LPC liposome preparation for brain-targeted delivery of chemotherapeutic drugs as well as preparation method and application of LPC liposome preparation

Abstract

The invention provides an LPC liposome preparation for brain-targeted delivery of chemotherapeutic drugs as well as a preparation method and application of the LPC liposome preparation. The LPC liposome preparation comprises L-alpha-lysophosphatidylcholine, egg yolk lecithin and cholesterol, and the mass of the L-alpha-lysophosphatidylcholine is 5%-30% of the total mass of the L-alpha-lysophosphatidylcholine, the egg yolk lecithin and the cholesterol. The constructed LPC lipidosome can accelerate endocytosis of the lipidosome in bECs, and the lipidosome is transferred to an endosome recovery way by up-regulating p62, so that the lipidosome is prevented from entering a lysosome degradation way, and the transendocytosis efficiency is remarkably improved.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and in particular relates to an LPC liposome preparation for brain-targeted delivery of chemotherapy drugs, and a preparation method and application thereof. Background Art

[0002] Glioblastoma (GBM) is the most prevalent and aggressive primary brain malignancy. Standard treatment includes surgical resection, followed by chemotherapy with temozolomide (TMZ), radiotherapy, and more recently, the introduction of tumor treating fields. Despite these interventions, GBM continues to challenge medical professionals, with median survival hovering around 15 months. A major obstacle to the application of many clinically approved drugs in glioma treatment is the difficulty in achieving therapeutic concentrations in the brain due to the restrictive nature of the blood-brain barrier (BBB). A typical example is paclitaxel (PTX), a drug that is 1,400 times more potent than TMZ but cannot be used in clinical glioma treatment due to insufficient BBB penetration. Therefore, developing technologies to overcome the BBB and enhance drug delivery is crucial to advancing GBM treatment.

[0003] The BBB is a unique, highly regulated cerebrovascular unit composed primarily of cerebrovascular endothelial cells (BECs). This barrier meticulously controls the exchange of substances to maintain brain homeostasis. Successfully delivering therapeutic agents across the BBB into the brain parenchyma is a critical but challenging goal. Current BBB penetration strategies utilize both passive and active mechanisms. Passive transport involves crossing the tight junctions between BECs, selectively allowing molecules of specific size and lipophilicity. Using focused ultrasound to transiently disrupt BBB tight junctions followed by drug injection has shown some promise, but this technique may disrupt brain homeostasis. Advances in nanotechnology have introduced nanoparticle-based delivery systems as a viable approach to enhance drug penetration across the BBB. Leveraging their capabilities for drug encapsulation, controlled release, and prolonged circulation, various nanoparticles, particularly liposomes, have been used to deliver drugs to the brain. Recent studies have demonstrated that up to 97% of nanoparticles cross the vascular barrier via active transport in endothelial cells. Current techniques primarily focus on enhancing receptor-mediated active transport across the BBB through ligand modification of nanoparticles. However, it is important to note that active transport across the BBB, also known as transcytosis, involves three steps: endocytosis, vesicle trafficking, and exocytosis. bECs have a unique characteristic of extremely low levels of vesicle formation and trafficking, which may significantly limit the efficiency of nanoparticles crossing the BBB. Furthermore, ligand-modified nanoparticles are easily embedded in lysosomes, which hinders efficient transcytosis.

[0004] Therefore, developing a robust carrier that effectively stimulates active transport of nanoparticles across the BBB is crucial for enhancing drug delivery and efficacy in GBM treatment. The core of this carrier is to optimize the endocytosis and directed transcytosis of nanoparticles in bECs. Summary of the Invention

[0005] In view of this, the present invention aims to overcome the defects in the prior art and proposes an LPC (lysophosphatidylcholine) liposome preparation for brain-targeted delivery of chemotherapy drugs, as well as a preparation method and application thereof.

[0006] To achieve the above object, the technical solution of the present invention is achieved as follows:

[0007] In a first aspect, the present invention provides an LPC liposome preparation for brain-targeted delivery of chemotherapy drugs, comprising L-α-lysophosphatidylcholine, egg yolk lecithin, and cholesterol, wherein the mass fractions of the L-α-lysophosphatidylcholine, egg yolk lecithin, and cholesterol are as follows:

[0008] 5-30 parts of L-α-lysophosphatidylcholine;

[0009] 70-90 parts of egg yolk lecithin;

[0010] 1 part cholesterol;

[0011] The total number of parts of L-α-lysophosphatidylcholine, egg yolk lecithin and cholesterol is 100 parts.

[0012] Preferably, the mass fraction of the L-α-lysophosphatidylcholine is 5 parts, 10 parts, 20 parts, 25 parts or 30 parts.

[0013] Preferably, the L-α-lysophosphatidylcholine is L-α-lysophosphatidylcholine derived from bovine brain.

[0014] In a second aspect, the present invention further provides a method for preparing the above-mentioned LPC liposome preparation for brain-targeted delivery of chemotherapy drugs, comprising the following steps:

[0015] Egg yolk lecithin and cholesterol are dissolved in an organic solvent and evaporated under reduced pressure to form a film, and then ultrapure water is added for ultrasonication; after being treated with ultrasonication and a liposome extruder, a freeze-drying protective agent is added and freeze-dried.

[0016] Preferably, the liposome extruder is a polycarbonate membrane with a pore size of 200 nm.

[0017] Preferably, the organic solvent is chloroform.

[0018] In a third aspect, the present invention also provides the use of the above-mentioned LPC liposome preparation for brain-targeted delivery of chemotherapy drugs in the preparation of drugs that cross the blood-brain barrier.

[0019] In a fourth aspect, the present invention further provides a pharmaceutical composition for treating brain glioma, comprising the above-mentioned LPC liposome preparation and paclitaxel.

[0020] In a fifth aspect, the present invention further provides a method for preparing the above-mentioned pharmaceutical composition for treating brain glioma, comprising the following steps:

[0021] Egg yolk lecithin, cholesterol, L-α-lysophosphatidylcholine and paclitaxel are dissolved in an organic solvent and evaporated under reduced pressure to form a film, and then ultrapure water is added for ultrasonic treatment; the film is treated with ultrasound and a liposome extruder; the film is dialyzed through a nanodialysis membrane to remove unloaded paclitaxel; and finally, a freeze-drying protective agent is added and freeze-dried.

[0022] The present invention aims to enhance the endocytosis and transcytosis of liposomes across the BBB, with the goal of improving drug delivery to GBM tissue. Phospholipids are key components of cell membranes and vesicles, playing a crucial role in regulating the formation and trafficking of intracellular vesicles. Our previous studies have identified the significant influence of L-α-lysophosphatidylcholine (LPC), a single-tailed lipid, in regulating membrane-related physiological processes, including endocytosis and exocytosis. We found that LPC can stimulate the formation of endocytic vesicles in bECs, thereby increasing cargo uptake and enhancing transcytosis. Leveraging these findings, we explored the use of LPC to modulate the active transport behavior of conventional liposome delivery systems across the BBB to enhance drug delivery efficiency. We demonstrated that the addition of LPC to a conventional liposome formulation, constructing LPC liposomes, accelerated endocytosis of liposomes in bECs and, by upregulating p62, redirected them to the recycling endosome pathway, avoiding lysosomal degradation, thereby significantly improving transcytosis efficiency. Using a GBM patient-derived xenograft (PDX) model, we demonstrated that LPC liposomes can cross the BBB, leading to higher PTX concentrations within GBM tissue and enhanced chemotherapeutic and immunotherapy efficacy. Furthermore, we validated the use of LPC to enhance a commercial PTX liposomal formulation. Comparable therapeutic outcomes to TMZ could be achieved in GBM, highlighting the potential of this novel technology in GBM treatment.

[0023] Compared with the prior art, the present invention has the following advantages:

[0024] This invention aims to improve the ability of glioma therapeutic drugs to cross the blood-brain barrier and enhance drug treatment effects. Combining cell biology and nanomaterials methods, this invention studies the effects of phospholipid LPC on liposome blood-brain barrier penetration and accumulation in glioma tissues. It also clarifies the regulatory effects and mechanisms of LPC modification on the intracellular transport of liposomes in vascular endothelial cells. Furthermore, LPC-modified liposome drug delivery systems are constructed for chemotherapy drugs and small molecule drugs. The drug delivery efficacy, glioma treatment efficacy, and safety are verified, and preclinical studies are conducted. The implementation of this patent is expected to establish a nano-drug delivery system with transformational potential for glioma treatment, provide a new solution for enhancing glioma drug treatment, and bring technological innovation to clinical glioma treatment through the integration of medicine and engineering. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 LPC enhances cargo uptake and transcellular transport in bECs. Figure: (A) LPC-treated bEnd.3 cell membrane fluidity is enhanced; (B) LPC-treated bEnd.3 cell membrane deformability is enhanced; (C) The density of pits on the bEnd.3 cell membrane increases after LPC treatment; (D) The density of early endosomes in bEnd.3 cells increases after LPC treatment; (EF) Molecular dynamics simulations show that the rate of protein penetration through the cell membrane is enhanced in the presence of LPC; (G) Cellular uptake of fluorescently labeled transferrin (Cy5-Tfn) is enhanced in LPC-treated bEnd.3 cells; (H) LPC treatment improves the efficiency of Cy5-Tfn transport to the lower chamber in an in vitro BBB model; (I) Schematic diagram of LPC-enhanced endocytosis and transcellular transport in bECs.

[0026] Figure 2 LPC modification enhances liposome endocytosis and transcytosis across the blood-brain barrier. Figures: (A) Molecular dynamics simulations show that LPC-containing lipid vesicles have stronger membrane tensile traction during transmembrane transport; (B) Schematic diagram of the synthesis of Lipo and LPC-Lipo and their particle size distribution; (C) Flow cytometry analysis of the time-dependent endocytosis of Lipo and LPC-Lipo in bEnd.3 cells demonstrates that LPC-Lipo has a higher endocytosis efficiency; (D) The cumulative transcellular transport efficiency of Lipo and LPC-Lipo at different incubation times in an in vitro BBB model demonstrates that LPC-Lipo has a higher transport efficiency; (E) Schematic diagram of how the addition of LPC components enhances liposome endocytosis and transport across the BBB.

[0027] Figure 3Comparison of LPC-Lipo with different LPC ratios; Figure: (A) Particle size distribution data of LPC-Lipo with LPC ratios ranging from 0% to 20%, with an average particle size of 130nm; (B) When the LPC ratio reaches 25% and 30%, uniformly dispersed liposomes cannot be formed; (C) Zeta potential data of LPC-Lipo with LPC ratios ranging from 0% to 20%; (D) There is no obvious cytotoxicity from LPC ratios ranging from 0% to 20%; (E) Representative confocal images and flow cytometry analysis of LPC-Lipo endocytosis with different LPC ratios in bEnd.3 cells, with the highest efficiency when the LPC ratio is 10%;

[0028] Figure 4 Figure 3: The blood-brain barrier permeation effect of LPC-Lipo in vivo and the internalization efficiency of GBM cells in vitro. Figure 3: (AB) Real-time fluorescence tracking images and fluorescence intensity quantification of DiR-labeled Lipo and LPC-Lipo in the brain region of mice carrying TBD0220, demonstrating that LPC-Lipo has a higher brain enrichment efficiency in vivo. (C) In vitro fluorescence images and relative fluorescence intensity quantification of brain tissue collected with and without perfusion in mice injected with Lipo and LPC-Lipo, demonstrating that LPC-Lipo has a higher brain enrichment. (D) Confocal images of the GBM region in brain slices, with blood vessels marked in green and liposomes marked in purple, demonstrating that LPC-Lipo effectively penetrates blood vessels and enters tumor tissue. (EJ) LPC-Lipo has a higher GBM cell internalization efficiency in U87 (E, G, and I) and TBD0220 (F, H, and J) cells.

[0029] Figure 5 Figure 3: Blood-brain barrier penetration and GBM therapeutic effect of LPC-Lipo-encapsulated paclitaxel (LPC-LPTX); Figure: (A) Particle size distribution of LPC-LPTX; (B) Compared with native liposomes, LPC-LPTX has a higher PTX drug concentration at the tumor site; (C) Schematic diagram of TBD0220-Luc tumor implantation and dosing timeline in PDX model; (D) Representative tumor bioluminescence images in mice 7, 14, and 21 days after tumor implantation; (EF) Individual tumors obtained by quantification of bioluminescence signal intensity. Figure 3 Total (E) and summary (F) tumor growth curves; (G) survival curves of TBD0220 tumor-bearing mice that received treatment; (H) Schematic diagram of the CT2A intracranial tumor model for studying the role of LPC-LPTX in GBM immunotherapy; (I) Representative bioluminescence images of CT2A tumor-bearing mice at 5, 10, and 17 days after tumor implantation; (J) Tumor growth curves plotted by quantifying bioluminescence imaging signal intensity; (K) Survival curves of CT2A tumor-bearing mice that received the indicated treatments; (L) Intratumoral CD8 +Representative flow cytometry results of T cell infiltration and their corresponding quantitative analysis;

[0030] Figure 6 Modification of LPC to enable commercialization of paclitaxel liposomal formulations It has therapeutic efficacy for GBM; in the figure: (A) Schematic diagram of the timeline of TBD0220-Luc tumor transplantation and LPC-Lipusu administration in PDX models; (B) Bioluminescence images of tumors in mice treated with LPC-Lipusu 7, 14, and 21 days after tumor transplantation; (C) Individual and aggregated (C) tumor growth curves obtained by quantifying the standardized bioluminescence signal intensity; (D) Survival curves of tumor-bearing mice in different treatment groups; (E) H&E staining images of different treatment groups. DETAILED DESCRIPTION

[0031] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be understood as limiting the present invention.

[0032] Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0033] Where values ​​are described herein as ranges, it should be understood that such disclosure includes disclosure of all possible sub-ranges within that range, as well as specific values ​​falling within that range, regardless of whether a specific value or sub-range is explicitly stated.

[0034] In this document, "a plurality of" and the like, unless otherwise specified, refer to a number greater than or equal to 2. For example, "one or more" means one or more than or equal to two.

[0035] In this document, the terms “preferably” and “more preferably” are only used to describe implementation methods or examples with better effects. It should be understood that they do not limit the scope of protection of the present invention.

[0036] In this document, the word "further" and the like are used for descriptive purposes to indicate differences in content, but should not be construed as limiting the scope of protection of the present invention.

[0037] In this article, the term "and / or" is used to describe the association relationship between objects, indicating that three relationships can exist. For example, A and / or B means: A or B, or A and B.

[0038] As used herein, the term "about" means + / - 10%, preferably + / - 5%, more preferably + / - 1% of the specified value.

[0039] In this document, the terms “include,” “including,” “have,” “contain,” etc. are open-ended terms, meaning including but not limited to.

[0040] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention.

[0041] The present invention will be described in detail below with reference to the embodiments.

[0042] Example 1 Construction of LPC liposomes

[0043] Egg yolk lecithin, 1% cholesterol, and L-α-lysophosphatidylcholine (derived from bovine brain, added in amounts of 5%, 10%, 20%, 25%, and 30%) were dissolved in 3 mL of chloroform and evaporated under reduced pressure in a sample vial to form a film. Ultrapure water was added for sonication. The film was then treated with sonication and a liposome extruder (polycarbonate membrane, 200 nm pore size). Finally, deionized water was added to bring the volume to 5 mL. A lyoprotectant was added and lyophilization was performed.

[0044] Example 2 Construction of LPC liposome-encapsulated paclitaxel preparation

[0045] 89% lecithin, 1% cholesterol, L-α-lysophosphatidylcholine (derived from bovine brain, added at 10%), and paclitaxel were dissolved in 12 mL of chloroform and evaporated under reduced pressure in a sample vial to form a film. Ultrapure water was added and sonicated. The solution was then treated with sonication and a liposome extruder (polycarbonate membrane, 200 nm pore size). Unloaded paclitaxel was then dialyzed using a nanodialysis device (polycarbonate membrane, 30 nm pore size). Finally, deionized water was added to the volume to 20 mL. A lyoprotectant was added and lyophilization was performed.

[0046] The formulations of Examples 1 and 2 above were tested.

[0047] Example 3 Establishment of animal model and experimental methods

[0048] 1. Establishment of mouse model:

[0049] GBM intracranial orthotopic mouse model:

[0050] Using an established GBM primary cell line, a needle was used to drill through the skull 1-2 mm to the right of the midline of the anterior and posterior bregma. After counting the cells, the tumor cells were implanted into the nude mouse skull using a stereotaxic apparatus. After cell implantation, the incision was sutured and the skin was disinfected before the mouse was returned to the SPF animal breeding room. Subsequent treatment experiments were conducted seven days after the intracranial inoculation surgery.

[0051] 2. Test method:

[0052] 1) Analyze the regulatory effect of phospholipid LPC on the endocytosis-efflux function of vascular endothelial cells

[0053] (1) Regulation of LPC on endocytosis- and efflux-related proteins: LPC from three sources (soybean, egg yolk, and bovine brain) was selected and prepared into solutions below their respective critical micelle concentrations. bEnd.3 cells were treated with a concentration gradient. Western blot and immunofluorescence (IF) were used to verify the effects of LPC on the protein levels and cellular localization of EEA1, RAB11, RAB7, and RAB27, and to determine the optimal LPC treatment concentration.

[0054] (2) Effect of LPC on cellular endocytosis: Transferrin (Tfn) was selected as a model molecule and labeled with Cy5 fluorescence. Cy5-Tfn was co-incubated with bEnd.3 and TBD0220 cells, and co-localization with early endosomes (EEA1) was analyzed by IF experiments. Cells were treated with LPC, and the fluorescence intensity of intracellular Tfn was analyzed by flow cytometry. Dynasore was further used to inhibit cell endocytosis, and the above flow cytometry analysis was repeated to verify the effect of LPC on endocytosis.

[0055] (3) Effect of LPC on transendothelial transport: The colocalization of Cy5-Tfn and circulating endosomes (RAB11) was analyzed in endothelial cells by IF. An in vitro BBB model was constructed using a Transwell chamber. bEnd.3 cells were seeded into the upper layer of the chamber. The transendothelial resistance of the bEnd.3 monolayer was measured using a resistance meter. When the resistance reached 200-300 Ω·cm 2 When the cells were 100 μm, a dense monolayer was formed, indicating successful model establishment. bEnd.3 monolayers were treated with LPC, and then Cy5-Tfn was added to the upper chamber. After 12 hours of incubation, the fluorescence intensity of the fluid in the lower chamber was measured using a fluorescence spectrophotometer to calculate the penetration efficiency. In the BBB model, TBD0220 cells were seeded into the well plate in the lower chamber of the chamber. The above experiment was repeated. Confocal microscopy and flow cytometry were used to observe and quantify the fluorescence intensity of Cy5-Tfn in the tumor cells in the lower chamber, comprehensively analyzing the regulatory effects of LPC.

[0056] 2) Construct LPC liposomes and investigate the structure-activity relationship between LPC incorporation, blood-brain barrier penetration, and glioma cell internalization

[0057] (1) Construction and characterization of LPC-Lipo: Traditional liposomes (Lipo) were prepared using PC and cholesterol molecules via a thin film hydration method. LPC liposomes (LPC-Lipo) were prepared using PC, LPC, and cholesterol molecules with an LPC ratio of 5%-30% via a thin film hydration method. The particle size distribution, zeta potential, morphology, and stability of Lipo and LPC-Lipo were characterized by dynamic light scattering and transmission electron microscopy.

[0058] (2) Analysis of endocytosis-efflux regulation: LPC-Lipo with different LPC ratios was constructed according to the above steps. Lipo without LPC was used as a control. Western blot and IF were used to verify the effect of LPC-Lipo treatment on the protein levels and cellular localization of transmembrane transport markers EEA1, RAB11, RAB7, and RAB27 in bEnd.3 and TBD0220 cells. The optimal LPC modification ratio was determined based on the above results.

[0059] (3) Endocytosis-efflux pathway analysis: LPC-Lipo was labeled with DiI fluorescent dye, and co-localization with EEA1 and RAB11 in bEnd.3 endothelial cells was confirmed by IF experiments to verify the transcytosis pathway. bEnd.3 and TBD0220 cells were pretreated with Dynasore, low temperature (4°C), and endocytosis pathway inhibitors (Filipin, Nystatin, Chlorpromazine, Amiloride). LPC-Lipo was co-incubated with the cells, and the endocytosis pathway was analyzed by flow cytometry.

[0060] (4) Analysis of transmembrane transport kinetics: An in vitro BBB model was established, and fluorescently labeled LPC-Lipo was added to the upper chamber. The culture medium in the lower chamber was collected at predetermined time intervals and supplemented with an equal volume of culture medium. The fluorescence intensity was measured using a fluorescence spectrophotometer, and the penetration efficiency was calculated to plot the BBB permeation kinetics curve. Fluorescently labeled LPC-Lipo was co-incubated with TBD0220 cells, and the cells were collected at predetermined time intervals. The intracellular fluorescence intensity was analyzed by flow cytometry to plot the tumor internalization kinetics curve.

[0061] 3) Elucidate the regulatory mechanism of LPC component incorporation on the transmembrane transport pathway of liposomes in vascular endothelial cells

[0062] (1) Effect of LPC on cell membrane structure: cells were seeded in culture dishes, and LPC or LPC-Lipo was added. The following experiments were performed: ①Simulation of ion conductance microscopy (SICM): performed in L15 culture medium, cells were observed in real time using SICM, and 3D images of cells were reconstructed after scanning to analyze membrane disturbances; ②Fluorescence recovery after photobleaching (FRAP) experiment: the cell membrane of living cells was stained with DiI, and a defined area of ​​the cell membrane was photobleached under a confocal microscope. The fluorescence intensity was recorded every 10 seconds after bleaching, and the membrane fluidity was evaluated by the fluorescence recovery rate; ③Transmission electron microscopy (TEM): cells were collected and fixed with glutaraldehyde, and the formation and density of pits on the membrane surface were observed and analyzed by TEM;

[0063] (2) Molecular dynamics (MD) simulations: MD simulations were used to capture the endocytic process of cells treated with LPC and the interaction between LPC-liposomes and the cell membrane. A 60 nm × 60 nm planar membrane and a 20 nm radius vesicle were used to simulate the cell membrane and liposomes, respectively. Constant-rate stretching and constant-force stretching modes were used to pull protein molecules through the model cell membrane with or without LPC, or to pull model liposomes with or without LPC through the model cell membrane. The data were recorded and analyzed.

[0064] (3) Proteomic analysis: After treating cells with LPC, protein extraction was performed, and the protein samples were enzymatically hydrolyzed, desalted, concentrated, and separated by high-performance liquid chromatography to collect the components. The pre-separated components were repeatedly loaded three times using ultra-high pressure liquid chromatography and high-resolution mass spectrometry for identification. Data independent scanning mode (DIA) was used for data acquisition and quantification. After data processing and retrieval, comparative analysis was performed to identify differentially expressed proteins and screen for key factors mediating vesicle transport processes;

[0065] (4) Verification of transmembrane transport mechanism: The CRISPR-Cas9 gene editing system was used to knock out the above key molecules in the cells. After the cells were treated with LPC or LPC-Lipo, the key vesicle transport pathways in the cells were labeled with immunofluorescence and lysosomal probes. The changes in the intracellular transport pathways of liposomes were observed by confocal microscopy combined with 3D reconstruction. The BBB penetration and tumor intracellular enrichment efficiency were quantitatively analyzed by flow cytometry. The above results were combined to analyze the regulatory mechanism of LPC on the transmembrane transport process of liposomes.

[0066] 4) In vitro and in vivo experiments validated the drug delivery efficacy and GBM treatment effects of LPC-modified liposomes for the chemotherapy drug paclitaxel

[0067] (1) Drug loading evaluation: During liposome preparation, PTX was added to the formulation to construct PTX-loaded LPC-Lipo. The loading conditions were investigated by characterizing the particle size and morphology. After drug loading, the drug was purified by adding a methanol / acetonitrile mixture to the drug-loaded liposome solution to extract the drug. After vortexing and centrifugation, the supernatant was collected for liquid chromatography-tandem mass spectrometry analysis to calculate the drug loading efficiency.

[0068] (2) Evaluation of in vitro tumor inhibition effect: TBD0220 cells were treated with drug-loaded LPC-Lipo according to a concentration gradient. The naked drug group and the unmodified drug-loaded liposome group were used as controls. The tumor inhibition effects were compared by cell plate colony formation assay, CCK-8 assay, and Western blot analysis.

[0069] (3) Evaluation of drug delivery efficacy: DiR-labeled LPC-Lipo was injected into GBM intracranial orthotopic model mice through the tail vein. The bioluminescence signal and fluorescence signal of the brain were monitored by in vivo imaging and two-photon imaging system at the predetermined time points. The mice were euthanized 24 hours after injection, and the brain tissue was obtained and subjected to in vitro fluorescence imaging using an imaging system. In addition, drug-loaded LPC-Lipo was injected, and the mice were killed 12 hours later. The brain tissue on the tumor side, normal brain tissue and other organs were collected. The tissues were homogenized and drug extraction was performed. The supernatant was sampled for liquid chromatography-mass spectrometry analysis to calculate the drug enrichment in the tissue.

[0070] (5) In vivo evaluation of tumor inhibition: GBM intracranial orthotopic model mice were randomly divided into four groups: control group, PTX, PC-Lipo-PTX, and LPC-Lipo-PTX. The drugs were injected into the tail vein. After the treatment cycle, the brain tissue distribution of the vector was observed by immunofluorescence. The tumor proliferation inhibition effect was detected by combining in vivo small animal imaging, animal survival observation, HE staining, and immunohistochemistry.

[0071] Example 4 Regulatory Effects of LPC on Endocytosis-Exocytosis Function of Vascular Endothelial Cells

[0072] In mouse brain microvascular endothelial cells (bEnd.3), LPC treatment was found to increase the expression levels of the early endosomal marker EEA1 and the circulating endosomal marker RAB11. Based on this, the following studies were conducted:

[0073] 1) In bEnd.3 cells, the effects of LPC on cell membrane structure and fluidity were observed using ion conductivity microscopy and fluorescence recovery after photobleaching experiments, and the effects on the formation and density of pits on the membrane surface were observed using transmission electron microscopy.

[0074] 2) Study the effect of LPC on the expression levels of transmembrane transport markers EEA1, RAB11, RAB7 (lysosomes), and RAB27 (exocrine) according to the concentration gradient;

[0075] 3) Molecular dynamics simulations were used to investigate the effect of LPC treatment on the kinetics of model molecule internalization into cells;

[0076] 4) Using transferrin (Tfn) as a model molecule, flow cytometry was used to analyze the effect of LPC on the endocytic efficiency of Tfn. An in vitro BBB model was established to quantitatively analyze the effect of LPC on the transendothelial transport efficiency of Tfn.

[0077] Example 5 Structure-activity relationship between blood-brain barrier penetration and glioma cell internalization of LPC-Lipo

[0078] The following studies were conducted on the LPC liposomes (LPC-Lipo) constructed in Example 1:

[0079] 1) Characterize the size, potential, morphology, and stability of LPC-Lipo at different LPC ratios through dynamic light scattering, nanoparticle tracer analysis, and transmission electron microscopy.

[0080] 2) After treating cells with LPC-Lipo at different LPC ratios, investigate the changes in endocytosis and efflux markers (EEA1, RAB11, RAB7, and RAB27) and determine the optimal LPC modification ratio based on the above results;

[0081] 3) Comparative analysis of the endocytic efficiency of LPC-Lipo in bEnd.3 endothelial cells and TBD0220 glioma cells by confocal microscopy and flow cytometry, and analysis of the endocytic pathway in combination with endocytic pathway inhibitors;

[0082] 4) Quantitatively analyze the changes in the transmembrane transport of LPC-Lipo in cells over time, and draw the BBB penetration kinetics curves and tumor intracellular enrichment kinetics curves of the two to clarify the effect of LPC modification on the blood-brain barrier penetration of nanovesicles and the internalization effect of glioma cells.

[0083] Example 6: Mechanism of Regulation of Transmembrane Transport Pathway of Liposomes in Vascular Endothelial Cells by Incorporation of LPC Components

[0084] Transmembrane transport is the process of intracellular vesicle transport: substances are wrapped in the cell membrane and invaginated to form early endosomes. Early endosomes can undergo two transport pathways: one is to form recycling endosomes and fuse with the cell membrane for transport, and the other is to mature into late endosomes and fuse with lysosomes for degradation. Therefore, the transmembrane transport pathway of LPC liposomes is studied:

[0085] 1) Immunofluorescence combined with confocal and super-resolution microscopy was used to observe the colocalization of LPC liposomes with early endosomes, recycling endosomes, and lysosomes. The colocalization ratio was calculated to analyze the effect of LPC on transmembrane transport pathways.

[0086] 2) Use proteomics to analyze differentially expressed proteins in cells treated with LPC to identify key molecules that mediate vesicle transport. This will be further validated through biological experiments to clarify the molecular mechanisms by which LPC regulates lipid transport to circulating endosomes or lysosomes.

[0087] 3) Interfere with the expression of the above key molecules in cells, combine immunofluorescence and confocal microscopy to observe the intracellular transport pathway of nanovesicles, and quantitatively analyze the transendothelial transcytosis efficiency by flow cytometry. Based on the above results, analyze the regulatory mechanism of LPC modification on the transmembrane transport pathway of liposomes.

[0088] Example 7 Establishment of LPC liposome drug delivery system and verification of its drug delivery efficacy, GBM treatment effect and safety in vitro and in vivo

[0089] Based on the above mechanism, a new drug formulation (LPC-Lipo(PTX)) was developed by encapsulating PTX with the synthesized LPC liposomes, targeting paclitaxel (PTX), a chemotherapy drug that is highly effective but cannot penetrate the BBB. The drug delivery efficiency of LPC liposomes and the drug therapy synergistic effect of the new drug formulation were verified using a PDX model.

[0090] 1) Liquid chromatography-mass spectrometry analysis of the encapsulation conditions and drug loading efficiency of PTX on LPC-Lipo;

[0091] 2) Treat TBD0220 cells with LPC-Lipo (PTX) and detect the cell proliferation inhibition efficiency to evaluate and compare the inhibitory effects of the two novel drug preparations on glioma cell proliferation;

[0092] 3) Establish an orthotopic intracranial glioma model in mice using TBD0220 cells. The GBM-targeting efficiency of LPC-Lipo was evaluated using in vivo small animal imaging and two-photon imaging. Liquid chromatography-mass spectrometry was used to detect drug distribution and evaluate drug delivery efficacy.

[0093] 4) Investigate tumor growth and survival after LPC-Lipo (PTX) injection using small animal imaging, survival analysis, and tissue section analysis. Evaluate the synergistic effect of a novel drug formulation constructed using LPC-Lipo on PTX therapy for GBM and assess the biosafety of the system.

[0094] from Figure 1 The results showed that LPC enhanced endocytic vesicle formation, cargo uptake and transcytosis in brain vascular endothelial cells.

[0095] from Figure 2The results show that in vitro experiments have shown that LPC liposomes formed by adding LPC to traditional liposome formulas have enhanced endocytosis and transcytosis in vascular endothelial cells, the main component of the blood-brain barrier, and the transcytosis efficiency can be increased by up to 3.3 times.

[0096] from Figure 3 The results show that when the LPC content is within 20%, LPC liposomes have better size distribution, stability and biosafety; in vitro experiments show that LPC liposomes with 10% LPC have the highest transcytosis efficiency.

[0097] from Figure 4 The results show that in vivo experiments have shown that LPC liposomes have enhanced blood-brain barrier penetration effect in vivo in the in situ glioma model, with the in vivo fluorescence enhanced by up to 3.8 times and the in vitro fluorescence enhanced by up to 2.5 times, and have good glioma cell internalization effect.

[0098] from Figure 5 The results show that a new paclitaxel liposome preparation (LPC-LPTX) was constructed by encapsulating paclitaxel using LPC liposomes, which can cross the blood-brain barrier and enhance the accumulation of paclitaxel in glioma sites, thereby achieving effective chemotherapy and immunotherapy effects on brain gliomas.

[0099] from Figure 6 The results show that LPC is modified into commercial paclitaxel liposome formulations. The resulting LPC-Lipusu can achieve therapeutic outcomes comparable to the gold standard TMZ in GBM.

[0100] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. An LPC liposome preparation for brain-targeted delivery of chemotherapy drugs, characterized by: It comprises L-α-lysophosphatidylcholine, egg yolk lecithin, and cholesterol, wherein the mass fractions of the L-α-lysophosphatidylcholine, egg yolk lecithin, and cholesterol are as follows: 5-30 parts of L-α-lysophosphatidylcholine; 70-90 parts of egg yolk lecithin; 1 part cholesterol; The total number of parts of L-α-lysophosphatidylcholine, egg yolk lecithin and cholesterol is 100 parts.

2. The LPC liposome preparation according to claim 1, wherein: The mass fraction of the L-α-lysophosphatidylcholine is 5 parts, 10 parts, 20 parts, 25 parts or 30 parts.

3. The LPC liposome preparation according to claim 1, wherein: The L-α-lysophosphatidylcholine is L-α-lysophosphatidylcholine derived from bovine brain.

4. The method for preparing the LPC liposome preparation for brain-targeted delivery of chemotherapy drugs according to any one of claims 1 to 3, characterized in that: The steps include: Egg yolk lecithin and cholesterol are dissolved in an organic solvent and evaporated under reduced pressure to form a film, and then ultrapure water is added for ultrasonication; after being treated with ultrasonication and a liposome extruder, a freeze-drying protective agent is added and freeze-dried.

5. Use of the LPC liposome preparation for brain-targeted delivery of chemotherapy drugs according to any one of claims 1 to 3 in the preparation of drugs that cross the blood-brain barrier.

6. A pharmaceutical composition for treating brain glioma, characterized in that: The invention comprises the LPC liposome preparation according to any one of claims 1 to 3 and paclitaxel.

7. The method for preparing the pharmaceutical composition for treating brain glioma according to claim 6, characterized in that: The steps include: Egg yolk lecithin, cholesterol, L-α-lysophosphatidylcholine and paclitaxel are dissolved in an organic solvent and evaporated under reduced pressure to form a film, and then ultrapure water is added for ultrasonic treatment; the film is treated with ultrasound and a liposome extruder; the film is dialyzed through a nanodialysis membrane to remove unloaded paclitaxel; and finally, a freeze-drying protective agent is added and freeze-dried.

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