Multiple rotor-based photothermal agents for NIR-I/NIR-II fluorescence imaging-guided tumor phototherapy

Abstract

Photothermal therapy (PTT) is emerging as a promising alternative therapy for tumor ablation through spatiotemporally controlled hyperthermia. However, designing near-infrared photothermal agents with a high photothermal conversion efficiency (PCE) and high photostability remains considerably challenging. To address this limitation, we engineered optimal molecule 3TPA by employing CF₃-BODIPY as an electron-deficient core and incorporating three electron-donating triphenylamine rotors, which showed an extended absorption wavelength and improved PCE. To optimize the therapeutic performance, 3TPA was further encapsulated into a DSPE-mPEG_5k-based amphiphilic polymer to form 3TPA NPs, which exhibited high PCE (η = 57.2%) and remarkable photostability. In vivo studies revealed that 3TPA NPs selectively accumulated in tumor sites under NIR-I/NIR-II fluorescence imaging guidance, simultaneously enabling effective photo-mediated tumor ablation through a precision PTT effect. This work not only presents a robust NIR-II therapeutic agent but also opens up more possibilities for its application in photothermal therapy in the field of biomedicine.

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1. Introduction

Photothermal therapy (PTT), a noninvasive cancer treatment strategy that induces localized hyperthermia through light-to-heat conversion in tumor tissues, has gained significant attention in biomedical research.^1–7 The critical challenge lies in developing photothermal agents (PTAs) capable of selectively eliminating cancer cells while minimizing adverse effects on healthy tissues.^8–13 Although inorganic nanomaterials such as gold nanorods,^14–21 carbon nanotubes,^22–30 and metal oxides^31–40 have demonstrated therapeutic potential through their strong near-infrared absorption properties, small-molecule organic photothermal agents offer distinct advantages including enhanced biodegradability, reduced systemic toxicity, and enhanced structural versatility, making them particularly promising candidates for advancing precision photothermal therapeutics.^4,41–53 However, these PTAs still suffer from low photothermal conversion efficiency (PCE) and poor photostability.^54–59 For example, clinical indocyanine green (ICG), an FDA-approved agent widely used in biomedical imaging, often faces limitations in PTT applications due to the lack of tumor selectivity and thermal-induced decomposition and photobleaching.^58,60–69 Therefore, the development of PTAs with NIR absorption, high photothermal conversion efficiency, and high photostability is of great importance.^70–79

In recent years, the investigation of excited-state intramolecular motion has provided an alternative approach for designing efficient PTAs.^80–94 The systematic engineering of PTAs incorporating adequate molecular rotors that drive vigorous intramolecular motion to amplify nonradiative decay pathways has been proposed to enhance heat generation and PCE.^1,95–100 Moreover, the introduction of bulky steric donor rotors within the molecular scaffold can effectively avoid fluorescence quenching in the aggregated state while maintaining the conjugation degree of PTAs.^101,102 For example, Tang and co-workers reported butterfly-shaped NIR-II aggregation-induced emission luminogens (AIEgens) featuring multiple triphenylamine (TPA) or tetraphenylethylene (TPE) rotors for photothermal combat of bacterial biofilms.¹⁰³ Liu's group presented a cyanine dye (IRLy) employing an “extended conjugation & molecular rotor” molecular design strategy to optimize the PTT efficacy and the balance with the NIR-II fluorescence intensity for image-guided therapy of glioblastoma.¹⁰⁴ Fortunately, meso-trifluoromethyl-BODIPY (CF₃-BODIPY)-based probes have emerged as powerful electron-withdrawing moieties for developing NIR emitting small-molecule organic PTAs, in which the –CF₃ moiety shows a free rotational mode that inhibits parallel ladder-like π–π stacking (H-aggregation) to maximize light-to-heat conversion efficiency.¹⁰⁵ Furthermore, when electron-donating groups are introduced at the α-position of CF₃-BODIPY, an electron transfer occurs within the molecule, resulting in a push–pull electronic effect that allows effective modulation of the absorption wavelength.^83,106–108 Therefore, it is worth exploring the introduction of multifunctional electron-donating rotors into the α- and β-sites of the CF₃-BODIPY-based probe to establish a robust non-radiative energy dissipation pathway, enabling the development of excellent PTAs with superior photostability and photothermal PCE.

In this work, a novel PTA named 3TPA for cancer treatment was designed with a high PCE based on a CF₃-BODIPY scaffold, by introducing a –CF₃ moiety as a “barrier-free” rotor on the meso-position and three TPA groups at the α- and β-sites (Scheme 1). The unhindered rotational characteristics of both –CF₃ and TPA components provided synergistic nonradiative decay pathways, thereby achieving exceptional photothermal conversion performance. To further improve the tumor targeting ability, 3TPA was encapsulated into the DSPE-mPEG_5k-based amphiphilic polymer to yield 3TPA NPs, which exhibited significantly better photostability than the FDA-approved ICG dye and a high PCE of 57.2%. In vivo studies revealed tumor-specific accumulation of 3TPA NPs under NIR-I/II fluorescence imaging guidance, enabling precision-targeted tumor photothermal inhibition, with nearly complete tumor eradication and no observed recurrence. This study not only provides a potent NIR-II therapeutic agent but also expands the biomedical application potential of photothermal therapy platforms.

Scheme 1 Design and biological application of the multiple rotor-based photothermal agents named 3TPA NPs.

2. Results and discussion

2.1. Spectroscopic studies of 2TPA and 3TPA

To construct near-infrared (NIR) photothermal agents, multiple triphenylamine rotors were introduced into the CF₃-BODIPY-based skeleton for the synthesis of 2TPA and 2TPA, as fully characterized by ¹ H NMR and ¹³C NMR in Scheme S1, Fig. S13–S20 (ESI†). Firstly, the photophysical characteristics of 2TPA and 3TPA were systematically investigated through absorption and fluorescence spectroscopy. As depicted in Fig. 1, 3TPA exhibited obvious redshifted absorption and intensive mole absorptivity (λ_abs = 806 nm, ε = 1.26 × 10⁵ M⁻¹ cm⁻¹) compared to 2TPA (λ_abs = 787 nm, ε = 1.18 × 10⁵ M⁻¹ cm⁻¹) in tetrahydrofuran solvent (Fig. 1b and f). The emission spectra of 3TPA exhibited two distinct peaks at 840 nm (NIR-I region, Fig. 1g) and 931 nm (NIR-II region, Fig. 1h), accompanied by a weak broad shoulder around 970 nm. In contrast, 2TPA showed relatively weak emission intensity, with peaks at 827 nm (NIR-I, Fig. 1c) and 927 nm (NIR-II, Fig. 1d). A similar spectral characteristic was also observed in other solvents (Fig. S1, ESI†). Notably, 3TPA showed a fluorescence quantum yield (Φ) of 1.28% relative to ICG (Φ_ICG = 13.2%, Fig. S2a, ESI†), while it displayed undetectable singlet oxygen (¹O₂) generation under 808 nm laser irradiation, as assessed by recording the decreased absorbance of 1,3-diphenylisobenzofuran (DPBF) (Fig. S2b and c, ESI†) and the increased fluorescence intensity of SOSG (Figure S2d–i, ESI†). The findings demonstrated that the extended absorption/emission wavelengths, stronger molar extinction coefficient (ε), and low ¹O₂ production synergistically rendered 3TPA a superior candidate for NIR fluorescence imaging and photothermal therapy applications.

Fig. 1 Photophysical properties of 2TPA and 3TPA. (a) Chemical structures, (b) absorption spectra, (c) fluorescence spectra (λ_ex = 764 nm) and (d) fluorescence spectra (λ_ex = 825 nm) of 2TPA (5 μM); (e) chemical structures, (f) absorption spectra, (g) fluorescence spectra (λ_ex = 767 nm) and (h) fluorescence spectra (λ_ex = 825 nm) of 3TPA (5 μM).

2.2. Preparation and photothermal performance of 2TPA/3TPA NPs

To further improve the tumor targeting ability, 3TPA/2TPA was further encapsulated into the amphiphilic polymer DSPE-mPEG_5k through a nanoprecipitation method, yielding monodisperse nanoparticles (named as 3TPA NPs and 2TPA NPs, respectively, Fig. 2a). The UV-Vis spectrophotometric quantification revealed the encapsulation efficiencies of 3TPA NPs and 2TPA NPs to be 94.4% and 93.7%, respectively (Fig. S3 and S4, ESI†). Transmission electron microscopy (TEM) and dynamic light scattering (DLS) analyses provided detailed characterization of the morphologies and sizes of the nanoparticles. TEM images showed that 3TPA NPs and 2TPA NPs exhibited spherical shapes with diameters of approximately 145 nm and 140 nm, respectively (Fig. 2b and Fig. S6a, ESI†). The nanoparticles remained stable for 9 days after being stored at 4 °C, as confirmed through size distribution measurements by DLS (Fig. S5b and S6c, ESI†). And there was no significant decrease in absorbance during the 9 days of storage (Fig. S5c, d and S6d, e, ESI†). In PBS buffer solution, the absorption peaks of 3TPA NPs and 2TPA NPs were centered at 825 nm and 787 nm, consistent with the characteristic peak positions of their single-molecule counterparts (Fig. 2c). Notably, following the formation of the nanoparticles, the NIR-I region emission peaks of 3TPA NPs and 2TPA NPs remained unchanged, while the emission peak near 970 nm became more pronounced in the NIR-II region (Fig. 2d and e).

Fig. 2 Preparation and characterization of nanoparticles. (a) Preparation of 2TPA NPs and 3TPA NPs; (b) particle size distribution of 3TPA NPs measured by DLS with a mean diameter of 145 nm and a PDI of 0.15, Inset: TEM image (top right) of 3TPA NPs; (c) absorption spectra, (d) NIR-I emission spectra and (e) NIR-II emission spectra of 2TPA NPs and 3TPA NPs (5 μM) in PBS; photothermal conversion of 2TPA NPs (f) and 3TPA NPs (h) at different concentrations (6.25–100 μM) under 808 nm (0.5 W cm⁻²) laser irradiation; infrared imaging of 2TPA NPs (g) and 3TPA NPs (i) dispersions; photothermal conversion of 2TPA NPs (j) and 3TPA NPs (k) (75 μM) under 808 nm laser irradiation with different exposure intensity (0.1–0.6 W cm⁻²); (l) photothermal stability study in PBS of heating–cooling processes upon 808 nm laser irradiation of 0.5 W cm⁻² for six on/off cycles; (m) photothermic heating curves of ICG, 3TPA NPs and 2TPA NPs (50 μM) dispersions under 808 nm irradiation (0.75 W cm⁻²) with 100 μL followed by cooling to room temperature.

Owing to the excellent light absorption in the NIR region and high stability of 3TPA NPs and 2TPA NPs, they could serve as promising candidates for PTT. Next, the photothermal properties of 3TPA NPs and 2TPA NPs were evaluated by monitoring the temperature change in PBS solution. Under 808 nm laser irradiation (0.5 W cm⁻²), both 3TPA NPs and 2TPA NPs demonstrated a pronounced dose-dependent temperature elevation (Fig. 2f and h), with corresponding infrared thermal images displayed in Fig. 2g and i. It was worth noting that 3TPA exhibited superior photothermal performance compared to 2TPA, as evidenced by the 77 °C rise for 3TPAversus 60 °C for 2TPA at a concentration of 100 μM. Meanwhile, with the increase of the laser power density from 0.1 to 0.6 W cm⁻², the temperature of the dispersed solutions (75 μM) of 3TPA NPs and 2TPA NPs also significantly increased (Fig. 2j and k), revealing their laser power-controllable photothermal behaviors. Remarkably, both 3TPA and 2TPA NPs maintained their structural integrity without detectable degradation over six consecutive laser-induced heating–cooling cycles (Fig. 2l), demonstrating much better photostability than FDA-approved indocyanine green (ICG). The PCEs of the 3TPA NPs and 2TPA NPs were quantified as 57.2% and 44.8%, respectively, through photothermal response profiles and thermal relaxation parameters (Fig. 2m and Fig. S7, S8, ESI†), surpassing the 20.4% PCE observed in the clinically benchmarked ICG agent. These findings illustrated that 3TPA NPs and 2TPA NPs could serve as efficient PTAs for tumor-specific photothermal ablation through localized hyperthermia generation.

2.3. Cytotoxicity assessment of nanoparticles in tumor cells

Subsequently, we carefully investigated the cytotoxicity and photothermal killing activity of 3TPA NPs and 2TPA NPs to MCF-7 cancer cells by a standard methyl thiazolyl tetrazolium (MTT) assay. As shown in Fig. 3a, the cell viability was over 80% when the cells were cultured with 3TPA NPs (up to 16 μM) without irradiation. However, 3TPA NPs exhibited dose-dependent toxicity upon 808 nm laser irradiation with IC₅₀ values of 6.07 ± 0.41 μM. In contrast, 2TPA NPs (IC₅₀ = 7.83 ± 0.45 μM) showed weaker cell killing activity compared to 3TPA NPs (Fig. S9a, ESI†), a disparity likely originating from its diminished PCE as evidenced by in vitro experiments.

Fig. 3 Cytotoxicity assessment of 3TPA NPs. (a) Cell viability of MCF-7 cells incubated with 3TPA NPs at various concentrations in the dark and after laser irradiation (808 nm, 0.75 W cm⁻², 10 min); (b) calcein AM (green) and propidium iodide (red) co-staining fluorescence imaging of MCF-7 cells after different treatments. Laser irradiation (808 nm, 0.75 W cm⁻², 10 min) was conducted after cells were incubated with 3TPA NPs (10 μM). Scale bar: 100 μm; (c) apoptosis and necrosis analysis using flow cytometry toward MCF-7 cells after different treatments. Laser irradiation (808 nm, 0.75 W cm⁻², 10 min) was conducted after cells were incubated with 3TPA NPs (10 μM).

To evaluate the phototherapeutic efficacy of 3TPA NPs, live-dead cell staining was conducted by using calcein AM (green) and propidium iodide (red) as fluorescent markers. 3TPA NPs triggered almost complete destruction of MCF-7 cells upon 0.75 W cm⁻² NIR laser irradiation for 10 min, as indicated by the intense red fluorescence and absence of green fluorescence (Fig. 3b). However, exclusively green fluorescence was detected in the control group under dark conditions. Additionally, flow cytometric analysis was performed to elucidate apoptotic pathways using the Annexin-V/FITC-PI detection kit. As illustrated in Fig. 3c, 3TPA NPs treatment combined with 808 nm photoirradiation (10 μM, 0.75 W cm⁻², 10 min) resulted in apoptotic progression in >30% of the MCF-7 cellular population. Similarly, 2TPA NPs also showed comparable in vitro effects, with detailed results presented in Fig. S9b (ESI†). These findings indicated that 3TPA NPs could serve as an effective phototherapeutic agent with negligible dark toxicity.

2.4. Imaging of 4T1 tumor xenograft mice in vivo

Next, the in vivo biodistribution and fluorescence imaging performance of 3TPA NPs and 2TPA NPs were systematically evaluated in MCF-7 tumor-bearing BALB/c mice xenograft models. Following tail vein administration of 3TPA or 2TPA NPs (100 μM in 150 μL PBS) in tumor-bearing mice, fluorescence images (filter: 745 ± 20 nm) were captured at different time points to determine the optimal time window for PTT. As illustrated in Fig. 4a and b, the fluorescence signals at the tumor site gradually increased, reaching a plateau at 12 h with marked differentiation from neighboring regions. Surprisingly, the nanoparticles exhibited prolonged tumor retention for up to 6 days post-administration, with sustained fluorescence signals remaining detectable throughout this period. The mice were euthanized 24 h post-injection, and their dissected organs and tumors were harvested for ex vivo imaging. As shown in Fig. 4c, the nanoparticles displayed distinct fluorescence in the tumor tissue and could be cleared out through hepatic excretion. As shown in Fig. 4d–f, similar results were also observed in the NIR-II imaging window (filter: >1000 nm), where 3TPA NPs exhibited stronger fluorescence intensity at tumor sites than that of 2TPA NPs, which could be linked to their differential aggregation state and cellular uptake within tumors.

Fig. 4 Imaging of 4T1 tumor xenograft mice in vivo. (a) NIR-I fluorescence imaging in vivo (filter: 745 ± 20 nm), (b) time profile of mean fluorescence intensities at the tumor site of the mice, and (c) fluorescence intensities of ex vivo organs and tumors from the mice of PBS, 2TPA NPs and 3TPA NPs-treated groups. (d) NIR-II fluorescence imaging in vivo (filter: >1000 nm), (e) time profile of mean fluorescence intensities at the tumor site of the mice, and (f) fluorescence intensities of ex vivo organs and tumors from the mice of PBS, 2TPA NPs and 3TPA NPs-treated groups. (g) Infrared thermal imaging of 4T1 tumor-bearing mice treated with different solutions exposed to an 808 nm laser (0.6 W cm⁻²) recorded at different time intervals, respectively; (h) temperature profiles of the tumor-site as a function of irradiation time.

To further validate the photothermal effects of 3TPA NPs and 2TPA NPsin vivo, 4T1 tumor-bearing mice intravenously injected with 3TPA NPs and 2TPA NPs, respectively, were used as experimental subjects exposed to 808 nm laser irradiation at a power density of 0.6 W cm⁻² for 10 min, and real-time temperature changes were recorded using a FLIR thermal mapping camera. As shown in Fig. 4g and h, the mean intratumoral temperature in 3TPA NPs-administered mice rapidly reached 57 °C, whereas the temperature increase in the PBS-treated mice was negligible. 2TPA NPs exhibited weaker temperature enhancement than 3TPA NPs with a maximum intra-tumoral temperature of 48 °C under identical experimental conditions. This significant thermal differential may be related to their respective photothermal conversion efficiencies. Meanwhile, as shown in Fig. S10 (ESI†), when the laser intensity increased from 0.6 W cm⁻² to 0.8 W cm⁻², the temperature rise at the tumor sites demonstrated a power-dependent correlation. Consequently, multimodal imaging modalities (NIR-I/NIR-II FI and PTI) targeting nanoparticles at tumor sites could offer crucial guidance for follow-up phototherapy.

2.5. In vivo PTT therapy evaluation

Finally, the antitumor efficacy of 3TPA NPs and 2TPA NPs under NIR irradiation was systematically evaluated in vivo, with the corresponding treatment schedule as detailed in Fig. 5a. Forty MCF-7 tumor-bearing mice were randomly divided into five groups, including “PBS”, “2TPA NPs + dark”, “2TPA NPs + light”, “3TPA NPs + dark” and “3TPA NPs + light”, and the therapeutic efficacy of each group was assessed by tracking the tumor volumes at two-day intervals post-treatment over a 14 day period. Among these groups, the “3TPA NPs + light” and “2TPA NPs + light” groups were treated with 808 nm laser irradiation at a power density of 0.75 W cm⁻² for 10 min. As displayed in Fig. 5b–d, the average tumor volumes increased 6–7-fold for the control groups (“PBS”, “2TPA NPs + dark” and “3TPA NPs + dark”), demonstrating negligible activity for the suppression of tumor growth. Although the “2TPA NPs + light” group exhibited moderate tumor inhibitory activity, the average tumor volume still increased to approximately twice the initial tumor volume. In comparison, irradiation of the 3TPANPs-treated group led to significant tumor growth inhibition, with nearly complete tumor eradication and no recurrence observed. These findings were consistent with the phototoxicity assays obtained from in vitro cell experiments, further confirming the excellent photothermal therapeutic ability of 3TPA NPs. Moreover, the body weights of the mice remained almost unchanged during the process of phototherapy (Fig. 5e). Subsequently, following the 14 day therapeutic period, the mice were sacrificed and their tumors were weighed, which further validating the therapeutic effect of 3TPA NPs (Fig. 5f). After the treatment, histological analysis of H&E-stained sections from major organs (heart, liver, spleen, lung, kidney) and tumors were measured and revealed no detectable organ damage or inflammatory responses in the control groups. In contrast, distinct nucleus dissociation and necrosis were found in the 3TPA NPs plus laser treated mice (Fig. S12, ESI†), ensuring the excellent in vivo biocompatibility of the nanoparticles. These results demonstrated that 3TPA NPs represented a promising phototherapeutic agent for tumor imaging and ablation, characterized by tumor-targeted phototherapeutic effects and excellent biocompatibility, with minimal toxicity to normal tissues.

Fig. 5 PTT in vivo. (a) Schematic of the in vivo tumor treatment plan for PTT; (b) photographs of living mice tumors from different treatment groups after 14 day treatment; (c) photographs of tumors from different treatment groups after 14 day treatment; (d) the tumor growth in 4T1 tumor-bearing mice treated with different formulations (100 μM, 200 μL 2TPA NPs and 3TPA NPs in PBS; in the dark or under 808 nm laser 0.75 W cm⁻² for 10 min); (e) body weight of the mice in different groups over time (mean ± SD, n = 5); (f) tumor weight of the mice in different groups over time (mean ± SD, n = 5).

3. Conclusion

In summary, a novel PTA (named 3TPA) was designed based on the CF₃-BODIPY scaffold for cancer treatment by introducing a –CF₃ portion as a “barrier-free” rotor at the meso-position and three TPA groups at the α- and β-sites. 3TPA exhibited an absorption band in the NIR-I region (λ_em = 840 nm) and a fluorescence emission peak extending into the NIR-II region (λ_em = 931 nm). The PCE could be optimized by systematically adjusting the number of triphenylamine (TPA) rotors that drive vigorous intramolecular motion to amplify nonradiative decay pathways. Encapsulating 3TPA in an amphiphilic polymer based on DSPE-mPEG_5k to further enhance tumor targeting ability resulted in 3TPA NPs with significantly better photostability than FDA approved ICG and a PCE of up to 57.2%. After exposing experimental mice to 808 nm laser irradiation with a power density of 0.6 W cm⁻² for 10 min, the average intra-tumoral temperature of 3TPA NPs-treated mice rapidly reached 57 °C, while the temperature increase of PBS-treated mice could be ignored. Experimental results in vitro and in vivo demonstrated that 3TPA NPs had excellent biocompatibility and photostability, and the tumor was almost completely eradicated with no recurrence observed under the guidance of NIR-I/II fluorescence imaging. This study provides a robust platform for the development of effective PTAs, thereby advancing the field of tumor-specific diagnosis and treatment.

Author contributions

Z. J. Guo, Y. C. Chen and S. K. Yao conceived the project and designed the experiments. N. W. Shi and Q. Sun synthesized the probes; N. W. Shi and X. Z. Yang prepared nanoparticles; N. W. Shi and Y. P. Wu performed MTT experiments; N. W. Shi and Y. Ying performed flow cytometry; N. W. Shi and Y. H. Tan performed co-staining fluorescence imaging; N. W. Shi, R. X. Zhang and J. W. Zhang performed the in vivo experiments; N. W. Shi and S. K. Yao wrote the manuscript with the help of all authors. All authors discussed the results and commented on the paper. All authors have given approval to the final version of the manuscript.

Conflicts of interest

The authors declare no competing interests.

Data availability

The data supporting this article have been included as part of the ESI.†

Acknowledgements

This work was financially supported by the National Natural Science Foundation of China (22377050, 22407060, 22293050, 22293051, 22477054), the Natural Science Foundation of Jiangsu Province (BK20232020), Jiangsu Funding Program for Excellent Postdoctoral Talent (2024ZB642), and the Excellent Research Program of Nanjing University (ZYJH004).

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Footnote

† Electronic supplementary information (ESI) available: Experimental materials, as shown in Scheme S1 and Fig. S1–S20. See DOI: https://doi.org/10.1039/d5tb01221j

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