Response liposome for treating traumatic brain injury by targeting blood-brain barrier loaded drug and preparation method thereof

Abstract

The invention provides a response liposome for treating traumatic brain injury by targeting a blood-brain barrier loaded drug and a preparation method of the response liposome, and belongs to the field of traumatic brain injury treatment.The response liposome is prepared by preparing TfR-T12 target peptide firstly, then preparing nano-particles with hydrophobic molecular drugs wrapped inside, and finally preparing the TfR-T12 target peptide with the hydrophobic molecular drugs wrapped inside. And finally, preparing the response liposome for treating traumatic brain injury by the targeted blood-brain barrier loaded medicine. According to the application, the TfR-T12 target peptide which is highly expressed in cerebral cortex microvessels and can help a nano-drug to pass through a blood brain barrier through endocytosis is prepared as a target spot, meanwhile, the liposome is subjected to polypeptide modification, and the self-assembly driving force of the nano-particles is improved through process control; the outer surface of the response lipidosome is a target peptide, and the response lipidosome is internally coated with a nano-drug; the liposome can repair the blood brain barrier after injury, improve the blood flow of the brain, improve the drainage of the colloidal lymphatic system, improve the motor and cognitive functions of mice after injury, and reduce the dosage of drugs and various possible side effects caused by the drug dosage.

Description

Technical Field

[0001] The present invention relates to the technical field of treating traumatic brain injury, and in particular to a responsive liposome for treating traumatic brain injury by loading a drug targeting the blood-brain barrier and a preparation method thereof. Background Art

[0002] Traumatic brain injury (TBI) has placed a serious burden on the health system due to its high mortality and disability rates, and has placed heavy economic and physical burdens on patients and their families. According to statistics, 60 million people are affected by TBI every year worldwide. Traumatic brain injury can cause damage to the blood-brain barrier (BBB), which can cause brain edema and neuroinflammation, leading to increased intracranial pressure. Treatment targeting the BBB has become a potential new treatment option for TBI, and some studies have confirmed that brain edema after TBI can be reduced by restoring the integrity of the BBB.

[0003] In addition to BBB disorders, dysfunction of the brain lymphatic system is another cause of cerebral edema. The glymphatic system is one of the two components of the brain lymphatic drainage system, the other component being the meningeal lymphatic vessels; the glymphatic system is a network of brain-wide paravascular pathways dependent on aquaporin-4 (AQP4) formed by astrocytes, which can effectively remove interstitial solutes and brain metabolic wastes from the central nervous system (CNS) through the exchange of fluid between cerebrospinal fluid (CSF) and interstitial fluid (ISF).

[0004] Fluvoxamine (Flv) is one of the 5-hydroxytryptamine selective reuptake inhibitors (SSRIs) and has been shown to have the highest affinity for sigma-1 receptors (Sig-1R) among SSRIs, so it has strong anti-inflammatory ability both in vivo and in vitro. Studies have shown that Flv can alleviate BBB dysfunction, cerebrovascular injury and brain edema formation after brain trauma by promoting the transformation of microglia from M1 phenotype to M2 phenotype. However, in the treatment of TBI, due to the existence of BBB, about 98% of small molecule drugs and nearly 100% of macromolecule drugs cannot be transported into brain tissue, resulting in insufficient targeting and retention of drugs in the brain injury area. Therefore, in the research on the treatment of brain injury, large doses of drugs have to be used to treat TBI in many cases, which may be accompanied by corresponding side effects and cause other damage to human health. Summary of the invention

[0005] In view of the technical problems existing in the background technology, the present application provides a responsive liposome targeting the blood-brain barrier and loading drugs to treat traumatic brain injury and a preparation method thereof, aiming to solve the technical problem that drugs cannot be transported into brain tissue and it is difficult to achieve high expression in the existing treatment of TBI.

[0006] In a first aspect, the present invention provides a method for preparing a responsive liposome that targets the blood-brain barrier and carries a drug for treating traumatic brain injury, comprising the following steps:

[0007] S1, preparing TfR-T12 target peptide by solid phase peptide synthesis process;

[0008] S2, dissolving phosphatidylcholine, cholesterol, hydrophobic molecular drugs, amphiphilic polymer molecules and the TfR-T12 target peptide prepared in step S1 in chloroform, evaporating under reduced pressure to form a film, then dehydrating to hydrate the film and self-assemble it, and then ultrasonically treating it, and using a liposome extruder to obtain nanoparticles containing hydrophobic molecular drugs;

[0009] S3. Using dialysis or centrifugal filtration to remove the hydrophobic molecular drugs or amphiphilic polypeptide molecules not loaded on the surface of the nanoparticles, to obtain responsive liposomes that target the blood-brain barrier and load drugs to treat traumatic brain injury.

[0010] In the technical scheme of the embodiment of the present application, a TfR-T12 target peptide that is highly expressed in the microvessels of the cerebral cortex and can help nano-drugs to cross the blood-brain barrier through endocytosis is prepared as a target, and the liposome is modified with polypeptides so that it can efficiently target the target peptide, thereby preparing a targeting peptide; and the self-assembly driving force of the nanoparticles is improved through process control. When the solvent evaporates and rehydrates, the lipid molecules self-assemble to form a bilayer structure, and the hydrophobic molecular drug interacts with the hydrophobic tail of the amphiphilic polymer molecule to form a stable embedding, which is wrapped in the hydrophobic core, and the TfR-T12 target peptide is fixed to the outer surface of the liposome by chemical bonds. Finally, a responsive liposome with a TfR-T12 target peptide on the outer surface and a drug for treating traumatic brain injury coated inside is synthesized. The responsive liposome meets the standards of nano-drugs, exhibits biosafety in both in vitro and in vivo studies, and can be efficiently taken up by vascular endothelial cells.

[0011] In some embodiments, in step S2, the molar ratio of the phosphatidylcholine, cholesterol, hydrophobic molecular drug, amphiphilic polymer molecule and TfR-T12 target peptide is (50-60): (30-40): (3-8): (2.5-7.5): (1-2). The method of dehydration is to add deionized water, and the amount of deionized water added is 0.6-1.5 times the volume of the chloroform. The temperature of the dehydration is 20-25°C, the time of ultrasonic treatment is 5-15min, and the frequency is 25-35kHz. The hydrophobic molecular drug is fluvoxamine, and the amphiphilic polymer molecule is phospholipid-polyethylene glycol-maleimide or phospholipid-polyethylene glycol-carboxyl.

[0012] In this embodiment, each raw material is dissolved in an organic solvent, and after uniform distribution, the solvent is removed by evaporation to form a homogeneous film. After rehydration, the hydrophobic DSPE (phosphatidylethanolamine) in the amphiphilic polymer molecules will spontaneously gather together to form a core, and the hydrophobic molecular drug is captured in the hydrophobic core. At the same time, the hydrophilic PEG (polyethylene glycol) extends into the aqueous phase and is distributed on the outer surface to reduce the free energy of the system and form a thermodynamically stable nanoparticle structure, so that the TfR-T12 target peptide at the end of the PEG is fixed to the outer surface of the liposome by a chemical bond for targeted recognition. Therefore, by adjusting the ratio of the liposome raw materials, the temperature of hydration, and the conditions of ultrasonic treatment, the process of self-assembly of the nanoparticles is guaranteed, thereby improving the loading efficiency of the drug and optimizing the encapsulation efficiency.

[0013] In some embodiments, in step S2, the pore size of the liposome extruder is 80-120 nm. By using a liposome extruder with nano-scale pore size and polycarbonate membrane as material, nano-scale targeted blood-brain barrier loaded drug-responsive liposomes for treating traumatic brain injury are prepared.

[0014] In some embodiments, in step S1, the specific preparation process of the TfR-T12 target peptide is: using Fmoc-protected amino acids, and synthesizing the target peptide by coupling with Rink amide AM resin or Wang resin. The TfR-T12 target peptide includes an Fmoc-modified amino acid matrix, and several amino acids with specific sequences modified on the Fmoc-modified amino acid matrix. The amino acid sequence of the TfR-T12 target peptide is THRPPMWSPVWP.

[0015] In the second aspect, the embodiment of the present application provides a responsive liposome targeting the blood-brain barrier loaded with drugs for treating traumatic brain injury, which is prepared by the preparation method of the responsive liposome targeting the blood-brain barrier loaded with drugs for treating traumatic brain injury described in any one of the above. The responsive liposome is a nanoparticle with the TfR-T12 target peptide exposed on the outer surface and the drug for treating traumatic brain injury coated inside.

[0016] In the technical scheme of the embodiment of the present application, the responsive liposomes that target the blood-brain barrier and load drugs to treat traumatic brain injury use the TfR-T12 target peptide exposed on the outer surface as a target during the treatment of traumatic brain injury, so that the drug can be efficiently delivered to the damaged cranial brain area, thereby improving the therapeutic ability of the drug, and can repair the damaged BBB to a certain extent, improve brain blood flow, increase the drainage of the glymphatic system, and enhance motor and cognitive functions after injury; the synthesized drug can also improve the drainage of the glymphatic system and cognitive function after TBI, which not only improves the efficiency of drug use, but also reduces the dosage of the drug and the various side effects that may be caused thereby.

[0017] The above description is only an overview of the technical solution of the present application. In order to more clearly understand the technical means of the present application, it can be implemented in accordance with the contents of the specification. In order to make the above and other purposes, features and advantages of the present application more obvious and easy to understand, the specific implementation methods of the present application are listed below. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the technical solution of the present application, the following is a brief introduction to the drawings used in the present application. Obviously, the drawings described below are only some embodiments of the present application, and for ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0019] Figure 1 Characterization diagram of the physical and chemical properties of the nanoparticles prepared in Example 1 of the present application;

[0020] Figure 2 A schematic diagram of the synthesis structure of the responsive liposomes loaded with drugs for treating traumatic brain injury and its therapeutic effect prepared in Example 1 of the present application;

[0021] Figure 3 In order to verify the TPL-Flv prepared in Example 1, an in vitro cell uptake experiment and cytotoxicity analysis result diagram are performed;

[0022] Figure 4 This is a graph showing the dose-dependent acute toxicity results of TPL-Flv prepared in Example 1 in C57BL / 6 mice;

[0023] Figure 5 This is a diagram showing the therapeutic effect of TPL-Flv prepared in Study Example 1 on TBI mice;

[0024] Figure 6 The results of verifying that the TPL-Flv prepared in Example 1 can alleviate the dysfunction of the glymphatic system in TBI mice;

[0025] Figure 7 The results of preparing TPL-Flv in Example 1 to verify that the treatment alleviated motor coordination disorders and improved cognitive functions in TBI mice. DETAILED DESCRIPTION

[0026] The following embodiments of the technical solution of the present application are described in detail in conjunction with the accompanying drawings. The following embodiments are only used to more clearly illustrate the technical solution of the present application, and are therefore only used as examples, and cannot be used to limit the scope of protection of the present application.

[0027] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by technicians in the technical field to which this application belongs; the terms used herein are only for the purpose of describing specific embodiments and are not intended to limit this application; the terms "including" and "having" in the specification and claims of this application and the above-mentioned figure descriptions and any variations thereof are intended to cover non-exclusive inclusions.

[0028] In the description of the embodiments of the present application, the technical terms "first", "second", etc. are only used to distinguish different objects, and cannot be understood as indicating or implying relative importance or implicitly indicating the number, specific order or primary and secondary relationship of the indicated technical features. In the description of the embodiments of the present application, the meaning of "multiple" is more than two, unless otherwise clearly and specifically defined.

[0029] Reference to "embodiments" herein means that a particular feature, structure, or characteristic described in conjunction with the embodiments may be included in at least one embodiment of the present application. The appearance of the phrase in various locations in the specification does not necessarily refer to the same embodiment, nor is it an independent or alternative embodiment that is mutually exclusive with other embodiments. It is explicitly and implicitly understood by those skilled in the art that the embodiments described herein may be combined with other embodiments.

[0030] In the description of the embodiments of the present application, the term "and / or" is only a description of the association relationship of the associated objects, indicating that there may be three relationships. For example, A and / or B can represent: A exists alone, A and B exist at the same time, and B exists alone. In addition, the character " / " in this article generally indicates that the associated objects before and after are in an "or" relationship.

[0031] In the description of the embodiments of the present application, the term "multiple" refers to more than two (including two). Similarly, "multiple groups" refers to more than two groups (including two groups), and "multiple pieces" refers to more than two pieces (including two pieces).

[0032] For those of ordinary skill in the art, the specific meanings of the above terms in the embodiments of the present application can be understood according to specific circumstances.

[0033] Traumatic brain injury (TBI) has brought a serious burden to the health system due to its high mortality and disability rates, and has placed a heavy economic and physical burden on patients and their families. Traumatic brain injury can cause damage to the blood-brain barrier (BBB), leading to brain edema and neuroinflammation, which in turn leads to increased intracranial pressure. Studies have shown that fluvoxamine (Flv) can reduce BBB dysfunction, cerebrovascular injury, and brain edema after brain trauma by promoting the transformation of microglia from M1 phenotype to M2 phenotype. However, in the treatment of TBI, due to the existence of the BBB, about 98% of small molecule drugs and nearly 100% of macromolecule drugs cannot be transported into brain tissue, resulting in insufficient targeting and retention of drugs in the brain injury area. Therefore, in the study of the treatment of brain injury, large doses of drugs have to be used to treat TBI in many cases, which may be accompanied by corresponding side effects and cause other damage to human health.

[0034] In order to solve the technical problem that drugs cannot be transported into brain tissue and high expression is difficult to achieve in the existing treatment of TBI, the present application provides a responsive liposome for targeting the blood-brain barrier and loading drugs to treat traumatic brain injury and a preparation method thereof, wherein the preparation method of the present application prepares a TfR-T12 target peptide that is highly expressed in the microvessels of the cerebral cortex and can help nano-drugs pass through the blood-brain barrier through endocytosis as a target, and at the same time, the liposome is modified with polypeptides so that it can efficiently target the target peptide, thereby preparing a targeting peptide containing the TfR-T12 targeting sequence, and improving the self-assembly driving force of the nanoparticles through process control. When the solvent evaporates and dehydrates, the lipid molecules self-assemble to form a bilayer structure, and the hydrophobic molecular drug interacts with the hydrophobic tail of the amphiphilic polymer molecule to form a stable embedding, which is wrapped in the hydrophobic core, and the TfR-T12 target peptide is fixed to the outer surface of the liposome by chemical bonds, and finally a responsive liposome with the TfR-T12 target peptide on the outer surface and the traumatic brain injury drug coated inside is synthesized.

[0035] For the convenience of description, the following examples are described by taking a method for preparing a responsive liposome targeting the blood-brain barrier and loaded with drugs for treating traumatic brain injury according to an embodiment of the present application as an example.

[0036] In a first aspect, the present invention provides a method for preparing a responsive liposome that targets the blood-brain barrier and carries a drug for treating traumatic brain injury, comprising the following steps:

[0037] S1, preparing TfR-T12 target peptide by solid phase peptide synthesis process;

[0038] S2, dissolving phosphatidylcholine, cholesterol, hydrophobic molecular drugs, amphiphilic polymer molecules and the TfR-T12 target peptide prepared in step S1 in chloroform, evaporating under reduced pressure to form a film, then dehydrating to hydrate the film and self-assemble it, and then ultrasonically treating it, and using a liposome extruder to obtain nanoparticles containing hydrophobic molecular drugs;

[0039] S3. Use dialysis or centrifugal filtration to remove the hydrophobic molecular drugs or amphiphilic polypeptide molecules not loaded on the surface of the nanoparticles, and obtain responsive liposomes that target the blood-brain barrier and load drugs to treat traumatic brain injury.

[0040] The preparation method prepares the TfR-T12 target peptide, which is highly expressed in the microvessels of the cerebral cortex and can help nano drugs pass through the blood-brain barrier through endocytosis, as a target, and at the same time modifies the liposome with peptides so that it can efficiently target the target peptide; the TfR-T12 target peptide is connected to the hydrophilic end group of the amphiphilic polymer molecule through a chemical bond, and the molecular structure is controlled to ensure that it is located on the outer surface during self-assembly, thereby forming a DSPE-PEG2K-TfR-T12 nanocarrier. In addition, the self-assembly driving force of the nanoparticles is improved through process control. When the solvent evaporates and rehydrates, the lipid molecules self-assemble to form a bilayer structure, and the hydrophobic molecular drug interacts with the hydrophobic tail of the amphiphilic polymer molecule to form a stable embedding, which is wrapped in the hydrophobic core. The hydrophilic head of the amphiphilic polymer molecule and the TfR-T12 target peptide fixed by chemical bonds extend to the water phase, and finally synthesizes a responsive liposome with the TfR-T12 target peptide on the outer surface and the internal coating of the drug for treating traumatic brain injury. The responsive liposomes meet the standards of nanomedicine, demonstrated biosafety in both in vitro and in vivo studies, and can be efficiently taken up by vascular endothelial cells.

[0041] Further, in some embodiments, in step S2, the molar ratio of phosphatidylcholine, cholesterol, hydrophobic molecular drug, amphiphilic polymer molecule and TfR-T12 target peptide is (50-60): (30-40): (3-8): (2.5-7.5): (1-2). The method of dehydration is to add deionized water, and the amount of deionized water added is 0.6-1.5 times the volume of chloroform. The temperature of dehydration is 20-25°C, the time of ultrasonic treatment is 5-15min, and the frequency is 25-35kHz. The hydrophobic molecular drug is fluvoxamine, and the amphiphilic polymer molecule is phospholipid-polyethylene glycol-maleimide or phospholipid-polyethylene glycol-carboxyl.

[0042] In the technical scheme of the embodiment of the present application, each raw material is dissolved in an organic solvent, evenly distributed, and then the solvent is removed by evaporation to form a homogeneous film. After dehydration, the hydrophobic DSPE (phosphatidylethanolamine) in the amphiphilic polymer molecules will spontaneously aggregate together to form an inner core, and the hydrophobic molecular drug is captured in the hydrophobic inner core. At the same time, the hydrophilic PEG (polyethylene glycol) extends into the aqueous phase and is distributed on the outer surface to reduce the free energy of the system and form a thermodynamically stable nanoparticle structure. In this way, the TfR-T12 target peptide at the end of PEG (the amino end or carboxyl end of the TfR-T12 target peptide is coupled to the active group (such as maleimide or carboxyl) on DSPE-PEG to achieve bonding between the two) is fixed on the outer surface of the liposome for targeted recognition. Therefore, by adjusting the proportion of liposome raw materials, the hydration temperature and the conditions of ultrasonic treatment, the self-assembly process of nanoparticles is guaranteed, the loading efficiency of drugs is improved, and the encapsulation efficiency is optimized; and dialysis or centrifugal filtration methods are used to remove unloaded free drugs or polymer molecules to ensure that the drugs in the final nanoparticle structure are mainly located in the inner core, thereby achieving effective drug transport during the treatment of traumatic brain injury.

[0043] Furthermore, in some embodiments, in step S2, the pore size of the liposome extruder is 80-120 nm. By using a liposome extruder with nano-scale pore size and polycarbonate membrane as material, nano-scale blood-brain barrier-targeted drug-loaded responsive liposomes for treating traumatic brain injury are prepared.

[0044] Further, in some embodiments, in step S1, the specific preparation process of the TfR-T12 target peptide is: using Fmoc-protected amino acids, and synthesizing the target peptide by coupling with Rink amide AM resin or Wang resin. The TfR-T12 target peptide includes an Fmoc-modified amino acid matrix, and several amino acids with specific sequences modified on the Fmoc-modified amino acid matrix. The amino acid sequence of the TfR-T12 target peptide is THRPPMWSPVWP (Thr-His-Arg-Pro-Pro-Met-Trp-Ser-Pro-Val-Trp-Pro), wherein Thr is threonine, His is histidine, Arg is arginine, Pro is proline, Met is methionine, Trp is tryptophan, Ser is serine, Val is valine, and Trp is tryptophan.

[0045] Specifically, the synthesis process of the TfR-T12 target peptide includes the following steps:

[0046] SS1, first remove the Fmoc protecting group by treating the Fmoc protected amino acid with a solution of piperazine (5% w / v) and DBU (1,8-diazabicyclo[5.4.0]undec-7-ene) (2% v / v) in DMF (N,N-dimethylformamide);

[0047] SS2. In the coupling reaction, the resin was mixed with Fmoc-protected amino acids (10 equivalents) in DMF, and HBTU (peptide coupling reagent, (5 equivalents), HOBt (1-hydroxybenzotriazole, 5 equivalents) and 4-methylmorpholine (50 equivalents) were added to modify the amino acids; and all amino acids were coupled and grafted in sequence according to the specific amino acid sequence of the TfR-T12 target peptide;

[0048] SS3. After synthesis, the peptide was deprotected (all side chain protecting groups on the peptide chain were removed to make the peptide reach its target chemical structure) by treating the resin with a mixture of TFA (trifluoroacetic acid, 95% v / v), triisopropylsilane (2.5% v / v) and water (2.5% v / v) at room temperature for 2 hours, and cleaved from the resin to obtain the intact free peptide. The cleavage solution was then filtered and concentrated under a nitrogen stream, and the free peptide was precipitated in cold ether;

[0049] SS4. The free peptide obtained in step SS3 was then purified using preparative liquid chromatography (Waters 2535Q) using acetonitrile and distilled water containing 0.1% TFA as the solvent system to obtain the purified TfR-T12 target peptide.

[0050] It should be noted that in the TfR-T12 target peptide synthesis process, the unit w / v is the ratio of the mass of the solute to the volume of the solution, and v / v is the volume percentage concentration, that is, the ratio of the volume of the solute to the volume of the solution; in step SS2, each time a new amino acid is coupled, it is necessary to first use a deprotection reagent to remove the Fmoc protecting group (step S1) to expose the free amino group for the next coupling reaction; n times equivalent represents the equivalent of the reagent relative to the active site on the resin.

[0051] In the second aspect, the present application provides a responsive liposome that targets the blood-brain barrier and loads drugs to treat traumatic brain injury, which is prepared by the above-mentioned preparation method of the responsive liposome that targets the blood-brain barrier and loads drugs to treat traumatic brain injury. The responsive liposome is a nanoparticle with the TfR-T12 target peptide exposed on the outer surface and the drug for treating traumatic brain injury coated inside.

[0052] Among them, the responsive liposomes that target the blood-brain barrier and load drugs to treat traumatic brain injury, DSPE provides the membrane anchoring function of the liposomes, PEG is used to increase water solubility and prolong circulation time, and TfR-T12 provides targeting function; thus, when treating traumatic brain injury, the TfR-T12 target peptide is exposed on the outer surface of the liposome as a target, which crosses the blood-brain barrier and enables the drug to be efficiently delivered to the damaged cranial brain area, thereby improving the therapeutic ability of the drug, and can repair the damaged BBB to a certain extent, improve brain blood flow, increase the drainage of the glymphatic system, and enhance the motor and cognitive functions after injury; the synthesized drug can also improve the drainage of the glymphatic system and cognitive functions after TBI, which not only improves the efficiency of drug use, but also reduces the dosage of the drug and the various side effects that may be caused thereby.

[0053] Some specific embodiments are listed below. It should be noted that the embodiments described below are exemplary and are only used to explain the present application, and should not be construed as limiting the present application. If specific techniques or conditions are not specified in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. The reagents or instruments used without specifying the manufacturer are all conventional products that can be obtained commercially.

[0054] Example 1

[0055] This embodiment provides a method for preparing a responsive liposome that targets the blood-brain barrier and loads drugs to treat traumatic brain injury, comprising the following steps:

[0056] S1, preparing TfR-T12 target peptide by solid phase peptide synthesis process;

[0057] SS1, first remove the Fmoc protecting group by treating the Fmoc protected amino acid with a solution of piperazine (5% w / v) and DBU (1,8-diazabicyclo[5.4.0]undec-7-ene) (2% v / v) in DMF (N,N-dimethylformamide);

[0058] SS2. In the coupling reaction, Rink amide AM resin was mixed with Fmoc-protected amino acids (10 equivalents) in DMF, and HBTU (peptide coupling reagent, (5 equivalents), HOBt (1-hydroxybenzotriazole, 5 equivalents) and 4-methylmorpholine (50 equivalents) were added to modify the amino acids; and all amino acids were coupled and grafted in sequence according to the specific amino acid sequence (THRPPMWSPVWP) of the TfR-T12 target peptide;

[0059] SS3. After synthesis, the peptide was deprotected (all side chain protecting groups on the peptide chain were removed to make the peptide reach its target chemical structure) by treating the resin with a mixture of TFA (trifluoroacetic acid, 95% v / v), triisopropylsilane (2.5% v / v) and water (2.5% v / v) at room temperature for 2 hours, and cleaved from the resin to obtain the intact free peptide. The cleavage solution was then filtered and concentrated under a nitrogen stream, and the free peptide was precipitated in cold ether;

[0060] SS4, the free peptide obtained in step SS3 was subsequently purified using preparative liquid chromatography (Waters 2535Q) using acetonitrile and distilled water containing 0.1% TFA as the solvent system to obtain the purified TfR-T12 target peptide;

[0061] S2, dissolving phosphatidylcholine, cholesterol, fluvoxamine, phospholipid-polyethylene glycol-carboxyl (DSPE-PEG2K-COOH) and the TfR-T12 target peptide prepared in step S1 in 3 mL of chloroform at a molar ratio of 55:35:5:4.5:0.5, evaporating under reduced pressure to form a film, adding 2 mL of deionized water for dehydration (temperature of 20°C), hydrating the film and self-assembling, and then performing ultrasonic treatment (time of 10 min, frequency of 30 kHz), and using a liposome extruder (polycarbonate membrane, pore size of 100 nm) to obtain nanoparticles containing hydrophobic molecular drugs;

[0062] S3. Use dialysis or centrifugal filtration to remove the hydrophobic molecular drugs or amphiphilic polypeptide molecules not loaded on the surface of the nanoparticles, and obtain responsive liposomes (TPL-Flv) that target the blood-brain barrier and load drugs to treat traumatic brain injury.

[0063] See also Figure 1 The figure shows the physical and chemical properties of the nanoparticles prepared in Example 1. Figure 1 A is the chemical formula for the TfR-T12 target peptide modification reaction on the liposome surface, Figure 2 B is the synthetic structure diagram of the target peptide detected by high performance liquid chromatography. Figure 1 C is the one-dimensional 1H-NMR spectra of TPL-Flv (one-dimensional hydrogen spectrum, green) and PEG-2K polymer (red) recorded in a 500 MHz magnetic field. Figure 1 D is a transmission electron microscopy image of TPL-Flv, scale bar = 50 μm. Figure 1 A~ Figure 1 C It can be seen that during the preparation of nanoparticles in step S2, the TfR-T12 target peptide was successfully modified onto the liposome surface to form DSPE-PEG2K-TfR-T12, which improved the ability to penetrate the blood-brain barrier; Figure 1As can be seen from the image of D, TPL-Flv has a relatively uniform spherical morphology with an average outer diameter of 60 ± 5 nm, which meets the standards of nanomedicine.

[0064] Please refer to Figure 2 As shown, Figure 2 A is a schematic diagram of the synthetic structure of the responsive liposome (TPL-Flv) targeting the blood-brain barrier and loading drugs for treating traumatic brain injury in Example 1, Figure 2 B is a schematic diagram of how TPL-Flv promotes recovery from TBI by targeting and crossing the BBB and releasing Flv, activating Treg cells, thereby repairing the blood-brain barrier and the lymphoid system.

[0065] In order to verify the performance of the responsive liposomes (TPL-Flv) prepared in Example 1 targeting the blood-brain barrier and loaded with drugs for treating traumatic brain injury, the following experimental studies were conducted.

[0066] (1) In vitro cellular uptake experiment and in vitro biosafety assessment of TPL-Flv

[0067] In vitro cell uptake experiment: HUVEC cells were seeded in 6-well plates (5×10 5 cells / well), incubated for 24 h, and then treated with PBS, TL-Cy5 (liposomes encapsulating Cy5 dye but without targeting peptide) or TPL-Cy5 (liposomes encapsulating Cy5 dye and containing targeting peptide, i.e., replacing the encapsulated Flv in TPL-Flv with Cy5 fluorescent dye, so as to observe whether the liposomes are effectively targeted to cells) (1×10 -6 After washing with PBS, the cells were trypsinized and suspended in cold PBS buffer, and the fluorescence intensity of intracellular Cy5 was evaluated by flow cytometry (n=4).

[0068] Cytotoxicity studies: The in vitro cytotoxicity of TPL-Flv was evaluated using the cell counting kit 8 (CCK-8) method. HUVEC cells were obtained from the American Type Culture Collection (ATCC) and seeded in 96-well plates (6 × 10 3 cells / well) and cultured for 24 hours; then the medium was replaced with fresh medium containing each sample, and after incubation for 24 hours, cell viability was assessed by CCK-8 method (n=6).

[0069] See also Figure 3 The results of in vitro cell uptake experiment and cytotoxicity analysis are shown in Figure 2. Figure 3A Cellular uptake analysis was performed by flow cytometry. After human umbilical vein endothelial cells (HUVEC) were incubated with each sample for 2 hours, the fluorescence intensity of intracellular Cy5 was analyzed by flow cytometry (n=4), where None is the non-drug group, Control is the control group (drug without targeting peptide modification), MFI is the mean fluorescence intensity, and the data are expressed as mean ± standard deviation. ****p<0.0001 compared with the control group (Control). ####p<0.0001 compared with the non-drug group (None); Figure 3 B is the cytotoxicity of TPL-Flv prepared in Example 1 to HUVEC cells (n=6). Figure 3 A shows that due to the presence of the targeting peptide motif, TPL-Cy5 significantly increased the cellular uptake in cultured HUVEC cells compared with drugs without targeting peptide modification; Figure 3 B, The cytotoxicity of TPL-Flv on HUVEC cells was studied by the cell counting kit-8 (CCK-8) method, and the results showed that at a concentration of at least 2000 μg / mL, the cytotoxicity to cultured HUVEC cells was very low, indicating that TPL-Flv has a large therapeutic window.

[0070] (2) Biosafety Assessment

[0071] See also Figure 4 As shown, the dose-dependent acute toxicity of TPL-Flv was evaluated in C57BL / 6 mice, and the mice were hit on the head to simulate TBI. Each figure shows the blood routine and blood biochemical analysis results of C57BL / 6 mice 24 hours after administration of 1 and 5 times the TPL-Flv treatment dose (equivalent to 2 mg / kg and 10 mg / kg Flv) (n=3), including hematopoietic function, liver function and kidney function, where the vehicle (control group) was not hit on the head of the mice, and the data were expressed as mean ± standard deviation. As can be seen from the figure, compared with the control group, the drug-treated groups did not show obvious abnormalities, which indicates that the responsive liposomes (TPL-Flv) targeting the blood-brain barrier loaded with drugs to treat traumatic brain injury have good biocompatibility and a large therapeutic window.

[0072] See also Figure 5 As shown, this is a graph for studying the therapeutic effect of TPL-Flv on TBI mice. The mice were subjected to head blows to simulate TBI, wherein the sham group was a group without head blows, the TBI+vehicle group was a group with head blows but no medication, the TBI+TPL-Flv group was a group treated with TPL-Flv prepared in Example 1, and the TBI+Flv group was a group treated with ordinary drugs; Figure 5 A~ Figure 5C: The protein levels of ZO-1 and Occludin in the perilesional cortex of mice were assessed by Western blotting 3 days after TBI, and the intensity was quantitatively analyzed using ImageJ software (n=6); Figure 5 D. Figure 5 E is a representative immunofluorescence photograph of ZO-1 and quantitative analysis of the mean fluorescence intensity of ZO-1 positive cells (n=6), cell nuclei were stained with DAPI, scale bar=80 μm. Figure 5 F. Figure 5 G is a representative image of laser speckle contrast imaging and quantitative analysis of cortical cerebral blood flow in mice 3 days after TBI (n=6); data are expressed as mean±SD, *p<0.05, **p<0.01, ***p<0.001 compared with the sham group; #p<0.05 compared with the TBI+vehicle group; +p<0.05 compared with the TBI+Flv group.

[0073] Specific dosing regimen: TPL-Flv was dissolved in 0.9% saline, Flv maleate was dissolved in 0.5% DMSO (dimethyl sulfoxide), and then diluted in 0.9% saline; the mice in the sham group underwent sham surgery, i.e., saline (0.2 mL) was injected through the tail vein; after establishing TBI simulation, the TBI+vehicle group, TBI+TPL-Flv group, and TBI+Flv group were injected with saline (0.2 mL), TPL-Flv (2 mg / kg, 0.2 mL), or Flv solution (2 mg / kg, 0.2 mL) through the tail vein 1 hour after the mice were injured, respectively.

[0074] Laser speckle experiment: The perfusion of cerebral cortex was monitored by laser speckle imager (PeriCam PSI system, Perimed AB, Sweden); the mice were first anesthetized and placed prone in a stereotaxic head frame, and then a midline incision was made above the skull to expose the skull. The perfusion of cerebral cortex was continuously measured for 60 seconds through this exposed site. The measurement height was 10 cm, the laser irradiation area was 2×2 square centimeters, and the PSI system was set to 1388×1038 pixels. The regional spatial contrast was calculated based on a 3×3 secondary matrix; after the measurement, the skin was sutured and disinfected. In order to monitor the changes in blood perfusion in the cortical injury area, the average value of the region of interest (ROI) was calculated; the perfusion data were evaluated by PIMsoft software (version 1.4; Perimed) provided by the supplier.

[0075] from Figure 5As can be seen in the figure, the expression of tight junction proteins (ZO-1 and Occludin) was significantly reduced after TBI in mice, indicating that the BBB was damaged after TBI. However, compared with the TBI+vehicle group and the TBI+Flv group, the expression level of tight junction proteins in the TBI+TPL-Flv group was higher ( Figure 5 A- Figure 5 C), the results of immunofluorescence also showed a similar phenomenon. Laser speckle imaging and its data analysis showed that compared with the sham group, 3 days after TBI, the cerebral blood flow in the cortical injury area of ​​mice treated with TBI+vehicle group and TBI+Flv group was significantly reduced, while the treatment of TBI+TPL-Flv group improved this reduced cerebral blood flow. This shows that TPL-Flv can alleviate the destruction of BBB after TBI and promote blood perfusion in the cerebral cortex.

[0076] See also Figure 6 The figure shows the results of verifying that TPL-Flv treatment alleviates the dysfunction of the glymphatic system in TBI mice. Figure 6 A: 3 days after simulated TBI in mice, cerebrospinal fluid tracer (rhodamine, 10kD) was injected into the cisterna magna of mice; Figure 6 B is a representative image of rhodamine (red) entering the brain parenchyma in TBI mouse brain slices, shown on 4 different coronal slices, located at bregma +1.0 mm, -1.5 mm, -2.0 mm, and -2.5 mm, scale bar = 1500 μm; Figure 6 C is the average quantitative analysis of the percentage of tracer coverage, four sections were analyzed for each animal (n=4-6); Figure 6 D, rhodamine was injected into the brain parenchyma of mice 3 days after TBI; Figure 6 E is the staining of TBI mouse brain sections to show the clearance of rhodamine (red) in the nuclei (blue) and brain parenchyma, shown in four sections at bregma -1.0mm, -1.5mm, -2.0mm and -2.5mm; Figure 6 F is the average quantitative analysis of the percentage of residual tracer coverage area in four sections (n=5-6); data are expressed as mean ± SD, *p<0.05, **p<0.01 compared with the sham group, #p<0.05, ##p<0.01 compared with the TBI+vehicle group, +p<0.05 compared with the TBI+Flv group.

[0077] Among them, occipital cistern injection: the fluorescent cerebrospinal fluid tracer (rhodamine isothiocyanate B-dextran; R8881, SigmaAldrich, 10kD, RITC-dextran) was dissolved in artificial cerebrospinal fluid at a concentration of 2.5%. The mouse was anesthetized and fixed in a stereotaxic frame, and the scalp was incised in the midline to expose the posterior atlanto-occipital membrane covering the cistern. Then, using a 25-μl syringe (#710RN, inner diameter 0.485 mm; Hamilton) and a 33-gauge needle (15 mm, pst4-12, Hamilton), cerebrospinal fluid was injected into the subarachnoid space at a rate of 1 μl / min through the cistern puncture, with a total volume of 10 μl, and injected using a microinjection pump (KDS LEGATO 130, RWD Life Science). After the injection, the tracer circulated in the brain for 30 minutes. During the injection, the inserted needle remained in the cistern until the circulation was completed to prevent leakage of the solution when the needle was removed. Subsequently, the anesthetized mice were perfused through the heart and fixed with 4% paraformaldehyde for 24 hours. Afterwards, the brain tissues were cut into 100 μm coronal sections and observed and photographed under an inverted fluorescence microscope.

[0078] Intraparenchymal injection: To evaluate the function of the interstitial metabolite clearance pathway, fluorescent cerebrospinal fluid tracers were stereotaxically microinjected into the hippocampus of mice. The pretreatment of mice was the same as that of the cisternal injection. Then, 0.5 μl of 2.5% concentration of 10kD rhodamine tracer was injected into the hippocampus (2.00 mm posterior, 1.50 mm lateral, and 2.00 mm below the brain surface relative to the midpoint of the microscope) at a rate of 0.2 μl / min using a 5 μl syringe (#75RN, inner diameter 0.343 mm, Hamilton) and a 33-gauge needle. After injection, the needle remained at the injection site for an additional 5 minutes and was then slowly withdrawn to prevent tracer leakage. Subsequently, the tracer circulated in the brain for 60 minutes, and subsequent treatment was the same as that of the cisternal injection.

[0079] Depend on Figure 6 It can be seen that 10 μL of 10 kD rhodamine was injected into the cisterna magna of mice 3 days after simulated TBI to evaluate the therapeutic effect of lateral vascular CSF penetration ( Figure 6 A); After 30 minutes of circulation, brain tissue was perfusion fixed and whole brain sections were collected; the penetration of cerebrospinal fluid tracers into the brain parenchyma was studied by conventional fluorescence microscopy, and the results showed that the influx of tracers into the brain was significantly increased in the TBI+TPL-Flv group compared with the TBI+vehicle group and the TBI+Flv group ( Figure 6 B. Figure 6 C). In addition, the efficacy of TPL-Flv on interstitial solute clearance after TBI was also evaluated. 0.5 μL of 10 kD rhodamine was injected into the ipsilateral hippocampus of mice 3 days after TBI and circulated for 1 hour ( Figure 6 D), the residual fluorescent tracer in the brain tissue of the TBI+vehicle group and the TBI+Flv group was significantly higher than that of the sham group, while the TBI+TPL-Flv group showed a better trend of tracer excretion than them ( Figure 6 E. Figure 6 F).

[0080] See also Figure 7 The figure below is a result diagram verifying that TPL-Flv treatment alleviates motor coordination disorders and improves cognitive function in TBI mice. Figure 7 A. Figure 7 B is the mNSS score and fall latency in the rotarod test of mice during the 14-day follow-up period after TBI (n=6-8); Figure 7 C is a representative swimming trajectory of mice in the MWM (Morris water maze) test (90 seconds); Figure 7 D is the learning curve of the training phase (n=6-8); Figure 7 E. Figure 7 F is the number of times TBI mice passed through the target platform and the latency to stay in the quadrant where the platform was located (n=6-8); Figure 7 G is a representative thermal imaging tracking image of mice after the novel object recognition test. Figure 7 H is the analysis of the discrimination index to evaluate the memory ability of TBI mice (n=6-8); data are expressed as mean ± SD, compared with the sham group, *p<0.05, **p<0.01, ***p<0.001, ****p<0.000, compared with the TBI+vehicle group, #p<0.05, compared with the TBI+Flv group, +p<0.05, ++p<0.01.

[0081] Among them, the modified neurological severity score (mNSS): In order to evaluate neurological function, the mNSS scoring system including motor, sensory, reflex and balance tests was used; a baseline assessment was performed before modeling, and scores were performed on the 1st, 3rd, 5th, 7th and 14th days after modeling; the mNSS score ranged from 0 to 18 points, and the higher the score, the more severe the neurological deficit. This comprehensive scoring system can monitor the functional impairment over time in detail.

[0082] Fatigue Rotarod test: On the 1st, 3rd, 5th, 7th and 14th day after TBI simulation or sham operation (sham group, 0.2 mL of saline injected through the tail vein) in mice, the rotarod (YLS-4C, Beijing) was used to evaluate the changes in the motor coordination and balance of mice. Before the test, all mice were trained on the rotarod for 300 seconds (5-10 rpm); on the day of the test, the rotarod was accelerated from 4 rpm to 40 rpm at a linear speed; the mice ran for a maximum of 5 minutes per test, with a 5-minute rest period between each test, and the latency before falling was recorded. The average latency of three consecutive tests was used as the result of statistical analysis.

[0083] Morris water maze (MWM) experiment: On days 15 to 20 after TBI, the spatial learning and memory abilities of mice were assessed by the MWM experiment. The experiment was divided into a training phase (days 15-19) and a test phase (day 20); in the training phase, the circular pool was divided into four quadrants, and the hidden platform was located in the third quadrant. Each mouse had 90 seconds to find the platform and stay on it for 3 seconds; if the platform was not found, it was guided to the platform and a latency of 90 seconds was recorded. Mice were trained four times a day, starting from a different quadrant each time; on day 20, the platform was removed for testing, and the software recorded and analyzed the latency of the training phase, the swimming path of the test phase, the number of times the platform position was crossed, and the time spent in the target quadrant.

[0084] Novel object recognition (NOR) experiment: The mice were placed in a 40×40×40cm 3 The mice were placed in an environment with two identical objects equidistant from the side walls. The mice explored in a quiet environment for 5 minutes. After a 4-hour interval, the mice underwent a short-term memory test, during which one of the familiar objects was replaced with a new object, and the mice explored again for 5 minutes. The exploration time and total exploration time of the new object were recorded and analyzed by the EthoVisionXT 13 video tracking system, and the discrimination index (i.e., the ratio of the exploration time of the new object to the total exploration time) was calculated.

[0085] from Figure 7 It can be seen that the motor function of mice was severely affected on the first day after TBI simulation and lasted until the 14th day. TPL-Flv treatment significantly improved the motor coordination ability of mice ( Figure 7 A. Figure 7 B); In the water maze test, the latency of mice in the TBI+vehicle and TBI+Flv groups to reach the target platform was longer than that in the sham group, while injection of the TBI+TPL-Flv group reduced the latency on days 18 and 19 after injury ( Figure 7D); After the platform was removed on the 20th day, the frequency of mice crossing the platform position was analyzed. The results showed that the number of crossings in the TBI+TPL-Flv group increased significantly, which was better than that in the TBI+vehicle group and the TBI+Flv group ( Figure 7 C. Figure 7 E); In addition, the TBI+TPL-Flv group spent longer time in the target quadrant after TBI in mice than the TBI+vehicle group and the TBI+Flv group ( Figure 7 F); In the novel object test, TBI mice in the TBI+vehicle and TBI+Flv groups spent a lower percentage of time around the novel object, while TBI+TPL-Flv treatment alleviated this phenomenon ( Figure 7 G. Figure 7 H). These results demonstrate that the administration of TPL-Flv can improve motor function, spatial learning and memory ability, and relieve anxiety in mice after TBI injury.

[0086] Comparative Example 1 to Comparative Example 2

[0087] Comparative Examples 1 to 2 provide a method for preparing a responsive liposome targeting the blood-brain barrier and loading drugs for treating traumatic brain injury. Compared with Example 1, the difference is that phosphatidylcholine, cholesterol, fluvoxamine, phospholipid-polyethylene glycol (DSPE-PEG2K) and TfR-T12 target peptide are used in different molar ratios of 55:35:10:4.5:0.5 and 55:35:3:4.5:0.5, respectively. The rest is roughly the same as Example 1 and will not be repeated here.

[0088] Comparative Example 3

[0089] Comparative Example 3 provides a method for preparing responsive liposomes that target the blood-brain barrier and load drugs to treat traumatic brain injury. Compared with Example 1, the difference is that in step S2, no reduced pressure evaporation film formation and heavy hydration treatment are performed, and nanoparticles are prepared by using a liposome extruder after direct ultrasonic treatment. The rest is roughly the same as Example 1 and will not be repeated here.

[0090] Comparative Examples 4 to 9

[0091] Comparative Examples 4 to 9 provide a method for preparing responsive liposomes that target the blood-brain barrier and load drugs for treating traumatic brain injury. Compared with Example 1, the difference is that in step S2, the temperature of dehydration, the ultrasonic treatment time and the frequency are different, as shown in Table 1. The rest is roughly the same as Example 1 and will not be repeated here.

[0092] Table 1 Parameter settings of Comparative Examples 4 to 9

[0093] Dehydration temperature / ℃ Ultrasonic time / min Frequency / kHz Comparative Example 4 5 10 30 Comparative Example 5 35 10 30 Comparative Example 6 20 3 30 Comparative Example 7 20 18 30 Comparative Example 8 20 10 20 Comparative Example 9 20 10 40

[0094] In the process of preparing responsive liposomes targeting the blood-brain barrier and loaded with drugs for treating traumatic brain injury by using Example 1 and Comparative Examples 1 to 9, the encapsulation efficiency and drug loading of Flv were analyzed, and the results are shown in the following table.

[0095] Table 2 Characterization of encapsulation efficiency and drug loading of Example 1 and Comparative Examples 1 to 9

[0096]

[0097]

[0098] As can be seen from Table 2, the liposomes prepared in Comparative Example 1 did not significantly increase the drug loading due to the increase in drug dosage, and the drug loading efficiency was not significantly improved in Comparative Example 2 due to the reduction in drug ratio; in Comparative Example 3, no reduced pressure evaporation film forming and dehydration treatment were performed, resulting in a significant decrease in the encapsulation rate of the synthesized liposomes; in Comparative Examples 4 and 5, due to the low or high dehydration temperature, the encapsulation rate and loading of the drug were significantly reduced; in Comparative Example 6, due to insufficient ultrasonic time, the drug was not fully encapsulated; in Comparative Example 7, due to the long ultrasonic time, more lipids were involved in the synthesis, reducing the drug loading; in Comparative Examples 8 and 9, due to the low or high ultrasonic frequency, the encapsulation efficiency and drug loading of the synthesized liposomes did not reach the level in Example 1.

[0099] It should be noted that the present application is not limited to the above-mentioned embodiments. The above-mentioned embodiments are only examples, and the embodiments having the same structure as the technical idea and exerting the same effect within the scope of the technical solution of the present application are all included in the technical scope of the present application. In addition, without departing from the scope of the main purpose of the present application, various modifications that can be thought of by those skilled in the art to the embodiments and other methods of combining some of the constituent elements in the embodiments are also included in the scope of the present application.

Claims

1. A method for preparing a responsive liposome targeting the blood-brain barrier and loading a drug for treating traumatic brain injury, characterized in that: The following steps are involved: S1, preparing TfR-T12 target peptide by solid phase peptide synthesis process; S2, dissolving phosphatidylcholine, cholesterol, hydrophobic molecular drugs, amphiphilic polymer molecules and the TfR-T12 target peptide prepared in step S1 in chloroform, evaporating under reduced pressure to form a film, then dehydrating to hydrate the film and self-assemble it, and then ultrasonically treating it, and using a liposome extruder to obtain nanoparticles containing hydrophobic molecular drugs; S3. Using dialysis or centrifugal filtration to remove the hydrophobic molecular drugs or amphiphilic polypeptide molecules not loaded on the surface of the nanoparticles, to obtain responsive liposomes that target the blood-brain barrier and load drugs to treat traumatic brain injury.

2. The method for preparing the responsive liposomes targeting the blood-brain barrier and loading drugs for treating traumatic brain injury according to claim 1, characterized in that: In step S2, the molar ratio of phosphatidylcholine, cholesterol, hydrophobic molecular drug, amphiphilic polymer molecule and TfR-T12 target peptide is (50-60):(30-40):(3-8):(2.5-7.5):(1-2).

3. The method for preparing the responsive liposomes targeting the blood-brain barrier and loading drugs for treating traumatic brain injury according to claim 1, characterized in that: In step S2, the method of dehydration is to add deionized water, and the amount of deionized water added is 0.6 to 1.5 times the volume of the chloroform.

4. The method for preparing the responsive liposomes targeting the blood-brain barrier and loading drugs for treating traumatic brain injury according to claim 3, characterized in that: In step S2, the temperature of the dehydration is 20-25°C, the time of ultrasonic treatment is 5-15 minutes, and the frequency is 25-35 kHz.

5. The method for preparing the responsive liposomes targeting the blood-brain barrier and loading drugs for treating traumatic brain injury according to claim 1, characterized in that: In step S2, the hydrophobic molecular drug is fluvoxamine, and the amphiphilic polymer molecule is phospholipid-polyethylene glycol-maleimide or phospholipid-polyethylene glycol-carboxyl.

6. The method for preparing the responsive liposomes targeting the blood-brain barrier and loading drugs for treating traumatic brain injury according to claim 1, characterized in that: In step S2, the pore size of the liposome extruder is 80-120 nm.

7. The method for preparing the responsive liposomes targeting the blood-brain barrier and loaded with drugs for treating traumatic brain injury according to claim 1, characterized in that: In step S1, the specific preparation process of the TfR-T12 target peptide is: using Fmoc-protected amino acids to synthesize the target peptide by coupling with Rink amide AM resin or Wang resin.

8. The method for preparing the responsive liposomes targeting the blood-brain barrier and loading drugs for treating traumatic brain injury according to claim 7, characterized in that: The TfR-T12 target peptide includes an Fmoc-modified amino acid matrix and several amino acids with specific sequences modified on the Fmoc-modified amino acid matrix. The amino acid sequence of the TfR-T12 target peptide is THRPPMWSPVWP.

9. A responsive liposome targeting the blood-brain barrier and loading drugs for treating traumatic brain injury, characterized in that: The responsive liposome is prepared by the method for preparing the responsive liposome targeting the blood-brain barrier and loading drugs for treating traumatic brain injury according to any one of claims 1 to 8.

10. The responsive liposome targeting the blood-brain barrier and loaded with drugs for treating traumatic brain injury according to claim 9, characterized in that: The responsive liposome is a nanoparticle with the TfR-T12 target peptide exposed on the outer surface and the drug for treating traumatic brain injury encapsulated inside.

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