An engineered nanovesicle and a preparation method and application thereof

Abstract

The application discloses an engineered nanovesicle and a preparation method and application thereof. The engineered nanovesicle is an extruded nanovesicle, and a VAMP3 transmembrane scaffold protein is integrated on the membrane of the nanovesicle. The amino acid sequence of the VAMP3 is shown as SEQ ID NO. 1, or a conservative variant thereof which retains the transmembrane scaffold function. The N terminal or C terminal of the VAMP3 protein can be connected to a targeting peptide, a functional protein, a therapeutic polypeptide or a tag protein, such as a cartilage targeting peptide CAP. The preparation method comprises constructing an expression plasmid, transfecting a host cell, and obtaining the vesicle through a physical extrusion method. The engineered nanovesicle provided by the application has good membrane integration, strong targeting, and high biological safety, and can be used for preparing a product of targeted delivery, functional molecule delivery or a drug carrier, and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to an engineered nanovesicle, its preparation method and application, and particularly to an extruded nanovesicle based on VAMP3 transmembrane scaffold protein, its preparation method, and its application in targeted delivery and drug carriers. Background Technology

[0002] Extracellular vesicles (EVs) are nanoscale lipid bilayer vesicles secreted by cells. Due to their natural ability to carry biomolecules such as proteins and nucleic acids and their intercellular communication functions, they are considered ideal candidates for next-generation drug delivery carriers. EVs can be classified into subclasses such as exosomes, microvesicles, and apoptotic bodies based on their origin. Among these, exosomes (30-150 nm) have received the most attention in the field of drug delivery due to their uniform size, low immunogenicity, and ability to cross biological barriers. However, the secretion yield of natural exosomes is extremely low, typically only micrograms of exosomal protein can be harvested per milliliter of cell culture medium. Furthermore, the composition of their contents is greatly affected by the state of the parent cells, making batch-to-batch consistency difficult to guarantee. These yield and quality control bottlenecks severely restrict the translation of exosomes from basic research to clinical applications.

[0003] To overcome the limitations of natural exosome production, researchers have developed various techniques for preparing artificial nanovesicles. Among them, the physical extrusion method is favored due to its simplicity, high yield, and lack of complex purification procedures. This method involves repeatedly extruding cells through polycarbonate membranes of varying pore sizes, using mechanical shear force to break down and reassemble the cell membrane and subcellular organelle membranes into nanoscale vesicles. The resulting products are typically called cell-derived nanovesicles (CDNVs) or extruded nanovesicles (ENVs). Compared to natural exosomes, extruded nanovesicles can increase yield by more than two orders of magnitude, with milligram-level vesicle proteins obtained in a single preparation. More importantly, extruded nanovesicles retain the membrane proteome and lipid composition of the parent cell, providing a basis for their functional engineering.

[0004] In the engineering of vesicles (EVs) and enzymatic vesicles (ENVs), the selection of transmembrane scaffold proteins is one of the core steps. The role of scaffold proteins is to anchor target peptides, functional proteins, or therapeutic peptides to the vesicle membrane surface or vesicle lumen, thereby achieving targeted delivery and functional enhancement of vesicles. Ideal scaffold proteins should possess the following characteristics: small molecular weight to minimize interference with vesicle membrane structure, clear transmembrane topology to facilitate the directional fusion of functional molecules, and efficient integration into the target vesicle type. Currently, the most commonly used scaffold proteins in EV engineering include the tetratransmembrane protein family (CD63, CD9, CD81) and type I transmembrane proteins (Lamp2b).

[0005] The physical fragmentation and recombination processes during the fabrication of extruded nanovesicles lead to rearrangements in membrane topology and the spatial positioning of transmembrane proteins, resulting in significant differences in the integration and localization mechanisms of membrane proteins compared to the natural exosome formation mechanism. Consequently, transmembrane scaffold proteins already established in exosome systems (such as CD63 and Lamp2b) often exhibit unstable membrane localization, poor integration efficiency, and unpredictable function in extruded nanovesicle systems. Therefore, there is an urgent need to develop novel and efficient transmembrane scaffold proteins for constructing engineered nanovesicle platforms capable of stably integrating functional molecules and possessing clear targeting. Summary of the Invention

[0006] To address the technical shortcomings of existing scaffold proteins in extruded nanovesicles, such as unstable membrane localization, poor integration efficiency, and lack of systematic verification of targeting functions, this paper provides an engineered extruded nanovesicle based on VAMP3 transmembrane scaffold protein, along with its preparation method and applications. This scaffold protein exhibits excellent membrane integration efficiency in extruded nanovesicles and can efficiently load targeting peptides or functional proteins, enabling targeted and functional modification of extruded nanovesicles.

[0007] To achieve the above objectives, the present invention provides the following technical solution: First, this invention provides an engineered nanovesicle, which is a squeeze-type nanovesicle with VAMP3 transmembrane scaffold protein integrated on its membrane. VAMP3 (vesicle-associated membrane protein 3, whose amino acid sequence is shown in SEQ ID NO.1) is a member of the SNARE protein family, with 100 amino acids and a molecular weight of approximately 11.3 kDa, belonging to a small C-terminal single-pass transmembrane protein. The C-terminus of VAMP3 contains a transmembrane domain (TMD), which anchors it to the membrane with a topology in which the N-terminus is located on the cytoplasmic side of the vesicle membrane and the C-terminus is located on the luminal side of the vesicle. This topology is well-defined and fixed, providing an ideal structural basis for the targeted fusion of functional molecules (such as targeting peptides, functional proteins, therapeutic peptides, or tag proteins): when fused at the N-terminus, the functional molecule is located on the cytoplasmic side, and when fused at the C-terminus, the functional molecule is located on the luminal side or the vesicle surface.

[0008] Furthermore, the N-terminus or C-terminus of the VAMP3 transmembrane scaffold protein can also be linked to a targeting peptide, functional protein, therapeutic peptide, or tag protein. Even further, this invention fuses the chondrocyte-targeting peptide CAP (a peptide with targeting affinity for chondrocytes) to the N-terminus or C-terminus of VAMP3 to construct a VAMP3-CAP fusion protein. When the C-terminus of this fusion protein expresses the chondrocyte-targeting peptide CAP, the VAMP3-CAP fusion protein can be integrated onto the vesicle membrane.

[0009] Furthermore, the cartilage-targeting peptide CAP is linked to the extracellular domain of VAMP3 via a flexible linker peptide, the amino acid sequence of which is shown in SEQ ID NO.2, and the amino acid sequence of the cartilage-targeting peptide CAP is shown in SEQ ID NO.3.

[0010] After transfecting the expression plasmid encoding VAMP3-CAP into HEK 293T cells, engineered nanovesicles were prepared using a physical extrusion method. Experimental data showed that the integration efficiency of the VAMP3-CAP fusion protein onto the extruded nanovesicle membrane was significantly better than that of corresponding extruded nanovesicles using CD63 or Lamp2b as scaffold proteins. In the uptake experiment targeting chondrocytes (ATDC5 cells), the chondrocyte uptake efficiency of the VAMP3-CAP engineered nanovesicles was also significantly better than that of the CD63-CAP and Lamp2b-CAP controls.

[0011] The VAMP3 scaffold protein platform provided by this invention is not limited to the fusion of CAP cartilage-targeting peptides; it can also link other targeting peptides, functional proteins, therapeutic peptides, or tag proteins to its N-terminus or C-terminus, achieving multifunctional modification of extruded nanovesicles. Simultaneously, the engineered nanovesicles can be loaded with therapeutic molecules, including but not limited to nucleic acid drugs, small molecule drugs, protein drugs, or peptide drugs, forming a nanodelivery platform integrating targeting and therapy. The particle size of these engineered nanovesicles is typically 50-100 nm.

[0012] The present invention also provides a method for preparing the engineered nanovesicles, comprising the following steps: First, constructing an expression plasmid encoding the VAMP3 transmembrane scaffold protein, wherein the nucleotide sequence encoding the VAMP3 is shown in SEQ ID NO.4; then, transfecting the expression plasmid into host cells to express the VAMP3 transmembrane scaffold protein; finally, collecting the expressed cells and preparing nanovesicles by physical extrusion to obtain engineered nanovesicles with the VAMP3 transmembrane scaffold protein integrated on the membrane.

[0013] During preparation, the host cells can be selected from any of the following: HEK293 cells, HEK293T cells, CHO cells, stem cells, immune cells, or tumor cells. The physical extrusion method typically involves sequentially extruding the expressed cells through polyester carbonate membranes with pore sizes of 5 μm, 1 μm, and 0.4 μm.

[0014] The present invention also provides the use of the above-described engineered nanovesicles in the preparation of products for targeted delivery, functional molecule delivery or drug carriers, particularly in cartilage targeted delivery.

[0015] Compared with existing technologies, the engineered nanovesicles provided by this invention have the following beneficial effects: Highly efficient membrane integration: As a transmembrane scaffold protein, VAMP3 can achieve stable and efficient integration on the membrane of extruded nanovesicles, providing a solid foundation for subsequent functionalization modifications.

[0016] Flexible functionalization platform: By fusing the C-terminus or N-terminus of the VAMP3 protein with the expression of target peptides or functional proteins, the targeted functionalization modification of nanovesicles can be achieved, which has a wide range of applications.

[0017] Significant targeting ability: When linked with the cartilage-targeting peptide CAP, this engineered nanovesicle exhibits excellent targeting and cellular uptake efficiency for chondrocytes, which is superior to traditional scaffold proteins CD63 and Lamp2b.

[0018] Excellent biocompatibility: In vivo experiments have verified that the engineered nanovesicles have good biocompatibility and no significant toxicity to major organs. Attached Figure Description

[0019] Figure 1 This study describes the acquisition, sequencing, and candidate protein screening of HEK 293T cells and their extruded nanovesicles. A shows a schematic diagram of the preparation of cartilage-targeted engineered nanovesicles; B shows a transmission electron microscopy (TEM) image of the extruded nanovesicles; C shows the particle size distribution of the extruded nanovesicles; D shows the Western blotting results of exosome marker proteins (ALIX, CD81, CD9, CD63, TSG101) from the extruded nanovesicles; E shows the identification results of membrane proteins extracted from HEK 293T cells and extruded nanovesicles; and F shows a Venn diagram of shared membrane structural proteins between cells and nanovesicles, revealing 577 shared membrane structural proteins.

[0020] Figure 2 This is a list of detailed information for the 28 candidate membrane proteins initially selected.

[0021] Figure 3Representative images of fluorescence signal intensity and transfection efficiency of 1-14 candidate EGFP fusion proteins observed under a fluorescence microscope in HEK 293T cells.

[0022] Figure 4 Representative images of the fluorescence signal intensity and transfection efficiency of 15-28 candidate EGFP fusion proteins observed under a fluorescence microscope in HEK 293T cells.

[0023] Figure 5 The proportion of cells positive for 28 candidate proteins - EGFP was determined by flow cytometry.

[0024] Figure 6 Quantitative results for detecting the fluorescence intensity of 28 candidate proteins-EGFP positive cells by flow cytometry.

[0025] Figure 7 Images of 1-14 candidate proteins finely distributed on the cell membrane and subcellular organelle membranes for super-resolution microscopy.

[0026] Figure 8 To provide images for super-resolution microscopy observation of the fine distribution of 15-28 candidate proteins on the cell membrane and subcellular organelle membranes.

[0027] Figure 9 The results were validated by Western blotting analysis of the expression levels of 28 candidate EGFP fusion proteins and the membrane marker protein A1A1.

[0028] Figure 10 This is a list of seven preferred candidate proteins selected based on a comprehensive evaluation of various indicators (membrane localization, EGFP fluorescence intensity, positive expression efficiency, and molecular weight accuracy of protein bands).

[0029] Figure 11 To obtain co-localization images of EGFP / PKH26 dual-fluorescent nanovesicles for super-resolution microscopy.

[0030] Figure 12 Statistical results for the quantitative detection of the proportion of EGFP-positive vesicles using fluorescent nanoflow cytometry.

[0031] Figure 13 The results of the intersection analysis between the 7 preferred candidate proteins and 213 known conserved exosome proteins in the NCI-60 database are shown. Figure 1 .

[0032] Figure 14 The results of the intersection analysis between the 7 preferred candidate proteins and 213 known conserved exosome proteins in the NCI-60 database are shown. Figure 2 .

[0033] Figure 15 Comparison of VAMP3, CD63, and Lamp2b nanovesicle uptake rates in ATDC5 chondrocytes.

[0034] Figure 16 The structure and validation of VAMP3-CAP nanovesicles are shown in Figure A, which is an agarose gel electrophoresis image and sequencing validation result of the reverse transcription PCR amplification product of the VAMP3-CAP fusion expression plasmid, indicating that the fusion sequence was constructed correctly; Figure B is the expression result of VAMP3-CAP fusion protein in HEK 293T cell membrane detected by Western Blot.

[0035] Figure 17 The structure and validation of VAMP3-CAP nanovesicles are shown in Figure A, which is a transmission electron microscope image of VAMP3-CAP engineered nanovesicles, showing their lipid bilayer membrane structure; and Figure B is a particle size distribution map of VAMP3-CAP engineered nanovesicles, showing their particle size range.

[0036] Figure 18 These are representative images for confocal microscopy observation of VAMP3 and VAMP3-CAP nanovesicle uptake in ATDC5 chondrocytes.

[0037] Figure 19 The results of the cartilage-targeting function verification and biosafety assessment of VAMP3-CAP nanovesicles are shown in Figure A, where flow cytometry is used to quantitatively detect the uptake rate of VAMP3 and VAMP3-CAP nanovesicles in ATDC5 chondrocytes; and C is an image of the aggregation of VAMP3 and VAMP3-CAP nanovesicles in the joint region of mice detected by a small animal in vivo imaging system.

[0038] Figure 20 HE staining results of major organs (heart, liver, spleen, lung, and kidney) of mice 7 days after joint injection (D) showed that there were no significant organic changes in any of the nanovesicle groups compared with the PBS group. Detailed Implementation

[0039] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments, but this does not limit the present invention in any way. Any modifications or improvements made based on the teachings of the present invention shall fall within the protection scope of the present invention.

[0040] The processes, conditions, reagents, and experimental methods used in implementing this invention, except as specifically mentioned below, are all common knowledge and general knowledge in the field, and this invention does not have any particular limitations. Experimental methods in the embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the manufacturer.

[0041] Unless otherwise stated, all technical terms and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. However, in the event of any conflict, the specification containing the definitions shall prevail.

[0042] Example 1: Acquisition, validation, and proteomics detection of HEK 293T cells and their extruded nanovesicle membrane proteins. (1) Acquisition of cell membrane proteins ① Take healthy HEK 293T cells, discard the culture medium in the culture dish, add 3 mL of membrane protein extraction reagent A, scrape the cells off the surface of the culture dish with a pipette tip, and resuspend the cells.

[0043] ② Transfer the cell suspension to a centrifuge tube, centrifuge at 300×g for 5 min, and discard the supernatant; ③ Resuspend the cell pellet in 1 mL of membrane protein extraction reagent A, transfer it to a 1.5 mL centrifuge tube, centrifuge at 300×g for 5 min, and discard the supernatant; ④ Add 0.75 mL of membrane protein extraction reagent B to the cell pellet, gently pipette and agitate several times to obtain a homogeneous cell suspension, place it on a rotary mixer and incubate at 4°C for 10 min. ⑤ Centrifuge the incubated cells for 15 min (16,000×g, 4℃), and carefully aspirate the supernatant containing cytoplasmic proteins. ⑥ Add 200 μL of membrane protein extraction reagent C to the cell pellet, gently pipette and agitate several times to obtain a homogeneous cell suspension, place it on a rotary mixer and incubate at 4°C for 45 min. ⑦ Centrifuge the incubated cells for 15 min (16,000×g, 4℃), and transfer the supernatant containing soluble membrane proteins and membrane-associated proteins to a new centrifuge tube. Place on ice or aliquot and store at -80℃ for later use.

[0044] (2) Acquisition and identification of extruded nanovesicles ① Connect all components of the squeezer and install a 5 μm pore size polyester carbonate membrane. Use a Pasteur pipette to aspirate HEK293T cell suspension (resuspended in 10 ml PBS) and collect the cell suspension passing through the 5 μm polyester carbonate membrane. Repeat this process at least three times; replace the polyester carbonate membrane with a 1 μm pore size and process the collected suspension. Transfer the collected suspension passing through the 5 μm polyester carbonate membrane to the squeezer, and repeat this process at least three times to ensure effective separation; replace the polyester carbonate membrane with a 0.4 μm pore size membrane and separate again. Replace with a 0.4 μm pore size polyester carbonate membrane, and re-absorb the collected liquid into the squeezer, purge with nitrogen gas, and collect the liquid passing through the 0.4 μm membrane. This process should also be repeated at least three times to further purify the suspension; obtain the final vesicle solution by filtration and concentration. Filter the final collected suspension through a 0.22 μm pore size filter membrane to remove larger impurities. Next, the filtered suspension was concentrated using a 30 kDa ultrafiltration tube, and centrifuged at 5000×g for 20 min. The supernatant was then collected. This supernatant is the processed HEK293T cell membrane self-assembled nanovesicle carrier.

[0045] ② A suitable amount of vesicle sample was dropped onto the surface of a carbon film copper mesh. After natural adsorption, excess liquid was removed, and a negative staining treatment was performed. The sample was then observed under a transmission electron microscope. The results showed that the obtained vesicles exhibited a spherical or vesicular structure with clear boundaries, consistent with the typical morphological characteristics of nanovesicles. (See figure) Figure 1 B.

[0046] ③ To detect the particle size distribution of nanovesicles, nanoparticle size analysis was used to measure the sample size. The results showed that the prepared vesicles were mainly distributed in the nanoscale range, and the particle size distribution was relatively concentrated, indicating that the mechanical extrusion method can stably prepare nanoscale vesicles. (See figure) Figure 1 C.

[0047] ④ Western blot analysis of exosome marker proteins (ALIX, CD81, CD9, CD63, TSG101) showed that all nanovesicles expressed exosome marker proteins. (See attached image) Figure 1 D.

[0048] (3) Obtaining vesicle membrane proteins ① Add 3 mL of membrane protein extraction reagent A to the nanovesicle suspension, mix well, transfer to a 30 kDa centrifuge tube, centrifuge at 5000×g for 10 min, discard the lower filtrate, and keep the upper liquid within 300 μL; ② Add 1 mL of membrane protein extraction reagent A to the upper centrifuge column, centrifuge at 5000×g for 10 min, discard the lower filtrate, and keep the upper liquid within 300 μL; ③ Subsequent experimental steps are the same as for the extraction of cell membrane proteins; ④ Add 120 μL of membrane protein extraction reagent C to the precipitate, gently pipette and agitate several times to obtain a homogeneous cell suspension, place it on a rotary mixer and incubate at 4°C for 45 min. ⑤ Centrifuge the incubated suspension for 15 min (16000×g, 4℃), and transfer the supernatant containing soluble membrane proteins and membrane-associated proteins to a new centrifuge tube. Place it on ice or aliquot it and store it at -80℃ for later use.

[0049] (4) Western Blot verification of membrane protein extraction ① Mix the protein and loading buffer directly; ② Routine electrophoresis, membrane transfer, blocking, and incubation with primary antibody (cytoplasmic marker protein: GAPDH; cell membrane marker protein: A1A1) Western blot results showed that both total cell and vesicle proteins expressed GAPDH and A1A1 markers, while cell and vesicle membrane proteins significantly expressed the cell membrane marker protein A1A1 but did not express the cytoplasmic protein GAPDH, indicating successful membrane protein extraction. (See attached results). Figure 1 E in Chinese.

[0050] (5) Proteomics and preliminary screening of membrane proteins ① Protein extraction Samples were removed from -80℃, and each group of samples was added with 4 times the volume of lysis buffer (8 M urea, 1% protease inhibitor), lysed by sonication, and the protein concentration was determined using a BCA kit.

[0051] ② Pancreatic enzyme digestion Equal volumes of protein from each sample were digested enzymatically. The volumes were adjusted to be uniform using lysis buffer, and then dithiothreitol (DTT) was added to a final concentration of 5 mM. Reduction was performed at 56°C for 30 min. Iodoacetamide (IAA) was then added to a final concentration of 11 mM, and incubation was carried out at room temperature in the dark for 15 min. Urea was diluted with TEAB to ensure a concentration below 2 M. Trypsin was added at a ratio of 1:50 (protease:protein, m / m), and digestion was carried out overnight. Trypsin was then added again at a ratio of 1:100 (protease:protein, m / m), and digestion continued for 4 h.

[0052] ③ Liquid chromatography-mass spectrometry analysis Peptides were dissolved in mobile phase A of liquid chromatography and then separated using a Vanquish neo ultra-high performance liquid chromatography (UHPLC) system. Mobile phase A was an aqueous solution containing 0.1% formic acid and 2% acetonitrile; mobile phase B was an aqueous solution containing 0.1% formic acid and 90% acetonitrile. The liquid phase gradient settings were: 0–22.5 min, 6%–22% B; 22.5–26.5 min, 22%–34% B; 26.5–28.5 min, 34%–80% B; 28.5–30 min, 80% B, with a flow rate maintained at 700 nL / min. After separation by the UHPLC system, the peptides were injected into an NSI ion source for ionization and then analyzed by an Orbitrap Exploris 480 mass spectrometer. The ion source voltage was set to 2300 V, and the FAIMS compensation voltage (CV) was set to -45 V. The precursor ion and its secondary fragments were detected and analyzed using a high-resolution Orbitrap mass spectrometer. The primary mass spectrometry (PMS) scan range was set to 350–1400 m / z, with a scan resolution of 60,000. The secondary mass spectrometry (SMS) scan range had a fixed starting point of 120 m / z, with a secondary scan resolution of 15,000. Data acquisition was performed using a data-independent scanning (DIA) procedure, where peptide ions from multiple consecutive m / z windows were followed by fragmentation in the HCD collision cell after the primary scan, using 27% of the fragmentation energy, and then analyzed sequentially by secondary mass spectrometry. To improve the efficiency of the mass spectrometer, automatic gain control (AGC) was set to 1E6, and the maximum injection time was set to 22 ms.

[0053] ④ Proteins commonly found in the cell and nanovesicle membrane structures were selected and Venn diagrams were plotted. The results showed that a total of 577 proteins shared membrane structures in the cell and nanovesicles were identified. (See figure) Figure 1 Middle F.

[0054] ⑤ Analysis was performed on the above 577 membrane structural proteins. Proteins with fewer than 150 amino acids, single-channel transmembrane proteins or anchoring proteins, and no significant biological activity reported in the literature were selected. A preliminary screening identified 28 membrane proteins. For details, please refer to [link to relevant information]. Figure 2 .

[0055] Example 2: EGFP fusion expression and membrane localization verification of 28 candidate proteins in HEK 293T cells ① Plasmid construction Twenty-eight candidate scaffold protein coding sequences were constructed into the pcDNA3.4 eukaryotic expression vector and fused with EGFP to form recombinant expression plasmids. These plasmids were then introduced into HEK 293T cells via transient transfection for expression. EGFP fluorescent markers were fused to the C-terminus of the proteins to detect protein localization within cells and on vesicle membranes. The plasmid sequences were sequenced to confirm the absence of mutations, ensuring complete protein expression.

[0056] ② Cell culture and transfection HEK 293T cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin, and kept in logarithmic growth phase at 37°C and 5% CO2. Cells were seeded into well plates and transfected when they reached approximately 80% confluence. The old medium was discarded, the cells were washed with PBS, and 900 μL of basal medium was added. After culturing at 37°C for 30 min, DNA / transfection reagent complex was added. After 24 h, the medium was replaced with complete medium without antibiotics. Samples were collected after another 24 h.

[0057] DNA / transfection reagent complex preparation: DNA reagent (add 3 μg DNA to 50 μL of basal medium and incubate at room temperature for 5 min), transfection reagent (add 15 μL of polyfast Transfection Reagent (MCE, HY-K1014) to 50 μL of basal medium and incubate at room temperature for 5 min), mix DNA reagent and transfection reagent 1:1, incubate at room temperature for 15 min, and then transfect the plasmid into HEK 293T cells.

[0058] ③ Observe the fluorescence signal intensity and transfection efficiency of EGFP in cells using a fluorescence microscope, see [link to relevant documentation]. Figure 3-4 Collect transfected cells, digest them appropriately into a single-cell suspension, wash them, and resuspend them in PBS. Flow cytometry was used to detect EGFP fluorescence intensity and the proportion of positive cells. See [link to relevant documentation]. Figure 5-6 Candidate proteins with good membrane localization and stable expression were screened based on fluorescence positivity rate and fluorescence intensity.

[0059] ④ Super-resolution microscopy: Enables high-resolution observation of the fine distribution of proteins on the membrane, confirming whether proteins are integrated into the membrane structure. (See [link to article]). Figure 7-8 Collect transfected cells and extract total or membrane proteins; separate proteins by SDS-PAGE and transfer them to a PVDF membrane; use anti-EGFP antibody to confirm the correct expression level and size of candidate proteins; use membrane marker protein A1A1 to verify the reliability of protein expression and membrane localization (see [link to relevant documentation]). Figure 9 .

[0060] Based on the above experimental results, and considering the following criteria: localization to the membrane (cell membrane or subcellular organelle membrane); strong and uniform EGFP signal with no obvious aggregation or nonspecific distribution; high positive expression efficiency shown by flow cytometry; and protein bands consistent with predicted molecular weights and no degradation band characteristics shown by Western blotting, seven proteins were selected as candidate proteins for extruded nanovesicles (DDOST, APMAP, VAMP3, STX7, SGCB, ST3GAL4, GINM1). Figure 10 .

[0061] Example 3: Verification of Nanovesicle Membrane Integration and Fluorescence Expression ① Obtaining candidate scaffold protein overexpression + EGFP nanovesicles The target protein coding sequence was constructed into the pcDNA3.4 eukaryotic expression vector and expressed in HEK 293T cells via transient transfection. After culturing for 48 hours, the cells were collected to prepare engineered cells for subsequent extrusion nanovesicle construction (preparation method is the same as in Example 1).

[0062] ② The engineered nanovesicles obtained were labeled with PKH26 dye. The specific operation steps are as follows: 50 μL of NVs / Exos was added to 1 mL of Diluent C, mixed well, and then 4 μL of PKH26 dye was added. After mixing well, the mixture was allowed to stand at room temperature for 5 min. The mixture was then added to a 30 kDa ultrafiltration tube and 10 mL of 1% BSA was added to bind excess dye. The mixture was centrifuged at 4℃, 5000×g for 20 min. The upper layer of successfully labeled PKH26 engineered nanovesicles was collected to obtain EGFP / PKH26 dual-fluorescent nanovesicles. Subsequently, the dual-fluorescent nanovesicles were spread on a glass slide, and the co-localization of EGFP and PKH26 signals was observed using a super-resolution microscope to evaluate the degree of integration and uniformity of the scaffold protein on the vesicle membrane. See [link to relevant documentation]. Figure 11 Simultaneously, dual-fluorescent nanovesicles were diluted to a concentration suitable for flow cytometry analysis, and the proportion of EGFP-positive vesicles was quantitatively detected using a fluorescent nanoflow cytometer to evaluate protein membrane integration and stability. (See [link to relevant documentation]). Figure 12 The experimental results, combining spatial distribution information from super-resolution microscopy with quantitative data from nanoflow cytometry, provide a basis for screening well-integrated and stable scaffold proteins.

[0063] Example 4: Intersection screening of candidate proteins and conserved exosome proteins and VAMP3 optimization First, the seven preferred candidate proteins selected in the previous step were compiled into a list, and a list of 213 known conserved exosomal proteins was obtained from the National Cancer Institute (NCI-60) database. Then, an intersection analysis was performed between the seven candidate proteins and the 213 conserved exosomal proteins to screen for proteins that are present in both extruded nanovesicles and conservedly expressed in exosomes. Statistical analysis revealed that among the intersection proteins, VAMP3 is a conserved and highly expressed protein that is expressed in both extruded nanovesicle membranes and exosomes. Therefore, VAMP3 was selected as the core scaffold protein for subsequent functionalization and targeting validation. (See...) Figure 13-14 This invention utilizes an intersection screening strategy to systematically identify preferred scaffold proteins that exhibit cross-platform conservation and are suitable for constructing engineered nanovesicles, providing a reliable foundation for subsequent functionalization experiments.

[0064] Example 5: Comparison of VAMP3, CD63, and Lamp2b nanovesicle uptake rates in ATDC5 chondrocytes The plasmids of pcDNA3.4 overexpressing VAMP3, CD63, and Lamp2b were transiently transfected into HEK 293T cells, and corresponding engineered nanovesicles were prepared (preparation method as in Example 1). The nanovesicle membranes were labeled with PKH67 green fluorescent dye, as described above for PKH26-labeled nanovesicles. Two control groups were set up: a blank control group (BC) without any nanovesicles to exclude interference from cell autofluorescence; and a negative control group (NC) transfected with empty vector pcDNA3.4 and nanovesicles were prepared using the same method to assess the background of nonspecific uptake.

[0065] Meanwhile, ATDC5 cells were subjected to chondrogenic culture to achieve a stable chondrogenic differentiation state. The cell density was then adjusted and seeded into culture plates to achieve a suitable co-incubation state. Subsequently, equal amounts of VAMP3, CD63, Lamp2b-modified PKH67-labeled nanovesicles and NC group nanovesicles were added to the ATDC5 cell culture system (BC group was added with an equal volume of PBS). The cells were co-incubated at 37°C and 5% CO2 for 1 h to allow the nanovesicles to fully contact the cells and undergo uptake.

[0066] After co-incubation, the culture medium was discarded, and the cells were thoroughly washed with PBS buffer to remove untaken or unbound nanovesicles. The cells were then gently digested with trypsin to prepare a single-cell suspension, which was resuspended in PBS buffer. The proportion and fluorescence intensity of PKH67-positive cells were detected by flow cytometry to reflect the uptake efficiency of nanovesicles in ATDC5 cells. The BC group was used to set the negative gating for flow cytometry, and the NC group was used to correct for non-specific binding background. The differences in the uptake capacity of nanovesicles modified with different scaffold proteins in chondrocytes were evaluated by comparing the PKH67 positivity rates of each experimental group (VAMP3, CD63, Lamp2b) with the NC group. The results showed that VAMP3-modified nanovesicles had higher cellular uptake efficiency and targeting potential. Figure 15 .

[0067] Example 6: Structure and Validation of VAMP3-CAP Nanovesicles (1) To further verify the feasibility of transmembrane scaffold proteins carrying functional peptides, a flexible linker peptide was introduced into the extracellular domain of VAMP3 and linked to the cartilage-targeting peptide CAP to construct a VAMP3-CAP fusion expression system. The system was transfected into HEK 293T cells using pcDNA3.4 as a vector, and its expression and sequence correctness were verified. At the same time, a negative control group (NC) was set up, which was transfected with the empty vector pcDNA3.4 and used as a background control in subsequent Western blot and nanovesicle preparation.

[0068] The nucleotide sequence encoding VAMP3 is shown in SEQ ID NO.4, the nucleotide sequence encoding the flexible linker peptide is shown in SEQ ID NO.5, the nucleotide sequence encoding CAP (cartilage affinity peptide) is shown in SEQ ID NO.6, and the nucleotide sequence of the VAMP3-CAP fusion plasmid is shown in SEQ ID NO.7.

[0069] (2) Simultaneously, cellular RNA was extracted and reverse transcription PCR amplification was performed. The amplification products were detected by agarose gel electrophoresis and sequenced for verification. The results confirmed that the constructed sequence was correct. Figure 16 (A). The specific operating steps are as follows: ① RNA was extracted from HEK 293T cells transfected for 48 h using an RNA extraction kit (Aikerui Biotechnology, AG21024); ② The reverse transcription reaction was performed according to the PrimeScript™ RT Master Mix (Perfect Real Time) (TAKARA, RR036A) instructions; ③ PCR Cloning: Based on the above expression cassette, design upstream and downstream primers, and synthesize upstream primer (SEQ ID NO.5: gctgtagggccggtgctc), downstream primer (SEQ ID NO.6: gcgactggcgggtgatca), and template DNA (the reversed DNA from above). In a 0.2 mL EP tube, add 45 μL of DNA polymerase (1.1×T3 Super PCR Mix / PAGE dedicated PCR, TSE030), 2 μL of upstream primer, 2 μL of downstream primer, and 1 μL of template DNA. Add enzyme-free water to a final volume of 50 μL, mix well, and place in a PCR instrument. Design the amplification program according to the primer annealing temperature. ④ The PCR products and primers were sent to Shanghai Sangon Biotech Co., Ltd. for sequence detection.

[0070] (3) After transfecting HEK 293T cells with the plasmid according to the method in Example 2, the cell membrane protein was extracted and Western blot was performed to detect the expression of VAMP3 and the position of the protein band. The results showed that the target protein was successfully expressed. Figure 16 (B) Membrane protein extraction was performed according to the instructions. (4) Engineered nanovesicles were prepared according to the method in Example 1 (see schematic diagram of preparation). Figure 1 (A) The extracted nanovesicles were subjected to transmission electron microscopy and particle size analysis, and all showed a lipid bilayer membrane structure and a particle size range of 50-100 nm. Figure 17 (AB).

[0071] Example 7: Validation of VAMP3-CAP nanovesicle cartilage-targeting function and assessment of biosafety. (1) In vitro uptake experiment The engineered nanovesicles (empty carrier nanovesicles, VAMP3 nanovesicles, and VAMP3-CAP nanovesicles) prepared in Examples 5 and 6 were labeled with PKH67 dye. After incubation at a concentration of 10 μg / mL with ATDC5 cells induced to become chondrocytes for 30 min, the number of nanovesicles taken up by chondrocytes at different time points was quantitatively analyzed using flow cytometry, and the differences in uptake among the blank group, blank transfection group, and scaffold protein group were compared. The results are as follows: Figure 18 and Figure 19 As shown in Figure A, flow cytometry yielded similar results to confocal microscopy, demonstrating that the engineered nanovesicles of the scaffold protein VAMP3-CAP group exhibited stronger cartilage targeting.

[0072] (2) In vivo targeted experiments To better observe the distribution of engineered nanovesicles using in vivo small animal imaging, 30 μg of engineered nanovesicles were labeled with far-infrared fluorescent-DID dye. Detailed labeling procedures are described in the instruction manual. The labeled engineered nanovesicles were injected into the joint region of C57BL / 6 mice, and the aggregation of nanovesicles in the cartilage region was observed using a small animal in vivo imaging system on days 3 and 7. The targeting ability of each group of engineered nanovesicles was evaluated based on the imaging results. Results are as follows: Figure 19 As shown in Figure B, the imaging results indicate that the engineered nanovesicles injected into the joint showed fluorescence signals only in the joint region. The engineered nanovesicles in the VAMP3-CAP scaffold protein group showed stronger fluorescence signals in the mouse cartilage region, and the fluorescence signal of the engineered nanovesicles in the VAMP3-CAP scaffold protein group remained significant at 7 days.

[0073] (3) Biosafety assessment After dehydrating the main organs (heart, liver, spleen, lung, and kidney) for in vivo imaging using a sucrose concentration gradient, they were embedded in paraffin, sectioned, and stained according to the following steps: distilled water for 2 min; hematoxylin staining for 10 min; distilled water for 2 min; hydrochloric acid-alcohol differentiation for 3 sec; warm water bluening for 2 min; 70% ethanol for 2 min; eosin staining for 25 sec; 80% ethanol for 2 min; 95% ethanol for 2 min; anhydrous ethanol I for 2 min; anhydrous ethanol II for 2 min; air-dried, mounted, and scanned. Results are as follows: Figure 20 As shown, the safety of each group of engineered nanovesicles in vivo was tested. The major organs (heart, lung, liver, spleen, and kidney) of C57BL / 6 mice 7 days after joint injection were stained with HE. The results showed that compared with the PBS group, there were no significant organic changes in the major organs of each engineered nanovesicle group.

[0074] 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 of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. An engineered nanovesicle, characterized in that, The nanovesicles are extruded nanovesicles with VAMP3 transmembrane scaffold protein integrated on their membranes. The amino acid sequence of VAMP3 is shown in SEQ ID NO.1, or a conserved variant thereof that retains the transmembrane scaffold function.

2. The engineered nanovesicles according to claim 1, characterized in that, The VAMP3 transmembrane scaffold protein has a targeting peptide, functional protein, therapeutic peptide, or tag protein attached to its N-terminus or C-terminus.

3. The engineered nanovesicles according to claim 2, characterized in that, The targeting peptide is the cartilage-targeting peptide CAP, and the C-terminus of VAMP3 is fused with CAP to form the VAMP3-CAP fusion protein.

4. The engineered nanovesicles according to claim 3, characterized in that, The cartilage-targeting peptide CAP is linked to the extracellular domain of VAMP3 via a flexible linker peptide. The amino acid sequence of the flexible linker peptide is shown in SEQ ID NO.2, and the amino acid sequence of the cartilage-targeting peptide CAP is shown in SEQ ID NO.

3.

5. The engineered nanovesicles according to claim 1, characterized in that, The nanovesicles have a particle size of 50-100 nm and are loaded with therapeutic molecules inside or on their membranes. The therapeutic molecules are selected from one or more of nucleic acid drugs, small molecule drugs, protein drugs, or peptide drugs.

6. A method for preparing the engineered nanovesicles according to any one of claims 1 to 5, characterized in that, Includes the following steps: (1) Construct an expression plasmid encoding the VAMP3 transmembrane scaffold protein, the nucleotide sequence encoding the VAMP3 being shown in SEQ ID NO.4; (2) Transfect the expression plasmid into host cells to express the VAMP3 transmembrane scaffold protein; (3) Collect the expressed cells and prepare nanovesicles by physical extrusion to obtain engineered nanovesicles with VAMP3 transmembrane scaffold protein integrated on the membrane.

7. The preparation method according to claim 6, characterized in that, The VAMP3 transmembrane scaffold protein described in step (1) is also fused to the N-terminus or C-terminus with a targeting peptide, functional protein or tag protein.

8. The preparation method according to claim 6, characterized in that, The host cell mentioned in step (2) is selected from any one of HEK293 cells, HEK293T cells, CHO cells, stem cells, immune cells or tumor cells; The physical compression method described in step (3) involves sequentially compressing the expressed cells with carbonated polyester membranes with pore sizes of 5 μm, 1 μm, and 0.4 μm.

9. The use of the engineered nanovesicles according to any one of claims 1 to 5 in the preparation of products for targeted delivery, functional molecule delivery or drug carriers.

10. The application according to claim 9, characterized in that, The targeted delivery is cartilage-targeted delivery.

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