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CN-122005834-A - Stable GE11m peptide modified extracellular vesicle anticancer targeting nano delivery system, preparation method and application thereof

CN122005834ACN 122005834 ACN122005834 ACN 122005834ACN-122005834-A

Abstract

The invention discloses a stable GE11m peptide modified extracellular vesicle anticancer targeting nano-delivery system, a preparation method and application thereof, belonging to the technical field of biological medicine preparations, wherein the nano-delivery system comprises extracellular vesicles which can be engineered by TRAIL gene and are derived from mammalian cells, the surface is connected with a D-type amino acid modified GE11m targeting peptide through a CP05 anchoring peptide, the amino acid sequence of the GE11m targeting peptide is Y D HWY D GYT D PQNVI, and the protease resistance and serum stability of the targeting peptide are obviously enhanced by replacing the 1 st tyrosine, the 4 th tyrosine and the 7 th threonine in the GE11m peptide sequence with the D-type amino acid.

Inventors

  • YUAN ZHENGQIANG
  • JIANG JIAHONG
  • WU ZELIN
  • PENG JUN
  • XU NUO
  • XIE BIN

Assignees

  • 广东工业大学

Dates

Publication Date
20260512
Application Date
20260414

Claims (10)

  1. 1. A stable GE11m peptide modified extracellular vesicle targeted nano delivery system is characterized by comprising extracellular vesicles which are derived from mammalian cells and can be engineered by TRAIL genes, wherein the surfaces of the extracellular vesicles are connected with GE11m targeted peptides through the binding action of CP05 anchor peptides and CD63 proteins, the GE11m targeted peptides are stable derivatives obtained by replacing GE11m peptides with D-type amino acids, the amino acid sequence of the stable derivatives is Y D HWY D GYT D PQNVI, tyrosine 1, tyrosine 4 and threonine 7 are D-type amino acids, the rest amino acids are L-type amino acids, the amino acid sequence of the CP05 anchor peptides is CRHSQMTVTSRL, the particle size of the nano delivery system is 70-150nm, and the binding efficiency of the GE11m targeted peptides is not lower than 90%.
  2. 2. The nano-delivery system according to claim 1, wherein the GE11m targeting peptide is linked to the CP05 anchor peptide via a peptide bond to form a GE11m-CP05 fusion peptide, wherein the fusion peptide has an amino acid sequence of Y D HWY D GYT D PQNVIPQNVI-CRHSQMTVTSRL and a purity of not less than 95%.
  3. 3. The nano-delivery system according to claim 1, wherein the GE11m targeting peptide has a main HPLC peak area ratio of not less than 90% after incubation in mouse serum at 37 ℃ for 1h, which is significantly improved over the native L-type GE11m peptide.
  4. 4. The nano-delivery system according to claim 1, wherein the extracellular vesicles are extracellular vesicles EV-T expressing TRAIL protein, the EV-T is derived from mesenchymal stem cells transfected by TRAIL gene, and the membrane surface of the EV-T carries TRAIL protein with biological activity.
  5. 5. The nano-delivery system according to claim 4, wherein the capsule cavity of EV-T is loaded with TRAIL-sensitized drug, which is an active substance capable of enhancing sensitivity of tumor cells to TRAIL-induced apoptosis.
  6. 6. The nano-delivery system according to claim 5, wherein the TRAIL-sensitized drug is a PI3K/Akt/mTOR signaling pathway inhibitor.
  7. 7. The nano-delivery system according to claim 1, wherein the mammalian cells capable of being engineered by TRAIL gene comprise mesenchymal stem cells, wherein the mesenchymal stem cells are one of umbilical cord mesenchymal stem cells, bone marrow mesenchymal stem cells or adipose mesenchymal stem cells, the mesenchymal stem cells are 3 rd-5 th generation cells, and the extracellular vesicles express exosome specific positive markers TSG101 and CD63.
  8. 8. The preparation method of the stable GE11m peptide modified extracellular vesicle targeted nano-delivery system according to any one of claims 1 to 7 is characterized by comprising the following steps of firstly, separating and extracting extracellular vesicles, namely inoculating mesenchymal stem cells with the density of 5X 10 5 cells/T75 culture flasks, continuously culturing for 24 to 48 hours by changing the culture medium into serum-free culture medium when the cell fusion degree reaches 80%, collecting the conditioned medium, and sequentially performing low-speed centrifugation, 0.22 mu m filter membrane filtration, 100kDa ultrafiltration tube concentration and 120000X g ultracentrifugation for 2 hours to obtain extracellular vesicles; synthesizing GE11m-CP05 fusion peptide, namely adopting Fmoc/T-Bu solid phase peptide synthesis strategy, introducing D-type amino acid into the 1 st tyrosine, 4 th tyrosine and 7 th threonine positions by using D-type Fmoc-amino acid raw material to synthesize the GE11m-CP05 fusion peptide, carrying out step three, namely carrying out surface GE11m modification on the extracellular vesicles prepared in step one and the GE11m-CP05 fusion peptide synthesized in step two according to the mass ratio of 0.5-1.1:0.5-1.1, incubating for 4-6h in a 4 ℃ table, and removing unbound free peptide fragments by ultrafiltration centrifugation to obtain the GE11m modified extracellular vesicles GE11m@EV, wherein the mesenchymal stem cells in step one are the mesenchymal stem cells transfected by TRAIL genes, the extracellular vesicles are the EV-T expressing TRAIL proteins, and the product obtained in step three is the GE11m@EV-T.
  9. 9. The preparation method of the medicine for treating the diabetes mellitus, the method is characterized by further comprising the step of loading the TRAIL sensitization medicine, wherein the GE11m@EV-T prepared in the step of mixing the TRAIL sensitization medicine with the TRAIL sensitization medicine, and loading the TRAIL sensitization medicine into an EV-T capsule cavity by adopting an ultrasonic method, wherein the ultrasonic method is carried out under the conditions that ultrasonic power is 20-30%, the temperature is 20-30 ℃, ultrasonic circulation is carried out for 4-8 times, each time is 30-40 seconds, the interval is 30-40 seconds, the exosome membrane structure is repaired after the ultrasonic treatment is completed, the free medicine is removed by ultracentrifugation, and the GE1m@EV-T carrying the medicine is obtained.
  10. 10. The use of the stabilized GE11m peptide-modified extracellular vesicle-targeted nano-delivery system according to any one of claims 1 to 7 for preparing a drug delivery preparation for targeting EGFR-high-expression tumors, wherein the tumors are solid tumors with EGFR high expression, including breast cancer, lung cancer, colorectal cancer or pancreatic cancer, and the drug delivery preparation specifically recognizes and binds to EGFR receptors with high expression on the surface of tumor cells through the GE11m targeting peptide, and mediates endocytic uptake of the nano-delivery system by the tumor cells, thereby realizing targeted delivery of antitumor active substances.

Description

Stable GE11m peptide modified extracellular vesicle anticancer targeting nano delivery system, preparation method and application thereof Technical Field The invention belongs to the technical field of biological medicine preparations, and particularly relates to a stable GE11m peptide modified extracellular vesicle targeted nano delivery system, a preparation method thereof and application thereof in preparation of an antitumor drug delivery preparation. Background Tumors are one of serious diseases seriously threatening human health, and traditional chemotherapeutic drugs often cause serious toxic and side effects on normal tissues while killing tumor cells due to lack of targeting. In recent years, nanotechnology-based targeted drug delivery systems have become a research hotspot in the field of tumor therapy. Extracellular vesicles (Extracellular Vesicles, EV) are considered ideal drug delivery vehicles (Weng Z et al ,Therapeutic roles of mesenchymal stem cell-derived extracellular vesicles in cancer,Journal of hematology & oncology,2021, volume 14, page 136) as a class of natural nanoscale membrane vesicles secreted by cells, typically 30-150nm in particle size, with good biocompatibility, low immunogenicity, and natural ability to cross biological barriers. The extracellular vesicles from mesenchymal stem cells are an important research object for tumor targeted therapy because of the advantages of easy acquisition, large-scale amplification culture, certain tumor chemotaxis and the like. However, unmodified natural extracellular vesicles lack active targeting ability and are easily cleared by reticuloendothelial system in vivo, resulting in lower drug enrichment efficiency at tumor sites. Thus, surface engineering modifications of extracellular vesicles to confer active targeting functions are a key strategy to enhance 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). The Epidermal Growth Factor Receptor (EGFR) is a transmembrane tyrosine kinase receptor and is in a high over-expression state on the surfaces of tumor cells of various solid tumors such as breast cancer, lung cancer, colorectal cancer and the like. GE11m is a dodecapeptide (sequence YHWYGYTPQNVI) obtained by phage display technology screening, can specifically bind to EGFR receptor, does not activate downstream signal pathway compared with natural ligand EGF, and is a safe and effective EGFR targeting ligand. Research shows that the GE11m modified nano delivery system can remarkably improve the tumor targeting delivery efficiency, and has good application prospect in the treatment of EGFR high-expression tumors such as lung cancer (Gaurav I et al ,Peptide-Conjugated Vascular Endothelial Extracellular Vesicles Encapsulating Vinorelbine for Lung Cancer Targeted Therapeutics,Nanomaterials,2024, volume 14, 20 and 1669). However, the use of native L-type GE11m peptides in vivo presents significant challenges. The GE11m peptide is easily and rapidly degraded in vivo by various proteases, resulting in poor serum stability and short half-life. This inherent defect severely restricts the clinical transformation application prospect of the GE11m peptide modified nano-delivery system. In the prior art, although there have been attempts to improve the stability of polypeptides by chemical modification or PEGylation, etc., these methods often affect the binding affinity of the polypeptide to the target or significantly increase the molecular weight and immunogenicity. Therefore, there is a need to develop an innovative modification strategy that can significantly improve the stability of GE11m peptides while maintaining their EGFR targeting activity. D-form amino acids are chiral isomers of L-form amino acids that occur in nature in certain microorganisms and specific biomolecules. Substitution of L-type amino acids at specific sites in the polypeptide chain with D-type amino acids is a classical strategy for improving the resistance of polypeptides to proteases. D-type amino acid is not easy to be recognized and cut by protease due to the fact that the chiral configuration of the D-type amino acid is opposite to that of a natural substrate, so that the in vivo half-life of the polypeptide is remarkably prolonged. However, D-type amino acid substitutions may affect the three-dimensional conformation of the polypeptide, which in turn affects its binding activity to the target. Therefore, how to achieve the optimal balance of stability and activity through reasonable D-type amino acid substitution design on the premise of maintaining the targeting activity of the polypeptide is a technical challenge facing the field. The extracellular vesicle surface modification method mainly comprises two major types of genetic engineering m