CN114146064A - Genetically engineered cell membrane bionic nano-microsphere with pancreatic cancer microenvironment targeting function and method thereof - Google Patents
Genetically engineered cell membrane bionic nano-microsphere with pancreatic cancer microenvironment targeting function and method thereof Download PDFInfo
- Publication number
- CN114146064A CN114146064A CN202111368423.5A CN202111368423A CN114146064A CN 114146064 A CN114146064 A CN 114146064A CN 202111368423 A CN202111368423 A CN 202111368423A CN 114146064 A CN114146064 A CN 114146064A
- Authority
- CN
- China
- Prior art keywords
- pancreatic cancer
- cell membrane
- gemcitabine
- solution
- m2pep
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 210000000170 cell membrane Anatomy 0.000 title claims abstract description 61
- 230000008685 targeting Effects 0.000 title claims abstract description 60
- 239000004005 microsphere Substances 0.000 title claims abstract description 57
- 206010061902 Pancreatic neoplasm Diseases 0.000 title claims abstract description 54
- 208000015486 malignant pancreatic neoplasm Diseases 0.000 title claims abstract description 54
- 201000002528 pancreatic cancer Diseases 0.000 title claims abstract description 54
- 208000008443 pancreatic carcinoma Diseases 0.000 title claims abstract description 54
- 239000011664 nicotinic acid Substances 0.000 title claims abstract description 32
- 238000000034 method Methods 0.000 title claims abstract description 16
- 210000004027 cell Anatomy 0.000 claims abstract description 54
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 35
- SDUQYLNIPVEERB-QPPQHZFASA-N gemcitabine Chemical compound O=C1N=C(N)C=CN1[C@H]1C(F)(F)[C@H](O)[C@@H](CO)O1 SDUQYLNIPVEERB-QPPQHZFASA-N 0.000 claims abstract description 32
- 229960005277 gemcitabine Drugs 0.000 claims abstract description 32
- 210000002540 macrophage Anatomy 0.000 claims abstract description 22
- 241000713666 Lentivirus Species 0.000 claims abstract description 10
- 238000005119 centrifugation Methods 0.000 claims abstract description 8
- 239000000243 solution Substances 0.000 claims description 36
- 229920001577 copolymer Polymers 0.000 claims description 32
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 15
- 230000002018 overexpression Effects 0.000 claims description 12
- 239000011259 mixed solution Substances 0.000 claims description 11
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 10
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 10
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 claims description 10
- 108090000623 proteins and genes Proteins 0.000 claims description 8
- 239000011258 core-shell material Substances 0.000 claims description 7
- 239000012634 fragment Substances 0.000 claims description 7
- 238000002156 mixing Methods 0.000 claims description 7
- 239000002077 nanosphere Substances 0.000 claims description 7
- 108010076504 Protein Sorting Signals Proteins 0.000 claims description 6
- 239000013592 cell lysate Substances 0.000 claims description 6
- 239000002502 liposome Substances 0.000 claims description 6
- 238000002360 preparation method Methods 0.000 claims description 6
- 238000009210 therapy by ultrasound Methods 0.000 claims description 6
- 230000003592 biomimetic effect Effects 0.000 claims description 5
- 108010048367 enhanced green fluorescent protein Proteins 0.000 claims description 5
- 238000001914 filtration Methods 0.000 claims description 5
- 239000007788 liquid Substances 0.000 claims description 5
- 239000012528 membrane Substances 0.000 claims description 5
- 229910052757 nitrogen Inorganic materials 0.000 claims description 5
- 108091005703 transmembrane proteins Proteins 0.000 claims description 5
- 102000035160 transmembrane proteins Human genes 0.000 claims description 5
- 238000000108 ultra-filtration Methods 0.000 claims description 5
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 4
- 238000010353 genetic engineering Methods 0.000 claims description 4
- 239000000203 mixture Substances 0.000 claims description 4
- 102000004169 proteins and genes Human genes 0.000 claims description 4
- 238000010276 construction Methods 0.000 claims description 3
- 230000008014 freezing Effects 0.000 claims description 3
- 238000007710 freezing Methods 0.000 claims description 3
- 230000014509 gene expression Effects 0.000 claims description 3
- 229920000515 polycarbonate Polymers 0.000 claims description 3
- 239000004417 polycarbonate Substances 0.000 claims description 3
- 239000002244 precipitate Substances 0.000 claims description 3
- 238000000746 purification Methods 0.000 claims description 3
- 238000000926 separation method Methods 0.000 claims description 3
- 239000010414 supernatant solution Substances 0.000 claims description 3
- 238000010257 thawing Methods 0.000 claims description 3
- 102000000588 Interleukin-2 Human genes 0.000 claims description 2
- 108010002350 Interleukin-2 Proteins 0.000 claims description 2
- 229920000954 Polyglycolide Polymers 0.000 claims description 2
- 238000004873 anchoring Methods 0.000 claims description 2
- 230000006037 cell lysis Effects 0.000 claims description 2
- 239000003550 marker Substances 0.000 claims description 2
- 150000003906 phosphoinositides Chemical class 0.000 claims description 2
- 229920000747 poly(lactic acid) Polymers 0.000 claims description 2
- 239000004633 polyglycolic acid Substances 0.000 claims description 2
- 239000004626 polylactic acid Substances 0.000 claims description 2
- 230000000694 effects Effects 0.000 abstract description 13
- 206010028980 Neoplasm Diseases 0.000 abstract description 4
- 238000009825 accumulation Methods 0.000 abstract description 3
- 230000008901 benefit Effects 0.000 abstract description 3
- 238000001727 in vivo Methods 0.000 abstract description 3
- 229920000642 polymer Polymers 0.000 abstract description 3
- 230000000973 chemotherapeutic effect Effects 0.000 abstract description 2
- 239000003814 drug Substances 0.000 abstract description 2
- 238000004945 emulsification Methods 0.000 abstract description 2
- 238000001890 transfection Methods 0.000 abstract description 2
- 238000011354 first-line chemotherapy Methods 0.000 abstract 1
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 4
- 230000005540 biological transmission Effects 0.000 description 4
- 229910052802 copper Inorganic materials 0.000 description 4
- 239000010949 copper Substances 0.000 description 4
- 210000000496 pancreas Anatomy 0.000 description 4
- 108010052285 Membrane Proteins Proteins 0.000 description 3
- 102000018697 Membrane Proteins Human genes 0.000 description 3
- 201000011510 cancer Diseases 0.000 description 3
- 238000002512 chemotherapy Methods 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- 238000000338 in vitro Methods 0.000 description 3
- 239000002539 nanocarrier Substances 0.000 description 3
- 210000004881 tumor cell Anatomy 0.000 description 3
- 239000011248 coating agent Substances 0.000 description 2
- 238000000576 coating method Methods 0.000 description 2
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 238000000684 flow cytometry Methods 0.000 description 2
- 239000002953 phosphate buffered saline Substances 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- XBBVURRQGJPTHH-UHFFFAOYSA-N 2-hydroxyacetic acid;2-hydroxypropanoic acid Chemical compound OCC(O)=O.CC(O)C(O)=O XBBVURRQGJPTHH-UHFFFAOYSA-N 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- COQLPRJCUIATTQ-UHFFFAOYSA-N Uranyl acetate Chemical compound O.O.O=[U]=O.CC(O)=O.CC(O)=O COQLPRJCUIATTQ-UHFFFAOYSA-N 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- 239000003519 biomedical and dental material Substances 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 229940044683 chemotherapy drug Drugs 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 231100000517 death Toxicity 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 238000013399 early diagnosis Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000001125 extrusion Methods 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 238000001215 fluorescent labelling Methods 0.000 description 1
- 230000003211 malignant effect Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000002105 nanoparticle Substances 0.000 description 1
- 230000000242 pagocytic effect Effects 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 150000003905 phosphatidylinositols Chemical class 0.000 description 1
- 238000001338 self-assembly Methods 0.000 description 1
- 238000000527 sonication Methods 0.000 description 1
- 238000005728 strengthening Methods 0.000 description 1
- 230000004614 tumor growth Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/51—Nanocapsules; Nanoparticles
- A61K9/5107—Excipients; Inactive ingredients
- A61K9/5176—Compounds of unknown constitution, e.g. material from plants or animals
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
- A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
- A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
- A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
- A61K31/7068—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines having oxo groups directly attached to the pyrimidine ring, e.g. cytidine, cytidylic acid
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/51—Nanocapsules; Nanoparticles
- A61K9/5107—Excipients; Inactive ingredients
- A61K9/513—Organic macromolecular compounds; Dendrimers
- A61K9/5146—Organic macromolecular compounds; Dendrimers obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, polyamines, polyanhydrides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/18—Drugs for disorders of the alimentary tract or the digestive system for pancreatic disorders, e.g. pancreatic enzymes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/86—Viral vectors
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0693—Tumour cells; Cancer cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2510/00—Genetically modified cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2740/00—Reverse transcribing RNA viruses
- C12N2740/00011—Details
- C12N2740/10011—Retroviridae
- C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
- C12N2740/15041—Use of virus, viral particle or viral elements as a vector
- C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Veterinary Medicine (AREA)
- Zoology (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Pharmacology & Pharmacy (AREA)
- Biotechnology (AREA)
- Wood Science & Technology (AREA)
- General Engineering & Computer Science (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Epidemiology (AREA)
- Molecular Biology (AREA)
- Nanotechnology (AREA)
- Optics & Photonics (AREA)
- Microbiology (AREA)
- Biophysics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Virology (AREA)
- Cell Biology (AREA)
- Oncology (AREA)
- Plant Pathology (AREA)
- Botany (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicinal Preparation (AREA)
Abstract
The invention discloses a genetically engineered cell membrane bionic nano-microsphere with pancreatic cancer microenvironment targeting and a method thereof. Expressing tumor-related macrophage targeting peptide on the surface of pancreatic cancer cells KPC in a lentivirus transfection mode, and constructing a pancreatic cancer cell line over-expressed by macrophage targeting peptide M2 pep; loading gemcitabine (a first-line chemotherapy medicament for pancreatic cancer) in a polylactic acid-glycolic acid polymer by using a multiple emulsion method, and self-assembling to form polylactic acid-glycolic acid nano microspheres; and (3) extracting the cell membrane vesicles of the pancreatic cancer cell line by using a gradient centrifugation method, and encapsulating polylactic acid-glycollic acid nano microspheres to obtain the pancreatic cancer microenvironment reinforced targeted genetically engineered cell membrane bionic nano microspheres. The cell membrane bionic nano-microsphere can be enriched in pancreatic cancer tissues in a large amount, reduces non-specific accumulation of other tissues in vivo, realizes a specific targeting effect on the pancreatic cancer tissues, and has the advantage of accurately delivering a large amount of chemotherapeutic gemcitabine to the pancreatic cancer tissues.
Description
Technical Field
The invention relates to a biomedical material, in particular to a genetically engineered cell membrane bionic nano-microsphere with pancreatic cancer microenvironment targeting and a preparation method thereof, belonging to the technical field of preparation of bionic nano-carriers.
Background
Pancreatic cancer is a malignant tumor that occurs in the pancreas, and accounts for about 1% to 2% of the total malignant tumor, and has been increasing in recent years. The most common part of the tumor growth is the head of the pancreas, which is about more than 60%, and the tail part of the pancreas, which is the tail part of the pancreas, and the total pancreatic cancer is the least, which only accounts for about 5%. Men are more than women, and the age of high incidence is 40-65 years. The disease is the most malignant, and the number of deaths ranks the fourth malignant tumor. Because the clinical manifestations are hidden, the onset is rapid, and the early diagnosis is very difficult, most of them are diagnosed at the late stage. Chemotherapy based on a nano system is becoming the popular direction in the research field of nano drug tumor treatment in recent years, and has the characteristics of high treatment accuracy, small damage to normal tissues and high biological safety. The treatment effect of the existing chemotherapy is limited, and the side effect is large. The efficient targeting nano-carrier is used for carrying a great amount of chemotherapeutic drug molecules to be enriched in pancreatic cancer, and the method is one of strategies for improving the treatment effect. The tumor cell membrane bionic nano-microsphere is a bionic nano-microsphere with a tumor cell membrane coating, and the membrane protein of tumor cells provides the functions of camouflage and active targeting of tumor tissues for a nano-carrier. The targeting peptide is a short peptide with high affinity for specific cellular components. Through a gene engineering mode, the targeting peptide is expressed on the surface of the cell membrane so as to realize the superposition of targeting effects, and hopefully construct the cell membrane bionic nano-microsphere for strengthening the pancreatic cancer targeting effect.
Disclosure of Invention
In order to solve the problems in the background art, the invention aims to provide a genetically engineered cell membrane bionic nano-microsphere with pancreatic cancer targeting and a preparation method thereof. The homologous homing targeting effect of the pancreatic cancer cell membrane bionic nano system and the active affinity targeting effect of the tumor-related macrophage targeting peptide are combined together to realize the double targeting enhancement effect. Meanwhile, the nano system with the chemotherapy function is efficiently delivered to pancreatic cancer tissues by utilizing the targeting capability to pancreatic cancer, so that the pancreatic cancer is treated.
The method comprises the steps of obtaining a genetically engineered cell membrane in a cell culture mode, and wrapping the cell membrane on the surface of the polylactic acid-glycolic acid copolymer nano microsphere in a co-extrusion mode of a liposome extruder to form the genetically engineered cell membrane bionic nano microsphere with pancreatic cancer microenvironment targeting. The operation is simple and easy, and the process parameters are easy to control. The cell membrane bionic nano-microspheres can be enriched in pancreatic cancer tissues in a large amount, reduce non-specific accumulation of other tissues in vivo and realize a specific targeting effect on the pancreatic cancer tissues. .
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
a genetically engineered cell membrane bionic nano-microsphere with pancreatic cancer microenvironment targeting comprises:
the nano system is a core-shell structure, the diameter of the nano system is about 100nm, and the outer shell of the core-shell structure is KPC of pancreatic cancer cell line KPC of genetic engineering over-expression transmembrane proteinM2pep+A cell membrane; the inner core of the core-shell structure is a poly lactic acid-glycolic acid copolymer microsphere for encapsulating gemcitabine.
The over-expressed transmembrane protein is over-expressed macrophage targeting peptide M2pep and mainly comprises a signal peptide, a targeting peptide, enhanced green fluorescent protein, 3XFLAG marker peptide and glycosylated phosphoinositide anchoring protein which are connected in sequence; the signal peptide is an IL-2 transmembrane protein signal peptide, and the targeting peptide is a macrophage specific targeting peptide.
The Enhanced Green Fluorescent Protein (EGFP) is used for membrane protein fluorescent labeling, and the amino acid sequence of the Enhanced green fluorescent protein is 'MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK'.
3XFLAG labeled peptide is used for detecting target protein by Western Blot.
Glycosylated phosphatidylinositol anchor proteins are used to stabilize membrane protein structures.
The amino acid sequence of the macrophage specificity targeting peptide is YEQDPWGVKWWY, such as SEQ ID NO. 1.
In the gemcitabine-encapsulated polylactic acid-glycolic acid copolymer microspheres, the ratio of polylactic acid: the mass ratio of the polyglycolic acid is 1: 1, the mass ratio of the polylactic acid-glycolic acid copolymer to the gemcitabine is 5: 1-1: 5.
secondly, a preparation method of the genetically engineered cell membrane bionic nano-microsphere, which specifically comprises the following steps:
the method comprises the following steps: cell line KPC for genetically engineering expression of M2pep targeting peptideM2pep+The construction of (1):
constructing an overexpression macrophage targeting peptide M2pep and a corresponding lentivirus thereof, transfecting the overexpression macrophage targeting peptide M2pep to pancreatic cancer cells through the lentivirus, and establishing a pancreatic cancer cell line which stably expresses the overexpression macrophage targeting peptide M2pep on cell membranes;
in the first step, pancreatic cancer cells are treatedLaying KPC in a 6-hole plate, culturing for 24 hours in an incubator at 37 ℃, adding lentivirus corresponding to the overexpression macrophage targeting peptide M2pep with 20 microliter/hole, and culturing for 24 hours at 37 ℃ to obtain the pancreatic cancer cell line KPC of the genetic engineering overexpression macrophage targeting peptide M2pepM2pep+。
Step two: KPC (Key performance control)M2pep+Cell membrane separation and purification:
collecting pancreatic cancer cells stably expressing over-expressed macrophage targeting peptide M2pep on cell membranes, using cell lysate containing 1% of phenylmethylsulfonyl fluoride by mass fraction to perform cell lysis, repeatedly freezing and thawing cell solution at normal temperature in liquid nitrogen, and using a gradient centrifugation method to obtain cell membrane fragment solution;
in the second step, the cells are scraped off by using a cell scraper, the cells are collected by centrifuging at 1000 rpm for 5 minutes, then 1 ml of cell lysate containing 1% by mass of phenylmethylsulfonyl fluoride is added, and the cell lysate is sequentially placed in liquid nitrogen and repeatedly frozen and thawed at normal temperature until the cells are broken; followed by centrifugation at 4 ℃ and 700g for 10 minutes, collection of the supernatant solution, and finally 14000g for 30 minutes, collection of the precipitate as KPCM2pep+Cell membrane fragments.
Step three: preparing the gemcitabine molecule-entrapped polylactic acid-glycolic acid copolymer nano microspheres:
step four: preparing a genetically engineered cell membrane bionic nano microsphere:
according to the mass ratio of 1: and 2, mixing the cell membrane fragment solution and the polylactic acid-glycolic acid copolymer nano microspheres containing gemcitabine molecules, extruding the mixture through a polycarbonate porous membrane with the thickness of 200nm by using a liposome extruder after ultrasonic treatment, and repeatedly processing the mixture by using the liposome extruder to obtain the genetically engineered cell membrane bionic nano microspheres with uniform size distribution.
And step three, specifically, dropwise adding the acetonitrile solution of the polylactic acid-glycolic acid copolymer into the ethanol solution of gemcitabine by using an ultrasonic instrument for ultrasonic treatment to obtain a mixed solution, centrifuging the obtained mixed solution by using an ultrafiltration tube to remove free gemcitabine molecules, and filtering by using a needle filter to remove the polylactic acid-glycolic acid copolymer microspheres with larger diameters to obtain the polylactic acid-glycolic acid copolymer nanospheres containing gemcitabine molecules with uniform size distribution.
The ultrasonic instrument is specifically an ultrasonic cell disruptor.
Preparing an 80 wt% acetonitrile solution of 2 g/l polylactic acid-glycolic acid copolymer and a 4 wt% ethanol solution of 0.75 g/l gemcitabine, and dropwise adding the polylactic acid-glycolic acid copolymer solution into the gemcitabine solution under the ultrasonic wave of 20kHz frequency and 40W power, wherein the mass mixing ratio is 1: 1, carrying out ultrasonic treatment for 10 minutes to obtain a mixed solution, centrifuging the obtained mixed solution for 30 minutes by using an Amicon ultra-4 ultrafiltration tube at 3000 rpm, taking the upper layer solution, and filtering the upper layer solution for 3 times by using a 0.22 micron needle filter to obtain the gemcitabine molecule-encapsulated polylactic acid-glycolic acid copolymer nanospheres with the average diameter of 0.22 micron.
In the fourth step, an ultrasonic cell disruptor is used for disrupting the cells at the frequency of 20kHz and the power of 20W according to the mass ratio of 1: 2, ultrasonically mixing the cell membrane fragment solution and the polylactic acid-glycolic acid copolymer nano microsphere containing gemcitabine molecules.
The invention expresses pancreatic cancer targeted peptide on the surface of pancreatic cancer KPC in a lentivirus transfection mode, constructs KPC over-expressed by pancreatic cancer targeted peptide M2pepM2pep+Pancreatic cancer cell lines. Gemcitabine is loaded in polylactic-co-glycolic acid (PLGA) polymer by using a multiple emulsion method, and the polylactic-co-glycolic acid nanometer microsphere is formed by self-assembly. Extraction of KPC using gradient centrifugationM2pep+The cell membrane vesicles of the pancreatic cancer cell line entrap the polylactic acid-glycollic acid nano microspheres to obtain the pancreatic cancer enhanced targeted genetically engineered cell membrane bionic nano microspheres. The nano-microsphere has the advantage of accurately delivering the chemotherapeutic gemcitabine to pancreatic cancer tissues in a large quantity.
The invention has the advantages that:
the cell membrane bionic nano-microsphere can be enriched in pancreatic cancer tissues in a large amount, reduces non-specific accumulation of other tissues in vivo and realizes a specific targeting effect on the pancreatic cancer tissues.
Drawings
Fig. 1 is a schematic structural view of the present invention.
FIG. 2 is a transmission electron microscope and particle size plot of the present invention.
FIG. 3 is an in vitro targeting map of the present invention.
FIG. 4 is an in vitro targeting map of the present invention.
Detailed Description
The invention will be further described with reference to the following examples and the accompanying drawings.
Example 1:
the method comprises the following steps: cell line KPC for genetically engineering expression of M2pep targeting peptideM2pep+Construction of
Pancreatic cancer cell line KPC was plated at a density of 1X 105/well in 6-well plates and cultured routinely at 37 ℃ for 24 hours in an incubator. Transfecting KPC cells by using an M2pep targeting peptide overexpression virus packaged by lentiviruses, adding 20 microliters of lentiviruses into each hole, and culturing at 37 ℃ for 24 hours to obtain KPC expressing the M2pep targeting peptideM2pep+A cell line.
Step two: KPC (Key performance control)M2pep+Cell membrane separation and purification
Culture of KPC Using a 10cm diameter Petri dishM2pep+Cells, after 80% of the culture dish was filled with cells, the medium was removed, washed once with phosphate buffered saline solution, the cells were gently scraped with a cell scraper, and the cells were transferred to a 15 ml centrifuge tube. The cells were collected by centrifugation at 1000 rpm for 5 minutes, the supernatant was aspirated and then gently resuspended in phosphate buffered saline, a small amount of the cells was counted, and 600g of the remaining cells were centrifuged for 5 minutes to remove the supernatant. 1 ml of cell lysate containing 1% phenylmethylsulfonyl fluoride was added to the centrifuge tube, and the cells were thoroughly suspended by pipetting with a pipette and allowed to stand on an ice bath for 15 minutes to swell the cells. And then repeatedly freezing and thawing the centrifuge tube in liquid nitrogen at normal temperature until the cells are broken. Followed by centrifugation at 4 ℃ and 700g for 10 minutes, the supernatant solution was collected and finally 14000g for 30 minutes to collect the precipitate as cell membrane debris.
Step three: preparing the poly (lactic acid-glycolic acid) copolymer nano-microspheres encapsulating gemcitabine molecules.
2 mg of polylactic acid-glycolic acid copolymer was dissolved in 1 ml of 80% acetonitrile solution, and 0.75 mg of gemcitabine was dissolved in 1 ml of 4% ethanol solution. The polylactic acid-glycolic acid copolymer solution was added dropwise to the gemcitabine solution using an ultrasonic cell disruptor at a frequency of 20kHz and a power of 40W to give 2 ml of a mixed solution, and sonicated for 10 minutes. The resulting mixed solution was centrifuged at 3000 rpm for 30 minutes using an Amicon ultra-4 ultrafiltration tube. And filtering the upper solution by using a 0.22 micron needle filter for 3 times to obtain the gemcitabine molecule-encapsulated polylactic acid-glycolic acid copolymer nano microspheres with the average diameter of 0.22 micron.
Step four: preparing the gene engineering cell membrane bionic nano-microsphere.
Mixing a polylactic acid-glycolic acid copolymer solution with the gemcitabine of which the mass is 0.2 mg with a cell membrane solution of which the mass is 0.1 mg, and metering to 1 ml. Using an ultrasonic cell disruptor, sonication was carried out at a frequency of 20kHz and a power of 20W for 5 minutes to mix well. The mixed solution is extruded through a polycarbonate porous membrane with the diameter of 200 nanometers by using an Avanti liposome extruder, and the process is repeated for 11 times to ensure that the cell membrane can be fully coated on the surface of the polylactic acid-glycolic acid copolymer nanometer microsphere, so as to form the gene engineering cell membrane bionic nanometer microsphere with the diameter of about 200 nanometers.
And (3) using a transmission electron microscope to represent the morphology of the genetically engineered cell membrane bionic nano-microspheres. Absorbing 10 mu L of nano microsphere solution, gently dripping the nano microsphere solution on a transmission electron microscope carbon supporting film copper mesh, drying the water on the copper mesh by using an infrared lamp, and dripping 5 mu L of 2% uranyl acetate solution to carry out copper mesh negative dyeing. And (3) drying by using an infrared lamp again, putting the copper mesh into a sample inlet hole of a transmission electron microscope, and taking a picture to obtain the shape of the nano system as shown in figure 2. The result shows that the gene engineering cell membrane bionic nano-microsphere is of a core-shell structure, the diameter is distributed about 100 nanometers, and the thickness of the cell membrane coating is about 7 nanometers. The cell membrane vesicle is positioned on the surface of the nano microsphere, and the core of the nano microsphere is polylactic acid-glycolic acid copolymer encapsulating gemcitabine molecules.
Flow cytometry detection of phagocytic capacity was performed using pancreatic cancer cell lines KPC. The cells were seeded in 96-well plates at 1 × 104/well, 200 μ l of genetically engineered cell membrane biomimetic nanoparticles with a concentration of 20 μmol were added, and the average fluorescence intensity was measured by flow cytometry after 3 hours of culture, as shown in fig. 3 and 4. The results show that the average fluorescence intensity of the genetically engineered cell membrane bionic nano-microspheres is 58586 which is 2.26 times and 3.95 times the average fluorescence intensity of the common cell membrane bionic nano-microspheres and the polymer nano-microspheres respectively, and the genetically engineered cell membrane bionic nano-microspheres are proved to have the effect of efficient in-vitro targeting.
Therefore, the genetically engineered cell membrane bionic nano-microsphere obtained by the embodiment of the invention has a specific targeting effect on pancreatic cancer tissues.
The foregoing is considered as illustrative and not restrictive, and any modifications, equivalents and improvements made within the spirit and scope of the present invention are intended to be included therein.
The sequence related by the invention is as follows:
SEQ ID NO.1：
name: the source of the amino acid sequence of the macrophage specific targeting peptide is as follows: artificial Sequence (Artificial Sequence) YEQDPWGVKWWY.
Sequence listing
<110> Zhejiang University (Zhejiang University)
<120> genetically engineered cell membrane bionic nano-microsphere with pancreatic cancer microenvironment targeting and method thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 36
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
tacgagcagg acccttgggg cgtgaagtgg tggtac 36
Claims (10)
1. A genetically engineered cell membrane bionic nano-microsphere with pancreatic cancer microenvironment targeting is characterized in that: the nano system is of a core-shell structure, and the outer shell of the core-shell structure is a cell membrane of a pancreatic cancer cell which expresses transmembrane protein through genetic engineering; the inner core of the core-shell structure is a poly lactic acid-glycolic acid copolymer microsphere for encapsulating gemcitabine.
2. The genetically engineered cell membrane biomimetic nanosphere with pancreatic cancer microenvironment targeting of claim 1, wherein: the over-expressed transmembrane protein is over-expressed macrophage targeting peptide M2pep and mainly comprises a signal peptide, a targeting peptide, enhanced green fluorescent protein, 3XFLAG marker peptide and glycosylated phosphoinositide anchoring protein which are connected in sequence; the signal peptide is an IL-2 transmembrane protein signal peptide, and the targeting peptide is a macrophage specific targeting peptide.
3. The genetically engineered cell membrane biomimetic nanosphere with pancreatic cancer microenvironment targeting of claim 1, wherein: the amino acid sequence of the macrophage specific targeting peptide is YEQDPWGVKWWY.
4. The genetically engineered cell membrane biomimetic nanosphere with pancreatic cancer microenvironment targeting of claim 1, wherein: in the gemcitabine-encapsulated polylactic acid-glycolic acid copolymer microspheres, the ratio of polylactic acid: the mass ratio of the polyglycolic acid is 1: 1, the mass ratio of the polylactic acid-glycolic acid copolymer to the gemcitabine is 5: 1-1: 5.
5. the preparation method of the genetically engineered cell membrane biomimetic nano-microsphere according to any one of claims 1 to 4, characterized in that: the preparation method specifically comprises the following steps:
the method comprises the following steps: cell line KPC for genetically engineering expression of M2pep targeting peptideM2pep+The construction of (1):
constructing an overexpression macrophage targeting peptide M2pep and a corresponding lentivirus thereof, transfecting the overexpression macrophage targeting peptide M2pep to pancreatic cancer cells through the lentivirus, and establishing a pancreatic cancer cell line which stably expresses the overexpression macrophage targeting peptide M2pep on cell membranes;
step two: KPC (Key performance control)M2pep+Cell membrane separation and purification:
collecting pancreatic cancer cells stably expressing over-expressed macrophage targeting peptide M2pep on cell membranes, using cell lysis solution containing phenylmethylsulfonyl fluoride to lyse the cells, repeatedly freezing and thawing the cell solution at normal temperature in liquid nitrogen, and using a gradient centrifugation method to obtain cell membrane debris solution;
step three: preparing the gemcitabine molecule-entrapped polylactic acid-glycolic acid copolymer nano microspheres:
step four: preparing a genetically engineered cell membrane bionic nano microsphere:
mixing the cell membrane fragment solution and the polylactic acid-glycolic acid copolymer nano microspheres containing gemcitabine molecules, extruding the mixture through a polycarbonate porous membrane by using a liposome extruder after ultrasonic treatment, and repeatedly processing the mixture by using the liposome extruder to obtain the genetically engineered cell membrane bionic nano microspheres with uniform size distribution.
6. The method of claim 5, wherein:
in the first step, pancreatic cancer cells KPC are paved in a 6-hole plate, cultured in an incubator at 37 ℃ for 24 hours, added with lentivirus corresponding to overexpression macrophage targeting peptide M2pep in 20 microliter/hole, and cultured at 37 ℃ for 24 hours to obtain the pancreatic cancer cell lines KPC of the genetic engineering overexpression macrophage targeting peptide M2pepM2pep+。
7. The method of claim 5, wherein:
in the second step, the cells are scraped off by using a cell scraper, the cells are collected by centrifuging at 1000 rpm for 5 minutes, then 1 ml of cell lysate containing 1% by mass of phenylmethylsulfonyl fluoride is added, and the cell lysate is sequentially placed in liquid nitrogen and repeatedly frozen and thawed at normal temperature until the cells are broken; followed by centrifugation at 4 ℃ and 700g for 10 minutes, collection of the supernatant solution, and finally 14000g for 30 minutes, collection of the precipitate as KPCM2pep+Cell membrane fragments.
8. The method of claim 5, wherein:
and step three, specifically, dropwise adding the acetonitrile solution of the polylactic acid-glycolic acid copolymer into the ethanol solution of gemcitabine by using an ultrasonic instrument for ultrasonic treatment to obtain a mixed solution, centrifuging the obtained mixed solution by using an ultrafiltration tube to remove free gemcitabine molecules, and filtering by using a needle filter to remove the polylactic acid-glycolic acid copolymer microspheres with larger diameters to obtain the polylactic acid-glycolic acid copolymer nanospheres containing gemcitabine molecules with uniform size distribution.
9. The method of claim 5, wherein:
preparing an 80 wt% acetonitrile solution of 2 g/l polylactic acid-glycolic acid copolymer and a 4 wt% ethanol solution of 0.75 g/l gemcitabine, and dropwise adding the polylactic acid-glycolic acid copolymer solution into the gemcitabine solution under the ultrasonic wave of 20kHz frequency and 40W power, wherein the mass mixing ratio is 1: 1, carrying out ultrasonic treatment for 10 minutes to obtain a mixed solution, centrifuging the obtained mixed solution for 30 minutes by using an Amicon ultra-4 ultrafiltration tube at 3000 rpm, taking the upper layer solution, and filtering the upper layer solution for 3 times by using a 0.22 micron needle filter to obtain the gemcitabine molecule-encapsulated polylactic acid-glycolic acid copolymer nanospheres with the average diameter of 0.22 micron.
10. The method of claim 5, wherein:
in the fourth step, an ultrasonic cell disruptor is used for disrupting the cells at the frequency of 20kHz and the power of 20W according to the mass ratio of 1: 2, ultrasonically mixing the cell membrane fragment solution and the polylactic acid-glycolic acid copolymer nano microsphere containing gemcitabine molecules.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111368423.5A CN114146064A (en) | 2021-11-18 | 2021-11-18 | Genetically engineered cell membrane bionic nano-microsphere with pancreatic cancer microenvironment targeting function and method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111368423.5A CN114146064A (en) | 2021-11-18 | 2021-11-18 | Genetically engineered cell membrane bionic nano-microsphere with pancreatic cancer microenvironment targeting function and method thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN114146064A true CN114146064A (en) | 2022-03-08 |
Family
ID=80456786
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111368423.5A Pending CN114146064A (en) | 2021-11-18 | 2021-11-18 | Genetically engineered cell membrane bionic nano-microsphere with pancreatic cancer microenvironment targeting function and method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114146064A (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108837154A (en) * | 2018-08-31 | 2018-11-20 | 中国药科大学 | A kind of preparation and application of cellular membrane biomimetic lipoprotein targeted nano delivery system |
| CN111249478A (en) * | 2020-03-16 | 2020-06-09 | 浙江大学 | Decitabine nano-carrier and application thereof in preparation of tumor fluorescence imaging agent |
| CN111733139A (en) * | 2020-01-13 | 2020-10-02 | 西安电子科技大学 | Functionalized macrophage/monocyte-based targeted delivery system and construction and application thereof |
| CN112569189A (en) * | 2019-09-29 | 2021-03-30 | 复旦大学 | Low-toxicity biomimetic nano system capable of simultaneously regulating tumor microenvironment and killing tumor cells in targeted manner and construction method |
| CN113499317A (en) * | 2021-07-09 | 2021-10-15 | 郑州大学 | Preparation method and application of multifunctional bionic nano-drug |
-
2021
- 2021-11-18 CN CN202111368423.5A patent/CN114146064A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108837154A (en) * | 2018-08-31 | 2018-11-20 | 中国药科大学 | A kind of preparation and application of cellular membrane biomimetic lipoprotein targeted nano delivery system |
| CN112569189A (en) * | 2019-09-29 | 2021-03-30 | 复旦大学 | Low-toxicity biomimetic nano system capable of simultaneously regulating tumor microenvironment and killing tumor cells in targeted manner and construction method |
| CN111733139A (en) * | 2020-01-13 | 2020-10-02 | 西安电子科技大学 | Functionalized macrophage/monocyte-based targeted delivery system and construction and application thereof |
| CN111249478A (en) * | 2020-03-16 | 2020-06-09 | 浙江大学 | Decitabine nano-carrier and application thereof in preparation of tumor fluorescence imaging agent |
| CN113499317A (en) * | 2021-07-09 | 2021-10-15 | 郑州大学 | Preparation method and application of multifunctional bionic nano-drug |
Non-Patent Citations (2)
| Title |
|---|
| LIANG PANG等: "Surface modification of polymeric nanoparticles with M2pep peptide for drug delivery to tumor-associated macrophages", 《HHS PUBLIC ACCESS》 * |
| 乔治•C.普伦德加斯特等: "《癌症的免疫治疗》", 30 November 2019, 东南大学出版社 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109666695B (en) | Targeted integrin alphavbeta 3 exosome vector and preparation method and application thereof | |
| CN111603454A (en) | Multi-targeting fusion cell membrane modified bionic nano delivery system and preparation method and application thereof | |
| CN114225027B (en) | Genetically engineered liver cancer targeting cell membrane bionic nano microsphere and preparation method thereof | |
| CN112168973B (en) | Aptamer delivery vehicle, preparation method and application thereof | |
| CN112760293B (en) | Method for preparing high-activity exosomes by 3D culture of MSC stem cells by using xeno-free serum | |
| CN111110855A (en) | Targeting vesicle medicine prepared from erythrocytes | |
| CN112494495B (en) | Preparation method of cancer cell membrane chimeric liposome nano drug delivery system | |
| WO2024050923A1 (en) | Neuronal transfection method based on adipose-derived exosomes and use thereof | |
| CN115960838A (en) | Internal and external engineered exosome and preparation method and application thereof | |
| CN118001416B (en) | PD-1 modified extracellular vesicle with dihydroartemisinin entrapped therein and preparation method thereof | |
| CN115029320A (en) | Engineered exosome for tumor radiotherapy sensitization, preparation method and application | |
| CN114146064A (en) | Genetically engineered cell membrane bionic nano-microsphere with pancreatic cancer microenvironment targeting function and method thereof | |
| CN110075122B (en) | Liver cancer therapeutic exosome medicine | |
| CN115737826A (en) | Extracellular vesicle loaded with polydopamine nanoparticles and preparation method thereof | |
| CN114869911A (en) | Application of PD-1 cell membrane nano vesicle combined stem cell membrane in postoperative treatment of malignant melanoma | |
| CN116763938A (en) | Extracellular vesicle nucleic acid nano-drug delivery system, preparation method and application thereof | |
| CN113041358A (en) | Superparamagnetic modified neutrophil exosome bionic vesicle drug delivery biological agent and preparation method thereof | |
| CN115887679B (en) | Gene-chemotherapy nano drug co-delivery system, preparation method and application thereof | |
| CN111378685B (en) | Method for preparing medicine-carrying exosome and medicine-carrying exosome | |
| CN115998709B (en) | Membrane fusion nano nucleic acid vector and preparation method and application thereof | |
| CN115252555B (en) | Membrane fusion liposome, preparation method and application thereof in protein delivery | |
| CN117363652B (en) | Dual-transcription factor regulated dual-start plasmid, nano material, preparation method and application thereof | |
| CN115404212B (en) | Small-particle-size nano cell membrane vesicle, preparation method, composition and kit | |
| CN117530932B (en) | Artificial exosome for inhibiting tumor growth and lung metastasis and application thereof | |
| CN114369575B (en) | Brain glioma cell-derived exosome without tumor promotion function, and preparation method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20220308 |
|
| RJ01 | Rejection of invention patent application after publication |