Abstract

Organic phosphorescence materials offer significant advantages for bioimaging applications. However, most of these materials are excited exclusively by ultraviolet (UV) light, which poses risks to living organisms. Herein, six donor–acceptor-type compounds incorporating triazine groups are designed as guests within doped systems. White-light excitable phosphorescent guests enable doped materials to show efficient afterglow under white-light excitation. By leveraging the ability of white-light to penetrate biological tissues, a bioimaging mode in which the materials are first concentrated within the organism and then excited was developed, yielding superior imaging effects compared with the traditional method in which materials are first excited and then concentrated. Furthermore, these materials are applied in imaging diagnosis of atherosclerosis plaques (male Apoe^−/− mice) and intestinal diseases (female BALB/c-nude mice), as well as in navigation for in situ liver tumor surgery (female BALB/c-nude mice), achieving excellent imaging outcomes. This work addresses the limitations of phosphorescent materials that rely on UV-light, significantly enhancing their potential for practical applications in clinical imaging.

Introduction

Optical imaging is becoming increasingly important in the visualization and detection of biological tissues because of its advantages of nonionizing radiation, high spatiotemporal resolution, and real-time diagnosis^1,2,3,4,5,6. Phosphorescence emission, which is an optical phenomenon originating from the radiative transition of excitons from triplet excited states to ground states, features emission lifetimes of milliseconds to seconds. This property effectively mitigates the issue of low image clarity caused by spontaneous fluorescence in biological tissues^{7,8,9,10,11,12,13,14,15}. Until now, inorganic luminophores that incorporate heavy metals or rare-earth metals, such as chromium, europium, dysprosium, and praseodymium, have been employed for tumor and vascular imaging, as well as for in vivo cell targeting and tracking^16,17. However, concerns regarding the long-term biotoxicity of these inorganic luminophores, resulting from the potential leakage of heavy-metal ions and their non-biodegradable nature, remain unresolved. Organic phosphorescent compounds, which are characterized by high biocompatibility, low toxicity, and easy functional group modification, present natural advantages for diagnosis and treatment of biological tissue^{18,19,20,21,22}. Despite lacking heavy-metal atoms, organic materials can be endowed with room-temperature phosphorescence (RTP) through a series of feasible strategies, such as crystal engineering, H-aggregation, polymerization, and host?guest doping. These advances have significantly expanded the properties and types of organic RTP materials^{23,24,25,26,27,28,29,30,31}. However, the majority of organic RTP materials are excitable only by short-wavelength ultraviolet (UV) light, which causes considerable damage to biological tissues. This limitation hinders the practical application of organic RTP materials in clinical imaging. Wu et al. developed self-assembled organic nanoparticles from difluoroboron β-diketonate compounds in aqueous solution³². Ma et al. and Liu et al. developed visible light or two-photon excited organic RTP in aqueous solution via supramolecular macrocyclic assembling strategy^33,34. The above materials rely on strict supramolecular assembly and the luminescence performance may further improvement. In addition, the above materials are only used for simple cell imaging. Recently, Li et al. successfully constructed deep-red afterglow materials that can be excited by visible light, including sunlight and mobile phone flashlight, through the rational combination of electron donor (D) and acceptor (A) with strong electronic pull-push effect, and successfully used the materials for in vivo imaging^35,36. The above work has preliminarily verified the advantages of white-light-excited phosphorescent materials in bioimaging, but there is a lack of systematic and in-depth research on the application of white-light-excited materials in biological tissue imaging, lesion tissue diagnosis, and surgical navigation. In addition, although researchers have discovered several phosphorescent materials that can be excited by white light^{37,38,39,40,41}, the clear and simple design concept for constructing white-light-excited phosphorescence materials are still lacking. In most doped systems, the phosphorescence emission originates primarily from guest molecules, with the host matrix assisting in the process^{42,43,44,45,46,47,48,49,50,51,52,53,54,55}. Consequently, the optical properties of guests determine the luminescent properties of doped materials. Thus, designing guest molecules that can be excited by white-light is crucial for achieving white-light-excited phosphorescence emission from doped materials.

Two modes currently exist for the use of organic phosphorescent materials in bioimaging. The first involves concentration of the materials at the target position before excitation to collect signals (concentration–excitation mode)^35,36. The second mode entails first exciting the materials and then concentrating them at the target position to collect signals (excitation–concentration mode)^56,57. The concentration–excitation mode offers clear advantages, such as real-time excitation and imaging. However, the use of UV-light, which is damaging to biological tissues, must be strictly limited, significantly reducing the imaging efficacy. Even at a reduced intensity, UV-light can still harm living organisms. In contrast, the excitation–concentration mode avoids the damage caused by excitation light to biological tissues but necessitates materials with long phosphorescence lifetimes. This mode often results in weak signals due to photon loss during the concentration process. White-light-excited phosphorescent materials can be employed in the concentration–excitation mode since white light can directly irradiate biological tissues. Therefore, the design and preparation of phosphorescent materials that can be excited by white-light would undoubtedly expand the use of bioimaging.

In this work, six donor–acceptor (D–π–A)-type compounds (TRZ-1 to TRZ-6) with a triazine group as the acceptor are designed as guests. The D–π–A structure is advantageous for increasing the absorption and emission wavelengths of the materials, whereas the triazole group, with its strong absorption capability, enhances the molar extinction coefficient of the molecules (Fig. 1a)^58,59,60. Upon excitation by white-light, all six guests exhibit efficient afterglow. Subsequently, doped systems are constructed using benzophenone (BPO) as the host (Fig. 1b). As expected, the doped materials demonstrate a bright afterglow of 3–5 s when excited by white-light, indicating excellent white-light-excited RTP performance. The phosphorescence quantum yield (Q.Y.) of the doped systems range from 15% to 75%, with a phosphorescence lifetime between 176 and 401 ms. Notably, even at excitation wavelengths of 460–500 nm, the doped materials maintain a phosphorescence lifetime of 81–239 ms. The doped material TRZ-1/BPO, with the strongest phosphorescence intensity, is selected for in vivo biological imaging. Systematic experiments confirm that the concentration–excitation bioimaging mode yields significantly better imaging results than the excitation–concentration mode. Moreover, the doped material is used for imaging tumors and atherosclerosis (AS) in mice, resulting in a high signal-to-background ratio (SBR) of 46.1. Moreover, based on the quenching effects of Fe²⁺ and Fe³⁺ on the phosphorescence signal of TRZ-1/BPO⁶¹, this material is applied for image-guided liver cancer surgery in mice. Furthermore, TRZ-1/BPO is prepared as an oral gel (O-TRZ-1/BPO-gel) for imaging cecum disease. These white-light-excited RTP materials exhibit excellent imaging effects in the aforementioned experiments, clearly demonstrating that white or visible light can replace UV-light as an excitation source for optical imaging. This work provides a strategy to mitigate the harm of excitation light to biological tissues in clinical imaging, significantly enhancing the practical application potential of organic RTP materials for living organisms and potentially human organs. However, this work is currently only in the laboratory basic research stage, and can only image animal tumor/lesion tissues in specific models. Navigation for animal tumor surgery is also only applicable to early-stage tumors. In the follow-up work, we will collaborate with the clinical department of the hospital to explore the further use of phosphorescence materials for the diagnosis of human pathological tissues and imaging navigation of diffuse tumor surgery.

Fig. 1: Schematic diagram.

Results

Synthesis and photophysical properties of guests

Guests TRZ-1 to TRZ-6 were synthesized according to established methods (Fig. 2a and Supplementary Fig. 1). The molecular structures and purities were confirmed via nuclear magnetic resonance spectroscopy, high-resolution mass spectrometry, single-crystal X-ray diffraction, and high-performance liquid chromatography (Supplementary Figs. 2 and 3, and 42–59). These guests were designed with a D–π–A molecular structure, using diphenylamine derivative groups as donors and triazole groups as acceptors. For TRZ-1, TRZ-2 and TRZ-3, the donor group was connected to the acceptor group through a benzene ring, whereas in TRZ-4, TRZ-5 and TRZ-6, the connection was made through a more conjugated naphthalene ring, further increasing the absorption and emission wavelengths. The results indicated that the absorption and phosphorescence wavelengths (at 77 K) of TRZ-1, TRZ-2, and TRZ-3 ranged from 320–440 nm and 510–526 nm, respectively (Fig. 2b and Supplementary Fig. 4). In contrast, TRZ-4, TRZ-5 and TRZ-6 presented absorption wavelengths ranging from 345–480 nm and phosphorescence wavelengths ranging from 590–638 nm (Fig. 2b and Supplementary Fig. 4). Additionally, the excitation spectra of the phosphorescence emission demonstrated that the guest molecules could be excited by light with a wide range of wavelengths from 350–480 nm (Fig. 2c), indicating that they could be activated by non-UV-light. The experimental results further confirmed that upon white-light excitation, TRZ-1 to TRZ-3 presented a bright green afterglow lasting 6–10 s, whereas TRZ-4 to TRZ-6 presented an orange–red afterglow lasting 3–4 s (Fig. 2e and Supplementary Fig. 5). The phosphorescence intensity attenugation curves under 420 nm excitation revealed phosphorescence lifetimes of 0.48–1.41 s for the six guests (Fig. 2d). These results collectively demonstrate that the designed molecules exhibit excellent white-light-excited phosphorescence activity. To gain deeper insight into the mechanism of white-light-excited phosphorescence, density functional theory (DFT) and time-dependent density functional theory (TD-DFT) calculations were carried out using the B3LYP/6-311 G(d) function. The TD-DFT simulated UV-Vis absorption spectra were almost consistent with the experiments, all guest showed effective absorption at wavelengths above 400 nm (Supplementary Fig. 6). Moreover, the molecular orbitals results shown that the electron clouds of the highest occupied molecular orbital (HOMO) were predominantly localized on the diphenylamine derivative groups, and the lowest unoccupied molecular orbital (LUMO) was distributed on the triazine groups (Supplementary Fig. 7). The electronic clouds of HOMO and LUMO had obvious separation, which can effectively narrow the ΔE_ST facilitating the phosphorescence emission. Moreover, compared to the molecules (TRZ-1, TRZ-2 and TRZ-3), the guest molecules (TRZ-4, TRZ-5 and TRZ-6) linked via the more conjugated naphthalene ring can effectively lower the triplet energy level, leading to long-wavelength phosphorescence emission, which is consistent with the excited state energy level results obtained through theoretical calculations (Supplementary Table 1). Moreover, the ΔE_ST (Energy gap between the lowest singlet state and lowest triplet state) of six guests were 0.41–0.62 eV, this is very close to the measurement results calculated based on the actual emission wavelength. (0.37–0.58 eV) (Supplementary Fig. 8), the smaller energy gaps are conducive to the intersystem crossing of excitons, resulting in the phosphorescence activity (77 K) of the guests.

Fig. 2: Photophysical property of the guests.

Discussion

In summary, six D–π–A-type compounds with white-light-excited phosphorescence properties were designed as guest molecules to establish doped system. The guests successfully induced the doped materials to emit afterglow under white-light excitation at room temperature. Under white-light excitation, the doped materials exhibited the bright and persistent bright blue or orange–red afterglow. The imaging effects of both the concentration–excitation and excitation–concentration imaging modes using the doped materials were systematically compared, confirming the advantages of white-light-excited phosphorescence materials in bioimaging. The white-light-excited phosphorescence material has not only improved the clarity and accuracy of imaging, but also achieved real-time, non-invasive diagnosis. Specifically, the application of these doped materials in imaging diagnosis of AS plaques and intestinal diseases demonstrated their ability to visualize complex biological structures with high contrast. Furthermore, their use in in situ liver tumor surgery navigation proved their potential to significantly improve surgical precision and reduce operative risks. This work further advanced the clinical application of organic phosphorescence materials, their ability to provide high-resolution images in real-time is expected to enhance current imaging technologies. However, the methods used in this work are currently in the basic research stage and can only be used to image animal tumors/lesion tissues in specific models, the animal tumor surgical navigation is also only applicable to early-stage tumors. In the future, we will increase the research efforts on the use of white-light activated phosphorescence materials for the diagnosis and treatment of complex human tumors/lesions.

Methods

Ethical regulations

All animal studies were performed according to the guidelines set by the Nankai University Animal Resources Center (The animal license number is SYXK (Jin) 2019-0003 promulgated by Tianjin Science and Technology Commission). The overall project plan has been approved by the Animal Ethics Committee of Nankai University, with the number 2022-SYDWLL-0000186. A humane endpoint was determined according to animal welfare standards, including the maximal tumor volume allowed was 2000 mm³ and the maximal tumor size in this study was not exceeded. Approval for these humane endpoints was granted by the Certification and Accreditation Administration of the People’s Republic of China (RB/T 173-2018).

Synthesis or purchase of guest compounds

In a round bottom flask, 2-(4-Bromophenyl)-4,6-diphenyl-1,3,5-triazine (851.5 mg, 2.2 mmol) or 2-(4-Bromonaphthalen-1-yl)-4,6-diphenyl-1,3,5-triazine (961.5 mg, 2.2 mmol) was reacted with Aromatic amine derivatives (2.0 mmol) under Buchwald-Hartwig coupling reaction using Pd₂(dba)₃ (36.6 mg, 0.04 mmol), HP^tBu₃BF₄ (34.8 mg, 0.12 mmol) and NaO^tBu (230.6 mg, 2.4 mmol) as catalysts in 20 ml toluene (reflux under nitrogen), and the mixture was stirred at 110 °C for 12 h. After cooling to room temperature, the reaction mixture was poured into CH₂Cl₂ (100 mL), the organic layer was washed with water (100 mL) three times and then dried over anhydrous Na₂SO₄. After removing the solvent under reduced pressure, the residue was purified by column chromatography on silica gel with petroleum ether/dichloromethane (10:1 v/v) to obtain target products TRZ-1, TRZ-2, TRZ-3, TRZ-4, TRZ-5 and TRZ-6.

Characterization

The molecular structures and purities of compounds were confirmed via nuclear magnetic resonance spectroscopy, high-resolution mass spectrometry, single-crystal X-ray diffraction, and high-performance liquid chromatography.

Preparation of doped materials

Place a certain amount of pre-weighed host and guest together and heat them in the ambient environment to 60–80 °C. After the host is completely melted, slowly stir with a glass rod until the guest is dissolved in the melted host, then the mixed system is cooled to room temperature, and the mixed system is crystallized to obtain the doped material. The doped materials with high guest-host molar ratio (1:20–1:500) are using direct weighing method. While, for the doped materials with a low molar ratio of guest-host (1:1000–1:100000), indirect dilution method is required, such as continuing to mix the doped material (1:100) with the corresponding amount of pure host.

Theoretical calculation methods

The density functional theory (DFT) and time-dependent density functional theory (TD-DFT) calculations were performed on Gaussian 09 program. The ground state (S₀) geometry optimizations were calculated using DFT at the B3LYP/6-311 G(d) level. Based on the specific optimized geometries, the excited state energies for S₁ and T_n were calculated using TD-DFT with B3LYP/6-311 G(d) level. The simulated absorption spectra were from TD-DFT calculation under B3LYP/6-311 G(d) level.

Preparation of phosphorescence NPs

Pluronic® F127 (10 mg) and TRZ-1/BPO crystals (5 mg) were sequentially added to 5 mL of ultrapure water. The mixture was then sonicated using a microtip probe sonicator (output power 60 W) in an ice bath for 30 min, and the resulting suspension was filtered through a 0.45-μm membrane and concentrated by ultrafiltration to 1 mL, resulting in the phosphorescent nanoprobes of TRZ-1/BPO, which were named as TRZ-1/BPO NPs.

Preparation of phosphorescence oral gel

TRZ-1 (2.61 mg) and BPO (500 mg) were taken in a 10 mL cilindrical vial, subsequently PEG20000 (21 mg) and gelatin (3 mg) were added to the vial, heated to a molten state, mixed well and then cooled to crystallize, subsequently tannic acid (7 mg) was added to the system, heated again to a molten state, cooled, and then the solid samples were crumbled and stirred uniformly. The above operation was repeated five times to obtain TRZ-1/BPO oral gel, named O-TRZ-1/BPO-gel.

White-light lamp

The white-light used in the experiment comes from common LED lamps. As white light is a composite light and located in the visible light region, its wavelength is between 400-700 nm (Supplementary Fig. 41).

In vitro phosphorescence imaging

In vitro afterglow imaging of TRZ-1/BPO nanoprobes was performed using 0.2 W/cm² white-light as the excitation light source. TRZ-1/BPO NPs (200 μL, 5 mg/mL) were placed in a 96-well plate and excited for 120 s. The afterglow signals were collected by using IVIS in the bioluminescence mode. Chicken breast tissue was purchased from marketing approach.

Cell culture

Mouse 4T1 breast cancer cells were purchased from the American Typical Culture Collection (ATCC). Cells were cultured using RPMI-1640 medium containing penicillin (10 U/mL), fetal bovine serum (10%), and streptomycin (10 mg/mL). Cells were periodically checked for mycoplasma contamination during culture and were stored in 5% carbon dioxide and 95% humidified air at 37 ^oC.

Cytotoxicity assessment

First, a density of 5000 4T1 breast cancer cells per well was spread in a 96-well plate and incubated overnight in an incubator to ensure cell attachment. When the cell density reached 70%, different concentrations of TRZ-1/BPO NPs were added (concentrations of 0, 5, 10, 20, 50, 100, 150, and 200 μM, respectively, calculated based on the concentration of BPO in the system). Next, the cells were incubated for 24 h. The next day, the cell growth status was observed and examined under a microscope. Subsequently, MTT solution (0.5 mg/mL) was added and incubated again for 4 h. When MTT combined with live cells to produce a purple precipitate, the supernatant was aspirated and 100 μL of dimethyl sulfoxide was added to each well to dissolve the precipitate. After the precipitates were solubilized, absorbance values were measured using an enzyme marker (wavelength 490 nm). Cell activity was then measured by the ratio of the absorbance value at each concentration to the absorbance value of cells cultured using only the medium.

Blood routine and liver function assessment

Healthy BALB/c mice were taken, and TRZ-1/BPO NPs (5 mg/mL, 100 μL) were injected into the mice via tail vein, and a group of mice injected with 100 μL of physiological saline in the tail vein was used as control. After 7 days, BALB/c mice were subjected to routine blood tests and blood biochemical tests. Tests included leukocytes, lymphocytes, monocytes, hemoglobin, hemocytes, hemoglobin, platelets, alanine aminotransferase, aspartate aminotransferase, albumin, blood urea nitrogen, creatinine, and uric acid. Blood routine and liver function were evaluated by the same method as above in mice after gavage of O-TRZ-1/BPO-gel (5 mg).

Histological study

Heart, liver, spleen, lung, and kidney tissues from normal mice, TRZ-1/BPO NPs (5 mg/mL, 100 μL) administered mice were fixed in 10% neutral buffered formalin and routinely processed into paraffin sections of 5-μm thickness and stained with hematoxylin and eosin (H&E), and the sections were examined by a digital microscope and evaluated.

Animals

Vital River Laboratory Animal Technology Co., Ltd (Beijing, China) is supplier of BALB/c-nude and apolipoprotein E-deficient (apoe^−/−) mice. The mice are housed in a Specefic Pathogen Free (SPF) grade barrier environment.

Anesthesiology

All mice were anesthetized using isoflurane via the inhalation system. Induction was performed in an induction chamber using 3–4% isoflurane and 100% oxygen, and anesthesia was maintained at 1.5–2% isoflurane concentration via a nose cone during the course of the experiment. Depth of anesthesia was monitored by checking pedal reflexes and respiratory rate. Body temperature was maintained at 37 °C throughout the experiment using a heating pad.

Subcutaneous tumor model

1.5 × 10⁵ 4T1 cancer cells were added to 0.1 mL of PBS buffer and injected subcutaneously into 5-week-old female BALB/c-nude mice. After approximately 7 days of tumor growth, the subcutaneous tumor mouse model was formed for imaging experiments.

Mouse liver cancer model

The subcutaneous tumor tissue of the above mouse model was removed, cut into tissue fragments, and the abdominal cavity of healthy female BALB/c-nude mice was opened, and the tissue fragments were sutured into the liver of the mice using surgical sutures, and the abdominal wounds were cleaned up with iodine povidone on the 2nd day after the abdominal wounds were sutured, and the tumors were formed into a model after about 7 days of growth.

Mouse AS model

Male 5-week-old Apoe^−/− mice were fed a high-fat diet (HFD) for 4 months to form a mouse model of AS for imaging experiments.

Mouse cecum obstruction model

Female 5-week-old BALB/c-nude mice, the upper part of the cecum is ligated with a surgical suture after the abdomen is opened, and then the abdominal wound is sutured, which leads to a mouse cecum obstruction model for imaging experiments.

In vivo phosphorescence imaging (concentration–excitation mode)

In situ subcutaneous injection imaging

TRZ-1/BPO NPs (50 μL, 5 mg/mL) were injected subcutaneously into the right dorsal side of mice, excited by a white-light source (0.2 W/cm²) for 120 s, and the afterglow signals were collected by using IVIS in bioluminescence mode with an exposure time of 10 s.

Lymph node imaging

TRZ-1/BPO NPs (50 μL, 5 mg/mL) were injected into the left hind paw of mice and delivered for 15 min. The ipsilateral inguinal lymph node of the hind paw was excited for 120 s using a white-light source, and afterglow signals were collected in bioluminescence mode using IVIS with a 10 s exposure time.

Subcutaneous tumor imaging

TRZ-1/BPO NPs (100 μL, 5 mg/mL) were injected into the tail vein of mice and delivered for 4 h. A white-light source was used to excite mice at the subcutaneous tumor, and afterglow signals were collected in bioluminescence mode with an exposure time of 10 s using IVIS.

AS plaque imaging

TRZ-1/BPO NPs (100 μL, 5 mg/mL) were injected into the tail vein of a mouse model of AS and delivered for 4 h. The necks of defatted mice were stimulated with a white-light source for 120 s, and the afterglow signals were collected by applying IVIS in bioluminescence mode with an exposure time of 10 s. The afterglow signals were collected by applying IVIS in bioluminescence mode.

Liver cancer imaging

TRZ-1/BPO NPs (100 μL, 5 mg/mL) were injected into the tail vein of the liver cancer mouse model and delivered for 4 h. The abdominal cavities of the mice were dissected, and the location of the livers of the mice was irradiated using a white-light source, and the afterglow signals were collected in bioluminescence mode with an exposure time of 10 s using IVIS for imaging or surgical treatment experiments.

Cecum imaging

O-TRZ-1/BPO-gel (5 mg) was placed in a 10 mL vial, and 200 μL of ultrapure water was added and sonicated until the O-TRZ-1/BPO-gel absorbed water and swelled up and dispersed uniformly in the aqueous system. The drug was administered by gavage, and the lower abdomen of mice was excited with a white-light source, and the afterglow signal was collected in bioluminescence mode with an exposure time of 10 s using IVIS for imaging.

In vivo phosphorescence imaging (excitation–concentration mode)

TRZ-1/BPO NPs (50 μL, 5 mg/mL) were excited by a white-light source (0.2 W/cm²) for 120 s and injected subcutaneously into the right dorsal side of mice. The afterglow signals were collected by using IVIS in bioluminescence mode with an exposure time of 10 s.

Statistical analysis

Quantitative data were expressed as mean ± standard deviation (s.d.). Statistical comparisons were performed using analysis of variance and two independent samples t test, p values < 0.05 were considered statistically significant. The “n” means the number of tested mice when we indicate how many data points were considered.