Use of a near-infrared fluorescent probe WL-808 in preparation of a drug for targeted treatment of heterotopic ossification

Abstract

The application discloses application of a near-infrared fluorescent probe WL-808 in preparation of a drug for targeted treatment of heterotopic ossification, wherein the near-infrared fluorescent probe WL-808 can kill chondrocytes loaded with the probe under irradiation of 808 nm excitation light; the energy of photons is transmitted to oxygen through the probe molecules, thereby generating a substance active oxygen (ROS) which is toxic to cells; a large amount of ROS induces chondrocyte apoptosis and extracellular matrix degradation at a heterotopic ossification (HO) lesion site, thereby inhibiting generation of HO chondral and bony lesions, and finally realizing targeted photodynamic treatment of HO.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of a near-infrared fluorescent probe WL-808 in the preparation of drugs for targeted treatment of heterotopic ossification. Background Technology

[0002] Heterotopic ossification (HO) refers to the abnormal formation of bone and cartilage matrix in soft tissues. It is a serious sequela of traumatic injuries such as fractures or dislocations, arthroplasty, brain and spinal cord injuries, and severe burns. If not diagnosed and intervened in a timely manner, complications such as chronic pain, impaired prosthesis fitting, decreased limb mobility, and vascular, nerve, and skin damage are likely to occur, significantly reducing the patient's quality of life. Currently, surgical resection is the only treatment for symptomatic HO. However, surgical resection has drawbacks such as insufficient restoration of mobility and a high recurrence rate. Other non-specific preventative measures, including anti-inflammatory drug therapy and radiation therapy, may lead to other serious complications in patients without HO.

[0003] Therefore, there is an urgent need to develop a drug for targeted treatment of heterotopic ossification. Summary of the Invention

[0004] The purpose of this invention is to provide an application of the near-infrared fluorescent probe WL-808 in the preparation of drugs for targeted treatment of heterotopic ossification. This invention discovers that WL-808 can target cartilage lesions of HO, and under near-infrared light excitation, it generates a large number of reactive oxygen species (ROS) to induce apoptosis of chondrocytes and degradation of extracellular matrix at the HO lesion site, inhibiting the formation of cartilage and bone lesions in HO, and ultimately achieving targeted photodynamic therapy for HO.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] In a first aspect of the present invention, an application of a near-infrared fluorescent probe WL-808 in the preparation of a drug for targeted treatment of heterotopic ossification is provided, wherein the structural formula of the near-infrared fluorescent probe WL-808 is as follows:

[0007]

[0008] Furthermore, the near-infrared fluorescent probe WL-808 induces chondrocyte death at the site of ectopic ossified cartilage lesions by triggering the generation of ROS during photodynamic therapy.

[0009] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0010] 1. The application of the near-infrared fluorescent probe WL-808 provided by this invention in the preparation of drugs for targeted treatment of heterotopic ossification. This invention discovers a new use for the near-infrared fluorescent probe WL-808: when irradiated with 808nm excitation light, the near-infrared fluorescent probe WL-808 can kill chondrocytes loaded with this probe. The energy of the photons is transferred to oxygen through the probe molecules, producing ROS, a substance that is toxic to cells. A large amount of ROS induces apoptosis of chondrocytes and degradation of the extracellular matrix at the HO lesion site, inhibits the formation of HO cartilage and bone lesions, and ultimately achieves targeted photodynamic therapy for HO.

[0011] 2. This invention has found that WL-808 has few toxic side effects. It can only induce the production of cell-killing ROS after being irradiated with a laser of a specific wavelength (808nm), thereby enabling more accurate control of the treatment area. Attached Figure Description

[0012] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0013] Figure 1 Results showing the localization of WL-808 in chondrocyte mitochondria;

[0014] Figure 2 This is the result of WL-808 inducing ROS generation in chondrocytes after irradiation with 808nm excitation light;

[0015] Figure 3 This is the result of WL-808 inducing apoptosis in chondrocytes after irradiation with 808nm excitation light;

[0016] Figure 4 This is the result of WL-808 inducing ROS generation at the HO lesion site in mice after irradiation with 808nm excitation light;

[0017] Figure 5 This is the result of WL-808 inducing apoptosis of chondrocytes at the HO lesion site in mice after irradiation with 808nm excitation light;

[0018] Figure 6 The results show that WL-808 combined with 808nm excitation light irradiation inhibited the formation of ectopic cartilage lesions in mice 4 weeks after surgery (black dashed circle: ectopic cartilage formation area);

[0019] Figure 7The results show the inhibition of ectopic bone lesion formation in mice 10 weeks post-surgery by WL-808 combined with 808nm excitation light. (A) Micro-CT reconstructed image of the left hind limb of a mouse (red dashed circle: ectopic bone formation area). (B) Masson staining image of the left hind limb of a mouse (red dashed circle: ectopic bone formation area).

[0020] Figure 8 The results of H&E staining toxicity testing of organs after WL-808 combined with laser irradiation treatment. Detailed Implementation

[0021] The present invention will be described in detail below with reference to specific embodiments and examples, thereby making the advantages and various effects of the present invention more clearly apparent. Those skilled in the art should understand that these specific embodiments and examples are for illustrative purposes only and are not intended to limit the present invention.

[0022] Throughout this specification, unless otherwise specified, the terminology used herein should be understood as having the meaning commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In the event of any conflict, this specification shall prevail.

[0023] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be obtained by purchasing them from the market or by existing methods.

[0024] To address the technical problem of this invention, the overall concept of this invention is as follows:

[0025] WL-808 is a near-infrared fluorescent probe that can target HO cartilage lesions, enabling early diagnosis of heterotopic ossification. The parent fluorescent dye IR-808 of WL-808 has been shown to have good photodynamic effects (see patent CN116462736A).

[0026] The inventors of this application accidentally discovered through experiments that the near-infrared fluorescent probe WL-808 can locate mitochondria. Figure 1 It achieves the effect of a photosensitizer. Subsequent experiments revealed that WL-808 combined with laser irradiation significantly induced chondrocyte apoptosis, demonstrating that the photodynamic effect of WL-808 can effectively induce chondrocyte apoptosis. Figure 3 WL-808 exhibits high specificity and ideal photodynamic effects on HO cartilage lesions. Figure 4 WL-808 photodynamic therapy can effectively induce apoptosis of chondrocytes at the HO site in vivo. Figure 5 WL-808 photodynamic therapy can effectively inhibit ectopic cartilage formation in vivo. Figure 6Due to impaired cartilage template formation, the photodynamic effect of WL-808 can effectively inhibit ultimate ectopic bone formation in vivo. Figure 7 ).

[0027] The above results indicate a novel application for the near-infrared fluorescent probe WL-808: its potential to be used in the preparation of drugs for targeted therapy of heterotopic ossification.

[0028] The following will describe in detail the application of a near-infrared fluorescent probe WL-808 of this application in the preparation of a drug for targeted treatment of heterotopic ossification, with reference to embodiments and experimental data.

[0029] Example 1: The near-infrared fluorescent probe WL-808 can locate mitochondria and achieve the effect of a photosensitizer.

[0030] 1. WL-808 chondrocyte mitochondrial localization assay

[0031] Mouse chondrocytes were seeded at a density of 10,000 cells / well in 20 mm confocal laser culture dishes. After co-incubating with 20 μM WL-808 for 2 h, the supernatant was removed, and the cells were washed three times with PBS. Cell nuclei were then stained with Hoechst 33342 for 10 min and mitochondria with Mito-Tracker Green for 30 min, respectively. The mitochondrial localization of WL-808 cells was observed using a confocal laser scanning microscope (CLSM).

[0032] The results are as follows Figure 1 As shown, WL-808 exhibits bright near-infrared fluorescence within the mitochondria of chondrocytes, indicating that WL-808 has good mitochondrial localization ability in chondrocytes.

[0033] 2. Assay on the induction of ROS generation in chondrocytes by WL-808 after laser irradiation

[0034] Mouse chondrocytes were seeded into 24-well plates at a density of 10,000 cells / well. After incubation with 20 μM WL-808 for 2 h, the supernatant was removed, and the cells were washed three times with PBS. ROS were then labeled with DCFH-DA (10 μM DCFH-DA) for 20 min. Subsequently, the DCFH-DA was removed, and the cells were irradiated with an 808 nm laser for 3 min at a power of 2 W / cm². 2 During irradiation, the orifice plate was placed on ice to eliminate the photothermal effect. ROS generation was detected using a CLSM after irradiation.

[0035] The results are as follows Figure 2As shown, chondrocytes treated with WL-808 in combination with laser irradiation exhibited a significant DCFH-DA green fluorescence signal, indicating that WL-808 has a good ability to induce ROS generation in chondrocytes.

[0036] Example 2: Experiment on chondrocyte apoptosis induced by WL-808 after laser irradiation

[0037] Chondrocytes were seeded at a density of 10,000 cells / well in 6-well plates. After incubation with 20 μM WL-808 for 2 h, the cells were washed three times with PBS after removing the supernatant, and then irradiated with an 808 nm laser for 3 min at a power of 2 W / cm². 2 During irradiation, the well plates were placed on ice to eliminate the photothermal effect. After irradiation, the complete culture medium was replaced and the cells were cultured for another 24 hours. Subsequently, chondrocytes were digested with trypsin, and the pellet was collected by centrifugation. Apoptosis analysis was performed using the Annexin V / Binding Buffer Apoptosis Detection Kit, and the proportion of apoptotic cells was detected by flow cytometry. Results are as follows: Figure 3 As shown, chondrocytes treated with WL-808 in combination with laser irradiation showed significant apoptosis, indicating that the photodynamic effect of WL-808 can effectively induce chondrocyte apoptosis.

[0038] Example 3: In vivo experiment on ROS generation induced at HO lesion sites by WL-808 after laser irradiation.

[0039] All animal experiments were conducted in accordance with the National Institutes of Health (NIH) Guidelines for the Care and Use of Laboratory Animals and approved by the Laboratory Animal Welfare and Ethics Committee of Zhongnan Hospital, Wuhan University. Twelve 8-week-old male wild-type C57BL / 6 mice underwent midpoint transection of the left hind limb Achilles tendon to induce homeostasis (HO), and the wound was sutured and disinfected. One week later, the twelve mice were randomly divided into four groups: Control group, Laser group, IR-808+Laser group, and WL-808+Laser group, with three mice in each group. The WL-808+Laser group and the IR-808+Laser group received 100 μL of WL-808 (5 mg / kg) or IR-808 (2.5 mg / kg) dissolved in fat emulsion via tail vein injection, respectively. The Control and Laser groups received the same dose of fat emulsion in the same manner. Six hours after drug administration, the Achilles tendon incision site was irradiated with an 808nm laser for 2 minutes (1W / cm). 2After a 5-minute interval, a second round of irradiation was performed in the same manner. The drug was administered twice weekly for a total of 3 weeks. Immediately after the final irradiation, the mice were sacrificed, and frozen sections of the surgical site were collected. The generation of ROS in vivo was observed using a DHE assay kit.

[0040] The results are as follows Figure 4 As shown, the surgical sites of mice in the Control group, Laser group, and IR-808+Laser group showed almost no DHE (ROS) red fluorescence, while the WL-808+Laser group showed obvious DHE red fluorescence at the surgical sites, indicating that WL-808 has high specificity and ideal targeted photodynamic effect in inducing ROS generation in HO cartilage lesions.

[0041] Example 4: In vivo experiment on the induction of chondrocyte apoptosis at HO lesion sites by WL-808 after laser irradiation.

[0042] Twelve 8-week-old male wild-type C57BL / 6 mice underwent midpoint transection of the left hind limb Achilles tendon to induce homeostasis (HO). The wound was sutured and disinfected. One week later, the twelve mice were randomly divided into four groups: Control group, Laser group, IR-808+Laser group, and WL-808+Laser group, with three mice in each group. The WL-808+Laser group and the IR-808+Laser group received 100 μL of WL-808 (5 mg / kg) or IR-808 (2.5 mg / kg) dissolved in fat emulsion via tail vein injection, respectively. The Control and Laser groups received the same dose of fat emulsion in the same manner. Six hours after administration, the Achilles tendon incision site was irradiated with an 808 nm laser for 2 minutes (1 W / cm²). 2 After a 5-minute interval, a second round of irradiation was performed in the same manner. The drug was administered twice weekly for a total of 3 weeks. Three days after the final irradiation, the mice were sacrificed, and tissue samples from the surgical site were collected for routine pathological sections. Cell apoptosis was detected using the TUNEL assay.

[0043] The results are as follows Figure 5 As shown, almost no green fluorescence of apoptotic cells was observed at the surgical site in mice in the Control group, Laser group, and IR-808+Laser group, while obvious green fluorescence of apoptotic cells was observed at the surgical site in the WL-808+Laser group, indicating that the photodynamic effect of WL-808 can effectively induce apoptosis of chondrocytes at the HO site in vivo.

[0044] Example 5: In vivo experiment on the inhibition of ectopic cartilage lesion formation by WL-808 through photodynamic effect.

[0045] Twelve 8-week-old male wild-type C57BL / 6 mice underwent midpoint transection of the left hind limb Achilles tendon to induce homeostasis (HO). The wound was sutured and disinfected. One week later, the twelve mice were randomly divided into four groups: Control group, Laser group, IR-808+Laser group, and WL-808+Laser group, with three mice in each group. The WL-808+Laser group and the IR-808+Laser group received 100 μL of WL-808 (5 mg / kg) or IR-808 (2.5 mg / kg) dissolved in fat emulsion via tail vein injection, respectively. The Control and Laser groups received the same dose of fat emulsion in the same manner. Six hours after administration, the Achilles tendon incision site was irradiated with an 808 nm laser for 2 minutes (1 W / cm²). 2 After a 5-minute interval, a second round of irradiation was performed in the same manner. The medication was administered twice weekly for a total of 3 weeks. Three days after the final irradiation, the mice were sacrificed, and tissue from the left hind limb was collected for routine pathological sectioning. Safranin-Fix-Green staining was used to observe the formation of ectopic cartilage.

[0046] The results are as follows Figure 6 As shown, there was no significant difference in the area of ​​cartilage stained orange-red in the left hind limb of mice in the Control group, Laser group, and IR-808+Laser group. Compared with the Control group, Laser group, and IR-808+Laser group, the area of ​​cartilage stained orange-red in the left hind limb of mice in the WL-808+Laser group was significantly smaller, indicating that the photodynamic effect of WL-808 can effectively inhibit ectopic cartilage formation in vivo.

[0047] Example 6: In vivo experiment on the inhibition of ectopic bone lesion formation by WL-808 through photodynamic effect.

[0048] Twelve 8-week-old male wild-type C57BL / 6 mice underwent midpoint transection of the left hind limb Achilles tendon to induce homeostasis (HO). The wound was sutured and disinfected. One week later, the twelve mice were randomly divided into four groups: Control group, Laser group, IR-808+Laser group, and WL-808+Laser group, with three mice in each group. The WL-808+Laser group and the IR-808+Laser group received 100 μL of WL-808 (5 mg / kg) or IR-808 (2.5 mg / kg) dissolved in fat emulsion via tail vein injection, respectively. The Control and Laser groups received the same dose of fat emulsion in the same manner. Six hours after administration, the Achilles tendon incision site was irradiated with an 808 nm laser for 2 minutes (1 W / cm²).2 After a 5-minute interval, a second round of irradiation was performed in the same manner. The medication was administered twice weekly for a total of 3 weeks. Six weeks after the final irradiation, the mice were sacrificed, and tissue from the left hind limb was harvested for micro-CT scanning. Subsequent routine pathological sections were prepared, and Masson staining was used to observe the formation of ectopic bone.

[0049] The results are as follows Figure 7 As shown, there were no significant differences in the volume and area of ​​ectopic bone at the surgical site in the left hind limb of mice in the Control group, Laser group, and IR-808+Laser group. Compared with the Control group, Laser group, and IR-808+Laser group, the volume and area of ​​ectopic bone at the surgical site in the left hind limb of mice in the WL-808+Laser group were smaller, indicating that the photodynamic effect of WL-808 can effectively inhibit the final ectopic bone formation in vivo.

[0050] Example 7: In vivo toxicity experiment of WL-808 combined with laser treatment for HO

[0051] After the mice in the Control group and WL-808+Laser group of Example 6 were sacrificed, their major organs (heart, liver, spleen, lung, and kidney) were harvested and fixed with 4% paraformaldehyde (PFA) solution for 30 minutes. The tissues were then embedded in paraffin and cut into 5 μm thin sections, stained with H&E, and observed under a microscope.

[0052] The results are as follows Figure 8 As shown, compared with the organs of mice in the Control group, no significant damage was observed in the organs and tissues of mice in the WL-808+Laser group, indicating that WL-808 combined with laser treatment for HO has good biocompatibility.

[0053] Finally, it should be noted that the terms “comprising,” “including,” or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0054] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0055] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. The application of a near-infrared fluorescent probe WL-808 in the preparation of a drug for targeted treatment of heterotopic ossification, characterized in that, The structural formula of the near-infrared fluorescent probe WL-808 is as follows:

2. The application according to claim 1, characterized in that, The near-infrared fluorescent probe WL-808 induces the death of chondrocytes at the site of ectopic ossification by triggering the generation of reactive oxygen species during photodynamic therapy.

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