3.1 Synthesis and Characterization of SPN-GIP and 177Lu-SPN-GIP
Scheme 1a presents the synthetic procedures of SPN-GIP and 177Lu-SPN-GIP. SPN-GIP was prepared using a nanoprecipitation method. A semiconducting polymer poly[2,1,3-benzothiadiazole-4,7-diy1[4,4-bis(2-ethylhexyl)-4H-cyclopenta[2,1-b:3,4-b’] dithiophene-2,6-diyl]] (PCPDTBT) was encapsulated with functionalized amphiphilic polymer (GIP-PEG12-DSPE and DOTA-PEG45-DSPE) to obtain water-soluble SPN-GIP. Further radiolabeling of SPN-GIP was performed through chelating 177Lu with DOTA on its surface to get the final radioactive nanoagent 177Lu-SPN-GIP. Transmission electron microscopy (TEM) revealed the spherical morphology of SPN-GIP with an average size of 207.70 nm (Fig. 1a). The hydrodynamic diameter of SPN-GIP in PBS was measured to be 187.8 ± 2.26 nm (Fig. 1a). SPN-GIP had an absorption in the NIR region with a maximum peak at 680 nm, which was originated from PCPDTBT (Fig. 1b). SPN-GIP had no obvious change in absorption intensity after 808 nm laser irradiation, suggesting its excellent photostability for further PTT study (Fig. 1b). The radiolabeling efficiency of 177Lu-SPN-GIP was 86.36 ± 6.12% with a high radiochemical purity of 97.48 ± 1.90%. After a 48-h incubation, the radiochemical purity of 177Lu-SPN-GIP remained to be 89.78 ± 0.75% in saline and 84.22 ± 2.60% in FBS (Fig. 1c), indicating its suitable stability for in vivo study. The photothermal performance of SPN-GIP at various concentrations (0, 12.5, 25, and 50 μg mL-1) was investigated with an 808 nm laser at 0.5 W cm−2, which was recorded by an infrared thermal mapping apparatus (Fig. S1, Supporting Information). Under continuous laser irradiation at 808 nm, the temperature of SPN-GIP solutions gradually increased with increasing its concentrations. The maximum temperature increment of SPN-GIP at 12.5, 25, and 50 μg mL-1 after laser irradiation for 270 s reached about 8.5, 16.8, and 27.1 °C, respectively (Fig. 1d). In contrast, the temperature increment of PBS solution was only 0.3 °C with the same laser irradiation density and time, indicating the capability of SPN-GIP for PTT. Remarkably, the photothermal conversion efficiency (η) reached approximately 42.84%, [25,36], which was comparable to the previously reported photothermal agents (Table S1, Supporting information). After three cycles of heating-cooling manipulation, no reduction of maximum temperature at each cycle was observed, indicating the perfect photothermal stability of SPN-GIP (Fig. 1e, f).
3.2 In Vitro RT and PTT Capability of SPN-GIP and 177Lu-SPN-GIP
Before investigating the RT and PTT performance, cell biocompatibility of SPN-GIP towards CFPAC-1 cells was firstly tested using cell counting kit-8 (CCK8) assay. As shown in Fig. 2a, SPN-GIP exhibited no appreciable toxicity to cells even at a high concentration of 50 µg mL− 1, showing its great biocompatibility. However, after labeling with 177Lu, 177Lu-SPN-GIP showed dose-dependent decrease in cell viability (Fig. 2b). In contrast, free 177LuCl3 showed a significant decrease in cancer cell killing ability, which probably because the nanoparticle is a better carrier in favor of the cell uptake of 177Lu and thereafter facilitates RT within cells (Fig. 2b, Fig. S2a, b in the Supporting Information). Besides, if the incubation time was extended from 24 to 96 h after replacing the medium containing 177Lu-SPN-GIP with fresh medium, the cell viability could be apparently decreased, indicating RT necessitates enough time to exert the killing effect. Then, a combination of RT and PTT was further investigated. After co-incubation with SPN-GIP (50 µg mL− 1) and 177Lu-SPN-GIP at different radioactive dosage ranging from 0 to 11.1 MBq/mL for 24 h, CFPAC-1 cells were irradiated by an 808 nm laser (1 W cm− 2, 5 min) and then incubated for another 24 h. After convergence with PTT, cell viability could be inhibited more effectively relative to RT alone. Specifically, after treatment with PTT along with RT at the dosage of 11.1 MBq mL− 1 (1.11MBq, 0.1 mL), cell viability could be decreased to 4.33 ± 3.03%, which was 7.74-fold and 18.58-fold lower than those of PTT and RT alone, respectively (Fig. 2b). Such combination of RT and PTT was demonstrated to possess outstanding effect on destructing cancer cells, which was further validated by fluorescence imaging using calcine AM and propidium iodide (PI) to stain cells after different therapeutic treatments (Fig. 2c). Therefore, the combined RT and PTT offers remarkably enhanced killing effect on cancer cells relative to RT or PTT alone.
3.3 In Vivo Therapeutic Effect of Combined RT and PTT
As β-emitting radiopharmaceuticals for molecular RT, 177Lu is routinely used to treat patients suffering from tumors with somatostatin receptor overexpression in Europe, which invariably consists of 2 or 4 intravenous administrations of 177Lu-DOTATATE at a high dosage (7.4 GBq) [37, 38]. Such strategy can significantly improve progression-free survival, but it needs several injections due to the fast efflux-induced short retention of radiopharmaceuticals within tumor. In this study, after intratumoral injection of 177Lu-SPN-GIP, a strong radioactive signal was observed in tumor and the signal kept unchanged even at t = 4-day post-injection, indicating 177Lu-SPN-GIP had an excellent accumulation and retention effect in tumor region. By contrast, 177LuCl3-treated group showed a diffused radioactive signal in the whole body within 1-day post-injection and nearly all 177LuCl3 was excreted from the body at t = 2-day post-injection (Fig. 3a), further validating the SPNs as a perfect nanocarrier to favor the accumulation and retention of radiopharmaceuticals for therapy.
As it well known, heat causes irreversible damage to cancer cell membranes and initiates protein denaturation [16, 39]. In order to avoid unnecessary thermal effect to proximal normal tissues, the temperature of PTT was held around 45°C [40, 41]. The temperature changes of the tumor area were recorded by IR thermal mapping apparatus. According to the IR thermographic images (Fig. S3, Supporting Information), the temperature at tumor area rapidly reached 45°C after treatment with SPN-GIP under the laser irradiation for 30 s. The temperature of the tumor without treatment with SPNs presented subtle changes under the same irradiation conditions for 5 min, thereby demonstrating that the high in vivo photothermal effect of SPN-GIP.
Therapeutic efficacy of combined RT and PTT was then evaluated by monitoring the tumor growth after various treatments including saline, SPN-GIP, 177LuCl3, SPN-GIP with PTT, 177Lu-SPN-GIP, and 177Lu-SPN-GIP with PTT (Fig. 3b-d). As shown in Fig. 3b, tumor volume in the saline-treated group grew over time. Though the tumor size of 177LuCl3-treated group at t = 7-day post-injection was slightly suppressed relative to those at t = 4-day post-injection probably due to the inherent killing effect of β-rays emitted by 177Lu, but there was no significant difference in comparison to the saline group post-treatment at day 21 (P = 0.2077). Compared with 177LuCl3-treated group, 177Lu-SPN-GIP showed obviously improved therapeutic capability in suppressing the tumor growth, attributing to increased tumor accumulation and retention effect of 177Lu-SPN-GIP relative to free 177LuCl3. Owing to ideal photothermal effect of SPN-GIP, SPN-GIP with PTT-treated group apparently inhibited the tumor growth after PTT while no therapeutic effect was observed for the group without laser irradiation (SPN-GIP group). Unfortunately, due to the low efficiency and incomplete killing of cancer cells, tumor recurrence was observed in both RT and PTT groups. By contrast, combined RT and PTT-treated group successfully inhibited the tumor growth within 21-day observation window. On the 21th day after treatment, mice of different groups were sacrificed and the residual tumor tissues were collected (Fig. 3c). Combine RT and PTT group presented the complete elimination of tumors while residual tumors were still observed in other groups receiving RT and PTT alone (Fig. 3c).
Tumors from various treatment groups were also collected for H&E staining (Fig. 3d). A mass of tumor cells was found almost in all the field of views in the control groups (including saline, SPN-GIP only, and 177LuCl3 group). As a comparison, the number of tumor cells was greatly reduced but a tiny of tumor cells was still observed in the RT and PTT alone-treated groups, indicating the incomplete killing effect of these groups. By contrast, no tumor cells were residual in combined PTT and RT group, which was in agreement with in vivo therapeutic result shown in Fig. 3b.
Besides, for mice in all groups, no significant weight loss was observed during 21-day observation period (Fig. S4, Supporting Information), and no noticeable histopathological abnormalities were found in heart, liver, spleen, lung, kidney, pancreas and intestine (Fig. S5, Supporting Information). In addition, serum biochemistry assay and blood glucose (GLU) test were also carried out for mice in all groups at t = 21-day post-treatment. There was no significant difference in GLU level between all treatment groups, suggesting there was no islet injury in all groups (Fig. S5a, Supporting Information). The liver (aspartate transaminase, AST and alanine transaminase, ALT) and kidney function markers (creatinine, CRE and blood urea nitrogen, URE) were measured and no significant difference was observed between treated groups and saline-treated group (Fig. S5b&c, Supporting Information). Therefore, these results verified the high therapeutic efficacy of synergistic PTT and RT with negligible risks towards the living organisms.
3.4 Mechanistic Study on Therapeutic Effect
Epithelial-mesenchymal transition (EMT) is an important process to transform epithelial cells into mesenchymal cells, which is involved in tumorigenesis, invasion and metastasis [42, 43]. In the process of EMT, the epithelial marker E-cadherin is downregulated, while the mesenchymal markers N-cadherin, fibronectin, and vimentin are upregulated significantly [44]. By comparing with the expression of E-cadherin, N-cadherin, vimentin, and fibronectin in pancreatic tumor tissue with that in normal pancreas, EMT process was verified to occur in PDAC (Fig. S6, Supporting Information). Therefore, we utilized the method to evaluate the EMT process after various therapeutic treatments. Remarkably, the expression of the epithelial marker E-cadherin was upregulated meanwhile mesenchymal markers such as N-cadherin, vimentin, and fibronectin were downregulated in RT and combined RT/PTT-treated groups (Fig. 4). Such phenomenon verified that the EMT process was effectively reversed and thus resulted in reduced risk of tumor metastasis and invasion in both treatment groups [44]. Despite endowing the fast ablation of tumor, PTT-treated group showed similar expression of mesenchymal markers to saline-treated group, implying its inability to reverse EMT process. The imbalanced of upregulated epithelial E-cadherin in PTT-treated group was probably due to high temperature-induced the lysosome inactivation of the inhibition pathway or degradative pathway of E-cadherin [45]. Thus, the combination of PTT and RT not only facilitates efficient reversion of EMT process derived from RT but also enables fast tumor ablation contributed from PTT.
Histological changes after different treatment were further analyzed (Fig. 5). As shown in Fig. 5, after treatment with PTT, RT, or the combination of PTT and RT, CD31 was changed from strong positive to weakly positive as compared with saline-treated group, indicating the successful anti-angiogenic effect of all therapeutic groups [46]. As a characteristic marker of tumor proliferation and aggressiveness, Ki67 was apparently decreased in the RT and RT/PTT-combined treatment group, indicating that the proliferation ability of tumor cells was greatly reduced [47]. However, there was no observable changes in the expression of Ki67 between PTT-treated group and saline-treated group although the tumors were rapidly ablated by elevated temperature. This was consistent with slow increase of tumor volume post-PPT treatment alone during observation window in Fig. 3. Such proliferating tendency post PTT treatment was possibly induced by its low efficiency to suppress the growth of the tumor margin and thus cause tumor proliferation and aggressiveness [14]. Thus, H&E staining was utilized to further investigate the tumor proliferation at its margin. As an expectation, an extensive generation of new capillaries was presented in the PTT-treated group relative to other groups. Such resulted abundance of capillaries could become a microenvironment conducive to tumor growth and spread. Besides, CD44 related to cancer stem cells (CSC) was also analyzed [44]. CSC is an important factor to predict the difficulty of tumor curability. Some studies showed that CSCs are closely related to EMT, resulted in expression of stem cell markers [43, 48, 49]. Compared with saline-treated group, the expression of CD44 was still observable in PTT-treated group, suggesting the existence of CSC and thereby implying a potential for recurrence. By contrast, the expression of CD44 decreased significantly in RT-treated and RT/PTT-combined-treated groups, validating the effective killing of CSC via 177Lu-induced damage of DNA double strands, which was also confirmed by positive immunofluorescence of γ-H2AX in cells and tissues of both groups (Figure S7, Supporting Information) [38]. In addition, CD90 expression was found increasing remarkably in the RT and RT/PTT-combined-treated groups, indicating the effective tumor-suppression while PTT-treated group showed its inferiority in the expression level [50]. All the data proved that combined PTT with RT not only achieved rapid tumor ablation derived from PTT, but also reversed EMT process and reduced the risk of invasiveness attributing to RT, providing a new attempt for effective tumor treatment.