A sonosensitizer for regulating morphology and function of lymphatic vessels by ultrasound, a composition thereof and application thereof

Abstract

The application provides a kind of sound sensitizer for binding ultrasound to regulate lymphatic vessel morphology and function, its composition and application.The LYVE-1 antibody is coupled with Ce6 to prepare LYVE1-Ce6, to improve the lymphatic endothelial cell targeting of Ce6 molecule.The application discloses a kind of application method of the above-mentioned sound sensitizer, by mouse cerebellopontine pool, skin or intestinal tract respectively injection PEG-Ce6 and LYVE1-Ce6, transcranial or local tissue uses ultrasonic instrument excitation Ce6 to regulate meningeal lymphatic vessel and peripheral lymphatic vessel morphology and function.The application proves by experiment that ultrasound combined with sound sensitizer can partially destroy meningeal lymphatic vessel and peripheral lymphatic vessel morphology, and inhibit meningeal lymphatic vessel drainage function.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and to a sonosensitive agent that combines ultrasound to regulate the morphology and function of lymphatic vessels, its composition and application, and more particularly to a method for regulating the morphology and function of lymphatic vessels by ultrasound combined with a sonosensitive agent, specifically a method for regulating the morphology and function of meningeal lymphatic vessels and peripheral lymphatic vessels (skin and intestines). Background Technology

[0002] Lymphatic vessels are distributed throughout the body, unidirectionally collecting tissue fluid from the interstitial spaces into the lumen, propelling it to lymphatic organs and then into the bloodstream to maintain homeostasis. Previous studies have confirmed the important role of lymphatic vessels in various diseases, such as skin inflammation, inflammatory bowel disease, and rheumatoid arthritis. Meningeallymphatic vessels (MLVs) are a central lymphatic network discovered in recent years that regulates the transport of cerebrospinal fluid and immune cells in the central nervous system (CNS). They take up cerebrospinal fluid and metabolic waste from the CNS through the subarachnoid space and participate in the development of various CNS diseases. For example, in aged mice, MLVs alleviated cognitive impairment and other behaviors by regulating the deposition of β-amyloid protein; in addition, in multiple sclerosis, MLVs regulated the efflux of brain-derived antigens in brain tissue, and the ablation of MLVs alleviated the progression of neuronal demyelinating disease in EAE mice. These findings demonstrate that MLVs' regulation of the CNS microenvironment may intervene in the homeostasis of CNS neurons. To further investigate the crucial role of meningeal lymphatic vessels in various central nervous system diseases, the development of meningeal lymphatic vessel intervention methods is essential.

[0003] Currently, intervention methods for meningeal and peripheral lymphatic vessels mainly include photosensitizer photodynamic therapy, lymphatic vessel ligation, and gene intervention. Photosensitizer photodynamic therapy utilizes transcranial laser irradiation of specific wavelengths to stimulate photosensitizers in the brain, generating reactive oxygen species (ROS) that damage meningeal lymphatic vessels. However, the photosensitizers used in this method lack specific cell targeting, and the laser irradiation time is relatively long, causing thermal pain effects on tissues such as the skull. Lymphatic vessel ligation involves ligating the lymphatic vessels connecting the meningeal lymphatic vessels to their draining lymph nodes (deep cervical lymph nodes), thereby blocking the transport of substances from the meningeal lymphatic vessels to the deep cervical lymph nodes (dCLNs). However, meningeal lymphatic vessels have other compensatory pathways for transporting substances from cerebrospinal fluid to the periphery, thus this method has limitations. The vascular endothelial growth factor C (VEGF-C) and vascular endothelial growth factor receptor 3 (VEGFR3) signaling pathway is crucial for lymphatic vessel development. Some studies have used adeno-associated virus 9 (AAV9) to deliver gene sequences from the soluble VEGFR-3 D1-D4 region to construct VEGF-C traps (VEGFC-trap), thereby inhibiting the VEGF-C / VEGFR3 signaling pathway and leading to functional decline in lymphatic vessels. However, this gene intervention method targets lymphatic vessels in all parts of the body and lacks tissue / organ specificity. Therefore, developing targeted, tissue / organ specific methods to intervene in meningeal lymphatic vessels and lymphatic vessels in specific peripheral sites is essential. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide a sonosensitive agent, its composition, and its application that combines ultrasound to modulate the morphology and function of lymphatic vessels. This provides an innovative method for intervening in the morphology and function of meningeal and peripheral lymphatic vessels, offering a research tool for understanding the role of meningeal and peripheral lymphatic vessels in treating central nervous system diseases. This invention provides the application of ultrasound combined with a sonosensitive agent in intervening in the morphology and function of meningeal and peripheral lymphatic vessels, enabling targeted and organ-specific damage to lymphatic vessel morphology and blocking the drainage function of meningeal lymphatic vessels. This technology has promising applications in studying the function and role of meningeal and peripheral lymphatic vessels.

[0005] The present invention provides a sonosensitive agent that combines ultrasound to regulate the morphology and function of lymphatic vessels, its composition and application, wherein the sonosensitive agent is a conjugate of LYVE1 antibody and dihydroporphyrin E6 (Ce6) LYVE1-Ce6.

[0006] Another aspect of the present invention provides a composition for regulating the morphology and function of lymphatic vessels, comprising any one of the following sound-sensitive agents: 1) LYVE1-Ce6 as described in claim 1, 2) PEG-Ce6, 3) Ce6, And pharmaceutically acceptable carriers or excipients.

[0007] In another aspect, the present invention provides the use of the above-mentioned acoustic sensitizer or the above-mentioned composition in the preparation of a reagent for regulating the morphology and function of lymphatic vessels.

[0008] Preferably, the lymphatic vessels include meningeal lymphatic vessels and peripheral lymphatic vessels. The meningeal lymphatic vessels and peripheral lymphatic vessels are selected from cutaneous lymphatic vessels and mesenteric lymphatic vessels, etc.

[0009] Preferably, the regulation of lymphatic vessel morphology specifically involves partially reducing the area of ​​meningeal lymphatic vessels or peripheral lymphatic vessels.

[0010] Preferably, the regulation of lymphatic vessel function specifically involves reducing the drainage capacity of the meningeal lymphatic vessels.

[0011] Another aspect of the present invention provides a method for preparing a mouse model of lymphatic vessel injury using ultrasound combined with a sound-sensitizing agent, comprising the following steps: 1) Inject mice with an effective dose of PEG-Ce6 or LYVE1-Ce6 via the cerebellomedullary cistern (foramen magnum), skin, or intestine; 2) Place the probe of the ultrasound device on the head, skin, and abdomen of the mouse, and turn on the power to continuously output ultrasound energy to activate the sonicator.

[0012] Preferably, the ultrasonic parameters are: operating frequency 1 MHz, continuous mode, and output power density 1.0 W / cm². 2 The stimulation lasts for 3 minutes.

[0013] In some preferred embodiments, the method for intervening in the morphology and function of meningeal lymphatic vessels and peripheral lymphatic vessels according to the present invention includes the following steps: PEG-Ce6, which has high water solubility, is prepared by coupling polyethylene glycol (PEG) with dihydroporphyrin E6 (Ce6), or LYVE1-Ce6, which has lymphatic vessel targeting, is prepared by coupling a lymphatic vessel endothelial hyaluronic acid receptor 1 (LYVE-1) antibody with Ce6. PEG-Ce6 or LYVE1-Ce6 is injected into the cerebellomedullary cistern (foramen magnum), skin, or intestine. After 15 minutes, the probe of an ultrasound device is placed on the head, skin, and abdomen of the organism, and the power is turned on to provide continuous ultrasound energy output to achieve regulation of meningeal lymphatic vessels and peripheral lymphatic vessels (skin and intestine).

[0014] Preferably, the acoustic sensitizer includes Ce6, PEG conjugated with Ce6, LYVE1 antibody conjugated with Ce6, and other acoustic sensitizers capable of generating ROS upon ultrasonic energy excitation. The acoustic sensitizer, when excited by ultrasonic energy, is used to intervene in the morphology and function of meningeal lymphatic vessels and peripheral lymphatic vessels.

[0015] In some specific embodiments, the photosensitizer is delivered to the meningeal lymphatic vessels via injection into the cerebellomedullary cistern.

[0016] In some specific embodiments, the photosensitizer is delivered to peripheral lymphatic vessels (skin and intestines) via local injection.

[0017] Preferably, the ultrasound operation involves placing the head, skin, abdomen, etc. of the organism at the center of the ultrasound probe, turning on the instrument power, and continuously outputting ultrasound energy stimulation.

[0018] Preferably, the continuous ultrasonic energy output parameters are: operating frequency 1 MHz, continuous mode, in which ultrasonic energy is continuously output with a duty cycle of 100%, no pulse cycle parameters need to be set, and the output power density is 1.0 W / cm². 2 The stimulation time is 3 minutes, thereby activating the aforementioned sound-sensitive agent.

[0019] The beneficial effects of this invention are as follows: It uses ultrasound energy to activate a sonosensitive agent to regulate meningeal lymphatic vessels and peripheral lymphatic vessels. Experiments have confirmed that ultrasound energy, by activating a sonosensitive agent targeting lymphatic vessels, disrupts the morphology of meningeal lymphatic vessels and peripheral (skin and intestine) lymphatic vessels and inhibits the drainage capacity of meningeal lymphatic vessels, thus providing an effective technical means for studying the role of meningeal lymphatic vessels and peripheral lymphatic vessels in diseases. Attached Figure Description

[0020] Figure 1 The chemical reaction equation for preparing the sound-sensitive agent PEG-Ce6; Figure 2 The chemical reaction equation for preparing the sound-sensitive agent LYVE1-Ce6; Figure 3 shows the morphology of meningeal lymphatic vessels damaged by ultrasound-activated acoustic sensitizer; among which... Figure 3A Effects on the meningeal lymphatic vessels of LYVE1+ mice; Figure 3B This indicates that the effect of the sound sensitizer in disrupting the meningeal lymphatic vessels in mice can last for up to two weeks. Figure 3C The results showed that transcranial ultrasound stimulation of LYVE1-Ce6 significantly reduced LYVE1+ meningeal lymphatic vessels in mice; Figure 4 To activate the acoustic sensitizer to inhibit the meningeal lymphatic drainage function; Figure 5Ultrasound-activated sonic sensitizers damage the morphology of skin and intestinal lymphatic vessels. Detailed Implementation

[0021] The following embodiments illustrate the present invention, but the present invention is not limited to these embodiments. Modifications to the specific embodiments of the present invention or equivalent substitutions for some technical features, without departing from the spirit of the present invention, should all be covered within the scope of the technical solutions claimed in the present invention.

[0022] Specific techniques or conditions are not specified in this embodiment; the operation is carried out in accordance with conventional techniques and instrument manuals in this field. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0023] Example 1: Preparation of the sound-sensitizing agent PEG-Ce6 The chemical reaction equation for the preparation of compound PEG-Ce6 is as follows: Figure 1 As shown, the specific method is as follows: Add Ce6 (100.0 mg, 0.17 mmol) and PEG to a 25 mL Schlenk reaction tube. 2000 -NH2 (340.0 mg, 0.17 mmol), DCC (35.0 mg, 0.17 mmol), and DMSO 2 mL were added. The mixture was stirred overnight at room temperature and the target compound PEG-Ce6 was prepared by preparative high performance liquid chromatography.

[0024] The synthesis method of PEG-Ce6 can be referred to in the following paper.

[0025] Xu B , Hou H , Xu W ,et al.An all-in-one polymeric nanomedicine as insitu cascade cancer vaccine for potent cancer immunotherapy[J]. Nano Today, 2025, 62:102691.DOI:10.1016 / j.nantod.2025.102691. Xu W , Wang J , Jin L ,et al.A tumor acidity-driven transformablepolymeric nanoassembly with deep tumor penetration and membrane-anchoringcapability for targeted photodynamic therapy[J].Biomaterials, 2021:121024.DOI:10.1016 / j.biomaterials.2021.121024. Example 2: Preparation of the sound-sensitizing agent LYVE1-Ce6 The chemical reaction equation for the preparation of compound Ce6-NHS is as follows: Figure 2 As shown, the specific method is as follows: Ce6 (129.0 mg, 0.22 mmol), NHS (25.3 mg, 0.22 mmol), and DCC (136.2 mg, 0.66 mmol) were added to a 25 mL Schlenk reaction tube, followed by 2 mL of DMSO. The mixture was stirred overnight at room temperature. The mixture was diluted with 10 mL of ethyl acetate and washed three times with 10 mL of water each time. The organic phase was separated by a separatory funnel, dried over anhydrous sodium sulfate, filtered, mixed with silica gel, separated by column chromatography, and evaporated to dryness using a rotary evaporator to obtain 100.2 mg of brown solid, with a yield of 65.6%.

[0026] The preparation method of its sound-sensitizing agent LYVE1-Ce6 is as follows: Prepare a 20 mg / mL DMSO solution of Ce6-NHS obtained above. Add 100 μL of LYVE1 antibody (0.5 mg / mL) to a 1.5 mL centrifuge tube, then add 1x PBS buffer solution (pH 8.2), followed by 50 μL of the prepared Ce6-NHS DMSO solution. Mix well and react at room temperature in the dark for 4 h. Centrifuge the protein concentration ultrafiltration tube at 15000 r / min, wash three times with PBS, and obtain the antibody-conjugated sonosensitive agent LYVE1-Ce6.

[0027] Example 3: Ultrasound activation of the morphology of meningeal lymphatic vessels damaged by a sonosensitive agent To investigate the effect of ultrasound-activated sonosensitive agents on the morphology of meningeal lymphatic vessels, 8-week-old SPF-grade C57BL / 6 mice were randomly divided into three groups. The experimental group mice were injected with 5 μL of different doses of PEG-Ce6 sonosensitive agent (3 mg / ml, 6 mg / ml; dissolved in physiological saline containing 5% sucrose) through the cerebellomedullary cistern. The control group mice were injected with the same volume of physiological saline (vehicle). Fifteen minutes after injection, the ultrasound probe was placed directly above the skull, and ultrasound was continuously applied for 3 minutes (operating frequency 1 MHz, output power density 1.0 W / cm²). 2 One week later, mouse meninges were collected for meningeal immunofluorescence staining. The results showed that transcranial ultrasound stimulation with a somatosensitizing agent (6 mg / ml PEF-Ce6) significantly reduced the number of LYVE1+ meningeal lymphatic vessels in mice. Figure 3A To further demonstrate this effect, mice were injected with 5 μL of the sonosensitive agent PEG-Ce6 (6 mg / ml) or saline (Vehicle) into the cerebellomedullary cistern. Immunofluorescence staining of the meninges was performed 1 and 2 weeks after ultrasound stimulation. The results showed that the effect of transcranial ultrasound stimulation of the sonosensitive agent in disrupting the meningeal lymphatic vessels of mice could last for 2 weeks (Figure 3B).

[0028] To improve the targeting of the sonosensitive agent to meningeal lymphatic vessels, an antibody against the meningeal lymphatic vessel-specific marker LYVE-1 was conjugated with Ce6 to prepare a new targeting sonosensitive agent, LYVE1-Ce6 (see Example 2). Subsequently, 5 μL of LYVE1-ce6 or physiological saline was injected into the cerebellomedullary cistern of mice, and the sonosensitive agent was stimulated by ultrasound or laser. One week later, the meninges of the mice were harvested for immunofluorescence staining. The experimental results showed that transcranial ultrasound stimulation of LYVE1-Ce6 significantly reduced LYVE1+ meningeal lymphatic vessels in mice, with a very significant effect (Figure 3C).

[0029] Example 4: Ultrasound activation of a sonosensitive agent to inhibit meningeal lymphatic transport function Previous studies have reported that meningeal lymphatic vessels transport fluids and substances from cerebrospinal fluid to cervical lymph nodes. To investigate the effect of ultrasound-activated sonostatin on meningeal lymphatic vessel function, 8-week-old SPF-grade C57BL / 6 mice were randomly divided into three groups. The experimental group mice were injected with 5 μL of different doses of PEG-Ce6 sonostatin (3 mg / ml, 6 mg / ml, dissolved in 5% sucrose saline) through the cerebellomedullary cistern, while the control group mice were injected with the same volume of saline (vehicle). Fifteen minutes after injection, an ultrasound probe was placed directly above the skull, and ultrasound was continuously performed for 3 minutes (operating frequency 1 MHz, output power density 1.0 W / cm²). 2One week later, OVA-647 was injected through the cerebellomedullary cistern, and the level of OVA-647 in the neck was detected using in vivo imaging 30 minutes after injection. The experimental results showed that transcranial ultrasound stimulation of somatosensitizers could reduce the ability of meningeal lymphatic vessels to drain biomolecules to the cervical lymph nodes, especially at a concentration of 6 mg / ml. Figure 4 A).

[0030] Example 5: Ultrasound activation of a sonosensitive agent damaging the morphology of skin and intestinal lymphatic vessels To further verify the effect of ultrasound combined with a sonostatin in peripheral lymphatic vessels, 8-week-old SPF-grade C57BL / 6 mice were randomly divided into three groups. Mice in the experimental group received an injection of 5 μL of different doses of PEG-Ce6 sonostatin (3 mg / ml, 6 mg / ml, dissolved in physiological saline containing 5% sucrose) into the ear skin. Mice in the control group received the same volume of physiological saline (vehicle). An ultrasound probe was then placed close to the injection site on the ear skin, and ultrasound was performed continuously for 3 minutes. One week later, the lymphatic vessels in the ear were detected by immunofluorescence. The experimental results showed that (…) Figure 5 A) Local ultrasound stimulation of the ear with a somatosensory agent significantly reduced the lymphatic vessels in the ear, especially with 6 mg / ml PEG-Ce6. Furthermore, the effect of ultrasound combined with a somatosensory agent was further verified in the mesenteric lymphatic vessels. Eight-week-old SPF-grade C57BL / 6 mice were randomly divided into two groups. Mice in the experimental group were injected with 3 μL of PEG-Ce6 somatosensory agent (6 mg / ml) through the Pell's node of the intestine, while mice in the control group were injected with the same volume of saline (vehicle). An ultrasound probe was then placed close to the mesentery, and ultrasound was performed continuously for 3 minutes. One week later, the mesenteric lymphatic vessels were detected by immunofluorescence. The results showed that ( Figure 5 B), Local ultrasound-activated sonosensitive agents can also significantly reduce mesenteric lymphatic vessels.

[0031] The specific steps of the experimental method used in the above embodiments are as follows: (1) Immunofluorescence staining: Mice were perfused with PBS and 4% PFA. The skin and muscles of the mouse skull were peeled off, and the mandible and the maxilla on the rostral side of the skull were removed. The top of the skull was then removed with surgical scissors. The skull connected to the meninges was fixed with 4% PFA at 4 °C for 24 h. The dura mater was then carefully peeled off from the skull. The entire meninges were incubated at room temperature for 1 h in blocking solution (PBS containing 0.5% Triton-x-100, 3% BSA and 2% fetal bovine serum). The LYVE-1 antibody was diluted with PBS containing 3% BSA and 0.5% Triton-x-100 and incubated overnight at 4 °C with an appropriate dilution of primary antibody. The mice were then washed 3 times with PBS, incubated at room temperature for 1 h with an appropriate dilution of fluorescently labeled secondary antibody, washed once with PBS, incubated at room temperature for 10 h with DAPI (1:1000 dilution), washed 3 times with PBS, and then the meninges were laid flat on a slide and mounted with an anti-quenching agent. Fluorescence images were acquired using a confocal microscope.

[0032] (2) In vivo imaging in mice: Hair was removed from the ventral neck of mice to prevent weakening of the fluorescence signal. The dorsal neck skin was then cut open, and 5 ml of OVA-647 (2 mg / ml) was injected into the cerebellomedullary cistern at a rate of 1 ml / min. The fluorescence intensity of OVA-647 in the neck was then measured using an in vivo imaging system at 30 min. The results were analyzed using LivingImage 4.7.3.

[0033] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A sonosensitive agent that combines ultrasound to regulate the morphology and function of lymphatic vessels, characterized in that, The sonosensitive agent is LYVE1-Ce6, a conjugate of LYVE1 antibody and dihydroporphyrin E6 (Ce6).

2. A composition for regulating the morphology and function of lymphatic vessels, characterized in that, It contains any of the following sound-sensitive agents. 1) LYVE1-Ce6 as described in claim 1, 2) PEG-Ce6, 3) Ce6, And pharmaceutically acceptable carriers or excipients.

3. The use of the acoustic sensitizer according to claim 1 or the composition according to claim 2 in the preparation of reagents for regulating the morphology and function of lymphatic vessels.

4. The application according to claim 3, characterized in that, The lymphatic vessels include meningeal lymphatic vessels and peripheral lymphatic vessels.

5. The application according to claim 3, characterized in that, The regulation of lymphatic vessel morphology specifically involves partially reducing the area of ​​meningeal lymphatic vessels or peripheral lymphatic vessels.

6. The application according to claim 3, characterized in that, Specifically, regulating lymphatic vessel function involves reducing the drainage capacity of the meningeal lymphatic vessels.

7. A method for preparing a mouse model of lymphatic vessel injury using ultrasound combined with a sonosensitive agent, characterized in that, Includes the following steps: 1) Inject mice with an effective dose of PEG-Ce6 or LYVE1-Ce6 via the cerebellomedullary cistern (foramen magnum), skin, or intestine; 2) Place the probe of the ultrasound device on the head, skin, and abdomen of the mouse, and turn on the power to continuously output ultrasound energy to activate the sonicator.

8. The method according to claim 7, characterized in that, The parameters of the ultrasound are: operating frequency 1 MHz, continuous mode, and output power density 1.0 W / cm². 2 The stimulation lasts for 3 minutes.

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