A porphyrin microbubble for treating lymphatic malformations and a preparation method and application thereof

Abstract

This invention discloses a targeted porphyrin microbubble for the treatment of lymphangiogenic malformations, its preparation method, and its application, belonging to the field of targeted nanomedicine technology. The targeted porphyrin microbubble provided by this invention consists of a bilayer shell and a bio-inert gas encapsulated within the bilayer shell. The bilayer shell is formed of amphiphilic molecules, and drug particles are dispersed within it. The porphyrin microbubble provided by this invention can achieve targeted sustained release of bleomycin at the lesion site, solving the problems of low efficacy and high toxicity in clinical sclerotherapy, while achieving photodynamic-sclerotherapy synergistic treatment and improving therapeutic efficacy. Furthermore, the ultrasound signal from the porphyrin microbubble enables ultrasound-guided local drug injection, monitoring precise drug injection into the lesion cavity and improving injection safety.

Description

Technical Field

[0001] This invention belongs to the field of targeted nanomedicine in the field of biomedical technology, specifically relating to a targeted porphyrin microbubble for the treatment of lymphatic malformations, its preparation method, and its application. Background Technology

[0002] Lymphatic malformations (LMs) are congenital developmental abnormalities of the lymphatic system, with an incidence rate as high as 1 / 4000-1 / 2000 in newborns, severely impacting patients' health and quality of life. Treatment of lymphatic malformations requires comprehensive consideration of clinical manifestations, lesion size, and anatomical location. Currently, the most commonly used treatments are sclerotherapy and surgical resection, with sclerotherapy being the first-line clinical approach. Sclerotherapy involves the local injection of sclerosing agents such as bleomycin into the lesion cavity, inducing an inflammatory response and endothelial damage. However, local injection of sclerosing agents has drawbacks, including low efficacy, the need for repeated injections, and significant side effects. Excessive use of bleomycin, the most widely used drug in sclerotherapy, carries the risk of inducing pulmonary fibrosis, and excessive infiltration of sclerosing agents into tissues can also lead to muscle atrophy and contractures. Therefore, improving the utilization rate of bleomycin at the lesion site, enhancing the efficacy of sclerotherapy, and improving the safety of sclerotherapy have become urgent priorities.

[0003] Liposomes are biocompatible drug carriers that are biodegradable and can achieve targeted and sustained drug release in diseased tissues, effectively improving drug utilization and reducing toxic side effects. While liposomes are primarily used as drug delivery materials in cancer treatment, their application in the treatment of lymphangiocarcinoma scleroderma (LCS) is rarely reported. Therefore, this invention utilizes liposomes to encapsulate bleomycin for the treatment of LCS, potentially addressing the problems of low efficacy and significant toxic side effects in current clinical scleroderma treatments.

[0004] Furthermore, mixing and shaking liposomes with inert gas forms microbubbles (MBs) with high drug loading capacity. Drug-loaded liposomes entering the body in microbubble form offer several advantages: First, MBs can be ruptured using ultrasound-targeted microbubbles destruction (UTMD) to form smaller nanoliposomes; second, under the influence of UTMD, MBs can enhance cell and vascular permeability, promote local drug accumulation and penetration, and improve cellular drug uptake; additionally, the optical properties of the microbubbles themselves enable precise local drug injection guided by ultrasound imaging.

[0005] Furthermore, studies have shown that lymphatic endothelial cells highly express the lymphatic vessel endothelial hyaluronic acid receptor 1 (LYVE-1), which is specifically expressed on lymphatic vessel endothelial cells but expressed at very low levels on vascular endothelial cells. Based on this, this invention utilizes hyaluronic acid (HA) as a targeted molecular modification drug carrier to achieve LYVE-1-mediated targeted drug delivery, enabling targeted drug release at the lesion site.

[0006] The clinical treatment of lymphangiogenic malformations is highly challenging, especially for refractory lymphangiogenic malformations characterized by extensive and diffuse expansive growth, for which effective treatments are lacking. This invention, while improving the efficacy of sclerotherapy, combines it with other novel treatment methods to bring new hope to the clinical treatment of lymphangiogenic malformations. The novel treatment method described is photodynamic therapy (PDT). Photodynamic therapy (PDT) is a clinically approved tumor treatment method that uses a laser of appropriate wavelength to excite a photosensitizer to generate reactive oxygen species, primarily singlet oxygen, which kills target cells. Compared to traditional therapies, PDT has advantages such as high tissue selectivity, high safety, good reproducibility, and low likelihood of developing drug resistance. Currently, PDT has been approved by the FDA for the clinical treatment of various tumors, including skin cancer, esophageal cancer, and non-small cell lung cancer; however, research on the application of PDT in the treatment of lymphangiogenic malformations has not yet been reported.

[0007] In the field of integrated diagnosis and treatment research of lymphatic malformations, this invention addresses the urgent need to improve the efficacy and safety of sclerotherapy, a first-line clinical treatment, and the lack of effective treatments for refractory lymphatic malformations. It develops a porphyrin liposome microvesicle capable of targeted drug release, introducing photodynamic-sclerotherapy synergistic therapy into lymphatic malformation treatment research. This approach, while improving the efficacy of sclerotherapy, is synergistic with novel treatment methods, and is expected to bring new hope to the clinical treatment of lymphatic malformations. Summary of the Invention

[0008] Based on the above background, this invention prepares porphyrin liposome microvesicles that encapsulate bleomycin using hyaluronic acid as the targeting molecule. This enables targeted and sustained release of bleomycin at the lesion site, addressing the issues of low efficacy and significant toxic side effects in clinical sclerotherapy while achieving synergistic photodynamic-sclerotherapy, thus improving treatment efficacy. Furthermore, the ultrasound signal from the porphyrin liposome microvesicles allows for ultrasound-guided local drug injection, monitoring precise drug delivery to the lesion cavity and improving injection safety.

[0009] One object of the present invention is to provide a targeted porphyrin microbubble for the treatment of lymphatic malformations; another object of the present invention is to provide a method for preparing the porphyrin microbubble; and yet another object of the present invention is to provide a pharmaceutical use of the porphyrin microbubble.

[0010] The objective of this invention is achieved through the following technical solution: In a first aspect, the present invention provides a targeted porphyrin microbubble for the treatment of lymphatic malformations, the microbubble being composed of a bilayer shell and a bio-inert gas encapsulated inside the bilayer shell, the bilayer shell being formed of amphiphilic molecules, and drug particles being dispersed within the bilayer shell.

[0011] The amphiphilic molecule is a phospholipid and porphyrin-hyaluronic acid conjugate (PL-HA).

[0012] In this invention, the porphyrin-hyaluronic acid conjugate (PL-HA) is obtained by coupling porphyrin and hyaluronic acid using an EDC-NHS amide reaction. The activated hyaluronic acid is reacted with an amino-modified porphyrin overnight, followed by dialyzing and lyophilization. The mass ratio of hyaluronic acid to porphyrin is (2-6):1.

[0013] The phospholipid is selected from one or more of lecithin, cholesterol, distearylphosphatidylcholine (DSPC), and distearylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG).

[0014] In a specific embodiment of the present invention, the phospholipid is a combination of distearylphosphatidylcholine (DSPC) and distearylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG).

[0015] In some embodiments of the present invention, the bilayer shell is composed of porphyrin-hyaluronic acid coupling compound (PL-HA), distearate phosphatidylcholine (DSPC), and distearate phosphatidylethanolamine-polyethylene glycol (DSPE-PEG) in a mass ratio of (1-5):(4-8):1; preferably (1.25-2):(7-7.75):1; and most preferably 1.25:7.75:1.

[0016] The drug particles are a hardening agent, which is selected from one or more of the following: sodium morrhuate, alum, bleomycin (PYM), bleomycin (BLM), ethanolamine, diatrizoate, quinoline, tetracycline, doxycycline, and urea.

[0017] In some embodiments of the present invention, the hardening agent is selected from bleomycin and / or phenytoin.

[0018] In a preferred embodiment of the present invention, the hardening agent is bleomycin, and the amount of hardening agent added is 5-30% of the liposome membrane material.

[0019] The bio-inert gas used in this invention is selected from at least one of perfluoropropane, perfluorobutane, and sulfur hexafluoride.

[0020] In a specific embodiment of the present invention, the biological inert gas is perfluoropropane.

[0021] The average particle size of the porphyrin microbubbles is 1-5 μm.

[0022] Secondly, the present invention provides a method for preparing targeted porphyrin microvesicles for the treatment of lymphatic malformations, the preparation method comprising the following steps: (1) Porphyrin is coupled with hyaluronic acid to form porphyrin-hyaluronic acid conjugate (PL-HA). (2) Using the thin-film hydration method or the ethanol injection method, phospholipids and the porphyrin-hyaluronic acid coupling compound prepared in step (1) are used as membrane materials and a hardener is used as a drug to prepare drug-loaded liposomes; (3) Introduce a bio-inert gas into the drug-loaded liposomes and shake to obtain targeted porphyrin microbubbles.

[0023] Preferably, in step (1), the porphyrin is coupled to hyaluronic acid by EDC-NHS amide reaction. The activated hyaluronic acid is reacted with amino-modified porphyrin overnight, dialyzed, and lyophilized to obtain the porphyrin-hyaluronic acid conjugate.

[0024] In a specific embodiment of the present invention, the mass ratio of hyaluronic acid to porphyrin is (2-6):1.

[0025] Preferably, the drug-loaded liposomes described in step (2) are prepared by the following method: Using a thin-film hydration method, porphyrin-hyaluronic acid coupling compound, distearate phosphatidylcholine (DSPC), and distearate phosphatidylethanolamine-polyethylene glycol (DSPE-PEG) were dissolved in chloroform and / or methanol solution at a mass ratio of (1-5):(4-8):1. A hardening agent was added, the solvent was removed by rotary evaporation, hydration solution was added, and the mixture was sonicated to obtain drug-loaded liposomes.

[0026] Preferably, the mass ratio of porphyrin-hyaluronic acid coupling compound, distearylphosphatidylcholine (DSPC), and distearylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG) is (1.25-2):(7-7.75):1.

[0027] In the most preferred embodiment of the present invention, the mass ratio of porphyrin-hyaluronic acid coupling compound, distearate phosphatidylcholine (DSPC), and distearate phosphatidylethanolamine-polyethylene glycol (DSPE-PEG) is 1.25: 7.75:1.

[0028] The amount of hardener added is 5-30% of the liposome membrane material, specifically 5%, 10%, 15%, 20%, 25%, and 30%.

[0029] The hydration solution is selected from phosphate buffer (PBS) or a mixture of anhydrous sodium acetate and anhydrous calcium chloride.

[0030] Preferably, in step (3), drug-loaded liposomes, glycerol, and 1,2-propanediol are mixed in a volume ratio of 10:(1-2):(1-2), a biological inert gas is introduced, and the mixture is mechanically stirred for 45-100s using a vibrating screen to obtain targeted porphyrin microbubbles.

[0031] Conventional liposome membranes are composed of amphiphilic phospholipids, which have a hydrophilic phosphate head and a hydrophobic tail consisting of two fatty acid chains. The inventors have creatively coupled porphyrins with hyaluronic acid to form an amphiphilic molecule with the same properties as phospholipids, which is used as a raw material for preparing liposome membranes. The advantage is that it ensures that porphyrin, which exerts the photodynamic therapy effect, and hyaluronic acid, which exerts the drug targeting effect, are uniformly dispersed in the microbubble shell. Subsequently, in vivo ultrasonic targeted demodulation (UTMD) of the microbubbles results in a more uniform distribution of porphyrins and hyaluronic acid in the nanoliposomes, enhancing drug targeting and photodynamic therapy effects. According to the specification, when porphyrin is directly dispersed in microbubbles, due to its lipophilic properties, it tends to disperse on the inner side of the lipid bilayer shell. Due to the randomness of drug encapsulation in liposomes, uniform dispersion of porphyrin cannot be guaranteed; in most cases, porphyrin is encapsulated in an aggregated form within the liposomes, which severely affects the in vivo photodynamic therapy effect.

[0032] Similarly, hyaluronic acid, due to its water-soluble properties, cannot be encapsulated in liposomes. If it does not form an amphiphilic molecule with porphyrin, it must be coupled to the microvessel surface through an additional pathway. According to the description of this invention, in the first case, after the liposomes are prepared, they are incubated and coupled with hyaluronic acid. The result is that the amount of hyaluronic acid coupled to the formed microvessel surface is relatively small, failing to guarantee the drug's targeted effect. In the second case, before liposome preparation, hyaluronic acid is coupled with DSPE-PEG, and then liposomes are prepared. The result is that the amount of hyaluronic acid added is affected by DSPE-PEG, and the porphyrin cannot be evenly distributed, affecting the photodynamic therapy effect.

[0033] Based on the above analysis and verification by the technical personnel of this invention, the optimal method is to first couple porphyrin with hyaluronic acid to form an amphiphilic molecule, and then mix it with phospholipids to prepare a liposome membrane. This method ensures the uniform distribution of porphyrin, which is beneficial to the subsequent photodynamic therapy effect in vivo. Secondly, the amount of porphyrin and hyaluronic acid added can be adjusted according to needs without being affected by other liposome membrane materials, thus ensuring the targeted effect of the drug.

[0034] Thirdly, the present invention provides the application of targeted porphyrin microbubbles in the preparation of drugs for treating lymphatic malformations.

[0035] The technical solution provided by this invention has the following technical advantages: The targeted porphyrin microbubbles (HA-BLM@CP MBs) prepared in this invention, encapsulating a sclerosing agent, can be used to treat lymphatic malformations. Due to the presence of hyaluronic acid, a targeting molecule, on the microbubble surface, the sclerosing agent can be released targetedly and sustainably at the lesion site. Furthermore, the porphyrin molecules are uniformly dispersed within the microbubble shell, enabling photodynamic therapy in vivo. Therefore, the targeted porphyrin microbubbles provided by this invention address the problems of low efficacy and significant toxic side effects in clinical sclerotherapy while achieving synergistic photodynamic-sclerotherapy, thus improving treatment efficacy.

[0036] The extensive use of sclerosing agents such as bleomycin carries the risk of inducing pulmonary fibrosis. Excessive tissue infiltration can lead to muscle atrophy and spasms, and sclerosing agents pose a risk of extravasation, potentially causing local soft tissue damage or systemic venous return pathway embolism. Therefore, to ensure treatment safety, sclerotherapy requires image-guided injection before treatment. This invention utilizes the ultrasound signal of targeted porphyrin microbubbles to achieve ultrasound imaging-guided local drug injection, monitoring precise drug delivery to the lesion cyst cavity and improving injection safety. Attached Figure Description

[0037] Figure 1 White light photographs of HA-BLM@CP MBs and HA-BLM@CP NPs prepared by this invention.

[0038] Figure 2 Particle size distribution of HA-BLM@CP MBs prepared in Example 2.

[0039] Figure 3 Transmission electron microscopy image of HA-BLM@CP MBs prepared in Example 2.

[0040] Figure 4 The NMR characterization image of the PL-HA prepared in this invention.

[0041] Figure 5 Morphological observation and immunofluorescence imaging of primary cultured lymphatic endothelial cells.

[0042] Figure 6Results of reactive oxygen species assay after cell therapy experiment.

[0043] Figure 7 Results of CCK8 assay after cell therapy experiment.

[0044] Figure 8 Diagram of a real-time ultrasound imaging-guided local injection drug delivery process.

[0045] Figure 9 Image showing the effect of in vivo photodynamic-sclerosis synergistic treatment. Detailed Implementation

[0046] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0047] In the technical solution provided by this invention, the porphyrin can be any compound having a porphyrin ring. In a specific embodiment of this invention, the porphyrin structural formula used is as follows: Preparation of HA-BLM@CP MBs Targeted Microbubbles Step 1: Preparation of PL-HA amphiphilic molecules Weigh 40 mg of hyaluronic acid (HA, molecular weight 10000) and dissolve it in DMF. Add 1.6 mg of EDC in DMF solution and stir for 10 min. Then add 1 mg of NHS in DMF solution and stir for 1 h. Finally, add 10 mg of amino-modified porphyrin molecule PL-NH2 in DMF solution and react overnight (at least 8 h). Evaporate the DMF to dryness using a rotary evaporator (air pump, water bath 40 ℃), add 1 mL of water, dissolve under ultrasonic conditions in a water bath, dialyze through a 5500-6000 Da membrane in ultrapure water for 3 h, changing the water every 1 h, and freeze-dry for 24 h to obtain PL-HA powder for later use. The NMR characterization of PL-HA is shown below. Figure 4 As shown.

[0048] Step 2: Preparation of HA-BLM@CP NPs BLM, PL-HA, DSPC, and DSPE-PEG2000 were dissolved in chloroform to form a lipid mixture. The chloroform was evaporated to dryness using a rotary evaporator (water pump, water bath 40°C). An appropriate amount of PBS was injected, and the lipids were dissolved under ultrasonic conditions in a water bath to obtain bleomycin-loaded hyaluronic acid-porphyrin liposomes (HA-BLM@CP NPs).

[0049] The amount of liposome membrane material used in this step, based on mass fractions, is shown in the table below. The amount of bleomycin, the curing agent, added is 10% of the mass of the liposome membrane material.

[0050] Step 3: Preparation of HA-BLM@CP MBs Glycerol and 1,2-propanediol were mixed with the above HA-BLM@CP NPs dispersion at a volume ratio of 1:1:10 in 3 mL glass vials, totaling 1.2 mL. The sample vials were filled with perfluoropropane (C3F8) gas and mechanically stirred for 45 s using a Vialmix vibrating screen to obtain bleomycin-loaded hyaluronic acid-porphyrin microbubbles (HA-BLM@CP MBs).

[0051] The porphyrin microbubbles prepared in Examples 1-7 were left to stand at room temperature for 24 hours. The solution was observed to separate into layers, and the stability of the microbubbles was determined by observing the morphology of the microbubbles at different time points under a microscope. The results showed that the microbubbles prepared in Examples 2-4 had better stability. Among them, the porphyrin microbubbles prepared in Example 2 had the best morphological uniformity. Therefore, the most preferred liposome membrane material formulation of this invention is shown in Example 2.

[0052] Comparative Example 1 Step 1: Coupling HA with DSPE-PEG Weigh 30 mg of HA (M=10000), dissolve it in 10 mL of distilled water by sonication, add 60 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 90 mg of N-hydroxythiosuccinimide (NHS), adjust the pH to 7.5 with sodium hydroxide solution, activate in a 37℃ water bath for 24 h, add a suspension containing 72 mg of DSPE-PEG-NH2 dropwise with continuous stirring, react overnight, and remove excess reactants and byproducts from the reaction solution by dialysis using a 5000 pore size membrane to prepare DSPE-PEG-HA; Step 2: Preparation of drug-loaded liposomes BLM, PL, DSPC, and DSPE-PEG-HA were dissolved in chloroform to form a lipid mixture. The chloroform was evaporated to dryness using a rotary evaporator (water pump, water bath 40°C). An appropriate amount of PBS was injected, and the lipids were dissolved under ultrasonic conditions in a water bath to obtain bleomycin-loaded liposomes. The amounts of BLM and DSPC added were the same as in Example 2, and the amounts of PL and HA added after conversion were also the same as in Example 2.

[0053] Step 3: Preparation of porphyrin microbubbles Glycerol and 1,2-propanediol were mixed with the above liposome dispersion at a volume ratio of 1:1:10 in a 3 mL glass vial, for a total volume of 1.2 mL. The vial was filled with perfluoropropane gas and mechanically stirred for 45 s using a Vialmix vibrating screen to obtain bleomycin-loaded porphyrin microbubbles.

[0054] Comparative Example 2 Step 1: Preparation of drug-loaded liposomes BLM, PL, DSPC, and DSPE-PEG-NH2 were dissolved in chloroform to form a lipid mixture. The chloroform was evaporated to dryness using a rotary evaporator (water pump, water bath 40°C). An appropriate amount of PBS was injected, and the lipids were dissolved under ultrasonic conditions in a water bath to obtain bleomycin-loaded liposomes. The amounts of BLM and DSPC added were the same as in Example 2, and the amount of PL added after conversion was also the same as in Example 2.

[0055] Step 2: Conjugation of drug-loaded liposomes with HA Weigh 30 mg of HA (M=10000), dissolve it in 10 mL of distilled water by sonication after swelling, add 60 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 90 mg of N-hydroxythiosuccinimide (NHS), adjust the pH to 7.5 with sodium hydroxide solution, activate in a water bath at 37°C for 24 h, incubate with the drug-loaded liposomes prepared in step 1 at 37°C overnight, and remove excess reactants and byproducts from the reaction solution by dialyzing with a 5000 pore size dialysis membrane to obtain HA-coupled drug-loaded liposomes; Step 3: Preparation of porphyrin microbubbles Glycerol and 1,2-propanediol were mixed with the above liposome dispersion at a volume ratio of 1:1:10 in a 3 mL glass vial, for a total volume of 1.2 mL. The vial was filled with perfluoropropane gas and mechanically stirred for 45 s using a Vialmix vibrating screen to obtain bleomycin-loaded porphyrin microbubbles.

[0056] Effect Example (1) Morphological characterization of HA-BLM@CP MBs Take the HA-BLM@CP MBs solution (1 mg / mL) prepared in Example 2 of this invention, add an appropriate amount of PBS to dilute, mix thoroughly, and use a Coulter multiplier 4 to detect the particle size of HA-BLM@CP MBs. The results are as follows. Figure 2 As shown.

[0057] The HA-BLM@CP MBs solution prepared in Example 2 of this invention was diluted with deionized water and mixed thoroughly for later use. 4 μL of the above aqueous solution was dropped onto a 300-mesh polyvinyl acetate coated copper mesh. After incubation for 10 min, excess sample solution was absorbed with filter paper. The copper mesh loaded with the sample was stained with 4% uranium acetate for 10 min. It was then washed several times with distilled water, air-dried at room temperature, and observed and photographed under a transmission electron microscope. The results are as follows: Figure 3 As shown.

[0058] (2) Cell experiments (2.1) Primary culture of lymphatic endothelial cells Clinical patients undergoing lymphatic vessel malformation resection surgery were collected for subsequent primary cell culture. Fresh tissue samples were used to separate lymphatic vessels from surrounding tissue. Lymphatic vessel tissue was immersed in PBS buffer for 3 min to restore morphology. The tissue was then minced using sterile scissors, and collagenase solution was added to the minced tissue pieces. The mixture was incubated at 37 °C for 60 min, with the tissue pieces resuspended every 10 min to ensure adequate contact between the collagenase and the tissue. The digestion products were filtered through a nylon sieve. The filtrate was transferred to cell culture dishes, EGM complete medium was added, and the dishes were incubated at 37 °C in a 5% CO2 incubator. Half the medium was replaced after 48 h, and the entire medium was replaced after 72 h. Cell growth was observed after 3 days. Peripheral cell culture: Peripheral lymphatic tissue was collected and cultured using the same method as above, in DMEM high-glucose medium containing 10% fetal bovine serum. The expression of LYVE-1, CD31, VEGFR-3, and Podoplanin receptors in primary cultured lymphatic endothelial cells and frozen tissue sections was detected to verify the purity of primary cultured lymphatic endothelial cells and the feasibility of this project.

[0059] The results are as follows Figure 5As shown in Figure 5A, primary cultured lymphatic endothelial cells exhibited a regular spindle-shaped morphology under a light microscope. Immunofluorescence imaging revealed high expression of CD31 and LYVE-1 receptors in the primary cultured lymphatic endothelial cells (Figures 5B-C). Frozen sections prepared from clinical lymphatic malformation tissue were also observed to highly express CD31 and LYVE-1 receptors using immunofluorescence imaging (Figures 5D-E). These results demonstrate that primary cultured lymphatic endothelial cells can be used for subsequent cell experiments in this study, and that the preparation of a targeted thermosensitive nanogel system based on HA is feasible.

[0060] (2.2) Cell therapy research Logarithmic growth phase lymphatic endothelial cells were seeded in 96-well cell culture plates at a density of 1 × 10⁶ cells / well. 4 Cells / wells were placed in EGM complete culture medium and cultured in an incubator for 24 hours. The cell therapy experiment was conducted in 3 groups, using microbubbles prepared in Example 2 of this invention, Comparative Example 1, and Comparative Example 2, with a blank control group also included.

[0061] The three types of porphyrin microvesicles were prepared into 1 mM culture medium solutions using EGM complete medium. 200 μL of culture medium was added to each well, and the cells were incubated at 37 ℃ in a 5% CO2 incubator for 8 h. After incubation, the culture medium was discarded, the cells were washed with PBS, and then EGM complete medium was added. The photothermal therapy (+laser) group was irradiated for 10 min with a 658 nm laser at a power density of 200 mW. After irradiation, the reactive oxygen species (ROS) levels in the cells of the three groups were detected using a reactive oxygen species (ROS) detection kit (chemifluorescence method). The results are as follows: Figure 6 As shown in the figure; subsequently, the cells were cultured for another 24 h in a 37 ℃, 5% CO2 incubator, and the cell viability was detected using the CCK8 assay kit. The results are shown in the figure. Figure 7 As shown.

[0062] The cell experiment results show that the porphyrin microvesicles prepared in Example 2 of this invention have the best cell therapy effect. The technicians analyzed that, in Comparative Example 2, due to the coupling of HA on the liposome surface, the actual coupling rate was not high, resulting in poor targeting of the porphyrin microvesicles prepared in Comparative Example 2 and insufficient uptake in cells, leading to insufficient drug killing effect on cells. Therefore, the product prepared in Comparative Example 2 was excluded. Compared with Example 2 and Comparative Example 1, the technicians believe that the porphyrin in Comparative Example 1 could not be uniformly dispersed, resulting in poor photodynamic therapy effect and a significantly higher cell survival rate compared to the Example group. This analysis is also verified by the reactive oxygen species (ROS) detection results, where the ROS level in Comparative Example 1 was significantly lower than that in the Example group.

[0063] (3) In vivo test (3.1) Construction of a mouse model of lymphatic malformation Two subcutaneous injections were administered to the left neck of BALB / c mice, and the mice were observed for three weeks. Obvious clear or translucent cystic lesions were observed in the subcutaneous tissue of the left neck, morphologically resembling microcystic lymphatic malformations. The larger vesicles were of moderately firm texture, completely encapsulated, and could be completely separated from the surrounding soft tissue. Several smaller vesicles with diameters of 1-3 mm were observed around the larger vesicles. Hematoxylin and eosin (HE) staining of tissue sections from the subcutaneous lesions revealed under a microscope that the lesions consisted of vesicles formed by dilated, irregular lymphatic vessels of varying thicknesses. The larger cysts had thicker vesicle walls, and the matrix was composed of fibrous connective tissue with varying degrees of lymphocyte infiltration. These results demonstrate that we successfully induced the formation of lymphatic malformations in the head and neck of mice.

[0064] (3.2) Ultrasound imaging test - real-time ultrasound imaging-guided local injection drug delivery The HA-BLM@CP MBs prepared in Example 2 of this invention were configured as a 1 mM saline solution. 100 μL was drawn and percutaneously inserted into the lesion under ultrasound guidance. The direction of needle movement and the depth reached by the needle tip were monitored, aiming to puncture the cyst cavity while avoiding surrounding blood vessels and normal tissue. The injection process was guided by ultrasound imaging. Simultaneously, the diffusion range of HA-BLM@CP MBs was observed under ultrasound imaging until it infiltrated the entire lesion cavity.

[0065] Using mice with lymphatic malformations, ultrasound imaging-guided local injection was performed, and the results... Figure 8 As shown, the ultrasound signal enhancement can be clearly seen during the injection process, and the ultrasound signal can be used to precisely control the injection location and depth.

[0066] (3.3) Evaluation of HA-BLM@CP MBs in vivo photodynamic-sclerotherapy synergistic treatment Mice with lymphangiogenic malformations were divided into three groups of five mice each. One group served as a blank control group, while the other two groups received local injections of porphyrin microbubbles prepared in Example 2 and Comparative Example 1, respectively, at doses of 100 μL and 100 μg. Following injection, the mice were irradiated with a 658 nm laser (200 mW, 20 min). The size of the lesions and the mice's weight were recorded before and after treatment. Treatment was repeated after 10 days until the lesions completely disappeared. Before the first injection and after the end of the treatment cycle, MRI (T2 signal) was used to observe changes in the lesions before and after treatment to confirm complete lesion regression. Treatment records for the experimental groups are as follows: Figure 9 As shown.

[0067] The experimental results show that the porphyrin microbubbles prepared in Example 2 of this invention have a better therapeutic effect in mice compared with Comparative Example 1. Technical personnel analyzed that the porphyrin microbubbles prepared by the method provided in Example 2 of this invention have porphyrin uniformly dispersed on the surface of the microbubbles. When the microbubbles enter the body and are burst by ultrasound, the porphyrin is uniformly dispersed into each nanoliposome, resulting in a better in vivo photodynamic therapy effect. In contrast, the porphyrin microbubbles prepared in Comparative Example 1 have porphyrin in a freely dispersed or aggregated state, resulting in uneven dispersion after bursting by ultrasound in the body, leading to poor photodynamic therapy effects.

[0068] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A targeted porphyrin microbubble for the treatment of lymphatic malformations, characterized in that, The microbubbles consist of a bilayer shell and a bio-inert gas encapsulated inside the bilayer shell. The bilayer shell is formed by amphiphilic molecules, and drug particles are dispersed within the bilayer shell. The drug particles are a hardening agent selected from bleomycin or bleomycin. The bilayer shell is composed of porphyrin-hyaluronic acid coupling compound, distearylphosphatidylcholine and distearylphosphatidylethanolamine-polyethylene glycol in a mass ratio of (1-5):(4-8):1; the porphyrin-hyaluronic acid coupling compound is obtained by EDC-NHS amide reaction, which involves reacting activated hyaluronic acid with amino-modified porphyrin overnight, dialyzing and lyophilizing, with a mass ratio of hyaluronic acid to porphyrin of (2-6):

1.

2. The targeted porphyrin microbubbles for the treatment of lymphatic malformations according to claim 1, characterized in that, The bio-inert gas is selected from at least one of perfluoropropane, perfluorobutane, and sulfur hexafluoride.

3. A method for preparing the targeted porphyrin microbubbles for the treatment of lymphatic malformations as described in claim 1, the preparation method comprising the following steps: (1) The activated hyaluronic acid was reacted with amino-modified porphyrin overnight using the EDC-NHS amide reaction, and then dialyzed and lyophilized to obtain porphyrin-hyaluronic acid conjugate. The mass ratio of hyaluronic acid to porphyrin was (2-6):

1. (2) Using the thin-film hydration method, porphyrin-hyaluronic acid coupling compound, distearylphosphatidylcholine, and distearylphosphatidylethanolamine-polyethylene glycol were dissolved in chloroform and / or methanol solution at a mass ratio of (1-5):(4-8):

1. A hardening agent was added, the solvent was removed by rotary evaporation, hydration solution was added, and the mixture was sonicated to obtain drug-loaded liposomes. (3) Mix drug-loaded liposomes, glycerol and 1,2-propanediol in a volume ratio of 10:(1-2):(1-2), introduce biological inert gas, and mechanically stir with a vibrating screen for 45-100s to obtain targeted porphyrin microbubbles.

4. The preparation method according to claim 3, characterized in that, The amount of hardener added is 5-30% of the liposome membrane material, and the hardener is selected from bleomycin or bleomycin.

5. The use of the targeted porphyrin microbubbles according to claim 1 in the preparation of a drug for treating lymphatic malformations.

Images