Fibroblast activation protein α activatable theranostic pro-photosensitizer for accurate tumor imaging and highly-specific photodynamic therapy

Abstract

The development of activatable photosensitizers (aPSs) responding to tumor-specific biomarkers for precision photodynamic therapy (PDT) is urgently required. Due to the unique proteolytic activity and highly restricted distribution of tumor-specific enzymes, enzyme activatable photosensitizers display superior selectivity. Methods: Herein, a series of novel Fibroblast Activation Protein α (FAPα) activatable theranostic pro-photosensitizers were designed by conjugating the different N-terminal blocked FAPα-sensitive dipeptide substrates with a clinical PS, methylene blue (MB), through a self-immolative linker, which resulting in the annihilation of the photoactivity (fluorescence and phototoxicity). The best FAPα-responsive pro-photosensitizer was screened out through hydrolytic efficiency and blood stability. Subsequently, a series of in vitro and in vivo experiments were carried out to investigate the FAPα responsiveness and enhanced PDT efficacy. Results: The pro-photosensitizers could be effectively activated by tumor-specific FAPα in the tumor sites. After response to FAPα, the "uncaged" MB can recover its fluorescence and phototoxicity for tumor imaging and cytotoxic singlet oxygen (¹O₂) generation, eventually achieving accurate imaging-guided PDT. Simultaneously, the generated azaquinone methide (AQM) could serve as a glutathione (GSH) scavenger to rapidly and irreversibly weaken intracellular antioxidant capacity, realizing synergistic oxidative stress amplification and enhanced PDT effect. Conclusion: This novel FAPα activatable theranostic pro-photosensitizers allow for accurate tumor imaging and admirable PDT efficacy with minimal systemic side effects, offering great potential in clinical precision antitumor application.

Keywords: Fibroblast activation protein α; activatable photosensitizer; oxidative stress amplification; photodynamic therapy; tumor imaging.

Scheme 1

Illustration of FAPα activatable “one for all” theranostic pro-photosensitizer for in vivo accurate cancer diagnosis and high-specific (imaging-guided/enhanced) PDT.

Figure 1

(A) The design strategy and mechanism of FAPα activatable theranostic photosensitizer FAP-MB-1~10. (B) Evaluation of the hydrolytic efficiency of pro-photosensitizer FAP-MB-1~10 by rhFAPα via HPLC. (C) The photograph of pro-photosensitizer FAP-MB-1~10 after hydrolysis by rhFAPα. (D) The catalytic efficiency of rhFAPα towards FAP-MB-1, FAP-MB-5 and FAP-MB-8. (E) The fluorescence intensity of FAP-MB-1, FAP-MB-5 and FAP-MB-8 incubated with or without serum. Results are described as mean ± SD, n = 3. *p < 0.05, **p < 0.01, and ***p < 0.001.

Figure 2

(A) Optical characterization of fluorescence emission of FAP-MB-5 and MB in MeOH. (B) Fluorescence intensity of FAP-MB-5 (200 μM) pre-incubated with or without talabostat (100 μM) for 1 h towards various concentration of rhFAPα (0.225, 0.3, 0.375, 0.45 μg/mL) over time. Excitation: 630 nm. (C) UV-vis absorption spectra change of FAP-MB-5 (500 μM) towards various concentration of rhFAPα (0.225, 0.3, 0.375, 0.45 μg/mL). As inhibition group, rhFAPα (0.45 μg/mL) was pre-incubated with talabostat (100 μM) for 1 h, and then incubated with FAP-MB-5. (D) HPLC chromatogram of rhFAPα-mediated hydrolysis of FAP-MB-5 over time. As inhibition group, rhFAPα was pre-incubated with talabostat (100 μM) for 1 h. (E) Fluorescence response of FAP-MB-5 (200 μM) treated with the indicated protein (0.45 μg/mL unless otherwise specified), metal iron (50 μM) and other analytes (50 μM) for 1 h. 1, control; 2, Na⁺; 3, Mg²⁺; 4, Fe³⁺; 5, H₂O₂; 6, GSH; 7, ascorbic acid; 8, Gly; 9, Pro; 10, Cys; 11, BSA; 12, esterase; 13, collagenase; 14, Legumain; 15, APN; 16, DPPIV; 17, rhFAPα (0.225 μg/mL) pre-incubated with talabostat; 18, rhFAPα (0.225 μg/mL). (F) DPBF attenuation by ¹O₂ generation with different treatments in MeOH at 415 nm. (G) ESR spectra of different reaction systems with TEMP as the spin trap. (H) Docking analysis of the interactions of FAP-MB-5 with FAPα. (I) Detailed interactions between FAP-MB-5 and FAPα in a three-dimensional view. (+) and (-) refer to the treatment with or without irradiation, respectively. Results are described as mean ± SD, n = 3.

Figure 3

(A) CLSM images of Mia-paca-2 cells incubated with FAP-MB-5 (40 μg/mL) for 1 h, 2 h and 4 h. As inhibition group, the cells were pre-incubated with talabostat (50 μM) for 1 h, and then incubated FAP-MB-5 for 4 h. The blue fluorescence represents the cell nuclei stained with DAPI (excitation: 405 nm) and the red fluorescence represents MB liberated from FAP-MB-5 (excitation: 633 nm). Scale bar: 30 μm. Intracellular ROS generation of Mia-paca-2 cell treated with FAP-MB-5 (40 μg/mL) or N-FAP-MB (40 μg/mL) under dark or irradiation condition observed by CLSM (B) and flow cytometry analysis (C). The inhibition group was pre-incubated with talabostat for 1 h and treated with FAP-MB-5 with irradiation. Scale bar: 30 μm. (+) and (-) refer to the treatment with or without irradiation, respectively.

Figure 4

(A) Intracellular GSH levels of Mia-paca-2 cells treated with various concentrations of FAP-MB-5 (10, 20, 40 μg/mL) and N-FAP-MB (40 μg/mL). The inhibition group was pre-incubated with talabostat for 1 h and then incubated with FAP-MB-5 (40 μg/mL). (B) The cytotoxicity of Mia-paca-2 cells under dark or irradiation condition (633 nm, 100 mW/cm², 5 min) after treatment with FAP-MB-5, N-FAP-MB or FAP-MB-5 pre-incubated with talabostat, respectively. (C) Live/dead cell staining assay of Mia-paca-2 cells after incubating with FAP-MB-5 and N-FAP-MB (40 μg/mL) under irradiation (633 nm, 100 mW/cm², 5 min). Live cells and dead cells were stained with FDA (green) and PI (red), respectively. Scale bar: 200 μm. (+) and (-) refer to the treatment with or without irradiation, respectively. Results are described as mean ± SD, n = 3. ***p < 0.001.

Figure 5

(A) In vivo fluorescence images of 4T1 tumor-bearing mice after intravenous injection of FAP-MB-5 (7mg/kg). (B) Ex vivo fluorescence images of tumor and major organs at 12 h post-injection of FAP-MB-5 (7 mg/kg). 1, heart; 2, liver; 3, spleen; 4, lung; 5, kidney; 6, tumor. (C) The mean fluorescence intensity (MFI) of major organs and tumor at 12 h poet injection of FAP-MB-5. (D) Real-time in vivo fluorescence imaging of FAP-MB-5 (7 mg/kg) after injection into the right tumor (right red circle) and the left flank of hypoderm (left red circle) in the tumor-bearing mice, respectively.

Figure 6

Relative tumor growth curve (A) and average body weight (B) of 4T1 tumor-bearing mice after various treatments indicated at an equivalent dosage of MB in 18 days. Corresponding average tumor weights (C) and representative tumor photographs (D) of 4T1 tumor-bearing mice after treatments on 18^th day. Intratumoral GSH level (E) and ROS generation (F) isolated from 4T1 tumor- bearing mice with different treatments. (G) The H&E and TUNEL staining of tumor sections after treatments on 18^th day. Scale bar: H&E staining 400 μm; TUNEL staining 100 μm. (+) and (-) refer to the treatment with or without NIR irradiation, respectively. Results are described as mean ± SD, n = 6. *p < 0.05, **p < 0.01, and ***p < 0.001.

Figure 7

Serum biochemistry analysis ALB (A), ALT (B), AST (C), BUN (D), CREA (E) of different groups after the 18-day treatment. (F) The H&E staining of major organs slices in various groups on 18^th day. Scale bar: 200 μm. (+) and (-) refer to the treatment with or without NIR irradiation, respectively. Results are described as mean ± SD, n = 6.