Exosome-based ligand delivery system and uses thereof

Abstract

The application discloses an exosome-based ligand delivery system and application thereof. The system comprises an artificially designed polypeptide expression element, which sequentially comprises an N-terminal fusion peptide, a first connecting peptide, a modified CD63, a second connecting peptide and a C-terminal fusion peptide from the N-terminal to the C-terminal. The modified CD63 is formed by extending an intracellular region, a transmembrane region and an extracellular region from the N-terminal and the C-terminal of the natural CD63 respectively, so that the modified CD63 becomes a 6-transmembrane protein, and the N-terminal and the C-terminal of the modified CD63 are located on the outside of the membrane of the exosome. The application further provides a nucleic acid encoding the system, a recombinant vector, an engineered cell strain, an engineered exosome and a preparation method and application thereof. The system can simultaneously complete the loading of the targeting peptide and the functional ligand on the outside of the membrane during the process of exosome secretion by cells, realizes the integration of the targeting recognition and the ligand delivery, and has important application value in targeting the hair follicle stem cells and activating the Wnt signal pathway.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to an exosome-based ligand delivery system and its applications. Background Technology

[0002] Exosomes are lipid-containing, bilayered membrane structures secreted by cells and containing biomolecules such as proteins, nucleic acids, and lipids. They are primarily produced by exocytosis, the release of multiple vesicles formed by the invagination of intracellular lysosomal microparticles into the extracellular environment. As messengers for intercellular communication, exosomes, relying on their unique lipid bilayer structure and carrying signaling molecules, can communicate over long distances between different cells, thereby influencing gene expression and function. Due to their low immunogenicity, good biocompatibility, ability to cross the blood-brain barrier, and targeted delivery to specific cells or tissues, as well as their ability to prolong the half-life of encapsulated drugs in vivo, exosomes are currently being engineered as drug delivery carriers.

[0003] Ligands are molecules that bind to receptors on the cell surface; these molecules can be proteins / peptides, steroids, or other small molecules. Ligands influence cell function by initiating signal transduction processes through receptor binding. However, protein drugs that act on receptors on the cell membrane have the following limitations: (1) Short half-life: Some protein drugs are easily degraded or rapidly cleared by the kidneys, resulting in a short half-life in the body; (2) Poor solubility and stability: Protein drugs usually have poor solubility and stability and are easily inactivated in complex biological environments; (3) Immunogenicity: Protein drugs may be recognized as foreign substances by the human immune system, triggering an immune response; (4) Poor targeting.

[0004] Currently, engineered exosome surface display technology mainly relies on the following scaffold proteins: the tetraspannome protein family (CD63, CD9, CD81), type I transmembrane proteins (Lamp2b, PTGFRN, etc.), and perimembrane proteins (MFGE8, etc.). However, these scaffold proteins have significant limitations: the N-terminus and C-terminus of tetraspannome proteins are located on the inner side of the membrane (cytoplasmic side), so when displaying proteins on the exosome surface, only the extracellular loop structure of the exosome can be fused and modified, which greatly increases the difficulty of molecular design and validation; although Lamp2b is suitable for N-terminal fusion display, its C-terminal fusion affects the exosome sorting ability, and the displayed peptides are easily degraded by endosomal proteases; although novel scaffold proteins such as PTGFRN can support high-density display, their development is mainly limited to specific production cell backgrounds.

[0005] Therefore, there is an urgent need in the field for a universal ligand delivery system that can simultaneously display functional proteins at both the N-terminus and C-terminus on the outer side of the exosome membrane while retaining efficient exosome sorting capabilities. Summary of the Invention

[0006] The present invention aims to solve the technical problem that the display of scaffold protein topology on the surface of exosomes is limited and that functional proteins cannot be displayed at both ends of the outer membrane at the same time in the prior art.

[0007] In a first aspect, the present invention provides an exosome-based ligand delivery system comprising an artificially designed polypeptide expression element, which, from the N-terminus to the C-terminus, comprises: an N-terminal fusion peptide, a first linker peptide, a modified CD63, a second linker peptide, and a C-terminal fusion peptide.

[0008] The modified CD63 is formed by extending an intracellular region, a transmembrane region, and an extracellular region from the N-terminus and C-terminus of the natural CD63, respectively, making the modified CD63 a 6-transmembrane protein, with its N-terminus and C-terminus both located on the outer membrane of the exosome.

[0009] Therefore, the "modified CD63" is the core of this invention. Natural CD63 is a four-transmembrane protein widely distributed in the lipid bilayer of exosomes and is one of the classic markers of exosomes; its N-terminus and C-terminus are both located on the inner side of the exosome membrane. This invention extends an intracellular region, a transmembrane region, and an extracellular region from the N-terminus and C-terminus of CD63, respectively, making the modified CD63 a six-transmembrane protein, with its N-terminus and C-terminus both located on the outer side of the exosome membrane. After fusing with other proteins, these proteins are localized to the outer side of the exosome membrane.

[0010] In this invention, the protein amino acid sequence of the "first linker peptide-modified CD63-second linker peptide" is as follows: gggsggggsggggsATPGSLLPVVIIAVGVFLFLVAFVGCCGACKENAVEGGMKCVKFLLYVLLLAFCACAVGLIAVGVGAQLVLSQTIIQGATPGSLLPVVIIAVGVFLFLVAFVGCCGACKENYCLMITFAIFSLIMLVEVAAAIAGYVFRDKVMSEFNNNFRQQ MENYPKNNHTASILDRMQADFKCCGAANYTDWEKIPSMSKNRVPDSCCINVTVGCGINFNEKAIHKEGCVEKIGGWLRKNVLVVAAAALGIAFVEVLGIVFACCLVKSIRSGAEVMYCLMITFAIFLSLIMLVEVAAAIAGYVFRDKVMSEFNNggggsggggsggggs.

[0011] In the above sequence, the lowercase letters "ggggsggggsggggs" at both ends represent the amino acid sequences encoding the first and second linker peptides; the remaining uppercase letters in the middle represent the amino acid sequence of the modified CD63 protein, namely "ATPGSLLPVVIIAVGVFLFLVAFVGCCGACKENAVEGGMKCVKFLLYVLLLAFCACAVGLIAVGVGAQLVLSQTIIQGATPGSLLPVVIIAVGVFLFLVAFVGCCGACKENYCLMITFAIFLSLIMLVEVAAAIAGYVFRDKVMSEFNNNFRQQMENYPKNNHTASILDRMQADFKCCGAANYTDWEKIPSMSKNRVPDSCCINVTVGCGINFNEKAIHKEGCVEKIGGWLRKNVLVVAAAALGIAFVEVLGIVFACCLVKSIRSGAEVMYCLMITFAIFLSLIMLVEVAAAIAGYVFRDKVMSEFNN", which is the amino acid sequence of the modified CD63 protein.

[0012] In practice, restriction enzyme sites can be added to the front and back ends of the nucleotide sequence encoding the ligand delivery system as needed. Therefore, this invention can obtain different nucleotide sequences encoding the ligand delivery system by changing the sequences of different restriction enzyme sites. Moreover, the design of each restriction enzyme site in the nucleotide sequence encoding the same ligand delivery system is generally different, so as to facilitate precise control of the digested fragments. If the same restriction enzyme site is designed, the enzyme recognition point cannot be precisely on a certain segment during digestion. The amino acid sequence of the linker peptide "ggggsggggsggggs" is not unique and can also be replaced with other suitable amino acid sequences with linking functions.

[0013] The N-terminal and C-terminal fusion peptides can be flexibly designed according to different needs, and each can be independently selected from homing peptides, targeting peptides, or protein ligands. For example, they can be designed as various combinations such as "N-terminal: homing peptide + C-terminal: ligand", "N-terminal: ligand + C-terminal: homing peptide" or "N-terminal: ligand + C-terminal: ligand".

[0014] In some embodiments, the first and second linker peptides are flexible linker peptides, for example, containing a repeating sequence of Gly-Gly-Gly-Gly-Ser, wherein the number of repeats is 1-5, preferably 3.

[0015] In some specific embodiments, the N-terminal fusion peptide is a TWY targeting peptide that can specifically bind to the hair follicle stem cell surface marker alpha 6-Integrin, with the amino acid sequence: TWYKIAFQRNRK; the C-terminal fusion peptide is a Wnt3a protein carrying a 6×His tag.

[0016] Secondly, the present invention provides a nucleic acid molecule encoding the above-mentioned ligand delivery system.

[0017] Thirdly, the present invention provides a recombinant expression vector comprising the above-mentioned nucleic acid molecules. The vector may be a lentiviral vector (such as pCDH-CMV-MCS-EF1-Puro) or a mammalian expression vector (such as pcDNA3.1).

[0018] Fourthly, the present invention provides an engineered cell comprising the above-mentioned recombinant expression vector, wherein the endogenous CD63 gene in the cell has been knocked out. Preferably, the cell is a 293T cell. CD63-KO Cells were obtained by knocking out the CD63 gene in 293T cells using CRISPR / Cas9 gene editing technology. Knocking out endogenous CD63 eliminates interference from endogenous CD63 expression, ensuring that the fusion proteins displayed on the exosome surface are all derived from exogenously expressed ligand delivery systems.

[0019] Fifthly, the present invention provides an engineered exosome (Eguli) secreted by the aforementioned engineered cells, wherein the N-terminal fusion peptide and the C-terminal fusion peptide are simultaneously displayed on the outer membrane.

[0020] Sixthly, the present invention provides a method for preparing the above-mentioned engineered exosomes, comprising: The recombinant expression vector was introduced into host cells in which the endogenous CD63 gene had been knocked out to obtain engineered cells; the engineered cells were cultured and the culture supernatant was collected; exosomes were isolated and purified from the culture supernatant.

[0021] The introduction method includes lentiviral infection or plasmid transfection; the separation and purification method includes gradient differential centrifugation.

[0022] In a seventh aspect, the present invention provides the application of the above-described engineered exosomes in the preparation of drugs that target hair follicle stem cells and activate the Wnt signaling pathway.

[0023] In an eighth aspect, the present invention provides a pharmaceutical composition comprising the above-described engineered exosomes and a pharmaceutically acceptable carrier.

[0024] The main advantages of this invention include: (1) Bi-terminal extramembrane display: By modifying the transmembrane topology of CD63, the N-terminal targeting peptide and C-terminal functional ligand were simultaneously displayed on the extramembrane side of the exosome for the first time, overcoming the limitation that both ends of the existing four-transmembrane protein are inside the membrane and can only be modified by loop region.

[0025] (2) Integrated self-assembly: Exosomes carry target peptides and ligands and complete the process of cell generation of exosomes. The secreted exosomes are already fully functional Eguli, which can be used after enrichment and purification without additional artificial intervention.

[0026] (3) High versatility: In theory, it can carry and deliver any protein ligand (except transmembrane proteins). By changing different N-terminal / C-terminal fusion peptides, it can be adapted to different target organs and different therapeutic proteins.

[0027] (4) High purity system: The CD63-KO engineered cell line was used to eliminate the interference of endogenous CD63 and ensure the uniformity of exosome surface display. Attached Figure Description

[0028] Figure 1 This is a schematic diagram of the structure of an exosome-based ligand delivery system; Figure 2 This is a schematic diagram of the present invention; Figure 3The plasmid maps of PX458-CD63-sgRNA1 (a) and PX458-CD63-sgRNA2 (b) provided in Example 1; Figure 4 This is a schematic diagram of the two sgRNA designs provided in Example 1. Uppercase letters indicate exon sequences; lowercase letters indicate intron sequences; "_" indicates the sgRNA position; red letters indicate the spliced ​​sequences. Figure 5 a represents 293T in Example 1. CD63-KO Results of Western blot analysis of cell proteins; Figure 5 b shows the results of immunoprecipitation detection of exosomes TWY-Wnt3a-Exo, Scr-Eguli-Exo, and exosomes TWY-Wnt3a-Exo-2 and Scr-Eguli-Exo-2. Figure 5 c represents the detection results of fluorescently labeled exosomes uptake by cells in Experiment Example 2; Figure 5 d represents the detection results of the Wnt pathway activation level experiment in Experiment Example 3.

[0029] Figure 6 a represents the detection results of fluorescently labeled exosomes uptake by cells in Experiment Example 5; Figure 6 b represents the detection results of the Wnt pathway activation level experiment in Experiment Example 6. Detailed Implementation

[0030] To enable those skilled in the art to understand the present invention more clearly and intuitively, the present invention will be further described below with reference to the accompanying drawings.

[0031] This invention addresses the technical problem in existing technologies where the topological display of scaffold proteins on the exosome surface is limited, preventing the simultaneous display of functional proteins at both ends of the membrane. Firstly, this invention proposes a modified CD63, with the following amino acid sequence: ATPGSLLPVVIIAVGVFLFLVAFVGCCGACKENAVEGGMKCVKFLLYVLLLAFCACAVGLIAVGVGAQLVLSQTIIQGATPGSLLPVVIIAVGVFLFLVAFVGCCGACKENYCLMITFAIFLSLIMLVEVAAAIAGYVFRDKVMSEFNNNFRQQME NYPKNNHTASILDRMQADFKCCGAANYTDWEKIPSMSKNRVPDSCCINVTVGCGINFNEKAIHKEGCVEKIGGWLRKNVLVVAAAALGIAFVEVLGIVFACCLVKSIRSGAEVMYCLMITFAIFLSLIMLVEVAAAIAGYVFRDKVMSEFNN (SEQ ID NO:1).

[0032] Based on this "modified CD63", this invention proposes a ligand delivery system, the structural schematic of which is shown below. Figure 1 As shown, from the N-terminus to the C-terminus, it sequentially comprises: an N-terminal fusion peptide, a first linker peptide, a modified CD63, a second linker peptide, and a C-terminal fusion peptide. The protein amino acid sequence of "first linker peptide-modified CD63-second linker peptide" is shown below: ggggsggggsggggsATPGSLLPVVIIAVGVFLFLVAFVGCCGACKENAVEGGMKCVKFLLYVLLLAFCACAVGLIAVGVGAQLVLSQTIIQGATPGSLLPVVIIAVGVFLFLVAFVGCCGACKENYCLMITFAIFLSLIMLVEVAAAIAGYVFRDKVMSEFNNNFRQQMENYPKNNHTASILDRMQADFKCCGAANYTDWEKIPSMSKNRVPDSCCINVTVGCGINFNEKAIHKEGCVEKIGGWLRKNVLVVAAAALGIAFVEVLGIVFACCLVKSIRSGAEVMYCLMITFAIFLSLIMLVEVAAAIAGYVFRDKVMSEFNNggggsggggsggggs (SEQ ID NO:2), wherein ggggsggggsggggs (SEQ ID NO:3) is the amino acid sequence of the first linker peptide and the second linker peptide.

[0033] It should be noted that in ligand delivery systems, the first linker peptide, the second linker peptide, the N-terminal fusion peptide, and the C-terminal fusion peptide are not unique. Correspondingly, their amino acid sequences are not unique, and the nucleotide sequences encoding them are not unique either.

[0034] This invention extends an intracellular region, a transmembrane region, and an extracellular region from the N-terminus and C-terminus of CD63, respectively, making the modified CD63 a six-transmembrane protein. Both its N-terminus and C-terminus are located on the extracellular side of the exosome. After fusing with other proteins, these proteins are localized to the extracellular side of the exosome. The schematic diagram is shown below. Figure 2 As shown.

[0035] Example 1: Constructing a 293T specifically for producing Eguli CD63-KO cell lines The commercially available PX458-EF1a-pSpCas9(BB)-2A-GFP plasmid (Addgen, 75232); all restriction endonucleases and T4 ligases were purchased from Thermo Fisher Scientific; plasmid mini-extraction and purification kits, plasmid mass extraction kits, DNA gel recovery and purification kits, and PCR purification and recovery kits were purchased from Axygen; and DH5α chemically competent cells were purchased from Shanghai Sangon Biotech Co., Ltd.

[0036] 1.1 gRNA Sequence Design: The coding region of the human gene CD63 was retrieved from the NCBI website. Based on the CD63 gene structure, exon 3 of CD63 gene transcript 201 (NM 001780.6) was selected to design the guide RNA (gRNA) sequence for the CRISPR / Cas9 gene editing system. The online software CRISPOR (http: / / crispor.tefor.net / ) was used to select high-scoring sgRNAs, obtaining the sgRNA sequences. The designed sgRNA (design diagram shown in Figure 1) was then presented. Figure 4 The corresponding oligos were synthesized by adding BbsI restriction endonuclease sites to the oligos and their complementary sequences (as shown in Table 1).

[0037] Table 1. Oligo sequences of sgRNA

[0038] 1.2 sgRNA oligo annealing: The oligo powder was diluted with ddH2O and the annealing system was prepared as shown in Table 2. After mixing, DNA strands with sticky ends of BbsI endonuclease were synthesized using a PCR instrument by gradually lowering the temperature. The cooling program was: 37℃ for 30 min, 95℃ for 5 min, then lowered by 0.1℃ per second until reaching 25℃, 25℃ for 5 min, and then stored at -20℃ for later use.

[0039] Table 2

[0040] 1.3 Vector digestion: The PX458-EF1a-pSpCas9(BB)-2A-GFP plasmid (Addgene, 75232) was digested using BbsI restriction enzyme. The digestion system is shown in Table 3.

[0041] Table 3

[0042] After thorough mixing, the enzyme digestion system was incubated at 37°C for 2 hours. Then, 2 μL of the digestion system was mixed with DNA loading buffer and subjected to agarose gel electrophoresis to verify successful digestion. The digested product was purified and recovered using a DNA gel recovery and purification kit to obtain the linear PX458 plasmid vector.

[0043] 1.4 Ligation and transformation: The linear PX458 plasmid vector was ligated with the oligo annealing product. The ligation system is shown in Table 4.

[0044] Table 4

[0045] After thorough mixing, the mixture was ligated overnight at 16°C and then transformed into DH5α competent cells. The transformation procedure was as follows: competent cells were removed from the -80°C freezer and thawed on ice. All ligation products were gently mixed with 50 μl of competent cells. The mixture was placed on ice for 30 min, then heat-shocked in a 42°C water bath for 30 s, and immediately placed on ice for 5 min. In a laminar flow hood, 400 μl of sterile antibiotic-free LB liquid medium was added to the mixture, and the mixture was gently pipetted and incubated in a shaker at 37°C and 170 rpm for 1 h. After incubation, 150 μl of the mixture was added to 150 μg / mL LB solid medium in a laminar flow hood for plating, and then incubated overnight at 37°C.

[0046] 1.5 Bacterial Picking Verification: Add 1 ml of LB liquid medium containing ampicillin to a 1.5 ml centrifuge tube, pick several single colonies, and incubate at 37°C and 200 rpm for 6 h with shaking. Then, take 200 μl of bacterial culture and add 5 ml of LB liquid medium containing ampicillin, and incubate overnight at 37°C and 200 rpm with shaking. Extract a small amount of plasmid and send it for sequencing to verify successful ligation. Select hU6-F (TAATTTCTTGGGTAGTTTGC, SEQ ID NO:8) as the sequencing primer. Expand the culture of correctly sequenced colonies and use a plasmid large-scale extraction kit to extract the gene knockout plasmids PX458-CD63-sgRNA1 and PX458-CD63-sgRNA2, which have been confirmed to be successfully ligated. The plasmid maps are shown below. Figure 3 a and Figure 3 As shown in b.

[0047] 1.6 Cell Transfection: HEK293T cells were seeded into six-well plates and cultured in DMEM medium containing 10% fetal bovine serum. When the cell confluence reached 40%-50%, transfection was performed according to the EZ Trans instructions, simultaneously transfecting HEK293T cells with the gene knockout plasmids PX458-CD63-sgRNA1 and PX458-CD63-sgRNA2. The culture medium was replaced with fresh medium 24 hours after transfection. 48-72 hours after transfection, the expression of green fluorescence was observed using an inverted fluorescence microscope, and cells exhibiting green fluorescence were sorted using a flow cytometer (FACSAria IIU, BD Biosciences) and seeded into 10cm culture dishes. After 7-10 days, single-clone cells were seeded into 96-well cell culture plates using a pipette tip. When the cell density reached approximately 80%, the clones were digested and divided into two aliquots: one for PCR identification and the other for expansion culture.

[0048] 1.7 Obtained 293T CD63-KO Cell lines: DNA was extracted from each monoclonal cell. Two primers for identification were designed based on the upstream and downstream regions of the gene knockout sequence (the sequences of the two identification primers are shown in Table 5). PCR was used to preliminarily determine whether the knockout was successful. For clones with successful gene knockout, the PCR result should show a 179 bp band; for clones with unsuccessful knockout, a 326 bp band should be shown. Monoclonal cells that tested positive by PCR were selected for further sequencing verification (results are shown in Table 5). Figure 4 (See figure below). After PCR identification and amplification of correctly identified clones, cell DNA was extracted again, and the PCR products were sequenced.

[0049] Table 5. Sequences of PCR identification primers

[0050] 1.8 Identification of 293T CD63-KO Cell lines: Detection of 293T and 293T cells by Western blotting CD63-KOCD63 protein expression levels in cells. Specific steps included: lysing cells with RIPA buffer (Solarbio, R0010) and a cocktail of phosphatase inhibitors (bimake, B15001) and protease inhibitors (bimake, B14001); scraping the lysate from the plate and centrifuging at 12,000×g for 15 min at 4°C, collecting the supernatant. Protein concentration was determined using a modified Bradford protein assay kit (Sangon, C503041). Equal volumes of protein were separated by SDS-PAGE and transferred to a polyvinylidene fluoride membrane (PVDF, Merck Millipore, ISEQ00010), which was then blocked with 5% skim milk at room temperature for 1 hour. The protein was then incubated overnight at 4°C with CD63 antibody (ABclonal, A5271) and GAPDH antibody (Proteintech, 60004-1), followed by incubation with secondary antibodies of HRP-conjugated affinipure goat anti-rabbit IgG (H+L) and HRP-conjugated affinipure goat anti-mouse IgG (H+L). Protein expression levels were detected using Plus high-sensitivity chemiluminescence reagent (Shanghai Qinxiang, 1810212), and bands were observed using a ChemiScope 6100 chemiluminescence plate imaging system. Results are as follows: Figure 5 As shown in a, 293T CD63-KO The failure to detect CD63 in the cells indicates that the engineered cells provided in this application do not express CD63.

[0051] Example 2: Production of engineered exosomes targeting hair follicle stem cells and loaded with Wnt3a protein using a lentiviral expression system 2.1 Construction of pCDH-TWY-Eguli-Wnt3a lentiviral plasmid 2.1.1 Artificially synthesized DNA fragments: The fragment contains, from the 5' end to the 3' end, the coding sequence of the hair follicle targeting peptide (i.e., the TWY targeting peptide), the coding sequence of the ligand delivery system, the coding sequence of the NsiI, the coding sequence of the Wnt3a protein carrying a 6×His tag, and the coding sequence of the NotI; the above sequences are in the same open reading frame and are co-expressed as a fusion protein.

[0052] The amino acid sequence of the hair follicle targeting peptide (TWY targeting peptide) is as follows: Thr Trp Tyr Lys Ile Ala Phe Gln Arg Asn Arg Lys, single-letter code: TWYKIAFQRNRK (SEQ ID NO:12).

[0053] The amino acid sequence of the Wnt3a protein carrying a 6×His tag is as follows: APLGYFLLLCSLKQALGSYPIWWSLAVGPQYSSLGSQPILCASIPGLVPKQLRFCRNYVEIMPSVAEGIKIGIQECQHQFRGRRWNCTTVHDSLAIFGPVLDKATRESAFVHAIASAGVAFAVTRSCAEGTAAICGCSSRHQGSPGKGWKWGGCSEDIEFGGMVSREFADARENRPDARS AMNRHNNEAGRQAIASHMHLKCKCHGLSGSCEVKTCWWSQPDFRAIGDFLKDKYDSASEMVVEKHRESRGWVETLRPRYTYFKVPTERDLVYYEASPNFCEPNPETGSFGTRDRTCNVSSHGIDGCDLLCCGRGHNARAERRREKCRCVFHWCCYVSCQECTRVYDVHTCKHHHHHH (SEQ ID NO:13).

[0054] 2.1.2 Using the pCDH-CMV-MCS-EF1-Puro vector (HonorGene, HG-VMS0750), the 7.3kb fragment was recovered after double digestion with XbaI and NotI. The DNA fragment synthesized in step 2.1.1 was then double digested with XbaI and NotI. The synthesized DNA fragment was cloned into the digested vector pCDH-CMV-MCS-EF1-Puro through the same restriction sites. Ligation was performed using T4 DNA ligase ligase. The ligation product was transformed into competent DH5α cells and sequenced. The results were confirmed by alignment with SnapGene software, yielding a positive clone pCDH-TWY-Eguli-Wnt3a.

[0055] 2.2 Preparation of 293T exosomes with high efficiency in secreting TWY-Eguli-Wnt3a CD63-KO cell 2.2.1 293T CD63-KO Establishment of TWY-Eguli-Wnt3a cells: 293T cells were infected with pCDH-TWY-Eguli-Wnt3a lentivirus. CD63-KO Cells were obtained that highly expressed 293T cells carrying the Wnt3a gene, a gene for the hair follicle-targeting peptide-ligand delivery system. CD63-KO Cells. Specifically: Lentiviral Packaging: (1) Inoculate HEK293T cells into 15cm culture dishes and culture them in D10 medium. When the cell confluence reaches 70%-80%, prepare for transfection. (2) 1 hour before transfection, discard the original culture medium and add 15mL of preheated D10 medium. According to the instructions, use EZ Trans cell transfection reagent (Liji Biotechnology, AC04L091) to co-transfect HEK293T cells with pCDH-TWY-Eguli-Wnt3a (15μg), pMD2G (6μg), and psPAX2 (9μg). (3) Replace with fresh D10 medium after 6-18 hours. (4) Collect the culture medium after about 24 hours of culture, and repeat the collection once. Centrifuge the culture supernatant at 3000rpm and 4℃ for 10min and collect the supernatant. Filter the supernatant through a 0.45μm low protein binding filter membrane (Millipore Steriflip HV / PVDF). Mix the virus-containing culture medium with 5× lentivirus concentrate (GM-040801) at a volume ratio of 4:1, incubate overnight at 4°C, and centrifuge at 4000g and 4°C for 25 min. Carefully discard the supernatant, invert the centrifuge tube onto absorbent paper to drain for 3 min, add 1×PBS to resuspend and obtain concentrated virus solution, and store at -80°C.

[0056] 2.2.2 Lentiviral infection: 293T CD63-KO Cells were seeded in six-well plates and cultured in D10 medium. Cells were passaged every 3 days or when they reached 70%-80% confluence. Cells were then cultured at 293T. CD63-KO When the cell confluence reached 70%-80%, the cells were infected with the lentivirus prepared in step (4). The multiplicity of infection (MOI) was approximately 0.3-0.5. 24 hours after infection, the culture medium was replaced with fresh medium. After 2 days of culture, the culture medium was replaced with a medium containing 2 μg / ml of puromycin (InvivoGen) for screening. After 3-4 days of screening, the 293T cell line, which stably highly expressed the TWY-Eguli-Wnt3a gene, was obtained. CD63-KO That is, 293T CD63-KO -TWY-Wnt3a.

[0057] 2.2.3 Exosome isolation and purification: 293T CD63-KO TWY-Wnt3a cells were seeded into 15cm culture dishes and D10 medium was added. When cell confluence reached 70%-80%, the medium was removed, and the cells were washed twice with PBS. The medium was then replaced with exosome-free serum-based medium, and cultured for another 3 days. The culture supernatant was collected. Exosomes were purified by gradient differential centrifugation (4℃ throughout). The supernatant was centrifuged at 300g for 10 min to remove intact cells and collected; centrifuged at 2000g for 20 min to remove cell debris and large vesicles and collected; centrifuged at 10000g for 30 min to remove apoptotic bodies and collected; and centrifuged at 120000g for 90 min to precipitate crude exosomes. The precipitate was resuspended in 1mL of pre-chilled PBS to obtain the exosome suspension (TWY-Wnt3a-Exo). The exosome suspension was quantified using a BCA protein quantification kit and used for subsequent experiments or aliquoted and stored at -80℃.

[0058] Experimental Example 1: Identification of Exosome Marker Proteins Protein lysis was performed by adding 100 μL of RIPA lysis buffer containing protease inhibitors and phosphatase inhibitors to the exosome particles. The sample was vortexed and shaken on ice for 20 minutes, then vortexed again. The sample was collected by centrifugation at 12000g for 10 minutes at 4°C, transferred to a new 1.5 mL microcentrifuge tube, and stored at -80°C until analysis.

[0059] Immunoblot procedure: Protein concentration was determined using a modified Bradford protein assay kit (C503041, Shanghai Sangon Biotech). The exosome-containing sample was mixed with 5× loading buffer and boiled at 95°C for 5–10 min. Equal volumes of protein were separated by SDS-PAGE, followed by thorough electroporation and blocking, and then washing with TBST. Western blot analysis was used to determine the expression of the purified exosome-specific markers CD9, CD63, and TSG101.

[0060] See results Figure 5 b. Western blot analysis revealed the TWY-Wnt3a-Exo exosome markers CD9, CD63, and TSG101, indicating that the isolated exosomes were of high purity and met the requirements for subsequent experiments.

[0061] Experimental Example 2: Cell Uptake Experiment Targeting Hair Follicle Stem Cells This experiment aimed to detect the targeting binding ability and cellular uptake efficiency of exosomes carrying hair follicle-targeting peptides to hair follicle stem cells. Exosomes modified with random peptides were used as a negative control. A multi-cell co-culture system was used to simulate the scalp microenvironment, and flow cytometry was employed to detect the targeted uptake ability of exosomes. The specific experimental steps are as follows: 1) Following the method in Example 2, a scrambled peptide (Scr) of the hair follicle targeting peptide without targeting function was inserted between the restriction enzyme sites XbaⅠ and AgeⅠ to construct the pCDH-Scr-Eguli lentiviral plasmid. The downstream Wnt3a protein coding sequence was deleted. Except for the above two modifications, the rest of the vector backbone and regulatory elements are completely identical to pCDH-TWY-Eguli-Wnt3a. This plasmid only replaces the targeting peptide with a scrambled sequence, causing it to lose its ability to target hair follicle stem cells and not carry the Wnt3a fragment, but it can normally express the Eguli ligand delivery backbone system. The successfully constructed pCDH-Scr-Eguli plasmid was packaged into lentivirus, and the resulting viral fluid was used to infect 293T cells. CD63-KO Screening and establishing a stable high expression of Scr-Eguli 293T CD63-KO Cells were collected, cell culture supernatant was collected, and exosomes were purified. The resulting negative control exosomes were named Scr-Eguli-Exo. Similarly, Western blot analysis was performed on Scr-Eguli-Exo, and the results are shown below. Figure 5 b. Western blot detected the Scr-Eguli-Exo exosome markers CD9, CD63, and TSG101.

[0062] The amino acid sequence of the scrambled peptide Scr, a hair follicle targeting peptide, is as follows: Lys Arg Ala Phe Thr Asn Trp Lys Ile Arg Gln Tyr, single-letter code: KRAFTNWKIRQY (SEQ ID NO:14).

[0063] 2) The total protein content of the purified exosomes was quantified using the BCA protein quantification kit. The dosage of exosomes was standardized across groups to ensure uniformity of experimental conditions. The quantified exosomes TWY-Wnt3a-Exo and Scr-Eguli-Exo were labeled with red fluorescence using PKH26 fluorescent dye. The PKH26 dye was diluted with Diluent C according to the kit, and the dye dilution was mixed with the exosome suspension. The mixture was incubated at 37°C in the dark for 5 min to achieve fluorescent labeling. After incubation, PBS containing 10% BSA was added to stop the labeling. The exosomes were then centrifuged at 3000g for 10 min using a 100kDa ultrafiltration tube to remove free dye. The exosomes were collected and resuspended in PBS, and washed once more to completely remove residual free dye. The PKH26-labeled red fluorescent TWY-Wnt3a-Exo and Scr-Eguli-Exo were then prepared and stored at 4°C in the dark for later use.

[0064] 3) Human hair follicle stem cells (HFSCs) were fluorescently labeled by infecting them with a lentivirus carrying the GFP fluorescent labeling gene. HFSCs were seeded in six-well plates, and when the cell confluence reached 50%-60%, GFP lentivirus was added to infect the cells. After 24 hours of infection, the culture medium was replaced with fresh medium. After 2 days of culture, GFP-positive cells were screened by flow cytometry to obtain GFP fluorescently labeled hair follicle stem cells (HFSC-GFP).

[0065] 4) Prepare single-cell suspensions and count cells separately for GFP-labeled human hair follicle stem cells (HFSC-GFP), human epidermal keratinocytes (HEK), and human skin fibroblasts (HDF). Mix them according to a cell ratio of HFSC-GFP:HEK:HDF = 1:1:1. Pour the thoroughly mixed cell suspensions into a solution at 1×10⁻⁶ cells / cells. 6 Cells were seeded at a density of 1 cell per well in six-well plates, and experiments were conducted after the cells had fully adhered to the plates and their growth had stabilized.

[0066] 5) Cell Culture Conditions: When culturing the three cell types individually, their respective dedicated culture media were used to maintain cell biological characteristics. For co-culture experiments, all groups used high-glucose DMEM medium containing 10% exosome-free fetal bovine serum. The experiment included a blank control group, a TWY-Wnt3a-Exo experimental group, and a Scr-Eguli-Exo negative control group. Equal volumes of PKH26-labeled TWY-Wnt3a-Exo and Scr-Eguli-Exo were added to the experimental and negative control groups, respectively. An equal volume of high-glucose DMEM blank medium containing 10% exosome-free fetal bovine serum was added to the blank control group. All groups were incubated in the dark for 24 h.

[0067] 6) After incubation, discard the cell culture medium for each group. Gently wash the cells twice with PBS buffer to thoroughly remove untaken free exosomes and residual culture medium components from the cell surface. Add an appropriate amount of trypsin to digest the cells in each group, collect the cell suspension from each group, centrifuge at 400g for 5 min, and discard the supernatant. Resuspend the cell pellet twice with PBS buffer, and finally resuspend the cells with PBS buffer to prepare a homogeneous single-cell suspension. Filter the suspension through a cell strainer to remove cell clumps, incubate on ice in the dark, and detect the GFP green fluorescence signal and PKH26 red fluorescence signal in the samples by flow cytometry.

[0068] 7) Flow cytometry results ( Figure 5c) shows that in the co-culture system of the three cell types, the background fluorescence signal of the blank control group cells was weak, confirming that there was no background interference such as dye residue or cell autoluminescence in the experimental system. The fluorescence peak of the Scr-Eguli-Exo group shifted slightly to the right, with only a small amount of low-intensity fluorescence signal, suggesting that the random peptide does not have the ability to target and recognize hair follicle stem cells. The cells only take up a small amount of exosomes through non-specific action, without specific enrichment effect. In contrast, the main fluorescence peak of HFSC-GFP cells in the TWY-Wnt3a-Exo experimental group shifted significantly to the right, and the overall fluorescence intensity was significantly higher than that of the Scr-Eguli-Exo group. The results indicate that under the same cell culture and intervention conditions, exosomes carrying the TWY targeting peptide can specifically recognize and bind to hair follicle stem cells, and the uptake efficiency is much higher than that of exosomes modified with random peptides without targeting function.

[0069] In summary, TWY targeting peptides can significantly enhance the ability of exosomes to target and take up hair follicle stem cells, effectively mediating the targeted enrichment of exosomes in hair follicle stem cells. This provides important experimental support for subsequent use of TWY targeting peptides to modify exosomes for delivery of Wnt3a protein and specifically regulate the biological functions of hair follicle stem cell proliferation and differentiation.

[0070] Experimental Example 3: Detection of Wnt Pathway Activation Level This experiment aimed to verify that engineered exosomes can stably load the C-terminus of the Wnt3a protein, and that the fusion protein possesses complete biological activity. Exosomes modified with a randomized peptide were selected as a negative control, and lithium chloride as a positive control. The activation level of the classical Wnt pathway was detected using the TOPflash dual-luciferase reporter system. The specific experimental steps are as follows: 1) Preparation of negative control exosomes: Following the method described in Example 2, 293T exosomes that stably highly express Scr-Eguli and do not express Wnt3a fusion protein were collected. CD63-KO The cell culture supernatant was purified, and the resulting product was the negative control exosome Scr-Eguli-Exo.

[0071] 2) Cell transfection: HEK293T cells were transfected at a concentration of 3.5 × 10⁻⁶ cells / cell. 6 Cells were seeded at a density of 10 cells / dish in 10 cm cell culture dishes and cultured for 24 h as usual. Then, the cells were co-transfected with the TOPFlash firefly luciferase reporter plasmid and the pRL-TK Renilla luciferase internal control plasmid at a plasmid ratio of 10:1. After transfection, the cells were incubated at 37°C and 5% CO2 for 24 h.

[0072] 3) Cell Plating: Replace the culture medium with fresh medium within 16–24 hours after cell transfection. Discard the original culture medium, gently wash the cells once with PBS, add 0.05% trypsin for cell digestion, and after the cells detach, add complete culture medium to stop digestion. Gently pipette to prepare a homogeneous single-cell suspension. Spread the transfected HEK293T cells at a concentration of 1×10⁻⁶ cells / mL. 4 Cells were seeded at a density of 1 cell per well in 96-well cell culture plates and cultured for 24 hours to ensure full cell adhesion and stable cell condition.

[0073] 4) After 24 hours of cell plating and culture, cells were uniformly treated according to the experimental groups. The exosome treatment group was treated with an equal volume of exosomes with the same total protein content for 24 hours; the LiCl positive group was treated with 20 mM LiCl medium for 24 hours; and the Mock blank control group was treated with an equal volume of basal medium without any intervention. The intervention duration, culture conditions, and liquid addition system were completely consistent for all groups.

[0074] 5) Dual-luciferase assay: This experiment used the Beyotime Dual-luciferase Reporter Gene Detection Kit (Catalog No.: RG089S) for detection. After cell intervention, the TOPFlash transfection system cells were tested strictly according to the kit instructions. Firefly luciferase assay reagent and Renilla luciferase assay reagent were added to each well in sequence. After sufficient reaction at room temperature in the dark, the fluorescence intensity values ​​of firefly luciferase (TOPFlash) and Renilla luciferase (Renilla) in each group of cells were detected by a multi-functional microplate reader.

[0075] 6) Calculate the ratio of the luminescence value of firefly luciferase to that of Renilla luciferase in each well of the TOPFlash system. The internal control ratio method is used to eliminate inter-group errors related to cell plating density and transfection efficiency. This ratio represents the specific relative activity of the Wnt / β-catenin pathway and is used for subsequent statistical analysis of differences in pathway activation levels between groups.

[0076] 7) Test results as follows Figure 5The results showed that the relative luciferase activity in the Mock blank control group remained at a very low baseline level, and the background Wnt pathway activation signal was negligible, indicating that the classical Wnt pathway in HEK293T cells was in a quiescent state without exogenous activation stimulation. The LiCl treatment group, as a positive control for pathway activation, showed a significant increase in relative luciferase activity, verifying that this assay can stably and sensitively reflect the activation status of the classical Wnt / β-catenin pathway. The Scr-Eguli-Exo group showed only a weak pathway activation signal similar to that of the Mock group, with no obvious pathway activation effect. The relative luciferase activity in the TWY-Wnt3a-Exo experimental group was significantly increased, significantly higher than that in the Scr-Eguli-Exo negative control group, and the statistical difference between the two groups was p < 0.001, indicating a highly significant difference.

[0077] The above results demonstrate that the TWY-targeted modified exosomes of the present invention can stably load biologically active Wnt3a fusion proteins and deliver functional Wnt3a into the cell to activate the classical Wnt pathway. The random peptide control exosomes without Wnt3a loading do not have the ability to significantly activate the Wnt pathway.

[0078] Example 3: Production of exosomes carrying target ligands using a mammalian expression system 3.1 Construction of pcDNA-TWY-Eguli-Wnt3a expression plasmid 3.1.1 Artificially synthesized DNA fragments: The fragment contains, from the 5' end to the 3' end, the coding sequence for the hair follicle targeting peptide (TWY targeting peptide), the coding sequence for the ligand delivery system, the coding sequence for the EcorI restriction site, the coding sequence for the Wnt3a protein carrying a 6×His tag, and the coding sequence for the NotI restriction site. All of these sequences are in the same open reading frame and are co-expressed as a fusion protein.

[0079] 3.1.2 Using the pcDNA3.1 vector (HonorGene, HG-VPI0001), the 5.3kb fragment was recovered after double digestion with NheⅠ and NotⅠ. The DNA fragment synthesized in step 3.1.1 was then double digested with NheⅠ and NotⅠ. The synthesized DNA fragment was cloned into the digested vector pcDNA3.1 through the same restriction sites and ligated using T4 DNA ligase. The ligation product was transformed into competent DH5α cells and sequenced. The results were confirmed by comparison with SnapGene software, and a positive clone was obtained, which is the pcDNA-TWY-Eguli-Wnt3a carrying the target ligand expression plasmid.

[0080] 3.2 Transfection of 293T CD63-KO And collect and cultivate the upper purine 293T CD63-KO Cells were seeded on 15cm culture dishes and cultured in D10 medium. Transfection was performed when the cell confluence reached 80-90%. One hour before transfection, the original culture medium was discarded, and 15mL / dish of preheated D10 medium was added. pcDNA-TWY-Eguli-Wnt3a was transfected using EZ Trans cell transfection reagent (Liji Biotechnology, AC04L091) according to the manufacturer's instructions. After 6-18 hours, the cells were washed twice with PBS, and the culture medium was replaced with exosome-free serum-free medium. After 72 hours, the culture medium was collected, centrifuged at 300g, 4℃ for 10 min, and filtered through a 0.45µm low protein binding membrane (Millipore Steriflip HV / PVDF) to collect the supernatant.

[0081] 3.3 Exosome isolation and purification Exosomes were purified by gradient differential centrifugation (4°C throughout). The supernatant was centrifuged at 300g for 10 min to remove intact cells and the supernatant was collected. Centrifugation at 2000g for 20 min removed cell debris and large vesicles, and the supernatant was collected again. Centrifugation at 10000g for 30 min removed apoptotic bodies, and the supernatant was collected again. Centrifugation at 120000g for 90 min precipitated crude exosomes. The precipitate was resuspended in 1 mL of pre-chilled PBS to obtain the exosome suspension (TWY-Wnt3a-Exo-2). After quantification using a BCA protein concentration assay kit, the exosome suspension was used for subsequent experiments or aliquoted and stored at -80°C.

[0082] Experiment 4: Identification of exosome marker proteins 1) Add 100 μL of RIPA lysis buffer containing protease inhibitors and phosphatase inhibitors to the exosome particles for protein lysis. Vortex, shake on ice for 20 minutes, then vortex again. Centrifuge at 12000g for 10 minutes at 4°C to collect the sample, transfer to a new 1.5 mL microcentrifuge tube, and store at -80°C until analysis.

[0083] 2) Immunoblot procedure: Protein concentration was determined using a modified Bradford Protein Assay Kit (C503041, Shanghai Sangon Biotech). The exosome-containing sample was mixed with 5× loading buffer and boiled at 95°C for 5–10 min. Equal volumes of protein were separated by SDS-PAGE, followed by thorough electroporation and blocking, and then washing with TBST. Western blot analysis was used to determine the expression of the purified exosome-specific markers CD9, CD63, and TSG101.

[0084] See results Figure 5 b. Western blot detected TWY-Wnt3a-Exo-2 and Scr-Eguli-Exo-2 exosome markers CD9, CD63 and TSG101, indicating that the exosomes we isolated were of high purity and met the requirements for subsequent experiments.

[0085] Experimental Example 5 This experiment aimed to detect the targeting binding ability and cellular uptake efficiency of exosomes carrying hair follicle-targeting peptides to hair follicle stem cells. Exosomes modified with random peptides were used as a negative control. A multi-cell co-culture system was used to simulate the scalp microenvironment, and flow cytometry was employed to detect the targeted uptake ability of exosomes. The specific experimental steps are as follows: 1) Following the method in Example 3, the sequence of the scrambled peptide Scr, encoding a non-targeting hair follicle-targeting peptide, was inserted between the restriction enzyme sites NheⅠ and HindⅠ to construct the pCDNA-Scr-Eguli plasmid. The coding sequence for the downstream Wnt3a protein was deleted. Except for the two modifications mentioned above, the rest of the vector backbone and regulatory elements are completely identical to pCDNA-TWY-Eguli-Wnt3a. This plasmid only replaces the targeting peptide with a scrambled sequence, thus eliminating its ability to target hair follicle stem cells and preventing it from carrying the Wnt3a fragment, but it can still express the Eguli ligand delivery backbone system normally. The successfully constructed pCDNA-Scr-Eguli plasmid was transfected into 293T cells. CD63-KO Cell culture supernatant was collected and exosomes were purified. The resulting negative control exosomes were named Scr-Eguli-Exo-2.

[0086] 2) The total protein content of the purified exosomes was quantified using the BCA protein quantification kit. The dosage of exosomes was standardized across groups to ensure uniformity of experimental conditions. The quantified exosomes TWY-Wnt3a-Exo-2 and Scr-Eguli-Exo-2 were labeled with red fluorescence using PKH26 fluorescent dye. The PKH26 dye was diluted with Diluent C according to the kit, and the dye dilution was mixed with the exosome suspension. The mixture was incubated at 37°C in the dark for 5 min to achieve fluorescent labeling. After incubation, PBS containing 10% BSA was added to stop the labeling. The exosomes were then centrifuged at 3000g for 10 min using a 100kDa ultrafiltration tube to remove free dye. The exosomes were collected and resuspended in PBS, and washed once more to thoroughly remove residual free dye. The PKH26-labeled red fluorescent TWY-Wnt3a-Exo-2 and Scr-Eguli-Exo-2 were then stored at 4°C in the dark for later use.

[0087] 3) Human hair follicle stem cells (HFSCs) were fluorescently labeled by infecting them with a lentivirus carrying the GFP fluorescent labeling gene. HFSCs were seeded in six-well plates, and when the cell confluence reached 50%-60%, GFP lentivirus was added to infect the cells. After 24 hours of infection, the culture medium was replaced with fresh medium. After 2 days of culture, GFP-positive cells were screened by flow cytometry to obtain GFP fluorescently labeled hair follicle stem cells (HFSC-GFP).

[0088] 4) Prepare single-cell suspensions and count cells by GFP-labeled human hair follicle stem cells (HFSC-GFP), human epidermal keratinocytes (HEK), and human skin fibroblasts (HDF). To simulate the abundance and microenvironment of human scalp cells, mix them according to a cell ratio of hair follicle stem cells: epidermal keratinocytes: human skin fibroblasts = 1:1:1. The thoroughly mixed cell suspensions are then diluted with water at a concentration of 1 × 10⁻⁶ cells / mL. 6Cells were seeded at a density of 1 cell per well in six-well plates, and experiments were conducted after the cells had fully adhered to the plates and their growth had stabilized.

[0089] 5) Cell Culture Conditions: When culturing the three cell types individually, their respective dedicated culture media were used to maintain cell biological characteristics. For co-culture experiments, all groups used high-glucose DMEM medium containing 10% exosome-free fetal bovine serum. The experiment included a blank control group, a TWY-Wnt3a-Exo-2 experimental group, and a Scr-Eguli-Exo-2 negative control group. Equal volumes of PKH26-labeled TWY-Wnt3a-Exo-2 and Scr-Exo-2 were added to the experimental and negative control groups, respectively. An equal volume of high-glucose DMEM blank medium containing 10% exosome-free fetal bovine serum was added to the blank control group. All groups were incubated in the dark for 24 h.

[0090] 6) After incubation, discard the cell culture medium for each group. Gently wash the cells twice with PBS buffer to thoroughly remove untaken free exosomes and residual culture medium components from the cell surface. Add an appropriate amount of trypsin to digest the cells in each group, collect the cell suspension from each group, centrifuge at 400g for 5 min, and discard the supernatant. Resuspend the cell pellet twice with PBS buffer, and finally resuspend the cells with PBS buffer to prepare a homogeneous single-cell suspension. Filter the suspension through a cell strainer to remove cell clumps, incubate on ice in the dark, and detect the GFP green fluorescence signal and PKH26 red fluorescence signal in the samples by flow cytometry.

[0091] 7) Flow cytometry results ( Figure 6 a) The results showed that in the co-culture system of the three cell types, the blank control group only exhibited basal background fluorescence, further eliminating the interference of cell autofluorescence on the detection results. In the Scr-Eguli-Exo-2 group, the fluorescence peak shifted to the right compared to the blank control group, producing only a moderate level of non-specific uptake fluorescence signal, failing to achieve targeted enrichment of hair follicle stem cells. In contrast, the fluorescence peak of the TWY-Wnt3a-Exo-2 experimental group shifted significantly to the right to the high fluorescence range, and the proportion of high fluorescence cells was significantly increased. The results indicate that under the same cell culture and intervention conditions, exosomes carrying TWY targeting peptides can be efficiently recognized, bound, and actively taken up by hair follicle stem cells, with an uptake efficiency far exceeding that of exosomes modified with random peptides without targeting function.

[0092] Experimental Example 6 This experiment aimed to verify that engineered exosomes can stably load the C-terminus of the Wnt3a protein, and that the fusion protein possesses complete biological activity. Exosomes modified with random peptides were used as a negative control, and lithium chloride as a positive control. The activation level of the classical Wnt pathway was detected using the TOPflash dual-luciferase reporter system. The specific experimental steps are as follows: 1) Following the method described in Experiment Example 5, the culture supernatant of 293TCD63-KO cells transfected with pCDNA-Scr-Eguli plasmid was collected and the exosomes were purified. The resulting product was the negative control exosome Scr-Eguli-Exo-2.

[0093] 2) Cell transfection: HEK293T cells were transfected at a concentration of 3.5 × 10⁻⁶ cells / cell. 6 Cells were seeded at a density of 10 cells / dish in 10cm cell culture dishes and cultured for 24 hours as usual. Then, the TOPFlash firefly luciferase reporter plasmid and pRL-TK Renilla luciferase internal control plasmid were co-transfected at a plasmid ratio of 10:1. After transfection, the cells were incubated at 37°C and 5% CO2 for 24 hours.

[0094] 3) Cell Plating: Replace the culture medium with fresh medium within 16–24 hours after cell transfection. Discard the original culture medium, gently wash the cells once with PBS, add 0.05% trypsin for cell digestion, and after the cells detach, add complete culture medium to stop digestion. Gently pipette to prepare a homogeneous single-cell suspension. Spread the transfected HEK293T cells at a concentration of 1×10⁻⁶ cells / mL. 4 Cells were seeded at a density of 1 cell per well in 96-well cell culture plates and cultured for 24 hours to ensure full cell adhesion and stable cell condition.

[0095] 4) After 24 hours of cell plating and culture, cells were uniformly treated according to the experimental groups. The exosome treatment group was treated with an equal volume of exosomes with the same total protein content for 24 hours; the LiCl positive group was treated with 20 mM LiCl medium for 24 hours; and the Mock blank control group was treated with an equal volume of basal medium without any intervention. The intervention duration, culture conditions, and liquid addition system were completely consistent for all groups.

[0096] 5) Dual-luciferase assay: This experiment used the Beyotime Dual-luciferase Reporter Gene Detection Kit (Catalog No.: RG089S) for detection. After cell intervention, the TOPFlash transfection system cells were tested strictly according to the kit instructions. Firefly luciferase assay reagent and Renilla luciferase assay reagent were added to each well in sequence. After sufficient reaction at room temperature in the dark, the fluorescence intensity values ​​of firefly luciferase (TOPFlash) and Renilla luciferase (Renilla) in each group of cells were detected by a multi-functional microplate reader.

[0097] 6) Calculate the ratio of the luminescence value of firefly luciferase to that of Renilla luciferase in each well of the TOPFlash system. Use the internal control ratio to eliminate inter-group errors related to cell plating density and transfection efficiency. This ratio represents the specific relative activity of the Wnt / β-catenin pathway and is used for subsequent differential analysis of pathway activation levels between groups.

[0098] Test results as follows Figure 6 b shows that the relative luciferase activity in the Mock blank control group was at a low baseline value, indicating no spontaneous Wnt pathway activation interference. The LiCl-treated group showed a significant increase in relative luciferase activity, demonstrating that the TOPFlash detection system effectively reflects the degree of Wnt pathway activation. The Scr-Eguli-Exo-2 group only detected a weak basal pathway activation signal, failing to effectively activate the Wnt / β-catenin pathway. The TWY-Wnt3a-Exo-2 experimental group showed significantly higher relative luciferase activity than the Scr-Eguli-Exo-2 group (p < 0.001), indicating a highly statistically significant difference. These results confirm that the TWY-modified engineered exosomes of this invention can load biologically functional Wnt3a protein and efficiently activate the classical Wnt signaling pathway after acting on cells, while the random peptide-modified control exosomes do not exhibit a significant Wnt pathway activation effect.

Claims

1. A modified CD63, characterized in that, The modified CD63 is formed by extending an intracellular region, a transmembrane region, and an extracellular region from the N-terminus and C-terminus of the natural CD63, respectively, making the modified CD63 a 6-transmembrane protein, with its N-terminus and C-terminus located on the outer membrane of the exosome. The amino acid sequence of the modified CD63 is shown in SEQ ID NO:

1.

2. An exosome-based ligand delivery system, characterized in that, It contains an artificially designed polypeptide expression element, which consists of the following components from N-terminus to C-terminus: N-terminal fusion peptide, first linker peptide, modified CD63, second linker peptide, and C-terminal fusion peptide. The modified CD63 is formed by extending an intracellular region, a transmembrane region, and an extracellular region from the N-terminus and C-terminus of natural CD63, respectively, making the modified CD63 a 6-transmembrane protein, with its N-terminus and C-terminus both located on the outer membrane of the exosome; the amino acid sequence of the modified CD63 is shown in SEQ ID NO:

1. The N-terminal fusion peptide and the C-terminal fusion peptide are each independently selected from homing peptides, targeting peptides, or protein ligands; Preferably, the first linker peptide and the second linker peptide each independently contain a repeating sequence of ggggs, with the number of repetitions being an integer from 1 to 5.

3. The ligand delivery system according to claim 2, characterized in that, The N-terminal fusion peptide is a TWY targeting peptide that can specifically bind to the hair follicle stem cell surface marker alpha 6-Integrin, and its amino acid sequence is shown in SEQ ID NO:

12. And / or, the C-terminal fusion peptide is a Wnt3a protein carrying a 6×His tag, the amino acid sequence of which is shown in SEQ ID NO:

13.

4. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the ligand delivery system of claim 2 or 3.

5. A recombinant expression vector, characterized in that, It includes the nucleic acid molecule as described in claim 4.

6. An engineered cell, characterized in that, The cell contains the recombinant expression vector of claim 5, and the endogenous CD63 gene in the cell has been knocked out; preferably, the cell is a 293T cell. CD63-KO cell.

7. An engineered exosome, characterized in that, Produced by the engineered cells of claim 6, the engineered exosomes simultaneously display the N-terminal fusion peptide and the C-terminal fusion peptide on their outer membrane.

8. A method for preparing engineered exosomes as described in claim 7, characterized in that, Includes the following steps: (a) The recombinant expression vector of claim 7 is introduced into host cells in which the endogenous CD63 gene has been knocked out to obtain engineered cells; (b) Culture the engineered cells and collect the culture supernatant; (c) Isolate and purify exosomes from the culture supernatant; In step (a), the recombinant expression vector is introduced into the host cell via lentiviral infection or plasmid transfection; In step (c), exosomes are purified by gradient differential centrifugation; The host cell was a 293T cell, and the CD63 gene of the 293T cell was knocked out using CRISPR / Cas9 gene editing technology to obtain the 293T cells. CD63-KO cell.

9. The use of the engineered exosomes of claim 7 in the preparation of a drug that targets hair follicle stem cells and activates the Wnt signaling pathway.

10. A pharmaceutical composition, characterized in that, It includes the engineered exosomes as described in claim 7 and pharmaceutically acceptable carriers.

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