A Strategy to Design Non-Symmetric Compound by Modifying the End-Group Functional Atoms for Photothermal and Photodynamic Therapy of Tumor

Introduction

Compared with surgery, radiotherapy and chemotherapy, phototherapy is a promising treatment method for tumors due to the advantages of minimal invasiveness, excellent spatiotemporal controllability, minimal toxic side effect and low drug resistance.^1–5 Phototherapy involves photothermal therapy (PTT) and photodynamic therapy (PDT) (Table S1), which plays an antitumor role by producing local high temperature^6–8 and toxic reactive oxygen species (ROS) to kill tumor cells.^9–11 In recent years, to promote the accuracy and efficacy of therapy, fluorescence imaging (FLI) is commonly used in guiding tumor therapy by determining the distribution and metabolism of the therapeutic agents to achieve real-time monitor.^12–14 Therefore, as an important part of phototherapy, the design of photosensitizer (PSs) with outstanding performance is crucial to the tumor therapy. However, the design of single PSs which simultaneously possessed PTT, PDT and FLI still faces massive challenge.

In the past several years, tremendous development had been achieved in PSs which mainly involved inorganic PSs and organic PSs.^15–18 Inorganic PSs including gold-based nanomaterials,^19,20 carbon-based nanostructures,^21,22 and other nanomaterials^23,24 were used to tumor therapy due to the high performance in near-infrared (NIR) absorbance. However, the low biocompatibility of inorganic PSs greatly limited its application in tumor therapy. Luckily, organic PSs have much irreplaceable superior characteristics of the biodegradability, low mammalian toxicity, and flexible design.^25–28 Thus, enormous efforts have been made to develop highly efficient organic PSs. To date, some organic PSs have been approved by the US Food and Drug Administration (FDA) as clinical drugs such as indocyanine green (ICG),^29,30 while these organic PSs have some disadvantages such as poor photostability, single phototherapy mode^31,32 or poor light harvesting capability^33,34 and so on. Thus, to synthesize organic PSs with superior nature, non-symmetric organic compound has attracted great attention in the last decade (Table S2). Peng et al designed and synthesized non-symmetric organic compound called IR-Y6, which possessed high photothermal conversion effect (PCE) by rational manipulation of nonradiative decay channels.³⁵ Tang and Wang et al prepared the non-symmetric organic compound IDT-TPE with an order of magnitude higher ε to 8.9×10⁴ m⁻¹ cm⁻¹, which exhibited an extremely light harvesting capacity.³⁶ Meng et al modified the compound to obtain the new symmetric organic compound T780T-TPP-C₁₂ possessing excellent photostability.³⁷ Tang et al designed and synthesized the symmetrical organic compound Y₅-2BO-2BTF by introducing a high-plane A-D-A skeleton with a high molar absorption coefficient of 1.0⁶×10⁵ M⁻¹ cm⁻¹ and a high photothermal conversion efficiency (77.8%).³⁸ Despite these PSs achieved excellent results, the design of most of the PSs were based on end-group symmetric compound^39,40 and the study on the aspect of the organic compound with non-symmetric end groups for PSs is still not reported.

Herein, we designed and synthesized the end-group symmetric compounds (Y-4Cl and Y-2Br) and end-group non-symmetric compounds (Y-2Cl-Br) with fused - ring benzothiazole - pyrrole - thiophene - indenone constructure to explore its phototherapy properties, respectively. Experimental results demonstrated Y-2Cl-Br had higher molar extinction coefficient (ε) of 3.0×10⁵ M⁻¹cm⁻¹. After self-assembly of Y-2Cl-Br with DSPE-PEG₂₀₀₀, Y-2Cl-Br nanoparticles (NPs) exhibited the highest temperature increase (ΔT) of 34 °C and PCE of 61.2% compared with the Y-4Cl NPs and Y-2Br NPs. Meanwhile, the Y-2Cl-Br NPs showed the strong ROS generation ability which was 9.8 folds of ICG. Given the superior performance in non-symmetric organic compound, Y-2Cl-Br NPs was selected as phototherapy agent for tumors. As expected, Y-2Cl-Br NPs showed good biocompatibility and outstanding antitumor efficacy both in vitro and in vivo (Scheme 1 and Figure S1). Collectively, this study illustrated that the design strategy of non-symmetric organic compound is a possible ideal class of PSs for tumor therapy with excellent photothermal and photodynamic performance under FLI guidance.

Scheme 1 Non-symmetric modified organic compound PSs for PTT and PDT of tumor.

Results and Discussion

The synthesis routes of the organic compounds Y-4Cl, Y-2Cl-Br, and Y-2Br were described in the Supporting Information (Figure S2). Their chemical structures were confirmed by nuclear magnetic resonance (NMR) (Figure S3–S5) and the curves of Y-4Cl, Y-2Cl-Br, and Y-2Br on the same graph (Figure S6). Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-TOF-MS) (Figure S7−S9). To explore the difference in photophysical features between non-symmetric compound (Y-2Cl-Br) and symmetric compounds (Y-4Cl and Y-2Br), the light absorption ability was measured by the Ultraviolet-Visible Near-infrared (UV−VIS-NIR) spectrophotometer. The Y-4Cl, Y-2Cl-Br and Y-2Br exhibited the obvious absorption within 400 ~ 800 nm (Figure 1a) and the ε were determined as 1.7×10⁵ M⁻¹cm⁻¹, 3.0×10⁵ M⁻¹cm⁻¹ and 2.0×10⁵ M⁻¹cm⁻¹, respectively (Figure S10–S12), which indicates the non-symmetric compounds possess the excellent light absorption. Subsequently, Y-4Cl, Y-2Cl-Br and Y-2Br were fabricated into Y-4Cl NPs, Y-2Cl-Br NPs and Y-2Br NPs by one-step nanoprecipitation method,^41,42 which effectively improved the biocompatibility and biostability. The final concentration of Y-4Cl NPs, Y-2Cl-Br NPs and Y-2Br NPs was determined by the standard curves of the absorbance of Y-4Cl, Y-2Cl-Br and Y-2Br in CHCl₃, respectively. Then, the photothermal property of Y-4Cl NPs, Y-2Cl-Br NPs and Y-2Br NPs were monitored under the same laser irradiation. As shown in Figure 1b–d and Figure S13, all three NPs had a certain temperature rise after irradiation. However, Y-2Cl-Br NPs showed the highest temperature increase (ΔT= 34 °C) compared with Y-4Cl NPs (ΔT= 24 °C) and Y-2Br NPs (ΔT= 22 °C). Finally, based on the reported method,^43,44 the PCE of Y-4Cl NPs, Y-2Cl-Br NPs and Y-2Br NPs were determined as 58.6%, 61.2% and 51.9% (Figure 1e and Figure S14), respectively. According to the results of ε, ΔT and PCE, the conclusion can be deduced that non-symmetric compound (Y-2Cl-Br) have a more excellent light absorption, ΔT as well as photothermal conversion capacity compared with symmetrical organic compounds (Y-4Cl and Y-2Br).

Figure 1 (a) Molar extinction coefficient of Y-4Cl, Y-2Cl-Br and Y-2Br. Photothermal property of Y-4Cl NPs (b), Y-2Cl-Br NPs (c) and Y-2Br NPs (d) at different concentrations (0, 13, 26, 39 and 52 μg mL−1) under 808 nm laser irradiation (1.0 W cm−2). (e) Photothermal conversion efficiency of Y-4Cl NPs, Y-2Cl-Br NPs and Y-2Br NPs. (f) DLS data of Y-2Cl-Br NPs (inset graph: transmission electron microscopy image of Y-2Cl-Br NPs; scale bar:100 nm).

Due to the remarkable photophysical features, Y-2Cl-Br NPs have the great potential in tumor phototherapy. Therefore, we chose Y-2Cl-Br NPs as the tumor phototherapy agent. Firstly, the size and morphology of Y-2Cl-Br NPs were characterized by dynamic light scattering (DLS) and transmission electron microscopy. As shown in Figure 1f, the Y-2Cl-Br NPs had the uniform spherical morphology with a hydrodynamic diameter of around 128.1 nm. Considering the importance of biostability of NPs in tumor phototherapy, Y-2Cl-Br NPs were incubated in acid solution PBS which mimics the acidic properties in the tumor microenvironment, and assessed the changes in nanoparticle size. As displayed in Figure S15 and S16, the size of Y-2Cl-Br NPs almost was unchanged in acid solution PBS for 21 days. Besides, the size of Y-2Cl-Br NPs almost was unchanged in 1640 medium (10% FBS) for 14 days, suggesting excellent size stability (Figure S17). Furthermore, Y-2Cl-Br NPs retained the similar ΔT after five cool/heating cycles (Figure S18). These results indicate that Y-2Cl-Br NPs possess the excellent biostability for biological applications.

Based on the superior photothermal property of Y-2Cl-Br NPs + NIR, the photodynamic performance was further assayed. First, 1.3-Diphenylisobenzofuran (DPBF) was selected as a probe to examine the level of total ROS in cells (Figure S19). When the time extended to 90s after irradiation, the absorption intensity obviously decreased at 415 nm in DPBF + Y-2Cl-Br NPs + NIR (Figure 2a). However, the absorption intensity of DPBF + ICG + NIR had no significant change in the same way (Figure S20), suggesting Y-2Cl-Br NPs + NIR possessed the superior ROS production ability, which was 9.8 folds of ICG (Figure 2b). In addition, the ability of Y-4Cl NPs and Y-2Br NPs to generate ROS under 808 nm laser irradiation was also detected (Figure S21). However, among various photosensitizers of ICG, Y-2Cl-Br NPs, Y-2Br NPs and Y-4Cl NPs, Y-2Cl-Br NPs + NIR produced the most ROS in the same condition. To further verify the ROS generation capability of Y-2Cl-Br NPs + NIR, the SOSG, HPF and DHE were used to detect the ability to produce ¹O₂, ^● OH and O₂^{• −}, respectively. As shown in Figure S22–S24, upon the NIR irradiation, compared with SOSG, APF and DHE alone, the curve of Y-2Cl-Br NPs + SOSG, Y-2Cl-Br NPs + HPF, Y-2Cl-Br NPs + DHE have gone up significantly. These results further indicated Y-2Cl-Br NPs + NIR possessed excellent PDT performance.

Figure 2 (a) ROS generation of Y-2Cl-Br NPs mixed with DPBF under laser irradiation (808 nm, 1.0 W cm−2). (b) The changes in absorption of DPBF mixed with ICG or Y-2Cl-Br NPs solution at 415 nm. (c) Intracellular ROS in 4T1 cells treated with Y-2Cl-Br NPs + NIR under normoxia or hypoxia conditions. DCFH-DA, SOSG, HPF and DHE were used to detect total ROS, 1O2, ●OH and O2•−, respectively, scale bar: 100 μm.

Next, we further distinguished the ROS categories in cells by using different fluorescence probes. The total ROS, ¹O₂, ^● OH and O₂^{• −} were detected by DCFH-DA, SOSG, HPF and DHE under both normoxic and hypoxic environment, respectively. Among them, the hypoxia environment is created by a three-gas incubator (1% O₂, 5% CO₂) (Figure S25). As shown in Figure 2c, under the normoxic environment, the images showed bright fluorescence signals in cells cultivated with Y-2Cl-Br NPs and irradiated at 808 nm, indicating that Y-2Cl-Br NPs + NIR generated ROS, ¹O₂, ^● OH and O₂^{• −}. However, under the hypoxic condition, ^● OH and O₂^{• −} was still detected in cells treated with Y-2Cl-Br NPs + NIR suggesting Y-2Cl-Br NPs + NIR possessed type I PDT therapy ability. Besides, the 4T1 cell treated with Y-4Cl NPs + NIR and Y-2Br NPs + NIR also observed fluorescence signal, indicating Y-4Cl NPs and Y-2Br NPs possessed ability to generate ROS (Figure S26 and S27). On the contrary, no fluorescence signal was observed in cells treated with PBS, NIR and Y-2Cl-Br NPs, indicating intracellular ROS (¹O₂, ^● OH and O₂^{• −}) was not increased after these treatments (Figure S28). The above results demonstrate that Y-2Cl-Br NPs could be a superior photosensitizer for tumor PDT.

Due to the excellent photothermal and photodynamic performance of Y-2Cl-Br NPs under the 808 nm laser irradiation, we further assessed the antitumor effect in vitro. First, to detect the internalization of Y-2Cl-Br NPs in tumor cells, 4T1 cells was incubated with FITC-labeled Y-2Cl-Br NPs for different duration (0.5, 2, 4, 6 and 8 h) as the previous report.⁴⁵ As shown in Figure 3a, the fluorescence signal reached the strongest at 4 h, which demonstrated FITC-Y-2Cl-Br NPs could be efficiently internalized into tumor cells and provided an optimal time for phototherapy in vitro. Then, we used the Cell Counting Kit-8 (CCK-8) assays to evaluate the photodynamic and photothermal effects of Y-2Cl-Br NPs (0, 13, 26, 39 and 54 μg mL⁻¹) on 4T1 and B16F10 cells. Cell viability was dramatically decreased in both 4T1 and B16F10 cell lines after Y-2Cl-Br NPs + NIR. As shown in Figure 3b and c, cell viability was decreased to nearly 20.6% and 22.9% in 4T1 and B16F10 cells treated with 0 μg mL⁻¹ ~ 39 μg mL⁻¹of Y-2Cl-Br NPs, respectively. Moreover, the cell viability was further decreased approximately 95% at the 52 μg mL⁻¹ of Y-2Cl-Br NPs under the 808 nm (1.0 W cm⁻²) irradiation. Besides, the cell ability of 4T1 and B16F10 cells still decreases at the power with 0.5 W cm⁻² (Figure S29). And compared with ICG + NIR, Y-2Cl-Br NPs + NIR can kill more tumor cells (Figure S30). Meanwhile, under hypoxia, the cell viability of 4T1 cells also exhibited an apparent decrease after Y-2Cl-Br NPs + NIR (Figure S31). The above results show Y-2Cl-Br NPs + NIR possesses a strong inhibitory effect on proliferation of tumor cells. In contrast, cell viability nearly maintained at 100% after treatment of Y-2Cl-Br NPs without NIR irradiation. The similar results were observed in H9c2, NIH 3T3 and HEK 293T cells without NIR irradiation (Figure S32). In addition, the antitumor effect of Y-2Cl-Br NPs + NIR was evaluated by calcein-AM (green fluorescence) and propidium iodide (red fluorescence) double staining to distinguish live and dead cells as previously reported.⁴⁶ As shown in Figure 3d and e, Figure S33 and S34, only bright green fluorescent was seen in the PBS, NIR or Y-2Cl-Br NPs treated 4T1 and B16F10 cells. Red fluorescence signal in dead cells was observed in the group with Y-2Cl-Br NPs + NIR. The result of live/dead double staining was consistent with the CCK-8 results. Subsequently, the flow cytometry analysis was conducted. As shown in Figure 4, cell apoptosis (85.77%) was significantly increased in Y-2Cl-Br NPs + NIR group compared with other groups (PBS, NIR and Y-2Cl-Br NPs) in 4T1 cells. Taken together, the results of CCK-8 assay, live/dead double staining and flow cytometry analysis indicated Y-2Cl-Br NPs + NIR possessed remarkable ability to ablate tumor cells.

Figure 3 (a) The uptake efficiency of FITC-labeled Y-2Cl-Br NPs in 4T1 cells, scale bar: 20 μm. The viability of 4T1 (b) or B16F10 (c) cells incubated with Y-2Cl-Br NPs after different phototherapy modes. n = 4, “ns” means “no significance”. ****p < 0.0001, t-test. The fluorescence images of 4T1 (d) and B16F10 (e) cells treated with PBS, NIR, Y-2Cl-Br NPs, and Y-2Cl-Br NPs + NIR after calcein AM (green)/propidium iodide (red) double-staining. Scale bar: 100 μm.

Figure 4 The results of flow cytometry were obtained from 4T1 cells stained with Annexin V-FITC/PI after treatment with (a) PBS, (b) NIR, (c) Y-2Cl-Br NPs or (d) Y-2Cl-Br NPs + NIR, respectively.

It is well known that mitochondria are the energy factories of the cell and involve in many other functions of the cellular activities. Considering the significance of mitochondria, we examined mitochondria function and morphology after different treatments. Firstly, Mito-SOX was employed to detect mitochondrial ROS in 4T1 cells as the previous. As displayed in Figure 5a, bright red fluorescence was observed in the group treated with Y-2Cl-Br NPs upon the 808 nm laser irradiation, demonstrating the increase of mitochondria ROS in Y-2Cl-Br NPs + NIR treated cells. To investigate whether the ROS and heat had an effect on mitochondria, the mitochondrial function and morphology in 4T1 cells was further assessed. JC-1 staining assay was conducted to detect mitochondrial membrane potential after different treatments as the previous reports. Green fluorescence signals of JC-1 in the group treated with Y-2Cl-Br NPs + NIR (Figure 5b) suggested the decrease of mitochondrial membrane potential and mitochondrial dysfunction. In addition, mitochondrial morphology was also monitored by transfection of Mito-DsReds2 in cells.⁴⁷ As shown in Figure 5c, the mitochondrial morphology in 4T1 cells treated with Y-2Cl-Br NPs was dot-like structures or fragmented network after 808 nm laser irradiation, while mitochondria in cells with the other treatments (PBS, NIR and Y-2Cl-Br NPs) was thread-like or tubular structures. These results suggested that Y-2Cl-Br NPs + NIR can cause mitochondrial dysfunction and structure breakdown and induce the death of tumor cells.

Figure 5 (a) Mitochondrial ROS in 4T1 cells treated with PBS, NIR, Y-2Cl-Br NPs or Y-2Cl-Br NPs + NIR, scale bar: 100 μm. (b) JC-1 staining of 4T1 cells treated with PBS, NIR, Y-2Cl-Br NPs or Y-2Cl-Br NPs + NIR, scale bar: 100 μm. (c) Mitochondrial morphology in 4T1 cells after different treatments, scale bar: 10 μm.

Furthermore, the in vivo anticancer performances were evaluated in 4T1 tumor-bearing mice. Firstly, we evaluated the hemocompatibility of Y-2Cl-Br NPs in vitro. The hemolysis ratios were less than 5% in red blood cells (RBCs) incubated with the mixture of Y-2Cl-Br NPs at different concentrations (0, 0.15, 0.3, 0.45 and 0.6 mg mL⁻¹). Meanwhile, the supernatant of the RBCs suspension was clear (Figure S35). These results demonstrated the splendid hemocompatibility of Y-2Cl-Br NPs. Next, the FLI performance of Y-2Cl-Br NPs was also investigated both in vitro and in vivo. The in vitro fluorescence signal was shown in Figure S36, which showed the concentration-dependent relationship between fluorescence intensity and nanoparticle concentrations. To study the distribution and tumor accumulation of Y-2Cl-Br NPs in vivo, the Y-2Cl-Br NPs were intratumorally injected into 4T1-tumor-bearing BALB/c mice as the previous report.^48–50 Subsequently, the fluorescence images of mice were collected at 0, 3, 6, 9, 12 and 24 h postinjection (Figure S37a and b). The fluorescence signals from Y-2Cl-Br NPs at the tumor site reached the maximum value at 0 h postinjection, then followed by a gradual fluorescence decay because of the possible metabolism of Y-2Cl-Br NPs. To further assess the biodistribution of Y-2Cl-Br NPs in vivo, the major organs (heart, liver, spleen, lung and kidney) and tumors were isolated from the mice for FLI and quantitative analysis at 24 h postinjection. As illustrated in Figure S37c and d, the strong fluorescence signals in tumor tissue demonstrated the efficient accumulation of Y-2Cl-Br NPs. By contrast, no fluorescence signals were detected in the major organs. These results indicated that Y-2Cl-Br NPs can be served as a potential agent for tumor imaging guided phototherapy.

Based on the results above, we further evaluated the antitumor efficacy of Y-2Cl-Br NPs + NIR in the 4T1 tumor-bearing mice (Figure 6a). First, the 4T1 tumor-bearing mice were randomly divided into four groups: PBS, NIR, Y-2Cl-Br NPs and Y-2Cl-Br NPs + NIR. After different treatments, mice body weight, tumor relative volumes and variation in tumor site were recorded and monitored every three days. As shown in Figure 6b, no significant change in body weight was observed during the treatments, suggesting the low toxicity of Y-2Cl-Br NPs in vivo. From the observation of tumor site (Figure S38) and the measurement of tumor volume (Figure 6c), it was showed that the tumors in mice injected with PBS, NIR or Y-2Cl-Br NPs exhibited a rapid growth, while the tumor growth showed entirely ablation without recurrence in the mice treated with Y-2Cl-Br NPs + NIR. Furthermore, the tumors from all groups were collected (Figure 6d) and weighed (Figure 6e) at 14 days after the indicated treatments. The tumor completely disappeared in Y-2Cl-Br NPs + NIR group, which reached 100% tumor inhibition rate. Besides, hematoxylin and eosin (H&E) staining as well as Ki67 staining were used to assess antitumor effect. As displayed in Figure 6f, H&E staining revealed abundant nucleus absence and vacuolization in the Y-2Cl-Br NPs + NIR group, suggesting Y-2Cl-Br NPs + NIR can severely destruct tumor tissues. Meanwhile, Ki67 staining showed that the proliferation of tumor cells was significantly inhibited (Y-2Cl-Br NPs + NIR). These results illustrate that Y-2Cl-Br NPs + NIR can effectively suppress tumors growth in vivo, which suggests non-symmetric structure (Y-2Cl-Br) can be a promising photosensitizer for PTT/PDT of tumors.

Figure 6 (a) Schematic diagram of Y-2Cl-Br NPs + NIR for the tumor therapy. (b) The changes in body weights of 4T1 tumor-bearing mice during the treatment process. (c) Relative tumor volume (V/V0) in mice after different treatments. n=6, *p < 0.05, t-test. (d) The excised tumors and (e) weight of tumors from 4T1 tumor-bearing mice after different treatments in fifteenth day. (f) H&E and Ki67 staining analyses of tumor tissues. Scale bar: 100 μm.

To further assess the biosafety of photosensitizer, the biocompatibility of Y-2Cl-Br NPs was examined in vivo by blood routine test (Table S3), serum biochemical indexes (Table S4), and H&E staining of main organs from mice. Blood routine index including white blood cell (WBC) and red blood cell (RBC) counts had no significant difference between the Y-2Cl-Br NPs + NIR group and other treatment groups (Figure 7a and b). Moreover, the alanine aminotransferase (ALT), aspartate aminotransferase (AST), urea (UREA) and creatinine (CREA) (Figure 7c–f) also were within the normal range in all mice. Besides, H&E staining of major organs (heart, liver, spleen, lung and kidney) revealed the normal histological features without histological damage or inflammatory lesions in all groups (Figure 7g). The myocardial fibers of cardiomyocytes were pink, the nuclear structure was clear, and no obvious lesions were observed. The hepatic lobule structure was clear and complete, and the hepatic cord was empty. No obvious abnormality was found in the white pulp, red pulp and marginal area of spleen. The alveolar structure was intact, the alveolar cavity was empty, and no abnormal changes were observed in the lung interstitial. The normal glomerular structure was clear, the contour of the renal balloon was complete, and no obvious injury was observed. All these results indicated the excellent biocompatibility of Y-2Cl-Br NPs in tumor-bearing mice, highlighting its potential for clinical application of tumor PTT/PDT in the future.

Figure 7 Blood routine parameters (a) WBC and (b) RBC, hepatic function markers (c) ALT, (d) AST, (e) UREA and (f) CREA as well as (g) H&E staining of major organs (heart, liver, spleen, lung and kidney) collected from health mice (saline) and mice treated with Y-2Cl-Br NPs + NIR. Scale bar: 50μm. n = 4, “ns” means “no significance”.

Conclusions

In summary, we have designed and synthesized three organic compounds (Y-4Cl, Y-2Br and Y-2Cl-Br). The non-symmetric structure of Y-2Cl-Br possessed the most excellent performance (ε=3.0× 10⁵ M⁻¹cm⁻¹). In addition, Y-2Cl-Br NPs also showed ΔT=34 °C, PCE=61.2% and 9.7-fold higher ROS generation efficiency than ICG. More importantly, benefiting from the admirable PTT and PDT capability, Y-2Cl-Br NPs guided by FLI exhibited completely tumor ablation ability with good biosafety. These results indicate that the design strategy of non-symmetric structure PSs offers a new avenue for antitumor study.