A nano-drug-loaded vesicle capable of crossing blood-brain barrier in a graded response and a preparation method and application thereof

Abstract

The application relates to a nano-drug-loaded vesicle for crossing a blood-brain barrier in a graded response and a preparation method and application thereof. The preparation method of the nano-drug-loaded vesicle comprises the following steps: S1. mixing a solution containing a polymer DSPE-PEG-BEAGA2, a polymer DSPE-PEG-N3, an anticancer drug and a liposome raw material with a solution containing a photosensitizer under ultrasonic conditions to form an emulsion; S2. under ultrasonic conditions, the emulsion prepared in step S1 is added into water to induce self-assembly to form a basic vesicle, and then the basic vesicle is incubated with ApoE-DBCO and a polymer 4-arm-PEG-phe-DBCO, so that the nano-drug-loaded vesicle is obtained. After the nano-drug-loaded vesicle efficiently crosses the BBB, self-assembly clusters are formed, membrane fusion between the nano-drug-loaded vesicles is triggered, the nano-drug-loaded vesicles become micrometer-level large vesicles, and then cavitation effect can be realized under the action of ultrasonic waves, so that drug penetration to deep tumor is promoted.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, and more specifically, relates to a graded response nanodrug-loaded vesicle that crosses the blood-brain barrier, its preparation method, and its application. Background Technology

[0002] Gliomas are a type of highly malignant intracranial tumor with an extremely poor prognosis. Current clinical treatment primarily involves surgical resection and chemotherapy. However, due to the invasive growth of these tumors and their indistinct boundaries with normal brain tissue, complete surgical removal is difficult, resulting in a high recurrence rate. Furthermore, chemotherapy drugs lack tumor targeting, damaging normal tissues while killing tumor cells, leading to significant toxic side effects. Affected by these factors, the median survival for patients is only 12-18 months, with a 5-year survival rate of less than 5%, and treatment-related complications are quite severe.

[0003] The development of nanomedicine has provided new avenues for improving drug efficacy and reducing toxic side effects, but it still faces a key obstacle in the treatment of gliomas—the blood-brain barrier (BBB). The BBB, composed of brain capillary endothelial cells, tight junctions, the basement membrane, and pericytes, protects the central nervous system from harmful substances, but also prevents almost all macromolecular drugs and over 98% of small molecule drugs from effectively penetrating the brain. Furthermore, the uneven distribution of nanomedicines within gliomas further affects efficacy. Gliomas possess a unique tumor microenvironment characterized by uneven blood perfusion, absence of lymphatic vessels, dense extracellular matrix, and high interstitial pressure. In advanced stages, central necrosis and avascular distribution in the tumor center lead to nanomedicines remaining largely at the tumor periphery, hindering their penetration. To accelerate drug release and deep penetration into the tumor, sonodynamic therapy is commonly used. However, the efficacy of sonodynamic therapy is closely related to the degree of cavitation. Effective cavitation requires the use of micron-sized vesicles, but these are easily cleared rapidly by the reticuloendothelial system, reducing the efficiency of the blood-brain barrier. If high-power focused ultrasound is used to enhance the cavitation effect, it can easily cause cerebral vascular damage, rupture and hemorrhage, and even lead to serious side effects such as protein extravasation, cerebral edema and inflammation.

[0004] Prior art, disclosed in publication number CN114452404A, describes a vesicle with anchoring and charge-flipping functions, its preparation, and its application. This vesicle is prepared through a series of reactions including polymerization, ammonolysis, amidation, and deprotection, exhibiting excellent anchoring and charge-flipping capabilities. The anchoring peptide ApoE linked to this vesicle enables it to efficiently anchor to the surface of intracranial capillary endothelium. Under the influence of GTT on the BBB surface, specific segments on its surface are cleaved, exposing amino groups and significantly increasing the charge, promoting AMT-mediated endocytosis and allowing the vesicle to efficiently cross the BBB. Furthermore, this vesicle is dually sensitive to pH and enzymes, and can expand to over 500 nm in the slightly acidic environment of the tumor site, enhancing the cavitation effect under ultrasound and promoting drug penetration into deeper tumors. However, while this technique can increase the vesicle size through expansion, the final vesicle size remains at the nanometer level, resulting in limited cavitation effects, which require further improvement.

[0005] Therefore, there is an urgent need to develop a technology that can cross the blood-brain barrier and achieve effective cavitation using micron-sized vesicles. Summary of the Invention

[0006] The primary objective of this invention is to provide a method for preparing drug-loaded nanovesicles, addressing the shortcomings or defects of existing technologies.

[0007] Another objective of this invention is to provide drug-loaded nanovesicles prepared by the above-described method. Under ultrasound, these drug-loaded nanovesicles self-assemble into clusters and fuse with a membrane to form micron-sized vesicles, enhancing the ultrasonic cavitation effect and thereby accelerating drug release and penetration into deeper tumors, thus improving therapeutic efficacy.

[0008] Another object of the present invention is to provide the application of the above-mentioned drug-loaded nanovesicles in the preparation of drugs for treating glioma.

[0009] To achieve the above objectives, the present invention employs the following technical solution: A method for preparing drug-loaded nanovesicles includes the following steps: S1. Under ultrasonic conditions, a solution containing polymers DSPE-PEG-BEAGA2 and DSPE-PEG-N3, anticancer drugs and liposome raw materials is mixed with a solution containing a sound-sensitive agent to form an emulsion; S2. Under ultrasonic conditions, the emulsion obtained in step S1 is added dropwise to water to induce self-assembly and form basic vesicles. The basic vesicles are then incubated with ApoE-DBCO and polymer 4-arm-PEG-phe-DBCO to obtain the drug-loaded nanovesicles. The general structural formula of the polymer DSPE-PEG-BEAGA2 is: Where n is a positive integer from 6 to 10, and m is a positive integer from 35 to 55; The general structural formula of the polymer 4-arm-PEG-phe-DBCO is: , where a is a positive integer from 10 to 15.

[0010] This invention involves loading polymers DSPE-PEG-BEAGA2 and 4-arm-PEG-phe-DBCO onto the surface of nanovesicles. Under the targeting action of ApoE peptides, the nanovesicles are targeted and anchored on the surface of the blood-brain barrier (BBB). Then, through the action of γ-glutamyl transferase (GGT) on the BBB surface, polymer DSPE-PEG-BEAGA2 undergoes an enzymatic response and releases primary amines, increasing surface positive charge and promoting AMT-mediated endocytosis. This allows the nanovesicles to efficiently cross the BBB and is less susceptible to clearance by the reticuloendothelial system. Simultaneously, polymer 4-arm-PEG-phe-DBCO can also limit the fusion of nanovesicles with the blood-brain barrier (BCECs) membrane during crossing, thus reducing penetration efficiency. After crossing the BBB, the nanovesicles accumulate at the tumor tissue site. Polymer 4-arm-PEG-phe-DBCO triggers an acid response and bond breaking, while polymer DSPE-PEG-BEAGA2... 2k Under the influence of the remaining segments of -BEAGA2, nanovesicles assemble into clusters and trigger membrane fusion between nanovesicles, becoming micron-sized large vesicles. This allows for cavitation under ultrasound, promoting drug penetration into the depths of the tumor.

[0011] In the vesicle membrane fusion process, the DSPE segment in the polymer DSPE-PEG-BEAGA2 plays a crucial role. Containing phospholipid groups, it exhibits good compatibility with liposomes, thereby enhancing the degree of vesicle membrane fusion and enabling vesicles to fuse into micron-sized vesicles. If the DSPE segment in polymer DSPE-PEG-BEAGA2 is replaced with other unsuitable segments, such as polyethylene glycol derivatives (e.g., mPEG-OH), random polyethers, or PDHD groups, these segments lack long-chain hydrophobic tails and cannot effectively insert into the hydrophobic bilayer of the liposome membrane. They can only be adsorbed onto the membrane surface through weak van der Waals forces. This shallow adsorption is insufficient to provide adequate membrane perturbation and curvature deformation energy, resulting in limited membrane fusion and failing to drive vesicles to become micron-sized vesicles.

[0012] The polymer 4-arm-PEG-phe-DBCO also plays a crucial role in vesicle membrane fusion. Targeting the slightly acidic tumor microenvironment of gliomas, this invention employs a pH-responsive design. This not only ensures a more stable response but also allows the amide bonds on the polymer 4-arm-PEG-phe-DBCO to rapidly break under slightly acidic conditions. This removes the long PEG chains that restrict membrane fluidity, relieves inhibition of membrane fusion, and drives vesicles to rapidly complete membrane fusion, ultimately growing into large micron-sized vesicles. If other response mechanisms (such as enzyme responses or ROS responses) are used, insufficient stability of the triggering and unlocking mechanisms often leads to incomplete shedding of groups restricting membrane fusion, limiting the degree of membrane fusion and ultimately making it difficult for vesicles to reach the required micron-sized dimensions.

[0013] Preferably, the average particle size of the drug-loaded nanovesicles is 50~200 nm.

[0014] Preferably, the mass ratio of the polymer DSPE-PEG-BEAGA2, polymer DSPE-PEG-N3, liposome raw material and anticancer drug is 2.5~3:1.5~2:36~45:1.

[0015] In this invention, the liposome raw material can be selected from common liposome raw materials in the art, such as: lecithin, cholesterol, phosphatidylethanolamine, dioleoylphosphatidylethanolamine, dimyristoylphosphatidylethanolamine, distearate phosphatidylethanolamine, dimyristoylphosphatidylglycerol, or dioleoylphosphatidylglycerol. Preferably, the liposome raw material is one or more of lecithin, cholesterol, and phosphatidylethanolamine. More preferably, the liposome raw material is lecithin, cholesterol, and phosphatidylethanolamine.

[0016] Preferably, the mass ratio of lecithin, cholesterol, and phosphatidylethanolamine is 1~3:1~2:1.

[0017] Preferably, the molar ratio of N3 groups in the polymer DSPE-PEG-N3 to DBCO groups in the polymer ApoE-DBCO to DBCO groups in the polymer 4-arm-PEG-phe-DBCO is 8:3~5:1~3.

[0018] In this invention, the "DSPE-PEG-N3" can be derived from commercially available products or prepared in-house. The preparation method is not particularly limited, as long as it can produce the DSPE-PEG-N3. Preferably, the degree of polymerization of the PEG segments in the DSPE-PEG-N3 is 77, meaning the DSPE-PEG-N3 is DSPE-PEG. 3.4k -N3.

[0019] In this invention, "ApoE-DBCO" refers to the product of chemically coupling an apolipoprotein E (ApoE) peptide with a dibenzocyclooctylene (DBCO) group. The ApoE-DBCO can be derived from commercially available products or prepared in-house; the preparation method is not particularly limited, as long as it can be prepared.

[0020] In this invention, the anticancer drug can be selected from commonly used anticancer drugs in the field for the treatment of glioma, such as decitabine, temozolomide or lomustine.

[0021] Preferably, the solvent in the solution containing polymer DSPE-PEG-BEAGA2, polymer DSPE-PEG-N3, the anticancer drug, and the liposome raw material is one or more of dimethyl sulfoxide, N,N-dimethylformamide, N,N-dimethylacetamide, chloroform, or methanol. A mixed solution of chloroform and methanol is preferred. More preferably, the volume ratio of chloroform to methanol is 1:1.

[0022] In this invention, the sound sensitizer can be a commonly used sound sensitizer in the art, such as iRGD-ILD, hematoporphyrin, or indocyanine green (ICG). Preferably, the sound sensitizer is iRGD-ILD.

[0023] Preferably, in step S1, the emulsion further includes an ultrasound contrast agent. After adding the ultrasound contrast agent, the drug-loaded nanovesicles can be used for MR imaging.

[0024] In this invention, the ultrasound contrast agent can be a commonly used ultrasound contrast agent in the art, such as one of pentafluorobutane, perfluoropropane, or perfluorohexane. Preferably, the ultrasound contrast agent is pentafluorobutane.

[0025] Preferably, the polymer DSPE-PEG-BEAGA2 is prepared by the following steps: S11. DSPE-PEG-NH2 is reacted with N,N'-difluorenemethoxycarbonyl-D-lysine via an amidation reaction to obtain DSPE-PEG-lys-Fmoc; S12. Remove the Fmoc protecting group from DSPE-PEG-lys-Fmoc to obtain DSPE-PEG-lys, and then react it with N-fluorenmethoxycarbonyl-L-2-aminobutyric acid to obtain DSPE-PEG-(ABU-Fmoc)2; S13. Remove the Fmoc protecting group from DSPE-PEG-(ABU-Fmoc)2 to obtain DSPE-PEG-ABU2, and then react it with Boc-Glu to obtain DSPE-PEG-(ABU-Glu-Boc)2. S14. Remove the Boc protecting group from DSPE-PEG-(ABU-Glu-Boc)2 to obtain the polymer DSPE-PEG-BEAGA2.

[0026] Preferably, in step S11, the degree of polymerization of the PEG segments in the DSPE-PEG-NH2 is 40~50.

[0027] More preferably, in step S11, the degree of polymerization of the PEG segment in the DSPE-PEG-NH2 is 45, that is, the DSPE-PEG-NH2 is DSPE-PEG. 2k -NH2.

[0028] Preferably, in step S11, the molar ratio of DSPE-PEG-NH2 to N,N'-difluorenemethoxycarbonyl-D-lysine is 1:2~4.

[0029] Preferably, in step S11, the amidation reaction takes 20-24 hours.

[0030] Preferably, in step S11, after the amidation reaction, a post-processing is required. The post-processing involves transferring the reaction solution to a dialysis bag, dialyzing it with methanol and ultrapure water for 6-8 hours respectively, and then freeze-drying the dialyzed product.

[0031] More preferably, in step S11, the amidation reaction is as follows: DSPE-PEG-NH2 and N,N'-difluorenemethoxycarbonyl-D-lysine are dissolved in an organic solvent, an activator and a stabilizer are added, and the reaction is stirred.

[0032] In the amidation reaction described in this invention, the activator can be selected from activators well known in the art that can be used in amidation reactions, preferably EDC. Those skilled in the art know that, in the amidation reaction, the conventional amount of the activator is 1.0 to 1.5 times the molar amount of the carboxyl substrate.

[0033] In the amidation reaction described in this invention, the stabilizer can be selected from stabilizers well known in the art that can be used in amidation reactions, preferably NHS. Those skilled in the art know that, in the amidation reaction, the conventional amount of the stabilizer is 1.0 to 1.5 times the molar amount of the carboxyl base.

[0034] In the amidation reaction described in this invention, the organic solvent can be a common organic solvent in the art, such as one or more of dichloromethane, trichloromethane, dimethyl sulfoxide, or N,N-dimethylformamide. Preferably, the organic solvent is dichloromethane and dimethyl sulfoxide. More preferably, the volume ratio of dichloromethane to dimethyl sulfoxide is 5:2.

[0035] Preferably, the reaction equation for step S11 is as follows: .

[0036] Preferably, in step S12, the method for removing the Fmoc protecting group from DSPE-PEG-lys-Fmoc is as follows: dissolve the Fmoc of DSPE-PEG-lys-Fmoc in an organic solvent, add piperidine and react for 20-30 minutes to obtain DSPE-PEG-lys.

[0037] More preferably, after the reaction is completed, post-processing is required. The post-processing involves transferring the reaction solution to a dialysis bag, dialyzing it with methanol and ultrapure water for 6-8 hours respectively, and then freeze-drying the dialyzed product.

[0038] More preferably, the organic solvent includes dimethyl sulfoxide.

[0039] More preferably, the piperidine has a volume percentage of 15-25% in the organic solvent. Most preferably, it has a volume percentage of 20%.

[0040] More preferably, the reaction time is 30 minutes.

[0041] Preferably, in step S12, the molar ratio of DSPE-PEG-lys to N-fluorenemethoxycarbonyl-L-2-aminobutyric acid is 1:2~4.

[0042] Preferably, in step S12, the amidation reaction takes 20-24 hours.

[0043] Preferably, in step S12, after the amidation reaction, a post-processing is required. The post-processing involves transferring the reaction solution to a dialysis bag, dialyzing it with methanol and ultrapure water for 6-8 hours respectively, and then freeze-drying the dialyzed product.

[0044] More preferably, in step S12, the amidation reaction is as follows: DSPE-PEG-lys and N-fluorenylmethoxycarbonyl-L-2-aminobutyric acid (Fmoc-ABU-OH) are dissolved in an organic solvent, an activator and a stabilizer are added, and the mixture is stirred to react.

[0045] In the amidation reaction described in this invention, the activator can be selected from activators well known in the art that can be used in amidation reactions, preferably EDC. Those skilled in the art know that, in the amidation reaction, the conventional amount of the activator is 1.0 to 1.5 times the molar amount of the carboxyl substrate.

[0046] In the amidation reaction described in this invention, the stabilizer can be selected from stabilizers well known in the art that can be used in amidation reactions, preferably NHS. Those skilled in the art know that, in the amidation reaction, the conventional amount of the stabilizer is 1.0 to 1.5 times the molar amount of the carboxyl base.

[0047] In the amidation reaction described in this invention, the organic solvent can be a common organic solvent in the art, such as one or more of dichloromethane, trichloromethane, dimethyl sulfoxide, or N,N-dimethylformamide. Preferably, the organic solvent is dichloromethane and dimethyl sulfoxide.

[0048] Preferably, the reaction equation for step S12 is as follows: .

[0049] Preferably, in step S13, the method for removing the Fmoc protecting group from DSPE-PEG-(ABU-Fmoc)2 is as follows: dissolve DSPE-PEG-(ABU-Fmoc)2 in an organic solvent, add piperidine and react for 20-30 minutes to obtain DSPE-PEG-ABU2.

[0050] More preferably, after the reaction is completed, post-processing is required. The post-processing involves transferring the reaction solution to a dialysis bag, dialyzing it with methanol and ultrapure water for 6-8 hours respectively, and then freeze-drying the dialyzed product.

[0051] More preferably, the organic solvent includes dimethyl sulfoxide.

[0052] More preferably, the piperidine has a volume percentage of 15-25% in the organic solvent. Most preferably, it has a volume percentage of 20%.

[0053] More preferably, the reaction time is 30 minutes.

[0054] Preferably, in step S13, the molar ratio of DSPE-PEG-ABU2 to Boc-Glu is 1:2~4.

[0055] Preferably, in step S13, the amidation reaction takes 20-24 hours.

[0056] Preferably, in step S13, after the amidation reaction, a post-processing is required. The post-processing involves transferring the reaction solution to a dialysis bag, dialyzing it with methanol and ultrapure water for 6-8 hours respectively, and then freeze-drying the dialyzed product.

[0057] More preferably, in step S13, the amidation reaction is as follows: dissolve DSPE-PEG-ABU2 and Boc-Glu in an organic solvent, add an activator and a stabilizer, and stir the reaction.

[0058] In the amidation reaction described in this invention, the activator can be selected from activators well known in the art that can be used in amidation reactions, preferably EDC. Those skilled in the art know that, in the amidation reaction, the conventional amount of the activator is 1.0 to 1.5 times the molar amount of the carboxyl substrate.

[0059] In the amidation reaction described in this invention, the stabilizer can be selected from stabilizers well known in the art that can be used in amidation reactions, preferably NHS. Those skilled in the art know that, in the amidation reaction, the conventional amount of the stabilizer is 1.0 to 1.5 times the molar amount of the carboxyl base.

[0060] In the amidation reaction described in this invention, the organic solvent can be a common organic solvent in the art, such as one or more of dichloromethane, trichloromethane, dimethyl sulfoxide, or N,N-dimethylformamide. Preferably, the organic solvent is dichloromethane and dimethyl sulfoxide.

[0061] Preferably, the reaction equation for step S13 is as follows: .

[0062] Preferably, in step S14, the method for removing the Boc protecting group from DSPE-PEG-(ABU-Glu-Boc)2 is as follows: DSPE-PEG-(ABU-Glu-Boc)2 is dissolved in trifluoroacetic acid (TFA) and stirred to react; then, the reaction solution is added dropwise to a poor solvent until the precipitate is completely precipitated, the precipitate is collected and dissolved again in an organic solvent, dialyzed, and freeze-dried.

[0063] More preferably, the reaction takes 30-60 minutes.

[0064] More preferably, the undesirable solvent is diethyl ether.

[0065] More preferably, collecting the precipitate also includes a washing step.

[0066] More preferably, the organic solvent is dichloromethane.

[0067] As a specific example, step S14 is as follows: dissolve DSPE-PEG-(ABU-Glu-Boc)2 in trifluoroacetic acid (TFA) and stir for 30-60 min; then, add the reaction solution dropwise to diethyl ether under stirring until the precipitate is completely precipitated; wash and collect the precipitate, add dichloromethane to redissolve the precipitate, and dialyze with methanol and ultrapure water for 6-8 h in sequence, and freeze-dry the dialyzed product.

[0068] Preferably, the polymer 4-arm-PEG-phe-DBCO is prepared by the following steps: S21. The carboxyl group in 2,5-dihydroxy-4-methyl-2,5-dioxy-3-furanpropionic acid is activated to an acyl chloride group to obtain 2,5-dihydroxy-4-methyl-2,5-dioxy-3-furanpropionic acid chloride; S22. Dissolve 4-arm-PEG-OH in a solvent, add 2,5-dihydroxy-4-methyl-2,5-dioxy-3-furanopropionyl chloride to undergo esterification reaction, and separate to obtain 4-arm-PEG-CDM; S23. Dissolve 4-arm-PEG-CDM in a solvent, add DBCO-NH2 to undergo a ring-opening reaction, and separate to obtain the polymer 4-arm-PEG-phe-DBCO.

[0069] Preferably, in step S21, the degree of polymerization of the PEG segment in the 4-arm-PEG-OH is 11~13.

[0070] More preferably, in step S21, the degree of polymerization of the PEG segment in the 4-arm-PEG-OH is 11, that is, the 4-arm-PEG-OH is 4-arm-PEG. 0.5k -OH.

[0071] Preferably, in step S21, the method for activating the carboxyl group to an acyl chloride group is as follows: 2,5-dihydroxy-4-methyl-2,5-dioxy-3-furanpropionic acid and the catalyst are dissolved in an organic solvent, oxalyl chloride is added dropwise to the system, the reaction is stirred, and rotary evaporation is performed to obtain 2,5-dihydroxy-4-methyl-2,5-dioxy-3-furanpropionic acid chloride.

[0072] More preferably, the organic solvent is anhydrous dichloromethane.

[0073] More preferably, the catalyst is N,N-dimethylformamide.

[0074] More preferably, the reaction is carried out for 3 to 5 hours.

[0075] Preferably, in step S22, the molar ratio of 4-arm-PEG-CDM to 2,5-dihydroxy-4-methyl-2,5-dioxy-3-furanopropionyl chloride is 1:4~7.

[0076] Preferably, in step S22, the solvent is dichloromethane.

[0077] Preferably, in step S22, the catalyst for the esterification reaction is pyridine.

[0078] Preferably, in step S22, the esterification reaction takes 3 to 5 hours.

[0079] Preferably, in step S22, the separation includes the following steps: adding a terminating agent, separating to obtain an organic phase, drying, filtering to obtain a filtrate, and then adding the filtrate dropwise to a poor solvent until the precipitate is completely precipitated, obtaining the precipitate and drying it.

[0080] More preferably, the terminating agent is saturated ammonium chloride.

[0081] More preferably, the undesirable solvent is anhydrous diethyl ether.

[0082] As a specific example, the separation in step S22 is as follows: saturated ammonium chloride is added to terminate the reaction, and the organic phase is separated by a separatory funnel; excess Na2SO4 is added to the organic phase for drying, and the filtrate is obtained by filtration; the filtrate is added dropwise to anhydrous diethyl ether until the precipitate is completely precipitated, the precipitate is obtained by centrifugation, and dried for more than 12 hours.

[0083] Preferably, in step S23, the molar ratio of 4-arm-PEG-CDM to DBCO-NH2 is 1:1~3.

[0084] Preferably, in step S23, the solvent is dichloromethane.

[0085] Preferably, in step S23, the catalyst for the ring-opening reaction is N,N-dimethylformamide.

[0086] Preferably, in step S23, the ring-opening reaction time is 24-26 hours.

[0087] Preferably, in step S23, the separation includes the following steps: adding the reaction solution dropwise into a poor solvent until the precipitate is completely precipitated, separating the precipitate, and drying it.

[0088] Preferably, the reaction equation for the polymer 4-arm-PEG-phe-DBCO is as follows: .

[0089] Preferably, in step S2, after the induced self-assembly to form basic vesicles, a dialysis purification step is also included.

[0090] Preferably, in step S2, the mixing process further includes a low-temperature ultrafiltration concentration step.

[0091] The drug-loaded nanovesicles prepared by the above method are also within the scope of protection of this invention.

[0092] The application of the aforementioned drug-loaded nanovesicles in the preparation of drugs for treating gliomas is also within the scope of protection of this invention.

[0093] Preferably, the above-mentioned drug-loaded nanovesicles are used in the preparation of drugs for treating gliomas under ultrasound.

[0094] Preferably, the above-mentioned drug-loaded nanovesicles are used in the preparation of drugs for treating gliomas by fusing energy membranes into micron-sized vesicles and then using ultrasound.

[0095] More preferably, the average particle size of the micron-sized vesicles is 1~2μm.

[0096] Compared with the prior art, the beneficial effects of the present invention include: The nanovesicles prepared in this invention possess excellent anchoring, charge reversal, and membrane fusion capabilities. The polymers DSPE-PEG-BEAGA2 and 4-arm-PEG-phe-DBCO loaded onto the surface of the nanovesicles not only anchor the nanovesicles to the surface of the BBB under the targeting effect of the ApoE peptide and facilitate their smooth and efficient crossing of the BBB, but also, after the nanovesicles cross the BBB, the specific segments of polymer DSPE-PEG-BEAGA2 and the pH-responsive design of polymer 4-arm-PEG-phe-DBCO promote the self-assembly of nanovesicles into clusters and trigger membrane fusion, transforming them into micron-sized large vesicles. This allows for cavitation under ultrasonic irradiation, thereby enhancing therapeutic efficacy. Attached Figure Description

[0097] Figure 1 For polymer DSPE-PEG 2k -H NMR spectrum of BEAGA2.

[0098] Figure 2 Polymer 4-arm-PEG 0.5k -phe-DBCO 1H NMR spectrum.

[0099] Figure 3 The ability of the drug-loaded nanovesicles prepared in Example 1 and Comparative Example 1 to cross the BBB was tested.

[0100] Figure 4 This is a comparison of the surface Zata potentials of the drug-loaded nanovesicles prepared in Example 1 under different concentrations of GGT enzyme treatment.

[0101] Figure 5 TEM images of the drug-loaded nanovesicles prepared in Example 1 after incubation with GGT enzyme for different times and ultrasonic treatment.

[0102] Figure 6The images show fluorescence images of mice injected via the tail vein into the nano-drug-loaded vesicles prepared in Example 1 and Comparative Example 1; where (a) is a fluorescence image of mice at different time points; and (b) is a fluorescence image of mice with and without ultrasound applied at 12 h and an in vitro fluorescence image of major organs; red circles or white arrows in the images indicate the location of tumors.

[0103] Figure 7 The images are MR images of mice after the nano-drug-loaded vesicles prepared in Example 1 were injected into the tail vein; (a) are MR images of the mouse brain in cross-section and coronal plane at different time points; (b) are MR images with and without ultrasound applied at 12h. Detailed Implementation

[0104] The present invention will be further described below with reference to embodiments and comparative examples. These embodiments are merely typical descriptions of the present invention, but the present invention is not limited thereto. Unless otherwise specified, the test methods used in the following embodiments and comparative examples are conventional methods, and the raw materials and reagents used are commercially available from conventional commercial sources.

[0105] The reagents used in this invention are described below: Polymer DSPE-PEG 2k -NH2, polymer 4-arm-PEG 0.5k -OH,DSPE-PEG 3.4k -N3 and ApoE-DBCO are both commercially available products.

[0106] Preparation Example 1 This preparation example provides a polymer DSPE-PEG. 2k -BEAGA2, the polymer DSPE-PEG 2k The structural formula for -BEAGA2 is: .

[0107] The polymer DSPE-PEG 2k The preparation method of -BEAGA2 includes the following steps: (1) Preparation of DSPE-PEG 2k -lys-Fmoc: S11. Weigh 450 mg (0.2 mmol) of DSPE-PEG. 2k-NH2 and 295.5 mg (0.5 mmol) of N,N'-difluorenemethoxycarbonyl-D-lysine (Fmoc-Lys) were placed in a reaction flask, along with 5 mL of dichloromethane and 2 mL of dimethyl sulfoxide. After stirring and mixing, 57.6 mg of NHS and 95 mg of EDC were added, and the mixture was stirred at 23 °C for at least 20 h. After the reaction was completed, the reaction solution was transferred to a dialysis bag and dialyzed against methanol and ultrapure water for 6 hours each to remove unreacted impurities. The dialyzed product was then freeze-dried to obtain the product DSPE-PEG. 2k -lys-Fmoc; (2) Preparation of DSPE-PEG 2k -(ABU-Fmoc)2: S12.1 Weigh out 45 mg (0.1 mmol) of DSPE-PEG. 2k The -lys-Fmoc solution was placed in a reaction flask, and 4 mL of dichloromethane and 3 mL of dimethyl sulfoxide were added. After stirring and mixing, 1.2 mL of piperidine (the piperidine volume percentage in the organic solvent was 20%) was added, and the mixture was stirred at 23°C for 30 min. After the reaction was completed, the reaction solution was transferred to a dialysis bag and dialyzed against methanol and ultrapure water for 6 hours respectively to remove unreacted impurities. The dialyzed product was then freeze-dried to obtain the product DSPE-PEG. 2k -lys; S12.2 Weigh out 250 mg (0.12 mmol) of DSPE-PEG. 2k 100 mg (0.3 mmol) of N-fluorenylmethoxycarbonyl-L-2-aminobutyric acid (Fmoc-ABU-OH) was placed in a reaction flask, along with 3 mL of dichloromethane and 2 mL of dimethyl sulfoxide. After stirring and mixing, 40 mg of NHS and 40 mg of EDC were added, and the mixture was stirred at 23 °C for at least 20 h. After the reaction was completed, the reaction solution was transferred to a dialysis bag and dialyzed against methanol and ultrapure water for 6 hours each to remove unreacted impurities. The dialyzed product was then freeze-dried to obtain the product DSPE-PEG. 2k -(ABU-Fmoc)2; (3) Preparation of DSPE-PEG 2k -(ABU-Glu-Boc)2: S13.1 Weigh out 250 mg (0.1 mmol) of DSPE-PEG. 2k-(ABU-Fmoc)2 was placed in a reaction flask, and 3 mL of dichloromethane and 2 mL of dimethyl sulfoxide were added. After stirring and mixing, 1 mL of piperidine (the volume percentage of piperidine in the organic solvent was 20%) was added, and the mixture was stirred at 25 °C for 30 mins. After the reaction was completed, the reaction solution was transferred to a dialysis bag and dialyzed with methanol and ultrapure water for 6 hours respectively to remove unreacted impurities. The dialyzed product was then freeze-dried to obtain the product DSPE-PEG. 2k -ABU-NH2; S13.2 Weigh 160 mg (0.2 mol) of DSPE-PEG. 2k -ABU-NH2, 158 mg (0.52 mmol) of Boc-Glu were placed in a reaction flask, along with 4 mL of dichloromethane and 3 mL of dimethyl sulfoxide. After stirring and mixing, 19.5 mg of NHS and 32.5 mg of EDC were added, and the mixture was stirred at 23 °C for at least 20 h. After the reaction was completed, the reaction solution was transferred to a dialysis bag and dialyzed against methanol and ultrapure water for 6 hours each to remove unreacted impurities. The dialyzed product was then freeze-dried to obtain the product DSPE-PEG. 2k -(ABU-Glu-Boc)2; (4) Preparation of DSPE-PEG 2k -BEAGA2: S14. Weigh 100 mg (0.03 mol) of DSPE-PEG. 2k -(ABU-Glu-Boc)2 was placed in a reaction flask, trifluoroacetic acid was added, and the mixture was stirred for 30 min. Then, the reaction solution was added dropwise to a reaction flask containing 50 mL of diethyl ether while stirring until the precipitate was completely precipitated. The precipitate was washed twice with diethyl ether, collected by centrifugation, and redissolved in dichloromethane. The precipitate was then dialyzed against methanol and ultrapure water for 6 h in sequence. The dialyzed product was freeze-dried to obtain the polymer DSPE-PEG. 2k -BEAGA2.

[0108] The polymer DSPE-PEG prepared in this example 2k -BEAGA2 was characterized by proton NMR spectroscopy, and the test results are as follows: Figure 1 As shown.

[0109] The 1H NMR data are as follows: 1 H NMR (400 MHz, DMSO-d 6, 298 K): δ (ppm) = 0.86 ppm(CH3CH2(CH2CH2)7CH2- , a), 1.24 ppm (CH3CH2(CH2CH2)7CH2-, b), 1.51 ppm (-CHCH2CH2CH2CH2NHCOO-, o), 1.64 ppm (-OCH2CH2NHCO- of DSPE, c), 3.51 ppm (-NHCO-(CH2CH2O) 45 - of PEG, f), 4.03 ppm (-COOCH2CH(OCOCH2-)CH2O- of DSPE, g). The results show that the polymer DSPE-PEG prepared in this example is... 2k The characteristic peaks of -BEAGA2 match the expected chemical shifts well.

[0110] Preparation Example 2 This preparation example provides a polymer 4-arm-PEG. 0.5k -phe-DBCO, the polymer 4-arm-PEG 0.5k The structural formula for -phe-DBCO is: .

[0111] The polymer 4-arm-PEG 0.5k The preparation method of -phe-DBCO includes the following steps: S21. Weigh 276 mg (1.5 mmol) of 2,5-dihydroxy-4-methyl-2,5-dioxy-3-furanopropionic acid and 10 μL of N,N-dimethylformamide into a reaction flask, add 10 mL of anhydrous dichloromethane, stir and mix in an ice bath, and after mixing evenly, add 378 mg (3 mmol) of oxaloyl chloride dropwise to the system, stir and react at 23 °C for 3 hours, remove dichloromethane and oxaloyl chloride by rotary evaporation to obtain 2,5-dihydroxy-4-methyl-2,5-dioxy-3-furanopropionic acid chloride; S22. Weigh 1500 mg (0.2 mmol) of 4-arm-PEG. 0.5k-OH and 30 μL of piperidine were placed in a reaction flask, and 10 mL of anhydrous dichloromethane was added and stirred until homogeneous. Then, 200 mg (1 mmol) of 2,5-dihydroxy-4-methyl-2,5-dioxy-3-furanopropionyl chloride was added dropwise to the system. The reaction was stirred at 23 °C for 3 hours. The reaction was terminated by adding 30 µL of saturated NH4Cl. The organic phase was separated using a separatory funnel. Excess Na2SO4 was added to the organic phase for drying, and the filtrate was obtained by filtration. The filtrate was added dropwise to anhydrous diethyl ether until the precipitate was completely formed. The precipitate was obtained by centrifugation and dried for at least 12 hours to obtain 4-arm-PEG. 0.5k -CDM; S23. Weigh 600 mg (0.12 mmol) of 4-arm-PEG 0.5 k-CDM into a reaction flask, add 5 mL of dichloromethane and stir to mix. After mixing evenly, add 63 mg (0.23 mmol) of DBCO-NH2 and 0.2 mL of N,N-dimethylformamide. Stir the reaction mixture at 23 °C for 24 hours. Add the reaction solution dropwise to anhydrous diethyl ether until the precipitate is completely formed. Centrifuge to obtain the precipitate and dry it for 12 hours to obtain the polymer 4-arm-PEG. 0.5k -phe-DBCO.

[0112] The polymer 4-arm-PEG prepared in this example 0.5k -phe-DBCO was used for proton NMR characterization, and the test results are as follows: Figure 2 As shown.

[0113] The 1H NMR data are as follows: 1 H NMR (400 MHz, CDCl3, 298 K) peaks at 2.13 ppm (-C(CO)=CCH3, a), 2.74 ppm (-COCH2CH2C(CO)=C-, b, c), 3.41 ppm (-OCH2CH2OCH2C-, f), 3.64 ppm (-OCH2CH2O- of PEG, e), 4.23 ppm (-CH2CH2OCO-, d), 7.38-7.67 ppm (iq in DBCO), 8.09 ppm (-CONHCH2-, g). The results show that the polymer 4-arm-PEG prepared in this example is 0.5k The characteristic peaks of -phe-DBCO match the expected chemical shifts well.

[0114] Example 1 This embodiment provides a drug-loaded nanovesicle, abbreviated as BAPD-4ARM@ILD. The preparation method of the drug-loaded nanovesicle includes the following steps: S1. Weigh 2.7 mg of the polymer DSPE-PEG. 2k -BEAGA2, 1.8 mg polymer DSPE-PEG 3.4k -N3, 12 mg of lecithin, 12 mg of cholesterol, 12 mg of phosphatidylethanolamine, and 1 mg of decitabine were placed in a reaction flask, dissolved in a chloroform / methanol (1:1 v / v) solution, and stirred to mix. S2. Dissolve 5 mg of iRGD-ILD in 200 μL of ultrapure water, add the solution obtained in step S1, stir and mix under ultrasonic conditions, cool in an ice bath, and add 200 μL of the ultrasound contrast agent pentafluorobutane dropwise under low temperature to form an emulsion. S3. Under sonication, the emulsion obtained in step S2 was dropped into 15 mL of deionized water placed in an ice-water bath to induce self-assembly and form basic vesicles; then, large aggregates were removed using a 0.45 μm syringe, followed by dialyzing with PBS (pH 7.4) at 15°C for 12 hours to remove free polymers and drugs; 0.6 mg (0.0008 mol) of ApoE-DBCO was added and incubated for 4 hours; subsequently, 0.23 mg (0.0001 mmol) of polymer 4-arm-PEG was added. 0.5k -phe-DBCO, continue incubation for 4 hours; after the final solution is concentrated, it is washed three times with PBS using a centrifugal filtration device (MWCO 100 kDa) at 4°C and 500 rpm, and then filtered through a 0.45 μm syringe filter to remove large aggregates, thus obtaining the drug-loaded nanovesicles.

[0115] Comparative Example 1 This comparative example provides a drug-loaded nanovesicle, abbreviated as BAPD@ILD. The preparation method of the drug-loaded nanovesicle is basically the same as that of Example 1, except that step S3 is different, specifically, the polymer 4-arm-PEG is not added in step S3. 0.5k -phe-DBCO.

[0116] Performance testing (1) Performance test across the BBB The drug-loaded nanovesicles (BAPD-4ARM@ILD) prepared in Example 1 and the drug-loaded nanovesicles (BAPD@ILD) prepared in Comparative Example 1 were subjected to BBB crossing performance tests. The test results are as follows: Figure 3As shown. The specific testing method is as follows: Two types of drug-loaded nanovesicles were labeled with Cy3 and co-incubated with brain capillary endothelial cells (BCECs) for 1 hour. The BCEC cell membrane was stained with DiO and the nucleus was stained with DAPI. Finally, the cells were observed using a laser confocal microscope.

[0117] from Figure 3 It can be seen that BAPD@ILD is complexed on the cell membrane of BCECs, and according to the fluorescence colocalization curve, the fluorescence of Cy3 overlaps with that of the cell membrane, indicating that BAPD@ILD fuses with BCECs. However, due to the presence of the polymer 4-arm-PEG... 0.5k 4-arm-PEG0.5k-phe-DBCO and BAPD-4ARM@ILD cannot fuse with BCECs membranes. Instead, they enter the cytoplasm of BCECs via endocytosis rather than localizing to the cell. This indicates that 4-arm-PEG0.5k-phe-DBCO can effectively restrict the fusion of nanovesicles with BCEC membranes, thereby enabling drug-loaded nanovesicles to cross the BBB smoothly and efficiently.

[0118] (2) Surface Zeta potential test The drug-loaded nanovesicles prepared in Example 1 were co-incubated with different concentrations of GGT enzyme, and their surface Zeta potentials were then measured. The test results are as follows: Figure 4 As shown.

[0119] from Figure 4 It can be seen that as the concentration of GGT enzyme increases, the surface of the drug-loaded nanovesicles becomes more positively charged, indicating that DSPE-PEG... 2k -The BEAGA2 chain segment is highly sensitive to GGT enzymes.

[0120] (3) Transmission electron microscopy test The drug-loaded nanovesicles prepared in Example 1 were co-incubated with GGT enzyme at pH 6.5. The effects of incubation time and sonication on the morphology of the drug-loaded nanovesicles were investigated. The results are as follows: Figure 5 As shown.

[0121] Figure 5 In this text, "pre" represents non-acidic incubation conditions. Under these conditions, the drug-loaded nanovesicles prepared in Example 1 were monodisperse with an average particle size of approximately 100 nm. After incubation at pH 6.5 for 1 hour, the drug-loaded nanovesicles clustered, and some began membrane fusion. With increasing incubation time, at 6 hours, membrane fusion occurred between the drug-loaded nanovesicles, forming a large number of large vesicles with an average particle size of 1 μm. Subsequently, after ultrasonic treatment, the micron-sized large vesicles ruptured.

[0122] (4) Fluorescence image analysis in mice The drug-loaded nanovesicles prepared in Example 1 were injected into mice via the tail vein, and fluorescence image analysis was performed. The results are as follows: Figure 6 As shown.

[0123] from Figure 6 It was found that after tail vein injection of BAPD@ILD, significant fluorescent signals appeared in the mouse brain 12 hours later. The fluorescence gradually decreased after 24 hours. Furthermore, when ultrasound was applied 12 hours after injection, no further accumulation was observed in the mouse brain 1 hour later.

[0124] In contrast, after tail vein injection of BAPD-4ARM@ILD, significant fluorescent signals appeared in the mouse brain 6 hours later, persisting for up to 12 hours. Furthermore, with ultrasound applied 12 hours after injection, the tumor portion of the mouse brain showed even stronger fluorescent signals 1 hour later. Simultaneously, fluorescent images of ex vivo organs from mice also confirmed this phenomenon.

[0125] (5) MR image analysis The drug-loaded nanovesicles prepared in Example 1 were injected into mice via the tail vein, and MR imaging analysis was performed. The results are as follows: Figure 7 As shown.

[0126] from Figure 7 It is known that, under the targeting effect of ApoE peptide, the drug-loaded nanovesicles provided by this invention can be rapidly anchored in brain capillary endothelial cells (BCECs). Then, through the action of GGT enzyme on the BBB surface, specific chain segments on the surface of the drug-loaded nanovesicles are cleaved, exposing amino groups and thus increasing their charge, promoting AMT-mediated endocytosis; simultaneously, in 4-arm-PEG... 0.5k Under the action of -phe-DBCO, membrane fusion of vesicles with BCECs is effectively inhibited during BBB crossing, resulting in more efficient BBB crossing. In the acidic tumor microenvironment, the polymer 4-arm-PEG, which restricts membrane fusion, further enhances this effect. 0.5k -phe-DBCO triggers an acid response and bond breaking / unlocking, causing the drug-loaded nanovesicles to fuse and release the drug under ultrasound. A significant T1 signal appeared at the tumor site at 1 hour, gradually increasing in intensity, reaching its peak at 12 hours, and then gradually weakening. However, a relatively strong signal was still detectable at 24 hours.

[0127] The specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art can make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A method for preparing drug-loaded nanovesicles, characterized in that, Includes the following steps: S1. Under ultrasonic conditions, a solution containing polymers DSPE-PEG-BEAGA2 and DSPE-PEG-N3, anticancer drugs and liposome raw materials is mixed with a solution containing a sound-sensitive agent to form an emulsion; S2. Under ultrasonic conditions, the emulsion obtained in step S1 is added dropwise to water to induce self-assembly and form basic vesicles. The basic vesicles are then incubated with ApoE-DBCO and polymer 4-arm-PEG-phe-DBCO to obtain the drug-loaded nanovesicles. The general structural formula of the polymer DSPE-PEG-BEAGA2 is: Where n is a positive integer from 6 to 10, and m is a positive integer from 35 to 55; The general structural formula of the polymer 4-arm-PEG-phe-DBCO is: , where a is a positive integer from 10 to 15.

2. The preparation method according to claim 1, characterized in that, The mass ratio of the polymer DSPE-PEG-BEAGA2, polymer DSPE-PEG-N3, liposome raw material and anticancer drug is 2.5~3:1.5~2:36~45:

1.

3. The preparation method according to claim 1, characterized in that, The liposome raw materials include lecithin, cholesterol, and phosphatidylethanolamine; preferably, the mass ratio of lecithin to cholesterol to phosphatidylethanolamine is 1~3:1~2:

1.

4. The preparation method according to claim 1, characterized in that, The molar ratio of the N3 group in the polymer DSPE-PEG-N3 to the DBCO group in the polymer ApoE-DBCO to the DBCO group in the polymer 4-arm-PEG-phe-DBCO is 8:3~5:1~3.

5. The preparation method according to claim 1, characterized in that, The polymer DSPE-PEG-BEAGA2 was prepared by the following steps: S11. Amidation reaction of DSPE-PEG-NH2 with N,N'-difluorenemethoxycarbonyl-D-lysine yields DSPE-PEG-lys-Fmoc; S12. Remove the Fmoc protecting group from DSPE-PEG-lys-Fmoc to obtain DSPE-PEG-lys, and then react it with N-fluorenmethoxycarbonyl-L-2-aminobutyric acid to obtain DSPE-PEG-(ABU-Fmoc)2; S13. Remove the Fmoc protecting group from DSPE-PEG-(ABU-Fmoc)2 to obtain DSPE-PEG-ABU2, and then react it with Boc-Glu to obtain DSPE-PEG-(ABU-Glu-Boc)2. S14. Remove the Boc protecting group from DSPE-PEG-(ABU-Glu-Boc)2 to obtain the polymer DSPE-PEG-BEAGA2.

6. The preparation method according to claim 1, characterized in that, The polymer 4-arm-PEG-phe-DBCO was prepared by the following steps: S21. The carboxyl group in 2,5-dihydroxy-4-methyl-2,5-dioxy-3-furanpropionic acid is activated to an acyl chloride group to obtain 2,5-dihydroxy-4-methyl-2,5-dioxy-3-furanpropionic acid chloride; S22. Dissolve 4-arm-PEG-OH in a solvent, add 2,5-dihydroxy-4-methyl-2,5-dioxy-3-furanopropionyl chloride to undergo esterification reaction, and separate to obtain 4-arm-PEG-CDM; S23. Dissolve 4-arm-PEG-CDM in a solvent, add DBCO-NH2 to undergo a ring-opening reaction, and separate to obtain the polymer 4-arm-PEG-phe-DBCO.

7. The drug-loaded nanovesicles prepared by any of the preparation methods described in claims 1 to 6.

8. The use of the drug-loaded nanovesicles according to claim 7 in the preparation of drugs for treating glioma.

9. The use of the nano-drug-loaded vesicles according to claim 8 in the preparation of a drug for treating glioma under ultrasound.

10. The application of the nano-drug-loaded vesicles of claim 9 in the preparation of a drug for treating glioma by fusing energy membranes into micron-sized vesicles and then using ultrasound.

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