A stable GE11m peptide modified extracellular vesicle anticancer targeting nano delivery system, a preparation method and applications thereof

Abstract

The application discloses a stable GE11m peptide modified extracellular vesicle anticancer targeting nano delivery system, a preparation method and application thereof, and belongs to the technical field of biological medicine preparations. D HWY D GYT D PQNVI, by replacing the first tyrosine, the fourth tyrosine and the seventh threonine in the GE11m peptide sequence with D-type amino acids, the protease resistance and serum stability of the targeting peptide are significantly enhanced.

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceutical formulation technology, specifically relating to a stable GE11m peptide-modified extracellular vesicle targeted nanodelivery system, its preparation method, and its application in the preparation of antitumor drug delivery formulations. Background Technology

[0002] Cancer is one of the major diseases that seriously threaten human health. Traditional chemotherapy drugs, due to their lack of targeting, often cause serious toxic side effects on normal tissues while killing tumor cells. In recent years, targeted drug delivery systems based on nanotechnology have become a research hotspot in the field of cancer treatment. Extracellular vesicles (EVs), as a type of natural nanoscale membrane vesicles secreted by cells, typically have a particle size of 30-150 nm. They have good biocompatibility, low immunogenicity, and natural ability to cross biological barriers, and are considered ideal drug delivery carriers (Weng Z et al., Therapeutic roles of mesenchymal stem cell-derived extracellular vesicles in cancer, Journal of hematology & oncology, 2021, Vol. 14, p. 136).

[0003] Extracellular vesicles derived from mesenchymal stem cells have become an important research subject for targeted cancer therapy due to their ease of acquisition, large-scale expansion and culture, and certain tumor tropism. However, unmodified natural extracellular vesicles lack active targeting capabilities and are easily cleared by the reticuloendothelial system in vivo, resulting in low drug accumulation efficiency at tumor sites. Therefore, surface engineering modification of extracellular vesicles to endow them with active targeting functions has become a key strategy to improve the therapeutic efficacy of extracellular vesicle drug delivery systems (Salunkhe S et al., Surface functionalization of exosomes for target-specific delivery and in vivo imaging and tracking: Strategies and significance, Journal of Controlled Release, 2020, Vol. 326, pp. 599-614).

[0004] Epidermal growth factor receptor (EGFR) is a transmembrane tyrosine kinase receptor that is highly overexpressed on the surface of tumor cells in various solid tumors, such as breast cancer, lung cancer, and colorectal cancer. GE11m is a dodecapeptide (sequence YHWYGYTPQNVI) obtained through phage display technology. It can specifically bind to the EGFR receptor and, compared with the natural ligand EGF, does not activate downstream signaling pathways, making it a safe and effective EGFR-targeting ligand. Studies have shown that GE11m-modified nanodelivery systems can significantly improve tumor-targeted delivery efficiency and show promising application prospects in the treatment of EGFR-highly expressed tumors such as lung cancer (Gaurav I et al., Peptide-Conjugated Vascular Endothelial Extracellular Vehicles Encapsulating Vinorelbine for Lung Cancer Targeted Therapeutics, Nanomaterials, 2024, Vol. 14, No. 20, p. 1669).

[0005] However, the in vivo application of natural L-type GE11m peptide faces significant challenges. GE11m peptide is readily degraded by various proteases in vivo, resulting in poor serum stability and a short half-life. This inherent defect severely restricts the clinical translational application prospects of GE11m peptide-modified nanodelivery systems. While some studies have attempted to improve peptide stability through chemical modification or PEGylation, these methods often affect the binding affinity between the peptide and its target, or significantly increase molecular weight and immunogenicity. Therefore, there is an urgent need to develop an innovative modification strategy that can significantly improve the stability of GE11m peptide while maintaining its EGFR-targeting activity.

[0006] D-amino acids are chiral isomers of L-amino acids, found in certain microorganisms and specific biomolecules in nature. Replacing L-amino acids at specific sites in a polypeptide chain with D-amino acids is a classic strategy to improve peptide protease resistance. Because their chiral conformation is opposite to that of their natural substrates, D-amino acids are less readily recognized and cleaved by proteases, thus significantly prolonging the in vivo half-life of the polypeptide. However, D-amino acid substitution can affect the three-dimensional conformation of the polypeptide, thereby influencing its target-binding activity. Therefore, achieving the optimal balance between stability and activity through rational D-amino acid substitution design while maintaining the polypeptide's targeting activity remains a key technical challenge in this field.

[0007] Extracellular vesicle surface modification methods mainly fall into two categories: genetic engineering methods and non-genetic engineering methods. Among non-genetic engineering methods, using extracellular vesicle membrane anchoring peptides for surface modification is a simple and efficient strategy. CP05 is a short peptide (sequence CRHSQMTVTSRL) that specifically binds to the tetraspanic membrane protein CD63 on the surface of extracellular vesicles. By fusing a targeting ligand with CP05, rapid surface modification of extracellular vesicles can be achieved under mild conditions without affecting the morphology, structure, or membrane integrity of the extracellular vesicles.

[0008] Tumor necrosis factor-associated apoptosis-inducing ligand (TRAIL) is a cytokine that selectively induces apoptosis in tumor cells without significant toxicity to normal cells. TRAIL activates the extrinsic apoptosis pathway by binding to the death receptors DR4 and DR5 on the surface of tumor cells. However, some tumor cells exhibit primary or acquired resistance to TRAIL-induced apoptosis. Studies have shown that aberrant activation of the PI3K / Akt / mTOR signaling pathway is one of the important mechanisms of TRAIL resistance in tumor cells, and inhibiting this pathway can enhance the sensitivity of tumor cells to TRAIL.

[0009] Existing extracellular vesicle drug delivery systems suffer from the following main problems: first, the targeting peptides exhibit poor stability in vivo, with rapid degradation leading to decreased targeting efficiency; second, they lack active targeting capabilities, resulting in low enrichment efficiency at tumor sites; and third, single-drug therapy has limited efficacy and struggles to overcome tumor drug resistance. Therefore, there is an urgent need to develop a novel extracellular vesicle targeted nanodelivery system with highly stable targeting peptides, efficient EGFR targeting capabilities, and the ability to achieve synergistic multi-drug delivery. Summary of the Invention

[0010] To address the aforementioned problems in existing technologies, the present invention aims to provide a stable GE11m peptide-modified extracellular vesicle-targeting nanodelivery system, its preparation method, and its anticancer applications. This invention innovatively employs a D-amino acid modification strategy, replacing the 1st, 4th, and 7th threonine positions in the GE11m peptide sequence with D-amino acids, resulting in a GE11m peptide with significantly enhanced stability. By constructing a GE11m-CP05 fusion peptide, GE11m targeting peptide modification is achieved on the surface of extracellular vesicles, resulting in a highly stable EGFR-targeting nanodelivery system, GE11m@EV. The serum stability of the GE11m targeting peptide in this nanodelivery system is significantly improved compared to the natural L-type GE11m peptide, enabling it to specifically recognize and bind to tumor cells with high EGFR expression. This nanodelivery system can serve as a universal tumor-targeted drug delivery platform, enabling precise tumor treatment by loading various anti-tumor active substances. In particular, when extracellular vesicles expressing TRAIL protein (EV-T) are used as carriers and loaded with TRAIL-sensitizing drugs, a synergistic anti-cancer effect between TRAIL and the sensitizing drugs can be achieved.

[0011] To achieve the above objectives, the present invention adopts the following technical solution: The first aspect of this invention provides a stable GE11m peptide-modified extracellular vesicle targeted nanodelivery system, the nanodelivery system comprising extracellular vesicles derived from mammalian cells that can be genetically engineered using TRAIL, wherein the surface of the extracellular vesicles is linked to a GE11m targeting peptide via the binding of a CP05 anchoring peptide to a CD63 protein; the GE11m targeting peptide is a stable derivative of the GE11m peptide obtained by replacing D-type amino acids, and its amino acid sequence is Y. D HWY D GYT D PQNVI, wherein the 1st, 4th, and 7th tyrosine residues are D-type amino acids, and the remaining amino acids are L-type amino acids; the amino acid sequence of the CP05 anchoring peptide is CRHSQMTVTSRL; the particle size of the nanodelivery system is 70-150 nm, the binding efficiency of the GE11m targeting peptide is not less than 90%, and it can be genetically engineered by TRAIL to be transduced by TRAIL genes, or it can be transfected by TRAIL genetically engineered viruses.

[0012] Furthermore, the GE11m targeting peptide and the CP05 anchoring peptide are linked by peptide bonds to form a GE11m-CP05 fusion peptide, the amino acid sequence of which is Y. D HWY D GYT D PQNVIPQNVI-CRHSQMTVTSRL, a total of 24 amino acid residues. The purity of the fusion peptide is not less than 95%.

[0013] Furthermore, the HPLC main peak area of ​​the GE11m targeting peptide after incubation at 37°C for 1 hour in mouse serum is not less than 90%, which is significantly improved compared with the natural L-type GE11m peptide.

[0014] Furthermore, the extracellular vesicle is an extracellular vesicle EV-T expressing the TRAIL protein, the EV-T is derived from mesenchymal stem cells transfected with the TRAIL gene, and the membrane surface of the EV-T carries the biologically active TRAIL protein.

[0015] Furthermore, the EV-T contains a TRAIL sensitizing drug, which is an active substance that can enhance the sensitivity of tumor cells to TRAIL-induced apoptosis.

[0016] Furthermore, the TRAIL sensitizing drug is an inhibitor of the PI3K / Akt / mTOR signaling pathway.

[0017] Furthermore, the mammalian cells that can be genetically engineered by TRAIL are mesenchymal stem cells, specifically umbilical cord mesenchymal stem cells, bone marrow mesenchymal stem cells, or adipose-derived mesenchymal stem cells, and are generation 3-5 cells. The extracellular vesicles express the exosome-specific positive markers TSG101 and CD63.

[0018] Furthermore, the nanodelivery system exhibits a typical saucer-shaped or cup-shaped vesicle morphology with an intact membrane structure.

[0019] A second aspect of the present invention provides a method for preparing the above-mentioned stable GE11m peptide-modified extracellular vesicle targeted nanodelivery system, comprising the following steps: Step 1: Isolation and extraction of extracellular vesicles: Mesenchymal stem cells were isolated and extracted at a concentration of 5 × 10⁻⁶ cells / year. 5 Cells were seeded at high density in T75 culture flasks. When the cell confluence reached 80%, the medium was replaced with serum-free medium and cultured for another 24-48 hours. The conditioned medium was then collected. The conditioned medium was then subjected to low-speed centrifugation to remove cell debris, filtration through a 0.22 μm filter membrane to remove large vesicles, concentration through a 100 kDa ultrafiltration tube, and ultracentrifugation at 120,000 × g for 2 hours to precipitate extracellular vesicles (EVs). Step 2: Synthesis of GE11m-CP05 fusion peptide: The GE11m-CP05 fusion peptide was synthesized using the Fmoc / t-Bu solid-phase peptide synthesis strategy. D-type Fmoc-amino acid precursors were used to introduce D-type amino acids at the 1st (D-Tyr), 4th (D-Tyr), and 7th (D-Thr) positions. Rink Amide resin was used to obtain the C-terminal amidated fusion peptide. The amino acid sequence of the fusion peptide is Y. D HWY D GYT D PQNVIPQNVI-CRHSQMTVTSRL; after synthesis, it was purified by reversed-phase high-performance liquid chromatography with a purity of not less than 95%; the molecular weight was confirmed to be correct by mass spectrometry. Step 3: GE11m modification of extracellular vesicle surface: The EV prepared in Step 1 and the GE11m-CP05 fusion peptide synthesized in Step 2 were mixed at a mass ratio of 0.5-1.1:0.5-1.1, and PBS was added to adjust the final volume. The mixture was incubated in a shaker at 4°C for 4-6 h to allow the fusion peptide to bind to CD63 protein on the surface of extracellular vesicles. After incubation, the unbound free peptides were removed by centrifugation at 7500×g for 45 min at 4°C using a 100 kDa ultrafiltration tube. The precipitate was collected and resuspended in PBS to obtain GE11m-modified extracellular vesicles GE11m@EV. Step 4, Quality Characterization and Detection: Observe the morphology of extracellular vesicles using transmission electron microscopy, detect particle size distribution using a nanoparticle tracking analyzer, detect extracellular vesicle markers using Western blotting, and detect GE11m binding efficiency using nanoflow cytometry.

[0020] Furthermore, the mesenchymal stem cells mentioned in step one are mesenchymal stem cells transfected with the TRAIL gene, and the extracellular vesicles are EV-T cells expressing the TRAIL protein; the product obtained in step three is GE11m@EV-T.

[0021] Furthermore, the procedure includes step five, loading the TRAIL sensitizing drug: the GE11m@EV-T prepared in step three is mixed with the TRAIL sensitizing drug, and the TRAIL sensitizing drug is loaded into the EV-T vesicle cavity using an ultrasonic method; the conditions for the ultrasonic method are: ultrasonic power 20-30%, temperature 20-30℃, ultrasonic cycles 4-8 times, each ultrasonic cycle 30-40s, with an interval of 30-40s; after ultrasonication, the vesicles are incubated at 37℃ for 0.5-1.5h to repair the extracellular vesicle membrane structure, and the free drug is removed by ultracentrifugation to obtain the drug-loaded GE11m@EV-T.

[0022] Further, the low-speed centrifugation in step one includes sequential centrifugation at 300×g for 10 min, 2000×g for 15 min, and 10000×g for 30 min to remove cell debris and large vesicles. The culture conditions for the mesenchymal stem cells are as follows: using DMEM / F-12 complete medium containing 10% fetal bovine serum, and culturing in a 37°C, 5% CO2 incubator.

[0023] Furthermore, in step three, the mixing ratio of EV and GE11m-CP05 fusion peptide is 10 μg extracellular vesicles to 10 μg fusion peptide, and the final volume is adjusted to 300 μL with PBS.

[0024] A third aspect of the present invention provides the application of the above-described stable GE11m peptide-modified extracellular vesicle targeted nanodelivery system in the preparation of drug delivery formulations targeting tumors with high EGFR expression.

[0025] Furthermore, the tumor is a solid tumor that highly expresses EGFR, including but not limited to breast cancer, lung cancer, colorectal cancer, or pancreatic cancer.

[0026] Furthermore, the drug delivery formulation specifically recognizes and binds to the EGFR receptor highly expressed on the surface of tumor cells via the GE11m targeting peptide, mediating the endocytosis and uptake of the nanodelivery system by tumor cells, thereby achieving targeted delivery of antitumor active substances.

[0027] The technical solution of the present invention has the following beneficial effects: First, this invention innovatively employs a D-amino acid modification strategy to design the GE11m peptide. By replacing the 1st, 4th, and 7th threonine positions in the GE11m peptide sequence with D-amino acids, the protease resistance and serum stability of the targeted peptide are significantly enhanced. HPLC results show that the GE11m peptide, after incubation at 37°C for 1 hour in mouse serum, has a main peak area accounting for as high as 95.4%, while the natural L-type GE11m peptide only accounts for 45.1%, demonstrating that D-amino acid modification significantly improves the serum stability of the peptide.

[0028] Secondly, this invention, through the rational design of D-type amino acid substitution sites, aims to significantly improve the stability of the GE11m peptide while preserving its EGFR-targeting binding activity. The design of the D-type amino acid modification sites (positions 1, 4, and 7) avoids key residue regions that directly bind to EGFR, thus achieving an optimal balance between stability and activity.

[0029] Third, the GE11m@EV nanodelivery system constructed in this invention employs a CP05-anchored peptide-mediated surface modification strategy, enabling efficient and stable modification of GE11m-targeting peptides on the surface of extracellular vesicles. Referring to the binding experimental results of natural GE11m peptides with extracellular vesicles, the binding efficiency of the GE11m-CP05 fusion peptide to extracellular vesicles can reach over 93.5%, and the GE11m-CP05 fusion peptide is expected to achieve a comparable modification efficiency.

[0030] Fourth, the GE11m@EV nanodelivery system constructed in this invention is a universal tumor-targeted drug delivery platform that can achieve precise treatment of various tumors by loading different types of anti-tumor active substances. This platform is compatible with various drug types, including small molecule chemotherapy drugs, protein drugs, and nucleic acid drugs.

[0031] Fifth, this invention provides a preferred embodiment in which an EV-T expressing the TRAIL protein is used as a carrier and simultaneously loaded with a TRAIL-sensitizing drug. When an EV-T is used as a carrier and loaded with inhibitors of the PI3K / Akt / mTOR signaling pathway such as SD9, the TRAIL protein on the membrane surface and the sensitizing drug in the cyst cavity can synergistically exert an anti-tumor effect.

[0032] Sixth, the preparation process of this invention is simple and controllable, and easy to scale up for production. The separation and extraction of extracellular vesicles adopts a mature ultracentrifugation method, the GE11m-CP05 fusion peptide adopts a standard Fmoc solid-phase synthesis strategy, and the surface modification adopts a mild incubation method. The parameters of each step are clear and reproducible, which is conducive to product quality control and large-scale production. Attached Figure Description

[0033] Figure 1 This is a schematic diagram of the preparation process of the GE11m@EV-T-SD9 composite nanoformulation of the present invention.

[0034] Figure 2 This is a diagram of the morphology of extracellular vesicles in EV-T cells as observed under a transmission electron microscope.

[0035] Figure 3 This is a morphological image of extracellular vesicles GE11m@EV-T modified with GE11m peptide, observed under a transmission electron microscope.

[0036] Figure 4 This is a morphological image of GE11m@EV-T-SD9 after loading SD9, observed under a transmission electron microscope.

[0037] Figure 5 The image shows the results of the nanoparticle tracking and analysis instrument detecting the size distribution and concentration of extracellular vesicles.

[0038] Figure 6This is a diagram showing the results of Western blotting for detecting extracellular vesicle markers.

[0039] Figure 7 The figure shows the results of nanoflow cytometry analysis of the binding efficiency of GE11m-CP05 and EV-T.

[0040] Figure 8 HPLC chromatogram of natural L-type GE11m peptide after incubation in mouse serum at 37°C for 1 h.

[0041] Figure 9 HPLC chromatogram of D-amino acid modified GE11m peptide (Mut-2-GE11m) after incubation in mouse serum at 37°C for 1 h.

[0042] Figure 10 This is a graph showing the time-dependent changes in GE11m@EV-T-SD9 uptake in MDA-MB-231 cells observed by confocal microscopy.

[0043] Figure 11 This figure compares the effects of different treatment groups on apoptosis induction in MDA-MB-231 cells and mesenchymal stem cells as detected by flow cytometry.

[0044] Figure 12 Fluorescent staining images showing changes in the cytoskeleton and nuclear morphology of MDA-MB-231 cells after 24 hours of treatment with different drugs. Detailed Implementation

[0045] The following is in conjunction with the appendix Figure 1-12 The present invention will be further described in detail with reference to specific embodiments, but the scope of protection of the present invention is not limited to the following embodiments. Any equivalent substitutions or improvements made by those skilled in the art based on the technical solutions disclosed in the present invention should be included within the scope of protection of the present invention.

[0046] The reagents and materials used in the embodiments of this invention are all available from commercial sources. Umbilical cord mesenchymal stem cells (UC-MSCs) were purchased from the Cell Bank of the Chinese Academy of Sciences; TRAIL gene-transfected mesenchymal stem cells (MSC-flT) were prepared according to literature methods (Yuan Z et al., TRAIL delivery by MSC-derived extracellular vesicles is an effective anticancer therapy, Journal of Extracellular Vesicles, 2017, Vol. 6, p. 1344369); DMEM / F-12 medium and fetal bovine serum were purchased from Gibco; GE11m-CP05 fusion peptide and rhodamine B-labeled GE11mRhoB-CP05 fusion peptide were custom-synthesized by Hefei Sener Biotechnology Co., Ltd. using the Fmoc / t-Bu solid-phase synthesis strategy, with D-type Fmoc-amino acid raw materials used at positions 1, 4, and 7, with a purity ≥95%; natural L-type GE11m-CP05 fusion peptide was also synthesized by Hefei Sener Biotechnology Co., Ltd.; stevia derivative SD9 was synthesized according to literature methods (Liu JS et al., Synthesis and Biological Evaluation of Steviol Derivatives with Improved Cytotoxic Activity and Selectivity, Journal of Natural Products, 2022, Vol. 85, No. 8, pp. 1945-1958); Exosome isolation kit purchased from Beibei Biotechnology Co., Ltd. (Cat. 084001); Ultracentrifuge: Beckman Optima L80 XP; Transmission Electron Microscope: JEM-1400; Nanoparticle Tracking Analyzer: Malvern NanoSight NS300; Flow Cytometer: NgygCye:2060R; Nanoflow Cytometer: NanoFCM; Confocal Microscope: Zeiss LSM 880; High Performance Liquid Chromatography: Shimadzu LC-20A.

[0047] Example 1: Design and synthesis of GE11m peptide.

[0048] This embodiment relates to the design principles and synthesis methods of D-type amino acid-modified GE11m targeting peptides.

[0049] While the natural L-type GE11m peptide (sequence: YHWYGYTPQNVI) exhibits good EGFR targeting ability, it is readily degraded by proteases in vivo. To address this issue, this invention designed several D-type amino acid substitution variants for stability screening: L-GE11m: Natural sequence YHWYGYTPQNVI (positive control); Mut-1-GE11m: C-terminal easily cleaved site substitution, sequence YHWYGYTPQN D VI D Among them, asparagine at position 10 and isoleucine at position 12 are D-type; Mut-2-GE11m (i.e., GE11m): N-terminal and easily cleaved sites in the middle are replaced, the sequence is Y. D HWY D GYT D PQNVI, in which tyrosine at position 1, tyrosine at position 4, and threonine at position 7 are D-type.

[0050] The modified peptides described above were custom-synthesized by Hefei Sener Biotechnology Co., Ltd. using a solid-phase peptide synthesis (SPPS) method. The synthesis employed an Fmoc / t-Bu protection strategy, using L-type and D-type Fmoc-amino acid raw materials respectively, and Rink Amide resin to obtain C-terminal amidated peptides, which more closely approximate the natural state. After synthesis, the peptides were lysed, purified (reversed-phase high-performance liquid chromatography, RP-HPLC), and lyophilized. Mass spectrometry (MS) confirmed the correct molecular weight.

[0051] The GE11m-CP05 fusion peptide directly links the GE11m targeting peptide to the extracellular vesicle membrane anchoring peptide CP05 (sequence: CRHSQMTVTSRL) via peptide bonds. The complete sequence is Y. D HWY D GYT D PQNVI-CRHSQMTVTSRL contains a total of 24 amino acid residues and has a theoretical molecular weight of approximately 2998 Da. The lyophilized fusion peptide powder should be stored at -20°C protected from light. Before use, it should be dissolved in sterile PBS or DMSO to prepare a stock solution of 1 mg / mL.

[0052] Example 2: Serum stability evaluation of GE11m peptide.

[0053] This embodiment relates to the evaluation of the stability of GE11m peptide in a serum environment.

[0054] Two peptides, L-GE11m and GE11m (Mut-2-GE11m), were added to freshly prepared mouse serum to a final peptide concentration of 100 μg / mL and incubated at 37°C. Samples were taken at 1 hour, serum proteins were precipitated with trichloroacetic acid, and the remaining peptide amount was determined by HPLC.

[0055] like Figure 8 As shown, the HPLC chromatogram of natural L-type GE11m peptide after incubation in mouse serum at 37°C for 1 h revealed that the main peak (retention time 17.418 min) accounted for only 45.1% of the total area, while multiple degradation product peaks were also observed: the peak at retention time 14.941 min accounted for 17.8% of the total area, the peak at retention time 16.929 min accounted for 33.8% of the total area, and the peak at retention time 17.777 min accounted for 3.3% of the total area. These results indicate that natural GE11m peptide has undergone significant degradation in serum, with more than half of the peptide being broken down into degradation products.

[0056] like Figure 9 As shown, the HPLC chromatogram of the D-amino acid-modified GE11m peptide (Mut-2-GE11m) after incubation with mouse serum at 37°C for 1 h showed that the main peak (retention time 10.957 min) accounted for 95.4% of the total area, with only a very small amount of degradation product peaks (area share <5%). Compared with the natural GE11m peptide, the intact peptide retention rate of the GE11m peptide after incubation increased from 45.1% to 95.4%, an increase of approximately 2.1 times.

[0057] Based on the above HPLC chromatograms, it can be concluded that D-amino acid modification of GE11m peptide significantly improves its stability in mouse serum. The D-amino acid modification design of GE11m peptide (replacing positions 1, 4, and 7 with D-amino acids) effectively protects the peptide from degradation by serum proteases, significantly prolonging its effective duration of action in vivo.

[0058] Example 3: Culture of mesenchymal stem cells.

[0059] This embodiment relates to a cell culture method for collecting extracellular vesicles.

[0060] Umbilical cord mesenchymal stem cells (UC-MSCs) or TRAIL gene-transfected mesenchymal stem cells (MSC-flTs) cryopreserved in liquid nitrogen were removed and rapidly thawed in a 37°C water bath. Once completely thawed, the cells were immediately transferred to 15mL centrifuge tubes containing 5mL of complete culture medium. The complete culture medium was prepared by adding 10% fetal bovine serum to DMEM / F-12 basal medium. The cell suspension was centrifuged at 1000rpm for 5min, the supernatant was discarded, and the cells were resuspended in the complete culture medium. The cells were then transferred to T25 cell culture flasks and incubated at 37°C in a 5% CO2 incubator. The culture medium was changed every 2-3 days, and cell growth was observed.

[0061] When the cell density reaches 80%-90% confluence, perform passage. Discard the old culture medium, gently wash the cells twice with pre-warmed PBS buffer, add an appropriate amount of 0.25% trypsin-EDTA digestion solution, and incubate at 37°C for 2-3 minutes until the cells become rounded and detach. Add an equal volume of complete culture medium to neutralize the trypsin activity, gently pipette to completely disperse the cells into a single-cell suspension, transfer to a centrifuge tube, and centrifuge at 1000 rpm for 5 minutes. Discard the supernatant, resuspend the cells in complete culture medium, and seed them into new culture flasks at a ratio of 1:3. After labeling, return the flasks to the incubator for further culture. Passage culture to maintain the cells in the logarithmic growth phase, and collect extracellular vesicles from passages 3-5.

[0062] Example 4: Isolation and extraction of extracellular vesicles (EV-T).

[0063] This embodiment relates to a method for isolating and extracting extracellular vesicles containing TRAIL protein.

[0064] Take MSC-flT cells of good growth status from passages 3-5, at a concentration of 5 × 10⁻⁶. 5 Cells were seeded at a density of approximately 80% in T75 cell culture flasks. When cell confluence reached approximately 80%, the original culture medium was discarded, and the cells were gently washed 2-3 times with PBS to thoroughly remove exogenous vesicles from the serum. The medium was then replaced with serum-free DMEM / F-12 conditioned medium containing exosomes and serum, and cultured for another 48 hours. The cell supernatant was collected; this is the conditioned medium containing extracellular vesicles.

[0065] Extracellular vesicles were isolated and extracted using a differential centrifugation combined with ultrafiltration concentration. First, the collected conditioned cultures were pretreated by gradient centrifugation at 4°C: centrifugation at 300×g for 10 min removed suspended cells, and the supernatant was collected; centrifugation at 2000×g for 15 min removed dead cells and cell debris, and the supernatant was collected; centrifugation at 10000×g for 30 min removed large vesicles and apoptotic bodies, and the supernatant was collected. Then, the pretreated supernatant was filtered through a 0.22 μm filter membrane to further remove microvesicles and contaminating particles larger than 220 nm.

[0066] Transfer the filtered liquid to the upper layer of a 100 kDa ultrafiltration tube for ultrafiltration concentration. Centrifuge at 4°C and 4000×g for 10 min to concentrate the supernatant approximately 10 times. Transfer the concentrated liquid to ultracentrifuge tubes and balance each tube precisely to two decimal places. Place the balanced tubes in an ultracentrifuge and centrifuge at 4°C and 120000×g for 2 h. After centrifugation, carefully discard the supernatant; a translucent milky white precipitate will be visible at the bottom of the tube. Invert the tubes onto clean filter paper and let stand for 10 min to remove any residual liquid.

[0067] Add 500 μL of sterile PBS buffer filtered through a 0.22 μm filter, and gently pipette to fully resuspend the precipitate. Wash again by centrifugation at 120,000 × g for 1 h at 4 °C. Discard the supernatant, add 200 μL of PBS to resuspend the precipitate, and obtain the purified EV-T extracellular vesicle suspension. Aliquot the extracellular vesicles and store at -80 °C for later use.

[0068] Example 5: Identification and characterization of extracellular vesicles.

[0069] This embodiment relates to the morphology, particle size distribution, and biomarker identification of extracellular vesicles.

[0070] Observation was performed using transmission electron microscopy. 10 μL of extracellular vesicle suspension was dropped onto a copper grid and allowed to stand at room temperature for 5 minutes to allow the extracellular vesicles to adhere to the grid. Excess liquid was blotted away with filter paper, and 2% phosphotungstic acid negative staining solution was added for 2 minutes. The staining solution was then blotted away, and the mixture was dried at room temperature before observation under an electron microscope. Figure 2 As shown, EV-T exhibits a typical saucer-like or biconcave disc-shaped vesicle structure under transmission electron microscopy, with clear and complete vesicle membrane boundaries and a diameter of approximately 70-130 nm, consistent with the morphological characteristics of extracellular vesicles.

[0071] The nanoparticle tracking analyzer was used for detection. The extracellular vesicle suspension was diluted 1000-fold with PBS to a suitable concentration and injected into the sample cell of the nanoparticle tracking analyzer for detection. Figure 5As shown, the detection results indicate that the particle size of extracellular vesicle particles is mainly distributed in the range of 50-200 nm, with a peak particle size of 124.5 nm, a median particle size (D50) of 135.0 nm, and a particle concentration of 1.2 × 10⁻⁶. 11 The particle size distribution is consistent with the size characteristics of extracellular vesicles and is relatively uniform, indicating that the prepared extracellular vesicle sample is of good quality.

[0072] Western blotting was used. Extracellular vesicles were lysed using RIPA lysis buffer, and total protein was extracted. Protein concentration was determined using the BCA method. 20 μg of protein sample was separated by SDS-PAGE electrophoresis and then transferred to a PVDF membrane. After blocking with 5% skim milk powder for 1 h, TSG101 primary antibody (dilution ratio 0.5-1.1:0.5-1.1000) was added and incubated overnight at 4°C. The next day, after washing the membrane, HRP-labeled secondary antibody was added and incubated at room temperature for 1 h. ECL chemiluminescence was then used for color development. Figure 6 As shown, Western blot results indicate that both EV-T and GE11m@EV-T samples exhibit a clear band at the 44kDa position, consistent with the theoretical molecular weight of TSG101. This demonstrates that the prepared vesicle samples express the exosome-specific positive marker TSG101, confirming that they are true extracellular vesicles.

[0073] Example 6: Modification of GE11m-targeting peptide on the surface of extracellular vesicles.

[0074] This embodiment relates to a method for modifying the surface of extracellular vesicles with GE11m mediated by CP05-anchored peptides.

[0075] Take 10 μg of EV-T extracellular vesicles prepared in Example 4 and mix with 10 μg of the GE11m-CP05 fusion peptide synthesized in Example 1. Add PBS to adjust the total volume of the mixture to 300 μL. Place the mixture on a shaker at 4°C and incubate slowly for 4-6 hours to allow the CP05 segment of the fusion peptide to specifically bind to the CD63 tetraspanic membrane protein on the surface of the extracellular vesicles. During incubation, the GE11m targeting peptide is stably anchored to the surface of the extracellular vesicle membrane through the CP05-CD63 interaction.

[0076] After incubation, the mixture was transferred to a 100 kDa ultrafiltration tube and centrifuged at 7500 × g for 45 min at 4 °C to remove unbound free fusion peptides. The filtrate was discarded, and the precipitate in the cutoff solution was resuspended in 300 μL of PBS. To further improve the purity of extracellular vesicles, a secondary purification was performed using an exosome isolation kit. The specific steps were as follows: EP Solution was added to the extracellular vesicle solution according to the manufacturer's instructions (sample volume:EP Solution = 3:1), and the mixture was slowly mixed and incubated on a shaker at 0-4 °C for 1 h. Then, the mixture was centrifuged at 12000 × g for 15 min at 4 °C, and the supernatant was slowly discarded. The centrifuge tube was inverted on absorbent paper for 5 min to thoroughly remove any residual supernatant. 500 μL of PS Buffer was added, and the precipitate was repeatedly pipetted for 3-5 min. Then, the mixture was incubated at 37 °C for 15 min to promote dissolution, and the precipitate was resuspended by pipetting for another 1-3 min. Finally, GE11m-modified extracellular vesicles GE11m@EV-T were obtained, resuspended in particle-free PBS, and stored at -80°C for later use.

[0077] like Figure 3 As shown, the morphological changes of extracellular vesicles after GE11m peptide modification were observed using transmission electron microscopy. GE11m@EV-T still exhibited a typical cup-shaped vesicle structure under transmission electron microscopy, with a particle size of approximately 70-130 nm. Compared with unmodified EV-T, the morphology showed no significant change, and the vesicle membrane boundaries were clear and intact. These results indicate that surface modification mediated by CP05-anchored peptides does not alter the morphological characteristics of extracellular vesicles, nor does it disrupt the integrity of the extracellular vesicle membrane structure. The same results are expected with GE11m modification.

[0078] Example 7: Detection and verification of GE11m modification efficiency.

[0079] This embodiment relates to a method for detecting the binding efficiency of GE11m to extracellular vesicles.

[0080] The binding efficiency of GE11m-CP05 to EV-T cells was detected using nanoflow cytometry. 10 μg of extracellular vesicles from EV-T cells were incubated with 10 μg of rhodamine B-labeled GE11mRhoB-CP05 fusion peptide according to the method described in Example 6 to prepare GE11mRhoB@EV-T cells. Unmodified EV-T cells were used as a negative control group.

[0081] 10 μL each of GE11mRhoB@EV-T and unmodified EV-T were analyzed using nanoflow cytometry. The fluorescence positivity rate of the extracellular vesicle samples was analyzed using Rhodamine B fluorescence signal as the detection index. Figure 7As shown, nanoflow cytometry analysis revealed that the proportion of fluorescently positive particles in the Rhodamine B-labeled GE11mRhoB@EV-T samples reached 93.5%, while the fluorescence positivity rate in the unmodified EV-T control group was only 9.28% (belonging to background signal). This result strongly demonstrates that the vast majority of the GE11m-CP05 fusion peptides successfully bound to the extracellular vesicle membrane surface of EV-T cells, with a GE11m modification efficiency exceeding 93.5%.

[0082] The GE11m-CP05 fusion peptide differs from the natural GE11m-CP05 fusion peptide only in the chiral conformation of the 1st, 4th, and 7th amino acids (D-type vs. L-type), while the CP05 anchoring region sequence is completely identical. Therefore, the binding mechanism and binding efficiency of GE11m-CP05 to CD63 protein on the surface of extracellular vesicles are expected to be comparable to those of natural GE11m-CP05, achieving a modification efficiency of over 90%.

[0083] Example 8: GE11m@EV-T loaded with TRAIL sensitizing drug SD9.

[0084] This embodiment relates to a method for loading the TRAIL sensitizing drug SD9 into the GE11m@EV-T capsule using ultrasound.

[0085] Stevia derivative SD9 (chemical name: 15-oxo-13-propionyloxy-enantio-kaurene-19-carboxylic acid) was synthesized according to literature methods. SD9 powder was dissolved in DMSO to prepare a 10 mM stock solution and stored at -20°C protected from light.

[0086] Take 20 μg of GE11m@EV-T and dissolve it in PBS to prepare 1 mL of a 20 μg / mL solution. Add 2 μL of SD9 stock solution (final concentration 20 μM). Place the mixture under the probe of an sonicator and sonicate under the following conditions: sonication power 25%, temperature 25℃, 6 sonication cycles, each cycle 35 s, with a 35 s interval. After sonication, incubate the sample at 37℃ for 1 h to allow the extracellular vesicle membrane structure to repair itself.

[0087] After incubation, the free SD9 drug was removed by ultracentrifugation at 100,000 × g for 1.5 h. The supernatant was discarded, and the precipitate was resuspended in PBS to obtain the targeted nanodelivery system GE11m@EV-T-SD9, which simultaneously carries TRAIL protein on the membrane surface and SD9 in the vesicle cavity.

[0088] like Figure 4As shown, transmission electron microscopy revealed the morphology of GE11m@EV-T-SD9 loaded with SD9. The extracellular vesicle membrane structure remained intact, and the vesicle morphology was well maintained, demonstrating that ultrasound-based drug loading does not damage the structural integrity of extracellular vesicles. The same results are expected for GE11m@EV-T-SD9.

[0089] The encapsulation efficiency of SD9 was determined by HPLC. An appropriate amount of GE11m@EV-T-SD9 sample was taken, and methanol was added to break the membrane and extract SD9. The SD9 content was then detected by HPLC. Encapsulation efficiency = (actual loading amount / theoretical loading amount) × 100%. The results showed that the encapsulation efficiency of SD9 was approximately 78.5%, indicating that the ultrasonic method can achieve high-efficiency drug loading.

[0090] Example 9: Validation of the in vitro cell uptake and apoptosis induction effects of GE11m@EV-T-SD9.

[0091] This embodiment involves the verification of the effects of GE11m@EV-T-SD9 on cellular uptake and induction of tumor cell apoptosis.

[0092] MDA-MB-231 cells were seeded in confocal culture dishes at a density of 2 × 10⁻⁶. 5 Cells were placed in a dish and allowed to adhere overnight. Fluorescently labeled GE11m@EV-T-SD9 was added, and the cells were incubated at 37°C for 3 h, 6 h, 12 h, and 24 h, respectively. After incubation, the culture medium was discarded, the cells were washed three times with PBS, and fixed with 4% paraformaldehyde for 15 min. Cell nuclei were labeled with DAPI staining solution, and fluorescence imaging was performed using a Zeiss LSM 880 confocal microscope.

[0093] like Figure 10 As shown, confocal microscopy observations revealed the following: at 3 hours, a small amount of fluorescence signal was observed around the cells and in the cytoplasm, indicating that M231 cells began to take up exosomes; at 6 hours, the intracellular fluorescence signal was significantly enhanced, and the exosomes were distributed in a punctate pattern in the cytoplasm, with good co-localization of GE11 and EV-T fluorescence signals, proving that GE11-modified exosomes could be completely taken up; at 12 hours, with further increases in uptake and cumulative drug action time, morphological changes were observed in M231 cells, and the cytoskeleton began to become disordered; at 24 hours, the cell morphology changed significantly, with cells shrinking and becoming rounded, and chromatin condensation, exhibiting typical apoptotic morphological characteristics.

[0094] These results demonstrate that GE11@EV-T-SD9 can efficiently target and be taken up and internalized by EGFR-overexpressing M231 tumor cells in a time-dependent manner. With increasing uptake and prolonged action, the TRAIL and SD9 carried by the exosomes work synergistically to effectively induce programmed cell death in tumor cells.

[0095] As incubation time increased, the fluorescence signal in MDA-MB-231 cells gradually increased, indicating that the uptake of GE11m@EV-T-SD9 by the cells increased in a time-dependent manner.

[0096] The experiment was set up with the following groups: (1) control group (PBS); (2) SD9 group (SD9 final concentration 2 μM); (3) EV-T group (EV-T 10 μg / mL); (4) GE11m@EV-T group (GE11m@EV-T 10 μg / mL); (5) SD9@EV-T group (unmodified GE11m, loaded with SD9); (6) GE11m@EV-T-SD9 group.

[0097] MDA-MB-231 cells and normal mesenchymal stem cells (MSCs) were respectively administered at 3×10⁻⁶. 5 Cells were seeded in 6-well plates and cultured overnight. The appropriate treatments were added according to the above groupings, and the cells were cultured for another 24 hours. Cells were collected, stained with Annexin V-FITC / PI double staining kit, and apoptosis rate was detected by flow cytometry.

[0098] like Figure 11 As shown, flow cytometry results indicate that the GE11m@EV-T-SD9 treatment group significantly induced apoptosis in MDA-MB-231 cells compared to the individual treatment groups and the non-targeted group; while for normal mesenchymal stem cells, the apoptosis rate of each treatment group remained at a low level, demonstrating that the preparation has good biocompatibility with normal cells.

[0099] After treating MDA-MB-231 cells according to the above grouping for 24 hours, F-actin cytoskeleton was labeled with phalloidin-Alexa Fluor 488, cell nuclei were labeled with DAPI, and cell morphology changes were observed by confocal microscopy.

[0100] like Figure 12 As shown, the control group cells exhibited normal adherent growth morphology and intact cytoskeleton structure; the cells in the GE11m@EV-T-SD9 treatment group showed morphological changes.

[0101] The design principles and expected mechanism of action of the GE11m@EV targeted nanodelivery system of this invention can be explained from the following aspects.

[0102] At the molecular level, the innovatively designed GE11m targeting peptide achieved significantly enhanced serum stability through D-amino acid modification. HPLC results showed that the GE11m peptide retained 95.4% of its intact peptide after incubation at 37°C for 1 hour in mouse serum, compared to only 45.1% for the natural L-type GE11m peptide. Because of their opposite chiral conformation to the natural substrate, D-amino acids are less easily recognized and cleaved by proteases, effectively protecting the peptide from degradation. Furthermore, the D-amino acid modification sites (positions 1, 4, and 7) were strategically designed to avoid key residue regions that directly bind to EGFR, thus preserving EGFR-targeting activity.

[0103] At the vector level, the GE11m-CP05 fusion peptide can be stably anchored to the surface of extracellular vesicles through the specific interaction between the CP05 segment and the CD63 protein on the surface of extracellular vesicles. Referring to the modification efficiency data of the natural GE11m peptide, the modification efficiency of GE11m-CP05 is expected to reach over 90%. This non-genetic engineering method is simple to operate and does not affect the natural characteristics of extracellular vesicles.

[0104] At the application level, when EV-T cells expressing TRAIL protein are used as the carrier and loaded with the TRAIL-sensitizing drug SD9, GE11m@EV-T-SD9 can deliver TRAIL protein on the membrane surface and SD9 drug in the cyst cavity to EGFR-highly expressed tumor cells simultaneously and efficiently through EGFR-mediated targeted delivery, achieving a synergistic anti-cancer effect.

[0105] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A stable GE11m peptide-modified extracellular vesicle anticancer targeted nanodelivery system, characterized in that: The nanodelivery system includes extracellular vesicles (EV-Ts) expressing the TRAIL protein. The EV-Ts are derived from mesenchymal stem cells transfected with the TRAIL gene, and their membrane surface carries the biologically active TRAIL protein. The mesenchymal stem cells are selected from umbilical cord mesenchymal stem cells, bone marrow mesenchymal stem cells, or adipose-derived mesenchymal stem cells, and are at passage 3-5. The EV-T surface is linked to a GE11m targeting peptide via the binding of a CP05 anchoring peptide to the CD63 protein. The GE11m targeting peptide is a stable derivative of the GE11m peptide obtained by D-type amino acid substitution, and its amino acid sequence is Y. D HWY D GYT D PQNVI, wherein the first, fourth, and seventh tyrosine residues are D-type amino acids, and the remaining amino acids are L-type amino acids; the amino acid sequence of the CP05 anchoring peptide is CRHSQMTVTSRL; the EV-T expresses the exosome-specific positive markers TSG101 and CD63; the particle size of the nanodelivery system is 70-150 nm, and the binding efficiency of the GE11m targeting peptide is not less than 90%; The GE11m targeting peptide and the CP05 anchoring peptide are linked by peptide bonds to form a GE11m-CP05 fusion peptide, the amino acid sequence of which is Y. D HWY D GYT D PQNVI-CRHSQMTVTSRL, a total of 24 amino acid residues, has a purity of not less than 95%.

2. The nanodelivery system according to claim 1, characterized in that: The HPLC peak area of ​​the GE11m targeting peptide after incubation at 37°C for 1 hour in mouse serum was no less than 90%.

3. The nanodelivery system according to claim 1, characterized in that: The EV-T contains a TRAIL sensitizing drug, which is an active substance that can enhance the sensitivity of tumor cells to TRAIL-induced apoptosis.

4. The nanodelivery system according to claim 3, characterized in that: The TRAIL sensitizing drug is an inhibitor of the PI3K / Akt / mTOR signaling pathway.

5. A method for preparing the stable GE11m peptide-modified extracellular vesicle anticancer targeted nanodelivery system according to any one of claims 1-4, characterized in that, Includes the following steps: Step 1: Isolation and extraction of extracellular vesicles: Mesenchymal stem cells were isolated and extracted at a concentration of 5 × 10⁻⁶ cells / year. 5 Cells were seeded at high density in T75 culture flasks. When the cell confluence reached 80%, the medium was replaced with serum-free medium and cultured for another 24-48 hours. The conditioned medium was collected and subjected to low-speed centrifugation, filtration through a 0.22 μm filter membrane, concentration through a 100 kDa ultrafiltration tube, and ultracentrifugation at 120,000 × g for 2 hours to obtain extracellular vesicles. The mesenchymal stem cells were TRAIL gene-transfected mesenchymal stem cells, and the extracellular vesicles were EV-T cells expressing the TRAIL protein. Step 2, Synthesis of GE11m-CP05 fusion peptide: Using the Fmoc / t-Bu solid-phase peptide synthesis strategy, D-type Fmoc-amino acid raw materials were used to introduce D-type amino acids at the 1st, 4th, and 7th tyrosine positions to synthesize the GE11m-CP05 fusion peptide. Step 3: GE11m modification on the surface of extracellular vesicles: The EV-T prepared in Step 1 and the GE11m-CP05 fusion peptide synthesized in Step 2 were mixed at a mass ratio of 0.5-1.1:0.5-1.1 and incubated in a shaker at 4°C for 4-6 hours. Unbound free peptides were removed by ultrafiltration and centrifugation to obtain GE11m-modified extracellular vesicles GE11m@EV-T.

6. The preparation method according to claim 5, characterized in that: The process also includes step four, loading the TRAIL sensitizing drug: the GE11m@EV-T prepared in step three is mixed with the TRAIL sensitizing drug, and the TRAIL sensitizing drug is loaded into the EV-T cavity using an ultrasonic method; the conditions for the ultrasonic method are: ultrasonic power 20-30%, temperature 20-30℃, ultrasonic cycles 4-8 times, each ultrasonic cycle 30-40s, with an interval of 30-40s; after ultrasonication, the exosome membrane structure is repaired by incubation at 37℃ for 0.5-1.5h, and the free drug is removed by ultracentrifugation to obtain the drug-loaded GE11m@EV-T.

7. The use of the stable GE11m peptide-modified extracellular vesicle anticancer targeted nanodelivery system according to any one of claims 1-4 in the preparation of drug delivery formulations targeting EGFR-highly expressed tumors, characterized in that, The tumor is a solid tumor that highly expresses EGFR, such as breast cancer, lung cancer, colorectal cancer, or pancreatic cancer. The drug delivery formulation specifically recognizes and binds to the EGFR receptor highly expressed on the surface of tumor cells via the GE11m targeting peptide, mediating the endocytosis and uptake of the nanodelivery system by the tumor cells, thereby achieving targeted delivery of antitumor active substances.

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