Exosome-based brain cell specific targeted delivery

Abstract

The invention provides brain cell specific targeting delivery based on exosome, and the exosome secreted by 293T cells is used for engineering transformation, so that the capabilities of penetrating BBB and specifically targeting neurons are achieved. According to the present invention, the L1CAM is loaded on the exosome, the exosome is obtained by transfecting a 293T cell of a Lamp2b-L1CAM PCDH-HygroHA plasmid through a 293T-Lamp2b-L1CAM cell line, and the BBB cell-penetrating peptide 4F-T7 and the neuronal targeting protein L1CAM are loaded on the surface of the exosome so as to treat the neurodegenerative diseases such as the Alzheimer's disease and the Parkinson's disease.

Description

Technical Field

[0001] The present invention relates to the technical field of exosome carrier construction, and in particular to brain cell-specific targeted delivery based on exosomes. Background Art

[0002] As the global population ages, the incidence of neurodegenerative diseases is showing a significant upward trend. According to the World Health Organization, the prevalence of Alzheimer's disease in people over 65 is as high as 5%-10%, and the prevalence of Parkinson's disease exceeds 1%. The global number of patients is projected to exceed 150 million by 2050. These diseases, characterized by irreversible neuronal degeneration, not only lead to progressive loss of cognitive and motor function but also impose a significant socioeconomic burden—Alzheimer's disease-related medical care costs alone account for 1% of global GDP, and there is no cure. The hallmarks of Alzheimer's disease (AD) are neurofibrillary tangles (NFTs) formed by hyperphosphorylated tau protein within neurons and extracellular deposition of β-amyloid (Aβ) plaques. Clinical manifestations include memory loss, spatial disorientation, and loss of executive function. In advanced stages, patients completely lose the ability to care for themselves, with a median survival of only 4-8 years after diagnosis. Parkinson's disease (PD) is characterized by the accumulation of Lewy bodies of α-synuclein within dopaminergic neurons in the substantia nigra. Patients experience motor symptoms such as resting tremor, bradykinesia, and muscle rigidity, along with non-motor symptoms such as depression and loss of smell. In the late stages, death often results from dysphagia or lung infection. Current clinical medications (such as cholinesterase inhibitors for AD and levodopa for PD) only provide temporary relief of symptoms and are unable to halt disease progression.

[0003] While targeted therapies for pathological protein clearance (such as the Tau monoclonal antibody Donanemab and α-synuclein-targeted siRNA) have demonstrated significant degradation of abnormal proteins in preclinical studies, their efficacy in Phase II / III clinical trials has generally failed to meet primary endpoints. A key obstacle is the difficulty in drug delivery across the blood-brain barrier (BBB)—a dynamic interface composed of tight junctions between brain capillary endothelial cells, pericytes, and astrocyte endfeet. This interface allows only passive diffusion of lipophilic molecules smaller than 500 Da, while large-molecule therapeutics (such as antibodies and nucleic acid drugs) must rely on active transport mechanisms. Furthermore, even if drugs enter the brain parenchyma, they must be specifically targeted to diseased neurons to avoid off-target toxicity. In recent years, exosomes (EXOs)—natural nanovesicles with a diameter of 30-150 nm, released extracellularly by fusion of multivesicular bodies with the plasma membrane—have been considered ideal drug carriers for traversing the BBB due to their low immunogenicity, high biocompatibility, and natural transmembrane transport capacity. Its lipid bilayer membrane structure can protect the contents from degradation, and the surface proteins (such as CD9 and CD63) give cells targeting potential. It can also be loaded with various therapeutic molecules (such as small molecule compounds, nucleic acids, and proteins) through mother cell engineering or drug loading process optimization.

[0004] However, existing exosome delivery vehicles face two key limitations in the treatment of neurological diseases. First, natural exosomes are inefficient at penetrating the blood-brain barrier (BBB), preventing effective brain accumulation. The BBB, a highly selective and dynamic interface, strictly restricts the entry of large molecules into the brain parenchyma through tight junction proteins (such as claudin-5 and occludin) and active efflux pumps (such as P-glycoprotein). While unengineered natural exosomes can partially penetrate the BBB through passive diffusion or receptor-mediated transcytosis, their efficiency is extremely low: following intravenous injection, approximately 80%-90% of exosomes are rapidly cleared by the mononuclear phagocytic system (MPS) in the liver and spleen, leaving less than 0.5% of the dose accessible to the brain. Even if a small number of exosomes do cross the BBB, their diffusion is limited by the high viscosity and complex microenvironment of the brain's interstitial fluid, making effective accumulation in diseased areas (such as the hippocampus in Alzheimer's disease or the substantia nigra pars compacta in Parkinson's disease) difficult. For example, experiments examining the distribution of natural exosomes loaded with tau antibodies in the mouse brain showed that only 0.2%-0.4% of the injected dose reached the target brain region, and only 10%-15% of these exosomes were able to enter neuronal cell bodies, resulting in drug concentrations far below the therapeutic threshold. This deficiency directly results in existing exosome delivery systems being unable to meet the dosage requirements required to clear pathological proteins.

[0005] The second is that natural exosomes lack neuronal targeting specificity, leading to off-target effects and insufficient efficacy. The surface molecular composition of natural exosomes is determined by their source cells, but whether they are derived from mesenchymal stem cells, immune cells, or other cell types, they lack targeting ligands that specifically recognize neuronal surface receptors. Therefore, after entering the brain parenchyma, exosomes are mainly taken up by neurons through nonspecific endocytosis. This process is inefficient and cannot distinguish between healthy neurons and diseased neurons. For example, in Parkinson's disease models, α-synuclein aggregates are mainly present in dopaminergic neurons, but natural exosomes are non-selectively taken up by all types of neurons (such as glutamatergic neurons and GABAergic neurons), resulting in insufficient local concentration of drugs in the lesion area and potentially interfering with normal neuronal function. In addition, adhesion molecules on the surface of exosomes (such as integrins and CD47) may cross-react with non-target cells (such as astrocytes and microglia), further reducing delivery efficiency. Studies have shown that the non-targeted distribution of natural exosomes in the brain results in the delivery of α-synuclein siRNA to inhibit pathological protein expression by less than 20%, while clinical treatment requires a knockdown efficiency of at least 60%. These limitations severely limit the translational application of exosome-based delivery vehicles in the treatment of neurodegenerative diseases. Summary of the Invention

[0006] To address the shortcomings of the aforementioned issues, the present invention provides exosome-based brain cell-specific targeted delivery. This method utilizes exosomes secreted by 293T cells and is engineered to achieve the ability to penetrate the BBB and specifically target neurons. The method involves loading the exosomes with the BBB-penetrating peptide 4F-T7 and the neuron-targeting protein L1CAM for the treatment of neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease.

[0007] The present invention provides an exosome, wherein: the exosome is loaded with L1CAM.

[0008] Preferably, the exosomes are obtained by 293T-Lamp2b-L1CAM cell line, and the 293T-Lamp2b-L1CAM cell line is obtained by transfecting 293T cells with Lamp2b-L1CAM PCDH-Hygro-HA plasmid.

[0009] The present invention provides a 4F-T7 engineered exosome, wherein: the 4F-T7 engineered exosome is obtained by loading the exosomes of claim 1 with a 4F-T7 chimeric peptide.

[0010] Preferably, the sequence of the 4F-T7 chimeric peptide is DWFKAFYDKVAEKFKEAFGGHAIYPRH.

[0011] The present invention provides a method for preparing the above-mentioned exosomes, which comprises the following steps: transfecting the Lamp2b-L1CAMPCDH-Hygro-HA plasmid into 293T cells via liposome Lip800, replacing the culture medium containing 100 μg / mL hygromycin after 48 hours, and continuing to culture for a period of time, and then extracting the exosomes.

[0012] The present invention provides a method for preparing the above-mentioned 4F-T7 engineered exosomes, which comprises the following steps: synthesizing 4F-T7 chimeric peptides by solid-phase synthesis, incubating 293T-Lamp2b-L1CAM exosomes and 4F-T7 chimeric peptides at a molar ratio of 1:50 at 37°C for 2 hours, and removing unbound free peptides by ultrafiltration and centrifugation (100 kDa) to obtain 4F-T7 engineered exosomes.

[0013] As a key physiological barrier of the central nervous system, the BBB is composed of brain capillary endothelial cells, tight junction proteins, pericytes and astrocyte end-feet. Its highly selective permeability mechanism is crucial for maintaining brain homeostasis, but it also becomes a major obstacle to drug delivery. Traditional delivery strategies (such as nanoparticles and liposomes) are difficult to meet the treatment needs of neurodegenerative diseases due to their lack of specific targeting ability and insufficient penetration efficiency. Exosomes, as naturally derived nanovesicles, have low immunogenicity, high biocompatibility and transmembrane transport potential, but their natural properties still present significant bottlenecks in penetrating the BBB. To address this challenge, the present invention innovatively designed the membrane-penetrating peptide 4F-T7 to construct an exosome delivery system with both efficient penetration and targeting specificity. Its core advantages are as follows: The membrane-penetrating peptide 4F-T7 is an artificially synthesized chimeric peptide composed of two functional domains connected in series by a flexible connecting peptide, which are optimized for exosome membrane stability and BBB penetration ability respectively. The 4F peptide structure consists of hydrophobic and hydrophilic surfaces, enabling it to selectively insert into the hydrophobic core of the exosome lipid bilayer, achieving stable anchoring through hydrophobic interactions and electrostatic adsorption. Compared to traditional chemical conjugation modifications (such as NHS-PEG cross-linking), the physical insertion mechanism of the 4F peptide avoids the disruption of membrane protein structure by covalent modification, thereby maximally preserving the natural biological activity of exosomes. Furthermore, the 4F peptide can enhance the mechanical strength of the exosome membrane by regulating the density of membrane lipids, thereby reducing drug leakage caused by shear forces or enzymatic degradation during circulation. The T7 peptide is a high-affinity ligand for the transferrin receptor (TfR). It specifically recognizes TfR, which is highly expressed on the surface of BBB endothelial cells, activating the receptor-mediated transcytosis pathway. TfR expression in BBB endothelial cells is 5-10 times higher than in peripheral vascular endothelial cells, and its endocytosis-recycling pathway efficiently drives exosomes across the endothelial layer and into the brain parenchyma. The T7 peptide binds to the TfR with high selectivity, avoiding cross-reactions with non-target cells in other tissues (such as the liver and kidney) that have low TfR expression, thereby significantly reducing the risk of drug accumulation in peripheral organs. After insertion into the exosome membrane, the positively charged lysine residues on its hydrophilic face neutralize the negative charge on the membrane surface, reducing nonspecific adsorption of exosomes to plasma proteins and prolonging their circulation half-life. Furthermore, optimized charge distribution enhances the binding affinity of the T7 peptide for the TfR, thereby improving the efficiency of receptor-mediated endocytosis. The TfR endocytic pathway activated by the T7 peptide directs exosomes into the early endosomes of endothelial cells, where they are rapidly transported to the basolateral membrane via Rab11-dependent recycling vesicles, avoiding capture and degradation in lysosomes. This pathway is more efficient than traditional transmembrane peptides, and exosomes maintain structural integrity during BBB crossing, ensuring the activity of the loaded drug is not compromised.Compared with the widely used TAT peptide or RVG peptide, the design of 4F-T7 has the following advantages: ① The T7 peptide only binds to TfR, which is highly expressed on BBB endothelial cells, avoiding uptake by non-target tissues; ② The physical insertion mechanism of the 4F peptide has no chemical modification residues, and the natural surface proteins of exosomes (such as CD9 and CD63) are retained.

[0014] Neuron-specific targeting is one of the core challenges in the treatment of neurodegenerative diseases. Traditional delivery systems (such as antibody conjugation or ligand modification) often lead to non-specific accumulation of drugs in glial cells or vascular endothelial cells due to the widespread expression of target molecules in non-neuronal cells, which not only reduces the efficacy but also may cause off-target toxicity. To address this problem, the present invention innovatively utilizes the biological properties of L1 cell adhesion molecule (L1CAM) and anchors its ligand binding domain to the surface of exosomes through genetic engineering to construct a highly selective and pathologically responsive neuron-targeted delivery system. Its core mechanism and advantages are as follows. L1CAM is a member of the immunoglobulin superfamily. As a type I transmembrane protein, its extracellular domain contains 6 immunoglobulin (Ig)-like domains and 5 fibronectin type III (FnⅢ) repeat domains, of which the Ig1-Ig4 domains are responsible for mediating the binding of homologous or heterologous molecules. L1CAM is specifically and highly expressed in the presynaptic membrane of neurons, the surface of axons, and growth cones in the central nervous system (CNS), and participates in key physiological processes such as synapse formation, axon guidance, and neuronal migration. However, L1CAM is hardly expressed in glial cells (such as astrocytes and microglia) and vascular endothelial cells. This characteristic makes it an ideal molecular tag for neuronal targeting. The present invention uses gene fusion technology to fuse L1CAM with the C-terminus of the transmembrane protein Lamp2b, and stably expresses it on the surface of the exosome membrane. As a lysosomal membrane protein, Lamp2b has its N-terminus located on the outside of the exosome membrane and its C-terminus located on the inside. This topological structure ensures that the Ig domain of L1CAM is fully exposed on the surface of the exosome, forming a homologous trans-binding with the FnⅢ domain of L1CAM on the surface of the neuronal membrane. This binding mode has the following advantages: ① High affinity: the equilibrium dissociation constant of L1CAM homologous binding is significantly higher than that of antibody-antigen interaction); ② Low steric hindrance: the flexible hinge region of the Ig domain allows exosomes to multivalently bind to L1CAM on the neuronal membrane surface, enhancing anchoring stability; ③ High internalization efficiency: after binding, the clathrin-dependent endocytic pathway on the neuronal membrane is triggered, allowing the exosomes to quickly enter the cell and avoid drug loss caused by exocytosis.

[0015] The beneficial effects of the present invention are as follows:

[0016] The present invention discloses that the exosomes secreted by 293T cells are engineered as natural exosomes and loaded with the BBB-penetrating peptide 4F-T7 and the neuron-targeting protein L1CAM, respectively. Through the dual-modification system, the exosomes can penetrate the blood-brain barrier and be precisely delivered to brain neurons, thereby providing a more precise and effective treatment tool for neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 To detect the 293T cell line transfected with Lamp2b-L1CAM and its secreted exosomes by WB and NTA, it was demonstrated that the exosomes were successfully extracted and L1CAM was successfully loaded on the exosomes.

[0018] Figure 2 In vivo imaging of mice was used to demonstrate that 4F-T7 promotes exosomes to penetrate the BBB and enter the brain.

[0019] Figure 3 Confocal staining of brain sections demonstrated that L1CAM promotes the specific targeting of exosomes to neurons. DETAILED DESCRIPTION

[0020] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present application will be further described below in conjunction with specific embodiments.

[0021] Example 1

[0022] Construction of 293T transfected Lamp2b-L1CAM cell line: Lamp2b-L1CAM PCDH-Hygro-HA plasmid and empty vector plasmid PCDH-Hygro-HA were transfected into 293T cells via liposome Lip800. After 48 hours, the culture medium containing 100 μg / mL hygromycin was replaced and cultured for about 2 weeks. 293T cells successfully transfected with Lamp2b-L1CAM (referred to as 293T-Lamp2b-L1CAM) and 293T cells transfected with the empty vector (referred to as 293T-Vector) were screened. Western blotting confirmed that 293T cells were successfully loaded with L1CAM (see results). Figure 1 A).

[0023] Exosome extraction from 293T-Lamp2b-L1CAM cell line: 293T-Vector cells and 293T-Lamp2b-L1CAM cells were seeded into exosome-free serum medium (e.g., EXO-FBS) and cultured for 48 hours. The conditioned medium was collected and centrifuged at 300 × g for 10 minutes to remove cell debris; 2000 × g for 20 minutes to remove apoptotic bodies; 10,000 × g for 30 minutes to remove large vesicles; and 100,000 × g for 70 minutes (4°C) to pellet exosomes and resuspend in PBS.

[0024] Figure 1 A, WB detection of 293T cells transfected with empty vector Vector and Lamp2b-L1CAM plasmid (293T-cells), with GAPDH as an internal control, showed that 293T cells were successfully loaded with L1CAM. NTA detection of extracted 293T-Vector exosomes and 293T-Lamp2b-L1CAM exosomes showed that their average particle size was around 100nm, consistent with the size of exosomes. The results are shown in Figure 1 B. WB detection of 293T-Vector exosomes and 293T-Lamp2b-L1CAM exosomes showed the expression of exosome-specific markers Alix and CD63, proving that the exosomes were successfully extracted. It also proved that L1CAM was successfully loaded on 293T-Lamp2b-L1CAM exosomes (see the results). Figure 1 C.

[0025] Example 2

[0026] Exosomes were loaded with 4F-T7: A 4F-T7 chimeric peptide (sequence: DWFKAFYDKVAEKFKEAFGGHAIYPRH) was synthesized via solid-phase synthesis. 293T-Vector exosomes and 293T-Lamp2b-L1CAM exosomes were incubated with the 4F-T7 chimeric peptide (molar ratio 1:50) at 37°C for 2 hours. The 4F peptide segment was inserted into the exosome membrane through hydrophobic interactions, while the T7 peptide segment was exposed on the membrane surface. Unbound free peptide was removed by ultrafiltration and centrifugation (100 kDa), yielding 4F-T7 engineered exosomes, namely 293T-Vector+T7 exosomes and 293T-L1CAM+T7 exosomes.

[0027] Example 3

[0028] Engineered exosome labeling with DiD and in vivo targeting verification: 1 μM DiD dye was incubated with 100 μg of 293T-Vector-loaded 4F-T7 engineered exosomes (hereinafter referred to as 293T-Vector+T7 exosomes) and 100 μg of 293T-Lamp2b-L1CAM-loaded 4F-T7 engineered exosomes (hereinafter referred to as 293T-L1CAM+T7 exosomes) at 37°C in the dark for 30 min, and free dye was removed by ultrafiltration and centrifugation.

[0029] Tail vein injection and in vivo imaging: 6-8 week-old C57BL / 6 mice were injected via the tail vein with 100 μg of 293T-Vector exosomes, 293T-Vector-T7 exosomes, 293T-L1CAM exosomes, and 293T-L1CAM-T7 exosomes (each dissolved in 200 μL PBS). Brain fluorescence signals were detected using an IVIS Spectrum Imaging System 1, 3, and 6 hours after injection. Mice were sacrificed, and brain tissue was prepared for frozen sections. Colocalization of DiD (red) with the neuronal marker NeuN (green) was observed using confocal microscopy.

[0030] Figure 2 The 6-8 week old C57BL / 6 mice were divided into Blank group (blank group, without any drug injection), PBS group (200 μl PBS injection through tail vein), 293T-EXOs-Vector group (100 μg 293T-Vector exosomes injection through tail vein), 293T-EXOs-Vector+T7 group (100 μg 293T-Vector exosomes injection through tail vein and loaded with 4F-T7 chimeric peptide), 293T-EXOs-L1CAM group (100 μg 293T-L1CAM exosomes injection through tail vein), 293T-EXOs-L1CAM+T7 group (100 μg 293T-L1CAM exosomes loaded with 4F-T7 chimeric peptides) were injected into the mouse brain using in vivo imaging. Fluorescence intensity in the mouse brain was observed 1, 3, and 6 hours after injection. The results showed that exosomes loaded with 4F-T7 chimeric peptides significantly improved their ability to penetrate the BBB and enter the brain. This example demonstrates that 4F-T7 promotes exosome penetration of the BBB and brain entry through in vivo imaging of mice.

[0031] Figure 3 Lieutenant General Figure 2 The brains of mice were removed and frozen sections were prepared. Confocal multicolor fluorescence staining was used to mark the nuclei (DAPI, blue fluorescence), neurons (Neun, green fluorescence), exosomes (EXOs, red fluorescence), and all fluorescence images were superimposed together (Merge) and magnified to 40 times (40X). The results showed that the number of 293T-L1CAM+T7 exosomes expressing L1CAM entering brain neurons increased significantly, demonstrating that L1CAM promotes the specific targeting of exosomes to brain neurons. This example demonstrates that L1CAM promotes the specific targeting of exosomes to neurons by confocal staining of brain sections.

[0032] The above preparation steps and conditions were used to test the ability of engineered exosomes loaded with the cell-penetrating peptide 4F-T7 and the targeting protein L1CAM to precisely target brain neurons.

[0033] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. An exosome, characterized by: The exosomes are loaded with L1CAM.

2. The exosome according to claim 1, wherein: The exosomes were obtained from the 293T-Lamp2b-L1CAM cell line, which was obtained by transfecting 293T cells with the Lamp2b-L1CAM PCDH-Hygro-HA plasmid.

3. A 4F-T7 engineered exosome, characterized by: The 4F-T7 engineered exosomes are obtained by loading the exosomes in claim 1 with the 4F-T7 chimeric peptide.

4. The 4F-T7 engineered exosomes according to claim 3, characterized in that: The sequence of the 4F-T7 chimeric peptide is DWFKAFYDKVAEKFKEAFGGHAIYPRH.

5. The method for preparing exosomes according to claim 1, wherein: The method comprises the following steps: transfecting Lamp2b-L1CAMPCDH-Hygro-HA plasmid into 293T cells via liposome Lip800, replacing the culture medium containing 100 μg / mL hygromycin after 48 hours, and continuing to culture for a period of time, and then extracting the obtained product.

6. The method for preparing the 4F-T7 engineered exosomes according to claim 3, characterized in that: The method comprises the following steps: synthesizing 4F-T7 chimeric peptide by solid phase synthesis, incubating 293T-Lamp2b-L1CAM exosomes with 4F-T7 chimeric peptide at a molar ratio of 1:50 at 37°C for 2 hours, and removing unbound free peptide by ultrafiltration centrifugation (100 kDa) to obtain 4F-T7 engineered exosomes.

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