1. Introduction
Photosensitizer (PS)-based photodynamic therapy (PDT) has been widely adopted as a safe therapeutic modality for tumors [
1]. Commonly, PSs are organic chromophores that generate reactive oxygen species (ROS) upon light irradiation [
2], [
3]. However, the limited penetration depth of light has restricted their widespread clinical use for the treatment of diverse cancers. In contrast to light, X-rays have almost unlimited tissue penetration and have been used clinically for diagnostic imaging and radiotherapy for more than 100 years [
4], [
5]. Inspired by the radio-induced luminescence of scintillators, X-rays have been exploited to replace light to generate photons for bioimaging and therapy of deep-seated tumors [
6], [
7]. As not all organic PSs are activated by X-rays to generate a sufficient amount of ROS to induce cell death, inorganic transducers are commonly employed to transform X-ray energy into singlet emissions that then excite the PSs, as part of the X-ray-induced PDT (X-PDT) process [
6], [
7]. However, inorganic transducers with high light yields are difficult to produce because of the challenges in precisely controlling their structures and surface modifications [
8]. Most importantly, the complicated multi-step transfer mechanisms inevitably lose energy and reduce the efficiency of X-PDT.
The study of organic scintillators has a long history. Anthracene and stilbene are both pure organic crystals that have been widely exploited in radiation detection applications. Organic scintillators are mainly composed of elements of low atomic numbers, such as H, C, N, and O. These conventional organic scintillators only generate fluorescence from single excitons by ionized excitation [
9], [
10], [
11]. A dominant scintillation normally takes place in heavily ionized regions, and the excitations are ultimately populated by ions and secondary electrons [
8]. In principle, singlets and triplets are generated at a ratio of 1:3 by following the rule of spin conservation [
12], [
13], [
14]. This means that approximately three-fourths of the triplets in most organic scintillators are not well utilized. With recent progress in pure organic room-temperature phosphorescence (RTP), strategies that achieve effective intersystem crossing (ISC) for triplet exciton generation or that possibly surpass non-radiation processes are expected to facilitate RTP [
15], [
16]. Carbazole-based organic molecule crystals have achieved efficient X-ray-excited luminescence by introducing heavy atoms to enhance spin-orbit coupling and rigid hydrophobic structures to suppress non-radiative energy dissipation [
17], [
18], [
19]. An acceptor-phenoxy-adamantylidene scaffold was designed that exhibited light and ultrasound-induced luminescence for tumor-specific theranostics and also produced radiation-induced luminescence and a therapeutic effect on deep-seated tumors [
20], [
21], [
22]. In the latter case, chemiluminescence was induced by singlet oxygen (radiation-generated singlet oxygen and an induced cycloaddition process, thereby releasing ring tension and luminescence) [
22]. The molecules discussed in these manuscripts have very complex structures. Consequently, rare organic molecules with simple structures have been reported to promote triplet excitons and generate singlet oxygen for X-PDT processes [
23], [
24]. Therefore, the need to develop innovative ionized energy-induced RTP organic molecules is urgent. To differentiate from the term radiosensitizer (also called radio-enhancer), we named this type of molecule an X-ray-sensitizer (XS), which is a molecule that is activated directly by X-rays for the efficient generation of triplet excitons.
Herein, we screened pharmaceutical drug intermediates that are derivatives of thioxanthone (TX) and investigated TX-derived organic molecules that efficiently underwent X-sensitization to populate singlet oxygen for cancer therapeutics when exposed to low-dose X-ray irradiation. By modifying alkoxy side chain substitutions at the 2-position of TX to tune the molecular packing and intermolecular interactions, the fluorescence and RTP of a series of TX derivatives were assessed under X-ray irradiation. Furthermore, the ability to generate singlet oxygen and the potential for therapy of tumors were evaluated. These results provide new opportunities for developing organic molecules in which a large number of triplets can be populated directly under low-dose X-ray.
2. Materials and methods
2.1. Materials, cell lines, and animals
1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)] (DSPE-PEG-NH2; ammonium salt) was purchased from Shanghai Ponsure Biotech, Inc (China). N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC; 99%) and N-hydroxysuccinimide (NHS; 98%) were purchased from J&K Scientific, Ltd. (China). Verteporfin, tetrahydrofuran (THF), dimethyl formamide (DMF), singlet oxygen sensor green (SOSG), and 4-nitrophenyl chloroformate 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich (USA). Fetal bovine serum (FBS) was purchased from HyClone Inc. (USA). Dichlorodihydrofluorescein diacetate (DCFH-DA) was purchased from Shanghai Maokang Biotechnology Co., Ltd. (China). Histone family member X (H2AX) phosphorylated on Ser-139 (γH2AX) antibody was purchased from Absin Bioscience Inc. (China). BODIPY™ 581/591 C11 was purchased from Thermo Fisher Scientific (USA). A JC-1 mitochondrial membrane potential assay kit was purchased from MedChemExpress Co., Ltd. (USA). Fluorescein isothiocyanate (FITC)-conjugated Annexin V (Annexin V-FITC) apoptosis detection kit was purchased from Beyotime Biotech Inc. (China). The 4T1 mouse breast cancer cell line was obtained from the Institute of Biochemistry and Cell Biology (China). Female BALB/c mice (5-6 weeks old) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (China) and Shanghai SLAC Laboratory Animal Co., Ltd. (China).
2.2. Preparation of XS molecules
TX and 2-positioned alkoxy-tailored TX derivatives were synthesized. TX, 2-hydroxythioxanthone, and 2-alkxxythioxanthone (R = -CH3 (M-TX), -CH2CH3 (E-TX), -CH2CH2CH3 (np-TX), and -CH(CH3)2 (ip-TX)) were synthesized and purified for further investigation.
2 -Chlorothioxanthone (4.9 g, 20.0 mmol), KOH (2.5 g, 44 mmol), Pd2(dba)3 (365 mg, 0.4 mmol), and t-Bu XPhos (680 mg, 1.6 mmol) were placed in an oven-dried Schlenk flask under an argon atmosphere. Dioxane (18 mL) and H2O (10 mL) were added to the mixture. The resulting solution was stirred at 100 °C for 16 h. The reaction mixture was cooled to room temperature and acidified with dilute aqueous HCl. The resulting yellow precipitate was filtered and washed with water and Et2O to create 2-hydroxythioxanthone (3.9 g, 16.95 mmol, 85% yield).
2 -Hydroxythioxanthone (500 mg, 2.19 mmol), MeI (777 mg, 5.47 mmol), and K2CO3 (1.51 g, 10.95 mmol) were placed in an oven-dried Schlenk flask under an argon atmosphere. Acetone (15 mL) was subsequently added to this mixture, and the resulting solution was refluxed for 3 h. The reaction mixture was then diluted with water and extracted with dichloromethane. The combined organic extracts were dried over anhydrous MgSO4. After solvent removal, the crude product was further purified by column chromatography on silica gel using dichloromethane/petroleum ether as the eluent. The product was isolated as a yellowish-green solid (460 mg, 1.9 mmol, 88% yield). Other 2-positioned alkoxy-tailored TX derivatives were synthesized using the same procedure.
2.3. Preparation of XS particles
XS particles were synthesized using nanoprecipitation method. Briefly, XS molecules (1 mg) were dissolved in THF (1 mL) containing DSPE-PEG-NH2 (7 mg) overnight at room temperature with constant stirring to obtain ip-TX nanoparticles (termed ip-TX). The nanoparticles were purified by centrifugation and washed three times with a Millipore ultrafiltration centrifugal filter (molecular weight cut off (MWCO): 3000 kD). The final product was suspended in 1 mL of distilled water as a stock solution for further use.
To conjugate the PSs, nanoparticles were reacted with the PSs using the EDC-NHS process. Briefly, 1 mg of verteporfin was dissolved in 1 mL of DMF and then diluted with 10 mL of deionized water, followed by the addition of 21 mg of NHS and 36 mg of EDC as covalent coupling reagents. The solution was stirred at room temperature for 30 min. After adjusting the pH to 7.0-7.4, 200 μL of the prepared ip-TX was added to the activated solution. The mixture was allowed to react for 24 h at room temperature with continuous stirring. Afterward, verteporfin-conjugated ip-TX (Vip-TX) was purified using Millipore ultrafiltration centrifugal filter (MWCO: 3000 kD) and washed with phosphate-buffered saline (PBS) for three times. No unusually high safety hazards were encountered during the synthesis process.
2.4. Loading rates of ip-TX and verteporfin in Vip-TX
Verteporfin molecules encapsulated in DSPE-PEG-NH
2 (termed as VP) and Vip-TX were concentrated with Millipore ultrafiltration centrifugal filter at 3000 r∙min
−1, and the supernatant was collected and dissolved in dimethyl sulfoxide (DMSO) to proceed with the standard absorption curve method for calculating the ip-TX or verteporfin loading rate using the following equation:
where
Wip-TX is the weight of ip-TX and
WVip-TX is the weight of Vip-TX.
2.5. Generation of singlet oxygen (1O2) in solution under X-ray irradiation
To confirm the generation of 1O2 in solution, SOSG was used as a detection probe based on its variation in its absorption spectrum. Specifically, ip-TX and Vip-TX were separately suspended in water (15 μg ip-TX∙mL−1) in the presence of SOSG (5 μmol∙L−1) and then irradiated with X-ray at a dose of 2 Gy (160 kV), after which the fluorescence spectra were measured.
2.6. Cellular uptake
All cell lines were used without further authentication, but they were routinely tested for mycoplasma contamination. The 4T1 cells were cultured in medium supplemented with 10% FBS, 100 U∙mL−1 of penicillin G sodium, and 100 µg∙mL−1 of streptomycin sulfate in a humidified atmosphere with 5% CO2 at 37 °C.
The 4T1 cells were seeded on 12-well plates at 5 × 105 cells per well and further cultured for 12 h. Vip-TX (15 μg ip-TX∙mL−1) was added to the cells and incubated for different lengths of time. Fluorescence images were acquired using an Olympus FV1200 laser scanning confocal microscope.
2.7. Cytotoxicity
The 4T1 cells were cultured in Dulbecco’s Modified Eagle Medium containing 10% FBS in a humidified atmosphere with 5% carbon dioxide at 37 °C for 24 h. The viability of the cells was using standard MTT assays. The cells were seeded on 96-well plates (104 cells per well) and incubated for 24 h prior to the next experiments. Vip-TX nanoparticles at different concentrations (0, 3.6, 7.2, 15, 30, and 60 μg ip-TX∙mL−1) were then added to the medium and incubated with the cells for 24 h. The MTT assay was then conducted.
2.8. In vitro 1O2 generation
DCFH-DA reagent was utilized for the detection of singlet oxygen. The 4T1 cells (105 cells per well) were seeded on 12-well plates overnight after which ip-TX, VP, and Vip-TX (15 μg ip-TX∙mL−1) were added to the cells. Cells incubated with PBS served as the control. After incubating for 6 h and washing with PBS again, 10 μmol∙L−1 DCFH-DA was added and the mixture was incubated for 30 min. After another wash with PBS, the cells were irradiated with X-rays at a dose of 2 Gy (160 kV). Luminescence images were captured on an Olympus FV1200 laser scanning confocal microscope.
2.9. Lipid peroxidation measurement
The 4T1 cells (2 × 105 cells) were seeded in glass bottom cell culture dishes (diameter: 10 mm) and incubated for 24 h. The cells were randomly divided into four groups: a blank control group (PBS only), a PBS + X-ray group, a Vip-TX (15 μg ip-TX∙mL−1) group, and a Vip-TX (15 μg ip-TX∙mL−1) + X-ray group. The two treatment groups were irradiated with X-rays (2 Gy, 160 kV). Next, confocal microscopic analysis using 1 μmol∙L−1 BODIPY™ 581/591 C11 staining was used to evaluate the lipid peroxidation levels.
2.10. Mitochondrial membrane potential measurement
The 4T1 cells (2 × 105 cells) were seeded in glass-bottom cell culture dishes (diameter: 10 mm) and incubated for 24 h. The cells were randomly divided into four groups: a blank control group (PBS only), a PBS + X-ray group, a Vip-TX (15 μg ip-TX∙mL−1) group, and a Vip-TX (15 μg ip-TX∙mL−1) + X-ray group. The two treatment groups were subsequently irradiated with X-rays (2 Gy, 160 kV). Next, the cells were tested for mitochondrial membrane potential changes using a JC-1 mitochondrial membrane potential assay kit by following the vendor’s protocol.
2.11. DNA damage measurement
The 4T1 cells (2 × 105 cells) were seeded in a glass-bottom cell culture dishes (diameter: 10 mm) and incubated for 24 h. The cells were randomly divided into four groups: a blank control group (PBS only), a PBS + X-ray group, a Vip-TX (15 μg ip-TX∙mL−1) group, and a Vip-TX (15 μg ip-TX∙mL−1) + X-ray group. The two treatment groups were then irradiated with X-rays (2 Gy, 160 kV). Next, confocal microscopic analysis with γH2AX staining was used to evaluate the DNA damage.
2.12. Cell apoptosis evaluation
Cells were treated under different conditions to evaluate apoptosis. The various treatments included PBS (control), Vip-TX (15 μg ip-TX∙mL−1), X-rays, Vip-TX (15 μg ip-TX∙mL−1) + X-rays. After these treatments, the cells were incubated for 4 h and rinsed twice with PBS. The treated cells were then incubated for 4 h and were subsequently harvested through trypsinization and centrifugation. The precipitated cells were dispersed with PBS, labeled with Annexin V-FITC and propidium iodide (PI) for 15 min and then analyzed using flow cytometry.
2.13. Animal experiments
Female BALB/c mice (5-6 weeks old) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (China) and Shanghai SLAC Laboratory Animal Co., Ltd. All animal experimental procedures were performed following the guidelines of the Regional Ethics Committee for Animal Experiments and the Care Regulations approved by the Institutional Animal Care and Use Committee of Xiamen University.
2.14. In vivo imaging, biodistribution, and blood clearance evaluation of Vip-TX
Tumor-bearing mice were established by subcutaneously injecting 2 × 106 4T1 cells. When the tumors reached a size of 60-80 mm3, Vip-TX (1.5 mg ip-TX∙kg−1) was injected intravenously. Fluorescent images were obtained at 0, 1, 2, 4, 8, 12, 18, and 24 h post-injection utilizing an IVIS Lumina II in vivo imaging system. Ex vivo imaging was also conducted. The 4T1 tumor-bearing mice were euthanized and dissected at 24 h after injection. The dissected main organs and tumors were analyzed using an IVIS Lumina II in vivo imaging system. The blood (200 µL) was also extracted and analyzed for fluorescence intensity. The in vivo blood circulation half-life of Vip-TX was calculated using a single-component pharmacokinetic model.
2.15. In vivo X-PDT tests
For tumor therapy, 4T1 tumor-bearing mice were randomly divided into groups: PBS only, Vip-TX, X-ray, and Vip-TX + X-ray groups. The mice were injected with Vip-TX (1.5 mg ip-TX∙kg−1) on the first day. X-ray irradiation at a dose of 1 Gy (160 kV) was applied to tumor areas 2 h post-injection, while the rest of each mouse’s body was shielded by lead. The body weights and tumor sizes of each group were recorded every other day during the 14 d treatment. Tumor volume was determined using the following equation: tumor volume = (width2 × length)/2. After tumor therapy, all mice were euthanized. The tumors were then excised and photographed. The excised tumors and major organs were subsequently sliced and subjected to standard hematoxylin and eosin staining for histological analysis.
2.16. Serum chemistry and hematological analysis
Mice were intravenously treated with Vip-TX (1.5 mg ip-TX∙kg−1). Blood samples were collected for serum chemistry analysis and complete blood analysis before treatment (day 0) and on days 3, 7, and 14 post-treatment. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), serum albumin (ALB), blood urea nitrogen (UREA), creatine kinase (CK), lactate dehydrogenase (LDH), creatinine (CREA), and creatine kinase-MB (CK-MB) in mouse serum were measured using an automatic biochemistry analyzer (Mindray BS-220; Mindray, China). White blood cell count (WBC), red blood cell count (RBC), hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), RBC distribution width (RDW-SD), platelets (PLT), and mean platelet volume (MPV) in mouse blood were measured using an automatic tri-classification blood cell analyzer (Mindray BC-2600).
2.17. Statistical analysis
All data were calculated as mean values ± standard deviations (SDs). Comparisons of data were conducted to calculate P values using one-way analysis of variance (ANOVA; SPSS Statistics 2). Differences were considered statistically significant as follows: *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.
3. Results and discussion
3.1. Synthesis and characterization of XS nanoparticles
To screen organic molecules with simple structures, two pharmaceutical intermediates, phenothiazine and TX, were chosen for a comparison of luminescence when exposed to X-ray irradiation [
25], [
26], [
27]. Molecular structural analysis revealed that even a minor structural difference led to significant variations in luminescence behavior under X-ray irradiation (Fig. S1 in Appendix A). Compared with the 2-positioned halogenated derivatives of phenothiazine, the halogenated derivatives of TX emitted radioluminescence around 480 nm (fluorescence emissions) and over 20 fold radioluminescence at 527 and 577 nm (RTP emissions) (Fig. S1). To fully investigate the biological effects of fluorescence and RTP emissions under X-ray irradiation, a series of TX molecules were tailored with 2-positioned alkoxy substituents, including methoxy (-OCH
3, termed M-TX), ethoxy (-OCH
2CH
3, termed E-TX), n-propoxy (-OCH
2CH
2CH
3, termed np-TX), and isopropoxy (-OCH(CH
3)
2, termed ip-TX) (Fig. S2 in Appendix A). TX has a limited photoluminescence efficiency (
Φ = 1.5%), and 2-positioned derivatives of TX efficiently increased photoluminescence efficiency [
28]. In our investigation, under X-ray irradiation, all 2-positioned TX derivative powders exhibited clear singlet emission (around 465 nm, assigned to fluorescence) and triplet emission (585 nm, assigned to phosphorescence) (
Fig. 1(a)). Notably, completely different from other 2-positioned TX derivatives, triplet emissions of M-TX and ip-TX were higher than singlet emissions, and ip-TX showed both the highest singlet and triplet emissions among all 2-positioned TX derivative powders (
Fig. 1(a)). In particular, ip-TX formed a typical J-aggregation, which increased ISC to promote triplet emission [
28]. Furthermore, multiple hydrogen bond interactions and the rigid environment of the isopropoxy group along the short axis of molecular packing efficiently restricted nonradiative vibrations and greatly enhanced triplet emission (
Fig. 1(b)) [
28]. The efficient population of triplet excitons then transferred energy to oxygen in a solution through phosphorescence (
Fig. 1(b)) [
28]. This process is very similar to the photodynamic mechanism in a photochemical field. To differentiate and compare the definitions of a PS and radiosensitizer (also called radio-enhancer), we define the organic molecule activated directly by X-rays to efficiently populate triplet excitons as an XS.
3.2. Radioluminescence photophysical properties and 1O2 generation in an aqueous solution
Under the same conditions, ip-TX exhibited the highest fluorescence efficiency (
ΦF = 12.7%) and phosphorescence efficiency (
ΦP = 32.3%) [
16]. For further investigation in biomedical applications, ip-TX, as a typical XS, was used. Nanoprecipitation was used to transform XS into water-soluble nanoparticles in the presence of DSPE-PEG-NH
2. Monodispersed spherical nanoparticles were obtained with an encapsulation ratio of ip-TX of approximately 15 wt% and a uniform size of around 105 nm (
Fig. 1(c)). Under 365 nm ultraviolet light excitation, the relative intensities of the singlet emission were higher than those of the triplet emission (black curve,
Fig. 1(d)). Notably, under X-ray excitation, the triplet emission increased sharply, with relative intensities that were higher than those of the singlet emission (red curve,
Fig. 1(d)). More interestingly, the relative intensity of phosphorescence increased dramatically under X-ray excitation (
Fig. 1(e)), clearly indicating that substantial proportion of the triplet states was directly populated by X-rays [
17], [
18]. In other words, 2-positioned tailored TX molecules activated by X-rays directly generated triplet states. We therefore inferred that in TX, the excitations under X-ray irradiation were dominated by ionized excitations.
X-ray excited triplet states from an XS transfer energy to oxygen in a solution based on phosphorescence. To amplify the energy transfer for the generation of
1O
2, singlet emissions (fluorescence) can be fully used by conjugating a PS to ip-TX and exciting the PS for more generation of
1O
2. A benzoporphyrin derivative verteporfin (trade name Visudyne) is a second-generation US Food and Drug Administration (FDA)-approved PS (2000) for PDT that triggers mitochondrial photochemical damage through ROS accretion [
29], [
30], [
31]. In 2014, a phase I/II study was conducted using verteporfin-mediated PDT to treat locally advanced pancreatic cancer, and the safety of the approach was confirmed [
32]. Verteporfin is also approved for the treatment of recurrent high-grade epidermal growth factor receptor (EGFR)-mutated glioblastoma (GBM) in a clinical trial (NCT04590664), and published data suggest that verteporfin is a promising therapeutic agent for EGFR-amplified and mutated GBM [
33]. Most importantly, the absorbance of verteporfin is well matched with the singlet emission (
Fig. 1(f)). After conjugation, Vip-TX maintained its spherical structure with a core size of around 93 nm and a hydrodynamic diameter of around 161 nm (
Fig. 1(g)). The loading rate of verteporfin in Vip-TX was approximately 8.6 wt%, and the surface conjugation had minimal effect on zeta potential (
Fig. 1(h)). The successful conjugation was further confirmed by Fourier-transform infrared (FT-IR) measurements (
Fig. 1(i)).
3.3. In vitro evaluation of direct radiation-activated generation of triplet excitons
The triplet-state emission was verified by observing
1O
2 production from the excited ip-TX and Vip-TX, in which the triplet state transferred its energy to the normal triplet state O
2, giving rise to singlet state
1O
2. To quantify
1O
2, SOSG was applied to test the generation of
1O
2 during X-ray irradiation. After Vip-TX radiation irradiation of 2 Gy, the fluorescent intensities of SOSG increased 1.8 fold, and radiation irradiation of only 2 Gy showed a minor increase (
Fig. 2(a)). Vip-TX and SOSG exhibited dose-dependence fluorescence enhancements (
Fig. 2(b)). Electron spin resonance (ESR) spectroscopy was used to explore the ROS generation. Compared with the weak and very small peaks of radiation-only treatment, clear and sharp peaks of
1O
2 were produced by radiation-triggered singlet and triplet emissions from Vip-TX (
Fig. 2(c)).
After confirming its stability in media (Fig. S3 in Appendix A), the cellular uptake of Vip-TX was explored by the verteporfin fluorescence of Vip-TX. After incubation with 4T1 cells for 4 h, clear and sharp red fluorescent signals were evenly distributed in the cells (
Fig. 2(d)). Time-course flow cytometry analysis further confirmed the high cellular uptake after a 4 h incubation (
Fig. 2(e)). Excellent cellular uptake would be beneficial for generating more
1O
2 in intracellular organelles. Under the same dosage of X-rays (2 Gy), 4T1 cells treated with ip-TX exhibited stronger green fluorescence signals than the controls (Fig. S4(a) in Appendix A). An over 4.0-fold enhancement clearly indicated that the triplet emission efficiently generated
1O
2 compared with the radiation group (Fig. S4(b) in Appendix A). A combination of singlet and triplet emissions (i.e., Vip-TX + X-ray irradiation) achieved stronger green fluorescence signals (
Fig. 2(f), Fig. S5 in Appendix A), and over 3.5 fold enhancement was achieved compared with that of the radiation group. Notably, the combination of singlet and triplet emissions (Vip-TX + X-ray irradiation) generated 1.3 fold more
1O
2 than did the triplet emission (ip-TX + X-ray irradiation) (
P = 0.024) (Figs. S6 and S7 in Appendix A). The ratio of triplet emission between the ip-TX + X-ray irradiation and Vip-TX + X-ray irradiation groups was calculated as 0.79 (3/3.8), which is extremely close to 0.75 (3/4). This indicates that most of the generated singlet and triplet emissions in our design were well utilized [
12], [
13], [
14]. The generated
1O
2 and heavy-atom effects of elemental S in TX induced lipid peroxidation, DNA damage, and mitochondrial dysfunction in 4T1 cells (Figs. 2(g)-(i), Figs. S8-S10 in Appendix A). Vip-TX + X-ray irradiation induced significant differences in lipid peroxidation, DNA damage, and mitochondrial dysfunction (Figs. S8-S10), which caused significant cell death (Fig. S11(a) in Appendix A). Using ip-TX concentrations of 0-60 µg∙mL
−1, Vip-TX in the absence of X-ray irradiation showed extremely low cytotoxicity in tumor cells, although it had a high cellular uptake (
Fig. 2(d), Fig. S11(a)). Notably, in the presence of X-ray irradiation, cellular viability decreased sharply (Fig. S11 in Appendix A). The therapeutic effects were further confirmed using flow cytometry analysis and fluorescent imaging with Annexin V-FITC/PI and Calcein-AM/PI staining (Figs. S11(b) and (c)).
3.4. In vivo evaluation of direct radiation activation for the efficient generation of triplet excitons
The pharmacokinetics of intravenously injected Vip-TX were examined by measuring the plasma concentrations of Vip-TX using the fluorescence of verteporfin in tumor-free mice. The elimination half-life (
t1/2) of Vip-TX was (163.8 ± 10.4) min (
Fig. 3(a)). The biodistribution
in vivo and
ex vivo and quantification of Vip-TX throughout the organs of tumor-bearing mice demonstrated high accumulation and long retention in tumors with an accumulation peak at 2 h post-injection (
Figs. 3(b) and (c)). The fluorescent signals were quickly attenuated from normal organs and were long and sharp in tumor regions (
Fig. 3(c)). Results from organs
ex vivo clearly exhibited 2.9 and 4.3 fold longer retention times in tumors than in livers or kidneys, respectively (
Figs. 3(d) and (e)). Most importantly, the high accumulation and long retention had no clear influence on blood parameter analyses or kidney and liver functions of healthy mice (Table S1 in Appendix A).
The high accumulation and long retention of the bright luminescence emitted from the extracted tumors under X-ray irradiation (Fig. S12 in Appendix A) indicated a high accumulation of TX in tumors. Encouraged by these
in vitro and
in vivo results, we performed
in vivo experiments to explore the ability of Vip-TX as XS to be directly activated by high-energy radiation and efficiently generate triplet excitons to suppress tumor growth in a subcutaneous 4T1-tumor model in BALB/c mice. When the tumors reached 60-80 mm
3, VP, ip-TX, and Vip-TX (1.5 mg ip-TX∙kg
−1) were injected intravenously, and X-rays (1 Gy, 160 kV) were irradiated 2 h post-injection. The quantified tumor volumes of each group showed that rapid tumor growth was observed in all controls (PBS, radiation group (1 Gy), and Vip-TX) (
Fig. 4(a)). In sharp contrast, there were significant decreases in the tumor volumes in the Vip-TX + X-ray irradiation group (1 Gy) (
Fig. 4 (a), Fig. S13 in Appendix A).
The quantitative analysis of the tumor volumes further verified the complete inhibition of tumor growth by Vip-TX + X-ray irradiation (
Fig. 4(b)). The tumor inhibition ratios based on tumor volumes were calculated as 86.4% (
Fig. 4(c)). Photographs of typical tumor-bearing mice showed a similar trend of treatment efficiency (Figs. S14 and S15 in Appendix A), and the excised tumors at the end time point were clearly smaller in the group treated with X-ray-irradiated Vip-TX (
Fig. 4(d)). The tumor weights further confirmed the excellent anti-tumor efficiency in the X-ray-activated Vip-TX group (
Fig. 4(e)). The tumor inhibition ratios based on tumor weight was calculated as 85.6% (
Fig. 4(f)), which is consistent with the calculation using tumor volumes. The hematoxylin and eosin staining of the tumors further indicated that the mice treated with X-ray-activated Vip-TX had significant damage in the tumor regions (
Fig. 4(g)) and showed fewer tumor nodules and looser tissue. Notably, there was no obvious body weight loss in mice treated with X-ray irradiated Vip-TX and the other treatments (Fig. S16 in Appendix A), and there were no effects on other organs (Fig. S17 in Appendix A), demonstrating that the treatment caused few side effects in mice. Together, these results show that X-ray-activated TX derivatives efficiently populate triplet excitons under low-dose X-ray irradiation and are safe and low-toxic
in vivo X-ray-activated XSs for cancer treatment.
4. Conclusions
We screened pharmaceutical drug intermediates that are derivatives of TX and investigated TX-derived organic molecules that efficiently underwent X-ray-sensitization to populate singlet oxygen for use as cancer therapeutics alongside exposure to low-dose X-ray irradiation. We assessed their luminescence under X-ray irradiation. TX structures exhibited higher radioluminescence than phenothiazine with the same position substitutions. After modifying alkoxy side chain substitutions at the 2-position to tune the molecular packing and intermolecular interactions, the fluorescence and RTP of a series of TX derivatives were assessed under low-dose X-ray irradiation, and TX-derived organic molecules efficiently underwent X-sensitization to populate singlet oxygen as a cancer therapeutic. The generation of singlet oxygen and potential for tumor treatment in vitro and in vivo provide new opportunities for developing an additional new class of organic molecules in which large numbers of triplets can be populated directly under low-dose X-ray.
Acknowledgments
The work was supported by the National Natural Science Foundation of China (82172007, 81771977, 52103209, and 52373183), the Science Fund for Distinguished Young Scholars of Fujian Province (2021J06007), the Scientific Research Foundation of Xiang An Biomedicine Laboratory (2023XAKJ0101023), the Open Research Fund of the National Facility for Translational Medicine (Shanghai) (TMSK-2021-102), and the Open Project of State Key Laboratory of Supramolecular Structure and Materials (sklssm2023026). All animal experiments were approved by the Animal Management and Ethics Committee of Xiamen University. We thank LetPub for its linguistic assistance during the preparation of this manuscript.
Compliance with ethics guidelines
Nuo Lin, Han Xu, Haichao Liu, Xiaoqian Ma, Qunying Shi, Qing Yang, Yating Wen, Huanglei Wei, Ke Hu, Bing Yang, and Hongmin Chen declare that they have no conflict of interest or financial conflicts to disclose.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.org/10.1016/j.eng.2024.06.010.
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