation for in vivo applications.³⁴

Fig. 1 (a) UV-Vis-NIR absorption spectra recorded for C₈-NBDP-OEG₄ and C₈-NBDP-OEG₄@NO in DMSO or PBS. (b) Fluorescence emission spectra (λ_ex = 808 nm) recorded for C₈-NBDP-OEG₄ (10 μM) in DMSO and PBS (pH 7.4, 10 mM). (c) Intensity-average hydrodynamic diameter distribution, f(D_h), and the TEM image (magnification: 40 000×) recorded for C₈-NBDP-OEG₄@NO nanoparticles. (d) Photo-stability characterization of C₈-NBDP-OEG₄ and ICG in PBS under 808 nm laser irradiation (1.8 W cm⁻²). (e) Normalized intensities recorded for C₈-NBDP-OEG₄ and ICG in PBS upon incubation with various ROSs (1 mM H₂O₂, 1 mM O₂⁻, 100 μM ClO⁻ and 100 μM ONOO⁻) in PBS (pH 7.4, 10 mM) for 12 h. (f) Diameters of C₈-NBDP-OEG₄@NO nanoparticles in deionized water, PBS (pH 7.4, 10 mM) and FBS (10% v/v) at different time points.

3.2. In vitro photothermal effect and NO release

The photothermal effects of C₈-NBDP-OEG₄ nanoparticles were systematically evaluated. Under 808 nm laser irradiation (1.8 W cm⁻²) for 10 minutes, the temperature increased from 37.4 °C to 60.8 °C as the nanoparticle concentration increased from 0.0125 g L⁻¹ to 0.05 g L⁻¹, whereas no significant temperature change was observed in pure water under identical conditions (Fig. 2a). Furthermore, the temperature elevation profile demonstrated the power intensity-dependent manner (Fig. 2b). These results collectively suggested that C₈-NBDP-OEG₄ nanomaterials exhibit promising photothermal performance. Besides, the photothermal conversion efficiency (PCE) of C₈-NBDP-OEG₄ was calculated to be 39.8% by previously reported literature methods³⁵ (Fig. 2c and d), which is comparable to the most reported photothermal agents.³⁶ In addition, the stability of C₈-NBDP-OEG₄ was further evaluated. C₈-NBDP-OEG₄ nanomaterials exhibited high photostability after 5 cycles of heating and cooling upon 808 nm laser irradiation, while indocyanine green (ICG) exhibited a dramatic absorption decrease under the same conditions (Fig. 2e and f).

Fig. 2 (a) The concentration-dependent and (b) the laser power-dependent temperature elevation curves of C₈-NBDP-OEG₄ nanoparticles under 808 nm laser irradiation. (c) Temperature increase/decrease curves and (d) cooling time vs. −ln(θ) plot (θ) (θ: temperature drive force) recorded for C₈-NBDP-OEG₄ nanoparticles (0.025 g L⁻¹, 1.8 W cm⁻²). (e) Photo-thermal stabilities recorded for C₈-NBDP-OEG₄ nanoparticles and ICG during 5 cycles of heating–cooling (0.05 g L⁻¹, 1.8 W cm⁻²). (f) The UV-Vis-NIR spectra recorded for C₈-NBDP-OEG₄ nanoparticles before and after 808 nm laser irradiation for five cycles (0.05 g L⁻¹, 1.8 W cm⁻²). (g) The NO release profile from C₈-NBDP-OEG₄@NO nanoparticles (0.05 g L⁻¹) at different temperatures. (h) The kinetics of the NO release profile from C₈-NBDP-OEG₄@NO nanoparticles (0.05 g L⁻¹) at different temperatures. (i) The 808 nm laser power-dependent NO release from C₈-NBDP-OEG₄@NO nanoparticles (0.05 g L⁻¹) for 10 min (environment temperature is 22 °C).

Subsequently, a thermal-sensitive NO donor, SNAP, was co-assembled within C₈-NBDP-OEG₄ to form C₈-NBDP-OEG₄@NO. The thermal-triggered NO release originates from the cleavage of the S–N bond in S-nitroso-N-acetylpenicillamine (SNAP) upon localized photothermal heating. In vitro NO release studies revealed that SNAP remained stable at 37 °C (<20% release), but exhibited a substantial release above 40 °C, and over 60% NO was released from the nanoparticles at 60 °C (Fig. 2g and h), highlighting the potential for photothermal-triggered NO release. Furthermore, the NO release profile demonstrated a power intensity-dependent response under 808 nm laser irradiation, with approximately 60% NO being released from C₈-NBDP-OEG₄@NO nanoparticles (Fig. 2i). These findings underscore the potential of C₈-NBDP-OEG₄@NO as a promising platform for photothermal-driven photothermal/gas therapy.

3.3. Cellular cytotoxicity and intracellular NO detection

To assess the therapeutic efficacy of C₈-NBDP-OEG₄@NO nanoparticles, in vitro cellular cytotoxicity was evaluated in 4T1 cells using the MTT assay. As shown in Fig. 3a, the viability of 4T1 cells remained above 93% after 12 hours of incubation with C₈-NBDP-OEG₄@NO nanoparticles at a concentration of up to 0.2 g L⁻¹, demonstrating their high biocompatibility. In the absence of laser irradiation, the cell viability remained above 90% for both C₈-NBDP-OEG₄@NO and DSPE-PEG_2k@NO nanoparticles, suggesting that SNAP is relatively stable under physiological conditions without significant NO release. Upon 808 nm laser irradiation for 5 minutes, C₈-NBDP-OEG₄ alone induced approximately 65% cell death in 4T1 cells, demonstrating the potent photothermal effect in killing cancer cells. Notably, C₈-NBDP-OEG₄@NO nanoparticles resulted in approximately 80% cell death under the same conditions, highlighting the synergistic enhancement of cytotoxicity through the combined effects of photothermal therapy and NO gas therapy. Besides, the cytotoxic effect of C₈-NBDP-OEG₄@NO on 4T1 cells exhibited a distinct concentration-dependent manner, demonstrating significant cell-killing efficacy (60% mortality) at a concentration of 100 μg mL⁻¹ (Fig. 3b). These findings underscore the potential of C₈-NBDP-OEG₄@NO nanoparticles as a PTT/gas synergistic therapeutic agent, leveraging both photothermal and NO-mediated cytotoxic mechanisms.

Fig. 3 (a) The relative viability of the 4T1 cells after incubation with different formulation nanoparticles (0.2 g L⁻¹) for 12 h. (b) The relative viability of the 4T1 cells after incubation with different concentrations of C₈-NBDP-OEG₄@NO nanoparticles for 12 h. (c) Confocal fluorescence images of NO generation in 4T1 cells under different treatment conditions (green fluorescence indicates the presence of NO). (d) The corresponding fluorescence images (scale bar: 80 μm) of the cells from various groups stained with calcein-AM (living cells, green fluorescence) and PI (dead cells, red fluorescence). The experimental group was subjected to laser irradiation using an 808 nm laser with a power density of 1.8 W cm⁻², and the irradiation time was 5 minutes.

To further investigate the release of NO from C₈-NBDP-OEG₄@NO nanoparticles in 4T1 cells, we employed a commercially available fluorescent NO probe, 3-amino-4-aminomethyl-2′,7′-difluorescein diacetate (DAF-FM DA) to detect the cellular NO level by confocal laser scanning microscopy (CLSM) (Fig. 3c). In both the DSPE-PEG_2k@NO and C₈-NBDP-OEG₄@NO groups without 808 nm laser irradiation, no significant green fluorescence signals were observed following 1 h of incubation with 4T1 cells. Similar results were also observed for C₈-NBDP-OEG₄ groups subjected solely to 808 nm laser irradiation. By contrast, the irradiated C₈-NBDP-OEG₄@NO group exhibited a pronounced ∼3.7-fold increase in green fluorescence intensity (Fig. S18), indicating effective NO release at the cellular level with 808 nm laser irradiation.

The therapeutic efficiency was further confirmed through the co-staining experiment employing calcein AM and propidium iodide (PI), wherein the green and red fluorescent signals corresponded to live and dead cells, respectively. As shown in Fig. 3d, obvious green fluorescent signals could be observed in the control, DSPE-PEG_2k@NO and C₈-NBDP-OEG₄@NO groups, indicating negligible cytotoxicity under these experimental conditions. However, the DSPE-PEG_2k@NO + laser and C₈-NBDP-OEG₄ + laser groups displayed green and red fluorescent signals, with the red signal of the C₈-NBDP-OEG₄ + laser group being stronger than that of the DSPE-PEG_2k@NO + laser group. As for the C₈-NBDP-OEG₄@NO + laser group, a very small amount of green signal was observed, demonstrating that most cells were dead or underwent apoptosis.

3.4. In vivo photothermal and photoacoustic imaging

After 12 h post intravenous injection, the 4T1-tumor bearing mice were irradiated with an 808 nm laser (1.8 W cm⁻²) for 10 min. As shown in Fig. 4a and b, the tumor region temperature in the laser-only group increased to 44.6 °C, whereas the C₈-NBDP-OEG₄ and C₈-NBDP-OEG₄@NO groups exhibited significantly higher temperatures of 53.9 °C and 57.3 °C, respectively. These results demonstrated that C₈-NBDP-OEG₄@NO nanoparticles served as highly effective near-infrared organic photothermal agents for cancer theranostics.

Fig. 4 (a) Photothermal images of the 4T1 tumor sites after intravenous administration of the indicated nanoparticles (0.2 g L⁻¹, 0.1 mL) or PBS with 808 nm laser irradiation (1.8 W cm⁻²) for different times. (b) In vivo photothermal temperature elevation curves in different groups. (c) Photoacoustic images of the tumor sites after intravenous administration of the indicated nanoparticles at different time points. (d) Time-dependence of the normalized photoacoustic intensity ratio at the tumour area between the C₈-NBDP-OEG₄@NO and C₈-NBDP-OEG₄ groups.

Given that vaporization enhances photoacoustic contrast, we further evaluated the in vivo accumulation of C₈-NBDP-OEG₄@NO and C₈-NBDP-OEG₄via photoacoustic imaging (Fig. 4c). After intravenous injection of C₈-NBDP-OEG₄@NO into 4T1 tumor-bearing mice, photoacoustic imaging was performed at 0, 1, 4, 8, 12, and 24 h post-injection. The results showed a significant increase in the photoacoustic signal at the tumor site in the C₈-NBDP-OEG₄@NO group, likely due to the laser-induced decomposition of SNAP within C₈-NBDP-OEG₄@NO, leading to NO gas production. The localized accumulation of NO gas enhanced the photoacoustic signal by inducing thermoelastic expansion and increasing the acoustic impedance contrast within the tumor microenvironment. In contrast, the C₈-NBDP-OEG₄ group exhibited only minimal accumulation, primarily attributed to passive enhanced permeability and retention (EPR) effects, resulting in a weaker and more transient photoacoustic signal. Quantitatively, the photoacoustic signal intensities of the C₈-NBDP-OEG₄@NO group at the tumor site were around 1.6-fold higher than those of the C₈-NBDP-OEG₄ group at 12 h post-injection, suggesting improved tumor diagnosis capability due to NO production.

3.5. In vivo tumor inhibition

Finally, in vivo tumor inhibition of C₈-NBDP-OEG₄@NO nanoparticles was examined on 4T1 tumor-bearing mice (Fig. 5a and b). For in vivo experiments, 4T1 tumor-bearing mice were randomly divided into six groups: PBS control (group 1); C₈-NBDP-OEG₄@NO (group 2); laser only (group 3); DSPE-PEG_2k@NO + laser (group 4); C₈-NBDP-OEG₄ + laser (group 5); and C₈-NBDP-OEG₄@NO + laser (group 6). After being subjected to different treatment protocols, the therapeutic efficacy was assessed by monitoring the changes in tumor volumes (Fig. 5c–f and Fig. S19). Groups 1, 2, 3, and 4 all exhibited minimal tumor inhibition, while Group 5 exhibited no obvious tumor growth in the entire treatment process. Only Group 6, C₈-NBDP-OEG₄@NO nanoparticles under 808 nm laser irradiation, exhibited the most efficient tumor inhibition; the tumor inhibition ratio reached up to ∼90%, along with the longest lifespan. These results are in agreement with the above discussion that the released NO gas reversed multidrug resistance and augmented the combined photothermal/gas dual-model therapy efficiency. Besides, no obvious body weight changes were observed during the whole therapeutic process (Fig. 5g). The proliferative activity of tumor cells was assessed by Ki67 immunohistochemical staining (Fig. 5h), revealing significantly reduced Ki67 expression in Group 6 compared to the other groups. Furthermore, H&E analysis revealed that tumor tissues treated with C₈-NBDP-OEG₄@NO nanoparticles displayed much more highly condensed nuclei, indicative of apoptosis or necrosis (Fig. 5h). The hepatic function indexes, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), urea nitrogen (BUN) and others, exhibited negligible changes between the group treated with 808-irradiated C₈-NBDP-OEG₄@NO nanoparticles and the other groups (Fig. 6). Moreover, there existed unnoticeable abnormalities for all major organs, including the heart, liver, spleen, lungs and kidneys (Fig. 7), indicating the high biocompatibility of C₈-NBDP-OEG₄@NO nanoparticles.

Fig. 5 (a) Illustration of the therapeutic procedures of C₈-NBDP-OEG₄@NO nanoparticles against a 4T1 tumor-bearing mouse model (n = 5 for each group). (b) The experiment groups used in the therapeutic procedures. (c) Photographs of the mice during the treatment process at 7, 14 and 21 days after intravenous injection of the corresponding dispersion (0.2 g L⁻¹, 100 μL) or PBS (100 μL). (d) Typical photographs of cancer issues collected on the 21^st day after different treatment procedures. (e) Cancer growth curve for each treatment group. (f) The tumor weight after different treatment procedures. (g) The mouse body weight for each treatment group during the therapeutic process. (h) Ki67 and H&E staining of tumors (scale bar: 40 μm) in mice from different treatment groups. Experiments were performed three times with similar results, and the data are presented as mean ± S. D.

Fig. 6 Hepatic/renal function analyses of 4T1 tumor-bearing mice after different treatments. (a-group names; b-ALT: alanine aminotransferase; c-AST: aspartate transaminase; d-BUN: blood urea nitrogen; e-CR: creatinine; f-WBC: white blood cell; g-RBC: red blood cell; h-HGB: hemoglobin; i-HCT: hematocrit; j-MCV: mean corpuscular volume; k-MCHC: mean corpuscular hemoglobin concentration; and l-PLT: platelet.)

Fig. 7 Representative hematoxylin and eosin (H&E)-stained histological sections of major organs from 4T1 tumor-bearing mice following various treatments: (1) control; (2) C₈-NBDP-OEG₄@NO; (3) laser; (4) DSPE-PEG_2k@NO + laser; (5) C₈-NBDP-OEG₄ + laser; and (6) C₈-NBDP-OEG₄@NO + laser.

4. Conclusions

In summary, we successfully developed a novel amphiphilic aza-BODIPY-based photothermal agent, C₈-NBDP-OEG₄, which self-assembles into uniform and stable nanoparticles exhibiting high photothermal conversion efficiency and excellent photostability. By co-encapsulating the NO donor S-nitroso-N-acetylpenicillamine (SNAP), the resulting C₈-NBDP-OEG₄@NO nanoparticles enabled NIR-triggered dual-modal therapy, synergistically combining photothermal ablation and NO-mediated tumor microenvironment modulation. This strategy led to enhanced tumor inhibition both in vitro and in vivo, confirming its therapeutic efficacy. Importantly, the released NO not only contributed to gas therapy but also amplified the photoacoustic signal intensity, facilitating photoacoustic imaging-guided therapy. This theranostic capability enabled precise diagnosis and real-time treatment monitoring, further supporting the translational potential of the platform. The integration of PTT, gas therapy, and imaging guidance within a single nanosystem represents a robust approach for advancing precision oncology. Looking forward, future research should aim to further red-shift the absorption of the aza-BODIPY core to maximize tissue penetration and explore active targeting strategies to enhance tumor specificity while minimizing off-target effects. Additionally, elucidating the precise cytotoxic mechanisms, such as DNA damage induction and mitochondrial dysfunction, will deepen our understanding of the therapeutic effects. A thorough evaluation of long-term accumulation, pharmacokinetics, clearance profile, and potential systemic toxicity will be essential to ensure the safety and clinical feasibility of this nanoplatform.^34,37,38

While the presented photothermal and NO-based therapy demonstrates promising efficacy, we acknowledge that the inherent physiological heterogeneity of solid tumors—particularly variations in vascular density, permeability, and elevated interstitial fluid pressure (IFP)—can significantly influence nanoparticle delivery and intratumoral distribution. These factors may impede uniform extravasation and penetration of our nanoparticles via the enhanced permeability and retention (EPR) effect, potentially leading to suboptimal drug/NOS loading in hypovascular regions or areas of high IFP. This heterogeneity could create spatial variations in photothermal heating efficiency and NO generation, thereby impacting the overall therapeutic outcome. Highlighting this challenge underscores that the efficacy observed in our model system may require further optimization or combination strategies (e.g., vascular normalization) to ensure robust performance across more complex, heterogeneous tumor microenvironments encountered clinically. Addressing this limitation is crucial for broader applicability.

Author contributions

Qian He: sample synthesis and characterization, formal analysis, and investigation. Jiawen He: cellular and animal experiments, formal analysis, and investigation. Panyue Wen: investigation and review & editing; Haochen Guo: investigation and review & editing; Shizhong Luo: supervision; Masaru Tanaka: review & editing; Junjie Li: review & editing, projection administration, supervision, and funding acquisition; Ye Liu: resources, projection administration, supervision, and funding acquisition; Liewei Wen: resources, projection administration, supervision, and funding acquisition. Chenzhi Yao: writing – review & editing, visualization, investigation, supervision, project administration, and funding acquisition.

Conflicts of interest

The authors declare no conflicts of interest.

Data availability

All data supporting the findings of this work are presented within the article and its SI. SI includes the instruments section, statistics section, synthesis method for compound A–D, and Figs. S1–S19. See DOI: https://doi.org/10.1039/d5bm00937e.

Acknowledgements

This work was financially supported by the National Natural Science Foundation of China (Grant No. 82302368), the Changjiang Scholars Program, the Anhui Provincial Natural Science Foundation (2408085QB056), the Guangdong Basic and Applied Basic Research Foundation (2022A1515220167), the Zhuhai Basic and Applied Basic Research Foundation (2320004002697), and the Grants-in-Aid for Scientific Research (23H03740, 23K19220, and 24K21109) from the Japan Society for the Promotion of Science (JSPS).

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Footnote

† These authors contributed equally to this work.

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