A novel organic room-temperature phosphorescent probe targeting fibroblast activation protein (FAP) and applications thereof

Abstract

This invention discloses a novel organic room-temperature phosphorescent probe targeting fibroblast activating protein (FAP) and its applications, belonging to the field of molecular imaging. This invention obtains an organic room-temperature phosphorescent probe targeting FAP by coupling a target FAP inhibitor with a polymer shell encapsulating a novel near-infrared organic room-temperature phosphorescent material, TPE-4BT. This probe can be used for liver cancer imaging or for preparing reagents or drugs for liver cancer imaging. The organic room-temperature phosphorescent probe of this invention can specifically bind to target fibroblast cells, and can even be localized to the cell nucleus. In vivo observation shows that the probe is significantly enriched at the tumor site without background interference. The organic room-temperature phosphorescent probe of this invention can provide accurate preoperative localization and precise intraoperative delineation of tumor lesion boundaries, providing multi-dimensional information for precision tumor diagnosis and treatment, helping to solve clinical visualization problems in liver cancer, and advancing translational applications in oncology.

Description

Technical Field

[0001] This invention belongs to the field of molecular imaging and relates to a novel organic room temperature phosphorescent probe that targets fibroblast activation protein (FAP) and its construction. This invention also relates to the application of this probe in liver cancer imaging. Background Technology

[0002] Liver cancer is the third leading cause of cancer-related deaths worldwide and ranks sixth among the most common diagnoses. More than 70% of cases are discovered at an advanced stage. Although liver resection is a key treatment option for liver cancer, the 5-year recurrence rate can be as high as 40% to 60%. Therefore, developing advanced visualization technologies and applying them to real-time surgical navigation has significant clinical implications.

[0003] Molecular imaging visualization technology holds great potential for precise navigation during liver resection. In particular, optical imaging strategies can enhance the contrast between tumors and normal tissues by utilizing optical probes activated by tumor-specific signals. Since 2008, indocyanine green (ICG), represented by near-infrared (NIR) fluorescent imaging probes, has been applied to liver resection. Currently, the application of ICG in liver resection faces two major limitations. First, ICG fluorescence is easily quenched, resulting in a limited signal-to-noise ratio (SNR). Second, ICG fluorescence can also be observed in benign lesions, leading to a false positive rate of 40-50%. To address these limitations, there is increasing interest in the design and development of various types of probes. To date, the development of these probes has focused on the field of fluorescence. However, achieving accurate and highly specific imaging remains challenging due to the inherent limitations of fluorescence, such as small Stokes shifts and short emission lifetimes. These factors, coupled with interference from background autofluorescence, degrade image quality. Organic room-temperature phosphorescence (RTP), with its unique three-state emission mechanism, large Stokes shifts, and long excited-state lifetimes, has attracted considerable attention as an advanced material for bioimaging. The time-resolved properties of RTP materials can effectively eliminate background interference, distinguishing them from fluorescent materials and enabling highly specific and sensitive bioimaging without continuous external stimulation. Developing target-specific RTP materials is crucial for achieving precise liver resection.

[0004] Fibroblast activating protein (FAP) is a specific marker of cancer-associated fibroblasts (CAFs) in the tumor microenvironment, playing a role in stromal remodeling and fibrosis formation. Studies have shown that FAP is highly expressed in hepatocellular carcinoma (HCC) and serves as an independent adverse prognostic factor. Clinical studies have confirmed that targeting FAP can enhance the molecular imaging diagnosis of HCC, unaffected by cirrhosis. Therefore, phosphorescent probes targeting FAP hold promise as a valuable adjunct to navigation-guided surgery for HCC. Summary of the Invention

[0005] The purpose of this invention is to provide a novel organic room-temperature phosphorescent probe targeting FAP, its preparation and application. By imaging the optical signals provided by the probe's specific binding with tumor markers, precise localization of liver cancer and differentiation of tumor boundaries can be achieved, enabling precise navigation for liver cancer surgery.

[0006] The objective of this invention is achieved through the following technical solution:

[0007] A novel organic room-temperature phosphorescent probe targeting fibroblast activation protein (FAP) is a nanoparticle encapsulated or loaded with an organic room-temperature phosphorescent material and surface-linked fibroblast activation protein inhibitor (FAPI). The organic room-temperature phosphorescent material is preferably TPE-4BT with the structure shown below. The preparation of TPE-4BT can be found in the literature Gu Juqing, Li Qianqian, Li Zhen, et al. Organic Materials with Ultrabright Phosphorescence at Room Temperature under Physiological Conditions for Bioimaging. Angew Chem Int Ed Engl, 2024, e202415637.

[0008]

[0009] The preferred fibroblast activating protein inhibitor is FAPI-04.

[0010] Preferably, the novel organic room-temperature phosphorescent probe targeting FAP is a nanoparticle containing a polymer shell encapsulating TPE-4BT and a surface-linked fibroblast activation protein inhibitor (FAPI). Further, the hydrophilic-hydrophobic interactions between the amphiphilic polymers DSPE-PEG2000 and DSPE-PEG2000-COOH in the aqueous phase constitute the polymer shell encapsulating TPE-4BT; the fibroblast activation protein inhibitor can be linked to the polymer shell surface via a reaction between amino and carboxyl groups.

[0011] The above-mentioned method for preparing novel organic room-temperature phosphorescent probes targeting FAP preferably includes the following steps (see schematic diagram). Figure 1 As shown in A):

[0012] (1) TPE-4BT, DSPE-PEG2000 and DSPE-PEG2000-COOH were added to an organic solvent and mixed to obtain solution 1. Solution 1 was added to water and ultrasonically treated. After removing the organic solvent, TPE-4BTCOOH nanoparticles encapsulating TPE-4BT were obtained.

[0013] (2) The TPE-4BT COOH obtained in step (1) and NH2-FAPI-04 are reacted by dehydration condensation to obtain the novel organic room temperature phosphorescent probe TPE-4BT FAPI targeting FAP.

[0014] In step (1), the mass ratio of TPE-4BT, DSPE-PEG2000, and DSPE-PEG2000-COOH is preferably 1:5-15:0.5-1.5, and more preferably 1:10:1. The organic solvent includes THF; the volume ratio of solution 1 to water is preferably 1:3-5, and more preferably 1:3.

[0015] In step (2), the dehydration condensation reaction preferably uses EDC / NHS as the condensing agent.

[0016] The application of the aforementioned novel organic room-temperature phosphorescent probe targeting FAP in liver cancer imaging.

[0017] The above-mentioned novel organic room-temperature phosphorescent probes targeting FAP are used in the preparation of reagents or drugs for liver cancer imaging.

[0018] The present invention has the following advantages and beneficial effects:

[0019] 1. This invention is the first to couple a novel near-infrared organic room-temperature phosphorescent material with a fibroblast activating protein inhibitor (FAPI), endowing the material with targeted intelligent properties and significantly improving its tumor targeting ability;

[0020] 2. The probe provided by this invention can specifically bind to target cells fibroblasts, and can even be located in the cell nucleus. In vivo observation shows that the probe can be significantly enriched at the tumor site, enabling precise localization diagnosis of subcutaneous liver cancer models. Furthermore, the unique time-resolved properties of phosphorescence can almost completely eliminate the interference of autofluorescence background in biological samples, achieving high-quality imaging.

[0021] 3. The probe provided by this invention can provide accurate preoperative localization and precise intraoperative delineation of cancerous lesion boundaries, providing multi-dimensional information for precision diagnosis and treatment of tumors, helping to solve clinical problems of liver cancer visualization, and realizing real-time navigation of in situ liver cancer resection surgery. Attached Figure Description

[0022] Figure 1The fabrication process and characterization results of TPE-4BT FAPI, a novel organic room-temperature phosphorescent probe targeting FAP, are presented below. A shows the fabrication process flowchart; B shows the transmission electron microscopy (TEM) images and dimensions of TPE-4BT COOH and TPE-4BT FAPI; C shows the Zeta potentials of TPE-4BTCOOH and TPE-4BT FAPI; D shows the imaging and parameters of TPE-4BT COOH and TPE-4BT FAPI; E shows the photoluminescence spectra of TPE-4BT COOH and TPE-4BT FAPI in PBS; F shows the phosphorescence spectra of TPE-4BT COOH and TPE-4BT FAPI; and G and H show the phosphorescence decay curves of TPE-4BT COOH and TPE-4BT FAPI.

[0023] Figure 2 The images show the in vivo imaging results of TPE-4BT FAPI, an organic room-temperature phosphorescent probe targeting FAP, in Example 2. A is the imaging flowchart for liver cancer-bearing mice; B and D show the subcutaneous tumor imaging and quantitative analysis of liver cancer in mice at different time points; C shows the signal-to-noise ratio for each group; and E shows the quantitative analysis of major organs and tumors in vitro.

[0024] Figure 3 The comparison results of in vivo imaging between the organic room temperature phosphorescent probe TPE-4BT FAPI targeting FAP in Example 3 and the probe used in clinical applications are shown.

[0025] Figure 4 The results of tumor resection guided by the organic room-temperature phosphorescent probe TPE-4BT FAPI targeting FAP in Example 4 are shown. A represents the surgical resection procedure; B represents the quantitative analysis of phosphorescence intensity in the liver and tumor during surgery; and C represents the pathological results of the resected and remaining liver tissue. Detailed Implementation

[0026] The following embodiments further illustrate the content of the present invention, but should not be construed as limiting the present invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the present invention.

[0027] Example 1: Preparation of TPE-4BT FAPI, an organic room-temperature phosphorescent probe targeting FAP

[0028] 1. Probe preparation and characterization

[0029] (1) TPE-4BT (1 mg), DSPE-PEG2000 (10 mg), and DSPE-PEG2000-COOH (1 mg) were resuspended in THF (1 mL). The resulting solution was rapidly added dropwise to deionized water (3 mL) and then sonicated for 1 minute using a probe sonicator equipped with a microtip (Scientz-IID). The mixture was then left to stand overnight at 50 °C to remove the organic solvent, yielding TPE-4BT COOH nanoparticles encapsulated with TPE-4BT. The aqueous solution was concentrated to 1 mg / mL by sieving through a PES syringe-driven filter (0.22 μm).

[0030] (2) Mix 0.5 mL of 1 mg / mL TPE-4BT COOH obtained in step (1), 1.303 mg N-hydroxysuccinimide (NHS), and 0.383 mg 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC), and stir slowly at room temperature for 30 min. Add 0.54 mg NH2-FAPI-04, stir at room temperature for 12 h, centrifuge, wash three times with PBS, and resuspend to obtain the organic room temperature phosphorescent probe TPE-4BT FAPI.

[0031] (3) The prepared TPE-4BT COOH and TPE-4BT FAPI were characterized as follows.

[0032] Photoluminescence and excitation spectra were recorded using a Hitachi F-4700 fluorescence spectrophotometer or an FLS980 spectrometer. Photoluminescence lifetime and quantum yield were determined using the FLS980 spectrometer. Dynamic light scattering and zeta potential measurements were performed using a Malvern Nano-ZS90 Zetasizer. Transmission electron microscopy (TEM) images were acquired using a JEM-2100Plus TEM. In vitro and in vivo phosphorescence imaging was performed using an IVIS spectral imaging system (PerkinElmer Inc.; charge-coupled device lens cooling temperature -85°C).

[0033] 2. Results and Analysis

[0034] The successful bonding of NH2-FAPI-O4 was verified by transmission electron microscopy (TEM). The diameter of TPE-4BT FAPI was slightly larger than that of TPE-4BT COOH. Dynamic light scattering (DLS) analysis showed that after FAPI bonding, the average diameter of TPE-4BT COOH increased from 63.31 nm to 66.57 nm. Figure 1 B). Furthermore, the zeta potentials of TPE-4BT COOH and TPE-4BT FAPI nanoparticles are -29 and -2.19 mV, respectively. Figure 1C). The higher charge of TPE-4BT FAPI verifies the conjugation of FAPI. Meanwhile, the phosphorescence intensity of TPE-4BT FAPI is essentially equivalent to that of TPE-4BT COOH, indicating that FAPI coupling does not affect its phosphorescence performance. The photoluminescence and phosphorescence spectra and lifetimes after FAPI coupling show no significant changes. Figure 1 D-1H).

[0035] Example 2: In vivo imaging of TPE-4BT FAPI, an organic room-temperature phosphorescent probe targeting FAP.

[0036] 1. Method

[0037] 4-week-old male nude mice, weighing 20-30g, were injected subcutaneously into the right axilla with 100μL of a solution containing 5×10⁻⁶ g of [unspecified substance]. 6 Suspension of Huh-7 cells, a personal liver malignant tumor cell line. Nude mice continued to be housed under specific pathogen-free conditions, maintaining a stable rearing environment. Tumor formation was observed every 2 days after inoculation. After tumor formation, the tumor diameter was measured using calipers to calculate the tumor volume. Mice bearing liver cancer were injected intravenously via tail vein with 100 μL of TPE-4BT COOH or TPE-4BT FAPI at a concentration of 1000 μg / mL. Phosphorescent biological images were obtained at 4, 6, 8, 10, 24, and 48 hours after injection. Phosphorescent images were immediately acquired using a live imaging system (filter on, exposure time 10 seconds) after 20 seconds of exposure to a conventional flashlight.

[0038] 2. Results and Analysis

[0039] Figure 2 A illustrates the imaging process. No phosphorescence signal was detected in the TPE-4BT COOH group before 10 hours due to its increased permeability and retention (EPR). A weak phosphorescence signal was detected in the tumor at 24 hours. Compared to the TPE-4BT COOH group, a weak signal was detected in the TPE-4BT FAPI group at 4 hours. The probe gradually accumulated within the tumor over time, peaking at 48 hours. Simultaneously, probe accumulation within the tumor was more pronounced in the TPE-4BT FAPI group at each time point. Figure 2 B Figure 2 D). The average signal-to-noise ratio of probes coupled with the target fragment FAPI is 16 ( Figure 2 C Figure 2 E). In vivo flashlight-induced imaging further validated the advantages of phosphorescent targeted imaging. In vitro phosphorescent imaging 48 hours after injection showed that tumors induced by TPE-4BT FAPI had stronger signals than those induced by TPE-4BT COOH. The quantitative phosphorescence intensity of TPE-4BT FAPI in tumors was 8.53 × 10⁻⁶. 5 ps -1 cm -2sr -1 .

[0040] Example 3: Comparison of TPE-4BT FAPI, an organic room-temperature phosphorescent probe targeting FAP, with clinically applied probe imaging.

[0041] 1. Method

[0042] Imaging comparisons were performed between the novel organic room-temperature phosphorescent probe targeting FAP of this invention and the existing clinical probe ICG, as well as the ICG coupled with FAPI-04. The structure of FAPI-ICG is as follows:

[0043]

[0044] The in vivo imaging method is described in Example 2.

[0045] 2. Results and Analysis

[0046] like Figure 3 As shown, the tumor-targeting imaging capabilities of ICG and FAPI-ICG remain limited, and the fluorescence intensity of the tumor is indistinguishable from the background, which is not conducive to observing the tumor location. In addition, the coupling of ICG and FAPI will adversely affect the inherent fluorescence properties of ICG. The organic room temperature phosphorescent probe TPE-4BT FAPI of this invention, which targets FAPI, shows a clear contrast between the tumor and the background in phosphorescent mode and has very good imaging quality.

[0047] Example 4: TPE-4BT, an organic room-temperature phosphorescent probe targeting FAPI, guides tumor resection.

[0048] 1. Method

[0049] Male nude mice, weighing 20-30g, were used at 4 weeks gestation. After anesthesia, the mice were fixed to the operating table. The abdominal surgical area was disinfected, and the skin was incised along the midline of the abdomen. The abdominal fascia was opened to enter the abdominal cavity. The costal arches were gently pressed to expel part of the left lobe of the liver. An insulin needle was then inserted at approximately a 20° angle to the liver plane along the long axis of the liver lobe, advancing parallel to the liver plane for about 1cm. 20μL of Huh-7 cell suspension was slowly injected, held for 3 seconds, and then withdrawn. Simultaneously, a sterile cotton swab was used to gently press until no active bleeding occurred. The liver was carefully returned to the abdominal cavity, and the abdomen was closed layer by layer, with wound disinfection performed. High-frequency ultrasound was used to assess the successful establishment of the orthotopic hepatocellular carcinoma model approximately 2 weeks later.

[0050] After the in situ liver cancer model is successfully constructed, the probe TPE-4BT FAPI of this invention is used to perform phosphorescence imaging to determine the tumor margin. Under the guidance of the phosphorescently displayed tumor margin, the liver cancer tissue is removed until the phosphorescence imaging can no longer detect the tumor tissue signal. After the removed tumor tissue and the remaining liver are fixed, pathological tissue examination is performed to determine whether it is a Huh-7 tumor and normal liver.

[0051] 2. Results and Analysis

[0052] Figure 4 A demonstrated the surgical procedure: after visually observing the tumor location, phosphorescence imaging was performed. The tumor was then removed under navigation based on the tumor boundaries determined by phosphorescence imaging, until no phosphorescence signal remained in the remaining liver. Statistical analysis showed that the phosphorescence signal intensity in the tumor was significantly higher than that in normal liver tissue. Figure 4 B), the pathological results confirmed that the excised tissue was indeed a tumor, and the resection margins were negative, confirming complete tumor resection. Figure 4 C).

[0053] The above embodiments are only used to help illustrate the present invention. The implementation of the present invention is not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention should be considered as equivalent substitutions and are included within the protection scope of the present invention.

Claims

1. An organic room-temperature phosphorescent probe targeting fibroblast activation proteins, characterized in that: The probe is a polymer shell encapsulating TPE-4BT and nanoparticles with fibroblast activation protein inhibitors linked to the surface; the polymer shell is composed of hydrophilic and hydrophobic interactions between DSPE-PEG2000 and DSPE-PEG2000-COOH in an aqueous phase. The structure of the TPE-4BT is shown below: ； The fibroblast activation protein inhibitor mentioned is FAPI-04; The fibroblast activation protein inhibitor is attached to the surface of the polymer shell through a reaction between amino and carboxyl groups.

2. The method for preparing the organic room-temperature phosphorescent probe targeting fibroblast activation proteins according to claim 1, characterized in that: Includes the following steps (1) TPE-4BT, DSPE-PEG2000 and DSPE-PEG2000-COOH were added to an organic solvent and mixed to obtain solution 1. Solution 1 was added to water and ultrasonically treated. After removing the organic solvent, TPE-4BTCOOH nanoparticles encapsulating TPE-4BT were obtained. (2) The organic room temperature phosphorescent probe targeting fibroblast activation protein obtained in step (1) is obtained by dehydration condensation reaction of TPE-4BT COOH and NH2-FAPI-04.

3. The method for preparing the organic room-temperature phosphorescent probe targeting fibroblast activation proteins according to claim 2, characterized in that: In step (1), the mass ratio of TPE-4BT, DSPE-PEG2000 and DSPE-PEG2000-COOH is 1:5-15:0.5-1.

5.

4. The method for preparing the organic room-temperature phosphorescent probe targeting fibroblast activation proteins according to claim 2, characterized in that: In step (1), the organic solvent includes THF; the volume ratio of solution 1 to water is 1:3-5.

5. The method for preparing the organic room-temperature phosphorescent probe targeting fibroblast activation proteins according to claim 2, characterized in that: In step (2), the dehydration condensation reaction uses EDC / NHS as the condensing agent.

6. The use of the organic room-temperature phosphorescent probe targeting fibroblast activation proteins as described in claim 1 in the preparation of reagents for liver cancer imaging.

7. The use of the organic room-temperature phosphorescent probe targeting fibroblast activation proteins as described in claim 1 in the preparation of a drug for liver cancer imaging.

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