CN117159707A - Application of near infrared fluorescent probe WL-808 in preparation of medicine for targeted treatment of ectopic ossification - Google Patents
Application of near infrared fluorescent probe WL-808 in preparation of medicine for targeted treatment of ectopic ossification Download PDFInfo
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Abstract
The application discloses an application of a near infrared fluorescent probe WL-808 in preparing a medicament for targeted treatment of ectopic ossification, wherein the near infrared fluorescent probe WL-808 can kill chondrocytes loaded with the probe through 808nm excitation light irradiation, photon energy is transferred to oxygen through probe molecules to generate substance active oxygen (reactive oxygen species, ROS) which is toxic to cells, a large amount of ROS induce apoptosis of chondrocytes and extracellular matrix degradation of the damaged parts of the ectopic ossification (heterotopic ossification, HO), HO cartilage and bone lesion generation is inhibited, and finally targeted photodynamic treatment of HO is realized.
Description
Technical Field
The application relates to the technical field of biological medicines, in particular to application of a near infrared fluorescent probe WL-808 in preparation of a drug for targeted treatment of ectopic ossification.
Background
Ectopic ossification (heterotopic ossification, HO) refers to the process of abnormal formation of bone and cartilage matrix in soft tissues, which is a serious sequelae of injury diseases such as fracture or dislocation, arthroplasty, brain and spinal cord injury, and serious burns. If not diagnosed early and intervening in time, complications such as chronic pain, impaired prosthesis assembly, reduced limb mobility, vascular nerve and skin damage may occur, which may lead to a significant reduction in the quality of life of the patient. Surgical excision is currently the only treatment for symptomatic HO that has developed. However, surgical excision treatment has the disadvantages of insufficient recovery of mobility, high recurrence rate, and the like. Other non-specific preventative measures, including anti-inflammatory treatment and radiation treatment, may lead to other serious complications in patients who do not develop HO.
Therefore, there is an urgent need to develop a drug for targeted treatment of ectopic ossification.
Disclosure of Invention
The application aims to provide an application of a near infrared fluorescent probe WL-808 in preparing a medicament for targeted treatment of ectopic ossification, and the application discovers that the WL-808 can target HO cartilage damage, generate a large amount of active oxygen (reactive oxygen species, ROS) under the excitation of near infrared light to induce the apoptosis of cartilage cells and the degradation of extracellular matrixes at the HO damage position, inhibit the generation of HO cartilage and bone damage and finally realize the targeted photodynamic treatment of HO.
In order to achieve the above purpose, the present application adopts the following technical scheme:
in a first aspect of the application, there is provided the use of a near infrared fluorescent probe WL-808 for the preparation of a medicament for targeted treatment of ectopic ossification, said near infrared fluorescent probe WL-808 having the structural formula:
further, the near infrared fluorescent probe WL-808 induces chondrocyte death at ectopic ossified cartilage lesions by inducing ROS production during photodynamic therapy.
One or more technical solutions in the embodiments of the present application at least have the following technical effects or advantages:
1. the application provides application of a near infrared fluorescent probe WL-808 in preparing a medicament for targeted treatment of ectopic ossification, and finds new application of the near infrared fluorescent probe WL-808: the near infrared fluorescent probe WL-808 can kill the cartilage cells loaded with the probe through 808nm excitation light irradiation, photon energy is transferred to oxygen through probe molecules, and generates substance ROS which causes toxicity to cells, a large amount of ROS induce cartilage cell apoptosis and extracellular matrix degradation at the HO lesion part, inhibit HO cartilage and bone lesion generation, and finally realize HO targeted photodynamic therapy.
2. The application discovers that WL-808 has small toxic and side effects, and can induce the generation of ROS killing cells only after being irradiated by laser with specific wavelength (808 nm), thereby being capable of controlling the treatment area more accurately.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings required for the description of the embodiments will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present application, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a graph showing the localization of WL-808 to chondrocyte mitochondria;
FIG. 2 shows the results of WL-808 induced ROS production in chondrocytes after irradiation with 808nm excitation light;
FIG. 3 shows the results of WL-808 induced chondrocyte apoptosis after irradiation with 808nm excitation light;
FIG. 4 shows the results of the induction of ROS production by WL-808 at the HO lesion site in mice after irradiation with 808nm excitation light;
FIG. 5 shows the results of induction of chondrocyte apoptosis by WL-808 at HO lesion in mice after irradiation with 808nm excitation light;
FIG. 6 shows the results of inhibition of ectopic cartilage lesion formation in mice (black dashed circle: ectopic cartilage formation region) by WL-808 in combination with 808nm excitation light irradiation at 4 weeks after surgery;
FIG. 7 shows the results of inhibition of ectopic bone lesion formation in mice by WL-808 in combination with 808nm excitation light irradiation 10 weeks after surgery. (A) Mice left hind limb micro-CT reconstructed images (red dotted circles: ectopic bone forming areas). (B) Left hind limb Masson stained image of mice (red dotted circle: ectopic bone forming area).
FIG. 8 shows the results of H & E staining toxicity test of in vivo organs after treatment with WL-808 combined with laser irradiation.
Detailed Description
The advantages and various effects of the present application will be more clearly apparent from the following detailed description and examples. It will be understood by those skilled in the art that these specific embodiments and examples are intended to illustrate the application, not to limit the application.
Throughout the specification, unless specifically indicated otherwise, the terms used herein should be understood as meaning as commonly used in the art. Accordingly, unless defined otherwise, 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 application belongs. In case of conflict, the present specification will control.
Unless specifically indicated otherwise, the various raw materials, reagents, instruments, equipment, etc., used in the present application are commercially available or may be obtained by existing methods.
In order to solve the technical problem of the application, the general idea of the application is as follows:
WL-808 is used as a near infrared fluorescent probe capable of targeting HO cartilage lesions, and early diagnosis of ectopic ossification can be achieved. The parent fluorescent dye IR-808 of WL-808 has been shown to have a good photodynamic effect (see patent CN 116462736A).
The inventor of the application unexpectedly discovers through experiments that a near infrared fluorescent probe WL-808 can locate mitochondria (figure 1) and achieve the effect of a photosensitizer, and then discovers that the apoptosis of chondrocytes after the treatment of WL-808 combined with laser irradiation is obvious through experiments, and the photodynamic effect of WL-808 can effectively induce the apoptosis of chondrocytes (figure 3). WL-808 had high specificity and ideal photodynamic effect on HO cartilage lesions (fig. 4). The WL-808 photodynamic effect was able to effectively induce chondrocyte apoptosis at HO sites in vivo (fig. 5). The WL-808 photodynamic effect was able to effectively inhibit ectopic cartilage production in vivo (FIG. 6). Due to the cartilage template formation disorder, WL-808 photodynamic effect was able to effectively inhibit final ectopic osteogenesis in vivo (fig. 7).
The results show that the near infrared fluorescent probe WL-808 has new application: can be used for preparing medicaments for targeted treatment of ectopic ossification.
The use of a near infrared fluorescent probe WL-808 of the present application for preparing a medicament for targeted treatment of ectopic ossification will be described in detail with reference to examples and experimental data.
Example 1 near infrared fluorescent probes WL-808 were able to localize mitochondria and achieve the effect of photosensitizers
1. Chondrocyte mitochondrial localization assay of WL-808
Mouse chondrocytes were seeded into 20mm confocal laser petri dishes at a density of 1 ten thousand cells/well. After 2h incubation of cells with 20. Mu.M WL-808, the supernatant was removed and washed 3 times with PBS, and the Hoechst was incubated for 33342 10 min to stain nuclei and Mito-Tracker Green for 30 min to stain cell mitochondria in sequence. The mitochondrial localization of WL-808 was observed with confocal laser scanning microscopy (confocal laser scanning microscope, CLSM).
As shown in FIG. 1, WL-808 showed bright near infrared fluorescence in the chondrocyte mitochondria, indicating that WL-808 has good chondrocyte mitochondrial localization ability.
2. Test for inducing ROS formation in chondrocytes after laser irradiation of WL-808
Mouse chondrocytes were seeded in 24-well plates at a density of 1 ten thousand cells/well. After 2h incubation of cells with 20. Mu.M WL-808, the supernatant was removed and then washed 3 times with PBS. ROS were labeled with DCFH-DA, i.e. incubated with 10 μm DCFH-DA for 20min. Subsequently, DCFH-DA was removed and irradiated with 808nm laser light at a power of 2W/cm for 3 minutes 2 . During irradiation, the well plate is placed on ice to eliminate photo-thermal effects. ROS production was detected using CLSM after the end of irradiation.
The results are shown in FIG. 2, and the WL-808 combined with the laser irradiation shows obvious DCFH-DA green fluorescence signals in the chondrocytes, so that the WL-808 has good capability of inducing ROS production in the chondrocytes.
EXAMPLE 2 test for inducing chondrocyte apoptosis after laser irradiation of WL-808
Chondrocytes were seeded at a density of 1 ten thousand cells/well in 6-well plates. After incubation of cells with 20. Mu.M WL-808 for 2h, the supernatant was removed and washed 3 times with PBS, and irradiated with 808nm laser for 3 min at a power of 2W/cm 2 . During irradiation, the well plate is placed on ice to eliminate photo-thermal effects. The complete medium was replaced again after the irradiation was completed and cultivation was continued for 24 hours. Chondrocytes were then digested with pancreatin, and after centrifugation to collect the pellet, apoptosis analysis was performed using an Annexin V/Binding Buffer apoptosis detection kit, and the proportion of apoptotic cells was detected with a flow cytometer. As shown in FIG. 3, the apoptosis of the chondrocytes after the treatment of WL-808 combined with laser irradiation is obvious, which indicates that the photodynamic effect of WL-808 can effectively induce the apoptosis of the chondrocytes.
EXAMPLE 3 in vivo WL-808 test for ROS production at HO lesion site induced by laser irradiation
All animal experiments were performed in accordance with the national institutes of health, national institutes of Care and use for laboratory animals, and were approved by the animal welfare ethics Committee of the south China Hospital, university of Wuhan. Left hind limb achilles tendon midpoint cutting was performed on 12 8 week old male wild type C57BL/6 mice to induce HO, suture and disinfect the wound. After 1 week, 12 mice were randomly divided into four groups, 3 mice each, a blank (Control) group, a simple Laser (Laser) group, an IR-808+ Laser (IR-808+ Laser) group, and a WL-808+ Laser (WL-808+ Laser) group. WL-808+Laser group and IR-808+Laser were injected into mice by tail vein injection with 100. Mu.L of WL-808 (5 mg/kg) or IR-808 (2.5 mg/kg) dissolved in fat emulsion, respectively, and Control and Laser groups were administered with the same dose of fat emulsion in the same manner. 6 hours after administration, the achilles tenascopening site was irradiated with 808nm laser for 2 minutes (1W/cm) 2 ) The second round was irradiated in the same way after 5 minutes of interval. The administration was 2 times per week and laser irradiation was performed in the same manner for a total of 3 weeks. Immediately after the end of the last irradiation, mice were sacrificed, tissues from the surgical site were frozen and sectioned, and ROS production in vivo was observed using DHE assay kit.
As shown in FIG. 4, the Control, laser and IR-808+laser groups showed little DHE (ROS) red fluorescence at the surgical site, while the WL-808+laser group showed significant DHE red fluorescence at the surgical site, indicating that WL-808 had highly specific and ideal targeted photodynamic effects on HO cartilage lesions that induced ROS production.
EXAMPLE 4 in vivo test of apoptosis of cartilage cells at HO lesion site induced by laser irradiation of WL-808
Left hind limb achilles tendon midpoint cutting was performed on 12 8 week old male wild type C57BL/6 mice to induce HO, suture and disinfect the wound. After 1 week, 12 mice were randomly divided into four groups, 3 mice each, a blank (Control) group, a simple Laser (Laser) group, an IR-808+ Laser (IR-808+ Laser) group, and a WL-808+ Laser (WL-808+ Laser) group. WL-808+Laser group and IR-808+Laser were injected into mice by tail vein injection with 100. Mu.L of WL-808 (5 mg/kg) or IR-808 (2.5 mg/kg) dissolved in fat emulsion, respectively, and Control and Laser groups were administered with the same dose of fat emulsion in the same manner. 6 hours after administration, the achilles tenascopening site was irradiated with 808nm laser for 2 minutes (1W/cm) 2 ) The second round was irradiated in the same way after 5 minutes of interval. The administration was 2 times per week and laser irradiation was performed in the same manner for a total of 3 weeks. Mice were sacrificed 3 days after the end of the last irradiation, and tissues at the surgical site were taken for routine pathological section, and apoptosis was detected using TUNEL method.
As shown in FIG. 5, the Control, laser and IR-808+laser groups showed almost no green fluorescence of apoptotic cells at the surgical site, while the WL-808+laser group observed significant green fluorescence of apoptotic cells at the surgical site, indicating that the WL-808 photodynamic effect was able to effectively induce chondrocyte apoptosis at the HO site in vivo.
Example 5 in vivo WL-808 inhibition of ectopic cartilage lesion formation assay by photodynamic effect
Left hind limb achilles tendon midpoint cutting was performed on 12 8 week old male wild type C57BL/6 mice to induce HO, suture and disinfect the wound. After 1 week, 12 mice were randomly divided into four groups, a blank (Control), a Laser only (Laser), and IR-808+ LaserLight (IR-808+Laser) groups and WL-808+laser (WL-808+Laser) groups, each group of 3 mice. WL-808+Laser group and IR-808+Laser were injected into mice by tail vein injection with 100. Mu.L of WL-808 (5 mg/kg) or IR-808 (2.5 mg/kg) dissolved in fat emulsion, respectively, and Control and Laser groups were administered with the same dose of fat emulsion in the same manner. 6 hours after administration, the achilles tenascopening site was irradiated with 808nm laser for 2 minutes (1W/cm) 2 ) The second round was irradiated in the same way after 5 minutes of interval. The administration was 2 times per week and laser irradiation was performed in the same manner for a total of 3 weeks. Mice were sacrificed 3 days after the end of the last irradiation, left hind limb tissues of the mice were taken for conventional pathological sections, and the formation of ectopic cartilage was observed using safranin fast green staining.
As a result, as shown in FIG. 6, there was no significant difference in the areas of orange-colored cartilage in the left hind limbs of the mice of the Control group, the Laser group, and the IR-808+laser group. Compared with the Control group, the Laser group and the IR-808+laser group, the area of the orange-colored cartilage in the left hind limb of the mouse of the WL-808+laser group is obviously smaller, which indicates that the WL-808 photodynamic effect can effectively inhibit ectopic cartilage generation in vivo.
Example 6 in vivo WL-808 inhibition of ectopic bone lesion formation assay by photodynamic effect
Left hind limb achilles tendon midpoint cutting was performed on 12 8 week old male wild type C57BL/6 mice to induce HO, suture and disinfect the wound. After 1 week, 12 mice were randomly divided into four groups, 3 mice each, a blank (Control) group, a simple Laser (Laser) group, an IR-808+ Laser (IR-808+ Laser) group, and a WL-808+ Laser (WL-808+ Laser) group. WL-808+Laser group and IR-808+Laser were injected into mice by tail vein injection with 100. Mu.L of WL-808 (5 mg/kg) or IR-808 (2.5 mg/kg) dissolved in fat emulsion, respectively, and Control and Laser groups were administered with the same dose of fat emulsion in the same manner. 6 hours after administration, the achilles tenascopening site was irradiated with 808nm laser for 2 minutes (1W/cm) 2 ) The second round was irradiated in the same way after 5 minutes of interval. The administration was 2 times per week and laser irradiation was performed in the same manner for a total of 3 weeks. Mice were sacrificed 6 weeks after the end of the last irradiation, and left hind limb tissues of the mice were scanned for micro-CT. Conventional pathological sections were then performed and the ectopic sites were observed using Masson stainingBone formation.
As shown in FIG. 7, there was no significant difference in the volume and area of the ectopic bone at the left hind limb surgical site in the Control group, the Laser group, and the IR-808+laser group mice. Compared with the Control group, the Laser group and the IR-808+laser group, the WL-808+laser group has smaller volume and area of the ectopic bone at the left hindlimb operation position of the mouse, which indicates that the WL-808 photodynamic effect can effectively inhibit the final ectopic bone generation in vivo.
Example 7, WL-808 in vivo toxicity experiment after HO in combination with laser therapy
After the Control group and WL-808+laser group mice in example 6 were sacrificed, the major organs (heart, liver, spleen, lung, kidney) were taken and fixed with 4% Paraformaldehyde (PFA) solution for 30 minutes. The tissues were then embedded in paraffin and cut into 5 μm slices, which were visualized under a microscope after staining with H & E.
As shown in FIG. 8, the organ tissue of the WL-808+laser group mice was not significantly damaged compared to the Control group mice, indicating that the WL-808 combined laser therapy HO had good biosafety.
Finally, it is also noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
While preferred embodiments of the present application have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. It is therefore intended that the following claims be interpreted as including the preferred embodiments and all such alterations and modifications as fall within the scope of the application.
It will be apparent to those skilled in the art that various modifications and variations can be made to the present application without departing from the spirit or scope of the application. Thus, it is intended that the present application also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.
Claims (2)
1. The application of a near infrared fluorescent probe WL-808 in preparing a medicament for targeted treatment of ectopic ossification is characterized in that the structural formula of the near infrared fluorescent probe WL-808 is as follows:
2. the use according to claim 1, wherein said near infrared fluorescent probe WL-808 induces chondrocyte death at ectopic ossified cartilage lesions by inducing the production of reactive oxygen species during photodynamic therapy.
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