Nanobody Nb.TB1, engineered plant exosomes, their preparation methods and applications

Abstract

This invention discloses a nanobody Nb.TB1, engineered plant exosomes, their preparation methods, and applications, belonging to the field of biomedical technology. The amino acid sequence of the nanobody Nb.TB1 is shown in Seq ID No. 1. Furthermore, this invention proposes a method for preparing engineered plant exosomes, comprising the following steps: S1, extraction of Gastrodia elata exosomes: extracting Gastrodia elata exosomes from Gastrodia elata using an enzymatic method; S2, expression and purification of the blood-brain barrier-crossing nanobody fusion protein with Sortase A ligase; S3, surface modification of the Gastrodia elata exosomes. In addition, this invention also proposes an engineered plant exosome prepared by the above method. Furthermore, this invention proposes the application of the above-mentioned engineered plant exosomes in the preparation of targeted therapeutic drugs for central nervous system diseases. The engineered plant exosomes prepared by this invention exhibit high blood-brain barrier crossing efficiency.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a nanobody Nb.TB1, engineered plant exosomes, their preparation methods, and applications. Background Technology

[0002] With the increasing aging of the global population, the incidence of central nervous system diseases has risen significantly. The development of drugs for the treatment of nervous system diseases faces multiple challenges: (1) unclear disease mechanisms and targets: the pathogenesis of most neurodegenerative diseases has not been elucidated, and there is a lack of clear molecular targets and reliable biomarkers; (2) defects in preclinical models: existing disease models cannot fully simulate human pathophysiological characteristics, resulting in poor efficacy of preclinical drugs in the human body; (3) limitations of the blood-brain barrier (BBB). The BBB can block 98% of small molecules and almost all biological drugs from entering the central nervous system, making the efficiency of drug delivery to the nervous system extremely low. Among them, the structural barrier of the blood-brain barrier puts central nervous system diseases into a double dilemma of "drug scarcity-inefficient delivery". On the one hand, there is an extreme shortage of therapeutic drugs, and on the other hand, even if the drugs are successfully delivered, they are difficult to reach therapeutic concentrations due to their transport efficiency being generally less than 0.1%.

[0003] Plant-derived exosome-like nanovesicles (PELNVs) have been considered ideal carriers for targeted therapy of neurological diseases due to their low immunogenicity, high biocompatibility, and inherent potential to cross the blood-brain barrier. Gastrodia elata, a traditional Chinese medicine, has shown clear neuroprotective effects from its active ingredient gastrodin in various central nervous system disease models, including epilepsy, Alzheimer's disease, and Parkinson's disease. Furthermore, the plant secretes gastrodia exosomes (GE EVs), providing a high-quality material for the development of drug delivery carriers derived from traditional Chinese medicine. However, current technologies suffer from low efficiency in crossing the blood-brain barrier, unclear molecular mechanisms and receptor targets, and a lack of effective surface modification techniques for targeted modification, severely hindering its transformation from a traditional Chinese medicine to a modern targeted delivery system. Currently, PELNV research faces a dual bottleneck: the molecular mechanisms and receptor targets across the BBB remain unclear, and the transport efficiency is far from reaching the clinical treatment threshold, restricting its progress from proof-of-concept to clinical application.

[0004] Currently, surface modification of plant exosomes mostly employs traditional dual-enzyme-linked immunosorbent assays (DELISA), which suffer from cumbersome reaction steps, demanding conditions, long production cycles, and high costs. Furthermore, it is difficult to achieve the directional and stable display of target molecules on the surface, resulting in limited improvement in the targeting ability of modified exosomes and failing to meet the needs of clinical treatment. Therefore, developing a simplified, mild, and highly efficient engineering technology for Gastrodia elata exosomes to achieve efficient functional enhancement across the blood-brain barrier is a pressing technical problem to be solved in the field of drug delivery for central nervous system diseases. Summary of the Invention

[0005] To address the problems of low efficiency of Gastrodia elata exosomes in crossing the blood-brain barrier, lack of efficient engineering modification technology, and cumbersome and demanding traditional exosome modification methods in existing technologies, the present invention aims to provide a nanobody Nb.TB1, engineered plant exosomes, their preparation methods and applications, providing a novel and efficient drug delivery carrier for targeted therapy of central nervous system diseases.

[0006] To achieve the above-mentioned technical objectives, the present invention provides a nanobody Nb.TB1, the amino acid sequence of which is shown in Seq ID No. 1.

[0007] Furthermore, this invention also proposes a method for preparing engineered plant exosomes, comprising the following steps:

[0008] S1. Extraction of exosomes: Exosomes were extracted from plants using an enzymatic hydrolysis method;

[0009] S2. Expression and purification of the cross-blood-brain barrier nanobody fusion protein and Sortase A ligase: The pet28a-Nb.TB1-SpyC recombinant vector and the pet30a-Sortase A recombinant vector were constructed, transformed into BL21 Escherichia coli for expression, and purified by Ni-beads and concentrated by ultrafiltration to obtain the Nb.TB1-SpyC fusion protein and the His-tagged Sortase A ligase.

[0010] S3. Surface modification of exosomes: The adaptor peptide SpyT-LPETG containing SpyTag and LPETG recognition motifs was designed and synthesized. It was covalently coupled to the surface of the exosome membrane obtained in step S1 using the Sortase A ligase obtained in step S1 to obtain Gastrodia elata exosomes modified with the adaptor peptide. Then, the Nb.TB1-SpyC fusion protein was mixed with the above-mentioned adaptor peptide modified exosomes. By utilizing the covalent binding characteristics of SpyTag and SpyCatcher, the nanobody was directionally displayed on the surface of the exosome. After washing and resuspending, engineered plant exosomes Nb.TB1-GE EVs were obtained.

[0011] In any embodiment, in step S1, the plant is Gastrodia elata.

[0012] In any embodiment, the extraction of exosomes in step S1 specifically includes: taking Gastrodia elata, washing and peeling it, cutting it into slices, adding PBS to extract the juice, filtering the juice through gauze to collect the juice; centrifuging and filtering the juice at 3-5℃ to obtain exosomes.

[0013] In any embodiment, in step S2, the amino acid sequence of the Nb.TB1-SpyC fusion protein is: N-terminus-His tag-GSG linker peptide-Nb.TB1-GSG linker peptide-Flag tag-SGSGSSGAS linker peptide-SpyCatcher-C-terminus.

[0014] In any implementation, the amino acid sequence of SpyCatcher is shown in Seq ID No. 2.

[0015] In any embodiment, in step S3, the amino acid sequence of the adaptor peptide SpyT-LPETG is as shown in Seq ID No. 3; and / or, in step S3, the binding conditions of the Nb.TB1-SpyC fusion protein to the adaptor peptide-modified Gastrodia elata exosomes are as follows: add 1 mM Nb.TB1-SpyC fusion protein, total volume 40-45 μL, incubate at room temperature at 30-40 rpm for 3-3.5 h; wash with PBS after binding, and finally resuspend the precipitate with PBS.

[0016] In addition, the present invention also proposes an engineered plant exosome, which is prepared by the above preparation method.

[0017] Furthermore, the present invention also proposes a central nervous system drug delivery carrier comprising the aforementioned engineered plant exosomes.

[0018] Furthermore, this invention also proposes the application of the above-mentioned nanobody Nb.TB1 or the above-mentioned engineered plant exosomes in the preparation of targeted therapeutic drugs for central nervous system diseases.

[0019] In any implementation, the central nervous system disease includes Alzheimer's disease, Parkinson's disease, epilepsy, and ischemic stroke.

[0020] Compared with the prior art, the beneficial effects of the present invention include: the engineered plant exosomes Nb.TB1-GE EVs prepared by the present invention, verified by an in vitro blood-brain barrier model, show a significantly improved blood-brain barrier crossing efficiency compared with wild-type Gastrodia elata exosomes; at the same time, the engineered plant exosomes inherit the low immunogenicity and high biocompatibility of exosomes, as well as the natural neuroprotective properties of plants such as Gastrodia elata, and can efficiently load various neuroprotective drugs and active ingredients, realizing precise targeted delivery of drugs to the central nervous system, providing a new strategy and carrier for the treatment of central nervous system diseases such as Alzheimer's disease and Parkinson's disease, with extremely high clinical translational value and market application prospects. Attached Figure Description

[0021] Figure 1 This is a schematic diagram of the structure of an in vitro blood-brain barrier model, showing that bEnd.3 cells form a dense monolayer on the Transwell membrane, simulating the structure of the blood-brain barrier in vivo.

[0022] Figure 2 The image shows the efficiency of the nanobody Nb.TB1 and the control nanobody Nb.Ctrl in crossing the blood-brain barrier in Example 2. The results show that Nb.TB1 can cross the blood-brain barrier efficiently in vitro, while Nb.Ctrl can hardly cross it.

[0023] Figure 3 The transmission electron microscope image of the engineered Gastrodia elata exosomes Nb.TB1-GE EVs in Example 4 shows that they have a typical roundish vesicle structure with intact morphology.

[0024] Figure 4 The NTA particle size distribution diagram of the engineered Gastrodia elata exosomes Nb.TB1-GE EVs in Example 4 shows that its particle size distribution is concentrated, which is consistent with the characteristics of exosomes.

[0025] Figure 5 The Western blot image shows the modification of engineered Gastrodia elata exosomes, revealing a specific band with a FLAG tag, proving the successful modification of the nanobody.

[0026] Figure 6 The image shows a comparison of the blood-brain barrier crossing efficiency between engineered Gastrodia elata exosomes and wild-type Gastrodia elata exosomes. The fluorescence intensity of Nb.TB1-GE EVs increased significantly over time, and its blood-brain barrier crossing efficiency was significantly better than that of WT-GE EVs. Detailed Implementation

[0027] The "range" disclosed in this application is defined by a lower limit and an upper limit. A given range is defined by selecting a lower limit and an upper limit, which define the boundaries of a particular range. Ranges defined in this way can include or exclude endpoints and can be arbitrarily combined; that is, any lower limit can be combined with any upper limit to form a range. For example, if ranges of 60~120 and 80~110 are listed for a specific parameter, it is also expected that ranges of 60~110 and 80~120 are also included. Furthermore, if minimum range values ​​of 1 and 2 are listed, and if maximum range values ​​of 3, 4, and 5 are listed, then the following ranges are all expected: 1~3, 1~4, 1~5, 2~3, 2~4, and 2~5. In this application, unless otherwise stated, the numerical range "a~b" represents a shortened representation of any combination of real numbers between a and b, where a and b are real numbers. For example, the numerical range "0~5" indicates that all real numbers between "0~5" have been listed in this article; "0~5" is simply a shortened representation of these numerical combinations. Furthermore, when a parameter is stated as an integer ≥2, it is equivalent to disclosing that the parameter is, for example, an integer such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, etc.

[0028] Unless otherwise specified, the terms "comprising" and "including" as used in this application can be open-ended or closed-ended. For example, "comprising" and "including" can mean that other components not listed may also be included, or that only the listed components may be included.

[0029] Unless otherwise specified, the term "or" is inclusive in this application. For example, the phrase "A or B" means "A, B, or both A and B". More specifically, the condition "A or B" is satisfied by any of the following conditions: A is true (or exists) and B is false (or does not exist); A is false (or does not exist) and B is true (or exists); or both A and B are true (or exist).

[0030] This specific embodiment provides a nanobody Nb.TB1, whose amino acid sequence is shown in Seq ID No.1.

[0031] This specific embodiment also proposes a method for preparing engineered plant exosomes, including the following steps:

[0032] S1. Extraction of Gastrodia elata exosomes: Gastrodia elata exosomes were extracted from Gastrodia elata using an enzymatic hydrolysis method. The extraction of Gastrodia elata exosomes specifically includes: taking fresh Gastrodia elata, washing and peeling it, cutting it into slices, adding PBS for juicing, filtering the juice through gauze to collect the juice; centrifuging and filtering the juice at 3-5℃ to obtain Gastrodia elata exosomes.

[0033] S2. Expression and purification of the blood-brain barrier nanobody fusion protein and Sortase A ligase: The pet28a-Nb.TB1-SpyC recombinant vector and the pet30a-Sortase A recombinant vector were constructed and transformed into BL21 *E. coli* for induced expression. After purification with Ni-beads and concentration by ultrafiltration, the Nb.TB1-SpyC fusion protein and the His-tagged Sortase A ligase were obtained. The amino acid sequence of the Nb.TB1-SpyC fusion protein is: N-terminus-His tag-GSG linker peptide-Nb.TB1-GSG linker peptide-Flag tag-SGSGSSGAS linker peptide-SpyCatcher-C-terminus. The amino acid sequence of SpyCatcher is shown in Seq ID No. 2.

[0034] S3. Surface modification of Gastrodia elata exosomes: The adaptor peptide SpyT-LPETG containing SpyTag and LPETG recognition motifs was designed and synthesized. It was covalently coupled to the surface of the Gastrodia elata exosome membrane obtained in step S1 using the Sortase A ligase obtained in step S1, resulting in adaptor peptide-modified Gastrodia elata exosomes. Then, the Nb.TB1-SpyC fusion protein was mixed with the adaptor peptide-modified Gastrodia elata exosomes. Utilizing the covalent binding property of SpyTag and SpyCatcher, nanobodies were directionally displayed on the surface of the Gastrodia elata exosomes. After washing and resuspending, engineered Gastrodia elata exosomes Nb.TB1-GE EVs (i.e., one type of engineered plant exosomes) were obtained. The amino acid sequence of the adaptor peptide SpyT-LPETG is shown in Seq ID No. 3. The binding conditions between the Nb.TB1-SpyC fusion protein and the adaptor peptide-modified Gastrodia elata exosomes were: the addition of 1 mM... Nb.TB1-SpyC fusion protein, total volume 40-45 μL, incubated at room temperature at 30-40 rpm for 3-3.5 h; after binding, wash with PBS, and finally resuspend the precipitate with PBS.

[0035] Furthermore, this specific embodiment also proposes an engineered Gastrodia elata exosome, which is prepared by the above-described preparation method.

[0036] Furthermore, this specific embodiment also proposes a central nervous system drug delivery carrier, including the above-mentioned engineered Gastrodia elata exosomes.

[0037] Furthermore, this specific embodiment also proposes the application of the above-mentioned nanobody Nb.TB1 or the above-mentioned engineered Gastrodia elata exosomes in the preparation of targeted therapeutic drugs for central nervous system diseases, including Alzheimer's disease, Parkinson's disease, epilepsy and ischemic stroke.

[0038] This invention uses nanobodies capable of crossing the blood-brain barrier to engineer Gastrodia elata exosomes, significantly improving their blood-brain barrier crossing efficiency and constructing a functional carrier for targeted drug delivery to the nervous system.

[0039] This invention utilizes Sortase A ligase derived from Staphylococcus aureus to establish a nanobody modification technology for the surface of Gastrodia elata exosomes. This Sortase A ligase specifically recognizes the LXTG motif (X being any amino acid) and covalently couples it to the surface of a protein containing a free glycine residue at its N-terminus. Specifically, SpyTag is fused with an LXTG linker peptide, and then covalently modified onto the surface of Gastrodia elata exosomes via Sortase A catalysis. Simultaneously, a blood-brain barrier-permeable nanobody is coupled to SpyCatcher. Leveraging the rapid covalent binding of SpyTag and SpyCatcher under mild conditions, highly efficient and targeted display of the nanobody on the surface of Gastrodia elata exosomes is achieved.

[0040] Other beneficial effects of the present invention include:

[0041] 1) This invention integrates a dual-system modification strategy of Sortase A-mediated linker peptide coupling and SpyTag-SpyCatcher covalent binding for the directional display of nanobodies on the surface of Gastrodia elata exosomes, avoiding multi-step enzymatic reactions.

[0042] 2) The engineered plant exosomes with surface-stabilized modified trans-blood-brain barrier nanobodies proposed in this invention endow them with efficient blood-brain barrier penetration function.

[0043] 3) The application of engineered plant exosomes proposed in this invention as drug delivery carriers for the central nervous system in the loading of neuroprotective drugs and their transport across the blood-brain barrier.

[0044] 4) Simplified process and mild conditions: Compared with the traditional double enzyme-linked method, this invention only requires one Sortase A enzymatic reaction, and the subsequent SpyTag-SpyCatcher spontaneously binds. The reaction conditions are mild and uniform, the operation is simple, the production cycle is shortened, and the cost is reduced.

[0045] 5) Enhanced function and broad application prospects: Engineered exosomes fully inherit the blood-brain barrier crossing characteristics of nanobodies, significantly enhancing brain targeting capabilities. They can be used for the efficient delivery of neuroprotective drugs, providing new strategies for the treatment of neurological diseases such as Alzheimer's disease and stroke.

[0046] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0047] In this invention, the terms "some embodiments," "this embodiment," and examples are used to describe a subset of all possible embodiments. However, it is understood that "some embodiments" can be the same subset or different subsets of all possible embodiments and can be combined with each other without conflict.

[0048] If the application documents contain similar descriptions such as "first / second", the following explanation shall be added: In the following description, the terms "first / second / third" are used only to distinguish similar objects and do not represent a specific ordering of objects. It is understood that "first / second / third" may be interchanged in a specific order or sequence where permitted, so that the embodiments described herein can be implemented in an order other than that illustrated or described herein.

[0049] In this embodiment, the term "and / or" is merely a description of the relationship between related objects, indicating that there can be three relationships. For example, object A and / or object B can represent three situations: object A exists alone, object A and object B exist simultaneously, and object B exists alone.

[0050] The following describes embodiments of this application. The embodiments described below are exemplary and are only used to explain this application, and should not be construed as limiting this application. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all conventional products that can be obtained commercially.

[0051] Example 1

[0052] This embodiment presents a Gastrodia elata exosome, which is prepared by the following steps:

[0053] 1) Take fresh Gastrodia elata, wash it, peel it, and weigh it (194.7g). Cut the Gastrodia elata into 1mm thick slices, add 1*pbs of 50% of the weight of the ingredients (if the peeled Gastrodia elata weighs 194.7g, add 92mlpbs), juice it with a juicer, filter it with gauze and collect the Gastrodia elata juice (about 160ml).

[0054] 2) Differential centrifugation for impurity removal. Place fresh Gastrodia elata juice into four 50ml centrifuge tubes and balance them precisely. Perform differential centrifugation at 4°C under the following conditions: 1000g, 10min; 3000g, 20min; 10000g, 60min. Collect the supernatant after each centrifugation.

[0055] 3) Ultracentrifugation. Divide the supernatant into 6 ultracentrifuge tubes, balance them strictly on an analytical balance, and centrifuge at 150,000g for 90 minutes at 4°C.

[0056] 4) Collection. Discard the supernatant, resuspend the precipitate in the centrifuge tube by blowing with an appropriate amount of PBS (about 4 ml in total), and repeat the operation on another tube. Finally, collect the precipitate from 6 tubes into 1 tube with 4 ml of PBS.

[0057] 5) Filtration. Filter the suspension through a 0.22µm filter membrane in a clean bench and collect the suspension in four 1.5ml centrifuge tubes.

[0058] 6) Concentration determination and particle size analysis were performed using nanoparticle tracking analysis (NTA).

[0059] Example 2: Expression and purification of Nb.TB1-SpyC fusion protein with Sortase A ligase

[0060] 1) Recombinant vector construction: Based on the designed amino acid sequence, the Nb.TB1-SpyC fusion gene was synthesized, with the sequence being N-terminal - His tag - GSG - Nb.TB1 - GSG - Flag tag - SGSGSSGAS - SpyCatcher - C-terminus. This gene was cloned into the pet28a vector to obtain the pet28a-Nb.TB1-SpyC recombinant vector. Simultaneously, the Sortase A gene was cloned into the pet30a vector to obtain the His-tagged pet30a-Sortase. A recombinant vector; wherein, the amino acid sequence of Nb.TB1 is: QVQLQESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAISWSGGRTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADRRGSGSSSPRRYDYWGQGTQVTVSS; the amino acid sequence of SpyCatcher is: VTTLSGLSGEQGPSGDMTTEEDSATHIKFSKRDEDGRELAGATMELRDSSGKTISTWISDGHVKDFYLYPGKYTFVETAAPDGYEVATPIEFTVNEDGQVTVDGEATEGDAHT;

[0061] 2) E. coli transformation: The two recombinant vectors were transformed into BL21 competent E. coli cells, and single clones were picked after plating and cultured overnight until saturation.

[0062] 3) Induction of expression: Saturated bacterial culture was inoculated into 50 mL of LB liquid medium at OD600nm=0.1, and the culture was shaken at 37℃ until OD600nm=0.5~0.6. 0.1 mM IPTG was added, and expression was induced overnight at 16℃ and 200 rpm.

[0063] 4) Protein purification: Collect the bacterial culture after induction of expression, centrifuge and resuspend the bacterial cells and sonicate to disrupt them. Take the supernatant and mix it with Ni-beads for incubation to purify the Nb.TB1-SpyC fusion protein carrying the His tag and Sortase A ligase.

[0064] 5) Concentration and impurity removal: The purified protein solution is concentrated through a 10 kDa ultrafiltration tube to remove impurities and small molecules. The protein concentration is determined by the BCA method and stored for later use.

[0065] The nanobody Nb.TB1 in this embodiment was prepared according to the preparation method of CN116813781A, and the nanobody was obtained by obtaining the corresponding DNA information through next-generation sequencing.

[0066] Example 3 Preparation of engineered Gastrodia elata exosomes Nb.TB1-GE EVs

[0067] Pre-incubation of adaptor peptide with Sortase A: The adaptor peptide SpyT-LPETG (amino acid sequence RGVPHIVMVDAYKRYKGGSGGSLPETG) was designed and synthesized. 20 μM Sortase A and 500 μM SpyT-LPETG were mixed in 1×Sortase buffer (50 mM Tris-HCl, 12 mM CaCl2, 150 mM NaCl, pH=6.5) and incubated at room temperature for 30 min.

[0068] Conjugation of the linker peptide to Gastrodia elata exosomes: Add 5 × 10⁻⁶ peptides to the above pre-incubated mixture. 10 The gastrodia elata exosomes prepared in Example 1 were adjusted to a total volume of 40 μL and incubated at room temperature at 20 rpm for 1 h. After incubation, they were centrifuged at 150000 g and 4 °C for 60 min, the supernatant was discarded, and the exosomes were washed twice with PBS at pH=7 to remove unbound linker peptides, thus obtaining linker peptide-modified gastrodia elata exosomes.

[0069] Directional display of nanobodies: 1 mM of Nb.TB1-SpyC fusion protein prepared in Example 2 was added to the Gastrodia elata exosomes modified with the linker peptide, and the total volume was adjusted to 40 μL. The mixture was then incubated at room temperature at 30 rpm for 3 h.

[0070] Purification and collection: After incubation, wash three times with PBS, centrifuge at 150,000g and 4℃ for 60 min each time to remove unbound fusion protein, and finally resuspend the precipitate with 1mL PBS to obtain engineered Gastrodia elata exosomes Nb.TB1-GE EVs.

[0071] Example 4 Characterization and Blood-Brain Barrier Crossing Efficiency Detection of Engineered Gastrodia elata Exosomes

[0072] Transmission electron microscopy characterization: A small amount of Nb.TB1-GE EVs prepared in Example 3 was dropped onto a copper mesh, negatively stained, and observed under a transmission electron microscope. Figure 3 It can be seen that the engineered Gastrodia elata exosomes have a typical roundish vesicle structure, with complete morphology and uniform particle size;

[0073] NTA particle size distribution analysis: The particle size of Nb.TB1-GE EVs was determined using nanoparticle tracking analysis, combined with... Figure 4 The results showed that its particle size distribution was concentrated, which was consistent with the typical particle size characteristics of exosomes. Compared with the unmodified Gastrodia elata exosomes, there was no significant change, indicating that the modification process did not destroy the structure of the exosomes.

[0074] Western blotting to verify the modification: Nb.TB1-GE EVs were lysed with RIPA buffer, 2×SDS loading buffer was added, and the samples were loaded onto a 10% polyacrylamide gel for electrophoresis. Western blotting was performed based on the FLAG tags carried by the nanobodies. Figure 5 The results showed the presence of specific migration bands, proving that the nanobody was successfully modified on the surface of Gastrodia elata exosomes.

[0075] Validation of nanobody crossing the blood-brain barrier

[0076] 1) The variable region of Nb.TB1 was scrambled to obtain the control nanobody Nb.Ctrl. The sequence of the nanobody Nb.Ctrl is as follows:

[0077] QVQLQESGGGLVQAGGSLRLSCAASGSSTRAFYMGWFRQAPGKEREFVAAITWRSGYSGYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAASRSRGRDPSYSGYRDWGQGTQVTVSS.

[0078] 2) Construction of an in vitro blood-brain barrier model. An in vitro blood-brain barrier model was constructed using mouse brain endothelial cells (bEnd.3) in Transwell culture dishes (see schematic diagram). Figure 1 ).

[0079] 3) 2*10 4 10 bEnd.3 cells were seeded into the upper chamber of a 24-well Transwell dish. 200 μL of upper culture medium. 600 μL of lower culture medium.

[0080] 4) After the cells adhered and grew for 24 hours, equal amounts of 50 μg each of Nb.Ctrl and Nb.TB1, which were coupled with Cy5.5 fluorescent labels, were added to the upper layer.

[0081] 5) Every 30 minutes, take 50 μL of the lower culture medium and divide it into three wells for fluorescence detection. Results are as follows: Figure 2 As shown, Nb.Ctrl is almost undetectable in the lower layer, while Nb.TB1 gradually accumulates in the lower culture medium over time.

[0082] Blood-brain barrier crossing efficiency test:

[0083] Construction of an in vitro blood-brain barrier model: 2×10 4 Mouse brain endothelial cells (bEnd.3) were seeded into the upper chamber of a 24-well Transwell culture dish. 200 μL of culture medium was added to the upper chamber and 700 μL of culture medium was added to the lower chamber. The cells were cultured for 24 h until they fused to form a dense cell monolayer, thus constructing an in vitro blood-brain barrier model.

[0084] Exosome labeling: Wild-type Gastrodia elata exosomes (WT-GEEVs) from Example 1 and engineered Gastrodia elata exosomes (Nb.TB1-GEEVs) from Example 3 were labeled with PKH67 fluorescent dye, respectively. After incubation for 5 min, the free dye was removed by ultrafiltration in a 10 kDa tube and the cells were resuspended in PBS.

[0085] Transmembrane experiment: Equal amounts of labeled WT-GE EVs and Nb.TB1-GE EVs (5 × 10⁻⁶ each) were added to the upper chamber. 9 (50 μL of sample was taken from the lower culture medium every 30 min and divided into 3 wells for fluorescence intensity detection);

[0086] The results are as follows Figure 6 As shown, the fluorescence intensity of Nb.TB1-GE EVs in the lower culture medium accumulated significantly over time, while the fluorescence intensity of WT-GE EVs remained almost unchanged. Both Nb.TB1-GE EVs and WT-GE EVs showed accumulation of fluorescence intensity over time in the lower culture medium. However, compared to WT-GE EVs, the accumulation rate of fluorescence intensity for Nb.TB1-GE EVs was significantly higher. This demonstrates that the blood-brain barrier crossing efficiency of the engineered Gastrodia elata exosomes prepared in this invention is significantly improved compared to unmodified Gastrodia elata exosomes.

[0087] The present invention provides a highly efficient method for preparing engineered plant exosomes that cross the blood-brain barrier. The method is simplified, the conditions are mild, and the operation is simple, enabling large-scale preparation. The engineered plant exosomes prepared have high blood-brain barrier crossing efficiency, good biocompatibility, and low immunogenicity. They can be used as highly efficient targeted delivery carriers for drugs of the central nervous system and can be widely applied in the research and development of drugs for the treatment of various central nervous system diseases such as Alzheimer's disease, Parkinson's disease, epilepsy, and ischemic stroke. It has significant industrial application value and clinical translation prospects.

[0088] The specific embodiments of the present invention described above do not constitute a limitation on the scope of protection of the present invention. Any other corresponding changes and modifications made in accordance with the technical concept of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A nanobody Nb.TB1, characterized in that, Its amino acid sequence is shown in Seq ID No.

1.

2. A method for preparing engineered plant exosomes, characterized in that, Includes the following steps: S1. Extraction of exosomes: Exosomes were extracted from plants using an enzymatic hydrolysis method; S2. Expression and purification of the cross-blood-brain barrier nanobody fusion protein and Sortase A ligase: The pet28a-Nb.TB1-SpyC recombinant vector and the pet30a-Sortase A recombinant vector were constructed, transformed into BL21 Escherichia coli for expression, and purified by Ni-beads and concentrated by ultrafiltration to obtain the Nb.TB1-SpyC fusion protein and the His-tagged Sortase A ligase. S3. Surface modification of exosomes: The adaptor peptide SpyT-LPETG containing SpyTag and LPETG recognition motifs was designed and synthesized. It was covalently coupled to the surface of the exosome membrane obtained in step S1 using the Sortase A ligase obtained in step S1 to obtain adaptor peptide-modified exosomes. Then, the Nb.TB1-SpyC fusion protein was mixed with the above adaptor peptide-modified exosomes. By utilizing the covalent binding characteristics of SpyTag and SpyCatcher, the nanobody was directionally displayed on the surface of the exosome. After washing and resuspending, engineered plant exosomes Nb.TB1-GE EVs were obtained.

3. The preparation method according to claim 2, characterized in that, In step S1, the plant is Gastrodia elata.

4. The preparation method according to claim 3, characterized in that, In step S1, the extraction of exosomes specifically includes: taking Gastrodia elata, washing and peeling it, cutting it into slices, adding PBS to extract the juice, filtering the juice through gauze to collect the juice; centrifuging and filtering the juice at 3-5℃ to obtain exosomes.

5. The preparation method according to claim 2, characterized in that, In step S2, the amino acid sequence of the Nb.TB1-SpyC fusion protein is: N-terminus-His tag-GSG linker peptide-Nb.TB1-GSG linker peptide-Flag tag-SGSGSSGAS linker peptide-SpyCatcher-C-terminus; and / or, in step S3, the amino acid sequence of the adaptor peptide SpyT-LPETG is as shown in Seq ID No. 3; and / or, in step S3, the binding conditions of the Nb.TB1-SpyC fusion protein to the adaptor peptide-modified exosomes are: adding 1 mM Nb.TB1-SpyC fusion protein, total volume 40-45 μL, incubating at room temperature at 30-40 rpm for 3-3.5 h; washing with PBS after binding, and finally resuspending the precipitate with PBS.

6. The preparation method according to claim 5, characterized in that, The amino acid sequence of SpyCatcher is shown in Seq ID No.

2.

7. An engineered plant exosome, characterized in that, It is prepared by the preparation method according to any one of claims 2-6.

8. A central nervous system drug delivery carrier, characterized in that, Includes the engineered plant exosomes as described in claim 7.

9. The use of the nanobody Nb.TB1 of claim 1 or the engineered plant exosomes of claim 7 in the preparation of targeted therapeutic drugs for central nervous system diseases.

10. The application according to claim 9, characterized in that, The central nervous system diseases mentioned include Alzheimer's disease, Parkinson's disease, epilepsy, and ischemic stroke.

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