Highly efficient targeting structure, nano-preparation, preparation method and application across blood-brain barrier

Abstract

This invention belongs to the field of functional pharmaceutical materials and nanotechnology, disclosing a highly efficient targeting structure, nano-formulation, preparation method, and application for crossing the blood-brain barrier. The polymer prodrug carrier is constructed using rapid nano-complexation technology from a copolymer of borneol (a drug-carrying agent) and two monomers covalently bound to PEG with ROS responsiveness. The borneol structure, with its hydrophobic region responding to dissociation, can exert anticoagulant, antioxidant, anti-inflammatory, and BBB-protective effects, while the hydrophilic terminal borneol enhances the rapid and efficient entry of nano-formulations across the BBB into the ischemic area. This invention achieves the combination of high-molecular polymers and traditional Chinese medicine molecules, enabling the polymer prodrug to simultaneously possess multiple effects as a drug carrier, a neurotargeting transporter crossing the blood-brain barrier, and a neuroprotective agent. This cutting-edge technology has unique characteristics and broad translational potential, providing new ideas for the treatment of acute and severe diseases.

Description

Technical Field

[0001] This invention belongs to the field of functional pharmaceutical materials and nanotechnology, and particularly relates to highly efficient cross-blood-brain barrier targeting structures, nano-formulations, preparation methods and applications. Background Technology

[0002] Stroke is a cerebrovascular disease caused by stenosis or occlusion of arteries supplying the brain, leading to insufficient blood and oxygen supply. This triggers a series of biochemical reactions resulting in neuronal cell death and severe neuroinflammation, ultimately causing severe and irreversible brain damage and posing a serious threat to human life and health. Ischemic stroke causes a cascade of damage to neurons, involving a series of cellular and tissue biochemical metabolic processes, including intracellular calcium overload, energy metabolism disorders, oxidative stress damage, and inflammatory responses. Among these, multiple interactive mechanisms induce intracellular calcium overload. 2+ Overload is a common pathway leading to brain cell damage from various causes. Therefore, rapidly reducing the concentration of free calcium in brain cells in the early stages of stroke is an important intervention to save endangered brain cells and reduce cell damage.

[0003] The blood-brain barrier (BBB) ​​is a physiological barrier that effectively prevents toxins and other exogenous harmful substances from entering the brain and damaging nerve cells. It has been reported that approximately 98% of small-molecule and almost 100% of large-molecule drugs cannot cross the BBB to reach the brain via peripheral administration. In the later stages of stroke reperfusion, inflammation increases BBB permeability, and drug transport efficiency across the damaged BBB remains very low. Therefore, there is an urgent need to design nanoparticle formulations capable of crossing the blood-brain barrier and their preparation methods. Current drug delivery strategies across the blood-brain barrier include adsorption-mediated endocytosis, receptor-mediated endocytosis, and cell-mediated transport. However, these strategies have various limitations, such as poor cell selectivity, high cytotoxicity, widespread receptor distribution, low crossing efficiency, and limitations in the preparation and stability of biomimetic nanoparticles.

[0004] Therefore, an ideal trans-BBB intracerebral drug delivery carrier should possess the following characteristics: 1) small size, uniform structural components, and good stability in blood circulation; 2) clear brain targeting mechanism, high BBB targeting enrichment efficiency, and reversible BBB opening to promote enhanced drug transport and penetration; 3) good biocompatibility, biodegradability, and low toxicity of degradation products; 4) simple carrier preparation process, large-scale batch preparation capability, and good batch-to-batch reproducibility.

[0005] 2-Borneol, commonly known as borneol (dextral borneol), is a representative traditional Chinese medicine for resuscitation, possessing the functions of opening the orifices, reviving the mind, clearing heat, and relieving pain. It has been used in clinical practice for over 1500 years. Borneol, with its low molecular weight and high lipid solubility, can rapidly distribute to the brain (within just 5 minutes), attracting increasing attention as a drug that can easily and rapidly cross the brain barrier (BBB). The mechanism by which borneol enhances BBB permeability is as follows: 1) Borneol can inhibit the expression of a series of ATP-dependent drug efflux proteins on the BBB; 2) It can rapidly induce the translocation of tight junction proteins from the cell membrane to the cytoplasm on the BBB, increasing paracellular permeability and reducing the BBB barrier function. Therefore, compared to traditional methods of increasing BBB penetration and targeting, borneol's enhancement of BBB permeability is physiological, does not damage the structural integrity of the BBB, and is reversible. Furthermore, borneol possesses neuroprotective effects such as anti-inflammatory, antioxidant, and anticoagulant properties. In the later stages of ischemia-reperfusion, borneol has been shown to restore the morphological changes of microglia and may reduce brain injury-induced BBB damage, with good safety and synergistic therapeutic effects.

[0006] Currently, solutions for restoring blood supply to ischemic areas, a crucial step, mainly include thrombolytic therapy and mechanical thrombectomy. However, these approaches are limited by strict treatment time windows and remain immature in terms of dosage. Furthermore, patients who undergo thrombolysis may also face risks such as reperfusion injury and bleeding, so in actual clinical practice, only a small number of patients can receive and benefit from thrombolytic therapy in a timely manner. Compared to intravenous thrombolysis, mechanical thrombectomy has a significantly longer time window (6-24 hours) and a significantly lower patient mortality rate. However, interventional thrombectomy is expensive and requires highly specialized equipment and skilled physicians; therefore, this technology is not yet widely used in clinical practice.

[0007] Stroke patients require protection of cranial nerves during the thrombolysis phase and subsequent stages to reduce brain damage, promote brain function recovery, and prevent sequelae. While currently available drugs have shown some efficacy in ischemic stroke trials, they require large doses, long treatment cycles (exceeding 14 days), and have unclear adverse reactions and drug incompatibilities. Neuroprotective drugs such as glutamate receptor antagonists have shown potential in preclinical trials, but most have failed to demonstrate effectiveness in clinical trials. The main reasons are their singular mechanism of action, unclear therapeutic window, low drug solubility, and lack of brain targeting. These issues limit the dosage and therapeutic effect of these drugs.

[0008] Therefore, there is an urgent clinical need to develop neuroprotective agents that are highly safe, break through the limitations of the treatment time window, have a short treatment cycle, and provide comprehensive treatment targeting the pathophysiological process of brain injury. These agents can block the activation of multiple cell death signaling pathways at the source, effectively restore neuronal function, rapidly reverse brain tissue damage, and thus significantly improve the survival rate of patients.

[0009] Based on the above analysis, the problems and shortcomings of the existing technology are as follows:

[0010] (1) Existing nanoparticle-based drug delivery methods across the blood-brain barrier are complex and time-consuming; physical methods such as hypertonic solutions, microbubbles and focused ultrasound may temporarily disrupt the blood-brain barrier and cause neurological problems and ion imbalances; nasal administration to bypass the BBB has problems such as limited dosage, short residence time and low total drug amount (especially for polar macromolecular drugs).

[0011] (2) In drug delivery across the blood-brain barrier, coating polymer particles with polyethylene glycol layers increases complexity, leading to problems such as uncontrollable coating and increased particle size; current functional targeting ligands or chemical groups have low targeting of universal receptors throughout the body and are subject to efficiency controversy; polymer nanomedicine carriers have problems such as large particle size, uncontrollable release, short cycle time, poor physiological stability, poor targeting, and difficulty in mass production, especially dendritic macromolecules have long-term safety issues.

[0012] (3) Among the solutions for restoring blood supply to ischemic areas, thrombolytic therapy and mechanical thrombectomy are subject to strict treatment time window limitations and are still immature in terms of dosage; patients who have undergone thrombolysis may have risks such as reperfusion injury and bleeding; interventional thrombectomy is expensive and requires extremely high levels of equipment and doctor's operating skills. At present, this technology has not been widely used in clinical practice.

[0013] (4) Existing neuroprotective drugs can only play a single role in anti-oxidative damage and anti-inflammation, while the neurodegenerative cascade is complex and a single mechanism may not be effective; the optimal treatment window is unknown, making it difficult to reverse the structure and function of damaged brain tissue; the drugs have low solubility, short half-life, and lack brain-targeting specificity, making it difficult for them to cross the BBB to reach the ischemic penumbra to exert their effects. In addition, the toxicity of the drugs and the adverse reactions caused by their accumulation in non-target organs also limit their dosage, thus seriously affecting their treatment results. Summary of the Invention

[0014] To overcome the problems existing in related technologies, the present invention discloses embodiments that provide highly efficient targeting structures, nano-formulations, preparation methods, and applications for crossing the blood-brain barrier. The technical solutions are as follows:

[0015] This invention is achieved by using a highly efficient targeting structure that crosses the blood-brain barrier, specifically targeting inflammation or brain cells by compounds or peptides that open the blood-brain barrier without damaging its structure.

[0016] Another objective of this invention is to provide a highly efficient blood-brain barrier-crossing nanoformulation that implements the aforementioned highly efficient blood-brain barrier-crossing targeting structure. The nanoformulation is composed of random copolymers and block copolymers; wherein the hydrophobic region contains ROS-sensitive groups, unsaturated alkanes capable of free radical polymerization, and a pharmacologically active structure with therapeutic effects upon dissociation; the hydrophilic region contains polyethylene glycol and zwitterionic segments, and the borneol at the hydrophilic chain terminus enhances the brain-targeting ability of the nanoformulation.

[0017] Furthermore, the core of this nano-formulation is a sensitive group that responds to ROS. This sensitive group breaks chemical bonds with high concentrations of ROS. The ROS-responsive bond is any one or more combinations of thioacetal / aldehyde bonds, borate ester bonds, diselenyl bonds, or oxalate ester bonds. After the ROS response and bond breaking, the active pharmaceutical ingredient is generated.

[0018] The hydrophilic segment of this nanoformulation is composed of ethylene glycol repeating units of different chain lengths, including tetraethylene glycol, nonaethylene glycol, PEG1K, PEG2K and PEG4K; the relative molecular mass range of the hydrophilic segment is 2-40 kDa, and the relative molecular mass range of the hydrophobic segment is 5-80 kDa; the ratio of hydrophilic region to hydrophobic region is 10:30, 10:45, 10:60, 20:30, 30:30 and 15:45.

[0019] Furthermore, the nano-formulation has the following characteristics: spherical particle shape, particle size of 40-200 nm, polydispersity index (PDI) of 0.1-0.4, and zeta potential of -7 mV to -35 mV.

[0020] Another objective of this invention is to provide a preparation method for preparing the aforementioned highly efficient blood-brain barrier nanoparticle formulation. This preparation method involves free radical polymerization to polymerize two types of monomers of different hydrophilic and hydrophobic classes into extended polymeric segments, and obtaining a highly efficient drug-loaded nanoparticle formulation through rapid nanocomplexation.

[0021] Furthermore, this method uses a polymer polymerization initiator to initiate the polymerization of monomers, and the polymer is any one of the polymerization methods of ATRP, NMP, ATRP, IPT or SETRP.

[0022] Polymers include random copolymers and block polymers;

[0023] Chain transfer agents include 4-cyano-4-(phenylthiocarbamoylthio)valerate and PEG. 1k Modified 4-cyano-4-(phenylthiocarbamoylthio)valerate, PEG 2k Modified 4-cyano-4-(phenylthiocarbamoylthio)valerate and PEG 4kModified 4-cyano-4-(phenylthiocarbamoylthio)valerate.

[0024] Furthermore, this method uses a rapid nano-complexation method to aggregate particles into nanoparticles; for (BO-PEG) 1K ) 10 -ran-(TK-Bo) 30 In the process of forming NPs through a rapid nano-complexation method, the solvent is one of dimethyl sulfoxide, acetonitrile, or tetrahydrofuran.

[0025] The oil phase and aqueous phase were subjected to nano-precipitation at ratios of 1:6, 1:7, 1:8, and 1:9;

[0026] The flow rate ratios of oil phase:water phase:water phase were 0.2:0.9:0.9, 4:1.8:1.8, 1:4.5:4.5, 2:9:9, 4:18:18, and 5:22.5:22.5. The polymer dissolved in the oil phase was mixed with the water phase to form (BO-PEG)-ran-(TK-Bo)NPs nanoprecipitates.

[0027] Solvent removal is achieved through one or a combination of dialysis, rotary evaporation, vacuum drying, or lyophilization.

[0028] Furthermore, the nano-formulation is in the form of a lyophilized formulation. The nano-precipitated (BO-PEG)-ran-(TK-Bo)NPs are removed by rotary evaporation and then mixed with a lyophilization protectant. The mixture is then frozen at -80°C. The frozen mixture is placed in a lyophilizer in the dark to produce lyophilized products. Salts are used as formulation resuspension buffers to balance the pH value during the freeze-drying preservation process.

[0029] The freeze-drying protectant is selected from alcohol-based freeze-drying protectants and sugar-based freeze-drying protectants. The alcohol-based freeze-drying protectant is any one or a combination of xylitol, mannitol, sorbitol, or glycine. The sugar-based freeze-drying protectant is any one or a combination of glucose, sucrose, lactose, trehalose, maltose, malt polysaccharide, fructan, or inulin.

[0030] The concentration of the freeze-drying protectant is selected from 0%, 5%, 10%, 15%, 20%, and 25%; the salt buffer is any one or a combination of two of sodium phosphate or potassium phosphate.

[0031] Furthermore, this nano-formulation is in injectable form, and a rapid nano-complexation method is used to precipitate and polymerize nanopolymers that reversibly open the blood-brain barrier, forming nanoparticles with a dense structure that efficiently loads calcium ion scavengers. Specifically, it includes:

[0032] S1, Synthesis of hydrophobic and hydrophilic monomers: After synthesizing the ROS-responsive structure and performing methacrylation, post-modification of the small molecule hydrophobic monomer and post-modification of the hydrophilic end were performed to obtain the therapeutic hydrophobic monomer and the targeted hydrophilic monomer. The ROS-responsive structure is ketethiocyanate.

[0033] S2, RAFT polymerization of hydrophobic and hydrophilic monomers to obtain an amphiphilic polymer with ROS responsive characteristics: DMF solution of hydrophobic monomer, hydrophilic monomer, chain transfer agent and AIBN is added to a Shrek bottle, deoxygenated and placed in an oil bath for reaction overnight, and the solid is collected by sedimentation.

[0034] S3, Preparation of nano-formulations: After fully dissolving the ROS-responsive amphiphilic polymer and the hydrophobic calcium ion scavenger in an organic solvent, the nano-polymers are then nano-precipitated in proportion using a rapid nano-complexation platform, allowing them to self-assemble in the aqueous phase based on hydrophilic and hydrophobic interactions, thus obtaining nano-formulations loaded with calcium ion scavenger drugs.

[0035] Another objective of this invention is to provide the application of the aforementioned highly efficient blood-brain barrier-crossing nanoformulation in the preparation of a highly efficient blood-brain barrier-crossing brain-targeting nanomedicine composition loaded with a calcium ion scavenger. The polymer concentration of the nanoformulation in brain cells is in the range of 1.6-4.8 μg / mL. The model drug is selected from BAPTA-AM, amlodipine, nifedipine, or nimodipine, and the concentration range of the model drug is 200-600 ng / mL.

[0036] Combining all the above technical solutions, the advantages and positive effects of this invention are as follows:

[0037] 1. This invention utilizes the high concentration of ROS microenvironment at the lesion site of ischemic stroke and the brain-awakening function of borneol to construct a stimulus-responsive nanoparticle for treating ischemic stroke. This invention uses reversible addition-fragmentation chain transfer polymerization to prepare a borneol-based prodrug polymer capable of loading the calcium ion chelator BAPTA-AM. The core of the particle uses ROS-responsive thioketol (TK) and borneol, which has therapeutic and targeting properties, as the main raw materials, and constructs two hydrophilic and hydrophobic monomers through a distribution esterification method. This invention uses RAFT polymerization, with a hydrophobic monomer:hydrophilic monomer ratio of 30:10, to finally obtain an amphiphilic polymer; wherein, the rigid structure of borneol and the alkyl chain together act as the hydrophobic cavity of the nanoparticle, ensuring the internal stability of the structure. To achieve better therapeutic effects and effectively target the drug for treating ischemic stroke to the ischemic penumbra region, this invention uses a borneol-modified polyethylene glycol derivative as the hydrophilic segment, which can effectively avoid uptake by the reticuloendothelial system and achieve long-term circulation in vivo. This invention uses a Flash Nanocomplexation (FNC) platform to control the parameters of nanoparticles, ultimately obtaining nanoparticles with uniform size and good colloidal stability.

[0038] 2. This invention employs chemical methods such as esterification, condensation, and nucleophilic substitution to synthesize monomers, and uses column chromatography, extraction, and precipitation to purify and separate the products. The monomers of this invention have a wide range of applications; the polymerization method is simple, low-cost, and does not introduce other impurities; they have a narrow molecular weight distribution and strong molecular design capabilities, enabling the preparation of block, graft, and star copolymers. The nanoparticles of this invention possess advantages such as good biocompatibility and a long half-life, enabling them to accumulate in the brain of a mouse model of stroke induced by ischemia-reperfusion, reducing ineffective accumulation in other vital organs. BAPTA-AM can be released responsively at the target site and efficiently enters the brain via the permeation of borneol, entering damaged neurons and rapidly reducing intracellular calcium overload while blocking multiple cell death signaling pathways. The borneol unit, after the core responds, also possesses anti-inflammatory and antioxidant neuroprotective effects, forming a synergistic therapeutic strategy with BAPTA-AM to provide comprehensive protection for the central nervous system. The technology of this invention has good versatility and can flexibly adjust the ratio of the two monomers to construct a series of drug delivery platforms loaded with different types of neuropharmaceutical agents (proteins, small molecules, and nucleic acids, etc.). It also has the advantages of being continuous, large-scale, having controllable particle size, narrow distribution, and high batch-to-batch reproducibility, providing a new approach for the treatment of acute critical illnesses.

[0039] 3. This invention includes the synthesis of an amphiphilic polyethylene glycol-borneol random copolymer with a pathological microenvironment (reactive oxygen species) responsiveness, and the self-assembly of the polymer based on rapid nanotechnology to efficiently load the hydrophobic drug calcium ion chelator BAPTA-AM (1,2-bis-(o-Aminophenoxy)-ethane-N,N,N',N'-tetraacetic acidtetraacetoxymethyl ester). On the one hand, the borneol structure on the carrier surface can achieve enrichment and enhanced penetration of the blood-brain barrier, achieving highly efficient targeting of the brain injury site; on the other hand, the borneol structure inside the carrier endows the carrier with hydrophobic regions to achieve loading of hydrophobic drugs, and at the same time, borneol exerts a synergistic therapeutic effect on stroke disease upon carrier dissociation. This invention utilizes the efficient blood-brain barrier crossing capability of borneol to promote drug accumulation in the brain injury area, thereby increasing drug concentration. In the presence of highly reactive oxygen species (ROS) at the lesion site, the responsive carrier dissociates, releasing BAPTA-AM. This rapidly reduces intracellular free calcium concentration in the early stages of ischemic stroke, alleviating or eliminating existing calcium overload and inhibiting the cellular damage cascade and local inflammatory cascade. Simultaneously, after the drug penetrates the blood-brain barrier, the barrier at the brain injury site returns to its closed state, preventing subsequent brain leakage and cerebral edema. This invention, through efficient blood-brain barrier crossing and microenvironment-responsive carrier disintegration, achieves rapid, targeted, and controllable precise drug release, further providing a potential therapeutic strategy for acute stroke by chelating calcium ions at the source to intervene and inhibit the development of downstream diseases. Attached Figure Description

[0040] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments consistent with this disclosure and, together with the description, serve to explain the principles of this disclosure;

[0041] Figure 1 This is a flowchart of the preparation method of nano-formulations that can open the blood-brain barrier provided in the embodiments of the present invention;

[0042] Figure 2 This is a roadmap for the preparation of nano-formulations capable of opening the blood-brain barrier provided in the embodiments of the present invention;

[0043] Figure 3 This is the 1H NMR spectrum of the hydrophobic monomer in deuterated chloroform (CDCl3) provided in an embodiment of the present invention;

[0044] Figure 4 This is the 1H NMR spectrum of the hydrophilic monomer in deuterated chloroform (CDCl3) provided in an embodiment of the present invention;

[0045] Figure 5 This is the NMR spectrum of the polymer provided in the embodiments of the present invention;

[0046] Figure 6 This is a schematic diagram of the GPC of the polymer provided in an embodiment of the present invention, with PDI = 1.46;

[0047] Figure 7 This is the decomposition NMR spectrum of the polymer provided in the embodiments of the present invention;

[0048] Figure 8 This is a graph showing the relationship between drug concentration and particle size, particle size distribution, and drug loading provided in an embodiment of the present invention.

[0049] Figure 9 These are TEM images of the polymer provided in embodiments of the present invention in water (Figure A) and in PBS (Figure B);

[0050] Figure 10 This is a schematic diagram of the stability (7 days) of nanoparticles in water provided in an embodiment of the present invention;

[0051] Figure 11 These are particle size distribution diagrams and drug release curves at different time points after exposure to 500 μmmol H2O2, provided in embodiments of the present invention.

[0052] Figure 12 These are cytotoxicity images provided in embodiments of the present invention;

[0053] Figure 13 These are polymer hemolysis data images provided in embodiments of the present invention;

[0054] Figure 14 This is a schematic diagram of intracellular calcium levels in TLCS-induced damaged PACs after different treatments following incubation with the Fluo-4 AM probe, provided by an embodiment of the present invention, n=3;

[0055] Figure 15 These are flow cytometry images of ROS levels in different types of nano-formulations provided in the embodiments of the present invention.

[0056] Figure 16 This is a schematic diagram of the biodistribution of the nano-formulation provided in this invention in important organs (heart, liver, spleen, lungs, kidneys, and brain);

[0057] Figure 17 These are images of the in vivo therapeutic effects of the nano-formulation provided in this embodiment of the invention;

[0058] Figure 18The following are the hematoxylin-eosin (H&E) staining and alanine aminotransferase (ALT, Figure A), aspartate aminotransferase (AST, Figure B), serum creatinine level (Figure C), and blood urea nitrogen level (Figure D) of various important isolated organs (heart, liver, spleen, lung, and kidney) provided in the embodiments of the present invention.

[0059] Figure 19 This is a schematic diagram of superoxide DHE fluorescence staining of brain tissue provided in an embodiment of the present invention; the scale bar is 100 μm.

[0060] Figure 20 This is a TUNEL fluorescence staining of brain tissue provided in an embodiment of the present invention; the scale bar is 100 nm.

[0061] Figure 21 The levels of malondialdehyde (MDA), glutathione (GSH), and superoxide dismutase (SOD) in the tissue homogenate provided in the embodiments of the present invention are given. Detailed Implementation

[0062] To make the above-mentioned objects, features, and advantages of the present invention more apparent and understandable, specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. Many specific details are set forth in the following description to provide a thorough understanding of the present invention. However, the present invention can be practiced in many other ways different from those described herein, and those skilled in the art can make similar modifications without departing from the spirit of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0063] The innovations of this invention regarding the highly efficient blood-brain barrier-crossing nanoparticle formulation, its preparation method, and its application are as follows: This invention includes a highly efficient blood-brain barrier-crossing targeting structure, nanoparticles constructed based on pathological microenvironment-responsive polymers, a drug, and a formulation preparation method. The highly efficient blood-brain barrier-crossing targeting structure of this invention comprises compounds or peptides that specifically open the blood-brain barrier without damaging its structure, targeting inflammation or brain cells. The nanoparticles of this invention consist of two parts: a hydrophilic region and a hydrophobic region. The hydrophobic region contains ROS-sensitive groups, unsaturated alkanes polymerized by free radicals, and small hydrophobic drug molecules with therapeutic effects. The hydrophilic region contains polyethylene glycol and zwitterionic segments. The drug of this invention is a calcium scavenging drug targeting the pathological calcium overload mechanism of brain injury. The scaffold preparation mechanism of this invention involves polymerizing two types of monomers of different hydrophilic and hydrophobic classes into extended polymer segments through free radical polymerization, and obtaining a highly efficient drug-loaded nanoparticle formulation based on hydrophilic-hydrophobic interactions through a rapid nano-complexation method.

[0064] Example 1, such as Figure 1As shown, the method for preparing highly efficient blood-brain barrier nanoparticles provided in this embodiment of the invention includes the following steps:

[0065] S1, Synthesis of monomers: Ketothiols are synthesized and then methallylated, followed by post-modification with borneol and post-modification with PEG to obtain hydrophobic and hydrophilic monomers, respectively.

[0066] S2, RAFT polymerization: A DMF solution of hydrophobic monomer, hydrophilic monomer, chain transfer agent and AIBN is added to Shrek, deoxygenated and placed in an oil bath for reaction overnight, and the solid is collected by sedimentation.

[0067] S3, Preparation of nano-formulations: Using the FNC platform, the amphiphilic polymer (Bo-PEG) was prepared. 1k ) 10 -ran-(TK-Bo) 30 Self-assembly was performed to obtain nanoparticles loaded with BAPTA-AM.

[0068] This nanoformulation consists of a series of random copolymers and block copolymers with different hydrophilicity and hydrophobicity; wherein, the block copolymer contains ROS-sensitive groups, unsaturated alkanes that can be polymerized by free radicals, and drug-active structures that have therapeutic effects after dissociation; the random copolymer contains polyethylene glycol and zwitterionic segments, which can effectively avoid uptake by the reticuloendothelial system and achieve long-term circulation in vivo.

[0069] In step S1, the synthesis of ketthioglycolate includes: dissolving mercaptopropionic acid a in acetone, adding trifluoroacetic acid while stirring at room temperature, fixing the container, and continuing to stir at room temperature for 8 hours to obtain a white solid; adding n-hexane to slurry, then filtering to obtain a white precipitate, and washing the precipitate with n-hexane and ice water to finally obtain a white powder TK.

[0070] In step S1, the methacrylation of ketithiolide includes: dissolving TK in DCM, adding DMAP and hydroxyethyl methacrylate, stirring in an ice bath for 10 min, slowly adding EDCI in DCM solvent, and stirring at room temperature for 12 h after the addition is complete; washing the organic phase three times with water and saturated brine, collecting the organic phase, drying it with anhydrous sodium sulfate, and purifying it by TLC column chromatography to finally obtain a colorless and transparent oily liquid (TK-MA).

[0071] Further purification by TLC column chromatography yielded: PE:EA = 4:1, 1% AcOH, Rf = 0.45.

[0072] In step S1, the post-modification of borneol includes: dissolving compound c, DMAP, and 2-carboxylic acid in 10 mL of DCM solution, stirring slowly in an ice bath for 15 min, then slowly adding EDCI in DCM solution using a constant pressure dropping funnel, stirring overnight at room temperature, concentrating to remove the solvent, and resuspending in ethyl acetate. The organic phase is washed once each with 1 M hydrochloric acid aqueous solution, water, and saturated saline solution. The organic phase is collected, dried over anhydrous sodium sulfate, and purified by TLC column chromatography to obtain the final product (MA-TK-Bo).

[0073] Furthermore, the TLC column chromatography purification parameters were: PE:EA = 12:1, Rf = 0.32.

[0074] In step S1, the post-modification of PEG includes: dissolving methacrylic anhydride in DCM, adding a DCM solution of Borneol and DMAP dropwise under stirring at room temperature, and continuing the reaction by stirring for 12 hours after the addition is completed in an ice bath; and then continuing to add the reaction solution dropwise to the PEG in an ice bath. 1k The reaction was continued for 12 hours at room temperature in a DCM solution with stirring. After the reaction was complete, the product was precipitated in cold diethyl ether, centrifuged, and vacuum dried to obtain the final product Bo-PEG. 1k .

[0075] In step S2, the RAFT polymerization includes: adding a DMF solution of hydrophobic monomer, hydrophilic monomer, chain transfer agent and AIBN to 10 mL of Shrek, performing double oxygen removal three times, and then reacting overnight in an oil bath at 75°C. The solid (MA-TK-Bo) is collected by sedimentation with ice-cold methanol. 30 -ran-(MA-PEG-Bo) 10 .

[0076] In step S3, the preparation of nanoparticles includes: using an FNC platform to prepare an amphiphilic polymer (MA-TK-Bo) at a flow rate of oil:water:water = 2:9:9 with an oil phase:water phase ratio of 1:9. 30 -ran-(MA-PEG-Bo) 10 Nanoparticles loaded with BAPTA-AM were obtained through self-assembly via hydrophilic-hydrophobic interactions.

[0077] This invention also provides the application of a highly efficient blood-brain barrier-crossing nanoformulation in the preparation of a brain-targeting nanomedicine composition loaded with a calcium ion scavenger. Based on the brain-targeting function of borneol and the highly efficient blood-brain barrier-crossing function of borneol molecules, the agent can rapidly cross the blood-brain barrier to reach the brain injury site. In response to the ROS hydrophobic monomer cleavage, the loaded calcium scavenging drug is released, rapidly clearing excess calcium ions and restoring brain cell function. At the same time, it releases a small molecule hydrophobic monomer drug with anti-inflammatory and antioxidant properties, which synergistically exerts a therapeutic effect. The drug composition is used for drug loading and provides therapeutic functions to prevent or treat diseases.

[0078] The reversibly blood-brain barrier-penetrating molecule is borneol, the therapeutic molecules are calcium ion scavengers and hydrophobic small molecule groups with anti-inflammatory and antioxidant properties, and the disease is stroke in the subjects; stroke includes hemorrhagic stroke and ischemic stroke; the subjects are mammals, including rodents, primates, canines, and swine. The polymer concentration range for therapeutic nanoparticles in brain cells is 1-100 μg / mL; the concentration range of the calcium ion chelating agent is 100-800 ng / mL.

[0079] Example 2: Composition and Method of Nanoparticles

[0080] Borneol is a terpene and bicyclic organic compound, including dextrorotatory borneol (natural borneol), levorotatory borneol (caryophylle), and synthetic borneol, which can be extracted from plants or chemically synthesized. Borneol has a low molecular weight and high lipid solubility, enabling rapid distribution to the brain (within just 5 minutes), and its distribution in the brain is second only to the liver. Borneol can also affect the sleep time of mice under the influence of phenobarbital sodium and pentobarbital sodium, playing a central regulatory role. Borneol can inhibit the expression of multidrug resistance protein P-gp and other ATP-dependent drug efflux proteins on the brain border (BBB), reducing efflux and allowing drug accumulation in the brain, thus achieving efficient drug delivery to the site of brain injury. Borneol can rapidly induce the translocation of tight junction proteins on the BBB from the cell membrane to the cytoplasm, redefining epithelial cell polarity, increasing paracellular permeability, and reducing the barrier function of the BBB. Furthermore, borneol also has neuroprotective effects such as anti-inflammatory, antioxidant, and anticoagulant properties, and can significantly increase sodium levels in brain tissue. + -K + -ATP, Ca 2+ The expression of enzymes such as ATP can improve energy metabolism disorders, and it has good safety and can exert a synergistic therapeutic effect.

[0081] In reversible addition-fragmentation chain transfer polymerization (RAFT) systems, dithioester or trithioester derivatives are typically added as chain transfer agents. During polymerization, the chain transfer agent forms a dormant intermediate (SC(Z)S-Pn) with the growing chain radical Pn·, which limits the irreversible bimolecular termination side reactions between the growing chain radicals, thus enabling effective control of the polymerization reaction.

[0082] Preparation of nanoparticles: The BAPTA-AM-loaded nanoparticles were prepared using the FNC platform with an amphiphilic polymer (MA-TK-Bo). 30 -ran-(MA-PEG-Bo) 10 Self-assembly occurs through hydrophilic-hydrophobic interactions.

[0083] Example 3: Structure and property characterization of nanoparticles

[0084] 1. Preparation of raw materials

[0085] 1.1 Synthesis of Monomers and Polymers

[0086] The hydrophobic monomer was synthesized using a two-step sequential esterification method. First, hydroxyethyl methacrylate was reacted with TK for the first esterification, followed by a reaction with 2-carboxyl alcohol to obtain the final hydrophobic monomer. Post-treatment of both reactions was performed using column chromatography for purification and separation. The 1H NMR spectrum of the hydrophobic monomer was obtained (see...). Figure 3 The olefin proton peaks on the double bond and the three methyl groups of 2-oxool can all be assigned to their corresponding positions in the spectrum.

[0087] like Figure 4 As shown, the proton peaks of the olefin (5.5ppm-6.2ppm) and the proton peak of the borneol methyl group (0.8ppm) can be seen in the 1H NMR spectrum of the hydrophilic monomer.

[0088] The NMR spectrum of the polymer is as follows Figure 5 As shown.

[0089] 1.2 Gel permeation chromatography (GPC)

[0090] GPC of polymers such as Figure 6 As shown, PDI = 1.46.

[0091] 1. ROS responsiveness of polymers

[0092] This invention disperses the polymer in 1 eq of H2O2 to simulate the response environment, reacts at 37°C for a period of time, and then purifies and separates the crude product, such as... Figure 7 As shown, through 1The H NMR spectrum shows a significant decrease in the absorption peak content of the side chain methyl groups at around 1.5 ppm, indicating that the polymer has degraded.

[0093] 2. Structural identification of nanoparticles

[0094] Nanoparticles loaded with BAPTA-AM were self-assembled using FNC to control the oil-water ratio. The hydration dynamic size was around 80 nm and the surface potential was around -10 mV.

[0095] 2.1 Drug loading and encapsulation efficiency

[0096] Furthermore, this invention further analyzed the effect of different total flow rates on particle size and particle size distribution in a flow-limited impingement jet micromixing chamber under other experimental conditions, with an organic phase to aqueous phase feed ratio of 1:9. The prepared nanoparticles were separated in aqueous solution using a G50 gel column, and the drug and encapsulation efficiency were evaluated by high-performance liquid chromatography. Figure 8 It can be seen that as the total flow rate increases from 2.25 mL / min to 20 mL / min, the average size of the nanoparticles decreases (from 78 nm to 46 nm). Under the action of rapid mixing, smaller and more uniform nanoparticles are formed. When the optimal parameter is a total volumetric flow rate of 20 mL / min (2:9:9), the encapsulation efficiency and drug loading reach the highest values, which are 86.5% and 9.7%, respectively.

[0097] 2.2 Transmission Electron Microscopy (TEM)

[0098] like Figure 9 As shown in the TEM images, the nanoparticles in water and 1×PBS are essentially the same size.

[0099] 3. Performance Analysis of Polyborneol Nanoparticles

[0100] 3.1 Stability

[0101] A certain concentration of nanoparticles was uniformly dispersed in water, and the particle size and dispersibility of the nanoparticles were monitored for 7 consecutive days. The results are as follows: Figure 10 As shown.

[0102] 3.2 Response and Drug Release

[0103] First, the particle size distribution of BAPTA-AM-loaded nanoparticles in an in vivo ROS-responsive microenvironment (PBS + 500 μmol H2O2) was simulated using DLS monitoring, as shown in the figure. Figure 11 As shown in Figure A, specific volumes of the solution were then taken at 1, 3, 6, 12, and 24 hours, and the absorbance of the free drug (BAPTA-AM) at 256 nm was detected using high-performance liquid chromatography (HPLC). Figure 11As shown in Figure B, the absorbance decreased with increasing time at a 500 μmol concentration. These results indicate that the hydrophobic regions of the amphiphilic polymer structure may be disrupted by ROS-simulated TK bond breaking in H₂O₂ solution, leading to a more loose and unstable nanoparticle structure and resulting in significant leakage of the effective load. This lays the foundation for the successful release of the loaded drug. The active pharmaceutical ingredient generated after bond breaking following the ROS response can exert its neuroprotective effects, including anti-inflammatory, antioxidant, and anticoagulant properties.

[0104] In the above embodiments, the descriptions of each embodiment have different focuses. For parts that are not described in detail or recorded in a certain embodiment, please refer to the relevant descriptions of other embodiments.

[0105] To further demonstrate the positive effects of the above embodiments, the present invention conducts the following effect experiments based on the above technical solutions.

[0106] 1. Characterization of the therapeutic effects of nanoparticles at the cellular level

[0107] 1.1 Cytotoxicity Detection

[0108] The isolated primary acinar cells were seeded and cultured for 6 hours, and then different concentrations of (Bo-PEG) were added. 1k ) 10 -ran-(TK-Bo) 30 @BANPs and equivalent concentrations of (Bo-PEG) 1k ) 10 -ran-(TK-Bo) 30 NPs were co-cultured for 12 hours (concentrations listed represent the concentration of nanoparticles in all formulations). For example... Figure 12 As shown, exposure to (Bo-PEG) 1k ) 10 -ran-(TK-Bo) 30 @BANPs and (Bo-PEG 1k ) 10 -ran-(TK-Bo) 30 NP dosage (from 0 to 800 μg·mL) -1 No cytotoxicity was observed at 800 μg / mL. -1 Even at high equivalent drug concentrations, cell viability remained good, and the survival rate exceeded 96%, indicating that the formulation has good cell compatibility.

[0109] 1.2 Nanoparticle Hemolysis Detection

[0110] Nanoparticles of different concentrations were dispersed in blood samples of a certain volume fraction and incubated at room temperature for 3 hours. Samples treated with surfactant (TX-100) and 1×PBS served as negative and positive controls, respectively. The hemolysis of different doses of nanoparticles was determined using an ELISA reader in the separated supernatant. Figure 13 It can be seen that even blood cells incubated with nanoparticles at a concentration of 800 μg / mL still retain intact cell membranes, indicating that the polymer-prepared nanoparticles have good blood compatibility and meet the safety standards for in vivo application.

[0111] 1.3 Calcium ion scavenging effect at the cellular level

[0112] Calcium overload and excessive ROS production are key cellular factors inducing brain cell apoptosis and transmitting necrosis signals. According to Figure 14 As shown, glutamate induces Ca2+ in isolated shsy5y cells. 2+ Signal abnormality, Ca 2+ The level increased rapidly by 33.10 times. Free BA drugs can only partially inhibit Ca. 2+ Overload occurred when glutamate-stimulated cells were treated with a high dose (4.8 μg / mL) of the empty carrier, resulting in increased intracellular calcium levels. 2+ The level decreased by 76.7%, while the therapeutic effect of the formulation loaded with a medium dose of drug was comparable to that of the high-dose empty carrier. The improvement in the therapeutic effect of both the empty carrier and the drug-loaded carrier was mainly attributed to the accumulation of the carrier in the brain. Notably, intracellular calcium in brain cells treated with a high dose of 600 ng / mL of BA-loaded nanoparticles... 2+ The level decreased by as much as 92.7%. These results indicate that the synergistic treatment of the carrier and the drug can leverage the advantages of the carrier to cross the blood-brain barrier, and the rapid and efficient release of BA maintains the homeostasis of intracellular calcium ions.

[0113] 1.4 ROS scavenging effect at the cellular level

[0114] Following cerebral ischemia-reperfusion, the injured side of the brain produces large amounts of hydrogen peroxide, superoxide anions / hydroxyl radicals, etc., causing oxidative stress. Excessive ROS severely damages the structure and function of cell membranes, mitochondria, and endoplasmic reticulum. Figure 15 As shown, the borneol molecules released after the empty vector responds to ROS can exert anti-inflammatory and antioxidant neuroprotective effects, which explains why they can successfully reduce the ROS level in brain cells by at least 56.9% after cells are attacked by glutamate. Compared to the model group, the drug-loaded NPs showed a significant ROS scavenging effect; among all tested concentrations, 600 ng / mL showed the best ROS scavenging ability (90.1%), greatly promoting the restoration of cellular redox state.

[0115] 2. Characterization of the therapeutic effect of nanoparticles in an animal model of glutamate-induced ischemic stroke

[0116] 2.1 Biodistribution within the body

[0117] In clinical treatment, it is necessary to promptly remove excess ROS to inhibit neuroinflammation and neuronal apoptosis. Therefore, the ability of drugs to cross the blood-brain barrier and precisely target the site of injury is crucial. The BBB is not only an important protective mechanism of the brain but also the biggest obstacle to drug delivery to the brain. 2-Cyclonol can inhibit the expression of multidrug resistance protein P-gp and ATP-dependent drug efflux protein on the BBB, temporarily increasing BBB cell permeability, which greatly improves the efficiency of nanomaterials crossing the BBB. Using chemical methods, Cy5.5 fluorescent molecules that can exhibit color at specific wavelengths are grafted onto the side chains of polymers, with ungrafted polymers as a control. To verify this hypothesis, this invention established a transient ischemic reperfusion mouse model. Two hours after reperfusion, two types of nanoparticles were injected into mice via tail vein injection. Organs (brain, heart, liver, spleen, lung, and kidney) were collected in vitro at 1, 3, 6, 9, and 12 hours after injection using an in vivo imaging system in a dark environment to observe the fluorescence signals of each organ. Figure 16 The results show that the nanoparticles modified with the target exhibit strong fluorescence signals in the brain, primarily located in the cortex and hippocampus of the ischemic hemisphere. (Bo-PEG) 1k ) 10 -ran-(TK-Bo) 30 -g-Cy5.5NPs showed significantly higher fluorescence signal intensity in the brain than (PEG). 1k ) 10 -ran-(TK-Bo) 30 -g-Cy5.5 NPs, and showed enhanced retention of brain tissue within 1h and 3h, with a fluorescence intensity ratio (PEG) 1k ) 10 -ran-(TK-Bo) 30 The -g-Cy5.5NPs treatment group showed 2.46 and 3.05 times higher fluorescence intensity. As the brain metabolized and circulated, the fluorescence intensity trended downwards over subsequent time periods, eventually recovering to levels comparable to (Bo-PEG). 1k ) 10 -ran-(TK-Bo) 30 -g-Cy5.5 NPs are almost identical. However, injected (PEG) 1k ) 10 -ran-(TK-Bo) 30 The fluorescence signal distribution of -g-Cy5.5 NPs remained essentially unchanged within 12 hours, indicating no difference in fluorescence signal distribution between the ischemic and normal hemispheres. (Bo-PEG)1k ) 10 -ran-(TK-Bo) 30 The fluorescence intensity of NPs was mainly concentrated in the ischemic hemisphere; in other organs, (PEG) 1k ) 10 -ran-(TK-Bo) 30 -g-Cy5.5 and (Bo-PEG) 1k ) 10 -ran-(TK-Bo) 30 Both types of nanoparticles, 2-g-Cy5.5, primarily accumulate in the liver. In summary, these data indicate that 2-Cy5.5 promotes the cross-brain barrier (BBB) ​​process of NPs, and modifying the particle surface with it can achieve effective brain targeting capabilities.

[0118] 2.2 In vivo therapeutic effect

[0119] Given the effective in vivo targeting, the MCAO model was used to evaluate (Bo-PEG). 1k ) 10 -ran-(TK-Bo) 30 @BA NPs treatment effect, such as Figure 17 As shown in Figure A, 24 hours after MCAO, the mouse MCAO model, stained with 2,3,5-triazole chloride (TTC), exhibited extensive infarction compared to the normal group. This was confirmed by intravenous injection of BA-loaded (Bo-PEG). 1k ) 10 -ran-(TK-Bo) 30 Nanoparticle formulations were used to assess infarct volume to determine the optimal dosage. Compared to the model group, empty vectors (Blank NPs), low doses (100 μg / kg), medium doses (200 μg / kg), and high doses (400 μg / kg) all reduced cerebral infarction area. Data showed that infarct area decreased progressively with increasing dose, with the low dose showing an infarct area similar to that of the model, while mice in the high-dose group showed the lowest infarct area (<1.5%). Notably, the empty vector was more effective than the medium dose.

[0120] To further confirm (Bo-PEG) 1k ) 10 -ran-(TK-Bo) 30 Its neuroprotective effects in vivo, such as Figure 17 As shown in Figure B, changes in cerebral perfusion were detected using a laser speckle imaging system to ensure the accuracy of the treatment. Six hours after intravenous injection, blood flow in the treatment group (BA-loaded NPs) recovered to over 90%, while the blood flow in the model group was only 61.2%.

[0121] 2.3 Biocompatibility assessment

[0122] To assess the in vivo toxicity of the nanoparticles, H&E staining of relevant biomedical indicators and major organs was performed, such as... Figure 18 As shown in Figure A, no pathological tissue abnormalities or necrosis were caused in any of the treatment groups, indicating the in vivo safety of the nanoplatform. Simultaneously, the results showed that all biomedical indicators, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (CRE), and blood urea nitrogen (BUN), were within the normal range, indicating that no systemic toxicity was observed with any treatment. Figure 18 As shown in BE.

[0123] 2.5 Inhibition of tissue cell apoptosis at the tissue level

[0124] The MCAO model led to ROS accumulation at the CIRI site in rats and increased apoptosis levels, compared to the model group (see [link to model group]). Figure 19 Blank NPs reduced the relative apoptotic cell ratio by 35.9%, while BA-loaded NPs reduced apoptosis levels to varying degrees in mice, with the high-dose group (400 μg / kg) showing a significant reduction in the number of Tunel-positive cells in brain tissue, reaching as high as 81.3%. In summary, (Bo-PEG) 1k ) 10 -ran-(TK-Bo) 30 @BA NPs, when targeted to the CIRI site, effectively inhibited neuronal apoptosis.

[0125] 2.6 Alleviating oxidative stress at the tissue level

[0126] The pathophysiological process of CIRI is accompanied by the production of large amounts of ROS, which not only induces damage to organelles and plasma membranes, but also accelerates calcium metabolism. 2+ The inflow of [unclear] led to severe Ca [unclear] 2+ The imbalance, a snowball effect of oxidative stress and calcium overload, amplifies and reinforces each other, inducing a severe inflammatory cascade and exacerbating brain cell damage. Elevated ROS levels are a marker of oxidative stress damage. As expected (see...) Figures 20-21Glutamate induces the production of large amounts of superoxide in cells, leading to the collapse of the antioxidant system. Dihydroethidium (DHE) stained sections in the CIRI model group showed a strong red fluorescence signal. In contrast, the DHE level at 400 μg / kg was significantly reduced by 70.68%. Furthermore, compared to the model group, the fluorescence intensity of DHE treated with the empty vector was reduced, indicating that oxidative stress damage was alleviated to some extent. Simultaneously, compared to the model group, the disruption of the oxidative system led to a 1.7-fold increase in MDA levels, while GSH and SOD decreased by 37.61% and 49.06%, respectively. Both were increased to some extent in the BA-loaded group, and the increasing trend was dose-dependent. This means that BA-treated nanoparticles can reduce MDA production in brain tissue and restore SOD activity. In particular, superoxide levels in mice treated with a dose of 400 μg / kg were close to normal, restoring the brain's antioxidant function. Based on these findings, it is further demonstrated that the prepared nanoparticles exhibit a powerful function in alleviating oxidative stress damage in damaged brain cells.

[0127] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications, equivalent substitutions and improvements made by those skilled in the art within the scope of the technology disclosed in the present invention and within the spirit and principles of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A highly efficient nano-formulation for crossing the blood-brain barrier, characterized in that, The nano-formulation consists of nanoparticles loaded with the calcium ion chelating agent BAPTA-AM. The nanoparticles are formed by the self-assembly of amphiphilic random copolymers through rapid nanocomplexation technology; The amphiphilic random copolymer has the structure shown in formula (I): (Bo-PEG)-ran-(TK-Bo) (I) Wherein, Bo is borneol, PEG is polyethylene glycol segment, TK is thioketal bond, and ran indicates random copolymerization; in the copolymer, the degree of polymerization ratio of the hydrophilic segment (Bo-PEG) to the hydrophobic segment (TK-Bo) is 10:

30. The polyethylene glycol segment is polyethylene glycol 1000.

2. The highly efficient blood-brain barrier-crossing nanoformulation according to claim 1, characterized in that, The nano-formulation has a spherical particle shape, a particle size of 40-200 nm, a polydispersity index (PDI) of 0.1-0.4, and a zeta potential of -7 mV to -35 mV.

3. The highly efficient blood-brain barrier-crossing nanoformulation according to claim 1, characterized in that, The nanoparticles were prepared by rapid nanocomplexation technology, wherein the volume ratio of the organic phase to the aqueous phase was 1:6 to 1:

9.

4. The highly efficient blood-brain barrier-crossing nanoformulation according to claim 3, characterized in that, In the rapid nanocomplexation technology, the flow rate ratio of the organic phase to the two aqueous phases is 2:9:

9.

5. The highly efficient blood-brain barrier-crossing nanoformulation according to claim 1, characterized in that, This nano-formulation is a lyophilized formulation containing a lyophilization protectant. The lyophilization protectant is selected from alcohol-based and sugar-based lyophilization protectants. The alcohol-based lyophilization protectant is any one or more combinations of xylitol, mannitol, sorbitol, or glycine. The sugar-based lyophilization protectant is any one or more combinations of glucose, sucrose, lactose, trehalose, maltose, malt polysaccharide, fructan, or inulin. The concentration of the lyophilization protectant is selected from 0%, 5%, 10%, 15%, 20%, and 25%.

6. The highly efficient blood-brain barrier-crossing nanoformulation according to claim 1, characterized in that, This nano-formulation is an injectable dosage form.

7. A method for preparing a highly efficient blood-brain barrier-crossing nanoformulation as described in any one of claims 1-6, characterized in that, The method includes the following steps: S1. Synthesize the hydrophobic monomer MA-TK-Bo and the hydrophilic monomer Bo-PEG; S2. The hydrophobic monomer, hydrophilic monomer, chain transfer agent and initiator are subjected to RAFT polymerization in a solvent to obtain the amphiphilic random copolymer (Bo-PEG)-ran-(TK-Bo); S3. Dissolve the amphiphilic random copolymer and BAPTA-AM in an organic solvent, and use a rapid nanocomplexation platform to mix the resulting organic phase with the aqueous phase, so that the polymer self-assembles to form nanoparticles loaded with BAPTA-AM.

8. The use of a highly efficient blood-brain barrier-crossing nanoformulation as described in any one of claims 1-6 in the preparation of a highly efficient blood-brain barrier-crossing brain-targeting nanomedicine composition loaded with a calcium ion scavenger.

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