Photothermal therapy (PTT) is a cutting-edge cancer treatment that uses high-efficiency photothermal conversion materials to convert light energy into heat energy, effectively killing cancer cells with near-infrared light [1-8]. PTT is unique because it can kill cancer cells in hypoxic environments without relying on oxygen [9-13]. When the photothermal agent absorbs energy, it generates heat that kills cancer cells and produces photoacoustic (PA) signals, enhancing its diagnostic capabilities [14-21].

Recently, continuous work has been focused on developing photothermal conversion agents to discover the more effective phototherapy treatment [22,23]. Generally, the ideal photothermal agents must have a high absorption coefficient in the near-infrared region, and a high excellent photothermal conversion efficiency, as well as excellent biocompatibility [24-28]. The excellent properties of fluorescent dyes have promoted the progress of bioimaging technology [29,30]. Among them, rhodamine dyes have received great attentions due to their advantages, such as large molar absorbance coefficient, high fluorescence quantum yield, excellent lightfastness, and adjustable excitation and emission wavelengths [31,32]. Moreover, it has been demonstrated that modifying the rhodamine core with heteroatoms can redshift their absorbance and emissions [33,34]. For example, substitution with sulfone can achieve an impressive absorption wavelength around 700 nm, making it an excellent candidate for biological imaging and phototherapy [35,36]. However, such an electron-withdrawing sulfone group can cause the C-9 position of the rhodamine core to become more susceptible to nucleophiles, such as water and thiols, resulting in instability and rendering them unsuitable for further biological applications. Therefore, developing high-stability sulfone rhodamine dyes is in high demand.

In this study, we synthesized five sulfone rhodamines and investigated the substitution effects on their stability. As shown in Fig. 1, the ortho-substituent is crucial for protecting the C-9 site from nucleophilic attack. Among them, SO₂R2, which contains a CF₃ group, showed high stability and strong absorbance at 714 nm. In vitro studies revealed that this compound exhibited excellent photothermal conversion efficiency of 53.06% and good biocompatibility, making it a promising therapeutic diagnostic agent. Furthermore, in vivo studies confirmed that SO₂R2 produced a robust photoacoustic signal (PA) and achieved local heating upon laser irradiation, leading to inhibition of tumor growth without significant side effects.

As depicted in Fig. S2A (Supporting information), all compounds exhibited absorption peaks around 700 nm in water solution. The emission spectrum of SO₂R1–SO₂R5 were measured in CH₂Cl₂, CH₃CN and H₂O. The emission peaks of SO₂R1–SO₂R5 were centered around 730 nm. We determined their fluorescence quantum yields (Ф_f) by taking Rhodamine B as the control [37]. As shown in Fig. S3 (Supporting information), all dyes showed high Ф_f in CH₂Cl₂, but low Ф_f in H₂O. For example, the Ф_f of SO₂R2 was determined to be 0.703 in CH₂Cl₂ and 0.09 in H₂O. Their stability in various solutions and photo-stabilities were investigated (Figs. S4–S18 in Supporting information). The para-substituted compounds, SO₂R3 and SO₂R4, showed a dramatic decrease in their absorbance along with a color change from green to colorless and poor photostability. This indicated that nucleophilic attack easily occurred in the presence of most common solvents, such as water, phosphate buffered saline (PBS), methanol, ethanol, isopropanol, N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), and tetrahydrofuran (THF). On the other hand, ortho-substituted compounds displayed considerable stability. SO₂R1 showed moderate stability, while SO₂R2 and SO₂R5 showed high stability and photostability in various solutions. Our findings reveal that the stability of the compound is related to the steric hindrance caused by the substituents. The larger the steric hindrance and more hydrophobic nature of the ortho-substitute, the higher the stability of the dye. We then carried out stability experiments under different pH conditions. As shown in Figs. S2D–F (Supporting information) and Figs. S19–S26 (Supporting information), SO₂R2 and SO₂R5 showed high pH tolerance in a broad pH range from 2 to 10 for a long period of time (24 h). This indicates that these compounds have potential for long-term tracking applications without pH influence.

As previously mentioned, both SO₂R2 and SO₂R5 showed ideal stability and pH tolerance. Considering the higher absorbance of SO₂R2 compared to SO₂R5, we selected SO₂R2 for further photothermal transition experiments. When the PBS solution (pH 7.4) of SO₂R2 was exposed to the light emitting diode (LED) lamp, the temperature increased drastically from 22 ℃ to 71 ℃, as shown in Figs. 2A and B. We investigated the effects of LED lamp power and dye concentration on the photothermal conversion efficiency, as depicted in Figs. 2C and D. After five irradiation/cooling cycles, the compound remained stable, as demonstrated in Figs. 2E and F. Furthermore, we calculated the photothermal conversion efficiency of SO₂R1 and SO₂R2 to be 38.04% and 53.06% respectively. We tested the dynamic light scattering (DLS) of SO₂R2 in water, the data indicated the formation of nanoparticle with a dimeter of 100 nm by self-assembly of SO₂R2, as described in Fig. S27 (Supporting information). The nano self-assembly may benefit to higher PTT performance [38].

We evaluated the cytotoxicity of SO₂R2 to HeLa cells using the cell counting kit-8 (CCK-8) method (Fig. 3B). Results showed that even under dark conditions, the survival rate was over 90%, indicating good cell compatibility and no significant threat to cell viability. To assess the photothermal therapy (PTT) capability of SO₂R2, we performed fluorescence microimaging by staining live and dead cells with calcein-AM (AM) and propidium iodide (PI), respectively. Interestingly, the control group for SO₂R2 showed strong green fluorescence, indicating no apoptosis (Fig. 3A). However, under LED irradiation, almost all HeLa cells in the SO₂R2 + Laser group emitted a bright red fluorescence signal, indicating that SO₂R2 displayed efficient PTT effect. Furthermore, additional experiments were performed to determine the dose dependence of HeLa cell survival after 10 min of irradiation with a 730 nm laser (1.0 W/cm²). The half maximal inhibitory concentration (IC₅₀) of SO₂R2 was calculated to be 65.82 µmol/L, further highlighting the potency of this compound for PTT.

The strong PA and good biocompatibility of SO₂R2 make it a promising theragnostic agent. In vitro photoacoustic imaging experiments demonstrated a positive correlation between the compound concentration and PA intensity, indicating its potential for high-resolution and high-contrast PA-guided imaging (Fig. 3E). To evaluate the targeting ability of SO₂R2 in vivo, we established a 4T1 tumor-bearing BALB/c mouse model and injected the compound into mice. Photoacoustic imaging revealed a gradual increase in PA intensity at the tumor site, reaching a maximum value after 6 h post-injection, followed by a gradual decrease over time (Figs. 3C and D). The PA values of normal tissue and tumor tissue at different times are shown in the Fig. S28 (Supporting information). These findings demonstrate the potential of SO₂R2 for non-invasive monitoring of pathological processes in deep tissues.

Subsequently, we conducted anti-tumor studies of SO₂R2 in nude mice bearing tumors (Fig. 4B). The animal experiments were approved by the Animal Ethical Committee of Medical College of Qingdao University (QDU-AEC-2022040). In vivo experiments demonstrated that continuous LED radiation for 10 min caused a rapid increase in temperature near the solid tumor from 34 ℃ to 54 ℃, while the temperature near the mouse tumor without injecting the photothermal reagent remained relatively unchanged (Figs. 4A and C). Randomly divide all tumor-bearing mice into 4 groups of 3 mice each. The mice were then divided into four groups and subjected to 15 days of photothermal treatment. The results showed that the tumor volume of the SO₂R2 + laser group was significantly reduced, while the tumor volume of control groups increased significantly. These findings clearly demonstrate the excellent in vivo photothermal effect of SO₂R2, which effectively kills tumor cells and inhibits tumor growth (Figs. 4D and E, Fig. S29 in Supporting information). These results indicate that SO₂R2 has great potential for use in cancer therapy. The biosafety evaluation involved monitoring various blood biochemical parameters of mice after 20 days of SO₂R2 injection [39]. The results showed that all markers in the SO₂R2 + laser group were similar to those in the control group. Notably, red blood cells (RBC), white blood cells (WBC), and platelets (PLT) were all within the normal range, indicating that SO₂R2 had negligible long-term in vivo toxicity (Fig. 4F). These findings establish SO₂R2 as a highly promising imaging-assisted PTT agent with minimal systemic toxicity.

In this study, we synthesized five sulfone rhodamines and investigated their substitution effects on stability. The data showed that the ortho-substituent is crucial for protecting the 9-site of the rhodamine core, preventing nucleophilic attack. SO₂R2 demonstrated high stability and strong absorbance at 714 nm. It also showed excellent photothermal conversion efficiency of 53.06% and generated strong PA. The excellent safety profile of SO₂R2, coupled with its impressive photothermal effect and in vivo targeting ability, make it an ideal candidate for imaging-assisted PTT in the near-infrared window.

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Prof. Xin Zhou acknowledges the National Natural Science Foundation of China (Nos. 21762045, 21911540466), Shandong Provincial Natural Science Foundation (No. ZR2019YQ12), China Postdoctoral Science Foundation (No. 219M652306), and Taishan Scholar Project (No. tsqn201812049) for supporting this work.

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.cclet.2023.108595.