Extracellular vesicle-anchored nanobodies and uses thereof

Abstract

The application discloses extracellular vesicle-anchored nanobodies and application thereof, and relates to the field of biological medicine engineering. The extracellular vesicle-anchored nanobodies are obtained by using a self-constructed nanobody phage library to screen extracellular vesicles. The extracellular vesicle-anchored nanobodies can be combined with extracellular vesicles, realize efficient loading of proteins and nucleic acids on the surface of the extracellular vesicles, and can be used for disease treatment. The extracellular vesicle-anchored nanobodies can capture extracellular vesicles and can be used as a capture and liquid biopsy tool. The extracellular vesicle-anchored nanobodies can be combined with circulating extracellular vesicles in vivo, capture and modify the extracellular vesicles in vivo, and can be used for drug delivery and disease treatment. The extracellular vesicle-anchored nanobodies can capture, modify and functionalize extracellular vesicles in vitro and in vivo, and can be used for constructing novel biological nanodrugs and treating diseases.

Description

Technical Field

[0001] This invention relates to the field of biomedical engineering technology, and in particular to extracellular vesicle-anchored nanobodies and their applications. Background Technology

[0002] Extracellular vesicles are nanoscale vesicles with a lipid bilayer structure secreted by living cells, widely present in various bodily fluids such as blood, urine, and saliva. Due to their excellent biocompatibility, low immunogenicity, and ability to cross biological barriers, extracellular vesicles show great promise for applications in drug delivery and disease diagnosis. As drug delivery carriers, extracellular vesicles can protect loaded bioactive molecules from degradation and target them to recipient cells; as diagnostic biomarkers, the proteins and nucleic acids carried by extracellular vesicles can reflect the physiological and pathological state of the source cells, providing important detection targets for liquid biopsies.

[0003] However, current methods for functionalizing extracellular vesicles still have significant shortcomings. Existing technologies are mainly divided into two categories: source cell modification and post-isolation modification. Source cell modification uses genetic engineering to enable source cells to express target proteins, thereby causing their secreted extracellular vesicles to carry functional molecules. However, this method is complex and inefficient, and it is difficult to achieve effective modification for extracellular vesicles derived from bodily fluids such as blood or urine. Post-isolation modification is currently a widely used strategy, mainly including chemical coupling and physical loading methods. However, these methods often damage the lipid bilayer structure of extracellular vesicles during operation, affecting the integrity and bioactivity of extracellular vesicles. At the same time, the preparation process is cumbersome, and the loading efficiency is difficult to meet the needs of practical applications. Therefore, there is an urgent need to develop a technology that minimizes damage to extracellular vesicles, is simple to operate, and has high functionalization efficiency.

[0004] Therefore, this invention is proposed. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides extracellular vesicle-anchored nanobodies and their applications. Using extracellular vesicles as the target, a self-constructed nanobody phage library is used for screening to obtain nanobodies with bioaffinity to extracellular vesicles. These extracellular vesicle nanobodies can bind to extracellular vesicles, achieving efficient loading of proteins and nucleic acids onto exosomes for disease treatment. They can also capture exosomes for liquid biopsy applications. More importantly, these extracellular vesicles can bind to circulating extracellular vesicles in vivo, enabling modification of in vivo extracellular vesicles for disease treatment. In summary, extracellular vesicle-anchored nanobodies can modify and functionalize in vitro and in vivo extracellular vesicles to construct novel biopharmaceuticals; they can also be used for exosome isolation and purification to achieve extracellular vesicle-based disease diagnosis.

[0006] In order to achieve the objective of this invention, the following technical solution is adopted: This invention provides a nanobody comprising three complementarity-determining regions, CDR1, CDR2, and CDR3, wherein the amino acid sequences of the CDRs are selected from the following group: The amino acid sequences are as shown in SEQ ID NO.1 for CDR1, SEQ ID NO.2 for CDR2, and SEQ ID NO.3 for CDR3; or, The amino acid sequences are as shown in SEQ ID NO.4 for CDR1, SEQ ID NO.5 for CDR2, and SEQ ID NO.6 for CDR3; or, The amino acid sequences are as shown in SEQ ID NO.7 for CDR1, SEQ ID NO.8 for CDR2, and SEQ ID NO.9 for CDR3.

[0007] SEQ ID NO.1 FIFSRYA SEQ ID NO.2 GIRRNGASIT SEQ ID NO.3 VGRRRGGDYPYRY SEQ ID NO.4 STYYWDV SEQ ID NO.5 GWPNWFT SEQ ID NO.6 PMPHTFFAPVTQ SEQ ID NO.7 RISTRSG SEQ ID NO.8 GISRTSGSNT SEQ ID NO.9 TRRFGFTGYTPRY Furthermore, the amino acid sequence of the nanobody is shown in SEQ ID NO.10, SEQ ID NO.11 or SEQ ID NO.12.

[0008] SEQ ID NO.10 MAQVQLQASGGGFVQPGGSLRLSCAASGFIFSRYAMGWFRQAPGKEREFVSAISGIRRNGASITYYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTATYYCAVGRRRGGDYPYRYYWGQGTQVTVSS SEQ ID NO.11 MAQVQLQASGGGFVQPGGSLRLSCAASGSTYYWDVMGWFRQAPGKEREFVSAISGWPNWFTTYYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTATYYCAPMPHTFFAPVTQYWGQGTQVTVSS SEQ ID NO.12 MAQVQLQASGGGFVQPGGSLRLSCAASGRISTRSGMGWFRQAPGKEREFVSAISGISRTSGSNTTYYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTATYYCATRRRFGFTGYTPRYYWGQGTQVTVSS The present invention also provides biomaterials, said biomaterials comprising at least one of (a)-(e): (a) Polynucleotides encoding the above amino acids; (b) An expression cassette containing the polynucleotide described in (a); (c) A recombinant vector containing the polynucleotide described in (a); (d) Recombinant microorganisms containing the polynucleotides described in (a); (e) Recombinant microorganisms containing the recombinant vector described in (c).

[0009] The present invention also provides a delivery system comprising the above-described nanobody and an active molecule bound to the nanobody, the nanobody being used to bind extracellular vesicles.

[0010] Furthermore, the active molecule is selected from protein or nucleic acid molecules.

[0011] Furthermore, the protein is selected from: mCherry protein, Cre protein, nucleic acid binding protein or Cas9 protein, wherein the amino acid sequence of the mCherry protein is shown in SEQ ID NO.13, the amino acid sequence of the Cre protein is shown in SEQ ID NO.14, the amino acid sequence of the nucleic acid binding protein is shown in SEQ ID NO.15, and the amino acid sequence of the Cas9 protein is shown in SEQ ID NO.16; the nucleic acid molecule is selected from: siRNA or sgRNA, wherein the nucleotide sequence of the siRNA is shown in SEQ ID NO.17, and the nucleotide sequence of the sgRNA is shown in SEQ ID NO.18.

[0012] SEQ ID NO.13 MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSL QDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYK SEQ ID NO.14 MSNLLTVHQNLPALPVDATSDEVRKNLMDMFRDRQAFSEHTWKMLLSVCRSWAAWCKLNNRKWFPAEPEDVRDYLLYLQARGLAVKTIQQHLGQLNMLHRRSGLPRPSDSNAVSLVMRRIRKENVDAGERAKQALAFERTDFDQVRSLMENSDRCQDIRNLAFLGIAYNTL LRIAEIARIRVDISRTDGGRMLIHIGRTKTLVSTAGVEKALSLGVTKLVERWISVSGVADDPNNYLFCRVRKNGVAAPSATSQLSTRALEGIFEATHRLIYGAKDDSGQRYLAWSGHSARVGAARDAMARAGVSIPEIMQAGGWTNVNIVMNYIRNLDSETGAMVRLLEDG SEQ ID NO.15 FFMEELNTYRQKQGVVLKYGELPNSGPPHDRRFTFQVIIDGREFPEGEGRSKKEAKNAAAKLAVEILNKE SEQ ID NO.16 SEQ ID NO.17 UGACCUCAACUACAUGGUUTT AACCAUGUAGUUGAGGGUCATT SEQ ID NO.18 GGGGCCACUAGGGACAGGAUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC The present invention also provides a kit comprising the above-mentioned nanobody for immobilizing and capturing extracellular vesicles.

[0013] The present invention also provides a pharmaceutical composition comprising the above-described nanobody, or the above-described delivery system, and a pharmaceutically acceptable carrier or excipient.

[0014] The present invention also provides the application of the above-mentioned nanobodies, or delivery systems, or kits, or pharmaceutical compositions in the preparation of biological drugs, the separation and purification of extracellular vesicles to prepare drugs for disease diagnosis and prognosis monitoring.

[0015] Furthermore, the nanobody, delivery system, kit, or pharmaceutical composition is used in any one of (1)-(6): (1) Modification of extracellular vesicles in vivo or in vitro; (2) Tracing extracellular vesicles in vivo or in vitro; (3) Loading protein and / or nucleic acid molecules onto extracellular vesicles; (4) Capture extracellular vesicles; (5) Isolate extracellular vesicles; (6) or any combination thereof.

[0016] The present invention has the following technical effects: Extracellular vesicle-anchored nanobodies can bind to extracellular vesicles, enabling the loading of molecules such as proteins and nucleic acids onto these vesicles, facilitating the functional modification of extracellular vesicles, and achieving intracellular delivery to different organisms. Moreover, these nanobodies can be used not only for modifying "in vitro" extracellular vesicles but also for modifying "in vivo" extracellular vesicles, avoiding the need for extracellular vesicle purification. Furthermore, these nanobodies can isolate extracellular vesicles, providing high-quality extracellular vesicles for disease diagnosis. Attached Figure Description

[0017] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0018] Figure 1 The screening process for exosome-anchored nanobodies includes: a) showing the amount of phage recovered in each of the 8 rounds of screening; b) showing the sequencing of the phages recovered in the 7th round, which were then sorted according to their amino acid composition; and c) showing the affinity test results for the top-ranked nanobodies. Figure 2 The results of loading and intracellular delivery of mCherry protein onto extracellular vesicles derived from 293 cells mediated by exosome-anchored nanobodies are shown in Figure a. (a) shows that mCherry protein was loaded onto extracellular vesicles derived from 293 cells; (b) shows that Nexo3 and Nexo11 can transport mCherry protein into cells. Figure 3 The results of loading and intracellular delivery of mCherry protein onto extracellular vesicles from different sources mediated by exosome-anchored nanobodies. Among them, a shows mCherry protein loaded onto extracellular vesicles derived from 3T3 cells; b shows mCherry protein loaded onto extracellular vesicles derived from DC cells; c shows mCherry protein loaded onto extracellular vesicles derived from mouse serum. Figure 4 The results of loading and intracellular delivery of Cre protein onto extracellular vesicles from different sources mediated by exosome-anchored nanobodies. Among them, a shows that Cre protein was loaded onto extracellular vesicles from 293 cells; b shows that Cre protein was transported into cells by extracellular vesicles; c shows that Cre protein was transported into the tibialis anterior muscle by extracellular vesicles and was able to exert its function. Figure 5 The results of exosome-anchored nanobody-mediated loading and intracellular delivery of Cas9 protein onto extracellular vesicles from different sources are shown in the following figures: a) shows that Cas9 protein can be loaded onto extracellular vesicles derived from 293 cells via Nexo3; b) shows that flow cytometry detected successful delivery of Cas9 protein into 293 cells; c) shows that confocal fluorescence microscopy detected successful delivery of Cas9 protein into 293 cells; and d) shows that the Cas9 protein delivered to the target cells successfully exerted its function. Figure 6The results of loading and intracellular delivery of nucleic acid siRNA on extracellular vesicles mediated by extracellular vesicle-anchored nanobodies are shown in the following figures: a) shows siRNA binding to Nexo3; b) shows siRNA being loaded onto extracellular vesicles; c) shows siRNA on extracellular vesicles being transported into cells; and d) shows that the loaded siRNA is functional. Figure 7 Results of in vivo extracellular vesicle loading of extracellular vesicles mediated by extracellular antibody-anchored proteins, where a shows that Nexo3 mediates the loading of mCherry on extracellular vesicles in culture medium; b shows that Nexo3 mediates the loading of mCherry on extracellular vesicles in plasma; c shows that Nexo3 can achieve in vivo extracellular vesicle modification. Figure 8 The results of the modification of circulating extracellular vesicles in vivo by the Nexo3-anchored exosome-mediated transferrin receptor antibody anti-TfR1 were shown in Figure a. The modified Nexo3 nanobody was able to be delivered to the target tissue. Frozen tissue sections showed that the modified Nexo3 nanobody delivered to the target tissue could exert a therapeutic effect. Muscle grip strength test in MDX mice showed that the modified Nexo3 nanobody delivered to the target tissue could exert a therapeutic effect. Figure 9 Results of the separation of extracellular vesicles by extracellular vesicle-anchored nanobodies. Detailed Implementation

[0019] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0020] In a first aspect, the present invention provides that the nanobody comprises three complementarity-determining regions CDR1, CDR2, and CDR3, wherein the amino acid sequence of the CDRs is selected from the following group: The amino acid sequences are as shown in SEQ ID NO.1 for CDR1, SEQ ID NO.2 for CDR2, and SEQ ID NO.3 for CDR3; or, The amino acid sequences are as shown in SEQ ID NO.4 for CDR1, SEQ ID NO.5 for CDR2, and SEQ ID NO.6 for CDR3; or, The amino acid sequences are as shown in SEQ ID NO.7 for CDR1, SEQ ID NO.8 for CDR2, and SEQ ID NO.9 for CDR3.

[0021] In some embodiments, the amino acid sequence of the nanobody is shown in SEQ ID NO.10, SEQ ID NO.11 or SEQ ID NO.12.

[0022] Secondly, the present invention also provides biomaterials, said biomaterials comprising at least any one of (a)-(e): (a) Polynucleotides encoding the above amino acids; (b) An expression cassette containing the polynucleotide described in (a); (c) A recombinant vector containing the polynucleotide described in (a); (d) Recombinant microorganisms containing the polynucleotides described in (a); (e) Recombinant microorganisms containing the recombinant vector described in (c).

[0023] Thirdly, the present invention also provides a delivery system comprising the above-described nanobody and an active molecule bound to the nanobody, the nanobody being used to bind extracellular vesicles.

[0024] In some embodiments, the active molecule is selected from protein or nucleic acid molecules.

[0025] In some embodiments, the protein is selected from mCherry protein, Cre protein, nucleic acid binding protein or Cas9 protein, and the nucleic acid molecule is selected from siRNA or sgRNA.

[0026] Fourthly, the present invention also provides a kit containing the above-mentioned nanobody for immobilizing and capturing extracellular vesicles.

[0027] Fifthly, the present invention also provides a pharmaceutical composition comprising the above-described nanobody, or the above-described delivery system, and a pharmaceutically acceptable carrier or excipient.

[0028] In a sixth aspect, the present invention also provides the application of the above-mentioned nanobody, or delivery system, or kit, or pharmaceutical composition in the preparation of biological drugs, the separation and purification of extracellular vesicles to prepare drugs for disease diagnosis and prognosis monitoring.

[0029] In some embodiments, the nanobody, delivery system, kit, or pharmaceutical composition is used in any one of (1)-(6): (1) Modification of extracellular vesicles in vivo or in vitro; (2) Tracing extracellular vesicles in vivo or in vitro; (3) Loading protein and / or nucleic acid molecules onto extracellular vesicles; (4) Capture extracellular vesicles; (5) Isolate extracellular vesicles; (6) or any combination thereof.

[0030] The following is a detailed explanation using specific embodiments: The main equipment involved in the following embodiments is shown in Table 1.

[0031] Table 1: Instrument and Equipment Description Example 1: Screening process for extracellular vesicle-anchored nanobodies The phage display library was used for screening, and a total of 8 rounds of screening were conducted. The amount of phage recovered in each round was calculated. The experimental results are as follows: Figure 1 As shown in Figure a, the phages recovered in the 7th round were used to infect E. coli, plated, and 20 single clones were selected. The genes displaying the phage sequences were sequenced, and the sequenced gene sequences were converted into amino acid sequences and sorted according to their proportions. The experimental results are shown in Figure a. Figure 1 As shown in b. Nexo3: FIFSRYA, GIRRNGASIT, VGRRRGGDYPYRY; Nexo11: STYYWDV, GWPNWFT, PMPHTFFAPVTQ; Nexo9: RISTRSG, GISRTSGSNT, TRRFGFTGYTPRY; Nexo12: RTFRRNT, TINTGSSTTY, GRSVASLNTART; Nexo13: RTLRYYD, AIRRGGRTIT, AGRVSLLNYDAY; Nexo20: RTFSYST, AISRSGSTTT, RIGAGGVRDGRY. The top three nanobodies, Nexo3, Nexo11, and Nexo9, were purified and labeled. Flow cytometry was used to test the affinity of the nanobodies for exosomes, showing that all three had affinity for exosomes, with Nexo3 exhibiting the strongest affinity. The experimental results are shown in the figure. Figure 1 As shown in c.

[0032] Example 2: Experiments on loading and intracellular delivery of mCherry protein into extracellular vesicles from different sources mediated by exosome-anchored nanobodies. Exosome-anchored nanobodies Nexo3, Nexo11, and NGFP (control) were fused with mCherry protein (amino acid sequence shown in SEQ ID NO. 13) to purify the fusion proteins. These three fusion proteins were then mixed with extracellular vesicles derived from 293 cells and incubated at 4°C. Flow cytometry was used to detect the fluorescence signal of mCherry protein bound to the extracellular vesicles. Compared with the control group, mCherry protein could be loaded onto extracellular vesicles derived from 293 cells via Nexo3 or Nexo11. The experimental results are as follows: Figure 2 As shown in Figure a. Based on this, mCherry protein was further loaded onto extracellular vesicles using Nexo3 or Nexo11, respectively. These mCherry protein-loaded extracellular vesicles were then added to cells and co-cultured for 48 hours. Flow cytometry was used to detect the mCherry protein uptake in the cells. The results showed that a fluorescent signal of mCherry protein could be detected in the cells, indicating that Nexo3 and Nexo11 can transport mCherry protein into the cells. The experimental results are shown in Figure a. Figure 2 As shown in b.

[0033] Exosome-anchored nanobody Nexo3, NGFP (control), and mCherry protein were fused for expression and the fusion protein was purified. The fusion protein was then mixed with extracellular vesicles derived from 3T3 cells, extracellular vesicles derived from DC cells, and extracellular vesicles derived from mouse serum, respectively, and incubated at 4°C. Flow cytometry was used to detect the fluorescence signal of mCherry protein bound to extracellular vesicles from different sources. Compared with the control group, mCherry protein could be loaded onto extracellular vesicles derived from 3T3 cells via Nexo3. Figure 3 a) Extracellular vesicles derived from DC cells ( Figure 3 b) and extracellular vesicles derived from mouse serum ( Figure 3 c).

[0034] Example 3: Exosome-anchored nanobody-mediated loading and intracellular delivery of Cre protein in extracellular vesicles from different sources The exosome-anchored nanobody Nexo3, NGFP (control), and Cre protein (amino acid sequence shown in SEQ ID NO. 14) were fused for expression and the fusion protein was purified. The fusion protein was then labeled with the fluorescent dye FITC. The FITC-labeled fusion protein was mixed with extracellular vesicles derived from 293 cells and incubated at 4°C. Flow cytometry was used to detect the fluorescence signal of Cre protein bound to the extracellular vesicles. Compared with the control group, Cre protein could be loaded onto extracellular vesicles derived from 293 cells via Nexo3. The experimental results are as follows: Figure 4 As shown in a.

[0035] Based on this, Cre protein was loaded onto extracellular vesicles via Nexo3, and then these Cre-loaded extracellular vesicles were added to loxP cells (this cell model expresses mCherry red fluorescent protein in its untreated state; after Cre protein enters the cells and cleaves the loxP gene, it expresses GFP green fluorescent protein). After co-culturing the cells for 48 hours, the expression of mCherry protein and GFP protein in the cells was observed using confocal fluorescence microscopy. The results showed that GFP protein expression could be detected after the cells were treated with Cre-loaded extracellular vesicles, indicating that Cre protein was transported into the cells by the extracellular vesicles and successfully cleaved the loxP gene in the cells. The experimental results are as follows: Figure 4 As shown in b.

[0036] Further, extracellular vesicles loaded with Cre protein were injected into the tibialis anterior muscle of a loxP mouse model (in this untreated mouse model, all tissues express mCherry red fluorescent protein; Cre protein, upon entering the tissue, cleaves the loxP gene and expresses GFP green fluorescent protein). One week after injection, the tibialis anterior muscle of the treated mice was recovered, and small animal in vivo imaging was used to observe the expression of mCherry and GFP proteins in the muscle. The results showed that GFP protein expression was detectable after the injection of extracellular vesicles loaded with Cre protein into the tibialis anterior muscle, indicating that Cre protein was transported to the tibialis anterior muscle by extracellular vesicles and successfully cleaved the loxP gene in the muscle fibers. The experimental results are as follows: Figure 4 As shown in c.

[0037] Example 5: Exosome-anchored nanobody-mediated loading and intracellular delivery of Cas9 protein in extracellular vesicles from different sources The exosome-anchored nanobody Nexo3 was fused with Cas9 protein (amino acid sequence shown in SEQ ID NO. 16) for expression and purification. The fusion protein was then labeled with the fluorescent dye Cy5. The Cy5-labeled fusion protein was mixed with extracellular vesicles derived from 293 cells and incubated at 4°C. Flow cytometry was used to detect the fluorescence signal of the Cas9 protein bound to the extracellular vesicles. Compared with the control group containing only Cas9 protein, Cas9 protein could be loaded onto extracellular vesicles derived from 293 cells via Nexo3. The experimental results are as follows: Figure 5 As shown in a.

[0038] Based on this, Cy5-labeled Cas9 protein was loaded onto extracellular vesicles using Nexo3, and then these Cy5-labeled Cas9 protein-loaded extracellular vesicles were added to 293 cells. After co-incubation, flow cytometry and confocal fluorescence microscopy were used to detect the Cas9 protein uptake by the cells. Cy5-labeled exosome fluorescence signals were observed in 293 cells, indicating that the Cas9 protein loaded onto the extracellular vesicles was successfully delivered to the 293 cells. The experimental results are as follows: Figure 5 As shown in b and 5c.

[0039] Furthermore, the complex formed by Cas9 protein and gRNA (Cas9RNP) was loaded onto extracellular vesicles via Nexo3 and then added to a Cas9 reporter cell line (this cell model does not express dTomato protein without Cas9RNP treatment; after Cas9RNP enters the cells and cleaves the gene, it expresses dTomato red fluorescent protein). After co-culturing the cells for 48 hours, the expression of dTomato protein in the cells was observed using confocal fluorescence microscopy. The results showed that dTomato protein expression could be detected after treatment with extracellular vesicles loaded with Cas9RNP, indicating that Cas9RNP was transported into the cells by extracellular vesicles and successfully edited the reporter gene in the cells. The experimental results are as follows: Figure 5 As shown in d.

[0040] Example 6: Experiment on loading and intracellular delivery of nucleic acid siRNA in extracellular vesicles mediated by extracellular vesicle-anchored nanobodies The extracellular vesicle-anchored nanobody Nexo3 was fused with DRBD protein (RNA-binding protein) for expression (DRBD-Nexo3), and then mixed with siRNA (UGACCUCAACUACAUGGUUTT AACCAUGUAGUUGAGGUCATT). The conjugation products were then subjected to agarose gel electrophoresis. Agarose gel migration assays showed that siRNA binds to Nexo3 by binding to DRBD protein. The experimental results are as follows: Figure 6 As shown in Figure a, Nexo3 cells containing FITC-labeled siRNA were mixed with extracellular vesicles and incubated. Flow cytometry was used to detect the FITC fluorescence signal of the siRNA on the extracellular vesicles, demonstrating that the siRNA was loaded onto the extracellular vesicles. The experimental results are shown in Figure a. Figure 6 As shown in b.

[0041] After loading GAPDH-targeting siRNA onto extracellular vesicles using Nexo3, the siRNA was added to cells for co-incubation. Flow cytometry was used to detect the FITC fluorescence signal of the siRNA in the cells, showing that the siRNA loaded onto the extracellular vesicles was transported into the cells. The experimental results are as follows: Figure 6 As shown in Figure c. Western blot analysis of the activity of siRNA added to cells revealed that siRNA loaded onto extracellular vesicles by Nexo3 interfered with GAPDH expression, significantly reducing GAPDH expression levels. The experimental results are shown in Figure c. Figure 6 As shown in d.

[0042] Example 7: Results of loading extracellular vesicles with nanobody-mediated proteins in vivo. Extracellular vesicles were anchored to a fusion protein of the nanobody Nexo3 and mCherry. This fusion was added to cell culture supernatant, followed by ultrafiltration. The extracellular vesicles were then recovered, and Western blotting was used to detect the marker proteins Alix and CD81, as well as mCherry, on the extracellular vesicles. The results showed that mCherry was detectable on the isolated extracellular vesicles, indicating that Nexo3 mediates the loading of mCherry onto extracellular vesicles in the culture medium. The experimental results are as follows: Figure 7 As shown in a.

[0043] The fusion protein of Nexo3 and mCherry was added to mouse plasma, followed by ultrafiltration. Extracellular vesicles were recovered, and Western blot analysis was performed to detect the marker proteins CD63 and CD81 on the extracellular vesicles, as well as mCherry. The results showed that mCherry was detectable on the isolated extracellular vesicles, indicating that Nexo3 mediates the loading of mCherry onto extracellular vesicles in plasma. The experimental results are as follows: Figure 7As shown in b, the fusion protein of Nexo3 and mCherry was injected into mice via the tail vein system. Mouse plasma was then separated, and exosomes were isolated by ultrafiltration. Flow cytometry was used to detect the mCherry signal on the isolated extracellular vesicles. The results showed that mCherry signal was detected on the extracellular vesicles, indicating that Nexo3 can mediate the loading of mCherry protein on extracellular vesicles in the blood. This demonstrates that Nexo3 can achieve in vivo extracellular vesicle modification. The experimental results are shown in b. Figure 7 As shown in c.

[0044] Example 8: Modification of circulating extracellular vesicles in vivo by exosome-anchored nanobody Nexo3-mediated transferrin receptor antibody anti-TfR1 The exosome-anchored nanobody Nexo3 was fused with Protein A protein that binds to the Fc terminus of the antibody. The fused PA-Nexo3 protein was then bound to the transferrin receptor antibody anti-TfR1. DiR-labeled extracellular vesicles and the PA-Nexo3-anti-TfR1 complex were subsequently injected into C57 mice. Small animal fluorescence imaging was performed on the tissues. The results showed that the extracellular vesicle-anchored nanobody Nexo3 mediated the loading of anti-TfR1 onto circulating extracellular vesicles and delivered these vesicles to muscle tissue. Figure 8 a). PA-Nexo3 protein, expressed in a fusion, was bound to the transferrin receptor antibody anti-TfR1 and administered systemically to MDX mice, a DMD disease model. Frozen sections of muscle tissue from MDX mice after treatment were then subjected to immunofluorescence staining for β-dystroglycan and α-sarcoglycan. The staining showed restoration of function of the dystroglycan-associated complex in the muscle cell membrane of MDX mice, demonstrating that circulating extracellular vesicles were successfully delivered to muscle tissue to exert a therapeutic effect. Figure 8 b). The muscle grip strength of MDX mice after treatment was measured using a mouse grip strength analyzer. The results showed increased muscle grip strength, indicating that the extracellular vesicle-anchored nanobody Nexo3 mediated the loading of anti-TfR1 onto circulating extracellular vesicles, delivering these vesicles to muscle tissue and exerting a therapeutic effect. Figure 8 c).

[0045] Example 9: Results of the separation of extracellular vesicles by extracellular vesicle-anchored nanobodies The extracellular vesicle-anchoring nanobody Nexo3 was conjugated onto agarose beads and added to cell culture supernatant. The agarose beads were then recovered, and the presence of extracellular vesicles on the beads was analyzed. Western blotting was used to detect the marker proteins Alix and CD81 of extracellular vesicles. The results showed that markers of extracellular vesicles were detectable on the agarose beads, indicating that Nexo3 can effectively separate extracellular vesicles. The experimental results are as follows: Figure 9 As shown.

[0046] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the technical solutions of the embodiments of the present invention.

Claims

1. Extracellular vesicle-anchored nanobodies, characterized in that, The nanobody comprises three complementarity-determining regions, CDR1, CDR2, and CDR3, and the amino acid sequences of the CDRs are selected from the following group: The amino acid sequences are CDR1 as shown in SEQ ID NO.1, CDR2 as shown in SEQ ID NO.2, and CDR3 as shown in SEQ ID NO.

3.

2. The extracellular vesicle-anchored nanobody according to claim 1, characterized in that, The amino acid sequence of the nanobody is as shown in SEQ ID NO.

10.

3. A biomaterial, characterized in that, The biomaterial is any one of (a)-(e): (a) A polynucleotide encoding an extracellular vesicle-anchored nanobody as described in any one of claims 1-2; (b) An expression cassette containing the polynucleotide described in (a); (c) A recombinant vector containing the polynucleotide described in (a); (d) Recombinant microorganisms containing the polynucleotides described in (a); (e) Recombinant microorganisms containing the recombinant vector described in (c).

4. A delivery system, characterized in that, The nanobody comprises the nanobody according to any one of claims 1 or 2 and an active molecule bound to the nanobody, wherein the nanobody is used to bind extracellular vesicles; The active molecule is selected from: protein or nucleic acid molecules; The protein is selected from mCherry protein, Cre protein, nucleic acid-binding protein or Cas9 protein, and the nanobody is fused with the protein for expression. The amino acid sequence of the mCherry protein is shown in SEQ ID NO.13, the amino acid sequence of the Cre protein is shown in SEQ ID NO.14, the amino acid sequence of the nucleic acid binding protein is shown in SEQ ID NO.15, and the amino acid sequence of the Cas9 protein is shown in SEQ ID NO.

16. The nucleic acid molecule is siRNA, which binds to the nucleic acid binding protein expressed in fusion with the nanobody. The nucleotide sequence of the siRNA is shown in SEQ ID NO.17, SEQ ID NO.17: UGACCUCAACUACAUGGUUTTAACCAUGUAGUUGAGGUCATT.

5. A reagent kit, characterized in that, The kit contains the nanobody as described in any one of claims 1 or 2, for immobilizing and capturing extracellular vesicles.

6. A drug or drug composition, characterized in that, The drug or pharmaceutical composition comprises a nanobody as described in any one of claims 1 or 2, or a delivery system as described in claim 4, and a pharmaceutically acceptable carrier or excipient.

7. The application of a nanobody as described in any one of claims 1 or 2 in the isolation and purification of extracellular vesicles, characterized in that, Apply to any one of (1)-(2): (1) Capture extracellular vesicles; (2) Isolate extracellular vesicles.

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